Acacia Monograph and Spray-Dried Acacia Monograph

1-1
Monographs
Medicinal and Pharmaceutical Substances
1-2
Acacia
revised 1/01
Acacia complies with the requirements of the 3rd edition of the European Pharmacopoeia [0307]. These
requirements are reproduced after the heading Definition below.
Action and use Bulk-forming laxative; pharmaceutical aid.
When Powdered Acacia is prescribed or demanded, material complying with the requirements below
with the exception of Identification test A shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Acacia is the air-hardened, gummy exudate flowing naturally from or obtained by incision of the
trunk and branches of Acacia senegal L. Willdenow and other species of Acacia of African origin.
CHARACTERS
Acacia is almost completely but very slowly soluble, after about 2 h, in twice its mass of water leaving
only a very small residue of vegetable particles; the liquid obtained is colourless or yellowish, dense,
viscous, adhesive, translucent and weakly acid to blue litmus paper. Acacia is practically insoluble in
alcohol.
It has the macroscopic and microscopic characters described under identification tests A and B.
IDENTIFICATION
A. Acacia occurs as yellowish-white, yellow or pale amber, sometimes with a pinkish tint, friable,
opaque, spheroidal, oval or reniform pieces (tears) of a diameter from about 1 cm to 3 cm, frequently
with a cracked surface, easily broken into irregular, whitish or slightly yellowish angular fragments
with conchoidal fracture and a glassy and transparent appearance. In the centre of an unbroken tear
there is sometimes a small cavity.
B. Reduce to a powder (355). The powder is white or yellowish-white. Examine under a microscope
using glycerol R (50 per cent V/V). The powder presents angular, irregular, colourless, transparent
fragments. Only traces of starch or vegetable tissues are visible. No stratified membrane is apparent.
C. To 2 ml of solution S (see Tests) add 8 ml of water R; the solution is laevorotatory.
D. Examine the chromatograms obtained in the test for glucose and fructose. The chromatogram
obtained with the test solution shows three zones due to galactose, arabinose and rhamnose. No
other important zones are visible, particularly in the upper part of the chromatogram.
E. Dissolve 1 g of the powdered drug (355) in 2 ml of water R by stirring frequently for 2 h. Add 2 ml
of alcohol R. After shaking, a white, gelatinous mucilage is formed which becomes fluid on adding
10 ml of water R.
TESTS
Solution S Dissolve 3.0 g of the powdered drug (355) in 25 ml of water R by stirring for 30 min.
Allow to stand for 30 min and dilute to 30 ml with water R.
Insoluble matter To 5.0 g of the powdered drug (355) add 100 ml of water R and 14 ml of dilute
hydrochloric acid R, boil gently for 15 min, shaking frequently, and filter while hot through a tared
sintered-glass filter. Wash with hot water R and dry at 100C to 105C. The residue weighs not more
than 25 mg (0.5 per cent).
Glucose and fructose Examine by thin-layer chromatography (2.2.27), using a TLC silica gel
plate R.
Test solution. To 0.100 g of the powdered drug (355) in a thick-walled centrifuge tube add 2 ml of a
100 g/l solution of trifluoroacetic acid R, shake vigorously to dissolve the forming gel, stopper the tube
and heat the mixture at 120C for 1 h. Centrifuge the hydrolysate, transfer the clear supernatant
carefully into a 50 ml flask, add 10 ml of water R and evaporate the solution to dryness under
reduced pressure. To the resulting clear film add 0.1 ml of water R and 0.9 ml of methanol R. Centri-
fuge to separate the amorphous precipitate. Dilute the supernatant, if necessary, to 1 ml with
methanol R.
Reference solution. Dissolve 10 mg of arabinose R, 10 mg of galactose R, 10 mg of glucose R, 10 mg of
rhamnose R and 10 mg of xylose R in 1 ml of water R and dilute to 10 ml with methanol R.
Apply to the plate as bands 10 l of each solution. Develop over a path of 10 cm using a mixture of
10 volumes of a 16 g/l solution of sodium dihydrogen phosphate R, 40 volumes of butanol R and 50
volumes of acetone R. Dry the plate in a current of warm air for a few minutes and develop again over
a path of 15 cm using the same mobile phase. Dry the plate at 110C for 10 min, spray with
1-3
anisaldehyde solution R and heat again at 110C for 10 min. The chromatogram obtained with the
reference solution shows five clearly separated coloured zones due to galactose (greyish-green to
green), glucose (grey), arabinose (yellowish-green) xylose (greenish-grey to yellowish-grey) and
rhamnose (yellowish-green), in order of increasing R
f
value. The chromatogram obtained with the
test solution shows no grey zone and no greyish-green zone between the zones corresponding to
galactose and arabinose in the chromatogram obtained with the reference solution.
Starch, dextrin and agar To 10 ml of solution S previously boiled and cooled add 0.1 ml of 0.05M
iodine. No blue or reddish-brown colour develops.
Sterculia gum
A. Place 0.2 g of the powdered drug (355) in a 10 ml ground-glass-stoppered cylinder graduated in
0.1 ml. Add 10 ml of alcohol (60 per cent V/V) R and shake. Any gel formed occupies not more than
1.5 ml.
B. To 1.0 g of the powdered drug (355) add 100 ml of water R and shake. Add 0.1 ml of methyl red
solution R. Not more than 5.0 ml of 0.01M sodium hydroxide is required to change the colour of the
indicator.
Tannins To 10 ml of solution S add 0.1 ml of ferric chloride solution R1. A gelatinous precipitate is
formed, but neither the precipitate nor the liquid shows a dark blue colour.
Tragacanth Examine the chromatograms obtained in the test for Glucose and fructose. The
chromatogram obtained with the test solution shows no greenish-grey to yellowish-grey zone corres-
ponding to the zone of xylose in the chromatogram obtained with the reference solution.
Loss on drying (2.2.32). Not more than 15.0 per cent, determined on 1.000 g of the powdered drug
(355) by drying in an oven at 100C to 105C.
Total ash (2.4.16). Not more than 4.0 per cent.
Microbial contamination Total viable aerobic count (2.6.12) not more than 10
4
micro-organisms
per gram, determined by plate count. It complies with the test for Escherichia coli (2.6.13).
STORAGE
Store in a well-closed container, protected from light.
_______________________________________________________________________ Ph Eur
1-4
Spray-dried Acacia
revised 1/01
Spray-dried Acacia complies with the requirements of the 3rd edition of the European Pharmacopoeia [0308].
These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Spray-dried acacia is obtained from a solution of acacia.
CHARACTERS
It has the characters described in the monograph on Acacia (307). It dissolves completely and
rapidly, after about 20 min, in twice its mass of water.
IDENTIFICATION
Examined under a microscope, in alcohol R, the powder is seen to consist predominantly of
spheroidal particles about 4 m to 40 m in diameter, with a central cavity containing one or several
air-bubbles; a few minute flat fragments are present. No starch granules showing birefringence are
visible in polarised light. No vegetable tissue is seen.
It also complies with identification tests C, D and E prescribed in the monograph on Acacia (307),
with the following modification:
E. Dissolve 1 g of the drug to be examined in 2 ml of water R by stirring frequently for 20 min. Add
2 ml of alcohol R. After shaking a white gelatinous mucilage is formed which becomes fluid on adding
10 ml of water R.
TESTS
With the exception of the test for insoluble matter, which is not carried out, it complies with the tests
prescribed in the monograph on Acacia (307), with the following modifications:
Solution S Dissolve 3.0 g of the drug to be examined in 25 ml of water R by stirring for 10 min.
Allow to stand for 20 min and dilute to 30 ml with water R.
Loss on drying (2.2.32). Not more than 10.0 per cent, determined on 1.000 g by drying in an oven
at 100C to 105C.
STORAGE
_______________________________________________________________________ Ph Eur
1-5
Acebutolol Hydrochloride
CH
3
O NHPr
i
N
OH H
CH
3
O
O
and enantiomer
H
,HCl
C
18
H
28
N
2
O
4
,HCl 372.9 34381-68-5
Acebutolol Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
[0871]. These requirements are reproduced after the heading Definition below.
Action and use Beta-adrenoceptor antagonist.
Preparations
Acebutolol Capsules
Acebutolol Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Acebutolol hydrochloride contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of (RS)-3-acetyl-4-[2-hydroxy-3-(1-methylethylamino)propoxy]butyranilide hydro-
chloride, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, freely soluble in water and in alcohol, very slightly
soluble in acetone and in methylene chloride, practically insoluble in ether.
It melts at about 143C.
IDENTIFICATION
First identification: B, D.
Second identification: A, C, D.
A. Dissolve 20.0 mg in a 0.1 per cent V/V solution of hydrochloric acid R and dilute to 100.0 ml with
the same acid solution. Dilute 5.0 ml to 100.0 ml with a 0.1 per cent V/V solution of hydrochloric
acid R. Examined between 220 nm and 350 nm (2.2.25), the solution shows two absorption maxima,
at 233 nm and 322 nm. The specific absorbance at the maximum at 233 nm is 555 to 605.
B. Examine by infrared absorption spectrophotometry (2.2.24) comparing with the spectrum
obtained with acebutolol hydrochloride CRS. Examine the substances prepared as discs.
C. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable
octadecylsilyl silica gel with a fluorescent indicator having an optimal intensity at 254 nm.
Test solution. Dissolve 20 mg of the substance to be examined in methanol R and dilute to 20 ml with
the same solvent.
Reference solution (a). Dissolve 20 mg of acebutolol hydrochloride CRS in methanol R and dilute to
20 ml with the same solvent.
Reference solution (b). Dissolve 20 mg of pindolol CRS in methanol R and dilute to 20 ml with the same
solvent. To 1 ml add 1 ml of reference solution (a).
Apply separately to the plate 10 l of each solution. Develop over a path of 15 cm using a mixture of
0.5 volume of perchloric acid R, 40 volumes of methanol R and 60 volumes of water R. Allow the plate
to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram
obtained with the test solution is similar in position and size to the principal spot in the chromato-
gram obtained with reference solution (a). The test is not valid unless the chromatogram obtained
with reference solution (b) shows two clearly separated principal spots.
D. It gives reaction (a) of chlorides (2.3.1).
TESTS
Appearance of solution Dissolve 0.5 g in water R and dilute to 10 ml with the same solvent. The
solution is not more opalescent than reference suspension II (2.2.1) and not more intensely coloured
than reference solution BY
5
(Method II, 2.2.2).
1-6
pH (2.2.3). Dissolve 0.20 g in carbon dioxide-free water R and dilute to 20 ml with the same solvent.
The pH of the solution is 5.0 to 7.0.
Related substances Examine by thin-layer chromatography (2.2.27), using two plates coated with
silica gel GF
254
R as the coating substance.
Test solution. Dissolve 0.10 g of the substance to be examined in methanol R and dilute to 10 ml with
the same solvent.
Reference solution (a). Dilute 3 ml of the Test solution to 100 ml with methanol R. Dilute 1 ml to
10 ml with methanol R.
Reference solution (b). Dilute 5 ml of reference solution (a) to 15 ml with methanol R.
A. Apply separately to the first plate 20 l of each solution. Develop over a path of 15 cm using the
upper layer from a mixture of 10 volumes of glacial acetic acid R, 40 volumes of butanol R and 50
volumes of water R.
B. Apply separately to the second plate 20 l of each solution. Develop over a path of 15 cm using a
mixture of 10 volumes of glacial acetic acid R and 90 volumes of 2-propanol R.
Allow the plates to dry in air and examine in ultraviolet light at 254 nm. For each plate, any spot in
the chromatogram obtained with the test solution, apart from the principal spot, is not more intense
than the spot in the chromatogram obtained with reference solution (a) (0.3 per cent) and at most
two such spots are more intense than the spot in the chromatogram obtained with reference solution
(b) (0.1 per cent). Disregard any spot on the starting-line.
Heavy metals (2.4.8). 1.0 g complies with limit test D for heavy metals (20 ppm). Prepare the
standard using 2 ml of lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32). Not more than 1.0 per cent, determined on 1.000 g by drying in an oven at
100C to 105C for 3 h.
Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g.
ASSAY
Dissolve 0.300 g in 50 ml of alcohol R and add 1 ml of 0.1M hydrochloric acid. Carry out a
potentiometric titration (2.2.20), using 0.1M sodium hydroxide. Read the volume added between the
two points of inflexion.
1 ml of 0.1M sodium hydroxide is equivalent to 37.29 mg of C
18
H
29
ClN
2
O
4
.
STORAGE
IMPURITIES
3C N
O
O
CH
3
O
O
H
A. 3-acetyl-4-(2,3-epoxypropoxy)butyranilide,
H
NHPr
i
O
OH
O
CH3
O
N 3C
B. 3-acetyl-4-[2-hydroxy-3-(1-methylethylamino)propoxy]-acetanilide (diacetolol),
O
N
H
O
CH
3
OH
H
3
C
C. 3-acetyl-4-hydroxybutyranilide,
NHPr
i
O
OH
O
CH
3
2N
D. 5-amino-2-[2-hydroxy-3-(1-methylethylamino)propoxy]acetophenone,
1-7
H
NHPr
i
O
OH
O
N H3C
E. 4-[2-hydroxy-3-(1-methylethylamino)propoxy]butyranilide.
_______________________________________________________________________ Ph Eur
1-8
Aceclofenac
N
O
O
COOH
Cl
Cl
H
C
16
H
13
Cl
2
NO
4
354.2 89796-99-6
Aceclofenac complies with the requirements of the 3rd edition of the European Pharmacopoeia [1281]. These
Action and use Analgesic; anti-inflammatory.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Aceclofenac contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of 2-[[2-[2-[(2,6-dichlorophenyl)amino]phenyl]acetyl]oxy]acetic acid, calculated with reference to
the dried substance.
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water, freely soluble in acetone
and in dimethylformamide, soluble in alcohol and in methanol.
IDENTIFICATION
First identification: B.
Second identification: A, C.
A. Dissolve 50.0 mg in methanol R and dilute to 100.0 ml with the same solvent. Dilute 2.0 ml of the
solution to 50.0 ml with methanol R. Examined between 220 nm and 370 nm (2.2.25), the solution
shows an absorption maximum at 275 nm. The specific absorbance at the maximum is 320 to 350.
B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum
obtained with aceclofenac CRS.
C. Dissolve about 10 mg in 10 ml of alcohol R. To 1 ml of the solution, add 0.2 ml of a mixture
prepared immediately before use of equal volumes of a 6 g/l solution of potassium ferricyanide R and a
9 g/l solution of ferric chloride R. Allow to stand protected from light for 5 min. Add 3 ml of a 10.0 g/l
solution of hydrochloric acid R. Allow to stand protected from light for 15 min. A blue colour develops
and a precipitate is formed.
TESTS
Related substances Examine by liquid chromatography (2.2.29).
Test solution. Dissolve 0.10 g of the substance to be examined in the mobile phase and dilute to
50.0 ml with the mobile phase.
Reference solution (a). Dissolve a quantity of sodium diclofenac CRS corresponding to 5.0 mg of
diclofenac in the mobile phase and dilute to 50.0 ml with the mobile phase.
Reference solution (b). Dilute 2.0 ml of reference solution (a) to 50.0 ml with the mobile phase.
Reference solution (c). To 5.0 ml of reference solution (a), add 0.25 ml of the test solution and dilute
to 50.0 ml with the mobile phase.
The chromatographic procedure may be carried out using:
a stainless steel column 0.15 m long and 4.0 mm in internal diameter packed with butylsilyl silica
gel for chromatography R (5 m),
as mobile phase at a flow rate of 1 ml per minute a mixture of 225 volumes of acetonitrile R, 225
volumes of tetrahydrofuran R and 550 volumes of a 1.2 g/l solution of glacial acetic acid R
adjusted to pH 3.5 with a 40 g/l solution of sodium hydroxide R,
as detector a spectrophotometer set at 275 nm.
Inject 10 l of reference solution (c). When the chromatograms are recorded in the prescribed
conditions the retention times are: aceclofenac about 4 min, diclofenac about 7 min. Adjust the
1-9
sensitivity of the system so that the height of the two principal peaks in the chromatogram obtained
with reference solution (c) is at least 50 per cent of the full scale of the recorder. The test is not valid
unless the resolution between the peaks corresponding to aceclofenac and diclofenac is at least 8.0.
Inject 10 l of the test solution and 10 l of reference solution (b). Continue the chromatography
for ten times the retention time of the principal peak. In the chromatogram obtained with the test
solution: the area of any peak apart from the principal peak is not greater than the area of the
principal peak in the chromatogram obtained with reference solution (b) (0.2 per cent); the sum of
the areas of all the peaks, apart from the principal peak, is not greater than 2.5 times the area of the
principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent). Disregard
any peak with an area less than 0.2 times that of the principal peak in the chromatogram obtained
with reference solution (b).
Heavy metals (2.4.8). To 2.0 g in a silica crucible, add 2 ml of sulphuric acid R to wet the substance.
Heat progressively to ignition and continue heating until an almost white or at most a greyish residue
is obtained. Carry out the ignition at a temperature not exceeding 800C. Allow to cool. Add 3 ml of
hydrochloric acid R and 1 ml of nitric acid R. Heat and evaporate slowly to dryness. Cool, add 1 ml of
a 100 g/l solution of hydrochloric acid R, 10.0 ml of distilled water R and neutralise with a 1.0 g/l
solution of ammonia R using 0.1 ml of phenolphthalein solution R as indicator. Add 2.0 ml of a 60 g/l
solution of anhydrous acetic acid R and dilute to 20 ml with distilled water R. 12 ml of the solution
complies with limit test A for heavy metals (10 ppm). Prepare the standard using lead standard
solution (1 ppm Pb) R.
Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying at 100C to
105C.
ASSAY
Dissolve 0.300 g in 40 ml of methanol R. Titrate with 0.1M sodium hydroxide, determining the end-
point potentiometrically (2.2.20).
16
H
13
Cl
2
NO
4
.
STORAGE
IMPURITIES
NH
COOR
Cl Cl
A. R = H: 2-[2-[(2,6-dichlorophenyl)amino]phenyl]acetic acid, (diclofenac),
B. R = CH
3
: methyl 2-[2-[(2,6-dichlorophenyl)amino]phenyl]acetate (methyl ester of diclofenac),
C. R = C
2
H
5
: ethyl 2-[2-[(2,6-dichlorophenyl)amino]phenyl]acetate (ethyl ester of diclofenac),
NH
Cl Cl
O COOR
O
D. R = CH
3
: methyl 2-[[2-[2-[(2,6-dichlorophenyl)amino]phenyl]acetyl]oxy]acetate (methyl
ester of aceclofenac),
E. R = C
2
H
5
: ethyl 2-[[2-[2-[(2,6-dichlorophenyl)amino]phenyl]acetyl]oxy]acetate (ethyl ester of
aceclofenac),
F. R = CH
2
-C
6
H
5
: benzyl 2-[[2-[2-[(2,6-dichlorophenyl)amino]-phenyl]acetyl]oxy]acetate (benzyl
ester of aceclofenac).
_______________________________________________________________________ Ph Eur
1-10
Acenocoumarol / Nicoumalone
For the purposes of product labelling in the United Kingdom, the pair of names given above shall be used
together when the substance is stated as an active ingredient in a formulated preparation (see Supplementary
Chapter II A, Changes in title).
O O NO
2
CH
3
O
OH
H
and enantiomer
C
19
H
15
NO
6
353.3 152-72-7
Definition Acenocoumarol is (RS)-4-hydroxy-3-(1-p-nitrophenyl-3-oxobutyl)coumarin. It contains
not less than 98.5% and not more than 100.5% of C
19
H
15
NO
6
, calculated with reference to the dried
substance.
Characteristics An almost white to buff powder; odourless or almost odourless.
Practically insoluble in water and in ether; slightly soluble in ethanol (96%) and in chloroform. It
dissolves in aqueous solutions of the alkali hydroxides. It exhibits polymorphism.
Identification The infrared absorption spectrum, Appendix II A, is concordant with the reference
spectrum of acenocoumarol (RS 001). If the spectra are not concordant, dissolve 0.1 g of the
substance being examined in 10 ml of acetone and add water dropwise until the solution becomes
turbid. Heat on a water bath until the solution is clear and allow to stand. Filter, wash the crystals
with a mixture of equal volumes of acetone and water and dry at 100 at a pressure of 2 kPa for 30
minutes. Prepare a new spectrum of the residue.
Clarity and colour of solution
A. A 2.0% w/v solution in acetone is clear, Appendix IV A.
B. The absorbance of a 4-cm layer of a 2.0% w/v solution in acetone at 460 nm is not more than 0.12,
Appendix II B.
C. A 2.0% w/v solution in 0.1M sodium hydroxide is clear, Appendix IV A, and yellow.
Light absorption Absorbance of a 0.001% w/v solution in a mixture of 1 volume of 1M hydrochloric
acid and 9 volumes of methanol at the maximum at 306 nm, 0.50 to 0.54, calculated with reference to
the dried substance, Appendix II B.
Related substances Carry out the method for thin-layer chromatography, Appendix III A, using silica
gel GF
254
as the coating substance and a mixture of 50 volumes of chloroform, 50 volumes of
cyclohexane and 20 volumes of glacial acetic acid as the mobile phase. Apply separately to the plate
20 l of each of two solutions of the substance being examined in acetone containing (1) 2.0% w/v
and (2) 0.0020% w/v. After removal of the plate, allow it to dry in air and immediately examine
under ultraviolet light (254 nm). Any secondary spot in the chromatogram obtained with solution (1) is
not more intense than the spot in the chromatogram obtained with solution (2).
Loss on drying When dried to constant weight at 105, loses not more than 0.5% of its weight. Use
1 g.
Sulphated ash Not more than 0.1%, Appendix IX A.
Assay Dissolve 0.6 g in 50 ml of acetone and titrate with 0.1M sodium hydroxide VS using bromothymol
blue solution R3 as indicator. Repeat the operation without the substance being examined. The
difference between the titrations represents the amount of sodium hydroxide required. Each ml of
0.1M sodium hydroxide VS is equivalent to 35.33 mg of C
19
H
15
NO
6
.
Action and use Anticoagulant.
Preparation
Acenocoumarol Tablets/Nicoumalone Tablets
1-11
Acesulfame Potassium
NK
SO
2
O
O
Me
C
4
H
4
KNO
4
S 201.2 55589-62-3
Acesulfame Potassium complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Sweetening agent.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Acesulfame potassium contains not less than 99.0 per cent and not more than the equivalent of 101.0
per cent of 6-methyl-4-oxo-1,2,3-oxathiazin-3(4H)-one potassium 2,2-dioxide, calculated with
reference to the dried substance.
CHARACTERS
A white, crystalline powder or colourless crystals, soluble in water, very slightly soluble in acetone
and in alcohol.
IDENTIFICATION
First identification: A, C.
Second identification: B, C.
A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum
obtained with acesulfame potassium CRS. Examine the substances prepared as discs.
B. Examine by thin-layer chromatography (2.2.27), using cellulose R as the coating substance.
Test solution. Dissolve 5 mg of the substance to be examined in water R and dilute to 5 ml with the
same solvent.
Reference solution (a). Dissolve 5 mg of acesulfame potassium CRS in water R and dilute to 5 ml with
the same solvent.
Reference solution (b). Dissolve 5 mg of acesulfame potassium CRS and 5 mg of saccharin sodium R in
water R and dilute to 5 ml with the same solvent.
Apply to the plate as bands 5 l of each solution. Develop twice over a path of 15 cm using a mixture
of 10 volumes of concentrated ammonia R, 60 volumes of acetone R and 60 volumes of ethyl acetate R.
Dry the plate in a current of warm air and examine in ultraviolet light at 254 nm. The principal band
in the chromatogram obtained with the test solution is similar in position and size to the principal
band in the chromatogram obtained with reference solution (a). The test is not valid unless the
chromatogram obtained with reference solution (b) shows two clearly separated bands.
C. 5 ml of solution S (see Tests) gives reaction (b) of potassium (2.3.1).
TESTS
Solution S Dissolve 10.0 g in carbon dioxide-free water R and dilute to 50 ml with the same solvent.
Appearance of solution Solution S is clear (2.2.1) and colourless (Method II, 2.2.2).
Acidity or alkalinity To 20 ml of solution S add 0.1 ml of bromothymol blue solution R1. Not more
than 0.2 ml of 0.01M hydrochloric acid or 0.01M sodium hydroxide is required to change the colour of
the indicator.
Acetylacetamide Examine by thin-layer chromatography (2.2.27), using a suitable silica gel as the
coating substance.
Test solution. Dissolve 0.80 g of the substance to be examined in water R and dilute to 10 ml with the
same solvent.
Reference solution (a). Dissolve 50 mg of acetylacetamide R in water R and dilute to 25 ml with the
same solvent. To 5 ml of the solution add 45 ml of water R and dilute to 100 ml with methanol R.
Reference solution (b). To 10 ml of reference solution (a) add 1 ml of the test solution and dilute to
1-12
Apply separately to the plate 5 l of each solution. Develop over a path of 15 cm using a mixture of 2
volumes of water R, 15 volumes of alcohol R and 74 volumes of ethyl acetate R. Allow the plate to dry
in air until the solvents are completely removed. Spray the plate with vanillin phosphoric solution R and
heat at 120C for about 10 min. Examine in daylight. Any spot corresponding to acetylacetamide in
the chromatogram obtained with the test solution is not more intense than the spot in the chroma-
togram obtained with reference solution (a) (0.125 per cent). The test is not valid unless the chroma-
togram obtained with reference solution (a) shows a clearly visible spot and the chromatogram
obtained with reference solution (b) shows two clearly separated spots.
Impurity B and related substances Examine by liquid chromatography (2.2.29).
Test solution. Dissolve 0.100 g of the substance to be examined in water R and dilute to 10.0 ml with
the same solvent.
Reference solution (a). Dissolve 20.0 mg of the acesulfame potassium impurity B CRS in water R and
dilute to 500.0 ml with the same solvent. Dilute 0.5 ml of this solution to 100.0 ml with water R.
Reference solution (b). Dissolve 10.0 mg of the acesulfame potassium impurity B CRS and 10.0 mg of
acesulfame potassium CRS in water R and dilute to 500.0 ml with the same solvent. Dilute 5.0 ml of
this solution to 100.0 ml with water R.
a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl
silica gel for chromatography R (3 m),
as mobile phase at a flow rate of 1 ml per minute a mixture of 40 volumes of acetonitrile R and 60
volumes of a 3.3 g/l solution of tetrabutylammonium hydrogen sulphate R,
Inject 20 l of reference solution (b). Adjust the sensitivity of the system so that the height of the
two principal peaks is at least 50 per cent of the full scale of the recorder. The test is not valid unless
the resolution between the two peaks due to acesulfame and impurity B is at least 3.
Inject 20 l of the test solution and 20 l of reference solution (a). Continue the chromatography of
the test solution for at least three times the retention time of the principal peak. In the chromatogram
obtained with the test solution the area of any peak apart from the principal peak is not greater than
the area of the principal peak in the chromatogram obtained with reference solution (a) (20 ppm).
Fluorides Not more than 3 ppm of F, determined potentiometrically (Method I, 2.2.36) using as
indicator electrode a fluoride selective-membrane electrode and as reference electrode a silver-silver
chloride electrode.
Test solution. Dissolve 3.000 g of the substance to be examined in distilled water R, add 15.0 ml of total
ionic strength adjustment buffer R1 and dilute to 50.0 ml with distilled water R.
Reference solutions. To 0.5 ml, 1.0 ml, 1.5 ml and 3.0 ml of fluoride standard solution (10 ppm F) R add
15.0 ml of total ionic strength adjustment buffer R1 and dilute to 50.0 ml with distilled water R.
Carry out the measurements of each solution.
Heavy metals (2.4.8). 12 ml of solution S complies with the limit test A for heavy metals (5 ppm).
Prepare the standard using lead standard solution (1 ppm Pb) R.
100C to 105C for 3 h.
ASSAY
Dissolve 0.150 g in 50 ml of anhydrous acetic acid R. Titrate with 0.1M perchloric acid, determining the
end-point potentiometrically (2.2.20).
1 ml of 0.1M perchloric acid is equivalent to 20.12 mg of C
4
H
4
KNO
4
S.
STORAGE
Store in a well-closed container.
IMPURITIES
NH
2
O Me
O
A. 3-oxobutanamide (acetylacetamide),
NH
SO
2
O
O
Me
Cl
B. 5-chloro-6-methyl-1,2,3-oxathiazin-4(3H)-one 2,2-dioxide.
_______________________________________________________________________ Ph Eur
1-13
Acetazolamide
N N
S
NHAc S
O O
H
2
N
C
4
H
6
N
4
O
3
S
2
222.2 59-6-5
Acetazolamide complies with the requirements of the 3rd edition of the European Pharmacopoeia [0454]. These
Action and use Carbonic anhydrase inhibitor; used in treatment of glaucoma.
Preparation
Acetazolamide Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Acetazolamide contains not less than 98.5 per cent and not more than the equivalent of 101.0 per
cent of N-(5-sulphamoyl-1,3,4-thiadiazol-2-yl)acetamide, calculated with reference to the dried
substance.
CHARACTERS
A white or almost white, crystalline powder, very slightly soluble in water, slightly soluble in alcohol,
practically insoluble in ether. It dissolves in dilute solutions of alkali hydroxides.
IDENTIFICATION
First identification: A, B.
A. Dissolve 30.0 mg in 0.01M sodium hydroxide and dilute to 100.0 ml with the same solvent. Dilute
10.0 ml of the solution to 100.0 ml with 0.01M sodium hydroxide (solution a). Examined between
230 nm and 260 nm (2.2.25), solution (a) shows an absorption maximum at 240 nm. The specific
absorbance at the maximum is 162 to 176. Dilute 25.0 ml of solution (a) to 100.0 ml with 0.01M
sodium hydroxide. Examined between 260 nm and 350 nm, the solution shows an absorption
maximum at 292 nm. The specific absorbance at the maximum is 570 to 620.
B. Examine by infrared absorption spectrophotometry (2.2.24) comparing with the spectrum
obtained with acetazolamide CRS. If the spectra obtained in the solid state with the substance to be
examined and the reference substance show differences, dissolve separately the substance to be
examined and the reference substance in alcohol R, evaporate to dryness, prepare new discs and
record the spectra.
C. Introduce about 20 mg into a test-tube and add 4 ml of dilute hydrochloric acid R and 0.2 g of zinc
powder R. Immediately place a piece of lead acetate paper R over the mouth of the tube. The paper
shows a brownish-black colour.
D. Dissolve about 25 mg in a mixture of 0.1 ml of dilute sodium hydroxide solution R and 5 ml of
water R. Add 0.1 ml of copper sulphate solution R. A greenish-blue precipitate is formed.
TESTS
Appearance of solution Dissolve 1.0 g in 10 ml of 1M sodium hydroxide. The solution is not more
opalescent than reference suspension II (2.2.1) and not more intensely coloured than reference
solution Y
5
or BY
5
(Method II, 2.2.2).
Related substances Examine by thin-layer chromatography (2.2.27), using silica gel GF
254
R as the
coating substance.
Test solution. Dissolve 50 mg of the substance to be examined in a mixture of equal volumes of
alcohol R and ethyl acetate R and dilute to 10 ml with the same mixture of solvents.
Reference solution. Dilute 1 ml of the Test solution to 100 ml with a mixture of equal volumes of
alcohol R and ethyl acetate R.
Apply separately to the plate 20 l of each solution. Use the tank without lining the walls and allow to
saturate for 1 h before development. Develop over a path of 15 cm using a freshly prepared mixture
of 20 volumes of concentrated ammonia R, 30 volumes of ethyl acetate R and 50 volumes of 2-prop-
anol R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any spot in the
1-14
chromatogram obtained with the test solution, apart from the principal spot, is not more intense
than the spot in the chromatogram obtained with the reference solution (1.0 per cent).
Sulphates (2.4.13). To 0.4 g add 20 ml of distilled water R and dissolve by heating to boiling. Allow
to cool with frequent shaking and filter. 15 ml of the filtrate complies with the limit test for sulphates
(500 ppm).
Heavy metals (2.4.8). 1.0 g complies with limit test C for heavy metals (20 ppm). Prepare the
100C to 105C.
ASSAY
Dissolve 0.200 g in 25 ml of dimethylformamide R. Titrate with 0.1M ethanolic sodium hydroxide,
determining the end-point potentiometrically (2.2.20).
1 ml of 0.1M ethanolic sodium hydroxide is equivalent to 22.22 mg of C
4
H
6
N
4
O
3
S
2
.
_______________________________________________________________________ Ph Eur
1-15
Glacial Acetic Acid
H
3
C COOH
C
2
H
4
O
2
60.1 64-19-7
Glacial Acetic Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia [0590].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Glacial acetic acid contains not less than 99.0 per cent m/m and not more than the equivalent of
100.5 per cent m/m of C
2
H
4
O
2
.
CHARACTERS
A crystalline mass or a clear, colourless, volatile liquid, miscible with water, with alcohol, with ether
and with methylene chloride.
IDENTIFICATION
A. A 100 g/l solution is strongly acid (2.2.4).
B. To 0.03 ml add 3 ml of water R and neutralise with dilute sodium hydroxide solution R. The solution
gives reaction (b) of acetates (2.3.1).
TESTS
Solution S Dilute 20 ml to 100 ml with distilled water R.
Appearance It is clear (2.2.1) and colourless (Method II, 2.2.2).
Freezing point (2.2.18). Not less than 14.8C.
Reducing substances To 5.0 ml add 10.0 ml of water R and mix. To 5.0 ml of the solution add
6 ml of sulphuric acid R, cool and add 2.0 ml of 0.0167M potassium dichromate. Allow to stand for
1 min and add 25 ml of water R and 1 ml of a freshly prepared 100 g/l solution of potassium iodide R.
Titrate with 0.1M sodium thiosulphate, using 1.0 ml of starch solution R as indicator. Not less than
1.0 ml of 0.1M sodium thiosulphate solution is required.
Chlorides (2.4.4). 10 ml of solution S diluted to 15 ml with water R complies with the limit test for
chlorides (25 mg/l).
Sulphates (2.4.13). 15 ml of solution S complies with the limit test for sulphates (50 mg/l).
Iron (2.4.9). 5.0 ml of solution (a) obtained in the test for heavy metals diluted to 10.0 ml with
water R complies with the limit test for iron (5 ppm).
Heavy metals (2.4.8). Dissolve the residue obtained in the test for residue on evaporation by heating
with two quantities, each of 15 ml, of water R and dilute to 50.0 ml (solution (a)). 12 ml of solution
(a) complies with limit test A for heavy metals (5 ppm). Prepare the standard using lead standard
Residue on evaporation Evaporate 20 g to dryness on a water-bath and dry at 100C to 105C.
The residue weighs not more than 2.0 mg (0.01 per cent).
ASSAY
Weigh accurately a conical flask with a ground-glass stopper containing 25 ml of water R. Add 1.0 ml
of the substance to be examined and weigh again accurately. Add 0.5 ml of phenolphthalein solution R
and titrate with 1M sodium hydroxide.
1 ml of 1M sodium hydroxide is equivalent to 60.1 mg of C
2
H
4
O
2
.
STORAGE
Store in an airtight container.
_______________________________________________________________________ Ph Eur
1-16
Acetic Acid (33 per cent)
Acetic Acid
Definition Acetic Acid (33 per cent) contains not less than 32.5% and not more than 33.5%
w/w of acetic acid, C
2
H
4
O
2
.
Characteristics A clear, colourless liquid; odour, pungent.
Miscible with water, with ethanol (96%) and with glycerol.
Identification
A. Strongly acidic, even when diluted freely.
B. When neutralised, yields the reactions characteristic of acetates, Appendix VI.
Weight per ml 1.040 to 1.042 g, Appendix V G.
Heavy metals Evaporate 10.0 ml to dryness and add 20 ml of water. 12 ml of the resulting
solution complies with limit test A for heavy metals, Appendix VII. Use lead standard solution (1 ppm
Pb) to prepare the standard (2 ppm).
Chloride Dilute 5.0 ml with sufficient water to produce 100 ml. 15 ml of the resulting solution
complies with the limit test for chlorides, Appendix VII (70 ppm).
Sulphate 12.5 ml of the solution used in the test for Chloride, diluted to 15 ml with water,
complies with the limit test for sulphates, Appendix VII (240 ppm).
Aldehydes Distil 15 ml. To the first 5 ml of the distillate add 10 ml of a 5% w/v solution of
mercury(II) chloride, make alkaline with 5M sodium hydroxide, allow to stand for 5 minutes and make
acidic with 1M sulphuric acid. The solution shows not more than a faint turbidity.
Formic acid and oxidisable impurities Mix 5 ml with 6 ml of sulphuric acid and cool to 20.
Add 2 ml of 0.0167M potassium dichromate VS, allow to stand for 1 minute, add 25 ml of water and
1 ml of freshly prepared dilute potassium iodide solution and titrate the liberated iodine with 0.1M
sodium thiosulphate VS using starch mucilage as indicator. Not less than 1.0 ml of 0.1M sodium
thiosulphate VS is required.
Readily oxidisable impurities To 5.0 ml add 20 ml of water and 0.2 ml of 0.02M potassium
permanganate VS and allow to stand for 1 minute. The pink colour is not entirely discharged.
Non-volatile matter When evaporated to dryness and dried at 105, leaves not more than 0.01%
w/w of residue.
Assay Weigh 5 g into a stoppered flask containing 50 ml of water and titrate with 1M sodium
hydroxide VS using phenolphthalein solution R1 as indicator. Each ml of 1M sodium hydroxide VS is
equivalent to 60.05 mg of C
2
H
4
O
2
.
Preparation
1-17
Dilute Acetic Acid
Definition Acetic Acid (6 per cent) contains not less than 5.7% and not more than 6.3% w/w of
acetic acid, C
2
H
4
O
2
. It may be prepared by mixing 182 g of Acetic Acid (33 per cent) with 818 g of
Purified Water.
Identification
A. Strongly acidic.
B. When neutralised, yields the reactions characteristic of acetates, Appendix VI.
Weight per ml About 1.005 g, Appendix V G.
Heavy metals Evaporate 20.0 ml to dryness and add 20 ml of water. 12 ml of the resulting
Chloride Dilute 5.0 ml with sufficient water to produce 100 ml. 15 ml of the resulting solution
Sulphate 12.5 ml of the solution used in the test for Chloride, diluted to 15 ml with water,
complies with the limit test for sulphates, Appendix VII (240 ppm).
Aldehydes Distil 75 ml. To the first 5 ml of the distillate add 10 ml of a 5% w/v solution of
mercury(II) chloride, make alkaline with 5M sodium hydroxide, allow to stand for 5 minutes and acidify
with 1M sulphuric acid. The solution shows not more than a faint turbidity.
Formic acid and oxidisable impurities Mix 5 ml with 6 ml of sulphuric acid and cool to 20.
Add 0.4 ml of 0.0167M potassium dichromate VS, allow to stand for 1 minute, add 25 ml of water
and 1 ml of freshly prepared dilute potassium iodide solution and titrate the liberated iodine with 0.1M
sodium thiosulphate VS using starch mucilage as indicator. Not less than 0.2 ml of 0.1M sodium
thiosulphate VS is required.
Readily oxidisable impurities To 25 ml add 0.2 ml of 0.02M potassium permanganate VS and
allow to stand for 1 minute. The pink colour is not entirely discharged.
Non-volatile matter When evaporated to dryness and dried at 105, leaves not more than 0.01%
w/w of residue.
Assay Add 30 ml of water to 20 g in a stoppered flask and titrate with 1M sodium hydroxide VS
using phenolphthalein solution R1 as indicator. Each ml of 1M sodium hydroxide VS is equivalent to
60.05 mg of C
2
H
4
O
2
.
1-18
Acetone
H
3
C CH
3
O
C
3
H
6
O 58.08 67-64-1
Acetone complies with the requirements of the 3rd edition of the European Pharmacopoeia [0872]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Acetone is propan-2-one.
CHARACTERS
A volatile clear, colourless liquid, miscible with water, with alcohol and with ether. The vapour is
flammable.
IDENTIFICATION
A. To 1 ml, add 3 ml of dilute sodium hydroxide R and 0.3 ml of a 25 g/l solution of sodium nitro-
prusside R. An intense red colour is produced which becomes violet with the addition of 3.5 ml of
acetic acid R.
B. To 10 ml of a 0.1 per cent V/V solution in alcohol (50 per cent V/V) R, add 1 ml of a 10 g/l solution
of nitrobenzaldehyde R in the same solvent and 0.5 ml of strong sodium hydroxide solution R. Allow to
stand for about 2 min and acidify with acetic acid R. A greenish-blue colour is produced.
TESTS
Appearance of solution To 10 ml add 10 ml of water R. The solution is clear (2.2.1) and colourless
(Method II, 2.2.2).
Acidity or alkalinity To 5 ml add 5 ml of carbon dioxide-free water R, 0.15 ml of phenolphthalein
solution R and 0.5 ml of 0.01M sodium hydroxide. The solution is pink. Add 0.7 ml of 0.01M hydro-
chloric acid and 0.05 ml of methyl red solution R. The solution is red or orange.
Relative density (2.2.5). 0.790 to 0.793.
Related substances Examine by gas chromatography (2.2.28).
Test solution. The substance to be examined.
Reference solution. To 0.5 ml of methanol R add 0.5 ml of 2-propanol R and dilute to 100.0 ml with the
test solution. Dilute 1.0 ml to 10.0 ml with the test solution.
a fused-silica column 50 m long and 0.3 mm in internal diameter coated with a film (0.5 m) of
macrogol 20,000 R,
helium for chromatography R as the carrier gas with a split ratio of about 50:1 and at a linear flow
of 21 cm/s,
a flame-ionisation detector,
maintaining the temperature of the column at 45C until injection, then raising the temperature at a
rate of 5C per minute to 100C and maintaining the temperature of the injection port at 150C and
that of the detector at 250C.
Inject 1 l of the test solution and 1 l of the reference solution.
When the chromatograms are recorded in the conditions described above, the substances are
eluted in the following order: acetone, methanol, 2-propanol.
Continue the chromatography for three times the retention time of acetone (which is about
5.3 min).
The test is not valid unless, in the chromatogram obtained with the reference solution, the resolu-
tion between the peaks corresponding to methanol and 2-propanol is at least 1.0.
In the chromatogram obtained with the test solution: the area of any peak corresponding to
methanol or 2-propanol is not greater than the difference between the areas of the corresponding
peaks in the chromatogram obtained with the reference solution and the areas of the corresponding
peaks in the chromatogram obtained with the test solution (0.05 per cent V/V for each impurity); the
area of any peak, apart from the principal peak and any peaks corresponding to methanol and
2-propanol, is not greater than the difference between the area of the methanol peak in the chromato-
gram obtained with the reference solution and the area of the corresponding peak in the chromato-
gram obtained with the test solution (0.05 per cent V/V for each of the additional impurities).
1-19
Matter insoluble in water To 1 ml add 19 ml of water R. The solution is clear (2.2.1).
Reducing substances To 30 ml add 0.1 ml of 0.02M potassium permanganate and allow to stand in
the dark for 2 h. The mixture is not completely decolorised.
Residue on evaporation Evaporate 20.0 g to dryness on a water-bath and dry at 100C to 105C.
The residue weighs not more than 1 mg (50 ppm).
Water (2.5.12). Not more than 3 g/l, determined on 10.0 ml by the semi-micro determination of
water. Use 20 ml of anhydrous pyridine R as solvent.
STORAGE
Store protected from light.
IMPURITIES
A. methanol,
B. 2-propanol.
__________________________________________________________________________________________________________ Ph Eur
1-20
Acetylcholine Chloride
1/01
Me O
NMe
3
O
Cl
C
7
H
16
ClNO
2
181.7 60-31-1
Acetylcholine Chloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Cholinergic
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
2-(Acetyloxy)-N,N,N-trimethylethanaminium chloride.
Content: 98.5 per cent to 101.5 per cent (dried substance).
CHARACTERS
Appearance: white crystalline powder or colourless crystals, very hygroscopic.
Solubility: very soluble in water, freely soluble in alcohol, slightly soluble in methylene chloride.
IDENTIFICATION
First identification: B, E.
Second identification: A, C, D, E.
A. Melting point (2.2.14): 149C to 152C.
Introduce the substance to be examined into a capillary tube. Dry in an oven at 100-105C for 3 h.
Seal the tube and determine the melting point.
B. Infrared absorption spectrophotometry (2.2.24).
Comparison: acetylcholine chloride CRS.
C. Examine the chromatograms obtained in the test for related substances.
Results: the principal band in the chromatogram obtained with test solution (b) is similar in position,
colour and size to the principal band in the chromatogram obtained with reference solution (b).
D. To 15 mg add 10 ml of dilute sodium hydroxide solution R, 2 ml of 0.02M potassium permanganate
and heat. The vapours formed change the colour of red litmus paper R to blue.
E. 0.5 ml of solution S (see Tests) gives reaction (a) of chlorides (2.3.1).
TESTS
Appearance of solution Solution S is clear (2.2.1) and not more intensely coloured than reference
solution Y
6
or BY
6
(2.2.2, Method II).
Acidity Dilute 1 ml of solution S to 10 ml with carbon dioxide-free water R. Add 0.05 ml of
phenolphthalein solution R. Not more than 0.4 ml of 0.01M sodium hydroxide is required to change the
colour of the indicator to pink.
Related substances Thin-layer chromatography (2.2.27).
Prepare the solutions immediately before use.
Test solution (a). Dissolve 0.20 g of the substance to be examined in methanol R and dilute to 2.0 ml
with the same solvent.
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with methanol R.
Reference solution (a). Dilute 1 ml of test solution (a) to 100 ml with methanol R.
Reference solution (b). Dissolve 20.0 mg of acetylcholine chloride CRS in methanol R and dilute to 2.0 ml
Reference solution (c). Dissolve 20 mg of choline chloride R in methanol R, add 0.4 ml of test solution (a)
and dilute to 2.0 ml with methanol R.
Plate: TLC silica gel plate R.
Mobile phase: mix 20 volumes of a 40 g/l solution of ammonium nitrate R, 20 volumes of methanol R
and 60 volumes of acetonitrile R.
Application: 5 l as bands of 10 mm by 2 mm.
1-21
Development: over 2/3 of the plate.
Detection: spray with potassium iodobismuthate solution R3.
System suitability: the test is not valid unless the chromatogram obtained with reference solution (c)
shows 2 clearly separated bands.
Limits:
any impurity: any bands in the chromatogram obtained with test solution (a), apart from the
principal band, are not more intense than the principal band in the chromatogram obtained with
reference solution (a) (1 per cent).
Trimethylamine Dissolve 0.1 g in 10 ml of sodium carbonate solution R and heat to boiling. No
vapours appear which turn red litmus paper R blue.
Heavy metals (2.4.8): maximum 10 ppm.
12 ml of solution S complies with limit test A. Prepare the standard using lead standard solution
(1 ppm Pb) R.
Loss on drying (2.2.32): maximum 1.0 per cent, determined on 1.000 g by drying in an oven at
100-105C for 3 h.
Sulphated ash (2.4.14): maximum 0.1 per cent, determined on the residue obtained in the test for
loss on drying.
ASSAY
Dissolve 0.200 g in 20 ml of carbon dioxide-free water R. Neutralise with 0.01M sodium hydroxide using
0.15 ml of phenolphthalein solution R as indicator. Add 20.0 ml of 0.1M sodium hydroxide and allow to
stand for 30 min. Titrate with 0.1M hydrochloric acid.
7
H
16
ClNO
2
.
STORAGE
In ampoules, protected from light.
IMPURITIES
HO
NMe
3 Cl
A. 2-hydroxy-N,N,N-trimethylethanaminium chloride (choline chloride),
AcO
NHMe
2 Cl
B. 2-(acetyloxy)-N,N-dimethylethanaminium chloride,
Me
3
C. N,N-dimethylmethanamine.
__________________________________________________________________________________________________________ Ph Eur
1-22
Acetylcysteine
COOH
NHAc H
HS
C
5
H
9
NO
3
S 163.2 616-91-1
Acetylcysteine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0967]. These
Action and use Antidote for paracetamol poisoning; mucolytic.
Preparation
Acetylcysteine Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Acetylcysteine contains not less than 98.0 per cent and not more than the equivalent of 101.0 per
cent of (R)-2-acetamido-3-mercaptopropanoic acid, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder or colourless crystals, freely soluble in water and in alcohol, practically
insoluble in methylene chloride.
IDENTIFICATION
Second identification: A, B, D, E.
A. It complies with the test for specific optical rotation (see Tests).
B. Melting point (2.2.14): 104C to 110C.
C. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum
obtained with acetylcysteine CRS. Examine the substances prepared as discs using potassium bromide R.
D. Examine the chromatograms obtained in the test for related substances. The retention time and
size of the principal peak in the chromatogram obtained with test solution (b) are approximately the
same as those of the principal peak in the chromatogram obtained with reference solution (b).
E. To 0.5 ml of solution S (see Tests) add 0.05 ml of a 50 g/l solution of sodium nitroprusside R and
0.05 ml of concentrated ammonia R. A dark violet colour develops.
TESTS
pH (2.2.3). To 2 ml of solution S add 8 ml of carbon dioxide-free water R and mix. The pH of the
solution is 2.0 to 2.8.
Specific optical rotation (2.2.7). In a 25 ml volumetric flask, mix 1.25 g with 1 ml of a 10 g/l
solution of sodium edetate R. Add 7.5 ml of a 40 g/l solution of sodium hydroxide R, mix and dissolve.
Dilute to 25.0 ml with phosphate buffer solution pH 7.0 R2. The specific optical rotation is +21.0 to
+27.0, calculated with reference to the dried substance.
Related substances Examine by liquid chromatography (2.2.29). Except where otherwise prescribed,
prepare the solutions immediately before use.
Test solution (a). Suspend 0.80 g of the substance to be examined in 1 ml of 1M hydrochloric acid and
dilute to 100.0 ml with water R.
Test solution (b). Dilute 5.0 ml of test solution (a) to 100.0 ml with water R. Dilute 5.0 ml of the
solution to 50.0 ml with water R.
Test solution (c). Use test solution (a) after storage for at least 1 h.
Reference solution (a). Suspend 4.0 mg of acetylcysteine CRS, 4.0 mg of L-cystine R, 4.0 mg of
L-cysteine R, 4.0 mg of acetylcysteine impurity C CRS and 4.0 mg of acetylcysteine impurity D CRS in
1 ml of 1M hydrochloric acid and dilute to 100.0 ml with water R.
Reference solution (b). Suspend 4.0 mg of acetylcysteine CRS in 1 ml of 1M hydrochloric acid and dilute
to 100.0 ml with water R.
1-23
a stainless steel column 0.25 m long and 4 mm in internal diameter packed with octadecylsilyl
as mobile phase at a flow rate at 1.0 ml per minute a mixture prepared as follows: stir 3 volumes
of acetonitrile R and 97 volumes of water R in a beaker; adjust to pH 3.0 with concentrated
phosphoric acid R,
When the chromatograms are recorded in the prescribed conditions, the retention times are: L-
cystine, about 2.2 min; L-cysteine, about 2.4 min; 2-methyl-2-thiazoline-4-carboxylic acid,
originating in test solution (c), about 3.3 min; acetylcysteine, about 6.4 min; acetylcysteine impurity
C, about 12 min; acetylcysteine impurity D, about 14 min. Inject 20 l of reference solution (a). The
test is not valid unless:
in the chromatogram obtained with reference solution (a), the resolution between the peaks due
to L-cystine and L-cysteine is at least 1.5,
in the chromatogram obtained with reference solution (a), the resolution between the peaks due
to acetylcysteine impurity C and acetylcysteine impurity D is at least 2.0.
Inject 20 l of 0.01M hydrochloric acid as a blank. Inject three times 20 l of reference solution (a),
20 l of reference solution (b) and 20 l of each test solution. Continue the chromatography for five
times the retention time of acetylcysteine which is about 30 min.
From the chromatogram obtained with test solution (a), calculate the percentage content of the
known impurities and the unknown impurities using the following expressions:
1 2
2 1
100
impurity known
m A
m A
1 4
3 3
100
impurity unknown
m A
m A
A
1
=peak area of individual impurity (L-cystine, L-cysteine, acetylcysteine impurity C and
acetylcysteine impurity D) in the chromatogram obtained with test solution (a),
A
2
=peak area of the corresponding individual impurity (L-cystine, L-cysteine, acetylcysteine
impurity C and acetylcysteine impurity D) in the chromatogram obtained with reference
solution (a),
A
3
=peak area of unknown impurity in the chromatogram obtained with test solution (a),
A
4
=peak area of acetylcysteine in the chromatogram obtained with reference solution (b),
m
1
=mass of the substance to be examined in test solution (a),
m
2
=mass of the individual impurity in reference solution (a),
m
3
=mass of acetylcysteine in reference solution (b).
The percentage content of each known impurity and of each unknown impurity is not greater than
0.5 per cent. The sum of the calculated percentage contents of known and unknown impurities is not
greater than 0.5 per cent. Disregard any peak due to the solvent, any peak appearing at retention time
of about 3.3 min corresponding to 2-methyl-2-thiazoline-4-carboxylic acid and any peak with an-area
less than 0.1 times that of the principal peak in the chromatogram obtained with reference solution
(b).
standard using 2 ml of lead standard (10 ppm Pb) R.
Zinc Not more than 10 ppm of Zn, determined by atomic absorption spectrometry (Method II,
2.2.23).
Test solution. Dissolve 1.00 g in 0.001M hydrochloric acid and dilute to 50.0 ml with the same acid.
Reference solutions. Prepare the reference solutions using zinc standard solution (5 mg/ml Zn) R, diluted
with 0.001M hydrochloric acid.
Measure the absorbance at 213.8 nm using a zinc hollow-cathode lamp as the source of radiation, an
air-acetylene flame and a correction procedure for non-specific absorption.
Loss on drying (2.2.32). Not more than 1.0 per cent, determined on 1.000 g by drying in an oven in
vacuo at 70C for 3 h.
ASSAY
Dissolve 0.140 g in 60 ml of water R and add 10 ml of dilute hydrochloric acid R. After cooling in iced
water, add 10 ml of potassium iodide solution R and titrate with 0.05M iodine, using 1 ml of starch
solution R as indicator.
1 ml of 0.05M iodine is equivalent to 16.32 mg of C
5
H
9
NO
3
S.
1-24
STORAGE
IMPURITIES
NH
2
H
S
HOOC S
COOH
H NH
2
A. L-cystine,
HS
COOH
H NH
2
B. L-cysteine,
COOH
H NHAc
NHAc H
S
S
HOOC
C. N,N-diacetyl-L-cystine,
AcS
COOH
H NHAc
D. N,S-diacetyl-L-cysteine.
_______________________________________________________________________ Ph Eur
1-25
Acetyltryptophan
revised 1/01
H
COOH
N
H
NHAc
and enantiomer
C
13
H
14
N
2
O
3
246.3 87-32-1
Acetyltryptophan complies with the requirements of the 3rd edition of the European Pharmacopoeia for
N-Acetyltryptophan [1383]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
N-Acetyltryptophan contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of (RS)-2-acetylamino-3-(1H-indol-3-yl)propanoic acid, calculated with reference to
PRODUCTION
When N-acetyltryptophan is produced by a process involving fermentation steps, it complies with the
requirements of the monograph on Products of fermentation (1468).
Tryptophan used for the production of N-acetyltryptophan complies with the test for related
substances in the monograph on Tryptophan (1272).
CHARACTERS
A white or almost white, crystalline powder, or colourless crystals, slightly soluble in water, very
soluble in alcohol. It dissolves in dilute solutions of alkali hydroxides.
IDENTIFICATION
A. It complies with the test for optical rotation (see Tests).
obtained with N-acetyltryptophan CRS.
C. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel F
254
plate R.
Test solution. Dissolve 50 mg of the substance to be examined in 0.2 ml of concentrated ammonia R
and dilute to 10 ml with water R.
Reference solution (a). Dissolve 50 mg of N-acetyltryptophan CRS in 0.2 ml of concentrated ammonia R
Reference solution (b). Dissolve 10 mg of tryptophan R in the test solution and dilute to 2 ml with the
same solution.
Apply to the plate 2 l of each solution. Develop over a path of 10 cm using a mixture of 25 volumes
of glacial acetic acid R, 25 volumes of water R and 50 volumes of butanol R. Dry the plate in an oven
at 100C to 105C for 15 min and examine in ultraviolet light at 254 nm. The principal spot in the
chromatogram obtained with the test solution is similar in position and size to the principal spot in
the chromatogram obtained with reference solution (a). The test is not valid unless the chromato-
gram obtained with reference solution (b) shows two clearly separated spots.
D. Dissolve about 2 mg in 2 ml of water R. Add 2 ml of dimethylaminobenzaldehyde solution R6. Heat
on a water-bath. A blue or greenish-blue colour develops.
E. It gives the reaction of acetyl (2.3.1). Proceed as described for substances hydrolysable only with
difficulty.
TESTS
Appearance of solution Dissolve 1.0 g in a 40 g/l solution of sodium hydroxide R and dilute to
100 ml with the same alkaline solution. The solution is clear (2.2.1) and not more intensely coloured
than reference solution Y
7
or GY
7
(Method II, 2.2.2).
1-26
Optical rotation (2.2.7). Dissolve 2.50 g in a 40 g/l solution of sodium hydroxide R and dilute to
25.0 ml with the same alkaline solution. The angle of optical rotation is 0.1 to +0.1.
Buffer solution pH 2.3. Dissolve 3.90 g of sodium dihydrogen phosphate R in 1000 ml of water R. Add
about 700 ml of a 2.9 g/l solution of phosphoric acid R and adjust the pH to 2.3 with the same acidic
solution.
Test solution. Dissolve 0.10 g of the substance to be examined in a mixture of 50 volumes of aceto-
nitrile R and 50 volumes of water R and dilute to 20.0 ml with the same mixture of solvents.
Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with a mixture of 10 volumes of
acetonitrile R and 90 volumes of water R.
Reference solution (b). Dissolve 1.0 mg of 1,1-ethylidenebis(tryptophan) CRS in a mixture of 10
volumes of acetonitrile R and 90 volumes of water R and dilute to 100.0 ml with the same mixture of
solvents.
Reference solution (c). To 4.0 ml of reference solution (a), add 20.0 ml of reference solution (b) and
dilute to 100.0 ml with a mixture of 10 volumes of acetonitrile R and 90 volumes of water R.
as mobile phase at a flow rate of 0.7 ml/min:
Mobile phase A. A mixture of 115 volumes of acetonitrile R and 885 volumes of buffer solution
pH 2.3,
Mobile phase B. A mixture of 350 volumes of acetonitrile R and 650 volumes of buffer solution
pH 2.3,
as detector a spectrophotometer set at 220 nm,
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
100 0 Equilibration
0 10 100 0 Isocratic
10 45 100 0 0 100 Linear gradient
45 65 0 100
Isocratic
66 80 100 0 Re-equilibration
maintaining the temperature of the column at 40C.
When the chromatograms are recorded in the prescribed conditions, the retention times are about
29 min for N-acetyltryptophan and about 34 min for 1,1-ethylidenebis(tryptophan). Adjust the
sensitivity of the system so that the height of the peak due to N-acetyltryptophan in the chromato-
gram obtained with reference solution (a) is at least 50 per cent of the full scale of the recorder.
Inject 20 l of reference solution (c). The test is not valid unless in the chromatogram obtained, the
resolution between the peaks corresponding to N-acetyltryptophan and 1,1-ethylidene-
bis(tryptophan) is at least 8.0. If necessary, adjust the time programme for the elution gradient. An
increase in the duration of elution with mobile phase A produces longer retention times and a better
resolution.
the test solution for 1.8 times the retention time of N-acetyltryptophan. In the chromatogram
obtained with the test solution: the area of any peak, apart from the principal peak, is not greater than
0.25 times the area of the principal peak in the chromatogram obtained with reference solution (a)
(0.25 per cent); the sum of the areas of all the peaks, apart from the principal peak, is not greater
than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution
(a) (0.5 per cent). Disregard any peak due to the solvent and any peak with an area less than 0.01
times that of the peak due to N-acetyltryptophan in the chromatogram obtained with reference
solution (a).
Ammonium (2.4.1).0.10 g complies with limit test B for ammonium (200 ppm). Prepare the
standard using 0.2 ml of ammonium standard solution (100 ppm NH
4
) R.
Iron (2.4.9). Dissolve 1.0 g in 50 ml of hydrochloric acid R1, with heating at 50C. Allow to cool. In a
separating funnel, shake with three quantities, each of 10 ml, of methyl isobutyl ketone R1, shaking for
3 min each time. To the combined organic layers add 10 ml of water R and shake for 3 min. The
aqueous layer complies with the limit test for iron (10 ppm).
1-27
100C to 105C.
ASSAY
Dissolve 0.200 g in 5 ml of methanol R. Add 50 ml of ethanol R. Titrate with 0.1M sodium hydroxide,
13
H
14
N
2
O
3
.
STORAGE
IMPURITIES
A. tryptophan,
*
and epimer at C*
H
N
COOH
H NH
2
OH
O
B. (S)-2-amino-3-[(3RS)-3-hydroxy-2-oxo-2,3-dihydro-1H-indol-3-yl]propanoic acid
(dioxyindolylalanine),
O
COOH
NH
2
NH
2
H
C. (S)-2-amino-4-(2-aminophenyl)-4-oxobutanoic acid (kynurenine),
H
N
COOH
H NH
2
HO
D. (S)-2-amino-3-(5-hydroxy-1H-indol-3-yl)propanoic acid (5-hydroxytryptophan),
N
H
O
COOH
NH
2
CHO
H
E. (S)-2-amino-4-[2-(formylamino)phenyl]-4-oxobutanoic acid (N-formylkynurenine),
H
N
COOH
NH
2
H
F. (S)-2-amino-3-(phenylamino)propanoic acid (3-phenylaminoalanine),
H
N
COOH
H NH
2
OH
G. (S)-2-amino-3-(2-hydroxy-1H-indol-3-yl)propanoic acid (2-hydroxytryptophan),
H
N
COOH
NH
H
and enantiomer
H. (3RS)-1,2,3,4-tetrahydro-9H--carboline-3-carboxylic acid,
H
N
COOH
NH
Me
I. 1-methyl-1,2,3,4-tetrahydro-9H--carboline-3-carboxylic acid,
1-28
H
N
OH
OH
COOH
H
2
N
N
H
H
*
*
J. (S)-2-amino-3-[2-[2,3-dihydroxy-1-(1H-indol-3-yl)propyl]-1H-indol-3-yl]propanoic acid,
H
N
COOH
H
2
N
N
H
H
K. (S)-2-amino-3-[2-(1H-indol-3-ylmethyl)-1H-indol-3-yl]propanoic acid,
H
N
NH
COOH
H
N
L. 1-(1H-indol-3-ylmethyl)-1,2,3,4-tetrahydro-9H--carboline-3-carboxylic acid.
__________________________________________________________________________________________________________ Ph Eur
1-29
Acetyltyrosine
COOH
O
H NHAc
C
11
H
13
NO
4
223.2 537-55-3
Acetyltyrosine complies with the requirements of the 3rd edition of the European Pharmacopoeia for N-
Acetyltyrosine [1384]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
N-Acetyltyrosine contains not less than 98.5 per cent and not more than the equivalent of 101.0 per
cent of (2S)-2-(acetylamino)-3-(4-hydroxyphenyl)propanoic acid, calculated with reference to the
dried substance.
CHARACTERS
A white, crystalline powder or colourless crystals, freely soluble in water, practically insoluble in
cyclohexane.
IDENTIFICATION
obtained with N-acetyltyrosine CRS. Examine the substances prepared as discs.
C. Examine the chromatograms obtained in the test for related substances in ultraviolet light at
254 nm. The principal spot obtained with test solution (b) is similar in position and size to the
principal spot in the chromatogram obtained with reference solution (a).
D. Solution S (see Tests) is strongly acid (2.2.4).
TESTS
Solution S Dissolve 2.50 g in water R and dilute to 100.0 ml with the same solvent.
Specific optical rotation (2.2.7). Dilute 10.0 ml of solution S to 25.0 ml with water R. The specific
optical rotation is +46 to +49 calculated with reference to the dried substance.
Related substances Examine by thin-layer chromatography (2.2.27), using a TLC silica gel F
254
plate R.
Test solution (a). Dissolve 0.80 g of the substance to be examined in 6 ml of a mixture of equal
volumes of glacial acetic acid R and water R and dilute to 10 ml with ethanol R.
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with ethanol R.
Reference solution (a). Dissolve 80 mg of N-acetyltyrosine CRS in a mixture of 3 volumes of water R, 3
volumes of glacial acetic acid R and 94 volumes of ethanol R and dilute to 10 ml with the same
mixture of solvents.
Reference solution (b). Dilute 0.5 ml of test solution (b) to 10 ml with ethanol R.
Reference solution (c). Dissolve 40 mg of tyrosine CRS in 20 ml of a mixture of equal volumes of
water R and glacial acetic acid R and dilute to 50 ml with ethanol R.
10 volumes of water R, 15 volumes of glacial acetic acid R and 75 volumes of ethyl acetate R. Allow the
plate to dry in air. Examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained
with test solution (a), apart from the principal spot, is not more intense than the spot in the chroma-
togram obtained with reference solution (b) (0.5 per cent). Spray with ninhydrin solution R and heat
at 100C to 105C for 10 min. Examine in daylight. Any spot corresponding to tyrosine is not more
intense that the spot in the chromatogram obtained with reference solution (c) (1 per cent).
Chlorides (2.4.4). Dilute 10 ml of solution S to 15 ml with water R. The solution complies with the
limit test for chlorides (200 ppm).
Sulphates (2.4.13). Dissolve 1.0 g in distilled water R and dilute to 20 ml with the same solvent. The
solution complies with the limit test for sulphates (200 ppm).
1-30
Ammonium Prepare a cell consisting of two watch-glasses 60 mm in diameter placed edge to
edge. To the inner wall of the upper watch-glass stick a piece of red litmus paper R 5 mm square and
wetted with a few drops of water R. Finely powder the substance to be examined, place 50 mg in
the lower watch-glass and dissolve in 0.5 ml of water R. To the solution add 0.30 g of heavy
magnesium oxide R. Briefly triturate with a glass rod. Immediately close the cell by putting the two
watch-glasses together. Heat at 40C for 15 min. The litmus paper is not more intensely blue
coloured than a standard prepared at the same time and in the same manner using 0.1 ml of
ammonium standard solution (100 ppm NH
4
) R, 0.5 ml of water R and 0.30 g of heavy magnesium oxide R
(200 ppm).
Iron (2.4.9). In a separating funnel, dissolve 0.5 g in 10 ml of dilute hydrochloric acid R. Shake with
three quantities, each of 10 ml, of methyl isobutyl ketone R1, shaking for 3 min each time. To the
combined organic layers add 10 ml of water R and shake for 3 min. The aqueous layer complies with
the limit test for iron (20 ppm).
Heavy metals (2.4.8). Dissolve 2.0 g in water R and dilute to 20 ml with the same solvent. 12 ml of
the solution complies with limit test A for heavy metals (10 ppm). Prepare the standard using lead
standard solution (1 ppm Pb) R.
100C to 105C.
Sterility (2.6.1). If intended for use in the manufacture of parenteral dosage forms without a further
appropriate sterilisation procedure, it complies with the test for sterility.
Pyrogens (2.6.8). If intended for use in the manufacture of parenteral dosage forms without a
further appropriate procedure for the removal of pyrogens, it complies with the test for pyrogens.
Inject per kilogram of the rabbits mass 1.0 ml of a freshly prepared solution in water for injections R
containing per millilitre 10.0 mg of the substance to be examined and 9.0 mg of pyrogen-free sodium
chloride R.
ASSAY
Dissolve 0.180 g in 50 ml of carbon dioxide-free water R. Titrate with 0.1M sodium hydroxide, deter-
mining the end-point potentiometrically (2.2.20).
11
H
13
NO
4
.
STORAGE
Store in a well-closed container, protected from light. If the substance is sterile, store in a sterile,
airtight, tamper-proof container.
LABELLING
The label states:
where applicable, that the substance is sterile,
where applicable, that the substance is apyrogenic.
IMPURITIES
COOH
HO
H NH
2
A. (S)-2-amino-3-[4(hydroxy)phenyl]propanoic acid (tyrosine),
COOH
cO
H NHAc
B. (2S)-2-(acetylamino)-3-[4-(acetoxy)phenyl]propanoic acid (diacetyltyrosine).
__________________________________________________________________________________________________________ Ph Eur
1-31
Aciclovir
N
N
N
O
HN
H
2
N
O
OH
C
8
H
11
N
5
O 225.2 59277-89-3
Aciclovir complies with the requirements of the 3rd edition of the European Pharmacopoeia [0968]. These
Action and use Antiviral.
Preparations
Aciclovir Cream
Aciclovir Eye Ointment
Aciclovir Intravenous Infusion
Aciclovir Oral Suspension
Aciclovir Tablets
Dispersible Aciclovir Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Aciclovir contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of
2-amino-9-[(2-hydroxyethoxy)methyl]-1,9-dihydro-6H-purin-6-one, calculated with reference to the
anhydrous substance.
CHARACTERS
A white or almost white, crystalline powder, slightly soluble in water, freely soluble in dimethyl
sulphoxide, very slightly soluble in alcohol. It dissolves in dilute solutions of mineral acids and alkali
hydroxides.
IDENTIFICATION
Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained
with aciclovir CRS.
TESTS
Appearance of solution Dissolve 0.25 g in 0.1M sodium hydroxide and dilute to 25 ml with the same
solvent. The solution is clear (2.2.1) and not more intensely coloured than reference solution Y
7
(Method II, 2.2.2).
Related substances
A. Examine by thin-layer chromatography (2.2.27), using silica gel GF
254
Test solution. Dissolve 0.1 g of the substance to be examined in dimethyl sulphoxide R and dilute to
Reference solution. Dissolve 10 mg of aciclovir impurity A CRS in dimethyl sulphoxide R and dilute to
20 ml with the same solvent. Dilute 1 ml of the solution to 10 ml with dimethyl sulphoxide R.
Apply separately to the plate 10 l of each solution. Keep the spots compact by drying in a current of
warm air. Allow the plate to cool and develop over a path of 10 cm with a mixture of 2 volumes of
concentrated ammonia R, 20 volumes of methanol R and 80 volumes of methylene chloride R. Allow the
plate to dry in air and examine in ultraviolet light at 254 nm. In the chromatogram obtained with the
test solution, any spot with an R
f
value greater than that of the principal spot is not more intense than
the spot in the chromatogram obtained with the reference solution (0.5 per cent).
B. Examine by liquid chromatography (2.2.29).
Test solution. Dissolve 50.0 mg of the substance to be examined in 10 ml of a mixture of 20 volumes
of glacial acetic acid R and 80 volumes of water R and dilute to 100.0 ml with the mobile phase.
Reference solution (a). Dilute 1.0 ml of the test solution to 200.0 ml with the mobile phase.
1-32
Reference solution (b). Dissolve 20 mg of aciclovir CRS and 20 mg of aciclovir impurity A CRS in a
mixture of 20 volumes of glacial acetic acid R and 80 volumes of water R and dilute to 100.0 ml with
the same mixture of solvents. Dilute 1.0 ml of the solution to 10.0 ml with the mobile phase.
Reference solution (c). Dissolve 7 mg of guanine R in 0.1M sodium hydroxide and dilute to 100.0 ml with
the same solution. Dilute 1.0 ml to 20.0 ml with the mobile phase.
as mobile phase at a flow rate of 2 ml/min a mixture prepared as follows: dissolve 6.0 g of sodium
dihydrogen phosphate R and 1.0 g of sodium decanesulphonate R in 900 ml of water R and adjust to
pH3 0.1 with phosphoric acid R; add 40 ml of acetonitrile R and dilute to 1 litre with water R,
a loop injector.
Inject 20 l of each solution. Record the chromatograms for seven times the retention time of
aciclovir. The test is not valid unless in the chromatogram obtained with reference solution (b), the
number of theoretical plates calculated for the peak due to aciclovir impurity A is at least 1500 and its
mass distribution ratio is at least 7 (V
0
can be calculated using dimethyl sulphoxide R). In the chroma-
togram obtained with the test solution: the area of any peak corresponding to guanine is not greater
than that of the peak in the chromatogram obtained with reference solution (c) (0.7 per cent); the
area of any peak apart from the principal peak and any peak corresponding to guanine is not greater
than the area of the peak in the chromatogram obtained with reference solution (a) (0.5 per cent) and
the sum of the areas of such peaks is not greater than twice the area of the peak in the chromatogram
obtained with reference solution (a) (1 per cent). Disregard any peak with an area less than 0.05
times that of the principal peak in the chromatogram obtained with reference solution (a).
Water (2.5.12). Not more than 6.0 per cent, determined on 0.500 g by the semi-micro deter-
mination of water.
ASSAY
end-point potentiometrically (2.2.20). Carry out a blank titration.
8
H
11
N
5
O
3
.
STORAGE
IMPURITIES
HN
N
N
N
O
OAc
O
H
2
N
A. 2-[(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methoxy]ethyl acetate,
HN
N
H
N
N
O
2
N
B. 2-amino-1,7-dihydro-6H-purin-6-one (guanine),
HN
N
N
N
O
2
N
O
OH
C. 2-amino-7-[(2-hydroxyethoxy)methyl]-1,7-dihydro-6H-purin-6-one,
HN
N
N
N
O
OCOPh
O
H
2
N
D. 2-[(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methoxy]ethyl benzoate,
1-33
HN
N
N
N
O
OH
O
NH
2
E. 6-amino-9-[(2-hydroxyethoxy)methyl]-1,9-dihydro-2H-purin-2-one,
HN
N
N
N
O
OH
O
AcHN
F. N-[9-[(2-hydroxyethoxy)methyl]-6-oxo-6,9-dihydro-1H-purin-2-yl]acetamide,
HN
N
N
N
O
OAc
O
AcHN
G. 2-[[2-(acetylamino)-6-oxo-1,6-dihydro-9H-purin-9-yl]methoxy]ethyl acetate,
HN
N
N
N
O
OCOPh
O
AcHN
H. 2-[[2-(acetylamino)-6-oxo-1,6-dihydro-9H-purin-9-yl]methoxy]ethyl benzoate.
__________________________________________________________________________________________________________ Ph Eur
1-34
Acitretin
corrected 1/01
COOH
Me
Me
MeO Me
Me Me
C
21
H
26
O
3
326.4 55079-83-9
Acitretin complies with the requirements of the 3rd edition of the European Pharmacopoeia [1385]. These
Action and use Antipsoriatic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Acitretin contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent of
(2E,4E,6E,8E)-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-dimethylnona-2,4,6,8-tetraenoic acid,
calculated with reference to the dried substance.
CHARACTERS
A yellow or greenish-yellow, crystalline powder, practically insoluble in water, very slightly soluble in
cyclohexane, slightly soluble in acetone and in alcohol, sparingly soluble in tetrahydrofuran. It is
sensitive to air, heat and light, especially in solution.
Carry out all operations as rapidly as possible and avoid exposure to actinic light; use freshly prepared
solutions.
IDENTIFICATION
A. Dissolve 15.0 mg in 10 ml of tetrahydrofuran R and dilute immediately to 100.0 ml with the same
solvent. Dilute 2.5 ml of this solution to 100.0 ml with tetrahydrofuran R. Examined between 300 nm
and 400 nm (2.2.25), the solution shows an absorption maximum at 358 nm. The specific
absorbance at the maximum is 1350 to 1475.
obtained with acitretin CRS. Examine the substances prepared as discs.
C. Examine the chromatograms obtained in the assay. The principal peak in the chromatogram
obtained with the test solution has a retention time similar to that of the principal peak in the
chromatogram obtained with reference solution (a).
TESTS
Related substances Examine by liquid chromatography (2.2.29) as described under Assay.
Inject separately 10 l each of reference solutions (b) and (c) and of the test solution. Adjust the
sensitivity of the system so that the height of the principal peak in the chromatogram obtained with
reference solution (b) is not less than 40 per cent of the full scale of the recorder. The test is not valid
unless the resolution between the peaks due to acitretin and tretinoin in the chromatogram obtained
with reference solution (b) is at least 2.0. If necessary, adjust the concentration of ethanol R.
Continue the chromatography of the test solution for 2.5 times the retention time of the principal
peak. In the chromatogram obtained with the test solution, the area of any peak, apart from the
principal peak, is not greater than the area of the peak due to acitretin in the chromatogram obtained
with reference solution (c) (0.3 per cent); the sum of the areas of any peaks, apart from the principal
peak, is not more than the area of the peak due to acitretin in the chromatogram obtained with
reference solution (b) (1.0 per cent). Disregard any peak with an area less than 0.1 times that of the
principal peak in the chromatogram obtained with reference solution (c).
Palladium Not more than 10 ppm of Pd, determined by atomic absorption spectrometry (Method I,
2.2.23).
Test solution. Introduce 2.0 g of the substance to be examined into a quartz beaker and add 3 ml of
magnesium nitrate solution R. Heat in a muffle furnace to 350C at a rate of 40C per minute to
1-35
incinerate the content. Ignite at about 450C for 8 h and then at 550C for a further hour.
Dissolve the residue in a mixture of 0.75 ml of hydrochloric acid R and 0.25 ml of nitric acid R
warming gently. Cool, transfer the solution into a volumetric flask with water R and dilute to
50.0 ml with the same solvent.
Reference solution. Dissolve 0.163 g of heavy magnesium oxide R in a mixture of 0.5 ml of nitric acid R,
1.5 ml of hydrochloric acid R and 50 ml of water R, add 2.0 ml of palladium standard solution (20 ppm
Pd) R and dilute to 100.0 ml with water R.
Measure the absorbance at 247.6 nm using a palladium hollow-cathode lamp as source of radiation
and an air-acetylene flame.
Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in vacuo at
100C for 4 h.
ASSAY
Carry out the assay protected from light, use amber volumetric flasks and prepare the solutions immediately
before use.
Examine by liquid chromatography (2.2.29).
Test solution. Dissolve 25.0 mg of the substance to be examined in 5 ml of tetrahydrofuran R and
dilute immediately to 100.0 ml with ethanol R.
Reference solution (a). Dissolve 25.0 mg of acitretin CRS in 5 ml of tetrahydrofuran R and dilute
immediately to 100.0 ml with ethanol R.
Reference solution (b). Dissolve 1.0 mg of tretinoin CRS in ethanol R and dilute to 20.0 ml with the
same solvent. Mix 5.0 ml of the solution with 1.0 ml of reference solution (a) and dilute to 100.0 ml
with ethanol R.
Reference solution (c). Dilute 2.5 ml of reference solution (a) to 50.0 ml with ethanol R. Dilute 3.0 ml
of the solution to 50.0 ml with ethanol R.
a stainless steel column 0.25 m long and 4 mm in internal diameter packed with
microparticulate octadecylsilyl silica gel for chromatography R (5 m), with a carbon loading of
20 per cent, a specific surface area of 200 m
2
/g and a pore size of 15 nm,
as mobile phase at a flow rate of 0.6 ml/min a 0.3 per cent V/V solution of glacial acetic acid R in
a mixture of 8 volumes of water R and 92 volumes of ethanol R,
a sampler kept at 4C,
When the chromatograms are recorded in the prescribed conditions, the retention times are: impurity
A about 4.8 min, tretinoin about 5.2 min, acitretin about 6.2 min and impurity B about 10.2 min.
Inject 10 l of reference solution (a) six times. The test is not valid unless the relative standard
deviation of the peak area for acitretin is at most 1.0 per cent. If necessary, adjust the integration
parameters. Inject alternately the test solution and reference solution (a).
STORAGE
Store in an airtight container, protected from light, at a temperature between 2C and 8C.
It is recommended that the contents of an opened container be used as soon as possible and any
unused part be protected by an atmosphere of inert gas.
IMPURITIES
COOH
Me Me Me
MeO Me
Me
A. (2Z,4E,6E,8E)-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-dimethylnona-2,4,6,8-tetraenoic
acid,
Me Me Me
eO Me
COOEt
Me
B. ethyl (2E,4E,6E,8E)-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-dimethylnona-2,4,6,8-
tetraenoate.
__________________________________________________________________________________________________________ Ph Eur
1-36
Adenine
HN
N
N
N
NH
2
C
5
H
5
N
5
135.1 73-24-5
Adenine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0800]. These
Action and use Constituent of anticoagulant and preservative solutions for blood.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Adenine contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of
(7H-purin-6-yl)amine, calculated with reference to the dried substance.
CHARACTERS
A white powder, very slightly soluble in water and in alcohol, practically insoluble in ether. It
dissolves in dilute mineral acids and in dilute solutions of alkali hydroxides.
IDENTIFICATION
First identification: A.
obtained with adenine CRS. Examine the substances prepared as discs.
B. Examine the chromatograms obtained in the test for related substances. The principal spot in the
chromatogram obtained with test solution (b) is similar in position and size to the principal spot in
the chromatogram obtained with reference solution (a).
C. To 1 g add 3.5 ml of propionic anhydride R and boil for 15 min with stirring. Cool. To the
resulting crystalline mass add 15 ml of light petroleum R and heat to boiling with vigorous stirring.
Cool and filter. Wash the precipitate with two quantities, each of 5 ml, of light petroleum R. Dissolve
the precipitate in 10 ml of water R and boil for 1 min. Filter the mixture at 30C to 40C. Allow to
cool. Filter, and dry the precipitate at 100C to 105C for 1 h. The melting point (2.2.14) of the
precipitate is 237C to 241C.
TESTS
Solution S Suspend 2.5 g in 50 ml of distilled water R and boil for 3 min. Cool and dilute to 50 ml
with distilled water R. Filter. Use the filtrate as solution S.
Appearance of solution Dissolve 0.5 g in dilute hydrochloric acid R and dilute to 50 ml with the
same acid. The solution is clear (2.2.1) and colourless (Method II, 2.2.2).
Acidity or alkalinity To 10 ml of solution S add 0.1 ml of bromothymol blue solution R1 and 0.2 ml
of 0.01M sodium hydroxide. The solution is blue. Add 0.4 ml of 0.01M hydrochloric acid. The solution is
yellow.
254
R as the
coating substance.
Test solution (a). Dissolve 0.10 g of the substance to be examined in dilute acetic acid R, with heating if
necessary, and dilute to 10 ml with the same acid.
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with dilute acetic acid R.
Reference solution (a). Dissolve 10 mg of adenine CRS in dilute acetic acid R, with heating if necessary,
and dilute to 10 ml with the same acid.
Reference solution (b). Dilute 1 ml of Test solution (b) to 20 ml with dilute acetic acid R.
Reference solution (c). Dissolve 10 mg of adenine CRS and 10 mg of adenosine R in dilute acetic acid R,
with heating if necessary, and dilute to 10 ml with the same acid.
20 volumes of concentrated ammonia R, 40 volumes of ethyl acetate R and 40 volumes of propanol R.
1-37
Dry the plate in a current of warm air and examine in ultraviolet light at 254 nm. Any spot in the
chromatogram obtained with test solution (a), apart from the principal spot, is not more intense than
the spot in the chromatogram obtained with reference solution (b) (0.5 per cent). The test is not
valid unless the chromatogram obtained with reference solution (c) shows two clearly separated
spots.
Chlorides (2.4.4). To 10 ml of solution S add 1 ml of concentrated ammonia R and 3 ml of silver
nitrate solution R2. Filter. Wash the precipitate with a little water R and dilute the filtrate to 15 ml with
water R. The solution complies with the limit test for chlorides (100 ppm). When carrying out the
test, add 2 ml of dilute nitric acid R instead of 1 ml of dilute nitric acid R.
Sulphates (2.4.13). Dilute 10 ml of solution S to 15 ml with distilled water R. The solution complies
with the limit test for sulphates (300 ppm).
Ammonium Prepare a cell consisting of two watch-glasses 60 mm in diameter placed edge to edge.
To the inner wall of the upper watch-glass stick a piece of red litmus paper R 5 mm square and wetted
with a few drops of water R. Finely powder the substance to be examined, place 0.5 g in the lower
watch-glass and suspend in 0.5 ml of water R. To the suspension add 0.30 g of heavy magnesium
oxide R. Briefly triturate with a glass rod. Immediately close the cell by putting the two watch-glasses
together. Heat at 40C for 15 min. The litmus paper is not more intensely blue coloured than a
standard prepared at the same time and in the same manner using 0.05 ml of ammonium standard
solution (100 ppm NH
4
) R, 0.5 ml of water R and 0.30 g of heavy magnesium oxide R (10 ppm).
100C to 105C.
ASSAY
Dissolve 0.100 g in a mixture of 20 ml of acetic anhydride R and 30 ml of anhydrous acetic acid R.
Titrate with 0.1M perchloric acid, determining the end-point potentiometrically (2.2.20).
5
H
5
N
5
.
STORAGE
_______________________________________________________________________ Ph Eur
1-38
Adenosine
1/01
N
N
N
N
NH
2
O
HO
OH OH
C
10
H
13
N
5
O
4
267.2 58-61-7
Adenosine complies with the requirements of the 3rd edition of the European Pharmacopoeia [1486]. These
Action and use Antiarrythmic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Adenosine contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of
9--D-ribofuranosyl-9H-purin-6-amine, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder slightly soluble in water, soluble in hot water, practically insoluble in
alcohol and in methylene chloride. It dissolves in dilute mineral acids.
IDENTIFICATION
with adenosine CRS.
TESTS
Solution S Suspend 5.0 g in 100 ml of distilled water R and heat to boiling. Allow to cool, filter with
the aid of vacuum and dilute to 100 ml with distilled water R.
Appearance of solution Solution S is colourless (Method II, 2.2.2).
Acidity or alkalinity To 10 ml of solution S, add 0.1 ml of bromocresol purple solution R and 0.1 ml
of 0.01M hydrochloric acid. The solution is yellow. Add 0.4 ml of 0.01M sodium hydroxide, the solution
is violet-blue.
Specific optical rotation (2.2.7). Dissolve 1.25 g in 1M hydrochloric acid and dilute to 50.0 ml with
the same acid. Determined within 10 min and calculated with reference to the dried substance, the
specific optical rotation is 45 to 49.
254
plate R.
Test solution. Dissolve 0.20 g of the substance to be examined in dilute acetic acid R with slight heating
and dilute to 5 ml with the same acid.
Reference solution (a). Dilute 1 ml of the test solution to 100 ml with water R.
Reference solution (b). Dissolve 10 mg of adenosine CRS and 10 mg of adenine CRS in dilute acetic
acid R, with heating if necessary, and dilute to 10 ml with the same acid.
of water R, 30 volumes of concentrated ammonia R and 60 volumes of propanol R. Allow the plate to
dry in a current of warm air and examine in ultraviolet light at 254 nm. Any spot in the chromato-
gram obtained with the test solution, apart from the principal spot, is not more intense than the spot
in the chromatogram obtained with reference solution (a) (1 per cent). Spray with a 5 g/l solution of
potassium permanganate R in 1M sodium hydroxide. Allow the plate to dry in a current of warm air and
examine in daylight. Any spot in the chromatogram obtained with the test solution, apart from the
principal spot, is not more intense than the spot in the chromatogram obtained with reference
1-39
solution (a) (1 per cent). The test is not valid unless the chromatogram obtained with reference
solution (b) shows two clearly separated spots.
Sulphates (2.4.13). 15 ml of solution S complies with the limit test for sulphates (200 ppm).
Ammonium (2.4.1). 0.5 g complies with limit test B for ammonium (10 ppm). Prepare the standard
using 5 ml of ammonium standard solution (1 ppm NH
4
) R.
100C to 105C.
ASSAY
Dissolve 0.200 g, warming slightly if necessary, in a mixture of 20 ml of acetic anhydride R and 30 ml
of anhydrous acetic acid R. Titrate with 0.1M perchloric acid, determining the end-point potentiometric-
ally (2.2.20).
10
H
13
N
5
O
4
.
STORAGE
IMPURITIES
A. 1H-purin-6-amine (adenine),
O
O
OH OH
OH
B. D-ribose,
N
N
N
N
NH
2
O
HO
O OH
P
O
R
O
OH
C. R = H: adenosine 3-(dihydrogen phosphate),
D. R = PO
3
H
2
: adenosine 3-(trihydrogen diphosphate),
E. R = PO
2
H-O-PO
3
H
2
: adenosine 3-(tetrahydrogen triphosphate).
__________________________________________________________________________________________________________ Ph Eur
1-40
Adrenaline / Epinephrine
NHMe
OH H
HO
HO
C
9
H
13
NO
3
183.2 51-43-4
Definition Adrenaline is (R)-1-(3,4-dihydroxyphenyl)-2-methylaminoethanol. It contains not less
than 98.5% and not more than 101.0% of C
9
H
13
NO
3
substance.
Production It may be prepared by synthesis or isolated from the medulla of the suprarenal glands
of certain mammals and substantially freed from noradrenaline.
Characteristics A white or creamy white, sphaero-crystalline powder. It darkens in colour on
exposure to air and light.
Sparingly soluble in water; practically insoluble in ethanol (96%) and in ether. It dissolves in
solutions of mineral acids, of potassium hydroxide and of sodium hydroxide, but not in solutions of
ammonia or of the alkali carbonates.
It is not stable in neutral or alkaline solutions, which rapidly become red on exposure to air.
Identification
A. The infrared absorption spectrum, Appendix II A, is concordant with the reference spectrum of
adrenaline (RS 004).
B. To 1 ml of a 0.1% w/v solution add 1 ml of a 1.0% v/v solution of 2,5-diethoxytetrahydrofuran
in glacial acetic acid. Heat at 80 for 2 minutes, cool in ice and add 3 ml of a 2.0% w/v solution of
4-dimethylaminobenzaldehyde in a mixture of 1 volume of hydrochloric acid and 19 volumes of glacial
acetic acid. Mix and allow to stand for 2 minutes. The solution shows a yellow colour which is
similar to that of a solution prepared in the same manner but omitting the substance being exam-
ined (distinction from noradrenaline).
Specific optical rotation In a freshly prepared 4% w/v solution in 1M hydrochloric acid, 50 to
53, calculated with reference to the dried substance, Appendix V F.
Noradrenaline Carry out the method for liquid chromatography, Appendix III D, using the
following solutions. Solution (1) contains 0.0018% w/v of noradrenaline acid tartrate in the mobile
phase. Solution (2) contains 0.10% w/v of the substance being examined and 1% v/v of hydrochloric
acid in the mobile phase. Solution (3) contains 0.0018% w/v of noradrenaline acid tartrate and
0.0018% w/v of adrenaline acid tartrate BPCRS in the mobile phase.
The chromatographic procedure may be carried out using (a) a stainless steel column
(10 cm 4.6 mm) packed with stationary phase C (5 m) (Nucleosil ODS is suitable), (b) as the
mobile phase with a flow rate of 2 ml per minute a solution containing 4.0 g of tetramethyl-
ammonium hydrogen sulphate, 1.1 g of sodium heptanesulphonate and 2 ml of 0.1M disodium edetate in
a mixture of 950 ml of water and 50 ml of methanol, the pH of the mixture being adjusted to 3.5
with 1M sodium hydroxide, and (c) a detection wavelength of 205 nm.
The test is not valid unless the resolution factor between the two principal peaks in the chromato-
gram obtained with solution (3) is at least 2.0.
In the chromatogram obtained with solution (2) the area of any peak corresponding to
noradrenaline is not greater than the area of the principal peak in the chromatogram obtained with
solution (1) (1%).
Phenones Absorbance of a 0.2% w/v solution in 0.1M hydrochloric acid at 310 nm, not more than
0.20, calculated with reference to the dried substance, Appendix II B.
Loss on drying When dried over phosphorus pentoxide at a pressure not exceeding 0.7 kPa for 18
hours, loses not more than 1.0% of its weight. Use 1 g.
Assay Carry out Method I for non-aqueous titration, Appendix VIII A, using 0.3 g and crystal violet
solution as indicator. Each ml of 0.1M perchloric acid VS is equivalent to 18.32 mg of C
9
H
13
NO
3
.
Storage Adrenaline should be kept in a well-closed container, which is preferably filled with
nitrogen, and protected from light.
Action and use Beta-adrenoceptor agonist; used in treatment of glaucoma.
1-41
Preparations
Adrenaline Eye Drops/Epinephrine Eye Drops
Dilute Adrenaline Injection (1 in 10,000)/Dilute Epinephrine Injection (1 in 10,000)
1-42
Adrenaline Acid Tartrate / Epinephrine Acid Tartrate
HOOC
COOH
OH H
H OH
,
NHMe
OH H
HO
HO
C
9
H
13
NO
3
,C
4
H
6
O
6
333.3 51-42-3
Adrenaline Acid Tartrate complies with the requirements of the 3rd edition of the European Pharmacopoeia
for Adrenaline Tartrate [0254]. These requirements are reproduced after the heading Definition below.
Action and use Beta-adrenoceptor agonist.
Preparations
Adrenaline Injection/Epinephrine Injection
Dilute Adrenaline Injection (1 in 10,000)/Dilute Epinephrine Injection (1 in 10,000)
Adrenaline Solution/Epinephrine Solution
Bupivacaine and Adrenaline Injection/Bupivacaine and Epinephrine Injection
Lidocaine and Adrenaline Injection/Lignocaine and Epinephrine Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Adrenaline tartrate contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of (R)-1-(3,4-dihydroxyphenyl)-2-(methylamino)ethanol hydrogen tartrate, calculated
with reference to the dried substance.
CHARACTERS
A white to greyish-white, crystalline powder, freely soluble in water, slightly soluble in alcohol,
practically insoluble in ether.
IDENTIFICATION
First identification: A, C, F.
Second identification: A, B, D, E, F.
A. Dissolve 2 g in 20 ml of a 5 g/l solution of sodium metabisulphite R and make alkaline by addition of
ammonia R. Keep the mixture in iced water for 1 h and filter. Reserve the filtrate for identification
test F. Wash the precipitate with three quantities, each of 2 ml, of water R, with 5 ml of alcohol R and
finally with 5 ml of ether R and dry in vacuo for 3 h. The specific optical rotation (2.2.7) of the
precipitate (adrenaline base) is 50 to 54, determined using a 20.0 g/l solution in 0.5M hydrochloric
acid.
B. Dissolve 50.0 mg in 0.01M hydrochloric acid and dilute to 100.0 ml with the same acid. Dilute
10.0 ml of the solution to 100.0 ml with 0.01M hydrochloric acid. Examined between 250 nm and
300 nm (2.2.25), the solution shows an absorption maximum at 279 nm. The specific absorbance at
the maximum is 79 to 85.
obtained with adrenaline base prepared by the same method from a suitable amount of adrenaline
tartrate CRS. Use adrenaline base prepared as described under identification test A. Examine the
substances prepared as discs.
D. Dissolve about 5 mg in 5 ml of water R. To 1 ml of the solution add 10 ml of buffer solution pH
3.6 R and 1 ml of 0.05M iodine. Allow to stand for 5 min. Add 2 ml of 0.1M sodium thiosulphate. An
intense violet-red colour is produced.
E. To 1 ml of the solution prepared for identification test D add 1 ml of a 1 per cent V/V solution of
diethoxytetrahydrofuran R in glacial acetic acid R. Heat at 80C for 2 min, cool in iced water and add
3 ml of a 20 g/l solution of dimethylaminobenzaldehyde R in a mixture of 1 volume of hydrochloric
acid R and 19 volumes of glacial acetic acid R. Mix and allow to stand for 2 min. The solution shows a
yellow colour which is similar to that of a blank solution.
F. 0.2 ml of the filtrate obtained in identification test A gives reaction (b) of tartrates (2.3.1).
1-43
TESTS
Appearance of solution Dissolve 0.5 g in water R and dilute to 10 ml with the same solvent.
Examine the solution immediately. The solution is not more opalescent than reference suspension II
(2.2.1) and is not more intensely coloured than reference solution BY
5
(Method II, 2.2.2).
Adrenalone Dissolve 50.0 mg in 0.01M hydrochloric acid and dilute to 25.0 ml with the same acid.
The absorbance (2.2.25) of the solution measured at 310 nm is not greater than 0.10.
Noradrenaline Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating
substance.
same solvent. Prepare immediately before use.
Reference solution (a). Dissolve 12.5 mg of noradrenaline tartrate CRS in water R and dilute to 10 ml
with the same solvent. Prepare immediately before use.
Reference solution (b). Dilute 2 ml of reference solution (a) to 10 ml with water R.
Reference solution (c). Mix 2 ml of the Test solution with 2 ml of reference solution (b).
Apply separately to the plate as bands 20 mm by 2 mm 6 l of the test solution, 6 l of reference
solution (a), 6 l of reference solution (b) and 12 l of reference solution (c). Allow to dry and spray
the bands with a saturated solution of sodium bicarbonate R. Allow the plate to dry in air and spray the
bands twice with acetic anhydride R, drying between the two sprayings. Heat the plate at 50C for
90 min. Develop over a path of 15 cm using a mixture of 0.5 volumes of anhydrous formic acid R, 50
volumes of acetone R and 50 volumes of methylene chloride R. Allow the plate to dry in air and spray
with a solution freshly prepared by mixing 2 volumes of ethylenediamine R and 8 volumes of
methanol R and adding 2 volumes of a 5 g/l solution of potassium ferricyanide R. Dry the plate at 60C
for 10 min and examine in ultraviolet light at 254 nm and 365 nm. In the chromatogram obtained
with the test solution, any zone situated between the two most intense zones is not more intense than
the corresponding zone in the chromatogram obtained with reference solution (b) (1.0 per cent). The
test is not valid unless the chromatogram obtained with reference solution (c) shows between the two
most intense zones a clearly separated zone corresponding to the most intense zone in the chromato-
gram obtained with reference solution (a).
Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.00 g by drying in vacuo for
18 h.
ASSAY
Dissolve 0.300 g in 50 ml of anhydrous acetic acid R, heating gently if necessary. Titrate with 0.1M
perchloric acid until a bluish-green colour is obtained using 0.1 ml of crystal violet solution R as
indicator.
13
H
19
NO
9
.
STORAGE
Store in an airtight container, or preferably in a sealed tube under vacuum or under an inert gas,
protected from light.
_______________________________________________________________________ Ph Eur
1-44
Agar
Agar complies with the requirements of the 3rd edition of the European Pharmacopoeia [0030]. These require-
ments are reproduced after the heading Definition below.
Action and use Pharmaceutical aid.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Agar consists of the polysaccharides from various species of Rhodophyceae mainly belonging to the
genus Gelidium. It is prepared by treating the algae with boiling water; the extract is filtered whilst
hot, concentrated and dried.
CHARACTERS
Agar has a mucilaginous taste.
It occurs in the form of a powder or in crumpled strips 2 mm to 5 mm wide or sometimes in flakes,
colourless to pale yellow, translucent, somewhat tough and difficult to break, becoming more brittle
on drying.
It has the microscopic characters described under identification test A.
IDENTIFICATION
A. Examine under a microscope. When mounted in 0.005M iodine, the strips or flakes are partly
stained brownish-violet. Magnified 100 times, they show numerous minute, colourless, ovoid or
rounded grains on an amorphous background; occasional brown, round to ovoid spores with a
reticulated surface, measuring up to 60 m, may be present. Reduce to a powder, if necessary. The
powder is yellowish-white. Examine under a microscope using 0.005M iodine. The powder presents
angular fragments with numerous grains similar to those seen in the strips and flakes; some of the
fragments are stained brownish-violet.
B. Dissolve 0.1 g with heating in 50 ml of water R. Cool. To 1 ml of the mucilage carefully add 3 ml
of water R so as to form two separate layers. Add 0.1 ml of 0.05M iodine. A dark brownish-violet
colour appears at the interface. Mix. The liquid becomes pale yellow.
C. Heat 5 ml of the mucilage prepared for identification test B on a water-bath with 0.5 ml of hydro-
chloric acid R for 30 min. Add 1 ml of barium chloride solution R1. A white turbidity develops within
30 min.
D. Heat 0.5 g with 50 ml of water R on a water-bath until dissolved. Only a few fragments remain
insoluble. During cooling, the solution gels between 35C and 30C. Heat the gel thus obtained on a
water-bath; it does not liquefy below 80C.
TESTS
Swelling index (2.8.4). The swelling index, determined on the powdered drug (355), is not less
than 10 and is within 10 per cent of the value stated on the label.
Insoluble matter To 5.00 g of the powdered drug (355) add 100 ml of water R and 14 ml of dilute
hydrochloric acid R. Boil gently for 15 min with frequent stirring. Filter the hot liquid through a tared,
sintered-glass filter (160), rinse the filter with hot water R and dry at 100C to 105C. The residue
weighs not more than 50 mg (1.0 per cent).
Gelatin To 1.00 g add 100 ml of water R and heat on a water-bath until dissolved. Allow to cool to
50C. To 5 ml of the solution add 5 ml of picric acid solution R. No turbidity appears within 10 min.
3
micro-
organisms per gram, determined by plate-count. It complies with the tests for Escherichia coli and
Salmonella (2.6.13).
STORAGE
LABELLING
The label states the swelling index.
_______________________________________________________________________ Ph Eur
1-45
Medical Air
Medical Air complies with the requirements of the 3rd edition of the European Pharmacopoeia [1238]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Medical air is compressed ambient air containing not less than 20.4 per cent V/V and not more than
21.4 per cent V/V of oxygen (O
2
).
CHARACTERS
A colourless, odourless gas.
At a temperature of 20C and a pressure of 101 kPa, 1 volume dissolves in about 50 volumes of
water.
PRODUCTION
Carbon dioxide Not more than 500 ppm V/V, determined using an infrared analyser (2.5.24).
Gas to be examined. Use the substance to be examined. It must be filtered to avoid stray light
phenomena.
Reference gas (a). Use a mixture of 79 per cent V/V of nitrogen R1 and 21 per cent V/V of oxygen R
containing less than 1 ppm of carbon dioxide R1.
Reference gas (b). Use a mixture of 79 per cent V/V of nitrogen R1 and 21 per cent V/V of oxygen R
containing 500 ppm of carbon dioxide R1.
Calibrate the apparatus and set the sensitivity using reference gases (a) and (b). Measure the content
of carbon dioxide in the gas to be examined.
Carbon monoxide Not more than 5 ppm V/V, determined using an infrared analyser (2.5.25).
Gas to be examined. Use the substance to be examined. It must be filtered to avoid stray light
phenomena.
containing less than 1 ppm of carbon monoxide R.
containing 5 ppm of carbon monoxide R.
of carbon monoxide in the gas to be examined.
Sulphur dioxide Not more than 1 ppm V/V, determined using an ultraviolet fluorescence analyser
(Fig. 12381).
1-46
Fig. 12381 UV Fluorescence Analyser
The apparatus consists of the following:
a system generating ultraviolet radiation with a wavelength of 210 nm, made up of an ultraviolet
lamp, a collimator, and a selective filter; the beam is blocked periodically by a chopper rotating
at high speed,
a reaction chamber, through which flows the gas to be examined,
a system that detects radiation emitted at a wavelength of 350 nm, made up of a selective filter, a
photomultiplier tube and an amplifier.
Gas to be examined. Use the substance to be examined. It must be filtered.
Reference gas (a). Use a mixture of 79 per cent V/V of nitrogen R1 and 21 per cent V/V of oxygen R.
containing 0.5 ppm V/V to 2 ppm V/V of sulphur dioxide R1.
of sulphur dioxide in the gas to be examined.
Oil Not more than 0.1 mg per cubic metre, at atmospheric pressure and at 0C, determined using a
measuring system such as described in Fig. 12382.
an on-off valve (1),
a three-way valve (2),
an oil cone (3),
a bypass line (4),
a pressure-regulator (5),
a flow-metering device (6),
All of the apparatus is cleaned beforehand using trichlorotrifluoroethane R free from oil and grease.
Fig. 12382 Measuring system for oil
Place a micro fibre-glass filter in the oil cone (3); this filter has the following characteristics: 100 per
cent borosilicate glass without binder; resistant to heat treatment at 500C (to eliminate organic
1-47
traces); 99.999 per cent retention efficiency for NaCl particles with a diameter of 0.6 m. Close the
on-off valve (1); the substance to be examined enters the bypass line (4) and purges the three-way
valve (2), the pressure-regulator (5) and the flow-metering device (6). Close the inlet valve of the
compression and filtration system: open the on-off valve and set the three-way valve to the position
allowing passage between the oil cone and the pressure-regulator. Open the inlet valve and set the
pressure-regulator so that the flow indicated by the flow-metering device is 20 litres per minute. Pass
100.0 litres of the substance to be examined through the system.
Remove the micro fibre-glass filter and place in an airtight container. Carefully cut up the micro
fibre-glass filter and place the pieces in 25.0 ml of trichlorotrifluoroethane R (test solution).
Prepare the reference solutions with quantities of oil (used for the lubrication of the compression
system) ranging from 0.05 g per millilitre to 0.5 g per millilitre in trichlorotrifluoroethane R. Measure
the absorbance of the test solution and the reference solutions, using an appropriate infrared
spectrophotometer, at 2960.3 cm
1
, 2927.7 cm
1
and 2855.0 cm
1
. The sum of the three
absorbances gives the absorbance of the oil. Use potassium bromide cells with a pathlength of several
centimetres.
Plot the calibration curve from the absorbances obtained with the reference solutions and
determine the quantity of oil from this curve.
Nitrogen monoxide and nitrogen dioxide Not more than a total of 2 ppm V/V, determined using
a chemiluminescence analyser (2.5.26).
Gas to be examined. Use the substance to be examined.
containing less than 0.05 ppm of nitrogen monoxide and nitrogen dioxide.
Reference gas (b). Use a mixture of 2 ppm V/V of nitrogen monoxide R in nitrogen R1.
of nitrogen monoxide and nitrogen dioxide in the gas to be examined.
1-48
Fig. 12383 Gas burette
Water Not more than 67 ppm V/V, determined using an electrolytic hygrometer (2.5.28).
Assay Determine the concentration of oxygen in air using a paramagnetic analyser (2.5.27).
IDENTIFICATION
First identification: C.
Second identification: A, B.
A. In a conical flask containing the substance to be examined, place a glowing wood splinter. The
splinter remains glowing.
B. Use a gas burette (Fig. 12383) of 25 ml capacity in the form of a chamber in the middle of which
is a tube graduated in 0.2 per cent between 19.0 per cent and 23.0 per cent, and isolated at each end
by a tap with a conical barrel. The lower tap is joined to a tube with an olive-shaped nozzle and is
used to introduce the gas into the apparatus. A cylindrical funnel above the upper tap is used to
introduce the absorbent solution. Wash the burette with water R and dry. Open the two taps.
Connect the nozzle to the source of the substance to be examined and set the flow rate to 1 litre per
minute. Flush the burette by passing the substance to be examined through it for 1 min. Close the
lower tap of the burette and immediately afterwards the upper tap. Rapidly disconnect the burette
from the source of the substance to be examined. Rapidly give a half turn to the upper tap to
1-49
eliminate any excess pressure in the burette. Keeping the burette vertical, fill the funnel with a
freshly prepared mixture of 21 ml of a 560 g/l solution of potassium hydroxide R and 130 ml of a 200
g/l solution of sodium dithionite R. Open the upper tap slowly. The solution absorbs the oxygen and
enters the burette. Allow to stand for 10 min without shaking. Read the level of the liquid meniscus
on the graduated part of the burette. This figure represents the percentage V/V of oxygen. The value
read is 20.4 to 21.4.
C. It complies with the limits of the assay.
TESTS
Carbon dioxide Not more than 500 ppm V/V, determined using a carbon dioxide detector tube
(2.1.6).
Sulphur dioxide Not more than 1 ppm V/V, determined using a sulphur dioxide detector tube
(2.1.6).
Oil Not more than 0.1 mg per cubic metre, determined using an oil detector tube (2.1.6).
Nitrogen monoxide and nitrogen dioxide Not more than 2 ppm V/V, determined using a
nitrogen monoxide and nitrogen dioxide detector tube (2.1.6).
Carbon monoxide Not more than 5 ppm V/V, determined using a carbon monoxide detector tube
(2.1.6).
Water vapour Not more than 67 ppm V/V, determined using a water vapour detector tube (2.1.6).
STORAGE
Store as a gas in suitable containers complying with the legal regulations or as a gas supplied by a
pipe network.
IMPURITIES
A. carbon dioxide,
B. sulphur dioxide,
C. nitrogen monoxide,
D. nitrogen dioxide,
E. oil,
F. carbon monoxide,
G. water.
_______________________________________________________________________ Ph Eur
1-50
Synthetic Air
Definition Synthetic Air is a mixture of Nitrogen and Oxygen containing not less than 19.9% and
not more than 21.9% v/v of oxygen (O
2
).
Characteristics A colourless, odourless gas. At a temperature of 20 and a pressure of 101 kPa, 1
volume dissolves in about 50 volumes of water.
Production
Carbon dioxide Not more than 300 ppm v/v, determined using an infrared analyser, Appendix
IX G.
Sample Use the gas being examined filtered to avoid stray light phenomena.
Reference gas (a) Use a mixture of 79% v/v of nitrogen R1 and 21% v/v of oxygen containing less than
1 ppm of carbon dioxide R1.
Reference gas (b) Use a mixture of 79% v/v of nitrogen R1 and 21% v/v of oxygen containing 500 ppm
of carbon dioxide R1.
of carbon dioxide in the gas being examined.
Carbon monoxide Not more than 5 ppm v/v, determined using an infrared analyser, Appendix
IX G.
Sample Use the gas being examined filtered to avoid stray light phenomena.
Reference gas (a) Use a mixture of 79% v/v of nitrogen R1 and 21% v/v of oxygen containing less than
1 ppm of carbon monoxide.
Reference gas (b) Use a mixture of 79% v/v of nitrogen R1 and 21% v/v of oxygen containing 5 ppm of
carbon monoxide.
of carbon monoxide in the gas to be examined.
Water Not more than 67 ppm v/v, determined using an electrolytic hygrometer, Appendix IX J.
Assay Determine the concentration of oxygen in the air using a paramagnetic analyser, Appendix
IX H.
Identification
A. In a conical flask containing the substance to be examined, place a glowing wood splinter. The
splinter remains glowing.
B. Complies with the Assay.
Carbon dioxide Not more than 300 ppm v/v, determined using a carbon dioxide detector tube,
Appendix IX K.
Carbon monoxide Not more than 5 ppm v/v, determined using a carbon monoxide detector tube,
Appendix IX K.
Water vapour Not more than 67 ppm v/v, determined using a water vapour detector tube,
Appendix IX K.
Assay Use a gas burette (Fig. 12383) of 25 ml capacity in the form of a chamber in the middle of
which is a tube graduated in 0.2% divisions between 19.0% and 23.0%, and isolated at each end by a
tap with a conical barrel. The lower tap is joined to a tube with an olive-shaped nozzle and is used to
introduce the gas into the apparatus. A cylindrical funnel above the upper tap is used to introduce the
absorbent solution. Wash the burette with water and dry. Open the two taps. Connect the nozzle to
the source of the substance to be examined and set the flow rate to 1 litre per minute. Flush the
burette by passing the substance to be examined through it for 1 minute. Close the lower tap of the
burette and immediately afterwards the upper tap. Rapidly disconnect the burette from the source of
the substance to be examined. Rapidly give a half turn to the upper tap to eliminate any excess
pressure in the burette. Keeping the burette vertical, fill the funnel with a freshly prepared mixture of
21 ml of a 56% w/v solution of potassium hydroxide and 130 ml of a 20% w/v solution of sodium
dithionite.Open the upper tap slowly. The solution absorbs the oxygen and enters the burette. Allow
to stand for 10 minutes without shaking. Read the level of the liquid meniscus on the graduated part
of the burette. This figure represents the percentage v/v of oxygen.
IMPURITIES
A. carbon dioxide,
B. carbon monoxide,
C. water.
1-51
Alanine
H C COOH
NH
2
H
C
3
H
7
NO
2
89.1 56-41-7
Alanine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0752]. These
Action and use Amino acid.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Alanine contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of
(S)-2-aminopropanoic acid, calculated with reference to the dried substance.
PRODUCTION
When Alanine is produced by a process involving fermentation steps, it complies with the
CHARACTERS
White or almost white, crystalline powder or colourless crystals, freely soluble in water, very slightly
soluble in alcohol, practically insoluble in ether.
IDENTIFICATION
obtained with alanine CRS. Examine the substances prepared as discs.
C. Examine the chromatograms obtained in the test for ninhydrin-positive substances. The principal
spot in the chromatogram obtained with test solution (b) is similar in position, colour and size to the
D. Dissolve 0.5 g in a mixture of 1 ml of water R, 0.5 ml of a 100 g/l solution of sodium nitrite R and
0.25 ml of hydrochloric acid R1. Shake. Gas is given off. Add 2 ml of dilute sodium hydroxide solution R,
followed by 0.25 ml of iodinated potassium iodide solution R. After about 30 min, a yellow precipitate
with a characteristic odour is formed.
TESTS
Solution S Dissolve 2.5 g in distilled water R and dilute to 50 ml with the same solvent.
Appearance of solution Dilute 10 ml of solution S to 20 ml with water R. The solution is clear
(2.2.1) and not more intensely coloured than reference solution BY
6
(Method II, 2.2.2).
Specific optical rotation (2.2.7). Dissolve 2.50 g in hydrochloric acid R1 and dilute to 25.0 ml with
the same acid. The specific optical rotation is +13.5 to +15.5, calculated with reference to the dried
substance.
Ninhydrin-positive substances Examine by thin-layer chromatography (2.2.27), using a TLC silica
gel G plate R.
Test solution (a). Dissolve 0.10 g in water R and dilute to 10 ml with the same solvent.
Test solution (b). Dilute 1 ml of test solution (a) to 50 ml with water R.
Reference solution (a). Dissolve 10 mg of alanine CRS in water R and dilute to 50 ml with the same
solvent.
Reference solution (b). Dilute 5 ml of Test solution (b) to 20 ml with water R.
Reference solution (c). Dissolve 10 mg of alanine CRS and 10 mg of glycine CRS in water R and dilute
to 25 ml with the same solvent.
Apply separately to the plate 5 l of each solution. Allow the plate to dry in air. Develop over a path
of 15 cm with a mixture of 20 volumes of glacial acetic acid R, 20 volumes of water R and 60 volumes
of butanol R. Allow the plate to dry in air. Spray with ninhydrin solution R. Heat the plate at 100C to
105C for 15 min. Any spot in the chromatogram obtained with test solution (a), apart from the
1-52
solution (b) (0.5 per cent). The test is not valid unless the chromatogram obtained with reference
solution (c) shows two clearly separated spots.
with a few drops of water R. Finely powder the substance to be examined, place 50 mg in the lower
watch-glass and dissolve it in 0.5 ml of water R. To the solution add 0.30 g of heavy magnesium
4
100C to 105C.
ASSAY
Dissolve 80.0 mg in 3 ml of anhydrous formic acid R. Add 30 ml of anhydrous acetic acid R. Using
0.1 ml of naphtholbenzein solution R as indicator, titrate with 0.1M perchloric acid, until the colour
changes from brownish-yellow to green.
3
H
7
NO
2
.
STORAGE
_______________________________________________________________________ Ph Eur
1-53
Albendazole
N
H
N
S
H
3
C
NHCOOMe
C
12
H
15
N
3
O
2
S 265.3 54965-21-8
Albendazole complies with the requirements of the 3rd edition of the European Pharmacopoeia [1386]. These
Action and use Anthelmintic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Albendazole contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent
of methyl [5-(propylsulphanyl)-1H-benzimidazol-2-yl]carbamate, calculated with reference to the
dried substance.
CHARACTERS
A white or faintly yellowish powder, practically insoluble in water, freely soluble in anhydrous formic
acid, very slightly soluble in methylene chloride, practically insoluble in alcohol.
IDENTIFICATION
with albendazole CRS. Examine the substances prepared as discs.
TESTS
Appearance of solution Dissolve 0.10 g in a mixture of 1 volume of anhydrous formic acid R and 9
volumes of methylene chloride R and dilute to 10 ml with the same mixture of solvents. The solution is
clear (2.2.1) and not more intensely coloured than reference solution BY
6
(Method II, 2.2.2).
Test solution. Dissolve 25.0 mg of the substance to be examined in 5 ml of methanol R containing
1 per cent V/V of sulphuric acid R and dilute to 50.0 ml with the mobile phase.
Reference solution (a). Dissolve 10.0 mg of the substance to be examined in 10 ml of methanol R
containing 1 per cent V/V of sulphuric acid R and dilute to 100.0 ml with the mobile phase. Dilute
0.5 ml of this solution to 20.0 ml with the mobile phase.
Reference solution (b). Dissolve 50.0 mg of the substance to be examined and 50 mg of oxybendazole
CRS in 5 ml of methanol R containing 1 per cent V/V of sulphuric acid R and dilute to 100.0 ml with
the mobile phase.
as the mobile phase at a flow rate of 0.7 ml/min a mixture of 300 volumes of a 1.67 g/l solution
of ammonium dihydrogen phosphate R and 700 volumes of methanol R,
Inject 20 l of reference solution (a). Adjust the sensitivity of the system so that the height of the
principal peak in the chromatogram obtained is at least 50 per cent of the full scale of the recorder.
Inject 20 l of reference solution (b). The test is not valid unless the resolution between the peaks
corresponding to albendazole and oxybendazole is at least 3.0.
Inject 20 l of the test solution. Continue the chromatography for 1.5 times the retention time of
albendazole. When the chromatograms are recorded in the prescribed conditions, the approximate
relative retention times are: 0.80 for impurity A, 0.43 for impurities B and C, 0.40 for impurity D,
0.47 for impurity E and 0.57 for impurity F. In the chromatogram obtained with the test solution the
area of any peak, apart from the principal peak, is not greater than 1.5 times the area of the principal
peak in the chromatogram obtained with reference solution (a) (0.75 per cent) and the sum of the
areas of any such peaks is not greater than three times the area of the principal peak in the chromato-
gram obtained with reference solution (a) (1.5 per cent). Disregard any peak with an area less than
0.1 times the area of the principal peak in the chromatogram obtained with reference solution (a).
100C to 105C for 4 h.
1-54
ASSAY
In order to avoid overheating during the titration, mix thoroughly throughout and stop the titration immediately
after the end-point has been reached.
Dissolve 0.250 g in 3 ml of anhydrous formic acid R and add 40 ml of anhydrous acetic acid R. Titrate
with 0.1M perchloric acid, determining the end-point potentiometrically (2.2.20).
12
H
15
N
3
O
2
S.
STORAGE
IMPURITIES
H
N
N
NH
2
A. R = S-CH
2
-CH
2
-CH
3
: 5-(propylsulphanyl)-1H-benzimidazol-2-amine,
H
N
N
NHCOOMe
B. R = SO-CH
2
-CH
2
-CH
3
: methyl [5-(propylsulphinyl)-1H-benzimidazol-2-yl]carbamate,
C. R = SO
2
-CH
2
-CH
2
-CH
3
: methyl [5-(propylsulphonyl)-1H-benzimidazol-2-yl]carbamate,
H
N
N
NH
2
D. R = SO
2
-CH
2
-CH
2
-CH
3
: 5-(propylsulphonyl)-1H-benzimidazol-2-amine,
H
N
N
NHCOOMe
E. R = H: methyl (1H-benzimidazol-2-yl)carbamate,
F. R = S-CH
3
: methyl [5-(methylsulphanyl)-1H-benzimidazol-2-yl]carbamate.
__________________________________________________________________________________________________________ Ph Eur
1-55
Alchemilla
Alchemilla complies with the requirements of the 3rd edition of the European Pharmacopoeia [1387]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Alchemilla consists of the whole or cut, dried, flowering, aerial parts of Alchemilla
xanthochlora Rothm. (A. vulgaris L. sensu latiore). It contains not less than 7.5 per cent of tannins,
expressed as pyrogallol (C
6
H
6
O
3
, M
r
126.1), calculated with reference to the dried drug.
CHARACTERS
IDENTIFICATION
A. The greyish-green, partly brownish-green, radical leaves which are the main part of the drug are
reniform to slightly semicircular with a diameter generally up to 8 cm, seldom up to 11 cm and have
seven to nine or eleven lobes and a long petiole. The smaller, cauline leaves, which have a pair of
large stipules at the base, have five to nine lobes and a shorter petiole or they are sessile. The leaves
are densely pubescent especially on the lower surface and have a coarsely serrated margin. Young
leaves are folded with a white-silvery pubescence; older leaves are slightly pubescent and have a finely
meshed venation, prominent on the lower surface. The greyish-green to yellowish-green petiole is
pubescent, about 1 mm in diameter, with an adaxial groove. The apetalous flowers are yellowish-
green to light green and about 3 mm in diameter. The calyx is double with four small segments of the
epicalyx alternating with four larger sepals, subacute to triangular. They are four short stamens and a
single carpel with a capitate sigma. The greyish-green to yellowish-green stem is pubescent, more or
less longitudinally wrinkled and hollow.
B. Reduce to a powder (355). The powder is greyish-green. Examine under a microscope, using
chloral hydrate solution R. The powder shows unicellular narrow trichomes up to 1 mm long partly
tortuous, acuminate and bluntly pointed at the apex, with thick lignified walls, somewhat enlarged
and pitted at the base; fragments of leaves with two layers of palisade parenchyma, the upper layer of
which is two to three times longer than the lower layer and with spongy parenchyma, containing
scattered cluster crystals of calcium oxalate, up to 25 m in diameter; leaf fragments in surface view
with sinuous to wavy epidermal cells, the anticlinal walls unevenly thickened and beaded, anomocytic
stomata (2.8.3); groups of vascular tissue and lignified fibres from the petioles and stems, the vessels
spirally thickened or with bordered pits; occasional thin-walled conical trichomes, about 300 m
long; thin-walled parenchyma containing cluster crystals of calcium oxalate; spherical pollen grains,
about 15 m in diameter, with three distinct pores and a granular exine; occasional fragments of the
ovary wall with cells containing a single crystals of calcium oxalate.
C. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R.
Test solution. To 0.5 g of the powdered drug (355) add 5 ml of methanol R and heat in a water-bath at
70C under a reflux condenser for 5 min. Cool and filter.
Reference solution. Dissolve 1.0 mg of caffeic acid R and 1.0 mg of chlorogenic acid R in 10 ml of
methanol R.
Apply separately to the plate as bands 20 l of the test solution and 10 l of the reference solution.
Develop over a path of 10 cm using a mixture of 8 volumes of anhydrous formic acid R, 8 volumes of
water R and 84 volumes of ethyl acetate R. Dry the plate at 100C to 105C for 5 min and spray the
warm plate with a 10 g/l solution of diphenylboric acid aminoethyl ester R in methanol R. Spray the plate
with a 50 g/l solution of macrogol 400 R in methanol R. Allow to stand for 30 min and examine in
ultraviolet light at 365 nm. The chromatogram obtained with the reference solution shows in the
middle part a light blue fluorescent zone (chlorogenic acid) and in the upper part, a light blue
fluorescent zone (caffeic acid). The chromatogram obtained with the test solution shows an intense
yellow to orange fluorescent zone corresponding in position to the zone of chlorogenic acid in the
chromatogram obtained with the reference solution; above it one or several intense green to greenish-
yellow fluorescent zones; above it one or two intense light blue fluorescent zones and two red
fluorescent zones (chlorophyll) above the zone of caffeic acid in the chromatogram obtained with the
reference solution near the front. Some fainter bluish fluorescent zones and a faint yellowish-brown
fluorescent zone a little above the start, are present.
TESTS
Foreign matter (2.8.2). It complies with the test for foreign matter.
Loss on drying (2.2.32). Not more than 10.0 per cent, determined on 1.000 g of powdered drug
(355) by drying in an oven at 100C to 105C for 2 h.
1-56
ASSAY
Carry out the determination of tannins in herbal drugs (2.8.14). Use 0.50 g of the powdered drug
(355).
STORAGE
__________________________________________________________________________________________________________ Ph Eur
1-57
Alcuronium Chloride
revised 1/01
N
N
N
N
OH
H
2
C
HO
CH
2
H
H
H
H
H
H
+
+
2Cl
_
C
44
H
50
Cl
2
N
4
O
2
738.0 151180-03-7
Alcuronium Chloride complies with the requirements of the 3rd edition of the European Pharmacopoeia [1285].
Action and use Neuromuscular blocking agent.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Alcuronium chloride contains not less than 98.0 per cent and not more than the equivalent of
102.0 per cent of (23E,26E)-(1R,3aS,10S,11aS,12R,14aS,19aS,20bS,21S,22aS)-23,26-bis(2-
hydroxyethylidene-1,12-diprop-2-enyl-2,3,11,11a,13,14,22,22a-octahydro-10H,21H-1,21: 10,12-
diethano-19aH,20bH-[1,5]diazocino[1,2,3-lm: 5,6,7-lm]dipyrrolo[2,3-d: 2,3-d]dicarbazolium
dichloride (4,4-didemethyl-4,4-diprop-2-enyltoxiferin I dichloride), calculated with reference to the
anhydrous, propan-2-ol-free substance.
CHARACTERS
A white or slightly greyish-white, crystalline powder, freely soluble in water and in methanol, soluble
in alcohol, practically insoluble in cyclohexane.
Carry out the identification, tests and assay as rapidly as possible avoiding exposure to actinic light.
IDENTIFICATION
obtained with alcuronium chloride CRS.
B. Examine by thin-layer chromatography (2.2.27) using a TLC silica gel plate R.
the same solvent.
Reference solution. Dissolve 10 mg of alcuronium chloride CRS in methanol R and dilute to 10 ml with
the same solvent.
Apply to the plate 10 l of each solution. Develop over a path of 15 cm using a mixture of 15
volumes of a 58.4 g/l solution of sodium chloride R, 35 volumes of dilute ammonia R2 and 50 volumes
of methanol R. Allow the plate to dry in air for 10 min and then spray with 0.1M ammonium and cerium
nitrate. The principal spot in the chromatogram obtained with the test solution is similar in position,
colour and size to the principal spot in the chromatogram obtained with the reference solution.
C. It gives reaction (a) of chlorides (2.3.1).
TESTS
Solution S Dissolve 0.250 g in carbon dioxide-free water R and dilute to 25.0 ml with the same
solvent.
solution Y
6
, BY
6
or B
6
(Method I, 2.2.2).
1-58
Acidity or alkalinity To 10 ml of solution S add 0.1 ml of methyl red solution R and 0.2 ml of 0.01M
hydrochloric acid. The solution is red. Add 0.4 ml of 0.01M sodium hydroxide. The solution is yellow.
Specific optical rotation (2.2.7). 430 to 451, determined on solution S and calculated with
reference to the anhydrous, propan-2-ol-free substance.
Propan-2-ol Not more than 1.0 per cent (2.4.24, System A).
Solvent mixture. Mix 100 ml of methanol R, 200 ml of acetonitrile R and 200 ml of a 6.82 g/l solution
of potassium dihydrogen phosphate R. Dissolve 1.09 g of sodium laurylsulphonate for chromatography R in
the mixture and adjust the apparent pH to 8.0 with a 100 g/l solution of sodium hydroxide R.
Test solution. Dissolve 0.20 g of the substance to be examined in the solvent mixture and dilute to
100.0 ml with the solvent mixture.
Reference solution (a). Dilute 0.5 ml of the test solution to 100.0 ml with the solvent mixture.
Reference solution (b). Dilute 4.0 ml of reference solution (a) to 10.0 ml with the solvent mixture.
Reference solution (c). Dilute 1.0 ml of reference solution (a) to 10.0 ml with the solvent mixture.
Reference solution (d). To 5.0 ml of the test solution add 5.0 mg of N-allylstrychnine bromide CRS,
dissolve in the solvent mixture and dilute to 100.0 ml with the solvent mixture.
a stainless steel column 0.25 m long and 4 mm in internal diameter packed with octylsilyl silica gel
for chromatography R (5 m),
as mobile phase at a flow rate of 1.2 ml/min a solution prepared as follows: mix 200 ml of
methanol R, 400 ml of acetonitrile R and 400 ml of a 6.82 g/l solution of potassium dihydrogen
phosphate R. Dissolve 2.18 g of sodium laurylsulphonate for chromatography R in the mixture and
adjust the apparent pH to 5.4 with a 100 g/l solution of phosphoric acid R,
peak due to alcuronium is at least 10 per cent of the full scale of the recorder. Inject 10 l of reference
solution (d). The test is not valid unless the resolution between the peak corresponding to N-allyl-
strychnine and alcuronium is at least 4.0. Inject 10 l of the test solution, 10 l of reference solution
(a) and 10 l of reference solution (c). Continue the chromatography of the test solution for twice the
retention time of the peak corresponding to alcuronium. In the chromatogram obtained with the test
solution the area of any peak, apart from the principal peak, is not greater than the area of the
principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent) and not more
than one such peak has an area greater than the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.2 per cent); the sum of the areas of all the peaks, apart from
the principal peak, is not greater than twice the area of the principal peak in the chromatogram
obtained with reference solution (a) (1 per cent). Disregard any peak with an area less than the area
of the principal peak in the chromatogram obtained with reference solution (c) (0.05 per cent).
mination of water.
ASSAY
Dissolve 0.300 g by stirring in 70 ml of acetic anhydride R for 1 min. Titrate with 0.1M perchloric acid
until the colour changes from violet-blue to greenish-blue, using 0.1 ml of crystal violet solution R as
indicator.
44
H
50
Cl
2
N
4
O
2
.
STORAGE
Store in an airtight container under nitrogen, protected from light, at a temperature of 2C to 8C.
IMPURITIES
N
N
N
N
H
2
C
CH
2
H
H
H
H
O
O
H
H
+
+
2Cl
_
A. (1R,3aS,9R,9aR,10R,11aS,12R,14aS,19aS,20R,20aR,20bS,21R,22aS)-1,12-diprop-2-enyl-
2,3,9a,11,11a,13,14,19a,20a,20b,22,22a-dodecahydro-10H,21H-1,23: 12,27-dimethano-
1-59
9,10: 20,21-bis(epoxyprop[2]eno)-9H,20H-[1,5]diazocino[1,2,3-lm: 5,6,7-
lm]dipyrrolo[2,3-d: 2,3-d]dicarbazolium dichloride (4,4-diallylcaracurin V dichloride),
HN
N
CH
2
H
H
O
H
H
HO
+
Cl
_
B. (4bS,7R,7aS,8aR,13R,13aR,13bS)-13-hydroxy-7-prop-2-enyl-
5,6,7a,8,8a,11,13,13a,13b,14-decahydro-7,9-methano-7H-oxepino[3,4-a]pyrrolo[2,3-
d]carbazolium chloride ((4R,17R)-4-allyl-17,18-epoxy-17-hydroxy-19,20-didehydro-
curanium chloride) ((17S)-4-allyl-19,20-didehydro-17,18-epoxycuran-17-ol chloride).
__________________________________________________________________________________________________________ Ph Eur
1-60
Alfacalcidol
corrected 1/01
Me
Me
Me
Me
H
H
H
CH
2
HO
HO
C
27
H
44
O
2
400.6 41294-56-8
Alfacalcidol complies with the requirements of the 3rd edition of the European Pharmacopoeia [1286]. These
Action and use Vitamin D analogue.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Alfacalcidol contains not less that 97.0 per cent and not more than the equivalent of 102.0 per cent
of (5Z,7E)-9,10-secocholesta-5,7,10(19)-triene-1,3-diol.
CHARACTERS
White or almost white crystals, practically insoluble in water, freely soluble in alcohol, soluble in fatty
oils. It is sensitive to air, heat and light.
A reversible isomerisation to pre-alfacalcidol takes place in solution, depending on temperature and
time. The activity is due to both compounds.
IDENTIFICATION
A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph. Eur. reference
spectrum of alfacalcidol, prepared using 2 mg of the substance to be examined and 150 mg of potassium
bromide R.
B. Examine the chromatograms obtained in the assay. The principal peak in the chromatogram
obtained with the test solution is similar in retention time and size to the principal peak in the
TESTS
Calculate the percentage content of related substances, apart from pre-alfacalcidol, that are eluted
within twice the retention time of alfacalcidol from the areas of the peaks in the chromatogram
obtained with the test solution by the normalisation procedure. The content of any individual related
substance is not greater than 0.5 per cent and the sum of the related substances is not greater than
1.0 per cent. Disregard any peak below 0.1 per cent.
ASSAY
Carry out the assay as rapidly as possible, avoiding exposure to actinic light and air.
Test solution. Dissolve 1.0 mg of the substance to be examined without heating in 10.0 ml of the
mobile phase.
Reference solution (a). Dissolve 1.0 mg of alfacalcidol CRS without heating in 10.0 ml of the mobile
phase.
Reference solution (b). Dilute reference solution (a) 100 times with the mobile phase.
Reference solution (c). Heat 2 ml of reference solution (a) in a water-bath at 80C under a reflux
condenser for 2 h and cool.
a column 0.25 m long and 4.0 mm in internal diameter packed with octadecylsilyl silica gel for
chromatography R2 (5 m),
1-61
as mobile phase at a flow rate of 2.0 ml/min a mixture of 1 volume of ammonia R, 200 volumes
of water R and 800 volumes of acetonitrile R,
a loop injector.
Inject 100 l of reference solution (c) and record the chromatogram. Make a total of six injections.
When the chromatograms are recorded in the prescribed conditions, the retention time for pre-
alfacalcidol, relative to alfacalcidol, is about 1.3. The assay is not valid unless the relative standard
deviation of the response for alfacalcidol is at most 1 per cent and the resolution between the peaks
due to pre-alfacalcidol and alfacalcidol is at least 4.0; adjust the proportions of the constituents of the
mobile phase, if necessary, to obtain this resolution.
Inject 100 l of reference solution (a) and 100 l of reference solution (b) and record the chroma-
tograms. Inject 100 l of the test solution and record the chromatogram in the same manner,
continuing the chromatography for twice the retention time of the principal peak.
STORAGE
Store under nitrogen, in an airtight container, protected from light, at a temperature of 2C to 8C.
The contents of an opened container are to be used immediately.
IMPURITIES
H
CH
2
O
OH
Me
Me
Me
Me
H
H
A. (5E,7E)-9,10-secocholesta-5,7,10(19)-triene-1,3-diol (trans-alfacalcidol),
H
HO
OH
Me
Me
Me
Me
H
H
B. (5Z,7E)-9,10-secocholesta-5,7,10(19)-triene-1,3-diol (1-calcidol),
H
N
N
Me
Me
Me
Me
H
H
Me
OH HO
N
O
O
C. triazoline adduct of pre-alfacalcidol.
__________________________________________________________________________________________________________ Ph Eur
1-62
Alfadex
Alphacyclodextrin
1/01
OH
OH
o
o
o
o
o
o o
o
o
o
OH
OH
HO
O
OH
OH
HO
OH
HO
HO
OH
OH
OH
o
OH
HO
O
HO
[C
6
H
10
O
5
]
6
973 10016-20-3
Alfadex complies with the requirements of the 3rd edition of the European Pharmacopoeia [1487]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Alfadex contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent of
cyclomaltohexaose, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, amorphous or crystalline powder, freely soluble in water and in propylene
glycol, practically insoluble in ethanol and in methylene chloride.
IDENTIFICATION
B. Examine the chromatograms obtained in the assay. The retention time and size of the principal
peak in the chromatogram obtained with test solution (b) are approximately the same as those of the
C. Dissolve 0.2 g in 2 ml of iodine solution R4 by warming on a water-bath, and allow to stand at
room temperature; a yellowish-brown precipitate is formed.
TESTS
solvent.
Appearance of solution Solution S is clear (2.2.1).
pH (2.2.3). The pH of a mixture of 30 ml of solution S and 1 ml of a 223.6 g/l solution of potassium
chloride R is 5.0 to 8.0.
Specific optical rotation (2.2.7). +147 to +152, determined on solution S and calculated with
Reducing sugars
Test solution. To 1 ml of solution S add 1 ml of cupri-tartaric solution R4. Heat on a water-bath for
10 min, cool to room temperature. Add 10 ml of ammonium molybdate reagent R1 and allow to stand
for 15 min.
Reference solution. Prepare a reference solution at the same time and in the same manner as the test
solution, using 1 ml of a 0.02 g/l solution of glucose R.
1-63
Measure the absorbance of the test solution and the reference solution (2.2.25) at the maximum
at 740 nm using water R as the compensation liquid. The absorbance of the test solution is not
greater than that of the reference solution (0.2 per cent).
Light-absorbing impurities Examine solution S between 230 nm and 750 nm (2.2.25). Between
230 nm and 350 nm, the absorbance is not greater than 0.10. Between 350 nm and 750 nm, the
absorbance is not greater than 0.05.
Related substances Examine by liquid chromatography (2.2.29) as described under Assay. Inject
test solution (a) and reference solution (b). In the chromatogram obtained with test solution (a): the
areas of any peaks corresponding to betadex or gammacyclodextrin are not greater than half of the
area of the corresponding peaks in the chromatogram obtained with reference solution (b) (0.25 per
cent); the sum of the areas of all the peaks, apart from the principal peak and any peaks correspond-
ing to betadex or gammacyclodextrin, is not greater than half of the area of the peak corresponding to
alfadex in the chromatogram obtained with reference solution (b) (0.5 per cent).
Residual solvents Not more than 10 ppm of trichloroethylene and not more than 10 ppm of
toluene. Examine by head-space gas chromatography (2.2.28), using the method of standard
additions and ethylene chloride R as the internal standard.
Test solution. In each of four identical 20 ml flasks, dissolve 500 mg of the substance to be examined
in water R and add 0.10 g of calcium chloride R and 30 l of -amylase solution R. To three of the
flasks, add 1 ml of one of the reference solutions, adding a different solution to each flask. Dilute to
10 ml with water R.
Reference solutions. Prepare solutions containing per litre: 3 l, 10 l and 15 l of trichloroethylene R
and of toluene R and 10 l of ethylene chloride R.
a fused-silica column 25 m long and 0.32 mm in internal diameter coated with a layer about
1 m thick of macrogol 20,000 R,
helium for chromatography R as the carrier gas,
maintaining the temperature of the column at 50C, that of the injection port at 140C and that of
the detector at 280C. Place the samples in a thermostated chamber at 45C for 2 h. Inject 200 l of
the head-space of each flask and repeat each test at least three times. The retention time of toluene is
about 10 min. The test is not valid unless: the resolutions between the peaks corresponding to
trichloroethylene and toluene and between the peaks corresponding to toluene and ethylene chloride
are greater than 1.1 and the relative standard deviations of the ratios of the areas of the peaks corres-
ponding to trichloroethylene and toluene to that of the peak corresponding to ethylene chloride are
less than 5 per cent.
Calculate the content of trichloroethylene and of toluene taking their relative densities to be 1.46
and 0.87 respectively.
at 120C for 2 h.
ASSAY
Test solution (a). Dissolve 0.25 g of the substance to be examined in water R with heating, cool and
dilute to 25.0 ml with the same solvent.
Test solution (b). Dilute 5.0 ml of test solution (a) to 50.0 ml with water R.
Reference solution (a). Dissolve 25.0 mg of betadex CRS, 25.0 mg of gammacyclodextrin CRS and
50.0 mg of alfadex CRS in water R and dilute to 50.0 ml with the same solvent.
Reference solution (b). Dilute 5.0 ml of reference solution (a) to 50.0 ml with water R.
Reference solution (c). Dissolve 25.0 mg of alfadex CRS in water R and dilute to 25.0 ml with the same
solvent.
as mobile phase at a flow rate of 1.5 ml/min a mixture of 10 volumes of methanol R and 90
volumes of water R,
as detector a differential refractometer,
a 50 l loop injector.
Equilibrate the column with the mobile phase at a flow rate of 1.5 ml/min for about 3 h. Inject
1-64
reference solution (a) five times and record the chromatograms for 3.5 times the retention time of
alfadex. Adjust the sensitivity of the system so that the height of the peak corresponding to
gammacyclodextrin is 55 per cent to 75 per cent of the full scale of the recorder. The retention time
of alfadex is about 4.5 min, the relative retention of gammacyclodextrin is about 0.7 and that of
betadex is about 2.2. The test is not valid unless the resolution between the peaks corresponding to
gammacyclodextrin and alfadex is at least 1.5 and the relative standard deviation for the area of the
peak corresponding to alfadex is less than 2.0 per cent. If necessary, adjust the concentration of
methanol in the mobile phase to achieve the required resolution. Inject alternately test solution (b)
and reference solution (c). Calculate the percentage content of [C
6
H
10
O
5
]
6
from the area of the
principal peak in each of the chromatograms obtained and from the declared content of alfadex CRS.
STORAGE
IMPURITIES
A. betadex,
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
HO
HO
HO
HO
OH
OH
OH
OH
HO
OH
OH
HO
HO
HO
HO
HO
HO
OH
OH
HO
HO
HO
HO
HO
B. gammacyclodextrin.
__________________________________________________________________________________________________________ Ph Eur
1-65
Alfentanil Hydrochloride
,HCl
N N
N
O
CH3
N
N
O
OMe
Et
C
21
H
32
N
6
O,HCl 453.0 69049-06-5
Alfentanil Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Opioid analgesic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Alfentanil hydrochloride contains not less than 98.5 per cent and not more than the equivalent of
101.5 per cent of N-[1-[2-(4-ethyl-4, 5-dihydro-5-oxo-1H-tetrazol-1-y1)-ethyl]-4-(methoxy-
methyl)piperidin-4-yl]-N-phenylpropanamide hydrochloride, calculated with reference to the anhy-
drous substance.
CHARACTERS
A white or almost white powder, freely soluble in water, in alcohol and in methanol.
It melts at about 140C, with decomposition.
IDENTIFICATION
spectrum of alfentanil hydrochloride.
B. Dissolve 50 mg in a mixture of 0.4 ml of ammonia R and 2 ml of water R. Mix, allow to stand for 5
min and filter. Acidify the filtrate with dilute nitric acid R. It gives reaction (a) of chlorides (2.3.1).
TESTS
solution is clear (2.2.1) and colourless (Method II, 2.2.2).
Test solution. Dissolve 0.100 g of the substance to be examined in methanol R and dilute to 10.0 ml
Reference solution (a). In order to prepare the in situ degradation compound (alfentanil impurity E),
dissolve 10 mg of the substance to be examined in 10.0 ml of dilute hydrochloric acid R. Heat on a
water-bath under a reflux condenser for 4 h. Neutralise with 10.0 ml of dilute sodium hydroxide R.
Evaporate to dryness on a water-bath. Cool and take up the residue in 10 ml of methanol R. Filter.
Reference solution (b). Dilute 1.0 ml of the Test solution to 100.0 ml with methanol R. Dilute 5.0 ml of
this solution to 20.0 ml with methanol R.
a stainless steel column 0.1 m long and 4.6 mm in internal diameter packed with octadecylsilica
gel for chromatography (3 m),
as mobile phase at a flow rate of 1.5 ml per minute, a gradient programme using the following
conditions:
Mobile phase A. A 5 g/l solution of ammonium carbonate R in a mixture of 10 volumes of
tetrahydrofuran R and 90 volumes of water R.
Mobile phase B. Acetonitrile R.
1-66
Time
(min)
Mobile
phase A
(per cent V/V)
Mobile
phase B
(per cent V/V)
Comment
015 9040 1060 linear gradient
1520 40 60 isocratic elution
2025 90 10 switch to initial
composition
25 = 0 90 10 restart gradient
Equilibrate the column for at least 30 min with acetonitrile R and then equilibrate at the initial
eluent composition for at least 5 min. Adjust the sensitivity of the system so that the height of the
principal peak in the chromatogram obtained with 10 l of reference solution (b) is at least 50 per
cent of the full scale of the recorder.
Inject 10 l of reference solution (a). When the chromatograms are recorded in the prescribed
conditions the retention times are: alfentanil impurity E, about 6 min and alfentanil hydrochloride,
about 7 min. Disregard any other peak. The test is not valid unless the resolution between the peaks
corresponding to alfentanil hydrochloride and alfentanil impurity E is at least 4.0. If necessary, adjust
the concentration of acetonitrile in the mobile phase or adjust the time programme for the linear-
gradient elution.
Inject separately 10 l of methanol R as a blank, 10 l of the test solution and 10 l of reference
solution (b). In the chromatogram obtained with the test solution: the area of any peak, apart from
the principal peak, is not greater than the area of the principal peak in the chromatogram obtained
with reference solution (b) (0.25 per cent); the sum of the areas of all such peaks is not greater than
twice the area of the principal peak in the chromatogram obtained with reference solution (b)
(0.5 per cent). Disregard any peak obtained with the blank-run, and any peak with an area less than
0.2 times the area of the principal peak in the chromatogram obtained with reference solution (b).
Water (2.5.12). 3.0 per cent to 4.0 per cent, determined on 0.500 g by the semi-micro
determination of water.
ASSAY
Dissolve 0.350 g in 50 ml of a mixture of 1 volume of alcohol R and 4 volumes of water R and add
5.0 ml of 0.01M hydrochloric acid. Titrate with 0.1M sodium hydroxide, determining the end-point
potentiometrically (2.2.20). Read the volume added between the two points of inflexion.
21
H
33
CIN
6
O
3
.
STORAGE
IMPURITIES
N N
N
O
CH
3
N
N
O
OMe
O
Et
and enanti ome
A. cis-N-[1-[2-(4-ethyl-4,5-dihydro-5-oxo-1H-tetrazol-1-yl)ethyl]-4-(methoxymethyl)-
piperidin-4-yl]-N-phenylpropanamide N-oxide,
N N
N
O
CH
3
N
N
O
OMe
O
Et
and enanti ome
B. trans-N-[1-[2-(4-ethyl-4,5-dihydro-5-oxo-1H-tetrazol-1-yl)ethyl]-4-(methoxymethyl)-
piperidin-4-yl]-N-phenylpropanamide N-oxide,
1-67
O
CH
3
N
HN
OMe
C. N-[4-(methoxymethyl)piperidin-4-yl]-N-phenylpropanamide,
N N
N
O
CH
3
N
N
O
OMe
Et
D. N-[1-[2-(4-ethyl-4,5-dihydro-5-oxo-1H-tetrazol-1-yl)ethyl]-4-(methoxymethyl)piperidin-
4-yl]-N-phenylacetamide,
N N
N
O
Et
OMe
N
NH
E. 1-ethyl-1,4-dihydro-4-[2-[[4-(methoxymethyl)-4-phenylamino]piperidin-1-yl]ethyl]-5H-
tetrazol-5-one,
O
CH
3
N
N
OMe
HO
F. N-[1-(2-hydroxyethyl)-4-(methoxymethyl)piperidin-4-yl]-N-phenylpropanamide,
N N
N
O
CH
3
N
N
O
O
CH
3
O
Et
G. N-[1-[2-(4-ethyl-4,5-dihydro-5-oxo-1H-tetrazol-1-yl)ethyl]-4-(propanoyloxymethyl)-
piperidin-4-yl]-N-phenylpropanamide,
N N
N
O
N
N
O
OMe
CH
3
Et
H. N-[1-[2-(4-ethyl-4,5-dihydro-5-oxo-1H-tetrazol-1-yl)ethyl]-4-(methoxymethyl)piperidin-4-
yl]-N-phenylbutanamide.
_______________________________________________________________________ Ph Eur
1-68
Alfuzosin Hydrochloride
H
N
O
O
N
H
Me
N
N
NH
2
OMe
OMe
and enantiomer
,HCl
C
19
H
27
N
5
O
4
,HCl 425.9 81403-68-1
Alfuzosin Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Alpha-adrenoceptor antagonist.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Alfuzosin hydrochloride contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of (RS)-N-[3-[(4-amino-6,7-dimethoxyquinazolin-2-yl)(methyl)amino]propyl]-
tetrahydrofuran-2-carboxamide hydrochloride, calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white, crystalline powder, slightly hygroscopic, freely soluble in water, sparingly
soluble in alcohol, practically insoluble in methylene chloride.
IDENTIFICATION
obtained with alfuzosin hydrochloride CRS. Examine the substances prepared as discs.
B. To 1 ml of solution S (see Tests) add 1 ml of water R. The solution gives reaction (a) of chlorides
(2.3.1).
TESTS
solvent.
pH (2.2.3). The pH of freshly prepared solution S is 4.0 to 6.0.
Optical rotation (2.2.7). The angle of optical rotation, determined on solution S, is 0.10 to
+0.10.
Test solution. Dissolve 20.0 mg of the substance to be examined in the mobile phase and dilute to
Reference solution (a). Dilute 1.0 ml of the test solution to 50.0 ml with the mobile phase. Dilute
5.0 ml of the solution to 20.0 ml with the mobile phase.
Reference solution (b). Dissolve 5 mg of alfuzosin impurity A CRS in the mobile phase and dilute to
25 ml with the mobile phase. To 1 ml of the solution, add 1 ml of the test solution and dilute to
100 ml with the mobile phase.
a stainless steel column 0.15 m long and 4.6 mm in internal diameter packed with microparticu-
late octadecylsilyl silica gel for chromatography R (5 m), with a carbon loading of 18.5 per cent, a
specific surface area of 320 m
2
/g, a pore size of 15 nm, and end-capped with hexamethyldisilane,
as mobile phase at a flow rate of 1.5 ml/min a mixture of 1 volume of tetrahydrofuran R, 20
volumes of acetonitrile R and 80 volumes of a solution of sodium perchlorate prepared as follows:
dilute 5.0 ml of perchloric acid R in 900 ml of water R, adjust to pH 3.5 with dilute sodium
hydroxide solution R and dilute to 1000 ml with water R,
two peaks in the chromatogram obtained is at least 50 per cent of the full scale of the recorder. The
1-69
test is not valid unless the resolution between the peaks corresponding to alfuzosin and alfuzosin
impurity A is at least 3.0. Inject 20 l of the test solution and 20 l of reference solution (a). In the
chromatogram obtained with the test solution: the area of any peak, apart from the principal peak, is
not greater than 0.6 times the area of the principal peak in the chromatogram obtained with reference
solution (a) (0.3 per cent); the sum of the areas of all the peaks, apart from the principal peak, is not
greater than the area of the principal peak in the chromatogram obtained with reference solution (a)
(0.5 per cent). Disregard any peak with an area less than 0.025 times that of the principal peak in the
mination of water.
ASSAY
Dissolve 0.300 g in a mixture of 40 ml of anhydrous acetic acid R and 40 ml of acetic anhydride R.
19
H
28
ClN
5
O
4
.
STORAGE
Store in an airtight container, protected from light.
IMPURITIES
N
N
NH
2
OMe
OMe
R =
H
O
N
O
N
Me
R
A. N-[3-[(4-amino-6,7-dimethoxyquinazolin-2-yl)(methyl)amino]propyl]furan-2-
carboxamide,
B. R-Cl : 2-chloro-6,7-dimethoxyquinazolin-4-amine,
O
N
O
N
R
H
Me
H
and enantiomer
C. (RS)-N-[3-[(4-amino-6,7-dimethoxyquinazolin-2-yl)amino]propyl]-N-
methyltetrahydrofuran-2-carboxamide,
N
Me
R
H
2
N
D. N-(4-amino-6,7-dimethoxyquinazolin-2-yl)-N-methylpropane-1,3-diamine,
H
N
O
N
Me
R
H
E. N-[3-[(4-amino-6,7-dimethoxyquinazolin-2-yl)(methyl)amino]propyl]formamide.
__________________________________________________________________________________________________________ Ph Eur
1-70
Alginic Acid
Alginic Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia [0591]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Alginic acid is a mixture of polyuronic acids [(C
6
H
8
O
6
)
n
] composed of residues of D-mannuronic and
L-guluronic acid and is obtained mainly from algae belonging to the Phaeophyceae. A small
proportion of the carboxyl groups may be neutralised. It contains not less than 19.0 per cent and not
more than 25.0 per cent of carboxyl groups (COOH), calculated with reference to the dried
substance.
CHARACTERS
A white or pale yellowish-brown, crystalline or amorphous powder, very slightly soluble or practically
insoluble in alcohol, practically insoluble in organic solvents. It swells in water but does not dissolve;
it dissolves in solutions of alkali hydroxides.
IDENTIFICATION
A. To 0.2 g add 20 ml of water R and 0.5 ml of sodium carbonate solution R. Shake and filter. To 5 ml
of the filtrate add 1 ml of calcium chloride solution R. A voluminous gelatinous mass is formed.
B. To 5 ml of the filtrate obtained in identification test A add 0.5 ml of a 123 g/l solution of
magnesium sulphate R. No voluminous gelatinous mass is formed.
C. To 5 mg add 5 ml of water R, 1 ml of a freshly prepared 10 g/l solution of 1,3-dihydroxy-
naphthalene R in alcohol R and 5 ml of hydrochloric acid R. Boil gently for 3 min, cool, add 5 ml of
water R, and shake with 15 ml of di-isopropyl ether R. Carry out a blank test. The upper layer obtained
with the substance to be examined exhibits a deeper bluish-red colour than that obtained with the
blank.
TESTS
Chlorides Not more than 1.0 per cent. To 2.50 g add 50 ml of dilute nitric acid R, shake for 1 h and
dilute to 100.0 ml with dilute nitric acid R. Filter. To 50.0 ml of the filtrate add 10.0 ml of 0.1M silver
nitrate and 5 ml of toluene R. Titrate with 0.1M ammonium thiocyanate, using 2 ml of ferric ammonium
sulphate solution R2 as indicator and shaking vigorously towards the end point.
1 ml of 0.1M silver nitrate is equivalent to 3.545 mg of Cl.
Heavy metals (2.4.8). 1.0 g complies with the limit test F for heavy metals (20 ppm). Prepare the
Loss on drying (2.2.32). Not more than 15.0 per cent, determined on 0.1000 g by drying in an
oven at 100C to 105C for 4 h.
Sulphated ash (2.4.14). Not more than 8.0 per cent, determined on 0.100 g and calculated with
2
micro-
organisms per gram, determined by plate-count. It complies with the tests for Escherichia coli and
ASSAY
To 0.2500 g add 25 ml of water R, 25.0 ml of 0.1M sodium hydroxide and 0.2 ml of phenolphthalein
solution R. Titrate with 0.1M hydrochloric acid.
1 ml of 0.1M sodium hydroxide is equivalent to 4.502 mg of carboxyl groups (COOH).
__________________________________________________________________________________________________________ Ph Eur
1-71
Alimemazine Tartrate / Trimeprazine Tartrate
S
N
Me H
NMe
2
and enantiomer
2
, HOOC
COOH
OH H
OH H
(C
18
H
22
N
2
S)
2
,C
4
H
6
O
6
747.0 4330-99-8
Definition Alimemazine Tartrate is (RS)-dimethyl(2-methyl-3-phenothiazin-10-ylpropyl)amine
(2R,3R)-tartrate. It contains not less than 99.0% and not more than 101.0% of
(C
18
H
22
N
2
S)
2
,C
4
H
6
O
6
, calculated with reference to the dried substance.
Characteristics A white or slightly cream powder; odourless or almost odourless. It darkens on
exposure to light.
Freely soluble in water and in chloroform; sparingly soluble in ethanol (96%); very slightly soluble in
ether.
Identification A. The light absorption, Appendix II B, in the range 230 to 350 nm of a 0.0008% w/v
solution in a mixture of 1 volume of 5M ammonia and 99 volumes of methanol exhibits a maximum at
255 nm and a less well-defined maximum at 301 nm. The absorbance at the maximum at 255 nm is
about 0.70.
B. Dissolve 0.1 g in 10 ml of water and add 2 ml of 1M sodium hydroxide. Extract with 25 ml of ether,
wash the extract with 5 ml of water, dry over anhydrous sodium sulphate, evaporate to dryness and
dissolve the residue in 1 ml of chloroform. The infrared absorption spectrum of the resulting solution,
Appendix II A, is concordant with the reference spectrum of alimemazine (RS 005).
C. To 1 mg add 0.05 ml of a mixture of equal volumes of formaldehyde solution and sulphuric acid. A
purple colour is produced.
D. Dissolve 0.5 g in 5 ml of water, add 1.5 ml of 5M sodium hydroxide and extract with two 10-ml
quantities of ether. To 2 ml of the aqueous layer add 6M acetic acid until it is acidic to litmus paper and
add 1 ml of ammonium metavanadate solution. An orange-red colour is produced.
Acidity pH of a 2% w/v solution, 5.0 to 6.5, Appendix V L.
Melting point 159 to 163, Appendix V A.
Related substances Complies with the test for related substances in phenothiazines, Appendix III A,
using mobile phase A.
Loss on drying When dried to constant weight at 100 at a pressure not exceeding 0.7 kPa, loses
not more than 0.5% of its weight. Use 1 g.
Assay Carry out Method I for non-aqueous titration, Appendix VIII A, using 1 g and crystal violet
solution as indicator. Each ml of 0.1M perchloric acid VS is equivalent to 37.35 mg of
(C
18
H
22
N
2
S)
2
,C
4
H
6
O
6
.
Storage Alimemazine Tartrate should be protected from light.
Action and use Histamine H
1
-receptor antagonist; sedative.
Preparations
Paediatric Alimemazine Oral Solution/Paediatric Trimeprazine Oral Solution
Strong Paediatric Alimemazine Oral Solution/Strong Paediatric Trimeprazine Oral Solution
Alimemazine Tablets/Trimeprazine Tablets
1-72
Allantoin
revised 1/01
N
N
N H
2
N
O
O
O H
H
H
H
and enantiomer
C
4
H
6
N
4
O
3
158.1 97-59-6
Allantoin complies with the requirements of the 3rd edition of the European Pharmacopoeia [1288]. These
Action and use Treatment of skin disorders.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Allantoin contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of
(RS)-(2,5-dioxoimidazolidin-4-yl)urea.
CHARACTERS
A white, crystalline powder, slightly soluble in water, very slightly soluble in alcohol.
IDENTIFICATION
Second identification: B, C, D.
obtained with allantoin CRS.
chromatogram obtained with test solution (b) is similar in position, colour and size to the principal
spot in the chromatogram obtained with reference solution (a).
C. Boil 20 mg with a mixture of 1 ml of dilute sodium hydroxide solution R and 1 ml of water R. Allow
to cool. Add 1 ml of dilute hydrochloric acid R. To 0.1 ml of the solution add 0.1 ml of a 100 g/l
solution of potassium bromide R, 0.1 ml of a 20 g/l solution of resorcinol R and 3 ml of sulphuric acid R.
Heat for 5 min to 10 min on a water-bath. A dark blue colour develops, which becomes red after
cooling and pouring into about 10 ml of water R.
D. Heat about 0.5 g. Ammonia vapour is evolved, which turns red litmus paper R blue.
TESTS
Solution S Dissolve 0.5 g in carbon dioxide-free water R, with heating if necessary, and dilute to
Acidity or alkalinity To 5 ml of solution S add 5 ml of carbon dioxide-free water R, 0.1 ml of methyl
red solution R and 0.2 ml of 0.01M sodium hydroxide. The solution is yellow. Add 0.4 ml of 0.01M
hydrochloric acid. The solution is red.
+0.10.
Reducing substances Shake 1.0 g with 10 ml of water R for 2 min. Filter. Add 1.5 ml of 0.02M
potassium permanganate. The solution must remain violet for at least 10 min.
Related substances Examine by thin-layer chromatography (2.2.27), using a suitable cellulose for
chromatography R as the coating substance.
Test solution (a). Dissolve 0.10 g of the substance to be examined in 5.0 ml of water R with heating.
Allow to cool. Dilute to 10 ml with methanol R. Use the solution immediately after preparation.
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with a mixture of 1 volume of methanol R
and 1 volume of water R.
Reference solution (a). Dissolve 10 mg of allantoin CRS in a mixture of 1 volume of methanol R and 1
volume of water R and dilute to 10 ml with the same mixture of solvents.
1-73
Reference solution (b). Dissolve 10 mg of urea R in 10 ml of water R. Dilute 1 ml of this solution to
Reference solution (c). Mix 1 ml of reference solution (a) and 1 ml of reference solution (b).
Apply separately to the plate 10 l of test solution (a) and 5 l each of test solution (b), reference
solution (a), reference solution (b) and reference solution (c). Develop over a path of 10 cm using a
mixture of 15 volumes of glacial acetic acid R, 25 volumes of water R and 60 volumes of butanol R.
Allow the plate to dry in air. Spray the plate with a 5 g/l solution of dimethylaminobenzaldehyde R in a
mixture of 1 volume of hydrochloric acid R and 3 volumes of methanol R. Dry the plate in a current of
hot air. Examine in daylight after 30 min. Any spot in the chromatogram obtained with test solution
(a), apart from the principal spot, is not more intense than the spot in the chromatogram obtained
with reference solution (b) (0.5 per cent). The test is not valid unless the chromatogram obtained
with reference solution (c) shows two clearly separated principal spots.
100C to 105C.
ASSAY
Dissolve 120.0 mg in 40 ml of water R. Titrate with 0.1M sodium hydroxide, determining the end-point
potentiometrically (2.2.20).
4
H
6
N
4
O
3
.
IMPURITIES
H COOH
O
A. glyoxylic acid,
B. urea.
__________________________________________________________________________________________________________ Ph Eur
1-74
Allergen Products
Allergen Products comply with the requirements of the 3rd edition of the European Pharmacopoeia [1063].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Allergen products are pharmaceutical preparations derived rom extracts of naturally occurring source
materials containing allergens, which are substances that cause or provoke allergic (hypersensitivity)
disease. The allergenic components are most often of a proteinaceous nature. Allergen products are
intended for in vivo diagnosis or treatment of allergic (hypersensitivity) diseases attributed to these
allergens.
Allergen products are available as finished products, as bulk preparations in dried form, solutions
or suspensions intended to be further concentrated or diluted prior to use or as final preparations in
solutions, suspensions or freeze-dried. Allergen products intended for parenteral, bronchial and
conjunctival administration are sterile.
For diagnostic use, allergen products are usually prepared as unmodified extracts in a 50 per cent
V/V solution of glycerol for skin-prick testing. For intradermal diagnosis or for provocation tests by
nasal, ocular or bronchial routes, suitable dilutions of allergen products may be prepared by dilution
of aqueous or glycerinated extracts, or by reconstitution immediately before use of unmodified freeze-
dried extracts.
For immunotherapy, allergen products may be either unmodified extracts or extracts modified
chemically and/or by adsorption onto different carriers (for example, aluminium hydroxide, calcium
phosphate or tyrosine).
This monograph does not apply: to chemicals that are used solely for diagnosis of contact dermatitis; to
chemically synthesised products; to allergens derived by rDNA technology; to finished products used on a
named-patient basis. It does not necessarily apply to allergen products for veterinary use.
PRODUCTION
Allergen products are derived from a wide range of allergenic source materials. They are often
prepared as bulk products intended to be further diluted or concentrated prior to use. They may be
treated to modify or reduce the allergenic activity or remain unmodified.
SOURCE MATERIALS
Source materials for the preparation of allergen products are mostly pollens, moulds, mites, animal
epithelia, hymenoptera venoms and certain foods.
They are described by their origin, nature, method of collection or production and pretreatment,
and are stored under defined conditions which minimise deterioration.
The collection or production, as well as the handling of the source materials are such that uniform
qualitative and quantitative composition is ensured as far as possible from batch to batch.
Pollens Potential chemical contaminants, such as pesticides and heavy metals, must be minimised.
Pollens contain not more than 1 per cent of foreign pollens as determined by microscopic
examination. Pollens contain not more than 1 per cent of mould spores as determined by
microscopic examination.
Mites and moulds Biologically active contaminants such as mycotoxins in moulds must be
minimised and any presence justified. Care must be taken to minimise any allergenic constituents of
the media used for the cultivation of mites and moulds as source materials. Culture media that
contain substances of human or animal origin must be justified and, when required must be suitably
treated to ensure the inactivation or elimination of possible transmissible agents of disease.
Animal epithelia Animal epithelia must be obtained from healthy animals selected to avoid possible
transmissible agents of disease.
MANUFACTURING PROCESS
Allergen products are generally obtained by extraction, and may be purified, from the source
materials using appropriate methods shown to preserve the biological properties of the allergenic
components. Allergen products are manufactured under conditions designed to minimise microbial
growth and enzymatic degradation.
A purification procedure, if any, is designed to minimise the content of any potential irritant low-
molecular-mass components or other non-allergenic components.
Allergen products may contain a suitable antimicrobial preservative. The nature and the concentra-
tion of the antimicrobial preservative have to be justified.
1-75
The manufacturing process comprises various stages.
Native allergen extracts result after separation from the extracted source materials
Intermediate allergen products are obtained by further processing or modification of the native
allergen extracts. The modification may be achieved by chemical processes (chemical conjugation) or
physical processes (physical adsorption onto different carriers, for example, aluminium hydroxide,
calcium phosphate or tyrosine). They may also be modified by inclusion into such vehicles as
liposomes or microspheres, or by the addition of other biologically active agents to enhance efficacy
or safety. Intermediate allergen products may be freeze-dried.
Bulk allergen preparations consist of products in solution or suspension which will not be further
processed or modified, and are ready for dilution or filling into final containers.
IN-HOUSE REFERENCE PREPARATION
An appropriate representative preparation is selected as the In-House Reference Preparation (IHRP),
characterised and used to verify batch-to-batch consistency. The IHRP is stored in suitably sized
aliquots under conditions ensuring its stability, usually freeze-dried.
Characterisation of the In-House Reference Preparation The extent of characterisation of the
IHRP depends on the nature of the allergenic source material, knowledge of the allergenic components and
availability of suitable reagents, as well as the intended use. The characterised IHRP is used as reference in
the batch control of native allergen extracts or intermediate allergen products and, if possible, in the batch
control of final allergen preparations.
The In-House Reference Preparation (IHRP) is characterised by the protein content determination
and a protein profile using relevant methods (such as isoelectric focusing, polyacrylamide gel
electrophoresis, immunoelectrophoresis or molecular-mass profiling). Allergenic components may be
detected by appropriate methods (for example immunoblotting or crossed radio-immunoelectro-
phoresis). Characterisation of the allergenic components may include identification of relevant
allergens based on serological or others techniques using a pool or individual sera from allergic
patients, or allergen-specific polyclonal or monoclonal antibodies. When allergen reference
substances are available determination of the content of individual allergens may be performed.
Individual allergens are identified according to internationally established nomenclature whenever
possible.
Where possible, the biological potency of the IHRP is established by in vivo techniques such as skin
testing, and expressed in units of biological activity. If not, for certain extracts, potency may be
established by suitable immunoassays (for example, those based on the inhibition of the binding
capacity of specific immunoglobulin E antibodies) or by quantitative techniques for a single major
component.
IDENTIFICATION
Identity is confirmed at the intermediate or other applicable stage by comparison with the IHRP
using protein profiling by appropriate methods (for example, isoelectric focusing, sodium dodecyl
sulphate-polyacrylamide gel electrophoresis or immunoelectrophoresis).
TESTS
Various biochemical and immunological tests have been developed in order to characterise allergens
qualitatively and quantitatively. However, some of the methods, particularly for the determination of
allergenic activity and allergen profile, are not applicable to all products at present. This is because knowledge
of the allergenic components or the required reagents is not available. Accordingly, allergen products have been
classified in different categories with increasing test requirements, according to quality and intended use.
Where possible the following tests are applied to the final preparations. If not, they must be performed on the
extracts as late as possible in the manufacturing process, for example, at the stage immediately prior to that
stage (modification, dilution etc.) which renders the test not feasible on the final preparation.
Water (2.5.12). Not more than 5 per cent for freeze-dried products.
Sterility (2.6.1). Allergen products intended for parenteral, bronchial and conjunctival
administration comply with the test for sterility.
Protein content 80 per cent to 120 per cent of the stated content of a given batch. If the biological
potency can be determined then the test for protein content may be omitted.
Protein profile The protein composition determined by suitable methods corresponds to that of the
IHRP.
Abnormal toxicity (2.6.9). Allergen products obtained from moulds and intended for parenteral
administration (except skin-prick tests) comply with the test for abnormal toxicity for immunosera
and vaccines for human use.
Various additional tests, some with increasing selectivity, depending on the allergen product concerned can be
1-76
applied, but in any case for allergen products intended for therapeutic use a validated test measuring the
potency (total allergenic activity, determination of individual allergens or any other justified tests) must be
applied.
Aluminium (2.5.13). Not less than 80 per cent and not more than 120 per cent of the stated
amount but in any case not more than 1.25 mg per human dose unless otherwise justified and
authorised, when aluminium hydroxide or aluminium phosphate is used as adsorbent.
Calcium (2.5.14). Not less than 80 per cent and not more than 120 per cent of the stated amount
when calcium phosphate is used as adsorbent.
Antigen profile The antigens are identified by means of suitable techniques using antigen-specific
animal antibodies.
Allergen profile Relevant allergenic components are identified by means of suitable techniques
using allergen-specific human antibodies.
Total allergenic activity The activity is 50 per cent to 200 per cent of the stated amount as assayed
by inhibition of the binding capacity of specific immunoglobulin E antibodies or a suitable equivalent
in vitro method.
Individual allergens 50 per cent to 200 per cent of the stated amount, determined by a suitable
method.
STORAGE
Adsorbed allergen products should not be frozen.
LABELLING
The label states:
the biological potency and/or the protein content and/or the extraction concentration;
the route of administration and the intended use;
the storage conditions;
where applicable, the name and amount of added antimicrobial preservative;
for freeze-dried preparations:
the name, composition and volume of the reconstituting liquid to be added
the period of time within which the preparation is to be used after reconstitution;
where applicable, that the preparation is sterile;
where applicable the name and amount of adsorbent.
_______________________________________________________________________ Ph Eur
1-77
Allopurinol
N
N
N
N
O
H
C
5
H
4
N
4
O 136.1 315-30-0
Allopurinol complies with the requirements of the 3rd edition of the European Pharmacopoeia [0576]. These
Action and use Used in treatment of gout.
Preparation
Allopurinol Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Allopurinol contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent
of 1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one, calculated with reference to the dried substance.
CHARACTERS
A white or almost white powder, very slightly soluble in water and in alcohol. It dissolves in dilute
solutions of alkali hydroxides.
IDENTIFICATION
A. Dissolve 10 mg in 1 ml of 0.1M sodium hydroxide and dilute to 100.0 ml with 0.1M hydrochloric acid.
Dilute 10.0 ml of this solution to 100.0 ml with 0.1M hydrochloric acid. Examined between 220 nm
and 350 nm (2.2.25), the solution shows an absorption maximum at 250 nm and a minimum at
231 nm. The ratio of the absorbance measured at the minimum at 231 nm to that measured at the
maximum at 250 nm is 0.52 to 0.62.
obtained with allopurinol CRS. Examine the substances prepared as discs.
C. Dissolve 0.3 g in 2.5 ml of dilute sodium hydroxide solution R and add 50 ml of water R. Add slowly
and with shaking 5 ml of silver nitrate solution R1. A white precipitate is formed which does not
dissolve on the addition of 5 ml of ammonia R.
D. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel F
254
plate R.
Test solution. Dissolve 20 mg of the substance to be examined in concentrated ammonia R and dilute to
Reference solution. Dissolve 20 mg of allopurinol CRS in concentrated ammonia R and dilute to 10 ml
volumes of ethanol R and 60 volumes of methylene chloride R. Allow the plate to dry in air and
examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test
solution is similar in position and size to the principal spot in the chromatogram obtained with the
reference solution.
TESTS
Appearance of solution Dissolve 1.0 g in 20 ml of dilute sodium hydroxide solution R. The solution is
clear (2.2.1) and not more intensely coloured than reference solution Y
6
or GY
6
(Method II, 2.2.2).
Related substances Examine by liquid chromatography (2.2.29), as prescribed under Assay.
Inject 20 l of reference solution (b). When the chromatogram is recorded in the prescribed
conditions, the retention times are: impurity A about 4.2 min, impurities B and C about 6.1 min,
allopurinol about 7.7 min, impurity D about 26.1 min, impurity E about 27.8 min. The test is not
valid unless the resolution between the peaks corresponding to impurity A and allopurinol is at least
3.0.
Inject 20 l of test solution (a) and 20 l of reference solution (a). Continue the chromatography of
test solution (a) for five times the retention time of allopurinol. In the chromatogram obtained with
test solution (a): the area of any peak corresponding to impurity A is not greater than the area of the
1-78
corresponding peak in the chromatogram obtained with reference solution (b) (0.2 per cent); the
area of any unresolved double peak corresponding to impurities B and C is not greater than the
area of the corresponding double peak in the chromatogram obtained with reference solution (b)
(0.2 per cent); the area of any peaks corresponding to impurity D or impurity E is not greater than
the area of the corresponding peaks in the chromatogram obtained with reference solution (b)
(0.1 per cent); the area of any peak apart from the principal peak and peaks corresponding to
impurities A, B, C, D and E, is not greater than the area of the peak due to allopurinol in the
chromatogram obtained with reference solution (a) (0.1 per cent); the sum of the areas of these
unknown peaks is not greater than three times the area of the peak due to allopurinol in the
chromatogram obtained with reference solution (a) (0.3 per cent). Disregard any peak with an
area less than 0.2 times that of the peak due to allopurinol in the chromatogram obtained with
reference solution (a).
100C to 105C.
ASSAY
Examine by liquid chromatography (2.2.29). Prepare the solutions immediately before use.
Solvent mixture. Mix 2 volumes of mobile phase B and 8 volumes of mobile phase A.
Test solution (a). Dissolve 50.0 mg of the substance to be examined in 5.0 ml of 0.1M sodium hydroxide
and immediately dilute to 100.0 ml with the solvent mixture.
Test solution (b). Dissolve 20.0 mg in 5.0 ml of 0.1M sodium hydroxide and immediately dilute to
Reference solution (a). Dilute 2.0 ml of test solution (a) to 100.0 ml with the solvent mixture. Dilute
5.0 ml of this solution to 100.0 ml with the solvent mixture.
Reference solution (b). Dissolve 10.0 mg of allopurinol impurity A CRS, 5.0 mg of allopurinol impurity B
CRS, 5.0 mg of allopurinol impurity C CRS, 5.0 mg of allopurinol impurity D CRS and 5.0 mg of
allopurinol impurity E CRS in 5.0 ml of 0.1M sodium hydroxide. Add 20.0 ml of test solution (a) and
dilute immediately to 100.0 ml with the solvent mixture. Dilute 1.0 ml of this solution to 100.0 ml
with the solvent mixture.
Reference solution (c). Dissolve 20.0 mg of allopurinol CRS in 5.0 ml of 0.1M sodium hydroxide and
immediately dilute to 250.0 ml with the solvent mixture.
Mobile phase A. A 1.25 g/l solution of potassium dihydrogen phosphate R,
Mobile phase B. Methanol R,
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
0 30 90 70 10 30 linear gradient
Inject 20 l of reference solution (c). Adjust the sensitivity of the system so that the height of the
principal peak is at least 50 per cent of the full scale of the recorder. Inject 20 l of test solution (b).
Calculate the percentage content of allopurinol using the chromatogram obtained with reference
solution (c).
IMPURITIES
N
H
R
2
R
1
N
N
O
A. R
1
= NH
2
,R
2
= H: 5-amino-1H-pyrazole-4-carboxamide,
B. R
1
= NH
2
,R
2
= CHO: 5-(formylamino)-1H-pyrazole-4-carboxamide,
D. R
1
= -O-CH
2
-CH
3
, R
2
= H: ethyl 5-amino-1H-pyrazole-4-carboxylate,
E. R
1
= -O-CH
2
-CH
3
, R
2
= CHO: ethyl 5-(formylamino)-1H-pyrazole-4-carboxylate,
1-79
N
N
N
NH
2
N
N
O
C. N-(4H-1,2,4-triazol-4-yl)-1H-pyrazole-4-carboxamide.
__________________________________________________________________________________________________________ Ph Eur
1-80
Refined Almond Oil
revised 1/01
Refined Almond Oil complies with the requirements of the 3rd edition of the European Pharmacopoeia [1064].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Refined almond oil is the fatty oil from the ripe seeds of Prunus dulcis (Miller) D. A. Webb var. dulcis
or Prunus dulcis (Miller) D. A. Webb var. amara (D. C.) Buchheim or a mixture of both varieties by
cold expression. It is then refined. A suitable antioxidant may be added.
CHARACTERS
A pale yellow, clear liquid, slightly soluble in alcohol, miscible with light petroleum.
It solidifies at about 18C and has a relative density of about 0.916.
IDENTIFICATION
A. Carry out the identification of fatty oils by thin-layer chromatography (2.3.2). The chromatogram
obtained is comparable with the type chromatogram for almond oil.
B. It complies with the test for composition of fatty acids (see Tests).
TESTS
Absorbance (2.2.25). Dissolve 0.100 g in cyclohexane R and dilute to 10.0 ml with the same solvent.
The absorbance measured at the maximum between 264 nm and 276 nm is 0.2 to 6.0.
Acid value (2.5.1). Not more than 0.5, determined on 5.0 g.
Peroxide value (2.5.5). Not more than 5.0.
Unsaponifiable matter (2.5.7). Not more than 0.7 per cent, determined on 5.0 g.
Composition of fatty acids Carry out the test for foreign oils in fatty oils by gas chromatography
(2.4.22, Method A). The fatty-acid fraction of the oil has the following composition:
saturated fatty acids of chain length less than C
16
: not more than 0.1 per cent,
palmitic acid: 4.0 per cent to 9.0 per cent,
palmitoleic acid (equivalent chain length on polyethyleneglycol adipate 16.3): not more than
0.6 per cent,
margaric acid: not more than 0.2 per cent,
stearic acid: not more than 3.0 per cent,
oleic acid (equivalent chain length on polyethyleneglycol adipate 18.3): 62.0 per cent to 86.0 per
cent,
linoleic acid (equivalent chain length on polyethyleneglycol adipate 18.9): 20.0 per cent to
30.0 per cent,
linolenic acid (equivalent chain length on polyethyleneglycol adipate 19.7): not more than
0.4 per cent,
arachidic acid: not more than 0.2 per cent,
eicosenoic acid (equivalent chain length on polyethyleneglycol adipate 20.3): not more than
0.3 per cent,
behenic acid: not more than 0.2 per cent,
erucic acid (equivalent chain length on polyethyleneglycol adipate 22.3): not more than 0.1 per
cent.
Sterols Carry out the test for sterols in fatty oils (2.4.23). The sterol fraction of the oil has the
following composition:
cholesterol: not more than 0.7 per cent,
campesterol: not more than 5.0 per cent,
stigmasterol: not more than 4.0 per cent,
-sitosterol: 73.0 per cent to 87.0 per cent,

5
-avenasterol: at least 5.0 per cent,

7
-avenasterol: not more than 3.0 per cent,

7
-stigmastenol: not more than 3.0 per cent,
brassicasterol: not more than 0.3 per cent.
Water (2.5.32). If intended for use in the manufacture of parenteral dosage forms, not more than
0.1 per cent, determined on 5.00 g by the micro-determination of water.
1-81
STORAGE
Store in a well-filled container, protected from light.
LABELLING
The label states:
where applicable, that the substance is suitable for use in the manufacture of parenteral dosage
forms,
the name and concentration of any added antioxidant.
_______________________________________________________________________ Ph Eur
1-82
Virgin Almond Oil
Almond Oil revised 1/01
Virgin Almond Oil complies with the requirements of the 3rd edition of the European Pharmacopoeia [0261].
Preparation
Almond Oil Ear Drops
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Virgin almond oil is the fatty oil obtained by cold expression from the ripe seeds of Prunus dulcis
(Miller) D. A. Webb var. dulcis or Prunus dulcis (Miller) D. A. Webb var. amara (D. C.) Buchheim or
a mixture of both varieties.
CHARACTERS
A yellow, clear, liquid, slightly soluble in alcohol, miscible with light petroleum.
It solidifies at about 18C and has a relative density of about 0.916.
IDENTIFICATION
A. It complies with the test for absorbance (see Tests).
B. Carry out the identification of fatty oils by thin-layer chromatography (2.3.2). The chromatogram
obtained is comparable with the type chromatogram for almond oil.
C. It complies with the test for composition of fatty acids (see Tests).
TESTS
Absorbance (2.2.25). Dissolve 0.100 g in cyclohexane R and dilute to 10.0 ml with the same solvent.
The absorbance measured at the maximum between 264 nm and 276 nm is not greater than 0.2.
The ratio of the absorbance at 232 nm to that at 270 nm is greater than 7.
(2.4.22, Method A). The fatty-acid fraction of the oil has the following composition:
16
palmitoleic acid (equivalent chain length on polyethyleneglycol adipate 16.3): not more than
0.6 per cent,
margaric acid: not more than 0.2 per cent,
cent,
30.0 per cent,
0.4 per cent,
eicosenoic acid (equivalent chain length on polyethyleneglycol adipate 20.3): not more than
0.3 per cent,
cent.
Sterols Carry out the test for sterols in fatty oils (2.4.23). The sterol fraction of the oil has the
cholesterol: not more than 0.7 per cent,
campesterol: not more than 4.0 per cent,
stigmasterol: not more than 3.0 per cent,
-sitosterol: 73.0 per cent to 87.0 per cent,
1-83

5
-avenasterol: at least 10.0 per cent,

7
-avenasterol: not more than 3.0 per cent,

7
-stigmastenol: not more than 3.0 per cent,
brassicasterol: not more than 0.3 per cent.
STORAGE
_______________________________________________________________________ Ph Eur
1-84
Barbados Aloes
Curaao Aloes
Barbados Aloes complies with the requirements of the 3rd edition of the European Pharmacopoeia [0257].
Action and use Laxative.
Preparation
Standardised Aloes Dry Extract
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Barbados aloes consists of the concentrated and dried juice of the leaves of Aloe barbadensis Miller. It
contains not less than 28.0 per cent of hydroxyanthracene derivatives, expressed as barbaloin
(C
21
H
22
O
9
; M
r
418.4) and calculated with reference to the dried drug.
CHARACTERS
Dark brown masses, slightly shiny or opaque with a conchoidal fracture, or a brown powder, soluble
in hot alcohol, partly soluble in boiling water, practically insoluble in ether.
IDENTIFICATION
A. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance.
Test solution. To 0.25 g of the powdered drug add 20 ml of methanol R and heat to boiling in a water-
bath. Shake for a few minutes and decant the solution. Store at about 4C and use within 24 h.
Reference solution. Dissolve 25 mg of barbaloin R in methanol R and dilute to 10 ml with the same
solvent.
Apply separately to the plate as bands 20 mm by not more than 3 mm 10 l of each solution.
Develop over a path of 10 cm using a mixture of 13 volumes of water R, 17 volumes of methanol R
and 100 volumes of ethyl acetate R. Allow the plate to dry in air, spray with a 100 g/l solution of
potassium hydroxide R in methanol R and examine in ultraviolet light at 365 nm. The chromatogram
obtained with the test solution shows in the central part a zone of yellow fluorescence (barbaloin)
similar in position to the zone corresponding to barbaloin in the chromatogram obtained with the
reference solution and in the lower part a zone of light-blue fluorescence (aloesine). Heat the plate at
110C for 5 min. In the chromatogram obtained with the test solution, a zone of violet fluorescence
appears just below the zone corresponding to barbaloin.
B. Shake 1 g of the powdered drug with 100 ml of boiling water R. Cool, add 1 g of talc R and filter.
To 10 ml of the filtrate add 0.25 g of disodium tetraborate R and heat to dissolve. Pour 2 ml of the
solution into 20 ml of water R. Yellowish-green fluorescence appears which is particularly marked in
ultraviolet light at 365 nm.
C. To 5 ml of the filtrate obtained in identification test B add 1 ml of freshly prepared bromine
water R. A brownish-yellow precipitate is formed and the supernatant liquid is violet.
TESTS
by drying in an oven at 100C to 105C.
ASSAY
Carry out the assay protected from bright light.
Introduce 0.300 g of powdered drug (180) into a 250 ml conical flask. Moisten with 2 ml of
methanol R, add 5 ml of water R warmed to about 60C, mix, add a further 75 ml of water R at about
60C and shake for 30 min. Cool, filter into a volumetric flask, rinse the conical flask and filter with
20 ml of water R, add the rinsings to the volumetric flask and dilute to 1000.0 ml with water.
Transfer 10.0 ml of this solution to a 100 ml round-bottomed flask containing 1 ml of a 600 g/l
solution of ferric chloride R and 6 ml of hydrochloric acid R. Heat in a water-bath under a reflux
condenser for 4 h, with the water level above that of the liquid in the flask. Allow to cool, transfer the
solution to a separating funnel, rinse the flask successively with 4 ml of water R, 4 ml of 1M sodium
hydroxide and 4 ml of water R and add the rinsings to the separating funnel. Shake the contents of the
separating funnel with three quantities, each of 20 ml, of ether R. Wash the combined ether layers
with two quantities, each of 10 ml, of water R. Discard the washings and dilute the organic phase to
100.0 ml with ether R. Evaporate 20.0 ml carefully to dryness on a water-bath and dissolve the
1-85
residue in 10.0 ml of a 5 g/l solution of magnesium acetate R in methanol R. Measure the absorbance
(2.2.25) at 512 nm using methanol R as the compensation liquid.
Calculate the percentage content of hydroxyanthracene derivatives, as barbaloin, from the
expression:
m
A 6 . 19
i.e. taking the specific absorbance of barbaloin to be 255.
A=absorbance at 512 nm,
m=mass of the substance to be examined in grams.
STORAGE
_______________________________________________________________________ Ph Eur
1-86
Cape Aloes
Cape Aloes complies with the requirements of the 3rd edition of the European Pharmacopoeia [0258]. These
Preparation
Standardised Aloes Dry Extract
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cape aloes consists of the concentrated and dried juice of the leaves of various species of Aloe, mainly
Aloe ferox Miller and its hybrids. It contains not less than 18.0 per cent of hydroxyanthracene
derivatives, expressed as barbaloin (C
21
H
22
O
9
; M
r
418.4) and calculated with reference to the dried
drug.
CHARACTERS
Dark brown masses tinged with green and having a shiny conchoidal fracture, or a greenish-brown
powder, soluble in hot alcohol, partly soluble in boiling water, practically insoluble in ether.
IDENTIFICATION
A. Examine the chromatograms obtained in the test for Aloe barbadensis. The chromatogram obtained
with the test solution shows in the central part a zone of yellow fluorescence (barbaloin) similar in
position to the zone corresponding to barbaloin in the chromatogram obtained with the reference
solution and in the lower part two zones of yellow fluorescence (aloinosides A and B) and a zone of
blue fluorescence (aloesine).
B. Shake 1 g of the powdered drug with 100 ml of boiling water R. Cool, add 1 g of talc R and filter.
To 10 ml of the filtrate add 0.25 g of disodium tetraborate R and heat to dissolve. Pour 2 ml of the
solution into 20 ml of water R. A yellowish-green fluorescence appears which is particularly marked in
ultraviolet light at 365 nm.
C. To 5 ml of the filtrate obtained in identification test B add 1 ml of freshly prepared bromine
water R. A yellow precipitate is formed. The supernatant liquid is not coloured violet.
TESTS
Barbados aloes Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating
substance.
Test solution. To 0.25 g of the powdered drug add 20 ml of methanol R and heat to boiling in a water-
bath. Shake for a few minutes and decant the solution. Store at about 4C and use within 24 h.
Reference solution. Dissolve 25 mg of barbaloin R in methanol R and dilute to 10 ml with the same
solvent.
Apply separately to the plate as bands 20 mm by not more than 3 mm 10 l of each solution.
Develop over a path of 10 cm using a mixture of 13 volumes of water R, 17 volumes of methanol R
and 100 volumes of ethyl acetate R. Allow the plate to dry in air, spray with a 100 g/l solution of
potassium hydroxide R in methanol R. Heat the plate at 110C for 5 min and examine in ultraviolet
light at 365 nm. The chromatogram obtained with the test solution shows no zone of violet
fluorescence just below the zone corresponding to barbaloin.
ASSAY
Introduce 0.400 g of powdered drug (180) into a 250 ml conical flask. Moisten with 2 ml of
methanol R, add 5 ml of water R warmed to about 60C, mix, add a further 75 ml of water R at about
60C and shake for 30 min. Cool, filter into a volumetric flask, rinse the conical flask and filter with
20 ml of water R, add the rinsings to the volumetric flask and dilute to 1000.0 ml with water R.
Transfer 10.0 ml of this solution to a 100 ml round-bottomed flask containing 1 ml of a 600 g/l
solution of ferric chloride R and 6 ml of hydrochloric acid R. Heat in a water-bath under a reflux
condenser for 4 h, with the water level above that of the liquid in the flask. Allow to cool, transfer the
solution to a separating funnel, rinse the flask successively with 4 ml of water R, 4 ml of 1M sodium
hydroxide and 4 ml of water R and add the rinsings to the separating funnel. Shake the contents of the
1-87
separating funnel with three quantities, each of 20 ml, of ether R. Wash the combined ether layers
with two quantities, each of 10 ml, of water R. Discard the washings and dilute the organic phase to
100.0 ml with ether R. Evaporate 20.0 ml carefully to dryness on a water-bath and dissolve the
residue in 10.0 ml of a 5 g/l solution of magnesium acetate R in methanol R. Measure the absorbance
(2.2.25) at 512 nm using methanol R as the compensation liquid.
Calculate the percentage content of barbaloin from the expression:
m
A 6 . 19
i.e. taking the specific absorbance of hydroxyanthracene derivatives, as barbaloin, to be 255.
STORAGE
_______________________________________________________________________ Ph Eur
1-88
Aloxiprin
9014-67-9
Definition Aloxiprin is a polymeric condensation product of aluminium oxide and O-
acetylsalicylic acid. It contains not less than 7.5% and not more than 8.5% of aluminium, Al, and
not less than 79.0% and not more than 87.4% of total salicylates, calculated as O-acetylsalicylic
acid, C
9
H
8
O
4
, both calculated with reference to the dried substance.
Characteristics A fine, white or slightly pink powder; odourless or almost odourless.
Practically insoluble in water; slightly soluble in chloroform; practically insoluble in ethanol (96%)
and in ether.
Identification
A. Boil 1 g with 20 ml of 2M hydrochloric acid, cool, filter and reserve the filtrate. Dissolve the
residue in 10 ml of 0.1M sodium hydroxide and neutralise with 1M acetic acid. 1 ml of the resulting
solution yields reaction A characteristic of salicylates, Appendix VI.
B. The filtrate reserved in test A yields the reaction characteristic of aluminium salts, Appendix VI.
Heavy metals Carefully ignite 2.0 g at a low temperature until completely charred, cool, add 2 ml
of nitric acid and 0.25 ml of sulphuric acid, heat cautiously until white fumes are evolved and ignite
at 500 to 600. Cool, add 2 ml of hydrochloric acid, evaporate to dryness on a water bath and carry
out the procedure for limit test C for heavy metals, Appendix VII, beginning at the words Dissolve
the residue .... Use 2 ml of lead standard solution (10 ppm Pb) to prepare the standard (10 ppm).
Free acetylsalicylic acid To a quantity containing the equivalent of 1.0 g of total salicylates add
50 ml of dry ether and shake for 30 minutes. Filter quickly through fluted filter paper, wash the
paper with several portions of dry ether and dilute the combined filtrate and washings to 100 ml
with dry ether. The absorbance of the solution at the maximum at 278 nm is not more than 0.36,
Appendix II B (0.5%, calculated with reference to the content of total salicylates).
Salicylic acid The absorbance of the solution used in the test for Free acetylsalicylic acid at the
maximum at 308 nm is not more than 0.50, Appendix II B (0.15%, calculated with reference to the
content of total salicylates).
Combined salicylate Not more than 9.5%, calculated as salicylic acid, C
7
H
6
O
3
, with reference to
the content of total salicylates calculated as O-acetylsalicylic acid when determined in the following
manner. To 0.1 g add 40 ml of a 0.5% w/v solution of sodium fluoride in 0.1M hydrochloric acid and
shake for 5 minutes. Allow the solution to stand for 10 minutes, shaking at frequent intervals.
Extract with six 20-ml quantities of chloroform, filter the combined extracts through a layer of
anhydrous sodium sulphate, wash with 30 ml of chloroform and dilute the combined filtrate and
washings to 200 ml with chloroform. Dilute 20 ml of the solution to 50 ml with chloroform and
measure the absorbance of the resulting solution at the maximum at 308 nm, Appendix II B.
Calculate the content of C
7
H
6
O
3
taking 293 as the value of A(1%, 1 cm) at the maximum at
308 nm.
Loss on drying When dried to constant weight over phosphorus pentoxide at a pressure not
exceeding 0.7 kPa, loses not more than 2.0% of its weight. Use 1 g.
Assay
For aluminium Ignite 2 g in a tared silica crucible, heat gently until the organic matter is
destroyed and then ignite to constant weight at 1000. Each g of residue is equivalent to 0.5292 g
of Al.
For total salicylates To 0.25 g add 50 ml of 1M sodium hydroxide and boil gently until dissolved.
Cool, add 50 ml of water, adjust the pH to between 2.40 and 2.50 with 1M hydrochloric acid and
dilute to 500 ml with water. To 5 ml add 4 ml of iron(III) chloride solution, allow to stand for 30
minutes, dilute to 50 ml with water and measure the absorbance of the resulting solution at the
maximum at 530 nm, Appendix II B, using in the reference cell a solution prepared by diluting 4 ml
of iron(III) chloride solution to 50 ml with water. Calculate the content of total salicylates as C
9
H
8
O
4
from the absorbance obtained by repeating the procedure using 4 ml of a 0.05% w/v solution of
salicylic acid in place of the solution being examined and beginning at the words add 4 ml of iron(III)
chloride solution .... Each g of salicylic acid is equivalent to 1.305 g of C
9
H
8
O
4
.
Storage Aloxiprin should be kept in a well-closed container.
Preparation
Aloxiprin Tablets
1-89
Alprazolam
N
N
N
N
Cl
Me
C
17
H
13
ClN
4
308.8 28981-97-7
Alprazolam complies with the requirements of the 3rd edition of the European Pharmacopoeia [1065]. These
Action and use Anxiolytic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Alprazolam contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of 8-chloro-1-methyl-6-phenyl-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepine, calculated with
CHARACTERS
A white, crystalline powder, practically insoluble in water, freely soluble in methylene chloride
sparingly soluble in acetone and in alcohol.
It shows polymorphism.
IDENTIFICATION
A. Dissolve the substance to be examined in the smallest necessary quantity of ethyl acetate R and
evaporate to dryness on a water-bath. Thoroughly mix 5.0 mg of the substance to be examined with
5.0 mg of alprazolam CRS. The melting point (2.2.14) of the mixture does not deviate from the
melting point of the substance to be examined by more than 2C.
obtained with alprazolam CRS. If the spectra obtained in the solid state show differences, dissolve the
substance to be examined and the reference substance separately in the minimum volume of ethyl
acetate R, evaporate to dryness on a water-bath and record new spectra using the residues. Examine
the substances prepared as discs.
C. Examine by thin-layer chromatography (2.2.27), using silica gel GF
254
the same solvent.
Reference solution (a). Dissolve 10 mg of alprazolam CRS in methanol R and dilute to 10 ml with the
same solvent.
Reference solution (b). Dissolve 10 mg of alprazolam CRS and 10 mg of midazolam CRS in methanol R
and dilute to 10 ml with the same solvent.
Apply separately to the plate 5 l of each solution. Develop over a path or 12 cm using a mixture of 2
volumes of glacial acetic acid R, 15 volumes of water R, 20 volumes of methanol R and 80 volumes of
ethyl acetate R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal
spot in the chromatogram obtained with the test solution is similar in position and size to the
principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless
the chromatogram obtained with reference solution (b) shows two clearly separately spots.
TESTS
1-90
Test solution. Dissolve 0.100 g of the substance to be examined in dimethylformamide R and dilute to
Reference solution (a). Dissolve 2 mg of alprazolam CRS and 2 mg of triazolam CRS in
dimethylformamide R and dilute to 100.0 ml with the same solvent.
Reference solution (b). Dilute 5.0 ml of the Test solution to 100.0 ml with dimethylformamide R. Dilute
0.5 ml of this solution to 10.0 ml with dimethylformamide R.
a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with phenylsilyl
silica gel for chromatography R1 (5 m),
as mobile phase at a flow rate of 2 ml per minute and at a temperature of 40C a gradient
programme using the following conditions:
Mobile phase A. A mixture of 44 volumes of buffer solution and 56 volumes of methanol R.
Mobile phase B. A mixture of 5 volumes of buffer solution and 95 volumes of methanol R.
Buffer solution. Dissolve 7.7 g of ammonium acetate R in 1000 ml of water R and adjust to pH 4.2
with glacial acetic acid R,
Time
(min)
Mobile
phase A
(per cent V/V)
Mobile
phase B
(per cent V/V)
Comment
0 98 2 Isocratic
15 98 2 Begin linear
gradient
35 1 99 Begin isocratic
40 1 99 End chromatogram
and switch to
initial equilibration
41 98 2 Begin equilibration
50 = 0 98 2 End equilibration
Equilibrate the column for at least 30 min with the initial eluent composition. For subsequent
chromatograms, use the conditions described from 40 min to 50 min. Adjust the sensitivity of the
system so that the height of the principal peak in the chromatogram obtained with reference solution
(b) is at least 50 per cent of the full scale of the recorder.
Inject 10 l of reference solution (a). When the chromatogram is recorded in the prescribed
conditions the retention times are: triazolam, about 9 min and alprazolam, about 10 min. The test is
not valid unless the resolution between the peaks corresponding to alprazolam and triazolam is at
least 1.5.
Inject separately 10 l of dimethylformamide R as a blank, 10 l of the test solution and 10 l of
reference solution (b). In the chromatogram obtained with the test solution, the sum of the areas of
the peaks, apart from the principal peak, is not greater than the area of the principal peak in the
chromatogram obtained with reference solution (b) (0.25 per cent). Disregard any peak in the
chromatogram obtained with the blank and any peak with an area less than 0.2 times the area of the
principal peak in the chromatogram obtained with reference solution (b).
100C to 105C.
ASSAY
Dissolve 0.140 g in 50 ml of a mixture of 3 volumes of glacial acetic acid R and 2 volumes of acetic
anhydride R. Titrate with 0.1M perchloric acid, determining the end-point potentiometrically (2.2.20).
Titrate to the second point of inflexion.
17
H
13
CIN
4
.
STORAGE
1-91
IMPURITIES
N N
NH
2
Me
OH
Cl
A. 3-amino-6-chloro-2-methyl-4-phenyl-3,4-dihydroquinazolin-4-ol,
N N
N O
Cl
OH
Me
B. 5-chloro-2-(3-hydroxymethyl-5-methyl-4H-[1,2,4]triazol-4-yl)benzophenone.
N N
N O
Cl
Me
C. 5-chloro-2-(5-methyl-4H-[1,2,4]triazol-4-yl)benzophenone.
N
N
Cl
N
N
H
2
C
D. 8-chloro-6-phenyl-1-vinyl-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepine,
NH
2
O
Cl
E. 2-amino-5-chlorobenzophenone,
N N
N O
Cl
Cl
Me
F. 5-chloro-2-(3-chloromethyl-5-methyl-4H-[1,2,4]triazol-4-yl)benzophenone.
N
N
N
NH
2
Me
Cl
G. 4-amino-7-chloro-1-methyl-5-phenyl-4H-[1,2,4]triazolo[4,3-a]quinoline,
1-92
N N
N O
Cl
N
Cl
O N
N N
Me Me
H
H. bis[[4-(2-benzoyl-4-chlorophenyl)-5-methyl-4H-[1,2,4]triazol-3-yl]methyl]amine,
Cl
O N
N N
Me
N
N
Cl N
N
OH
Me
I. 5[[4-(2-benzoyl-4-chlorophenyl)-5-methyl-4H-[1,2,4]triazol-3-yl]methyl]-8-chloro-1-
methyl-6-phenyl-5,6-dihydro-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin-6-ol,
N
O
N N
N
N N
N
Ph Ph
Cl Cl
Me Me
J. condensed tertiary amine dimer.
_______________________________________________________________________ Ph Eur
1-93
Alprenolol Benzoate
O NHPr
i
OH H
CH
2
and enantiomer
COOH
,
C
22
H
29
NO
4
371.5
Alprenolol Benzoate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1066].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Alprenolol benzoate contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of (RS)-1-(2-allylphenoxy)-3-(isopropylamino)propan-2-ol benzoate, calculated with
CHARACTERS
A white, crystalline powder or colourless crystals, slightly soluble in water, soluble in alcohol.
IDENTIFICATION
obtained with alprenolol benzoate CRS.
C. Examine the chromatogram obtained in test A for related substances in daylight, after exposure to
iodine vapour for at least 2 h. The principal spot in the chromatogram obtained with test solution (b)
is similar in position, colour and size to the principal spot in the chromatogram obtained with
reference solution (b).
D. Place 0.5 g of the substance to be examined in a vessel for sublimation. Moisten with 0.5 ml of
hydrochloric acid R and gently warm the bottom of the vessel. Collect the white sublimate deposited
on the cold finger. The white sublimate (benzoic acid) melts (2.2.14) at 120C to 124C.
TESTS
Appearance of solution Dissolve 0.50 g in methanol R and dilute to 25.0 ml with the same solvent.
The solution is clear (2.2.1) and colourless (Method II, 2.2.2).
1-(Isopropylamino)-3-[2-(1-propenyl)phenoxy]propan-2-ol Dissolve 0.25 g in alcohol R and
dilute to 25.0 ml with the same solvent. The absorbance (2.2.25) at 297 nm is not more than 0.20
(0.1 per cent).
Related substances
A. Examine by thin-layer chromatography (2.2.27), using a suitable silica gel as the coating
substance.
Test solution (a). Dissolve 0.60 g of the substance to be examined in methanol R and dilute to 10 ml
Reference solution (a). Dissolve 10 mg of alprenolol benzoate CRS and 10 mg of oxprenolol hydrochloride
CRS in methanol R and dilute to 10 ml with the same solvent.
Reference solution (b). Dissolve 12 mg of alprenolol benzoate CRS in methanol R and dilute to 10 ml
Reference solution (c). Dilute 5 ml of Test solution (b) to 50 ml with methanol R.
Place two beakers, each containing 30 volumes of concentrated ammonia R on the bottom of a
chromatographic tank containing a mixture of 5 volumes of methanol R and 95 volumes of ethyl
1-94
acetate R and allow to saturate for at least 1 h before use. Apply separately to the plate 5 l of each
solution. Develop over a path of 15 cm. Allow the plate to dry in air for at least 3 h. Expose the plate
to iodine vapour for at least 6 h. Any spot in the chromatogram obtained with test solution (a) with
an R
f
value higher than the principal spot due to alprenolol is not more intense than the spot in the
chromatogram obtained with reference solution (c) (0.2 per cent). The test is not valid unless the
chromatogram obtained with reference solution (a) shows two clearly separated principal spots.
Reference solution (a). Dissolve 20.0 mg of alprenolol benzoate CRS and 4.0 mg of 4-isopropylphenol R in
the mobile phase and dilute to 100.0 ml with the mobile phase. Dilute 4.0 ml of the solution to
Reference solution (b). Dilute 4.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute
as mobile phase at a flow rate of 1 ml per minute a mixture prepared as follows: dissolve 0.45 g
of sodium octanesulphonate R in 150 ml of acetonitrile R and dilute to 500 ml with phosphate
buffer pH 2.8 (prepared by dissolving 1.78 g of phosphoric acid R and 15.6 g of sodium dihydrogen
phosphate R in water R and diluting to 2000 ml),
Equilibrate the column with the mobile phase at a flow rate of 1 ml per minute for about 1 h.
Adjust the sensitivity so that the height of the principal peak in the chromatogram obtained with
20 l of reference solution (b) is about 50 per cent to 70 per cent of the full scale of the recorder.
conditions, the retention times are: benzoic acid, about 4 min; alprenolol, about 10 min and 4-iso-
propylphenol, about 16 min. The test is not valid unless in the chromatogram obtained with
reference solution (a), the resolution between the peaks corresponding to alprenolol and 4-isopropyl-
phenol is at least 5; if necessary, adjust the concentration of sodium octanesulphonate and/or aceto-
nitrile in the mobile phase (increase the concentration of sodium octanesulphonate to increase the
retention time for alprenolol and increase the concentration of acetonitrile to decrease the retention
time for both compounds).
Inject separately 20 l of the test solution and 20 l of reference solution (b). Continue the chro-
matography for twice the retention time of alprenolol. In the chromatogram obtained with the test
solution, the sum of the areas of any peaks, apart from the peaks due to alprenolol and benzoic acid,
is not greater than the area of the peak due to alprenolol in the chromatogram obtained with refer-
ence solution (b) (0.4 per cent). Disregard any peak due to benzoic acid and any peak with an area
less than 0.1 times the area of the alprenolol peak in the chromatogram obtained with reference
solution (b).
60C over diphosphorus pentoxide R for 4 h.
ASSAY
The mixing must be thorough and continuous otherwise low or inconsistent results may occur.
Dissolve 0.275 g in about 50 ml of alcohol R. Titrate with 0.1M sodium hydroxide, determining the
22
H
29
NO
4
.
STORAGE
IMPURITIES
A. 3-(2-allyl)phenoxy])propane-1,2-diol,
B. 2-allylphenol,
C. 1-(isopropylamino)-3-[2-(propyl)phenoxy]propan-2-ol,
D. N-isopropylamino-1,1-bis[3-(2-allylphenoxy)propan-2-ol],
E. 1-(isopropylamino)-3-(2-(1-propenyl)phenoxy)propan-2-ol.
_______________________________________________________________________ Ph Eur
1-95
Alprenolol Hydrochloride
O NHPr
i
OH H
CH
2
and enantiomer
,HCl
C
15
H
24
ClNO
2
285.8 13707-88-5
Alprenolol Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Alprenolol hydrochloride contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of (RS)-1-(2-allylphenoxy)-3-isopropylamino-propan-2-ol hydrochloride, calculated
CHARACTERS
A white, crystalline powder or colourless crystals, very soluble in water, freely soluble in alcohol and
in methylene chloride.
IDENTIFICATION
obtained with alprenolol hydrochloride CRS.
C. Examine the chromatograms obtained in test A for related substances in daylight, after exposure
to iodine vapour for 30 min. The principal spot in the chromatogram obtained with test solution (b)
TESTS
solution B
9
(Method II, 2.2.2).
1-Isopropylamino-3-(2-prop-1-enylphenoxy)propan-2-ol Dissolve 0.25 g in alcohol R and
dilute to 25 ml with the same solvent. The absorbance (2.2.25) measured at 297 nm is not greater
than 0.20 (0.1 per cent).
Related substances
Reference solution (a). Dissolve 10 mg of alprenolol hydrochloride CRS in methanol R and dilute to 10 ml
Reference solution (b). Dissolve 10 mg of alprenolol hydrochloride CRS and 10 mg of oxprenolol hydro-
chloride CRS in methanol R and dilute to 10 ml with the same solvent.
Reference solution (c). Dilute 5 ml of Test solution (b) to 50 ml with methanol R.
1-96
Place two beakers each containing 30 ml of ammonia R at the bottom of the chamber which is
saturated with the mobile phase and ammonia R for at least 1 h before use. Apply separately to the
plate 5 l of each solution. Develop over a path of 15 cm using a mixture of 5 volumes of methanol R
and 95 volumes of ethyl acetate R. Dry the plate at 100C for 15 min. Expose the plate to iodine
vapour for up to 6 h. In the chromatogram obtained with test solution (a) any spot with an R
f
value
higher than that of the principal spot is not more intense than the spot in the chromatogram obtained
with reference solution (c). The test is not valid unless the chromatogram obtained with reference
Reference solution (a). Dissolve 4.0 mg of alprenolol hydrochloride CRS and 0.8 mg of 4-isopropyl-
phenol R in the mobile phase and dilute to 100.0 ml with the mobile phase.
as mobile phase at a flow rate of 1 ml per minute a mixture prepared as follows: mix 0.656 g of
sodium octanesulphonate R with 150 ml of acetonitrile R and dilute to 500 ml with phosphate
buffer pH 2.8 (prepared from 1.78 g of phosphoric acid R and 15.6 g of sodium dihydrogen
phosphate R diluted to 2000 ml),
Equilibrate the column with the mobile phase at a flow rate of 1 ml per minute for about 1 h.
Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram
obtained with 20 l of reference solution (b) is at least 50 per cent of the full scale of the recorder.
Inject 20 l of reference solution (a). When the chromatograms are recorded under the prescribed
conditions, the retention times are: alprenolol hydrochloride about 11 min; 4-isopropylphenol about
18 min. The test is not valid unless, in the chromatogram obtained with reference solution (a), the
resolution between the peaks corresponding to alprenolol hydrochloride and 4-isopropylphenol is at
least 5; if necessary, adjust the concentration of sodium octanesulphonate and/or acetonitrile in the
mobile phase (increase the concentration of sodium octanesulphonate to increase the retention time
of alprenolol hydrochloride and increase the concentration of acetonitrile to decrease the retention
times of both compounds).
Inject separately 20 l of the test solution and 20 l of reference solution (b). Continue the
chromatography for twice the retention time of the principal peak. In the chromatogram obtained
with the test solution, the sum of the areas of any peaks apart from the principal peak is not greater
than the area of the principal peak in the chromatogram obtained with reference solution (b) (0.4 per
cent). Disregard any peak with an area less than 0.1 times the area of the principal peak in the
chromatogram obtained with reference solution (b).
Heavy metals (2.4.8). Dissolve 2.0 g in 20 ml of water R. 12 ml of the solution complies with limit
test A for heavy metals (10 ppm). Prepare the standard using lead standard solution (1 ppm Pb) R.
Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying over
diphosphorus pentoxide R at a pressure not exceeding 2.7 kPa.
ASSAY
Dissolve 0.400 g in 25 ml of a mixture of equal volumes of ethanol R and water R. Add 10 ml of
0.01M hydrochloric acid. Carry out a potentiometric titration (2.2.20), using 0.1M sodium
hydroxide. Read the volume added between the two points of inflexion.
15
H
24
ClNO
2
.
STORAGE
IMPURITIES
A. 3-(2-allylphenoxy)propane-1,2-diol,
B. 2-allylphenol,
C. 1-isopropylamino-3-[2-(1-propenyl)phenoxy]propan-2-ol,
D. N, N-bis[2-(2-allylphenoxy)-2-hydroxypropyl]isopropylamine.
_______________________________________________________________________ Ph Eur
1-97
Alprostadil
1/01
COOH
CH
3
O
OH
H
H
H
OH H
C
20
H
34
O
5
354.5 745-65-3
Alprostadil complies with the requirements of the 3rd edition of the European Pharmacopoeia [1488]. These
Action and use Prostanoid vasodilator.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
7-[(1R,2R,3R)-3-Hydroxy-2-[(1E,3S)-3-hydroxyoct-1-enyl]-5-oxocyclopentyl]heptanoic acid.
Content: 95.0 per cent to 102.5 per cent (anhydrous substance).
CHARACTERS
Appearance: a white or slightly yellowish, crystalline powder.
Solubility: practically insoluble in water, freely soluble in alcohol, soluble in acetone, slightly soluble in
ethyl acetate.
IDENTIFICATION
A. Specific optical rotation (2.2.7): 60 to 70 (anhydrous substance).
Immediately before use, dissolve 50 mg in alcohol R and dilute to 10.0 ml with the same solvent.
B. Infrared absorption spectrophotometry (2.2.24):
Preparation: discs.
Comparison: alprostadil CRS.
C. Examine the chromatograms obtained in the assay.
Results: the principal peak in the chromatogram obtained with the test solution is similar in retention
time and size to the principal peak in the chromatogram obtained with the reference solution.
TESTS
Related substances Liquid chromatography (2.2.29).
Prepare the solutions protected from light.
Test solution. Dissolve 10.0 mg of the substance to be examined in a mixture of equal volumes of
acetonitrile R1 and water R and dilute to 10.0 ml with the same mixture of solvents.
Reference solution (a). Dilute 100 l of the test solution to 20.0 ml with a mixture of equal volumes of
acetonitrile R1 and water R.
Reference solution (b). Dissolve 1.0 mg of alprostadil impurity H CRS and 1.0 mg of alprostadil CRS in a
mixture of equal volumes of acetonitrile R1 and water R and dilute to 20.0 ml with the same mixture
of solvents.
Reference solution (c). In order to prepare in situ the degradation compounds (impurity A and impurity
B), dissolve 1 mg of the substance to be examined in 100 l of 1M sodium hydroxide (the solution
becomes brownish-red), wait for 3 min and add 100 l of 1M phosphoric acid (yellowish-white
opalescent solution); dilute to 5.0 ml with a mixture of equal volumes of acetonitrile R1 and water R.
System A
Column:
size: l = 0.25 m, = 4.0 mm,
stationary phase: octylsilyl base-deactivated silica gel for chromatography R (4 m), pore size 6 nm,
temperature: 35C.
Mobile phase:
mobile phase A. Dissolve 3.9 g of sodium dihydrogen phosphate R in water R and dilute to
1000.0 ml with the same solvent; adjust to pH 2.5 with a 2.9 g/l solution of phosphoric acid R
(approximately 600 ml is required); to 740 ml of the buffer solution add 260 ml of aceto-
nitrile R1,
1-98
mobile phase B. Dissolve 3.9 g of sodium dihydrogen phosphate R in water R and dilute to 1000.0 ml
with the same solvent; adjust to pH 2.5 with a 2.9 g/l solution of phosphoric acid R (approxi-
mately 600 ml is required); to 200 ml of the buffer solution add 800 ml of acetonitrile R1.
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
0 75 100 0
75 76 100 0 0 100
76 86 0 100
86 87 0 100 100 0
87 102 100 0
Flow rate: 1 ml/min.
Detection: spectrophotometer at 200 nm.
Injection: 20 l loop injector.
System suitability:
retention time: alprostadil = about 63 min,
resolution: minimum of 1.5 between the peaks due to impurity H and alprostadil in the chroma-
togram obtained with reference solution (b).
The following chromatogram is given for information.
0.00 20.00 40.00 60.00
minutes
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
m
V
Syst. A
I
m
p
u
r
i
t
y

G
I
m
p
u
r
i
t
y

F
I
m
p
u
r
i
t
y

D
I
m
p
u
r
i
t
y

H
I
m
p
u
r
i
t
y

E
Fig. 14881 Chromatogram for the test for related substances (system A)
System B
Use the same conditions as for system A with the following mobile phase and elution programme:
mobile phase A. Dissolve 3.9 g of sodium dihydrogen phosphate R in water R and dilute to
1000.0 ml with the same solvent; adjust to pH 2.5 with a 2.9 g/l solution of phosphoric acid R
(approximately 600 ml is required); to 600 ml of the buffer solution add 400 ml of aceto-
1-99
nitrile R1,
mobile phase B. Use mobile phase B as described under system A.
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
0 50 100 0
50 51 100 0 0 100
51 61 0 100
61 62 0 100 100 0
62 72 100 0
System suitability:
relative retentions with reference to alprostadil (retention time = about 7 min): impurity A =
about 2.4; impurity B = about 2.6,
resolution: minimum of 1.5 between the peaks due to impurity A and impurity B in the chroma-
togram obtained with reference solution (c).
0.00 10.00 20.00 30.00 40.00
minutes
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
m
V
Syst. B
I
m
p
u
r
i
t
y

C
I
m
p
u
r
i
t
y

B
I
m
p
u
r
i
t
y

A
I
m
p
u
r
i
t
y

I
I
m
p
u
r
i
t
y

J
A
l
p
r
o
s
t
a
d
i
l
Fig. 14882 Chromatogram for the test for related substances (system B)
Carry out the test according to system A and B.
1-100
Table 14881
Impurity Relative
retention
(system A)
Relative
retention
(system B)
Correction
factor
Impurity G 0.80 0.7
Impurity F 0.88 0.8
Impurity D 0.90 1.0
Impurity H 0.96 0.7
Impurity E 1.10 0.7
Impurity C 1.36 1.9
Impurity K 1.85 0.06
Impurity A 2.32 0.7
Impurity B 2.45 1.5
Impurity I 4.00 1.0
Impurity J 5.89 1.0
Limits:
correction factors: multiply the areas of the corresponding peaks using the correction factors in
Table 1488.-1 to obtain the corrected areas,
impurity A (corrected area): not more than 3 times the area of the principal peak in the chromato-
gram obtained with reference solution (a) (1.5 per cent),
impurity B (corrected area): not more than the area of the principal peak in the chromatogram
obtained with reference solution (a) ( 0.5 per cent ),
any other impurity (corrected area): not more than 1.8 times the area of the principal peak in the
chromatogram obtained with reference solution (a) (0.9 per cent ), and not more than 1 such
peak has an area greater than the area of the principal peak in the chromatogram obtained with
reference solution (a) (0.5 per cent). Evaluate impurities appearing at relative retentions less than
1.2 by system A and impurities appearing at relative retentions greater than 1.2 by system B,
total (corrected area): not more than 3 times the area of the principal peak in the chromatogram
obtained with reference solution (a) (1.5 per cent ),
disregard limit: 0.1 times the area of the principal peak in the chromatogram obtained with
reference solution (a) (0.05 per cent).
Water (2.5.32): maximum 0.5 per cent, determined on 50 mg.
ASSAY
Liquid chromatography (2.2.29) as described in the test for related substances, system A.
Prepare the solutions protected from light.
acetonitrile R1 and water R and dilute to 25.0 ml with the same mixture of solvents. Dilute 3.0 ml of
the solution to 20.0 ml with a mixture of equal volumes of acetonitrile R1 and water R.
Reference solution. Dissolve 10.0 mg of alprostadil CRS in a mixture of equal volumes of acetonitrile R1
and water R and dilute to 25.0 ml with the same mixture of solvents. Dilute 3.0 ml of the solution to
20.0 ml with a mixture of equal volumes of acetonitrile R1 and water R.
Injection: 20 l.
Calculate the percentage content of C
20
H
34
O
5
.
STORAGE
At a temperature of 2C to 8C.
IMPURITIES
COOH
CH
3
O
H
H
H OH
A. 7-[(1R,2S)-2-[(1E,3S)-3-hydroxyoct-1-enyl]-5-oxocyclopent-3-enyl]heptanoic acid
(prostaglandin A
1
),
COOH
CH
3
O
H OH
B. 7-[2-[(1E,3S)-3-hydroxyoct-1-enyl]-5-oxocyclopent-1-enyl]heptanoic acid (prostaglandin
B
1
),
1-101
COOH
CH
3
O
H
H
O
H
O
C. 7-[(1R,2R,3R)-3-hydroxy-2-[(1E)-3-oxo-oct-1-enyl]-5-oxocyclopentyl]heptanoic acid (15-
oxoprostaglandin E
1
),
COOH
CH
3
O
H
H
HO
H
H OH
D. 7-[(1R,2R,3R)-3-hydroxy-2-[(1E,3R)-3-hydroxyoct-1-enyl]-5-oxocyclopentyl]heptanoic
acid (15-epiprostaglandin E
1
),
COOH
CH
3
O
H
H
HO
H
H OH
E. 7-[(1R,2R,3S)-3-hydroxy-2-[(1E,3S)-3-hydroxyoct-1-enyl]-5-oxocyclopentyl]heptanoic
1
),
COOH
CH
3
O
H
H
O
H
H OH
F. 7-[(1S,2R,3R)-3-hydroxy-2-[(1E,3S)-3-hydroxyoct-1-enyl]-5-oxocyclopentyl]heptanoic
1
),
CH
3
O
H
H
O
H
H OH
COOH
G. (5Z)-7-[(1R,2R,3R)-3-hydroxy-2-[(1E,3S)-3-hydroxyoct-1-enyl]-5-oxocyclopentyl]hept-5-
enoic acid (dinoprostone),
COOH
CH
3
O
H
H
O
H OH
H. (5E)-7-[(1R,2R,3R)-3-hydroxy-2-[(1E,3S)-3-hydroxyoct-1-enyl]-5-oxocyclopentyl]hept-5-
enoic acid ((5E)-prostaglandin E
2
),
COOR
CH
3
O
H
H
O
H OH
I. R = CH
2
-CH
3
: ethyl 7-[(1R,2R,3R)-3-hydroxy-2-[(1E,3S)-3-hydroxyoct-1-enyl]-5-
oxocyclopentyl]heptanoate (prostaglandin E
1
ethyl ester),
J. R = CH(CH
3
)
2
: 1-methylethyl 7-[(1R,2R,3R)-3-hydroxy-2-[(1E,3S)-3-hydroxyoct-1-enyl]-5-
oxocyclopentyl]heptanoate (prostaglandin E
1
isopropyl ester),
P
O
K. triphenylphosphine oxide.
__________________________________________________________________________________________________________ Ph Eur
1-102
Alteplase for Injection
SYQVICRDEK TQMIYQQHQS WLRPVLRSNR
VEYCWCNSGR AQCHSVPVKS CSEPRCFNGG
TCQQALYFSD FVCQCPEGFA GKCCEIDTRA
TCYEDQGISY RGTWSTAESG AECTNWNSSA
LAQKPYSGRR PDAIRLGLGN HNYCRNPDRD
SKPWCYVFKA GKYSSEFCST PACSEGNSDC
YFGNGSAYRG THSLTESGAS CLPWNSMILI
GKVYTAQNPS AQALGLGKHN YCRNPDGDAK
PWCHVLKNRR LTWEYCDVPS CSTCGLRQYS
QPQFR
IKGGLFADIA SHPWQAAIFA KHRRSPGERF
LCGGILISSC WILSAAHCFQ ERFPPHHLTV
ILGRTYRVVP GEEEQKFEVE KYIVHKEFDD
DTYDNDIALL QLKSDSSRCA QESSVVRTVC
LPPADLQLPD WTECELSGYG KHEALSPFYS
ERLKEAHVRL YPSSRCTSQH LLNRTVTDNM
LCAGDTRSGG PQANLHDACQ GDSGGPLVCL
NDGRMTLVGI ISWGLGCGQK DVPGVYTKVT
NYLDWIRDNM RP
C
2736
H
4174
N
914
O
824
S
45
(non-glycosylated protein) 105857-23-6
Alteplase for Injection complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Fibrinolytic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Alteplase for injection is a sterile, freeze-dried preparation of alteplase, a tissue plasminogen activator
produced by recombinant DNA technology. It has a potency of not less than 500,000 I.U. per
milligram of protein.
Tissue plasminogen activator binds to fibrin clots and activates plasminogen, leading to the
generation of plasmin and to the degradation of fibrin clots or blood coagulates.
Alteplase consists of 527 amino acids with a calculated relative molecular mass of 59,050 without
consideration of the carbohydrate moieties attached at positions Asn 117, Asn 184 and Asn 448. The
total relative molecular mass is approximately 65,000. Alteplase is cleaved by plasmin between
amino-acids 275 and 276 into a two-chain form (A chain and B chain) that are connected by a
disulphide bridge between Cys 264 and Cys 395. The single-chain form and the two-chain form
show comparable fibrinolytic activity in vitro.
PRODUCTION
Alteplase is produced by recombinant DNA synthesis in cell culture; the fermentation takes place in
serum-free medium. It complies with the monograph on Products of recombinant DNA technology
(784).
The purification process is designed to remove efficiently potential impurities, such as antibiotics,
DNA and protein contaminants derived both from the host cell and from the production medium,
and potential viral contaminants.
If alteplase is stored in bulk form, stability (maintenance of potency) in the intended storage
conditions must be demonstrated.
The production, purification and product consistency are checked by a number of analytical
methods described below, carried out routinely as in-process controls.
Protein content The protein concentration of alteplase solutions is determined by measuring the
absorbance (2.2.25) of the protein solution at 280 nm and at 320 nm, using formulation buffer as the
1-103
compensation liquid. If dilution of alteplase samples is necessary, the samples are diluted in
formulation buffer. The absorbance of the formulation buffer is subtracted from the absorbance of
the samples for calculation of the protein content. For the calculation of the alteplase concentra-
tion the absorbance value A
280
A
320
is divided by the specific absorption coefficient for alteplase of
1.9.
Potency The potency of alteplase is determined in an in-vitro clot-lysis assay as described under
Assay. The specific activity of bulk alteplase is approximately 580,000 I.U. per milligram of alteplase.
N-terminal sequence N-terminal sequencing is applied to determine the correct N-terminal
sequence and to determine semiquantitatively additional cleavage sites in the alteplase molecule, for
example at position AA 275-276 or at position AA 27-28. The N-terminal sequence must conform
with the sequence of human tissue plasminogen activator.
Isoelectric focusing The consistency in the microheterogeneity of glycosylation of the alteplase
molecule can be demonstrated by isoelectric focusing (IEF). A complex banding pattern with ten
major and several minor bands in the pH range 6.5 - 8.5 is observed. Denaturing conditions are
applied to achieve a good separation of differently charged variants of alteplase. The broad charge
distribution observed is due to a population of molecules, which differ in the fine structure of
biantenary and triantenary complex-type carbohydrate residues, with different degrees of substitution
with sialic acids. The banding pattern of alteplase test samples must be consistent with the pattern of
alteplase reference standard.
Single-chain alteplase content The alteplase produced by CHO (Chinese hamster ovary) cells in
serum-free medium is predominantly single-chain alteplase. The single-chain form can be separated
from the two-chain form by gel-permeation liquid chromatography under reducing conditions as
described under Single-chain content (see Tests). The single-chain alteplase content in bulk samples
must be higher than 60 per cent.
Tryptic-peptide mapping The primary structure of the alteplase molecule is verified by tryptic-
peptide mapping as described under Identification B. The reduced and carboxymethylated molecule
is cleaved by trypsin into about fifty peptides, which are separated by reverse-phase liquid chromato-
graphy. A characteristic chromatogram (fingerprint) is obtained. The identity of the tryptic-peptide
map of a given alteplase sample with the profile of a well-characterised reference standard is an
indirect confirmation of the amino-acid sequence, because even single amino-acid exchanges in
individual peptides can be detected by this sensitive technique. In addition, complex peaks of the
glycopeptides can be isolated from the tryptic-peptide map and separated in a second dimension,
either by reverse-phase liquid chromatography under modified conditions or by capillary electro-
phoresis. By this two-dimensional separation of glycopeptide variants, lot-to-lot consistency of the
microheterogeneity of glycosylation can be demonstrated.
The tryptic-peptide map of alteplase samples must be consistent with the tryptic-peptide map of
alteplase reference standard.
Monomer content The monomer content of alteplase is measured by gel-permeation liquid
chromatography under non-reduced conditions as described under Monomer content (see Tests).
The monomer content of alteplase bulk samples must be higher than 95 per cent.
Type I/Type II alteplase content CHO cells produce two glycosylation variants of alteplase. Type I
alteplase contains one polymannose-type glycosylation at position Asn 117 and two complex-type
glycosylation sites at positions Asn 184 and Asn 448. Type II alteplase is only glycosylated at
positions Asn 117 and Asn 448.
The ratio of Type I/Type II alteplase is constant in the range of 45 to 65 per cent of Type I and 35
to 55 per cent of Type II. The content of alteplase Type I and Type II can be determined by a
densitometric scan of SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) gel.
Plasmin-treated samples of alteplase, which are reduced and carboxymethylated before loading on
the gel, are separated into three bands: Type I alteplase A-chain (AA 1-275), Type II alteplase A-
chain (AA 1-275) and alteplase B-chain (AA 276-527). The ratio of Type I/Type II alteplase is
determined from a calibration curve, which is obtained by a densitometric scan of defined mixtures of
purified Type I alteplase and Type II alteplase standards.
SDS-PAGE SDS-PAGE (silver staining) is used to demonstrate purity of the alteplase bulk material
and the integrity of the alteplase molecule. For alteplase bulk samples, no additional protein bands
compared to reference standard or degradation products must occur in SDS-PAGE gels at a loading
amount of 2.5 g alteplase protein per lane and a limit of detection of 5 ng per protein (BSA) band.
Bacterial endotoxins (2.6.14). Not more than 1 I.U. of bacterial endotoxin per milligram of
alteplase.
Sialic acids Dialyse samples and alteplase reference standard against enzyme buffer (8.9 g/l sodium
chloride R, 4.1 g/l sodium acetate R, pH 5.5) using a membrane with a cut-off point corresponding to a
relative molecular mass of 10,000 for globular proteins. After dialysis, determine the protein concen-
tration. Add 5 l of calcium chloride solution (19.98 per cent m/m calcium chloride R) to 1 ml of
protein solution. Add 10 milliunits of neuraminidase per milligram of protein. Incubate this solution
1-104
at 37C for about 17 h.
Prepare standard dilutions between 1.56 mg/ml and 25.0 mg/ml from an N-acetylneuraminic acid R
reference stock solution with a concentration of 50 mg/ml. Pipette 0.2 ml of each sample and
protein reference standard in duplicate into reagent tubes. Pipette also 0.2 ml of N-acetylneuraminic
acid R standard dilutions into reagent tubes. Add 0.25 ml of periodate reagent (5.4 g/l of
sodium periodate R in a 1.25 per cent V/V solution of sulphuric acid R), mix and incubate for 30 min at
37C. Add 0.2 ml of arsenite reagent (20 g/l sodium arsenite R in a 1.55 per cent V/V solution of
hydrochloric acid R) and mix. A yellowish-brown colour develops and disappears. Add 2.0 ml of a
28.9 g/l solution of thiobarbituric acid R and mix. Heat the closed tubes in boiling water for 7.5 min
and then cool them in an ice-bath for 5 min. Add 2.0 ml of a mixture of butanol R and hydrochloric
acid R (95:5) and mix. Centrifuge the tubes at 3000 r/min for 3 min. Measure the absorbance of
the butanol-HCl layer at 552 nm within 30 min using the butanol-HCl mixture as the compensation
solution. Perform a linear-regression analysis for the N-acetylneuraminic acid standard. The molar
content of N-acetylneuraminic acid for the samples and for alteplase reference standard is
calculated from the calibration curve. The sialic acid content for the test samples must be within
the range 70 to 130 per cent of alteplase reference standard, which contains about 3 moles of sialic
acid per mole of alteplase.
Neutral sugars Dilute alteplase samples and the reference standard in the assay buffer, containing
34.8 g/l of arginine R, 0.1 g/l of polysorbate 80 R and adjusted to pH 7.4 with phosphoric acid R, to a
protein concentration of 50 g/ml. Prepare the following concentrations of mannose in the same
assay buffer for a calibration curve: 20, 30, 40, 50 and 60 g/ml. Pipette 2 ml of alteplase samples
and reference standard, as well as 2 ml of each mannose concentration in duplicate in reagent tubes.
Add 50 l of phenol R, followed by 5 ml of sulphuric acid R in each reagent tube. Incubate the mixture
for 30 min at room temperature. Measure the absorbance at 492 nm for each tube. Read the content
of neutral sugars from the mannose calibration curve. The neutral sugar content is expressed in moles
of neutral sugar per mole of alteplase, taking into account the dilution factor for alteplase samples
and reference standard and using a relative molecular mass of 180.2 for mannose and a relative
molecular mass of 59,050 for the alteplase protein moiety. The neutral sugar content of the alteplase
samples must be in the range of 70 to 130 per cent compared to alteplase reference standard, which
contains about 12 moles of neutral sugar per mole of alteplase.
CHARACTERS
A white or slightly yellow powder or solid friable mass.
Reconstitute the preparation as stated on the label immediately before carrying out the Identification, Tests
(except those for solubility and water) and Assay.
IDENTIFICATION
A. The assay serves also to identify the preparation.
B. Tryptic-peptide mapping. Examine by liquid chromatography (2.2.29).
Test solution. Dilute the preparation to be examined with water R to obtain a solution containing
about 1 mg of alteplase per millilitre. Dialyse about 2.5 ml of the solution for at least 12 h into a
solution containing 480 g/l of urea R, 44 g/l of tris(hydroxymethylamino)methane R and 1.5 g/l of
disodium edetate R and adjusted to pH 8.6, using a membrane with a cut-off point corresponding to a
relative molecular mass of 10,000 for globular proteins. Measure the volume of the solution, transfer
it to a clean test-tube and add per millilitre 10 l of a 156 g/l solution of dithiothreitol R. Allow to
stand for 4 h, cool in iced water and add per millilitre of solution 25 l of a freshly prepared 190 g/l
solution of iodoacetic acid R. Allow to stand in the dark for 30 min. Add per millilitre 50 l of
dithiothreitol solution to stop the reaction. Dialyse for 24 h against an 8 g/l solution of ammonium
hydrogen carbonate R. Add 1 part of trypsin for peptide mapping R to 100 parts of the protein and allow
to stand for 6 h to 8 h. Repeat the addition of trypsin and allow to stand for a total of 24 h.
Reference solution. Prepare as for the test solution using alteplase CRS instead of the preparation to be
examined.
chromatography R (5 m to 10 m),
Mobile phase A. A 8 g/l solution of sodium dihydrogen phosphate R, adjusted to pH 2.85 with
phosphoric acid R, filtered and degassed,
Mobile phase B. Acetonitrile R 75 per cent V/V in mobile phase A,
Equilibrate the system with mobile phase A at a flow rate of 1 ml/min. After injection of the
solution, increase the proportion of mobile phase B at a rate of 0.44 per cent per minute until the
ratio of mobile phase A to mobile phase B is 60:40 and then increase the proportion of mobile phase
B at a rate of 1.33 per cent per minute until the ratio of mobile phase A to mobile phase B is 20:80
and then continue elution with this mixture for a further 10 min. Record the chromatogram for the
1-105
reference solution: the test is not valid unless the resolution of peaks 6 (peptides 268-275) and 7
(peptides 1-7) is at least 1.5; b
0.5a
and b
0.5b
are not more than 0.4 min. Inject about 100 l of the test
solution and record the chromatogram. Verify the identity of the peaks by comparison with the
chromatograms of the reference solution. There should not be any additional significant peaks or
shoulders, a significant peak or shoulder being defined as one with an area response equal to or
greater than 5 per cent of peak 19 (peptides 278-296); no significant peak is missing. A type chroma-
togram for identification of the peaks cited is given at the end of the monograph (see Fig. 1170-1).
TESTS
Appearance of solution The reconstituted preparation is clear (2.2.1) and not more intensely
coloured than reference solution Y
7
(Method II, 2.2.2).
pH (2.2.3). 7.1 to 7.5.
Solubility Add the volume of the liquid stated on the label. The preparation dissolves completely
within 2 min at 20C to 25C.
Protein content Prepare a solution of the substance to be examined with an accurately known
concentration of about 1 g/l. Using a 34.8 g/l solution of arginine R adjusted to pH 7.3 with
phosphoric acid R, dilute an accurately measured volume of the solution of the substance to be
examined so that the absorbance measured at the maximum at about 280 nm is 0.5 to 1.0 (test
solution). Measure the absorbance (2.2.25) of the solution at the maximum at about 280 nm and at
320 nm using the arginine solution as the compensation liquid. Calculate the protein content in the
portion of alteplase taken from the expression:
V A A ( )
280 320
9
1.
in which V is the volume of arginine solution required to prepare the test solution, A
280
is the
absorbance at the maximum at about 280 nm and A
320
is the absorbance at 320 nm.
Single-chain content Examine by liquid chromatography (2.2.29).
Test solution. Dissolve the preparation to be examined in water R to obtain a solution containing about
1 mg of alteplase per millilitre. Place about 1 ml of the solution in a tube, add 3 ml of a 3 g/l solution
of dithiothreitol R in the mobile phase, place a cap on the tube and heat at about 80C for 3 min to
5 min.
a column 0.6 m long and 7.5 mm in internal diameter packed with silica-based rigid, hydrophilic
gel with spherical particles 10 m to 13 m in diameter, suitable for size-exclusion chromato-
graphy,
as mobile phase at a flow rate of 0.5 ml/min a solution containing 30 g/l of sodium dihydrogen
phosphate R and 1 g/l of sodium dodecyl sulphate R, adjusted to pH 6.8 with dilute sodium hydroxide
solution R,
Inject about 50 l of the test solution and record the chromatogram. The chromatogram shows two
major peaks corresponding to single-chain and two-chain alteplase. Calculate the relative amount of
single-chain alteplase from the peak area values.
The test is not valid unless: the number of theoretical plates calculated on the basis of the single-
chain alteplase peak is at least 1000. The content of single-chain alteplase is not less than 60 per cent
of the total amount of alteplase-related substances found.
Monomer content Examine by liquid chromatography (2.2.29).
Test solution. Reconstitute the preparation to be examined to obtain a solution containing about
1 mg per millilitre.
a column 0.6 m long and 7.5 mm in internal diameter packed with silica-based rigid, hydrophilic
gel with spherical particles 10 m to 13 m in diameter, suitable for size-exclusion chromato-
graphy,
as mobile phase at a flow rate of 0.5 ml/min a solution containing 30 g/l of sodium dihydrogen
phosphate R and 1 g/l of sodium dodecyl sulphate R, adjusted to pH 6.8 with dilute sodium hydroxide
solution R,
Inject the test solution and record the chromatogram. The test is not valid unless the number of
theoretical plates calculated for the alteplase monomer peak is at least 1000. Measure the response
for all peaks, i.e. peaks corresponding to alteplase species of different molecular masses. Calculate the
relative content of monomer from the area values of these peaks. The monomer content for alteplase
must be at least 95 per cent.
Water (2.5.12). Not more than 4.0 per cent, determined by the semi-micro determination of water.
Bacterial endotoxins (2.6.14). Not more than 1 I.U. of bacterial endotoxin per milligram of
1-106
protein.
Sterility (2.6.1). It complies with the test for sterility.
ASSAY
The potency of alteplase is determined by comparing its ability to activate plasminogen to form
plasmin with the same capacity of a reference preparation calibrated in International Units. The
formation of plasmin is measured by the determination of the lysis time of a fibrin clot in given
conditions.
The International Unit is the activity of a stated quantity of the International Standard of alteplase.
The equivalence in International Units of the International Standard is stated by the World Health
Organisation.
Solvent buffer. A solution containing 1.38 g/l of sodium dihydrogen phosphate monohydrate R, 7.10 g/l of
anhydrous disodium hydrogen phosphate R, 0.20 g/l of sodium azide R and 0.10 g/l of polysorbate 80 R.
Human thrombin solution. A solution of human thrombin R containing 33 I.U. per millilitre in solvent
buffer.
Human fibrinogen solution. A 2 g/l solution of fibrinogen R in solvent buffer.
Human plasminogen solution. A 1 g/l solution of human plasminogen R in solvent buffer.
Test solutions. Using a solution of the substance to be examined containing 1 g/l, prepare serial
dilutions using solvent buffer, for example 1:5000, 1:10,000, 1:20,000.
Reference solutions. Using a solution of alteplase CRS having an accurately known concentration of
about 1 g/l (580,000 I.U. of alteplase per millilitre), prepare five serial dilutions using water R to
obtain reference solutions having known concentrations in the range 9.0 I.U./ml to 145 I.U./ml.
To each of a set of labelled glass test-tubes, add 0.5 ml of human thrombin solution. Allocate each
test and reference solution to a separate tube and add to each tube 0.5 ml of the solution allocated to
it. To each of a second set of labelled glass tubes, add 20 l of human plasminogen solution, and
1 ml of human fibrinogen solution, mix and store on ice. Beginning with the reference/thrombin
mixture containing the lowest number of International Units per millilitre, record the time and
separately add 200 l of each of the thrombin mixtures to the test tubes containing the plasminogen-
fibrinogen mixture. Using a vortex mixture, intermittently mix the contents of each tube for a total of
15 s and carefully place in a rack in a circulating water-bath at 37C. A visibly turbid clot forms
within 30 s and bubbles subsequently form within the clot. Record the clot-lysis time as the time
between the first addition of alteplase solution and the moment when the last bubble rises to the
surface. Using a least-squares fit, determine the equation of the line using the logarithms of the
concentrations of the reference preparation in International Units per millilitre versus the logarithms
of the values of their clot-lysis times in seconds, according to the equation:
log t = a + b(logU
S
)
in which t is the clot-lysis time, U
S
the activity in International Units per millilitre of the reference
preparation, b is the slope and a the y-intercept of the line. The test is not valid unless the correlation
coefficient is 0.9900 to 1.0000. From the line equation and the clot-lysis time for the test solution,
calculate the logarithm of the activity U
A
from the equation:
b
a t
U
] ) [(log
log
A
Calculate the alteplase activity in International Units per millilitre from the expression:
A
U D
in which D is the dilution factor for the test solution. Calculate the specific activity in the portion of
the substance to be examined from the expression:
P
U
A
in which P is the concentration of protein obtained in the test for protein content.
The estimated potency is not less than 90 per cent and not more than 110 per cent of the stated
potency.
STORAGE
Store in a colourless, glass container, under vacuum or under an inert gas, protected from light, at a
temperature between 2C and 30C.
LABELLING
The label states:
the number of International Units per container,
the amount of protein per container.
1-107
The following type chromatogram is given for information and guidance; it does not form a mandatory part of the
monograph.
Fig. 11701 Type chromatogram for tryptic-peptide mapping of alteplase
__________________________________________________________________________________________________________ Ph Eur
2-1
Alum
Potash Alum; Aluminium Potassium Sulphate
AlK(SO
4
)
2
,12H
2
O 474.4 7784-24-9
Alum complies with the requirements of the 3rd edition of the European Pharmacopoeia [0006]. These require-
Action and use Astringent.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Alum contains not less than 99.0 per cent and not more than the equivalent of 100.5 per cent of
AlK(SO
4
)
2
,12H
2
O.
CHARACTERS
A granular powder or colourless, transparent, crystalline masses, freely soluble in water, very soluble in
boiling water, soluble in glycerol, practically insoluble in alcohol.
IDENTIFICATION
A. Solution S (see Tests) gives the reactions of sulphates (2.3.1).
B. Solution S gives the reaction of aluminium (2.3.1).
C. Shake 10 ml of solution S with 0.5 g of sodium bicarbonate R and filter. The filtrate gives reaction
(a) of potassium (2.3.1).
TESTS
Solution S Dissolve 2.5 g in water R and dilute to 50 ml with the same solvent.
pH (2.2.3). Dissolve 1.0 g in carbon dioxide-free water R and dilute to 10 ml with the same solvent. The
pH of the solution is 3.0 to 3.5.
Ammonium (2.4.1). To 1 ml of solution S add 4 ml of water R. Dilute 0.5 ml of the solution to
14 ml with the same solvent. The solution complies with the limit test for ammonium (0.2 per cent).
Heavy metals (2.4.8). 12 ml of solution S complies with limit test A for heavy metals (20 ppm).
Iron (2.4.9). 2 ml of solution S diluted to 10 ml with water R complies with the limit test for iron
(100 ppm). Use in this test 0.3 ml of thioglycollic acid R.
ASSAY
Dissolve 0.900 g in 20 ml of water R and carry out the complexometric titration of aluminium
(2.5.11).
1 ml of 0.1M sodium edetate is equivalent to 47.44 mg of AIK(SO
4
)
2
,12H
2
O.
STORAGE
_______________________________________________________________________ Ph Eur
2-2
Aluminium Chloride Hexahydrate
AlCl
3
,6H
2
O 241.4 7784-13-6
Aluminium Chloride Hexahydrate complies with the requirements of the 3rd edition of the European
Pharmacopoeia [0971]. These requirements are reproduced after the heading Definition below.
Preparation
Aluminium Chloride Solution
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Aluminium chloride hexahydrate contains not less than 95.0 per cent and not more than the equiva-
lent of 101.0 per cent of AlCl
3
,6H
2
O.
CHARACTERS
A white or slightly yellow, crystalline powder or colourless crystals, deliquescent, very soluble in
water, freely soluble in alcohol, soluble in glycerol.
IDENTIFICATION
A. Dilute 0.1 ml of solution S2 (see Tests) to 2 ml with water R. The solution gives reaction (a) of
chlorides (2.3.1).
B. Dilute 0.3 ml of solution S2 to 2 ml with water R. The solution gives the reaction of aluminium
(2.3.1).
TESTS
Solution S1 Dissolve 10.0 g in distilled water R and dilute to 100 ml with the same solvent.
Solution S2 Dilute 50 ml of solution S1 to 100 ml with water R.
Appearance of solution Solution S2 is clear (2.2.1) and not more intensely coloured than reference
solution B
7
(Method II, 2.2.2).
Sulphates (2.4.13). 15 ml of solution S1 complies with the limit test for sulphates (100 ppm).
Alkali and alkaline-earth metals To 20 ml of solution S2 add 100 ml of water R and heat to
boiling. To the hot solution add 0.2 ml of methyl red solution R. Add dilute ammonia R1 until the
colour of the indicator changes to yellow and dilute to 150 ml with water R. Heat to boiling and filter.
Evaporate 75 ml of the filtrate to dryness on a water-bath and ignite to constant mass. The residue
weighs not more than 2.5 mg (0.5 per cent).
Heavy metals (2.4.8). 12 ml of solution S1 complies with limit test A for heavy metals (20 ppm).
Iron (2.4.9). 10 ml of solution S1 complies with the limit test for iron (10 ppm).
Water (2.5.12). 42.0 per cent to 48.0 per cent, determined on 50.0 mg by the semi-micro
ASSAY
Dissolve 0.500 g in 25.0 ml of water R. Carry out the complexometric titration of aluminium
(2.5.11). Titrate with 0.1M zinc sulphate until the colour of the indicator changes from greyish-green
to pink. Carry out a blank titration.
1 ml of 0.1M sodium edetate is equivalent to 24.14 mg of AlCl
3
,6H
2
O.
STORAGE
_______________________________________________________________________ Ph Eur
2-3
Aluminium Glycinate
O
Al
NH
2
O
OH
OH
.x H
2
O
C
2
H
6
AlNO
4
,xH
2
O 135.1 41354-48-7
Definition Aluminium Glycinate is a basic aluminium monoglycinate, partly hydrated. It contains
not less than 34.5% and not more than 38.5% of Al
2
O
3
and not less than 9.9% and not more than
10.8% of N, both calculated with reference to the dried substance.
Characteristics A white or almost white powder; odourless or almost odourless.
Practically insoluble in water and in organic solvents. It dissolves in dilute mineral acids and in
aqueous solutions of the alkali hydroxides.
Identification
A. Add 0.1 g to 10 ml of a solution prepared by dissolving 0.84 g of citric acid in 8 ml of 1M sodium
hydroxide and diluting to 20 ml with water. Add 0.5 ml of a 0.1% w/v solution of ninhydrin in
methanol and warm. A purple colour is produced.
B. Suspend 1 g in 25 ml of 0.5M hydrochloric acid and heat gently until a clear solution is produced.
Reserve half of the solution. To 2 ml of the solution add 0.15 ml of liquefied phenol, shake and add
carefully without shaking 5 ml of dilute sodium hypochlorite solution. A blue colour is produced.
C. The solution reserved in test B yields the reaction characteristic of aluminium salts, Appendix VI.
Acidity or alkalinity pH of a suspension of 1 g in 25 ml of carbon dioxide-free water, 6.5 to 7.5,
Appendix V L.
Neutralising capacity Shake 0.2 g vigorously with 25 ml of 0.1M hydrochloric acid for 5 minutes
and allow to stand for 5 minutes. The pH of the mixture is greater than 3.0, Appendix V L.
Arsenic Dissolve 2.0 g in 18 ml of brominated hydrochloric acid and 32 ml of water. 25 ml of the
resulting solution complies with the limit test for arsenic, Appendix VII (1 ppm).
Heavy metals Dissolve 1.5 g in 20 ml of 2M hydrochloric acid and 10 ml of water, add 0.5 ml of
nitric acid and boil for about 30 seconds. Cool, add 2 g of ammonium chloride and 2 g of ammonium
thiocyanate and extract with two 10-ml quantities of a mixture of equal parts of isoamyl alcohol and
ether. To the aqueous layer add 2 g of citric acid. 12 ml of the resulting solution complies with limit
test A for heavy metals, Appendix VII. Use lead standard solution (1 ppm Pb) to prepare the standard
(20 ppm).
Mercuric salts Dissolve 2.0 g in 10 ml of 1M sulphuric acid, transfer to a separating funnel with the
aid of water, dilute to about 50 ml with water and add 50 ml of 0.5M sulphuric acid. Add 100 ml of
water, 2 g of hydroxylamine hydrochloride, 1 ml of 0.05M disodium edetate and 1 ml of glacial acetic
acid. Add 5 ml of chloroform, shake, allow to separate and discard the chloroform layer. Titrate the
aqueous layer with a solution of dithizone in chloroform containing 8 g per ml until the chloroform
layer remains green. After each addition, shake vigorously, allow the layers to separate and discard
the chloroform layer. Repeat the operation using a solution prepared by diluting 1 ml of mercury
standard solution (5 ppm Hg) to 100 ml with 0.5M sulphuric acid and beginning at the words Add
100 ml of water . The volume of the dithizone solution required by the substance being exam-
ined does not exceed that required by the mercury standard solution.
Chloride Dissolve 1.0 g in 10 ml of 2M nitric acid and dilute to 100 ml with water. 15 ml of the
resulting solution complies with the limit test for chlorides, Appendix VII (330 ppm).
Loss on drying When dried to constant weight at 130, loses not more than 12.0% of its weight.
Use 1 g.
Assay For Al
2
O
3
Dissolve 0.25 g in a mixture of 3 ml of 1M hydrochloric acid and 50 ml of water, add
50 ml of 0.05M disodium edetate VS and neutralise with 1M sodium hydroxide using methyl red solution
as indicator. Heat the solution to boiling, allow to stand for 10 minutes on a water bath, cool rapidly,
add about 50 mg of xylenol orange triturate and 5 g of hexamine and titrate the excess of disodium
edetate with 0.05M lead nitrate VS until the solution becomes red. Each ml of 0.05M disodium
edetate VS is equivalent to 2.549 mg of Al
2
O
3
.
Storage Aluminium Glycinate should be kept in a well-closed container.
Action and use Antacid.
2-4
Dried Aluminium Hydroxide
Dried Aluminium Hydroxide complies with the requirements of the 3rd edition of the European Pharmacopoeia
for Hydrated Aluminium Oxide [0311]. These requirements are reproduced after the heading Definition
below.
Preparations
Aluminium Hydroxide Tablets
Co-magaldrox Oral Suspension
Co-magaldrox Tablets
Compound Magnesium Trisilicate Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hydrated aluminium oxide contains the equivalent of not less than 47.0 per cent and not more than
60.0 per cent of Al
2
O
3
(M
r
102.0).
CHARACTERS
A white, amorphous powder, practically insoluble in water. It dissolves in dilute mineral acids and in
IDENTIFICATION
Solution S (see Tests) gives the reaction of aluminium (2.3.1).
TESTS
Solution S Dissolve 2.5 g in 15 ml of hydrochloric acid R, heating on a water-bath. Dilute to 100 ml
with distilled water R.
Appearance of solution Solution S is not more opalescent than reference suspension II (2.2.1) and
not more intensely coloured than reference solution GY
6
(Method II, 2.2.2).
Alkaline impurities Shake 1.0 g with 20 ml of carbon dioxide-free water R for 1 min and filter. To
10 ml of the filtrate add 0.1 ml of phenolphthalein solution R. Any pink colour disappears on the
addition of 0.3 ml of 0.1M hydrochloric acid.
Neutralising capacity Carry out the test at 37C. Disperse 0.5 g in 100 ml of water R, heat, add
100.0 ml of 0.1M hydrochloric acid, previously heated, and stir continuously; the pH (2.2.3) of the
solution after 10 min, 15 min and 20 min is not less than 1.8, 2.3 and 3.0 respectively and is at no
time greater than 4.5. Add 10.0 ml of 0.5M hydrochloric acid, previously heated, stir continuously for
1 h and titrate with 0.1M sodium hydroxide to pH 3.5; not more than 35.0 ml of 0.1M sodium hydroxide
is required.
Chlorides (2.4.4). Dissolve 0.1 g with heating in 10 ml of dilute nitric acid R and dilute to 100 ml
with water R. 5 ml of the solution diluted to 15 ml with water R complies with the limit test for
chlorides (1 per cent).
Sulphates (2.4.13). Dilute 4 ml of solution S to 100 ml with distilled water R. 15 ml of the solution
complies with the limit test for sulphates (1 per cent).
Arsenic (2.4.2). 10 ml of solution S complies with limit test A for arsenic (4 ppm).
Heavy metals (2.4.8). Neutralise 20 ml of solution S with concentrated ammonia R, using metanil
yellow solution R as an external indicator. Filter, if necessary, and dilute to 30 ml with water R. 12 ml
of the solution complies with limit test A for heavy metals (60 ppm). Prepare the standard using
10 ml of lead standard solution (1 ppm Pb) R.
Microbial contamination. Total viable aerobic count (2.6.12) not more than 10
3
micro-
organisms per gram, determined by plate-count. It complies with the tests for Enterobacteria and
certain other gram-negative bacteria and with the test for Escherichia coli (2.6.13).
ASSAY
Dissolve 0.800 g in 10 ml of hydrochloric acid R1, heating on a water-bath. Cool and dilute to 50.0 ml
with water R. To 10.0 ml of the solution, add dilute ammonia R1 until a precipitate begins to appear.
Add the smallest quantity of dilute hydrochloric acid R needed to dissolve the precipitate and dilute to
20 ml with water R. Carry out the complexometric titration of aluminium (2.5.11).
1 ml of 0.1M sodium edetate is equivalent to 5.098 mg of Al
2
O
3
.
STORAGE
Store in an airtight container, at a temperature below 30C.
_______________________________________________________________________ Ph Eur
2-5
Aluminium Magnesium Silicate
corrected 1/01
12511-31-8
Aluminium Magnesium Silicate complies with the requirements of the 3rd edition of the European
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Aluminium magnesium silicate is a mixture of particles with colloidal particle size of montmorillonite
and saponite, free from grit and nonswellable ore. It contains not less than 95.0 per cent and not
more than 105.0 per cent of the amount of aluminium and magnesium stated on the label.
CHARACTERS
Almost white powder, granules or plates, practically insoluble in water and in organic solvents.
It swells in water to produce a colloidal dispersion.
IDENTIFICATION
A. Fuse 1 g with 2 g of anhydrous sodium carbonate R. Warm the residue with water R and filter.
Acidify the filtrate with hydrochloric acid R and evaporate to dryness on a water-bath. 0.25 g of the
residue gives the reaction of silicates (2.3.1).
B. Dissolve the remainder of the residue obtained in identification test A in a mixture of 5 ml of dilute
hydrochloric acid R and 10 ml of water R. Filter and add ammonium chloride buffer solution pH 10.0 R. A
white, gelatinous precipitate is formed. Centrifuge, keep the supernatant for identification C.
Dissolve the remaining precipitate in dilute hydrochloric acid R. The solution gives the reaction of
aluminium (2.3.1).
C. The supernatant liquid obtained after centrifugation in identification test B gives the reaction of
magnesium (2.3.1).
TESTS
pH (2.2.3). Disperse 5.0 g in 100 ml of carbon dioxide-free water R. The pH of the dispersion is 9.0 to
10.0.
Arsenic (2.4.2). Transfer 16.6 g to a 250 ml beaker containing 100 ml of dilute hydrochloric acid R.
Mix, cover with a watch glass and boil gently, with occasional stirring, for 15 min. Allow the
insoluble matter to settle and decant the supernatant liquid through a rapid-flow filter paper into a
250 ml volumetric flask, retaining as much sediment as possible in the beaker. To the residue in the
beaker add 25 ml of hot dilute hydrochloric acid R, stir, heat to boiling, allow the insoluble matter to
settle and decant the supernatant liquid through the filter into the volumetric flask. Repeat the
extraction with four additional quantities, each of 25 ml, of hot dilute hydrochloric acid R, decanting
each supernatant liquid through the filter into the volumetric flask. At the last extraction, transfer as
much of the insoluble matter as possible onto the filter. Allow to cool the combined filtrates to room
temperature and dilute to 250.0 ml with dilute hydrochloric acid R. Dilute 5.0 ml of the solution to
25.0 ml with dilute hydrochloric acid R. The solution complies with limit test A for arsenic (3 ppm).
Lead Not more than 15 ppm of Pb, determined by atomic absorption spectrometry (Method I,
2.2.23).
Test solution. Transfer 10.0 g to a 250 ml beaker containing 100 ml of dilute hydrochloric acid R. Mix,
cover with a watch glass and boil for 15 min. Allow to cool to room temperature and allow the
insoluble matter to settle. Decant the supernatant liquid through a rapid-flow filter paper into a
400 ml beaker. To the insoluble matter in the 250 ml beaker add 25 ml of hot water R. Stir, allow the
insoluble matter to settle and decant the supernatant liquid through the filter into the 400 ml
beaker. Repeat the extraction with two additional quantities, each of 25 ml, of water R, decanting
each supernatant liquid through the filter into the 400 ml beaker. Wash the filter with 25 ml of hot
water R, collecting this filtrate in the 400 ml beaker. Concentrate the combined filtrates by gently
boiling to about 20 ml. If a precipitate appears, add about 0.1 ml of nitric acid R, heat to boiling and
allow to cool to room temperature. Filter the concentrated extracts through a rapid-flow filter paper
into a 50 ml volumetric flask. Transfer the remaining contents of the 400 ml beaker through the filter
paper and into the flask with water R. Dilute to 50.0 ml with water R.
Reference solutions. Prepare the reference solutions using lead standard solution (10 ppm Pb) R, diluted
if necessary with water R.
2-6
Measure the absorbance at 217 nm, using a lead hollow-cathode lamp as source of radiation and
an oxidising air-acetylene flame.
100C to 105C.
3
micro-organisms
per gram determined by plate count. It complies with the test for Escherichia coli (2.6.13).
ASSAY
Aluminium Determine by atomic absorption spectrometry (Method I, 2.2.23).
Test solution. In a platinum crucible mix 0.200 g with 1.0 g of lithium metaborate R. Heat slowly at first
and ignite at 1000C to 1200C for 15 min. Cool, place the crucible in a 100 ml beaker containing
25 ml of dilute nitric acid R and add an additional 50 ml of dilute nitric acid R, filling and submerging
the crucible. Place a polytetrafluoroethylene coated magnetic stirring bar in the crucible and stir
gently with a magnetic stirrer until dissolution is complete. Pour the contents into a 250 ml beaker
and remove the crucible. Warm the solution and transfer through a rapid-flow filter paper into a
250 ml volumetric flask, wash the filter and beaker with water R and dilute to 250.0 ml with water R
(solution A). To 20.0 ml of this solution add 20 ml of a 10 g/l solution of sodium chloride R and dilute
to 100.0 ml with water R.
Reference solutions. Dissolve 1.000 g of aluminium R in a mixture of 10 ml of hydrochloric acid R and
10 ml of water R by gentle heating. Cool and dilute to 1000.0 ml with water R (1 mg of aluminium
per millilitre). Into three identical volumetric flasks, each containing 0.20 g of sodium chloride R,
introduce 2.0 ml, 5.0 ml and 10.0 ml of the solution, respectively and dilute to 100.0 ml with
water R.
Measure the absorbance at 309 nm using an aluminium hollow-cathode lamp as the source of
radiation and an oxidising acetylene-nitrous oxide flame.
Calculate the content of aluminium in the substance to be examined.
Magnesium Determine by atomic absorption spectrometry (Method I, 2.2.23).
Test solution. Dilute 25.0 ml of solution A, prepared in the assay for aluminium, to 50.0 ml with
water R. To 5.0 ml of this solution add 20.0 ml of lanthanum chloride solution R and dilute to 100.0 ml
with water R.
Reference solutions. Place 1.000 g of magnesium R in a 250 ml beaker containing 20 ml of water R and
carefully add 20 ml of hydrochloric acid R, warming if necessary to dissolve. Transfer the solution to a
volumetric flask and dilute to 1000.0 ml with water R (1 mg of magnesium per millilitre). Dilute
5.0 ml of the solution to 250.0 ml with water R. Into four identical volumetric flasks, introduce
5.0 ml, 10.0 ml, 15.0 ml and 20.0 ml of the solution, respectively. To each flask add 20.0 ml of
lanthanum nitrate solution R and dilute to 100.0 ml with water R.
Measure the absorbance at 285 nm using a magnesium hollow-cathode lamp as source of radiation
and a reducing air-acetylene flame.
Calculate the content of magnesium in the substance to be examined.
STORAGE
LABELLING
The label states the content of aluminium and magnesium.
__________________________________________________________________________________________________________ Ph Eur
2-7
Dried Aluminium Phosphate
Definition Dried Aluminium Phosphate consists mainly of hydrated aluminium orthophosphate.
It contains not less than 80.0% of AlPO
4
.
Production It may be prepared by drying under suitable conditions the product of interaction in
aqueous solution of an aluminium salt with an alkali phosphate such as sodium phosphate.
Characteristics A white powder containing some friable aggregates.
Practically insoluble in water; soluble in dilute mineral acids; practically insoluble in ethanol
(96%) and in solutions of the alkali hydroxides.
Identification
A. A solution in 2M hydrochloric acid yields the reaction characteristic of aluminium salts, Appendix
VI.
B. A solution in 2M nitric acid yields the reactions characteristic of phosphates, Appendix VI.
Acidity pH of a 4% w/v suspension in carbon dioxide-free water, 5.5 to 6.5, Appendix V L.
Neutralising capacity Pass a sufficient quantity, triturated if necessary, through a sieve of
nominal mesh aperture 75 m, add 0.5 g to 30 ml of 0.1M hydrochloric acid previously heated to 37
and maintain at 37 for 30 minutes, stirring continuously. The pH of the mixture at 37 is 2.0 to
2.5, Appendix V L.
Arsenic Dissolve 0.40 g in 18 ml of brominated hydrochloric acid and dilute to 50 ml with water.
25 ml of the resulting solution complies with the limit test for arsenic, Appendix VII (5 ppm).
Heavy metals Dissolve 1.2 g in 20 ml of 1M hydrochloric acid and 10 ml of water, add 0.5 ml of
nitric acid and boil for about 30 seconds. Cool, add 2 g of ammonium chloride and 2 g of ammonium
thiocyanate and extract with two 10-ml quantities of a mixture of equal parts of isoamyl alcohol and
ether. To the aqueous layer add 2 g of citric acid and dilute to 40 ml with water. 12 ml of the
resulting solution complies with limit test A for heavy metals, Appendix VII. Use lead standard
solution (2 ppm Pb) to prepare the standard (70 ppm).
Chloride Dissolve 0.24 g in 10 ml of 2M nitric acid, boil, cool, dilute to 1000 ml with water and
filter. 15 ml of the filtrate complies with the limit test for chlorides, Appendix VII (1.4%).
Sulphate Dissolve 0.17 g in 10 ml of 2M hydrochloric acid, boil, cool, dilute to 100 ml with water
and filter. 15 ml of the filtrate, with the addition of 2 ml of 2M hydrochloric acid, complies with the
limit test for sulphates, Appendix VII (0.6%).
Soluble phosphate Not more than 1.0% w/w, calculated as PO
4
, when determined by the
following method. Shake 5.0 g with 150 ml of water for 2 hours, filter until the filtrate is clear and
wash the filter with 50 ml of water. Add to the combined filtrate and washings sufficient water to
produce 250 ml, mix well and dilute 10 ml to 100 ml with water. To 5 ml of the resulting solution
add 4 ml of 1M sulphuric acid, 1 ml of ammonium molybdatesulphuric acid solution, 2 ml of
methylaminophenolsulphite reagent and 5 ml of water. Mix well, allow to stand for 15 minutes and
add sufficient water to produce 25 ml. Allow to stand for a further 15 minutes and measure the
absorbance of a 2-cm layer of the resulting solution at 730 nm, Appendix II B. Calculate the content
of soluble phosphate from a calibration curve prepared by treating suitable aliquots of a 0.00286%
w/v solution of potassium dihydrogen orthophosphate in the same manner, beginning at the words add
4 ml of 1M sulphuric acid . A 0.00286% w/v solution of potassium dihydrogen orthophosphate is
equivalent to 0.0020% w/v of PO
4
.
Assay Dissolve 0.8 g in 15 ml of 2M hydrochloric acid, warming if necessary to effect solution, cool
and add sufficient 2M hydrochloric acid to produce 100 ml. To 10 ml add 25 ml of 0.05M disodium
edetate VS then add 13.5M ammonia dropwise until the solution is just alkaline to litmus paper.
Boil gently for 5 minutes, cool and add 10 ml of a solution prepared by dissolving 7.7 g of
ammonium acetate in 50 ml of water and adding 6 ml of glacial acetic acid and sufficient water to
produce 100 ml. Adjust the pH to 4.5 with glacial acetic acid and add 2 ml of a 0.025% w/v solution
of dithizone in ethanol (96%). Add sufficient ethanol (96%) to double the volume of the solution and
titrate with 0.05M zinc chloride VS until the colour changes to red. Repeat the operation without the
substance being examined. The difference between the titrations represents the amount of
disodium edetate required. Each ml of 0.05M disodium edetate VS is equivalent to 6.098 mg of
AlPO
4
.
Storage Dried Aluminium Phosphate should be kept in a well-closed container and stored at a
temperature not exceeding 30.
2-8
Aluminium Powder
Al 26.98 7429-90-5
Definition Aluminium Powder consists mainly of metallic aluminium in the form of very small
flakes, usually with an appreciable proportion of aluminium oxide; it is lubricated with stearic acid
to prevent oxidation. It contains not less than 86.0% of Al, calculated with reference to the
substance freed from lubricant and volatile matter.
Characteristics A silvery grey powder; odourless or almost odourless.
Practically insoluble in water and in ethanol (96%). It dissolves in dilute acids and in aqueous
solutions of alkali hydroxides, with the evolution of hydrogen.
Identification A solution in 2M hydrochloric acid yields the reaction characteristic of aluminium
salts, Appendix VI.
Surface-covering power Not less than 4000 cm
2
per g when determined by the following
method. Fill with water a shallow trough measuring approximately 60 cm 12 cm 1.5 cm, fitted
with a movable partition so constructed that it is a sliding fit and can be used to divide the trough
into two rectangular areas. Place the movable partition near one end and sprinkle 50 mg of the
substance being examined on the surface of the liquid confined in the smaller area. Using a glass
rod, spread the powder evenly over the liquid surface until an unbroken film covers the entire
surface. Move the partition so as to increase the area confined and again spread the powder to
cover the increased surface. Continue this process and determine the maximum unbroken surface
area obtained. The surface-covering power is the area covered per g of the powder at the breaking
point of the film.
Iron Dissolve 10 mg in 20 ml of 2M hydrochloric acid and dilute to 100 ml with water. 10 ml of the
resulting solution complies with the limit test for iron, Appendix VII (1.0%).
Lead Use two solutions prepared in the following manner. For solution (1) boil 0.40 g with 20 ml
of 2M hydrochloric acid and 10 ml of water until effervescence ceases, add 0.5 ml of nitric acid, boil
for 30 seconds and cool; add 2 g of ammonium chloride and 2 g of ammonium thiocyanate, extract
with three 10-ml quantities of a mixture of equal volumes of amyl alcohol and ether, discard the
extracts and add 2 g of citric acid. For solution (2) dissolve 2 g of citric acid in 10 ml of 2M hydro-
chloric acid and add 4 ml of lead standard solution (10 ppm Pb). Make solutions (1) and (2) alkaline
with 5M ammonia and to each add 1 ml of potassium cyanide solution PbT. The solutions should be
not more than faintly opalescent. If the colours of the solutions differ, equalise by the addition of
about 0.2 ml of a highly diluted solution of burnt sugar or other non-reactive substance. Dilute
each solution to 50 ml with water, add 0.1 ml of a 10% w/v solution of sodium sulphide to each and
mix thoroughly. The colour produced in solution (1) is not more intense than that produced in
solution (2), when viewed against a white background (100 ppm).
Other metals Dissolve 2 g in 40 ml of 2M hydrochloric acid. Dilute 20 ml of the solution to 100 ml
with water, make alkaline to litmus paper by the addition of 5M ammonia, boil and filter. Evaporate
the filtrate to dryness, add 0.05 ml of sulphuric acid and ignite. The residue weighs not more than
2 mg.
Lubricant To 2 g add 100 ml of hot water, cover and add, dropwise, sufficient of a mixture of
equal volumes of hydrochloric acid and water to dissolve the metal almost completely. Heat to
complete dissolution, cool, filter through a hardened filter paper and wash the vessel and filter
paper thoroughly with water; dry both the vessel and paper at room temperature. Extract the paper
with three 100-ml quantities of boiling, freshly distilled acetone, using the original vessel to contain
the solvent and then wash the paper with five 10-ml quantities of freshly distilled acetone. Evaporate
the combined filtrate and washings to dryness using a rotary evaporator. The residue, after drying
at 105 for 30 minutes and allowing to cool, weighs 10 to 60 mg.
When the basin containing the residue is floated in a beaker of water suitably stirred and heated,
the residue melts between 40 and 60. The residue is almost completely soluble, with
effervescence, in hot dilute sodium carbonate solution.
Volatile matter When heated to constant weight at 105, loses not more than 0.5% of its weight.
Use 1 g.
Assay Transfer 0.2 g, previously freed from lubricant by successive washing with acetone and
drying, to a three-necked 500-ml flask fitted with a 150-ml dropping funnel, an inlet tube
connected to a cylinder of carbon dioxide and an outlet tube dipping into a water trap. Add 60 ml of
water and disperse the substance being examined; replace the air by carbon dioxide and add 100 ml
of a solution containing 56 g of ammonium iron(III) sulphate and 7.5 ml of sulphuric acid in water.
While maintaining an atmosphere of carbon dioxide in the flask, heat to boiling, boil for 5 minutes
after the sample has dissolved, cool rapidly to 20 and dilute to 250 ml with water. To 50 ml add
15 ml of orthophosphoric acid and titrate with 0.02M potassium permanganate VS. Each ml of 0.02M
potassium permanganate VS is equivalent to 0.8994 mg of Al.
2-9
Storage Aluminium Powder should be kept in a well-closed container.
Preparation
Compound Aluminium Paste
Action and use Topical protective.
2-10
Aluminium Sulphate
Aluminium Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0165].
Preparation
Aluminium Acetate Ear Drops
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Aluminium sulphate contains not less than 51.0 per cent and not more than the equivalent of
59.0 per cent of Al
2
(SO
4
)
3
(M
r
342.1). It contains a variable quantity of water of crystallisation.
CHARACTERS
Colourless, lustrous crystals or crystalline masses, soluble in cold water, freely soluble in hot water,
practically insoluble in alcohol.
IDENTIFICATION
A. Solution S (see Tests) gives reaction (a) of sulphates (2.3.1).
B. Solution S gives the reaction of aluminium (2.3.1).
TESTS
Appearance of solution Solution S is not more opalescent than reference suspension III (2.2.1) and
is colourless (Method II, 2.2.2).
Alkali and alkaline-earth metals To 20 ml of solution S add 100 ml of water R, heat and add
0.1 ml of methyl red solution R. Add dilute ammonia R1 until the colour of the indicator changes to
yellow. Dilute to 150 ml with water R, heat to boiling and filter. Evaporate 75 ml of the filtrate to
dryness on a water-bath and ignite. The residue weighs not more than 2 mg (0.4 per cent).
Ammonium (2.4.1). 0.4 ml of solution S diluted to 14 ml with water R complies with the limit test
for ammonium (500 ppm).
Heavy metals (2.4.8). Dilute 8 ml of solution S to 20 ml with water R. 12 ml of the solution
(100 ppm). Use 0.3 ml of thioglycollic acid R in this test.
ASSAY
Dissolve 0.500 g in 20 ml of water R. Carry out the complexometric titration of aluminium (2.5.11).
1 ml of 0.1M sodium edetate is equivalent to 17.11 mg of Al
2
(SO
4
)
3
.
STORAGE
_______________________________________________________________________ Ph Eur
2-11
Amantadine Hydrochloride
,HCl
NH
2
C
10
H
18
ClN 187.7 665-67-7
Amantadine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Anticonvulsant; antiviral.
Preparations
Amantadine Capsules
Amantadine Oral Solution
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Amantadine hydrochloride contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of tricyclo[3.3.1.13,7]dec-1-ylamine hydrochloride, calculated with reference to the
CHARACTERS
A white or almost white, crystalline powder, freely soluble in water and in alcohol, practically
insoluble in ether. It sublimes on being heated.
IDENTIFICATION
First identification: A, D.
obtained with amantadine hydrochloride CRS. Examine the substances prepared as discs.
B. To 0.1 g add 1 ml of pyridine R, mix and add 0.1 ml of acetic anhydride R. Heat to boiling for
about 10 s. Pour the hot solution into 10 ml of dilute hydrochloric acid R, cool to 5C and filter. The
precipitate, washed with water R and dried in vacuo at 60C for 1 h, melts (2.2.14) at 147C to
151C.
C. Dissolve 0.2 g in 1 ml of 0.1M hydrochloric acid. Add 1 ml of a 500 g/l solution of sodium nitrite R.
A white precipitate is formed.
D. 1 ml of solution S (see Tests) gives reaction (a) of chlorides (2.3.1).
TESTS
solution Y
7
(Method II, 2.2.2).
Acidity or alkalinity Dilute 2 ml of solution S to 10 ml with carbon dioxide-free water R. Add 0.1 ml
of methyl red solution R and 0.2 ml of 0.01M sodium hydroxide. The solution is yellow. Add 0.4 ml of
0.01M hydrochloric acid. The solution is red.
Test solution. Dissolve 0.10 g of the substance to be examined in 2 ml of water R. Add 2 ml of a
200 g/l solution of sodium hydroxide R and 2 ml of chloroform R. Shake for 10 min. Separate the
chloroform layer, dry over anhydrous sodium sulphate R and filter.
a glass column 1.8 m long and 2 mm in internal diameter with a packing prepared as follows:
mix 19.5 g of silanised diatomaceous earth for gas chromatography R with 60 ml of a 3.3 g/l solution
of potassium hydroxide R in methanol R and evaporate the solvent under reduced pressure while
rotating the mixture slowly (support); dissolve 0.4 g of low-vapour-pressure hydrocarbons (type
L) R in 60 ml of toluene R (dissolution requires up to 5 h), add this solution to the support and
evaporate the solvent under reduced pressure while rotating the mixture slowly,
nitrogen for chromatography R as the carrier gas at a flow rate of 30 ml per minute,
2-12
a flame-ionisation detector.
Programme the temperature of the column linearly at a rate of 6C per minute from 100C to
200C. Maintain the temperature of the injection port at 220C and that of the detector at 300C.
Inject 1 l or the chosen volume of the test solution. Continue the chromatography for a period at
least 2.5 times the retention time of the principal peak. In the chromatogram the sum of the area of
any peaks apart from that corresponding to amantadine does not exceed 1 per cent of the total area
of the peaks; no peak, apart from that corresponding to amantadine, has an area exceeding 0.3 per
cent of the total area. Disregard the peak corresponding to the solvent during the evaluation.
Water (2.5.12). Not more than 0.5 per cent, determined on 2.000 g by the semi-micro
ASSAY
Dissolve 0.150 g in a mixture of 5.0 ml of 0.01M hydrochloric acid and 50 ml of alcohol R. Carry out a
10
H
18
ClN.
_______________________________________________________________________ Ph Eur
2-13
Ambroxol Hydrochloride
1/01
,HCl
N
H
r
Br
NH
2
OH
C
13
H
18
Br
2
N
2
O,HCl 414.6 23828-92-4
Ambroxol Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Expectorant.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
trans-4-[(2-Amino-3,5-dibromobenzyl)amino]cyclohexanol hydrochloride.
CHARACTERS
Appearance: white or yellowish crystalline powder.
Solubility: sparingly soluble in water, soluble in methanol, practically insoluble in methylene chloride.
IDENTIFICATION
A. Dissolve 20.0 mg in 0.05M sulphuric acid and dilute to 100.0 ml with the same acid. Dilute 2.0 ml
of the solution to 10.0 ml with 0.05M sulphuric acid. Examined between 200 nm and 350 nm (2.2.25),
the solution shows two absorption maxima at 245 nm and 310 nm. The ratio of the absorbance
measured at 245 nm to that measured at 310 nm is 3.2 to 3.4.
Comparison: ambroxol hydrochloride CRS.
C. Examine by thin-layer chromatography (2.2.27).
the same solvent.
Reference solution. Dissolve 50 mg of ambroxol hydrochloride CRS in methanol R and dilute to 5 ml with
the same solvent.
Plate: TLC silica gel F
254
plate R.
Mobile phase: concentrated ammonia R, 1-propanol R, ethyl acetate R, hexane R (1:10:20:70 V/V/V/V).
Application: 10 l.
Drying: in air.
Detection: examine in ultraviolet light at 254 nm.
Results: the principal spot in the chromatogram obtained with the test solution is similar in position
and size to the principal spot in the chromatogram obtained with the reference solution.
D.Dissolve 25 mg in 2.5 ml of water R, mix with 1.0 ml of dilute ammonia R1 and allow to stand for
5 min. Filter and acidify the filtrate with dilute nitric acid R. The filtrate gives reaction (a) of chlorides
(2.3.1).
TESTS
Solution S Dissolve 0.75 g in methanol R and dilute to 15 ml with the same solvent.
solution Y
6
(Method II, 2.2.2).
pH (2.2.3): 4.5 to 6.0.
Dissolve 0.2 g in carbon dioxide-free water R and dilute to 20 ml with the same solvent.
2-14
Test solution. Dissolve 50.0 mg of the substance to be examined in water R and dilute to 50.0 ml with
the same solvent.
Reference solution (a). Dilute 5.0 ml of the test solution to 250.0 ml with water R. Dilute 1.0 ml of this
solution to 20.0 ml with the mobile phase.
Reference solution (b). Dissolve 5 mg of the substance to be examined in 0.2 ml of methanol R and add
0.04 ml of a mixture of 1 volume of formaldehyde solution R and 99 volumes of water R. Heat at 60C
for 5 min. Evaporate to dryness under a current of nitrogen. Dissolve the residue in 5 ml of water R
and dilute to 20 ml with the mobile phase.
Column:
size: l = 0.25 m, = 4.0 mm,
stationary phase: octadecylsilyl silica gel for chromatography R (5 m).
Mobile phase: a mixture of equal volumes of acetonitrile R and a solution prepared as follows: dissolve
1.32 g of ammonium phosphate R in 900 ml of water R, adjust to pH 7.0 with phosphoric acid R and
dilute to 1000 ml with water R.
Injection: 20 l.
Sensitivity: reference solution (a).
Run time: 3 times the retention time of the principal peak in the chromatogram obtained with the test
solution.
System suitability:
resolution: minimum of 4.0 between the peaks due to impurity B and ambroxol in the chromato-
gram obtained with reference solution (b).
Limits:
any impurity: not more than the area of the principal peak in the chromatogram obtained with
reference solution (a) (0.1 per cent),
total: not more than 3 times the area of the principal peak in the chromatogram obtained with
1.0 g complies with limit test C. Prepare the standard using 2 ml of lead standard solution (10 ppm
Pb) R.
100-105C.
Sulphated ash (2.4.14): maximum 0.1 per cent, determined on 1.0 g.
ASSAY
Dissolve 0.300 g in 70 ml of alcohol R and add 5 ml of 0.01M hydrochloric acid. Carry out a potentio-
metric titration (2.2.20), using 0.1M sodium hydroxide. Read the volume added between the two
points of inflexion.
13
H
19
Br
2
ClN
2
O.
STORAGE
IMPURITIES
r- =
Br
Br
NH
2
A. Ar-CH
2
OH: (2-amino-3,5-dibromophenyl)methanol,
N
N
H
r
Br
OH
B. trans-4-(6,8-dibromo-1,4-dihydroquinazolin-3(2H)-yl)cyclohexanol,
2-15
OH
N Ar
C. trans-4-[[(E)-2-amino-3,5-dibromobenzyliden]amino]cyclohexanol,
OH
N Ar
D. cis-4-[(2-amino-3,5-dibromobenzyl)amino]cyclohexanol,
E. Ar-CH = O: 2-amino-3,5-dibromobenzaldehyde.
__________________________________________________________________________________________________________ Ph Eur
2-16
Amfetamine Sulphate
NH
2
Me H
2
,H
2
SO
4
and enantiomer
C
18
H
28
N
2
O
4
S 368.5 60-13-9
Amfetamine Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Amphetamine Sulphate [0368]. These requirements are reproduced after the heading Definition below.
Action and use Central nervous system stimulant.
When amphetamine sulphate is prescribed or demanded, Amfetamine Sulphate shall be dispensed or
supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Amphetamine sulphate contains not less than 99.0 per cent and not more than the equivalent of
100.5 per cent of bis[(RS)-1-methyl-2-phenylethylamine] sulphate, calculated with reference to the
dried substance.
CHARACTERS
A white powder, freely soluble in water, slightly soluble in alcohol, practically insoluble in ether.
IDENTIFICATION
First identification: A, B, E.
A. The angle of optical rotation (2.2.7) of solution S (see Tests), measured in a 2 dm tube, is 0.04
to +0.04.
B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph. Eur. reference
spectrum of amphetamine sulphate. Examine as a mull in liquid paraffin R.
C. To 50 ml of solution S add 5 ml of strong sodium hydroxide solution R and 0.5 ml of benzoyl
chloride R and shake. Continue to add benzoyl chloride R in portions of 0.5 ml until no further
precipitate is formed. Filter, wash the precipitate with water R and recrystallise twice from a mixture
of equal volumes of alcohol R and water R and dry at 100C to 105C. The crystals melt (2.2.14) at
131C to 135C.
D. To about 2 mg add 1 ml of sulphuric acid-formaldehyde reagent R. An orange colour develops and
quickly becomes dark-brown.
E. Solution S gives reaction (a) of sulphates (2.3.1).
TESTS
Appearance of solution Solution S is clear (2.2.1) and colourless (Method II, 2.2.2)
Acidity or alkalinity To 25 ml of solution S add 0.1 ml of methyl red solution R. Not more than
0.1 ml of 0.01M hydrochloric acid or 0.01M sodium hydroxide is required to change the colour of the
indicator.
100C to 105C.
ASSAY
18
H
28
N
2
O
4
S.
STORAGE
_______________________________________________________________________ Ph Eur
2-17
Amidotrizoic Acid Dihydrate
COOH
I I
I
NHAc AcHN
,2H2O
C
11
H
9
I
3
N
2
O
4
,2H
2
O 650 50978-11-5
Amidotrizoic Acid Dihydrate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Radio-opaque substance.
Preparation
Meglumine Amidotrizoate Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Amidotrizoic acid dihydrate contains not less than 98.5 per cent and not more than the equivalent
of 101.0 per cent of 3,5-di(acetylamino)-2,4,6-tri-iodobenzoic acid, calculated with reference to
CHARACTERS
A white or almost white, crystalline powder, very slightly soluble in water and in alcohol, practically
insoluble in ether. It dissolves in dilute solutions of alkali hydroxides.
IDENTIFICATION
obtained with amidotrizoic acid dihydrate CRS.
B. Examine the chromatograms obtained in the test for related substances (see Tests). The principal
spot in the chromatogram obtained with test solution (b) is similar in position and size to the
principal spot in the chromatogram obtained with reference solution (b).
C. Heat 50 mg gently in a small porcelain dish over a naked flame. Violet vapour is evolved.
TESTS
Appearance of solution Dissolve 1.0 g in dilute sodium hydroxide solution R and dilute to 20 ml with
the same solvent. The solution is clear (2.2.1) and colourless (Method II, 2.2.2).
254
R as the
coating substance.
Test solution (a). Dissolve 0.50 g of the substance to be examined in a 3 per cent V/V solution of
ammonia R in methanol R and dilute to 10 ml with the same solvent.
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with a 3 per cent V/V solution of ammonia R
in methanol R.
Reference solution (a). Dilute 1 ml of test solution (b) to 50 ml with a 3 per cent V/V solution of
ammonia R in methanol R.
Reference solution (b). Dissolve 50 mg of amidotrizoic acid dihydrate CRS in a 3 per cent V/V solution
of ammonia R in methanol R and dilute to 10 ml with the same solvent.
20 volumes of anhydrous formic acid R, 25 volumes of methyl ethyl ketone R and 60 volumes of
toluene R. Allow the plate to dry until the solvents have evaporated and examine in ultraviolet light at
254 nm. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot,
is not more intense than the spot in the chromatogram obtained with reference solution (a) (0.2 per
cent).
Halides Dissolve 0.55 g in a mixture of 4 ml of dilute sodium hydroxide solution R and 15 ml of
water R. Add 6 ml of dilute nitric acid R and filter. 15 ml of the filtrate complies with the limit test for
chlorides (2.4.4) (150 ppm expressed as chloride).
2-18
Free aromatic amines Maintain the solutions and reagents in iced water protected from bright light. To
0.50 g in a 50 ml volumetric flask add 15 ml of water R. Shake and add 1 ml of dilute sodium hydroxide
solution R. Cool in iced water, add 5 ml of a freshly prepared 5 g/l solution of sodium nitrite R and
12 ml of dilute hydrochloric acid R. Shake gently and allow to stand for exactly 2 min after adding the
hydrochloric acid. Add 10 ml of a 20 g/l solution of ammonium sulphamate R. Allow to stand for
5 min, shaking frequently, and add 0.15 ml of a 100 g/l solution of -naphthol R in alcohol R. Shake
and allow to stand for 5 min. Add 3.5 ml of buffer solution pH 10.9 R, mix and dilute to 50.0 ml with
water R. The absorbance (2.2.25), measured within 20 min at 485 nm using as the compensation
liquid a solution prepared at the same time and in the same manner but omitting the substance to be
examined, is not greater than 0.30.
Heavy metals (2.4.8). Dissolve 2.0 g in 4 ml of dilute sodium hydroxide solution R and dilute to 20 ml
with water R. 12 ml of this solution complies with limit test A for heavy metals (20 ppm). Prepare the
standard using lead standard solution (2 ppm Pb) R.
Loss on drying (2.2.32). 4.5 per cent to 7.0 per cent, determined on 0.500 g by drying in an oven at
100C to 105C.
ASSAY
To 0.150 g in a 250 ml round-bottomed flask add 5 ml of strong sodium hydroxide solution R, 20 ml of
water R, 1 g of zinc powder R and a few glass beads. Boil under a reflux condenser for 30 min. Allow
to cool and rinse the condenser with 20 ml of water R, adding the rinsings to the flask. Filter through
a sintered-glass filter and wash the filter with several quantities of water R. Collect the filtrate and
washings. Add 40 ml of dilute sulphuric acid R and titrate immediately with 0.1M silver nitrate. Deter-
mine the end-point potentiometrically (2.2.20), using a suitable electrode system such as silver-
mercurous sulphate.
1 ml of 0.1M silver nitrate is equivalent to 20.47 mg of C
11
H
9
I
3
N
2
O
4
.
STORAGE
IMPURITIES
COOH
I I
I
NH
2
AcHN
A. 5-(acetylamino)-3-amino-2,4,6-tri-iodobenzoic acid.
__________________________________________________________________________________________________________ Ph Eur
2-19
Amikacin
N
NH
2
O
H OH
H
2
N
HO
HO
HO
HO
O
O
HO
H
2
N
O
HO
H
2
N
OH
O
H
C
22
H
43
N
5
O
13
585.6 37517-28-5
Amikacin complies with the requirements of the 3rd edition of the European Pharmacopoeia [1289]. These
Action and use Antibiotic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Amikacin is 6-O-(3-amino-3-deoxy--D-glucopyranosyl)-4-O-(6-amino-6-deoxy--D-
glucopyranosyl)-N
1
-[(2S)-4-amino-2-hydroxybutanoyl]-2-deoxy-D-streptamine, an antimicrobial
substance obtained from kanamycin A. It contains not less than 96.5 per cent and not more than the
equivalent of 102.5 per cent of C
22
H
43
N
5
O
13,
calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white powder, sparingly soluble in water, slightly soluble in methanol, practically
insoluble in acetone and in alcohol.
IDENTIFICATION
A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum of
amikacin CRS.
B. Examine by thin-layer chromatography (2.2.27) using a TLC silica gel plate R.
same solvent.
Reference solution (a). Dissolve 25 mg of amikacin CRS in water R and dilute to 10 ml with the same
solvent.
Reference solution (b). Dissolve 5 mg of kanamycin monosulphate CRS in 1 ml of the test solution and
Apply to the plate 5 l of each solution. Develop over a path of 15 cm using the lower layer of a
mixture of equal volumes of concentrated ammonia R, methanol R and methylene chloride R. Allow the
plate to dry in air and spray with ninhydrin solution R1. Heat at 110C for 5 min. The principal spot
in the chromatogram obtained with the test solution is similar in position, colour and size to the
principal spot in the chromatogram obtained with the reference solution (a). The identification test is
not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated
spots.
TESTS
Specific optical rotation (2.2.7). Dissolve 0.50 g in water R and dilute to 25.0 ml with the same
solvent. The specific optical rotation is +97 to +105, calculated with reference to the anhydrous
substance.
Inject 20 l of reference solution (c). The test is not valid unless, in the chromatogram obtained, the
resolution between the peaks corresponding to amikacin and impurity A is at least 3.5. Inject 20 l of
2-20
test solution (a). Continue the chromatography for four times the retention time of amikacin. In
the chromatogram obtained with test solution (a): the area of any peak corresponding to impurity
A is not greater than the area of the principal peak in the chromatogram obtained with reference
solution (a) (1 per cent); the area of any peak, apart from the principal peak and any peak corres-
ponding to impurity A, is not greater than half the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.5 per cent); the sum of the areas of any such peaks is not
greater than 1.5 times the area of the principal peak in the chromatogram obtained with reference
solution (a) (1.5 per cent). Disregard any peak due to the blank and any peak with an area less than
0.1 times that of the principal peak in the chromatogram obtained with reference solution (a).
mination of water.
ASSAY
Test solution (a). Dissolve 0.100 g of the substance to be examined in water R and dilute to 10.0 ml
with the same solvent. In a round-glass-stoppered vial, add 0.2 ml of this solution to 2.0 ml of a
10 g/l solution of 2,4,6-trinitrobenzene sulphonic acid R. Then add 3.0 ml of pyridine R and close the
vial tightly. Shake vigorously for 30 s and heat in a water-bath at 75C for 45 min. Cool in cold water
for 2 min and add 2 ml of glacial acetic acid R. Shake vigorously for 30 s.
Test solution (b). Dissolve 50.0 mg of the substance to be examined in water R and dilute to 50.0 ml
with the same solvent. Then prepare as prescribed for test solution (a).
Reference solution (a). Dissolve 10.0 mg of amikacin impurity A CRS in water R and dilute to 100.0 ml
Reference solution (b). Dissolve 50.0 mg of amikacin CRS in water R and dilute to 50.0 ml with the
same solvent. Then prepare as prescribed for test solution (a).
Reference solution (c). Dissolve 5 mg of amikacin CRS and 5 mg of amikacin impurity A CRS in water R
and dilute to 50 ml with the same solvent. Then prepare as prescribed for test solution (a).
Blank solution. Prepare as described for test solution (a) using 0.2 ml of water R.
as mobile phase at a flow rate of 1 ml/min a mixture of 30 volumes of a 2.7 g/l solution of
potassium dihydrogen phosphate R adjusted to pH 6.5 with a 22 g/l solution of potassium
hydroxide R and 70 volumes of methanol R,
maintaining the temperature of the column at 30C and that of the solutions to be examined at 10C.
principal peak is at least 50 per cent of the full scale of the recorder. Inject reference solution (b) six
times. The assay is not valid unless the relative standard deviation of the peak area for amikacin is
2.0 per cent. Inject test solution (b) and reference solution (b).
IMPURITIES
NHR
1
R
3
HN
HO
HO
HO
HO
O
O
HO
R
2
HN
O
HO
R4HN
OH
O
NH
2
O
H OH
acyl =
A. R
1
= R
3
= R
4
= H, R
2
= acyl : 4-O-(3-amino-3-deoxy--D-glucopyranosyl)-6-O-(6-amino-6-
deoxy--D-glucopyranosyl)-N
1
-[(2S)4-amino-2-hydroxybutanoyl]-2-deoxy-L-streptamine,
B. R
1
= R
2
= acyl, R
3
= R
4
= H : 4-O-(3-amino-3-deoxy--D-glucopyranosyl)-6-O-(6-amino-6-
1
,N
3
-bis[(2S)-4-amino-2-hydroxybutanoyl]-2-deoxy-L-
streptamine,
2-21
C. R
1
= R
2
= R
3
= H, R
4
= acyl : 4-O-(6-amino-6-deoxy--D-glucopyranosyl)-6-O-[3-[[(2S)-4-
amino-2-hydroxybutanoyl]-amino]-3-deoxy--D glucopyranosyl]-2-deoxy-D-streptamine,
D. R
1
= R
2
= R
3
= R
4
= H : 4-O-(3-amino-3-deoxy--D-glucopyranosyl)-6-O-(6-amino-6-deoxy-
-D-glucopyranosyl)-2-deoxy-L-streptamine (kanamycin).
The following type chromatogram is given for information and guidance only; it does not form a mandatory
part of the monograph.
0.00 20.00 40.00
0.01800
0.01600
0.01400
0.01200
0.01000
0.00800
0.00600
0.00400
0.00200
0.00000
-0.00200
-0.00400
Channel Descr.: PDA 340.0 nm
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__________________________________________________________________________________________________________ Ph Eur
2-22
Amikacin Sulphate
N
NH
2
O
H OH
H
2
N
HO
HO
HO
HO
O
O
HO
H
2
N
O
HO
H
2
N
OH
O
H
,2H2SO4
(C
22
H
43
N
5
O
13
),2H
2
SO
4
782 39831-55-5
Amikacin Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1290].
Action and use Antibiotic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Amikacin sulphate is 6-O-(3-amino-3-deoxy--D-glucopyranosyl)-4-O-(6-amino-6-deoxy--D-
glucopyranosyl)-N
1
-[(2S)-4-amino-2-hydroxybutanoyl]-2-deoxy-D-streptamine sulphate, an
antimicrobial substance obtained from kanamycin A. It contains not less than 72.3 per cent and not
more than the equivalent of 76.8 per cent of C
22
H
43
N
5
O
13
substance.
CHARACTERS
A white or almost white powder, freely soluble in water, practically insoluble in acetone and in
alcohol.
IDENTIFICATION
obtained with amikacin sulphate CRS.
B. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R.
same solvent.
Reference solution (a). Dissolve 25 mg of amikacin sulphate CRS in water R and dilute to 10 ml with
the same solvent.
Reference solution (b). Dissolve 5 mg of kanamycin monosulphate CRS in 1 ml of the test solution and
mixture of equal volumes of concentrated ammonia R, methanol R and methylene chloride R. Allow the
plate to dry in air and spray with ninhydrin solution R1. Heat at 110C for 5 min. The principal spot
principal spot in the chromatogram obtained with the reference solution (a). The identification test is
spots.
C. It gives reaction (a) of sulphates (2.3.1).
TESTS
solvent. The specific optical rotation is +76 to +84, calculated with reference to the dried
substance.
2-23
Inject 20 l of reference solution (c). The test is not valid unless in the chromatogram obtained, the
resolution between the peaks corresponding to amikacin and impurity A is at least 3.5. Inject 20 l of
test solution (a). Continue the chromatography for four times the retention time of amikacin. In the
chromatogram obtained with test solution (a): the area of any peak corresponding to impurity A is
not greater than the area of the principal peak in the chromatogram obtained with reference solution
(a) (1 per cent); the area of any peak, apart from the principal peak and any peak corresponding to
impurity A, is not greater than half the area of the principal peak in the chromatogram obtained with
reference solution (a) (0.5 per cent); the sum of the areas of any such peaks is not greater than 1.5
times the area of the principal peak in the chromatogram obtained with reference solution (a) (1.5
per cent). Disregard any peak due to the blank and any peak with an area less than 0.1 times that of
the principal peak in the chromatogram obtained with reference solution (a).
Sulphates 23.3 per cent to 25.8 per cent of sulphate (SO
4
), calculated with reference to the dried
substance. Dissolve 0.250 g in 100 ml of water R and adjust the solution to pH 11 using concentrated
ammonia R. Add 10.0 ml of 0.1M barium chloride and about 0.5 mg of phthalein purple R. Titrate with
0.1M sodium edetate adding 50 ml of alcohol R when the colour of the solution begins to change and
continue the titration until the violet-blue colour disappears.
1 ml of 0.1M barium chloride is equivalent to 9.606 mg of sulphate (SO
4
).
at 100C to 105C at a pressure not exceeding 0.7 kPa for 3 h.
Inject per kilogram of the rabbits mass 5 ml of a solution containing 25 mg of the substance to be
examined in water for injections R.
ASSAY
with the same solvent. In a round-glass-stoppered vial, add 0.2 ml of this solution to 2.0 ml of a
10 g/l solution of 2,4,6-trinitrobenzene sulphonic acid R. Then add 3.0 ml of pyridine R and close the
vial tightly. Shake vigorously for 30 s and heat on a water-bath at 75C for 45 min. Cool in cold
water for 2 min and add 2 ml of glacial acetic acid R. Shake vigorously for 30 s.
Test solution (b). Dissolve 50.0 mg of the substance to be examined in water R and dilute to 50.0 ml
Reference solution (a). Dissolve 10.0 mg of amikacin impurity A CRS in water R and dilute to 100.0 ml
Reference solution (b). Dissolve 50.0 mg of amikacin sulphate CRS in water R and dilute to 50.0 ml
Reference solution (c). Dissolve 5 mg of amikacin sulphate CRS and 5 mg of amikacin impurity A CRS
in water R and dilute to 50 ml with the same solvent. Then prepare as prescribed for test solution (a).
Blank solution. Prepare as described for test solution (a) using 0.2 ml of water R.
potassium dihydrogen phosphate R adjusted to pH 6.5 with a 22 g/l solution of potassium hydroxide
R and 70 volumes of methanol R,
maintaining the temperature of the column at 30C and that of the solutions to be examined at 10C.
principal peak is at least 50 per cent of the full scale of the recorder. Inject reference solution (b) six
times. The assay is not valid unless the relative standard deviation of the peak area for amikacin is
2.0 per cent. Inject test solution (b) and reference solution (b).
STORAGE
If the substance is sterile, store in a sterile, airtight, tamper-proof container.
LABELLING
The label states:
2-24
IMPURITIES
NHR
1
R
3
HN
HO
HO
HO
HO
O
O
HO
R
2
HN
O
HO
R4HN
OH
O
NH
2
O
H OH
acyl =
A. R
1
= R
3
= R
4
= H, R
2
= acyl : 4-O-(3-amino-3-deoxy--D-glucopyranosyl)-6-O-(6-amino-6-
1
-[(2S)4-amino-2-hydroxybutanoyl]-2-deoxy-L-streptamine,
B. R
1
= R
2
= acyl, R
3
= R
4
= H : 4-O-(3-amino-3-deoxy--D-glucopyranosyl)-6-O-(6-amino-6-
1
,N
3
-bis[(2S)-4-amino-2-hydroxybutanoyl]-2-deoxy-L-
streptamine,
C. R
1
= R
2
= R
3
= H, R
4
= acyl : 4-O-(6-amino-6-deoxy--D-glucopyranosyl)-6-O-[3-[[(2S)-4-
amino-2-hydroxybutanoyl]-amino]-3-deoxy--D glucopyranosyl]-2-deoxy-D-streptamine,
D. R
1
= R
2
= R
3
= R
4
= H : 4-O-(3-amino-3-deoxy--D-glucopyranosyl)-6-O-(6-amino-6-deoxy-
-D-glucopyranosyl)-2-deoxy-L-streptamine (kanamycin).
0.00 20.00 40.00
0.01800
0.01600
0.01400
0.01200
0.01000
0.00800
0.00600
0.00400
0.00200
0.00000
-0.00200
-0.00400
Channel Descr.: PDA 340.0 nm
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__________________________________________________________________________________________________________ Ph Eur
2-25
Amiloride Hydrochloride
1/01
,HCl H
NH
2
N NH
2
O NH
N
N
Cl
H
2
N
C
6
H
9
Cl
2
N
7
O,2H
2
O 302.1 17440-83-4
Amiloride Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Diuretic.
Preparations
Amiloride Tablets
Co-amilofruse Tablets
Co-amilozide Oral Solution
Co-amilozide Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Amiloride hydrochloride contains not less than 98.0 per cent and not more than the equivalent of
101.0 per cent of N-amidino-3,5-diamino-6-chloropyrazine-2-carboxamide hydrochloride, calculated
with reference to the anhydrous substance.
CHARACTERS
A pale-yellow to greenish-yellow powder, slightly soluble in water and in ethanol.
IDENTIFICATION
obtained with amiloride hydrochloride CRS.
B. Examine by thin-layer chromatography (2.2.27), using a suitable silica gel as the coating
substance.
the same solvent.
Reference solution. Dissolve 40 mg of amiloride hydrochloride CRS in methanol R and dilute to 10 ml
Apply to the plate 5 l of each solution. Develop over a path of 12 cm using a freshly prepared
mixture of 6 volumes of dilute ammonia R1, 6 volumes of water R and 88 volumes of dioxan R. Allow
the plate to dry in air and examine in ultraviolet light at 365 nm. The principal spot in the chromato-
gram obtained with the test solution is similar in position, fluorescence and size to the principal spot
in the chromatogram obtained with the reference solution.
C. Dissolve about 10 mg in 10 ml of water R. Add 10 ml of a 200 g/l solution of cetrimide R, 0.25 ml
of dilute sodium hydroxide solution R and 1 ml of bromine water R. A greenish-yellow colour is
produced. Add 2 ml of dilute hydrochloric acid R. The solution becomes deep yellow and shows blue
fluorescence in ultraviolet light at 365 nm.
D. It gives reaction (b) of chlorides (2.3.1).
TESTS
Free acid Dissolve 1.0 g in a mixture of 50 ml of methanol R and 50 ml of water R and titrate with
0.1M sodium hydroxide, determining the end-point potentiometrically (2.2.20). Not more than 0.3 ml
of 0.1M sodium hydroxide is required to reach the end-point.
Test solution. Dissolve 20.0 mg of the substance to be examined in a mixture of 1 volume of aceto-
Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with a mixture of 1 volume of
2-26
acetonitrile R and 3 volumes of water R.
Reference solution (b). Dilute 1.0 ml of reference solution (a) to 10.0 ml with a mixture of 1 volume
of acetonitrile R and 3 volumes of water R.
Reference solution (c). Dissolve 20.0 mg of amiloride impurity A CRS in a mixture of 1 volume of
acetonitrile R and 3 volumes of water R and dilute to 20.0 ml with the same mixture of solvents.
Dilute 1.0 ml of the solution to 100.0 ml with a mixture of 1 volume of acetonitrile R and 3 volumes
of water R.
as mobile phase at a flow rate of 1 ml/min a mixture of 5 volumes of tetramethylammonium
hydroxide solution R, 250 volumes of acetonitrile R and 745 volumes of water R; adjust to pH 7.0
using a mixture of 1 volume of phosphoric acid R and 9 volumes of water R,
Inject 20 l of reference solution (c). Adjust the concentration of acetonitrile in the mobile phase so
that the retention time of impurity A is 5 min to 6 min (an increase in the concentration of aceto-
nitrile results in a shorter retention time). Inject 20 l of reference solution (a). Adjust the concentra-
tion of tetramethylammonium hydroxide and of phosphoric acid keeping the pH at 7.0 so that the
retention time of amiloride is 9 min to 12 min (an increase in the concentration results in a shorter
retention time for amiloride). Inject 20 l of reference solution (b). The test is not valid unless the
signal-to-noise ratio for the peak due to amiloride is at least 5.0.
Inject 20 l of the test solution and 20 l of reference solution (c). Record the chromatograms for
five times the retention time of amiloride. In the chromatogram obtained with the test solution, the
sum of the areas of the peaks, apart from the peak corresponding to amiloride, is not greater than the
area of the peak corresponding to impurity A in the chromatogram obtained with reference solution
(c) (0.5 per cent). Disregard any peak with an area less than 10 per cent of the area of the peak
corresponding to impurity A in the chromatogram obtained with reference solution (c).
Water (2.5.12).11.0 per cent to 13.0 per cent, determined on 0.200 g by the semi-micro deter-
mination of water.
ASSAY
6
H
9
Cl
2
N
7
O.
STORAGE
IMPURITIES
N
N
NH
2 2
N
Cl COOMe
A. methyl 3,5-diamino-6-chloropyrazine-2-carboxylate.
_______________________________________________________________________ Ph Eur
2-27
Aminobenzoic Acid
COOH
H
2
N
C
7
H
7
NO
2
137.1 150-13-0
Definition Aminobenzoic Acid is 4-aminobenzoic acid. It contains not less than 98.5% and not
more than 100.5% of C
7
H
7
NO
2
Characteristics White or slightly yellow crystals or crystalline powder, gradually darkening on
exposure to air and light; odourless or almost odourless.
Slightly soluble in water; freely soluble in ethanol (96%). It dissolves in solutions of alkali
hydroxides and carbonates.
Identification
aminobenzoic acid (RS 007).
B. The light absorption, Appendix II B, in the range 230 to 350 nm of a 0.0005% w/v solution in
ethanol (96%) exhibits a maximum at 280 nm; the absorbance at the maximum is about 0.48. A
0.0005% w/v solution in 0.1M sodium hydroxide exhibits a maximum at 265 nm; the absorbance at
the maximum is about 0.53.
C. Dissolve 0.1 g in 2 ml of 2M hydrochloric acid, heating if necessary. Cool in ice, add 4 ml of a
freshly prepared 1% w/v solution of sodium nitrite and pour the mixture into 2 ml of 2-naphthol
solution containing 1 g of sodium acetate. A bright red precipitate is produced.
Iron Ignite 0.25 g with 0.25 g of anhydrous sodium carbonate, dissolve the residue in 4 ml of 2M
hydrochloric acid and dilute to 10 ml with water. The solution complies with the limit test for iron,
Appendix VII (40 ppm).
Aromatic amines Carry out the method for thin-layer chromatography, Appendix III A, using
silica gel G as the coating substance and a mixture of 90 volumes of toluene, 5 volumes of ethyl
acetate and 5 volumes of glacial acetic acid as the mobile phase. Apply separately to the plate 10 l of
each of the following solutions. For solution (1) dissolve 2.0 g of the substance being examined in
10 ml of 2M sodium hydroxide and extract with two 10-ml quantities of 1,2-dichloroethane. Wash the
combined dichloroethane extracts with 5 ml of 2M sodium hydroxide followed by 5 ml of water,
shake with anhydrous sodium sulphate and filter. Evaporate to a low volume using a rotary evaporator
and dilute to 2 ml with 1,2-dichloroethane. Solution (2) contains 0.0020% w/v of aniline in
1,2-dichloroethane. Solution (3) contains 0.0020% w/v of ethyl 4-aminobenzoate in 1,2-dichloroethane.
After removal of the plate, dry it in a current of warm air and spray with dimethylaminobenzaldehyde
solution R1. Any spots corresponding to aniline and ethyl 4-aminobenzoate in the chromatogram
obtained with solution (1) are not more intense than the spots in the chromatograms obtained with
solutions (2) and (3) respectively.
4-Nitrobenzoic acid Carry out the method for thin-layer chromatography, Appendix III A, using
cellulose F
254
as the coating substance and the upper layer obtained by mixing 70 volumes of
2-methylpropan-1-ol, 30 volumes of water and 15 volumes of 5M ammonia as the mobile phase.
Apply separately to the plate 5 l of each of two solutions in ethanol (96%) containing (1) 10% w/v
of the substance being examined and (2) 0.020% w/v of 4-nitrobenzoic acid. After removal of the
plate, allow it to dry in a current of air and examine under ultraviolet light (254 nm). Any spot
corresponding to 4-nitrobenzoic acid in the chromatogram obtained with solution (1) is not more
intense than the spot in the chromatogram obtained with solution (2).
Assay Dissolve 0.3 g in 60 ml of ethanol (50%) and titrate with 0.1M sodium hydroxide VS using
phenolphthalein solution R1 as indicator. Each ml of 0.1M sodium hydroxide VS is equivalent to
13.71 mg of C
7
H
7
NO
2
.
Storage Aminobenzoic Acid should be kept in a well-closed container and protected from light.
Action and use Skin protective.
2-28
Aminocaproic Acid
H
2
N COOH
C
6
H
13
NO
2
131.2 60-32-2
Aminocaproic Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia [0874].
Action and use Antifibrinolytic; haemostatic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Aminocaproic acid contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of 6-aminohexanoic acid, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder or colourless crystals, freely soluble in water, slightly soluble in alcohol,
It melts at about 205C with decomposition.
IDENTIFICATION
obtained with aminocaproic acid CRS. Examine the substances prepared as discs.
B. Examine the chromatograms obtained in the test for ninhydrin-positive substances. The principal
spot in the chromatogram obtained with the test solution (b) is similar in position, colour and size to
the principal spot in the chromatogram obtained with reference solution (a).
C. Dissolve 0.5 g in 4 ml of a mixture of equal volumes of dilute hydrochloric acid R and water R.
Evaporate to dryness by heating on a water-bath. Dry the residue in a desiccator. Dissolve the residue
in about 2 ml of boiling ethanol R. Allow to cool and maintain at 4C to 8C for 3 h. Filter under
reduced pressure. The residue washed with about 10 ml of acetone R and dried at 60C for 30 min,
melts (2.2.14) at 131C to 133C.
D. Dissolve about 5 mg in 0.5 ml of distilled water R. Add 3 ml of dimethylformamide R and 2 ml of
ascorbic acid solution R. Heat on a water-bath. An orange colour develops.
TESTS
Solution S Dissolve 10.0 g in carbon dioxide-free water R and dilute to 50.0 ml with the same solvent.
Appearance of solution Solution S is colourless (Method II, 2.2.2) and remains clear (2.2.1) on
standing for 24 h.
pH (2.2.3). The pH of solution S is 7.5 to 8.0.
Absorbance (2.2.25).
A. The absorbance of solution S at 287 nm is not more than 0.10 and at 450 nm is not more than
0.03.
B. Place 2.0 g in an even layer in a shallow dish 9 cm in diameter, cover and allow to stand at 98C
to 102C for 72 h. Dissolve in water R and dilute to 10.0 ml with the same solvent. The absorbance
of the solution at 287 nm is not more than 0.15 and at 450 nm is not more than 0.03.
Ninhydrin-positive substances Examine by thin-layer chromatography (2.2.27), using a suitable
silica gel as the coating substance.
Test solution (a). Dissolve 0.10 g of the substance to be examined in water R and dilute to 10 ml with
the same solvent.
Reference solution (a). Dissolve 10 mg of aminocaproic acid CRS in water R and dilute to 50 ml with
the same solvent.
Reference solution (b). Dilute 5 ml of Test solution (b) to 20 ml with water R.
Reference solution (c). Dissolve 10 mg of aminocaproic acid CRS and 10 mg of leucine CRS in water R
2-29
of 15 cm using a mixture of 20 volumes of glacial acetic acid R, 20 volumes of water R and 60
volumes of butanol R. Dry the plate in a current of warm air. Spray with ninhydrin solution R and heat
at 100C to 105C for 15 min. Any spot in the chromatogram obtained with the test solution (a),
apart from the principal spot, is not more intense than the spot in the chromatogram obtained with
reference solution (b) (0.5 per cent). The test is not valid unless the chromatogram obtained with
reference solution (c) shows two clearly separated principal spots.
100C to 105C.
ASSAY
Dissolve 0.100 g in 20 ml of anhydrous acetic acid R. Using 0.1 ml of crystal violet solution R as
indicator, titrate with 0.1M perchloric acid until the colour changes from bluish-violet to bluish-green.
6
H
13
NO
2
.
STORAGE
_______________________________________________________________________ Ph Eur
2-30
Aminoglutethimide
corrected 1/01
HN
NH
2
Et
O
O
and enanti omer
C
13
H
16
N
2
O
2
232.3 125-84-8
Aminoglutethimide complies with the requirements of the 3rd edition of the European Pharmacopoeia [1291].
Action and use Inhibitor of adrenal corticosteroid synthesis.
Preparation
Aminoglutethimide Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Aminoglutethimide contains not less than 98.0 per cent and not more than the equivalent of
101.5 per cent of (3RS)-3-(4-aminophenyl)-3-ethylpiperidin-2,6-dione, calculated with reference to
CHARACTERS
A white or slightly yellow, crystalline powder, practically insoluble in water, freely soluble in acetone,
soluble in methanol.
IDENTIFICATION
A. Melting point (2.2.14). 150C to 154C.
obtained with aminoglutethimide CRS. Examine the substances prepared as discs.
C. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable silica gel
with a fluorescent indicator having an optimal intensity at 254 nm.
Test solution. Dissolve 25 mg of the substance to be examined in acetone R and dilute to 5 ml with the
same solvent.
Reference solution (a). Dissolve 25 mg of aminoglutethimide CRS in acetone R and dilute to 5 ml with
the same solvent.
Reference solution (b). Dissolve 25 mg of aminoglutethimide CRS and 25 mg of glutethimide CRS in
acetone R and dilute to 5 ml with the same solvent.
Apply to the plate 5 l of each solution. Develop over a path of 15 cm using a mixture of 0.5 volumes
of glacial acetic acid R, 15 volumes of methanol R and 85 volumes of ethyl acetate R. Allow the plate to
dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram
gram obtained with reference solution (a). The identification is not valid unless the chromatogram
TESTS
Solution S Dissolve 1.0 g in methanol R and dilute to 20.0 ml with the same solvent.
solution Y
7
(Method II, 2.2.2).
+0.10.
3-Aminoglutethimide and other related substances Examine by liquid chromatography
(2.2.29).
2-31
Test solution. Dissolve 0.10 g of the substance to be examined in a mixture of equal volumes of
methanol R and acetate buffer solution pH 5.0 R and dilute to 50.0 ml with the same mixture of
solvents.
Reference solution (a). Dissolve 5.0 mg of aminoglutethimide impurity A CRS in a mixture of equal
volumes of methanol R and acetate buffer solution pH 5.0 R and dilute to 25.0 ml with the same
Reference solution (b). Dilute 1.0 ml of reference solution (a) to 10.0 ml with a mixture of equal
volumes of methanol R and acetate buffer solution pH 5.0 R.
Reference solution (c). Dilute 1.0 ml of the test solution to 100.0 ml with a mixture of equal volumes
of methanol R and acetate buffer solution pH 5.0 R.
Reference solution (d). Dilute 1.0 ml of the test solution to 10.0 ml with reference solution (a).
volumes of acetate buffer solution pH 5.0 R,
Inject 10 l of reference solution (d). When the chromatograms are recorded in the prescribed
conditions, the retention times are: aminoglutethimide about 9 min and impurity A about 12 min.
obtained with reference solution (d) is at least 60 per cent of the full scale of the recorder. The test is
not valid unless in the chromatogram obtained with reference solution (d), the resolution between the
peaks corresponding to aminoglutethimide and impurity A is at least 2.0.
Inject 10 l of the test solution, 10 l of reference solution (b) and 10 l of reference solution (c).
Continue the chromatography of the test solution for four times the retention time of the principal
peak. In the chromatogram obtained with the test solution: the area of any peak due to impurity A is
not greater than twice the area of the principal peak in the chromatogram obtained with reference
solution (b) (2 per cent); the sum of the areas of any peaks apart from the principal peak and the
peak due to impurity A is not greater than the area of the principal peak in the chromatogram
obtained with reference solution (c) (1 per cent); the sum of the contents of all the impurities is not
greater than 2.0 per cent. Disregard any peak with an area less than 0.05 times that of the principal
peak in the chromatogram obtained with reference solution (c).
Azo-glutethimide Not more than 300 ppm, determined by liquid chromatography (2.2.29).
Carry out the test protected from light. Use shaking, not sonication or heat, to dissolve the reference substance
and the substance to be examined.
Test solution. Dissolve 0.100 g of the substance to be examined in dimethyl sulphoxide R and dilute to
Reference solution. Dissolve 3.0 mg of aminoglutethimide impurity D CRS in dimethyl sulphoxide R and
dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of this solution to 100.0 ml with dimethyl
sulphoxide R.
as mobile phase at a flow of 1.0 ml/min a mixture prepared as follows: dissolve 0.285 g of sodium
edetate R in water R, add 7.5 ml of dilute acetic acid R and 50 ml of 0.1M potassium hydroxide and
dilute to 1000 ml with water R; adjust to pH 5.0 with glacial acetic acid R; mix 350 ml of this
solution with 650 ml of methanol R,
Inject 10 l of each solution. The test is not valid unless: in the chromatogram obtained with the
test solution the number of theoretical plates calculated for the principal peak is at least 3300; the
mass distribution ratio of the principal peak is 2.0 to 5.0 and the symmetry factor of the principal
peak is less than 1.2. The area of the peak due to impurity D in the chromatogram obtained with the
test solution is not greater than that of the principal peak in the chromatogram obtained with the
reference solution.
Heavy metals (2.4.8). Dissolve 2.0 g in 15 ml of acetone R and dilute to 20 ml with water R. 12 ml
of the solution complies with limit test B for heavy metals (10 ppm). Prepare the standard using lead
standard solution (1 ppm Pb) obtained by diluting lead standard solution (100 ppm Pb) R with a
mixture of 15 ml of acetone R and 5 ml of water R.
100C to 105C.
2-32
ASSAY
Dissolve 0.180 g in 50 ml of anhydrous acetic acid R and titrate with 0.1M perchloric acid, determining
the end-point potentiometrically (2.2.20).
13
H
16
N
2
O
2
.
IMPURITIES
HN
R
2
Et
O
R
1
and enantiomer
A. R
1
= NH
2
, R
2
= H: (3RS)-3-(3-aminophenyl)-3-ethylpiperidine-2,6-dione
(3-aminoglutethimide),
B. R
1
= NO
2
, R
2
= H: (3RS)-3-ethyl-3-(3-nitrophenyl)piperidine-2,6-dione,
C. R
1
= H, R
2
= NO
2:
(3RS)-3-ethyl-3-(4-nitrophenyl)piperidine-2,6-dione,
O
O
Et
N
HN
N
O
O
Et
NH
D. 3,3-[diazenediyldi(4,1-phenylene)]bis(3-ethylpiperidine-2,6-dione) (azoglutethimide).
__________________________________________________________________________________________________________ Ph Eur
2-33
Aminophylline
N
N
N
O
O
Me
MeN
H
2
NH
2
NH
2
C
16
H
24
N
10
O
4
420.4 317-34-0
Aminophylline complies with the requirements of the 3rd edition of the European Pharmacopoeia for
TheophyllineEthylenediamine [0300]. These requirements are reproduced after the heading Definition
below.
Action and use Xanthine bronchodilator.
Preparations
Aminophylline Injection
Aminophylline Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Theophylline-ethylenediamine contains not less than 84.0 per cent and not more than the equivalent
of 87.4 per cent of theophylline (C
7
H
8
N
4
O
2
; M
r
180.2) and not less than 13.5 per cent and not more
than the equivalent of 15.0 per cent of ethylenediamine (C
2
H
8
N
2
; M
r
60.1), both calculated with
reference to the anhydrous substance.
CHARACTERS
A white or slightly yellowish powder, sometimes granular, freely soluble in water (the solution
becomes cloudy through absorption of carbon dioxide), practically insoluble in ethanol.
IDENTIFICATION
First identification: B, C, E.
Second identification: A, C, D, E, F.
Dissolve 1.0 g in 10 ml of water R and add 2 ml of dilute hydrochloric acid R dropwise with shaking.
Filter. Use the precipitate for identification tests A, B, D and F and the filtrate for identification test
C.
A. The precipitate, washed with water R and dried at 100C to 105C, melts (2.2.14) at 270C to
274C.
B. Examine the precipitate, washed with water R and dried at 100C to 105C, by infrared
absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with theophylline
CRS.
C. To the filtrate add 0.2 ml of benzoyl chloride R, make alkaline with dilute sodium hydroxide solution R
and shake vigorously. Filter the precipitate, wash with 10 ml of water R, dissolve in 5 ml of hot
alcohol R and add 5 ml of water R. A precipitate is formed, which when washed and dried at 100C to
105C, melts (2.2.14) at 248C to 252C.
D. Heat about 10 mg of the precipitate with 1.0 ml of a 360 g/l solution of potassium hydroxide R in a
water-bath at 90C for 3 min, then add 1.0 ml of diazotised sulphanilic acid solution R. A red colour
slowly develops. Carry out a blank test.
E. It complies with the test for water (see Tests).
F. The precipitate gives the reaction of xanthines (2.3.1).
TESTS
Appearance of solution Dissolve 0.5 g with gentle warming in 10 ml of carbon dioxide-free water R.
The solution is not more opalescent than reference suspension II (2.2.1) and not more intensely
coloured than reference solution GY
6
(Method II, 2.2.2).
Related substances Examine by thin-layer chromatography (2.2.27), using as the coating substance
a suitable silica gel with a fluorescent indicator having an optimal intensity at 254 nm.
2-34
Test solution. Dissolve 0.2 g of the substance to be examined in 2 ml of water R with heating and
dilute to 10 ml with methanol R.
Reference solution. Dilute 0.5 ml of the test solution to 100 ml with methanol R.
10 volumes of concentrated ammonia R, 30 volumes of acetone R, 30 volumes of chloroform R and 40
volumes of butanol R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any
spot in the chromatogram obtained with the test solution, apart from the principal spot, is not more
intense than the spot in the chromatogram obtained with the reference solution (0.5 per cent).
Water (2.5.12). Not more than 1.5 per cent, determined on 2.00 g dissolved in 20 ml of anhydrous
pyridine R, by the semi-micro determination of water.
ASSAY
Ethylenediamine Dissolve 0.250 g in 30 ml of water R. Add 0.1 ml of bromocresol green solution R.
Titrate with 0.1M hydrochloric acid until a green colour is obtained.
1 ml of 0.1M hydrochloric acid is equivalent to 3.005 mg of C
2
H
8
N
2
.
Theophylline Heat 0.200 g to constant mass in an oven at 135C. Dissolve the residue with heating
in 100 ml of water R, allow to cool, add 20 ml of 0.1M silver nitrate and shake. Add 1 ml of
bromothymol blue solution R1. Titrate with 0.1M sodium hydroxide.
7
H
8
N
4
O
2
.
STORAGE
Store in airtight container, protected from light.
_______________________________________________________________________ Ph Eur
2-35
Aminophylline Hydrate
5877-66-5
Aminophylline complies with the requirements of the 3rd edition of the European Pharmacopoeia for
TheophyllineEthylenediamine Hydrate [0301]. These requirements are reproduced after the heading
Definition below.
Preparations
Aminophylline Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Theophylline and ethylenediamine hydrate contains not less than 84.0 per cent and not more than
the equivalent of 87.4 per cent of theophylline (C
7
H
8
N
4
O
2
; M
r
180.2) and not less than 13.5 per
cent and not more than the equivalent of 15.0 per cent of ethylenediamine (C
2
H
8
N
2
; M
r
60.1), both
CHARACTERS
It has the characters described in the monograph on Theophylline-Ethylenediamine (300).
IDENTIFICATION
It complies with the identification tests prescribed in the monograph on Theophylline-Ethylenediamine
(300) except for identification test E which is replaced by the following:
E. It contains 3.0 per cent to 8.0 per cent of water (see Tests).
TESTS
It complies with the tests prescribed in the monograph on Theophylline-Ethylenediamine (300) with the
following modification.
Water (2.5.12). 3.0 per cent to 8.0 per cent, determined on 0.50 g dissolved in 20 ml of pyridine R,
by the semi-micro determination of water.
ASSAY
Ethylenediamine Carry out the assay prescribed in the monograph on Theophylline-Ethylenediamine
(300).
2
H
8
N
2
.
Theophylline Carry out the assay prescribed in the monograph on Theophylline-Ethylenediamine
(300).
7
H
8
N
4
O
2
.
STORAGE
Store in a well-filled airtight container, protected from light.
_______________________________________________________________________ Ph Eur
2-36
Amiodarone Hydrochloride
O
O
O
NEt
2
I
I
Bu
n
,HCl
C
25
H
30
ClI
2
NO
3
682 199774-82-4
Amiodarone Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Antiarrhythmic.
Preparations
Amiodarone Intravenous Infusion
Amiodarone Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Amiodarone hydrochloride contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of 2-butyl-3-benzofuranyl 4-(2-diethylaminoethoxy)-3,5-di-iodophenyl ketone hydro-
CHARACTERS
A white or almost white, fine crystalline powder, very slightly soluble in water, freely soluble in
methylene chloride, soluble in methanol, sparingly soluble in alcohol, very slightly soluble in hexane.
IDENTIFICATION
First identification: A, B, D.
obtained with amiodarone hydrochloride CRS. Examine the substances prepared as discs.
254 nm. The principal spot in the chromatogram obtained with test solution (b) is similar in position
and size to the principal spot in the chromatogram obtained with reference solution (a).
TESTS
Appearance of solution Dissolve 1.0 g in methanol R and dilute to 20 ml with the same solvent.
The solution is clear (2.2.1) and not more intensely coloured than reference solution GY
5
(Method II,
2.2.2).
pH (2.2.3). Dissolve 1.0 g in carbon dioxide-free water R, heating at 80C, cool and dilute to 20 ml
with the same solvent. The pH of the solution is 3.2 to 3.8.
254
R as the
coating substance.
Prepare the solutions immediately before use and keep protected from bright light.
Test solution (a). Dissolve 0.50 g of the substance to be examined in methylene chloride R and dilute to
Test solution (b). Dilute 1 ml of test solution (a) to 20 ml with methylene chloride R.
Reference solution (a). Dissolve 25 mg of amiodarone hydrochloride CRS in methylene chloride R and
dilute to 5 ml with the same solvent.
Reference solution (b). Dilute 1 ml of Test solution (b) to 10 ml with methylene chloride R.
Reference solution (c). Dilute 5 ml of reference solution (b) to 10 ml with methylene chloride R.
Reference solution (d). Dissolve 10 mg of (2-chloroethyl)di-ethylamine hydrochloride R in methylene
chloride R and dilute to 50 ml with the same solvent.
2-37
5 volumes of anhydrous formic acid R, 10 volumes of methanol R and 85 volumes of methylene
chloride R. Dry the plate in a current of cold air until the odour of the solvents is no
longer perceptible and examine in ultraviolet light at 254 nm. Any spot in the chromatogram
obtained with test solution (a), apart from the principal spot, is not more intense than the spot in
the chromatogram obtained with reference solution (b) (0.5 per cent) and not more than one such
spot is more intense than the spot in the chromatogram obtained with reference solution (c)
(0.25 per cent). Spray with potassium iodobismuthate solution R1 and then with dilute hydrogen peroxide
solution R. Examine immediately in daylight. Any spot corresponding to (2-chloroethyl)diethylamine
in the chromatogram obtained with test solution (a) is not more intense than the spot in the
chromatogram obtained with reference solution (d) (0.2 per cent).
Iodides Prepare the test and reference solutions simultaneously. Add 1.50 g to 40 ml of water R at 80C
and shake until completely dissolved. Cool and dilute to 50.0 ml with water R (solution (a)).
Test solution. To 15.0 ml of solution (a) add 1.0 ml of 0.1M hydrochloric acid and 1.0 ml of 0.05M
potassium iodate. Dilute to 20.0 ml with water R. Allow to stand protected from light for 4 h.
Reference solution. To 15.0 ml of solution (a) add 1.0 ml of 0.1M hydrochloric acid, 1.0 ml of an
88.2 ppm solution of potassium iodide R and 1.0 ml of 0.05M potassium iodate. Dilute to 20.0 ml with
water R. Allow to stand protected from light for 4 h.
Measure the absorbances (2.2.25) of the solutions at 420 nm, using as the compensation liquid a
mixture of 15.0 ml of solution (a) and 1.0 ml of 0.1M hydrochloric acid diluted to 20.0 ml with
water R. The absorbance of the test solution is not greater than half the absorbance of the reference
solution (150 ppm).
Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying at 50C at a
pressure not exceeding 0.3 kPa for 4 h.
ASSAY
25
H
30
ClI
2
NO
3
.
STORAGE
Store in well-closed container, protected from light, at a temperature not exceeding 30C.
_______________________________________________________________________ Ph Eur
2-38
Amisulpride
1/01
O O
S
N
H
OMe
3
C
H
2
N
O
N
H
Et
and enantiomer
C
17
H
27
N
3
O
4
S 369.5 71675-85-9
Amisulpride complies with the requirements of the 3rd edition of the European Pharmacopoeia [1490]. These
Action and use Dopamine D
2
/D
3
-receptor antagonist; treatment of schizophrenia.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
4-Amino-N-[[(2RS)-1-ethylpyrrolidin-2-yl]methyl]-5-(ethylsulphonyl)-2-methoxybenzamide.
CHARACTERS
Appearance: white or almost white, crystalline powder.
Solubility: practically insoluble in water, freely soluble in methylene chloride, sparingly soluble in
ethanol.
mp: about 126C.
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison: amisulpride CRS.
TESTS
Appearance of solution The solution is not more opalescent than reference suspension II (2.2.1)
and not more intensely coloured than reference solution Y
6
(2.2.2, Method II).
Dissolve 1.0 g in 3 ml of a mixture of 1 volume of acetic acid R and 4 volumes of water R and dilute to
20 ml with water R.
Optical rotation (2.2.7): 0.10 to +0.10.
Dissolve 5.0 g in dimethylformamide R and dilute to 50.0 ml with the same solvent.
Impurity A Thin-layer chromatography (2.2.27).
Test solution. Dissolve 0.20 g in methanol R and dilute to 10 ml with the same solvent.
Reference solution (a). Dissolve 20 mg of amisulpride impurity A CRS in methanol R and dilute to
100 ml with the same solvent. Dilute 2 ml of the solution to 20 ml with methanol R.
Reference solution (b). Dilute 1 ml of the test solution to 10 ml with methanol R. Dilute 1 ml of the
solution to 10 ml with reference solution (a).
Plate: TLC silica gel G plate R.
Mobile phase: the upper layer obtained after shaking a mixture of a 50 per cent V/V solution of
concentrated ammonia R, ethanol R and di-isopropyl ether R (10:25:65 V/V/V).
Application: 10 l.
Development: over a path of 12 cm.
Drying: in air.
Detection: spray with ninhydrin solution R and heat at 100-105C for 15 min.
System suitability: the test is not valid unless the chromatogram obtained with reference solution (b)
shows 2 clearly separated spots.
Limits:
impurity A: any spot corresponding to impurity A in the chromatogram obtained with the test
solution is not more intense than the spot in the chromatogram obtained with reference solution
(a) (0.1 per cent).
2-39
Test solution. Dissolve 0.10 g in 30 ml of methanol R and dilute to 100.0 ml with mobile phase B.
mobile phase A and 70 volumes of mobile phase B. Dilute 1.0 ml of the solution to 25.0 ml with a
mixture of 30 volumes of mobile phase A and 70 volumes of mobile phase B.
Reference solution (b). Dissolve 5 mg of amisulpride impurity B CRS in 5 ml of the test solution and
dilute to 50 ml with a mixture of 30 volumes of mobile phase A and 70 volumes of mobile phase B.
Dilute 1 ml of the solution to 10 ml with a mixture of 30 volumes of mobile phase A and 70 volumes
of mobile phase B.
Column:
size: l = 0.25 m, = 4.6 mm,
stationary phase: octylsilyl silica gel for chromatography R (5 m) with a carbon loading of 16 per
cent, a specific surface area of 330 m
2
/g and a pore size of 7.5 nm.
Mobile phase:
mobile phase A: methanol R,
mobile phase B: a 0.7 g/l solution of sodium octanesulphonate R in a 0.25 per cent V/V solution of
dilute sulphuric acid R.
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
0 18 30 36 70 64
18 35 36 52 64 48
35 45 52 48
45 46 52 30 48 70
46 56 30 70
Flow rate: 1.5 ml/min.
Injection: 10 l loop injector; inject the test solution and reference solutions (a) and (b).
System suitability:
resolution: minimum of 2.0 between the peaks due to amisulpride and to impurity B in the
Limits:
any impurity: not more than 0.5 times the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.1 per cent),
total: not more than 1.5 times the area of the principal peak in the chromatogram obtained with
reference solution (a) (0.02 per cent).
Chlorides (2.4.4): maximum 200 ppm.
Shake 0.5 g with 30 ml of water R for 10 min. Filter. 15 ml of the filtrate complies with the limit test
for chlorides.
Dissolve 4.0 g by gently heating in 5 ml of dilute acetic acid R. Allow to cool and dilute to 20 ml with
water R. 12 ml of the solution complies with limit test A. Prepare the standard using lead standard
100-105C for 3 h.
ASSAY
Dissolve 0.300 g with shaking in a mixture of 5 ml of acetic anhydride R and 50 ml of anhydrous acetic
acid R. Titrate with 0.1M perchloric acid, determining the end-point potentiometrically (2.2.20).
17
H
27
N
3
O
4
S.
IMPURITIES
and enantiomer
N
H
Et
2
N
A. [(2RS)-1-ethylpyrrolidin-2-yl]methanamine,
2-40
and enantiomer
N
H
R
1 2
N
O
N
H
Et
R
2
B. R
1
= OH, R
2
= SO
2
-CH
2
-CH
3
: 4-amino-N-[[(2RS)-1-ethylpyrrolidin-2-yl]methyl]-5-
(ethylsulphonyl)-2-hydroxybenzamide,
C. R
1
= OCH
3
, R
2
= I: 4-amino-N-[[(2RS)-1-ethylpyrrolidin-2-yl]methyl]-5-iodo-2-
methoxybenzamide,
D. R
1
= OCH
3
, R
2
= SO
2
-CH
3
: 4-amino-N-[[(2RS)-1-ethylpyrrolidin-2-yl]methyl]-2-methoxy-5-
(methylsulphonyl)benzamide,
O O
S
3
C
H
2
N
COOH
OMe
E. 4-amino-5-(ethylsulphonyl)-2-methoxybenzoic acid.
__________________________________________________________________________________________________________ Ph Eur
2-41
Amitriptyline Embonate
2
CH
2
COOH
COOH
OH
OH
,
NMe
2
(C
20
H
23
N)
2
,C
23
H
16
O
6
943.2 17086-03-2
Definition Amitriptyline Embonate is 3-(10,11-dihydro-5H-dibenzo[a,d]cyclohept-5-
ylidene)propyldimethylamine 4,4-methylenebis(3-hydroxy-2-naphthoate). It contains not less than
98.5% and not more than 101.0% of (C
20
H
23
N)
2
,C
23
H
16
O
6
, calculated with reference to the
Characteristics A pale yellow to brownish yellow powder; odourless or almost odourless.
Practically insoluble in water; freely soluble in chloroform; slightly soluble in ethanol (96%).
Identification
A. Dissolve 40 mg in 100 ml of methanol. To 1 ml of the solution add 1 ml of a 2.5% w/v solution
of sodium hydrogen carbonate, 1 ml of a 2.0% w/v solution of sodium periodate and 1 ml of a 0.3% w/v
solution of potassium permanganate, shake and allow to stand for 15 minutes. Acidify with 1M
sulphuric acid, extract with 10 ml of 2,2,4-trimethylpentane and filter. The light absorption of the
filtrate, Appendix II B, in the range 230 to 350 nm exhibits a maximum only at 265 nm.
B. Dissolve 0.2 g in 10 ml of dichloromethane, add 5 ml of 1.25M sodium hydroxide and shake. The
aqueous layer exhibits a green fluorescence when examined under ultraviolet light (365 nm).
Chloride Not more than 0.2% when determined by the following method. Dissolve 1 g in a
mixture of 50 ml of acetone and 50 ml of water, add 2 ml of nitric acid and 75 ml of acetate buffer pH
5.0 and titrate with 0.01M silver nitrate VS determining the end point potentiometrically. Each ml of
0.01M silver nitrate VS is equivalent to 0.3545 mg of Cl.
Related substances Carry out the method for thin-layer chromatography, Appendix III A, protected
from light, using silica gel G as the coating substance and a mixture of 3 volumes of diethylamine, 15
volumes of ethyl acetate and 85 volumes of cyclohexane as the mobile phase but allowing the solvent
front to ascend 14 cm above the line of application in an unlined tank. Apply separately to the plate
10 l of each of three solutions in chloroform containing (1) 3.3% w/v of the substance being
examined, (2) 0.0010% w/v of dibenzosuberone EPCRS and (3) 0.0040% w/v of cyclobenzaprine hydro-
chloride EPCRS. After removal of the plate, allow it to dry in air, spray with a freshly prepared
mixture of 4 volumes of formaldehyde and 96 volumes of sulphuric acid, heat at 100 to 105 for 10
minutes and examine under ultraviolet light (365 nm). Any spot corresponding to dibenzosuberone in
the chromatogram obtained with solution (1) is not more intense than the spot in the chromatogram
obtained with solution (2) (0.05%, with reference to amitriptyline). Examine the plate under
ultraviolet light (254 nm). Any other secondary spot in the chromatogram obtained with solution (1) is
not more intense than the spot in the chromatogram obtained with solution (3) (0.2%, with reference
to amitriptyline).
Water Not more than 5.0% w/w, Appendix IX. C Use 0.5 g.
Assay Dissolve 0.6 g in 50 ml of acetic anhydride and carry out Method I for non-aqueous titration,
Appendix VIII A, using 1-naphtholbenzein solution as indicator. Each ml of 0.1M perchloric acid VS is
equivalent to 47.16 mg of (C
20
H
23
N)
2
,C
23
H
16
O
6
.
Storage Amitriptyline Embonate should be protected from light.
Action and use Antidepressant.
IMPURITIES
O
A. dibenzosuberone
2-42
NMe
2
B. cyclobenzaprine.
2-43
Amitriptyline Hydrochloride
,HCl
NMe
2
C
20
H
24
ClN 313.9 549-18-8
Amitriptyline Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Amitriptyline Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Amitriptyline hydrochloride contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of 3-(10,11-dihydro-5H-dibenzo-[a,d]-cyclohepten-5-ylidene)-N,N-dimethylpropan-
1-amine hydrochloride, calculated with reference to the dried substance.
CHARACTERS
A white or almost white powder or colourless crystals, freely soluble in water, in alcohol and in
methylene chloride.
IDENTIFICATION
First identification: C, E.
B. Dissolve 25.0 mg in methanol R and dilute to 100.0 ml with the same solvent. Dilute 5.0 ml of the
C. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph. Eur. reference
spectrum of amitriptyline hydrochloride.
D. Dissolve 0.1 g in 10 ml of dilute sulphuric acid R. Add 2 ml of a saturated solution of potassium
permanganate R. The violet colour of the solution disappears quickly. Heat on a water-bath until the
brown precipitate is almost completely dissolved. Cool, shake with 15 ml of ether R to remove the
white turbidity and discard the ether layer. To the aqueous layer add 5 ml of concentrated ammonia R
and shake for 2 min. Add 3 ml of methylene chloride R and shake again. A violet-red colour is
produced in the lower layer.
E. 50 mg gives reaction (b) of chlorides (2.3.1).
TESTS
solution is clear (2.2.1) and not more intensely coloured than reference solution B
7
(Method II,
2.2.2).
Acidity or alkalinity Dissolve 0.20 g in carbon dioxide-free water R and dilute to 10 ml with the same
solvent. Add 0.1 ml of methyl red solution R and 0.2 ml of 0.01M sodium hydroxide. The solution is
yellow. Add 0.4 ml of 0.01M hydrochloric acid. The solution is red.
Related substances Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R as
the coating substance.
Prepare the solutions in subdued light and develop the chromatograms protected from light.
Test solution. Dissolve 0.20 g of the substance to be examined in alcohol R and dilute to 10 ml with
the same solvent.
Reference solution (a). Dissolve 10 mg of dibenzosuberone CRS in alcohol R and dilute to 10 ml with the
same solvent. Dilute 1 ml of the solution to 100 ml with alcohol R.
2-44
Reference solution (b). Dissolve 10 mg of cyclobenzaprine hydrochloride CRS in alcohol R and dilute to
10 ml with the same solvent. Dilute 2 ml of the solution to 50 ml with alcohol R.
Apply separately to the plate 5 l of each solution. Develop in an unsaturated tank over a path of
14 cm using a mixture of 3 volumes of diethylamine R, 15 volumes of ethyl acetate R and 85 volumes
of cyclohexane R. Allow the plate to dry in air and spray with a freshly prepared mixture of 4 volumes
of formaldehyde solution R and 96 volumes of sulphuric acid R. Heat at 100C to 105C for 10 min and
examine in ultraviolet light at 365 nm. In the chromatogram obtained with the test solution: any
spots corresponding to dibenzosuberone and cyclobenzaprine hydrochloride are not more intense
than the spots in the chromatograms obtained with reference solutions (a) (0.05 per cent) and (b)
(0.2 per cent), respectively; any spot, apart from the principal spot and any spots corresponding to
dibenzosuberone and cyclobenzaprine hydrochloride, is not more intense than the spot in the
chromatogram obtained with reference solution (b) (0.2 per cent).
Heavy metals (2.4.8). 1.0 g complies with limit test F for heavy metals (20 ppm). Prepare the
100C to 105C for 2 h.
ASSAY
Dissolve 0.250 g in 30 ml of alcohol R. Titrate with 0.1M sodium hydroxide, determining the end-point
20
H
24
ClN.
STORAGE
IMPURITIES
O
A. dibenzosuberone,
NMe
2
B. cyclobenzaprine,
NHMe
C. 3-(10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-ylidene)-N-methylpropylamine,
NMe
2
HO
D. 5-[3-(dimethylamino)propyl]-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-ol,
NMe
2
E. 1,2,3,4,4a,10,11,11a-octahydro-3-(5H-dibenzo[a,d]cyclohepten-5-ylidene)-N,N-
dimethylpropylamine,
2-45
NMe
2
OH
H
and enantiomer
F. (RS)-5-[3-(dimethylamino)propylidene]-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-10-
ol.
_______________________________________________________________________ Ph Eur
2-46
Amlodipine Besilate
1/01
H
N
eOOC
H
Cl
O
NH
2
COOEt
Me
SO
3
H
,
and enantiomer
C
20
H
25
ClN
2
O
5
,C
6
H
6
O
3
S 567.1 111470-99-6
Amlodipine Besilate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1491].
Action and use Calcium antagonist.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Amlodipine besilate contains not less than 97.0 per cent and not more than the equivalent of
102.0 per cent of 3-ethyl 5-methyl (4RS)-2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-6-methyl-
1,4-dihydropyridine-3,5-dicarboxylate benzenesulphonate, calculated with reference to the anhy-
drous substance.
CHARACTERS
A white or almost white powder, slightly soluble in water, freely soluble in methanol, sparingly
soluble in ethanol, slightly soluble in 2-propanol.
IDENTIFICATION
obtained with amlodipine besilate CRS. Examine the substances as mulls.
B. Examine the chromatograms obtained in test A for related substances in ultraviolet light at
366 nm. The principal spot in the chromatogram obtained with test solution (b) is similar in position,
colour and size to the principal spot in the chromatogram obtained with reference solution (a).
C. Dissolve 5.0 mg in a 1 per cent V/V solution of 0.1M hydrochloric acid in methanol R and dilute to
100.0 ml with the same acid solution. Examined between 300 nm and 400 nm the solution shows an
absorption maximum at 360 nm. The specific absorbance at the maximum is 113 to 121.
TESTS
Optical rotation (2.2.7). Dissolve 0.250 g in methanol R and dilute to 25.0 ml with the same
solvent. The angle of optical rotation is 0.10 to +0.10.
Related substances
A. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel F
254
plate R.
Test solution (b). Dilute 1.0 ml of test solution (a) to 10.0 ml with methanol R.
Reference solution (a). Dissolve 7.0 mg of amlodipine besilate CRS in 1.0 ml of methanol R.
Reference solution (b). Dilute 3.0 ml of test solution (b) to 100.0 ml with methanol R.
Reference solution (c). Dilute 1.0 ml of test solution (b) to 100.0 ml with methanol R.
Reference solution (d). Dissolve 1.0 mg of amlodipine impurity C CRS in 5 ml of reference solution (b)
and dilute to 10.0 ml with methanol R.
Apply to the plate 10 l of each solution. Develop over a path of 15 cm using the upper layer of a
mixture of 25 volumes of glacial acetic acid R, 25 volumes of water R and 50 volumes of methyl isobutyl
ketone R. Dry the plate for 15 min at 80C. Examine the plate in ultraviolet light at 254 nm and
366 nm. In the chromatogram obtained with test solution (a) any spot, apart from the principal spot,
2-47
is not more intense than the spot in the chromatogram obtained with reference solution (b)
(0.3 per cent), and at most two spots are more intense than the spot obtained with reference
solution (c) (0.1 per cent). The test is not valid unless the chromatogram obtained with reference
solution (d) shows two clearly separated spots.
B.Examine by liquid chromatography (2.2.29) as described under Assay. Inject test solution (a),
test solution (b) and reference solutions (c), (d) and (e). Continue the chromatography of test
solution (a) for 3 times the retention time of amlodipine besilate. In the chromatogram obtained
with test solution (a): the area of any peak corresponding to impurity D is not greater than the area
of the principal peak in the chromatogram obtained with reference solution (c) (0.3 per cent); the
sum of the areas of all the peaks, apart from the principal peak and the peak corresponding to
amlodipine impurity D, is not greater than the area of the principal peak in the chromatogram
obtained with reference solution (d) (0.3 per cent). Disregard any peak with an area less than 0.1
times the area of the principal peak in the chromatogram obtained with reference solution (d)
(0.03 per cent).
mination of water.
ASSAY
Test solution (a). Dissolve 50.0 mg of the substance to be examined in the mobile phase and dilute to
Test solution (b). Dilute 5.0 ml of test solution (a) to 100.0 ml with the mobile phase.
Reference solution (a). Dissolve 50.0 mg of amlodipine besilate CRS in the mobile phase and dilute to
50.0 ml with the mobile phase. Dilute 5.0 ml of the solution to 100.0 ml with the mobile phase.
Reference solution (b). Dissolve 1.0 mg of amlodipine impurity D CRS in the mobile phase and dilute to
Reference solution (c). Dilute 3.0 ml of reference solution (b) to 100.0 ml with the mobile phase.
Reference solution (d). Dilute 3.0 ml of test solution (a) to 100.0 ml with the mobile phase and dilute
Reference solution (e). Dilute 5 ml of reference solution (b) and 0.5 ml of test solution (a) to 50 ml
with the mobile phase.
as mobile phase at a flow rate of 1.0 ml/min a mixture of 15 volumes of acetonitrile R, 35
volumes of methanol R and 50 volumes of a solution prepared as follows: dissolve 7.0 ml of
triethylamine R in 1 litre of water R and adjust the pH to 3.0 0.1 with phosphoric acid R,
Inject 10 l of test solution (b), 10 l of reference solution (a) and 10 l of reference solution (e).
Adjust the sensitivity of the system so that the heights of the peaks obtained are about 80 per cent of
the full scale of the recorder. The test is not valid unless, in the chromatogram obtained with
reference solution (e), the resolution between the peaks corresponding to amlodipine and impurity D
is at least 4.5. Calculate the content of amlodipine besilate from the areas of the peaks and the
declared content of C
26
H
31
ClN
2
O
8
S in amlodipine besilate CRS.
STORAGE
IMPURITIES
N
H
N
Me
eOOC
Cl
COOEt
O
O
O
H
and enantiomer
A. 3-ethyl 5-methyl (4RS)-4-(2-chlorophenyl)-2-[[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-
yl)ethoxy]methyl]-6-methyl- 1,4-dihydropyridine-3,5-dicarboxylate,
2-48
and enantiomer
H
N
Me
eOOC
Cl
COOEt
O
H
H
N
O
CONHMe
B. 5-ethyl 3-methyl (4RS)-4-(2-chlorophenyl)-2-methyl-6-[[2-[[2-
(methylcarbamoyl)benzoyl]amino]ethoxy]methyl]- 1,4-dihydropyridine-3,5-dicarboxylate,
H
N
MeOOC
Cl
COOEt
O
NH
2
O
2
N
H
C. ethyl methyl (4RS)-2,6-bis-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-1,4-
dihydropyridine-3,5-dicarboxylate,
N Me
eOOC
Cl
COOEt
O
NH
2
D. 3-ethyl 5-methyl 2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-6-methylpyridine-3,5-
dicarboxylate.
__________________________________________________________________________________________________________ Ph Eur
2-49
Strong Ammonia Solution
Strong Ammonia Solution complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Concentrated Ammonia Solution [0877]. These requirements are reproduced after the heading Definition
below.
Preparation
Dilute Ammonia Solution
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Concentrated ammonia solution contains not less than 25.0 per cent m/m and not more than
30.0 per cent m/m of ammonia (NH
3
; M
r
17.03).
CHARACTERS
A clear, colourless liquid, very caustic, miscible with water and with alcohol.
IDENTIFICATION
A. Relative density (2.2.5): 0.892 to 0.910.
B. It is strongly alkaline (2.2.4).
C. To 0.5 ml add 5 ml of water R. Bubble air through the solution and lead the gaseous mixture
obtained over the surface of a solution containing 1 ml of 0.1M hydrochloric acid and 0.05 ml of methyl
red solution R. The colour changes from red to yellow. Add 1 ml of sodium cobaltinitrite solution R. A
yellow precipitate is formed.
TESTS
Solution S Evaporate 220 ml almost to dryness on a water-bath. Cool, add 1 ml of dilute acetic R
and dilute to 20 ml with distilled water R.
(Method II, 2.2.2).
Oxidisable substances Cautiously add, whilst cooling, 8.8 ml to 100 ml of dilute sulphuric acid R.
Add 0.75 ml of 0.002M potassium permanganate. Allow to stand for 5 min. The solution remains
faintly pink.
Pyridine and related substances Measure the absorbance (2.2.25) at 252 nm using water R as the
compensation liquid. The absorbance is not greater than 0.06 (2 ppm calculated as pyridine).
Carbonates To 10 ml in a test-tube with a ground-glass neck add 10 ml of calcium hydroxide
solution R. Stopper immediately and mix. Any opalescence in the solution is not more intense than
that in a standard prepared at the same time and in the same manner using 10 ml of a 0.1 g/l solution
of anhydrous sodium carbonate R (60 ppm).
complies with limit test A for heavy metals (1 ppm). Prepare the standard using lead standard solution
(2 ppm Pb) R.
Iron (2.4.9). Dilute 4 ml of solution S to 10 ml with water R. The solution complies with the limit
test for iron (0.25 ppm).
Residue on evaporation Evaporate 50 ml to dryness on a water-bath and dry at 100C to 105C
for 1 h. The residue weighs not more than 1 mg (0.02 g/l).
ASSAY
Weigh accurately a flask with a ground-glass neck containing 50.0 ml of 1M hydrochloric acid. Add
2 ml of the substance to be examined and re-weigh. Add 0.1 ml of methyl red solution R as indicator.
Titrate with 1M sodium hydroxide until the colour changes from red to yellow.
1 ml of 1M hydrochloric acid is equivalent to 17.03 mg of NH
3
.
STORAGE
Store protected from air, at a temperature not exceeding 20C.
_______________________________________________________________________ Ph Eur
2-50
Ammonium Bicarbonate
NH
4
HCO
3
79.1 1066-33-7
Ammonium Bicarbonate complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Ammonium Hydrogen Carbonate [1390]. These requirements are reproduced after the heading Definition
below.
Preparations
Aromatic Ammonia Solution
Strong Ammonium Acetate Solution
Aromatic Ammonia Spirit
When ammonium carbonate is prescribed or demanded, Ammonium Bicarbonate shall be
dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ammonium hydrogen carbonate contains not less than 98.0 per cent and not more than 101.0 per
cent of the equivalent of ammonium hydrogen carbonate.
CHARACTERS
A fine, white, crystalline powder or white crystals, slightly hygroscopic, freely soluble in water,
It volatilises rapidly at 60C. The volatilisation takes place slowly at ambient temperatures if the
substance is slightly moist. It is in a state of equilibrium with ammonium carbamate.
IDENTIFICATION
A. It gives the reaction of carbonates and bicarbonates (2.3.1).
B. Dissolve 50 mg in 2 ml of water R. The solution gives the reaction of ammonium salts (2.3.1).
TESTS
Solution S Dissolve 14.0 g in 100 ml of distilled water R. Boil to remove the ammonia, allow to cool
and dilute to 100.0 ml with distilled water R.
Iron (2.4.9). Dilute 1.8 ml of solution S to 10 ml with water R. The solution complies with the limit
test for iron (40 ppm).
Heavy metals (2.4.8). Dissolve cautiously 2.5 g in 25 ml of 1M hydrochloric acid. 12 ml of the
solution complies with limit test A for heavy metals (10 ppm). Prepare the standard using lead
ASSAY
Dissolve cautiously 1.0 g in 20.0 ml of 0.5M sulphuric acid and dilute to 50 ml with water R. Boil, cool
and titrate the excess of acid with 1M sodium hydroxide, using 0.1 ml of methyl red solution R as
indicator.
1 ml of 0.5M sulphuric acid is equivalent to 79.1 mg of NH
4
HCO
3
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
2-51
Ammonium Bromide
NH
4
Br 97.9 12124-97-9
Ammonium Bromide complies with the requirements of the 3rd edition of the European Pharmacopoeia [1389].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ammonium bromide contains not less than 98.5 per cent and not more than the equivalent of
100.5 per cent of NH
4
Br, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder or colourless crystals, hygroscopic, freely soluble in water,
sparingly soluble in alcohol.
It becomes yellow when exposed to light or air.
IDENTIFICATION
A. It gives reaction (a) of bromides (2.3.1).
B. 10 ml of solution S (see Tests) gives the reaction of ammonium salts (2.3.1).
TESTS
Solution S Dissolve 10.0 g in carbon dioxide-free water R prepared from distilled water R and dilute to
indicator.
Bromates To 5 ml of solution S add 5 ml of water R, 1 ml of dilute sulphuric acid R, 1 ml of methylene
chloride R and shake vigorously. The lower layer remains colourless (Method I, 2.2.2).
Chlorides Not more than 0.6 per cent. In a conical flask, dissolve 1.000 g in 20 ml of dilute nitric
acid R. Add 5 ml of strong hydrogen peroxide solution R and heat on a water-bath until the solution is
completely decolorised. Rinse the sides of the flask with a little water R and heat on a water-bath for
15 min. Allow to cool, dilute to 50 ml with water R and add 5.0 ml of 0.1M silver nitrate and 1 ml of
dibutyl phthalate R. Shake and titrate with 0.1M ammonium thiocyanate using 5 ml of ferric ammonium
sulphate solution R2 as indicator.
1 ml of 0.1M silver nitrate is equivalent to 3.545 mg of Cl.
Iodides To 5 ml of solution S add 0.15 ml of ferric chloride solution R1 and 2 ml of methylene
chloride R. Shake and allow to separate. The lower layer is colourless (Method I, 2.2.2).
Calcium (2.4.3). 10 ml of solution S diluted to 15 ml with distilled water R complies with the limit
test for calcium (100 ppm).
(20 ppm).
Heavy metals (2.4.8) 12 ml of solution S complies with limit test A for heavy metals (10 ppm).
Prepare the standard using lead standard solution (1ppm Pb) R.
100C to 105C.
ASSAY
Dissolve 1.500 g in water R and dilute to 100.0 ml with the same solvent. To 10.0 ml of the solution
add 50 ml of water R, 5 ml of dilute nitric acid R, 25.0 ml of 0.1M silver nitrate and 2 ml of dibutyl
phthalate R. Shake. Titrate with 0.1M ammonium thiocyanate using 2 ml of ferric ammonium sulphate
solution R2 as indicator and shaking vigorously towards the end-point.
1 ml of 0.1M silver nitrate is equivalent to 9.794 mg of NH
4
Br.
Calculate the percentage content of NH
4
Br from the expression:
a2.763b
a=percentage content of NH
4
Br and NH
4
Cl obtained in the assay and calculated as NH
4
Br,
b =percentage content of Cl obtained in the test for chlorides.
2-52
STORAGE
__________________________________________________________________________________________________________ Ph Eur
2-53
Ammonium Chloride
NH
4
Cl 53.49 12125-02-9
Ammonium Chloride complies with the requirements of the 3rd edition of the European Pharmacopoeia [0007].
Action and use Used for the acidification of urine and to correct metabolic alkalosis.
Preparation
Ammonium Chloride Mixture
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ammonium chloride contains not less than 99.0 per cent and not more than the equivalent of
100.5 per cent of NH
4
Cl, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder or colourless crystals, freely soluble in water.
IDENTIFICATION
A. It gives the reactions of chlorides (2.3.1).
B. It gives the reaction of ammonium salts (2.3.1).
TESTS
indicator.
Bromides and iodides To 10 ml of solution S add 0.1 ml of dilute hydrochloric acid R and 0.05 ml
of chloramine solution R. After 1 min, add 2 ml of chloroform R and shake vigorously. The chloroform
layer remains colourless (Method I, 2.2.2).
Sulphates (2.4.13). 10 ml of solution S diluted to 15 ml with distilled water R complies with the limit
test for sulphates (150 ppm).
Calcium (2.4.3). 5 ml of solution S diluted to 15 ml with distilled water R complies with the limit test
for calcium (200 ppm).
(20 ppm).
100C to 105C for 2 h.
ASSAY
Dissolve 1.000 g in 20 ml of water R and add a mixture of 5 ml of formaldehyde solution R, previously
neutralised to phenolphthalein solution R, and 20 ml of water R. After 1 min to 2 min, titrate slowly
with 1M sodium hydroxide, using a further 0.2 ml of the same indicator.
1 ml of 1M sodium hydroxide is equivalent to 53.49 mg of NH
4
Cl.
_______________________________________________________________________ Ph Eur
2-54
Ammonium Glycyrrhizinate
Me Me
O
H Me
Me
H
Me COO
_
O
Me
H
HO
HO
HO
O
O
O
COO
_
HO
HO
COO
_
NH
4
+
, 2H
+
C
42
H
65
NO
16
840 53956-04-0
Definition Ammonium Glycyrrhizinate is monoammonium 20-carboxy-11-oxo-30-norolean-12-
en-3-D-glucopyranuronosyl--D-glucopyranosiduronate. It contains not less than 98.0% and not
42
H
65
NO
16
, calculated with reference to the dried substance. It exists as a
mixture of the 18- and 18-isomers in which the -isomer predominates.
Characteristics A white or faintly yellow, fine powder.
Slightly soluble in ethanol (50%); sparingly soluble in ethanol (96%); practically insoluble in
dichloromethane. It swells in cold water, the resulting mixture foaming when poured; a clear solution is
produced on warming which reverts to a gel on cooling.
Dissolve 0.50 g of the substance being examined in water at about 60 and add sufficient water at the same
temperature to produce 50 ml. The solution should be maintained at this temperature when used in the
following tests (solution S).
Identification
ammonium glycyrrhizinate (RS 009).
B. Yields the reaction characteristic of ammonium salts and salts of volatile bases, Appendix VI.
Appearance of solution Solution S is clear, Appendix IV A, and not more intensely coloured than
reference solution BY
7
, Appendix IV B, Method II.
Specific optical rotation In a 1% w/v solution in ethanol (50%), +47 to +54, calculated with
reference to the dried substance, Appendix V F.
Heavy metals 2.0 g complies with limit test C for heavy metals, Appendix VII (10 ppm). Use 2 ml of
lead standard solution (10 ppm Pb) to prepare the standard.
Loss on drying When dried to constant weight at a temperature of 100 to 105, loses not more
than 6.0% of its weight. Use 1 g.
Sulphated ash Not more than 0.1%, Appendix IX A, Method II. Use 1 g.
Assay Dissolve 0.6 g in 50 ml of anhydrous acetic acid, warming to about 50 to effect dissolution and
carry out Method I for non-aqueous titration, Appendix VIII A, using 0.2 ml of naphtholbenzein solution
as indicator. Each ml of 0.1M perchloric acid VS is equivalent to 84.0 mg of C
42
H
65
NO
16
.
2-55
Amobarbital
NH
N O O
O
Me
Et
Me
H
C
11
H
18
N
2
O
3
226.3 57-43-2
Amobarbital complies with the requirements of the 3rd edition of the European Pharmacopoeia [0594]. These
Action and use Sedative and hypnotic.
When amylobarbitone is prescribed or demanded, Amobarbital shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Amobarbital contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of 5-ethyl-5-isopentyl-1H,3H,5H-pyrimidine-2,4,6-trione, calculated with reference to the dried
substance.
CHARACTERS
A white, crystalline powder, very slightly soluble in water, freely soluble in alcohol and in ether,
soluble in methylene chloride. It forms water-soluble compounds with alkali hydroxides and
carbonates and with ammonia.
IDENTIFICATION
A. Determine the melting point (2.2.14) of the substance to be examined. Mix equal parts of the
substance to be examined and amobarbital CRS and determine the melting point of the mixture. The
difference between the melting points (which are about 157C) is not greater than 2C.
obtained with amobarbital CRS.
254
the same solvent.
Reference solution. Dissolve 0.1 g of amobarbital CRS in alcohol R and dilute to 100 ml with the same
solvent.
Apply separately to the plate 10 l of each solution. Develop over a path of 18 cm using the lower
layer from a mixture of 5 volumes of concentrated ammonia R, 15 volumes of alcohol R and 80 volumes
of chloroform R. Examine immediately in ultraviolet light at 254 nm. The principal spot in the
the chromatogram obtained with the reference solution.
D. It gives the reaction of non-nitrogen substituted barbiturates (2.3.1).
TESTS
Appearance of solution Dissolve 1.0 g in a mixture of 4 ml of dilute sodium hydroxide solution R and
6 ml of water R. The solution is clear (2.2.1) and not more intensely coloured than reference solution
Y
6
(Method II, 2.2.2).
Acidity or alkalinity To 1.0 g add 50 ml of water R and boil for 2 min. Allow to cool and filter. To
10 ml of the filtrate add 0.15 ml of methyl red solution R and 0.1 ml of 0.01M sodium hydroxide. The
solution is yellow. Add 0.2 ml of 0.01M hydrochloric acid. The solution is red.
254
R as the
coating substance.
the same solvent.
2-56
Reference solution. Dilute 0.5 ml of the Test solution to 100 ml with alcohol R.
layer from a mixture of 5 volumes of concentrated ammonia R, 15 volumes of alcohol R and 80 volumes
of chloroform R. Examine the plate immediately in ultraviolet light at 254 nm. Any spot in the
chromatogram obtained with the test solution, apart from the principal spot, is not more intense than
the spot in the chromatogram obtained with the reference solution. Spray with diphenylcarbazone
mercuric reagent R. Allow the plate to dry in air and spray with freshly prepared alcoholic potassium
hydroxide solution R diluted 1 in 5 with aldehyde-free alcohol R. Heat at 100C to 105C for 5 min and
examine immediately. Any spot in the chromatogram obtained with the test solution, apart from the
principal spot, is not more intense than the spot in the chromatogram obtained with the reference
solution (0.5 per cent).
100C to 105C.
ASSAY
Dissolve 0.100 g in 5 ml of pyridine R. Add 0.5 ml of thymolphthalein solution R and 10 ml of silver
nitrate solution in pyridine R. Titrate with 0.1M ethanolic sodium hydroxide until a pure blue colour is
obtained. Carry out a blank titration.
11
H
18
N
2
O
3
.
_______________________________________________________________________ Ph Eur
2-57
Amobarbital Sodium
N
N O
O
Me
Et
Me
ONa
H
C
11
H
17
N
2
NaO
3
248.3 64-43-7
Amobarbital Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia [0166].
Action and use Sedative and hypnotic.
When amylobarbitone sodium is prescribed or demanded, Amobarbital Sodium shall be dispensed
or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Amobarbital sodium contains not less than 98.5 per cent and not more than the equivalent of
102.0 per cent of the sodium derivative of 5-ethyl-5-isopentyl-1H,3H,5H-pyrimidine-2,4,6-trione,
CHARACTERS
A white, granular powder, hygroscopic, very soluble in carbon dioxide-free water (a small fraction
may be insoluble), freely soluble in alcohol, practically insoluble in ether.
IDENTIFICATION
A. Acidify 10 ml of solution S (see Tests) with dilute hydrochloric acid R and shake with 20 ml of
ether R. Separate the ether layer, wash with 10 ml of water R, dry over anhydrous sodium sulphate R
and filter. Evaporate the filtrate to dryness and dry the residue at 100C to 105C (test
residue). Repeat the operations using 0.1 g of amobarbital sodium CRS (reference residue). Determine
the melting point (2.2.14) of the test residue. Mix equal parts of the test residue and the reference
residue and determine the melting point of the mixture. The difference between the melting points
(which are about 157C) is not greater than 2C.
B. Examine by infrared absorption spectrophotometry (2.2.24), comparing the spectrum obtained
with the reference residue prepared from amobarbital sodium CRS with that obtained with the test
residue (see identification test A).
254
the same solvent.
Reference solution. Dissolve 0.1 g of amobarbital sodium CRS in alcohol R and dilute to 100 ml with the
same solvent.
layer of a mixture of 5 volumes of concentrated ammonia R, 15 volumes of alcohol R and 80 volumes of
chloroform R. Examine immediately in ultraviolet light at 254 nm. The principal spot in the chromato-
gram obtained with the test solution is similar in position and size to the principal spot in the chroma-
togram obtained with the reference solution.
E. It gives reaction (a) of sodium (2.3.1).
TESTS
Solution S Dissolve 5.0 g in alcohol (50 per cent V/V) R and dilute to 50 ml with the same solvent.
solution Y
7
(Method II, 2.2.2).
Disregard any slight residue. The pH of the solution is not more than 11.0.
2-58
254
R as the
coating substance.
the same solvent.
Reference solution. Dilute 0.5 ml of the Test solution to 100 ml with alcohol R.
chloroform R. Examine the plate immediately in ultraviolet light at 254 nm. Spray with
diphenylcarbazone mercuric reagent R. Allow the plate to dry in air and spray with freshly prepared
alcoholic potassium hydroxide solution R diluted 1 in 5 with aldehyde-free alcohol R. Heat at 100C to
105C for 5 min and examine immediately. When examined in ultraviolet light and after spraying,
any spot in the chromatogram obtained with the test solution, apart from the principal spot, is not
more intense than the spot in the chromatogram obtained with the reference solution (0.5 per cent).
Disregard any spot at the starting-point.
130C.
ASSAY
Dissolve 0.200 g in 5 ml of ethanol R. Add 0.5 ml of thymolphthalein solution R and 10 ml of silver
11
H
17
N
2
NaO
3
.
STORAGE
_______________________________________________________________________ Ph Eur
2-59
Amoxicillin Sodium
1/00
N
S
O
H
Me
Me
N
H H
O
NH
2 H
O
COONa
C
16
H
18
N
3
NaO
5
S 387.4 34642-77-8
Amoxicillin Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia [0577].
Action and use Antibacterial.
Preparation
Amoxicillin Injection
When amoxycillin sodium is prescribed or demanded, Amoxicillin Sodium shall be dispensed or
supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Amoxicillin sodium contains not less than 89.0 per cent and not more than the equivalent of
100.5 per cent of sodium (2S,5R,6R)-6-[[(2R)-2-amino-2-(4-hydroxyphenyl)acetyl]amino]-3,3-
dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate, calculated with reference to the
PRODUCTION
If manufactured by a process that may leave residues of 2-ethylhexanoic acid in the product, it
complies with the following test.
2-Ethylhexanoic acid (2.4.28). Not more than 0.8 per cent m/m.
If manufactured by a process that may leave residues of dimethylaniline in the substance and/or by a process
using starting materials or intermediates which contain residues of dimethylaniline, it complies with the
following test.
N,N-Dimethylaniline (2.4.26, Method B). Not more than 20 ppm.
CHARACTERS
A white or almost white powder, very hygroscopic, very soluble in water, sparingly soluble in ethanol,
very slightly soluble in acetone.
IDENTIFICATION
A. Dissolve 0.250 g in 5 ml of water R, add 0.5 ml of dilute acetic acid R, swirl and allow to stand for
10 min in iced water. Filter the crystals and wash with 2 ml to 3 ml of a mixture of 1 volume of
water R and 9 volumes of acetone R, then dry in an oven at 60C for 30 min. Examine by infrared
absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with amoxicillin
trihydrate CRS.
B. Examine by thin-layer chromatography (2.2.27), using a TLC silanised silica gel plate R.
Test solution. Dissolve 25 mg of the substance to be examined in 10 ml of sodium hydrogen carbonate
solution R.
Reference solution (a). Dissolve 25 mg of amoxicillin trihydrate CRS in 10 ml of sodium hydrogen
carbonate solution R.
Reference solution (b). Dissolve 25 mg of amoxicillin trihydrate CRS and 25 mg of ampicillin trihydrate
CRS in 10 ml of sodium hydrogen carbonate solution R.
of acetone R and 90 volumes of a 154 g/l solution of ammonium acetate R the pH of which has been
adjusted to 5.0 with glacial acetic acid R. Allow the plate to dry in air and expose it to iodine vapour
until the spots appear. Examine in daylight. The principal spot in the chromatogram obtained with
the test solution is similar in position, colour and size to the principal spot in the chromatogram
2-60
obtained with reference solution (a). The test is not valid unless the chromatogram obtained with
reference solution (b) shows two clearly separated spots.
C. Place about 2 mg in a test-tube about 150 mm long and 15 mm in diameter. Moisten with
0.05 ml of water R and add 2 ml of sulphuric acid-formaldehyde reagent R. Mix the contents of the tube
by swirling; the solution is practically colourless. Place the test-tube in a water-bath for 1 min; a dark
yellow colour develops.
D. It gives reaction (a) of sodium (2.3.1).
TESTS
Appearance of solution Dissolve 1.0 g in water R and dilute to 10.0 ml with the same solvent.
Immediately after dissolution, the solution is not more opalescent than reference suspension II
(2.2.1). The solution may show an initial, but transient, pink colour. After 5 min, the absorbance
measured at 430 nm (2.2.25) is not greater than 0.20.
Specific optical rotation (2.2.7). Dissolve 62.5 mg in a 4 g/l solution of potassium hydrogen
phthalate R and dilute to 25.0 ml with the same solution. The specific optical rotation is +240 to
+290, calculated with reference to the anhydrous substance.
50 l of reference solution (d) and elute isocratically until elution of the amoxicillin peak. Inject 50 l
of test solution (b) and start the elution isocratically. Immediately after elution of the amoxicillin peak
start the following linear gradient. If the mobile phase composition has been adjusted to achieve the
required resolution, the adjusted composition will apply at time zero in the gradient.
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
025 920 8100
linear gradient
2540 0 100 isocratic
4055 92 8
re-equilibration
Inject mobile phase A and use the same elution pattern to obtain a blank. Inject reference solution
(e). The three principal peaks eluted after the main peak correspond to amoxicillin diketopiperazine,
amoxicillin dimer (impurity J; n = 1) and amoxicillin trimer (impurity J; n = 2) and, compared to the
principal peak they have a relative retention time of about 3.4, 4.1 and 4.5, respectively. In the
chromatogram obtained with test solution (b): the area of any peak corresponding to amoxicillin
dimer is not greater than three times the area of the principal peak in the chromatogram obtained
with reference solution (d) (3 per cent); the area of any peak, apart from the principal peak and any
peak corresponding to amoxicillin dimer, is not greater than twice the area of the principal peak in
the chromatogram obtained with reference solution (d) (2 per cent); the sum of the areas of all the
peaks, apart from the principal peak, is not greater than nine times the area of the principal peak in
the chromatogram obtained with reference solution (d) (9 per cent). Disregard any peak with an area
(d).
mination of water.
Bacterial endotoxins (2.6.14, Method C). If intended for use in the manufacture of parenteral
dosage forms without a further appropriate procedure for the removal of bacterial endotoxins, not
more than 0.25 I.U./mg.
ASSAY
Test solution (a). Dissolve 30.0 mg of the substance to be examined in mobile phase A and dilute to
50.0 ml with the same mobile phase.
Test solution (b). Prepare immediately before use. Dissolve 30.0 mg of the substance to be examined in
mobile phase A and dilute to 20.0 ml with the same mobile phase.
Reference solution (a). Dissolve 30.0 mg of amoxicillin trihydrate CRS in mobile phase A and dilute to
Reference solution (b). Dissolve 4.0 mg of cefadroxil CRS in mobile phase A and dilute to 50 ml with
2-61
the same mobile phase. To 5.0 ml of this solution add 5.0 ml of reference solution (a) and dilute to
100 ml with mobile phase A.
Reference solution (c). Dilute 1.0 ml of reference solution (a) to 20.0 ml with mobile phase A. Dilute
1.0 ml of the solution to 50.0 ml with mobile phase A.
Reference solution (d). Dilute 2.0 ml of reference solution (a) to 20.0 ml with mobile phase A. Dilute
Reference solution (e). To 0.20 g of amoxicillin trihydrate R add 1.0 ml of water R. Shake and add
dropwise dilute sodium hydroxide solution R to obtain a solution. The pH of the solution is about 8.5.
Store the solution at room temperature for 4 h. Dilute 0.5 ml of the solution to 50.0 ml with mobile
phase A.
chromatography R (5 m),
Mobile phase A. Mix 1 volume of acetonitrile R and 99 volumes of a 25 per cent V/V solution of
0.2M potassium dihydrogen phosphate R adjusted to pH 5.0 with dilute sodium hydroxide solution R,
Mobile phase B. Mix 20 volumes of acetonitrile R and 80 volumes of a 25 per cent V/V solution of
0.2M potassium dihydrogen phosphate R adjusted to pH 5.0 with dilute sodium hydroxide solution R,
Equilibrate the column with a mobile phase ratio A:B of 92:8. Inject 50 l of reference solution
(b). The test is not valid unless in the chromatogram obtained, the resolution between the peaks
corresponding to amoxicillin and cefadroxil is at least 2.0 (if necessary, adjust the ratio A:B of the
mobile phase) and the mass distribution ratio for the first peak (amoxicillin) is 1.3 to 2.5. Inject
reference solution (c). Adjust the system to obtain a peak with a signal-to-noise ratio of at least 3.
Inject reference solution (a) six times. The test is not valid unless the relative standard deviation for
the area of the principal peak is at most 1.0 per cent. Inject test solution (a) and reference solution
(a).
Calculate the percentage content of amoxicillin sodium by multiplying the percentage content of
amoxicillin by 1.060.
STORAGE
Store in an airtight container. If the substance is sterile, store in a sterile, airtight, tamper-proof
container.
LABELLING
The label states:
where applicable, that the substance is free from bacterial endotoxins.
IMPURITIES
N
S
O
H
Me
Me
H H
COOH
H
2
N
A. (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic
acid (6-aminopenicillanic acid),
N
O
H H
2
N
HO
N
S
O
H
Me
Me
H H
COOH
H
B. (2S,5R,6R)-6-[[(2S)-2-amino-2-(4-hydroxyphenyl)acetyl]amino]-3,3-dimethyl-7-oxo-4-thia-
l-azabicyclo[3.2.0]heptane-2-carboxylic acid (L-amoxicillin),
HN
S
H
Me
Me
COOH
N
N
HO
O
O
H
*
*
*
H
C. (4S)-2-[5-(4-hydroxyphenyl)-3,6-dioxopiperazin-2-yl]-5,5-dimethylthiazolidine-4-
carboxylic acid (amoxicillin diketopiperazines),
2-62
N
O
H H
2
N
HO
COOH
Me
Me
H
S
HN
COOH
H
*
*
D. (4S)-2-[[[(2R)-2-amino-2-(4-hydroxyphenyl)acetyl]amino]carboxymethyl]-5,5-
dimethylthiazolidine-4-carboxylic acid (penicilloic acids of amoxicillin),
N
O
H H
2
N
HO
COOH
Me
Me
H
S
HN
H
H
*
and epimer at C*
E. (2RS,4S)-2-[[[(2R)-2-amino-2-(4-hydroxyphenyl)acetyl]amino]methyl]-5,5-dimethyl-
thiazolidine-4-carboxylic acid (penilloic acids of amoxicillin),
N
N
OH
OH
F. 3-(4-hydroxyphenyl)pyrazin-2-ol,
N
O
H HN
HO
N
S
O
H
Me
Me
H H
COOH
O
NH
2
H HO
H
G. (2S,5R,6R)-6-[[(2R)-2-[[(2R)-2-amino-2-(4-hydroxyphenyl)-acetyl]amino]-2-(4-hydroxy-
phenyl)acetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic
acid (L-(4-hydroxyphenyl)glycylamoxicillin),
HO
COOH
NH Me
O
Me
Me
H
H. (2R)-2-[(2,2-dimethylpropanoyl)amino]-2-(4-hydroxyphenyl)acetic acid,
HO
COOH
NH
2
H
I. (2R)-2-amino-2-(4-hydroxyphenyl)acetic acid,
n
H
N
O
HO
N
S
O
H
Me
Me
H H
COOH
HN
H
H
H
HN
N
O
HO
O
HN
S
H
Me
Me
COOH
H
*
*
J. co-oligomers of amoxicillin and penicilloic acids of amoxicillin,
2-63
N
O
HO
COOH
Me
Me
H
S
N
HN
H
O
HO
H
*
*
H
n
K. oligomers of penicilloic acids of amoxicillin.
__________________________________________________________________________________________________________ Ph Eur
2-64
Amoxicillin Trihydrate
N
S
O
H COOH
Me
Me
N
H H
O
NH
2
H
HO
H
,3H
2
O
C
16
H
19
N
3
O
5
S,3H
2
O 419.4 61336-70-7
Amoxicillin Trihydrate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparations
Amoxicillin Capsules
Amoxicillin Oral Suspension
Co-amoxiclav Tablets
When amoxycillin trihydrate is prescribed or demanded, Amoxicillin Trihydrate shall be dispensed or
supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Amoxicillin trihydrate contains not less than 95.0 per cent and not more than the equivalent of 100.5
per cent of (2S,5R,6R)-6-[(R)-2-amino-2-(4-hydroxyphenyl)acetamido]-3,3-dimethyl-7-oxo-4-thia-
1-azabicyclo[3.2.0]heptane-2-carboxylic acid, calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white, crystalline powder, slightly soluble in water and in alcohol, practically
insoluble in ether and in fatty oils. It dissolves in dilute acids and dilute solutions of alkali hydroxides.
IDENTIFICATION
obtained with amoxicillin trihydrate CRS.
B. Examine by thin-layer chromatography (2.2.27), using silanised silica gel H R as the coating
substance.
solution R.
Reference solution (a). Dissolve 25 mg of amoxicillin trihydrate CRS in 10 ml of sodium hydrogen
10 volumes of acetone R and 90 volumes of a 154 g/l solution of ammonium acetate R, the pH of which
has been adjusted to 5.0 with glacial acetic acid R. Allow the plate to dry in air and expose it to iodine
vapour until the spots appear. Examine in daylight. The principal spot in the chromatogram obtained
with the test solution is similar in position, colour and size to the principal spot in the chromatogram
TESTS
Solution S With the aid of ultrasound or gentle heating, dissolve 0.100 g in carbon dioxide-free
water R and dilute to 50.0 ml with the same solvent.
2-65
Appearance of solution Dissolve 1.0 g in 10 ml of 0.5M hydrochloric acid. Dissolve separately 1.0 g
in 10 ml of dilute ammonia R2. Immediately after dissolution, the solutions are not more opalescent
than reference suspension II (2.2.1).
Related substances Examine by liquid chromatography (2.2.29) adjusting the ratio A:B of the
mobile phase and the attenuation as described under Assay. Inject reference solution (d). Inject a
freshly prepared test solution (b) and start the elution isocratically with the chosen mobile phase.
Immediately after elution of the amoxicillin peak start a linear gradient elution to reach a mobile
phase A:B of 0:100 over a period of 25 min. Continue the chromatography with mobile phase B for
15 min, then equilibrate the column for 15 min with the mobile phase chosen originally. Inject
mobile phase A and use the same elution gradient to obtain a blank. In the chromatogram obtained
with test solution (b), the area of any peak, apart from the principal peak and any peak observed in
the blank chromatogram, is not greater than the area of the principal peak in the chromatogram
obtained with reference solution (d) (1 per cent).
Dimethylaniline Not more than 20 ppm, determined by gas chromatography (2.2.28) using
naphthalene R as the internal standard.
Internal standard solution. Dissolve 50.0 mg of naphthalene R in cyclohexane R and dilute to 50.0 ml
with the same solvent. Dilute 5.0 ml of this solution to 100.0 ml with cyclohexane R.
Test solution. To 1.00 g of the substance to be examined in a ground-glass-stoppered tube add 5 ml of
1M sodium hydroxide and 1.0 ml of the internal standard solution. Stopper the tube and shake
vigorously for 1 min. Centrifuge if necessary and use the upper layer.
Reference solution. To 50.0 mg of dimethylaniline R add 2 ml of hydrochloric acid R and 20 ml of
water R, shake to dissolve and dilute to 50.0 ml with water R. Dilute 5.0 ml of this solution to
250.0 ml with water R. To 1.0 ml of the latter solution in a ground-glass-stoppered tube add 5 ml of
a glass column 2 m long and 2 mm in internal diameter packed with silanised diatomaceous earth
for gas chromatography R impregnated with 3 per cent m/m of polymethylphenylsiloxane R,
maintaining the temperature of the column at 120C and that of the injection port and of the
detector at 150C. Inject separately 1 l of the test solution and 1 l of the reference solution.
ASSAY
Test solution (b). Dissolve 30.0 mg of the substance to be examined in mobile phase A and dilute to
Reference solution (a). Dissolve 30.0 mg of amoxicillin trihydrate CRS in mobile phase A and dilute to
Reference solution (b). Dissolve 4.0 mg of cefadroxil CRS in mobile phase A and dilute to 50 ml with
the same mobile phase. To 5.0 ml of this solution add 5.0 ml of reference solution (a) and dilute to
100 ml with mobile phase A.
1.0 ml of this solution to 50.0 ml with mobile phase A.
Reference solution (d). Dilute 2.0 ml of reference solution (a) to 20.0 ml with mobile phase A. Dilute
5.0 ml of this solution to 20.0 ml with mobile phase A.
as mobile phase at a flow rate of 1.0 ml per minute:
Mobile phase A. A mixture of 1 volume of acetonitrile R and 99 volumes of buffer solution pH
5.0,
2-66
Mobile phase B. A mixture of 20 volumes of acetonitrile R and 80 volumes of buffer solution pH
5.0,
Prepare the buffer solution as follows: to 250 ml of 0.2M potassium dihydrogen phosphate R add dilute
sodium hydroxide solution R to pH 5.0 and dilute to 1000.0 ml with water R,
Equilibrate the column with a mobile phase with ratio A : B of 92:8. Inject reference solution (b).
The assay is not valid unless the resolution between the two principal peaks is at least 2.0. If
necessary, adjust the ratio A : B of the mobile phase. The mass distribution ratio for the first peak
(amoxicillin) is 1.3 to 2.5. Inject reference solution (c). Adjust the system to obtain a peak with a
signal-to-noise ratio of at least 3. Inject reference solution (a) six times. The test is not valid unless
the relative standard deviation for the area of the principal peak is at most 1.0 per cent. Inject
alternately test solution (a) and reference solution (a).
STORAGE
IMPURITIES
The impurities shown under Amoxicillin Sodium apply.
_______________________________________________________________________ Ph Eur
2-67
Amphotericin
O
OH OH OH OH
O
OH
OH
O O
Me
H H H H
H
H
COOH
H
H
HO
H H
Me
OH
Me
H
H
O
OH
OH NH
2
Me
C
47
H
73
NO
17
924 1397-89-3
Amphotericin complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Amphotericin B [1292]. These requirements are reproduced after the heading Definition below.
Action and use Antifungal.
Preparation
Amphotericin Lozenges
Amphotericin Oral Suspension
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Amphotericin B is a mixture of antifungal polyenes produced by the growth of certain strains of
Streptomyces nodosus or by any other means. It consists mainly of amphotericin B which is
(19E,21E,23E,25E,27E,29E,31E)-(1R,3S,5R,6R,9R,11R,15S,16R,17R,18S,33R,35S,36R,37S)-33-
[(3-amino-3,6-dideoxy--D-mannopyranosyl)oxy]-1,3,5,6,9,11,17,37-octahydroxy-15,16,18-
trimethyl-13-oxo-14,39-dioxabicyclo-[33.3.1]nonatriaconta-19,21,23,25,27,29,31-heptaene-36-
carboxylic acid. The potency is not less than 750 I.U. per milligram, calculated with reference to the
dried substance.
CHARACTERS
A yellow or orange powder, practically insoluble in water, soluble in dimethyl sulphoxide and in
propylene glycol, slightly soluble in dimethylformamide, very slightly soluble in methanol, practically
insoluble in alcohol.
It is sensitive to light in dilute solutions and is inactivated at low pH values.
IDENTIFICATION
A. Dissolve 25 mg in 5 ml of dimethyl sulphoxide R and dilute to 50 ml with methanol R. Dilute 2 ml
of the solution to 200 ml with methanol R. Examined between 300 nm and 450 nm (2.2.25), the
solution shows three absorption maxima at 362 nm, 381 nm and 405 nm. The ratio of the
absorbance measured at 362 nm to that measured at 381 nm is 0.57 to 0.61. The ratio of the
absorbance measured at 381 nm to that measured at 405 nm is 0.87 to 0.93.
obtained with amphotericin B CRS. If the spectra obtained show differences dry the substance to be
examined at 60C at a pressure not exceeding 0.7 kPa for 1 h and prepare a new spectrum.
C. To 1 ml of a 0.5 g/l solution in dimethyl sulphoxide R, add 5 ml of phosphoric acid R to form a lower
layer, avoiding mixing the two liquids. A blue ring is immediately produced at the junction of the
liquids. Mix, an intense blue colour is produced. Add 15 ml of water R and mix; the solution
becomes pale yellow.
TESTS
Content of tetraenes. Not more than 10.0 per cent and not more than 5.0 per cent if intended for
use in the manufacture of parenteral dosage forms, determined by the following method.
Test solution. Dissolve 50.0 mg in 5 ml of dimethyl sulphoxide R and dilute to 50.0 ml with methanol R.
Dilute 4.0 ml of the solution to 50.0 ml with methanol R.
2-68
Reference solution (a). Dissolve 50.0 mg of amphotericin B CRS in 5 ml of dimethyl sulphoxide R and
dilute to 50.0 ml with methanol R. Dilute 4.0 ml of the solution to 50.0 ml with methanol R.
Reference solution (b). Dissolve 25.0 mg of nystatin CRS in 25 ml of dimethyl sulphoxide R, dilute to
250.0 ml with methanol R. Dilute 4.0 ml of the solution to 50.0 ml with methanol R.
Measure the absorbances (2.2.25) of the test solution and of reference solutions (a) and (b) at the
maxima at 282 nm and 304 nm respectively, using a 0.8 per cent V/V solution of dimethyl
sulphoxide R in methanol R as the compensation liquid. Calculate the specific absorbance of the
substance being examined, of nystatin CRS and of amphotericin B CRS at both wavelengths, each with
reference to the dried substance, and calculate the percentage content of tetraenes using the following
expression:
) (
) ( 100
2 1 1 2
1 2 2 1
B N B N
S B S B
F
+
S
1
and S
2
=specific absorbances of the substance to be examined at 282 nm and 304 nm
respectively,
N
1
and N
2
=specific absorbance of nystatin CRS at 282 nm and 304 nm respectively,
B
1
and B
2
=specific absorbance of amphotericin B CRS at 282 nm and 304 nm respectively,
F=declared content of tetraenes in amphotericin B CRS.
60C at a pressure not exceeding 0.7 kPa.
Sulphated ash (2.4.14). Not more than 3.0 per cent and not more than 0.5 per cent if intended for
use in the manufacture of parenteral dosage forms, determined on 1.0 g.
Bacterial endotoxins (2.6.14). If intended for use in the manufacture of parenteral dosage forms
without a further appropriate procedure for removal of bacterial endotoxins, not more than 1.0 I.U.
of endotoxin per milligram.
ASSAY
Triturate 60 mg with dimethylformamide R and dilute, with shaking, to 100 ml with the same solvent.
Dilute 10 ml of the solution to 100 ml with dimethylformamide R. Carry out the microbiological assay
of antibiotics (2.7.2).
STORAGE
Store in a well-closed container, protected from light, at a temperature of 2C to 8C. If the
substance is sterile, store in a sterile, airtight, tamper-proof container.
LABELLING
The label states:
where applicable, that the substance is free from bacterial endotoxins,
forms.
IMPURITIES
A. amphotericin A (tetraene).
__________________________________________________________________________________________________________ Ph Eur
2-69
Ampicillin
H
O
NH
2
H
N
O
N
S
H COOH
Me
Me
H H
C
16
H
19
N
3
O
4
S 349.4 69-53-4
Ampicillin complies with the requirements of the 3rd edition of the European Pharmacopoeia for Anhydrous
Ampicillin [0167]. These requirements are reproduced after the heading Definition below.
Preparations
Ampicillin Capsules
Ampicillin Oral Suspension
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Anhydrous ampicillin contains not less than 96.0 per cent and not more than the equivalent of
100.5 per cent of (2S,5R, 6R)-6-[(R)-2-amino-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-
azabicyclo[3.2.0]heptane-2-carboxylic acid, calculated with reference to the anhydrous substance.
CHARACTERS
A white, crystalline powder, sparingly soluble in water, practically insoluble in acetone, in alcohol, in
ether and in fatty oils. It dissolves in dilute solutions of acids and of alkali hydroxides.
IDENTIFICATION
obtained with anhydrous ampicillin CRS. Examine the substances prepared as discs using potassium
bromide R.
substance.
solution R.
Reference solution (a). Dissolve 25 mg of anhydrous ampicillin CRS in 10 ml of sodium hydrogen
Reference solution (b). Dissolve 25 mg of amoxicillin trihydrate CRS and 25 mg of anhydrous ampicillin
D. It complies with the test for water (see Tests).
2-70
TESTS
Appearance of solution Dissolve 1.0 g in 10 ml of 1M hydrochloric acid. Separately dissolve 1.0 g in
10 ml of dilute ammonia R2. Immediately after dissolution, the solutions are not more opalescent than
reference suspension II (2.2.1).
Specific optical rotation (2.2.7). Dissolve 62.5 mg in water R and dilute to 25.0 ml with the same
substance.
reference solution (c) and elute isocratically. Inject freshly prepared test solution (b) and start the
elution isocratically. Immediately after elution of the ampicillin peak start the following linear
gradient. If the mobile phase composition has been adjusted to achieve the required resolution, the
adjusted composition will apply at time zero in the gradient.
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
0 85 15
30 0 100
45 0 100
Equilibrate the column with the originally chosen mobile phase for 15 min. Inject mobile phase A
and use the same elution gradient to obtain a blank. In the chromatogram obtained with test solution
(b), the area of any peak, apart from the principal peak and any peak observed in the blank chroma-
togram, is not greater than the area of the principal peak in the chromatogram obtained with refer-
ence solution (c) (1.0 per cent).
detector at 150C. Inject separately 1 l of the test solution and of the reference solution.
ASSAY
Reference solution (a). Dissolve 27.0 mg of anhydrous ampicillin CRS in mobile phase A and dilute to
Reference solution (b). Dissolve 2.0 mg of cefradine CRS in mobile phase A and dilute to 50 ml with
the same solvent. To 5.0 ml of this solution add 5.0 ml of reference solution (a).
2-71
Reference solution (c). Dilute 1.0 ml of reference solution (a) to 20.0 ml with mobile phase A.
Reference solution (d). Dilute 1.0 ml of reference solution (c) to 25.0 ml with mobile phase A.
Mobile phase A. A mixture of 0.5 ml of dilute acetic acid R, 50 ml of 0.2M potassium dihydrogen
phosphate and 50 ml of acetonitrile R, diluted to 1000 ml with water R,
Mobile phase B. A mixture of 0.5 ml of dilute acetic acid R, 50 ml of 0.2M potassium dihydrogen
The test is not valid unless the resolution between the two principal peaks is at least 3.0. If necessary,
adjust the ratio A : B of the mobile phase. The mass distribution ratio for the first peak (ampicillin) is
2.0 to 2.5. Inject reference solution (d). Adjust the system to obtain a peak with a signal-to-noise
ratio of at least 3. Inject reference solution (a) six times. The test is not valid unless the relative
standard deviation for the area of the principal peak is at most 1.0 per cent. Inject alternately test
solution (a) and reference solution (a).
Calculate the percentage content of ampicillin.
STORAGE
Store in an airtight container, at a temperature not exceeding 30C.
IMPURITIES
N
S
O
H
Me
Me
H H
COOH
H
2
N
N
O
H H
2
N
N
S
O
H
Me
Me
H H
COOH
H
B. (2S,5R,6R)-6-[(S)-2-amino-2-phenyl(acetamido)]-3,3-dimethyl-7-oxo-4-thia-1-
azabicyclo[3.2.0]heptane-2-carboxylic acid (L-ampicillin),
HN
S
H
Me
Me
COOH
N
N O
O
H
H
H
*
and epimer at C*
C. (RS)-5,5-dimethyl-2-[(3,6-dioxo-5-phenylpiperazin-2-yl)thiazole-4-carboxylic acid,
N
O
H H
2
N
COOH
Me
Me
H
S
HN
COOH
H
*
*
D. (2R,4S)-2-[(RS)-1-carboxy-1-[(R)-2-amino-2-phenylacetamido]methyl]-5,5-dimethyl-
thiazolidine-4-carboxylic acid (penicilloic acids of ampicillin),
2-72
N
O
H H
2
N
N
S
O
H
Me
Me
H H
NH
O
COOH
H
H
E. ampicillin phenylglycylamide,
N
O
H H
2
N
COOH
Me
Me
H
S
HN
H
H
*
and epimer at C*
F. (2RS,4S)-2-[(R)-2-amino-2-phenylacetamido]methyl-5,5-dimethylthiazolidine-4-
carboxylic acid (penilloic acids of ampicillin),
NH
HN
O
O
H
H
G. 3, 6-diphenylpiperazine-2, 5-dione,
N
N OH
H. 3-phenylpyrazin-2-ol,
N
O
H HN
N
S
O
H
Me
Me
H H
COOH
O
NH
2
H
H
I. phenylglycylampicillin,
N
O
N
S
O
H
Me
Me
H H
COOH
Me
Me
Me
H
J. (2S,5R,6R)-6-(2,2-dimethylpropanamido)-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]-
heptane-2-carboxylic acid,
COOH
NH Me
O
Me
Me
H
K. 2-(2,2-dimethylpropanamido)-2-phenylacetic acid,
COOH
NH
2
H
L. 2-phenylglycine,
2-73
n
N
S
N
O
NH
Me
Me
COOH
O
N
O
NH2
CO
HN
S
Me
Me
COOH
N
O
NH
CO
HN
S
Me
Me
COOH
M. oligomers of ampicillin and penicilloic acids of ampicillin.
_______________________________________________________________________ Ph Eur
2-74
Ampicillin Sodium
corrected 1/01
O
NH
2
H
N
O
N
S
H COONa
Me
Me
H H
H
C
16
H
18
N
3
NaO
4
S 371.4 69-52-3
Ampicillin Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia [0578].
Preparation
Ampicillin Injection
Ph Eur _______________________________________________________________________________________________________________
DEFINITION
Ampicillin sodium contains not less than 91.0 per cent and not more than the equivalent of 100.5 per
cent of sodium (2S,5R,6R)-6-[[(2R)-2-amino-2-phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-
azabicyclo[3.2.0]heptane-2-carboxylate, calculated with reference to the anhydrous substance.
PRODUCTION
complies with the following test:
2-Ethylhexanoic acid Not more than 0.8 per cent m/m, determined by gas chromatography
(2.2.28), using a suitable, validated method.
CHARACTERS
A white powder, hygroscopic, freely soluble in water, sparingly soluble in acetone, practically
insoluble in ether, in fatty oils and in liquid paraffin.
IDENTIFICATION
A. Dissolve 0.250 g in 5 ml of water R, add 0.5 ml of dilute acetic acid R, swirl and allow to stand for
10 min in iced water. Filter the crystals through a small sintered-glass filter (40), applying suction,
wash with 2 ml to 3 ml of a mixture of 1 volume of water R and 9 volumes of acetone R and dry in an
oven at 60C for 30 min. Examine the crystals by infrared absorption spectrophotometry (2.2.24),
comparing with the spectrum obtained with ampicillin trihydrate CRS.
substance.
solution R.
Reference solution (a). Dissolve 25 mg of ampicillin trihydrate CRS in 10 ml of sodium hydrogen
2-75
TESTS
Appearance of solution Place 1.0 g in a conical flask and add slowly and with continuous swirling
10 ml of 1M hydrochloric acid. Separately dissolve 1.0 g in water R and dilute to 10.0 ml with the same
solvent. Immediately after dissolution, the solutions are not more opalescent than reference
suspension II (2.2.1). The absorbance (2.2.25) of the solution in water measured at 430 nm is not
greater than 0.15.
The pH of the solution, measured 10 min after dissolution, is 8.0 to 10.0.
Specific optical rotation (2.2.7). Dissolve 62.5 mg in a 4 g/l solution of potassium hydrogen
phthalate R and dilute to 25.0 ml with the same solvent. The specific optical rotation is +258 to
+287, calculated with reference to the anhydrous substance.
50 l of reference solution (d) and elute isocratically until elution of the ampicillin peak. Inject 50 l
of test solution (b) and start the elution isocratically. Immediately after elution of the ampicillin peak
start the following linear gradient. If the mobile phase composition has been adjusted to achieve the
required resolution, the adjusted composition will apply at time zero in the gradient.
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
30 45 0 100 isocratic
45 60 85 15 re-equilibration
Inject mobile phase A and use the same elution pattern to obtain a blank. Inject reference solution
(e) and use the same elution pattern. The chromatogram obtained with reference solution (e), shows
an ampicillin peak and a dimer peak, with a retention time of 2.8 relative to ampicillin. In the
chromatogram obtained with test solution (b), the area of any peak corresponding to ampicillin dimer
is not greater than 4.5 times the area of the principal peak in the chromatogram obtained with
reference solution (d) (4.5 per cent); the area of any peak, apart from the principal peak and any peak
corresponding to ampicillin dimer, is not greater than twice the area of the principal peak in the
chromatogram obtained with reference solution (d) (2 per cent). Disregard any peak due to the
blank.
Dimethylaniline Not more than 20 ppm, determined by gas chromatography (2.2.28), using
nitrogen for chromatography R as the carrier gas at a flow rate of 30 ml/min,
detector at 150C. Inject separately, 1 l of the test solution and 1 l of the reference solution.
Methylene chloride Not more than 0.2 per cent m/m, determined by gas chromatography (2.2.28),
using ethylene chloride R as the internal standard.
Internal standard solution. Dissolve 1.0 ml of ethylene chloride R in water R and dilute to 500.0 ml with
the same solvent.
Test solution (a). Dissolve 1.0 g of the substance to be examined in water R and dilute to 10.0 ml with
the same solvent.
Test solution (b). Dissolve 1.0 g of the substance to be examined in water R, add 1.0 ml of the internal
standard solution and dilute to 10.0 ml with water R.
Reference solution. Dissolve 1.0 ml of methylene chloride R in water R and dilute to 500.0 ml with the
2-76
same solvent. To 1.0 ml of the solution add 1.0 ml of the internal standard solution and dilute to
10.0 ml with water R.
a glass column 1.5 m long and 4 mm in internal diameter packed with diatomaceous earth for gas
chromatography R impregnated with 10 per cent m/m of macrogol 1000 R,
the detector at 150C. Calculate the content of methylene chloride taking its density at 20C to be
1.325 g/ml.
mination of water.
without a further appropriate procedure for the removal of bacterial endotoxins, not more than
0.15 I.U. of endotoxin per milligram.
ASSAY
50.0 ml with mobile phase A.
mobile phase A and dilute to 10.0 ml with mobile phase A.
mobile phase A. To 5.0 ml of the solution add 5.0 ml of reference solution (a).
Reference solution (d). Dilute 1.0 ml of reference solution (a) to 20.0 ml with mobile phase A.
Reference solution (e). To 0.20 g of the substance to be examined add 1.0 ml of water R. Heat the
solution at 60C for 1 h. Dilute 0.5 ml of the solution to 50.0 ml with mobile phase A.
Equilibrate the column with a mobile phase with A:B ratio of 85:15. Inject 50 l of reference solution
(b). The assay is not valid unless in the chromatogram obtained, the resolution between the two
principal peaks is at least 3.0. If necessary, adjust the A:B ratio of the mobile phase. The mass
distribution ratio for the first peak (ampicillin) is 2.0 to 2.5. Inject 50 l of reference solution (c).
Adjust the system to obtain a peak with a signal-to-noise ratio of at least 3. Inject reference solution
(a) six times. The test is not valid unless the relative standard deviation for the area of the principal
peak is at most 1.0 per cent. Inject alternately test solution (a) and reference solution (a).
Calculate the percentage content of ampicillin sodium by multiplying the percentage content of
ampicillin by 1.063.
STORAGE
container.
LABELLING
The label states:
2-77
IMPURITIES
N
S
O
H
Me
Me
H H
COOH
H
2
N
N
O
H H
2
N
N
S
O
H
Me
Me
H H
COOH
H
B. (2S,5R,6R)-6-[[(2S)-2-amino-2-phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-
azabicyclo[3.2.0]heptane-2-carboxylic acid (L-ampicillin),
HN
S
H
Me
Me
COOH
N
N O
O
H
H
*
*
*
C. (4S)-5,5-dimethyl-2-(3,6-dioxo-5-phenylpiperazin-2-yl)thiazolidine-4-carboxylic acid
(diketopiperazines of ampicillin),
N
O
H H
2
N
COOH
Me
Me
H
S
HN
COOH
H
*
*
D. (4S)-2-[[[(2R)-2-amino-2-phenylacetyl]amino]carboxymethyl]-5,5-dimethylthiazolidine-4-
carboxylic acid (penicilloic acids of ampicillin),
N
O
H H
2
N
N
S
O
H
Me
Me
H H
NH
O
COOH
H
H
E. (R)-2-[[[(2S,5R,6R)-6-[[(2R)-2-amino-2-phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-
1-azabicyclo[3.2.0]hept-2-yl]carbonyl]amino]-2-phenylacetic acid (ampicillinyl-D-
phenylglycine),
N
O
H H
2
N
COOH
Me
Me
H
S
HN
H
H
*
and epimer at C*
F. (2RS,4S)-2-[[[(2R)-2-amino-2-phenylacetyl]amino]methyl]-5,5-dimethylthiazolidine-4-
NH
HN
O
O
H
H
G. (3R,6R)-3,6-diphenylpiperazine-2,5-dione,
2-78
N
N OH
H. 3-phenylpyrazin-2-ol,
N
O
H HN
N
S
O
H
Me
Me
H H
COOH
O
NH
2
H
H
I. (2S,5R,6R)-6-[[(2R)-2-[[(2R)-2-amino-2-phenylacetyl]amino]-2-phenylacetyl]amino]-
3,3-dimethyl-7-oxo-4-thia-1azabicyclo[3.2.0]heptane-2-carboxylic acid
(D-phenylglycylampicillin),
N
O
N
S
O
H
Me
Me
H H
COOH
Me
Me
Me
H
J. (2S,5R,6R)-6-[(2,2-dimethylpropanoyl)amino]-3,3-dimethyl-7-oxo-4-thia-1-
azabicyclo[3.2.0]heptane-2-carboxylic acid,
COOH
NH Me
O
Me
Me
H
K. (2R)-2-[(2,2-dimethylpropanoyl)amino]-2-phenylacetic acid,
COOH
NH
2
H
L. (2R)-2-amino-2-phenylacetic acid (D-phenylglycine),
n
H
N
O
N
S
O
H
Me
Me
H H
COOH
HN
H
H
H
HN
N
O
O
HN
S
H
Me
Me
COOH
H
*
*
M. co-oligomers of ampicillin and of penicilloic acids of ampicillin,
N
O
COOH
Me
Me
H
S
HN
H
O
HO
H
*
*
HN
H
n
N. oligomers of penicilloic acids of ampicillin.
__________________________________________________________________________________________________________ Ph Eur.
2-79
Ampicillin Trihydrate
H
O
NH
2
H
N
O
N
S
H COOH
Me
Me
H H
,3H
2
O
C
16
H
19
N
3
O
4
S,3H
2
O 403.5 7177-48-2
Ampicillin Trihydrate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparations
Ampicillin Capsules
Ampicillin Oral Suspension
Co-fluampicil Capsules
Co-fluampicil Oral Suspension
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ampicillin trihydrate contains not less than 96.0 per cent and not more than the equivalent of
100.5 per cent of (2S,5R,6R)-6-[(R)-2-amino-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-
azabicylo[3.2.0]heptane-2-carboxylic acid, calculated with reference to the anhydrous substance.
CHARACTERS
A white, crystalline powder, slightly soluble in water, practically insoluble in alcohol, in ether and in
fatty oils. It dissolves in dilute solutions of acids and of alkali hydroxides.
IDENTIFICATION
obtained with ampicillin trihydrate CRS.
substance.
solution R.
Reference solution (a). Dissolve 25 mg of ampicillin trihydrate CRS in 10 ml of sodium hydrogen
D. It complies with the test for water (see Tests).
2-80
TESTS
Appearance of solution Dissolve 1.0 g in 10 ml of 1M hydrochloric acid. Separately dissolve 1.0 g in
10 ml of dilute ammonia R2. Immediately after dissolution, the solutions are not more opalescent than
reference suspension II (2.2.1).
Specific optical rotation (2.2.7). Dissolve 62.5 mg in water R and dilute to 25.0 ml with the same
substance.
reference solution (c) and elute isocratically. Inject freshly prepared test solution (b) and start the
elution isocratically. Immediately after elution of the ampicillin peak start the following linear
gradient. If the mobile phase composition has been adjusted to achieve the required resolution, the
adjusted composition will apply at time zero in the gradient.
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
0 85 15
30 0 100
45 0 100
Equilibrate the column with the originally chosen mobile phase for 15 min. Inject mobile phase A
and use the same elution gradient to obtain a blank. In the chromatogram obtained with test solution
(b), the area of any peak, apart from the principal peak and any peak observed in the blank chroma-
togram, is not greater than the area of the principal peak in the chromatogram obtained with refer-
detector at 150C. Inject separately 1 l of the test solution and of the reference solution.
ASSAY
the same solvent. To 5.0 ml of this solution add 5.0 ml of reference solution (a).
2-81
Reference solution (c). Dilute 1.0 ml of reference solution (a) to 20.0 ml with mobile phase A.
Reference solution (d). Dilute 1.0 ml of reference solution (c) to 25.0 ml with mobile phase A.
phosphate, 50 ml of acetonitrile R diluted to 1000 ml with water R,
phosphate and 400 ml of acetonitrile R diluted to 1000 ml with water R,
The test is not valid unless the resolution between the two principal peaks is at least 3.0. If necessary,
adjust the ratio A : B of the mobile phase. The mass distribution ratio for the first peak (ampicillin) is
2.0 to 2.5. Inject reference solution (d). Adjust the system to obtain a peak with a signal-to-noise ratio
of at least 3. Inject reference solution (a) six times. The test is not valid unless the relative standard
deviation for the area of the principal peak is at most 1.0 per cent. Inject alternately test solution (a)
and reference solution (a).
Calculate the percentage content of ampicillin.
STORAGE
Store in an airtight container, at a temperature not exceeding 30C.
IMPURITIES
The impurities shown under Ampicillin apply
_______________________________________________________________________ Ph Eur
2-82
Amylmetacresol
C
5
H
11
OH
Me
C
12
H
18
O 178.3 1300-94-3
Definition Amylmetacresol is 5-methyl-2-pentylphenol. It contains not less than 96.0% and not
12
H
18
O.
Characteristics A clear or almost clear liquid or a solid crystalline mass, colourless or slightly
yellow when freshly prepared, which darkens on keeping; odour, characteristic.
Practically insoluble in water; soluble in ethanol (96%), in ether and in fixed and volatile oils.
Identification
amylmetacresol (RS 014).
absolute ethanol containing 2% v/v of 0.1M hydrochloric acid exhibits a maximum at 278 nm and a
less well-defined maximum at 286 nm. The absorbance at the maximum at 278 nm is about 0.53.
C. To 5 ml of a 0.5% v/v solution in methanol add 5 ml of bromine water. A yellow precipitate is
produced.
D. Freezing point, about 22, Appendix V B.
Related substances Carry out the method for gas chromatography, Appendix III B, using the
following solutions. Solution (1) contains 1% w/v of the substance being examined in chloroform.
Solution (2) is the substance being examined.
The chromatographic procedure may be carried out using a glass column (1.8 m 3 mm) packed
with acid-washed, silanised diatomaceous support (80 to 100 mesh) coated with 10% w/w of
silicone grease (Apiezon L is suitable) and maintained at 180.
In the chromatogram obtained with solution (2) the area of any secondary peak is not greater than
2% and the sum of the areas of any secondary peaks is not greater than 3% by normalisation.
Assay Dissolve 0.2 g in absolute ethanol containing 2% v/v of 0.1M hydrochloric acid and dilute to
100 ml with the same solvent. Dilute 2 ml of the solution to 100 ml with the same solvent and
12
H
18
O taking 132 as the value of A(1%, 1 cm) at the maximum at
278 nm.
Storage Amylmetacresol should be kept in a well-closed container and protected from light.
Action and use Antiseptic.
2-83
Anise Oil
Aniseed Oil
Anise Oil complies with the requirements of the 3rd edition of the European Pharmacopoeia [0804]. These
Action and use Flavour.
Preparation
Concentrated Anise Water
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Anise oil is obtained by steam distillation from the dry ripe fruits of Pimpinella anisum L. or of Illicium
verum Hook. fil.
CHARACTERS
A clear, colourless or pale yellow liquid, solidifying on cooling, practically insoluble in water, miscible
with alcohol, with ether, with light petroleum and with methylene chloride.
IDENTIFICATION
Second identification: A.
254
Test solution. Dissolve 1 g of the substance to be examined in toluene R and dilute to 10 ml with the
same solvent.
Reference solution (a). Dissolve 80 l of anethole R in toluene R and dilute to 1 ml with the same
solvent.
Reference solution (b). Dissolve 3 l of anisaldehyde R in toluene R and dilute to 1 ml with the same
solvent.
Reference solution (c). Dissolve 1 l of linalol R in toluene R and dilute to 1 ml with the same solvent.
volumes of ethyl acetate R and 93 volumes of toluene R. Allow the plate to dry in air and examine in
ultraviolet light at 254 nm. The chromatogram obtained with the test solution shows a quenching
spot which is similar in position to the spot in the chromatogram obtained with reference solution
(b). In the upper third of the chromatogram obtained with the test solution, there is a quenching spot
corresponding to anethole. Spray with vanillin reagent R. Heat at 100C to 105C for 10 min.
Examine the chromatograms in daylight within 10 min. The chromatogram obtained with the test
solution shows a blue spot similar in position and colour to the spot in the chromatogram obtained
with reference solution (c) and an orange-pink spot similar in position and colour to the spot in the
chromatogram obtained with reference solution (a). It also shows a violet spot near the solvent front
(monoterpene hydrocarbons); the oil from P. anisum may show an additional brown spot just above
the spot corresponding to anisaldehyde.
B. Examine the chromatograms obtained in the test for chromatographic profile. The chromatogram
obtained with the test solution shows six peaks similar in retention time to the six peaks in the
chromatogram obtained with the reference solution.
TESTS
Relative density (2.2.5): 0.978 to 0.994.
Refractive index (2.2.6): 1.552 to 1.561.
Freezing point (2.2.18): 15C to 19C.
Acid value (2.5.1). Not more than 1.0, determined on 5.0 g dissolved in 50 ml of the prescribed
Fatty oils and resinified essential oils (2.8.7). It complies with the test for fatty oils and resinified
essential oils.
Chromatographic profile Examine by gas chromatography (2.2.28).
Reference solution. Prepare the following mixture by weighing the indicated quantities to within 20 per
cent. To 1 g of hexane R, add 20 mg of linalol R, 20 mg of estragole R, 20 mg of -terpineol R, 10 mg
of cis-anethole R, 60 mg of anethole R and 30 mg of anisaldehyde R.
2-84
a glass column 30 m to 60 m long and about 0.3 mm in internal diameter coated with macrogol
20, 000 R,
maintaining the temperature of the column at 60C for 4 min, then raising the temperature at a
rate of 2C per minute to 210C and maintaining at 210C for 15 min and maintaining the tempera-
ture of the injection port at 180C to 200C and that of the detector at 220C to 250C.
Inject about 0.2 l of the reference solution. When the chromatograms are recorded in the
prescribed conditions, the components elute in the order indicated in the composition of the
reference solution. Record the retention times of these substances.
The test is not valid unless: the number of theoretical plates is at least 30, 000, calculated from the
peak due to estragole at 120C; the resolution, calculated at 130C, between the peaks due to
estragole and -terpineol is at least 1.3.
Inject about 0.2 l of the test solution. Using the retention times determined from the chromato-
gram obtained with the reference solution, locate the six components of the reference solution on the
chromatogram obtained with the test solution (disregard the peak due to hexane).
Determine the percentage of each of these six components by the normalisation procedure using
the chromatogram obtained with the test solution.
The percentages are within the following ranges:
Linalol 0.1 to 1.5 per cent
Estragole 0.5 to 6.0 per cent
-Terpineol 0.1 to 1.5 per cent
cis-Anethole less than 0.5 per cent
trans-Anethole 84 to 93 per cent
Anisaldehyde 0.1 to 3.5 per cent
STORAGE
Store in a well-filled, airtight container, protected from light and heat.
LABELLING
The label states whether the anise oil is obtained from P. anisum or from I. verum.
The type chromatograms are given for information and guidance only; they do not form a mandatory part of
the monograph.
Fig. 804-1-Type chromatogram of the essential oil from Illicium verum
2-85
Fig. 804-2-Type chromatogram of the essential oil from Pimpinella anisum
_______________________________________________________________________ Ph Eur
2-86
Star Anise
Star Anise complies with the requirements of the 3rd edition of the European Pharmacopoeia [1153]. These
Action and use Carminative.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Star anise consists of the dried composite fruit of Illicium verum Hooker fil. It contains not less than
70 ml/kg of essential oil, with reference to the anhydrous drug.
CHARACTERS
Star anise is a composite fruit, consisting of follicles. Each follicle is about 12 mm to 20 mm long and
6 mm to 11 mm high. The seeds are up to 8 mm long.
The fruit carpels are brown and have an odour of anethol.
It has the macroscopic and microscopic characters described under Identification A and B.
IDENTIFICATION
A. The fruit is formed of six to eleven (usually eight), often unequally developed, boat-shaped
follicles, radially arranged around a short, central, blunt-ending columella. Separated follicles and
seeds may occur.
The distal portion of each follicle is prolonged to a blunt beak. The outer surface is reddish-brown
to greyish-brown and roughly wrinkled. The inner surface is shiny, reddish-brown and smooth.
Ripe follicles have a split ventral suture uppermost, revealing a single hard ovoid, compressed,
shiny, brown seed. The fruit stalk, often absent, is small and strongly curved at the distal end.
B. Reduce to a powder (355). The powder is reddish-brown. Examine under a microscope using
chloral hydrate solution R. The powder shows the following diagnostic characters: brown epicarpal
cells, polygonal in surface view, with strongly striated cuticles and occasional anomocytic stomata
(2.8.3); fragments of the endocarp with palisade-like cells up to about 600 m long; fragments of the
mesocarp with large parenchymatous cells, vessels, oil-containing cells and groups of large very
elongated stone cells with thickened and pitted walls; fragments of the seed with palisade-like, yellow
stone cells up to 200 m long, with strongly pitted walls; fragments of the endosperm containing
droplets of oil and aleurone grains; separate fragments of the columella and the fruit stalk with
strongly thickened irregular stone cells up to 400 m long and about 150 m wide, with pointed,
partly star-shaped projections (astrosclereids), rhomboidal or rectangular crystals of calcium oxalate.
C. Examine the chromatograms obtained in the test for Illicium anisatum. The chromatogram
obtained with the test solution shows a blue zone that is similar to the anethole zone in the chromato-
gram obtained with reference solution (a) and a zone that is similar to the triglycerides zone in the
TESTS
Illicium anisatum Examine by thin-layer chromatography (2.2.27), using a suitable silica gel as the
coating substance.
Test solution. 100 l of xylene solution obtained in the assay of the essential oil.
Reference solution (a). Dissolve 3 l of anethole R and 50 l of olive oil R in 1 ml of toluene R.
Reference solution (b). Dissolve 1 l of myristicine R in 10 ml of toluene R.
Apply separately to the plate as bands 10 l of each solution. Develop over a path of 15 cm using a
mixture of 50 volumes of hexane R and 50 volumes of toluene R. Allow the plate to dry in air. Spray
with a freshly prepared 200 g/l solution of phosphomolybdic acid R in alcohol R and heat at 120C for
5 min. The zones corresponding to the constituents of the reference solutions are blue. The zone
corresponding to myristicine in the chromatogram obtained with reference solution (b) is located at
an R
f
value intermediate between that of the zone corresponding to anethole and that of the zone
corresponding to triglycerides of olive oil in the chromatogram obtained with reference solution (a).
The chromatogram obtained with the test solution shows no zone corresponding to myristicine.
Foreign matter (2.8.2). It complies with the test for foreign matter. The fruit should not contain
follicles with an upwardly turned beak and straight fruit stalks not curved at the distal end (fruits of
Illicium anisatum L. syn I. religiosum Sieb. et Zucc.).
Water (2.2.13). Not more than 10.0 per cent, determined by distillation on 20.0 g of the powdered
drug.
2-87
ASSAY
Carry out the determination of essential oils in vegetable drugs (2.8.12). Use a 250 ml round-
bottomed flask and 100 ml of water R as the distillation liquid. Immediately before the determination,
reduce 50.0 g of the drug to a coarse powder (1400) and mix. Further reduce about 10.0 g of this
mixture to a finer powder (710). Use 2.50 g of the powdered drug for the determination. Introduce
0.50 ml of xylene R into the graduated tube. Distil at a rate of 2 ml to 3 ml per minute for 2 h.
STORAGE
_______________________________________________________________________ Ph Eur
2-88
Aniseed
Anise
Aniseed complies with the requirements of the 3rd edition of the European Pharmacopoeia [0262]. These
When Powdered Aniseed is prescribed or demanded, material complying with the requirements
below with the exception of Identification test A and the test for Foreign matter shall be dispensed or
supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Aniseed consists of the whole dry cremocarp of Pimpinella anisum L. It contains not less than
20 ml/kg of essential oil.
CHARACTERS
Aniseed has an odour reminiscent of anethole.
The fruit is a cremocarp and generally entire; a small fragment of the thin, rigid, slightly curved
pedicel is frequently attached.
IDENTIFICATION
A. The cremocarp is ovoid or pyriform and slightly compressed laterally, yellowish-green or greenish-
grey, 3 mm to 5 mm long and up to 3 mm wide, surmounted by a stylopod with two short, reflexed
stylar points. The mericarps are attached by their tops to the carpophore with a plane commissural
surface and a convex dorsal surface, the latter being covered with short, warty trichomes visible using
a lens; the fruit shows five primary ridges, running longitudinally, comprising three dorsal ridges and
two lateral ridges, non-prominent, and lighter in colour.
B. Reduce to a powder (355). The powder is greenish-yellow to brownish-green. Examine under a
microscope using chloral hydrate solution R. The powder shows the following diagnostic characters:
whole or broken trichomes, mostly unicellular, sometimes curved, with blunt apex and warty cuticle;
fragments of epidermis with striated cuticle, occasional anomocytic stomata; fragments of numerous
narrow, branched vittae, fragments of endosperm containing aleurone grains and micro-rosettes of
calcium oxalate; oblong sclereids from the commissural zone and bundles of sclerenchymatous fibres
from the carpophore and the pedicel. Starch is absent.
254
Test solution. Shake 0.10 g of the powdered drug (1500) with 2 ml of methylene chloride R for 15 min.
Filter and carefully evaporate the filtrate to dryness on a water-bath at 60C. Dissolve the residue in
0.5 ml of toluene R.
Reference solution. Dissolve 3 l of anethole R and 40 l of olive oil R in 1 ml of toluene R.
Apply separately to the plate at 2 cm intervals 2 l and 3 l of the test solution and 1 l, 2 l and 3 l
of the reference solution. Develop over a path of 10 cm using toluene R. Allow the plate to dry in air
and examine in ultraviolet light at 254 nm. The chromatograms show a quenching zone (anethole) in
the central part against a light background. Spray the plate with a freshly prepared 200 g/l solution of
phosphomolybdic acid R in alcohol R, using 10 ml for a plate 200 mm square, and heat at 120C for
5 min. The spots corresponding to anethole appear blue against a yellow background. In the chroma-
togram obtained with 2 l of the test solution, the spot corresponding to anethole is intermediate in
size between the spots corresponding to anethole in the chromatograms obtained with 1 l and 3 l of
the reference solution. The chromatograms obtained with the test solution show in the lower third a
blue spot (triglycerides) similar in position to the spot in the lower third of the chromatograms
obtained with the reference solution (triglycerides of olive oil).
TESTS
drug.
Ash insoluble in hydrochloric acid (2.8.1). Not more than 2.5 per cent.
2-89
ASSAY
bottomed flask, 100 ml of water R as the distillation liquid and 0.50 ml of xylene R in the graduated
tube. Reduce the drug to a coarse powder and immediately use 10.0 g for the determination. Distil at
a rate of 2.5 ml to 3.5 ml per minute for 2 h.
STORAGE
_______________________________________________________________________ Ph Eur
2-90
Antazoline Hydrochloride
N
N
NH
,HCl
C
17
H
20
ClN
3
301.8 2508-72-7
Antazoline Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
1
-receptor antagonist.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Antazoline hydrochloride contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of 4,5-dihydro-2-(N-benzyl-N-phenylaminomethyl)imidazole hydrochloride,
CHARACTERS
A white or almost white, crystalline powder, sparingly soluble in water, soluble in alcohol, slightly
soluble in methylene chloride.
IDENTIFICATION
obtained with antazoline hydrochloride CRS. Examine the substances as discs prepared using potassium
chloride R.
B. Examine the chromatograms obtained in the test for related substances in daylight after spraying.
The principal spot in the chromatogram obtained with test solution (b) is similar in position, colour
and size to the principal spot in the chromatogram obtained with reference solution (b).
C. To 5 ml of solution S (see Tests) add, drop by drop, dilute sodium hydroxide solution R until an
alkaline reaction is produced. Filter. The precipitate, washed with two quantities, each of 10 ml, of
water R and dried in a desiccator under reduced pressure, melts (2.2.14) at 119C to 123C.
TESTS
Solution S Dissolve 2.0 g in carbon dioxide-free water R prepared from distilled water R, heating at
60C if necessary. Allow to cool and dilute to 100 ml with the same solvent.
solution Y
7
(Method II, 2.2.2).
indicator.
254
R as the
coating substance. Heat the plate at 110C for 15 min before using.
Reference solution (a). Dilute 0.5 ml of Test solution (a) to 100 ml with methanol R.
Reference solution (b). Dissolve 20 mg of antazoline hydrochloride CRS in methanol R and dilute to 5 ml
Reference solution (c). Dissolve 20 mg of xylometazoline hydrochloride CRS in 1 ml of Test solution (a)
and dilute to 5 ml with methanol R.
2-91
5 volumes of diethylamine R, 10 volumes of methanol R and 85 volumes of ethyl acetate R. Dry the
plate in a current of warm air for 15 min. Examine in ultraviolet light at 254 nm. The test is not
principal spots. Spray with a mixture of equal volumes of a 200 g/l solution of ferric chloride R and a
5 g/l solution of potassium ferricyanide R. Examine immediately in daylight. Any spot in the chromato-
gram obtained with test solution (a), apart from the principal spot, is not more intense than the
spot in
the chromatogram obtained with reference solution (a) (0.5 per cent).
100C to 105C for 3 h.
Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on the residue obtained in the test
for loss on drying.
ASSAY
Dissolve 0.250 g in 100 ml of alcohol R. Add 0.1 ml of phenolphthalein solution R1. Titrate with 0.1M
alcoholic potassium hydroxide.
1 ml of 0.1M alcoholic potassium hydroxide is equivalent to 30.18 mg of C
17
H
20
ClN
3
.
STORAGE
IMPURITIES
N
O
H
N
NH
2
A. N-(2-aminoethyl)-2-[benzyl(phenyl)amino]acetamide
__________________________________________________________________________________________________________ Ph Eur
3-1
Apomorphine Hydrochloride
NMe
HO
HO
H
,HCl
C
17
H
18
ClNO
2
,H
2
O 312.8 41372-20-7
Apomorphine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Emetic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Apomorphine hydrochloride contains not less than 98.0 per cent and not more than the equivalent
of 101.0 per cent of (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol
hydrochloride, calculated with reference to the dried substance.
CHARACTERS
White or faintly yellow to green-tinged greyish, crystalline powder or crystals; on exposure to air
and light, the green tinge becomes more pronounced; sparingly soluble in water and in alcohol, very
slightly soluble in ether.
IDENTIFICATION
A. Dissolve 10.0 mg in 0.1M hydrochloric acid and dilute to 100.0 ml with the same acid. Dilute
350 nm (2.2.25), the solution shows an absorption maximum at 273 nm and a shoulder at 300 nm
to 310 nm. The specific absorbance at the maximum is 530 to 570.
B. To 5 ml of solution S (see Tests) add a few millilitres of sodium hydrogen carbonate solution R
until a permanent, white precipitate is formed. The precipitate slowly becomes greenish. Add
0.25 ml of 0.05M iodine and shake. The precipitate becomes greyish-green. Collect the precipitate.
The precipitate dissolves in ether R giving a purple solution, in chloroform R giving a violet-blue
solution and in alcohol R giving a blue solution.
C. To 2 ml of solution S add 0.1 ml of nitric acid R. Mix and filter. The filtrate gives reaction (a) of
chlorides (2.3.1).
TESTS
Solution S Dissolve 0.25 g without heating in carbon dioxide-free water R and dilute to 25 ml with
the same solvent.
Appearance of solution 10 ml of solution S is clear (2.2.1) and not more intensely coloured than
a reference solution prepared as follows: dissolve 5 mg of the substance to be examined in 100 ml
of water R, transfer 1 ml of the solution to a test-tube and add 6 ml of water R, 1 ml of sodium
hydrogen carbonate solution R and 0.5 ml of 0.05M iodine; allow to stand for 30 s, add 0.6 ml of 0.1M
sodium thiosulphate and dilute to 10 ml with water R.
Specific optical rotation (2.2.7). Dissolve 0.25 g in 0.02M hydrochloric acid and dilute to 25.0 ml
with the same acid. The specific optical rotation is 48 to 52, calculated with reference to the
dried substance.
Morphine Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating
substance.
Test solution. Dissolve 20 mg of the substance to be examined in methanol R and dilute to 10 ml
Reference solution. Dilute 1 ml of the test solution to 50 ml with methanol R.
5 volumes of anhydrous formic acid R, 5 volumes of water R, 30 volumes of acetonitrile R, 30 volumes
3-2
of ethyl acetate R and 30 volumes of methylene chloride R. Dry the plate in a current of cold air until
all traces of solvent have disappeared and spray with a 30 g/l solution of sodium nitrite R. Expose the
plate to ammonia vapour for a few minutes and allow to stand in daylight for about 1 h. In the
chromatogram obtained with the test solution, no reddish-orange spot with an R
f
value 0.3 to 0.5
times that of the principal spot appears (about 2 per cent of morphine). The test is not valid unless
there is a clearly visible spot in the chromatogram obtained with the reference solution.
Loss on drying (2.2.32). 2.5 per cent to 4.2 per cent, determined on 0.50 g by drying in an oven
at 100C to 105C.
ASSAY
Dissolve 0.250 g in 5 ml of anhydrous formic acid R. Add 30 ml of anhydrous acetic acid R and 7 ml
of mercuric acetate solution R. Titrate with 0.1M perchloric acid, determining the end-point potentio-
metrically (2.2.20).
17
H
18
ClNO
2
STORAGE
__________________________________________________________________________________________________________ Ph Eur
3-3
Aprotinin
Aprotinin complies with the requirements of the 3rd edition of the European Pharmacopoeia [0580]. These
Action and use Proteolytic enzyme inhibitor.
Preparation
Aprotinin Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Aprotinin is a polypeptide consisting of a chain of fifty-eight amino acids. It inhibits
stoichiometrically the activity of several proteolytic enzymes such as chymotrypsin, kallikrein, plasmin
and trypsin. It contains not less than 3.0 European Pharmacopoeia Units of aprotinin activity per
milligram, calculated with reference to the dried substance.
PRODUCTION
Where applicable, it complies with the monograph on Products with risk of transmitting agents of animal
spongiform encephalopathies (1483).
The animals from which aprotinin is derived must fulfil the requirements for the health of animals
suitable for human consumption to the satisfaction of the competent authority.
It must have been shown to what extent the method of production allows inactivation or removal
of any contamination by viruses or other infectious agents.
The method of manufacture is validated to demonstrate that the product, if tested, would comply
with the following tests:
Abnormal toxicity (2.6.9). Inject into each mouse a quantity of the substance to be examined
containing 2 Ph. Eur. U. dissolved in a sufficient quantity of water for injections R to give a volume of
0.5 ml.
Histamine (2.6.10). Not more than 0.2 g of histamine base per 3 Ph. Eur. U.
CHARACTERS
An almost white powder, hygroscopic, soluble in water and in isotonic solutions, practically insoluble
in organic solvents.
IDENTIFICATION
A. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel G plate R.
Test solution. Use solution S (see Tests).
Reference solution. Use aprotinin solution BRP.
Apply separately to the plate 10 l of each solution. Develop over a path of 12 cm using a mixture
of 80 volumes of water R and 100 volumes of glacial acetic acid R containing per millilitre of mixture
0.10 g of sodium acetate R. Allow the plate to dry in air and spray with a solution of 0.1 g of
ninhydrin R in a mixture of 6 ml of a 10 g/l solution of cupric chloride R, 21 ml of glacial acetic acid R
and 70 ml of ethanol R. Dry the plate at 60C. The principal spot in the chromatogram obtained with
obtained with the reference solution.
B. Determine the ability of the substance to be examined to inhibit trypsin activity using the method
described below.
Test solution. Dilute 1 ml of solution S to 50 ml with buffer solution pH 7.2 R.
Trypsin solution. Dissolve 10 mg of trypsin CRS in 0.002M hydrochloric acid and dilute to 100 ml with
the same acid.
Casein solution. Dissolve 0.2 g of casein R in buffer solution pH 7.2 R and dilute to 100 ml with the
same buffer solution.
Precipitating solution. Mix 1 volume of glacial acetic acid R, 49 volumes of water R and 50 volumes of
ethanol R.
Mix 1 ml of the test solution with 1 ml of the trypsin solution. Allow to stand for 10 min and add
1 ml of the casein solution. Incubate at 35C for 30 min. Cool in iced water and add 0.5 ml of the
precipitating solution. Shake and allow to stand at room temperature for 15 min. The solution is
cloudy. Carry out a blank test under the same conditions using buffer solution pH 7.2 R instead of the
test solution. The solution is not cloudy.
TESTS
Solution S Prepare a solution of the substance to be examined containing 15 Ph. Eur. U. per
millilitre, calculated from the activity stated on the label.
3-4
Absorbance (2.2.25). Prepare a solution of the substance to be examined containing 3.0 Ph. Eur. U.
per millilitre. The solution shows an absorption maximum at 277 nm. The absorbance at the
maximum is not greater than 0.80.
Protein impurities of higher molecular mass Examine by size-exclusion chromatography
(2.2.30), using cross-linked dextran for chromatography R2. Use a 180 g/l solution of anhydrous acetic
acid R to swell the gel and as the eluent. Prepare a column of gel 0.8 m to 1.0 m long and 25 mm in
diameter, taking care to avoid the introduction of air bubbles. Place at the top of the column a
quantity of the substance to be examined containing 300 Ph. Eur. U. dissolved in 1 ml of a 180 g/l
solution of anhydrous acetic acid R and allow to elute. Collect the eluate in fractions of 2 ml. Measure
the absorbance (2.2.25) of each fraction at the absorption maximum at 277 nm and plot the values
on a graph. The chromatogram obtained does not present an absorption maximum before the elution
of aprotinin.
Loss on drying (2.2.32). Not more than 6.0 per cent, determined on 0.100 g by drying in vacuo.
Inject per kilogram of the rabbits mass 1 ml of a solution of the substance to be examined containing
15 Ph. Eur. U. per millilitre.
ASSAY
The activity of aprotinin is determined by measuring its inhibitory action on a solution of trypsin of
known activity. The inhibiting activity of aprotinin is calculated from the difference between the
initial activity and the residual activity of trypsin.
The inhibiting activity of aprotinin is expressed in Ph. Eur. Units. 1 Ph. Eur. U. inhibits 50 per
cent of the enzymatic activity of 2 microkatals of trypsin.
Use a reaction vessel with a capacity of about 30 ml and provided with: a device that will maintain
a temperature of 25 0.1C; a stirring device, such as a magnetic stirrer; a lid with five holes for
accommodating the electrodes, the tip of a burette, a tube for the admission of nitrogen and the
introduction of the reagents. An automatic or manual titration apparatus may be used. In the latter
case the burette is graduated in 0.05 ml and the pH meter is provided with a wide reading scale and
glass and calomel electrodes.
Test solution. Prepare a solution of the substance to be examined in 0.0015M borate buffer solution pH
8.0 R expected to contain 1.67 Ph. Eur. U. per millilitre (about 0.6 mg (m mg) per millilitre).
Trypsin solution. Prepare a solution of trypsin CRS containing about 0.8 microkatals per millilitre
(about 1 mg per millilitre), using 0.001M hydrochloric acid as the solvent. Use a freshly prepared
solution and keep in iced water.
Trypsin and aprotinin solution. To 4.0 ml of the trypsin solution add 1.0 ml of the test solution. Dilute
immediately to 40.0 ml with 0.0015M borate buffer solution pH 8.0 R. Allow to stand at room tempera-
ture for 10 min and then keep in iced water. Use within 6 h of preparation.
Dilute trypsin solution. Dilute 0.5 ml of the trypsin solution to 10.0 ml with 0.0015M borate buffer
solution pH 8.0 R. Allow to stand at room temperature for 10 min and then keep in iced water.
Maintain an atmosphere of nitrogen in the reaction flask and stir continuously; introduce 9.0 ml of
0.0015M borate buffer solution pH 8.0 R and 1.0 ml of a freshly prepared 6.9 g/l solution of
benzoylarginine ethyl ester hydrochloride R. Adjust to pH 8.0 by the addition of 0.1M sodium hydroxide.
When the temperature has reached equilibrium at 25 0.1C, add 1.0 ml of the trypsin and
aprotinin solution and start a timer. Maintain at pH 8.0 by the addition of 0.1M sodium hydroxide and
note the volume added every 30 s. Continue the reaction for 6 min. Determine the number of
millilitres of 0.1M sodium hydroxide used per second (n
1
ml). Carry out, under the same conditions, a
titration using 1.0 ml of the dilute trypsin solution. Determine the number of millilitres of 0.1M
sodium hydroxide used per second (n
2
ml).
Calculate the aprotinin activity in Ph. Eur. Units per milligram from the expression:
m
n n ) 2 ( 4000
1 2

The estimated activity is not less than 90 per cent and not more than 110 per cent of the activity
stated on the label.
STORAGE
Store in an airtight, tamper-proof container, protected from light.
3-5
LABELLING
The label states:
the number of Ph. Eur. Units of aprotinin activity per milligram,
__________________________________________________________________________________________________________ Ph Eur
3-6
Aprotinin Concentrated Solution
Aprotinin Concentrated Solution complies with the requirements of the 3rd edition of the European
Action and use Proteolytic enzyme inhibitor.
Preparation
Aprotinin Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Aprotinin concentrated solution is a solution of aprotinin, a polypeptide consisting of a chain of fifty-
eight amino acids, which inhibits stoichiometrically the activity of several proteolytic enzymes such as
chymotrypsin, kallikrein, plasmin and trypsin. It contains not less than 15.0 European
Pharmacopoeia Units of aprotinin activity per millilitre.
PRODUCTION
The animals from which aprotinin is derived must fulfil the requirements for the health of animals
It must have been shown to what extent the method of production allows inactivation or removal
of any contamination by viruses or other infectious agents.
with the following tests:
Abnormal toxicity (2.6.9). Inject into each mouse a quantity of the preparation to be examined
containing 2 Ph. Eur. U. diluted with a sufficient quantity of water for injections R to give a volume of
0.5 ml.
Histamine (2.6.10). Not more than 0.2 g of histamine base per 3 Ph. Eur. U.
CHARACTERS
A clear and colourless solution.
IDENTIFICATION
Test solution. Use solution S (see Tests).
Reference solution. Use aprotinin solution BRP.
of 80 volumes of water R and 100 volumes of glacial acetic acid R containing per millilitre of mixture
0.10 g of sodium acetate R. Allow the plate to dry in air and spray with a solution of 0.1 g of
ninhydrin R in a mixture of 6 ml of a 10 g/l solution of cupric chloride R, 21 ml of glacial acetic acid R
and 70 ml of ethanol R. Dry the plate at 60C. The principal spot in the chromatogram obtained with
B. Determine the ability of the preparation to be examined to inhibit trypsin activity using the
method described below.
Test solution. Dilute 1 ml of solution S to 50 ml with buffer solution pH 7.2 R.
Trypsin solution. Dissolve 10 mg of trypsin CRS in 0.002M hydrochloric acid and dilute to 100 ml with
the same acid.
Casein solution. Dissolve 0.2 g of casein R in buffer solution pH 7.2 R and dilute to 100 ml with the
same buffer solution.
Precipitating solution. Mix 1 volume of glacial acetic acid R, 49 volumes of water R and 50 volumes of
ethanol R.
Mix 1 ml of the test solution with 1 ml of the trypsin solution. Allow to stand for 10 min and add
1 ml of the casein solution. Incubate at 35C for 30 min. Cool in iced water and add 0.5 ml of the
precipitating solution. Shake and allow to stand at room temperature for 15 min. The solution is
cloudy. Carry out a blank test under the same conditions using buffer solution pH 7.2 R instead of the
test solution. The solution is not cloudy.
TESTS
Solution S A solution containing 15 Ph. Eur. U. per millilitre. Prepare it if necessary by dilution on
the basis of the activity stated on the label.
3-7
Absorbance (2.2.25). Prepare from the concentrated solution a dilution containing 3.0 Ph. Eur. U.
per millilitre. The solution shows an absorption maximum at 277 nm. The absorbance at the
Protein impurities of higher molecular mass Freeze-dry the preparation to be examined using a
pressure of 2.7 Pa and a temperature of 30C; the operation, including freeze-drying and a period of
drying at 15C to 25C, takes 6 h to 12 h. Examine by size-exclusion chromatography (2.2.30), using
cross-linked dextran for chromatography R2. Use a 180 g/l solution of anhydrous acetic acid R to swell the
gel and as the eluent. Prepare a column of gel 0.8 m to 1.0 m long and 25 mm in diameter, taking
care to avoid the introduction of air bubbles. Place at the top of the column a quantity of the prepara-
tion to be examined containing 300 Ph. Eur. U. dissolved in 1 ml of a 180 g/l solution of anhydrous
acetic acid R and allow to elute. Collect the eluate in fractions of 2 ml. Measure the absorbance
(2.2.25) of each fraction at the absorption maximum at 277 nm and plot the values on a graph. The
chromatogram obtained does not present an absorption maximum before the elution of aprotinin.
Specific activity of the dry residue Not less than 3.0 Ph. Eur. U. of aprotinin activity per
milligram of dry residue. Evaporate 25.0 ml to dryness in a water-bath, dry the residue at 110C for
15 h and weigh. From the mass of the residue and the activity determined as described below,
calculate the number of Ph. Eur. Units per milligram of dry residue.
Inject per kilogram of the rabbits mass 1 ml of a solution of the preparation to be examined contain-
ing 15 Ph. Eur. U. per millilitre.
ASSAY
The activity of aprotinin is determined by measuring its inhibitory action on a solution of trypsin of
known activity. The inhibiting activity of aprotinin is calculated from the difference between the
initial activity and the residual activity of trypsin.
The inhibiting activity of aprotinin is expressed in Ph. Eur. Units.1 European Pharmacopoeia Unit
inhibits 50 per cent of the enzymatic activity of 2 microkatals of trypsin.
Use a reaction vessel with a capacity of about 30 ml and provided with: a device that will maintain
a temperature of 25 0.1C; a stirring device, such as a magnetic stirrer; a lid with five holes for
accommodating the electrodes, the tip of a burette, a tube for the admission of nitrogen and the
introduction of the reagents. An automatic or manual titration apparatus may be used. In the latter
case the burette is graduated in 0.05 ml and the pH meter is provided with a wide reading scale and
glass and calomel electrodes.
Test solution. With 0.0015M borate buffer solution pH 8.0 R prepare an appropriate dilution (D) of the
concentrated solution expected on the basis of the stated potency to contain 1.67 Ph. Eur. U. per
millilitre.
Trypsin solution. Prepare a solution of trypsin CRS containing about 0.8 microkatals per millilitre
(about 1 mg per millilitre), using 0.001M hydrochloric acid as the solvent. Use a freshly prepared
solution and keep in iced water.
Trypsin and aprotinin solution. To 4.0 ml of the trypsin solution add 1.0 ml of the test solution. Dilute
immediately to 40.0 ml with 0.0015M borate buffer solution pH 8.0 R. Allow to stand at room tempera-
ture for 10 min and then keep in iced water. Use within 6 h of preparation.
Dilute trypsin solution. Dilute 0.5 ml of the trypsin solution to 10.0 ml with 0.0015M borate buffer
solution pH 8.0 R. Allow to stand at room temperature for 10 min and then keep in iced water.
Maintain an atmosphere of nitrogen in the reaction flask and stir continuously; introduce 9.0 ml of
0.0015M borate buffer solution pH 8.0 R and 1.0 ml of a freshly prepared 6.9 g/l solution of
benzoylarginine ethyl ester hydrochloride R. Adjust to pH 8.0 by the addition of 0.1M sodium hydroxide.
When the temperature has reached equilibrium at 25 0.1C, add 1.0 ml of the trypsin and
aprotinin solution and start a timer. Maintain at pH 8.0 by the addition of 0.1M sodium hydroxide and
note the volume added every 30 s. Continue the reaction for 6 min. Determine the number of
millilitres of 0.1M sodium hydroxide used per second (n
1
ml). Carry out, under the same conditions, a
titration using 1.0 ml of the dilute trypsin solution. Determine the number of millilitres of 0.1M
sodium hydroxide used per second (n
2
ml).
Calculate the aprotinin activity in Ph. Eur. Units per millilitre from the expression:
4000 (2n
2
-n
1
) D
D=dilution factor of the concentrated solution to be examined in order to obtain a solution
containing 1.67 Ph. Eur. Units per millilitre.
The estimated activity is not less than 90 per cent and not more than 110 per cent of the activity
stated on the label.
3-8
STORAGE
Store in an airtight, tamper-proof container, protected from light.
LABELLING
The label states:
the number of Ph. Eur. Units of aprotinin activity per millilitre.
where applicable, that the solution is sterile,
where applicable, that the solution is apyrogenic.
__________________________________________________________________________________________________________ Ph Eur
3-9
Arachis Oil
Ground-nut Oil; Peanut Oil
Arachis Oil complies with the requirements of the 3rd edition of the European Pharmacopoeia [0263]. These
Preparation
Arachis Oil Enema
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Arachis oil is the refined fatty oil obtained from the shelled seeds of Arachis hypogaea L.
CHARACTERS
A clear, yellowish, viscous liquid, very slightly soluble in alcohol, miscible with ether and with light
petroleum.
It solidifies at about 2C.
IDENTIFICATION
Carry out the identification of fatty oils by thin-layer chromatography (2.3.2). The chromatogram
obtained is similar to the typical chromatogram for arachis oil.
TESTS
Acid value (2.5.1): Not more than 0.6.
If intended for use in the manufacture of parenteral dosage forms it complies with the following
test. Not more than 0.5.
Unsaponifiable matter (2.5.7). Not more than 1.0 per cent m/m, determined on 5.0 g.
Alkaline impurities (2.4.19). It complies with the test for alkaline impurities in fatty oils.
Foreign fatty oils Carry out the test for foreign oils in fatty oils by gas chromatography (2.4.22).
The fatty-acid fraction of the oil has the following composition:
16
stearic acid: 1.3 per cent to 6.5 per cent,
cent,
43.0 per cent,
0.6 per cent,
arachidic acid: 1.0 per cent to 3.0 per cent,
gadoleic acid (equivalent chain length on polyethyleneglycol adipate 20.3): 0.5 per cent to
2.1 per cent,
behenic acid: 1.0 per cent to 5.0 per cent,
cent,
lignoceric acid: 0.5 per cent to 3.0 per cent.
The ratio linoleic acid:behenic acid is not more than 15.
Semi-drying oils Boil 1.0 g under a reflux condenser with 5 ml of a mixture of 1 volume of
alcohol R and 3 volumes of 2M alcoholic potassium hydroxide R for 5 min. Add 1.5 ml of acetic acid R
and 50 ml of alcohol (70 per cent V/V) R and heat until the solution is clear. Allow to cool or cool
very slowly, with a thermometer in the liquid. The temperature at which the liquid begins to
become cloudy is not lower than 36C.
Sesame oil In a ground-glass-stoppered graduated cylinder, shake 10 ml for about 1 min with a
mixture of 0.5 ml of a 0.35 per cent V/V solution of furfural R in acetic anhydride R and 4.5 ml of
acetic anhydride R. Filter through a filter paper impregnated with acetic anhydride R.To the filtrate
add 0.2 ml of sulphuric acid R. No bluish-green colour develops.
Water (2.5.12). If intended for use in the manufacture of parenteral dosage forms it complies with
the following test. Not more than 0.3 per cent, determined on 3.00 g by the semi-micro
3-10
STORAGE
Store in a well-filled, well-closed container, protected from light.
LABELLING
The label states:
forms,
__________________________________________________________________________________________________________ Ph Eur
3-11
Hydrogenated Arachis Oil
Hydrogenated Arachis Oil complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hydrogenated arachis oil is the product obtained by refining, bleaching, hydrogenating and
deodorising oil obtained from the shelled seeds of Arachis hypogaea L. Each type of hydrogenated
arachis oil is characterised by its nominal drop point.
CHARACTERS
A white or faintly yellowish, soft mass which melts to a clear, pale yellow liquid when heated,
practically insoluble in water, freely soluble in methylene chloride and in light petroleum (bp: 65C to
70C), very slightly soluble in alcohol.
IDENTIFICATION
A. It complies with the test for drop point.
B. Carry out the identification of fatty oils by thin-layer chromatography (2.3.2). The chromatogram
obtained is similar to the typical chromatogram for arachis oil.
C. It complies with the test for foreign fatty oils.
TESTS
Drop point (2.2.17). 32C to 43C. Within this range the drop point does not differ by more than
3C from the nominal value.
Acid value (2.5.1). Not more than 0.5. Dissolve 10.0 g in 50 ml of the prescribed solvent by heating
on a water-bath.
Peroxide value (2.5.5). Not more than 5.0. Dissolve 5.0 g in 30 ml of the prescribed solvent by
heating on a water-bath.
Unsaponifiable matter (2.5.7). Not more than 1.0 per cent.
Alkaline impurities (2.4.19). It complies with the test for alkaline impurities in fatty oils.
Foreign fatty oils Carry out the test for foreign fatty oils in fatty oils by gas chromatography
(2.4.22).
a capillary fused-silica column 25 m long and 0.25 mm in internal diameter, coated with
poly(cyanopropyl)siloxane R (film thickness 0.2 m),
helium for chromatography R as the carrier gas at a flow rate of 0.7 ml per minute,
a split injector (1:100),
maintaining the temperature of the column at 180C for 20 min and that of the injection port and of
the detector at 250C.
The fatty acid fraction of the oil has the following composition:
14
myristic acid: not more than 0.5 per cent,
oleic acid and isomers (C
18:1
, equivalent chain length on poly(cyanopropyl)siloxane 18.5 to
18.8): 54.0 per cent to 78.0 per cent,
linoleic acid and isomers (C
18:2
19.8): not more than 10.0 per cent,
arachidic acid: 1.0 per cent to 3.0 per cent,
eicosenoic acids (C
20:1
, equivalent chain length on poly(cyanopropyl)siloxane 20.4 to 20.7): not
more than 2.1 per cent,
behenic acid: 1.0 per cent to 5.0 per cent,
erucic acid and isomers (C
22:1
lignoceric acid: 0.5 per cent to 3.0 per cent.
Nickel Not more than 1 ppm of Ni, determined by atomic absorption spectrometry (Method II,
2.2.23).
3-12
Test solution. Into a platinum or silica crucible previously tared after ignition introduce 5.0 g.
Cautiously heat and introduce into the substance a wick formed from twisted ashless filter paper.
Ignite the wick. When the substance has ignited stop heating. After combustion, ignite in a muffle
furnace at about 600C. Continue ignition until white ash is obtained. After cooling, take up the
residue with two quantities, each of 2 ml, of dilute hydrochloric acid R and transfer into a 25 ml
graduated flask. Add 0.3 ml of nitric acid R and dilute to 25.0 ml with water R.
Reference solutions. Prepare three reference solutions by adding 1.0 ml, 2.0 ml and 4.0 ml of nickel
standard solution (0.2 ppm Ni) R to 2.0 ml of the test solution and diluting to 10.0 ml with water R.
Measure the absorbance at 232 nm using a nickel hollow-cathode lamp as a source of radiation, a
graphite furnace as an atomic generator and argon R as the carrier gas.
Water (2.5.12). Not more than 0.3 per cent, determined on 1.000 g by the semi-micro determination
of water.
STORAGE
LABELLING
The label states the nominal drop point.
__________________________________________________________________________________________________________ Ph Eur
3-13
Arginine
H
2
N N
COOH
NH
2
H
NH
H
C
6
H
14
N
4
O
2
174.2 74-79-3
Arginine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0806]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Arginine contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of
(S)-2-amino-5-guanidinopentanoic acid, calculated with reference to the dried substance.
PRODUCTION
When Arginine is produced by a process involving fermentation steps, it complies with the
CHARACTERS
A white or almost white, crystalline powder or colourless crystals, freely soluble in water, very
slightly soluble in alcohol, practically insoluble in ether.
IDENTIFICATION
B. Solution S (see Tests) is strongly alkaline (2.2.4).
obtained with arginine CRS. Examine the substances prepared as discs.
D. Examine the chromatograms obtained in the test for ninhydrin-positive substances. The
principal spot in the chromatogram obtained with test solution (b) is similar in position, colour and
size to the principal spot in the chromatogram obtained with reference solution (a).
E. Dissolve about 25 mg in 2 ml of water R. Add 1 ml of -naphthol solution R and 2 ml of a
mixture of equal volumes of strong sodium hypochlorite solution R and water. A red colour develops.
TESTS
solution BY
6
(Method II, 2.2.2).
Specific optical rotation (2.2.7). Dissolve 2.00 g in hydrochloric acid R1 and dilute to 25.0 ml
with the same acid. The specific optical rotation is +25.5 to +28.5, calculated with reference to
Ninhydrin-positive substances Examine by thin-layer chromatography (2.2.27), using a TLC
silica gel plate R.
Test solution (a). Dissolve 0.10 g of the substance to be examined in dilute hydrochloric acid R and
dilute to 10 ml with the same acid.
Reference solution (a). Dissolve 10 mg of arginine CRS in 0.1M hydrochloric acid and dilute to 50 ml
with the same acid.
Reference solution (b). Dilute 5 ml of test solution (b) to 20 ml with water R.
Reference solution (c). Dissolve 10 mg of arginine CRS and 10 mg of lysine hydrochloride CRS in 0.1M
hydrochloric acid and dilute to 25 ml with the same acid.
of 15 cm using a mixture of 30 volumes of concentrated ammonia R and 70 volumes of 2-propanol R.
Dry the plate at 100C to 105C until the ammonia disappears completely. Spray with ninhydrin
3-14
solution R and heat at 100C to 105C for 15 min. Any spot in the chromatogram obtained with
test solution (a), apart from the principal spot, is not more intense than the spot in the chromato-
gram obtained with reference solution (b) (0.5 per cent). The test is not valid unless the chromato-
gram obtained with reference solution (c) shows two clearly separated spots.
Chlorides (2.4.4). To 5 ml of solution S add 0.5 ml of dilute nitric acid R and dilute to 15 ml with
water R. The solution complies with the limit test for chlorides (200 ppm).
Sulphates (2.4.13). To 10 ml of solution S, add 1.7 ml of dilute hydrochloric acid R and dilute to
15 ml with distilled water R. The solution complies with the limit test for sulphates (300 ppm).
the lower watch-glass and dissolve in 0.5 ml of water R. To the solution add 0.30 g of heavy
4
) R, 0.5 ml of water R and 0.30 g of heavy magnesium
oxide R (200 ppm).
combined organic layers add 10 ml of water R and shake for 3 min. The aqueous layer complies
with the limit test for iron (10 ppm).
Heavy metals (2.4.8). Dissolve 2.0 g in water R and dilute to 20 ml with the same solvent. 12 ml
lead standard solution (1 ppm Pb) R.
at 100C to 105C.
ASSAY
Dissolve 0.150 g in 50 ml of water R. Using 0.2 ml of methyl red mixed solution R as indicator, titrate
with 0.1M hydrochloric acid until the colour changes from green to violet-red.
6
H
14
N
4
O
2
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
3-15
Arginine Hydrochloride
H
2
N N
COOH
NH
2
H
NH
H
,HCl
C
6
H
15
ClN
4
O
2
210.7 1119-34-2
Arginine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Arginine Hydrochloride Intravenous Infusion
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Arginine hydrochloride contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of the hydrochloride of (S)-2-amino-5-guanidinopentanoic acid, calculated with
PRODUCTION
When Arginine Hydrochloride is produced by a process involving fermentation steps, it complies with
the requirements of the monograph on Products of fermentation (1468).
CHARACTERS
A white or almost white, crystalline powder or colourless crystals, freely soluble in water, very
slightly soluble in alcohol, practically insoluble in ether.
IDENTIFICATION
obtained with arginine hydrochloride CRS. Examine the substances prepared as discs.
C. Examine the chromatograms obtained in the test for ninhydrin-positive substances. The
D. Dissolve about 25 mg in 2 ml of water R. Add 1 ml of -naphthol solution R and 2 ml of a
mixture of equal volumes of strong sodium hypochlorite solution R and water R. A red colour develops.
E. About 20 mg gives reaction (a) of chlorides (2.3.1).
TESTS
solution BY
6
(Method II, 2.2.2).
silica gel plate R
Test solution (a). Dissolve 0.10 g of the substance to be examined in water R and dilute to 10 ml
Reference solution (a). Dissolve 10 mg of arginine hydrochloride CRS in water R and dilute to 50 ml
Reference solution (c). Dissolve 10 mg of arginine hydrochloride CRS and 10 mg of lysine hydrochloride
CRS in water R and dilute to 25 ml with the same solvent.
3-16
of 15 cm using a mixture of 30 volumes of concentrated ammonia R and 70 volumes of 2-propanol R.
Dry the plate at 100C to 105C until the ammonia disappears completely. Spray with ninhydrin
solution R and heat at 100C to 105C for 15 min. Any spot in the chromatogram obtained with test
solution (a), apart from the principal spot, is not more intense than the spot in the chromatogram
obtained with reference solution (b) (0.5 per cent). The test is not valid unless the chromatogram
obtained with reference solution (c) shows two clearly separated spots.
To the inner wall of the upper watch glass stick a piece of red litmus paper R 5 mm square and wetted
watch-glass and dissolve in 0.5 ml of water R. To the solution add 0.30 g of heavy magnesium oxide R.
Briefly triturate with a glass rod. Immediately close the cell by putting the two watch-glasses
4
at 100C to 105C.
ASSAY
Dissolve 0.180 g in 3 ml of anhydrous formic acid R. Add 30 ml of anhydrous acetic acid R. Using
0.1 ml of naphtholbenzein solution R as indicator, titrate with 0.1M perchloric acid until the colour
6
H
15
ClN
4
O
2
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
3-17
Arnica Flower
corrected 1/01
Arnica Flower complies with the requirements of the 3rd edition of the European Pharmacopoeia [1391]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Arnica flower consists of the whole or partially broken, dried flower-heads of Arnica montana L. It
contains not less than 0.40 per cent m/m of total lactone sesquiterpenes expressed as helenalin tiglate,
calculated with reference to the dried drug.
CHARACTERS
It has an aromatic odour.
The capitulum when spread out, is about 20 mm in diameter and about 15 mm deep, and has a
peduncle of 2 cm to 3 cm in length. The involucre consists of eighteen to twenty-four elongated
lanceolate bracts, with acute apices, arranged in one or two rows: the bracts, 8 mm to about 10 mm
long, are green with yellowish-green external hairs visible under a lens. The receptacle, about 6 mm
in diameter, is convex, alveolate and covered with hairs. Its periphery bears about twenty ligulate
florets 20 mm to 30 mm long; the disc bears a greater number of tubular florets about 15 mm long.
The ovary, 4 mm to 8 mm long, is crowned by a pappus of whitish bristles 4 mm to 8 mm long.
Some brown achenes, crowned or not by a pappus, may be present.
IDENTIFICATION
A. The involucre consists of elongated oval bracts with acute apices; the margin is ciliated. The
ligulate floret has a reduced calyx crowned by fine, shiny, whitish bristles, bearing small coarse
trichomes. The orange-yellow corolla bears seven to ten parallel veins and ends in three small lobes.
The stamens, with free anthers, are incompletely developed. The narrow, brown ovary bears a stigma
divided into two branches curving outwards. The tubular floret is actinomorphic. The ovary and the
calyx are similar to those of the ligulate floret. The short corolla has five reflexed triangular lobes; the
five fertile stamens are fused at the anthers.
B. Separate the capitulum into its different parts. Reduce to a powder (355). Examine under a
microscope using chloral hydrate solution R. The powder shows the following characteristics. The
epidermises of the bracts of the involucre have stomata and trichomes, which are more abundant on
the outer (abaxial) surface. There are several different types of trichomes: uniseriate multicellular
covering trichomes, varying in length from 50 m to 500 m, particularly abundant on the margins of
the bract; secretory trichomes with uni-or biseriate multicellular stalks and with multicellular,
globular heads, about 300 m long, abundant on the outer surface of the bract; secretory trichomes
with uniseriate multicellular stalks and with multicellular, globular heads, about 80 m long,
abundant on the inner surface of the bract. The epidermis of the ligulate corolla consists of lobed or
elongated cells, a few stomata and trichomes of different types: covering trichomes, with very sharp
ends, whose length may exceed 500 m, consisting of one to three proximal cells with thickened walls
and two to four distal cells with thin walls; secretory trichomes with biseriate multicellular heads;
secretory trichomes with multicellular stalks and multicellular globular heads. The ligule ends in
rounded papillose cells. The epidermis of the ovary is covered with trichomes: secretory trichomes
with short stalks and multicellular globular heads; twinned covering trichomes usually consisting of
two longitudinally united cells, with common punctuated walls; their ends are sharp and sometimes
bifid. The epidermises of the calyx consist of elongated cells bearing short, unicellular, covering
trichomes pointing towards the upper end of the bristle. The pollen grains have a diameter of about
30 m and are rounded, with a spiny exine; they have three germinal pores.
C. Examine the chromatograms obtained in the test for Calendula officinalis L. Heterotheca inuloides.
The chromatogram obtained with the test solution shows, in the middle, a fluorescent blue zone
corresponding to the zone due to chlorogenic acid in the chromatogram obtained with the reference
solution; it shows, above this zone, three fluorescent yellowish-brown to orange-yellow zones, and
above these three zones a fluorescent greenish-yellow zone corresponding to astragalin. The zone
located below the astragalin zone corresponds to isoquercitroside; the zone located just below this
zone corresponds to luteolin-7-glucoside. It also shows a fluorescent greenish-blue zone below the
zone due to caffeic acid in the chromatogram obtained with the reference solution.
TESTS
Foreign matter (2.8.2). Not more than 5.0 per cent.
3-18
Calendula officinalis L Heterotheca inuloides Examine by thin-layer chromatography (2.2.27),
using a TLC silica gel plate R.
Test solution. To 2.00 g of the powdered drug (710) add 10 ml of methanol R. Heat in a water-bath at
60C for 5 min with shaking. Cool and filter.
Reference solution. Dissolve 2.0 mg of caffeic acid R, 2.0 mg of chlorogenic acid R and 5.0 mg of rutin R
in methanol R and dilute to 30 ml with the same solvent.
Apply separately to the plate, as bands, 15 l of each solution. Develop over a path of 15 cm using
a mixture of 10 volumes of anhydrous formic acid R, 10 volumes of water R, 30 volumes of methyl ethyl
ketone R and 50 volumes of ethyl acetate R. Allow the plate to dry in air for a few minutes. Spray with
a 10 g/l solution of diphenylboric acid aminoethyl ester R in methanol R and then with a 50 g/l solution
of macrogol 400 R in methanol R. Heat for 5 min at 100C to 105C. Allow the plate to dry in air and
examine in ultraviolet light at 365 nm. The chromatogram obtained with the reference solution
shows in the lower part an orange-yellow fluorescent zone (rutin), in the middle part a fluorescent
zone due to chlorogenic acid and in the upper part a light bluish fluorescent zone (caffeic acid). The
chromatogram obtained with the test solution does not show a fluorescent orange-yellow zone
corresponding to rutin in the chromatogram obtained with the reference solution, nor does it show a
zone below the zone corresponding to rutin.
ASSAY
Examine by liquid chromatography (2.2.29), using santonin R as the internal standard.
Internal standard solution. Dissolve immediately before use 0.010 g of santonin R accurately weighed in
10.0 ml of methanol R.
Test solution. In a 250 ml round-bottomed flask introduce 1.00 g of the powdered drug (355), add
50 ml of a mixture of equal volumes of methanol R and water R and heat under a reflux condenser in
a water-bath at 50C to 60C for 30 min, shaking frequently. Allow to cool and filter through a paper
filter. Add the paper filter, cut in pieces, to the residue in the round-bottomed flask, add 50 ml of a
mixture of equal volumes of methanol R and water R and heat under a reflux condenser in a water-
bath at 50C to 60C for 30 min, shaking frequently. Repeat this procedure twice. To the combined
filtrate add 3.00 ml of the internal standard solution and evaporate to 18 ml under reduced
pressure. Rinse the round-bottomed flask with water R and dilute, with the washings, to 20.0 ml.
Transfer the solution to a chromatography column about 0.15 m long and about 30 mm in internal
diameter containing 15 g of kieselguhr for chromatography R. Allow to stand for 20 min. Elute with
200 ml of a mixture of equal volumes of ethyl acetate R and methylene chloride R. Evaporate the eluate
to dryness in a 250 ml round-bottomed flask. Dissolve the residue in 10.0 ml of methanol R and add
10.0 ml of water R. Add 7.0 g of neutral aluminium oxide R, shake for 120 s, centrifuge (at 5000 g for
10 min) and filter through a paper filter. Evaporate 10.0 ml of the filtrate to dryness. Dissolve the
residue in 3.0 ml of a mixture of equal volumes of methanol R and water R and filter.
Mobile phase A. Water R,
Time
(min)
Mobile
phase A
(per cent V/V)
Mobile
phase B
(per cent V/V)
Comment
0 3 62 38 isocratic
Calculate the percentage of total lactone sesquiterpenes, expressed as helenalin tiglate, from the
expression:
3-19
1000
100 187 . 1
S
LS

m S
V C S
S
LS
=area of all peaks corresponding to lactone sesquiterpenes appearing after the santonin
peak in the chromatogram obtained with the test solution,
S
S
=area of the peak corresponding to santonin in the chromatogram obtained with the test
solution,
m=mass of the drug to be examined, in grams,
C=concentration of santonin in the internal standard solution used for the test solution, in
milligrams per millilitre,
V=volume of the internal standard solution used for the test solution, in millilitres,
1. 187=peak correlation factor between helenalin tiglate and santonin.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
3-20
Ascorbic Acid
O
O
OH HO
HCOH
CH
2
OH
C
6
H
8
O
6
176.1 50-81-7
Ascorbic Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia [0253]. These
Action and use Used in the treatment of vitamin C deficiency.
Preparations
Ascorbic Acid Injection
Ascorbic Acid Tablets
Vitamins B and C Injection
When vitamin C is prescribed or demanded, Ascorbic Acid shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ascorbic acid contains not less than 99.0 per cent and not more than the equivalent of 100.5 per
cent of (R)-5-[(S)-1,2-dihydroxyethyl]-3,4-dihydroxy-5H-furan-2-one.
CHARACTERS
A white or almost white, crystalline powder or colourless crystals, becoming discolored on
exposure to air and moisture, freely soluble in water, soluble in alcohol, practically insoluble in
ether.
IDENTIFICATION
First identification: B, C.
A. Dissolve 0.10 g in water R and dilute immediately to 100.0 ml with the same solvent. To 10 ml of
0.1M hydrochloric acid, add 1.0 ml of the solution and dilute to 100.0 ml with water R. Measure the
absorbance (2.2.25) at the maximum at 243 nm immediately after dissolution. The specific
obtained with ascorbic acid CRS. Examine the substance prepared as discs containing 1 mg.
C. The pH (2.2.3) of solution S (see Tests) is 2.1 to 2.6.
D. To 1 ml of solution S add 0.2 ml of dilute nitric acid R and 0.2 ml of silver nitrate solution R2. A
grey precipitate is formed.
TESTS
Solution S. Dissolve 1.0 g in carbon dioxide-free water R and dilute to 20 ml with the same solvent.
solution BY
7
(Method II, 2.2.2).
solvent. The specific optical rotation is +20.5 to +21.5.
Oxalic acid Dissolve 0.25 g in 5 ml of water R. Neutralise to red litmus paper R using dilute sodium
hydroxide solution R and add 1 ml of dilute acetic acid R and 0.5 ml of calcium chloride solution R (test
solution). Prepare a reference solution as follows: dissolve 70 mg of oxalic acid R in water R and dilute
to 500 ml with the same solvent; to 5 ml of this solution add 1 ml of dilute acetic acid R and 0.5 ml of
calcium chloride solution R (reference solution). Allow the solutions to stand for 1 h. Any opalescence
in the test solution is not more intense than that in the reference solution (0.2 per cent).
Copper Not more than 5 ppm of Cu, determined by atomic absorption spectrophotometry (Method
I, 2.2.23).
3-21
Test solution. Dissolve 2.0 g of the substance to be examined in 0.1M nitric acid and dilute to 25.0 ml
with the same acid.
Reference solutions. Prepare reference solutions containing 0.2 ppm, 0.4 ppm and 0.6 ppm of Cu by
diluting copper standard solution (10 ppm Cu) R with 0.1M nitric acid.
Measure the absorbance at 324.8 nm using a copper hollow-cathode lamp as a source of radiation
and an air-acetylene flame. Adjust the zero of the apparatus using 0.1M nitric acid.
Iron. Not more than 2 ppm of Fe, determined by atomic absorption spectrophotometry (Method I,
2.2.23).
Test solution. Dissolve 5.0 g of the substance to be examined in 0.1M nitric acid and dilute to 25.0 ml
with the same acid.
Reference solutions. Prepare reference solutions containing 0.2 ppm, 0.4 ppm and 0.6 ppm of Fe by
diluting iron standard solution (20 ppm Fe) R with 0.1M nitric acid.
Measure the absorbance at 248.3 nm using an iron hollow-cathode lamp as a source of radiation and
an air-acetylene flame. Adjust the zero of the apparatus using 0.1M nitric acid.
Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g
ASSAY
Dissolve 0.150 g in a mixture of 10 ml of dilute sulphuric acid R and 80 ml of carbon dioxide-free
water R. Add 1 ml of starch solution R. Titrate with 0.05M iodine until a persistent violet-blue colour is
obtained.
6
H
8
O
6
.
STORAGE
Store in a well-closed, non-metallic container, protected from light.
__________________________________________________________________________________________________________ Ph Eur
3-22
Ascorbyl Palmitate
O
O
OH HO
HCOH
CH
2
OCO[CH
2
]
14
CH
3
C
22
H
38
O
7
414.5 137-66-6
Ascorbyl Palmitate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0807].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ascorbyl palmitate contains not less than 98.0 per cent and not more than the equivalent of
100.5 per cent of (S)-2-[(R)-3,4-dihydroxy-5-oxo-2,5-dihydro-2-furyl]-2-hydroxyethyl
hexadecanoate, calculated with reference to the dried substance.
CHARACTERS
A white or yellowish-white powder, practically insoluble in water, freely soluble in alcohol and in
methanol, practically insoluble in methylene chloride and in fatty oils.
IDENTIFICATION
B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph. Eur.
reference spectrum of ascorbyl palmitate.
C. Dissolve about 10 mg in 5 ml of methanol R. The solution decolourises dichlorophenolindophenol
standard solution R.
TESTS
solution BY
4
(Method I, 2.2.2).
60C for 5 h.
ASSAY
Dissolve 0.160 g in 50 ml of methanol R. Add 30 ml of water R and 1 ml of starch solution R. Titrate
with 0.05M iodine until a persistent violet-blue colour is obtained.
22
H
38
O
7
.
STORAGE
Store in an airtight container, protected from light, at a temperature of 8C to 15C.
__________________________________________________________________________________________________________ Ph Eur
3-23
Aspartame
HOOC N
NH
2
H
O
H COOMe
H
C
14
H
18
N
2
O
5
294.3 53906-69-7
Aspartame complies with the requirements of the 3rd edition of the European Pharmacopoeia [0973]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Aspartame contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent
of (S)-3-amino-N-[(S)-1-(methoxycarbonyl)-2-phenylethyl]succinamic acid, calculated with
CHARACTERS
A white, crystalline powder, slightly hygroscopic, sparingly soluble or slightly soluble in water and
in alcohol, practically insoluble in hexane and in methylene chloride.
IDENTIFICATION
A. Dissolve 0.1 g in alcohol R and dilute to 100 ml with the same solvent. Examined between
230 nm and 300 nm (2.2.25), the solution shows absorption maxima at 247 nm, 252 nm, 258 nm
and 264 nm.
obtained with aspartame CRS. Examine the substances prepared as discs.
C. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance.
Test solution. Dissolve 15 mg of the substance to be examined in 2.5 ml of water R and dilute to
10 ml with acetic acid R.
Reference solution. Dissolve 15 mg of aspartame CRS in 2.5 ml of water R and dilute to 10 ml with
acetic acid R.
of 2 volumes of water R, 4 volumes of anhydrous formic acid R, 30 volumes of methanol R and 64
volumes of methylene chloride R. Allow the plate to dry in air. Spray with ninhydrin solution R and
heat at 100C to 105C for 15 min. The spot in the chromatogram obtained with the test solution
is similar in position, colour and size to the spot in the chromatogram obtained with the reference
solution.
D. Dissolve about 20 mg in 5 ml of methanol R and add 1 ml of alkaline hydroxylamine solution R1.
Heat on a water-bath for 15 min. Allow to cool and adjust to about pH 2 with dilute hydrochloric
acid R. Add 0.1 ml of ferric chloride solution R1. A brownish-red colour is produced.
TESTS
solution GY
6
(Method II, 2.2.2).
Conductivity (2.2.38). Not more than 30 S.cm
1
. Dissolve 0.80 g in carbon dioxide-free water R
prepared from distilled water R and dilute to 100.0 ml with the same solvent. Measure the
conductivity of the solution (C
1
) and that of the water used for preparing the solution (C
2
). The
readings must be stable within 1 per cent over a period of 30 s. Calculate the conductivity of the
solution of the substance to be examined from the expression:
C
1
0.992 C
2
Specific optical rotation (2.2.7). Dissolve 2.00 g in a 690 g/l solution of anhydrous formic acid R
3-24
and dilute to 50.0 ml with the same solution. The specific optical rotation is +14.5 to +16.5,
measured within 30 min of preparation of the solution and calculated with reference to the dried
substance.
Test solution. Dissolve 0.60 g of the substance to be examined in a mixture of 1.5 volumes of glacial
acetic acid R and 98.5 volumes of water R and dilute to 100.0 ml with the same mixture of solvents.
Reference solution (a). Dissolve 9.0 mg of aspartame impurity A CRS in a mixture of 1.5 volumes of
glacial acetic acid R and 98.5 volumes of water R and dilute to 100.0 ml with the same mixture of
solvents.
Reference solution (b). Dissolve 30.0 mg of phenylalanine R in a mixture of 15 volumes of glacial
acetic acid R and 85 volumes of water R and dilute to 100.0 ml with the same mixture of solvents.
Dilute 1.0 ml of the solution to 10.0 ml with water R.
Reference solution (c). Dilute 5.0 ml of the test solution to 10.0 ml with water R. Dilute 3.0 ml of the
Reference solution (d). Dissolve 30.0 mg of L-aspartyl-L-phenylalanine R in a mixture of 15 volumes of
glacial acetic acid R and 85 volumes of water R and dilute to 100.0 ml with the same mixture of
solvents. Dilute 1.0 ml of the solution to 10.0 ml with water R. Mix 1.0 ml of this solution with
1.0 ml of reference solution (b).
silica gel for chromatography R (5 m to 10 m),
volumes of a 6.8 g/l solution of potassium dihydrogen phosphate R previously adjusted to pH 3.7
with phosphoric acid R,
principal peak in the chromatogram obtained is not less than 50 per cent of the full scale of the
recorder. Inject 20 l of reference solution (d). The test is not valid unless in the chromatogram
obtained, the resolution between the peaks due to phenylalanine and L-aspartyl-L-phenylalanine is
at least 3.5. Inject separately 20 l of each solution. Continue the chromatography for twice the
retention time of aspartame. In the chromatogram obtained with the test solution: the area of any
peak corresponding to aspartame impurity A is not greater than the area of the principal peak in the
chromatogram obtained with reference solution (a) (1.5 per cent); the area of any peak correspond-
ing to phenylalanine is not greater than the area of the principal peak in the chromatogram obtained
with reference solution (b) (0.5 per cent); the sum of the areas of any other peaks, apart from the
principal peak, is not greater than the area of the principal peak in the chromatogram obtained with
reference solution (c) (1.5 per cent). Disregard any peak due to the solvent.
at 100C to 105C.
ASSAY
Dissolve 0.250 g in 1.5 ml of anhydrous formic acid R and 60 ml of glacial acetic acid R. Titrate
immediately with 0.1M perchloric acid, determining the end-point potentiometrically (2.2.20).
14
H
18
N
2
O
5.
STORAGE
IMPURITIES
N
N O
O
HOOC
H
A. 2-(5-benzyl-3,6-dioxopiperazin-2-yl)acetic acid (diketopiperazine),
3-25
N HOOC
COOH H
O
NH
2
H
H
N HOOC
COOH H
O
NH
2
H
H
N HOOC
COOH H
O
NH
2
H
H
B. L-aspartyl-L-phenylalanine,
H
2
N
COOH H
C. phenylalanine.
__________________________________________________________________________________________________________ Ph Eur
3-26
Aspartic Acid
COOH
NH
2
H
HOOC
C
4
H
7
NO
4
133.1 6899-03-2
Aspartic Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia [0797]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Aspartic acid contains not less than 98.5 per cent and not more than the equivalent of 101.5 per
cent of (S)-2-aminobutanedioic acid, calculated with reference to the dried substance.
PRODUCTION
When Aspartic Acid is produced by a process involving fermentation steps, it complies with the
CHARACTERS
A white or almost white, crystalline powder or colourless crystals, slightly soluble in water,
practically insoluble in alcohol and in ether. It dissolves in dilute solutions of alkali hydroxides and
in dilute mineral acids.
IDENTIFICATION
Second identification: A, B, D.
B. A suspension of 1 g in 10 ml of water R is strongly acid (2.2.4).
obtained with aspartic acid CRS. Examine the substances prepared as discs.
D. Examine the chromatograms obtained in the test for ninhydrin-positive substances. The
TESTS
Appearance of solution Dissolve 0.5 g in 1M hydrochloric acid and dilute to 10 ml with the same
acid. The solution is clear (2.2.1) and not more intensely coloured than reference solution BY
6
(Method II, 2.2.2).
silica gel plate R.
Test solution (a). Dissolve 0.10 g of the substance to be examined in 2 ml of ammonia R and dilute
to 10 ml with water R.
Reference solution (a). Dissolve 10 mg of aspartic acid CRS in 2 ml of dilute ammonia R1 and dilute
Reference solution (c). Dissolve 10 mg of aspartic acid CRS and 10 mg of glutamic acid CRS in 2 ml of
dilute ammonia R1 and dilute to 25 ml with water R.
of 15 cm using a mixture of 20 volumes of glacial acetic acid R, 20 volumes of water R and 60
volumes of butanol R. Allow the plate to dry in air, spray with ninhydrin solution R. Heat at 100C to
105C for 15 min. Any spot in the chromatogram obtained with test solution (a), apart from the
3-27
solution (c) shows two clearly separated principal spots.
Chlorides (2.4.4). Dissolve 0.25 g in 3 ml of dilute nitric acid R and dilute to 15 ml with water R.
The solution, without the further addition of dilute nitric acid R, complies with the limit test for
chlorides (200 ppm).
Sulphates (2.4.13). Dissolve 0.5 g in 4 ml of hydrochloric acid R and dilute to 15 ml with distilled
water R. The solution complies with the limit test for sulphates (300 ppm). Carry out the evaluation
of the test after 30 min.
the lower watch-glass and suspend in 0.5 ml of water R. To the suspension add 0.30 g of heavy
coloured than a standard prepared at the same time in the same manner using 0.1 ml of ammonium
standard solution (100 ppm NH
4
) R, 0.5 ml of water R and 0.30 g of heavy magnesium oxide R
(200 ppm).
at 100C to 105C.
ASSAY
Dissolve 0.100 g in 50 ml of carbon dioxide-free water R, with slight heating if necessary. Cool and
add 0.1 ml of bromothymol blue solution R1. Titrate with 0.1M sodium hydroxide until the colour
changes from yellow to blue.
4
H
7
NO
4
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
3-28
Aspirin
NOTE The name Aspirin may be freely used in many countries including the United Kingdom. In countries
where exclusive proprietary rights in this name are claimed, the official title is Acetylsalicylic Acid.
O
O
CH
3
COOH
C
9
H
8
O
4
180.2 50-78-2
Aspirin complies with the requirements of the 3rd edition of the European Pharmacopoeia for Acetylsalicylic
Acid [0309]. These requirements are reproduced after the heading Definition below.
Action and use Analgesic; antipyretic.
Preparations
Aspirin Tablets
Dispersible Aspirin Tablets
Effervescent Soluble Aspirin Tablets
Enteric-coated Aspirin Tablets
Aspirin and Caffeine Tablets
Co-codaprin Tablets
Dispersible Co-codaprin Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Acetylsalicylic acid contains not less than 99.5 per cent and not more than the equivalent of
101.0 per cent of 2-(acetyloxy)benzoic acid, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder or colourless crystals, slightly soluble in water, freely soluble in alcohol,
soluble in ether.
It melts at about 143C (instantaneous method).
IDENTIFICATION
obtained with acetylsalicylic acid CRS.
B. To 0.2 g add 4 ml of dilute sodium hydroxide solution R and boil for 3 min. Cool and add 5 ml of
dilute sulphuric acid R. A crystalline precipitate is formed. Filter, wash the precipitate and dry at
100C to 105C. The melting point (2.2.14) is 156C to 161C.
C. In a test tube mix 0.1 g with 0.5 g of calcium hydroxide R. Heat the mixture and expose to the
fumes produced a piece of filter paper impregnated with 0.05 ml of nitrobenzaldehyde solution R. A
greenish-blue or greenish-yellow colour develops on the paper. Moisten the paper with dilute hydro-
chloric acid R. The colour becomes blue.
D. Dissolve with heating about 20 mg of the precipitate obtained in identification test B in 10 ml of
water R and cool. The solution gives reaction (a) of salicylates (2.3.1).
TESTS
Appearance of solution Dissolve 1.0 g in 9 ml of alcohol R. The solution is clear (2.2.1) and
colourless (Method II, 2.2.2).
Related substance Examine by liquid chromatography (2.2.29). Prepare the solutions immediately
before use.
Test solution. Dissolve 0.10 g of the substance to be examined in acetonitrile for chromatography R and
Reference solution (a). Dissolve 50.0 mg of salicylic acid R in the mobile phase and dilute to 50.0 ml
with the mobile phase. Dilute 1.0 ml of this solution to 100.0 with the mobile phase.
Reference solution (b). Dissolve 10.0 mg of salicylic acid R in the mobile phase and dilute to 10.0 ml
with the mobile phase. To 1.0 ml of this solution add 0.2 ml of the test solution and dilute to
3-29
as mobile phase at a flow rate of 1 ml/min a mixture of 2 volumes of phosphoric acid R, 400
volumes of acetonitrile for chromatography R and 600 volumes of water R,
Inject 10 l of each solution. Continue the chromatography of the test solution for seven times the
retention time of acetylsalicylic acid. The test is not valid unless in the chromatogram obtained with
reference solution (b), the resolution between the two principal peaks is at least 6.0.
In the chromatogram obtained with the test solution the area of any peak, apart from the principal
peak, is not greater than the area of the principal peak in the chromatogram obtained with reference
solution (a) (0.1 per cent); the sum of the areas of all the peaks is not greater than 2.5 times the area
of the principal peak in the chromatogram obtained with reference solution (a) (0.25 per cent).
Disregard any peak with an area less than 0.25 times the area of the principal peak in the chromato-
Heavy metals (2.4.8). Dissolve 1.0 g in 12 ml of acetone R and dilute to 20 ml with water R. 12 ml
of this solution complies with limit test B for heavy metals (20 ppm). Prepare the standard using lead
standard solution (1 ppm Pb) obtained by diluting lead standard solution (100 ppm Pb) R with a
mixture of 6 volumes of water R and 9 volumes of acetone R.
ASSAY
In a flask with a ground-glass stopper, dissolve 1.000 g in 10 ml of alcohol R. Add 50.0 ml of 0.5M
sodium hydroxide. Close the flask and allow to stand for 1 h. Using 0.2 ml of phenolphthalein solution R
as indicator, titrate with 0.5M hydrochloric acid. Carry out a blank titration.
9
H
8
O
4
.
STORAGE
IMPURITIES
HO
COOH
A. 4-hydroxybenzoic acid,
COOH
OH
OOC
B. 4-hydroxybenzene-1,3-dicarboxylic acid (4-hydroxyisophthalic acid),
C. 2-hydroxybenzoic acid (salicylic acid),
O
OAc
O
COOH
D. 2-[[2-(acetyloxy)benzoyl]oxy]benzoic acid (acetylsalicylsalicylic acid),
O
OH
O
COOH
E. 2-[(2-hydroxybenzoyl)oxy]benzoic acid (salicylsalicylic acid),
O
OAc AcO
O O
F. 2-(acetyloxy)benzoic anhydride (acetylsalicylic anhydride).
__________________________________________________________________________________________________________ Ph Eur
3-30
Astemizole
N
N
F
N
N
OMe
H
C
28
H
31
FN
4
O 458.6 68844-77-9
Astemizole complies with the requirements of the 3rd edition of the European Pharmacopoeia [1067]. These
1
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Astemizole contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of 1-[(4-fluorophenyl)-methyl]-N-[1-[2-(4-methoxyphenyl)ethyl]piperidin-4-yl]-1H-benzimidazol-
2-amine, calculated with reference to the dried substance.
CHARACTERS
A white or almost white powder, practically insoluble in water, freely soluble in methylene chloride
and in methanol, soluble in alcohol.
IDENTIFICATION
obtained with astemizole CRS. Examine the substances prepared as discs.
C. Examine by thin layer chromatography (2.2.27), using a suitable octadecylsilyl silica gel as the
coating substance.
the same solvent.
Reference solution (a). Dissolve 30 mg of astemizole CRS in methanol R and dilute to 5 ml with the
same solvent.
Reference solution (b). Dissolve 30 mg of astemizole CRS and 30 mg of ketoconazole CRS in
methanol R and dilute to 5 ml with the same solvent.
20 volumes of ammonium acetate solution R, 40 volumes of dioxan R and 40 volumes of methanol R.
Dry the plate in a current of warm air for 15 min and expose it to iodine vapour until the spots
appear. Examine in daylight. The principal spot in the chromatogram obtained with the test
solution is similar in position and size to the principal spot in the chromatogram obtained with
reference solution (a). The test is not valid unless the chromatogram obtained with reference
D. Mix about 5 mg with 45 mg of heavy magnesium oxide R and ignite in a crucible until an almost
white residue is obtained (usually less than 5 min). Allow to cool, add 1 ml of water R, 0.05 ml of
phenolphthalein solution R1 and about 1 ml of dilute hydrochloric acid R to render the solution
colourless. Filter. To a freshly prepared mixture of 0.1 ml of alizarin S solution R and 0.1 ml of
zirconyl nitrate solution R, add 1 ml of the filtrate. Mix, allow to stand for 5 min and compare the
colour of the solution with that of a blank prepared in the same manner. The test solution is yellow
and the blank is red.
TESTS
The solution is clear (2.2.1) and is not more intensely coloured than reference solution Y
7
(Method
II, 2.2.2).
3-31
Related substances Examine by liquid chromatography (2.2.29). Prepare the solutions immediately
before use.
Reference solution (a). Dissolve 2.5 mg of astemizole CRS and 25 mg of ketoconazole CRS in
methanol R and dilute to 100.0 ml with the same solvent.
Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with methanol R. Dilute 5.0 ml
of this solution to 20.0 ml with methanol R.
a stainless steel column 0.1 m long and 4.6 mm in internal diameter packed with base-deactivated
octadecylsilyl silica gel for chromatography R (3 m),
as mobile phase at a flow rate of 1.5 ml per minute, a linear gradient programme as follows:
Mobile phase A. A 17 g/l solution of tetrabutylammonium hydrogen sulphate R,
Mobile phase B. Acetonitrile R,
Time
(min)
Mobile
phase A
(per cent V/V)
Mobile
phase B
(per cent V/V)
Comment
0 95 5 Start gradient
15 80 20 End gradient
18 0 100 Purge time
23 95 5 Switch to initial
calibration
28 = 0 95 5 Start of next
chromatogram
Equilibrate the column for at least 30 min with acetonitrile R and then equilibrate the initial
elution composition for at least 5 min.
conditions the retention times are: ketoconazole, about 8 min and astemizole, about 9 min. The
test is not valid unless the resolution between the peaks corresponding to ketoconazole and
astemizole is a least 1.5. If necessary, adjust the final concentration of acetonitrile or the percentage
of tetrabutylammonium hydrogen sulphate in the mobile phase or adjust the time programme for
the linear gradient elution.
with reference solution (b) (0.25 per cent); the sum of the areas of all the peaks, apart from the
principal peak, is not greater than twice the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.5 per cent). Disregard any peak obtained with the blank and
any peak with an area less than 0.2 times the area of the principal peak in the chromatogram
obtained with reference solution (b).
at 100C to 105C.
ASSAY
Dissolve 0.200 g in 50 ml of a mixture of 1 volume of glacial acetic acid R and 7 volumes of methyl
ethyl ketone R. Titrate with 0.1M perchloric acid, using 0.2 ml of naphtholbenzein solution R as
indicator.
28
H
31
FN
4
O.
STORAGE
IMPURITIES
3-32
N
N
F
N
NH
H
A. 1-[(4-fluorophenyl)methyl]-N-(piperidin-4-yl)-1H-benzi-midazol-2-amine,
N
N N
N
OMe
F
H
B. 1-[(2-fluorophenyl)methyl]-N-[1-[2-(4-methoxyphenyl)-ethyl]piperidin-4-yl]-1H-
benzimidazol-2-amine,
N
N N
N
OMe
H
C. N-[1-[2-(4-methoxyphenyl)ethyl]piperidin-4-yl]-1-(phenylme-thyl)-1H-benzimidazol-2-
amine,
N
N N
N
OMe
F
H
D. 1-[(3-fluorophenyl)methyl]-N-[1-[2-(4-methoxyphenyl)-ethyl]piperidin-4-yl]-1H-
benzimidazol-2-amine,
N
N N
N
OMe
O
H
E. cis-1-[(4-fluorophenyl)methyl]-N-[1-[2-(4-methoxyphenyl)-ethyl]piperidin-4-yl]-1H-
benzimidazol-2-amine N-oxide,
N
N
F
N
N
OMe
O
H
F. trans-1-[(4-fluorophenyl)methyl]-N-[1-[2-(4-methoxyphenyl)-ethyl]piperidin-4-yl]-1H-
benzimidazol-2-amine N-oxide,
N
N
i
Pr
N
N
OMe
H
G. N-[1-[2-(4-methoxyphenyl)ethyl]piperidin-4-yl]-1-[[4-(1-methylethoxy)phenyl]methyl]-
1H-benzimidazol-2-amine,
3-33
N
N
F
N
N O
O
OMe
H
H. 2-(4-methoxyphenyl)ethyl 4-[[1-[(4-fluorophenyl)methyl]-1H-benzimidazol-2-
yl]amino]piperidine-1-carboxylate.
__________________________________________________________________________________________________________ Ph Eur
3-34
Atenolol
H
2
N
O
NHPr
i
O
OH H
and enantiomer
C
14
H
22
N
2
O
3
266.3 29122-68-7
Atenolol complies with the requirements of the 3rd edition of the European Pharmacopoeia [0703]. These
Preparations
Atenolol Injection
Atenolol Oral Solution
Atenolol Tablets
Co-tenidone Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Atenolol contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of
(RS)-4-[2-(hydroxy-3-isopropylaminopropoxy)phenyl]acetamide, calculated with reference to the
dried substance.
CHARACTERS
A white or almost white powder, sparingly soluble in water, soluble in ethanol, slightly soluble in
methylene chloride, practically insoluble in ether.
IDENTIFICATION
B. Dissolve 0.100 g in methanol R and dilute to 100 ml with the same solvent. Dilute 10.0 ml of this
solution to 100 ml with methanol R. Examined between 230 nm and 350 nm (2.2.25), the solution
shows two absorption maxima, at 275 nm and 282 nm. The ratio of the absorbance measured at
the maximum at 275 nm to that measured at the maximum at 282 nm is 1.15 to 1.20.
obtained with atenolol CRS.
D. Examine by thin-layer chromatography (2.2.27), using silica gel GF
254
R as the coating
substance.
Test solution. Dissolve 10 mg of the substance to be examined in 1 ml of methanol R.
Reference solution. Dissolve 10 mg of atenolol CRS in 1 ml of methanol R.
of 1 volume of concentrated ammonia R1 and 99 volumes of methanol R. Allow the plate to dry in air
and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with
the test solution is similar in position and size to the principal spot in the chromatogram obtained
with the reference solution.
TESTS
Appearance of solution Solution S is clear (2.2.1) and not more intensely coloured than degree 6
of the range of reference solutions of the most appropriate colour (Method II, 2.2.2).
Optical rotation (2.2.7). Determined on solution S, the angle of optical rotation is +0.10 to
0.10.
Test solution (a). Dissolve 50.0 mg of the substance to be examined in 20 ml of the mobile phase
and dilute to 25.0 ml with the mobile phase.
Test solution (b). Dissolve 50.0 mg of the substance to be examined in 0.1 ml of dimethyl
3-35
sulphoxide R, if necessary applying gentle heat by placing the containing vessel in a water-bath for
a few seconds, and dilute to 25.0 ml with the mobile phase.
Reference solution (a). Dilute 0.5 ml of test solution (a) to 100.0 ml with the mobile phase.
Reference solution (b). Dissolve 50.0 mg of atenolol for column validation CRS in 0.1 ml of dimethyl
sulphoxide R, if necessary applying gentle heat by placing the containing vessel in a water-bath for a
few seconds, and dilute to 25.0 ml with the mobile phase.
as mobile phase at a flow rate of 1.0 ml per minute a mixture prepared as follows: dissolve 1.0 g
of sodium octanesulphonate R and 0.4 g of tetrabutylammonium hydrogen sulphate R in 1 litre of a
mixture of 20 volumes of tetrahydrofuran R, 180 volumes of methanol R and 800 volumes of a
3.4 g/l solution of potassium dihydrogen phosphate R; adjust to pH 3.0 with phosphoric acid R,
Equilibrate the column with the mobile phase at a flow rate of 1.0 ml per minute for about 30
min.
obtained with 10 l of reference solution (a) is at least 50 per cent of the full scale of the recorder.
Inject 10 l of reference solution (b). The resulting chromatogram is similar to that of the
specimen chromatogram provided with atenolol for column validation CRS in that the peak due to
bis-ether precedes and is separated from that due to tertiary amine which normally appears as a
doublet. If necessary, adjust the concentration of sodium octanesulphonate in the mobile phase.
Increasing the concentration of sodium octanesulphonate increases the retention time of the tertiary
amine.
Inject separately 10 l of test solution (a) and reference solution (a). Continue the chromato-
graphy for four times the retention time of the principal peak. In the chromatogram obtained with
test solution (a): the area of any peak apart from the principal peak is not greater than half the area
of the principal peak in the chromatogram obtained with reference solution (a) (0.25 per cent); the
sum of the areas of all the peaks, apart from the principal peak, is not greater than the area of the
principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent). Disregard
any peak with an area less than 0.1 times of that of the principal peak in the chromatogram
obtained with reference solution (a).
If the substance to be examined is found to contain more than 0.15 per cent of bis-ether, its
compliance is confirmed by repeating the chromatography using 10 l of test solution (b).
Chlorides (2.4.4). Dissolve 50 mg in a mixture of 1 ml of dilute nitric acid R and 15 ml of water R.
The solution, without further addition of dilute nitric acid R, complies with the limit test for
chlorides (0.1 per cent).
at 100C to 105C.
ASSAY
Dissolve 0.200 g in 80 ml of glacial acetic acid R. Titrate with 0.1M perchloric acid, determining the
14
H
22
N
2
O
3
.
IMPURITIES
OH
H
2
N
O
A. 2-(4-hydroxyphenyl)acetamide,
H
2
N
O
O OH
OH
B. 2-[4-(2,3-dihydroxypropoxy)phenyl]acetamide,
O
H
2
N
O
O
3-36
C. 2-[4-(2,3-epoxypropoxy)phenyl]acetamide,
H
2
N
O
O Cl
OH
D. 2-[4-(2-hydroxy-3-chloropropoxy)phenyl]acetamide,
H
2
N
O CH
2
CHOH
O
2
E. 4,4-(2-hydroxypropane-1,3-diyldioxy)bis(phenylacetamide),
2
H
2
N
O
O
OH
NPr
i
F. 4,4-[N-isopropyl-3,3-iminobis(2-hydroxypropoxy)]bis(phenylacetamide),
O NHPr
i
OH
HOOC
G. 2-[4-(2-hydroxy-3-isopropylaminopropoxy)phenyl]acetic acid,
O NHPr
i
OH
NC
H. 2-[4-(2-hydroxy-3-isopropylaminopropoxy)phenyl]acetonitrile.
__________________________________________________________________________________________________________ Ph Eur
3-37
Atracurium Besilate
N
MeO
MeO Me
MeO
OMe
O
O
[CH
2
]
5
N
OMe
OMe Me
OMe
OMe
O
O
S
O
O O
2
mixture of stereoisomers
C
65
H
82
N
2
O
18
S
2
1243 64228-81-5
Definition Atracurium Besilate is (RS)-2,2-(3,11-dioxo-4,10-dioxatridecamethylene)bis(1,2,3,4-
tetrahydro-6,7-dimethoxy-2-methyl-1-veratrylisoquinolinium) di(benzenesulphonate). It contains not
less than 97.0% and not more than 103.0% of C
65
H
82
N
2
O
18
S
2
substance. It contains about 58% of the cis-cis isomer, about 36% of the cis-trans isomer and about
6% of the trans-trans isomer.
Characteristics A white to pale yellow powder or flaky crystals; hygroscopic.
Very soluble in acetonitrile and chloroform; soluble in water and practically insoluble in ether.
Identification
A. The light absorption, Appendix II B, in the range 250 to 350 nm of a 0.005% w/v solution in 0.1M
hydrochloric acid exhibits a maximum at 280 nm. The absorbance at the maximum is about 0.4.
B. The infrared absorption spectrum, Appendix II A, is concordant with the spectrum of atracurium
besilate (Fig. 1).
Fig. 1 Typical infrared spectrum of atracurium besilate (phase: potassium bromide disc)
3-38
Appearance of solution A 1.0% w/v solution is clear, Appendix IV A, and not more intensely
7
, Appendix IV B, Method I.
Methyl benzenesulphonate Prepare a 0.1% w/v solution of methyltoluene-4-sulphonate (internal
standard) in a mixture of 45 volumes of acetonitrile and 55 volumes of water (solution A). Carry out
the method for liquid chromatography, Appendix III D, using the following solutions. For solution (1)
dissolve 1 g of the substance being examined in 30 ml of water, add 25 ml of solution A and 25 ml of
n-hexane, shake vigorously for 15 seconds and allow to separate. Repeat the extraction with four
25-ml quantities of n-hexane, evaporate the combined extracts to dryness under reduced pressure at a
temperature of 40 and dissolve the residue in 5 ml of the mobile phase. For solution (2) dilute 40 ml
of a solution containing 0.0001% v/v of methyl benzenesulphonate in acetonitrile and 100 ml of solution
A to 20 ml with the mobile phase.
(25 cm 4.6 mm) packed with stationary phase C (5 m) (Spherisorb ODS is suitable), (b) as the
mobile phase with a flow rate of 2 ml per minute a mixture of 45 volumes of acetonitrile and 55
volumes of water and (c) a detection wavelength of 217 nm.
Inject 20 l of each solution. The test is not valid unless, in the chromatogram obtained with
solution (2), the resolution factor between the two principal peaks is at least 4.
In the chromatogram obtained with solution (1) the ratio of the area of any peak corresponding to
methyl benzenesulphonate to the area of the internal standard is not greater than the corresponding
ratio in the chromatogram obtained with solution (2) (10 ppm).
Toluene Not more than 0.2% when determined by the following method. Carry out the method for
liquid chromatography, Appendix III D, using the following solutions. Solution (1) is a 3.5% w/v
solution of the substance being examined in the mobile phase. For solution (2) dilute 80 l of toluene
to 100 ml with the mobile phase and dilute 1 volume of this solution to 10 volumes with the mobile
phase.
The chromatographic conditions described under Methyl benzenesulphonate may be used but
using 261 nm as the detection wavelength.
In the chromatogram obtained with solution (1) the area of any peak corresponding to toluene is
not greater than the area of the principal peak in the chromatogram obtained with solution (2).
Related substances Carry out the method for liquid chromatography, Appendix III D, using a
solution prepared immediately before use containing 0.1% w/v solution of the substance being
examined in the mobile phase.
(25 cm 4.6 mm) packed with silica gel for chromatography (5 m) (Lichrosorb Si 60 is suitable), (b)
as the mobile phase with a flow rate of 2 ml per minute a mixture of 10 volumes of orthophosphoric
acid, 100 volumes of water and 900 volumes of acetonitrile and (c) a detection wavelength of 280 nm.
The chromatogram obtained exhibits a principal peak at about 12 minutes and a smaller peak due
to [2-methyl-2,2-(1,5-pentandiylbis[oxy-(3-oxo-3,1-propandiyl)]bis(1-[3,4-dimethoxyphenyl)-
methyl]-1,2,3,4-tetrahydro-6,7-dimethoxyisokinoline) dibenzenesulphonate. The test is not valid
unless the resolution factor between these peaks is at least 1. If necessary, adjust the composition of the
mobile phase (reducing the concentration of orthophosphoric acid increases retention times).
In the chromatogram obtained with solution (1) the area of any peak corresponding to [2-methyl-
2,2-(1,5-pentandiylbis[oxy-(3-oxo-3,1-propandiyl)]bis(1-[3,4-dimethoxyphenyl)methyl]-1,2,3,4-
tetrahydro-6,7-dimethoxyisokinoline) dibenzenesulphonate is not more than 3.0%, the area of any
other secondary peak is not greater than 2.0% and the area of the principal peak is not less than 93.0%
by normalisation.
Isomer ratio 56.0% to 60.0% of the cis-cis isomer, 34.5% to 37.5% of the cis-trans isomer and 5.0%
to 6.5% of the trans-trans isomer determined using the following method. Carry out the method for
liquid chromatography, Appendix III D, using a solution containing 0.05% w/v of the substance being
examined in the mobile phase.
(25 cm 4.6 mm) packed with silica gel for chromatography (5 m) (Lichrosorb Si 60 is suitable), (b)
as the mobile phase with a flow rate of 1 ml per minute a mixture of 1 volume of sulphuric acid, 4
volumes of trifluoroacetic acid and 1000 volumes of methanol and (c) a detection wavelength of
280 nm.
The order of elution of the peaks is the cis-cis isomer (retention time, about 8 minutes), the cis-trans
isomer (retention time, about 10 minutes) and the trans-trans isomer, (retention time, about 13
minutes). The test is not valid unless the resolution factor between the peaks due to the cis-cis isomer
and the cis-trans isomer is at least 1.5. Adjust the composition of the mobile phase and the flow rate
to obtain satisfactory resolution (increasing the content of methanol increases the retention times).
Continue the chromatography for a period equivalent to 1.5 times the retention time of the trans-
trans isomer.
In the chromatogram obtained calculate the content of each isomer by normalisation.
3-39
Assay Measure the absorbance of a 0.010% w/v solution of the substance being examined in 0.1M
hydrochloric acid at the maximum at about 280 nm, Appendix II B. Calculate the content of
C
65
H
82
N
2
O
18
S
2
taking 85.5 as the value of A (1%, 1 cm) at the maximum at 280 nm.
Storage Atracurium Besilate should be kept in an airtight container at a temperature of 2 to 8,
IMPURITIES
N
MeO
MeO
MeO
OMe
O O
O
CH
2
O
Me
SO
3
A. 2-(3,11-dioxo-4,10-dioxatridec-12-enyl)-2-methyl-1,2,3,4-tetrahydropropapaverinium
benzenesulphonate,
N
MeO
MeO
MeO
OMe
COOH
Me
SO
3
B. 2-(2-carboxyethyl)-2-methyl-1,2,3,4-tetrahydropapaverinium benzenesulphonate,
N
MeO
MeO
MeO
OMe
Me
C. 2-methyl-1,2,3,4-tetrahydropapaverine,
N
MeO
MeO
MeO
OMe
O OH
O
Me
SO
3
D. 2-(9-hydroxy-3-oxo-4-oxanonyl)-2-methyl-1,2,3,4-tetrahydropapaverinium
benzenesulphonate,
NMe
2
MeO
MeO
MeO
OMe
SO
3
E. 2,2-dimethyl-1,2,3,4-tetrahydropapaverinium benzenesulphonate,
3-40
N
OMe
OMe
OMe
OMe
O
O
H
N
MeO
MeO
MeO
OMe
O
O
[CH
2
]
5
Me
SO
3
2
F. 2-methyl-2,2-(3,11-dioxo-4,10-dioxatridecamethylene)-bis-(1,2,3,4-
tetrahydropapaverinium benzenesulphonate.
3-41
Atropine Methobromide
and epimer at C*
N
Me
H Ph
O
OH
O
H
Me
+
Br
*
C
18
H
26
BrNO
3
384.3 2870-71-5
Atropine Methobromide complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Methylatropine Bromide [0511]. These requirements are reproduced after the heading Definition below.
Action and use Antispasmodic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Methylatropine bromide contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of (1R,3r,5S)-3-[(RS)-(3-hydroxy-2-phenylpropionyl)oxy]-8,8-dimethyl-8-
azoniabicyclo[3.2.1]octane bromide, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder or colourless crystals, freely soluble in water, sparingly soluble in alcohol.
IDENTIFICATION
obtained with methylatropine bromide CRS.
C. To 5 ml of solution S (see Tests) add 2 ml of dilute sodium hydroxide solution R. No precipitate is
formed.
D. To about 1 mg add 0.2 ml of fuming nitric acid R and evaporate to dryness on a water-bath.
Dissolve the residue in 2 ml of acetone R and add 0.1 ml of a 30 g/l solution of potassium hydroxide R
in methanol R. A violet colour develops.
E. It gives reaction (a) of bromides (2.3.1).
TESTS
solution B
9
(Method II, 2.2.2).
Acidity or alkalinity To 10 ml of solution S add 0.1 ml of phenolphthalein solution R; the solution is
colourless. Add 0.5 ml of 0.01M sodium hydroxide; the solution is red.
Optical rotation (2.2.7). Dissolve 2.50 g in water R and dilute to 25.0 ml with the same solvent.
The angle of optical rotation, measured in a 2 dm tube, is 0.25 to +0.05.
Related substances Examine by thin-layer chromatography (2.2.27), using a TLC silica gel G plate R.
Test solution. Dissolve 0.2 g of the substance to be examined in a mixture of 1 volume of water R and
9 volumes of methanol R and dilute to 5 ml with the same mixture of solvents.
Reference solution. Dilute 0.5 ml of the test solution to 100 ml with a mixture of 1 volume of water R
and 9 volumes of methanol R.
of methanol R, 15 volumes of anhydrous formic acid R, 15 volumes of water R and 60 volumes of ethyl
acetate R. Dry the plate at 100C to 105C until the odour of the solvent is no longer perceptible,
allow to cool and spray with dilute potassium iodobismuthate solution R until the spots appear. Any spot
3-42
in the chromatogram obtained with the test solution, apart from the principal spot, is not more
Apomethylatropine Dissolve 0.10 g in 0.01M hydrochloric acid and dilute to 100.0 ml with the same
acid. Measure the absorbances (2.2.25) at the maxima at 252 nm and 257 nm. The ratio of the
absorbance at 257 nm to that at 252 nm is at least 1.19.
100C to 105C.
for loss on drying.
ASSAY
Dissolve 0.300 g in 50 ml of anhydrous acetic acid R, warming slightly if necessary. Titrate with 0.1M
perchloric acid, determining the end-point potentiometrically (2.2.20).
18
H
26
BrNO
3
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
3-43
Atropine Methonitrate
and epimer at C*
N
Me
H Ph
O
OH
O
H
Me
+
*
NO
3
_
C
18
H
26
N
2
O
6
366.4 52-88-0
Atropine Methonitrate complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Methylatropine Nitrate [0512]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Methylatropine nitrate contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of (1R,3r,5S)-3-[(RS)-(3-hydroxy-2-phenylpropionyl)oxy]-8,8-dimethyl-8-
azoniabicyclo[3.2.1]octane nitrate, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder or colourless crystals, freely soluble in water, soluble in alcohol,
IDENTIFICATION
obtained with methylatropine nitrate CRS.
C. To a mixture of 2.5 ml of solution S (see Tests) and 2.5 ml of water R add 2 ml of dilute sodium
hydroxide solution R. No precipitate is formed.
E. To 0.05 ml of diphenylamine solution R add 0.05 ml of a 1 in 10 dilution of solution S. An intense
blue colour is produced.
TESTS
solution B
9
(Method II, 2.2.2).
Acidity or alkalinity To 10 ml of solution S add 0.1 ml of phenolphthalein solution R; the solution is
colourless. Add 0.5 ml of 0.01M sodium hydroxide; the solution is red.
The angle of optical rotation, measured in a 2 dm tube, is 0.25 to +0.05.
Related substances Examine by thin-layer chromatography (2.2.27), using silica gel G R as the
coating substance.
3-44
10 volumes of methanol R, 15 volumes of anhydrous formic acid R, 15 volumes of water R and 60
volumes of ethyl acetate R. Dry the plate at 100C to 105C until the odour of the solvent is no
longer perceptible, allow to cool and spray with dilute potassium iodobismuthate solution R until the
spots appear. Any spot in the chromatogram obtained with the test solution, apart from the principal
spot, is not more intense than the spot in the chromatogram obtained with the reference solution
(0.5 per cent).
Apomethylatropine Dissolve 0.10 g in 0.01M hydrochloric acid and dilute to 100.0 ml with the same
acid. Measure the absorbances (2.2.25) at the maxima at 252 nm and 257 nm. The ratio of the
absorbance at 257 nm to that at 252 nm is not less than 1.17.
Halides 15 ml of solution S complies with the limit test for chlorides (2.4.4). Prepare the standard
using 1.5 ml of chloride standard solution (5 ppm Cl) R (10 ppm).
Silver Dissolve 1.0 g in 10 ml of water R and add 0.1 ml of sodium sulphide solution R. Allow to stand
for 2 min. The solution is not more intensely coloured than reference solution B
8
(Method II, 2.2.2)
(10 ppm).
100C to 105C.
for loss on drying.
ASSAY
Dissolve 0.300 g in 50 ml of anhydrous acetic acid R. Titrate with 0.1M perchloric acid determining the
18
H
26
N
2
O
6
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
3-45
Atropine Sulphate
and epimer at C*
N
Me
H Ph
O
OH
O
H
2
,H
2
SO
4
*
C
34
H
48
N
2
O
10
S,H
2
O 695 5908-99-6
Atropine Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0068].
Preparations
Atropine Eye Drops
Atropine Eye Ointment
Atropine Injection
Atropine Tablets
Morphine and Atropine Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Atropine sulphate contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of bis (1R,3r,5S)-3-[(RS)-(3-hydroxy-2-phenylpropionyl)oxy]-8-methyl-8-
azabicyclo[3.2.1]octane sulphate, calculated with reference to the anhydrous substance.
CHARACTERS
A white, crystalline powder or colourless crystals, very soluble in water, freely soluble in alcohol,
It melts at about 190C with decomposition, determined on the substance dried at 135C for 15
min.
IDENTIFICATION
Second identification: C, D, E, F.
A. An aqueous solution shows almost no optical rotation (see Tests).
obtained with atropine sulphate CRS.
C. Dissolve about 50 mg in 5 ml of water R and add 5 ml of picric acid solution R. The precipitate,
washed with water R and dried at 100C to 105C for 2 h, melts (2.2.14) at 174C to 179C.
D. To about 1 mg add 0.2 ml of fuming nitric acid R and evaporate to dryness in a water-bath.
E. It gives the reactions of sulphates (2.3.1).
F. It gives the reaction of alkaloids (2.3.1).
TESTS
The angle of optical rotation measured in a 2-dm tube is 0.50 to +0.05.
Foreign alkaloids and decomposition products Examine by thin-layer chromatography
(2.2.27), using silica gel G R as the coating substance.
the same solvent.
3-46
Reference solution (a). Dilute 1 ml of the test solution to 100 ml with methanol R.
of 90 volumes of acetone R, 7 volumes of water R and 3 volumes of concentrated ammonia R. Dry the
plate at 100C to 105C for 15 min. Allow to cool and spray with dilute potassium iodobismuthate
solution R until the spots appear. Any spot in the chromatogram obtained with the test solution,
reference solution (a) (1.0 per cent) and not more than one such spot is more intense than the spot
in the chromatogram obtained with reference solution (b) (0.5 per cent).
Apoatropine Dissolve 0.10 g in 0.01M hydrochloric acid and dilute to 100.0 ml with the same acid.
Determine the absorbance (2.2.25) at 245 nm. The specific absorbance is not greater than 4.0,
calculated with reference to the anhydrous substance (about 0.5 per cent).
ASSAY
Dissolve 0.500 g in 30 ml of anhydrous acetic acid R, warming if necessary. Cool the solution.
Titrate with 0.1M perchloric acid and determining the end-point potentiometrically (2.2.20).
34
H
48
N
2
O
10
S.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
3-47
Attapulgite
Definition Attapulgite is a purified native hydrated magnesium aluminium silicate essentially
consisting of the clay mineral palygorskite.
Characteristics A light, cream or buff, very fine powder, free or almost free from gritty particles.
Identification
A. Ignite 0.5 g with 2 g of anhydrous sodium carbonate for 20 minutes, cool and extract with 25 ml of
boiling water. Cool, filter, wash the residue with water and add the washings to the filtrate. Reserve
the residue for test B. Cautiously acidify the combined filtrate and washings with hydrochloric acid,
evaporate to dryness, moisten the residue with 0.2 ml of hydrochloric acid, add 10 ml of water and stir.
A white, gelatinous precipitate is produced.
B. Wash the residue reserved in test A with water and dissolve in 10 ml of 2M hydrochloric acid. To
2 ml of the solution add a 10% w/v solution of ammonium thiocyanate. An intense red colour is
produced.
C. To 2 ml of the solution obtained in test B add 1 ml of strong sodium hydroxide solution and filter.
To the filtrate add 3 ml of ammonium chloride solution. A gelatinous white precipitate is produced.
D. To 2 ml of the solution obtained in test B add ammonium chloride and an excess of 13.5M ammonia
and filter. To the filtrate add 0.15 ml of magneson reagent and an excess of 5M sodium hydroxide. A
blue precipitate is produced.
Acidity or alkalinity pH of a 5% w/v suspension in carbon dioxide-free water, after shaking for 5
minutes, 7.0 to 9.5, Appendix V L.
Adsorptive capacity Moisture adsorption, 5 to 14% when determined by the following method.
Dry in air and powder a sufficient quantity of the substance being examined and pass through a sieve
with a nominal mesh aperture of 150 m. Spread 0.5 g as a thin layer on a previously weighed piece
of aluminium foil (60 mm 50 mm) of nominal gauge 17.5 m and transfer to a desiccator contain-
ing a dish of sodium chloride crystals partially immersed in saturated brine at 25. After 4 hours,
remove from the desiccator and weigh immediately. Dry in an oven at 110 for 4 hours, allow to cool
in a desiccator and weigh. The moisture adsorption is the gain in weight of the substance being exam-
ined expressed as a percentage of its oven-dried weight.
Arsenic To 0.13 g add 5 ml of water, 2 ml of sulphuric acid and 10 ml of sulphur dioxide solution and
evaporate on a water bath until the sulphur dioxide solution is removed and the volume reduced to
about 2 ml. Transfer the solution to the generator flask with the aid of 5 ml of water. The resulting
solution complies with the limit test for arsenic, Appendix VII (8 ppm).
Heavy metals
A. Not more than 20 ppm when determined by the following method. Shake 6.0 g with 40 ml of
0.5M hydrochloric acid at 37 for 30 minutes, cool and filter. Wash the residue with water and dilute
the combined filtrate and washings to 50 ml with water. To 20 ml add 2 g each of ammonium chloride
and ammonium thiocyanate and dissolve. Shake the solution with 80 ml of a mixture of equal volumes
of ether and isoamyl alcohol and separate, retaining the aqueous layer. Extract with a further 80 ml of
the mixture. To the aqueous layer add 2 g of citric acid, neutralise with 13.5M ammonia and dilute to
25 ml with water. 12 ml of the resulting solution complies with limit test A for heavy metals, Appendix
VII. Use lead standard solution (2 ppm Pb) to prepare the standard.
B. Not more than 10 ppm when determined by the following method. Shake 6.0 g with 40 ml of
0.5M sodium hydroxide at 37 for 30 minutes, cool and filter. Wash the residue with water and dilute
the combined filtrate and washings to 50 ml with water. Neutralise 20 ml of the solution with hydro-
chloric acid and dilute to 25 ml with water. 12 ml of the resulting solution complies with limit test A for
heavy metals, Appendix VII. Use lead standard solution (1 ppm Pb) to prepare the standard.
Acid-soluble matter Boil 2 g with 100 ml of 0.2M hydrochloric acid under a reflux condenser for 5
minutes, cool and filter. Evaporate 50 ml of the filtrate to dryness. The residue, after ignition at
about 600 for 30 minutes, weighs not more than 0.25 g.
Water-soluble matter Boil 10 g with 100 ml of water under a reflux condenser for 5 minutes, cool
and filter. Evaporate 50 ml of the filtrate to dryness. The residue, after ignition at 600 for 30
minutes, weighs not more than 50 mg.
Use 1 g.
Loss on ignition When ignited at 600, loses 15.0 to 27.0% of its weight. Use 1 g.
3-48
Activated Attapulgite
Definition Activated Attapulgite is a purified native hydrated magnesium aluminium silicate
essentially consisting of the clay mineral palygorskite that has been carefully heated to increase its
adsorptive capacity.
Characteristics A light, cream or buff, very fine powder, free or almost free from gritty particles.
Identification Complies with tests A to D for Identification stated under Attapulgite.
Acidity or alkalinity; Arsenic; Heavy metals; Acid-soluble matter; Water-soluble matter
Complies with the requirements stated under Attapulgite.
Adsorptive capacity In a stoppered bottle shake 1.0 g, in very fine powder, with 50 ml of a 0.12%
w/v solution of methylene blue for 5 minutes, allow to settle and centrifuge. The colour of the clear
supernatant solution is not more intense than that of a 0.0012% w/v solution of methylene blue.
1 g.
Loss on ignition When ignited at 600, loses not more than 9.0% of its weight. Use 1 g.
Action and use Antidiarrhoeal.
3-49
Azapropazone
N
N
N
O
O
Me
Pr
n
NMe
2
C
16
H
20
N
4
O
2
,2H
2
O 336.4 13539-59-8 (anhydrous)
Definition Azapropazone is 5-dimethylamino-9-methyl-2-propylpyrazolo[1,2-
a][1,2,4]benzotriazine-1,3(2H)-dione dihydrate. It contains not less than 99.0% and not more than
101.0% of C
16
H
20
N
4
O
2
, calculated with reference to the anhydrous substance.
Characteristics A white to pale yellow, crystalline powder.
Very slightly soluble in water and chloroform; soluble in ethanol (96%); it dissolves in solutions of
alkali hydroxides.
Identification
azapropazone (RS 016).
B. The light absorption, Appendix II B, in the range 230 to 350 nm of a 0.0008% w/v solution in 0.1M
sodium hydroxide exhibits two maxima, at 255 nm and 325 nm. The absorbances at 255 nm and
325 nm are about 0.86 and 0.18 respectively.
Acetic acid Not more than 0.2%, determined by the following method. Dissolve 10 g in 25 ml of
methanol, add 75 ml of water and carry out a potentiometric titration, Appendix VIII B, using 0.1M
sodium hydroxide VS as titrant to a pH of 5.9. Each ml of 0.1M sodium hydroxide VS is equivalent to
6.005 mg of acetic acid, C
2
H
4
O
2
.
Related substances Carry out the following operations in subdued light using low-actinic glassware
without delay. Carry out the method for liquid chromatography, Appendix III D, using the following
solutions in a mixture of 1 volume of phosphate buffer pH 4.0 and 3 volumes of methanol. Solution (1)
contains 0.10% w/v of the substance being examined. Solution (2) contains 0.00010% w/v of
azapropazone impurity A BPCRS. Solution (3) contains 0.00025% w/v of azapropazone impurity B
BPCRS. Solution (4) contains 0.00025% w/v of azapropazone impurity C BPCRS. Solution (5)
contains 0.0001% w/v of the substance being examined. Solution (6) contains 0.00005% w/v of the
substance being examined. Solution (7) contains 0.1% w/v of azapropazone impurity standard BPCRS.
(30 cm 3.9 mm) packed with stationary phase C (10 m) (Bondapak C18 is suitable), (b) a
mixture of 1 volume of glacial acetic acid, 36 volumes of methanol and 63 volumes of a 0.068% w/v
solution of sodium butanesulphonate in water as the mobile phase with a flow rate of 2.5 ml per minute
and (c) a detection wavelength of 254 nm.
Inject solution (7) and continue the chromatography for 5 times the retention time of the principal
peak. The test is not valid unless the chromatogram obtained with solution (7) closely resembles the
reference chromatogram supplied with the azapropazone impurity standard. If necessary adjust the
proportion of methanol in the mobile phase to give the required retention times.
In the chromatogram obtained with solution (1) the areas of any peaks corresponding to
azapropazone impurities A, B or C are not greater than the areas of the corresponding peaks in the
chromatograms obtained with solutions (2), (3) and (4) respectively (0.1%, 0.25% and 0.25%). The
area of any other secondary peak is not greater than the area of the peak in the chromatogram
obtained with solution (5) (0.1%). Calculate the content of impurities A, B and C using the
respective reference solutions and the content of any unnamed impurities using solution (5). The
total nominal content of impurities is not greater than 0.5%. Disregard any peak with an area less
than the area of the peak in the chromatogram obtained with solution (6) (0.05%).
Heavy metals Dissolve 3.125 g in 20 ml of water, boil for 10 minutes, dilute to 50 ml and filter
(solution A). 12 ml of solution A complies with limit test A for heavy metals, Appendix VII. Use 10 ml
of lead standard solution (1 ppm Pb) to prepare the standard (16 ppm).
Chloride A mixture of 10 ml of solution A and 5 ml of water complies with the limit test for chlorides,
Sulphate A mixture of 10 ml of solution A and 5 ml of water complies with the limit test for sulphates,
Water 10.0 to 11.5% w/w, Appendix IX C. Use 0.25 g.
3-50
Assay Carry out Method I for non-aqueous titration, Appendix VIII A, using 0.25 g and determining
the end point potentiometrically. Each ml of 0.1M perchloric acid VS is equivalent to 30.04 mg of
C
16
H
20
N
4
O
2
.
Preparations
Azapropazone Capsules
Azapropazone Tablets
IMPURITIES
N
N
N NMe
2
Me
A. 3-dimethylamino-7-methyl-1,2,4-benzotriazine (impurity A)
H
N
N
N NMe
2
Me
Bu
n
O
B. 3-dimethylamino-1,2-dihydro-7-methyl-2-valeryl-1,2,4-benzotriazine
N
N
N
O
O
Me
Pr
n
OH
C. 5-hydroxy-9-methyl-2-propylpyrazolo[1,2-a][1,2,4]benzotriazine-1,3(2H)-dione
(impurity B)
N
N
N NMe
2
Me
H
HOOC Pr
n
O
D. -(3-dimethylamino-7-methyl-1,2-dihydro-1,2,4-benzotriazin-2-ylcarbonyl)valeric acid
(impurity C)
3-51
Azathioprine
N
N
H
N
N
S
N
N
NO
2
Me
C
9
H
7
N
7
O
2
S 277.3 446-86-6
Azathioprine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0369]. These
Action and use Immunosuppressant.
Preparation
Azathioprine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Azathioprine contains not less than 98.5 per cent and not more than the equivalent of 101.0 per
cent of 6-[(1-methyl-4-nitro-1H-imidazol-5-yl)thio]-1H-purine, calculated with reference to the
dried substance.
CHARACTERS
A pale-yellow powder, practically insoluble in water and in alcohol. It is soluble in dilute solutions
of alkali hydroxides and sparingly soluble in dilute mineral acids.
IDENTIFICATION
A. Dissolve 0.150 g in 30 ml of dimethyl sulphoxide R and dilute to 500.0 ml with 0.1M hydrochloric
acid. Dilute 25.0 ml of the solution to 1000.0 ml with 0.1M hydrochloric acid. Examined between
230 nm and 350 nm (2.2.25), the solution shows an absorption maximum at 280 nm. The specific
obtained with azathioprine CRS.
C. To about 20 mg add 100 ml of water R, heat and filter. To 5 ml of the filtrate add 1 ml of
hydrochloric acid R and about 10 mg of zinc powder R. Allow to stand for 5 min. The solution
becomes yellow. Filter, cool in iced water, add 0.1 ml of sodium nitrite solution R and 0.1 g of
sulphamic acid R and shake until the bubbles disappear. Add 1 ml of n-naphthol solution R. A pale-
pink precipitate is formed.
TESTS
Acidity or alkalinity To 0.5 g add 25 ml of carbon dioxide-free water R, shake for 15 min and filter.
To 20 ml of the filtrate add 0.1 ml of methyl red solution R. Not more than 0.2 ml of 0.01M hydro-
chloric acid or 0.01M sodium hydroxide is required to change the colour of the indicator.
Chloromethylnitroimidazole and mercaptopurine Examine by thin-layer chromatography
(2.2.27), using cellulose for chromatography F
254
Test solution. Dissolve 0.2 g of the substance to be examined in dilute ammonia R1 and dilute to
10 ml with the same solvent. Prepare immediately before use.
Reference solution (a). Dissolve 10 mg of chloromethyl-nitroimidazole CRS in dilute ammonia R1 and
dilute to 50 ml with the same solvent. Prepare immediately before use.
Reference solution (b). Dissolve 10 mg of mercaptopurine R in dilute ammonia R1 and dilute to 50 ml
with the same solvent. Prepare immediately before use.
Apply separately to the plate 5 l of each solution. Develop over a path of 15 cm using butanol R
saturated with dilute ammonia R1. Dry the plate at 50C and examine in ultraviolet light at 254 nm.
In the chromatogram obtained with the test solution, any spots corresponding to chloromethyl-
nitroimidazole and mercaptopurine are not more intense than the spots in the chromatograms
obtained with reference solution (a) (1.0 per cent) and reference solution (b) (1.0 per cent),
respectively.
at 100C to 105C.
3-52
ASSAY
Dissolve 0.250 g in 25 ml of dimethylformamide R. Titrate with 0.1M tetrabutylammonium hydroxide,
1 ml of 0. 1M tetrabutylammonium hydroxide is equivalent to 27.73 mg of C
9
H
7
N
7
O
2
S.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
3-53
Azelastine Hydrochloride
NMe
N
N
O
Cl
H
,HCl
C
22
H
24
ClN
3
O,HCl 418.4 79307-93-0
Definition Azelastine Hydrochloride is (RS)-4-(4-chlorobenzyl)-2-(1-methylazepan-4-
yl)phthalazin-1(2H)-one hydrochloride. It contains not less than 98.5% and not more than
101.0% of C
22
H
24
ClN
3
O,HCl, calculated with reference to the dried substance.
Characteristics A white, crystalline powder.
Slightly soluble in water; soluble in absolute ethanol; practically insoluble in ether, in n-hexane and in
toluene.
Identification
azelastine hydrochloride (RS 018).
B. Yields reaction A characteristic of chlorides, Appendix VI.
C. Complies with the test for Optical rotation.
Acidity or alkalinity To 10 ml of a 1.0% w/v solution in carbon dioxide-free water add 0.2 ml of
bromothymol blue solution R1. Not more than 0.1 ml of 0.01M hydrochloric acid VS or 0.01M sodium
hydroxide VS is required to change the colour of the solution.
Optical rotation 0.05 to +0.05, Appendix V F, in a 5% w/v solution in dichloromethane.
1-Methyl-4-(2-benzoylhydrazino)azepan Carry out the method for liquid chromatography,
Appendix III D, using the following solutions. For solution (1) dissolve 0.1 g of the substance being
examined in 12 ml of methanol and dilute to 20 ml with water. Solution (2) contains 0.0005% w/v of
1-methyl-4-(2-benzoylhydrazino)azepan hydrochloride BPCRS in methanol (60%).
The chromatographic procedure may be carried out using (a) a stainless steel column (25 cm
4.6 mm) packed with octadecylsilyl gel for chromatography (10 m) (Nucleosil C18 10 is suitable), (b)
as the mobile phase with a flow rate of 1.5 ml per minute 0.02M sodium octanesulphonic acid in a
mixture of 1 volume of glacial acetic acid, 40 volumes of water and 60 volumes of methanol and (c) a
detection wavelength of 254 nm.
In the chromatogram obtained with solution (1) the area of any peak corresponding to 1-methyl-4-
(2-benzoylhydrazino)azepan is not greater than the area of the principal peak in the chromatogram
obtained with solution (2) (0.1%).
Related substances Carry out the method for thin-layer chromatography, Appendix III A, using TLC
silica gel F
254
plate (Merck silica gel 60 F
254
plates are suitable) and a mixture of 1 volume of 18M
ammonia, 40 volumes of ethanol (96%) and 60 volumes of toluene as the mobile phase. Apply
separately to the plate 10 l of each of four solutions in a mixture of equal volumes of dichloromethane
and methanol containing (1) 5.0% w/v and (2) 0.0050% w/v of the substance being examined, (3)
0.0050% w/v of 4-chlorobenzylphthalazinone BPCRS and (4) 0.0050% w/v of 1-methylazepan-4-one
hydrochloride BPCRS. After removal of the plate, heat at 115 for 1 hour, cool and examine under
ultraviolet light (254 nm). Spray the plate with sodium iodobismuthate solution followed by a 5% w/v
solution of sodium nitrite and re-examine. In the chromatogram obtained with solution (1) any spot
corresponding to either of the two named impurities is not more intense than the corresponding spot
in the chromatograms obtained with solution (3) and (4), respectively (0.1% of each) and any other
secondary spot is not more intense than the spot in the chromatogram obtained with solution (2)
(0.1%). Disregard any spot remaining on the line of application.
Loss on drying When dried to constant weight at 100 to 105 loses not more than 1% of its weight.
Use 1 g.
Assay Dissolve 0.3 g in 5 ml of anhydrous formic acid, add 30 ml of acetic anhydride and carry
out Method I for non-aqueous titration, Appendix VIII A, determining the end-point potentio-
metrically. Each ml of 0.1M perchloric acid VS is equivalent to 41.84 mg of C
22
H
24
ClN
3
O,HCl.
3-54
Storage Azelastine Hydrochloride should be kept in an airtight container and protected from light.
Action and use Prophylaxis of allergic conditions.
IMPURITIES
NH
N
Cl
O
A. 4-chlorobenzylphthalazinone
N Me
HN
NH
O
B. 1-methyl-4-(2-benzoylhydrazino)azepan
N Me
O
C. 1-methylazepan-4-one
3-55
Bacampicillin Hydrochloride
N
S
O
N
H H
O
NH
2 H
O O
H
O
O
Me H
O
CH
3
Me
Me
H
and enanti omer at C*
,HCl
*
C
21
H
28
ClN
3
O
7
S 502.0 37661-08-8
Bacampicillin Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Bacampicillin hydrochloride contains not less than 95.0 per cent and not more than the equivalent
of 102.0 per cent of (1RS)-1-[(ethoxycarbonyl)oxy]ethyl (2S,5R,6R)-6-[[(2R)-2-amino-2-
phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate hydro-
chloride, calculated with reference to the anhydrous, solvent-free substance.
PRODUCTION
If manufactured by a process that may leave residues of dimethylaniline in the substance and/or by a
process using starting materials or intermediates which contain residues of dimethylaniline, it
N,N-Dimethylaniline (2.4.26, Method A). Not more than 20 ppm.
CHARACTERS
A white or almost white powder or granules, hygroscopic, soluble in water, freely soluble in alcohol,
IDENTIFICATION
obtained with bacampicillin hydrochloride CRS.
B. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel silanised plate R.
Reference solution (a). Dissolve 10 mg of bacampicillin hydrochloride CRS in 2 ml of methanol R.
Reference solution (b). Dissolve 10 mg each of bacampicillin hydrochloride CRS, talampicillin hydro-
chloride CRS and pivampicillin CRS in 2 ml of methanol R.
of a 272 g/l solution of sodium acetate R, the pH of which has been adjusted to 5.0 with glacial acetic
acid R, 40 volumes of water R and 50 volumes of alcohol R. Dry the plate in a current of warm air,
then spray with ninhydrin solution R1 and heat at 60C for 10 min. The principal spot in the chroma-
togram obtained with the test solution is similar in position, colour and size to the principal spot in
gram obtained with reference solution (b) shows three clearly separated spots.
D. Dissolve about 25 mg in 2 ml of water R. Add 2 ml of dilute sodium hydroxide solution R and shake.
Wait a few minutes and add 3 ml of dilute nitric acid R and 0.5 ml of silver nitrate solution R1. A white
precipitate is formed. Add 0.5 ml of concentrated ammonia R. The precipitate dissolves.
TESTS
3-56
Appearance of solution. Dissolve 0.200 g in 20 ml of water R; the solution is not more opalescent
than reference suspension II (2.2.1). Dissolve 0.500 g in 10 ml of water R; the absorbance (2.2.25) of
the solution measured at 430 nm is not greater than 0.10.
solvent. The specific optical rotation is +175 to +195, calculated with reference to the anhydrous,
solvent-free substance.
Butyl acetate and ethyl acetate Not more than 2.0 per cent m/m of butyl acetate, not more than
4.0 per cent m/m of ethyl acetate and not more than 5.0 per cent m/m for the sum determined by
head-space gas chromatography (2.2.28), using the method of standard additions.
Sample solution. Dissolve 50.0 mg of the substance to be examined in water R and dilute to 10.0 ml
The chromatographic procedure may be carried out using System A of the test for residual solvents
(2.4.24) and the following static head-space injection conditions:
equilibration temperature: 60C,
equilibration time: 20 min.
Related substances. Examine by liquid chromatography (2.2.29) as prescribed under Assay. Inject
20 l of reference solution (b). Adjust the sensitivity of the system so that the height of the principal
peak in the chromatogram obtained is at least 50 per cent of the full scale of the recorder. Inject 20 l
of reference solution (d). The test is not valid unless in the chromatogram obtained, the peak
corresponding to ampicillin is separated from the peaks due to the solvent. Inject 20 l of the test
solution and continue the chromatography for 3.5 times the retention time of the principal peak. In
the chromatogram obtained with the test solution: the area of any peak, apart from the principal
peak, is not greater than 1.5 times the area of the principal peak in the chromatogram obtained with
reference solution (b) (1.5 per cent); the sum of the areas of all the peaks, apart from the principal
peak, is not greater than three times the area of the principal peak in the chromatogram obtained with
reference solution (b) (3 per cent). Disregard any peak with an area less than 0.1 times the area of the
mination of water.
ASSAY
Examine by liquid chromatography (2.2.29). Prepare the test solution and reference solutions (a), (b) and
(d) immediately before use.
Phosphate buffer A. Dissolve 1.4 g of sodium dihydrogen phosphate monohydrate R in water R and dilute
to about 800 ml with the same solvent. Adjust to pH 3.0 with dilute phosphoric acid R and dilute to
Phosphate buffer B. Dissolve 2.75 g of sodium dihydrogen phosphate monohydrate R and 2.3 g of disodium
hydrogen phosphate dihydrate R in water R and dilute to about 1800 ml with the same solvent. Adjust
to pH 6.8, if necessary, using dilute phosphoric acid R or dilute sodium hydroxide solution R and dilute to
Test solution. Dissolve 30.0 mg of the substance to be examined in phosphate buffer A and dilute to
100.0 ml with the same solution.
Reference solution (a). Dissolve 30.0 mg of bacampicillin hydrochloride CRS in phosphate buffer A and
dilute to 100.0 ml with the same solution.
Reference solution (b). Dilute 1.0 ml of reference solution (a) to 100.0 ml with phosphate buffer A.
Reference solution (c). Dissolve 30 mg of the substance to be examined in phosphate buffer B and
dilute to 100 ml with the same solution. Heat the solution at 80C for about 30 min.
Reference solution (d). Dissolve 20 mg of ampicillin trihydrate CRS in phosphate buffer A and dilute to
250 ml with the same solution. Dilute 5 ml of the solution to 100 ml with phosphate buffer A.
as mobile phase at a flow rate of 1.0 ml/min a mixture of 30 volumes of acetonitrile R and 70
volumes of a 0.06 per cent m/m solution of tetrahexylammonium hydrogen sulphate R in phosphate
buffer B,
Inject 20 l of reference solution (c). The test is not valid unless, in the chromatogram obtained, the
retention time of a degradation product eluting just after bacampicillin is 1.12 to 1.38 relative to
3-57
bacampicillin. If necessary adjust the concentration of tetrahexylammonium hydrogen sulphate in the
mobile phase. Inject reference solution (a) six times. The test is not valid unless the relative standard
deviation of the peak area for bacampicillin is at most 1.0 per cent. Inject alternately the test solution
and reference solution (a). Calculate the percentage content of bacampicillin hydrochloride.
STORAGE
IMPURITIES
N
S
O
H
Me
Me
H H
COOH
H
2
N
A. (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo-[3.2.0]heptane-2-carboxylic
COOH
NH
2
H
B. (2R)-2-amino-2-phenylacetic acid (D-phenylglycine),
*
N
O
H H
2
N
COOH
Me
Me
H
S
HN
H
H
and epimer at C*
C. (2RS,4S)-2-[[[(2R)-2-amino-2-phenylacetyl]amino]methyl]-5,5-dimethylthiazolidine-4-
N
O
H H
2
N
COOH
Me
Me
H
S
HN
COOH
H
*
*
D. (4S)-2-[[[(2R)-2-amino-2-phenylacetyl]amino]carboxymethyl]-5,5-dimethylthiazolidine-4-
carboxylic acid (penicilloic acids of ampicillin),
HN
S
H
Me
Me
COOH
N
N O
O
H
H
*
*
*
E. (4S)-2-(3,6-dioxo-5-phenylpiperazin-2-yl)-5,5-dimethylthiazolidine-4-carboxylic acid
(diketopiperazines of ampicillin),
H
3
C
COOH
HS
CH
3
H NH
2
and enantiomer
F. (2RS)-2-amino-3-methyl-3-sulphanylbutanoic acid (DL-penicillamine),
COOMe
NH
2
H
G. methyl (2R)-2-amino-2-phenylacetate (methyl D-phenylglycinate),
3-58
*
N
O
H AcHN
N
S
O
H H
O O O
Me H
Me
Me
OEt
O
H
H and epimer at C*
H. (1RS)-1-[(ethoxycarbonyl)oxy]ethyl (2S,5R,6R)-6-[[(2R)-2-(acetylamino)-2-
phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate
(N-acetyl-bacampicillin),
N
O
H H
2
N
N
S
O
H
Me
Me
H H
COOH
H
I. ampicillin.
__________________________________________________________________________________________________________ Ph Eur
3-59
Bacitracin
1405-87-4
Bacitracin complies with the requirements of the 3rd edition of the European Pharmacopoeia [0465]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Bacitracin consists of one or more of the antimicrobial polypeptides produced by certain strains of
Bacillus licheniformis and by Bacillus subtilis var. Tracy and yielding on hydrolysis the amino acids
L-cysteine, D-glutamic acid, L-histidine, L-isoleucine, L-leucine, L-lysine, D-ornithine,
D-phenylalanine and DL-aspartic acid. The potency is not less than 60 I.U. per milligram, calculated
CHARACTERS
A white or almost white powder, hygroscopic, freely soluble in water and in alcohol, practically
insoluble in ether.
IDENTIFICATION
Test solution. Dissolve 5 mg of the substance to be examined in a mixture of 0.5 ml of hydrochloric
acid R and 0.5 ml of water R. Heat in a sealed tube at 135C for 5 h, evaporate to dryness on a
water-bath and continue heating until the odour of hydrogen chloride is no longer detectable.
Dissolve the residue in 0.5 ml of water R.
Reference solution. Prepare as described for the test solution using 5 mg of bacitracin zinc CRS
instead of the substance to be examined.
Carry out the following procedure protected from light. Apply separately to the plate as 10 mm bands
5 l of each solution. Place the plate in the chromatographic tank so that it is not in contact with
the mobile phase consisting of a mixture of 25 parts of water R and 75 parts of phenol R. Leave the
plate to become impregnated with the vapour of the solvent for at least 12 h. Develop over a path
of 12 cm using the same mobile phase. Dry the plate at 100C to 105C and spray with ninhydrin
solution R1. Heat at 110C for 5 min. The chromatogram obtained with the test solution shows
zones corresponding to those in the chromatogram obtained with the reference solution.
B. Dissolve about 5 mg in 3 ml of water R and add 3 ml of dilute sodium hydroxide solution R. Shake
and add 0.5 ml of a 10 g/l solution of copper sulphate R. A violet colour develops.
C. Ignite 0.2 g. An insignificant residue remains which is not yellow at high temperature. Allow to
cool. Dissolve the residue in 0.1 ml of dilute hydrochloric acid R. Add 5 ml of water R and 0.2 ml of
strong sodium hydroxide solution R. No white precipitate is formed.
TESTS
solution BY
5
(Method II, 2.2.2).
Bacitracin F and related substances Dissolve 0.15 g in 0.05M sulphuric acid and dilute to
100.0 ml with the same acid. Dilute 2.0 ml of this solution to 10.0 ml with 0.05M sulphuric acid.
The ratio of the absorbance (2.2.25) at 290 nm to that at 252 nm is not greater than 0.20.
Loss on drying (2.2.32). Not more than 5.0 per cent, determined on 1.000 g by drying at 60C
over diphosphorus pentoxide R at a pressure not exceeding 0.1 kPa for 3 h.
Sterility (2.6.1). If intended for the preparation of eye-drops without a further appropriate
sterilisation procedure, it complies with the test for sterility.
ASSAY
Carry out the microbiological assay of antibiotics (2.7.2). Use bacitracin zinc CRS as the reference
substance.
STORAGE
Store in an airtight container at 8C to 15C. If the substance is sterile, store in a sterile, airtight,
tamper-proof container.
3-60
LABELLING
The label states, where applicable, that the substance is sterile.
__________________________________________________________________________________________________________ Ph Eur
3-61
Bacitracin Zinc
1405-49-6
Bacitracin Zinc complies with the requirements of the 3rd edition of the European Pharmacopoeia [0466].
Preparation
Polymyxin and Bacitracin Eye Ointment
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Bacitracin zinc is the zinc complex of bacitracin, which consists of one or more of the antimicrobial
polypeptides produced by certain strains of Bacillus licheniformis and by Bacillus subtilis var. Tracy
and which on hydrolysis yields the amino acids L-cysteine, D-glutamic acid, L-histidine, L-isoleucine,
L-leucine, L-lysine, D-ornithine, D-phenylalanine and DL-aspartic acid. The potency is not less than
60 I.U. per milligram, calculated with reference to the dried substance.
CHARACTERS
A white or light-yellowish-grey powder, hygroscopic, slightly soluble in water and in alcohol, very
slightly soluble in ether.
IDENTIFICATION
Test solution. Dissolve 5 mg of the substance to be examined in a mixture of 0.5 ml of hydrochloric
acid R and 0.5 ml of water R. Heat in a sealed tube at 135C for 5 h, evaporate to dryness on a
water-bath and continue heating until the odour of hydrogen chloride is no longer detectable.
Dissolve the residue in 0.5 ml of water.
Reference solution. Prepare as described for the test solution using 5 mg of bacitracin zinc CRS
Carry out the following procedure protected from light. Apply separately to the plate as 10 mm bands
5 l of each solution. Place the plate in the chromatographic tank so that it is not in contact with
the mobile phase consisting of a mixture of 25 parts of water R and 75 parts of phenol R. Leave the
plate to become impregnated with the vapour of the mobile phase for at least 12 h. Develop over a
path of 12 cm using the same mobile phase. Dry the plate at 100C to 105C and spray with
ninhydrin solution R1. Heat at 110C for 5 min. The chromatogram obtained with the test solution
shows zones corresponding to those in the chromatogram obtained with the reference solution.
B. Dissolve about 5 mg in 3 ml of water R and add 3 ml of dilute sodium hydroxide solution R. Shake
and add 0.5 ml of a 10 g/l solution of copper sulphate R. A violet colour develops.
C. Ignite about 0.15 g, allow to cool and dissolve the residue in 1 ml of dilute hydrochloric acid R.
Add 4 ml of water R. The solution gives the reaction of zinc (2.3.1).
TESTS
pH (2.2.3). Shake 1.0 g for about 1 min with 10 ml of carbon dioxide-free water R and filter. The pH
of the filtrate is 6.0 to 7.5.
Bacitracin F and related substances Dissolve 0.15 g in 0.05M sulphuric acid and dilute to
100.0 ml with the same acid. Dilute 2.0 ml of this solution to 10.0 ml with 0.05M sulphuric acid.
The ratio of the absorbance (2.2.25) at 290 nm to that at 252 nm is not greater than 0.15.
Zinc 4.0 per cent to 6.0 per cent, calculated with reference to the dried substance. Dissolve 0.200 g
in a mixture of 2.5 ml of dilute acetic acid R and 2.5 ml of water. Add 50 ml of water R, 50 mg of
xylenol orange triturate R and sufficient hexamethylenetetramine R to produce a red colour. Add 2 g of
hexamethylenetetramine R in excess. Titrate with 0.01M sodium edetate until a yellow colour is
obtained.
1 ml of 0.01M sodium edetate is equivalent to 0.654 mg of Zn.
diphosphorus pentoxide R at 60C at a pressure not exceeding 0.1 kPa for 3 h.
Sterility (2.6.1). If intended for administration by spraying into internal body cavities without a
further appropriate sterilisation procedure, it complies with the test for sterility.
Pyrogens (2.6.8). If intended for administration by spraying into internal body cavities without a
Inject per kilogram of the rabbits mass 1 ml of the supernatant liquid obtained by centrifuging a
3-62
suspension containing 11 mg per millilitre in a 9 g/l solution of sodium chloride R.
ASSAY
Suspend 50.0 mg in 5 ml of water R, add 0.5 ml of dilute hydrochloric acid R and dilute to 100.0 ml
with water R. Allow the solution to stand for 30 min. Carry out the microbiological assay of
antibiotics (2.7.2).
STORAGE
container.
LABELLING
The label states, where applicable, that the substance is sterile.
__________________________________________________________________________________________________________ Ph Eur
3-63
Baclofen
Cl
COOH
CH
2
NH
2
H
and enantiomer
C
10
H
12
ClNO
2
213.7 1134-47-0
Baclofen complies with the requirements of the 3rd edition of the European Pharmacopoeia [0653]. These
Action and use Skeletal muscle relaxant.
Preparations
Baclofen Oral Solution
Baclofen Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Baclofen contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent of
(RS)-4-amino-3-(4-chlorophenyl)butyric acid, calculated with reference to the anhydrous
substance.
CHARACTERS
A white or almost white powder, slightly soluble in water, very slightly soluble in alcohol, practically
insoluble in acetone and in ether. It dissolves in dilute mineral acids and in dilute solutions of alkali
hydroxides.
IDENTIFICATION
A. Dissolve 70 mg in water R and dilute to 100.0 ml with the same solvent. Examined between
220 nm and 320 nm (2.2.25), the solution shows three absorption maxima, at 259 nm, 266 nm and
275 nm. The specific absorbances at these maxima are 9.8 to 10.8, 11.5 to 12.7 and 8.4 to 9.3,
respectively. The test is not valid unless in the test for resolution (2.2.25) the ratio of absorbances is
at least 1.5.
obtained with baclofen CRS. Examine as discs prepared using 3 mg of substance and 300 mg of
potassium bromide R. If the spectra obtained with the substance to be examined and the reference
substance show differences, dissolve 0.1 g of each of the substances separately in 1 ml of dilute
sodium hydroxide solution R and add 10 ml of alcohol R and 1 ml of dilute acetic acid R. Allow to
stand for 1 h. Filter, wash the precipitate with alcohol R and dry in vacuo. Prepare new discs and
record the spectra.
Test solution. Dissolve 10 mg of the substance to be examined in the mobile phase (see below) and
dilute to 10 ml with the mobile phase.
Reference solution. Dissolve 10 mg of baclofen CRS in the mobile phase and dilute to 10 ml with the
mobile phase.
5 volumes of anhydrous formic acid R, 5 volumes of water R, 20 volumes of methanol R, 30 volumes
of chloroform R and 40 volumes of ethyl acetate R. Allow the solvents to evaporate. Spray with
ninhydrin solution R3 until the plate is slightly wet. Place the plate in an oven maintained at 100C
for 10 min. Examine in daylight. The principal spot in the chromatogram obtained with the test
solution is similar in position, colour and size to the principal spot in the chromatogram obtained
TESTS
Appearance of solution Dissolve 0.50 g in 1M sodium hydroxide and dilute to 25 ml with the same
3-64
solvent. The solution is not more opalescent than reference suspension II (2.2.1) and not more
intensely coloured than reference solution BY
5
(Method II, 2.2.2).
Reference solution (a). Dissolve 25.0 mg of baclofen impurity A CRS in the mobile phase and dilute to
Reference solution (c). Dilute 2.0 ml of the test solution to 100.0 ml with the mobile phase.
Reference solution (d). Dilute 2.0 ml of the test solution and 2.0 ml of reference solution (a) to
as mobile phase at a flow-rate of 2.0 ml per minute a mixture prepared as follows: dissolve
1.822 g of sodium hexanesulphonate R in 1 litre of a mixture of 560 volumes of water R, 440
volumes of methanol R and 5 volumes of glacial acetic acid R,
obtained with 20 l of reference solution (c) is at least 50 per cent of the full scale of the recorder.
Inject 20 l of reference solution (d). The test is not valid unless the resolution between the peaks
corresponding to baclofen and baclofen impurity A is at least 2.0. Inject separately 20 l of the test
solution, reference solution (b) and reference solution (c). Continue the chromatography for five
times the retention time of the principal peak. In the chromatogram obtained with the test solution:
the area of the peak corresponding to baclofen impurity A is not greater than the area of the
the areas of all the peaks, apart from the principal peak, is not greater than the area of the principal
peak in the chromatogram obtained with reference solution (c) (2.0 per cent).
ASSAY
Dissolve 0.1500 g in 50 ml of anhydrous acetic acid R. Titrate with 0.1M perchloric acid, determining
10
H
12
ClNO
2
.
IMPURITIES
Cl
NH
O
A. (RS)-4-amino-3-(4-chlorophenyl)butyric acid lactam,
Cl
CONH2
COOH
B. 3-(4-chlorophenyl)glutaric acid monoamide.
__________________________________________________________________________________________________________ Ph Eur
3-65
Bambuterol Hydrochloride
NHBu
t
OH H
O
O Me
2
N
NMe
2
O
O
and enanti omer
,HCl
C
18
H
29
N
3
O
5
,HCl 403.9 81732-46-9
Bambuterol Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Beta
2
-adrenoceptor agonist; bronchodilator.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Bambuterol hydrochloride contains not less than 98.5 per cent and not more than the equivalent of
101.5 per cent of 5-[(1RS)-2-[(1,1-dimethylethyl)amino]-1-hydroxyethyl]-1,3-phenylene
bis(dimethylcarbamate) hydrochloride, calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white, crystalline powder, freely soluble in water, soluble in alcohol.
IDENTIFICATION
obtained with bambuterol hydrochloride CRS. Examine the substances prepared as discs. If the spectra
obtained show differences, dissolve the substance to be examined and the reference substance in a
mixture of 1 volume of water R and 6 volumes of acetone R, cool in ice to precipitate and dry both
precipitates in vacuo at 50C to constant weight. Record new spectra using the residues.
B. It gives reaction (a) of chlorides (2.3.1).
TESTS
Solution S. Dissolve 4.0 g in carbon dioxide-free water R and dilute to 20.0 ml with the same solvent.
Optical rotation (2.2.7). Dilute 1 ml of solution S to 10 ml with carbon dioxide-free water R. The
angle of optical rotation is 0.10 to +0.10.
Reference solution (a). Dissolve 1.0 mg of formoterol fumarate dihydrate CRS in the mobile phase and
dilute to 10.0 ml with the mobile phase. Mix 0.8 ml of the obtained solution with 0.4 ml of the test
solution and dilute to 100.0 ml with the mobile phase.
2.0 ml of the obtained solution to 20.0 ml with the mobile phase.
a stainless steel column 0.15 m long and 4.6 mm in internal diameter packed with base-
deactivated octadecylsilyl silica gel for chromatography R (5 m),
as mobile phase at flow rate of 1.5 ml/min a mixture prepared as follows: dissolve 1.3 g of sodium
octanesulphonate R in 430 ml of a mixture of 25 volumes of acetonitrile R1 with 75 volumes of
methanol R; then mix this solution with 570 ml of 0.050M phosphate buffer pH 3.0 (dissolve
6.90 g of sodium dihydrogen phosphate monohydrate R in water R and dilute to 1000 ml with
water R, adjust the pH to 3.0 with a 50 g/l solution of dilute phosphoric acid R),
3-66
obtained with 20 l of reference solution (b) is about 50 per cent of the full scale of the recorder.
conditions, the retention times are: formoterol, about 7 min and bambuterol, about 9 min. The test
is not valid unless the resolution between the peaks corresponding to bambuterol and formoterol is at
least 5.0. Continue the chromatography of the test solution for 1.5 times the retention time of
bambuterol. If necessary adjust the composition of the mobile phase. Increase the content of
phosphate buffer to increase the retention time.
Inject separately 20 l of the mobile phase, 20 l of the test solution and 20 l of reference solution
(b). In the chromatogram obtained with the test solution the area of any peak, apart from the
principal peak, is not greater than the area of the principal peak obtained with reference solution (b)
(0.2 per cent); the sum of the areas of all the peaks, apart from the principal peak, is not greater than
three times the area of the principal peak in the chromatogram obtained with reference solution (b)
(0.6 per cent). Disregard any peak due to the mobile phase and any peak with an area less than 0.25
times the area of the principal peak obtained with reference solution (b).
mination of water.
ASSAY
Dissolve 0.320 g in 50 ml of alcohol R and add 5 ml of 0.01M hydrochloric acid. Carry out a potentio-
metric titration (2.2.20), using 0.1M sodium hydroxide. Read the volume added between the two points
of inflexion.
18
H
30
ClN
3
O
5
.
STORAGE
IMPURITIES
and enantiomer
NHBu
t
OH H
HO
OH
A. (1RS)-1-(3,5-dihydroxyphenyl)-2-[(1,1-dimethylethyl)amino]ethanol (terbutaline),
OH
OH H
O
O Me
2
N
NMe
2
O
O
and enantiomer
B. 5-[(1RS)-1,2-dihydroxyethyl]-1,3-phenylene bis(dimethylcarbamate),
and enantiomer
NHBu
t
OH H
O
O Me
2
N
OH
C. 3-[(1RS)-2-[(1,1-dimethylethyl)amino]-1-hydroxyethyl]-5-hydroxyphenyl
dimethylcarbamate,
CH
3
OH H
O
O Me
2
N
NMe
2
O
O
and enantiomer
D. 5-[(1RS)-1-hydroxyethyl]-1,3-phenylene bis(dimethylcarbamate),
3-67
CH
3
O
O Me
2
N
NMe
2
O
O
O
and enantiomer
E. 5-acetyl-1,3-phenylene bis(dimethylcarbamate),
NHBu
t
O
O Me
2
N
NMe
2
O
O
O
and enantiomer
F. 5-[[(1,1-dimethylethyl)amino]acetyl]-1,3-phenylene bis(dimethylcarbamate).
__________________________________________________________________________________________________________ Ph Eur
3-68
Barbital
NH
N
H
O O
O
Et
Et
C
8
H
12
N
2
O
3
184.2 57-44-3
Barbital complies with the requirements of the 3rd edition of the European Pharmacopoeia [0170]. These
When barbitone is prescribed or demanded, Barbital shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Barbital contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of
5,5-diethyl-1H,3H,5H-pyrimidine-2,4,6 trione, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder or colourless crystals, slightly soluble in water, soluble in boiling water,
in alcohol and in ether. It forms water-soluble compounds with alkali hydroxides and carbonates
and with ammonia.
IDENTIFICATION
substance to be examined and barbital CRS and determine the melting point of the mixture. The
obtained with barbital CRS.
254
R as the coating
substance.
Test solution. Dissolve 75 mg of the substance to be examined in alcohol R and dilute to 25 ml with
the same solvent.
Reference solution. Dissolve 75 mg of barbital CRS in alcohol R and dilute to 25 ml with the same
solvent.
layer of a mixture of 5 volumes of concentrated ammonia R, 15 volumes of alcohol R and 80 volumes
of chloroform R. Examine immediately in ultraviolet light at 254 nm. The principal spot in the
TESTS
Appearance of solution Dissolve 1.0 g in a mixture of 4 ml of dilute sodium hydroxide solution R
and 6 ml of water R. The solution is clear (2.2.1) and not more intensely coloured than reference
solution Y
6
(Method II, 2.2.2).
Acidity Boil 1.0 g with 50 ml of water R for 2 min, allow to cool and filter. To 10 ml of the filtrate
add 0.15 ml of methyl red solution R. The solution is orange-yellow. Not more than 0.1 ml of 0. 1M
sodium hydroxide is required to produce a pure yellow colour.
254
R as the
coating substance.
the same solvent.
Reference solution. Dilute 0.5 ml of the test solution to 100 ml with alcohol R.
3-69
layer of a mixture of 5 volumes of concentrated ammonia R, 15 volumes of alcohol R and 80 volumes
of chloroform R. Examine immediately in ultraviolet light at 254 nm. Spray with diphenylcarbazone
mercuric reagent R. Allow the plate to dry in air and spray with freshly prepared alcoholic potassium
hydroxide solution R diluted 1 in 5 with aldehyde-free alcohol R. Heat at 100C to 105C for 5 min
and examine immediately. When examined in ultraviolet light and after spraying, any spot in the
at 100C to 105C.
ASSAY
Dissolve 85.0 mg in 5 ml of pyridine R. Add 0.5 ml of thymolphthalein solution R and 10 ml of silver
8
H
12
N
2
O
3
.
__________________________________________________________________________________________________________ Ph Eur
3-70
Barium Sulphate
BaSO
4
233.4 7727-43-7
Barium Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0010].
These requirements are reproduced after the heading Characters below.
Action and use Radio-opaque substance used in the investigation of the gastro-intestinal tract.
Preparation
Barium Sulphate for Suspension
Ph Eur ___________________________________________________________________________________________________________
CHARACTERS
A fine, heavy, white powder, free from gritty particles, practically insoluble in water and in organic
solvents. It is very slightly soluble in acids and in solutions of alkali hydroxides.
IDENTIFICATION
A. Boil 0.2 g with 5 ml of a 500 g/l solution of sodium carbonate R for 5 min, add 10 ml of water R,
filter and acidify a part of the filtrate with dilute hydrochloric acid R. The solution gives the reactions
of sulphates (2.3.1).
B. Wash the residue collected in the preceding test with three successive small quantities of water R.
To the residue add 5 ml of dilute hydrochloric acid R, filter and add to the filtrate 0.3 ml of dilute
sulphuric acid R. A white precipitate is formed that is insoluble in dilute sodium hydroxide solution R.
TESTS
Solution S To 20.0 g add 40 ml of distilled water R and 60 ml of dilute acetic acid R. Boil for 5 min,
filter and dilute the cooled filtrate to 100 ml with distilled water R.
Acidity or alkalinity Heat 5.0 g with 20 ml of carbon dioxide-free water R on a water-bath for 5 min
and filter. To 10 ml of the filtrate add 0.05 ml of bromothymol blue solution R1. Not more than 0.5 ml
of 0.01M hydrochloric acid or 0.01M sodium hydroxide is required to change the colour of the indicator.
Acid-soluble substances Evaporate 25 ml of solution S to dryness on a water-bath and dry to
constant mass at 100C to 105C. The residue weighs not more than 15 mg (0.3 per cent).
Oxidisable sulphur compounds Shake 1.0 g with 5 ml of water R for 30 s and filter. To the filtrate
add 0.1 ml of starch solution R, dissolve 0.1 g of potassium iodide R in the mixture, add 1.0 ml of a
freshly prepared 3.6 mg/l solution of potassium iodate R and 1 ml of 1M hydrochloric acid and shake
well. The colour of the solution is more intense than that of a standard prepared at the same time in
the same manner omitting the potassium iodate.
Soluble barium salts To 10 ml of solution S add 1 ml of dilute sulphuric acid R. After 1 h, any
opalescence in the solution is not more intense than that in a mixture of 10 ml of solution S and 1 ml
of distilled water R.
Phosphates To 1.0 g add a mixture of 3 ml of dilute nitric acid R and 7 ml of water R. Heat on a
water-bath for 5 min, filter and dilute the filtrate to 10 ml with water R. Add 5 ml of molybdovanadic
reagent R. After 5 min, any yellow colour in the test solution is not more intense than that in a
standard prepared at the same time in the same manner using 10 ml of phosphate standard solution
(5 ppm PO
4
) R (50 ppm).
Arsenic (2.4.2). In a small long-necked combustion flask, shake 0.5 g with 2 ml of nitric acid R and
30 ml of water R, insert a small funnel in the neck of the flask, heat in an inclined position on a
water-bath for 2 h, allow to cool, adjust to the original volume with water R and filter. Wash the
residue by decantation with three quantities, each of 5 ml, of water R. Combine the filtrate and
washings, add 1 ml of sulphuric acid R, evaporate to dryness on a water-bath, and heat until copious
white fumes are evolved. Dissolve the residue in 10 ml of dilute sulphuric acid R and add 10 ml of
water R. The solution complies with limit test A for arsenic (2 ppm).
Heavy metals (2.4.8). Dilute 10 ml of solution S to 20 ml with water R, 12 ml of this solution
Loss on ignition Not more than 2.0 per cent, determined on 1.0 g at 600C.
Sedimentation Place 5.0 g in a glass-stoppered, 50 ml graduated cylinder having the 50 ml
graduation mark 14 cm from the base. Add water R to 50 ml, shake the mixture for 5 min and allow
to stand for 15 min. The barium sulphate does not settle below the 15 ml mark.
__________________________________________________________________________________________________________ Ph Eur
3-71
Barium Sulphate for Suspension
Definition Barium Sulphate for Suspension is a dry mixture of Barium Sulphate with suitable
flavours, suitable antimicrobial preservatives and a suitable dispersing agent. It contains not less than
85.0% w/w of barium sulphate, BaSO
4
.
Characteristics A fine, white or creamy white powder.
Identification
A. Ignite 1 g to constant weight. To 0.2 g of the residue add 5 ml of a 50% w/v solution of sodium
carbonate and boil for 5 minutes. Add 10 ml of water and filter. Reserve the residue for test B. Acidify
a portion of the filtrate with 2M hydrochloric acid. The solution yields the reactions characteristic of
sulphates, Appendix VI.
B. Wash the residue reserved in test A with water, add 5 ml of 2M hydrochloric acid, mix well and
filter. Add 0.3 ml of 1M sulphuric acid to the filtrate. A white precipitate is produced which is insoluble
in 2M hydrochloric acid.
Acidity or alkalinity pH of an aqueous suspension containing the equivalent of 75% w/v of Barium
Sulphate, 4.5 to 7.0, Appendix V L.
Loss on drying When dried at 105 for 4 hours, loses not more than 1.0% of its weight. Use 1 g.
Assay To 0.75 g in a platinum dish add 5 g of sodium carbonate and 5 g of potassium carbonate
sesquihydrate and mix. Heat to 1000 and maintain at this temperature for 15 minutes. Allow to cool
and suspend the residue in 150 ml of water. Wash the dish with 2 ml of 6M acetic acid and add the
washings to the suspension. Cool in ice and decant the supernatant liquid, transferring as little of the
solid matter as possible to the filter. Wash the residue with successive quantities of a 2% w/v solution
of sodium carbonate until the washings are free from sulphate and discard the washings. Add 5 ml of
2M hydrochloric acid to the filter, wash through into the vessel containing the bulk of the solid matter
with water, add 5 ml of hydrochloric acid and dilute to 100 ml with water. Add 10 ml of a 40% w/v
solution of ammonium acetate, 25 ml of a 10% w/v solution of potassium dichromate and 10 g of urea.
Cover and digest in a hot-air oven at 80 to 85 for 16 hours. Filter whilst still hot through a sintered-
glass filter (BS porosity No. 4), washing the precipitate initially with a 0.5% w/v solution of potassium
dichromate and finally with 2 ml of water. Dry to constant weight at 105. Each g of the residue is
equivalent to 0.9213 g of barium sulphate, BaSO
4
.
Action and use Radio-opaque preparation used in the investigation of the gastro-intestinal tract.
Preparation
Barium Sulphate Oral Suspension
3-72
Bearberry Leaf
Bearberry Leaf complies with the requirements of the 3rd edition of the European Pharmacopoeia [1054].
Action and use Astringent; diuretic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Bearberry leaf consists of the whole or cut, dry leaf of Arctostaphylos uva-ursi (L.) Spreng. It
contains not less than 8.0 per cent of hydroquinone derivatives, expressed as anhydrous arbutin
(C
12
H
16
O
7
; M
r
CHARACTERS
IDENTIFICATION
A. The leaf, shiny and dark green on the adaxial surface, lighter on the abaxial surface, is normally
7 mm to 30 mm long and 5 mm to 12 mm wide. The entire leaf is obovate with smooth margins,
somewhat reflexed downwards, narrowing at the base into a short petiole. The leaf is obtuse or
retuse at its apex. The lamina is thick and coriaceous. The venation, pinnate and finely reticulate, is
clearly visible on both surfaces. The adaxial surface is marked with sunken veinlets, giving it a
characteristic grainy appearance. Only the young leaf has ciliated margins. Old leaves are glabrous.
B. Reduce to a powder (355). The powder is green to greenish-grey or yellowish-green. Examine
under a microscope using chloral hydrate solution R. The powder consists of fragments of
epidermises which, seen in surface view, show polygonal cells covered by a thick smooth cuticle,
and with straight, thick and irregularly pitted walls; anomocytic stomata (2.8.3), surrounded by five
to eleven subsidiary cells and scars of hair bases only on the abaxial epidermis; fragments of
palisade parenchyma, with three or four layers of cells of unequal lengths, and spongy parenchyma;
groups of lignified fibres from the pericycle, with rows of cells containing prisms of calcium oxalate;
occasional conical, unicellular covering trichomes.
Test solution. To 0.5 g of the powdered drug (355) add 5 ml of a mixture of equal volumes of
methanol R and water R, and heat under a reflux condenser for 10 min. Filter whilst hot. Wash the
flask and the filter with a mixture of equal volumes of methanol R and water R and dilute to 5 ml
with the same mixture of solvents.
Reference solution. Dissolve 25 mg of arbutin R, 25 mg of gallic acid R and 25 mg of hydroquinone R
in methanol R and dilute to 10.0 ml with the same solvent.
Apply separately to the plate as bands 10 l of the reference solution and 20 l of the test solution.
water R and 88 volumes of ethyl acetate R. Dry the plate at 105C to 110C until the odour of
formic acid disappears. Spray with a 10 g/l solution of dichloroquinonechlorimide R in methanol R.
Next spray with a 20 g/l solution of anhydrous sodium carbonate R. The chromatogram obtained with
the test solution shows in its lower third a light blue band similar in position and colour to one of
the bands in the chromatogram obtained with the reference solution (arbutin). In its upper third, it
shows two bands similar in position and colour to the other two bands in the chromatogram
obtained with the reference solution, one is brownish (gallic acid) and the other is blue
(hydroquinone). There may also be two or three other blue bands and several brown or brownish-
grey bands.
TESTS
Foreign matter (2.8.2). Not more than 8 per cent of which not more than 5 per cent of stems and
not more than 3 per cent of other foreign matter.
Leaves of different colour Not more than 10 per cent, determined in the same manner as foreign
matter (2.8.2).
Loss on drying (2.2.32). Not more than 10.0 per cent, determined on 1.000 g of the powdered
drug (355) by drying in an oven at 100C to 105C for 2 h.
ASSAY
In a 250 ml flask with a ground-glass neck introduce 0.400 g (m g) of the powdered drug (250).
Add 50 ml of water R and heat under a reflux condenser for 30 min. Allow to cool and dilute to
3-73
250.0 ml with water R. Allow the particles to settle and introduce 5.0 ml of the solution into a
separating funnel. Add successively, mixing after each addition, 45 ml of water R, 1.0 ml of a 20 g/l
solution of aminopyrazolone R, 0.5 ml of dilute ammonia R2 and 1.0 ml of an 80 g/l solution of
potassium ferricyanide R. Allow to stand for 5 min and shake with 25 ml of methylene chloride R.
Filter the methylene chloride layer through a plug of absorbent cotton soaked in methylene chloride R
into a 100 ml volumetric flask. Shake the aqueous layer again with three quantities, each of 25 ml,
of methylene chloride R and dilute the combined methylene chloride layers to 100.0 ml with the same
solvent. Measure the absorbance (2.2.25) of the solution at 455 nm using water R as the compensa-
tion liquid.
Calculate the percentage content of hydroquinone derivatives as anhydrous arbutin from the
expression:
m
A 716 . 7
taking the specific absorbance of anhydrous arbutin to be 648.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
4-1
Beclometasone Dipropionate
H
O
Cl
HO
O
OCOEt
OCOEt
Me
Me
Me H
C
28
H
37
ClO
7
521.1 5534-09-8
Beclometasone Dipropionate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Corticosteroid.
Preparations
Beclometasone Cream
Beclometasone Nasal Spray
Beclometasone Ointment
Beclometasone Pressurised Inhalation
When beclomethasone dipropionate is prescribed or demanded. Beclometasone Dipropionate shall
be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Beclometasone dipropionate contains not less than 96.0 per cent and not more than the equivalent
of 103.0 per cent of 9-chloro-11,17,21-trihydroxy-16-methylpregna-1,4-diene-3,20-dione 17,21-
dipropionate, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water, freely soluble in acetone,
IDENTIFICATION
obtained with beclometasone dipropionate CRS. If the spectra obtained in the solid state with the
substance to be examined and the reference substance show differences, record further spectra
using 50 g/l solutions in chloroform R.
B. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable silica
gel with a fluorescent indicator having an optimal intensity at 254 nm.
Test solution (a). Dissolve 25 mg of the substance to be examined in methanol R with gentle heating
and dilute to 5 ml with the same solvent. (This solution is also used to prepare test solution (b)).
Dilute 2 ml of the solution to 10 ml with chloroform R.
Test solution (b). Transfer 2 ml of the solution obtained during preparation of test solution (a) to a
15 ml glass tube with a ground-glass stopper or a polytetrafluoroethylene cap. Add 10 ml of
saturated methanolic potassium hydrogen carbonate solution R and immediately pass a stream of
nitrogen R briskly through the solution for 5 min. Stopper the tube. Heat in a water-bath at 45C,
protected from light, for 3 h. Allow to cool.
Reference solution (a). Dissolve 25 mg of beclometasone dipropionate CRS in methanol R with gentle
heating and dilute to 5 ml with the same solvent. (This solution is also used to prepare reference
solution (b)). Dilute 2 ml of the solution to 10 ml with chloroform R.
Reference solution (b). Transfer 2 ml of the solution obtained during preparation of reference
solution (a) to a 15 ml glass tube with a ground-glass stopper or a polytetrafluoroethylene cap. Add
10 ml of saturated methanolic potassium hydrogen carbonate solution R and immediately pass a stream
of nitrogen R briskly through the solution for 5 min. Stopper the tube. Heat in a water-bath at 45C,
4-2
Apply separately to the plate 5 l of each solution. Prepare the mobile phase by mixing 1.2
volumes of water R and 8 volumes of methanol R and adding the mixture to a mixture of 15
volumes of ether R and 77 volumes of methylene chloride R. Develop over a path of 15 cm. Allow
the plate to dry in air and examine in ultraviolet light at 254 nm. The chromatogram obtained
with test solution (b) shows not fewer than two distinct spots which are similar in position and
size to the spots in the chromatogram obtained with reference solution (b). The principal spot in
the chromatogram obtained with test solution (a) is similar in position and size to the principal
spot in the chromatogram obtained with reference solution (a). Spray with alcoholic solution of
sulphuric acid R. Heat at 120C for 10 min or until the spots appear. Allow to cool. Examine the
plate in daylight and in ultraviolet light at 365 nm. The chromatogram obtained with test solution
(b) shows not fewer than two distinct spots, similar in position, colour in daylight, fluorescence in
ultraviolet light at 365 nm and size to the spots in the chromatogram obtained with reference
solution (b). The principal spot in the chromatogram obtained with test solution (a) is similar in
position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to the principal
spot in the chromatogram obtained with reference solution (a). The principal spots in the chrom-
atograms obtained with test solution (b) and reference solution (b) have R
f
values distinctly lower
than the principal spots in the chromatograms obtained with test solution (a) and reference
solution (a).
C. Add about 2 mg to 2 ml of sulphuric acid R and shake to dissolve. Within 5 min, a deep reddish-
brown colour develops. Add the solution to 10 ml of water R and mix. The colour is discharged and
a clear solution remains.
D. Treat 25 mg by the oxygen-flask method (2.5.10). Use a mixture of 1 ml of 1M sodium hydroxide
and 20 ml of water R to absorb the combustion products. The solution gives reaction (a) of
chlorides (2.3.1).
TESTS
Specific optical rotation (2.2.7). Dissolve 0.250 g in dioxan R and dilute to 25.0 ml with the
same solvent. The specific optical rotation is +88 to +94, calculated with reference to the dried
substance.
Absorbance (2.2.25). Dissolve 50.0 mg in alcohol R and dilute to 100.0 ml with the same solvent.
Dilute 2.0 ml of the solution to 50.0 ml with alcohol R. The specific absorbance at the maximum at
238 nm is 284 to 302, calculated with reference to the dried substance.
Reference solution (a). Dissolve 2 mg of beclometasone 17-propionate CRS and 2 mg of beclometasone
21-propionate CRS in the mobile phase and dilute to 50.0 ml with the mobile phase.
Reference solution (b). Dilute 1.0 ml of the test solution to 50.0 ml with the mobile phase.
as mobile phase at a flow rate of 1 ml per minute a mixture prepared as follows: mix 600 ml of
acetonitrile R and 350 ml of water R, allow to cool, dilute to 1000 ml with water R and mix again,
as detector a spectrophotometer set at 254 nm and with the sensitivity adjusted so that the
height of the principal peak in the chromatogram obtained with reference solution (b) is 70 per
cent to 90 per cent of the full scale.
Equilibrate the column with the mobile phase at a flow rate of 1 ml per minute for about 45 min.
When the chromatograms are recorded in the conditions described above, the retention times are:
beclometasone 17-propionate, about 6.2 min; beclometasone 21-propionate, about 6.7 min;
beclometasone dipropionate, 13 min to 14 min. Inject 20 l of reference solution (a). The system is
not suitable unless the resolution between the peaks corresponding to beclometasone 17-propionate
and beclometasone 21-propionate is at least 1.4; if this resolution is not achieved, adjust the
concentration of acetonitrile in the mobile phase. Verify the repeatability by making five separate
injections of 20 l of reference solution (b); the system is not suitable unless the relative standard
deviation for the area of the principal peak in the chromatograms obtained with reference solution
(b) is less than 2.0 per cent.
with the test solution: the area of any peak, apart from the principal peak, is not greater than the
area of the principal peak in the chromatogram obtained with reference solution (b) (2.0 per cent)
and not more than one such peak has an area greater than half the area of the principal peak in the
chromatogram obtained with reference solution (b) (1.0 per cent); the sum of the areas of all the
peaks, apart from the principal peak, is not greater than 1.25 times the area of the principal peak in
the chromatogram obtained with reference solution (b) (2.5 per cent). Disregard any peak due to
4-3
the solvent and any peak with an area less than 0.025 times the area of the principal peak in the
ASSAY
Protect the solutions from light throughout the assay. The coloured reaction products tend to adsorb to the
surface of glassware. To avoid low results, it is recommended that the glassware be treated with the coloured
reaction products before use and that the treated glassware be reserved for this assay and washed only with
water R between assays.
Dissolve an accurately weighed quantity in sufficient aldehyde-free alcohol R to produce a solution
containing 340 g to 360 g in 10.0 ml. Prepare a reference solution at the same time and in the
same manner, using beclometasone dipropionate CRS. Into two 25 ml volumetric flasks, introduce
respectively 10.0 ml of each solution. Into a third identical flask, introduce 10 ml of aldehyde-free
alcohol R. To each flask add 2.0 ml of triphenyltetrazolium chloride solution R. Displace the air from
the flasks using oxygen-free nitrogen R, immediately add 2.0 ml of dilute tetramethylammonium
hydroxide solution R and again displace the air with oxygen-free nitrogen R. Stopper the flasks, mix the
contents by gentle swirling and allow to stand in a water-bath at 35C for 2 h. Cool rapidly and
dilute to 25.0 ml with aldehyde-free alcohol R. Immediately measure the absorbance (2.2.25) of the
test solution and of the reference solution at the maximum at 485 nm, using a closed 1 cm cell and
as the compensation liquid the solution prepared from 10 ml of aldehyde-free alcohol R. Treat the
test solution and the reference solution in such a manner that the period of time that elapses
between the addition of the dilute tetramethylammonium hydroxide solution R and measurement of the
absorbance is exactly the same for each solution.
28
H
37
ClO
7
from the absorbances measured and the concentrations of
the solutions.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
4-4
Beclometasone Dipropionate Monohydrate
H
O
Cl
HO
O
OCOEt
OCOEt
Me
Me
Me H
,H
2
O
C
28
H
37
ClO
7
,H
2
O 539.1 77011-63-3
Definition Beclometasone Dipropionate Monohydrate is 9-chloro-11-hydroxy-16-methyl-
3,20-dioxopregna-1,4-diene-17,21-diyl dipropionate monohydrate. It contains not less than
96.0% and not more than 103.0% of C
28
H
37
ClO
7
Characteristics A white or almost white, crystalline powder. It exhibits polymorphism.
Practically insoluble in water; freely soluble in acetone and in chloroform; sparingly soluble in ethanol
(96%).
Identification
beclometasone dipropionate monohydrate (RS 021). If the spectra are not concordant, prepare a new
spectrum using a 5% w/v solution in chloroform IR. The infrared absorption spectrum is concordant with
the reference spectrum of beclometasone dipropionate (1) (RS 020)
B. Carry out the method for thin-layer chromatography, Appendix III A, using as the coating substance
a suitable silica gel containing a fluorescent indicator with an optimal intensity at 254 nm (Merck
silica gel 60 F
254
is suitable) and a mixture of 1.2 volumes of water, 8 volumes of methanol, 15
volumes of ether and 77 volumes of dichloromethane as the mobile phase. Apply separately to the plate
5 ml of each of the following solutions. For solution (1) dissolve 25 mg of the substance being exam-
ined in methanol with gentle heating and dilute to 5 ml with the same solvent (solution A); dilute 2 ml
of solution A to 10 ml with chloroform. For solution (2) transfer 2 ml of solution A to a 15-ml glass
tube with a ground-glass stopper or a polytetrafluoroethylene cap, add 10 ml of saturated methanolic
potassium hydrogen carbonate solution and immediately pass a current of nitrogen briskly through the
solution for 5 minutes. Stopper the tube, heat in a water bath at 45 protected from light for 3 hours
and allow to cool. For solution (3) dissolve 25 mg of beclometasone dipropionate EPCRS in methanol
with gentle heating and dilute to 5 ml with the same solvent (solution B); dilute 2 ml of solution B to
10 ml with chloroform. Prepare solution (4) in the same manner as solution (2) but using solution B in
place of solution A.
After removal of the plate, allow it to dry in air and examine under ultraviolet light (254 nm). The
principal spot in the chromatogram obtained with solution (1) is similar in position and size to that in
the chromatogram obtained with solution (3). In the chromatogram obtained with solution (2) at
least two distinct spots are similar in position and size to the spots in the chromatogram obtained
with solution (4). Spray the plate with ethanolic sulphuric acid (20%), heat at 120 for 10 minutes, or
until spots appear, and allow to cool. Examine the plate in daylight and under ultraviolet light
(365 nm). The principal spot in the chromatogram obtained with solution (1) is similar in colour in
daylight, fluorescence under ultraviolet light (365 nm), position and size to that in the chromatogram
obtained with solution (3). The Rf values of the principal spots in the chromatograms obtained with
solutions (2) and (4) are distinctly lower than those of the principal spots in the chromatograms
obtained with solutions (1) and (3) respectively. In the chromatogram obtained with solution (2) at
least two distinct spots are similar in colour in daylight, fluorescence under ultraviolet light (365 nm),
position and size to the spots in the chromatogram obtained with solution (4).
Light absorption Dissolve 50 mg in sufficient ethanol (96%) to produce 100 ml and dilute 2 ml of
this solution to 50 ml with the same solvent. The A(1%, 1 cm) of the resulting solution at the
maximum at 238 nm is 284 to 302, calculated with reference to the dried substance, Appendix II B.
Specific optical rotation In a 1% w/v solution in 1,4-dioxan, +88 to +94, calculated with refer-
ence to the dried substance, Appendix V F.
Related substances Carry out the method for liquid chromatography, Appendix III D, using three
solutions in the mobile phase containing (1) 0.0050% w/v of the substance being examined, (2)
0.25% w/v of the substance being examined and (3) 0.0040% w/v each of beclometasone 17-propionate
EPCRS and beclometasone 21-propionate EPCRS. The chromatographic procedure may be carried out
using (a) a stainless steel column (25 cm 4.6 mm) packed with stationary phase C (5 m)
4-5
(Lichrosorb R18 or Lichrosphere RP18 is suitable), (b) as the mobile phase with a flow rate of
1 ml per minute, a cooled mixture of 350 volumes of water and 600 volumes of acetonitrile diluted to
1000 volumes with water and (c) a detection wavelength of 254 nm. Adjust the sensitivity so that the
height of the principal peak in the chromatogram obtained with solution (1) is 70 to 90% of full-scale
deflection on the chart paper. Equilibrate the column with the mobile phase at a flow rate of 1 ml per
minute for about 45 minutes. When the chromatograms are recorded under the conditions described
above, the retention time for beclometasone 17-propionate is about 6.2 minutes, for beclometasone
21-propionate, about 6.7 minutes and for beclometasone dipropionate, 13 to 14 minutes. Inject 20 l
of solution (3). The test is not valid unless the resolution factor between the peaks due to
beclometasone 17-propionate and beclometasone 21-propionate is at least 1.4. If this resolution is
not achieved, adjust the concentration of acetonitrile in the mobile phase. Verify the repeatability by
making five separate 20-l injections of solution (1). The system is not suitable unless the relative
standard deviation for the area of the principal peak in the chromatogram obtained with solution (1)
is less than 2.0%. Inject separately 20 l of each of solutions (1) and (2) and record the chromato-
grams for twice the retention time of the principal peak.
the area of the principal peak in the chromatogram obtained with solution (1) (2.0%), not more than
one such peak has an area greater than half the area of the principal peak in the chromatogram
obtained with solution (1) (1.0%) and the sum of the areas of any secondary peaks is not greater than
1.25 times the area of the principal peak in the chromatogram obtained with solution (1) (2.5%).
Disregard any peak the area of which is less than 0.025 times the area of the principal peak in the
chromatogram obtained with solution (1) (0.05%).
Loss on drying When dried at 100 to 105 for 3 hours, loses not less than 2.8% and not more than
3.8% of its weight.
Assay Carry out the tetrazolium assay of steroids, Appendix VIII J, allowing the reaction to proceed at
35 for 2 hours, and calculate the content of C
28
H
37
ClO
7
from the absorbance obtained by repeating
the operation using beclometasone dipropionate EPCRS in place of the substance being examined.
Storage Beclometasone Dipropionate Monohydrate should be kept in a well-closed container and
When beclomethasone dipropionate monohydrate is prescribed or demanded Beclometasone
Dipropionate Monohydrate shall be dispensed or supplied.
IMPURITIES
Me
Cl
HO
Me
H
Me
O
OCOEt
OH
H
A. beclometasone 21-propionate
O
Me
Cl
HO
Me
H
Me
O
OH
OCOEt
H
B. beclometasone 17-propionate
O
Me
Cl
HO
Me
H
Me
O
OCOMe
OCOEt
H
C. beclometasone 17-propionate 21-acetate
O
Me
Cl
HO
Me
H
Me
O
OCOPr
n
OCOEt
H
4-6
D. beclometasone 17-propionate 21-butyrate
O
Me
Cl
Pr
n
COO
Me
H
Me
O
OCOEt
OCOEt
H
E. beclometasone 11,17,21-tripropionate
Me
Cl
HO
H
H O
O
O
Me Me
OMe
Me
F. 9-chloro-11-hydroxy-17,21-(1-methoxypropylidene)dioxy-16-methyl-3,20-
dioxopregna-1,4-diene.
4-7
White Beeswax
1/01
White Beeswax complies with the requirements of the 3rd edition of the European Pharmacopoeia [0069].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
White beeswax is obtained by bleaching yellow beeswax.
CHARACTERS
White or yellowish-white pieces or plates, translucent when thin, with a fine-grained, matt and non-
crystalline fracture; when warmed in the hand they become soft and malleable. It has an odour
similar to that of yellow beeswax, though fainter and never rancid. It is tasteless and does not stick to
the teeth. It is practically insoluble in water, partially soluble in hot alcohol (90 per cent V/V) and
completely soluble in fatty and essential oils.
It has a relative density of about 0.960.
TESTS
Drop point (2.2.17). 61C to 65C. Melt the beeswax by heating on a water-bath, pour on a glass
plate and allow to cool to a semi-solid mass. Fill the metal cup by inserting the wider end into the
beeswax and repeating the procedure until beeswax extrudes from the narrow opening. Remove the
excess with a spatula and insert the thermometer immediately. Remove the beeswax displaced. Allow
to stand at room temperature for at least 12 h before determining the drop point.
Acid value 17.0 to 24.0. To 2.00 g (m g) in a 250 ml conical flask fitted with a reflux condenser add
40 ml of xylene R and a few glass beads and heat until the substance is dissolved. Add 20 ml of
alcohol R and 0.5 ml of phenolphthalein solution R1 and titrate the hot solution with 0.5M alcoholic
potassium hydroxide until a red colour persists for at least 10 s (n
1
ml). Carry out a blank test (n
2
ml).
m
n n ) ( 05 . 28
value Acid
2 1

Ester value (2.5.2). 70 to 80.

Ratio number The ratio of the ester value to the acid value is 3.3 to 4.3.
Saponification value 87 to 104. To 2.00 g (m g) in a 250 ml conical flask fitted with a reflux
condenser add 30 ml of a mixture of equal volumes of alcohol R and xylene R and a few glass beads
and heat until the substance is dissolved. Add 25.0 ml of 0.5M alcoholic potassium hydroxide and heat
under a reflux condenser for 3 h. Titrate the hot solution immediately with 0.5M hydrochloric acid,
using 1 ml of phenolphthalein solution R1 as indicator (n
1
ml). Reheat the solution to boiling several
times during the course of the titration. Carry out a blank test (n
2
ml).
m
n n ) ( 05 . 28
value tion Saponifica
1 2

Ceresin, paraffins and certain other waxes To 3.0 g in a 100 ml round-bottomed flask add
30 ml of a 40 g/l solution of potassium hydroxide R in aldehyde-free alcohol R and boil gently under a
reflux condenser for 2 h. Remove the condenser and immediately insert a thermometer. Place the
flask in a water-bath at 80C and allow to cool, swirling the solution continuously. No precipitate is
formed until 65C is reached, although the solution may be slightly opalescent. Beginning with the
temperature of 65C, the solution may become cloudy and precipitates may be formed. At 59C, the
solution is cloudy.
Glycerol and other polyols To 0.20 g add 10 ml of alcoholic potassium hydroxide solution R and heat
on a water-bath under a reflux condenser for 30 min. Add 50 ml of dilute sulphuric acid R, cool and
filter. Rinse the flask and the filter with dilute sulphuric acid R. Combine the filtrate and washings and
dilute to 100.0 ml with dilute sulphuric acid R. Place 1.0 ml of the solution in a test-tube, add 0.5 ml
of a 10.7 g/l solution of sodium periodate R, mix and allow to stand for 5 min. Add 1.0 ml of decolorised
fuchsin solution R and mix. Any precipitate disappears. Place the tube in a beaker containing water at
40C and during cooling observe for 10 min to 15 min. Any bluish-violet colour in the solution is not
more intense than that in a standard prepared at the same time and in the same manner using 1.0 ml
of a 10 mg/l solution of glycerol R in dilute sulphuric acid R (0.5 per cent m/m, calculated as glycerol).
__________________________________________________________________________________________________________ Ph Eur
4-8
Yellow Beeswax
1/01
Yellow Beeswax complies with the requirements of the 3rd edition of the European Pharmacopoeia [0070].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Yellow beeswax is the wax obtained by melting the walls of the honeycomb made by the honey-bee,
Apis mellifera L., with hot water and removing foreign matter.
CHARACTERS
Yellow or light-brown pieces or plates with a fine-grained, matt and non-crystalline fracture; when
warmed in the hand they become soft and malleable. It has a faint odour, characteristic of honey. It is
tasteless and does not stick to the teeth. Practically insoluble in water, partially soluble in hot alcohol
(90 per cent V/V) and in ether, completely soluble in fatty and essential oils.
TESTS
Drop point (2.2.17). 61C to 65C. Melt the beeswax by heating on a water-bath, pour on a glass
plate and allow to cool to a semi-solid mass. Fill the metal cup by inserting the wider end into the
beeswax and repeating the procedure until beeswax extrudes from the narrow opening. Remove the
excess with a spatula and insert the thermometer immediately. Remove the beeswax displaced. Allow
to stand at room temperature for at least 12 h before determining the drop point.
Acid value 17.0 to 22.0. To 2.00 g (m g) in a 250 ml conical flask fitted with a reflux condenser add
40 ml of xylene R and a few glass beads and heat until the substance is dissolved. Add 20 ml of
alcohol R and 0.5 ml of phenolphthalein solution R1 and titrate the hot solution with 0.5M alcoholic
potassium hydroxide until a red colour persists for at least 10 s (n
1
2
ml).
m
n n ) ( 05 . 28
value Acid
2 1

Ester value (2.5.2). 70 to 80.

Ratio number The ratio of the ester value to the acid value is 3.3 to 4.3.
Saponification value 87 to 102. To 2.00 g (m g) in a 250 ml conical flask fitted with a reflux
condenser add 30 ml of a mixture of equal volumes of alcohol R and xylene R and a few glass beads
and heat until the substance is dissolved. Add 25.0 ml of 0.5M alcoholic potassium hydroxide and heat
under a reflux condenser for 3 h. Titrate the hot solution immediately with 0.5M hydrochloric acid,
using 1 ml of phenolphthalein solution R1 as indicator (n
1
ml). Reheat the solution to boiling several
times during the course of the titration. Carry out a blank test (n
2
ml).
m
n n ) ( 05 . 28
value tion Saponifica
1 2

Ceresin, paraffins and certain other waxes To 3.0 g in a 100 ml round-bottomed flask add
30 ml of a 40 g/l solution of potassium hydroxide R in aldehyde-free alcohol R and boil gently under a
reflux condenser for 2 h. Remove the condenser and immediately insert a thermometer. Place the
flask in a water-bath at 80C and allow to cool, swirling the solution continuously. No precipitate is
formed until 65C is reached, although the solution may be slightly opalescent. Beginning with the
temperature of 65C, the solution may become cloudy and precipitates may be formed. At 59C, the
solution is cloudy.
Glycerol and other polyols To 0.20 g add 10 ml of alcoholic potassium hydroxide solution R and heat
on a water-bath under a reflux condenser for 30 min. Add 50 ml of dilute sulphuric acid R, cool and
filter. Rinse the flask and the filter with dilute sulphuric acid R. Combine the filtrate and washings and
dilute to 100.0 ml with dilute sulphuric acid R. Place 1.0 ml of the solution in a test-tube, add 0.5 ml
of a 10.7 g/l solution of sodium periodate R, mix and allow to stand for 5 min. Add 1.0 ml of decolorised
fuchsin solution R and mix. Any precipitate disappears. Place the tube in a beaker containing water at
40C and during cooling observe for 10 min to 15 min. Any bluish-violet colour in the solution is not
more intense than that in a standard prepared at the same time and in the same manner using 1.0 ml
of a 10 mg/l solution of glycerol R in dilute sulphuric acid R (0.5 per cent m/m, calculated as glycerol).
__________________________________________________________________________________________________________ Ph Eur
4-9
Belladonna Herb
Belladonna Herb complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Belladonna Leaf [0221]. These requirements are reproduced after the heading Definition below.
Preparations
Prepared Belladonna Herb
Standardised Belladonna Leaf Dry Extract
Belladonna Tincture
When Belladonna Herb, Belladonna Leaf or Powdered Belladonna Herb is prescribed, Prepared
Belladonna Herb shall be supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Belladonna leaf consists of the dried leaf or of the dried leaf and flowering, and occasionally fruit-
bearing, tops of Atropa belladonna L. It contains not less than 0.30 per cent of total alkaloids,
calculated as hyoscyamine (M
r
289.4) with reference to the drug dried at 100C to 105C. The
alkaloids consist mainly of hyoscyamine together with small quantities of hyoscine (scopolamine).
CHARACTERS
Belladonna leaf has a slightly nauseous odour.
IDENTIFICATION
A. The leaves are green to brownish-green, slightly darker on the upper surface, often crumpled
and rolled and partly matted together in the drug. The leaf is petiolate and the base of the lamina is
acute and decurrent and the margin entire. The flowering stems are flattened and bear at each node
a pair of leaves unequal in size, in the axils of which occur singly the flowers or occasionally fruits.
The flowers have a gamosepalous calyx and campanulate corolla. The fruits are globular berries,
green to brownish-black and surrounded by the persistent calyx with widely spread lobes.
B. Reduce to a powder (355). The powder is dark green. Examine under a microscope, using
chloral hydrate solution R. The powder shows the following diagnostic characters: fragments of leaf
lamina showing sinuous-walled epidermal cells, a striated cuticle and numerous stomata
predominantly present on the lower epidermis (anisocytic and also some anomocytic); multicellular
uniseriate covering trichomes with smooth cuticle, glandular trichomes with unicellular heads and
multicellular, uniseriate stalks or with multicellular heads and unicellular stalks; parenchyma cells
including rounded cells containing microsphenoidal crystals of calcium oxalate; annular and spirally
thickened vessels. The powdered drug may also show the following: fibres and reticulately
thickened vessels from the stems; subspherical pollen grains, 40 m to 50 m in diameter, with
three germinal pores, three furrows and an extensively pitted exine; fragments of the corolla, with a
papillose epidermis or bearing numerous covering or glandular trichomes of the types previously
described; brownish-yellow seed fragments containing irregularly sclerified and pitted cells of the
testa.
C. Shake 1 g of powdered drug with 10 ml of 0.05M sulphuric acid for 2 min. Filter and add to the
filtrate 1 ml of concentrated ammonia R and 5 ml of water R. Shake cautiously with 15 ml of ether R,
avoiding formation of an emulsion. Separate the ether layer and dry over anhydrous sodium
sulphate R. Filter and evaporate the ether in a porcelain dish. Add 0.5 ml of fuming nitric acid R and
evaporate to dryness on a water-bath. Add 10 ml of acetone R and, dropwise, a 30 g/l solution of
potassium hydroxide R in alcohol R. A deep violet colour develops.
D. Examine the chromatogram obtained in the chromatography test. The principal zones in the
chromatogram obtained with the test solution are similar in position, colour and size to the
principal zones in the chromatogram obtained with the same volume of the reference solution.
TESTS
Chromatography Examine by thin-layer chromatography (2.2.27), using silica gel G R as the
coating substance.
Test solution. To 0.6 g of powdered drug (180) add 15 ml of 0.05M sulphuric acid, shake for 15 min
and filter. Wash the filter with 0.05M sulphuric acid until 20 ml of filtrate is obtained. To the filtrate
add 1 ml of concentrated ammonia R and shake with two quantities, each of 10 ml, of peroxide-free
ether R. If necessary, separate by centrifugation. Dry the combined ether layers over anhydrous
sodium sulphate R, filter and evaporate to dryness on a water-bath. Dissolve the residue in 0.5 ml of
methanol R.
4-10
Reference solution. Dissolve 50 mg of hyoscyamine sulphate R in 9 ml of methanol R. Dissolve 15 mg
of hyoscine hydrobromide R in 10 ml of methanol R. Mix 1.8 ml of the hyoscine hydrobromide
solution and 8 ml of the hyoscyamine sulphate solution.
Apply separately to the plate as bands 20 mm by 3 mm 10 l and 20 l of each solution, leaving
1 cm between the bands. Develop over a path of 10 cm using a mixture of 3 volumes of concentrated
ammonia R, 7 volumes of water and 90 volumes of acetone R. Dry the plate at 100C to 105C for
15 min, allow to cool and spray with potassium iodobismuthate solution R2, using about 10 ml for a
plate 200 mm square, until the orange or brown zones become visible against a yellow background.
The zones in the chromatograms obtained with the test solution are similar to those in the chrom-
atograms obtained with the reference solution with respect to their position (hyoscyamine in the
lower third, hyoscine in the upper third of the chromatogram) and their colour. The zones in the
chromatograms obtained with the test solution are at least equal in size to the corresponding zones
in the chromatogram obtained with the same volume of the reference solution. Faint secondary
zones may appear, particularly in the middle of the chromatogram obtained with 20 l of the test
solution or near the starting-point in the chromatogram obtained with 10 l of the test solution.
Spray the plate with sodium nitrite solution R until the coating is transparent. Examine after 15 min.
The zones corresponding to hyoscyamine in the chromatograms obtained with the test solution and
the reference solution change from brown to reddish-brown but not to greyish-blue (atropine) and
any secondary zones disappear.
Foreign matter (2.8.2). Not more than 3 per cent of stem having a diameter exceeding 5 mm.
Ash insoluble in hydrochloric acid (2.8.1). Not more than 4.0 per cent
ASSAY
Take about 50 g of drug and completely reduce it to a powder (180). Using the powder, determine
the loss on drying and the total alkaloids.
a) Determine the loss on drying (2.2.32) on 2.000 g, by drying in an oven at 100C to 105C.
b) Moisten 10.00 g of powder with a mixture of 5 ml of ammonia R, 10 ml of alcohol R and 30 ml
of peroxide-free ether R and mix thoroughly. Transfer the mixture to a suitable percolator, if
necessary with the aid of the extracting mixture. Allow to macerate for 4 h and percolate with a
mixture of 1 volume of chloroform R and 3 volumes of peroxide-free ether R until the alkaloids are
completely extracted. Evaporate to dryness a few millilitres of the liquid flowing from
the percolator, dissolve the residue in 0.25M sulphuric acid and verify the absence of alkaloids using
potassium tetraiodomercurate solution R. Concentrate the percolate to about 50 ml by distilling on a
water-bath and transfer it to a separating funnel, rinsing with peroxide-free ether R. Add a quantity
of peroxide-free ether R equal to at least 2.1 times the volume of the percolate to produce a liquid of
a density well below that of water R. Shake the solution with no fewer than three quantities, each of
20 ml, of 0.25M sulphuric acid, separate the two layers by centrifugation if necessary and transfer the
acid layers to a second separating funnel. Make the acid layer alkaline with ammonia R and shake
with three quantities, each of 30 ml, of chloroform R. Combine the chloroform layers, add 4 g of
anhydrous sodium sulphate R and allow to stand for 30 min with occasional shaking. Decant the
chloroform and wash the sodium sulphate with three quantities, each of 10 ml, of chloroform R. Add
the washings to the chloroform extract, evaporate to dryness on a water-bath and heat in an oven at
100C to 105C for 15 min. Dissolve the residue in a few millilitres of chloroform R, add 20.0 ml of
0.01M sulphuric acid and remove the chloroform by evaporation on a water-bath. Titrate the excess
of acid with 0.02M sodium hydroxide using methyl red mixed solution R as indicator.
Calculate the percentage content of total alkaloids, expressed as hyoscyamine, from the
expression:
5788 20
100
. ( )
( )

n
d m
d=loss on drying as a percentage,
n=volume of 0.02M sodium hydroxide used in millilitres,
m=mass of drug used in grams.
STORAGE
______________________________________________________________________________________________________________ Ph Eur
4-11
Prepared Belladonna Herb
Prepared Belladonna Herb complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Prepared Belladonna [0222]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Prepared belladonna is belladonna leaf powder (180) adjusted if necessary by adding powdered
lactose or belladonna leaf powder with a lower alkaloidal content to contain 0.28 per cent to
0.32 per cent of total alkaloids, calculated as hyoscyamine (M
r
289.4) with reference to the dried
drug.
CHARACTERS
It presents the characters of the powder described in the monograph on Belladonna leaf (221).
IDENTIFICATION
It complies with identification tests B, C and D in the monograph on Belladonna leaf (221). Exam-
ined in glycerol (85 per cent) R, it may be seen to contain lactose crystals.
TESTS
It complies with the test for chromatography, for total ash and for ash insoluble in hydrochloric
acid prescribed in the monograph on Belladonna leaf (221) and with the following additional test.
at 100C to 105C.
ASSAY
Carry out the assay prescribed in the monograph on Belladonna leaf (221).
Calculate the percentage content of total alkaloids, expressed as hyoscyamine, from the expression:
5788 20
100
. ( )
( )

n
d m
STORAGE
__________________________________________________________________________________________________________ Ph Eur
4-12
Bendroflumethiazide / Bendrofluazide
S
NH
N
O O
S
H
2
N
O O
F
3
C
H H
and enantiomer
C
15
H
14
F
3
N
3
O
4
S
2
421.4 73-48-3
Bendroflumethiazide complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Bendroflumethiazide Tablets/Bendrofluazide Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Bendroflumethiazide contains not less than 98.0 per cent and not more than the equivalent of
102.0 per cent of 3-benzyl-3,4-dihydro-6-(trifluoromethyl)-2H-1,2,4-benzothiadiazine-7-
sulphonamide 1,1-dioxide, calculated with reference to the dried substance.
CHARACTERS
soluble in alcohol.
IDENTIFICATION
obtained with bendroflumethiazide CRS. Examine the substances prepared in the form of discs.
B. Examine the chromatograms obtained in the test for related substances. The principal spot in
the chromatogram obtained with 4 l of the test solution is similar in position, colour and size to
C. In a test-tube, heat 0.5 ml of a saturated solution of chromium trioxide R in sulphuric acid R in a
naked flame until white fumes appear in the upper part of the tube. The solution wets the side of
the tube and there is no appearance of greasiness. Add about 2 mg of the substance to be examined
and heat again in a naked flame until white fumes appear. The solution does not wet the side of the
tube and does not pour easily from the tube.
D. Heat about 5 mg with a mixture of 5 ml of potassium permanganate solution R and 1 ml of
sulphuric acid R. An odour of benzaldehyde is produced.
TESTS
coating substance.
Test solution. Dissolve 25 mg of the substance to be examined in acetone R and dilute to 5 ml with
the same solvent.
Reference solution (a). Dissolve 25 mg of bendroflumethiazide CRS in acetone R and dilute to 5 ml
Reference solution (b). Dilute 1 ml of reference solution (a) to 100 ml with acetone R.
Apply separately to the plate 4 l and 20 l of the test solution, 4 l of reference solution (a) and
20 l of reference solution (b). Develop over a path of 15 cm using ethyl acetate R. Dry the plate in
a current of air for 10 min and spray with a mixture of equal volumes of alcoholic solution of sulphuric
acid R and alcohol R; use about 10 ml for a plate 200 mm square and spray in small portions,
allowing the solvent to evaporate each time to avoid excessive wetting. Heat at 100C to 105C for
4-13
30 min and immediately place the plate above, but not in contact with, 10 ml of a saturated
solution of sodium nitrite R in a glass tank. Carefully add 0.5 ml of sulphuric acid R to the sodium
nitrite solution, close the tank, and allow to stand for 15 min. Remove the plate, heat in a ventilated
oven at 40C for 15 min and spray with three quantities, each of 5 ml, of a freshly prepared 5 g/l
solution of naphthylethylenediamine dihydrochloride R in alcohol R. Examine the plate by transmitted
light. Any spot in the chromatogram obtained with 20 l of the test solution, apart from the
solution (b) (1.0 per cent).
at 100C to 105C.
Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g
ASSAY
Dissolve 0.200 g in 50 ml of anhydrous pyridine R. Titrate with 0.1M tetrabutylammonium hydroxide,
determining the end-point potentiometrically at the second point of inflexion (2.2.20). Carry out a
blank titration.
1 ml of 0.1M tetrabutylammonium hydroxide is equivalent to 21.07 mg of C
15
H
14
F
3
N
3
O
4
S
2.
__________________________________________________________________________________________________________ Ph Eur
4-14
Benethamine Penicillin
H
HN
Ph
Ph
,
N
S
O
H COOH
Me
Me
N
H H
O
C
15
H
17
N,C
16
H
18
N
2
O
4
S 545.7 751-84-8
Definition Benethamine Penicillin is benzyl(phenethyl)ammonium (6R)-6-(2-phenylacetamido)-
penicillanate. It contains not less than 96.0% and not more than 102.0% of penicillins, calculated as
C
15
H
17
N,C
16
H
18
N
2
O
4
S, and not less than 36.5% and not more than 39.0% of benethamine
[benzyl(phenethyl)amine], C
15
H
17
N, both calculated with reference to the dried substance.
Very slightly soluble in water; sparingly soluble in acetone and in chloroform; slightly soluble in
ethanol (96%).
Identification
benethamine penicillin (RS 022).
B. Carry out the method for thin-layer chromatography, Appendix III A, using a TLC silica gel silanised
plate (Merck silanised silica gel 60 plates are suitable) and a mixture of 30 volumes of acetone and 70
volumes of a 15.4% w/v solution of ammonium acetate adjusted to pH 7.0 with 13.5M ammonia or
glacial acetic acid as the mobile phase. Apply separately to the plate 1 l of each of two solutions in a
mixture of equal volumes of acetone and methanol containing (1) 0.5% w/v of the substance being
examined and (2) 0.5% w/v of benethamine penicillin BPCRS. After removal of the plate, allow it to
dry in air, expose to iodine vapour until spots appear and examine in daylight. The two principal
spots in the chromatogram obtained with solution (1) are similar in position, colour and size to the
two principal spots in the chromatogram obtained with solution (2). The test is not valid unless the
chromatogram obtained with solution (2) shows two clearly separated spots.
Specific optical rotation In a 1% w/v solution in chloroform, +120 to +125, calculated with the
dried substance, Appendix V F.
Loss on drying When dried at 60 at a pressure not exceeding 0.7 kPa for 4 hours, loses not more
Assay
For penicillins Dissolve 80 mg in 20 ml of methanol, add 5 ml of water and 5 ml of 1M sodium
hydroxide and allow to stand for 15 minutes. Add 5 ml of 1M nitric acid, 20 ml of acetate buffer pH 4.6
and 20 ml of water and titrate at 35 to 40 with 0.02M mercury nitrate VS. Titrate slowly so that the
titration takes about 15 minutes. Determine the end point potentiometrically using a platinum or
mercury indicator electrode and a mercurymercury(I) sulphate reference electrode. Disregard any
preliminary inflection on the titration curve. Each ml of 0.02M mercury nitrate VS is equivalent to
10.91 mg of total penicillins, calculated as C
15
H
17
N, C
16
H
18
N
2
O
4
S.
To a further 0.25 g dissolved in 50 ml of methanol add 25 ml of acetate buffer pH 4.6 and titrate
immediately at room temperature with 0.02M mercury nitrate VS. Determine the end point
potentiometrically using a platinum or mercury indicator electrode and a mercurymercury(I)
sulphate reference electrode. Each ml of 0.02M mercury nitrate VS is equivalent to 10.91 mg of
degradation products, calculated as C
15
H
17
N,C
16
H
18
N
2
O
4
S.
Calculate the percentage content of total penicillins and the percentage content of degradation
products. The difference between the two percentages is the content of penicillins.
For benethamine To 0.5 g add 30 ml of a saturated solution of sodium chloride and 10 ml of 5M
sodium hydroxide and extract with four 50-ml quantities of ether. Wash the combined extracts with
three 10-ml quantities of water, extract the combined washings with 25 ml of ether and add the
extract to the main ether solution. Evaporate the ether solution to a small volume, add 2 ml of
absolute ethanol and evaporate to dryness. Dissolve the residue in 50 ml of anhydrous acetic acid and
titrate with 0.1M perchloric acid VS using 1 ml of 1-naphtholbenzein solution as indicator. Repeat the
operation without the substance being examined. The difference between the titrations represents the
amount of perchloric acid required to neutralise the liberated base. Each ml of 0.1M perchloric acid VS
is equivalent to 21.13 mg of C
15
H
17
N.
Storage Benethamine Penicillin should be kept in an airtight container and stored at a temperature
not exceeding 30. If the substance is sterile, the container should be sterile, tamper-evident and
sealed so as to exclude micro-organisms.
Labelling The label states (1) the date after which the material is not intended to be used; (2) the
4-15
conditions under which it should be stored; (3) where applicable, that it is sterile; (4) where
applicable, that it is apyrogenic.
Benethamine Penicillin intended for use in the manufacture of a parenteral dosage form without a further
appropriate procedure for the removal of pyrogens complies with the following additional requirement.
Pyrogens Complies with the test for pyrogens, Appendix XIV D. Use per kg of the rabbits weight
1 ml of the supernatant liquid obtained by suspending 40 mg in 20 ml of water for injections, shaking
thoroughly and centrifuging.
Benethamine Penicillin intended for use in the manufacture of a parenteral dosage form without a further
appropriate sterilisation procedure complies with the following additional requirement.
Sterility Complies with the test for sterility, Appendix XVI A.
4-16
Benorilate
O
O OAc
NHAc
C
17
H
15
NO
5
313.3 5003-48-5
Definition Benorilate is 4-acetamidophenyl O-acetylsalicylate. It contains not less than 99.0% and
not more than 100.5% of C
17
H
15
NO
5
Characteristics A white or almost white, crystalline powder; odourless or almost odourless.
Practically insoluble in water; soluble in acetone and in chloroform; sparingly soluble in ethanol (96%)
and in methanol.
Identification
benorilate (RS 023).
B. To 10 mg add 10 ml of 6M hydrochloric acid and boil until completely dissolved. To 5 ml of the
resulting solution add 0.1 ml of strong 1-naphthol solution, mix and add sufficient 1M sodium hydroxide
to make the solution just alkaline. A blue colour is produced which is extracted into butan-1-ol.
C. The light absorption, Appendix II B, in the range 230 to 350 nm of a 0.001% w/v solution in
absolute ethanol exhibits a maximum only at 240 nm. The absorbance at 240 nm is about 0.74.
Heavy metals 1.0 g complies with limit test C for heavy metals, Appendix VII. Use 2 ml of lead
standard solution (10 ppm Pb) to prepare the standard (20 ppm).
4-Aminophenol Shake 2.5 g with 100 ml of water for 15 minutes and filter. To 20 ml of the filtrate
add 0.2 ml of sodium nitroprussidecarbonate solution, mix and allow to stand for 30 minutes. The
solution is not more intensely coloured than a solution prepared at the same time and in the same
manner but using 2 ml of a solution of 4-aminophenol containing 5 g per ml and 18 ml of water in
place of the filtrate (20 ppm).
Salicylic acid Shake 0.50 g with 20 ml of water for 15 minutes and filter. Transfer 10 ml of the
filtrate to a Nessler cylinder, dilute to 50 ml with water, add 0.2 ml of a 0.5% w/v solution of iron(III)
chloride and allow to stand for 1 minute. The colour obtained is not more intense than that of a
solution prepared at the same time by adding 0.2 ml of a 0.5% w/v solution of iron(III) chloride to a
mixture of 1 ml of a 0.025% w/v solution of salicylic acid in ethanol (96%) and sufficient water to
produce 50 ml (0.1%).
Related substances Carry out the method for thin-layer chromatography, Appendix III A, using a
silica gel HF
254
precoated plate (Analtech plates are suitable) and a mixture of 80 volumes of
dichloromethane, 15 volumes of ether and 5 volumes of glacial acetic acid as the mobile phase. Apply
separately to the plate 10 l of each of four solutions in a mixture of 9 volumes of chloroform and 1
volume of methanol containing (1) 4.0% w/v of the substance being examined, (2) 0.040% w/v of the
substance being examined, (3) 0.0080% w/v of the substance being examined and (4) 0.0080% w/v
of paracetamol. After removal of the plate, allow it to dry in air and develop in a second mobile phase
consisting of a mixture of 45 volumes of ether, 45 volumes of 2,2,4-trimethylpentane and 10 volumes
of formic acid. After removal of the plate, allow it to dry in air and examine under ultraviolet light
(254 nm).
Any spot corresponding to paracetamol in the chromatogram obtained with solution (1) is not
more intense than the spot in the chromatogram obtained with solution (4) (0.2%). Any secondary
spot in the chromatogram obtained with solution (1) with an Rf value slightly higher than that of the
principal spot is not more intense than the principal spot in the chromatogram obtained with solution
(2) (1%). Any other secondary spot in the chromatogram obtained with solution (1) is not more
intense than the spot in the chromatogram obtained with solution (3) (0.2%).
1 g.
Assay Carry out Method I for the determination of nitrogen, Appendix VIII H, using 0.6 g and 10 ml
of nitrogen-free sulphuric acid. Each ml of 0.05M sulphuric acid VS is equivalent to 31.33 mg of
C
17
H
15
NO
5
.
Action and use Anti-inflammatory analgesic.
4-17
Preparations
Benorilate Oral Suspension
Benorilate Tablets
When benorylate is prescribed or demanded, Benorilate shall be dispensed or supplied.
4-18
Benperidol
H
N
N
N
O
F
O
C
22
H
24
FN
3
O
2
381.4 2062-84-2
Benperidol complies with the requirements of the 3rd edition of the European Pharmacopoeia [1172]. These
Action and use Antipsychotic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Benperidol contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of
1-[1-[4-(4-fluorophenyl)-4-oxobutyl]piperidin-4-yl]-1,3-dihydro-2H-benzimidazol-2-one, calculated
CHARACTERS
A white or almost white powder, practically insoluble in water, freely soluble in dimethylformamide,
soluble in methylene chloride, slightly soluble in alcohol.
IDENTIFICATION
obtained with benperidol CRS. Examine the substances prepared as discs. If the spectra obtained in
the solid state show differences, dissolve the substance to be examined and the reference substance
separately in the minimum volume of methyl isobutyl ketone R, evaporate to dryness and record new
spectra using the residues.
B. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable silica gel
Test solution. Dissolve 30 mg of the substance to be examined in a mixture of 1 volume of acetone R
and 9 volumes of methanol R and dilute to 10 ml with the same mixture of solvents.
Reference solution (a). Dissolve 30 mg of benperidol CRS in a mixture of 1 volume of acetone R and 9
volumes of methanol R and dilute to 10 ml with the same mixture of solvents.
Reference solution (b). Dissolve 30 mg of benperidol CRS and 30 mg of droperidol CRS in a mixture of 1
volume of acetone R and 9 volumes of methanol R and dilute to 10 ml with the same mixture of
solvents.
1 volume of acetone R and 9 volumes of methanol R. Allow the plate to dry in air and examine in
ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is
similar in position and size to the principal spot in the chromatogram obtained with reference solu-
tion (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows
two clearly separated spots.
C. Dissolve about 10 mg in 5 ml of ethanol R. Add 0.5 ml of dinitrobenzene solution R and 0.5 ml of
2M alcoholic potassium hydroxide R. A violet colour is produced and becomes brownish-red after
20 min.
zirconyl nitrate solution R, add 1.0 ml of the filtrate. Mix, allow to stand for 5 min and compare the
4-19
TESTS
Reference solution (a). Dissolve 2.5 mg of benperidol CRS and 2.5 mg of droperidol CRS in
Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with dimethylformamide R. Dilute
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
015 10060 040 Linear gradient
1520 60 40 Isocratic elution
eluent
composition
25=0 100 0 Re-start gradient
Equilibrate the column for at least 30 min with acetonitrile R and then equilibrate with the initial
eluent composition for at least 5 min.
conditions, the retention times are: benperidol about 6.5 min and droperidol about 7 min. The test is
not valid unless the resolution between the peaks corresponding to benperidol and droperidol is at
least 2.0. If necessary, adjust the concentration of acetonitrile in the mobile phase or adjust the time
programme for the linear gradient.
Inject 10 l of dimethylformamide R as a blank, 10 l of the test solution and 10 l of reference
principal peak, is not greater than twice the area of the principal peak in the chromatogram obtained
with reference solution (b) (0.5 per cent). Disregard any peak obtained with the blank and any peak
with an area less then 0.2 times the area of the principal peak in the chromatogram obtained with
100C to 105C.
Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g in a platinum crucible.
ASSAY
Dissolve 0.300 g in 50 ml of a mixture of 1 volume of anhydrous acetic acid R and 7 volumes of methyl
ethyl ketone R and titrate with 0.1M perchloric acid, using 0.2 ml of naphtholbenzein solution R as
indicator.
22
H
24
FN
3
O
2
.
STORAGE
4-20
IMPURITIES
H
N
N
HN
O
A. 1-(piperidin-4-yl)-1,3-dihydro-2H-benzimidazol-2-one,
H
N
N
N
O
O
F
B. 1-[1-[4-(2-fluorophenyl)-4-oxobutyl]piperidin-4-yl]-1,3-dihydro-2H-benzimidazol-2-one,
N
H
N
N
N
H
N
N
O
O
O
C. 1-[1-[4-oxo-4-[4-[4-(2-oxo-2,3-dihydro-1H-benzimidazol-1-yl)piperidin-1-yl]-
phenyl]butyl]piperidin-4-yl]-1,3-dihydro-2H-benzimidazol-2-one,
H
N
N
N
O
F
O
O
D. cis-1-[1-[4-(4-fluorophenyl)-4-oxobutyl]piperidin-4-yl 1-oxide]-1,3-dihydro-2H-
benzimidazol-2-one,
H
N
N
N
O
F
O
O
E. trans-1-[1-[4-(4-fluorophenyl)-4-oxobutyl]piperidin-4-yl 1-oxide]-1,3-dihydro-2H-
benzimidazol-2-one.
__________________________________________________________________________________________________________ Ph Eur
4-21
Benserazide Hydrochloride
N
N
O
H NH
2
OH
HO OH
OH
H
H
and enantiomer
C
10
H
15
N
3
O
5
,HCl 293.7 14046-64-1
Benserazide Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Dopa-decarboxylase inhibitor.
Preparations
Co-beneldopa Tablets
Dispersible Co-beneldopa Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Benserazide hydrochloride contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of (RS)-2-amino-3-hydroxy-2-(2,3,4-trihydroxybenzyl)propanohydrazide hydro-
chloride, calculated with reference to the anhydrous substance.
CHARACTERS
A white or yellowish-white or orange-white, crystalline powder, freely soluble in water, sparingly
soluble in acetone, slightly soluble in ethanol.
IDENTIFICATION
obtained with benserazide hydrochloride CRS. Examine the substances prepared as discs. If the spectra
obtained show differences, dissolve the substance to be examined and the reference substance
separately in hot methanol R, evaporate to dryness and record new spectra using the residues.
B. Solution S (see Tests) gives reaction (a) of chlorides (2.3.1).
TESTS
solution BY
6
(Method II, 2.2.2).
+0.05.
Prepare the solutions using the mobile phase cooled to 4C and inject immediately.
Reference solution. Dissolve 5.0 mg of benserazide impurity A CRS and 5.0 mg of benserazide hydro-
chloride CRS in the mobile phase and dilute to 50.0 ml with the mobile phase. Dilute 5.0 ml of the
solution to 100.0 ml with the mobile phase.
a stainless steel column 0.125 m long and 4 mm in internal diameter packed with octylsilyl silica
of potassium dihydrogen phosphate R in 800 ml of water R; add 200 ml of acetonitrile R and 1.22 g
of sodium decanesulphonate R; adjust to pH 3.5 with phosphoric acid R,
4-22
Inject 20 l of the reference solution. The test is not valid unless the resolution between the peaks
corresponding to benserazide impurity A (first peak) and benserazide (second peak) is at least 2.0.
Inject 20 l of the test solution. Continue the chromatography for nine times the retention time of
benserazide. In the chromatogram obtained with the test solution: the area of any peak due to
benserazide impurity A is not greater than the area of the corresponding peak in the chromatogram
obtained with the reference solution (0.5 per cent); the area of any peak, apart from the principal
peak and any peak due to benserazide impurity A, is not greater than the area of the peak due to
benserazide in the chromatogram obtained with the reference solution (0.5 per cent); the sum of the
areas of all the peaks, apart from the principal peak and any peak due to benserazide impurity A, is
not greater than twice the area of the peak due to benserazide in the chromatogram obtained with the
reference solution (1 per cent). Disregard any peak with an area less than 0.1 times that of the peak
due to benserazide in the chromatogram obtained with the reference solution.
Water (2.5.12). Not more than 1.0 per cent determined on 0.500 g by the semi-micro determination
of water.
ASSAY
In order to avoid overheating during the titration, mix thoroughly throughout and stop the titration immedi-
ately after the end-point has been reached.
Dissolve 0.250 g in 5 ml of anhydrous formic acid R. Add 70 ml of anhydrous acetic acid R. Titrate
immediately with 0.1M perchloric acid, determining the end-point potentiometrically (2.2.20).
10
H
16
CIN
3
O
5
.
STORAGE
IMPURITIES
and enantiomer
H
2
N
H
N
O
H NH
2
OH
A. (RS)-2-amino-3-hydroxypropanohydrazide,
N
N
O
H NH
2
OH
HO
HO
OH
CH
2
OH
OH
OH
H
and enantiomer
B. (RS)-2-amino-3-hydroxy-2,2-bis(2,3,4-trihydroxybenzyl)propanohydrazide,
N
N
O
H NH
2
OH
HO OH
OH
H
and enantiomer
C. (RS)-2-amino-3-hydroxy-2-(2,3,4-trihydroxybenzylidene)propanohydrazide.
__________________________________________________________________________________________________________ Ph Eur
4-23
Bentonite
1302-78-9
Bentonite complies with the requirements of the 3rd edition of the European Pharmacopoeia [0467]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Bentonite is a natural clay containing a high proportion of montmorillonite, a native hydrated
aluminium silicate in which some aluminium and silicon atoms may be replaced by other atoms
such as magnesium and iron.
CHARACTERS
A very fine, homogeneous, greyish-white powder with a more or less yellowish or pinkish tint,
practically insoluble in water and in aqueous solutions. It swells with a little water forming a
malleable mass.
IDENTIFICATION
A. To 0.5 g in a metal crucible add 1 g of potassium nitrate R and 3 g of sodium carbonate R and heat
until the mixture melts. Allow to cool. To the residue add 20 ml of boiling water R, mix and filter.
Wash the residue with 50 ml of water R. To the residue add 1 ml of hydrochloric acid R and 5 ml of
water R. Filter. To the filtrate add 1 ml of strong sodium hydroxide solution R and filter. To the filtrate
add 3 ml of ammonium chloride solution R. A gelatinous white precipitate is formed.
B. It complies with the test for swelling power with water (see Tests).
C. 0.25 g gives the reaction of silicates (2.3.1).
TESTS
Alkalinity To 2 g add 100 ml of carbon dioxide-free water R and shake for 5 min. To 5 ml of the
suspension add 0.1 ml of thymolphthalein solution R. The liquid becomes bluish. Add 0.1 ml of 0.1M
hydrochloric acid. The liquid is decolourised within 5 min.
Coarse particles To 20 g add 1000 ml of water R and mix for 15 min using a high-speed mixer
capable of operating at not less than 5000 r/min. Transfer to a wet sieve (75), tared after drying at
100C to 105C. Wash with three quantities, each of 500 ml, of water R, ensuring that any
agglomerates have been dispersed. Dry at 100C to 105C and weigh. The particles on the sieve
weigh not more than 0.1 g (0.5 per cent).
Sedimentation volume To 6.0 g add 200 ml of water R and mix for 20 min using a high-speed
mixer capable of operating at 10 000 r/min. Transfer 100 ml of the suspension to a graduated
cylinder. Allow to stand for 24 h. The volume of the clear supernatant liquid is not greater than
2 ml.
Swelling power with water Add 2.0 g in twenty portions to 100 ml of a 10 g/l solution of sodium
lauryl sulphate R in a 100 ml graduated cylinder about 30 mm in diameter. Allow 2 min between
additions for each portion to settle. Allow to stand for 2 h. The apparent volume of the sediment is
not less than 22 ml.
Heavy metals (2.4.8). To 5.0 g add 7.5 ml of dilute hydrochloric acid R and 27.5 ml of water R.
Boil for 5 min. Centrifuge and filter the supernatant liquid. Wash the centrifugation residue with
water R and filter. Dilute the combined filtrates to 50.0 ml with water R. To 5 ml of the solution
add 5 ml of water R, 10 ml of hydrochloric acid R and 25 ml of methyl isobutyl ketone R and shake for
2 min. Separate the layers. Evaporate the aqueous layer to dryness on a water-bath. Dissolve the
residue in 1 ml of acetic acid R, dilute to 25 ml with water R and filter. 12 ml of the filtrate complies
with limit test A for heavy metals (50 ppm). Prepare the standard using lead standard solution
(1 ppm Pb) R.
Loss on drying (2.2.32). Not more than 15 per cent, determined on 1.000 g by drying in an oven
at 100C to 105C.
3
micro-
organisms per gram, determined by plate-count.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
4-24
Benzaldehyde
CHO
C
7
H
6
O 106.1 100-52-7
Definition Benzaldehyde contains not less than 98.0% w/w and not more than 100.5% w/w of
C
7
H
6
O.
Characteristics A clear, colourless liquid; odour, characteristic of bitter almonds.
Slightly soluble in water; miscible with ethanol (96%) and with ether.
Refractive index 1.544 to 1.546, Appendix V E.
Free acid Not more than 1.0% w/v, calculated as benzoic acid, C
7
H
6
O
2
, when determined by the
following method. To 10 ml add 20 ml of ethanol (96%) previously neutralised to phenolphthalein
solution R1 and titrate with 0.1M sodium hydroxide VS using phenolphthalein solution R1 as indicator.
Each ml of 0.1M sodium hydroxide VS is equivalent to 12.21 mg of C
7
H
6
O
2
.
Chlorinated compounds Not more than 0.05% w/v, calculated as Cl, when determined by the
following method. To 5 ml add 50 ml of isoamyl alcohol and 3 g of sodium and boil under a reflux
condenser for 1 hour. Cool, add 50 ml of water and 15 ml of nitric acid, cool, add 5 ml of 0.1M silver
nitrate VS, shake and titrate the excess silver nitrate with 0.1M ammonium thiocyanate VS using
ammonium iron(III) sulphate solution R2 as indicator. Repeat the procedure without the substance
being examined. The difference between the titrations represents the amount of silver nitrate
required. Each ml of 0.1M silver nitrate VS is equivalent to 3.545 mg of Cl.
Assay Carry out the method for determination of aldehydes, Appendix X K, using 0.5 g. Each ml of
0.5M potassium hydroxide in ethanol (60%) VS is equivalent to 53.06 mg of C
7
H
6
O.
Storage Benzaldehyde should be kept in a well-filled, well-closed container, protected from light and
stored at a temperature not exceeding 15.
4-25
Benzalkonium Chloride
8001-54-5
Benzalkonium Chloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Antiseptic detergent.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Benzalkonium chloride is a mixture of alkylbenzyldimethylammonium chlorides, the alkyl groups
having chain lengths of C
8
to C
18
. It contains not less than 95.0 per cent and not more than the
equivalent of 104.0 per cent of alkylbenzyldimethylammonium chlorides, calculated as C
22
H
40
ClN
(M
r
354.0) with reference to the anhydrous substance.
CHARACTERS
A white or yellowish-white powder or gelatinous, yellowish-white fragments, hygroscopic, soapy to
the touch, very soluble in water and in alcohol. On heating it forms a clear molten mass. An aqueous
solution froths copiously when shaken.
IDENTIFICATION
A. Dissolve 80 mg in water R and dilute to 100 ml with the same solvent. Examined between 220 nm
and 350 nm (2.2.25), the solution shows three absorption maxima, at 257 nm, 263 nm and 269 nm,
and a shoulder at about 250 nm.
B. To 2 ml of solution S (see Tests) add 0.1 ml of glacial acetic acid R and, dropwise, 1 ml of sodium
tetraphenylborate solution R. A white precipitate is formed. Filter. Dissolve the precipitate in a mixture
of 1 ml of acetone R and 5 ml of alcohol R, heating to not more than 70C. Add water R dropwise to
the warm solution until a slight opalescence forms. Heat gently until the solution is clear and allow to
cool. White crystals separate. Filter, wash with three quantities, each of 10 ml, of water R and dry in
vacuo over diphosphorus pentoxide R or anhydrous silica gel R at a temperature not exceeding 50C. The
crystals melt (2.2.14) at 127C to 133C.
C. To 5 ml of dilute sodium hydroxide solution R add 0.1 ml of bromophenol blue solution R1 and 5 ml of
chloroform R and shake. The chloroform layer is colourless. Add 0.1 ml of solution S and shake. The
chloroform layer becomes blue.
D. To 2 ml of solution S add 1 ml of dilute nitric acid R. A white precipitate is formed which dissolves
on the addition of 5 ml of alcohol R. The solution gives reaction (a) of chlorides (2.3.1).
TESTS
solution Y
6
(Method II, 2.2.2).
Acidity or alkalinity To 50 ml of solution S add 0.1 ml of bromocresol purple solution R. Not more
than 0.1 ml of 0.1M hydrochloric acid or 0.1M sodium hydroxide is required to change the colour of the
indicator.
Amines and amine salts Dissolve 5.0 g with heating in 20 ml of a mixture of 3 volumes of 1M
hydrochloric acid and 97 volumes of methanol R and add 100 ml of 2-propanol R. Pass a stream of
nitrogen R slowly through the solution. Gradually add 12.0 ml of 0.1M tetrabutylammonium hydroxide
and record the potentiometric titration curve (2.2.20). If the curve shows two points of inflexion, the
volume of titrant added between the two points is not greater than 5.0 ml. If the curve shows no
point of inflexion, the substance to be examined does not comply with the test. If the curve shows
one point of inflexion, repeat the test but add 3.0 ml of a 25.0 g/l solution of dimethyldecylamine R in
2-propanol R before the titration. If the titration curve after addition of 12.0 ml of the titrant shows
only one point of inflexion, the substance to be examined does not comply with the test.
Water (2.5.12). Not more than 10 per cent, determined on 0.300 g by the semi-micro determination
of water.
ASSAY
Dissolve 2.00 g in water R and dilute to 100.0 ml with the same solvent. Transfer 25.0 ml of the
solution to a separating funnel, add 25 ml of chloroform R, 10 ml of 0.1M sodium hydroxide and
10.0 ml of a freshly prepared 50 g/l solution of potassium iodide R. Shake well, allow to separate and
discard the chloroform layer. Shake the aqueous layer with three quantities, each of 10 ml, of
4-26
chloroform R and discard the chloroform layers. To the aqueous layer add 40 ml of hydrochloric
acid R, allow to cool and titrate with 0.05M potassium iodate until the deep-brown colour is almost
discharged. Add 2 ml of chloroform R and continue the titration, shaking vigorously, until the
chloroform layer no longer changes colour. Carry out a blank titration on a mixture of 10.0 ml of
the freshly prepared 50 g/l solution of potassium iodide R, 20 ml of water R and 40 ml of hydrochloric
acid R.
1 ml of 0.05M potassium iodate is equivalent to 35.4 mg of C
22
H
40
ClN.
__________________________________________________________________________________________________________ Ph Eur
4-27
Benzalkonium Chloride Solution
Benzalkonium Chloride Solution complies with the requirements of the 3rd edition of the European
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Benzalkonium chloride solution is an aqueous solution of a mixture of alkylbenzyldimethyl-
ammonium chlorides, the alkyl groups having chain lengths of C
8
to C
18
. Benzalkonium chloride
solution contains not less than 475 g/l and not more than 525 g/l of alkylbenzyldimethylammonium
chlorides, calculated as C
22
H
40
ClN (M
r
354.0). The solution may contain alcohol.
CHARACTERS
A clear, colourless or slightly yellowish liquid, miscible with water and with alcohol. It froths
copiously when shaken.
IDENTIFICATION
A. Dilute 0.3 ml to 100 ml with water R. Examined between 220 nm and 350 nm (2.2.25), the
solution shows three absorption maxima, at 257 nm, 263 nm and 269 nm, and a shoulder at about
250 nm.
B. To 0.05 ml add 2 ml of water R, 0.1 ml of glacial acetic acid R and, dropwise, 1 ml of sodium
tetraphenylborate solution R. A white precipitate is formed. Filter. Dissolve the precipitate in a mixture
of 1 ml of acetone R and 5 ml of alcohol R, heating to not more than 70C. Add water R dropwise to
the warm solution until a slight opalescence forms. Heat gently until the solution is clear and allow to
cool. White crystals separate. Filter, wash with three quantities, each of 10 ml, of water R and dry in
vacuo over diphosphorus pentoxide R or anhydrous silica gel R at a temperature not exceeding 50C. The
crystals melt (2.2.14) at 127C to 133C.
chloroform R and shake. The chloroform layer is colourless. Add 0.05 ml of the solution to be exam-
ined and shake. The chloroform layer becomes blue.
D. To 0.05 ml add 1 ml of dilute nitric acid R. A white precipitate is formed which dissolves on the
addition of 5 ml of alcohol R. The solution gives reaction (a) of chlorides (2.3.1).
TESTS
Solution S Dilute 2.0 g to 100 ml with carbon dioxide-free water R.
solution Y
6
(Method II, 2.2.2).
indicator.
Amines and amine salts Mix 10.0 g, while heating, with 20 ml of a mixture of 3 volumes of 1M
point of inflexion, the solution to be examined does not comply with the test. If the curve shows one
point of inflexion, repeat the test but add 3.0 ml of a 25.0 g/l solution of dimethyldecylamine R in
2-propanol R before the titration. If the titration curve after the addition of 12.0 ml of the titrant
shows only one point of inflexion, the solution to be examined does not comply with the test.
ASSAY
Determine the density (2.2.5) of the solution to be examined. Dilute 4.00 g to 100.0 ml with water R.
Transfer 25.0 ml of the solution to a separating funnel, add 25 ml of chloroform R, 10 ml of 0.1M
sodium hydroxide and 10.0 ml of a freshly prepared 50 g/l solution of potassium iodide R. Shake well,
allow to separate and discard the chloroform layer. Shake the aqueous layer with three quantities,
each of 10 ml, of chloroform R and discard the chloroform layers. To the aqueous layer add 40 ml of
hydrochloric acid R, allow to cool and titrate with 0.05M potassium iodate until the deep-brown colour is
almost discharged. Add 2 ml of chloroform R and continue the titration, shaking vigorously, until the
chloroform layer no longer changes colour. Carry out a blank titration on a mixture of 10.0 ml of the
freshly prepared 50 g/l solution of potassium iodide R, 20 ml of water R and 40 ml of hydrochloric
4-28
acid R.
22
H
40
ClN.
LABELLING
The label states the content of alcohol, if any.
______________________________________________________________________________________________________________ Ph Eur
4-29
Benzathine Benzylpenicillin
1/01
H
N
S
O
H COOH
Me
Me
N
H H
O ,
N
N
Ph
Ph
2
H
H
(C
16
H
18
N
2
O
4
S)
2
.C
16
H
20
N
2
909 1538-09-6
Benzathine Benzylpenicillin complies with the requirements of the 3rd edition of the European Pharmacopoeia
When benzathine penicillin is prescribed or demanded, Benzathine Benzylpenicillin shall be
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Benzathine benzylpenicillin is (2S,5R,6R)-3,3-dimethyl-7-oxo-6-[(phenylacetyl)amino]-4-thia-1-
azabicyclo[3.2.0]heptane-2-carboxylic acid compound with N,N-dibenzylethylenediamine (2:1). It
contains not less than 96.0 per cent and not more than the equivalent of 102.0 per cent of benzathine
benzylpenicillin and not less than 24.0 per cent and not more than 27.0 per cent of N,N-
dibenzylethylenediamine (benzathine C
16
H
20
N
2
; 240.3), both calculated with reference to the
anhydrous substance. It contains a variable quantity of water. Dispersing or suspending agents may
be added.
PRODUCTION
Benzathine benzylpenicillin complies with the requirements of the monograph on Products of
fermentation (1468).
CHARACTERS
A white powder, very slightly soluble in water, freely soluble in dimethylformamide and in
formamide, slightly soluble in alcohol.
IDENTIFICATION
obtained with benzathine benzylpenicillin CRS.
Reference solution. Dissolve 25 mg of benzathine benzylpenicillin CRS in 5 ml of methanol R.
of acetone R and 70 volumes of a 154 g/l solution of ammonium acetate R, the pH of which has been
adjusted to 7.0 with ammonia R. Allow the plate to dry in air and expose it to iodine vapour until the
spots appear. Examine in daylight. The two principal spots in the chromatogram obtained with the
test solution are similar in position, colour and size to the two principal spots in the chromatogram
obtained with the reference solution. The test is not valid unless the chromatogram obtained with the
reference solution shows to clearly separated spots.
by swirling; the solution is practically colourless. Place the test-tube on a water-bath for 1 min; a
reddish-brown colour develops.
D. To 0.1 g add 2 ml of 1M sodium hydroxide and shake for 2 min. Shake the mixture with two
quantities, each of 3 ml, of ether R. Evaporate the combined ether layers to dryness and dissolve the
residue in 1 ml of alcohol (50 per cent V/V) R. Add 5 ml of picric acid solution R, heat at 90C for 5
min and allow to cool slowly. Separate the crystals and recrystallise from alcohol (25 per cent V/V) R
containing 10 g/l of picric acid R. The crystals melt (2.2.14) at about 214C.
4-30
TESTS
Acidity or alkalinity To 0.50 g add 100 ml of carbon dioxide-free water R and shake for 5 min. Filter
through a sintered-glass filter. To 20 ml of the filtrate add 0.1 ml of bromothymol blue solution R1. The
solution is green or yellow. Not more than 0.2 ml of 0.02M sodium hydroxide is required to change the
colour of the indicator to blue.
Prepare the solutions immediately before use, using sonication (for about 2 min) to dissolve the samples.
Avoid any over heating during the sample preparation.
Test solution. Dissolve 70.0 mg of the substance to be examined in 25 ml of methanol R and dilute to
50.0 ml with a solution containing 6.8 g/l of potassium dihydrogen phosphate R and 1.02 g/l of disodium
hydrogen phosphate R.
Reference solution (a). Dissolve 70.0 mg of benzathine benzylpenicillin CRS in 25 ml of methanol R and
dilute to 50.0 ml with a solution containing 6.8 g/l of potassium dihydrogen phosphate R and 1.02 g/l of
disodium hydrogen phosphate R.
Reference solution (b). Dilute 1.0 ml of reference solution (a) to 100.0 ml with mobile phase A.
Column:
size: l = 0.25 m, = 4.0 mm,
stationary phase: end-capped octadecylsilyl silica gel for chromatography R (5 m),
temperature: 40C.
Mobile phase:
mobile phase A: mix 10 volumes of a 34 g/l solution of potassium dihydrogen phosphate R adjusted
to pH 3.5 with phosphoric acid R, 30 volumes of methanol R and 60 volumes of water R,
mobile phase B: mix 10 volumes of a 34 g/l solution of potassium dihydrogen phosphate R adjusted
to pH 3.5 with phosphoric acid R, 30 volumes of water R and 60 volumes of methanol R,
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
0 10 75 25
10 20 75 0 25 100
20 55 0 100
55 70 75 25
Injection: 20 l; inject the test solution and the reference solutions.
System suitability: reference solution (a)
relative retention with reference to benzylpenicillin: benzathine = 0.3 to 0.4; impurity C = about
2.4. If necessary, adjust the concentration of methanol in the mobile phase.
Limits:
impurity C: not more than twice the sum of the areas of the 2 principal peaks in the chromato-
gram obtained with reference solution (b) (2 per cent),
any other impurity: not more than the sum of the areas of the 2 principal peaks in the chromato-
gram obtained with reference solution (b) (1 per cent),
disregard limit: 0.05 times the sum of the areas of the 2 principal peaks in the chromatogram
obtained with reference solution (b) (0.05 per cent).
Water (2.5.12). 5.0 per cent to 8.0 per cent, determined on 0.300 g by the semi-micro deter-
mination of water.
Bacterial endotoxins (2.6.14, Method E). Suspend 20 mg of the substance to be examined in 20 ml
of a solution of 0.1M sodium hydroxide diluted 1 to 100, shake thoroughly and centrifuge. The super-
natant contains less than 0.13 I.U. per millilitre, if intended for use in the manufacture of parenteral
dosage forms without a further appropriate procedure for the removal of bacterial endotoxins.
ASSAY
Liquid chromatography (2.2.29) as described in the test for related substances.
Mobile phase: phosphate buffer solution pH 3.5 R, methanol R, water R (10: 35: 55 V/V/V).
Injection: 20 l; inject the test solution and reference solution (a).
Calculate the percentage contents of benzathine and of benzathine benzylpenicillin. Calculate the
latter by multiplying the percentage content of benzylpenicillin by 1.36.
4-31
STORAGE
container.
LABELLING
The label states:
where applicable, the name and quantity of any added dispersing or suspending agent,
IMPURITIES
NH
2
H
N
Ph
A. monobenzylethylenediamine,
COOH
B. phenylacetic acid,
HN
S
H
N
O
O N
H
N
Ph
Ph
Me
Me
H COOH
*
*
C. benzathine benzylpenicillin acid,
N
N
S
Ph H COOH
Me
Me
H
H
HOOC
D. (3S,7R,7aR)-5-benzyl-2,2-dimethyl-2,3,7,7a-tetrahydroimidazo[5,1-b]thiazole-3,7-
dicarboxylic acid (penillic acid of benzylpenicillin),
HN
S
H
N
O
Me
Me
H COOH
COOH
*
*
E. (4S)-2-[carboxy[(phenylacetyl)amino]methyl]-5,5-dimethylthiazolidine-4-carboxylic acid
(penicilloic acids of benzylpenicillin),
HN
S
H
N
O
Me
Me
H COOH
H
*
and epimer at C*
F. (2RS,4S)-2-[[(phenylacetyl)amino]methyl]-5,5-dimethylthiazolidine-4-carboxylic acid
(penilloic acids of benzylpenicillin).
__________________________________________________________________________________________________________ Ph Eur
4-32
Benzatropine Mesilate
N
Me
H
OCHPh
2
,CH
3
SO
3
H
C
21
H
25
NO,CH
4
O
3
S 403.5 132-17-2
Definition Benzatropine Mesilate is (1R,3r,5S)-3-benzhydryloxytropane methanesulphonate. It
contains not less than 98.0% and not more than 100.5% of C
21
H
25
NO, CH
4
O
3
S, calculated with
Characteristics A white, crystalline powder; odourless or almost odourless. It melts at about 144.
Very soluble in water; freely soluble in ethanol (96%); practically insoluble in ether.
Identification
A. Dry the substance at 105 for 3 hours. The infrared absorption spectrum, Appendix II A, is
concordant with the reference spectrum of benzatropine mesilate (RS 026).
B. The light absorption, Appendix II B, in the range 230 to 350 nm of a 0.1% w/v solution in 2M
hydrochloric acid exhibits two maxima, at 253 and 258 nm. The absorbance at 253 nm is about 0.96
and at 258 nm is about 1.1.
C. Dissolve 10 mg in 2 ml of water, pour into 5 ml of hot picric acid solution R1 and allow to cool. The
melting point of the precipitate, after drying at 105, is about 185, Appendix V A.
Tropine Carry out the method for thin-layer chromatography, Appendix III A, using silica gel G as the
coating substance and a mixture of 75 volumes of ethanol (96%) and 15 volumes of 13.5M ammonia
as the mobile phase. Apply separately to the plate 10 l of each of two solutions in acetone containing
(1) 4.0% w/v of the substance being examined and (2) 0.020% w/v of tropine. After removal of the
plate, allow it to dry in air and spray with sodium iodobismuthate solution and then with a 0.4% w/v
solution of sulphuric acid. Any spot corresponding to tropine in the chromatogram obtained with
solution (1) is not more intense than the spot in the chromatogram obtained with solution (2).
Related substances Carry out the method for liquid chromatography, Appendix III D, using the
following solutions. For solution (1) mix with the aid of ultrasound 50 mg of the substance being
examined with 15 ml of mobile phase A, dilute to 50 ml with the same solvent and filter. For solution
(2) dilute 1 volume of solution (1) to 100 volumes with mobile phase A and further dilute 1 volume
of the resulting solution to 5 volumes with the same solvent. For solution (3) mix with the aid of
ultrasound 50 mg of desmethyl benzatropine BPCRS with 15 ml of mobile phase A, dilute to 100 ml
and dilute 1 volume of the resulting solution to 100 volumes with the same solvent. Solution (4)
contains 0.1% w/v each of benzatropine mesilate BPCRS and desmethyl benzatropine hydrochloride
BPCRS in mobile phase A.
4.6 mm) packed with phenylsilyl silica gel for chromatography (5 m) (Zorbax SB-Phenyl 5 is suitable).
Carry out a linear gradient elution with a flow rate of 1 ml per minute using the following conditions.
Use a detection wavelength of 220 nm.
Mobile phase A: A mixture of 5 volumes of a 1M potassium phosphate buffer prepared as described for
mobile phase B, 20 volumes of acetonitrile and 75 volumes of water.
Mobile phase B: A mixture of 35 volumes of water, 60 volumes of acetonitrile and 5 volumes of a 1M
potassium phosphate buffer prepared in the following manner: dissolve 136.1 g of potassium
dihydrogen orthophosphate in 900 ml of water, add 5 ml of orthophosphoric acid (85%) and dilute to
1000 ml.
Time
(minutes)
Mobile phase A
% v/v
Mobile phase B
% v/v
0 70 30
20 30 70
30 0 100
50 0 100
55 70 30
65 70 30
Inject 20 l of solution (4). The test is not valid unless the resolution factor between the two
4-33
principal peaks is at least 1. If necessary adjust the concentration of acetonitrile or adjust the time
program of the linear gradient elution.
Inject separately 20 l of mobile phase A as a blank and 20 l each of solutions (1), (2) and (3). In
the chromatogram obtained with solution (1) the area of any peak corresponding to desmethyl
benzatropine is not greater than the area of the principal peak in the chromatogram obtained with
solution (3) (0.5%), the area of any other secondary peak is not greater that the area of the principal
peak in the chromatogram obtained with solution (2) (0.2%) and the sum of the areas of any such
peaks is not greater than 2.5 times the area of the principal peak in the chromatogram obtained with
solution (2) (0.5%). In solution (1) disregard any peaks corresponding to the peaks in the chrom-
atogram obtained with the blank solution.
1 g.
Assay Dissolve 0.6 g in 25 ml of water, add 5 ml of dilute sodium carbonate solution and extract with
four 10-ml quantities of chloroform. Wash the combined extracts with 10 ml of water, extract the
washings with 5 ml of chloroform and add the chloroform to the combined extracts. Filter and wash
the filter with 5 ml of chloroform. To the combined filtrate and washings add 25 ml of 1,4-dioxan and
titrate with 0.1M perchloric acid VS using 0.15 ml of a 0.1% w/v solution of methyl red in methanol as
indicator. Each ml of 0.1M perchloric acid VS is equivalent to 40.35 mg of C
21
H
25
NO,CH
4
O
3
S.
Storage Benzatropine Mesylate should be kept in a well-closed container.
Action and use Used in treatment of Parkinsons disease.
Preparations
Benzatropine Injection
Benzatropine Tablets
When benztropine mesylate is prescribed or demanded, Benzatropine Mesilate shall be dispensed or
supplied.
4-34
Benzbromarone
O Et
O
OH
Br
Br
C
17
H
12
Br
2
O
3
424.1 3562-84-3
Benzbromarone complies with the requirements of the 3rd edition of the European Pharmacopoeia [1393].
Action and use Uricosuric.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Benzbromarone contains not less than 98.0 and not more than the equivalent of 101.0 per cent of
(3,5-dibromo-4-hydroxyphenyl)(2-ethylbenzofuran-3-yl)methanone, calculated with reference to the
dried substance.
CHARACTERS
and in methylene chloride, sparingly soluble in alcohol.
IDENTIFICATION
A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph. Eur reference
spectrum of benzbromarone.
B. By means of a copper wire, previously ignited, introduce a small amount of the substance into the
non-illuminated part of a flame. The colour of the flame becomes green.
TESTS
Appearance of solution Dissolve 1.25 g in dimethylformamide R and dilute to 25 ml with the same
5
(Method II, 2.2.2).
Acidity or alkalinity Shake 0.5 g with 10 ml of carbon dioxide-free water R for 1 min and filter. To
2.0 ml of the filtrate add 0.1 ml of methyl red solution R and 0.1 ml of 0.01M hydrochloric acid. The
solution is red. Add 0.3 ml of 0.01M sodium hydroxide. The solution is yellow.
Test solution. Dissolve 0.125 g of the substance in 30 ml of methanol R and dilute to 50.0 ml with the
mobile phase.
Reference solution (a). Dilute 1.0 ml of the test solution to 100 ml in the mobile phase. Dilute 1 ml of
this solution to 10 ml with the mobile phase.
Reference solution (b). Dissolve 10 mg of benzarone CRS in the mobile phase and dilute to 20 ml with
the mobile phase.
Reference solution (c). To 5 ml of reference solution (b) add 1 ml of the test solution and dilute to
as mobile phase at a flow rate of 1.5 ml/min a mixture of 5 volumes of glacial acetic acid R, 25
volumes of acetonitrile R, 300 volumes of water R and 990 volumes of methanol R,
Inject 20 l of reference solution (c). Adjust the sensitivity of the system so that the heights of the
principal peaks in the chromatogram obtained are at least 50 per cent of the full scale of the recorder.
The test is not valid unless the resolution between the first peak (impurity C) and the second peak
(benzbromarone) is at least 10.0.
the test solution for 2.5 times the retention time of benzbromarone. If peaks other than the principal
4-35
peak are observed in the chromatogram obtained with the test solution, these may be due to
impurity A or to impurity B. When the chromatograms are recorded in the prescribed conditions,
the relative retention times are: impurity A about 0.6 and impurity B about 2.
In the chromatogram obtained with the test solution: the area of the peak corresponding to
impurity A is not greater than four times the area of the principal peak in the chromatogram obtained
with reference solution (a) (0.4 per cent); the area of the peak corresponding to impurity B is not
greater than ten times the area of the principal peak in the chromatogram obtained with reference
solution (a) (1 per cent); the area of any peak, apart from the principal peak and the peaks corres-
ponding to impurity A and impurity B is not greater than the area of the principal peak in the
chromatogram obtained with reference solution (a) (0.1 per cent) and the sum of the areas of any of
such peaks is not greater than twice the area of the principal peak in the chromatogram obtained with
reference solution (a)( 0.2 per cent). Disregard any peak with an area less than 0.2 times that of the
principal peak in the chromatogram obtained with reference solution (a).
Halides expressed as chlorides (2.4.4). Shake 1.25 g of the substance to be examined with a
mixture of 5 ml of dilute nitric acid R and 15 ml of water R. Filter. Rinse the filter with water R and
dilute the filtrate to 25 ml with the same solvent. Dilute 2.5 ml to 15 ml with water R. The solution
obtained complies with the limit test for chlorides (400 ppm).
Iron (2.4.9). Moisten the residue obtained in the test for sulphated ash with 2 ml of hydrochloric
acid R and evaporate to dryness on a water-bath. Add 0.05 ml of hydrochloric acid R and 10 ml of
water R and heat until boiling for 1 min. Allow to cool. Rinse the crucible with water R, collect the
rinsings and dilute to 25 ml with water R. Dilute 2 ml of this solution to 10 ml with water R. The
solution complies with the limit test for iron (125 ppm).
ASSAY
Dissolve 0.300 g in 60 ml of methanol R. Stir until completely dissolved and add 10 ml of water R.
Titrate with 0.1M sodium hydroxide. Determine the end-point potentiometrically (2.2.20).
17
H
12
BR
2
O
3
.
STORAGE
IMPURITIES
O Et
O
OH
R
4
R
3
1
R
2
A. R
1
= R
2
= R
4
= H, R
3
= Br: (3-bromo-4 hydroxyphenyl)(2 ethylbenzofuran-3-yl)methanone,
B. R
1
= R
3
= R
4
= Br, R
2
= H : (6-bromo-2-ethylbenzofuran-3-yl)(3,5-dibromo-4-
hydroxyphenyl)methanone,
C. R
1
= R
2
= R
3
= R
4
= H: (2-ethylbenzofuran-3-yl)(4-hydroxyphenyl)methanone (benzarone).
__________________________________________________________________________________________________________ Ph Eur
4-36
Benzethonium Chloride
O
O
N
Me Me
Me Me
Me
Me
Me
Cl
+
C
27
H
42
ClNO
2
448.1 121-54-0
Benzethonium Chloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Anti-infective.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Benzethonium chloride contains not less than 97.0 per cent and not more than the equivalent of
103.0 per cent of benzyldimethyl[2-[2-[4-(1,1,3,3-tetramethylbutyl)-
phenoxy]ethoxy]ethyl]ammonium chloride, calculated with reference to the dried substance.
CHARACTERS
A white or yellowish-white powder, very soluble in water and in alcohol, freely soluble in methylene
chloride.
An aqueous solution froths copiously when shaken.
IDENTIFICATION
A. Melting point (2.2.14): 158C to 164C, after drying at 105C for 4 h.
B. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable silica
same solvent.
Reference solution. Dissolve 25 mg of benzethonium chloride CRS in water R and dilute to 5 ml with
the same solvent.
of 5 volumes of water R, 5 volumes of glacial acetic acid R and 100 volumes of methanol R. Dry the
plate in a current of warm air and examine in ultraviolet light at 254 nm. The principal spot in the
C. To 5 ml of dilute sodium hydroxide solution R add 0.1 ml of bromophenol blue solution R1 and 5 ml
of methylene chloride R and shake. The lower layer is colourless. Add 0.1 ml of solution S (see Tests)
and shake. A blue colour develops in the lower layer.
D. To 2 ml of solution S add 1 ml of dilute nitric acid R. A white precipitate is formed which
dissolves upon addition of 5 ml of alcohol R. The solution gives reaction (a) of chlorides (2.3.1).
TESTS
solution Y
6
(Method II, 2.2.2).
Acidity or alkalinity To 25 ml of solution S add 0.1 ml of phenolphthalein solution R. The solution
is colourless. Add 0.3 ml of 0.01M sodium hydroxide. The solution is pink. Add 0.1 ml of methyl red
solution R and 0.5 ml of 0.01M hydrochloric acid. The solution is orange-red.
Volatile bases and salts of volatile bases (2.4.1). 0.20 g complies with limit test B for
ammonium (50 ppm). Prepare the standard using 0.1 ml of ammonium standard solution (100 ppm
NH
4
) R. Replace heavy magnesium oxide by 2.0 ml of strong sodium hydroxide solution R.
at 100C to 105C for 4 h.
ASSAY
4-37
solution to a separating funnel, add 10 ml of a 4 g/l solution of sodium hydroxide R, 10.0 ml of a
freshly prepared 50 g/l solution of potassium iodide R and 25 ml of methylene chloride R. Shake
vigorously, allow to separate and discard the lower layer. Shake the upper layer with three quanti-
ties, each of 10 ml, of methylene chloride R and discard the lower layers. To the upper layer add
40 ml of hydrochloric acid R, allow to cool and titrate with 0.05M potassium iodate until the deep
brown colour is almost discharged. Add 4 ml of methylene chloride R and continue the titration,
shaking vigorously, until the lower layer is no longer brown. Carry out a blank titration using a
mixture of 10.0 ml of a freshly prepared 50 g/l solution of potassium iodide R, 20 ml of water R and
40 ml of hydrochloric acid R.
27
H
42
ClNO
2
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
4-38
Benzocaine
COOEt
H
2
N
C
9
H
11
NO
2
165.2 94-09-7
Benzocaine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0011]. These
Action and use Local anaesthetic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Benzocaine contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of ethyl 4-aminobenzoate, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder or colourless crystals, very slightly soluble in water, freely soluble in
alcohol and in ether.
IDENTIFICATION
obtained with benzocaine CRS.
C. To about 50 mg in a test-tube add 0.2 ml of a 500 g/l solution of chromium trioxide R. Cover the
mouth of the tube with a piece of filter paper moistened with a freshly prepared mixture of equal
volumes of a 50 g/l solution of sodium nitroprusside R and a 200 g/l solution of piperazine hydrate R.
Boil gently for at least 30 s. A blue colour develops on the filter paper.
D. Dissolve about 50 mg in alcohol R and dilute to 100 ml with the same solvent. 2 ml of the
solution gives the reaction of primary aromatic amines (2.3.1).
TESTS
Appearance of solution Dissolve 1.0 g in alcohol R and dilute to 20 ml with the same solvent. The
Acidity or alkalinity Dissolve 0.5 g in 10 ml of alcohol R previously neutralised to 0.05 ml of
phenolphthalein solution R. Add 10 ml of carbon dioxide-free water R. The solution remains colourless
and not more than 0.5 ml of 0.01M sodium hydroxide is required to change the colour of the
indicator.
ASSAY
Dissolve 0.400 g in a mixture of 25 ml of hydrochloric acid R and 50 ml of water R. Carry out the
determination of primary aromatic amino-nitrogen (2.5.8).
1 ml of 0.1M sodium nitrite is equivalent to 16.52 mg of C
9
H
11
NO
2
STORAGE
__________________________________________________________________________________________________________ Ph Eur
4-39
Benzoic Acid
COOH
C
7
H
6
O
2
122.1 65-95-0
Benzoic Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia [0066]. These
Action and use Antimicrobial preservative.
Preparations
Compound Benzoic Acid Ointment
Benzoic Acid Solution
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Benzoic acid contains not less than 99.0 per cent and not more than the equivalent of 100.5 per
cent of benzenecarboxylic acid.
CHARACTERS
A white, crystalline powder or colourless crystals, odourless or with a very slight characteristic
odour, slightly soluble in water, soluble in boiling water, freely soluble in alcohol, in ether and in
fatty oils.
IDENTIFICATION
B. Solution S (see Tests) gives reaction (a) of benzoates (2.3.1).
TESTS
Solution S Dissolve 5.0 g in alcohol R and dilute to 100 ml with the same solvent.
Carbonisable substances Dissolve 0.5 g with shaking in 5 ml of sulphuric acid R. After 5 min, the
solution is not more intensely coloured than reference solution Y
5
(Method I, 2.2.2).
Oxidisable substances Dissolve 0.2 g in 10 ml of boiling water R. Cool, shake and filter. To the
filtrate add 1 ml of dilute sulphuric acid R and 0.2 ml of 0.02M potassium permanganate. After 5 min,
the solution is still coloured pink.
Halogenated compounds and halides
All glassware used must be chloride-free and may be prepared by soaking overnight in a 500 g/l solution of
nitric acid R, rinsed with water R and stored full of water R. It is recommended that glassware be reserved
for this test.
Solution (a). Dissolve 6.7 g of the substance to be examined in a mixture of 40 ml of 1M sodium
hydroxide and 50 ml of alcohol R and dilute to 100.0 ml with water R. To 10.0 ml of this solution
add 7.5 ml of dilute sodium hydroxide solution R and 0.125 g of nickel-aluminium alloy R and heat on
a water-bath for 10 min. Allow to cool to room temperature, filter into a 25 ml volumetric flask and
wash with three quantities, each of 2 ml, of alcohol R. Dilute the filtrate and washings to 25.0 ml
with water R. This solution is used to prepare solution A.
Solution (b). In the same manner, prepare a similar solution without the substance to be examined.
This solution is used to prepare solution B.
In four 25 ml volumetric flasks, place separately 10 ml of solution (a), 10 ml of solution (b), 10 ml
of chloride standard solution (8 ppm Cl) R (used to prepare solution C) and 10 ml of water R. To
each flask add 5 ml of ferric ammonium sulphate solution R5, mix and add dropwise and with swirling
2 ml of nitric acid R and 5 ml of mercuric thiocyanate solution R. Shake. Dilute the contents of each
flask to 25.0 ml with water R and allow the solutions to stand in a water-bath at 20C for 15 min.
Measure at 460 nm the absorbance (2.2.25) of solution A using solution B as the compensation
liquid, and the absorbance of solution C using the solution obtained with 10 ml of water R as the
compensation liquid. The absorbance of solution A is not greater than that of solution C
(300 ppm).
Heavy metals (2.4.8). 12 ml of solution S complies with limit test B for heavy metals (10 ppm).
Prepare the standard using a mixture of 5 ml of lead standard solution (1 ppm Pb) R and 5 ml of
alcohol R.
4-40
ASSAY
Dissolve 0.200 g in 20 ml of alcohol R and titrate with 0.1M sodium hydroxide, using 0.1 ml of phenol
red solution R as indicator, until the colour changes from yellow to violet-red.
7
H
6
O
2
.
__________________________________________________________________________________________________________ Ph Eur
4-41
Sumatra Benzoin
Benzoin
Definition Sumatra Benzoin is the balsamic resin obtained from the incised stem of Styrax benzoin
Dryand. and of S. paralleloneurus Perkins. It contains not less than 25% of total balsamic acids,
calculated as cinnamic acid, C
9
H
8
O
2
, with reference to the dried material.
Characteristics Hard, brittle masses consisting of whitish tears embedded in a greyish brown to
reddish brown translucent matrix, known in commerce as block benzoin. It also occurs in the form of
tears with cream coloured surfaces and, when broken, exhibiting surfaces having a milky white
colour. Odour, agreeable and balsamic.
Identification
A. Heat 0.5 g gently in a dry test tube. It melts and white fumes are evolved which form a white,
crystalline sublimate.
B. Warm gently 1 g, in powder, with 5 ml of dilute potassium permanganate solution. A distinct odour
of benzaldehyde is produced.
C. Triturate 0.1 g, in powder, with 5 ml of ethanol (96%), filter and add 0.5 ml of a 5% w/v solution
of anhydrous iron(III) chloride in ethanol (96%). No bright green colour is produced.
Matter insoluble in ethanol (90%) Not more than 20.0% when determined by the following
method. Warm 2 g, in coarse powder, with 25 ml of ethanol (90%), filter, wash the residue with hot
ethanol (90%) until extraction is complete and dry to constant weight at 100.
Dammar gum Carry out the method for thin-layer chromatography, Appendix III A, using aluminium
oxide G as the coating substance and a mixture of 60 volumes of ether and 40 volumes of petroleum
spirit (boiling range, 80 to 100) as the mobile phase but allowing the solvent front to ascend 10 cm
above the line of application. Apply to the plate 5 l of a solution prepared by warming 0.2 g of the
substance being examined with 10 ml of ethanol (90%) and centrifuging. After removal of the plate,
allow it to dry in air, spray with anisaldehyde solution and heat at 100 to 105 for 5 minutes. The
chromatogram does not show any prominent spot with an Rf value between 0.4 and 1.0.
Loss on drying Not more than 10.0% when determined by the following method. Spread evenly
2 g, in coarse powder, in a flat-bottomed dish (9 cm 1.5 cm) and dry over phosphorus pentoxide at a
pressure not exceeding 2.7 kPa for 4 hours.
Ash Not more than 2.0%, Appendix XI J.
Assay Boil 1.25 g with 25 ml of alcoholic potassium hydroxide solution under a reflux condenser for 1
hour. Evaporate the ethanol, disperse the residue in 50 ml of hot water, cool, add 80 ml of water and
1.5 g of magnesium sulphate dissolved in 50 ml of water. Mix thoroughly and allow to stand for 10
minutes. Filter, wash the residue on the filter with 20 ml of water, acidify the combined filtrate and
washings with hydrochloric acid and extract with four 40-ml quantities of ether. Discard the aqueous
solution, combine the ether extracts and extract with successive quantities of 20, 20, 10, 10 and
10 ml of sodium hydrogen carbonate solution, washing each aqueous extract with the same 20 ml of
ether. Discard the ether layers, carefully acidify the combined aqueous extracts with hydrochloric acid
and extract with successive quantities of 30, 20, 20 and 10 ml of chloroform, filtering each extract
through anhydrous sodium sulphate supported on absorbent cotton. Distil the chloroform from the
combined filtrates until 10 ml remains and remove the remainder in a current of air. Dissolve the
residue, with the aid of gentle heat, in 10 ml of ethanol (96%), previously neutralised to phenol red
solution, cool and titrate with 0.1M sodium hydroxide VS using phenol red solution as indicator. Each ml
of 0.1M sodium hydroxide VS is equivalent to 14.82 mg of total balsamic acids, calculated as cinnamic
acid, C
9
H
8
O
2
.
Storage Sumatra Benzoin should be kept in a well-closed container, protected from light and stored
at a temperature not exceeding 25.
Preparations
Benzoin Inhalation
Compound Benzoin Tincture
4-42
Hydrous Benzoyl Peroxide
O
O
O
O
C
14
H
10
O
4
242.2 94-36-0
Hydrous Benzoyl Peroxide complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Used topically in the treatment of acne.
Preparations
Benzoyl Peroxide Cream
Benzoyl Peroxide Gel
Benzoyl Peroxide Lotion
Potassium Hydroxyquinoline Sulphate and Benzoyl Peroxide Cream
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hydrous benzoyl peroxide contains not less than 70.0 per cent and not more than 77.0 per cent of
dibenzoyl peroxide (C
14
H
10
O
4
; M
r
242.2) and not less than 20.0 per cent of water.
CHARACTERS
A white, amorphous or granular powder, practically insoluble in water, soluble in acetone, soluble
in methylene chloride with separation of water, slightly soluble in alcohol. It loses water rapidly on
exposure to air.
Mix the entire sample thoroughly before carrying out the following tests.
IDENTIFICATION
A. Dissolve 80.0 mg in alcohol R and dilute to 100.0 ml with the same solvent. Dilute 10.0 ml of
the solution to 100.0 ml with alcohol R (solution A). Examined between 250 nm and 300 nm
(2.2.25), solution A shows an absorption maximum at 274 nm and a shoulder at about 282 nm.
Dilute 10.0 ml of solution A to 100.0 ml with alcohol R (solution B). Examined between 220 nm
and 250 nm, solution B shows an absorption maximum at 235 nm. The ratio of the absorbance at
the maximum at 235 nm (solution B) to that at the maximum at 274 nm (solution A) is 1.17 to
1.21.
B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph. Eur. refer-
ence spectrum of benzoyl peroxide.
C. Dissolve about 25 mg in 2 ml of acetone R. Add 1 ml of a 10 g/l solution of diethylphenylene-
diamine sulphate R and mix. A red colour develops which quickly darkens and becomes dark violet
within 5 min.
D. To 1 g add 5 ml of alcohol R, 5 ml of dilute sodium hydroxide solution R and 10 ml of water R. Boil
the mixture under reflux for 20 min. Cool. The solution gives reaction (c) of benzoates (2.3.1).
TESTS
Acidity Dissolve a quantity of the substance to be examined containing the equivalent of 1.0 g of
dibenzoyl peroxide in 25 ml of acetone R, add 75 ml of water R and filter. Wash the residue with
two quantities, each of 10 ml, of water R. Combine the filtrate and washings and add 0.25 ml of
phenolphthalein solution R1. Not more than 1.25 ml of 0.1M sodium hydroxide is required to change
the colour of the indicator. Carry out a blank test.
254
R as the
coating substance.
Test solution. Dissolve a quantity of the substance to be examined containing the equivalent of
0.20 g of dibenzoyl peroxide in acetone R and dilute to 5 ml with the same solvent.
Reference solution (a). Dilute 1 ml of the test solution to 100 ml with acetone R.
4-43
Reference solution (b). Dissolve 30 mg of benzoic acid R in acetone R and dilute to 50 ml with the
same solvent.
Reference solution (c). Mix 0.4 ml of benzyl benzoate R with 5 ml of acetone R and dilute to 10 ml
with the same solvent. To 1 ml of this solution add 1 ml of the test solution and dilute to 10 ml
with acetone R.
1 volume of anhydrous acetic acid R, 15 volumes of acetone R, 20 volumes of toluene R and 40
volumes of light petroleum R. Allow the plate to dry in air for 20 min and examine in ultraviolet light
at 254 nm. In the chromatogram obtained with the test solution: any spot corresponding to benzoic
acid is not more intense than the spot in the chromatogram obtained with reference solution (b)
(1.5 per cent); any spot apart from the principal spot and any spot corresponding to benzoic acid is
not more intense than the spot in the chromatogram obtained with reference solution (a) (1.0 per
cent). The test is not valid unless the chromatogram obtained with reference solution (c) shows two
clearly separated principal spots.
Chlorides (2.4.4). Dissolve a quantity of the substance to be examined containing the equivalent
of 0.5 g of dibenzoyl peroxide in 15 ml of acetone R. Add, while stirring, 50 ml of 0.05M nitric acid.
Allow to stand for 10 min and filter. Wash the residue with two quantities, each of 10 ml, of 0.05M
nitric acid. Combine the filtrate and the washings and dilute to 100 ml with 0.05M nitric acid. 2.5 ml
of the solution diluted to 15.0 ml with water R complies with the limit test for chlorides (0.4 per
cent).
ASSAY
Dissolve 2.500 g immediately before use in 75 ml of dimethylformamide R and dilute to 100.0 ml
with the same solvent (solution a).
Dibenzoyl peroxide To 5.0 ml of solution (a) add 20 ml of acetone R and 3 ml of a 500 g/l
solution of potassium iodide R and mix. Allow to stand for 1 min. Titrate with 0.1M sodium
thiosulphate until the solution is colourless. Carry out a blank titration.
1 ml of 0.1M sodium thiosulphate is equivalent to 12.11 mg of C
14
H
10
O
4
.
Water (2.5.12). Carry out the semi-micro determination of water, using 5.0 ml of solution (a). Use
as solvent 20.0 ml of anhydrous methanol R and 3.0 ml of a 100 g/l solution of potassium iodide R in
dimethylformamide R. After adding solution (a), stir for 5 min before starting the titration. Carry out
a blank determination.
Calculate the percentage content of water using the expression:
) 0744 0 (
2
2 1
. p
m
w ) n n (
+

n
1
=number of millilitres of iodosulphurous reagent R used in the sample determination,
n
2
=number of millilitres of iodosulphurous reagent R used in the blank determination,
w=the water equivalent of the iodosulphurous reagent R in milligrams of water per millilitre
of reagent,
m=mass in grams of the substance to be examined used for the preparation of solution (a),
p=percentage content of dibenzoyl peroxide.
STORAGE
Store in a container that has been treated to reduce static discharge and that has a device for release
of excess pressure. Store at a temperature between 2C and 8C, protected from light.
CAUTION
Hydrous benzoyl peroxide may explode at temperatures higher than 60C or if its water content
becomes too low. It may burst into flame in the presence of reducing substances. Unused material
must not be returned to the original container but destroyed by treating with a 100 g/l solution of
sodium hydroxide R until addition of a crystal of potassium iodide R does not liberate iodine after
acidification with dilute hydrochloric acid R.
__________________________________________________________________________________________________________ Ph Eur
4-44
Benzydamine Hydrochloride
N
N
O NMe
2
,HCl
C
19
H
23
N
3
O,HCl 345.9 132-69-4
Definition Benzydamine Hydrochloride is 3-(1-benzylindazol-3-yloxy)propyldimethylamine
hydrochloride. It contains not less than 99.0% and not more than 101.0% of C
19
H
23
N
3
O,HCl,
Production The method of manufacture is such that the level of 3-chloropropyl(dimethyl)amine
hydrochloride is not more than 5 ppm when determined by a suitable method.
Characteristics A white crystalline powder.
Very soluble in water; freely soluble in ethanol (96%) and in chloroform; practically insoluble in ether.
Identification
benzydamine hydrochloride (RS 027).
Clarity and colour of solution A 10.0% w/v solution is clear, Appendix IV A, and not more
intensely coloured than reference solution Y
6
Heavy metals 12 ml of a 10% w/v solution complies with limit test A for heavy metals, Appendix VII
(10 ppm). Use 1 ml of lead standard solution (10 ppm Pb) to prepare the standard.
following solutions in methanol (50%). Solution (1) contains 0.25% w/v of the substance being
examined. Solution (2) contains 0.0005% w/v of 3-dimethylaminopropyl 2-benzylaminobenzoate
hydrochloride BPCRS (impurity A) and 0.00125% w/v of 3-(1,5-dibenzyl-1H-indazole-3-yl)oxy-
propyldimethylamine hydrochloride BPCRS (impurity B). Solution (3) contains 0.00025% w/v of
1-benzyl-1H-indazol-3-ol BPCRS (impurity C). Solution (4) contains 0.00025% w/v of the substance
being examined. Solution (5) contains equal volumes of solutions (1), (2) and (3).
The chromatographic procedure may be carried out using a stainless steel column (25 cm
4.6 mm) packed with stationary phase C (5 m) (Suplex pkb100 is suitable), fitted with a stainless
steel guard column (2 cm 4.6 mm) packed with the same material. Use as the initial mobile phase
with a flow rate of 1.5 ml per minute, a mixture of 50 volumes of solution A and 50 volumes of
methanol. Solution A contains 0.01M potassium dihydrogen orthophosphate and 0.005M sodium octyl
sulphate in water, adjusted to pH 3.0 0.1 with orthophosphoric acid. Carry out a linear gradient
elution increasing the percentage of methanol to 70% over 20 minutes from the moment of injection
and then decreasing the percentage of methanol to 50% over 2 minutes and maintain the final mobile
phase until the completion of that run. Use a detection wavelength of 320 nm.
Inject 20 l of solution (5) and modify the rate of change of the mobile phase, if necessary, to
obtain a retention time of about 10 minutes for the substance being examined.
The test is not valid unless, in the chromatogram obtained with solution (5), the resolution factor
between any two adjacent peaks is at least 2.5.
Inject 20 l of solution (1) and allow the chromatography to proceed for 30 minutes. In the chrom-
atogram obtained the areas of any peak corresponding to impurity A or impurity B is not greater than
the area of the corresponding peak in the chromatogram obtained with solution (2) (0.2% of
impurity A and 0.5% of impurity B), the area of any peak corresponding to impurity C is not greater
than the area of the peak in the chromatogram obtained with solution (3) (0.1%) and the area of any
other secondary peak is not greater than the area of the peak in the chromatogram obtained with
solution (4) (0.1%). The sum of the areas of any such peaks is not greater than 1%.
Primary amines Dissolve 50 mg of the substance being examined in 10 ml of ethanol (96%), add
0.1 ml of hydrochloric acid and 2 ml of a 5% w/v solution of 4-dimethylaminobenzaldehyde in ethanol
(96%). The yellow colour obtained is not more intense than that obtained by treating 10 ml of a
4-45
0.00005% w/v solution of 2-aminobenzoic acid in ethanol (96%) in the same manner.
Loss on drying When dried for 3 hours at 100 to 105 at a pressure not exceeding 0.7 kPa, loses
Sulphated ash Not more than 0.1%, Appendix IX A. Use 1 g.
Assay Dissolve 0.3 g in 100 ml of anhydrous acetic acid and carry out Method I for non-aqueous
titration, Appendix VIII A, determining the end point potentiometrically. Each ml of 0.1M perchloric
acid VS is equivalent to 34.59 mg of C
19
H
23
N
3
O,HCl.
Storage Benzydamine Hydrochloride should be kept in a well-closed container.
Action and use Anti-inflammatory; analgesic.
Preparations
Benzydamine Cream
Benzydamine Mouthwash
Benzydamine Oromucosal Spray
IMPURITIES
NH
O
O
NMe
2
A. 3-dimethylaminopropyl 2-benzylaminobenzoate
N
N
O NMe
2
B. 3-(1,5-dibenzyl-1H-indazol-3-yl)oxypropyldimethylamine
N
N
OH
C. 1-benzyl-1H-indazol-3-ol
4-46
Benzyl Alcohol
CH
2
OH
C
7
H
8
O 108.1 100-51-6
Benzyl Alcohol complies with the requirements of the 3rd edition of the European Pharmacopoeia [0256].
Action and use Local anaesthetic; disinfectant.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Benzyl alcohol contains not less than 97.0 per cent and not more than the equivalent of 100.5 per
cent of phenylmethanol.
CHARACTERS
A clear colourless, refringent, oily liquid, soluble in water, miscible with alcohol, with ether and
with fatty and essential oils.
IDENTIFICATION
Add 0.1 ml to 5 ml of potassium permanganate solution R. Add 1 ml of dilute sulphuric acid R. The
characteristic odour of benzaldehyde is produced.
TESTS
Solubility Shake 2 ml with 60 ml of water R. It dissolves completely giving a clear solution (2.2.1).
Acidity. To 10 ml add 10 ml of alcohol R and 1 ml of phenolphthalein solution R. Not more than
1 ml of 0.1M sodium hydroxide is required to change the colour of the indicator to pink.
Benzaldehyde and other related substances Examine by gas chromatography (2.2.28).
Test solution. Use the substance to be examined.
Reference solution. Dissolve 0.1 g of dibutyl phthalate R and 0.1 g of benzaldehyde R in the substance
to be examined and dilute to 100 ml with the same solvent.
a column 2 m long and 3 mm in internal diameter packed with silanised diatomaceous earth for gas
chromatography R impregnated with 3 per cent m/m of polymethylphenylsiloxane R,
Use a glass column and injector. Programme the temperature of the column linearly at 10C per
minute from 85C to 290C. Maintain the temperature of the injection port at 210C and that of
the detector at 275C. Inject 1 l of each solution at the top of the column or via a glass-lined
injector.
benzaldehyde is not greater than 1.5 times the area of the peak corresponding to dibutyl phthalate
in the chromatogram obtained with the reference solution (0.15 per cent); the sum of the areas of
the peaks, apart from those corresponding to benzyl alcohol and benzaldehyde, is not greater than
twice the area of the peak corresponding to dibutyl phthalate in the chromatogram obtained with
the reference solution (0.2 per cent).
Benzyl alcohol intended for use in the manufacture of parenteral dosage forms contains not more than
0.05 per cent of benzaldehyde and not more than 0.1 per cent of other related substances.
Halogenated compounds and halides.
All glassware must be chloride-free and may be prepared by soaking overnight in a 500 g/l solution of nitric
acid R, rinsed with water R and stored full of water R. It is recommended that glassware be reserved for this
test.
Solution (a). Dissolve 6.7 g of the substance to be examined in 50 ml of alcohol R and dilute to
100.0 ml with water R. To 10.0 ml of this solution add 7.5 ml of dilute sodium hydroxide solution R
and 0.125 g of nickel-aluminium alloy R and heat on a water-bath for 10 min. Allow to cool to room
temperature, filter into a 25 ml volumetric flask and wash with three quantities, each of 2 ml, of
4-47
alcohol R. Dilute the filtrate and washings to 25.0 ml with water R. This solution is used to prepare
solution A.
Solution (b). In the same manner, prepare a similar solution without the substance to be examined.
This solution is used to prepare solution B.
In four 25 ml volumetric flasks, place separately 10 ml of solution (a), 10 ml of solution (b), 10 ml
of chloride standard solution (8 ppm Cl) R (used to prepare solution C) and 10 ml of water R. To
each flask add 5 ml of ferric ammonium sulphate solution R5, mix and add dropwise and with swirling
2 ml of nitric acid R and 5 ml of mercuric thiocyanate solution R. Shake. Dilute the contents of each
flask to 25.0 ml with water R and allow the solutions to stand in a water-bath at 20C for 15 min.
Measure at 460 nm the absorbance (2.2.25) of solution A using solution B as the compensation
liquid, and the absorbance of solution C using the solution obtained with 10 ml of water R as the
compensation liquid. The absorbance of solution A is not greater than that of solution C
(300 ppm).
Peroxide value (2.5.5). Not more than 5.
Residue on evaporation Evaporate 2.0 g to dryness on a water-bath, dry at 100C to 105C for
1 h and allow to cool in a desiccator. The residue weighs not more than 1 mg (0.05 per cent).
ASSAY
To 0.900 g (m g) add 15.0 ml of a freshly prepared mixture of 1 volume of acetic anhydride R and 7
volumes of pyridine R and boil under a reflux condenser on a water-bath for 30 min. Cool and add
25 ml of water R. Using 0.25 ml of phenolphthalein solution R as indicator, titrate with 1M sodium
hydroxide (n
1
ml). Carry out a blank titration (n
2
ml).
7
H
8
O from the expression:
10 81
2 1
. ( ) n n
m
STORAGE
Store in a completely filled and well-closed container, protected from light.
LABELLING
The label states, where applicable, that the substance is suitable for use in the manufacture of
parenteral dosage forms.
__________________________________________________________________________________________________________ Ph Eur
4-48
Benzyl Benzoate
O
O
C
14
H
12
O
2
212.2 120-51-4
Benzyl Benzoate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0705].
Action and use Used topically in the treatment of scabies.
Preparation
Benzyl Benzoate Application
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Benzyl benzoate contains not less than 99.0 per cent and not more than the equivalent of 100.5 per
cent of phenylmethyl benzoate.
CHARACTERS
Colourless or almost colourless crystals or a colourless or almost colourless, oily liquid, practically
insoluble in water, miscible with alcohol, with ether, with methylene chloride and with fatty and
essential oils.
It boils at about 320C.
IDENTIFICATION
A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph. Eur. refer-
ence spectrum of benzyl benzoate.
B. To 2 g add 25 ml of alcoholic potassium hydroxide solution R and boil under a reflux condenser for
2 h. Remove the ethanol on a water-bath, add 50 ml of water R and distil; collect about 25 ml of
distillate and use it for identification test C. Acidify the liquid remaining in the distillation flask with
dilute hydrochloric acid R; a white precipitate is formed, which, when washed with water R and dried
in vacuo, melts (2.2.14) at 121C to 124C.
C. To the distillate obtained in identification test B add 2.5 g of potassium permanganate R and 5 ml
of dilute sodium hydroxide solution R. Boil under a reflux condenser for 15 min, cool and filter.
Acidify the filtrate with dilute hydrochloric acid R; a white precipitate is formed which, when washed
with water R and dried in vacuo, melts (2.2.14) at 121C to 124C.
TESTS
Acidity Dissolve 2.0 g in alcohol R and dilute to 10 ml with the same solvent. Titrate with 0.1M
sodium hydroxide using phenolphthalein solution R as indicator. Not more than 0.2 ml is required to
change the colour of the indicator to pink.
ASSAY
To 2.000 g add 50.0 ml of 0.5M alcoholic potassium hydroxide and boil gently under a reflux
condenser for 1 h. Titrate the hot solution with 0.5M hydrochloric acid using 1 ml of phenolphthalein
solution R as indicator. Carry out a blank determination.
14
H
12
O
2
.
STORAGE
Store in an airtight, well-filled container, protected from light.
__________________________________________________________________________________________________________ Ph Eur
05-1
Benzyl Hydroxybenzoate
Benzylparaben
HO
O
O
C
14
H
12
O
3
228.3 94-18-8
Definition Benzyl Hydroxybenzoate is benzyl 4-hydroxybenzoate. It contains not less than 99.0%
and not more than 101.0% of C
14
H
12
O
3
.
Characteristics A white to creamy white, crystalline powder; odourless or almost odourless.
Practically insoluble in water; freely soluble in ethanol (96%) and in ether. It dissolves in solutions
of alkali hydroxides.
Identification
A. The infrared absorption spectrum, Appendix II A, is concordant with the reference spectrum of benzyl
hydroxybenzoate (RS 028).
ethanol (96%) exhibits a maximum only at 260 nm. The absorbance at the maximum at 260 nm is
about 0.76.
C. Dissolve 0.1 g in 2 ml of ethanol (96%), boil and add 0.5 ml of nitric acid solution of mercury. A
precipitate is produced slowly and the supernatant liquid becomes red.
D. Melting point, about 112, Appendix V A.
Acidity Dissolve 0.2 g in 10 ml of ethanol (50%) previously neutralised to methyl red solution and
titrate with 0.1M sodium hydroxide VS using methyl red solution as indicator. Not more than 0.1 ml of
0.1M sodium hydroxide VS is required to change the colour of the solution.
plate precoated with silica gel F
254
the surface of which has been modified with chemically-bonded
octadecylsilyl groups (Whatman KC18F plates are suitable) and a mixture of 70 volumes of methanol,
30 volumes of water and 1 volume of glacial acetic acid as the mobile phase. Apply separately to the
plate 2 l of each of two solutions of the substance being examined in acetone containing (1) 1.0% w/v
and (2) 0.010% w/v. After removal of the plate, allow it to dry in air and examine under ultraviolet
light (254 nm). Any secondary spot in the chromatogram obtained with solution (1) is not more intense
than the spot in the chromatogram obtained with solution (2).
Assay Gently boil 0.12 g under a reflux condenser with 20 ml of 2M sodium hydroxide for 30 minutes.
Cool and extract with three 20-ml quantities of 1,2-dichloroethane. Wash the combined extracts with
20 ml of 0.1M sodium hydroxide and add the washings to the main aqueous phase, discarding the
organic layer. To the aqueous solution add 25 ml of 0.0333M potassium bromate VS, 5 ml of a 12.5%
w/v solution of potassium bromide and 10 ml of hydrochloric acid and immediately stopper the flask.
Shake for 15 minutes and allow to stand for 15 minutes. Add 25 ml of dilute potassium iodide solution
and shake vigorously. Titrate the liberated iodine with 0.1M sodium thiosulphate VS using starch
mucilage, added towards the end of the titration, as indicator. Repeat the operation without the
substance being examined. The difference between the titrations represents the amount of potassium
bromate required. The volume of 0.0333M potassium bromate VS used is equivalent to half of the
volume of 0.1M sodium thiosulphate VS required for the titration. Each ml of 0.0333M potassium
bromate VS is equivalent to 7.608 mg of C
14
H
12
O
3
.
05-2
Benzylpenicillin Potassium
1/01
N
S
O
H
Me
Me
H H
COOK
N
O
H
C
16
H
17
KN
2
O
4
S 372.5 113-98-4
Benzylpenicillin Potassium complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparations
Benzylpenicillin Injection
Fortified Procaine Benzylpenicillin Injection/Fortified Procaine Penicillin Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Benzylpenicillin potassium is potassium (2S,5R,6R)-3,3-dimethyl-7-oxo-6-[(phenylacetyl)amino]-4-
thia-1-azabicyclo[3.2.0]heptane-2-carboxylate, a substance produced by the growth of certain strains
of Penicillium notatum or related organisms, or obtained by any other means. It contains not less than
96.0 per cent and not more than the equivalent of 102.0 per cent of benzylpenicillin potassium,
PRODUCTION
Benzylpenicillin potassium complies with the requirements of the monograph on Products of
fermentation (1468).
CHARACTERS
A white or almost white, crystalline powder, very soluble in water, practically insoluble in fatty oils
and in liquid paraffin.
IDENTIFICATION
obtained with benzylpenicillin potassium CRS.
Test solution. Dissolve 25 mg of the substance to be examined in 5 ml of water R.
Reference solution (a). Dissolve 25 mg of benzylpenicillin potassium CRS in 5 ml of water R.
Reference solution (b). Dissolve 25 mg of benzylpenicillin potassium CRS and 25 mg of
phenoxymethylpenicillin potassium CRS in 5 ml of water R.
D. It gives reaction (a) of potassium (2.3.1).
05-3
TESTS
Specific optical rotation (2.2.7). Dissolve 0.500 g in carbon dioxide-free water R and dilute to
25.0 ml with the same solvent. The specific optical rotation is +270 to +300, calculated with
Absorbance (2.2.25). Dissolve 94.0 mg in water R and dilute to 50.0 ml with the same solvent.
Measure the absorbance of the solution at 325 nm, 280 nm and at the maximum at 264 nm, diluting
the solution, if necessary, for the measurement at 264 nm. The absorbances at 325 nm and 280 nm
do not exceed 0.10 and that at the maximum at 264 nm is 0.80 to 0.88, calculated on the basis of the
undiluted (1.88 g/l) solution. Verify the resolution of the apparatus (2.2.25); the ratio of the
absorbances is at least 1.7.
Related substances Examine by liquid chromatography (2.2.29) as described under Assay. Inject 20
l of reference solution (d) and elute isocratically with the chosen mobile phase. Inject 20 l of test
solution (b) and start the elution isocratically. Immediately after elution of the benzylpenicillin peak
start the following linear gradient:
Time
(min)
Mobile phase A
(% V/V)
Mobile phase B
(%V/V)
Comment
Inject water R and use the same elution pattern to obtain a blank. In the chromatogram obtained
with test solution (b), the area of any peak, apart from the principal peak, is not greater than the area
of the principal peak in the chromatogram obtained with reference solution (d) (1 per cent).
100C to 105C.
Bacterial endotoxins (2.6.14, Method E): less than 0.16 I.U. per milligram, if intended for use in
the manufacture of parenteral dosage forms without a further appropriate procedure for the removal
of bacterial endotoxins.
ASSAY
Test solution (a). Dissolve 50.0 mg of the substance to be examined in water R and dilute to 50.0 ml
Test solution (b). Prepare the solution immediately before use. Dissolve 80.0 mg of the substance to be
examined in water R and dilute to 20.0 ml with the same solvent.
Reference solution (a). Dissolve 50.0 mg of benzylpenicillin sodium CRS in water R and dilute to 50.0 ml
Reference solution (b). Dissolve 10 mg of benzylpenicillin sodium CRS and 10 mg of phenylacetic acid
Reference solution (c). Dilute 1.0 ml of reference solution (a) to 20.0 ml with water R. Dilute 1.0 ml of
the solution to 50.0 ml with water R.
Reference solution (d). Dilute 4.0 ml of reference solution (a) to 100.0 ml with water R.
Mobile phase A. Mix 10 volumes of a 68 g/l solution of potassium dihydrogen phosphate R adjusted to
pH 3.5 with a 500 g/l solution of dilute phosphoric acid R, 30 volumes of methanol R and 60 volumes of
water R,
Mobile phase B. Mix 10 volumes of a 68 g/l solution of potassium dihydrogen phosphate R adjusted to
pH 3.5 with a 500 g/l solution of dilute phosphoric acid R, 40 volumes of water R and 50 volumes of
methanol R,
Equilibrate the column with a mobile phase ratio A:B of 70:30. Inject 20 l of reference solution (b).
05-4
The test is not valid unless the resolution between the two principal peaks is at least 6.0 (if
necessary, adjust the ratio A: B of the mobile phase) and the mass distribution ratio for the second
peak (benzylpenicillin) is 4.0 to 6.0. Inject 20 l of reference solution (c). Adjust the system to
obtain a peak with a signal-to-noise ratio of at least 3. Inject reference solution (a) six times. The
test is not valid unless the relative standard deviation for the area of the principal peak is at most
1.0 per cent. Inject alternately test solution (a) and reference solution (a).
Calculate the percentage content of benzylpenicillin potassium by multiplying the percentage
content of benzylpenicillin sodium by 1.045.
STORAGE
container.
LABELLING
The label states:
IMPURITIES
N
S
Me
Me
H COOH
O
H H
2
N
COOH
N
S
Me
Me
H COOH
O
H H
H
N
O
O
C. (2S,5R,6R)-6-[[(4-hydroxyphenyl)acetyl]amino]-3,3-dimethyl-7-oxo-4-thia- 1-
N
N
S
H COOH
Me
Me
H
H
HOOC
HN
S
H
N
O
Me
Me
H COOH
COOH
*
*
HN
S
H
N
O
Me
Me
H COOH
H
*
and epimer at C*
__________________________________________________________________________________________________________ Ph Eur
05-5
Benzylpenicillin Sodium
1/01
N
S
O
H
Me
Me
H H
COONa
N
O
H
C
16
H
17
N
2
NaO
4
S 356.4 69-57-8
Benzylpenicillin Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparations
Benzylpenicillin Injection
Fortified Procaine Benzylpenicillin Injection/Fortified Procaine Penicillin Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Benzylpenicillin sodium is sodium (2S,5R,6R)-3,3-dimethyl-7-oxo-6-[(phenylacetyl)amino]-4-thia-1-
azabicyclo[3.2.0]heptane-2-carboxylate, a substance produced by the growth of certain strains of
Penicillium notatum or related organisms, or obtained by any other means. It contains not less than
96.0 per cent and not more than the equivalent of 102.0 per cent of benzylpenicillin sodium,
PRODUCTION
Benzylpenicillin sodium complies with the requirements of the monograph on Products of fermentation
(1468).
If manufactured by a process that may leave residues of 2-ethylhexanoic acid in the substance, it
complies with the following test.
CHARACTERS
A white or almost white, crystalline powder, very soluble in water, practically insoluble in fatty oils
and in liquid paraffin.
IDENTIFICATION
obtained with benzylpenicillin sodium CRS.
Reference solution (a). Dissolve 25 mg of benzylpenicillin sodium CRS in 5 ml of water R.
Reference solution (b). Dissolve 25 mg of benzylpenicillin sodium CRS and 25 mg of
phenoxymethylpenicillin potassium CRS in 5 ml of water R.
05-6
TESTS
Specific optical rotation (2.2.7). Dissolve 0.500 g in carbon dioxide-free water R and dilute to
Measure the absorbance of the solution at 325 nm, at 280 nm and at the maximum at 264 nm,
diluting the solution, if necessary, for the measurement at 264 nm. The absorbances at 325 nm and
280 nm are not greater than 0.10 and the absorbance at the maximum at 264 nm is 0.80 to 0.88,
calculated on the basis of the undiluted (1.80 g/l) solution. Verify the resolution of the apparatus
(2.2.25); the ratio of the absorbances is at least 1.7.
20 l of reference solution (d) and elute isocratically with the chosen mobile phase. Inject 20 l of test
solution (b) and start the elution isocratically. Immediately after elution of the benzylpenicillin peak
start the following linear gradient:
Time
(min)
Mobile phase A
(%V/V)
Mobile phase B
(% V/V)
Comment
Inject water R and use the same elution pattern to obtain a blank. In the chromatogram obtained
with test solution (b), the area of any peak, apart from the principal peak, is not greater than the area
of the principal peak in the chromatogram obtained with reference solution (d) (1 per cent).
100C to 105C.
Bacterial endotoxins (2.6.14, Method E): less than 0.16 I.U. per milligram, if intended for use in
the manufacture of parenteral dosage forms without a further appropriate procedure for the removal
of bacterial endotoxins.
ASSAY
Test solution (a). Dissolve 50.0 mg of the substance to be examined in water R and dilute to 50.0 ml
Test solution (b). Prepare the solution immediately before use. Dissolve 80.0 mg of the substance to be
examined in water R and dilute to 20.0 ml with the same solvent.
Reference solution (a). Dissolve 50.0 mg of benzylpenicillin sodium CRS in water R and dilute to 50.0 ml
Reference solution (b). Dissolve 10 mg of benzylpenicillin sodium CRS and 10 mg of phenylacetic acid
Reference solution (c). Dilute 1.0 ml of reference solution (a) to 20.0 ml with water R. Dilute 1.0 ml of
the solution to 50.0 ml with water R.
Mobile phase A. Mix 10 volumes of a 68 g/l solution of potassium dihydrogen phosphate R adjusted to
pH 3.5 with a 500 g/l solution of dilute phosphoric acid R, 30 volumes of methanol R and 60 volumes of
water R,
Mobile phase B. Mix 10 volumes of a 68 g/l solution of potassium dihydrogen phosphate R adjusted to
pH 3.5 with a 500 g/l solution of dilute phosphoric acid R, 40 volumes of water R and 50 volumes of
methanol R,
Equilibrate the column with a mobile phase ratio A: B of 70: 30. Inject 20 l of reference solution
05-7
(b). The test is not valid unless the resolution between the two principal peaks is at least 6.0 (if
necessary, adjust the ratio A: B of the mobile phase) and the mass distribution ratio for the second
peak (benzylpenicillin) is 4.0 to 6.0. Inject 20 l of reference solution (c). Adjust the system to obtain
a peak with a signal-to-noise ratio of at least 3. Inject reference solution (a) six times. The test is not
valid unless the relative standard deviation for the area of the principal peak is at most 1.0 per cent.
Inject alternately test solution (a) and reference solution (a).
STORAGE
container.
LABELLING
The label states:
IMPURITIES
N
S
Me
Me
H COOH
O
H H
2
N
COOH
N
S
Me
Me
H COOH
O
H H
H
N
O
O
C. (2S,5R,6R)-6-[[(4-hydroxyphenyl)acetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-
N
N
S
H COOH
Me
Me
H
H
HOOC
HN
S
H
N
O
Me
Me
H COOH
COOH
*
*
HN
S
H
N
O
Me
Me
H COOH
H
*
and enantiomer at C*
__________________________________________________________________________________________________________ Ph Eur
05-8
Betacarotene
Me Me
Me Me
Me Me
Me
Me Me
Me
C
40
H
56
536.9 7235-40-7
Betacarotene complies with the requirements of the 3rd edition of the European Pharmacopoeia [1069]. These
Action and use Precursor of vitamin A.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Betacarotene contains not less than 96.0 per cent and not more than the equivalent of 101.0 per cent
of (all-E)-1,19-(3,7,12,16-tetramethyl-1,3,5,7,9,11,13,15,17-octadecanonaene-1,18-diyl)bis[2,6,6-
trimethylcyclohexene], calculated with reference to the dried substance.
CHARACTERS
A brown-red or brownish-red, crystalline powder, practically insoluble in water, slightly soluble in
cyclohexane and in ether, practically insoluble in ethanol. It is sensitive to air, heat and light,
especially in solution.
Carry out all operations as rapidly as possible avoiding exposure to actinic light; use freshly prepared solutions.
IDENTIFICATION
Dissolve 50.0 mg in 10 ml of chloroform R and dilute immediately to 100.0 ml with cyclohexane R.
Dilute 5.0 ml of this solution to 100.0 ml with cyclohexane R (solution A; use solution A also for the
test for related substances). Dilute 5.0 ml of solution A to 50.0 ml with cyclohexane R. (Solution B;
use solution B also for the test for related substances and for the assay). Determine the absorbance
(2.2.25) of solution B at 455 nm and at 483 nm using cyclohexane R as the compensation liquid. The
ratio of the absorbance at 455 nm to that at 483 nm is between 1.14 and 1.18.
TESTS
Related substances Determine the absorbance (2.2.25) of solution B at 455 nm and that of solu-
tion A at 340 nm, used in Identification. The ratio of the absorbance at 455 nm to that at 340 nm is
not less than 1.5.
Loss on drying (2.2.32). Not more than 0.2 per cent, determined on 1.000 g by drying in vacuo
over diphosphorus pentoxide R at 40C for 4 h.
Sulphated ash (2.4.14). Not more than 0.2 per cent, determined on 1.0 g, moistened with a
mixture of 2 ml of dilute sulphuric acid R and 5 ml of alcohol R.
ASSAY
Measure the absorbance (2.2.25) of solution B used in Identification at the maximum at 455 nm,
using cyclohexane R as the compensation liquid.
40
H
56
taking the specific absorbance to be 2500.
STORAGE
Store in an airtight container, protected from light, at a temperature not exceeding 25C.
__________________________________________________________________________________________________________ Ph Eur
05-9
Betadex
Betacyclodextrin corrected1/01
O
O
O
O
O
O
O
O
O
O
O
O
O
OH
OH
OH
HO
OH
HO
OH
OH
OH
HO
OH
HO
OH
HO
OH
OH
HO
HO
HO
HO
HO
[C
6
H
10
O
5
]
7
1135 68168-23-0 (monohydrate)
Betadex complies with the requirements of the 3rd edition of the European Pharmacopoeia [1070]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Betadex (betacyclodextrin) contains not less than 98.0 per cent and not more than the equivalent of
101.0 per cent of cyclo--(14)-D-heptaglucopyranoside, calculated with reference to the dried
substance.
CHARACTERS
A white or almost white, amorphous or crystalline powder, sparingly soluble in water, freely soluble
in propylene glycol, practically insoluble in ethanol and in methylene chloride.
IDENTIFICATION
C. Dissolve 0.2 g in 2 ml of iodine solution R4 by warming on a water-bath, and allow to stand at
room temperature. A yellowish-brown precipitate is formed.
TESTS
Solution S Dissolve 1.000 g in carbon dioxide-free water R with heating, allow to cool and dilute to
pH (2.2.3). To 10 ml of solution S add 0.1 ml of a saturated solution of potassium chloride R. The pH
of the solution is 5.0 to 8.0.
Reducing sugars
Test solution. To 1 ml of solution S add 1 ml of cupri-tartaric solution R4. Heat on a water-bath for
10 min, cool to room temperature. Add 10 ml of ammonium molybdate reagent R1 and allow to stand
for 15 min.
Reference solution. Prepare a reference solution at the same time and in the same manner as the test
solution, using 1 ml of a 0.02 g/l solution of glucose R.
Measure the absorbance of the test solution and the reference solution (2.2.25) at the maximum at
740 nm using water R as the compensation liquid. The absorbance of the test solution is not greater
05-10
than that of the reference solution (0.2 per cent).
Light-absorbing impurities Examine solution S between 230 nm and 750 nm (2.2.25). Between
230 nm and 350 nm, the absorbance is not greater than 0.10. Between 350 nm and 750 nm, the
absorbance is not greater than 0.05.
Related substances Examine by liquid chromatography (2.2.29), as described under Assay. Inject
separately test solution (a) and reference solution (b). In the chromatogram obtained with test
solution (a): the areas of any peaks corresponding to gammacyclodextrin and alphacyclodextrin are
not greater than half of the area of the corresponding peaks in the chromatogram obtained with
peak and any peaks corresponding to alphacyclodextrin and gammacyclodextrin, is not greater than
half of the area of the peak corresponding to betadex in the chromatogram obtained with reference
Residual solvents Not more than 10 ppm of trichloroethylene and not more than 10 ppm of
toluene. Examine by head-space gas chromatography (2.2.28), using the standard additions method
and ethylene chloride R as the internal standard.
Test solution. In each of four identical 20 ml flasks, dissolve 500 mg of the substance to be examined
in water R and add 0.10 g of calcium chloride R and 30 l of -amylase solution R. To three of the
flasks, add 1 ml of one of the reference solutions, adding a different solution to each flask. Dilute to
10 ml with water R.
Reference solutions. Prepare solutions containing per litre: 5 l, 10 l and 15 l each of
trichloroethylene R and of toluene R and 10 l of ethylene chloride R.
a fused-silica column 25 m long and 0.32 mm in internal diameter coated with a layer about
1 m thick of macrogol 20,000 R,
the detector at 280C. Place the samples in a thermostated chamber at 45C for 2 h. Inject 200 l of
the head-space of each flask and repeat each test at least three times. The retention time of toluene is
about 10 min. The test is not valid unless: the resolutions between the peaks corresponding to
trichloroethylene and toluene and between the peaks corresponding to toluene and ethylene chloride
are greater than 1.1 and the relative standard deviations of the ratios of the areas of the peaks corres-
ponding to trichloroethylene and toluene to that of the peak corresponding to ethylene chloride are
Calculate the content of trichloroethylene and of toluene taking their relative densities to be 1.46
and 0.87, respectively.
at 120C for 2 h.
ASSAY
Test solution (a). Dissolve 0.25 g of the substance to be examined in water R with heating, cool and
Reference solution (a). Dissolve 25.0 mg of alfadex CRS, 25.0 mg of gammacyclodextrin CRS and
50.0 mg of betadex CRS in water R and dilute to 50.0 ml with the same solvent.
Reference solution (c). Dissolve 25.0 mg of betadex CRS in water R and dilute to 25.0 ml with the same
solvent.
volumes of water R,
as detector a differential refractometer,
Equilibrate the column with the mobile phase at a flow rate of 1.5 ml/min for about 3 h. Inject each
solution. Record the chromatograms for 1.5 times the retention time of betadex. Adjust the
05-11
sensitivity of the detector so that the height of the peak corresponding to gammacyclodextrin, in
the chromatogram obtained with reference solution (a), is 55 per cent to 75 per cent of the full
scale of the recorder. The retention time of betadex is about 10 min, the relative retention time of
gammacyclodextrin is about 0.3 and that of alfadex is about 0.45. The test is not valid unless the
resolution between the peaks corresponding to gammacyclodextrin and alfadex is not less than 1.5,
and the relative standard deviation of the area of the peak corresponding to betadex is less than
2.0 per cent. If necessary, adjust the concentration of methanol in the mobile phase to achieve the
required resolution. Calculate the percentage content of [C
6
H
10
O
5
]
7
from the area of the principal
peak in each of the chromatograms obtained with test solution (b) and reference solution (c) and the
declared content of betadex CRS.
STORAGE
IMPURITIES
A. alfadex,
B. gammacyclodextrin.
__________________________________________________________________________________________________________ Ph Eur
05-12
Betahistine Mesilate
N NHMe
, CH
3
SO
3
H
C
10
H
20
N
2
O
6
S
2
328.4
Betahistine Mesilate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1071].
Action and use Vasodilator.
When betahistine mesylate is prescribed or demanded, Betahistine Mesilate shall be dispensed or
supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Betahistine mesilate contains not less than 98.0 per cent and not more than the equivalent of
101.0 per cent of 2-[(2-methylamino)ethyl]pyridinebis(methanesulphonate), calculated with
reference to the anhydrous, 2-propanol-free substance.
CHARACTERS
A white, crystalline powder, very hygroscopic, very soluble in water, freely soluble in alcohol, very
slightly soluble in 2-propanol.
IDENTIFICATION
Second identification A, C, D.
obtained with betahistine mesilate CRS. Examine the substances prepared as discs.
C. Examine by thin-layer chromatography (2.2.27), using a suitable silica gel with a fluorescent
indicator having an optimal intensity at 254 nm as the coating substance.
Test solution. Dissolve 10 mg of the substance to be examined in alcohol R and dilute to 2 ml with the
same solvent.
Reference solution. Dissolve 10 mg of betahistine mesilate CRS, in alcohol R and dilute to 2 ml with the
same solvent.
0.75 volumes of concentrated ammonia R, 15 volumes of ethyl acetate R and 30 volumes of methanol R.
Dry the plate at 110C for 10 min and examine in ultraviolet light at 254 nm. The principal spot in
the chromatogram obtained with the test solution is similar in position and size to the principal spot
D. To 0.1 g add 5 ml of dilute hydrochloric acid R and shake for about 5 min. Add 1 ml of barium
chloride solution R1. The solution remains clear. To a further 0.1 g add 0.5 g of anhydrous sodium
carbonate R, mix and ignite until a white residue is obtained. Allow to cool and dissolve the residue in
7 ml of water R. The solution gives reaction (a) of sulphates (2.3.1).
TESTS
Solution S Dissolve 5.0 g in carbon dioxide-free water R prepared from distilled water R, and dilute to
Related substances. Examine by liquid chromatography (2.2.29).
Test solution. Dissolve 50 mg of the substance to be examined in the mobile phase and dilute to
Reference solution (a). Dissolve 10 mg of betahistine mesilate CRS and 10 mg of 2-vinylpyridine R in the
mobile phase and dilute to 50.0 ml with the mobile phase. Dilute 2.0 ml of the solution to 50.0 ml
05-13
Reference solution (c). Dilute 2.0 ml of reference solution (b) to 10.0 ml with the mobile phase.
as mobile phase at a flow rate of 1 ml per minute a mixture prepared as follows: dissolve 2.0 g of
sodium dodecyl sulphate R in a mixture of 15 volumes of a 10 per cent V/V solution of sulphuric
acid R, 35 volumes of a 17 g/l solution of tetrabutylammonium dihydrogen sulphate R and 650
volumes of water R; adjust to pH 3.3 using dilute sodium hydroxide solution R and mix with 300
volumes of acetonitrile R,
Inject 20 l of reference solution (a). When using a recorder, adjust the sensitivity of the system so
that the height of the first peak in the chromatogram obtained with reference solution (a) is not less
than 70 per cent of the full scale of the recorder. The test is not valid unless: in the chromatogram
obtained with reference solution (a), the resolution between the peaks corresponding to 2-vinyl-
pyridine and betahistine mesilate is at least 3.5.
Inject 20 l of the test solution and of reference solutions (b) and (c). Continue the chromato-
graphy for three times the retention time of betahistine mesilate (which is about 8 min). In the
chromatogram obtained with the test solution the area of any peak, apart from the principal peak, is
(c) (0.2 per cent); the sum of the areas of any peaks, apart from the principal peak, is not greater than
half of the area of the principal peak in the chromatogram obtained with reference solution (b)
(0.5 per cent).
Disregard any peak with an area less than 0.025 times that of the principal peak in the chromato-
2-Propanol Not more than 0.5 per cent, determined by the test for residual solvents (2.4.24).
Chlorides (2.4.4). To 14 ml of solution S add 1 ml of water R. The solution complies with the limit
test for chlorides (35 ppm).
of water.
ASSAY
Dissolve 0.140 g in 50 ml of a mixture of 1 volume of glacial acetic acid R and 7 volumes of acetic
anhydride R. Titrate with 0.1M perchloric acid, determining the end-point potentiometrically (2.2.20).
10
H
20
N
2
O
6
S
2
.
STORAGE
IMPURITIES
N
CH
2
A. 2-vinylpyridine.
__________________________________________________________________________________________________________ Ph Eur
05-14
Betamethasone
H
O
HO
O
OH
F
OH
Me
Me
Me H
C
22
H
29
FO
5
392.5 378-44-9
Betamethasone complies with the requirements of the 3rd edition of the European Pharmacopoeia [0312].
Preparation
Betamethasone Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Betamethasone contains not less than 97.0 per cent and not more than the equivalent of 103.0 per
cent of 9-fluoro-11,17,21-trihydroxy-16-methylpregna-1,4-diene-3,20-dione, calculated with
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water, sparingly soluble in
ethanol, very slightly soluble in methylene chloride.
IDENTIFICATION
A. Dissolve 10.0 mg in ethanol R and dilute to 100.0 ml with the same solvent. Place 2.0 ml of the
solution in a stoppered tube, add 10.0 ml of phenylhydrazine-sulphuric acid solution R, mix and heat in
a water-bath at 60C for 20 min. Cool immediately. The absorbance (2.2.25) of the solution
measured at 419 nm is not greater than 0.10.
obtained with betamethasone CRS. If the spectra obtained in the solid state with the substance to be
examined and the reference substance show differences, dissolve the substance to be examined and
the reference substance separately in the smallest necessary quantity of methylene chloride R and
evaporate to dryness on a water-bath. Using the residues, record the spectra again.
Test solution. Dissolve 10 mg of the substance to be examined in a mixture of 1 volume of methanol R
and 9 volumes of methylene chloride R and dilute to 10 ml with the same mixture of solvents.
Reference solution (a). Dissolve 20 mg of betamethasone CRS in a mixture of 1 volume of methanol R
Reference solution (b). Dissolve 10 mg of dexamethasone CRS in reference solution (a) and dilute to
10 ml with the same solution.
volumes of butanol R saturated with water R, 10 volumes of toluene R and 85 volumes of ether R.
Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the
the chromatogram obtained with reference solution (a). Spray with alcoholic solution of sulphuric
acid R. Heat at 120C for 10 min or until the spots appear. Allow to cool. Examine the chromato-
grams in daylight and in ultraviolet light at 365 nm. The principal spot in the chromatogram
obtained with the test solution is similar in position, colour in daylight, fluorescence in ultraviolet
light at 365 nm and size to the principal spot in the chromatogram obtained with reference solution
(a). The test is not valid unless the chromatogram obtained with reference solution (b) shows two
spots which may however not be completely separated.
05-15
colourless. Filter. Add 1.0 ml of the filtrate to a freshly prepared mixture of 0.1 ml of alizarin S
solution R and 0.1 ml of zirconyl nitrate solution R. Mix, allow to stand for 5 min and compare the
E. Add about 2 mg to 2 ml of sulphuric acid R and shake to dissolve. Within 5 min, a deep reddish-
brown colour develops. Add the solution to 10 ml of water R and mix. The colour is discharged and a
clear solution remains.
TESTS
Specific optical rotation (2.2.7). Dissolve 0.125 g in methanol R and dilute to 25.0 ml with the
substance.
acetonitrile R and methanol R and dilute to 10.0 ml with the same solvent.
Reference solution (a). Dissolve 2 mg of betamethasone CRS and 2 mg of methylprednisolone CRS in
mobile phase A and dilute to 100.0 ml with the same mobile phase.
Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with mobile phase A.
a stainless steel column 0.25 m long and 4.6 mm internal diameter packed with octadecylsilyl
as mobile phase at a flow rate of 2.5 ml per minute, a linear-gradient programme using the
following conditions:
Mobile phase A. In a 1000 ml volumetric flask mix 250 ml of acetonitrile R with 700 ml of water R
and allow to equilibrate; adjust the volume to 1000 ml with water R and mix again,
Time
(min)
Mobile
phase A
Mobile
phase B
Comment
0 100 0 isocratic
15 100 0 begin linear gradient
40 0 100 end chromatogram, return to
100A
41 100 0 begin equilibration with A
46 = 0 100 0 end equilibration, begin next
chromatogram
Equilibrate the column with mobile phase B at a flow rate of 2.5 ml per minute for at least 30 min
and then with mobile phase A for 5 min. For subsequent chromatograms, use the conditions
described from 40 min to 46 min.
obtained with 20 l of reference solution (b) is not less than 50 per cent of the full scale of the
recorder.
Inject 20 l of reference solution (a). When the chromatograms are recorded in the conditions
described above, the retention times are: methylprednisolone, about 11.5 minutes and
betamethasone, about 12.5 minutes. The test is not valid unless the resolution between the peaks
corresponding to methylprednisolone and betamethasone is at least 1.5; if necessary, adjust the
concentration of acetonitrile in mobile phase A.
Inject separately 20 l of the mixture of equal volumes of acetonitrile R and methanol R as a blank,
20 l of the test solution and 20 l of reference solution (b). In the chromatogram obtained with the
test solution: the area of any peak, apart from the principal peak, is not greater than the area of the
principal peak in the chromatogram obtained with reference solution (b) (1.0 per cent) and not more
than one such peak has an area greater than half the area of the principal peak in the chromatogram
obtained with reference solution (b) (2.0 per cent). Disregard any peak due to the blank and any
peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained
05-16
at 100C to 105C.
ASSAY
Dissolve 0.100 g in alcohol R and dilute to 100.0 ml with the same solvent. Dilute 2.0 ml of the
solution to 100.0 ml with alcohol R. Measure the absorbance (2.2.25) at the maximum at 238.5 nm.
22
H
29
FO
5
STORAGE
IMPURITIES
A. dexamethasone,
H
O
HO
O
OH
F
Me
Me
Me H
Cl
B. 21-chloro-9-fluoro-11,17-dihydroxy-16-methylpregna-1,4-diene-3,20-dione,
H
O
O
OH
Me
Me
Me H
OH
C. 17,21-dihydroxy-16-methylpregna-1,4,9(11)-triene-3,20-dione,
H
O
HO
O
OH
F
Me
Me
Me H
O O CH
3
O
D. 9-fluoro-11,17-dihydroxy-21-ethoxycarbonyloxy-16-methylpregna-1,4-diene-
3,20-dione,
H
O
O
OH
Me
Me H
OH
O
Me
E. 17,21-dihydroxy-9,11-epoxy-16-methylpregna-1,4-diene-3,20-dione,
H
O
O
OH
H
Me
Me
Me H
OH
F. 17,21-dihydroxy-16-methylpregna-1,4,11-triene-3,20-dione,
H
O
O
OH
H
Me
Me
Me H
OH
HO
G. 11,17,21-trihydroxy-16-methylpregna-1,4-diene-3,20-dione,
05-17
O
HO
O
OH
H
Me
Me
Me
OH
F
H
H. 14-fluoro-11,17,21-trihydroxy-16-methyl-8,9-pregna-1,4-diene-3,20-
dione,
O
HO
O
OH
H
Me
Me
Me
OH
H
F
I. 8-fluoro-11,17,21-trihydroxy-16-methyl-9-pregna-1,4-diene-3,20-dione,
H
O
O
OH
H
Me
Me
Me H
OH
J. 17,21-dihydroxy-16-methylpregna-1,4-diene-3,20-dione
__________________________________________________________________________________________________________ Ph Eur
05-18
Betamethasone Acetate
H
O
HO
O
OH
F
OAc
Me
Me
Me H
C
24
H
31
FO
6
434.5 987-24-6
Betamethasone Acetate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Betamethasone acetate contains not less than 97.0 per cent and not more than the equivalent of
103.0 per cent of 9-fluoro-11,17,21-trihydroxy-16-methylpregna-1,4-diene-3,20-dione 21-acetate,
CHARACTERS
soluble in alcohol and in methylene chloride.
IDENTIFICATION
A. Dissolve 10.0 mg in ethanol R and dilute to 100.0 ml with the same solvent. Place 2.0 ml of this
solution in a ground-glass-stoppered tube, add 10.0 ml of phenylhydrazine-sulphuric acid solution R,
mix and heat in a water-bath at 60C for 20 min. Cool immediately. The absorbance (2.2.25) of the
solution measured at 419 nm is not more than 0.10.
obtained with betamethasone acetate CRS. If the spectra obtained in the solid state show differences,
dissolve the substance to be examined and the reference substance separately in the minimum
volume of methanol R, evaporate to dryness on a water-bath and record new spectra using the
residues.
Reference solution (a). Dissolve 20 mg of betamethasone acetate CRS in a mixture of 1 volume of
methanol R and 9 volumes of methylene chloride R and dilute to 20 ml with the same mixture of
solvents.
Reference solution (b). Dissolve 10 mg of prednisolone acetate CRS in reference solution (a) and dilute
to 10 ml with the same solution.
Apply separately to the plate 5 l of each solution. Prepare the mobile phase by adding a mixture of
1.2 volumes of water R and 8 volumes of methanol R to a mixture of 15 volumes of ether R and 77
volumes of methylene chloride R. Develop over a path of 15 cm. Allow the plate to dry in air and
reference solution (a). Spray with alcoholic solution of sulphuric acid R. Heat at 120C for 10 min or
until the spots appear. Allow to cool. Examine the plate in daylight and in ultraviolet light at 365 nm.
The principal spot in the chromatogram obtained with the test solution is similar in position, colour
in daylight, fluorescence in ultraviolet light at 365 nm and size to the principal spot in the chromato-
with reference solution (b) shows two clearly separated spots.
05-19
D. Add about 2 mg to 2 ml of sulphuric acid R and shake to dissolve. Within 5 min, a deep reddish-
E. Mix about 5 mg with 45 mg of heavy magnesium oxide R and ignite in a crucible until an almost
F. About 10 mg gives the reaction of acetyl (2.3.1).
TESTS
Specific optical rotation (2.2.7). Dissolve 0.250 g in dioxan R and dilute to 25.0 ml with the same
substance.
Test solution. Dissolve 25.0 mg of the substance to be examined in 4 ml of acetonitrile R and dilute to
Reference solution (a). Dissolve 2 mg of betamethasone acetate CRS and 2 mg of dexamethasone acetate
CRS in the mobile phase and dilute to 100.0 ml with the mobile phase.
as mobile phase at a flow rate of 1 ml per minute a mixture prepared as follows: in a 1000 ml
volumetric flask mix 380 ml of acetonitrile R with 550 ml of water R and allow to equilibrate;
dilute to 1000 ml with water R and mix again,
conditions, the retention times are: betamethasone acetate, about 19 min and dexamethasone
acetate, about 22 min. The test is not valid unless the resolution between the peaks due to
betamethasone acetate and dexamethasone acetate is at least 3.3; if necessary, adjust slightly the
concentration of acetonitrile in the mobile phase.
chromatography for 2.5 times the retention time of the principal peak in the chromatogram obtained
with the test solution. In the chromatogram obtained with the test solution: the area of any peak,
apart from the principal peak, is not greater than half the area of the principal peak in the chromato-
gram obtained with reference solution (b) (0.5 per cent); the sum of the areas of all the peaks, apart
from the principal peak, is not greater than the 1.25 times the area of the principal peak in the
chromatogram obtained with reference solution (b) (1.25 per cent). Disregard any peak with an area
less than 0.05 times the area of the principal peak obtained in the chromatogram with reference
solution (b).
ASSAY
solution to 100.0 ml with alcohol R. Measure the absorbance (2.2.25) at the maximum at 240 nm.
24
H
31
FO
6
STORAGE
IMPURITIES
H
O
HO
O
OH
F
Me
Me
Me H
OH
A. betamethasone,
05-20
H
O
HO
O
OH
F
OAc
Me
Me
Me H
B. dexamethasone acetate,
H
O
AcO
O
OH
F
OAc
Me
Me
Me H
C. betamethasone 11,21-diacetate,
H
O
H
O
OH
OAc
Me
Me
Me
H
O
D. 9,11-epoxy-17,21-dihydroxy-16-methyl-9-pregna-1,4-diene-3,20-dione 21-
acetate.
__________________________________________________________________________________________________________ Ph Eur
05-21
Betamethasone Dipropionate
H
O
HO
O
OCOEt
OCOEt
Me
Me
H
F
H
H
Me
C
28
H
37
FO
7
504.6 5593-20-4
Betamethasone Dipropionate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Betamethasone dipropionate contains not less than 97.0 per cent and not more than the equivalent
of 103.0 per cent of 9-fluoro-11,17,21-trihydroxy-16-methylpregna-1,4-diene-3,20-dione
17,21-dipropionate, calculated with reference to the dried substance.
CHARACTERS
IDENTIFICATION
Second identification: A, D, E, F.
obtained with betamethasone dipropionate CRS.
Reference solution (a). Dissolve 10 mg of betamethasone dipropionate CRS in a mixture of 1 volume of
solvents.
Reference solution (b). Dissolve 10 mg of deoxycortone acetate CRS in a mixture of 1 volume of
solvents. Dilute 5 ml of this solution to 10 ml with reference solution (a).
reference solution (a). Spray the plate with alcoholic solution of sulphuric acid R. Heat at 120C for
10 min or until the spots appear. Allow to cool. Examine in daylight and in ultraviolet light at
365 nm. The principal spot in the chromatogram obtained with the test solution is similar in
position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to the principal spot
in the chromatogram obtained with reference solution (a). The test is not valid unless the chromato-
D. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable silica gel
05-22
and dilute to 5 ml with the same solvent. This solution is also used to prepare test solution (b).
Dilute 2 ml of the solution to 10 ml with methylene chloride R.
15 ml glass tube with a ground-glass stopper or a polytetrafluoroethylene cap. Add 10 ml of saturated
methanolic potassium hydrogen carbonate solution R and immediately pass a current of nitrogen R briskly
through the solution for 5 min. Stopper the tube. Heat in a water-bath at 45C, protected from light,
for 2 h. Allow to cool.
Reference solution (a). Dissolve 25 mg of betamethasone dipropionate CRS in methanol R with gentle
heating and dilute to 5 ml with the same solvent. This solution is also used to prepare reference
solution (b). Dilute 2 ml of the solution to 10 ml with methylene chloride R.
Reference solution (b). Transfer 2 ml of the solution obtained during preparation of reference solution
(a) to a 15 ml glass tube with a ground-glass stopper or a polytetrafluoroethylene cap. Add 10 ml of
saturated methanolic potassium hydrogen carbonate solution R and immediately pass a current of
examine in ultraviolet light at 254 nm. The principal spot in each of the chromatograms obtained
with the test solutions is similar in position and size to the principal spot in the chromatogram
obtained with the corresponding reference solution. Spray the plate with alcoholic solution of sulphuric
acid R. Heat at 120C for 10 min or until the spots appear. Allow to cool. Examine in daylight and in
ultraviolet light at 365 nm. The principal spot in each of the chromatograms obtained with the test
solutions is similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size
to the principal spot in the chromatogram obtained with the corresponding reference solution. The
principal spot in each of the chromatograms obtained with test solution (b) and reference solution (b)
has an R
f
value distinctly lower than that of the principal spots in each of the chromatograms
obtained with test solution (a) and reference solution (a).
E. Add about 2 mg to 2 ml of sulphuric acid R and shake to dissolve. Within 5 min, a deep reddish-
F. Mix about 5 mg with 45 mg of heavy magnesium oxide R and ignite in a crucible until an almost
TESTS
substance.
Reference solution (a). Dissolve 2.5 mg of betamethasone dipropionate CRS and 2.5 mg of beclometasone
dipropionate CRS in the mobile phase and dilute to 50.0 ml with the same solvent.
as mobile phase at a flow rate of 1 ml per minute a mixture prepared as follows: mix carefully
350 ml of water R with 600 ml of acetonitrile R and allow to equilibrate; adjust the volume to
1000 ml with water R and mix again,
reference solution (b) is 70 per cent to 90 per cent of the full scale of the recorder.
conditions, the retention times are: betamethasone dipropionate, about 9 min; beclometasone
dipropionate, about 10.7 min. The test is not valid unless the resolution between the peaks corres-
ponding to betamethasone dipropionate and beclometasone dipropionate is at least 2.5; if necessary,
05-23
adjust the concentration of acetonitrile in the mobile phase.
chromatography for 2.5 times the retention time of the principal peak. In the chromatogram obtained
with the test solution: the area of any peak apart from the principal peak is not greater than 0.75
times the area of the principal peak in the chromatogram obtained with reference solution (b)
(1.5 per cent) and not more than one such peak has an area greater than half the area of the principal
peak in the chromatogram obtained with reference solution (b) (1 per cent); the sum of the areas of
all the peaks, apart from the principal peak, is not greater than 1.25 times the area of the principal
peak in the chromatogram obtained with reference solution (b) (2.5 per cent). Disregard any peak
with an area less than 0.025 times the area of the principal peak in the chromatogram obtained with
100C to 105C.
ASSAY
Dissolve 50.0 mg in alcohol R and dilute to 100.0 ml with the same solvent. Dilute 2.0 ml of the
28
H
37
FO
7
STORAGE
__________________________________________________________________________________________________________ Ph Eur
05-24
Betamethasone Sodium Phosphate
corrected 1/01
H
O
HO
O
OH
F
ONa
ONa
O P O
H Me
Me H
H
Me
C
22
H
28
FNa
2
O
8
P 516.4 151-73-5
Betamethasone Sodium Phosphate complies with the requirements of the 3rd edition of the European
Preparations
Betamethasone Eye Drops
Betamethasone Injection
Betamethasone Sodium Phosphate Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Betamethasone sodium phosphate contains not less than 96.0 per cent and not more than the
equivalent of 103.0 per cent of 9-fluoro-11,17-dihydroxy-16-methyl-3,20-dioxopregna-1,4-diene-
21-yl disodium phosphate, calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white powder, very hygroscopic, freely soluble in water, slightly soluble in alcohol,
practically insoluble in ether and in methylene chloride.
IDENTIFICATION
A. Dissolve 10.0 mg in 5 ml of water R and dilute to 100.0 ml with ethanol R. Place 2.0 ml of this
solution measured at the maximum at 450 nm is not more than 0.10.
obtained with betamethasone sodium phosphate CRS. If the spectra obtained in the solid state show
differences, dissolve the substance to be examined and the reference substance separately in the
minimum volume of alcohol R, evaporate to dryness on a water-bath and record new spectra using the
residues.
the same solvent.
Reference solution (a). Dissolve 10 mg of betamethasone sodium phosphate CRS in methanol R and dilute
Reference solution (b). Dissolve 10 mg of prednisolone sodium phosphate CRS in methanol R and dilute to
10 ml with the same solvent. Dilute 5 ml of this solution to 10 ml with reference solution (a).
20 volumes of glacial acetic acid R, 20 volumes of water R and 60 volumes of butanol R. Allow the
plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram
gram obtained with reference solution (a). Spray the plate with alcoholic solution of sulphuric acid R.
Heat at 120C for 10 min or until the spots appear. Allow to cool. Examine in daylight and in
similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to the
05-25
principal spot in the chromatogram obtained with reference solution (a). The test is not valid
unless the chromatogram obtained with reference solution (b) shows two spots which may
however not be completely separated.
D. Add about 2 mg to 2 ml of sulphuric acid R and shake to dissolve. Within 5 min, an intense
reddish-brown colour develops. Add the solution to 10 ml of water R and mix. The colour is
discharged and a clear solution remains.
F. To about 40 mg add 2 ml of sulphuric acid R and heat gently until white fumes are evolved. Add
nitric acid R dropwise, continue the heating until the solution is almost colourless and cool. Add 2 ml
of water R, heat until white fumes are again evolved, cool, add 10 ml of water R and neutralise to red
litmus paper R with dilute ammonia R1. The solution gives reaction (a) of sodium (2.3.1) and reaction
(b) of phosphates (2.3.1).
TESTS
solution B
7
(Method II, 2.2.2).
pH (2.2.3). Dilute 1 ml of solution S to 5 ml with carbon dioxide-free water R. The pH of the solution
is 7.5 to 9.0.
substance.
Reference solution (a). Dissolve 25 mg of betamethasone sodium phosphate CRS and 25 mg of
dexamethasone sodium phosphate CRS in the mobile phase and dilute to 25.0 ml with the mobile phase.
Dilute 1.0 ml of this solution to 25.0 ml with the mobile phase.
as mobile phase at a flow rate of 1 ml/min a mixture prepared as follows: in a 250 ml conical
flask, weigh 1.360 g of potassium dihydrogen phosphate R and 0.600 g of hexylamine R, mix and
allow to stand for 10 min and then dissolve in 185 ml of water R; add 65 ml of acetonitrile R, mix
and filter (0.45 m),
Equilibrate the column with the mobile phase at a flow rate of 1 ml/min for about 45 min.
obtained with reference solution (b) is 70 per cent to 90 per cent of the full scale of the recorder.
described above, the retention times are: betamethasone sodium phosphate about 14 min;
dexamethasone sodium phosphate about 15.5 min. The test is not valid unless the resolution
between the peaks corresponding to betamethasone sodium phosphate and dexamethasone sodium
phosphate is at least 2.0; if necessary, increase the concentration of acetonitrile or increase the
concentration of water in the mobile phase.
with the test solution: the area of any peak, apart from the principal peak, is not greater than the area
of the principal peak in the chromatogram obtained with reference solution (b) (2 per cent) and not
more than one such peak has an area greater than half the area of the principal peak in the chromato-
gram obtained with reference solution (b) (1 per cent); the sum of the areas of all the peaks, apart
from the principal peak, is not greater than 1.5 times the area of the principal peak in the chromato-
gram obtained with reference solution (b) (3 per cent). Disregard any peak with an area less than
Inorganic phosphate Dissolve 50 mg in water R and dilute to 100 ml with the same solvent. To
05-26
10 ml of this solution add 5 ml of molybdovanadic reagent R, mix and allow to stand for 5 min. Any
yellow colour in the solution is not more intense than that in a standard prepared at the same time
and in the same manner using 10 ml of phosphate standard solution (5 ppm PO
4
) R (1 per cent).
mination of water.
ASSAY
Dissolve 0.100 g in water R and dilute to 100.0 ml with the same solvent. Dilute 5.0 ml of the
solution to 250.0 ml with water R. Measure the absorbance (2.2.25) at the maximum at 241 nm.
22
H
28
FNa
2
O
8
P taking the specific absorbance to be 297.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
05-27
Betamethasone Valerate
H
H
O
HO
O
F
OH
Me
Me
Me
H
H
OCO[CH
2
]
3
CH
3
C
27
H
37
FO
6
476.6 2152-44-5
Betamethasone Valerate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparations
Betamethasone Valerate Scalp Application
Betamethasone Valerate Cream
Betamethasone and Clioquinol Cream
Betamethasone Valerate Lotion
Betamethasone Valerate Ointment
Betamethasone and Clioquinol Ointment
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Betamethasone valerate contains not less than 97.0 per cent and not more than the equivalent of
103.0 per cent of 9-fluoro-11,17,21-trihydroxy-16-methylpregna-1,4-diene-3,20-dione 17-
valerate, calculated with reference to the dried substance.
CHARACTERS
and in methylene chloride, soluble in alcohol.
IDENTIFICATION
First identification: C, D.
Second identification: A, B, E, F, G.
B. Dissolve 10.0 mg in ethanol R and dilute to 100.0 ml with the same solvent. Place 2.0 ml of this
obtained with betamethasone 17-valerate CRS. If the spectra obtained in the solid state show
minimum volume of chloroform R, evaporate to dryness on a water-bath and record new spectra using
the residues.
Reference solution (a). Dissolve 10 mg of betamethasone 17-valerate CRS in a mixture of 1 volume of
solvents.
Reference solution (b). Dissolve 10 mg of betamethasone 21-valerate CRS in a mixture of 1 volume of
solvents. Dilute 5 ml of this solution to 10 ml with reference solution (a).
05-28
reference solution (a). Spray the plate with alcoholic solution of sulphuric acid R. Heat at 120C for
10 min or until the spots appear. Allow to cool. Examine in daylight and in ultraviolet light at
E. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable silica gel
methanolic potassium hydrogen carbonate solution R and immediately pass a current of nitrogen R briskly
for 3 h. Allow to cool.
Reference solution (a). Dissolve 25 mg of betamethasone 17-valerate CRS in methanol R with gentle
heating and dilute to 5 ml with the same solvent. This solution is also used to prepare reference
solution (b). Dilute 2 ml of the solution to 10 ml with methylene chloride R.
(a) to a 15 ml glass tube with a ground glass-stopper or a polytetrafluoroethylene cap. Add 10 ml of
saturated methanolic potassium hydrogen carbonate solution R and immediately pass a current of
examine under ultraviolet light at 254 nm. The principal spot in each of the chromatograms obtained
obtained with the corresponding reference solution. Spray with alcoholic solution of sulphuric acid R.
Heat at 120C for 10 min or until the spots appear. Allow to cool. Examine in daylight and in ultra-
violet light at 365 nm. The principal spot in each of the chromatograms obtained with the test solu-
tions is similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to
the principal spot in the chromatograms obtained with the corresponding reference solution. The
principal spot in each of the chromatograms obtained with test solution (b) and reference solution (b)
has an R
f
value distinctly lower than that of the principal spots in each of the chromatograms
F. Add about 2 mg to 2 ml of sulphuric acid R and shake to dissolve. Within 5 min, a deep reddish-
G. Mix about 5 mg with 45 mg of heavy magnesium oxide R and ignite in a crucible until an almost
TESTS
substance.
Reference solution (a). Dissolve 2 mg of betamethasone 17-valerate CRS and 2 mg of betamethasone
21-valerate CRS in the mobile phase and dilute to 50.0 ml with the mobile phase.
05-29
as mobile phase at a flow rate of 1 ml per minute a mixture prepared as follows: mix carefully
350 ml of water R with 600 ml of acetonitrile R and allow to equilibrate; adjust the volume to
conditions, the retention times are: betamethasone 17-valerate, about 7 min; betamethasone
21-valerate, about 9 min. The test is not valid unless the resolution between the peaks corresponding
to betamethasone 17-valerate and betamethasone 21-valerate is at least 5.0; if necessary, adjust the
with the test solution: the area of any peak apart from the principal peak is not greater than 0.75
times the area of the principal peak in the chromatogram obtained with reference solution (b)
(1.5 per cent) and not more than one such peak has an area greater than half the area of the principal
peak in the chromatogram obtained with reference solution (b) (1.0 per cent); the sum of the areas of
100C to 105C.
ASSAY
27
H
37
FO
6
STORAGE
__________________________________________________________________________________________________________ Ph Eur
05-30
Betanidine Sulphate
N NHMe
NMe
2
,H
2
SO
4
H
C
20
H
32
N
6
O
4
S 452.6 114-85-2
Betanidine Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0374].
Action and use Antihypertensive.
When bethanidine sulphate is prescribed or demanded, Betanidine Sulphate shall be dispensed or
supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Betanidine sulphate contains not less than 98.0 per cent and not more than the equivalent of
101.0 per cent of di(1-benzyl-2,3-dimethylguanidine) sulphate, calculated with reference to the dried
substance.
CHARACTERS
A white powder, freely soluble in water, sparingly soluble in alcohol, practically insoluble in ether.
IDENTIFICATION
A. Dissolve 50 mg in 100 ml of water R. Examined between 230 nm and 350 nm (2.2.25), the solu-
tion shows three absorption maxima, at 251 nm, 257 nm and 263 nm, the maximum at 257 nm
being more intense than the other two.
obtained with betanidine sulphate CRS. Examine the substances prepared as mulls in liquid paraffin R
and compare over the wave-number range 2000 cm
1
to 600 cm
1
.
C. Dissolve 0.1 g in 10 ml of water R and add 20 ml of picric acid solution R. The precipitate, washed
with water R and dried at 80C for 30 min, melts (2.2.14) at 147C to 152C.
D. Dissolve about 25 mg in 5 ml of water R. Add 1 ml of strong sodium hydroxide solution R, 1 ml of -
naphthol solution R and, dropwise with shaking, 1 ml of strong sodium hypochlorite solution R. A bright-
pink precipitate is formed and becomes violet-red on standing.
E. Dissolve 50 mg in 5 ml of water R. The solution gives reaction (a) of sulphates (2.3.1).
TESTS
solvent. Add 0.1 ml of phenolphthalein solution R; the solution is colourless. Add 0.2 ml of 0.01M
sodium hydroxide; the solution becomes red. Add 0.4 ml of 0.01M hydrochloric acid and 0.25 ml of
methyl red solution R; the solution becomes red or orange.
Trimethylguanidine Examine by thin-layer chromatography (2.2.27), using silica gel G R as the
coating substance.
Test solution. Dissolve 40 mg of the substance to be examined in 1.0 ml of methanol R.
Reference solution. Dissolve 40 mg of betanidine sulphate CRS in 1.0 ml of a solution of 3 mg of
trimethylguanidine sulphate CRS in 10 ml of methanol R.
10 volumes of alcohol R, 16 volumes of water R, 24 volumes of glacial acetic acid R and 50 volumes of
ethyl acetate R. Allow the plate to dry in air and spray with potassium iodobismuthate solution R. Any
spot corresponding to trimethylguanidine in the chromatogram obtained with the test solution is not
more intense than the smaller spot in the chromatogram obtained with the reference solution
(0.75 per cent).
05-31
100C to 105C.
ASSAY
20
H
32
N
6
O
4
S.
__________________________________________________________________________________________________________ Ph Eur
05-32
Betaxolol Hydrochloride
O
O NHPr
i
H OH
and enantiomer
,HCl
C
18
H
30
ClNO
3
343.9 63659-19-8
Betaxolol Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparations
Betaxolol Eye Drops, Solution
Betaxolol Eye Drops, Suspension
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Betaxolol hydrochloride contains not less than 98.5 per cent and not more than the equivalent of
101.5 per cent of (RS)-1-[4-[2-(cyclopropylmethoxy)ethyl]phenoxy]-3-[(1-methylethyl)amino]-
propan-2-ol hydrochloride, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, very soluble in water, freely soluble in alcohol, soluble in
IDENTIFICATION
obtained with betaxolol hydrochloride CRS.
C. Examine by thin-layer chromatography (2.2.27), using as the coating substance octadecylsilyl silica
gel for chromatography R with a fluorescent indicator having an optimal intensity at 254 nm.
Reference solution (a). Dissolve 20 mg of betaxolol hydrochloride CRS in 2 ml of methanol R.
Reference solution (b). Dissolve 10 mg of oxprenolol hydrochloride CRS in 1 ml of reference solution (a).
0.5 volumes of perchloric acid R, 50 volumes of methanol R and 50 volumes of water R. Allow the
gram obtained with reference solution (a). Spray the plate with a 50 g/l solution of vanillin R in a
mixture of 5 volumes of sulphuric acid R, 10 volumes of glacial acetic acid R and 85 volumes of
methanol R. Heat the plate at 100C to 105C until the colour of the spots reaches maximum
intensity (10 min to 15 min). Examine in daylight. The principal spot in the chromatogram obtained
TESTS
solvent. Add 0.2 ml of methyl red solution R and 0.2 ml of 0.01M hydrochloric acid. The solution is red.
Add 0.4 ml of 0.01M sodium hydroxide. The solution is yellow.
05-33
Reference solution (a). Dissolve 8 mg of the substance to be examined and 4 mg of betaxolol impurity A
CRS in 20.0 ml of the mobile phase.
as the mobile phase at a flow rate of 1.5 ml per minute a mixture prepared as follows: mix
175 ml of acetonitrile R with 175 ml of methanol R and dilute the mixture to 1 litre with a 3.4 g/l
solution of potassium dihydrogen phosphate R, previously adjusted to pH 3.0 with phosphoric
acid R,
as the detector a spectrophotometer set at 273 nm,
a loop injector.
Inject 20 l of each solution. Continue the chromatography for at least four times the retention
time of the principal peak in the chromatogram obtained with the test solution. In the chromatogram
obtained with the test solution: the area of any peak apart from the principal peak is not greater than
0.3 times the area of the peak in the chromatogram obtained with reference solution (b) (0.3 per
cent) and the sum of the areas of such peaks is not greater than the area of the peak in the chromato-
gram obtained with reference solution (b) (1 per cent). The test is not valid unless the resolution
between the peaks due to betaxolol impurity A and betaxolol in the chromatogram obtained with
reference solution (a) is at least 2.0. Disregard any peak with an area less than 0.025 times that of the
peak in the chromatogram obtained with reference solution (b).
test A for heavy metals (10 ppm). Prepare the standard using 10 ml of lead standard solution (1 ppm
Pb) R.
100C to 105C.
ASSAY
Dissolve 0.300 g in a mixture of 10.0 ml of 0.01M hydrochloric acid and 50 ml of alcohol R. Carry out
a potentiometric titration (2.2.20), using 0.1M sodium hydroxide. Read the volume added between the
18
H
30
ClNO
3
.
STORAGE
IMPURITIES
A. (RS)-3-(4-ethylphenoxy)-1-[(1-methylethyl)amino]propan-2-ol,
B. (RS)-3-[4-(2-hydroxyethyl)phenoxy]-1-[(1-methylethyl)amino]propan-2-ol,
C. (RS)-[4-(2-cyclopropylmethoxyethyl)-phenoxy]-2,3-epoxypropane,
D. 4-[2-(cyclopropylmethoxy)ethyl]phenol,
E. (RS)-3-[4-(2-butyloxyethyl)phenoxy)]-1-[(1-methylethyl)amino]propan-2-ol.
__________________________________________________________________________________________________________ Ph Eur
05-34
Bezafibrate
corrected 1/01
N
H
O COOH
Cl
O
Me Me
C
19
H
20
ClNO
4
361.8 41859-67-0
Bezafibrate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1394]. These
Action and use Hypolipidaemic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Bezafibrate contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent
of 2-[4-[2-[(4-chlorobenzoyl)amino]ethyl]phenoxy]-2-methylpropanoic acid, calculated with
CHARACTERS
A white or almost white crystalline powder, practically insoluble in water, freely soluble in
dimethylformamide, sparingly soluble in acetone and in alcohol. It dissolves in dilute solutions of
alkali hydroxides.
IDENTIFICATION
obtained with bezafibrate CRS. Examine the substances prepared as discs. If the spectra obtained
show differences, dissolve the substance to be examined and the reference substance separately in
methanol R and evaporate to dryness. Dry the residues in vacuo at 80C for 1 h and record new
254
plate R.
the same solvent.
Reference solution. Dissolve 10 mg of bezafibrate CRS in methanol R and dilute to 5 ml with the same
solvent.
2.7 volumes of glacial acetic acid R, 30 volumes of methyl ethyl ketone R and 60 volumes of xylene R.
Dry the plate at 120C for at least 15 min and examine in ultraviolet light at 254 nm. The principal
spot in the chromatogram obtained with the test solution is similar in position and size to the
principal spot in the chromatogram obtained with the reference solution.
TESTS
Solution S Dissolve 1.0 g in dimethylformamide R and dilute to 20 ml with the same solvent.
solution BY
5
(Method II, 2.2.2).
Reference solution (c). To 1 ml of the test solution, add 1 ml of 0.1M hydrochloric acid and evaporate to
dryness on a hot plate. Dissolve the residue in 20 ml of the mobile phase.
05-35
potassium dihydrogen phosphate R adjusted to pH 2.3 with phosphoric acid R and 60 volumes of
methanol R,
Inject separately 20 l of the test solution and 20 l of reference solutions (a), (b) and (c). When
the chromatogram is recorded in the prescribed conditions, the retention times are: impurity A about
3 min, impurity B about 3.5 min, bezafibrate about 6.0 min, impurity C about 9 min, impurity D
about 14 min and impurity E about 37 min. Continue the chromatography for the time necessary to
detect the ester, which, depending on the route of synthesis, may be impurity C, D or E. The test is
not valid unless: in the chromatogram obtained with reference solution (c) the resolution between the
two principal peaks is at least 5.0 and the principal peak in the chromatogram obtained with
reference solution (b) has a signal-to-noise ratio of at least 5. In the chromatogram obtained with the
test solution: the area of any peak, apart from the principal peak, is not greater than the area of the
principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent); the sum of
any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained
with reference solution (a).
Chlorides (2.4.4). Dilute 10 ml of solution S to 50 ml with water R. Filter the resultant suspension
through a wet filter previously washed with water R until free from chlorides. 15 ml of the filtrate
complies with the limit test for chlorides (300 ppm). Prepare the standard using 9 ml of chloride
standard solution (5 ppm Cl) R and 6 ml of water R.
100C to 105C.
ASSAY
Dissolve 0.300 g in 50 ml of a mixture of 25 volumes of water R and 75 volumes of alcohol R. Using
0.1 ml of phenolphthalein solution R as indicator, titrate with 0.1M sodium hydroxide until a pink colour
is obtained. Carry out a blank titration.
19
H
20
ClNO
4
.
IMPURITIES
N
H
OH
O
Cl
A. 4-chloro-N-[2-(4-hydroxyphenyl)ethyl]benzamide (chlorobenzoyltyramine),
l
COOH
B. 4-chlorobenzoic acid,
N
H
O
O
Cl
COOR
Me Me
C. R = CH
3
:

methyl 2-[4-[2-[(4-chlorobenzoyl)amino]ethyl]phenoxy]-2 methylpropanoate,
D. R = CH
2
-CH
3
:

ethyl 2-[4-[2-[(4-chlorobenzoyl)amino]ethyl]phenoxy]-2-methylpropanoate,
E. R = CH
2
-CH
2
-CH
2
-CH
3
: butyl 2-[4-[2-[(4-chlorobenzoyl)amino]ethyl]phenoxy]-2-
methylpropanoate.
__________________________________________________________________________________________________________ Ph Eur
05-36
Bifonazole
N
N
H
and enantiomer
C
22
H
18
N
2
310.4 60628-96-8
Bifonazole complies with the requirements of the 3rd edition of the European Pharmacopoeia [1395]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Bifonazole contains not less than 98.0 per cent and not more than the equivalent of 100.5 per cent of
1-[(RS)-(biphenyl-4-yl)phenylmethyl]-1H-imidazole, calculated with reference to the dried
substance.
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water, soluble in ethanol.
IDENTIFICATION
with bifonazole CRS. If the spectra obtained in the solid state show differences, dissolve the substance
to be examined and the reference substance separately in the minimum volume of 2-propanol R,
evaporate to dryness and record new spectra using the residues.
TESTS
Optical rotation (2.2.7). Dissolve 0.20 g of the substance to be examined in 20.0 ml of methanol R.
The angle of optical rotation is 0.10 to +0.10.
Buffer solution pH 3.2. Mix 2.0 ml of phosphoric acid R with water R and dilute to 1000.0 ml with the
same solvent. Adjust to pH 3.2 (2.2.3) with triethylamine R.
50.0 ml with buffer solution pH 3.2.
Reference solution (a). Dilute 0.25 ml of the test solution to 50.0 ml with buffer solution pH 3.2.
Reference solution (b). Dissolve 25.0 mg of imidazole R (impurity C) in acetonitrile R and dilute to
25.0 ml with the same solvent. Dilute 0.25 ml of the solution to 100.0 ml with buffer solution pH
3.2.
Reference solution (c). Dissolve 34.2 mg of 4-[(RS)(biphenyl-4-yl)phenylmethyl]-4H-imidazole
trifluoroacetate CRS (corresponding to 25.0 mg of impurity B base) in acetonitrile R and dilute to
Reference solution (d). Dilute 0.25 ml of reference solution (c) to 50.0 ml with buffer solution pH 3.2.
Reference solution (e). Mix 0.25 ml of the test solution and 0.25 ml of reference solution (c) and dilute
to 50.0 ml with buffer solution pH 3.2.
as mobile phase at a flow rate of 1 ml/min a gradient programme using the following conditions:
Mobile phase A. A mixture of 20 volumes of acetonitrile R and 80 volumes of buffer solution pH
3.2,
05-37
Mobile phase B. A mixture of 20 volumes of buffer solution pH 3.2 and 80 volumes of aceto-
nitrile R,
Time
(min)
Mobile
phase A
(per cent V/V)
Mobile
phase B
(per cent V/V)
Comment
0 8 60 40 isocratic elution
30 32 10 60 90 40 switch to initial
eluent
composition
32 40 60 40 equilibration
40 = 0 60 40 restart isocratic
elution
maintaining the column temperature at 40C.
Adjust the sensitivity of the system so that the height of the bifonazole peak in the chromatogram
obtained with 50 l of reference solution (e) is at least 50 per cent of the full scale of the recorder.
Inject 50 l of reference solution (e). When the chromatogram is recorded in the prescribed
conditions the retention times are: impurity B about 4 min and bifonazole about 4.5 min. The test is
not valid unless the resolution between the peaks corresponding to impurity B and bifonazole is at
least 2.5.
Inject separately 50 l of the test solution and 50 l each of reference solutions (a), (b) and (d). In
the chromatogram obtained with the test solution: the area of any peak corresponding to impurity C
is not greater than the corresponding peak in the chromatogram obtained with reference solution (b)
(0.25 per cent); the area of any peak corresponding to impurity B is not greater than three times the
area of the corresponding peak in the chromatogram obtained with reference solution (d) (1.5 per
cent); none of the peaks apart from the principal peak and the peaks corresponding to impurities B
and C, has an area greater than the area of the peak in the chromatogram obtained with reference
solution (a) (0.5 per cent); the sum of the areas of all the peaks apart from the principal peak, is not
greater than four times the area of the principal peak in the chromatogram obtained with reference
solution (a) (2 per cent). Disregard any peak whose area is less than 0.1 times the area of the
100C to 105C.
ASSAY
22
H
18
N
22
.
IMPURITIES
Qualified impurities
H OH
and enantiomer
A. (RS)-(biphenyl-4-yl)phenylmethanol,
H
and enantiomer
N
H
N
B. 4-[(RS)-(biphenyl-4-yl)phenylmethyl]-1H-imidazole,
N
H
N
C. 1H-imidazole,
05-38
N
+
N
Cl
_
D. 1,3-bis[(biphenyl-4-yl)phenylmethyl]-1H-imidazolium chloride.
__________________________________________________________________________________________________________ Ph Eur
05-39
Biotin
corrected 1/01
S
N
H
H
N
H
H
H
COOH
C
10
H
16
N
2
O
3
S 244.3 58-85-5
Biotin complies with the requirements of the 3rd edition of the European Pharmacopoeia [1073]. These
Action and use Vitamin.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Biotin contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of 5-
[(3aS,4S,6aR)-2-oxohexahydrothieno[3,4-d]imidazol-4-yl]pentanoic acid, calculated with reference
to the dried substance.
CHARACTERS
A white, crystalline powder or colourless crystals, very slightly soluble in water and in alcohol,
practically insoluble in acetone. It dissolves in dilute solutions of alkali hydroxides.
IDENTIFICATION
obtained with biotin CRS.
B. Examine the chromatograms obtained in the test for related substances (see Tests). The principal
C. Dissolve about 10 mg in 20 ml of water R with heating. Allow to cool. Add 0.1 ml of bromine
water R. The bromine water is decolorised.
TESTS
Solution S Dissolve 0.250 g in a 4 g/l solution of sodium hydroxide R and dilute to 25.0 ml with the
same alkaline solution.
Specific optical rotation (2.2.7). The specific optical rotation is +89 to +93, determined on
solution S and calculated with reference to the dried substance.
a suitable silica gel (5 m).
Prepare the solutions immediately before use and keep protected from bright light.
Test solution (a). Dissolve 50 mg of the substance to be examined in glacial acetic acid R and dilute to
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with glacial acetic acid R.
Reference solution (a). Dissolve 5 mg of biotin CRS in glacial acetic acid R and dilute to 10 ml with the
same solvent.
Reference solution (b). Dilute 1 ml of test solution (b) to 20 ml with glacial acetic acid R.
Reference solution (c). Dilute 1 ml of test solution (b) to 40 ml with glacial acetic acid R.
of 5 volumes of methanol R, 25 volumes of glacial acetic acid R and 75 volumes of toluene R. Dry the
plate in a current of warm air. Allow to cool and spray with 4-dimethylaminocinnamaldehyde solution R.
Examine immediately in daylight. Any spot in the chromatogram obtained with test solution (a),
reference solution (b) (0.5 per cent) and at most one such spot is more intense than the spot in the
chromatogram obtained with reference solution (c) (0.25 per cent).
05-40
100C to 105C.
ASSAY
Suspend 0.200 g in 5 ml of dimethylformamide R. Heat until the substance has dissolved completely.
Add 50 ml of ethanol R and titrate with 0.1M tetrabutylammonium hydroxide, determining the end-
10
H
16
N
2
O
3
S.
STORAGE
IMPURITIES
N
N
S O
H
H
H
H
H
R =
R
R
COOH
A. di[3-[(3aS,4S,6aR)-2-oxohexahydrothieno[3,4-d]imidazol-4-yl]propyl]acetic acid,
COOH
R
COOH
B. 4-[(3aS,4S,6aR)-2-oxohexahydrothieno[3,4-d]imidazol-4-yl]butane-1,1-dicarboxylic acid,
S
COOH
NH
2
NH
2
C. 5-(3,4-diamino-2-thienyl)pentanoic acid,
R
COOH
H Me
and enantiomer
D. 2-methyl-5-[(3aS,4S,6aR)-2-oxohexahydrothieno[3,4-d]imidazol-4-yl]pentanoic acid,
N
N
S O
H
H
H
COOH
H
and
N
N
S O
H
H
H
COOH H
E. 5-[(3aS,4S,6aR)-3-benzyl-2-oxohexahydrothieno[3,4-d]imidazol-4-yl]pentanoic acid and
5-[(3aS,4S,6aR)-1-benzyl-2-oxohexahydrothieno[3,4-d]imidazol-4-yl]pentanoic acid.
__________________________________________________________________________________________________________ Ph Eur
05-41
Biperiden Hydrochloride
HO
N
,HCl
C
21
H
29
NO,HCl 347.9 1235-82-1
Biperiden Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Anticholinergic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Biperiden hydrochloride contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of (RS)-1-[(1RS,2SR,4RS)-bicyclo[2.2.1]hept-5-en-2-yl]-1-phenyl-3-(piperidin-1-
yl)propan-1-ol hydrochloride, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder, slightly soluble in water and in alcohol, very slightly soluble in methylene
chloride, practically insoluble in ether.
IDENTIFICATION
obtained with biperiden hydrochloride CRS.
the same solvent.
Reference solution (a). Dissolve 25 mg of biperiden hydrochloride CRS in methanol R and dilute to 5 ml
Reference solution (b). Dissolve 5 mg of biperiden impurity A CRS in reference solution (a) and dilute to
volume of diethylamine R, 1 volume of methanol R and 20 volumes of toluene R. Allow the plate to dry
in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained
with the test solution is similar in position and size to the principal spot in the chromatogram
obtained with reference solution (a). Spray with dilute potassium iodobismuthate solution R and then
with sodium nitrite solution R and examine in daylight. The principal spot in the chromatogram
obtained with the test solution is similar in position, colour and size to the principal spot obtained
with reference solution (a). The test is not valid unless the chromatogram obtained with reference
C. To about 20 mg add 5 ml of phosphoric acid R. A green colour develops.
TESTS
Solution S Dissolve 0.10 g in carbon dioxide-free water R, heating gently if necessary, and dilute to
05-42
the same solvent.
Reference solution (a). Dilute 0.5 ml of the test solution to 100 ml with methanol R. Dilute 10 ml of
this solution to 50 ml with methanol R.
Reference solution (b). Dissolve 10 mg of the substance to be examined and 10 mg of biperiden impurity
A CRS and dilute to 10 ml with methanol R. Dilute 1 ml of the solution to 10 ml with methanol R.
a fused-silica capillary column 50 m long and 0.25 mm in internal diameter coated with
poly(vinylphenylmethyl)siloxane R (film thickness 0.25 m),
nitrogen for chromatography R as the carrier gas at a flow rate of 0.4 ml per minute and a split
ratio of 1:250,
maintaining the temperature of the column at 200C for 5 min, then raising the temperature at a rate
of 2C per minute to 270C and maintaining the temperature of the injection port at 250C and that
of the detector at 300C.
Inject 2 l of reference solution (a) and 2 l of reference solution (b). When using a recorder,
adjust the sensitivity of the system so that the height of the two principal peaks in the chromatogram
obtained with reference solution (b), are not less than 50 per cent of the full scale of the recorder.
The test is not valid unless: in the chromatogram obtained with reference solution (b), the resolution
between the first peak (biperiden) and the second peak (biperiden impurity A) is at least 2.5; the
principal peak in the chromatogram obtained with reference solution (a) has a signal- to-noise ratio of
at least 6.
Inject 2 l of the test solution. Continue the chromatography for twice the retention time of
biperiden hydrochloride. For peaks with a retention time relative to biperiden of 0.95 to 1.05: the
area of any peak, apart from the principal peak, is not greater than 0.5 per cent of the area of the
principal peak and the sum of the area of any such peaks is not greater than 1.0 per cent of the area
of the principal peak. For peaks with relative retention times outside the above-mentioned range: the
area of any peak is not greater than 0.1 per cent of the area of the principal peak and the sum of the
areas of such peaks is not greater than 0.5 per cent of the area of the principal peak. Disregard any
peak with an area less than 0.05 per cent of the area of the principal peak in the chromatogram
obtained with the test solution.
ASSAY
Dissolve 0.200 g in 60 ml of alcohol R. In a closed vessel, titrate with 0.1M alcoholic potassium
hydroxide, determining the end-point potentiometrically (2.2.20).
21
H
30
ClNO.
STORAGE
IMPURITIES
A. (SR)-1-[(1RS,2RS,4RS)-bicyclo[2.2.1]hept-5-en-2-yl]-1-phenyl-3-(piperidin-1-
yl)propan-1-ol (endo form),
B. (SR)-1-[(1RS,2SR,4RS)-bicyclo[2.2.1]hept-5-en-2-yl]-1-phenyl-3-(piperidin-1-
yl)propan-1-ol,
C. (RS)-1-[(1RS,2RS,4RS)-bicyclo[2.2.1]hept-5-en-2-yl]-1-phenyl-3-(piperidin-1-
yl)propan-1-ol,
D. 1-[(1RS,2SR,4RS)-bicyclo[2.2.1]hept-5-en-2-yl]-3-(piperidin-1-yl)propan-1-one,
E. 1-[(1RS,2RS,4RS)-bicyclo[2.2.1]hept-5-en-2-yl]-3-(piperidin-1-yl)propan-1-one.
__________________________________________________________________________________________________________ Ph Eur
05-43
Birch Leaf
Birch Leaf complies with the requirements of the 3rd edition of the European Pharmacopoeia [1174]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Birch leaf consists of the whole or fragmented dried leaves of Betula pendula Roth and/or Betula
pubescens Ehrh. as well as hybrids of both species. It contains not less than 1.5 per cent of flavonoids,
calculated as hyperoside (C
22
H
20
O
12
; M
r
464.4) with reference to the dried drug.
CHARACTERS
IDENTIFICATION
A. The leaves of both species are dark green on the adaxial surface and a lighter greenish-grey colour
on the abaxial surface; they show a characteristic dense reticulate venation. The veins are light brown
to almost white.
The leaves of Betula pendula are glabrous and show closely spaced glandular pits on both surfaces.
The leaves of Betula pendula are 3 cm to 7 cm long and 2 cm to 5 cm wide; the petiole is long and the
doubly dentate lamina is triangular to rhomboid and broadly cuneate or truncate at the base. The
angle on each side is unrounded or slightly rounded, and the apex is long and acuminate.
The leaves of Betula pubescens show few glandular trichomes and are slightly pubescent on both
surfaces. The abaxial surface shows small bundles of yellowish-grey trichomes at the branch points of
the veins. The leaves of Betula pubescens are slightly smaller, oval to rhomboid and more rounded.
They are more roughly and more regularly dentate. The apex is neither long nor acuminate.
B. Reduce to a powder (355). The powder is greenish-grey. Examine under a microscope using
chloral hydrate solution R. The powder shows numerous fragments of lamina with straight-walled
epidermal cells and cells of the lower epidermis surrounding anomocytic stomata (2.8.3). Peltate
large glands usually measuring 100 m to 120 m on the upper and lower epidermises. The
mesophyll fragments contain calcium oxalate crystals. Fragments of radial vascular bundles and
sclerenchyma fibres are accompanied by crystal sheaths. If Betula pubescens is present, the powder also
contains unicellular covering trichomes with very thick walls, about 80 m to 600 m long, usually
between 100 m and 200 m.
C. Examine by thin-layer chromatography (2.2.27) using as the coating substance a suitable silica gel.
Test solution. To 1 g of the powdered drug (355) add 10 ml of methanol R. Heat in a water-bath at
60C for 5 min. Cool and filter the solution.
Reference solution. Dissolve 1 mg of chlorogenic acid R, 1 mg of caffeic acid R, 2.5 mg of hyperoside R
and 2.5 mg of rutin R in 10 ml of methanol R.
Apply separately to the plates as bands, 10 l of each solution. Develop over a path of 10 cm using a
mixture of 10 volumes of anhydrous formic acid R, 10 volumes of water R, 30 volumes of methyl ethyl
ketone R and 50 volumes of ethyl acetate R. Dry the plate in a current of warm air. Spray with a 10 g/l
solution of diphenylboric acid aminoethyl ester R in methanol R. Subsequently spray the plate with a
50 g/l solution of macrogol 400 R in methanol R. Allow the plate to dry in air for 30 min and examine
in ultraviolet light at 365 nm. The chromatogram obtained with the reference solution shows three
zones in its lower half: in increasing order of R
f
a yellowish-brown fluorescent zone (rutin), a light
blue fluorescent zone (chlorogenic acid) and a yellowish-brown fluorescent zone (hyperoside), and in
its upper third, a light blue fluorescent zone (caffeic acid).
The chromatogram obtained with the test solution shows three zones similar in position and
fluorescence to the zones due to rutin, chlorogenic acid and hyperoside in the chromatogram
obtained with the reference solution. The zone corresponding to rutin is very faint and the zone
corresponding to hyperoside is intense. It shows also other yellowish-brown faint fluorescence zones
between the zones corresponding to caffeic acid and chlorogenic acid in the chromatogram obtained
with the reference solution. Near the solvent front, the red fluorescent zone due to chlorophylls is
visible. In the chromatogram obtained with the test solution, between this zone and the zone corres-
ponding to caffeic acid in the chromatogram obtained with the reference solution, there is a
brownish-yellow zone corresponding to quercetin.
TESTS
Foreign matter (2.8.2). Not more than 3 per cent of fragments of female catkins and not more than
3 per cent of other foreign matter.
Loss on drying (2.2.32). Not more than 10.0 per cent, determined on 1.000 g of powered drug
05-44
ASSAY
Stock solution. In a 100 ml round-bottomed flask introduce 0.200 g of the powdered drug (355), 1 ml
of a 5 g/l solution of hexamethylenetetramine R, 20 ml of acetone R and 2 ml of hydrochloric acid R1.
Boil the mixture under a reflux condenser for 30 min. Filter the liquid through a plug of absorbent
cotton in a 100 ml flask. Add the absorbent cotton to the residue in the round-bottomed flask and
extract with two quantities, each of 20 ml, of acetone R, each time boiling under a reflux condenser
for 10 min. Allow to cool to room temperature, filter the liquid through a plug of absorbent cotton
then filter the solution through a filter-paper in the volumetric flask, and dilute to 100.0 ml with
acetone R by rinsing of the flask and filter. Introduce 20.0 ml of the solution into a separating funnel,
add 20 ml of water R and extract the mixture with one quantity of 15 ml and then three quantities,
each of 10 ml, of ethyl acetate R. Combine the ethyl acetate extracts in a separating funnel, rinse with
two quantities, each of 50 ml, of water R, and filter the extract over 10 g of anhydrous sodium
sulphate R into a 50 ml volumetric flask and dilute to 50.0 ml with ethyl acetate R.
Test solution. To 10.0 ml of the stock solution add 1 ml of aluminium chloride reagent R and dilute to
25.0 ml with a 5 per cent V/V solution of glacial acetic acid R in methanol R.
Compensation solution. Dilute 10.0 ml of the stock solution to 25.0 ml with a 5 per cent V/V solution
of glacial acetic acid R in methanol R.
Measure the absorbance (2.2.25) of the test solution after 30 min, by comparison with the
compensation solution at 425 nm.
Calculate the percentage content of flavonoids, calculated as hyperoside, from the expression:
m
A
25 . 1
i.e. taking the specific absorbance of hyperoside to be 500.

STORAGE
__________________________________________________________________________________________________________ Ph Eur
05-45
Bisacodyl
N
H
OAc AcO
C
22
H
19
NO
4
361.4 603-50-9
Bisacodyl complies with the requirements of the 3rd edition of the European Pharmacopoeia [0595]. These
Action and use Stimulant laxative.
Preparations
Bisacodyl Suppositories
Bisacodyl Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Bisacodyl contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent of
4,4-(2-pyridylmethylene)diphenyl diacetate, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water, soluble in acetone,
sparingly soluble in alcohol, slightly soluble in ether. It dissolves in dilute mineral acids.
IDENTIFICATION
B. Dissolve 10.0 mg in a 6 g/l solution of potassium hydroxide R in methanol R and dilute to 100.0 ml
with the same alkaline solvent. Dilute 10.0 ml of this solution to 100.0 ml with a 6 g/l solution of
potassium hydroxide R in methanol R. Examined between 220 nm and 350 nm (2.2.25), the solution
shows an absorption maximum at 248 nm and a shoulder at about 290 nm. The specific absorbance
at the maximum is 632 to 672.
obtained with bisacodyl CRS. If the spectra obtained with the substance to be examined and the
reference substance show differences, dissolve separately the substance to be examined and the
reference substance in chloroform R, evaporate to dryness and record the spectra again.
D. Spray the chromatograms obtained in the test for related substances with a mixture of equal
volumes of 0.05M iodine and dilute sulphuric acid R. Examine in daylight. The principal spot in the
TESTS
Acidity or alkalinity To 1.0 g add 20 ml of carbon dioxide-free water R, shake, heat to boiling, cool
and filter. Add 0.2 ml of 0.01M sodium hydroxide and 0.1 ml of methyl red solution R. The solution is
yellow. Not more than 0.4 ml of 0.01M hydrochloric acid is required to change the colour of the
indicator to red.
254
R as the
coating substance.
Test solution (a). Dissolve 0.20 g of the substance to be examined in acetone R and dilute to 10 ml
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with acetone R.
Reference solution (a). Dissolve 20 mg of bisacodyl CRS in acetone R and dilute to 10 ml with the same
solvent.
Reference solution (b). Dilute 1 ml of test solution (a) to 100 ml with acetone R.
05-46
Reference solution (c). Dilute 5 ml of reference solution (b) to 10 ml with acetone R.
of 50 volumes of xylene R and 50 volumes of methyl ethyl ketone R. Allow the plate to dry in air, if
necessary heating at 100C to 105C, and examine in ultraviolet light at 254 nm. Any spot in the
the spot in the chromatogram obtained with reference solution (b) (1.0 per cent) and not more than
one such spot is more intense than the spot in the chromatogram obtained with reference solution (c)
(0.5 per cent).
100C to 105C.
ASSAY
22
H
19
NO
4
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
05-47
Bismuth Subcarbonate
Bismuth Carbonate
Bismuth Subcarbonate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Bismuth subcarbonate contains not less than 80.0 per cent and not more than 82.5 per cent of Bi (A
r
209.0), calculated with reference to the dried substance.
CHARACTERS
A white or almost white powder, practically insoluble in water, in alcohol and in ether. It dissolves
with effervescence in mineral acids.
IDENTIFICATION
A. It gives the reaction of carbonates (2.3.1).
B. It gives the reactions of bismuth (2.3.1).
TESTS
Solution S Shake 5.0 g with 10 ml of water R and add 20 ml of nitric acid R. Heat to dissolve, cool
Chlorides (2.4.4). To 6.6 ml of solution S add 4 ml of nitric acid R and dilute to 50 ml with water R.
15 ml of the solution complies with the limit test for chlorides (500 ppm).
Nitrates To 0.25 g in a 125 ml conical flask, add 20 ml of water R, 0.05 ml of indigo carmine solu-
tion R1 and then, as a single addition but with caution, 30 ml of sulphuric acid R. Titrate immediately
with indigo carmine solution R1 until a stable blue colour is obtained. Not more than n ml of the titrant
is required, n ml being the volume corresponding to 1 mg of NO
3
(0.4 per cent).
Alkali and alkaline-earth metals To 1.0 g add 10 ml of water R and 10 ml of acetic acid R. Boil for
2 min, cool and filter. Wash the residue with 20 ml of water R. To the combined filtrate and washings
add 2 ml of dilute hydrochloric acid R and 20 ml of water R. Boil and pass hydrogen sulphide R through
the boiling solution until no further precipitate is formed. Filter, wash the residue with water R,
evaporate the combined filtrate and washings to dryness on a water-bath and add 0.5 ml of sulphuric
acid R. Ignite gently and allow to cool. The residue weighs not more than 10 mg (1.0 per cent).
Arsenic (2.4.2). To 0.5 g in a distillation flask add 5 ml of water R and 7 ml of sulphuric acid R, allow
to cool and add 5 g of reducing mixture R and 10 ml of hydrochloric acid R. Heat the contents of the
flask to boiling gradually over 15 min to 30 min and continue heating at such a rate that the
distillation proceeds steadily until the volume in the flask is reduced by half or until 5 min after the
air-condenser has become full of steam. It is important that distillation be discontinued before fumes
of sulphur trioxide appear. Collect the distillate in a tube containing 15 ml of water R cooled in ice-
water. Wash down the condenser with water R and dilute the distillate to 25 ml with the same
solvent. The solution complies with limit test A for arsenic (5 ppm). Prepare the standard using a
mixture of 2.5 ml of arsenic standard solution (1 ppm As) R and 22.5 ml of water R.
Copper To 5 ml of solution S, add 2 ml of ammonia R and dilute to 50 ml with water R. Filter. To
10 ml of the filtrate add 1 ml of a 1 g/l solution of sodium diethyldithiocarbamate R. The solution is not
more intensely coloured than a standard prepared at the same time in the same manner using a
mixture of 0.25 ml of copper standard solution (10 ppm Cu) R and 9.75 ml of water R instead of 10 ml
of the filtrate (50 ppm).
Lead Not more than 20 ppm of Pb, determined by atomic absorption spectrometry (Method II,
2.2.23).
Test solution. Dissolve 12.5 g of the substance to be examined in 75 ml of a mixture of equal volumes
of lead-free nitric acid R and water R. Boil for 1 min, cool and dilute to 100.0 ml with water R.
Reference solutions. Prepare the reference solutions using appropriate quantities of lead standard
solution and a 37 per cent V/V solution of lead-free nitric acid R.
Measure the absorbance at 283.3 nm, using a hollow-cathode lamp as source of radiation and an air-
acetylene flame. Depending on the apparatus, the line at 217.0 nm may be used.
Silver To 2.0 g add 1 ml of water R and 4 ml of nitric acid R. Heat gently until dissolved and dilute
05-48
to 11 ml with water R. Cool and add 2 ml of 1M hydrochloric acid. Allow to stand for 5 min, protected
from light. Any opalescence in the solution is not more intense than that in a standard prepared at
the same time in the same manner using a mixture of 10 ml of silver standard solution (5 ppm Ag) R,
1 ml of nitric acid R and 2 ml of 1M hydrochloric acid (25 ppm).
100C to 105C.
ASSAY
Dissolve 0.500 g in 3 ml of nitric acid R and dilute to 250 ml with water R. Carry out the complexo-
metric titration of bismuth (2.5.11).
1 ml of 0.1M sodium edetate is equivalent to 20.90 mg of Bi.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
05-49
Bismuth Subgallate
1/01
O
Bi
O
O
COOH
OH
C
7
H
5
BiO
6
394.1 149-91-7
Bismuth Subgallate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1493].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Bismuth subgallate contains not less than 48.0 per cent and not more than 51.0 per cent of Bi (A
r
CHARACTERS
A yellow powder, practically insoluble in water and in alcohol. It dissolves in mineral acids with
decomposition and in solutions of alkali hydroxides, producing a reddish-brown liquid.
IDENTIFICATION
A. Mix 0.1 g with 5 ml of water R and 0.1 ml of phosphoric acid R. Heat to boiling and maintain
boiling for 2 min. Cool and filter. To the filtrate, add 1.5 ml of ferric chloride solution R1, a blackish-
blue colour develops.
B. It gives reaction (b) of bismuth (2.3.1).
TESTS
Solution S Place 1.00 g in a 20 ml volumetric flask and add 2.0 ml of lead-free nitric acid R. Allow
acid attack to take place without heating and if necessary warm slightly at the end to dissolve the test
sample completely. Add 10 ml of water R, shake and add, in small fractions, 4.5 ml of lead-free
ammonia R; shake, allow to cool, dilute to 20.0 ml with water R, shake again and allow the solids to
settle. The clear supernatant solution is solution S.
Acidity Shake 1.0 g with 20 ml of water R for 1 min and filter. To the filtrate add 0.1 ml of methyl
red solution R. Not more than 0.15 ml of 0.1M sodium hydroxide is required to change the colour of the
indicator to yellow (0.25 per cent).
Chlorides (2.4.4). To 0.5 g add 10 ml of dilute nitric acid R. Heat on a water-bath for 5 min and
filter. Dilute 5 ml of the filtrate to 15 ml with water R. The solution complies with the limit test for
Nitrates To 1.0 g add 25 ml of water R then 25 ml of a mixture of 2 volumes of sulphuric acid R and
9 volumes of water R. Heat at about 50C for 1 min with stirring and filter. To 10 ml of the filtrate,
carefully add 30 ml of sulphuric acid R. The solution is not more intensely brownish-yellow coloured
than 10 ml of a reference solution prepared at the same time as follows: to 0.4 g of gallic acid R, add
20 ml of nitrate standard solution (100 ppm NO
3
) R and 30 ml of a mixture of 2 volumes of sulphuric
acid R and 9 volumes of water R, then filter (0.2 per cent).
Copper Not more than 50 ppm of Cu, determined by atomic absorption spectrometry (Method I,
2.2.23).
Test solution. Use solution S.
Reference solutions. Prepare the reference solutions using copper standard solution (10 ppm Cu) R and
diluting with a 37 per cent V/V solution of lead-free nitric acid R.
Measure the absorbance at 324.7 nm using a copper hollow-cathode lamp as source of radiation
2.2.23).
Reference solutions. Prepare the reference solutions using lead standard solution (10 ppm Pb) R and
Measure the absorbance at 283.3 nm, using a lead hollow-cathode lamp as source of radiation and
an air-acetylene flame. Depending on the apparatus, the line at 217.0 nm may be used.
05-50
Silver Not more than 25 ppm of Ag, determined by atomic absorption spectrometry (Method I,
2.2.23).
Reference solutions. Prepare the reference solutions using silver standard solution (5 ppm Ag) R and
Measure the absorbance at 328.1 nm using a silver hollow-cathode lamp as source of radiation and
an air-acetylene flame.
Substances not precipitated by ammonia In a porcelain or quartz dish, ignite 2.0 g by increasing
the temperature very gradually; allow to cool. Moisten the residue with 2 ml of nitric acid R, evapor-
ate to dryness on a water-bath and carefully heat and ignite once more. After cooling, dissolve the
residue in 5 ml of nitric acid R and dilute to 20 ml with water R. To 10 ml of this solution, add
concentrated ammonia R until alkaline and filter. Wash the residue with water R, evaporate the
combined filtrate and washings to dryness on a water-bath and add 0.3 ml of dilute sulphuric acid R.
Ignite, the residue weighs not more than 10 mg (1.0 per cent).
100C to 105C for 3h.
ASSAY
To 0.300 g add 10 ml of a mixture of equal volumes of nitric acid R and water R, heat to boiling and
maintain boiling for 2 min. Add 0.1 g of potassium chlorate R, heat to boiling and maintain boiling for
1 min. Add 10 ml of water R and heat until the solution becomes colourless. To the hot solution, add
200 ml of water R and 50 mg of xylenol orange triturate R. Titrate with 0.1M sodium edetate until a
yellow colour is obtained.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
05-51
Heavy Bismuth Subnitrate
1/01
4[BiNO
3
(OH)
2
],BiO(OH) 1462 1304-85-4
Heavy Bismuth Subnitrate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Heavy bismuth subnitrate contains not less than 71.0 per cent and not more than 74.0 per cent of Bi
(A
r
CHARACTERS
A white powder, practically insoluble in water and in alcohol. It dissolves in mineral acids with
decomposition.
IDENTIFICATION
A. Dilute 1 ml of solution S1 (see Tests) to 5 ml with water R, add 0.3 ml of potassium iodide
solution R. A black precipitate is formed which dissolves into an orange solution with the addition of
2 ml of potassium iodide solution R.
B. It gives reaction (b) of bismuth (2.3.1).
C. It gives the reaction of nitrates (2.3.1).
D. The pH (2.2.3) of solution S2 (see Tests) is not more than 2.0.
TESTS
Solution S1 Shake 5.0 g by gently heating in 10 ml of water R, add 20 ml of nitric acid R. Heat until
dissolution, cool and dilute to 100 ml with water R.
Solution S2. Place 1.00 g in a 20 ml volumetric flask and add 2.0 ml of lead-free nitric acid R. Allow
settle. The clear supernatant solution is solution S2.
Acidity Suspend 1.0 g in 15 ml of water R and shake several times. Allow to stand for 5 min and
filter. To 10 ml of the filtrate, add 0.5 ml of phenolphthalein solution R1. Not more than 0.5 ml of
0.1M sodium hydroxide is required to change the colour of the indicator to pink.
Chlorides (2.4.4). To 5.0 ml of solution S1, add 3 ml of nitric acid R and dilute to 15 ml with
2.2.23).
Test solution. Solution S2.
2.2.23).
Reference solutions. Prepare the reference solutions using appropriate quantities of lead standard solution
(10 ppm Pb) R and diluting with a 37 per cent V/V solution of lead-free nitric acid R.
2.2.23).
Substances not precipitated by ammonia To 20 ml of solution S1, add concentrated ammonia R
05-52
until alkaline and filter. Wash the residue with water R, and evaporate the combined filtrate and
washings to dryness on a water-bath. Add 0.3 ml of dilute sulphuric acid R and ignite. The residue
100C to 105C.
ASSAY
Dissolve with heating 0.250 g in 10 ml of a mixture of 2 volumes of perchloric acid R and 5 volumes of
water R. To the hot solution, add 200 ml of water R and 50 mg of xylenol orange triturate R. Titrate
with 0.1M sodium edetate until a yellow colour is obtained.
__________________________________________________________________________________________________________ Ph Eur
05-53
Bismuth Subsalicylate
1/01
C
7
H
5
BiO
4
362.1 14882-18-9
Bismuth Subsalicylate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Bismuth subsalicylate contains not less than 57.0 per cent and not more than 60.0 per cent of Bi (A
r
CHARACTERS
A white powder, practically insoluble in water and in alcohol. It dissolves in mineral acids with
decomposition.
IDENTIFICATION
A. To 0.5 g add 10 ml of hydrochloric acid R1. Heat on a boiling water-bath for 5 min. Cool and
filter. Retain the filtrate for identification test B. Wash the residue with dilute hydrochloric acid R and
then with water R. Dissolve the residue in 0.5 ml to 1 ml of dilute sodium hydroxide solution R. Add
15 ml of water R. Neutralise with dilute hydrochloric acid R. The solution gives reaction (a) of
salicylates (2.3.1).
B. The filtrate obtained in identification test A gives reaction (b) of bismuth (2.3.1).
TESTS
Solution S Place 1.00 g in a 20 ml volumetric flask and add 2.0 ml of lead-free nitric acid R. Allow
settle. The clear supernatant solution is solution S.
Acidity Shake 2.0 g with 30 ml of ether R for 1 min and filter. To the filtrate add 30 ml of alcohol R
and 0.1 ml of thymol blue solution R. Not more than 0.35 ml of 0.1M sodium hydroxide is required to
change the colour of the indicator to blue (0.25 per cent).
Chlorides (2.4.4). Dissolve 0.250 g in a mixture of 2 ml of nitric acid R, 5 ml of water R and 8 ml of
methanol R. The solution complies with the limit test for chlorides (200 ppm).
Nitrates To 0.1 g add 10 ml of water R and, with caution, 20 ml of sulphuric acid R and stir. The
solution is not more intensely yellow coloured than a reference solution prepared at the same time
using 0.1 g of salicylic acid R, 6 ml of water R, 4 ml of nitrate standard solution (100 ppm NO
3
) R and
20 ml of sulphuric acid R (0.4 per cent).
2.2.23).
Test solution. Solution S.
2.2.23).
2.2.23).
05-54
Soluble bismuth Not more than 40 ppm of Bi determined by atomic absorption spectrometry
(Method I, 2.2.23).
Test solution. Suspend 5.0 g in 100 ml of water R. Stir constantly for 2 h at a temperature between
20C and 23C. Filter through filter paper (slow filtration) then through a cellulose micropore
membrane filter (0.1 m). To 10.0 ml of clear filtrate, add 0.1 ml of nitric acid R.
Reference solutions. Prepare the reference solutions using bismuth standard solution (100 ppm Bi) R and
diluting with a mixture of equal volumes of dilute nitric acid R and water R.
Measure the absorbance at 223.06 nm using a bismuth hollow-cathode lamp as source of radiation
100C to 105C.
ASSAY
Dissolve with heating 0.300 g in 10 ml of a mixture of 2 volumes of perchloric acid R and 5 volumes of
water R. To the hot solution, add 200 ml of water R and 50 mg of xylenol orange triturate R. Titrate
with 0.1M sodium edetate until a yellow colour is obtained.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
05-55
Black Currant
Ribes Nigrum
Definition Black Currant consists of the fresh ripe fruits of Ribes nigrum L., together with their
pedicels and rachides.
Characteristics Odour, strong and characteristic; taste, pleasantly acidic.
Macroscopical Berries: globose, ranging in diameter from about 7 to 15 mm, occurring in pendulous
racemes; epicarp shiny black externally, enclosing a yellowish green translucent pulp containing
numerous flattened ovoid seeds, about 2.5 mm long, 1.25 mm wide and 1 mm thick; berry crowned
with withered remains of five-cleft calyx; pedicels thin, up to about 10 mm long, attached to a rachis
of variable length.
Microscopical Epicarp: glands yellow, disc-shaped, roughly circular or broadly elliptical, varying in
diameter from about 140 to 240 m, each consisting of a single layer of cells attached in the centre to
the epicarp by means of a short, multiseriate stalk. Calyx: trichomes unicellular, blunt-ended with
thin, crooked walls, about 10 to 14 m wide and averaging about 350 m in length. Seed: testa with
pigment layer composed of small cells with horseshoe-shaped wall thickenings as seen in cross
section, each cell containing one or two prismatic crystals of calcium oxalate; endosperm cells with
irregularly thickened walls.
Preparation
Black Currant Syrup
Action and use Flavour; source of vitamin C.
05-56
Bleomycin Sulphate
H
H
H
O
O
HO
OH
OH
N N
HN Me
O
O HO
N Me
O
N
HO Me
NH N
S
H2N
Me O
H
H
N
S
O R
N
NH2
NH2
O
NH2 H
O
H
O
N
NH
O
OH
O
CONH2
OH
OH
H
,xH2SO4
A2: R =
_
NH[CH2]3SMe2
+
B2: R =
_
NH[CH2]4NH

C
NH
NH2
9041-34-4
Bleomycin Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0976].
Action and use Antineoplastic.
Preparation
Bleomycin Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Bleomycin sulphate is the sulphate of a mixture of glycopeptides produced by Streptomyces verticillus
or by any other means; the two principal components of the mixture are N
1
-[3-
(dimethylsulphonio)propyl]bleomycin-amide (bleomycin A
2
) and N
1
-4-(guanidobutyl)bleo-
mycinamide (bleomycin B
2
). The potency is not less than 1500 I.U. per milligram, calculated with
CHARACTERS
A white or yellowish-white powder, very hygroscopic, very soluble in water, slightly soluble in
ethanol, practically insoluble in acetone and in ether.
IDENTIFICATION
A. Examine the chromatograms obtained in the test for composition. The retention times and sizes of
the two principal peaks in the chromatogram obtained with the test solution are approximately the
same as those of the two principal peaks in the chromatogram obtained with reference solution (a).
B. It gives the reactions of sulphates (2.3.1).
TESTS
Appearance of solution Dissolve 0.200 g in water R and dilute to 10.0 ml with the same solvent.
The solution is clear (2.2.1). The absorbance (2.2.25) measured at 430 nm is not greater than 0.10.
pH (2.2.3). Dissolve 50 mg in carbon dioxide-free water R and dilute to 10 ml with the same solvent.
Composition Examine by liquid chromatography (2.2.29).
the same solvent.
Reference solution (a). Dissolve 25.0 mg of bleomycin sulphate CRS in water R and dilute to 50.0 ml
gradient elution at a flow rate of 1.2 ml per minute with a mobile phase initially composed of
10 per cent V/V of methanol R and 90 per cent V/V of a mixture prepared as follows: dissolve
0.960 g of sodium pentanesulphonate R in 900 ml of acetic acid (4.8 g/l C
2
H
4
O
2
), add 1.86 g of
sodium edetate R, dilute to 1000 ml with the same solvent and adjust to pH 4.3 using ammonia R;
05-57
increasing the proportion of methanol R to 40 per cent V/V over 60 min and continuing with
the final mixture for about 20 min, until demethylbleomycin A
2
is eluted (retention time 1.5 to
2.5, relative to bleomycin A
2
),
Inject reference solution (a). The test is not valid unless the resolution between the two principal
peaks is at least 5. Inject reference solution (b). The test is not valid unless the signal-to-noise ratio
calculated for the principal peak is at least 20.
Inject reference solution (a) six times. The test is not valid unless the relative standard deviation of
the area of the principal peak is at most 2 per cent.
Inject the test solution. The composition, calculated by the normalisation procedure and
disregarding any peak with an area less than 0.1 per cent of the total, is: bleomycin A
2
(first principal
peak) 55 per cent to 70 per cent; bleomycin B
2
(second principal peak) 25 per cent to 32 per cent;
sum of bleomycin A
2
and bleomycin B
2
not less than 85 per cent; demethylbleomycin A
2
(retention
time relative to bleomycin A
2
1.5 to 2.5) not more than 5.5 per cent; other related substances not
more than 9.5 per cent.
2.2.23).
Test solution. Dissolve 50 mg in water R and dilute to 10.0 ml with the same solvent.
Reference solution. Dilute 1.0 ml of copper standard solution (10 ppm Cu) R to 10.0 ml with water R.
Measure the absorbance at 324.7 nm using a copper hollow-cathode lamp as source of radiation and
Loss on drying (2.2.32). Not more than 3.0 per cent, determined on 50 mg by drying at 60C at a
pressure not exceeding 670 Pa for 3 h.
without a further appropriate procedure for the removal of bacterial endotoxins, not more than 5 I.U.
ASSAY
Carry out the microbiological assay of antibiotics (2.7.2), using the diffusion method. Use bleomycin
sulphate CRS as the reference substance.
STORAGE
Store in an airtight container, at a temperature of 2C to 8C. If the substance is sterile, store in a
sterile, airtight, tamper-proof container.
LABELLING
The label states:
IMPURITIES
A. bleomycinic acid (R = OH),
B. bleomycin A
5
(R = NH(CH
2
)
3
NH(CH
2
)
4
NH
2
),
C. bleomycin B
4
(R = NH(CH
2
)
4
NHC(=NH)NH(CH
2
)
4
NHC(=NH)NH
2
),
D. demethylbleomycin A
2
(R = NH(CH
2
)
4
SCH
3
).
__________________________________________________________________________________________________________ Ph Eur
05-58
Boldo Leaf
corrected 1/01
Boldo Leaf complies with the requirements of the 3rd edition of the European Pharmacopoeia [1396]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Boldo leaf consists of the whole or fragmented dried leaf of Peumus boldus Molina. The whole drug
contains not less than 20.0 ml/kg and not more than 40.0 ml/kg and the fragmented drug not less
than 15.0 ml/kg of essential oil. It contains not less than 0.1 per cent of total alkaloids, expressed as
boldine, (C
19
H
21
NO
4
;

327.4), calculated with reference to the anhydrous drug.
CHARACTERS
Boldo leaf has an aromatic odour especially when rubbed.
It has the microscopic and macroscopic characters described under Identification tests A and B.
IDENTIFICATION
A. The leaf is oval or elliptical usually 5 cm long with a short petiole, an obtuse or slightly emarginate
or mucronate apex and an equal and rounded base; the margin is entire and slightly undulate and the
thickened edges are more or less revolute. The lamina is greyish-green, thick, tough and brittle. The
upper surface is rough with numerous prominent small protuberances and a depressed venation. The
lower surface is finely pubescent, with the protuberances less well-marked, and a prominent, pinnate
venation.
chloral hydrate solution R. The powder shows fragments of the upper epidermis and underlying
hypodermis with straight or slightly sinuous thickened and beaded walls, those of the lower epidermis
with numerous stomata surrounded by four to seven subsidiary cells; solitary, bifurcated or stellate
clustered unicellular covering trichomes with more or less thickened and lignified wall; fragments of
the lamina showing a two-layered palisade; debris of the spongy mesophyll including numerous, large
rounded oil cells and parenchyma containing fine needle-shaped crystals; thick walled fibres and
lignified, pitted parenchymatous cells associated with vascular tissue from the veins.
Test solution. Add to 0.5 g of the powdered drug (355) a mixture of 1 ml of dilute hydrochloric acid R
and 20 ml of water R and heat on a water-bath under reflux for 10 min. Cool and filter. Add to the
filtrate 2 ml of dilute ammonia R1 and extract with two quantities, each of 20 ml of ether R avoiding
emulsifying. Combine the organic layers and evaporate the solvent on a water-bath. Dissolve the
residue in 1.0 ml of methanol R.
Reference solution. Dissolve 2 mg of boldine R in 5 ml of methanol R.
Apply to the plate as bands 20 l of the test solution and 10 l of the reference solution. Develop over
a path of 15 cm using a mixture of 10 volumes of methanol R, 10 volumes of diethylamine R and 80
volumes of toluene R. Allow the plate to dry in air. Spray the plate with potassium iodobismuthate
solution R2. Allow the plate to dry in air for 5 min and then spray the plate with sodium nitrite
solution R. Examine in daylight. The chromatograms show in the lower third the brown to reddish-
brown zone of boldine. The chromatogram obtained with the test solution shows several brownish
zones above and below the zone corresponding to boldine.
TESTS
Foreign matter (2.8.2). Not more than 4 per cent of twigs and 2 per cent of other foreign matter.
Water (2.2.13). Not more than 10.0 per cent determined by distillation of 20.0 g of the powdered
drug (355).
ASSAY
Essential oil Carry out the determination of essential oils in vegetable drugs (2.8.12). Use 10.0 g of
the freshly crushed drug, a 1000 ml flask and 300 ml of water R as the distillation liquid. Distil at a
rate of 2 ml/min to 3 ml/min for 3 h.
Alkaloids Examine by liquid chromatography (2.2.29).
Test solution. To 1.000 g (m
1
) of the powdered drug (355) add 50 ml of dilute hydrochloric acid R.
Shake in a water-bath at 80C for 30 min. Filter, take up the residue with 50 ml of dilute hydrochloric
acid R and shake in a water-bath at 80C for 30 min. Filter and repeat the operation once on the
05-59
residue obtained. Filter. Combine the cooled filtrates and shake with 100 ml of a mixture of equal
volumes of ethyl acetate R and hexane R. Adjust the aqueous layer to pH 9.5 with dilute ammonia R1.
Shake successively with 100 ml, 50 ml and 50 ml of methylene chloride R and combine the lower layers
and evaporate to dryness under reduced pressure. In a 10.0 ml volumetric flask dilute the residue to
Reference solution. In a 100.0 ml volumetric flask dissolve 12 mg (m
2
) of boldine R in 100.0 ml of the
mobile phase. Dilute 1.0 ml of the solution to 10.0 ml with the mobile phase.
as mobile phase at a flow rate of 1.5 ml/min a mixture of 16 volumes of solution A and 84
volumes of solution B,
Solution A. Mix 99.8 ml of acetonitrile R and 0.2 ml of diethylamine R,
Solution B. Mix 99.8 ml of water R and 0.2 ml of diethylamine R, adjusted to pH 3 with formic acid R,
Inject 20 l of each solution. When the chromatograms are recorded in the prescribed conditions,
the retention times relative to boldine are: isoboldine about 0.9; isocorydine N-oxide about 1.8;
laurotetanine about 2.2; isocorydine about 2.8 and N-methyllaurotetanine about 3.2. Additional
peaks may be present.
Calculate the percentage content of total alkaloids expressed as boldine from the expression:
1 2
2 1
m A
m A
m
1
=mass of the substance to be examined in grams,
m
2
=mass of boldine R in grams,
A
1
=sum of the areas of the peaks due to the six alkaloids identified in the chromatogram
obtained with the test solution,
A
2
=area of the peak due to boldine in the chromatogram obtained with the reference solution.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
05-60
Borax
1/01
Sodium Borate; Sodium Tetraborate
Na
2
B
4
O
7
,10H
2
O 381.4 1303-96-4
Borax complies with the requirements of the 3rd edition of the European Pharmacopoeia [0013]. These require-
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Borax contains not less than 99.0 per cent and not more than the equivalent of 103.0 per cent of
disodium tetraborate decahydrate.
CHARACTERS
A white, crystalline powder, colourless crystals or crystalline masses, efflorescent, soluble in water,
very soluble in boiling water, freely soluble in glycerol.
IDENTIFICATION
A. To 1 ml of solution S (see Tests) add 0.1 ml of sulphuric acid R and 5 ml of methanol R and ignite.
The flame has a green border.
B. To 5 ml of solution S add 0.1 ml of phenolphthalein solution R. The solution is red. On the addition
of 5 ml of glycerol (85 per cent) R the colour disappears.
C. Solution S gives the reactions of sodium (2.3.1).
TESTS
Sulphates (2.4.13).15 ml of solution S complies with the limit test for sulphates (50 ppm). Use
1.0 ml of acetic acid R instead of the 0.5 ml prescribed. Prepare the standard using a mixture of 3 ml
of sulphate standard solution (10 ppm SO
4
) R and 12 ml of distilled water R.
Ammonium (2.4.1).6 ml of solution S diluted to 14 ml with water R complies with the limit test for
ammonium (10 ppm). Prepare the standard using a mixture of 2.5 ml of ammonium standard solution
(1 ppm NH
4
) R and 7.5 ml of water R.
Arsenic (2.4.2).5 ml of solution S complies with limit test A for arsenic (5 ppm).
Calcium (2.4.3).15 ml of solution S complies with the limit test for calcium (100 ppm). Prepare the
standard using a mixture of 6 ml of calcium standard solution (10 ppm Ca) R and 9 ml of distilled
water R.
Heavy metals (2.4.8).12 ml of solution S complies with limit test A for heavy metals (25 ppm).
ASSAY
Dissolve 20 g of mannitol R in 100 ml of water R, heating if necessary, cool, add 0.5 ml of
phenolphthalein solution R and neutralise with 0.1M sodium hydroxide until a pink colour is obtained. To
this solution add 3.00 g of the substance to be examined, heat until dissolution is complete, cool, and
titrate with 1M sodium hydroxide until the pink colour reappears.
1 ml of 1M sodium hydroxide is equivalent to 0.1907 g of Na
2
B
4
O
7
,10H
2
O.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
05-61
Boric Acid
H
3
BO
3
61.8 10043-35-3
Boric Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia [0001]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Boric acid contains not less than 99.0 per cent and not more than the equivalent of 100.5 per cent of
H
3
BO
3
.
CHARACTERS
A white, crystalline powder, colourless, shiny plates greasy to the touch, or white crystals, soluble in
water and in alcohol, freely soluble in boiling water and in glycerol (85 per cent).
IDENTIFICATION
A. Dissolve 0.1 g by gently heating in 5 ml of methanol R, add 0.1 ml of sulphuric acid R and ignite the
solution. The flame has a green border.
B. Solution S (see Tests) is acid (2.2.4).
TESTS
Solution S Dissolve 3.3 g in 80 ml of boiling distilled water R, cool and dilute to 100 ml with carbon
dioxide-free water R prepared from distilled water R.
Solubility in alcohol Dissolve 1.0 g in 10 ml of boiling alcohol R. The solution is not more
opalescent than reference suspension II (2.2.1) and is colourless (Method II, 2.2.2).
Organic matter It does not darken on progressive heating to dull redness.
Prepare the standard using a mixture of 2.5 ml of lead standard solution (2 ppm Pb) R and 7.5 ml of
water R.
ASSAY
Dissolve 1.000 g with heating in 100 ml of water R containing 15 g of mannitol R. Titrate with 1M
sodium hydroxide, using 0.5 ml of phenolphthalein solution R as indicator, until a pink colour is
obtained.
1 ml of 1M sodium hydroxide is equivalent to 61.8 mg of H
3
BO
3
.
__________________________________________________________________________________________________________ Ph Eur
05-62
Bretylium Tosilate
NMe
2
Et
Br
Me
SO
3
_
+
C
18
H
24
BrNO
3
S 414.4 61-75-6
Definition Bretylium Tosilate is 2-bromobenzyl-N-ethyldimethylammonium-p-toluenesulphonate.
It contains not less than 99.0% and not more than 101.0% of C
18
H
24
BrNO
3
S, calculated with
Characteristics A white, crystalline powder. It melts at about 98. It exhibits polymorphism.
Freely soluble in water, in ethanol (96%) and in methanol.
Identification
bretylium tosilate (RS 030). If the spectra are not concordant, dissolve a quantity of the substance
being examined in the minimum volume of acetone by heating on a water bath at 50, evaporate to
dryness at room temperature under a current of nitrogen and prepare a new spectrum of the residue.
B. Carry out the method for thin-layer chromatography, Appendix III A, using a silica gel F
254
pre-
coated plate (Merck plates are suitable) and a mixture of 15 volumes of glacial acetic acid, 30 volumes
of water and 75 volumes of butan-1-ol as the mobile phase. Apply separately to the plate 10 l of each
of two solutions in water containing (1) 0.5% w/v of the substance being examined and (2) 0.5% w/v
of bretylium tosilate BPCRS. After removal of the plate, dry it in a current of air and examine under
ultraviolet light (254 nm). In the chromatogram obtained with solution (1) the two principal spots
correspond to those in the chromatogram obtained with solution (2).
Acidity pH of a 5.0% w/v solution, 5.0 to 6.5, Appendix V L.
Clarity and colour of solution A 5.0% w/v solution is clear, Appendix IV A, and colourless,
Appendix IV B, Method II.
Related substances Carry out the method for liquid chromatography, Appendix III D, using solu-
tions in the mobile phase containing (1) 0.20% w/v of the substance being examined, (2) 0.002% w/v
of the substance being examined and (3) 0.05% w/v of bretylium tosilate BPCRS and 0.05% w/v of
2-bromobenzyldimethylamine BPCRS.
4.6 mm) packed with particles of silica the surface of which has been modified by chemically bonded
phenyl groups (5 m) (Spherisorb Phenyl is suitable), (b) as the mobile phase with a flow rate of
2 ml per minute a mixture of 0.5 volume of triethylamine, 2 volumes of glacial acetic acid, 19 volumes
of acetonitrile and 81 volumes of 0.01M sodium octanesulphonate and (c) a detection wavelength of
265 nm.
between the two principal peaks is at least 6.
In the chromatogram obtained with solution (1), the area of any secondary peak is not greater than
half the area of the peak in the chromatogram obtained with solution (2) (0.5%) and the sum of the
areas of all such peaks is not greater than the area of the principal peak in the chromatogram obtained
with solution (2) (1%). Disregard the peak due to tosilate (retention time, about 2 minutes) and any
peak with an area less than 0.05 times the area of the peak in the chromatogram obtained with solu-
tion (2) (0.05%).
Loss on drying When dried to constant weight over phosphorus pentoxide at 60 at a pressure not
Assay Dissolve 0.2 g in 50 ml of 1,4-dioxan and carry out Method I for non-aqueous titration,
Appendix VIII A, using 0.025M perchloric acid VS as titrant and determining the end-point potentio-
18
H
24
BrNO
3
S.
Storage Bretylium Tosilate should be kept in an airtight container, protected from light and stored
at a temperature not exceeding 25.
Action and use Adrenergic neurone blocker.
Preparation
Bretylium Injection
When bretylium tosylate is prescribed or demanded Bretylium Tosilate shall be dispensed or
supplied.
05-63
IMPURITIES
NMe
2
Br
A. 2-bromobenzyldimethylamine
NMe
2
Br
B. 3-bromobenzyldimethylamine
NMe
2
Br
C. 4-bromobenzyldimethylamine
05-64
Bromazepam
N
N
N
Br
O H
C
14
H
10
BrN
3
O 316.2 1812-30-2
Bromazepam complies with the requirements of the 3rd edition of the European Pharmacopoeia [0879]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Bromazepam contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of 7-bromo-2,3-dihydro-5-pyrid-2-yl-1H-1,4-benzodiazepin-2-one, calculated with reference
CHARACTERS
A white or yellowish, crystalline powder, practically insoluble in water, sparingly soluble in alcohol
and in methylene chloride.
IDENTIFICATION
A. Dissolve 50.0 mg in methanol R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of the
shows an absorption maximum at 233 nm and a shoulder at about 260 nm and may show a broad
absorption maximum at about 325 nm. The specific absorbance at the maximum at 233 nm is 980 to
1080.
obtained with bromazepam CRS. Examine the substances prepared as discs.
Reference solution (a). Dissolve 10 mg of bromazepam CRS in a mixture of 1 volume of methanol R and
9 volumes of methylene chloride R and dilute to 10 ml with the same mixture of solvents.
Reference solution (b). Dissolve 10 mg of bromazepam CRS and 10 mg of temazepam CRS in a mixture
of 1 volume of methanol R and 9 volumes of methylene chloride R and dilute to 10 ml with the same
30 volumes of diethylamine R and 70 volumes of ether R. Dry the plate in a current of air and examine
in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution
is similar in position and size to the principal spot in the chromatogram obtained with reference
solution (a). The test is not valid unless the chromatogram obtained with reference solution (b)
shows two clearly separated principal spots.
D. Dissolve about 20 mg in 5 ml of methanol R. Add 5 ml of water R and 1 ml of a 10 g/l solution of
ferrous ammonium sulphate R. A violet colour develops.
E. To 0.15 g in a porcelain crucible add 0.5 g of anhydrous sodium carbonate R. Heat over an open
flame for 10 min. Allow to cool. Take up the residue in 10 ml of dilute nitric acid R and filter. To 1 ml
of the filtrate add 1 ml of water R. The solution gives reaction (a) of bromides (2.3.1).
05-65
TESTS
Appearance of solution Dissolve 0.5 g in a mixture of 1 volume of methanol R and 4 volumes of
tetrahydrofuran R and dilute to 20 ml with the same mixture of solvents. The solution is clear (2.2.1).
254
R as the
coating substance. Prepare the solutions immediately before use and carry out the test protected from light.
Reference solution. Dilute 1 ml of the test solution to 20 ml with a mixture of 1 volume of methanol R
and 9 volumes of methylene chloride R. Dilute 2 ml of the solution to 50 ml with a mixture of 1
volume of methanol R and 9 volumes of methylene chloride R.
Apply separately to the plate 5 l of each solution. Develop over a path of 7.5 cm using a mixture of 5
volumes of alcohol R, 5 volumes of triethylamine R, 20 volumes of methylene chloride R and 70 volumes
of light petroleum R1. Dry the plate in a current of air for 20 min and examine in ultraviolet light at
254 nm. Any spot in the chromatogram obtained with the test solution, apart from the principal spot,
is not more intense than the spot in the chromatogram obtained with the reference solution (0.2 per
cent).
ASSAY
Dissolve 0.250 g in 20 ml of anhydrous acetic acid R. Add 50 ml of acetic anhydride R. Titrate with
0.1M perchloric acid, determining the end-point potentiometrically (2.2.20).
14
H
10
BrN
3
O.
STORAGE
IMPURITIES
N
Br
NH
2
O
A. 2-amino-5-bromophenyl pyrid-2-yl ketone,
N
r
NH
O
Cl
O
B. N-[4-bromo-2-(pyrid-2-ylcarbonyl)phenyl-2-chloroacetamide,
H
N
N
N
O
Me
Br
C. 7-bromo-2,3-dihydro-5-(6-methylpyrid-2-yl)-1H-1,4-benzodiazepin-2-one,
H
N
r
O
NH
2
D. 3-amino-6-bromo-1,2-dihydro-4-pyrid-2-ylquinolin-2-one.
__________________________________________________________________________________________________________ Ph Eur
05-66
Bromhexine Hydrochloride
N
Me
NH
2
Br
Br
,HCl
C
14
H
21
Br
2
ClN
2
412.6 611-75-6
Bromhexine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Bromhexine hydrochloride contains not less than 98.5 per cent and not more than the equivalent of
101.5 per cent of N-(2-amino-3,5-dibromophenylmethyl)-N-methylcyclohexylamine hydrochloride,
CHARACTERS
A white or almost white, crystalline powder, very slightly soluble in water, slightly soluble in alcohol
IDENTIFICATION
First identification: A, E.
Second identification: B, C, D, E.
obtained with bromhexine hydrochloride CRS. If the spectra obtained in the solid state with the
substance to be examined and the reference substance show differences, dissolve the substance to be
examined and the reference substance separately in the smallest necessary volume of methanol R and
evaporate to dryness on a water-bath. Using the residues, prepare halide salt discs and record the
spectra again.
B. Examine the chromatograms obtained in the test for related substances in ultraviolet light at
and size to the principal spot in the chromatogram obtained with reference solution (c).
C. Dissolve about 25 mg in a mixture of 1 ml of dilute sulphuric acid R and 50 ml of water R. Add
2 ml of methylene chloride R and 5 ml of chloramine solution R and shake. A brownish-yellow colour
develops in the lower layer.
D. Dissolve about 1 mg in 3 ml of 0.1M hydrochloric acid. The solution gives the reaction of primary
aromatic amines (2.3.1).
E. Dissolve about 20 mg in 1 ml of methanol R and add 1 ml of water R. The solution gives reaction
(a) of chlorides (2.3.1).
TESTS
254
R as the
coating substance.
Test solution (b). Dilute 1.0 ml of test solution (a) to 10 ml with methanol R.
Reference solution (a). Dilute 0.5 ml of test solution (b) to 20 ml with methanol R.
Reference solution (b). Dilute 7.5 ml of reference solution (a) to 10 ml with methanol R.
Reference solution (c). Dissolve 20 mg of bromhexine hydrochloride CRS in methanol R and dilute to
Apply separately to the plate in small quantities a total of 20 l of each solution. Develop over a path
of 15 cm using a mixture of 17 volumes of glacial acetic acid R, 17 volumes of water R and 66 volumes
of butanol R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any spot in the
05-67
chromatogram obtained with test solution (a), apart from the principal spot, is not more intense
than the spot in the chromatogram obtained with reference solution (a) (0.25 per cent). The test is
not valid unless the chromatogram obtained with reference solution (b) shows a clearly visible spot.
at 100C to 105C.
ASSAY
14
H
21
Br
2
ClN
2
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
05-68
Bromocriptine Mesilate
1/01
N
N
O
HN
N
H
N
r
H
Me
H
O
O
OH
O
Me
Me
Pr
i
H
,CH
3
SO
3
H
C
33
H
44
BrN
5
O
8
S 751 22260-51-1
Bromocriptine Mesilate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Dopamine agonist.
Preparations
Bromocriptine Capsules
Bromocriptine Tablets
When bromocriptine mesylate is prescribed or demanded, Bromocriptine Mesilate shall be dispensed
or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Bromocriptine mesilate contains not less than 98.0 per cent and not more than the equivalent of
101.0 per cent of (6aR,9R)-5-bromo-N-[(2R,5S,10aS,10bS)-10b-hydroxy-2-(1-methylethyl)-5-(2-
methylpropyl)- 3,6-dioxo-octahydro-8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-
4,6,6a,7,8,9-hexahydroindolo[4,3-fg]quinoline-9-carboxamide monomethanesulphonate, calculated
CHARACTERS
A white or slightly coloured, fine crystalline powder, very sensitive to light, practically insoluble in
water, freely soluble in methanol, soluble in alcohol, sparingly soluble in methylene chloride.
The identification, tests and assay are to be carried out as rapidly as possible, protected from light.
IDENTIFICATION
A. Dissolve 10.0 mg in 10 ml of methanol R and dilute to 200.0 ml with 0.01M hydrochloric acid.
Examined between 250 nm and 380 nm (2.2.25), the solution shows an absorption maximum at
305 nm and a minimum at 270 nm. The specific absorbance at the maximum is 120 to 135,
obtained with bromocriptine mesilate CRS.
C. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel G plate R.
Test solution. Dissolve 10 mg of the substance to be examined in a mixture of 3 volumes of alcohol R,
3 volumes of methanol R and 4 volumes of methylene chloride R and dilute to 10 ml with the same
Reference solution. Dissolve 10 mg of bromocriptine mesilate CRS in a mixture of 3 volumes of alcohol R,
3 volumes of methanol R and 4 volumes of methylene chloride R and dilute to 10 ml with the same
Apply to the plate 10 l of each solution. Develop immediately in an unsaturated tank over a path of
15 cm using a mixture of 0.1 volumes of concentrated ammonia R, 1.5 volumes of water R, 3 volumes
of 2-propanol R, 88 volumes of methylene chloride R and 100 volumes of ether R. Dry the plate in a
current of cold air for 2 min. Spray with ammonium molybdate solution R3. Dry the plate at 100C
until the spots appear (about 10 min). The principal spot in the chromatogram obtained with the test
05-69
D. To 0.1 g add 5 ml of dilute hydrochloric acid R and shake for about 5 min. Filter and add 1 ml of
barium chloride solution R1. The filtrate remains clear. To a further 0.1 g add 0.5 g of anhydrous sodium
carbonate R, mix and ignite until a white residue is obtained. Allow to cool and dissolve the residue in
7 ml of water R (solution A). Solution A gives reaction (a) of sulphates (2.3.1).
E. Solution A obtained in identification test D gives reaction (a) of bromides (2.3.1).
TESTS
The solution is clear (2.2.1) and not more intensely coloured than reference solution B
5
, BY
5
or Y
5
(Method II, 2.2.2).
pH (2.2.3). Dissolve 0.2 g in a mixture of 2 volumes of methanol R and 8 volumes of carbon dioxide-
free water R and dilute to 20 ml with the same mixture of solvents. The pH of the solution is 3.1 to
3.8.
Specific optical rotation (2.2.7). Dissolve 0.100 g in a mixture of equal volumes of methanol R and
methylene chloride R and dilute to 10.0 ml with the same mixture of solvents. The specific optical
rotation is +95 to +105, calculated with reference to the dried substance.
Test solution. Dissolve 0.500 g of the substance to be examined in 5.0 ml of methanol R and dilute to
10.0 ml with buffer solution pH 2.0 R.
Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with a mixture of equal volumes
of buffer solution pH 2.0 R and methanol R.
volumes of buffer solution pH 2.0 R and methanol R.
Reference solution (c). Dissolve 5.0 mg of bromocriptine impurity A CRS in a mixture of equal volumes
of buffer solution pH 2.0 R and methanol R and dilute to 5.0 ml with the same mixture of solvents.
Reference solution (d). Dissolve 5.0 mg of bromocriptine impurity B CRS in a mixture of equal volumes
of buffer solution pH 2.0 R and methanol R and dilute to 5.0 ml with the same mixture of solvents.
Reference solution (e). Mix 0.5 ml of reference solution (c) and 0.5 ml of reference solution (d) and
dilute to 10.0 ml with a mixture of equal volumes of buffer solution pH 2.0 R and methanol R.
Reference solution (f). Dilute 1.0 ml of reference solution (c) to 100.0 ml with a mixture of equal
volumes of buffer solution pH 2.0 R and methanol R.
as mobile phase at a flow rate of 2 ml/min:
Mobile phase A. A 0.791 g/l solution of ammonium carbonate R,
Time
(min)
Mobile phase A
(% V/V)
Mobile phase B
(% V/V)
Comment
Inject 20 l of reference solution (e) and adjust the sensitivity of the system so that the heights of
the two peaks are about 20 per cent of the full scale of the recorder. The test is not valid unless the
resolution between the peaks corresponding to impurity A and impurity B is at least 1.1.
Inject 20 l of all other solutions. In the chromatogram obtained with the test solution: the area of
the peak corresponding to impurity A is not greater than the area of the principal peak in the chroma-
togram obtained with reference solution (f) (0.02 per cent) and the area of the peak corresponding to
impurity C, with a relative retention of about 1.2, is not greater than 4 times the area of the principal
peak in the chromatogram obtained with reference solution (b) (0.4 per cent); the area of any peak,
apart from the principal peak and the peak corresponding to impurity C, is not greater than twice the
and not more than one such peak has an area greater than the area of the principal peak in the
chromatogram obtained with reference solution (b) (0.1 per cent); the sum of the areas of all the
peaks, apart from the principal peak, is not greater than 1.5 times the area of the principal peak in the
chromatogram obtained with reference solution (a) (1.5 per cent). Disregard any peak, apart from
the peak due to impurity A, with an area less than half the area of the principal peak in the chromato-
gram obtained with reference solution (b) (0.05 per cent).
05-70
80C for 5 h.
ASSAY
Dissolve 0.500 g in 80 ml of a mixture of 10 volumes of anhydrous acetic acid R and 70 volumes of
acetic anhydride R. Titrate with 0.1M perchloric acid, determining the end-point potentiometrically
(2.2.20).
33
H
44
BrN
5
O
8
S.
STORAGE
IMPURITIES
N
N
O
N
N
H
N
Br
H
Me
H
O
O
O
Pr
i
Pr
i
H
A. (6aR,9R)-5-bromo-N-[(2R,5S)-2-(1-methylethyl)-5-(2-methylpropyl)-3,6-dioxo-
2,3,5,6,9,10-hexahydro-8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-
4,6,6a,7,8,9-hexahydroindolo[4,3-fg]quinoline-9-carboxamide (2-bromodehydro--
ergocriptine),
N
N
O
N
N
H
N
H
Me
H
O
O
O
Pr
i
Pr
i
H
OH
H
B. (6aR,9R)-N-[(2R,5S,10aS,10bS)-10b-hydroxy-2-(1-methylethyl)-5-(2-methylpropyl)- 3,6-
dioxo-octahydro-8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl- 4,6,6a,7,8,9-
hexahydroindolo[4,3-fg]quinoline-9-carboxamide (-ergocriptine),
N
N
O
N
N
H
N
Br
H
Me
H
O
O
O
Pr
i
Pr
i
H
OH
H
C. (6aR,9S)-5-bromo-N-[(2R,5S,10aS,10bS)-10b-hydroxy-2-(1-methylethyl)-5-(2-
methylpropyl)- 3,6-dioxo-octahydro-8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-
methyl-4,6,6a,7,8,9-hexahydroindolo[4,3-fg]quinoline-9-carboxamide ((9S)-2-bromo--
ergocriptine),
N
N
Br
H
Me
R
O
H
D. R = OH: (6aR,9R)-5-bromo-7-methyl-4,6,6a,7,8,9-hexahydroindolo[4,3-fg]quinoline-9-
carboxylic acid,
E. R = NH
2:
(6aR,9R)-5-bromo-7-methyl-4,6,6a,7,8,9-hexahydroindolo[4,3-fg]quinoline-9-
carboxamide,
N
N
O
N
N
H
N
H
Me
H
O
O
O
Pr
i
Pr
i
H
OH
H
Br
F. (6aR,9R)-5-bromo-N-[(2S,5S,10aS,10bS)-10b-hydroxy-2-(1-methylethyl)-5-(2-
methylpropyl)- 3,6-dioxo-octahydro-8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-
methyl-4,6,6a,7,8,9-hexahydroindolo[4,3-fg]quinoline-9-carboxamide ((2S)-2-bromo--
ergocriptine),
05-71
N
N
O
N
N
H
N
H
Me
H
O
O
O
Pr
i
Pr
i
H
H
MeO
Br
G. 6aR,9R)-5-bromo-N-[(2R,5S,10aS,10bS)-10b-methoxy-2-(1-methylethyl)-5-(2-
methylpropyl)-3,6-dioxo-octahydro-8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-
methyl-4,6,6a,7,8,9-hexahydroindolo[4,3-fg]quinoline-9-carboxamide (2-bromo-10b-O-
methyl--ergocriptine).
__________________________________________________________________________________________________________ Ph Eur
6-1
Bromperidol
N
O
F
HO
Br
C
21
H
23
BrFNO
2
420.3 10457-90-6
Bromperidol complies with the requirements of the 3rd edition of the European Pharmacopoeia [1178]. These
Ph Eur _________________________________________________________________________________________________________________________________________
DEFINITION
Bromperidol contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of 4-[4-(4-bromophenyl)-4-hydroxypiperidin-l-yl]-1-(4-fluorophenyl)butan-1-one, calculated with
CHARACTERS
A white or almost white powder, practically insoluble in water, sparingly soluble in methanol and in
methylene chloride, slightly soluble in alcohol.
IDENTIFICATION
obtained with bromperidol CRS. Examine the substances prepared as discs.
C. Examine by thin layer chromatography (2.2.27), using as the coating substance a suitable
octadecylsilyl silica gel.
the same solvent.
Reference solution (a). Dissolve 10 mg of bromperidol CRS in methanol R and dilute to 10 ml with the
same solvent.
Reference solution (b). Dissolve 10 mg of bromperidol CRS and 10 mg of haloperidol CRS in methanol R
15 cm using a mixture of 10 volumes of tetrahydrofuran R, 45 volumes of methanol R and 45 volumes
of a 58 g/l solution of sodium chloride R. Allow the plate to dry in air and examine in ultraviolet light at
254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position
and size to the principal spot in the chromatogram obtained with reference solution (a). The test is
not valid unless the chromatogram obtained with the reference solution (b) shows two spots which
may, however, not be completely separated.
D. Dissolve about 10 mg in 5 ml of ethanol R. Add 0.5 ml of dinitrobenzene solution R and 0.5 ml of
2M alcoholic potassium hydroxide solution R. A violet colour is produced that becomes brownish-red
after 20 min.
flame for 10 min. Allow to cool. Take up the residue with 5 ml of dilute nitric acid R and filter. To
1 ml of the filtrate add 1 ml of water R. The solution gives reaction (a) of bromides (2.3.1).
TESTS
Appearance of solution Dissolve 0.2 g in 20 ml of a 1 per cent V/V solution of lactic acid R. The
solution is clear (2.2.1) and not more intensely coloured than reference solution Y
7
(Method II,
2.2.2).
6-2
Reference solution (a). Dissolve 2.5 mg of bromperidol CRS and 5.0 mg of haloperidol CRS in
Reference solution (b). Dilute 5.0 ml of the test solution to 100.0 ml with methanol R. Dilute 1.0 ml of
octadecylsilylsilica gel for chromatography R (3 m),
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
eluent
composition
25 = 0 90 10 Re-start gradient
Equilibrate the column for at least 30 min with acetonitrile R and then equilibrate with the initial
conditions, the retention times are: haloperidol, about 5.5 min and bromperidol, about 6 min. The
test is not valid unless the resolution between the peaks due to haloperidol and bromperidol is at least
3.0. If necessary, adjust the concentration of acetonitrile in the mobile phase or adjust the time
programme for the linear gradient elution.
with reference solution (b) (0.5 per cent); the sum of the areas of all peaks, apart from the principal
peak, is not greater than twice the area of the principal peak in the chromatogram obtained with
reference solution (b) (1 per cent). Disregard any peak obtained with the blank and any peak with an
area less than 0.1 times the area of the principal peak in the chromatogram obtained with reference
solution (b).
100C to 105C.
ASSAY
ethyl ketone R. Titrate with 0.1M perchloric acid, using 0.2 ml of naphtholbenzein solution R as indicator.
21
H
23
BrFNO
2
.
STORAGE
IMPURITIES
N
OH
O
F
A. 1-(4-fluorophenyl)-4-(4-hydroxy-4-phenylpiperidin-1-yl)butan-1-one,
6-3
N
Br
OH
O
F
B. 4-[4-(4-bromophenyl)-4-hydroxypiperidin-1-yl]-1-(2-fluorophenyl)butan-1-one,
N
OH
O
F
C. 4-[4-(biphenyl-4-yl)-4-hydroxypiperidin-1-yl]-1-(4-fluorophenyl)butan-1-one,
N
Br
OH
O
F
Et
D. 4-[4-(4-bromophenyl)-4-hydroxypiperidin-1-yl]-1-(3-ethyl-4-fluorophenyl) butan-1-one,
N
Br
HO
N
Br
OH
O
E. 4-[4-(4-bromophenyl)-4-hydroxypiperidin-1-yl]-1-[4-[4-(4-bromophenyl)- 4-hydroxy-
piperidin-1-yl]phenyl]butan-1-one,
N
OH
O
F
Br
F. 4-[4-(4-bromobiphenyl-4-yl)-4-hydroxypiperidin-1-yl]-1-(4- fluorophenyl)butan-1-one.
_________________________________________________________________________________________________________________________________________ Ph Eur
6-4
Bromperidol Decanoate
N
O
F
H
3
C[ CH
2
]
8
COO
Br
C
31
H
14
BrFNO
3
574.6 75067-66-2
Bromperidol Decanoate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ________________________________________________________________________________________________________________
DEFINITION
Bromperidol decanoate contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of 4-(4-bromophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]piperidin-4-yl decanoate,
CHARACTERS
A white or almost white powder, practically insoluble in water, very soluble in methylene chloride,
soluble in alcohol.
IDENTIFICATION
obtained with bromperidol decanoate CRS. Examine the substances prepared as mulls in liquid
paraffin R.
B. To 0.1 g in a porcelain crucible add 0.5 g of anhydrous sodium carbonate R. Heat over an open
1 ml of the filtrate add 1 ml of water R. The solution gives reaction (a) of bromides (2.3.1).
TESTS
Appearance of solution Dissolve 2.0 g in methylene chloride R and dilute to 20 ml with the same
solvent. The solution is clear (2.2.1) and not more intensely coloured than reference solution B
5
(Method II, 2.2.2).
before use and protect from light.
Reference solution (a). Dissolve 2.5 mg of bromperidol decanoate CRS and 2.5 mg of haloperidol
decanoate CRS in methanol R and dilute to 50.0 ml with the same solvent.
Reference solution. (b). Dilute 5.0 ml of the test solution to 100.0 ml with methanol R. Dilute 1.0 ml of
6-5
Time
(min)
Mobile
phase A
(per cent V/V)
Mobile
phase B
(per cent V/V)
Comment
eluent
composition
conditions, the retention times are: haloperidol decanoate about 24 min; bromperidol decanoate
about 24.5 min. The test is not valid unless the resolution between the peaks due to haloperidol
decanoate and bromperidol decanoate is at least 1.5. If necessary, adjust the gradient or the time
solution (b). In the chromatogram obtained with the test solution: the area of any peak apart from
with reference solution (b) (0.5 per cent); the sum of the areas of all the peaks apart from the
principal peak is not greater than three times the area of the principal peak in the chromatogram
obtained with reference solution (b) (1.5 per cent). Disregard any peak due to the blank and any
30C.
ASSAY
ethyl ketone R. Titrate with 0.1M perchloric acid using 0.2 ml of naphtholbenzein solution R as indicator.
31
H
41
BrFNO
3
.
STORAGE
Store in a well-closed container at a temperature below 25C, protected from light.
IMPURITIES
F
Ar =
Br
N
H
3
C[CH
2
]
8
COO
R =
N
H
3
C[CH
2
]
8
COO
Ar
O
A. 1-[4-(4-fluoropheyl)-4-oxobutyl]-4-phenylpiperidin-4-yl decanoate,
F
O
R
B. 4-(4-bromophenyl)-1-[4-(2-fluorophenyl)-4-oxobutyl]piperidin-4-yl decanoate,
O
R
F
Et
C. 4-(4-bromophenyl)-1-[4-(3-ethyl-4-fluorophenyl)-4-oxobutyl]piperidin-4-yl decanoate,
6-6
O
R
N
Br
OH
D. 4-(4-bromophenyl)-1-[4-[4-[4-(4-bromophenyl)-4-hydroxypiperidin-1-yl]phenyl]-4-
oxobutyl]piperidin-4-yl decanoate,
N
H
3
C[CH
2
]
8
COO
Ar
O
Br
E. 4-(4-bromobiphenyl-4-yl)-1-[4-(4-fluorophenyl)-4-oxobutyl]piperidin-4-yl decanoate,
N
H
3
C[CH
2
]
8
COO
Ar
O
F. 4-(biphenyl-4-yl)-1-[4-(4-fluorophenyl)-4-oxobutyl]piperidin-4-yl decanoate,
G. 4-[4-(4-bromophenyl)-4-hydroxypiperidin-1-yl]-1-(4-fluorophenyl)butan-1-one
(bromperidol),
N
H
3
C[CH
2
]
6
COO
Ar
O
Br
H. 4-(4-bromophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]piperidin-4-yl octanoate,
N
H[CH
2
]
8
COO
Ar
O
Br
I. 4-(4-bromophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]piperidin-4-yl nonanoate,
N
H[CH
2
]
10
COO
Ar
O
Br
J. 4-(4-bromophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]piperidin-4-yl undecanoate,
N
H
3
C[CH
2
]
10
COO
Ar
O
Br
K. 4-(4-bromophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]piperidin-4-yl dodecanoate.
Other detectable impurities
H
3
C Ar
O
L. 1-(4-fluorophenyl)ethanone.
_______________________________________________________________________________________________________________ Ph Eur
6-7
Brompheniramine Maleate
COOH
COOH
,
and enanti omer
N
Br
NMe
2
H
C
16
H
19
BrN
2
,C
4
H
4
O
4
435.3 980-71-2
Brompheniramine Maleate complies with the requirements of the 3rd edition of the European Pharmacopoeia
1
Preparation
Brompheniramine Tablets
Ph Eur _________________________________________________________________________________________________________________________________________
DEFINITION
Brompheniramine maleate contains not less than 98.0 per cent and not more than the equivalent of
101.0 per cent of (RS)-[3-(4-bromophenyl)-3-(pyridin-2-yl)propyl]dimethylamine (Z)-butenedioate,
CHARACTERS
A white or almost white, crystalline powder, soluble in water, freely soluble in alcohol, in methanol
IDENTIFICATION
First identification: A, B, C, D, E.
Second identification: A, B, E, F.
B. Dissolve 65 mg in 0.1M hydrochloric acid and dilute to 100.0 ml with the same acid. Dilute 5.0 ml
of this solution to 100.0 ml with 0.1M hydrochloric acid. Examined between 220 nm and 320 nm
(2.2.25), the solution shows an absorption maximum at 265 nm. The specific absorbance at the
maximum is 190 to 210.
obtained with brompheniramine maleate CRS. Examine the substances prepared as discs using
potassium bromide R.
D. Examine the chromatograms obtained in the test for related substances. The retention time and
size of the principal peak in the chromatogram obtained with the test solution are approximately the
same as those of the principal peak in the chromatogram obtained with reference solution (a). The
chromatogram obtained with reference solution (c) shows two principal peaks with retention times
corresponding to the retention times of the peaks obtained with reference solutions (a) and (b).
E. Examine by thin layer chromatography (2.2.27), using as the coating substance a suitable silica gel
Test solution. Dissolve 0.10 g of the substance to be examined in methanol R and dilute to 5.0 ml with
the same solvent.
Reference solution. Dissolve 56 mg of maleic acid R in methanol R and dilute to 10 ml with the same
solvent.
volumes of water R, 7 volumes of anhydrous formic acid R, 20 volumes of methanol R and 70 volumes
of di-isopropyl ether R. Allow the plate to dry in a current of air for a few minutes and examine in
ultraviolet light at 254 nm. The chromatogram obtained with the test solution shows two clearly
separated spots. The upper spot is similar in position and size to the spot in the chromatogram
F. To 0.15 g in a porcelain crucible add 0.5 g of anhydrous sodium carbonate R. Heat over an open
flame for 10 min. Allow to cool. Take up the residue in 10 ml of dilute nitric acid R and filter. To 1 ml
of the filtrate add 1 ml of water R. The solution gives reaction (a) of bromides (2.3.1).
6-8
TESTS
The solution is clear (2.2.1) and not more intensely coloured than reference solution BY
6
(Method II,
2.2.2).
pH (2.2.3). Dissolve 0.20 g in 20 ml of carbon dioxide-free water R. The pH of the solution is 4.0 to
5.0.
Optical rotation (2.2.7). Dissolve 2.5 g in water R and dilute to 25.0 ml with the same solvent. The
angle of optical rotation measured in a 2 dm tube is 0.2 to +0.2.
Test solution. Dissolve 0.10 g of the substance to be examined in 10 ml of methylene chloride R.
Reference solution (a). Dissolve 10 mg of brompheniramine maleate CRS in methylene chloride R and
Reference solution (b). Dissolve 5 mg of chlorphenamine maleate CRS in methylene chloride R and dilute
Reference solution (c). To 0.5 ml of the test solution add 0.5 ml of reference solution (b).
a glass column 2.3 m long and 2 mm in internal diameter packed with acid- and base-washed
silanised diatomaceous earth for gas chromatography R (135 m to 175 m) impregnated with 3 per
cent m/m of polymethylphenylsiloxane R,
detector at 250C.
Inject 1 l of each solution. The test is not valid unless: in the chromatogram obtained with
reference solution (c), the resolution between the peaks corresponding to brompheniramine and
chlorphenamine is at least 1.5. After injecting the test solution, continue the chromatography for at
least 2.5 times the retention time of the principal peak. In the chromatogram obtained with the test
solution: the sum of the areas of the peaks, apart from the principal peak, is not greater than 1 per
cent of the area of the principal peak; none of the peaks, apart from the principal peak, has an area
greater than 0.4 per cent of the area of the principal peak. Disregard any peak with an area less than
0.1 per cent of that of the peak corresponding to brompheniramine in the chromatogram obtained
with the test solution.
100C to 105C for 3 h.
ASSAY
20
H
23
BrN
2
O
4
.
STORAGE
IMPURITIES
N
Cl
NMe
2
A. chlorphenamine,
N
Cl
NMe
2
H
B. dexchlorpheniramine,
N
NMe
2
H
and enantiomer
C. (3RS)-N,N-dimethyl-3-phenyl-3-(pyridin-2-yl)propan-1-amine.
_________________________________________________________________________________________________________________________________________ Ph Eur
6-9
Bronopol
HO OH
NO
2
Br
C
3
H
6
BrNO
4
200.0 52-51-7
Definition Bronopol is 2-bromo-2-nitropropane-1,3-diol. It contains not less than 99.0% and not
3
H
6
BrNO
4
Characteristics White or almost white crystals or crystalline powder; odourless or almost odourless.
Freely soluble in water and in ethanol (96%); slightly soluble in glycerol and in liquid paraffin.
Identification
bronopol (RS 031).
B. Dissolve 0.1 g in 10 ml of water, add 10 ml of 7.5M sodium hydroxide and, carefully with constant
stirring and cooling, 0.5 g of nickelaluminium alloy. Allow the reaction to subside, filter and
carefully neutralise with nitric acid. The resulting solution yields reaction A characteristic of bromides,
Appendix VI.
C. Melting point, after drying over phosphorus pentoxide at a pressure not exceeding 0.7 kPa, about
130, Appendix V A.
Acidity or alkalinity pH of a 1% w/v solution, 5.0 to 7.0, Appendix V L.
Related substances Prepare a 0.05% w/v solution of biphenyl (internal standard) in pyridine
(solution A) and a mixture of 9 volumes of bis(trimethylsilyl)trifluoroacetamide and 1 volume of
trimethylchlorosilane (solution B). Carry out the method for gas chromatography, Appendix III B, using
the following solutions. For solution (1) add 1 ml of solution A and 1 ml of solution B to 1 ml of a
0.05% w/v solution of the substance being examined in pyridine, mix and heat at 70 for 20 minutes.
For solution (2) add 2 ml of pyridine to 0.1 g of the substance being examined, shake to dissolve, add
1 ml of solution B, mix and heat at 70 for 20 minutes. For solution (3) add 1 ml of solution A and
1 ml of pyridine to 0.1 g of the substance being examined, shake to dissolve, add 1 ml of solution B,
mix and heat at 70 for 20 minutes.
with acid-washed, silanised diatomaceous support (80 to 100 mesh) coated with 3% w/w of phenyl
methyl silicone fluid (50% phenyl) (OV-17 is suitable) and maintained at 115.
Calculate the ratio (r) of the area of the peak corresponding to bronopol to the area of the peak due
to the internal standard in the chromatogram obtained with solution (1). In the chromatogram
obtained with solution (3) the ratio of the area of any secondary peak to the area of the peak due to the
internal standard is not greater than r and the ratio of the sum of the areas of any secondary peaks to
the area of the peak due to the internal standard is not greater than 2r.
Water Not more than 0.5% w/w, Appendix IX C, Method I B. Use 5 g.
Assay In a flask fitted with a reflux condenser dissolve 0.4 g in 15 ml of water and add 15 ml of 7.5M
sodium hydroxide. Slowly, with caution, add 2 g of nickelaluminium alloy through the reflux
condenser, agitating the flask whilst cooling under running water. Allow the mixture to stand for 10
minutes and boil for 1 hour. Cool and filter under reduced pressure, washing the condenser, flask
and residue with 150 ml of water. Combine the filtrate and washings, add 25 ml of nitric acid and
40 ml of 0.1M silver nitrate VS, shake vigorously and titrate with 0.1M ammonium thiocyanate VS
using ammonium iron(III) sulphate solution R2 as indicator. Repeat the operation without the substance
being examined. The difference between the titrations represents the amount of silver nitrate
required. Each ml of 0.1M silver nitrate VS is equivalent to 20.00 mg of C
3
H
6
BrNO
4
.
Storage Bronopol should be kept in a well-closed container and protected from light.
Action and use Preservative; mild antiseptic.
6-10
Buclizine Hydrochloride
N
N
H
Bu
t
Cl
,2HCl
C
28
H
33
ClN
2
,2HCl 506.0 129-74-8
Definition Buclizine Hydrochloride is (RS)-1-(4-tert-butylbenzyl)-4-(4-chlorobenzhydryl)piperazine
dihydrochloride. It contains not less than 99.0% and not more than 100.5% of C
28
H
33
ClN
2
,2HCl,
Characteristics A white or slightly yellowish, crystalline powder.
Practically insoluble in water; sparingly soluble in chloroform and in propane-1,2-diol; very slightly
soluble in ethanol (96%).
Identification
buclizine hydrochloride (RS 032).
B. A 0.25% w/v solution in ethanol (50%) yields reaction A characteristic of chlorides, Appendix VI.
Related substances Carry out the method for liquid chromatography, Appendix III D, using four
solutions in the initial mobile phase containing (1) 0.0010% w/v of the substance being examined,
(2) 0.50% w/v of the substance being examined, (3) 0.0010% w/v of 1,4-bis(4-chlorobenzhydryl)-
piperazine BPCRS and (4) 0.50% w/v of buclizine hydrochloride impurity standard BPCRS.
The chromatographic procedure may be carried out using a stainless steel column (20 cm 4 mm)
packed with stationary phase C (10 m) (Nucleosil C18 is suitable). Use as the initial mobile phase
0.01M sodium heptanesulphonate in a mixture of 55 volumes of water and 45 volumes of acetonitrile and
as the final mobile phase 0.01M sodium heptanesulphonate in a mixture of 20 volumes of water and 80
volumes of acetonitrile. Before use, adjust the pH of both the initial and final mobile phases to 4.0
with 1M orthophosphoric acid. Carry out a linear gradient elution with a flow rate of 2 ml per minute
for 30 minutes and maintain the final mobile phase for 10 minutes with the same flow rate. Use a
The test is not valid unless the chromatogram obtained with solution (4) closely resembles the
chromatogram supplied with buclizine hydrochloride impurity standard BPCRS.
In the chromatogram obtained with solution (2) the area of any peak corresponding to 1,4-bis(4-
chlorobenzhydryl)piperazine is not greater than the area of the peak obtained in the chromatogram
with solution (3) and the area of any other secondary peak is not greater than the area of the peak in
the chromatogram obtained with solution (1).
Loss on drying When dried to constant weight at 100 to 105, loses not more than 1.0% of its
weight. Use 1 g.
C
28
H
33
ClN
2
,2HCl.
1
-receptor antagonist; antiemetic.
IMPURITIES
A. 1,4-bis(4-chlorobenzhydryl)piperazine,
B. 4-chlorobenzhydrol, 1-(4-chlorobenzhydryl)piperazine,
C. 4-chlorobenzophenone.
6-11
Budesonide
Me
H
H Me
O
O
O
O
Pr
n
H
HO
H
H
H
OH
and epimer at C*
*
C
25
H
34
O
6
430.5 51333-22-3
Budesonide complies with the requirements of the 3rd edition of the European Pharmacopoeia [1075]. These
Ph Eur _________________________________________________________________________________________________________________________________________
DEFINITION
Budesonide contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent
of a mixture of the C-22S (epimer A) and the C-22R (epimer B) epimers of 16,17-
butylidenedioxy-11,21-dihydroxypregna-1,4-diene-3,20-dione, calculated with reference to the
dried substance.
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water, freely soluble in methylene
chloride, sparingly soluble in alcohol.
IDENTIFICATION
obtained with budesonide CRS. Examine the substances prepared as discs.
Reference solution (a). Dissolve 25 mg budesonide CRS in a mixture of 1 volume of methanol R and 9
volumes of methylene chloride R and dilute to 10 ml with the same mixture of solvents.
Reference solution (b). Dissolve 12.5 mg triamcinolone acetonide CRS in reference solution (a) and
dilute to 5 ml with the same solution.
until spots appear. Allow to cool. Examine in daylight and in ultraviolet light at 365 nm. The
principal spot in the chromatogram obtained with the test solution is similar in position, colour in
daylight, fluorescence in ultraviolet light at 365 nm and size to the principal spot in the chromato-
gram obtained with the reference solution (a). The test is not valid unless the chromatogram
C. Dissolve about 2 mg in 2 ml of sulphuric acid R. Within 5 min, a yellow colour develops. Within
30 min, the colour changes to brown or reddish-brown. Add cautiously the solution to 10 ml of
water R and mix. The colour fades and a clear solution remains.
D. Dissolve about 1 mg in 2 ml of a solution containing 2 g of phosphomolybdic acid R dissolved in a
mixture of 10 ml of dilute sodium hydroxide solution R, 15 ml of water R and 25 ml of glacial acetic
acid R. Heat for 5 min on a water-bath. Cool in iced water for 10 min and add 3 ml of dilute sodium
hydroxide solution R. The solution is blue.
6-12
TESTS
peak corresponding to epimer B (the first of the two principal peaks) in the chromatogram obtained
is not less than 50 per cent of the full scale of the recorder. Inject 20 l of the test solution, 20 l of
reference solution (a) and 20 l of reference solution (b). Continue the chromatography for 1.5 times
the retention time of epimer B. In the chromatogram obtained with the test solution: the area of any
peak, apart from the peaks corresponding to epimer A and epimer B, is not greater than the sum of
the areas of the epimer peaks in the chromatogram obtained with reference solution (b) (0.5 per
cent); the sum of the areas of any such peaks is not greater than the sum of the areas of the epimer
peaks in the chromatogram obtained with reference solution (a) (1.5 per cent). Disregard any peak
with an area less than 0.1 times the sum of the areas of the epimer peaks in the chromatogram
Epimer A Examine by liquid chromatography (2.2.29) as described under Assay.
Inject 20 l of the test solution. The content of epimer A (second peak) is 40.0 per cent to 51.0 per
cent of the sum of the areas of the two epimer peaks of budesonide.
Methanol Not more than 0.1 per cent, determined by head-space gas chromatography using the
standard addition method (2.2.28).
Test solution. Dissolve 2.000 g of the substance to be examined in dimethylacetamide R and dilute to
Reference solution. Dilute 0.500 g of methanol R in dimethylacetamide R and dilute to 100.0 ml with the
same solvent.
The following head-space conditions may be used:
equilibration time: 30 min,
transfer-line temperature: 85C,
pressurisation time: 10 s,
injection time: 10 s.
a fused-silica capillary column 30 m long and 0.32 mm in internal diameter coated with macrogol
20,000 R (film thickness 1 m),
nitrogen for chromatography R as the carrier gas at a pressure of 55 kPa,
of 30C per minute to 220C and maintaining at 220C for 2 min, and maintaining the temperature
of the injection port at 250C and that of the detector at 300C.
at 100C to 105C.
ASSAY
Protect the solutions from light throughout the assay.
50.0 ml with phosphate buffer solution pH 3.2 R. Allow to stand for at least 15 min before use.
Reference solution (c). Dissolve 25.0 mg of budesonide CRS in 15 ml of acetonitrile R and dilute to
50.0 ml with phosphate buffer solution pH 3.2 R.
as mobile phase at a flow rate of 1.5 ml per minute a mixture of 32 volumes of acetonitrile R and
68 volumes of phosphate buffer solution pH 3.2 R,
peak corresponding to epimer B (the first of the two principal peaks) in the chromatogram obtained
is not less than 50 per cent of the full scale of the recorder. When the chromatograms are recorded in
the prescribed conditions, the retention time for epimer B is about 16 min. If necessary, adjust the
concentration of acetonitrile in the mobile phase (increasing the concentration to decrease the
retention time). The test is not valid unless: in the chromatogram obtained with reference solution
6-13
(c), the resolution between the peaks corresponding to epimer A and epimer B is not less than 1.5;
the number of theoretical plates determined from the epimer B peak is at least 4000; the symmetry
factor for the same peak is less than 1.5.
Inject 20 l of reference solution (c) six times. The assay is not valid unless the relative standard
deviation of the sum of the peak areas of the two epimers is at most 1.0 per cent. Inject alternatively
the test solution and reference solution (c).
25
H
34
O
6
from the sum of the areas of the two epimer peaks.
IMPURITIES
Me
H
H Me
O
O
OH
OH
HO
H
H
H
OH
A. 11,16,17,21-tetrahydroxypregna-1,4-diene-3,20-dione (16-hydroxyprednisolone),
Me
H
H Me
O
O
O
O
H
Me
HO
H
H
H
OH
B. 16,17-ethylidenedioxy-11,21-dihydroxypregna-1,4-diene-3,20-dione (22-methyl
homologue of budesonide),
H
H Me
O
HO
H
H
O
O
O
OH
Pr
n
C. 16,17-butylidenedioxy-11-hydroxy-17-(2-hydroxymethyl)-D-homoandrosta-1,4-
diene-3,17-dione (D-homobudesonide),
Me
H
H Me
O
O
O
O
Pr
n
H
HO
H
H
H
CHO
D. 16,17-butylidenedioxy-11-hydroxy-3,20-dioxopregna-1,4-diene-21-al
(21-dehydrobudesonide),
Me
H Me
O
O
O
O
Pr
n
H
HO
H
H
H
OH
E. 16,17-butylidenedioxy-11,21-dihydroxypregna-1,4,14-triene-3,20-dione
(14,15-dehydrobudesonide),
Me
H
H Me
O
O
O
O
Me
Me
HO
H
H
H
OH
F. 16,17 -methylethylidenedioxy-11,21-dihydroxypregna-1,4-diene-3,20-dione (desonide).
_________________________________________________________________________________________________________________________________________ Ph Eur
6-14
Bufexamac
N
OH
O H
3
C
O
H
C
12
H
17
NO
3
223.3 2438-72-4
Bufexamac complies with the requirements of the 3rd edition of the European Pharmacopoeia [1179]. These
Ph Eur _________________________________________________________________________________________________________________________________________
DEFINITION
Bufexamac contains not less than 98.5 per cent and not more than the equivalent of 101.5 per cent
of 2-(4-butoxyphenyl)-N-hydroxyacetamide, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water, soluble in
dimethylformamide, slightly soluble in ethyl acetate and in methanol.
IDENTIFICATION
A. Dissolve 20 mg in methanol R and dilute to 20 ml with the same solvent. Dilute 1 ml of the
solution to 50 ml with methanol R. Examined between 210 nm and 360 nm (2.2.25), the solution
shows three absorption maxima, at 228 nm, 277 nm and 284 nm.
obtained with bufexamac CRS. Examine the substances prepared as discs.
the same solvent.
Reference solution (a). Dissolve 20 mg of bufexamac CRS in methanol R and dilute to 10 ml with the
same solvent.
Reference solution (b). Dissolve 10 mg of salicylic acid R in reference solution (a) and dilute to 5 ml
with the same solution.
of glacial acetic acid R, 20 volumes of dioxan R and 90 volumes of toluene R. Dry the plate in a current
of warm air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram
TESTS
Reference solution (b). Dissolve 5 mg of bufexamac CRS and 5 mg of salicylic acid R in the mobile phase
and dilute to 10 ml with the mobile phase. Dilute 1 ml of the solution to 10 ml with the mobile
phase.
silica gel for chromatography R (5 m) having a specific surface area of 350 m
2
/g and a pore size of
10 nm,
as mobile phase at a flow rate of 1 ml per minute a mixture of 30 volumes of a 1.4 g/l solution of
6-15
dipotassium hydrogen phosphate R and 70 volumes of methanol R adjusted to pH to 3.6 with dilute
phosphoric acid R,
Inject 20 l of reference solution (a) and 20 l of reference solution (b). Adjust the sensitivity of the
(a) is at least 50 per cent of the full scale of the recorder. The test is not valid unless in the chromato-
gram obtained with reference solution (b) the resolution between the peaks corresponding to salicylic
acid and to bufexamac is at least 2.0.
Inject 20 l of the test solution. Continue the chromatography for four times the retention time of
bufexamac. In the chromatogram obtained with the test solution: the area of any peak, apart from the
principal peak is not greater than the area of the principal peak in the chromatogram obtained with
reference solution (a) (0.2 per cent); the sum of the areas of all the peaks, apart from the principal
peak, is not greater than 2.5 times the area of the principal peak in the chromatogram obtained with
reference solution (a) (0.5 per cent). Disregard any peak due to the solvent and any peak with an area
(a).
Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g in vacuo at 80C for
3 h.
ASSAY
Dissolve 0.200 g in 50 ml of dimethylformamide R. Titrate with 0.1M lithium methoxide, determining
1 ml of 0.1M lithium methoxide is equivalent to 22.33 mg of C
12
H
17
NO
3
.
STORAGE
IMPURITIES
O
R
n
BuO
A. R = OH: 2-(4-butoxyphenyl)acetic acid,
B. R = OCH
3
: methyl 2-(4-butoxyphenyl)acetate,
C. R = OC
4
H
9
: butyl 2-(4-butoxyphenyl)acetate,
D. R = NH
2
: 2-(4-butoxyphenyl)acetamide.
_________________________________________________________________________________________________________________________________________ Ph Eur
6-16
Buflomedil Hydrochloride
corrected 1/01
,HCl
N
OMe
MeO OMe
O
C
17
H
26
ClNO
4
343.9 35543-24-9
Buflomedil Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ________________________________________________________________________________________________________________
DEFINITION
Buflomedil hydrochloride contains not less than 98.5 per cent and not more than the equivalent of
101.5 per cent of 4-(pyrrolidin-1-yl)-1-(2,4,6-trimethoxyphenyl)butan-1-one hydrochloride,
CHARACTERS
A white or almost white, microcrystalline powder, freely soluble in water, soluble in alcohol, very
slightly soluble in acetone.
IDENTIFICATION
A. Dissolve 25.0 mg in alcohol R and dilute to 50 ml with the same solvent. Dilute 1.0 ml to 25.0 ml
with alcohol R. Examined between 220 nm and 350 nm (2.2.25), the solution shows an absorption
obtained with buflomedil hydrochloride CRS. Examine the substances prepared as discs.
254
plate R.
the same solvent.
Reference solution. Dissolve 40 mg of buflomedil hydrochloride CRS in methanol R and dilute to 2 ml
of triethylamine R, 50 volumes of 2-propanol R and 50 volumes of toluene R. Allow the plate to dry in
air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with
the test solution is similar in position and size to the principal spot in the chromatogram obtained
TESTS
solution B
6
(Method II, 2.2.2).
Reference solution (b). Dissolve 2 mg of buflomedil impurity B CRS in the mobile phase. Add 0.5 ml of
the test solution and dilute to 100 ml with the mobile phase.
6-17
a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with end-capped
as mobile phase at a flow rate of 1 ml/min a mixture of 45 volumes of acetonitrile R and 55
volumes of a 9.25 g/l solution of potassium dihydrogen phosphate R adjusted to pH 2.5 with
concentrated phosphoric acid R,
principal peaks in the chromatogram obtained are at least 50 per cent of the full scale of the recorder.
The test is not valid unless in the chromatogram obtained, the resolution between the peaks corres-
ponding to buflomedil and impurity B is at least 5.0. Inject 10 l of the test solution and 10 l of
reference solution (a). Continue the chromatography of the test solution for twice the retention time
of buflomedil which is about 5 min. In the chromatogram obtained with the test solution: the area of
any peak apart from the principal peak, is not greater than the area of the principal peak in the
chromatogram obtained with reference solution (a) (0.25 per cent); the sum of the area of all the
peaks apart from the principal peak, is not greater than twice the area of the principal peak in the
chromatogram obtained with reference solution (a) (0.5 per cent). Disregard any peak with an area
(a).
100C to 105C for 2 h.
ASSAY
Dissolve 0.300 g in 15 ml of anhydrous acetic acid R and add 35 ml of acetic anhydride R. Titrate with
17
H
26
ClNO
4
.
IMPURITIES
N
O R
1
R
2
MeO
A. R
1
= OH, R
2
= OCH
3
: 4-(pyrrolidin-1-yl)-1-(2-hydroxy-4,6-dimethoxyphenyl)butan-1-one,
B. R
1
= OCH
3
, R
2
= OH: 4-(pyrrolidin-1-yl)-1-(4-hydroxy-2,6-dimethoxyphenyl)butan-1-one,
C. R
1
= OH, R
2
= OH: 4-(pyrrolidin-1-yl)-1-(2,4-dihydroxy-6-methoxyphenyl)butan-1-one.
_______________________________________________________________________________________________________________ Ph Eur
6-18
Bumetanide
S
NH
2
Bu
n
HN
O O
COOH
O
C
17
H
20
N
2
O
5
S 364.4 28395-03-1
Bumetanide complies with the requirements of the 3rd edition of the European Pharmacopoeia [1076]. These
Preparations
Bumetanide Injection
Bumetanide Oral Solution
Bumetanide Tablets
Bumetanide and Slow Potassium Tablets
Ph Eur _________________________________________________________________________________________________________________________________________
DEFINITION
Bumetanide contains not less than 99.0 per cent and not more than the equivalent of 101.5 per cent
of 3-butylamino-4-phenoxy-5-sulphamoylbenzoic acid, calculated with reference to the dried
substance.
CHARACTERS
A white, crystalline powder, practically insoluble in water, soluble in acetone and in alcohol, slightly
soluble in ether and in methylene chloride. It dissolves in dilute solutions of alkali hydroxides. It
shows polymorphism.
IDENTIFICATION
A. Dissolve 0.100 g in a 4 g/l solution of sodium hydroxide R and dilute to 100.0 ml with the same
alkaline solution. Dilute 1.0 ml of the solution to 100.0 ml with a 4 g/l solution of sodium hydroxide R.
Examined between 210 nm and 350 nm (2.2.25), the solution shows two absorption maxima, at
224 nm and 320 nm, and a shoulder at 255 nm to 265 nm. The specific absorbance at the maximum
at 224 nm is 720 to 760.
B. Examine by infrared absorption spectrophotometry (2.2.24), comparing the spectrum obtained
with bumetanide CRS. If the spectra in the solid state obtained show differences, dissolve the
substance to be examined and the reference substance separately in acetone R, evaporate to dryness
and record new spectra using the residues.
D. To about 20 mg add 0.1 g of anhydrous sodium carbonate R. Add 0.1 ml of water R and heat.
Ammonia vapour is evolved, which turns red litmus paper R blue.
TESTS
Appearance of solution Dissolve 0.1 g in a 6 g/l solution of potassium hydroxide R and dilute to
20 ml with the same solution. The solution is clear (2.2.1) and colourless (Method II, 2.2.2).
Related substances Prepare the solutions immediately before use. Examine by thin-layer chromato-
graphy (2.2.27), using silica gel GF
254
6-19
Reference solution (a). Dissolve 25 mg of bumetanide CRS in methanol R and dilute to 10 ml with the
same solvent.
Reference solution (b). Dilute 1 ml of test solution (b) to 50 ml with methanol R.
Reference solution (c). Dilute 5 ml of reference solution (b) to 10 ml with methanol R.
2.5 volumes of methanol R, 10 volumes of glacial acetic acid R, 10 volumes of cyclohexane R and 80
volumes of methylene chloride R. Allow the plate to dry in air and examine in ultraviolet light at
254 nm. In the chromatogram obtained with test solution (a): any spot, apart from the principal spot,
is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.2 per
cent) and at most three such spots are more intense than the spot in the chromatogram obtained with
reference solution (c) (0.1 per cent).
ASSAY
Dissolve 0.300 g in 50 ml of alcohol R. Add 0.1 ml of phenol red solution R. Titrate with 0.1M sodium
hydroxide until a violet-red colour is obtained. Carry out a blank titration.
17
H
20
N
2
O
5
S.
STORAGE
IMPURITIES
A. 3-nitro-4-phenoxy-5-sulphamoylbenzoic acid,
B. 3-amino-4-phenoxy-5-sulphamoylbenzoic acid,
C. butyl 3-(butylamino)-4-phenoxy-5-sulphamoylbenzoate,
D. 3-(2-ethylhexylamino)-4-phenoxy-5-sulphamoylbenzoic acid.
_________________________________________________________________________________________________________________________________________ Ph Eur
6-20
Bupivacaine Hydrochloride
N
N
Me
Me
O
Bu
n
H
H
,HCl
and enantiomer
C
18
H
29
ClN
2
O,H
2
O 342.9 14252-80-3
Bupivacaine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparations
Bupivacaine Injection
Bupivacaine and Adrenaline Injection/Bupivacaine and Epinephrine Injection
Ph Eur _________________________________________________________________________________________________________________________________________
DEFINITION
Bupivacaine hydrochloride contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of (RS)-1-butyl-2,6-dimethylpiperidine-2-carboxanilide hydrochloride, calculated
CHARACTERS
A white, crystalline powder or colourless crystals, soluble in water, freely soluble in alcohol.
IDENTIFICATION
obtained with bupivacaine hydrochloride CRS. Examine the substances as mulls in liquid paraffin R.
C. Dissolve 0.1 g in 10 ml of water R, add 2 ml of dilute sodium hydroxide solution R and shake with
two quantities, each of 15 ml, of ether R. Dry the combined ether layers over anhydrous sodium
sulphate R and filter. Evaporate the ether, recrystallise the residue from alcohol (90 per cent V/V) R and
dry under reduced pressure. The crystals melt (2.2.14) at 105C to 108C.
TESTS
Acidity or alkalinity To 10 ml of solution S add 0.2 ml of 0.01M sodium hydroxide; the pH (2.2.3) is
not less than 4.7. Add 0.4 ml of 0.01M hydrochloric acid; the pH is not greater than 4.7.
coating substance.
Reference solution (a). Dissolve 25 mg of bupivacaine hydrochloride CRS in methanol R and dilute to
Reference solution (b). Dilute 0.5 ml of test solution (a) to 100 ml with methanol R.
0.1 volumes of concentrated ammonia R and 100 volumes of methanol R. Dry the plate in a current of
air and spray with dilute potassium iodobismuthate solution R. Any spot in the chromatogram obtained
togram obtained with reference solution (b) (0.5 per cent).
6-21
2,6-Dimethylaniline Dissolve 0.50 g in methanol R and dilute to 10 ml with the same solvent. To
2 ml of the solution add 1 ml of a freshly prepared 10 g/l solution of dimethylaminobenzaldehyde R in
methanol R and 2 ml of glacial acetic acid R and allow to stand for 10 min. Any yellow colour in the
solution is not more intense than that in a standard prepared at the same time and in the same
manner using 2 ml of a 5 mg/l solution of 2,6-dimethylaniline R in methanol R (100 ppm).
Heavy metals (2.4.8). Dissolve 2.0 g in a mixture of 15 volumes of water R and 85 volumes of
methanol R and dilute to 20 ml with the same mixture of solvents. 12 ml of the solution complies with
limit test B for heavy metals (10 ppm). Prepare the standard using lead standard solution (1 ppm Pb)
obtained by diluting lead standard solution (100 ppm Pb) R with a mixture of 15 volumes of water R
100C to 105C.
ASSAY
Dissolve 0.250 g in a mixture of 20 ml of water R and 25 ml of alcohol R. Add 5.0 ml of 0.01M hydro-
chloric acid. Carry out a potentiometric titration (2.2.20), using 0.1M ethanolic sodium hydroxide. Read
the volume added between the two points of inflexion.
18
H
29
ClN
2
O.
STORAGE
_________________________________________________________________________________________________________________________________________ Ph Eur
6-22
Buprenorphine
CH
2
H
HO
N
O
MeO
Me
HO
Bu
t
C
29
H
41
NO
4
467.6 52485-79-7
Buprenorphine complies with the requirements of the 3rd edition of the European Pharmacopoeia [1180].
Ph Eur _________________________________________________________________________________________________________________________________________
DEFINITION
Buprenorphine contains not less than 98.5 per cent and not more than the equivalent of 101.0 per
cent of (2S)-2-[17-(cyclopropylmethyl)-4,5-epoxy-3-hydroxy-6-methoxy-6,14-ethano-14-
morphinan-7-yl]-3,3-dimethylbutan-2-ol, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, very slightly soluble in water, freely soluble in acetone,
soluble in methanol, slightly soluble in cyclohexane. It dissolves in dilute solutions of acids.
IDENTIFICATION
Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph. Eur. reference
spectrum of buprenorphine.
TESTS
Solution S. Dissolve 0.250 g in ethanol R and dilute to 25.0 ml with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and colourless (Method II, 2.2.2).
Related substances. Examine by liquid chromatography (2.2.29).
Reference solution (a). Dissolve 5 mg of the substance to be examined in 2.0 ml of methanol R. Add
0.25 ml of 2M hydrochloric acid.
Reference solution (d). Dilute 4.0 ml of reference solution (b) to 10.0 ml with the mobile phase.
as mobile phase at a flow rate of about 1 ml per minute, a mixture of 10 volumes of a 10 g/l
solution of ammonium acetate R and 60 volumes of methanol R,
Inject 20 l of reference solution (a). Adjust the flow rate so that the retention time of the peak
corresponding to buprenorphine is about 15 min. The test is not valid unless the chromatogram
obtained with reference solution (a) presents two peaks, the first having a retention time of 0.93
relative to the second peak (buprenorphine). Inject separately 20 l of each solution. Record the
chromatogram of the test solution for 2.5 times the retention time of the main peak. In the chromato-
6-23
gram obtained with the test solution: the area of any peak, apart from the principal peak, is not
greater than the area of the principal peak in the chromatogram obtained with reference solution (b)
(0.25 per cent); and the sum of the areas of such peaks is not greater than the area of the peak in the
chromatogram obtained with reference solution (c) (0.65 per cent). Disregard any peak with an area
less than that of the principal peak in the chromatogram obtained with reference solution (d).
100C to 105C.
ASSAY
Dissolve 0.400 g in 40 ml of anhydrous acetic acid R. Titrate with 0.1M perchloric acid until the colour
changes from violet-blue to green, using 0.1 ml of crystal violet solution R as indicator.
29
H
41
NO
4
.
STORAGE
IMPURITIES
H
R
1
O
NR
2
O
MeO
HO Bu
t
Me
A. R
1
= H, R
2
= CH
2
-CH
2
-CH=CH
2
:
(2S)-2-[17-(but-3-enyl)-4,5-epoxy-3-hydroxy-6-methoxy-6,14-ethano-14-morphinan-
7-yl]-3,3-dimethylbutan-2-ol,
B. R
1
= H, R
2
= H:
(2S)-2-(4,5-epoxy-3-hydroxy-6-methoxy-6,14-ethano-14-morphinan-7-yl)-3,3-
dimethylbutan-2-ol,
C. R
1
= CH
3,
R
2
= CN:
4,5-epoxy-7-[(1S)-1-hydroxy-1,2,2-trimethylpropyl]-3,6-dimethoxy-6,14-ethano-14-
morphinan-17-carbonitrile.
_________________________________________________________________________________________________________________________________________ Ph Eur
6-24
Buprenorphine Hydrochloride
CH
2
H
HO
N
O
MeO
Me
HO
Bu
t
,HCl
C
29
H
41
NO
4
,HCl 504.1 53152-21-9
Buprenorphine Hydrochloride complies with the requirements of the 3rd edition of the European
Ph Eur _________________________________________________________________________________________________________________________________________
DEFINITION
Buprenorphine hydrochloride contains not less than 98.5 per cent and not more than the equivalent
of 101.0 per cent of (2S)-2-[17-(cyclopropylmethyl)-4,5-epoxy-3-hydroxy-6-methoxy-6,14-
ethano-14-morphinan-7-yl]-3,3-dimethylbutan-2-ol hydrochloride, calculated with reference to
CHARACTERS
A white or almost white, crystalline powder, sparingly soluble in water, freely soluble in methanol,
soluble in alcohol, practically insoluble in cyclohexane.
IDENTIFICATION
spectrum of buprenorphine hydrochloride.
B. 3 ml of solution S (see Tests) gives reaction (a) of chlorides (2.3.1).
TESTS
Solution S Dissolve 0.250 g in 5.0 ml of methanol R and while stirring dilute to 25.0 ml with carbon
dioxide-free water R.
Acidity or alkalinity To 10.0 ml of solution S add 0.05 ml of methyl red solution R. Not more than
0.2 ml of 0.02M sodium hydroxide or 0.02M hydrochloric acid is required to change the colour of the
indicator.
same solvent. The specific optical rotation is 92 to 98, calculated with reference to the dried
substance.
Reference solution (a). Dissolve 5 mg of the substance to be examined in 2.0 ml of methanol R. Add
0.25 ml of 2M hydrochloric acid.
Reference solution (d). Dilute 4.0 ml of reference solution (b) to 10.0 ml with the mobile phase.
as the mobile phase at a flow rate of about 1 ml per minute, a mixture of 10 volumes of a 10 g/l
solution of ammonium acetate R and 60 volumes of methanol R,
6-25
Inject 20 l of reference solution (a). Adjust the flow rate so that the retention time of the peak
corresponding to buprenorphine is about 15 min. The test is not valid unless the chromatogram
obtained with reference solution (a) presents two peaks, the first having a retention time of 0.93
relative to the second peak (buprenorphine). Inject separately 20 l of each solution. Record the
chromatogram of the test solution for 2.5 times the retention time of the main peak. In the chromato-
gram obtained with the test solution: the area of any peak, apart from the principal peak, is not
(0.25 per cent); and the sum of the areas of such peaks is not greater than the area of the peak in the
less than that of the principal peak in the chromatogram obtained with reference solution (d).
Loss on drying (2.2.32). Not more than 1.0 per cent, determined on 1.000 g by heating in an oven
at 115C to 120C.
ASSAY
29
H
42
CINO
4
.
STORAGE
IMPURITIES
H
R
1
O
NR
2
O
MeO
HO Bu
t
Me
A. R
1
= H, R
2
= CH
2
-CH
2
-CH=CH
2
:
(2S)-2-[17-(but-3-enyl)-4,5-epoxy-3-hydroxy-6-methoxy-6,14-ethano-14-morphinan-
7-yl]-3,3-dimethylbutan-2-ol,
B. R
1
= H, R
2
= H:
(2S)-2-(4,5-epoxy-3-hydroxy-6-methoxy-6,14-ethano-14-morphinan-7-yl)-3,3-
dimethylbutan-2-ol,
C. R
1
= CH
3,
R
2
= CN:
4,5-epoxy-7-[(1S)-1-hydroxy-1,2,2-trimethylpropyl]-3,6-dimethoxy-6,14-ethano-14-
morphinan-17-carbonitrile.
_________________________________________________________________________________________________________________________________________ Ph Eur
6-26
Buserelin
1/01
5oxoProHisTrpSerTyrDSer(tertBu)
LeuArgProNHEt
C
60
H
86
N
16
O
13
1239.5 68630-75-1
Buserelin complies with the requirements of the 3rd edition of the European Pharmacopoeia [1077]. These
Action and use Gonadorelin analogue.
Ph Eur _________________________________________________________________________________________________________________________________________
DEFINITION
Buserelin is a synthetic nonapeptide analogue of human gonadotrophin-releasing hormone GnRH
with agonistic activity to gonadorelin. It is obtained by chemical synthesis and is available as an
acetate form. It contains not less than 95.0 per cent and not more than the equivalent of 102.0 per
cent of the peptide C
60
H
86
N
16
O
13
, calculated with reference to the anhydrous, acetic acid-free
substance.
CHARACTERS
A white or slightly yellowish powder, hygroscopic, sparingly soluble in water and in dilute acids.
IDENTIFICATION
A. Examine the chromatograms obtained in the assay. The retention time of the principal peak in the
chromatogram obtained with the test solution is approximately the same as that of the principal peak
in the chromatogram obtained with reference solution (b).
B. The
1
H nuclear magnetic resonance spectrum (2.2.33) of a 4 mg/ml solution in a mixture of 20
volumes of deuterated acetic acid R and 80 volumes of deuterium oxide R is qualitatively similar to the
Ph. Eur. reference spectrum of buserelin.
TESTS
Appearance of solution A 10 g/l solution is clear (2.2.1) and not more intensely coloured than
reference solution Y
7
(Method II, 2.2.2).
Specific optical rotation (2.2.7). The specific optical rotation of a 10 g/l solution is 49 to 58,
calculated with reference to the anhydrous, acetic acid-free substance.
Absorbance (2.2.25). Dissolve 10.0 mg in 100.0 ml of 0.01M hydrochloric acid. The specific
absorbance at the maximum at 278 nm is 49 to 56, calculated with reference to the anhydrous, acetic
acid-free substance.
Amino acids Examine by means of an amino-acid analyser. Standardise the apparatus with a
mixture containing equimolar amounts of ammonia, glycine and the L-form of the following amino
acids:
Lysine Threonine Alanine Leucine
Histidine Serine Valine Tyrosine
Arginine Glutamic acid Methionine Phenylalanine
Aspartic acid Proline Isoleucine
together with half the equimolar amount of L-cystine. For the validation of the method, an
appropriate internal standard, such as DL-norleucine R, is used.
Test solution. Place 1.0 mg of the substance to be examined in a rigorously cleaned hard-glass tube
100 mm long and 6 mm in internal diameter. Add a suitable amount of a 50 per cent V/V solution of
hydrochloric acid R. Immerse the tube in a freezing mixture at 5C, reduce the pressure to below
133 Pa and seal. Heat at 110C to 115C for 16 h. Cool, open the tube, transfer the contents to a
10 ml flask with the aid of five quantities, each of 0.2 ml, of water R and evaporate to dryness over
potassium hydroxide R under reduced pressure. Take up the residue in water R and evaporate to
dryness over potassium hydroxide R under reduced pressure; repeat these operations once. Take up the
residue in a buffer solution suitable for the amino-acid analyser used and dilute to a suitable volume
with the same buffer solution. Apply a suitable volume to the amino-acid analyser.
Express the content of each amino acid in moles. Calculate the relative proportions of the amino
acids, taking one-sixth of the sum of the number of moles of glutamic acid, histidine, tyrosine,
leucine, arginine, proline as equal to one. The values fall within the following limits: serine 1.4 to 2.0;
proline 0.8 to 1.2; glutamic acid 0.9 to 1.1; leucine 0.9 to 1.1; tyrosine 0.9 to 1.1; histidine 0.9 to 1.1
6-27
and arginine 0.9 to 1.1. Not more than traces of other amino acids are present, with the exception of
tryptophan.
Related peptides Examine by liquid chromatography (2.2.29) as described under Assay.
Inject 10 l of reference solution (c). Record the chromatogram for about twice the retention time
of the principal peak. Adjust the sensitivity of the system so that the height of the principal peak in
the chromatogram obtained with reference solution (c) is 50 per cent to 70 per cent of the full scale
of the recorder. Inject 10 l of the test solution. In the chromatogram obtained with the test solution:
the area of any peak, apart from the principal peak, is not greater than 3 times the area of the
principal peak in the chromatogram obtained with reference solution (c) (3 per cent); the sum of the
areas of such peaks is not greater than 5 times the area of the principal peak in the chromatogram
obtained with reference solution (c) (5 per cent). Disregard any peak due to the solvent and any peak
with an area less than 0.1 times that of the principal peak in the chromatogram obtained with
reference solution (c) (0.1 per cent).
Acetic acid (2.5.34). 3.0 per cent to 7.0 per cent.
Test solution. Dissolve 20.0 mg of the substance to be examined in a mixture of 5 volumes of mobile
phase B and 95 volumes of mobile phase A and dilute to 10.0 ml with the same mixture of solvents.
Water (2.5.12). Not more than 4.0 per cent, determined on 80.0 mg by the semi-micro deter-
mination of water.
ASSAY
Test solution. Dissolve 5.0 mg of the substance to be examined in 5.0 ml of the mobile phase.
Reference solution (a). Dissolve the contents of a vial of D-His-buserelin CRS in the mobile phase.
Dilute an appropriate volume of this solution in the mobile phase to obtain a final concentration of
1 mg/ml. Add 1.0 ml of the test solution to 1.0 ml of this solution.
Reference solution (b). Dissolve the contents of a vial of buserelin CRS in the mobile phase. Dilute an
appropriate volume of this solution in the mobile phase to obtain a final concentration of 1.0 mg/ml.
as mobile phase at a flow rate of 0.8 ml/min a mixture of 200 ml of acetonitrile R and 700 ml of
an 11.2 g/l solution of phosphoric acid R; adjust the mixture to pH 2.5 with triethylamine R,
Inject 10 l of reference solution (a). The test is not valid unless the resolution between the peaks
corresponding to D-His-buserelin and buserelin is at least 1.5.
Inject 10 l of the test solution and 10 l of reference solution (b).
Calculate the content of buserelin (C
60
H
86
N
16
O
13
) from the areas of the peaks in the chromato-
grams obtained with the test solution and reference solution (b) and the stated content of
C
60
H
86
N
16
O
13
in buserelin CRS.
STORAGE
If the substance is sterile, store in an airtight, sterile, tamper-proof container.
LABELLING
The label states:
the mass of peptide in the container,
where applicable, that the substance is sterile.
_________________________________________________________________________________________________________________________________________ Ph Eur
6-28
Busulfan
H
3
C
S
O
[CH
2
]
4
O O
O
S
CH
3
O O
C
6
H
14
O
6
S
2
246.3 55-98-1
Busulfan complies with the requirements of the 3rd edition of the European Pharmacopoeia [0542]. These
Action and use Cytotoxic.
Preparation
Busulfan Tablets
When busulphan is prescribed or demanded, Busulfan shall be dispensed or supplied.
Ph Eur _________________________________________________________________________________________________________________________________________
DEFINITION
Busulfan contains not less than 99.0 per cent and not more than the equivalent of 100.5 per cent of
tetramethylene di(methanesulphonate), calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, very slightly soluble in water, freely soluble in acetone
and in acetonitrile, very slightly soluble in alcohol and in ether.
IDENTIFICATION
obtained with busulfan CRS.
B. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance.
Test solution. Dissolve 20 mg of the substance to be examined in 2 ml of acetone R.
Reference solution. Dissolve 20 mg of busulfan CRS in 2 ml of acetone R.
equal volumes of acetone R and toluene R. Dry the plate in a stream of hot air and spray with
anisaldehyde solution R. Heat the plate at 120C. The principal spot in the chromatogram obtained
C. To 0.1 g add 5 ml of 1M sodium hydroxide. Heat until a clear solution is obtained. Allow to cool.
To 2 ml of the solution add 0.1 ml of potassium permanganate solution R. The colour changes from
purple through violet to blue and finally to green. Filter and add 1 ml of ammoniacal silver nitrate
solution R. A precipitate is formed.
D. To 0.1 g add 0.1 g of potassium nitrate R and 0.25 g of sodium hydroxide R, mix and heat to fusion.
Allow to cool and dissolve the residue in 5 ml of water R. Adjust to pH 1 to 2 using dilute hydrochloric
acid R. The solution gives reaction (a) of sulphates (2.3.1).
TESTS
Appearance of solution Dissolve 0.25 g in 20 ml of acetonitrile R, dilute to 25 ml with water R and
examine immediately. The solution is clear (2.2.1) and not more intensely coloured than reference
solution B
7
(Method II, 2.2.2).
Acidity Dissolve 0.20 g with heating in 50 ml of ethanol R. Add 0.1 ml of methyl red solution R. Not
more than 0.05 ml of 0.1M sodium hydroxide is required to change the colour of the indicator.
60C.
ASSAY
To 0.250 g add 50 ml of water R. Shake. Boil under a reflux condenser for 30 min and, if necessary,
make up to the initial volume with water R. Allow to cool. Using 0.3 ml of phenolphthalein solution R
as indicator, titrate with 0.1M sodium hydroxide until a pink colour is obtained.
6-29
6
H
14
O
6
S
2
.
STORAGE
_________________________________________________________________________________________________________________________________________ Ph Eur
6-30
Butyl Hydroxybenzoate
HO
COOBu
n
C
11
H
14
O
3
194.2 94-26-8
Butyl Hydroxybenzoate complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Butyl Parahydroxybenzoate [0881]. These requirements are reproduced after the heading Definition below.
Ph Eur ________________________________________________________________________________________________________________
DEFINITION
Butyl parahydroxybenzoate contains not less than 99.0 per cent and not more than the equivalent of
100.5 per cent of butyl 4-hydroxybenzoate.
CHARACTERS
A white or almost white, crystalline powder or colourless crystals, very slightly soluble in water, freely
soluble in alcohol and in methanol.
IDENTIFICATION
obtained with butyl parahydroxybenzoate CRS.
C. Examine the chromatograms obtained in the test for related substances. The principal spot in the
the chromatogram obtained with reference solution (b).
D. To about 10 mg in a test-tube add 1 ml of sodium carbonate solution R, heat to boiling for 30 s and
cool (solution a). To about 10 mg in a similar test-tube add 1 ml of sodium carbonate solution R; the
substance partly dissolves (solution b). Add at the same time to solutions (a) and (b) 5 ml of
aminopyrazolone solution R and 1 ml of potassium ferricyanide solution R and mix. Solution (b) is yellow
to orange-brown. Solution (a) is orange to red, the colour being clearly more intense than any similar
colour that may be obtained with solution (b).
TESTS
Solution S. Dissolve 1.0 g in alcohol R and dilute to 10 ml with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference
solution BY
6
(Method II, 2.2.2).
Acidity. To 2 ml of solution S add 3 ml of alcohol R, 5 ml of carbon dioxide-free water R and 0.1 ml of
bromocresol green solution R. Not more than 0.1 ml of 0.1M sodium hydroxide is required to change the
a suitable octadecylsilyl silica gel with a fluorescent indicator having an optimal intensity at 254 nm.
Reference solution (a). Dilute 0.5 ml of test solution (a) to 100 ml with acetone R.
Reference solution (b). Dissolve 10 mg of butyl parahydroxybenzoate CRS in acetone R and dilute to
Reference solution (c). Dissolve 10 mg of propyl parahydroxybenzoate R in 1 ml of test solution (a) and
dilute to 10 ml with acetone R.
Apply to the plate 2 l of each solution. Develop over a path of 15 cm using a mixture of 1 volume of
glacial acetic acid R, 30 volumes of water R and 70 volumes of methanol R. Allow the plate to dry in air
and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with test solution
with reference solution (a) (0.5 per cent). The test is not valid unless the chromatogram obtained
6-31
ASSAY
Place 2.000 g in a ground-glass-stoppered flask and add 40.0 ml of 1M sodium hydroxide. Boil under a
reflux condenser for 1 h. Allow to cool and rinse the condenser with water R. Titrate the excess
sodium hydroxide with 0.5M sulphuric acid, continuing the titration until the second point of inflexion
and determining the end-point potentiometrically (2.2.20). Carry out a blank titration.
11
H
14
O
3
.
IMPURITIES
COOR
HO
A. R = H: 4-hydroxybenzoic acid,
B. R = CH
3
: methyl 4-hydroxybenzoate,
C. R = CH
2
-CH
3
: ethyl 4-hydroxybenzoate,
D. R = CH
2
-CH
2
-CH
3
: propyl 4-hydroxybenzoate.
_______________________________________________________________________________________________________________ Ph Eur
6-32
Butylated Hydroxyanisole
MeO
OH
Bu
t
C
11
H
16
O
2
180.3 25013-16-5
Butylated Hydroxyanisole complies with the requirements of the 3rd edition of the European Pharmacopoeia
for Butylhydroxyanisole [0880]. These requirements are reproduced after the heading Definition below.
Action and use Antioxidant.
Ph Eur _________________________________________________________________________________________________________________________________________
DEFINITION
Butylhydroxyanisole is 2-(1,1-dimethylethyl)-4-methoxyphenol containing not more than 10 per cent
of 3-(1,1-dimethylethyl)-4-methoxyphenol.
CHARACTERS
A white, yellowish or slightly pinkish, crystalline powder, practically insoluble in water, very soluble
in methylene chloride, freely soluble in alcohol and in fatty oils. It dissolves in dilute solutions of
alkali hydroxides.
IDENTIFICATION
A. Examine the chromatograms obtained in the test for related substances. The principal spot in the
B. To 0.5 ml of solution S (see Tests) add 10 ml of aminopyrazolone solution R and 1 ml of potassium
ferricyanide solution R. Mix and add 10 ml of methylene chloride R. Shake vigorously. After separation,
the organic layer is red.
C. Dissolve about 10 mg in 2 ml of alcohol R. Add 1 ml of a 1.0 g/l solution of testosterone propionate R
in alcohol R and 2 ml of dilute sodium hydroxide solution R. Heat in a water-bath at 80C for 10 min
and allow to cool. A red colour develops.
TESTS
Appearance of solution Solution S is clear (2.2.1) and not more intensely coloured than intensity 5
coating substance.
Reference solution (a). Dissolve 25 mg of butylhydroxyanisole CRS in methylene chloride R and dilute to
Reference solution (b). Dilute 1 ml of reference solution (a) to 20 ml with methylene chloride R.
Reference solution (c). Dissolve 50 mg of hydroquinone R in 5 ml of alcohol R and dilute to 100 ml with
methylene chloride R. Dilute 1 ml of this solution to 10 ml with methylene chloride R.
Apply separately to the plate 5 l of each solution. Develop over a path of 10 cm using methylene
chloride R. Allow the plate to dry in air and spray with a freshly prepared mixture of 10 volumes of
potassium ferricyanide solution R, 20 volumes of ferric chloride solution R1 and 70 volumes of water R. In
the chromatogram obtained with test solution (a): any violet-blue spot with an R
f
value of about 0.35
(corresponding to 3-(1,1-dimethylethyl)-4-methoxyphenol) is not more intense than the principal
spot in the chromatogram obtained with reference solution (a) (10 per cent); any spot corresponding
to hydroquinone is not more intense than the principal spot in the chromatogram obtained with
reference solution (c) (0.2 per cent); any spot, apart from the principal spot and any spots corres-
ponding to 3-(1,1-dimethylethyl)-4-methoxyphenol and hydroquinone, is not more intense than the
principal spot in the chromatogram obtained with reference solution (b) (0.5 per cent).
6-33
STORAGE
IMPURITIES
A. Hydroquinone.
_________________________________________________________________________________________________________________________________________ Ph Eur
6-34
Butylated Hydroxytoluene
OH
Bu
t
Bu
t
Me
C
15
H
24
O 220.4 128-37-0
Butylated Hydroxytoluene complies with the requirements of the 3rd edition of the European Pharmacopoeia
for Butylhydroxytoluene [0581]. These requirements are reproduced after the heading Definition below.
Ph Eur _________________________________________________________________________________________________________________________________________
DEFINITION
Butylhydroxytoluene is 2,6-di(1,1-dimethylethyl)-4-methylphenol.
CHARACTERS
A white or yellowish-white, crystalline powder, practically insoluble in water, very soluble in acetone
and in ether, freely soluble in alcohol and in vegetable oils.
IDENTIFICATION
A. It complies with the test for the freezing-point (see Tests).
B. Dissolve 0.500 g in ethanol R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of this
solution to 100.0 ml with ethanol R. Examined between 230 nm and 300 nm (2.2.25), the solution
obtained with butylhydroxytoluene CRS.
D. Dissolve about 10 mg in 2 ml of alcohol R. Add 1 ml of a 1 g/l solution of testosterone propionate R
in alcohol R and 2 ml of dilute sodium hydroxide solution R. Heat in a water-bath at 80C for 10 min
and allow to cool. A blue colour develops.
TESTS
The solution is clear (2.2.1) and not more intensely coloured than reference solution Y
5
or BY
5
(Method II, 2.2.2).
Freezing-point (2.2.18). 69C to 70C.
coating substance.
the same solvent.
Apply separately to the plate 10 l of each solution. Develop over a path of 15 cm using methylene
chloride R. Dry the plate in air and spray with a freshly prepared mixture of 10 volumes of potassium
ferricyanide solution R, 20 volumes of ferric chloride solution R1 and 70 volumes of water R. Any spot in
_________________________________________________________________________________________________________________________________________ Ph Eur
6-35
Caffeine
Anhydrous Caffeine
N
N
N
O
O
Me
MeN
Me
C
8
H
10
N
4
O
2
194.2 56-08-2
Caffeine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0267]. These
Action and use Central nervous stimulant.
Preparation
Aspirin and Caffeine Tablets
Ph Eur _________________________________________________________________________________________________________________________________________
DEFINITION
Caffeine contains not less than 98.5 per cent and not more than the equivalent of 101.5 per cent of
1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6-dione, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder or silky, white crystals, sublimes readily, sparingly soluble in water, freely
soluble in boiling water, slightly soluble in ethanol and in ether. It dissolves in concentrated solutions
of alkali benzoates or salicylates.
IDENTIFICATION
obtained with caffeine CRS.
C. To 2 ml of a saturated solution add 0.05 ml of iodinated potassium iodide solution R. The solution
remains clear. Add 0.1 ml of dilute hydrochloric acid R. A brown precipitate is formed. Neutralise with
dilute sodium hydroxide solution R. The precipitate dissolves.
D. In a glass-stoppered tube, dissolve about 10 mg in 0.25 ml of a mixture of 0.5 ml of
acetylacetone R and 5 ml of dilute sodium hydroxide solution R. Heat in a water-bath at 80C for 7 min.
Cool and add 0.5 ml of dimethylaminobenzaldehyde solution R2. Heat again in a water-bath at 80C for
7 min. Allow to cool and add 10 ml of water R. An intense blue colour develops.
E. It complies with the test for loss on drying (see Tests).
F. It gives the reaction of xanthines (2.3.1).
TESTS
Solution S Dissolve 0.5 g with heating in 50 ml of carbon dioxide-free water R prepared from distilled
water R, cool and dilute to 50 ml with the same solvent.
Acidity To 10 ml of solution S add 0.05 ml of bromothymol blue solution R1. The solution is green or
yellow. Not more than 0.2 ml of 0.01M sodium hydroxide is required to change the colour of the
indicator to blue.
Related substances Examine by thin-layer chromatography (2.2.27) using silica gel GF
254
R as the
coating substance.
Test solution. Dissolve 0.2 g of the substance to be examined in a mixture of 4 volumes of methanol R
and 6 volumes of chloroform R and dilute to 10 ml with the same mixture of solvents.
Reference solution. Dilute 0.5 ml of the test solution to 100 ml with a mixture of 4 volumes of
methanol R and 6 volumes of chloroform R.
10 volumes of concentrated ammonia R, 30 volumes of acetone R, 30 volumes of chloroform R and 40
6-36
volumes of butanol R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any
Sulphates (2.4.13). 15 ml of solution S complies with the limit test for sulphates (500 ppm). Prepare
the standard using a mixture of 7.5 ml of sulphate standard solution (10 ppm SO
4
) R and 7.5 ml of
distilled water R.
100C to 105C for 1 h.
ASSAY
Dissolve 0.170 g with heating in 5 ml of anhydrous acetic acid R. Allow to cool, add 10 ml of acetic
anhydride R and 20 ml of toluene R. Titrate with 0.1M perchloric acid determining the end-point
8
H
10
N
4
O
2
.
_________________________________________________________________________________________________________________________________________ Ph Eur
6-37
Caffeine Hydrate
C
8
H
10
N
4
O
2
,H
2
O 212.2 5743-12-4
Caffeine Hydrate complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Caffeine Monohydrate [0268]. These requirements are reproduced after the heading Definition below.
Action and use Central nervous stimulant.
Ph Eur _________________________________________________________________________________________________________________________________________
DEFINITION
Caffeine monohydrate contains not less than 98.5 per cent and not more than the equivalent of
101.5 per cent of 1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6-dione, calculated with reference to the
dried substance.
CHARACTERS
It has the characters described in the monograph on Caffeine (267).
IDENTIFICATION
It complies with the identification tests prescribed in the monograph on Caffeine (267); dry the
substance to be examined at 100C to 105C before carrying out identification tests A and B.
TESTS
It complies with the tests prescribed in the monograph on Caffeine (267) with the following
modification.
100C to 105C for 1 h.
ASSAY
Carry out the assay prescribed in the monograph on Caffeine (267) using the substance to be
examined previously dried at 100C to 105C.
8
H
10
N
4
O
2
.
_________________________________________________________________________________________________________________________________________ Ph Eur
6-38
Calamine
Prepared Calamine
Definition Calamine is a basic zinc carbonate suitably coloured with iron(III) oxide.
Characteristics An amorphous, impalpable, pink or reddish brown powder, the colour depending
on the variety and amount of iron(III) oxide present and the process by which it is incorporated.
Practically insoluble in water. It dissolves with effervescence in hydrochloric acid.
Identification
A. Yields the reactions characteristic of carbonates, Appendix VI.
B. To 2 g add 5 ml of hydrochloric acid and heat to boiling; if necessary, add hydrochloric acid dropwise
until a bright yellow solution is obtained. Cool and add 13.5M ammonia until the first sign of
precipitate (solution A). The solution yields reaction B characteristic of iron salts, Appendix VI.
Dilute 1 ml of solution A to 5 ml with water; the solution yields the reaction characteristic of zinc
salts, Appendix VI.
Calcium Dissolve 0.50 g in a mixture of 10 ml of water and 2.5 ml of glacial acetic acid and filter. To
0.5 ml of the filtrate add 15 ml of 5M ammonia and 2 ml of a 2.5% w/v solution of ammonium oxalate
and allow to stand for 2 minutes. The solution remains clear.
Soluble barium salts To the remainder of the filtrate obtained in the test for Calcium add 2 ml of
1M sulphuric acid and allow to stand for 5 minutes. The solution remains clear.
Lead Not more than 150 ppm when determined by atomic absorption spectrophotometry, Appendix
II D, Method II, measuring at 283.3 nm or 217 nm and using an airacetylene flame. Carefully add
5 g of the substance being examined to 25 ml of hydrochloric acid and allow to stand for 18 hours.
Add 5 ml of nitric acid and sufficient water to produce 200 ml. Use lead standard solution (100 ppm Pb)
suitably diluted with a 3.5% v/v solution of nitric acid to prepare the standard solution.
Chloride Dissolve 0.15 g in water with the addition of 1 ml of nitric acid, filter and dilute to 30 ml
with water. The resulting solution complies with the limit test for chlorides, Appendix VII (0.07%).
Sulphate Dissolve 0.1 g in water with the addition of 3 ml of 2M hydrochloric acid, filter and dilute to
60 ml with water. The resulting solution complies with the limit test for sulphates, Appendix VII
(0.6%).
Ethanol-soluble dyes Shake 1.0 g with 10 ml of ethanol (90%) and filter. The filtrate is colourless,
Matter insoluble in hydrochloric acid Dissolve 1 g in 20 ml of warm 2M hydrochloric acid and
filter. The residue, when washed with water and dried to constant weight at 105, weighs not more
than 10 mg.
Water-soluble dyes Shake 1.0 g with 10 ml of water and filter. The filtrate is colourless, Appendix
IV B, Method II.
Residue on ignition 68.0 to 74.0%, when ignited at a temperature not lower than 900 until, after
further ignition, two successive weighings do not differ by more than 0.2% of the weight of the
residue. Use 1 g.
Action and use Antipruritic.
Preparations
Aqueous Calamine Cream
Calamine Lotion
Calamine Ointment
Calamine and Coal Tar Ointment
6-39
Calcifediol
corrected 1/01
OH Me
Me
H
H
H
CH
2
HO
Me
Me
C
27
H
44
O
2
,H
2
O 418.7 63283-36-3
Calcifediol complies with the requirements of the 3rd edition of the European Pharmacopoeia [1295]. These
Action and use Calcium regulator.
Ph Eur
DEFINITION
Calcifediol is the monohydrate of (5Z,7E)-9,10-secocholesta-5,7,10(19)-triene-3,25-diol. It
contains not less than 97.0 per cent and not more than the equivalent of 102.0 per cent of C
27
H
44
O
2
,
CHARACTERS
A reversible isomerisation to pre-calcifediol takes place in solution, depending on temperature and
IDENTIFICATION
spectrum of calcifediol, prepared using 2 mg of the substance to be examined and 225 mg of potassium
bromide R.
obtained with the test solution is similar in retention time and approximate size to the principal peak
in the chromatogram obtained with reference solution (a).
TESTS
Calculate the percentage content of related substances, apart from pre-calcifediol, that are eluted
within twice the retention time of calcifediol from the areas of the peaks in the chromatogram
obtained with the test solution by the normalisation procedure. The content of any individual related
1.0 per cent. Disregard any peak below 0.1 per cent.
Water (2.5.32). 3.8 per cent to 5.0 per cent, determined on 10.0 mg by the micro determination of
water.
ASSAY
mobile phase.
Reference solution (a). Dissolve 1.0 mg of calcifediol CRS without heating in 10.0 ml of the mobile
phase.
Reference solution (b). Dilute reference solution (a) 100 times with the mobile phase.
Reference solution (c). Heat 2 ml of reference solution (a) in a water-bath at 80C under a reflux
condenser for 2 h and cool.
6-40
a column 0.15 m long and 4.6 mm in internal diameter packed with octylsilyl silica gel for chroma-
tography R1 (5 m),
as mobile phase at a flow rate of 1.5 ml/min a mixture of 200 volumes of water R and 800
volumes of methanol R,
a loop injector.
Inject 50 l of reference solution (c) and record the chromatogram. Make a total of six injections.
When the chromatograms are recorded in the prescribed conditions, the retention time for pre-
calcifediol, relative to calcifediol, is about 1.3. The assay is not valid unless the relative standard
deviation of the response for calcifediol is at most 1 per cent and the resolution between the peaks
due to pre-calcifediol and calcifediol is at least 5.0; adjust the proportions of the constituents of the
mobile phase, if necessary, to obtain this resolution.
Inject 50 l of reference solution (a) and 50 l of reference solution (b) and record the chromato-
grams. Inject 50 l of the test solution and record the chromatogram in the same manner, continuing
the chromatography for twice the retention time of the principal peak.
STORAGE
IMPURITIES
Me
H H
Me
Me
Me
OH Me
HO
H
A. 9,10-cholesta-5,7-diene-3,25-diol,
Me
H H
Me
Me
Me
OH Me
HO
H
B. cholesta-5,7-diene-3,25-diol,
H
Me
Me
Me
OH Me
H
Me
HO
C. (6E)-9,10-secocholesta-5(10),6,8-triene-3,25-diol,
H
Me
Me
Me
OH Me
H
CH
2
OH
D. (5E,7E)-9,10-secocholesta-5,7,10(19)-triene-3,25-diol.
_______________________________________________________________________________________________________________ Ph Eur
6-41
Calcitonin (Salmon) / Salcatonin
1/01
CysSerAsnLeuSerThrCysVal
LeuGlyLysLeuSerGlnGluLeu
HisLysLeuGlnThrTyrProArg
ThrAsnThrGlySerGlyThrProNH
2
C
145
H
240
N
44
O
48
S
2
3432 47931-85-1
Calcitonin (Salmon) complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Hypocalcaemic hormone.
Preparation
Calcitonin (Salmon) Injection/Salcatonin Injection
Ph Eur ________________________________________________________________________________________________________________
DEFINITION
Calcitonin (salmon) is a synthetic polypeptide having the structure determined for salmon calcitonin
I. It lowers the calcium concentration in plasma of mammals by diminishing the rate of bone
resorption. It is obtained by chemical synthesis and is available as an acetate. Calcitonin (salmon)
contains not less than 90.0 per cent and not more than the equivalent of 105.0 per cent of the
peptide C
145
H
240
N
44
O
48
S
2
, calculated with reference to the anhydrous, acetic acid-free substance.
By convention, for the purpose of labelling calcitonin (salmon) preparations, 1 mg of calcitonin
(salmon) (C
145
H
240
N
44
O
48
S
2
) is equivalent to 6000 I.U. of biological activity.
CHARACTERS
A white or almost white powder, freely soluble in water.
IDENTIFICATION
A. Examine by thin-layer chromatography (2.2.27), using cellulose for chromatography R1 as the
coating substance.
Test solution. Dissolve 4 mg of the substance to be examined in 2 ml of a mixture of 2 volumes of
acetic acid R and 98 volumes of water R. Prepare immediately before use.
Reference solution. Dissolve the contents of a vial of calcitonin (salmon) CRS in a mixture of 2 volumes
of acetic acid R and 98 volumes of water R to obtain a concentration of 2.0 mg/ml. Prepare immediately
before use.
of glacial acetic acid R, 20 volumes of pyridine R, 24 volumes of water R and 30 volumes of butanol R.
Allow the plate to dry in air for 1 h, heat at 110C for 10 min and spray the hot plate with strong
sodium hypochlorite solution R diluted with water R immediately before use to contain 5 g/l of available
chlorine. Dry in a current of cold air until a sprayed area of the plate below the line of application
gives at most a very faint blue colour with a drop of potassium iodide and starch solution R; avoid
prolonged exposure to cold air. Spray with potassium iodide and starch solution R until the spots are
clearly visible. The principal spot in the chromatogram obtained with the test solution is similar in
position, colour and size to the principal spot in the chromatogram obtained with the reference
solution.
B. Examine the chromatograms obtained in the Assay. The retention time of the principal peak in the
chromatogram obtained with the test solution is similar to that of the principal peak in the chromato-
gram obtained with the reference solution.
TESTS
Acetic acid (2.5.34). 4.0 per cent to 15.0 per cent.
acids:
6-42
Lysine Histidine Arginine
Serine Methionine Glutamic acid
Isoleucine Proline Leucine
Aspartic acid Alanine Tyrosine
Threonine Valine Phenylalanine
acids taking as equivalent to 1 the sum, divided by 20, of the number of moles of aspartic acid,
glutamic acid, proline, glycine, valine, leucine, histidine, arginine and lysine. The values fall within
the following limits: aspartic acid 1.8 to 2.2; glutamic acid 2.7 to 3.3; proline 1.7 to 2.3; glycine 2.7
to 3.3; valine 0.9 to 1.1; leucine 4.5 to 5.3; histidine 0.9 to 1.1; arginine 0.9 to 1.1; lysine 1.8 to 2.2;
serine 3.2 to 4.2; threonine 4.2 to 5.2; tyrosine 0.7 to 1.1; half-cystine 1.4 to 2.1.
Related peptides Examine by liquid chromatography (2.2.29) as described under Assay.
Inject 20 l of the test solution. In the chromatogram obtained, the area of any peak apart from the
principal peak is not greater than 3.0 per cent of the total area of the peaks. The sum of the areas of
all the peaks, apart from the principal peak, is not greater than 5.0 per cent of the total area of the
peaks. Disregard any peak due to the solvent and any peak with an area less than 0.1 per cent of that
of the principal peak.
Water (2.5.32). Not more than 10.0 per cent, determined by the micro determination of water.
Acetic acid and water Not more than 20 per cent, calculated by adding together the percentage
contents of acetic acid and water determined by the methods described above.
1000 I.U. of endotoxin per milligram.
ASSAY
Test solution. Prepare a 1.0 mg/ml solution of the substance to be examined in mobile phase A.
Reference solution. Dissolve the contents of a vial of calcitonin (salmon) CRS in mobile phase A to
obtain a concentration of 1.0 mg/ml.
Resolution solution. Dissolve the contents of a vial of N-acetyl-cys
1
calcitonin CRS in 400 l of mobile
phase A and add 100 l of the test solution.
Mobile phase A. Dissolve 3.26 g of tetramethylammonium hydroxide R in 900 ml of water R, adjust the
pH to 2.5 with phosphoric acid R and mix with 100 ml of acetonitrile for chromatography R; filter and
degas,
Mobile phase B. Dissolve 1.45 g of tetramethylammonium hydroxide R in 400 ml of water R, adjust the
pH to 2.5 with phosphoric acid R and mix with 600 ml of acetonitrile for chromatography R; filter and
degas,
Time
(min)
Mobile phase A
(% V/V)
Mobile phase B
(% V/V)
Comment
eluent conditions
6-43
Equilibrate the column with a mixture of 72 volumes of mobile phase A and 28 volumes of mobile
phase B.
Inject 20 l of the resolution solution. When the chromatogram is recorded in the prescribed
conditions, the relative retention of N-acetyl-cys
1
calcitonin is about 1.15 relative to the principal
peak. The test is not valid unless the resolution between the peaks corresponding to calcitonin and
N-acetyl-cys
1
calcitonin is at least 5.0 and the symmetry factor for the N-acetyl-cys
1
calcitonin peak is
not greater than 2.5. If necessary, adjust the initial A: B ratio of the mobile phase.
Calculate the content of calcitonin (salmon) (C
145
H
240
N
44
O
48
S
2
) from the peak areas in the
chromatograms obtained with the test solution and the reference solution and the declared content of
C
145
H
240
N
44
O
48
S
2
in calcitonin (salmon) CRS. Proceed with tangential integration of the peak areas.
STORAGE
Store in a well-closed container, protected from light at a temperature between 2C and 8C. If the
LABELLING
The label states:
the calcitonin peptide content (C
145
H
240
N
44
O
48
S
2
),
_______________________________________________________________________________________________________________ Ph Eur
6-44
Calcitriol
1/01
Me
Me
Me
Me
H
H
H
OH
CH
2
HO
H
HO
H
H
H
C
27
H
44
O
3
416.6 32222-06-3
Calcitriol complies with the requirements of the 3rd edition of the European Pharmacopoeia [0883]. These
Action and use Vitamin D analogue.
Preparation
Calcitriol Capsules
Ph Eur _________________________________________________________________________________________________________________________________________
DEFINITION
Calcitriol contains not less than 97.0 per cent and not more than the equivalent of 103.0 per cent of
(5Z,7E)-9,10-secocholesta-5,7,10(19)-triene-1,3,25-triol.
CHARACTERS
A reversible isomerisation to pre-calcitriol takes place in solution, depending on temperature and
IDENTIFICATION
spectrum of calcitriol.
peak in the chromatogram obtained with the test solution are approximately the same as those of the
TESTS
Calculate the percentage content of related substances, apart from pre-calcitriol, that are eluted
within twice the retention time of calcitriol from the areas of the peaks in the chromatogram obtained
with the test solution by the normalisation procedure. The content of any individual related
1.0 per cent. Disregard any peak with an area less than 0.1 times that of the peak in the chromato-
ASSAY
mobile phase.
Reference solution (a). Dissolve 1.000 mg of calcitriol CRS without heating in 10.0 ml of the mobile
phase.
Reference solution (c). Keep 2 ml of reference solution (a) for 30 min at 80C.
tography R1 (5 m),
6-45
as mobile phase at a flow rate of 1.0 ml/min, a mixture of 450 volumes of a solution containing
1.0 g/l of tris(hydroxymethyl)aminomethane R adjusted to pH 7.0 to 7.5 with phosphoric acid R,
and of 550 volumes of acetonitrile R,
a loop injector,
Inject 50 l of reference solution (a) and six times 50 l of reference solution (c). When the
chromatograms are recorded in the prescribed conditions, the retention time for pre-calcitriol,
relative to calcitriol, is about 0.9. The assay is not valid unless the number of theoretical plates
calculated for the peak due to calcitriol in the chromatogram obtained with reference solution (a) is at
least 10,000, the relative standard deviation of the peak area due to calcitriol is at most 1 per cent and
the resolution between the peaks due to calcitriol and pre-calcitriol is at least 3.5.
Inject 50 l of reference solution (b) and 50 l of the test solution. Continue the chromatography
for twice the retention time of calcitriol.
Calculate the percentage content of calcitriol.
STORAGE
IMPURITIES
H
Me
H Me
Me
OH
Me
H H
2
C
HO
HO
A. (5E,7E)-9,10-secocholesta-5,7,10(19)-triene-1,3,25-triol (trans-calcitriol),
H
Me
H Me
Me
OH
Me
H
HO
CH
2
OH
B. (5Z,7E)-9,10-secocholesta-5,7,10(19)-triene-1,3,25-triol (1-calcitriol),
H
Me
H Me
Me
OH
Me
H
N
N
N
O
O
Me
OH HO
C. (6aR,7R,9aR)-11-[(3S,5R)-3,5-dihydroxy-2-methylcyclohex-1-enyl]-7-[(1R)-5-hydroxy-
1,5-dimethylhexyl]-6a-methyl-2-phenyl-5,6,6a,7,8,9,9a,11-octahydro- 1H,4aH-
cyclopenta[f]1,2,4]triazolo[1,2-a]cinnoline-1,3(2H)-dione (triazoline adduct of pre-calcitriol).
_________________________________________________________________________________________________________________________________________ Ph Eur
6-46
Calcium Acetate
C
4
H
6
CaO
4
158.2 62-54-4
Definition Calcium Acetate contains not less than 98.0% and not more than 100.5% of C
4
H
6
CaO
4
,
Characteristics A white powder; odourless or almost odourless; hygroscopic.
Freely soluble in water; slightly soluble in ethanol (96%).
Identification Yields the reactions characteristic of calcium salts and of acetates, Appendix VI.
Alkalinity pH of a 5% w/v solution, 7.2 to 8.2, Appendix V L.
Aluminium Dissolve 4.0 g in 100 ml of water and add 10 ml of acetate buffer pH 6.0. The resulting
solution complies with the limit test for aluminium, Appendix VII (1 ppm).
Arsenic 0.50 g dissolved in 25 ml of water complies with the limit test for arsenic, Appendix VII
(2 ppm).
Barium Not more than 50 ppm of Ba when determined by Method II for atomic emission
spectrophotometry, Appendix II D, measuring at 455.5 nm with a nitrous oxideacetylene flame.
Prepare a 5.0% w/v solution of the substance being examined and use barium standard solution (0.1%
Ba), suitably diluted with water, to prepare the standard solutions.
Heavy metals Dissolve 2.0 g in 20 ml of water. 12 ml of the resulting solution complies with limit
test A for heavy metals, Appendix VII. Use lead standard solution (2 ppm Pb) to prepare the standard
(20 ppm).
Magnesium Not more than 500 ppm of Mg when determined by Method II, for atomic absorption
spectrophotometry, Appendix II D, measuring at 285.2 nm. Prepare a 0.20% w/v solution and use
magnesium standard solution (0.1% Mg), suitably diluted with water, to prepare the standard solutions.
Potassium Not more than 0.1% of K when determined by Method II for atomic emission
potassium standard solution (600 ppm K), suitably diluted with water, to prepare the standard solutions.
Sodium Not more than 0.5% of Na when determined by Method II for atomic emission
sodium solution (200 ppm Na), suitably diluted with water, to prepare the standard solutions.
Chloride 0.15 g complies with the limit test for chlorides, Appendix VII (330 ppm).
Nitrate Dissolve 1.0 g in 10 ml of water, add 5 mg of sodium chloride, 0.05 ml of indigo carmine
solution and, with stirring, 10 ml of nitrogen-free sulphuric acid. The blue colour remains for at least 10
minutes.
Sulphate 0.25 g complies with the limit test for sulphates, Appendix VII (600 ppm).
Readily oxidisable substances Dissolve 2.0 g in 100 ml of boiling water, add a few anti-bumping
granules, 6 ml of 5M sulphuric acid and 0.3 ml of 0.02M potassium permanganate, mix, boil gently for 5
minutes and allow the precipitate to settle. The pink colour in the supernatant liquid is not
completely discharged.
Water Not more than 7.0% w/w, Appendix IX C. Use 0.7 g and add 20 ml of anhydrous acetic acid
to the titration vessel in addition to the methanol.
Assay Dissolve 0.6 g in 15 ml of water, add 5 ml of diethylamine and titrate with 0.1M disodium
edetate VS using methyl thymol blue mixture as indicator. Each ml of 0.1M disodium edetate VS is
4
H
6
CaO
4
.
Storage Calcium Acetate should be kept in a well-closed container.
Action and use Used in solutions for haemodialysis and peritoneal dialysis.
6-47
Calcium Ascorbate
O
O
OH O
OH H
CH
2
OH
_
2
Ca
2+
C
12
H
14
CaO
12
,2H
2
O 426.3 5743-28-2
Calcium Ascorbate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1182].
Ph Eur _________________________________________________________________________________________________________________________________________
DEFINITION
Calcium ascorbate contains not less than 99.0 per cent and not more than the equivalent of 100.5
per cent of calcium di[(R)-2-[(S)-1,2-dihydroxyethyl]-4-hydroxy-5-oxo-2H-furan-3-olate] dihydrate.
CHARACTERS
A white or slightly yellowish, crystalline powder, freely soluble in water, practically insoluble in
alcohol.
IDENTIFICATION
spectrum of calcium ascorbate.
C. Dilute 1 ml of solution S (see Tests) to 10 ml with water R. To 2 ml of the solution add 0.2 ml of
a 100 g/l solution of ferrous sulphate R. A deep violet colour develops.
D. To 1 ml of solution S add 0.2 ml of dilute nitric acid R and 0.2 ml of silver nitrate solution R2. A
grey precipitate is formed.
E. The substance gives reaction (b) of calcium (2.3.1).
TESTS
solution Y
6
(Method II, 2.2.2). Examine the colour of the solution immediately after preparation of
the solution.
Specific optical rotation (2.2.7). +95 to +97, determined using freshly prepared solution S and
Fluorides Not more than 10 ppm of F, determined potentiometrically (Method I, 22.36) using as
indicator electrode a fluoride-selective electrode and as reference electrode a silver-silver chloride
electrode.
Test solution. In a 50 ml volumetric flask, dissolve 1.000 g in a 10.3 g/l solution of hydrochloric acid R,
add 5.0 ml of fluoride standard solution (1 ppm F) R and dilute to 50.0 ml with a 10.3 g/l solution of
hydrochloric acid R. To 20.0 ml of the solution add 20.0 ml of total-ionic-strength-adjustment buffer R
and 3 ml of an 82 g/l solution of anhydrous sodium acetate R. Adjust to pH 5.2 with ammonia R and
dilute to 50.0 ml with distilled water R.
Reference solutions. To 0.25 ml, 0.5 ml, 1.0 ml, 2.0 ml and 5.0 ml of fluoride standard solution (10 ppm
F) R add 20.0 ml of total-ionic-strength-adjustment buffer R and dilute to 50.0 ml with distilled water R.
Carry out the measurements for each solution. Calculate the concentration of fluorides using the
calibration curve, taking into account the addition of fluoride to the test solution.
Copper Not more than 5 ppm of Cu, determined by atomic absorption spectrometry (Method 1,
2.2.23).
6-48
Test solution. Dissolve 2.0 g of the substance to be examined in a 9.7 g/l solution of nitric acid R and
dilute to 25.0 ml with the same acid solution.
Reference solutions. Prepare the reference solutions using copper standard solution (10 ppm Cu) R,
diluting with a 9.7 g/l solution of nitric acid R.
Measure the absorbance at 324.8 nm using a copper hollow cathode lamp as source of radiation and
Iron. Not more than 2 ppm of Fe, determined by atomic absorption spectrometry (Method, 2.2.23).
Test solution. Dissolve 5.0 g of the substance to be examined in a 9.7 g/l solution of nitric acid R and
dilute to 25.0 ml with the same acid solution.
Reference solutions. Prepare the reference solutions using iron standard solution (10 ppm Fe) R, diluting
with a 9.7 g/l solution of nitric acid R.
Measure the absorbance at 248.3 nm using an iron hollow-cathode lamp as the radiation source and
standard using 2.0 ml of lead standard solution (10 ppm Pb) R.
100C to 105C for 2 h.
ASSAY
Dissolve 80.0 mg in a mixture of 10 ml of dilute sulphuric acid R and 80 ml of carbon dioxide-free
water R. Add 1 ml of starch solution R. Titrate with 0.05M iodine until a persistent violet-blue colour is
obtained.
12
H
14
CaO
12
,2H
2
O.
STORAGE
Store in a well-closed, non-metallic container, protected from light.
_________________________________________________________________________________________________________________________________________ Ph Eur
6-49
Calcium Carbonate
CaCO
3
100.1 471-34-1
Calcium Carbonate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0014].
Preparations
Calcium Carbonate Chewable Tablets
Calcium and Colecalciferol Tablets
Ph Eur _________________________________________________________________________________________________________________________________________
DEFINITION
Calcium carbonate contains not less than 98.5 per cent and not more than the equivalent of
100.5 per cent of CaCO
3
CHARACTERS
A white powder, practically insoluble in water.
IDENTIFICATION
A. It gives the reaction of carbonates (2.3.1).
B. 0.2 ml of solution S (see Tests) gives the reactions of calcium (2.3.1).
TESTS
Solution S Dissolve 5.0 g in 80 ml of dilute acetic acid R. When the effervescence ceases, boil the
solution for 2 min, allow to cool, dilute to 100 ml with dilute acetic acid R and filter, if necessary,
through a sintered-glass filter.
Substances insoluble in acetic acid Wash any residue obtained during the preparation of solution
S with four quantities, each of 5 ml, of hot water R and dry at 100C to 105C for 1 h. The residue
Sulphates (2.4.13). 1.2 ml of solution S diluted to 15 ml with distilled water R complies with the
limit test for sulphates (0.25 per cent).
Barium To 10 ml of solution S add 10 ml of calcium sulphate solution R. After at least 15 min, any
opalescence in the solution is not more intense than that in a mixture of 10 ml of solution S and
10 ml of distilled water R.
Iron (2.4.9). Dissolve 50 mg in 5 ml of dilute hydrochloric acid R and dilute to 10 ml with water R.
The solution complies with the limit test for iron (200 ppm).
Magnesium and alkali metals Dissolve 1.0 g in 12 ml of dilute hydrochloric acid R. Boil the solution
for about 2 min and add 20 ml of water R, 1 g of ammonium chloride R and 0.1 ml of methyl red
solution R. Add dilute ammonia R1 until the colour of the indicator changes and then 2 ml in excess.
Heat to boiling and add 50 ml of hot ammonium oxalate solution R. Allow to stand for 4 h, dilute to
100 ml with water R and filter through a suitable filter. To 50 ml of the filtrate add 0.25 ml of
sulphuric acid R. Evaporate to dryness on a water-bath and ignite to constant mass at 600C. The
residue weighs not more than 7.5 mg (1.5 per cent).
200C.
ASSAY
Dissolve 0.150 g in a mixture of 3 ml of dilute hydrochloric acid R and 20 ml of water R. Boil for
2 min, allow to cool and dilute to 50 ml with water R. Carry out the complexometric titration of
calcium (2.5.11).
1 ml of 0.1M sodium edetate is equivalent to 10.01 mg of CaCO
3
.
_________________________________________________________________________________________________________________________________________ Ph Eur
6-50
Calcium Chloride
Calcium Chloride Dihydrate
CaCl
2
,2H
2
O 147.0 10035-04-8
Calcium Chloride complies with the requirements of the 3rd edition of the European Pharmacopoeia [0015].
Preparation
Calcium Chloride Injection
Compound Sodium Lactate Intravenous Infusion
Ph Eur _________________________________________________________________________________________________________________________________________
DEFINITION
Calcium chloride contains not less than 97.0 per cent and not more than the equivalent of 103.0 per
cent of CaCl
2
,2H
2
O.
CHARACTERS
A white, crystalline powder, hygroscopic, freely soluble in water, soluble in alcohol.
IDENTIFICATION
A. Solution S (see Tests) gives reaction (a) of chlorides (2.3.1).
B. It gives the reactions of calcium (2.3.1).
TESTS
solution Y
6
(Method II, 2.2.2).
Acidity or alkalinity To 10 ml of freshly prepared solution S add 0.1 ml of phenolphthalein
solution R. If the solution is red, not more than 0.2 ml of 0.01M hydrochloric acid is required to
discharge the colour and if the solution is colourless, not more than 0.2 ml of 0.01M sodium hydroxide
is required to turn it red.
Aluminium (2.4.17). To 10 ml of solution S add 2 ml of ammonium chloride solution R and 1 ml of
dilute ammonia R1 and boil the solution. No turbidity or precipitate is formed.
If intended for use in the manufacture of dialysis solutions, it complies with the following require-
ment which replaces the test for aluminium prescribed above.
Dissolve 4 g in 100 ml of water R and add 10 ml of acetate buffer solution pH 6.0 R. The solution
complies with the limit test for aluminium (1 ppm). Use as the reference solution a mixture of 2 ml of
aluminium standard solution (2 ppm Al) R, 10 ml of acetate buffer solution pH 6.0 R and 98 ml of
water R. To prepare the blank use a mixture of 10 ml of acetate buffer solution pH 6.0 R and 100 ml of
water R.
Iron (2.4.9). 10 ml of solution S complies with the limit test for iron (10 ppm).
Magnesium and alkali metals To a mixture of 20 ml of solution S and 80 ml of water R add 2 g of
ammonium chloride R and 2 ml of dilute ammonia R1, heat to boiling and pour into the boiling solution
a hot solution of 5 g of ammonium oxalate R in 75 ml of water R. Allow to stand for 4 h, dilute to
residue weighs not more than 5 mg (0.5 per cent).
ASSAY
Dissolve 0.280 g in 100 ml of water R and carry out the complexometric titration of calcium (2.5.11).
1 ml of 0.1M sodium edetate is equivalent to 14.70 mg of CaCl
2
,2H
2
O.
6-51
STORAGE
LABELLING
The label states, where applicable, that the substance is suitable for use in the manufacture of dialysis
solutions.
_________________________________________________________________________________________________________________________________________ Ph Eur
6-52
Calcium Chloride Hexahydrate
CaCl
2
,6H
2
O 219.1 7774-34-7
Calcium Chloride Hexahydrate complies with the requirements of the 3rd edition of the European
Ph Eur _________________________________________________________________________________________________________________________________________
DEFINITION
Calcium chloride hexahydrate contains not less than 97.0 per cent and not more than the equivalent
of 103.0 per cent of CaCl
2
,6H
2
O.
CHARACTERS
A white, crystalline mass or colourless crystals, very soluble in water, freely soluble in alcohol.
It freezes at about 29C.
IDENTIFICATION
A. Solution S (see Tests) gives reaction (a) of chlorides (2.3.1).
B. It gives the reactions of calcium (2.3.1).
TESTS
solution Y
6
(Method II, 2.2.2).
Acidity or alkalinity To 10 ml of freshly prepared solution S add 0.1 ml of phenolphthalein
solution R. If the solution is red, not more than 0.2 ml of 0.01M hydrochloric acid is required to
discharge the colour and if the solution is colourless, not more than 0.2 ml of 0.01M sodium hydroxide
is required to turn it red.
Aluminium (2.4.17). To 10 ml of solution S add 2 ml of ammonium chloride solution R and 1 ml of
dilute ammonia R1. Heat to boiling. No turbidity or precipitate is formed.
If intended for use in the manufacture of dialysis solutions, it complies with the following test which
replaces the test for aluminium prescribed above.
Dissolve 6 g in 100 ml of water R and add 10 ml of acetate buffer solution pH 6.0 R. The solution
complies with the limit test for aluminium (1 ppm). Use as the reference solution a mixture of 2 ml of
aluminium standard solution (2 ppm Al) R, 10 ml of acetate buffer solution pH 6.0 R and 98 ml of
water R. To prepare the blank use a mixture of 10 ml of acetate buffer solution pH 6.0 R and 100 ml of
water R.
Magnesium and alkali metals To a mixture of 20 ml of solution S and 80 ml of water R add 2 g of
ammonium chloride R and 2 ml of dilute ammonia R1, heat to boiling and pour into the boiling solution
a hot solution of 5 g of ammonium oxalate R in 75 ml of water R. Allow to stand for 4 h, dilute to
ASSAY
Dissolve 0.200 g in 100 ml of water R. Carry out the complexometric titration of calcium (2.5.11).
1 ml of 0.1M sodium edetate is equivalent to 21.91 mg of CaCl
2
,6H
2
O.
STORAGE
LABELLING
The label states, where applicable, that the substance is suitable for use in the manufacture of dialysis
solutions.
_________________________________________________________________________________________________________________________________________ Ph Eur
6-53
Calcium Dobesilate Monohydrate
SO
3
OH
HO
_
2
Ca
2+
C
12
H
10
CaO
10
S
2
,H
2
O 436.4 20123-80-2 (anhydrous)
Calcium Dobesilate Monohydrate complies with the requirements of the 3rd edition of the European
Ph Eur _________________________________________________________________________________________________________________________________________
DEFINITION
Calcium dobesilate monohydrate contains not less than 99.0 per cent and not more than the equiva-
lent of 102.0 per cent of calcium di(2,5-dihydroxybenzenesulphonate), calculated with reference to
the anhydrous substance.
CHARACTERS
A white or almost white, hygroscopic powder, very soluble in water, freely soluble in ethanol, very
slightly soluble in 2-propanol, practically insoluble in methylene chloride.
IDENTIFICATION
A. Dissolve 0.100 g in water R and dilute to 200.0 ml with the same solvent. Dilute 5.0 ml of the
solution to 100.0 ml with water R. Examined between 210 nm and 350 nm (2.2.25), the solution
shows two absorption maxima, at 221 nm and 301 nm. The specific absorbance at the maximum at
301 nm is 174 to 181.
B. Mix 1 ml of ferric chloride solution R2, 1 ml of a freshly prepared 10 g/l solution of potassium
ferricyanide R and 0.1 ml of nitric acid R. To this mixture add 5 ml of freshly prepared solution S (see
Tests): a blue colour and a precipitate are immediately produced.
C. 2 ml of freshly prepared solution S (see Tests) gives reaction (b) of calcium (2.3.1).
TESTS
Appearance of solution Solution S, when freshly prepared, is clear (2.2.1.) and colourless (Method
II, 2.22).
Hydroquinone Examine by thin-layer chromatography (2.2.27), using as the coating substance a
suitable silica gel with a fluorescent indicator having an optimal intensity at 254 nm.
same solvent.
Reference solution. Dissolve 10 mg of hydroquinone R in water R and dilute to 50 ml with the same
solvent.
Apply to the plate 10 l of each solution and dry the starting points in a current of cool air. Develop
over a path of 15 cm using a mixture of 20 volumes of methylene chloride R, 30 volumes of methyl
acetate R and 50 volumes of ethyl acetate R. Dry the plate in a current of hot air and examine in
ultraviolet light at 254 nm. Any spot corresponding to hydroquinone in the chromatogram obtained
with the test solution is not more intense than the principal spot in the chromatogram obtained with
ASSAY
Dissolve 0.200 g in a mixture of 10 ml of water R and 40 ml of dilute sulphuric acid R. Titrate with
0.1M cerium sulphate, determining the end-point potentiometrically (2.2.20).
1 ml of 0.1M cerium sulphate is equivalent to 10.45 mg of C
12
H
10
CaO
10
S
2
.
6-54
STORAGE
IMPURITIES
OH
HO
A. benzene-1,4-diol (hydroquinone).
_________________________________________________________________________________________________________________________________________ Ph Eur
6-55
Calcium Folinate
N
N
H
N
N
H
N
H
N
H
COO
H
2
N
O CHO
H
O
COO H
Ca
2
*
and epimer at C*
C
20
H
21
CaN
7
O
7
,xH
2
O 511.5 1492-18-8
(anhydrous) (anhydrous)
Calcium Folinate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0978].
Action and use Antidote to folic acid antagonists.
Preparations
Calcium Folinate Injection
Calcium Folinate Tablets
Ph Eur ________________________________________________________________________________________________________________
DEFINITION
Calcium folinate contains not less than 97.0 per cent and not more than the equivalent of 102.0 per
cent of calcium (2S)-2-[[4-[[[(6RS)-2-amino-5-formyl-4-oxo-1,4,5,6,7,8-hexahydropteridin-6-
yl]methyl]amino]benzoyl]amino]pentanedioate and not less than 7.54 per cent and not more than
the equivalent of 8.14 per cent of Ca, both calculated with reference to the anhydrous and solvent-
free substance.
CHARACTERS
A white or light yellow, amorphous or crystalline powder, sparingly soluble in water, practically
insoluble in acetone and in alcohol. The amorphous form may produce supersaturated solutions in
water.
IDENTIFICATION
obtained with calcium folinate CRS. Examine the substances prepared as discs. If the spectra obtained
show differences, dissolve the substance to be examined and the reference substance separately in the
minimum quantity of water R and add dropwise sufficient acetone R to produce sufficient precipitate.
Allow to stand for 15 min, collect the precipitate by centrifugation, wash the precipitate with two
small quantities of acetone R and dry. Record new spectra using the residues.
C. Examine by thin-layer chromatography (2.2.27), using cellulose for chromatography F
254
R as the
coating substance.
Test solution. Dissolve 15 mg of the substance to be examined in a 3 per cent V/V solution of
ammonia R and dilute to 5 ml with the same solvent.
Reference solution. Dissolve 15 mg of calcium folinate CRS in a 3 per cent V/V solution of ammonia R
mixture of 1 volume of isoamyl alcohol R and 10 volumes of a 50 g/l solution of citric acid R previously
adjusted to pH 8 with ammonia R. Allow the plate to dry in air and examine in ultraviolet light at
D. It gives reaction (b) of calcium (2.3.1).
Carry out the tests and the assay as rapidly as possible, protected from actinic light.
TESTS
Solution S Dissolve 1.25 g in carbon dioxide-free water R heating at 40C if necessary, and dilute to
6-56
Appearance of solution Solution S is clear (2.2.1). The absorbance (2.2.25) of solution S,
measured at 420 nm using water R as the compensation liquid, is not greater than 0.60.
Specific optical rotation (2.2.7). The specific optical rotation is +14.4 to +18.0, determined on
solution S and calculated with reference to the anhydrous and solvent-free substance.
Acetone, ethanol and methanol Not more than 0.5 per cent of acetone, not more than 3.0 per
cent of ethanol and not more than 0.5 per cent of methanol. Examine by head-space gas chromato-
graphy (2.2.28, Method B).
the same solvent.
Reference solution. Dilute 0.125 g of acetone R, 0.750 g of ethanol R and 0.125 g of methanol R in
water R and dilute to 1000.0 ml with water R.
a fused-silica column 10 m long and 0.32 mm in internal diameter coated with styrene-
divinylbenzene copolymer R,
raising the temperature of the column from 125C to 185C at a rate of 10C per min and
maintaining at 185C until the total run time is 15 min. Maintain the temperature of the injection
port at 250C and that of the detector at 250C. Place the samples in a thermostatically controlled
chamber at 80C for 20 min and pressurise them for 30 s. Repeat the injections at least three times.
Related substances Examine the chromatograms obtained in the Assay. In the chromatogram
obtained with the test solution: the area of any peak corresponding to formylfolic acid is not greater
than the area of the principal peak in the chromatogram obtained with reference solution (c) (1 per
cent); the area of any peak, apart from the principal peak and any peak corresponding to formylfolic
acid, is not greater than the area of the principal peak in the chromatogram obtained with reference
solution (b) (1 per cent); the sum of the areas of all the peaks, apart from the principal peak, is not
solution (b) (2.5 per cent). Disregard any peak with an area less than that of the principal peak in the
chromatogram obtained with reference solution (d).
Chlorides (2.4.4). Dissolve 67 mg in 10 ml of water R and add 3 ml of acetic acid R. Filter and wash
the precipitate with five quantities, each of 5 ml, of water R. Collect the filtrate and washings and
dilute to 100 ml with water R. 15 ml of this solution complies with the limit test for chlorides (0.5 per
cent).
Heavy metals (2.4.8) 1.0 g complies with limit test F for heavy metals (50 ppm). Prepare the
To 12 ml of the above solution add 2 ml of buffer solution pH 3.5 R. Mix and add to 1.2 ml of
thioacetamide reagent R. Mix immediately. The solution is not more intensely coloured than a
standard prepared as follows: dilute 4 ml of lead standard solution (10 ppm Pb) R to 25 ml with
water R, adjust to pH 3.0 to 4.0 using dilute acetic acid R and dilute to 40 ml with water R. To 12 ml
of this solution add 2 ml of buffer solution pH 3.5 R, mix and add the mixture to 1.2 ml of
thioacetamide reagent R (50 ppm).
Platinum Not more than 20 ppm, determined by atomic absorption spectrometry (Method II,
2.2.23).
Test solution. Dissolve 1.00 g in water R and dilute to 100.0 ml with the same solvent.
Reference solutions. Prepare the reference solutions using platinum standard solution (30 ppm Pt) R,
diluted as necessary with a mixture of 1 volume of nitric acid R and 99 volumes of water R.
Measure the absorbance at 265.9 nm using a platinum hollow-cathode lamp as source of radiation.
Water (2.5.12). Not more than 17.0 per cent, determined on 0.200 g of the substance to be
examined, ground to a very fine powder. Stir the substance to be examined in the titration solvent for
about 6 min before titrating and use a suitable titrant that does not contain pyridine.
ASSAY
Calcium Dissolve 0.400 g in 150 ml of water R and dilute to 300 ml with the same solvent. Carry
out the complexometric titration of calcium (2.5.11).
1 ml of 0.1M sodium edetate is equivalent to 4.008 mg of Ca.
Calcium folinate Examine by liquid chromatography (2.2.29).
6-57
the same solvent.
Reference solution (a). Dissolve 10.0 mg of calcium folinate CRS in water R and dilute to 10.0 ml with
the same solvent.
Reference solution (c). Dissolve 10.0 mg of formylfolic acid CRS in water R and dilute to 100.0 ml with
the same solvent. Dilute 1.0 ml of this solution to 10.0 ml with water R.
Reference solution (d). Dilute 1.0 ml of reference solution (b) to 10.0 ml with water R.
Reference solution (e). Dilute 5.0 ml of reference solution (c) to 10.0 ml with reference solution (b).
as mobile phase at a flow rate of 1 ml/min a mixture prepared as follows: mix 220 ml of
methanol R and 780 ml of a solution containing 2.0 ml of tetrabutylammonium hydroxide solution R
and 2.2 g of disodium hydrogen phosphate R previously adjusted to pH 7.8 with phosphoric acid R,
Inject 10 l of each solution. Continue the chromatography for 2.5 times the retention time of the
principal peak in the chromatogram obtained with the test solution. The assay is not valid unless, in
the chromatogram obtained with reference solution (e), the resolution between the peaks correspond-
ing to calcium folinate and formylfolic acid is at least 2.2 and the relative standard deviation of the
area of the principal peak for six replicate injections of reference solution (a) is at most 2.0 per cent.
20
H
21
CaN
7
O
7
from the peak areas and the declared content
of calcium folinate CRS.
STORAGE
Store in an airtight container, protected from light. If the substance is sterile, store in a sterile,
LABELLING
The label states:
IMPURITIES
H
2
N
N COOH
COOH H
O
H
A. (2S)-2[(4-aminobenzoyl)amino]pentanedioic acid,
N
N COOH
COOH H
O
N
N
N
N H
2
N
CHO O
CHO
H
H H
H
and epimer at C*
*
B. (2S)-2-[[4-[[[(6RS)-2-amino-5-formyl-4-oxo-1,4,5,6,7,8 hexahydropteridin-6-yl] methyl]
formylamino]benzoyl]-amino]pentanedioic acid (5,10-diformyltetrahydrofolic acid),
C. folic acid,
N
N COOH
COOH H
O
N
N
N
N H
2
N
O
CHO
H
D. (2S)-2-[[4-[[(2-amino-4-oxo-1,4-dihydropteridin-6-yl)methyl]-
formylamino]benzoyl]amino]pentanedioic acid (10-formylfolic acid),
6-58
H
N
N COOH
COOH H
O
N
N
N
N H
2
N
O
CHO
H
and enantiomer
E. 4-[[[(6RS)-2-amino-5-formyl-4-oxo-1,4,5,6,7,8-hexahydropteridin-6-yl]methyl]amino]-
benzoic acid (5-formyltetra-hydropteroic acid),
N
N COOH
COOH H
O
N
N
N
N H
2
N
O
R
H
H
F. R = CHO: (2S)-2-[[4-[[(2-amino-4-oxo-1,4,7,8-tetrahydropteridin-6-yl)methyl]formyl-
amino]benzoyl]amino]pentanedioic acid (10-formyldihydrofolic acid),
G. R = H: (2S)-2-[[4-[[(2-amino-4-oxo-1,4,7,8-tetrahydropteridin-6-yl)methyl]amino]-
benzoyl]amino]pentanedioic acid (dihydrofolic acid).
_______________________________________________________________________________________________________________ Ph Eur
6-59
Calcium Glucoheptonate
1/01
H OH
H HO
OH H
H OH
CO
2
_
OH H
OH
*
2
Ca
2+
and epimer at C*
C
14
H
26
CaO
16
490.4 29039-00-7
Calcium Glucoheptonate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Used in treatment of calcium deficiency.
Ph Eur ________________________________________________________________________________________________________________
DEFINITION
Calcium glucoheptonate is a mixture, in variable proportions, of calcium di(D-glycero-D-gulo-
heptonate) and calcium di(D-glycero-D-ido-heptonate). It contains not less than 98.0 per cent and not
more than the equivalent of 102.0 per cent of calcium 2,3,4,5,6,7-hexahydroxyheptanoate, calculated
CHARACTERS
A white or very slightly yellow, amorphous powder, hygroscopic, very soluble in water, practically
insoluble in acetone and in alcohol.
IDENTIFICATION
A. Examine by thin-layer chromatography (2.2.27), using cellulose for chromatography R1 as the
coating substance.
Reference solution (a). Dissolve 20 mg of calcium glucoheptonate CRS in 1 ml of water R.
Reference solution (b). Dissolve 10 mg of calcium gluconate CRS in 0.5 ml of the test solution and dilute
Apply separately to the plate, as bands 20 mm by 2 mm, 10 l of each solution. Allow the tank to
saturate for 10 min. Develop over a path of 12 cm using a freshly prepared mixture of 20 volumes of
anhydrous formic acid R, 20 volumes of water R, 30 volumes of acetone R and 30 volumes of butanol R.
Allow the plate to dry in air and spray with 0.02M potassium permanganate. The principal spot in the
B. 0.2 ml of solution S (see Tests) gives reaction (b) of calcium (2.3.1).
TESTS
solution Y
6
(Method II, 2.2.2).
Reducing sugars Dissolve 1.0 g in 5 ml of water R with the aid of gentle heat. Cool and add 20 ml
of cupri-citric solution R and a few glass beads. Heat so that boiling begins after 4 min and maintain
boiling for 3 min. Cool rapidly and add 100 ml of a 2.4 per cent V/V solution of glacial acetic acid R
and 20.0 ml of 0.025M iodine. With continuous shaking, add 25 ml of a mixture of 6 volumes of
hydrochloric acid R and 94 volumes of water R until the precipitate dissolves, titrate the excess of
iodine with 0.05M sodium thiosulphate using 1 ml of starch solution R added towards the end of the
titration, as indicator. Not less than 12.6 ml of 0.05M sodium thiosulphate is required (1 per cent
expressed as glucose).
6-60
Cyanide Dissolve 5.0 g in 50 ml of water R and add 2.0 g of tartaric acid R. Place this solution in a
distillation apparatus (2.2.11). The plain bend adapter attached to the end of the condenser has a
vertical part that is long enough to extend to 1 cm from the bottom of a 50 ml test-tube used as a
receiver. Place 10 ml of water R and 2 ml of 0.1M sodium hydroxide into the receiver. Distil, collect
25 ml of distillate and dilute to 50 ml with water R. To 25 ml of this solution add 25 mg of ferrous
sulphate R and boil for a short time. After cooling to about 70C add 10 ml of hydrochloric acid R1.
After 30 min, filter the solution and wash the filter. A yellow spot appears on the filter; there is no
blue or green spot.
Chlorides (2.4.4). To 5 ml of solution S, add 10 ml of water R. The solution complies with the limit
Heavy metals (2.4.8). Dissolve 2.0 g in 10 ml of buffer solution pH 3.5 R and dilute to 20 ml with
water R.12 ml of the solution complies with limit test A for heavy metals (10 ppm). Prepare the
100C to 105C for 3 h.
167 I.U. of endotoxin per gram.
ASSAY
Dissolve 0.800 g in a mixture of 150 ml of water R and 2 ml of 3M hydrochloric acid. While stirring,
add 12.5 ml of 0.1M sodium edetate, 15 ml of 1M sodium hydroxide and 0.3 g of hydroxynaphthol blue,
sodium salt R. Titrate with 0.1M sodium edetate until the colour changes from violet to pure blue.
1 ml of 0.1M sodium edetate is equivalent to 49.04 mg of C
14
H
26
CaO
16
.
STORAGE
In an airtight container. If the substance is sterile, store in a sterile, airtight, tamper-proof container.
LABELLING
The label states:
_______________________________________________________________________________________________________________ Ph Eur
6-61
Calcium Gluconate
2
Ca
2+
COO
_
OH H
H HO
H OH
H OH
CH
2
OH
C
12
H
22
CaO
14
,H
2
O 448.4 18016-24-5
Calcium Gluconate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0172].
Preparations
Calcium Gluconate Tablets
Effervescent Calcium Gluconate Tablets
Ph Eur _________________________________________________________________________________________________________________________________________
DEFINITION
Calcium gluconate contains not less than 98.5 per cent and not more than the equivalent of
102.0 per cent of calcium D-gluconate monohydrate.
CHARACTERS
A white, crystalline or granular powder, sparingly soluble in water, freely soluble in boiling water.
IDENTIFICATION
Test solution. Dissolve 20 mg of the substance to be examined in 1 ml of water R, heating if necessary
in a water-bath at 60C.
Reference solution. Dissolve 20 mg of calcium gluconate CRS in 1 ml of water R, heating if necessary in a
water-bath at 60C.
10 volumes of ethyl acetate R, 10 volumes of concentrated ammonia R, 30 volumes of water R and 50
volumes of alcohol R. Dry the plate at 100C for 20 min. Allow to cool and spray with a 50 g/l
solution of potassium dichromate R in a 40 percent m/m solution of sulphuric acid R. After 5 min, the
principal spot in the chromatogram obtained with the test solution is similar in position, colour and
size to the principal spot in the chromatogram obtained with the reference solution.
B. Solution S (see Tests) gives the reactions of calcium (2.3.1).
TESTS
Solution S Dissolve 1.0 g in water R heated to 60C and dilute to 50 ml with the same solvent.
Appearance of solution At 60C, solution S is not more intensely coloured than reference solution
Y
6
(Method II, 2.2.2). After cooling, it is not more opalescent than reference suspension II (2.2.1).
Organic impurities and boric acid Introduce 0.5 g into a porcelain dish previously rinsed with
sulphuric acid R and placed in a bath of iced water. Add 2 ml of cooled sulphuric acid R and mix. No
yellow or brown colour develops. Add 1 ml of chromotrope II B solution R. A violet colour develops
and does not become dark blue. Compare the colour obtained with that of a mixture of 1 ml of
chromotrope II B solution R and 2 ml of cooled sulphuric acid R.
Sucrose and reducing sugars Dissolve 0.5 g in a mixture of 2 ml of hydrochloric acid R1 and 10 ml
of water R. Boil for 5 min, allow to cool, add 10 ml of sodium carbonate solution R and allow to stand.
Dilute to 25 ml with water R and filter. To 5 ml of the filtrate add 2 ml of cupri-tartaric solution R and
boil for 1 min. Allow to stand for 2 min. No red precipitate is formed.
Chlorides (2.4.4). 12.5 ml of solution S diluted to 15 ml with water R complies with the limit test
for chlorides (200 ppm).
Sulphates (2.4.13). Dissolve 10.0 g with heating in a mixture of 10 ml of acetic acid R and 90 ml of
distilled water R. 15 ml of the solution complies with the limit test for sulphates (100 ppm).
6-62
Heavy metals (2.4.8). 2.0 g complies with limit test D for heavy metals (10 ppm). Heat the
substance to be examined gradually and with care until it is almost completely transformed into a
white mass and then ignite. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R.
Magnesium and alkali metals Dissolve 1.00 g in 100 ml of boiling water R, add 10 ml of
ammonium chloride solution R, 1 ml of ammonia R and, dropwise, 50 ml of hot ammonium oxalate
solution R. Allow to stand for 4 h, dilute to 200 ml with water R and filter. Evaporate 100 ml of the
filtrate to dryness and ignite. The residue weighs not more than 2 mg (0.4 per cent).
3
micro-
organisms per gram, determined by plate count.
ASSAY
Dissolve 0.8000 g in 20 ml of hot water R, allow to cool and dilute to 300 ml with water R. Carry out
the complexometric titration of calcium (2.5.11).
12
H
22
CaO
14
,H
2
O.
_________________________________________________________________________________________________________________________________________ Ph Eur
6-63
Calcium Gluconate for Injection
C
12
H
22
CaO
14
,H
2
O 448.4 18016-24-5
Calcium Gluconate for Injection complies with the requirements of the 3rd edition of the European
Preparation
Calcium Gluconate Injection
Ph Eur _________________________________________________________________________________________________________________________________________
DEFINITION
Calcium gluconate for injection contains not less than 99.0 per cent and not more than the equiva-
lent of 101.0 per cent of calcium D-gluconate monohydrate.
CHARACTERS
A white, crystalline or granular powder, sparingly soluble in water, freely soluble in boiling water.
IDENTIFICATION
Reference solution. Dissolve 20 mg of calcium gluconate CRS in 1 ml of water R, heating if necessary in a
water-bath at 60C.
10 volumes of concentrated ammonia R, 10 volumes of ethyl acetate R, 30 volumes of water R and 50
volumes of alcohol R. Dry the plate at 100C for 20 min. Allow to cool and spray with a 50 g/l
solution of potassium dichromate R in a 40 per cent m/m solution of sulphuric acid R. After 5 min, the
B. About 20 mg gives reaction (b) of calcium (2.3.1).
TESTS
Solution S To 10.0 g add 90 ml of boiling distilled water R and boil with stirring, for not more than
10 s, until completely dissolved. Dilute to 100.0 ml with the same solvent.
Appearance of solution Solution S at 60C is not more intensely coloured than reference solution
B
7
(Method II, 2.2.2). After cooling to 20C, it is not more opalescent than reference suspension II
(2.2.1).
pH (2.2.3). Dissolve 1.0 g in 20 ml of carbon dioxide-free water R, heating on a water-bath. The pH of
the solution is 6.4 to 8.3.
Organic impurities and boric acid Introduce 0.5 g into a porcelain dish previously rinsed with
sulphuric acid R and placed in a bath of iced water. Add 2 ml of cooled sulphuric acid R and mix. No
yellow or brown colour develops. Add 1 ml of chromotrope II B solution R. A violet colour develops
and does not become dark blue. The solution is not more intensely coloured than that of a mixture of
1 ml of chromotrope II B solution R and 2 ml of cooled sulphuric acid R.
Oxalate Not more than 100 ppm, determined by liquid chromatography (2.2.29).
Test solution. Dissolve 1.00 g of the substance to be examined in water for chromatography R and dilute
to 100.0 ml with the same solvent.
Reference solution. Dissolve 1.00 g of the substance to be examined in water for chromatography R, add
0.5 ml of a 0.152 g/l solution of sodium oxalate R in water for chromatography R and dilute to 100.0 ml
a guard column 30 mm long and 4 mm in internal diameter packed with a suitable strong anion
exchange resin (30 m to 50 m),
two columns each 0.25 m long and 4 mm in internal diameter packed with a suitable strong
anion exchange resin (30 m to 50 m),
a micromembrane anion-suppressor column, connected in series with the guard and analytical
columns; the anion-suppressor column is equipped with a micromembrane that separates the
mobile phase from the suppressor regeneration solution, flowing countercurrent to the mobile
phase at a rate of 4 ml per minute,
6-64
as mobile phase at a flow rate of 2 ml per minute a solution prepared as follows: dissolve 0.212 g
of anhydrous sodium carbonate R and 63 mg of sodium hydrogen carbonate R in water for chromato-
graphy R and dilute to 1000.0 ml with the same solvent,
as suppressor regeneration solution a 1.23 g/l solution of sulphuric acid R in water for chromato-
graphy R,
a conductance detector,
a loop injector.
Inject 50 l of the reference solution five times. The test is not valid unless the relative standard
deviation of the peak area for oxalate in the chromatogram obtained with the reference solution is at
most 2.0 per cent.
Inject 50 l of each solution three times. Calculate the content of oxalate in parts per million using
the following expression:
T R
T
S S
S
50
S
T
=area of the oxalate peak in the chromatogram obtained with the test solution,
S
R
=area of the oxalate peak in the chromatogram obtained with the reference solution.
Sucrose and reducing sugars Dissolve 0.5 g in a mixture of 2 ml of hydrochloric acid R1 and 10 ml
of water R. Boil for 5 min, allow to cool, add 10 ml of sodium carbonate solution R and allow to stand
for 10 min. Dilute to 25 ml with water R and filter. To 5 ml of the filtrate add 2 ml of cupri-tartaric
solution R and boil for 1 min. Allow to stand for 2 min. No red precipitate is formed.
Chlorides (2.4.4). To 10 ml of previously filtered solution S add 5 ml of water R. The solution
complies with the limit test for chlorides (50 ppm).
Phosphates (2.4.11). Dilute 1 ml of solution S to 100 ml with water R. The solution complies with
the limit test for phosphates (100 ppm).
Sulphates (2.4.13). 15 ml of previously filtered solution S complies with the limit test for sulphates
(50 ppm). Prepare the standard using a mixture of 7.5 ml of sulphate standard solution (10 ppm
SO
4
) R and 7.5 ml of distilled water R.
Iron Not more than 5 ppm of Fe, determined by atomic absorption spectrometry (Method I, 2.2.23).
Test solution. Introduce 2.0 g into a 100 ml polytetrafluoroethylene beaker and add 5 ml of nitric
acid R. Boil, evaporating almost to dryness. Add 1 ml of strong hydrogen peroxide solution R and
evaporate again almost to dryness. Repeat the hydrogen peroxide treatment until a clear solution is
obtained. Using 2 ml of nitric acid R, transfer the solution into a 25 ml volumetric flask. Dilute to
25.0 ml with dilute hydrochloric acid R. In the same manner, prepare a compensation solution using
0.65 g of calcium chloride R1 instead of the substance to be examined.
Reference solutions. Prepare the reference solutions from iron solution (20 ppm Fe) R diluted with dilute
hydrochloric acid R.
Measure the absorbance at 248.3 nm, using an iron hollow-cathode lamp as source of radiation and
an air-acetylene flame. Carry out a basic correction using a deuterium lamp.
Magnesium and alkali metals To 0.50 g add a mixture of 1.0 ml of dilute acetic acid R and 10.0 ml
of water R and rapidly boil, whilst shaking, until completely dissolved. To the boiling solution add
5.0 ml of ammonium oxalate solution R and allow to stand for at least 6 h. Filter through a sintered-
glass filter (1.6) into a porcelain crucible. Carefully evaporate the filtrate to dryness and ignite. The
Bacterial endotoxins (2.6.14). Not more than 167 I.U. per gram.
2
micro-
organisms per gram, determined by plate count. It complies with the tests for Escherichia coli,
Pseudomonas aeruginosa and Staphylococcus aureus (2.6.13).
ASSAY
Dissolve 0.350 g in 20 ml of hot water R, allow to cool and dilute to 300 ml with water R. Carry out
the complexometric titration of calcium (2.5.11). Use 50 mg of calconecarboxylic acid triturate R.
12
H
22
CaO
14
,H
2
O.
STORAGE
_________________________________________________________________________________________________________________________________________ Ph Eur
6-65
Calcium Glycerophosphate
C
3
H
7
CaO
6
P 210.1 27214-00-2
Calcium Glycerophosphate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur _________________________________________________________________________________________________________________________________________
DEFINITION
Calcium glycerophosphate is a mixture in variable proportions of calcium (RS)-2,3-dihydroxypropyl
phosphate, and of calcium 2-hydroxy-1-(hydroxymethyl)ethyl phosphate, which may be hydrated.
Calcium glycerophosphate contains not less than 18.6 per cent and not more than 19.4 per cent of
Ca, calculated with reference to the dried substance.
CHARACTERS
A white powder, hygroscopic, sparingly soluble in water, practically insoluble in alcohol.
IDENTIFICATION
A. Mix 1 g with 1 g of potassium hydrogen sulphate R in a test tube fitted with a glass tube. Heat
strongly and direct the white vapour towards a piece of filter paper impregnated with a freshly
prepared 10 g/l solution of sodium nitroprusside R. The filter paper develops a blue colour in contact
with piperidine R.
B. Ignite 0.1 g in a crucible. Take up the residue with 5 ml of nitric acid R and heat on a water-bath
for 1 min. Filter. The filtrate gives reaction (b) of phosphates (2.3.1).
C. It gives reaction (b) of calcium (2.3.1).
TESTS
Solution S Dissolve 1.5 g at room temperature in carbon dioxide-free water R prepared from distilled
Appearance of solution Solution S is not more opalescent than reference suspension III (2.2.1).
Acidity or alkalinity To 100 ml of solution S add 0.1 ml of phenolphthalein solution R. Not more
than 1.5 ml of 0.1M hydrochloric acid or 0.5 ml of 0.1M sodium hydroxide is required to change the
colour of the indicator.
Citric acid Shake 5.0 g with 20 ml of carbon dioxide-free water R and filter. To the filtrate add
0.15 ml of sulphuric acid R and filter again. To the filtrate add 5 ml of mercuric sulphate solution R and
heat to boiling. Add 0.5 ml of a 3.2 g/l solution of potassium permanganate R and again heat to
boiling. No precipitate is formed.
Glycerol and alcohol-soluble substances Shake 1.000 g with 25 ml of alcohol R for 1 min. Filter.
Evaporate the filtrate to dryness on a water-bath and dry the residue at 70C for 1 h. The residue
Chlorides (2.4.4). Dissolve 0.1 g in a mixture of 2 ml of acetic acid R and 8 ml of water R and dilute
to 15 ml with water R. The solution complies with the limit test for chlorides (500 ppm).
Phosphates (2.4.11). Dilute 2.5 ml of solution S to 100 ml with water R. The solution complies
with the limit test for phosphates (400 ppm).
Sulphates (2.4.13). 15 ml of solution S complies with the limit test for sulphates (0.1 per cent).
Arsenic (2.4.2). Dissolve 0.33 g in water R and dilute to 25 ml with the same solvent. The solution
complies with limit test A for arsenic (3 ppm).
Heavy metals (2.4.8). Dissolve 2.0 g in 10 ml of buffer solution pH 3.5 R and dilute to 20 ml with
water R. 12 ml of the solution complies with limit test A for heavy metals (20 ppm). Prepare the
Iron (2.4.9). 0.20 g complies with the limit test for iron (50 ppm).
at 150C for 4 h.
ASSAY
Dissolve 0.200 g in water R. Carry out the complexometric titration of calcium (2.5.11).
STORAGE
_________________________________________________________________________________________________________________________________________ Ph Eur
6-66
Anhydrous Calcium Hydrogen Phosphate
CaHPO
4
136.1 7757-93-9
Anhydrous Calcium Hydrogen Phosphate complies with the requirements of the 3rd edition of the European
Ph Eur _________________________________________________________________________________________________________________________________________
DEFINITION
Calcium hydrogen phosphate, anhydrous contains not less than 98.0 per cent and not more than the
equivalent of 101.0 per cent of CaHPO
4
CHARACTERS
A white, crystalline powder or colourless crystals, practically insoluble in water and in alcohol. It
dissolves in dilute hydrochloric acid and in dilute nitric acid.
IDENTIFICATION
A. It complies with the test for monocalcium phosphate and tricalcium phosphate (see Tests).
B. It complies with the test for loss on drying (see Tests).
C. Dissolve 0.1 g in a mixture of 5 ml of water R and 5 ml of dilute nitric acid R. The solution gives
reaction (b) of phosphates (2.3.1).
D. Dissolve about 5 mg in 5 ml of acetic acid R. Add 0.5 ml of potassium ferrocyanide solution R. The
solution remains clear. Add about 50 mg of ammonium chloride R and allow to stand for 5 min. A
white, crystalline precipitate is formed.
TESTS
Solution S Dissolve 2.5 g in 20 ml of dilute hydrochloric acid R. Filter if necessary. Add dilute
ammonia R1 until a precipitate is formed. Dissolve by adding the minimum quantity needed of dilute
hydrochloric acid R and dilute to 50 ml with distilled water R.
Monocalcium phosphate and tricalcium phosphate Dissolve 2.00 g in 30.0 ml of 1M hydro-
chloric acid. Add 20 ml of water R and 0.05 ml of bromophenol blue solution R. Titrate the excess hydro-
chloric acid with 1M sodium hydroxide. The quantity of 1M hydrochloric acid required is 14.0 ml to
15.5 ml.
Carbonates Shake 0.5 g with 5 ml of carbon dioxide-free water R and add 1 ml of hydrochloric acid R.
No effervescence is produced.
Chlorides (2.4.4). Dissolve 0.5 g in a mixture of 1 ml of nitric acid R and 10 ml of water R and dilute
to 50 ml with water R. 15 ml of the solution complies with the limit test for chlorides (330 ppm).
Fluorides (2.4.5). 0.5 g complies with the limit test for fluorides (100 ppm).
Sulphates (2.4.13). To 1 ml of solution S add 2 ml of dilute hydrochloric acid R and dilute to 25 ml
with distilled water R. 15 ml of the solution complies with the limit test for sulphates (0.5 per cent).
Barium To 10 ml of filtered solution S add 0.5 ml of dilute sulphuric acid R. Allow to stand for
15 min. Any opalescence in the solution is not more intense than that in a mixture of 10 ml of
solution S and 0.5 ml of distilled water R.
150C for 2 h.
ASSAY
Dissolve 0.250 g in a mixture of 5 ml of water R and 1 ml of hydrochloric acid R1. Add 25.0 ml of
0.1M sodium edetate and dilute to 200 ml with water R. Neutralise with concentrated ammonia R, add
10 ml of ammonium chloride buffer solution pH 10.0 R and about 50 mg of mordant black 11 triturate R.
Titrate the excess of sodium edetate with 0.1M zinc sulphate until the colour changes from blue to
violet. Carry out a blank titration.
1 ml of 0.1M sodium edetate is equivalent to 13.61 mg of CaHPO
4
.
STORAGE
_________________________________________________________________________________________________________________________________________ Ph Eur
6-67
Calcium Hydrogen Phosphate
Dibasic Calcium Phosphate
CaHPO
4
,2H
2
O 172.1 7789-77-7
Calcium Hydrogen Phosphate complies with the requirements of the 3rd edition of the European
Pharmacopoeia for Calcium Hydrogen Phosphate Dihydrate [0116]. These requirements are reproduced after
the heading Definition below.
Ph Eur _________________________________________________________________________________________________________________________________________
DEFINITION
Calcium hydrogen phosphate dihydrate contains not less than 98.0 per cent and not more than the
equivalent of 105.0 per cent of CaHPO
4
,2H
2
O.
CHARACTERS
A white, crystalline powder, practically insoluble in cold water and in alcohol. It dissolves in dilute
hydrochloric acid and in dilute nitric acid.
IDENTIFICATION
A. Dissolve 0.1 g in a mixture of 5 ml of dilute nitric acid R and 5 ml of water R. The solution gives
reaction (b) of phosphates (2.3.1).
B. It gives reaction (b) of calcium (2.3.1).
TESTS
Solution S Dissolve 2.5 g in 20 ml of dilute hydrochloric acid R, filter if necessary and add dilute
ammonia R1 until a precipitate is formed. Add just sufficient dilute hydrochloric acid R to dissolve the
precipitate and dilute to 50 ml with distilled water R.
Monocalcium and tricalcium phosphates Dissolve 2.00 g in 30.0 ml of 1M hydrochloric acid and
add 20 ml of water R and 0.05 ml of methyl orange solution R. Titrate the excess hydrochloric acid
with 1M sodium hydroxide. The quantity of 1M hydrochloric acid required is 11.0 ml to 12.5 ml.
Carbonates Shake 0.5 g with 5 ml of carbon dioxide-free water R and add 1 ml of hydrochloric acid R.
No effervescence is produced.
Chlorides (2.4.4). Dissolve 0.5 g in a mixture of 1 ml of nitric acid R and 10 ml of water R and dilute
to 50 ml with water R. 15 ml of the solution complies with the limit test for chlorides (330 ppm).
with distilled water R. 15 ml of the solution complies with the limit test for sulphates (0.5 per cent).
Barium To 10 ml of solution S add 0.5 ml of dilute sulphuric acid R. After 15 min, any opalescence
in the solution is not more intense than that in a mixture of 10 ml of solution S and 0.5 ml of distilled
water R.
Iron (2.4.9). 0.5 ml of solution S diluted to 10 ml with water R complies with the limit test for iron
(400 ppm).
ASSAY
Dissolve 0.300 g in a mixture of 1 ml of hydrochloric acid R1 and 5 ml of water R. Add 25.0 ml of
0.1M sodium edetate and dilute to 200 ml with water R. Neutralise with concentrated ammonia R and
add 10 ml of ammonium chloride buffer solution pH 10.0 R and about 50 mg of mordant black 11
triturate R. Titrate the excess of sodium edetate with 0.1M zinc sulphate until the colour changes from
blue to violet. Carry out a blank titration.
1 ml of 0.1M sodium edetate is equivalent to 17.21 mg of CaHPO
4
,2H
2
O.
STORAGE
_________________________________________________________________________________________________________________________________________ Ph Eur
6-68
Calcium Hydroxide
Ca(OH)
2
74.1 1305-62-0
Calcium Hydroxide complies with the requirements of the 3rd edition of the European Pharmacopoeia [1078].
Preparation
Calcium Hydroxide Solution
Ph Eur _________________________________________________________________________________________________________________________________________
DEFINITION
Calcium hydroxide contains not less than 95.0 per cent and not more than the equivalent of
100.5 per cent of Ca(OH)
2
.
CHARACTERS
A white, fine powder, practically insoluble in water.
IDENTIFICATION
A. To 0.80 g in a mortar, add 10 ml of water R and 0.5 ml of phenolphthalein solution R and mix. The
suspension turns red. On addition of 17.5 ml of 1M hydrochloric acid, the suspension becomes
colourless without effervescing. The red colour occurs again when the mixture is triturated for 1 min.
On addition of a further 6 ml of 1M hydrochloric acid and triturating, the solution becomes colourless.
B. Dissolve about 0.1 g in dilute hydrochloric acid R and dilute to 10 ml with water R. 5 ml of the
solution gives reaction (b) of calcium (2.3.1).
TESTS
Matter insoluble in hydrochloric acid Dissolve 2.0 g in 30 ml of hydrochloric acid R. Boil the
solution and filter. Wash the residue with hot water R. The residue weighs not more than 10 mg
(0.5 per cent).
Carbonates Not more than 5.0 per cent of CaCO
3
.
Add 5.0 ml of 1M hydrochloric acid to the titrated solution obtained under Assay and titrate with 1M
sodium hydroxide using 0.5 ml of methyl orange solution R as indicator.
1 ml of 1M hydrochloric acid is equivalent to 50.05 mg of CaCO
3
.
Chlorides (2.4.4). Dissolve 0.30 g in a mixture of 2 ml of nitric acid R and 10 ml of water R and
dilute to 30 ml with water R. 15 ml of the solution complies with the limit test for chlorides
(330 ppm).
Sulphates (2.4.13). Dissolve 0.15 g in a mixture of 5 ml of dilute hydrochloric acid R and 10 ml of
water R and dilute to 60 ml with distilled water R. 15 ml of the solution complies with the limit test for
sulphates (0.4 per cent).
Arsenic (2.4.2). Dissolve 0.50 g in 5 ml of brominated hydrochloric acid R and dilute to 50 ml with
water R. 25 ml of the solution complies with the limit test for arsenic (4 ppm).
Magnesium and alkali metals Dissolve 1.0 g in a mixture of 10 ml of hydrochloric acid R and 40 ml
of water R. Boil and add 50 ml of a 63 g/l solution of oxalic acid R. Neutralise with ammonia R and
dilute to 200 ml with water R. Allow to stand for 1 h and filter through a suitable filter. To 100 ml of
the filtrate, add 0.5 ml of sulphuric acid R. Cautiously evaporate to dryness and ignite. The residue
weighs not more than 20 mg (4.0 per cent calculated as sulphates).
Heavy metals (2.4.8). Dissolve 1.0 g in 10 ml of hydrochloric acid R1 and evaporate to dryness on a
water-bath. Dissolve the residue in 20 ml of water R and filter. 12 ml of the filtrate complies with
limit test A for heavy metals (20 ppm). Prepare the standard using lead standard solution (1 ppm
Pb) R.
ASSAY
To 1.500 g in a mortar, add 20 ml to 30 ml of water R and 0.5 ml of phenolphthalein solution R.
Titrate with 1M hydrochloric acid by triturating the substance until the red colour disappears. The final
solution is used in the tests for carbonates.
1 ml of 1M hydrochloric acid is equivalent to 37.05 mg of Ca(OH)
2
.
STORAGE
_________________________________________________________________________________________________________________________________________ Ph Eur
6-69
Calcium Lactate Pentahydrate
Calcium Lactate
H
3
C
COO
H OH
2
Ca
2
+
C
6
H
10
CaO
6
,5H
2
O (approx.) 218.2 (anhydrous) 814-80-2(anhydrous)
Calcium Lactate Pentahydrate complies with the requirements of the 3rd edition of the European
Preparations
Calcium and Ergocalciferol Tablets
Calcium Lactate Tablets
Ph Eur _________________________________________________________________________________________________________________________________________
DEFINITION
Calcium lactate pentahydrate contains not less than 98.0 per cent and not more than the equivalent
of 102.0 per cent of calcium (RS)-2-hydroxypropionate or of a mixture of calcium (R)-, (S)- and
(RS)-2-hydroxypropionates, calculated with reference to the dried substance. It contains not less than
22.0 per cent and not more than 27.0 per cent of water, determined by the loss on drying.
CHARACTERS
A white or almost white, crystalline or granular powder, slightly efflorescent, soluble in water, freely
soluble in boiling water, very slightly soluble in alcohol.
IDENTIFICATION
A. It complies with the test for loss on drying (see Tests).
B. It gives the reaction of lactates (2.3.1).
TESTS
Solution S Dissolve 5.0 g with heating in carbon dioxide-free water R prepared from distilled water R,
allow to cool and dilute to 100 ml with the same solvent.
not more intensely coloured than reference solution BY
6
(Method II, 2.2.2).
Acidity or alkalinity To 10 ml of solution S add 0.1 ml of phenolphthalein solution R and 0.5 ml of
0.01M hydrochloric acid. The solution is colourless. Not more than 2.0 ml of 0.01M sodium hydroxide is
required to change the colour of the indicator to pink.
Volatile fatty acids In a 100 ml ground-glass-stoppered flask stir 0.5 g with 1 ml of phosphoric
acid R. Close the flask. Heat cautiously at 50C for 10 min. No unpleasant odour resembling that of
the lower fatty acids is recognisable immediately after opening the flask.
limit test for sulphates (400 ppm).
Barium To 10 ml of solution S add 1 ml of calcium sulphate solution R. Allow to stand for 15 min.
Any opalescence in the solution is not more intense than that in a mixture of 1 ml of distilled water R
and 10 ml of solution S.
(50 ppm).
Magnesium and alkali salts To 20 ml of solution S add 20 ml of water R, 2 g of ammonium
chloride R and 2 ml of dilute ammonia R1. Heat to boiling and rapidly add 40 ml of hot ammonium
oxalate solution R. Allow to stand for 4 h, dilute to 100.0 ml with water R and filter. To 50.0 ml of the
filtrate add 0.5 ml of sulphuric acid R, evaporate to dryness and ignite the residue to constant mass at
600C. The residue weighs not more than 5 mg (1 per cent).
at 125C.
6-70
ASSAY
Dissolve 0.200 g in water R and dilute to 300 ml with the same solvent. Carry out the complexo-
metric titration of calcium (2.5.11).
6
H
10
CaO
6
.
STORAGE
_________________________________________________________________________________________________________________________________________ Ph Eur
6-71
Calcium Lactate Trihydrate
C
6
H
10
CaO
6
,3H
2
O (approx.) 218.2 (anhydrous) 814-80-2 (anhydrous)
Calcium Lactate Trihydrate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Calcium Lactate Tablets
Ph Eur _________________________________________________________________________________________________________________________________________
DEFINITION
Calcium lactate trihydrate contains not less than 98.0 per cent and not more than the equivalent of
102.0 per cent of calcium (RS)-2-hydroxypropionate or of a mixture of calcium (R)-, (S)- and (RS)-2-
hydroxypropionates, calculated with reference to the dried substance. It contains not less than
15.0 per cent and not more than 20.0 per cent of water, determined by the loss on drying.
CHARACTERS
A white or almost white, crystalline or granular powder, soluble in water, freely soluble in boiling
water, very slightly soluble in alcohol.
IDENTIFICATION
A. It complies with the test for loss on drying (see Tests).
B. It gives the reaction of lactates (2.3.1).
TESTS
Solution S Dissolve 5.0 g with heating in carbon dioxide-free water R prepared from distilled water R,
allow to cool and dilute to 100 ml with the same solvent.
6
(Method II, 2.2.2).
Acidity or alkalinity To 10 ml of solution S add 0.1 ml of phenolphthalein solution R and 0.5 ml of
0.01M hydrochloric acid. The solution is colourless. Not more than 2.0 ml of 0.01M sodium hydroxide is
required to change the colour of the indicator to pink.
Volatile fatty acids In a 100 ml ground-glass-stoppered flask stir 0.5 g with 1 ml of phosphoric acid R.
Close the flask. Heat cautiously at 50C for 10 min. No unpleasant odour resembling that of the
lower fatty acids is recognisable immediately after opening the flask.
Sulphates (2.4.13). 7.5 ml of solution S diluted to 15 ml with distilled water R complies with the limit
Barium To 10 ml of solution S add 1 ml of calcium sulphate solution R. Allow to stand for 15 min.
Any opalescence in the solution is not more intense than that in a mixture of 1 ml of distilled water R
and 10 ml of solution S.
(50 ppm).
Magnesium and alkali salts To 20 ml of solution S add 20 ml of water R, 2 g of ammonium
chloride R and 2 ml of dilute ammonia R1. Heat to boiling and rapidly add 40 ml of hot ammonium
oxalate solution R. Allow to stand for 4 h, dilute to 100.0 ml with water R and filter. To 50.0 ml of the
filtrate add 0.5 ml of sulphuric acid R, evaporate to dryness and ignite the residue to constant mass at
600C. The residue weighs not more than 5 mg (1 per cent).
at 125C.
ASSAY
Dissolve 0.200 g in water R and dilute to 300 ml with the same solvent. Carry out the complexometric
titration of calcium (2.5.11).
6
H
10
CaO
6
.
STORAGE
_________________________________________________________________________________________________________________________________________ Ph Eur
6-72
Calcium Levulinate Dihydrate
H
3
C COO
_
O
2
Ca
2+
,2H
2
O
C
10
H
14
CaO
6
,2H
2
O 306.3 5743-49-7
Calcium Levulinate Dihydrate complies with the requirements of the 3rd edition of the European
Ph Eur ________________________________________________________________________________________________________________
DEFINITION
Calcium levulinate dihydrate contains not less than 98.0 per cent and not more than the equivalent of
101.0 per cent of calcium di(4-oxopentanoate), calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, freely soluble in water, very slightly soluble in alcohol,
practically insoluble in methylene chloride.
IDENTIFICATION
First identification: A, D, E.
obtained with calcium levulinate dihydrate CRS.
substance.
same solvent.
Reference solution. Dissolve 60 mg of calcium levulinate dihydrate CRS in water R and dilute to 1 ml
volumes of alcohol R. Dry the plate at 100C to 105C for 20 min and allow to cool. Spray with a
30 g/l solution of potassium permanganate R. Dry the plate in a current of warm air for about 5 min or
until the spots become yellow. Examine in daylight. The principal spot in the chromatogram
obtained with the test solution is similar in position, colour and size to the principal spot in the
C. To 1 ml of solution S (see Tests), add 20 ml of a 2.5 g/l solution of dinitrophenylhydrazine R in
dilute hydrochloric acid R. Allow to stand for 15 min. Filter, wash the precipitate with water R. Dry the
precipitate in an oven at 100C to 105C. The melting point (2.2.14) is 203C to 210C.
D. It gives reaction (b) of calcium (2.3.1).
E. It complies with the test for loss on drying (see Tests).
TESTS
solution Y
6
(Method II, 2.2.2).
Oxidisable substances To 1 ml of solution S, add 10 ml of water R, 1 ml of dilute sulphuric acid R
and 0.25 ml of a 3.0 g/l solution of potassium permanganate R. Mix. After 5 min, the violet colour of
the mixture is still visible.
Sucrose and reducing sugars To 5 ml of solution S, add 2 ml of hydrochloride acid R1 and dilute to
10 ml with water R. Heat to boiling for 5 min and allow to cool. Add 10 ml of sodium carbonate
solution R. Allow to stand for 5 min, dilute to 25 ml with water R and filter. To 5 ml of the filtrate,
add 2 ml of cupri-tartaric solution R and heat to boiling for 1 min. No red precipitate is formed.
6-73
Sulphates (2.4.13). Dilute 7.5 ml of solution S to 15 ml with distilled water R. The solution complies
Magnesium and alkali metals To 10 ml of solution S, add 80 ml of water R, 10 ml of ammonium
chloride solution R and 1 ml of ammonia R. Heat to boiling. To the boiling solution, add dropwise
50 ml of warm ammonium oxalate solution R. Allow to stand for 4 h, then dilute to 200 ml with
water R and filter. To 100 ml of the filtrate, add 0.5 ml of sulphuric acid R. Evaporate to dryness on a
water-bath and ignite to constant mass at 600C. The residue weighs not more than 5.0 mg (1.0 per
cent).
Loss on drying (2.2.32). 11.0 per cent to 12.5 per cent, determined on 0.200 g by drying at 100C
to 105C.
Inject per kilogram of the rabbits mass 4 ml of a solution containing per millilitre 50 mg of the
substance to be examined.
ASSAY
1 ml of 0.1M sodium ededate is equivalent to 27.03 mg of C
10
H
14
CaO
6
.
STORAGE
LABELLING
The label states, where applicable, that the substance is apyrogenic.
_______________________________________________________________________________________________________________ Ph Eur
6-74
Calcium Pantothenate
N
COO
Me Me
OH H
O
HO
H
2
Ca
2
+
C
18
H
32
CaN
2
O
10
476.5 137-08-6
Calcium Pantothenate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Component of vitamin B.
Ph Eur _________________________________________________________________________________________________________________________________________
DEFINITION
Calcium pantothenate contains not less than 98.0 per cent and not more than the equivalent of
101.0 per cent of calcium bis[(R)-3-(2,4-dihydroxy-3,3-dimethylbutyramido) propionate], calculated
CHARACTERS
A white powder, slightly hygroscopic, freely soluble in water, slightly soluble in alcohol, practically
insoluble in ether.
IDENTIFICATION
B. Examine the chromatograms obtained in the test for 3-aminopropionic acid. The principal spot in
the chromatogram obtained with test solution (b) is similar in position, colour and size to the
C. To 1 ml of solution S (see Tests) add 1 ml of dilute sodium hydroxide solution R and 0.1 ml of copper
sulphate solution R. A blue colour develops.
D. It gives reaction (a) of calcium (2.3.1).
TESTS
Specific optical rotation (2.2.7). +25.5 to +27.5, determined on solution S and calculated with
3-Aminopropionic acid Examine by thin-layer chromatography (2.2.27), using silica gel G R as the
coating substance.
the same solvent.
Reference solution (a). Dissolve 20 mg of calcium pantothenate CRS in water R and dilute to 5 ml with
the same solvent.
Reference solution (b). Dissolve 10 mg of 3-aminopropionic acid R in water R and dilute to 50 ml with
the same solvent.
35 volumes of water R and 65 volumes of ethanol R. Dry the plate in a current of air and spray with
ninhydrin solution R1. Heat at 110C for 10 min. Any spot corresponding to 3-aminopropionic acid in
the chromatogram obtained with test solution (a) is not more intense than the spot in the chromato-
100C to 105C.
6-75
ASSAY
Dissolve 0.180 g in 50 ml of glacial acetic acid R. Titrate with 0.1M perchloric acid determining the
18
H
32
CaN
2
O
10
.
STORAGE
_________________________________________________________________________________________________________________________________________ Ph Eur
6-76
Calcium Phosphate
Tribasic Calcium Phosphate
1/01
7758-23-8
Calcium Phosphate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1052].
Preparation
Ph Eur _________________________________________________________________________________________________________________________________________
DEFINITION
Calcium phosphate consists of a mixture of calcium phosphates. It contains not less than 35.0 per
cent and not more than the equivalent of 40.0 per cent of Ca (A
r
40.08).
CHARACTERS
A white or almost white powder, practically insoluble in water. It dissolves in dilute hydrochloric acid
and in dilute nitric acid.
IDENTIFICATION
A. Dissolve 0.1 g in 5 ml of a 25 per cent V/V solution of nitric acid R. The solution gives reaction (b)
of phosphates (2.3.1).
B. It gives reaction (b) of calcium (2.3.1). Filter before adding potassium ferrocyanide solution R.
TESTS
Solution S Dissolve 2.50 g in 20 ml of dilute hydrochloric acid R. If the solution is not clear, filter it.
Add dilute ammonia R1 dropwise until a precipitate is formed. Dissolve the precipitate by adding
dilute hydrochloric acid R and dilute to 50 ml with distilled water R.
Chlorides (2.4.4). Dissolve 0.22 g in a mixture of 1 ml of nitric acid R and 10 ml of water R and
dilute to 100 ml with water R.15 ml of the solution complies with the limit test for chlorides (0.15 per
cent).
Fluorides Not more than 75 ppm of F, determined potentiometrically (Method I, 2.2.36) using a
fluoride-selective indicator electrode and a silver-silver chloride reference electrode.
Test solution. In a 50 ml volumetric flask, dissolve 0.250 g in 0.1M hydrochloric acid, add 5.0 ml of
fluoride standard solution (1 ppm F) R and dilute to 50.0 ml with 0.1M hydrochloric acid. To 20.0 ml of
the solution add 20.0 ml of total-ionic-strength-adjustment buffer R and 3 ml of an 82 g/l solution of
anhydrous sodium acetate R. Adjust to pH 5.2 with ammonia R and dilute to 50.0 ml with distilled
water R.
Reference solutions. To 5.0 ml, 2.0 ml, 1.0 ml, 0.5 ml and 0.25 ml of fluoride standard solution (10 ppm
F) R add 20.0 ml of total-ionic-strength-adjustment buffer R and dilute to 50.0 ml with distilled water R.
Carry out the measurements on 20.0 ml of each solution. Calculate the concentration of fluorides
using the calibration curve, taking into account the addition of fluoride to the test solution.
complies with the limit test for sulphates (0.5 per cent).
Arsenic (2.4.2).5 ml of solution S complies with limit test A for arsenic (4 ppm).
Acid-insoluble matter Dissolve 5.0 g in a mixture of 10 ml of hydrochloric acid R and 30 ml of
water R. Filter, wash the residue with water R and dry to constant mass at 100C to 105C. The
Loss on ignition Not more than 8.0 per cent, determined on 1.000 g by ignition at 800C for 30
min.
ASSAY
Dissolve 0.200 g in a mixture of 1 ml of hydrochloric acid R1 and 5 ml of water R. Add 25.0 ml of
0.1M sodium edetate and dilute to 200 ml with water R. Adjust to about pH 10 with concentrated
6-77
ammonia R. Add 10 ml of ammonium chloride buffer solution pH 10.0 R and a few milligrams of
mordant black 11 triturate R. Titrate the excess sodium edetate with 0.1M zinc sulphate until the colour
changes from blue to violet.
STORAGE
_________________________________________________________________________________________________________________________________________ Ph Eur
6-78
Calcium Polystyrene Sulphonate
Definition Calcium Polystyrene Sulphonate is a cation-exchange resin prepared in the calcium form
containing not less than 6.5% w/w and not more than 9.5% w/w of calcium, calculated with reference
to the dried substance. Each g exchanges not less than 1.3 mEq and not more than 2.0 mEq of
potassium, calculated with reference to the dried substance.
Characteristics A cream to light brown, fine powder.
Practically insoluble in water and in ethanol (96%).
Identification
calcium polystyrene sulphonate (RS 037).
B. Yields reaction C characteristic of calcium salts, Appendix VI.
Particle size Not more than 1% w/w is retained on a 150-m sieve, Appendix XVII B. Use 20 g and
sieve for 5 minutes.
Potassium Not more than 0.1% of K when determined by atomic emission spectrophotometry,
Appendix II D, measuring at 766.5 nm and using a solution prepared in the following manner. To
1.1 g of the substance being examined add 5 ml of hydrochloric acid, heat to boiling, cool and add
10 ml of water. Filter, wash the filter and residue with water and dilute the filtrate and washings to
25 ml with water. Use potassium standard solution (100 ppm K), suitably diluted with water, to prepare
the standard solutions.
Sodium Not more than 0.1% of Na when determined by atomic emission spectrophotometry, Appendix
II D, measuring at 589.0 nm and using a solution prepared in the following manner. To 1.1 g of the
substance being examined add 5 ml of hydrochloric acid, heat to boiling, cool and add 10 ml of water.
Filter, wash the filter and residue with water and dilute the filtrate and washings to 25 ml with water.
Use sodium solution (200 ppm Na), suitably diluted with water, to prepare the standard solutions.
Arsenic 1 g complies with the limit test for arsenic, Appendix VII (1 ppm).
Heavy metals Heat 4 g until charred, cool, add 4 ml of lead-free nitric acid and 0.5 ml of sulphuric
acid dropwise and heat cautiously until white fumes are no longer evolved. Ignite in a muffle furnace
at 500 to 600 until a white residue is obtained. Cool, add 4 ml of hydrochloric acid and dilute to
20 ml. The resulting solution complies with limit test A for heavy metals, Appendix VII. Use 2 ml of
lead standard solution (10 ppm Pb) to prepare the standard (10 ppm).
Styrene Carry out the method for liquid chromatography, Appendix III D, using the following
solutions. For solution (1) shake 10 g of the substance being examined with 10 ml of acetone for 30
minutes, centrifuge and use the supernatant liquid. Solution (2) contains 0.0001% w/v of styrene in
acetone.
(25 cm 4 mm) packed with stationary phase C (Bondapak C18 is suitable), (b) a mixture of equal
volumes of acetonitrile and water as the mobile phase with a flow rate of 2 ml per minute and (c) a
Inject separately 20 l of each solution. The area of the peak corresponding to styrene in the
chromatogram obtained with solution (1) is not greater than the area of the principal peak in the
chromatogram obtained with solution (2) (1 ppm).
Potassium exchange capacity To 3 g of the substance being examined in a dry 250-ml glass-
stoppered flask add 100 ml of a solution containing 0.7455% w/v of potassium chloride and 0.4401%
w/v of potassium hydrogen carbonate in water (solution A), stopper and shake for 15 minutes. Filter and
dilute 2 ml of the filtrate to 1000 ml with water. Determine the concentration of unbound potassium
in this solution by atomic emission spectrophotometry, Appendix II D, measuring at 766.5 nm and using
solution A suitably diluted with water, to prepare the standard solutions. Calculate the potassium
exchange capacity of the substance being examined in milliequivalents taking the concentration of
potassium in solution A as 144 milliequivalents of K per litre.
Microbial contamination Carry out a quantitative evaluation for Enterobacteria and certain other
Gram-negative bacteria, Appendix XVI B1. 0.01 g of the substance being examined gives a negative
result, Table I (most probable number of bacteria per gram fewer than 10
2
).
Assay For calcium Carefully heat 1 g in a platinum crucible until a white ash is obtained and
dissolve in 10 ml of 2M hydrochloric acid with the aid of heat. Transfer the resulting solution to a
conical flask using 20 ml of water. Add 50 ml of 0.05M disodium edetate VS, 20 ml of ammonia buffer
pH 10.9 and titrate the excess of disodium edetate with 0.02M zinc sulphate VS, using a 0.5% w/v
solution of mordant black 11 in ethanol (96%) as indicator to a red purple end point. Each ml of
0.05M disodium edetate VS is equivalent to 2.004 mg of Ca.
Storage Calcium Polystyrene Sulphonate should be kept in an airtight container.
Action and use Used in the treatment of hyperkalaemia.
6-79
Calcium Sodium Lactate
2C
3
H
5
NaO
3
,(C
3
H
5
O
3
)
2
Ca,4H
2
O 514.4
Definition Calcium Sodium Lactate contains not less than 7.5% and not more than 8.5% of
calcium, Ca, and not less than 8.5% and not more than 10.0% of sodium, Na.
Characteristics A white powder or granules; odour, slight and characteristic; deliquescent.
Soluble in water and in boiling ethanol (96%); practically insoluble in ether.
Identification
A. Heat 1 g gently with 10 ml of 1M sulphuric acid and 0.1 g of potassium permanganate. The odour of
acetaldehyde is produced.
B. Yields the reactions characteristic of calcium salts and of sodium salts, Appendix VI.
Acidity or alkalinity Dissolve 5 g in 100 ml of hot carbon dioxide-free water. The solution is not
alkaline to phenolphthalein solution R1 and not more than 2.5 ml of 0.1M sodium hydroxide VS is
required to change the colour of the solution to pink.
Heavy metals A 10.0% w/v solution complies with limit test A for heavy metals, Appendix VII. Use
Chloride Dissolve 0.50 g in 100 ml of water containing 1.5 ml of nitric acid. 15 ml of the resulting
solution complies with the limit test for chlorides, Appendix VII (700 ppm).
Sulphate Dissolve 0.25 g in 60 ml of water containing 3 ml of 2M hydrochloric acid. 15 ml of the
resulting solution complies with the limit test for sulphates, Appendix VII (0.24%).
Reducing sugars 1.0 g dissolved in 10 ml of water and boiled with 5 ml of cupri-tartaric solution R1
yields not more than the slightest trace of a red precipitate.
Assay
For calcium Dissolve 0.5 g in a few ml of water, acidify if necessary and dilute to about 50 ml with
water. Titrate with 0.05M disodium edetate VS to within a few ml of the expected end point, add 4 ml
of 10M sodium hydroxide and 0.1 g of solochrome dark blue mixture and continue the titration until the
colour changes from pink to full blue. Each ml of 0.05M disodium edetate VS is equivalent to
2.004 mg of Ca.
For sodium Ignite 2 g gently until carbonised, cool, boil the residue with 50 ml of water and 50 ml
of 0.5M hydrochloric acid VS and filter. Wash the residue with water and titrate the excess of acid in
the combined filtrate and washings with 0.5M sodium hydroxide VS using methyl orange solution as
indicator. Each ml of 0.5M hydrochloric acid VS, after the subtraction of one fifth of the volume of
0.05M disodium edetate VS that would be required by the calcium in the weight of sample taken, is
equivalent to 11.49 mg of Na.
Storage Calcium Sodium Lactate should be kept in a well-closed container.
6-80
Calcium Stearate
1592-23-0
Calcium Stearate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0882].
Ph Eur ________________________________________________________________________________________________________________
DEFINITION
Calcium stearate is a mixture of calcium salts of different fatty acids consisting mainly of stearic acid
[(C
17
H
35
COO)
2
Ca; 607] and palmitic acid [(C
15
H
31
COO)
2
Ca; 550.9] with minor proportions of
other fatty acids. It contains not less than 6.4 per cent and not more than 7.4 per cent of Ca (A
r
40.08), calculated with reference to the dried substance. The fatty acid fraction contains not less than
40.0 per cent of stearic acid and the sum of stearic acid and palmitic acids is not less than 90.0 per
cent.
PRODUCTION
CHARACTERS
A fine, white or almost white, crystalline powder, practically insoluble in water, in alcohol and in
ether.
IDENTIFICATION
A. The residue obtained in the preparation of solution S (see Tests) has a freezing point (2.2.18) not
lower than 53C.
B. The acid value of the fatty acids (2.5.1) is 195 to 210, determined on 0.200 g of the residue
obtained in the preparation of solution S dissolved in 25 ml of the prescribed mixture of solvents.
C. Examine the chromatograms obtained in the test for fatty acid composition. The retention times
of the principal peaks in the chromatogram obtained with the test solution are approximately the
same as those of the principal peaks in the chromatogram obtained with the reference solution.
D. Neutralise 5 ml of solution S to red litmus paper R using strong sodium hydroxide solution R. The
solution gives reaction (b) of calcium (2.3.1).
TESTS
Solution S To 5.0 g add 50 ml of peroxide-free ether R, 20 ml of dilute nitric acid R and 20 ml of
distilled water R. Boil under a reflux condenser until dissolution is complete. Allow to cool. In a
separating funnel, separate the aqueous layer and shake the ether layer with two quantities, each of
5 ml, of distilled water R. Combine the aqueous layers, wash with 15 ml of peroxide-free ether R and
dilute the aqueous layer to 50 ml with distilled water R (solution S). Evaporate the ether layer to
dryness and dry the residue at 100C to 105C. Keep the residue for identification tests A and B.
Acidity or alkalinity To 1.0 g add 20 ml of carbon dioxide-free water R and boil for 1 min with
continuous shaking. Cool and filter. To 10 ml of the filtrate add 0.05 ml of bromothymol blue
solution R1. Not more than 0.5 ml of 0.01M hydrochloric acid or 0.01M sodium hydroxide is required to
change the colour of the indicator.
Chlorides (2.4.4). Dilute 0.5 ml of solution S to 15 ml with water R. The solution complies with the
limit test for chlorides (0.1 per cent).
Sulphates (2.4.13). Dilute 0.5 ml of solution S to 15 ml with distilled water R. The solution complies
with the limit test for sulphates (0.3 per cent).
Cadmium Not more than 3 ppm of Cd, determined by atomic absorption spectrometry (Method II,
2.2.23).
Test solution. Place 50.0 mg of the substance to be examined in a polytetrafluoroethylene digestion
bomb and add 0.5 ml of a mixture of 1 volume of hydrochloric acid R and 5 volumes of cadmium- and
lead-free nitric acid R. Allow to digest at 170C for 5 h. Allow to cool. Dissolve the residue in water R
and dilute to 5.0 ml with the same solvent.
Reference solutions. Prepare the reference solutions using cadmium standard solution (10 ppm Cd) R,
diluted if necessary with a 1 per cent V/V solution of hydrochloric acid R.
Measure the absorbance at 228.8 nm using a cadmium hollow-cathode lamp as source of radiation
6-81
2.2.23).
Test solution. Use the solution described in the test for cadmium.
Reference solutions. Prepare the reference solutions using lead standard solution (10 ppm Pb) R, diluted
Measure the absorbance at 283.3 nm using a lead hollow-cathode lamp as source of radiation and
2.2.23).
Test solution. Use the solution described in the test for cadmium.
Reference solutions. Prepare the reference solutions using nickel standard solution (10 ppm Ni) R, diluted
Measure the absorbance at 232.0 nm using a nickel hollow-cathode lamp as source of radiation
100C to 105C.
3
micro-organisms
per gram, determined by plate count. It complies with the test for Escherichia coli (2.6.13).
ASSAY
Calcium To 0.500 g in a 250 ml conical flask add 50 ml of a mixture of equal volumes of butanol R
and ethanol R, 5 ml of concentrated ammonia R, 3 ml of ammonium chloride buffer solution pH 10.0 R,
30.0 ml of 0.1M sodium edetate and 15 mg of mordant black 11 triturate R. Heat to 45C to 50C until
the solution is clear. Cool and titrate with 0.1M zinc sulphate until the colour changes from blue to
violet. Carry out a blank titration.
Fatty acid composition Examine by gas chromatography (2.2.28).
Test solution. In a conical flask fitted with a reflux condenser, dissolve 0.10 g of the substance to be
examined in 5 ml of boron trifluoride-methanol solution R. Boil under a reflux condenser for 10 min.
Add 4 ml of heptane R through the condenser and boil again under a reflux condenser for 10 min.
Allow to cool. Add 20 ml of a saturated solution of sodium chloride R. Shake and allow the layers to
separate. Remove about 2 ml of the organic layer and dry over 0.2 g of anhydrous sodium sulphate R.
Dilute 1.0 ml of the solution to 100.0 ml with heptane R.
Reference solution. Prepare the reference solution in the same manner as the test solution using
50.0 mg of palmitic acid CRS and 50.0 mg of stearic acid CRS instead of calcium stearate.
a fused-silica column 30 m long and 0.32 mm in internal diameter coated with macrogol
20,000 R (film thickness 0.5 m),
helium for chromatography R as the carrier gas at a flow rate of 2.4 ml/min,
with the following temperature programme:
Time
(min)
Temperature
(C)
Rate
(C/min)
Comment
Column 0 2 70 isothermal
2 36 70 240 5 linear
gradient
36 41 240 isothermal
Injection port 220
Detector 260
Inject 1 l of the reference solution. When the chromatogram is recorded in the prescribed
conditions, the retention time of methyl palmitate relative to that of methyl stearate is about 0.88.
The test is not valid unless, in the chromatogram obtained with the reference solution, the resolution
between the peaks corresponding to methyl stearate and methyl palmitate is at least 5.0.
Inject 1 l of the test solution. Calculate the percentage content of stearic acid and palmitic acid
from the areas of the peaks in the chromatogram obtained with the test solution by the normalisation
procedure, disregarding the peak due to the solvent.
STORAGE
_______________________________________________________________________________________________________________ Ph Eur
07-1
Dried Calcium Sulphate
Exsiccated Calcium Sulphate; Plaster of Paris
CaSO
4
,H
2
O 145.1 26499-65-0
Definition Dried Calcium Sulphate is prepared by heating powdered gypsum, CaSO
4
,2H
2
O, at
about 150 in a controlled manner such that it is substantially converted into the hemihydrate,
CaSO
4
,H
2
O, with minimum production of the anhydrous phases of calcium sulphate. It may
contain suitable setting accelerators or decelerators.
Characteristics A white or almost white powder; odourless or almost odourless; hygroscopic.
Slightly soluble in water; more soluble in dilute mineral acids; practically insoluble in ethanol
(96%).
Identification Yields the reactions characteristic of calcium salts and of sulphates, Appendix VI.
Setting properties 20 g mixed with 10 ml of water at 15 to 20 in a cylindrical mould about 2.4 cm
in diameter sets in 4 to 11 minutes. The mass thus produced, after standing for 3 hours, possesses
sufficient hardness to resist pressure of the fingers at the edges, which retain their sharpness of outline
and do not crumble.
Loss on ignition When ignited to constant weight at red heat, loses 4.5% to 8.0% of its weight. Use
1 g.
Storage Dried Calcium Sulphate should be kept in a well-closed container.
07-2
Calcium Sulphate Dihydrate
CaSO
4
,2H
2
O 72.2 10101-41-4
Calcium Sulphate Dihydrate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Calcium sulphate dihydrate contains not less than 98.0 per cent and not more than the equivalent
of 102.0 per cent of CaSO
4
,2H
2
O.
CHARACTERS
A white, fine powder, very slightly soluble in water, practically insoluble in alcohol.
IDENTIFICATION
A. It complies with the test for loss on ignition (see Tests).
B. Solution S (see Tests) gives reaction (a) of sulphates (2.3.1).
C. Solution S gives reaction (a) of calcium (2.3.1).
TESTS
Solution S Dissolve 1.0 g in 50 ml of a 10 per cent V/V solution of hydrochloric acid R by heating at
50C for 5 min. Allow the solution to cool.
Acidity or alkalinity Shake 1.5 g with 15 ml of carbon dioxide-free water R for 5 min. Allow to stand
for 5 min and filter. To 10 ml of the filtrate, add 0.1 ml of phenolphthalein solution R and 0.25 ml of
0.01M sodium hydroxide. The solution is red. Add 0.30 ml of 0.01M hydrochloric acid. The solution is
colourless. Add 0.2 ml of methyl red solution R. The solution is reddish-orange.
Chlorides (2.4.4). Shake 0.5 g with 15 ml of water R for 5 min. Allow to stand for 15 min and filter.
Dilute 5 ml of the filtrate to 15 ml with water R. The solution complies with the limit test for
Heavy metals (2.4.8). To 2.5 g add a mixture of 2 ml of hydrochloric acid R and 15 ml of water R.
Heat to boiling. Cool and then add 0.5 ml of phenolphthalein solution R. Cautiously add concentrated
ammonia R until the colour changes to pink. Add 0.5 ml of glacial acetic acid R and dilute to 25 ml
with water R. Filter. 12 ml of the filtrate complies with limit test A for heavy metals (20 ppm).
Iron (2.4.9). To 0.25 g add a mixture of 5 ml of hydrochloric acid R and 20 ml of water R. Heat to
boiling, cool and filter. 10 ml of the filtrate complies with the limit test for iron (100 ppm).
Loss on ignition 18.0 per cent to 22.0 per cent, determined on 1.000 g by ignition to constant mass
at 800C.
ASSAY
1 ml of 0.1M sodium edetate is equivalent to 17.22 mg of CaSO
4
,2H
2
O.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
07-3
Calendula Flower
Calendula Flower complies with the requirements of the 3rd edition of the European Pharmacopoeia [1297].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Calendula flower consists of the whole or cut, dried, and fully opened flowers which have been
detached from the receptacle of the cultivated, double-flowered varieties of Calendula officinalis L. It
contains not less than 0.4 per cent of flavonoids, calculated as hyperoside (C
21
H
20
O
12
M
r
464.4)
with reference to the dried drug.
CHARACTERS
IDENTIFICATION
A. The ligulate florets consist of a yellow or orange-yellow ligule, about 3 mm to 5 mm wide and
about 7 mm in the middle part, with a three toothed apex and a hairy, partly sickle-shaped yellowish-
brown to orange-brown tube with a projecting style and a bifid stigma occasionally with a partly bent
yellowish-brown to orange-brown ovary. The tubular florets, about 5 mm long, are present and
consist of the yellow, orange-red or red-violet five lobed corolla and the yellowish-brown or orange-
brown tube, hairy in its lower part, mostly with a partly bent yellowish-brown to orange-brown ovary.
B. Reduce to a powder (355). The powder is yellowish-brown. Examine under a microscope using
chloral hydrate solution R. The powder shows fragments of the corollas containing light yellow oil
droplets, some with fairly large anomocytic stomata (2.8.3), others containing prisms and very small
cluster crystals of calcium oxalate; covering trichomes biseriate, multicellular and conical, glandular
trichomes with a uniseriate or biseriate, multicellular biseriate stalk and a large, ovoid, biseriate and
multicellular head; spherical pollen grains up to about 40 m in diameter with a sharply spiny exine
and three germinal pores; occasional fragments of the stigmas with short, bulbous papillae.
C. Examine by thin-layer chromatography (2.2.27), using a suitable silica gel as the coating
substance.
Test solution. To 1.0 g of the powdered drug (500) add 10 ml of methanol R and heat on a water-bath
under a reflux condenser for 10 min. Cool and filter.
Reference solution. Dissolve 1.0 mg of caffeic acid R, 1.0 mg of chlorogenic acid R and 2.5 mg of rutin R
in 10 ml of methanol R.
Apply separately to the plate, as bands, 20 l of the test solution and 10 l of the reference solution.
Develop over a path of 10 cm using a mixture of 10 volumes of anhydrous formic acid R, 10 volumes
of water R and 80 volumes of ethyl acetate R. Allow the plate to dry at 100C to 105C and spray the
still warm plate with a 10 g/l solution of diphenylboric acid aminoethyl ester R in methanol R and then
spray with a 50 g/l solution of macrogol 400 R in methanol R. Allow the plate to dry in air for 30 min
and examine in ultraviolet light at 365 nm. The chromatogram obtained with the reference solution
shows in the lower part a yellowish-brown fluorescent zone (rutin), in the middle part a light bluish
fluorescent zone (chlorogenic acid) and in the upper part a light bluish fluorescent zone (caffeic acid).
The chromatogram obtained with the test solution shows a yellowish-brown fluorescent zone corres-
ponding in position to the zone due to rutin in the chromatogram obtained with the reference
solution, below and directly above it, it shows a yellowish-green fluorescent zone and a light bluish
fluorescent zone corresponding to the zone due to chlorogenic acid in the chromatogram obtained
with the reference solution, a yellowish-green fluorescent zone above it and a light bluish fluorescent
zone shortly below the zone due to caffeic acid in the chromatogram obtained with the reference
solution. Further zones are present.
TESTS
Foreign matter (2.8.2). Not more than 5 per cent of bracts and not more than 2 per cent of other
foreign matter.
ASSAY
Stock solution. In a 100 ml round-bottomed flask introduce 0.800 g of the powdered drug (500), 1 ml
of a 5 g/l solution of hexamethylenetetramine R, 20 ml of acetone R and 7 ml of hydrochloric acid R1.
Boil the mixture under a reflux condenser for 30 min. Filter the liquid through a plug of absorbent
07-4
cotton in a 100 ml flask. Add the absorbent cotton to the residue in the round-bottomed flask and
extract with two quantities, each of 20 ml, of acetone R, each time boiling under a reflux condenser
for 10 min. Allow to cool to room temperature, filter the liquid through a plug of absorbent cotton
then filter the combined acetone solution through a filter-paper in the volumetric flask and dilute to
100.0 ml with acetone R by rinsing of the flask and the filter. Introduce 20.0 ml of the solution into a
separating funnel, add 20 ml of water R and extract the mixture with one quantity of 15 ml and then
three quantities, each of 10 ml, of ethyl acetate R. Combine the ethyl acetate extracts in a separating
funnel, rinse with two quantities, each of 50 ml, of water R, filter the extract over 10 g of anhydrous
sodium sulphate R in to a 50 ml volumetric flask and dilute to 50.0 ml with ethyl acetate R.
compensation solution at 425 nm.
Calculate the percentage content of flavonoids, calculated as hyperoside, from the expression:
m
A 25 . 1
i.e. taking the specific absorbance of hyproside to be 500.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
07-5
Natural Camphor
Me Me
Me
O
C
10
H
16
O 152.2 464-49-3
Natural Camphor complies with the requirements of the 3rd edition of the European Pharmacopoeia for
D-Camphor [1400]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
D-Camphor is (1R,4R)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-one.
CHARACTERS
A white, crystalline powder or friable, crystalline masses, highly volatile even at room temperature,
slightly soluble in water, very soluble in alcohol and in light petroleum, freely soluble in fatty oils,
very slightly soluble in glycerol.
IDENTIFICATION
obtained with racemic camphor CRS.
D. Dissolve 1.0 g in 30 ml of methanol R. Add 1.0 g of hydroxylamine hydrochloride R and 1.0 g of
anhydrous sodium acetate R. Boil under a reflux condenser for 2 h. Allow to cool and add 100 ml of
water R. Filter, wash the precipitate obtained with 10 ml of water R and recrystallise from 10 ml of a
mixture of 4 volumes of alcohol R and 6 volumes of water R. The crystals, dried in vacuo, melt
(2.2.14) at 118C to 121C.
TESTS
Carry out the weighings rapidly.
Solution S Dissolve 2.50 g in 10 ml of alcohol R and dilute to 25.0 ml with the same solvent.
Acidity or alkalinity To 10 ml of solution S add 0.1 ml of phenolphthalein solution R1. The solution
is colourless. Not more than 0.2 ml of 0.1M sodium hydroxide is required to change the colour of the
indicator.
Optical rotation (2.2.7). +40.0 to +43.0, determined on solution S.
Test solution. Dissolve 50 mg of the substance to be examined in hexane R and dilute to 50.0 ml with
the same solvent.
Reference solution (a). Dissolve 50 mg of the substance to be examined and 50 mg of bornyl acetate R
in hexane R and dilute to 50.0 ml with the same solvent.
Reference solution (b). Dilute 1.0 ml of the test solution to 200.0 ml with hexane R.
a column 2 m long and 2 mm in internal diameter packed with diatomaceous earth for gas chroma-
tography R impregnated with 10 per cent m/m of macrogol 20,000 R,
maintaining the temperature of the column at 130C and that of the injection port and the detector
at 200C.
Inject 1 l of each solution and adjust the sensitivity of the system so that the height of the principal
peak in the chromatogram obtained with the test solution is about 80 per cent of the full scale of the
recorder. Record the chromatogram for three times the retention time of camphor. The test is not
valid unless: in the chromatogram obtained with reference solution (a), the resolution between the
07-6
peaks corresponding to camphor and bornyl acetate is at least 1.5; in the chromatogram obtained
with reference solution (b), the principal peak has a signal-to-noise ratio of at least 5. In the chroma-
togram obtained with the test solution: the sum of the areas of the peaks, apart from the principal
peak, is not greater than 4 per cent of the area of the principal peak; none of the peaks, apart from the
principal peak, has an area greater than 2 per cent of the area of the principal peak. Disregard any
peak with an area less than one tenth of the peak in the chromatogram obtained with reference
solution (b).
Halogens Dissolve 1.0 g in 10 ml of 2-propanol R in a distillation flask. Add 1.5 ml of dilute sodium
hydroxide solution R and 50 mg of nickel-aluminium alloy R. Heat on a water-bath until the
2-propanol R has evaporated. Allow to cool and add 5 ml of water R. Mix and filter through a wet
filter previously washed with water R until free from chlorides. Dilute the filtrate to 10.0 ml with
water R. To 5.0 ml of the solution, add nitric acid R dropwise until the precipitate which forms is
redissolved and dilute to 15 ml with water R. The solution complies with the limit test for chlorides
(2.4.4) (100 ppm).
Water Dissolve 1 g in 10 ml of light petroleum R. The solution is clear (2.2.1).
Residue on evaporation Evaporate 2.0 g on a water-bath and dry at 100C to 105C for 1 h. The
STORAGE
__________________________________________________________________________________________________________ Ph Eur
07-7
Racemic Camphor
Me Me
Me
O
and enantiomer
C
10
H
16
O 152.2 76-22-2
Racemic Camphor complies with the requirements of the 3rd edition of the European Pharmacopoeia [0655].
Action and use Counter-irritant.
Preparations
Camphorated Opium Tincture
Concentrated Camphorated Opium Tincture
Concentrated Camphor Water
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Racemic camphor is (1RS,4SR)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-one.
CHARACTERS
A white, crystalline powder or friable, crystalline masses, highly volatile even at room temperature,
slightly soluble in water, very soluble in alcohol, in ether and in light petroleum, freely soluble in fatty
oils, very slightly soluble in glycerol.
IDENTIFICATION
obtained with racemic camphor CRS. Examine the substances as mulls in liquid paraffin R.
D. Dissolve 1.0 g in 30 ml of methanol R. Add 1.0 g of hydroxylamine hydrochloride R and 1.0 g of
anhydrous sodium acetate R. Boil under a reflux condenser for 2 h. Allow to cool and add 100 ml of
water R. A precipitate is formed. Filter, wash with 10 ml of water R and recrystallise from 10 ml of a
mixture of 4 volumes of alcohol R and 6 volumes of water R. The crystals, dried in vacuo, melt
(2.2.14) at 118C to 121C.
TESTS
Carry out the weighings rapidly.
Acidity or alkalinity Dissolve 1.0 g in 10 ml of alcohol R and add 0.1 ml of phenolphthalein
solution R1. The solution is colourless. Not more than 0.2 ml of 0.1M sodium hydroxide is required to
change the colour of the indicator.
Optical rotation (2.2.7). +0.15 to 0.15, determined on solution S.
Test solution. Dissolve 50 mg of the substance to be examined in hexane R and dilute to 50.0 ml with
the same solvent.
Reference solution (a). Dissolve 50 mg of the substance to be examined and 50 mg of bornyl acetate R
in hexane R and dilute to 50.0 ml with the same solvent.
Reference solution (b). Dilute 1.0 ml of the test solution to 200.0 ml with hexane R.
a column 2 m long and 2 mm in internal diameter packed with diatomaceous earth for gas
chromatography R impregnated with 10 per cent m/m of macrogol 20,000 R,
07-8
at 200C.
Inject 1 l of each solution and adjust the sensitivity of the system so that the height of the principal
peak in the chromatogram obtained with the test solution is about 80 per cent of the full scale of the
recorder. Record the chromatograms for three times the retention time of camphor.
The test is not valid unless in the chromatogram obtained with reference solution (a), the
resolution between the peaks corresponding to camphor and bornyl acetate is not less than 1.5; in the
chromatogram obtained with reference solution (b), the principal peak has a signal-to-noise ratio of
at least 5. In the chromatogram obtained with the test solution: the sum of the areas of the peaks,
apart from the principal peak, is not greater than 4 per cent of the area of the principal peak; none of
the peaks, apart from the principal peak, has an area greater than 2 per cent of the area of the
principal peak. Disregard any peak with an area less than that of the peak in the chromatogram
Halogens Dissolve 1.0 g in 10 ml of 2-propanol R in a distillation flask. Add 1.5 ml of dilute sodium
hydroxide solution R and 50 mg of nickel-aluminium alloy R. Heat on a water-bath until the
2-propanol R has evaporated. Allow to cool and add 5 ml of water R. Mix and filter through a wet
filter previously washed with water R until free from chlorides. Dilute the filtrate to 10.0 ml with
water R. To 5.0 ml of the solution, add nitric acid R dropwise until the precipitate which forms is
redissolved and dilute to 15 ml with water R. The solution complies with the limit test for chlorides
(2.4.4) (100 ppm).
Water Dissolve 1 g in 10 ml of light petroleum R. The solution is clear (2.2.1).
Residue on evaporation Evaporate 2.0 g on a water-bath and dry at 100C to 105C for 1 h. The
STORAGE
__________________________________________________________________________________________________________ Ph Eur
07-9
Capreomycin Sulphate
1405-37-4
Definition Capreomycin Sulphate is a mixture of the sulphates of antimicrobial substances
produced by certain strains of Streptomyces capreolus. The potency is not less than 700 IU per mg.
Characteristics A white, or almost white, solid.
Very soluble in water; practically insoluble in chloroform, in ethanol (96%) and in ether.
Identification
hydrochloric acid exhibits a maximum only at 268 nm. The absorbance at the maximum is about 1.2.
sodium hydroxide exhibits a maximum only at 287 nm. The absorbance at the maximum is about 0.8.
C. Carry out the method for thin-layer chromatography, Appendix III A, using microcrystalline cellulose
as the coating substance and a mixture of 60 volumes of propan-2-ol, 25 volumes of 1M hydrochloric
acid and 15 volumes of butan-2-one as the mobile phase. Apply separately to the plate 5 l of each of
the following solutions. For solution (1) dissolve 5 mg of the substance being examined in a mixture
of 0.5 ml of hydrochloric acid and 0.5 ml of water, heat in a sealed tube at 100 for 16 hours, evaporate
to dryness on a water bath, continue to heat until any residual hydrogen chloride has been removed
and dissolve the residue in 1 ml of water. Prepare solution (2) in the same manner, but using
capreomycin sulphate BPCRS in place of the substance being examined. After removal of the plate, dry
it in a current of cold air for 15 minutes and then at 100 for 15 minutes. Allow to cool, spray with
cadmium and ninhydrin solution and heat at 100 for 30 minutes. The spots in the chromatogram
obtained with solution (1) correspond to those in the chromatogram obtained with solution (2).
D. Yields the reactions characteristic of sulphates, Appendix VI.
Capreomycin I Carry out the method for liquid chromatography, Appendix III D, using 20 l of each
of two solutions in water containing (1) 0.025% w/v of the substance being examined and (2) 0.025%
w/v of capreomcyin sulphate BPCRS.
The chromatographic procedure may be carried out using (a) a stainless steel column (15 cm 4.6
mm) packed with particles of silica, the surface of which has been modified with chemically-bonded
cyano groups (Spherisorb CN is suitable) (5 m), (b) a mixture of 450 volumes of methanol and 550
volumes of a 0.05% w/v solution of ammonium hydrogen sulphate as the mobile phase at a flow rate of
1.5 ml per minute and (c) a detection wavelength of 268 nm.
The test is not valid unless the resolution factor between the two principal peaks in the chromato-
gram obtained with solution (2) is at least 1.5.
In the chromatogram obtained with solution (1) the sum of the areas of the two principal peaks,
due to capreomycins IA and IB, is not less than 90% of the total area of all the peaks.
Assay Carry out the biological assay of antibiotics, Appendix XIV A. The precision of the assay is such
that the fiducial limits of error are not less than 95% and not more than 105% of the estimated
potency.
Storage Capreomycin Sulphate should be kept in a well-closed container and stored at a
temperature not exceeding 25. If the material is sterile, the container should be sterile, tamper-
evident and sealed so as to exclude micro-organisms.
Labelling The label states (1) the number of IU (Units) per mg; (2) the date after which the material
is not intended to be used; (3) the conditions under which it should be stored; (4) where applicable,
that it is sterile; (5) where applicable, that it is apyrogenic.
Preparation
Capreomycin Injection
Action and use Antituberculous.
Capreomycin Sulphate intended intended for use in the manufacture of a parenteral dosage form without a
further appropriate procedure for the removal of pyrogens complies with the following additional requirement.
Bacterial endotoxins Carry out the test for bacterial endotoxins, Appendix XIV C. Prepare a solution
in tris-chloride buffer pH 7.4 prepared using water BET containing 7,000 IU of Capreomycin Sulphate
per ml (solution A). The endotoxin limit concentration of solution A is 2.5 IU of endotoxin per ml.
Carry out the test using the maximum valid dilution of solution A calculated from the declared
sensitivity of the lysate used in the test.
07-10
Capreomycin Sulphate intended for use in the manufacture of a parenteral dosage form without a further
sterilisation procedure complies with the following additional requirement.
Sterility Complies with the test for sterility, Appendix XVI A.
07-11
Caprylocaproyl Macrogolglycerides
corrected 1/01
Caprylocaproyl Macrogolglycerides comply with the requirements of the 3rd edition of the European
___________________________________________________________________________________________________________ Ph Eur
DEFINITION
Caprylocaproyl macrogolglycerides are mixtures of monoesters, diesters and triesters of glycerol and
monoesters and diesters of macrogols with a mean relative molecular mass between 200 and 400.
They are obtained by partial alcoholysis of medium-chain triglycerides using macrogol or by
esterification of glycerol and macrogol with caprylic acid and capric acid or a mixture of glycerol
esters and condensates of ethylene oxide with caprylic acid (octanoic acid) and capric acid (decanoic
acid). They may contain free macrogols.
CHARACTERS
Pale-yellow oily liquids, dispersible in hot water, freely soluble in methylene chloride.
The relative density at 20C is about 1.0, the refractive index at 20C is about 1.4.
IDENTIFICATION
substance.
Test solution. Dissolve 1.0 g of the substance to be examined in methylene chloride R and dilute to
Apply to the plate 50 l of the test solution. Develop over a path of 15 cm using a mixture of 30
volumes of hexane R and 70 volumes of ether R. Allow the plate to dry in air. Spray with a 0.1 g/l
solution of rhodamine B R in alcohol R and examine in ultraviolet light at 365 nm. The chromatogram
shows a spot corresponding to triglycerides with an R
f
value of about 0.9 (R
st
1) and spots corres-
ponding to 1,3-diglycerides (R
st
0.7), to 1,2-diglycerides (R
st
0.6), to monoglycerides (R
st
0.1) and to
esters of macrogol (R
st
0).
B. They comply with the test for hydroxyl value (see Tests).
C. They comply with the test for saponification value (see Tests).
D. They comply with the test for fatty acid composition (see Tests).
TESTS
Viscosity (2.2.9). The ranges are presented in Table 11841, determined at 20 0.5C.
Table 11841
Ethylene oxide units
per molecule
(nominal value)
Type of macrogol Viscosity
(mPas)
4 200 30 to 50
6 300 60 to 80
8 400 80 to 110
Hydroxyl value (2.5.3, Method A). The ranges are presented in Table 11842, determined on 1.0 g.
Table 11842
per molecule
(nominal value)
Type of macrogol Hydroxyl value
4 200 80 to 120
6 300 140 to 180
8 400 170 to 205
Peroxide value (2.5.5). Not more than 6.0, determined on 2.0 g.
Saponification value (2.5.6). The ranges are presented in Table 11843, determined on 2.0 g.
07-12
Table 11843
per molecule
(nominal value)
Type of macrogol Saponification
value
4 200 265 to 285
6 300 170 to 190
8 400 85 to 105
Alkaline impurities Introduce into a test-tube 5.0 g and carefully add a mixture, neutralised if
necessary with 0.01M hydrochloric acid or with 0.01M sodium hydroxide, of 0.05 ml of a 0.4 g/l solution
of bromophenol blue R in alcohol R, 0.3 ml of water R and 10 ml of alcohol R. Shake and allow to stand.
Not more than 1.0 ml of 0.01M hydrochloric acid is required to change the colour of the upper layer to
yellow.
Free glycerol Not more than 5.0 per cent. Dissolve 1.20 g in 25.0 ml of methylene chloride R. Heat
if necessary. After cooling, add 100 ml of water R. Shake and add 25.0 ml of a 6 g/l solution of
periodic acid R. Shake and allow to stand for 30 min. Add 40 ml of a 75 g/l solution of potassium
iodide R. Allow to stand for 1 min. Add 1 ml of starch solution R. Titrate the iodine with 0.1M sodium
thiosulphate. Carry out a blank titration.
1 ml of 0.1M sodium thiosulphate is equivalent to 2.3 mg of glycerol.
Fatty acid composition Examine by gas chromatography (2.4.22). The fatty acid fraction has the
caproic acid: not more than 2.0 per cent,
caprylic acid: 50.0 per cent to 80.0 per cent,
capric acid: 20.0 per cent to 50.0 per cent,
lauric acid: not more than 3.0 per cent,
myristic acid: not more than 1.0 per cent.
Residual ethylene oxide and dioxan (2.4.25). Not more than 1 ppm of residual ethylene oxide
and not more than 10 ppm of residual dioxan. When determining the dioxan content, apply a
correction factor of 1/5 in the calculation.
of water. Use a mixture of 30 volumes of anhydrous methanol R and 70 volumes of methylene chloride R
as solvent.
Total ash (2.4.16). Not more than 0.1 per cent, determined on 1.0 g.
LABELLING
The label states the type of macrogol used (mean relative molecular mass) or the number of ethylene
oxide units per molecule (nominal value).
___________________________________________________________________________________________________________ Ph Eu
07-13
Captopril
N HS
O
H Me
H COOH
C
9
H
15
NO
3
S 217.3 62571-86-2
Captopril complies with the requirements of the 3rd edition of the European Pharmacopoeia [1079]. These
Action and use Angiotensin-converting enzyme inhibitor.
Preparation
Captopril Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Captopril contains not less than 98.0 per cent and not more than the equivalent of 101.5 per cent of
(2S)-1-[(2S)-3-mercapto-2-methylpropanoyl]pyrrolidine-2-carboxylic acid, calculated with reference
CHARACTERS
A white or almost white crystalline powder, freely soluble in water, in methylene chloride and in
methanol. It dissolves in dilute solutions of alkali hydroxides.
IDENTIFICATION
obtained with captopril CRS.
the same solvent.
Reference solution. Dissolve 10 mg of captopril CRS in methanol R and dilute to 2 ml with the same
solvent.
volume of anhydrous acetic acid R and 3 volumes of toluene R. Dry the plate in a stream of cold air.
Spray with a 20 g/l solution of 5,5-dithiobis(2-nitrobenzoic acid) R in methanol R, adjusted to pH 8
with dilute ammonia R. The principal spot in the chromatogram obtained with the test solution is
similar in position, colour and size to the principal spot in the chromatogram obtained with the
reference solution.
D. Dissolve about 20 mg in 2 ml of water R. Add 0.5 ml of 0.05M iodine. The colour is discharged
immediately.
TESTS
Specific optical rotation (2.2.7). The specific optical rotation is 156 to 161, determined on
Reference solution (b). Dissolve 10 mg of the substance to be examined in the mobile phase, add 1 ml
of 0.05M iodine and dilute to 100.0 ml with the mobile phase. Dilute 10.0 ml of the solution to
07-14
as mobile phase at a flow rate of 1 ml per minute a mixture of 0.05 volumes of concentrated
phosphoric acid R, 50 volumes of methanol R and 50 volumes of water R,
principal peak obtained with reference solution (a) is not less than 40 per cent of the full scale of the
recorder. Inject 20 l of reference solution (b). The test is not valid unless the chromatogram
obtained with reference solution (b) shows three peaks and the resolution between the last two
eluting principal peaks is at least 2.0. Inject separately 20 l of the test solution and 20 l of reference
solution (a). Continue the chromatography for three times the retention time of the principal peak in
the chromatogram obtained with the test solution. In the chromatogram obtained with the test
solution: the area of any peak apart from the principal peak is not greater than half the area of the
peak in the chromatogram obtained with reference solution (a) (1.0 per cent), and the sum of the
areas of any peaks, apart from the principal peak, is not greater than the area of the peak in the
chromatogram obtained with reference solution (a) (2.0 per cent). Disregard any peak with a
retention time less than 1.4 min or with an area less than 0.1 times that of the peak in the chromato-
Loss on drying (2.2.32). Not more than 1.0 per cent, determined on 1.000 g by drying under high
vacuum at 60C for 3 h.
ASSAY
Dissolve 0.150 g in 30 ml of water R. Titrate with 0.05M iodine, determining the end-point potentio-
metrically (2.2.20). Use a combined platinum electrode.
9
H
15
NO
3
S.
STORAGE
IMPURITIES
A. (2S,2S)-1,1-[3,3-dithiobis[(2S)-2-methylpropanoyl]]bispyrrolidine-2-carboxylic acid
(captopril disulphide).
__________________________________________________________________________________________________________ Ph Eur
07-15
Caraway
Caraway complies with the requirements of the 3rd edition of the European Pharmacopoeia for Caraway Fruit
When Powdered Caraway is prescribed or demanded, material complying with the appropriate
requirements below and containing not less than 2.5% v/w (25 ml/kg) of essential oil shall be
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Caraway fruit consists of the whole, dry mericarp of Carum carvi L. It contains not less than 30 ml/kg
of essential oil, calculated with reference to the anhydrous drug.
CHARACTERS
Caraway fruit has an odour reminiscent of carvone.
IDENTIFICATION
A. The fruit is a cremocarp of almost cylindrical shape. It is generally 3 mm to 6.5 mm long and
1 mm to 1.5 mm wide. The mericarps, usually free, are greyish-brown to brown, glabrous, mostly
sickle-shaped, with both ends sharply terminated. Each bears five prominent narrow ridges. When cut
transversely the profile shows an almost regular pentagon and four vittae on the dorsal surface and
two on the commissural surface may be seen with a lens.
chloral hydrate solution R. The powder shows the following diagnostic characters: fragments of the
secretory cells composed of yellowish-brown to brown, thin-walled, polygonal secretory cells,
frequently associated with a layer of thin-walled transversely elongated cells, 8-12 m wide; fragments
of the epicarp with thick-walled cells and occasional anomocytic stomata (2.8.3); numerous
endosperm fragments containing aleurone grains, droplets of fatty oil and microcrystals of calcium
oxalate in rosette formation; spiral vessels accompanied by sclerenchymatous fibres; rarely some fibre
bundles from the carpophore; groups of rectangular to sub-rectangular sclereids from the mesocarp
with moderately thickened and pitted walls may be present.
C. Examine by thin-layer chromatography (2.2.27) using a suitable silica gel as the coating substance.
Test solution. Shake 0.5 g of the powdered drug (710) with 5.0 ml of ethyl acetate R for 2 min to 3 min.
Filter over 2 g of anhydrous sodium sulphate R. Use the filtrate as the test solution.
Reference solution. Dissolve 2 l of carvone R and 5 l of olive oil R in 1.0 ml of ethyl acetate R.
a path of 10 cm using a mixture of 5 volumes of ethyl acetate R and 95 volumes of toluene R. Allow the
plate to dry in air and examine in ultraviolet light at 254 nm. The chromatograms of the test solution
and of the reference solution show a quenching zone (carvone) in the central part against a light
background. Spray with anisaldehyde solution R and heat under observation at 100C to 105C for 2 to
4 min. Examine in daylight. The zones corresponding to carvone appear strong orange-brown. The
chromatogram obtained with the test solution shows above the zone corresponding to carvone a violet
zone corresponding to triglycerides similar in position and colour to the zone in the chromatogram
obtained with the reference solution (triglycerides of olive oil). The chromatogram obtained with the
test solution shows close to the solvent front a weak violet zone corresponding to terpene hydro-
carbons and in the lower part some weak, mostly violet-greyish and brownish zones.
TESTS
Water (2.2.13). Not more than 10.0 per cent, determined on 10.0 g of powdered drug by distillation.
ASSAY
tube. Reduce the drug to a powder (710) and immediately use 10.0 g for the determination. Distil at a
rate of 2 ml to 3 ml per minute for 90 min.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
07-16
Caraway Oil
Definition Caraway Oil is obtained by distillation from Caraway.
Characteristics A clear, colourless or pale yellow liquid, visibly free from water; odour, that of
caraway.
Optical rotation +74 to +80, Appendix V F.
Solubility in ethanol Soluble, at 20, in 7 volumes of ethanol (80%), Appendix X M.
Content of ketones 53.0 to 63.0% w/w, calculated as carvone, C
10
H
14
O, when determined by the
following method. To 1.5 g in a glass-stoppered tube (approximately 150 mm 25 mm) add 10 ml
of a solution prepared in the following manner. Dissolve 7.0 g of hydroxylamine hydrochloride in 90 ml
of ethanol (90%), warming gently if necessary, add 1.6 ml of dimethyl yellow solution and sufficient 1M
potassium hydroxide in ethanol (90%) to produce a pure yellow colour and dilute to 100 ml with
ethanol (90%). Titrate with 1M potassium hydroxide in ethanol (90%) VS until the red colour changes
to yellow. Place the tube in a water bath at 75 to 80 and, at 5-minute intervals, neutralise with 1M
potassium hydroxide in ethanol (90%) VS; after 40 minutes complete the titration to the full yellow
colour of the indicator. This procedure gives an approximate value for the carvone content of the oil.
Repeat the procedure, using as the colour standard for the end point of the titration the titrated liquid
of the first determination with the addition of 0.5 ml of 1M potassium hydroxide in ethanol (90%) VS.
Calculate the content of carvone from the second determination. Each ml of 1M potassium hydroxide
in ethanol (90%) VS is equivalent to 151.4 mg of carvone, C
10
H
14
O.
Storage Caraway Oil should be kept in a well-filled, well-closed container, protected from light and
Action and use Carminative and flavour.
07-17
Carbamazepine
1/01
N
CONH
2
C
15
H
12
N
2
O 236.3 298-46-4
Carbamazepine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0543].
Action and use Anticonvulsant.
Preparation
Carbamazepine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
5H-Dibenzo[b,f]azepine-5-carboxamide.
CHARACTERS
Appearance: white or almost white crystalline powder.
Solubility: very slightly soluble in water, freely soluble in methylene chloride, sparingly soluble in
acetone and in alcohol.
It shows polymorphism; the acceptable crystalline form corresponds to carbamazepine CRS.
IDENTIFICATION
Comparison: carbamazepine CRS.
Preparation: examine the substances as discs without prior treatment.
TESTS
To 10 ml of the filtrate add 0.05 ml of phenolphthalein solution R1 and 0.5 ml of 0.01M sodium
hydroxide; the solution is red. Add 1.0 ml of 0.01M hydrochloric acid; the solution is colourless. Add
0.15 ml of methyl red solution R; the solution is red.
Test solution (a). Dissolve 0.150 g of the substance to be examined in methanol R2 and dilute to
50.0 ml with the same solvent. Sonicate. Dilute 10.0 ml of this solution to 20.0 ml with water R.
Test solution (b). Dilute 10.0 ml of test solution (a) to 50.0 ml with a mixture of equal volumes of
methanol R2 and water R.
Reference solution (a). Dissolve 7.5 mg of carbamazepine CRS, 7.5 mg of carbamazepine impurity A
CRS and 7.5 mg of iminodibenzyl R (impurity E) in methanol R2 and dilute to 100.0 ml with the same
solvent. Dilute 1.0 ml of this solution to 50.0 ml with a mixture of equal volumes of methanol R2 and
water R.
Reference solution (b). Dissolve 0.150 g of carbamazepine CRS in methanol R2 and dilute to 50.0 ml
with the same solvent. Dilute 5.0 ml of this solution to 50.0 ml with a mixture of equal volumes of
methanol R2 and water R.
Column:
size: l = 0.25 m, = 4.6 mm,
stationary phase: nitrile silica gel for chromatography R1 (10 m).
Mobile phase: tetrahydrofuran R, methanol R2, water R (3: 12: 85 V/V/V). To 1000 ml of this solution
add 0.2 ml of anhydrous formic acid R and 0.5 ml of triethylamine R.
Detection: a spectrophotometer at 230 nm.
07-18
Injection: 20 l; inject test solution (a) and reference solution (a).
Run time: 6 times the retention time of carbamazepine which is about 10 min.
Relative retention with reference to carbamazepine: impurity B = about 0.7; impurity A = about 0.9;
impurity C = about 1.6; impurity D = about 3.5; impurity E = about 5.1.
System suitability:
resolution: minimum of 1.7 between the peaks due to carbamazepine and impurity A in the
Limits:
impurity A: not more than the area of the corresponding peak in the chromatogram obtained
with reference solution (a) (0.1 per cent),
impurity E: not more than the area of the corresponding peak in the chromatogram obtained with
any other impurity: not more than the area of the peak due to carbamazepine in the chromato-
gram obtained with reference solution (a) (0.1 per cent),
total: not more than 5 times the area of the peak due to carbamazepine in the chromatogram
obtained with reference solution (a) (0.5 per cent),
disregard limit: 0.5 times the area of the peak due to carbamazepine in the chromatogram
obtained with reference solution (a) (0.05 per cent).
Chlorides (2.4.4): maximum 140 ppm.
Suspend 0.715 g in 20 ml of water R and boil for 10 min. Cool and dilute to 20 ml with water R.
Filter through a membrane filter (nominal pore size: 0.8 m). Dilute 10 ml of the filtrate to 15 ml
with water R. This solution complies with the limit test for chlorides.
Pb) R.
100-105C for 2h.
ASSAY
Injection: test solution (b) and reference solution (b).
System suitability:
repeatability: reference solution (b).
Calculate the percentage content m/m of dried substance.
STORAGE
In an airtight container.
IMPURITIES
N
R
A. R = CO-NH
2
: 10,11-dihydro-5H-dibenz[b,f]azepine-5-carboxamide (10,11-dihydro-
carbamazepine),
E. R = H: 10,11-dihydro-5H-dibenz[b,f]azepine (iminodibenzyl),
N
Me
B. 9-methylacridine,
N
R
C. R = CO-NH-CO-NH
2
:

(5H-dibenz[b,f]azepin-5-ylcarbonyl)urea
(N-carbamoylcarbamazepine),
D. R = H: 5H-dibenz[b,f]azepine (iminostilbene).
__________________________________________________________________________________________________________ Ph Eur
07-19
Carbaryl
O NHMe
O
C
12
H
11
NO
2
201.2 63-25-2
Definition Carbaryl is 1-naphthyl methylcarbamate. It contains not less than 98.0% and not more
than 102.0% of C
12
H
11
NO
2
Characteristics A white to off-white or light grey powder which darkens on exposure to light. It
melts at about 142.
Very slightly soluble in water; soluble in acetone and in ethanol (96%).
spectrum of carbaryl (RS 039).
1-Naphthol Carry out the method for liquid chromatography, Appendix III D, using the following
solutions. Solution (1) contains 0.1% w/v of the substance being examined in acetonitrile. Solution (2)
contains 0.001% w/v of 1-naphthol in a 75% v/v solution of acetonitrile in water. Solution (3) contains
0.005% w/v of the substance being examined and 0.005% w/v of 1-naphthol in the mobile phase.
The chromatographic conditions described under Assay may be used.
between the two principal peaks is at least 2.0.
In the chromatogram obtained with solution (1) the area of any peak corresponding to 1-naphthol
is not greater than the area of the principal peak in the chromatogram obtained with solution (2)
(1%).
Loss on drying When dried to constant weight over phosphorus pentoxide at a pressure not exceeding
0.7 kPa, loses not more than 0.5% of its weight. Use 1 g.
Assay Carry out the method for liquid chromatography, Appendix III D, using the following solutions.
Solution (1) contains 0.005% w/v of the substance being examined in methanol. Solution (2) contains
0.005% w/v of carbaryl BPCRS in methanol. Solution (3) contains 0.005% w/v of the substance being
examined and 0.005% w/v of 1-naphthol in the mobile phase.
(10 cm 4.6 mm) packed with stationary phase C (5 m) (Spherisorb ODS 2 is suitable), (b) a
mixture of 1 volume of glacial acetic acid, 25 volumes of acetonitrile and 75 volumes of water as the
mobile phase with a flow rate of 2.5 ml per minute and (c) a detection wavelength of 280 nm.
Inject 20 l of each solution. The test is not valid unless in the chromatogram obtained with
solution (3) the resolution factor between the two principal peaks is at least 2.0.
12
H
11
NO
2
using the declared content of C
12
H
11
NO
2
in carbaryl BPCRS.
Storage Carbaryl should be kept in a well-closed container, protected from light and stored at at
Action and use Insecticide.
Preparation
Carbaryl Lotion
IMPURITIES
OH
A. 1-naphthol
07-20
Carbasalate Calcium
COO
_
OAc
2
Ca
2+
H
2
N NH
2
O
.
C
19
H
18
CaN
2
O
9
458.4 5749-67-7
Carbasalate Calcium complies with the requirements of the 3rd edition of the European Pharmacopoeia [1185].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Carbasalate calcium contains not less than 99.0 per cent and not more than the equivalent of 101.0
per cent of an equimolecular compound of calcium di[2-(acetyloxy)benzoate] and urea, calculated
CHARACTERS
A white, crystalline powder, freely soluble in water and dimethylformamide, practically insoluble in
acetone and in anhydrous methanol.
Protect the substance from moisture during handling. Examination in aqueous solutions has to be performed
immediately after preparation.
IDENTIFICATION
A. Dissolve 0.250 g in water R and dilute to 100.0 ml with the same solvent. To 1.0 ml of the
solution add 75 ml of water R and 5 ml of dilute hydrochloric acid R, mix and dilute with water R to
100.0 ml. Examined immediately after preparation between 220 nm and 350 nm (2.2.25), the
solution shows two absorption maxima, at 228 nm and 276 nm. The specific absorbances at the
maxima are 363 to 379 and 49 to 53, respectively.
B. Examine by infrared spectrophotometry (2.2.24), comparing with the Ph. Eur. reference spectrum of
carbasalate calcium.
C. Dissolve 0.1 g in 10 ml of water R, boil for 2 min and cool. The solution gives reaction (a) of
salicylates (2.3.1).
D. Heat 0.2 g with 0.2 g of sodium hydroxide R; a yellow to yellowish-brown colour is produced and
the vapour turns red litmus paper R blue.
E. It gives reaction (a) of calcium (2.3.1).
TESTS
Appearance of solution Dissolve 2.5 g in 50 ml of water R. The solution is not more opalescent
than reference suspension II (2.2.1) and is colourless (Method II, 2.2.2).
Related substances In a 100 ml volumetric flask, dissolve 0.150 g in 10 ml of 0.1M
tetrabutylammonium hydroxide in 2-propanol. Allow to stand for 10 min shaking occasionally. Add
8.0 ml of 0.1M hydrochloric acid and 20.0 ml of a 19 g/l solution of disodium tetraborate R and mix.
While swirling continuously, add 2.0 ml of a 10 g/l solution of aminopyrazolone R and 2.0 ml of a
10 g/l solution of potassium ferricyanide R. Allow to stand for 2 min, dilute to 100.0 ml with water R,
mix and allow to stand for 20 min. Measure the absorbance (2.2.25) of the solution at the maximum
at 505 nm using water R as the compensation liquid. The absorbance is not greater than 0.125
(0.1 per cent, expressed as acetylsalicylsalicylic acid).
Salicylic acid In a 100 ml volumetric flask, dissolve 0.200 g in 80 ml of water R and add 10 ml of a
10 g/l solution of ferric nitrate R in a 80 g/l solution of dilute nitric acid R. Dilute to 100.0 ml with
water R. Immediately after preparation, measure the absorbance (2.2.25) of the solution at the
maximum of 525 nm using water R as the compensation liquid. The absorbance is not greater than
0.115 (0.5 per cent, expressed as salicylic acid).
Sodium Not more than 0.1 per cent, determined by atomic emission spectrometry (Method I,
2.2.22) using 1.0 g dissolved in 500.0 ml of water R.
07-21
Heavy metals (2.4.8). Dissolve 2.0 g in 8 ml of water R, with heating, cool and add 12 ml of
acetone R. 12 ml of the solution complies with limit test B for heavy metals (10 ppm). Prepare the
determination of water. Use a mixture of 15 ml of anhydrous methanol R and 15 ml of
dimethylformamide R as the solvent.
ASSAY
In a flask with a ground-glass stopper, dissolve 0.400 g in 25 ml of water R. Add 25.0 ml of 0.1M
sodium hydroxide. Close the flask and allow to stand for 2 h. Titrate the excess of alkali with 0.1M
hydrochloric acid, using 0.2 ml of phenolphthalein solution R. Carry out a blank titration.
19
H
18
CaN
2
O
9
.
STORAGE
IMPURITIES
AcO
O
AcO
O O
A. 2-(acetyloxy)benzoic anhydride,
OAc
O
COOH
O
B. 2-[[2-(acetyloxy)benzoyl]oxy]benzoic acid (acetylsalicylsalicylic acid),
C. 2-hydroxybenzoic acid (salicylic acid).
__________________________________________________________________________________________________________ Ph Eur
07-22
Carbenicillin Sodium
N
S
O
H
Me
Me
N
H H
O
COONa
H NaOOC
H
and epimer at C*
*
C
17
H
16
N
2
Na
2
O
6
S 422.4 4800-94-6
Carbenicillin Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia [0812].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Carbenicillin sodium contains not less than 89.0 per cent and not more than the equivalent of
101.0 per cent of disodium (2S,5R,6R)-6-[(RS)-2-carboxylato-2-phenylacetylamino]-3,3-dimethyl-
7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate, calculated with reference to the anhydrous
substance.
PRODUCTION
process using starting materials or intermediates which contain residue of dimethylaniline, it complies
with the following test:
CHARACTERS
A white or slightly yellowish powder, hygroscopic, freely soluble in water, soluble in alcohol and in
methanol.
IDENTIFICATION
obtained with carbenicillin sodium CRS.
the same solvent.
Reference solution (a). Dissolve 25 mg of carbenicillin sodium CRS in methanol R and dilute to 5 ml with
the same solvent.
Reference solution (b). Dissolve 25 mg of carbenicillin sodium CRS and 25 mg of ampicillin sodium CRS
in methanol R and dilute to 5 ml with the same solvent.
of acetone R and 90 volumes of a 154 g/l solution of ammonium acetate R, adjusted to pH 5.0 with
glacial acetic acid R. Allow the plate to dry in a current of warm air and expose it to iodine vapour.
spots.
by swirling; the solution is practically colourless. Place the test-tube in a water-bath for 1 min; a
yellowish-brown colour develops.
07-23
TESTS
solution Y
5
(Method II, 2.2.2).
substance.
Inject 20 l of test solution (b) and start the elution isocratically. Immediately after elution of the
carbenicillin peak start the following linear gradient. If the mobile phase composition has been
adjusted to achieve the required resolution, the adjusted composition will apply at time zero in the
gradient.
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
4560 085 10015 re-equilibration
Equilibrate the column with the mobile phase originally chosen for 15 min. Inject 20 l of reference
solution (b) and use the same elution pattern. Inject 20 l of the dissolution mixture and use the
same elution pattern to obtain a blank. In the chromatogram obtained with test solution (b): the area
of any peak corresponding to benzylpenicillin sodium is not greater than five times the area of the
principal peak in the chromatogram obtained with reference solution (b) (5 per cent); the area of any
peak, apart from the principal peak and any peak due to benzylpenicillin, is not greater than three
times the area of the principal peak in the chromatogram obtained with reference solution (b) (3 per
cent); the sum of the areas of all the peaks, apart from the principal peak, is not greater than eleven
cent). Disregard any peak due to the solvent and any peak with an area less than 0.1 times that of the
mination of water.
ASSAY
Dissolution mixture. Dissolve 7.8 g of sodium dihydrogen phosphate R in 900 ml of water R. Adjust to
pH 6.4 with dilute sodium hydroxide solution R and dilute to 1000 ml with water R.
Test solution (a). Dissolve 25.0 mg of the substance to be examined in the dissolution mixture and
dilute to 50.0 ml with the same mixture.
the dissolution mixture and dilute to 20.0 ml with the same mixture.
Reference solution (a). Dissolve 25.0 mg of carbenicillin sodium CRS in the dissolution mixture and
dilute to 50.0 ml with the same mixture.
Reference solution (b). Dissolve 30.0 mg of benzylpenicillin sodium CRS in the dissolution mixture and
dilute to 20.0 ml with the same mixture. Dilute 1.0 ml of the solution to 50.0 ml with the dissolution
mixture.
Reference solution (c). Dissolve 2.0 mg of cefixime CRS in the dissolution mixture and dilute to 50.0 ml
with the same mixture. To 5.0 ml of the solution add 5.0 ml of reference solution (a).
Reference solution (d). Dilute 1.0 ml of reference solution (a) to 25.0 ml with the dissolution mixture.
Dilute 1.0 ml of the solution to 100.0 ml with the dissolution mixture.
Mobile phase A. Mix 980 ml of a 15.6 g/l solution of sodium dihydrogen phosphate R, adjusted to
07-24
pH 4.3, if necessary, with dilute phosphoric acid R or dilute sodium hydroxide solution R and 20 ml of
acetonitrile R,
Mobile phase B. Mix 600 ml of a 15.6 g/l solution of sodium dihydrogen phosphate R, adjusted to
pH 4.3, if necessary, with dilute phosphoric acid R or dilute sodium hydroxide solution R and 400 ml
of acetonitrile R,
Equilibrate the column with a mobile phase ratio A:B of 85:15. Inject 20 l of reference solution (c)
and 20 l of reference solution (d). The test is not valid unless: in the chromatogram obtained with
reference solution (c), the resolution between the two principal peaks is at least 9.0 (if necessary,
adjust the ratio A:B of the mobile phase) and the mass distribution ratio for the second peak
(carbenicillin) is 3.5 to 4.8; in the chromatogram obtained with reference solution (d), the principal
peak has a signal-to-noise ratio of at least three.
Inject 20 l of reference solution (a) six times. The assay is not valid unless the relative standard
deviation of the peak area for the principal peak is at most 1.0 per cent. Inject alternately 20 l of test
solution (a) and 20 l of reference solution (a).
STORAGE
Store in an airtight container, protected from light, at a temperature of 2C to 8C. If the substance
is sterile, store in a sterile, airtight, tamper-proof container.
LABELLING
The label states:
IMPURITIES
N
S
O
H
Me
Me
H H
COOH
H
2
N
A. (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo-[3.2.0]heptane-2-carboxylic
COOH
COOH
COOH
C. 2-phenylpropanedioic acid (phenylmalonic acid),
N
O
COOH
Me
Me
H
S
HN
COOH
H
*
*
COOH
*
D. (4S)-2-[carboxy((2-carboxy-2-phenylacetyl)amino]methyl]-5,5-dimethylthiazolidine-4-
carboxylic acid (penicilloic acids of carbenicillin),
N
O
COOH
Me
Me
H
S
HN
H
*
COOH
*
E. (4S)-2-[[(2-carboxy-2-phenylacetyl)amino]methyl]-5,5-dimethylthiazolidine-4-carboxylic
acid (penilloic acids of carbenicillin),
F. (2S,5R,6R)-3,3-dimethyl-7-oxo-6-[(phenylacetyl)amino]-4-thia-1-azabicyclo[3.2.0]heptane-
2-carboxylic acid (benzylpenicillin),
07-25
N
O
COOH
Me
Me
H
S
HN
COOH
H
*
*
G. (4S)-2-[carboxy[(phenylacetyl)amino]methyl]-5,5-dimethylthiazolidine-4-carboxylic acid
*
N
O
COOH
Me
Me
H
S
HN
H
H
and epimer at C*
H. (2RS,4S)-2-[[(phenylacetyl)amino]methyl-5,5-dimethylthiazolidine-4-carboxylic acid
(penilloic acids of benzylpenicillin),
N
S
H
Me
Me
COOH
N
H
HOOC
H
I. (3S,7R,7aR)-5-benzyl-2,2-dimethyl-2,3,7,7a-tetrahydroimidazo[5,1-b]thiazole-3,7-
dicarboxylic acid (penillic acid of benzylpenicillin).
__________________________________________________________________________________________________________ Ph Eur
07-26
Carbenoxolone Sodium
H
Me
Me
Me Me
Me COONa
H
O
O O
COONa
H
Me
Me
C
34
H
48
Na
2
O
7
614.7 7421-40-1
Definition Carbenoxolone Sodium is disodium 3-(3-carboxylatopropionyloxy)-11-oxo-olean-12-
en-30-oate. It contains not less than 97.0% and not more than 103.0% of C
34
H
48
Na
2
O
7
, calculated
Characteristics A white or pale cream powder; hygroscopic. The powder is irritant to nasal
membranes.
Freely soluble in water; sparingly soluble in ethanol (96%); practically insoluble in chloroform and in
ether.
Identification
A. Dissolve 0.1 g in 5 ml of water and make just acid with 2M hydrochloric acid, stir well and filter.
Wash the residue with water until the washings are no longer acidic and dry to constant weight at
105. The infrared absorption spectrum of the residue, Appendix II A, is concordant with the reference
spectrum of carbenoxolone (RS 041).
B. Yields the reactions characteristic of sodium salts, Appendix VI.
Specific optical rotation In a 1% w/v solution in a mixture of equal volumes of methanol and 0.02M
sodium carbonate, +132 to +140, calculated with reference to the anhydrous substance, Appendix
V F.
silica gel F
254
precoated plate (Merck silica gel 60 F
254
plates are suitable) and a mixture of 60
volumes of ethyl acetate, 20 volumes of methanol, 11 volumes of water and 1 volume of 13.5M
ammonia as the mobile phase. Apply separately to the plate 5 l of each of two solutions of the
substance being examined in methanol containing (1) 1.50% w/v and (2) 0.030% w/v. After removal
of the plate, allow it to dry in air and examine under ultraviolet light (254 nm). Spray with a 1.5% w/v
solution of vanillin in sulphuric acid (60%) and heat at 105 for 10 to 15 minutes. By each method of
visualisation, any secondary spot in the chromatogram obtained with solution (1) is not more intense
Water Not more than 4.0% w/w, Appendix IX C. Use 0.6 g.
Assay Dissolve 1 g in 30 ml of water, add 30 ml of chloroform and 15 ml of a mixture of 1 volume of
2M hydrochloric acid and 9 volumes of water, shake and allow to separate. Add the chloroform layer to
40 ml of a 20% w/v solution of sodium chloride, shake and allow to separate. Repeat the extraction
with four 15-ml quantities of chloroform, combine the chloroform extracts and add sufficient chloro-
form to produce 100 ml. Evaporate 25 ml, dry the residue at 100 at a pressure of 2 kPa, dissolve in
10 ml of dimethylformamide and carry out Method II for non-aqueous titration, Appendix VIII A, using
0.1M tetrabutylammonium hydroxide VS as titrant and thymol blue solution as indicator. Each ml of 0.1M
tetrabutylammonium hydroxide VS is equivalent to 30.73 mg of C
34
H
48
Na
2
O
7
.
Storage Carbenoxolone Sodium should be kept in a well-closed container.
Action and use Used in treatment of gastric ulcer.
07-27
Carbidopa
HO
HO COOH
NHNH
2
Me
C
10
H
14
N
2
O
4
,H
2
O 244.2 38821-49-7
Carbidopa complies with the requirements of the 3rd edition of the European Pharmacopoeia [0755]. These
Preparation
Co-careldopa Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Carbidopa contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of
(S)-3-(3,4-dihydroxyphenyl)-2-hydrazino-2-methylpropionic acid, calculated with reference to the
dried substance.
CHARACTERS
A white or yellowish-white powder, slightly soluble in water, very slightly soluble in alcohol,
practically insoluble in methylene chloride. It dissolves in dilute solutions of mineral acids.
IDENTIFICATION
B. Dissolve 50.0 mg in a 8.5 g/l solution of hydrochloric acid R in methanol R and dilute to 100.0 ml
with the same acid. Dilute 10.0 ml of this solution to 100.0 ml with a 8.5 g/l solution of hydrochloric
acid R in methanol R. Examined between 230 nm and 350 nm (2.2.25), the solution shows an
absorption maximum at 283 nm. The specific absorbance at the maximum is 135 to 150, calculated
obtained with carbidopa CRS. Examine the substances prepared as discs.
D. Shake vigorously about 5 mg with 10 ml of water R for 1 min and add 0.3 ml of ferric chloride
solution R2. An intense green colour is produced, which quickly turns to reddish-brown.
E. Suspend about 20 mg in 5 ml of water R and add 5 ml of cupri-tartaric solution R. On heating, the
colour of the solution changes to dark brown and a red precipitate is formed.
TESTS
Appearance of solution Dissolve 0.25 g in 25 ml of 1M hydrochloric acid. The solution is clear
6
or B
6
(Method II, 2.2.2).
Specific optical rotation (2.2.7). With the aid of an ultrasonic bath, dissolve completely 0.250 g in
aluminium chloride solution R and dilute to 25.0 ml with the same solution. The specific optical
rotation is 22.5 to 26.5, calculated with reference to the dried substance.
Hydrazine Examine by thin-layer chromatography (2.2.27), using silanised silica gel H R as the
coating substance.
Test solution (a). Dissolve 0.50 g in dilute hydrochloric acid R and dilute to 2.0 ml with the same acid.
Test solution (b). Place 25 g of strongly basic anion exchange resin R into each of two conical flasks with
ground-glass stoppers. To each, add 150 ml of carbon dioxide-free water R and shake from time to
time during 30 min. Decant the liquid from both flasks and repeat the process with further quanti-
ties, each of 150 ml, of carbon dioxide-free water R.
Take two 100 ml measuring cylinders 3.5 cm to 4.5 cm in internal diameter and label these A and
B. Into cylinder A, transfer as completely as possible the resin from one conical flask using 60 ml of
carbon dioxide-free water R; into cylinder B, transfer the second quantity of resin, this time using 20 ml
of carbon dioxide-free water R.
Into each cylinder, insert a gas-inlet tube, the end of which has an internal diameter of 2 mm to
3 mm and which reaches almost to the bottom of the cylinder. Pass a rapid stream of nitrogen for
chromatography R through each mixture so that homogeneous suspensions are formed. After 30 min,
07-28
without interrupting the gas flow, add 1.0 ml of test solution (a) to cylinder A; after 1 min stop the
gas flow into cylinder A and transfer the contents, through a moistened filter paper, into cylinder B.
After 1 min, stop the gas flow to cylinder B and pour the solution immediately through a moistened
filter paper into a freshly prepared mixture of 1 ml of a 200 g/l solution of salicylaldehyde R in
methanol R and 20 ml of phosphate buffer solution pH 5.5 R in a conical flask, shake thoroughly for
1 min and heat in a water-bath at 60C for 15 min. The liquid becomes clear. Allow to cool, add
2.0 ml of toluene R and shake vigorously for 2 min. Transfer the mixture into a centrifuge tube and
centrifuge.
Separate the toluene layer in a 100 ml separating funnel and shake vigorously with two quantities,
each of 20 ml, of a 200 g/l solution of sodium metabisulphite R and finally with two quantities, each of
50 ml, of water R. Separate the toluene layer.
Reference solution (a). Dissolve 10 mg of hydrazine sulphate R in dilute hydrochloric acid R and dilute to
50 ml with the same acid. Dilute 1.0 ml of this solution to 10.0 ml with dilute hydrochloric acid R.
Reference solution (b). Prepare the solution at the same time and in the same manner as described for
test solution (b) using 1.0 ml of reference solution (a) in place of 1.0 ml of test solution (a).
Apply separately to the plate 10 l of test solution (b) and 10 l of reference solution (b). Develop
over a path of 10 cm using a mixture of 1 volume of water R and 2 volumes of methanol R. Allow the
with test solution (b) showing a yellow fluorescence is not more intense than the corresponding spot
in the chromatogram obtained with reference solution (b) (20 ppm of hydrazine).
Methyldopa and methylcarbidopa Examine by liquid chromatography (2.2.29).
Test solution. Dissolve 0.100 g of the substance to be examined in 0.1M hydrochloric acid and dilute to
10.0 ml with the same acid.
Reference solution (a). Dissolve 5 mg of methylcarbidopa CRS and 5 mg of methyldopa CRS in 0.1M
hydrochloric acid and dilute to 100.0 ml with the same acid.
Reference solution (b). Dissolve 5 mg of carbidopa CRS and 5 mg of methyldopa CRS in 0.1M hydro-
chloric acid and dilute to 10.0 ml with the same acid.
a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octylsilyl silica
as mobile phase at a flow rate of 1 ml per minute a mixture of 2 volumes of methanol R and 98
volumes of a 14 g/l solution of potassium dihydrogen phosphate R,
a loop injector.
Inject 20 l of each solution. The test is not valid unless the resolution between the peaks corres-
ponding to methyldopa and carbidopa in the chromatogram obtained with reference solution (b) is
greater than 4.0. In the chromatogram obtained with the test solution, the areas of any peaks corres-
ponding to methyldopa and methylcarbidopa are not greater than the areas of the corresponding
peaks in the chromatogram obtained with reference solution (a) (0.5 per cent).
100C to 105C.
ASSAY
Dissolve 0.150 g with gentle heating in 75 ml of anhydrous acetic acid R. Titrate with 0.1M perchloric
acid, determining the end-point potentiometrically (2.2.20).
10
H
14
N
2
O
4
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
07-29
Carbimazole
NMe
N
COOEt
S
C
7
H
10
N
2
O
2
S 186.2 22232-54-8
Carbimazole complies with the requirements of the 3rd edition of the European Pharmacopoeia [0884]. These
Action and use Antithyroid.
Preparation
Carbimazole Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Carbimazole contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent
of ethyl 3-methyl-2-thioxo-4-imidazoline-1-carboxylate, calculated with reference to the dried
substance.
CHARACTERS
A white or yellowish-white, crystalline powder, slightly soluble in water, soluble in acetone and in
alcohol.
IDENTIFICATION
obtained with carbimazole CRS. Examine the substances prepared as discs.
C. Examine the chromatograms obtained in the test for thiamazole and other related substances. The
principal spot in the chromatogram obtained with test solution (b) is similar in position and size to
D. Dissolve about 10 mg in a mixture of 50 ml of water R and 0.05 ml of dilute hydrochloric acid R.
Add 1 ml of potassium iodobismuthate solution R. A red precipitate is formed.
TESTS
Thiamazole and other related substances Examine by thin-layer chromatography (2.2.27), using
silica gel GF
254
Reference solution (a). Dissolve 10 mg of carbimazole CRS in methylene chloride R and dilute to 10 ml
Reference solution (b). Dissolve 5 mg of thiamazole R in methylene chloride R and dilute to 100 ml with
the same solvent.
Reference solution (c). Dilute 1 ml of test solution (b) to 20 ml with methylene chloride R.
20 volumes of acetone R and 80 volumes of methylene chloride R. Allow the plate to dry in air for
30 min and examine in ultraviolet light at 254 nm. In the chromatogram obtained with test solution
(a): any spot corresponding to thiamazole is not more intense than the spot in the chromatogram
obtained with reference solution (b) (0.5 per cent); any spot, apart from the principal spot and any
spot corresponding to thiamazole, is not more intense than the spot in the chromatogram obtained
with reference solution (c) (0.5 per cent).
Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in a
desiccator over diphosphorus pentoxide R at a pressure not exceeding 670 Pa for 24 h.
07-30
ASSAY
Dissolve 50.0 mg in water R and dilute to 500.0 ml with the same solvent. To 10.0 ml add 10 ml of
dilute hydrochloric acid R and dilute to 100.0 ml with water R. Measure the absorbance (2.2.25) at the
maximum at 291 nm. Calculate the content of C
7
H
10
N
2
O
2
S taking the specific absorbance to be
557.
STORAGE
IMPURITIES
N
N
Me
SH
A. 1-methyl-1H-imidazole-2-thiol (thiamazole).
__________________________________________________________________________________________________________ Ph Eur
07-31
Carbocisteine
S
COOH
HOOC
NH
2
H
C
5
H
9
NO
4
S 79.2 2387-59-9
Carbocisteine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0885]. These
Action and use Mucolytic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Carbocisteine contains not less than 98.5 per cent and not more than the equivalent of 101.0 per
cent of (R)-2-amino-3-[(carboxymethyl)thio]propanoic acid, calculated with reference to the dried
substance.
CHARACTERS
A white, crystalline powder, practically insoluble in water, in alcohol and in ether. It dissolves in
dilute mineral acids and in dilute solutions of alkali hydroxides.
IDENTIFICATION
obtained with carbocisteine CRS. Examine the substances prepared as discs.
D. Dissolve 0.1 g in 4.5 ml of dilute sodium hydroxide solution R. Heat on a water-bath for 10 min.
Cool and add 1 ml of a 25 g/l solution of sodium nitroprusside R. A dark red colour is produced, which
changes to brown and then to yellow within a few minutes.
TESTS
Solution S Disperse 5.00 g in 20 ml of water R and add dropwise with shaking 2.5 ml of strong
sodium hydroxide solution R. Adjust to pH 6.3 with 1M sodium hydroxide and dilute to 50.0 ml with
water R.
pH (2.2.3). Shake 0.2 g with 20 ml of carbon dioxide-free water R. The pH of the suspension is 2.8 to
3.0.
Specific optical rotation (2.2.7). 32.5 to 35.5, determined on solution S and calculated with
Ninhydrin-positive substances Examine by thin-layer chromatography (2.2.27), using a suitable
silica gel as the coating substance.
Test solution (a). Dissolve 0.10 g of the substance to be examined in dilute ammonia R2 and dilute to
Reference solution (a). Dissolve 10 mg of carbocisteine CRS in dilute ammonia R2 and dilute to 50 ml
Reference solution (c). Dissolve 10 mg of carbocisteine CRS and 10 mg of arginine hydrochloride CRS in
5 ml of dilute ammonia R2 and dilute to 25 ml with water R.
of butanol R. Dry the plate in a current of warm air. Spray with ninhydrin solution R and heat at 100C
to 105C for 15 min. Any spot in the chromatogram obtained with test solution (a), apart from the
07-32
Chlorides (2.4.4). Dissolve 33 mg in 5 ml of dilute nitric acid R and dilute to 15 ml with water R.
The solution, without further addition of nitric acid, complies with the limit test for chlorides
(0.15 per cent).
Sulphates (2.4.13). Dissolve 0.5 g in 5 ml of dilute hydrochloric acid R and dilute to 15 ml with
distilled water R. The solution complies with the limit test for sulphates (300 ppm).
100C to 105C for 2 h.
ASSAY
Dissolve 0.150 g in 10 ml of anhydrous formic acid R with slight heating and shake until dissolution is
complete. Add 50 ml of anhydrous acetic acid R. Titrate with 0.1M perchloric acid, determining the end-
5
H
9
NO
4
S.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
07-33
Carbomers
corrected 1/01
Carbomers comply with the requirements of the 3rd edition of the European Pharmacopoeia [1299]. These
Action and use Pharmaceutical aids.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Carbomers are high molecular weight polymers of acrylic acid cross-linked with polyalkenyl ethers of
sugars or polyalcohols. They contain not less than 56.0 per cent and not more than 68.0 per cent of
carboxylic acid (-COOH) groups, calculated with reference to the dried substance. The apparent
viscosity is not less than 70.0 per cent and not more than 130.0 per cent of the value stated on the
label for carbomers with a nominal apparent viscosity of 20,000 mPas or more, and not less than
50.0 per cent and not more than 150.0 per cent of the value stated on the label for carbomers with a
nominal apparent viscosity less than 20,000 mPas.
CHARACTERS
A white, fluffy powder, hygroscopic, swelling in water and other polar solvents after dispersion and
neutralisation with sodium hydroxide solution.
IDENTIFICATION
A. Examine by infrared absorption spectrophotometry (2.2.24). The spectrum has main bands at
2960 cm
1
, 1720 cm
1
, 1455 cm
1
, 1415 cm
1
, 1250 cm
1
, 1175 cm
1
and 800 cm
1
, with the
strongest band at 1720 cm
1
.
B. Adjust a 10 g/l dispersion to about pH 7.5 with 1M sodium hydroxide. A very viscous gel is formed.
C. Add 2 ml of a 100 g/l solution of calcium chloride R with continuous stirring to 10 ml of the gel
from test B. A white precipitate is immediately produced.
D. Add 0.5 ml of thymol blue solution R to 10 ml of a 10 g/l dispersion. An orange colour is produced.
Add 0.5 ml of cresol red solution R to 10 ml of a 10 g/l dispersion. A yellow colour is produced.
E. It complies with the viscosity indicated on the label.
TESTS
Apparent viscosity Dry the substance to be examined in vacuum at 80C for 1 h. Carefully add
2.50 g of the previously dried substance to 500 ml of water R in a 1000 ml beaker while stirring
continuously at 1000 50 rpm, with the stirrer shaft set at an angle of 60 to one side of the beaker.
Add the previously dried substance over a period of 45 s to 90 s, at a uniform rate, ensuring that
loose aggregates of powder are broken up and continue stirring at 1000 50 rpm

for
15 min. Remove the stirrer, and place the beaker containing the dispersion in a water-bath at a
temperature of 25 0.2C for 30 min. Insert the stirrer to a depth necessary to ensure that air is not
drawn into the dispersion, and while stirring at 300 25 rpm, titrate with a glass-calomel electrode
system to a pH of between 7.3 and 7.8 by adding a 180 g/l solution of sodium hydroxide R below the
surface, determining the end-point potentiometrically (2.2.20). The total volume of the 180 g/l
solution of sodium hydroxide R used is about 6.2 ml. Allow 2 min to 3 min before the final pH
determination. If the final pH exceeds 7.8, discard the preparation, and prepare another using a
smaller amount of sodium hydroxide for titration. Return the neutralised preparation to the water-
bath at 25C for 1 h, then perform the viscosity determination without delay to avoid slight viscosity
changes that occur 75 min after neutralisation. Determine the viscosity (2.2.10) with a rotating
viscometer with a spindle rotating at 20 rpm, using a suitable spindle for the range of the expected
apparent viscosity. The apparent viscosity is 300 mPas to 115,000 mPas.
Free acrylic acid Examine by liquid chromatography (2.2.29).
Test solution. Mix 0.125 g of the substance to be examined with a 25 g/l solution of aluminium
potassium sulphate R and dilute to 25.0 ml with the same solution. Heat the suspension at 50C for
20 min with occasional shaking. Then shake the suspension continuously at room temperature for
60 min. Centrifuge and use the clear supernatant solution as the test solution.
Reference solution. Dissolve 62.5 mg of acrylic acid R in a 25 g/l solution of aluminium potassium
sulphate R and dilute to 100.0 ml with the same solution. Dilute 1.0 ml of this solution to 50.0 ml
with a 25 g/l solution of aluminium potassium sulphate R.
07-34
as mobile phase at a flow rate of 1 ml/min a gradient prepared as follows:
Mobile phase A. A 1.361 g/l solution of potassium dihydrogen phosphate R,
Mobile phase B. Equal volumes of a 1.361 g/l solution of potassium dihydrogen phosphate R and
acetonitrile for chromatography R,
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
0 100 0
8 100 0
9 0 100
20 0 100
21 100 0
30 100 0
Inject about 20 l of each solution. When the chromatograms are recorded in the prescribed
conditions, the retention time of acrylic acid is about 6.0 min.
In the chromatogram obtained with the test solution, the area of any peak corresponding to acrylic
acid is not greater that the area of the corresponding peak in the chromatogram obtained with the
reference solution (0.25 per cent).
Benzene Examine according to the general method for the determination of residual solvents
(2.4.24) system A.
Solvent solution. Dissolve 0.100 g of benzene R in dimethyl sulphoxide R and dilute to 100.0 ml with the
same solvent. Dilute 1.0 ml of this solution to 100.0 ml with water R. Dilute 1.0 ml of this solution to
Test solution. Weigh 50.0 mg of the sample to be examined into an injection vial and add 5.0 ml of
water R and 1.0 ml of dimethyl sulphoxide R.
Reference solution. Weigh 50.0 mg of the sample to be examined into an injection vial and add 4.0 ml
of water R, 1.0 ml of dimethyl sulphoxide R and 1.0 ml of the solvent solution.
Close the vials with a tight rubber membrane slighter stopper coated with polytetrafluoroethylene and secure
with an aluminium crimped cap. Shake to obtain a homogeneous dispersion.
The following head space conditions may be used:
transfer line temperature: 90C.
Inject 1 ml of the gaseous phase of the reference solution into the column described for System A.
If necessary, adjust the sensitivity of the system so that the height of the peak corresponding to
benzene is at least 20 per cent of the full scale of the recorder.
Inject 1 ml of the gaseous phase of the test solution and 1 ml of the gaseous phase of reference
solution into the column. Repeat these injections twice more.
The mean area of the peak corresponding to benzene in the chromatograms obtained with the test
solution is not greater than half the mean area of the peak corresponding to benzene in the chromato-
grams obtained with the reference solution.
The test is not valid unless the relative standard deviation of the differences in areas between the
analyte peaks obtained from the three replicate paired injections of the reference solution and the test
solution is at most 15 per cent.
The content of benzene is not more than 2 ppm.
80C for 60 min.
ASSAY
Slowly add 0.120 g to 400 ml of water R stirring vigorously and continue stirring for 15 min. Reduce
the stirring speed and titrate with 0.2M sodium hydroxide to pH 10.0 determining the end-point
potentiometrically (2.2.20) using a glass-calomel electrode system. Stir for about 1 min after each
addition of 0.2M sodium hydroxide before reading the pH.
1 ml of 0.2M sodium hydroxide is equivalent to 9.0 mg of carboxylic acid (-COOH) groups.
07-35
STORAGE
LABELLING
The label states the apparent nominal viscosity.
__________________________________________________________________________________________________________ Ph Eur
07-36
Carbon Dioxide
CO
2
44.01 124-38-9
Carbon Dioxide complies with the requirements of the 3rd edition of the European Pharmacopoeia [0375].
Carbon Dioxide should be kept in approved metal cylinders which are painted grey and carry a label
stating Carbon Dioxide. In addition, Carbon Dioxide or the symbol CO
2
should be stencilled in
paint on the shoulder of the cylinder.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Carbon dioxide contains not less than 99.5 per cent V/V of CO
2
in the gaseous phase.
CHARACTERS
A colourless gas. At 20C and at a pressure of 101 kPa, 1 volume dissolves in about 1 volume of
water.
PRODUCTION
Examine the gaseous phase.
If the test is performed on a cylinder of gas, keep the cylinder of the substance to be examined at room
temperature for not less than 6 h before carrying out the tests. Keep the cylinder in the vertical position with
the outlet valve uppermost.
Carbon monoxide Not more than 5 ppm V/V, determined by gas chromatography (2.2.28).
Gas to be examined. The substance to be examined.
Reference gas. Use a mixture containing 5 ppm V/V of carbon monoxide R in carbon dioxide R1.
a stainless steel column 2 m long and 4 mm in internal diameter packed with an appropriate
molecular sieve for chromatography (0.5 nm),
helium for chromatography R as the carrier gas at a flow rate of 60 ml/min,
a flame-ionisation detector with methaniser,
maintaining the temperature of the column at 50C and that of the injection port and of the detector
at 130C.
Inject the gas to be examined and the reference gas. Adjust the injected volumes and the operating
conditions such that the height of the peak due to carbon monoxide in the chromatogram obtained
with the reference gas is at least 35 per cent of the full scale of the recorder.
Calculate the carbon monoxide content from the area of the peak due to carbon monoxide in the
chromatogram obtained with the reference gas.
Nitrogen monoxide and nitrogen dioxide Not more than a total of 2 ppm V/V, determined using
a chemiluminescence analyser (2.5.26).
Reference gas (a). Use carbon dioxide R1.
Reference gas (b). Use a mixture containing 2 ppm V/V of nitrogen monoxide R in carbon dioxide R1.
of nitrogen monoxide and nitrogen dioxide in the gas to be examined.
07-37
Fig.3751 UV Fluorescence Analyser
Total sulphur Not more than 1 ppm V/V, determined using an ultraviolet fluorescence analyser
after oxidation of the sulphur compounds by heating at 1000C (Figure 3751).
a system generating ultraviolet radiation with a wavelength of 210 nm, made up of an ultraviolet
lamp, a collimator, and a selective filter; the beam is blocked periodically by a chopper rotating
at high speed,
a reaction chamber through which flows the previously filtered gas to be examined,
a system that detects radiation emitted at a wavelength of 350 nm, made up of a selective filter, a
photomultiplier tube and an amplifier.
Reference gas (b). Use a mixture containing between 0.5 ppm V/V and 2 ppm V/V of hydrogen
sulphide R1 in carbon dioxide R1.
Calibrate the apparatus and set the sensitivity using reference gases (a) and (b). Pass the gas to be
examined through a quartz oven heated to 1000C. Oxygen R is circulated in the oven at a tenth of
the flow rate of the gas to be examined. Measure the sulphur dioxide content in the gaseous mixture
leaving the oven.
Water Not more than 67 ppm V/V, determined using an electrolytic hygrometer (2.5.28).
Assay Carry out the assay using an infrared analyser (2.5.24).
Gas to be examined. The substance to be examined. It must be filtered to avoid stray light phenomena.
Reference gas (b). Use a mixture containing 95.0 per cent V/V of carbon dioxide R1 and 5.0 per cent
V/V of nitrogen R1.
of carbon dioxide in the gas to be examined.
IDENTIFICATION
spectrum of carbon dioxide.
B. Place a glowing splinter of wood in an atmosphere of the substance to be examined. It is
extinguished.
C. Pass a stream of the substance to be examined through barium hydroxide solution R. A white
07-38
precipitate is formed which dissolves with effervescence in dilute acetic acid R.
TESTS
Examine the gaseous phase.
If the test is performed on a cylinder of gas, keep the cylinder of the substance to be examined at room tempera-
ture for not less than 6 h before carrying out the tests. Keep the cylinder in the vertical position with the outlet
valve uppermost.
Carbon monoxide Not more than 5 ppm V/V, determined using a carbon monoxide detector tube
(2.1.6).
Hydrogen sulphide Not more than 1 ppm V/V, determined using a hydrogen sulphide detector
tube (2.1.6).
Nitrogen monoxide and nitrogen dioxide Not more than 2 ppm V/V, determined using a
nitrogen monoxide and nitrogen dioxide detector tube (2.1.6).
Sulphur dioxide Not more than 2 ppm V/V, determined using a sulphur dioxide detector tube
(2.1.6).
Water vapour Not more than 67 ppm V/V, determined using a water vapour detector tube (2.1.6).
STORAGE
Store liquefied under pressure in suitable containers complying with the legal regulations.
IMPURITIES
A. nitrogen monoxide,
B. nitrogen dioxide,
C. carbon monoxide,
D. total sulphur,
E. water.
__________________________________________________________________________________________________________ Ph Eur
07-39
Carboplatin
O
O
Pt
O
O
NH
3
NH
3
C
6
H
12
N
2
O
4
Pt 371.3 41575-94-4
Carboplatin complies with the requirements of the 3rd edition of the European Pharmacopoeia [1081]. These
Preparation
Carboplatin Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Carboplatin contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent
of cis-diammine[cyclobutane-1,1-dicarboxylato(2)-O,O]platinum, calculated with reference to the
dried substance.
CHARACTERS
A colourless, crystalline powder, sparingly soluble in water, very slightly soluble in acetone and in
alcohol.
IDENTIFICATION
spectrum of carboplatin.
TESTS
Solution S1 Dissolve 0.25 g in carbon dioxide-free water R and dilute to 25 ml with the same solvent.
Solution S2 Dissolve 1.0 g in water R, heating slightly if necessary, and dilute to 40 ml with the same
solvent. Filter if necessary.
Appearance of solution Solution S1 is clear (2.2.1) and colourless (Method II, 2.2.2).
Acidity To 10 ml of solution S1 add 0.1 ml of phenolphthalein solution R1. The solution is colourless.
Not more than 0.7 ml of 0.01M sodium hydroxide is required to change the colour of the indicator to
pink.
acetonitrile R and water R and dilute to 20.0 ml with the same mixture.
Reference solution. Dilute 0.5 ml of the test solution to 200.0 ml with the mobile phase.
a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with
aminopropylsilyl silica gel for chromatography R (5 m),
as mobile phase at a flow rate of 2 ml per minute a mixture of 130 volumes of water R and 870
a 10 l loop injector,
Inject the test solution. The test is not valid unless:
the mass distribution ratio, D
m
, is not less than 4.0,
the number of theoretical plates, n, is not less than 5000,
the symmetry factor is not more than 2.0.
If necessary adjust the concentration of acetonitrile in the mobile phase.
Inject the test solution and the reference solution. Continue the chromatography for 2.5 times the
retention time of the principal peak. In the chromatogram obtained with the test solution; the area of
any peak apart from the principal peak is not greater than the area of the principal peak in the
chromatogram obtained with the reference solution (0.25 per cent); the sum of the areas of all the
07-40
peaks apart from the principal peak is not greater than twice the area of the principal peak in the
chromatogram obtained with the reference solution (0.5 per cent). Disregard any peak with an area
less than 0.2 times the area of the principal peak in the chromatogram obtained with the reference
solution.
Chlorides (2.4.4). Dilute 10 ml of solution S2 to 15 ml with water R. The solution complies with
the limit test for chlorides (100 ppm). Prepare the standard using 5 ml of chloride standard solution
(5 ppm Cl) R.
Ammonium (2.4.1). 0.20 g complies with the limit test B for ammonium (100 ppm). Prepare the
4
) R.
Silver Not more than 10 ppm of Ag, determined by atomic emission spectrometry (Method I,
2.2.22).
Test solution. Dissolve 0.50 g in a 1 per cent V/V solution of nitric acid R and dilute to 50.0 ml with
the same acid.
Reference solutions. Prepare the reference solutions using silver standard solution (5 ppm Ag) R, diluted
with a 1 per cent V/V solution of nitric acid R.
Measure the intensity emitted at 328.1 nm.
Soluble barium Not more than 10 ppm of Ba, determined by atomic emission spectrometry
(Method I, 2.2.22).
Test solution. Use the solution described in the test for silver.
Reference solutions. Prepare the reference solutions using barium standard solution (50 ppm Ba) R,
diluted with a 1 per cent V/V solution of nitric acid R.
Measure the intensity emitted at 455.4 nm.
Heavy metals (2.4.8). 12 ml of solution S2 complies with limit test A for heavy metals (20 ppm).
Prepare the standard using 5 ml of lead standard solution (1 ppm Pb) R.
at 100C to 105C.
ASSAY
Use the residue obtained in the test for loss on drying.
Ignite 0.200 g of the residue to constant mass at 800C.
1 mg of the residue is equivalent to 1.903 mg of C
6
H
12
N
2
O
4
Pt.
STORAGE
IMPURITIES
A. cisplatin,
B. cyclobutane-1,1-dicarboxylic acid.
__________________________________________________________________________________________________________ Ph Eur
07-41
Cardamom Fruit
Definition Cardamom Fruit consists of the dried, nearly ripe fruit of Elettaria cardamomum Maton
var. minuscula Burkill.
Characteristics Odour and taste of the seeds, strongly aromatic.
Macroscopical Fruit: a trilocular inferior capsule, up to about 2 cm long, ovoid or oblong, dull green
to pale buff, plump or slightly shrunken, obtusely triangular in cross section, nearly smooth or
longitudinally striated. Seeds in each loculus in two rows, forming an adherent mass attached to the
axile placenta. Seed: pale to dark reddish brown, about 4 mm long and 3 mm broad, irregularly
angular, marked with six to eight transverse wrinkles, with a longitudinal channel containing the
raphe, each seed enveloped by a colourless, membranous aril. Transversely cut surface of seed
showing a brown testa, white starchy perisperm, grooved on one side, yellowish endosperm and a
paler embryo.
Microscopical Seed: aril composed of flattened, thin-walled, parenchymatous cells. Testa composed of
the following layers: (i) outer epidermis of thick-walled, narrow, axially elongated cells; (ii) a layer of
collapsed parenchyma subjacent to the outer epidermis; (iii) a single layer (two or three layers near
the raphe) of large, thin-walled, rectangular cells containing volatile oil; (iv) two or three layers of
parenchyma; (v) layers of thin-walled, flattened cells; (vi) distinctive sclerenchymatous layer of closely
packed brown, thick-walled cells, each with a bowl-shaped cavity in the upper part containing a warty
silica body; (vii) inner layer consisting of flattened cells. Perisperm: cells thin-walled, packed with
numerous starch granules up to 6 m in diameter and, in a small cavity, one to seven prisms of
calcium oxalate about 10 to 30 m long. Endosperm parenchymatous, thin-walled, with a granular
hyaline mass of protein in each cell. Embryo: cells small, containing aleurone grains.
Foreign matter Of the fruit, not more than 1.0%; of the separated seeds, not more than 3.0%,
Appendix XI D.
Volatile oil In the seeds, not less than 4.0% v/w, Appendix XI E, Method I. Use 20 g of the
unground seeds and distil for 5 hours.
Acid-insoluble ash Of the seeds, not more than 3.5%, Appendix XI K.
Ash Of the seeds, not more than 6.0%, Appendix XI J.
In making preparations of Cardamom, only the seed is used. The seed is removed from the fruit,
immediately powdered or bruised and used immediately in making the preparation. Cardamom seed,
after removal from the fruit, should not be stored.
07-42
Cardamom Oil
Definition Cardamom Oil is obtained by distillation from crushed Cardamom Fruit.
Characteristics A clear, colourless or pale yellow liquid, visibly free from water; odour, that of
Cardamom Fruit.
Ester value 90 to 156, Appendix X C.
Storage Cardamom Oil should be kept in a well-filled, well-closed container, protected from light
and stored at a temperature not exceeding 25.
Action and use Carminative and flavour.
Preparations
Aromatic Cardamom Tincture
Compound Cardamom Tincture
07-43
Carmellose Calcium
9050-04-8
Carmellose Calcium complies with the requirements of the 3rd edition of the European Pharmacopoeia [0886].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Carmellose calcium (carboxymethylcellulose calcium) is the calcium salt of a partly O-carboxy-
methylated cellulose. It may contain not more than 0.6 per cent of silica (SiO
2
).
CHARACTERS
A white or yellowish-white powder, hygroscopic after drying, practically insoluble in acetone, in
alcohol and in toluene. It swells with water to form a suspension.
IDENTIFICATION
A. Shake 0.1 g thoroughly with 10 ml of water R. Add 12 ml of dilute sodium hydroxide solution R and
allow to stand for 10 min (solution A). Dilute 1 ml of solution A to 5 ml with water R. To 0.05 ml
add 0.5 ml of a 0.5 g/l solution of chromotropic acid, sodium salt R in a 75 per cent m/m solution of
sulphuric acid R and heat on a water-bath for 10 min. A reddish-violet colour develops.
B. Shake 5 ml of solution A obtained in identification test A with 10 ml of acetone R. A white,
flocculent precipitate is produced.
C. Shake 5 ml of solution A obtained in identification test A with 1 ml of ferric chloride solution R1. A
brown, flocculent precipitate is formed.
D. Ignite 1 g and dissolve the residue in a mixture of 5 ml of acetic acid R and 10 ml of water R. Filter
if necessary and boil the filtrate for a few minutes. Cool and neutralise with dilute ammonia R1. The
solution gives reaction (a) of calcium (2.3.1).
TESTS
Solution S Shake 1.0 g with 50 ml of distilled water R, add 5 ml of dilute sodium hydroxide solution R
and dilute to 100 ml with distilled water R.
Alkalinity Shake 1.0 g thoroughly with 50 ml of carbon dioxide-free water R and add 0.05 ml of
phenolphthalein solution R. No red colour develops.
Silica Not more than 0.6 per cent. To the residue obtained in the test for sulphated ash add
sufficient alcohol R to moisten the residue completely. Add 6 ml of hydrofluoric acid R in small
portions. Evaporate to dryness at 95C to 105C, taking care to avoid loss from sputtering. Cool and
rinse the wall of the platinum crucible with 6 ml of hydrofluoric acid R. Add 0.5 ml of sulphuric acid R
and evaporate to dryness. Progressively increase the temperature, ignite at 900C, allow to cool in a
desiccator and weigh. The difference between the mass of the final residue obtained in the test for
sulphated ash and the mass of the final residue is equal to the amount of silica in the substance to be
examined.
Chlorides (2.4.4). Heat 20 ml of solution S with 10 ml of dilute nitric acid R on a water-bath until a
flocculent precipitate is produced. Cool, centrifuge and separate the supernatant liquid. Wash the
precipitate with three quantities, each of 10 ml, of water R, centrifuging each time. Combine the
supernatant liquid and the washings and dilute to 100 ml with water R. To 25 ml add 6 ml of dilute
nitric acid R and dilute to 50 ml with water R. 10 ml of the solution diluted to 15 ml with water R
complies with the limit test for chlorides (0.5 per cent).
Sulphates (2.4.13). Heat 20 ml of solution S with 1 ml of hydrochloric acid R on a water-bath until a
flocculent precipitate is produced. Cool, centrifuge and separate the supernatant liquid. Wash the
precipitate with three quantities, each of 10 ml, of distilled water R, centrifuging each time. Combine
the supernatant liquid and the washings and dilute to 100 ml with distilled water R. To 25 ml add
1 ml of dilute hydrochloric acid R and dilute to 50 ml with distilled water R. 15 ml of the solution
complies with the limit test for sulphates (1 per cent).
Sulphated ash (2.4.14). 10.0 per cent to 20.0 per cent, determined on 1.0 g in a platinum crucible
and calculated with reference to the dried substance. Moisten with a mixture of equal volumes of
07-44
water R and sulphuric acid R.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
07-45
Carmellose Sodium
1/01
9050-32-4
Carmellose Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia [0472].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Carmellose sodium (carboxymethylcellulose sodium) is the sodium salt of a partly O-carboxy-
methylated cellulose. It contains not less than 6.5 per cent and not more than 10.8 per cent of
sodium (Na), calculated with reference to the dried substance.
CHARACTERS
A white or almost white, granular powder, hygroscopic after drying, practically insoluble in acetone,
in ethanol, in ether and in toluene. It is easily dispersed in water giving colloidal solutions.
IDENTIFICATION
A. To 10 ml of solution S (see Tests) add 1 ml of copper sulphate solution R. A blue, cotton-like
precipitate is formed.
B. Boil 5 ml of solution S for a few minutes. No precipitate is formed.
C. The solution prepared from the sulphated ash in the test for heavy metals gives the reactions of
sodium (2.3.1).
TESTS
Solution S Sprinkle a quantity of the substance to be examined equivalent to 1.0 g of the dried
substance onto 90 ml of carbon dioxide-free water R at 40C to 50C stirring vigorously. Continue
stirring until a colloidal solution is obtained, cool and dilute to 100 ml with carbon dioxide-free
water R.
not more intensely coloured than reference solution Y
6
(Method II, 2.2.2).
Apparent viscosity While stirring, introduce a quantity of the substance to be examined equivalent
to 2.00 g of the dried substance into 50 ml of water R heated to 90C. For a product of low viscosity,
use if necessary, the quantity required to give the concentration indicated on the label. Allow to cool,
dilute to 100.0 ml with water R and stir until dissolution is complete. Determine the viscosity (2.2.10)
using a rotating viscometer at 20C and a shear rate of 10 s
1
. If it is impossible to obtain a shear rate
of exactly 10 s
1
, use a shear rate slightly higher and a rate slightly lower and interpolate. The
apparent viscosity is not less than 75 per cent and not more than 140 per cent of the value stated on
the label.
Sodium glycollate Place a quantity of the substance to be examined equivalent to 0.500 g of dried
substance in a beaker. Add 5 ml of acetic acid R and 5 ml of water R. Stir until dissolution is complete
(about 30 min). Add 80 ml of acetone R and 2 g of sodium chloride R. Filter through a fast filter paper
impregnated with acetone R into a volumetric flask, rinse the beaker and filter with acetone R and
dilute the filtrate to 100.0 ml with the same solvent. Allow to stand for 24 h without shaking. Use the
clear supernatant liquid to prepare the test solution.
In a volumetric flask, dissolve 0.310 g of glycollic acid R, previously dried in vacuo over diphosphorus
pentoxide R, in water R and dilute to 1000.0 ml with the same solvent. Place 5.0 ml of this solution in
a volumetric flask, add 5 ml of acetic acid R and allow to stand for about 30 min. Add 80 ml of
acetone R and 2 g of sodium chloride R and dilute to 100.0 ml with acetone R. Use this solution to
prepare the reference solution.
Place 2.0 ml of each solution in a separate 25 ml volumetric flask. Heat on a water-bath to
eliminate acetone. Cool to room temperature and add 5.0 ml of 2,7-dihydroxynaphthalene solution R
to each flask. Shake and add 15.0 ml of 2,7-dihydroxynaphthalene solution R. Close the flasks with
aluminium foil and heat on a water-bath for 20 min. Cool under running water and dilute to 25.0 ml
with sulphuric acid R. Within 10 min, transfer 10.0 ml of each solution to a flat-bottomed tube.
Examine the solutions viewing vertically. The test solution is not more intensely coloured than the
07-46
Heavy metals (2.4.8). To the residue obtained in the determination of the sulphated ash, add 1 ml
of hydrochloric acid R and evaporate on a water-bath. Take up the residue in 20 ml of water R.12 ml
of the solution complies with limit test A for heavy metals (20 ppm). Prepare the standard using lead
at 100C to 105C.
Sulphated ash (2.4.14). 20.0 per cent to 33.3 per cent, determined on 1.0 g using a mixture of
equal volumes of sulphuric acid R and water R and calculated with reference to the dried substance.
These limits correspond to a content of 6.5 per cent to 10.8 per cent of sodium (Na).
STORAGE
LABELLING
The label states the apparent viscosity in millipascal seconds for a 20 g/l solution; for a product of low
viscosity, the label states the concentration of the solution to be used and the apparent viscosity in
millipascal seconds.
__________________________________________________________________________________________________________ Ph Eur
07-47
Low-substituted Carmellose Sodium
9050-32-4
Low-substituted Carmellose Sodium complies with the requirements of the 3rd edition of the European
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Low-substituted carmellose sodium (sodium carboxymethylcellulose) is the sodium salt of a partly
O-(carboxymethylated) cellulose. It contains not less than 2.0 per cent and not more than 4.5 per
cent of sodium (Na), calculated with reference to the dried substance.
CHARACTERS
A white or almost white powder or short fibres, practically insoluble in acetone, in ethanol and in
toluene. It swells in water to form a gel.
IDENTIFICATION
A. Shake 1 g with 100 ml of a 100 g/l solution of sodium hydroxide R. A suspension is produced.
B. Shake 1 g with 50 ml of water R. Transfer 1 ml of the mixture to a test tube, add 1 ml of water R
and 0.05 ml of a freshly prepared 40 g/l solution of -naphthol R in methanol R. Incline the test tube
and add carefully 2 ml of sulphuric acid R down the side so that it forms a lower layer. A reddish-
purple colour develops at the interface.
C. It complies with the test for sulphated ash (2.4.14) (see Tests).
D. The solution prepared from the sulphated ash for the test for heavy metals gives reaction (a) of
sodium (2.3.1).
TESTS
pH (223). Shake 1 g with 100 ml of carbon dioxide-free water R for 5 min. Centrifuge. The pH of the
suspension is 6.0 to 8.5
Sodium chloride and sodium glycollate The sum of the percentages of sodium chloride and
sodium glycollate is not more than 0.5 per cent, calculated with reference to the dried substance.
Sodium chloride. Place 5.00 g in a 250 ml conical flask, add 50 ml of water R and 5 ml of strong
hydrogen peroxide solution R and heat on a water bath for 20 min, stirring occasionally to ensure total
hydration. Cool, add 100 ml of water R and 10 ml of nitric acid R. Titrate with 0.05M silver nitrate
determining the end-point potentiometrically (2.2.20) using a silver-based indicator electrode and a
double-junction reference electrode containing a 100 g/l solution of potassium nitrate R in the outer
jacket and a standard filling solution in the inner jacket.
1 ml of 0.05M silver nitrate is equivalent to 2.922 mg of NaCl.
Sodium glycollate. Place a quantity of the substance to be examined equivalent to 0.500 g of the dried
substance in a beaker. Add 5 ml of glacial acetic acid R and 5 ml of water R and stir to ensure total
hydration (about 30 min). Add 80 ml of acetone R and 2 g of sodium chloride R. Stir for several
minutes to ensure complete precipitation of the carboxymethylcellulose. Filter through a fast filter
paper impregnated with acetone R into a volumetric flask, rinse the beaker and filter with acetone R
and dilute the filtrate to 100.0 ml with the same solvent. Allow to stand for 24 h without shaking.
Use the clear supernatant as the test solution.
Prepare the reference solutions as follows: in a 100 ml volumetric flask, dissolve 0.100 g of glycollic
acid R, previously dried in vacuo over diphosphorus pentoxide R, in water R and dilute to 100.0 ml with
the same solvent. Transfer 0.5 ml, 1.0 ml, 1.5 ml and 2.0 ml of the solution to separate volumetric
flasks; dilute the contents of each flask to 5.0 ml with water R, add 5 ml of glacial acetic acid R, dilute
to 100.0 ml with acetone R and mix.
Transfer 2.0 ml of the test solution and 2.0 ml of each of the reference solutions to separate 25 ml
volumetric flasks. Heat the uncovered flasks in a water-bath to eliminate the acetone. Allow to cool
and add 5.0 ml of 2,7-dihydroxynaphthalene solution R to each flask. Mix, add a further 15.0 ml of
2,7-dihydroxynaphthalene solution R and mix again. Close the flasks with aluminium foil and heat in a
water-bath for 20 min. Cool and dilute to 25.0 ml with sulphuric acid R.
Measure the absorbance (2.2.25) of each solution at 540 nm. Prepare a blank using 2.0 ml of a
solution containing 5 per cent V/V each of glacial acetic acid R and water R in acetone R. Prepare a
standard curve using the absorbances obtained with the reference solutions. From the standard curve
and the absorbance of the test solution, determine the mass (a), in milligrams, of glycollic acid in the
substance to be examined and calculate the content of sodium glycollate from the formula:
07-48
10
100

1.29 a
b m ( )
1.29=the factor converting glycollic acid to sodium glycollate,
b=the loss on drying as a percentage,
m=the mass of the substance to be examined in grams.
Water-soluble substances Not more than 70.0 per cent. Disperse 5.00 g in 400.0 ml of water R
and stir for 1 min every 10 min during the first 30 min. Allow to stand for 1 h and centrifuge, if
necessary. Decant 100.0 ml of the supernatant liquid onto a fast filter paper in a vacuum filtration
funnel, apply vacuum and collect 75.0 ml of the filtrate. Evaporate to dryness and dry the residue at
100C to 105C for 4 h.
Heavy metals (2.4.8). To the residue obtained in the determination of the sulphated ash add 1 ml
of hydrochloric acid R and evaporate on a water-bath. Take up the residue in 20 ml of water R. 12 ml
at 100C to 105C.
Sulphated ash (2.4.14). 6.5 per cent to 13.5 per cent, determined on 1.0 g using a mixture of equal
volumes of sulphuric acid R and water R and calculated with reference to the dried substance. These
limits correspond to a content of 2.0 per cent to 4.5 per cent of Na.
The following test concerning the pharmaco-technological properties may be carried out depending on the
intended formulation. It is not a mandatory requirement.
Settling volume In a 100 ml graduated cylinder, place 20 ml of 2-propanol R, add 5.0 g of the
substance to be examined and shake vigorously. Dilute to 30 ml with 2-propanol R then to 50 ml with
water R and shake vigorously. Within 15 min, repeat the shaking three times. Allow to stand for 4 h
and determine the volume of the settled mass (15.0 ml to 35.0 ml).
STORAGE
__________________________________________________________________________________________________________ Ph Eur
07-49
Carmustine
Cl
N N
Cl
O
N
O
H
C
5
H
9
Cl
2
N
3
O
2
214.1 154-93-8
Carmustine complies with the requirements of the 3rd edition of the European Pharmacopoeia [1187]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Carmustine contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent
of 1,3-bis(2-chloroethyl)-1-nitrosourea, calculated with reference to the anhydrous substance.
CHARACTERS
A yellowish, granular powder, very slightly soluble in water, very soluble in ether and in methylene
chloride, freely soluble in ethanol.
IDENTIFICATION
spectrum of carmustine. Examine the melted substances prepared as films.
TESTS
1,3-bis(2-chloroethyl)urea (impurity A) Examine by thin-layer chromatography (2.2.27), using a
suitable silica gel as the coating substance.
Reference solution (a). Dissolve 2 mg of carmustine impurity A CRS in methylene chloride R and dilute to
Reference solution (b). Dilute 1 ml of the test solution to 10 ml with methylene chloride R. To 5 ml of
this solution, add 5 ml of reference solution (a).
10 volumes of methanol R and 90 volumes of methylene chloride R. Allow the plate to dry in air. Spray
with diethylamine R and heat at 125C for 10 min. Allow to cool and spray with silver nitrate
solution R2. Expose to ultraviolet light at 365 nm until brown to black spots appear. Any spot corres-
ponding to carmustine impurity A in the chromatogram obtained with the test solution is not more
intense than the spot in the chromatogram obtained with reference solution (a) (1 per cent). The test
is not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated
spots.
of water.
ASSAY
Dissolve 0.100 g in 30 ml of ethanol R and dilute to 100.0 ml with water R. Dilute 3.0 ml of the
5
H
9
C
l2
N
3
O
2
STORAGE
IMPURITIES
Cl
N N
Cl
O
H H
A. 1,3-bis(2-chloroethyl)urea.
__________________________________________________________________________________________________________ Ph Eur
07-50
Carnauba Wax
Carnauba Wax complies with the requirements of the 3rd edition of the European Pharmacopoeia [0597].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Carnauba wax is the purified wax obtained from the leaves of Copernicia cerifera Mart.
CHARACTERS
Pale-yellow or yellow powder, flakes or hard masses, practically insoluble in water and in alcohol,
soluble on warming in ethyl acetate and in xylene.
IDENTIFICATION
Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance.
Test solution. Dissolve 0.10 g of the substance to be examined with warming in 5 ml of chloroform R.
Use the warm solution.
Reference solution. Dissolve 5 mg of menthol R, 5 l of menthyl acetate R and 5 mg of thymol R in 10 ml
of toluene R.
Apply separately to the plate as bands 20 mm by 3 mm, 30 l of the test solution and 10 l of the
reference solution. Develop over a path of 10 cm using a mixture of 2 volumes of ethyl acetate R and
98 volumes of chloroform R. Allow the plate to dry and spray with a freshly prepared 200 g/l solution
of phosphomolybdic acid R in alcohol R, using about 10 ml for a plate 200 mm square and heat at
100C to 105C for 10 min to 15 min. The chromatogram obtained with the reference solution
shows in the lower part a dark blue zone (menthol), above this zone a reddish zone (thymol) and in
the upper part a dark blue zone (menthyl acetate). The chromatogram obtained with the test solution
shows a large blue zone (triacontanol = melissyl alcohol) at a level between the thymol and menthol
zones in the chromatogram obtained with the reference solution. Further blue zones are visible in the
upper part of the chromatogram obtained with the test solution, at levels between those of the
menthyl acetate and thymol zones in the chromatogram obtained with the reference solution; above
these zones further zones are visible in the chromatogram obtained with the test solution; the zone
with the highest R
f
value is very pronounced. A number of faint zones are visible below the triacon-
tanol zone and the starting point is coloured blue.
TESTS
Appearance of solution Dissolve 0.10 g with heating in chloroform R and dilute to 10 ml with the
same solvent. The solution is clear (2.2.1) and not more intensely coloured than a 0.05 g/l solution of
potassium dichromate R (Method II, 2.2.2).
Melting point (2.2.15). 80C to 88C. Melt the substance carefully on a water-bath before
introduction into the capillary tubes. Allow the tubes to stand at a temperature not exceeding 10C
for 24 h or at 0C for 2 h.
Acid value 2 to 7. To 2.000 g (m g) in a 250 ml conical flask fitted with a reflux condenser add
40 ml of xylene R and a few glass beads and heat until the substance is completely dissolved. Add
20 ml of alcohol R and 1 ml of phenolphthalein solution R1 and titrate the hot solution with 0.5M
alcoholic potassium hydroxide until a pink colour persists for at least 10 s (n
1
ml). Carry out a blank test
(n
2
ml). Calculate the acid value from the expression:
m
n n ) ( 05 . 28
2 1
Saponification value 78 to 95. To the titrated solution from the determination of the acid value,
add 20.0 ml of 0.5M alcoholic potassium hydroxide and boil under a reflux condenser for 3 h. Add 1 ml
of phenolphthalein solution R1 and titrate the hot solution immediately with 0.5M hydrochloric acid until
the red colour disappears. Reheat the solution to boiling and continue the titration, if necessary, until
the red colour disappears; repeat the heating and titration until the colour no longer reappears on
heating (n
3
4
ml). Calculate the saponification value from the
expression:
value acid
) ( 05 . 28
3 4
+
m
n n
07-51
STORAGE
__________________________________________________________________________________________________________ Ph Eur
07-52
Carteolol Hydrochloride
and enantiomer
N
O
O
NHBu
t
H OH
H
,HCl
C
16
H
24
N
2
O
3
,HCl 328.8 51781-21-6
Definition Carteolol Hydrochloride is (RS)-5-(3-tert-butylamino-2-hydroxypropoxy)-3,4-
dihydroquinolin-2(1H)-one hydrochloride. It contains not less than 98.5% and not more than
101.5% of C
16
H
24
N
2
O
3
,HCl, calculated with reference to the dried substance.
Characteristics White crystals or a white, crystalline powder.
Soluble in water; very slightly soluble in ethanol (96%) and in glacial acetic acid; sparingly soluble in
methanol; practically insoluble in ether.
Identification
carteolol hydrochloride (RS 043).
B. Yields the reactions characteristic of chlorides, Appendix VI.
Clarity and colour of solution A 3.0% w/v solution is clear, Appendix IV A, and colourless,
Heavy metals 2.0 g complies with limit test A for heavy metals, Appendix VII (10 ppm). Use 2 ml of
Related substances Carry out the method for liquid chromatography, Appendix III D, using
solutions of the substance being examined in the mobile phase containing (1) 0.20% w/v, (2)
0.0020% w/v and (3) 0.00020% w/v, respectively.
(25 cm 4.6 mm) packed with stationary phase C (5 m) (YMC-Pack ODS-A is suitable), (b) as the
mobile phase a mixture of 1 volume of methanol, 20 volumes of acetonitrile and 79 volumes of a
0.282% w/v solution of sodium hexanesulphonate at a flow rate such that the retention time of carteolol
hydrochloride is about 14 minutes (1 ml per minute may be suitable) and (c) a detection wavelength
of 252 nm.
The test is not valid unless the column efficiency, determined on the principal peak in the
chromatogram obtained with solution (2), is at least 6000 theoretical plates per metre.
the area of the peak in the chromatogram obtained with solution (3) (0.1%) and the sum of the areas
of any such peaks is not greater than half the area of the peak in the chromatogram obtained with
solution (2) (0.5%).
Assay Dissolve 0.25 g in 30 ml of glacial acetic acid by heating on a water bath with the aid of
ultrasound if necessary, cool, add 70 ml of acetic anhydride and titrate with 0.1M perchloric acid VS
determining the end point potentiometrically, Appendix VIII B. Each ml of 0.1M perchloric acid VS is
16
H
24
N
2
O
3
,HCl.
Storage Carteolol Hydrochloride should be kept in an airtight container.
Preparation
Carteolol Eye Drops
IMPURITIES
N
O
H
A. 3,4,5,6,7,8-hexahydroquinolin-2(1H),5-dione
07-53
N O
OH
H
B. 1,2,3,4-tetrahydro-2-oxoquinolin-5-ol
N O
O
O
H
C. 5-(2,3-epoxy)propoxy-1,2,3,4-tetrahydro-2-oxoquinoline
H
N O
O
OH
Cl
D. 5-(3-chloro-2-hydroxypropoxy)-1,2,3,4-tetrahydro-2-oxoquinoline
H
N O
O
OH
O NH
O
E. 1,3-bis-(1,2,3,4-tetrahydro-2-oxoquinolin-5-oxy)propan-2-ol
H
N O
O
OH
OMe
F. 5-(2-hydroxy-3-methoxy-propoxy)-1,2,3,4-tetrahydro-2-oxoquinoline
H
N O
O
OH
OH
G. 5-(2,3-dihydroxypropoxy)-1,2,3,4-tetrahydro-2-oxoquinoline
N
O
O
NHBu
t
H OH
H
H. 5-(3-tert-butylamino-2-hydroxypropoxy)quinolin-2(1H)-one
07-54
Cascara
Cascara complies with the requirements of the 3rd edition of the European Pharmacopoeia [0105]. These
Preparation
Cascara Dry Extract
When Powdered Cascara is prescribed or demanded, material complying with the requirements
supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cascara consists of the dried, whole or fragmented bark of Rhamnus purshianus D. C. (Frangula
purshiana (D. C.) A. Gray ex J. C. Cooper). It contains not less than 8.0 per cent of
hydroxyanthracene glycosides of which not less than 60 per cent consists of cascarosides, both
expressed as cascaroside A (C
27
H
32
O
14
; M
r
CHARACTERS
IDENTIFICATION
A. The bark occurs in slightly channelled or nearly flat pieces, usually 1 mm to 5 mm in thickness,
usually varying greatly in length and width. The outer surface is grey or dark greyish-brown and
shows occasional lenticels that are orientated transversally. It is usually more or less completely
covered by a whitish coat of lichens, epiphytic moss and foliaceous liverwort. The inner surface is
yellow to reddish-brown or almost black with fine longitudinal striations; it turns red when treated
with alkali. The yellow fracture is short and granular in the outer part and somewhat fibrous at the
inner part.
chloral hydrate solution R. The powder shows: bundles of partly lignified phloem fibres accompanied
by crystal sheaths containing prisms of calcium oxalate; groups of sclereids accompanied by crystal
sheaths; cluster crystals of calcium oxalate; some parenchymatous cells contain a yellow substance
which becomes deep red when treated with alkali; cork cells and, frequently, epiphytes; the latter may
be liverworts, entire or in fragments, having a lamina one cell thick without a midrib and composed
of isodiametric cells, or leaves of mosses, having a lamina one cell thick composed of elongated cells
and possessing a midrib several cells thick.
C. Examine the chromatograms obtained in the test for Other species of Rhamnus; anthrones after
spraying with a 50 g/l solution of potassium hydroxide R in alcohol (50 per cent V/V) R and heating. The
chromatogram obtained with the test solution shows several reddish-brown zones with different
intensities: there are four faint zones, three being situated at about the mid-point of the chromato-
gram and one in the lower third and there is a strong zone in the upper third of the chromatogram.
Examine in ultraviolet light at 365 nm. The chromatogram obtained with the test solution shows
several zones with the same fluorescence, situated above and particularly below (cascarosides) that
corresponding to barbaloin in the chromatogram obtained with the reference solution.
D. Heat 0.2 g of the powdered drug (180) with 50 ml of water R on a water-bath for 15 min. Allow
to cool and filter. To 10 ml of the filtrate add 20 ml of hydrochloric acid R1 and heat on a water-bath
for 15 min. Allow to cool, transfer to a separating funnel and shake with three quantities, each of
20 ml, of ether R. Reserve the aqueous layer (solution A).
(a) Combine the three ether extracts and shake with 10 ml of dilute ammonia R2. The aqueous layer
becomes reddish-violet.
(b) Transfer solution A to a small flask, add 5 g of ferric chloride R and heat on a water-bath for
30 min. Allow to cool, transfer to a separating funnel and shake with 15 ml of ether R. Wash the ether
layer with 10 ml of water R, discard the water and shake the ether layer with 5 ml of dilute
ammonia R2. A red colour develops in the aqueous layer.
TESTS
Other species of Rhamnus; anthrones Examine by thin-layer chromatography (2.2.27), using a
Test solution. To 0.5 g of the powdered drug (180) add 5 ml of alcohol (70 per cent V/V) R and heat to
boiling. Cool and centrifuge. Decant the supernatant solution immediately and use within 30 min.
07-55
Reference solution. Dissolve 20 mg of barbaloin R in alcohol (70 per cent V/V) R and dilute to 10 ml with
the same solvent.
mixture of 13 volumes of water R, 17 volumes of methanol R and 100 volumes of ethyl acetate R.
Allow the plate to dry for 5 min, spray with about 10 ml of a 50 g/l solution of potassium hydroxide R
in alcohol (50 per cent V/V) R and heat at 100C to 105C for 15 min. Examine the chromatograms
immediately after heating. The chromatogram obtained with the reference solution shows, in the
central part, a reddish-brown zone corresponding to barbaloin. Examine in ultraviolet light at
365 nm. The zone corresponding to barbaloin shows intense yellowish-brown fluorescence. In the
chromatogram obtained with the test solution, no zone with orange-brown fluorescence is seen
between the zone of barbaloin and the zones of cascarosides.
Apply to another plate as a band 10 l of the test solution and develop as described above. Allow
the plate to dry for not longer than 5 min and spray immediately with a 5 g/l solution of
nitrotetrazolium blue R in methanol R. Examine the chromatogram immediately. No violet or greyish-
blue zones appear.
Foreign matter (2.8.2). Not more than 1 per cent.
ASSAY
Carry out the assay protected from bright light in one day.
Stir 1.00 g of the powdered drug (180) into 100 ml of boiling water R and continue boiling and
stirring for 5 min. Allow to cool, dilute to 100.0 ml with water R, shake, filter and discard the first
20 ml of filtrate. Transfer 10.0 ml of the filtrate to a separating funnel, add 0.1 ml of 1M hydrochloric
acid and shake with two quantities, each of 20 ml, of a mixture of 1 volume of ether R and 3 volumes
of hexane R. Wash the combined organic extracts with 5 ml of water R, discard the organic layer and
return the rinsings to the aqueous layer. Shake the combined aqueous layers with four quantities,
each of 30 ml, of ethyl acetate R freshly saturated with water R, (to 150 ml of ethyl acetate R add 15 ml
of water R, shake for 3 min and allow to stand) on each occasion allowing separation to take place
until the organic layer is clear. Combine the ethyl acetate extracts. Use the aqueous layer for the assay
for cascarosides and the organic layer for the assay for hydroxyanthracene glycosides other than
cascarosides.
Hydroxyanthracene glycosides other than cascarosides. Transfer the organic layer to a suitable flask and
remove the solvent by distillation, evaporating almost to dryness. Dissolve the residue in 0.3 ml to
0.5 ml of methanol R and transfer to a volumetric flask, rinsing the first flask with warm water R and
adding the rinsings to the methanolic solution. Allow to cool and dilute to 50.0 ml with water R.
Transfer 20.0 ml of the solution to a 100 ml round-bottomed flask with a ground-glass neck and
containing 2 g of ferric chloride R and 12 ml of hydrochloric acid R. Attach a reflux condenser and place
the flask in a water-bath so that the level of the water is above that of the liquid in the flask and heat
for 4 h. Allow to cool, transfer the solution to a separating funnel and rinse the flask successively with
3 ml to 4 ml of 1M sodium hydroxide and 3 ml to 4 ml of water R, adding the rinsings to the separating
funnel. Shake the contents of the separating funnel with three quantities, each of 30 ml, of a mixture
of 1 volume of ether R and 3 volumes of hexane R. Wash the combined organic layers with two
quantities, each of 10 ml, of water R and discard the rinsings. Dilute the organic phase to 100.0 ml
with the mixture of ether and hexane. Take 20.0 ml, evaporate carefully to dryness on a water-bath
and dissolve the residue in 10.0 ml of a 5 g/l solution of magnesium acetate R in methanol R. Measure
the absorbance (2.2.25) at 515 nm using methanol R as the compensation liquid.
Calculate the percentage content of hydroxyanthracene glycosides, expressed as cascaroside A,
from the expression:
m
A 95 . 6
i.e. taking the specific absorbance to be 180.
m=mass of the sample in grams.
Measure the absorbance of the test solution at 440 nm. If the ratio of the absorbance at 515 nm to
that at 440 nm is less than 2.4, the assay is not valid and must be repeated.
Cascarosides. Dilute the aqueous layer reserved for this assay to 50.0 ml with water R. Treat 20.0 ml
of the solution as described above in the assay of hydroxyanthracene glycosides other than
cascarosides.
Calculate the percentage content of hydroxyanthracene glycosides, expressed as cascaroside A from
the expression:
07-56
m
A 95 . 6
i.e. taking the specific absorbance to be 180.
Measure the absorbance of the test solution at 440 nm. If the ratio of the absorbance at 515 nm to
that at 440 nm is less than 2.7, the assay is not valid and must be repeated.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
07-57
Cassia Oil
1/01
8007-80-5
Cassai Oil complies with the requirements of the 3rd edition of the European Pharmacopoeia [1496]. These
Action and use Carminative; flavouring agent.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cassia oil is obtained by steam distillation of the leaves and young branches of Cinnamomum cassia
Blume (C. aromaticum Nees).
CHARACTERS
A clear, mobile, yellow to reddish-brown liquid, with a characteristic odour reminiscent of cinnamic
aldehyde.
IDENTIFICATION
A. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R.
Test solution. Dissolve 0.5 ml of the substance to be examined in acetone R and dilute to 10 ml with
the same solvent.
Reference solution. Dissolve 50 l of trans-cinnamic aldehyde R, 10 l of eugenol R and 50 mg of
coumarin R in acetone R and dilute to 10 ml with the same solvent.
Apply to the plate, as bands, 10 l of each solution. Develop over a path of 15 cm using a mixture of
10 volumes of methanol R and 90 volumes of toluene R. Allow the plate to dry in air and examine in
ultraviolet light at 365 nm. The zone of blue fluorescence in the chromatogram obtained with the test
solution is similar in position and colour to the zone in the chromatogram obtained with the reference
solution (coumarin). Spray the plate with anisaldehyde solution R. Examine in daylight while heating at
100C to 105C for 5 min to 10 min. The chromatogram obtained with the reference solution shows
in its upper part a violet zone (eugenol) and above this zone a greenish-blue zone (trans-cinnamic
aldehyde). The chromatogram obtained with the test solution shows a zone similar in position and
colour to the zone due to trans-cinnamic aldehyde in the chromatogram obtained with the reference
solution and may show a very faint zone corresponding to eugenol. Other faint zones are present.
B. Examine the chromatograms obtained in the test for chromatographic profile. The retention times
of the principal peaks in the chromatogram obtained with the test solution are similar to those in the
chromatogram obtained with the reference solution. Eugenol may be absent from the chromatogram
obtained with the test solution.
TESTS
Optical rotation (2.2.7): 1 to +1.
Reference solution. Dissolve 100 l of trans-cinnamic aldehyde R, 10 l of cinnamyl acetate R, 10 l of
eugenol R, 20 mg of coumarin R and 10 l of trans-2-methoxycinnamaldehyde R in 1 ml of acetone R.
a fused-silica column 60 m long and about 0.25 mm in internal diameter coated with macrogol
20,000 R as the bonded phase,
a split ratio of 1:100,
07-58
Time
(min)
Temperature
(C)
Rate (C
per min)
Comment
10 75 60 190 2 linear gradient
Injection port 200
Detector 240
Inject 0.2 l of the reference solution. When the chromatogram is recorded in the prescribed
conditions, the components elute in the order indicated in the composition of the reference
solution. Record the retention times of these substances.
The test is not valid unless the resolution between the peaks corresponding to coumarin and trans-
2-methoxycinnamaldehyde is at least 1.5.
Inject 0.2 l of the test solution. Using the retention times determined from the chromatogram
obtained with the reference solution, locate the components of the reference solution in the chroma-
togram obtained with the test solution. Determine the percentage content of each of these
components by the normalisation procedure.
trans-cinnamic aldehyde 70 per cent to 90 per cent
cinnamyl acetate 1.0 per cent to 6.0 per cent
eugenol less than 0.5 per cent
coumarin 1.5 per cent to 4.0 per cent
trans-2-methoxycinnamaldehyde 3.0 per cent to 15 per cent
STORAGE
0
0
5
1
0
1
5
2
0
2
5
3
0
3
5
4
0
4
5
5
0
5
5
6
0
6
5
7
0
7
5
8
0
8
5
9
0
9
5
1
0
0
1
0
5
1
1
0
1
1
5
1
2
0
1
2
5
1
3
0
1
3
5
1
4
0
1
4
5
1
5
0
1
5
5
1
6
0
10
20
30
40
50
mVolts 1
2
3
5
4
1. trans-cinnamic aldehyde
2. cinnamyl acetate
3. eugenol
4. trans-2-methoxycinnamaldehyde
5. coumarin
Depending on operating conditions and the state of the column, coumarin may elute before or after
trans-2-methoxycinnamaldehyde.
Fig. 14961 Chromatographic profile of cassia oil
__________________________________________________________________________________________________________ Ph Eur
07-59
Hydrogenated Castor Oil
1/01
Hydrogenated Castor Oil complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hydrogenated castor oil is the oil obtained by hydrogenation of Virgin Castor oil (0051). It consists
mainly of the triglyceride of 12-hydroxystearic acid.
CHARACTERS
A fine, almost white or pale yellow powder or almost white or pale yellow masses or flakes, practically
insoluble in water, freely soluble in methylene chloride, slightly soluble in light petroleum and very
slightly soluble in ethanol.
IDENTIFICATION
B. It complies with the test for hydroxyl value (see Tests).
TESTS
Acid value (2.5.1). Not more than 4.0, determined on 10.0 g dissolved in 75 ml of hot alcohol R.
Hydroxyl value (2.5.3, Method A). 145 to 165, determined on a warm solution.
Iodine value (2.5.4). Not more than 5.0.
Alkaline impurities Dissolve 1.0 g by gentle heating in a mixture of 1.5 ml of alcohol R and 3 ml of
toluene R. Add 0.05 ml of a 0.4 g/l solution of bromophenol blue R in alcohol R. Not more than 0.2 ml
of 0.01M hydrochloric acid is required to change the colour of the indicator to yellow.
(2.4.22).
Test solution. Introduce 75 mg of the substance to be examined in a 10 ml centrifuge tube with a
screw cap. Dissolve in 2 ml of 1,1-dimethylethyl methyl ether R1 by shaking and heat gently (50C to
60C). Add, when still warm, 1 ml of a 12 g/l solution of sodium R in anhydrous methanol R, prepared
with the necessary precautions, and mix vigorously for at least 5 min. Add 5 ml of distilled water R
and mix vigorously for about 30 s. Centrifuge for 15 min at 1500 g. Use the upper layer.
Reference solution. Dissolve 50 mg of methyl 12-hydroxystearate CRS and 50 mg of methyl stearate CRS
in 10.0 ml of 1,1-dimethylethyl methyl ether R1.
maintaining the temperature of the column at 215C for 55 min and maintaining the temperature of
the injection port and that of the detector at 250C.
Inject 1 l of each solution.
Calculate the fraction of each fatty acid using the following expression:
m m A A / cent per 100 /

s,c x, s,c x,
A
x,s,c
=corrected peak area of the fatty acid in the test solution:
A
x,s,c
= A
x,s
R
c
R
c
=relative correction factor for the peaks corresponding to methyl 12-hydroxystearate and
methyl 9,10-dihydroxystearate:
r 2, r l,
r 2, r l,
c
m A
A m
R
R
c
=1 for peaks corresponding to each of the other specified fatty acids or any unspecified
fatty acid,
m
1,r
=mass of methyl 12-hydroxystearate in the reference solution,
07-60
m
2,r
=mass of methyl stearate in the reference solution,
A
1,r
=area of any peak corresponding to methyl 12-hydroxystearate in the chromatogram
obtained with the reference solution,
A
2,r
=area of any peak corresponding to methyl stearate in the chromatogram obtained with the
reference solution,
A
x,s
=area of the peaks corresponding to any specified or unspecified fatty acid methyl esters.
palmitic acid: not more than 2.0 per cent,
12-oxostearic acid (equivalent chain length on macrogol 20,000: 22.7): not more than 5.0 per
cent,
12-hydroxystearic acid (equivalent chain length on macrogol 20,000: 23.9): 78.0 per cent to
91.0 per cent,
9,10-dihydroxystearic acid (equivalent chain length on macrogol 20,000: 25.8): 0.3 per cent to
0.7 per cent,
any other fatty acid: not more than 3.0 per cent.
Nickel (2.4.27). Not more than 1 ppm of Ni.
STORAGE
Store in a well-filled container.
IMPURITIES
H
3
C
COOH
O
A. 12-oxostearic acid.
__________________________________________________________________________________________________________ Ph Eur
07-61
Virgin Castor Oil
Castor Oil 1/01
Virgin Castor Oil complies with the requirements of the 3rd edition of the European Pharmacopoeia [0051].
Action and use Stimulant laxative; emollient.
Preparation
Zinc and Castor Oil Ointment
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Virgin castor oil is the fatty oil obtained by cold expression from the seeds of Ricinus communis L. A
suitable antioxidant may be added.
CHARACTERS
A clear, almost colourless or slightly yellow, viscous, hygroscopic liquid, slightly soluble in light
petroleum, miscible with alcohol and with glacial acetic acid.
It has a refractive index of about 1.479 and a relative density of about 0.958.
IDENTIFICATION
First identification: D.
Second identification: A, B, C.
B. It complies with the test for hydroxyl value (see Tests).
C. It complies with the test for iodine value (see Tests).
D. It complies with the test for composition of fatty acids (see Tests).
TESTS
Optical rotation (2.2.7): +3.5 to +6.0.
Absorbance (2.2.25). To 1.0 g add alcohol R and dilute to 100.0 ml with the same solvent. The
solution shows an absorption maximum at 269 nm. The specific absorbance measured at this
Acid value (2.5.1). Dissolve 5.0 g in 25 ml of the prescribed mixture of solvents. The acid value is
not greater than 2.0.
Hydroxyl value (2.5.3, Method A). Not less than 150.
Iodine value (2.5.4): 82 to 90.
Peroxide value (2.5.5). Not greater than 10.0.
(2.4.22).
Test solution. Introduce 75 mg of the substance to be examined in a 10 ml centrifuge tube with a
screw cap. Dissolve in 2 ml of 1,1-dimethylethyl methyl ether R1 with shaking and heat gently (50C to
60C). Add, when still warm, 1 ml of a 12 g/l solution of sodium R in anhydrous methanol R, prepared
with the necessary precautions, and mix vigorously for at least 5 min. Add 5 ml of distilled water R
and mix vigorously for about 30 s. Centrifuge for 15 min at 1500 g. Use the upper layer.
Reference solution. Dissolve 50 mg of methyl ricinoleate CRS and 50 mg of methyl stearate CRS in
10.0 ml of 1,1-dimethylethyl methyl ether R1.
maintaining the temperature of the column at 215C for 55 min and maintaining the temperature of
the injection port and that of the detector at 250C.
Calculate the fraction of each fatty acid using the following expression:
m m A / A / cent per 100

s,c x, s,c x,
07-62
A
x,s,c
=corrected peak area of the fatty acid in the test solution:
c s x, s,c x,
R A A
R
c
=relative correction factor:
r 2, r l,
r 2, r l,
c
m A
A m
R
for the peaks corresponding to methyl ricinoleate and methyl 9,10-dihydroxystearate.

R
c
=1 for peaks corresponding to each of the other specified fatty acids or any unspecified
fatty acid,
m
1,r
=mass of methyl ricinoleate in the reference solution,
m
2,r
=mass of methyl stearate in the reference solution,
A
1,r
=area of any peak corresponding to methyl ricinoleate in the chromatogram obtained with
the reference solution,
A
2,r
=area of any peak corresponding to methyl stearate in the chromatogram obtained with the
reference solution,
A
x,s
=area of the peaks corresponding to any specified or unspecified fatty acid methyl esters.
oleic acid and isomers (C18: 1 equivalent chain length on macrogol 20,000: 18.3): 2.5 per cent
to 6.0 per cent,
linoleic acid (C18:2 equivalent chain length on macrogol 20,000: 18.8): 2.5 per cent to 7.0 per
cent,
linolenic acid (C18:3 equivalent chain length on macrogol 20,000: 19.2): not more than 1.0 per
cent,
eicosenoic acid (C20:1 equivalent chain length on macrogol 20,000: 20.2): not more than
1.0 per cent,
ricinoleic acid (equivalent chain length on macrogol 20,000: 23.9): 85.0 per cent to 92.0 per
cent,
9,10-dihydroxystearic acid (equivalent chain length on macrogol 20,000: 25.8): 0.3 per cent to
0.7 per cent,
any other fatty acid: not more than 1.0 per cent.
of water.
STORAGE
Store in a well-filled container, protected from light, at a temperature not exceeding 25C.
LABELLING
The label states the name and concentration of any added antioxidant.
__________________________________________________________________________________________________________ Ph Eur
07-63
Polyoxyl Castor Oil
Polyoxyl Castor Oil complies with the requirements of the 3rd edition of the European Pharmacopoeia [1082].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Polyoxyl castor oil contains mainly ricinoleyl glycerol ethoxylated with thirty to fifty molecules of
ethylene oxide (nominal value), with small amounts of macrogol ricinoleate and of the corresponding
free glycols. It results from the reaction of castor oil with ethylene oxide.
CHARACTERS
A clear, yellow viscous liquid, freely soluble in water, very soluble in methylene chloride, freely
soluble in alcohol.
It has a relative density of about 1.05 and a viscosity of 500 mPa.s to 800 mPa.s at 25C.
IDENTIFICATION
A. It complies with the test for iodine value (see Tests).
B. It complies with the test for saponification value (see Tests).
C. Examine by thin-layer chromatography (2.2.27), using a suitable octadecylsilyl silica gel as the
coating substance.
Test solution. To 1 g of the substance to be examined add 100 ml of a 100 g/l solution of potassium
hydroxide R and boil under a reflux condenser for 30 min. Allow to cool. Acidify the solution with
20 ml of hydrochloric acid R. Shake the mixture with 50 ml of ether R and allow to stand until a
separation of the layers is obtained. Transfer 10 ml of the clear upper layer to a suitable tube and
evaporate to dryness on a water-bath. Dissolve the residue in 1 ml of ether R.
Reference solution. Dissolve 40 mg of ricinoleic acid R in methylene chloride R and dilute to 2 ml with the
same solvent.
volumes of methylene chloride R, 40 volumes of glacial acetic acid R and 50 volumes of acetone R. Dry
the plate in a current of cold air, spray with a 200 g/l solution of phosphomolybdic acid R in alcohol R
and heat at 120C for about 10 min. The spots in the chromatogram obtained with the test solution
are similar in position and colour to the spots in the chromatogram obtained with the reference
solution.
D. Introduce into a test-tube about 2 g of the substance to be examined and add 0.2 ml of sulphuric
acid R. Close the tube using a stopper fitted with a glass tube bent twice at right angles. Heat the tube
until white fumes appear. Collect the fumes in 1 ml of mercuric chloride solution R. A white precipitate
is formed and the fumes turn the colour of a filter-paper impregnated with alkaline potassium
tetraiodomercurate solution R to black.
TESTS
Appearance of solution. Solution S is not more opalescent than reference suspension III (2.2.1)
and not more intensely coloured than reference solution BY
6
(Method II, 2.2.2).
Alkalinity. Dissolve 2.0 g in a hot mixture of 10 ml of water R and 10 ml of alcohol R. Add 0.1 ml of
bromothymol blue solution R1. Not more than 0.5 ml of 0.1M hydrochloric acid is required to change the
colour of the indicator to yellow.
Hydroxyl value (2.5.3, Method A). See Table 1082-1.
Iodine value (2.5.4). 25 to 35.
Saponification value (2.5.6). See Table 1082-1.
07-64
Table 10821
Ethylene oxide
units per molecule
(nominal value)
Hydroxyl
value
Saponification
value

7 115 to 135 125 to 140
25 70 to 90 70 to 90
40 60 to 80 45 to 69
60 45 to 67 40 to 51
1,4-Dioxan. Not more than 10 ppm, determined by the method for residual solvents (2.4.24).
Free ethylene oxide. Not more than 1 ppm, determined by head-space gas chromatography
(2.2.28).
Test solution. Weigh 1.00 g (M
T
) of the substance to be examined in a vial of appropriate volume and
add 1 ml of water R. If necessary, heat to 50C for about 10 min.
Reference solution (a). Weigh 1.00 g (M
R
) of the substance to be examined in a vial of appropriate
volume and add 0.20 g of cooled ethylene oxide solution R and 0.8 ml of water R. If necessary, heat to
50C for about 10 min.
Reference solution (b). To 0.1 g of ethylene oxide solution R in a vial of appropriate volume, add 0.1 ml
of a freshly prepared 0.01 g/l solution of acetaldehyde R.
Close the vials with a butyl rubber membrane stopper, coated with an aluminium or teflon layer
and secure with an aluminium crimped cap. Mix to obtain a homogeneous solution.
The following static head-space injection conditions may be used:
equilibration temperature: at least 70C,
transfer line temperature: 75C,
carrier gas: helium for chromatography R or nitrogen for chromatography R,
injection volume: 1 ml.
a glass or quartz column 30 m long and 0.32 mm in internal diameter, the inner surface of which
is coated with a 1.0 m thick layer of polydimethylsiloxane R,
helium for chromatography R or nitrogen for chromatography R as the carrier gas with a linear
velocity of about 20 cm per second and a split ratio of 1:20,
of 30C per minute to 230C and maintaining at 230C for 5 min; maintaining the temperature of
the injection port at 150C and that of the detector at 250C.
Inject 1 ml of the gaseous phase of reference solution (b). Adjust the sensitivity of the system so
that the heights of the two principal peaks in the chromatogram obtained are at least 15 per cent of
the full scale of the recorder. The test is not valid unless: in the chromatogram obtained with refer-
ence solution (b), the resolution between the peaks corresponding to acetaldehyde and ethylene oxide
is at least 2.0; the peak due to ethylene oxide in the chromatogram obtained with reference solution
(b) has a signal- to-noise ratio of at least ten.
Inject separately suitable volumes (for example 1 ml) of the gaseous phases of the test solution and
reference solution (a). Repeat the procedure twice more. The mean area of the ethylene oxide peak in
the chromatogram obtained with the test solution is not greater than half the mean area of the corres-
ponding peak in the chromatogram obtained with reference solution (a).
The test is not valid unless the relative standard deviation of the area for three injections of refer-
ence solution (a) is not greater than 15 per cent.
The content of ethylene oxide in parts per million may be calculated from the expression:
) ( ) (
R T T R
T
M A M A
A

A
T
=area of the peak corresponding to ethylene oxide in the chromatogram obtained with the
test solution,
A
R
=area of the peak corresponding to ethylene oxide in the chromatogram obtained with
Heavy metals (2.4.8). 12 ml of solution S, filtered if necessary, complies with limit test A for heavy
metals (10 ppm). Prepare the standard using lead standard solution (2 ppm Pb) R.
of water.
07-65
STORAGE
Store in a well closed container, protected from light.
LABELLING
The label states the amount of ethylene oxide reacted with castor oil (nominal value).
__________________________________________________________________________________________________________ Ph Eur
07-66
Hydrogenated Polyoxyl Castor Oil
Hydrogenated Polyoxyl Castor Oil complies with the requirements of the 3rd edition of the European
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Polyoxyl hydrogenated castor oil contains mainly tri-hydroxystearyl glycerol ethoxylated with seven to
sixty molecules of ethylene oxide (nominal value), with small amounts of macrogol hydroxystearate
and of the corresponding free glycols. It results from the reaction of hydrogenated castor oil with
ethylene oxide.
CHARACTERS
Polyoxyl hydrogenated castor oil with less than ten units of ethylene oxide per molecule is a
yellowish, turbid, viscous liquid, practically insoluble in water, soluble in acetone and dispersible in
alcohol.
Polyoxyl hydrogenated castor oil with more than twenty units of ethylene oxide per molecule is a
white or yellowish semi-liquid or pasty mass, freely soluble in water, in alcohol and in acetone,
practically insoluble in light petroleum.
IDENTIFICATION
C. Examine by thin-layer chromatography (2.2.27) using a suitable octadecylsilyl silica gel as the
coating substance.
Test solution. To 1 g of the substance to be examined, add 100 ml of a 100 g/l solution of potassium
hydroxide R and boil under a reflux condenser for 30 min. Allow to cool. Acidify the solution with
20 ml of hydrochloric acid R. Shake the mixture with 50 ml of ether R and allow to stand until a
separation of the layers is obtained. Transfer 10 ml of the clear upper layer to a suitable tube and
evaporate to dryness on a water-bath. Dissolve the residue in 1.0 ml of ether R.
Reference solution. Dissolve 40 mg of 12-hydroxystearic acid R in methylene chloride R and dilute to 2 ml
volumes of methylene chloride R, 40 volumes of glacial acetic acid R and 50 volumes of acetone R. Dry
the plate in a current of cold air, spray with a 200 g/l solution of phosphomolybdic acid R in alcohol R
and heat at 120C for about 10 min. The principal spot in the chromatogram obtained with the test
solution is similar in position and colour to the principal spot in the chromatogram obtained with the
reference solution.
D. Place about 2 g in a test-tube and add 0.2 ml of sulphuric acid R. Close the tube using a stopper
fitted with a glass tube bent twice at right angles. Heat the tube until white fumes appear. Collect the
fumes in 1 ml of mercuric chloride solution R. A white precipitate is formed and the fumes turn the
colour of a filter-paper impregnated with alkaline potassium tetraiodomercurate solution R to black.
TESTS
Solution S. Dissolve 5.0 g of polyoxyl hydrogenated castor oil with less than ten units of ethylene
oxide per molecule in a mixture of 50 volumes of acetone R and 50 volumes of ethanol R and dilute to
50 ml with the same mixture of solvents.
Dissolve 5.0 g of polyoxyl hydrogenated castor oil with more than twenty units of ethylene
oxide per molecule in carbon dioxide-free water R and dilute to 50 ml with the same solvent.
Appearance of solution. Solution S is not more opalescent than reference suspension III (2.2.1)
6
(Method II, 2.2.2).
Alkalinity. To 2 ml of solution S add 0.5 ml of bromothymol blue solution R1. The solution is not
blue.
Hydroxyl value (2.5.3, Method A). See Table 1083-1.
Saponification value (2.5.6). See Table 1083-1.
07-67
Table 10831
Ethylene oxide
units per molecule
(nominal value)
Hydroxyl
value
Saponification
value

7 115 to 135 125 to 140
25 70 to 90 70 to 90
40 60 to 80 45 to 69
60 45 to 67 40 to 51
1,4-Dioxan. Not more than 10 ppm, determined by the method for residual solvents (2.4.24).
Free ethylene oxide. Not more than 1 ppm, determined by head-space gas chromatography
(2.2.28).
Test solution. Weigh 1.00 g (M
T
) of the substance to be examined in a vial of appropriate volume and
add 1 ml of water R. If necessary, heat to 50C for about 10 min.
Reference solution (a). Weigh 1.00 g (M
R
) of the substance to be examined in a vial of appropriate
volume and add 0.20 g of cooled ethylene oxide solution R and 0.8 ml of water R. If necessary, heat to
50C for about 10 min.
Reference solution (b). To 0.1 g of ethylene oxide solution R in a vial of appropriate volume, add 0.1 ml
of a freshly prepared 0.01 g/l solution of acetaldehyde R.
Close the vials with a butyl rubber membrane stopper, coated with an aluminium or teflon layer
and secure with an aluminium crimped cap. Mix to obtain a homogeneous solution.
equilibration temperature: at least 70C,
transfer line temperature: 75C,
a glass or quartz column 30 m long and 0.32 mm in internal diameter, the inner surface of which
is coated with a 1.0 m thick layer of polydimethylsiloxane R,
velocity of about 20 cm per second and a split ratio of 1:20,
Inject 1 ml of the gaseous phase of reference solution (b). Adjust the sensitivity of the system so
that the heights of the two principal peaks in the chromatogram obtained are not less than 15 per
cent of the full scale of the recorder. The test is not valid unless: in the chromatogram obtained with
reference solution (b), the resolution between the peaks corresponding to acetaldehyde and ethylene
oxide is at least 2.0; the peak due to ethylene oxide in the chromatogram obtained with reference
solution (b) has a signal- to-noise ratio of at least ten.
Inject separately suitable volumes (for example 1 ml) of the gaseous phases of the test solution and
reference solution (a). Repeat the procedure twice more. The mean area of the ethylene oxide peak in
the chromatogram obtained with the test solution is not greater than half the mean area of the corres-
ponding peak in the chromatogram obtained with reference solution (a).
The test is not valid unless the relative standard deviation of the area for three injections of refer-
ence solution (a) is not greater than 15 per cent.
The content of ethylene oxide in parts per million may be calculated from the expression:
) ( ) (
R T T R
T
M A M A
A

A
T
=area of the peak corresponding to ethylene oxide in the chromatogram obtained with the
test solution,
A
R
Heavy metals (2.4.8). Dissolve 5.0 g in a mixture of equal volumes of acetone R and ethanol R and
dilute to 50 ml with the same mixture of solvents. 12 ml of this solution complies with limit test A for
heavy metals (10 ppm). Prepare the standard using lead standard solution (2 ppm Pb) R.
of water.
07-68
STORAGE
LABELLING
The label states the amount of ethylene oxide reacted with hydrogenated castor oil (nominal value).
__________________________________________________________________________________________________________ Ph Eur
08-1
Cefaclor
corrected 1/01
O
O
N
N
H H
S
Cl
COOH
H NH
2
H
C
15
H
14
ClN
3
O
4
S,H
2
O 385.8 53994-73-3
Cefaclor complies with the requirements of the 3rd edition of the European Pharmacopoeia [0986]. These
Preparations
Cefaclor Capsules
Cefaclor Oral Suspension
Slow Cefaclor Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cefaclor is the monohydrate of (6R,7R)-7-[[(2R)-2-amino-2-phenylacetyl]amino]-3-chloro-8-oxo-5-
thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid. It contains not less than 96.0 per cent and not
more than the equivalent of 102.0 per cent of C
15
H
14
ClN
3
O
4
S, calculated with reference to the
CHARACTERS
A white or slightly yellow powder, slightly soluble in water, practically insoluble in methanol and in
methylene chloride.
IDENTIFICATION
obtained with cefaclor CRS.
B. Examine by thin-layer chromatography (2.2.27), using silanised silica gel HF
254
R as the coating
substance.
Test solution. Dissolve 10 mg of the substance to be examined in 5 ml of a mixture of equal volumes of
methanol R and 0.067M phosphate buffer solution pH 7.0 R.
Reference solution (a). Dissolve 10 mg of cefaclor CRS in 5 ml of a mixture of equal volumes of
Reference solution (b). Dissolve 10 mg of cefaclor CRS and 10 mg of cefalexin CRS in 5 ml of a mixture
of equal volumes of methanol R and 0.067M phosphate buffer solution pH 7.0 R.
15 volumes of methanol R and 85 volumes of a 154 g/l solution of ammonium acetate R previously
adjusted to pH 6.2 with acetic acid R. Dry the plate in a current of warm air and examine in ultraviolet
light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in
position and size to the principal spot in the chromatogram obtained with reference solution (a). The
test is not valid unless the chromatogram obtained with reference solution (b) shows two clearly
separated spots.
C. Place about 2 mg in a test-tube about 150 mm long and 15 mm in diameter. Moisten with 0.05 ml
of water R and add 2 ml of sulphuric acid-formaldehyde reagent R. Mix the contents of the tube by
swirling; the solution is colourless. Place the test-tube on a water-bath for 1 min; a yellowish-brown
colour develops.
TESTS
pH (2.2.3). Suspend 0.250 g in carbon dioxide-free water R and dilute to 10 ml with the same solvent.
The pH of the suspension is 3.0 to 4.5.
Specific optical rotation (2.2.7). Dissolve 0.250 g in a 10 g/l solution of hydrochloric acid R and
08-2
dilute to 25.0 ml with the same solution. The specific optical rotation is +101 to +111, calculated
Test solution. Dissolve 50.0 mg of the substance to be examined in 10.0 ml of a 2.7 g/l solution of
sodium dihydrogen phosphate R adjusted to pH 2.5 with phosphoric acid R.
Reference solution (a). Dissolve 2.5 mg of cefaclor CRS and 5.0 mg of delta-3-cefaclor CRS in 100.0 ml
of a 2.7 g/l solution of sodium dihydrogen phosphate R adjusted to pH 2.5 with phosphoric acid R.
Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with a 2.7 g/l solution of sodium
dihydrogen phosphate R adjusted to pH 2.5 with phosphoric acid R.
Mobile phase A. Adjust a 7.8 g/l solution of sodium dihydrogen phosphate R to pH 4.0 with
phosphoric acid R,
Mobile phase B. Mix 450 ml of acetonitrile R with 550 ml of mobile phase A,
Equilibrate the column with a mixture of 5 volumes of mobile phase B and 95 volumes of mobile
phase A for at least 15 min between each analysis. Inject the solutions. Operate by gradient elution
increasing the concentration of mobile phase B continuously and linearly by 0.67 per cent V/V per
minute for 30 min (25 per cent V/V). Then increase the concentration of mobile phase B
continuously and linearly by 5 per cent V/V per minute for 15 min (100 per cent V/V). Finally elute
with mobile phase B for 10 min. Change the composition to a mixture of 5 volumes of mobile phase
B and 95 volumes of mobile phase A to re-equilibrate the column.
Inject reference solution (a). The test is not valid unless the resolution between the peaks due to
cefaclor and delta-3-cefaclor is at least 2 and the symmetry factor of the cefaclor peak is at most 1.2.
If necessary, adjust the acetonitrile content of the mobile phase.
Inject the test solution and reference solution (b). In the chromatogram obtained with the test
solution: the area of any peak, apart from the principal peak and any peaks due to the mobile phase,
is not greater than 0.5 times the area of the principal peak in the chromatogram obtained with
reference solution (b) (0.5 per cent); and the sum of all such peaks is not more than twice the area of
the principal peak in the chromatogram obtained with reference solution (b) (2 per cent). Disregard
mination of water.
ASSAY
Reference solution (a). Dissolve 15.0 mg of cefaclor CRS in the mobile phase and dilute to 50.0 ml with
the mobile phase.
Reference solution (b). Dissolve 15.0 mg of cefaclor CRS and 15.0 mg of delta-3-cefaclor CRS in the
mobile phase and dilute to 50.0 ml with the mobile phase.
as mobile phase at a flow rate of 1.5 ml/min a mixture prepared by adding 220 ml of methanol R
to a mixture of 780 ml of water R, 10 ml of triethylamine R and 1 g of sodium pentanesulphonate R,
adjusted to pH 2.5 with phosphoric acid R,
Inject reference solution (b). The assay is not valid unless the resolution between the peaks due to
cefaclor and delta-3-cefaclor is at least 2.5. Adjust the concentration of methanol in the mobile
phase, if necessary. The assay is not valid unless the symmetry factor of the cefaclor peak is at most
1.5. Inject reference solution (a) six times. The assay is not valid unless the relative standard
deviation of the peak area of cefaclor is at most 1.0 per cent. Inject alternately the test solution and
08-3
STORAGE
IMPURITIES
COOH
H NH
2
A. (2R)-2-amino-2-phenylacetic acid (phenylglycine),
N
S
COOH
Cl
H H
2
N
O
B. (6R,7R)-7-amino-3-chloro-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid,
N
S
COOH
Cl
H H
H
N
O
H
2
N H
O
C. (6R,7R)-7-[[(2S)-2-amino-2-phenylacetyl]amino]-3-chloro-8-oxo-5-thia-1-
azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid,
N
S
Cl
H H
H
N
O
H
2
N H
O
H COOH
*
and epimer at C*
D. (2R,6R,7R)-and (2S,6R,7R)-7-[[(2R)-2-amino-2-phenylacetyl]amino]-3-chloro-8-oxo-5-
thia-1-azabicyclo[4.2.0]oct-3-ene-2-carboxylic acid (delta-3-cefaclor),
H
N
S
HN
H
2
N H
O
O
Cl
COOH
*
*
E. 2-[[(2R)-2-amino-2-phenylacetyl]amino]-2-(5-chloro-3,4-dihydro-2H-4-oxo-1,3-thiazin-2-
yl)acetic acid,
N
N OH
F. 3-phenylpyrazin-2-ol.
__________________________________________________________________________________________________________ Ph Eur
08-4
Cefadroxil
H NH
2
Me
COOH
S
O
H H
N
O
N
HO
H
C
16
H
17
N
3
O
5
S 363.4 50370-12-2
Cefadroxil complies with the requirements of the 3rd edition of the European Pharmacopoeia [0813]. These
Preparations
Cefadroxil Capsules
Cefadroxil Oral Suspension
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cefadroxil contains not less than 95.0 per cent and not more than the equivalent of 101.0 per cent of
(6R,7R)-7-[(R)-2-amino-2-(4-hydroxyphenyl)acetamido]-3-methyl-8-oxo-5-thia-1-
azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white powder, slightly soluble in water, very slightly soluble in alcohol, practically
insoluble in ether.
IDENTIFICATION
obtained with cefadroxil CRS.
254
R as the coating
substance.
Test solution. Dissolve 20 mg of the substance to be examined in 5 ml of a mixture of equal volumes
of methanol R and 0.067M phosphate buffer solution pH 7.0 R.
Reference solution (a). Dissolve 20 mg of cefadroxil CRS in 5 ml of a mixture of equal volumes of
Reference solution (b). Dissolve 20 mg of cefadroxil CRS and 20 mg of cefalexin CRS in 5 ml of a
mixture of equal volumes of methanol R and 0.067M phosphate buffer solution pH 7.0 R.
15 volumes of methanol R and 85 volumes of a 154 g/l solution of ammonium acetate R previously
adjusted to pH 6.2 with acetic acid R. Allow the plate to dry and examine in ultraviolet light at
spots.
by swirling; the solution is yellow. Place the test-tube in a water-bath for 1 min; an orange colour
develops.
TESTS
substance.
08-5
Absorbance (2.2.25). Dissolve 20.0 mg in phosphate buffer solution pH 6.0 R and dilute to 100.0 ml
with the same solvent. The absorbance of the solution determined at 330 nm is not greater than 0.05.
Dilute 10.0 ml of the solution to 100.0 ml with phosphate buffer solution pH 6.0 R. Examined between
235 nm and 340 nm, the diluted solution shows an absorption maximum at 264 nm. The specific
absorbance at this maximum is 225 to 250, calculated with reference to the anhydrous substance.
254
R as the
coating substance.
Test solution. Dissolve 0.25 g of the substance to be examined in a mixture of 3 volumes of dilute
hydrochloric acid R, 22 volumes of water R and 75 volumes of alcohol R and dilute to 10 ml with the
same mixture of solvents.
Reference solution (a). Dilute 1 ml of the test solution to 100 ml with a mixture of 3 volumes of dilute
hydrochloric acid R, 22 volumes of water R and 75 volumes of alcohol R.
Reference solution (b). Dissolve 25 mg of 7-aminodesacetoxycephalosporanic acid CRS in a mixture of 3
volumes of dilute hydrochloric acid R, 22 volumes of water R and 75 volumes of alcohol R and dilute to
Reference solution (c). Dissolve 25 mg of D--(4-hydroxy-phenyl)glycine CRS in a mixture of 3 volumes
of dilute hydrochloric acid R, 22 volumes of water R and 75 volumes of alcohol R and dilute to 100 ml
Reference solution (d). To 1.0 ml of the test solution add 0.5 ml of reference solution (b) and 0.5 ml of
reference solution (c).
Apply separately to the plate 2 l of the test solution, 2 l of each of reference solutions (a), (b) and
(c) and 4 l of reference solution (d). Develop over a path of 10 cm using a mixture of 4 volumes of
anhydrous formic acid R, 20 volumes of alcohol R, 20 volumes of water R and 56 volumes of ethyl
acetate R. Heat the plate at 105C to 110C for 10 min and spray with ninhydrin solution R2. In the
chromatogram obtained with the test solution: any spot corresponding to 7-aminodesacetoxycephalo-
sporanic acid is not more intense than the spot in the chromatogram obtained with reference solution
(b) (1.0 per cent); any spot corresponding to D--(4-hydroxyphenyl)glycine is not more intense than
the spot in the chromatogram obtained with reference solution (c) (1.0 per cent); any spot, apart
from the principal spot and those already mentioned, is not more intense than the spot in the chro-
matogram obtained with reference solution (a) (1.0 per cent). The test is not valid unless the
chromatogram obtained with reference solution (d) shows three clearly separated spots.
Dimethylaniline. Not more than 20 ppm, determined by gas chromatography (2.2.28) using
naphthalene R as internal standard.
250.0 ml with water R. To 1.0 ml of the solution in a ground-glass-stoppered tube add 5 ml of 1M
sodium hydroxide and 1.0 ml of the internal standard solution. Stopper the tube and shake vigorously
for 1 min. Centrifuge if necessary and use the upper layer.
mination of water.
ASSAY
Reference solution (a). Dissolve 50.0 mg of cefadroxil CRS in the mobile phase and dilute to 100.0 ml
Reference solution (b). Dissolve 5 mg of cefadroxil CRS and 50 mg of amoxicillin trihydrate CRS in the
mobile phase and dilute to 100 ml with the mobile phase.
08-6
silica gel for chromatography R (5 m or 10 m),
as mobile phase, at a flow rate of 1 ml per minute, a mixture of 4 volumes of acetonitrile R and 96
volumes of a 2.72 g/l solution of potassium dihydrogen phosphate R,
Inject reference solution (b). Adjust the sensitivity to obtain peaks with a height corresponding to
at least half the full-scale deflection. The test is not valid unless the resolution between the peaks
corresponding to cefadroxil and amoxicillin is at least 5.0. If necessary, adjust the acetonitrile content
in the mobile phase. Inject reference solution (a) six times. The test is not valid unless the relative
standard deviation of the peak area of cefadroxil is at most 1.0 per cent. Inject alternately the test
solution and reference solution (a). Calculate the percentage content of cefadroxil.
STORAGE
Store in a well-closed container, protected from light and at a temperature not exceeding 30C.
__________________________________________________________________________________________________________ Ph Eur
08-7
Cefalexin
H
H NH
2
Me
COOH
S
O
H H
N
O
N
C
16
H
17
N
3
O
4
S,H
2
O 365.4 23325-78-2
Cefalexin complies with the requirements of the 3rd edition of the European Pharmacopoeia [0708]. These
Preparations
Cefalexin Capsules
Cefalexin Oral Suspension
Cefalexin Tablets
When cephalexin is prescribed or demanded. Cefalexin shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cefalexin contains not less than 95.0 per cent and not more than the equivalent of 101.0 per cent of
(6R,7R)-7-[(R)-2-amino-2-phenylacetamido]-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-
carboxylic acid, calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white, crystalline powder, soluble in about 100 parts of water, practically insoluble
in alcohol and in ether.
IDENTIFICATION
obtained with cefalexin CRS.
254
R as the coating
substance.
Test solution. Dissolve 20 mg of the substance to be examined in 5.0 ml of a mixture of equal volumes
Reference solution (a). Dissolve 20 mg of cefalexin CRS in 5.0 ml of a mixture of equal volumes of
Reference solution (b). Dissolve 20 mg of cefalexin CRS and 20 mg of cefradine CRS in 5.0 ml of a
15 volumes of acetone R and 85 volumes of a 154 g/l solution of ammonium acetate R previously
spots.
by swirling; the solution is pale yellow. Place the test-tube in a water-bath for 1 min; a dark yellow
colour develops.
TESTS
08-8
Specific optical rotation (2.2.7). Dissolve 0.125 g in phthalate buffer solution pH 4.4 R and dilute to
Absorbance (2.2.25). Dissolve 50 mg in water R and dilute to 100.0 ml with the same solvent. The
absorbance of the solution determined at 330 nm is not greater than 0.05. Dilute 2.0 ml of the
solution to 50.0 ml with water R. Examined between 220 nm and 300 nm, the diluted solution shows
an absorption maximum at 262 nm. The specific absorbance at this maximum is 220 to 245,
coating substance. Impregnate the plate by development with a 5 per cent V/V solution of
tetradecane R in hexane R. Allow the solvent to evaporate and carry out the chromatography in the
same direction as the impregnation.
Test solution. Dissolve 0.25 g of the substance to be examined in dilute hydrochloric acid R and dilute to
10 ml with the same acid.
Reference solution (a). Dilute 1 ml of the test solution to 100 ml with dilute hydrochloric acid R.
Reference solution (b). Dissolve 25 mg of 7-aminodesacetoxycephalosporanic acid CRS in dilute hydro-
chloric acid R and dilute to 10 ml with the same acid (reference solution b). Dilute 1 ml of this solution
to 10 ml with dilute hydrochloric acid R.
Reference solution (c). Dissolve 25 mg of -phenylglycine R in dilute hydrochloric acid R and dilute to
10 ml with the same acid (reference solution c). Dilute 1 ml of this solution to 10 ml with dilute hydro-
chloric acid R.
Reference solution (d). Dissolve 0.25 g of the substance to be examined in a mixture of 1 ml of refer-
ence solution b and 1 ml of reference solution c and dilute to 10 ml with dilute hydrochloric acid R.
volumes of acetone R, 80 volumes of a 72 g/l solution of disodium hydrogen phosphate R and 120
volumes of a 21 g/l solution of citric acid R. Dry the plate by heating at 90C for 3 min. Spray the hot
plate with a 1 g/l solution of ninhydrin R in the mobile phase. Heat the plate at 90C for 15 min and
allow to cool. In the chromatogram obtained with the test solution: any spot corresponding to
7-aminodesacetoxy-cephalosporanic acid is not more intense than the spot in the chromatogram
obtained with reference solution (b) (1.0 per cent); any spot corresponding to -phenylglycine (the
position of which is defined by comparison with the chromatogram obtained with reference solution
(d)), is not more intense than the spot in the chromatogram obtained with reference solution (c)
(1.0 per cent); any spot apart from the principal spot and the spots corresponding to 7-aminode-
sacetoxycephalosporanic acid and -phenylglycine is not more intense than the principal spot in the
chromatogram obtained with reference solution (a) (1.0 per cent). The test is not valid unless the
chromatogram obtained with reference solution (d) shows three clearly separated spots.
at 150C. Inject separately 1 l of the test solution and 1 l of the reference solution.
mination of water.
ASSAY
08-9
Test solution. Dissolve 50.0 mg of the substance to be examined in water R and dilute to 100.0 ml
Reference solution (a). Dissolve 50.0 mg of cefalexin CRS in water R and dilute to 100.0 ml with the
same solvent.
Reference solution (b). Dissolve 10 mg of cefalexin CRS and 10 mg of cefradine CRS in water R and
a column 0.25 m long and 4.6 mm in internal diameter, packed with octadecylsilyl silica gel for
chromatography R (5 m or 10 m),
as mobile phase, at a flow rate of 1.5 ml per minute, a mixture of 2 volumes of methanol R, 5
volumes of acetonitrile R, 10 volumes of a 13.6 g/l solution of potassium dihydrogen phosphate R
and 83 volumes of water R,
Inject reference solution (b). Adjust the sensitivity to obtain peaks with a height of at least half the
full-scale deflection. The test is not valid unless the resolution between the peaks corresponding to
cefalexin and cefradine is at least four. If necessary, adjust the acetonitrile content in the mobile
phase. Inject reference solution (a) six times. The test is not valid unless the relative standard
deviation for the peak area of cefalexin is at most 1.0 per cent. Inject alternately the test solution and
reference solution (a). Calculate the percentage content of cefalexin.
STORAGE
Store in a well-closed container, protected from light and at a temperature not exceeding 30C.
__________________________________________________________________________________________________________ Ph Eur
08-10
Cefalotin Sodium
1/01
H
COONa
N
O
H H
S
OAc
S
O
N
C
16
H
15
N
2
NaO
6
S
2
418.4 58-71-9
Cefalotin Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia [0987].
Preparation
Cefalotin Injection
When cephalothin sodium is prescribed or demanded, Cefalotin Sodium shall be dispensed or
supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cefalotin sodium contains not less than 96.0 per cent and not more than the equivalent of 101.0 per
cent of sodium (6R,7R)-3-[(acetyloxy)methyl]-8-oxo-7-[[2-(thiophen-2-yl)acetyl]-amino]-5-thia-1-
azabicyclo[4.2.0]oct-2-ene-2-carboxylate, calculated with reference to the anhydrous substance.
PRODUCTION
process using starting materials or intermediates that may contain residues of dimethylaniline, it
CHARACTERS
A white or almost white powder, freely soluble in water, slightly soluble in ethanol.
IDENTIFICATION
obtained with cefalotin sodium CRS.
B. It gives reaction (a) of sodium (2.3.1).
TESTS
Appearance of solution Solution S is clear (2.2.1). The absorbance (2.2.25) of solution S measured
at 450 nm is not greater than 0.20.
substance.
the test solution and reference solution (b). Continue the chromatography for at least four times the
retention time of the principal peak. In the chromatogram obtained with the test solution: the area of
any peak, apart from the principal peak, is not greater than the area of the principal peak in the
chromatogram obtained with reference solution (b) (1 per cent) and the sum of the areas of any such
peaks is not greater than three times the area of the principal peak in the chromatogram obtained
with reference solution (b) (3 per cent). Disregard any peak with an area less than 0.1 times the area
of the principal peak in the chromatogram obtained with reference solution (b) (0.1 per cent).
08-11
mination of water.
ASSAY
Reference solution (a). Dissolve 25.0 mg of cefalotin sodium CRS in the mobile phase and dilute to
Reference solution (c). Heat 5 ml of reference solution (a) in a water-bath at 90C for 10 min. Cool
and inject immediately.
as mobile phase at a flow rate of 1.0 ml/min a solution prepared as follows: dissolve 17 g of
sodium acetate R in 790 ml of water R, add 0.6 ml of glacial acetic acid R and adjust if necessary to
pH 5.8 to 6.0 with dilute sodium hydroxide solution R or glacial acetic acid R, add 150 ml of
acetonitrile R and 70 ml of ethanol R and mix,
Inject reference solution (c). Adjust the sensitivity of the system to obtain peaks with a height
corresponding to at least half the full scale of the recorder. The chromatogram obtained shows two
principal peaks corresponding to cefalotin and desacetylcefalotin. The assay is not valid unless the
resolution between these two peaks is at least 9.0. Adjust the concentration of acetonitrile in the
mobile phase if necessary. The assay is not valid unless the symmetry factor of the cefalotin peak is at
most 1.8. Inject reference solution (a) six times. The assay is not valid unless the relative standard
deviation of the peak area for cefalotin is at most 1.0 per cent. Inject alternately the test solution and
Calculate the percentage content of cefalotin sodium.
STORAGE
LABELLING
The label states:
IMPURITIES
S
N
S
H
N
O
O
H H
COOH
Me
A. (6R,7R)-3-methyl-8-oxo-7-[[2-(thiophen-2-yl)acetyl]amino]-5-thia-1-azabicyclo[4.2.0]oct-
2-ene- 2-carboxylic acid (desacetoxycefalotin).
__________________________________________________________________________________________________________ Ph Eur
08-12
Cefamandole Nafate
N
H
O O
H
N
H
S
S
COONa
N
N N
N
Me
O
H
O
C
19
H
17
N
6
NaO
6
S
2
512.5 42540-40-9
Cefamandole Nafate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1402].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cefamandole nafate is sodium (6R,7R)-7-[[(2R)-2-(formyloxy)-2-phenylacetyl]amino]-3-[[(1-
methyl-1H-tetrazol-5-yl)sulphanyl]methyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate.
It contains sodium carbonate. It contains not less than 84.0 per cent and not more than 93.0 per cent
of cefamandole (C
18
H
18
N
6
O
5
S
2
), calculated with reference to the anhydrous and sodium carbonate-
free substance.
PRODUCTION
CHARACTERS
A white or almost white powder, freely soluble in water, sparingly soluble in methanol.
IDENTIFICATION
obtained with cefamandole nafate CRS. Examine the substances prepared as discs.
TESTS
Appearance of solution Solution S is clear (2.2.1). The absorbance of solution S measured at
475 nm (2.2.25) is not greater than 0.03.
pH The pH of solution S, measured after 30 min, is 6.0 to 8.0.
Specific optical rotation (2.2.7). Dissolve 1.00 g in acetate buffer pH 4.7 R and dilute to 10.0 ml
with the same solvent. The specific optical rotation is 25.0 to 33.0, calculated with reference to
the anhydrous and sodium carbonate-free substance.
Related substances Examine by liquid chromatography (2.2.29). Prepare the solution immediately
before use.
Solvent mixture. Mix 18 volumes of acetonitrile R and 75 volumes of a 10 per cent V/V solution of
triethylamine R previously adjusted to pH 2.5 with phosphoric acid R.
Test solution. Dissolve 0.100 g of the substance to be examined in the solvent mixture and dilute to
Reference solution (a). Dilute 1 ml of the test solution to 10 ml with the solvent mixture, then heat the
solution at 60C for 30 min.
Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with the solvent mixture.
Triethylamine phosphate solution: dissolve 2.0 g of sodium pentanesulphonate R in 350 ml of
water R, add 40 ml of triethylamine R, adjust to pH 2.5 with phosphoric acid R and dilute to
700 ml with water R,
08-13
Mobile phase A. Mix 1 volume of triethylamine phosphate solution and 2 volumes of water R,
Mobile phase B. Mix equal volumes of triethylamine phosphate solution, methanol R and aceto-
nitrile R,
Time
(min)
Mobile
phase A
(per cent V/V)
Mobile
phase B
(per cent V/V)
Comment
0 1 100 0 isocratic
1 35 1000 0100 linear gradient
Inject 20 l of reference solution (a). Adjust the sensitivity of the system so that the heights of the
two principal peaks are at least 50 per cent of the full scale of the recorder. The test is not valid unless
the resolution between the peaks corresponding to cefamandole and cefamandole nafate is at least
5.0. Inject 20 l of the test solution and 20 l of reference solution (b). In the chromatogram
obtained with the test solution: the area of any peak, apart from the principal peak and the peak due
to cefamandole, is not greater than the area of the principal peak in the chromatogram obtained with
reference solution (b) (1 per cent) and the sum of the areas of any such peaks is not greater than five
Cefamandole free acid Not more than 9.5 per cent, calculated with reference to the anhydrous and
sodium carbonate-free substance and determined by liquid chromatography as described under
Assay.
Sodium carbonate 4.8 per cent to 6.4 per cent. Dissolve 0.500 g in 50 ml of water R. Titrate with
0.1M hydrochloric acid, determining the end-point potentiometrically (2.2.20).
1 ml of 0.1M hydrochloric acid is equivalent to 10.6 mg of Na
2
CO
3
.
mination of water.
ASSAY
Reference solution (a). Dissolve 50.0 mg of cefamandole nafate CRS in the mobile phase and dilute to
Reference solution (b). Dilute 1 ml of the test solution to 10 ml with the mobile phase, then heat the
solution at 60C for 30 min.
volumes of a 10 per cent V/V solution of triethylamine R previously adjusted to pH 2.5 with
phosphoric acid R,
Inject 20 l of reference solution (b). Adjust the sensitivity of the system so that the heights of the
two peaks are at least 50 per cent of the full scale of the recorder. The test is not valid unless the
resolution between the two peaks is at least 7.0. Adjust the concentration of acetonitrile in the mobile
phase if necessary. Inject reference solution (a) six times. The test is not valid unless the relative
standard deviation of the peak area for cefamandole nafate is at most 1.0 per cent. Inject the test
solution and reference solution (a).
Calculate the percentage content of cefamandole (C
18
H
18
N
6
O
5
S
2
) from the sum of the contents of
cefamandole nafate and cefamandole free acid. 1 mg of cefamandole nafate is equivalent to
0.9025 mg of cefamandole.
08-14
STORAGE
LABELLING
The label states:
that the substance contains sodium carbonate,
IMPURITIES
N
S
H
N
O
H O
O
H
H H
O
COOH
CH
3
A. (6R,7R)-7-[[(2R)-2-(formyloxy)-2-phenylacetyl]amino]-3-methyl-8-oxo-5-thia-1-
azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (formylmandeloyl-7-aminodesacetoxy-
cephalosporanic acid),
N
N MeN
N N
S
H
N
O
H OR
H H
O
COOH
S
B. R = H : (6R,7R)-7[[(2R)-2-hydroxy-2-phenylacetyl]amino]-3-[[(1-methyl-1H-tetrazol-5-
yl)sulphanyl]methyl]-8-oxo-5 thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid
(cefamandole),
C. R = H
3
C-CO : (6R,7R)-7-[[(2R)-2-(acetyloxy)-2-phenylacetyl]amino]-3-[[(1-methyl-1H-
tetrazol-5-yl)sulphanyl]methyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid
(O-acetylcefamandole),
N
N MeN
N S
D. 1-methyl-1H-tetrazole-5-thiol,
N
S
H
N
O
H O
O
H
H H
O
COOH
CH
2
OAc
E. (6R,7R)-7-[[(2R)-2-(formyloxy)-2-phenylacetyl]amino]-3-[(acetyloxy)methyl]-8-oxo-5-
thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (formylmandeloyl-7-ACA).
__________________________________________________________________________________________________________ Ph Eur
08-15
Cefatrizine Propylene Glycol
corrected 1/01
N
N
H
N
N
S
S
H
N
O
H NH
2
O
O
H H
COOH
HO
OH
CH
3
C
18
H
18
N
6
O
5
S
2
,(C
3
H
8
O
2
)
n
538.5 51627-14-6 (cefatrizine)
Cefatrizine Propylene Glycol complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cefatrizine propylene glycol contains not less than 95.0 per cent and not more than the equivalent
of 102.0 per cent of (6R,7R)-7-[[(2R)-2-amino-2-(4-hydroxyphenyl)acetyl]amino]-8-oxo-3-[[(1H-
1,2,3-triazol-4-yl)sulphanyl]methyl]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, calculated
with reference to the anhydrous and propylene glycol free substance. It consists of cefatrizine and
propane-1,2-diol (molecular proportions about 1:1). It contains not less than 13.0 per cent and not
more than 18.0 per cent of propylene glycol.
CHARACTERS
A white or almost white powder, slightly soluble in water, practically insoluble in alcohol and in
methylene chloride.
IDENTIFICATION
obtained with cefatrizine propylene glycol CRS.
B. Examine the chromatograms obtained in the test for propylene glycol. The retention time and size
of the principal peak in the chromatogram obtained with the test solution are approximately the same
as those of the principal peak in the chromatogram obtained with reference solution (b).
TESTS
Specific optical rotation (2.2.7). Dissolve 0.400 g of the substance to be examined in 1M hydro-
chloric acid and dilute to 20.0 ml with the same solvent. The specific optical rotation is +63 to +69,
calculated with reference to the anhydrous, propylene glycol free substance.
Propylene glycol 13.0 per cent to 18.0 per cent, determined by gas chromatography (2.2.28), using
dimethylacetamide R as the internal standard.
Internal standard solution. Dissolve 1.0 g of dimethylacetamide R in a mixture of 20 volumes of
acetone R and 80 volumes of water R, and dilute to 50.0 ml with the same mixture of solvents.
Test solution. Introduce 0.40 g of the substance to be examined into a ground-glass-stoppered test
tube. Add 3.0 ml of the internal standard solution, 1.0 ml of a mixture of 20 volumes of acetone R
and 80 volumes of water R and 2.0 ml of hydrochloric acid. Seal the test tube and stir the solution.
Reference solution (a). Dissolve 2.0 g of propylene glycol R in a mixture of 20 volumes of acetone R and
80 volumes of water R and dilute to 100.0 ml with the same solvent.
Reference solution (b). Introduce into a ground-glass-stoppered test tube 1.0 ml of reference solution
(a) and 1.0 ml of the internal standard solution.
a stainless steel column 2 m long and 2 mm in internal diameter packed with ethylvinylbenzene-
divinylbenzene copolymer R (150 m to 180 m),
nitrogen for chromatography R as the carrier gas at a flow rate of about 30 ml/min,
detector at 250C.
Inject separately 1 l of the test solution and 1 l of reference solution (b).
08-16
7-ACA triazole and other related substances Examine by liquid chromatography (2.2.29) as
described under Assay. Adjust the sensitivity of the system so that the height of the principal peak
obtained with 20 l of reference solution (c) is at least 50 per cent of the full scale of the recorder.
Inject 20 l of the test solution and continue the chromatography for at least twice the retention time
of the principal peak. In the chromatogram obtained with the test solution: the area of any peak
corresponding to cefatrizine impurity A is not greater than that of the peak due to cefatrizine impurity
A in the chromatogram obtained with reference solution (d) (0.5 per cent); the area of any peak apart
from the principal peak and any peak corresponding to cefatrizine impurity A is not greater than the
area of the principal peak in the chromatogram obtained with reference solution (c) (0.6 per cent);
the sum of the areas of any such peaks is not greater than 3.5 times the area of the principal peak in
the chromatogram obtained with reference solution (c) (2.1 per cent). Disregard any peak with an
area less than 0.05 times that of the principal peak in the chromatogram obtained with reference
solution (c).
mination of water.
ASSAY
Reference solution (a). Dissolve 60.0 mg of cefatrizine propylene glycol CRS in the mobile phase and
dilute to 100.0 ml with the mobile phase.
Reference solution (b). Dissolve 30.0 mg of cefatrizine impurity A CRS in buffer solution pH 7.0 R and
dilute to 100.0 ml with the same buffer solution.
Reference solution (c). Dilute 0.6 ml of reference solution (a) to 100.0 ml with the mobile phase.
Reference solution (d). Dilute 1.0 ml of reference solution (b) to 100.0 ml with buffer solution pH 7.0 R.
Reference solution (e). To 1.0 ml of reference solution (a) add 1.0 ml of reference solution (b) and
volumes of a 2.72 g/l solution of potassium dihydrogen phosphate R in water R,
Adjust the sensitivity of the system so that the height of the principal peak obtained with 20 l of
reference solution (a) is at least 50 per cent of the full scale of the recorder. Inject 20 l of reference
solution (a) six times. The test is not valid unless the relative standard deviation is at most 1.0 per
cent. Inject reference solution (e). The test is not valid unless the resolution between the peaks
corresponding to cefatrizine and cefatrizine impurity A is at least 5.0.
Inject alternately 20 l of the test solution and 20 l of reference solution (a) and calculate the
percentage content of cefatrizine using the chromatogram obtained with reference solution (a).
IMPURITIES
N
NH
N N
S
H H
O
COOH
S
H
2
N
A. 7-amino-(6R,7R)-3-[(1H-1,2,3-triazol-4-yl)sulphanyl]methyl-8-oxo-5-thia-1-
azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (7-ACA triazole).
__________________________________________________________________________________________________________ Ph Eur
08-17
Cefazolin Sodium
H
O
O
N
N
H H
S
N
N
N
N S
COONa N
N
S
Me
C
14
H
13
N
8
NaO
4
S
3
476.5 27164-46-1
Cefazolin Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia [0988].
Action and use Antibacterial
Preparation
Cefazolin Injection
When cephazolin sodium is prescribed or demanded, Cefazolin Sodium shall be dispensed or
supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cefazolin sodium contains not less than 95.0 per cent and not more than the equivalent of 101.0 per
cent of sodium (6R,7R)-3-[(5-methyl-1,3,4-thiadiazol-2-yl)thiomethyl]-8-oxo-7-[2-(1H-tetrazol-1-
yl)acetamido]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate, calculated with reference to the
CHARACTERS
A white or almost white powder, very hygroscopic, freely soluble in water, very slightly soluble in
alcohol, practically insoluble in ether.
IDENTIFICATION
A. Examine by thin-layer chromatography (2.2.27), using silanised silica gel HF
254
R as the coating
substance.
Reference solution (a). Dissolve 20 mg of cefazolin CRS in 5 ml of a mixture of equal volumes of
Reference solution (b). Dissolve 20 mg of cefazolin CRS and 20 mg of cefoxitin sodium CRS in 5 ml of a
15 volumes of acetonitrile R and 85 volumes of a 150 g/l solution of ammonium acetate R, previously
adjusted to pH 6.2 with acetic acid R. Dry the plate in a current of warm air and examine in ultra-
violet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is
similar in position and size to the principal spot in the chromatogram obtained with reference
shows two clearly separated spots.
B. Place about 2 mg in a test-tube about 150 mm long and 15 mm in diameter. Moisten with
by swirling; the solution is pale yellow. Place the test-tube in a water-bath for 1 min; a yellow colour
develops.
C. It gives reaction (a) of sodium (2.3.1).
TESTS
08-18
solvent. The specific optical rotation is 15 to 24, calculated with reference to the anhydrous
substance.
Absorbance (2.2.25). Dissolve 0.100 g in water R and dilute to 100.0 ml with the same solvent.
Dilute 2.0 ml of the solution to 100.0 ml with sodium hydrogen carbonate solution R. Examined
between 220 nm and 350 nm, the solution shows an absorption maximum at 272 nm. The specific
absorbance at the maximum is 260 to 300, calculated with reference to the anhydrous substance.
254
R as the
coating substance.
same solvent.
Reference solution. Dilute 1.0 ml of the test solution to 100 ml with water R.
Apply separately to the plate 5 l of each solution under a current of nitrogen. Develop over a path of
15 cm using a mixture of 10 volumes of water R, 10 volumes of glacial acetic acid R, 20 volumes of
acetone R and 50 volumes of ethyl acetate R. Allow the plate to dry in air and examine in ultraviolet
light at 254 nm. Expose the plate to iodine vapour in an airtight tank until the spots appear. Any spot
mination of water.
ASSAY
Reference solution (a). Dissolve 25.0 mg of cefazolin CRS in the mobile phase and dilute to 25.0 ml
Reference solution (b). Dissolve 5.0 mg of cefuroxime sodium CRS in 10.0 ml of reference solution (a)
chromatography R (5 m to 10 m),
90 volumes of a solution containing 2.77 g/l of disodium hydrogen phosphate R and 1.86 g/l of
citric acid R,
Inject reference solution (b). Adjust the attenuation to obtain peaks with a height corresponding to
at least half the full scale deflection. The test is not valid unless the resolution between the peaks
08-19
corresponding to cefazolin and cefuroxime is at least 2.0. Adjust the concentration of acetonitrile in
the mobile phase, if necessary. Inject reference solution (a) six times. The test is not valid unless the
relative standard deviation of the peak area for cefazolin is at most 1.0 per cent. Inject alternately the
test solution and reference solution (a).
Calculate the percentage content of cefazolin sodium.
STORAGE
Store in an airtight container, protected from light, at a temperature not exceeding 30C. If the
LABELLING
The label states:
IMPURITIES
S
S
N
COOH
O
H H
N
N
S
Me
H
2
N
A. (6R,7R)-7-amino-3-[(5-methyl-1,3,4-thiadiazol-2-yl)thio-methyl]-8-oxo-5-thia-1-
S
S
N O
N Me
3
C
COOH
O
H H
N
N
S
Me
H
B. (6R,7R)-3-[(5-methyl-1,3,4-thiadiazol-2-yl)thiomethyl]-8-oxo-7-trimethylacetamido-5-
thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid,
O
H H
O
N
N
N
N
N
O N
S
O
H
C. (6R,7R)-3-hydroxymethyl-8-oxo-7-[2-(1H-tetrazol-1-yl)acetamido]-5-thia-1-azabicyclo-
[4.2.0]oct-2-ene-2-carboxylic acid lactone,
H
S
N O
N
N
N
N
N
COOH
O
H H
OAc
D. (6R,7R)-3-acetoxymethyl-8-oxo-7-[2-(1H-tetrazol-1-yl)acetamido]-5-thia-1-azabicyclo-
[4.2.0]oct-2-ene-2-carboxylic acid,
N
N
S
Me
HS
E. 5-methyl-1,3,4-thiadiazole-2-thiol,
N
N
N
N COOH
F. 2-(1H-tetrazol-1-yl)acetic acid,
H
S
N O
N
N
N
N
N
COOH
O
H H
Me
G. (6R,7R)-3-methyl-8-oxo-7-[2-(1H-tetrazol-1-yl)acetamido]-5-thia-1-azabicyclo[4.2.0]oct-
2-ene-2-carboxylic acid.
__________________________________________________________________________________________________________ Ph Eur
08-20
Cefixime
H
S
N O
N
N
O
COOH
H
2
N
CH
2
S
H H
O
N
COOH
C
16
H
15
N
5
O
7
S
2
,3H
2
O 507.5 79350-37-1
Cefixime complies with the requirements of the 3rd edition of the European Pharmacopoeia [1188]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cefixime is the trihydrate of (6R,7R)-7-[[(Z)-2-(2-aminothiazol-4-yl)-2-
[(carboxymethoxy)imino]acetyl]amino]-3-ethenyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-
carboxylic acid. It contains not less than 95.0 per cent and not more than the equivalent of 101.0 per
cent of C
16
H
15
N
5
O
7
S
2
, calculated with reference to the anhydrous and ethanol-free substance.
CHARACTERS
A white or almost white powder, slightly hygroscopic, slightly soluble in water, freely soluble in
methanol, sparingly soluble in ethanol, practically insoluble in ethyl acetate.
IDENTIFICATION
obtained with cefixime CRS. If the spectra obtained show differences, dissolve the substance to be
examined and the reference substance separately in methanol R, evaporate to dryness and record new
B. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel F
254
silanised plate R.
Reference solution (a). Dissolve 20 mg of cefixime CRS in 5 ml of a mixture of equal volumes of
Reference solution (b). Dissolve 20 mg of cefixime CRS and 20 mg of ceftriaxone sodium CRS in 5 ml of
a mixture of equal volumes of methanol R and 0.067M phosphate buffer solution pH 7.0 R.
of methyl acetate R and 90 volumes of a 154 g/l solution of ammonium acetate R previously adjusted to
pH 6.2 with acetic acid R. Allow the plate to dry and examine in ultraviolet light at 254 nm. The
principal spot in the chromatogram obtained with the test solution is similar in position and size to
the principal spot in the chromatogram obtained with reference solution (a). The test is not valid
unless the chromatogram obtained with reference solution (b) shows two clearly separated spots.
by swirling; the solution is yellow. Place the test-tube in a water-bath for 1 min; an orange colour
develops.
TESTS
reference solution (b). Adjust the sensitivity of the system so that the height of the principal peak in
the chromatogram obtained is at least 50 per cent of the full scale of the recorder. Inject the test
08-21
solution and continue the chromatography for three times the retention time of the principal peak.
In the chromatogram obtained with the test solution: the area of any peak, apart from the principal
peak, is not greater than half the area of the principal peak obtained in the chromatogram with
peak, is not greater than three times the area of the principal peak obtained with reference solution
(b) (3 per cent). Disregard any peak with an area less than 0.1 times that of the principal peak in the
Ethanol (2.4.24). Not more than 1.0 per cent m/m, determined by head-space gas chromatography
(2.2.28), using the method of standard additions.
Sample solution. Dissolve 0.250 g of the substance to be examined in a mixture of 1 volume of
dimethylacetamide R and 4 volumes of water R and dilute to 25.0 ml with the same mixture of
solvents.
mination of water.
ASSAY
Reference solution (a). Dissolve 25.0 mg of cefixime CRS in the mobile phase and dilute to 25.0 ml
Reference solution (c). Dissolve 10 mg of cefixime CRS in 10 ml of water R. Heat on a water-bath for
45 min. Cool and inject immediately.
a column 0.125 m long and 4 mm in internal diameter packed with octadecylsilyl silica gel for
volumes of a tetrabutylammonium hydroxide solution prepared as follows: dissolve 8.2 g of
tetrabutylammonium hydroxide R in water R and dilute to 800 ml with the same solvent; adjust to
pH 6.5 with dilute phosphoric acid R and dilute to 1000 ml with water R,
Inject reference solution (c). Adjust the sensitivity of the system so that the heights of the principal
peaks are at least 20 per cent of the full scale of the recorder. The test is not valid unless the
resolution between the two principal peaks (cefixime and E-isomer) is at least 2.0. If necessary, adjust
the concentration of acetonitrile in the mobile phase. Inject reference solution (a) six times. The test
is not valid unless the relative standard deviation of the peak area for cefixime is at most 1.0 per cent.
Inject alternately the test solution and reference solution (a).
STORAGE
IMPURITIES
H
*
O
H
COOH
N
S
N O
N
O
COOH
H
2
N
S
N
O
Me
*
A. 2-[[(Z)-2-(2-aminothiazol-4-yl)-2-[(carboxymethoxy)imino]acetyl]amino]-2-[(2R)-5-
methyl-7-oxo-1,2,5,7-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl]acetic acid,
08-22
H
and epimer at C*
O
H
N
S
N O
N
O
COOH
H
2
N
S
N
O
Me
H
*
B. 2-[[[(Z)-1-(2-aminothiazol-4-yl)-2-[[[(2R,5 RS)-5-methyl-7-oxo-1,2,5,7-tetrahydro-4H-
furo[3,4-d][1,3]thiazin-2-yl]methyl]amino]-2-oxoethylidene]amino]oxy]acetic acid,
N
S
N
S
N
COOH
CH
2
H H
O
O
N
O
COOH
H
2
N
H
C. (6R,7S)-7-[[(Z)-2-(2-aminothiazol-4-yl)-2-[(carboxymethoxy)imino]acetyl]amino]-3-
ethenyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid
(cefixime 7-epimer),
N
S
N
S
N
COOH
CH
2
H H
O
O
N
H
2
N
O
COOH
H
D. (6R,7R)-7-[[(E)-2-(2-aminothiazol-4-yl)-2-[(carboxymethoxy)imino]acetyl]amino]-3-
ethenyl-8-oxo-5-thia-1-azabicyclo-[4.2.0]oct-2-ene-2-carboxylic acid
(cefixime E-isomer),
N
S
N
S
N
COOH
Me
H H
O
O
N
O
COOH
H
2
N
H
E. (6R,7R)-7-[[(Z)-2-(2-aminothiazol-4yl)-2-(carboxymethoxyimino)acetyl]amino]-3-methyl-
8-oxo-5-thia-1-azabicyclo-[4.2.0]oct-2-ene-2-carboxylic acid.
__________________________________________________________________________________________________________ Ph Eur
08-23
Cefoperazone Sodium
1/01
N
H
HN
N
H
O
tN
O
O
OH
N
H H
S
S
COONa
N
N N
N
Me
O
O
C
25
H
26
N
9
NaO
8
S
2
668 62893-19-0
Cefoperazone Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia [1404].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cefoperazone sodium contains not less than 95.0 per cent and not more than the equivalent of
102.0 per cent of sodium (6R,7R)-7-[[(2R)-2-[[(4-ethyl-2,3-dioxopiperazin-1-yl)carbonyl]amino]- 2-
(4-hydroxyphenyl)acetyl]amino]-3-[[(1-methyl-1H-tetrazol-5-yl)sulphanyl]methyl]-8-oxo-5-thia-1-
azabicyclo[4.2.0]oct-2-ene-2-carboxylate, calculated with reference to the anhydrous and acetone-
free substance.
CHARACTERS
A white or slightly yellow powder, hygroscopic, freely soluble in water, soluble in methanol, slightly
soluble in alcohol.
If crystalline, it shows polymorphism.
IDENTIFICATION
A. Dissolve the substance to be examined in methanol R and evaporate to dryness. Examine by
infrared absorption spectrophotometry (2.2.24), comparing with the Ph. Eur. reference spectrum of
cefoperazone sodium.
peak in the chromatogram obtained with test solution (a) are approximately the same as those of the
TESTS
Appearance of solution Dissolve 2.5 g in water R and dilute to 25.0 ml with the same solvent. The
solution is clear (2.2.1). The absorbance of the solution measured at 430 nm (2.2.25) is not greater
than 0.15.
Related substances Examine by liquid chromatography (2.2.29) as prescribed under Assay. Inject
20 l of reference solution (b) and adjust the sensitivity of the system so that the height of the
Inject 20 l of test solution (b). Continue the chromatography for at least 2.5 times the retention time
of the principal peak. In the chromatogram obtained with test solution (b): the area of any peak,
apart from the principal peak, is not greater than 1.5 times the area of the principal peak in the
chromatogram obtained with reference solution (b) (1.5 per cent); the sum of the areas of any such
peaks is not greater than 4.5 times the area of the principal peak in the chromatogram obtained with
reference solution (b) (4.5 per cent). Disregard any peak with an area less than 0.1 times the area of
the principal peak in the chromatogram obtained with reference solution (b).
08-24
Acetone Not more than 2.0 per cent, determined by head-space gas chromatography (2.2.28),
using the method of standard additions.
Sample solution. Dissolve 0.500 g of the substance to be examined in water R and dilute to 10.0 ml
Solvent solution. Dissolve 0.350 g of acetone R in water R and dilute to 100.0 ml with the same solvent.
Dilute 10.0 ml of the solution to 100.0 ml with water R.
Prepare each of four injection vials as shown in the table below:
Vial
No.
Sample
solution
(ml)
Solvent
solution
(ml)
Water R
(ml)
1 1.0 0 4.0
2 1.0 1.0 3.0
3 1.0 2.0 2.0
4 1.0 3.0 1.0
The chromatographic procedure may be carried out using System B of the test for residual solvents
(2.4.24) and the following static head-space injection conditions:
maintaining the temperature of the column at 40C for 10 min.
mination of water.
ASSAY
Test solution (b). Dissolve 25.0 mg of the substance to be examined in the mobile phase and dilute to
Reference solution (a). Dissolve 25.0 mg of cefoperazone dihydrate CRS in the mobile phase and dilute
as mobile phase at a flow rate of 1 ml/min a mixture of 884 volumes of water R; 110 volumes of
acetonitrile R; 3.5 volumes of a 60 g/l solution of acetic acid R; 2.5 volumes of a
triethylammonium acetate solution prepared by diluting 14 ml of triethylamine R and 5.7 ml of
glacial acetic acid R to 100 ml with water R,
conditions, the retention time is about 15 minutes for cefoperazone. Adjust the sensitivity of the
system so that the height of the principal peak in the chromatogram obtained is at least 50 per cent of
the full scale of the recorder. The test is not valid unless in the chromatogram obtained with
reference solution (a), the number of theoretical plates calculated for the principal peak is at least
5000 and its symmetry factor is at most 1.6. If necessary adjust the content of acetonitrile R in the
mobile phase. Inject reference solution (a) six times. The test is not valid unless the relative standard
deviation of the peak area is at most 1.0 per cent. Inject alternately test solution (a) and reference
solution (a).
Calculate the percentage content of cefoperazone sodium by multiplying the percentage content of
cefoperazone by 1.034.
STORAGE
08-25
LABELLING
The label states:
IMPURITIES
O
N
S
H
N
O
H H
O
O
H HN
N
O
EtN
O
O
O
A. (2R)-N-[(5aR,6R)-1,7-dioxo-1,4,6,7-tetrahydro-3H,5aH-azeto[2,1-b]furo[3,4-d][1,3]-
thiazin-6-yl]-2-[[(4-ethyl-2,3-dioxopiperazin-1-yl)carbonyl]amino]-2-(4-hydroxyphenyl)
acetamide,
COOH
N
S
H
N
O
H H
O
H HN
N
O
EtN
O
O
N N
N
N S
Me
O
B. (6R,7R)-7-[[(2R)-2-[[(4-ethyl-2,3-dioxopiperazin-1-yl)carbonyl]amino]-2-(4-
hydroxyphenyl)acetyl]amino]-3-[(4-methyl-5-thioxo-4,5-dihydro-1H-tetrazol-1-yl)methyl]-
8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid,
N
N MeN
N S
C. 1-methyl-1H-tetrazole-5-thiol,
N
N
H
N
COOH
N
S
H
2
N
H H
O
S
D. (6R,7R)-3-[(1H-1,2,3-triazol-4-yl)thio]methyl]-8-oxo-7-amino-5-thia-1-
azabicyclo[4.2.0]oct-2-ene- 2-carboxylic acid (7-TACA),
COOH
N
S
H
2
N
H H
O
O CH
3
O
E. 3-acetoxymethyl-8-oxo-7-amino-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (7-
ACA).
__________________________________________________________________________________________________________ Ph Eur
08-26
Cefotaxime Sodium
H
N
S
OMe
N
N
O
H
2
N
COOH
N
H H
S
OAc
O
C
16
H
16
N
5
NaO
7
S
2
477.4 64485-93-4
Cefotaxime Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia [0989].
Preparation
Cefotaxime Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cefotaxime sodium contains not less than 96.0 per cent and not more than the equivalent of
101.0 per cent of sodium (6R,7R)-3-[(acetyloxy)methyl]-7-[[(Z)-2-(2-aminothiazol-4-yl)-2-
(methoxyimino)acetyl]amino]-8-oxo-5-thia-1-azabicyclo[4.2.0]-oct-2-ene-2-carboxylate, calculated
PRODUCTION
CHARACTERS
A white or slightly yellow powder, hygroscopic, very soluble in water, sparingly soluble in methanol,
IDENTIFICATION
obtained with cefotaxime sodium CRS.
254
R as the coating
substance.
Test solution. Dissolve 20 mg of the substance to be examined in 5.0 ml of a mixture of equal volumes
Reference solution (a). Dissolve 20 mg of cefotaxime sodium CRS in 5.0 ml of a mixture of equal
volumes of methanol R and 0.067M phosphate buffer solution pH 7.0 R.
Reference solution (b). Dissolve 20 mg of cefotaxime sodium CRS and 20 mg of cefoxitin sodium CRS in
5.0 ml of a mixture of equal volumes of methanol R and 0.067M phosphate buffer solution pH 7.0 R.
spots.
by swirling; the solution is bright yellow. Place the test-tube in a water-bath for 1 min; a brown
colour develops.
08-27
TESTS
Appearance of solution Solution S is clear (2.2.1). Add 1 ml of glacial acetic acid R to 10 ml of
solution S. The solution, examined immediately, is clear. The absorbance (2.2.25) of solution S
measured at 430 nm is not greater than 0.20.
substance.
Dilute 10.0 ml of this solution to 100.0 ml with water R. The specific absorbance determined at the
maximum at 235 nm is 360 to 390, calculated with reference to the dried substance.
Inject the test solution and reference solution (b). Continue the chromatogram for at least eight
times the retention time of the main peak. In the chromatogram obtained with the test solution: the
area of any peak, apart from the principal peak, is not greater than the area of the principal peak in
the chromatogram obtained with reference solution (b) (1 per cent); the sum of the areas of such
peaks is at most three times the area of the principal peak in the chromatogram obtained with
reference solution (b) (3 per cent).
maintaining the temperature of the column at 120 C and that of the injection port and the detector
at 150 C.
Inject separately 1 l of the test solution and 1 l of the reference solution.
100C to 105C.
ASSAY
Reference solution (a). Dissolve 25.0 mg of cefotaxime sodium CRS in the mobile phase and dilute to
Reference solution (c). Add 1.0 ml of dilute hydrochloric acid R to 4.0 ml of the test solution. Heat the
solution at 40C for 2 h. Add 5.0 ml of buffer solution pH 6.6 R and 1.0 ml of dilute sodium hydroxide
solution R.
08-28
as mobile phase at a flow rate of 1.0 ml/min a mixture prepared as follows: dissolve 3.5 g of
potassium dihydrogen phosphate R and 11.6 g of disodium hydrogen phosphate R in 1000 ml of
water R at pH 7.0 and add 180 ml of methanol R,
an injector with a fixed loop of 10 l.
Inject reference solution (a) and reference solution (c). Adjust the attenuation to obtain principal
peaks from reference solution (c) with a height of at least 50 per cent of the full-scale deflection of the
recorder. The test is not valid unless cefotaxime is eluted as the second of the principal peaks and the
resolution between the two principal peaks is at least 3.5. If necessary, use another stationary phase
or adjust the methanol content of the mobile phase. The test is not valid if the symmetry factor of the
cefotaxime peak is greater than 2.0. Inject reference solution (a) six times. The test is not valid unless
the relative standard deviation of the area of the cefotaxime peak is at most 1.0 per cent. Inject
alternately the test solution and reference solution (a).
STORAGE
Store in an airtight container, protected from light at a temperature not exceeding 30C. If the
substance is sterile, store in an sterile, airtight, tamper-proof container.
LABELLING
The label states:
IMPURITIES
N
S
N
S
N
COOH
H H
O
N
R'HN
R
OMe
O
H
A. R = R = H:
(6R,7R)-7-[[(Z)-2-(2-aminothiazol-4-yl)-2-methoxyimino)acetyl]amino]-3-methyl-8-oxo-
5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (desacetoxycefotaxime),
B. R = OH, R = H:
(6R,7R)-7-[[(Z)-2-(2-aminothiazol-4-yl)-2-methoxyimino)acetyl]amino]-3-(hydroxymethyl)-
8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (desacetylcefotaxime),
C. (6R,7R)-3-[(acetyloxy)methyl]-7-[[(Z)-2-[2-(formylamino)-thiazol-4-yl]-2-(methoxyimino)-
acetyl]amino]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid
(N -formylcefotaxime),
N
S
N
S
N
COOH
H H
O
O
N
H
2
N
MeO
OAc
H
D. (6R,7R)-3-[(acetyloxy)methyl]-7-[[(E)-2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetyl]-
amino]-8-oxo-5-thia-1-azabicyclo-[4.2.0]oct-2-ene-2-carboxylic acid
(E-cefotaxime),
H
O
O
H
2
N
OMe
N
O
H H
N
S
N
S
N
O
E. (Z)-2-(2-aminothiazol-4-yl)-N -[(5aR,6R)-1,7-dioxo-1,4,6,7-tetrahydro-3H,5aH-azeto[2,1-
b]furo[3,4-d][1,3]thiazin-6-yl]-2-(methoxyimino)acetamide (desacetylcefotaxime lactone).
__________________________________________________________________________________________________________ Ph Eur
08-29
Cefoxitin Sodium
H
S
N
O
O NH
2
H
O
COONa
N
S
O
MeO
C
16
H
16
N
3
NaO
7
S
2
449.4 33564-33-6
Cefoxitin Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia [0990].
Preparation
Cefoxitin Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cefoxitin sodium contains not less than 97.5 per cent and not more than the equivalent of 101.0 per
cent of sodium (6R,7S)-3-carbamoyloxymethyl-7-methoxy-8-oxo-7-[2-(2-thienyl)acetamido]-5-thia-
1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate, calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white powder, very hygroscopic, very soluble in water, sparingly soluble in alcohol,
IDENTIFICATION
obtained with cefoxitin sodium CRS.
254
R as the coating
substance.
Reference solution (a). Dissolve 20 mg of cefoxitin sodium CRS in 5 ml of a mixture of equal volumes of
Reference solution (b). Dissolve 20 mg of cefoxitin sodium CRS and 20 mg of cefazolin CRS in 5 ml of a
10 volumes of tetrahydrofuran R and 90 volumes of a 154 g/l solution of ammonium acetate R
previously adjusted to pH 6.2 with acetic acid R. Dry the plate in a current of warm air and examine
by swirling; the solution is pale brown. Place the test-tube in a water-bath for 1 min; a dark reddish-
brown colour develops.
TESTS
pH (2.2.3). Dilute 2 ml of solution S to 20 ml with carbon dioxide-free water R. The pH of the
08-30
same solvent. The specific optical rotation is +206 to +214, calculated with reference to the anhy-
drous substance.
Absorbance (2.2.25). Dissolve 0.100 g in water R and dilute to 100.0 ml with the same solvent.
Dilute 2.0 ml of the solution to 100.0 ml with sodium hydrogen carbonate solution R. Examined
between 220 nm and 350 nm, the diluted solution shows an absorption maximum at 236 nm and a
broad absorption maximum at about 262 nm. The specific absorbance at this broad maximum is 190
to 210, calculated with reference to the anhydrous substance.
254
R as the
coating substance.
same solvent.
Reference solution. Dilute 0.5 ml of the test solution to 100 ml with water R.
10 volumes of glacial acetic acid R, 10 volumes of water R, 20 volumes of acetone R and 50 volumes of
ethyl acetate R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any spot in
mination of water.
ASSAY
the same solvent.
Reference solution (a). Dissolve 25.0 mg of cefoxitin sodium CRS in water R and dilute to 25.0 ml with
the same solvent.
Reference solution (b). Dissolve 20.0 mg of 2-(2-thienyl)acetic acid R in water R and dilute to 25.0 ml
Reference solution (c). Mix 1.0 ml of reference solution (a) and 5.0 ml of reference solution (b).
as mobile phase at a flow rate of 1.0 ml per minute a mixture of 1 volume of acetic acid R, 19
volumes of acetonitrile R and 81 volumes of water R,
Inject reference solution (c). Adjust the sensitivity of the detector so that the height of the peaks is
at least half the full scale of the recorder. The test is not valid unless the resolution between the two
principal peaks is at least 3.5. Adjust the concentration of acetonitrile in the mobile phase, if
necessary. Inject reference solution (a) six times. The test is not valid unless the relative standard
deviation of the peak area of cefoxitin is at most 1.0 per cent. Inject alternately the test solution and
Calculate the percentage content of cefoxitin sodium.
STORAGE
Store in an airtight container, at a temperature not exceeding 30C. If the substance is sterile, store in
a sterile, airtight, tamper-proof container.
LABELLING
The label states:
08-31
IMPURITIES
O
S
N
COO
-
O
H
NH
2
O N
O
H
2
N
HN
O
S
O
O
Me
OMe
H
NH
2
+
A. 3-carbamoyloxymethyl-7-[5-carbamoyl-5-(tosylamino)-pentanamido]-7-methoxy-8-oxo-
5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid bis(dicyclohexylamine) salt,
H
S
N
O
S
N O
COOH
O
H
N O
N
COOMe
OMe
H
H
B. (6R,7S)-7-methoxy-3-methoxycarbonylaminomethyl-carbamoyloxymethyl-8-oxo-7-[2-
(2-thienyl)acetamido]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid,
H
S
N
O
S
N O
COOH
O
H
NH
2
O
OMe
C. (6R,7S)-3-carbamoyloxymethyl-7-methoxy-8-oxo-7-[2-(2-thienyl)acetamido]-5-thia-1-
S
COOH
D. 2-(2-thienyl)acetic acid.
__________________________________________________________________________________________________________ Ph Eur
08-32
Cefradine
H
H NH
2
O
N
N
O
H H
S
COOH
Me
C
16
H
19
N
3
O
4
S 349.4 38821-53-3
Cefradine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0891]. These
Preparation
Cefradine Capsules
When cephradine is prescribed or demanded, Cefradine shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cefradine contains not less than 90.0 per cent of (6R,7R)-7-[(R)-2-amino-2-(cyclohexa-1,4-dienyl)-
acetamido]-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, calculated with
reference to the anhydrous substance. The sum of the percentage contents of C
16
H
19
N
3
O
4
S and of
cefalexin (C
16
H
17
N
3
O
4
S; M
r
347.4) is not less than 95.0 per cent and not more than the equivalent
of 102.0 per cent, calculated with reference to the anhydrous substance.
CHARACTERS
A white or slightly yellow, hygroscopic powder, sparingly soluble in water, practically insoluble in
alcohol and in ether.
IDENTIFICATION
obtained with cefradine CRS. If the spectra obtained in the solid state show differences, dissolve
30 mg of the substance to be examined and of the reference substance separately in 10 ml of
methanol R, evaporate to dryness at 40C at a pressure less than 2 kPa and record new spectra using
the residues.
254
R as the coating
substance.
Reference solution (a). Dissolve 20 mg of cefradine CRS in 5 ml of a mixture of equal volumes of
Reference solution (b). Dissolve 20 mg of cefradine CRS and 20 mg of cefalexin CRS in 5 ml of a
adjusted to pH 6.2 with glacial acetic acid R. Allow the plate to dry and examine in ultraviolet light at
spots.
by swirling; the solution is pale yellow. Place the test-tube in a water-bath for 1 min; a dark yellow
colour develops.
08-33
TESTS
Solution S Dissolve 2.50 g in sodium carbonate solution R and dilute to 25.0 ml with the same solvent.
Appearance of solution Solution S is not more opalescent than reference suspension II (2.2.1).
Allow solution S to stand for 5 min. The absorbance of solution S measured at 450 nm (2.2.25) is
not greater than 0.20.
Specific optical rotation (2.2.7). Dissolve 0.250 g in acetate buffer solution pH 4.6 R and dilute to
25.0 ml with the same solvent. The specific optical rotation is +80 to +90, calculated with reference
to the anhydrous substance.
The absorbance measured at 330 nm is not greater than 0.05. Dilute 2.0 ml of the solution to
50.0 ml with water R. Examined between 220 nm and 300 nm, the diluted solution shows an
absorption maximum at 262 nm. The specific absorbance at this maximum is 215 to 240, calculated
coating substance. Impregnate the plate by development with a 5 per cent V/V solution of
tetradecane R in hexane R. Allow the solvent to evaporate and carry out the chromatography in the
same direction as the impregnation.
Test solution. Dissolve 0.25 g of the substance to be examined in dilute hydrochloric acid R and dilute to
Reference solution (a). Dilute 1 ml of the test solution to 100 ml with dilute hydrochloric acid R.
Reference solution (b). Dissolve 25 mg of 7-amino-desacetoxycephalosporanic acid CRS in dilute hydro-
chloric acid R and dilute to 10 ml with the same acid (reference solution b). Dilute 1 ml of this solution
to 10 ml with dilute hydrochloric acid R.
Reference solution (c). Dissolve 25 mg of cyclohexa-1,4-dienylglycine CRS in dilute hydrochloric acid R
and dilute to 10 ml with the same acid (reference solution c). Dilute 1 ml of this solution to 10 ml with
dilute hydrochloric acid R.
Reference solution (d). Dissolve 0.25 g of the substance to be examined in a mixture of 1 ml of refer-
ence solution b and 1 ml of reference solution c and dilute to 10 ml with dilute hydrochloric acid R.
volumes of acetone R, 80 volumes of a 72 g/l solution of disodium hydrogen phosphate R and 120
volumes of a 21 g/l solution of citric acid R. Dry the plate by heating at 90C for 3 min. Spray the hot
plate with a 1 g/l solution of ninhydrin R in the mobile phase. Heat the plate at 90C for 15 min and
allow to cool. In the chromatogram obtained with the test solution: any spot corresponding to 7-
aminodesacetoxycephalosporanic acid is not more intense than the spot in the chromatogram
obtained with reference solution (b) (1.0 per cent); any spot corresponding to cyclohexa-1,4-
dienylglycine (the position of which is defined by comparison with the chromatogram obtained with
reference solution (d)), is not more intense than the spot in the chromatogram obtained with refer-
ence solution (c) (1.0 per cent); any spot apart from the principal spot and the spots corresponding
to 7-aminodesacetoxycephalosporanic acid and to cyclohexa-1,4-dienylglycine is not more intense
than the principal spot in the chromatogram obtained with reference solution (a) (1.0 per cent). The
test is not valid unless the chromatogram obtained with reference solution (d) shows three clearly
separated spots.
Cefalexin Not more than 5.0 per cent, calculated with reference to the anhydrous substance and
determined by liquid chromatography (2.2.29), as prescribed under Assay. Inject separately the test
solution and reference solution (b).
250.0 ml with water R. To 1.0 ml of the solution in a ground-glass-stoppered tube add 5.0 ml of 1M
08-34
detector at 150C.
Inject separately 1 l of the test solution and 1 l of the reference solution.
mination of water.
ASSAY
Reference solution (a). Dissolve 50.0 mg of cefradine CRS in the mobile phase and dilute to 100.0 ml
Reference solution (b). Dissolve 10.0 mg of cefradine CRS and 10.0 mg of cefalexin CRS in the mobile
phase and dilute to 100.0 ml with the mobile phase.
a column 0.25 m long and 4.6 mm in internal diameter, packed with octadecylsilyl silica gel for
chromatography R (5 m or 10 m),
as mobile phase at a flow rate of 1.0 ml per minute a mixture of 1 volume of dilute acetic acid R,
17 volumes of a 36.2 g/l solution of sodium acetate R, 200 volumes of methanol R and 782
volumes of water R,
Inject reference solution (b). Adjust the sensitivity of the detector so that the height of the peaks is
at least half the full scale of the detector. The test is not valid unless the resolution between the peaks
corresponding to cefalexin and cefradine is at least four. If necessary, adjust the methanol content of
the mobile phase. Inject reference solution (a) six times. The test is not valid unless the relative
standard deviation of the peak area for cefradine is at most 1.0 per cent. Inject separately the test
Calculate the percentage content of cefradine and cefalexin.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
08-35
Ceftazidime
1/01
N
S
O
N
N
H
N
O
Me COOH
Me
N
S
2
N
O
H H
COO
_
C
22
H
22
N
6
O
7
S
2
,5H
2
O 636.6 72558-82-8
Ceftazidime complies with the requirements of the 3rd edition of the European Pharmacopoeia [1405]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ceftazidime is (6R,7R)-7-[[(Z)-2-(2-aminothiazol-4-yl)-2-[(1-carboxy-1-
methylethoxy)imino]acetyl]amino]-8-oxo- 3-[(1-pyridinio)methyl]-5-thia-1-azabicyclo[4.2.0]oct-2-
ene-2-carboxylate pentahydrate. It contains not less than 95.0 per cent and not more than 102.0 per
cent of C
22
H
22
N
6
O
7
S
2
CHARACTERS
A white or almost white, crystalline powder, slightly soluble in water and in methanol, practically
insoluble in acetone and in alcohol. It dissolves in acid and alkali solutions.
IDENTIFICATION
with ceftazidime CRS.
TESTS
Related substances
254
plate R.
Test solution. Dissolve 0.100 g of the substance to be examined in a 36 g/l solution of disodium
hydrogen phosphate R and dilute to 2.0 ml with the same solution.
Reference solution. Dilute 1 ml of the test solution to 200 ml with a 36 g/l solution of disodium hydrogen
phosphate R.
of butanol R, 26 volumes of sodium acetate buffer solution pH 4.5 R, 32 volumes of butyl acetate R and
32 volumes of glacial acetic acid R. Dry the plate in a current of warm air and examine in ultraviolet
light at 254 nm. Any spot with an R
f
value greater than that of the principal spot in the chromato-
gram obtained with the test solution is not more intense than the spot in the chromatogram obtained
with the reference solution (0.5 per cent).
Reference solution (a). Dissolve 5.0 mg of ceftazidime impurity A CRS in the mobile phase and dilute to
Reference solution (b). Dissolve 5 mg of ceftazidime impurity A CRS and 5 mg of ceftazidime CRS in the
08-36
volumes of a 22.6 g/l solution of ammonium dihydrogen phosphate R, adjusted to pH 3.9 with a
10 per cent V/V solution of phosphoric acid R,
two peaks in the chromatogram obtained are at least 50 per cent of the full scale of the recorder. The
test is not valid unless in the chromatogram obtained, the resolution between the peaks correspond-
ing to ceftazidime and impurity A is at least 5.9. Inject 20 l of the test solution and 20 l of reference
solution (a). Continue the chromatography of the test solution for three times the retention time of
ceftazidime. In the chromatogram obtained with the test solution: the area of any peak, apart from
the principal peak, is not greater than half the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.5 per cent); the sum of the areas of all the peaks, apart from
obtained with reference solution (a) (2 per cent). Disregard any peak with an area less than 0.1 times
the area of the principal peak in the chromatogram obtained with reference solution (a).
Pyridine Not more than 500 ppm, determined by liquid chromatography (2.2.29). Prepare the
solutions immediately before use.
Test solution. Dissolve 0.500 g of the substance to be examined in a 10 per cent V/V solution of
phosphate buffer solution pH 7.0 R4 and dilute to 100.0 ml with the same solvent.
Reference solution. Dissolve 1.00 g of pyridine R in water R and dilute to 100.0 ml with the same
solvent. Dilute 5.0 ml of the solution to 200.0 ml with water R. To 1.0 ml of the solution, add 10 ml
of phosphate buffer solution pH 7.0 R4 and dilute to 100.0 ml with water R.
as mobile phase at a flow rate of 1.6 ml/min a mixture of 10 volumes of a 28.8 g/l solution of
ammonium dihydrogen phosphate R previously adjusted to pH 7.0 with ammonia R, 30 volumes of
acetonitrile R and 60 volumes of water R,
Inject 20 l of the reference solution. Adjust the sensitivity of the system so that the height of the
Inject the reference solution six times. The test is not valid unless the relative standard deviation of
the area of the principal peak is at most 3.0 per cent. Inject alternately 20 l of the test solution and
20 l of the reference solution.
mination of water.
ASSAY
Reference solution (a). Dissolve 25.0 mg of ceftazidime CRS in the mobile phase and dilute to 25.0 ml
Reference solution (b). Dissolve 5 mg of ceftazidime impurity A CRS in 5.0 ml of reference solution (a).
a column 0.15 m long and 4.6 mm in internal diameter packed with hexylsilyl silica gel for
as mobile phase at a flow rate of 2 ml/min a mixture prepared as follows: dissolve 4.26 g of
disodium hydrogen phosphate R and 2.73 g of potassium dihydrogen phosphate R in 980 ml of
water R, then add 20 ml of acetonitrile R,
two principal peaks in the chromatogram obtained are at least 50 per cent of the full scale of the
recorder. The test is not valid unless, in the chromatogram obtained, the resolution between the
08-37
peaks corresponding to ceftazidime and impurity A is at least 1.0. Inject alternately the test solution
and reference solution (a). Calculate the percentage content of ceftazidime.
STORAGE
container.
LABELLING
The label states:
IMPURITIES
By liquid chromatography (Related substances test): A,B,C.
By thin-layer chromatography (Related substances test): D,E.
By liquid chromatography (Pyridine test): F.
N
S
N
S
H
N
O
H H
O
COO
_
N N
O
Me COOH
Me
2
N
A. (6R,7R)-7-[[(Z)-2-(2-aminothiazol-4-yl)-2-[(1-carboxy-1-
methylethoxy)imino]acetyl]amino]-8-oxo-3-[(1-pyridinio)methyl]-5-thia-1-
azabicyclo[4.2.0]oct-3-ene-2-carboxylate (-2-ceftazidime),
O
Me COOH
Me
N
S
N
S
H
N
O
H H
O
COO
_
N N
2
N
B. (6R,7R)-7-[[(E)-2-(2-aminothiazol-4-yl)-2-[(1-carboxy-1-
methylethoxy)imino]acetyl]amino]-8-oxo- 3-[(1-pyridinio)methyl]-5-thia-1-
azabicyclo[4.2.0]oct-2-ene-2-carboxylate,
N
S
H H
O
COOH
N
3
N
2Cl
_
C. (6R,7R)-2-carboxy-8-oxo-3-(pyridiniomethyl)-5-thia-1-azabicyclo[4.2.0]oct-2-en-7-
aminium dichloride,
N
S
N
S
H
N
O
H H
O
COO
_
N N
O
Me
Me
N
H
O
O
Me
Me
Me
Ph
3
C
D. (6R,7R)-7-[[(Z)-2-[[2-(1,1-dimethylethoxy)-1,1-dimethyl-2-oxoethoxy]imino]- 2-[2-
[(triphenylmethyl)amino]thiazol-4-yl]acetyl]amino]-8-oxo-3-(pyridiniomethyl)-5-thia-1-
azabicyclo[4.2.0]oct-2-ene-2-carboxylate,
N
S
N
S
H
N
O
H H
O
COO
_
N N
O
Me
Me
O
O
Me
Me
Me
3
N
Cl
_
E. (6R,7R)-7-[[(Z)-2-(2-ammoniothiazol-4-yl)-2-[[2-(1,1-dimethylethoxy)-1,1-dimethyl- 2-
oxoethoxy]imino]acetyl]amino]-8-oxo-3-(pyridiniomethyl)-5-thia-1-azabicyclo[4.2.0]oct-
2-ene-2-carboxylate chloride,
08-38
N
F. pyridine.
__________________________________________________________________________________________________________ Ph Eur
08-39
Ceftriaxone Sodium
H
N
O
N
OMe
S
N
H
2
N
Me
O
ONa
N
N
N COONa
S
S
H H
N
O
C
18
H
16
N
8
Na
2
O
7
S
3
,3H
2
O 662 104376-79-6
Ceftriaxone Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia [0981].
Preparation
Ceftriaxone Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ceftriaxone sodium contains not less than 96.0 per cent and not more than the equivalent of 102.0
per cent of disodium (Z)-(6R,7R)-7-[2-(2-amino-1,3-thiazol-4-yl)-2-(methoxy-imino)acetamido]-8-
oxo-3-[(2,5-dihydro-2-methyl-6-oxido-5-oxo-1,2,4-triazin-3-yl)thiomethyl]-5-thia-1-
azabicyclo[4.2.0]oct-2-ene-2-carboxylate, calculated with reference to the anhydrous substance.
CHARACTERS
An almost white or yellowish, crystalline powder, slightly hygroscopic, very soluble in water, sparingly
soluble in methanol, very slightly soluble in ethanol.
IDENTIFICATION
obtained with ceftriaxone sodium CRS.
254
R as the coating
substance.
Reference solution (a). Dissolve 20 mg of ceftriaxone sodium CRS in 5 ml of a mixture of equal volumes
Reference solution (b). Dissolve 20 mg of ceftriaxone sodium CRS and 20 mg of cefradine CRS in 5 ml of
a mixture of equal volumes of methanol R and 0.067M phosphate buffer solution pH 7.0 R.
10 volumes of methyl acetate R and 90 volumes of a 150 g/l solution of ammonium acetate R previously
adjusted to pH 6.2 with acetic acid R. Dry the plate in a current of warm air and examine in ultra-
violet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is
C. Place about 2 mg in a test-tube 150 mm long and 15 mm in diameter. Moisten with 0.05 ml of
water R and add 2 ml of sulphuric acid-formaldehyde reagent R. Mix the contents of the tube by
swirling; the solution is greenish-yellow. Place the test-tube in a water-bath for 1 min; a yellow colour
develops.
TESTS
(2.2.1) and not more intensely coloured than reference solution Y
5
or BY
5
(2.2.2).
08-40
substance.
the test solution and reference solution (c). Continue the chromatography for at least twice the
retention time of the principal peak. In the chromatogram obtained with the test solution, the area of
any peak, apart from the principal peak, is not greater than the area of the principal peak obtained in
the chromatogram with reference solution (c) (1.0 per cent) and the sum of the area of such peaks is
at most four times the area of the principal peak in the chromatogram obtained with reference
solution (c) (4.0 per cent). Disregard any peak with an area less than 10 per cent of the area of the
Water (2.5.12). 8.0 per cent to 11.0 per cent, determined on 0.100 g by semi-micro determination
of water.
ASSAY
Reference solution (a). Dissolve 30.0 mg of ceftriaxone sodium CRS in the mobile phase and dilute to
Reference solution (b). Dissolve 5.0 mg of ceftriaxone sodium CRS and 5.0 mg of ceftriaxone impurity A
as mobile phase at a flow rate of 1.5 ml per minute, a mixture prepared as follows: dissolve 2.0 g
of tetradecylammonium bromide R and 2.0 g of tetraheptylammonium bromide R in a mixture of
440 ml of water R, 55 ml of 0.067M phosphate buffer solution pH 7.0 R, 5.0 ml of citrate buffer
solution pH 5.0 prepared by dissolving 20.17 g of citric acid R in 800 ml of water R, adjusting to
pH 5.0 with strong sodium hydroxide solution R and diluting to 1000.0 ml with water R, and
500 ml of acetonitrile R,
a 20 l injector loop.
Inject reference solution (b). Adjust the sensitivity of the detector to obtain peaks with a height
corresponding to at least half the full-scale deflection. The test is not valid unless the resolution
between the two principal peaks is at least 3.0. Inject reference solution (a) six times. The test is not
valid unless the relative standard deviation of the peak area for ceftriaxone sodium is at most 1.0 per
cent. Inject alternately the test solution and reference solution (a).
Calculate the percentage content of ceftriaxone sodium.
STORAGE
Store in a well-closed container, protected from light, at a temperature not exceeding 30C. If the
LABELLING
The label states:
IMPURITIES
08-41
S
N
N
N
S
N O
N
S
N
ONa
Me
COONa
O
N
MeO
O
H
2
N
H H
A. disodium (E)-(6R,7R)-7-[(2-amino-1,3-thiazol-4-yl) (methoxyimino)acetamido]-3-[[2,5-
dihydro-2-methyl-6-oxido-5-oxo-1,2,4-triazin-3-yl)thio]methyl]-8-oxo-5-thia-1-
azabicyclo[4.2.0]oct-2-ene-2-carboxylate (E-isomer),
S
N O
N
S
N
O
N
H
2
N
H H
OMe
O
O
B. (Z)-2-(2-amino-1,3,-thiazol-4-yl)-N-[(5aR,6R)-1,7-dioxo-1,4,6,7-tetrahydro-3H,5aH-
azeto[2,1-b]furo[3,4-d][1,3]thiazin-6-yl]-2-(methoxyimino)acetamide,
N
N
N HS
OH
Me
O
C. 6-hydroxy-2-methyl-3-mercapto-1,2,4-triazin-5(2H)-one,
O
S
S
N
N
H
2
N
OMe
N
S
D. S-(1,3-benzothiazol-2-yl)(Z)-(2-amino-1,3-thiazol-4-yl)(methoxyimino)thioacetate,
S
N
N
N
S
N
OH
Me
COOH
O
H
2
N
H H
O
E. (6R,7R)-7-amino-3-[[(2,5,-dihydro-6-hydroxy-2-methyl-5-oxo-1,2,4-triazin-3-
yl)thio]methyl]-8-oxo-5-thia-1-azabicyclo-[4.2.0]oct-2-ene-2-carboxylic acid.
__________________________________________________________________________________________________________ Ph Eur
08-42
Cefuroxime Axetil
corrected 1/01
H
*
and epimer at C*
CH
3
O
O
H
Me
O O
O
N
H H
S
O
OMe
N
O
N O NH
2
O
C
20
H
22
N
4
O
10
S 510.5 64544-07-6
Cefuroxime Axetil complies with the requirements of the 3rd edition of the European Pharmacopoeia [1300].
Preparation
Cefuroxime Axetil Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cefuroxime axetil contains not less than 96.0 per cent and not more than the equivalent of 102.0 per
cent of a mixture of the two diastereoisomers of (1RS)-1-[(acetyl)oxy]ethyl (6R,7R)-3-
[(carbamoyloxy)methyl]-7-[[(Z)-2-(furan-2-yl)-2-(methoxyimino)acetyl]amino]-8-oxo-5-thia-1-
azabicyclo[4.2.0]oct-2-ene-2-carboxylate, calculated with reference to the anhydrous and acetone-
free substance.
CHARACTERS
A white or almost white, amorphous powder, slightly soluble in water, soluble in acetone, in ethyl
acetate and in methanol, slightly soluble in alcohol.
IDENTIFICATION
obtained with cefuroxime axetil CRS.
B. Examine the chromatograms obtained in the Assay. The retention time and size of the principal
peaks in the chromatogram obtained with the test solution are the same as those of the peaks due to
diastereoisomers A and B of cefuroxime axetil in the chromatogram obtained with reference solution
(d).
TESTS
Diastereoisomer ratio Examine by liquid chromatography (2.2.29) as described under Assay. In
the chromatogram obtained with the test solution, the ratio of the peak due to cefuroxime axetil
diastereoisomer A to the sum of the peaks due to cefuroxime axetil diastereoisomers A and B is
between 0.48 and 0.55 by the normalisation procedure.
Calculate the percentage content of related substances from the areas of the peaks in the chromato-
gram obtained with the test solution by the normalisation procedure, disregarding any peak with an
area less than 0.05 times that of the two principal peaks in the chromatogram obtained with reference
solution (a). The percentage sum of the pair of peaks corresponding to the (E)-isomers located by
comparison with the chromatogram obtained with reference solution (c) is not greater than 1.0 per
cent, the percentage sum of the pair of peaks corresponding to the
3
-isomers located by comparison
with the chromatogram obtained with reference solution (b) is not greater than 1.5 per cent and the
area of any other secondary peak is not greater than 0.5 per cent. The sum of related substances is
not greater than 3.0 per cent.
Acetone (2.4.24). Not more than 1.1 per cent.
mination of water.
08-43
ASSAY
Test solution. Prepare the solution immediately before use. Dissolve 10.0 mg of the substance to be
examined in the mobile phase and dilute to 50.0 ml with the mobile phase.
Reference solution (b). Heat 5 ml of the test solution at 60C for 1 h to generate the
3
-isomers.
Reference solution (c). Expose 5 ml of the test solution to ultraviolet light at 254 nm for 24 h to
generate (E) isomers.
Reference solution (d). Prepare the solution immediately before use. Dissolve 10.0 mg of cefuroxime axetil
a column 0.25 m long and 4.6 mm in internal diameter packed with trimethylsilyl silica gel for
volumes of a 23 g/l solution of ammonium dihydrogen phosphate R,
Inject 20 l each of reference solutions (a), (b), (c) and (d). When the chromatograms are recorded
in the prescribed conditions the retention times relative to cefuroxime axetil diastereoisomer A
(second peak) are approximately 0.9 for cefuroxime axetil diastereoisomer B, 1.2 for the cefuroxime
axetil
3
-isomers and 1.7 and 2.1 for the (E) isomers. The test is not valid unless in the chromato-
gram obtained with reference solution (d), the resolution between the peaks corresponding to
cefuroxime axetil diastereoisomers A and B is at least 1.5. In the chromatogram obtained with
reference solution (b), the resolution between the peaks corresponding to cefuroxime axetil
diastereoisomer A and cefuroxime axetil
3
-isomer is at least 1.5.
Inject reference solution (d) solution six times. The assay is not valid unless the relative standard
deviation of the sum of the peaks corresponding to cefuroxime axetil diastereoisomers A and B is at
most 2.0 per cent.
20
H
22
N
4
O
10
S from the sum of areas of the two
diastereoisomer peaks and the declared content of C
20
H
22
N
4
O
10
S in cefuroxime axetil CRS.
STORAGE
IMPURITIES

*
*
N
S
N
H H
O O
N
OMe
O
O NH
2
O
H
O
O CH
3
O O
Me
H
A. (1RS)-1-[(acetyl)oxy]ethyl (2RS,6R,7R)-3-[(carbamoyloxy)methyl]-7-[[(Z)-2-(furan-2-yl)-2-
(methoxyimino)acetyl]amino]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-3-ene-2-carboxylate (
3
-isomers),
N
S
O
N
H H
O O
N
O
MeO
NH
2
O
H
O
O CH
3
O O
Me
H
*
B. (1RS)-1-[(acetyl)oxy]ethyl (6R,7R)-3-[(carbamoyloxy)methyl]-7-[[(E)-2-(furan-2-yl)-2-
(methoxyimino)acetyl]amino]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate
((E)-isomers),
08-44
N
S
O
N
H H
O O
N
O
N
O
OMe
CCl
3
O
H
COOH
H
C. (6R,7R)-7-[[(Z)-2-(furan-2-yl)-2-(methoxyimino)acetyl]amino]-8-oxo-3-
[[[(trichloroacetyl)carbamoyl]oxy]methyl]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic
acid,
N
S
O
N
H H
O O
N
O
NH
2
O
OMe
H
COOH
D. (6R,7R)-3-[(carbamoyloxy)methyl]-7-[[(Z)-2-(furan-2-yl)-2-
(methoxyimino)acetyl]amino]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid
(cefuroxime).
__________________________________________________________________________________________________________ Ph Eur
08-45
Cefuroxime Sodium
N
O
H
N
OMe
O
O
COONa
S
H H
N
O
NH
2
O
C
16
H
15
N
4
NaO
8
S 446.4 56238-63-2
Cefuroxime Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia [0992].
Preparation
Cefuroxime Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cefuroxime sodium contains not less than 96.0 per cent and not more than the equivalent of 101.0
per cent of sodium (Z)-(6R,7R)-3-(carbamoyloxymethyl)-7-[2-(2-furyl)-2-(methoxy-
imino)acetamido]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate, calculated with reference
CHARACTERS
A white or almost white powder, slightly hygroscopic, freely soluble in water, very slightly soluble in
IDENTIFICATION
obtained with cefuroxime sodium CRS.
254
R as the coating
substance.
Reference solution (a). Dissolve 20 mg of cefuroxime sodium CRS in 5 ml of a mixture of equal volumes
Reference solution (b). Dissolve 20 mg of cefuroxime sodium CRS and 20 mg of cefoxitin sodium CRS in
5 ml of a mixture of equal volumes of methanol R and 0.067M phosphate buffer solution pH 7.0 R.
10 volumes of tetrahydrofuran R and 90 volumes of a 150 g/l solution of ammonium acetate R
previously adjusted to pH 6.2 with acetic acid R. Dry the plate in a current of warm air and examine
is similar in position, colour and size to the principal spot in the chromatogram obtained with the
reference solution (a). The test is not valid unless the chromatogram obtained with reference solution
(b) shows two clearly separated spots.
by swirling; the solution is pale brown. Place the test-tube in a water-bath for 1 min; a reddish-brown
colour develops.
D. It gives the reaction (a) of sodium (2.3.1).
TESTS
The absorbance (2.2.25) of solution S measured at 450 nm is not greater than 0.25.
08-46
Specific optical rotation (2.2.7). Dissolve 0.500 g in acetate buffer solution pH 4.6 R and dilute to
25.0 ml with the same solvent. The specific optical rotation is +59 to +66, calculated with reference
the test solution and reference solutions (b) and (c). Continue the chromatography for at least three
times the retention time of the principal peak. In the chromatogram obtained with the test solution:
the area of any descarbamoylcefuroxime peak (located by comparison with the chromatogram
obtained with reference solution (b)) is not greater than the area of the principal peak in the chroma-
togram obtained with reference solution (c) (1.0 per cent); the area of any peak, apart from the
principal peak and any peak corresponding to descarbamoylcefuroxime, is not greater than the area of
the principal peak in the chromatogram obtained with reference solution (c) (1.0 per cent); the sum
of the areas of all the peaks, apart from the principal peak, is at most three times the area of the
principal peak in the chromatogram obtained with reference solution (c) (3.0 per cent). Disregard
any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained
with reference solution (c).
mination of water.
ASSAY
the same solvent.
Reference solution (a). Dissolve 25.0 mg of cefuroxime sodium CRS in water R and dilute to 25.0 ml
Reference solution (b). Place 20 ml of reference solution (a) in a water-bath at 60C for 10 min. Cool
and inject immediately.
Reference solution (c). Dilute 1.0 ml of the test solution to 100.0 ml with water R.
a column 0.125 m long and 4.6 mm in internal diameter packed with hexylsilyl silica gel for
as mobile phase at a flow rate of 1.5 ml per minute a mixture of 1 volume of acetonitrile R and 99
volumes of acetate buffer solution pH 3.4, prepared by dissolving 6.01 g of glacial acetic acid R
and 0.68 g of sodium acetate R in water R and diluting to 1000 ml with the same solvent,
Inject reference solution (b). The chromatogram shows peaks corresponding to cefuroxime and
descarbamoylcefuroxime. The test is not valid unless the resolution between these peaks is at least
2.0. Adjust the concentration of acetonitrile in the mobile phase, if necessary. Inject reference
solution (c). Adjust the sensitivity of the detector so that the height of the principal peak is at least
one quarter of the full scale of the recorder. The assay is not valid unless the symmetry factor of the
cefuroxime peak is at most 1.5. Inject reference solution (a) six times. The assay is not valid unless
the relative standard deviation of the peak area of cefuroxime is at most 1.0 per cent. Inject
Calculate the percentage content of cefuroxime sodium.
STORAGE
LABELLING
The label states:
08-47
IMPURITIES
O
N
O
S
N O
N
OMe
COOH
H H
OH
H
A. (Z)-(6R,7R)-7-[2-(2-furyl)-2-(methoxyimino)acetamido]-3-hydroxymethyl-8-oxo-5-thia-
1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid,
O
N
O
S
N O
N
OMe
COOH
H H
OAc
H
B. (Z)-(6R,7R)-3-acetoxymethyl-7-[2-(2-furyl)-2-(methoxy-imino)acetamido]-8-oxo-5-thia-
1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (cefuracetim),
O
N
O
S
N O
N
OMe
COOH
Me
H H H
C. (Z)-(6R,7R)-7-[2-(2-furyl)-2-(methoxyimino)acetamido]-3- methyl-8-oxo-5-thia-1-
O
N
O
S
N O
N
OMe
COOH
H H
O N
O
CCl
3
O
H
D. (Z)-(6R,7R)-7-[2-(2-furyl)-2-(methoxyimino)acetamido]-8-oxo-3-trichloroacetyl-
carbamoyloxymethyl-5-thia-1-azabicyclo-[4.2.0]oct-2-ene-2-carboxylic acid,
O
N
O
S
N O
N
OMe
COOH
H H
O NH
2
O
H
E. (Z)-(6R,7R)-3-carbamoyloxymethyl-7-[2-(2-furyl)-2-(methoxyimino)acetamido]-8-oxo-5-
thia-1-azabicyclo-[4.2.0]oct-2-ene-2-carboxylic acid (cefuroxime),
O
N
O
S
N O
N
COOH
H H
OH
MeO
H
F. (E)-(6R,7R)-7-[2-(2-furyl)-2-(methoxyimino)acetamido]-3-hydroxymethyl-8-oxo-5-thia-1-
O
N
O
S
N O
N
COOH
H H
OAc
MeO
H
G. (E)-(6R,7R)-3-acetoxymethyl-7-[2-(2-furyl)-2-(methoxy-imino)acetamido]-8-oxo-5-thia-1-
08-48
O
H H
OMe
N
O N
S
O
N
O
O
H
H. (Z)-(6R,7R)-7-[2-(2-furyl)-2-(methoxyimino)acetamido]-3-hydroxymethyl-8-oxo-5-thia-
1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid lactone,
OMe
N
O
COOH
I. (Z)-2-(2-furyl)-2-(methoxyimino)acetic acid.
__________________________________________________________________________________________________________ Ph Eur
08-49
Cellacefate
Cellacefate complies with the requirements of the 3rd edition of the European Pharmacopoeia for Cellulose
Acetate Phthalate [0314]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cellulose acetate phthalate is a partly O-acetylated and O-phthalylated cellulose. It contains not less
than 30.0 per cent and not more than 36.0 per cent of phthaloyl groups (C
8
H
5
O
3
, relative mass of
the group 149.1) and not less than 21.5 per cent and not more than 26.0 per cent of acetyl groups
(C
2
H
3
O, relative mass of the group 43.05), both calculated with reference to the anhydrous and acid-
free substance.
CHARACTERS
A white, free-flowing powder or colourless flakes, hygroscopic, practically insoluble in water, freely
soluble in acetone, soluble in diethylene glycol, practically insoluble in ethanol and in methylene
chloride. It dissolves in dilute solutions of alkalis.
IDENTIFICATION
spectrum of cellulose acetate phthalate.
B. Dissolve about 150 mg in 1 ml of acetone R. Allow the solution to flow over a glass plate and dry.
A thin, transparent, glossy film is formed.
TESTS
Viscosity (2.2.9). Dissolve 15 g, calculated with reference to the anhydrous substance, in 85 g of a
mixture of 1 volume of water R and 249 volumes of acetone R. Determined at 25C, the apparent
viscosity is 45 mPas to 90 mPas.
Free acid Not more than 3.0 per cent (S), calculated as phthalic acid with reference to the anhy-
drous substance. Shake 3.0 g for 2 h with 100 ml of a mixture of 35 volumes of water R and 65
volumes of methanol R and filter. Wash the flask and the filter with two quantities, each of 10 ml, of a
mixture of 35 volumes of water R and 65 volumes of methanol R. Combine the filtrate and washings,
add phenolphthalein solution R and titrate with 0.1M sodium hydroxide until a faint pink colour is
1 ml of 0.1M sodium hydroxide is equivalent to 8.3 mg of free acid, calculated as phthalic acid.
mination of water. Carry out the test using a mixture of 2 volumes of methylene chloride R and 3
volumes of ethanol R.
ASSAY
Phthaloyl groups Dissolve 1.000 g in 50 ml of a mixture of 2 volumes of acetone R and 3 volumes of
alcohol R. Add 0.1 ml of phenolphthalein solution R and titrate with 0.1M sodium hydroxide until a faint
pink colour is obtained. Carry out a blank titration.
Calculate the percentage content of phthaloyl groups (P) from the expression:
) 100 (
5 . 179
) 100 )( 100 (
900 , 14
S
S
m S a
n

a=percentage content of water,
m=mass in grams of the substance to be examined,
n=number of millilitres of 0.1M sodium hydroxide used,
S=percentage content of free acid (see Tests).
Acetyl groups To 0.100 g add 25.0 ml of 0.1M sodium hydroxide and heat on a water-bath under a
reflux condenser for 30 min. Cool, add 0.1 ml of phenolphthalein solution R and titrate with 0.1M
hydrochloric acid until the colour is discharged. Carry out a blank titration.
Calculate the percentage content of acetyl groups from the expression:
08-50
P
S
S
m S a
n n
578 . 0
100
8 . 51
) 100 )( 100 (
) ( 4300
1 2
1
]
1

n
1
=number of millilitres of 0.1M hydrochloric acid used in the test,
n
2
=number of millilitres of 0.1M hydrochloric acid used in the blank titration,
P=percentage content of phthaloyl groups,
S=percentage content of free acid (see Tests).
STORAGE
__________________________________________________________________________________________________________ Ph Eur
08-51
Dispersible Cellulose
Definition Dispersible Cellulose is a colloid-forming, attrited mixture of Microcrystalline Cellulose
and Carmellose Sodium.
Content of carmellose sodium 75.0 to 125.0% w/w of the stated amount.
Characteristics A white or off-white, coarse or fine powder; odourless or almost odourless.
Disperses in water producing a white, opaque dispersion or gel; practically insoluble in organic
solvents and in dilute acids.
Identification
A. Mix 6 g with 300 ml of water stirring at 18,000 revolutions per minute for 5 minutes. A white,
opaque dispersion is obtained which does not produce a supernatant liquid.
B. Add several drops of the dispersion obtained in test A to a 10% w/v solution of aluminium chloride.
Each drop forms a white, opaque globule which does not disperse on standing.
C. Add 2 ml of iodinated potassium iodide solution to the dispersion obtained in test A. No blue or
purplish colour is produced.
D. The solution obtained in the test for Heavy metals yields the reactions characteristic of sodium
salts, Appendix VI, except that in test A the white precipitate produced may not be dense.
Acidity or alkalinity pH of the dispersion obtained in the test for Apparent viscosity, 6.0 to 8.0,
Appendix V L.
Solubility Add 50 mg to 10 ml of ammoniacal solution of copper tetrammine and shake. It dissolves
completely leaving no residue.
Apparent viscosity 60 to 140% of the declared value when determined by the following method.
Calculate the quantity (x g) needed to prepare exactly 600 g of a dispersion of the stated percentage
w/w, with reference to the dried substance. To (600 x) g of water at 23 to 25 contained in a 1000-
ml high-speed blender bowl add x g of the substance being examined, stirring at reduced speed,
taking care to avoid contacting the sides of the bowl with the powder. Continue stirring at low speed
for 15 seconds after the addition and then stir at 18,000 revolutions per minute for exactly 2 minutes.
Immerse the appropriate spindle of a rotational viscometer, switch on after 30 seconds and after a
further 30 seconds determine the viscosity, Appendix V H, Method III, using a speed of 20
revolutions per minute (2.09 radians per second).
Heavy metals To the residue obtained in the test for Sulphated ash add 1 ml of hydrochloric acid,
evaporate to dryness on a water bath and dissolve the residue in 20 ml of water. 12 ml of the resulting
1 g.
Assay Heat 2 g with 75 ml of anhydrous acetic acid under a reflux condenser for 2 hours, cool and
carry out Method I for non-aqueous titration, Appendix VIII A, determining the end point
potentiometrically. Each ml of 0.1M perchloric acid VS is equivalent to 29.6 mg of carmellose sodium.
Storage Dispersible Cellulose should be kept in a well-closed container and stored at a temperature
of 8 to 15.
Labelling The label states (1) the percentage w/w of Carmellose Sodium; (2) the viscosity of a
dispersion in water of a stated percentage w/w of Carmellose Sodium.
08-52
Microcrystalline Cellulose
9004-34-6
Microcrystalline Cellulose complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Microcrystalline cellulose is a purified, partly depolymerised cellulose prepared by treating alpha-
cellulose, obtained as a pulp from fibrous plant material, with mineral acids.
CHARACTERS
A white or almost white, fine or granular powder, practically insoluble in water, in acetone, in
ethanol, in toluene and in dilute acids and in a 50 g/l solution of sodium hydroxide.
IDENTIFICATION
A. Place about 10 mg on a watch-glass and disperse in 2 ml of iodinated zinc chloride solution R. The
substance becomes violet-blue.
B. Transfer 1.300 g to a 125 ml conical flask. Add 25.0 ml of water R and 25.0 ml of 1M
cupriethylenediamine hydroxide solution. Immediately purge the solution with nitrogen R, insert the
stopper and shake until completely dissolved. Transfer 7.0 ml of the solution to a suitable capillary
viscometer (2.2.9). Equilibrate the solution at 25 0.1C for not less than 5 min. Record the flow
time, t
1
, in seconds, between the two marks on the viscometer. Calculate the kinematic viscosity
1
of
the solution using the formula:
t
1
(k
1
),
where k
1
is the viscometer constant.
Dilute a suitable volume of 1M cupriethylenediamine hydroxide solution with an equal volume of water R
and measure the flow time, t
2
, using a suitable capillary viscometer. Calculate the kinematic viscosity
2
of the solvent using the formula:
t
2
(k
2
),
where k
2
Determine the relative viscosity R
el
of the substance to be examined using the formula:
1
/
2
.
Determine the intrinsic viscosity, []c, by interpolation, using the Intrinsic Viscosity Table (Table
315-1 see Cellulose, powdered).
Calculate the degree of polymerisation P using the formula:
95
100 100]
[ ]
[( ) /
c
m b
where m is the mass in grams, of the substance to be examined and b is the loss on drying as
a percentage.
The degree of polymerisation is not more than 350.
TESTS
Solubility Dissolve 50 mg in 10 ml of ammoniacal solution of copper tetrammine R. It dissolves
completely, leaving no residue.
pH (2.2.3). Shake 5 g with 40 ml of carbon dioxide-free water R for 20 min and centrifuge. The pH of
the supernatant liquid is 5.0. to 7.5.
Ether-soluble substances Prepare a column using 10.0 g in a tube about 20 mm in internal
diameter. Pass 50 ml of peroxide-free ether R through the column. Evaporate the eluate to dryness.
Water-soluble substances Shake 5.0 g with 80 ml of water R for 10 min. Filter with the aid of
vacuum into a tared flask. Evaporate to dryness on a water-bath and dry at 100C to 105C for 1 h.
Starch To 10 g add 90 ml of water R and boil for 5 min. Filter whilst hot. Cool and add to the
filtrate 0.1 ml of 0.05M iodine. No blue colour is produced.
100C to 105C for 3 h.
08-53
3
micro-
organisms per gram and with a limit for fungi of 10
2
per gram, determined by plate-count. It
complies with the tests for Escherichia coli, for Pseudomonas aeruginosa, for Staphylococcus aureus and
for Salmonella (2.6.13).
__________________________________________________________________________________________________________ Ph Eur
08-54
Powdered Cellulose
(C
6
H
10
O
5
)
n
Powdered Cellulose complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Cellulose Powder [0315]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Powdered cellulose is purified, mechanically disintegrated cellulose prepared by processing alpha-
cellulose obtained as a pulp from fibrous plant material.
CHARACTERS
A white or almost white, fine or granular powder, practically insoluble in water, in acetone, in
ethanol, in toluene, in dilute acids and in most organic solvents, slightly soluble in a 50 g/l solution of
sodium hydroxide.
IDENTIFICATION
A. Place about 10 mg on a watch-glass and disperse in 2 ml of iodinated zinc chloride solution R. The
substance becomes violet-blue.
B. Transfer 0.250 g to a 125 ml conical flask. Add 25.0 ml of water R and 25.0 ml of 1M
cupriethylenediamine hydroxide solution. Immediately purge the solution with nitrogen R, insert the
stopper and shake until completely dissolved. Transfer 7.0 ml of the solution to a suitable capillary
viscometer (2.2.9). Equilibrate the solution at 25 0.1C for not less than 5 min. Record the flow
time t
1
, in seconds, between the two marks on the viscometer. Calculate the kinematic viscosity
1
of
the solution using the formula:
t
1
(k
1
),
where k
1
Dilute a suitable volume of 1M cupriethylenediamine hydroxide solution with an equal volume of water R
and measure the flow time t
2
using a suitable capillary viscometer. Calculate the kinematic viscosity
2
of the solvent using the formula:
t
2
(k
2
),
where k
2
Determine the relative viscosity
Rel
of the substance to be examined using the formula:
1
/
2
.
Determine the intrinsic viscosity, []c, by interpolation, using the Intrinsic Viscosity Table.
Calculate the degree of polymerisation, P, using the formula:
95
100 100]
[ ]
[( ) /
c
m b
where m is the mass in grams, of the substance to be examined and b is the loss on drying as
a percentage.
The degree of polymerisation is not less than 440.
08-55
Table 315-1 INTRINSIC VISCOSITY TABLE
Intrinsic viscosity, []c, at Different Values of Relative Viscosity,
rel
[]c
rel
0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09
1.1 0.098 0.106 0.115 0.125 0.134 0.143 0.152 0.161 0.170 0.180
1.2 0.189 0.198 0.207 0.216 0.225 0.233 0.242 0.250 0.259 0.268
1.3 0.276 0.285 0.293 0.302 0.310 0.318 0.326 0.334 0.342 0.350
1.4 0.358 0.367 0.375 0.383 0.391 0.399 0.407 0.414 0.422 0.430
1.5 0.437 0.445 0.453 0.460 0.468 0.476 0.484 0.491 0.499 0.507
1.6 0.515 0.522 0.529 0.536 0.544 0.551 0.558 0.566 0.573 0.480
1.7 0.587 0.595 0.602 0.608 0.615 0.622 0.629 0.636 0.642 0.649
1.8 0.656 0.663 0.670 0.677 0.683 0.690 0.697 0.704 0.710 0.717
1.9 0.723 0.730 0.736 0.743 0.749 0.756 0.762 0.769 0.775 0.782
2.0 0.788 0.795 0.802 0.809 0.815 0.821 0.827 0.833 0.840 0.846
2.1 0.852 0.858 0.864 0.870 0.876 0.882 0.888 0.894 0.900 0.906
2.2 0.912 0.918 0.924 0.929 0.935 0.941 0.948 0.953 0.959 0.965
2.3 0.971 0.976 0.983 0.988 0.994 1.000 1.006 1.011 1.017 1.002
2.4 1.028 1.033 1.039 1.044 1.050 1.056 1.061 1.067 1.072 1.078
2.5 1.083 1.089 1.094 1.100 1.105 1.111 1.116 1.121 1.126 1.131
2.6 1.137 1.142 1.147 1.153 1.158 1.163 1.169 1.174 1.179 1.184
2.7 1.190 1.195 1.200 1.205 1.210 1.215 1.220 1.225 1.230 1.235
2.8 1.240 1.245 1.250 1.255 1.260 1.265 1.270 1.275 1.280 1.285
2.9 1.290 1.295 1.300 1.305 1.310 1.314 1.319 1.324 1.329 1.243
3.0 1.338 1.343 1.348 1.352 1.357 1.362 1.367 1.371 1.376 1.381
3.1 1.386 1.390 1.395 1.400 1.405 1.409 1.414 1.418 1.423 1.427
3.2 1.432 1.436 1.441 1.446 1.450 1.455 1.459 1.464 1.468 1.473
3.3 1.477 1.482 1.486 1.491 1.496 1.500 1.504 1.508 1.513 1.517
3.4 1.521 1.525 1.529 1.533 1.537 1.542 1.546 1.550 1.554 1.558
3.5 1.562 1.566 1.570 1.575 1.579 1.583 1.587 1.591 1.595 1.600
3.6 1.604 1.608 1.612 1.617 1.621 1.625 1.629 1.633 1.637 1.642
3.7 1.646 1.650 1.654 1.658 1.662 1.666 1.671 1.675 1.679 1.683
3.8 1.687 1.691 1.695 1.700 1.704 1.780 1.712 1.715 1.719 1.723
3.9 1.727 1.731 1.735 1.739 1.742 1.746 1.750 1.754 1.758 1.762
4.0 1.765 1.769 1.773 1.777 1.781 1.785 1.789 1.792 1.796 1.800
4.1 1.804 1.808 1.881 1.815 1.819 1.822 1.826 1.830 1.833 1.837
4.2 1.841 1.845 1.848 1.852 1.856 1.859 1.863 1.867 1.870 1.874
4.3 1.878 1.882 1.885 1.889 1.893 1.896 1.900 1.904 1.907 1.911
4.4 1.914 1.918 1.921 1.925 1.929 1.932 1.936 1.939 1.943 1.946
4.5 1.950 1.954 1.957 1.961 1.964 1.968 1.971 1.975 1.979 1.982
4.6 1.986 1.989 1.993 1.996 2.000 2.003 2.007 2.010 2.013 2.017
4.7 2.020 2.023 2.027 2.030 2.033 2.037 2.040 2.043 2.047 2.050
4.8 2.053 2.047 2.060 2.063 2.67 2.070 2.073 2.077 2.080 2.083
4.9 2.087 2.090 2.093 2.097 2.100 2.103 2.107 2.110 2.113 2.116
5.0 2.119 2.122 2.125 2.129 2.132 2.135 2.139 2.142 2.145 2.148
5.1 2.151 2.154 2.158 2.160 2.164 2.167 2.170 2.173 2.176 2.180
5.2 2.183 2.186 2.190 2.192 2.195 2.197 2.200 2.203 2.206 2.209
5.3 2.212 2.215 2.218 2.221 2.224 2.227 2.230 2.233 2.236 2.240
5.4 2.243 2.245 2.249 2.252 2.255 2.258 2.261 2.264 2.267 2.270
5.5 2.273 2.276 2.279 2.282 2.285 2.288 2.291 2.294 2.297 2.300
5.6 2.303 2.306 2.309 2.312 2.315 2.318 2.320 2.324 2.326 2.329
5.7 2.332 2.335 2.338 2.341 2.344 2.347 2.350 2.353 2.355 2.358
5.8 2.361 2.364 2.367 2.370 2.373 2.376 2.379 2.382 2.384 2.387
5.9 2.390 2.393 2.396 2.400 2.403 2.405 2.408 2.411 2.414 2.417
08-56
Table 315-1 (Contd) INTRINSIC VISCOSITY TABLE
Intrinsic viscosity, []c, at Different Values of Relative Viscosity,
rel
[]c
6.0 2.419 2.422 2.425 2.428 2.431 2.433 2.436 2.439 2.442 2.444
6.1 2.447 2.450 2.453 2.456 2.458 2.461 2.464 2.467 2.470 2.472
6.2 2.475 2.478 2.481 2.483 2.486 2.489 2.492 2.494 2.497 2.500
6.3 2.503 2.505 2.508 2.511 2.513 2.516 2.518 2.521 2.524 2.526
6.4 2.529 2.532 2.534 2.537 2.540 2.542 2.545 2.547 2.550 2.553
6.5 2.555 2.558 2.561 2.563 2.566 2.568 2.571 2.574 2.576 2.579
6.6 2.581 2.584 2.587 2.590 2.592 2.595 2.597 2.600 2.603 2.605
67 2.608 2.610 2.613 2.615 2.618 2.620 2.623 2.625 2.627 2.630
6.8 2.633 2.635 2.637 2.640 2.643 2.645 2.648 2.650 2.653 2.655
6.9 2.658 2.660 2.663 2.665 2.668 2.670 2.673 2.675 2.678 2.680
7.0 2.683 2.685 2.687 2.690 2.693 2.695 2.698 2.700 2.702 2.705
7.1 2.707 2.710 2.712 2.714 2.717 2.719 2.721 2.724 2.726 2.729
7.2 2.731 2.733 2.736 2.738 2.740 2.743 2.745 2.748 2.750 2.752
7.3 2.755 2.757 2.760 2.762 2.764 2.767 2.769 2.771 2.774 2.776
7.4 2.779 2.781 2.783 2.786 2.788 2.790 2.793 2.795 2.798 2.800
7.5 2.802 2.805 2.807 2.809 2.812 2.814 2.816 2.819 2.821 2.823
7.6 2.826 2.828 2.830 2.833 2.835 2.837 2.840 2.842 2.844 2.847
7.7 2.849 2.851 2.854 2.856 2.858 2.860 2.863 2.865 2.868 2.870
7.8 2.873 2.875 2.877 2.879 2.881 2.884 2.887 2.889 2.891 2.893
7.9 2.895 2.898 2.900 2.902 2.905 2.907 2.909 2.911 2.913 2.915
8.0 2.918 2.920 2.922 2.924 2.926 2.928 2.931 2.933 2.935 2.937
8.1 2.939 2.942 2.944 2.946 2.948 2.950 2.952 2.955 2.957 2.959
8.2 2.961 2.963 2.966 2.968 2.970 2.972 2.974 2.976 2.979 2.981
8.3 2.983 2.985 2.987 2.990 2.992 2.994 2.996 2.998 3.000 3.002
8.4 3.004 3.006 3.008 3.010 3.012 3.015 3.017 3.019 3.021 3.023
8.5 3.025 3.027 3.029 3.031 3.033 3.035 3.037 3.040 3.042 3.044
8.6 3.046 3.048 3.050 3.052 3.054 3.056 3.058 3.060 3.062 3.064
8.7 3.067 3.069 3.071 3.073 3.075 3.077 3.079 3.081 3.083 3.085
8.8 3.087 3.089 3.092 3.094 3.096 3.098 3.100 3.102 3.104 3.106
8.9 3.108 3.110 3.112 3.114 3.116 3.118 3.120 3.122 3.124 3.126
9.0 3.128 3.130 3.132 3.134 3.136 3.138 3.140 3.142 3.144 3.146
9.1 3.148 3.150 3.152 3.154 3.156 3.158 3.160 3.162 3.164 3.166
9.2 3.168 3.170 3.172 3.174 3.176 3.178 3.180 3.182 3.184 3.186
9.3 3.188 3.190 3.192 3.194 3.196 3.198 3.200 3.202 3.204 3.206
9.4 3.208 3.210 3.212 3.214 3.215 3.217 3.219 3.221 3.223 3.225
9.5 3.227 3.229 3.231 3.233 3.235 3.237 3.239 3.241 3.242 3.244
9.6 3.246 3.248 3.250 3.252 3.254 3.256 3.258 3.260 3.262 3.264
9.7 3.266 3.268 3.269 3.271 3.273 3.275 3.277 3.279 3.281 3.283
9.8 3.285 3.287 3.289 3.291 3.293 3.295 3.297 3.298 3.300 3.302
9.9 3.304 3.305 3.307 3.309 3.311 3.313 3.316 3.318 3.320 3.321
rel
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
10 3.32 3.34 3.36 3.37 3.39 3.41 3.43 3.45 3.46 3.48
11 3.50 3.52 3.53 3.55 3.56 3.58 3.60 3.61 3.63 3.64
12 3.66 3.68 3.69 3.71 3.72 3.74 3.76 3.77 3.79 3.80
13 3.80 3.83 3.85 3.86 3.88 3.89 3.90 3.92 3.93 3.95
14 3.96 3.97 3.99 4.00 4.02 4.03 4.04 4.06 4.07 4.09
15 4.10 4.11 4.13 4.14 4.15 4.17 4.18 4.19 4.20 4.22
16 4.23 4.24 4.25 4.27 4.28 4.29 4.30 4.31 4.33 4.34
17 4.35 4.36 4.37 4.38 4.39 4.41 4.42 4.43 4.44 4.45
18 4.46 4.47 4.48 4.49 4.50 4.52 4.53 4.55 4.45 4.56
19 4.57 4.58 4.59 4.60 4.61 4.62 4.63 4.65 4.65 4.66
08-57
TESTS
Solubility Dissolve 50 mg in 10 ml of ammoniacal solution of copper tetrammine R. It dissolves
completely, leaving no residue.
pH (2.2.3). Mix 10 g with 90 ml of carbon dioxide-free water R and allow to stand with occasional
stirring for 1 h. The pH of the supernatant liquid is 5.0 to 7.5.
Ether-soluble substances Prepare a column using 10.0 g in a tube about 20 mm in internal
diameter. Pass 50 ml of peroxide-free ether R through the column. Evaporate the eluate to dryness.
Water-soluble substances Mix 6.0 g with 90 ml of freshly boiled and cooled water R and allow to
stand for 10 min with occasional stirring. Filter. Discard the first 10 ml of the filtrate and pass the
filtrate through the same filter a second time, if necessary, to obtain a clear filtrate. Evaporate 15.0 ml
of the filtrate in a tared evaporating dish on a water bath to dryness and dry at 100C to 105C for
1 h. The residue weighs not more than 15.0 mg (1.5 per cent).
Starch To 10 g add 90 ml of water R and boil for 5 min. Filter whilst hot. Cool and add to the
filtrate 0.1 ml of 0.05M iodine. No blue colour is produced.
3
micro-
organisms per gram and with a limit for fungi of 10
2
per gram, determined by plate-count. It
complies with the tests for Escherichia coli, for Pseudomonas aeruginosa, for Staphylococcus aureus and
for Salmonella (2.6.13).
__________________________________________________________________________________________________________ Ph Eur
08-58
Cellulose Acetate
Cellulose Acetate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0887].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cellulose acetate is partly or completely O-acetylated cellulose. It contains not less than 29.0 per cent
and not more than 44.8 per cent of acetyl groups (C
2
H
3
O), calculated with reference to the dried
substance. The acetyl content is not less than 90.0 per cent and not more than 110.0 per cent of that
stated on the label, calculated with reference to the dried substance.
CHARACTERS
A white, yellowish-white or greyish-white powder or granules, hygroscopic, practically insoluble in
water and in alcohol, soluble in acetone, in formic acid and in a mixture of equal volumes of
methanol and methylene chloride.
IDENTIFICATION
spectrum of cellulose acetate.
TESTS
Free acid Not more than 0.1 per cent, calculated as acetic acid and with reference to the dried
substance. To 5.00 g in a 250 ml conical flask, add 150 ml of carbon dioxide-free water R, insert the
stopper, swirl the suspension gently and allow to stand for 3 h. Filter, wash the flask and the filter
with carbon dioxide-free water R. Combine the filtrate and washings. Add 0.1 ml of phenolphthalein
solution R1. Titrate with 0.01M sodium hydroxide until a faint pink colour is obtained.
1 ml of 0.01M sodium hydroxide is equivalent to 0.6005 mg of free acid, calculated as acetic acid.
100C to 105C for 3 h.
3
micro-
organisms per gram of which not more than 10
2
fungi per gram determined by plate count. It
complies with the tests for Escherichia coli and Salmonella (2.6.13).
ASSAY
A. For cellulose acetate containing not more than 42.0 per cent of acetyl groups.
To 2.000 g in a 500 ml conical flask add 100 ml of acetone R and 10 ml of water R. Close the flask
and stir with a magnetic stirrer until dissolution is complete. Add 30.0 ml of 1M sodium hydroxide with
constant stirring. Close the flask and stir with a magnetic stirrer for 30 min. Add 100 ml of water R at
80C, washing down the sides of the flask, stir for 2 min and cool to room temperature. Titrate with
0.5M sulphuric acid, using 0.1 ml of phenolphthalein solution R as indicator. Carry out a blank titration.
( )
( ) m d
n n

100
305 . 4
1 2
m=mass of the substance to be examined, in grams,
n
1
=number of millilitres of 0.5M sulphuric acid used in the test,
n
2
=number of millilitres of 0.5M sulphuric acid used in the blank titration.
B. For cellulose acetate containing more than 42.0 per cent of acetyl groups.
To 2.000 g in a 500 ml conical flask add 30 ml of dimethyl sulphoxide R and 100 ml of acetone R.
Close the flask and stir with a magnetic stirrer for 16 h. Add 30.0 ml of 1M sodium hydroxide with
constant stirring. Close the flask and stir with a magnetic stirrer for 6 min. Allow to stand without
stirring for 60 min. Add 100 ml of water R at 80C, washing down the sides of the flask, stir for 2 min
and cool to room temperature. Titrate with 0.5M hydrochloric acid, using 0.1 ml of phenolphthalein
solution R. Add 0.5 ml of 0.5M hydrochloric acid in excess, stir for 5 min and allow to stand for 30 min.
Titrate with 0.5M sodium hydroxide, until a persistent pink colour is obtained, stirring with a magnetic
stirrer. Calculate the net number of milliequivalents of sodium hydroxide consumed, taking the mean
of two blank titrations into consideration.
08-59
( ) m d
n

100
305 . 4
m=mass of the substance to be examined, in grams,
n=net number of milliequivalents of sodium hydroxide.
STORAGE
LABELLING
The label states the nominal percentage content of acetyl groups.
__________________________________________________________________________________________________________ Ph Eur
08-60
Cellulose Acetate Butyrate
Cellulose Acetate Butyrate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cellulose acetate butyrate is partly or completely O-acetylated and O-butyrated cellulose. It contains
not less than 2.0 per cent and not more than 30.0 per cent of acetyl groups (C
2
H
3
O) and not less
than 16.0 per cent and not more than 53.0 per cent of butyryl groups (C
4
H
7
O) calculated with
reference to the dried substance. Both the acetyl content and the butyryl content are not less than
90.0 per cent and not more than 110.0 per cent of those stated on the label, calculated with reference
CHARACTERS
A white, yellowish-white or greyish-white powder or granules, slightly hygroscopic, practically
insoluble in water and in alcohol, soluble in acetone, in formic acid and in a mixture of equal
volumes of methanol and methylene chloride.
IDENTIFICATION
spectrum of cellulose acetate butyrate. The intensities of the bands may vary according to the degree of
substitution.
B. It complies with the limits of the assay.
TESTS
Acidity To 5.00 g in a 250 ml conical flask, add 150 ml of carbon dioxide-free water R, insert the
stopper, swirl the suspension gently and allow to stand for 3 h. Filter, wash the flask and the filter
with carbon dioxide-free water R. Combine the filtrate and washings. Add 0.1 ml of phenolphthalein
solution R1. Not more than 3.0 ml of 0.01M sodium hydroxide is required to change the colour of the
indicator.
100C to 105C for 3 h.
ASSAY
Test solution. To 1.000 g in a 500 ml conical flask, add 100 ml of acetone R and 10 ml of water R.
Close the flask and stir with a magnetic stirrer until dissolution is complete. Add 30.0 ml of 1M
sodium hydroxide with constant stirring. Close the flask and stir with a magnetic stirrer for 30 min.
Add 100 ml of hot water R at 80C, washing down the sides of the flask and stir for 2 min. Cool,
centrifuge or filter the suspension and wash the residue with water R. Combine the filtrate and
washings, adjust the pH to 3 (2.2.3) using dilute phosphoric acid R and dilute to 500.0 ml with
water R.
Reference solution. Dissolve 0.200 g of glacial acetic acid R and 0.400 g of butyric acid R in water R,
adjust the pH to 3 (2.2.3) using dilute phosphoric acid R and dilute to 500.0 ml with water R.
volumes of phosphate buffer solution pH 3.0 R1, changing after 30 min, by linear gradient elution
over 5 min, to a mixture of 20 volumes of methanol R and 80 volumes of phosphate buffer solution
pH 3.0 R1 and eluting for a further 25 min. Restore the initial solvent mixture for 1 min,
Inject successively 20 l of the reference solution and 20 l of the test solution.
Calculate the percentage contents of acetic acid and butyric acid in the substance to be examined
from the peak areas in the chromatograms obtained with the two solutions.
To calculate the percentage contents of acetyl (C
2
H
3
O) and of butyryl (C
4
H
7
O) groups, multiply
the percentage contents of acetic acid and of butyric acid by 0.717 and 0.807, respectively.
08-61
STORAGE
Store in an air-tight container.
LABELLING
The label states the nominal percentage content of acetyl and butyryl groups.
__________________________________________________________________________________________________________ Ph Eur
08-62
Centaury
Centaury complies with the requirements of the 3rd edition of the European Pharmacopoeia [1301]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Centaury consists of the whole or cut dried flowering aerial parts of Centaurium erythraea Rafn. (C.
minus Moench, C. umbellatum Gilib., Erythraea centaurium (L.) Pers.).
CHARACTERS
Centaury has a very bitter taste.
IDENTIFICATION
A. The hollow cylindrical, light green to dark brown stem has longitudinal ridges, and is branched
only in its upper part. The sessile leaves are entire, decussately arranged, and have an ovate to
lanceolate lamina, up to about 3 cm long. Both surfaces are glabrous and green to brownish-green.
The inflorescence is diaxially branched. The tubular calyx is green and has five lanceolate, acuminate
teeth. The corolla consists of a whitish tube divided in five elongated lanceolate pink lobes, about
5 mm to 8 mm long. Five stamens are present attached to the top of the corolla tube. The ovary is
superior and has a short style, a broad bifid stigma and numerous ovules. Cylindrical capsules, about
7 mm to 10 mm long, with small brown markedly rough seeds are frequently present.
B. Reduce to a powder (355). The powder is greenish-yellow to brownish. Examine under a
microscope, using chloral hydrate solution R. The powder shows numerous fragments of stem contain-
ing sclerenchymatous fibres and narrow vessels with bordered pits or spiral of reticulate thickening, as
well as rectangular pitted cells of the pith and medullary rays; fragments of leaves with sinuous
epidermal cells and striated cuticle, especially over the margins and surrounding the stomata,
anisocytic stomata (2.8.3) and cells of the mesophyll with crystals of calcium oxalate of various types;
fragments of calyx and corolla, those of the calyx with straight-walled epidermal cells, epidermis of
the corolla with obtuse papillae and radially striated cuticle; parts of the endothecium with reticulate
or ridge-shaped wall thickenings; triangularly rounded to elliptical, yellow pollen grains, about 30 m
in diameter, with a finely grained exine and three germinal pores; fragments of the wall of the fruit
capsule composed of crossed layers of fusiform cells; small yellowish-brown seeds with a raised
reticulate, dark brown structure formed by the coarse lateral walls of their epidermis.
254
plate R.
Test solution. To 1.0 g of the powdered drug (355) add 20 ml of methanol R and heat under a reflux
condenser for 10 min. Cool and filter.
Reference solution. Dissolve 10 mg of rutin R in 10 ml of methanol R.
a path of 12 cm using a mixture of 16 volumes of anhydrous acetic acid R, 16 volumes of water R and
68 volumes of ethyl acetate R. Allow the plate to dry in a current of cold air and develop again in the
same manner. Allow the plate to dry in a current of cold air. Examine the plate in ultraviolet light at
254 nm. The chromatogram obtained with the test solution shows as the main quenching zone a
strong zone (swertiamarin) similar in position to the quenching zone of rutin in the chromatogram
obtained with the reference solution; further weaker quenching zones may also be present. Spray the
plate with anisaldehyde solution R. Examine in daylight whilst heating at 100C to 105C for 5 min to
10 min. The chromatogram obtained with the reference solution shows a yellowish-brown zone
(rutin). The chromatogram obtained with the test solution shows: a violet-brown zone
(swertiamarin); a little above this zone, a faint yellwish-brown zone; and above this a few, very faint,
mostly grey zones leading to a reddish-violet zone at the solvent front. Examine the plate in ultra-
violet light at 365 nm. The chromatogram obtained with the test solution shows: a strong brown to
brownish-yellow fluorescent zone (swertiamarin); a little above this zone, a blue to yellowish-green
fluorescent zone; and above this a few faint blue to yellow fluorescent zones, leading to a faint
reddish-violet fluorescent zone at the solvent front. Below the zone due to swertiamarin occur zones
of intense light green to yellowish-green fluorescence as well as a number of faint brown fluorescent
zones.
TESTS
Bitterness value Not less than 2000. The bitterness value is determined by comparison with
quinine hydrochloride, the bitterness value of which is set at 200,000; the bitterness value is the
reciprocal of the dilution that still has a bitter taste.
08-63
Quinine hydrochloride stock solution. Dissolve 0.100 g of quinine hydrochloride R in water R and dilute to
100.0 ml with the same solvent. Dilute 1.0 ml of the solution to 100.0 ml with water R.
Centaury extract. To 1.0 g of powdered drug (355) add 1000 ml of boiling water R. Heat on a water-
bath for 30 min, stirring continuously. Allow to cool and dilute to 1000 ml with water R. Shake
vigorously and filter, discarding the first 20 ml of filtrate.
Prepare a series of dilutions by placing in the first tube 4.2 ml of quinine hydrochloride stock
solution and increasing the volume by 0.2 ml in each subsequent tube to a total of 5.8 ml; dilute the
contents of each tube to 10.0 ml with water R. Determine as follows the dilution with the lowest
concentration that still has a bitter taste. Take 10.0 ml of the weakest solution into the mouth and
pass it from side to side over the back of the tongue for 30 s. If the solution is not found to be bitter,
spit it out and wait for 1 min. Rinse the mouth with water R. After 10 min, use the next dilution in
order of increasing concentration.
Calculate the correction factor k from the expression:
n
00 . 5
n=number of millilitres of quinine hydrochloride stock solution in the dilution of lowest
concentration that is bitter.
Dilute 10/k ml of the centaury extract to 20.0 ml with water R. 10.0 ml of this dilution has a bitter
taste.
(355) by drying in an oven at 100 C to 105 C for 2 h.
STORAGE
Store in well-closed container, protected from light.
__________________________________________________________________________________________________________ Ph Eur
08-64
Centella
1/01
Centella complies with the requirements of the 3rd edition of the European Pharmacopoeia [1498]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Centella consists of the dried, fragmented aerial parts of Centella asiatica (L.) Urban. It contains not
less than 6.0 per cent of total triterpenoid derivatives, expressed as asiaticoside (C
48
H
78
O
19
; M
r
CHARACTERS
The leaves are very variable in size; the petiole is usually 5 to 10, sometimes 15, times longer than the
lamina, which is 10 mm to 40 mm long and 20 mm to 40 mm, sometimes up to 70 mm, wide.
IDENTIFICATION
A. The leaves are alternate, sometimes grouped together at the nodes, reniform or orbicular or
oblong-elliptic and have palmate nervation, usually with seven veins, and a crenate margin. Young
leaves show a few trichomes on the lower surface while adult leaves are glabrous. The inflorescence, if
present, is a single umbel which usually consists of three flowers, rarely two or four; the flowers are
very small (about 2 mm) pentamerous and have an inferior ovary; the fruit, a brownish-grey,
orbicular cremocarp, up to 5 mm long, is very flattened laterally and has seven to nine prominent
curved ridges.
B. Reduce the drug to a powder (355). The powder is greenish-grey. Examine under a microscope
using chloral hydrate solution R. The powder shows numerous fragments of leaf epidermis with
polygonal cells having an irregularly striated cuticle, and paracytic stomata (2.8.3) that are more
numerous in the lower epidermis; fragments of petiole epidermis with elongated cells; uniseriate,
long, flexuous unicellular covering trichomes, occasionally multicellular; young leaves; spiral vessels;
resiniferous canals; calcium oxalate prisms and macles up to 40 m in diameter; bundles of narrow
septate fibres from the stem; fragments of the fruit: layers of wide cells in a parquetry arrangement,
annular vessels, parenchyma cells containing simple or compound starch granules.
Test solution. To 5.0 g of the powdered drug (355) add 50 ml of alcohol (30 per cent V/V) R; heat to
boiling under a reflux condenser and centrifuge.
Reference solution. Dissolve 5 mg of asiaticoside R in methanol R and dilute to 10 ml with the same
solvent.
11 volumes of acetic acid R, 11 volumes of formic acid R, 27 volumes of water R and 100 volumes of
ethyl acetate R. Allow the plate to dry in air. Spray with anisaldehyde solution R and heat at 100C to
105C. Examine in daylight. The chromatograms obtained with the reference and the test solutions
show in the lower third a greenish-blue zone (asiaticoside). The chromatogram obtained with the test
solution shows also below this zone a violet zone (madecassoside); near the solvent front it shows a
light blue zone (asiatic acid) and just below a pinkish-violet zone (madecassic acid); in the lower half
it shows brown, grey and brownish-green zones between the starting point and the zone due to
madecassoside, and other brownish-yellow or light yellow zones above the zone due to asiaticoside.
TESTS
Foreign matter (2.8.2). Not more than 7 per cent of which not more than 5 per cent of
underground organs and not more than 2 per cent of other foreign matter.
by drying in an oven at 100C to 105C for 2 h.
ASSAY
Test solution. Place 5.0 g of the powdered drug (355) in a cellulose fingerstall in a continuous
extraction apparatus (Soxhlet type). Add 100 ml of methanol R and heat for 8 h. Cool and dilute the
extract to 100.0 ml with methanol R. Filter through a 0.45 m filter. Dilute 2.0 ml of the filtrate and
dilute to 20.0 ml with methanol R.
08-65
Reference solution. Dissolve 20.0 mg of asiaticoside R in methanol R, if necessary using sonication, and
dilute to 20.0 ml with the same solvent. Dilute 2.0 ml of the solution to 100.0 ml with methanol R.
Mobile phase A. Acetonitrile for chromatography R,
Mobile phase B. Dilute 3 ml of phosphoric acid R to 1000 ml with water R,
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
0 65 22 78
65 66 55 45
66 76 95 5
76 85 22 78
Calculate the response factor of asiaticoside from the expression:
p 1
1 1
100
HPLC m
V A
R
f
A
1
=area of the peak corresponding to asiaticoside in the chromatogram obtained with the
reference solution,
V
1
=volume of the reference solution, in millilitres,
m
1
=mass of asiaticoside in the reference solution, in milligrams,
HPLC
P
=purity determined for asiaticoside.
Calculate the mean response factor for asiaticoside from the expression:
N
R
R
N
i
fi
f
N
i
fi
R
1
=sum of response factors of asiaticoside for the chromatograms obtained with the reference
solution,
N=number of injections of reference solution (N = 4, at least).
Calculate the percentage content of total triterpenoid derivatives, expressed as asiaticoside from the
expression:
1
1
]
1
+ + +
f
R
D C B A
m
V ) 509 . 0 ( ) 526 . 0 ( ) 017 . 1 (
V=volume of the test solution, in millilitres,
m=mass of the substance to be examined in the test solution, in milligrams,
A=area of the peak corresponding to asiaticoside in the chromatogram with the test solution,
B=area of the peak corresponding to madecassoside in the chromatogram with the test
solution,
C=area of the peak corresponding to madecassic acid in the chromatogram with the test
solution,
D=area of the peak corresponding to asiatic acid in the chromatogram with the test solution,
f
R =mean response factor of asiaticoside.
Relative retention with reference to the solvent: madecassoside = about 5.8; asiaticoside = about 8.1;
madecassic acid = about 17.6; asiatic acid = about 21.7.
STORAGE
08-66
1
2
3
4
5
1 : solvent
2 : madecassoside
3 : asiaticoside
4 : madecassic acid
5 : asiatic acid
Fig. 14981 Chromatogram of Centella
__________________________________________________________________________________________________________ Ph Eur
08-67
Cetirizine Hydrochloride
N
N
O COOH Cl
H
and enantiomer
,2HCl
C
21
H
27
Cl
3
N
2
O
3
461.8 83881-52-1
Cetirizine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Cetirizine Dihydrochloride [1084]. These requirements are reproduced after the heading Definition below.
1
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cetirizine dihydrochloride contains not less than 99.0 per cent and not more than the equivalent of
100.5 per cent of (RS)-2-[2-[4-[(4-chlorophenyl)phenylmethyl]piperazin- 1-yl]ethoxy]acetic acid
dihydrochloride, calculated with reference to the dried substance.
CHARACTERS
A white or almost white powder, freely soluble in water, practically insoluble in acetone and in
methylene chloride.
IDENTIFICATION
A. Dissolve 20.0 mg in 50 ml of 0.1M hydrochloric acid and dilute to 100.0 ml with the same acid.
Dilute 10.0 ml of the solution to 100.0 ml with 0.1M hydrochloric acid. Examined between 210 nm
and 350 nm (2.2.25), the solution shows an absorption maximum at 231 nm. The specific
obtained with cetirizine dihydrochloride CRS. Examine the substances as discs.
254
same solvent.
Reference solution (a). Dissolve 10 mg of cetirizine dihydrochloride CRS in water R and dilute to 5 ml
Reference solution (b). Dissolve 10 mg of chlorphenamine maleate CRS in water R and dilute to 5 ml
with the same solvent. To 1 ml of the solution add 1 ml of reference solution (a).
Apply separately to the plate 5 l of each solution. Develop over a path of 15 cm, using a mixture of 1
volume of ammonia R, 10 volumes of methanol R and 90 volumes of methylene chloride R. Allow the
plate to dry in a current of cold air. Examine in ultraviolet light at 254 nm. The principal spot in the
chromatogram obtained with the test solution is similar in position and size to that in the chromato-
TESTS
solution BY
7
(Method II, 2.2.2).
08-68
Reference solution (a). Dissolve 5.0 mg of cetirizine dihydrochloride CRS and 5.0 mg of cetirizine impurity
A CRS in the mobile phase and dilute to 25.0 ml with the mobile phase. Dilute 1.0 ml of the solution
The chromatography may be carried out using:
a stainless steel column, 0.25 m long and 4.6 mm in internal diameter, packed with silica gel for
as mobile phase, at a flow rate of 1 ml per minute, a mixture of 0.4 volumes of dilute sulphuric
acid R, 6.6 volumes of water R and 93 volumes of acetonitrile R,
peaks in the chromatogram obtained is about 50 per cent of the full scale of the recorder. The test is
not valid unless the resolution between the first peak (cetirizine) and the second peak (cetirizine
impurity A) is at least three and the symmetry factors are at most 2.0. Inject separately 20 l of the
test solution and 20 l of reference solution (b). Continue the chromatography for at least three times
the retention time of the peak corresponding to cetirizine. In the chromatogram obtained with the
test solution, the area of any peak apart from the principal peak is not greater than that of the peak in
the chromatogram obtained with reference solution (b) (0.2 per cent) and the sum of the areas of all
the peaks apart from the principal peak is not greater than 1.5 times that of the peak in the chromato-
gram obtained with reference solution (b) (0.3 per cent). Disregard any peak with an area less than
0.1 times that of the peak in the chromatogram obtained with reference solution (b).
the solution complies with limit test A of heavy metals (10 ppm). Prepare the standard using 1 ml of
Loss on drying (2.2.32). Not more than 0.5 per cent determined on 1.000 g by drying in an oven at
100C to 105C.
ASSAY
Dissolve 0.100 g in 70 ml of a mixture of 30 volumes of water R and 70 volumes of acetone R. Titrate
with 0.1M sodium hydroxide up to the second point of inflexion. Determine the end point potentio-
metrically (2.2.20). Carry out a blank titration.
21
H
27
Cl
3
N
2
O
3
.
STORAGE
IMPURITIES
N
NH
Cl
A. (RS)-1-[(4-chlorophenyl)phenylmethyl]piperazine,
N
N COOH
Cl
B. (RS)-2-[4-[(4-chlorophenyl)phenylmethyl]piperazin-1-yl]acetic acid,
N
N
Cl O COOH
C. (RS)-2-[2-[4-[(2-chlorophenyl)phenylmethyl]piperazin-1-yl]ethoxy]acetic acid,
08-69
Cl
N
N
Cl
D. bis[(4-chlorophenyl)phenylmethyl]piperazine,
N
N
Cl O
O COOH
E. (RS)-2-[2-[2-[4-[(4-chlorophenyl)phenylmethyl]piperazin-1-yl]ethoxy]acetic acid
ethoxycetirizine,
N
N
O COOH
F. (RS)-2-[2-[4-(diphenylmethyl)piperazin-1-yl]ethoxy]acetic acid.
__________________________________________________________________________________________________________ Ph Eur
08-70
Cetostearyl Alcohol
corrected 1/01
Cetostearyl Alcohol complies with the requirements of the 3rd edition of the European Pharmacopoeia [0702].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cetostearyl alcohol is a mixture of solid aliphatic alcohols. It contains not less than 40.0 per cent of
stearyl alcohol (C
18
H
38
O; 270.5) and the sum of the contents of cetyl alcohol (C
16
H
34
O; 242.4) and
of stearyl alcohol is not less than 90.0 per cent.
CHARACTERS
White or pale yellow, wax-like mass, plates, flakes or granules, practically insoluble in water, freely
soluble in ether, soluble in alcohol (90 per cent V/V) and in light petroleum; when melted, it is
miscible with fatty oils, with liquid paraffin and with melted wool fat.
IDENTIFICATION
Examine the chromatograms obtained in the assay. The two principal peaks in the chromatogram
obtained with the test solution have similar retention times to the principal peaks in the chromato-
grams obtained with reference solution (a) and reference solution (b), respectively.
TESTS
Appearance of solution Dissolve 0.50 g in 20 ml of boiling alcohol R. The solution is clear (2.2.1)
and not more intensely coloured than reference solution B
6
(Method II, 2.2.2).
Melting point (2.2.14). 49C to 56C.
Acid value (2.5.1). Not more than 1.0.
Hydroxyl value (2.5.3, Method A). 208 to 228.
Iodine value (2.5.4). Not more than 2.0, determined on 2.00 g, dissolved in 25 ml of chloroform R.
Saponification value (2.5.6). Not more than 2.0, determined on 2.00 g.
ASSAY
Examine by gas chromatography (2.2.28).
Test solution. Dissolve 0.100 g of the substance to be examined in ethanol R and dilute to 10.0 ml with
the same solvent.
Reference solution (a). Dissolve 60.0 mg of cetyl alcohol CRS in ethanol R and dilute to 10.0 ml with the
same solvent.
Reference solution (b). Dissolve 40.0 mg of stearyl alcohol CRS in ethanol R and dilute to 10.0 ml with
the same solvent.
Reference solution (c). Mix 1 ml of reference solution (a) and 1 ml of reference solution (b) and dilute
to 10.0 ml with ethanol R.
a stainless steel column 3 m long and 4 mm in internal diameter packed with diatomaceous earth
for gas chromatography R impregnated with 10 per cent m/m of poly(dimethyl)siloxane R,
at 250C.
Inject separately 2 l of each solution. Adjust the flow rate so that the resolution between the two
principal peaks in the chromatogram obtained with the test solution is not less than 1.25. The test is
not valid unless the chromatogram obtained with reference solution (c) shows two principal peaks
with a signal-to-noise ratio of at least five. Determine the content of cetyl alcohol and of stearyl
alcohol from the chromatogram obtained with the test solution, using the normalisation procedure
and identifying the peaks by comparison with the chromatograms obtained with reference solution
(a) and reference solution (b), respectively.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
08-71
Emulsifying Cetostearyl Alcohol (Type A)
Emulsifying Cetostearyl Alcohol (Type A) complies with the requirements of the 3rd edition of the European
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Emulsifying cetostearyl alcohol (type A) is a mixture which contains not less than 80.0 per cent of
cetostearyl alcohol and not less than 7.0 per cent of sodium cetostearyl sulphate, both calculated with
reference to the anhydrous substance. A suitable buffer may be added.
CHARACTERS
White or pale yellow, wax-like mass, plates, flakes or granules, soluble in hot water giving an
opalescent solution, practically insoluble in cold water, slightly soluble in alcohol.
IDENTIFICATION
First identification: B, C, D.
A. Examine by thin-layer chromatography (2.2.27), using a TLC silanised silica gel plate R.
Test solution (a). Dissolve 0.1 g of the substance to be examined in 10 ml of trimethylpentane R,
heating on a water-bath. Shake with 2 ml of alcohol (70 per cent V/V) R and allow to separate. Use the
lower layer as test solution (b). Dilute 1 ml of the upper layer to 8 ml with trimethylpentane R.
Test solution (b). Use the lower layer obtained in the preparation of test solution (a).
Reference solution (a). Dissolve 40 mg of cetostearyl alcohol R in 10 ml of trimethylpentane R.
Reference solution (b). Dissolve 20 mg of sodium cetostearyl sulphate R in 10 ml of alcohol (70 per cent
V/V) R, heating on a water-bath.
of water R, 40 volumes of acetone R and 40 volumes of methanol R. Allow the plate to dry in air and
spray with a 50 g/l solution of phosphomolybdic acid R in alcohol R. Heat at 120C until the appearance
of the spots (about 3 h). The two principal spots in the chromatogram obtained with test solution (a)
are similar in position and colour to the principal spots in the chromatogram obtained with reference
solution (a). Two of the spots in the chromatogram obtained with test solution (b) are similar in
position and colour to the principal spots in the chromatogram obtained with reference solution (b).
B. Examine the chromatograms obtained in the assay. The retention times of the two principal peaks
in the chromatogram obtained with test solution (b) are similar to those of the two principal peaks in
C. It gives a yellow colour to a non-luminous flame.
D. To 0.3 g add 20 ml of ethanol R and heat on a water-bath with shaking. Filter the mixture
immediately, evaporate to dryness and take up the residue in 7 ml of water R. To 1 ml of the solution
add 0.1 ml of a 1 g/l solution of methylene blue R, 2 ml of methylene chloride R and 2 ml of dilute
sulphuric acid R and shake. A blue colour develops in the lower layer.
TESTS
Iodine value (2.5.4). Not more than 3.0, determined on 2.00 g dissolved in 25 ml of methylene
chloride R.
of water.
ASSAY
Cetostearyl alcohol
Internal standard solution. Dissolve 0.60 g of heptadecanol CRS in ethanol R and dilute to 150 ml with
the same solvent.
Test solution (a). Dissolve 0.300 g of the substance to be examined in 50 ml of the internal standard
solution, add 50 ml of water R and shake with four quantities, each of 25 ml, of pentane R, adding
sodium chloride R, if necessary, to facilitate the separation of the layers. Combine the organic layers.
Wash with two quantities, each of 30 ml, of water R, dry over anhydrous sodium sulphate R and filter.
Test solution (b). Dissolve 0.300 g of the substance to be examined in 50 ml of ethanol R, add 50 ml of
08-72
water R and shake with four quantities, each of 25 ml, of pentane R, adding sodium chloride R, if
necessary, to facilitate the separation of the layers. Combine the organic layers. Wash with two
quantities, each of 30 ml, of water R, dry over anhydrous sodium sulphate R and filter.
Reference solution. Dissolve 50 mg of cetyl alcohol CRS and 50 mg of stearyl alcohol CRS in ethanol R
a fused-silica column 25 m long and 0.25 mm in internal diameter coated with
poly(dimethyl)siloxane R,
Time
(min)
Temperature
(C)
Rate
(C/min)
Comment
Column 020 150250 5 Linear gradient
Injection
port
250
detector 250
The substances are eluted in the following order: cetyl alcohol, heptadecanol (internal standard) and
stearyl alcohol.
Inject 1 l of test solution (a) and 1 l of test solution (b). If the chromatogram obtained with test
solution (b) shows a peak with the same retention time as the peak corresponding to the internal
standard in the chromatogram obtained with test solution (a), calculate the ratio:
i
ci
S
S
r
S
ci
=area of the peak corresponding to cetyl alcohol in the chromatogram obtained with
solution (b),
S
i
=area of the peak with the same rention time as the peak corresponding to the internal
standard in the chromatogram obtained with test solution (a).
If r is less than 300, calculate the corrected area S
Ha(corr)
of the peak corresponding to the internal
standard in the chromatogram obtained with test solution (a):
ci
c i
Ha Ha(corr)
S
S S
S S

S
Ha
=area of the peak corresponding to the internal standard in the chromatogram obtained
with test solution (a),
S
c
=area of the peak corresponding to cetyl acohol in the chromatogram obtained with test
solution (a).
Inject, under the same conditions, equal volumes of the reference solution and of test solution (a).
Identify the peaks in the chromatogram obtained with test solution (a) by comparing their retention
times with those of the peaks in the chromatogram obtained with the reference solution and determine
the area of each peak.
Calculate the percentage content of cetyl alcohol using the expression:
m S
m
S
Ha(corr)
H
A
100
S
A
solution (a).
m
H
=mass of the internal standard in test solution (a) in milligrams,
S
Ha(corr)
=corrected area of the peak corresponding to the internal standard in the chromatogram
obtained with test solution (a),
m=mass of the substance to be examined in test solution (a) in milligrams.
Calculate the percentage content of stearyl alcohol using the expression:
m S
m
S
Ha(corr)
H
B
100
S
B
=area of the peak corresponding to stearyl acohol in the chromatogram obtained with test
solution (a).
The percentage content of cetostearyl alcohol corresponds to the sum of the percentage content of
cetyl alcohol and of stearyl alcohol.
08-73
Sodium cetostearyl sulphate
Disperse 0.300 g in 25 ml of methylene chloride R. Add 50 ml of water R and 10 ml of dimidium
bromide-sulphan blue mixed solution R. Titrate with 0.004M benzethonium chloride, using sonication and
heating and allowing the layers to separate before each addition, until the colour of the lower layer
changes from pink to grey.
1 ml of 0.004M benzethonium chloride is equivalent to 1.434 mg of sodium cetostearyl sulphate.
LABELLING
The label states, where applicable, the name and concentration of any added buffer.
__________________________________________________________________________________________________________ Ph Eur
08-74
Emulsifying Cetostearyl Alcohol (Type B)
Emulsifying Cetostearyl Alcohol (Type B)complies with the requirements of the 3rd edition of the European
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Emulsifying cetostearyl alcohol (type B) is a mixture which contains not less than 80.0 per cent of
cetostearyl alcohol and not less than 7.0 per cent of sodium laurilsulfate, both calculated with
reference to the anhydrous substance. A suitable buffer may be added.
CHARACTERS
White or pale yellow, wax-like mass, plates, flakes or granules, soluble in hot water giving an
opalescent solution, practically insoluble in cold water, slightly soluble in alcohol.
IDENTIFICATION
A. Examine by thin-layer chromatography (2.2.27), using a TLC silanised silica gel plate R.
Test solution (a). Dissolve 0.1 g of the substance to be examined in 10 ml of trimethylpentane R,
heating on a water-bath. Shake with 2 ml of alcohol (70 per cent V/V) R and allow to separate. Use the
lower layer as test solution (b). Dilute 1 ml of the upper layer to 8 ml with trimethylpentane R.
Test solution (b). Use the lower layer obtained in the preparation of test solution (a).
Reference solution (a). Dissolve 40 mg of cetostearyl alcohol R in 10 ml of trimethylpentane R.
Reference solution (b). Dissolve 20 mg of sodium laurilsulfate R in 10 ml of alcohol (70 per cent V/V) R,
heating on a water-bath.
of water R, 40 volumes of acetone R and 40 volumes of methanol R. Allow the plate to dry in air and
spray with a 50 g/l solution of phosphomolybdic acid R in alcohol R. Heat at 120C until the appearance
of the spots (about 3 h). The two principal spots in the chromatogram obtained with test solution (a)
are similar in position and colour to the principal spots in the chromatogram obtained with reference
solution (a). One of the spots in the chromatogram obtained with test solution (b) is similar in
position and colour to the principal spot in the chromatogram obtained with reference solution (b).
B. Examine the chromatograms obtained in the assay. The retention times of the two principal peaks
in the chromatogram obtained with test solution (b) are similar to those of the two principal peaks in
C. It gives a yellow colour to a non-luminous flame.
D. To 0.3 g add 20 ml of ethanol R and heat to boiling on a water-bath with shaking. Filter the
mixture immediately, evaporate to dryness and take up the residue in 7 ml of water R. To 1 ml of the
solution add 0.1 ml of a 1 g/l solution of methylene blue R, 2 ml of methylene chloride R and 2 ml of
dilute sulphuric acid R and shake. A blue colour develops in the lower layer.
TESTS
Iodine value (2.5.4). Not more than 3.0, determined on 2.00 g dissolved in 25 ml of methylene
chloride R.
of water.
ASSAY
Cetostearyl alcohol
Internal standard solution. Dissolve 0.60 g of heptadecanol CRS in ethanol R and dilute to 150 ml with
the same solvent.
Test solution (a). Dissolve 0.300 g of the substance to be examined in 50 ml of the internal standard
solution, add 50 ml of water R and shake with four quantities, each of 25 ml, of pentane R, adding
sodium chloride R, if necessary, to facilitate the separation of the layers. Combine the organic layers.
Wash with two quantities, each of 30 ml, of water R, dry over anhydrous sodium sulphate R and filter.
08-75
Test solution (b). Dissolve 0.300 g of the substance to be examined in 50 ml of ethanol R, add 50 ml of
water R and shake with four quantities, each of 25 ml, of pentane R, adding sodium chloride R, if
necessary, to facilitate the separation of the layers. Combine the organic layers. Wash with two
quantities, each of 30 ml, of water R, dry over anhydrous sodium sulphate R and filter.
Reference solution. Dissolve 50 mg of cetyl alcohol CRS and 50 mg of stearyl alcohol CRS in ethanol R
polydimethylsiloxane R,
Time
(min)
Temperature
(C)
Rate
(C/min)
Comment
Column 020 150250 5 Linear gradient
Injection
port
250
detector 250
The substances are eluted in the following order: cetyl alcohol, heptadecanol (internal standard) and
stearyl alcohol.
Inject 1 l of test solution (a) and 1 l of test solution (b). If the chromatogram obtained with test
solution (b) shows a peak with the same retention time as the peak corresponding to the internal
standard in the chromatogram obtained with test solution (a), calculate the ratio:
i
ci
S
S
r
S
ci
=area of the peak corresponding to cetyl alcohol in the chromatogram obtained with
solution (b),
S
i
=area of the peak with the same rention time as the peak corresponding to the internal
standard in the chromatogram obtained with test solution (a).
If r is less than 300, calculate the corrected area S
Ha(corr)
of the peak corresponding to the internal
standard in the chromatogram obtained with test solution (a):
ci
c i
Ha Ha(corr)
S
S S
S S

S
Ha
=area of the peak corresponding to the internal standard in the chromatogram obtained
with test solution (a),
S
c
solution (a).
Inject, under the same conditions, equal volumes of the reference solution and of test solution (a).
Identify the peaks in the chromatogram obtained with test solution (a) by comparing their retention
times with those of the peaks in the chromatogram obtained with the reference solution and deter-
mine the area of each peak.
Calculate the percentage content of cetyl alcohol using the expression:
m S
m
S
Ha(corr)
H
A
100
S
A
solution (a).
m
H
=mass of the internal standard in test solution (a) in milligrams,
S
Ha(corr)
=corrected area of the peak corresponding to the internal standard in the chromatogram
m=mass of the substance to be examined in test solution (a) in milligrams.
Calculate the percentage content of stearyl alcohol using the expression:
m S
m
S
Ha(corr)
H
B
100
S
B
=area of the peak corresponding to stearyl acohol in the chromatogram obtained with test
solution (a).
The percentage content of cetostearyl alcohol corresponds to the sum of the percentage content of
cetyl alcohol and of stearyl alcohol.
08-76
Sodium laurilsulfate
Disperse 0.300 g in 25 ml of methylene chloride R. Add 50 ml of water R and 10 ml of dimidium
bromide-sulphan blue mixed solution R. Titrate with 0.004M benzethonium chloride, using sonication and
heating, and allowing the layers to separate before each addition, until the colour of the lower layer
changes from pink to grey.
1 ml of 0.004M benzethonium chloride is equivalent to 1.154 mg of sodium laurilsulfate.
LABELLING
The label states, where applicable, the name and concentration of any added buffer.
__________________________________________________________________________________________________________ Ph Eur
08-77
Cetostearyl Isononanoate
Cetostearyl Isononanoate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cetostearyl isononanoate is a mixture of esters of cetostearyl alcohol with isononanoic acid, mainly
3,5,5-trimethylhexanoic acid.
CHARACTERS
A clear, colourless or slightly yellowish liquid, practically insoluble in water, soluble in alcohol and in
light petroleum, miscible with fatty oils and with liquid paraffins.
It has a viscosity of 15 mPa.s to 30 mPa.s, a relative density of 0.85 to 0.86 and a refractive index
of 1.44 to 1.45.
IDENTIFICATION
A. On cooling, turbidity occurs below 15C.
spectrum of cetostearyl isononanoate.
TESTS
Appearance. The substance to be examined is clear (2.2.1) and not more intensely coloured than
6
(Method I, 2.2.2).
Hydroxyl value (2.5.3). Not more than 5.0 (Method A).
Saponification value (2.5.6). 135 to 148, determined on 1.0 g.
of water.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
08-78
Cetrimide
8044-71-1
Cetrimide complies with the requirements of the 3rd edition of the European Pharmacopoeia [0378]. These
Preparations
Cetrimide Cream
Cetrimide Emulsifying Ointment
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cetrimide consists of trimethyltetradecylammonium bromide and may contain smaller amounts of
dodecyl- and hexadecyl-trimethylammonium bromides. It contains not less than 96.0 per cent and
not more than the equivalent of 101.0 per cent of alkyltrimethylammonium bromides, calculated as
C
17
H
38
BrN (M
r
336.4) with reference to the dried substance.
CHARACTERS
A white or almost white, voluminous, free-flowing powder, freely soluble in water and in alcohol,
IDENTIFICATION
A. Dissolve 0.25 g in alcohol R and dilute to 25.0 ml with the same solvent. At wavelengths from
260 nm to 280 nm, the absorbance (2.2.25) of the solution is not greater than 0.05.
B. Dissolve about 5 mg in 5 ml of buffer solution pH 8.0 R. Add a strip of methyl green-iodomercurate
paper R. After 5 min, the solution shows a more intense greenish-blue colour than a blank solution
prepared at the same time in the same manner.
C. Solution S (see Tests) froths copiously when shaken.
D. Examine by thin-layer chromatography (2.2.27), using silanised silica gel HF
254
R as the coating
substance.
same solvent.
Reference solution. Dissolve 0.10 g of trimethyltetradecylammonium bromide CRS in water R and dilute to
20 volumes of acetone R, 35 volumes of a 270 g/l solution of sodium acetate R and 45 volumes of
methanol R. Dry the plate in a current of hot air. Allow to cool. Expose the plate to iodine vapour and
examine in daylight. The principal spot in the chromatogram obtained with the test solution is similar
in position, colour and size to the principal spot in the chromatogram obtained with the reference
solution.
TESTS
indicator.
Amines and amine salts Dissolve 5.0 g in 30 ml of a mixture of 1 volume of 1M hydrochloric acid
and 99 volumes of methanol R and add 100 ml of 2propanol R. Pass a stream of nitrogen R slowly
through the solution. Gradually add 15.0 ml of 0.1M tetrabutylammonium hydroxide and record the
potentiometric titration curve (2.2.20). If the curve shows two points of inflexion, the volume of
titrant added between the two points is not greater than 2.0 ml.
100C to 105C for 2 h.
ASSAY
08-79
10.0 ml of a freshly prepared 50 g/l solution of potassium iodide R. Shake, allow to separate and
discard the chloroform layer. Shake the aqueous layer with three quantities, each of 10 ml, of chloro-
form R and discard the chloroform layers. Add 40 ml of hydrochloric acid R, allow to cool and titrate
with 0.05M potassium iodate until the deep brown colour is almost discharged. Add 2 ml of chloro-
form R and continue the titration, shaking vigorously, until the colour of the chloroform layer no
longer changes. Carry out a blank titration on a mixture of 10.0 ml of the freshly prepared 50 g/l
solution of potassium iodide R, 20 ml of water R and 40 ml of hydrochloric acid R.
17
H
38
BrN.
__________________________________________________________________________________________________________ Ph Eur
08-80
Strong Cetrimide Solution
Definition Strong Cetrimide Solution is an aqueous solution of cetrimide. It contains 20 to 40%
w/v of cetrimide, calculated as C
17
H
38
BrN. It contains Ethanol (96 per cent) or Isopropyl Alcohol
or both. It may be perfumed and may contain colouring matter.
Production In making Strong Cetrimide Solution, Ethanol (96 per cent) may be replaced by
Industrial Methylated Spirit, provided that the law and the statutory regulations governing the use of
Industrial Methylated Spirit are observed.
Content of cetrimide, C
17
H
38
BrN 95.0 to 105.0% of the stated amount.
Identification
A. Dilute a volume of the solution containing 0.1 g of cetrimide to 5 ml with water and add 2 ml of a
5% w/v solution of potassium hexacyanoferrate(III). A yellow precipitate is produced.
B. Shake together 5 ml of water, 1 ml of 2M sulphuric acid, 2 ml of chloroform and 0.05 ml of methyl
orange solution; the chloroform layer is colourless. Add 0.1 ml of the solution being examined and
shake; a yellow colour is produced slowly in the chloroform layer.
C. Yields reaction A characteristic of bromides, Appendix VI..
Acidity or alkalinity Dilute a volume of the solution containing 10 g of cetrimide to 100 ml and
add 0.1 ml of bromocresol purple solution. Not more than 1.0 ml of either 0.1M hydrochloric acid VS or
0.1M sodium hydroxide VS is required to change the colour of the solution.
Miscibility with ethanol Mix a volume of the solution containing 1.6 g of cetrimide with a mixture
of 2 ml of water and 16 ml of ethanol (96%). The solution remains clear, Appendix IV A.
Neutral substances To a volume of the solution containing 10 g of cetrimide add 25 ml of ethanol
(50%), acidify to bromophenol blue solution by the dropwise addition of hydrochloric acid and add
0.05 ml in excess. Transfer quantitatively to the extraction compartment of an apparatus designed for
continuous liquidliquid extraction by fluids of a lesser density than water, washing out the beaker
with 10 ml ethanol (50%) and adding the washings to the bulk of the solution in the extractor. Add
sufficient ethanol (50%), if necessary, to half-fill the extraction chamber to the level of the overflow
limb. Add sufficient purified hexane to fill the extraction chamber, secure an overflow volume of about
30 ml in the ebullition flask and heat using an electrically heated mantle. Ensure that a continuous
flow of hexane through the aqueous ethanol layer is observed and continue the extraction for 16
hours. Transfer the hexane extract to a separating funnel, washing out the flask with 10 ml of purified
hexane. Shake the combined extract and washings with 25 ml of ethanol (50%) and discard the
aqueous ethanol layer. Filter the hexane layer through a dry filter paper (Whatman No. 1 is suitable)
into a tared flask and remove the solvent using a rotary evaporator at 40 and then at room
temperature at a pressure not exceeding 0.7 kPa for 2 hours. The residue weighs not more than
0.4 g.
Non-quaternised amines To a volume of the solution containing 10 g of cetrimide add a mixture
of 100 ml of propan-2-ol, 0.1 ml of hydrochloric acid and 20 ml of methanol. Titrate with 0.1M
tetrabutylammonium hydroxide VS passing a slow current of nitrogen through the solution and deter-
mining the end point potentiometrically using a platinumglass electrode system. Inflections in the
titration curve indicate (A) neutralisation of excess hydrochloric acid and (B) neutralisation of non-
quaternised amine salts. The difference between the volumes corresponding to A and B is not more
than 10 ml (2.4%, calculated as C
16
H
35
N).
Ethanol; Isopropyl alcohol Carry out one or both of the following methods according to the
declared alcohol content of the solution being examined.
Ethanol Not more than 10.0% v/v, by the method for the determination of ethanol, Appendix VIII F.
Use on-column injection and do not heat the injection port.
Isopropyl alcohol Not more than 10.0% v/v, by the method for the determination of ethanol,
Appendix VIII F, with the following modifications. For solution (1) use a solution containing 5.0%
v/v of propan-2-ol and 5.0% v/v of propan-1-ol (internal standard). For solution (2) use the solution
being examined, diluted with water, if necessary, to contain about 5.0% v/v of isopropyl alcohol.
Maintain the column temperature at 170, use on-column injection and do not heat the injection
port.
Assay Dilute a volume containing 4 g of cetrimide with sufficient water to produce 100 ml. Transfer
25 ml of the solution to a separating funnel and add 25 ml of chloroform, 10 ml of 0.1M sodium
hydroxide and 10 ml of a freshly prepared 8.0% w/v solution of potassium iodide. Shake well, allow to
separate and discard the chloroform layer. Wash the aqueous layer with three 10-ml quantities of
chloroform and discard the washings. Add 40 ml of hydrochloric acid, cool and titrate with 0.05M
potassium iodate VS until the deep brown colour is almost discharged. Add 2 ml of chloroform and
continue the titration, with shaking, until the chloroform layer becomes colourless. Carry out a blank
titration on a mixture of 10 ml of the freshly prepared potassium iodide solution, 20 ml of water and
40 ml of hydrochloric acid. The difference between the titrations represents the amount of potassium
08-81
iodate required. Each ml of 0.05M potassium iodate VS is equivalent to 33.64 mg of C
17
H
38
BrN.
Storage Strong Cetrimide Solution should be kept in a well-closed container and stored at a
temperature above 15.
Labelling The label states whether Ethanol, Isopropyl Alcohol or both are present and the
percentage of cetrimide, weight in volume.
Preparation
Cetrimide Solution
08-82
Cetyl Alcohol
36653-82-4
Cetyl Alcohol complies with the requirements of the 3rd edition of the European Pharmacopoeia [0540]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cetyl alcohol is a mixture of solid alcohols consisting mainly of hexadecanol (CH
3
(CH
2
)
14
CH
2
OH;
M
r
242.4).
CHARACTERS
White, unctuous mass, powder, flakes or granules, practically insoluble in water, freely to sparingly
soluble in alcohol, freely soluble in ether; when melted, it is miscible with vegetable and animal oils,
with liquid paraffin and with melted wool fat.
IDENTIFICATION
B. Hydroxyl value (2.5.3): 218 to 238.
TESTS
Appearance of solution Dissolve 0.5 g in boiling alcohol R, allow to cool and dilute to 20 ml with
the same solvent. The solution is clear (2.2.1) and not more intensely coloured than reference
solution B
6
(Method II, 2.2.2).
Iodine value (2.5.4). Not more than 2.0, determined on 2.00 g dissolved in 25 ml of chloroform R.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
08-83
Cetylpyridinium Chloride
N
[CH
2
]
15
CH
3
+
Cl
_
C
21
H
38
ClN,H
2
O 358.0 6004-24-6
Cetylpyridinium Chloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cetylpyridinium chloride contains not less than 96.0 per cent and not more than the equivalent of
101.0 per cent of 1-hexadecylpyridinium chloride, calculated with reference to the anhydrous
substance.
CHARACTERS
A white powder, slightly soapy to the touch, soluble in water and in alcohol, very slightly soluble in
ether. An aqueous solution froths copiously when shaken.
IDENTIFICATION
A. Dissolve 0.10 g in water R and dilute to 100.0 ml with the same solvent. Dilute 5.0 ml of this
shows an absorption maximum at 259 nm and two shoulders at about 254 nm and at about 265 nm.
The specific absorbance at the maximum is 126 to 134, calculated with reference to the anhydrous
substance.
obtained with cetylpyridinium chloride CRS. Examine the substances in the solid state.
chloroform R and shake. The chloroform layer is colourless. Add 0.1 ml of solution S (see Tests) and
shake. The chloroform layer becomes blue.
D. Solution S gives reaction (a) of chlorides (2.3.1).
TESTS
Acidity To 50 ml of solution S add 0.1 ml of phenolphthalein solution R. Not more than 2.5 ml of
0.02M sodium hydroxide is required to change the colour of the indicator.
Amines and amine salts Dissolve 5.0 g with heating in 20 ml of a mixture of 3 volumes of 1M
point of inflexion, the substance to be examined does not comply with the test. If the curve shows
one point of inflexion, repeat the test but add 3.0 ml of a 25.0 g/l solution of dimethyldecylamine R in
2-propanol R before the titration. If the titration curve after the addition of 12.0 ml of the titrant
shows only one point of inflexion, the substance to be examined does not comply with the test.
mination of water.
ASSAY
08-84
10.0 ml of a freshly prepared 50 g/l solution of potassium iodide R. Shake well, allow to separate and
discard the chloroform layer. Shake the aqueous layer with three quantities, each of 10 ml, of
chloroform R and discard the chloroform layers. To the aqueous layer add 40 ml of hydrochloric acid R,
allow to cool and titrate with 0.05M potassium iodate until the deep-brown colour is almost
discharged. Add 2 ml of chloroform R and continue the titration, shaking vigorously, until the chloro-
form layer no longer changes colour. Carry out a blank titration on a mixture of 10.0 ml of the freshly
prepared 50 g/l solution of potassium iodide R, 20 ml of water R and 40 ml of hydrochloric acid R.
21
H
38
ClN.
__________________________________________________________________________________________________________ Ph Eur
08-85
Chalk
Prepared Chalk
CaCO
3
100.1
Definition Chalk is a native form of calcium carbonate freed from most of its impurities by
elutriation and dried. It contains not less than 97.0% and not more than 100.5% of CaCO
3
,
Characteristics Odourless or almost odourless. It absorbs water readily.
Practically insoluble in water; slightly soluble in water containing carbon dioxide.
Macroscopical White or greyish white, small friable masses, usually conical in form, or in powder;
amorphous; earthy; soft to the touch.
Microscopical Consists of the calcareous shells and detritus of various foraminifera; the calcareous
shells vary from about 35 to 100 m in breadth and from about 50 to 180 m in length; among the
detritus are numerous small rings and discs about 5 to 10 m in diameter.
Identification
A. A solution in 6M acetic acid yields reaction C characteristic of calcium salts, Appendix VI.
B. Yields reaction A characteristic of carbonates, Appendix VI.
Acidity or alkalinity 1 g, boiled with 50 ml of water and filtered, yields a filtrate which is neutral to
bromothymol blue solution R3 or requires not more than 0.05 ml of 0.1M hydrochloric acid VS to make it
so.
Aluminium, iron, phosphate and matter insoluble in hydrochloric acid Dissolve 2 g in a
mixture of 5 ml of hydrochloric acid and 75 ml of water, boil to remove carbon dioxide and make
alkaline with 5M ammonia using methyl red solution as indicator. Boil for 1 minute, filter and wash the
precipitate with a hot 2% w/v solution of ammonium chloride. Dissolve the precipitate as completely as
possible by passing 20 ml of hot 2M hydrochloric acid through the filter and wash the filter with
sufficient hot water to adjust the volume of the solution to 50 ml. Boil the solution and make alkaline
with 5M ammonia using methyl red solution as indicator. Boil for 1 minute, filter through the same
filter, wash the precipitate with a hot 2% w/v solution of ammonium nitrate, dry and ignite at a
temperature not lower than 1000. The residue weighs not more than 40 mg.
Arsenic Dissolve 0.5 g in 5 ml of brominated hydrochloric acid and dilute to 50 ml with water. 25 ml of
the resulting solution complies with the limit test for arsenic, Appendix VII (4 ppm).
Heavy metals Dissolve 1.0 g in 10 ml of 2M hydrochloric acid, add 0.1 ml of nitric acid and boil to
remove carbon dioxide. Cool, make alkaline with 5M ammonia, filter and wash the precipitate with
water. Pass 5 ml of hot 2M hydrochloric acid through the filter, cool the filtrate, add 0.5 g of ammonium
thiocyanate and extract with two 5-ml quantities of a mixture of equal volumes of isoamyl alcohol and
ether. To the aqueous layer add 0.5 g of citric acid and dilute to 20 ml with water. 12 ml of the
resulting solution complies with limit test A for heavy metals, Appendix VII. Use lead standard solution
(2 ppm Pb) to prepare the standard (40 ppm).
Chloride Dissolve 0.3 g in 2 ml of nitric acid and 10 ml of water, filter and dilute the filtrate to 30 ml
with water. 15 ml of the resulting solution complies with the limit test for chlorides, Appendix VII
(330 ppm).
Sulphate Dissolve 0.25 g in 5.5 ml of 2M hydrochloric acid, dilute to 30 ml with water and filter.
15 ml of the resulting solution complies with the limit test for sulphates, Appendix VII (0.12%).
1 g.
Assay To 2 g in 100 ml of water add 50 ml of 1M hydrochloric acid VS, boil to remove carbon
dioxide, cool and titrate the excess of acid with 1M sodium hydroxide VS using methyl orange solution as
indicator. Each ml of 1M hydrochloric acid VS is equivalent to 50.04 mg of CaCO
3
.
08-86
Chamomile Flowers
Chamomile Flowers complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Roman Chamomile Flower [0380]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Roman chamomile flower consists of the dried flower-heads of the cultivated double variety of
Chamaemelum nobile (L.) All. (Anthemis nobilis L.). It contains not less than 7 ml/kg of essential oil.
CHARACTERS
Roman chamomile flower has a strong and characteristic odour.
It consists of flower-heads with a white to yellowish-grey colour, being composed of solitary
hemispherical capitula, made up of a solid conical receptacle bearing the florets, each subtended by a
transparent small palea.
IDENTIFICATION
A. The capitula have a diameter of 8 mm to 20 mm; the receptacle is solid; the base of the receptacle
is surrounded by an involucre consisting of two or three rows of compact and imbricated bracts with
scarious margins. Most florets are ligulate, but a few pale yellow tubular florets occur in the central
region. Ligulate florets are white, dull, lanceolate and reflexed with a dark brown, inferior ovary, a
filiform style and a bifid stigma; tubular florets have a five- toothed corolla tube, five syngenesious,
epipetalous stamens and a gynoecium similar to that of the ligulate florets.
B. Separate the capitulum into its different parts. Examine under a microscope, using chloral hydrate
solution R. All parts of the flower-heads are covered with numerous small yellow glistening glandular
trichomes. The involucral bracts and paleae have epidermal cells in longitudal rows, sclerified at the
base and they are covered with conical trichomes, about 500 m long, each composed of three or
four very short base cells and a long, bent, terminal cell about 20 m wide. The corolla of the ligulate
flowers consists of papillary cells with cuticular striations. The ovaries of both kinds of florets have at
their base a sclerous ring consisting of a single row of cells. The receptacle and the ovaries contain
small clusters of calcium oxalate. The pollen grains have a diameter of about 35 m and are rounded
and triangular with three germinal pores and a spiny exine.
substance.
Test solution. To 0.5 g of the powdered drug (710) add 10 ml of methanol R and heat with shaking in
a water-bath at 60C for 5 min. Allow to cool and filter.
Reference solution. Dissolve 2.5 mg of apigenin R and 2.5 mg of apigenin-7-glucoside R in 10 ml of
methanol R.
mixture of 17 volumes of glacial acetic acid R, 17 volumes of water R and 66 volumes of butanol R.
Dry the plate at 100C to 105C for 5 min and spray the warm plate with a 10 g/l solution of
diphenylboric acid aminoethyl ester R in methanol R, using about 10 ml for a plate 200 mm square.
Spray the plate with the same volume of a 50 g/l solution of macrogol 400 R in methanol R. Allow to
stand for about 30 min and examine in ultraviolet light at 365 nm. The chromatogram obtained with
the reference solution shows in the upper third a zone of yellowish-greenish fluorescence (apigenin)
and in the middle third a zone of yellowish fluorescence (apigenin-7-glucoside). The chromatogram
obtained with the test solution shows a zone of yellowish-green fluorescence corresponding in
position and fluorescence to the apigenin and a zone of yellowish fluorescence corresponding in
position and fluorescence to the apigenin-7-glucoside obtained with the reference solution; above the
apigenin-7-glucoside zone there is a zone of brownish fluorescence (luteolin); immediately below the
apigenin-7-glucoside zone there is a zone of light brownish fluorescence (apiin); immediately below
the apiin zone there is a zone of bright blue fluorescence and below this zone a zone of bright blue
fluorescence; other faint zones may be present.
TESTS
Diameter of the flower-heads. Not more than 3 per cent of flower-heads have a diameter smaller
than 8 mm.
Deteriorated flower-heads. Brown or darkened flower-heads are absent.
Water (2.2.13). Not more than 10.0 per cent, determined by distillation using 20.0 g of whole drug.
08-87
ASSAY
Carry out the determination of essential oils in vegetable drugs (2.8.12). Use 20.0 g of whole drug,
a 500 ml round-bottomed flask, 250 ml of water R as the distillation liquid and 0.50 ml of xylene R in
the graduated tube. Distil at a rate of 3 ml to 3.5 ml per minute for 3 h.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
08-88
Activated Charcoal
Decolorising Charcoal
Activated Charcoal complies with the requirements of the 3rd edition of the European Pharmacopoeia [0313].
Action and use Adsorbent.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Activated charcoal is obtained from vegetable matter by suitable carbonisation processes intended to
confer a high adsorption power.
CHARACTERS
A black, light powder free from grittiness, practically insoluble in all usual solvents.
IDENTIFICATION
A. When heated to redness it burns slowly without a flame.
B. It complies with the test for adsorption power (see Tests).
TESTS
Solution S To 2.0 g in a conical flask with a ground-glass neck add 50 ml of dilute hydrochloric
acid R. Boil gently under a reflux condenser for 1 h, filter and wash the filter with dilute hydrochloric
acid R. Evaporate the combined filtrate and washings to dryness on a water-bath, dissolve the residue
in 0.1M hydrochloric acid and dilute to 50.0 ml with the same acid.
Acidity or alkalinity To 2.0 g add 40 ml of water R and boil for 5 min. Cool, restore to the original
mass with carbon dioxide-free water R and filter. Reject the first 20 ml of the filtrate. To 10 ml of the
filtrate add 0.25 ml of bromothymol blue solution R1 and 0.25 ml of 0.02M sodium hydroxide. The
solution is blue. Not more than 0.75 ml of 0.02M hydrochloric acid is required to change the colour of
the indicator to yellow.
Acid-soluble substances To 1.0 g add 25 ml of dilute nitric acid R and boil for 5 min. Filter whilst
hot through a sintered-glass filter (10) and wash with 10 ml of hot water R. Evaporate the combined
filtrate and washings to dryness on a water-bath, add to the residue 1 ml of hydrochloric acid R,
evaporate to dryness again and dry the residue to constant mass at 100C to 105C. The residue
weighs not more than 30 mg (3 per cent).
Alkali-soluble coloured substances To 0.25 g add 10 ml of dilute sodium hydroxide solution R and
boil for 1 min. Cool, filter and dilute the filtrate to 10 ml with water R. The solution is not more
intensely coloured than reference solution GY
4
(Method II, 2.2.2).
Alcohol-soluble substances To 2.0 g add 50 ml of alcohol R and boil under a reflux condenser for
10 min. Filter immediately, cool, and dilute to 50 ml with alcohol R. The filtrate is not more intensely
6
or BY
6
(Method II, 2.2.2). Evaporate 40 ml of the filtrate to
dryness and dry to constant mass at 100C to 105C. The residue weighs not more than 8 mg
(0.5 per cent).
Fluorescent substances In an intermittent-extraction apparatus, treat 10.0 g with 100 ml of
cyclohexane R1 for 2 h. Collect the liquid and dilute to 100 ml with cyclohexane R1. Examine in
ultraviolet light at 365 nm. The fluorescence of the solution is not more intense than that of a
solution of 83 g of quinine R in 1000 ml of 0.005M sulphuric acid examined in the same manner.
Sulphides To 1.0 g in a conical flask add 5 ml of hydrochloric acid R1 and 20 ml of water R. Heat to
boiling. The fumes released do not turn lead acetate paper R brown.
2.2.23).
Reference solutions. Prepare the reference solutions using copper standard solution (0.1 per cent Cu) R
and diluting with 0.1M hydrochloric acid.
Measure the absorbance at 325.0 nm using a copper hollow-cathode lamp as source of radiation and
2.2.23).
diluting with 0.1M hydrochloric acid.
08-89
Measure the absorbance at 283.3 nm using a lead hollow-cathode lamp as source of radiation and an
air-acetylene flame. Depending on the apparatus the line at 217.0 nm may be used.
Zinc Not more than 25 ppm of Zn, determined by atomic absorption spectrometry (Method I,
2.2.23).
Reference solutions. Prepare the reference solutions using zinc standard solution (100 ppm Zn) R and
diluting with 0.1M hydrochloric acid.
Measure the absorbance at 214.0 nm using a zinc hollow-cathode lamp as source of radiation and an
air-acetylene flame.
Loss on drying (2.2.32). Not more than 15 per cent, determined on 1.00 g by drying in an oven at
120C for 4 h.
Adsorption power To 0.300 g in a 100 ml ground-glass-stoppered conical flask add 25.0 ml of a
freshly prepared solution of 0.5 g of phenazone R in 50 ml of water R. Shake thoroughly for 15 min.
Filter and reject the first 5 ml of filtrate. To 10.0 ml of the filtrate add 1.0 g of potassium bromide R
and 20 ml of dilute hydrochloric acid R. Using 0.1 ml of methyl red solution R as indicator, titrate with
0.0167M potassium bromate until the red colour is discharged. Titrate slowly (1 drop every 15 s)
towards the end of the titration. Carry out a blank titration using 10.0 ml of the phenazone solution.
Calculate the quantity of phenazone adsorbed per 100 g of activated charcoal from the expression:
2 353 . ( ) a b
m
a=number of millilitres of 0.0167M potassium bromate used for the blank,

b =number of millilitres of 0.0167M potassium bromate used for the test
m=mass in grams of the substance to be examined.
Not less than 40 g of phenazone is adsorbed per 100 g of activated charcoal, calculated with reference
3
micro-
organisms per gram, determined by plate count.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
08-90
Chenodeoxycholic Acid
HO
H
H H
Me
Me
H
COOH
H
Me
H
OH
C
24
H
40
O
4
392.6 474-25-9
Chenodeoxycholic Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Dissolution of cholesterol gallstones.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Chenodeoxycholic acid contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of 3,7-dihydroxy-5-cholan-24-oic acid, calculated with reference to the dried
substance.
CHARACTERS
A white or almost white powder, very slightly soluble in water, freely soluble in alcohol, soluble in
acetone, slightly soluble in methylene chloride.
IDENTIFICATION
obtained with chenodeoxycholic acid CRS. Examine the substances prepared as discs using potassium
bromide R.
C. Dissolve about 10 mg in 1 ml of sulphuric acid R. Add 0.1 ml of formaldehyde solution R and allow
to stand for 5 min. Add 5 ml of water R. The suspension obtained is greenish-blue.
TESTS
same solvent. The specific optical rotation is +11.0 to +13.0, calculated with reference to the dried
substance.
Related substances Examine by thin-layer chromatography (2.2.27), using a suitable silica gel as
Test solution (a). Dissolve 0.40 g of the substance to be examined in a mixture of 1 volume of water R
and 9 volumes of acetone R and dilute to 10 ml with the same mixture of solvents.
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with a mixture of 1 volume of water R and 9
volumes of acetone R.
Reference solution (a). Dissolve 40 mg of chenodeoxycholic acid CRS in a mixture of 1 volume of
water R and 9 volumes of acetone R and dilute to 10 ml with the same mixture of solvents.
Reference solution (b). Dissolve 20 mg of lithocholic acid CRS in a mixture of 1 volume of water R and 9
volumes of acetone R and dilute to 10 ml with the same mixture of solvents. Dilute 2 ml of the
solution to 100 ml with a mixture of 1 volume of water R and 9 volumes of acetone R.
Reference solution (c). Dissolve 20 mg of ursodeoxycholic acid CRS in a mixture of 1 volume of water R
Reference solution (d). Dissolve 20 mg of cholic acid CRS in a mixture of 1 volume of water R and 9
volumes of acetone R and dilute to 100 ml with the same mixture of solvents.
Reference solution (e). Dilute 0.5 ml of test solution (a) to 20 ml with a mixture of 1 volume of water R
08-91
and 9 volumes of acetone R. Dilute 1 ml of the solution to 10 ml with a mixture of 1 volume of
water R and 9 volumes of acetone R.
Reference solution (f). Dissolve 10 mg of chenodeoxycholic acid CRS in reference solution (c) and dilute
15 cm using a mixture of 1 volume of glacial acetic acid R, 30 volumes of acetone R and 60 volumes of
methylene chloride R. Dry the plate at 120C for 10 min. Spray the plate immediately with a 47.6 g/l
solution of phosphomolybdic acid R in a mixture of 1 volume of sulphuric acid R and 20 volumes of
glacial acetic acid R and heat again at 120C until blue spots appear on a lighter background. In the
chromatogram obtained with test solution (a): any spot corresponding to lithocholic acid is not more
intense than the principal spot in the chromatogram obtained with reference solution (b) (0.1 per
cent); any spot corresponding to ursodeoxycholic acid is not more intense than the principal spot in
the chromatogram obtained with reference solution (c) (1 per cent); any spot corresponding to cholic
acid is not more intense than the principal spot in the chromatogram obtained with reference solution
(d) (0.5 per cent); any spot apart from the principal spot and any spots corresponding to lithocholic
acid, ursodeoxycholic acid and cholic acid, is not more intense than the principal spot in the chroma-
togram obtained with reference solution (e) (0.25 per cent). The test is not valid unless the chroma-
togram obtained with reference solution (f) shows two clearly separated principal spots.
100C to 105C.
ASSAY
Dissolve 0.350 g in 50 ml of alcohol R, previously neutralised to 0.2 ml of phenolphthalein solution R.
Add 50 ml of water R and titrate with 0.1M sodium hydroxide until a pink colour is obtained.
24
H
40
O
4
.
IMPURITIES
OR
H
Me
O
H
H
R
2
R
1
H
HO
Me
H
H
Me
A. R = H, R
1
= OH, R
2
= H, R
3
= H:
ursodeoxycholic acid,
B. R = H, R
1
= H, R
2
= OH, R
3
= OH:
cholic acid (3,7,12-trihydroxy-5-cholan-24-oic acid),
C. R = H, R
1
= H, R
2
= H, R
3
= H:
lithocholic acid (3-hydroxy-5-cholan-24-oic acid),
D. R = H, R
1
= OH, R
2
= H, R
3
= OH:
ursocholic acid (3,7,12-trihydroxy-5-cholan-24-oic acid),
E. R = H, R
1
= H, R
2
= H, R
3
= OH:
deoxycholic acid (3,12-dihydroxy-5-cholan-24-oic acid),
F. R = H, R
1
, R
2
= O, R
3
= H:
3-hydroxy-7-oxo-5-cholan-24-oic acid,
G. R = CH
3
, R1 = OH, R2 = H, R3 = H:
methyl 3,7-dihydroxy-5-cholan-24-oate.
__________________________________________________________________________________________________________ Ph Eur
08-92
Chloral Hydrate
Cl
OH
Cl Cl
HO
H
C
2
H
3
Cl
3
O
2
165.4 302-17-0
Chloral Hydrate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0265].
Action and use Hypnotic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Chloral hydrate contains not less than 98.5 per cent and not more than the equivalent of 101.0 per
cent of 2,2,2-trichloroethane-1,1-diol.
CHARACTERS
Colourless, transparent crystals, very soluble in water, freely soluble in alcohol and in ether.
IDENTIFICATION
A. To 10 ml of solution S (see Tests) add 2 ml of dilute sodium hydroxide solution R. The mixture
becomes cloudy and, when heated, gives off an odour of chloroform.
B. To 1 ml of solution S add 2 ml of sodium sulphide solution R. A yellow colour develops which
quickly becomes reddish-brown. On standing for a short time, a red precipitate may be formed.
TESTS
Chloral alcoholate Warm 1.0 g with 10 ml of dilute sodium hydroxide solution R, filter the super-
natant solution and add 0.05M iodine dropwise until a yellow colour is obtained. Allow to stand for
1 h. No precipitate is formed.
Heavy metals (2.4.8). 10 ml of solution S diluted to 20 ml with water R complies with limit test A
for heavy metals (20 ppm). Prepare the standard using lead standard solution (1 ppm Pb) R.
Non-volatile residue Evaporate 2.000 g on a water-bath. The residue weighs not more than 2 mg
(0.1 per cent).
ASSAY
Dissolve 4.000 g in 10 ml of water R and add 40.0 ml of 1M sodium hydroxide. Allow to stand for
exactly 2 min and titrate with 0.5M sulphuric acid, using 0.1 ml of phenolphthalein solution R as
indicator. Titrate the neutralised solution with 0.1M silver nitrate, using 0.2 ml of potassium chromate
solution R as indicator. Calculate the number of millilitres of 1M sodium hydroxide used by deducting
from the volume of 1M sodium hydroxide added at the beginning of the titration the volume of 0.5M
sulphuric acid used in the first titration and two-fifteenths of the volume of 0.1M silver nitrate used in
the second titration.
1 ml of 1M sodium hydroxide is equivalent to 0.1654 g of C
2
H
3
Cl
3
O
2
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
08-93
Chlorambucil
COOH
N
Cl
Cl
C
14
H
19
Cl
2
NO
2
304.2 305-03-3
Chlorambucil complies with the requirements of the 3rd edition of the European Pharmacopoeia [0137]. These
Preparation
Chlorambucil Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Chlorambucil contains not less than 98.5 per cent and not more than the equivalent of 101.0 per
cent of 4-4-[di(2-chloroethyl)amino]phenylbutyric acid, calculated with reference to the anhydrous
substance.
CHARACTERS
A white, crystalline powder, practically insoluble in water; freely soluble in acetone and in alcohol.
IDENTIFICATION
obtained with chlorambucil CRS.
C. To 0.4 g add 10 ml of dilute hydrochloric acid R, mix and allow to stand for 30 min, shaking from
time to time. Filter and wash the precipitate with two quantities, each of 10 ml, of water R. To 10 ml
of the combined filtrate and washings add 0.5 ml of potassium tetraiodomercurate solution R. A pale-
brown precipitate is formed. To another 10 ml of the combined filtrate and washings add 0.2 ml of
potassium permanganate solution R. The colour of the latter is discharged immediately.
D. Dissolve 50 mg in 5 ml of acetone R and dilute to 10 ml with water R. Add 0.05 ml of dilute nitric
acid R and 0.2 ml of silver nitrate solution R2. No opalescence is produced immediately. Heat the
solution on a water-bath; an opalescence develops.
TESTS
254
R as the
coating substance.
Carry out all operations as rapidly as possible protected from light. Prepare the solutions immediately before
use.
Test solution. Dissolve 0.2 g of the substance to be examined in acetone R and dilute to 10 ml with the
same solvent.
Reference solution (a). Dilute 1 ml of the test solution to 50 ml with acetone R.
Reference solution (b). Dilute 25 ml of reference solution (a) to 100 ml with acetone R.
20 volumes of methyl ethyl ketone R, 20 volumes of heptane R, 25 volumes of methanol R and 40
volumes of toluene R. Examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained
with the test solution, apart from the principal spot, is not more intense than the spot in the chroma-
togram obtained with reference solution (a) (2.0 per cent) and at most one such spot is more intense
than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent).
mination of water.
08-94
ASSAY
Dissolve 0.200 g in 10 ml of acetone R and add 10 ml of water R. Titrate with 0.1M sodium hydroxide,
using 0.1 ml of phenolphthalein solution R as indicator.
14
H
19
Cl
2
NO
2.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
09-1
Chloramphenicol
OH
OH H
H NHCOCHCl
2
O
2
N
C
11
H
12
Cl
2
N
2
O
5
323.1 56-75-7
Chloramphenicol complies with the requirements of the 3rd edition of the European Pharmacopoeia [0071].
Preparations
Chloramphenicol Capsules
Chloramphenicol Ear Drops
Chloramphenicol Eye Drops
Chloramphenicol Eye Ointment
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Chloramphenicol is 2,2-dichloro-N-[(1R,2R)-2-hydroxy-1-hydroxymethyl-2-(4-nitrophenyl)-
ethyl]acetamide, produced by the growth of certain strains of Streptomyces venezuelae in a suitable
medium. It is normally prepared by synthesis. It contains not less than 98.0 per cent and not more
than the equivalent of 102.0 per cent of C
11
H
12
Cl
2
N
2
O
5
substance.
CHARACTERS
A white, greyish-white or yellowish-white, fine, crystalline powder or fine crystals, needles or
elongated plates, slightly soluble in water, freely soluble in alcohol and in propylene glycol, slightly
soluble in ether.
A solution in ethanol is dextrorotatory and a solution in ethyl acetate is laevorotatory.
IDENTIFICATION
obtained with chloramphenicol CRS.
chromatogram obtained with 1 l of the test solution is similar in position and size to the principal
D. Dissolve about 10 mg in 1 ml of alcohol (50 per cent V/V) R, add 3 ml of a 10 g/l solution of
calcium chloride R and 50 mg of zinc powder R and heat on a water-bath for 10 min. Filter the hot
solution and allow to cool. Add 0.1 ml of benzoyl chloride R and shake for 1 min. Add 0.5 ml of ferric
chloride solution R1 and 2 ml of chloroform R and shake. The aqueous layer is coloured light violet-red
to purple.
E. To 50 mg in a porcelain crucible add 0.5 g of anhydrous sodium carbonate R. Heat over an open
1 ml of the filtrate add 1 ml of water R. The solution gives reaction (a) of chlorides (2.3.1).
TESTS
Acidity or alkalinity To 0.1 g add 20 ml of carbon dioxide-free water R, shake and add 0.1 ml of
bromothymol blue solution R1. Not more than 0.1 ml of 0.02M hydrochloric acid or 0.02M sodium
hydroxide is required to change the colour of the indicator.
Specific optical rotation (2.2.7). Dissolve 1.50 g in ethanol R and dilute to 25.0 ml with the same
254
R as the
coating substance.
Test solution. Dissolve 0.10 g of the substance to be examined in acetone R and dilute to 10 ml with
the same solvent.
09-2
Reference solution (a). Dissolve 0.10 g of chloramphenicol CRS in acetone R and dilute to 10 ml with the
same solvent.
Reference solution (b). Dilute 0.5 ml of reference solution (a) to 100 ml with acetone R.
Apply separately to the plate 1 l and 20 l of the test solution, 1 l of reference solution (a) and 20 l
of reference solution (b). Develop over a path of 15 cm using a mixture of 1 volume of water R, 10
volumes of methanol R and 90 volumes of chloroform R. Allow the plate to dry in air and examine in
ultraviolet light at 254 nm. Any spot in the chromatogram obtained with 20 l of the test solution,
reference solution (b) (0.5 per cent).
Chlorides (2.4.4). To 1.00 g add 20 ml of water R and 10 ml of nitric acid R and shake for 5 min.
Filter through a filter paper previously washed by filtering 5 ml portions of water R until 5 ml of
filtrate no longer becomes opalescent on addition of 0.1 ml of nitric acid R and 0.1 ml of silver nitrate
solution R1. 15 ml of the filtrate complies with the limit test for chlorides (100 ppm).
100C to 105C.
Sterility (2.6.1). If intended for use in the manufacture of a parenteral or ophthalmic dosage form
without a further appropriate sterilisation procedure, it complies with the test for sterility.
Pyrogens (2.6.8). If intended for use in the manufacture of a parenteral dosage form without a
Inject per kilogram of the rabbits mass 2.5 ml of a solution containing per millilitre 2 mg of the
ASSAY
Dissolve 0.100 g in water R and dilute to 500.0 ml with the same solvent. Dilute 10.0 ml of this
11
H
12
Cl
2
N
2
O
5
STORAGE
Store protected from light. If the substance is sterile, store in a sterile, airtight, tamper-proof
container.
LABELLING
The label states:
__________________________________________________________________________________________________________ Ph Eur
09-3
Chloramphenicol Palmitate
OH H
H NHCOCHCl
2
O
2
N
OCO[CH
2
]
14
CH
3
C
27
H
42
Cl
2
N
2
O
6
561.6 530-43-8
Chloramphenicol Palmitate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Chloramphenicol palmitate contains not less than 98.0 per cent and not more than the equivalent of
102.0 per cent of (2R,3R)-2-(2,2-dichloroacetamido)-3-hydroxy-3-(4-nitrophenyl)propyl
hexadecanoate, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, fine, unctuous powder, practically insoluble in water, freely soluble in
acetone, soluble in ether, sparingly soluble in alcohol, very slightly soluble in hexane.
It melts at 87C to 95C.
It shows polymorphism and the thermodynamically stable form has low bioavailability following
oral administration.
IDENTIFICATION
A. Examine by thin-layer chromatography (2.2.27), using silanised silica gel H R as the coating
substance.
Test solution. Dissolve 50 mg of the substance to be examined in a mixture of 1 ml of 1M sodium
hydroxide and 5 ml of acetone R and allow to stand for 30 min. Add 1.1 ml of 1M hydrochloric acid and
3 ml of acetone R.
Reference solution (a). Dissolve 10 mg of chloramphenicol CRS in acetone R and dilute to 5 ml with the
same solvent.
Reference solution (b). Dissolve 10 mg of palmitic acid R in acetone R and dilute to 5 ml with the same
solvent.
Reference solution (c). Dissolve 10 mg of the substance to be examined in acetone R and dilute to 5 ml
30 volumes of a 100 g/l solution of ammonium acetate R and 70 volumes of alcohol R. Allow the plate
to dry in air and spray with a solution containing 0.2 g/l of dichlorofluorescein R and 0.1 g/l of
rhodamine B R in alcohol R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm.
The chromatogram obtained with the test solution shows three spots corresponding in position to the
principal spots in the chromatograms obtained with reference solutions (a), (b) and (c).
B. Dissolve 0.2 g in 2 ml of pyridine R, add 2 ml of a 100 g/l solution of potassium hydroxide R and
heat on a water-bath. A red colour is produced.
C. Dissolve 10 mg in 5 ml of alcohol R and add 4.5 ml of dilute sulphuric acid R and 50 mg of zinc
powder R. Allow to stand for 10 min and if necessary decant the supernatant liquid or filter. Cool the
solution in iced water and add 0.5 ml of sodium nitrite solution R. Allow to stand for 2 min and add 1 g
of urea R, 2 ml of strong sodium hydroxide solution R and 1 ml of -naphthol solution R. A red colour
develops.
TESTS
Acidity Dissolve 1.0 g in 5 ml of a mixture of equal volumes of alcohol R and ether R, warming to
35C. Add 0.2 ml of phenolphthalein solution R. Not more than 0.4 ml of 0.1M sodium hydroxide is
required to produce a pink colour persisting for 30 s.
Free chloramphenicol Not more than 450 ppm. Dissolve 1.0 g, with gentle heating, in 80 ml of
xylene R. Cool and shake with three quantities, each of 15 ml, of water R. Dilute the combined
09-4
aqueous extracts to 50 ml with water R and shake with 10 ml of toluene R. Allow to separate and
discard the toluene layer. Centrifuge a portion of the aqueous layer and measure the absorbance (A)
(2.2.25) at the maximum at 278 nm using as the compensation liquid a blank solution having an
absorbance not greater than 0.05.
Calculate the content of free chloramphenicol in parts per million from the expression:
A10
596
4
.
254
R as the
coating substance.
same solvent.
Reference solution (a). Dissolve 20 mg of chloramphenicol palmitate isomer CRS in acetone R and dilute
to 10 ml with the same solvent. Dilute 1 ml of this solution to 10 ml with acetone R.
Reference solution (b). Dissolve 20 mg of chloramphenicol dipalmitate CRS in acetone R and dilute to
10 ml with the same solvent. Dilute 1 ml of this solution to 10 ml with acetone R.
Reference solution (c). Dissolve 5 mg of chloramphenicol CRS in acetone R and dilute to 10 ml with the
same solvent. Dilute 1 ml of this solution to 10 ml with acetone R.
10 volumes of methanol R, 40 volumes of chloroform R and 50 volumes of cyclohexane R. Allow the
plate to dry in air and examine in ultraviolet light at 254 nm. In the chromatogram obtained with the
test solution, any spots corresponding to chloramphenicol palmitate isomer and chloramphenicol
dipalmitate are not more intense than the corresponding spots in the chromatograms obtained with
reference solutions (a) and (b) respectively (2.0 per cent) and any spot, apart from the principal spot
and the spots corresponding to chloramphenicol palmitate isomer and chloramphenicol dipalmitate,
is not more intense than the principal spot in the chromatogram obtained with reference solution (c)
(0.5 per cent).
Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by heating at 80C
over diphosphorus pentoxide R at a pressure not exceeding 0.1 kPa for 3 h.
ASSAY
Dissolve 90.0 mg in alcohol R and dilute to 100.0 ml with the same solvent. Dilute 10.0 ml of this
solution to 250.0 ml with alcohol R. Measure the absorbance (2.2.25) of the solution at the maximum
at 271 nm.
27
H
42
Cl
2
N
2
O
6
STORAGE
IMPURITIES
A. (1R,2R)-2-(2,2-dichloroacetamido)-3-hydroxy-1-(4-nitrophenyl)propyl hexadecanoate
(chloramphenicol palmitate isomer),
B. chloramphenicol dipalmitate.
__________________________________________________________________________________________________________ Ph Eur
09-5
Chloramphenicol Sodium Succinate
O COONa
O
O
2
N
NHCOCHCl
2
H
H OH
C
15
H
15
Cl
2
N
2
NaO
8
445.2 982-57-0
Chloramphenicol Sodium Succinate complies with the requirements of the 3rd edition of the European
Preparation
Chloramphenicol Sodium Succinate Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Chloramphenicol sodium succinate, a mixture in variable proportions of sodium (2R,3R)-2-(2,2-
dichloroacetamido)-3-hydroxy-3-(4-nitrophenyl)propyl succinate (3 isomer) and of sodium (1R,2R)-
2-(2,2-dichloroacetamido)-3-hydroxy-1-(4-nitrophenyl)propyl succinate (1 isomer), contains not less
than 98.0 per cent and not more than the equivalent of 102.0 per cent of C
15
H
15
Cl
2
N
2
NaO
8
,
CHARACTERS
A white or yellowish-white powder, hygroscopic, very soluble in water, freely soluble in alcohol,
IDENTIFICATION
254
Test solution. Dissolve 20 mg of the substance to be examined in 2 ml of acetone R.
Reference solution (a). Dissolve 20 mg of chloramphenicol sodium succinate CRS in 2 ml of acetone R.
Reference solution (b). Dissolve 20 mg of chloramphenicol CRS in 2 ml of acetone R.
volume of dilute acetic acid R, 14 volumes of methanol R and 85 volumes of chloroform R. Allow the
plate to dry in air and examine in ultraviolet light at 254 nm. The two principal spots in the chroma-
togram obtained with the test solution are similar in position and size to those in the chromatogram
obtained with reference solution (a). Their positions are different from that of the principal spot in
B. Dissolve about 10 mg in 1 ml of alcohol (50 per cent V/V) R, add 3 ml of a 10 g/l solution of
calcium chloride R and 50 mg of zinc powder R and heat on a water-bath for 10 min. Filter the hot
solution and allow to cool. Add 0.1 ml of benzoyl chloride R and shake for 1 min. Add 0.5 ml of ferric
chloride solution R1 and 2 ml of chloroform R and shake. The upper layer is light violet-red to purple.
C. Dissolve 50 mg in 1 ml of pyridine R. Add 0.5 ml of dilute sodium hydroxide solution R and 1.5 ml of
water R. Heat in a water-bath for 3 min. A red colour develops. Add 2 ml of nitric acid R and cool
under running water. Add 1 ml of 0.1M silver nitrate. A white precipitate is formed slowly.
TESTS
solvent. The specific optical rotation is +5.0 to +8.0, calculated with reference to the anhydrous
substance.
Chloramphenicol and chloramphenicol disodium disuccinate Examine by liquid chromato-
graphy (2.2.29).
Reference solution (a). Dissolve 10.0 mg of chloramphenicol CRS in the mobile phase and dilute to
100.0 ml with the mobile phase (solution a). Dilute 5.0 ml of this solution to 100.0 ml with the
mobile phase.
09-6
Reference solution (b). Dissolve 10.0 mg of chloramphenicol disodium disuccinate CRS in the mobile
phase and dilute to 100.0 ml with the mobile phase (solution b). Dilute 5.0 ml of this solution to
Reference solution (c). Dissolve 25 mg of the substance to be examined in the mobile phase, add 5 ml
of solution (a) and 5 ml of solution (b) and dilute to 100 ml with the mobile phase.
as mobile phase at a flow rate of 1.0 ml per minute a mixture of 5 volumes of a 20 g/l solution of
phosphoric acid R, 40 volumes of methanol R and 55 volumes of water R,
a 20 l fixed-loop injector.
Inject the test solution and each of the reference solutions. The test is not valid unless, in the
chromatogram obtained with reference solution (c), the two peaks corresponding to those in the
chromatograms obtained with reference solutions (a) and (b) are clearly separated from the peaks
corresponding to the two principal peaks in the chromatogram obtained with the test solution. If
necessary, adjust the methanol content of the mobile phase.
In the chromatogram obtained with the test solution, the area of any peak corresponding to
chloramphenicol is not greater than the area of the principal peak in the chromatogram obtained with
reference solution (a) (2.0 per cent); the area of any peak corresponding to chloramphenicol
disodium disuccinate is not greater than the area of the principal peak in the chromatogram obtained
with reference solution (b) (2.0 per cent).
Sterility (2.6.1). If intended for use in the manufacture of a parenteral dosage form without a further
Pyrogens (2.6.8). If intended for use in the manufacture of a parenteral dosage form without a
further appropriate procedure for removal of pyrogens, it complies with the test for pyrogens.
Inject per kilogram of the rabbits mass 2.5 ml of a solution in water for injections R containing 2 mg of
the substance to be examined per millilitre.
ASSAY
Dissolve 0.200 g in water R and dilute to 500.0 ml with the same solvent. Dilute 5.0 ml of this
15
H
15
Cl
2
N
2
NaO
8
, taking the specific absorbance to be 220.
STORAGE
airtight, tamper-proof container, protected from light.
LABELLING
The label states:
__________________________________________________________________________________________________________ Ph Eur
09-7
Chlorcyclizine Hydrochloride
corrected 1/01
N
NMe
Cl
,HCl
C
18
H
21
ClN
2
,HCl 337.3 1620-21-9
Chlorcyclizine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
1
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Chlorcyclizine hydrochloride contains not less than 99.0 per cent and not more than the equivalent
of 101.0 per cent of (RS)-1-[(4-chlorophenyl)phenylmethyl]-4-methylpiperazine hydrochloride,
CHARACTERS
A white, crystalline powder, freely soluble in water and in methylene chloride, soluble in alcohol,
IDENTIFICATION
A. Dissolve 10.0 mg in a 5 g/l solution of sulphuric acid R and dilute to 100.0 ml with the same acid.
Dilute 10.0 ml of the solution to 100.0 ml with a 0.5 g/l solution of sulphuric acid R. Examined
between 215 nm and 300 nm (2.2.25), the solution shows an absorption maximum at 231 nm. The
specific absorbance at the maximum is 475 to 525, calculated with reference to the dried substance.
obtained with chlorcyclizine hydrochloride CRS. Examine the substances prepared as discs.
C. Examine the chromatograms obtained in the test for related substances (see Tests). The principal
TESTS
Related substances Examine by thin-layer chromatography (2.2.27), using a plate coated with a
suitable silica gel.
Reference solution (a). Dissolve 10 mg of chlorcyclizine hydrochloride CRS in methanol R and dilute to
Reference solution (b). Dissolve 5 mg of methylpiperazine R in methanol R and dilute to 50 ml with the
same solvent.
Reference solution (c). Dilute 1 ml of test solution (b) to 25 ml with methanol R.
Reference solution (d). Dissolve 10 mg of hydroxyzine hydrochloride CRS and 10 mg of chlorcyclizine
hydrochloride CRS in methanol R and dilute to 10 ml with the same solvent.
Apply separately to the plate 10 l of each solution and develop over a path of 15 cm using a
09-8
mixture of 2 volumes of concentrated ammonia R, 13 volumes of methanol R and 85 volumes of
methylene chloride R. Allow the plate to dry in air and expose it to iodine vapour for 10 min. In the
chromatogram obtained with test solution (a): any spot corresponding to methylpiperazine is not
more intense than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent);
any spot, apart from the principal spot and any spot corresponding to methylpiperazine, is not more
intense than the spot in the chromatogram obtained with reference solution (c) (0.2 per cent). The
test is not valid unless the chromatogram obtained with reference solution (d) shows two clearly
separated spots.
130C.
ASSAY
Dissolve 0.200 g in a mixture of 1 ml of 0.1M hydrochloric acid and 50 ml of methanol R. Carry out a
18
H
22
Cl
2
N
2
.
STORAGE
IMPURITIES
HN
NMe
A. N-methylpiperazine.
__________________________________________________________________________________________________________ Ph Eur
09-9
Chlordiazepoxide
corrected 1/01
N
N
NHMe
Cl
O
C
16
H
14
ClN
3
O 299.8 58-25-3
Chlordiazepoxide complies with the requirements of the 3rd edition of the European Pharmacopoeia [0656].
Action and use Hypnotic; anxiolytic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Chlordiazepoxide contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of 7-chloro-2-methylamino-5-phenyl-3H-1,4-benzodiazepine 4-oxide, calculated with reference
CHARACTERS
An almost white or light yellow, crystalline powder, practically insoluble in water, sparingly soluble in
alcohol, slightly soluble in ether.
IDENTIFICATION
B. Prepare the solutions protected from bright light and immediately before use.
Dissolve 10.0 mg in 0.1M hydrochloric acid and dilute to 200.0 ml with the same acid. Dilute 10.0 ml
(2.2.25), the solution shows two absorption maxima, at 246 nm and 308 nm. The specific
absorbances at the maxima are 1120 to 1190 and 316 to 336, respectively.
obtained with chlordiazepoxide CRS. Examine the substances prepared as discs.
D. Examine the chromatograms obtained in the test for related substances in ultraviolet light at
E. Dissolve about 20 mg in a mixture of 5 ml of hydrochloric acid R and 10 ml of water R. Boil for
5 min, cool and add 2 ml of a 1 g/l solution of sodium nitrite R. Allow to stand for 1 min and add 1 ml
of a 5 g/l solution of sulphamic acid R and mix. Allow to stand for 1 min and add 1 ml of a 1 g/l
solution of naphthylethylenediamine dihydrochloride R. A reddish-violet colour is produced.
TESTS
Related substances Carry out the test protected from bright light and prepare the solutions immediately
before use.
Examine by thin-layer chromatography (2.2.27), using silica gel GF
254
Test solution (a). Dissolve 0.10 g in a mixture of 8 volumes of toluene R and 12 volumes of methanol R
and dilute to 5 ml with the same mixture of solvents.
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with a mixture of 8 volumes of toluene R and
12 volumes of methanol R.
Reference solution (a). Dissolve 5 mg of aminochlorobenzophenone R in a mixture of 8 volumes of
toluene R and 12 volumes of methanol R and dilute to 100 ml with the same mixture of solvents.
Reference solution (b). Dissolve 20 mg of chlordiazepoxide CRS in a mixture of 8 volumes of toluene R
09-10
Reference solution (c). Dilute 0.5 ml of test solution (a) to 100 ml with a mixture of 8 volumes of
toluene R and 12 volumes of methanol R.
Apply separately to the plate 5 l of reference solution (a), 5 l of reference solution (b), 5 l of
reference solution (c) and 5 l of test solution (b). To a fifth starting point, apply five quantities each
of 5 l of test solution (a), allowing to dry between applications. Develop over a path of 15 cm using
a mixture of 1 volume of water R, 4 volumes of diethylamine R, 10 volumes of alcohol R, 15 volumes
of ethyl acetate R and 70 volumes of toluene R. Allow the plate to dry in air. Examine in ultraviolet
light at 254 nm. Any spot in the chromatogram obtained with test solution (a), apart from the
solution (c) (0.1 per cent). Spray with a freshly prepared 10 g/l solution of sodium nitrite R in 1M
hydrochloric acid (about 10 ml). Dry the plate in a current of cold air and spray with a 4 g/l solution of
naphthylethylenediamine dihydrochloride R in alcohol R. Any violet spot corresponding to
aminochlorobenzophenone in the chromatogram obtained with test solution (a) is not more intense
than the spot in the chromatogram obtained with reference solution (a) (0.05 per cent).
100C to 105C.
ASSAY
Dissolve 0.2500 g, with heating if necessary, in 80 ml of anhydrous acetic acid R. Titrate with 0.1M
perchloric acid, determining the end-point potentiometrically (2.2.20).
16
H
14
ClN
3
O.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
09-11
Chlordiazepoxide Hydrochloride
corrected 1/01
N
N
NHMe
Cl
O
,HCl
C
16
H
14
ClN
3
O,HCl 336.2 438-41-5
Chlordiazepoxide Hydrochloride complies with the requirements of the 3rd edition of the European
Preparations
Chlordiazepoxide Capsules
Chlordiazepoxide Hydrochloride Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Chlordiazepoxide hydrochloride contains not less than 99.0 per cent and not more than the equiva-
lent of 101.0 per cent of 7-chloro-2-methylamino-5-phenyl-3H-1,4-benzodiazepine 4-oxide hydro-
CHARACTERS
A white or slightly yellow, crystalline powder, soluble in water, sparingly soluble in alcohol,
IDENTIFICATION
A. Prepare the solutions protected from bright light and immediately before use.
of the solution to 100.0 ml with 0.1M hydrochloric acid. Examined between 230 nm and 320 nm
absorbances at the maxima are 996 to 1058 and 280 to 298, respectively.
obtained with chlordiazepoxide hydrochloride CRS. Examine the substances prepared as discs.
D. Dissolve about 20 mg in a mixture of 5 ml of hydrochloric acid R and 10 ml of water R. Boil for
5 min, cool and add 2 ml of a 1 g/l solution of sodium nitrite R. Allow to stand for 1 min. Add 1 ml of
a 5 g/l solution of sulphamic acid R and mix. Allow to stand for 1 min and add 1 ml of a 1 g/l solution
of naphthylethylenediamine dihydrochloride R. A violet-red colour is produced.
E. Dissolve 50 mg in 5 ml of water R, add 1 ml of dilute ammonia R1 mix, allow to stand for 5 min
and filter. Acidify the filtrate with dilute nitric acid R. The solution gives reaction (a) of chlorides
(2.3.1).
TESTS
solution GY
6
(Method II, 2.2.2).
Related substances Carry out the following operations protected from bright light and prepare the
09-12
254
Test solution (a). Dissolve 0.1 g of the substance to be examined in a mixture of 3 volumes of dilute
ammonia R1 and 97 volumes of methanol R and dilute to 5 ml with the same mixture of solvents.
Test solution (b). Dilute 1.0 ml of test solution (a) to 10 ml with methanol R.
Reference solution (a). Dissolve 10 mg of aminochlorobenzophenone R in methanol R and dilute to
Reference solution (b). Dissolve 20 mg of chlordiazepoxide hydrochloride CRS in a mixture of 3 volumes
of dilute ammonia R1 and 97 volumes of methanol R and dilute to 10 ml with the same mixture of
solvents.
Reference solution (c). Dilute 0.5 ml of test solution (a) to 100 ml with methanol R.
Apply on a first starting point 5 l of reference solution (a), on a second starting point five quantities,
each of 5 l, of test solution (a), allowing to dry between applications, on a third starting point 5 l of
reference solution (c), on a fourth starting point 5 l of test solution (b) and on a fifth starting point
5 l of reference solution (b). Develop over a path of 15 cm using a mixture of 1 volume of
concentrated ammonia R, 14 volumes of methanol R and 85 volumes of chloroform R. Allow the plate to
dry in air and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with
test solution (a), apart from the principal spot, is not more intense than the spot in the chromatogram
obtained with reference solution (c) (0.1 per cent). Spray with a freshly prepared 10 g/l solution of
sodium nitrite R in 1M hydrochloric acid (about 10 ml), dry the plate in a current of cold air and spray
with a 4 g/l solution of naphthylethylenediamine dihydrochloride R in alcohol R. Any violet spot corres-
ponding to aminochlorobenzophenone in the chromatogram obtained with test solution (a) is not
more intense than the spot in the chromatogram obtained with reference solution (a) (0.1 per cent).
60C for 4 h.
ASSAY
Dissolve 0.250 g in 80 ml of anhydrous acetic acid R, heating if necessary. Cool and add 10 ml of
mercuric acetate solution R. Titrate with 0.1M perchloric acid, determining the end-point potentiometric-
ally (2.2.20).
16
H
15
Cl
2
N
3
O.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
09-13
Chlorhexidine Acetate
[CH
2
]
6
HN
HN
N N
N N
NH NH
NH NH
Cl
Cl
H H
H H
CH
3
COOH ,2
C
22
H
30
Cl
2
N
10
,2C
2
H
4
O
2
625.6 56-95-1
Chlorhexidine Acetate complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Chlorhexidine Diacetate [0657]. These requirements are reproduced after the heading Definition below.
Action and use Disinfectant.
Preparations
Chlorhexidine Irrigation Solution
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Chlorhexidine diacetate contains not less than 98.0 per cent and not more than the equivalent of
101.0 per cent of 1,1-hexamethylenebis[5-(4-chlorophenyl)biguanide] diacetate, calculated with
CHARACTERS
A white or almost white, microcrystalline powder, sparingly soluble in water, soluble in alcohol,
slightly soluble in glycerol and in propylene glycol.
IDENTIFICATION
obtained with chlorhexidine diacetate CRS.
B. Dissolve about 5 mg in 5 ml of a warm 10 g/l solution of cetrimide R and add 1 ml of strong sodium
hydroxide solution R and 1 ml of bromine water R. A deep red colour is produced.
C. Dissolve 0.3 g in 10 ml of a mixture of equal volumes of hydrochloric acid R and water R. Add
40 ml of water R, filter if necessary and cool in ice water. Make alkaline to titan yellow paper R by
adding dropwise and with stirring strong sodium hydroxide solution R and add 1 ml in excess. Filter,
wash the precipitate with water R until the washings are free from alkali and recrystallise from alcohol
(70 per cent V/V) R. Dry at 100C to 105C. The residue melts (2.2.14) at 132C to 136C.
D. It gives reaction (a) of acetates (2.3.1).
TESTS
Chloroaniline Dissolve 0.20 g of the substance to be examined in 25 ml of water R with shaking if
necessary. Add 1 ml of hydrochloric acid R and dilute to 30 ml with water R. Add rapidly and with
thorough mixing after each addition: 2.5 ml of dilute hydrochloric acid R, 0.35 ml of sodium nitrite
solution R, 2 ml of a 50 g/l solution of ammonium sulphamate R, 5 ml of a 1.0 g/l solution of
naphthylethylenediamine dihydrochloride R and 1 ml of alcohol R, dilute to 50.0 ml with water R and
allow to stand for 30 min. Any reddish-blue colour in the solution is not more intense than that in a
standard prepared at the same time in the same manner using a mixture of 10.0 ml of a 0.010 g/l
solution of chloroaniline R in dilute hydrochloric acid R and 20 ml of dilute hydrochloric acid R instead of
the solution of the substance to be examined (500 ppm).
Reference solution (a). Dissolve 15 mg of chlorhexidine for performance test CRS in the mobile phase and
09-14
Reference solution (b). Dilute 2.5 ml of the test solution to 100 ml with the mobile phase.
Reference solution (c). Dilute 2.0 ml of reference solution (b) to 10 ml with the mobile phase. Dilute
1.0 ml of this solution to 10 ml with the mobile phase.
a stainless steel column 0.2 m long and 4 mm in internal diameter, packed with octadecylsilyl
as mobile phase at a flow-rate of 1.0 ml per minute, a solution of 2.0 g of sodium
octanesulphonate R in a mixture of 120 ml of glacial acetic acid R, 270 ml of water R and 730 ml of
methanol R,
Equilibrate the column with the mobile phase for at least 1 h. Adjust the sensitivity of the system so
that the height of the principal peak in the chromatogram obtained with 10 l of reference solution
Inject 10 l of reference solution (a). The test is not valid unless the resulting chromatogram is
similar to the specimen chromatogram provided with chlorhexidine for performance test CRS in that the
peaks due to impurity-A and impurity-B precede that due to chlorhexidine. If necessary, adjust the
concentration of acetic acid in the mobile phase (increasing the concentration decreases the retention
times).
Inject separately 10 l of the test solution and 10 l each of reference solutions (b) and (c). Record
the chromatograms of reference solutions (b) and (c) until the peak due to chlorhexidine has been
eluted and record the chromatogram of the test solution for six times the retention time of the peak
due to chlorhexidine. In the chromatogram obtained with the test solution, the sum of the areas of all
the peaks, apart from the principal peak is not greater than the area of the principal peak in the
chromatogram obtained with reference solution (b) (2.5 per cent). Disregard any peak with a with a
relative retention time of 0.25 or less with respect to the principal peak and any peak whose area is
less than that of the principal peak in the chromatogram obtained with reference solution (c).
100C to 105C.
ASSAY
Dissolve 0.140 g in 100 ml of anhydrous acetic acid R and titrate with 0.1M perchloric acid. Determine
26
H
38
Cl
2
N
10
O
4
.
IMPURITIES
l
H
N
H
N
H
N
N
H
N
H
NH NH
NH
N
A. 1-(4-chlorophenyl)-5-[6-(3-cyanoguanidino)hexyl]biguanide,
Cl
H
N
H
N
H
N
N
H
N
H
NH NH
NH
NH
2
O
B. 1-[N-[6-[5-(4-chlorophenyl)biguanidino]hexyl]amidino]urea,
Cl
H
N
H
N
H
N
N
H
N
H
NH NH
NH
N
H
O
Cl
C. N,N-hexamethylenebis[1-amidino-3-(4-chlorophenyl)urea],
H
N
N
H
N
H
NH
N
H
O
Cl
Cl
H
N
N
NH
D. 5,5-di(4-chlorophenyl)-1,1-[6,6-[[[(4-chlorophenyl-amino)(imino)methyl]imino]-
methylenediamino]dihexamethylene]di(biguanide).
__________________________________________________________________________________________________________ Ph Eur
09-15
Chlorhexidine Gluconate Solution
C
22
H
30
Cl
2
N
10.
2C
6
H
12
O
7
898 18472-51-0
Chlorhexidine Gluconate Solution complies with the requirements of the 3rd edition of the European
Pharmacopoeia for Chlorhexidine Digluconate Solution [0658]. These requirements are reproduced after the
heading Definition below.
Preparations
Chlorhexidine Irrigation Solution
Chlorhexidine Mouthwash
Lidocaine and Chlorhexidine Gel/Lignocaine and Chlorhexidine Gel
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Chlorhexidine digluconate solution is an aqueous solution which contains not less than 190 g/l and
not more than 210 g/l of 1,1-hexamethylenebis[5-(4-chlorophenyl)-biguanide] di(D-digluconate).
CHARACTERS
An almost colourless or pale-yellowish liquid, miscible with water, soluble in acetone and in alcohol.
IDENTIFICATION
A. To 1 ml add 40 ml of water R, cool in iced water, make alkaline to titan yellow paper R by adding
dropwise and with stirring strong sodium hydroxide solution R and add 1 ml in excess. Filter, wash the
precipitate with water R until the washings are free from alkali and recrystallise from alcohol (70 per
cent V/V) R. Dry at 100C to 105C. Examine the residue by infrared absorption spectrophotometry
(2.2.24), comparing with the spectrum obtained with chlorhexidine CRS.
Test solution. Dilute 10.0 ml of the solution to be examined to 50 ml with water R.
Reference solution. Dissolve 25 mg of calcium gluconate CRS in 1 ml of water R.
volumes of alcohol R. Dry the plate at 100C for 20 min, allow to cool and spray with a 50 g/l
solution of potassium dichromate R in a 40 per cent m/m solution of sulphuric acid R. After 5 min, the
C. To 1 ml add 40 ml of water R, cool in iced water, make alkaline to titan yellow paper R by adding
dropwise and with stirring strong sodium hydroxide solution R and add 1 ml in excess. Filter, wash the
precipitate with water R until the washings are free from alkali and recrystallise from alcohol (70 per
cent V/V) R. Dry at 100C to 105C. The residue melts (2.2.14) at 132C to 136C.
D. To 0.05 ml add 5 ml of a 10 g/l solution of cetrimide R, 1 ml of strong sodium hydroxide solution R
and 1 ml of bromine water R; a deep red colour is produced.
TESTS
pH (2.2.3). Dilute 5.0 ml to 100 ml with carbon dioxide-free water R. The pH of the solution is 5.5 to
7.0.
Chloroaniline Dilute 2.0 ml to 100 ml with water R. To 10 ml of the solution add 2.5 ml of dilute
hydrochloric acid R and dilute to 20 ml with water R. Add rapidly and with thorough mixing after each
addition: 0.35 ml of sodium nitrite solution R, 2 ml of a 50 g/l solution of ammonium sulphamate R,
5 ml of a 1 g/l solution of naphthylethylenediamine dihydrochloride R, 1 ml of alcohol R, dilute to
50.0 ml with water R and allow to stand for 30 min. Any reddish-blue colour in the solution is not
greater than that in a standard prepared at the same time in the same manner using a mixture of
10.0 ml of a 0.010 g/l solution of chloroaniline R in dilute hydrochloric acid R and 10 ml of water R
instead of the dilution of the solution to be examined (0.25 per cent with reference to chlorhexidine
digluconate at a nominal concentration of 200 g/l).
Related substances Examined by liquid chromatography (2.2.29).
Test solution. Dilute 5.0 ml of the sample to be examined to 50.0 ml with the mobile phase. Dilute
09-16
Reference solution (c). Dilute 1.0 ml of reference solution (b) to 50 ml with the mobile phase.
as mobile phase at a flow-rate of 1.0 ml per minute, a solution of 2.0 g of sodium
octanesulphonate R in a mixture of 120 ml of glacial acetic acid R, 270 ml of water R and 730 ml of
methanol R,
Equilibrate the column with mobile phase for at least 1 hour. Adjust the sensitivity of the system so
peaks due to impurity-A and impurity-B precede that due to chlorhexidine. If necessary, adjust the
times).
the chromatograms of reference solutions (b) and (c) until the peak due to chlorhexidine has been
eluted and record the chromatogram of the test solution for six times the retention time of the peak
due to chlorhexidine. In the chromatogram obtained with the test solution, the sum of the areas of
the peaks, apart from the principal peak is not greater than the area of the principal peak in the
chromatogram obtained with reference solution (b) (3.0 per cent). Disregard any peak with a relative
retention time of 0.25 or less with respect to the principal peak and any peak whose area is less than
that of the principal peak in the chromatogram obtained with reference solution (c).
ASSAY
Determine the density (2.2.5) of the solution to be examined. Transfer 1.00 g to a 250 ml beaker and
add 50 ml of glacial acetic acid R. Titrate with 0.1M perchloric acid. Determine the end-point
34
H
54
Cl
2
N
10
O
14
.
STORAGE
IMPURITIES
H H
Cl
N N
NH NH
N
N
N
NH
H
H
H H
Cl
N N
NH NH
N
N
NH
NH
2
O
H
H
B. 1-[N-[6-[5-(4-chlorophenyl)biguanidino]hexyl]amidino]urea,
H H
Cl
N N
O NH
N
N
NH
N
O
Cl
H
H
H
N
H
N
H
NH
N
H
NH
Cl
N
H
N
H
N
H
NH
N
H
NH
Cl
N
H
N
NH
NH
Cl
2 2
D. 5,5-di(4-chlorophenyl)-1,1-[6,6-[[[(4-chlorophenylamino)(imino)methyl]imino]-
methylenediamino]dihexamethylene]di(biguanide).
__________________________________________________________________________________________________________ Ph Eur
09-17
Chlorhexidine Hydrochloride
1/01
C
22
H
30
Cl
2
N
10
,2HCl 578.4 3697-42-5
Chlorhexidine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
for Chlorhexidine Dihydrochloride [0659]. These requirements are reproduced after the heading Definition
below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Chlorhexidine dihydrochloride contains not less than 98.0 per cent and not more than the equiva-
lent of 101.0 per cent of 1,1-hexamethylenebis[5-(4-chlorophenyl)biguanide] dihydrochloride,
CHARACTERS
A white or almost white, crystalline powder, sparingly soluble in water and in propylene glycol,
very slightly soluble in alcohol.
IDENTIFICATION
obtained with chlorhexidine dihydrochloride CRS.
B. Dissolve about 5 mg in 5 ml of a warm 10 g/l solution of cetrimide R and add 1 ml of strong sodium
hydroxide solution R and 1 ml of bromine water R. A dark red colour is produced.
C. Dissolve 0.3 g in 10 ml of a mixture of equal volumes of hydrochloric acid R and water R. Add
40 ml of water R, filter if necessary and cool in iced water. Make alkaline to titan yellow paper R by
adding dropwise, and with stirring, strong sodium hydroxide solution R and add 1 ml in excess. Filter,
wash the precipitate with water R until the washings are free from alkali and recrystallise from alcohol
(70 per cent V/V) R. Dry at 100C to 105C. The residue melts (2.2.14) at 132C to 136C.
TESTS
Chloroaniline Disperse 0.20 g of the substance to be examined in 25 ml of water R and add 1 ml of
hydrochloric acid R. Shake to dissolve and dilute to 30 ml with water R. Add rapidly and with
thorough mixing after each addition: 2.5 ml of dilute hydrochloric acid R, 0.35 ml of sodium nitrite
solution R, 2 ml of a 50 g/l solution of ammonium sulphamate R, 5 ml of a 1.0 g/l solution of
naphthylethylenediamine dihydrochloride R and 1 ml of alcohol R; dilute to 50.0 ml with water R and
allow to stand for 30 min. Any reddish-blue colour in the solution is not more intense than that in a
standard prepared at the same time and in the same manner using a mixture of 10.0 ml of a 0.010 g/l
solution of chloroaniline R in dilute hydrochloric acid R and 20 ml of dilute hydrochloric acid R instead of
the solution of the substance to be examined (500 ppm).
Reference solution (c). Dilute 2.0 ml of reference solution (b) to 10 ml with the mobile phase. Dilute
1.0 ml of the solution to 10 ml with the mobile phase.
as mobile phase at a flow rate of 1.0 ml/min, a solution of 2.0 g of sodium octanesulphonate R in a
mixture of 120 ml of glacial acetic acid R, 270 ml of water R and 730 ml of methanol R,
Equilibrate the column with the mobile phase for at least 1 h. Adjust the sensitivity of the system so
09-18
peaks due to impurity A and impurity B precede that due to chlorhexidine. If necessary, adjust the
times).
the chromatograms of reference solutions (b) and (c) until the peak due to chlorhexidine is eluted
and record the chromatogram of the test solution for six times the retention time of the peak due to
chlorhexidine. In the chromatogram obtained with the test solution, the sum of the areas of the
peaks, apart from the principal peak, is not greater than the area of the principal peak in the chroma-
togram obtained with reference solution (b) (2.5 per cent). Disregard any peak with a relative
retention time of 0.25 or less, with respect to chlorhexidine, and any peak with an area less than that
of the principal peak in the chromatogram obtained with reference solution (c).
100C to 105C.
ASSAY
Dissolve 100.0 mg in 5 ml of anhydrous formic acid R and add 70 ml of acetic anhydride R. Titrate with
22
H
32
Cl
4
N
10
.
IMPURITIES
l
H
N
H
N
H
N
N
H
N
H
NH NH
NH
N
Cl
H
N
H
N
H
N
N
H
N
H
NH NH
NH
NH
2
O
B. 1-[N[6-[5-(4-chlorophenyl)biguanidino]hexyl]amidino]urea,
Cl
H
N
H
N
H
N
N
H
N
H
O NH
NH
N
H
O
Cl
N
H
N
H
NH
N
H
NH
Cl
N
H
N
H
N
H
NH
N
H
NH
Cl
N
H
N
NH
NH
Cl
2 2
D. 5,5-di(4-chlorophenyl)-1,1-[6,6-[[[(4-
chlorophenylamino)(imino)methyl]imino]methylenediamino]dihexamethylene]di(biguanide).
__________________________________________________________________________________________________________ Ph Eur
09-19
Chlorinated Lime
Definition Chlorinated Lime contains not less than 30.0% w/w of available chlorine, Cl.
Characteristics A dull white powder; odour, characteristic.
Partly soluble in water and in ethanol (96%).
Identification
A. Evolves chlorine copiously on the addition of 2M hydrochloric acid.
B. When shaken with water and filtered, the filtrate yields reaction C characteristic of calcium salts
and reaction A characteristic of chlorides, Appendix VI.
Assay Triturate 4 g with successive small quantities of water, dilute to 1000 ml with water and shake
thoroughly. Mix 100 ml of the resulting suspension with a solution containing 3 g of potassium iodide
in 100 ml of water, acidify with 5 ml of 6M acetic acid and titrate the liberated iodine with 0.1M
sodium thiosulphate VS. Each ml of 0.1M sodium thiosulphate VS is equivalent to 3.545 mg of available
chlorine, Cl.
Storage Chlorinated Lime should be kept in a well-closed container. On exposure to air it becomes
moist and gradually decomposes, carbon dioxide being absorbed and chlorine evolved.
09-20
Chlormethine Hydrochloride / Mustine Hydrochloride
Cl
N
Cl
Me
,HCl
C
5
H
11
Cl
2
N,HCl 192.5 55-86-7
Definition Chlormethine Hydrochloride is bis(2-chloroethyl)methylamine hydrochloride. It contains
5
H
11
Cl
2
N,HCl.
Characteristics A white or almost white crystalline powder or mass; hygroscopic; vesicant.
Very soluble in water.
Identification
A. Dissolve 50 mg in 5 ml of water and add 1 ml of 5M sodium hydroxide. Oily globules are produced
which dissolve on warming.
B. Dissolve 50 mg in 5 ml of water and add 0.02 ml of potassium tetraiodomercurate solution. A cream
precipitate is produced.
C. Melting point, about 108, Appendix V A.
Assay To 0.2 g add 15 ml of 1M ethanolic potassium hydroxide and 15 ml of water and boil under a
reflux condenser for 2 hours. Evaporate the solution to half its volume on a water bath, dilute to
150 ml with water, add 3 ml of nitric acid and 50 ml of 0.1M silver nitrate VS, shake vigorously and
filter. Wash the residue with water and titrate the excess of silver nitrate in the combined filtrate and
washings with 0.1M ammonium thiocyanate VS using 1 ml of ammonium iron(III) sulphate solution R2 as
indicator. Each ml of 0.1M silver nitrate VS is equivalent to 6.418 mg of C
5
H
11
Cl
2
N,HCl.
Storage Chlormethine Hydrochloride should be kept in a well-closed container and stored at a
temperature of 8 to 15.
Labelling The label states that the contents of the container are strongly vesicant.
Preparation
Chlormethine Injection/Mustine Injection
09-21
Chlorobutanol
Cl
3
C OH
Me Me
C
4
H
7
Cl
3
O,H
2
O 186.5 6001-64-5
Chlorobutanol complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Chlorobutanol Hemihydrate [0383]. These requirements are reproduced after the heading Definition below.
When chlorbutol is demanded, Chlorobutanol shall be supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Chlorobutanol hemihydrate contains not less than 98.0 per cent and not more than the equivalent of
101.0 per cent of 1,1,1-trichloro-2-methylpropan-2-ol, calculated with reference to the anhydrous
substance.
CHARACTERS
A white, crystalline powder or colourless crystals, sublimes readily, slightly soluble in water, very
soluble in alcohol and in ether, soluble in glycerol (85 per cent).
It melts at about 78C, determined without previous drying.
IDENTIFICATION
A. Add about 20 mg to a mixture of 1 ml of pyridine R and 2 ml of strong sodium hydroxide solution R.
Heat in a water-bath and shake. Allow to stand. The pyridine layer becomes red.
B. Add about 20 mg to 5 ml of ammoniacal silver nitrate solution R and warm slightly. A black
C. To about 20 mg add 3 ml of 1M sodium hydroxide and shake to dissolve. Add 5 ml of water R and
then, slowly, 2 ml of iodinated potassium iodide solution R. An odour of iodoform is evolved and a
yellowish precipitate is formed.
D. It contains 4.5 per cent to 5.5 per cent of water (see Tests).
TESTS
Solution S Dissolve 5 g in alcohol R and dilute to 10 ml with the same solvent.
5
(Method II, 2.2.2).
Acidity To 4 ml of solution S add 15 ml of alcohol R and 0.1 ml of bromothymol blue solution R1. Not
more than 1.0 ml of 0.01M sodium hydroxide is required to change the colour of the indicator to blue.
Chlorides (2.4.4). To 1 ml of solution S add 4 ml of alcohol R and dilute to 15 ml with water R. The
solution complies with the limit test for chlorides (100 ppm). When preparing the standard, replace
the 5 ml of water R by 5 ml of alcohol R.
ASSAY
Dissolve 0.100 g in 20 ml of alcohol R. Add 10 ml of dilute sodium hydroxide solution R, heat in a
water-bath for 5 min and cool. Add 20 ml of dilute nitric acid R, 25.0 ml of 0.1M silver nitrate and 2 ml
of dibutyl phthalate R and shake vigorously. Add 2 ml of ferric ammonium sulphate solution R2 and
titrate with 0.1M ammonium thiocyanate until an orange colour is obtained.
4
H
7
Cl
3
O.
STORAGE
Store in an airtight container, at a temperature of 8C to 15C.
__________________________________________________________________________________________________________ Ph Eur
09-22
Anhydrous Chlorobutanol
C
4
H
7
Cl
3
O 177.5 57-15-8
Anhydrous Chlorobutanol complies with the requirements of the 3rd edition of the European Pharmacopoeia
When anhydrous chlorbutol is demanded, Anhydrous Chlorobutanol shall be supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Anhydrous chlorobutanol contains not less than 98.0 per cent and not more than the equivalent of
101.0 per cent of 1,1,1-trichloro-2-methylpropan-2-ol, calculated with reference to the anhydrous
substance.
CHARACTERS
It has the characters described in the monograph on Chlorobutanol hemihydrate (383).
IDENTIFICATION
It complies with the identification tests prescribed in the monograph on Chlorobutanol hemihydrate
(383) except for identification test D which is replaced by the following:
D. It contains not more than 1.0 per cent of water (see Tests).
TESTS
It complies with the tests prescribed in the monograph on Chlorobutanol hemihydrate (383) with the
following modifications:
Chlorides (2.4.4). Dissolve 0.17 g in 5 ml of alcohol R and dilute to 15 ml with water R. The
solution complies with the limit test for chlorides (300 ppm). When preparing the standard, replace
the 5 ml of water R by 5 ml of alcohol R.
of water.
ASSAY
Carry out the assay prescribed in the monograph on Chlorobutanol hemihydrate (383).
4
H
7
Cl
3
O.
STORAGE
Store in an airtight container, at a temperature of 8C to 15C.
__________________________________________________________________________________________________________ Ph Eur
09-23
Chlorocresol
OH
Cl
Me
C
7
H
7
ClO 142.6 59-50-7
Chlorocresol complies with the requirements of the 3rd edition of the European Pharmacopoeia [0384]. These
Action and use Antiseptic; antimicrobial preservative.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Chlorocresol contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent
of 4-chloro-3-methylphenol.
CHARACTERS
A white or almost white, crystalline powder or compacted crystalline masses supplied as pellets or
colourless or white crystals, slightly soluble in water, very soluble in alcohol, freely soluble in ether
and in fatty oils. It dissolves in solutions of alkali hydroxides.
IDENTIFICATION
B. To 0.1 g add 0.2 ml of benzoyl chloride R and 0.5 ml of dilute sodium hydroxide solution R. Shake
vigorously until a white, crystalline precipitate is formed. Add 5 ml of water R and filter. The
precipitate, recrystallised from 5 ml of methanol R and dried at 70C, melts (2.2.14) at 85C to 88C.
C. To 5 ml of solution S (see Tests) add 0.1 ml of ferric chloride solution R1. A bluish colour is
produced.
TESTS
Solution S To 3.0 g, finely powdered, add 60 ml of carbon dioxide-free water R, shake for 2 min and
filter.
solution is clear (2.2.1) and not more intensely coloured than reference solution BY
6
(Method II,
2.2.2).
Acidity To 10 ml of solution S add 0.1 ml of methyl red solution R. The solution is orange or red. Not
more than 0.2 ml of 0.01M sodium hydroxide is required to produce a pure yellow colour.
the same solvent.
a glass column 1.80 m long and 3 mm to 4 mm in internal diameter packed with silanised
diatomaceous earth for gas chromatography R impregnated with 3 per cent m/m to 5 per cent m/m
of polymethylphenylsiloxane R,
the detector at 230C. Continue the chromatography for three times the period of time (about 8 min)
required for the appearance of the peak corresponding to chlorocresol. In the chromatogram
obtained, the sum of the areas of the peaks apart from that corresponding to chlorocresol does not
exceed 1 per cent of the total area of the peaks. Disregard the peak corresponding to the solvent.
Non-volatile matter Evaporate 2.0 g to dryness on a water-bath and dry the residue at 100C to
105C. The residue weighs not more than 2 mg (0.1 per cent).
ASSAY
In a ground-glass-stoppered flask, dissolve 70.0 mg in 30 ml of glacial acetic acid R. Add 25.0 ml of
09-24
0.0167M potassium bromate, 20 ml of a 150 g/l solution of potassium bromide R and 10 ml of hydrochloric
acid R. Allow to stand protected from light for 15 min. Add 1 g of potassium iodide R and 100 ml of
water R. Titrate with 0.1M sodium thiosulphate, shaking vigorously and using 1 ml of starch solution R,
added towards the end of the titration, as indicator. Carry out a blank titration.
1 ml of 0.0167M potassium bromate is equivalent to 3.565 mg of C
7
H
7
ClO.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
09-25
Chloroform
CHCl
3
119.4 67-66-3
Definition Chloroform is trichloromethane to which either 1.0 to 2.0% of ethanol or 50 mg per
litre of amylene has been added.
Characteristics A colourless, volatile liquid; odour, characteristic.
Slightly soluble in water; miscible with absolute ethanol, with ether, with fixed and volatile oils and
with most organic solvents.
Identification The infrared absorption spectrum, Appendix II A, of the substance being examined after
washing with water and drying with anhydrous sodium sulphate is concordant with the reference spectrum
of chloroform (RS 053).
Distillation range Not more than 5.0% v/v distils below 60 and the remainder distils at 60 to 62,
Appendix V C.
Acidity or alkalinity Shake 10 ml with 20 ml of freshly boiled and cooled water for 3 minutes and
allow to separate. To 5 ml of the aqueous layer add 0.1 ml of neutral litmus solution. The colour
produced is the same as that produced on adding 0.1 ml of the neutral litmus solution to 5 ml of
freshly boiled and cooled water.
Chloride To 5 ml of the aqueous layer obtained in the test for Acidity or alkalinity add 5 ml of water
and 0.2 ml of silver nitrate solution. The solution is clear, Appendix IV A.
Free chlorine To 10 ml of the aqueous layer obtained in the test for Acidity or alkalinity add 1 ml of
a 5.0% w/v solution of zinc iodide and 0.1 ml of starch mucilage. No blue colour is produced.
Aldehyde Shake 5 ml with 5 ml of water and 0.2 ml of alkaline potassium tetraiodomercurate solution in
a glass-stoppered flask and allow to stand in the dark for 15 minutes. Not more than a pale yellow
colour is produced.
Foreign chlorine compounds Shake 20 ml for 5 minutes with 10 ml of sulphuric acid in a glass-
stoppered flask previously rinsed with sulphuric acid, allow to stand in the dark for 30 minutes and
discard the acid layer. Shake 15 ml of the chloroform layer with 30 ml of water in a glass-stoppered
flask for 3 minutes and allow to separate. To the aqueous layer add 0.2 ml of silver nitrate solution and
allow to stand in the dark for 5 minutes. No opalescence is produced.
Related substances Carry out the method for gas chromatography, Appendix III B, injecting 0.1 l
of each of the following solutions. Solution (1) contains 0.2% v/v of carbon tetrachloride, 0.2% v/v of
1,1,1-trichloroethane (internal standard), 0.2% v/v of dichloromethane, 0.2% v/v of ethanol, 0.5% v/v of
bromochloromethane and 0.2% v/v of the substance being examined in propan-1-ol. Solution (2) is the
substance being examined. Solution (3) contains 0.2% v/v of the internal standard in the substance
being examined. Solution (4) is propan-1-ol.
The chromatographic procedure may be carried out using a glass column (4 m 3.0 mm) packed
with acid-washed kieselguhr (60 to 100 mesh) coated with 15% w/w of di-2-cyanoethyl ether and
maintained at 40 with both the inlet port and the detector at 100 and a flow rate of 30 ml per
minute for the carrier gas.
The test is not valid unless the column efficiency, determined using the chloroform peak in the
chromatogram obtained with solution (1), is greater than 700 plates per metre and the total number
of plates is greater than 2,500.
In the chromatogram obtained with solution (1) the peaks, in order of emergence, are due to
carbon tetrachloride, 1,1,1-trichloroethane, dichloromethane, chloroform, ethanol,
bromochloromethane and propan-1-ol. (solvent).
Using the chromatogram obtained with solution (4), make any corrections due to the contribution
of secondary peaks from the solvent to the peaks in the chromatogram obtained with solution (1).
In the chromatogram obtained with solution (3), the ratio of the areas of any peaks due to carbon
tetrachloride, dichloromethane and bromochloromethane to the area of the peak due to the internal
standard is not greater than the corresponding ratios in the chromatogram obtained with solution (1)
and the ratio of the area of any other secondary peak that elutes prior to the solvent peak, except for
the peak corresponding to ethanol, to the area of the peak due to the internal standard is not greater
than the ratio of the area of the peak due to chloroform to the area of the peak due to the internal
standard in the chromatogram obtained with solution (1).
Calculate the percentage content of each of the specified impurities and also calculate the
percentage content of each of any other impurities assuming the same response per unit volume as for
chloroform. The total content of all impurities is not more than 1.0% v/v.
Ethanol Carry out the following test for Chloroform that contains ethanol. Carry out the method for
gas chromatography, Appendix III B, injecting 0.1 l of each of the following solutions. Solution (1)
contains 1.0% v/v of absolute ethanol and 1.0% v/v of propan-1-ol (internal standard) in water. Solution
(2) is the substance being examined. Solution (3) contains 1.0% v/v of the internal standard in the
substance being examined.
09-26
The chromatographic procedure described under Related substances may be used.
The test is not valid unless the height of the trough separating the ethanol peak from the chloro-
form peak in the chromatogram obtained with solution (2) is less than 15% of the height of the
ethanol peak.
Calculate the percentage content of ethanol from the areas of the peaks due to ethanol and the
internal standard in the chromatograms obtained with solutions (1) and (3).
Non-volatile matter 25 ml, when evaporated to dryness and dried at 105, leaves not more than
1 mg of residue.
Storage Chloroform should be kept in a well-closed container with a glass stopper or other suitable
closure and protected from light.
Labelling The label states whether it contains ethanol or amylene.
Action and use General anaesthetic; antimicrobial preservative.
Preparations
Chloroform Spirit
Chloroform Water
Double-strength Chloroform Water
IMPURITIES
A. carbon tetrachloride,
B. dichloromethane,
C. bromochloromethane
09-27
Chloroquine Phosphate
N Cl
HN
H Me
NEt
2
and enantiomer

,2H
3
PO
4
C
18
H
26
ClN
3
,2H
3
PO
4
515.9 50-63-5
Chloroquine Phosphate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Antimalarial.
Preparation
Chloroquine Phosphate Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Chloroquine phosphate contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of (RS)-7-chloro-4-(4-diethylamino-1-methylbutylamino)quinoline bis(dihydrogen
phosphate), calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white, crystalline powder, hygroscopic, freely soluble in water, very slightly soluble
in alcohol, in ether and in methanol.
It exists in two forms, one of which melts at about 195C and the other at about 218C.
IDENTIFICATION
shows absorption maxima at 220 nm, 235 nm, 256 nm, 329 nm and 342 nm. The specific
absorbances at the maxima are respectively 600 to 660, 350 to 390, 300 to 330, 325 to 355 and 360
to 390.
obtained with the base isolated from chloroquine sulphate CRS. Record the spectra using solutions
prepared as follows: dissolve separately 0.1 g of the substance to be examined and 80 mg of the
reference substance in 10 ml of water R, add 2 ml of dilute sodium hydroxide solution R and shake with
two quantities, each of 20 ml, of chloroform R; combine the chloroform layers, wash with water R, dry
over anhydrous sodium sulphate R evaporate to dryness and dissolve the residues separately each in
2 ml of chloroform R.
C. Dissolve 25 mg in 20 ml of water R and add 8 ml of picric acid solution R1. The precipitate, washed
with water R, with alcohol R and finally with ether R, melts (2.2.14) at 206C to 209C.
D. Dissolve 0.1 g in 10 ml of water R, add 2 ml of dilute sodium hydroxide solution R and shake with
two quantities, each of 20 ml, of chloroform R. The aqueous layer, acidified by the addition of nitric
acid R, gives reaction (b) of phosphates (2.3.1).
TESTS
solution BY
5
or GY
5
(Method II, 2.2.2).
254
R as the
coating substance.
same solvent.
09-28
10 volumes of diethylamine R, 40 volumes of cyclohexane R and 50 volumes of chloroform R. Allow the
togram obtained with reference solution (a) (1.0 per cent) and not more than one such spot is more
intense than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent).
Heavy metals (2.4.8). Dissolve 2.0 g in 10 ml of water R. Add 5 ml of concentrated ammonia R and
shake with 40 ml of ether R. Filter the aqueous layer and neutralise the filtrate with glacial acetic
acid R. Heat on a water-bath to eliminate ether, allow to cool and dilute to 20.0 ml with water R.
12 ml of this solution complies with limit test A for heavy metals (20 ppm). Prepare the standard
using lead standard solution (2 ppm Pb) R.
ASSAY
18
H
32
ClN
3
O
8
P
2
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
09-29
Chloroquine Sulphate
N
Cl
HN
H Me
NEt
2
and enantiomer

,H
2
SO
4
C
18
H
26
ClN
3
,H
2
SO
4
,H
2
O 436.0 132-73-0
Chloroquine Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0545].
Preparations
Chloroquine Sulphate Injection
Chloroquine Sulphate Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Chloroquine sulphate contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of (RS)-7-chloro-4-(4-diethylamino-1-methylbutylamino)quinoline sulphate,
CHARACTERS
A white or almost white, crystalline powder, freely soluble in water and in methanol, very slightly
It melts at about 208C (instantaneous method).
IDENTIFICATION
shows absorption maxima at 220 nm, 235 nm, 256 nm, 329 nm and 342 nm. The specific
absorbances at the maxima are respectively 730 to 810, 430 to 470, 370 to 410, 400 to 440 and 430
to 470.
obtained with the base isolated from chloroquine sulphate CRS. Record the spectra using solutions
prepared as follows: dissolve separately 0.1 g of the substance to be examined and of the reference
substance in 10 ml of water R, add 2 ml of dilute sodium hydroxide solution R and shake with two
quantities, each of 20 ml, of chloroform R; combine the chloroform layers, wash with water R, dry over
anhydrous sodium sulphate R, evaporate to dryness and dissolve the residues separately each in 2 ml of
chloroform R.
C. Dissolve 25 mg in 20 ml of water R and add 8 ml of picric acid solution R1. The precipitate, washed
with water R, with alcohol R and finally with ether R, melts (2.2.14) at 206C to 209C.
D. It gives reaction (a) of sulphates (2.3.1).
TESTS
solution BY
5
or GY
5
(Method II, 2.2.2).
254
R as the
coating substance.
same solvent.
09-30
10 volumes of diethylamine R, 40 volumes of cyclohexane R and 50 volumes of chloroform R. Allow the
togram obtained with reference solution (a) (1.0 per cent) and not more than one such spot is more
intense than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent).
Heavy metals (2.4.8). Dissolve 2.0 g in 10 ml of water R. Add 5 ml of concentrated ammonia R and
shake with 40 ml of ether R. Filter the aqueous layer and neutralise the filtrate with glacial acetic
acid R. Heat on a water-bath to eliminate ether, allow to cool and dilute to 20.0 ml with water R.
12 ml of this solution complies with limit test A for heavy metals (20 ppm). Prepare the standard
using lead standard solution (2 ppm Pb) R.
ASSAY
18
H
28
ClN
3
O
4
S.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
09-31
Chlorothiazide
S
NH
N
O O
S
H
2
N
O O
Cl
C
7
H
6
ClN
3
O
4
S
2
295.7 58-94-6
Chlorothiazide complies with the requirements of the 3rd edition of the European Pharmacopoeia [0385]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Chlorothiazide contains not less than 98.0 per cent and not more than the equivalent of 102.0 per
cent of 6-chloro-2H-1,2,4-benzothiadiazine-7-sulphonamide-1,1-dioxide, calculated with reference
CHARACTERS
A white or almost white, crystalline powder, very slightly soluble in water, sparingly soluble in
acetone, slightly soluble in alcohol. It dissolves in dilute solutions of alkali hydroxides.
IDENTIFICATION
A. Dissolve 80.0 mg in 100 ml of 0.1M sodium hydroxide and dilute to 1000.0 ml with water R. Dilute
10.0 ml of this solution to 100.0 ml with 0.01M sodium hydroxide. Examined between 220 nm and
320 nm (2.2.25), the solution shows two absorption maxima, at 225 nm and 292 nm, and a shoulder
at about 310 nm. The specific absorbances at the maxima are 725 to 800 and 425 to 455,
respectively.
obtained with chlorothiazide CRS.
254
same solvent.
Reference solution. Dissolve 25 mg of chlorothiazide CRS in acetone R and dilute to 5 ml with the same
solvent.
Apply separately to the plate 2 l of each solution. Develop over a path of 10 cm using ethyl acetate R.
Dry the plate in a current of air and examine in ultraviolet light at 254 nm. The principal spot in the
D. To 0.1 g add a pellet of sodium hydroxide R and heat strongly. Gas is evolved which turns red litmus
paper R blue. After cooling, take up the residue with 10 ml of dilute hydrochloric acid R. Gas is evolved
which turns lead acetate paper R black.
TESTS
Solution S To 1.0 g of the powdered substance to be examined add 50 ml of water R, shake for
2 min and filter.
Acidity or alkalinity To 10 ml of solution S add 0.2 ml of 0.01M sodium hydroxide and 0.15 ml of
methyl red solution R. The solution is yellow. Not more than 0.4 ml of 0.01M hydrochloric acid is
required to change the colour of the indicator to red.
coating substance.
same solvent.
Reference solution. Dilute 1 ml of the test solution to 100 ml with acetone R.
15 volumes of 2-propanol R and 85 volumes of ethyl acetate R. Dry the plate in a current of air until
09-32
the odour of solvent is no longer perceptible (about 10 min) and spray with a mixture of equal
volumes of alcoholic solution of sulphuric acid R and alcohol R; use about 10 ml for a plate 200 mm
square and spray in small portions, allowing the solvent to evaporate each time to avoid excessive
wetting. Heat at 100C to 105C for 30 min and immediately place the plate above, but not in
contact with, 10 ml of a saturated solution of sodium nitrite R in a glass tank. Carefully add 0.5 ml of
sulphuric acid R to the sodium nitrite solution, close the tank, and allow to stand for 15 min. Remove
the plate, heat in a ventilated oven at 40C for 15 min and spray with three quantities, each of 5 ml,
of a freshly prepared 5 g/l solution of naphthylethylenediamine dihydrochloride R in alcohol R. Examine
the plate by transmitted light. Any spot in the chromatogram obtained with the test solution, apart
from the principal spot, is not more intense than the spot in the chromatogram obtained with the
Chlorides (2.4.4). 15 ml of solution S complies with the limit test for chlorides (160 ppm).
100C to 105C.
ASSAY
Dissolve 0.250 g in 50 ml of dimethylformamide R. Titrate with 0.1M tetrabutylammonium hydroxide
determining the end-point potentiometrically (2.2.20) at the first point of inflexion. Carry out a blank
titration.
7
H
6
ClN
3
O
4
S
2
.
__________________________________________________________________________________________________________ Ph Eur
09-33
Chloroxylenol
OH
Cl
Me Me
C
8
H
9
ClO 156.6 88-04-0
Definition Chloroxylenol is 4-chloro-3,5-xylenol. It contains not less than 98.0% and not more than
103.0% of C
8
H
9
ClO.
Characteristics White or cream crystals or crystalline powder with a characteristic odour. It is
volatile in steam.
Very slightly soluble in water; freely soluble in ethanol (96%); soluble in ether, in terpenes and in
fixed oils. It dissolves in solutions of the alkali hydroxides.
Identification
chloroxylenol (RS 055).
B. Dissolve 0.1 g in 5 ml of chloroform and add 0.5 ml of a filtered 1% w/v solution of anhydrous
iron(III) chloride in chloroform and 0.1 ml of pyridine. A blue colour is produced.
C. To 5 ml of a saturated solution in water add 0.5 ml of iron(III) chloride solution R1. No blue colour
is produced.
D. Mix 50 mg with 0.5 g of anhydrous sodium carbonate and ignite strongly, cool, boil the residue with
5 ml of water, acidify with nitric acid, filter and add silver nitrate solution. A white precipitate is
produced.
Related substances Carry out the method for gas chromatography, Appendix III B, using solutions
in chloroform containing (1) 2.0% w/v of the substance being examined and (2) 2.0% w/v of the
substance being examined and 0.040% w/v of 4-chloro-o-cresol (internal standard).
with acid-washed diatomaceous support (80 to 100 mesh) coated with 3% w/w of polyethylene glycol
(Carbowax 20M is suitable) and maintained at 160.
In the chromatogram obtained with solution (2) the sum of the areas of any secondary peaks is not
greater than the area of the peak due to the internal standard.
Assay Dissolve 70 mg in 30 ml of glacial acetic acid, add 25 ml of 0.0167M potassium bromate VS,
20 ml of a 15% w/v solution of potassium bromide and 10 ml of hydrochloric acid, stopper the flask and
allow to stand protected from light for 15 minutes. Add 1 g of potassium iodide and 100 ml of water
and titrate with 0.1M sodium thiosulphate VS, shaking vigorously and using 1 ml of starch solution,
added towards the end of the titration, as indicator. Repeat the operation without the substance
being examined. The difference between the titrations represents the amount of potassium bromate
required. Each ml of 0.0167M potassium bromate VS is equivalent to 3.915 mg of C
8
H
9
ClO.
Preparation
Chloroxylenol Solution
Action and use Antiseptic; disinfectant.
09-34
Chlorphenamine Maleate / Chlorpheniramine Maleate
corrected 1/01
COOH
COOH
and enantiomer
,
N
Cl
NMe
2
H
C
16
H
19
ClN
2
,C
4
H
4
O
4
390.9 113-92-8
Chlorphenamine Maleate complies with the requirements of the 3rd edition of the European Pharmacopoeia
1
Preparations
Chlorphenamine Injection/Chlorpheniramine Injection
Chlorphenamine Oral Solution/Chlorpheniramine Oral Solution
Chlorphenamine Tablets/Chlorpheniramine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Chlorphenamine maleate contains not less than 98.0 per cent and not more than the equivalent of
101.0 per cent of (RS)-3-(4-chlorophenyl)-N,N-dimethyl-3-pyrid-2-ylpropanamine hydrogen (Z)-
butenedioate, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder, freely soluble in water, soluble in alcohol and slightly soluble in ether.
IDENTIFICATION
B. Dissolve 30.0 mg in 0.1M hydrochloric acid and dilute to 100.0 ml with the same acid. Dilute
10.0 ml of this solution to 100.0 ml with 0.1M hydrochloric acid. Examined between 230 nm and
obtained with chlorphenamine maleate CRS. Examine the substances prepared as discs.
D. Dissolve 0.1 g in 10 ml of water R and add dropwise and with shaking 25 ml of picric acid
solution R. Collect the precipitate on a sintered-glass filter. The precipitate, washed with 3 ml of
alcohol R, recrystallised from a mixture of equal volumes of alcohol R and water R and dried at 100C
to 105C, melts (2.2.14) at 196C to 200C.
E. To 0.2 g add 3 ml of water R and 1 ml of strong sodium hydroxide solution R. Shake with three
quantities, each of 5 ml, of ether R. To 0.1 ml of the aqueous layer add a solution of 10 mg of
resorcinol R in 3 ml of sulphuric acid R. Heat on a water-bath for 15 min. No colour develops. To the
remainder of the aqueous layer add 2 ml of bromine solution R. Heat in a water-bath for 15 min, heat
to boiling and cool. To 0.2 ml of the solution add a solution of 10 mg of resorcinol R in 3 ml of
sulphuric acid R and heat on a water-bath for 15 min. A blue colour develops.
TESTS
solution BY
6
(Method II, 2.2.2).
254
R as the
coating substance.
09-35
Test solution. Dissolve 0.5 g of the substance to be examined in chloroform R and dilute to 10 ml with
the same solvent.
Reference solution. Dilute 1 ml of the test solution to 50 ml with chloroform R. Dilute 1 ml of this
solution to 10 ml with chloroform R.
of 10 volumes of diethylamine R, 40 volumes of chloroform R and 50 volumes of cyclohexane R. Allow
the plate to dry in air and examine in ultraviolet light at 254 nm. Any spot in the chromatogram
obtained with the test solution, apart from the principal spot, is not more intense than the spot in the
chromatogram obtained with the reference solution (0.2 per cent). Disregard any spots remaining on
the starting-line.
100C to 105C for 4 h.
ASSAY
20
H
23
ClN
2
O
4.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
09-36
Chlorpromazine
N
S
Cl
NMe
2
C
17
H
19
ClN
2
S 318.9 50-53-3
Definition Chlorpromazine is [3-(2-chlorophenothiazin-10-yl)propyl]dimethylamine. It contains not
17
H
19
ClN
2
S, calculated with reference to the dried
substance.
Characteristics A white or creamy white powder or waxy solid; odourless or almost odourless.
Practically insoluble in water; very soluble in chloroform; freely soluble in ethanol (96%) and in ether.
Identification
chlorpromazine (RS 056).
B. Complies with the test for identification of phenothiazines, Appendix III A, using chlorpromazine
hydrochloride EPCRS to prepare solution (2).
Related substances Complies with the test for related substances in phenothiazines, Appendix III A,
using mobile phase A.
Loss on drying When dried to constant weight over phosphorus pentoxide at a pressure not exceeding
0.7 kPa, loses not more than 0.5% of its weight. Use 1 g.
Assay Dissolve 0.8 g in 300 ml of acetone and carry out Method I for non-aqueous titration, Appendix
VIII A, using 3 ml of a saturated solution of methyl orange in acetone as indicator. Each ml of 0.1M
perchloric acid VS is equivalent to 31.89 mg of C
17
H
19
ClN
2
S.
Storage Chlorpromazine should be kept in a well-closed container and protected from light.
Preparation
Chlorpromazine Suppositories
09-37
Chlorpromazine Hydrochloride
N
S
Cl
NMe
2
,HCl
C
17
H
19
ClN
2
S,HCl 355.3 69-09-0
Chlorpromazine Hydrochloride complies with the requirements of the 3rd edition of the European
Action and use Antipsychotic; antiemetic.
Preparations
Chlorpromazine Injection
Chlorpromazine Oral Solution
Chlorpromazine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Chlorpromazine hydrochloride contains not less than 99.0 per cent and not more than the equivalent
of 101.0 per cent of 2-chloro-10-(3-dimethylaminopropyl)phenothiazine hydrochloride, calculated
CHARACTERS
A white or almost white, crystalline powder, very soluble in water, freely soluble in alcohol and
practically insoluble in ether. It decomposes on exposure to air and light.
IDENTIFICATION
A. Prepare the solutions protected from bright light and measure the absorbances immediately.
of the solution to 100.0 ml with 0.1M hydrochloric acid. Examined between 230 nm and 340 nm
(2.2.25), the solution shows two absorption maxima, at 254 nm and 306 nm respectively. The
specific absorbance at the maximum at 254 nm is 890 to 960.
obtained with chlorpromazine hydrochloride CRS. Examine the substances as 60 g/l solutions in
methylene chloride R using a 0.1 mm cell.
C. It complies with the identification test for phenothiazines by thin-layer chromatography (2.3.3).
TESTS
The pH of the freshly prepared solution is 3.5 to 4.5.
Related substances Carry out the test protected from bright light.
254
Test solution. Dissolve 0.2 g of the substance to be examined in a mixture of 5 volumes of
diethylamine R and 95 volumes of methanol R and dilute to 10 ml with the same mixture of solvents.
Prepare immediately before use.
Reference solution. Dilute 1 ml of the test solution to 200 ml with a mixture of 5 volumes of
diethylamine R and 95 volumes of methanol R.
10 volumes of acetone R, 10 volumes of diethylamine R and 80 volumes of cyclohexane R. Allow the
plate to dry in air and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained
togram obtained with the reference solution (0.5 per cent). Disregard any spot at the starting point.
09-38
100C to 105C.
ASSAY
potentiometric titration (2.2.20), using 0.1M sodium hydroxide. Read the volume of 0.1M sodium
hydroxide added between the two points of inflexion.
17
H
20
Cl
2
N
2
S.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
09-39
Chlorpropamide
S
N
O
O O
Cl
NHPr
n
H
C
10
H
13
ClN
2
O
3
S 276.7 94-20-2
Chlorpropamide complies with the requirements of the 3rd edition of the European Pharmacopoeia [1087].
Action and use Hypoglycaemic.
Preparation
Chlorpropamide Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Chlorpropamide contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of 1-[(4-chlorophenyl)sulphonyl]-3-propylurea, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder, practically insoluble in water, freely soluble in acetone and in methylene
chloride, soluble in alcohol. It dissolves in dilute solutions of alkali hydroxides.
IDENTIFICATION
B. Dissolve 0.10 g in methanol R and dilute to 50.0 ml with the same solvent. Dilute 5.0 ml of the
solution to 100.0 ml with 0.01M hydrochloric acid. Dilute 10.0 ml of the solution to 100.0 ml with
0.01M hydrochloric acid. Examined between 220 nm and 350 nm (2.2.25), the solution shows an
absorption maximum at 232 nm. The specific absorption at the maximum is 570 to 630.
obtained with chlorpropamide CRS. Examine the substances prepared as discs. If the spectra obtained
show differences, dissolve the substance to be examined and the reference substance in methylene
chloride R, evaporate to dryness and record the new spectra using the residues.
D. Heat 0.1 g with 2 g of anhydrous sodium carbonate R until a dull red colour appears for 10 min.
Allow to cool, extract the residue with about 5 ml of water R, dilute to 10 ml with water R and filter.
The solution gives the reaction (a) of chloride (2.3.1).
TESTS
the same solvent.
Reference solution (a). Dissolve 15 mg of 4-chlorobenzenesulphonamide R (chlorpropamide impurity A)
in acetone R and dilute to 100 ml with the same solvent.
Reference solution (b). Dissolve 15 mg of chlorpropamide impurity B CRS in acetone R and dilute to
Reference solution (c). Dilute 0.3 ml of the test solution to 100 ml with acetone R.
Reference solution (d). Dilute 5 ml of reference solution (c) to 15 ml with acetone R.
Reference solution (e). Dissolve 0.10 g of the substance to be examined, 5 mg of 4-chlorobenzene-
sulphonamide R and 5 mg of chlorpropamide impurity B CRS in acetone R and dilute to 10 ml with the
same solvent.
Apply to the plate 5 l of each solution. Develop over a path of 15 cm using a mixture of 11.5
volumes of concentrated ammonia R, 30 volumes of cyclohexane R, 50 volumes of methanol R and 100
volumes of methylene chloride R. Allow the plate to dry in a current of cold air, heat at 110C for
09-40
10 min. At the bottom of a chromatography tank, place an evaporating dish containing a mixture of
1 volume of hydrochloric acid R, 1 volume of water R and 2 volumes of a 50 g/l solution of potassium
permanganate R, close the tank and allow to stand for 15 min. Place the dried hot plate in the tank
and close the tank. Leave the plate in contact with the chlorine vapour for 2 min. Withdraw the plate
and place it in a current of cold air until the excess of chlorine is removed and an area of coating
below the points of application does not give a blue colour with a drop of potassium iodide and starch
solution R. Spray with potassium iodide and starch solution R. In the chromatogram obtained with the
test solution: any spot corresponding to impurity A is not more intense than the spot in the chroma-
togram obtained with reference solution (a) (0.3 per cent); any spot corresponding to impurity B is
not more intense than the spot in the chromatogram obtained with reference solution (b) (0.3 per
cent); any spot, apart from the principal spot and any spot corresponding to impurity A and B, is not
more intense than the spot in the chromatogram obtained with reference solution (c) (0.3 per cent);
not more than two such spots are more intense than the spot in the chromatogram obtained with
reference solution (d) (0.1 per cent). The test is not valid unless the chromatogram obtained with
reference solution (e) shows three clearly separated spots with approximate R
f
values of 0.4, 0.6 and
0.9 corresponding to chlorpropamide, impurity A and impurity B respectively.
acetone R and dilute to 20 ml with the same mixture of solvents. 12 ml of solution complies with limit
test B for heavy metals (20 ppm). Prepare the standard using lead standard solution (2 ppm Pb)
prepared by diluting lead standard solution (100 ppm Pb) R with a mixture of 15 volumes of water R
and 85 volumes of acetone R.
100C to 105C.
ASSAY
Dissolve 0.250 g in 50 ml of alcohol R previously neutralised using phenolphthalein solution R1 as
indicator and add 25 ml of water R. Titrate with 0.1M sodium hydroxide until a pink colour is
obtained.
10
H
13
ClN
2
O
3
S.
STORAGE
IMPURITIES
Cl
SO
2
NH
2
A. 4-chlorobenzenesulphonamide,
H
3
C
N N
CH
3
O
H H
B. 1,3-dipropylurea,
Cl
S
N NH
2
O
O O
C. [(4-chlorophenyl)sulphonyl]urea.
__________________________________________________________________________________________________________ Ph Eur
09-41
Chlorprothixene Hydrochloride
1/01
S
Cl
H NMe
2
,HCl
C
18
H
18
ClNS,HCl 352.3 6469-93-8
Chlorprothixene Hydrochloride complies with the requirements of the 3rd edition of the European
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Chlorprothixene hydrochloride is (Z)-3-(2-chloro-9H-thioxanthen-9-ylidene)-N,N-dimethylpropan-
1-amine hydrochloride. It contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of C
18
H
19
Cl
2
NS, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, soluble in water and in alcohol, slightly soluble in
methylene chloride.
IDENTIFICATION
obtained with chlorprothixene hydrochloride CRS. Examine the substances prepared as discs.
B. Dissolve 0.2 g in a mixture of 5 ml of dioxan R and 5 ml of a 1.5 g/l solution of sodium nitrite R.
Add 0.8 ml of nitric acid R. After 10 min add the solution to 20 ml of water R.1 h later filter the
precipate formed. The filtrate is used immediately for identification test C. Dissolve the precipitate by
warming in about 15 ml of alcohol R and add the solution to 10 ml of water R. Filter and dry the
precipitate at 100C to 105C for 2 h. The melting point (2.2.14) is 152C to 154C.
C. To 1 ml of the filtrate obtained in identification test B, add 0.2 ml of a suspension of 50 mg of fast
red B salt R in 1 ml of alcohol R. Add 1 ml of 0.5M alcoholic potassium hydroxide. A dark red colour is
produced. Carry out a blank test.
D. Dissolve about 20 mg in 2 ml of nitric acid R. A red colour is produced. Add 5 ml of water R and
examine in ultraviolet light at 365 nm. The solution shows green fluorescence.
E. It gives reaction (a) of chlorides (2.3.1).
TESTS
Related substances Carry out the test protected from bright light. Examine by liquid chromatography
(2.2.29).
Reference solution (a). Dissolve 20.0 mg of chlorprothixene hydrochloride CRS (with a defined content of
E-isomer) in the mobile phase and dilute to 20.0 ml with the mobile phase.
deactivated octadecylsilyl silica gel for chromatography R (3 m or 5 m),
09-42
as mobile phase at a flow rate of 1.5 ml/min, a solution containing 6.0 g/l of potassium dihydrogen
phosphate R, 2.9 g/l of sodium lauryl sulphate R and 9 g/l of tetrabutylammonium bromide R in a
mixture of 50 volumes of methanol R, 400 volumes of acetonitrile R and 550 volumes of distilled
water R,
Equilibrate the column with the mobile phase at a flow rate of 1.5 ml/min for about 30 min.
Inject 20 l of reference solution (a). The test is not valid unless the relative retention time of the
E-isomer to the principal peak is about 1.35 and the retention time of the principal peak is about
10 min.
chlorprothixene. From the chromatograms obtained with the test solution and reference solution (a),
determine the percentage content of E-isomer, taking into account the assigned content of this
isomer in chlorprothixene hydrochloride CRS. The content of E-isomer in the substance to be examined
is not greater than 2.0 per cent. In the chromatogram obtained with the test solution: the area of any
peak with a relative retention time of about 1.55 (impurity E), is not greater than three times the area
of the principal peak in the chromatogram obtained with reference solution (b) (0.3 per cent of
impurity E taking into account a response factor of 3); the area of any other peak, apart from the
principal peak, the peak due to the E-isomer and any peak corresponding to impurity E, is not greater
than the area of the principal peak in the chromatogram obtained with reference solution (b) (0.3 per
cent). The sum of the areas of all the peaks, apart from the principal peak, the peak due to the
E-isomer and any peak corresponding to impurity E, is not greater than 2.33 times the area of the
Heavy metals (2.4.8).1.0 g complies with limit test F for heavy metals (20 ppm). Prepare the
60C for 3 h.
ASSAY
18
H
19
Cl
2
NS.
STORAGE
IMPURITIES
S
NMe
2
OH
Cl
and enantiomer
A. (RS)-2-chloro-9-[3-(dimethylamino)propyl]-9H-thioxanthen-9-ol,
S
R
2
R
1
R
3
B. R
1
= H, R
2
= CH-CH
2
-CH
2
-N(CH
3
)
2
, R
3
= H: N,N-dimethyl-3-(9H-thioxanthen-9-
ylidene)propan-1-amine,
C. R
1
=

Cl, R
2
= CH-CH
2
-CH
2
-NH-CH
3
, R
3
= H: (Z)-3-(2-chloro-9H-thioxanthen-9-ylidene)-N-
methylpropan-1-amine,
D. R
1
=

H, R
2
= CH-CH
2
-CH
2
-N(CH
3
)
2
, R
3
= Cl: (Z)-3-(4-chloro-9H-thioxanthen-9-ylidene)-N,N-
dimethylpropan-1-amine,
E. R
1
=

Cl, R
2
= O, R
3
= H: 2-chloro-9H-thioxanthen-9-one,
09-43
S
Cl
NMe
2
F. (E)-3-(2-chloro-9H-thioxanthen-9-ylidene)-N,N-dimethylpropan-1-amine (E-isomer).
__________________________________________________________________________________________________________ Ph Eur
09-44
Chlortalidone
NH
O
OH
Cl
SO
2
NH
2
and enantiomer
C
14
H
11
ClN
2
O
4
S 338.8 77-36-1
Chlortalidone complies with the requirements of the 3rd edition of the European Pharmacopoeia [0546]. These
Preparations
Chlortalidone Tablets
Co-tenidone Tablets
When chlorthalidone is prescribed or demanded, Chlortalidone shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Chlortalidone contains not less than 98.0 per cent and not more than the equivalent of 102.0 per
cent of 2-chloro-5-[(1RS)-1-hydroxy-3-oxo-2,3-dihydro-1H- isoindol-1-yl]benzenesulphonamide,
CHARACTERS
A white or yellowish-white powder, practically insoluble in water, soluble in acetone and in methanol,
slightly soluble in alcohol, practically insoluble in methylene chloride. It dissolves in dilute solutions
of the alkali hydroxides.
IDENTIFICATION
A. Dissolve 50.0 mg in alcohol R and dilute to 50.0 ml with the same solvent. Dilute 10.0 ml of this
solution to 100.0 ml with alcohol R. Examined between 230 nm and 340 nm (2.2.25), the solution
shows two absorption maxima, at 275 nm and 284 nm. The ratio of the absorbance measured at the
maximum at 284 nm to that measured at the maximum at 275 nm is 0.73 to 0.88.
obtained with chlortalidone CRS. Examine the substances prepared as discs using potassium bromide R.
254
the same solvent.
Reference solution (a). Dissolve 10 mg of chlortalidone CRS in acetone R and dilute to 10 ml with the
same solvent.
Reference solution (b). Dissolve 10 mg of chlortalidone CRS and 10 mg of hydrochlorothiazide CRS in
1.5 volumes of water R and 98.5 volumes of ethyl acetate R. Allow the plate to dry in air and examine
D. Dissolve about 10 mg in 1 ml of sulphuric acid R. An intense yellow colour develops.
E. It complies with the test for optical rotation (see Tests).
09-45
TESTS
Optical rotation (2.2.7). Dissolve 0.20 g in methanol R and dilute to 20 ml with the same solvent.
Appearance of solution Dissolve 1.0 g in dilute sodium hydroxide solution R and dilute to 10 ml with
the same solution. The solution is clear (2.2.1) and not more intensely coloured than intensity 6 of
the range of reference solutions of the most appropriate colour (Method II, 2.2.2).
Acidity Dissolve 1.0 g with heating in a mixture of 25 ml of acetone R and 25 ml of carbon dioxide-free
water R. Cool. Titrate with 0.1M sodium hydroxide, determining the end-point potentiometrically
(2.2.20). Not more than 0.75 ml of 0.1M sodium hydroxide is required.
4 volumes of acetone R and dilute to 5 ml with the same mixture of solvents.
Reference solution (a). Dissolve 20 mg of chlortalidone impurity B CRS and 20 mg of chlortalidone CRS
in a mixture of 1 volume of water R and 4 volumes of acetone R and dilute to 50 ml with the same
Reference solution (b). Dilute 1 ml of the test solution to 200 ml with a mixture of 1 volume of water R
volumes of toluene R, 10 volumes of xylene R, 20 volumes of concentrated ammonia R, 30 volumes of
dioxan R and 30 volumes of 2-propanol R. Allow the plate to dry in a current of warm air and examine
in ultraviolet light at 254 nm. In the chromatogram obtained with the test solution, any spot corres-
ponding to chlortalidone impurity B is not more intense than the corresponding spot in the chroma-
togram obtained with reference solution (a) (1 per cent) and any spot, apart from the principal spot
and the spot corresponding to chlortalidone impurity B, is not more intense than the spot in the
chromatogram obtained with reference solution (b) (0.5 per cent). The test is not valid unless the
chromatogram obtained with reference solution (a) shows two clearly separated spots.
Chlorides (2.4.4). Triturate 0.3 g, add 30 ml of water R, shake for 5 min and filter. 15 ml of the
filtrate complies with the limit test for chlorides (350 ppm). Prepare the standard using 10 ml of
chloride standard solution (5 ppm Cl) R.
100C to 105C.
ASSAY
Dissolve 0.200 g in 50 ml of acetone R. In an atmosphere of nitrogen, titrate with 0.1M
tetrabutylammonium hydroxide, determining the end-point potentiometrically (2.2.20). Carry out a
blank titration.
14
H
11
ClN
2
O
4
S.
IMPURITIES
Cl
SO
2
R'
O
O RO
A. R = H, R = OH: 2-(4-chloro-3-sulphobenzoyl)benzoic acid,
B. R = H, R= NH
2
: 2-(4-chloro-3-sulphamoylbenzoyl)benzoic acid,
C. R = CH
2
-CH
3
, R = NH
2
: ethyl 2-(4-chloro-3-sulphamoylbenzoyl)benzoate,
NH
SO
2
NH
2
Cl
O
R
and enantiomer
D. R = O-CH
2
-CH
3
: 2-chloro-5-[(1RS)-1-ethoxy-3-oxo-2,3-dihydro-1H-isoindol-1-yl]benzene-
sulphonamide,
E. R = H : 2-chloro-5[(1RS)-3-oxo-2,3-dihydro-1H-isoindol-1-yl]benzenesulphonamide,
09-46
NH
SO
2
Cl
O
OH
HN
O
2
S
Cl
O
HO
N
H
F. bis[2-chloro-5-(1-hydroxy-3-oxo-2,3-dihydro-1H-isoindol-1-yl)benzenesulphonyl]amine.
__________________________________________________________________________________________________________ Ph Eur
09-47
Chlortetracycline Hydrochloride
corrected 1/01
OH OH
OH
OH
Cl
O O
CONH
2
H H
OH
Me
NMe
2
,HCl
C
22
H
23
ClN
2
O
8
,HCl 515.3 64-72-2
Chlortetracycline Hydrochloride complies with the requirements of the 3rd edition of the European
Preparations
Chlortetracycline Eye Ointment
Chlortetracycline Ointment
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Chlortetracycline hydrochloride is the hydrochloride of (4S,4aS,5aS,6S,12aS)-7-chloro-4-
dimethylamino-1,4,4a,5,5a,6,11,12a-octahydro-3,6,10,12,12a-pentahydroxy-6-methyl-1,11-
dioxonaphthacene-2-carboxamide, a substance produced by the growth of certain strains of
Streptomyces aureofaciens or obtained by any other means. The content of chlortetracycline hydro-
chloride is not less than 89.5 per cent and the sum of the contents of chlortetracycline hydrochloride
and tetracycline hydrochloride is not less than 94.5 per cent and not more than the equivalent of
100.5 per cent of chlortetracycline hydrochloride, calculated with reference to the anhydrous
substance.
CHARACTERS
A yellow powder, slightly soluble in water and in alcohol. It dissolves in solutions of alkali hydroxides
and carbonates.
IDENTIFICATION
A. Examine by thin-layer chromatography (2.2.27), using silica gel H R as the coating substance.
Adjust a 100 g/l solution of sodium edetate R to pH 8.0 with strong sodium hydroxide solution R and
spray the solution evenly onto the plate (about 10 ml for a plate 100 mm 200 mm). Allow the plate
to dry in a horizontal position for at least 1 h. At the time of use, dry the plate in an oven at 110C
for 1 h.
the same solvent.
Reference solution (a). Dissolve 5 mg of chlortetracycline hydrochloride CRS in methanol R and dilute to
Reference solution (b). Dissolve 5 mg of chlortetracycline hydrochloride CRS, 5 mg of tetracycline hydro-
chloride CRS and 5 mg of metacycline hydrochloride CRS in methanol R and dilute to 10 ml with the
same solvent.
6 volumes of water R, 35 volumes of methanol R and 59 volumes of methylene chloride R. Dry the plate
in a stream of air and examine in ultraviolet light at 365 nm. The principal spot in the chromatogram
chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram
obtained with reference solution (b) shows three clearly separated spots.
B. To about 2 mg add 5 ml of sulphuric acid R. A deep blue colour develops which becomes bluish-
green. Add the solution to 2.5 ml of water R. The colour becomes brownish.
TESTS
pH (2.2.3). Dissolve 0.1 g in 10 ml of carbon dioxide-free water R, heating slightly. The pH of the
09-48
substance.
Absorbance (2.2.25). Dissolve 0.125 g in water R and dilute to 25.0 ml with the same solvent. The
absorbance, measured at 460 nm, is not greater than 0.40.
the test solution and reference solutions (e) and (f). The test is not valid unless the peak in the
chromatogram obtained with reference solution (f) is properly integrated. In the chromatogram
obtained with the test solution: the area of the peak corresponding to 4-epichlortetracycline is not
greater than the area of the peak corresponding to 4-epichlortetracycline in the chromatogram
obtained with reference solution (e) (4 per cent); the total area of all the peaks appearing between the
solvent peak and the peak corresponding to chlortetracycline, apart from the peaks due to tetracycline
and 4-epichlortetracycline, is not greater than 25 per cent of the area of the peak corresponding to
4-epichlortetracycline in the chromatogram obtained with reference solution (e) (1 per cent).
Disregard any peak with an area smaller than that of the principal peak in the chromatogram
obtained with reference solution (f).
Tetracycline hydrochloride Not more than 8.0 per cent, calculated with reference to the anhy-
drous substance and determined by liquid chromatography (2.2.29), as described under Assay. Inject
separately the test solution and reference solution (e).
mination of water.
without a further appropriate procedure for the removal of bacterial endotoxins, not more than 1 I.U.
of bacterial endotoxins per milligram.
ASSAY
Test solution. Dissolve 25.0 mg of the substance to be examined in 0.01M hydrochloric acid and dilute
to 25.0 ml with the same acid.
Reference solution (a). Dissolve 25.0 mg of chlortetracycline hydrochloride CRS in 0.01M hydrochloric acid
and dilute to 25.0 ml with the same acid.
Reference solution (b). Dissolve 10.0 mg of 4-epichlortetracycline hydrochloride CRS in 0.01M hydrochloric
acid and dilute to 25.0 ml with the same acid.
Reference solution (c). Dissolve 20.0 mg of tetracycline hydrochloride CRS in 0.01M hydrochloric acid and
dilute to 25.0 ml with the same acid.
Reference solution (d). Mix 5.0 ml of reference solution (a) and 10.0 ml of reference solution (b) and
dilute to 25.0 ml with 0.01M hydrochloric acid.
Reference solution (e). Mix 5.0 ml of reference solution (b) and 5.0 ml of reference solution (c) and
Reference solution (f). Dilute 1.0 ml of reference solution (c) to 20.0 ml with 0.01M hydrochloric acid.
Dilute 5.0 ml of this solution to 200.0 ml with 0.01M hydrochloric acid.
tography R (5 m or 10 m), and maintained at 35C,
as mobile phase at a flow rate of 1 ml/min a mixture prepared as follows: to 500 ml of water R,
add 50 ml of perchloric acid solution R, shake and add 450 ml of dimethyl sulphoxide R,
an electronic integrator.
Inject reference solution (d). Adjust the attenuation to obtain peaks with a height corresponding to
at least 50 per cent of the full scale deflection of the recorder. The test is not valid unless the
resolution between the first peak (4-epichlortetracycline) and the second (chlortetracycline) is not less
than 2.0. Adapt the dimethyl sulphoxide content in the mobile phase if necessary. The symmetry
factor for the second peak is not greater than 1.3. Inject reference solution (a) six times. The test is
not valid unless the relative standard deviation of the peak area for chlortetracycline hydrochloride is
not greater than 1.0 per cent. If necessary, adjust the integrator parameters. Inject alternately the test
09-49
Calculate the percentage content of chlortetracycline hydrochloride.
STORAGE
LABELLING
The label states:
IMPURITIES
OH HO
OH
Cl
O O
CONH
2
H H
OH
NMe
2
OH
Me
A. 4-epichlortetracycline,
B. tetracycline.
__________________________________________________________________________________________________________ Ph Eur
09-50
Cholesterol
Me
Me
H
H H
Me
Me Me
H
HO
C
27
H
46
O 386.7 57-88-5
Cholesterol complies with the requirements of the 3rd edition of the European Pharmacopoeia [0993]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cholesterol contains not less than 95.0 per cent of cholest-5-en-3-ol and not less than 97.0 per cent
and not more than 103.0 per cent of total sterols, calculated with reference to the dried substance.
PRODUCTION
CHARACTERS
acetone and in alcohol. It is sensitive to light.
IDENTIFICATION
B. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel G plate R. Prepare the
Test solution. Dissolve 10 mg of the substance to be examined in ethylene chloride R and dilute to 5 ml
Reference solution. Dissolve 10 mg of cholesterol CRS in ethylene chloride R and dilute to 5 ml with the
same solvent.
Apply separately to the plate 20 l of each solution. Develop immediately, protected from light,
over a path of 15 cm using a mixture of 33 volumes of ethyl acetate R and 66 volumes of toluene R.
Allow the plate to dry in air and spray three times with antimony trichloride solution R. Examine the
chromatograms 3 min to 4 min after spraying. The principal spot in the chromatogram obtained with
C. Dissolve about 5 mg in 2 ml of methylene chloride R. Add 1 ml of acetic anhydride R, 0.01 ml of
sulphuric acid R and shake. A pink colour is produced which rapidly changes to red, then to blue and
finally to brilliant green.
TESTS
Solubility in alcohol In a stoppered flask, dissolve 0.5 g in 50 ml of alcohol R at 50C. Allow to
stand for 2 h. No deposit or turbidity is formed.
Acidity Dissolve 1.0 g in 10 ml of ether R, add 10.0 ml of 0.1M sodium hydroxide and shake for about
1 min. Heat gently to eliminate ether and then boil for 5 min. Cool, add 10 ml of water R and 0.1 ml
of phenolphthalein solution R as indicator and titrate with 0.1M hydrochloric acid until the pink colour
just disappears, stirring the solution vigorously throughout the titration. Carry out a blank titration.
The difference between the volumes of 0.1M hydrochloric acid required to change the colour of the
indicator in the blank and in the test is not more than 0.3 ml.
60C for 4 h.
ASSAY
09-51
Examine by gas chromatography (2.2.28), using pregnenolone isobutyrate CRS as the internal standard.
Internal standard solution. Dissolve 0.100 g of pregnenolone isobutyrate CRS in heptane R and dilute to
Test solution. Dissolve 25.0 mg of the substance to be examined in the internal standard solution and
dilute to 25.0 ml with the same solution.
Reference solution. Dissolve 25.0 mg of cholesterol CRS in the internal standard solution and dilute to
poly(dimethyl)siloxane R (film thickness 1.5 m),
Inject 0.5 l of each solution. The assay is not valid unless the resolution between the peaks due to
pregnenolone isobutyrate and cholest-5-en-3-ol in the chromatogram obtained with the reference
solution is at least 3.5.
Calculate the percentage content of cholest-5-en-3-ol, using the declared content of cholest-5-en-
3-ol in cholesterol CRS.
Calculate the percentage content of total sterols by adding together the contents of cholest-5-en-
3-ol and other substances with a retention time less than or equal to 1.5 times the retention time of
cholest-5-en-3-ol.
Disregard the peaks due to the internal standard and the solvent.
STORAGE
IMPURITIES
Me
Me
H H
Me
Me Me
H
H
HO
H
A. cholest-7-en-3-ol (lathosterol),
Me
Me
H H
Me
Me Me
H
H
H
HO
B. cholesta-5,24-dien-3-ol (desmosterol),
Me
Me
H H
Me
Me Me
H
H
HO
H
C. cholesta-7,24-dien-3-ol.
__________________________________________________________________________________________________________ Ph Eur
10-1
Choline Salicylate Solution
Definition Choline Salicylate Solution is an aqueous solution of choline salicylate. It contains not
less than 47.5% w/v and not more than 52.5% w/v of choline salicylate, C
12
H
19
NO
4
. It may contain
a suitable antimicrobial preservative.
Characteristics A clear, colourless liquid.
Identification
A. Mix 0.5 ml with 10 ml of methanol, dry with anhydrous sodium sulphate, filter and evaporate the
methanol to dryness. The infrared absorption spectrum of the residue, Appendix II A, is concordant with
the reference spectrum of choline salicylate (RS 059).
B. Dilute 5 ml to 25 ml with water. The resulting solution yields the reactions characteristic of
salicylates, Appendix VI.
Acidity Dilute 4 ml to 20 ml with water and add 0.1 ml of phenol red solution. The solution is yellow
and not more than 0.4 ml of 0.1M sodium hydroxide VS is required to change the colour of the
solution to reddish violet.
Clarity and colour of solution Dilute 1 volume of the solution to 5 volumes with water. The
resulting solution is clear, Appendix IV A, and colourless, Appendix IV B, Method II.
Chloride Mix 0.2 ml with 10 ml of water and add carefully, with mixing, 0.1 ml of a mixture of 10
volumes of silver nitrate solution and 1 volume of nitric acid. The resulting solution is not more
opalescent than a standard prepared by treating 10 ml of a 0.00164% w/v solution of sodium chloride
in the same manner beginning at the words add carefully (0.1%).
Assay To 1 g add 50 ml of 1,4-dioxan and 5 ml of acetic anhydride and carry out Method I for non-
aqueous titration, Appendix VIII A, using 0.25 ml of methyl orangexylene cyanol FF solution as
12
H
19
NO
4
. Use the weight
per ml to calculate the percentage of C
12
H
19
NO
4
, weight in volume.
Storage Choline Salicylate Solution should be kept in a well-closed container.
Preparations
Choline Salicylate Ear Drops
Choline Salicylate Dental Gel
10-2
Choline Theophyllinate
+
Me
3
N
OH
N
O
O
Me
N
MeN
_
C
12
H
21
N
5
O
3
283.3 4499-40-5
Definition Choline Theophyllinate is choline 1,2,3,6-tetrahydro-1,3-dimethyl-2,6-dioxo-7H-
purin-7-ide. It contains not less than 41.9% and not more than 43.6% of choline, C
5
H
15
NO
2
, and
not less than 61.7% and not more than 65.5% of theophylline, C
7
H
9
N
4
O
2
, each calculated with
Characteristics A white, crystalline powder; odourless or with a faint amine-like odour. It melts
between 187 and 192, Appendix V A.
Very soluble in water; soluble in ethanol (96%); very slightly soluble in chloroform and in ether.
Identification
sodium hydroxide exhibits a maximum only at 275 nm. The absorbance at 275 nm is about 0.83.
B. The infrared absorption spectrum, Appendix II A, is concordant with the reference spectrum of choline
theophyllinate (RS 060).
Clarity and colour of solution 50 ml of a 10% w/v solution is clear, Appendix IV A, and not more
intensely coloured than reference solution GY
4
gel HF
254
as the coating substance and a mixture of 95 volumes of chloroform and 5 volumes of ethanol
(96%) as the mobile phase. Apply separately to the plate 5 l of each of two solutions of the
substance being examined in ethanol (96%) containing (1) 1.0% w/v and (2) 0.010% w/v. After
removal of the plate, allow it to dry in air and examine under ultraviolet light (254 nm). Any secondary
spot in the chromatogram obtained with solution (1) is not more intense than the spot in the chroma-
togram obtained with solution (2).
1 g.
Assay
For choline Dissolve 0.6 g in 50 ml of water and titrate with 0.05M sulphuric acid VS, using methyl
red mixed solution as indicator, until a violet end point is obtained. Each ml of 0.05M sulphuric acid VS
is equivalent to 12.12 mg of choline, C
5
H
15
NO
2
.
For theophylline To the solution obtained in the Assay for choline, add 25 ml of 0.1M silver nitrate
VS and warm on a water bath for 15 minutes. Cool in ice for 30 minutes, filter and wash the residue
with three 10-ml quantities of water. Titrate the combined filtrate and washings with 0.1M sodium
hydroxide VS. Each ml of 0.1M sodium hydroxide VS is equivalent to 18.02 mg of theophylline,
C
7
H
8
N
4
O
2
.
Storage Choline Theophyllinate should be kept in a well-closed container, protected from light and
Preparation
Choline Theophyllinate Tablets
10-3
Chorionic Gonadotrophin
Chorionic Gonadotrophin complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Gonadotrophic hormone.
Preparation
Chorionic Gonadotrophin Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Chorionic gonadotrophin is a dry preparation of placental glycoproteins which have luteinising
activity. The potency is not less than 2500 I.U. per milligram.
PRODUCTION
Chorionic gonadotrophin is extracted from the urine of pregnant women using a suitable
fractionation procedure. It is either dried under reduced pressure or freeze-dried. It is prepared in
conditions designed to minimise or eliminate microbial and viral contamination. The manufacturing
process must have been shown to reduce any viral contamination such as hepatitis virus or HIV by
appropriate validated methods.
CHARACTERS
A white to yellowish-white, amorphous powder, soluble in water.
IDENTIFICATION
When administered to immature rats as prescribed in the assay, it causes an increase in the mass of
the seminal vesicles and of the prostate gland.
TESTS
Water Not more than 5 per cent m/m, determined by gas chromatography (2.2.28), using anhydrous
methanol R as the internal standard.
Use dry glassware (which may be silicone-treated).
Internal standard solution. Dilute 15 l of anhydrous methanol R to 100 ml with 2-propanol R1.
Test solution (a). Dissolve 4 mg of the substance to be examined in 0.5 ml of 2-propanol R1.
Test solution (b). Dissolve 4 mg of the substance to be examined in 0.5 ml of the internal standard
solution.
Reference solution. Add 10 l of water R to 50 ml of the internal standard solution.
a stainless steel column 1 m long and 2 mm in internal diameter packed with styrene-
divinylbenzene co-polymer R (180 m to 250 m),
a thermal-conductivity detector.
Maintain the temperature of the column at 120C and that of the detector at 150C. Inject the
chosen volumes of each solution. Calculate the content of water assuming its density (2.2.5) at 20C
to be 0.9972 g per millilitre and taking into account any water detectable in the internal standard
solution.
Inject per kilogram of the rabbits mass a quantity equivalent to 300 I.U., dissolved in not more than
1 ml of a sterile, pyrogen-free 9 g/l solution of sodium chloride.
ASSAY
The potency of chorionic gonadotrophin is estimated by comparing under given conditions its effect
of increasing the mass of the seminal vesicles (or the prostate gland) of immature rats with the same
effect of the International Standard of chorionic gonadotrophin or of a reference preparation
calibrated in International Units.
The International Unit is the activity contained in a stated amount of the International Standard,
which consists of a mixture of a freeze-dried extract of chorionic gonadotrophin from the urine of
pregnant women with lactose. The equivalence in International Units of the International Standard is
stated by the World Health Organisation.
10-4
Use immature male rats of the same strain, 19 to 28 days old, differing in age by not more than 3
days and having body masses such that the difference between the heaviest and the lightest rat is not
more than 10 g. Assign the rats at random to six equal groups of at least five animals. If sets of six
litter mates are available, assign one litter mate from each set to each group and mark according to
litter.
Choose three doses of the reference preparation and three doses of the preparation to be examined
such that the smallest dose is sufficient to produce a positive response in some of the rats and the
largest dose does not produce a maximal response in all the rats. Use doses in geometric progression
and as an initial approximation total doses of 4 I.U., 8 I.U. and 16 I.U. may be tried although the
dose will depend on the sensitivity of the animals used, which may vary widely.
Dissolve separately the total quantities of the preparation to be examined and of the reference
preparation corresponding to the daily doses to be used in sufficient phosphate-albumin buffered saline
pH 7.2 R such that the daily dose is administered in a volume of about 0.5 ml. Add a suitable
antimicrobial preservative such as 4 g/l of phenol or 0.02 g/l of thiomersal. Store the solutions at
5 3C.
Inject subcutaneously into each rat the daily dose allocated to its group, on four consecutive days at
the same time each day. On the fifth day, about 24 h after the last injection, kill the rats and remove
the seminal vesicles. Remove any extraneous fluid and tissue and weigh the vesicles immediately.
Calculate the results by the usual statistical methods, using the mass of the vesicles as the response.
(The precision of the assay may be improved by a suitable correction of the organ mass with refer-
ence to the body mass of the animal from which it was taken; an analysis of covariance may be used).
potency. The fiducial limits of error (P = 0.95) of the estimated potency are not less than 64 per cent
and not more than 156 per cent of the stated potency.
STORAGE
Store in an airtight, tamper-proof container, protected from light at a temperature of 2C to 8C. If
the substance is sterile, store in a sterile, airtight, tamper-proof container.
LABELLING
The label states:
the number of International Units per container,
the potency in International Units per milligram,
__________________________________________________________________________________________________________ Ph Eur
10-5
Chymotrypsin
Chymotrypsin complies with the requirements of the 3rd edition of the European Pharmacopoeia [0476]. These
Action and use Proteolytic enzyme.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Chymotrypsin is a proteolytic enzyme obtained by the activation of chrymotrypsinogen extracted
from the pancreas of beef (Bos taurus L.). It has an activity not less than 5.0 microkatals per
milligram. In solution it has maximal enzymic activity at about pH 8; the activity is reversibly
inhibited at pH 3, at which pH it is most stable.
PRODUCTION
The animals from which chymotrypsin is derived must fulfil the requirements for the health of
animals suitable for human consumption to the satisfaction of the competent authority; furthermore
the tissues used shall not include any specified risk material as defined by any relevant international
or, where appropriate, national legislation. It must have been shown to what extent the method of
production allows inactivation or removal of any contamination by viruses or other infectious agents.
with the following test:
Histamine (2.6.10). Not more than 1 g (calculated as histamine base) per 5 microkatals of
chymotrypsin activity. Before carrying out the test, heat the solution of the substance to be examined
on a water-bath for 30 min.
CHARACTERS
A white, crystalline or amorphous powder, sparingly soluble in water; the amorphous form is
hygroscopic.
IDENTIFICATION
A. Dilute 1 ml of solution S (see Tests) to 10 ml with water R. In a depression in a white spot plate,
mix 0.05 ml of this solution with 0.2 ml of substrate solution. A purple colour develops.
Substrate solution. To 24.0 mg of acetyltyrosine ethyl ester R add 0.2 ml of alcohol R, and swirl until
solution is effected. Add 2.0 ml of 0.067M phosphate buffer solution pH 7.0 R and 1 ml of methyl red
mixed solution R and dilute to 10.0 ml with water R.
B. Dilute 0.5 ml of solution S to 5 ml with water R. Add 0.10 ml of a 20 g/l solution of tosylphenyl-
alanylchloromethane R in alcohol R. Adjust to pH 7.0 and shake for 2 h. In a depression in a white spot
plate, mix 0.05 ml of this solution with 0.2 ml of the substrate solution (see Identification test A). No
colour develops within 3 min of mixing.
TESTS
Absorbance (2.2.25). Dissolve 30.0 mg in 0.001M hydrochloric acid and dilute to 100.0 ml with the
same acid. The solution shows an absorption maximum at 281 nm and a minimum at 250 nm. The
specific absorbance at the maximum is 18.5 to 22.5 and that at the minimum is not greater than 8.
Trypsin Transfer to a depression in a white spot plate 0.05 ml of tris(hydroxymethyl)aminomethane
buffer solution pH 8.1 R and 0.1 ml of solution S. Add 0.2 ml of substrate solution (test solution). At
the same time and in the same manner, prepare a reference solution using the substance to be
examined to which not more than 1 per cent m/m of trypsin BRP has been added. Start a timer. No
colour appears in the test solution within 3 min to 5 min after the addition of the substrate solution.
A purple colour is produced in the control solution.
Substrate solution. To 98.5 mg of tosylarginine methyl ester hydrochloride R, suitable for assaying trypsin,
add 5 ml of tris(hydroxymethyl)aminomethane buffer solution pH 8.1 R and swirl to dissolve. Add 2.5 ml
of methyl red mixed solution R and dilute to 25.0 ml with water R.
ASSAY
10-6
The activity of chymotrypsin is determined by comparing the rate at which it hydrolyses
acetyltyrosine ethyl ester R with the rate at which chymotrypsin BRP hydrolyses the same substrate
under the same conditions.
Apparatus. Use a reaction vessel of about 30 ml capacity provided with:
a device that will maintain a temperature of 25.0 0.1C,
a stirring device, for example a magnetic stirrer,
a lid with holes for the insertion of electrodes, the tip of a burette, a tube for the admission of
nitrogen and the introduction of reagents.
An automatic or manual titration apparatus may be used. For the latter the burette is graduated in
0.005 ml and the pH meter is provided with a wide scale and glass-calomel electrodes.
Reference solution. Dissolve 25.0 mg of chymotrypsin BRP in 0.001M hydrochloric acid and dilute to
250.0 ml with the same acid.
Store the solutions at 0C to 5C. Warm 1 ml of each solution to about 25C over 15 min and use 50
l of each solution (corresponding to about 25 nanokatals) for each titration. Carry out the titration
in an atmosphere of nitrogen. Transfer 10.0 ml of 0.01M calcium chloride solution R to the reaction
vessel and, while stirring, add 0.35 ml of 0.2M acetyltyrosine ethyl ester solution R. When the tempera-
ture is steady at 25.0 0.1C (after about 5 min) adjust the pH to exactly 8.0 with 0.02M sodium
hydroxide. Add 50 l of the test solution (equivalent to about 5 g of the substance to be examined)
and start a timer. Maintain the pH at 8.0 by the addition of 0.02M sodium hydroxide, noting the
volume added every 30 s. Calculate the volume of 0.02M sodium hydroxide used per second between
30 s and 210 s. Carry out a titration in the same manner using the reference solution and calculate
the volume of 0.02M sodium hydroxide used per second.
Calculate the activity in microkatals per milligram using the expression:
A
V m
V m

m=mass of the substance to be examined, in milligrams,
m =mass of chymotrypsin BRP, in milligrams,
V=volume of 0.02M sodium hydroxide used per second by the test solution,
V =volume of 0.02M sodium hydroxide used per second by the reference solution,
A=activity of chymotrypsin BRP, in microkatals per milligram.
STORAGE
Store in an airtight container at 2C to 8C, protected from light.
LABELLING
The label states:
the quantity of chymotrypsin and the total activity in microkatals per container,
for the amorphous substance, that it is hygroscopic.
__________________________________________________________________________________________________________ Ph Eur
10-7
Ciclopirox
N
OH
O
Me
C
12
H
17
NO
2
207.3 29342-05-0
Ciclopirox complies with the requirements of the 3rd edition of the European Pharmacopoeia [1407]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ciclopirox contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent of
6-cyclohexyl-1-hydroxy-4-methylpyridin-2(1H)-one, calculated with reference to the dried substance.
CHARACTERS
A white or yellowish-white, crystalline powder, slightly soluble in water, freely soluble in ethanol and
IDENTIFICATION
A. Melting point (2.2.14). 140C to 145C.
obtained with ciclopirox CRS.
254
plate R.
the same solvent.
Reference solution. Dissolve 20 mg of ciclopirox CRS in methanol R and dilute to 10 ml with the same
solvent.
Before use predevelop the plate with a mixture of 10 volumes of concentrated ammonia R, 15 volumes
of water R and 75 volumes of alcohol R until the solvent front has migrated to the top of the plate.
Allow the plate to dry in air for 5 min.
of 10 volumes of concentrated ammonia R, 15 volumes of water R and 75 volumes of alcohol R. Allow
the plate to dry in air for 10 min and examine in ultraviolet light at 254 nm. The principal spot in the
the chromatogram obtained with the reference solution. Spray the plate with a 20 g/l solution of ferric
chloride R in ethanol R. The principal spot in the chromatogram obtained with the test solution is
reference solution.
TESTS
5
(Method II,
2.2.2).
Carry out the operations avoiding exposure to actinic light. All materials which are in direct contact with the
substance to be examined like column materials, reagents, solvents and others should contain only very low
amounts of extractable metal cations.
Solvent mixture. Mix 1 volume of acetonitrile R and 9 volumes of the mobile phase.
Test solution. Dissolve 30.0 mg of the substance to be examined in 15 ml of the solvent mixture. If
necessary, use an ultrasonic bath. Dilute to 20.0 ml with the solvent mixture.
Reference solution (a). Dissolve 15.0 mg of ciclopirox impurity A CRS and 15.0 mg of ciclopirox impurity
10-8
B CRS in the solvent mixture and dilute to 10.0 ml with the same solvent mixture.
Reference solution (c). Dilute 2.0 ml of reference solution (b) to 10.0 ml with the solvent mixture.
Reference solution (d). Mix 5 ml of reference solution (a) with 5 ml of the test solution.
a stainless steel column 80 mm long and 4 mm in internal diameter packed with nitrile silica gel
for chromatography R2 (5 m),
as rinsing solution a mixture of 1 volume of glacial acetic acid R, 1 volume of acetylacetone R, 500
as mobile phase at a flow rate of 0.7 ml/min a mixture of 0.1 ml of glacial acetic acid R, 230 ml of
acetonitrile R and 770 ml of a 0.96 g/l solution of sodium edetate R. If the retention time of the
principal peak in the chromatogram obtained with the test solution is not between 8 min and
11 min adjust the ratio of the 0.96 g/l solution of sodium edetate: acetonitrile accordingly,
as detector a spectrophotometer set at 220 nm and 298 nm.
In order to ensure desorption of disruptive metal ions, every new column is to be rinsed with the
rinsing solution over a period of not less than 15 h and then with the mobile phase for not less than
5 h at a flow rate of 0.2 ml/min.
Inject 10 l each of the solvent mixture, the test solution and the reference solutions (b), (c) and
(d) and record the chromatograms at 220 nm and 298 nm. Continue the chromatography of the test
solution for 2.5 times the retention time of the principal peak.
The relative retention times are:
impurity A: 0.5
impurity C: 0.9
ciclopirox: 1
impurity B: 1.3
The test is not valid unless: in the chromatogram obtained with reference solution (d), the
resolution between the peaks corresponding to impurity B and ciclopirox is not less than 2.0; the
chromatogram obtained with reference solution (c) shows at 298 nm a peak corresponding to
impurity B with a signal-to-noise ratio of not less than three; in the chromatogram obtained with the
test solution the symmetry factor of the principal peak is between 0.8 and 2.0.
In the chromatogram obtained with the test solution at 220 nm, the area of the peak due to
impurity A is not greater than the area of the corresponding peak in the chromatogram obtained with
reference solution (b) at the same wavelength (0.5 per cent). In the chromatogram obtained with the
test solution at 298 nm, the area of any peak apart from the principal peak is not greater than the area
of the peak due to impurity B in the chromatogram obtained with reference solution (b) at the same
wavelength (0.5 per cent); the sum of all peak areas at 298 nm, apart from the principal peak and any
peak due to impurity B, is not greater than the area of the peak due to impurity B in the chromato-
gram obtained with reference solution (b) (0.5 per cent). At 298 nm disregard any peak due to the
solvent and any peak with an area less than half the area of the peak due to impurity B in the chroma-
togram obtained with reference solution (c) at the same wavelength.
60C over diphosphorus pentoxide R.
Sulphated ash (2.4.14). Not more than 0.1 per cent determined on 1.0 g.
ASSAY
Dissolve 0.150 g in 20 ml of methanol R. Add 20 ml of water R and titrate with 0.1M sodium hydroxide,
determining the end-point potentiometrically (2.2.20). Carry out a blank titration.
12
H
17
NO
2
.
STORAGE
IMPURITIES
N
O
Me
COOH
and enantiomer
A. (RS)-2-(3-cyclohexyl-5-methyl-4,5-dihydroisoxazol-5-yl)acetic acid,
10-9
O O
Me
B. 6-cyclohexyl-4-methyl-2H-pyran-2-one,
H
N
O
Me
C. 6-cyclohexyl-4-methylpyridin-2(1H)-one.
__________________________________________________________________________________________________________ Ph Eur
10-10
Ciclopirox Olamine
N
OH
O
Me
H
2
N
OH
.
C
12
H
17
NO
2
.C
2
H
7
NO 268.4 41621-49-2
Ciclopirox Olamine complies with the requirements of the 3rd edition of the European Pharmacopoeia [1302].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ciclopirox olamine contains not less than 76.0 per cent and not more than the equivalent of 78.5 per
cent of ciclopirox (6-cyclohexyl-1-hydroxy-4-methylpyridin-2(1H)-one, C
12
H
17
NO
2
; M
r
207.3) and
not less than 22.2 per cent and not more than the equivalent of 23.3 per cent of 2-aminoethanol
(C
2
H
7
NO; M
r
61.1), both calculated with reference to the dried substance.
CHARACTERS
A white or pale yellow, crystalline powder, slightly soluble in water, very soluble in alcohol and
methylene chloride, slightly soluble in ethyl acetate, practically insoluble in cyclohexane.
IDENTIFICATION
Second identification: B.
obtained with ciclopirox olamine CRS. If the spectra obtained in the solid state show differences,
volume of ethyl acetate R, evaporate to dryness on a water-bath and record new spectra using the
residues.
the same solvent.
Reference solution. Dissolve 25 mg of ciclopirox olamine CRS in methanol R and dilute to 10 ml with the
same solvent.
Before use wash two plates with a mixture of 10 volumes of concentrated ammonia R, 15 volumes of
water R and 75 volumes of ethanol R until the solvent front has migrated to the top of the plate. Allow
the plates to dry in air for 5 min.
Apply separately to the plates 10 l of each solution. Develop over a path of 15 cm using a mixture of
10 volumes of concentrated ammonia R, 15 volumes of water R and 75 volumes of ethanol R. Allow the
plates to dry in air for 10 min and examine in ultraviolet light at 254 nm. The principal spot in the
the chromatogram obtained with the reference solution. Spray one plate with ferric chloride
solution R3. The principal spot in the chromatogram obtained with the test solution is similar in
solution. Spray the second plate with ninhydrin solution R. Heat at 110C until the spots appear. The
TESTS
7
(Method II,
2.2.2).
10-11
Carry out the operations avoiding exposure to actinic light. All materials which are in direct contact with the
substance to be examined like column materials, reagents, solvents and others should contain only small
amounts of extractable metal cations.
Test solution. Dissolve 40.0 mg of the substance to be examined (corresponding to about 30 mg of
ciclopirox) in a mixture of 20 l of anhydrous acetic acid R, 2 ml of acetonitrile R, and 15 ml of mobile
phase. If necessary, use an ultrasonic bath. Dilute to 20.0 ml with the mobile phase.
Reference solution (a). Dissolve 15.0 mg of ciclopirox impurity A CRS and 15.0 mg of ciclopirox impurity
B CRS in 1 ml of acetonitrile R and 7 ml of the mobile phase. Dilute to 10.0 ml with the mobile
phase.
Reference solution (b). Dilute 1.0 ml of reference solution (a) to 200.0 ml with a mixture of 1 volume
of acetonitrile R and 9 volumes of the mobile phase.
Reference solution (c). Dilute 2.0 ml of reference solution (b) to 10.0 ml with a mixture of 1 volume of
acetonitrile R and 9 volumes of the mobile phase.
Reference solution (d). Mix 5 ml of reference solution (a) with 5 ml of the test solution.
a stainless steel column 80 mm long and 4 mm in internal diameter packed with nitrile silica gel
as rinsing solution a mixture of 1 volume of anhydrous acetic acid R, 1 volume of acetylacetone R,
500 volumes of water R and 500 volumes of acetonitrile R,
as mobile phase at a flow rate of 0.7 ml/min a mixture of 0.1 volumes of anhydrous acetic acid R,
230 volumes of acetonitrile R and 770 ml of a 0.96 g/l solution of sodium edetate R. If the
retention time of the principal peak in the chromatogram obtained with the test solution is not
between 8 min and 11 min adjust the ratio of the 0.96 g/l solution of sodium edetate: acetonitrile
accordingly,
as detector a spectrophotometer set at 220 nm and 298 nm.
In order to ensure desorption of disruptive metal ions, a new column must be rinsed with the rinsing
solution over a period of at least 15 h and then with the mobile phase for at least 5 h at a flow rate of
0.2 ml/min.
Inject 10 l each of the test solution and reference solutions (b), (c) and (d) and record the
chromatograms at 220 nm and 298 nm for 2.5 times the retention time of the principal peak of the
test solution.
The relative retention times are:
impurity A: 0.5
impurity C: 0.9
ciclopirox: 1.0
impurity B: 1.3
The test is not valid unless: in the chromatogram obtained with reference solution (d), the resolution
between the peaks corresponding to impurity B and ciclopirox is at least 2.0; the chromatogram
obtained with reference solution (c) shows at 298 nm a peak corresponding to impurity B with a
signal-to-noise ratio of at least 3; in the chromatogram obtained with the test solution the symmetry
factor of the principal peak is between 0.8 and 2.0.
In the chromatogram obtained with the test solution at 220 nm the area of the peak due to
impurity A is not greater than the area of the corresponding peak in the chromatogram obtained with
reference solution (b) at the same wavelength (0.5 per cent). In the chromatogram obtained with the
test solution at 298 nm the area of any peak apart from the principal peak is not greater than the area
of the peak due to impurity B in the chromatogram obtained with reference solution (b) at the same
wavelength (0.5 per cent); the sum of all peak areas at 298 nm apart from the principal peak and the
peak due to impurity B is not greater than the area of the peak due to impurity B in the chromato-
gram obtained with reference solution (b) (0.5 per cent). At 298 nm disregard any peak due to the
solvent and any peak with an area less than the area of the peak due to impurity B in the chromato-
gram obtained with reference solution (c) at the same wavelength (0.1 per cent).
45C to 50C over anhydrous calcium chloride R.
ASSAY
2-Aminoethanol Dissolve 0.250 g in 25 ml of anhydrous acetic acid R. Titrate with 0.1M perchloric
2
H
7
NO.
10-12
Ciclopirox Dissolve 0.200 g of the substance to be examined in 2 ml of methanol R. Add 38 ml of
water R, swirl and titrate immediately with 0.1M sodium hydroxide, determining the end-point
potentiometrically (2.2.20). Carry out a blank titration.
Use 0.1M sodium hydroxide, the titre of which has been determined under the conditions prescribed
above, using 0.100 g of benzoic acid RV.
1.0 ml of 0.1M sodium hydroxide is equivalent to 20.73 mg of C
12
H
17
NO
2
.
STORAGE
IMPURITIES
N
O
COOH
Me
and enantiomer
A. (RS)-2-(3-cyclohexyl-5-methyl-4,5-dihydroisoxazol-5-yl)acetic acid,
O
Me
O
B. 6-cyclohexyl-4-methyl-2H-pyran-2-one,
N
Me
O
H
C. 6-cyclohexyl-4-methylpyridin-2(1H)-one.
__________________________________________________________________________________________________________ Ph Eur
10-13
Ciclosporin
DAlaMeLeuMeLeuMeValNCCOAbuMeGlyMeLeuVal
Me
H
H
H
H
H Me
OH Me
Ala MeLeu
C
62
H
111
N
11
O
12
1203 59865-13-3
Ciclosporin complies with the requirements of the 3rd edition of the European Pharmacopoeia [0994]. These
Action and use Immunosuppressant.
When cyclosporin is prescribed or demanded Ciclosporin shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ciclosporin contains not less than 98.5 per cent and not more than the equivalent of 101.5 per cent
of cyclo[[(E)-(2S,3R,4R)-3-hydroxy-4-methyl-2-(methylamino)oct-6-enoyl]-L-2-amino-butyryl-N-
methylglycyl-N-methyl-L-leucyl-L-valyl-N-methyl-L-leucyl-L-alanyl-D-alanyl-N-methyl-L-leucyl-N-
methyl-L-leucyl-N-methyl-L-valyl], calculated with reference to the dried substance. Ciclosporin is a
substance produced by Beauveria nivea (Tolypocladium inflatum Gams) or obtained by any other
means.
CHARACTERS
A white or almost white powder, practically insoluble in water, freely soluble in ethanol and
methylene chloride.
IDENTIFICATION
obtained with ciclosporin CRS.
obtained with the test solution is similar in retention time to the principal peak in the chromatogram
TESTS
Appearance of solution Dissolve 1.5 g in ethanol R and dilute to 15 ml with the same solvent. The
5
, BY
5
or R
7
(Method II, 2.2.2).
same solvent. The specific optical rotation is 185 to 193, calculated with reference to the dried
substance.
separately the test solution and reference solution (b). Continue the chromatography for 1.7 times
the retention time of the principal peak. In the chromatogram obtained with the test solution: the
area of any peak, apart from the principal peak, is not greater than 0.7 times the area of the principal
due to the solvent and any peak with an area less than 0.05 times that of the principal peak in the
chromatogram with reference solution (b).
Heavy metals (2.4.8). The residue obtained in the test for loss on drying complies with limit test C
for heavy metals (20 ppm). Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32). Not more than 2.0 per cent, determined on 1.000 g at 60C at a pressure
not exceeding 15 Pa for 3 h.
10-14
0.84 I.U. of endotoxin per milligram. Dissolve 50 mg of the substance to be examined in a mixture of
280 mg of alcohol R and 650 mg of polyoxyethylated castor oil R and dilute to the required
concentration using water LAL.
ASSAY
acetonitrile R and water R and dilute to 25.0 ml with the same mixture of solvents.
Reference solution (a). Dissolve 30.0 mg of ciclosporin CRS in a mixture of equal volumes of aceto-
nitrile R and water R and dilute to 25.0 ml with the same mixture of solvents.
volumes of acetonitrile R and water R.
Reference solution (c). Dissolve 3 mg of cyclosporin U CRS in 2.5 ml of a mixture of equal volumes of
acetonitrile R and water R. Add 2.5 ml of reference solution (a).
silica gel for chromatography R (3 m to 5 m); the column is connected to the injection port by a
steel capillary tube about 1 m long and having an internal diameter of 0.25 mm,
as mobile phase at a flow rate of about 1.5 ml per minute a mixture of 1 volume of phosphoric
acid R, 50 volumes of 1,1-dimethylethylmethyl ether R, 430 volumes of acetonitrile R and 520
volumes of water R,
maintaining the temperature of the column and of the steel capillary tube at 80C.
Inject reference solution (c). The test is not valid unless the resolution between the two principal
peaks is 1.0 to 1.8. Adjust the ratio of 1,1-dimethylethylmethyl ether to acetonitrile, if necessary. The
test is not valid unless the retention time of the principal peak is 25 min to 30 min. Adjust the ratio of
acetonitrile to water, if necessary. Inject reference solution (a) six times. The test is not valid unless
the relative standard deviation of the area of the principal peak is at most 1.0 per cent. Inject
Calculate the percentage content of ciclosporin.
STORAGE
LABELLING
The label states:
IMPURITIES
A. cyclosporins B, C, D, E, G, H, L, T, U, V,
B. dihydrocyclosporin A,
C. isocyclosporin A.
__________________________________________________________________________________________________________ Ph Eur
10-15
Cilastatin Sodium
HOOC
S
COONa
HN
Me
Me
O
H
NH
2
H
C
16
H
25
N
2
NaO
5
S 380.4 81129-83-1
Cilastatin Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia [1408].
Action and use Dehydropeptidase inhibitor.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cilastatin sodium contains not less than 98.0 per cent and not more than the equivalent of 101.5 per
cent of sodium (Z)-7-[[(R)-2-amino-2-carboxyethyl]sulphanyl]-2-[[[(1S)-2,2-
dimethylcyclopropyl]carbonyl]amino]hept-2-enoate, calculated with reference to the anhydrous and
solvent-free substance.
CHARACTERS
A white or light yellow amorphous powder, hygroscopic, very soluble in water and in methanol,
soluble in dimethyl sulphoxide, slightly soluble in ethanol, practically insoluble in acetone and in
methylene chloride.
IDENTIFICATION
obtained with cilastatin sodium CRS.
TESTS
solution Y
6
(Method II, 2.2.2).
Specific optical rotation (2.2.7). Dissolve 0.250 g in a mixture of 1 volume of hydrochloric acid R
and 120 volumes of methanol R and dilute to 25.0 ml with the same mixture of solvents. The specific
optical rotation is +41.5 to +44.5, calculated with reference to the anhydrous and solvent-free
substance.
the same solvent.
Reference solution (a). Dilute 2.0 ml of the test solution to 100.0 ml with water R. Dilute 5.0 ml of this
Reference solution (b). Dilute 5.0 ml of the test solution to 100.0 ml with water R. Dilute 2.0 ml of this
Reference solution (c). Dissolve 16 mg of the substance to be examined in dilute hydrogen peroxide
solution R and dilute to 10.0 ml with the same solvent. Allow to stand for 30 min. Dilute 1 ml of this
solution to 100 ml with water R.
Reference solution (d). Dissolve 32 mg of mesityl oxide R in 100 ml of water R. Dilute 1 ml of the
as mobile phase at a flow rate of 2.0 ml/min, the following mixtures:
10-16
Mobile phase A. Prepare a mixture of 300 volumes of acetonitrile R and 700 volumes of a 0.1 per
cent V/V solution of phosphoric acid R in water R,
Mobile phase B. A 0.1 per cent V/V solution of phosphoric acid R in water R,
Time
(min)
Mobile
phase A
(per cent V/V)
Mobile
phase B
(per cent V/V)
15 85
0 30 15100 850
30 46 100 0
46 56 10015 085
Equilibrate the column with a mixture of 15 per cent V/V of mobile phase A and 85 per cent V/V
of mobile phase B. Inject separately each solution. Adjust the sensitivity of the system so that the
height of the principal peak in the chromatogram obtained with reference solution (b) is at least
15 per cent of the full scale of the recorder.
The test is not valid unless, the chromatogram obtained with reference solution (c) shows three
principal peaks: the first two peaks (cilastatin impurity A) may elute without being completely
resolved and the mass distribution ratio of the third peak (cilastatin) is at least ten; in the chromato-
gram obtained with reference solution (a), the principal peak has a signal-to-noise ratio of at least 5.0.
In the chromatogram obtained with the test solution, the area of any peak, apart from the principal
solution (b) (0.5 per cent); the sum of the areas of all peaks, apart from the principal peak, is not
greater than twice the area of the principal peak in the chromatogram obtained with reference
solution (b) (1 per cent). Disregard any peak due to the solvent, any peak with an area less than that
of the principal peak in the chromatogram obtained with reference solution (a) and any peak corres-
ponding to the principal peak in the chromatogram obtained with reference solution (d).
Mesityl oxide, acetone and methanol Not more than 1.0 per cent m/m of acetone, 0.5 per cent
m/m of methanol and 0.4 per cent m/m of mesityl oxide. Examine by gas chromatography (2.2.28)
using propanol R as the internal standard.
Internal standard solution. Dissolve 0.5 ml of propanol R in water R and dilute to 1000 ml with the
same solvent.
Test solution. Dissolve 0.200 g of the substance to be examined in water R, add 2.0 ml of the internal
Reference solution. Dissolve 2.0 ml of acetone R, 0.5 ml of methanol R and 0.5 ml of mesityl oxide R in
water R and dilute to 1000 ml with the same solvent. To 2.0 ml of this solution add 2.0 ml of the
internal standard solution and dilute to 10.0 ml with water R. This solution contains 316 g of
acetone, 79 g of methanol and 86 g of mesityl oxide per millilitre.
a fused silica column 30 m long and 0.53 mm in internal diameter coated with macrogol
Time
(min)
Temperature
(
o
C)
Rate
(
o
C/min)
Comment
Column 0 2.5
2.5 5
5 5.5
50
50 70
70
isothermal
linear
gradient
isothermal
Injection port 160
Detector 220
Inject 1 l of the reference solution and then 1 l of the test solution. Calculate the percentage
contents of acetone, methanol and mesityl oxide using the following expression:

s
u
R
R
W
C
in which C is the concentration in g/ml of the solvent in the reference solution, W is the quantity in
10-17
milligrams of cilastatin sodium in the test solution and R
u
and R
s
are the ratios of the corresponding
solvent peak areas to the propanol peak area in the test solution and the reference solution,
respectively.
of water.
ASSAY
Dissolve 0.300 g in 30 ml of methanol R and add 5 ml of water R. Add 0.1M hydrochloric acid to a pH
of about 3.0. Carry out a potentiometric titration (2.2.20), using 0.1M sodium hydroxide. Three jumps
of potential are observed. Titrate to the third equivalence point.
16
H
25
N
2
NaO
5
S.
STORAGE
LABELLING
The label states:
IMPURITIES
OOC
S
COOH
H
HN O
Me
Me
H
O
H
2
N
*
and epimer at S*
A. (Z)-7-[(RS)-[(R)-2-amino-2-carboxyethyl]sulphinyl]-2-[[[(1S)-2,2-
dimethylcyclopropyl]carbonyl]amino]hept-2-enoic acid,
HOOC
S
COOH
H
HN O
Me
Me
H
HN
3C Me
H
O
and epimer at C* *
B. (Z)-7[[(R)-2[[(1RS)-1-methyl-3-oxobutyl]amino]-2-carboxyethyl]sulphanyl]-2-[[[(1S)-2,2-
dimethycyclopropyl]carbonyl]amino]hept-2-enoic acid,
HOOC
S
COOH
H
HN O
Me
Me
H
HN
3C Me
Me
O
C. (Z)-7[[(R)-2-[(1,1-dimethyl-3-oxobutyl)amino]-2-carboxyethyl]sulphanyl]-2-[[[(1S)-2,2-
dimethylcyclopropyl]carbonyl]amino]hept-2-enoic acid,
CH3
O 3C
CH3
D. 4-methylpent-3-en-2-one (mesityl oxide).
__________________________________________________________________________________________________________ Ph Eur
10-18
Cilazapril
1/01
N
N
H COOH
O
H
N
EtOOC H
H
C
22
H
31
N
3
O
5
,H
2
O 435.5 92077-78-6
88768-40-5 (anhydrous)
Cilazapril complies with the requirements of the 3rd edition of the European Pharmacopoeia [1499]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cilazapril contains not less than 98.5 per cent and not more than the equivalent of 101.5 per cent of
(1S,9S)-9-[[(S)-1-ethoxycarbonyl-3-phenylpropyl]amino]-10-oxo-octahydro-6H-pyridazino-[1,2-
a][1,2]diazepine-1-carboxylic acid, calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white, crystalline powder, slightly soluble in water, freely soluble in methanol and
IDENTIFICATION
obtained with cilazapril CRS.
B. It complies with the test for specific optical rotation (see Tests).
TESTS
Specific optical rotation (2.2.7). Dissolve 0.200 g in 0.067M phosphate buffer solution pH 7.0 R, with
the aid of ultrasound if necessary and dilute to 50.0 ml with the same buffer solution. The specific
optical rotation is 383 to 399, determined at 365 nm and calculated with reference to the
Impurity A Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R.
the same solvent.
Reference solution (a). Dissolve 2 mg of cilazapril impurity A CRS in methanol R and dilute to 50.0 ml
Reference solution (b). Dissolve 5 mg of cilazapril impurity A CRS and 5 mg of the substance to be
examined in methanol R and dilute to 10.0 ml with the same solvent.
of glacial acetic acid R, 5 volumes of water R, 15 volumes of hexane R, 15 volumes of methanol R and
60 volumes of ethyl acetate R. Dry the plate in a current of cold air for 10 min. Spray the plate with a
freshly prepared mixture of 1 volume of potassium iodobismuthate solution R and 10 volumes of dilute
acetic acid R and then with dilute hydrogen peroxide solution R. Any spot corresponding to impurity A in
the chromatogram obtained with the test solution is not more intense than the spot in the chromato-
gram obtained with reference solution (a) (0.1 per cent). The test is not valid unless the chromato-
Other related substances Examine by liquid chromatography (2.2.29).
Reference solution (b). Dissolve 10.0 mg of cilazapril impurity D CRS in the test solution and dilute to
10-19
as mobile phase at a flow rate of 1.0 ml/min a mixture prepared as follows: mix 10 volumes of
triethylamine R and 750 volumes of water R, adjust to pH 2.30 with phosphoric acid R, and add
200 volumes of tetrahydrofuran R,
(a) is at least 50 per cent of the full scale of the recorder. The test is not valid unless the resolution
between the peaks corresponding to cilazapril and impurity D in the chromatogram obtained with
reference solution (b) is at least 2.5.
Inject 20 l of the test solution and 20 l of reference solution (a). Continue the chromatography
for twice the retention time of the principal peak. When impurity A is present (relative retention time
of 4 to 5), it may be necessary to continue the chromatography until it is eluted. When the chromato-
grams are recorded in the prescribed conditions, the retention times relative to cilazapril are: impurity
B about 0.6; impurity D about 0.9; impurity C about 1.6. In the chromatogram obtained with the
test solution: the area of any peak corresponding to impurity D is not greater than 0.4 times the area
area of any peak corresponding to impurity B is not greater than the area of the principal peak in the
chromatogram obtained with reference solution (a) (0.5 per cent); the area of any peak correspond-
ing to impurity C is not greater than 0.2 times the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.1 per cent); the area of any peak, apart from the principal
peak and the peaks corresponding to impurities B, C and D, is not greater than 0.2 times the area of
the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent); the sum
of the areas of all the peaks, apart from the principal peak, is not greater than twice the area of the
principal peak in the chromatogram obtained with reference solution (a) (1 per cent). Disregard any
peak with an area less than 0.1 times that of the principal peak in the chromatogram obtained with
reference solution (a) and any peak due to impurity A.
mination of water.
ASSAY
Dissolve 0.300 g in 10 ml of ethanol R and add 50 ml of water R. Titrate with 0.1M sodium hydroxide,
22
H
31
N
3
O
5
.
STORAGE
IMPURITIES
N
N
H COOBu
t
O
H
N
EtOOC H
H
A. 1,1-dimethylethyl (1S,9S)-9-[[(S)-1-ethoxycarbonyl-3-phenylpropyl]amino]-10-oxo-
octahydro- 6H-pyridazino[1,2-a][1,2]diazepine-1-carboxylate,
N
N
H COOH
O
H
N
HOOC H
H
B. (1S,9S)-9-[[(S)-1-carboxy-3-phenylpropyl]amino]-10-oxo-octahydro- 6H-pyridazino[1,2-
a][1,2]diazepine-1-carboxylic acid,
N
N
H COOEt
O
H
N
EtOOC H
H
C. ethyl (1S,9S)-9-[[(S)-1-ethoxycarbonyl-3-phenylpropyl]amino]-10-oxo-octahydro- 6H-
pyridazino[1,2-a][1,2]diazepine-1-carboxylate,
10-20
N
N
H COOH
O
H
N
EtOOC H
H
D. (1S,9S)-9-[[(R)-1-ethoxycarbonyl-3-phenylpropyl]amino]-10-oxo-octahydro-6H-
pyridazino- [1,2-a][1,2]diazepine-1-carboxylic acid.
__________________________________________________________________________________________________________ Ph Eur
10-21
Cimetidine
N
N
S
N
H
N
NHMe
CN
Me
C
10
H
16
N
6
S 252.3 51481-61-9
Cimetidine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0756]. These
2
Preparations
Cimetidine Injection
Cimetidine Oral Solution
Cimetidine Oral Suspension
Cimetidine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cimetidine contains not less than 98.5 per cent and not more than the equivalent of 101.5 per cent
of 2-cyano-1-methyl-3-[2-(5-methylimidazol-4-ylmethylthio)ethyl]guanidine, calculated with refer-
ence to the dried substance.
CHARACTERS
A white or almost white powder, slightly soluble in water, soluble in alcohol, practically insoluble in
ether and in methylene chloride. It dissolves in dilute mineral acids.
IDENTIFICATION
A. Melting point (2.2.14): 139C to 144C. If necessary, dissolve the substance to be examined in
2-propanol R, evaporate to dryness and determine the melting point again.
obtained with cimetidine CRS. If the spectra obtained in the solid state show differences, dissolve the
substance to be examined and the reference substance separately in 2-propanol R, evaporate to
dryness and record the spectra again.
spot in the chromatogram obtained with reference solution (d).
D. Dissolve about 1 mg in a mixture of 1 ml of ethanol R and 5 ml of a freshly prepared 20 g/l
solution of citric acid R in acetic anhydride R. Heat in a water-bath for 10 min to 15 min. A reddish-
violet colour develops.
TESTS
Appearance of solution Dissolve 3.0 g in 12 ml of 1M hydrochloric acid and dilute to 20 ml with
water R. The solution is clear (2.2.1) and not more intensely coloured than reference solution Y
5
(Method II, 2.2.2).
254
R as the
coating substance.
Reference solution (a). Dilute 1 ml of test solution (a) to 100 ml with methanol R. Dilute 20 ml of this
solution to 100 ml with methanol R.
Reference solution (d). Dissolve 10 mg of cimetidine CRS in 2 ml of methanol R.
A. Apply separately to the plate 4 l of each solution. Allow the plate to stand for 15 min in the
10-22
chromatographic tank saturated with vapour from the mobile phase which consists of a mixture of
15 volumes of concentrated ammonia R, 20 volumes of methanol R and 65 volumes of ethyl acetate R
and develop immediately over a path of 15 cm using the same mixture of solvents. Dry the plate in a
stream of cold air, expose to iodine vapour until maximum contrast of the spots has been obtained
(a), apart from the principal spot, is not more intense than the principal spot in the chromatogram
obtained with reference solution (a) (0.2 per cent) and not more than two such spots are more
intense than the principal spot in the chromatogram obtained with reference solution (b) (0.1 per
cent). The test is not valid unless the chromatogram obtained with reference solution (c) shows a
clearly visible spot.
B. Apply separately to the plate 4 l of each solution. Develop over a path of 15 cm using a mixture
of 8 volumes of concentrated ammonia R, 8 volumes of methanol R and 84 volumes of ethyl acetate R.
Dry the plate in a stream of cold air, expose to iodine vapour until maximum contrast of the spots has
been obtained and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained
with the test solution (a), apart from the principal spot, is not more intense than the principal spot in
the chromatogram obtained with reference solution (a) (0.2 per cent) and not more than two such
spots are more intense than the principal spot in the chromatogram obtained with reference solution
(b) (0.1 per cent). The test is not valid unless the chromatogram obtained with reference solution (c)
shows a clearly visible spot.
100C to 105C.
ASSAY
10
H
16
N
6
S.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
10-23
Cimetidine Hydrochloride
1/01
N
N
S
N
H
N
NHMe
CN
Me
,HCl
C
10
H
16
N
6
S,HCl 288.8 70059-30-2
Cimetidine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
2
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cimetidine hydrochloride contains not less than 98.5 per cent and not more than the equivalent of
101.5 per cent of 2-cyano-1-methyl-3-[2-[[(5-methyl-1H-imidazol-4-yl)methyl]sulphanyl]-
ethyl]guanidine hydrochloride, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, freely soluble in water, sparingly soluble in ethanol.
IDENTIFICATION
A. Dissolve 70 mg in 0.2M sulphuric acid and dilute to 100.0 ml with the same acid. Dilute 2.0 ml of
the solution to 100.0 ml with 0.2M sulphuric acid. Measure the absorbance (2.2.25) at the absorption
obtained with cimetidine hydrochloride CRS.
spot in the chromatogram obtained with reference solution (d).
D. Dissolve about 1 mg in a mixture of 1 ml of ethanol R and 5 ml of a freshly prepared 20 g/l
solution of citric acid R in acetic anhydride R. Heat on a water-bath for 10 min to 15 min. A reddish-
violet colour develops.
TESTS
Appearance of solution Dissolve 3.0 g in 12 ml of 1M hydrochloric acid and dilute to 20 ml with
5
(Method II, 2.2.2).
pH (2.2.3). Dissolve 100 mg in carbon dioxide-free water R and dilute to 10.0 ml with the same
solvent. The pH of the solution is 4.0 to 5.0.
Related substances Examine by thin-layer chromatography (2.2.27), using a TLC silica gel GF
254
plate R.
Reference solution (a). Dilute 2 ml of test solution (b) to 100 ml with methanol R.
Reference solution (d). Dissolve 10 mg of cimetidine hydrochloride CRS in 2 ml of methanol R.
A. Apply to the plate 4 l of each solution. Allow the plate to stand for 15 min in a chromatographic
tank saturated with vapour from the mobile phase, which consists of a mixture of 15 volumes of
concentrated ammonia R, 20 volumes of methanol R and 65 volumes of ethyl acetate R, and develop
immediately over a path of 15 cm using the same mixture of solvents. Dry the plate in a stream of
cold air, expose to iodine vapour until maximum contrast of the spots has been obtained and
examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with test solution (a),
10-24
apart from the principal spot, is not more intense than the principal spot in the chromatogram
obtained with reference solution (a) (0.2 per cent) and at most two such spots are more intense than
the principal spot in the chromatogram obtained with reference solution (b) (0.1 per cent). The test
is not valid unless the chromatogram obtained with reference solution (c) shows a clearly visible spot.
B. Apply to the plate 4 l of each solution. Develop over a path of 15 cm using a mixture of 8
volumes of concentrated ammonia R, 8 volumes of methanol R and 84 volumes of ethyl acetate R. Dry
the plate in a stream of cold air, expose to iodine vapour until maximum contrast of the spots has
been obtained and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained
with test solution (a), apart from the principal spot, is not more intense than the principal spot in the
chromatogram obtained with reference solution (a) (0.2 per cent) and at most two such spots are
more intense than the principal spot in the chromatogram obtained with reference solution (b)
(0.1 per cent). The test is not valid unless the chromatogram obtained with reference solution (c)
shows a clearly visible spot.
100C to 105C.
ASSAY
Dissolve 0.200 g of the substance to be examined in a mixture of 5 ml of 0.01M hydrochloric acid and
50 ml of alcohol R. Carry out a potentiometric titration (2.2.20), using 0.1M sodium hydroxide. Read
the volume added between the two points of inflexion.
10
H
17
ClN
6
S.
STORAGE
IMPURITIES
N
N
S
N
H
N
R
2
Me R
1
A. R
1
= CN, R
2
= SCH
3:
3-cyano-2-methyl-1-[2-[[(5-methyl-1H-imidazol-4-
yl)methyl]sulphanyl]ethyl]isothiourea,
B. R
1
= CN, R
2
= OCH
3:
3-cyano-2-methyl-1-[2-[[(5-methyl-1H-imidazol-4-
yl)methyl]sulphanyl]ethyl]isourea,
C. R
1
= CONH
2
, R
2
= NHCH
3:
1-[(methylamino)[[2-[[(5-methyl-1H-imidazol-4-
yl)methyl]sulphanyl]ethyl]amino]methylene]urea,
D. R
1
= H, R
2
= NHCH
3:
1-methyl-3-[2-[[(5-methyl-1H-imidazol-4-
yl)methyl]sulphanyl]ethyl]guanidine,
N
N
S
N
H
N
NHMe
Me
O
CN
E. 2-cyano-1-methyl-3-[2-[[(5-methyl-1H-imidazol-4-yl)methyl]sulphinyl]ethyl]guanidine,
N
N
S
N
H
N
H
N
Me
N
NH
S
Me
CN
F. 2-cyano-1,3-bis[2-[[(5-methyl-1H-imidazol-4-yl)methyl]-sulphanyl]ethyl]guanidine.
__________________________________________________________________________________________________________ Ph Eur
10-25
Cinchocaine Hydrochloride
N
N O
NEt
2
OBu
n
H
,HCl
C
20
H
29
N
3
O
2
,HCl 379.9 61-12-1
Cinchocaine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cinchocaine hydrochloride contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of 2-butoxy-N-(2-diethylaminoethyl)quinoline-4-carboxamide hydrochloride, calcu-
lated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder or colourless crystals, hygroscopic, very soluble in water,
freely soluble in acetone, in alcohol and in methylene chloride. It agglomerates very easily.
IDENTIFICATION
A. Dissolve 60.0 mg in 1M hydrochloric acid and dilute to 100 ml with the same acid. Dilute 2 ml of
the solution to 100 ml with 1M hydrochloric acid. Examined between 220 nm and 350 nm (2.2.25),
the solution shows two absorption maxima, at 246 nm and 319 nm. The ratio of the absorbance
measured at 246 nm to that measured at 319 nm is 2.7 to 3.0.
obtained with cinchocaine hydrochloride CRS. Examine the substances prepared as discs using
potassium chloride R.
D. Dissolve 0.5 g in 5 ml of water R. Add 1 ml of dilute ammonia R2. A white precipitate is formed.
Filter, wash the precipitate with five quantities, each of 10 ml, of water R and dry in a desiccator. It
melts at 64C to 66C (2.2.14).
TESTS
Solution S Dissolve 5.0 g in carbon dioxide-free water R prepared from distilled water R, and dilute to
solution Y
6
(Method II, 2.2.2).
Reference solution (a). Dissolve 20 mg of cinchocaine hydrochloride CRS in methanol R and dilute to
10-26
Reference solution (d). Dissolve 20 mg of benzocaine CRS in methanol R and dilute to 5 ml with the
same solvent. Dilute 1 ml of the solution and 1 ml of reference solution (a) to 20 ml with methanol R.
volume of ammonia R, 5 volumes of methanol R, 30 volumes of acetone R and 50 volumes of toluene R.
Dry the plate in a current of warm air for 15 min. Examine in ultraviolet light at 254 nm. Any spot in
the chromatogram obtained with test solution (a), apart from the principal spot, is not more intense
than the principal spot in the chromatogram obtained with reference solution (b) (0.5 per cent) and
at most one such spot is more intense than the spot in the chromatogram obtained with reference
solution (c) (0.2 per cent). The test is not valid unless the chromatogram obtained with reference
solution (d) shows two clearly separated spots.
60C.
ASSAY
20
H
30
ClN
3
O
2
.
STORAGE
IMPURITIES
N R
1
O R
2
A. R
1
= Cl; R
2
= NH(CH
2
)
2
N(C
2
H
5
)
2
: 2-chloro-N-(2-diethylaminoethyl)quinoline-4-carboxamide,
B. R
1
= OH; R
2
= OH: 2-hydroxyquinoline-4-carboxylic acid,
C. R
1
= OH; R
2
= NH(CH
2
)
2
N(C
2
H
5
)
2
: 2-hydroxy-N-(2diethylaminoethyl)quinoline-4-
carboxamide,
D. R
1
= O(CH
2
)
3
CH
3
; R
2
= OH: 2-butoxyquinoline-4-carboxylic acid.
__________________________________________________________________________________________________________ Ph Eur
10-27
Cinchona Bark
Cinchona; Red Cinchona Bark
Cinchona Bark complies with the requirements of the 3rd edition of the European Pharmacopoeia [0174]. These
Action and use Bitter.
When Powdered Cinchona is prescribed or demanded, material complying with the requirements
supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cinchona bark consists of the dried bark of Cinchona pubescens Vahl (Cinchona succirubra Pavon) or of
its varieties or hybrids. It contains not less than 6.5 per cent of total alkaloids, of which not less than
30 per cent and not more than 60 per cent consists of quinine-type alkaloids.
CHARACTERS
Cinchona bark has an intensely bitter, somewhat astringent taste.
IDENTIFICATION
A. The stem and branch bark is supplied in quilled or curved pieces 2 mm to 6 mm thick. The outer
surface is dull brownish-grey or grey and frequently bears lichens; it is usually rough, marked with
transverse fissures and longitudinally furrowed or wrinkled and fissured; exfoliation of the outer
surface occurs in some varieties. The inner surface is striated and deep reddish-brown; the fracture is
short in the outer part and fibrous in the inner part. The root bark occurs in irregularly channelled,
curved or twisted pieces. The outer surface is somewhat scaly and the inner surface more or less
striated. The colour of the outer and inner surfaces is similar to that of the inner surface of the stem
bark and the fracture is fibrous.
B. Reduce to a powder (355). The powder is reddish-brown. Examine under a microscope using
chloral hydrate solution R. The powder shows the following diagnostic characters: thin-walled cork
cells filled with reddish-brown contents; yellow, spindle-shaped striated phloem fibres up to 90 m in
diameter and up to 1300 m in length, very thick-walled with an uneven lumen and with
conspicuous, funnel-shaped pits; parenchymatous idioblasts filled with microprisms of calcium
oxalate. Examine under a microscope using glycerol (50 per cent V/V) R. The powder shows a few
starch granules 6 m to 10 m in diameter;
Test solution. To 0.10 g of the powdered drug (180) in a test-tube add 0.1 ml of concentrated
ammonia R and 5 ml of chloroform R. Shake vigorously occasionally during 30 min and filter.
Evaporate the filtrate to dryness on a water-bath and dissolve the residue in 1 ml of alcohol R.
Reference solution. Dissolve 17.5 mg of quinine R, 0.5 mg of quinidine R, 10 mg of cinchonine R and
10 mg of cinchonidine R in 5 ml of alcohol R.
Apply separately to the plate at 2 cm intervals 1 l and 2 l of each solution. Develop over a path of
15 cm using a mixture of 10 volumes of diethylamine R and 90 volumes of chloroform R. Dry the plate
at 100C to 105C until the odour of diethylamine is no longer detectable (about 10 min). Allow the
plate to cool. Spray with anhydrous formic acid R and examine in ultraviolet light at 365 nm. The
spots corresponding to quinine and quinidine show a distinct blue fluorescence. Spray with
iodoplatinate reagent R. The chromatograms obtained with the reference solution show three violet
spots, later becoming violet-grey, with R
f
values of 0.2 to 0.3 (quinine), 0.3 to 0.4 (quinidine) and 0.4
to 0.5 (cinchonine), and an intense dark blue spot (cinchonidine) with an R
f
value slightly lower than
that of quinidine. The chromatograms obtained with the test solution show spots similar in position,
colour and intensity to the spots in the chromatogram obtained with the same volume of the refer-
ence solution.
D. Carefully heat 0.5 g of the powdered drug (180) in a test-tube over an open flame. Blood-red
drops condense on the wall of the tube. Allow to cool and take up the drops in 10 ml of alcohol
(70 per cent V/V) R. The solution shows a blue fluorescence in ultraviolet light at 365 nm.
TESTS
10-28
ASSAY
In a 250 ml conical flask mix 1.000 g of the powdered drug (180) with 10 ml of water R and 7 ml of
dilute hydrochloric acid R. Heat in a water-bath for 30 min, allow to cool and add 25 ml of chloro-
form R, 50 ml of ether R and 5 ml of a 200 g/l solution of sodium hydroxide R. Shake the mixture
repeatedly for 30 min, add 3 g of tragacanth R powder and shake until the mixture becomes clear.
Filter through a plug of absorbent cotton and rinse the flask and the cotton with five quantities, each
of 20 ml, of a mixture of 1 volume of chloroform R and 2 volumes of ether R. Combine the filtrate and
washings, evaporate to dryness and dissolve the residue in 10.0 ml of ethanol R. Evaporate 5.0 ml of
the solution to dryness, dissolve the residue in 0.1M hydrochloric acid and dilute to 1000.0 ml with the
same acid. Prepare two reference solutions by dissolving separately 30.0 mg of quinine R and 30.0 mg
of cinchonine R in 0.1M hydrochloric acid and diluting each solution to 1000.0 ml with the same acid.
Measure the absorbances (2.2.25) of the three solutions at 316 nm and 348 nm.
Calculate the percentage content of alkaloids from the following equations:
x
A A A A
A A A A m
c c
q c c q

[ ] [ ]
[ ] [ ]
316 348 316 348
316 348 316 348
100 2
1000
1000
2 100
] [ ] [
] [ ] [
c 348 q 316 q 348 c 316
348 q 316 q 348 316

m A A A A
A A A A
y
m=mass of the drug used in grams,
x=percentage content of quinine-type alkaloids,
y =percentage content of cinchonine-type alkaloids,
A
316
=absorbance of the test solution at 316 nm,
A
348
=absorbance of the test solution at 348 nm,
A
316c =
absorbance of the reference solution containing cinchonine at 316 nm, corrected to a
concentration of 1 mg per 1000 ml,
A
316q
=absorbance of the reference solution containing quinine at 316 nm, corrected to a
A
348c
=absorbance of the reference solution containing cinchonine at 348 nm, corrected to a
A
348q
=absorbance of the reference solution containing quinine at 348 nm, corrected to a
concentration of 1 mg per 1000 ml.
Calculate the content of total alkaloids, (x and y), and the relative content of quinine-type
alkaloids, from the following expression:
100x
x y +
STORAGE
Store protected from light and moisture.
__________________________________________________________________________________________________________ Ph Eur
10-29
Cinnamic Acid
COOH
C
9
H
8
O
2
148.2 621-82-9
Definition Cinnamic Acid is (E)-3-phenylprop-2-enoic acid. It contains not less than 99.0% and
9
H
8
O
2
Characteristics Colourless crystals; odour, faint and balsamic.
Very slightly soluble in water; freely soluble in ethanol (96%); soluble in chloroform and in ether.
Identification
cinnamic acid (RS 062).
sodium hydroxide exhibits a maximum only at 267 nm. The absorbance at 267 nm is about 1.4.
Ethanol-insoluble matter A 10% w/v solution in ethanol (96%) is clear, Appendix IV A.
gel GF
254
as the coating substance and a mixture of 90 volumes of toluene and 10 volumes of glacial
acetic acid as the mobile phase. Apply separately to the plate 5 l of each of two solutions of the
substance being examined in methanol containing (1) 5.0% w/v and (2) 0.025% w/v. After removal of
the plate, allow it to dry in air and examine under ultraviolet light (254 nm). Any secondary spot in the
chromatogram obtained with solution (1) is not more intense than the spot in the chromatogram
obtained with solution (2).
Loss on drying When dried to constant weight at 60 at a pressure not exceeding 0.7 kPa, loses not
more than 1.0% of its weight. Use 1 g.
Assay Dissolve 0.5 g in 15 ml of ethanol (96%) previously neutralised to phenol red solution and titrate
with 0.1M sodium hydroxide VS using phenol red solution as indicator. Each ml of 0.1M sodium hydroxide
VS is equivalent to 14.82 mg of C
9
H
8
O
2
.
Action and use Antimicrobial preservative; pharmaceutical aid.
10-30
Cinnamon
Cinnamon Bark; Ceylon Cinnamon
Cinnamon complies with the requirements of the 3rd edition of the European Pharmacopoeia [0387]. These
When Powdered Cinnamon is prescribed or demanded, material complying with the requirements
below with the exception of Identification test A and containing not less than 1.0% v/w (10 ml/kg) of
essential oil shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cinnamon consists of the dried bark, freed from the outer cork and the underlying parenchyma, of
the shoots grown on cut stock of Cinnamomum zeylanicum Nees. It contains not less than 12 ml/kg of
essential oil.
CHARACTERS
Cinnamon has a characteristic, aromatic odour.
IDENTIFICATION
A. The bark is about 0.2 mm to 0.8 mm thick and occurs in closely-packed compound quills made
up of single or double quills. The outer surface is smooth, yellowish-brown with faint scars marking
the position of leaves and axillary buds and has fine, whitish and wavy longitudinal striations. The
inner surface is slightly darker and longitudinally striated. The fracture is short and fibrous.
B. Reduce to a powder (355). The powder is yellowish to reddish-brown. Examine under a
groups of rounded sclereids with pitted, channelled and moderately thickened walls; numerous
colourless single fibres, often whole with narrow lumen and thickened, lignified walls and few pits;
small acicular crystals of calcium oxalate. Examine under a microscope using a 50 per cent V/V
solution of glycerol R; the powder shows abundant starch granules. Cork fragments are absent or very
rare.
254
Test solution. Shake 0.1 g of powdered drug (500) with 2 ml of methylene chloride R for 15 min. Filter
and evaporate the filtrate carefully almost to dryness on a water-bath. Dissolve the residue in 0.4 ml
of toluene R.
Reference solution. Dissolve 50 l of cinnamic aldehyde R and 10 l of eugenol R in toluene R and dilute
Apply separately to the plate as bands 20 mm by 3 mm, 10 l of each solution. Develop over a path
of 10 cm using methylene chloride R. Allow the plate to dry in air, examine in ultraviolet light at
254 nm and mark the quenching zones. Examine the plate in ultraviolet light at 365 nm and mark
the fluorescent zones. In ultraviolet light at 254 nm, the chromatograms obtained with the test and
reference solutions show a quenching zone (cinnamaldehyde) in the median part and just above it a
weaker quenching zone (eugenol). In ultraviolet light at 365 nm, the chromatogram obtained with
the test solution shows a zone of light-blue fluorescence (o-methoxycinnamaldehyde) just below the
zone corresponding to cinnamaldehyde. Spray with phloroglucinol solution R. The zone corresponding
to cinnamaldehyde is yellowish-brown and the zone corresponding to o-methoxycinnamaldehyde is
violet.
TESTS
ASSAY
Carry out the determination of essential oils in vegetable drugs (2.8.12). Use a 500 ml flask, 200 ml
of 0.1M hydrochloric acid as the distillation liquid and 0.50 ml of xylene R in the graduated tube.
Powder the drug (710) and proceed immediately with the determination, using 20.0 g. Distil at a rate
of 2.5 ml to 3.5 ml per minute for 3 h.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
10-31
Ceylon Cinnamon Bark Oil
Cinnamon Oil 1/01
Cinnamon Oil complies with the requirements of the 3rd edition of the European Pharmacopoeia for Ceylon
Cinnamon Bark Oil [1501]. These requirements are reproduced after the heading Definition below.
Action and use Carminative; Flavouring agent.
Preparation
Concentrated Cinnamon Water
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ceylon cinnamon bark oil is obtained by steam distillation of the bark of the shoots of Cinnamomum
zeylanicum Nees (C. Verum J.S. Presl.).
CHARACTERS
A clear, mobile, light yellow liquid becoming reddish over time, with a characteristic odour
reminiscent of cinnamic aldehyde.
IDENTIFICATION
Test solution. Dissolve 1 ml of the substance to be examined in acetone R and dilute to 10 ml with the
same solvent.
Reference solution. Dissolve 50 l of trans-cinnamic aldehyde R, 10 l of eugenol R, 10 l of linalol R and
10 l of -caryophyllene R in alcohol R and dilute to 10 ml with the same solvent.
10 volumes of methanol R and 90 volumes of toluene R. Allow the plate to dry in air and spray with
anisaldehyde solution R. Examine in daylight while heating at 100C to 105C for 5 min to 10 min.
The chromatogram obtained with the test solution shows zones similar in position and colour to
those in the chromatogram obtained with the reference solution.
B. Examine the chromatograms obtained in the test for chromatographic profile. The retention times
of the principal peaks in the chromatogram obtained with the test solution are similar to those in the
chromatogram obtained with the reference solution. Safrole, coumarin and cineole may be absent
from the chromatogram obtained with the test solution.
TESTS
Optical rotation (2.2.7): 2 to +1.
Reference solution. Dissolve 10 l of cineole R, 10 l of linalol R, 10 l of -caryophyllene R, 10 l of
safrole R, 100 l of trans-cinnamic aldehyde R, 10 l of eugenol R, 20 mg of coumarin R, 10 l of trans-2-
methoxycinnamaldehyde R and 10 l of benzyl benzoate R in 1 ml of acetone R.
10-32
Time
(min)
Temperature
(C)
Rate (C
per min)
Comment
Column 010 60 isothermal
75200 190 isothermal
Injection port 200
Detector 240
Inject 0.2 l of the reference solution. When the chromatogram is recorded in the prescribed
conditions, the components elute in the order indicated in the composition of the reference
solution. Record the retention times of these substances.
The test is not valid unless the resolution between the peaks corresponding to linalol and
-caryophyllene is at least 1.5.
obtained with the reference solution, locate the components of the reference solution in the chroma-
togram obtained with the test solution. Determine the percentage content of each of these
components by the normalisation procedure.
cineole less than 3.0 per cent
linalol 1.0 per cent to 6.0 per cent
-caryophyllene 1.0 per cent to 4.0 per cent
safrole less than 0.5 per cent
trans-cinnamic aldehyde 55 per cent to 75 per cent
eugenol less than 7.5 per cent
coumarin less than 0.5 per cent
trans-2-methoxy-
cinnamaldehyde 0.1 per cent to 1.0 per cent
benzyl benzoate less than 1.0 per cent
STORAGE
10-33
05
1
0
1
5
2
0
2
5
3
0
3
5
4
0
4
5
5
0
5
5
6
0
6
5
7
0
7
5
8
0
8
5
9
0
9
5
1
0
0
1
0
5
1
1
0
1
1
5
1
2
0
1
2
5
1
3
0
1
3
5
1
4
0
1
4
5
1
5
0
1
5
5
1
6
0
1
6
5
1
7
0
1
7
5
1
8
0
1
8
5
1
9
0
1
9
5
2
0
0
9
7 8
6
4
2 3 5
1
0.0
0.1
0.2
0.3
0.4
0.5
Volts
1. cineole
2. linalol
3. -caryophyllene
4. safrole
5. trans-cinnamic aldehyde
6. eugenol
7. trans-2-methoxycinnamaldehyde
8. coumarin
9. benzyl benzoate
Depending on operating conditions and the state of the column, coumarin may elute before or
after trans-2-methoxycinnamaldehyde.
Fig. 15011 Chromatographic profile of Ceylon cinnamon bark oil.
__________________________________________________________________________________________________________ Ph Eur
10-34
Cinnarizine
N
N
H
H
C
26
H
28
N
2
368.5 298-57-7
Cinnarizine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0816]. These
1
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cinnarizine contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of (E)-1-(diphenylmethyl)-4-(3-phenylprop-2-enyl)piperazine, calculated with reference to the dried
substance.
CHARACTERS
A white or almost white powder, practically insoluble in water, freely soluble in methylene chloride,
soluble in acetone, slightly soluble in alcohol and in methanol.
IDENTIFICATION
obtained with cinnarizine CRS. Examine the substances prepared as discs.
C. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable
the same solvent.
Reference solution (a). Dissolve 10 mg of cinnarizine CRS in methanol R and dilute to 20 ml with the
same solvent.
Reference solution (b). Dissolve 10 mg of cinnarizine CRS and 10 mg of flunarizine hydrochloride CRS in
15 cm using a mixture of 20 volumes of 1M sodium chloride, 30 volumes of methanol R and 50 volumes
of acetone R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal
spot in the chromatogram obtained with the test solution is similar in position and size to the prin-
cipal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the
chromatogram obtained with reference solution (b) shows two clearly separated spots.
D. Dissolve 0.2 g of anhydrous citric acid R in 10 ml of acetic anhydride R in a water-bath at 80C and
maintain the temperature of the water-bath at 80C for 10 min. Add about 20 mg of the substance to
be examined. A purple colour develops.
TESTS
solvent. The solution is clear (2.2.1) and not more intensely coloured than reference solution BY
7
(Method II, 2.2.2).
Acidity or alkalinity Suspend 0.5 g in 15 ml of water R. Boil for 2 min. Cool and filter. Dilute the
filtrate to 20 ml with carbon dioxide-free water R. To 10 ml add 0.1 ml of phenolphthalein solution R and
0.25 ml of 0.01M sodium hydroxide. The solution is pink. To 10 ml add 0.1 ml of methyl red solution R
and 0.25 ml of 0.01M hydrochloric acid. The solution is red.
10-35
Test solution. Dissolve 25.0 mg of the substance to be examined in methanol R and dilute to 10.0 ml
Reference solution (a). Dissolve 12.5 mg of cinnarizine CRS and 15.0 mg of flunarizine hydrochloride
CRS in methanol R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of this solution to
20.0 ml with methanol R.
conditions,
Mobile phase A. A 10 g/l solution of ammonium acetate R,
Mobile phase B. A 0.2 per cent V/V solution of glacial acetic acid R in acetonitrile R,
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
2530 75 25
Switch to initial
eluent
composition
Equilibrate the column for a least 30 min at the initial eluent composition.
obtained with 10 l of reference solution (b) is at least 50 per cent of the full scale of the recorder. If
necessary, adjust the concentration of glacial acetic acid in mobile phase B to obtain a horizontal
baseline in the chromatogram.
conditions, the retention times are: cinnarizine, about 11 min and flunarizine, about 11.5 min. The
test is not valid unless the resolution between the peaks corresponding to cinnarizine and flunarizine
is at least 5.0. If necessary, adjust the time programme for the gradient elution.
with reference solution (b) (0.25 per cent); the sum of the areas of the peaks, apart from the principal
reference solution (b) (0.5 per cent). Disregard any peak due to the blank and any peak with an area
less than 0.2 times the area of the principal peak in the chromatogram obtained with reference
solution (b).
acetone R. Add dilute hydrochloric acid R until dissolution is complete. Dilute to 20 ml with the same
mixture of water R and acetone R. 12 ml of the solution complies with limit test B for heavy metals
(20 ppm). Prepare the standard using 10 ml of lead standard solution (1 ppm Pb) obtained by
diluting lead standard solution (100 ppm Pb) R with the mixture of water R and acetone R.
ASSAY
Dissolve 0.150 g in 50 ml of a mixture of 1 volume of anhydrous acetic acid R and 7 volumes of ethyl
methyl ketone R. Titrate with 0.1M perchloric acid, using 0.2 ml of naphtholbenzein solution R as
indicator.
26
H
28
N
2
.
STORAGE
IMPURITIES
10-36
Ar =
N
NH
Ar
Ar
A. 1-(diphenylmethyl)piperazine,
N
N
Ar
Ar
Ar
B. (Z)-1-(diphenylmethyl)-4-(3-phenylprop-2-enyl)piperazine,
N
N
Ar
Ar
Ar
Ar
+
Cl
_
C. (E)-1,1-bis(3-phenylprop-2-enyl)-4-(diphenylmethyl)piperazinium chloride,
N
N
Ar
Ar
Ar
Ar
D. (E)-1-(diphenylmethyl)-4-[4-phenyl-1-[(E)-2-phenylvinyl]but-3-enyl]piperazine,
N
N
Ar
Ar
Ar
Ar
E. 1,4-bis(diphenylmethyl)piperazine.
__________________________________________________________________________________________________________ Ph Eur
10-37
Ciprofloxacin
HN
N N
F
O
COOH
C
17
H
18
FN
3
O
3
331.4 885721-33-1
Ciprofloxacin complies with the requirements of the 3rd edition of the European Pharmacopoeia [1089]. These
Preparation
Ciprofloxacin Intravenous Infusion
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ciprofloxacin contains not less than 98.5 per cent and not more than the equivalent of 101.5 per cent
of 1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(piperazin-1-yl)-quinoline-3-carboxylic acid, calcu-
CHARACTERS
A pale yellow, crystalline powder, practically insoluble in water, very slightly soluble in ethanol and
methylene chloride. It is soluble in dilute acetic acid.
IDENTIFICATION
with ciprofloxacin CRS.
TESTS
Appearance of solution Dissolve 0.25 g in 0.1M hydrochloric acid and dilute to 20 ml with the same
solvent. The solution is clear (2.2.1) and not more intensely coloured than reference solution GY
4
(Method II, 2.2.2).
Fluoroquinolonic acid. Examine by thin-layer chromatography (2.2.27), using as the coating
substance a suitable silica gel with a fluorescent indicator having an optimal intensity at 254 nm.
Test solution. Dissolve 50 mg of the substance to be examined in dilute ammonia R1 and dilute to 5 ml
Reference solution. Dissolve 10 mg of fluoroquinolonic acid CRS in a mixture of 0.1 ml of dilute
ammonia R1 and 90 ml of water R and dilute to 100 ml with water R. Dilute 2 ml of this solution to
10 ml with water R.
Apply separately to the plate 5 l of each solution. At the bottom of a chromatographic tank, place an
evaporating dish containing 50 ml of concentrated ammonia R. Close the tank and expose the plate to
the ammonia vapour for 15 min. Withdraw the plate and transfer to a chromatographic tank and
develop over a path of 15 cm using a mixture of 10 volumes of acetonitrile R, 20 volumes of
concentrated ammonia R, 40 volumes of methanol R and 40 volumes of methylene chloride R. Allow the
plate to dry in air and examine in ultraviolet light at 254 nm. Any spot corresponding to
fluoroquinolonic acid in the chromatogram obtained with the test solution is not more intense than
Test solution (a). To 25 mg of the substance to be examined add 0.2 ml of dilute phosphoric acid R and
dilute to 50.0 ml with the mobile phase and treat in an ultrasonic bath until a clear solution is
obtained.
Reference solution (a). Dissolve 2.5 mg of ciprofloxacin impurity B CRS in the mobile phase and dilute
to 50.0 ml with the mobile phase. This solution is also used to prepare reference solution (d). Dilute
10-38
Reference solution (b). Dissolve 2.5 mg of ciprofloxacin impurity C CRS in the mobile phase and dilute
Reference solution (c). Dissolve 2.5 mg of ciprofloxacin impurity D CRS in the mobile phase and dilute
Reference solution (d). Mix 0.1 ml of the test solution (a), 1.0 ml of reference solution (a), 1.0 ml of
reference solution (b), 1.0 ml of reference solution (c) as described above and dilute to 50.0 ml with
the mobile phase.
a stainless steel column 0.25 m long and 4.6 mm in internal diameter, packed with base-
deactivated octadecylsilyl silica for chromatography R (5 m),
as mobile phase at a flow rate of about 1.5 ml per minute a mixture of 13 volumes of
acetonitrile R and 87 volumes of 0.025M phosphoric acid, previously adjusted to pH 3.0 with
triethylamine R,
maintaining the column temperature at 40C.
Inject separately 50 l of test solution (b) and 50 l of reference solutions (a), (b), (c) and (d).
When the chromatogram is recorded in the conditions described above, the retention time of
ciprofloxacin is about 9 min. Adjust the sensitivity of the system so that the height of the
ciprofloxacin impurity C peak in the chromatogram obtained with reference solution (d) is at least
40 per cent of the full scale deflection of the recorder. The test is not valid unless in the chromato-
gram obtained with reference solution (d) the resolution between the peaks corresponding to
ciprofloxacin impurity B and the ciprofloxacin impurity C is at least 1.3 and the resolution between
the peaks corresponding to ciprofloxacin and ciprofloxacin impurity D is at least 3.0.
Inject separately 50 l of test solution (a) and 50 l of reference solutions (b) and (c) and record
the chromatograms for twice the retention time of ciprofloxacin. In the chromatogram obtained with
test solution (a): the areas of the peaks corresponding to ciprofloxacin impurity C and ciprofloxacin
impurity D are not greater than the corresponding peaks in the chromatogram obtained with refer-
ence solution (b) and (c) (0.2 per cent); the areas of any other secondary peaks are not greater than
the peak in the chromatogram obtained with reference solution (b) (0.2 per cent). The sum of the
areas of all the peaks, apart from the principal peak, is not greater than 2.5 times the area of the peak
in the chromatogram obtained with reference solution (b) (0.5 per cent). Disregard any peak whose
area is less than 0.25 times the area of the peak in the chromatogram obtained with reference solution
(b).
Heavy metals (2.4.8). Dissolve 0.5 g in dilute acetic acid R and dilute to 30 ml with the same solvent.
Add 2 ml of water R instead of 2 ml of buffer solution pH 3.5 R. The filtrate complies with limit test E
for heavy metals (20 ppm). Prepare the standard using 5 ml of lead standard solution (2 ppm Pb) R.
Loss on drying (2.2.32). Not more than 1.0 per cent determined on 1.000 g by drying under
vacuum at 120C.
ASSAY
Dissolve 0.300 g in 80 ml glacial acetic acid R. Titrate with 0.1M perchloric acid, determining the end-
17
H
18
FN
3
O
3
.
STORAGE
IMPURITIES
N
COOH F
Cl
O
A. 7-chloro-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-3-quinoline-3-carboxylic acid
(fluoroquinolonic acid),
10-39
N
N
HN
COOH
O
B. 1-cyclopropyl-1,4-dihydro-4-oxo-7-(piperazin-1-yl)quinoline-3-carboxylic acid (desfluoro
compound).
N
COOH
F
HN
H
2
N
O
C. 7-[(2-aminoethyl)amino]-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-quinoline-3-carboxylic
acid (ethylenediamine compound),
N
COOH
N
Cl
HN O
D. 7-chloro-1-cyclopropyl-1,4-dihydro-4-oxo-6-(piperazin-1-yl)quinoline-3-carboxylic acid
(by-compound A),
N
F
N
HN
O
E. 1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(piperazin-1-yl)quinoline (decarboxylated
compound).
__________________________________________________________________________________________________________ Ph Eur
10-40
Ciprofloxacin Hydrochloride
HN
N N
F
O
COOH
,HCl
C
17
H
18
FN
3
O
3
,HCl,H
2
O 385.8 86393-32-0
Ciprofloxacin Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Ciprofloxacin Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ciprofloxacin hydrochloride contains not less than 98.0 per cent and not more than the equivalent of
102.0 per cent of 1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(piperazin-1-yl)quinoline-3-carboxylic
acid hydrochloride, calculated with reference to the anhydrous substance.
CHARACTERS
A pale yellow, crystalline powder, soluble in water, slightly soluble in methanol, very slightly soluble
in ethanol, practically insoluble in acetone, in ethyl acetate and in methylene chloride.
IDENTIFICATION
obtained with ciprofloxacin hydrochloride CRS. Examine the substances prepared as discs.
B. 0.1 g gives reaction (b) of chlorides (2.3.1).
TESTS
Appearance of solution Dilute 10 ml of solution S to 20 ml with carbon dioxide-free water R. The
solution is clear (2.2.1) and not more intensely coloured than reference solution GY
4
(Method II,
2.2.2).
Fluoroquinolonic acid (impurity A) Examine by thin-layer chromatography (2.2.27), using as the
coating substance a suitable silica gel with a fluorescent indicator having an optimal intensity at
254 nm.
same solvent.
Reference solution. Dissolve 10 mg of fluoroquinolonic acid CRS in a mixture of 0.1 ml of dilute
ammonia R1 and 90 ml of water R and dilute to 100 ml with water R. Dilute 2 ml of this solution to
10 ml with water R.
Apply separately to the plate 5 l of each solution. At the bottom of a chromatographic tank, place an
evaporating dish containing 50 ml of concentrated ammonia R. Close the tank and expose the plate to
the ammonia vapour for 15 min. Withdraw the plate, transfer to a chromatographic tank and develop
over a path of 15 cm using a mixture of 10 volumes of acetonitrile R, 20 volumes of concentrated
ammonia R, 40 volumes of methanol R and 40 volumes of methylene chloride R. Allow the plate to dry
in air and examine in ultraviolet light at 254 nm. Any spot corresponding to fluoroquinolonic acid in
the chromatogram obtained with the test solution is not more intense than the spot in the chromato-
gram obtained with the reference solution (0.2 per cent).
Related substances Examine by liquid chromatography as described under assay. Inject 50 l of the
test solution and 50 l of reference solution (e) and continue the chromatography for twice the
retention time of ciprofloxacin. In the chromatogram obtained with the test solution: the areas of any
peaks corresponding to ciprofloxacin impurity C and ciprofloxacin impurity D are not greater than
10-41
the corresponding peaks in the chromatogram obtained with reference solution (e) (0.2 per cent);
the areas of any other peaks are not greater than that of the peak due to ciprofloxacin impurity C in
the chromatogram obtained with reference solution (e) (0.2 per cent); the sum of the areas of all
the peaks, apart from the principal peak, is not greater than 2.5 times the area of the peak due to
ciprofloxacin impurity C in the chromatogram obtained with reference solution (e) (0.5 per cent).
Disregard any peak with an area less than 0.25 times the area of the peak due to ciprofloxacin
impurity C in the chromatogram obtained with reference solution (e).
Heavy metals (2.4.8). Dissolve 0.25 g in water R and dilute to 30 ml with the same solvent. Carry
out the prefiltration. The filtrate complies with limit test E for heavy metals (20 ppm). Prepare the
ASSAY
Reference solution (a). Dissolve 25.0 mg of ciprofloxacin hydrochloride CRS in the mobile phase and
Reference solution (b). Dissolve 2.5 mg of ciprofloxacin impurity B CRS in the mobile phase and dilute
Reference solution (c). Dissolve 2.5 mg of ciprofloxacin impurity C CRS in the mobile phase and dilute
Reference solution (d). Dissolve 2.5 mg of ciprofloxacin impurity D CRS in the mobile phase and dilute
Reference solution (e). Mix 0.1 ml of the test solution, 1.0 ml of reference solution (b), 1.0 ml of
reference solution (c), 1.0 ml of reference solution (d) and dilute to 50.0 ml with the mobile phase.
as mobile phase at a flow rate of about 1.5 ml per minute a mixture of 13 volumes of
acetonitrile R and 87 volumes of phosphoric acid (2.45 g/l H
3
PO
4
), previously adjusted to pH 3.0
with triethylamine R,
maintaining the column temperature at 40C. Equilibrate the column with the mobile phase at a flow
rate of 1.5 ml per minute for about 30 min. Inject 50 l of reference solution (e). When the chroma-
togram is recorded in the prescribed conditions, the substances are eluted in the following order:
ciprofloxacin impurity B, ciprofloxacin impurity C, ciprofloxacin and ciprofloxacin impurity D; the
retention time of ciprofloxacin is about 9 min. Adjust the sensitivity of the system so that the height
of the peak due to the ciprofloxacin impurity C is at least 40 per cent of the full scale of the recorder.
The test is not valid unless the resolution between the peaks due to ciprofloxacin impurity B and
ciprofloxacin impurity C is at least 1.3 and the resolution between the peaks due to ciprofloxacin and
ciprofloxacin impurity D is at least 3.0. If necessary, adjust slightly the composition of the mobile
phase. Inject 10 l of reference solution (a) six times. The test is not valid unless the relative standard
deviation of the peak area of ciprofloxacin is at most 1.0 per cent. Inject alternately 10 l of the test
solution and 10 l of reference solution (a).
Calculate the percentage content of ciprofloxacin hydrochloride.
STORAGE
IMPURITIES
N
COOH F
Cl
O
A. 7-chloro-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-3-quinoline-3-carboxylic acid
(fluoroquinolonic acid),
10-42
N
N
HN
COOH
O
B. 1-cyclopropyl-1,4-dihydro-4-oxo-7-(piperazin-1-yl)quinoline-3-carboxylic acid (desfluoro
compound),
N
COOH
F
HN
H
2
N
O
C. 7-[(2-aminoethyl)amino]-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-quinoline-3-carboxylic
acid (ethylenediamine compound),
N
COOH
N
Cl
HN O
D. 7-chloro-1-cyclopropyl-1,4-dihydro-4-oxo-6-(piperazin-1-yl)quinoline-3-carboxylic acid
(by-compound A),
N
F
N
HN
O
E. 1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(piperazin-1-yl)quinoline \ (decarboxylated
compound).
__________________________________________________________________________________________________________ Ph Eur
10-43
Cisapride
1/01
NH
N O
O
F
OMe
Cl
H
2
N
H
OMe
H
and enantiomer
C
23
H
29
ClFN
3
O
4
,H
2
O 484.0 81098-60-4
Cisapride complies with the requirements of the 3rd edition of the European Pharmacopoeia for Cisapride
Monohydrate [0995]. These requirements are reproduced after the heading Definition below.
Action and use Increases gastro-intestinal motility.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cisapride monohydrate contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of 4-amino-5-chloro-N-[(3RS,4SR)-1-[3-(4-fluorophenoxy)propyl]-3-
methoxypiperidin-4-yl]- 2-methoxybenzamide, calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white powder, practically insoluble in water, freely soluble in dimethylformamide,
soluble in methylene chloride, sparingly soluble in methanol.
IDENTIFICATION
with cisapride monohydrate CRS. Examine the substances prepared as discs. If the spectra obtained
minimum volume of methanol R, evaporate to dryness in a current of air and record new spectra using
the residues.
TESTS
Solution S Dissolve 0.20 g in methylene chloride R and dilute to 20.0 ml with the same solvent.
solution BY
6
(Method II, 2.2.2).
Optical rotation (2.2.7). The angle of optical rotation, determined on solution S, is 0.1 to +0.1.
Reference solution (a). Dissolve 5.0 mg of cisapride monohydrate CRS and 40.0 mg of haloperidol CRS
10-44
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
20 21 55 5 45 95
switch to next
step
21 25 5 95
isocratic
25 26 5 80 95 20
switch to initial
eluent
composition
30 = 0 80 20 re-start gradient
Equilibrate the column with the initial mobile phase composition for at least 5 min.
conditions, the retention times are: cisapride about 15 min and haloperidol about 16 min. The test is
not valid unless the resolution between the peaks due to cisapride and haloperidol is at least 2.5. If
necessary, adjust the concentration of methanol in the mobile phase or adjust the time programme
for the linear gradient.
with reference solution (b) (1 per cent). Disregard any peak due to the solvent (blank) and any peak
Water (2.5.12).3.4 per cent to 4.0 per cent, determined on 0.500 g by the semi-micro determination
of water.
ASSAY
ethyl ketone R and titrate with 0.1M perchloric acid. Determine the end-point potentiometrically
(2.2.20).
23
H
29
ClFN
3
O
4
.
STORAGE
IMPURITIES
Qualified impurities: A, B, C, E.
Other detectable impurities: D.
NH
N O
OMe
H
2
N
Cl
O
H OMe
H
and enantiomer
A. 4-amino-5-chloro-2-methoxy-N-[(3RS,4SR)-3-methoxy-1-(3-phenoxypropyl)piperidin-4-
yl]benzamide,
10-45
NH
N O
OMe
H
2
N
Cl
O
F
B. 4-amino-5-chloro-N-[1-[3-(4-fluorophenoxy)propyl]piperidin-4-yl]-2-
methoxybenzamide,
NH
N O
OMe
H
2
N
Cl
O
H OMe
H
F
and enantiomer
C. 4-amino-5-chloro-N-[(3RS,4RS)-1-[3-(4-fluorophenoxy)propyl]-3-methoxypiperidin-4-
yl-2-methoxybenzamide,
NH
N
H
OMe
H
2
N
Cl
O
N
Cl
O
O
F
OMe H
H
MeO
D 4-[(4-amino-5-chloro-2-methoxybenzoyl)amino]-5-chloro-N-[(3RS,4SR)-1-[3-(4-
fluorophenoxy)propyl]-3-methoxypiperidin-4-yl]-2-methoxybenzamide,
NH
N O
OMe
H
2
N
Cl
O
H OH
H
F
and enantiomer
E. 4-amino-5-chloro-N-[(3RS,4SR)-1-[3-(4-fluorophenoxy)propyl]-3-hydroxypiperidin-4-yl]-
2-methoxybenzamide.
_______________________________________________________________________________________________________________ Ph Eur
10-46
Cisapride Tartrate
1/01
NH
N O
O
F
OMe
Cl
H
2
N
H
OMe
H
and enantiomer
HOOC
COOH
OH H
H OH
,
C
23
H
29
ClFN
3
O
6
,C
4
H
6
O
6
616.0
Cisapride Tartrate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1503].
Action and use Increases gastro-intestinal motility.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cisapride tartrate contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of 4-amino-5-chloro-N-[(3RS,4SR)-1-[3-(4-fluorophenoxy)propyl]-3-methoxypiperidin-4-yl]-
2-methoxybenzamide (2R,3R)-2,3-dihydroxybutanedioate, calculated with reference to the dried
substance.
CHARACTERS
A white or almost white powder, slightly soluble in water, freely soluble in dimethylformamide,
slightly soluble in methanol, very slightly soluble in alcohol.
IDENTIFICATION
obtained with cisapride tartrate CRS. Examine the substances prepared as discs. If the spectra
separately in the minimum volume of alcohol R, evaporate to dryness in a current of air and record
new spectra using the residues.
TESTS
same solvent. The specific optical rotation is +5.0 to +10.0, calculated with reference to the dried
substance.
Test solution. Dissolve 0.100 g of the substance to be examined in a mixture of 10 volumes of water R
and 90 volumes of methanol R, with gentle warming if necessary, and dilute to 10.0 ml with the same
Reference solution (a). Dissolve 5.0 mg of cisapride tartrate CRS and 30.0 mg of haloperidol CRS in a
mixture of 10 volumes of water R and 90 volumes of methanol R and dilute to 100.0 ml with the same
Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with a mixture of 10 volumes of
water R and 90 volumes of methanol R. Dilute 5.0 ml of the solution to 20.0 ml with the same
10-47
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
20 21 55 5 45 95
switch to next
step
21 25 5 95
isocratic
25 26 5 80 95 20
switch to initial
eluent
composition
Equilibrate the column with the initial mobile phase composition for at least 5 min.
conditions, the retention times are: cisapride about 15 min and haloperidol about 16 min. The test is
not valid unless the resolution between the peaks due to cisapride and haloperidol is at least 2.5. If
necessary, adjust the concentration of methanol in the mobile phase or adjust the time programme
Inject separately 10 l of a mixture of 10 volumes of water R and 90 volumes of methanol R as a
blank, 10 l of the test solution and 10 l of reference solution (b). In the chromatogram obtained
of the principal peak in the chromatogram obtained with reference solution (b) (0.25 per cent); the
sum of the areas of all the peaks, apart from the principal peak, is not greater than twice the area of
the principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent).
Disregard any peak due to the solvent (blank) and any peak with an area less than 0.2 times the area
of the principal peak in the chromatogram obtained with reference solution (b).
100C to 105C.
ASSAY
Dissolve 0.500 g in 70 ml of glacial acetic acid R and titrate with 0.1M perchloric acid. Determine the
27
H
35
ClFN
3
O
10
.
STORAGE
IMPURITIES
Qualified impurities: A, C.
Other detectable impurities: B, D, E.
NH
N O
OMe
H
2
N
Cl
O
H OMe
H
and enantiomer
A. 4-amino-5-chloro-2-methoxy-N-[(3RS,4SR)-3-methoxy-1-(3-phenoxypropyl)piperidin-4-
yl]benzamide,
10-48
NH
N O
OMe
H
2
N
Cl
O
F
B. 4-amino-5-chloro-N-[1-[3-(4-fluorophenoxy)propyl]piperidin-4-yl]-2-
methoxybenzamide,
NH
N O
OMe
H
2
N
Cl
O
H OMe
H
F
and enantiomer
C. 4-amino-5-chloro-N-[(3RS,4RS)-1-[3-(4-fluorophenoxy)propyl]-3-methoxypiperidin-4-
yl]- 2-methoxybenzamide,
NH
N
H
OMe
H
2
N
Cl
O
N
Cl
MeO
O
O
F
OMe H
H
D. 4-[(4-amino-5-chloro-2-methoxybenzoyl)amino]-5-chloro-N-[(3RS,4SR)-1-[3-(4-
fluorophenoxy)propyl]- 3-methoxypiperidin-4-yl]-2-methoxybenzamide,
NH
N O
OMe
H
2
N
Cl
O
H OH
H
F
and enantiomer
E. 4-amino-5-chloro-N-[(3RS,4SR)-1-[3-(4-fluorophenoxy)propyl]-3-hydroxypiperidin-4-yl]-
2-methoxybenzamide.
__________________________________________________________________________________________________________ Ph Eur
10-49
Cisplatin
Pt
Cl
Cl
NH
3
NH
3
H
6
Cl
2
N
2
Pt 300.0 15663-27-1
Cisplatin complies with the requirements of the 3rd edition of the European Pharmacopoeia [0599]. These
Preparation
Cisplatin Injection
Ph Eur _______________________________________________________________________________________________________________
DEFINITION
Cisplatin contains not less than 97.0 per cent and not more than the equivalent of 102.0 per cent of
cis-diamminedichloroplatinum (II).
CHARACTERS
A yellow powder or yellow or orange-yellow crystals, slightly soluble in water, sparingly soluble in
dimethylformamide, practically insoluble in alcohol.
It decomposes with blackening at about 270C.
Carry out identification test B, the tests (except that for silver) and the assay protected from light.
IDENTIFICATION
obtained with cisplatin CRS. Examine the substances prepared as discs in potassium bromide R.
chromatogram obtained with test solution (a) is similar in position, colour and size to the principal
C. Add 50 mg to 2 ml of dilute sodium hydroxide solution R in a glass dish. Evaporate to dryness.
Dissolve the residue in a mixture of 0.5 ml of nitric acid R and 1.5 ml of hydrochloric acid R. Evaporate
to dryness. The residue is orange. Dissolve the residue in 0.5 ml of water R and add 0.5 ml of
ammonium chloride solution R. A yellow, crystalline precipitate is formed.
TESTS
Solution S1 Dissolve 25 mg in a 9 g/l solution of sodium chloride R prepared with carbon dioxide-free
Solution S2 Dissolve 0.20 g in dimethylformamide R and dilute to 10 ml with the same solvent.
Appearance of solution S1 Solution S1 is clear (2.2.1) and not more intensely coloured than
reference solution GY
5
(Method II, 2.2.2).
Appearance of solution S2 Solution S2 is clear (2.2.1).
pH (2.2.3). The pH of solution S1, measured immediately after preparation, is 4.5 to 6.0.
Related substances Examine by thin-layer chromatography (2.2.27), using cellulose for chromato-
graphy R1 as the coating substance. Activate the plate by heating at 150C for 1 h.
Test solution (a). Dilute 1 ml of solution S2 to 10 ml with dimethylformamide R.
Test solution (b). Use solution S2.
Reference solution (a). Dissolve 10 mg of cisplatin CRS in 5 ml of dimethylformamide R.
Reference solution (b). Dilute 1 ml of solution S2 to 50 ml with dimethylformamide R.
Apply separately to the plate 2.5 l of test solution (a), 2.5 l of reference solution (a), 5 l of test
solution (b) and 5 l of reference solution (b). Develop over a path of 15 cm using a mixture of 10
volumes of acetone R and 90 volumes of dimethylformamide R. Allow the plate to dry in air and spray
with a 50 g/l solution of stannous chloride R in a mixture of equal volumes of dilute hydrochloric acid R
and water R. After 1 h, the chromatogram obtained with test solution (b) shows no spot with an R
f
value less than that of the principal spot and any spot with an R
f
value greater than that of the
10-50
principal spot is not more intense than the spot in the chromatogram obtained with reference
solution (b).
2.2.23).
Test solution. Dissolve 0.100 g of the substance to be examined in 15 ml of nitric acid R, heating to
80C. Cool and dilute to 25.0 ml with water R.
Reference solutions. To suitable volumes (10 ml to 30 ml) of silver standard solution (5 ppm Ag) R add
50 ml of nitric acid R and dilute to 100.0 ml with water R.
Measure the absorbance at 328 nm using a silver hollow-cathode lamp as source of radiation, a
fuel-lean air-acetylene flame and, preferably, a spectral slit width of 0.5 nm. Carry out a blank
determination.
ASSAY
Test solution. Dissolve 50.0 mg of the substance to be examined in a 9 g/l solution of sodium chloride R
Reference solution. Dissolve 50.0 mg of cisplatin CRS in a 9 g/l solution of sodium chloride R and dilute
a column 0.25 m long and 4.6 mm in internal diameter packed with strong-anion-exchange silica
as mobile phase at a flow rate of 1.2 ml per minute a mixture of 10 volumes of a 9 g/l solution of
sodium chloride R and 90 volumes of methanol R,
Use a sample loop. Inject separately 20 l of the test solution and 20 l of the reference solution.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
10-51
Anhydrous Citric Acid
Citric Acid
HOOC COOH
COOH HO
C
6
H
8
O
7
192.1 77-92-9
Anhydrous Citric Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Anhydrous citric acid contains not less than 99.5 per cent and not more than the equivalent of
101.0 per cent of 2-hydroxypropane-1,2,3-tricarboxylic acid, calculated with reference to the
CHARACTERS
A white, crystalline powder, colourless crystals or granules, very soluble in water, freely soluble in
alcohol, sparingly soluble in ether. It melts at about 153C with decomposition.
IDENTIFICATION
A. Dissolve 1 g in 10 ml of water R. The solution is strongly acidic (2.2.4).
obtained with anhydrous citric acid CRS after drying both the substance being examined and the
reference substance at 100C to 105C for 24 h.
C. Add about 5 mg to a mixture of 1 ml of acetic anhydride R and 3 ml of pyridine R. A red colour
develops.
D. Dissolve 0.5 g in 5 ml of water R, neutralise using 1M sodium hydroxide (about 7 ml), add 10 ml of
calcium chloride solution R and heat to boiling. A white precipitate is formed.
TESTS
Appearance of solution. Dissolve 2.0 g in water R and dilute to 10 ml with the same solvent. The
7
, BY
7
or GY
7
(Method II, 2.2.2).
Readily carbonisable substances. To 1.0 g in a cleaned test tube add 10 ml of sulphuric acid R and
immediately heat the mixture in a water-bath at 90 1C for 60 min. Immediately cool rapidly. The
solution is not more intensely coloured than a mixture of 1 ml of red primary solution and 9 ml of
yellow primary solution (Method I, 2.2.2).
Oxalic acid. Dissolve 0.80 g in 4 ml of water R. Add 3 ml of hydrochloric acid R and 1 g of zinc R in
granules. Boil for 1 min. Allow to stand for 2 min. Transfer the supernatant liquid to a test-tube
containing 0.25 ml of a 10 g/l solution of phenylhydrazine hydrochloride R and heat to boiling. Cool
rapidly, transfer to a graduated cylinder and add an equal volume of hydrochloric acid R and 0.25 ml
of a 50 g/l solution of potassium ferricyanide R. Shake and allow to stand for 30 min. Any pink colour
in the solution is not more intense than that in a standard prepared at the same time in the same
manner using 4 ml of a 0.1 g/l solution of oxalic acid R (350 ppm, calculated as anhydrous oxalic
acid).
Heavy metals (2.4.8). Dissolve 5.0 g in several portions in 39 ml of dilute sodium hydroxide solution R
and dilute to 50 ml with distilled water R. 12 ml complies with limit test A for heavy metals (10 ppm).
10-52
Aluminium (2.4.17). If intended for use in the manufacture of dialysis solutions, it complies with
the test for aluminium. Dissolve 20 g in 100 ml of water R and add 10 ml of acetate buffer solution pH
6.0 R. The solution complies with the limit test for aluminium (0.2 ppm). Use as the reference
solution a mixture of 2 ml of aluminium standard solution (2 ppm Al) R, 10 ml of acetate buffer solution
pH 6.0 R and 98 ml of water R. To prepare the blank use a mixture of 10 ml of acetate buffer solution
pH 6.0 R and 100 ml of water R.
ASSAY
Dissolve 0.550 g in 50 ml of water R. Titrate with 1M sodium hydroxide, using 0.5 ml of
phenolphthalein solution R as indicator.
6
H
8
O
7
.
STORAGE
LABELLING
The label states:
where applicable, that the substance is intended for use in the manufacture of dialysis solutions.
__________________________________________________________________________________________________________ Ph Eur
10-53
Citric Acid Monohydrate
HOOC COOH
COOH HO
,H
2
O
C
6
H
8
O
7
,H
2
O 210.1 5949-29-1
Citric Acid Monohydrate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Citric acid monohydrate contains not less than 99.5 per cent and not more than the equivalent of
101.0 per cent of 2-hydroxypropane-1,2,3-tricarboxylic acid, calculated with reference to the
CHARACTERS
A white, crystalline powder, colourless crystals or granules, efflorescent, very soluble in water, freely
soluble in alcohol, sparingly soluble in ether.
IDENTIFICATION
obtained with citric acid monohydrate CRS after drying both the substance being examined and the
reference substance at 100C to 105C for 24 h.
C. Add about 5 mg to a mixture of 1 ml of acetic anhydride R and 3 ml of pyridine R. A red colour
develops.
D. Dissolve 0.5 g in 5 ml of water R, neutralise using 1M sodium hydroxide (about 7 ml), add 10 ml of
calcium chloride solution R and heat to boiling. A white precipitate is formed.
TESTS
7
, BY
7
or GY
7
(Method II, 2.2.2).
Readily carbonisable substances To 1.0 g in a cleaned test tube add 10 ml of sulphuric acid R and
immediately heat the mixture in a water-bath at 90 1C for 60 min. Immediately cool rapidly. The
solution is not more intensely coloured than a mixture of 1 ml of red primary solution and 9 ml of
yellow primary solution (Method I, 2.2.2).
10-54
Oxalic acid Dissolve 0.80 g in 4 ml of water R. Add 3 ml of hydrochloric acid R and 1 g of zinc R in
granules. Boil for 1 min. Allow to stand for 2 min. Transfer the supernatant liquid to a test-tube
containing 0.25 ml of a 10 g/l solution of phenylhydrazine hydrochloride R and heat to boiling. Cool
rapidly, transfer to a graduated cylinder and add an equal volume of hydrochloric acid R and 0.25 ml
of a 50 g/l solution of potassium ferricyanide R. Shake and allow to stand for 30 min. Any pink colour
in the solution is not more intense than that in a standard prepared at the same time in the same
manner using 4 ml of a 0.1 g/l solution of oxalic acid R (350 ppm, calculated as anhydrous oxalic
acid).
Aluminium (2.4.17). If intended for use in the manufacture of dialysis solutions, it complies with
the test for aluminium. Dissolve 20 g in 100 ml of water R and add 10 ml of acetate buffer solution pH
6.0 R. The solution complies with the limit test for aluminium (0.2 ppm). Use as the reference
solution a mixture of 2 ml of aluminium standard solution (2 ppm Al) R, 10 ml of acetate buffer solution
pH 6.0 R and 98 ml of water R. To prepare the blank use a mixture of 10 ml of acetate buffer solution
pH 6.0 R and 100 ml of water R.
Heavy metals (2.4.8). Dissolve 5.0 g in several portions in 39 ml of dilute sodium hydroxide solution R
and dilute to 50 ml with distilled water R. 12 ml complies with limit test A for heavy metals (10 ppm).
ASSAY
Dissolve 0.550 g in 50 ml of water R. Titrate with 1M sodium hydroxide, using 0.5 ml of
phenolphthalein solution R as indicator.
6
H
8
O
7
.
STORAGE
LABELLING
The label states:
where applicable, that the substance is intended for use in the manufacture of dialysis solutions.
__________________________________________________________________________________________________________ Ph Eur
10-55
Clebopride Malate
N
N
Cl
H
2
N OMe
O
H
,HOOC
COOH
H OH
and enantiomer
C
20
H
24
ClN
3
O
2
,C
4
H
6
O
5
508.0 57645-91-7
Clebopride Malate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1303].
Action and use Anti-emetic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Clebopride malate contains not less than 98.5 per cent and not more than the equivalent of 101.0 per
cent of 4-amino-N-(1-benzylpiperidin-4-yl)-5-chloro-2-methoxybenzamide acid (RS)-2-hydroxy-
butanedioate, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, sparingly soluble in water and in methanol, slightly
soluble in ethanol, practically insoluble in methylene chloride.
IDENTIFICATION
A. Dissolve 20.0 mg in water R and dilute to 100.0 ml with the same solvent. Dilute 10.0 ml of the
shows two absorption maxima, at 270 nm and 307 nm. The specific absorbances at the maxima are
252 to 278 and 204 to 226, respectively.
obtained with clebopride malate CRS. Examine the substances prepared as discs.
C. Dissolve 20 mg in 1 ml of sulphuric acid R, add 1 ml of -naphthol solution R1 and mix. The
solution examined in daylight has a yellow colour with blue fluorescence.
Test solution. Dissolve 5 mg of the substance to be examined in ethanol R and dilute to 10 ml with the
same solvent.
Reference solution (a). Dissolve 5 mg of clebopride malate CRS in ethanol R and dilute to 10 ml with the
same solvent.
Reference solution (b). Dissolve 5 mg of clebopride malate CRS and 5 mg of metoclopramide hydrochloride
CRS in ethanol R and dilute to 10 ml with the same solvent.
Apply to the plate as bands 10 mm by 3 mm, 5 l of each solution. Develop over a path of 15 cm
using a mixture of 2 volumes of concentrated ammonia R, 14 volumes of acetone R, 14 volumes of
methanol R and 70 volumes of toluene R. Allow the plate to dry in air and examine in ultraviolet light
at 254 nm. The principal band in the chromatogram obtained with the test solution is similar in
position and size to the principal band in the chromatogram obtained with the reference solution (a).
The identification is not valid unless the chromatogram obtained with reference solution (b) shows
two clearly separated bands.
TESTS
Appearance of solution Examined immediately after preparation, solution S is clear (2.2.1) and
colourless (Method I, 2.2.2).
10-56
Reference solution (b). Dissolve 10.0 mg of clebopride malate CRS and 10.0 mg of metoclopramide
hydrochloride CRS in the mobile phase and dilute to 100.0 ml with the mobile phase. Dilute 1.0 ml of
this solution to 10.0 ml with the mobile phase.
volumes of a 1 g/l solution of heptanesulphonate sodium R adjusted to pH 2.5 with phosphoric
acid R,
Equilibrate the column with the mobile phase for 30 min. Inject 20 l of reference solution (b).
Adjust the sensitivity of the system so that the heights of the peaks in the chromatogram obtained are
at least 30 per cent of the full scale of the recorder. The test is not valid unless the retention time of
the second peak (clebopride) is about 15 min and the relative retention time of the first peak is about
0.45. Inject 20 l of the test solution and 20 l of reference solution (a). Continue the chromato-
graphy of the test solution for twice the retention time of the principal peak. In the chromatogram
obtained with the test solution: the area of any peak, apart from the principal peak and the two peaks
eluting within 2 min, is not greater than the area of the principal peak in the chromatogram obtained
with reference solution (a) (0.1 per cent); the sum of the areas of all peaks, apart from the principal
peak and the two peaks eluting within 2 min, is not greater than three times the area of the principal
peak in the chromatogram obtained with reference solution (a) (0.3 per cent). Disregard any peak
Chlorides Prepare the solutions at the same time.
Test solution. Dissolve 0.530 g of the substance to be examined in 20.0 ml of anhydrous acetic acid R,
add 6 ml of dilute nitric acid R and dilute to 50.0 ml with water R.
Reference solution. To 1.5 ml of 0. 001M hydrochloric acid, add 20.0 ml of anhydrous acetic acid R and
6 ml of dilute nitric acid R and dilute to 50.0 ml with water R.
Transfer separately both recently prepared solutions to test tubes. Add to each tube 1 ml of silver
nitrate solution R2. Allow to stand for 5 min protected from light. Examine the tubes laterally against a
black background. Any opalescence in the test solution is not more intense than that in the reference
solution (100 ppm).
Sulphates Prepare the solutions at the same time.
Test solution. Dissolve 3.00 g of the substance to be examined in 20.0 ml of glacial acetic acid R,
heating gently if necessary. Allow to cool and dilute to 50.0 ml with water R.
Reference solution. To 9 ml of sulphate standard solution (10 ppm SO
4
) R1, add 6 ml of glacial acetic
acid R.
Into two test tubes introduce 1.5 ml of sulphate standard solution (10 ppm SO
4
) R1 and add 1 ml of a
250 g/l solution of barium chloride R. Shake and allow to stand for 1 min. To one of the tubes add
15 ml of the test solution and to the other one add 15 ml of the reference solution.
After 5 min, any opalescence in the tube containing the test solution is not more intense than that
containing the reference solution (100 ppm).
100C to 105C.
ASSAY
24
H
30
ClN
3
O
7
.
STORAGE
10-57
IMPURITIES
Cl COOH
OMe H
2
N
A. 4-amino-5-chloro-2-methoxybenzoic acid,
N
H
2
N
B. (1-benzylpiperidin-4-yl)amine,
N
N
H
2
N OMe
O
H
C. 4-amino-N-(1-benzylpiperidin-4-yl)-2-methoxybenzamide.
__________________________________________________________________________________________________________ Ph Eur
10-58
Clemastine Fumarate
O
N
H
Me
Cl
Me
COOH
HOOC
H
H
,
C
21
H
26
ClNO,C
4
H
4
O
4
460.0 14976-57-9
Clemastine Fumarate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Histamine-H
1
receptor antagonist.
Preparations
Clemastine Oral Solution
Clemastine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Clemastine fumarate contains not less than 98.5 per cent and not more than the equivalent of 101.0
per cent of (2R)-2-[2-[(R)-1-(4-chlorophenyl)-1-phenylethoxy]ethyl]-1-methylpyrrolidine (E)-
CHARACTERS
A white or almost white, crystalline powder, very slightly soluble in water, sparingly soluble in 70 per
cent V/V alcohol, slightly soluble in 50 per cent V/V alcohol and in methanol.
IDENTIFICATION
obtained with clemastine fumarate CRS.
C. Examine the chromatograms obtained in the test for related substances (see Tests). The principal
D. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance.
the same solvent.
Reference solution. Dissolve 50 mg of fumaric acid CRS in alcohol R and dilute to 10 ml with the same
solvent.
volumes of water R, 25 volumes of anhydrous formic acid R and 70 volumes of di-isopropyl ether R. Dry
the plate at 100C to 105C for 30 min, allow to cool and spray with a 16 g/l solution of potassium
permanganate R. Examine in daylight. In the chromatogram obtained with test solution the spot with
the highest R
f
value is similar in position, colour and size to the spot in the chromatogram obtained
TESTS
Solution S Dissolve 0.500 g of substance to be examined in methanol R and dilute to 50.0 ml with
the same solvent.
solution BY
7
(Method II, 2.2.2).
pH (2.2.3). Suspend 1.0 g of the substance to be examined in 10 ml of carbon dioxide-free water R.
10-59
coating substance.
Test solution (b). Dilute 1.0 ml of test solution (a) to 10.0 ml with methanol R.
Reference solution (a). Dissolve 20.0 mg of clemastine fumarate CRS in methanol R and dilute to
Reference solution (b). Dilute 1.5 ml of test solution (b) to 50.0 ml with methanol R.
Reference solution (c). Dilute 0.5 ml of test solution (b) to 50.0 ml with methanol R.
Reference solution (d). Dissolve 10.0 mg of diphenhydramine hydrochloride CRS in 5.0 ml of reference
solution (a).
volume of concentrated ammonia R, 20 volumes of methanol R and 80 volumes of tetrahydrofuran R.
Dry the plate in a current of cold air for 5 min, spray with a freshly prepared mixture of 1 volume of
potassium iodobismuthate solution R and 10 volumes of dilute acetic acid R and then with dilute hydrogen
peroxide solution R. Cover the plate immediately with a glass plate of the same size and examine the
chromatograms after 2 min. Any spot in the chromatogram obtained with test solution (a), apart
from the principal spot, is not more intense than the spot in the chromatogram obtained with refer-
ence solution (b) (0.3 per cent) and at most four such spots are more intense than the spot in the
chromatogram obtained with reference solution (c) (0.1 per cent). Disregard any spot remaining at
the starting point (fumaric acid). The test is not valid unless the chromatogram obtained with refer-
ence solution (d) shows two clearly separated spots.
1-(4-Chlorophenyl)-1-phenylethanol Examine by liquid chromatography (2.2.29).
Test solution. Dissolve 20 mg of the substance to be examined in a mixture of 25 volumes of aceto-
nitrile R and 75 volumes of a 10 g/l solution of ammonium dihydrogen phosphate R and dilute to 100 ml
Reference solution (a). Dissolve 6 mg of 1-(4-chlorophenyl)-1-phenylethanol CRS in a mixture of 25
volumes of acetonitrile R and 75 volumes of a 10 g/l solution of ammonium dihydrogen phosphate R and
dilute to 100 ml with the same mixture of solvents.
Reference solution (b). Dilute 1 ml of reference solution (a) to 100 ml with a mixture of 25 volumes of
acetonitrile R and 75 volumes of a 10 g/l solution of ammonium dihydrogen phosphate R.
Reference solution (c). Dissolve 10 mg of the substance to be examined in a mixture of 25 volumes of
acetonitrile R and 75 volumes of a 10 g/l solution of ammonium dihydrogen phosphate R and dilute to
100 ml with the same mixture of solvents. To 1 ml of the solution add 1 ml of reference solution (a)
and dilute to 100 ml with a mixture of 25 volumes of acetonitrile R and 75 volumes of a 10 g/l
solution of ammonium dihydrogen phosphate R.
a stainless steel column 0.1 m long and 4.6 mm in internal diameter packed with octadecyl silica
as mobile phase at a flow rate of 1 ml per minute a mixture of 0.1 volumes of phosphoric acid R,
45 volumes of acetonitrile R and 55 volumes of a 10 g/l solution of ammonium dihydrogen
phosphate R,
Inject 100 l of each solution. The test is not valid unless the resolution between the peaks due to
clemastine and 1-(4-chlorophenyl)-1-phenylethanol in the chromatogram obtained with reference
solution (c) is greater than 2.2. In the chromatogram obtained with the test solution: the area of any
peak corresponding to 1-(4-chlorophenyl)-1-phenylethanol is not greater than the area of the peak in
the chromatogram obtained with reference solution (b) (0.3 per cent).
Loss on drying (2.2.32). Not more than 0.5 per cent. determined on 1.000 g by drying in an oven at
100C to 105C for 6 h.
ASSAY
Dissolve 0.350 g of the substance to be examined in 60 ml of anhydrous acetic acid R. Titrate with
25
H
30
ClNO
5
.
IMPURITIES
10-60
O
N
H
Me
Cl
Me
O
*
and epimer at N*
A. (1RS,2R)-2-[2-[(R)-1-(4-chlorophenyl)-1-phenylethoxy]ethyl]-1-methylpyrrolidine
1-oxide,
O
Cl
NMe
Me
B. 4-[1-(4-chlorophenyl)-1-phenylethoxy]-1-methylazepane,
OH
Cl
Me
and enantiomer
C. (RS)-1-(4-chlorophenyl)-1-phenylethanol.
HO
N
H
Me
and enantiomer
D. 2-[(2RS)-1-methylpyrrolidin-2-yl]ethanol.
__________________________________________________________________________________________________________ Ph Eur
10-61
Clenbuterol Hydrochloride
,HCl
NHBu
t
Cl
Cl
H
2
N
H OH
and enantiomer
C
12
N
19
Cl
3
N
2
O 313.7 21898-19-1
Clenbuterol Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Clenbuterol hydrochloride contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of (RS)-1-(4-amino-3,5-dichlorophenyl)-2-[(1,1-dimethylethyl)amino]ethanol
hydrochloride, calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white, crystalline powder, soluble in water and in alcohol, slightly soluble in
acetone.
IDENTIFICATION
obtained with clenbuterol hydrochloride CRS.
254
plate R.
Reference solution. Dissolve 10 mg of clenbuterol hydrochloride CRS in 10 ml of methanol R.
0.15 volumes of ammonia R, 10 volumes of ethanol R and 15 volumes of toluene R. Allow the plate to
dry in air. Spray with a 10 g/l solution of sodium nitrite R in 1M hydrochloric acid and dip after 10 min
in a 4 g/l solution of naphthylethylenediamine dihydrochloride R in methanol R. Allow the plate to dry in
air. The principal spot in the chromatogram obtained with the test solution is similar in position,
TESTS
Solution S Dissolve 0.5 g in 10 ml of carbon dioxide-free water R.
6
(Method II, 2.2.2).
Optical rotation (2.2.7). Dissolve 0.30 g in water R and dilute to 10.0 ml with the same solvent. If
necessary filter. The angle of optical rotation is 0.10 to +0.10.
Test solution. Disperse 100.0 mg of the substance to be examined in the mobile phase and dilute to
Reference solution (a). Dilute 0.1 ml of the test solution to 100.0 ml with water R.
Reference solution (b). Dissolve 10 mg of clenbuterol impurity B CRS in 20 ml of the mobile phase, add
5 ml of the test solution and dilute to 50.0 ml with the mobile phase.
a stainless steel column 0.125 m long and 4 mm in internal diameter, packed with end-capped
10-62
as mobile phase at a flow rate of 0.5 ml/min a mixture of 200 volumes of acetonitrile R, 200
volumes of methanol R and 600 volumes of a solution prepared as follows: dissolve 3.0 g of
sodium decanesulphonate R and 5.0 g of potassium dihydrogen phosphate R in 900 ml of water R,
adjust to pH 3.0 with dilute phosphoric acid R and dilute to 1000 ml with water R,
(a) is at least 50 per cent of the full scale of the recorder. When the chromatogram are recorded in
the prescribed conditions, the retention time of clenbuterol is about 11 min. The test is not valid
until in the chromatogram obtained with reference solution (b) the resolution between the peaks
corresponding to impurity B and clenbuterol is at least 2.5.
Inject 5 l of the test solution and continue the chromatography for 1.5 times the retention time of
the principal peak. In the chromatogram obtained with the test solution: the area of any peak apart
from the principal peak is not greater than the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.1 per cent) and the sum of the areas of all the peaks apart
from the principal peak is not greater than 2 times the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.2 per cent). Disregard any peak with an area less than 0.1
times the area of the principal peak in the chromatogram obtained with reference solution (a).
mination of water.
ASSAY
Dissolve 0.250 g in 50 ml of alcohol R and add 5.0 ml of 0.01M hydrochloric acid. Titrate with 0.1M
sodium hydroxide, determining the end-point potentiometrically (2.2.20). Read the volume added
between the two points of inflexion.
12
H
19
Cl
3
N
2
O.
IMPURITIES
R
2
H
2
N
R
2
R
1
O
A. R
1
= H, R
2
= Cl: 4-amino-3,5-dichlorobenzaldehyde,
B. R
1
= CH
2
-NH-C(CH
3
)
3
, R
2
= Cl: 1-(4-amino-3,5-dichlorophenyl)-2-[(1,1-
dimethylethyl)amino]ethanone (clenbuterol-ketone),
C. R
1
= CH
3
, R
2
= Cl: 1-(4-amino-3,5-dichlorophenyl)ethanone,
D. R
1
= CH
3
, R
2
= H: 1-(4-aminophenyl)ethanone,
E. R
1
= CH
2
Br, R
2
= Cl: 1-(4-amino-3,5-dichlorophenyl)-2-bromoethanone.
__________________________________________________________________________________________________________ Ph Eur
10-63
Clindamycin Hydrochloride
OH
OH
O
HO
SMe
CONHCH
HCCl
CH
3
N
H
H
Me
Pr
n
,HCl
C
18
H
33
ClN
2
O
5
S,HCl 461.5 21462-39-5
Clindamycin Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Clindamycin Capsules
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Clindamycin hydrochloride is methyl 7-chloro-6,7,8-trideoxy-6-[(2S,4R)-1-methyl-4-
propylpyrrolidine-2-carboxamido]-1-thio--L-threo-D-galacto-octapyranoside hydrochloride. It
contains not less than 84.0 per cent and not more than 93.0 per cent of clindamycin
(C
18
H
33
ClN
2
O
5
S), calculated with reference to the anhydrous substance. It contains a variable
quantity of water.
CHARACTERS
A white or almost white, crystalline powder, very soluble in water, slightly soluble in alcohol.
IDENTIFICATION
obtained with clindamycin hydrochloride CRS.
the same solvent.
Reference solution (a). Dissolve 10 mg of clindamycin hydrochloride CRS in methanol R and dilute to
Reference solution (b). Dissolve 10 mg of clindamycin hydrochloride CRS and 10 mg of lincomycin hydro-
chloride CRS and in methanol R and dilute to 10 ml with the same solvent.
Apply separately to the plate 5 l of each solution. Develop over a path of 15 cm using the upper
layer from a mixture of 20 volumes of 2-propanol R, 40 volumes of a 150 g/l solution of ammonium
acetate R previously adjusted to pH 9.6 with ammonia R and 45 volumes of ethyl acetate R. Allow the
plate to dry in air and spray with a 1 g/l solution of potassium permanganate R. The principal spot in
the chromatogram obtained with the test solution is similar in position, colour and size to the prin-
cipal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the
C. Dissolve about 10 mg in 2 ml of dilute hydrochloric acid R and heat in a water-bath for 3 min. Add
3 ml of sodium carbonate solution R and 1 ml of a 20 g/l solution of sodium nitroprusside R. A violet-red
colour develops.
D. Dissolve 0.1 g in water R and dilute to 10 ml with the same solvent. The solution gives reaction
TESTS
10-64
substance.
Related substances Examine the chromatograms obtained in the assay with test solutions (c) and
(d). In the chromatogram obtained with test solution (c), the sum of the areas of the peaks apart
from that corresponding to clindamycin is not greater than 3 per cent of the total area of the peaks
and the area of any one of such peaks is not greater than 2 per cent of the total area. Disregard the
peak corresponding to the solvent. The test is not valid unless the peak corresponding to clindamycin
in the chromatogram obtained with test solution (d) occupies not less than 70 per cent of the scale.
ASSAY
Examine by gas chromatography (2.2.28), using hexacosane R as the internal standard.
Internal standard solution. Dissolve 0.15 g of hexacosane R in ethylene chloride R and dilute to 100 ml
Test solution (a). Dissolve 50.0 mg of the substance to be examined in 1.0 ml of trifluoroacetic
anhydride R, swirl to dissolve and allow to stand for 30 min. Add 20.0 ml of ethylene chloride R and
mix.
Test solution (b). Dissolve 50.0 mg of the substance to be examined in 1.0 ml of trifluoroacetic
anhydride R, swirl to dissolve and allow to stand for 30 min. Add 20.0 ml of the internal standard
solution and mix.
Test solution (c). Dissolve 50.0 mg of the substance to be examined in 0.5 ml of trifluoroacetic
anhydride R, allow to stand for 30 min and dilute to 5.0 ml with ethylene chloride R.
Test solution (d). Dilute 1 ml of test solution (c) to 20 ml with ethylene chloride R.
Reference solution. Dissolve 50.0 mg of clindamycin hydrochloride CRS in 1.0 ml of trifluoroacetic
anhydride R, swirl to dissolve and allow to stand for 30 min. Add 20.0 ml of the internal standard
solution and mix.
a glass column 1.5 m long and 4 mm in internal diameter packed with silanised diatomaceous
earth for gas chromatography R (180 m to 250 m) impregnated with 1 per cent m/m of
polymethylphenylsiloxane R,
nitrogen for chromatography R as the carrier gas at a flow rate of about 30 ml per minute,
detector between 200C and 210C. Inject the chosen volumes of the test solutions and the reference
solution. Continue the recording of the chromatogram obtained with test solution (c) for twice the
retention time of clindamycin.
STORAGE
Store in an airtight container at a temperature not exceeding 30C.
__________________________________________________________________________________________________________ Ph Eur
10-65
Clindamycin Phosphate
OH
O
HO
SMe
CONHCH
HCCl
CH
3
O
P
O
OH HO
N
H
H
Me
Pr
n
C
18
H
34
ClN
2
O
8
PS 505.0 24729-96-2
Clindamycin Phosphate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Clindamycin Injection
Ph Eur _______________________________________________________________________________________________________________
DEFINITION
Clindamycin phosphate contains not less than 95.0 per cent and not more than the equivalent of
100.5 per cent of methyl 7-chloro-6,7,8-trideoxy-6-[(2S,4R)-1-methyl-4-propylpyrrolidine-2-
carboxamido]-1-thio-L-threo--D-galacto-octopyranoside 2-(dihydrogen phosphate), calculated with
CHARACTERS
A white or almost white powder, slightly hygroscopic, freely soluble in water, very slightly soluble in
alcohol, practically insoluble in ether and in methylene chloride.
IDENTIFICATION
A. Examine by infrared spectrophotometry (2.2.24), comparing with the spectrum obtained with
clindamycin phosphate CRS. In two separate tubes place 50 mg of the substance to be examined and
50 mg of clindamycin phosphate CRS. Add 0.2 ml of water R and heat until completely dissolved.
Evaporate to dryness under reduced pressure and dry at 100C to 105C for 2 h. Examine the
substances prepared as discs using potassium bromide R.
B. Examine by thin-layer chromatography (2.2.27), using silica gel H R as the coating substance.
the same solvent.
Reference solution (a). Dissolve 20 mg of clindamycin phosphate CRS in methanol R and dilute to 10 ml
Reference solution (b). Dissolve 10 mg of lincomycin hydrochloride CRS in 5 ml of reference solution (a).
20 volumes of glacial acetic acid R, 20 volumes of water R and 60 volumes of butanol R. Dry the plate
at 100C to 105C for 30 min and spray with a 1 g/l solution of potassium permanganate R. The
size to the principal spot in the chromatogram obtained with reference solution (a). The test is not
valid unless the chromatogram obtained with reference solution (b) shows two principal spots.
4 ml of sodium carbonate solution R and 1 ml of a 20 g/l solution of sodium nitroprusside R. Prepare a
standard in the same manner using clindamycin phosphate CRS. The colour in the test solution
corresponds to that in the standard.
10-66
D. Boil 0.1 g under a reflux condenser with a mixture of 5 ml of strong sodium hydroxide solution R and
5 ml of water R for 90 min. Cool and add 5 ml of nitric acid R. Extract with three quantities, each of
15 ml, of methylene chloride R and discard the extracts. Filter the upper layer through a paper filter.
The filtrate gives reaction (b) of phosphates (2.3.1).
TESTS
Solution S Dissolve 1.00 g in carbon dioxide-free water R. Heat gently if necessary. Cool and dilute to
25.0 ml with carbon dioxide-free water R.
Appearance of the solution Solution S is clear (2.2.1) and colourless (Method II, 2.2.2).
pH (2.2.3). Dilute 5.0 ml of solution S to 20 ml with carbon dioxide-free water R. The pH of the
substance.
reference solution (c). Adjust the sensitivity of the detector so that the height of the principal peak is
not less than 50 per cent of the full scale of the recorder. Inject the test solution and continue the
chromatography for the retention time of clindamycin: the area of any peak, apart from the principal
peak and any peak due to the solvent, is not greater than 2.5 times the area of the peak corresponding
to clindamycin phosphate in the chromatogram obtained with reference solution (c) (2.5 per cent)
and the sum of the areas of all the peaks, apart from the principal peak and any peak due to the
solvent, is not greater than four times the area of the peak corresponding to clindamycin phosphate in
the chromatogram obtained with reference solution (c) (4.0 per cent). Disregard any peak with an
area less than 10.0 per cent of that of the principal peak in the chromatogram obtained with reference
solution (c).
ASSAY
Reference solution (a). Dissolve 75.0 mg of clindamycin phosphate CRS in the mobile phase and dilute
Reference solution (b). Dissolve 5.0 mg of lincomycin hydrochloride CRS and 15.0 mg of clindamycin
hydrochloride CRS in 5.0 ml of reference solution (a) and dilute to 100.0 ml with the mobile phase.
gel for chromatography R (5 m to 10 m),
as mobile phase at a flow rate of 1.0 ml per minute a mixture of 200 ml of acetonitrile R and
800 ml of a 13.6 g/l solution of potassium dihydrogen phosphate R, previously adjusted to pH 2.5
with phosphoric acid R,
Inject reference solution (b). Adjust the sensitivity of the detector so that the height of the peaks is
not less than 50 per cent of the full scale of the recorder. The assay is not valid unless the first peak
(lincomycin) is clearly separated from the solvent peak and the resolution between the second peak
(clindamycin phosphate) and the third peak (clindamycin) is at least 6.0. Adjust the concentration of
acetonitrile in the mobile phase, if necessary. The assay is not valid unless the symmetry factor of the
clindamycin phosphate peak is at most 1.5. Inject reference solution (a) six times. The test is not
valid unless the relative standard deviation of the peak area for clindamycin phosphate is at most
1.0 per cent. If necessary adjust the integrator parameters. Inject alternately the test solution and
Calculate the percentage content of clindamycin phosphate.
STORAGE
10-67
LABELLING
The label states:
IMPURITIES
A. lincomycin,
CH
3
Cl H
CONH H
HO OH
O
P
OH
O
SMe
O
Me
N
Et
HO
B. clindamycin B 2-(dihydrogen phosphate),
Me
N
O
SMe
OH
H CONH
H Cl
CH
3
OR
1
Pr
R
2
O
C. R
1
= PO
3
H
2
; R2 = H: clindamycin 3-(dihydrogen phosphate),
D. R
1
= H; R
2
= PO
3
H
2
: clindamycin 4-(dihydrogen phosphate),
E. clindamycin.
__________________________________________________________________________________________________________ Ph Eur
10-68
Clioquinol
N
OH
Cl
I
C
9
H
5
ClINO 305.5 130-26-7
Definition Clioquinol is 5-chloro-7-iodoquinolin-8-ol. It contains not less than 97.0% and not
more than 103.0% of total phenols, calculated as C
9
H
5
ClINO with reference to the dried
substance.
Characteristics A yellowish white to brownish yellow, voluminous powder; odour, faint and
characteristic.
Practically insoluble in water; freely soluble in dimethylformamide and in pyridine; practically
insoluble in ethanol (96%).
Identification
clioquinol (RS 065).
B. Burn 20 mg by the method for oxygen-flask combustion, Appendix VIII C, using 5 ml of 2M sodium
hydroxide as the absorbing liquid and dilute to 25 ml with water. To 5 ml add 1 ml of silver nitrate
solution; a yellow precipitate is produced. Add 5 ml of 5M ammonia, shake, filter and acidify the
filtrate with nitric acid; a white precipitate is produced.
Acidity or alkalinity Shake 0.5 g with 10 ml of water previously neutralised to phenolphthalein
solution R1. The solution is colourless and not more than 0.05 ml of 0.1M sodium hydroxide VS is
required to change the colour of the solution to pink.
Free iodine Shake 1.0 g with a solution of 1 g of potassium iodide in 20 ml of water for 30 seconds,
allow to stand for 5 minutes and filter. To 10 ml of the filtrate add 1 ml of 1M sulphuric acid and 2 ml
of chloroform and shake. Any colour in the chloroform layer is discharged on the addition of 0.1 ml of
0.005M sodium thiosulphate VS.
Halide ions Shake 0.50 g with 25 ml of water for 1 minute and filter. To the filtrate add 0.5 ml of
2M nitric acid and 0.5 ml of 0.1M silver nitrate and allow to stand for 5 minutes. Any opalescence
produced is not more intense than that obtained by adding 0.5 ml of 0.1M silver nitrate to 25 ml of
water containing 0.5 ml of 2M nitric acid and 0.2 ml of 0.01M hydrochloric acid VS and allowing to
stand for 5 minutes.
Related substances Carry out the method for gas chromatography, Appendix III B, protected from
light, using the following solutions. For solution (1) add 0.5 ml of N,O-bis(trimethylsilyl)acetamide to
0.5 ml of a solution in pyridine containing 0.40% w/v each of 5-chloro-8-hydroxyquinoline, 5,7-dichloro-
8-hydroxyquinoline and 5,7-di-iodo-8-hydroxyquinoline and 0.040% w/v of the substance being exam-
ined, mix, allow to stand for 15 minutes and add 5 ml of a 0.05% w/v solution of dibutyl phthalate
(internal standard) in n-hexane. For solution (2) add 0.5 ml of N,O-bis(trimethylsilyl)acetamide to a
mixture of 0.10 g of the substance being examined and 0.5 ml of pyridine, mix, allow to stand for 15
minutes and add 5 ml of n-hexane. For solution (3) treat a mixture of 0.10 g of the substance being
examined and 0.5 ml of pyridine as described for solution (1).
with silanised diatomaceous support (100 to 120 mesh) coated with 3% w/w of methyl silicone gum
(SE-30 is suitable) and maintained at 190.
In the chromatogram obtained with solution (1) the peaks following the solvent peak, in order of
emergence, are due to (a) 5-chloro-8-hydroxyquinoline, (b) 5,7-dichloro-8-hydroxyquinoline, (c) the
internal standard, (d) clioquinol and (e) 5,7-di-iodo-8-hydroxyquinoline.
In the chromatogram obtained with solution (3) calculate the content of 5-chloro-8-hydro-
xyquinoline, 5,7-dichloro-8-hydroxyquinoline and 5,7-di-iodo-8-hydroxyquinoline by reference to
the corresponding peaks in the chromatogram obtained with solution (1). Calculate the content of
any other impurity by reference to the peak derived from clioquinol in the chromatogram obtained
with solution (1). The content of the named impurities does not exceed 3.0% w/w, the content of any
other impurity does not exceed 0.2% w/w and the sum of the contents is not more than 4.0% w/v.
10-69
Assay Dissolve 0.6 g in 50 ml of anhydrous pyridine and carry out Method II for non-aqueous titration,
Appendix VIII A, using 0.1M tetrabutylammonium hydroxide VS as titrant and determining the end
point potentiometrically. Each ml of 0.1M tetrabutylammonium hydroxide VS is equivalent to
30.55 mg of total phenols, calculated as C
9
H
5
ClNO.
Storage Clioquinol should be protected from light.
Action and use Topical and intestinal antiseptic.
Preparations
Betamethasone and Clioquinol Cream
Betamethasone and Clioquinol Ointment
Hydrocortisone and Clioquinol Cream
Hydrocortisone and Clioquinol Ointment
10-70
Clobazam
N
N
Cl
O
Me
O
C
16
H
13
ClN
2
O
2
300.7 22316-47-8
Definition Clobazam is 7-chloro-1,5-dihydro-1-methyl-5-phenyl-1,5-benzodiazepine-2,4(3H)-
dione. It contains not less than 97.5% and not more than 102.5% of C
16
H
13
ClN
2
O
2
, calculated with
Very slightly soluble in water; soluble in ethanol (96%) and in methanol.
Identification
clobazam (RS 066).
B. In the test for Related substances the spot in the chromatogram obtained with solution (2)
corresponds to that in the chromatogram obtained with solution (5).
Acidity or alkalinity pH of a 1.0% w/v suspension in carbon dioxide-free water, 5.5 to 7.5, Appendix
V L.
Heavy metals Dissolve 1.0 g in 17 ml of acetone and dilute to 20 ml with water. 12 ml of the
solution complies with limit test B for heavy metals, Appendix VII. Use a solution prepared by diluting
lead standard solution (100 ppm Pb) with a mixture of 9 ml of acetone and 6 ml of water to contain 1 g
of Pb per ml as the standard (20 ppm).
gel GF
254
as the coating substance and a mixture of 40 volumes of acetone and 60 volumes of hexane
as the mobile phase. Use an unlined tank for the development of the plate. Apply separately to the
plate 20 l of each of five solutions in methanol containing (1) 2.0% w/v of the substance being exam-
ined, (2) 0.010% w/v of the substance being examined, (3) 0.0040% w/v of the substance being
examined, (4) 0.0040% w/v of 7-chloro-1,5-dihydro-5-phenyl-1,5-benzodiazepine-2,4(3H)-dione BPCRS
and (5) 0.010% w/v of clobazam BPCRS. After removal of the plate, allow it to dry in air and
examine under ultraviolet light (254 nm). Any spot corresponding to 7-chloro-1,5-dihydro-5-phenyl-
1,5-benzodiazepine-2,4(3H)-dione in the chromatogram obtained with solution (1) is not more
intense than the spot in the chromatogram obtained with solution (4) (0.2%). Any other secondary
spot in the chromatogram obtained with solution (1) is not more intense than the spot in the
chromatogram obtained with solution (3) (0.2%).
Loss on drying When dried to constant weight at 100 to 105, loses not more than 1.0% of its
weight. Use 1 g.
Solution (1) contains 0.005% w/v of the substance being examined in methanol. Solution (2) contains
0.005% w/v of clobazam BPCRS in methanol. Solution (3) contains 0.006% w/v of 7-chloro-1,5-
dihydro-5-phenyl-1,5-benzodiazepine-2,4(3H)-dione BPCRS (desmethylclobazam) and 0.005% w/v of
clobazam BPCRS in methanol.
The chromatographic procedure may be carried out using (a) a stainless steel column (20
cm 4.6 mm) packed with stationary phase C (5 m) (Nucleosil C18 is suitable), (b) a mixture of
470 volumes of acetonitrile and 530 volumes of water as the mobile phase with a flow rate of 0.7 ml
per minute and (c) a detection wavelength of 230 nm. Adjust the composition of the mobile phase, if
necessary, such that the resolution factor between the peaks corresponding to desmethylclobazam and
clobazam in the chromatogram obtained with solution (3) is at least 3.
16
H
13
ClN
2
O
2
16
H
13
ClN
2
O
2
in clobazam BPCRS.
Action and use Anxiolytic; anticonvulsant.
Preparation
Clobazam Capsules
10-71
IMPURITIES
N
N
Cl
O
O
H
A. 7-chloro-1,5-dihydro-5-phenyl-1,5-benzodiazepine-2,4(3H)-dione.
10-72
Clobetasol Propionate
H
O
HO
OCOEt
F
Cl
Me
Me
Me H
O
C
25
H
32
ClFO
5
467.0 25122-46-7
Definition Clobetasol Propionate is 21-chloro-9-fluoro-11-hydroxy-16-methyl-3,20-
dioxopregna-1,4-dien-17-yl propionate. It contains not less than 97.0% and not more than 102.0%
of C
25
H
32
ClFO
5
Characteristics A white or almost white, crystalline powder.
Practically insoluble in water; freely soluble in acetone and in dichloromethane; sparingly soluble in
ethanol (96%).
It melts at about 196, with decomposition.
Identification
clobetasol propionate (RS 067).
B. In the Assay, the chromatogram obtained with solution (2) shows a peak with the same retention
time as the peak due to clobetasol propionate in the chromatogram obtained with solution (1).
Related substances Carry out the method for liquid chromatography, Appendix III D, using 10 l of
each of the following solutions. Solution (1) contains 0.01% w/v of the substance being examined in
the mobile phase. Solution (2) contains 0.01% w/v of clobetasol propionate BPCRS and 0.005% w/v of
clobetasol impurity A BPCRS in the mobile phase. For solution (3) dilute 1 ml of solution (1) to 50 ml
(15 cm 4.6 mm) packed with stationary phase C (5 m) (Spherisorb ODS 2 is suitable), (b) as the
mobile phase with a flow rate of 1 ml per minute a solution prepared by mixing 425 volumes of
0.05M sodium dihydrogen orthophosphate adjusted to pH 2.5 with orthophosphoric acid with 475 volumes
of acetonitrile, allowing to cool and adding 100 volumes of methanol and (c) a detection wavelength of
240 nm.
Allow the chromatography to proceed for 2.5 times the retention time of the principal peak. The
test is not valid unless, in the chromatogram obtained with solution (2), the resolution factor between
the peaks corresponding to clobetasol propionate and clobetasol impurity A is at least 1.5.
0.5 times the area of the principal peak in the chromatogram obtained with solution (3) (1%). The
sum of the areas of any secondary peaks is not greater than 1.25 times the area of the principal peak in
the chromatogram obtained with solution (3) (2.5%). Disregard any peak with an area less than
0.025 times the area of the principal peak in the chromatogram obtained with solution (3) (0.05%).
Loss on drying When dried to constant weight at 100 to 105 for 3 hours, loses not more than
0.5% of its weight. Use 1 g.
Assay Prepare a 0.02% w/v solution of beclometasone dipropionate BPCRS (internal standard) in the
mobile phase. Carry out the method for liquid chromatography, Appendix III D, using 10 l of each of
the following solutions. For solution (1) dissolve 10 mg of the substance being examined in the
mobile phase, add 100 ml of the internal standard solution and dilute to 250 ml with the mobile
phase. For solution (2) dissolve 10 mg of clobetasol propionate BPCRS in the mobile phase, add
100 ml of the internal standard solution and dilute to 250 ml with the mobile phase. Solution (3)
contains 0.01% w/v of clobetasol propionate BPCRS and 0.005% w/v of clobetasol impurity A BPCRS in
the mobile phase.
Allow the chromatography to proceed for twice the retention time of the principal peak. The assay
is not valid unless, in the chromatogram obtained with solution (3), the resolution factor between the
peaks corresponding to clobetasol propionate and clobetasol impurity A is at least 3.
25
H
32
ClFO
5
in the substance being examined using the declared content
of C
25
H
32
ClFO
5
in clobetasol propionate BPCRS.
10-73
Storage Clobetasol Propionate should be kept in a well-closed container and protected from light.
Preparations
Clobetasol Cream
Clobetasol Ointment
IMPURITIES
O
H
H Me
Me
Me
F
HO
O
H
O
Cl
Et
A. clobetasol
H Me
Me
Me
F
O
H
O
Cl
OCOEt
O
B. clobetasone 17-propionate
O
H
H Me
Me
Me
F
HO
O
H
O
Cl
Et
C. 9-fluoro-11-hydroxy-16-methyl-3-oxoandrosta-1,4-diene-17(R)-spiro-2-[4-chloro-5-
ethylfuran-3-(2H)-one] (clobetasol impurity A)
10-74
Clobetasone Butyrate
H
O
O
F
Cl
Me
Me
Me H
O
OCOPr
n
C
26
H
32
ClFO
5
479.0 25122-57-0
Clobetasone Butyrate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1090].
Preparations
Clobetasone Cream
Clobetasone Ointment
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Clobetasone butyrate contains not less than 97.0 per cent and not more than the equivalent of
102.0 per cent of 21-chloro-9-fluoro-17-hydroxy-16-methylpregna-1,4-diene-3,11,20-trione 17-
butyrate, calculated with reference to the dried substance.
CHARACTERS
A white or almost white powder, practically insoluble in water, freely soluble in acetone and in
IDENTIFICATION
obtained with clobetasone butyrate CRS. Examine the substances prepared as discs.
methanol R and methylene chloride R and dilute to 10 ml with the same mixture of solvents.
Reference solution (a). Dissolve 10 mg of clobetasone butyrate CRS in a mixture of equal volumes of
Reference solution (b). Dissolve 10 mg of clobetasol propionate R in a mixture of equal volumes of
methanol R and methylene chloride R and dilute to 10 ml with the same mixture of solvents. Dilute
5 ml of this solution to 10 ml with reference solution (a).
equal volumes of cyclohexane R and methyl acetate R. Allow the plate to dry in air and examine in
C. Mix about 5 mg with 45 mg of heavy magnesium oxide R and ignite in a crucible until an almost
colourless. Filter. To a freshly prepared mixture of 0.1 ml alizarin S solution R and 0.1 ml of zirconyl
nitrate solution R, add 1.0 ml of the filtrate. Mix, allow to stand for 5 min and compare the colour of
the solution with that of a blank prepared in the same manner. The colour of the test solution is
yellow and that of the blank is red.
TESTS
substance.
10-75
Related substances Prepare the solutions immediately before use.
Test solution. Dissolve 50.0 mg of the substance to be examined in 5.0 ml of ethanol R and dilute to
Reference solution (a). Dissolve 2 mg of clobetasone butyrate CRS and 1.5 mg of clobetasol propionate R
in 5 ml of ethanol R and dilute to 100 ml with the mobile phase.
a stainless steel column 0.20 m long and 4.6 mm in internal diameter, packed with octadecylsilyl
as mobile phase at a flow rate of 1 ml per minute a mixture of 45 volumes of ethanol R and 55
volumes of water R,
maintaining the temperature of the column at 60C. Inject 20 l of reference solution (a). When
using a recorder, adjust the sensitivity of the system so that the height of the two principal peaks are
at least 50 per cent of the full scale of the recorder. The test is not valid unless the resolution between
the first peak (clobetasol propionate) and the second peak (clobetasone butyrate) is at least five.
for 2.5 times the retention time of the principal peak. In the chromatogram obtained with the test
solution: the area of any peak, apart from the principal peak, is not greater than the area of the prin-
cipal peak in the chromatogram obtained with reference solution (b) (1.0 per cent) and not more
than one such peak has an area greater than half the area of the principal peak in the chromatogram
the principal peak, is not greater than 1.5 times the area of the principal peak in the chromatogram
obtained with reference solution (b) (1.5 per cent). Disregard any peak with an area less than 0.05
times the area of the principal peak in the chromatogram obtained with reference solution (b).
at 100C to 105C.
ASSAY
26
H
32
ClFO
5
STORAGE
IMPURITIES
O
Me
O
Me
Cl
O
F H
H Me
OH
A. clobetasone,
O
Me H
H F
Me
O
Me
O
O
Cl
Pr
n
B. (17R)-spiro[9-fluoro-16-methyl-3,11-dioxoandrosta-1,4-diene-17,2-4-chloro-5-
propylfuran-3(2H)-one].
__________________________________________________________________________________________________________ Ph Eur
10-76
Clofazimine
N
N N
Cl
Cl
NPr
i
H
C
27
H
22
Cl
2
N
4
473.4 2030-63-9
Definition Clofazimine is 3-(4-chloranilino)-10-(4-chlorophenyl)-2,10-dihydrophenazin-2-
ylideneisopropylamine. It contains not less than 97.5% and not more than 102.5% of C
27
H
22
Cl
2
N
4
,
Characteristics A reddish brown, fine powder; odourless or almost odourless. It exhibits
polymorphism.
Practically insoluble in water; soluble in chloroform; slightly soluble in ethanol (96%); very slightly
soluble in ether.
Identification
clofazimine (RS 068).
methanolic hydrochloric acid exhibits two maxima, at 283 nm and 487 nm. The absorbance at 283 nm is
about 1.30 and at 487 nm is about 0.64.
C. Dissolve 2 mg in 3 ml of acetone and add 0.1 ml of hydrochloric acid; an intense violet colour is
produced. Add 0.5 ml of 5M sodium hydroxide; the colour changes to orange-red.
following solutions in the mobile phase. Solution (1) contains 0.005% w/v of the substance being
examined. Solution (2) contains 0.0000125% w/v of iminophenazine BPCRS. Solution (3) contains
0.0005% w/v of the substance being examined and 0.0005% w/v of iminophenazine BPCRS.
(25 cm 4.6 mm) packed with stationary phase B (5 m) (Nucleosil C8 is suitable), (b) as the mobile
phase with a flow rate of 1.5 ml per minute a mixture of 35 volumes of a solution prepared by
dissolving 2.25 g of sodium dodecyl sulphate, 0.85 g of tetrabutylammonium hydrogen sulphate and
0.885 g of disodium hydrogen orthophosphate in suffcient water to produce 500 ml and adjusting the pH
to 3.0 with orthophosphoric acid and 65 volumes of acetonitrile and (c) a detection wavelength of
280 nm.
principal peaks is at least 2, the relative retention time for iminophenazine is about 0.7 and the
column efficiency, determined on the peak due to clofazimine is at least 3000 theoretical plates.
the area of the principal peak in the chromatogram obtained with solution (2) (0.25%) and the sum
of the areas of all such peaks is not greater than twice the area of the peak in the chromatogram
1 g.
Assay Carry out the method for liquid chromatography, Appendix III D, using the following solutions
in the mobile phase. Solution (1) contains 0.005% w/v of the substance being examined. Solution (2)
contains 0.005% w/v of clofazimine BPCRS. Solution (3) contains 0.0005% w/v of the substance
being examined and 0.0005% w/v of iminophenazine BPCRS in the mobile phase.
principal peaks is at least 2, the relative retention time for iminophenazine is about 0.7 and the
column efficiency, determined on the peak due to clofazimine is at least 3000 theoretical plates.
27
H
22
Cl
2
N
4
27
H
22
Cl
2
N
4
in
clofazimine BPCRS.
Action and use Antileprotic.
10-77
Preparation
Clofazimine Capsules
IMPURITIES
N
N
NH
N
H
Cl
Cl
A. Iminophenazine
10-78
Clofibrate
O
Cl
COOEt
Me Me
C
12
H
15
ClO
3
242.7 637-07-0
Clofibrate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0318]. These
Action and use Antihyperlipidaemic.
Preparation
Clofibrate Capsules
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Clofibrate is ethyl 2-(4-chlorophenoxy)-2-methylpropionate.
CHARACTERS
A clear, almost colourless liquid, very slightly soluble in water, miscible with alcohol and with ether.
IDENTIFICATION
obtained with clofibrate CRS.
B. Dissolve 0.10 g in methanol R and dilute to 100.0 ml with the same solvent. Dilute 10.0 ml of the
solution to 100.0 ml with methanol R (solution a). Examined between 250 nm and 350 nm (2.2.25),
solution (a) shows two absorption maxima, at 280 nm and at 288 nm. The specific absorbances at
the maxima are about 44 and 31, respectively. Dilute 10.0 ml of solution (a) to 100.0 ml with
methanol R. Examined between 220 nm and 250 nm, the solution shows an absorption maximum at
226 nm. The specific absorbance at the maximum is about 460.
TESTS
Acidity To 1.0 g add 10 ml of ethanol R and 0.1 ml of phenol red solution R. Not more than 1.0 ml of
0.01M sodium hydroxide is required to change the colour of the indicator.
Volatile related substances Examine by gas chromatography (2.2.28).
Test solution. To 10.0 g of the substance to be examined add a mixture of 10 ml of dilute sodium
hydroxide solution R and 10 ml of water R. Shake, separate the lower (organic) layer, wash with 5 ml of
water R and add the washings to the aqueous layer. Dry the organic layer with anhydrous sodium
sulphate R and use as the test solution. Reserve the aqueous layer for the test for 4-chlorophenol.
Reference solution (a). Dissolve 0.12 g of the substance to be examined in chloroform R and dilute to
100.0 ml with the same solvent. Dilute 1.0 ml of the solution to 10.0 ml with chloroform R.
Reference solution (b). Dissolve 0.12 g of methyl 2-(4-chlorophenoxy)-2-methylpropionate CRS in the
substance to be examined and dilute to 10.0 ml with the same solvent. Dilute 1.0 ml of the solution
to 10.0 ml with the substance to be examined. Dilute 1.0 ml of this solution to 10.0 ml with the
a column 1.5 m long and 4 mm in internal diameter packed with either: silanised diatomaceous
polydimethylsiloxane R; or silanised diatomaceous earth for gas chromatography R (150 m to
180 m) impregnated with 10 per cent m/m of polydimethylsiloxane R,
nitrogen for chromatography R as the carrier gas,
Maintain the temperature of the column at 185C. Inject 2 l of each solution.
In the chromatogram obtained with the test solution, the sum of the areas of the peaks, except that
corresponding to clofibrate, is not greater than ten times the area of the peak corresponding to
clofibrate in the chromatogram obtained with reference solution (a) (0.1 per cent). In the chromato-
gram obtained with reference solution (b), measure from the baseline the height (A) of the peak
corresponding to methyl 2-(4-chlorophenoxy)-2-methylpropionate and the height (B) of the lowest
point of the curve separating this peak from the peak corresponding to clofibrate (see Figure 318-1).
10-79
The test is not valid unless A is equal to at least 30 per cent of the recorder-chart scale and A B is
greater than 75 per cent of A.
4-Chlorophenol Examine by gas chromatography (2.2.28).
Test solution. Shake the aqueous layer reserved in the test for volatile related substances with two
quantities, each of 5 ml, of chloroform R and discard the organic layers. Acidify the aqueous layer by
the dropwise addition of hydrochloric acid R. Shake with three quantities, each of 3 ml, of chloro-
form R. Combine the organic layers and dilute to 10.0 ml with chloroform R.
Reference solution. Dissolve 0.25 g of chlorophenol R in chloroform R and dilute to 100.0 ml with the
same solvent. Dilute 1.0 ml of the solution to 100.0 ml with chloroform R.
The chromatographic procedure may be carried out as described in the test for volatile related
substances. Inject 2 l of each solution.
The area of any peak corresponding to 4-chlorophenol in the chromatogram obtained with the test
solution is not greater than the area of the peak corresponding to 4-chlorophenol in the chromato-
gram obtained with the reference solution (25 ppm).
Fig. 3181 Typical chromatogram for the test for volatile related substances
__________________________________________________________________________________________________________ Ph Eur
10-80
Clomethiazole
Cl
Me
S
N
C
6
H
8
ClNS 161.6 533-45-9
Definition Clomethiazole is 5-(2-chloroethyl)-4-methylthiazole. It contains not less than 98.0%
6
H
8
ClNS.
Characteristics A colourless to slightly yellowish brown liquid; odour, characteristic.
Slightly soluble in water; miscible with chloroform, with ethanol (96%) and with ether.
Identification
B. The infrared absorption spectrum, Appendix II A, is concordant with the reference spectrum of
clomethiazole (RS 051).
C. Mix 0.1 g with 0.2 g of powdered sodium hydroxide, heat to fusion and continue heating for a
further few seconds. Cool, add 0.5 ml of water and a slight excess of 2M hydrochloric acid and warm.
Any fumes evolved do not turn moistened starch iodate paper blue (distinction from clomethiazole
edisilate).
Acidity or alkalinity pH of a 0.5% w/v solution, 5.5 to 7.0, Appendix V L.
Heavy metals Moisten the residue obtained in the test for Sulphated ash with 2 ml of hydrochloric
acid and evaporate to dryness. Dissolve the residue in water and add sufficient water to produce
20 ml. 12 ml of the resulting solution complies with limit test A for heavy metals, Appendix VII. Use
following solutions. Solution (1) contains 0.20% w/v of the substance being examined in the mobile
phase. For solution (2) dilute 1 volume of solution (1) to 1000 volumes with the mobile phase. For
solution (3) dilute 1 volume of a 0.030% w/v solution of 4-methyl-5-vinylthiazole edisilate BPCRS in
methanol (solution A) to 50 volumes with the mobile phase. For solution (4) dilute 1 volume of a
0.020% w/v solution of 5-(2-chloroethyl)-4-methyl-3-[2-(4-methylthiazol-5-yl)ethyl]thiazolium chloride
BPCRS (quaternary dimer) in methanol (solution B) to 50 volumes with the mobile phase. For
solution (5) dilute 1 volume of a 0.020% w/v solution of 4-methyl-5-(2-hydroxyethyl)thiazole BPCRS
in methanol (solution C) to 50 volumes with the mobile phase. For solution (6) add 1 ml each of
solutions A, B and C to 0.10 g of the substance being examined and dilute to 50 ml with the mobile
phase.
cm 4 mm) packed with stationary phase C (10 m) (Lichrosorb RP18 is suitable), (b) as the mobile
phase with a flow rate of 1 ml per minute, a mixture of 70 volumes of a solution containing 0.13%
w/v of sodium hexanesulphonate and 2.7% w/v of tetramethylammonium hydrogen sulphate, adjusted to
pH 2.0 with 5M sodium hydroxide, and 30 volumes of methanol and (c) a detection wavelength of
257 nm.
The test is not valid unless in the chromatogram obtained with solution (6) baseline separation is
achieved between the peaks due to the three specified impurities and also between the principal peak
and the two adjacent specified impurity peaks.
Calculate the content of each of the three specified impurities in the substance being examined
expressing the content of 4-methyl-5-vinylthiazole as the base (1 mg of 4-methyl-5-vinylthiazole
edisilate is equivalent to 0.568 mg of base). The total content of the three specified impurities is not
greater than 0.5%. In the chromatogram obtained with solution (1) the area of any other secondary
peak is not greater than the area of the peak in the chromatogram obtained with solution (2).
C
6
H
8
ClNS.
Storage Clomethiazole should be kept in a well-closed container and stored at a temperature of 2 to
8.
Action and use Hypnotic; sedative; anticonvulsant
Preparation
Clomethiazole Capsules.
When chlormethiazole is prescribed or demanded, Clomethiazole shall be dispensed or supplied.
10-81
Clomethiazole Edisilate
S
S
OH
OH
O O
O O
,
2
Cl
Me
S
N
(C
6
H
8
ClNS)
2
,C
2
H
6
O
6
S
2
513.5 1867-58-9
Definition Clomethiazole Edisilate is 5-(2-chloroethyl)-4-methylthiazole ethanedisulphonate. It
contains not less than 99.0% and not more than 101.0% of (C
6
H
8
ClNS)
2
,C
2
H
6
O
6
S
2
, calculated with
Characteristics A white, crystalline powder; odour, characteristic.
Freely soluble in water; soluble in ethanol (96%); practically insoluble in ether.
Identification
A. Melting point, about 128, Appendix V A.
C. The infrared absorption spectrum, Appendix II A, is concordant with the reference spectrum of
clomethiazole edisilate (RS 052).
D. Mix 0.1 g with 0.2 g of powdered sodium hydroxide, heat to fusion and continue heating for a
further few seconds. Cool, add 0.5 ml of water and a slight excess of 2M hydrochloric acid and warm.
Fumes are evolved which turn moistened starch iodate paper blue (distinction from clomethiazole).
Calcium 10 ml of a 10.0% w/v solution diluted to 15 ml with water complies with the limit test for
calcium, Appendix VII (100 ppm).
Heavy metals Moisten the residue obtained in the test for Sulphated ash with 2 ml of hydrochloric
acid and evaporate to dryness. Dissolve the residue in water and add sufficient water to produce
20 ml. 12 ml of the resulting solution complies with limit test A for heavy metals, Appendix VII. Use
Chloride 10 ml of a 10% w/v solution diluted to 15 ml with water complies with the limit test for
chlorides, Appendix VII (50 ppm).
Sulphate 10 ml of a 1.0% w/v solution diluted to 15 ml with distilled water complies with the limit test
for sulphates, Appendix VII (0.15%).
phase. For solution (2) dilute 1 volume of solution (1) to 1000 volumes with the mobile phase. For
solution (3) dilute 1 volume of a 0.030% w/v solution of 4-methyl-5-vinylthiazole edisilate BPCRS in
methanol (solution A) to 50 volumes with the mobile phase. For solution (4) dilute 1 volume of a
0.020% w/v solution of 5-(2-chloroethyl)-4-methyl-3-[2-(4-methylthiazol-5-yl)ethyl]thiazolium chloride
BPCRS (quaternary dimer) in methanol (solution B) to 50 volumes with the mobile phase. For
solution (5) dilute 1 volume of a 0.020% w/v solution of 4-methyl-5-(2-hydroxyethyl)thiazole BPCRS
in methanol (solution C) to 50 volumes with the mobile phase. For solution (6) add 1 ml each of
solutions A, B and C to 0.15 g of the substance being examined and dilute to 50 ml with the mobile
phase.
cm 4 mm) packed with stationary phase C (10 m) (Lichrosorb RP18 is suitable), (b) as the mobile
phase with a flow rate of 1 ml per minute, a mixture of 70 volumes of a solution containing 0.13%
w/v of sodium hexanesulphonate and 2.7% w/v of tetramethylammonium hydrogen sulphate, adjusted to
pH 2.0 with 5M sodium hydroxide, and 30 volumes of methanol and (c) a detection wavelength of 257
nm.
The test is not valid unless in the chromatogram obtained with solution (6) baseline separation is
achieved between the peaks due to the three specified impurities and also between the principal peak
and the two adjacent specified impurity peaks.
Calculate the content of each of the three specified impurities in the substance being examined
with reference to clomethiazole base (1 mg of clomethiazole edisilate is equivalent to 0.629 mg of
base) expressing the content of 4-methyl-5-vinylthiazole as the base (1 mg of 4-methyl-5-vinylthiazole
edisilate is equivalent to 0.568 mg of base). The total content of the three specified impurities is not
greater than 0.5%. In the chromatogram obtained with solution (1) the area of any other secondary
peak is not greater than the area of the peak in the chromatogram obtained with solution (2).
10-82
Assay Dissolve 0.4 g in 50 ml of water and titrate with 0.1M sodium hydroxide VS using
phenolphthalein solution R1 as indicator. Each ml of 0.1M sodium hydroxide VS is equivalent to
25.67 mg of (C
6
H
8
ClNS)
2
,C
2
H
6
O
6
S
2
.
Storage Clomethiazole Edisilate should be kept in a well-closed container.
Action and use Hypnotic; sedative; anticonvulsant.
Preparations
Clomethiazole Intravenous Infusion
Clomethiazole Oral Solution
When chlormethiazole edisylate is prescribed or demanded, Clomethiazole Edisilate shall be
10-83
Clomifene Citrate
O
Cl
NEt
2
Z-isomer
C COOH HO
CH
2
COOH
CH
2
COOH
,
C
26
H
28
ClNO,C
6
H
8
O
7
598.1 50-41-9
Clomifene Citrate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0997].
When clomiphene citrate is prescribed or demanded, Clomifene Citrate shall be dispensed or
supplied.
Action and use Induction of ovulation.
Preparation
Clomifene Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Clomifene citrate contains not less than 98.0 per cent and not more than the equivalent of 101.0 per
cent of a mixture of the E- and Z-isomers of 2-[4-(2-chloro-1,2-diphenylvinyl)-phenoxy]triethylamine
dihydrogen citrate, calculated with reference to the anhydrous substance.
CHARACTERS
A white or pale yellow, crystalline powder, slightly soluble in water, sparingly soluble in alcohol,
IDENTIFICATION
obtained with clomifene citrate CRS. Examine the substances as discs prepared using potassium
bromide R.
B. Dissolve about 5 mg in 5 ml of a mixture of 1 volume of acetic anhydride R and 5 volumes of
pyridine R and heat in a water-bath. A deep red colour is produced.
TESTS
Prepare the solutions protected from light in brown-glass vessels. Ensure minimum exposure of the solutions to
daylight until they are required for chromatography.
Reference solution (a). Dissolve 12.5 mg of clomifene citrate for performance test CRS in the mobile phase
a stainless steel column 0.25 m long and 4.6 mm internal diameter packed with butylsilyl silica gel
as mobile phase at a flow rate of 1.2 ml per minute a mixture prepared as follows: mix 400 ml of
acetonitrile R with 600 ml of water R and add 8.0 ml of diethylamine R; adjust the the mixture to
pH 6.2 by the addition of about 1 ml to 2 ml of phosphoric acid R taking care to reduce
progressively the volume of each addition as the required pH is approached,
Equilibrate the column with the mobile phase at a flow rate of 1.2 ml per minute for about 1 h.
recorder.
10-84
Inject 10 l of reference solution (a). Continue the chromatography for twice the retention time of
the principal peak. Measure the height (A) above the base-line of the peak due to clomifene impurity
A and the height (B) above the base-line of the lowest point of the curve separating this peak from
the peak due to clomifene. The test is not valid unless A is greater than fifteen times B and the
chromatogram obtained resembles the reference chromatogram. If necessary, adjust the
chromatography for four times the retention time of the principal peak. In the chromatogram
obtained with the test solution: the area of any peak due to 2-[4-(1,2-diphenylvinyl)phenoxy]triethyl-
amine is not greater than the area of the principal peak in the chromatogram obtained with reference
solution (b) (2.0 per cent); the area of any other peak, apart from the principal peak, is not greater
than half the area of the principal peak in the chromatogram obtained with reference solution (b)
(1.0 per cent); the sum of the areas of all the peaks, apart from the principal peak, is not greater than
1.25 times the area of the principal peak in the chromatogram obtained with reference solution (b)
(2.5 per cent). Disregard any peak with a retention time relative to the clomifene peak of 0.2 or less
and any peak with an area less than 0.025 times the area of the principal peak in the chromatogram
Z-isomer 30.0 per cent to 50.0 per cent, determined by liquid chromatography (2.2.29).
Test solution. Dissolve 25 mg of the substance to be examined in 25 ml of 0.1M hydrochloric acid, add
5 ml of 1M sodium hydroxide and shake with three quantities, each of 25 ml, of ethanol-free chloro-
form R. Wash the combined extracts with 10 ml of water R, dry over anhydrous sodium sulphate R and
dilute to 100 ml with ethanol-free chloroform R. To 20 ml of the solution add 0.1 ml of triethylamine R
and dilute to 100 ml with hexane R.
Reference solution. Dissolve 25 mg of clomifene citrate CRS in 25 ml of 0.1M hydrochloric acid, add 5 ml
of 1M sodium hydroxide and shake with three quantities, each of 25 ml, of ethanol-free chloroform R.
Wash the combined extracts with 10 ml of water R, dry over anhydrous sodium sulphate R and dilute to
100 ml with ethanol-free chloroform R. To 20 ml of the solution add 0.1 ml of triethylamine R and
dilute to 100 ml with hexane R.
a stainless steel column 0.3 m long and 4 mm in internal diameter packed with silica gel for
as mobile phase at a flow rate of 2 ml per minute a mixture of 1 volume of triethylamine R, 200
volumes of ethanol-free chloroform R and 800 volumes of hexane R,
Equilibrate the column with the mobile phase for about 2 h. Inject 50 l of the reference solution.
The chromatogram obtained shows a peak due to the E-isomer just before a peak due to the
Z-isomer. The test is not valid unless the resolution between the peaks corresponding to the E- and
Z-isomers is at least 1.0. If necessary, adjust the relative proportions of ethanol-free chloroform and
hexane in the mobile phase. Measure the area of the peak due to the Z-isomer in the chromatograms
obtained with the test solution and the reference solution. Calculate the content of the Z-isomer as
a percentage of the total clomifene citrate present using the declared content of clomifene citrate CRS.
ASSAY
32
H
36
C1NO
8
.
STORAGE
IMPURITIES
O
NEt
2
H
A. 2-[4-(1,2-diphenylvinyl)phenoxy]triethylamine,
10-85
O
NEt
2
O
B. 4-(2-diethylaminoethoxy)benzophenone,
O
NEt
2
O
C. 2-[4-[2-diethylaminoethoxy]phenyl]-2-phenylacetophenone,
O
NEt
2
O
O
Et
2
N
D. 2,2-bis[4-[2-diethylaminoethoxy]phenyl]-2-phenylacetophenone,
O
Cl
Cl
H
NEt
2
E. 2-[4-[1,2-bis(4-chlorophenyl)vinyl]phenoxy]triethylamine,
O
NEt
2
Cl
Cl
F. 2-[4-[2-chloro-2-(4-chlorophenyl)-1-phenylvinyl]phenoxy]triethylamine,
O
NEt
2
Cl
Cl
G. 2-[2-chloro-4-(2-chloro-1,2-diphenylvinyl)phenoxy]triethylamine (higher-melting point
isomer),
H. 2-[2-chloro-4-(2-chloro-1,2-diphenylvinyl)phenoxy]triethylamine (lower-melting point isomer).
__________________________________________________________________________________________________________ Ph Eur
10-86
Clomipramine Hydrochloride
N
Cl
NMe
2
,HCl
C
19
H
23
ClN
2
,HCl 351.3 17321-77-6
Clomipramine hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Clomipramine Capsules
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Clomipramine hydrochloride contains not less than 99.0 per cent and not more than the equivalent
of 101.0 per cent of 3-chloro-5-[3-(dimethylamino)propyl]-10,11-dihydro-5H-dibenz[b,f]azepine
CHARACTERS
A white or slightly yellow, crystalline powder, slightly hygroscopic, freely soluble in water and in
methylene chloride, soluble in alcohol, practically insoluble in ether.
IDENTIFICATION
obtained with clomipramine hydrochloride CRS. Examine the substances as discs prepared using
D. Dissolve about 5 mg in 2 ml of nitric acid R. An intense blue colour develops.
E. Dissolve about 50 mg in 5 ml of water R and add 1 ml of dilute ammonia R1. Mix, allow to stand
for 5 min and filter. Acidify the filtrate with dilute nitric acid R. The solution gives reaction (a) of
chlorides (2.3.1).
TESTS
solution Y
5
(Method I, 2.2.2).
Related substances Prepare the solutions immediately before use and protected from light.
Reference solution (a). Dissolve 20 mg of clomipramine hydrochloride CRS in methanol R and dilute to
Reference solution (b). Dissolve 20 mg of imipramine hydrochloride CRS in methanol R and dilute to
Reference solution (d). Dilute 1 ml of reference solution (b) to 5 ml with methanol R. To 1 ml add 1 ml
of reference solution (c).
10-87
volumes of concentrated ammonia R, 25 volumes of acetone R and 75 volumes of ethyl acetate R. Allow
the plate to dry in air. Spray with a 5 g/l solution of potassium dichromate R in a 20 per cent V/V
solution of sulphuric acid R. Examine the plate immediately. In the chromatogram obtained with test
solution (a): any spot corresponding to imipramine is not more intense than the spot in the chroma-
togram obtained with reference solution (b) (1.0 per cent); any spot, apart from the principal spot
and any spot corresponding to imipramine, is not more intense than the spot in the chromatogram
obtained with reference solution (c) (0.2 per cent). The test is not valid unless the chromatogram
obtained with reference solution (d) shows two clearly separated principal spots.
100C to 105C.
ASSAY
19
H
24
Cl
2
N
2
.
STORAGE
IMPURITIES
,HCl N
NMe
2
A. imipramine hydrochloride,
H
N
Cl
B. 3-chloro-10,11-dihydro-5H-dibenz[b,f]azepine.
__________________________________________________________________________________________________________ Ph Eur
10-88
Clonazepam
N
N
O
Cl
O
2
N
H
C
15
H
10
ClN
3
O
3
315.7 1622-61-3
Clonazepam complies with the requirements of the 3rd edition of the European Pharmacopoeia [0890]. These
Preparation
Clonazepam Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Clonazepam contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of 5-(2-chlorophenyl)-7-nitro-2,3-dihydro-1H-1,4-benzodiazepin-2-one, calculated with reference to
CHARACTERS
A slightly yellowish, crystalline powder, practically insoluble in water, slightly soluble in alcohol and
in methanol, very slightly soluble in ether.
IDENTIFICATION
A. Protect the solutions from light and carry out the measurement of the absorbance immediately. Dissolve
20.0 mg in methanol R and dilute to 100.0 ml with the same solvent. Dilute 5.0 ml of this solution to
100.0 ml with methanol R. Examined between 230 nm and 340 nm (2.2.25), the solution shows two
absorption maxima, at 248 nm and 310 nm. The specific absorbances at the maxima are 450 to 470
and 350 to 370, respectively.
obtained with clonazepam CRS. Examine the substances prepared as discs.
C. Carry out the test protected from light and prepare the solutions immediately before use. Examine by thin-
layer chromatography (2.2.27), using silica gel GF
254
same solvent.
Reference solution (a). Dissolve 8 mg of clonazepam CRS in acetone R and dilute to 10 ml with the
same solvent.
Reference solution (b). Dissolve 8 mg of clonazepam CRS and 8 mg of flunitrazepam CRS in acetone R
15 volumes of ethyl acetate R and 85 volumes of nitromethane R. Allow the plate to dry in air and
solution is similar in position and size to the principal spot in the chromatogram obtained with refer-
ence solution (a). The test is not valid unless the chromatogram obtained with reference solution (b)
D. Dissolve about 20 mg in a mixture of 5 ml of hydrochloric acid R and 10 ml of water R. Heat to
boiling for 5 min and cool. Add 2 ml of a 1 g/l solution of sodium nitrite R. Allow to stand for 1 min.
Add 1 ml of a 5 g/l solution of sulphamic acid R and mix. Allow to stand for 1 min. Add 1 ml of a 1 g/l
solution of naphthylethylene-diamine dihydrochloride R. A red colour develops.
10-89
TESTS
Related substances Carry out the test protected from light and prepare the solutions immediately before use.
Examine by thin-layer chromatography (2.2.27), using silica gel HF
254
the same solvent.
Reference solution (a). Dilute 1 ml of the test solution to 20 ml with acetone R. Dilute 1 ml of the
solution to 50 ml with acetone R.
Reference solution (b). Dissolve 10 mg of clonazepam impurity A CRS in acetone R and dilute to 100 ml
with the same solvent. Dilute 2 ml of the solution to 10 ml with acetone R.
Reference solution (c). Dissolve 10 mg of clonazepam impurity B CRS in acetone R and dilute to 100 ml
with the same solvent. Dilute 2 ml of the solution to 10 ml with acetone R.
Apply separately to the plate 10 l of the test solution and 10 l of each reference solution. Develop
over a path of 15 cm using a mixture of equal volumes of ethyl acetate R and 1,1,1-trichloroethane R.
Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any spot corresponding to
clonazepam impurity A in the chromatogram obtained with the test solution is not more intense than
the spot in the chromatogram obtained with reference solution (b) (0.1 per cent). Any spot corres-
ponding to clonazepam impurity B in the chromatogram obtained with the test solution is not more
intense than the spot in the chromatogram obtained with reference solution (c) (0.1 per cent). Any
spot in the chromatogram obtained with the test solution, apart from the principal spot and any spots
corresponding to clonazepam impurity A and clonazepam impurity B, is not more intense than the
spot in the chromatogram obtained with reference solution (a) (0.1 per cent).
100C to 105C for 4 h.
ASSAY
Dissolve 0.275 g in 50 ml of acetic anhydride R. Titrate with 0.1M perchloric acid, determining the end-
15
H
10
ClN
3
O
3
.
STORAGE
IMPURITIES
NH
2
O
O
2
N
Cl
A. 2-amino-2-chloro-5-nitrobenzophenone,
N O
O
2
N NH
2
Cl
H
B. 3-amino-4-(2-chlorophenyl)-6-nitro-1H-quinolin-2-one.
__________________________________________________________________________________________________________ Ph Eur
10-90
Clonidine Hydrochloride
NH
N
N
Cl
Cl
,HCl
H
C
9
H
9
Cl
2
N
3
,HCl 266.6 4205-91-8
Clonidine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Antihypertensive.
Preparations
Clonidine Injection
Clonidine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Clonidine hydrochloride contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of 2-[(2,6-dichlorophenyl)amino]-2-imidazoline hydrochloride, calculated with refer-
ence to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, soluble in water and in ethanol.
IDENTIFICATION
A. Dissolve 30.0 mg in 0.01M hydrochloric acid and dilute to 100.0 ml with the same acid. Examined
between 245 nm and 350 nm (2.2.25), the solution shows two absorption maxima, at 272 nm and at
279 nm, and a point of inflexion at 265 nm. The specific absorbances at the maxima are about 18
and about 16, respectively.
obtained with clonidine hydrochloride CRS.
TESTS
solution Y
7
(Method II, 2.2.2).
coating substance.
Reference solution (a). Dissolve 10 mg of clonidine hydrochloride CRS in methanol R and dilute to 10 ml
Reference solution (b). Dilute 1 ml of test solution (a) to 10 ml with methanol R. Dilute 5 ml of this
Apply separately to the plate 10 l of each solution. Shake a mixture of 10 volumes of glacial acetic
acid R, 40 volumes of butanol R and 50 volumes of water R. Allow to separate. Filter the upper layer
and use the filtrate as the mobile phase. Develop over a path of 15 cm. Allow the plate to dry in air
and spray with potassium iodobismuthate solution R2. Allow the plate to dry in air for 1 h, spray again
with potassium iodobismuthate solution R2 and then immediately spray with a 50 g/l solution of sodium
nitrite R. Any spot in the chromatogram obtained with test solution(a), apart from the principal spot,
10-91
is not more intense than the spot in the chromatogram obtained with reference solution (b)
(0.5 per cent).
at 100C to 105C.
ASSAY
Dissolve 0.200 g in 70 ml of alcohol R. Titrate with 0.1M ethanolic sodium hydroxide determining the
9
H
10
Cl
3
N
3
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
11-1
Clotrimazole
N
N
Cl
C
22
H
17
ClN
2
344.8 23593-75-1
Clotrimazole complies with the requirements of the 3rd edition of the European Pharmacopoeia [0757]. These
Preparations
Clotrimazole Cream
Clotrimazole Pessaries
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Clotrimazole contains not less than 98.5 per cent and not more than the equivalent of 100.5 per cent
of 1-[(2-chlorophenyl)diphenylmethyl]-1H-imidazole, calculated with reference to the dried
substance.
CHARACTERS
A white or pale yellow, crystalline powder, practically insoluble in water, soluble in alcohol and in
methylene chloride, slightly soluble in ether.
IDENTIFICATION
obtained with clotrimazole CRS.
C. Examine before spraying in ultraviolet light at 254 nm, the chromatograms obtained in the test for
(2-chlorophenyl)diphenylmethanol. The principal spot in the chromatogram obtained with test
solution (b) is similar in position and size to the principal spot in the chromatogram obtained with
D. Dissolve about 10 mg in 3 ml of sulphuric acid R. The solution is pale yellow. Add 10 mg of
mercuric oxide R and 20 mg of sodium nitrite R. Allow to stand with occasional shaking. An orange
colour develops, becoming orange-brown.
TESTS
solution is clear (2.2.1) and not more intensely coloured than reference solution BY
6
(Method II,
2.2.2).
(2-Chlorophenyl)diphenylmethanol Examine by thin-layer chromatography (2.2.27), using silica
gel GF
254
Test solution (a). Dissolve 0.50 g of the substance to be examined in alcohol R and dilute to 5 ml with
the same solvent.
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with alcohol R.
Reference solution (a). Dissolve 50 mg of clotrimazole CRS in alcohol R and dilute to 5 ml with the
same solvent.
Reference solution (b). Dissolve 10 mg of (2-chlorophenyl)diphenylmethanol CRS in alcohol R and dilute
to 5 ml with the same solvent. Dilute 1 ml of the solution to 10 ml with alcohol R.
0.5 volumes of concentrated ammonia R1, 10 volumes of propanol R and 90 volumes of toluene R.
11-2
Allow the plate to dry in air. Spray with a 10 per cent V/V solution of sulphuric acid R in alcohol R and
heat at 100C to 105C for 30 min. Any spot corresponding to (2-chlorophenyl)diphenylmethanol in
the chromatogram obtained with test solution (a) is not more intense than the spot in the chromato-
Imidazole Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating
substance.
the same solvent.
Reference solution. Dissolve 10 mg of imidazole R in alcohol R and dilute to 10 ml with the same
solvent. Dilute 1 ml of the solution to 10 ml with alcohol R.
0.5 volumes of concentrated ammonia R1, 10 volumes of propanol R and 90 volumes of toluene R.
Allow the plate to dry in air. At the bottom of a chromatography tank, place an evaporating dish
containing a mixture of 1 volume of hydrochloric acid R1, 1 volume of water R and 2 volumes of a
15 g/l solution of potassium permanganate R, close the tank and allow to stand for 15 min. Place the
dried plate in the tank and close the tank. Leave the plate in contact with the chlorine vapour for
5 min. Withdraw the plate and place it in a current of cold air until the excess of chlorine is removed
and an area of coating below the points of application does not give a blue colour with a drop of
potassium iodide and starch solution R. Spray with potassium iodide and starch solution R. Any spot
corresponding to imidazole in the chromatogram obtained with the test solution is not more intense
100C to 105C.
ASSAY
Dissolve 0.300 g in 80 ml of glacial acetic acid R. Titrate with 0.1M perchloric acid using 0.3 ml of
naphtholbenzein solution R as indicator until the colour changes from brownish-yellow to green.
22
H
17
ClN
2
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
11-3
Clove
1/01
Clove complies with the requirements of the 3rd edition of the European Pharmacopoeia [0376]. These require-
Action and use Carminative; flavour.
When Powdered Clove is prescribed or demanded, material complying with the requirements
below with the exception of Identification test A and the test for Foreign matter and containing not
less than 12.0% v/w (120 ml/kg) of essential oil shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Clove consists of the whole flower buds of Syzygium aromaticum (L.) Merill et L. M. Perry (Eugenia
caryophyllus (C. Spreng.) Bull. et Harr.) dried until they become reddish-brown. It contains not less
than 150 ml/kg of essential oil.
CHARACTERS
Clove has a characteristic, aromatic odour.
IDENTIFICATION
A. The flower bud is reddish-brown and consists of a quadrangular stalked portion, the hypanthium,
10 mm to 12 mm long and 2 mm to 3 mm in diameter, surmounted by four divergent lobes of sepals
which surround a globular head 4 mm to 6 mm in diameter. A bilocular ovary containing numerous
ovules is situated in the upper part of the hypanthium. The head is globular and dome-shaped,
composed of four imbricated petals that enclose numerous incurved stamens and a short, erect style
with a nectary disc at the base. The hypanthium exudes essential oil when indented with the finger-
nail.
B. Reduce to a powder (355). The powder is dark brown and has the odour and taste of the
unground drug. Examine under a microscope using chloral hydrate solution R. The powder shows the
following diagnostic characters: fragments of the hypanthium showing the epidermis and underlying
parenchyma containing large oil glands; short fibres occurring singly or in small groups, with
thickened, lignified walls and few pits; abundant fragments of parenchyma containing cluster crystals
of calcium oxalate; numerous triangular pollen grains about 15 m in diameter with three pores in
the angles. Starch granules are absent.
254
Test solution. Shake 0.1 g of the powdered drug (500) with 2 ml of methylene chloride R for 15 min.
Filter and carefully evaporate the filtrate to dryness on a water-bath. Dissolve the residue in 2 ml of
toluene R.
Reference solution. Dissolve 20 l of eugenol R in 2 ml of toluene R.
Apply to the plate as bands 20 mm by 3 mm, 10 l of the reference solution and 20 l of the test
solution. Develop in an unsaturated tank over a path of 10 cm using toluene R. Allow the plate to
stand for 5 min and develop again in the same manner. Allow the plate to dry in air, examine in
ultraviolet light at 254 nm and mark the quenching zones. In the chromatogram obtained with the
test solution there is in the median part a quenching zone (eugenol) similar in position to the
quenching zone in the chromatogram obtained with the reference solution and there may be a weak
quenching zone (acetyleugenol) just below the zone corresponding to eugenol. Spray the plate with
anisaldehyde solution R using 10 ml for a plate 200 mm square and examine in daylight while heating
at 100C to 105C for 5 min to 10 min. The zones corresponding to eugenol in the chromatograms
obtained with the test and reference solutions have a strong brownish-violet colour and the zone
corresponding to acetyleugenol in the chromatogram obtained with the test solution is faint violet-
blue. In the chromatogram obtained with the test solution there are other coloured zones, particularly
a faint red zone in the lower part and a reddish-violet zone (caryophyllene) in the upper part.
TESTS
Foreign matter (2.8.2). Not more than 6 per cent of peduncles, petioles and fruits, not more than
2 per cent of deteriorated cloves and not more than 0.5 per cent of other foreign matter.
ASSAY
Carry out the determination of essential oils in vegetable drugs (2.8.12). Use a 250 ml flask, 100 ml
11-4
of water R as the distillation liquid and 0.50 ml of xylene R in the graduated tube. Grind 5.0 g of the
drug with 5.0 g of diatomaceous earth R to form a fine, homogeneous powder and proceed
immediately with the determination using 4.0 g of the mixture. Distil at a rate of 2.5 ml/min to
3.5 ml/min for 2 h.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
11-5
Clove Oil
Clove Oil complies with the requirements of the 3rd edition of the European Pharmacopoeia [1091]. These
Action and use Local analgesic used in dentistry; flavour.
Ph Eur _______________________________________________________________________________________________________________
DEFINITION
Clove oil is obtained by steam distillation from the dried flower buds of Syzygium aromaticum (L.)
Merill and L. M. Perry (Eugenia caryophyllus C. Spring. Bull. et Harr.).
CHARACTERS
A clear, yellow liquid which becomes brown when exposed to air, miscible with methylene chloride,
ether, toluene and fatty oils.
IDENTIFICATION
A. Examine by thin-layer chromatography (2.2.27) using a suitable silica gel with a fluorescent
indicator having an optimal intensity at 254 nm as the coating substance.
Test solution. Dissolve 20 l of the substance to be examined in 2.0 ml of toluene R.
Reference solution. Dissolve 15 l of eugenol R and 15 l of acetyleugenol R in 2.0 ml of toluene R.
Develop in an unsaturated tank over a path of 10 cm using toluene R. Allow the plate to stand for
5 min and develop again in the same manner. Allow the plate to dry in air, examine in ultraviolet
light at 254 nm and mark the quenching zones. The chromatogram obtained with the test solution
shows in the medium part a quenching zone (eugenol) which corresponds in position to the
quenching zone in the chromatogram obtained with the reference solution; there is a weak quenching
zone (acetyleugenol) just below the quenching zone of eugenol which corresponds in position to the
zone of acetyleugenol in the chromatogram obtained with the reference solution. Spray the plate with
anisaldehyde solution R and examine in daylight while heating at 100C to 105C for 5 to 10 min. The
zones corresponding to eugenol in the chromatogram obtained with the test and reference solutions
have a strong brownish-violet colour and the zone corresponding to acetyleugenol in the chromato-
gram obtained with the test solution is faint violet-blue. In the chromatogram obtained with the test
solution there are other coloured zones particularly a faint red zone in the lower part and a reddish-
violet zone (-caryophyllene) in the upper part.
obtained with the test solution shows three main peaks similar in retention time to the three peaks
TESTS
Angle of optical rotation (2.2.7). 0 to 2.
oils.
Solubility in alcohol (2.8.10). 1.0 ml is soluble in 2.0 ml or more of alcohol (70 per cent V/V) R.
Test solution. Dissolve 0.2 g of the substance to be examined in 10 g of hexane R.
Reference solution. Dissolve 7 mg of -caryophyllene R, 80 mg of eugenol R and 4 mg of acetyleugenol R
in 10 g of hexane R.
a fused silica capillary column 60 m long and about 0.25 mm in internal diameter, coated with
macrogol 20,000 R,
helium for chromatography R as the carrier gas at a flow rate of 1.5 ml per min,
a split ratio of 1/100,
of 3C per min to 180C and maintaining at 180C for 5 min and maintaining the temperature of the
injection port and that of the detector at 270C.
11-6
Inject about 1.0 l of the reference solution. Identify the eluted components following the order
indicated in the composition of the reference solution. Record the retention times of these
substances.
The test is not valid unless: the number of theoretical plates is at least 30,000, calculated from
the -caryophyllene peak at 110C and the resolution between the peak corresponding to eugenol
and that corresponding to acetyleugenol is at least 1.5.
Inject about 1.0 l of the substance to be examined. Using the retention times determined from the
chromatogram obtained with the reference solution, locate the components of the reference solution
on the chromatogram obtained with the test solution. Disregard the peak due to the solvent.
Determine the percentage content of each of the three components by the normalisation
procedure. The contents are within the following ranges:
-caryophyllene 5.0 to 14.0 per cent,
eugenol 75.0 to 88.0 per cent,
acetyleugenol 4.0 to 15.0 per cent.
STORAGE
The type chromatogram is given for information and guidance in application of the analytical method. It is
not part of the requirements of the monograph.
__________________________________________________________________________________________________________ Ph Eur
11-7
Cloxacillin Sodium
H
N
S
O
H
Me
Me
N
H H
O
COONa
O
N
Me
Cl
C
19
H
17
ClN
3
NaO
5
S,H
2
O 475.9 7081-44-9
Cloxacillin Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia [0661].
Preparations
Cloxacillin Capsules
Cloxacillin Injection
Cloxacillin Oral Solution
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cloxacillin sodium contains not less than 95.0 per cent and not more than the equivalent of 101.0
per cent of sodium (2S,5R,6R)-6-[[[3-(2-chlorophenyl)-5-methylisoxazol-4-yl]carbonyl]amino]-3,3-
dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate, calculated with reference to the
PRODUCTION
2-Ethylhexanoic acid. Not more than 0.8 per cent m/m, determined by gas chromatography
(2.2.28), using a suitable validated method.
CHARACTERS
A white or almost white, crystalline powder, hygroscopic, freely soluble in water and in methanol,
soluble in alcohol.
IDENTIFICATION
obtained with cloxacillin sodium CRS. Examine the substances prepared as discs.
substance.
Reference solution (a). Dissolve 25 mg of cloxacillin sodium CRS in 5 ml of water R.
Reference solution (b). Dissolve 25 mg of each of cloxacillin sodium CRS, dicloxacillin sodium CRS and
flucloxacillin sodium CRS in 5 ml of water R.
reference solution (b) shows three clearly separated spots.
by swirling; the colour of the solution is slightly greenish-yellow. Place the test-tube in a water-bath
for 1 min; the solution becomes yellow.
11-8
TESTS
substance.
Related substances Examine by liquid chromatography (2.2.2.9) as prescribed under Assay. Inject
test solution (a) and continue the chromatography for five times the retention time of the principal
peak. Inject reference solution (b). In the chromatogram obtained with test solution (a): the area of
chromatogram obtained with reference solution (b) (1 per cent); the sum of the areas of all peaks,
apart from the principal peak, is not greater than five times the area of the principal peak in the
chromatogram obtained with reference solution (b) (5 per cent). Disregard any peak with an area less
(b).
1M sodium hydroxide and 1.0 ml of the internal standard solution. Close the tube and shake vigorously
water R. Shake to dissolve and dilute to 50.0 ml with water R. Dilute 5.0 ml of this solution to
detector at 150C. Inject 1 l of the test solution and 1 l of the reference solution.
mination of water.
ASSAY
Reference solution (a). Dissolve 50.0 mg of cloxacillin sodium CRS in the mobile phase and dilute to
Reference solution (b). Dilute 5.0 ml of test solution (b) to 50.0 ml with the mobile phase.
Reference solution (c). Dissolve 5 mg of flucloxacillin sodium CRS and 5 mg of cloxacillin sodium CRS in
the mobile phase and dilute to 50.0 ml with the mobile phase.
75 volumes of a 2.7 g/l solution of potassium dihydrogen phosphate R adjusted to pH 5.0 with
dilute sodium hydroxide solution R,
11-9
peaks in the chromatogram obtained are at least 50 per cent of the full scale of the recorder. The test
is not valid unless the resolution between the first peak (cloxacillin) and the second peak
(flucloxacillin) is at least 2.5. Inject reference solution (a) six times. The test is not valid unless the
relative standard deviation of the peak area for cloxacillin is at most 1.0 per cent. Inject alternately
test solution (b) and reference solution (a).
STORAGE
LABELLING
The label states:
IMPURITIES
HN
S
N
N
O
Cl
H COOH
Me
Me
COOH O
Me
H
*
*
A. (4S)-2-[carboxy[[[3-(2-chlorophenyl)-5-methylisoxazol-4-yl]carbonyl]amino]methyl]-5,5-
dimethylthiazolidine-4-carboxylic acid (penicilloic acids of cloxacillin),
HN
S
N
N
O
Cl
H COOH
Me
Me
O
Me
H
H
*
and epimer at C*
B. (2RS,4S)-2-[[[[3-(2-chlorophenyl)-5-methylisoxazol-4-yl]carbonyl]amino]methyl]-5,5-
dimethylthiazolidine-4-carboxylic acid (penilloic acids of cloxacillin),
N
S
H COOH
Me
Me
O
H H
H
2
N
C. (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia-1-aza-bicyclo[3.2.0]heptane-2-caboxylic
N
O
Cl
Me
COOH
D. 3-(2-chlorophenyl)-5-methylisoxazole-4-carboxylic acid.
__________________________________________________________________________________________________________ Ph Eur
11-10
Clozapine
H
N
N
Cl
NMe
N
C
18
H
19
ClN
4
326.8 5786-21-0
Clozapine complies with the requirements of the 3rd edition of the European Pharmacopoeia [1191]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Clozapine contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of
8-chloro-11-(4-methylpiperazin-1-yl)-5H-dibenzo[b,e][1,4]diazepine, calculated with reference to the
dried substance.
CHARACTERS
A yellow, crystalline powder, practically insoluble in water, freely soluble in methylene chloride,
soluble in alcohol. It dissolves in dilute acetic acid.
IDENTIFICATION
obtained with clozapine CRS. Examine the substances prepared as discs.
TESTS
a suitable silica gel with a fluorescent indicator having an optimal intensity at 254 nm. Before use,
develop the plate with a mixture of 25 volumes of methanol R and 75 volumes of methylene chloride R.
Allow the plate to dry in air for 15 min.
Reference solution (a). Dissolve 20 mg of clozapine CRS in methylene chloride R and dilute to 5 ml with
the same solvent.
Reference solution (b). Dilute 1.5 ml of test solution (b) to 50 ml with methylene chloride R.
Reference solution (c). Dilute 1 ml of test solution (b) to 50 ml with methylene chloride R.
Reference solution (d). Dilute 5 ml of reference solution (c) to 10 ml with methylene chloride R.
Reference solution (e). Dissolve 10 mg of clozapine CRS and 10 mg of oxazepam CRS in methylene
25 volumes of methanol R and 75 volumes of methylene chloride R. Allow the plate to dry in air and
reference solution (b) (0.3 per cent), at most one such spot is more intense than the spot in the
chromatogram obtained with reference solution (c) (0.2 per cent) and at most two such spots are
more intense than the spot in the chromatogram obtained with reference solution (d) (0.1 per cent).
The test is not valid unless the chromatogram obtained with reference solution (e) shows two clearly
separated spots and the chromatogram obtained with reference solution (d) shows a clearly visible
spot.
Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying at 100C to
105C.
11-11
ASSAY
18
H
19
ClN
4
.
IMPURITIES
H
N
N
Cl
R =
H
N
N
Cl
O
H
A. 8-chloro-5,10-dihydro-11H-dibenzo[b,e][1,4]diazepin-11-one,
N
N
R
R
B. 11,11-(piperazin-1,4-diyl)bis(8-chloro-5H-dibenzo[b,e][1,4]diazepine),
N
NH
R
C. 8-chloro-11-(piperazin-l-yl)-5H-dibenzo[b,e][1,4]diazepine)
__________________________________________________________________________________________________________ Ph Eur
11-12
Cocaine
N
Me
H
O
O
COOMe
H
C
17
H
21
NO
4
303.4 50-36-2
Definition Cocaine is (1R,2R,3s,5S)-2-methoxycarbonyltropan-3-yl benzoate and may be
obtained from the leaves of Erythroxylum coca Lam. and other species of Erythroxylum or by
synthesis. It contains not less than 98.0% and not more than 101.0% of C
17
H
21
NO
4
, calculated
Characteristics Colourless crystals or a white, crystalline powder. Slightly volatile.
Practically insoluble in water; very soluble in chloroform; freely soluble in ethanol (96%) and in
ether; soluble in arachis oil; slightly soluble in liquid paraffin.
Identification
cocaine (RS 071).
B. Complies with the test for Melting point.
C. Yields the reaction characteristic of alkaloids, Appendix VI.
Specific optical rotation In a solution of 0.6 g in 2.5 ml of 1M hydrochloric acid and sufficient water
to produce 25 ml, 79 to 81, calculated with reference to the dried substance, Appendix V F.
Cinnamylcocaine and other reducing substances Dissolve 0.30 g in 1 ml of 1M hydrochloric acid,
warming if necessary, and dilute to 15 ml with water. To 5 ml add 0.3 ml of 0.5M sulphuric acid and
0.5 ml of 0.004M potassium permanganate and reserve the remainder for the test for Truxillines. After
30 minutes the pink colour is not completely discharged.
Truxillines Dilute 5 ml of the solution reserved in the test for Cinnamylcocaine and other reducing
substances to 100 ml with water, add while stirring 0.2 ml of 6M ammonia and allow to stand for 15
minutes, scratching the side of the container occasionally with a glass rod. A crystalline precipitate is
produced which on settling leaves a clear supernatant liquid.
1 g.
Assay Carry out Method I for non-aqueous titration, Appendix VIII A, using 0.7 g dissolved in 50 ml
of 1,4-dioxan and crystal violet solution as indicator. Each ml of 0.1M perchloric acid VS is equivalent to
30.34 mg of C
17
H
21
NO
4
.
11-13
Cocaine Hydrochloride
N
Me
H
O
O
COOMe
H
,HCl
C
17
H
21
NO
4
,HCl 339.8 53-21-4
Cocaine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cocaine hydrochloride contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of (1R,2R,3S,5S)-3-benzoyloxy-2-methoxycarbonyl-8-methyl-8-
azabicyclo[3.2.1]octane hydrochloride, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder or colourless crystals, hygroscopic, with a bitter and sharp taste followed
by anaesthesia of the tongue, very soluble in water, freely soluble in alcohol, practically insoluble in
ether.
IDENTIFICATION
A. Dissolve 20 mg in 0.01M hydrochloric acid and dilute to 10.0 ml with the same acid. Dilute 1.0 ml
absorbances at the maxima are about 390 and 31, respectively.
B. Dissolve 0.1 g in 5 ml of water R and add 1 ml of dilute ammonia R2. A white precipitate is formed.
Initiate crystallisation by scratching the wall of the tube with a glass rod. The crystals, washed with
water R and dried in vacuo, melt (2.2.14) at 96C to 99C.
D. It gives the reaction of alkaloids (2.3.1).
TESTS
Acidity To 10 ml of solution S add 0.05 ml of methyl red solution R. Not more than 0.2 ml of 0.02M
sodium hydroxide is required to change the colour of the indicator.
solvent. The specific optical rotation is 70 to 73.
Readily carbonisable substances To 0.2 g add 2 ml of sulphuric acid R. After 15 min, the solution
is not more intensely coloured than reference solution BY
5
(Method I, 2.2.2).
Cinnamoylcocaine and reducing substances To 5 ml of solution S add 0.3 ml of 0.5M sulphuric
acid and 0.5 ml of 0.004M potassium permanganate. Allow to stand protected from light for 30 min.
The pink colour is not completely discharged.
Truxillines To 7.5 ml of solution S add 72.5 ml of water R and 0.2 ml of dilute ammonia R1. Allow
to stand for 15 min. Induce crystallisation by scratching the wall of the container with a glass rod. A
crystalline precipitate is formed which, on settling, leaves a clear supernatant liquid.
100C to 105C.
Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on the residue from the test for
loss on drying.
11-14
ASSAY
Dissolve 0.300 g in 10 ml of anhydrous acetic acid R. Add 20 ml of dioxan R and 7 ml of mercuric
acetate solution R. Titrate with 0.1M perchloric acid until a pure blue colour is obtained using 0.05 ml of
crystal violet solution R as indicator.
17
H
22
ClNO
4
.
STORAGE
Store in a well-closed container, protected from light and moisture.
__________________________________________________________________________________________________________ Ph Eur
11-15
Cochineal
Definition Cochineal is the dried female insect, Dactylopius coccus Costa, containing eggs and
larvae.
Characteristics Odour, characteristic.
Macroscopical Purplish black or purplish grey; about 3.5 to 5.5 mm long and 3 to 4.5 mm wide,
plano-convex and somewhat oval in outline; the convex dorsal surface is transversely wrinkled and
shows about 11 segments; the flat or slightly concave ventral surface carries upon the anterior part
two seven-jointed straight antennae, three pairs of short legs, each terminating in a single claw, and a
mouth from which projects the remains of a long filiform proboscis; these appendages are frequently
more or less broken. Easily reduced to powder, which is dark red or puce.
Microscopical Scattered irregularly over the whole dermis are numerous solitary and grouped, short,
tubular wax glands; within each insect are found numerous larvae, which are characterised by their
proboscides appearing as two circular coils.
Colour value To 0.5 g in moderately fine powder add 60 ml of phosphate buffer pH 8.0 and heat on a
water bath for 30 minutes. Cool, add sufficient phosphate buffer pH 8.0 to produce 100 ml and filter.
Dilute 5 ml of the filtrate to 100 ml with phosphate buffer pH 8.0. The absorbance of the resulting
solution at the maximum at 530 nm is not less than 0.25, Appendix II B.
Foreign matter Not more than 2.0%, Appendix XI D.
Water-insoluble matter When the insects are placed in water, no insoluble powder separates.
Microbial contamination 1 g is free from Escherichia coli; 10 g is free from Salmonella, Appendix
XVI B1.
Action and use Colouring agent.
11-16
Cocillana
Definition Cocillana is the dried bark of Guarea rusbyi (Britton) Rusby and closely related species.
Characteristics Odour, slight and characteristic.
Macroscopical Large, flattish or curved pieces up to about 60 cm long and 15 cm wide and from 5 to
20 mm in thickness; outer surface showing shallow or deep longitudinal fissures according to age,
colour greyish brown or orange-brown where cork has been removed and often bearing whitish
patches of lichen; inner surface longitudinally striated with straight or slightly wavy striae and easily
detachable fibre strands; fracture short and granular in outer part, coarsely splintery, fibrous and soft
in the much thicker inner part; transversely cut surface showing a narrow outer corky region and
wider inner region with dark-coloured, narrow, wavy medullary rays.
Microscopical Cork cells in layers alternating with parenchyma containing yellowish sclereids, angular
or irregular in shape and up to 150 m long; fibres straight, thick-walled and strongly
sclerenchymatous in numerous tangentially elongated groups alternating with tangential bands of
dark-coloured parenchyma and sieve tubes, each fibre group surrounded by a crystal sheath with
prisms of calcium oxalate from 10 to 25 m long; medullary rays one to three cells wide; cells of
medullary rays and parenchyma with reddish brown contents and single spheroidal or two- to four-
compound starch granules, individual granules 5 to 20 m in diameter.
Extractive soluble in ethanol (60%) Not less than 3.5%, Appendix XI B.
When Powdered Cocillana is prescribed or demanded, material complying with the appropriate
requirements above shall be dispensed or supplied.
11-17
Coconut Oil
Coconut Oil complies with the requirements of the 3rd edition of the European Pharmacopoeia for Refined
Coconut Oil [1410]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Refined Coconut oil is the refined fatty oil obtained from the dried, solid part of the endosperm of
Cocos nucifera L.
CHARACTERS
A white or almost white, unctuous mass, practically insoluble in water, freely soluble in methylene
chloride and in light petroleum (bp: 65C to 70C), very slightly soluble in alcohol.
The refractive index is about 1.449, determined at 40C.
IDENTIFICATION
A. It complies with the test for melting point (see Tests).
TESTS
Alkaline impurities in fatty oils (2.4.19). It complies with the test for alkaline impurities in fatty
oils.
(2.4.22, Method B). Coconut oil is melted under gentle heating to a homogeneous liquid prior to
sampling.
Reference solution. Dissolve 15.0 mg of tricaproin CRS, 80.0 mg of tristearin CRS, 0.150 g of tricaprin
CRS, 0.200 g of tricaprylin CRS, 0.450 g of trimyristin CRS and 1.25 g of trilaurin CRS in a mixture
of 2 volumes of methylene chloride R and 8 volumes of heptane R and dilute to 50 ml with the same
mixture of solvents heating at 45C to 50C. Transfer 2 ml to a 10 ml centrifuge tube with a screw
cap and evaporate the solvent in a current of nitrogen R. Dissolve with 1 ml of heptane R and 1 ml of
dimethyl carbonate R and mix vigorously under gentle heating (50C to 60C). Add, while still warm,
1 ml of a 12 g/l solution of sodium R in anhydrous methanol R, prepared with the necessary precautions
and mix vigorously for about 5 min. Add 3 ml of distilled water R and mix vigorously for about 30 s.
Centrifuge for 15 min at 1500 g. Inject 1 l of the organic phase.
Calculate the percentage content of each fatty acid using the following expression:
m m
A
A
c s x
c s x
/ cent per 100
, ,
, ,
A
x,s,c
is the corrected peak area of each fatty acid in the test solution:
A
x,s,c
= A
x,s
R
c
R
c
is the relative correction factor:
r r x
r r x
c
m A
A m
R
, 1 ,
, 1 ,
for the peaks corresponding to caproic, caprylic, capric, lauric and myristic acid methyl esters.
m
x,r
=mass of tricaproin, tricaprylin, tricaprin, trilaurin or trimyristin in the reference solution,
in milligrams,
m
l,r
=mass of tristearin in the reference solution, in milligrams,
A
x,r
=area of the peaks corresponding to caproic, caprylic, capric, lauric and myristic acid
methyl esters in the reference solution,
A
1,r
=area of the peak corresponding to stearic acid methyl ester in the reference solution,
A
x,s
=area of peaks corresponding to any specified or unspecified fatty acid methyl esters,
R
c
=1 for peaks corresponding to each of the remaining specified fatty acid methyl esters or
any unspecified fatty acid methyl ester.
11-18
caproic acid (R
Rt
caprylic acid (R
Rt
capric acid (R
Rt
lauric acid (R
Rt
myristic acid (R
Rt
palmitic acid (R
Rt
stearic acid (R
Rt
oleic acid and isomers (R
Rt
linoleic acid (R
Rt
linoleic acid (R
Rt
arachidic acid (R
Rt
eicosenoic acid (R
Rt
1.11): not more than 0.2 per cent.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
11-19
Cocoyl Caprylocaprate
corrected 1/01
Cocoyl Caprylocaprate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cocoyl caprylocaprate is a mixture of esters of saturated C
12
to C
18
alcohols with caprylic and capric
acids obtained by the reaction of these acids with vegetable saturated fatty alcohols.
CHARACTERS
A slightly yellowish liquid, practically insoluble in water, miscible with alcohol and with liquid
paraffin.
It has a relative density of about 0.86, a refractive index of about 1.445 and a viscosity of about
11 mPa
.
s.
IDENTIFICATION
A. Freezing point (2.2.18): not more than 15C.
obtained with cocoyl caprylocaprate CRS.
C. It complies with the test for composition of fatty acids and fatty alcohols (see Tests).
TESTS
Appearance The substance to be examined is not more intensely coloured than reference solution
Y
5
(Method I, 2.2.2).
Hydroxyl value (2.5.3, Method A). Not more than 5.0.
Saponification value (2.5.6): 160 to 173.
Composition of fatty acids and fatty alcohols Carry out the test for foreign oils in fatty oils by
gas chromatography (2.4.22, Method C), using the chromatogram obtained with the following
reference solution for identification of the peaks corresponding to the fatty alcohols.
Reference solution. Dissolve the following amounts of the substances listed in Table 1411-1 in 10 ml of
heptane R.
Consider the sum of the areas of the peaks due to the fatty acids listed below to be equal to 100 and
the sum of the areas of the peaks due to the fatty alcohols listed below to be equal to 100.
The fatty-acid fraction of the substance has the following composition:
caproic acid: not more than 2.0 per cent,
caprylic acid: 50.0 per cent to 80.0 per cent,
capric acid: 20.0 per cent to 50.0 per cent,
lauric acid: not more than 3.0 per cent,
myristic acid: not more than 1.0 per cent.
The fatty-alcohol fraction of the substance has the following composition:
capric alcohol: not more than 3.0 per cent,
lauryl alcohol: 48.0 per cent to 59.0 per cent,
myristyl alcohol: 18.0 per cent to 25.0 per cent,
cetyl alcohol: 6.0 per cent to 12.0 per cent,
stearyl alcohol: 9.0 per cent to 16.0 per cent.
11-20
Table 14111
Substance Amount (mg)
Methyl caproate R 10
Methyl caprylate R 90
Methyl caprate R 50
Methyl laurate R 20
Methyl myristate R 10
Methyl palmitate R 10
Methyl stearate R 10
Capryl alcohol R 10
Lauryl alcohol R 100
Myristyl alcohol R 40
Cetyl alcohol CRS 30
Stearyl alcohol CRS 20
mination of water.
__________________________________________________________________________________________________________ Ph Eur
11-21
Codeine
O
HO
H
NMe
MeO
C
18
H
21
NO
3
,H
2
O 317.4 76-57-3
Codeine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0076]. These
Action and use Antidiarrhoeal; cough suppressant; analgesic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Codeine contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of
(5R,6S)-4,5-epoxy-3-methoxy-N-methylmorphin-7-en-6-ol, calculated with reference to the dried
substance.
CHARACTERS
A white, crystalline powder or colourless crystals, soluble in boiling water, freely soluble in alcohol,
soluble in ether.
IDENTIFICATION
B. To 2.0 ml of solution S (see Tests) add 50 ml of water R then 10 ml of 1M sodium hydroxide and
dilute to 100.0 ml with water R. Examined between 250 nm and 350 nm (2.2.25), the solution shows
only one absorption maximum, at 284 nm. The specific absorbance at the maximum is about 50,
spectrum of codeine. Examine the dried substance prepared as a disc using potassium bromide R.
D. To about 10 mg add 1 ml of sulphuric acid R and 0.05 ml of ferric chloride solution R2 and heat on a
water-bath. A blue colour develops. Add 0.05 ml of nitric acid R. The colour changes to red.
E. It gives the reaction of alkaloids (2.3.1).
TESTS
Solution S Dissolve 50 mg in carbon dioxide-free water R and dilute to 10 ml with the same solvent.
pH (2.2.3). The pH of solution S is greater than 9.
Specific optical rotation (2.2.7). Dissolve 0.50 g in alcohol R and dilute to 25.0 ml with the same
solvent. The specific optical rotation is 142 to 146, calculated with reference to the dried
substance.
Foreign alkaloids Examine by thin-layer chromatography (2.2.27), using silica gel G R as the
coating substance.
Test solution. Dissolve 0.4 g of the substance to be examined in ethanol R and dilute to 10 ml with the
same solvent.
Reference solution (a). Dilute 1.5 ml of the test solution to 100 ml with ethanol R.
Reference solution (b). Dilute 1 ml of the test solution to 100 ml with ethanol R.
6 volumes of concentrated ammonia R, 30 volumes of cyclohexane R and 72 volumes of ethanol R.
Allow the plate to dry in air and spray with potassium iodobismuthate solution R. Any spot in the
chromatogram obtained with the test solution, apart from the principal spot, is not more intense that
the spot in the chromatogram obtained with reference solution (a) (1.5 per cent) and not more than
one such spot (situated above the principal spot) is more intense than the spot in the chromatogram
11-22
Morphine Dissolve 0.10 g in 0.1M hydrochloric acid and dilute to 5 ml with the same acid. Add 2 ml
of a 10 g/l solution of sodium nitrite R, allow to stand for 15 min and add 3 ml of dilute ammonia R1.
The solution is not more intensely coloured than reference solution B
4
(Method II, 2.2.2) (about
0.13 per cent of morphine).
100C to 105C.
ASSAY
Dissolve 0.250 g in 10 ml of anhydrous acetic acid R and add 20 ml of dioxan R. Titrate with 0.1M
perchloric acid using 0.05 ml of crystal violet solution R as indicator.
18
H
21
NO
3
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
11-23
Codeine Hydrochloride
,HCl
O
HO
H
NMe
eO
C
18
H
22
ClNO
3
,2H
2
O 371.9 1422-07-7
Codeine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Codeine Hydrochloride Dihydrate [1412]. These requirements are reproduced after the heading Definition
below.
Action and use Analgesic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Codeine hydrochloride dihydrate contains not less than 99.0 per cent and not more than the equiva-
lent of 101.0 per cent of (5R,6S)-4,5-epoxy-3-methoxy-N-methylmorphin-7-en-6-ol hydrochloride,
CHARACTERS
A white, crystalline powder or small, colourless crystals, soluble in water, slightly soluble in ethanol,
practically insoluble in cyclohexane.
IDENTIFICATION
spectrum of codeine hydrochloride dihydrate.
B. To 5 ml of solution S (see Tests) add 1 ml of a mixture of equal volumes of strong sodium hydroxide
solution R and water R and initiate crystallisation, if necessary, by scratching the wall of the tube with
a glass rod and cooling in iced water. The precipitate, washed with water and dried at 100C to
105C, melts (2.2.15) at 155C to 159C.
C. To about 10 mg add 1 ml of sulphuric acid R and 0.05 ml of ferric chloride solution R2 and heat on a
D. Solution S gives reaction (a) of chlorides (2.3.1).
E. It gives the reaction of alkaloids (2.3.1).
TESTS
solution Y
6
(Method II, 2.2.2).
Acidity or alkalinity To 5 ml of solution S add 5 ml of carbon dioxide-free water R. Add 0.05 ml of
methyl red solution R and 0.2 ml of 0.02M hydrochloric acid; the solution is red. Add 0.4 ml of 0.02M
sodium hydroxide; the solution becomes yellow.
optical rotation is 117

to 121 calculated with reference to the anhydrous substance.
Related substances. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel F
254
plate R.
methanol R and water R and dilute to 10 ml with the same mixture of solvents.
Reference solution (a). Dilute 1.5 ml of the test solution to 100 ml with a mixture of equal volumes of
methanol R and water R.
Reference solution (b). Dilute 1 ml of the test solution to 100 ml with a mixture of equal volumes of
11-24
Reference solution (c). Dilute 1.5 ml of the test solution to 50 ml with a mixture of equal volumes of
Reference solution (d). Dissolve 15 mg of dextromethorphan hydrobromide CRS in 10 ml of reference
solution (c).
obtained with reference solution (b) (1 per cent). The test is not valid unless the chromatogram
obtained with reference solution (d) shows two clearly separated principal spots.
of a 10 g/l solution of sodium nitrite R, allow to stand for 15 min add 3 ml of dilute ammonia R1. The
solution is not more intensely coloured than reference solution B
4
(Method II, 2.2.2) (about 0.13 per
cent of morphine).
mination of water.
ASSAY
Dissolve 0.300 g in a mixture of 5 ml of 0.01M hydrochloric acid R and 30 ml of alcohol R. Carry out a
18
H
22
ClNO
3
.
STORAGE
IMPURITIES
O
NMe
H
eO
eO
A. (5R,6S)-4,5-epoxy-3,6-dimethoxy-N-methylmorphin-7-en (6-0-methylcodeine),
B. morphine.
__________________________________________________________________________________________________________ Ph Eur
11-25
Codeine Phosphate
,H
3
PO
4
O
HO
H
NMe
MeO
C
18
H
21
NO
3
,H
3
PO
4
,H
2
O 406.4 41444-62-6
Codeine Phosphate complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Codeine Phosphate Hemihydrate [0074]. These requirements are reproduced after the heading Definition
below.
Preparations
Co-codamol Tablets
Co-codaprin Tablets
Dispersible Co-codaprin Tablets
Codeine Linctus
Paediatric Codeine Linctus
Codeine Phosphate Oral Solution
Codeine Phosphate Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Codeine phosphate hemihydrate contains not less than 98.5 per cent and not more than the equiva-
lent of 101.0 per cent of (5R,6S,9R,13S,14R)-4,5-epoxy-3-methoxy-9-methyl-morphin-7-en-6-ol
phosphate, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder or small, colourless crystals, freely soluble in water, slightly soluble in
IDENTIFICATION
A. Dilute 1.0 ml of solution S (see Tests) to 100.0 ml with water R. To 25.0 ml of this solution add
25 ml of water R and 10 ml of 1M sodium hydroxide and dilute to 100.0 ml with water R. Examined
between 250 nm and 350 nm (2.2.25), the solution shows only one absorption maximum, at
284 nm. The specific absorbance at the maximum is about 38, calculated with reference to the dried
substance.
B. Dissolve 0.20 g in 4 ml of water R. Add 1 ml of a mixture of equal volumes of strong sodium
hydroxide solution R and water R and initiate crystallisation, if necessary, by scratching the wall of the
tube with a glass rod and cooling in iced water. Wash the precipitate and dry at 100C to 105C.
spectrum of codeine. Examine the dried precipitate prepared as discs using potassium bromide R.
C. Dissolve 0.20 g in 4 ml of water R. Add 1 ml of a mixture of equal volumes of strong sodium
hydroxide solution R and water R and initiate crystallisation, if necessary, by scratching the wall of the
tube with a glass rod and cooling in iced water. The precipitate, washed and dried at 100C to
105C, melts (2.2.14) at 155C to 159C.
D. To about 10 mg add 1 ml of sulphuric acid R and 0.05 ml of ferric chloride solution R2 and heat on a
E. Solution S gives reaction (a) of phosphates (2.3.1).
F. It gives the reaction of alkaloids (2.3.1).
TESTS
11-26
solution Y
6
(Method II, 2.2.2).
optical rotation is 98 to 102, calculated with reference to the dried substance.
Foreign alkaloids Examine by thin-layer chromatography (2.2.27), using silica gel G R as the
coating substance.
Test solution. Dissolve 0.5 g of the substance to be examined in a mixture of 1 volume of ethanol R
and 4 volumes of 0.01M hydrochloric acid and dilute to 10 ml with the same mixture of solvents.
Reference solution (a). Dilute 1.5 ml of the test solution to 100 ml with a mixture of 1 volume of
ethanol R and 4 volumes of 0.01M hydrochloric acid.
Reference solution (b). Dilute 1 ml of the test solution to 100 ml with a mixture of 1 volume of
ethanol R and 4 volumes of 0.01M hydrochloric acid.
of a 10 g/l solution of sodium nitrite R, allow to stand for 15 min and add 3 ml of dilute ammonia R1.
The solution is not more intensely coloured than reference solution B
4
(Method II, 2.2.2) (about
0.13 per cent of morphine).
100C to 105C.
ASSAY
Dissolve 0.350 g in a mixture of 10 ml of anhydrous acetic acid R and 20 ml of dioxan R. Titrate with
0.1M perchloric acid using 0.05 ml of crystal violet solution R as indicator.
18
H
24
NO
7
P.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
11-27
Codeine Phosphate Sesquihydrate
C
18
H
21
NO
3
,H
3
PO
4
,1H
2
O 424.4 5913-76-8
Codeine Phosphate Sesquihydrate complies with the requirements of the 3rd edition of the European
Preparations
Codeine Linctus
Paediatric Codeine Linctus
Codeine Phosphate Oral Solution
Codeine Phosphate Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Codeine phosphate sesquihydrate contains not less than 98.5 per cent and not more than the equiva-
lent of 101.0 per cent of (5R,6S)-4,5-epoxy-3-methoxy-N-methylmorphin-7-en- 6-ol phosphate,
CHARACTERS
It has the characters described in the monograph on Codeine phosphate hemihydrate (74).
IDENTIFICATION
It complies with the identification tests prescribed in the monograph on Codeine phosphate hemihydrate
(74).
TESTS
It complies with the tests prescribed in the monograph on Codeine phosphate hemihydrate (74) with the
following modification.
100C to 105C.
ASSAY
Carry out the assay prescribed in the monograph on Codeine phosphate hemihydrate (74).
18
H
24
NO
7
P.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
11-28
Co-dergocrine Mesilate
N
O
N
N
O
OH
H
H
H
O
R
Pr
i
,CH
3
SO
3
H
NMe
HN
H
H
O
H
R
dihydroergocristine mesilate CH
2
Ph
dihydroergocryptine mesilate, CH
2
CHMe
2
dihydroergocryptine mesilate, CHMeCH
2
CH
3
dihydroergocornine mesilate CHMe
2
8067-24-1
Definition Co-dergocrine Mesilate consists of equal proportions of the methanesulphonates of
dihydroergocornine (C
31
H
41
N
5
O
5
,CH
4
O
3
S; 659.8), dihydroergocristine (C
35
H
41
N
5
O
5
,CH
4
O
3
S;
707.8) and dihydroergocryptine (C
32
H
43
N
5
O
5
,CH
4
O
3
S; 673.8), the dihydroergocryptine being
present as both the -and -forms in the ratio of not less than 1.5 to 1 and not more than 2.5 to 1. It
contains not less than 97.0% and not more than 103.0% of co-dergocrine mesilate, calculated with
reference to the dried substance, and not less than 30.0% and not more than 36.5% of each of the
three components.
Characteristics A white to yellowish white powder; odourless or almost odourless.
Sparingly soluble in water; soluble in acetone and in ethanol (96%); slightly soluble in chloroform;
Identification
A. In the Assay, the chromatogram obtained with solution (2) shows four major peaks with retention
times corresponding to the peaks due to co-dergocrine mesilate in the chromatogram obtained with
solution (1).
B. Dissolve 10 mg in 1 ml of methanol and add 5 ml of a 1% w/v solution of (+)-tartaric acid. To 1 ml
of the resulting solution, add slowly 2 ml of dimethylaminobenzaldehyde solution R6 and mix. A deep
blue colour is produced.
C. Mix 0.1 g with 0.5 g of powdered sodium hydroxide, heat to fusion and continue heating for a few
seconds. Cool, add 0.5 ml of water and a slight excess of 2M hydrochloric acid and warm. Sulphur
dioxide is evolved, which turns moistened starch iodate paper blue.
Related substances Carry out in subdued light the method for thin-layer chromatography, Appendix
III A, using a silica gel precoated plate (Merck silica gel 60 plates are suitable), scored at a distance of
15 cm from the line of application, and a freshly prepared mixture of 50 volumes of dichloromethane,
50 volumes of ethyl acetate, 3 volumes of methanol and 1 volume of 13.5M ammonia as the mobile
phase. Use an unlined tank closed with an ungreased lid and develop the chromatogram immediately
after introducing the mobile phase into the tank. Apply separately to the plate, in the order specified,
5 l of each of the following solutions prepared immediately before use in a mixture of 9 volumes of
chloroform and 1 volume of methanol containing (1) 0.0060% w/v of dihydroergocristine mesilate
BPCRS, (2) 0.0040% w/v of dihydroergocristine mesilate BPCRS, (3) 0.0020% w/v of dihydroergocristine
mesilate BPCRS and (4) 2.0% w/v of the substance being examined. Allow the solvent front to ascend
for exactly 90 minutes, remove the plate and dry it in a current of cold air for not longer than 1
minute. Using a freshly prepared mobile phase, repeat the development for a further 90 minutes,
remove the plate and dry it in a current of cold air for not longer than 1 minute. Spray with a 1% w/v
solution of dimethylaminobenzaldehyde in a mixture of equal volumes of hydrochloric acid and ethanol
(96%), dry in a current of cold air for not longer than 2 minutes and heat at 40 for 15 minutes. In
the chromatogram obtained with solution (4), any secondary spot is not more intense than the spot in
the chromatogram obtained with solution (1) (0.3%), not more than two such spots are more intense
than the spot in the chromatogram obtained with solution (2) (0.2%) and not more than a total of
four such spots are more intense than the spot in the chromatogram obtained with solution (3)
(0.1%). Disregard any spot remaining on the line of application.
11-29
Loss on drying When dried to constant weight at 120 at a pressure of 14 Pa, loses not more than
6.0% of its weight. Use 1 g.
Solution (1) contains 0.060% w/v of co-dergocrine mesilate BPCRS in a mixture of 2 volumes of a 1%
w/v solution of (+)-tartaric acid and 1 volume of absolute ethanol. Solution (2) contains 0.060% w/v
of the substance being examined in the same solvent mixture.
cm 4.6 mm) packed with stationary phase C (5 m) (Spherisorb ODS 1 is suitable), (b) a mixture of
150 volumes of water, 50 volumes of acetonitrile and 5 volumes of triethylamine as the mobile phase
with a flow rate of 1.5 ml per minute and (c) a detection wavelength of 280 nm. The components are
eluted in the following order: dihydroergocornine, dihydro--ergocryptine, dihydroergocristine and
dihydro--ergocryptine.
The resolution factor between the peaks due to dihydro--ergocryptine and dihydroergocristine
should be not less than 1.2; this value may be obtained by appropriate adjustment of the acetonitrile
content of the mobile phase.
Calculate the content of co-dergocrine mesilate in the substance being examined using the
expression:
] [ ] [
] [
L w M A M A M A M A
w M A M A M A M A
01 . 0 1
p
S R 2 4 3 3 2 2 1 1
R R S 2 4 3 3 2 2 1 1
+ + +
+ + +
whereA
1
, A
2
, A
3
, A
4
= the peak areas of the components in order of elution,
M
1
, M
2
, M
3
= the molecular weights of the methanesulphonates of dihydroergocornine,
dihydroergocryptine (- and -forms) and dihydroergocristine,
w
R
= the percentage w/v of co-dergocrine mesilate BPCRS in solution (1) (R),
w
S
= the percentage w/v of the substance being examined in solution (2) (S),
L = the percentage loss on drying of the substance being examined,
p
R
= the declared content of co-dergocrine mesilate in co-dergocrine mesilate BPCRS.
Calculate the percentage contents of the methanesulphonates of dihydroergocornine, of dihydroergo-
cryptine (- and -forms) and of dihydroergocristine with respect to the sum of these components in
solution (2), using the products of the molecular weights and peak area figures, and determine the
ratio of the peak area of dihydro--ergocryptine to that of dihydro--ergocryptine.
Storage Co-dergocrine Mesilate should be kept in a well-closed container, protected from light and
Preparation
Co-dergocrine Tablets
When co-dergocrine mesylate is prescribed or demanded, Co-dergocrine Mesilate shall be dispensed
or supplied.
11-30
Cod-liver Oil (Type A)
Cod-liver Oil (Type A)complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Source of vitamins A and D.
Each IU of vitamin D
3
is equivalent to 0.025 g of colecalciferol.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cod-liver oil (type A) is the purified fatty oil obtained from the fresh livers of Gadus morhua L. and
other species of Gadidae, solid substances being removed by cooling and filtering. It contains not less
than 600 I.U. (180 g) and not more than 2500 I.U. (750 g) of vitamin A per gram and not less
than 60 I.U. (1.5 g) and not more than 250 I.U. (6.25 g) of vitamin D
3
per gram.
Authorised antioxidants in concentrations not exceeding those prescribed by the competent
authority may be added.
CHARACTERS
A clear, yellowish, viscous liquid, practically insoluble in water, slightly soluble in alcohol, miscible
with light petroleum.
IDENTIFICATION
First identification: A, B, C.
Second identification: C, D.
A. In the assay for vitamin A using method A, the test solution shows an absorption maximum
(2.2.25) at 325 2 nm. In the assay for vitamin A using method B, the chromatogram obtained with
the test solution shows a peak corresponding to the peak of all-trans-retinol in the chromatogram
B. In the assay for vitamin D
3
, the chromatogram obtained with test solution (a) shows a peak
corresponding to the peak of cholecalciferol in the chromatogram obtained with reference solution
(b).
D. To 0.1 g add 0.5 ml of chloroform R and 1 ml of antimony trichloride solution R. Mix. A deep blue
colour develops in about 10 s.
TESTS
Colour It is not more intensely coloured than a reference solution prepared as follows: to 3.0 ml of
red primary solution add 25.0 ml of yellow primary solution and dilute to 50.0 ml with a 10 g/l
solution of hydrochloric acid R (Method II, 2.2.2).
Refractive index (2.2.6). 1.477 to 1.484.
Anisidine value Not more than 30.0.
The anisidine value is defined as 100 times the optical density measured in a 1 cm cell of a solution
containing 1 g of the substance to be examined in 100 ml of a mixture of solvents and reagents
according to the method described below.
Carry out the operations as rapidly as possible, avoiding exposure to actinic light.
Test solution (a). Dissolve 0.500 g of the substance to be examined in trimethylpentane R and dilute to
Test solution (b). To 5.0 ml of test solution (a) add 1.0 ml of a 2.5 g/l solution of p-anisidine R in
glacial acetic acid R, shake and store protected from light.
Reference solution. To 5.0 ml of trimethylpentane R add 1.0 ml of a 2.5 g/l solution of p-anisidine R in
glacial acetic acid R, shake and store protected from light.
Measure the absorbance of test solution (a) at 350 nm using trimethylpentane R as the
compensation liquid. Measure the absorbance of test solution (b) at 350 nm exactly 10 min after its
preparation, using the reference solution as the compensation liquid.
Calculate the anisidine value from the expression:
m
A A
b s
) 2 . 1 ( 25
A
s
=absorbance of test solution (b) at 350 nm,
11-31
A
b
=absorbance of test solution (a) at 350 nm,
m=mass of the substance to be examined in test solution (a) in grams.
Iodine value (2.5.4). 150 to 180.
Stearin 10 ml remains clear after cooling in iced water for 3 h.
Composition of fatty acids
Trivial name of
fatty acid
Shorthand
notation
Lower
limit area
(per cent)
Upper
limit area
(per cent)
Saturated fatty acids:
Myristic acid 14:0 2.0 6.0
Palmitic acid 16:0 7.0 14.0
Stearic acid 18:0 1.0 4.0
Mono-unsaturated fatty acids:
Palmitoleic acid 16:1 n-7 4.5 11.5
cis -Vaccenic acid 18:1 n-7 2.0 7.0
Oleic acid 18:1 n-9 12.0 21.0
Gadoleic acid 20:1 n-11 1.0 5.5
Gondoic acid 20:1 n-9 5.0 17.0
Erucic acid 22:1 n-9 0 1.5
Cetoleic acid
(22:1 n-11)
22:1 n-11+13 5.0 12.0
Poly-unsaturated fatty acids
Linoleic acid 18:2 n-6 0.5 3.0
-Linolenic acid 18:3 n-3 0 2.0
Moroctic acid 18:4 n-3 0.5 4.5
Timnodonic
eicosapentaenoic acid
(EPA)
20:5 n-3 7.0 16.0
Cervonic
docosahexaenoic acid
(DHA)
22:6 n-3 6.0 18.0
Test solution. Introduce about 0.45 g of the substance to be examined into a 10 ml volumetric flask,
dissolve in hexane R containing 50 mg of butylhydroxytoluene R per litre and dilute to 10.0 ml with the
same solvent. Transfer 2.0 ml of the solution into a quartz tube and evaporate the solvent with a
gentle current of nitrogen R. Add 1.5 ml of a 20 g/l solution of sodium hydroxide R in methanol R, cover
with nitrogen R, cap tightly with a polytetrafluoroethylene lined cap, mix and heat in a water-bath for
7 min. Cool, add 2 ml of boron trichloride-methanol solution R, cover with nitrogen R, cap tightly, mix
and heat in a water-bath for 30 min. Cool to 40C to 50C, add 1 ml of trimethylpentane R, cap and
vortex or shake vigorously for at least30 s. Immediately add 5 ml of a saturated solution of sodium
chloride R, cover with nitrogen R, cap and vortex or shake thoroughly for at least 15 s. Allow the upper
layer to become clear and transfer to a separate tube. Shake the methanol layer once more with 1 ml
of trimethylpentane R and combine the trimethylpentane extracts. Wash the combined extracts with
two quantities, each of 1 ml, of water R and dry over anhydrous sodium sulphate R. Prepare two
solutions for each sample.
a fused-silica capillary column at least 30 m long and 0.25 mm in internal diameter coated with
macrogol 20,000 R (film thickness 0.25 m),
hydrogen for chromatography R or helium for chromatography R as the carrier gas where oxygen
scrubber is applied,
a split mode injector of 1:200,
a suitable integrator,
Inject twice 1 l of the test solution.
11-32
The test is not valid unless:
the fifteen fatty acids to be tested are satisfactorily identified from the type chromatogram
(Figure 1192-1),
injection of a mixture of equal amounts of methyl palmitate R, methyl stearate R, methyl arachidate
R, and methyl behenate R give area percentages of 24.4, 24.8, 25.2 and 25.6, respectively,
the resolution between the peaks corresponding to methyl oleate and methyl cis-vaccenate is at
least 1.3 and the pair corresponding to methyl gadoleate and methyl gondoate is sufficient for
purposes of identification and area measurement.
Calculate the area per cent for each fatty acid methyl ester from the expression:
100 cent per Area
t
x
A
A
A
x
=peak area of fatty acid x,
A
t
=total area for the whole chromatogram.
The calculation is not valid unless:
the total area is based only on peaks corresponding to solely fatty acids methyl esters,
the number of fatty acid methyl ester peaks exceeding 0.05 per cent of the total area is at least
twenty four,
the twenty-four largest peaks of the methyl esters account for more than 90 per cent of the total
area. (These correspond to, in common elution order: 14:0, 15:0, 16:0, 16:1 n-7, 16:4 n-1,
18:0, 18:1 n-9, 18:1 n-7, 18:2 n-6, 18:3 n-3, 18:4 n-3, 20:1 n-11, 20:1 n-9, 20:1 n-7, 20:2 n-6,
20:4 n-6, 20:3 n-3, 20:4 n-3, 20:5 n-3, 22:1 n-11, 22:1 n-9, 21:5 n-3, 22:5 n-3, 22:6 n-3).
Fig. 11921 Type chromatogram for the test composition of fatty acids
ASSAY
Vitamin A Carry out the test as rapidly as possible, avoiding exposure to actinic light and air, oxidising
11-33
agents, oxidation catalysts (e.g. copper and iron) and acids.
Examine by ultraviolet absorption spectrophotometry (2.2.25) (Method A). If method A is found
not to be valid, examine by liquid chromatography (2.2.29) (Method B).
Method A
Test solution. To 1.00 g in a round-bottomed flask, add 3 ml of a freshly prepared 50 per cent m/m
solution of potassium hydroxide R and 30 ml of ethanol R. Boil under reflux in a current of nitrogen R
for 30 min. Cool rapidly and add 30 ml of water R. Extract with 50 ml of ether R. Repeat the
extraction three times and discard the lower layer after complete separation. Wash the combined
upper layers with four quantities, each of 50 ml, of water R and evaporate to dryness under a gentle
current of nitrogen R at a temperature not exceeding 30C or in a rotary evaporator at a temperature
not exceeding 30C under reduced pressure (water ejector). Dissolve the residue in sufficient
2-propanol R1 to give an expected concentration of vitamin A equivalent to 10 I.U. per millilitre to
15 I.U. per millilitre.
Measure the absorbances of the solution at 300 nm, 310 nm, 325 nm and 334 nm and at the
wavelength of maximum absorption with a suitable spectrophotometer in 1 cm specially matched
cells, using 2-propanol R1 as the compensation liquid.
Calculate the content of vitamin A, as all-trans-retinol, in International Units per gram from the
expression:
A
m
V
325
1830
100

A
325
=absorbance at 325 nm,
m=mass of the substance to be examined in grams,
V=total volume of solution containing 10 I.U. to 15 I.U. of vitamin A per millilitre,
1830=conversion factor for the specific absorbance of all-trans-retinol in International Units.
The above expression can be used only if A
325
has a value of not greater than A
325,corr
/0.970 where
A
325,corr
is the corrected absorbance at 325 nm and is given by the equation:
A
325, corr
= 6.815A
325
2.555A
310
4.260A
324
A designates the absorbance at the wavelength indicated by the subscript.
If A
325
has a value greater than A
325,corr
/0.970, calculate the content of vitamin A from the
expression:
A
m
V
325
1830
100
,corr

The assay is not valid unless:
the wavelength of maximum absorption lies between 323 nm and 327 nm,
the absorbance at 300 nm relative to that at 325 nm is at most 0.73.
Method B
Test solution. To 2.00 g in a round-bottomed flask, add 5 ml of a freshly prepared 100 g/l solution of
ascorbic acid R and 10 ml of a freshly prepared 800 g/l solution of potassium hydroxide R and 100 ml of
ethanol R. Boil under a reflux condenser on a water-bath for 15 min. Add 100 ml of a 10 g/l solution
of sodium chloride R and cool. Transfer the solution to a 500 ml separating funnel rinsing the round-
bottomed flask with about 75 ml of a 10 g/l solution of sodium chloride R and then with 150 ml of a
mixture of equal volumes of light petroleum R3 and ether R. Shake for 1 min. When the layers have
separated completely, discard the lower layer and wash the upper layer, first with 50 ml of a 30 g/l
solution of potassium hydroxide R in a 10 per cent V/V solution of ethanol R and then with three
quantities, each of 50 ml, of a 10 g/l solution of sodium chloride R. Filter the upper layer through 5 g
of anhydrous sodium sulphate R on a fast filter paper into a 250 ml flask suitable for a rotary
evaporator. Wash the funnel with 10 ml of fresh extraction mixture, filter and combine the upper
layers. Distil them at a temperature not exceeding 30C under reduced pressure (water ejector) and
fill with nitrogen R when evaporation is completed. Alternatively evaporate the solvent under a gentle
current of nitrogen R at a temperature not exceeding 30C. Dissolve the residue in 2-propanol R,
transfer to a 25 ml volumetric flask and dilute to 25 ml with 2-propanol R. Gentle heating in an
ultrasonic bath may be required. (A large fraction of the white residue is cholesterol, constituting
approximately 50 per cent of the unsaponifiable matter of cod-liver oil).
Reference solution (a). Prepare a solution of retinyl acetate CRS in 2-propanol R1 so that 1 ml contains
about 1000 I.U. of all-trans-retinol.
The exact concentration of reference solution (a) is assessed by ultraviolet absorption spectro-
photometry (2.2.25). Dilute reference solution (a) with 2-propanol R1 to a presumed concentration of
10 I.U. per millilitre to 15 1.U. per millilitre and measure the absorbance at 326 nm in matched
1 cm cells using 2-propanol R1 as the compensation liquid.
Calculate the content of vitamin A in International Units per millilitre of reference solution (a)
from the following expression, taking into account the assigned content of retinyl acetate CRS:
11-34
A
V
V
326
2
1
1900
100
A
326
V
2
=volume of the diluted solution,
V
1
=volume of reference solution (a) used,
1900=conversion factor for the specific absorbance of retinyl acetate CRS in International Units.
Reference solution (b). Proceed as described for the test solution but using 2.00 ml of reference
solution (a) in place of the substance to be examined.
The exact concentration of reference solution (b) is assessed by ultraviolet absorption spectro-
photometry (2.2.25). Dilute reference solution (b) with 2-propanol R1 to a presumed concentration of
10 I.U. per millilitre to 15 I.U. per millilitre of all-trans-retinol and measure the absorbance at
325 nm in matched 1 cm cells using 2-propanol R1 as the compensation liquid.
Calculate the content of all-trans-retinol in International Units per millilitre of reference solution
(b) from the expression:
A
V
V
325
4
3
1830
100
A
325
V
3
V
4
=volume of reference solution (b) used,
as mobile phase at a flow rate of 1 ml/min a mixture of 3 volumes of water R and 97 volumes of
methanol R,
Inject in triplicate the test solution and reference solution (b). The retention time of all-trans-
retinol is 5 1 min.
the chromatogram obtained with the test solution shows a peak corresponding to that of all-
trans-retinol in the chromatogram obtained with reference solution (b),
when using the method of standard additions to the test solution there is greater than 95 per
cent recovery of the added retinyl acetate CRS,
the recovery of all-trans-retinol in reference solution (b) as assessed by direct absorption
spectrophotometry is greater than 95 per cent.
Calculate the content of vitamin A using the following expression:
A
C V
A m
1
2
1
A
1
=area of the peak corresponding to all-trans-retinol in the chromatogram obtained with the
test solution,
A
2
=area of the peak corresponding to all-trans-retinol in the chromatogram obtained with
reference solution (b),
C=concentration of retinyl acetate CRS in reference solution (a) as assessed prior to the
saponification in International Units per millilitre (= 1000 I.U. per millilitre),
V=volume of reference solution (a) treated (2.00 ml),
m=mass of the substance to be examined in the test solution (2.00 g).
Vitamin D
3
Internal standard solution. Dissolve 0.50 mg of ergocalciferol CRS in 100 ml of ethanol R.
Test solution (a). To 4.00 g in a round-bottomed flask, add 5 ml of a freshly prepared 100 g/l solution
of ascorbic acid R, 10 ml of a freshly prepared 800 g/l solution of potassium hydroxide R and 100 ml of
of sodium chloride R and cool the solution to room temperature. Transfer the solution to a 500 ml
separating funnel rinsing the round-bottomed flask with about 75 ml of a 10 g/l solution of sodium
chloride R and then with 150 ml of a mixture of equal volumes of light petroleum R3 and ether R. Shake
for 1 min. When the layers have separated completely, discard the lower layer and wash the upper
layer, first with 50 ml of a 30 g/l solution of potassium hydroxide R in a 10 per cent V/V solution of
ethanol R, and then with three quantities, each of 50 ml, of a 10 g/l solution of sodium chloride R.
11-35
Filter the upper layer through 5 g of anhydrous sodium sulphate R on a fast filter paper into a 250 ml
flask suitable for a rotary evaporator. Wash the funnel with 10 ml of fresh extraction mixture, filter
and combine the upper layers. Distil them at a temperature not exceeding 30C under reduced
pressure (water ejector) and fill with nitrogen R when evaporation is completed. Alternatively evapor-
ate the solvent under a gentle current of nitrogen R at a temperature not exceeding 30C. Dissolve the
residue in 1.5 ml of the mobile phase described under Purification. Gentle heating in an ultrasonic
bath may be required. (A large fraction of the white residue is cholesterol, constituting approximately
50 per cent m/m of the unsaponifiable matter of cod-liver oil).
Test solution (b). To 4.00 g add 2.0 ml of the internal standard solution and proceed as described for
test solution (a).
Reference solution (a). Dissolve 0.50 mg of cholecalciferol CRS in 100.0 ml of ethanol R.
Reference solution (b). In a round-bottomed flask, add 2.0 ml of reference solution (a) and 2.0 ml of
the internal standard solution and proceed as described for test solution (a).
Purification
a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with cyanosilyl silica
as mobile phase at a flow rate of 1.1 ml/min a mixture of 1.6 volumes of isoamyl alcohol R and
98.4 volumes of hexane R,
Inject 350 l of reference solution (b). Collect the eluate from 2 min before until 2 min after the
retention time of cholecalciferol, in a ground-glass-stoppered tube containing 1 ml of a 1 g/l solution
of butylhydroxytoluene R in hexane R. Repeat the procedure with test solutions (a) and (b). Evaporate
the eluates obtained from reference solution (b) and from test solutions (a) and (b), separately, to
dryness at a temperature not exceeding 30C under a gentle current of nitrogen R. Dissolve each
residue in 1.5 ml of acetonitrile R.
Determination
as mobile phase at a flow rate of 1.0 ml/min a mixture of 0.2 volumes of phosphoric acid R and
99.8 volumes of a 96 per cent V/V solution of acetonitrile R,
Inject two quantities not exceeding 200 l of each of the three solutions obtained under
Purification.
in the chromatogram obtained with reference solution (b), the resolution between the peaks
corresponding to ergocalciferol and cholecalciferol is at least 1.4,
when using the method of standard additions to test solution (a) there is greater than 95 per cent
recovery of the added cholecalciferol CRS when due consideration has been given to correction by
the internal standard.
Calculate the content of vitamin D
3
in I.U. per gram using the following expression, taking into
account the assigned content of cholecalciferol CRS:
40
1
2
1
2
2
1
5
4
3
6
2

1
]
1
V
V
m
m
A
A
A
A
A
A
A
m
1
=mass of the substance to be examined in test solution (b) in grams,
m
2
=total mass of cholecalciferol CRS used for the preparation of reference solution (a) in
micrograms (500 g),
A
1
=area (or height) of the peak corresponding to cholecalciferol in the chromatogram
A
2
obtained with test solution (b),
A
3
=area (or height) of the peak corresponding to ergocalciferol in the chromatogram obtained
with reference solution (b),
A
4
with test solution (b),
A
5
=area (or height) of the peak corresponding to a possible peak in the chromatogram
obtained with test solution (a) with the same retention time as ergocalciferol co-eluting
with ergocalciferol in test solution (b),
A
6
obtained with reference solution (b),
11-36
V
1
=total volume of reference solution (a) (100 ml),
V
2
=volume of reference solution (a) used for preparing reference solution (b) (2.0 ml),
STORAGE
Store preferably under an inert gas in an airtight and well-filled container, protected from light.
Once the container has been opened, its contents are used as soon as possible and any part of the
contents not used at once is protected by an atmosphere of inert gas.
LABELLING
When the addition of antioxidants is authorised by the competent authority, the name and concentra-
tion of the added antioxidant are stated on the label.
The label states:
the number of International Units of vitamin A,
the number of International Units of vitamin D
3
.
_______________________________________________________________________ Ph Eur
11-37
Cod-liver Oil (Type B)
Cod-liver Oil (Type B)complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Source of vitamins A and D.
Each IU of vitamin D
3
is equivalent to 0.025 g of colecalciferol.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cod-liver oil (type B) is the purified fatty oil obtained from the fresh livers of Gadus morhua L. and
other species of Gadidae, solid substances being removed by cooling and filtering. It contains not less
than 600 I.U. (180 g) and not more than 2500 I.U. (750 g) of vitamin A per gram and not less
than 60 I.U. (1.5 g) and not more than 250 I.U. (6.25 g) of vitamin D
3
per gram.
Authorised antioxidants in concentrations not exceeding those prescribed by the competent
authority may be added.
CHARACTERS
A clear, yellowish, viscous liquid, practically insoluble in water, slightly soluble in alcohol, miscible
with light petroleum.
IDENTIFICATION
First identification: A, B, C.
A. In the assay for vitamin A using method A, the test solution shows an absorption maximum
(2.2.25) at 325 2 nm. In the assay for vitamin A using method B, the chromatogram obtained with
the test solution shows a peak corresponding to the peak of all-trans-retinol in the chromatogram
B. In the assay for vitamin D
3
, the chromatogram obtained with test solution (a) shows a peak
corresponding to the peak of cholecalciferol in the chromatogram obtained with reference solution
(b).
D. To 0.1 g add 0.5 ml of chloroform R and 1 ml of antimony trichloride solution R. Mix. A deep blue
colour develops in about 10 s.
TESTS
Colour It is not more intensely coloured than a reference solution prepared as follows: to 3.0 ml of
red primary solution add 25.0 ml of yellow primary solution and dilute to 50.0 ml with a 10 g/l
solution of hydrochloric acid R (Method II, 2.2.2).
Iodine value (2.5.4). 150 to 180.
Stearin 10 ml remains clear after cooling in iced water for 3 h.
11-38
Trivial name of
fatty acid
Shorthand
notation
Lower
limit area
(per cent)
Upper
limit area
(per cent)
Saturated fatty acids:
Myristic acid 14:0 2.0 6.0
Palmitic acid 16:0 7.0 14.0
Stearic acid 18:0 1.0 4.0
Mono-unsaturated fatty acids:
Palmitoleic acid 16:1 n-7 4.5 11.5
cis -Vaccenic acid 18:1 n-7 2.0 7.0
Oleic acid 18:1 n-9 12.0 21.0
Gadoleic acid 20:1 n-11 1.0 5.5
Gondoic acid 20:1 n-9 5.0 17.0
Erucic acid 22:1 n-9 0 1.5
Cetoleic acid
(22:1 n-11)
22:1 n-11+13 5.0 12.0
Poly-unsaturated fatty acids
Linoleic acid 18:2 n-6 0.5 3.0
-Linolenic acid 18:3 n-3 0 2.0
Moroctic acid 18:4 n-3 0.5 4.5
Timnodonic
eicosapentaenoic acid
(EPA)
20:5 n-3 7.0 16.0
Cervonic
docosahexaenoic acid
(DHA)
22:6 n-3 6.0 18.0
Test solution. Introduce about 0.45 g of the substance to be examined into a 10 ml volumetric flask,
dissolve in hexane R containing 50 mg of butylhydroxytoluene R per litre and dilute to 10.0 ml with the
same solvent. Transfer 2.0 ml of the solution into a quartz tube and evaporate the solvent with a
gentle current of nitrogen R. Add 1.5 ml of a 20 g/l solution of sodium hydroxide R in methanol R, cover
with nitrogen R, cap tightly with a polytetrafluoroethylene lined cap, mix and heat in a water-bath for
7 min. Cool, add 2 ml of boron trichloride-methanol solution R, cover with nitrogen R, cap tightly, mix
and heat in a water-bath for 30 min. Cool to 40C to 50C, add 1 ml of trimethylpentane R, cap and
vortex or shake vigorously for at least 30 s. Immediately add 5 ml of a saturated solution of sodium
chloride R, cover with nitrogen R, cap and vortex or shake thoroughly for at least 15 s. Allow the upper
layer to become clear and transfer to a separate tube. Shake the methanol layer once more with 1 ml
of trimethylpentane R and combine the trimethylpentane extracts. Wash the combined extracts with
two quantities, each of 1 ml, of water R and dry over anhydrous sodium sulphate R. Prepare two
solutions for each sample.
a fused-silica capillary column at least 30 m long and 0.25 mm in internal diameter coated with
macrogol 20,000 R (film thickness 0.25 m),
hydrogen for chromatography R or helium for chromatography R as the carrier gas where oxygen
scrubber is applied,
a suitable integrator,
Inject twice 1 l of the test solution.
The test is not valid unless:
the fifteen ftty acids to be tested are satisfactorily identified from the type chromatogram (Figure
1193.-1),
injection of a mixture of equal amounts of methyl palmitate R, methyl stearate R, methyl
arachidate R, and methyl behenate R give area percentages of 24.4, 24.8, 25.2 and 25.6,
respectively,
the resolution between the peaks corresponding to methyl oleate and methyl cis-vaccenate is at
least 1.3 and the pair corresponding to methyl gadoleate and methyl gondoate is sufficient for
purposes of identification and area measurement.
11-39
Calculate the area per cent for each fatty acid methyl ester from the expression:
Area per cent
A
A
x
t
100
A
x
=peak area of fatty acid x,
A
t
=total area for the whole chromatogram.
The calculation is not valid unless:
the total area is based only on peaks corresponding to solely fatty acids methyl esters,
the number of fatty acid methyl ester peaks exceeding 0.05 per cent of the total area is at least
twenty four,
the twenty four largest peaks of the methyl esters account for more than 90 per cent of the total
area. (These correspond to, in common elution order: 14:0, 15:0, 16:0, 16:1 n-7, 16:4 n-1,
18:0, 18:1 n-9, 18:1 n-7, 18:2 n-6, 18:3 n-3, 18:4 n-3, 20:1 n-11, 20:1 n-9, 20:1 n-7, 20:2 n-6,
20:4 n-6, 20:3 n-3, 20:4 n-3, 20:5 n-3, 22:1 n-11, 22:1 n-9, 21:5 n-3, 22:5 n-3, 22:6 n-3).
Fig. 11931 Type chromatogram for the test composition of fatty acids
ASSAY
Vitamin A Carry out the test as rapidly as possible, avoiding exposure to actinic light and air, oxidising
agents, oxidation catalysts (for example, copper and iron) and acids.
Examine by ultraviolet absorption spectrophotometry (2.2.25) (Method A). If method A is found not
to be valid, examine by liquid chromatography (2.2.29) (Method B).
Method A
Test solution. To 1.00 g in a round-bottomed flask, add 3 ml of a freshly prepared 50 per cent m/m
11-40
solution of potassium hydroxide R and 30 ml of ethanol R. Boil under reflux in a current of nitrogen R
for 30 min. Cool rapidly and add 30 ml of water R. Extract with 50 ml of ether R. Repeat the
extraction three times and discard the lower layer after complete separation. Wash the combined
upper layers with four quantities, each of 50 ml, of water R and evaporate to dryness under a gentle
current of nitrogen R at a temperature not exceeding 30C or in a rotary evaporator at a temperature
not exceeding 30C under reduced pressure (water ejector). Dissolve the residue in sufficient
2-propanol R1 to give an expected concentration of vitamin A equivalent to 10 I.U. per millilitre to
15 I.U. per millilitre.
Measure the absorbances of the solution at 300 nm, 310 nm, 325 nm and 334 nm and at the
wavelength of maximum absorption with a suitable spectrophotometer in 1 cm specially matched
cells, using 2-propanol R1 as the compensation liquid.
Calculate the content of vitamin A, as all-trans-retinol, in International Units per gram from the
expression:
A
m
V
325
1830
100

A
325
m=mass of the substance to be examined in grams,
V=total volume of solution containing 10 I.U. to 15 I.U. of vitamin A per millilitre,
325
325,corr
/0.970 where
A
325,corr
A
325, corr
= 6.815A
325
2.555A
310
4.260A
324
If A
325
325,corr
expression:
A
m
V
325
1830
100
,corr

the wavelength of maximum absorption lies between 323 nm and 327 nm,
the absorbance at 300 nm relative to that at 325 nm is at most 0.73.
Method B
Test solution. To 2.00 g in a round-bottomed flask, add 5 ml of a freshly prepared 100 g/l solution of
ascorbic acid R and 10 ml of a freshly prepared 800 g/l solution of potassium hydroxide R and 100 ml of
of sodium chloride R and cool. Transfer the solution to a 500 ml separating funnel rinsing the round-
bottomed flask with about 75 ml of a 10 g/l solution of sodium chloride R and then with 150 ml of a
mixture of equal volumes of light petroleum R3 and ether R. Shake for 1 min. When the layers have
separated completely, discard the lower layer and wash the upper layer, first with 50 ml of a 30 g/l
solution of potassium hydroxide R in a 10 per cent V/V solution of ethanol R and then with three
quantities, each of 50 ml, of a 10 g/l solution of sodium chloride R. Filter the upper layer through 5 g
of anhydrous sodium sulphate R on a fast filter paper into a 250 ml flask suitable for a rotary
evaporator. Wash the funnel with 10 ml of fresh extraction mixture, filter and combine the upper
layers. Distil them at a temperature not exceeding 30C under reduced pressure (water ejector) and
fill with nitrogen R when evaporation is completed. Alternatively evaporate the solvent under a gentle
current of nitrogen R at a temperature not exceeding 30C. Dissolve the residue in 2-propanol R,
transfer to a 25 ml volumetric flask and dilute to 25 ml with 2-propanol R. Gentle heating in an
ultrasonic bath may be required. (A large fraction of the white residue is cholesterol, constituting
approximately 50 per cent of the unsaponifiable matter of cod-liver oil).
Reference solution (a). Prepare a solution of retinyl acetate CRS in 2-propanol R1 so that 1 ml contains
about 1000 I.U. of all-trans-retinol.
The exact concentration of reference solution (a) is assessed by ultraviolet absorption spectro-
photometry (2.2.25). Dilute reference solution (a) with 2-propanol R1 to a presumed concentration of
10 I.U. per millilitre to 15 I.U. per millilitre and measure the absorbance at 326 nm in matched 1 cm
cells using 2-propanol R1 as the compensation liquid.
Calculate the content of vitamin A in International Units per millilitre of reference solution (a)
using the following expression, taking into account the assigned content of retinyl acetate CRS:
A
V
V
326
2
1
1900
100
A
326
V
2
V
1
1900=conversion factor for the specific absorbance of retinyl acetate CRS in International Units.
11-41
Reference solution (b). Proceed as described for the test solution but using 2.00 ml of reference
solution (a) in place of the substance to be examined.
The exact concentration of reference solution (b) is assessed by ultraviolet absorption spectro-
photometry (2.2.25). Dilute reference solution (b) with 2-propanol R1 to a presumed concentration of
10 I.U. per millilitre to 15 I.U. per millilitre of all-trans-retinol and measure the absorbance at
325 nm in matched 1 cm cells using 2-propanol R1 as the compensation liquid.
Calculate the content of all-trans-retinol in International Units per millilitre of reference solution
(b) from the expression:
A
V
V
325
4
3
1830
100
A
325
V
3
V
4
as mobile phase at a flow rate of 1 ml/min a mixture of 3 volumes of water R and 97 volumes of
methanol R,
Inject in triplicate the test solution and reference solution (b). The retention time of all-trans-
retinol is 5 1 min.
the chromatogram obtained with the test solution shows a peak corresponding to that of all-
trans-retinol in the chromatogram obtained with reference solution (b),
when using the method of standard additions to the test solution there is greater than 95 per
cent recovery of the added retinyl acetate CRS,
the recovery of all-trans-retinol in the reference solution (b) as assessed by direct absorption
spectrophotometry is greater than 95 per cent.
A
C V
A m
1
2
1
A
1
=area of the peak corresponding to all-trans-retinol in the chromatogram obtained with the
test solution,
A
2
reference solution (b),
C=concentration of retinyl acetate CRS in reference solution (a) as assessed prior to the
saponification in International Units per millilitre ( = 1000 I.U. per millilitre),
V=volume of reference solution (a) treated (2.00 ml),
m=mass of the substance to be examined in the test solution (2.00 g).
Vitamin D
3
Internal standard solution. Dissolve 0.50 mg of ergocalciferol CRS in 100 ml of ethanol R.
Test solution (a). To 4.00 g in a round-bottomed flask, add 5 ml of a freshly prepared 100 g/l solution
of ascorbic acid R, 10 ml of a freshly prepared 800 g/l solution of potassium hydroxide R and 100 ml of
of sodium chloride R and cool the solution to room temperature. Transfer the solution to a 500 ml
separating funnel rinsing the round-bottomed flask with about 75 ml of a 10 g/l solution of sodium
chloride R and then with 150 ml of a mixture of equal volumes of light petroleum R3 and ether R. Shake
for 1 min. When the layers have separated completely, discard the lower layer and wash the upper
layer, first with 50 ml of a 30 g/l solution of potassium hydroxide R in a 10 per cent V/V solution of
ethanol R, and then with three quantities, each of 50 ml, of a 10 g/l solution of sodium chloride R.
Filter the upper layer through 5 g of anhydrous sodium sulphate R on a fast filter paper into a 250 ml
flask suitable for a rotary evaporator. Wash the funnel with 10 ml of fresh extraction mixture, filter
and combine the upper layers. Distil them at a temperature not exceeding 30C under reduced
pressure (water ejector) and fill with nitrogen R when evaporation is completed. Alternatively evapor-
ate the solvent under a gentle current of nitrogen R at a temperature not exceeding 30C. Dissolve the
residue in 1.5 ml of the mobile phase described under Purification. Gentle heating in an ultrasonic
bath may be required. (A large fraction of the white residue is cholesterol, constituting approximately
50 per cent m/m of the unsaponifiable matter of cod-liver oil).
11-42
Test solution (b). To 4.00 g add 2.0 ml of the internal standard solution and proceed as described for
test solution (a).
Reference solution (a). Dissolve 0.50 mg of cholecalciferol CRS in 100.0 ml of ethanol R.
Reference solution (b). In a round-bottomed flask, add 2.0 ml of reference solution (a) and 2.0 ml of
the internal standard solution and proceed as described for test solution (a).
Purification
a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with cyanosilyl silica
as mobile phase at a flow rate of 1.1 ml/min a mixture of 1.6 volumes of isoamyl alcohol R and
98.4 volumes of hexane R,
Inject 350 l of reference solution (b). Collect the eluate from 2 min before until 2 min after the
retention time of cholecalciferol, in a ground-glass-stoppered tube containing 1 ml of a 1 g/l solution
of butylhydroxytoluene R in hexane R. Repeat the procedure with test solutions (a) and (b). Evaporate
the eluates obtained from reference solution (b) and from test solutions (a) and (b), separately, to
dryness at a temperature not exceeding 30C under a gentle current of nitrogen R. Dissolve each
residue in 1.5 ml of acetonitrile R.
Determination
as mobile phase at a flow rate of 1.0 ml/min a mixture of 0.2 volumes of phosphoric acid R and
99.8 volumes of a 96 per cent V/V solution of acetonitrile R,
Inject two quantities not exceeding 200 l of each of the three solutions obtained under
Purification.
in the chromatogram obtained with reference solution (b), the resolution between the peaks
corresponding to ergocalciferol and cholecalciferol is at least 1.4,
when using the method of standard additions to test solution (a) there is greater than 95 per cent
recovery of the added cholecalciferol CRS when due consideration has been given to correction by
Calculate the content of vitamin D
3
in I.U. per gram using the following expression, taking into
account the assigned content of cholecalciferol CRS:
40
1
2
1
2
2
1
5
4
3
6
2

1
]
1
V
V
m
m
A
A
A
A
A
A
A
m
1
=mass of the substance to be examined in test solution (b) in grams,
m
2
=total mass of cholecalciferol CRS used for the preparation of reference solution (a) in
micrograms (500 g),
A
1
A
2
obtained with test solution (b),
A
3
with reference solution (b),
A
4
with test solution (b),
A
5
=area (or height) of the peak corresponding to a possible peak in the chromatogram
obtained with test solution (a) with the same retention time as ergocalciferol co-eluting
with ergocalciferol in test solution (b),
A
6
obtained with reference solution (b),
V
1
=total volume of reference solution (a) (100 ml),
V
2
=volume of reference solution (a) used for preparing reference solution (b) (2.0 ml),
STORAGE
Store preferably under an inert gas in an airtight and well-filled container, protected from light.
Once the container has been opened, its contents are used as soon as possible and any part of the
contents not used at once is protected by an atmosphere of inert gas.
11-43
LABELLING
When the addition of antioxidants is authorised by the competent authority, the name and
concentration of the added antioxidant are stated on the label.
The label states:
the number of International Units of vitamin A,
the number of International Units of vitamin D
3
.
_______________________________________________________________________ Ph Eur
11-44
Cola
1/01
Cola complies with the requirements of the 3rd edition of the European Pharmacopoeia [1504]. These require-
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cola consists of the whole or fragmented dried seeds, freed from the testa, of Cola nitida (Vent.)
Schott et Endl. (C. vera K. Schum.) and its varieties, as well as of Cola acuminata (P. Beauv.) Schott
et Endl. (Sterculia acuminata P. Beauv.). It contains not less than 1.5 per cent of caffeine (194.2),
calculated with reference to the dried drug.
CHARACTERS
IDENTIFICATION
A. The kernels have an oblong, somewhat obtuse, sub-tetragonal shape, with deformations resulting
from mutual pressure inside the fruit; they vary in size and mass, ranging from 5 g to 15 g; the
outside is hard, smooth and very dark brown, the inside is more reddish-brown. In C. nitida and its
varieties, the kernels are divided in two parts, almost plano-convex, corresponding to the cotyledons
and usually occurring separated in the commercial drug; the cotyledons are 3 cm to 4 cm long, 2 cm
to 2.5 cm wide and 1 cm to 2 cm thick. In C. acuminata, the cotyledons are smaller and divided into
four to six irregular parts.
B. Reduce to a powder (355). The powder is reddish-brown. Examine under a microscope using a
50 per cent V/V solution of glycerol R. The powder shows numerous ovoid or reniform starch
granules, 5 m to 25 m in size, with concentric striations and a stellate, slightly eccentric hilum;
fragments of cotyledon tissue showing large, thick-walled, reddish polygonal cells filled with starch
granules; occasional fragments of the external epidermis of the cotyledons.
254
plate R.
Test solution. To 1.0 g of the powdered drug (355), add 5 ml of alcohol (60 per cent V/V) R. Shake
mechanically at 40C for 30 min and filter.
Reference solution (a). Dissolve 25 mg of caffeine R in 10 ml of alcohol (60 per cent V/V) R.
Reference solution (b). Dissolve 50 mg of theobromine R in 10 ml of a mixture of 10 volumes of water R,
13 volumes of methanol R and 77 volumes of ethyl acetate R. Filter.
10 volumes of water R, 13 volumes of methanol R and 77 volumes of ethyl acetate R. Allow the plate to
dry in air for 5 min. Examine in ultraviolet light at 254 nm. The chromatogram obtained with the test
solution shows two principal quenching zones which are similar in position to the zones in the
chromatograms obtained with reference solutions (a) and (b), respectively. Spray the plates with a
mixture of equal volumes of alcohol R and hydrochloric acid R and then with a solution prepared
immediately before use by dissolving 1 g of iodine R and 1 g of potassium iodide R in 100 ml of
alcohol R. The chromatogram obtained with the test solution shows a reddish-brown principal zone
similar in position and colour to the zone in the chromatogram obtained with reference solution (a).
TESTS
ASSAY
Test solution. To 1.00 g (m
1
) of the powdered drug (355), add 50 ml of methanol R. Heat the mixture
under a reflux condenser on a water-bath for 30 min. Allow to cool and filter. Rinse the filter with
10 ml of methanol R. Take up the residue with 50 ml of methanol R. Proceed as before. Combine the
filtrates and the washings in a 200.0 ml volumetric flask and dilute to 200.0 ml with methanol R.
Transfer 20.0 ml of this solution into a round-bottomed flask and evaporate to dryness under
reduced pressure. Take up the residue with the mobile phase, transfer to a 50.0 ml volumetric flask
Reference solution. In a 100.0 ml volumetric flask, dissolve 30.0 mg (m
2
) of caffeine R and 15.0 mg of
11-45
theobromine R in the mobile phase and dilute to 100.0 ml with the mobile phase. Transfer 10.0 ml of
this solution to a 100.0 ml volumetric flask and dilute to 100.0 ml with the mobile phase.
as mobile phase at a flow rate of 1 ml/min a mixture of 25 volumes of methanol R and 75
volumes of water R,
a loop injector.
Inject appropriate volumes of each solution. The test is not valid unless: in the chromatogram
obtained with the reference solution, the resolution between the peaks corresponding respectively to
cafeine and theobromine is at least 2.5. If necessary, adjust the volume of water R in the mobile
phase.
Calculate the caffeine content using the expression:
2 1
1 2
50
A m
A m

A
1
=area of the peak due to caffeine in the chromatogram obtained with the test solution,
A
2
=area of the peak due to caffeine in the chromatogram obtained with the reference
solution,
m
1
=mass of the drug to be examined in the test solution, in grams,
m
2
=mass of caffeine R in the reference solution, in grams.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
11-46
Colchicine
MeO
MeO
MeO
OMe
O
NHAc
H
C
22
H
25
NO
6
399.4 64-86-8
Colchicine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0758]. These
Action and use Used in treatment of gout.
Preparation
Colchicine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Colchicine contains not less than 97.0 per cent and not more than the equivalent of 102.0 per cent of
(S)-N-(5,6,7,9-tetrahydro-1,2,3,10-tetramethoxy-9-oxobenzo[a]heptalen- 7-yl)acetamide, calculated
with reference to the anhydrous, ethyl acetate-free substance.
CHARACTERS
A yellowish-white, amorphous or crystalline powder, very soluble in water, rapidly recrystallising
from concentrated solutions as the sesquihydrate, freely soluble in alcohol and in chloroform.
IDENTIFICATION
A. Dissolve 5 mg in alcohol R and dilute to 100.0 ml with the same solvent. Dilute 5.0 ml of the
shows two absorption maxima, at 243 nm and 350 nm. The ratio of the absorbance measured at
243 nm to that measured at 350 nm is 1.7 to 1.9.
obtained with colchicine CRS. Examine the substances in the form of discs prepared as follows:
dissolve the substance in 0.5 ml of chloroform R, sprinkle the solution on the potassium bromide R, mix
thoroughly and evaporate the solvent by first placing in a current of air and then by heating at 80C
for 60 min.
C. To 0.5 ml of solution S (see Tests) add 0.5 ml of dilute hydrochloric acid R and 0.15 ml of ferric
chloride solution R1. The solution is yellow and becomes dark green on boiling for 30 s. Cool, add
2 ml of chloroform R and shake. The organic layer is greenish-yellow.
D. Dissolve about 30 mg in 1 ml of alcohol R and add 0.15 ml of ferric chloride solution R1. A
brownish-red colour develops.
TESTS
solution GY
3
(Method II, 2.2.2).
Acidity or alkalinity To 10 ml of solution S add 0.1 ml of bromothymol blue solution R1. Either the
solution does not change colour or it becomes green. Not more than 0.1 ml of 0.01M sodium hydroxide
is required to change the colour of the indicator to blue.
Specific optical rotation (2.2.7). Dissolve 50.0 mg in alcohol R and dilute to 10.0 ml with the same
solvent. The specific optical rotation is 235 to 250, calculated with reference to the anhydrous,
ethyl acetate-free substance.
Related substances Carry out the test protected from bright light.
Examine by thin-layer chromatography (2.2.27), using silica gel HF
254
11-47
Test solution. Dissolve 50 mg of the substance to be examined in chloroform R and dilute to 5 ml with
the same solvent.
Reference solution (a). Dilute 2 ml of the test solution to 100 ml with chloroform R.
Reference solution (b). Dilute 5 ml of reference solution (a) to 10 ml with chloroform R.
1 volume of concentrated ammonia R, 25 volumes of ethylene chloride R and 50 volumes of acetone R.
Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any spot in the chromato-
in the chromatogram obtained with reference solution (a) (2.0 per cent) and at most one such spot is
more intense than the spot in the chromatogram obtained with reference solution (b) (1.0 per cent).
Chloroform Not more than 500 ppm, determined by head-space gas chromatography
(2.2.28, Method II).
the same solvent. In three suitable identical vials, introduce 1.0 ml of this solution and stopper the
vials.
Reference solution. Dilute 5 l of chloroform R to 10.0 ml with water R. In each of three suitable
identical vials, place 10 l of this solution, add 1.0 ml of the test solution and stopper the vials.
a fused-silica column, 50 m long and 0.32 mm in internal diameter, the internal wall of which is
covered with a 5 m layer of chemically bonded polydimethylsiloxane R,
programming the temperature of the column from 80C to 200C at a rate of 10C per minute and
maintaining the temperature of the detector at 250C.
Maintain each solution at 90C for 20 min, pressurise for 30 s and transfer onto the column at a
temperature of 120C. Carry out a blank test using a vial containing 1 ml of water R. Perform each
test three times.
From the chromatograms obtained with the test solution and the reference solution, calculate the
content (per cent m/m) of chloroform taking its density (2.2.5) to be 1.48 g per millilitre at 20C.
Colchiceine Dissolve 50 mg in water R and dilute to 5 ml with the same solvent. Add 0.1 ml of ferric
chloride solution R1. The solution is not more intensely coloured than a mixture of 1 ml of red primary
solution, 2 ml of yellow primary solution and 2 ml of blue primary solution (Method II, 2.2.2) (about
0.2 per cent).
Ethyl acetate Not more than 6.0 per cent m/m of ethyl acetate, determined by gas chromatography
(2.2.28) using ethanol R as internal standard.
Internal standard solution. Dilute 1.0 ml of ethanol R to 100.0 ml with water R. Dilute 10.0 ml of this
Test solution. Dissolve 0.10 g of the substance to be examined in water R. Add 5.0 ml of the internal
Reference solution. Dissolve 1.0 ml of ethyl acetate R in water R and dilute to 100.0 ml with the same
solvent. To 1.0 ml of this solution add 5.0 ml of the internal standard solution and dilute to 10.0 ml
with water R.
a glass or stainless steel column 1.5 m long and 4 mm in internal diameter, packed with silanised
diatomaceous earth for gas chromatography R impregnated with 10 per cent m/m of macrogol
1000 R,
From the chromatograms obtained with the test solution and the reference solution, calculate the
content (per cent m/m) of ethyl acetate, taking its density (2.2.5) to be 0.901 g per millilitre at 20C.
mination of water.
ASSAY
Dissolve 0.250 g with gentle heating in a mixture of 10 ml of acetic anhydride R and 20 ml of
toluene R. Titrate with 0.1M perchloric acid determining the end-point potentiometrically (2.2.20).
22
H
25
NO
6
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
11-48
Colecalciferol
1/01
Me
Me
Me
Me
H
H
H
H
2
C
HO
H
C
27
H
44
O 384.6 67-97-0
Colecalciferol complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Cholecalciferol [0072]. These requirements are reproduced after the heading Definition below.
Action and use Used in treatment of vitamin D deficiency.
Preparations
Calcium and Colecalciferol Tablets
Colecalciferol Injection
Colecalciferol Tablets
When cholecalciferol or vitamin D
3
is prescribed or demanded, Colecalciferol shall be dispensed or
supplied. When calciferol or vitamin D is prescribed or demanded, Colecalciferol or Ergocalciferol
shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cholecalciferol contains not less than 97.0 per cent and not more than the equivalent of 103.0 per
cent of (5Z,7E)-9,10-secocholesta-5,7,10(19)-trien-3-ol.
1 milligram of cholecalciferol is equivalent to 40,000 I.U. of antirachitic activity (vitamin D) in
rats.
CHARACTERS
oils. It is sensitive to air, heat and light. Solutions in volatile solvents are unstable and are to be used
immediately.
A reversible isomerisation to pre-cholecalciferol takes place in solution, depending on temperature
and time. The activity is due to both compounds.
IDENTIFICATION
with cholecalciferol CRS. Examine the substances prepared as discs.
TESTS
Specific optical rotation (2.2.7). Dissolve 0.200 g rapidly and without heating in aldehyde-free
alcohol R and dilute to 25.0 ml with the same solvent. The specific optical rotation, determined
within 30 min of preparing the solution, is +105 to +112.
ASSAY
Carry out the operations as rapidly as possible, avoiding exposure to actinic light and air.
Test solution. Dissolve 10.0 mg of the substance to be examined without heating in 10.0 ml of
toluene R and dilute to 100.0 ml with the mobile phase.
Reference solution (a). Dissolve 10.0 mg of cholecalciferol CRS without heating in 10.0 ml of toluene R
Reference solution (b). Dilute 1.0 ml of cholecalciferol for performance test CRS to 5.0 ml with the mobile
phase. Heat in a water-bath at 90C under a reflux condenser for 45 min and cool.
a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with a suitable
silica gel (5 m),
11-49
as mobile phase at a flow rate of 2 ml/min a mixture of 3 volumes of pentanol R and 997
volumes of hexane R,
An automatic injection device or a sample loop is recommended. Inject a suitable volume of reference
solution (b). Adjust the sensitivity of the system so that the height of the principal peak is at least
50 per cent of the full scale of the recorder. Inject reference solution (b) 6 times. When the chroma-
tograms are recorded in the prescribed conditions, the approximate relative retention times with
reference to cholecalciferol are 0.4 for pre-cholecalciferol and 0.5 for trans-cholecalciferol. The
relative standard deviation of the response for cholecalciferol is not greater than 1 per cent and the
resolution between the peaks due to pre-cholecalciferol and trans-cholecalciferol is not less than 1.0.
If necessary adjust the proportions of the constituents and the flow rate of the mobile phase to obtain
this resolution.
Inject a suitable volume of reference solution (a). Adjust the sensitivity of the system so that the
height of the principal peak is at least 50 per cent of the full scale of the recorder. Inject the same
volume of the test solution and record the chromatogram in the same manner.
Calculate the percentage content of cholecalciferol from the expression:
100
D
D
S
S
m
m
m =mass of cholecalciferol CRS in reference solution (a), in milligrams,
S
D
=area (or height) of the peak due to cholecalciferol in the chromatogram obtained with the
test solution,
S
D
=area (or height) of the peak due to cholecalciferol in the chromatogram obtained with
STORAGE
Store in an airtight container, under nitrogen, protected from light, at a temperature between 2C
and 8C.
IMPURITIES
H
Me
H Me
Me
Me
H H
2
C
O
H
A. (5E,7E)-9,10-secocholesta-5,7,10(19)-trien-3-ol (trans-cholecalciferol, trans-vitamin D
3
),
H
Me
H Me
Me
Me
H
O
H
Me
H
B. cholesta-5,7-dien-3-ol (7-dehydrocholesterol, provitamin D
3
),
H
Me
H Me
Me
Me
H
O
H
Me
H
C. (9,10)-cholesta-5,7-dien-3-ol (lumisterol
3
),
Me
H Me
Me
Me
H
HO
H
Me
D. (6E)-9,10-secocholesta-5(10),6,8(14)-trien-3-ol (iso-tachysterol
3
),
11-50
H
Me
H Me
Me
Me
H
HO
H
Me
E. (6E)-9,10-secocholesta-5(10),6,8-trien-3-ol (tachysterol
3
).
__________________________________________________________________________________________________________ Ph Eur
11-51
Colecalciferol Concentrate (Oily Form)
1/01
Colecalciferol Concentrate (Oily Form) complies with the requirements of the 3rd edition of the European
Pharmacopoeia for Cholecalciferol Concentrate (Oily Form) [0575]. These requirements are reproduced after
the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cholecalciferol concentrate (oily form) consists of a solution of Cholecalciferol (0072) in a suitable
vegetable oil, authorised by the competent authority.
The content of cholecalciferol stated on the label is not less than 500,000 I.U. per gram and the
concentrate contains not less than 90.0 per cent and not more than 110.0 per cent of the content
stated on the label. The concentrate may contain suitable stabilisers such as antioxidants.
CHARACTERS
A clear, yellow liquid, practically insoluble in water, slightly soluble in ethanol, miscible with solvents
of fats.
Partial solidification may occur, depending on the temperature.
IDENTIFICATION
Test solution. Dissolve an amount of the substance to be examined corresponding to 400,000 I.U. in
ethylene chloride R containing 10 g/l of squalane R and 0.1 g/l of butylhydroxytoluene R and dilute to
4 ml with the same solvent. Prepare immediately before use.
Reference solution (a). Dissolve 10 mg of cholecalciferol CRS in ethylene chloride R containing 10 g/l of
squalane R and 0.1 g/l of butylhydroxytoluene R and dilute to 4 ml with the same solvent. Prepare
immediately before use.
Reference solution (b). Dissolve 10 mg of ergocalciferol CRS in ethylene chloride R containing 10 g/l of
Apply to the plate 20 l of each solution. Develop immediately, protected from light, over a path of
15 cm using a mixture of equal volumes of cyclohexane R and peroxide-free ether R, the mixture
containing 0.1 g/l of butylhydroxytoluene R. Allow the plate to dry in air and spray with sulphuric
acid R. Compare the principal spot in the chromatogram obtained with the test solution with the
principal spot in each of the chromatograms obtained with reference solution (a) and reference
solution (b), respectively. The chromatogram obtained with the test solution shows immediately a
bright yellow principal spot which rapidly becomes orange-brown, then gradually greenish-grey and
remains so for 10 min. This spot is similar in position, colour and size to the spot in the chromato-
gram obtained with reference solution (a). The chromatogram obtained with reference solution (b)
shows immediately at the same level an orange principal spot which gradually becomes reddish-
brown and remains so for 10 min.
B. Prepare a solution in cyclohexane R containing the equivalent of about 400 I.U. per millilitre.
267 nm.
obtained with the test solution has a similar retention time to the principal peak in the chromatogram
TESTS
ASSAY
Test solution. Dissolve a quantity of the preparation to be examined, weighed with an accuracy of
0.1 per cent, equivalent to about 400,000 I.U., in 10.0 ml of toluene R and dilute to 100.0 ml with
the mobile phase.
11-52
Reference solution (c). Dissolve 0.10 g of cholecalciferol CRS without heating in toluene R and dilute to
Reference solution (d). Dilute 5.0 ml of reference solution (c) to 50.0 ml with the mobile phase. Keep
the solution in iced water.
Reference solution (e). Place 5.0 ml of reference solution (c) in a volumetric flask, add about 10 mg of
butylhydroxytoluene R and displace air from the flask with nitrogen R. Heat in a water-bath at 90C
under a reflux condenser protected from light and under nitrogen R for 45 min. Cool and dilute to
silica gel (5 m),
as mobile phase at a flow rate of 2 ml/min a mixture of 3 volumes of pentanol R and 997 volumes
of hexane R,
An automatic injection device or a sample loop is recommended. Inject a suitable volume of
reference solution (b). Adjust the sensitivity of the system so that the height of the principal peak is at
least 50 per cent of the full scale of the recorder. Inject reference solution (b) 6 times. When the
chromatograms are recorded in the prescribed conditions, the approximate relative retention times
with reference to cholecalciferol are 0.4 for pre-cholecalciferol and 0.5 for trans-cholecalciferol. The
this resolution.
Inject a suitable volume of reference solution (d) and of reference solution (e).
Calculate the conversion factor (f) from the expression:
M
L K
f

K=area (or height) of the peak due to cholecalciferol in the chromatogram obtained with
reference solution (d),
L=area (or height) of the peak due to cholecalciferol in the chromatogram obtained with
reference solution (e),
M=area (or height) of the peak due to pre-cholecalciferol in the chromatogram obtained with
reference solution (e).
The value of f determined in duplicate on different days may be used during the entire procedure.
Calculate the content of cholecalciferol in International Units per gram from the expression:
1000 000 , 40
) (

D
p D
S
S f S
m
V
V
m
m=mass of the preparation to be examined in the test solution, in milligrams,
V=volume of the test solution (100 ml),
V =volume of reference solution (a) (100 ml),
S
D
test solution,
S
D
reference solution (a),
S
p
=area (or height) of the peak due to pre-cholecalciferol in the chromatogram obtained with
the test solution,
f =conversion factor.
STORAGE
Store in an airtight, well-filled container, protected from light. The contents of an opened container
are to be used as soon as possible; any unused part is to be protected by an atmosphere of nitrogen.
LABELLING
The label states:
11-53
the number of International Units per gram,
the method of restoring the solution if partial solidification occurs,
the name of any added stabilisers.
IMPURITIES
H
Me
H Me
Me
Me
H H
2
C
O
H
3
),
H
Me
H Me
Me
Me
H
O
H
Me
H
3
),
H
Me
H Me
Me
Me
H
O
H
Me
H
3
),
Me
H Me
Me
Me
H
HO
H
Me
3
),
H
Me
H Me
Me
Me
H
HO
H
Me
3
).
__________________________________________________________________________________________________________ Ph Eur
11-54
Colecalciferol Concentrate (Powder Form)
1/01
Colecalciferol Concentrate (Powder Form) complies with the requirements of the 3rd edition of the European
Pharmacopoeia for Cholecalciferol Concentrate (Powder Form) [0574]. These requirements are reproduced
after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cholecalciferol concentrate (powder form) is obtained by dispersing an oily solution of Cholecalciferol
(0072) in an appropriate matrix which is usually based on a combination of gelatin and carbohydrates
of suitable quality, authorised by the competent authority.
The declared content of cholecalciferol is not less than 100,000 I.U. per gram and the concentrate
contains not less than 90.0 per cent and not more than 110.0 per cent of the content stated on the
label. The concentrate may contain suitable stabilisers such as antioxidants.
CHARACTERS
White or yellowish-white, small particles which, depending on their formulation, may be practically
insoluble in water or may swell or form a dispersion.
IDENTIFICATION
Test solution. Place 10.0 ml of the test solution prepared for the assay in a suitable flask and evaporate
to dryness under reduced pressure by swirling in a water-bath at 40C. Cool under running water
and restore atmospheric pressure with nitrogen R. Dissolve the residue immediately in 0.4 ml of
ethylene chloride R containing 10 g/l of squalane R and 0.1 g/l of butylhydroxytoluene R. Prepare
solution (b), respectively. The chromatogram obtained with the test solution shows immediately a
bright-yellow principal spot which rapidly becomes orange-brown, then gradually greenish-grey and
remains so for 10 min. This spot is similar in position, colour and size to the spot in the chromato-
gram obtained with reference solution (a). The chromatogram obtained with reference solution (b)
shows immediately at the same level an orange principal spot which gradually becomes reddish-
brown and remains so for 10 min.
B. Place 5.0 ml of the test solution prepared for the assay in a suitable flask and evaporate to dryness
under reduced pressure by swirling in a water-bath at 40C. Cool under running water and restore
atmospheric pressure with nitrogen R. Dissolve the residue immediately in 50.0 ml of cyclohexane R.
265 nm.
ASSAY
Test solution. Introduce into a saponification flask a quantity of the preparation to be examined,
weighed with an accuracy of 0.1 per cent, equivalent to about 100,000 I.U. Add 5 ml of water R,
20 ml of ethanol R, 1 ml of sodium ascorbate solution R and 3 ml of a freshly prepared 50 per cent m/m
11-55
solution of potassium hydroxide R. Heat under a reflux condenser on a water-bath for 30 min. Cool
rapidly under running water. Transfer the liquid to a separating funnel with the aid of two quantities,
each of 15 ml, of water R, one quantity of 10 ml of alcohol R and two quantities, each of 50 ml, of
pentane R. Shake vigorously for 30 s. Allow to stand until the two layers are clear. Transfer the lower
aqueous-alcoholic layer to a second separating funnel and shake with a mixture of 10 ml of alcohol R
and 50 ml of pentane R. After separation, transfer the aqueous-alcoholic layer to a third separating
funnel and the pentane layer to the first separating funnel, washing the second separating funnel with
two quantities, each of 10 ml, of pentane R and adding the washings to the first separating funnel.
Shake the aqueous-alcoholic layer with 50 ml of pentane R and add the pentane layer to the first
funnel. Wash the pentane layer with two quantities, each of 50 ml, of a freshly prepared 30 g/l
solution of potassium hydroxide R in alcohol (10 per cent V/V) R, shaking vigorously, then wash with
successive quantities, each of 50 ml, of water R until the washings are neutral to phenolphthalein.
Transfer the washed pentane extract to a ground-glass-stoppered flask. Evaporate the contents of the
flask to dryness under reduced pressure by swirling in a water-bath at 40C. Cool under running
water and restore atmospheric pressure with nitrogen R. Dissolve the residue immediately in 5.0 ml of
toluene R and add 20.0 ml of the mobile phase to obtain a solution containing about 4000 I.U. per
millilitre.
Reference solution (b). Dilute 1.0 g of cholecalciferol for performance test CRS to 5.0 ml with the mobile
silica gel (5 m),
as mobile phase at a flow rate of 2 ml/min, a mixture of 3 volumes of pentanol R and 997
volumes of hexane R,
this resolution.
Calculate the conversion factor ( f ) from the expression:
M
L K
f

1000 000 , 40
) (

D
p D
S
S f S
m
V
V
m
11-56
V =volume of reference solution (a) (100 ml),
S
D
test solution,
S
D
S
p
=rea (or height) of the peak due to pre-cholecalciferol in the chromatogram obtained with
the test solution,
STORAGE
Store in an airtight, well-filled container, protected from light. The contents of an opened container
are to be used as soon as possible; any unused part is to be protected by an atmosphere of nitrogen.
LABELLING
The label states:
the name of any added stabilisers.
IMPURITIES
H
Me
H Me
Me
Me
H H
2
C
O
H
3
),
H
Me
H Me
Me
Me
H
O
H
Me
H
3
),
H
Me
H Me
Me
Me
H
O
H
Me
H
3
),
Me
H Me
Me
Me
H
HO
H
Me
3
),
H
Me
H Me
Me
Me
H
HO
H
Me
3
).
__________________________________________________________________________________________________________ Ph Eur
11-57
Colecalciferol Concentrate (Water-dispersible Form)
1/01
Colecalciferol Concentrate (Water-dispersible Form) complies with the requirements of the 3rd edition of the
European Pharmacopoeia for Cholecalciferol Concentrate (Water-dispersible Form) [0598]. These require-
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cholecalciferol concentrate (water-dispersible form) consists of a solution of Cholecalciferol (0072) in
a suitable vegetable oil, authorised by the competent authority, to which suitable solubilisers have
been added.
The declared content of cholecalciferol is not less than 100,000 I.U. per gram and the concentrate
contains not less than 90.0 per cent and not more than 115.0 per cent of the content stated on the
label. The concentrate may contain suitable stabilisers such as antioxidants.
CHARACTERS
A slightly yellowish liquid of variable opalescence and viscosity. Highly concentrated solutions may
become cloudy at low temperatures or form a gel at room temperature.
IDENTIFICATION
First identification: A, C, D.
Test solution. Place 10.0 ml of the test solution prepared for the assay in a suitable flask and evaporate
to dryness under reduced pressure by swirling in a water-bath at 40C. Cool under running water
and restore atmospheric pressure with nitrogen R. Dissolve the residue immediately in 0.4 ml of
ethylene chloride R containing 10 g/l of squalane R and 0.1 g/l of butylhydroxytoluene R. Prepare
solution (b). The chromatogram obtained with the test solution shows immediately a bright-yellow
principal spot which rapidly becomes orange-brown, then gradually greenish-grey and remains so for
10 min. This spot is similar in position, colour and size to the principal spot in the chromatogram
obtained with reference solution (a). The chromatogram obtained with reference solution (b) shows
immediately at the same level an orange principal spot which gradually becomes reddish-brown and
remains so for 10 min.
B. Place 5.0 ml of the test solution prepared for the assay in a suitable flask and evaporate to dryness
under reduced pressure by swirling in a water-bath at 40C. Cool under running water and restore
atmospheric pressure with nitrogen R. Dissolve the residue immediately in 50.0 ml of cyclohexane R.
265 nm.
D. Mix about 1 g with 10 ml of water R previously warmed to 50C and cool to 20C. Immediately
after cooling, a uniform, slightly opalescent and slightly yellow dispersion is obtained.
ASSAY
Test solution. Introduce into a saponification flask a quantity of the preparation to be examined,
weighed with an accuracy of 0.1 per cent, equivalent to about 100,000 I.U. Add 5 ml of water R,
11-58
20 ml of ethanol R, 1 ml of sodium ascorbate solution R and 3 ml of a freshly prepared 50 per cent m/m
solution of potassium hydroxide R. Heat under a reflux condenser on a water-bath for 30 min. Cool
rapidly under running water. Transfer the liquid to a separating funnel with the aid of two quantities,
each of 15 ml, of water R, one quantity of 10 ml of alcohol R and two quantities, each of 50 ml, of
pentane R. Shake vigorously for 30 s. Allow to stand until the two layers are clear. Transfer the
aqueous-alcoholic layer to a second separating funnel and shake with a mixture of 10 ml of alcohol R
and 50 ml of pentane R. After separation, transfer the aqueous-alcoholic layer to a third separating
funnel and the pentane layer to the first separating funnel, washing the second separating funnel with
two quantities, each of 10 ml, of pentane R and adding the washings to the first separating funnel.
Shake the aqueous-alcoholic layer with 50 ml of pentane R and add the pentane layer to the first
funnel. Wash the pentane layer with two quantities, each of 50 ml, of a freshly prepared 30 g/l
solution of potassium hydroxide R in alcohol (10 per cent V/V) R, shaking vigorously, and then wash
with successive quantities, each of 50 ml, of water R until the washings are neutral to
phenolphthalein. Transfer the washed pentane extract to a ground-glass-stoppered flask. Evaporate
the contents of the flask to dryness under reduced pressure by swirling in a water-bath at 40C. Cool
under running water and restore atmospheric pressure with nitrogen R. Dissolve the residue
immediately in 5.0 ml of toluene R and add 20.0 ml of the mobile phase to obtain a solution contain-
ing about 4000 I.U. per millilitre.
Reference solution (b). Dilute 1.0 g of cholecalciferol for performance test CRS to 5.0 ml with the mobile
silica gel (5 m),
of hexane R,
this resolution.
Calculate the conversion factor (f) from the expression:
M
L K
f

1000 000 , 40
) (

D
p D
S
S f S
m
V
V
m
11-59
m=mass of the preparation to be examined in the test solution, in milligrams,
V =olume of reference solution (a) (100 ml),
S
D
test solution,
S
D
S
p
=area (or height) of the peak due to pre-cholecalciferol in the chromatogram obtained with
the test solution,
STORAGE
Store in an airtight, well-filled container, protected from light, at the temperature stated on the label.
Once the container has been opened, its contents are to be used as soon as possible; any part of the
contents not used at once is to be protected by an atmosphere of inert gas.
LABELLING
The label states:
the name of the principal solubiliser or solubilisers used and the name of any added stabilisers,
the storage temperature.
IMPURITIES
H
Me
H Me
Me
Me
H H
2
C
O
H
3
),
H
Me
H Me
Me
Me
H
O
H
Me
H
3
),
H
Me
H Me
Me
Me
H
O
H
Me
H
3
),
Me
H Me
Me
Me
H
HO
H
Me
3
),
H
Me
H Me
Me
Me
H
HO
H
Me
3
).
__________________________________________________________________________________________________________ Ph Eur
11-60
Colestipol Hydrochloride
37296-80-3
Definition Colestipol Hydrochloride is a co-polymer of diethylenetriamine and 1-chloro-2,3-
epoxypropane. Each g binds not less than 1.1 mEq and not more than 1.7 mEq of sodium cholate,
determined in the test for Cholate binding capacity and calculated with reference to the dried
material.
Characteristics Yellow to orange beads; hygroscopic.
Practically insoluble in ethanol (96%) and in dichloromethane; swells but does not dissolve in water
and dilute aqueous solutions of acids and alkalis.
Identification Carry out the method for gas chromatography, Appendix III B, using a suitable gas
chromatograph fitted with a pyrolysis unit.
To prepare the sample, mix 1 part of n-eicosane and 4 parts of the substance being examined and
grind the mixture in a mortar with chloroform until the substance being examined is uniformly coated
with the n-eicosane. Prepare the standard in the same manner but adding 4 parts of colestipol hydro-
chloride BPCRS in place of the substance being examined. Load the sample and the standard
separately into the pyrolysis unit.
with acid-washed, silanised diatomaceous support (80 to 100 mesh) (Chromosorb W is suitable) coated
with 0.25% w/w of potassium hydroxide and 5% w/w of polyethylene glycol 20,000 (Carbowax 20M is
suitable) maintained at about 85 with the detector at about 270. The pyrolysis unit is capable of
attaining a temperature of about 1000 when fitted with a platinum ribbon probe and using helium as
the carrier gas with a flow rate of 60 ml per minute. Operate the unit in accordance with the
manufacturers instructions to obtain a pyrogram for colestipol hydrochloride BPCRS that is similar to
that supplied with the reference material.
The pyrogram obtained with the substance being examined is concordant with that obtained with
colestipol hydrochloride BPCRS.
Acidity or alkalinity Shake a 10% w/w suspension in a stoppered vial at approximately 10-minute
intervals for 1 hour and centrifuge. Transfer a portion of the clear supernatant liquid to a suitable
container and record the pH as soon as the reading has stabilised. The pH is 6.0 to 7.5, Appendix
V L.
Water-soluble substances Place 5 g in a glass-stoppered, 125-ml conical flask, add 80 ml of water,
close the flask and shake in a water bath at 36 to 38 for 72 hours. Filter the contents of the flask
through a fine-porosity, sintered glass funnel or woven glass-fibre filter, collecting the filtrate in a
tared 125-ml conical flask. Rinse any residual contents in the flask with two 5-ml quantities of water,
filter the washings and combine the filtrates from the washings with the filtrate obtained previously.
Evaporate the filtrate to dryness, using filtered air or nitrogen, if necessary, to aid in the evaporation.
Dry the residue at 75 at a pressure of not more than 2 kPa for 1 hour, allow to cool in a desiccator
and weigh. Repeat the procedure at the same time without the substance being examined beginning
at the words add 80 ml of water . The difference in the weights of the residues is not more than
25 mg (0.5%).
Heavy metals Transfer 1.0 g to a suitable crucible, wet with sulphuric acid and carefully ignite at a
low temperature until thoroughly charred. To the carbonised substance add 2 ml of nitric acid and
0.25 ml of sulphuric acid and heat cautiously until white fumes are no longer evolved. Ignite,
preferably in a muffle furnace, at 500 to 600, until the carbon is completely removed and cool. Add
4 ml of 6M hydrochloric acid, cover, heat on a water bath for 15 minutes, uncover and slowly evaporate
to dryness. Dissolve the residue using two 5-ml quantities of 2M hydrochloric acid. Add 0.1 ml of
phenolphthalein solution and 13.5M ammonia dropwise until a pink colour is produced. Cool, add
glacial acetic acid until the solution is decolorised and add a further 0.5 ml. Filter if necessary and
dilute the solution to 20 ml with water. 12 ml of the resulting solution complies with limit test A for
heavy metals, Appendix VII (20 ppm). Prepare the standard using a mixture of 2 ml of the test
solution obtained above and 10 ml of lead standard solution (2 ppm Pb).
Loss on drying When dried at 75 at a pressure of not more than 2 kPa for 16 hours, loses not more
than 1.0% of its weight.
Chloride content Not less than 6.5% and not more than 9.0% calculated with reference to the dried
substance. Burn 20 mg by the method for oxygen-flask combustion, Appendix VIII C, using 10 ml of
0.05M sodium hydroxide as the absorbing liquid. When the process is complete shake the flask
vigorously, allow to stand with frequent shaking for about 40 minutes or until no cloudiness is
observed; add 20 ml of ethanol (96%) and 0.2 ml of nitric acid. Titrate the resulting solution with
0.05M silver nitrate VS, determining the end point potentiometrically using a silversilver chloride
electrode and a glass reference electrode (V ml). Repeat the procedure without the substance being
11-61
examined adding 10 ml of 0.0075M sodium chloride to the solution in the flask (V
1
ml). Add 10 ml of
0.0075M sodium chloride to a flask containing a mixture of 10 ml of water and 20 ml of ethanol (96%),
add 0.2 ml of nitric acid and titrate with 0.05M silver nitrate VS, determining the end point potentio-
metrically using a silversilver chloride electrode and a glass reference electrode (V
2
ml). Determine
the volume of 0.05M silver nitrate VS required by the substance being examined using the following
expression:
V (V
1
V
2
)
Each ml of 0.05M silver nitrate VS is equivalent to 1.773 mg of Cl.
Water absorption capacity Each g of Colestipol Hydrochloride absorbs not less than 3.3 g and not
more than 5.3 g of water when determined in the following manner. Transfer 5 g to a dry, plastic
container and add 80 g of water. Cover the container and allow the resulting suspension to equilibrate
for 72 hours. Filter the resulting slurry through a medium-porosity fritted-glass funnel (KIMAX 60-
ml-40M is suitable) at a pressure of 2kPa; collect the filtrate in a tared, plastic container,
disconnecting the vacuum 2 minutes after collection of the last portion of the filtrate. Immediately
weigh the container and the filtrate and determine the weight, in g, of the filtrate. Calculate the
weight of water absorbed per g from the difference between the weight of the filtrate and the original
weight of water used in the test.
Cholate binding capacity Prepare a solution containing 1.0% w/v of sodium cholate and 0.9% w/v
of sodium chloride and adjust to pH 6.4 by the dropwise addition of hydrochloric acid (solution A).
Transfer 1 g (m g) of the substance being examined to a ground-glass-stoppered flask, add 100 ml of
freshly prepared solution A and shake vigorously for 90 minutes with the flask positioned
horizontally. Remove the flask from the shaker and allow the contents to settle for 5 minutes. Adjust
the pH of a 20-ml aliquot of the supernatant liquid to 10.5 by the dropwise addition of 1M sodium
hydroxide and titrate potentiometrically with 0.1M hydrochloric acid VS to the second inflection point
of the pH curve. Determine the volume of titrant added between the inflection points. Carry out a
blank titration on 20 ml of freshly prepared solution A. The difference between the titrations
represents the amount of hydrochloric acid required (V ml). Calculate the cholate binding capacity of
the substance being examined in milliequivalents from the expression 0.5V/m.
Colestipol exchange capacity limit Not less than 9.0 mEq per g and not more than
11.0 mEq per g determined in the following manner. Transfer not less than 2 g and 100 ml of 1M
sodium hydroxide to a stoppered flask and shake for 4 hours. Filter the suspension through a coarse-
porosity sintered-glass funnel and wash the resin with 500 ml of water. Transfer the resin to a 1000-
ml beaker, add 200 ml of water and allow to stand for 10 minutes. Filter the suspension, check the
pH of the filtrate and repeat the washing procedure with 200-ml quantities of water until the pH of
the filtrate is less than 8 [5 litres may be required]. Dry the resin and funnel at 60 at a pressure of
2 kPa for at least 16 hours, breaking up any aggregates with a spatula and store in a dessicator.
Place 1 g (w g) of the free base resin prepared above and add 100 ml of 0.2M hydrochloric acid VS in
a stoppered flask and shake for not less than 2.5 hours. Filter a portion of the suspension through
glass wool. Titrate 8 ml of the filtrate with 0.2M sodium hydroxide VS, determining the end point
potentiometrically (a ml). Carry out a blank titration on 5 ml of the 0.2M hydrochloric acid VS used to
equilibrate the free-base resin diluted with 5 ml of water (b ml). Calculate the exchange capacity in
milliequivalents per g from the expression:
(20/w)(b/5a/8)
Storage Colestipol Hydrochloride should be kept in an airtight container.
Preparation
Colestipol Granules
11-62
Colestyramine
11041-12-6
Definition Colestyramine is a strongly basic anion-exchange resin in the chloride form, consisting
of styrene-divinylbenzene co-polymer with quaternary ammonium function groups. Each g
exchanges not less than 1.8 g and not more than 2.2 g of sodium glycocholate, calculated with
reference to the dried material.
Characteristics A white to buff-coloured, fine powder; hygroscopic.
Insoluble in water, in ethanol (96%), in chloroform and in ether.
spectrum of colestyramine (RS 369). Use the residue obtained in the test for Loss on drying.
Acidity Prepare a 1% w/v suspension and allow to stand for 10 minutes. The pH of the suspension is
4.0 to 6.0, Appendix V L.
Dialysable quaternary amines Place a 25-cm piece of cellulose dialysis tubing having a molecular
weight cut-off of 12,000 to 14,000 and an inflated diameter of 3 to 6 cm (flat width of 5 to 9 cm)
(Visking dialysis tubing is suitable) in water to hydrate until pliable, appropriately sealing one end.
For solution (1) introduce 2.0 g of the substance being examined into the tube and add 10 ml of
water. Seal the tube and completely immerse it in 100 ml of water in a suitable vessel and stir the
liquid for 16 hours to effect dialysis. Prepare solution (2) in a similar manner but using 10 ml of a
freshly prepared solution containing 0.010% w/v of benzyltrimethylammonium chloride.
Pipette 5.0 ml of solution (1) into a separating funnel and add 5 ml of a 0.38% w/v solution of
sodium tetraborate, 1 ml of a solution containing 0.150% w/v of bromothymol blue and 0.405% w/v of
sodium carbonate and 10 ml of chloroform. Shake the mixture vigorously for 1 minute, allow to separate
and transfer the clear organic layer to a 25-ml graduated flask. Repeat the extraction with a further
10 ml of chloroform and add sufficient chloroform to produce 25 ml. Measure the absorbance of the
resulting solution at the maximum at 420 nm, Appendix II B, using in the reference cell a solution
prepared in the same manner but using 5.0 ml of water and beginning at the words add 5 ml. The
absorbance obtained is not greater than that obtained by repeating the operation using 5.0 ml of
solution (2) (500 ppm).
Styrene Carry out the method for liquid chromatography, Appendix III D, using the following solu-
tions. For solution (1) shake 5 g of the substance being examined with 10 ml of acetone for 30
minutes, centrifuge and use the supernatant liquid. Solution (2) contains 0.00005% w/v of styrene in
acetone.
mixture of equal volumes of acetonitrile and water as the mobile phase with a flow rate of 2 ml per
minute and (c) a detection wavelength of 254 nm.
Inject separately 20 l of each solution. The area of the peak corresponding to styrene in the
chromatogram obtained with solution (1) is not greater than the area of the principal peak in the
chromatogram obtained with solution (2) (1 ppm).
Chloride content 13.0% to 17.0%, calculated with reference to the dried substance. To 0.2 g add
100 ml of water and 50 mg of potassium nitrate. Add, with stirring, 2 ml of nitric acid and titrate with
0.1M silver nitrate VS determining the end point potentiometrically. Each ml of 0.1M silver nitrate VS
is equivalent to 3.545 mg of Cl.
Exchange capacity Carry out the method for liquid chromatography, Appendix III D, using the
following solutions. Prepare a solution of sodium glycocholate by dissolving 1.50 g in sufficient of a
solution containing 0.4% w/v of potassium dihydrogen orthophosphate and 1.2% w/v of dipotassium
hydrogen orthophosphate to produce 100 ml (solution A). For solution (1), dilute 4 ml of solution A to
100 ml with water. For solution (2) add 20 ml of solution A to a quantity of the substance being
examined equivalent to about 0.1 g of anhydrous colestyramine, shake mechanically for 2 hours and
then centrifuge for 15 minutes. Dilute 5 ml of the supernatant liquid to 50 ml with water. Prepare
solution (3) in the same manner as solution (2) but using 0.1 g of colestyramine BPCRS, previously
dried at 70 at a pressure not exceeding 7 kPa for 16 hours, in place of the substance being
examined. Solution (4) contains 0.06% w/v of sodium glycocholate and 0.03% w/v of taurodeoxycholic
acid sodium salt in water.
cm 4.6 mm) containing stationary phase C (5 m), (b) as the mobile phase with a flow rate of
1.5 ml per minute a mixture of 35 volumes of acetonitrile and 65 volumes of 0.08M potassium
dihydrogen orthophosphate, the mixture adjusted to pH 3.0 with orthophosphoric acid and (c) a detection
wavelength of 214 nm.
11-63
Inject about 50 l of each solution. The test is not valid unless, in the chromatogram obtained with
solution (4), the resolution factor between the two principal peaks is at least 1.5.
Determine the area of the peak corresponding to sodium glycocholate in the chromatograms
obtained with each of solutions (1), (2) and (3) and calculate the exchange capacity in g of sodium
glycocholate per g of resin using the following expression:
t 3 1
s 2 1
) 5 . 2 (
) 5 . 2 (
W A A
W A A M
where M=stated value, in mg, of sodium glycocholate absorbed per g of colestyramine BPCRS,
A
1
, A
2
and A
3
=the areas of the peaks corresponding to sodium glycocholate in the chromatograms
obtained with solutions (1), (2) and (3), respectively,
W
s
=weight, in mg, of colestyramine BPCRS used in the preparation of solution (3),
W
t
=weight of the substance being examined used in the preparation of solution (2).
Loss on drying When dried at 70 at a pressure not exceeding 7 kPa for 16 hours, loses not more
than 12% of its weight. Use 1 g.
Storage Colestyramine should be kept in an airtight container.
Preparation
Colestyramine Oral Powder
When cholestyramine is prescribed or demanded, Colestyramine shall be dispensed or supplied.
IMPURITIES
A. Dialysable Quaternary Amines
CH
2
B. Styrene
11-64
Colistimethate Sodium
R CO-Dbu-Thr-Dbu-Dbu-Dbu-DLeu-Leu-DbuDbu-Thr
Dbu = L- 2,4-diaminobutyric acid
R = CH
2
SO
3
Na
N
4
N
4
R N
4
R N
4
R N
4
R N
4
R
R
Colistin A [CH
2
]
4
CHMeCH
2
CH
3
Colistin B [CH
2
]
4
CHMe
2
8068-28-8
Colistimethate Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Colistimethate Injection
When colistin sulphomethate sodium is prescribed or demanded, Colistimethate Sodium shall be
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Colistimethate sodium is prepared from colistin by the action of formaldehyde and sodium hydrogen
sulphite. The potency is not less than 11,500 I.U. per milligram, calculated with reference to the
dried substance.
CHARACTERS
A white or almost white powder, hygroscopic, very soluble in water, slightly soluble in alcohol,
practically insoluble in acetone and in ether.
IDENTIFICATION
hydrochloric acid R and water R. Heat at 135C in a sealed tube for 5 h. Evaporate to dryness on a
water-bath and continue the heating until the odour of hydrochloric acid has been completely
removed. Dissolve the residue in 0.5 ml of water R.
Reference solution (a). Dissolve 20 mg of leucine R in water R and dilute to 10 ml with the same
solvent.
Reference solution (b). Dissolve 20 mg of threonine R in water R and dilute to 10 ml with the same
solvent.
Reference solution (c). Dissolve 20 mg of phenylalanine R in water R and dilute to 10 ml with the same
solvent.
Reference solution (d). Dissolve 20 mg of serine R in water R and dilute to 10 ml with the same solvent.
Carry out the following procedures protected from light.
Apply separately to the plate as 10 mm bands 5 l of each solution. Place the plate in the chromato-
graphic tank so that it is not in contact with the mobile phase consisting of a mixture of 25 parts of
water R and 75 parts of phenol R. Leave the plate to become impregnated with the vapour of the
solvent for at least 12 h. Develop over a path of 12 cm using the same mobile phase. Dry the plate at
100C to 105C and spray with ninhydrin solution R1. Heat at 110C for 5 min. The chromatogram
obtained with the test solution shows zones corresponding to those in the chromatograms obtained
with reference solutions (a) and (b), but shows no zones corresponding to those in the chromato-
grams obtained with reference solutions (c) and (d). The chromatogram obtained with the test
solution also shows a zone with a very low R
f
value (2,4-diaminobutyric acid).
B. Dissolve about 5 mg in 3 ml of water R. Add 3 ml of dilute sodium hydroxide solution R. Shake and
add 0.5 ml of a 10 g/l solution of copper sulphate R. A violet colour is produced.
11-65
C. Dissolve about 50 mg in 1 ml of 1M hydrochloric acid and add 0.5 ml of 0.01M iodine. The solution
is decolourised and gives reaction (a) of sulphates (2.3.1).
D. It gives reaction (b) of sodium (2.3.1).
TESTS
Appearance of solution Dissolve 0.16 g in 10 ml of water R. The solution is clear (2.2.1).
solvent. The specific optical rotation is 46 to 51, calculated with reference to the dried substance.
Free colistin Dissolve 80 mg in 3 ml of water R. Add 0.1 ml of a 100 g/l solution of silicotungstic
acid R; 10 s to 20 s after addition of the reagent, the solution is not more opalescent than reference
suspension II (2.2.1).
Total sulphite Work in a fume cupboard. Dissolve 0.100 g in 50 ml of water R and add 5 ml of a
100 g/l solution of sodium hydroxide R and 0.3 g of potassium cyanide R. Boil gently for 3 min and then
cool. Neutralise with 0.5M sulphuric acid using 0.2 ml of methyl orange solution R as indicator. Add an
excess of 0.5 ml of the acid and 0.2 g of potassium iodide R. Titrate with 0.05M iodine using 1 ml of
starch solution R as indicator. The volume of 0.05M iodine used in the titration is 5.5 ml to 7.0 ml.
Loss on drying (2.2.32). Not more than 1.5 per cent, determined on 1.000 g by drying at 60C over
diphosphorus pentoxide R at a pressure not exceeding 670 Pa for 3 h.
Sulphated ash (2.4.14). 16 per cent to 21 per cent, determined on 0.50 g.
Inject per kilogram of the rabbits mass 1 ml of a solution in water for injections R containing 2.5 mg of
ASSAY
Carry out the microbiological assay of antibiotics (2.7.2).
STORAGE
LABELLING
The label states:
where applicable, that the substance is free from pyrogens.
__________________________________________________________________________________________________________ Ph Eur
11-66
Colistin Sulphate
R CO-Dbu-Thr-Dbu-Dbu-Dbu-DLeu-Leu-Dbu-Dbu-Thr
Dbu = L- 2,4-diaminobutyric acid
,2.5H
2
SO
4
N
4
R
Colistin A [CH
2
]
4
CHMeCH
2
CH
3
Colistin B [CH
2
]
4
CHMe
2
1264-72-8
Colistin Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0320].
Preparation
Colistin Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Colistin sulphate is a mixture of the sulphates of polypeptides elaborated by certain strains of Bacillus
polymyxa var. colistinus or obtained by any other means. The potency is not less than 19,000 I.U. per
CHARACTERS
A white or almost white powder, hygroscopic, freely soluble in water, slightly soluble in alcohol,
practically insoluble in acetone and in ether.
IDENTIFICATION
hydrochloric acid R and water R. Heat at 135C in a sealed tube for 5 h. Evaporate to dryness on a
water-bath and continue the heating until the odour of hydrochloric acid has been completely
removed. Dissolve the residue in 0.5 ml of water R.
Reference solution (a). Dissolve 20 mg of leucine R in water R and dilute to 10 ml with the same
solvent.
Reference solution (b). Dissolve 20 mg of threonine R in water R and dilute to 10 ml with the same
solvent.
Reference solution (c). Dissolve 20 mg of phenylalanine R in water R and dilute to 10 ml with the same
solvent.
Reference solution (d). Dissolve 20 mg of serine R in water R and dilute to 10 ml with the same solvent.
Carry out the following procedures protected from light.
Apply separately to the plate as 10 mm bands 5 l of each solution. Place the plate in the chromato-
graphic tank so that it is not in contact with the mobile phase consisting of a mixture of 25 parts of
water R and 75 parts of phenol R. Leave the plate to become impregnated with the vapour of the
mobile phase for at least 12 h. Develop over a path of 12 cm using the same mobile phase. Dry the
plate at 100C to 105C and spray with ninhydrin solution R1. Heat at 110C for 5 min. The chroma-
togram obtained with the test solution shows zones corresponding to those in the chromatograms
obtained with reference solutions (a) and (b), but shows no zones corresponding to those in the
chromatograms obtained with reference solutions (c) and (d). The chromatogram obtained with the
test solution also shows a zone with a very low R
f
value (2,4-diaminobutyric acid).
B. Dissolve about 5 mg in 3 ml of water R. Add 3 ml of dilute sodium hydroxide solution R. Shake and
add 0.5 ml of a 10 g/l solution of copper sulphate R. A violet colour is produced.
C. Dissolve about 50 mg in 1 ml of 1M hydrochloric acid and add 0.5 ml of 0.01M iodine. The solution
remains coloured.
11-67
TESTS
Sulphate 16.0 per cent to 18.0 per cent of sulphate (SO
4
0. 1M sodium edetate, adding 50 ml of alcohol R when the colour of the solution begins to change and
continuing the titration until the violet-blue colour disappears.
4
).
ASSAY
STORAGE
__________________________________________________________________________________________________________ Ph Eur
11-68
Colophony
Definition Colophony is the residue left after distilling the volatile oil from the oleo-resin obtained
from various species of Pinus.
Characteristics Translucent, pale yellow or brownish yellow, angular, brittle, readily fusible, glassy
masses; odour, faintly terebinthinate.
Practically insoluble in water; soluble in ethanol (96%), in carbon disulphide and in ether; partially
soluble in petroleum spirit (boiling range, 40 to 60).
Identification Dissolve 0.1 g in 10 ml of acetic anhydride with the aid of gentle heat, cool and add
0.05 ml of sulphuric acid. A bright purplish red colour, rapidly changing to violet, is produced.
Acid value 150 to 180, Appendix X B, using 1 g and 50 ml of ethanol (96%) as the solvent.
11-69
Copovidone
corrected 1/01
O O
CH
3
H
H CH
2
CH
O
N
m n
n = 1.2m
(C
6
H
9
NO)
n
+ (C
4
H
6
O
2
)
m
(111.1)
n
+ (86.1)
m
Copovidone complies with the requirements of the 3rd edition of the European Pharmacopoeia [0891]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Copovidone is a copolymer of 1-vinylpyrrolidin-2-one and vinyl acetate in the mass proportion 3:2. It
contains not less than 7.0 and not more than 8.0 per cent of nitrogen, and not less than 35.3 per cent
and not more than 42.0 per cent of the vinyl acetate component, both calculated with reference to
the dried substance. The K-value is not less than 90.0 per cent and not more than 110.0 per cent of
the value stated on the label.
CHARACTERS
A white or yellowish-white powder or flakes, hygroscopic, freely soluble in water, in alcohol and in
IDENTIFICATION
spectrum of copovidone.
B. To 1 ml of solution S (see Tests) add 5 ml of water R and 0.2 ml of 0.05M iodine. A red colour
appears.
C. Dissolve 0.7 g of hydroxylamine hydrochloride R in 10 ml of methanol R, add 20 ml of a 40 g/l
solution of sodium hydroxide R and filter if necessary. To 5 ml of the solution add 0.1 g of the
substance to be examined and boil for 2 min. Transfer 50 l to a filter paper and add 0.1 ml of a
mixture of equal volumes of ferric chloride solution R1 and hydrochloric acid R. A violet colour appears.
TESTS
Solution S Dissolve 10 g in water R and dilute to 100 ml with the same solvent. Add the substance
to be examined to the water R in small portions with constant stirring.
Appearance of solution Solution S is not more opalescent than reference suspension III (2.2.1)
and not more intensely coloured than reference solution B
5
, R
5
or BY
5
(Method II, 2.2.2).
Aldehydes To 20.0 g in a ground-glass-stoppered flask add 180 ml of water R. Boil under a reflux
condenser for 45 min. Allow to cool. Distil and collect about 60 ml of distillate in 20.0 ml of a 70 g/l
solution of hydroxylamine hydrochloride R, previously adjusted to pH 3.1 using 1M sodium hydroxide
and cooled in iced water. Titrate with 0.1M sodium hydroxide. Not more than 9.1 ml of 0.1M sodium
hydroxide is required to attain pH 3.1 (0.2 per cent expressed as acetaldehyde). Carry out a blank
determination.
Peroxides Dilute 10 ml of solution S to 25 ml with water R. Add 2 ml of titanium trichloride-
sulphuric acid reagent R and allow to stand for 30 min. The absorbance (2.2.25) of the solution,
measured at 405 nm using a mixture of 25 ml of a 40 g/l solution of the substance to be examined
and 2 ml of a 13 per cent V/V solution of sulphuric acid R as the compensation liquid, is not greater
than 0.35 (400 ppm, expressed as H
2
O
2
).
Hydrazine Examine by thin-layer chromatography (2.2.27), using a TLC silanised silica gel plate R.
Use freshly prepared solutions.
11-70
Test solution. To 25 ml of solution S add 0.5 ml of a 50 g/l solution of salicylaldehyde R in methanol R,
mix and heat in a water-bath at 60C for 15 min. Allow to cool, add 2.0 ml of xylene R, shake for
2 min and centrifuge. Use the clear supernatant layer.
Reference solution. Dissolve 9 mg of salicylaldehyde azine R in xylene R and dilute to 100 ml with the
same solvent. Dilute 1 ml of this solution to 10 ml with xylene R.
volumes of water R and 80 volumes of methanol R. Allow the plate to dry in air and examine in
ultraviolet light at 365 nm. Any spot corresponding to salicylaldehyde azine in the chromatogram
obtained with the test solution is not more intense than the spot in the chromatogram obtained with
the reference solution (1 ppm of hydrazine).
Monomers Dissolve 5.0 g in 15 ml of methanol R and add slowly 20.0 ml of iodine bromide
solution R. Allow to stand for 30 min protected from light with repeated shaking. Add 10 ml of a
100 g/l solution of potassium iodide R and titrate with 0.1M sodium thiosulphate until a yellow colour is
obtained. Continue the titration dropwise until the solution becomes colourless. Carry out a blank
titration. Not more than 3.6 ml of 0.1M sodium thiosulphate is used (0.4 per cent).
100C to 105C.
Viscosity, expressed as K-value Dilute 5.0 ml of solution S to 50.0 ml with water R. Allow to
stand for 1 h and determine the viscosity (2.2.9) of the solution at 25 0.1C using viscometer No. 1
with a minimum flow time of 100 s. Calculate the K-value from the expression:
2
2
003 0 15 0
5 1 ( 300
003 0 15 0
1 5 1
c . c .
log c . c log c
c . .
log .
+
) + +
+
+
c =percentage concentration (g/100 ml) of the substance to be examined, calculated with

reference to the dried substance,
=viscosity of the solution relative to that of water.
ASSAY
Nitrogen Carry out the determination of nitrogen (2.5.9) using 30.0 mg of the substance to be
examined and 1 g of a mixture of 3 parts of copper sulphate R and 997 parts of dipotassium sulphate R,
heating until a clear, light-green solution is obtained and then for a further 45 min.
Vinyl acetate component Determine the saponification value (2.5.6) on 2.00 g of the substance to
be examined. Multiply the result obtained by 0.1534 to obtain the percentage content of the vinyl
acetate component.
STORAGE
Store protected from moisture.
LABELLING
The label states the K-value.
_______________________________________________________________________ Ph Eur
11-71
Anhydrous Copper Sulphate
CuSO
4
159.6 7758-98-7
Anhydrous Copper Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Used in treatment of copper deficiency.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Anhydrous copper sulphate contains not less than 99.0 per cent and not more than the equivalent
of 101.0 per cent of CuSO
4
CHARACTERS
A greenish-grey powder, very hygroscopic, freely soluble in water, slightly soluble in methanol,
IDENTIFICATION
A. Add several drops of dilute ammonia R2 to 1 ml of solution S (see Tests). A blue precipitate is
formed on further addition of dilute ammonia R2, the precipitate dissolves and a dark blue colour is
produced.
C. Dilute 1 ml of solution S to 5 ml with water R. The solution gives reaction (a) of sulphates (2.3.1).
TESTS
limit test for chlorides (150 ppm). Examine the tubes laterally against a black background.
Iron Not more than 150 ppm of Fe, determined by atomic absorption spectrometry (Method I,
2.2.23).
Test solution. Dissolve 0.32 g in 10 ml of water R, add 2.5 ml of lead-free nitric acid R and dilute to
Reference solutions. Prepare the reference solutions using iron standard solution (20 ppm Fe) R, adding
2.5 ml of lead-free nitric acid R and diluting to 25.0 ml with water R.
an air-butane flame.
2.2.23).
Reference solutions. Prepare the reference solutions using lead standard solution (100 ppm Pb) R, adding
Measure the absorbance at 217.0 nm using a lead hollow-cathode lamp as a source of radiation and
250C.
ASSAY
Dissolve 0.125 g in 50 ml of water R. Add 2 ml of sulphuric acid R and 3 g of potassium iodide R.
Titrate with 0.1M sodium thiosulphate, using 1 ml of starch solution R, added towards the end of the
titration, as indicator.
1 ml of 0.1M sodium thiosulphate is equivalent to 15.96 mg of CuSO
4
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
11-72
Copper Sulphate Pentahydrate
Copper Sulphate
CuSO
4
,5H
2
O 249.7 7758-99-8
Copper Sulphate Pentahydrate complies with the requirements of the 3rd edition of the European
Action and use Used in treatment of copper deficiency.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Copper sulphate pentahydrate contains not less than 99.0 per cent and not more than the equivalent
of 101.0 per cent of CuSO
4
,5H
2
O.
CHARACTERS
A blue, crystalline powder or transparent, blue crystals, freely soluble in water, soluble in methanol,
IDENTIFICATION
A. Add several drops of dilute ammonia R2 to 1 ml of solution S (see Tests). A blue precipitate is
formed on further addition of dilute ammonia R2, the precipitate dissolves and a dark blue colour is
produced.
C. Dilute 1 ml of solution S to 5 ml with water R. The solution gives reaction (a) of sulphates (2.3.1).
TESTS
Solution S Dissolve 5 g in water R and dilute to 100 ml with the same solvent.
limit test for chlorides (100 ppm). Examine the tubes laterally against a black background.
Iron Not more than 100 ppm of Fe, determined by atomic absorption spectrometry (Method I,
2.2.23).
Reference solutions. Prepare the reference solutions using iron standard solution (20 ppm Fe) R, adding
2.2.23).
Reference solutions. Prepare the reference solutions using lead standard solution (100 ppm Pb) R, adding
Measure the absorbance at 217.0 nm using a lead hollow-cathode lamp as a source of radiation and
at 250C.
ASSAY
Dissolve 0.200 g in 50 ml of water R. Add 2 ml of sulphuric acid R and 3 g of potassium iodide R.
Titrate with 0.1M sodium thiosulphate, adding 1 ml of starch solution R towards the end of the titration.
1 ml of 0.1M sodium thiosulphate is equivalent to 24.97 mg of CuSO
4
,5H
2
O.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
11-73
Coriander
Coriander complies with the requirements of the 3rd edition of the European Pharmacopoeia [1304]. These
When Powdered Coriander is prescribed or demanded, material complying with the appropriate
requirements below but containing not less than 0.2% v/w of essential oil shall be dispensed or
supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Coriander consists of the dried cremocarp of Coriandrum sativum L. It contains not less than 3 ml/kg
of essential oil, calculated with reference to the dried drug.
CHARACTERS
The cremocarp is brown or light brown and is more or less spherical, about 1.5 mm to 5 mm in
diameter, or oval from 2 mm to 6 mm long.
IDENTIFICATION
A. The mericarps are usually tightly connected. The cremocarp is glabrous and has ten wavy, slightly
raised primary ridges and eight straight, more prominent secondary ridges. The stylopod crowns the
apex. The mericarps are concave on the internal surface. A small fragment of the pedicel may be
present.
B. Reduce to a powder (355). The powder is brown. Examine under a microscope using chloral
hydrate solution R. The powder shows numerous oil droplets; fragments of endosperm with small
thick-walled regular cells containing microcrystals and microrosettes of calcium oxalate and oil
droplets; fragments of endocarp with very narrow cells having a parquetry arrangement and usually
associated with a layer of thin-walled rectangular sclereids of the mesocarp; fragments from the
sclerenchymatous layer of the mesocarp with short, strongly thickened, pitted, fusiform cells
occurring in layers with the cells of adjacent layers approximately at right angles to one another;
fragments of parenchyma with small, thick-walled cells; occasional fragments of vascular bundles.
substance.
Test solution. Shake 0.50 g of the freshly powdered drug (355) with 5.0 ml of hexane R for 2 min to
3 min and filter over 2 g of anhydrous sodium sulphate R.
Reference solution. Dissolve 15 l of linalol R and 25 l of olive oil R in 5.0 ml of hexane R immediately
before use.
a path of 10 cm using a mixture of 5 volumes of ethyl acetate R and 95 volumes of toluene R. Allow
the plate to dry in air and develop again in the same conditions. Allow the plate to dry in air. Spray
the plate with anisaldehyde solution R and examine in daylight while heating at 100C to 105C for
5 min to 10 min. The chromatogram obtained with the reference solution shows in the lower half a
violet to greyish-violet zone (linalol) and in the upper half a bluish-violet zone (triglycerides). The
chromatogram obtained with the test solution shows zones similar in position and colour to the zones
in the chromatogram obtained with the reference solution. Several violet-grey to brownish zones,
including the zone corresponding to geraniol, are between the starting point and the zone due to
linalol in the chromatogram obtained with the reference solution. It may also show several faint
violet-grey zones between the zone due to triglycerides and that due to linalol in the chromatogram
TESTS
Foreign matter (2.8.2). It complies with the test. None of the cremocarps show perforations due to
animals.
ASSAY
tube. Reduce the drug to a coarse powder and immediately use 30.0 g for the determination. Distil at
a rate of 2 ml/min to 3 ml/min for 2 h.
11-74
STORAGE
_______________________________________________________________________ Ph Eur
11-75
Coriander Oil
Definition Coriander Oil is obtained by distillation from Coriander. It contains not less than
65.0% and not more than 75.0% w/w of linalool, C
10
H
18
0.
Characteristics A clear, colourless or pale yellow liquid, visibly free from water; odour and taste,
those of coriander.
Assay Carry out the method for gas chromatography, Appendix III B, using solutions in ethanol (96%)
containing (1) 0.20% w/v of linalool and 0.15% w/v of geraniol (internal standard), (2) 0.20% w/v of
the oil being examined and (3) 0.20% w/v of the oil being examined and 0.15% w/v of the internal
standard.
with acid-washed diatomaceous support (100 to 200 mesh) coated with 3% w/w of dimethyl silicone
fluid (OV-101 is suitable) and maintained at 70 for 12 minutes, then increased to 105 at a rate of
6 per minute and maintained at 105 for not less than 6 minutes with the inlet port at 220, the
detector at 300 and a flow rate of 40 ml per minute for the carrier gas.
Calculate the content of linalool in the oil being examined using the ratios of the area of the peak
corresponding to linalool to the area of the peak due to the internal standard in the chromatograms
obtained with solutions (1) and (3).
Storage Coriander Oil should be kept in a well-filled, well-closed container, protected from light and
11-76
Corticotropin
corrected 1/01
Corticotropin complies with the requirements of the 3rd edition of the European Pharmacopoeia [0759]. These
Action and use Corticotrophic hormone.
When corticotrophin is prescribed or demanded, Corticotropin shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Corticotropin is a substance obtained from the anterior lobe of the pituitary gland of pigs and
contains the corticotrophic peptide which increases the rate at which corticoid hormones are secreted
by the adrenal glands. The purified material is dried by a suitable method. The potency is not less
than 70 I.U./mg.
PRODUCTION
It is prepared under conditions designed to minimise the degree of microbial contamination.
CHARACTERS
White or almost white powder or flakes, hygroscopic. A 10 g/l solution is clear or not more than
slightly opalescent.
IDENTIFICATION
A. It depresses the concentration of ascorbic acid in the adrenal glands, when injected as prescribed
in potency (Method A or Method B).
Test solution. Dissolve 1 mg of the substance to be examined in 1.0 ml of 0.05M hydrochloric acid.
Reference solution. Dissolve 1 mg of corticotropin CRS in 1.0 ml of 0.05M hydrochloric acid.
as mobile phases at a flow rate of 1 ml/min:
Mobile phase A. A 15.6 g/l solution of sodium dihydrogen phosphate R, adjusted to pH 2.0 with
phosphoric acid R,
Equilibrate the column with a mixture of 20 volumes of mobile phase B and 80 volumes of mobile
phase A.
Record the chromatogram eluting with a mobile phase consisting of a mixture of 20 volumes of
mobile phase B and 80 volumes of mobile phase A for 5 min; then operate by gradient elution
increasing continuously and linearly the proportion of mobile phase B to 40 volumes by 0.33 per cent
V/V per minute for 60 min. Then finally elute using a mixture of 20 volumes of mobile phase B and
80 volumes of mobile phase A for 15 min.
Inject separately 100 l of each solution. The retention time of the principal peak in the chromato-
gram obtained with the test solution is similar to that of the principal peak in the chromatogram
obtained with the reference solution. The test is not valid unless, in the chromatogram obtained with
the reference solution: the symmetry factor of the principal peak is 0.8 to 2.0; the number of
theoretical plates calculated using the principal peak is at least 1000.
C. Examine the electrophoretograms obtained in the test for related proteins. The principal band in
the electrophoretogram obtained with the test solution corresponds in position to the principal band
in the electrophoretogram obtained with reference solution (a).
TESTS
pH (2.2.3). The pH of a 10 g/l solution is 3.0 to 5.0.
Impurities with greater molecular mass than corticotropin Examine by size-exclusion chroma-
tography (2.2.30).
Test solution. Dissolve 1 mg of the substance to be examined in 1 ml of acetic acid (60 g/l C
2
H
4
O
2
)
containing 10 g/l of sodium dodecyl sulphate R. Heat the solution at 100C for 10 min. Allow to cool.
a column about 0.85 m long and 10 mm in internal diameter packed with polyacrylamide or
11-77
cross-linked dextran for chromatography having a fractionation range for peptides with
relative molecular masses of approximately 1000 to 10,000,
as mobile phase at a flow rate of 7 ml per hour, acetic acid (60 g/l C
2
H
4
O
2
)
as detector a spectrophotometer set at 276 nm and connected to a strip-chart recorder, fitted
with a flow-cell suitable for liquid chromatography having a volume of not more than 1 ml. Set
the detector and chart recorder at a full-scale sensitivity of 0.5 absorbance unit.
Equilibrate the column with acetic acid (60 g/l C
2
H
4
O
2
). Apply 0.4 ml of the test solution per
square centimetre of column cross-sectional area to the head of the column and carry out the elution.
The sum of the areas of any peaks eluted in advance of the principal peak is not greater than 5.0 per
cent of the sum of the areas of all the peaks in the chromatogram.
Related proteins Examine by polyacrylamide gel electrophoresis (2.2.31), using rod gels 80 mm
long and 5 mm in diameter and, as buffer, tris-glycine buffer solution pH 8.3 R.
The electrode in the lower reservoir is the anode and that in the upper reservoir is the cathode.
Use the following gel mixture: mix 3 volumes of water R, 3 volumes of a solution containing, in
100 ml, 28 g of acrylamide R and 0.735 g of methylenebisacrylamide R and 1 volume of a solution
containing, in 100 ml, 36.6 g of tris(hydroxymethyl)aminomethane R, 0.23 ml of tetramethylethylene-
diamine R and 48.0 ml of hydrochloric acid R. Degas the solution and add 1 volume of a 5.6 g/l
solution of ammonium persulphate R.
Test solution. Dissolve 1 mg of the substance to be examined in 1 ml of tris-glycine buffer solution pH
8.3 R and dilute to 2 ml with a 400 g/l solution of sucrose R.
Reference solution (a). Dissolve 1 mg of corticotropin CRS in 1 ml of tris-glycine buffer solution pH 8.3 R
and dilute to 2 ml with a 400 g/l solution of sucrose R.
Reference solution (b). Dilute 1 ml of reference solution (a) to 2 ml with a mixture of equal volumes of
tris-glycine buffer solution pH 8.3 R and a 400 g/l solution of sucrose R.
Reference solution (c). Dilute 1 ml of reference solution (b) to 2 ml with a mixture in equal volumes of
tris-glycine buffer solution pH 8.3 R and a 400 g/l solution of sucrose R.
Apply 100 l of each solution separately to the surface of the gel. After the addition of the buffer, add
0.2 ml of bromophenol blue solution R. Allow electrophoresis to take place with a constant current of
1 mA per tube for 30 min and then increase the current to 3 mA per tube for 100 min. Immerse the
gels in a 10 g/l solution of amido black 10B R in a 7 per cent V/V solution of acetic acid R for
1 h. Remove the staining solution and wash for 24 h with 7 per cent V/V acetic acid R. Examine the
electrophoretograms using a cold-light illuminator. In the electrophoretogram obtained with the test
solution, no band is more intense than the one corresponding in mobility to the main band in the
electrophoretogram obtained with reference solution (a). The test is not valid unless: the
electrophoretogram obtained with reference solution (c) shows a visible band; a gradation of intensity
of staining is seen in the electrophoretograms obtained with reference solutions (a), (b) and (c).
Pressor activity The blood pressor activity in the rat of the preparation to be examined is compared
with that of the International Standard for lysine vasopressin

or of a reference preparation of lysine
vasopressin calibrated in International Units. The equivalence in International Units of the
International Standard is stated by the World Health Organization.
Inject slowly into a tail vein of a male albino rat weighing about 300 g, a solution of a suitable
-adrenergic blocking agent, for example 10 ml per kilogram of body mass of a solution prepared by
dissolving 5 mg of phenoxybenzamine hydrochloride R in 0.1 ml of alcohol R, adding 0.05 ml of 1M
hydrochloric acid and diluting to 5 ml with a 9 g/l solution of sodium chloride R. After 18 h, anaesthetise
the rat using an anaesthetic favourable to the maintenance of uniform blood pressure. After 45 min
to 60 min, tie the rat on its back by its hind legs on an operating table. Introduce a cannula consisting
of a short glass or polyethylene tube of 2.5 mm external diameter into the trachea and dissect a
carotid artery ready for cannulation. Cannulate the femoral vein close to the inguinal ligament,
retract the abdominal muscles to expose the inguinal ligament, retract the superficial pudendal vein
to one side and dissect the femoral vein towards the inguinal ligament from the corresponding artery.
When dissecting, locate and tie off a deep branch reaching the femoral vein to prevent bleeding
during cannulation. Tie a short polyethylene cannula of about 1 mm in external diameter into the
femoral vein by two ligatures and join by a short piece of flexible tubing to a 1 ml burette with an
attached thistle funnel containing a 9 g/l solution of sodium chloride R at about 37C. Fix a wet cotton
swab firmly to the thigh so as to cover the incision and cannula. At this stage 200 I.U. of heparin per
100 g of body mass, dissolved in a 9 g/l solution of sodium chloride R, may be injected through the
venous cannula. Tie in a carotid cannula, about 1 mm in external diameter, and connect it via a tube
containing a 9 g/l solution of sodium chloride R to which heparin has been added, to a suitable
pressure-measuring device. Leave intact the central and peripheral nervous system, including both
vagi and associated sympathetic nerves. No artificial respiration is necessary. Inject all solutions
through the venous cannula using a 1 ml syringe graduated in 0.01 ml and after each injection inject
0.2 ml of the 9 g/l solution of sodium chloride R from the burette. Take care that no air is injected.
Dilute the solution of the reference preparation and the preparation to be examined with a 9 g/l
11-78
solution of sodium chloride R so that the volume to be injected is 0.1 ml to 0.5 ml. Choose two doses
of the reference preparation such that the elevation of the blood pressure is about 4 kPa for the lower
dose and about 6.67 kPa, but always submaximal, for the higher dose; the ratio of low to high dose is
determined by the response and is usually 3 to 5. As an initial approximation, doses of 3 milliunits
and 5 milliunits may be tried. Determine the geometric mean number of units of lysine vasopressin of
the higher and the lower dose of the reference preparation. Prepare a dose of the preparation to be
examined such that it contains twenty times as many International Units of corticotropin as this
geometric mean number of units of lysine vasopressin, and that the volume to be injected lies
between 0.1 ml and 0.5 ml. Inject doses at intervals of 10 min to 15 min, the two doses of the
reference preparation and the dose of the preparation to be examined constituting one group of three
doses, and the order in which they are given being varied at random in successive groups to a total of
four or five groups (i.e. twelve to fifteen injections in all). Record the maximum rise in blood pressure
in response to each dose.
The mean response to the dose of the preparation being examined is less than the response
corresponding with the geometric mean of the two doses of the reference preparation (5 I.U. of
pressor activity per 100 I.U. of corticotropin activity).
Loss on drying (2.2.32). Not more than 7.0 per cent, determined by drying in an oven at 60C at a
pressure not exceeding 670 Pa.
appropriate procedure of sterilisation, it complies with the test for sterility.
318 I.U. of endotoxin per milligram.
POTENCY
The International Unit is the activity contained in a stated amount of the International Reference
Preparation. The equivalence in International Units of the International Standard is stated by the
World Health Organization.
potency. The fiducial limits of error (P = 0.95) of the estimated potency are not less than 64 per cent
Carry out the assay by method A or method B.
A. Carry out the assay of corticotropin (2.7.3). If required, hypophysectomised animals may be
replaced with animals whose endogenous corticotropic activity has been suppressed by a suitable
substance such as dexamethasone given in the following way: two doses, each of 0.5 ml of a
suspension containing 2 mg of dexamethasone and 50 g/l of acacia are administered to each animal,
the first being given subcutaneously 18 h before the assay and the second intraperitoneally at the time
of administration of the preparations being assayed. During the time between the two parenteral
doses, dexamethasone (20 g/ml) is included in the drinking water together with glucose (50 g/l).
B. The potency of corticotropin is estimated by comparing in given conditions its activity in
increasing the amount of corticosterone produced by isolated rat adrenal cells with that of the
International Reference Preparation of corticotropin or a reference preparation calibrated in
International Units.
Use siliconised glassware and rinse well with water R before use. Kill four male rats, each weighing
between 200 g and 400 g, by a method causing minimum stress. Remove the adrenal glands,
carefully free them of adhering fat and immerse in solution B maintained at 4C. Cut each gland into
four equal pieces and transfer to a suitable plastic stirring apparatus (see Fig. 07591) containing
5 ml of solution C maintained at 37C. Disperse the adrenal cells by stirring the mixture at 500
r/min. After 20 min, remove the supernatant liquid, cool to 4C, add 5 ml of solution C and repeat
the dispersal procedure. Repeat the operation a further three times. Combine the five supernatant
liquids, centrifuge at 4C in a polyethylene test-tube for 30 min after slow acceleration to 100 g.
Suspend the resulting pellet in 8 ml of solution D and centrifuge at 100 g for 30 min. Again suspend
the residue in 8 ml of solution D and filter the mixture through nylon gauze with 100 m pores into a
polyethylene beaker and add a suitable volume of solution D to the filtrate (a total volume of 65 ml
to 105 ml has been found suitable). Maintain the resulting solution at 4C.
11-79
A Pulley and stirring paddle,
B Bearing,
C Incubation medium.
Fig. 07591 Stirring Apparatus
Dimensions in millimetres
Prepare four independent dilutions, with two-fold dose intervals, from solutions of suitable
concentration of the substance to be examined and of the reference preparation using solution E as
diluent. Pipette 0.1 ml of each dilution into each of four polystyrene test-tubes and add 1.0 ml of
the cell suspensions prepared above to each tube. Incubate at 37C for 2 h and then cool to 4C.
Transfer 1 ml of the contents of each tube to glass tubes containing 1.4 ml of methylene chloride R,
mix in a vortex mixer for 10 s and centrifuge at 3000 g for 5 min. Transfer 1 ml of each of the
methylene chloride layers, avoiding taking up aqueous phase, to a glass tube containing 0.6 ml of a
mixture of 15 volumes of alcohol R and 35 volumes of sulphuric acid R. Mix in a vortex mixer for 10 s
and centrifuge at 1500 g for 5 min. Allow each tube to stand for 30 min. Examine by fluorimetry
(2.2.21), irradiating the lower layer with an excitant beam of suitable wavelength such as 436 nm or
470 nm and measuring the fluorescence at the maximum between 530 nm and 545 nm. If the
solutions are not transferred to measurement cells, select glass tubes that give fluorescence values for
a standard corticosterone that do not differ from each other by more than 5 per cent. Calculate the
result of the assay by the usual statistical methods using the linear portion of the log dose-response
curve.
Solution A
Sodium chloride R 6.60 g
Potassium chloride R 0.353 g
Sodium hydrogen carbonate R 0.840 g
Potassium dihydrogen phosphate R 0.161 g
Magnesium sulphate R 0.291 g
Calcium chloride R 0.373 g
2-[4-(2-Hydroxyethyl)piperazin-1-yl-
ethanesulphonic acid R 4.77 g
Dissolve the above ingredients in about 950 ml of water R, adjust to pH 7.4 with 1M sodium hydroxide
and add 60 mg of benzylpenicillin sodium R and a quantity of streptomycin sulphate R equivalent to
11-80
100 mg of streptomycin. Dilute to 1000 ml with water R.
Solution B
Add 2 g/l of glucose R to solution A.
Solution C
Add to solution B, 1 g/l of a preparation of collagenase obtained from Clostridium histolyticum and of
a grade suitable for the preparation of dispersed cells.
Solution D
Add 5 g/l of bovine albumin R to solution B.
Solution E
To a 9 g/l sterile solution of sodium chloride R add 1 g/l of bovine albumin R and adjust to pH 2.0 with
1M hydrochloric acid.
STORAGE
Store in a well-closed container, protected from light, at a temperature not exceeding 25C. If the
LABELLING
The label states:
the route or routes of injection used in determining the potency (when method A has been
used),
_______________________________________________________________________ Ph Eur
12-1
Cortisone Acetate
Me
H
H Me
O
O
OH
O
H
OAc
C
23
H
30
O
6
402.5 50-04-4
Cortisone Acetate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0321].
Preparation
Cortisone Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cortisone acetate contains not less than 97.0 per cent and not more than the equivalent of
103.0 per cent of 17,21-dihydroxypregn-4-ene-3,11,20-trione 21-acetate, calculated with reference
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water, freely soluble in
methylene chloride, soluble in dioxan, sparingly soluble in acetone, slightly soluble in alcohol and
in methanol.
IDENTIFICATION
Second identification: C, D, E.
obtained with cortisone acetate CRS. If the spectra obtained in the solid state show differences,
record new spectra using 50 g/l solutions of the substance to be examined and the reference
substance in methylene chloride R in a 0.2 mm cell.
254
plate R.
Test solution. Dissolve 10 mg of the substance to be examined in a mixture of 1 volume of
solvents.
Reference solution (a). Dissolve 20 mg of cortisone acetate CRS in a mixture of 1 volume of
solvents.
Reference solution (b). Dissolve 10 mg of hydrocortisone acetate R in reference solution (a) and dilute
to 10 ml with reference solution (a).
examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the
test solution is similar in position and size to the principal spot in the chromatogram obtained with
until the spots appear. Allow to cool. Examine the plate in daylight and in ultraviolet light at
in the chromatogram obtained with reference solution (a). The test is not valid unless the chroma-
togram obtained with reference solution (b) shows two clearly separated spots.
254
plate R.
12-2
potassium hydrogen carbonate solution, saturated methanolic R and immediately pass a stream of
nitrogen R briskly through the solution for 5 min. Stopper the tube. Heat in a water-bath at 45C
protected from light for 2 h 30 min. Allow to cool.
Reference solution (a). Dissolve 25 mg of cortisone acetate CRS in methanol R with gentle heating and
dilute to 5 ml with the same solvent. This solution is also used to prepare reference solution (b).
Reference solution (b). Transfer 2 ml of the solution obtained during preparation of reference
solution (a) to a 15 ml glass tube with a ground-glass stopper or a polytetrafluoroethylene cap. Add
10 ml of potassium hydrogen carbonate solution, saturated methanolic R and immediately pass a stream
of nitrogen R briskly through the solution for 5 min. Stopper the tube. Heat in a water-bath at 45C
obtained with the corresponding reference solution. Spray with alcoholic solution of sulphuric acid R
and heat at 120C for 10 min or until the spots appear. Allow to cool. Examine the plate in daylight
and in ultraviolet light at 365 nm. The principal spot in each of the chromatograms obtained with
the test solutions is similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm
and size to the principal spot in the chromatogram obtained with the corresponding reference
solution. The principal spots in the chromatograms obtained with test solution (b) and reference
solution (b) have an R
f
value distinctly lower than that of the principal spots in the chromatograms
D. Add about 2 mg to 2 ml of sulphuric acid R and shake to dissolve. Within 5 min, a faint yellow
colour develops. Add the solution to 10 ml of water R and mix. The colour is discharged and a clear
solution remains.
E. About 10 mg gives the reaction of acetyl (2.3.1).
TESTS
substance.
Test solution. Dissolve 25.0 mg of the substance to be examined in acetonitrile R and dilute to
Reference solution (a). Dissolve 2 mg of cortisone acetate CRS and 2 mg of hydrocortisone acetate CRS
in acetonitrile R and dilute to 100.0 ml with the same solvent.
Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with acetonitrile R.
as mobile phase at a flow rate of 1 ml/min a mixture prepared as follows: in a 1000 ml
adjust the volume to 1000 ml with water R and mix again,
conditions the retention times are: hydrocortisone acetate about 10 min and cortisone acetate about
12 min. The test is not valid unless the resolution between the peaks due to hydrocortisone acetate
and cortisone acetate is at least 4.2; if necessary, adjust the concentration of acetonitrile in the
mobile phase.
Inject 20 l of the test solution, 20 l of reference solution (b) and 20 l of acetonitrile as a blank.
Continue the chromatography for twice the retention time of the principal peak. In the chromato-
gram obtained with the test solution: the area of any peak apart from the principal peak, is not
greater than half the area of the principal peak in the chromatogram obtained with reference
solution (b) (0.5 per cent); the sum of the areas of all the peaks apart from the principal peak, is not
solution (b) (1.5 per cent). Disregard any peak with an area less than 0.05 times the area of the
12-3
at 100C to 105C.
ASSAY
solution to 100.0 ml with alcohol R. Measure the absorbance (2.2.25) at the maximum of 237 nm.
23
H
30
O
6
STORAGE
IMPURITIES
A. hydrocortisone acetate.
__________________________________________________________________________________________________________ Ph Eur
12-4
Hydrogenated Cottonseed Oil
Hydrogenated Cottonseed Oil complies with the requirements of the 3rd edition of the European
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hydrogenated cottonseed oil is the product obtained by refining and hydrogenation of oil obtained
from seeds of cultivated plants of various varieties of Gossypium hirsutum L. or of other species of
Gossypium. The product consists mainly of triglycerides of palmitic and stearic acids.
CHARACTERS
A white mass or powder which melts to a clear, pale yellow liquid when heated, practically insoluble
in water, freely soluble in methylene chloride and in toluene, very slightly soluble in alcohol.
IDENTIFICATION
B. It complies with the test for foreign fatty oils (see Tests).
TESTS
Acid value (2.5.1). Not more than 0.5, determined on 10.0 g. Dissolve the substance to be
examined in 50 ml of a hot mixture of equal volumes of alcohol R and toluene R, previously
neutralised with 0.1M potassium hydroxide using 0.5 ml of phenolphthalein solution R1 as indicator.
Titrate the solution immediately while still hot.
Alkaline impurities Dissolve by gentle heating 2.0 g of the substance to be examined in a mixture
of 1.5 ml of alcohol R and 3 ml of toluene R. Add 0.05 ml of a 0.4 g/l solution of bromophenol blue R
in alcohol R. Not more than 0.4 ml of 0.01M hydrochloric acid is required to change the colour to
yellow.
Foreign fatty oils Carry out the test for foreign oils in fatty oils by gas chromatography (2.4.22).
a fused-silica capillary column 25 m long and 0.25 mm in internal diameter coated on the inner
wall with poly(cyanopropyl)siloxane R (film thickness 0.2 m),
maintaining the temperature of the column at 180C for 35 min and that of the injection port and
14
myristic acid: not more than 1.0 per cent,
oleic acid and isomers (C
18:1
equivalent chain length on poly(cyanopropyl)siloxan 18.5 to 18.8):
not more than 4.0 per cent,
linoleic acid and isomers (C
18:2
equivalent chain length on poly(cyanopropyl)siloxan 19.4 to
lignoceric acid: not more than 0.5 per cent.
2.2.23).
Test solution. Introduce 5.0 g of the substance to be examined into a platinum or silica crucible
tared after ignition. Cautiously heat and introduce into the substance a wick formed from twisted
ashless filter paper. Ignite the wick. When the substance ignites, stop heating. After combustion,
ignite in a muffle furnace at about 600C. Continue the incineration until white ash is obtained.
After cooling, take up the residue with two quantities, each of 2 ml, of dilute hydrochloric acid R and
transfer into a 25 ml graduated flask. Add 0.3 ml of nitric acid R and dilute to 25.0 ml with distilled
water R.
12-5
Reference solutions. Prepare three reference solutions by adding 1.0 ml, 2.0 ml and 4.0 ml of nickel
standard solution (0.2 ppm Ni) R to 2.0 ml portions of the test solution, diluting to 10.0 ml with
distilled water R.
Measure the absorbance at 232 nm using a nickel hollow-cathode lamp as a source of radiation, a
graphite furnace as an atomic generator and argon R as the carrier gas.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
12-6
Couch Grass Rhizome
Couch Grass Rhizome complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Couch grass rhizome consists of the whole or cut, washed and dried rhizome of Agropyron repens
(L.) Beauv. (Elymus repens (L.) Gould); the adventitious roots are removed.
CHARACTERS
Couch grass rhizome has the macroscopic and microscopic characters described under
identification tests A and B.
IDENTIFICATION
A. The shiny yellowish, light brown or yellowish-brown pieces of the rhizome are 2 mm to 3 mm
thick and longitudinally furrowed. At the nodes are the remains of very thin, more or less branched
roots and whitish or brownish scale-like leaves; the internodes, up to 6 cm long, are furrowed and
hollow inside. The transverse section of the nodes shows a yellowish medulla.
B. Reduce to a powder (355). The powder is whitish-yellow. Examine under a microscope, using
chloral hydrate solution R. The powder shows fragments of the epidermis covered with a thick cuticle
and composed of rectangular and elongated thick-walled cells with pitted slightly wavy walls which
alternate with small rounded to almost square cells; U-shaped thickened endodermic cells;
numerous fragments of moderately thickened fibres and groups of vessels with slit-shaped pits or
with spiral and annular thickening.
TESTS
Cynodon dactilon, Imperata cylindrica Examined under a microscope using iodine solution R1.
No blue starch grains are visible and no U-shaped thickened endodermic cells are present.
Foreign matter (2.8.2). Not more than 15 per cent of greyish-black pieces of rhizomes in the cut
drug.
Water-soluble extractive To 5.0 g of powdered drug (355) add 200 ml of boiling water R. Allow
to stand for 10 min, shaking occasionally. Allow to cool, dilute to 200.0 ml with water R and filter.
Evaporate 20.0 ml of the filtrate to dryness on a water-bath. Dry the residue in an oven at 100C to
105C. The residue weighs not less than 0.125 g (25 per cent).
STORAGE
__________________________________________________________________________________________________________ Ph Eur
12-7
Cresol
Definition Cresol is a mixture of cresols and other phenols obtained from coal tar.
Characteristics An almost colourless to pale brownish yellow liquid; odour, resembling that of
phenol but more tarry.
Almost completely soluble in 50 volumes of water; freely soluble in chloroform, in ethanol (96%),
in ether and in fixed and volatile oils.
Identification Shake 0.5 ml with 300 ml of water and filter. The filtrate complies with the
following tests.
A. Add iron(III) chloride solution R1. A transient blue colour is produced.
B. Add bromine water. A pale yellow flocculent precipitate is produced.
Acidity A 2.0% w/v solution is neutral to bromocresol purple solution.
Distillation range Not more than 2% v/v distils below 188 and not less than 80% v/v distils
between 195 and 205, Appendix V C.
Hydrocarbons Place 50 ml in a 500-ml round-bottomed flask, add 150 ml of 5M sodium hydroxide
and 30 ml of water and mix thoroughly. Connect the flask to a splash-bulb and air-condenser about
60 cm long, with the end of the air-condenser fitting closely into the neck of a 250-ml pear-shaped
separating funnel and passing well into the separating funnel, which has a cylindrical graduated
portion above the stopcock. Fill the graduated portion of the separating funnel with water. Distil
rapidly until 75 ml of distillate has been collected, cooling the separating funnel in running water if
necessary. Allow the separating funnel to stand in a vertical position until separation is complete
and draw off the aqueous liquid into a titration flask for use in the test for Volatile bases.
Allow the separating funnel to stand for a few minutes, measure the volume of hydrocarbon oil in
the graduated portion and warm, if necessary, to keep the oil in the liquid state. Subtract the
volume of volatile bases in the hydrocarbon oil, as determined in the following test. Not more than
0.15% v/v of hydrocarbon oil is present.
Volatile bases To the aqueous liquid reserved in the test for Hydrocarbons add any aqueous liquid
still remaining in the separating funnel and neutralise, if necessary, with 0.1M hydrochloric acid using
phenolphthalein solution R1 as indicator. Titrate with 1M hydrochloric acid VS using methyl orange
solution as indicator. Wash the oil from the separating funnel into the titration flask with water and
again titrate with 1M hydrochloric acid VS. From the volume of additional 1M hydrochloric acid VS,
calculate the proportion of volatile bases in the hydrocarbon oil. From the total volume of 1M
hydrochloric acid VS used in both titrations calculate the volume of volatile bases in the substance
being examined. Each ml of 1M hydrochloric acid VS is equivalent to 0.080 ml of volatile bases. Not
more than 0.15% v/v of volatile bases is present.
Hydrocarbons and volatile bases The sum of the contents of hydrocarbon oil and volatile bases,
as determined in the tests for Hydrocarbons and for Volatile bases, does not exceed 0.25% v/v.
Sulphur compounds Place 20 ml in a small conical flask and over the mouth of the flask fix a
piece of filter paper moistened with a 10% w/v solution of lead(II) acetate. Heat the flask on a water
bath for 5 minutes. Not more than a light yellow colour is produced on the filter paper.
Non-volatile matter When evaporated on a water bath and dried at 105, leaves not more than
0.1% w/v of residue.
Storage Cresol should be kept in a well-closed container and protected from light. It darkens with
age or on exposure to light.
Action and use Antiseptic; antimicrobial preservative.
12-8
Croscarmellose Sodium
corrected 1/01
Croscarmellose Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ______________________________________________________________________________________________________________
DEFINITION
Croscarmellose sodium (cross-linked sodium carboxymethylcellulose) is the sodium salt of a cross-
linked, partly O-carboxymethylated cellulose.
CHARACTERS
A white or greyish-white powder, practically insoluble in acetone, in ethanol and in toluene.
IDENTIFICATION
A. Shake 1 g with 100 ml of a solution containing 4 ppm of methylene blue R and allow to settle.
The substance to be examined absorbs the methylene blue and settles as a blue, fibrous mass.
B. Shake 1 g with 50 ml of water R. Transfer 1 ml of the mixture to a test-tube, add 1 ml of water R
and 0.05 ml of a freshly prepared 40 g/l solution of -naphthol R in methanol R. Incline the test-
tube and add carefully 2 ml of sulphuric acid R down the side so that it forms a lower layer. A
reddish-violet colour develops at the interface.
TESTS
pH (2.2.3). Shake 1 g with 100 ml of carbon dioxide-free water R for 5 min. The pH of the
suspension is 5.0 to 7.0.
Degree of substitution Place 1.000 g in a 500 ml conical flask, add 300 ml of a 100 g/l solution
of sodium chloride R, 25.0 ml of 0.1M sodium hydroxide, stopper the flask and allow to stand for
5 min, shaking occasionally. Add 0.05 ml of m-cresol purple solution R and about 15 ml of 0.1M
hydrochloric acid from a burette. Insert the stopper and shake. If the solution is violet, add 0.1M
hydrochloric acid in 1 ml portions until the solution becomes yellow, shaking after each addition.
Titrate with 0.1M sodium hydroxide until the colour turns to violet.
Calculate the number of milliequivalents (M) of base required for the neutralisation equivalent to
1 g of dried substance.
Calculate the degree of acid carboxymethyl substitution (A) from the expression:
) 80 412 7102 (
1150
C M
M

C=sulphated ash as a percentage.
Calculate the degree of sodium carboxymethyl substitution (S) from the expression:
) 80 7102 (
) 58 162 (
C
C A
+
The degree of substitution is the sum of A +S and it is between 0.60 and 0.85, calculated with
Sodium chloride and sodium glycollate The sum of the percentage contents of sodium chloride
and sodium glycollate is not more than 0.5 per cent, calculated with reference to the dried
substance.
Sodium chloride. Place 5.00 g in a 250 ml conical flask, add 50 ml of water R and 5 ml of strong
hydrogen peroxide solution R and heat on a water-bath for 20 min, stirring occasionally to ensure
total hydration. Cool, add 100 ml of water R and 10 ml of nitric acid R. Titrate with 0.05M silver
nitrate determining the end-point potentiometrically (2.2.20) using a silver indicator electrode and
a double-junction reference electrode containing a 100 g/l solution of potassium nitrate R in the
outer jacket and a standard filling solution in the inner jacket, and stirring constantly.
Sodium glycollate. Place a quantity of the substance to be examined equivalent to 0.500 g of the
dried substance in a 100 ml beaker. Add 5 ml of glacial acetic acid R and 5 ml of water R and stir to
ensure total hydration (about 15 min). Add 50 ml of acetone R and 1 g of sodium chloride R. Stir for
several minutes to ensure complete precipitation of the carboxymethylcellulose. Filter through a
fast filter paper impregnated with acetone R into a volumetric flask, rinse the beaker and filter with
12-9
30 ml of acetone R and dilute the filtrate to 100.0 ml with the same solvent. Allow to stand for 24
h without shaking. Use the clear supernatant to prepare the test solution.
Prepare the reference solutions as follows: in a 100 ml volumetric flask, dissolve 0.100 g of
glycollic acid R, previously dried in vacuo over diphosphorus pentoxide R, in water R and dilute to
100.0 ml with the same solvent. Use the solution within 30 days. Transfer 1.0 ml, 2.0 ml, 3.0 ml
and 4.0 ml of the solution to separate volumetric flasks; dilute the contents of each flask to 5.0 ml
with water R, add 5 ml of glacial acetic acid R, dilute to 100.0 ml with acetone R and mix.
Transfer 2.0 ml of the test solution and 2.0 ml of each of the reference solutions to separate
25 ml volumetric flasks. Heat the uncovered flasks for 20 min on a water-bath to eliminate acetone.
Allow to cool and add 5.0 ml of 2,7-dihydroxynaphthalene solution R to each flask. Mix, add a
further 15.0 ml of 2,7-dihydroxynaphthalene solution R and mix again. Close the flasks with
aluminium foil and heat on a water-bath for 20 min. Cool and dilute to 25.0 ml with sulphuric
acid R.
Measure the absorbance (2.2.25) of each solution at 540 nm. Prepare a blank using 2.0 ml of a
solution containing 5 per cent V/V each of glacial acetic acid R and water R in acetone R. Prepare a
standard curve using the absorbances obtained with the reference solutions. From the standard
curve and the absorbance of the test solution, determine the mass a, in milligrams, of glycollic acid
in the substance to be examined, and calculate the content of sodium glycollate from the
expression:
m b
a
) 100 (
29 . 1 10

1.29=the factor converting glycollic acid to sodium glycollate,
b =loss on drying as a percentage,
m=mass of the substance to be examined, in grams.
Water-soluble substances Not more than 10.0 per cent. Disperse 10.00 g in 800.0 ml of water R
and stir for 1 min every 10 min during the first 30 min. Allow to stand for 1 h and centrifuge, if
necessary. Decant 200.0 ml of the supernatant liquid onto a fast filter paper in a vacuum filtration
funnel, apply vacuum and collect 150.0 ml of the filtrate. Evaporate to dryness and dry the residue
Heavy metals (2.4.8). To the residue obtained in the determination of the sulphated ash add 1 ml
of hydrochloric acid R and evaporate on a water-bath. Take up the residue in 20 ml of water R. 12 ml
Sulphated ash (2.4.14). 14.0 per cent to 28.0 per cent, determined on 2.000 g, using a mixture of
equal volumes of sulphuric acid R and water R, and calculated with reference to the dried substance.
Settling volume Place 75 ml of water R in a 100 ml graduated cylinder and add 1.5 g of the
substance to be examined in 0.5 g portions, shaking vigorously after each addition. Dilute to
100.0 ml with water R and shake again until the substance is homogeneously distributed. Allow to
stand for 4 h. The settling volume is between 10.0 ml and 30.0 ml.
3
bacteria and
10
2
fungi per gram, determined by plate count. It complies with the test for Escherichia coli (2.6.13).
STORAGE
__________________________________________________________________________________________________________ Ph Eur
12-10
Crospovidone
N
O
CH CH
2
H H
n
(C
6
H
9
NO)
n
(111.1)
n
9003-39-8
Crospovidone complies with the requirements of the 3rd edition of the European Pharmacopoeia [0892].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Crospovidone is a cross-linked homopolymer of 1-vinyl-pyrrolidin-2-one.
CHARACTERS
White or yellowish-white powder or flakes, hygroscopic, practically insoluble in water, in alcohol
IDENTIFICATION
A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph. Eur. refer-
ence spectrum of crospovidone.
B. Suspend 1 g in 10 ml of water R, add 0.1 ml of 0.05M iodine and shake for 30 s. Add 1 ml of
starch solution R and shake. No blue colour develops within 30 s.
C. It complies with the test for water-soluble substances (see Tests).
TESTS
Peroxides Suspend 2.0 g in 50 ml of water R. To 25 ml of this suspension add 2 ml of titanium
trichloride-sulphuric acid reagent R. Allow to stand for 30 min and filter. The absorbance (2.2.25) of
the filtrate, measured at 405 nm using a mixture of 25 ml of a filtered 40 g/l suspension of the
substance to be examined and 2 ml of a 13 per cent V/V solution of sulphuric acid R as the
compensation liquid, is not greater than 0.35 (400 ppm expressed as H
2
O
2
).
Water-soluble substances Transfer 25.0 g to a 400 ml beaker, add 200 ml of water R and stir for
1 h using a magnetic stirrer. Transfer the suspension to a 250 ml volumetric flask, rinsing with
water R, and dilute to 250.0 ml with the same solvent. Allow the bulk of the solids to settle. Filter
about 100 ml of the almost clear supernatant liquid through a 0.45 m membrane filter, protected
by superimposing a 3 m membrane filter. While filtering, stir the liquid above the filter manually
or by means of a mechanical stirrer, taking care not to damage the filter. Transfer 50.0 ml of the
clear filtrate to a tared 100 ml beaker, evaporate to dryness and dry at 105C to 110C for 3 h. The
Vinylpyrrolidone Suspend 4.0 g in 30 ml of water R and stir for 15 min. Centrifuge the
suspension and filter the supernatant liquid through a sintered-glass filter (10). Take up the
centrifugation residue with 50 ml of water R and stir for 15 min. Centrifuge and filter the super-
natant liquid through a sintered-glass filter (10). Repeat these operations once. To the combined
filtrates, add 0.5 g of sodium acetate R and titrate with 0.05M iodine until a persistent colour is
obtained. Add a further 3.0 ml of 0.05M iodine, allow to stand for 10 min, filter and titrate with
0.1M sodium thiosulphate, using 3 ml of starch solution R added towards the end of the titration.
Carry out a blank titration, using 30 ml of water R adjusted to the same pH as the solution to be
examined with acetic acid R and to which the same volume of 0.05M iodine as that used in the first
titration of the solution to be examined has been added. Not more than 0.72 ml of 0.05M iodine is
used (0.1 per cent).
Loss on drying (2.2.32). Not more than 5.0 per cent determined on 0.500 g by drying in an oven
at 100C to 105C.
STORAGE
Store protected from moisture.
__________________________________________________________________________________________________________ Ph Eur
12-11
Crotamiton
N Me
O H
H Et Me
C
13
H
17
NO 203.3 483-63-6
Crotamiton complies with the requirements of the 3rd edition of the European Pharmacopoeia [1194]. These
Action and use Antipruritic; used in treatment of scabies.
Preparations
Crotamiton Cream
Crotamiton Lotion
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Crotamiton contains not less than 96.0 per cent and not more than the equivalent of 101.0 per
cent of N-ethyl-N-(2-methylphenyl)but-2-enamide, calculated as the sum of the E- and Z-
isomers, and not more than 15.0 per cent of the Z-isomer.
CHARACTERS
A colourless or pale yellow, oily liquid, slightly soluble in water, miscible with alcohol. At low
temperatures it may partly or completely solidify.
IDENTIFICATION
A. Dissolve 25.0 mg in cyclohexane R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of
the solution to 10.0 ml with cyclohexane R. Examined between 220 nm and 300 nm (2.2.25), the
solution shows an absorption maximum at 242 nm. The specific absorbance at the maximum is
300 to 330.
obtained with crotamiton CRS.
C. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable silica
Test solution. Dissolve 25 mg of the substance to be examined in ethanol R and dilute to 10 ml with
the same solvent.
Reference solution. Dissolve 25 mg of crotamiton CRS in ethanol R and dilute to 10 ml with the same
solvent.
Apply separately to the plate 5 l of each solution. Develop over a path of 15 cm using the mixture
prepared as follows: shake 98 volumes of methylene chloride R with 2 volumes of concentrated
ammonia R, dry over anhydrous sodium sulphate R, filter and mix 97 volumes of the filtrate with 3
volumes of 2-propanol R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm.
The principal spot in the chromatogram obtained with the test solution is similar in position and
D. To 10 ml of a saturated solution add a few drops of a 3 g/l solution of potassium permanganate R.
A brown colour is produced and a brown precipitate is formed on standing.
TESTS
Free amines Dissolve 5.00 g in 16 ml of methylene chloride R and add 4.0 ml of acetic acid R. Add
0.1 ml of metanil yellow solution R and 1.0 ml of 0.02M perchloric acid. The solution is red (500 ppm
as ethylaminotoluene).
Chlorides Boil 5.0 g under a reflux condenser for 1 h with 25 ml of alcohol R and 5 ml of a 200 g/l
solution of sodium hydroxide R. Cool, add 5 ml of water R and shake with 25 ml of ether R. Dilute
the lower layer to 20 ml with water R; add 5 ml of nitric acid R, dilute to 50 ml with water R and add
12-12
1 ml of a freshly prepared 50 g/l solution of silver nitrate R. Any opalescence in the solution is not
more intense than that in a mixture of 1 ml of a freshly prepared 50 g/l solution of silver nitrate R
and a solution prepared by diluting 5 ml of a 200 g/l solution of sodium hydroxide R to 20 ml with
water R and adding 1.5 ml of 0.01M hydrochloric acid, 5 ml of nitric acid R and sufficient water R to
produce 50 ml (100 ppm).
Inject 20 l of test solution (a), reference solution (b) and reference solution (c). Continue the
chromatography for 2.5 times the retention time of the principal peak. In the chromatogram
obtained with test solution (a): the area of any peak corresponding to crotamiton impurity A is not
greater than the area of the corresponding peak in the chromatogram obtained with reference
solution (b) (3 per cent); the sum of the areas of any peaks, apart from the principal peak, the peak
corresponding to the Z-isomer and any peak corresponding to crotamiton impurity A, is not greater
than the sum of the areas of the peaks corresponding to the Z- and E-isomers in the chromatogram
obtained with reference solution (c) (1 per cent). Disregard any peak with an area less than 0.02
times the area of the principal peak in the chromatogram obtained with reference solution (c).
Sulphated ash (24.14). Not more than 0.1 per cent, determined on 1.0 g.
ASSAY
Test solution (a). Dissolve 50.0 mg of the substance to be examined in the mobile phase and dilute
Reference solution (a). Dissolve 50.0 mg of crotamiton CRS in the mobile phase and dilute to
Reference solution (b). Dissolve 15.0 mg of crotamiton impurity A CRS in the mobile phase and dilute
to 20.0 ml with the mobile phase. Dilute 1.0 ml of the solution to 50.0 ml with the mobile phase.
Reference solution (c). Dilute 1.0 ml of test solution (a) to 100.0 ml with the mobile phase.
Reference solution (d). Dissolve 15 mg of crotamiton impurity A CRS in the mobile phase and dilute
to 100 ml with the mobile phase. Dilute 1 ml of the solution to 10 ml with test solution (a).
a stainless steel column 0.25 m long and 4 mm in internal diameter packed with silica gel for
as mobile phase at a flow rate of 1.0 ml/min a mixture of 8 volumes of tetrahydrofuran R and 92
volumes of cyclohexane R,
Inject 20 l of reference solution (b) and 20 l of reference solution (d). When the chromato-
grams are recorded in the prescribed conditions, the retention times relative to the principal peak
(E-isomer) are: Z-isomer, about 0.5 and crotamiton impurity A, about 0.8. Adjust the sensitivity of
the system so that the height of the principal peak in the chromatogram obtained with reference
solution (b) is at least 70 per cent of the full scale of the recorder. The test is not valid unless, in the
chromatogram obtained with reference solution (d), the resolution between the peaks correspond-
ing to impurity A and the E-isomer is at least 4.5.
Inject alternately test solution (b) and reference solution (a). Calculate the percentage content of
C
13
H
17
NO from the sum of the areas of the peaks due to the Z- and E-isomers in the chromato-
grams obtained.
Calculate the content of the Z-isomer, as a percentage of the total content of the E- and Z-
isomers, from the chromatogram obtained with test solution (b).
STORAGE
Store in a well-filled, well-closed container, protected from light.
IMPURITIES
N
Me
Me
O
H
2
C
A. N-ethyl-N-(2-methylphenyl)but-3-enamide.
_____________________________________________________________________________________________________________ Ph Eur
12-13
Cyanocobalamin
N
Me
N
Me
Me
N
Me
Me
CH
2
CH
2
CONH
2
N
Me
Me
H
CH
2
CONH
2
CH
2
CH
2
CONH
2
H
2
NCOCH
2
CH
2
H
H
2
NCOCH
2
H
2
NCOCH
2
H
H
H
Me
N
O
H
Me H
N
N
Me
Me
O
O
O
O P O
H CH
2
OH
HO
H
H
H
Co
CN
+
C
63
H
88
CoN
14
O
14
P 1355 68-19-9
Cyanocobalamin complies with the requirements of the 3rd edition of the European Pharmacopoeia [0547].
Action and use Used in treatment of vitamin B
12
deficiency.
Preparation
Cyanocobalamin Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cyanocobalamin contains not less than 96.0 per cent and not more than the equivalent of
102.0 per cent of -(5,6-dimethylbenzimidazol-1-yl)cobamide cyanide, calculated with reference to
CHARACTERS
A dark-red, crystalline powder or dark-red crystals, sparingly soluble in water and in alcohol,
practically insoluble in acetone and in ether. The anhydrous substance is very hygroscopic.
IDENTIFICATION
A. Dissolve 2.5 mg in water R and dilute to 100.0 ml with the same solvent. Examined between
260 nm and 610 nm (2.2.25), the solution shows three absorption maxima, at 278 nm, 361 nm and
at 547 nm to 559 nm. The ratio of the absorbance at the maximum at 361 nm to that at the
maximum at 547 nm to 559 nm is 3.15 to 3.45. The ratio of the absorbance at the maximum at
361 nm to that at the maximum at 278 nm is 1.70 to 1.90.
B. Carry out the test protected from light.
of alcohol R and water R.
Reference solution. Dissolve 2 mg of cyanocobalamin CRS in 1 ml of a mixture of equal volumes of
alcohol R and water R.
Apply separately to the plate 10 l of each solution. Develop in a non-saturated tank over a path of
12 cm using a mixture of 9 volumes of dilute ammonia R1, 30 volumes of methanol R and 45
volumes of chloroform R. Allow the plate to dry in air. Examine in daylight. The principal spot in the
chromatogram obtained with the test solution is similar in position, colour and size to the principal
spot in the chromatogram obtained with the reference solution.
TESTS
10.0 ml with the same mixture of solvents. Use within 1 h.
Reference solution (a). Dilute 3.0 ml of the test solution to 100.0 ml with the mobile phase. Use
within 1 h.
1.0 ml of this solution to 100.0 ml with the mobile phase. Use within 1 h.
12-14
Reference solution (c). Dissolve 25 mg of the substance to be examined in 10 ml of water R, warming
if necessary. Allow to cool and add 5 ml of a 1.0 g/l solution of chloramine R and 0.5 ml of 0.05M
hydrochloric acid. Dilute to 25 ml with water R. Shake and allow to stand for 5 min. Dilute 1 ml of
this solution to 10 ml with the mobile phase and inject immediately.
as the mobile phase at a flow rate of 0.8 ml per minute a mixture prepared as follows: mix 26.5
volumes of methanol R and 73.5 volumes of a 10 g/l solution of disodium hydrogen phosphate R,
adjust to pH 3.5 using phosphoric acid R and use within 2 days,
a spectrophotometer set at 361 nm as detector,
a loop injector.
Inject separately 20 l of each solution and continue the chromatography for three times the
retention time of cyanocobalamin. In the chromatogram obtained with the test solution, the sum of
the areas of any peaks apart from the principal peak is not greater than the area of the principal
whose area is less than that of the principal peak in the chromatogram obtained with reference
solution (b). The test is not valid unless the chromatogram obtained with reference solution (c)
shows two principal peaks, the resolution between these peaks is not less than 2.5 and the chroma-
togram obtained with reference solution (b) shows one principal peak with a signal-to-noise ratio of
not less than five.
Loss on drying (2.2.32). Not more than 12.0 per cent, determined on 20.00 mg by drying in
vacuo at 100C to 105C for 2 h.
ASSAY
Dissolve 25.00 mg in water R and dilute to 1000.0 ml with the same solvent. Measure the
absorbance (2.2.25) of the solution at the maximum at 361 nm. Calculate the content of
C
63
H
88
CoN
14
O
14
P, taking the specific absorbance to be 207.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
12-15
Cyclizine
N
NMe
C
18
H
22
N
2
266.4 82-92-8
Definition Cyclizine is 1-benzhydryl-4-methylpiperazine. It contains not less than 98.5% and not
18
H
22
N
2
Characteristics A white or creamy white, crystalline powder.
Practically insoluble in water. It dissolves in most organic solvents and in dilute acids.
Identification
cyclizine (RS 075).
B. The light absorption, Appendix II B, in the range 220 to 350 nm of a freshly prepared 0.002%
w/v solution in 0.05M sulphuric acid exhibits a maximum at 227 nm with a series of ill-defined
maxima between 258 and 272 nm. The absorbance at 227 nm is about 0.87.
C. Melting point, about 107, Appendix V A.
Alkalinity Shake 1 g with 25 ml of carbon dioxide-free water for 5 minutes and filter. The pH of the
filtrate is 7.6 to 8.6, Appendix V L.
Clarity of solution A 1.0% w/v solution in ether and a 1.0% w/v solution in 2M hydrochloric acid
are clear, Appendix IV A.
Chloride Dissolve 0.20 g in 2 ml of methanol and dilute to 30 ml with 2M nitric acid. 15 ml of the
Related substances Carry out the method for thin-layer chromatography, Appendix III A, using
silica gel G as the coating substance and as the mobile phase the lower layer obtained after shaking
together a mixture of 2 volumes of 13.5M ammonia, 8 volumes of methanol and 90 volumes of
dichloromethane and allowing the layers to separate. Apply separately to the plate 20 l of each of
four freshly prepared solutions in methanol containing (1) 2.0% w/v of the substance being exam-
ined, (2) 0.010% w/v of the substance being examined, (3) 0.010% w/v of N-methylpiperazine and
(4) 0.10% w/v of each of the substance being examined and hydroxyzine hydrochloride EPCRS. After
removal of the plate, allow it to dry in air and expose to iodine vapour for 10 minutes. Any spot
corresponding to N-methylpiperazine in the chromatogram obtained with solution (1) is not more
intense than the spot in the chromatogram obtained with solution (3) (0.5%). Any other secondary
spot in the chromatogram obtained with solution (1) is not more intense than the spot in the
chromatogram obtained with solution (2) (0.5%). The test is not valid unless the chromatogram
obtained with solution (4) shows two clearly separated spots.
1 g.
C
18
H
22
N
2
.
Storage Cyclizine should be kept in a well-closed container.
Action and use Antiemetic.
Preparation
Cyclizine Injection
12-16
Cyclizine Hydrochloride
N
NMe
,HCl
C
18
H
22
N
2
,HCl 302.8 305-25-3
Cyclizine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
1
Preparations
Cyclizine Injection
Cyclizine Tablets
Dipipanone and Cyclizine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cyclizine hydrochloride contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of 1-(diphenylmethyl)-4-methylpiperazine hydrochloride, calculated with reference
CHARACTERS
A white, crystalline powder, slightly soluble in water and in alcohol, practically insoluble in ether.
IDENTIFICATION
A. Dissolve 20.0 mg in a 5 g/l solution of sulphuric acid R and dilute to 100.0 ml with the same acid
solution (solution A). Examined between 240 nm and 350 nm (2.2.25), solution A shows two
absorption maxima, at 258 nm and 262 nm. The ratio of the absorbance measured at the
maximum at 262 nm to that measured at the maximum at 258 nm is 1.0 to 1.1. Dilute 10.0 ml of
solution A to 100.0 ml with a 5 g/l solution of sulphuric acid R (solution B). Examined between
210 nm and 240 nm, solution B shows an absorption maximum at 225 nm. The specific
absorbance at the maximum is 370 to 410. Verify the resolution of the apparatus (2.2.25); the test
is not valid unless the ratio of the absorbances is at least 1.7.
obtained with cyclizine hydrochloride CRS. Examine the substances prepared as discs using potassium
chloride R.
C. Examine the chromatograms obtained in the test for related substances. The principal spot in
D. Dissolve 0.5 g in 10 ml of alcohol (60 per cent V/V) R using heat, if necessary. Cool in iced water.
Add 1 ml of dilute sodium hydroxide solution R and 10 ml of water R. Filter, wash the precipitate with
water R and dry it at 60C at a pressure not exceeding 0.7 kPa for 2 h. The melting point (2.2.14) is
105C to 108C.
TESTS
pH (2.2.3). Dissolve 0.5 g in a mixture of 40 volumes of alcohol R and 60 volumes of carbon dioxide-
free water R and dilute to 25 ml with the same mixture of solvents. The pH of the solution is 4.5 to
5.5.
Related substances Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R.
12-17
Reference solution (a). Dissolve 10 mg of cyclizine hydrochloride CRS in methanol R and dilute to
Reference solution (b). Dissolve 5 mg of methylpiperazine R in methanol R and dilute to 50 ml with the
same solvent.
Reference solution (d). Dissolve 10 mg of cyclizine hydrochloride CRS and 10 mg of hydroxyzine
volumes of concentrated ammonia R, 13 volumes of methanol R and 85 volumes of methylene
chloride R. Allow the plate to dry in air for 30 min. Expose the plate to iodine vapour for 10 min. In
the chromatogram obtained with test solution (a): any spot corresponding to 1-methylpiperazine is
cent); any spot, apart from the principal spot and any spot corresponding to 1-methylpiperazine, is
not more intense than the principal spot in the chromatogram obtained with reference solution (c)
(0.5 per cent). The test is not valid unless the chromatogram obtained with reference solution (d)
at 130C.
ASSAY
Dissolve 0.200 g in 15 ml of anhydrous formic acid R and add 40 ml of acetic anhydride R. Titrate
18
H
23
ClN
2
.
STORAGE
IMPURITIES
HN
NMe
A. 1-methylpiperazine.
__________________________________________________________________________________________________________ Ph Eur
12-18
Cyclopenthiazide
S
NH
N
O O
S
H
2
N
O O
Cl
H
C
13
H
18
ClN
3
O
4
S
2
379.9 742-20-1
Definition Cyclopenthiazide is 6-chloro-3-cyclopentylmethyl-3,4-dihydro-1,2,4-
benzothiadiazine-7-sulphonamide 1,1-dioxide. It contains not less than 98.0% and not more than
102.0% of C
13
H
18
ClN
3
O
4
S
2
Characteristics A white powder; odourless or almost odourless.
Practically insoluble in water; soluble in acetone and in ethanol (96%); very slightly soluble in
ether; practically insoluble in chloroform.
Identification
cyclopenthiazide (RS 077).
0.01M sodium hydroxide exhibits two maxima, at 273 nm and 320 nm. The absorbance at 273 nm is
about 0.88 and at 320 nm is about 0.12.
C. Carry out the method for thin-layer chromatography, Appendix III A, using silica gel GF
254
as the
coating substance and ethyl acetate as the mobile phase. Apply separately to the plate 5 l of each of
two solutions in acetone containing (1) 0.1% w/v of the substance being examined and (2) 0.1% w/v
of cyclopenthiazide BPCRS. After removal of the plate, dry it in a current of air, examine under
ultraviolet light (254 nm) and then reveal the spots by Method I. By each method of visualisation the
principal spot in the chromatogram obtained with solution (1) corresponds in colour and intensity
to that in the chromatogram obtained with solution (2).
silica gel G as the coating substance and ethyl acetate as the mobile phase. Apply separately to the
plate 5 l of each of two solutions of the substance being examined in acetone containing (1) 0.50%
w/v and (2) 0.0050% w/v. After removal of the plate, dry it in a current of air and reveal the spots
by Method I. Any secondary spot in the chromatogram obtained with solution (1) is not more intense
Use 1 g.
Assay Dissolve 0.5 g in 50 ml of butylamine and carry out Method II for non-aqueous titration,
Appendix VIII A, using 0.1M tetrabutylammonium hydroxide VS as titrant and magneson solution as
indicator; titrate to a pure blue end point. Each ml of 0.1M tetrabutylammonium hydroxide VS is
13
H
18
ClN
3
O
4
S
2
.
Preparation
Cyclopenthiazide Tablets
12-19
Cyclopentolate Hydrochloride
O
NMe
2
O
OH H
and enantiomer
C
17
H
25
NO
3
,HCl 327.9 5870-29-1
Cyclopentolate Hydrochloride complies with the requirements of the 3rd edition of the European
Preparation
Cyclopentolate Eye Drops
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cyclopentolate hydrochloride contains not less than 98.5 per cent and not more than the equiva-
lent of 101.5 per cent of 2-dimethylaminoethyl (RS)-2-(1-hydroxycyclopentyl)-2-phenylacetate
CHARACTERS
A white, crystalline powder, very soluble in water, freely soluble in alcohol.
IDENTIFICATION
A. Melting point (2.2.14) : 135C to 141C.
obtained with cyclopentolate hydrochloride CRS. Examine the substances as discs prepared using
potassium chloride R. If the spectra obtained show differences, dissolve the substance to be examined
and the reference substance separately in alcohol R, evaporate to dryness and record new spectra
using the residues.
the chromatogram obtained with test solution (b) is similar in position, fluorescence and size to the
TESTS
Related substances. Examine by thin-layer chromatography (2.2.27), using a suitable silica gel as
Test solution (a). Dissolve 0.20 g of the substance to be examined in alcohol R and dilute to 5 ml
Reference solution (a). Dilute 1 ml of test solution (a) to 200 ml with alcohol R.
Reference solution (b). Dissolve 10 mg of cyclopentolate hydrochloride CRS in alcohol R and dilute to
of 5 volumes of concentrated ammonia R, 15 volumes of water R, 30 volumes of butyl acetate R and
50 volumes of 2-propanol R. Allow the plate to dry in air. Spray with alcoholic solution of sulphuric
acid R and heat the plate at 120C for 30 min. Examine in ultraviolet light at 365 nm. Any spot in
than the spot in the chromatogram obtained with reference solution (a) (0.5 per cent).
12-20
ASSAY
17
H
26
ClNO
3
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
12-21
Cyclophosphamide
NH
P
O O
N[CH
2
CH
2
Cl]
2
C
7
H
15
Cl
2
N
2
O
2
P,H
2
O 279.1 6055-19-2
Cyclophosphamide complies with the requirements of the 3rd edition of the European Pharmacopoeia [0711].
Preparations
Cyclophosphamide Injection
Cyclophosphamide Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cyclophosphamide contains not less than 98.0 per cent and not more than the equivalent of
102.0 per cent of (RS)-2-[bis(2-chloroethyl)amino]tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide,
CHARACTERS
A white or almost white, crystalline powder, soluble in water, freely soluble in alcohol, slightly
soluble in ether.
IDENTIFICATION
substance to be examined and cyclophosphamide CRS and determine the melting point of the
mixture. The difference between the melting points (which are about 51C) is not greater than 2C.
obtained with cyclophosphamide CRS.
D. Dissolve 0.1 g in 10 ml of water R and add 5 ml of silver nitrate solution R; the solution remains
clear. Boil, a white precipitate is formed which dissolves in concentrated ammonia R and is
reprecipitated on the addition of dilute nitric acid R.
TESTS
solvent.
solution Y
6
(Method II, 2.2.2).
pH (2.2.3). The pH of solution S is 4.0 to 6.0, determined immediately after preparation of the
solution.
coating substance.
Test solution (a). Dissolve 0.10 g of the substance to be examined in alcohol R and dilute to 5 ml
Reference solution (a). Dissolve 10 mg of cyclophosphamide CRS in alcohol R and dilute to 5 ml with
the same solvent.
Reference solution (b). Dilute 0.1 ml of test solution (a) to 10 ml with alcohol R.
of 2 volumes of anhydrous formic acid R, 4 volumes of acetone R, 12 volumes of water R and 80
volumes of methyl ethyl ketone R. Dry the plate in a current of warm air and heat at 110C for
10 min. At the bottom of a chromatography tank, place an evaporating dish containing a 50 g/l
12-22
solution of potassium permanganate R and add an equal volume of hydrochloric acid R. Place the plate
whilst still hot in the tank and close the tank. Leave the plate in contact with the chlorine gas for
2 min. Withdraw the plate and place it in a current of cold air until the excess of chlorine is
removed and an area of coating below the points of application gives at most a very faint blue
colour with a drop of potassium iodide and starch solution R. Avoid prolonged exposure to cold air.
Spray with potassium iodide and starch solution R and allow to stand for 5 min. Any spot in the
than the spot in the chromatogram obtained with reference solution (b) (1.0 per cent). Disregard
any spot remaining at the starting-point.
Chlorides (2.4.4). Dissolve 0.15 g in water R and dilute to 15 ml with the same solvent. The
freshly prepared solution complies with the limit test for chlorides (330 ppm).
Phosphates (2.4.11). Dissolve 0.10 g in water R and dilute to 100 ml with the same solvent. The
solution complies with the limit test for phosphates (100 ppm).
mination of water.
ASSAY
Dissolve 0.100 g in 50 ml of a 1 g/l solution of sodium hydroxide R in ethylene glycol R and boil under
a reflux condenser for 30 min. Allow to cool and rinse the condenser with 25 ml of water R. Add
75 ml of 2-propanol R, 15 ml of dilute nitric acid R, 10.0 ml of 0.1M silver nitrate and 2.0 ml of ferric
ammonium sulphate solution R2 and titrate with 0.1M ammonium thiocyanate.
7
H
15
Cl
2
N
2
O
2
P.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
12-23
Cyproheptadine Hydrochloride
,HCl
N
Me
C
21
H
21
N,HCl,1H
2
O 350.9 969-33- 5
Cyproheptadine Hydrochloride complies with the requirements of the 3rd edition of the European
1
Preparation
Cyproheptadine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cyproheptadine hydrochloride contains not less than 98.5 per cent and not more than the equiva-
lent of 101.0 per cent of 4-(5H-dibenzo[a,d]cyclohepten-5-ylidene)-1-methylpiperidine hydro-
CHARACTERS
A white or slightly yellow, crystalline powder, slightly soluble in water, freely soluble in methanol,
sparingly soluble in alcohol, practically insoluble in ether.
IDENTIFICATION
A. Dissolve 50.0 mg in alcohol R and dilute to 50.0 ml with the same solvent. Dilute 2.0 ml of the
shows an absorption maximum at 286 nm. The specific absorbance at the maximum is 335 to 365,
obtained with cyproheptadine hydrochloride CRS. Examine the substances as mulls in liquid
paraffin R.
254
R as the coating
substance.
Test solution. Dissolve 25 mg of the substance to be examined in methanol R and dilute to 25 ml
Reference solution (a). Dissolve 10 mg of cyproheptadine hydrochloride CRS in methanol R and dilute
Reference solution (b). Dissolve 10 mg of imipramine hydrochloride CRS in methanol R and dilute to
10 ml with the same solvent. Dilute 1 ml to 2 ml with reference solution (a).
5 volumes of diethylamine R, 20 volumes of ether R and 75 volumes of cyclohexane R. Allow the plate
to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram
with reference solution (b) shows two clearly separated principal spots.
D. A saturated solution gives reaction (b) of chlorides (2.3.1).
TESTS
Acidity Dissolve 0.10 g in water R and dilute to 25 ml with the same solvent. Add 0.1 ml of methyl
12-24
red solution R. Not more than 0.15 ml of 0.01M sodium hydroxide is required to change the colour of
the indicator.
coating substance.
solvents.
Reference solution (a). Dissolve 10 mg of dibenzocycloheptene CRS (5H-dibenzo[a,d]cycloheptene) in
a mixture of 1 volume of methanol R and 9 volumes of methylene chloride R and dilute to 50 ml with
the same mixture of solvents. Dilute 1 ml to 10 ml with a mixture of 1 volume of methanol R and 9
volumes of methylene chloride R.
Reference solution (b). Dilute 1 ml of the test solution to 100 ml with a mixture of 1 volume of
methanol R and 9 volumes of methylene chloride R. Dilute 1 ml to 10 ml with a mixture of 1 volume
of methanol R and 9 volumes of methylene chloride R.
of 10 volumes of methanol R and 90 volumes of methylene chloride R. Allow the plate to dry in air
and spray with alcoholic solution of sulphuric acid R. Heat at 110C for 30 min and examine in ultra-
violet light at 365 nm while hot. In the chromatogram obtained with the test solution: any spot
corresponding to dibenzocycloheptene is not more intense than the spot in the chromatogram
obtained with reference solution (a) (0.2 per cent); any spot apart from the principal spot and any
spot corresponding to dibenzocycloheptene is not more intense than the spot in the chromatogram
at 100C to 105C at a pressure not exceeding 700 Pa.
ASSAY
Dissolve 0.250 g in a mixture of 5.0 ml of 0.01M hydrochloric acid and 50 ml of alcohol R. Carry out
a potentiometric titration (2.2.20), using 0.1M sodium hydroxide. Read the volume added between
the two points of inflexion.
21
H
22
ClN.
STORAGE
IMPURITIES
A. 5H-dibenzo[a,d]cycloheptene,
O
B. dibenzosuberone.
__________________________________________________________________________________________________________ Ph Eur
12-25
Cyproterone Acetate
corrected 1/01
O
Cl
H
H
H
Me
Me
CH
3
O
OAc
H
2
C
C
24
H
29
ClO
4
416.9 427-51-0
Cyproterone Acetate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Antiandrogen
Preparation
Cyproterone Tablets.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cyproterone acetate contains not less than 97.0 per cent and not more than the equivalent of
103.0 per cent of 6-chloro-1,2-methylene-3,20-dioxopregna-4,6-dien-17-yl acetate, calculated
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water, very soluble in
methylene chloride, freely soluble in acetone, soluble in methanol, sparingly soluble in ethanol.
IDENTIFICATION
obtained with cyproterone acetate CRS.
254
plate R.
Test solution. Dissolve 20 mg of the substance to be examined in methylene chloride R and dilute to
Reference solution. Dissolve 10 mg of cyproterone acetate CRS in methylene chloride R and dilute to
Apply to the plate 5 l of each solution. Develop over a path of 15 cm using a mixture of equal
volumes of cyclohexane R and ethyl acetate R. Allow the plate to dry in air. Repeat the development.
Allow the plate to dry in air. Examine in ultraviolet light at 254 nm. The principal spot in the
C. To about 1 mg add 2 ml of sulphuric acid R and heat on a water-bath for 2 min. A red colour
develops. Cool. Add the solution cautiously to 4 ml of water R and shake. The solution becomes
violet.
D. Incinerate about 30 mg with 0.3 g of anhydrous sodium carbonate R over a naked flame for about
10 min. Cool and dissolve the residue in 5 ml of dilute nitric acid R. Filter. To 1 ml of the filtrate
add 1 ml of water R. The solution gives reaction (a) of chlorides (2.3.1).
E. It gives the reaction of acetyl (2.3.1).
TESTS
Specific optical rotation (2.2.7). Dissolve 0.25 g in acetone R and dilute to 25.0 ml with the same
substance.
12-26
Test solution. Dissolve 10.0 mg of the substance to be examined in acetonitrile R and dilute to
Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with acetonitrile R.
Reference solution (b). Dissolve 5 mg of medroxyprogesterone acetate CRS in acetonitrile R and dilute to
50.0 ml with the same solvent. Dilute 1.0 ml of the solution to 10.0 ml with reference solution (a).
volumes of water R,
Inject 20 l of reference solution (a) and 20 l of reference solution (b). Adjust the sensitivity of
solution (a) is at least 50 per cent of the full scale of the recorder. The test is not valid unless, in the
chromatogram obtained with reference solution (b), the resolution between the peak corresponding
to cyproterone acetate and the peak corresponding to medroxyprogesterone acetate is at least 3.0.
cyproterone acetate. In the chromatogram obtained with the test solution, the sum of the areas of
Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying at 80C at
a pressure not exceeding 0.7 kPa.
ASSAY
Dissolve 50.0 mg in methanol R and dilute to 50.0 ml with the same solvent. Dilute 1.0 ml of the
solution to 100.0 ml with methanol R. Measure the absorbance (2.2.25) at the maximum at
282 nm.
24
H
29
ClO
4
STORAGE
IMPURITIES
O
H H
H Me
Me
R
H
2
C
O CH
3
OAc
A. R = H: 1,2-methylene-3,20-dioxopregna-4,6-dien-17-yl acetate,
B. R = OCH
3
: 6-methoxy-1,2-methylene-3,20-dioxopregna-4,6-dien-17-yl acetate.
__________________________________________________________________________________________________________ Ph Eur
12-27
Cysteine Hydrochloride
HS
COOH
NH
2
H
,HCl
C
3
H
7
NO
2
S,HCl,H
2
O 175.6 7048-04-6
Cysteine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Cysteine Hydrochloride Monohydrate [0895]. These requirements are reproduced after the heading
Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cysteine hydrochloride monohydrate contains not less than 98.5 per cent and not more than the
equivalent of 101.0 per cent of (R)-2-amino-3-mercaptopropanoic acid hydrochloride, calculated
PRODUCTION
When Cysteine Hydrochloride Monohydrate is produced by a process involving fermentation steps,
it complies with the requirements of the monograph on Products of Fermentation (1468).
CHARACTERS
A white, colourless powder or colourless crystals, freely soluble in water, slightly soluble in alcohol,
IDENTIFICATION
obtained with cysteine hydrochloride monohydrate CRS. Examine the substances prepared as discs.
C. Examine the chromatograms obtained in the test for ninhydrin-positive substances. The prin-
cipal spot in the chromatogram obtained with test solution (b) is similar in position, colour, and
size to the principal spot in the chromatogram obtained with reference solution (b).
D. Dissolve about 5 mg in 1 ml of dilute sodium hydroxide solution R. Add 1 ml of a 30 g/l solution of
sodium nitroprusside R. An intense violet colour develops which becomes brownish-red and then
orange. Add 1 ml of hydrochloric acid R. The solution becomes green.
TESTS
6
(Method II, 2.2.2).
with the same acid. The specific optical rotation is +5.5 to +7.0, calculated with reference to the
dried substance.
silica gel plate R.
Test solution (a). Dissolve 0.20 g of the substance to be examined in water R and dilute to 10 ml
with the same solvent. To 5 ml of the solution add 5 ml of a 20 g/l solution of N-ethylmaleimide R in
alcohol R. Allow to stand for 5 min.
Reference solution (a). Dissolve 20 mg of cysteine hydrochloride monohydrate CRS in water R and
dilute to 10 ml with the same solvent. Add 10 ml of a 20 g/l solution of N-ethylmaleimide R in
alcohol R. Allow to stand for 5 min.
Reference solution (c). Dilute 5 ml of test solution (b) to 20 ml with water R.
Reference solution (d). Dissolve 10 mg of tyrosine CRS in 10 ml of reference solution (a) and dilute to
25 ml with water R.
12-28
Apply separately to the plate 5 l of each test solution and reference solutions (b), (c), and (d).
Develop over a path of 15 cm using a mixture of 20 volumes of glacial acetic acid R, 20 volumes of
water R and 60 volumes of butanol R. Allow the plate to dry in air. Spray with ninhydrin solution R
and heat at 100C to 105C for 15 min. Any spot in the chromatogram obtained with test solution
with reference solution (c) (0.5 per cent). The test is not valid unless the chromatogram obtained
with reference solution (d) shows two clearly separated principal spots.
Sulphates (2.4.13). Dilute 10 ml of solution S to 15 ml with distilled water R. The solution
complies with the limit test for sulphates (300 ppm).
the lower watch-glass and dissolve it in 0.5 ml of water R. To the solution add 0.30 g of heavy
4
) R, 0.5 ml of water R and 0.30 g of heavy magnesium
oxide R (200 ppm).
Heavy metals (2.4.8). Dissolve 2.0 g in water R. Adjust the pH to 3 to 4 with concentrated
ammonia R and dilute to 20 ml with water R. 12 ml of the solution complies with limit test A for
Loss on drying (2.2.32). 8.0 per cent to 12.0 per cent, determined on 1.000 g by drying at a
ASSAY
In a flask with a ground-glass stopper dissolve 0.300 g of the substance to be examined and 4 g of
potassium iodide R in 20 ml of water R. Cool the solution in iced water and add 3 ml of hydrochloric
acid R1 and 25.0 ml of 0.05M iodine. Stopper the flask and allow to stand in the dark for 20 min.
Titrate with 0.1M sodium thiosulphate using 3 ml of starch solution R, added towards the end of the
titration, as indicator. Carry out a blank titration.
3
H
8
ClNO
2
S.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
12-29
Cystine
HOOC
S
S
COOH
NH
2
H
H NH
2
C
6
H
12
N
2
O
4
S
2
240.3 56-89-3
Cystine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0988]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cystine contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of
(R,R)-3,3-dithiobis(2-aminopropanoic acid), calculated with reference to the dried substance.
PRODUCTION
When Cystine is produced by a process involving fermentation steps, it complies with the
requirements of the monograph on Products of Fermentation (1468).
CHARACTERS
A white, crystalline powder, practically insoluble in water and in alcohol. It dissolves in dilute
IDENTIFICATION
obtained with cystine CRS. Examine the substances prepared as discs.
C. Examine the chromatograms obtained in the test for ninhydrin-positive substances. The prin-
cipal spot in the chromatogram obtained with test solution (b) is similar in position, colour and size
to the principal spot in the chromatogram obtained with reference solution (a).
D. To 0.1 g carefully add 1 ml of strong hydrogen peroxide solution R and 0.1 ml of ferric chloride
solution R1. Allow to cool. Add 1 ml of dilute hydrochloric acid R and 5 ml of water R. Add 1 ml of
barium chloride solution R1. Turbidity or a white precipitate develops within 3 min.
TESTS
Appearance of solution Dissolve 1.0 g in dilute hydrochloric acid R and dilute to 10 ml with the
same acid. The solution is clear (2.2.1) and not more intensely coloured than reference solution Y
7
(Method II, 2.2.2).
Specific optical rotation (2.2.7). Dissolve 0.50 g in 1M hydrochloric acid and dilute to 25.0 ml
dried substance.
silica gel plate R.
Test solution (a). Dissolve 0.10 g of the substance to be examined in 1M hydrochloric acid and dilute
to 10 ml with the same acid.
Reference solution (a). Dissolve 10 mg of cystine CRS in 1 ml of 1M hydrochloric acid and dilute to
50 ml with water R.
Reference solution (c). Dissolve 10 mg of cystine CRS and 10 mg of arginine hydrochloride CRS in 1 ml
of 1M hydrochloric acid and dilute to 25 ml with water R.
30 volumes of concentrated ammonia R and 70 volumes of 2-propanol R. Allow the plate to dry in air.
Spray with ninhydrin solution R and heat at 100C to 105C for 15 min. Any spot in the chromato-
gram obtained with test solution (a), apart from the principal spot, is not more intense than the
spot in the chromatogram obtained with reference solution (b) (0.2 per cent). The test is not valid
unless the chromatogram obtained with reference solution (c) shows two clearly separated spots.
12-30
The solution, without further addition of nitric acid, complies with the limit test for chlorides
(200 ppm).
Ammonium (2.4.1). 0.10 g complies with limit test B for ammonium (200 ppm). Prepare the
4
) R.
at 100C to 105C.
ASSAY
In a flask with a ground-glass stopper, dissolve 0.100 g in a mixture of 2 ml of dilute sodium
hydroxide solution R and 10 ml of water R. Add 10 ml of a 200 g/l solution of potassium bromide R,
50.0 ml of 0.0167M potassium bromate and 15 ml of dilute hydrochloric acid R. Stopper the flask and
cool in iced water. Allow to stand in the dark for 10 min. Add 1.5 g of potassium iodide R. After
1 min, titrate with 0.1M sodium thiosulphate, using 2 ml of starch solution R, added towards the end-
point, as indicator. Carry out a blank titration.
6
H
12
N
2
O
4
S
2
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
12-31
Cytarabine
N
N
NH
2
O
HOCH
2
HO
HO
O
C
9
H
13
N
3
O
5
243.2 147-94-4
Cytarabine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0760]. These
Preparation
Cytarabine Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Cytarabine contains not less than 99.0 per cent and not more than the equivalent of 100.5 per cent
of 4-amino-1--D-arabinofuranosyl-1H-pyrimidin-2-one, calculated with reference to the dried
substance.
CHARACTERS
A white or almost white, crystalline powder, freely soluble in water, very slightly soluble in alcohol
IDENTIFICATION
350 nm (2.2.25), the solution shows an absorption maximum at 281 nm. The specific absorbance
at the maximum is 540 to 570.
obtained with cytarabine CRS. Examine the substances prepared as discs.
254 nm. The principal spot in the chromatogram obtained with test solution (b) is similar in
position and size to the principal spot in the chromatogram obtained with reference solution (a).
TESTS
5
(Method II,
2.2.2).
substance.
254
R as the
coating substance.
the same solvent.
Reference solution (a). Dissolve 10 mg of cytarabine CRS in water R and dilute to 5 ml with the same
solvent.
Reference solution (b). Dilute 0.5 ml of test solution (a) to 100 ml with water R.
Reference solution (c). Dissolve 20 mg of uridine R and 20 mg of uracil arabinoside CRS in methanol R
12-32
15 volumes of water R, 20 volumes of acetone R and 65 volumes of methyl ethyl ketone R. Allow the
plate to dry in air and examine in ultraviolet light at 254 nm. Any spot in the chromatogram
obtained with test solution (a), apart from the principal spot, is not more intense than the spot in
the chromatogram obtained with reference solution (b) (0.5 per cent). The test is not valid unless
the chromatogram obtained with reference solution (c) shows two clearly separated spots.
diphosphorus pentoxide R at 60C at a pressure of 0.2 kPa to 0.7 kPa for 3 h.
ASSAY
Dissolve 0.200 g in 60 ml of anhydrous acetic acid R, warming if necessary. Titrate with 0.1M
perchloric acid determining the end-point potentiometrically (2.2.20).
9
H
13
N
3
O
5
.
STORAGE
IMPURITIES
A. uracil arabinoside,
B. uridine.
__________________________________________________________________________________________________________ Ph Eur
12-33
Dacarbazine
N
N
N
CONH
2
N NMe
2
H
C
6
H
10
N
6
O 182.2 4342-03-4
Definition Dacarbazine is 5-(3,3-dimethyltriazeno)imidazole-4-carboxamide. It contains not less
6
H
10
N
6
O, calculated with reference to the dried
substance.
Characteristics A colourless or pale yellow, crystalline powder.
Slightly soluble in water and in ethanol (96%).
Identification
dacarbazine (RS 082).
0.1M hydrochloric acid exhibits a maximum at 323 nm and a pronounced shoulder at 275 nm. The
absorbance at 323 nm is about 0.64.
Clarity and colour of solution A 2.0% w/v solution in 0.1M citric acid is clear, Appendix IV A,
6
5-Aminoimidazole-4-carboxamide hydrochloride Carry out the following procedure protected
from light and inject samples within 1 hour of preparation. Use low-actinic glassware. Carry out the
method for liquid chromatography, Appendix III D, using two solutions in 0.1M acetic acid containing
(1) 0.40% w/v of the substance being examined and (2) 0.0024% w/v of 5-aminoimidazole-4-
carboxamide hydrochloride.
The chromatographic procedure described under Related substances may be used but using
0.005M dioctyl sodium sulphosuccinate in a mixture of 3 volumes of glacial acetic acid, 87 volumes of
water and 110 volumes of methanol as the mobile phase.
The area of any peak corresponding to 5-aminoimidazole-4-carboxamide hydrochloride in the
chromatogram obtained with solution (1) is not greater than the area of the peak in the chromato-
gram obtained with solution (2) (0.6%).
Related substances Carry out the following procedure protected from light and inject samples
within 1 hour of preparation. Carry out the method for liquid chromatography, Appendix III D,
using two solutions in 0.25M acetic acid containing (1) 0.40% w/v of the substance being examined
and (2) 0.0040% w/v of 2-azahypoxanthine BPCRS.
The chromatographic procedure may be carried out using (a) a stainless steel column (20 cm 4
mm) packed with stationary phase C (10 m) (Nucleosil C18 is suitable), (b) 0.005M dioctyl sodium
sulphosuccinate in a mixture of 1.5 volumes of glacial acetic acid and 98.5 volumes of water as the
mobile phase with a flow rate of 1.5 ml per minute and (c) a detection wavelength of 254 nm. After
use the column should be thoroughly flushed with methanol to remove dacarbazine which does not
elute with the mobile phase.
the area of the principal peak in the chromatogram obtained with solution (2) (1%), not more than
one such peak has an area greater than half the area of the peak in the chromatogram obtained with
solution (2) (0.5%) and the sum of the areas of all such peaks is not greater than three times the
area of the peak in the chromatogram obtained with solution (2) (3%).
6
H
10
N
6
O.
Storage Dacarbazine should be kept in a well-closed container, protected from light and stored at
a temperature of 2 to 8.
Preparation
Dacarbazine Injection
12-34
Dalteparin Sodium
O
O
OH
NaO
3
SO
O
OR
O
NHR'
O
O
OR
R
2
O
HO
OSO
3
Na
HO
OH
R
3
OR
CO
2
Na
OH
n
n = 3 to 20, R = H or SO
3
Na, R = SO
3
Na or COCH
3
R
2
= H and R
3
= CO
2
Na or R
2
= CO
2
Na and R
3
= H
Dalteparin Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia [1195].
Action and use Low-molecular-weight heparin; anticoagulant.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Dalteparin sodium is the sodium salt of a low-molecular-mass heparin that is obtained by nitrous
acid depolymerisation of heparin from porcine intestinal mucosa. The majority of the components
have a 2-O-sulpho--L-idopyranosuronic acid structure at the non-reducing end and a 6-O-sulpho-
2,5-anhydro-D-mannitol structure at the reducing end of their chain.
Dalteparin sodium complies with the monograph on Low-molecular-mass heparins (828) with the
modifications and additional requirements below:
The mass-average molecular mass ranges between 5600 and 6400, with a characteristic value of
about 6000.
The degree of sulphatation is 2.0 to 2.5 per disaccharide unit.
The potency is not less than 110 I.U. and not more than 210 I.U. of anti-factor Xa activity per
milligram, calculated with reference to the dried substance. The anti-factor IIa activity is not less
than 35 I.U. and not more than 100 I.U. per milligram, calculated with reference to the dried
substance. The ratio of anti-factor Xa activity to anti-factor IIa activity is between 1.9 and 3.2.
PRODUCTION
Dalteparin sodium is produced by a validated manufacturing and purification procedure under
conditions designed to minimise the presence of NNO groups.
The manufacturing procedure must have been shown to reduce any contamination by NNO
groups to approved limits using an appropriate, validated quantification method.
IDENTIFICATION
Carry out identification test C as described in the monograph on Low-molecular-mass heparins
(828). The following requirements apply:
The mass-average molecular mass ranges between 5600 and 6400. The mass percentage of chains
lower than 3000 is not more than 13.0 per cent. The mass percentage of chains higher than 8000
ranges between 15.0 per cent and 25.0 per cent.
TESTS
Appearance of solution Dissolve 1 g in 10 ml of water R. The solution is clear (2.2.1) and not
more intensely coloured than intensity 5 of the range of reference solutions of the most appropriate
colour (Method II, 2.2.2).
Nitrite Not more than 5 ppm. Examine by liquid chromatography (2.2.29).
Rinse all volumetric flasks at least three times with water R before the preparation of the solutions.
Test solution. Dissolve 80.0 mg of the substance to be examined in water R and dilute to 10.0 ml
with the same solvent. Allow to stand for at least 30 min.
Reference solution (a). Dissolve 60.0 mg of sodium nitrite R in water R and dilute to 1000.0 ml with
the same solvent.
For the preparation of reference solution (b), use a pipette previously rinsed with reference solution (a).
12-35
Before preparing reference solutions (c), (d) and (e), rinse all pipettes with reference solution (b).
Reference solution (c). Dilute 1.00 ml of reference solution (b) to 100.0 ml with water R (1 ppm
nitrite).
Reference solution (d). Dilute 3.00 ml of reference solution (b) to 100.0 ml with water R (3 ppm
nitrite).
Reference solution (e). Dilute 5.00 ml of reference solution (b) to 100.0 ml with water R (5 ppm
nitrite).
a column 0.125 m long and 4.3 mm in internal diameter, packed with a strong anion-exchange
resin,
as mobile phase at a flow rate of 1.0 ml per minute a solution consisting of 13.61 g of sodium
acetate R dissolved in water R, adjusted to pH 4.3 with phosphoric acid R and diluted to 1000 ml
with water R,
as detector an appropriate electrochemical device with the following characteristics and settings:
a suitable working electrode, a detector potential of +1.00 V versus Ag/AgCl reference electrode
and a detector sensitivity of 0.1 A full scale.
Inject 100 l of reference solution (d). When the chromatograms are recorded in the prescribed
conditions, the retention time for nitrite is 3.3 to 4.0 min. The test is not valid unless:
the number of theoretical plates calculated for the nitrite peak is at least 7000 per metre per
column (dalteparin sodium will block the binding sites of the stationary phase, which will cause
shorter retention times and lower separation efficiency for the analyte; the initial performance of
the column may be partially restored using a 58 g/l solution of sodium chloride R at a flow rate of
1.0 ml per minute for 1 h; after regeneration the column is rinsed with 200 ml to 400 ml of
water R),
the symmetry factor for the nitrite peak is less than three,
the relative standard deviation of the peak area for nitrite obtained from six injections is less than
3.0 per cent.
Inject 100 l each of reference solutions (c) and (e). The test is not valid unless:
the correlation factor for a linear relationship between concentration and response for reference
solutions (c), (d) and (e) is at least 0.995,
the signal-to-noise ratio for reference solution (c) is not less than 5 (if the noise level is too high,
electrode recalibration is recommended),
a blank injection of water R does not give rise to spurious peaks.
Inject 100 l of the test solution. Calculate the content of nitrite from the peak areas in the
chromatogram obtained with reference solutions (c), (d) and (e).
Boron Not more than 1 ppm, determined by inductively coupled plasma atomic emission
spectroscopy.
Boron is determined by measurement of the emission from an inductively coupled plasma (ICP)
at a wavelength specific to boron. The emission line at 249.733 nm is used. Use an appropriate
apparatus, whose settings have been optimised as directed by the manufacturer.
Test solution. Dissolve 0.2500 g of the substance to be examined in about 2 ml of water for chromato-
graphy R, add 100 l of nitric acid R and dilute to 10.00 ml with the same solvent.
Reference solution (a). Prepare an 1 per cent V/V solution of nitric acid R in water for chromato-
graphy R (blank).
Reference solution (b). Prepare a 11.4 g/ml solution of boric acid R in a 1 per cent V/V solution of
nitric acid R in water for chromatography R (STD
CAL
).
Reference solution (c). Dissolve 0.2500 g of a reference dalteparin sodium with no detectable boron
in about 2 ml of water for chromatography R, add 100 l of nitric acid R and dilute to 10.00 ml with
the same solvent (STD
0
).
Reference solution (d). Dissolve 0.2500 g of a reference dalteparin sodium with no boron detected in
about 2 ml of a 1 per cent V/V solution of nitric acid R in water for chromatography R, add 10 l of a
5.7 mg/ml solution of boric acid R and dilute to 10.00 ml with the same solvent (STD
1
). This
solution contains 1 g/ml of boron.
Calculate the content of boron in the substance to be examined, using the following correction
factor:
f [ ] / [ ] STD STD STD blank
CAL 1 0
2
at 60C over diphosphorus pentoxide R at a pressure not exceeding 670 Pa for 3 h.
__________________________________________________________________________________________________________ Ph Eur
12-36
Dantron
OH OH O
O
C
14
H
8
O
4
240.2 117-10-2
Definition Dantron is mainly 1,8-dihydroxyanthraquinone. It contains not less than 98.0% and
not more than 102.0% of total phenols, calculated as C
14
H
8
O
4
and with reference to the dried
substance.
Characteristics An orange, crystalline powder; odourless or almost odourless.
Practically insoluble in water; soluble in chloroform; slightly soluble in ether; very slightly soluble in
ethanol (96%). It dissolves in solutions of alkali hydroxides.
Identification
dantron (RS 083).
chloroform exhibits maxima at 255 nm and 285 nm and a less well-defined maximum at 275 nm.
The absorbance at the maximum at 255 nm is about 0.82 and at the maximum at 285 nm is about
0.48, each calculated with reference to the dried substance.
C. Dissolve 5 mg in 5 ml of 1M sodium hydroxide. A clear red solution is produced immediately.
Mercury To 0.50 g in a Kjeldahl flask add 2.5 ml of nitric acid and allow to stand until the initial
vigorous reaction has subsided. Add 2.5 ml of sulphuric acid and heat until dense white fumes are
evolved. Cool, add 2.5 ml of nitric acid and heat until fumes are again evolved. Repeat the
procedure with a further 2.5 ml of nitric acid, cool, add 50 ml of water, boil the solution until the
volume has been reduced to about 25 ml and cool. Transfer to a separating funnel using water,
dilute to about 50 ml with water and add 50 ml of 0.5M sulphuric acid. Add 100 ml of water, 2 g of
hydroxylamine hydrochloride, 1 ml of 0.05M disodium edetate, 1 ml of glacial acetic acid and 5 ml of
chloroform, shake, allow to separate and discard the chloroform layer. Titrate the aqueous layer with
a 0.0008% w/v solution of dithizone in chloroform, shaking vigorously after each addition, allowing
the layers to separate and discarding the chloroform layer, until the chloroform layer remains green.
Repeat the operation using a solution prepared by diluting 1 ml of mercury standard solution (5 ppm
Hg) to 100 ml with 0.5M sulphuric acid and beginning at the words Add 100 ml of water . The
volume of the dithizone solution required by the substance being examined does not exceed that
required by the mercury standard solution.
following solutions. For solution (1) dissolve 50 mg of the substance being examined in 20 ml of
tetrahydrofuran and dilute to 100 ml with the mobile phase. For solution (2) dilute 1 volume of
solution (1) to 50 volumes with the mobile phase. For solution (3) dissolve 50 mg of dantron
impurity standard BPCRS in 20 ml of tetrahydrofuran and dilute to 100 ml with the mobile phase.
(20 cm 4.6 mm) packed with stationary phase C (5 m) (Nucleosil C18 is suitable), (b) a mixture
of 2.5 volumes of glacial acetic acid, 40 volumes of tetrahydrofuran and 60 volumes of water as the
mobile phase with a flow rate of 1 ml per minute and (c) a detection wavelength of 254 nm. For
solution (2) allow the chromatography to proceed for 1.5 times the retention time of the principal
peak.
The test is not valid unless, in the chromatogram obtained with solution (3), a peak due to
1-hydroxyanthraquinone appears immediately before the principal peak, as indicated in the
reference chromatogram supplied with dantron impurity standard BPCRS, and the height of the
trough separating the two peaks is not greater than one third of the height of the peak due to 1-
hydroxyanthraquinone.
1-hydroxyanthraquinone is not greater than 2.5 times the area of the principal peak in the chroma-
togram obtained with solution (2) (3.3%, taking into account the relative response factor of the
impurity) and the sum of the areas of any other secondary peaks is not greater than the area of the
principal peak in the chromatogram obtained with solution (2) (2%). Disregard any peak with a
retention time less than one third of that of the principal peak.
Use 1 g.
12-37
Assay Dissolve 0.2 g in 50 ml of anhydrous pyridine and carry out Method II for non-aqueous
titration, Appendix VIII A, using 0.1M tetrabutylammonium hydroxide VS as titrant and determining
the end point potentiometrically. Each ml of 0.1M tetrabutylammonium hydroxide VS is equivalent to
24.02 mg of total phenols, calculated as C
14
H
8
O
4
.
Preparation
Co-danthrusate Capsules
When danthron is prescribed or demanded, Dantron shall be dispensed or supplied.
12-38
Dapsone
S
O O
NH
2
H
2
N
C
12
H
12
N
2
O
2
S 248.3 80-08-0
Dapsone complies with the requirements of the 3rd edition of the European Pharmacopoeia [0077]. These
Action and use Antileprotic.
Preparation
Dapsone Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Dapsone contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of
4,4-sulphonyldianiline, calculated with reference to the dried substance.
CHARACTERS
A white or slightly yellowish-white, crystalline powder, very slightly soluble in water, freely soluble
in acetone, sparingly soluble in alcohol. It dissolves freely in dilute mineral acids.
IDENTIFICATION
B. Dissolve 50.0 mg in methanol R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of
this solution to 100.0 ml with methanol R. Examined between 230 nm and 350 nm (2.2.25), the
solution shows two absorption maxima, at 260 nm and 295 nm. The specific absorbances at these
maxima are 700 to 760 and 1150 to 1250, respectively.
TESTS
coating substance.
Reference solution (a). Dissolve 10 mg of dapsone CRS in methanol R and dilute to 10 ml with the
same solvent.
Apply separately to the plate 1 l of test solution (b), 1 l of reference solution (a), 10 l of test
solution (a), 10 l of reference solution (b) and 10 l of reference solution (c). Develop in an
unsaturated tank over a path of 15 cm using a mixture of 1 volume of concentrated ammonia R, 6
volumes of methanol R, 20 volumes of ethyl acetate R and 20 volumes of heptane R. Allow the plate
to dry in air. Spray the plate with a 1 g/l solution of dimethylaminocinnamaldehyde R in a mixture of
1 volume of hydrochloric acid R and 99 volumes of alcohol R. Examine in daylight. Any spot in the
than the spot in the chromatogram obtained with reference solution (b) (1.0 per cent) and not
more than two such spots are more intense than the spot in the chromatogram obtained with refer-
at 100C to 105C.
12-39
ASSAY
Dissolve 0.100 g in 50 ml of dilute hydrochloric acid R. Carry out the determination of primary
aromatic amino-nitrogen (2.5.8).
1 ml of 0.1M sodium nitrite is equivalent to 12.42 mg of C
12
H
12
N
2
O
2
S.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
12-40
Daunorubicin Hydrochloride
revised 4/01
O
O
O
CH
3
OH
OH
O
O
HO
Me
NH
2
H
MeO
OH
,HCl
C
27
H
30
ClNO
10
564.0 23541-50-6
Daunorubicin Hydrochloride complies with the requirements of the 3
rd
edition of the European Pharmacopoeia
Action and use Antineoplastic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
(8S,10S)-8-Acetyl-10-[(3-amino-2,3,6-trideoxy--L-lyxo-hexopyranosyl)oxy]-6,8,11-trihydroxy-
1-methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione hydrochloride, a substance produced by
certain strains of Streptomyces coeruleorubidus or of Streptomyces peucetius or obtained by any other
means.
Content: 95.0 per cent to 102.0 per cent (anhydrous and solvent-free substance).
PRODUCTION
It is produced by methods of manufacture designed to eliminate or minimise the presence of
histamine.
CHARACTERS
Appearance: crystalline, orange-red powder, hygroscopic.
Solubility: freely soluble in water and in methanol, slightly soluble in alcohol, practically insoluble in
acetone.
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
Comparison: daunorubicin hydrochloride CRS.
B. Dissolve about 10 mg in 0.5 ml of nitric acid R, add 0.5 ml of water R and heat over a flame for
2 min. Allow to cool and add 0.5 ml of silver nitrate solution R1. A white precipitate is formed.
TESTS
pH (2.2.3): 4.5 to 6.5.
Dissolve 50 mg in carbon dioxide-free water R and dilute to 10 ml with the same solvent.
Reference solution (a). Dissolve 50.0 mg of daunorubicin hydrochloride CRS in the mobile phase and
Reference solution (b). Dissolve 10 mg of doxorubicin hydrochloride CRS and 10 mg of epirubicin
hydrochloride CRS in the mobile phase and dilute to 100.0 ml with the mobile phase. Dilute 1.0 ml
of the solution to 10.0 ml with the mobile phase.
Reference solution (c). Dissolve 5.0 mg of daunorubicinone CRS and 5.0 mg of doxorubicin
hydrochloride CRS in the mobile phase and dilute to 100.0 ml with the mobile phase. Dilute 1.0 ml
12-41
of the solution to 10.0 ml with the mobile phase.
Reference solution (d). Dilute 1.0 ml of reference solution (a) to 200.0 ml with the mobile phase.
Column:
size: l = 0.25 m, = 4.0 mm,
stationary phase: end-capped octadecylsilyl silica gel for chromatography R (5 m).
Mobile phase: a mixture of equal volumes of acetonitrile R and a solution containing 2.88 g/l of
sodium laurilsulfate R and 2.25 g/l of phosphoric acid R.
Injection: 5l; inject the test solution and reference solutions (b), (c), and (d).
Run time: twice the retention time of daunorubicin.
Relative retention with reference to daunorubicin (retention time = about 15 min): impurity A =
about 0.4; impurity D = about 0.5; epirubicin = about 0.6; impurity B = about 0.7.
System suitability: reference solution (b):
resolution: minimum 2.0 between the peaks due to impurity D and to epirubicin.
Limits:
impurity A: not more than the area of the corresponding peak in the chromatogram obtained
with reference solution (c) (0.5 per cent),
impurity B: not more than 3 times the area of the principal peak in the chromatogram obtained
with reference solution (d) (1.5 per cent),
impurity D: not more than the area of the corresponding peak in the chromatogram obtained
with reference solution (c) (0.5 per cent),
any other impurity: not more than the area of the principal peak in the chromatogram obtained
with reference solution (d) (0.5 per cent),
total of other impurities: not more than 5 times the area of the principal peak in the chromatogram
obtained with reference solution (d) (2.5 per cent),
reference solution (d) (0.05 per cent).
Butanol (2.4.24, System B): maximum 1.0 per cent.
Water (2.5.12): maximum 3.0 per cent, determined on 0.100 g.
Sterility (2.6.1). If intended for use in the manufacture of parenteral dosage forms without a
Bacterial endotoxins (2.6.14): less than 4.3 IU per milligram, if intended for use in the
manufacture of parenteral dosage forms without a further appropriate procedure for the removal of
bacterial endotoxins.
ASSAY
Injection: test solution and reference solution (a).
27
H
30
ClNO
10
.
STORAGE
In an airtight container, protected from light. If the substance is sterile, store in a sterile, airtight,
tamper-proof container.
LABELLING
The label states:
IMPURITIES
OMe O
O OH
OH
R
OH
OH H
A. R = CO-CH
3
: (8S,10S)-8-acetyl-6,8,10,11-tetrahydroxy-1-methoxy-7,8,9,10-
tetrahydrotetracene-5,12-dione (daunorubicin aglycone, daunorubicinone),
E. R = CHOH-CH
3
: (8S,10S)-6,8,10,11-tetrahydroxy-8-[(1RS)-1-hydroxyethyl]-1-methoxy-
12-42
7,8,9,10-tetrahydrotetracene-5,12-dione (13-dihydrodaunorubicinone),
OMe O
O OH
OH
R
OH
O H
O
HO
NH
2
Me
B. R = CHOH-CH
3
: (8S,10S)-10-[(3-amino-2,3,6-trideoxy--L-lyxo-hexopyranosyl)oxy]-6,8,11-
tetrahydroxy-8-[(1RS)-1-hydroxyethyl]-1-methoxy-7,8,9,10-tetrahydrotetracene-5,12-
dione (daunorubicinol),
C. R = CH
2
-CO-CH
3
: (8S,10S)-10-[(3-amino-2,3,6-trideoxy--L-lyxo-hexopyranosyl)oxy]-
6,8,11-tetrahydroxy-1-methoxy-8-(2-oxopropyl)-7,8,9,10-tetrahydrotetracene-5,12-dione
(feudomycin B),
D. R = CO-CH
2
OH: doxorubicin,
F. R = CO-CH
2
-CH
3
: (8S,10S)-10-[(3-amino-2,3,6-trideoxy--L-lyxo-hexopyranosyl)oxy]-
6,8,11-tetrahydroxy-1-methoxy-8-propanoyl-7,8,9,10-tetrahydrotetracene-5,12-dione
(8-ethyldaunorubicin).
___________________________________________________________________________________________________________ PhEur
12-43
Debrisoquine Sulphate
N NH
2
NH
2
,H
2
SO
4
(C
10
H
13
N
3
)
2
,H
2
SO
4
448.5 581-88-4
Definition Debrisoquine Sulphate is 1,2,3,4-tetrahydroisoquinoline-2-carboxamidine sulphate.
It contains not less than 99.0% and not more than 101.0% of (C
10
H
13
N
3
)
2
,H
2
SO
4
, calculated with
Characteristics A white, crystalline powder; odourless or almost odourless.
Sparingly soluble in water; very slightly soluble in ethanol (96%); practically insoluble in chloro-
form and in ether.
Identification
A. The light absorption, Appendix II B, in the range 230 to 350 nm of a 0.05% w/v solution in
0.05M sulphuric acid exhibits two maxima, at 262 nm and 270 nm. The absorbance at the maximum
at 262 nm is about 0.69 and at the maximum at 270 nm is about 0.51.
B. Carry out the method described under Related substances, applying separately to the plate 10 l
of each of the following solutions in water. Solution (1) contains 0.25% w/v of the substance being
examined. Solution (2) contains 0.25% w/v of debrisoquine sulphate BPCRS. Solution (3) is a
mixture of equal volumes of solutions (1) and (2). The principal spot in the chromatogram
obtained with solution (1) corresponds to that in the chromatogram obtained with solution (2).
The principal spot in the chromatogram obtained with solution (3) appears as a single compact
spot.
C. Yields the reactions characteristic of sulphates, Appendix VI.
silica gel G as the coating substance and a mixture of 60 volumes of butan-1-ol, 25 volumes of water
and 15 volumes of glacial acetic acid as the mobile phase. Apply separately to the plate 10 l of each
of two solutions in water containing (1) 2.0% w/v of the substance being examined and (2) 0.010%
w/v of debrisoquine sulphate BPCRS. After removal of the plate, allow it to dry in air and spray with a
solution prepared by adding 1 ml of sulphuric acid to 40 ml of a freshly prepared mixture of equal
volumes of a 0.135% w/v solution of chloroplatinic(IV) acid and a 1.1% w/v solution of potassium
iodide. Any secondary spot in the chromatogram obtained with solution (1) is not more intense than
the spot in the chromatogram obtained with solution (2).
Use 1 g.
Assay Carry out Method I for non-aqueous titration, Appendix VIII A, using 1 g and determining
(C
10
H
13
N
3
)
2
,H
2
SO
4
.
Storage Debrisoquine Sulphate should be kept in a well- closed container and protected from
light.
Action and use Adrenergic neurone blocker.
12-44
Decyl Oleate
Decyl Oleate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1307].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Decyl oleate is a mixture consisting of decyl esters of fatty acids, mainly oleic acid. A suitable
antioxidant may be added.
PRODUCTION
Where applicable, it complies with the monograph on Products with risk of transmitting agents of
animal spongiform encephalopathies (1483).
CHARACTERS
A clear, pale yellow or colourless liquid, practically insoluble in water, miscible with alcohol, with
methylene chloride and with light petroleum (40C to 60C).
IDENTIFICATION
A. It complies with the test for relative density (see Tests).
C. It complies with the test for content of oleic acid (see Tests).
TESTS
Relative density (2.2.5).0.860 to 0.870.
Iodine value (2.5.4).55 to 70.
Saponification value (2.5.6).130 to 140, determined on 2.0 g.
Content of oleic acid Carry out the test for foreign oils in fatty oils by gas chromatography
(2.4.22, Method A). The fatty acid fraction of the substance contains at least 60.0 per cent of oleic
acid.
mination of water.
STORAGE
LABELLING
The label states, where applicable, the name and concentration of any added antioxidant.
__________________________________________________________________________________________________________ Ph Eur
12-45
Demeclocycline Hydrochloride
OH OH
OH
Cl
O O
CONH
2
H H
NMe
2
HO H
OH
H
,HCl
C
21
H
21
ClN
2
O
8
,HCl 501.3 64-73-3
Demeclocycline Hydrochloride complies with the requirements of the 3rd edition of the European
Preparation
Demeclocycline Capsules
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Demeclocycline hydrochloride is the hydrochloride of (4S,4aS,5aS,6S,12aS)-7-chloro-4-
dimethylamino-1,4,4a, 5,5a,6, 11,12a-octahydro-3,6,10,12,12a-pentahydroxy-1,11-
dioxonaphthacene-2-carboxamide, a substance produced by certain strains of Streptomyces
aureofaciens or obtained by any other means. It contains not less than 89.5 per cent and not more
than the equivalent of 100.5 per cent of demeclocycline hydrochloride, calculated with reference to
CHARACTERS
A yellow powder, soluble or sparingly soluble in water, slightly soluble in alcohol, very slightly
soluble in acetone, practically insoluble in ether. It dissolves in solutions of alkali hydroxides and
carbonates.
IDENTIFICATION
A. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R. Adjust a 100 g/l
solution of sodium edetate R to pH 7.0 with strong sodium hydroxide solution R and spray the solution
evenly onto the plate (about 10 ml for a plate 100 mm 200 mm). Allow the plate to dry in a
horizontal position for at least 1 h. Before use, dry the plate in an oven at 110C for 1 h.
the same solvent.
Reference solution (a). Dissolve 5 mg of demeclocycline hydrochloride CRS in methanol R and dilute to
Reference solution (b). Dissolve 5 mg of demeclocycline hydrochloride CRS, 5 mg of oxytetracycline
hydrochloride CRS and 5 mg of metacycline hydrochloride CRS in methanol R and dilute to 10 ml with
the same solvent.
6 volumes of water R, 35 volumes of methanol R and 59 volumes of methylene chloride R. Dry the
plate in a current of air and examine in ultraviolet light at 365 nm. The principal spot in the
spot in the chromatogram obtained with reference solution (a). The test is not valid unless the
chromatogram obtained with reference solution (b) shows three clearly separated spots.
B. To about 2 mg add 5 ml of sulphuric acid R. A violet colour develops. Add the solution to 2.5 ml
of water R. The colour becomes yellow.
TESTS
12-46
same acid. To 10.0 ml of the solution add 12 ml of dilute sodium hydroxide solution R and dilute to
100.0 ml with water R. The specific absorbance determined at the maximum at 385 nm is 340 to
370, calculated with reference to the anhydrous substance.
Inject the test solution and reference solution (d). In the chromatogram obtained with the test
solution: the area of any peak, apart from the principal peak and any peak due to the solvent, is not
greater than the area of the principal peak in the chromatogram obtained with reference solution
(d) (5 per cent); at most one such peak has an area greater than 0.8 times the area of the principal
peak in the chromatogram obtained with reference solution (d) (4 per cent) and the sum of the
areas of all such peaks is not greater than twice the area of the principal peak in the chromatogram
obtained with reference solution (d) (10 per cent).
mination of water.
ASSAY
Reference solution (a). Dissolve 25.0 mg of demeclocycline hydrochloride CRS in 0.01M hydrochloric acid
Reference solution (b). Dissolve 5.0 mg of 4-epidemeclocycline hydrochloride CRS in 0.01M hydrochloric
acid and dilute to 25.0 ml with the same acid.
Reference solution (c). Mix 1.0 ml of reference solution (a) and 5.0 ml of reference solution (b) and
Reference solution (d). Dilute 5.0 ml of reference solution (a) to 100.0 ml with 0.01M hydrochloric
acid.
a column 0.25 m long and 4.6 mm in internal diameter packed with styrene-divinylbenzene
copolymer R (8 m to 10 m),
as mobile phase at a flow rate of 1 ml/min a solution prepared as follows: weigh 80.0 g of
2-methyl-2-propanol R and transfer to a 1000 ml volumetric flask with the aid of 200 ml of
water R; add 100 ml of a 35 g/l solution of dipotassium hydrogen phosphate R adjusted to pH 9.0
with dilute phosphoric acid R, 150 ml of a 10 g/l solution of tetrabutylammonium hydrogen
sulphate R adjusted to pH 9.0 with dilute sodium hydroxide solution R and 10 ml of a 40 g/l
solution of sodium edetate R adjusted to pH 9.0 with dilute sodium hydroxide solution R; dilute to
an electronic integrator,
maintaining the temperature of the column at 60C. Inject reference solution (c). Adjust the
sensitivity of the detector so that the height of the peaks is not less than half the full scale of the
recorder. The test is not valid unless the resolution between the first peak (4-epidemeclocycline)
and the second peak (demeclocycline) is at least 2.8. If necessary, adjust the 2-methyl-2-propanol
content of the mobile phase or lower the pH of the mobile phase. The symmetry factor for the
second peak is at most 1.25. Inject reference solution (a) six times. The test is not valid unless the
relative standard deviation of the peak area for demeclocycline is at most 1.0 per cent. If necessary,
adjust the integrator parameters. Inject alternately the test solution and reference solution (a).
Calculate the percentage content of demeclocycline hydrochloride.
STORAGE
IMPURITIES
OH O O HO
OH
CONH
2
OH
NMe
2
H H
OH
H
A. demethyltetracycline,
12-47
OH O O HO
OH
CONH
2
OH
NMe
2
H H
OH
H
Cl
B. 4-epidemeclocycline.
__________________________________________________________________________________________________________ Ph Eur
12-48
Deptropine Citrate
N
H
Me
O
, HOOC COOH
COOH HO
C
23
H
27
NO,C
6
H
8
O
7
525.6 2169-75-7
Deptropine Citrate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1308].
Action and use Anticholinergic; histamine H
1
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Deptropine citrate contains not less than 98.0 per cent and not more than the equivalent of
101.0 per cent of (1R,3r,5S)-3-(10,11-dihydro-5H-dibenzo[a,d][7]annulen-5-yloxy)-8-methyl-8-
azabicyclo[3.2.1]octane dihydrogen citrate, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, microcrystalline powder, very slightly soluble in water and in ethanol,
IDENTIFICATION
obtained with deptropine citrate CRS.
C. To about 1 mg add 0.5 ml of sulphuric acid R. A stable red-orange colour develops.
D. Dissolve about 1 mg in 0.25 ml of perchloric acid R and warm gently until the solution becomes
turbid. Add 5 ml of glacial acetic acid R; a pink colour with an intense green fluorescence appears.
E. To about 5 mg add 1 ml of acetic anhydride R and 5 ml of pyridine R. A purple colour develops.
TESTS
pH (2.2.3). Suspend 0.25 g in carbon dioxide-free water R, dilute to 25 ml with the same solvent and
filter. The pH of the solution is 3.7 to 4.5.
Related substances Examine by thin-layer chromatography (2.2.27), using as the coating
substance a suitable silica gel with a fluorescent indicator having an optimal intensity at 254 nm.
Reference solution (a). Dilute 1.0 ml of test solution (a) to 100.0 ml with methanol R.
Reference solution (b). Dissolve 20 mg of deptropine citrate CRS in methanol R and dilute to 2 ml with
the same solvent. Dilute 1 ml of the solution to 10 ml with methanol R.
Reference solution (c). Dissolve 5 mg of tropine CRS in methanol R and dilute to 100.0 ml with the
same solvent.
Reference solution (d). Dissolve 10 mg of deptropine citrate CRS and 10 mg of tropine CRS in
of 8 volumes of concentrated ammonia R and 92 volumes of butanol R. Dry the plate at 100C to
105C until the ammonia has completely evaporated. Examine in ultraviolet light at 254 nm. Any
12-49
spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not
more intense than the spot in the chromatogram obtained with reference solution (a) (1 per
cent). Spray with dilute potassium iodobismuthate solution R and then with a 10 g/l solution of sodium
nitrite R. Expose the plate to iodine vapours. Examine in daylight and in ultraviolet light at
254 nm. In the chromatogram obtained with test solution (a): any spot corresponding to tropine
is not more intense than the spot in the chromatogram obtained with reference solution (c)
(0.5 per cent); any spot, apart from the principal spot and any spot corresponding to tropine, is
not more intense than the spot in the chromatogram obtained with reference solution (a) (1 per
cent). The test is not valid unless the chromatogram obtained with reference solution (d) shows
two clearly separated spots.
ASSAY
29
H
35
NO
8
.
STORAGE
IMPURITIES
N
H
Me
OH
A. (1R,3r,5S)-8-methyl-8-azabicyclo[3.2.1]octan-3-ol (tropine),
O
H
Me
N
B. (1R,3s,5S)-3-(10,11-dihydro-5H-dibenzo[a,d][7]annulen-5-yloxy)-8-methyl-8-
azabicyclo[3.2.1]octane (pseudodeptropine),
OH
C. 10,11-dihydro-5H-dibenzo[a,d][7]annulen-5-ol (dibenzocycloheptadienol),
Me
H
O
N
D. (1R,3r,5S)-3-(10,11-dihydro-5H-dibenzo[a,d][7]annulen-5-yloxy)-8-
azabicyclo[3.2.1]octane (demethyldeptropine).
__________________________________________________________________________________________________________ Ph Eur
12-50
Dequalinium Chloride
N
Me
2
N N
Me
NH
2
[CH
2
]
10
+ +
2Cl
C
30
H
40
Cl
2
N
4
527.6 522-51-1
Dequalinium Chloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Anti-infective.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Dequalinium chloride contains not less than 95.0 per cent and not more than the equivalent of
101.0 per cent of 1,1-(decane-1,10-diyl)bis(4-amino-2-methylquinolinium) dichloride, calculated
CHARACTERS
A white or yellowish-white powder, hygroscopic, slightly soluble in water and in alcohol.
IDENTIFICATION
A. Dissolve about 10 mg in water R and dilute to 100 ml with the same solvent. Dilute 10 ml to
100 ml with water R. Examined between 230 nm and 350 nm (2.2.25), the solution shows two
absorption maxima, at 240 nm and 326 nm and a shoulder at 336 nm. The ratio of the absorbance
measured at the maximum at 240 nm to that measured at the maximum at 326 nm is 1.56 to 1.80
and the ratio of the absorbance measured at the maximum at 326 nm to that measured at the
shoulder at 336 nm is 1.12 to 1.30.
B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph. Eur.
reference spectrum of dequalinium chloride.
C. To 5 ml of solution S (see Tests) add 5 ml of potassium ferricyanide solution R. A yellow
D. To 10 ml of solution S add 1 ml of dilute nitric acid R. A white precipitate is formed. Filter and
reserve the filtrate for identification test E.
E. The filtrate from identification test D gives reaction (a) of chlorides (2.3.1).
TESTS
Solution S Dissolve 0.2 g in 90 ml of carbon dioxide-free water R, heating if necessary, and dilute to
Acidity or alkalinity To 5 ml of solution S add 0.1 ml of bromothymol blue solution R1. Not more
than 0.2 ml of 0.01M hydrochloric acid or 0.01M sodium hydroxide is required to change the colour of
the indicator.
Reference solution (a). Dissolve 10.0 mg of dequalinium chloride for performance test CRS in the mobile
Reference solution (b). Dissolve 10.0 mg of dequalinium chloride CRS in the mobile phase and dilute
to 10.0 ml with the mobile phase. Dilute 1.0 ml to 50.0 ml with the mobile phase.
a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with an end-capped
octadecylsilyl silica gel for chromatography R,
as mobile phase at a flow rate of 1.5 ml/min the following solution: dissolve 2 g of sodium
12-51
hexanesulphonate R in 300 ml of water R. Adjust to pH 4.0 with acetic acid R and add 700 ml of
methanol R,
Adjust the sensitivity of the system so that the height of the peak due to impurity B in the
chromatogram obtained with 10 l of reference solution (a) is at least 25 per cent of the full scale of
the recorder. Measure the height (A) above the base-line of the peak due to impurity B and the
height (B) above the base-line at the lowest point of the curve separating this peak from the peak
due to dequalinium chloride. The test is not valid unless A is greater than twice B. If necessary,
adjust the concentration of methanol in the mobile phase.
of the test solution for five times the retention time of the peak due to chloride. In the chromato-
gram obtained with the test solution: the area of any peak due to impurity A is not greater than half
the area of the principal peak in the chromatogram of reference solution (b) (1 per cent); and the
sum of the areas of all the peaks, apart from the principal peak, is not greater than five times the
area of the principal peak in the chromatogram obtained with reference solution (b) (10 per cent).
Disregard any peak with an area less than 0.025 times the area of the principal peak in the chroma-
togram of reference solution (b).
Readily carbonisable substances Dissolve 20 mg in 2 ml of sulphuric acid R. After 5 min the
solution is not more intensely coloured than reference solution BY
4
(Method I, 2.2.2).
to 105C at a pressure not exceeding 0.7 kPa.
ASSAY
In order to avoid overheating in the reaction medium, mix thoroughly throughout and stop the titration
immediately after the end-point has been reached.
Dissolve 0.200 g in 5 ml of anhydrous formic acid R and add 50 ml of acetic anhydride R. Titrate with
30
H
40
Cl
2
N
4
.
STORAGE
IMPURITIES
N Me
NH
2
A. 2 methylquinolin-4-amine,
N
N Me
NH
2
NH
Me
Cl
_
B. 4-amino-1-[10-[(2-methylquinolin-4-yl)amino]decyl]-2-methylquinolinium chloride,
N HN
Me
N
Me
NH
2
5
2
3Cl
_
C. 1-[10-(4-amino-2-methylquinolinio)decyl]-4-[[10-(4-amino-2-
methylquinolinio)decyl]amino]-2-methylquinolinium trichloride.
__________________________________________________________________________________________________________ Ph Eur
12-52
Desferrioxamine Mesilate
O
O
H
3
C N N
O O
O
N N
N OH
NH
2
HO
OH
H
H
,
,H
3
C SO
3
H
C
25
H
48
N
6
O
8
,CH
4
SO
3
657 138-14-7
Desferrioxamine Mesilate complies with the requirements of the 3rd edition of the European Pharmacopoeia
for Deferoxamine Mesilate [0896]. These requirements are reproduced after the heading Definition below.
Action and use Iron-chelating agent.
Preparation
Desferrioxamine Injection
When desferrioxamine mesylate is prescribed or demanded, Desferrioxamine Mesilate shall be
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Deferoxamine mesilate contains not less than 98.0 per cent and not more than the equivalent of
102.0 per cent expressed as 30-amino-3,14,25-trihydroxy-3,9,14,20,25-penta-azatriacontane-
2,10,13,21,24-pentone methanesulphonate, calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white powder, freely soluble in water, slightly soluble in methanol, very slightly
IDENTIFICATION
obtained with deferoxamine mesilate CRS. Examine the substances prepared as discs. If the spectra
separately in alcohol R, evaporate to dryness and record new spectra using the residues.
B. Dissolve about 5 mg in 5 ml of water R. Add 2 ml of a 5 g/l solution of trisodium phosphate
dodecahydrate R and 0.5 ml of a 25 g/l solution of sodium naphthoquinonesulphonate R. A brownish-
black colour develops.
C. The titrated solution obtained in the Assay (solution (a)) is brownish-red. To 10 ml of solution
(a) add 3 ml of ether R. Shake. The organic layer is colourless. To 10 ml of solution (a) add 3 ml of
benzyl alcohol R. Shake. The organic layer is brownish-red.
D. Dissolve 0.1 g in 5 ml of dilute hydrochloric acid R. Add 1 ml of barium chloride solution R2. The
solution is clear. In a porcelain crucible, mix 0.1 g with 1 g of anhydrous sodium carbonate R, heat
and ignite over a naked flame. Allow to cool. Dissolve the residue in 10 ml of water R, heating if
necessary, and filter. The filtrate gives reaction (a) of sulphates (2.3.1).
TESTS
solution Y
5
(Method II, 2.2.2).
pH (2.2.3). The pH of freshly prepared solution S is 3.7 to 5.5.
Prepare the solutions immediately before use, protected from light.
12-53
Reference solution (a). Dissolve 10.0 mg of deferoxamine mesilate CRS in the mobile phase and dilute
as mobile phase at a flow rate of 2 ml per minute a solution prepared as follows: dissolve 1.32 g
of ammonium phosphate R and 0.37 g of sodium edetate R in 950 ml of water R; adjust to pH 2.8
with phosphoric acid R (about 3 ml to 4 ml) and add 55 ml of tetrahydrofuran R,
Inject 20 l of reference solution (a). If a recorder is used, adjust the sensitivity of the detector so
that the height of the peak with a relative retention time of about 0.8 is not less than 15 per cent of
the full scale of the recorder. The test is not valid unless the resolution between the peak with the
relative retention time of about 0.8 and the principal peak is at least 1.0.
for three times the retention time of deferoxamine. In the chromatogram obtained with the test
solution: the area of any peak, apart from the principal peak, is not greater than the area of the
the area of any such peaks is not greater than 1.75 times the area of the principal peak in the
less than 0.02 times that of the principal peak in the chromatogram obtained with reference
solution (b).
Chlorides (2.4.4). Dilute 2 ml of solution S to 20 ml with water R. 15 ml of the solution complies
with the limit test for chlorides (330 ppm).
complies with the limit test for sulphates (400 ppm).
mination of water.
without a further appropriate procedure for the removal of bacterial endotoxins, it complies with
the test for bacterial endotoxins. Not more than 0.025 I.U. of endotoxin per milligram.
ASSAY
Dissolve 0.500 g in 25 ml of water R. Add 4 ml of 0.05M sulphuric acid. Titrate with 0.1M ferric
ammonium sulphate. Towards the end of the titration, titrate uniformly and at a rate of about
0.2 ml per minute. Determine the end-point potentiometrically (2.2.20) using a platinum indicator
electrode and a calomel reference electrode. Retain the titrated solution for identification test C.
1 ml of 0.1M ferric ammonium sulphate is equivalent to 65.68 mg of C
26
H
52
N
6
O
11
S.
STORAGE
Store in a well-closed container, protected from light, at a temperature of 2C to 8C. If the
LABELLING
The label states:
IMPURITIES
A. desferrioxamine A
1
,
B. other desferrioxamines.
__________________________________________________________________________________________________________ Ph Eur
12-54
Desipramine Hydrochloride
,HCl
NHMe
C
18
H
22
N
2
,HCl 302.8 58-28-6
Desipramine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Desipramine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Desipramine hydrochloride contains not less than 99.0 per cent and not more than the equivalent
of 101.0 per cent of 10,11-dihydro-5-(3-methylaminopropyl)-5H-dibenz[b,f]azepine hydrochloride,
CHARACTERS
A white or almost white, crystalline powder, soluble in water and in alcohol.
IDENTIFICATION
350 nm (2.2.25), the solution shows an absorption maximum at 251 nm and a shoulder at 270 nm.
The specific absorbance at the maximum is 255 to 285.
obtained with desipramine hydrochloride CRS.
D. Dissolve about 50 mg in 3 ml of water R and add 0.05 ml of a 25 g/l solution of quinhydrone R in
methanol R. An intense pink colour develops within about 15 min.
E. To 0.5 ml of solution S (see Tests) add 1.5 ml of water R. The solution gives reaction (a) of
chlorides (2.3.1).
TESTS
Solution S Dissolve 1.25 g in carbon dioxide-free water R, warming to not more than 30C if
necessary, and dilute to 25 ml with the same solvent.
Appearance of solution Solution S, examined immediately after preparation, is not more
6
(Method II, 2.2.2).
Acidity or alkalinity To 10 ml of solution S add 0.1 ml of methyl red solution R and 0.3 ml of
0.01M sodium hydroxide. The solution is yellow. Not more than 0.5 ml of 0.01M hydrochloric acid is
Related substances Carry out the test protected from bright light. Examine by thin-layer chromato-
graphy (2.2.27), using a TLC silica gel plate R.
Test solution (a). Dissolve 0.10 g of the substance to be examined in a mixture of equal volumes of
ethanol R and methylene chloride R and dilute to 10 ml with the same mixture of solvents. Prepare
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with a mixture of equal volumes of
ethanol R and methylene chloride R.
Reference solution (a). Dissolve 25 mg of desipramine hydrochloride CRS in a mixture of equal
volumes of ethanol R and methylene chloride R and dilute to 25 ml with the same mixture of solvents.
12-55
Prepare immediately before use.
Reference solution (b). Dilute 1 ml of reference solution (a) to 50 ml with a mixture of equal
volumes of ethanol R and methylene chloride R.
Apply to the plate 5 l of each solution. Develop over a path of 7 cm using a mixture of 1 volume
of water R, 10 volumes of anhydrous acetic acid R and 10 volumes of toluene R. Dry the plate in a
current of air for 10 min, spray with a 5 g/l solution of potassium dichromate R in a mixture of 1
volume of sulphuric acid R and 4 volumes of water R and examine immediately. Any spot in the
at 100C to 105C.
ASSAY
Dissolve 0.2500 g in a mixture of 5 ml of 0.01M hydrochloric acid and 50 ml of alcohol R. Carry out a
18
H
23
ClN
2
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
12-56
Deslanoside
3
O
Me
OH
O
O
R
O
OH
OH
HOCH
2
HO
O
HO H
H
H
H
H
H
OH
Me
Me
O
R =
C
47
H
74
O
19
943.1 17598-65-1
Deslanoside complies with the requirements of the 3rd edition of the European Pharmacopoeia [0482]. These
Action and use Cardiac glycoside.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Deslanoside contains not less than 95.0 per cent and not more than the equivalent of 105.0 per
cent of 3-[(O--D-glucopyranosyl-(14)-O-2,6-dideoxy--D-ribo-hexopyranosyl-(14)-O-2,6-
dideoxy--D-ribo-hexopyranosyl-(14)-2,6-dideoxy--D-ribo-hexopyranosyl)oxy]-12,14-
dihydroxy-5,14-card-20(22)-enolide, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline or finely crystalline powder, hygroscopic, practically insoluble in water and in
ether, very slightly soluble in alcohol. In an atmosphere of low relative humidity, it loses water.
IDENTIFICATION
obtained with deslanoside CRS. When comparing the spectra, special attention is given to the
absence of a distinct absorption maximum at about 1260 cm
1
and to the intensity of the
absorption maximum at about 1740 cm
1
. Examine the substances in discs prepared by dissolving
1 mg of the substance to be examined or 1 mg of the reference substance in 0.3 ml of methanol R
and triturating with about 0.4 g of dry, finely powdered potassium bromide R until the mixture is
uniform and completely dry.
B. Examine the chromatograms obtained in the test for related substances. The principal band in
principal band in the chromatogram obtained with reference solution (a).
C. Suspend about 0.5 mg in 0.2 ml of alcohol (60 per cent V/V) R. Add 0.1 ml of dinitrobenzoic acid
solution R and 0.1 ml of dilute sodium hydroxide solution R. A violet colour develops.
D. Dissolve about 5 mg in 5 ml of glacial acetic acid R and add 0.05 ml of ferric chloride solution R1.
Cautiously add 2 ml of sulphuric acid R, avoiding mixing the two liquids. Allow to stand; a brown
but not reddish ring develops at the interface and a greenish-yellow, then bluish-green colour
diffuses from it to the upper layer.
TESTS
Solution S Dissolve 0.20 g in a mixture of equal volumes of chloroform R and methanol R and dilute
to 10 ml with the same mixture of solvents.
Specific optical rotation (2.2.7). Dissolve 0.200 g in anhydrous pyridine R and dilute to 10.0 ml
with the same solvent. The specific optical rotation is +6.5 to +8.5, calculated with reference to
coating substance.
12-57
Test solution (a). Use solution S.
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with a mixture of equal volumes of chloro-
form R and methanol R.
Reference solution (a). Dissolve 20 mg of deslanoside CRS in a mixture of equal volumes of chloro-
form R and methanol R and dilute to 10 ml with the same mixture of solvents.
Reference solution (b). Dilute 2.5 ml of reference solution (a) to 10 ml with a mixture of equal
volumes of chloroform R and methanol R.
Reference solution (c). Dilute 1 ml of reference solution (a) to 10 ml with a mixture of equal volumes
of chloroform R and methanol R.
Apply separately to the plate as 10 mm bands 5 l of each solution. Develop immediately over a
path of 15 cm using a mixture of 3 volumes of water R, 36 volumes of methanol R and 130 volumes
of methylene chloride R. Dry the plate in a current of warm air, spray with a mixture of 5 volumes of
sulphuric acid R and 95 volumes of alcohol R and heat at 140C for 15 min. Examine in daylight. In
the chromatogram obtained with test solution (a), any band, apart from the principal band, is not
more intense than the band in the chromatogram obtained with reference solution (b) (2.5 per
cent) and at most two such bands are more intense than the band in the chromatogram obtained
with reference solution (c) (1.0 per cent).
100C to 105C.
Sulphated ash (2.4.14) Not more than 0.1 per cent, determined on the residue obtained in the
test for loss on drying.
ASSAY
solution to 100.0 ml with alcohol R. Prepare a reference solution in the same manner, using
50.0 mg of deslanoside CRS (undried). To 5.0 ml of each solution add 3.0 ml of alkaline sodium
picrate solution R and allow to stand protected from bright light in a water-bath at 20 1C for
40 min. Measure the absorbance (2.2.25) of each solution at the maximum at 484 nm, using as the
compensation liquid a mixture of 3.0 ml of alkaline sodium picrate solution R and 5.0 ml of alcohol R
prepared at the same time.
47
H
74
O
19
the solutions.
STORAGE
Store in an airtight, glass container, protected from light, at a temperature below 10C.
__________________________________________________________________________________________________________ Ph Eur
12-58
Desmopressin
1/01
SCH
2
CH
2
CO-Tyr-Phe-Gln-Asn-Cys-Pro-
D-Arg-Gly-NH
2
C
46
H
64
N
14
O
12
S
2
1069 16679-58-6
Desmopressin complies with the requirements of the 3rd edition of the European Pharmacopoeia [0712].
Action and use Antidiuretic and factor VIII-increasing peptide.
Preparations
Desmopressin Injection
Desmopressin Intranasal Solution
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Desmopressin is a synthetic cyclic nonapeptide having an antidiuretic action. It contains not less
than 95.0 per cent

and not more than the equivalent of 105.0 per cent of the peptide
C
46
H
64
N
14
O
12
S
2
, calculated with reference to the anhydrous, acetic acid-free substance. It is
available as an acetate.
CHARACTERS
A white, fluffy powder, soluble in water, in alcohol and in glacial acetic acid.
IDENTIFICATION
Examine the chromatograms obtained in the assay. The retention time and size of the principal
peak in the chromatogram obtained with the test solution are approximately the same as those of
the principal peak in the chromatogram obtained with the reference solution.
TESTS
Specific optical rotation (2.2.7). Dissolve 10.0 mg in a 1 per cent V/V solution of glacial acetic
acid R and dilute to 5.0 ml with the same acid. The specific optical rotation is 72 to 82,
calculated with reference to the anhydrous, acetic acid-free substance.
acids:
100 mm long and 6 mm in internal diameter. Add a suitable amount of a 50 per cent V/V solution
of hydrochloric acid R. Immerse the tube in a freezing mixture at 5C, reduce the pressure to below
dryness over potassium hydroxide R under reduced pressure; repeat these operations once. Take up
the residue in a buffer solution suitable for the amino-acid analyser used and dilute to a suitable
volume with the same buffer solution. Apply a suitable volume to the amino-acid analyser.
acids, taking one-sixth of the sum of the number of moles of aspartic acid, glutamic acid, proline,
glycine, arginine and phenylalanine as equal to one. The values fall within the following limits:
aspartic acid 0.95 to 1.05; glutamic acid 0.95 to 1.05; proline 0.95 to 1.05; glycine 0.95 to 1.05;
arginine 0.95 to 1.05; phenylalanine 0.95 to 1.05; tyrosine 0.70 to 1.05; half-cystine 0.30 to 1.05.
Lysine, isoleucine and leucine are absent; not more than traces of other amino acids are present.
Related peptides Examine by liquid chromatography (2.2.29) as described under Assay, following
the elution conditions shown in the table below, at a flow rate of 1.5 ml/min:
12-59
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
0 4 76 24 Isocratic
35 40 48 76 52 24 Switch to initial
eluent composition
Inject 50 l of the resolution solution and identify the peaks due to desmopressin and oxytocin
(first and second peaks, respectively). If necessary, adjust the concentration of acetonitrile in the
mobile phase to obtain a retention time of about 16 min for the desmopressin peak. The test is not
valid unless the resolution between the peaks corresponding to desmopressin and oxytocin is at
least 1.5.
Inject 50 l of the test solution. In the chromatogram obtained the area of any peak, apart from
the principal peak, is not greater than 0.5 per cent of the total area of the peaks; the sum of the
areas of all the peaks, apart from the principal peak, is not greater than 1.5 per cent of the total area
of the peaks. Disregard any peak due to the solvent and any peak with an area less than 0.05 per
cent of that of the principal peak.
Acetic acid (2.5.34).3.0 per cent to 8.0 per cent.
500 I.U. of endotoxin per milligram of desmopressin.
ASSAY
Test solution. Dissolve 1.0 mg of the substance to be examined in 2.0 ml of water R.
Reference solution. Dissolve the contents of a vial of desmopressin CRS in water R to obtain a concen-
tration of 0.5 mg/ml.
Resolution solution. Dissolve the contents of a vial of oxytocin/desmopressin validation mixture CRS in
500 l of water R.
silica gel for chromatography R

(5 m),
as mobile phase at a flow rate of 2.0 ml/min a mixture of 60 volumes of mobile phase A and 40
volumes of mobile phase B:
Mobile phase A.0.067M phosphate buffer solution pH 7.0 R; filter and degas,
Mobile phase B. Mix equal volumes of mobile phase A and of acetonitrile for chromatography R;
filter and degas,
Inject 50 l of the resolution solution and identify the peaks due to desmopressin and oxytocin
(first and second peaks, respectively). If necessary, adjust the concentration of acetonitrile in the
mobile phase to obtain a retention time of about 5 min for the desmopressin peak. The test is not
valid unless the resolution between the peaks corresponding to desmopression and oxytocin is at
least 1.5.
Calculate the content of desmopressin (C
46
H
64
N
14
O
12
S
2
) from the peak areas in the chromato-
grams obtained with the test solution and the reference solution and the declared content of
C
46
H
64
N
14
O
12
S
2
in desmopressin CRS.
STORAGE
LABELLING
The label states:
the mass of peptide per container,
__________________________________________________________________________________________________________ Ph Eur
12-60
Desogestrel
H
H H
Et
C
OH
H
CH
H
2
C
C
22
H
30
O 310.5 54024-22-5
Definition Desogestrel is 13-ethyl-11-methylene-18,19-dinor-17-pregn-4-en-20-yn-17-ol. It
22
H
30
O, calculated with reference to
Practically insoluble in water; slightly soluble in ethanol (96%) and in ethyl acetate; sparingly
soluble in n-hexane.
Identification
desogestrel (RS 088).
B. Carry out the method for thin-layer chromatography, Appendix III A, using a silica gel 60 pre-
coated plate for high performance thin-layer chromatography (Merck silica gel 60 HPTLC plates
are suitable), a mixture of 20 volumes of ethyl acetate and 80 volumes of toluene as the mobile phase.
Apply separately to the plate 1 l of each of three solutions in dichloromethane containing (1) 0.01%
w/v of the substance being examined, (2) 0.01% w/v of desogestrel BPCRS and (3) 0.01% w/v each
of desogestrel BPCRS and lynestrenol EPCRS. After removal of the plate, spray it with ethanolic
sulphuric acid (2%), heat at 110 for 10 minutes and examine under ultraviolet light (365 nm). The
principal spot in the chromatogram obtained with solution (1) is similar in position and size to the
principal spot in the chromatogram obtained with solution (2). The test is not valid unless the
chromatogram obtained with solution (3) shows two clearly separated principal spots.
Specific optical rotation In a 1% w/v solution in chloroform, +53 to +57, Appendix V F, calcu-
silica gel 60 precoated plate for high performance thin-layer chromatography (Merck silica gel 60
HPTLC plates are suitable), a mixture of 25 volumes of n-heptane and 75 volumes of chloroform as
the mobile phase. Apply separately to the plate 1 l of each of three solutions of the substance being
examined in dichloromethane containing (1) 2.0% w/v, (2) 0.004% w/v and (3) 0.001% w/v
respectively. After removal of the plate, spray it with ethanolic sulphuric acid (2%), heat at 110 for 5
minutes and examine under ultraviolet light (365 nm). In the chromatogram obtained with solution
(1) any secondary spot is not more intense than the spot in the chromatogram obtained with solution
(2) (0.2%) and not more than five such spots are more intense than the spot in the chromatogram

3
-Isomer Carry out the method for gas chromatography, Appendix III B, using three solutions in
absolute ethanol containing (1) 1.0% w/v of the substance being examined, (2) 0.0020% w/v of
desogestrel-
3
-isomer BPCRS and (3) 1.0% w/v of the substance being examined and 0.00050% w/v
of desogestrel-
3
-isomer BPCRS.
The chromatographic procedure may be carried out using (a) a capillary glass column (10
m 0.32 mm) bonded with a film (0.25 m) of dimethylpolysiloxane (CP Sil 5 CB is suitable) at
an initial temperature of 140 increasing linearly to 240 at a rate of 10 per minute, with a split
injection system having a split ratio of 30:1 maintained at 280 and a detector temperature of 280
and (b) a carrier gas flow rate of 2 ml per minute. Inject 1 l of each of the solutions.
The test is not valid unless the chromatogram obtained with solution (3) shows two clearly
separated peaks.
desogestrel-
3
-isomer is not greater than the area of the peak in the chromatogram obtained with
solution (2) (0.2%).
Loss on drying When dried to constant weight at room temperature at a pressure of 1.5 kPa to
2.5 kPa, loses not more than 0.5% of its weight. Use 0.4 g.
Sulphated ash Not more than 0.1%, Appendix IX A, Method II. Use 0.5 g.
Assay Carry out the method for liquid chromatography, Appendix III D, using three solutions in
methanol containing (1) 0.02% w/v of the substance being examined, (2) 0.02% w/v of desogestrel
12-61
BPCRS and (3) 0.02% w/v each of the substance being examined and lynestrenol EPCRS. Inject 20
l of each of the solutions.
cm 4.6 mm) packed with stationary phase C (5 m) (Zorbax ODS is suitable), (b) methanol as the
mobile phase with a flow rate of 1 ml per minute and (c) a detection wavelength of 205 nm. Inject
20 l of each solution.
The assay is not valid unless, in the chromatogram obtained with solution (3), the resolution factor
between the peaks corresponding to desogestrel and lynestrenol is at least 2.0.
22
H
30
O using the declared content of C
22
H
30
O in desogestrel BPCRS.
Action and use Progestogen.
IMPURITIES
H
H H
Et
C
OH
H
CH
H
2
C
H
A. 13-ethyl-11-methylene-18,19-dinor-5,17-pregn-3-en-20-yn-17-ol (desogestrel D
3
-isomer)
H
H H
Et
C
HO
H
HC
CH
2
H
2
C
CH
H
OH
C
Et
H H
H
B. 13-ethyl-16-[13-ethyl-17-hydroxy-11-methylene-18,19-dinor-17-pregn-4-en-20-yn-16-
ylidene]-11-methylene-18,19-dinor-17-pregn-4-en-20-yn-17-ol
H
H H
Me
C
OH
H
CH
H
2
C
C. 11-methylene-19-nor-17-pregn-4-en-20-yn-17-ol
H
H H
Et
H
H
2
C
O
D. 13-ethyl-11-methylenegon-4-en-17-one
12-62
Desoxycortone Acetate
Me
H
H Me
O
O
H
OAc
C
23
H
32
O
4
372.5 56-47-3
Desoxycortone Acetate complies with the requirements of the 3rd edition of the European Pharmacopoeia
When deoxycortone acetate is prescribed or demanded, Desoxycortone Acetate shall be dispensed
or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Desoxycortone acetate contains not less than 97.0 per cent and not more than the equivalent of
103.0 per cent of 21-hydroxypregn-4-en-3,20-dione 21-acetate, calculated with reference to the
dried substance.
CHARACTERS
A white or almost white, crystalline powder or colourless crystals, practically insoluble in water,
freely soluble in methylene chloride, soluble in acetone, sparingly soluble in alcohol, slightly soluble
in propylene glycol and in fatty oils.
IDENTIFICATION
obtained with desoxycortone acetate CRS.
solvents.
Reference solution (a). Dissolve 20 mg of desoxycortone acetate CRS in a mixture of 1 volume of
solvents.
Reference solution (b). Dissolve 10 mg of cortisone acetate R in reference solution (a) and dilute to
10 ml with reference solution (a).
examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the
until the spots appear. Allow to cool. Examine the plate in daylight and in ultraviolet light at
in the chromatogram obtained with reference solution (a). The test is not valid unless the chroma-
togram obtained with reference solution (b) shows two clearly separated spots.
D. Add about 2 mg to 2 ml of sulphuric acid R and shake to dissolve. Within 5 min, a yellow colour
develops. Add the solution to 2 ml of water R and shake. The resulting solution is dichroic showing
an intense blue colour by transparency, and red fluorescence which is particularly intense in ultra-
violet light at 365 nm.
12-63
TESTS
substance.
Reference solution (a). Dissolve 2 mg of desoxycortone acetate CRS and 2 mg of betamethasone
17-valerate CRS in the mobile phase and dilute to 200.0 ml with the mobile phase.
volumetric flask mix 350 ml of water R with 600 ml of acetonitrile R and allow to equilibrate;
recorder.
conditions, the retention times are: betamethasone 17-valerate, about 7.5 min and desoxycortone
acetate, about 9.5 min. The test is not valid unless the resolution between the peaks due to
betamethasone 17-valerate and desoxycortone acetate is at least 4.5; if necessary, adjust the
chromatography for three times the retention time of the principal peak. In the chromatogram
obtained with the test solution, the sum of the areas of all the peaks, apart from the principal peak,
is not greater than the area of the principal peak in the chromatogram obtained with reference
solution (b) (0.5 per cent). Disregard any peak with an area less than 0.1 times the area of the
at 100C to 105C.
ASSAY
Dissolve 0.100 g in alcohol R and dilute to 100.0 ml with the same solvent. Dilute 2.0 ml to
100.0 ml with alcohol R. Measure the absorbance (2.2.25) at the maximum at 240 nm.
23
H
32
O
4
STORAGE
__________________________________________________________________________________________________________ Ph Eur
12-64
Devils Claw
Harpagophytum
Devils Claw complies with the requirements of the 3rd edition of the European Pharmacopoeia for Devils
Claw Root [1069]. These requirements are reproduced after the heading Definition below.
Action and use Antirheumatic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Devils claw root consists of the cut and dried tuberous, secondary roots of Harpagophytum
procumbens D.C.
It contains not less than 1.2 per cent of harpagoside (C
24
H
30
O
11
, M
r
494.5), calculated with
reference to the dried drug.
CHARACTERS
Devils claw root is greyish-brown to dark brown and it has a bitter taste.
It has the macroscopic and microscopic characters described in identification tests A and B.
IDENTIFICATION
A. It consists of thick, fan-shaped or rounded slices or of roughly crushed discs. The darker outer
surface is traversed by tortuous longitudinal wrinkles. The paler cut surface shows a dark cambial
zone and xylem bundles distinctly aligned in radial rows. The central cylinder shows fine concentric
striations. Seen under a lens, the cut surface presents yellow to brownish-red granules.
B. Reduce to a powder (355). The powder is brownish-yellow. Examine under a microscope using
chloral hydrate solution R. The powder shows the following diagnostic characters: fragments of cork
layer consisting of yellowish-brown, thin-walled cells; fragments of cortical parenchyma consisting
of large, thin-walled cells, sometimes containing reddish-brown granular inclusions and isolated
yellow droplets; fragments of reticulately thickened vessels and tracheidal vessels with associated
lignified parenchyma from the central cylinder; small needles and crystals of calcium oxalate are
present in the parenchyma. The powder may show rectangular or polygonal pitted sclereids with
dark reddish-brown contents. With a solution of phloroglucinol in hydrochloric acid, the
parenchyma turns green.
substance.
Test solution. Heat on a water-bath at 60C for 10 min 1.0 g of the powdered drug (355) with 10 ml
of methanol R. Filter and reduce the filtrate to about 2 ml under reduced pressure at a temperature
not exceeding 40C.
Reference solution. Dissolve 1 mg of harpagoside R in 1 ml of methanol R.
mixture of 8 volumes of water R, 15 volumes of methanol R and 77 volumes of ethyl acetate R. Dry
the plate in a current of warm air. Examine in ultraviolet light at 254 nm. The chromatograms
obtained with the test solution and the reference solution both show at the middle a quenching
zone corresponding to harpagoside. The chromatogram obtained with the test solution shows other
distinct bands, mainly above the zone corresponding to harpagoside. Spray with a 10 g/l solution of
phloroglucinol R in alcohol R and then with hydrochloric acid R. Dry the plate at 80C for 5 min to
10 min. In the chromatograms obtained with the reference solution and with the test solution the
zone corresponding to harpagoside is green. The chromatogram obtained with the test solution also
shows several yellow to brown zones below and above the zone corresponding to harpagoside.
TESTS
Starch Examine the powdered drug under a microscope using water R. Add iodine solution R1. No
drug (355) by drying in an oven at 100C to 105C.
ASSAY
Examine by liquid chromatography (2.2.29) using methyl cinnamate R as the internal standard.
Internal standard solution. Dissolve 0.130 g of methyl cinnamate R in 50 ml of methanol R and dilute
12-65
Test solution. To 0.500 g of the powdered drug (355) add 50 ml of methanol R. Shake for 1 h and
filter. Transfer the filter with the residue to a 100 ml flask, add 50 ml of methanol R and heat under
a reflux condenser for 1 h. Cool and filter. Rinse the flask and the filter with two quantities, each of
5 ml, of methanol R. Combine the filtrate and the rinsing solution and evaporate to dryness under
reduced pressure at a temperature not exceeding 40C. Take up the residue with three quantities,
each of 5 ml, of methanol R and filter the extracts in a 25 ml volumetric flask. Whilst washing the
filter, dilute to 25.0 ml with methanol R. To 10.0 ml of this solution add 1.0 ml of the internal
standard solution and dilute to 25.0 ml with methanol R.
Reference solution. Dilute 0.5 ml of the reference solution described in Identification test C to 2.0 ml
with methanol R.
as mobile phase at a flow rate of 1.5 ml per minute a mixture of equal volumes of methanol R and
water R,
Inject the test solution. Adjust the sensitivity of the detector so that the height of the peak due to
methyl cinnamate is about 50 per cent of the full scale of the recorder.
Determine the retention time of harpagoside using 10 l of the reference solution examined
under the same conditions as the test solution.
Calculate the percentage content of harpagoside from the expression:
m F
F m
2 1
2 1
7622
.
m
1
=mass of the drug in grams,
m
2
=mass of methyl cinnamate R in grams in 100.0 ml of the internal standard solution,
F
1
=area of the peak corresponding to harpagoside in the chromatogram of the test solution,
F
2
=area of the peak corresponding to methyl cinnamate, in the chromatogram of the test
solution.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
12-66
Dexamethasone
Me
H
H Me
O
O
OH
Me
F
HO
OH
C
22
H
29
FO
5
392.5 50-02-2
Dexamethasone complies with the requirements of the 3rd edition of the European Pharmacopoeia [0388].
Preparation
Dexamethasone Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Dexamethasone contains not less than 97.0 per cent and not more than the equivalent of 103.0 per
cent of 9-fluoro-11,17,21-trihydroxy-16-methylpregna-1,4-diene-3,20-dione, calculated with
CHARACTERS
ethanol, slightly soluble in methylene chloride.
IDENTIFICATION
A. Dissolve 10.0 mg in ethanol R and dilute to 100.0 ml with the same solvent. Place 2.0 ml of the
solution in a stoppered test tube, add 10.0 ml of phenylhydrazine-sulphuric acid solution R, mix and
heat in a water-bath at 60C for 20 min. Cool immediately. The absorbance (2.2.25) of the
solution at the maximum at 419 nm is not less than 0.4.
obtained with dexamethasone CRS.
solvents.
Reference solution (a). Dissolve 20 mg of dexamethasone CRS in a mixture of 1 volume of methanol R
Reference solution (b). Dissolve 10 mg of betamethasone CRS in reference solution (a) and dilute to
Apply separately to the plate 5 l of each solution. Develop over a path of 15 cm, using a mixture of
5 volumes of butanol R saturated with water R, 10 volumes of toluene R and 85 volumes of ether R.
the chromatogram obtained with reference solution (a). Spray with alcoholic solution of sulphuric
acid R. Heat at 120C for 10 min or until the spots appear. Allow to cool. Examine the chromato-
grams in daylight and in ultraviolet light at 365 nm. The principal spot in the chromatogram
obtained with the test solution is similar in position, colour in daylight, fluorescence in ultraviolet
light at 365 nm and size to the principal spot in the chromatogram obtained with reference solution
spots which may, however, not be completely separated.
D. Add about 2 mg to 2 ml of sulphuric acid R and shake to dissolve. Within 5 min, a faint reddish-
brown colour develops. Add the solution to 10 ml of water R and mix. The colour is discharged.
12-67
TESTS
substance.
Test solution. Dissolve 25.0 mg of the substance to be examined in a 50 per cent V/V solution of
acetonitrile R in methanol R and dilute to 10.0 ml with the same solvent.
Reference solution (a). Dissolve 2 mg of dexamethasone CRS and 2 mg of methylprednisolone CRS in
mobile phase A and dilute to 100.0 ml with the same solvent mixture.
Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with mobile phase A.
silica gel for chromatography R (5 m).
as mobile phase at a flow rate of 2.5 ml per minute a linear gradient programme using the follow-
ing conditions:
Mobile phase A. In a 1000 ml volumetric flask, mix 250 ml of acetonitrile R with 700 ml of water R
and allow to equilibrate; adjust the volume to 1000 ml with water R and mix again,
Time
(min)
Mobile
phase A (per
cent V/V)
Mobile
phase B (per
cent V/V)
Comment
0 100 0 Isocratic
15 1000 0100 Begin linear gradient
40 0 100 End chromatogram,
return to 100A
41 100 0 Begin equilibration
with A
460 100 0 End equilibration,
begin next
chromatogram
Equilibrate the column for at least 30 min with mobile phase B at a flow rate of 2.5 ml per minute
and then with the mobile phase A for 5 min. For subsequent chromatograms, use the conditions
described from 40.0 min to 46.0 min.
conditions, the retention times are: methylprednisolone, about 11.5 min and dexamethasone, about
13 min. The test is not valid unless the resolution between the peaks corresponding to
methylprednisolone and dexamethasone is at least 2.8; if necessary, adjust the concentration of
acetonitrile in mobile phase A.
Inject separately 20 l of the 50 per cent V/V solution of acetonitrile R in methanol R as a blank,
20 l of the test solution and 20 l of reference solution (b). Record the chromatogram of the test
solution for twice the retention time of the principal peak. In the chromatogram obtained with the
test solution: the area of any peak, apart from the principal peak, is not greater than 0.5 times the
area of the principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent);
the sum of the areas of all the peaks, apart from the principal peak, is not greater than the area of
the principal peak in the chromatogram obtained with reference solution (b) (1 per cent). Disregard
any peak due to the blank and any peak with the area less than 0.05 times the area of the principal
at 100C to 105C.
ASSAY
12-68
22
H
29
FO
5
STORAGE
__________________________________________________________________________________________________________ Ph Eur
13-1
Dexamethasone Acetate
Me
H
H Me
O
O
OH
Me
F
HO
OAc
C
24
H
31
FO
6
434.5 55812-90-3
Dexamethasone Acetate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Dexamethasone acetate contains not less than 97.0 per cent and not more than the equivalent of
103.0 per cent of 9-fluoro-11,17,21-trihydroxy-16-methylpregna-1,4-diene-3,20-dione 21-acetate,
CHARACTERS
and in alcohol, slightly soluble in methylene chloride.
Dexamethasone acetate shows polymorphism.
IDENTIFICATION
solution at the maximum at 419 nm is not less than 0.35.
obtained with dexamethasone acetate CRS. If the spectra obtained in the solid state with the substance
to be examined and the reference substance show differences, record further spectra using saturated
solutions (about 30 g/l) of the substance to be examined and of the reference substance in chloro-
form R in a 0.2 mm cell.
Reference solution (a). Dissolve 20 mg of dexamethasone acetate CRS in a mixture of 1 volume of
solvents.
ence solution (a). Spray with alcoholic solution of sulphuric acid R. Heat at 120C for 10 min or until
the spots appear. Allow to cool. Examine the plate in daylight and in ultraviolet light at 365 nm. The
D. Add about 2 mg to 2 ml of sulphuric acid R and shake to dissolve. Within 5 min, a faint reddish-
13-2
brown colour develops. Add the solution to 10 ml of water R and mix. The colour is discharged and
a clear solution remains.
F. About 10 mg gives the reaction of acetyl (2.3.1).
TESTS
substance.
Test solution. Dissolve 25.0 mg of the substance to be examined in about 4 ml of acetonitrile R and
Reference solution (a). Dissolve 2 mg of dexamethasone acetate CRS and 2 mg of betamethasone acetate
volumetric flask mix 380 ml of acetonitrile R with 550 ml of water R and allow to equilibrate; dilute
to 1000 ml with water R and mix again,
Equilibrate the column with the mobile phase at a flow rate of 1 ml per minute for about 30
minutes.
recorder.
conditions, the retention times are: betamethasone acetate, about 19 min and dexamethasone
acetate, about 22 min. The test is not valid unless the resolution between the peaks due to
betamethasone acetate and dexamethasone acetate is at least 3.3; if necessary, adjust the concentra-
tion of acetonitrile in the mobile phase.
with the test solution: the area of any peak, apart from the principal peak, is not greater than half the
the sum of the areas of all such peaks is not greater than the area of the principal peak in the chroma-
togram obtained with reference solution (b) (1.0 per cent). Disregard any peak with an area less than
Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 0.500 g by drying in vacuo in
an oven at 100C to 105C.
ASSAY
Dissolve 0.100 g in alcohol R and dilute to 100.0 ml with the same solvent. Dilute 2.0 ml to 100.0 ml
with alcohol R. Measure the absorbance (2.2.25) at the maximum at 238.5 nm.
24
H
31
FO
6
STORAGE
__________________________________________________________________________________________________________ Ph Eur
13-3
Dexamethasone Sodium Phosphate
corrected 1/01
H
O
O
OH
Me
F
HO
O P O
ONa
ONa
Me
Me
H
C
22
H
28
FNa
2
O
8
P 516.4 2392-39-4
Dexamethasone Sodium Phosphate complies with the requirements of the 3rd edition of the European
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Dexamethasone sodium phosphate contains not less than 97.0 per cent and not more than the
equivalent of 103.0 per cent of 9-fluoro-11,17,21-trihydroxy-16-methylpregna-1,4-diene-3,20-
dione disodium 21-phosphate, calculated with reference to the anhydrous, ethanol-free substance.
CHARACTERS
A white or almost white powder, very hygroscopic, freely soluble in water, slightly soluble in alcohol,
IDENTIFICATION
A. Dissolve 10.0 mg in 5 ml of water R and dilute to 100.0 ml with ethanol R. Place 2.0 ml of this
solution measured at the maximum at 419 nm is at least 0.20.
obtained with dexamethasone sodium phosphate CRS. If the spectra obtained in the solid state show
minimum volume of alcohol R, evaporate to dryness on a water-bath and record new spectra using the
residues.
the same solvent.
Reference solution (a). Dissolve 20 mg of dexamethasone sodium phosphate CRS in methanol R and dilute
Reference solution (b). Dissolve 10 mg of prednisolone sodium phosphate CRS in reference solution (a)
and dilute to 10 ml with the same solution.
Apply separately to the plate 5 l of each solution. Develop over a path of 15 cm, using a mixture
of 20 volumes of glacial acetic acid R, 20 volumes of water R and 60 volumes of butanol R. Allow the
gram obtained with reference solution (a). Spray with alcoholic solution of sulphuric acid R. Heat at
120C for 10 min or until the spots appear. Allow to cool. Examine in daylight and in ultraviolet light
at 365 nm. The principal spot in the chromatogram obtained with the test solution is similar in
gram obtained with reference solution (b) shows two spots which may, however, not be completely
separated.
13-4
D. Add about 2 mg to 2 ml of sulphuric acid R and shake to dissolve. Within 5 min, a faint yellowish-
brown colour develops. Add the solution to 10 ml of water R and mix. The colour fades and a clear
solution remains.
F. To 40 mg add 2 ml of sulphuric acid R and heat gently until white fumes are evolved, add nitric
acid R dropwise, continue the heating until the solution is almost colourless and cool. Add 2 ml of
water R, heat until white fumes are again evolved, cool, add 10 ml of water R and neutralise to red
litmus paper R with dilute ammonia R1. The solution gives reaction (a) of sodium (2.3.1) and reaction
(b) of phosphates (2.3.1).
TESTS
solution B
7
(Method II, 2.2.2).
pH (2.2.3). Dilute 1 ml of solution S to 5 ml with carbon dioxide-free water R. The pH of the solution
is 7.5 to 9.5.
solvent. The specific optical rotation is +75 to +83, calculated with reference to the anhydrous,
ethanol-free substance.
Reference solution (a). Dissolve 2 mg of dexamethasone sodium phosphate CRS and 2 mg of
betamethasone sodium phosphate CRS in the mobile phase and dilute to 100.0 ml with the mobile
phase.
as mobile phase at a flow rate of 1 ml/min a mixture prepared as follows: in a 250 ml conical
flask, weigh 1.360 g of potassium dihydrogen phosphate R and 0.600 g of hexylamine R, mix and
allow to stand for 10 min and then dissolve in 182.5 ml of water R; add 67.5 ml of acetonitrile R,
mix and filter (0.45 m),
conditions, the retention times are: betamethasone sodium phosphate, about 12.5 min and
dexamethasone sodium phosphate about 14 min. The test is not valid unless the resolution between
the peaks corresponding to betamethasone sodium phosphate and dexamethasone sodium phosphate
is at least 2.2. If necessary, adjust slightly the concentration of acetonitrile or increase the concentra-
tion of water in the mobile phase.
the sum of the areas of all such peaks is not greater than the area of the principal peak in the chroma-
togram obtained with reference solution (b) (1 per cent). Disregard any peak with an area less than
0.05 times that of the principal peak in the chromatogram obtained with reference solution (b).
Inorganic phosphates Dissolve 50 mg in water R and dilute to 100 ml with the same solvent. To
10 ml of this solution add 5 ml of molybdovanadic reagent R, mix and allow to stand for 5 min. Any
yellow colour in the solution is not more intense than that in a standard prepared at the same time in
the same manner using 10 ml of phosphate standard solution (5 ppm PO
4
) R (1 per cent).
Ethanol Not more than 3.0 per cent m/m, determined by gas chromatography (2.2.28), using
propanol R as the internal standard.
13-5
Internal standard solution. Dilute 1.0 ml of propanol R to 100.0 ml with water R.
Test solution. Dissolve 0.50 g of the substance to be examined in 5.0 ml of the internal standard
solution and dilute to 10.0 ml with water R.
Reference solution. Dilute 1.0 g of ethanol R to 100.0 ml with water R. To 2.0 ml of the solution add
5.0 ml of the internal standard solution and dilute to 10.0 ml with water R.
a column 1 m long and 3.2 mm in internal diameter packed with ethylvinylbenzene-divinylbenzene
copolymer R1 (150 m to 180 m),
the detector at 280C. Inject 2 l of each solution.
Ethanol and water Determine the water content using 0.200 g by the semi-micro determination of
water (2.5.12). Add the percentage content of water and the percentage content of ethanol obtained
in the test for ethanol. The sum of the percentage contents of water and ethanol is not greater than
13.0 per cent m/m.
ASSAY
Dissolve 0.100 g in water R and dilute to 100.0 ml with the same solvent. Dilute 10.0 ml of the
solution to 500.0 ml with water R. Measure the absorbance (2.2.25) at the maximum at 241.5 nm.
22
H
28
FNa
2
O
8
STORAGE
IMPURITIES
A. dexamethasone,
B. betamethasone sodium phosphate.
__________________________________________________________________________________________________________ Ph Eur
13-6
Dexamfetamine Sulphate
NH
2
Me H
2
,H
2
SO
4
(C
9
H
13
N)
2
,H
2
SO
4
368.5 51-63-8
Definition Dexamfetamine Sulphate is (S)--methylphenethylamine sulphate. It contains not less
than 99.0% and not more than 100.5% of (C
9
H
13
N)
2
,H
2
SO
4
substance.
Freely soluble in water; slightly soluble in ethanol (96%); practically insoluble in ether.
Identification
A. Dissolve 1 g in 50 ml of water, add 10 ml of 5M sodium hydroxide and 0.5 ml of benzoyl chloride and
shake. Repeat the addition of benzoyl chloride in 0.5-ml quantities until no further precipitate is
produced. The melting point of the precipitate, after recrystallising twice from ethanol (50%), is about
157, Appendix V A.
B. Dissolve 2 mg in 4 ml of water, add 1 ml of 1M hydrochloric acid, 2 ml of diazotised nitroaniline
solution, 4 ml of 1M sodium hydroxide and 2 ml of butan-1-ol, shake and allow to separate. A red colour
is produced in the butanol layer (distinction from methylamfetamine).
C. Yields the reactions characteristic of sulphates, Appendix VI.
Acidity or alkalinity Dissolve 0.5 g in 10 ml of water and titrate with 0.01M hydrochloric acid VS or
0.01M sodium hydroxide VS using methyl red solution as indicator. Not more than 0.1 ml of 0.01M
hydrochloric acid VS or 0.01M sodium hydroxide VS is required to change the colour of the solution.
Specific optical rotation In an 8.0% w/v solution, +19.5 to +22.0, calculated with reference to
the dried substance, Appendix V F.
1 g.
Assay Dissolve 0.4 g in 120 ml of water, add 2 ml of 5M sodium hydroxide and distil into 50 ml of
0.1M hydrochloric acid VS, continuing the distillation until only 5 ml of liquid is left in the distillation
flask. Titrate the excess of acid with 0.1M sodium hydroxide VS using methyl red solution as indicator.
Each ml of 0.1M hydrochloric acid VS is equivalent to 18.42 mg of (C
9
H
13
N)
2
,H
2
SO
4
.
Preparation
Dexamfetamine Tablets
Action and use Central nervous system stimulant.
When dexamphetamine sulphate is prescribed or demanded, Dexamfetamine Sulphate shall be
13-7
Dexchlorpheniramine Maleate
N
NMe
2
H
Cl
COOH
COOH
,
C
16
H
19
ClN
2
,C
4
H
4
O
4
390.9 2438-32-6
Dexchlorpheniramine Maleate complies with the requirements of the 3rd edition of the European
1
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Dexchlorpheniramine maleate contains not less than 98.0 per cent and not more than the equivalent
of 100.5 per cent of (3S)-3-(4-chlorophenyl)-3-(pyridin-2-yl)-N,N-dimethylpropan-1-amine (Z)-
CHARACTERS
A white crystalline powder, very soluble in water, freely soluble in alcohol, in methanol and in
methylene chloride.
IDENTIFICATION
First identification: A, C, E.
obtained with dexchlorpheniramine maleate CRS. Examine the substances prepared as discs using
potassium bromide R.
D. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel F
254
plate R.
the same solvent.
Reference solution. Dissolve 56 mg of maleic acid R in methanol R and dilute to 10 ml with the same
solvent.
of water R, 7 volumes of anhydrous formic acid R, 20 volumes of methanol R and 70 volumes of di-
isopropyl ether R. Allow the plate to dry in a current of air for a few minutes and examine in ultraviolet
light at 254 nm. The chromatogram obtained with the test solution shows two clearly separated
spots. The upper spot is similar in position and size to the spot in the chromatogram obtained with
the reference solution.
TESTS
solution BY
6
(Method II, 2.2.2).
pH (2.2.3). Dissolve 0.20 g in 20 ml of water R. The pH of the solution is 4.5 to 5.5.
Test solution. Dissolve 10.0 mg in 1.0 ml of methylene chloride R.
Reference solution. Dissolve 5.0 mg of brompheniramine maleate CRS in 0.5 ml of methylene chloride R
and add 0.5 ml of the test solution. Dilute 0.5 ml of this solution to 50.0 ml with methylene chloride R.
a glass column 2.3 m long and 2 mm in internal diameter packed with acid- and base-washed
13-8
silanised diatomaceous earth for gas chromatography R (135 m to 175 m) impregnated with 3 per
cent m/m of a mixture of 50 per cent of poly(dimethyl)siloxane and 50 per cent of poly-
(diphenyl)siloxane,
detector at 250C.
Inject 1 l of reference solution. The test is not valid unless, in the chromatogram obtained with
the reference solution, the resolution between the two peaks corresponding to dexchlorpheniramine
and brompheniramine is at least 1.5. Inject 1 l of the test solution. Continue the chromatography
for at least 2.5 times the retention time of the principal peak. In the chromatogram obtained with the
test solution: none of the peaks, apart from the principal peak, has an area greater than 0.8 times the
area of the peak corresponding to dexchlorpheniramine in the chromatogram obtained with reference
solution (0.4 per cent); the sum of the areas of all the peaks, apart from the principal peak, is not
greater than twice the area of the peak corresponding to dexchlorpheniramine in the chromatogram
obtained with reference solution (1 per cent).
Enantiomeric purity Examine by liquid chromatography (2.2.29).
Test solution. Dissolve 10.0 mg in 3 ml of water R. Add a few drops of concentrated ammonia R to
alkaline reaction. Shake with 5 ml of methylene chloride R. Separate the layers. Evaporate the lower,
methylene chloride layer to an oily residue on a water-bath. Dissolve the oily residue in 2-propanol R
Reference solution (a). Dissolve 10.0 mg of dexchlorpheniramine maleate CRS in 3 ml of water R. Add a
few drops of concentrated ammonia R to alkaline reaction. Shake with 5 ml of methylene chloride R.
Separate the layers. Evaporate the lower, methylene chloride layer to an oily residue on a water-bath.
Dissolve the oily residue in 2-propanol R and dilute to 10.0 ml with the same solvent.
Reference solution (b). Dissolve 10.0 mg of chlorphenamine maleate CRS in 3 ml of water R. Add a few
drops of concentrated ammonia R to alkaline reaction. Shake with 5 ml of methylene chloride R.
Separate the layers. Evaporate the lower, methylene chloride layer to an oily residue on a water-bath.
Dissolve the oily residue in 2-propanol R and dilute to 10.0 ml with the same solvent.
Reference solution (c). Dilute 1.0 ml of the test solution to 50 ml with 2-propanol R.
a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with amylose
derivative of silica gel for chromatography R,
as mobile phase at a flow rate of 1 ml/min a mixture of 3 volumes of diethylamine R, 20 volumes
of 2-propanol R and 980 volumes of hexane R,
Under these conditions the peak of the (S) isomer appears first.
reference solution (b), the resolution between the peaks corresponding to the (R)-enantiomer and to
the (S)-enantiomer is at least 1.5; the retention times of the principal peaks in the chromatograms
obtained with the test solution and reference solution (a) are identical ((S)-enantiomer). In the
chromatogram obtained with the test solution, the area of the peak corresponding to the (R)-
enantiomer is not greater than the area of the principal peak in the chromatogram obtained with
reference solution (c) (2 per cent) and the area of any peak, apart from the principal peak and any
peak corresponding to the (R)-enantiomer, is not greater than 0.25 times the area of the principal
peak in the chromatogram obtained with reference solution (c) (0.5 per cent).
65C for 4 h.
ASSAY
20
H
23
ClN
2
O
4
.
STORAGE
13-9
IMPURITIES
N
N
Me
Me
H
and enantiomer
A. (3RS)-3-phenyl-3-(pyridin-2-yl)-N,N-dimethylpropan-1-amine (pheniramine),
B. (3R)-3-(4-chlorophenyl)3-(pyridin-2-yl)-N,N-dimethylpropan-1-amine.
__________________________________________________________________________________________________________ Ph Eur
13-10
Dexpanthenol
HO
N
Me Me
OH H
OH
O
H
C
9
H
19
NO
4
205.3 81-13-0
Dexpanthenol complies with the requirements of the 3rd edition of the European Pharmacopoeia [0761]. These
Action and use Analogue of pantothenic acid.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Dexpanthenol contains not less than 98.0 per cent and not more than the equivalent of 101.0 per
cent of (R)-2,4-dihydroxy-N-(3-hydroxypropyl)-3,3-dimethylbutyramide, calculated with reference
CHARACTERS
A colourless or slightly yellowish, viscous hygroscopic liquid, or a white, crystalline powder, very
soluble in water, freely soluble in alcohol, slightly soluble in ether.
IDENTIFICATION
obtained with dexpanthenol CRS. Examine the substances using discs prepared by placing 0.05 ml of
a 50 g/l solution in ethanol R on a disc of potassium bromide R. Dry the disc at 100C to 105C for
15 min.
C. Examine the chromatograms obtained in the test for 3-aminopropanol. The principal spot in the
D. To 1 ml of solution S (see Tests) add 1 ml of dilute sodium hydroxide solution R and 0.1 ml of
copper sulphate solution R. A blue colour develops.
TESTS
solvent.
solution B
6
(Method II, 2.2.2).
pH (2.2.3). The pH of solution S is not greater than 10.5.
solution S and calculated with reference to the anhydrous substance.
3-Aminopropanol Examine by thin-layer chromatography (2.2.27), using silica gel G R as the
coating substance.
Reference solution (a). Dissolve 50 mg of dexpanthenol CRS in methanol R and dilute to 10 ml with the
same solvent.
Reference solution (b). Dissolve 25 mg of 3-aminopropanol R in methanol R and dilute to 100 ml with
the same solvent.
20 volumes of concentrated ammonia R, 25 volumes of methanol R and 55 volumes of butanol R. Allow
the plate to dry in air, spray with a 100 g/l solution of trichloroacetic acid R in methanol R and heat at
150C for 10 min. Spray with a 1 g/l solution of ninhydrin R in methanol R and heat at 120C until a
colour appears. Any spot corresponding to 3-aminopropanol in the chromatogram obtained with test
13-11
solution (a) is not more intense than the spot in the chromatogram obtained with reference
mination of water.
ASSAY
To 0.400 g add 50.0 ml of 0.1M perchloric acid. Boil under a reflux condenser for 5 h protected from
humidity. Allow to cool. Add 50 ml of dioxan R by rinsing the condenser, protected from humidity.
Add 0.2 ml of naphtholbenzein solution R and titrate with 0.1M potassium hydrogen phthalate until the
colour changes from green to yellow. Carry out a blank titration.
9
H
19
NO
4
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
13-12
Dextran 1 for Injection
1/01
Dextran 1 for Injection complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Dextran 1 for injection is a low molecular weight fraction of dextran, consisting of a mixture of
isomaltooligosaccharides. It is obtained by hydrolysis and fractionation of dextrans produced by
fermentation of sucrose using Leuconostoc mesenteroides strain NRRL B-512 = CIP 78.59 or substrains
thereof (for example L. mesenteroides B-512F = NCTC 10817). It is prepared in conditions designed
to minimise the risk of microbial contamination.
The average relative molecular mass is about 1000.
CHARACTERS
A white or almost white powder, hygroscopic, very soluble in water, very slightly soluble in alcohol.
IDENTIFICATION
A. Dissolve 3.000 g in water R, heat on a water-bath and dilute to 100.0 ml with the same solvent.
The specific optical rotation (2.2.7) is +148 to +164, calculated with reference to the dried
substance. Dry an aliquot of the solution first on a water-bath and then to constant weight in vacuo at
70C. Calculate the dextran content after correction for the content of sodium chloride.
obtained with dextran 1 CRS. Prepare the discs as follows: to 1-2 mg add one or a few drops of
water R; grind in an agate mortar for 1 min to 2 min; add about 300 mg of potassium bromide R and
mix to a slurry (do not grind); dry in vacuo at 40C for 15 min, crush the residue (if it is not dry, dry
for another 15 min). Prepare a disc using potassium bromide R. Run the infrared spectrum with a
blank disc using potassium bromide R in the reference beam.
C. It complies with the test for molecular-mass distribution (see Tests).
TESTS
Solution S Dissolve 7.5 g in carbon dioxide-free water R, heat on a water-bath and dilute to 50 ml
Absorbance (2.2.25). Measure the absorbance of solution S at 375 nm. The absorbance is not more
than 0.12.
Acidity or alkalinity To 10 ml of solution S add 0.1 ml of phenolphthalein solution R. The solution is
colourless. Add 0.2 ml of 0.01M sodium hydroxide. The solution is pink. Add 0.4 ml of 0.01M hydro-
chloric acid. The solution is colourless. Add 0.1 ml of methyl red solution R. The solution is red or
orange.
Nitrogen-containing substances Carry out the determination of nitrogen by sulphuric acid
digestion (2.5.9), using 0.200 g and heating for 2 h. Collect the distillate in a mixture of 0.5 ml of
bromocresol green solution R, 0.5 ml of methyl red solution R and 20 ml of water R. Titrate with 0.01M
hydrochloric acid. Not more than 0.15 ml of 0.01M hydrochloric acid is required to change the colour of
the indicator (110 ppm N
2
).
Sodium chloride Not more than 1.5 per cent. Accurately weigh 3 g to 5 g and dissolve in 100 ml of
water R. Add 0.3 ml of potassium chromate solution R and titrate with 0.1M silver nitrate until the
yellowish-white colour changes to reddish-brown.
Molecular-mass distribution The average molecular mass (M
w
) is 850 to 1150. The fraction with
less than 3 units of glucose is less than 15 per cent, the fraction with more than 9 units of glucose is
Examine by size-exclusion chromatography (2.2.30).
Test solution. Dissolve 6.0 mg to 6.5 mg of the substance to be examined in 1.0 ml of the mobile
phase.
Reference solution (a). Dissolve 6.0 mg to 6.5 mg of dextran 1 CRS in 1.0 ml of the mobile phase.
Reference solution (b). Dissolve the content of an ampoule of isomaltooligosaccharide CRS in 1 ml of the
mobile phase, and mix. This corresponds to approximately 45 g of isomaltotriose (3 glucose units),
approximately 45 g of isomaltononaose (9 glucose units), and approximately 60 g of sodium
chloride per 100 l.
13-13
2 columns, 30 cm long and 10 mm in internal diameter, in series, prepacked with a packing
material of dextran covalently bound to highly cross-linked porous agarose beads, allowing
resolution of oligosaccharides in the molecular mass range of 180 to 3000, kept at a temperature
of 20C to 25C,
as mobile phase at a flow rate of 0.07 ml/min to 0.08 ml/min maintained constant to 1 per
cent, a 2.92 g/l solution of sodium chloride R,
as detector a differential refractometer.
Inject 100 l of reference solution (b) and record the resulting chromatogram for definition of the
positions of isomaltotriose, isomaltononaose and sodium chloride. Inject 100 l of the test solution
and 100 l of reference solution (a) and record the chromatograms. Determine the peak areas.
Disregard any peak due to sodium chloride.
Calculate the average relative molecular mass M
w
, and the amount of the fraction with less than 3
and more than 9 glucose units, of dextran 1 CRS and of the substance to be examined. The test is not
valid unless the values obtained for dextran 1 CRS are within the values stated on the label.

i i w
m w M
M
w
=average molecular mass of the dextran,
m
i
=molecular mass of oligosaccharide i,
w
i
=weight proportion of oligosaccharide i.
Use the following molecular mass values for the calculation:
glucose 180
isomaltose 342
isomaltotriose 504
isomaltotetraose 666
isomaltopentaose 828
isomaltohexaose 990
isomaltoheptaose 1152
isomaltooctaose 1314
isomaltononaose 1476
isomaltodecaose 1638
isomaltoundecaose 1800
isomaltododecaose 1962
isomaltotridecaose 2124
isomaltotetradecaose 2286
isomaltopentadecaose 2448
isomaltohexadecaose 2610
isomaltoheptadecaose 2772
isomaltooctadecaose 2934
isomaltononadecaose 3096
Heavy metals (2.4.8). Dilute 10 ml of solution S to 15 ml with water R. 12 ml of this solution
100C to 105C for 5 h.
Bacterial endotoxins (2.6.14). Not more than 25 I.U. of endotoxin per gram.
2
micro-organisms
per gram, determined by plate-count. It complies with the test for Escherichia coli (2.6.13).
__________________________________________________________________________________________________________ Ph Eur
13-14
Action and use Plasma volume expander.
Preparation
Dextran 40 Intravenous Infusion
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Dextran 40 for injection is a mixture of polysaccharides, principally of the -1,6-glucan type,
obtained by hydrolysis and fractionation of dextrans produced by fermentation of sucrose using
Leuconostoc mesenteroides strain NRRL B-512 or substrains thereof (for example L. mesenteroides B-
512F = NCTC 10817). It is prepared in conditions designed to minimise the risk of microbial
contamination.
The average relative molecular mass is about 40,000.
CHARACTERS
A white or almost white powder, very soluble in water, slightly soluble in ether, very slightly soluble
in alcohol.
IDENTIFICATION
A. Dissolve 1.0 g in water R, heating on a water-bath, and dilute to 50.0 ml with the same solvent.
The specific optical rotation (2.2.7) of the solution is +195 to +201, calculated with reference to
obtained with dextran CRS.
TESTS
Solution S Dissolve 5.0 g in distilled water R, heating on a water-bath, and dilute to 50 ml with the
same solvent.
remains colourless. Add 0.2 ml of 0.01M sodium hydroxide. The solution is red. Add 0.4 ml of 0.01M
hydrochloric acid. The solution is colourless. Add 0.1 ml of methyl red solution R. The solution is red or
orange.
the indicator.
Residual solvents Examine by gas chromatography (2.2.28), using propanol R as internal standard.
Test solution. Dissolve 5 g of the substance to be examined in 100 ml of water R and distil. Collect the
first 45 ml of the distillate, add 1 ml of a 25 g/l solution of propanol R and dilute to 50 ml with
water R.
Reference solution. Mix 0.5 ml of a 25 g/l solution of ethanol R, 0.5 ml of a 25 g/l solution of propanol R
and 0.5 ml of a 2.5 g/l solution of methanol R and dilute to 25.0 ml with water R.
a stainless steel column 1.8 m long and 2 mm in internal diameter packed with ethylvinylbenzene-
the detector at 210C. Inject the chosen volume of each solution. In the chromatogram obtained with
the test solution, the area of any peak corresponding to methanol or ethanol is not greater than the
area of the corresponding peak in the chromatogram obtained with the reference solution (0.5 per
cent of ethanol and 0.05 per cent of methanol) and the sum of the areas of any peaks, apart from the
peaks corresponding to ethanol, methanol and the internal standard, is not greater than the area of
the peak corresponding to the internal standard (0.5 per cent calculated as propanol).
13-15
Molecular-mass distribution (2.2.39). The average molecular mass (M
w
) is 35,000 to 45,000.
The average molecular mass of the 10 per cent high fraction is not more than 110,000. The average
molecular mass of the 10 per cent low fraction is not less than 7000.
at 105 2C for 5 h.
2
micro-
organisms per gram, determined by plate-count. It complies with the test for Escherichia coli (2.6.13).
STORAGE
__________________________________________________________________________________________________________ Ph Eur
13-16
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
obtained by hydrolysis and fractionation of dextrans produced by fermentation of sucrose using
contamination.
CHARACTERS
in alcohol.
IDENTIFICATION
TESTS
same solvent.
orange.
the indicator.
water R.
w
) is 54,000 to 66,000.
molecular mass of the 10 per cent low fraction is not less than 14,000.
13-17
at 105 2C for 5 h.
Microbial contamination Total viable count (2.6.12) not more than 10
2
micro-organisms per
gram, determined by plate-count. It complies with the test for Escherichia coli (2.6.13).
STORAGE
__________________________________________________________________________________________________________ Ph Eur
13-18
Preparation
Dextran 70 Intravenous Infusion
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
obtained by hydrolysis and fractionation of dextrans obtained by fermentation of sucrose using
contamination.
CHARACTERS
in alcohol.
IDENTIFICATION
TESTS
same solvent.
orange.
the indicator.
water R.
13-19
w
) is 64,000 to 76,000.
molecular mass of the 10 per cent low fraction is not less than 15,000.
at 105 2C for 5 h.
Microbial contamination Total viable count (2.6.12) not more than 10
2
micro-organisms per
gram, determined by plate-count. It complies with the test for Escherichia coli (2.6.13).
STORAGE
__________________________________________________________________________________________________________ Ph Eur
13-20
Dextrin
1/01
Dextrin complies with the requirements of the 3rd edition of the European Pharmacopoeia [1507]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Dextrin is maize or potato starch partly hydrolysed and modified by heating with or without the
presence of acids, alkalis or pH control agents.
CHARACTERS
White or almost white, free-flowing powder, very soluble in boiling water forming a mucilaginous
solution, slowly soluble in cold water, practically insoluble in alcohol.
IDENTIFICATION
A. Suspend 1 g in 50 ml of water R, boil for 1 min and cool. To 1 ml of the solution add 0.05 ml of
iodine solution R1. A dark blue colour is produced which disappears on heating.
B. Centrifuge 5 ml of the mucilage obtained in identification test A. To the upper layer add 2 ml of
2M sodium hydroxide and, dropwise with shaking, 0.5 ml of copper sulphate solution R and boil. A red
C. It is very soluble in boiling water R, forming a mucilaginous solution.
TESTS
pH (2.2.3). Disperse 5.0 g in 100 ml of carbon dioxide-free water R. The pH is 2.0 to 8.0.
Chloride Dissolve 2.5 g in 50 ml of boiling water R, dilute to 100 ml with water R and filter. Dilute
1 ml of the filtrate to 15 ml, add 1 ml of 2M nitric acid, pour the mixture as a single addition into 1 ml
of silver nitrate solution R and allow to stand for 5 min protected from light. When viewed transversely
against a black background any opalescence produced is not more intense than that obtained by
treating a mixture of 10 ml of chloride standard solution (5 ppm Cl) R and 5 ml of water R, prepared in
the same manner (0.2 per cent).
Reducing sugars. To a quantity of dextrin equivalent to 2.0 g (dried substance) add 100 ml of
water R, shake for 30 min, dilute with water R to 200.0 ml and filter. To 10.0 ml of alkaline cupri-
tartaric solution R add 20.0 ml of the filtrate, mix, and heat on a hot plate adjusted to bring the
solution to boil within 3 min. Boil for 2 min, and cool immediately. Add 5 ml of a 300 g/l solution of
potassium iodide R and 10 ml of 1M sulphuric acid, mix, and titrate immediately with 0.1M sodium
thiosulphate, using starch solution R, added towards the end of the titration, as indicator. Repeat the
procedure beginning with To 10.0 ml of ..., using, in place of the filtrate, 20.0 ml of a 1 g/l
solution of glucose R, accurately prepared. Perform a blank titration. (V
B
V
U
) is not greater than (V
B
V
S
), in which V
B
, V
U
and V
S
are the number of millilitres of 0.1M sodium thiosulphate consumed in
the titrations of the blank, the dextrin and the glucose, respectively (10 per cent, calculated as glucose
C
6
H
12
O
6
).
to 135C for 90 min.
__________________________________________________________________________________________________________ Ph Eur
13-21
Dextromethorphan Hydrobromide
N
H
MeO
Me
,HBr
C
18
H
25
NO,HBr,H
2
O 370.3 6700-34-1
Dextromethorphan Hydrobromide complies with the requirements of the 3rd edition of the European
Action and use Cough suppressant.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Dextromethorphan hydrobromide contains not less than 99.0 per cent and not more than the equiva-
lent of 101.0 per cent of (9S,13S,14S)-3-methoxy-17-methylmorphinan hydrobromide, calculated
CHARACTERS
An almost white, crystalline powder, sparingly soluble in water, freely soluble in alcohol, practically
insoluble in ether.
IDENTIFICATION
obtained with dextromethorphan hydrobromide CRS. Examine the substances prepared as discs.
D. It gives reaction (a) of bromides (2.3.1).
TESTS
Acidity or alkalinity. Dissolve 0.4 g in carbon dioxide-free water R with gentle heating, cool and
dilute to 20 ml with the same solvent. Add 0.1 ml of methyl red solution R and 0.2 ml of 0.01M sodium
hydroxide. The solution is yellow. Not more than 0.4 ml of 0.01M hydrochloric acid is required to
change the colour of the indicator to red.
with the same acid. The specific optical rotation is +28 to +30, calculated with reference to the
coating substance.
Reference solution (a). Dissolve 25 mg of dextromethorphan hydrobromide CRS in methanol R and dilute
13-22
volumes of concentrated ammonia R, 10 volumes of methylene chloride R, 13 volumes of methanol R,
20 volumes of ethyl acetate R and 55 volumes of toluene R. Dry the plate in a current of air and spray
with potassium iodobismuthate solution R2 and then with dilute hydrogen peroxide solution R. Any spot in
than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent) and not more
than one such spot is more intense than the spot in the chromatogram obtained with reference
solution (c) (0.25 per cent).
N,N-Dimethylaniline Dissolve 0.5 g with heating in 20 ml of water R. Allow to cool, add 2 ml of
dilute acetic acid R and 1 ml of a 10 g/l solution of sodium nitrite R and dilute to 25 ml with water R.
The solution is not more intensely coloured than a reference solution prepared at the same time in
the same manner using 20 ml of a 0.25 mg/l solution of dimethylaniline R (10 ppm).
mination of water.
ASSAY
Dissolve 0.300 g in a mixture of 5.0 ml of 0.01M hydrochloric acid and 20 ml of alcohol R. Titrate with
0.1M sodium hydroxide, determining the end-point potentiometrically (2.2.20). Read the volume
added between the two points of inflexion.
18
H
26
BrNO.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
13-23
Dextromoramide Tartrate
HOOC
COOH
OH H
OH H
, N
O
Ph
O
H Me
N
Ph
C
25
H
32
N
2
O
2
,C
4
H
6
O
6
542.6 2922-44-3
Dextromoramide Tartrate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Narcotic analgesic.
Preparation
Dextromoramide Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Dextromoramide tartrate contains not less than 98.0 per cent and not more than the equivalent of
101.0 per cent of (R)-1-(3-methyl-4-morpholino-2,2-diphenylbutyryl)pyrrolidine hydrogen (2R,3R)-
tartrate, calculated with reference to the dried substance.
CHARACTERS
A white, amorphous or crystalline powder, soluble in water, sparingly soluble in alcohol, very slightly
soluble in ether.
It melts at about 190C, with slight decomposition.
IDENTIFICATION
A. Dissolve 75 mg in 1M hydrochloric acid and dilute to 100.0 ml with the same acid. Examined
between 230 nm and 350 nm (2.2.25), the solution shows three absorption maxima, at 254 nm,
259 nm and 264 nm. The specific absorbances at the maxima are about 6.9, 7.7 and 6.5,
respectively.
B. Dissolve about 50 mg in water R and dilute to 10 ml with the same solvent. To 2 ml of the
solution add 3 ml of ammoniacal silver nitrate solution R and heat on a water-bath. A grey or black
C. It gives reaction (b) of tartrates (2.3.1).
TESTS
with the same acid. The specific optical rotation is +21 to +23.
coating substance.
the same solvent.
Apply separately to the plate 10 l of each solution. Develop over a path of 15 cm using methanol R.
Allow the plate to dry in air and spray with dilute potassium iodobismuthate solution R. Any spot in the
100C to 105C.
ASSAY
Dissolve 0.250 g in 30 ml of anhydrous acetic acid R. Titrate with 0.05M perchloric acid using 0.15 ml
of naphtholbenzein solution R as indicator.
29
H
38
N
2
O
8
.
__________________________________________________________________________________________________________ Ph Eur
13-24
Dextropropoxyphene Hydrochloride
1/01
NMe
2
Me H
OCOEt Ph
,HCl
C
22
H
29
NO
2
,HCl 375.9 1639-60-7
Dextropropoxyphene Hydrochloride complies with the requirements of the 3rd edition of the European
Preparation
Co-proxamol Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Dextropropoxyphene hydrochloride contains not less than 98.5 per cent and not more than the
equivalent of 101.0 per cent of (1S,2R)-1-benzyl-3-dimethylamino-2-methyl-1-phenylpropyl
propionate hydrochloride, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, very soluble in water, freely soluble in alcohol.
IDENTIFICATION
B. Dissolve 50.0 mg in 0.01M hydrochloric acid and dilute to 100.0 ml with the same acid. Examined
between 220 nm and 360 nm (2.2.25), the solution shows three absorption maxima, at 252 nm,
257 nm and 263 nm and two shoulders, at 240 nm and 246 nm. The ratio of the absorbance at the
maximum at 257 nm to that at the maximum at 252 nm is 1.22 to 1.28. The ratio of the absorbance
at the maximum at 257 nm to that at the maximum at 263 nm is 1.29 to 1.35. The test is not valid
unless, in the test for resolution (2.2.25), the ratio of the absorbances is at least 1.5.
spectrum of dextropropoxyphene hydrochloride.
D. Solution S (see Tests) gives reaction (a) of chlorides (2.3.1).
TESTS
solvent. The specific optical rotation is +52 to +57.
Reference solution (b). Dissolve 50 mg of the substance to be examined in 2.5 ml of 2M alcoholic
potassium hydroxide R. Add 2.5 ml of water R and boil under a reflux condenser for 30 min. Add
2.5 ml of dilute hydrochloric acid R and dilute to 50 ml with the mobile phase.
a column 0.125 m long and 4.6 mm in internal diameter packed with silica gel for chromato-
graphy R (5 m),
a guard column packed with a suitable silica gel, equilibrated with the mobile phase and placed
between the pump and the injection device,
13-25
as mobile phase at a flow rate of 1.0 ml/min a mixture of 50 volumes of 0.2M phosphate buffer
solution pH 7.5 R, 84 volumes of tetrahydrofuran R, 350 volumes of methanol R and 516 volumes
of water R, containing 0.9 g/l of cetyltrimethylammonium bromide R,
a loop injector.
Equilibrate the chromatographic system by passage of the mobile phase for 16 h (the mobile phase
may be recirculated after 6 h).
Inject 20 l of each solution and record the chromatograms for twice the retention time of the
principal peak. The test is not valid unless the chromatogram obtained with reference solution (a)
shows a peak with a signal-to-noise ratio of at least 5 and the chromatogram obtained with reference
solution (b) shows two peaks with a resolution of at least 2.0. In the chromatogram obtained with the
test solution, the area of any peak apart from the principal peak, is not greater than the area of the
principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent).
100C to 105C.
ASSAY
Dissolve 0.270 g in 60 ml of acetic anhydride R. Titrate with 0.1M perchloric acid, using 0.1 ml of a
5 g/l solution of malachite green R in acetic anhydride R as the indicator.
22
H
30
ClNO
2
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
13-26
Dextropropoxyphene Napsilate
SO
3
H
,
NMe
2
Me H
OCOEt Ph
C
22
H
29
NO
2
,C
10
H
8
O
3
S,H
2
O 565.8 26570-10-5
Definition Dextropropoxyphene Napsilate is (1S,2R)-1-benzyl-3-dimethylamino-2-methyl-1-
phenylpropyl propionate naphthalene-2-sulphonate monohydrate. It contains not less than 98.0%
22
H
29
NO
2
,C
10
H
8
O
3
S, calculated with reference to the anhydrous
substance.
Characteristics A white powder; odourless or almost odourless. It exhibits polymorphism.
Practically insoluble in water; freely soluble in chloroform; soluble in ethanol (96%).
Identification
dextropropoxyphene napsilate (RS 092). If the spectra are not concordant, dissolve a sufficient
quantity in the minimum volume of chloroform IR, evaporate to dryness, dry the residue at 105 for 1
hour and prepare a new spectrum.
B. Dissolve 25 mg in 5 ml of chloroform, evaporate 0.05 ml of the solution in a porcelain dish and
streak the spot with sulphuric acid containing 5% v/v of formaldehyde solution. A purple colour is
produced.
C. Burn 20 mg by the method for oxygen-flask combustion, Appendix VIII C, using 5 ml of 1.25M
sodium hydroxide as the absorbing liquid. When the process is complete, dilute the liquid to 25 ml
with water. To 5 ml of the solution so obtained add 1 ml of hydrogen peroxide solution (100 vol) and
1 ml of 1M hydrochloric acid, mix and add 0.05 ml of barium chloride solution. The solution becomes
turbid.
Specific optical rotation In a 5% w/v solution in ethanol (96%), +26 to +31, calculated with
reference to the anhydrous substance, Appendix V F.
Related substances Dissolve 10 mg of triphenylamine (internal standard) in sufficient chloroform to
produce 50 ml (solution A). Carry out the method for gas chromatography, Appendix III B, using the
following solutions. For solution (1) add 5 ml of solution A, 10 ml of water, 2 ml of l.25M sodium
hydroxide and 15 ml of chloroform to 5 ml of a solution in chloroform containing 0.022% w/v of
(1S,2R)-1-benzyl-3-dimethylamino-2-methyl-1-phenylpropyl acetate BPCRS and 0.020% w/v of
4-dimethylamino-3-methyl-1,2-diphenylbutan-2-ol hydrochloride BPCRS and shake. Extract the aqueous
layer with two 20-ml quantities of chloroform. Shake the combined chloroform extracts with 5 g of
anhydrous sodium sulphate, filter and evaporate to dryness at a temperature not exceeding 40 using a
rotary evaporator. Dissolve the residue in 10 ml of chloroform. For solution (2) dissolve 0.3 g of the
substance being examined in 5 ml of chloroform, add 10 ml of water, 2 ml of 1.25M sodium hydroxide
and 15 ml of chloroform and shake. Extract the aqueous layer with two 20-ml quantities of chloroform.
Shake the combined chloroform extracts with 5 g of anhydrous sodium sulphate, filter and evaporate to
dryness at a temperature not exceeding 40 using a rotary evaporator. Dissolve the residue in 10 ml
of chloroform. Prepare solution (3) in the same manner as solution (2) but add 5 ml of solution A to
the initial solution of the substance being examined.
The chromatographic procedure may be carried out using a glass column (60 cm 3 mm) packed
with acid-washed, silanised diatomaceous support (100 to 120 mesh) coated with 3% w/w of dimethyl
silicone fluid (OV-101 is suitable) and maintained at 160 with an inlet port temperature of 150 and
using 60 ml per minute as the flow rate of the carrier gas.
The peaks, other than the solvent peak, in the chromatogram obtained with solution (1) are due, in
order of emergence, to (a) the internal standard, (b) (1S,2R)-1-benzyl-3-dimethylamino-2-methyl-1-
phenylpropyl acetate and (c) 4-dimethylamino-3-methyl-1,2-diphenylbutan-2-ol hydrochloride. In
the chromatogram obtained with solution (3) the ratio of the area of any peak corresponding to (b) to
that of the peak due to (a) and the ratio of the area of any peak corresponding to (c) to that of the
peak due to (a) are not greater than the corresponding ratios in the chromatogram obtained with
solution (1) (0.67% each).
Assay To 0.75 g add 50 ml of water, swirl to disperse, add 5 ml of 5M sodium hydroxide and extract
with five 25-ml quantities of chloroform, washing each extract with the same 20 ml of water. Dry the
combined extracts with anhydrous sodium sulphate, evaporate to about 3 ml on a water bath in a
13-27
current of air and allow to evaporate to dryness at room temperature. Carry out Method I for non-
aqueous titration on the residue, Appendix VIII A, using 1-naphtholbenzein solution as indicator. Each
ml of 0.1M perchloric acid VS is equivalent to 54.77 mg of C
22
H
29
NO
2
,C
10
H
8
O
3
S.
Preparation
Dextropropoxyphene Capsules
When dextropropoxyphene napsylate is prescribed or demanded, Dextropropoxyphene Napsilate
13-28
Diamorphine Hydrochloride
,HCl
O
N
H
H
H
AcO
AcO
Me
C
21
H
23
NO
5
,HCl,H
2
O 423.9 1502-95-0
Definition Diamorphine Hydrochloride is 4,5-epoxy-17-methylmorphinan-3,6-diyl diacetate
hydrochloride monohydrate.It contains not less than 98.0% and not more than 102.0% of
C
21
H
23
NO
5
Characteristics A white or almost white, crystalline powder; odourless when freshly prepared but
an odour characteristic of acetic acid develops on storage.
Freely soluble in water and in chloroform; soluble in ethanol (96%); practically insoluble in ether.
Identification
A. Dissolve a sufficient quantity in the minimum volume of dichloromethane and evaporate to dryness.
The infrared absorption spectrum of the residue, Appendix II A, is concordant with the reference
spectrum of diamorphine hydrochloride (RS 093).
Acidity Dissolve 0.2 g in 10 ml of carbon dioxide-free water and titrate with 0.02M sodium hydroxide
VS using methyl red solution as indicator. Not more than 0.2 ml of 0.02M sodium hydroxide VS is
required.
following solutions. Solution (1) contains 0.5% w/v of the substance being examined in water. For
solution (2) dilute 1 volume of solution (1) to 50 volumes with water. For solution (3) use a freshly
prepared solution containing 1% w/v of the substance being examined in 0.01M sodium hydroxide. For
solution (1) allow the chromatography to proceed for twice the retention time of the principal peak.
The chromatographic procedure may be carried out using (a) a stainless steel column (12.5 cm
4.6 mm) packed with stationary phase B (5 m) (Lichrosphere RP-select B 5 is suitable), (b) as the
mobile phase with a flow rate of 1 ml per minute a solution containing 0.11% w/v of sodium
octanesulphonate in 10 volumes of glacial acetic acid, 10 volumes of methanol, 115 volumes of aceto-
nitrile and 365 volumes of water and (c) a detection wavelength of 283 nm.
The chromatogram obtained with solution (3) exhibits two secondary peaks with retention times
relative to the principal peak of about 0.23 (morphine) and 0.43 (6-O-acetylmorphine). The test is
not valid unless the resolution factor between the peaks due to morphine and 6-O-acetylmorphine is at
least 2.
In the chromatogram obtained with solution (1) the area of any peak corresponding to 6-O-
acetylmorphine is not greater than the area of the peak in the chromatogram obtained with solution
(2) (2%) and the sum of the areas of any other secondary peaks is not greater than 0.25 times the area
of the peak in the chromatogram obtained with solution (2) (0.5%). Disregard any peak with an area
less than 0.05 times the area of the peak in the chromatogram obtained with solution (2) (0.1%).
Loss on drying When dried to constant weight at 105, loses 3.5 to 4.5% of its weight. Use 1 g.
Assay Dissolve 0.40 g in 50 ml of ethanol (96%) and add 5.0 ml of 0.01M hydrochloric acid VS.
Titrate with 0.1M sodium hydroxide VS, determining the end point potentiometrically. Measure the
volume of titrant required between the two points of inflection. Each ml of 0.1M sodium hydroxide VS
21
H
23
NO
5
,HCl.
Storage Diamorphine Hydrochloride should be kept in a well-closed container and protected from
light.
Preparation
Diamorphine Injection
IMPURITIES
The impurities listed by the requirements of this monograph include:
13-29
O
NHMe
H
H
HO
AcO
H
6-O-acetylmorphine
13-30
Diazepam
N
N
Cl
O
Me
C
16
H
13
ClN
2
O 284.7 439-14-5
Diazepam complies with the requirements of the 3rd edition of the European Pharmacopoeia [0022]. These
Preparations
Diazepam Injection
Diazepam Oral Solution
Diazepam Rectal Solution
Diazepam Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Diazepam contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of
7-chloro-1-methyl-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-2-one, calculated with reference to
CHARACTERS
A white or almost white, crystalline powder, very slightly soluble in water, soluble in alcohol.
IDENTIFICATION
B. Protect the solutions from bright light and measure the absorbances immediately. Dissolve 25 mg in a
5 g/l solution of sulphuric acid R in methanol R and dilute to 250.0 ml with the same acid solution
(solution A). Dilute 5.0 ml of solution A to 100.0 ml with a 5 g/l solution of sulphuric acid R in
methanol R. Examined between 230 nm and 330 nm (2.2.25), the solution shows two absorption
maxima, at 242 nm and 285 nm. The specific absorbance at the maximum at 242 nm is about 1020.
Dilute 25.0 ml of solution A to 100.0 ml with a 5 g/l solution of sulphuric acid R in methanol R. Exam-
ined between 325 nm and 400 nm, the solution shows a single absorption maximum, at 366 nm. The
specific absorbance at the maximum is 140 to 155.
C. Dissolve about 10 mg in 3 ml of sulphuric acid R. The solution shows greenish-yellow fluorescence
in ultraviolet light at 365 nm.
D. To 80 mg in a porcelain crucible add 0.3 g of anhydrous sodium carbonate R. Heat over an open
TESTS
Related substances and decomposition products Carry out the test protected from bright light.
254
Reference solution. Dilute 1 ml of the test solution to 100 ml with acetone R. Dilute 1 ml of this
equal volumes of ethyl acetate R and hexane R. Allow the plate to dry and examine in ultraviolet light
at 254 nm. Any spot in the chromatogram obtained with the test solution, apart from the principal
(0.1 per cent).
13-31
Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying at 60C in
vacuo for 4 h.
ASSAY
Dissolve 0.500 g in 50 ml of acetic anhydride R. Using 0.3 ml of Nile blue A solution R as indicator,
titrate with 0.1M perchloric acid until a yellowish-green colour is obtained.
16
H
13
ClN
2
O.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
13-32
Diazoxide
S
NH
N
O O
Cl
Me
C
8
H
7
ClN
2
O
2
S 230.7 364-98-7
Diazoxide complies with the requirements of the 3rd edition of the European Pharmacopoeia [0550]. These
Preparations
Diazoxide Injection
Diazoxide Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Diazoxide contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent of
7-chloro-3-methyl-2H-1,2,4-benzothiadiazine 1,1-dioxide, calculated with reference to the dried
substance.
CHARACTERS
A white or almost white, fine or crystalline powder, practically insoluble in water, freely soluble in
dimethylformamide, slightly soluble in alcohol, practically insoluble in ether. It is very soluble in
dilute solutions of the alkali hydroxides.
IDENTIFICATION
A. Dissolve 50.0 mg in 5 ml of 1M sodium hydroxide and dilute to 50.0 ml with water R. Dilute 1.0 ml
of this solution to 100.0 ml with 0.1M sodium hydroxide. Examined between 230 nm and 350 nm
(2.2.25), the solution shows an absorption maximum at 280 nm and a shoulder at 304 nm. The
obtained with diazoxide CRS. Examine the substances prepared as discs using potassium bromide R.
D. Dissolve about 20 mg in a mixture of 5 ml of hydrochloric acid R and 10 ml of water R. Add 0.1 g
of zinc powder R. Boil for 5 min, cool and filter. To the filtrate add 2 ml of a 1 g/l solution of sodium
nitrite R and mix. Allow to stand for 1 min and add 1 ml of a 5 g/l solution of naphthylethylenediamine
dihydrochloride R. A red or violet-red colour develops.
TESTS
Appearance of solution Dissolve 0.4 g in 2 ml of 1M sodium hydroxide and dilute to 20 ml with
7
(Method II, 2.2.2).
Acidity or alkalinity To 0.5 g of the powdered substance to be examined add 30 ml of carbon
dioxide-free water R, shake for 2 min and filter. To 10 ml of the filtrate add 0.2 ml of 0.01M sodium
hydroxide and 0.15 ml of methyl red solution R. The solution is yellow. Not more than 0.4 ml of 0.01M
hydrochloric acid is required to change the colour of the indicator to red.
254
R as the
coating substance.
Test solution (a). Dissolve 0.1 g of the substance to be examined in a mixture of 0.5 ml of 1M sodium
hydroxide and 1 ml of methanol R and dilute to 5 ml with methanol R.
Test solution (b). Dilute 1 ml of test solution (a) to 5 ml with a mixture of 1 volume of 1M sodium
hydroxide and 9 volumes of methanol R.
Reference solution (a). Dilute 0.5 ml of test solution (a) to 100 ml with a mixture of 1 volume of 1M
sodium hydroxide and 9 volumes of methanol R.
13-33
Reference solution (b). Dissolve 20 mg of diazoxide CRS in a mixture of 0.5 ml of 1M sodium hydroxide
and 1 ml of methanol R and dilute to 5 ml with methanol R.
volumes of concentrated ammonia R, 25 volumes of methanol R and 68 volumes of chloroform R. Allow
the plate to dry in air and examine in ultraviolet light at 254 nm. Any spot in the chromatogram
obtained with test solution (a), apart from the principal spot, is not more intense than the spot in the
chromatogram obtained with reference solution (a) (0.5 per cent).
100C to 105C for 2 h.
ASSAY
Dissolve 0.200 g with gentle heating in 50 ml of a mixture of 1 volume of water R and 2 volumes of
dimethylformamide R. Titrate with 0.1M sodium hydroxide, determining the end-point potentiometric-
ally (2.2.20). Carry out a blank titration.
8
H
7
ClN
2
O
2
S.
STORAGE
______________________________________________________________________________________________________________ Ph Eur
13-34
Dibrompropamidine Isetionate
,
OH
SO
3
H
2
Br
NH
NH
2
O O
H
2
N
NH
Br
C
17
H
18
Br
2
N
4
O
2
,2C
2
H
6
O
4
S 722.4 614-87-9
Definition Dibrompropamidine Isetionate is 3,3-dibromo-4,4-trimethylenedioxydibenzamidine
bis(2-hydroxyethanesulphonate). It contains not less than 97.0% and not more than 101.0% of
C
17
H
18
Br
2
N
4
O
2
, 2C
2
H
6
O
4
S, calculated with reference to the dried substance.
Freely soluble in water; soluble in glycerol; sparingly soluble in ethanol (96%); practically insoluble
in chloroform, in ether, in liquid paraffin and in fixed oils.
Identification
dibrompropamidine isetionate (RS 095).
B. Mix 0.1 g with 0.5 g of anhydrous sodium carbonate, ignite and extract the residue with 20 ml of
water. Filter and neutralise the filtrate to litmus paper with nitric acid. The solution yields reaction A
characteristic of bromides, Appendix VI.
Light absorption Absorbance of a 0.0015% w/v solution in 0.01M hydrochloric acid at the maximum
at 261 nm, 0.50 to 0.52, Appendix II B, calculated with reference to the dried substance.
Ammonium isetionate To 1 g in a boiling tube about 4 cm in diameter add 10 ml of water and
20 ml of 1M sodium hydroxide. Immediately insert a stopper carrying a splash head and an aspirator
tube about 5 mm in diameter. Connect the splash head to two test tubes in series, each containing
20 ml of 0.01M sulphuric acid VS. Heat the tube containing the substance being examined in a water
bath at 45 to 50 and, maintaining this temperature, draw a current of air, previously passed through
1M sulphuric acid, through the liquids in the series of tubes for 3 hours at such a rate that the bubbles
are just too rapid to count. Titrate the combined solutions from the two absorption tubes with 0.02M
sodium hydroxide VS using methyl red mixed solution as indicator. At least 36.5 ml is required.
silica gel F
254
254
plates are suitable) activated at 105 for 1
hour and a mixture of 90 volumes of propan-1-ol, 5 volumes of formic acid and 5 volumes of water as
the mobile phase. Apply separately to the plate 10 l of each of two solutions of the substance being
examined in methanol (90%) containing (1) 1.0% w/v and (2) 0.0050% w/v. After removal of the
plate, allow it to dry in air and examine under ultraviolet light (254 nm). Any secondary spot in the
chromatogram obtained with solution (1) is not more intense than the spot in the chromatogram
1 g.
Assay Heat 0.4 g with 70 ml of isoamyl alcohol and 4 g of sodium under a reflux condenser until the
sodium has dissolved. Continue heating for a further 30 minutes, cool, add 50 ml of water and shake.
Add 15 ml of nitric acid and 25 ml of 0.1M silver nitrate VS, keeping the mixture cold. Shake
vigorously and titrate the excess of silver nitrate with 0.1M ammonium thiocyanate VS using ammonium
iron(III) sulphate solution R2 as indicator. Each ml of 0.1M silver nitrate VS is equivalent to 36.12 mg of
C
17
H
18
Br
2
N
4
O
2
,2C
2
H
6
O
4
S.
Storage Dibrompropamidine Isetionate should be kept in a well-closed container.
When dibromopropamidine isethionate is demanded, Dibrompropamidine Isetionate shall be
supplied.
13-35
Dibutyl Phthalate
COOBu
n
COOBu
n
C
16
H
22
O
4
278.3 84-74-2
Dibutyl Phthalate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0762].
Action and use Insect repellent.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Dibutyl phthalate contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent m/m of dibutyl benzene-1,2-dicarboxylate.
CHARACTERS
A clear, oily liquid, colourless or very slightly yellow, practically insoluble in water, miscible with
alcohol and with ether.
IDENTIFICATION
Second identification: A, D, E.
B. Refractive index (2.2.6): 1.490 to 1.495.
obtained with dibutyl phthalate CRS.
254
Test solution. Dissolve 50 mg of the substance to be examined in ether R and dilute to 10 ml with the
same solvent.
Reference solution. Dissolve 50 mg of dibutyl phthalate CRS in ether R and dilute to 10 ml with the
same solvent.
30 volumes of heptane R and 70 volumes of ether R. Allow the plate to dry in air and examine in
similar in position and size to the principal spot in the chromatogram obtained with the reference
solution.
E. To about 0.1 ml add 0.25 ml of sulphuric acid R and 50 mg of resorcinol R. Heat in a water-bath for
5 min. Allow to cool. Add 10 ml of water R and 1 ml of strong sodium hydroxide solution R. The
solution becomes yellow or brownish-yellow and shows a green fluorescence.
TESTS
Appearance The substance to be examined is clear (2.2.1) and not more intensely coloured than
6
(Method II, 2.2.2).
Acidity Dissolve 20.0 g in 50 ml of alcohol R previously neutralised to phenolphthalein solution R1.
Add 0.2 ml of phenolphthalein solution R1. Not more than 0.50 ml of 0.1M sodium hydroxide is required
to change the colour of the indicator.
Related substances Examine by gas chromatography (2.2.28), using bibenzyl R as internal standard.
Internal standard solution. Dissolve 60 mg of bibenzyl R in methylene chloride R and dilute to 20 ml with
the same solvent.
Test solution (b). Dissolve 1.0 g of the substance to be examined in methylene chloride R, add 2.0 ml of
the internal standard solution and dilute to 20.0 ml with methylene chloride R.
Reference solution. To 1.0 ml of test solution (a) add 10.0 ml of the internal standard solution and
dilute to 100.0 ml with methylene chloride R.
13-36
a glass column 1.5 m long and 4 mm in internal diameter, packed with silanised diatomaceous earth
for gas chromatography R (150 m to 180 m) impregnated with 3 per cent m/m of
detector at 225C.
Inject 1 l of the reference solution. The substances elute in the following order: bibenzyl and
dibutyl phthalate. Adjust the sensitivity of the detector so that the height of the bibenzyl peak is not
less than 70 per cent of the full scale of the recorder. The test is not valid unless the resolution
between the peaks corresponding to bibenzyl and dibutyl phthalate is at least 12.
Inject 1 l of test solution (a). In the chromatogram obtained, verify that there is no peak with the
same retention time as the internal standard.
Inject separately 1 l of test solution (b) and the reference solution. Continue the chromatography
for 3 times the retention time of dibutyl phthalate, which is about 12 min. From the chromatogram
obtained with the reference solution, calculate the ratio (R) of the area of the peak due to dibutyl
phthalate to the area of the peak due to the internal standard. From the chromatogram obtained with
test solution (b), calculate the ratio of the sum of the areas of any peaks, apart from the principal
peak, the peak due to the internal standard and the peak due to the solvent, to the area of the peak
due to the internal standard: this ratio is not greater than R (1.0 per cent).
mination of water.
ASSAY
Introduce 0.750 g into a 250 ml borosilicate glass flask. Add 25.0 ml of 0.5M alcoholic potassium
hydroxide and a few glass beads. Heat in a water-bath under a reflux condenser for 1 h. Add 1 ml of
phenolphthalein solution R1 and titrate immediately with 0.5M hydrochloric acid until the colour changes
from red to colourless. Carry out a blank titration. Calculate the volume of potassium hydroxide used
in the saponification.
16
H
22
O
4
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
13-37
Dichloromethane
CH
2
Cl
2
84.9 75-09-02
Dichloromethane complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Methylene Chloride [0932]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Methylene chloride is dichloromethane. It may contain not more than 2.0 per cent V/V of ethanol
and/or not more than 0.03 per cent V/V of 2-methylbut-2-ene as stabiliser.
CHARACTERS
A clear, colourless, volatile liquid, sparingly soluble in water, miscible with alcohol and with ether.
IDENTIFICATION
B. It complies with the test for refractive index (see Tests).
C. Examine the chromatograms obtained in the test for ethanol, 2-methylbut-2-ene and other related
substances. The retention time and size of the principal peak in the chromatogram obtained with test
solution (b) are approximately the same as those of the principal peak in the chromatogram obtained
D. Heat 2 ml with 2 g of potassium hydroxide R and 20 ml of alcohol R under a reflux condenser for
30 min. Allow to cool. Add 15 ml of dilute sulphuric acid R and filter. To 1 ml of the filtrate add 1 ml
of a 15 g/l solution of chromotropic acid, sodium salt R, 2 ml of water R and 8 ml of sulphuric acid R. A
violet colour is produced.
E. 2 ml of the filtrate obtained in identification test D gives reaction (a) of chlorides (2.3.1).
TESTS
Acidity To 50 ml of methanol R previously neutralised to 0.1 ml of bromothymol blue solution R1, add
50 g of the substance to be examined. Not more than 0.15 ml of 0.1M sodium hydroxide is required to
change the colour of the indicator to blue.
Ethanol, 2-methylbut-2-ene and other related substances Examine by gas chromatography
(2.2.28).
Test solution (a). The substance to be examined.
Reference solution (a). Dilute 0.5 ml of methylene chloride CRS to 100.0 ml with water R.
Reference solution (b). Dilute 2.0 ml of test solution (b) to 10.0 ml with water R.
Reference solution (c). To 20.0 ml of ethanol R, add 0.3 ml of 2-methylbut-2-ene R and dilute to
100.0 ml with test solution (a). Dilute 1.0 ml of the solution to 10.0 ml with test solution (a).
Reference solution (d). Dilute 0.1 ml of methanol R and 0.1 ml of methylene chloride CRS to 100.0 ml
with water R.
a glass column 2 m long and 2 mm in internal diameter, packed with ethylvinylbenzene-divinyl-
benzene copolymer R (136 m to 173 m),
rate of 4C per minute to 190C and maintaining at 190C for 15 min and maintaining the tempera-
ture of the injection port and the detector at 240C.
Inject 1 l of reference solution (d). If a recorder is used, adjust the sensitivity of the detector such
that the height of the peak due to methanol is not less than 25 per cent of the full scale of the
recorder. The test is not valid unless: in the chromatogram obtained with reference solution (d), the
13-38
resolution between the peaks corresponding to methanol and methylene chloride is at least 3.0.
Inject twice 2 l of reference solution (c). If the peaks obtained show an area difference greater
than 1.0 per cent, verify the repeatability by making four separate injections of reference solution
(c); the test is not valid unless the relative standard deviation of the peak area is not more than
5.0 per cent.
Inject 2 l of test solution (a), 2 l of reference solution (b) and 2 l of reference solution (c). In
the chromatogram obtained with test solution (a): the areas of any peaks corresponding to ethanol
and 2-methylbut-2-ene respectively are not greater than the difference between the areas of the
peaks due to ethanol and 2-methylbut-2-ene in the chromatogram obtained with reference solution
(c) and those of the peaks due to ethanol and 2-methylbut-2-ene in the chromatogram obtained
with test solution (a) (2.0 per cent and 0.03 per cent, respectively).
In the chromatogram obtained with test solution (a), the sum of the areas of any peaks apart
from the principal peak and any peaks due to ethanol and 2-methylbut-2-ene, is not greater than
the area of the principal peak in the chromatogram obtained with reference solution (b) (0.1 per
cent).
Free chlorine Place 5 ml in a ground-glass-stoppered tube. Add 5 ml of a 100 g/l solution of
potassium iodide R and 0.2 g of soluble starch R. Shake for 30 s and allow to stand for 5 min. No blue
colour develops.
Heavy metals (2.4.8). Evaporate 25.0 g to dryness on a water-bath. Allow to cool. Add 1 ml of
hydrochloric acid R and evaporate again. Dissolve the residue in 1 ml of acetic acid R and dilute to
25 ml with water R. 12 ml of the solution complies with limit test A for heavy metals (1 ppm).
Residue on evaporation Evaporate 50.0 g to dryness on a water-bath and dry at 100C to 105C
for 30 min. The residue weighs not more than 1 mg (20 ppm).
Water (2.5.12). Not more than 0.05 per cent m/m, determined on 10.00 g by the semi-micro deter-
mination of water.
STORAGE
LABELLING
The label states the name and the concentration of any stabilisers.
IMPURITIES
A. carbon tetrachloride,
B. chloroform,
C. ethanol,
D. methanol,
E. 2-methylbut-2-ene.
_______________________________________________________________________________________________________________ _______________________________________________________________________________________________________________ _______________________________________________________________________________________________________________ _______________________________________________________________________________________________________________ _______________________________________________________________________________________________________________ Ph Eur
13-39
Dichlorophen
Cl Cl
OH OH
C
13
H
10
Cl
2
O
2
269.1 97-23-4
Definition Dichlorophen is 4,4-dichloro-2,2-methylenediphenol. It contains not less than 97.0%
13
H
10
Cl
2
O
2
Characteristics A white or not more than slightly cream powder; odour, not more than slightly
phenolic.
Practically insoluble in water; very soluble in ether; freely soluble in ethanol (96%).
Identification
sodium hydroxide exhibits two maxima, at 245 nm and 304 nm. The absorbances at the maxima are
about 1.3 and about 0.54, respectively.
B. Dissolve 0.2 g in a mixture of 5 ml of water and 5 ml of 5M sodium hydroxide, cool in ice and add a
solution prepared by mixing 1 ml of sodium nitrite solution with a cold solution containing 0.15 ml of
aniline in a mixture of 4 ml of water and 1 ml of hydrochloric acid. A reddish brown precipitate is
produced.
C. Fuse 0.5 g with 2 g of anhydrous sodium carbonate, cool, extract the residue with water and filter.
The filtrate yields reaction A characteristic of chlorides, Appendix VI.
D. Melting point, about 175, Appendix V A.
Chloride Dissolve 1.0 g in 2 ml of ethanol (96%), dilute to 100 ml with water, allow to stand for 5
minutes and filter through a slow filter paper (Whatman No. 42 is suitable). 15 ml of the filtrate
Sulphate Shake 0.8 g with 16 ml of water for 2 minutes, filter and dilute 5 ml of the filtrate to 15 ml
with water. The solution complies with the limit test for sulphates, Appendix VII (600 ppm).
solutions in the mobile phase containing (1) 1.0% w/v of dichlorophen impurity standard BPCRS, (2)
1.0% w/v of the substance being examined and (3) 0.0010% w/v of 4-chlorophenol.
cm 5 mm) packed with stationary phase C (10 m) (Spherisorb ODS 1 is suitable), (b) as the
mobile phase with a flow rate of 1.5 ml per minute a mixture of 25 volumes of water and 1 volume of
glacial acetic acid and sufficient methanol to produce a chromatogram with solution (1) closely
resembling the reference chromatogram supplied with the impurity standard (75 volumes of methanol
is usually suitable) and (c) a detection wavelength of 280 nm. Record the chromatograms until all of
the peaks named on the reference chromatogram have emerged.
In the chromatogram obtained with solution (2) the area of any peak corresponding to 4-
chlorophenol is not greater than the area of the principal peak in the chromatogram obtained with
solution (3) (0.1%). The content of 4,4-dichloro-2,2-(2-hydroxy-4-chloro-m-xylene-,-di-
yl)diphenol in the substance being examined does not exceed 8.0% w/w and the sum of the contents
of any other impurities, excluding 4-chlorophenol, is not greater than 2.0% w/w calculated using the
declared content of 4,4-dichloro-2,2-(2-hydroxy-4-chloro-m-xylene-,-diyl)diphenol in
dichlorophen impurity standard BPCRS.
1 g.
Assay Dissolve 0.5 g in 20 ml of propan-2-ol and carry out Method II for non-aqueous titration,
point potentiometrically. Each ml of 0.1M tetrabutylammonium hydroxide VS is equivalent to 26.91 mg
of C
13
H
10
Cl
2
O
2
.
Preparation
Dichlorophen Tablets
13-40
Diclofenac Diethylamine
N
COO
_
H Cl
Cl
Et
2
NH
+
C
18
H
22
Cl
2
N
2
O
2
369.29 78213-16-8
Definition Diclofenac Diethylamine is diethylammonium 2-[(2,6-dichloroanilino)phenyl]acetate.
18
H
22
Cl
2
N
2
O
2
, calculated with
Characteristics A white to light beige, crystalline powder.
Sparingly soluble in water and in acetone; freely soluble in ethanol (96%) and in methanol; practically
insoluble in 1M sodium hydroxide.
It melts at about 154, with decomposition.
Identification
diclofenac diethylamine (RS 371).
B. Carry out the method for thin-layer chromatography, Appendix III A, using a silica gel precoated
plate (Macherey Nagel SIL G-25 HR or silica gel 60F
254
HPTLC plates are suitable) and a mixture
of 1 volume of hydrochloric acid, 1 volume of water, 6 volumes of glacial acetic acid and 11 volumes of
ethyl acetate as the mobile phase. Apply separately to the plate 2 l of each of two solutions in
methanol containing (1) 5.0% w/v of the substance being examined, and (2) 5.0% w/v of diclofenac
diethylamine BPCRS. After removal of the plate, dry it in a stream of warm air for 10 minutes. Spray
with ninhydrin solution and heat at 110 for 15 minutes. The two principal spots in the chromatogram
obtained with solution (1) are similar in position, colour and size to the corresponding spots in the
chromatogram obtained with solution (2).
Acidity or alkalinity pH of a 1% w/v solution in ethanol (10%), 6.4 to 8.4, Appendix V L.
Clarity and colour of solution A 5% w/v solution in methanol is clear, Appendix IV A. The
absorbance of the solution measured at 440 nm is not greater than 0.05, Appendix II B.
Heavy metals 2 g complies with limit test C for heavy metals, Appendix VII. Use 2 ml of lead standard
phase. For solution (2) dilute 2 volumes of solution (1) to 100 volumes with the mobile phase and
dilute 1 volume of this solution to 10 volumes with the same solvent. For solution (3) dissolve 1 mg
of diclofenac impurity A EPCRS in the mobile phase, add 1 ml of solution (1) and dilute to 200 ml
(25 cm 4.6 mm) packed with end-capped octylsilyl silica gel for chromatography (5 m) (end-capped
Zorbax C8 is suitable), (b) as the mobile phase at a flow rate of 1 ml per minute a mixture of 34
volumes of a mixture of equal volumes of a 0.1% w/v solution of orthophosphoric acid and a 0.16% w/v
solution of sodium dihydrogen orthophosphate adjusted to pH 2.5 and 66 volumes of methanol and (c) a
Inject 20 l of solution (3). When the chromatograms are recorded in the prescribed conditions,
the retention times are about 25 minutes for diclofenac and about 12 minutes for diclofenac impurity
A. Adjust the sensitivity of the system so that the height of the peaks in the chromatogram obtained
with solution (3) is not less than 50% of the full scale of the recorder. Continue the chromatography
for 1.5 times the retention time of diclofenac. The test is not valid unless, in the chromatogram
obtained with solution (3), the resolution factor between the peaks corresponding to diclofenac and
diclofenac impurity A is at least 6.5.
Inject 20 l of solution (1) and 20 l of solution (2). In the chromatogram obtained with solution
(1) the area of any secondary peak is not greater than the area of the principal peak in the chromato-
gram obtained with solution (2) (0.2%) and the sum of the areas of any secondary peaks is not greater
than 2.5 times the area of the principal peak in the chromatogram obtained with solution (2) (0.5%).
gram obtained with solution (2) (0.05%).
Loss on drying When dried at a pressure not exceeding 1 kPa for 24 hours, loses not more than
0.5% of its weight. Use 1 g..
13-41
18
H
22
Cl
2
N
2
O
2
.
Storage Diclofenac Diethylamine should be kept in an airtight container and protected from light.
Preparation
Diclofenac Gel
IMPURITIES
The impurities limited by the requirements of this monograph include those listed under Diclofenac Sodium.
13-42
Diclofenac Potassium
1/01
NH
Cl Cl
COOK
C
14
H
10
Cl
2
KNO
2
334.2 15307-81-0
Diclofenac Potassium complies with the requirements of the 3rd edition of the European Pharmacopoeia [1508].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Potassium diclofenac contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of potassium [2-[(2,6-dichlorophenyl)amino]phenyl]acetate, calculated with reference
CHARACTERS
A white or slightly yellowish, crystalline powder, slightly hygroscopic, sparingly soluble in water,
freely soluble in methanol, soluble in alcohol, slightly soluble in acetone.
IDENTIFICATION
obtained with diclofenac potassium CRS. Examine the substances prepared as discs.
B. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel GF
254
plate R.
the same solvent.
Reference solution (a). Dissolve 25 mg of diclofenac potassium CRS in methanol R and dilute to 5 ml
Reference solution (b). Dissolve 10 mg of indometacin R in reference solution (a) and dilute to 2 ml
10 volumes of concentrated ammonia R, 10 volumes of methanol R and 80 volumes of ethyl acetate R.
C. Dissolve about 10 mg in 10 ml of alcohol R. To 1 ml of the solution add 0.2 ml of a mixture,
prepared immediately before use, of equal volumes of a 6 g/l solution of potassium ferricyanide R and a
9 g/l solution of ferric chloride R. Allow to stand protected from light for 5 min. Add 3 ml of a 10 g/l
solution of hydrochloric acid R. Allow to stand protected from light for 15 min. A blue colour develops
and a precipitate is formed.
D. Suspend 0.5 g of the substance to be examined in 10 ml of water R. Stir, add water R until the
substance is dissolved. Add 2 ml of hydrochloric acid R1, stir for 1 h and filter with the aid of vacuum.
Neutralise the solution with sodium hydroxide solution R. The solution gives reaction (b) of potassium
(2.3.1).
TESTS
13-43
Reference solution (a). Dilute 2.0 ml of the test solution to 100.0 ml with methanol R. Dilute 1.0 ml of
the solution to 10.0 ml with methanol R.
Reference solution (b). Dissolve 1.0 mg of diclofenac impurity A CRS in methanol R, add 1.0 ml of the
test solution and dilute to 200.0 ml with methanol R.
octylsilyl silica gel for chromatography R (5 m),
as mobile phase at a flow rate of 1 ml/min a mixture of 34 volumes of a solution containing
0.5 g/l of phosphoric acid R and 0.8 g/l of sodium dihydrogen phosphate R adjusted to pH 2.5 with
phosphoric acid R, and 66 volumes of methanol R,
Inject 20 l of reference solution (b). When the chromatograms are recorded in the prescribed
conditions, the retention times are: diclofenac, about 25 min and impurity A, about 12 min. Adjust
the sensitivity of the system so that the height of the peaks in the chromatogram obtained is at least
50 per cent of the full scale of the recorder. The test is not valid unless in the chromatogram obtained
the resolution between the peaks corresponding to diclofenac and impurity A is not less than 6.5.
Inject 20 l of the test solution and 20 l of reference solution (a). Continue the chromatography
for 1.5 times the retention time of the principal peak in the chromatogram obtained with the test
solution. In the chromatogram obtained with the test solution: the area of any peak, apart from the
principal peak, is not greater than the area of the principal peak in the chromatogram obtained with
reference solution (a) (0.2 per cent); the sum of the areas of all the peaks, apart from the principal
peak, is not greater than 2.5 times that of the principal peak in the chromatogram obtained with
reference solution (a) (0.5 per cent). Disregard any peak with an area less than 0.25 times the area of
the principal peak in the chromatogram obtained with reference solution (a).
100C to 105C for 3 h.
ASSAY
14
H
10
Cl
2
KNO
2
.
STORAGE
IMPURITIES
N
Cl Cl
O
A. 1-(2,6-dichlorophenyl)-1,3-dihydro-2H-indol-2-one,
NH
Cl R
2
R
1
B. R
1
= CHO, R
2
= Cl: 2-[(2,6-dichlorophenyl)amino]benzaldehyde,
C. R
1
= CH
2
OH, R
2
= Cl: [2-[(2,6-dichlorophenyl)amino]phenyl]methanol,
D. R
1
= CH
2
-CO
2
H, R
2
= Br: 2-[2-[(2-bromo-6-chlorophenyl)amino]phenyl]acetic acid,
N
H
O
E. 1,3-dihydro-2H-indol-2-one.
__________________________________________________________________________________________________________ Ph Eur
13-44
Diclofenac Sodium
N
Cl
Cl
COONa
H
C
14
H
10
Cl
2
NNaO
2
318.1 15307-79-6
Diclofenac Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia for Sodium
Diclofenac [1002]. These requirements are reproduced after the heading Definition below.
Preparations
Diclofenac Tablets
Slow Diclofenac Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium diclofenac contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of sodium 2-[(2,6-dichlorophenyl)amino]phenyl]acetate, calculated with reference to
CHARACTERS
A white or slightly yellowish, crystalline powder, slightly hygroscopic, sparingly soluble in water,
freely soluble in methanol, soluble in alcohol, slightly soluble in acetone, practically insoluble in
ether.
IDENTIFICATION
obtained with sodium diclofenac CRS. Examine the substances prepared as discs.
B. Examine by thin-layer chromatography (2.2.27), using silica gel GF
254
the same solvent.
Reference solution (a). Dissolve 25 mg of sodium diclofenac CRS in methanol R and dilute to 5 ml with
the same solvent.
Reference solution (b). Dissolve 10 mg of indometacin CRS in reference solution (a) and dilute to 2 ml
10 volumes of concentrated ammonia R, 10 volumes of methanol R and 80 volumes of ethyl acetate R.
the chromatogram obtained with the reference solution (a). The test is not valid unless the chromato-
C. Dissolve about 10 mg in 10 ml of alcohol R. To 1 ml of the solution add 0.2 ml of a mixture,
prepared immediately before use, of equal volumes of a 6 g/l solution of potassium ferricyanide R and a
9 g/l solution of ferric chloride R. Allow to stand protected from light for 5 min. Add 3 ml of a 10 g/l
solution of hydrochloric acid R. Allow to stand, protected from light, for 15 min. A blue colour
develops and a precipitate is formed.
D. Dissolve 60 mg in 0.5 ml of methanol R and add 0.5 ml of water R. The solution gives reaction (b)
of sodium (2.3.1).
TESTS
13-45
Reference solution (b). Dissolve 1.0 mg of diclofenac impurity A CRS in methanol R, add 1.0 ml of the
test solution and dilute to 200.0 ml with methanol R.
octylsilyl silica gel for chromatography R (5 m),
as mobile phase at a flow rate of 1 ml per minute a mixture of 34 volumes of a mixture of equal
volumes of a 1 g/l solution of phosphoric acid R and a 1.6 g/l solution of sodium dihydrogen
phosphate R adjusted to pH 2.5 and 66 volumes of methanol R,
conditions, the retention times are about 25 min for diclofenac and about 12 min for diclofenac
impurity A. Adjust the sensitivity of the system so that the height of the peaks in the chromatogram
obtained with reference solution (b) is not less than 50 per cent of the full scale of the recorder.
Continue the chromatography for 1.5 times the retention time of sodium diclofenac. The test is not
valid unless in the chromatogram obtained with reference solution (b) the resolution between the
peaks corresponding to diclofenac and diclofenac impurity A is at least 6.5.
Inject 20 l of the test solution and 20 l of reference solution (a). In the chromatogram obtained
with the test solution: the area of any peak apart from the principal peak is not greater than the area
sum of the areas of all the peaks apart from the principal peak is not greater than 2.5 times that of the
100C to 105C for 3 h.
ASSAY
14
H
10
Cl
2
NNaO
2
.
STORAGE
Store in a airtight container, protected from light.
IMPURITIES
N
Cl Cl
O
A. 1-(2,6-dichlorophenyl)indolin-2-one,
NH
CHO
Cl Cl
B. 2-[(2,6-dichlorophenyl)amino]benzaldehyde,
13-46
NH
OH
Cl Cl
C. [2-[(2,6-dichlorophenyl)amino]phenyl]methanol,
NH
COOH
Br Cl
D. 2-[2-[(2-bromo-6-chlorophenyl)amino]phenyl]acetic acid,
N
O
E. 1,3-dihydro-2H-ndolin-2-one.
__________________________________________________________________________________________________________ Ph Eur
13-47
Dicloxacillin Sodium
H
N
S
O
Me
Me
H H Cl
Cl
Me
N
O
O
N
H COONa
C
19
H
16
Cl
2
N
3
NaO
5
S,H
2
O 510.3 13412-64-1
Dicloxacillin Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia [0663].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Dicloxacillin sodium contains not less than 95.0 per cent and not more than the equivalent of
101.0 per cent of sodium (2S,5R,6R)-6-[[[3-(2,6-dichlorophenyl)-5-methylisoxazol-4-
yl]carbonyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate, calcu-
lated with reference to the anhydrous substance.
PRODUCTION
(2.2.28), using a suitable validated method.
CHARACTERS
A white or almost white, crystalline powder, hygroscopic, freely soluble in water, soluble in alcohol
and in methanol.
IDENTIFICATION
obtained with dicloxacillin sodium CRS. Examine the substances prepared as discs.
substance.
Reference solution (a). Dissolve 25 mg of dicloxacillin sodium CRS in 5 ml of water R.
Reference solution (b). Dissolve 25 mg of each of cloxacillin sodium CRS, dicloxacillin sodium CRS and
flucloxacillin sodium CRS in 5 ml of water R.
of acetone R and 70 volumes of a 154 g/l solution of ammonium acetate R the pH of which has been
for 1 min; a yellow colour develops.
TESTS
13-48
substance.
the chromatogram obtained is at least 50 per cent of the full scale of the recorder. Inject test solution
(a) and continue the chromatography for five times the retention time of the principal peak. In the
chromatogram obtained with test solution (a): the area of any peak, apart from the principal peak, is
(b) (1 per cent); the sum of the areas of all peaks, apart from the principal peak, is not greater than
five times the area of the principal peak in the chromatogram obtained with reference solution (b)
(5 per cent). Disregard any peak with an area less than 0.05 times the area of the principal peak in
mination of water.
Inject per kilogram of the rabbits mass 1 ml of a solution in water for injections R containing 20 mg of
ASSAY
Reference solution (a). Dissolve 50.0 mg of dicloxacillin sodium CRS in the mobile phase and dilute to
Reference solution (b). Dilute 5.0 ml of test solution (b) to 50.0 ml with the mobile phase.
Reference solution (c). Dissolve 5 mg of flucloxacillin sodium CRS and 5 mg of dicloxacillin sodium CRS
in the mobile phase and dilute to 50.0 ml with the mobile phase.
as mobile phase at a flow rate of 1.0 ml per minute a mixture of 75 volumes of a 2.7 g/l solution
of potassium dihydrogen phosphate R adjusted to pH 5.0 with dilute sodium hydroxide solution R and
25 volumes of acetonitrile R,
Inject reference solution (c). When the chromatograms are recorded in the prescribed conditions,
the retention time of dicloxacillin is about 10 min. Adjust the sensitivity of the system so that the
13-49
heights of the principal peaks in the chromatogram obtained are at least 50 per cent of the full scale
of the recorder. The test is not valid unless in the chromatogram obtained, the resolution between the
first peak (flucloxacillin) and the second peak (dicloxacillin) is at least 2.5. Inject reference solution
(a) six times. The test is not valid unless the relative standard deviation of the peak area for
dicloxacillin is at most 1.0 per cent. Inject alternately test solution (b) and reference solution (a).
STORAGE
a sterile, air-tight, tamper-proof container.
LABELLING
The label states:
IMPURITIES
H
HN
S
N
N
O
H COOH
Me
Me
COOH O
Me
Cl
Cl
*
*
A. (4S)-2-[carboxy[[[3-(2,6-dichlorophenyl)-5-methylisoxazol-4-yl]carbonyl]amino]methyl]-
5,5-dimethylthiazolidine-4-carboxylic acid (penicilloic acids of dicloxacillin),
and epimer at C*
HN
S
N
N
O
H COOH
Me
Me
O
Me
Cl
Cl
H
H
*
B. (2RS,4S)-2-[[[[3-(2,6-dichlorophenyl)-5-methylisoxazol-4-yl]carbonyl]amino]methyl]-5,5-
dimethylthiazolidine-4-carboxylic acid (penilloic acids of dicloxacillin),
N
S
H COOH
Me
Me
O
H H
H
2
N
C. (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[ 3.2.0]heptane-2-carboxylic
acid (6 aminopenicillanic acid),
N
O
Me
COOH
Cl
Cl
D. 3-(2,6-dichlorophenyl)-5-methylisoxazole-4-carboxylic acid.
__________________________________________________________________________________________________________ Ph Eur
13-50
Dicycloverine Hydrochloride / Dicyclomine Hydrochloride
O O
NEt
2
,HCl
C
19
H
35
NO
2
,HCl 346.0 67-92-5
Dicycloverine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparations
Dicycloverine Oral Solution/Dicyclomine Oral Solution
Dicyloverine Tablets/Dicyclomine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Dicycloverine hydrochloride contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of 2-(diethylamino)ethyl bicyclohexyl-1-carboxylate hydrochloride, calculated with
CHARACTERS
A white or almost white, crystalline powder, soluble in water, freely soluble in alcohol and in
methylene chloride.
IDENTIFICATION
obtained with dicycloverine hydrochloride CRS. Examine the substances prepared as discs using
potassium chloride R. If the spectra obtained show differences, dissolve the substance to be examined
and the reference substance separately in acetone R, evaporate to dryness and record new spectra
using the residues.
spot in the chromatogram obtained with reference solution (b).
C. To 3 ml of a 1.0 g/l solution of sodium lauryl sulphate R add 5 ml of methylene chloride R and
0.05 ml of a 2.5 g/l solution of methylene blue R, mix gently and allow to stand; the lower layer is blue.
Add 2 ml of a 20 g/l solution of the substance to be examined, mix gently and allow to stand; the
upper layer is blue and the lower layer is colourless.
TESTS
pH (2.2.3). Dissolve 0.5 g in water R and dilute to 50 ml with the same solvent. The pH of the
Test solution (a). Dissolve 0.25 g to the substance to be examined in methanol R and dilute to 5 ml
Reference solution (a). Dilute 1 ml of test solution (b) to 10 ml with methanol R.
Reference solution (b). Dissolve 10 mg of dicycloverine hydrochloride CRS in methanol R and dilute to
13-51
Reference solution (c). Dissolve 5 mg of tropicamide CRS in reference solution (b) and dilute to 5 ml
volumes of propanol R. Dry the plate in a current of warm air. Spray with dilute potassium iodo-
bismuthate solution R. Any spot in the chromatogram obtained with test solution (a), apart from the
solution (a) (0.2 per cent). The test is not valid unless the chromatogram obtained with reference
solution (c) shows two clearly separated spots.
100C to 105C.
ASSAY
19
H
36
ClNO
2
.
STORAGE
IMPURITIES
COOH
A. bicyclohexyl-1-carboxylic acid.
__________________________________________________________________________________________________________ Ph Eur
13-52
Dienestrol
OH
HO
H
3
C
CH
3
C
18
H
18
O
2
266.3 84-17-3
Dienestrol complies with the requirements of the 3rd edition of the European Pharmacopoeia [0483]. These
Action and use Oestrogen.
When dienoestrol is prescribed or demanded, Dienestrol shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Dienestrol contains not less than 98.5 per cent and not more than the equivalent of 101.5 per cent of
(E,E)-4,4-(1,2-diethylidene-ethylene)diphenol, calculated with reference to the dried substance.
CHARACTERS
and in alcohol, soluble in ether. It dissolves in dilute solutions of the alkali hydroxides.
IDENTIFICATION
obtained with dienestrol CRS. Examine the substances prepared as discs.
chromatogram obtained with test solution (b) is similar in position, colour and size to the spot in the
C. Dissolve about 1 mg in 5 ml of glacial acetic acid R, add 1 ml of a 1 per cent V/V solution of
bromine R in glacial acetic acid R and heat in a water-bath for 2 min. Place 0.5 ml of this solution in a
dry test-tube, add 0.5 ml of ethanol R, mix and add 10 ml of water R. A reddish-violet colour is
produced. Add 5 ml of chloroform R, shake vigorously and allow to separate. The chloroform layer is
red and the aqueous layer is almost colourless.
D. Dissolve about 0.5 mg in 0.2 ml of glacial acetic acid R, add 1 ml of phosphoric acid R and heat on a
water-bath for 3 min. A reddish-violet colour is produced.
TESTS
Melting range Determined by the capillary method (2.2.14), the melting point is 227C to 234C.
The temperature interval between the formation of a definite meniscus in the melt and the
disappearance of the last particle does not exceed 3C.
coating substance.
Test solution (a). Dissolve 0.2 g of the substance to be examined in 2 ml of alcohol R.
Reference solution (a). Dissolve 25 mg of dienestrol CRS in alcohol R and dilute to 5 ml with the same
solvent.
Reference solution (b). Dilute 1 ml of reference solution (a) to 10 ml with alcohol R.
Reference solution (c). Dissolve 10 mg of diethylstilbestrol CRS in 2 ml of alcohol R. To 1 ml of this
solution add 1 ml of reference solution (a).
10 volumes of diethylamine R and 90 volumes of toluene R. Allow the plate to dry in air, spray with
alcoholic solution of sulphuric acid R and heat at 120C for 10 min. Any spot in the chromatogram
chromatogram obtained with reference solution (c) shows at least two clearly separated spots
13-53
having approximately the same intensity.
at 100C to 105C.
ASSAY
Dissolve 25.0 mg in ethanol R and dilute to 100.0 ml with the same solvent. To 5.0 ml of this
solution add 10 ml of ethanol R and dilute to 250.0 ml with 0.1M sodium hydroxide. Prepare a refer-
ence solution in the same manner using 25.0 ml of dienestrol CRS. Measure the absorbance (2.2.25)
of the solutions at the maximum at 245 nm.
18
H
18
O
2
from the measured absorbances and the concentrations of the
solutions.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
13-54
Diethyl Phthalate
COOEt
COOEt
C
12
H
14
O
4
222.2 84-66-2
Diethyl Phthalate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0897].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Diethyl phthalate contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent m/m of diethyl benzene-1,2-dicarboxylate.
CHARACTERS
A clear, oily liquid, colourless or very slightly yellow, practically insoluble in water, miscible with
alcohol and with ether.
IDENTIFICATION
obtained with diethyl phthalate CRS. Examine as thin films.
254
Test solution. Dissolve 50 mg of the substance to be examined in ether R and dilute to 10 ml with the
same solvent.
Reference solution. Dissolve 50 mg of diethyl phthalate CRS in ether R and dilute to 10 ml with the same
solvent.
30 volumes of heptane R and 70 volumes of ether R. Allow the plate to dry in air and examine in
solution.
E. To about 0.1 ml add 0.25 ml of sulphuric acid R and 50 mg of resorcinol R. Heat on a water-bath
for 5 min. Allow to cool. Add 10 ml of water R and 1 ml of strong sodium hydroxide solution R. The
solution becomes yellow or brownish-yellow and shows green fluorescence.
TESTS
6
(Method II, 2.2.2).
Acidity Dissolve 20.0 g in 50 ml of alcohol R previously neutralised to phenolphthalein solution R1.
Add 0.2 ml of phenolphthalein solution R1. Not more than 0.1 ml of 0.1M sodium hydroxide is required
to change the colour of the indicator to pink.
Related substances Examine by gas chromatography (2.2.28), using naphthalene R as the internal
standard.
Internal standard solution. Dissolve 60 mg of naphthalene R in methylene chloride R and dilute to 20 ml
Test solution (b). Dissolve 1.0 g of the substance to be examined in methylene chloride R, add 2.0 ml of
the internal standard solution and dilute to 20.0 ml with methylene chloride R.
Reference solution. To 1.0 ml of test solution (a) add 10.0 ml of the internal standard solution and
dilute to 100.0 ml with methylene chloride R.
13-55
a glass column 2 m long and 2 mm in internal diameter packed with silanised diatomaceous earth for
gas chromatography R (150 m to 180 m) impregnated with 3 per cent m/m of
detector at 225C.
Inject 1 l of the reference solution. The substances are eluted in the following order: naphthalene
and diethyl phthalate. Adjust the sensitivity of the detector so that the height of the peak due to
naphthalene is not less than 50 per cent of the full scale of the recorder. The test is not valid unless
the resolution between the peaks corresponding to naphthalene and diethyl phthalate is at least 10.
Inject separately 1 l of test solution (b) and 1 l of the reference solution. Continue the chromato-
graphy for three times the retention time of diethyl phthalate. From the chromatogram obtained with
the reference solution, calculate the ratio (R) of the area of the peak due to diethyl phthalate to the
area of the peak due to the internal standard. From the chromatogram obtained with test solution
(b), calculate the ratio of the sum of the areas of any peaks, apart from the principal peak and the
peaks due to the internal standard and the solvent, to the area of the peak due to the internal
standard; this ratio is not greater than R (1.0 per cent).
of water.
ASSAY
Introduce 0.750 g into a 250 ml borosilicate glass flask. Add 25.0 ml of 0.5M alcoholic potassium
hydroxide and a few glass beads. Boil on a water-bath under a reflux condenser for 1 h. Add 1 ml of
phenolphthalein solution R1 and titrate immediately with 0.5M hydrochloric acid. Carry out a blank
titration. Calculate the volume of 0.5M alcoholic potassium hydroxide used in the saponification.
12
H
14
O
4
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
13-56
Diethylamine Salicylate
COOH
OH
Et
2
NH
C
11
H
17
NO
3
211.3 4419-92-5
Definition Diethylamine Salicylate contains not less than 99.0% and not more than 101.0% of
C
11
H
17
NO
3
.
Characteristics White or almost white crystals; odourless or almost odourless.
Very soluble in water; freely soluble in chloroform and in ethanol (96%).
Identification
diethylamine salicylate (RS 099).
B. To 0.2 g add 5 ml of 1M sodium hydroxide, heat to boiling point, cool and acidify with 2M
hydrochloric acid; a white precipitate is produced. The melting point of the precipitate, after
recrystallisation from water and drying at 105, is about 160, Appendix V A.
Acidity Dissolve 2 g in 25 ml of water and titrate with 0.1M sodium hydroxide VS using phenol red
solution as indicator. Not more than 0.2 ml of 0.1M sodium hydroxide VS is required to change the
colour of the solution.
Clarity and colour of solution A 50% w/v solution is clear, Appendix IV A, and not more intensely
coloured than reference solution BY
5
Heavy metals 12 ml of a 10.0% w/v solution complies with limit test A for heavy metals, Appendix
VII. Use lead standard solution (1 ppm Pb) to prepare the standard (10 ppm).
Sulphate 0.6 g complies with the limit test for sulphates, Appendix VII (250 ppm).
Assay Carry out Method I for non-aqueous titration, Appendix VIII A, using 0.4 g and 1-naphthol-
benzein solution as indicator. Each ml of 0.1M perchloric acid VS is equivalent to 21.13 mg of
C
11
H
17
NO
3
.
Storage Diethylamine Salicylate should be kept in a well-closed container and protected from light.
It should not be allowed to come into contact with iron or iron salts.
Action and use Counter-irritant.
Preparation
Diethylamine Salicylate Cream
13-57
Diethylcarbamazine Citrate
MeN
N
CONEt
2
HO C COOH
CH
2
COOH
CH
2
COOH
,
C
10
H
21
N
3
O,C
6
H
8
O
7
391.4 1642-54-2
Diethylcarbamazine Citrate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Diethylcarbamazine citrate contains not less than 98.0 per cent and not more than the equivalent of
101.0 per cent of N,N-diethyl-4-methylpiperazine-1-carboxamide dihydrogen 2-hydroxypropane-
1,2,3-tricarboxylate, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder, slightly hygroscopic, very soluble in water, soluble in alcohol, practically
insoluble in acetone and in ether.
IDENTIFICATION
obtained with diethylcarbamazine citrate CRS.
B. Examine the chromatograms obtained in the test for dimethylpiperazine and methylpiperazine.
The principal spot in the chromatogram obtained with the test solution corresponds in position,
colour and size to the principal spot in the chromatogram obtained with reference solution (a).
C. Dissolve 0.1 g in 5 ml of water R. The solution gives the reaction of citrates (2.3.1).
TESTS
Solution S Shake 2.5 g with water R until dissolved and dilute to 25 ml with the same solvent.
6
(Method II, 2.2.2).
Dimethylpiperazine and methylpiperazine Examine by thin-layer chromatography (2.2.27),
using silica gel G R as the coating substance.
the same solvent.
Reference solution (a). Dissolve 0.1 g of diethylcarbamazine citrate CRS in methanol R and dilute to
Reference solution (b). Dissolve 10 mg of methylpiperazine R in methanol R and dilute to 100 ml with
the same solvent.
Reference solution (c). Dissolve 10 mg of dimethylpiperazine R in methanol R and dilute to 100 ml with
the same solvent.
5 volumes of concentrated ammonia R, 30 volumes of methyl ethyl ketone R and 65 volumes of
methanol R. Dry the plate at 100C to 105C and expose to iodine vapour for 30 min. In the chroma-
togram obtained with the test solution, any spots corresponding to methylpiperazine and
dimethylpiperazine are not more intense than the spots in the chromatograms obtained with refer-
ence solutions (b) and (c) respectively (0.2 per cent).
60C for 4 h.
13-58
ASSAY
Dissolve 0.350 g in 25 ml of anhydrous acetic acid R and add 25 ml of acetic anhydride R. Using 0.2 ml
of crystal violet solution R as indicator, titrate with 0.1M perchloric acid until a greenish-blue colour is
obtained.
16
H
29
N
3
O
8.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
13-59
Diethylene Glycol Monoethyl Ether
O
O
OH
3
C
C
6
H
14
O
3
134.2 111-90-0
Diethylene Glycol Monoethyl Ether complies with the requirements of the 3rd edition of the European
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Diethylene glycol monoethyl ether contains not less than 99.0 per cent and not more than the equiva-
lent of 101.0 per cent of 2-(2-ethoxyethoxy)ethanol. It is produced by condensation of ethylene oxide
and alcohol, followed by distillation.
CHARACTERS
A clear, colourless, hygroscopic liquid, miscible with water, with acetone and with alcohol, miscible
in certain proportions with vegetable oils, not miscible with mineral oils.
IDENTIFICATION
A. Refractive index (2.2.6): 1.426 to 1.428.
obtained with diethylene glycol monoethyl ether CRS. Examine the substances between two potassium
bromide R plates.
C. Examine the chromatograms obtained in the assay. The retention time and size of the principal
peak in the chromatogram obtained with the test solution are similar to those of the principal peak in
TESTS
Acid value (2.5.1). Dissolve 30.0 ml in 30 ml of alcohol R previously neutralised with 0.1M potassium
hydroxide using phenolphthalein solution R as indicator. Titrate with 0.01M alcoholic potassium hydroxide.
The acid value is not greater than 0.1.
Ethylene glycol monoethyl ether, ethylene glycol and diethylene glycol Not more than
0.20 per cent of ethylene glycol monoethyl ether and not more than 0.50 per cent of ethylene glycol
and diethylene glycol combined, determined by gas chromatography (2.2.28) as prescribed under
Assay.
Inject 0.1 l of reference solution (a). When the chromatograms are recorded under the prescribed
conditions, the retention times are: ethylene glycol monoethyl ether, about 1 min; ethylene glycol,
about 7 min and diethylene glycol, about 14 min.
Inject 0.1 l of the test solution. Calculate the percentage content of ethylene glycol monoethyl
ether, ethylene glycol and diethylene glycol by the normalisation procedure.
Residual ethylene oxide (2.4.25). Not more than 1 ppm.
of water.
ASSAY
Reference solution (a). Dissolve 0.10 g of diethylene glycol R, 0.10 g of ethylene glycol R and 0.10 g of
ethylene glycol monoethyl ether R in methanol R and dilute to 100.0 ml with the same solvent.
Reference solution (b). Diethylene glycol monoethyl ether CRS.
a fused-silica capillary column 15 m long and 0.53 mm in internal diameter coated with a
0.5 m layer of macrogol 20,000 2-nitroterephthalate R,
raising the temperature of the column from 60C to 200C at a rate of 5C per minute, maintaining
the temperature of the injection port at 240C and that of the detector at 250C.
13-60
Inject 0.1 l of the test solution and calculate the content of C
6
H
14
O
3
from the areas of the peaks
in the chromatogram obtained, by the normalisation procedure.
STORAGE
Store under an inert gas, in an airtight container, at a temperature not exceeding 35C.
LABELLING
The label indicates that the substance is stored under an inert gas.
__________________________________________________________________________________________________________ Ph Eur
13-61
Diethylene Glycol Monopalmitostearate
Diethylene Glycol Monopalmitostearate complies with the requirements of the 3rd edition of the European
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Diethylene glycol monopalmitostearate is a mixture of diethylene glycol mono- and diesters of stearic
and palmitic acids. It contains not less than 45.0 per cent of monoesters produced from the
condensation of diethylene glycol and stearic acid 50 of vegetable or animal origin.
PRODUCTION
CHARACTERS
A white or almost white, waxy solid, practically insoluble in water, soluble in acetone and in hot
alcohol.
IDENTIFICATION
C. It complies with the assay (monoesters content).
TESTS
Melting point (2.2.15).43C to 50C.
(2.4.22, Method A). The fatty acid fraction has the following composition:
sum of contents of palmitic acid and stearic acid: not less than 90.0 per cent.
Free diethylene glycol Not more than 8.0 per cent, determined as prescribed under Assay.
ASSAY
Determine the free diethylene glycol content and monoesters content by size-exclusion chromato-
graphy (2.2.30).
Test solution. Into a 15 ml flask, weigh about 0.2 g (m), to the nearest 0.1 mg. Add 5.0 ml of
tetrahydrofuran R and shake to dissolve. Heat gently, if necessary. Reweigh the flask and calculate the
total mass of solvent and substance (M).
Reference solutions. Into four 15 ml flasks, respectively weigh, to the nearest 0.1 mg, about 2.5 mg,
5.0 mg, 10.0 mg and 20.0 mg of diethylene glycol R. Add 5.0 ml of tetrahydrofuran R and shake to
dissolve. Weigh the flasks again and calculate the concentration of diethylene glycol in milligrams per
gram for each reference solution.
a gel-permeation column 0.6 m long and 7 mm in internal diameter packed with styrene-
divinylbenzene copolymer R (particle diameter 5 m and pore size 10 nm),
as mobile phase at a flow rate of 1 ml/min tetrahydrofuran R,
a differential refractive index detector.
the retention times relative to diethylene glycol are about 0.84 for the monoesters and about 0.78 for
the diesters. From the calibration curve obtained with the reference solutions determine the concen-
tration (C) in milligrams per gram of diethylene glycol in the test solution.
Calculate the percentage content of free diethylene glycol in the substance to be examined using
the following expression:
10
m
M C
From the peak area of the monoesters (A) and the diesters (B), calculate the percentage content of
13-62
monoesters using the following expression:
) 100 ( D
B A
A

+
D=the percentage content of free diethylene glycol and the percentage content of free fatty
acids.
Calculate the percentage content of free fatty acids using the expression:
1 561
270
A
.
I
I
A
=acid value.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
13-63
Diethylstilbestrol
CH
3
H
3
C OH
HO
C
18
H
20
O
2
268.4 56-53-1
Diethylstilbestrol complies with the requirements of the 3rd edition of the European Pharmacopoeia [0484].
Action and use Oestrogen
Preparations
Diethylstilbestrol Pessaries
Diethylstilbestrol Tablets
When stilboestrol is prescribed or demanded, Diethylstilbestrol shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Diethylstilbestrol contains not less than 97.0 per cent and not more than the equivalent of 101.0 per
cent of (E)-4,4-(1,2-diethylvinylene)diphenol, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water, freely soluble in alcohol
and in ether. It dissolves in solutions of the alkali hydroxides.
IDENTIFICATION
A. Examined between 230 nm and 450 nm (2.2.25), the irradiated solution of the substance to be
examined prepared as prescribed in the assay shows two absorption maxima, at 292 nm and 418 nm.
obtained with diethylstilbestrol CRS. Examine the substances prepared as discs.
C. Examine the chromatograms obtained in the test for mono-and dimethyl ethers. The principal
D. Dissolve about 0.5 mg in 0.2 ml of glacial acetic acid R, add 1 ml of phosphoric acid R and heat on a
water-bath for 3 min. A deep-yellow colour develops.
TESTS
4,4-Dihydroxystilbene and related ethers Dissolve 0.100 g in ethanol R and dilute to 10.0 ml
with the same solvent. The absorbance (2.2.25) of the solution measured at 325 nm is not greater
than 0.50.
Mono- and dimethyl ethers Examine by thin-layer chromatography (2.2.27), using silica gel G R as
Test solution (a). Dissolve 0.2 g of the substance to be examined in 2 ml of alcohol R.
Reference solution (a). Dissolve 10 mg of diethylstilbestrol CRS in 2 ml of alcohol R.
Reference solution (b). Dissolve 5 mg of diethylstilbestrol monomethyl ether CRS in alcohol R and dilute to
Reference solution (c). Dissolve 5 mg of diethylstilbestrol dimethyl ether CRS in alcohol R and dilute to
Reference solution (d). Dissolve 10 mg of dienestrol CRS in 2 ml of alcohol R. To 1 ml of this solution
add 1 ml of reference solution (a).
13-64
10 volumes of diethylamine R and 90 volumes of toluene R. Allow the plate to dry in air, spray with
alcoholic solution of sulphuric acid R and heat at 120C for 10 min. In the chromatogram obtained with
test solution (a), any spots corresponding to diethylstilbestrol monomethyl ether and diethylstilbestrol
dimethyl ether are not more intense than the spots in the chromatograms obtained with reference
solutions (b) and (c) respectively (0.5 per cent). Diethylstilbestrol gives one or sometimes two spots.
The test is not valid unless the chromatogram obtained with reference solution (d) shows at least two
clearly separated spots having approximately the same intensity.
100C to 105C.
ASSAY
Dissolve 20.0 mg in ethanol R and dilute to 100.0 ml with the same solvent. Dilute 10.0 ml of the
solution to 100.0 ml with ethanol R. To 25.0 ml of the resulting solution add 25.0 ml of a solution of
1 g of dipotassium hydrogen phosphate R in 55 ml of water R. Prepare in the same manner a reference
solution using 20.0 mg of diethylstilbestrol CRS. Transfer an equal volume of each solution to separate
1 cm quartz cells and close the cells; place the two cells about 5 cm from a low-pressure, short-wave
2 W to 20 W mercury lamp and irradiate for about 5 min. Measure the absorbance (2.2.25) of the
irradiated solutions at the maximum at 418 nm, using water R as the compensation liquid. Continue
the irradiation for successive periods of 3 min to 15 min, depending on the power of the lamp, and
repeat the measurement of the absorbances at 418 nm until the maximum absorbance (about 0.7) is
obtained. If necessary, adjust the geometry of the irradiation apparatus to obtain a maximum,
reproducible absorbance at 418 nm.
18
H
20
O
2
from the measured absorbances and the concentrations of the
solutions.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
13-65
Diflucortolone Valerate
H
H
O
HO
O
F
O
Me
Me
H
F
Me
O
CH
3
C
27
H
36
F
2
O
5
478.6 59198-70-8
Definition Diflucortolone Valerate is 6,9-difluoro-3,20-dioxo-11-hydroxy-16-methylpregna-
1,4-dien-21-yl valerate. It contains not less than 97.0% and not more than 102.0% of C
27
H
36
F
2
O
5
,
Characteristics A white to creamy white, crystalline powder.
Practically insoluble in water; freely soluble in dichloromethane and in 1,4-dioxan; slightly soluble in
methanol; sparingly soluble in ether.
Identification
methanol exhibits a maximum only at 238 nm. The absorbance at the maximum at 238 nm is about
0.69.
diflucortolone valerate (RS 100).
Specific optical rotation In a 1% w/v solution in 1,4-dioxan, +98 to +103, calculated with
Related substances Carry out the method for liquid chromatography, Appendix III D, injecting 20 l
of each of the following solutions in a mixture of 25 volumes of water and 75 volumes of acetonitrile.
Solution (1) contains 0.060% w/v of the substance being examined. For solution (2) dilute 1 volume
of solution (1) to 100 volumes. Solution (3) contains 0.060% w/v of diflucortolone valerate impurity
standard BPCRS.
cm 4.6 mm) packed with stationary phase C (3 m) (Spherisorb ODS 2 is suitable), (b) a mixture of
equal volumes of acetonitrile and water as the mobile phase with a flow rate of 2 ml per minute and (c)
a detection wavelength of 238 nm. If necessary the composition of the mobile phase may be altered
so that the chromatogram obtained with solution (3) shows similar resolution to the reference
chromatogram supplied with diflucortolone valerate impurity standard BPCRS.
Continue the chromatography for twice the retention time of the principal peak. In the chromato-
gram obtained with solution (1) the area of any secondary peak is not greater than the area of the
principal peak in the chromatogram obtained with solution (2) (1%) and the sum of the areas of any
secondary peaks is not greater than twice the area of the principal peak in the chromatogram obtained
with solution (2) (2%).
1 g.
Assay Carry out the method for liquid chromatography, Appendix III D, using the following three
solutions in a mixture of 25 volumes of water and 75 volumes of acetonitrile containing (1) 0.03% w/v
of the substance being examined, (2) 0.03% w/v of diflucortolone valerate BPCRS and (3) 0.03% w/v
of diflucortolone valerate impurity standard BPCRS.
27
H
36
F
2
O
5
from the declared content of C
27
H
36
F
2
O
5
in diflucortolone
valerate BPCRS.
Storage Diflucortolone Valerate should be protected from light.
Preparations
Diflucortolone Cream
Diflucortolone Oily Cream
Diflucortolone Ointment
13-66
IMPURITIES
H
H
O
HO
O
OCO[CH
2
]
3
CH
3
Me
Me
Me
F
A. 6-fluoro-3,20-dioxo-11-hydroxy-16-methylpregna-1,4-dien-21-yl valerate
H
H
O
HO
O
OCO[CH
2
]
3
CH
3
Me
Me F
Me
F
B. 6,12-difluoro-3,20-dioxo-11-hydroxy-16-methylpregna-1,4-dien-21-yl valerate
H
H
O
HO
O
F
OCO[CH
2
]
3
CH
3
Me
Me
Me
F
C. 6,9-difluoro-3,20-dioxo-11-hydroxy-16-methylpregna-1,4-dien-21-yl valerate
13-67
Diflunisal
COOH
OH
F F
C
13
H
8
F
2
O
3
250.2 22494-42-4
Diflunisal complies with the requirements of the 3rd edition of the European Pharmacopoeia [0818]. These
Preparation
Diflunisal Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Diflunisal contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of
2,4-difluoro-4-hydroxybiphenyl-3-carboxylic acid, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water, soluble in alcohol and in
ether. It dissolves in dilute solutions of alkali hydroxides.
IDENTIFICATION
A. Dissolve 10 mg in a 0.3 per cent V/V solution of hydrochloric acid R in methanol R and dilute to
100.0 ml with the same acid solution. Dilute 2.0 ml of the solution to 10.0 ml with a 0.3 per cent
V/V solution of hydrochloric acid R in methanol R. Examined between 230 nm and 350 nm (2.2.25),
the solution shows two absorption maxima, at 251 nm and 315 nm. The ratio of the absorbance
measured at the maximum at 251 nm to that measured at the maximum at 315 nm is 4.2 to 4.6.
obtained with diflunisal CRS. Examine the substances prepared as discs. If the spectra obtained show
differences, dissolve the substance to be examined and the reference substance separately in alcohol R,
evaporate to dryness and record new spectra using the residues.
C. Dissolve about 2 mg in 10 ml of alcohol R and add 0.1 ml of ferric chloride solution R1. A violet-red
colour is produced.
TESTS
7
(Method II,
2.2.2).
Related substances
254
the same solvent.
Reference solution (a). Dissolve 30 mg of biphenyl-4-ol R in methanol R and dilute to 100 ml with the
same solvent. Dilute 1 ml of the solution to 10 ml with methanol R.
13-68
Reference solution (b). Dissolve 20 mg of biphenyl-4-ol R in methanol R, add 1 ml of the test solution
10 volumes of glacial acetic acid R, 20 volumes of acetone R and 70 volumes of methylene chloride R.
Dry the plate in a current of warm air and examine in ultraviolet light at 254 nm. Any spot in the
the spot in the chromatogram obtained with reference solution (a) (0.15 per cent). The test is not
valid unless the chromatogram obtained with reference solution (b) shows two clearly separated
principal spots.
Test solution. Dissolve 50.0 mg of the substance to be examined in the reference solution and dilute to
Reference solution. Dissolve 55.0 mg of fluoranthene R in a mixture of 1 volume of water R and 4
volumes of acetonitrile R and dilute to 100.0 ml with the same mixture of solvents. Dilute 1.0 ml to
100.0 ml with a mixture of 1 volume of water R and 4 volumes of acetonitrile R.
a stainless steel column 0.25 m long and 4 mm in internal diameter packed with octadecylsilyl silica
as mobile phase at a flow rate of 2 ml per minute a mixture of 2 volumes of glacial acetic acid R, 25
volumes of methanol R, 55 volumes of water R and 70 volumes of acetonitrile R,
Inject 20 l of the reference solution and adjust the sensitivity of the detector so that the height of
the principal peak in the chromatogram obtained is not less than 10 per cent of the full scale of the
recorder. Inject separately 20 l of the test solution and 20 l of the reference solution. Continue the
chromatography for three times the retention time of fluoranthene. In the chromatogram obtained
with the test solution, the sum of the areas of any peaks, apart from the principal peak, with a
retention time greater than that of fluoranthene is not greater than the area of the principal peak in
the chromatogram obtained with the reference solution (0.11 per cent). Disregard any peak with an
area less than 0.05 times that of the principal peak in the chromatogram obtained with the reference
solution.
Heavy metals (2.4.8). 2.0 g complies with limit test C for heavy metals (10 ppm). Use a platinum
crucible. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R.
ASSAY
Dissolve 0.200 g in 40 ml of methanol R. Add 5 ml of water R and 0.2 ml of phenol red solution R.
Titrate with 0.1M sodium hydroxide until the colour changes from yellow to reddish-violet.
13
H
8
F
2
O
3
.
STORAGE
IMPURITIES
A. biphenyl-4-ol,
B. 2,4-difluorobiphenyl-4-ol,
C. 2,4-difluorobiphenyl-4-yl acetate
D. condensation products.
__________________________________________________________________________________________________________ Ph Eur
14-1
Digitalis Leaf
Digitalis
Digitalis Leaf complies with the requirements of the 3rd edition of the European Pharmacopoeia [0117]. These
When Powdered Digitalis is prescribed or demanded, material complying with the requirements
supplied.
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Digitalis leaf consists of the dried leaf of Digitalis purpurea L. It contains not less than 0.3 per cent of
cardenolic glycosides, expressed as digitoxin (M
r
765), and calculated with reference to the drug dried
at 100C to 105C.
CHARACTERS
Digitalis leaf has a faint but characteristic odour. The whole leaf is about 10 cm to 40 cm long and
4 cm to 15 cm wide. The lamina is ovate lanceolate to broadly ovate. The winged petiole is from one
quarter as long as to equal in length to the lamina.
IDENTIFICATION
A. The leaf is brittle and often occurs broken. The upper surface is green and the lower surface is
greyish-green. The apex is subacute and the margin is irregularly crenate, dentate or serrate. The base
is decurrent. The venation is pinnate, the lateral veins being prominent especially on the lower
surface, leaving the midrib at about 45 and anastomosing near the margin; a veinlet terminates in
each tooth of the margin and the lower veins run down the winged petiole. The upper surface is
rugose and pubescent; the lower surface shows a network of raised veinlets and is densely pubescent.
B. Reduce to a powder (355). Examine under a microscope using chloral hydrate solution R. The
powder shows the following diagnostic characters: epidermal cells with anticlinal walls which are
straight or slightly sinuous on the upper surface and markedly sinuous on the lower surface; the
cuticle is smooth. Trichomes are of two types: uniseriate, bluntly pointed non-glandular, usually of
three to five cells, often with one or more collapsed cells, walls mostly finely warty or faintly striated;
glandular trichomes usually with a unicellular, sometimes a multicellular uniseriate stalk and a
unicellular or bicellular or exceptionally tetracellular head. Anomocytic stomata (2.8.3) are absent or
very rare on the upper surface, numerous on the lower surface. Calcium oxalate crystals and
sclerenchyma are absent.
Test solution. To 1.0 g of the powdered drug (180) add a mixture of 20 ml of alcohol (50 per cent
V/V) R and 10 ml of lead acetate solution R. Boil for 2 min, allow to cool and centrifuge. Shake the
supernatant solution with two quantities, each of 15 ml, of chloroform R; separate the two layers by
centrifugation if necessary. Dry the chloroform layers over anhydrous sodium sulphate R and filter.
Evaporate 10 ml of the solution to dryness on a water-bath and dissolve the residue in 1 ml of a
mixture of equal volumes of chloroform R and methanol R.
Reference solution. Dissolve 5 mg of purpureaglycoside A CRS, 2 mg of purpureaglycoside B CRS, 5 mg of
digitoxin R and 2 mg of gitoxin R in a mixture of equal volumes of chloroform R and methanol R and
Apply separately to the plate as bands 2 cm by 0.3 cm 20 l of each solution. Develop over a path of
10 cm using a mixture of 7.5 volumes of water R, 10 volumes of methanol R and 75 volumes of ethyl
acetate R. Allow the solvents to evaporate. Spray with a mixture of 2 volumes of a 10 g/l solution of
chloramine R and 8 volumes of a 250 g/l solution of trichloroacetic acid R in alcohol R. Heat at 100C to
105C for 10 min. Examine in ultraviolet light at 365 nm. The chromatogram obtained with the
reference solution shows a zone of light-blue fluorescence in the lower part of the chromatogram,
corresponding to purpureaglycoside B, and just above it a zone of brownish-yellow fluorescence,
corresponding to purpureaglycoside A. A zone of light-blue fluorescence, corresponding to gitoxin,
appears in the middle of the chromatogram and above it a zone of brownish-yellow fluorescence,
corresponding to digitoxin. The zones in the chromatogram obtained with the test solution are similar
in position, colour and size to the zones in the chromatogram obtained with the reference solution.
Other zones of fluorescence may also appear in the chromatogram obtained with the test solution.
D. Evaporate 5 ml of the chloroformic solution obtained in identification test C to dryness on a
14-2
water-bath. To the residue add 2 ml of dinitrobenzoic acid solution R and 1 ml of 1M sodium hydroxide.
A reddish-violet colour develops within 5 min.
E. Evaporate 5 ml of the chloroformic solution obtained in identification test C to dryness on a
water-bath. To the residue add 3 ml of xanthydrol solution R and heat on a water-bath for 3 min. A
red colour develops.
TESTS
Foreign matter (2.8.2). There are no leaves with few or no trichomes and epidermal cells showing,
in surface view, beaded anticlinal walls (Digitalis lanata).
ASSAY
Shake 0.250 g of the powdered drug (180) with 50.0 ml of water R for 1 h. Add 5.0 ml of a 150 g/l
solution of lead acetate R, shake and after a few minutes add 7.5 ml of a 40 g/l solution of disodium
hydrogen phosphate R. Filter through a pleated filter paper. Heat 50.0 ml of the filtrate with 5 ml of
hydrochloric acid (150 g/l HCl) under a reflux condenser on a water-bath for 1 h. Transfer to a
separating funnel, rinse the flask with two quantities, each of 5 ml, of water R and shake with three
quantities, each of 25 ml, of chloroform R. Dry the combined chloroform layers over anhydrous sodium
sulphate R and dilute to 100.0 ml with chloroform R. Evaporate 40.0 ml of the chloroformic solution
to dryness, dissolve the residue in 7 ml of alcohol (50 per cent V/V) R and add 2 ml of dinitrobenzoic
acid solution R and 1 ml of 1M sodium hydroxide. At the same time prepare a reference solution as
follows. Dissolve 50.0 mg of digitoxin CRS in alcohol R and dilute to 50.0 ml with the same solvent.
Dilute 5.0 ml of the solution to 50.0 ml with alcohol R. To 5.0 ml of the resulting solution add 25 ml
of water R and 3 ml of hydrochloric acid (150 g/l HCl). Heat the solution under a reflux condenser
on a water-bath for 1 h and complete the preparation as described above. Measure the absorbance of
the two solutions (2.2.25) at 540 nm several times during the first 12 min until the maximum is
reached, using as the compensation liquid a mixture of 7 ml of alcohol (50 per cent V/V) R, 2 ml of
dinitrobenzoic acid solution R and 1 ml of 1M sodium hydroxide.
From the absorbances measured and the concentrations of the solutions, calculate the content of
cardenolic glycosides, expressed as digitoxin.
STORAGE
___________________________________________________________________________________________________ Ph Eur
14-3
Digitoxin
1/01
2
O
Me
OH
O
O
R
O
Me
OH
HO
R=
OH H
H
H
H
H Me
Me
O
O
C
41
H
64
O
13
765 71-63-6
Digitoxin complies with the requirements of the 3rd edition of the European Pharmacopoeia [0078]. These
Preparation
Digitoxin Tablets
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Digitoxin contains not less than 95.0 per cent and not more than the equivalent of 103.0 per cent of
3-[(O-2,6-dideoxy--D-ribo-hexopyranosyl-(14)-O-2,6-dideoxy--D-ribo-hexopyranosyl-(14)-
2,6-dideoxy--D-ribo-hexopyranosyl)oxy]-14-hydroxy-5,14-card-20(22)-enolide, calculated with
CHARACTERS
A white or almost white powder, practically insoluble in water, freely soluble in a mixture of equal
volumes of methanol and methylene chloride, slightly soluble in alcohol and in methanol.
IDENTIFICATION
obtained with digitoxin CRS.
D. Dissolve about 0.5 mg in 1 ml of glacial acetic acid R, heating gently, allow to cool and add
0.05 ml of ferric chloride solution R1. Cautiously add 1 ml of sulphuric acid R, avoiding mixing the two
liquids. A brown ring develops at the interface and on standing a green, then blue colour passes to
the upper layer.
TESTS
Appearance of solution Dissolve 50 mg in a mixture of equal volumes of methanol R and methylene
chloride R and dilute to 10 ml with the same mixture of solvents. The solution is clear (2.2.1) and
Specific optical rotation (2.2.7). Dissolve 0.25 g in chloroform R and dilute to 10.0 ml with the
same solvent. The specific optical rotation is +16.0 to +18.5.
14-4
Reference solution (a). Dissolve 20 mg of digitoxin CRS in a mixture of equal volumes of methanol R
and methylene chloride R and dilute to 2 ml with the same mixture of solvents.
Reference solution (b). Dilute 0.5 ml of reference solution (a) to 50 ml with a mixture of equal volumes
of methanol R and methylene chloride R.
Reference solution (c). Dissolve 10 mg of gitoxin CRS with stirring in a mixture of equal volumes of
Reference solution (d). Dilute 1 ml of reference solution (b) to 2 ml with a mixture of equal volumes of
methanol R and methylene chloride R.
Reference solution (e). Mix 1 ml of reference solution (a) and 1 ml of reference solution (c).
Apply to the plate 5 l of each solution. Develop immediately over a path of 15 cm using a mixture of
15 volumes of methanol R, 40 volumes of cyclohexane R and 90 volumes of methylene chloride R. Dry
the plate in a stream of cold air for 5 min. Repeat the development and dry the plate in a stream of
cold air for 5 min. Spray with a mixture of 1 volume of sulphuric acid R and 9 volumes of alcohol R
and heat at 130C for 15 min. Examine in daylight.
Gitoxin. Any spot corresponding to gitoxin in the chromatogram obtained with the test solution is not
more intense than the spot in the chromatogram obtained with reference solution (c) (2.0 per cent).
Other glycosides. Any spot in the chromatogram obtained with the test solution, apart from the
principal spot and the spot corresponding to gitoxin, is not more intense than the spot in the chroma-
togram obtained with reference solution (b) (1.0 per cent).
The test is not valid unless the chromatogram obtained with reference solution (e) shows clearly
separated spots corresponding to digitoxin, gitoxin and other glycosides and the spot in the chroma-
togram obtained with reference solution (d) is clearly visible.
100C to 105C for 2 h.
loss on drying.
ASSAY
solution to 100.0 ml with alcohol R. Prepare a reference solution in the same manner, using 40.0 mg
of digitoxin CRS. To 5.0 ml of each solution add 3.0 ml of alkaline sodium picrate solution R, allow to
stand protected from bright light for 30 min and measure the absorbance (2.2.25) of each solution at
the maximum at 495 nm, using as the compensation liquid a mixture of 5.0 ml of alcohol R and
3.0 ml of alkaline sodium picrate solution R prepared at the same time.
41
H
64
O
13
from the absorbances measured and the concentrations of the
solutions.
STORAGE
___________________________________________________________________________________________________ Ph Eur
14-5
Digoxin
2
O
Me
OH
O
O
R
O
Me
OH
HO
R=
OH H
H
H
H
H Me
Me
O
O
HO
H
C
41
H
64
O
14
781 20830-75-5
Digoxin complies with the requirements of the 3rd edition of the European Pharmacopoeia [0079]. These
Preparations
Digoxin Injection
Paediatric Digoxin Injection
Paediatric Digoxin Oral Solution
Digoxin Tablets
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Digoxin contains not less than 95.0 per cent and not more than the equivalent of 103.0 per cent of
3-[(O-2,6-dideoxy--D-ribo-hexopyranosyl-(14)-O-2,6-dideoxy--D-ribo-hexopyranosyl-(14)-2,6-
dideoxy--D-ribo-hexopyranosyl)oxy]-12b,14-dihydroxy-5,14-card-20(22)-enolide, calculated with
CHARACTERS
A white or almost white powder or colourless crystals, practically insoluble in water, freely soluble in
a mixture of equal volumes of methanol and methylene chloride, slightly soluble in alcohol.
IDENTIFICATION
obtained with digoxin CRS.
D. Dissolve about 0.5 mg in 1 ml of glacial acetic acid R, heating gently, allow to cool and add
0.05 ml of ferric chloride solution R1. Cautiously add 1 ml of sulphuric acid R avoiding mixing the two
liquids. A brown ring develops at the interface and on standing a green, then blue colour passes to
the upper layer.
TESTS
Appearance of solution Dissolve 50 mg in a mixture of equal volumes of methanol R and methylene
chloride R and dilute to 10 ml with the same mixture of solvents. The solution is clear (2.2.1) and
Specific optical rotation (2.2.7). Dissolve 0.20 g in anhydrous pyridine R and dilute to 10.0 ml with
the same solvent. The specific optical rotation is +10.0 to +13.0, calculated with reference to the
dried substance.
14-6
Related substances Examine by thin-layer chromatography (2.2.27), using kieselguhr G R as the
coating substance. Impregnate the plate by placing it in a closed chromatographic tank containing the
necessary quantity of a mixture of 10 volumes of formamide R and 90 volumes of acetone R so that the
plate dips about 5 mm into the liquid. When the impregnation mixture has risen at least 15 cm from
the lower edge of the plate, remove the plate and allow the solvent to evaporate for 30 min. Use the
plate immediately.
Reference solution (a). Dissolve 20 mg of digoxin CRS in a mixture of equal volumes of methanol R and
methylene chloride R and dilute to 2 ml with the same mixture of solvents.
Reference solution (b). Dilute 1 ml of reference solution (a) to 50 ml with a mixture of equal volumes
Reference solution (c). Dilute 2 ml of reference solution (b) to 4 ml with a mixture of equal volumes of
Reference solution (d). Dissolve 5 mg of digitoxin CRS in a mixture of equal volumes of methanol R and
Reference solution (e). Dissolve 5 mg of gitoxin CRS in a mixture of equal volumes of methanol R and
volumes of formamide R, 50 volumes of methyl ethyl ketone R and 50 volumes of xylene R. Dry the
plate in a current of cold air until only the lower edge is still visibly moist. Repeat the development
and dry the plate at 115C for 20 min. Allow to cool, spray with a mixture of 1 volume of a freshly
prepared 30 g/l solution of chloramine R and 15 volumes of a 250 g/l solution of trichloroacetic acid R
in alcohol R and heat at 115C for 5 min. Examine in ultraviolet light at 365 nm. Any spot corres-
ponding to digitoxin in the chromatogram obtained with the test solution is not more intense than
the spot in the chromatogram obtained with reference solution (d) (1.0 per cent). Any spot corres-
ponding to gitoxin in the chromatogram obtained with the test solution is not more intense than the
spot in the chromatogram obtained with reference solution (e) (2.0 per cent). Any spot in the
chromatogram obtained with the test solution, apart from the principal spot and spots corresponding
to digitoxin and gitoxin, is not more intense than the spot in the chromatogram obtained with
reference solution (b) (2.0 per cent) and at most one such spot is more intense than the spot in the
loss on drying.
ASSAY
Dissolve 40.0 mg in alcohol R, heating if necessary, and dilute to 50.0 ml with the same solvent.
Dilute 5.0 ml of the solution to 100.0 ml with alcohol R. Prepare a reference solution in the same
manner, using 40.0 mg of digoxin CRS. To 5.0 ml of each solution add 3.0 ml of alkaline sodium
picrate solution R, allow to stand protected from bright light for 30 min and measure the absorbance
(2.2.25) of each solution at the maximum at 495 nm, using as the compensation liquid a mixture of
5.0 ml of alcohol R and 3.0 ml of alkaline sodium picrate solution R prepared at the same time.
41
H
64
O
14
solutions.
STORAGE
IMPURITIES
A. digitoxin,
B. gitoxin.
___________________________________________________________________________________________________ Ph Eur
14-7
Hydrated Dihydralazine Sulphate
N
N
HN
NH
2
HN
NH
2
, H
2
SO
4
C
8
H
10
N
6
,H
2
SO
4,
2H
2
O 333.3 7327-87-9 (anhydrous)
Hydrated Dihydralazine Sulphate complies with the requirements of the 3rd edition of the European
Pharmacopoeia for Dihydralazine Sulphate (Hydrated) [1310]. These requirements are reproduced after the
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Dihydralazine sulphate (hydrated) contains not less than 98.0 per cent and not more than the
equivalent of 102.0 per cent of (phthalazine-1,4-diyl)dihydrazine sulphate, calculated with reference
CHARACTERS
A white or slightly yellow, crystalline powder, slightly soluble in water, practically insoluble in
ethanol. It dissolves in dilute mineral acids.
IDENTIFICATION
spectrum of dihydralazine sulphate hydrated.
B. Dissolve about 50 mg in 5 ml of dilute hydrochloric acid R. The solution gives reaction (a) of
sulphates (2.3.1).
TESTS
Appearance of solution Dissolve 0.20 g in dilute nitric acid R and dilute to 10 ml with the same
6
(Method II, 2.2.2).
Prepare the soutions immediately before use.
Test solution. Dissolve 50.0 mg of the substance to be examined in a 6 g/l solution of glacial acetic
acid R and dilute to 50.0 ml with the same acidic solution.
Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase containing
0.5 g/l of sodium edetate R. Dilute 1.0 ml of the solution to 10.0 ml with the mobile phase containing
0.5 g/l of sodium edetate R.
Reference solution (b). Dissolve 10.0 mg of dihydralazine impurity A CRS in the mobile phase contain-
ing 0.5 g/l of sodium edetate R and dilute to 50.0 ml with the same solution. Dilute 2.0 ml of the
solution to 20.0 ml with the mobile phase containing 0.5 g/l of sodium edetate R.
Reference solution (c). Dilute 2 ml of the test solution to 10 ml with the mobile phase containing
0.5 g/l of sodium edetate R. Dilute 1 ml of the solution to 10 ml with reference solution (b).
a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with nitrile silica gel
for chromatography R (5 m - 10 m),
as mobile phase at a flow rate of 1.5 ml/min a mixture prepared as follows: to 22 volumes of
acetonitrile R add 78 volumes of a solution containing 1.44 g/l of sodium lauryl sulphate R and
0.75 g/l of tetrabutylammonium bromide R and adjust to pH 3.0 with 0.05M sulphuric acid,
two peaks in the chromatogram is at least 50 per cent of the full scale of the recorder. When the
chromatogram is recorded in the prescribed conditions, the retention time of impurity A relative to
dihydralazine is about 0.8. The test is not valid unless: in the chromatogram obtained, the resolution
14-8
between the peaks corresponding to impurity A and dihydralazine is at least 2.0. Inject 20 l of the
test solution and continue the chromatography for twice the retention time of dihydralazine. Inject
20 l of reference solution (a) and of reference solution (b). In the chromatogram obtained with the
test solution: the area of any peak corresponding to impurity A is not greater than that of the peak
due to impurity A in the chromatogram obtained with reference solution (b) (2 per cent); the area of
any peak, apart from the principal peak and the peak ue to impurity A, is not greater than the area of
the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent); the sum
of the areas of such peaks is not greater than five times the area of the principal peak in the chromato-
0.1 times that of the principal peak in the chromatogram obtained with reference solution (a).
Hydrazine. Examine by liquid chromatography (2.2.29).
Test solution. Dissolve 40.0 mg of hydrazine sulphate R in water R and dilute to 100.0 ml with the same
solvent. Dilute 1.0 ml of the solution to 25.0 ml with water R. To 0.50 ml of the solution, add
0.200 g of the substance to be examined and dissolve in 6 ml of dilute hydrochloric acid R, then dilute
to 10.0 ml with water R. In a centrifuge tube with a ground-glass stopper, place immediately 0.50 ml
of the solution and 2.0 ml of a 60 g/l solution of benzaldehyde R in a mixture of equal volumes of
methanol R and water R. Shake for 90 s. Add 1.0 ml of water R and 5.0 ml of heptane R. Shake for
1 min and centrifuge. Use the upper layer.
Reference solution. Dissolve 40.0 mg of hydrazine sulphate R in water R and dilute to 100.0 ml with the
same solvent. Dilute 1.0 ml of the solution to 25.0 ml with water R. To 0.50 ml of the solution, add
6 ml of dilute hydrochloric acid R and dilute to 10.0 ml with water R. In a centrifuge tube with a
ground-glass stopper, place 0.50 ml of the solution and 2.0 ml of a 60 g/l solution of benzaldehyde R
in a mixture of equal volumes of methanol R and water R. Shake for 90 s. Add 1.0 ml of water R and
5.0 ml of heptane R. Shake for 1 min and centrifuge. Use the upper layer.
Blank solution. Prepare in the same manner as for the reference solution but replacing the 0.50 ml of
hydrazine sulphate solution by 0.50 ml of water R.
as mobile phase at a flow rate of 1 ml/min a mixture of 30 volumes of a 0.3 g/l solution of sodium
edetate R and 70 volumes of acetonitrile R,
Inject 20 l each of the test solution, the reference solution and the blank solution. Comparing the
chromatograms obtained with the reference solution and the blank solution, identify the peak of
benzaldehyde azine (benzalazine) corresponding to hydrazine having a retention time relative to the
principal peak (benzaldehyde) of about 1.8. In the chromatogram obtained with the test solution, the
area of the peak corresponding to benzaldehyde azine is not greater than twice that of the corres-
ponding peak in the chromatogram obtained with the reference solution (10 ppm of hydrazine).
Iron (2.4.9). To the residue obtained in the test for sulphated ash add 0.2 ml of sulphuric acid R and
heat carefully until the acid is almost completely eliminated. Allow to cool and dissolve the residue
with heating in 5.5 ml of hydrochloric acid R1. Filter the hot solution through a filter previously
washed three times with dilute hydrochloric acid R. Wash the crucible and the filter with 5 ml of
water R. Combine the filtrate and the washings and neutralise with about 3.5 ml of strong sodium
hydroxide solution R. Adjust the pH to between 3 and 4 with acetic acid R and dilute to 20 ml with
water R. The solution complies with the limit test for iron (20 ppm). Prepare the standard with 5 ml
of iron standard solution (2 ppm Fe) R and 5 ml of water R.
at 50C at a pressure not exceeding 0.7 kPa for 5 h.
ASSAY
Dissolve 60.0 mg in 25 ml of water R. Add 35 ml of hydrochloric acid R and titrate slowly with 0.05M
potassium iodate, determining the end-point potentiometrically (2.2.20), using a calomel reference
electrode and a platinum indicator electrode.
8
H
12
N
6
O
4
S.
14-9
IMPURITIES
N
N
NH
2
HN
NH
2
A. 4-hydrazinophthalzin-1-amine,
H
2
N
NH
2
B. hydrazine,
N
N
HN
NH
2
C. (phthalazin-1-yl)hydrazine (hydralazine).
___________________________________________________________________________________________________ Ph Eur
14-10
Dihydrocodeine Tartrate
O
N
H
H
H
MeO
HO
Me
HOOC
COOH
OH H
OH H
,
C
18
H
23
NO
3
,C
4
H
6
O
6
451.5 5965-13-9
Definition Dihydrocodeine Tartrate is 4,5-epoxy-3-methoxy-17-methylmorphinan-6-ol hydrogen
(2R,3R)-tartrate. It contains not less than 98.0% and not more than 101.0% of C
18
H
23
NO
3
,C
4
H
6
O
6
,
Characteristics Colourless crystals or a white, crystalline powder; odourless or almost odourless.
Freely soluble in water; sparingly soluble in ethanol (96%); practically insoluble in ether.
Identification
A. The light absorption, Appendix II B, in the range 230 to 350 nm of a 0.02% w/v solution exhibits a
maximum only at 284 nm. The absorbance at the maximum at 284 nm is about 0.72.
B. Add 10 mg, in powder, to a mixture of 1 ml of sulphuric acid and 0.05 ml of formaldehyde solution. A
purple colour is produced (distinction from pholcodine).
C. Dissolve 10 mg in 0.05 ml of nitric acid. A yellow but no red colour is produced (distinction from
morphine).
D. Dissolve 0.1 g in 1 ml of sulphuric acid, add 0.05 ml of iron(III) chloride solution R1 and warm
gently. A brownish yellow colour is produced which does not become red on the addition of 0.05 ml
of 2M nitric acid (distinction from codeine and morphine).
E. Yields reaction B characteristic of tartrates, Appendix VI.
Colour of solution A 10% w/v solution is not more intensely coloured than reference solution BY
5
,
Specific optical rotation In a 5% w/v solution, 71.5 to 73.5, calculated with reference to the
dried substance, Appendix V F.
Morphine and non-hydrogenated morphine-type substances To 0.1 g add 5 ml of sulphuric acid
(80%) and 0.05 ml of iron(III) chloride solution R1 and heat on a water bath for 2 minutes. Any blue or
green colour produced is not more intense than that produced when a mixture of 0.5 mg of codeine
phosphate and 30 mg of (+)-tartaric acid is treated in the same manner, beginning at the words add
5 ml .
Other foreign alkaloids Carry out the method for thin-layer chromatography, Appendix III A, using
silica gel GF
254
as the coating substance and a mixture of 90 volumes of dichloromethane, 10 volumes of
methanol and 1 volume of 13.5M ammonia as the mobile phase. Apply separately to the plate 10 l of
each of three solutions of the substance being examined in water containing (1) 2.0% w/v, (2) 0.020%
w/v and (3) 0.010% w/v. After removal of the plate, allow it to dry in air and spray with dilute
potassium iodobismuthate solution. Any secondary spot in the chromatogram obtained with solution (1) is
not more intense than the spot in the chromatogram obtained with solution (2) and not more than
one such spot is more intense than the spot in the chromatogram obtained with solution (3).
1 g.
Assay Carry out Method I for non-aqueous titration, Appendix VIII A, using 0.5 g and crystal violet
solution as indicator. Each ml of 0.1M perchloric acid VS is equivalent to 45.15 mg of
C
18
H
23
NO
3
,C
4
H
6
O
6
.
Storage Dihydrocodeine Tartrate should be protected from light.
Preparations
Co-dydramol Tablets
Dihydrocodeine Injection
Dihydrocodeine Oral Solution
Dihydrocodeine Tablets
14-11
Dihydroergocristine Mesilate
corrected 1/01
N
N
O
O
H
O
OH
H
N
N
H
H
Me
H
N
O
Pr
i
H
SO
3
H
CH
3
,
C
36
H
45
N
5
O
8
S 708 24730-10-7
Dihydroergocristine Mesilate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Component of Co-dergocrine Mesilate; vasodilator.
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Dihydroergocristine mesilate contains not less than 98.0 per cent and not more than the equivalent
of 102.0 per cent of (6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-5-benzyl-10b-hydroxy-2-(1-
methylethyl)-3,6-dioxo-octahydro-8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-
4,6,6a,7,8,9,10,10a-octahydroindolo[4,3-fg]quinoline-9-carboxamide methanesulphonate,
CHARACTERS
A white or almost white, fine crystalline powder, slightly soluble in water, soluble in methanol.
IDENTIFICATION
obtained with dihydroergocristine mesilate CRS. Examine the substances prepared as discs.
254
plate R.
Test solution. Dissolve 0.10 g of the substance to be examined in a mixture of 1 volume of methanol R
Reference solution. Dissolve 0.10 g of dihydroergocristine mesilate CRS in a mixture of 1 volume of
solvents.
Apply immediately to the plate 5 l of each solution. Develop protected from light over a path of
15 cm using a mixture of 2 volumes of concentrated ammonia R, 15 volumes of dimethylformamide R
and 85 volumes of ether R. Dry the plate in a current of cold air for 5 min. Spray the plate with
dimethylaminobenzaldehyde solution R7 and dry in a current of hot air for 2 min. The principal spot in
the chromatogram obtained with the test solution is similar in position, colour and size to the
254
plate R.
Test solution. Dissolve 0.20 g of the substance to be examined in a mixture of 1 volume of methanol R
Reference solution. Dissolve 0.20 g of methanesulphonic acid R in a mixture of 1 volume of methanol R
Apply immediately to the plate 10 l of each solution. Develop protected from light over a path of
10 cm using a mixture of 5 volumes of water R, 10 volumes of concentrated ammonia R, 20 volumes of
butanol R and 65 volumes of acetone R. Dry the plate in a current of cold air for not more than 1 min.
Spray the plate with a 1 g/l solution of bromocresol purple R in methanol R, adjusting the colour to
violet-red with one drop of dilute ammonia R1 and dry the plate in a current of hot air at 100C. The
TESTS
14-12
The solution is clear (2.2.1) and not more intensely coloured than reference solution B
7
(Method II,
2.2.2).
with the same solvent. The specific optical rotation is 37 to 43, calculated with reference to the
dried substance.
Carry out the test and preparation of the solutions protected from bright light.
Test solution. Dissolve 75.0 mg of the substance to be examined in a mixture of 40 volumes of water R
and 60 volumes of acetonitrile R and dilute to 50.0 ml with the same mixture of solvents.
Reference solution. Dissolve 20.0 mg of codergocrine mesilate CRS in a mixture of 40 volumes of water R
and 60 volumes of acetonitrile R and dilute to 50.0 ml with the same mixture of solvents. Dilute
6.0 ml of the solution to 50.0 ml with a mixture of 40 volumes of water R and 60 volumes of aceto-
nitrile R.
as mobile phase at a flow rate of 1.5 ml/min a mixture of 2.2 volumes of diethylamine R, 400
volumes of acetonitrile R and 650 volumes of a 1.1 g/l solution of ammonium carbonate R,
Inject 10 l of the test solution and 10 l of the reference solution. Continue the chromatography
of the test solution for twice the retention time of the principal peak. When the chromatograms are
recorded in the prescribed conditions the elution order of the peaks is the following: impurity F,
impurity H, impurity I and dihydroergocristine.
The test is not valid unless: the chromatogram obtained with the reference solution exhibits four
clearly separated peaks; the symmetry factor of the peak corresponding to dihydroergocristine is not
greater than 2.5; the resolution between the peaks due to impurity I and dihydroergocristine is at
least 1.5.
In the chromatogram obtained with the test solution, the area of any peak apart from the principal
peak is not greater than the area of the peak corresponding to dihydroergocristine in the chromato-
gram obtained with the reference solution (1 per cent). The sum of the areas of all the peaks, apart
from the principal peak, is not greater than twice the area of the peak corresponding to dihydroergo-
cristine in the chromatogram obtained with the reference solution (2 per cent). Disregard any peak
with an area less than 0.1 times the area of the peak corresponding to dihydroergocristine in the
Loss on drying (2.2.32). Not more than 3.0 per cent, determined on 0.500 g by drying under high
vacuum at 80C.
ASSAY
Dissolve 0.300 g in 60 ml of pyridine R. Blow a stream of nitrogen R over the surface of the solution
and titrate with 0.1M tetrabutylammonium hydroxide determining the end-point potentiometrically
(2.2.20). Evaluate the volume of titrant required to the second point of inflexion.
36
H
45
N
5
O
8
S.
STORAGE
IMPURITIES
N
N
H
H
Me
H
CONH
2
A. (6aR,9R,10aR)-7-methyl-4,6,6a,7,8,9,10,10a-octahydroindolo[4,3-fg]quinoline-9-
carboxamide (6-methylergoline-8-carboxamide),
N
N
H
H
Me
H
CONH
2
B. (6aR,9S,10aS)-7-methyl-4,6,6a,7,8,9,10,10a-octahydroindolo[4,3-fg]quinoline-9-
carboxamide (6-methylisoergoline-8-carboxamide),
14-13
N
N
O
O
H
O
OH
H
N
HN
H
H
Me
H
N
O
Pr
i
H
C. (6aR,9R,10aR)-N-[(2S,5S,10aS,10bS)-5-benzyl-10b-hydroxy-2-(1-methylethyl)-3,6-dioxo-
octahydro-8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-4,6,6a,7,8,9,10,10a-
octahydroindolo[4,3-fg]quinoline-9-carboxamide (2-epidihydroergocristine),
N
N
O
R
1
O
H
O
OH
H
N
HN
H
H
Me
H
N
O
R
2
H
D. R
1
= CH(CH
3
)
2
, R
2
= CH
3
: (6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-10b-hydroxy-2-
methyl-5-(1-methylethyl)-3,6-dioxo-octahydro-8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-
yl]-7-methyl-4,6,6a,7,8,9,10,10a-octahydroindolo[4,3-fg]quinoline-9-carboxamide
(dihydroergosine),
E. R
1
= CH
2
-C
6
H
5
, R
2
= CH
3
: (6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-5-benzyl-10b-hydroxy-
2-methyl-3,6-dioxooctahydro-8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-
4,6,6a,7,8,9,10,10a-octahydroindolo[4,3-fg]quinoline-9-carboxamide (dihydroergotamine),
F. R
1
= R
2
= CH(CH
3
)
2
: (6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-10b-hydroxy-2,5-bis(1-
methylethyl)-3,6-dioxooctahydro-8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-
4,6,6a,7,8,9,10,10a-octahydroindolo[4,3-fg]quinoline-9-carboxamide (dihydroergocornine),
G. R
1
= CH
2
-C
6
H
5
, R
2
= CH
2
-CH
3
: (6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-5-benzyl-2-ethyl-
10b-hydroxy-3,6-dioxo-octahydro-8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-
4,6,6a,7,8,9,10,10a-octahydroindolo[4,3-fg]quinoline-9-carboxamide (dihydroergostine),
H. R
1
= CH
2
-CH(CH
3
)
2
, R
2
= CH(CH
3
)
2
: (6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-10b-
hydroxy-2-(1-methylethyl)-5-(2-methylpropyl)-3,6-dioxooctahydro-8H-oxazolo[3,2-
a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-4,6,6a,7,8,9,10,10a-octahydroindolo[4,3-
fg]quinoline-9-carboxamide (-dihydroergocryptine),
I. R
1
= CH(CH
3
)-CH
2
-CH
3
, R
2
= CH(CH
3
)
2
: (6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-10b-
hydroxy-2-(1-methylethyl)-5-[(1RS)-1-methylpropyl]-3,6-dioxo-octahydro-8H-oxazolo[3,2-
fg]quinoline-9-carboxamide (-dihydroergocryptine or epicriptine),
J. R
1
= CH
2
-C
6
H
5
, R
2
= CH(CH
3
)-CH
2
-CH
3
: (6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-5-
benzyl-10b-hydroxy-2-[(1RS)-1-methylpropyl]-3,6-dioxo-octahydro-8H-oxazolo[3,2-
fg]quinoline-9-carboxamide (dihydroergosedmine),
N
N
O
O
H
O
OH
H
N
N
H
Me
H
N
O
Pr
i
H
K. (6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-5-benzyl-10b-hydroxy-2-(1-methylethyl)-3,6-
dioxo-octahydro-8H-oxazolo-[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-4,6,6a,7,8,9-
hexahydroindolo[4,3-fg]quinoline-9-carboxamide (ergocristine),
N
N
O
O
H
O
OH
H
N
N
H
Me
H
N
O
Pr
i
H
H
O
L. (6aR,7RS,9R,10aR)-N-[(2R,5S,10aS,10bS)-5-benzyl-10b-hydroxy-2-(1-methylethyl)-3,6-
dioxo-octahydro-8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-
4,6,6a,7,8,9,10,10a-octahydroindolo[4,3-fg]quinoline-9-carboxamide 7-oxide
(dihydroergocristine 6-oxide).
___________________________________________________________________________________________________ Ph Eur
14-14
Dihydroergotamine Mesilate
NMe
HN
H
H NH
O
OH
O
O
H
H CH
2
Ph
O
N
N
Me
H .CH
3
SO
3
H
C
33
H
37
N
5
O
5
,CH
4
O
3
S 679.8 6190-39-2
Dihydroergotamine Mesilate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Used in treatment of migraine.
When dihydroergotamine mesylate is prescribed or demanded, Dihydroergotamine Mesilate shall
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Dihydroergotamine mesilate contains not less than 97.0 per cent and not more than the equivalent
of 103.0 per cent of (6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-5-benzyl-10b-hydroxy-2-methyl-3,6-
dioxo-octahydro-8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-4,6,6a,7,8,9,10,10a-
octahydro-indolo[4,3-fg]quinoline-9-carboxamide methanesulphonate, calculated with reference
CHARACTERS
A white or almost white, crystalline powder or colourless crystals, slightly soluble in water and in
alcohol, sparingly soluble in methanol.
IDENTIFICATION
A. Dissolve 5.0 mg in methanol R and dilute to 100.0 ml with the same solvent. Examined between
250 nm and 350 nm (2.2.25), the solution shows two absorption maxima, at 281 nm and 291 nm,
and a shoulder at 275 nm. Above 320 nm the absorbance is negligible. The specific absorbance at the
obtained with dihydroergotamine mesilate CRS. Examine the substances prepared as discs.
D. To 0.1 g add 5 ml of dilute hydrochloric acid R and shake for about 5 min. Filter and add 1 ml of
barium chloride solution R1. The filtrate remains clear. Mix 0.1 g with 0.4 g of powdered sodium
hydroxide R, heat to fusion and continue to heat for 1 min. Cool, add 5 ml of water R, boil and filter.
Acidify the filtrate by the addition of hydrochloric acid R1 and filter again. The filtrate gives reaction
(a) of sulphates (2.3.1).
TESTS
Appearance of solution Dissolve 0.10 g in a mixture of 0.1 ml of a 70 g/l solution of methane-
sulphonic acid R and 50 ml of water R. The solution is clear (2.2.1) and not more intensely coloured
7
or BY
7
(Method II, 2.2.2).
dried substance.
coating substance.
14-15
Prepare the reference solutions and the test solutions immediately before use and in the order indicated below..
Reference solution (a). Dissolve 20 mg of dihydroergotamine mesilate CRS in a mixture of 1 volume of
methanol R and 9 volumes of chloroform R and dilute to 10 ml with the same mixture of solvents.
Reference solution (b). Dilute 2.5 ml of reference solution (a) to 50 ml with a mixture of 1 volume of
Reference solution (c). Dilute 2 ml of reference solution (b) to 5 ml with a mixture of 1 volume of
Test solution (a). Dissolve 0.10 g of the substance to be examined in a mixture of 1 volume of
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with a mixture of 1 volume of methanol R
and 9 volumes of chloroform R.
Apply separately to the plate 5 l of each solution. Develop protected from light over a path of 15 cm
using a mixture of 1 volume of concentrated ammonia R, 6 volumes of methanol R, 50 volumes of ethyl
acetate R and 50 volumes of methylene chloride R. Dry the plate in a current of cold air for not longer
than 1 min. Repeat the development protected from light over a path of 15 cm using a freshly
prepared amount of the same mobile phase. Dry the plate in a current of cold air. Spray the plate
abundantly with dimethylaminobenzaldehyde solution R7 and dry in a current of warm air for about
2 min. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is
not more intense than the principal spot in the chromatogram obtained with reference solution (b)
(0.5 per cent) and at most two such spots are more intense than the principal spot in the chromato-
gram obtained with reference solution (c) (0.2 per cent).
to 105C at a pressure not exceeding 0.1 kPa for 5 h.
ASSAY
Dissolve 50.0 mg in 10 ml of methanol R and dilute to 200.0 ml with a 10 g/l solution of tartaric
acid R. Dilute 10.0 ml of this solution to 50.0 ml with a 10 g/l solution of tartaric acid R. Prepare a
reference solution in the same manner, using 50.0 mg of dihydroergotamine mesilate CRS. To 3.0 ml of
each solution add slowly and with gentle swirling 6.0 ml of dimethylaminobenzaldehyde solution R6.
Allow to stand protected from light for exactly 30 min and measure the absorbance (2.2.25) of each
solution at the maximum at 585 nm, using as the compensation liquid a mixture of 3.0 ml of a 10 g/l
solution of tartaric acid R and 6.0 ml of dimethylaminobenzaldehyde solution R6 prepared at the same
time.
34
H
41
N
5
O
8
S from the absorbances measured and the concentrations of
the solutions.
STORAGE
___________________________________________________________________________________________________ Ph Eur
14-16
Dihydroergotamine Tartrate
NMe
HN
H
H
NH
O
OH
O
O
H
H CH
2
Ph
O
N
N
Me
H
HOOC
COOH
OH H
H OH
,
(C
33
H
37
N
5
O
5
)
2
,C
4
H
6
O
6
1317 5989-77-5
Dihydroergotamine Tartrate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Dihydroergotamine tartrate contains not less than 97.0 per cent and not more than the equivalent of
103.0 per cent of di[(6aR,9R,10aR)-N-[(2R,5S,10aS, 10bS)-5-benzyl-10b-hydroxy-2-methyl-3,6-
dioxo-octahydro-8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl 4,6,6a,7,8,9,10, 10a-
octahydro-indolo[4,3-fg]quinoline-9-carboxamide] tartrate, calculated with reference to the dried
substance.
CHARACTERS
A white or almost white, crystalline powder or colourless crystals, very slightly soluble in water,
IDENTIFICATION
A. Dissolve 5.0 mg in methanol R and dilute to 100.0 ml with the same solvent. Examined between
250 nm and 350 nm (2.2.25), the solution shows two absorption maxima, at 281 nm and 291 nm,
and a shoulder at 275 nm. Above 320 nm the absorbance is negligible. The specific absorbance at the
obtained with dihydroergotamine tartrate CRS. Examine the substances as discs.
D. Suspend about 15 mg in 1 ml of water R. 0.1 ml of the suspension gives reaction (b) of tartrates
(2.3.1).
TESTS
Appearance of solution Dissolve 0.1 g in alcohol (85 per cent V/V) R warming carefully in a water-
bath at 40C and dilute to 50 ml with the same solvent. The solution is clear (2.2.1) and not more
7
or BY
7
(Method II, 2.2.2).
pH (2.2.3). Suspend 50 mg in 50 ml of carbon dioxide-free water R and shake for 10 min. Allow to
stand. The pH of the clear supernatant solution is 4.0 to 5.5.
dried substance.
coating substance.
14-17
Prepare the reference solutions and the test solutions immediately before use and in the order indicated.
Reference solution (a). Dissolve 20 mg of dihydroergotamine tartrate CRS in a mixture of 1 volume of
Reference solution (b). Dilute 2.5 ml of reference solution (a) to 50 ml with a mixture of 1 volume of
Reference solution (c). Dilute 2 ml of reference solution (b) to 5 ml with a mixture of 1 volume of
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with a mixture of 1 volume methanol R and
9 volumes of chloroform R.
Apply separately to the plate 5 l of each solution. Develop protected from light over a path of 15 cm
using a mixture of 1 volume of concentrated ammonia R, 6 volumes of methanol R, 50 volumes of ethyl
acetate R and 50 volumes of methylene chloride R. Dry the plate in a current of cold air for not longer
than 1 min. Repeat the development protected from light over a path of 15 cm using a freshly
prepared amount of the same mobile phase. Dry the plate in a current of cold air. Spray the plate
abundantly with dimethylaminobenzaldehyde solution R7 and dry in a current of warm air for about
2 min. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is
(0.5 per cent) and at most two such spots are more intense than the principal spot in the chromato-
gram obtained with reference solution (c) (0.2 per cent).
100C to 105C.
ASSAY
Dissolve 50.0 mg in 10 ml of methanol R and dilute to 200.0 ml with a 10 g/l solution of tartaric
acid R. Dilute 10.0 ml of this solution to 50.0 ml with a 10 g/l solution of tartaric acid R. Prepare a
reference solution in the same manner, using 50.0 mg of dihydroergotamine tartrate CRS. To 3.0 ml of
each solution add slowly and with gentle swirling 6.0 ml of dimethylaminobenzaldehyde solution R6.
Allow to stand protected from light for exactly 30 min and measure the absorbance (2.2.25) of each
solution at the maximum at 585 nm, using as the compensation liquid a mixture of 3.0 ml of a 10 g/l
solution of tartaric acid R and 6.0 ml of dimethylaminobenzaldehyde solution R6 prepared at the same
time.
70
H
80
N
10
O
16
the solutions.
STORAGE
___________________________________________________________________________________________________ Ph Eur
14-18
Dill Oil
Definition Dill Oil is obtained by distillation from the dried ripe fruits of Anethum graveolens L.
Characteristics A clear, colourless or pale yellow liquid, visibly free from water; odour,
characteristic of the crushed fruit.
Solubility in ethanol Soluble, at 20, in 1 volume or more of ethanol (90%) and in 10 volumes or
more of ethanol (80%), Appendix X M.
Content of carvone 43.0 to 63.0% w/w when determined by the following method. To 1.5 g in a
glass-stoppered tube (approximately 150 mm 25 mm) add 10 ml of a solution prepared in the
following manner. Dissolve 7.0 g of hydroxylamine hydrochloride in 90 ml of ethanol (90%), warming
gently if necessary, add 1.6 ml of dimethyl yellow solution and sufficient 1M potassium hydroxide in
ethanol (90%) to produce a pure yellow colour and dilute to 100 ml with ethanol (90%). Titrate with
1M potassium hydroxide in ethanol (90%) VS until the red colour changes to yellow. Place the tube in a
water bath at 75 to 80 and, at 5-minute intervals, neutralise with 1M potassium hydroxide in ethanol
(90%) VS; after 40 minutes complete the titration to the full yellow colour of the indicator. This
procedure gives an approximate value for the carvone content of the oil. Repeat the procedure, using
as the colour standard for the end point of the titration the titrated liquid of the first determination
with the addition of 0.5 ml of 1M potassium hydroxide in ethanol (90%) VS. Calculate the content of
carvone from the second determination. Each ml of 1M potassium hydroxide in ethanol (90%) VS is
equivalent to 151.4 mg of carvone, C
10
H
14
O.
Storage Dill Oil should be kept in a well-filled, well-closed container, protected from light and
stored at a temperature not exceeding 25. It darkens in colour on storage.
14-19
Diloxanide Furoate
N CHCl
2
O
O
O
O
Me
C
14
H
11
Cl
2
NO
4
328.2 3736-81-0
Definition Diloxanide Furoate is 4-(N-methyl-2,2-dichloroacetamido)phenyl 2-furoate. It
14
H
11
Cl
2
NO
4
, calculated with
Very slightly soluble in water; freely soluble in chloroform; slightly soluble in ethanol (96%) and in
ether.
Identification
diloxanide furoate (RS 103).
ethanol (96%) exhibits a maximum only at 258 nm. The absorbance at the maximum is about 0.98.
C. Burn 20 mg by the method for oxygen-flask combustion, Appendix VIII C, using 10 ml of 1M
sodium hydroxide as the absorbing liquid. When the process is complete, acidify the liquid with nitric
acid and add silver nitrate solution. A white precipitate is produced.
Free acidity Shake 3 g with 50 ml of water, filter and wash the residue with three 20-ml quantities of
water. Titrate the combined filtrate and washings with 0.1M sodium hydroxide VS using phenolphthalein
solution R1 as indicator. Not more than 1.3 ml is required.
gel HF
254
as the coating substance and a mixture of 96 volumes of dichloromethane and 4 volumes of
methanol as the mobile phase. Apply separately to the plate 5 l of each of two solutions of the
substance being examined in chloroform containing (1) 10.0% w/v and (2) 0.025% w/v. After removal
of the plate, allow it to dry in air and examine under ultraviolet light (254 nm). Any secondary spot in
1 g.
of C
14
H
11
Cl
2
NO
4
.
Storage Diloxanide Furoate should be protected from light.
Action and use Antiprotozoal.
Preparation
Diloxanide Tablets
14-20
Diltiazem Hydrochloride
S
N
H
H
OAc
O
Me
2
N
OMe
,HCl
C
22
H
26
N
2
O
4
S,HCl 451.0 33286-22-5
Diltiazem Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Diltiazem hydrochloride contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of (2S,3S)-3-acetyloxy-5-[2-(dimethyl-amino)ethyl]-2-(4-methoxyphenyl)-2,3-dihydro-
1,5-benzothiazepin-4(5H)-one hydrochloride, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder, freely soluble in water, in methanol and in methylene chloride, slightly
soluble in ethanol. It melts at about 213C with decomposition.
IDENTIFICATION
A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained
with diltiazem hydrochloride CRS. Examine the substances prepared as discs.
Reference solution. Dissolve 0.10 g of diltiazem hydrochloride CRS in methylene chloride R and dilute to
volume of acetic acid R, 3 volumes of water R, 10 volumes of methylene chloride R and 12 volumes of
ethanol R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in
the chromatogram obtained with the test solution is similar in position and size to the principal spot in
C. Dissolve 50 mg in 5 ml of water R. Add 1 ml of ammonium reineckate solution R. A pink precipitate is
produced.
TESTS
Solution S Dissolve 1.00 g in carbon-dioxide free water R and dilute to 20.0 ml with the same solvent.
pH (2.2.3). Dilute 2.0 ml of solution S to 10.0 ml with carbon dioxide-free water R. The pH of the
optical rotation is +115 to +120, calculated with reference to the dried substance.
Reference solution (a). Dissolve 50.0 mg of diltiazem hydrochloride CRS in the mobile phase and dilute to
14-21
Reference solution (b). Dissolve 3 mg of diltiazem impurity A CRS in the mobile phase and dilute to
10 ml with the mobile phase. To 1 ml of this solution, add 1.2 ml of reference solution (a) and dilute
as mobile phase at a flow rate of 1.5 ml per minute a mixture of 5 volumes of ethanol R, 25
volumes of acetonitrile R and 70 volumes of a solution containing, in 1 litre, 6.8 g of potassium
dihydrogen phosphate R and 0.1 ml of N,N-dimethyloctylamine R, adjusted to pH 4.5 using dilute
phosphoric acid R,
a loop injector.
Inject separately 20 l of each solution. Adjust the sensitivity of the detector so that the height of
the principal peak in the chromatogram obtained with reference solution (c) is at least 10 per cent of
the full scale of the recorder. Continue the chromatography for five times the retention time of the
principal peak. The test is not valid unless in the chromatogram obtained with reference solution (b),
the resolution between the peaks due to diltiazem impurity A and diltiazem is at least 4.0 and the
symmetry factors are not more than 2.0. If necessary, adjust the concentration of N,N-dimethyloctyl-
amine in the mobile phase. In the chromatogram obtained with the test solution, the sum of the areas
of any peaks, apart from the principal peak, is not greater than the area of the principal peak in the
less than 0.025 times that of the peak in the chromatogram obtained with reference solution (c).
Heavy metals (2.4.8). Dissolve 2.0 g in water R and dilute to 20.0 ml with the same solvent. 12 ml
100C to 105C for 2 h.
ASSAY
Dissolve 0.400 g in a mixture of 2 ml of anhydrous formic acid R and 60 ml of acetic anhydride R and
titrate with 0.1M perchloric acid, determining the end-point potentiometrically (2.2.20).
22
H
27
ClN
2
O
4
S.
STORAGE
IMPURITIES
S
N
H
H
OAc
O
Me
2
N
OMe
A. (2R,3S)-3-acetyloxy-5-[2-(dimethylamino)ethyl]-2-(4-methoxyphenyl)-2,3-dihydro-1,5-
benzothiazepin-4(5H)-one,
S
N
H
H
OAc
O
OMe
H
B. (2S,3S)-3-acetyloxy-2-(4-methoxyphenyl)-2,3-dihydro-1,5-benzothiazepin-4(5H)-one,
S
N
H
H
OAc
Me
2
N
O
OH
C. (2S,3S)-3-acetyloxy-5-[2-(dimethylamino)ethyl]-2-(4-hydroxyphenyl)-2,3-dihydro-1,5-
benzothiazepin-4(5H)-one,
14-22
S
N
H
H
OAc
O
MeNH
OMe
D. (2S,3S)-3-acetyloxy-2-(4-methoxyphenyl)-5-[2-(methylamino)ethyl]-2,3-dihydro-1,5-
benzothiazepin-4 (5H)-one,
S
N
H
H
OH
O
OMe
H
E. (2S,3S)-3-hydroxy-2-(4-methoxyphenyl)-2,3-dihydro-1,5-benzo-thiazepin-4(5H)-one,
S
N
H
H
OH
O
Me
2
N
OMe
F. (2S,3S)-5-[2-(dimethylamino)ethyl]-3-hydroxy-2-(4-methoxyphenyl)-2,3-dihydro-1,5-
benzothiazepin-4(5H)-one.
___________________________________________________________________________________________________ Ph Eur
14-23
Dimenhydrinate
MeN
N
N
N
Cl
O
O
Me
H
,
O
NMe
2
C
17
H
21
NO,C
7
H
7
ClN
4
O
2
470.0 523-87-5
Dimenhydrinate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0601].
Preparation
Dimenhydrinate Tablets
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Dimenhydrinate contains not less than 53.0 per cent and not more than 55.5 per cent of
diphenhydramine [2-(diphenylmethoxy)-N,N-dimethylethylamine, C
17
H
21
NO, M
r
255.4] and not
less than 44.0 per cent and not more than 46.5 per cent of 8-chlorotheophylline (8-chloro-3,7-
dihydro-1,3-dimethyl-1H-purine-2,6-dione, C
7
H
7
ClN
4
O
2
, M
r
214.6), both calculated with reference
CHARACTERS
A white, crystalline powder or colourless crystals, slightly soluble in water, freely soluble in alcohol,
sparingly soluble in ether.
IDENTIFICATION
B. Dissolve 0.1 g in a mixture of 3 ml of water R and 3 ml of alcohol R, add 6 ml of water R and 1 ml
of dilute hydrochloric acid R and cool in iced water for 30 min, scratching the wall of the tube with a
glass rod if necessary to initiate crystallisation. Dissolve about 10 mg of the precipitate obtained in
1 ml of hydrochloric acid R, add 0.1 g of potassium chlorate R and evaporate to dryness in a porcelain
dish. A reddish residue is obtained which becomes violet-red when exposed to ammonia vapour.
obtained with dimenhydrinate CRS.
D. Dissolve 0.2 g in 10 ml of alcohol R. Add 10 ml of picric acid solution R and initiate crystallisation
by scratching the wall of the tube with a glass rod. The precipitate, washed with water R and dried at
100C to 105C, melts (2.2.14) at 130C to 134C.
TESTS
pH (2.2.3). To 0.4 g add 20 ml of carbon dioxide-free water R, shake for 2 min and filter. The pH of
the filtrate is 7.1 to 7.6.
Theophylline and substances related to diphenhydramine Examine by thin-layer chromato-
254
Reference solution (a). Dissolve 20 mg of theophylline R in methylene chloride R and dilute to 100 ml
Reference solution (b). Dilute 5 ml of the test solution to 100 ml with methylene chloride R. Dilute
10 ml of this solution to 100 ml with methylene chloride R.
volume of concentrated ammonia R, 9 volumes of methanol R and 90 volumes of methylene chloride R.
14-24
Dry the plate in a current of cold air and examine in ultraviolet light at 254 nm. Any spot corres-
ponding to theophylline in the chromatogram obtained with the test solution is not more intense than
the spot in the chromatogram obtained with reference solution (a) (0.5 per cent). Spray with
potassium iodobismuthate solution R. Allow the plate to dry in air and spray with dilute hydrogen peroxide
solution R. Any spot in the chromatogram obtained with the test solution, apart from the principal
spot, is not more intense than the spot in the chromatogram obtained with reference solution (b)
(0.5 per cent). Disregard any spot extending from the starting point to an R
f
of about 0.1.
acetone R and dilute to 25 ml with the same mixture of solvents. The solution complies with limit test
B for heavy metals (20 ppm). Prepare the standard using lead standard solution (2 ppm Pb) prepared
by diluting lead standard solution (100 ppm Pb) R with a mixture of 15 volumes of water R and 85
volumes of acetone R.
ASSAY
Diphenhydramine Dissolve 0.200 g in 60 ml of anhydrous acetic acid R. Titrate with 0.1M perchloric
17
H
21
NO.
8-Chlorotheophylline To 0.800 g add 50 ml of water R, 3 ml of dilute ammonia R1 and 0.6 g of
ammonium nitrate R and heat on a water-bath for 5 min. Add 25.0 ml of 0.1M silver nitrate and
continue heating on a water-bath for 15 min with frequent swirling. Cool, add 25 ml of dilute nitric
acid R and dilute to 250.0 ml with water R. Filter and discard the first 25 ml of the filtrate. Using
5 ml of ferric ammonium sulphate solution R2 as indicator, titrate 100.0 ml of the filtrate with 0.1M
ammonium thiocyanate until a yellowish-brown colour is obtained.
7
H
7
ClN
4
O
2
.
___________________________________________________________________________________________________ Ph Eur
14-25
Dimercaprol
HO
SH H
SH
and enantiomer
C
3
H
8
OS
2
124.2 59-52-9
Dimercaprol complies with the requirements of the 3rd edition of the European Pharmacopoeia [0389]. These
Action and use Used in treatment of arsenic, gold and mercury poisoning.
Preparation
Dimercaprol Injection
When B.A.L. is prescribed or demanded. Dimercaprol shall be dispensed or supplied.
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Dimercaprol contains not less than 98.5 per cent and not more than the equivalent of 101.5 per cent
of (RS)-2,3-dimercaptopropanol.
CHARACTERS
A clear, colourless or slightly yellow liquid, soluble in water and in arachis oil, miscible with alcohol
and with benzyl benzoate.
IDENTIFICATION
A. Dissolve 0.05 ml in 2 ml of water R. Add 1 ml of 0.05M iodine. The colour of the iodine is
discharged immediately.
B. Dissolve 0.1 ml in 5 ml of water R and add 2 ml of copper sulphate solution R. A bluish-black
precipitate is formed which quickly becomes dark grey.
C. In a ground-glass-stoppered tube, suspend 0.6 g of sodium bismuthate R, previously heated to
200C for 2 h, in a mixture of 2.8 ml of dilute phosphoric acid R and 6 ml of water R. Add 0.2 ml of
the substance to be examined, mix and allow to stand for 10 min with frequent shaking. To 1 ml of
the supernatant liquid add 5 ml of a 4 g/l solution of chromotropic acid, sodium salt R in sulphuric acid R
and mix. Heat for 15 min in a water-bath. A violet-red colour develops.
TESTS
Appearance It is clear (2.2.1) and not more intensely coloured than reference solution B
6
or BY
6
(Method II, 2.2.2).
solvent. Add 0.25 ml of bromocresol green solution R and 0.3 ml of 0.01M hydrochloric acid. The solution
is yellow. Not more than 0.5 ml of 0.01M sodium hydroxide is required to change the colour of the
indicator to blue.
Halides To 2.0 g add 25 ml of alcoholic potassium hydroxide solution R and boil under a reflux
condenser for 2 h. Eliminate the alcohol by evaporation in a stream of hot air, add 20 ml of water R
and cool. Add 40 ml of water R and 10 ml of strong hydrogen peroxide solution R, boil gently for
10 min, cool and filter rapidly. Add 10 ml of dilute nitric acid R and 5.0 ml of 0.1M silver nitrate. Using
2 ml of ferric ammonium sulphate solution R2 as indicator, titrate with 0.1M ammonium thiocyanate until
a reddish-yellow colour is obtained. Carry out a blank titration. The difference between the titration
volumes is not greater than 1.0 ml.
ASSAY
Dissolve 0.100 g in 40 ml of methanol R. Add 20 ml of 0.1M hydrochloric acid and 50.0 ml of 0.05M
iodine. Allow to stand for 10 min and titrate with 0.1M sodium thiosulphate. Carry out a blank titration.
3
H
8
OS
2
.
STORAGE
Store in a well-filled, airtight container, protected from light, at a temperature of 2C to 8C.
___________________________________________________________________________________________________ Ph Eur
14-26
Dimethyl Phthalate
COOMe
COOMe
C
10
H
10
O
4
194.2 131-11-3
Definition Dimethyl Phthalate contains not less than 99.0% and not more than 100.5% w/w of
C
10
H
10
O
4
.
Characteristics A colourless or faintly coloured liquid; odourless or almost odourless.
Slightly soluble in water; miscible with ethanol (96%), with ether and with most organic solvents.
Identification
dimethyl phthalate (RS 105).
B. Gently boil 1 g with 5 ml of 2M methanolic potassium hydroxide for 10 minutes, add 5 ml of water,
evaporate the mixture to half its volume and cool. Add 1 ml of hydrochloric acid, filter, melt the dried
precipitate in a small tube, add 0.5 g of resorcinol and 0.05 ml of chloroform and heat to about 180 for
3 minutes. Cool, add 1 ml of 5M sodium hydroxide and pour into water. An intense yellowish green
fluorescence is produced.
Acidity Mix 20 ml with 50 ml of ethanol (96%) previously neutralised to phenolphthalein solution R1.
Not more than 0.1 ml of 0.1M sodium hydroxide VS is required to neutralise the solution using
phenolphthalein solution R1 as indicator.
Related substances Prepare a 0.075% w/v solution of phenyl benzoate (internal standard) in
chloroform (solution A). Carry out the method for gas chromatography, Appendix III B, using solutions
of the substance being examined containing (1) 0.10% w/v in solution A, (2) 5.0% w/v in chloroform
and (3) 5.0% w/v in solution A.
methyl silicone fluid (50% phenyl) (OV-17 is suitable) and maintained at 145.
In the chromatogram obtained with solution (3) the ratio of the sum of the areas of any secondary
peaks to the area of the peak due to the internal standard is not greater than the ratio of the area of
the peak due to dimethyl phthalate to the area of the peak due to the internal standard in the
Sulphated ash Not more than 0.1% w/w, Appendix IX A.
Water Not more than 0.1% w/w, Appendix IX C. Use 20 g.
Assay In a borosilicate glass flask dissolve 1.5 g of the substance being examined in 5 ml of carbon
dioxide-free ethanol prepared by boiling ethanol (96%) thoroughly and neutralising to phenolphthalein
solution R1. Neutralise the free acid in the solution with 0.1M ethanolic potassium hydroxide VS using
0.2 ml of phenolphthalein solution R1 as indicator. Add 50 ml of 0.5M ethanolic potassium hydroxide VS
and boil under a reflux condenser on a water bath for 1 hour. Add 20 ml of water and titrate the
excess of alkali with 0.5M hydrochloric acid VS using a further 0.2 ml of phenolphthalein solution R1 as
indicator. Repeat the operation without the substance being examined. The difference between the
titrations represents the alkali required to saponify the esters. Each ml of 0.5M ethanolic potassium
hydroxide VS is equivalent to 48.55 mg of C
10
H
10
O
4
.
Action and use Insect repellent.
14-27
Dimethyl Sulfoxide
corrected 1/01
H
3
C
S
CH
3
O
C
2
H
6
OS 78.1 67-68-5
Dimethyl Sulfoxide complies with the requirements of the 3rd edition of the European Pharmacopoeia [0763].
These require-ments are reproduced after the heading Definition below.
When dimethyl sulphoxide is demanded, Dimethyl Sulfoxide shall be supplied.
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Dimethyl sulfoxide is sulphinylbismethane.
CHARACTERS
A colourless liquid or colourless crystals, hygroscopic, miscible with water, with alcohol and with
ether.
IDENTIFICATION
B. It complies with the test for refractive index (see Tests).
obtained with dimethyl sulfoxide CRS.
D. Dissolve 50 mg of nickel chloride R in 5 ml of the substance to be examined. The solution is
greenish-yellow. Heat in a water-bath at 50C. The colour changes to green or bluish-green. Cool.
The colour changes to greenish-yellow.
TESTS
Acidity Dissolve 50.0 g in 100 ml of carbon dioxide-free water R. Add 0.1 ml of phenolphthalein
solution R1. Not more than 5.0 ml of 0.01M sodium hydroxide is required to produce a pink colour.
Absorbance (2.2.25). Purge with nitrogen R for 15 min. The absorbance, measured using water R as
the compensation liquid, is not more than 0.30 at 275 nm and not more than 0.20 at both 285 nm
and 295 nm. Examined between 270 nm and 350 nm, the substance to be examined shows no
absorption maximum.
Related substances Examine by gas chromatography (2.2.28), using bibenzyl R as the internal
standard.
Internal standard solution. Dissolve 0.125 g of bibenzyl R in acetone R and dilute to 50 ml with the
same solvent.
Test solution (a). Dissolve 5.0 g of the substance to be examined in acetone R and dilute to 10.0 ml
Test solution (b). Dissolve 5.0 g of the substance to be examined in acetone R, add 1.0 ml of the
internal standard solution and dilute to 10.0 ml with acetone R.
Reference solution. Dissolve 50.0 mg of the substance to be examined and 50 mg of dimethyl sulphone R
in acetone R, add 10.0 ml of the internal standard solution and dilute to 100.0 ml with acetone R.
a glass column 1.5 m long and 4 mm in internal diameter, packed with diatomaceous earth for gas
chromatography R (125 m to 180 m) impregnated with 10 per cent m/m of polyethyleneglycol
adipate R,
14-28
detector at 190C.
Inject 1 l of the reference solution and adjust the sensitivity of the system so that the heights of the
three peaks, apart from the solvent peak, are not less than 70 per cent of the full scale of the recorder.
The substances elute in the following order: dimethyl sulfoxide, dimethyl sulphone, bibenzyl. The
test is not valid unless, in the chromatogram obtained with the reference solution, the resolution
between the peaks due to dimethyl sulfoxide and dimethyl sulphone is at least 3.
Inject 1 l of test solution (b) and 1 l of the reference solution. Continue the chromatography for
four times the retention time of dimethyl sulfoxide, which is about 5 min. From the chromatogram
obtained with the reference solution, calculate the ratio (R) of the area of the peak due to dimethyl
sulfoxide to the area of the peak due to the internal standard. From the chromatogram obtained with
test solution (b), calculate the ratio of the sum of the areas of any peaks, apart from the principal
peak, the peak due to the internal standard and the peak due to the solvent, to the area of the peak
due to the internal standard; this ratio is not greater than R (0.1 per cent).
of water.
STORAGE
Store in an airtight, glass container, protected from light.
___________________________________________________________________________________________________ Ph Eur
14-29
Dimeticone
Si O Si Me
3
Me
Me
Me
n = 20 to 400
n+1
9006-75-9
Dimeticone complies with the requirements of the 3rd edition of the European Pharmacopoeia [0138]. These
When dimethicone is demanded, Dimeticone shall be supplied.
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Dimeticone is a poly(dimethylsiloxane) obtained by hydrolysis and polycondensation of dichloro-
dimethylsilane and chlorotrimethylsilane. Different grades exist which are distinguished by a number
indicating the nominal viscosity placed after the name.
Their degree of polymerisation (n = 20 to 400) is such that their kinematic viscosities are nominally
between 20 mm
2.
s
1
and 1300 mm
2.
s
1
.
Dimeticones with a nominal viscosity of 50 mm
2.
s
-1
or lower are intended for external use only.
CHARACTERS
Clear, colourless liquids of various viscosities, practically insoluble in water, very slightly soluble to
practically insoluble in ethanol, miscible with ethyl acetate, with methyl ethyl ketone and with
toluene.
IDENTIFICATION
A. It is identified by its kinematic viscosity at 25C (see Tests).
B. Examine by infrared spectrophotometry (2.2.24) comparing with the spectrum obtained with
dimeticone CRS. The region of the spectrum from 850 cm
-1
to 750 cm
-1
is not taken into account.
The spectrum obtained is identical to that of the material selected for the type sample.
C. Heat 0.5 g in a test-tube over a small flame until white fumes begin to appear. Invert the tube over
a second tube containing 1 ml of a 1 g/l solution of chromotropic acid, sodium salt R in sulphuric acid R
so that the fumes reach the solution. Shake the second tube for about 10 s and heat on a water-bath
for 5 min. The solution is violet.
D. In a platinum crucible, prepare the sulphated ash (2.4.14) using 50 mg. The residue is a white
powder that gives the reaction of silicates (2.3.1).
TESTS
Acidity To 2.0 g add 25 ml of a mixture of equal volumes of ethanol R and ether R, previously
neutralised to 0.2 ml of bromothymol blue solution R1 and shake. Not more than 0.15 ml of 0.01M
sodium hydroxide is required to change the colour of the solution to blue.
Viscosity (2.2.9). Determine the kinematic viscosity at 25C. For dimeticones, the kinematic
viscosity is not less than 90 per cent and not more than 110 per cent of the nominal viscosity stated
on the label.
Mineral oils Place 2 g in a test-tube and examine in ultraviolet light at 365 nm. The fluorescence is
not more intense than that of a solution containing 0.1 ppm of quinine sulphate R in 0.005M sulphuric
acid examined in the same conditions.
Phenylated compounds Dissolve 5.0 g with shaking in 10 ml of cyclohexane R. At wavelengths from
250 nm to 270 nm, the absorbance (2.2.25) of the solution is not greater than 0.2.
Heavy metals Mix 1.0 g with methylene chloride R and dilute to 20 ml with the same olvent. Add
1.0 ml of an extemporaneously prepared 0.02 g/l solution of dithizone R in methylene chloride R,
0.5 ml of water R and 0.5 ml of a mixture of 1 volume of dilute ammonia R2 and 9 volumes of a 2 g/l
solution of hydroxylamine hydrochloride R. At the same time, prepare a standard as follows: to 20 ml of
methylene chloride R add 1.0 ml of a freshly prepared 0.02 g/l solution of dithizone R in methylene
chloride R, 0.5 ml of lead standard solution (10 ppm Pb) R and 0.5 ml of a mixture of 1 volume of dilute
ammonia R2 and 9 volumes of a 2 g/l solution of hydroxylamine hydrochloride R. Immediately shake
14-30
each solution vigorously for 1 min. Any red colour in the test solution is not more intense than that
in the standard (5 ppm).
Volatile matter For dimeticones with a nominal viscosity greater than 50 mm
2.
s
-1
, not more than
0.3 per cent, determined on 1.00 g by heating in an oven at 150C for 2 h. Carry out the test using a
dish 60 mm in diameter and 10 mm deep.
LABELLING
The label states:
the nominal viscosity by a number placed after the name of the product,
where applicable, that the product is intended for external use.
___________________________________________________________________________________________________ Ph Eur
14-31
Dimetindene Maleate
N
NMe
2
Me
H
COOH
COOH
,
and enantiomer
C
24
H
28
N
2
O
4
408.5 3614-69-5
Dimetindene Maleate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Histamine-H
1
receptor antagonist.
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Dimetindene maleate contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of N,N-dimethyl-2-[3-[(RS)-1-(pyridin-2-yl)ethyl]-1H-inden-2-yl]ethanamine (Z)-
CHARACTERS
A white to almost white, crystalline powder, slightly soluble in water, soluble in methanol.
IDENTIFICATION
with dimetindene maleate CRS. Examine the substance prepared as discs.
TESTS
solution Y
6
(Method II, 2.2.2).
Optical rotation (2.2.7). Determined on solution S, the angle of optical rotation is 0.10 to
+0.10.
Related substances. Examine by gas chromatography (2.2.28).
acetone R and methylene chloride R and dilute to 5.0 ml with the same mixture of solvents.
Reference solution (a). Dilute 1 ml of the test solution to 100.0 ml with a mixture of equal volumes of
acetone R and methylene chloride R.
Reference solution (b). Dissolve 5.0 mg of 2-ethylpyridine R in a mixture of equal volumes of acetone R
and methylene chloride R and dilute to 50.0 ml with the same mixture of solvents. Dilute 10.0 ml of
this solution to 100.0 ml with the same mixture of solvents.
a fused-silica column 30 m long and 0.32 mm in internal diameter coated with polymethylphenyl-
siloxane R (film thickness 0.25 m),
helium for chromatography R as the carrier gas at a linear velocity of about 30 cm/s,
of 6C/min to 260C and maintaining at 260C for 12 min. Maintain the temperature of the
injection port at 240C and that of the detector at 260C.
Inject via a split injector with a split flow of 30 ml/min for 1.3 times the retention time of the
principal peak.
Inject 2 l of reference solution (a). The symmetry factor of the principal peak in the chromato-
gram obtained is not greater than 1.3.
Inject 2 l of test solution and 2 l of reference solution (b). In the chromatogram obtained with
the test solution the area of the peak corresponding to 2-ethylpyridine is not greater than the area of
the peak in the chromatogram obtained with reference solution (b) (0.1 per cent).
In the chromatogram obtained with the test solution the area of any peak, apart from the principal
14-32
peak and any peak appearing during the first 8 min (2-ethylpyridine, the solvent mixture and maleic
acid), is not greater than 0.2 times the area of the peak in the chromatogram with reference solution
(a) (0.2 per cent) and the sum of the areas of all peaks is not greater than 0.5 times the area of the peak
in the chromatogram obtained with reference solution (a) (0.5 per cent). Disregard any peak with an
solution (a).
100C to 105C for 2 h.
ASSAY
Dissolve 0.150 g in 80 ml of glacial acetic acid R. Titrate with 0.1M perchloric acid, determining the end-
24
H
28
N
2
O
4
.
STORAGE
IMPURITIES
N
CH
3
A. 2-ethylpyridine,
NMe
2
B. 2-(1H-inden-2-yl)-N,N-dimethylethanamine,
NMe
2
H COOEt
and enantiomer
C. ethyl (2RS)-2-benzyl-4-(dimethylamino)butanoate,
NMe
2
H COOH
and enantiomer
D. (2RS)-2-benzyl-4-(dimethylamino)butanoic acid,
NMe
2
O
H
and enantiomer
E. (2RS)-2-[2-(dimethylamino)ethyl]indan-1-one,
NMe
2
Bu
F. 2-(3-butyl-1H-inden-2-yl)-N,N-dimethylethanamine,
NMe
2
G. N,N-dimethyl-2-(3-phenyl-1H-inden-2-yl)ethanamine,
N
CH
2
Me
H
and enantiomer
H. 2-[(1RS)-1-(2-ethenyl-1H-inden-3-yl)ethyl]pyridine,
14-33
N
NHMe
Me
H
and enantiomer
I. N-methyl-2-[3-[(1RS)-1-(pyridin-2-yl)ethyl]-1H-inden-2-yl]ethanamine.
_______________________________________________________________________________________________________ Ph Eur
14-34
Dinoprost Trometamol
OH
OH
H
H
H OH
H
H
COOH
CH
3
CH
2
OH
C CH
2
OH
CH
2
OH
H
2
N
,
C
20
H
34
O
5
,C
4
H
11
NO
3
475.6 38562-01-5
Dinoprost Trometamol complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Inducer of uterine muscle contraction.
Preparation
Dinoprost Injection
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Dinoprost trometamol contains not less than 96.0 per cent and not more than the equivalent of
102.0 per cent of trometamol (Z)-7-[(1R-2R,3R,5S)-3,5-dihydroxy-2-[(E)-(3S)-3-hydroxyoct-1-
enyl]cyclopentyl]hept-5-enoate (PGF
2
), calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white powder, very soluble in water, freely soluble in alcohol, practically insoluble
in acetonitrile.
IDENTIFICATION
A. Dissolve 0.100 g in alcohol R and dilute to 10.0 ml with the same solvent. The specific optical
rotation (2.2.7) is +19 to +26, calculated with reference to the anhydrous substance.
B. Examine by infrared absorption spectophotometry (2.2.24), comparing with the spectrum
obtained with dinoprost trometamol CRS.
TESTS
Test solution. Dissolve 10.0 mg of the substance to be examined in a mixture of 23 volumes of
Reference solution (a). Degradation of dinoprost trometamol to impurity B. Dissolve 1 mg of the substance
to be examined in 1 ml of the mobile phase and heat the solution on a water-bath at 85C for 5 min
and cool.
acetonitrile R and 77 volumes of water R. Dilute 2.0 ml of the solution to 20.0 ml with a mixture of 23
volumes of acetonitrile R and 77 volumes of water R.
sodium dihydrogen phosphate R in water R and dilute to 1000 ml with water R; adjust the pH to
2.5 with phosphoric acid R (about 0.6 ml); mix 770 ml of the buffer solution with 230 ml of
acetonitrile R1,
obtained with 20 l of reference solution (b) is 25 per cent to 50 per cent of the full scale of the
recorder.
conditions, the retention times are: (15R)-PGF
2
(impurity B) about 55 min; 5,6-trans-PGF
2
(impurity A) about 60 min and dinoprost about 66 min. Continue the chromatography for 2.5 times
the retention time of the principal peak (to elute degradation products formed during heating). The
test is not valid unless the resolution between the peaks corresponding to impurity A and impurity B
is at least 1.5 and the resolution between the peaks corresponding to impurity A and dinoprost is at
least 2.0; the symmetry factor for the peaks corresponding to impurity A and impurity B is less than
14-35
1.2. If necessary, adjust the composition of the mobile phase by increasing the concentration of
acetonitrile to decrease the retention times.
chromatography of the test solution for 10 min after the elution of the principal peak. In the chroma-
togram obtained with the test solution the area of any peak corresponding to impurity A is not greater
than twice the area of the principal peak obtained with reference solution (b) (2 per cent); the area of
any other peak, apart from the principal peak and any peak corresponding to impurity A, is not
greater than 1.5 times the area of the principal peak obtained with reference solutio (b) (1.5 per cent)
and not more than one such peak has an area greater than half the area of the principal peak obtained
with reference solution (b) (0.5 per cent). The sum of the areas of all the peaks, apart from the
principal peak and the peak corresponding to impurity A, is not greater than twice the area of the
principal peak obtained with reference solution (b) (2 per cent). Disregard any peak due to the
mobile phase and any peak due to trometamol (retention time about 1.5 min) and also any peak with
an area of less than 0.05 times the area of the principal peak obtained with reference solution (b).
Water (2.5.12). Not more than 1.0 per cent, determined on 0.500 g by the semi-micro determina-
tion of water.
ASSAY
acetonitrile R and 77 volumes of water R and dilute to 10.0 ml with the same mixture.
Reference solution. Dissolve 10.0 mg of dinoprost trometamol CRS in a mixture of 23 volumes of
acetonitrile R and 77 volumes of water R and dilute to 10.0 ml with the same mixture.
sodium dihydrogen phosphate R in water R and dilute to 1000 ml with water R; adjust the pH to
2.5 with phosphoric acid R (about 0.6 ml); mix 730 ml of the buffer solution with 270 ml of
acetonitrile R1,
obtained with 20 l of the reference solution is 70 per cent to 90 per cent of the full scale of the
recorder. When the chromatogram is recorded under the conditions described above, the retention
time is about 23 min for dinoprost.
Inject the reference solution six times. The test is not valid unless the relative standard deviation of
the peak area for dinoprost is at most 2.0 per cent. Inject 20 l of the test solution. Calculate the
percentage of dinoprost trometamol.
STORAGE
IMPURITIES
COOH
CH
3
H OH
H
H
OH
OH
H
H
A. (E)-7-[(1R,2R,3R,5S)-3,5-dihydroxy-2-[(E)-(3S)-3-hydroxyoct-1-enyl]cyclopentyl]hept-5-
enoic acid ((5E)-PGF
2
; 5,6-trans-PGF
2
),
CH
3
H OH
H
H
OH
OH
H
H
COOH
B. (Z)-7-[(1R,2R,3R,5S)-3,5-dihydroxy-2-[(E)-(3R)-3-hydroxyoct-1-enyl]cyclopentyl]hept-5-
enoic acid ((15R)-PGF
2
; 15-epiPGF
2
),
CH
3
H OH
H
H
OH
OH
H
H
COOH
C. (Z)-7-[(1S,2R,3R,5S)-3,5-dihydroxy-2-[(E)-(3S)-3-hydroxyoct-1-enyl]cyclopentyl]hept-5-
enoic acid ((8S)-PGF
2
; 8-epiPGF
2
),
14-36
CH
3
H OH
H
H
OH
OH
H
H
COOH
D. (Z)-7-[(1R,2R,3S,5S)-3,5-dihydroxy-2-[(E)-(3S)-3-hydroxyoct-1-enyl]cyclopentyl]hept-5-
enoic acid (11-PGF
2
; 11-epiPGF
2
).
___________________________________________________________________________________________________ Ph Eur
14-37
Dinoprostone
OH
H
H OH
H
H
COOH
CH
3
O
C
20
H
32
O
5
352.5 363-24-6
Dinoprostone complies with the requirements of the 3rd edition of the European Pharmacopoeia [1311]. These
Action and use Inducer of uterine muscle contraction.
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Dinoprostone contains not less than 95.0 per cent and not more than the equivalent of 102.0 per
cent of (Z)-7-[(1R,2R,3R)-3-hydroxy-2-[(E)-(3S)-3-hydroxyoct-1-enyl]-5-oxocyclopentyl]hept-5-
enoic acid (PGE
2
CHARACTERS
A white or almost white, crystalline powder or colourless crystals, practically insoluble in water, very
soluble in methanol, freely soluble in alcohol. The substance degrades at room temperature.
IDENTIFICATION
A. Immediately before use, dissolve 50.0 mg in alcohol R and dilute to 10.0 ml with the same solvent.
The specific optical rotation (2.2.7) is 82 to 90, calculated with reference to the anhydrous
substance.
obtained with dinoprostone CRS.
TESTS
Related substances Examine by liquid chromatography (2.2.29) as prescribed under Assay.
Adjust the sensitivity of the system so that the height of the dinoprostone peak in the chromatogram
Inject separately 20 l of test solution (a), 20 l of reference solution (b) and 20 l of reference
solution (c). When the chromatograms are recorded under the prescribed conditions, the retention
times are: dinoprostone, about 18 min; 5-trans-PGE
2
(impurity C), about 21 min; PGA
2
(impurity
D) about 33 min and PGB
2
(impurity E), about 36 min.
Limits
Correction factors:
Peak corresponding to: Correction factor
15-oxo-PGE
2
(impurity F)
0.2
impurity D 0.2
impurity E 0.7
Impurity C
Not more than three times the area of the principal peak in the chromatogram obtained with
Impurity D (corrected area)
Not more than twice the area of the principal peak in the chromatogram obtained with the reference
Impurity E (corrected area)
Not more than the area of the principal peak in the chromatogram obtained with the reference
Any other impurity
Not more than the area of the principal peak in the chromatogram obtained with the reference
14-38
Total content of other impurities
Not more than twice the area of the principal peak in the chromatogram obtained with the reference
Disregard limit
Peaks due to the mobile phase or the solvent and any peak with an area less than 0.1 times the area of
If any peak with a relative retention time to dinoprostone of about 0.8 is greater than 0.5 per cent or
if the total content of other impurities is greater than 1.0 per cent, inject 20 l of test solution (a) and
record the chromatogram with a detector set at 230 nm. If the area of the peak at 230 nm is twice the
area of the peak at 210 nm multiply the area at 210 nm with the correction factor for impurity F.
of water.
ASSAY
Test solution (a). Dissolve 10.0 mg of the substance to be examined in a 58 per cent V/V solution of
methanol R2 and dilute to 2.0 ml with the same solvent.
Test solution (b). Dissolve 20.0 mg of the substance to be examined in a 58 per cent V/V solution of
Reference solution (a). Dissolve 1 mg of dinoprostone CRS and 1 mg of dinoprostone impurity C CRS in a
58 per cent V/V solution of methanol R2 and dilute to 10.0 ml with the same solvent. Dilute 4.0 ml of
the solution to 10.0 ml with a 58 per cent V/V solution of methanol R2.
Reference solution (b). Dilute 0.5 ml of test solution (a) to 10.0 ml with a 58 per cent V/V solution of
methanol R2. Dilute 1.0 ml of the solution to 10.0 ml with a 58 per cent V/V solution of methanol R2.
Reference solution (c). Degradation of dinoprostone to impurity D and impurity E for identification. Dissolve
1 mg dinoprostone in 100 l of 1M sodium hydroxide (the solution becomes brownish-red), wait 4 min,
add 150 l of 1M acetic acid (yellowish-white opalescent solution) and dilute to 5.0 ml with a 58 per
cent V/V solution of methanol R2.
Reference solution (d). Dissolve 20 mg of dinoprostone CRS in a 58 per cent V/V solution of
silica gel, end-capped for chromatography R,
as a mobile phase at a flow rate of 1.0 ml/min a mixture of 42 volumes of a 0.2 per cent V/V
solution of acetic acid R and 58 volumes of methanol R2,
maintaining the temperature of the column at about 30C.
conditions, the retention times are: dinoprostone, about 18 min and impurity C, about 21 min. The
tests for the assay and related substances are not valid unless the resolution between the peaks
corresponding to dinoprostone and impurity C is at least 3.8. If necessary, adjust the concentration
of the acetic acid solution and/or methanol (increase the concentration of the acetic acid solution to
increase the retention time for dinoprostone and impurity C and increase the concentration of
methanol to decrease the retention time for both compounds).
Adjust the sensitivity of the system so that the height of the principal peak obtained with 20 l of
reference solution (d) is at least 50 per cent of the full scale of the recorder.
The test is not valid unless the relative standard deviation of the peak area for dinoprostone in the
chromatogram obtained with reference solution (d) is at most 2.0 per cent.
Inject 20 l of test solution (b). Calculate the percentage content of dinoprostone.
STORAGE
Store in a well-closed container, at a temperature not exceeding 15C.
IMPURITIES
CH
3
H OH
H
H
OH
H
COOH
O
A. (Z)-7-[(1R,2R,3R)-3-hydroxy-2-[(E)-(3R)-3-hydroxyoct-1-enyl]-5-oxocyclopentyl]hept-5-
enoic acid (15-epiPGE
2
; (15R)-PGE
2
),
14-39
CH
3
H OH
H
H
COOH
O
OH
H
B. (Z)-7-[(1S,2R,3R)-3-hydroxy-2-[(E)-(3S)-3-hydroxyoct-1-enyl]-5-oxocyclopentyl]hept-5-
enoic acid (8-epiPGE
2
; (8S)-PGE
2
),
COOH
CH
3
H OH
H
H
OH
H
O
C. (E)-7-[(1R,2R,3R)-3-hydroxy-2-[(E)-(3S)-3-hydroxyoct-1-enyl]-5-oxocyclopentyl]hept-5-
enoic acid (5-trans-PGE
2
; (5E)-PGE
2
),
CH
3
H OH
H
H
COOH
O
D. (Z)-7-[(1R,2S)-2-[(E)-(3S)-3-hydroxyoct-1-enyl]-5-oxocyclopent-3-enyl]hept-5-enoic acid
(PGA
2
),
CH
3
H OH
COOH
O
E. (Z)-7-[-2-[(E)-(3S)-3-hydroxyoct-1-enyl]-5-oxocyclopent-1-enyl]hept-5-enoic acid
(PGB
2
),
CH
3
H
H
OH
H
COOH
O
O
F. (Z)-7-[(1R,2R,3R)-3-hydroxy-2-[(E)-3-oxooct-1-enyl]-5-oxocyclopentyl]hept-5-enoic acid
(15-oxo-PGE
2
; 15-keto-PGE
2
).
___________________________________________________________________________________________________ Ph Eur
14-40
Diphenhydramine Hydrochloride
1/01
O
NMe
2
H
,HCl
C
17
H
21
NO,HCl 291.8 147-24-0
Diphenhydramine Hydrochloride complies with the requirements of the 3rd edition of the European
1
Preparation
Diphenhydramine Oral Solution
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Diphenhydramine hydrochloride contains not less than 99.0 per cent and not more than the equiva-
lent of 101.0 per cent of 2-diphenylmethoxy-N,N-dimethylethylamine hydrochloride, calculated with
CHARACTERS
A white or almost white, crystalline powder, very soluble in water, freely soluble in alcohol.
IDENTIFICATION
B. Dissolve 50 mg in alcohol R and dilute to 100.0 ml with the same solvent. Examined between
230 nm and 350 nm, the solution shows 3 absorption maxima (2.2.25), at 253 nm, 258 nm and
264 nm. The specific absorbances at the maxima are about 12, 15 and 12, respectively.
obtained with diphenhydramine hydrochloride CRS. Examine the substances as discs prepared using
D. To 0.05 ml of solution S (see Tests) add 2 ml of sulphuric acid R. An intense yellow colour
develops and becomes red on the addition of 0.5 ml of nitric acid R. Add 15 ml of water R, cool, add
5 ml of chloroform R and shake. An intense violet colour develops in the chloroform layer.
E. It gives the reactions of chlorides (2.3.1).
TESTS
Appearance of solution Solution S and a fivefold dilution of solution S are clear (2.2.1). Solution S
6
(Method II, 2.2.2).
Related substances Examine by thin-layer chromatography (2.2.27), using a TLC silica gel H plate R.
the same solvent. Prepare immediately before use.
diethylamine R, 20 volumes of methanol R and 80 volumes of chloroform R. Allow the plate to dry in air
for 5 min, spray with sulphuric acid R and heat at 120C for 10 min or until the spots appear. Any
100C to 105C.
14-41
ASSAY
Dissolve 0.250 g in 50 ml of alcohol R and add 5.0 ml of 0.01M hydrochloric acid. Carry out a
potentiometric titration (2.2.20), using 0.1M sodium hydroxide. Read the volume added between the 2
17
H
22
ClNO.
STORAGE
___________________________________________________________________________________________________ Ph Eur
14-42
Diphenoxylate Hydrochloride
N
CN COOEt
,HCl
C
30
H
32
N
2
O
2
,HCl 489.1 3810-80-8
Diphenoxylate Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Diphenoxylate hydrochloride contains not less than 98.0 per cent and not more than the equiva-
lent of 102.0 per cent of ethyl 1-(3-cyano-3,3-diphenylpropyl)-4-phenylpiperidine-4-carboxylate
CHARACTERS
A white or almost white, crystalline powder, very slightly soluble in water, freely soluble in methylene
chloride, sparingly soluble in alcohol, practically insoluble in ether.
IDENTIFICATION
spectrum of diphenoxylate hydrochloride.
B. Dissolve about 30 mg in 5 ml of methanol R. Add 0.25 ml of nitric acid R and 0.4 ml of silver nitrate
solution R1. Shake and allow to stand. A curdled precipitate is formed. Centrifuge and rinse the
precipitate with three quantities, each of 2 ml, of methanol R. Carry out this operation rapidly and
protected from bright light. Suspend the precipitate in 2 ml of water R and add 1.5 ml of ammonia R.
The precipitate dissolves easily.
TESTS
6
(Method II, 2.2.2).
suitable octadecylsilyl silica gel (5 m) with a fluorescent indicator having an optimal intensity at
254 nm.
Test solution. Dissolve 1.0 g in a mixture of 1 volume of methanol R and 2 volumes of methylene
chloride R and dilute to 20 ml with the same mixture of solvents.
Reference solution (a). Dilute 0.5 ml of the test solution to 100 ml with a mixture of 1 volume of
methanol R and 2 volumes of methylene chloride R.
Reference solution (b). Dissolve 0.50 g of the substance to be examined in 25 ml of a 15 g/l solution of
potassium hydroxide R in methanol R and add 1 ml of water R. Heat on a water-bath under a reflux
condenser for 4 h. Cool and add 25 ml of 0.5M hydrochloric acid. Shake with 100 ml of methylene
chloride R. Evaporate the lower layer to dryness on a water-bath. Dissolve the residue in 10 ml of a
mixture of 1 volume of methanol R and 2 volumes of methylene chloride R, add 10 ml of the test
solution and dilute to 25 ml with a mixture of 1 volume of methanol R and 2 volumes of methylene
chloride R.
Apply separately to a plate (100 mm square) 1 l of each solution. Develop in an unsaturated tank
over a path of 7 cm using a mixture of 10 volumes of methanol R, 30 volumes of a 59 g/l solution of
sodium chloride R and 60 volumes of dioxan R. Allow the plate to dry in an oven at 160C for 15 min
and place the hot plate in a closed tank containing about 20 ml of fuming nitric acid R for 30 min.
14-43
Remove the plate and heat it again at 160C for 15 min. Allow to cool and examine immediately in
ultraviolet light at 254 nm. Any spot in the chromatogram obtained with the test solution, apart from
the principal spot, is not more intense than the spot in the chromatogram obtained with reference
solution (a) (0.5 per cent). The test is not valid unless the chromatogram obtained with reference
solution (b) shows two clearly separated principal spots.
100C to 105C.
ASSAY
potentiometric titration (2.2.20), using 0.1M ethanolic sodium hydroxide. Read the volume added
between the two points of inflexion.
30
H
33
ClN
2
O
2
.
STORAGE
___________________________________________________________________________________________________ Ph Eur
14-44
Diphenylpyraline Hydrochloride
O
NMe
H H
,HCl
C
19
H
23
NO,HCl 317.9 132-18-3
Definition Diphenylpyraline Hydrochloride is 4-benzhydryloxy-1-methylpiperidine hydrochloride.
19
H
23
NO,HCl, calculated with
Freely soluble in water, in chloroform and in ethanol (96%); practically insoluble in ether.
Identification
diphenylpyraline hydrochloride (RS 106).
following solutions. For solution (1) dissolve 45 mg of bibenzyl (internal standard) in sufficient
chloroform to produce 100 ml. For solution (2) dissolve 0.20 g of the substance being examined in
20 ml of water, make the solution alkaline with 5M ammonia and extract with three 25-ml quantities
of chloroform. Shake the combined extracts with 10 g of anhydrous sodium sulphate, filter, evaporate the
filtrate to dryness at about 30 and dissolve the residue in 2 ml of chloroform. Prepare solution (3) in
the same manner as solution (2) but dissolve the residue in 2 ml of solution (1).
methyl silicone fluid (50% phenyl) (OV-17 is suitable) and maintained at 165. After allowing
chromatography to proceed for 3 times the retention time of bibenzyl, increase the oven temperature
to 240 to elute the diphenylpyraline from the column.
greater than the area of the peak due to the internal standard.
1 g.
Assay Carry out Method I for non-aqueous titration, Appendix VIII A, using 0.2 g, adding 5 ml of
mercury(II) acetate solution and using oracet blue B solution as indicator. Each ml of 0.1M perchloric acid
VS is equivalent to 31.79 mg of C
19
H
23
NO,HCl.
1
14-45
Dipipanone Hydrochloride
N
CO
CH
3
Me H
and enantiomer
, HCl
C
24
H
31
NO,HCl,H
2
O 404.0 856-87-1
Definition Dipipanone Hydrochloride is (RS)-4,4-diphenyl-6-piperidinoheptan-3-one
hydrochloride monohydrate. It contains not less than 99.0% and not more than 101.0% of
C
24
H
31
NO,HCl, calculated with reference to the anhydrous substance.
Characteristics A white, crystalline powder; odourless or almost odourless.
Sparingly soluble in water; freely soluble in acetone and in ethanol (96%); practically insoluble in
ether.
Identification
A. Dissolve 20 mg in 5 ml of water and make alkaline to litmus paper with 2M sodium hydroxide.
Extract with two 10-ml quantities of chloroform, evaporate the chloroform extracts and dry at 50 at a
pressure not exceeding 0.7 kPa for 2 hours. The infrared absorption spectrum of a thin film of the oily
residue, Appendix II A, is concordant with the reference spectrum of dipipanone (RS 107).
Melting point 124 to 127, determined on the undried substance, Appendix V A.
silica gel precoated plate (Merck silica gel 60 plates are suitable) and methanol as the mobile phase.
Apply separately to the plate 5 l of each of two solutions freshly prepared in methanol containing (1)
2.0% w/v of the substance being examined and (2) 0.020% w/v of 3-methyl-2,2-diphenyl-4-
piperidinobutyronitrile BPCRS. After removal of the plate, allow it to dry in air and expose to iodine
vapour for 10 minutes. Any secondary spot in the chromatogram obtained with solution (1) is not
more intense than the spot in the chromatogram obtained with solution (2).
C
24
H
31
NO,HCl.
Preparation
Dipipanone and Cyclizine Tablets
14-46
Dipivefrine Hydrochloride
NHMe
H OH
O
O
O CMe
3
O
Me
3
C
and enantiomer
,HCl
C
19
H
29
NO
5
,HCl 387.9 64019-93-8
Definition Dipivefrine Hydrochloride is (RS)-4-[1-hydroxy-2-(methylamino)ethyl]-o-phenylene
dipivalate hydrochloride. It contains not less than 98.5% and not more than 101.5% of
C
19
H
29
NO
5
Characteristics A white, crystalline powder or small crystals. It melts at about 160.
Freely soluble in water, in ethanol (96%) and in methanol.
Identification
dipivefrine hydrochloride (RS 382).
Heavy metals A 12.0% w/v solution complies with limit test A for heavy metals, Appendix VII
(15 ppm). Use lead standard solution (2 ppm Pb) to prepare the standard.
Iron In a Nessler cylinder, dissolve 2.0 g of the substance being examined in a mixture of 45 ml of
water and 2 ml of hydrochloric acid, add 50 mg of ammonium persulphate and 3 ml of a 30% w/v
solution of ammonium thiocyanate. Any colour produced is not more intense than that produced by
treating a mixture of 1 ml of iron standard solution (10 ppm Fe), 44 ml of water and 2 ml of hydrochloric
acid in the same manner beginning at the words add 50 mg of ammonium persulphate (5 ppm).
following solutions. Solution (1) contains 0.5% w/v of the substance being examined in 0.0015M
hydrochloric acid. Solution (2) contains 0.001% w/v of the substance being examined in 0.0015M
hydrochloric acid. For solution (3) dissolve the contents of one vial of dipivefrine impurity standard
BPCRS (the impurity standard contains 99 mg of dipivefrine hydrochloride and 1 mg of dipivalyl-
adrenalone hydrochloride) in 0.0015M hydrochloric acid, wash the container with the same solvent and
dilute the solution and washings to 20 ml with the same solvent.
3.9 mm) packed with stationary phase C (10 m) (Bondapak C18 is suitable), (b) as the mobile
phase with a flow rate of 1 ml per minute a mixture of 1 volume of glacial acetic acid, 15 volumes of
0.014M sodium dodecyl sulphate and 24 volumes of acetonitrile, (c) a detection wavelength of 254 nm
and (d) an electronic integrator.
The test is not valid unless the chromatogram obtained with solution (3) resembles the appropriate
chromatogram provided with the impurity standard.
dipivalyladrenalone hydrochloride is not greater than 0.2 times the area of the peak due to dipivalyl-
adrenalone hydrochloride in the chromatogram obtained with solution (3) (0.2%), the area of any
other secondary peak is not greater than the area of the peak in the chromatogram obtained with
solution (2) (0.2%) and the sum of the areas of any such peaks is not greater than five times the area
of the peak in the chromatogram obtained with solution (2) (1%).
Loss on drying When dried over phosphorus pentoxide at 60 at a pressure of 2 kPa for 6 hours, loses
Solution (1) contains 0.1% w/v of the substance being examined in 0.0015M hydrochloric acid.
Solution (2) contains 0.1% w/v of dipivefrine hydrochloride BPCRS in 0.0015M hydrochloric acid. For
solution (3) dilute 1 volume of solution (3) prepared in the test for Related substances to 5 volumes
with 0.0015M hydrochloric acid.
The assay is not valid unless the chromatogram obtained with solution (3) resembles the
appropriate reference chromatogram provided with the impurity standard.
19
H
29
NO
5
,HCl in the substance being examined from the
14-47
chromatograms obtained and using the declared content of C
19
H
29
NO
5
,HCl in dipivefrine
hydrochloride BPCRS.
Action and use Sympathomimetic.
Preparation
Dipivefrine Eye Drops
IMPURITIES
NHMe O
O
O CMe
3
O
Me
3
C
O
A. Dipivalyladrenalone
14-48
Dipotassium Clorazepate
1/01
and enantiomer
N
N
Cl
Me
O
COOK
H
KOH
C
16
H
11
ClK
2
N
2
O
4
408.9 57109-90-7
Dipotassium Clorazepate complies with the requirements of the 3rd edition of the European Pharmacopoeia
When potassium clorazepate is prescribed or demanded, Dipotassium Clorazepate shall be dispensed
or supplied.
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Dipotassium clorazepate contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of potassium (3RS)-7-chloro-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepine-3-
carboxylate compound with potassium hydroxide (1:1), calculated with reference to the dried
substance.
CHARACTERS
A white or light yellow, crystalline powder, freely soluble or very soluble in water, very slightly soluble
in alcohol, practically insoluble in methylene chloride. Solutions in water and in alcohol are unstable
and are to be used immediately.
IDENTIFICATION
A. Dissolve 10.0 mg in a 0.3 g/l solution of potassium carbonate R and dilute to 100.0 ml with the
same solution (solution A). Examined between 280 nm and 350 nm (2.2.25), solution A shows a
broad absorption maximum at about 315 nm. The specific absorbance at the maximum is 49 to 56.
Dilute 10.0 ml of solution A to 100.0 ml with a 0.3 g/l solution of potassium carbonate R (solution B).
Examined between 220 nm and 280 nm (2.2.25), solution B shows an absorption maximum at
230 nm. The specific absorbance at the maximum is 800 to 870.
spectrum of dipotassium clorazepate. Examine the substance prepared as a disc.
C. Dissolve about 20 mg in 2 ml of sulphuric acid R. Observed in ultraviolet light at 365 nm, the
solution shows yellow fluorescence.
D. Dissolve 0.5 g in 5 ml of water R. Add 0.1 ml of thymol blue solution R. The solution is violet-blue.
E. Place 1.0 g of the substance to be examined in a crucible and add 2 ml of dilute sulphuric acid R.
Heat at first on a water-bath, then ignite until all black particles have disappeared. Allow to cool.
Take up the residue with water R and dilute to 20 ml with the same solvent. The solution gives
reaction (b) of potassium (2.3.1).
TESTS
Appearance of solution Rapidly dissolve with shaking 1.0 g in water R and dilute to 10.0 ml with
the same solvent. Observed immediately, the solution is clear (2.2.1) and not more intensely coloured
than reference solution GY
5
(Method II, 2.2.2).
254
plate R.
14-49
Prepare the solutions immediately before use and carry out the test protected from light.
Test solution. Dissolve 0.10 g of the substance to be examined in a 40 g/l solution of potassium
carbonate R and dilute to 5 ml with the same solution.
Reference solution (a). Dissolve 10 mg of aminochlorobenzophenone R in acetone R and dilute to 100 ml
with the same solvent. Dilute 5 ml of this solution to 25 ml with acetone R.
Reference solution (b). Dissolve 10 mg of nordazepam CRS in acetone R and dilute to 50 ml with the
same solvent. Dilute 5 ml of this solution to 25 ml with acetone R.
Reference solution (c). Dilute 0.5 ml of the test solution to 100 ml with a 40 g/l solution of potassium
carbonate R.
Reference solution (d). Dissolve 10 mg of nordazepam CRS and 10 mg of nitrazepam CRS in acetone R
of acetone R and 85 volumes of methylene chloride R. Allow the plate to dry in air and examine in
ultraviolet light at 254 nm. In the chromatogram obtained with the test solution: any spot corres-
ponding to nordazepam is not more intense than the spot in the chromatogram obtained with
reference solution (b) (0.2 per cent); any spot apart from the principal spot and any spot correspond-
ing to nordazepam is not more intense than the spot in the chromatogram obtained with reference
solution (c) (0.5 per cent).
Spray the plate with a freshly prepared 10 g/l solution of sodium nitrite R in dilute hydrochloric acid R.
Dry the plate in a current of warm air and spray with a 4 g/l solution of naphthylethylenediamine
dihydrochloride R in alcohol R. Any spot corresponding to aminochlorobenzophenone in the chromato-
gram obtained with the test solution is not more intense than the spot in the chromatogram obtained
with reference solution (d) shows two clearly separated spots.
60C for 4 h.
ASSAY
Dissolve 0.130 g in 10 ml of anhydrous acetic acid R. Add 30 ml of methylene chloride R. Titrate with
0.1M perchloric acid, determining the two points of inflexion by potentiometry (2.2.20). The ratio of
the volume of perchloric acid used up to the second point of inflexion to that used up to the first
point of inflexion is 1.48 to 1.52.
At the second point of inflexion, 1 ml of 0.1M perchloric acid is equivalent to 13.63 mg of
C
16
H
11
ClK
2
N
2
O
4
.
STORAGE
14-50
IMPURITIES
Cl
NH
2
O
A. (2-amino-5-chlorophenyl)phenylmethanone,
N
H
N
O
Cl
B. 7-chloro-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one (nordazepam).
___________________________________________________________________________________________________________________________________ Ph Eur
14-51
Dipotassium Hydrogen Phosphate
K
2
HPO
4
174.2 7758-11-4
Dipotassium Hydrogen Phosphate complies with the requirements of the 3rd edition of the European
Pharmacopoeia for Dipotassium Phosphate [1003]. These requirements are reproduced after the heading
Definition below.
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Dipotassium phosphate contains not less than 98.0 per cent and not more than the equivalent of
101.0 per cent of K
2
HPO
4
CHARACTERS
A white powder or colourless crystals, very hygroscopic, very soluble in water, very slightly soluble in
alcohol.
IDENTIFICATION
A. Solution S (see Tests) is slightly alkaline (2.2.4).
B. Solution S gives reaction (b) of phosphates (2.3.1).
C. Solution S gives reaction (a) of potassium (2.3.1).
TESTS
Reducing substances Heat on a water-bath for 5 min a mixture of 5 ml of solution S, 5 ml of dilute
sulphuric acid R and 0.25 ml of 0.02M potassium permanganate. The solution remains faintly pink.
Monopotassium phosphate Calculated from the number of millilitres of 1M hydrochloric acid
(10.0 ml) and of 1M sodium hydroxide (n
1
ml and n
2
ml) used in the assay, the ratio:
1
2
10
10
n
n
does not exceed 0.025 (2.5 per cent).

Chlorides (2.4.4). To 2.5 ml of solution S add 10 ml of dilute nitric acid R and dilute to 15 ml with
Sulphates (2.4.13). To 1.5 ml of solution S add 2 ml of dilute hydrochloric acid R and dilute to 15 ml
with distilled water R. The solution complies with the limit test for sulphates (0.1 per cent).
Heavy metals (2.4.8). Dissolve 2.0 g in 8 ml of water R. Acidify with about 6 ml of dilute hydrochloric
acid R (pH 3 to 4) and dilute to 20 ml with water R. 12 ml of the solution complies with limit test A
for heavy metals (10 ppm). Prepare the standard using lead standard solution (1 ppm Pb) R.
Sodium If intended for use in the manufacture of parenteral dosage forms, it contains not more than
0.1 per cent of Na, determined by atomic emission spectrometry (Method I, 2.2.22).
the same solvent.
Reference solutions. Prepare the reference solutions using sodium standard solution (200 ppm Na) R,
diluted as necessary with water R.
Measure the emission intensity at 589 nm.
125C to 130C.
without a further appropriate procedure for the removal of bacterial endotoxins, it contains not more
than 1.1 I.U. of endotoxin per milligram.
14-52
ASSAY
Dissolve 0.800 g (m) in 40 ml of carbon dioxide-free water R and add 10.0 ml of 1M hydrochloric acid.
Using 1M sodium hydroxide, titrate potentiometrically (2.2.20) to the first inflexion point of the pH
curve (n
1
ml). Continue the titration to the second inflexion point of the curve (total volume of 1M
sodium hydroxide required, n
2
ml).
Calculate the percentage content of K
2
HPO
4
) 100 (
) 10 ( 1742
1
d m
n
d=percentage loss on drying.

STORAGE
LABELLING
The label states:
forms,
___________________________________________________________________________________________________ Ph Eur
14-53
Diprophylline
N
N
N
O
O
Me
OH
H OH
MeN
and enantiomer
C
10
H
14
N
4
O
4
254.2 479-18-5
Diprophylline complies with the requirements of the 3rd edition of the European Pharmacopoeia [0486]. These
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Diprophylline contains not less than 98.5 per cent and not more than the equivalent of 101.0 per
cent of 7-[(2RS)-2,3-dihydroxypropyl]-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione, calculated
CHARACTERS
A white, crystalline powder, freely soluble in water, slightly soluble in alcohol.
IDENTIFICATION
obtained with diprophylline CRS. Examine the substances as discs prepared using 0.5 mg to 1 mg of
the substance to be examined in 0.3 g of potassium bromide R.
C. Dissolve 1 g in 5 ml of acetic anhydride R and boil under a reflux condenser for 15 min. Allow to
cool and add 100 ml of a mixture of 20 volumes of ether R and 80 volumes of light petroleum R. Cool
in iced water for at least 20 min, shaking from time to time. Filter, wash the precipitate with a
mixture of 20 volumes of ether R and 80 volumes of light petroleum R, recrystallise from alcohol R and
dry in vacuo. The crystals melt (2.2.14) at 142C to 148C.
D. It gives the reaction of xanthines (2.3.1).
TESTS
Acidity or alkalinity To 10 ml of solution S add 0.25 ml of bromothymol blue solution R1. The
solution is yellow or green. Not more than 0.4 ml of 0.01M sodium hydroxide is required to change the
Related substances Examine by thin-layer chromatography (2.2.27), using silica gel HF
254
R as the
coating substance.
and 30 volumes of methanol R and dilute to 10 ml with the same mixture of solvents. Prepare
Reference solution (b). Dilute 0.2 ml of the test solution to 100 ml with methanol R.
Reference solution (c). Dissolve 10 mg of theophylline R in methanol R, add 0.3 ml of the test solution
of concentrated ammonia R, 10 volumes of ethanol R and 90 volumes of chloroform R. Allow the plate
to dry in air and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with
the test solution, apart from the principal spot, is not more intense than the spot in the chromato-
gram obtained with reference solution (a) (1 per cent) and at most one such spot is more intense than
14-54
spots.
100C to 105C.
ASSAY
Dissolve 0.200 g in 3.0 ml of anhydrous formic acid R and add 50.0 ml of acetic anhydride R. Titrate
10
H
14
N
4
O
4
.
STORAGE
___________________________________________________________________________________________________ Ph Eur
14-55
Dipyridamole
HO N
HO
N
N
N
N
N
N
OH
OH
N
C
24
H
40
N
8
O
4
504.6 58-32-2
Dipyridamole complies with the requirements of the 3rd edition of the European Pharmacopoeia [1199]. These
Action and use Antiplatelet aggregating agent.
Preparation
Dipyridamole Tablets
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Dipyridamole contains not less than 98.5 per cent and not more than the equivalent of 101.5 per
cent of 2,2,2,2-[[4,8-di(piperidin-1-yl)pyrimido[5,4-d]pyrimidine-2,6-diyl]dinitrilo]tetraethanol,
CHARACTERS
A bright yellow, crystalline powder, practically insoluble in water, freely soluble in acetone, soluble in
ethanol, practically insoluble in ether. It dissolves in dilute solutions of mineral acids.
IDENTIFICATION
B. Dissolve 10 mg in a mixture of 1 volume of 0.1M hydrochloric acid and 9 volumes of methanol R and
dilute to 50.0 ml with the same mixture of solvents. Dilute 5.0 ml of this solution to 100.0 ml with a
mixture of 1 volume of 0.1M hydrochloric acid and 9 volumes of methanol R. Examined between
220 nm and 350 nm (2.2.25), the solution shows two absorption maxima at 232 nm and 284 nm.
The ratio of the absorbance measured at the maximum at 284 nm to that measured at the maximum
at 232 nm is 1.25 to 1.45.
obtained with dipyridamole CRS. Examine the substances as discs prepared using potassium bromide R.
D. Dissolve about 5 mg in a mixture of 0.1 ml of nitric acid R and 2 ml of sulphuric acid R. An intense
violet colour is produced.
TESTS
Test solution. Dissolve 10.0 mg in the mobile phase and dilute to 20.0 ml with the mobile phase.
Reference solution (b). Dissolve 10.0 mg of diltiazem hydrochloride CRS in the mobile phase and dilute
to 10.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 20.0 ml with reference solution
(a).
14-56
as mobile phase at a flow rate of 1.3 ml per minute a mixture prepared as follows: dissolve
0.504 g of potassium dihydrogen phosphate R in 370 ml of water R and adjust to pH 3.0 with
phosphoric acid R; add 80 ml of acetonitrile R and 550 ml of methanol R,
Inject 20 l of each solution and continue the chromatography of the test solution for nine times
the retention time of dipyridamole. The test is not valid unless, in the chromatogram obtained with
reference solution (b), the resolution between the peaks corresponding respectively to diltiazem and
dipyridamole is at least 2.0. In the chromatogram obtained with the test solution: the area of any
peak, apart from the principal peak is not greater than the area of the peak in the chromatogram
obtained with reference solution (a) (0.5 per cent); and the sum of the areas of all the peaks, apart
from the principal peak, is not greater than twice the area of the peak in the chromatogram obtained
with reference solution (a) (1 per cent). Disregard any peak with an area less than 0.1 times that of
the peak in the chromatogram obtained with reference solution (a).
Chlorides (2.4.4). To 0.250 g add 10 ml of water R and strongly shake. Filter, rinse the filter with
5 ml of water R and dilute to 15 ml with water R. The combined filtrates comply with the limit test
for chlorides (200 ppm).
100C to 105C.
ASSAY
Dissolve 0.400 g in 70 ml of methanol R. Titrate with 0.1M perchloric acid, determining the end-point
24
H
40
N
8
O
4
.
STORAGE
IMPURITIES
N
OH
OH
R
N
N
N
N
N
R
1
A. R = R
1
= NC
5
H
10
: 2,2-[[4,6,8-tri(piperidin-1-yl)pyrimido[5,4-d]pyrimidin-2-yl]nitrilo]-
diethanol,
B. R = R
1
= N(CH
2
-CH
2
OH)
2
:
2,2,2,2,2, 2-[[8-(piperidin-1-yl)pyrimido[5,4-d]pyrimidine-2,4,6-triyl]-
trinitrilo]hexaethanol,
C. R = NEt
2
, R
1
= Cl : 2,2-[[2-chloro-4,8-di(piperidin-1-yl)pyrimido[5,4-d]pyrimidin-6-
yl]nitrilo]diethanol.
___________________________________________________________________________________________________ Ph Eur
14-57
Dipyrone
N
N
O
Me
Ph Me
N NaO
3
S
Me
C
13
H
16
N
3
NaO
4
S,H
2
O 351.4 5907-38-0
Dipyrone complies with the requirements of the 3rd edition of the European Pharmacopoeia for Metamizole
Sodium [1346]. These requirements are reproduced after the heading Definition below.
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Metamizole sodium contains not less than 99.0 and not more than the equivalent of 100.5 per cent
of sodium [(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-N-methylamino]methane-
sulphonate, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, very soluble in water, soluble in alcohol.
IDENTIFICATION
obtained with metamizole sodium CRS.
B. Dissolve 50 mg in 1 ml of strong hydrogen peroxide solution R. A blue colour is produced which
fades rapidly and turns to intense red in a few minutes.
C. Place 0.10 g in a test tube, add some beads of glass and dissolve the substance in 1.5 ml of
water R. Add 1.5 ml of dilute hydrochloric acid R and place a filter paper wetted with a solution of
20 mg of potassium iodate R in 2 ml of starch solution R at the open end of the test tube. Heat gently,
the evolving vapour of sulphur dioxide colours the filter paper blue. After heating gently for 1 min
take a glass rod with a drop of a 10 g/l solution of chromotropic acid sodium salt R in sulphuric acid R
and place in the opening of the tube. Within 10 min, a blue-violet colour develops in the drop of the
reagent.
D. 0.5 ml of solution S (see Tests) gives reaction (a) of sodium (2.3.1).
TESTS
Appearance of solution Solution S is clear (2.2.1) and immediately after preparation, not more
6
(Method I, 2.2.2).
Acidity or alkalinity To 5 ml of solution S, add 0.1 ml of phenolphthalein solution R1. The solution
is colourless. Not more than 0.1 ml of 0.02M sodium hydroxide is required to change the colour of the
indicator to pink.
Reference solution (a). Dissolve 10.0 mg of metamizole impurity A CRS in methanol R and dilute to
Reference solution (b). Dilute 1.0 ml of reference solution (a) to 20.0 ml with methanol R.
Reference solution (c). Dissolve 40 mg of metamizole sodium CRS in methanol R and dilute to 20.0 ml
Reference solution (d). Take 10 ml of reference solution (c) and boil under a reflux condenser for
10 min. Allow to cool to room temperature and dilute to 20.0 ml with methanol R.
Reference solution (e). To 6 ml of reference solution (a) add 1 ml of reference solution (c).
14-58
volumes of a buffer solution prepared by adjusting a mixture of 1000 volumes of a 6.0 g/l
solution of sodium dihydrogen phosphate R and 1 volume of triethylamine R to pH 7.0 with strong
sodium hydroxide solution R,
When the chromatograms are recorded in the prescribed conditions, the substances elute in the
following order: impurity A, metamizole, impurity B, impurity C and impurity D. Inject 10 l of
the chromatogram obtained is at least 50 per cent of the full scale of the recorder.
Inject 10 l of reference solution (d). The chromatogram shows two principal peaks due to
metamizole and impurity C.
Inject 10 l of reference solution (e). The test is not valid unless in the chromatogram obtained the
resolution between the peaks corresponding to impurity A and metamizole is at least 2.5.
Inject 10 l of the test solution and 10 l of reference solution (b) and continue the chromato-
graphy for 3.5 times the retention time of metamizole. In the chromatogram obtained with the test
solution: the area of any peak corresponding to impurity C is not greater than the area of the
principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent), the area of
any peaks, apart from the principal peak and the peak due to impurity C is not greater than 0.4 times
cent). The sum of the areas of all the peaks, apart from the principal peak, is not greater than the area
of the principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent).
Disregard any peak with an area less than 0.05 times that of the principal peak in the chromatogram
obtained with the reference solution (b).
Sulphates (2.4.13). Dissolve 0.150 g in distilled water R and dilute to 15 ml with the same solvent.
The solution complies with the limit test for sulphates (0.1 per cent).
the freshly prepared solution complies with limit test A for heavy metals (20 ppm). Prepare the
100C to 105C.
ASSAY
Dissolve 0.200 g in 10 ml of 0.01M hydrochloric acid previously cooled in iced water and titrate
immediately, dropwise, with 0.05M iodine. Before each addition of 0.05M iodine dissolve the
precipitate by swirling. At the end of the titration add 2 ml of starch solution R and titrate until the
blue colour of the solution persists for at least 2 min. The temperature of the solution during the
titration must not exceed 10C.
13
H
16
N
3
NaO
4
S.
STORAGE
IMPURITIES
N
N
O
Ph
R
Me
Me
A. R = NHCHO: 4-formylamino-1,5-dimethyl-2-phenyl-1,2-dihydro-3H-pyrazol-3-one,
B. R = NH
2
: 4-amino-1,5-dimethyl-2-phenyl-1,2-dihydro-3H-pyrazol-3-one,
C. R = NHCH
3
: 4-methylamino-1,5-dimethyl-2-phenyl-1,2-dihydro-3H-pyrazol-3-one,
D. R = N(CH
3
)
2
: 4-dimethylamino-1,5-dimethyl-2-phenyl-1,2-dihydro-3H-pyrazol-3-one.
___________________________________________________________________________________________________ Ph Eur
14-59
Dirithromycin
Me
Me
HO
Me
O
Et
OH
Me
Me
O
O
O
OH
Me
NMe
2
Me
O
OMe
Me
HO
Me
O
O
NH O
O
H
3
C
H
*
C
42
H
78
N
2
O
14
835 62013-04-1
Dirithromycin complies with the requirements of the 3rd edition of the European Pharmacopoeia [1313]. These
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Dirithromycin is (1R,2S,3R,6R,7S,8S,9R,10R,12R,13S,15R,17S)-9-[[3-(dimethylamino)-3,4,6-
trideoxy--D-xylo-hexopyranosyl]oxy]-3-ethyl-2,10-dihydroxy-15-[(2-methoxyethoxy)methyl]-
2,6,8,10,12,17-hexamethyl-7-[(3-C-methyl-3-O-methyl-2,6-dideoxy--L-ribo-hexopyranosyl)oxy]-
4,16-dioxa-14-azabicyclo[11.3.1]heptadecan-5-one (or (9S)-9,11-[imino[(1R)-2-(2-methoxy-
ethoxy)ethylidene]oxy]-9-deoxo-11-deoxyerythromycin). The sum of the percentage contents of
C
42
H
78
N
2
O
14
and dirithromycin 15S-epimer is not less than 96.0 per cent and not more than the
equivalent of 102.0 per cent, calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white powder, very slightly soluble in water, very soluble in methanol and in
methylene chloride.
IDENTIFICATION
obtained with dirithromycin CRS. Examine the substance prepared as discs.
peak in the chromatogram obtained with test solution (a) are similar to those of the principal peak in
TESTS
peak is at least 50 per cent of the full scale of the recorder. Inject 10 l of test solution (b) and
continue the chromatography for three times the retention time of the principal peak. In the chroma-
togram obtained with test solution (b): the area of any peak corresponding to impurity A is not
solution (b) (1.5 per cent); the area of any peak, apart from the principal peak and any peak corres-
ponding to impurity A and any peak corresponding to 15S-epimer, is not greater than half the area of
the principal peak in the chromatogram obtained with reference solution (b) (1 per cent).
Dirithromycin 15S-epimer Not more than 1.5 per cent, determined by liquid chromatography
(2.2.29), as described under Assay. Inject 10 l of reference solution (b). Adjust the sensitivity of the
system so that the height of the principal peak is at least 50 per cent of the full scale of the recorder.
14-60
Inject reference solution (b) six times. The test is not valid unless the relative standard deviation of
the peak area for dirithromycin is at most 5.0 per cent. Inject alternately test solution (b) and
reference solution (b). Calculate the percentage content of dirithromycin 15S-epimer using the
Acetonitrile (2.4.24, System A). Not more than 0.1 per cent.
Prepare the solutions using dimethylformamide R instead of water R.
Sample solution. Dissolve 0.200 g of the substance to be examined in dimethylformamide R and dilute
transfer-line temperature: 125C.
Heavy metals (2.4.8). Dissolve 1.0 g in 20 ml of a mixture of equal volumes of methanol R and
water R. 12 ml of the solution complies with limit test B for heavy metals (20 ppm). Prepare the
standard using lead standard solution (1 ppm Pb) obtained by diluting lead standard solution (100 ppm
Pb) R with a mixture of equal volumes of methanol R and water R.
of water.
ASSAY
Solvent mixture. Mix 30 volumes of methanol R and 70 volumes of acetonitrile R.
Test solution (a). Dissolve 20.0 mg of the substance to be examined in the solvent mixture and dilute
to 10.0 ml with the solvent mixture.
Test solution (b). Dissolve 0.10 g of the substance to be examined in the solvent mixture and dilute to
Reference solution (a). Dissolve 20.0 mg of dirithromycin CRS in the solvent mixture and dilute to
Reference solution (c). Dissolve 20 mg of dirithromycin CRS in the mobile phase and dilute to 10 ml
with the mobile phase. Allow to stand for 24 h before use.
as mobile phase at a flow rate of 2.0 ml/min a mixture of: 9 volumes of water R; 19 volumes of
methanol R; 28 volumes of a solution containing 1.9 g/l of potassium dihydrogen phosphate R and
9.1 g/l of dipotassium hydrogen phosphate R adjusted to pH 7.5 if necessary with a 100 g/l solution
of potassium hydroxide R; 44 volumes of acetonitrile R,
maintaining the temperature of the column at 40C. Inject 10 l of reference solution (a). Adjust the
sensitivity of the system so that the height of the principal peak in the chromatogram obtained is at
least 50 per cent of the full scale of the recorder. Inject 10 l of reference solution (c). When the
chromatogram is recorded in the prescribed conditions, the retention times relative to dirithromycin
are: impurity A about 0.7, 15S-epimer about 1.1. The test is not valid unless, in the chromatogram
obtained with reference solution (c), the resolution between the peaks corresponding to dirithromycin
and its 15S-epimer is at least 2.0 (if necessary adjust the concentration of the organic modifiers in the
mobile phase). Inject reference solution (a) six times. The test is not valid unless the relative standard
deviation of the peak area for dirithromycin is at most 1.0 per cent. Inject alternately test solution (a)
14-61
IMPURITIES
O
Me
Me
HO
OMe R =
Me
O
NH
2
Me
OH
Me
OH
Et
O
Me
RO
Me
HO
Me
O
O
Me
OH
NMe2
A. (9S)-9-amino-9-deoxoerythromycin,
B. R = H : (9S)-9-amino-3-de(2,6-dideoxy-3-C-methyl-3-O-methyl--L-ribo-hexopyranosyl)-9-
deoxoerythromycin,
O
Me
Me
HO
Me
O
NH
Me
Me
R
2
Et
O
Me
O
Me
HO
Me
O
H
R
R
O
O
Me
OH
NMe2
OR3
and epimer at C*
*
C. R = CH
2
-O-CH
2
-CH
2
-O-CH
3
, R = H, R2 = H, R3 = CH
3
: (9S)-9,11-[imino[(1RS)-2-(2-methoxy-
ethoxy)ethylidene]oxy]-9-deoxo-11,12-dideoxyerythromycin (dirithromycin B),
D. R = CH
2
-O-CH
2
-CH
2
-O-CH
3
, R = H, R2 = OH, R3 = H : (9S)-9,11-[imino[(1RS)-2-(2-methoxy-
ethoxy)ethylidene]oxy]-3-O-demethyl-9-deoxo-11-deoxyerythromycin (dirithromycin C),
E. R = CH
3
, R = CH
3
, R
2
= OH, R
3
= CH
3
: 9,11-[imino(1-methylethylidene)oxy]-9-deoxo-11-
deoxyerythromycin.
___________________________________________________________________________________________________ Ph Eur
14-62
Disodium Edetate
N N COOH HOOC
NaOOC COONa
C
10
H
14
N
2
Na
2
O
8
,2H
2
O 372.2 139-33-3
Disodium Edetate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0232].
Action and use Chelating agent.
Preparation
Trisodium Edetate Intravenous Infusion
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Disodium edetate contains not less than 98.5 per cent and not more than the equivalent of 101.0 per
cent of disodium (ethylenedinitrilo)tetra-acetate dihydrate.
CHARACTERS
A white, crystalline powder, soluble in water, sparingly soluble in alcohol, practically insoluble in
ether.
IDENTIFICATION
obtained with disodium edetate CRS. Examine the substances prepared as discs.
B. Dissolve 2 g in 25 ml of water R, add 6 ml of lead nitrate solution R, shake and add 3 ml of
potassium iodide solution R. No yellow precipitate is formed. Make alkaline to red litmus paper R by the
addition of dilute ammonia R2 and add 3 ml of ammonium oxalate solution R. No precipitate is formed.
C. Dissolve 0.5 g in 10 ml of water R and add 0.5 ml of calcium chloride solution R. Make alkaline to
red litmus paper R by the addition of dilute ammonia R2 and add 3 ml of ammonium oxalate solution R.
No precipitate is formed.
D. It gives the reactions of sodium (2.3.1).
TESTS
Iron (2.4.9). 2.5 ml of solution S diluted to 10 ml with water R complies with the limit test for iron
(80 ppm). Add 0.25 g of calcium chloride R to each solution before the addition of the thioglycollic
acid R.
ASSAY
Dissolve 0.500 g in water R and dilute to 300 ml with the same solvent. Add 2 g of hexamethylene-
tetramine R and 2 ml of dilute hydrochloric acid R. Titrate with 0.1M lead nitrate, using about 50 mg of
xylenol orange triturate R as indicator.
1 ml of 0.1M lead nitrate is equivalent to 37.22 mg of C
10
H
14
N
2
Na
2
O
8
,2H
2
O.
___________________________________________________________________________________________________ Ph Eur
14-63
Anhydrous Disodium Hydrogen Phosphate
1/01
Na
2
HPO
4
142.0 7558-79-4
Anhydrous Disodium Hydrogen Phosphate complies with the requirements of the 3rd edition of the European
Pharmacopoeia for Anhydrous Disodium Phosphate [1509]. These requirements are reproduced after the
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Anhydrous disodium phosphate contains not less than 98.0 per cent and not more than the equiva-
lent of 101.0 per cent of Na
2
HPO
4
CHARACTERS
A white powder, hygroscopic, soluble in water, practically insoluble in alcohol.
IDENTIFICATION
C. Solution S (see Tests) gives the reactions of phosphates (2.3.1).
D. Solution S (see Tests) gives the reactions of sodium (2.3.1).
TESTS
Solution S Dissolve 5.0 g in distilled water R and dilute to 100.0 ml with the same solvent.
Reducing substances To 10 ml of solution S add 5 ml of dilute sulphuric acid R and 0.25 ml of
0.02M potassium permanganate and heat on a water-bath for 5 min. The solution retains a slight red
colour.
Monosodium phosphate Calculated from the number of millilitres of 1M hydrochloric acid (25 ml)
and of 1M sodium hydroxide (n
1
ml and n
2
1
2
25
25
n
n
does not exceed 0.025.

Chlorides (2.4.4). 5 ml of solution S diluted to 15 ml with dilute nitric acid R complies with the limit
Sulphates (2.4.13). To 6 ml of solution S add 0.5 ml of dilute hydrochloric acid R and dilute to 15 ml
with distilled water R. The solution complies with the limit test for sulphates (500 ppm).
Prepare the standard using 5 ml of lead standard solution (1 ppm Pb) R and 5 ml of water R.
100C to 105C for 4 h.
ASSAY
Dissolve 1.600 g (m) in 25.0 ml of carbon dioxide-free water R and add 25.0 ml of 1M hydrochloric acid.
Using 1M sodium hydroxide, titrate potentiometrically (2.2.20) to the first inflexion point (n
1
ml).
Continue the titration to the second inflexion point (total volume of 1M sodium hydroxide required, n
2
ml).
Calculate the percentage content of Na
2
HPO
4
) 100 (
) 25 ( 1420
1
d m
n

STORAGE
___________________________________________________________________________________________________ Ph Eur
14-64
Disodium Hydrogen Phosphate Dihydrate
Sodium Phosphate Dihydrate
Na
2
HPO
4
,2H
2
O 178.0 10028-24-7
Disodium Hydrogen Phosphate Dihydrate complies with the requirements of the 3rd edition of the European
Pharmacopoeia for Disodium Phosphate Dihydrate [0602]. These requirements are reproduced after the
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Disodium phosphate dihydrate contains not less than 98.0 per cent and not more than the equivalent
of 101.0 per cent of Na
2
HPO
4
CHARACTERS
A white or almost white powder or colourless crystals, soluble in water, practically insoluble in
alcohol.
IDENTIFICATION
C. Solution S gives reaction (b) of phosphates (2.3.1).
D. Solution S gives reaction (a) of sodium (2.3.1).
TESTS
Reducing substances To 5 ml of solution S add 5 ml of dilute sulphuric acid R and 0.25 ml of 0.02M
potassium permanganate and heat on a water-bath for 5 min. The solution retains a slight red colour.
1
ml and n
2
n
n
2
1
25
25

with distilled water R. The solution complies with the limit test for sulphates (0.1 per cent).
(40 ppm).
at 130C.
ASSAY
Dissolve 2.000 g (m g) in 50 ml of water R and add 25.0 ml of 1M hydrochloric acid. Using 1M sodium
hydroxide, titrate potentiometrically (2.2.20) to the first inflexion point of the pH curve (n
1
ml).
Continue the titration to the second inflexion point of the curve (total volume of 1M sodium hydroxide
required, n
2
ml).
2
HPO
4
1420 25
100
1
( )
( )
n
m d
14-65
STORAGE
___________________________________________________________________________________________________ Ph Eur
14-66
Disodium Hydrogen Phosphate Dodecahydrate
Na
2
HPO
4
,12H
2
O 358.1 7782-75-6
Disodium Hydrogen Phosphate Dodecahydrate complies with the requirements of the 3rd edition of the
European Pharmacopoeia for Disodium Phosphate Dodecahydrate [0118]. These requirements are reproduced
after the heading Definition below.
Preparation
Phosphates Enema
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Disodium phosphate dodecahydrate contains not less than 98.0 per cent and not more than the
equivalent of 101.0 per cent of Na
2
HPO
4
CHARACTERS
Colourless, transparent crystals, very efflorescent, very soluble in water, practically insoluble in
alcohol.
IDENTIFICATION
A. Solution S (see Tests) gives the reactions of phosphates (2.3.1).
B. Solution S gives the reactions of sodium (2.3.1).
TESTS
Reducing substances To 5 ml of solution S add 5 ml of dilute sulphuric acid R and 0.25 ml of 0.02M
potassium permanganate and heat on a water-bath for 5 min. The solution retains a slight red colour.
1
ml and n
2
1
2
25
25
n
n

with distilled water R. The solution complies with the limit test for sulphates (500 ppm).
(20 ppm).
Water (2.5.12). 57.0 per cent to 61.0 per cent, determined on 50.0 mg by the semi-micro deter-
mination of water. Use a mixture of 10 volumes of anhydrous methanol R and 40 volumes of
formamide R as solvent.
ASSAY
Dissolve 4.00 g (m g) in 25 ml of water R and add 25.0 ml of 1M hydrochloric acid. Using 1M sodium
hydroxide, titrate potentiometrically (2.2.20) to the first inflexion point of the pH curve (n
1
ml).
Continue the titration to the second inflexion point of the curve (total volume of 1M sodium hydroxide
required, n
2
ml).
2
HPO
4
) 100 (
) 25 ( 1420
1
d m
n
d=percentage water content.

STORAGE
___________________________________________________________________________________________________ Ph Eur
14-67
Disodium Pamidronate
C
O
OH
ONa
P
O
OH
ONa
P
HO
H
2
N
C
3
H
9
NNa
2
O
7
P
2
,5H
2
O 369.1 57248-88-1
Definition Disodium Pamidronate is disodium 3-amino-1-hydroxypropylidenebis(phosphonate)
pentahydrate. It contains not less than 98.0% and not more than 102.0% of C
3
H
9
NNa
2
O
7
P
2
,
Characteristics A white crystalline powder.
Soluble in water and in 2M sodium hydroxide; sparingly soluble in 0.1M hydrochloric acid; practically
insoluble in organic solvents.
Identification
disodium pamidronate (RS 109).
time as the principal peak in the chromatogram obtained with solution (2).
C. A 5% w/v solution yields reaction A characteristic of sodium salts, Appendix VI.
Clarity and colour of solution A 2.0% w/v solution in carbon dioxide-free water is clear, Appendix
IV A, and not more intensely coloured than reference solution Y
6
Heavy metals 1.0 g complies with limit test C for heavy metals, Appendix VII (20 ppm). Use 2 ml of
-Alanine Carry out the method for thin-layer chromatography under subdued light, Appendix III A,
using a silica gel precoated plate (Merck silica gel 60 plates are suitable) and a mixture of 4 volumes
of 13.5M ammonia, 8 volumes of di-isopropyl ether and 9 volumes of methanol as the mobile phase but
allowing the solvent front to ascend 7 cm above the line of application. Apply separately to the plate
10 l of each of two solutions in water containing (1) 0.30% w/v of the substance being examined and
(2) 0.0015% w/v of 3-aminopropionic acid. After removal of the plate, allow it to dry in a current of
warm air and spray with a 0.01% w/v solution of fluorescamine in acetone, immerse the plate for 2
seconds in a mixture of 1 volume of liquid paraffin and 2 volumes of n-hexane, allow to dry in air for
15 minutes and examine under ultraviolet light (365 nm). In the chromatogram obtained with solution
(1) any spot corresponding to -alanine (3-aminopropionic acid) is not more intense than the spot in
the chromatogram obtained with solution (2) (0.5%).
Phosphate and phosphite Carry out the method for liquid chromatography, Appendix III D, using
the following solutions. Solution (1) contains 0.20% w/v of the substance being examined in water.
For solution (2) dilute a mixture of 2 ml of a 0.030% w/v solution of orthophosphoric acid and 2 ml of
a 0.025% w/v solution of orthophosphorous acid to 50 ml with water.
4.6 mm) packed with an anion-exchange stationary phase (5 m) (IC Anion Super-sep is suitable)
and maintained at 35, (b) as the mobile phase with a flow rate of 1.0 ml per minute a solution
prepared by adding 0.47 ml of formic acid to 2500 ml of water and adjusting the solution to pH 3.5
with 2M sodium hydroxide and (c) a refractive index detector. Inject 100 l of each solution.
In the chromatogram obtained with solution (2) the peaks appear in the order orthophosphoric
acid (retention time, 12 to 15 minutes) and orthophosphorous acid (retention time, 16 to 20
minutes).
In the chromatogram obtained with solution (1), the area of any peaks corresponding to
orthophosphoric acid (phosphate) or orthophosphorous acid (phosphite) is not greater than the area
of the respective peak in the chromatogram obtained with solution (2) (0.5% of each).
Assay Carry out the method for liquid chromatography, Appendix III D, using solutions in water
containing (1) 0.20% w/v of the substance being examined and (2) 0.20% w/v of disodium
pamidronate BPCRS.
The chromatographic conditions described under Phosphate and phosphite may be used.
14-68
3
H
9
NNa
2
O
7
P
2
from the chromatograms obtained and using the
declared content of C
3
H
9
NNa
2
O
7
P
2
in disodium pamidronate BPCRS.
Action and use Used in the treatment of malignant hypercalcaemia.
Preparation
Disodium Pamidronate Intravenous Infusion
IMPURITIES
H
2
N
COOH
-alanine,
together with phosphate and phosphite impurities.
14-69
Disopyramide
N
Pr
i
2
N
CONH
2
and enantiomer
C
21
H
29
N
3
O 339.5 3737-09-5
Disopyramide complies with the requirements of the 3rd edition of the European Pharmacopoeia [1006]. These
Preparation
Disopyramide Capsules
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Disopyramide contains not less than 98.5 per cent and not more than the equivalent of 101.5 per
cent of (RS)-4-[bis(1-methylethyl)amino]-2-phenyl-2-(pyrid-2-yl)butyramide, calculated with
CHARACTERS
A white or almost white powder, slightly soluble in water, freely soluble in methylene chloride,
soluble in alcohol.
IDENTIFICATION
A. Dissolve 40.0 mg in a 5 g/l solution of sulphuric acid R in methanol R and dilute to 100.0 ml with
the same solution. Dilute 5.0 ml of this solution to 50.0 ml with a 5 g/l solution of sulphuric acid R in
methanol R. Examined between 240 nm and 350 nm (2.2.25), the solution shows an absorption
maximum at 269 nm and a shoulder at 263 nm. The specific absorbance at the maximum is 190 to
210.
obtained with disopyramide CRS. Examine the substances as discs prepared by placing 50 l of a
50 g/l solution in methylene chloride R on a disc of potassium bromide R. Dry the discs at 60C for 1 h
before use.
and size to the principal spot in the chromatogram obtained with reference solution (a). Spray with
dilute potassium iodobismuthate solution R. Examine in daylight. The principal spot in the chromato-
gram obtained with test solution (b) is similar in position, colour and size to the principal spot in the
TESTS
254
R as the
coating substance.
Reference solution (a). Dissolve 20 mg of disopyramide CRS in methanol R and dilute to 10 ml with the
same solvent.
Reference solution (b). Dilute 0.5 ml of test solution (b) to 20 ml with methanol R.
1 volume of concentrated ammonia R, 30 volumes of acetone R and 30 volumes of cyclohexane R. Dry
the plate in a current of warm air and examine in ultraviolet light at 254 nm. Any spot in the
the spot in the chromatogram obtained with reference solution (b) (0.25 per cent).
14-70
diphosphorus pentoxide R at a pressure not exceeding 0.7 kPa for 2 h.
ASSAY
Dissolve 0.130 g in 30 ml of anhydrous acetic acid R. Add 0.2 ml of naphtholbenzein solution R. Titrate
with 0.1M perchloric acid until the colour changes from yellow to green.
21
H
29
N
3
O.
STORAGE
IMPURITIES
N
CN
Pr
i
2
N
A. 4-[bis(1-methylethyl)amino]-2-phenyl-2-(pyrid-2-yl)butyronitrile (= di-isopyronitrile),
N
Pr
i
2
N
B. 3-phenyl-3-(pyrid-2-yl)-N,N-di(1-methylethyl)propylamine,
N
CONH
2
Pr
i
2
N
C. 4-(1-methylethylamino)-2-phenyl-2-(pyrid- 2-yl)butyramide,
N
H
CN
D. 2-phenyl-2-(pyrid-2-yl)acetonitrile (= pyronitrile).
___________________________________________________________________________________________________ Ph Eur
14-71
Disopyramide Phosphate
,H
3
PO
4
N
Pr
i
2
N
CONH
2
and enantiomer
C
21
H
29
N
3
O,H
3
PO
4
437.5 22059-60-5
Disopyramide Phosphate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Disopyramide Phosphate Capsules
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Disopyramide phosphate contains not less than 98.0 per cent and not more than the equivalent of
102.0 per cent of (RS)-4-[bis(1-methylethyl)amino]-2-phenyl-2-(pyrid-2-yl)butyramide dihydrogen
phosphate, calculated with reference to the dried substance.
CHARACTERS
A white or almost white powder, soluble in water, sparingly soluble in alcohol, practically insoluble in
methylene chloride.
IDENTIFICATION
A. Dissolve 50.0 mg in a 5 g/l solution of sulphuric acid R in methanol R and dilute to 100.0 ml with
the same solution. Dilute 5.0 ml of this solution to 50.0 ml with a 5 g/l solution of sulphuric acid R in
maximum at 269 nm and a shoulder at 263 nm. The specific absorbance at the maximum is 147 to
163.
obtained with disopyramide phosphate CRS. Examine the substances prepared as discs.
dilute potassium iodobismuthate solution R. Examine in daylight. The principal spot in the chromato-
D. Solution S (see Tests) gives reaction (a) of phosphates (2.3.1).
TESTS
254
R as the
coating substance.
Reference solution (a). Dissolve 25 mg of disopyramide phosphate CRS in methanol R and dilute to 10 ml
14-72
1 volume of concentrated ammonia R, 30 volumes of acetone R and 30 volumes of cyclohexane R. Dry
the plate in a current of warm air and examine in ultraviolet light at 254 nm. Any spot in the
the spot in the chromatogram obtained with reference solution (b) (0.5 per cent).
100C to 105C.
ASSAY
Dissolve 0.180 g in 30 ml of anhydrous acetic acid R. Add 0.2 ml of naphtholbenzein solution R. Titrate
with 0.1M perchloric acid until the colour changes from yellow to green.
21
H
32
N
3
O
5
P.
STORAGE
14-73
IMPURITIES
N
CN
Pr
i
2
N
A. 4-[bis(1-methylethyl)amino]-2-phenyl-2-(pyrid-2-yl)butyronitrile (= di-isopyronitrile),
N
Pr
i
2
N
B. 3-phenyl-3-(pyrid-2-yl)-N,N-di(1-methylethyl)propylamine,
N
CONH
2
Pr
i
2
N
C. 4-(1-methylethylamino)-2-phenyl-2-(pyrid-2-yl)butyramide,
N
H
CN
D. 2-phenyl-2-(pyrid-2-yl)acetonitrile (= pyronitrile).
___________________________________________________________________________________________________ Ph Eur
14-74
Disulfiram
Et
2
N S
S NEt
2
S
S
C
10
H
20
N
2
S
4
296.5 97-77-8
Disulfiram complies with the requirements of the 3rd edition of the European Pharmacopoeia [0603]. These
Action and use Used in treatment of alcoholism.
Preparation
Disulfiram Tablets
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Disulfiram contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of
bis(diethylthiocarbamoyl) disulphide, calculated with reference to the dried substance.
CHARACTERS
chloride, soluble in ether, sparingly soluble in alcohol.
IDENTIFICATION
obtained with disulfiram CRS. Examine the substances prepared as discs.
D. Dissolve about 10 mg in 10 ml of methanol R. Add 2 ml of a 0.5 g/l solution of cupric chloride R in
methanol R. A yellow colour develops which becomes greenish-yellow.
TESTS
Test solution (a). Dissolve 0.20 g of the substance to be examined in ethyl acetate R and dilute to
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with ethyl acetate R.
Reference solution (a). Dissolve 10 mg of disulfiram CRS in ethyl acetate R and dilute to 5 ml with the
same solvent.
Reference solution (b). Dilute 1 ml of test solution (b) to 20 ml with ethyl acetate R.
volumes of butyl acetate R and 70 volumes of hexane R. Allow the plate to dry in air and examine in
ultraviolet light at 254 nm. Any spot in the chromatogram obtained with test solution (a), apart from
Diethyldithiocarbamate Dissolve 0.20 g in 10 ml of peroxide-free ether R, add 5 ml of buffer solution
pH 8.0 R and shake vigorously. Discard the upper layer and wash the lower layer with 10 ml
of peroxide-free ether R. Add to the lower layer 0.2 ml of a 4 g/l solution of copper sulphate R and 5 ml
of cyclohexane R. Shake. Any yellow colour in the upper layer is not more intense than that of a
standard prepared at the same time using 0.2 ml of a freshly prepared 0.15 g/l solution of sodium
diethyldithiocarbamate R (150 ppm).
50C.
14-75
ASSAY
Dissolve 0.450 g in 80 ml of acetone R and add 20 ml of a 20 g/l solution of potassium nitrate R.
Titrate with 0.1M silver nitrate. Determine the end-point potentiometrically (2.2.20), using a silver
electrode and a silver-silver chloride double-junction electrode saturated with potassium nitrate.
10
H
20
N
2
S
4
.
STORAGE
IMPURITIES
A. sulfiram,
B. diethydithiocarbamate.
___________________________________________________________________________________________________ Ph Eur
14-76
Dithranol
O OH OH
C
14
H
10
O
3
226.2 1143-38-0
Dithranol complies with the requirements of the 3rd edition of the European Pharmacopoeia [1007]. These
Action and use Used in treatment of psoriasis.
Preparations
Dithranol Cream
Dithranol Ointment
Dithranol Paste
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Dithranol contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of
1,8-dihydroxyanthracen-9(10H)-one, calculated with reference to the dried substance.
CHARACTERS
A yellow or brownish-yellow, crystalline powder, practically insoluble in water, soluble in methylene
chloride, sparingly soluble in acetone, slightly soluble in alcohol. It dissolves in dilute solutions of
alkali hydroxides.
Carry out all tests protected from bright light and use freshly prepared solutions.
IDENTIFICATION
A. It melts (2.2.14) at 178C to 182C.
obtained with dithranol CRS.
C. Examine by thin-layer chromatography (2.2.27), using silica gel H R as the coating substance.
Reference solution (a). Dissolve 10 mg of dithranol CRS in methylene chloride R and dilute to 10 ml with
the same solvent.
Reference solution (b). Dissolve about 5 mg of dantron R in 5 ml of reference solution (a).
equal volumes of hexane R and methylene chloride R. Allow the plate to dry in air. Place the plate in a
tank saturated with ammonia vapour until the spots appear. Examine in daylight. The principal spot
the chromatogram obtained with reference solution (b) shows two clearly separated spots.
D. To 5 mg add 0.1 g of anhydrous sodium acetate R and 1 ml of acetic anhydride R. Boil for 30 s. Add
20 ml of alcohol R. Examined in ultraviolet light at 365 nm, the solution shows a blue fluorescence.
TESTS
Related substances
A. Examine by liquid chromatography (2.2.29).
Test solution. Dissolve 0.200 g of the substance to be examined in 20 ml of methylene chloride R, add
1.0 ml of glacial acetic acid R and dilute to 100.0 ml hexane R.
Reference solution. Dissolve 10.0 mg each of anthrone R, dantron R, dithranol impurity C CRS and
dithranol CRS in methylene chloride R and dilute to 10.0 ml with the same solvent. To 1.0 ml of the
solution, add 19.0 ml of methylene chloride R and 1.0 ml of glacial acetic acid R, and dilute to 50.0 ml
with hexane R.
14-77
a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with silica gel for
as mobile phase at a flow rate of 2 ml per minute a mixture of 1 volume of glacial acetic acid R, 5
volumes of methylene chloride R and 82 volumes of hexane R,
Inject 20 l of each solution. Continue the chromatography for 1.5 times the retention time of
dithranol impurity C. Adjust the sensitivity of the system so that the height of the principal peak in
the chromatogram obtained with the reference solution is about 70 per cent of the full scale of the
recorder. When the chromatograms are recorded in the prescribed conditions, the substances are
eluted in the following sequence: dithranol, dantron, anthrone, dithranol impurity C. The test is not
valid unless, in the chromatogram obtained with the reference solution, the resolution between the
peaks due to dithranol and dantron is greater than 2.0. In the chromatogram obtained with the test
solution: the area of any peak corresponding to anthrone, dantron or dithranol impurity C is not
greater than that of the corresponding peak in the chromatogram obtained with the reference solution
(1.0 per cent); the area of any peak apart from the principal peak and any peaks due to anthrone,
dantron and dithranol impurity C is not greater than that of the peak due to dithranol in the
chromatogram obtained with the reference solution (1.0 per cent).
Reference solution. Dissolve 25.0 mg of dithranol impurity D CRS and 25.0 mg of dithranol CRS in the
as mobile phase at a flow rate of 0.9 ml per minute a mixture of 2.5 ml of glacial acetic acid R, 40
volumes of tetrahydrofuran R and 60 ml of water R,
Inject 20 l of each solution. Continue the chromatography for three times the retention time of
the peak due to dithranol. Adjust the sensitivity of the system so that the height of the principal peak
in the chromatogram obtained with the reference solution is about 50 per cent of the full scale of the
recorder. The test is not valid unless, in the chromatogram obtained with the reference solution, the
resolution between the peaks due to dithranol impurity D and dithranol is greater than 2.5. In the
chromatogram obtained with the test solution: the area of any peak corresponding to dithranol
impurity D is not greater than that of the corresponding peak in the chromatogram obtained with the
The total content of related substances as determined in tests A and B is not more than 3.0 per
cent.
Chlorides (2.4.4). Shake 1.0 g with 20 ml of water R for 1 min and filter. Dilute 10 ml of the filtrate
to 15 ml with water R. The solution complies with the limit test for chlorides (100 ppm).
100C to 105C
ASSAY
Dissolve 0.200 g in 50 ml of anhydrous pyridine R. Titrate with 0.1M tetrabutylammonium hydroxide
under nitrogen R. Determine the end-point potentiometrically (2.2.20), using a glass indicator
electrode and a calomel reference electrode containing, as the electrolyte, a saturated solution of
potassium chloride R in methanol R.
14
H
10
O
3
.
STORAGE
IMPURITIES
O
A. anthrone,
14-78
O
O
OH OH
B. dantron,
O
O OH OH
OH OH
C. dithranol dimer,
O OH
D. 1-hydroxy-9-anthrone.
___________________________________________________________________________________________________ Ph Eur
14-79
Dobutamine Hydrochloride
N
HO
HO
H Me
OH
H
and enantiomer
,HCl
C
18
H
23
NO
3
,HCl 337.9 49745-95-1
Dobutamine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Cardiac stimulant.
Preparation
Dobutamine Intravenous Infusion
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Dobutamine hydrochloride contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of (RS)-4-[2-[[3-(4-hydroxyphenyl)-1-methylpropyl]amino]-ethyl]benzene-1,2-diol
CHARACTERS
A white or almost white, crystalline powder, sparingly soluble in water, soluble in methanol, sparingly
soluble in alcohol.
IDENTIFICATION
the solution to 100.0 ml with methanol R. Examined between 220 nm and 300 nm (2.2.25), the
solution shows two absorption maxima, at 223 nm and 281 nm. The ratio of the absorbance
measured at the maximum at 281 nm to that measured at the maximum at 223 nm is 0.34 to 0.36.
obtained with dobutamine hydrochloride CRS. Examine the substances prepared as discs.
D. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance.
Test solution. Dissolve 10 mg in a mixture of equal volumes of glacial acetic acid R and methanol R and
Reference solution (a). Dissolve 10.0 mg of dobutamine hydrochloride CRS in a mixture of equal
volumes of glacial acetic acid R and methanol R and dilute to 10 ml with the same mixture of solvents.
Reference solution (b). Dissolve 5.0 mg of dopamine hydrochloride CRS in 5 ml of the test solution.
5 volumes of water R, 15 volumes of glacial acetic acid R, 30 volumes of ether R and 45 volumes of
butanol R. Dry the plate and spray with a 1 g/l solution of potassium permanganate R. The principal
spot in the chromatogram obtained with the test solution is similar in position, colour and size to the
E. It gives reaction (a) of chlorides (2.3.1) using a mixture of equal volumes of methanol R and
water R.
TESTS
Acidity or alkalinity Dissolve 0.1 g in water R with gentle heating and dilute to 10 ml with the same
Optical rotation (2.2.7). Dissolve 0.50 g in methanol R and dilute to 10.0 ml with the same solvent.
14-80
Absorbance (2.2.25). Dissolve 0.5 g in a mixture of equal volumes of methanol R and of water R
with heating, if necessary, at 30C to 35C and dilute to 25 ml with the same mixture of solvents.
Cool quickly. Measure the absorbance immediately at 480 nm. The absorbance is not greater than
0.04.
Test solution. Dissolve 0.10 g in a mixture of 35 volumes of mobile phase B and 65 volumes of mobile
phase A and dilute to 20.0 ml with the same mixture of mobile phases.
Reference solution (a). Dilute 4.0 ml of the test solution to 100.0 ml with a 0.05 g/l solution of
anisaldehyde R in a mixture of 35 volumes of mobile phase B and 65 volumes of mobile phase A.
Dilute 1.0 ml of the solution to 10.0 ml with the same mixture of mobile phases.
mobile phase B and 65 volumes of mobile phase A. Dilute 1.0 ml of the solution to 10.0 ml with the
same mixture of mobile phases.
Mobile phase A. Dissolve 2.60 g of sodium octanesulphonate R in 1000 ml of water R and add 3 ml
of triethylamine R; adjust to pH 2.5 with phosphoric acid R,
Mobile phase B. A mixture of 18 volumes of acetonitrile R and 82 volumes of methanol R,
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
05 65 35
520 6520 3580
2025 20 80
Inject 20 l of reference solution (a). The test is not valid unless the resolution between the peaks
corresponding to dobutamine hydrochloride and anisaldehyde is at least 4.0. Inject 20 l of the test
solution and 20 l of reference solution (b). In the chromatogram obtained with the test solution: any
peak apart from the principal peak is not greater than the principal peak in the chromatogram
obtained with reference solution (b) (0.5 per cent); and the sum of the areas of all the peaks apart
from the principal peak is not greater than twice the area of the principal peak in the chromatogram
obtained with the reference solution (b) (1 per cent). Disregard any peak due to the solvent and any
100C to 105C.
ASSAY
Dissolve 0.250 g in 10 ml of anhydrous formic acid R. Add 50 ml of acetic anhydride R. Titrate with
18
H
24
ClNO
3
.
STORAGE
IMPURITIES
HO
HO NH
2
A. dopamine,
14-81
O
Me
HO
B. 4-(4-hydroxyphenyl)-2-butanone,
N
H
Me H
OCH
3
OCH
3
MeO
and enantiomer
C. (RS)-4-[2-[[3-(4-methoxyphenyl)-1-methylpropyl]amino]ethyl]-1,2-dimethoxybenzene.
___________________________________________________________________________________________________ Ph Eur
14-82
Docusate Sodium
Dioctyl Sodium Sulphosuccinate
H
3
C O
O CH
3

SO
3
Na H
O
O
Et H
Et H
mixture of 8 stereoisomers
* * *
C
20
H
37
NaO
7
S

444.6 577-11-7
Docusate Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia [1418].
Action and use Faecal softener.
Preparations
Co-danthrusate Capsules
Docusate Capsules
Compound Docusate Enema
Docusate Oral Solution
Paediatric Docusate Oral Solution
Docusate Tablets
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Docusate sodium contains not less than 98.0 per cent and not more than the equivalent of 100.5 per
cent of sodium 1,4-bis[(2-ethylhexyl)oxy]-1,4-dioxobutane-2-sulphonate, calculated with reference
CHARACTERS
White or almost white, waxy masses or flakes, hygroscopic, sparingly soluble in water, freely soluble
in alcohol and in methylene chloride.
IDENTIFICATION
spectrum of docusate sodium. Place about 3 mg of the substance to be examined on a sodium chloride
plate, add 0.05 ml of acetone R and immediately cover with another sodium chloride plate. Rub the
plates together to dissolve the substance to be examined, slide the plates apart and allow the acetone
to evaporate.
B. In a crucible ignite 0.75 g in the presence of dilute sulphuric acid R, until an almost white residue is
obtained. Allow to cool, and take up the residue with 5 ml of water R. Filter. 2 ml of filtrate gives
reaction (a) of sodium (2.3.1).
TESTS
Alkalinity Dissolve 1.0 g in 100 ml of a mixture of equal volumes of methanol R and water R,
previously neutralised to methyl red solution R. Add 0.1 ml of methyl red solution R. Not more than
0.2 ml of 0.1M hydrochloric acid is required to change the colour of the indicator to red.
Related non-ionic substances Examine by gas chromatography (2.2.28), using methyl behenate R as
Internal standard solution. Dissolve 10 mg of methyl behenate R in hexane R and dilute to 50 ml with
the same solvent.
Test solution (a). Dissolve 0.10 g of the substance to be examined in 2.0 ml of the internal standard
solution and dilute to 5.0 ml with hexane R. Pass the solution at a rate of about 1.5 ml/min, through a
column 10 mm in internal diameter, packed with 5 g of basic aluminium oxide R and previously
washed with 25 ml of hexane R. Elute with 5 ml of hexane R and discard the eluate. Elute with 20 ml
of a mixture of equal volumes of ether R and hexane R. Evaporate the eluate to dryness and dissolve
the residue in 2.0 ml of hexane R.
Test solution (b). Prepare as described for test solution (a) but dissolving 0.10 g of the substance to be
examined in hexane R and diluting to 5.0 ml with the same solvent, and using a new column.
Reference solution. Dilute 2.0 ml of the internal standard solution to 5.0 ml with hexane R.
14-83
nitrogen R as the carrier gas at a flow rate of 30 ml/min,
detector at 280C.
Inject 1 l of each solution. In the chromatogram obtained with test solution (b), verify that there is
no peak with the same retention time as the internal standard. In the chromatogram obtained with
test solution (a), the area of any peak, apart from the peak due to the internal standard, is not greater
than the area of the peak due to the internal standard (0.4 per cent). Continue the chromatography
for 2.5 times the retention time of the internal standard.
Chlorides Dissolve 5.0 g in 50 ml of alcohol (50 per cent V/V) R and add 0.1 ml of potassium
dichromate solution R. Not more than 0.5 ml of 0.1M silver nitrate is required to change the colour of
the indicator (350 ppm).
Sodium sulphate Dissolve 0.25 g in 40 ml of a mixture of 20 volumes of water R and 80 volumes of
2-propanol R. Adjust the pH to between 2.5 and 4.0 using perchloric acid solution R. Add 0.4 ml of
naphtharson solution R and 0.1 ml of a 0.125 g/l solution of methylene blue R. Not more than 1.5 ml of
0.025M barium perchlorate is required to change the colour of the indicator from yellowish-green to
yellowish-pink (2 per cent).
Heavy metals (2.4.8). Dissolve 4.0 g in alcohol (80 per cent V/V) R and dilute to 20 ml with the same
solvent. 12 ml of the solution complies with limit test B for heavy metals (10 ppm). Prepare the
standard using lead standard solution (2 ppm Pb) obtained by diluting lead standard solution (100 ppm
Pb) R with alcohol (80 per cent V/V) R.
mination of water.
ASSAY
Dissolve with heating 0.350 g of the substance to be examined in about 50 ml of water R. Allow to
cool and dilute to 250.0 ml with the same solvent. Transfer 25.0 ml of this solution to a 250 ml flask
and add 5 ml of water R, 15 ml of methylene chloride R and 10 ml of a 10 per cent V/V solution of
sulphuric acid R. Add 1 ml of a mixture of dimethyl yellow and oracet blue solution R, shake energetically
and titrate with 0.004M benzethonium chloride. Towards the end of the titration, titrate slowly, close
the flask and shake vigorously between each addition. The end of the titration is reached when the
solution in the methylene chloride changes from pink to green.
1 ml of 0.004M benzethonium chloride is equivalent to 1.778 mg of C
20
H
37
NaO
7
S.
STORAGE
Store in an air-tight container.
___________________________________________________________________________________________________ Ph Eur
14-84
Dodecyl Gallate
HO
HO
OH
COO[CH
2
]
11
CH
3
C
19
H
30
O
5
338.4 1166-52-5
Definition Dodecyl Gallate is dodecyl 3,4,5-trihydroxybenzoate.
Characteristics A white or creamy white powder; odourless or almost odourless.
Practically insoluble in water; freely soluble in acetone, in ethanol (96%), in ether and in methanol;
sparingly soluble in arachis oil, in chloroform and in propane-1,2-diol.
Identification
methanol exhibits a maximum only at 275 nm. The absorbance at 275 nm is about 0.60.
B. Carry out the method for gas chromatography, Appendix III B, using a solution prepared in the
following manner. Boil 0.5 g with 50 ml of 5M sodium hydroxide under a reflux condenser for 10
minutes, cool and extract with 50 ml of ether.
with acid-washed, silanised diatomaceous support (80 to 100 mesh) coated with 10% w/w of free fatty
acid phase (Supelco FFAP is suitable) and maintained at 180.
The principal peak in the chromatogram has the same retention time as that of dodecan-1-ol,
examined under the same conditions.
C. Dissolve 5 mg in a mixture of 25 ml of acetone and 25 ml of water and add 0.05 ml of iron(III)
chloride solution. A purplish blue colour is produced which rapidly becomes bluish black.
Acidity Dissolve 0.4 g in 50 ml of acetone, add 50 ml of carbon dioxide-free water and titrate with 0.1M
sodium hydroxide VS, using bromocresol green solution as indicator, to the blue colour indicative of pH
5.0. Repeat the operation without the substance being examined. The difference between the
titrations does not exceed 0.1 ml.
Melting point 96 to 97.5, Appendix V A.
Chloride Shake 0.50 g with 100 ml of water at 60 for 10 minutes, cool and filter. 15 ml of the
filtrate complies with the limit test for chlorides, Appendix VII (0.07%).
Sulphate Shake 0.25 g with 30 ml of water at 60 for 10 minutes, cool and filter. 15 ml of the filtrate
complies with the limit test for sulphates, Appendix VII (0.12%).
1 g.
Storage Dodecyl Gallate should be kept in a well-closed container and protected from light. Contact
with metals should be avoided.
14-85
Dog Rose
1/01
Dog Rose complies with the requirements of the 3rd edition of the European Pharmacopoeia [1510]. These
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Dog rose consists of the rose hips made up by the receptacle and the remains of the dried sepals
of Rosa canina L., R. pendulina L. and other Rosa species, with the achenes removed. It contains not
less than 0.3 per cent of ascorbic acid (C
6
H
8
O
6
; M
r
176.1), calculated with reference to the dried
drug.
CHARACTERS
IDENTIFICATION
A. It consists of fragments of the fleshy hollow urceolate receptacle, bearing the remains of the
reduced sepals, light pink to orange-pink, the convex outer surface shiny and strongly wrinkled;
bearing on its lighter inner surface abundant bristle-like hairs.
B. Reduce to a powder (355). The powder is orange-yellow. Examine under a microscope using
chloral hydrate solution R. The powder shows numerous fragments of receptacle, the outer epidermis
with orange-yellow contents and a thick cuticle, the inner epidermis composed of thin-walled cells
containing cluster crystals and occasional prisms of calcium oxalate; scattered lignified cells,
isodiametric, with thickened and pitted walls forming the trichome bases; abundant long, unicellar
trichomes, up to 2 mm long and 30 m to 45 m thick, tapering towards each end, walls heavily
thickened and with a waxy cuticle which may show fissures in a spiral arrangement; numerous oily
orange-yellow globules.
254
plate R.
Test solution. To 5 g of the powdered drug (355) add 25 ml of alcohol R. Shake for 30 min and filter.
Reference solution. Dissolve 10 mg of ascorbic acid R in 5.0 ml of alcohol (60 per cent V/V) R.
Apply to the plate 20 l of the test solution and 2 l of the reference solution. Develop over a path of
15 cm using a mixture of 5 volumes of acetic acid R, 5 volumes of acetone R, 20 volumes of
methanol R and 70 volumes of toluene R. Allow the plate to dry in air and examine in ultraviolet light
at 254 nm. The chromatogram obtained with the test solution shows a quenching zone similar in
position to the principal zone in the chromatogram obtained with the reference solution. Spray with a
0.2 g/l solution of sodium salt of dichlorophenolindophenol R in alcohol R. Examine in daylight. The
chromatogram obtained with the test solution shows a white zone on a pink background similar in
position and colour to the principal zone in the chromatogram obtained with the reference solution.
The chromatogram also shows an intense orange-yellow zone near the solvent front and a yellow
zone in the upper third (carotenoids).
TESTS
ASSAY
Test solution. In a round-bottomed flask, weigh 0.500 g of the freshly powdered drug (710). Add a
solution of 1.0 g of oxalic acid R in 50.0 ml of methanol R. Boil under a reflux condenser for 10 min,
and cool in iced water until the temperature reaches 15C to 20C. Filter. Transfer 2.0 ml of the
filtrate to a 50 ml conical flask. Add successively, with gentle shaking after each addition, 2.0 ml of
dichlorophenolindophenol standard solution R and then, exactly 60 s later, 0.5 ml of a 100 g/l solution of
thiourea R in alcohol (50 per cent V/V) R and 0.7 ml of dinitrophenylhydrazine-sulphuric acid solution R.
Heat under a reflux condenser at 50C for 75 min, and place immediately in iced water for 5 min.
Add dropwise 5.0 ml of a mixture of 12 ml of water R and 50 ml of sulphuric acid R, taking care to
carry out the addition over a period of not less than 90 s and not more than 120 s while maintaining
vigorous stirring in iced water. Allow to stand for 30 min at room temperature and measure the
absorbance (2.2.25) at 520 nm using solution A as compensation liquid.
Solution A. Treat 2.0 ml of the filtrate obtained during the preparation of the test solution as
described but adding the dinitrophenylhydrazine-sulphuric acid solution R just before the absorbance is
measured.
14-86
Reference solution. Dissolve 40.0 mg of ascorbic acid R in a freshly prepared 20 g/l solution of oxalic
acid R in methanol R and dilute to 100.0 ml with the same solvent. Dilute 5.0 ml of the solution to
100.0 ml with the methanolic solution of oxalic acid. Treat 2.0 ml of the solution as described above
for the filtrate obtained during the preparation of the test solution. Measure the absorbance (2.2.25)
at 520 nm using solution B as compensation liquid.
Solution B. Treat 2.0 ml of the reference solution as described above for solution A.
Calculate the percentage content of ascorbic acid from the expression:
1 2
2 1
5 . 2
m A
m A

A
1
=absorbance of the test solution,
A
2
=absorbance of the reference solution,
m
1
=mass of the substance to be examined, in grams,
m
2
=mass of ascorbic acid used, in grams.
STORAGE
___________________________________________________________________________________________________ Ph Eur
14-87
Domiphen Bromide
O
NMe
2
C
12
H
25
+
Br
C
22
H
40
BrNO 414.5 538-71-6
Definition Domiphen Bromide consists chiefly of dodecyldimethyl-2-phenoxyethylammonium
bromide. It contains not less than 97.0% and not more than 100.5% of C
22
H
40
BrNO, calculated
Characteristics Colourless or faintly yellow, crystalline flakes.
Freely soluble in water and in ethanol (96%); soluble in acetone.
Identification
domiphen bromide (RS 383).
B. Dissolve 10 mg in 10 ml of water and add 0.1 ml of a 0.5% w/v solution of eosin and 100 ml of
water. An intense pink colour is produced.
C. Yields the reactions characteristic of bromides, Appendix VI.
D. Melting point, 106 to 116, Appendix V A.
Acidity or alkalinity Add 0.5 ml of bromothymol blue solution to each of 10 ml of phosphate buffer pH
6.4 (solution A) and 10 ml of phosphate buffer pH 7.6 (solution B). Dissolve 0.10 g in 10 ml of carbon
dioxide-free water and add 0.5 ml of bromothymol blue solution R3. The resulting solution is not more
yellow than solution A and not more blue than solution B.
Clarity and colour of solution Dissolve 1.0 g in 10 ml of carbon dioxide-free water. The solution is
not more opalescent than reference suspension II, Appendix IV A, and not more intensely coloured
7
Non-quaternary amines Carry out the Assay described below using a further 25 ml of the original
solution and 10 ml of 0.1M hydrochloric acid in place of the 0.1M sodium hydroxide. The difference
between the volume of 0.05M potassium iodate VS required in this titration and that required in the
Assay is not more than 0.5 ml for each g of substance taken.
Assay Dissolve 2 g in sufficient water to produce 100 ml. Transfer 25 ml to a separating funnel and
add 25 ml of chloroform, 10 ml of 0.1M sodium hydroxide and 10 ml of a freshly prepared 5% w/v
solution of potassium iodide. Shake well, allow to separate and discard the chloroform layer. Wash the
aqueous layer with three 10-ml quantities of chloroform and discard the chloroform solutions. Add
40 ml of hydrochloric acid, allow to cool and titrate with 0.05M potassium iodate VS until the deep
brown colour is discharged. Add 2 ml of chloroform and continue the titration, shaking vigorously,
until the chloroform layer no longer changes colour. Carry out a blank titration on a mixture of 10 ml
of the freshly prepared potassium iodide solution, 20 ml of water and 40 ml of hydrochloric acid. The
difference between the titrations represents the amount of potassium iodate required. Each ml of
0.05M potassium iodate VS is equivalent to 41.45 mg of C
22
H
40
BrNO.
14-88
Domperidone
H
N
N
Cl O
N
N
N O
H
C
22
H
24
ClN
5
O
2
425.9 57808-66-9
Domperidone complies with the requirements of the 3rd edition of the European Pharmacopoeia [1009]. These
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Domperidone contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of 5-chloro-1-[1-[3-(2,3-dihydro-2-oxo-1H-benzimidazol-1-yl)propyl]piperidin-4-yl]-2,3-
dihydro-1H-benzimidazol-2-one, calculated with reference to the dried substance.
CHARACTERS
A white or almost white powder, practically insoluble in water, soluble in dimethylformamide, slightly
IDENTIFICATION
obtained with domperidone CRS. Examine the substances prepared as discs.
coating substance.
the same solvent.
Reference solution (a). Dissolve 20 mg of domperidone CRS in methanol R and dilute to 10 ml with the
same solvent.
Reference solution (b). Dissolve 20 mg of domperidone CRS and 20 mg of droperidol CRS in methanol R
appear. Examine in daylight. The principal spot in the chromatogram obtained with the test solution
TESTS
Appearance of solution Dissolve 0.20 g in dimethylformamide R and dilute to 20.0 ml with the same
6
(Method II, 2.2.2).
before use.
Reference solution (a). Dissolve 10.0 mg of domperidone CRS and 15.0 mg of droperidol CRS in
14-89
as mobile phase at a flow rate of 1.5 ml per minute a mixture of 3 volumes methanol R and 7
volumes of a 5 g/l solution of ammonium acetate R, changing by linear gradient to methanol R over
10 min, followed by elution with methanol R for 2 min,
Equilibrate the column for at least 30 min with methanol R and then Equilibrate at the initial
mobile phase composition for at least 5 min.
conditions, the retention times are: domperidone about 6.5 min; droperidol about 7 min. The test is
not valid unless the resolution between the peaks due to domperidone and droperidol is at least 2.0.
If necessary, adjust the concentration of methanol in the mobile phase or adjust the time programme
reference solution (b). In the chromatogram obtained with the test solution: the area of any peak,
apart from the principal peak, is not greater than the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.25 per cent); the sum of the areas of all peaks, apart from the
with reference solution (b) (0.5 per cent). Disregard any peak in the chromatogram obtained with the
blank run and any peak with an area less than 0.2 times the area of the principal peak in the
100C to 105C.
ASSAY
ethyl ketone R. Titrate with 0.1M perchloric acid until the colour changes from orange-yellow to green
using 0.2 ml of naphtholbenzein solution R as indicator.
22
H
24
ClN
5
O
2
.
STORAGE
IMPURITIES
Cl O
N
N
HN
H
A. 5-chloro-2,3-dihydro-1-(piperidin-4-yl)-1H-benzimidazol-2-one,
Cl O
N
N
N
OHC
H
B. 4-(5-chloro-2,3-dihydro-2-oxo-1H-benzimidazol-1-yl)-1-formyl-piperidine,
H
H
N
N
Cl O
N
N
N O
O
C. cis-5-chloro-1-[1-[3-(2,3-dihydro-2-oxo-1H-benzimidazol-1-yl)propyl]piperidin-4-yl]-2,3-
dihydro-1H-benzimidazol-2-one 1-oxide,
14-90
N
N
O N
N
N
O Cl
N
N
O
H
D. 5-chloro-3-[3-(2,3-dihydro-2-oxo-1H-benzimidazol-1-yl)propyl]-1-[1-[3-(2,3-dihydro-2-
oxo-1H-benzimidazol-1-yl)propyl]piperidin-4-yl]-2,3-dihydro-1H-benzimidazol-2-one,
N
N O
N
O N
N
O N
N
Cl
H
E. 1-[3-[4-(5-chloro-2,3-dihydro-2-oxo-1H-benzimidazol-1-yl)piperidin-1-yl]propyl]-3-[3-(2,3-
dihydro-2-oxo-1H-benzimidazol-1-yl)propyl]-2,3-dihydro-1H-benzimidazol-2-one,
O N
N
N
Cl
N
N O
N
O N
N
Cl
H
H
F 1,1-[(1,2-dihydro-2-oxo-1H-benzimidazole-1,3-diyl)di(propane-3,1-diyl)di(piperidine-1,4-
diyl)]bis[5-chloro-2,3-dihydro-1H-benzimidazol-2-one].
___________________________________________________________________________________________________ Ph Eur
14-91
Domperidone Maleate
H
N
N
Cl O
N
N
N O
H
COOH
COOH H
H
,
C
22
H
24
ClN
5
O
2
,C
4
H
4
O
4
542.0 99497-03-7
Domperidone Maleate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Domperidone Tablets
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Domperidone maleate contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of 5-chloro-1-[1-[3-(2,3-dihydro-2-oxo-1H-benzimidazol-1-yl)propyl]piperidin-4-yl]-
2,3-dihydro-1H-benzimidazol-2-one (Z)-butenedioate, calculated with reference to the dried
substance.
CHARACTERS
A white or almost white powder, very slightly soluble in water, sparingly soluble in
dimethylformamide, slightly soluble in methanol, very slightly soluble in alcohol.
IDENTIFICATION
obtained with domperidone maleate CRS. Examine the substances prepared as discs. If the spectra
separately in the minimum volume of 2-propanol R, evaporate to dryness on a water-bath and record
new spectra using the residues.
B. Examine by thin-layer chromatography (2.2.27), using a suitable octadecylsilyl silica gel as the
coating substance.
the same solvent.
Reference solution (a). Dissolve 20 mg of domperidone maleate CRS in methanol R and dilute to 10 ml
Reference solution (b). Dissolve 20 mg of domperidone maleate CRS and 20 mg of droperidol CRS in
C. Triturate 0.1 g with a mixture of 1 ml of strong sodium hydroxide solution R and 3 ml of water R.
Shake with three quantities, each of 5 ml, of ether R. To 0.1 ml of the aqueous layer add a solution of
10 mg of resorcinol R in 3 ml of sulphuric acid R. Heat on a water-bath for 15 min. No colour
develops. To the remainder of the aqueous layer add 2 ml of bromine solution R. Heat on a water-bath
for 15 min and then heat to boiling. Cool. To 0.1 ml of the solution add a solution of 10 mg of
resorcinol R in 3 ml of sulphuric acid R. Heat on a water-bath for 15 min. A violet colour develops.
14-92
TESTS
Appearance of solution Dissolve 0.20 g in dimethylformamide R and dilute to 20.0 ml with the same
6
(Method II, 2.2.2).
before use.
Reference solution (a). Dissolve 10.0 mg of domperidone maleate CRS and 15.0 mg of droperidol CRS in
as mobile phase at a flow rate of 1.5 ml per minute of a mixture of 3 volumes of methanol R and 7
volumes of a 5 g/l solution of ammonium acetate R, changing by linear gradient to methanol R over
10 min, followed by elution with methanol R for 2 min,
Equilibrate the column for at least 30 min with methanol R and then Equilibrate at the initial
conditions, the retention times are: domperidone maleate, about 6.5 min and droperidol, about
7 min. The test is not valid unless the resolution between the peaks due to domperidone maleate and
droperidol is at least 2.0. If necessary, adjust the concentration of methanol in the mobile phase or
adjust the time programme for the linear gradient.
with reference solution (b) (0.5 per cent). Disregard any peak in the chromatogram obtained with the
blank run and any peak with an area less than 0.2 times that of the principal peak in the chromato-
100C to 105C.
ASSAY
Dissolve 0.400 g in 50 ml of glacial acetic acid R. Using 0.2 ml of naphtholbenzein solution R as
indicator, titrate with 0.1M perchloric acid until the colour changes from orange-yellow to green.
26
H
28
ClN
5
O
6
.
STORAGE
IMPURITIES
Cl O
N
N
HN
H
A. 5-chloro-2,3-dihydro-1-(piperidin-4-yl)-1H-benzimidazol-2-one,
14-93
Cl O
N
N
N
OHC
H
B. 4-(5-chloro-2,3-dihydro-2-oxo-1H-benzimidazol-1-yl)-1-formyl-piperidine,
H
H
N
N
Cl O
N
N
N O
O
C. cis-5-chloro-1-[1-[3-(2,3-dihydro-2-oxo-1H-benzimidazol-1-yl)propyl]piperidin-4-yl]-2,3-
dihydro-1H-benzimidazol-2-one 1-oxide,
N
N
O N
N
N
O Cl
N
N
O
H
D. 5-chloro-3-[3-(2,3-dihydro-2-oxo-1H-benzimidazol-1-yl)propyl]-1-[1-[3-(2,3-dihydro-2-
oxo-1H-benzimidazol-1-yl)propyl]piperidin-4-yl]-2,3-dihydro-1H-benzimidazol-2-one,
N
N O
N
O N
N
O N
N
Cl
H
E. 1-[3-[4-(5-chloro-2,3-dihydro-2-oxo-1H-benzimidazol-1-yl)piperidin-1-yl]propyl]-3-[3-(2,3-
dihydro-2-oxo-1H-benzimidazol-1-yl)propyl]-2,3-dihydro-1H-benzimidazol-2-one,
O N
N
N
Cl
N
N O
N
O N
N
Cl
H
H
F. 1,1-[(1,2-dihydro-2-oxo-1H-benzimidazole-1,3-diyl)di(propane-3,1-diyl)di(piperidine-1,4-
diyl)]bis[5-chloro-2,3-dihydro-1H-benzimidazol-2-one].
___________________________________________________________________________________________________ Ph Eur
15-1
Dopamine Hydrochloride
HO
HO NH
2
,HCl
C
8
H
11
NO
2
,HCl 189.6 62-31-7
Dopamine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Sympathomimetic.
Preparation
Dopamine Intravenous Infusion
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Dopamine hydrochloride contains not less than 98.0 per cent and not more than the equivalent of
102.0 per cent of 4-(2-aminoethyl)benzene-1,2-diol hydrochloride, calculated with reference to the
dried substance.
CHARACTERS
A white or almost white, crystalline powder, freely soluble in water, soluble in alcohol, sparingly
soluble in acetone and in methylene chloride.
IDENTIFICATION
obtained with dopamine hydrochloride CRS. Examine the substances as discs prepared using potassium
chloride R.
C. Dissolve about 5 mg in a mixture of 5 ml of 1M hydrochloric acid and 5 ml of water R. Add 0.1 ml
of sodium nitrite solution R containing 100 g/l of ammonium molybdate R. A yellow colour develops
which becomes red on the addition of strong sodium hydroxide solution R.
D. Dissolve about 2 mg in 2 ml of water R and add 0.2 ml of ferric chloride solution R2. A green colour
develops which changes to bluish-violet on the addition of 0.1 g of hexamethylenetetramine R.
TESTS
6
or Y
6
(Method II,
2.2.2).
coating substance.
the same solvent.
Reference solution (a). Dissolve 7.5 mg of 4-O-methyldopamine hydrochloride R in methanol R and dilute
Reference solution (b). Dissolve 7.5 mg each of 3-O-methyldopamine hydrochloride R and 4-O-
methyldopamine hydrochloride R in methanol R and dilute to 100 ml with the same solvent.
2 volumes of anhydrous formic acid R, 7 volumes of water R, 36 volumes of methanol R and 52
volumes of chloroform R. Allow the plate to dry in air for 15 min. Spray evenly and abundantly with a
mixture of equal volumes of potassium ferricyanide solution R and ferric chloride solution R1, prepared
15-2
immediately before use. Any spot in the chromatogram obtained with the test solution with an R
f
value higher than that of the principal spot is not more intense than the spot in the chromatogram
obtained with reference solution (a) (0.25 per cent). The test is not valid unless the chromatogram
100C to 105C for 2 h.
ASSAY
Dissolve 0.1500 g in 10 ml of anhydrous formic acid R. Add 50 ml of acetic anhydride R. Titrate with
8
H
12
ClNO
2
.
STORAGE
IMPURITIES
H
3
CO
HO NH
2
A. 5-(2-aminoethyl)-2-methoxyphenol (4-O-methyldopamine).
__________________________________________________________________________________________________________ Ph Eur
15-3
Dosulepin Hydrochloride / Dothiepin Hydrochloride
,HCl
S
H Me
2
N
C
19
H
21
NS,HCl 331.9 1897-15-4
Dosulepin Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparations
Dosulepin Capsules/Dothiepin Capsules
Dosulepin Tablets/Dothiepin Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Dosulepin hydrochloride is (E)-3-(dibenzo[b,e]thiepin-11(6H)ylidene)-N,N-dimethylpropan-1-amine
hydrochloride. It contains not less than 98.0 per cent and not more than the equivalent of 101.0 per
cent of C
19
H
22
ClNS, calculated with reference to the dried substance.
CHARACTERS
A white or faintly yellow, crystalline powder, freely soluble in water, in alcohol and in methylene
chloride.
IDENTIFICATION
A. Dissolve 25.0 mg in a 1 g/l solution of hydrochloric acid R in methanol R and dilute to 100.0 ml
with the same acid solution. Dilute 2.0 ml to 50.0 ml with a 1 g/l solution of hydrochloric acid R in
methanol R. Examined between 220 nm and 350 nm (2.2.25), the solution shows two absorption
maxima at 231 nm and 306 nm, and a shoulder at about 260 nm. The specific absorbance at the
maximum at 231 nm is 660 to 730.
obtained with dosulepin hydrochloride CRS. Examine the substances prepared as discs.
C. Dissolve about 1 mg in 5 ml of sulphuric acid R. A dark red colour is produced.
TESTS
Appearance of solution Dissolve 1 g in water R and dilute to 20 ml with the same solvent. The
5
(Method II,
2.2.2).
pH (2.2.3). Dissolve 1 g in carbon dioxide-free water R and dilute to 10 ml with the same solvent. The
pH of the solution is between 4.2 and 5.2.
Z-Isomer and related substances Examine by liquid chromatography (2.2.29).
Reference solution (a). Dissolve 12.5 mg of dosulepin impurity A CRS in 5 ml of methanol R and dilute
to 50.0 ml with the mobile phase. Dilute 1.0 ml of the solution to 100.0 ml with the mobile phase.
Reference solution (b). Dissolve 20.0 mg of dosulepin hydrochloride CRS in 5 ml of methanol R and dilute
15-4
a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with nitrile silica gel
for chromatography R1 (5 m),
as mobile phase at a flow rate of 1 ml/min a mixture of 0.2 volumes of phosphoric acid R, 10
volumes of a 10 g/l solution of sodium butanesulphonate R, 35 volumes of methanol R and 55
volumes of water R,
conditions, the retention time of the Z-isomer relative to the main peak (E-isomer) is about 0.9.
Adjust the sensitivity of the system so that the height of the peak due to Z-isomer is at least 50 per
cent of the full scale of the recorder. Measure the height (A) above the base-line of the peak due to
the Z-isomer and the height (B) above the base-line of the lowest point of the curve separating this
peak from the peak due to the E-isomer. The test is not valid unless the ratio A/B is not less than 4.
with the test solution: the area of any peak corresponding to the Z-isomer is not greater than 5 per
cent of the sum of the areas of the two isomer peaks; the area of any peak corresponding to impurity
A is not greater than the area of the principal peak in the chromatogram obtained with reference
solution (a) (0.25 per cent); the area of any peak, apart from the principal peak and any peak corres-
ponding to the Z-isomer and to impurity A, is not greater than 0.4 times the area of the principal
peak in the chromatogram obtained with reference solution (a) (0.1 per cent); the sum of the areas of
any peak, apart from the principal peak and any peak corresponding to the Z-isomer, is not greater
than twice the area of the principal peak in the chromatogram obtained with reference solution (a)
100C to 105C.
ASSAY
19
H
22
ClNS
STORAGE
IMPURITIES
S
Me
2
N H
O
A. (E)-3-(dibenzo[b,e]thiepin-11(6H)-ylidene)-N,N-dimethylpropan-1-amine S-oxide,
S
O
B. dibenzo[b,e]thiepin-11(6H)-one,
S
Me
2
N
OH
and enantiomer
C. (11RS)-11-[3-(dimethylamino)propyl]-6,11-dihydrodibenzo[b,e]thiepin-11-ol,
15-5
S
Me
2
N H
O
O
D. (E)-3-(dibenzo[b,e]thiepin-11(6H)-ylidene)-N,N-dimethylpropan-1-amine S,S-dioxide,
S
H NMe
2
E. (Z)-3-(dibenzo[b,e]thiepin-11(6H)-ylidene)-N,N-dimethylpropan-1-amine.
__________________________________________________________________________________________________________ Ph Eur
15-6
Doxapram Hydrochloride
O
N
N
Ph
Ph
O
Et
H
,HCl
and enantiomer
C
24
H
30
N
2
O
2
,HCl,H
2
O 432.9 7081-53-0
Doxapram Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Respiratory stimulant.
Preparation
Doxapram Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Doxapram hydrochloride contains not less than 98.0 per cent and not more than the equivalent of
100.5 per cent of (RS)-1-ethyl-4-(2-morpholinoethyl)-3,3-diphenylpyrrolidin-2-one hydrochloride,
CHARACTERS
A white or almost white, crystalline powder, sparingly soluble in water, in alcohol and in methylene
chloride.
IDENTIFICATION
obtained with doxapram hydrochloride CRS. Examine the substances as discs prepared using potassium
chloride R.
substance.
the same solvent.
Reference solution. Dissolve 10 mg of doxapram hydrochloride CRS in methanol R and dilute to 10 ml
20 volumes of a 17 g/l solution of ammonia R and 80 volumes of 2-propanol R. Allow the plate to dry
in air. Spray with dilute potassium iodobismuthate solution R and examine immediately. The principal
TESTS
solvent.
(2.2.1) and colourless (Method II, 2.2.2).
+0.10.
15-7
as mobile phase at a flow rate of 1.5 ml per minute a mixture of 15 volumes of acetonitrile R, 20
volumes of a 1.1 g/l solution of ammonium dihydrogen phosphate R adjusted to pH 4.5 with dilute
phosphoric acid R, and 25 volumes of methanol R,
Inject 20 l of the reference solution (b). Adjust the sensitivity of the system so that the height of
the principal peak in the chromatogram obtained is at least 50 per cent of the full scale of the
recorder. When the chromatogram is recorded in the prescribed conditions, the retention time of
doxapram hydrochloride is about 10 min. Inject 20 l of the test solution. Continue the chromato-
graphy for twice the retention time of doxapram hydrochloride. In the chromatogram obtained with
the test solution: the area of any peak, apart from the principal peak, is not greater than the area of
the principal peak in the chromatogram obtained with reference solution (b) (0.2 per cent); the sum
of the areas of all the peaks, apart from the principal peak, is not greater than the area of the principal
peak in the chromatogram obtained with reference solution (a) (1 per cent). Disregard any peak due
to the solvent and any peak with an area less than 0.025 times that of the principal peak in the
obtained by diluting lead standard solution (100 ppm Pb) R with a mixture of 3 volumes of water R and
100C to 105C.
ASSAY
potentiometric titration (2.2.20) using 0.1M sodium hydroxide. Read the volume added between the
24
H
31
ClN
2
O
2
.
STORAGE
IMPURITIES
R
N
Ph
Ph
O
H
Et
and enantiomer
A. R = Cl : 4-(2-chloroethyl)-1-ethyl-3,3-diphenylpyrrolidin-2-one,
B. R = NH-CH
2
-CH
2
OH : 1-ethyl-4-[2-[(2-hydroxyethyl)amino]ethyl]-3,3-diphenylpyrrolidin-
2-one.
__________________________________________________________________________________________________________ Ph Eur
15-8
Doxepin Hydrochloride
corrected 1/01
O
H e
2
N
,HCl
C
19
H
21
NO,HCl 315.8 1229-29-4
Doxepin Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Doxepin Capsules
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Doxepin hydrochloride is (E)-3-(dibenzo[b,e]oxepin-11(6H)-ylidene)-N,N-dimethylpropan-1-amine
hydrochloride. It contains not less than 98.0 per cent and not more than the equivalent of 101.0 per
cent of C
19
H
22
ClNO, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, freely soluble in water, in alcohol and in methylene
chloride.
IDENTIFICATION
B. Dissolve 50.0 mg in a 1 g/l solution of hydrochloric acid R in methanol R and dilute to 100.0 ml with
the same acid solution. Dilute 5.0 ml to 50.0 ml with a 1 g/l solution of hydrochloric acid R in
spectrum of doxepin hydrochloride.
D. Dissolve about 5 mg in 2 ml of sulphuric acid R. A dark red colour is produced.
E. Solution S (see Tests) gives reaction (a) of chlorides (2.3.1).
TESTS
Solution S Dissolve 1 g in carbon dioxide-free water R and dilute to 20 ml with the same solvent.
(2.2.1) and colourless (Method II, 2.2.2).
Acidity To 10 ml of solution S add 0.1 ml of methyl red solution R. Not more than 0.1 ml of 0.1M
sodium hydroxide is required to change the colour of the indicator to yellow.
254
plate R (5 m) with a concentration zone.
the same solvent.
Reference solution (a). Dissolve 10.0 mg of doxepin impurity A CRS in methanol R, add 1 ml of the test
solution and dilute to 10 ml with methanol R. Dilute 1.0 ml of this solution to 50 ml with methanol R.
Reference solution (b). Dissolve 10.0 mg of doxepin impurity B CRS in methanol R, add 1 ml of the test
solution and dilute to 10 ml with methanol R. Dilute 1.0 ml of this solution to 50 ml with methanol R.
Reference solution (c). Dissolve 10.0 mg of doxepin impurity B CRS in methanol R and dilute to 200 ml
Apply to one plate (plate A) 2 l of the test solution and 2 l of reference solution (a). Develop over a
path of 5 cm using a mixture of 30 volumes of methyl ethyl ketone R and 60 volumes of heptane R.
Doxepin remains on the starting line
15-9
Apply to another plate (plate B) 2 l of the test solution and 2 l of reference solutions (b) and (c).
Develop over a path of 5 cm using a mixture of 10 volumes of methanol R and 90 volumes of
methylene chloride R. Allow the plates to dry at 120C for about 30 min. Spray with a solution
prepared as follows: dissolve 20 g of zinc chloride R in 30 ml of glacial acetic acid R, add 3 ml of
phosphoric acid R and 0.80 ml of strong hydrogen peroxide solution R and dilute to 60 ml with water R.
Heat the plates at 120C for 15 min and examine immediately in ultraviolet light at 365 nm.
Plate A. In the chromatogram obtained with the test solution: any spot corresponding to impurity A is
not more intense than the corresponding spot in the chromatogram obtained with reference solution
(a) (0.2 per cent), and any spot, apart from the principal spot and the spot corresponding to impurity
A, is not more intense than the spots in the chromatogram obtained with reference solution (a)
(0.2 per cent).
Plate B. In the chromatogram obtained with the test solution: any spot corresponding to impurity C
(R
f
about 0.12) is not more intense than the spot in the chromatogram obtained with reference
solution (c) (0.5 per cent), any spot corresponding to impurity B is not more intense than the
corresponding spot in the chromatogram obtained with reference solution (b) (0.2 per cent), and any
spot, apart from the principal spot, the spot corresponding to impurity B or the spot corresponding to
impurity C, is not more intense than the spots in the chromatogram obtained with reference solution
(b) (0.2 per cent).
The test is not valid unless the chromatograms obtained with reference solutions (a) and (b) show
clearly visible and separated principal spots.
Z-Isomer 13.0 per cent to 18.5 per cent of the Z-isomer, determined by liquid chromatography
(2.2.29).
20.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase.
a stainless steel column 0.12 m long and 4 mm in internal diameter packed with spherical
octylsilyl silica gel for chromatography R (5 m) with a specific surface area of 220 m
2
/g and a pore
size of 80 nm,
as mobile phase at a flow rate of 1 ml/min a mixture of 30 volumes of methanol R and 70 volumes
of a 30 g/l solution of sodium dihydrogen phosphate R, previously adjusted to pH 2.5 with
phosphoric acid R,
Inject 20 l of the test solution. Adjust the sensitivity of the system so that the height of the
principal peak is at least 50 per cent of the full scale of the recorder. The test is not valid unless the
resolution between the first peak (E-isomer) and the second peak (Z-isomer) is at least 1.5.
The ratio of the area of the peak due to the E-isomer to the area of the peak due to the Z-isomer is
4.4 to 6.7.
100C to 105C.
ASSAY
Titrate with 0.1M perchloric acid until the colour changes from blue to green, using 0.2 ml of crystal
violet solution R as indicator.
19
H
22
ClNO.
STORAGE
IMPURITIES
O
O
A. dibenzo[b,e]oxepin-11(6H)-one,
15-10
O
e
2
N
OH
and enantiomer
B. (11RS)-11-[3-(dimethylamino)propyl]-6,11-dihydrodibenzo[b,e]oxepin-11-ol,
O
eHN H
C. (E)-3-(dibenzo[b,e]oxepin-11(6H)-ylidene)-N-methylpropan-1-amine,
O
NMe
2
H
D. (Z)-3-(dibenzo[b,e]oxepin-11(6H)-ylidene)-N,N-dimethylpropan-1-amine.
__________________________________________________________________________________________________________ Ph Eur
15-11
Doxorubicin Hydrochloride
O
O
O
OH
OH
O
O
HO
Me
NH
2
H
MeO
CH
2
OH
OH
,HCl
C
27
H
29
NO
11
,HCl 580.0 25316-40-9
Doxorubicin Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Doxorubicin Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Doxorubicin hydrochloride is the hydrochloride of a substance produced by certain strains of
Streptomyces coeruleorubidus or Streptomyces peucetius or obtained by any other means. It contains not
less than 98.0 per cent and not more than the equivalent of 102.0 per cent of (8S,10S)-10-[(3-
amino-2,3,6-trideoxy--L-lyxo-hexopyranosyl)oxy]-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-
7,8,9,10-tetrahydronaphthacene-5,12-dione hydrochloride, calculated with reference to the anhy-
drous and solvent-free substance.
CHARACTERS
An orange-red, crystalline powder, hygroscopic, soluble in water, slightly soluble in methanol.
IDENTIFICATION
A. Dissolve 1 mg in alcohol R and dilute to 100 ml with the same solvent. Examined between 220 nm
and 550 nm (2.2.25), the solution shows six absorption maxima, at 234 nm, 252 nm, 288 nm,
475 nm, 495 nm and 530 nm.
obtained with doxorubicin hydrochloride CRS.
spot in the chromatogram obtained with reference solution (a) and differs in position from the prin-
cipal spot in the chromatogram obtained with reference solution (b).
D. Dissolve about 10 mg in 0.5 ml of nitric acid R, add 0.5 ml of water R and heat over a flame for
2 min. Allow to cool and add 0.5 ml of silver nitrate solution R1. A white precipitate is formed.
TESTS
Related substances Examine by thin-layer chromatography (2.2.27), using silica gel H R as the
coating substance.
Test solution (a). Dissolve 50 mg of the substance to be examined in 1 ml of water R and dilute to
Test solution (b). Dilute 1 ml of test solution (a) to 5 ml with a mixture of 10 volumes of water R and
15-12
Reference solution (a). Dissolve 10 mg of doxorubicin hydrochloride CRS in a mixture of 10 volumes of
water R and 90 volumes of methanol R and dilute to 5 ml with the same mixture of solvents.
Reference solution (b). Dissolve 10 mg of daunorubicin hydrochloride CRS in a mixture of 10 volumes of
Reference solution (c). Dissolve 5 mg of doxorubicin aglycone CRS (doxorubicinone) in methylene
Reference solution (d). Dilute 1 ml of reference solution (a) to 40 ml with methanol R.
Apply separately to the plate as 5 mm bands 10 l of each solution. Develop in an unsaturated tank
over a path of 15 cm using a mixture of 1 volume of water R, 2 volumes of anhydrous formic acid R, 15
volumes of methanol R and 82 volumes of methylene chloride R. Allow the plate to dry in air and
examine immediately. In the chromatogram obtained with test solution (a): any spot corresponding
to doxorubicin aglycone is not more intense than the principal spot in the chromatogram obtained
with reference solution (c) (0.5 per cent); any spot, apart from the principal spot and any spot corres-
ponding to doxorubicin aglycone, is not more intense than the principal spot in the chromatogram
obtained with reference solution (d) (0.5 per cent).
Acetone and ethanol Not more than a total of 2.0 per cent m/m, of which acetone is not more than
0.5 per cent m/m, determined by gas chromatography (2.2.28) using dioxan R as the internal
standard.
Test solution. Dissolve 50 mg of the substance to be examined in 1.0 ml of water R containing 1 g/l of
dioxan R.
Reference solution. Mix 0.50 g of ethanol R, 0.50 g of acetone R and 1.00 g of dioxan R and dilute to
100 ml with water R. Dilute 10 ml of this solution to 100 ml with water R.
a glass column 2 m long and 3 mm in internal diameter packed with diatomaceous earth for chroma-
tography R1 impregnated with 10 per cent m/m of macrogol 20,000 R,
at 125C. Inject 1 l of each solution.
mination of water.
without a further appropriate procedure for removal of pyrogens, it complies with the test for
bacterial endotoxins. Not more than 2.2 I.U. of endotoxin per milligram.
ASSAY
Reference solution (a). Dissolve 50.0 mg of doxorubicin hydrochloride CRS in the mobile phase and
Reference solution (b). Dissolve 10.0 mg of doxorubicin hydrochloride CRS and 10.0 mg of epirubicin
hydrochloride CRS in the mobile phase and dilute to 50.0 ml with the mobile phase. Dilute 10.0 ml of
as mobile phase at a flow rate of 0.8 ml per minute a mixture of 5 volumes of methanol R1, 45
volumes of acetonitrile R and 50 volumes of a solution containing 2.88 g/l of sodium lauryl
sulphate R and 2.30 g/l of phosphoric acid R,
Inject 20 l of reference solution (b) six times. The test is not valid unless the resolution between
the peaks corresponding to doxorubicin and epirubicin is not less than 2.0 and the relative standard
deviation for the peak area of doxorubicin is not more than 1.0 per cent.
If these criteria are not fulfilled, adjust the operating conditions. Inject 20 l of the test solution and
20 l of reference solution (a) and calculate the percentage content of C
27
H
30
ClNO
11
.
STORAGE
container.
15-13
LABELLING
The label states:
__________________________________________________________________________________________________________ Ph Eur
15-14
Doxycycline
OH OH
OH
CONH
2
H H
OH NMe
2
Me H
OH
H
O O
C
22
H
24
N
2
O
8
,H
2
O 462.5 17086-28-1
Doxycycline complies with the requirements of the 3rd edition of the European Pharmacopoeia [0820]. These
Preparation
Dispersible Doxycycline Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Doxycycline is the monohydrate of (4S,4aR,5S,5aR,6R, 12aS)-4-dimethylamino-
1,4,4a,5,5a,6,11,12a-octahydro-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxonaphthacene-2-
carboxamide, a substance obtained from oxytetracycline or metacycline or by any other means. It
contains not less than 95.0 per cent and not more than the equivalent of 100.5 per cent of
doxycycline (C
22
H
24
N
2
O
8
CHARACTERS
A yellow, crystalline powder, very slightly soluble in water and in alcohol, practically insoluble in
ether. It dissolves in dilute solutions of mineral acids and in solutions of alkali hydroxides and
carbonates.
IDENTIFICATION
Adjust the pH of a 100 g/l solution of sodium edetate R to 9.0 with strong sodium hydroxide solution R
and spray the solution evenly onto the plate (about 10 ml for a plate 100 mm 200 mm). Allow the
plate to dry in a horizontal position for at least 1 h. At the time of use, dry the plate in an oven at
110C for 1 h.
the same solvent.
Reference solution (a). Dissolve 5 mg of doxycycline hyclate CRS in methanol R and dilute to 10 ml with
the same solvent.
Reference solution (b). Dissolve 5 mg of doxycycline hyclate CRS and 5 mg of tetracycline hydrochloride
volumes of water R, 35 volumes of methanol R and 59 volumes of methylene chloride R. Dry the plate
in a stream of air. Examine in ultraviolet light at 365 nm. The principal spot in the chromatogram
B. To about 2 mg add 5 ml of sulphuric acid R. A yellow colour develops.
C. Dissolve 25 mg in a mixture of 0.2 ml of dilute nitric acid R and 1.8 ml of water R. The solution
does not give reaction (a) of chlorides (2.3.1).
TESTS
Specific optical rotation (2.2.7). Dissolve 0.250 g in a mixture of 0.5 volume of hydrochloric acid R
and 99.5 volumes of methanol R and dilute to 25.0 ml with the same mixture of solvents. The specific
optical rotation is 113 to 130, calculated with reference to the anhydrous substance.
Absorbance (2.2.25). Dissolve 25.0 mg in a mixture of 0.5 volumes of hydrochloric acid R and 99.5
volumes of methanol R and dilute to 50.0 ml with the same mixture of solvents. Dilute 2.0 ml of this
15-15
solution to 100.0 ml with a mixture of 0.5 volumes of 1M hydrochloric acid R and 99.5 volumes of
methanol R. The specific absorbance determined at the maximum at 349 nm is 325 to 363, calculated
with reference to the anhydrous substance. Carry out the measurement within l h of preparing the
solution.
Light-absorbing impurities Dissolve 0.10 g in a mixture of 0.5 volume of hydrochloric acid R and
99.5 volumes of methanol R and dilute to 10.0 ml with the same mixture of solvents. The absorbance
(2.2.25), determined at 490 nm and calculated with reference to the anhydrous substance, is not
greater than 0.07. Carry out the measurement within 1 h of preparing the solution.
Related substances Examine by liquid chromatography (2.2.29), as prescribed under Assay. Inject
the test solution and reference solution (e). In the chromatogram obtained with the test solution: the
area of any peak corresponding to metacycline or 6-epidoxycycline is not greater than the area of the
corresponding peak in the chromatogram obtained with reference solution (e) (2.0 per cent); the area
of any peak appearing between the solvent peak and the peak corresponding to metacycline and the
area of any peak appearing on the tail of the principal peak, is not greater than 25 per cent of the area
of the peak corresponding to 6-epidoxycycline in the chromatogram obtained with reference solution
(e) (0.5 per cent).
mination of water.
ASSAY
Reference solution (a). Dissolve 20.0 mg of doxycycline hyclate CRS in 0.01M hydrochloric acid and dilute
Reference solution (b). Dissolve 20.0 mg of 6-epidoxycycline hydrochloride CRS in 0.01M hydrochloric acid
Reference solution (c). Dissolve 20.0 mg of metacycline hydrochloride CRS in 0.01M hydrochloric acid and
Reference solution (d). Mix 4.0 ml of reference solution (a), 1.5 ml of reference solution (b) and 1.0 ml
of reference solution (c) and dilute to 25.0 ml with 0.01M hydrochloric acid.
a column 0.25 m long and 4.6 mm in internal diameter packed with styrene-divinylbenzene copoly-
mer R (8 m to 10 m),
as mobile phase at a flow rate of 1.0 ml per minute a solution prepared as follows: weigh 60.0 g of
2-methyl-2-propanol R and transfer to a 1000 ml volumetric flask with the aid of 200 ml of water R;
add 400 ml of buffer solution pH 8.0 R, 50 ml of a 10 g/l solution of tetrabutylammonium hydrogen
sulphate R adjusted to pH 8.0 with dilute sodium hydroxide solution R and 10 ml of a 40 g/l solution
of sodium edetate R adjusted to pH 8.0 with dilute sodium hydroxide solution R; dilute to 1000.0 ml
with water R,
a 20 l fixed loop injector,
maintaining the column at 60C. Inject reference solution (d). Adjust the sensitivity of the detector
so that the height of the peaks is not less than half the full-scale of the detector. The test is not valid
unless the resolution between the first peak (metacycline) and the second peak (6-epidoxycycline) is
at least l.25 and the resolution between the second peak and the third peak (doxycycline) is at least
2.0. Adjust the concentration of 2-methyl-2-propanol in the mobile phase if necessary. The test is not
valid unless the symmetry factor for the third peak is at most 1.25. Inject reference solution (a) six
times. The test is not valid unless the relative standard deviation of the peak area for doxycycline is at
most 1.0 per cent. If necessary, adjust the integrator parameters. Inject alternately the test solution
Calculate the percentage content of doxycycline.
STORAGE
15-16
IMPURITIES
A. 6-epidoxycycline,
B. metacycline,
C. 4-epidoxycycline,
D. 4,6-epidoxycycline,
E. oxytetracycline,
F. 2-acetyl-2-decarboxamidodoxycycline.
__________________________________________________________________________________________________________ Ph Eur
15-17
Doxycycline Hyclate
OH OH
OH
CONH
2
H H
OH NMe
2
Me H
OH
H
O O
,HCl, C
2
H
5
OH, H
2
O
C
22
H
24
N
2
O
8
,HC1,C
2
H
6
O,H
2
O 512.9 24390-14-5
Doxycycline Hyclate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0272].
Preparation
Doxycycline Capsules
When doxycycline hydrochloride is prescribed or demanded, Doxycycline Hyclate shall be
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Doxycycline hyclate is the hydrochloride hemiethanol hemihydrate of (4S,4aR,5S,5aR,6R,12aS)-
4-dimethylamino-1,4,4a,5,5a,6,11,12a-octahydro-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-
dioxonaphthacene-2-carboxamide, a substance obtained from oxytetracycline or metacycline or by
any other means. It contains not less than 88.0 per cent and not more than 94.0 per cent of
doxycycline (C
22
H
24
N
2
O
8
), calculated with reference to the anhydrous, ethanol-free substance.
CHARACTERS
A yellow, crystalline powder, hygroscopic, freely soluble in water and in methanol, sparingly soluble
in alcohol, practically insoluble in ether. It dissolves in solutions of alkali hydroxides and carbonates.
IDENTIFICATION
Adjust a 100 g/l solution of sodium edetate R to pH 9.0 with strong sodium hydroxide solution R and
spray the solution evenly onto the plate (about 10 ml for a plate 100 mm 200 mm). Allow the plate
to dry in a horizontal position for at least 1 h. At the time of use, dry the plate in an oven at 110C
for 1 h.
the same solvent.
Reference solution (a). Dissolve 5 mg of doxycycline hyclate CRS in methanol R and dilute to 10 ml with
the same solvent.
Reference solution (b). Dissolve 5 mg of doxycycline hyclate CRS and 5 mg of tetracycline hydrochloride
volumes of water R, 35 volumes of methanol R and 59 volumes of methylene chloride R. Dry the plate
in a stream of air and examine in ultraviolet light at 365 nm. The principal spot in the chromatogram
B. To about 2 mg add 5 ml of sulphuric acid R. A yellow colour develops.
TESTS
Specific optical rotation (2.2.7). Dissolve 0.250 g in a mixture of 1 volume of 1M hydrochloric acid
and 99 volumes of methanol R and dilute to 25.0 ml with the same mixture of solvents. The specific
15-18
optical rotation is 105 to 120, calculated with reference to the anhydrous, ethanol-free
substance. Carry out the measurement within 5 min of preparing the solution.
Absorbance (2.2.25). Dissolve 25.0 mg in a mixture of 1 volume of 1M hydrochloric acid and 99
volumes of methanol R and dilute to 25.0 ml with the same mixture of solvents. Dilute 1.0 ml of this
solution to 100.0 ml with a mixture of 1 volume of 1M hydrochloric acid and 99 volumes of
methanol R. The specific absorbance determined at the maximum at 349 nm is 300 to 335, calculated
with reference to the anhydrous, ethanol-free substance. Carry out the measurement within 1 h of
preparing the solution.
Light-absorbing impurities Dissolve 0.10 g in a mixture of 1 volume of 1M hydrochloric acid and 99
volumes of methanol R and dilute to 10.0 ml with the same mixture of solvents. The absorbance
(2.2.25), determined at 490 nm and calculated with reference to the anhydrous, ethanol-free
substance, is not greater than 0.07. Carry out the measurement within 1 h of preparing the solution.
the test solution and reference solution (e). In the chromatogram obtained with the test solution: the
area of any peak corresponding to metacycline or 6-epidoxycycline is not greater than the area of the
corresponding peak in the chromatogram obtained with reference solution (e) (2 per cent); the area
of any peak appearing between the solvent peak and the peak corresponding to metacycline and the
area of any peak appearing on the tail of the main peak is not greater than 25 per cent of the area of
the peak corresponding to 6-epidoxycycline in the chromatogram obtained with reference solution (e)
(0.5 per cent).
Ethanol 4.3 per cent m/m to 6.0 per cent m/m, determined by gas chromatography (2.2.28), using
Test solution (b). Dissolve 0.10 g of the substance to be examined in the internal standard solution
and dilute to 10.0 ml with the same solution.
Reference solution. Dilute 0.50 ml of ethanol R to 100.0 ml with the internal standard solution. Dilute
1.0 ml of the solution to 10.0 ml with the internal standard solution.
a column 1.5 m long and 4 mm in internal diameter packed with ethylvinylbenzene-divinylbenzene
copolymer R (150 m to 180 m),
detector at 150C. Inject the chosen volume of each of the test solutions and of the reference
solution. Calculate the content of ethanol taking the density (2.2.5) at 20C to be 0.790 g/ml.
Water (2.5.12). 1.4 per cent to 2.8 per cent, determined on 1.20 g by the semi-micro determination
of water.
ASSAY
Reference solution (a). Dissolve 20.0 mg of doxycycline hyclate CRS in 0.01M hydrochloric acid and dilute
Reference solution (b). Dissolve 20.0 mg of 6-epidoxycycline hydrochloride CRS in 0.01M hydrochloric acid
Reference solution (c). Dissolve 20.0 mg of metacycline hydrochloride CRS in 0.01M hydrochloric acid and
Reference solution (d). Mix 4.0 ml of reference solution (a), 1.5 ml of reference solution (b) and 1.0 ml
of reference solution (c) and dilute to 25.0 ml with 0.01M hydrochloric acid.
15-19
a column 0.25 m long and 4.6 mm in internal diameter packed with styrenedivinylbenzene
copolymer R (8 m to 10 m) and maintained at 60C,
as mobile phase at a flow rate of 1.0 ml/min a solution prepared as follows: weigh 60.0 g of
2-methyl-2-propanol R and transfer to a 1000 ml volumetric flask with the aid of 200 ml of
water R; add 400 ml of buffer solution pH 8.0 R, 50 ml of a 10 g/l solution of tetrabutylammonium
hydrogen sulphate R adjusted to pH 8.0 with dilute sodium hydroxide solution R and 10 ml of a
40 g/l solution of sodium edetate R adjusted to pH 8.0 with dilute sodium hydroxide solution R;
dilute to 1000 ml with water R,
an injector with a fixed loop of 20 l,
Inject reference solution (d). Adjust the attenuation to obtain peaks with a height corresponding to
at least half the full-scale deflection. The test is not valid unless the resolution between the first peak
(metacycline) and the second peak (6-epidoxycycline) is not less than 1.25 and the resolution
between the second peak and the third peak (doxycycline) is not less than 2.0. Adjust the 2-methyl-2-
propanol content in the mobile phase if necessary. The test is not valid unless the symmetry factor for
the third peak is not more than 1.25. Inject reference solution (a) six times. The test is not valid
unless the relative standard deviation of the peak area for doxycycline is not greater than 1.0 per cent.
If necessary adjust the integrator parameters. Inject alternately the test solution and reference
solution (a).
Calculate the percentage content of doxycycline.
STORAGE
LABELLING
The label states:
IMPURITIES
OH HO
OH
O
CONH
2
H
OH
O
OH
H
Me NMe
2
A. 6-epidoxycycline,
OH HO
OH
O
CONH
2
H
OH
O
OH
H
NMe
2
CH
2
B. metacycline,
OH HO
OH
O
CONH
2
H
OH
O
OH
H
Me NMe
2
C. 4-epidoxycycline,
OH HO
OH
O
CONH
2
H
OH
O
OH
H
Me NMe
2
D. 4,6-epidoxycycline,
15-20
E. oxytetracycline,
OH HO
OH
O
H
OH
O
OH
H
Me NMe
2
CH
3
O
F. 2-acetyl-2-decarboxamidodoxycycline.
__________________________________________________________________________________________________________ Ph Eur
15-21
Droperidol
N
N
N
F
O
O
H
C
22
H
22
FN
3
O
2
379.4 548-73-2
Droperidol complies with the requirements of the 3rd edition of the European Pharmacopoeia [1010]. These
Preparations
Droperidol Injection
Droperidol Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Droperidol contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of 1-[1-[4-(4-fluorophenyl)-4-oxobutyl]-1,2,3,6-tetrahydropyridin-4-yl]-2,3-dihydro-1H-
benzimidazol-2-one, calculated with reference to the dried substance.
CHARACTERS
A white or almost white powder, practically insoluble in water, freely soluble in dimethylformamide
IDENTIFICATION
obtained with droperidol CRS. Examine the substances prepared as discs. If the spectra obtained show
minimum volume of acetone R, evaporate to dryness on a water-bath and record new spectra using
the residues.
254
Test solution. Dissolve 30 mg of the substance to be examined in a mixture of 1 volume of acetone R
Reference solution (a). Dissolve 30 mg of droperidol CRS in a mixture of 1 volume of acetone R and 9
Reference solution (b). Dissolve 30 mg of droperidol CRS and 30 mg of benperidol CRS in a mixture of 1
volume of acetone R and 9 volumes of methanol R and dilute to 10 ml with the same mixture of
solvents.
1 volume of acetone R and 9 volumes of methanol R. Allow the plate to dry in air and examine in
C. Dissolve about 10 mg in 5 ml of ethanol R. Add 0.5 ml of dinitrobenzene solution R and 0.5 ml of
2M alcoholic potassium hydroxide R. A violet colour is produced and becomes brownish-red after
20 min.
15-22
TESTS
Appearance of solution Dissolve 0.20 g in methylene chloride R and dilute to 20.0 ml with the same
solvent. The solution is clear (2.2.1) and not more intensely coloured than reference solution BY
5
(Method II, 2.2.2).
before use.
Reference solution (a). Dissolve 2.5 mg of droperidol CRS and 2.5 mg of benperidol CRS in
a stainless steel column 0.10 m long and 4.6 mm in internal diameter packed with deactivated
as mobile phase at a flow rate of 1.5 ml per minute a 10 g/l solution of tetrabutylammonium
hydrogen sulphate R and acetonitrile R changing by linear gradient to a mixture of 6 volumes of a
10 g/l solution of tetrabutylammonium hydrogen sulphate R and 4 volumes of acetonitrile R over
15 min, followed by the final mixture for 5 min,
conditions, the retention times are: benperidol, about 6.5 min and droperidol, about 7 min. The test
is not valid unless the resolution between the peaks due to droperidol and benperidol is at least 2.0. If
necessary, adjust the final concentration of acetonitrile in the mobile phase or adjust the time
programme for the linear gradient.
Inject 10 l of dimethylformamide R as a blank, 10 l of the test solution and 10 l of reference
with reference solution (b) (0.5 per cent). Disregard any peak obtained with the blank and any peak
100C to 105C.
ASSAY
ethyl ketone R. Using 0.2 ml of naphtholbenzein solution R, titrate with 0.1M perchloric acid until the
colour changes from orange-yellow to green.
22
H
22
FN
3
O
2
.
STORAGE
IMPURITIES
N
N
HN
O
H
A. 2,3-dihydro-1-(1,2,3,6-tetrahydropyridin-4-yl)-1H-benzimidazol-2-one,
15-23
N
N
N
O
O
F
H
B. 1-[1-[4-(2-fluorophenyl)-4-oxobutyl]-1,2,3,6-tetrahydropyridin-4-yl]-2,3-dihydro-1H-
benzimidazol-2-one,
N
N
N
O
O
F
+
Cl
-
H
C. 4-(2,3-dihydro-2-oxo-1H-benzimidazol-1-yl)-1-[4-(4-fluorophenyl)-4-oxobutyl]pyridinium
chloride,
N
N
N
O
O
F
O
H
D. 1-[1-[4-(4-fluorophenyl)-4-oxobutyl]-1,2,3,6-tetrahydropyridin-4-yl]-2,3-dihydro-1H-
benzimidazol-2-one N-oxide,
N
H
N
N
N
H
N
N
O
O
O
E. 2,3-dihydro-1-[1-[4-[4-[4-(2,3-dihydro-2-oxo-1H-benzimidazol-1-yl)-1,2,3,6-tetra-
hydropyridin-1-yl]-1-oxobutyl]phenyl]-1,2,3,6-tetrahydropyridin-4-yl]-1H-benzimidazol-2-
one.
__________________________________________________________________________________________________________ Ph Eur
15-24
Dydrogesterone
H
H
Me
O
H
H
Me
CH
3
O
C
21
H
28
O
2
312.5 152-62-5
Definition Dydrogesterone is 9,10-pregna-4,6-diene-3,20-dione. It contains not less than
21
H
28
O
2
Practically insoluble in water; freely soluble in chloroform; soluble in acetone; sparingly soluble in
ethanol (96%) and in methanol; slightly soluble in ether and in fixed oils.
Identification
dydrogesterone (RS 118).
B. Complies with the test for identification of steroids, Appendix III A, using impregnating solvent II and
mobile phase E.
Light absorption The light absorption, Appendix II B, in the range 230 to 350 nm of the solution
obtained in the Assay exhibits a maximum only at 286 nm. The ratio of the absorbance at 240 nm to
that at the maximum at 286 nm is not more than 0.12.
Specific optical rotation In a 1% w/v solution in 1,4-dioxan, 446 to 464, calculated with refer-
Related substances Carry out the method for gas chromatography, Appendix III B, using three
solutions in chloroform containing (1) 0.040% w/v of the substance being examined and 0.020% w/v
of 1,2,3,4-tetraphenylcyclopenta-1,3-diene (internal standard), (2) 2.0% w/v of the substance being
examined and (3) 2.0% w/v of the substance being examined and 0.020% w/v of the internal
standard.
The chromatographic procedure may be carried out using a glass column (1.5 m 2.5 mm)
packed with acid-washed, silanised diatomaceous support (100 to 120 mesh) coated with 3% w/w of
cyanopropyl methyl silicone fluid (SP-2300 is suitable) and maintained at 240.
In the chromatogram obtained with solution (3) the ratio of the sum of the areas of any secondary
peaks to the area of the peak due to the internal standard is not greater than the ratio of the area of
the peak due to the substance being examined to the area of the peak due to the internal standard in
the chromatogram obtained with solution (1).
Assay Dissolve 40 mg in sufficient methanol to produce 200 ml, dilute 5 ml to 100 ml with methanol
and measure the absorbance of the resulting solution at the maximum at 286 nm, Appendix II B.
21
H
28
O
2
286 nm.
Storage Dydrogesterone should be kept in a well-closed container and protected from light.
Preparation
Dydrogesterone Tablets
15-25
Econazole Nitrate
,HNO
3
and enantiomer
Cl
O
Cl
Cl
N
N
H
C
18
H
15
Cl
3
N
2
O,HNO
3
444.7 68797-31-9
Econazole Nitrate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0665].
Preparations
Econazole Cream
Econazole Pessaries
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Econazole nitrate contains not less than 98.5 per cent and not more than the equivalent of 101.5 per
cent of (RS)-1-[2-(4-chlorobenzyloxy)-2-(2,4-dichlorophenyl)ethyl]imidazole nitrate, calculated with
CHARACTERS
A white or almost white, crystalline powder, very slightly soluble in water, soluble in methanol,
sparingly soluble in methylene chloride, slightly soluble in alcohol, practically insoluble in ether.
IDENTIFICATION
B. Dissolve 40 mg in a mixture of 1 volume of 0.1M hydrochloric acid and 9 volumes of 2-propanol R,
and dilute to 100.0 ml with the same mixture of solvents. Examined between 240 nm and 320 nm
(2.2.25), the solution shows three absorption maxima, at 265 nm, 271 nm and 280 nm. The ratio of
the absorbance measured at the maximum at 271 nm to that at the maximum at 280 nm is 1.55 to
1.70. The identification is not valid unless, in the test for resolution (2.2.25), the ratio of the
absorbances is at least 2.
obtained with econazole nitrate CRS. Examine the substances prepared as discs.
254 nm before spraying. The principal spot in the chromatogram obtained with test solution (b) is
solution (a).
E. It gives the reaction of nitrates (2.3.1).
TESTS
The solution is clear (2.2.1) and not more intensely coloured than degree 7 of the range of reference
solutions of the most appropriate colour (Method II, 2.2.2).
Test solution (a). Dissolve 0.25 g in a mixture of 1 volume of concentrated ammonia R and 9 volumes
of methanol R and dilute to 5 ml with the same mixture of solvents.
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with a mixture of 1 volume of concentrated
ammonia R and 9 volumes of methanol R.
Reference solution (a). Dissolve 25 mg of econazole nitrate CRS in a mixture of 1 volume of concentrated
15-26
ammonia R and 9 volumes of methanol R, and dilute to 5 ml with the same mixture of solvents.
Reference solution (b). Dilute 2.5 ml of test solution (b) to 100 ml with a mixture of 1 volume of
concentrated ammonia R and 9 volumes of methanol R.
1 volume of concentrated ammonia R, 40 volumes of toluene R and 60 volumes of dioxan R. Dry the
plate in a stream of air for 15 min. Examine in ultraviolet light at 254 nm. Any spot in the chromato-
gram obtained with test solution (a), apart from the principal spot, is not more intense than the spot
in the chromatogram obtained with reference solution (b) (0.25 per cent). Spray the plate with
potassium iodobismuthate solution R2 and examine in daylight. Any spot in the chromatogram obtained
togram obtained with reference solution (b) (0.25 per cent). The test is not valid unless the chroma-
togram obtained with reference solution (b) shows a clearly visible spot.
100C to 105C for 2 h.
ASSAY
the end-point potentiometrically (2.2.20). Carry out a blank titration.
18
H
16
Cl
3
N
3
O
4.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
15-27
Edetic Acid
N N COOH OOC
HOOC COOH
C
10
H
16
N
2
O
8
292.2 60-00-4
Definition Edetic Acid is ethylenediaminetetra-acetic acid. It contains not less than 98.0% and not
10
H
16
N
2
O
8
Characteristics A white, crystalline powder or colourless crystals. Practically insoluble in water, in
ethanol (96%) and in chloroform. It dissolves in dilute solutions of alkali hydroxides.
Dissolve 5.0 g of the substance being examined in 20 ml of 2M sodium hydroxide and add sufficient water to
produce 100 ml (solution S).
Identification
A. The infrared absorption spectrum, Appendix II A, is concordant with the reference spectrum of edetic
acid (RS 119).
B. To 10 ml of solution S add 0.5 ml of calcium chloride solution, make alkaline to red litmus paper with
2M ammonia and add 3 ml of ammonium oxalate solution. No precipitate is produced.
Appearance of solution Solution S is clear, Appendix IV A, and colourless, Appendix
IV B, Method II.
Nitrilotriacetic acid Carry out the method for liquid chromatography, Appendix III D, using the
following solutions. For solution (1) dissolve 0.1 g of the substance being examined in 0.7 ml of 1M
sodium hydroxide, warming if necessary to aid dissolution, and add sufficient of a 1% w/v solution of
copper(II) nitrate to produce 10 ml. For solution (2) dissolve, with the aid of ultrasound, 50 mg of
nitrilotriacetic acid in sufficient of a 1% w/v solution of copper(II) nitrate to produce 25 ml and dilute
1 ml of this solution to 100 ml with the same solvent. For solution (3) dissolve 0.1 g of the substance
being examined in 0.7 ml of 1M sodium hydroxide, warming if necessary to aid dissolution, add 5 ml of
solution (2) and sufficient of a 1% w/v solution of copper(II) nitrate to produce 10 ml.
4.6 mm) packed with stationary phase C ( 5 m) (Hypersil ODS is suitable) and a precolumn (1 cm
4.6 mm) packed with the same material, (b) as the mobile phase with a flow rate of 1 ml per minute a
mixture of 850 ml of a 0.5% w/v solution of tetrabutylammonium dihydrogen orthophosphate adjusted to
pH 7.7 with an 8.5% w/v solution of sodium hydroxide, 115 ml of methanol and sufficient water to
produce 1000 ml and (c) a detection wavelength of 254 nm.
Equilibrate the column by passing the mobile phase through it at a rate of 1 ml per minute in a
closed system for 1 hour before use.
Inject separately 50 l of a 1% w/v solution of copper(II) nitrate and of solution (2) and identify the
peak due to nitrilotriacetic acid from the chromatograms obtained. Inject separately 50 l of solution
(1) and of solution (3) allowing the chromatography to proceed until the peak due to edetic acid has
eluted. In the chromatogram obtained with solution (1) the area of any peak corresponding to
nitrilotriacetic acid is not greater than the difference between the area of the peak due to nitrilo-
triacetic acid in the chromatogram obtained with solution (3) and the area of any peak due to
nitrilotriacetic acid in the chromatogram obtained with solution (1) (0.1%).
Iron 2.5 ml of solution S diluted to 10 ml with water complies with the limit test for iron, Appendix
VII (80 ppm). Add 0.25 g of calcium chloride to both the test and standard solutions before addition
of the mercaptoacetic acid. Carry out the evaluation 5 minutes after the preparation of the test
solution.
Heavy metals 1.0 g complies with limit test D for heavy metals, Appendix VII (20 ppm). Use a quartz
crucible. If necessary during the ashing process, add a few drops of nitric acid and heat until a white
ash is obtained. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb).
Chloride Mix 10 ml of solution S with 10 ml of 2M nitric acid and allow to stand for 10 minutes.
Filter, wash the filter and dilute the combined filtrate and washings to 20 ml with water. 10 ml of this
solution diluted to 15 ml with water complies with the limit test for chlorides, Appendix VII (200 ppm).
Loss on drying When dried for 1 hour at 110, loses not more than 0.1% of its weight. Use 5 g.
Assay Dissolve 0.25 g in a mixture of 2 ml of 2M sodium hydroxide and 300 ml of water, add 2 g of
hexamine, 2 ml of 2M hydrochloric acid and 50 mg of xylenol orange triturate and titrate with 0.1M zinc
sulphate VS to a red colour. Each ml of 0.1M zinc sulphate VS is equivalent to 29.22 mg of
C
10
H
16
N
2
O
8
.
15-28
Storage Edetic Acid should be kept in a well-closed container.
Action and use Chelating agent; pharmaceutical aid.
IMPURITIES
N COOH
HOOC
COOH
A. nitrilotriacetic acid
15-29
Edrophonium Chloride
NMe
2
HO
Et
+
Cl
C
10
H
16
ClNO 201.7 16-38-1
Definition Edrophonium Chloride is ethyl(3-hydroxyphenyl)dimethylammonium chloride. It
10
H
16
ClNO, calculated with reference
Very soluble in water; freely soluble in ethanol (96%); practically insoluble in chloroform and in ether.
Identification
edrophonium chloride (RS 120).
Dimethylaminophenol Dissolve 0.1 g in 10 ml of water, add 5 ml of phosphate buffer pH 8.0 and
extract with two 20-ml quantities of chloroform. Wash the extracts successively with two 10-ml
quantities of water, extract with 10 ml of 0.1M sodium hydroxide and discard the chloroform. The
absorbance of the resulting solution at 293 nm is not greater than 0.25, Appendix II B.
Assay Carry out Method I for non-aqueous titration, Appendix VIII A, using 0.6 g, adding 20 ml of
mercury(II) acetate solution and using a 0.1% w/v solution of quinaldine red in absolute ethanol as
10
H
16
ClNO.
Storage Edrophonium Chloride should be kept in a well-closed container and protected from light.
Action and use Anticholinesterase.
Preparation
Edrophonium Injection
15-30
Elder Flower
Elder Flower complies with the requirements of the 3rd edition of the European Pharmacopoeia [1217]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Elder flower consists of the dried flowers of Sambucus nigra L. It contains not less than 0.80 per cent
of flavonoids, calculated as isoquercitroside (C
21
H
20
O
12
; M
r
CHARACTERS
IDENTIFICATION
A. The flower, about 5 mm in diameter, has three small bracts (hand lens) and may have a peduncle.
The five-toothed calyx is small; the corolla is light yellow, with five broadly oval petals fused at their
bases into a tube. The filaments of the five yellow stamens alternate with the petals. The corolla is
often isolated or attached to the stamens, to which it is fused at the base. The ovary is inferior with
three locules and it bears a short style with three obtuse stigmata.
B. Reduce to a powder (355). The powder is greenish-yellow. Examine under a microscope using
chloral hydrate solution R. The powder shows numerous spherical, sometimes ellipsoidal, pollen grains
about 30 m in diameter, with three germinal pores and very finely pitted exine; calyx epidermal cells
with a striated cuticle and occasional unicellular marginal teeth from the basal region; corolla
fragments with numerous small globules of volatile oil, those of the upper epidermis with slightly
thickened and beaded walls and a striated cuticle; mesophyll cells of petals and sepals with idioblasts
containing numerous sandy crystals of calcium oxalate.
C. Examine in ultraviolet light at 365 nm the chromatograms obtained in the test for Sambucus
ebulus. The chromatogram obtained with the test solution shows an intense zone of light blue
fluorescence due to chlorogenic acid, a zone of orange fluorescence due to rutin as well as a zone of
orange fluorescence due to isoquercitrin, which appears slightly above the zone due to hyperoside in
the chromatogram obtained with the reference solution. A zone of greenish-blue fluorescence appears
in the chromatogram obtained with the test solution a little below the caffeic acid zone in the
chromatogram obtained with the reference solution. Additional faint fluorescent zones may be
present. In daylight, only the orange fluorescent zones due to rutin and isoquercitrin in the chromato-
gram obtained with the test solution are clearly visible.
TESTS
Foreign matter (2.8.2). Determined on 10 g, not more than 8 per cent of fragments of coarse
pedicels and other foreign matter; not more than 15 per cent of discolored, brown flowers.
Sambucus ebulus L. Examine by thin-layer chromatography (2.2.27), using a suitable silica gel as
Test solution. To 0.5 g of the powdered drug (355) add 10 ml of methanol R and heat in a water bath
at 65C for 5 min, shaking frequently. Allow to cool and filter. Dilute the filtrate to 10 ml with
methanol R.
Reference solution. Dissolve 1 mg of caffeic acid R, 1 mg of chlorogenic acid R, 2.5 mg of hyperoside R
and 2.5 mg of rutin R in 10 ml of methanol R.
Apply separately to the plate, as bands, 10 l of each solution. Develop over a path of 15 cm using a
ketone R and 50 volumes of ethyl acetate R. Dry the plate at 100C to 105C and spray it while still
warm with a 10 g/l solution of diphenylboric acid aminoethyl ester R in methanol R. Subsequently spray
the plate with a 50 g/l solution of macrogol 400 R in methanol R. Allow the plate to dry in air for
30 min and examine in ultraviolet light at 365 nm. The chromatogram obtained with the reference
solution shows in the lower half, with increasing R
f
values, the zone of orange fluorescence due to
rutin, the zone of light blue fluorescence due to chlorogenic acid and the zone of orange-yellow to
orange-brown fluorescence due to hyperoside. The upper third presents a zone of greenish-blue
fluorescence due to caffeic acid. The chromatogram obtained with the test solution does not show a
pink zone below the zone due to rutin in the chromatogram obtained with the reference solution.
ASSAY
Stock solution. In a 100 ml round-bottomed flask, introduce 0.600 g of the powdered drug (355), add
15-31
1 ml of a 5 g/l solution of hexamethylenetetramine R, 20 ml of acetone R and 2 ml of hydrochloric
acid R1. Boil the mixture under a reflux condenser for 30 min. Filter the liquid through a plug of
absorbent cotton into a flask. Add the absorbent cotton to the residue in the round-bottomed flask
and extract with two quantities, each of 20 ml, of acetone R, each time boiling under a reflux
condenser for 10 min. Allow to cool, filter each extract through the plug of absorbent cotton into the
flask. After cooling, filter the combined acetone extracts through a paper-filter into a volumetric flask,
dilute to 100.0 ml with acetone R by rinsing the flask and the paper-filter. Introduce 20.0 ml of the
solution into a separating funnel, add 20 ml of water R and shake the mixture with one quantity of
15 ml and then three quantities, each of 10 ml, of ethyl acetate R. Combine the ethyl acetate extracts
in a separating funnel, wash with two quantities, each of 50 ml, of water R, and filter the extracts over
10 g of anhydrous sodium sulphate R into a volumetric flask and dilute to 50.0 ml with ethyl acetate R.
compensation solution at 425 nm. Calculate the percentage content of flavonoids, calculated as
isoquercitroside, from the expression:
m
. A 25 1
i.e. taking the specific absorbance of isoquercitrin to be 500.
A = absorbance at 425 nm,
m = mass of the substance to be examined in grams.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
15-32
Eleutherococcus
Siberian Ginseng
Eleutherococcus complies with the requirements of the 3rd edition of the European Pharmacopoeia [1419].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Eleutherococcus consists of the whole or cut, dried, underground organs of Eleutherococcus senticosus
(Rupr. et Maxim.) Maxim.
CHARACTERS
IDENTIFICATION
A. The rhizome is knotty, of irregular cylindrical shape, 1.5 cm to 4.0 cm in diameter. The surface is
rugged, longitudinally wrinkled and greyish-brown to blackish-brown. The bark, about 2 mm thick,
closely adheres to the xylem. The heartwood is light brown and the sapwood is pale yellow. The
fracture has short thin fibres in the bark and is coarsely fibrous, especially in the internal part of the
xylem. The lower surface bears numerous roots. The roots are cylindrical and knotty, 3.5 cm to
15 cm long and 0.3 cm to 1.5 cm in diameter. The smooth surface is greyish-brown to blackish-
brown. The bark is about 0.5 mm thick, closely adhering to the pale yellow xylem. The fracture is
slightly fibrous. In places where the epidermis is peeled off, the root is yellowish-brown.
B. Reduce to a powder (355). The powder is yellowish. Examine under a microscope, using chloral
hydrate solution R. The powder shows numerous groups of thick-walled, lignified fibres; fragments of
reticulate and bordered pitted vessels with a wide lumen; groups of secretory canals, up to 20 m in
diameter with brown contents; parenchymatous cells containing cluster crystals of calcium oxalate
10 m to 50 m in diameter. Examine under a microscope, using a 50 per cent V/V solution of
glycerol R. The powder shows small starch granules, rounded to slightly angular in outline, single or
in groups of two or three.
Test solution. Add to 1.0 g of the powdered drug (500) 10 ml of alcohol (50 per cent V/V) R and boil
under reflux for 1 h. Cool and filter. Evaporate the filtrate to dryness on a water-bath. Dissolve the
residue in 2.5 ml of a mixture of 5 volumes of water R and 20 volumes of alcohol (50 per cent V/V) R
and filter.
Reference solution. Dissolve 2.0 mg of esculin R in 10 ml of a mixture of 2 volumes of water R and 8
volumes of alcohol (50 per cent V/V)R.
Develop over a path of 10 cm using a mixture of 4 volumes of water R, 30 volumes of methanol R and
70 volumes of methylene chloride R. Allow the plate to dry in air. Examine the plate in ultraviolet light
at 365 nm. The chromatogram obtained with the reference solution shows in the upper half a blue
fluorescent zone (esculin). Spray the plate with anisaldehyde solution R and heat at 100C to 105C
for 5 min to 10 min. Examine in daylight. The chromatogram obtained with the test solution shows a
reddish zone (eleutheroside E) below the zone of esculin in the chromatogram obtained with the
reference solution and a brown zone (eleutheroside B) immediately above the zone of esculin in the
chromatogram obtained with the reference solution. Further zones are present.
TESTS
Extractable matter To 1.00 g of powdered drug (500) in a 250 ml round-bottomed flask add
50 ml of a mixture of 1 volume of water R and 4 volumes of alcohol (50 per cent V/V) R. Allow to
stand for 1 h. Boil under a reflux condenser for 2 h. Cool and filter the mixture in a volumetric flask
and dilute to 50.0 ml with the same solvent by rinsing the flask and filter. Evaporate 25.0 ml of this
solution to dryness on a water-bath. Dry the residue in an oven at 100C to 105C for 3 h. The
residue weighs not less than 30 mg (6.0 per cent).
STORAGE
__________________________________________________________________________________________________________ Ph Eur
15-33
Emetine Hydrochloride
1/01
N
H
Et
HN
OMe
OMe
MeO
MeO
H
H H
,2HCl
C
29
H
40
N
2
O
4
,2HCl,7H
2
O 679.7 316-42-7 (anhydrous)
Emetine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Emetine Hydrochloride Heptahydrate [0080]. These requirements are reproduced after the heading
Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Emetine hydrochloride heptahydrate contains not less than 98.0 per cent and not more than the
equivalent of 102.0 per cent of (2S,3R,11bS)-3-ethyl-1,3,4,6,7,11b-hexahydro-9,10-dimethoxy-2-
[[(R)-1,2,3,4-tetrahydro- 6,7-dimethoxy-1-isoquinolyl]methyl]-2H-benzo[a]quinolizine hydro-
CHARACTERS
A white or slightly yellowish, crystalline powder, freely soluble in water and in alcohol.
IDENTIFICATION
obtained with emetine hydrochloride CRS.
position, fluorescence and size to the spot in the chromatogram obtained with reference solution (a).
C. Dissolve about 10 mg in 2 ml of dilute hydrogen peroxide solution R, add 1 ml of hydrochloric acid R
and heat. An orange colour develops.
D. Sprinkle about 5 mg on the surface of 1 ml of sulphomolybdic reagent R2. A bright-green colour
develops.
TESTS
solution Y
5
or BY
5
(Method II, 2.2.2).
Specific optical rotation (2.2.7). Dissolve in water R a quantity of the substance to be examined
corresponding to 1.250 g of dried substance and dilute to 25.0 ml with the same solvent. The specific
optical rotation is +16 to +19, calculated with reference to the dried substance.
Test solution. Dissolve 50 mg of the substance to be examined in methanol R containing 1 per cent V/V
of dilute ammonia R2 and dilute to 100 ml with the same solvent.
15-34
Reference solution (a). Dissolve 50 mg of emetine hydrochloride CRS in methanol R containing 1 per cent
V/V of dilute ammonia R2 and dilute to 100 ml with the same solvent.
Reference solution (b). Dissolve 10 mg of isoemetine hydrobromide CRS in methanol R containing 1 per
cent V/V of dilute ammonia R2 and dilute to 100 ml with the same solvent. Dilute 5 ml of this
solution to 50 ml with methanol R containing 1 per cent V/V of dilute ammonia R2.
Reference solution (c). Dissolve 10 mg of cephaline hydrochloride CRS in methanol R containing 1 per
cent V/V of dilute ammonia R2 and dilute to 100 ml with the same solvent. Dilute 5 ml of this
solution to 50 ml with methanol R containing 1 per cent V/V of dilute ammonia R2.
Reference solution (d). Dilute 1 ml of reference solution (a) to 100 ml with methanol R containing 1 per
cent V/V of dilute ammonia R2.
Reference solution (e). To 1 ml of reference solution (a) add 1 ml of reference solution (b) and 1 ml of
reference solution (c).
Apply to the plate 10 l of the test solution and each of reference solutions (a), (b), (c) and (d) and
30 l of reference solution (e). Develop over a path of 15 cm using a mixture of 0.5 volumes of
diethylamine R, 2 volumes of water R, 5 volumes of methanol R, 20 volumes of ethylene glycol mono-
methyl ether R and 100 volumes of chloroform R. Allow the plate to dry in air until the odour of solvent
is no longer detectable. Spray in a well ventilated fume-cupboard with chloroformic solution of iodine R
and heat at 60C for 15 min. Examine in ultraviolet light at 365 nm. In the chromatogram obtained
with the test solution, any spots corresponding to isoemetine and cephaline are not more intense
than the spots in the chromatograms obtained with reference solutions (b) and (c) respectively
(2.0 per cent); any spot, apart from the principal spot and the spots corresponding to isoemetine and
cephaline, is not more intense than the spot in the chromatogram obtained with reference solution
(d) (1.0 per cent). The test is not valid unless the chromatogram obtained with reference solution (e)
shows three clearly separated spots.
Loss on drying (2.2.32).15.0 per cent to 19.0 per cent, determined on 1.00 g by drying in an oven
ASSAY
29
H
42
Cl
2
N
2
O
4
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
15-35
Emetine Hydrochloride Pentahydrate
C
29
H
40
N
2
O
4
,2HCl,5H
2
O 643.6 316-42-7 (anhydrous)
Emetine Hydrochloride Pentahydrate complies with the requirements of the 3rd edition of the European
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Emetine hydrochloride pentahydrate contains not less than 98.0 per cent and not more than the
equivalent of 102.0 per cent of (2S,3R,11bS)-3-ethyl-1,3,4,6,7,11b-hexahydro-9,10-dimethoxy-2-
[[(R)-1,2,3,4-tetrahydro-6,7-dimethoxy-1-isoquinolyl]methyl]-2H-benzo[a]quinolizine hydro-
CHARACTERS
It has the characters described in the monograph on Emetine hydrochloride heptahydrate (80).
IDENTIFICATION
It complies with the identification tests prescribed in the monograph on Emetine hydrochloride
heptahydrate (80).
TESTS
It complies with the tests prescribed in the monograph on Emetine hydrochloride heptahydrate (80)
with the following modification:
ASSAY
Carry out the assay prescribed in the monograph on Emetine hydrochloride heptahydrate (80).
29
H
42
Cl
2
N
2
O
4.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
15-36
Enalapril Maleate
H
N
N
H
COOH
O
H Me
EtOOC
H
,
COOH
COOH
C
24
H
32
N
2
O
9
492.5 76095-16-4
Enalapril Maleate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1420].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Enalapril maleate contains not less than 98.5 per cent and not more than the equivalent of 101.5 per
cent of (2S)-1-[(2S)-2-[[(1S)-1-(ethoxycarbonyl)-3-phenylpropy]amino]propanoyl]pyrrolidin-2-
carboxylic acid (Z)-butenedioate, calculated with reference to the dried substance.
CHARACTERS
A white or almost white crystalline powder, soluble in water, freely soluble in methanol, sparingly
soluble in methylene chloride. It dissolves in dilute solutions of alkali hydroxides.
IDENTIFICATION
obtained with enalapril maleate CRS.
C. Dissolve about 30 mg in 3 ml of water R. Add 1 ml of bromine water R and heat on a water bath
until bromine has disappeared completely and cool. To 0.2 ml of this solution, add 3 ml of a 3 g/l
solution of resorcinol R in sulphuric acid R and heat on a water bath for 15 min. A reddish-brown
colour develops.
D. To about 30 mg, add 0.5 ml of a 100 g/l solution of hydroxylamine hydrochloride R in methanol R
and 1.0 ml of a 100 g/l solution of potassium hydroxide R in alcohol R. Heat to boiling, allow to cool
and acidify with dilute hydrochloric acid R. Add 0.2 ml of ferric chloride solution R1 diluted 1 to 10; a
reddish-brown colour appears.
TESTS
Buffer solution A. Dissolve 2.8 g of sodium dihydrogen phosphate monohydrate R in 950 ml of water R.
Adjust to pH 2.5 with phosphoric acid R and dilute to 1000 ml with water R.
Buffer solution B. Dissolve 2.8 g of sodium dihydrogen phosphate monohydrate R in 950 ml of water R.
Adjust to pH 6.8 with strong sodium hydroxide solution R and dilute to 1000 ml with water R.
Dissolution mixture. Mix 50 ml of acetonitrile R and 950 ml of buffer solution A.
Test solution. Dissolve 30.0 mg of the substance to be examined in the dissolution mixture and dilute
to 100.0 ml with the same mixture.
Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with the dissolution mixture.
Reference solution (b). Dissolve 3.0 mg of enalapril for system suitability CRS in the dissolution mixture
and dilute to 10.0 ml with the same mixture.
a stainless steel column 0.15 m long and 4.1 mm in internal diameter packed with styrene-
divinylbenzene copolymer R (5 m),
as mobile phase at a flow rate of 1.4 ml/min,
15-37
Mobile phase A. Mix 50 ml of acetonitrile R and 950 ml of buffer solution B,
Mobile phase B. Mix 340 ml of buffer solution B and 660 ml of acetonitrile R,
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
020 9540 560
2025 40 60
2526 4095 605
2630 95 5
conditions, the retention times are: enalapril about 11 min and impurity A about 12 min. The test is
not valid unless the resolution between the peaks corresponding to enalapril and impurity A is at least
1.5. Inject 50 l of the test solution and 50 l of reference solution (a). In the chromatogram
obtained with the test solution: the area of any peak corresponding to impurity A is not greater than
that of the principal peak in the chromatogram obtained with reference solution (a) (1.0 per cent);
the area of any peak apart from the principal peak and any peak corresponding to impurity A, is not
solution (a) (0.3 per cent) and the sum of the areas of any such peaks is not greater than the area of
the principal peak in the chromatogram obtained with reference solution (a) (1.0 per cent). Disregard
Heavy metals (2.4.8). 2.0 g complies with limit test C for heavy metals (10 ppm). Use 2 ml of lead
Loss on drying (2.2.32). Not more than 1.0 per cent determined on 1.000 g by heating in an oven
ASSAY
Dissolve 0.100 g in carbon dioxide-free water R and dilute to 30 ml with the same solvent. Titrate with
0.1M sodium hydroxide determining the end point potentiometrically (2.2.20). Titrate up to the second
point of inflexion.
24
H
32
N
2
O
9
.
STORAGE
IMPURITIES
H
N
N
H
COOH
O
H Me EtOOC H
A. (2S)-1-[(2S)-2-[[(1R)-1-(ethoxycarbonyl)-3-phenylpropyl]amino]propanoyl]pyrrolidin-2-
carboxylic acid,
H
N
H Me
COOH
EtOOC H
B. (2S)-2-[[(1S)-1-(ethoxycarbonyl)-3-phenylpropyl]amino]propanoic acid,
H
N
N
H
COOH
O
H Me HOOC H
C. (2S)-1-[(2S)-2-[[(1S)-1-carboxy-3-phenylpropyl]amino]propanoyl]pyrrolidin-2-carboxylic
acid,
N
H EtOOC
N
H Me
O
O
H
D. ethyl (2S)-2-[(3S,8aS)-3-methyl-1,4-dioxo-octahydropyrrolo[1,2-a]pyrazin-2-y]-4-
phenylbutanoate,
15-38
H
N
N
H
COOH
O
H Me H ROOC
E. R = CH
2
-CH
2
-C
6
H
5
: (2S)-1-[(2S)-2-[[(1S)-3-phenyl-1-[(2-phenylethoxy)carbonyl]-
propyl]amino]propanoyl]pyrrolidin-2-carboxylic acid.
H
N
H Me
COOH
EtOOC H
F. R = CH
2
-CH
2
-CH
2
-CH
3
: (2S)-1-[(2S)-2-[[(1S)-1-(butoxycarbonyl)-3-phenylpropyl]-
amino]propanoyl]pyrrolidin-2-carboxylic acid.
H
N
N
H
COOH
O
H Me H EtOOC
G. 2S)-2-[[(1S)-3-cyclohexyl-1-(ethoxycarbony)propyl]amino]propanoic acid,
H. (2S)-1-[(2S)-3-cyclohexyl-2-[[(1S)-1-(ethoxycarbonyl)propyl]amino]propanoyl]pyrrolidin-
2-carboxylic acid,
N
N
I. 1H-imidazole.
__________________________________________________________________________________________________________ Ph Eur
15-39
Enoxaparin Sodium
O
OR
COONa
OH
OR
O
OR
O
NHR
1
O
O
OR
R
2
OH
n
OR
O
NHR
1
OH
O
OR
R
3
n = 1 to 21, R = H or SO
3
Na, R
1
= H or SO
3
Na or COCH
2

R
2
= H and R
3
= CO
2
Na or R
2
= CO
2
Na and R
3
= H
Enoxaparin Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Low-molecular weight heparin; anticoagulant.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Enoxaparin sodium is the sodium salt of a low-molecular-mass heparin that is obtained by alkaline
depolymerisation of the benzyl ester derivative of heparin from porcine intestinal mucosa. The
majority of the components have a 4-enopyranose uronate structure at the non-reducing end of their
chain.
Enoxaparin sodium complies with the monograph on Heparins, low-molecular-mass (828) with the
modifications and additional requirements below.
The mass-average molecular mass ranges between 3500 and 5500 with a characteristic value of about
4500.
The degree of sulphatation is about 2 per disaccharide unit.
The potency is not less than 90 I.U. and not more than 125 I.U. of anti-factor Xa activity per
milligram, calculated with reference to the dried substance. The ratio of anti-factor Xa activity to
anti-factor IIa activity is between 3.3 and 5.3.
IDENTIFICATION
Carry out identification test C as described in the monograph on Heparins, low-molecular-mass (828).
The following requirements apply.
The mass-average molecular mass ranges between 3500 and 5500. The mass percentage of chains
lower than 2000 ranges between 12.0 per cent and 20.0 per cent. The mass percentage of chains
between 2000 and 8000 ranges between 68.0 per cent and 88.0 per cent.
TESTS
Appearance of solution. Dissolve 1.0 g in 10 ml of water R. The solution is clear (2.2.1) and not
more intensely coloured than intensity 5 of the range of reference solutions of the most appropriate
colour (Method II, 2.2.2).
Absorbance (2.2.25). Dissolve 50.0 mg in 100 ml of 0.01M hydrochloric acid. The specific
absorbance at 231 nm is 14.0 to 20.0, calculated with reference to the dried substance.
Benzyl alcohol. Not more than 0.1 per cent m/m, determined by liquid chromatography (2.2.29).
Internal standard solution. Prepare a 1 g/l solution of 3,4-dimethylphenol R in methanol R.
Test solution. Dissolve about 0.500 g of the substance to be examined in 5.0 ml of 1M sodium
hydroxide. Allow to stand for 1 h. Add 1.0 ml of glacial acetic acid R and 1.0 ml of the internal
Reference solution. Prepare a 0.25 g/l solution of benzyl alcohol R in water R. Mix 0.50 ml of this
solution with 1.0 ml of the internal standard solution and dilute to 10.0 ml with water R.
gel for chromatography R (5 m) equipped with a guard column 20 mm long and 4.6 mm in
internal diameter, packed with the same material,
15-40
as mobile phase at a flow rate of 1 ml/min a mixture of 5 volumes of methanol R, 15 volumes of
acetonitrile R and 80 volumes of water R,
From the chromatogram obtained with the reference solution, calculate the ratio (R
1
) of the height of
the peak due to benzyl alcohol to the height of the peak due to the internal standard. From the
chromatogram obtained with the test solution, calculate the ratio (R
2
) of the height of the peak due
to benzyl alcohol to the height of the peak due to the internal standard.
Calculate the content (m/m) of benzyl alcohol from the following expression:
1
2
0125 0
R m
R .

Sodium 11.3 to 13.5 per cent of Na calculated with reference to the dried substance and determined
by atomic absorption spectrometry (Method I, 2.2.23).
__________________________________________________________________________________________________________ Ph Eur
15-41
Enoxolone
1/01
Me Me
Me H
O
H
Me
Me COOH
Me Me
HO
H H
C
30
H
46
O
4
470.7 471-53-4
Enoxolone complies with the requirements of the 3rd edition of the European Pharmacopoeia [1511]. These
Action and use Treatment of skin diseases.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
(20)-3-Hydroxy-11-oxo-olean-12-en-29-oic acid.
CHARACTERS
Appearance: white or almost white crystalline powder.
Solubility: practically insoluble in water, soluble in ethanol, sparingly soluble in methylene chloride.
IDENTIFICATION
A. Examine by infrared absorption spectrophotometry (2.2.24).
Comparison: enoxolone CRS.
If the spectra obtained in the solid state show differences, dissolve 0.2 g of the substance to be
examined and 0.2 g of the reference substance separately in 6 ml of ethanol R. Boil under a reflux
condenser for 1 h and add 6 ml of water R. A precipitate is formed. Cool to about 10C and filter
with the aid of vacuum. Wash the precipitate with 10 ml of alcohol R, dry in an oven at 80C and
record new spectra.
B. Thin-layer chromatography (2.2.27).
Reference solution. Dissolve 10 mg of enoxolone CRS in methylene chloride R and dilute to 10 ml with
the same solvent.
Plate: TLC silica gel plate R.
Mobile phase: glacial acetic acid R, acetone R, methylene chloride R (5:10:90 V/V/V).
Application: 5 l.
Drying: in air for 5 min.
Detection: spray with anisaldehyde solution R and heat at 100-105C for 10 min.
Results: the principal spot in the chromatogram obtained with the test solution is similar in position,
C. Dissolve 50 mg in 10 ml of methylene chloride R. To 2 ml of this solution, add 1 ml of acetic
anhydride R and 0.3 ml of sulphuric acid R. A pink colour is produced.
TESTS
15-42
Appearance of solution The solution is clear (2.2.1) and not more intensely coloured than
6
(2.2.2, Method II).
Dissolve 0.1 g in ethanol R and dilute to 10 ml with the same solvent.
Specific optical rotation (2.2.7): +145 to +154 (dried substance).
Dissolve 0.50 g in dioxan R and dilute to 50.0 ml with the same solvent.
Reference solution (c). Dissolve 0.1 g of 18-glycyrrhetinic acid R in tetrahydrofuran R and dilute to
100.0 ml with the same solvent. To 2.0 ml of the solution, add 2.0 ml of the test solution and dilute
Column:
size: l = 0.25 m, = 4.6 mm,
stationary phase: octadecylsilyl silica gel for chromatography R (5 m),
temperature: 30C.
Mobile phase: mix 430 volumes of tetrahydrofuran R and 570 volumes of a 1.36 g/l solution of sodium
acetate R adjusted to pH 4.8 with glacial acetic acid R.
Injection: 20 l loop injector; inject the test solution and the reference solutions.
Run time: 4 times the retention time of enoxolone.
System suitability:
resolution: minimum of 2.0 between the peaks due to enoxolone and to 18-glycyrrhetinic acid in
the chromatogram obtained with reference solution (c).
Limits:
any impurity: not more than 7 times the area of the principal peak in the chromatogram obtained
with reference solution (b) (0.7 per cent),
total: not more than the area of the principal peak in the chromatogram obtained with reference
solution (a) (2.0 per cent),
1.0 g complies with limit test F. Prepare the standard using 2 ml of lead standard solution (10 ppm
Pb) R.
100-105C for 4 h.
ASSAY
30
H
46
O
4
.
STORAGE
Protected from light.
IMPURITIES
Me Me
Me H
O
H
Me
Me COOH
Me Me
O
H H
A. (20)-3-hydroxy-11-oxo-18-olean-12-en-29-oic acid,
15-43
Me Me
Me H
O
H
Me
Me COOH
Me
O
H H
OH
B. (4,20)-3,23-dihydroxy-11-oxo-olean-12-en-29-oic acid.
__________________________________________________________________________________________________________ Ph Eur
15-44
Ephedrine
CH
3
OH H
NHMe H
C
10
H
15
NO,H
2
O 174.2 50906-05-3
Ephedrine complies with the requirements of the 3rd edition of the European Pharmacopoeia for Ephedrine
Hemihydrate [0489]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ephedrine hemihydrate contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of (1R,2S)-2-methylamino-1-phenylpropan-1-ol, calculated with reference to the
CHARACTERS
A white, crystalline powder or colourless crystals, soluble in water, very soluble in alcohol, freely
soluble in ether.
IDENTIFICATION
obtained with the base isolated from ephedrine hydrochloride CRS. Examine the substances in discs
prepared as follows: dissolve 40 mg of the substance to be examined in 1 ml of water R, add 1 ml of
dilute sodium hydroxide solution R and 4 ml of chloroform R and shake; dry the organic layer over 0.2 g
of anhydrous sodium sulphate R; prepare a blank disc using about 0.3 g of potassium bromide R; apply
dropwise to the disc 0.1 ml of the organic layer, allowing the solvent to evaporate between
applications; dry the disc at 50C for 2 min. Repeat the operations using 50 mg of ephedrine hydro-
chloride CRS.
D. Dissolve about 10 mg in 1 ml of water R. Add 0.2 ml of strong sodium hydroxide solution R and
0.2 ml of copper sulphate solution R. A violet colour is produced. Add 2 ml of ether R and shake. The
ether layer is purple and the aqueous layer blue.
TESTS
Specific optical rotation (2.2.7). Dissolve 2.25 g in 15 ml of dilute hydrochloric acid R and dilute to
50.0 ml with water R. The specific optical rotation is 41 to 43, calculated with reference to the
coating substance.
Reference solution (a). Dissolve 25 mg of ephedrine hydrochloride CRS in methanol R and dilute to 10 ml
15-45
5 volumes of chloroform R, 15 volumes of concentrated ammonia R and 80 volumes of 2-propanol R.
Allow the plate to dry in air and spray with ninhydrin solution R. Heat at 110C for 5 min. Any spot in
than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent). Disregard any
spot of lighter colour than the background.
Chlorides Dissolve 0.18 g in 10 ml of water R. Add 5 ml of dilute nitric acid R and 0.5 ml of silver
nitrate solution R1. Allow to stand for 2 min, protected from bright light. Any opalescence in the
manner using 10 ml of chloride standard solution (5 ppm Cl) R, 5 ml of dilute nitric acid R and 0.5 ml of
silver nitrate solution R1 (280 ppm).
mination of water.
ASSAY
Dissolve 0.200 g in 5 ml of alcohol R and add 20.0 ml of 0.1M hydrochloric acid. Using 0.05 ml of
methyl red solution R as indicator, titrate with 0.1M sodium hydroxide until a yellow colour is obtained.
10
H
15
NO.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
15-46
Anhydrous Ephedrine
C
10
H
15
No 165.2 299-42-3
Anhydrous Ephedrine complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Anhydrous ephedrine contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of (1R,2S)-2-methylamino-1-phenylpropan-1-ol, calculated with reference to the
CHARACTERS
A white, crystalline powder or colourless crystals, soluble in water, very soluble in alcohol, freely
soluble in ether.
IDENTIFICATION
obtained with the base isolated from ephedrine hydrochloride CRS. Examine the substances in discs
prepared as follows: dissolve 40 mg of the substance to be examined in 1 ml of water R, add 1 ml of
dilute sodium hydroxide solution R and 4 ml of chloroform R and shake; dry the organic layer over 0.2 g
of anhydrous sodium sulphate R; prepare a blank disc using about 0.3 g of potassium bromide R; apply
dropwise to the disc 0.1 ml of the organic layer, allowing the solvent to evaporate between
applications; dry the disc at 50C for 2 min. Repeat the operations using 50 mg of ephedrine hydro-
chloride CRS.
D. Dissolve about 10 mg in 1 ml of water R. Add 0.2 ml of strong sodium hydroxide solution R and
0.2 ml of copper sulphate solution R. A violet colour is produced. Add 2 ml of ether R and shake. The
ether layer is purple and the aqueous layer blue.
TESTS
Specific optical rotation (2.2.7). Dissolve 2.25 g in 15 ml of dilute hydrochloric acid R and dilute to
50.0 ml with water R. The specific optical rotation is 41 to 43, calculated with reference to the
coating substance.
5 volumes of chloroform R, 15 volumes of concentrated ammonia R and 80 volumes of 2-propanol R.
Allow the plate to dry in air and spray with ninhydrin solution R. Heat at 110C for 5 min. Any spot in
than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent). Disregard any
spot of lighter colour than the background.
Chlorides Dissolve 0.17 g in 10 ml of water R. Add 5 ml of dilute nitric acid R and 0.5 ml of silver
nitrate solution R1. Allow to stand for 2 min, protected from bright light. Any opalescence in the
15-47
manner using 10 ml of chloride standard solution (5 ppm Cl) R, 5 ml of dilute nitric acid R and 0.5 ml of
silver nitrate solution R1 (290 ppm).
mination of water.
ASSAY
Dissolve 0.200 g in 5 ml of alcohol R and add 20.0 ml of 0.1M hydrochloric acid. Using 0.05 ml of
methyl red solution R as indicator, titrate with 0.1M sodium hydroxide until a yellow colour is obtained.
10
H
15
NO.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
15-48
Ephedrine Hydrochloride
1/01
CH
3
OH H
NHMe H
,HCl
C
10
H
15
NO,HCl 201.7 50-98-6
Ephedrine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparations
Ephedrine Elixir
Ephedrine Nasal Drops
Ephedrine Hydrochloride Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ephedrine hydrochloride contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of (1R,2S)-2-methylamino-1-phenylpropan-1-ol hydrochloride, calculated with
CHARACTERS
A white, crystalline powder or colourless crystals, freely soluble in water, soluble in alcohol.
IDENTIFICATION
obtained with ephedrine hydrochloride CRS.
D. To 0.1 ml of solution S (see Tests) add 1 ml of water R, 0.2 ml of copper sulphate solution R and
1 ml of strong sodium hydroxide solution R. A violet colour is produced. Add 2 ml of ether R and shake.
The ether layer is purple and the aqueous layer blue.
E. To 5 ml of solution S add 5 ml of water R. The solution gives reaction (a) of chlorides (2.3.1).
TESTS
Solution S Dissolve 5.00 g in distilled water R and dilute to 50.0 ml with the same solvent.
sodium hydroxide. The solution is yellow. Add 0.4 ml of 0.01M hydrochloric acid. The solution is red.
optical rotation is 33.5 to 35.5, calculated with reference to the dried substance.
of chloroform R, 15 volumes of concentrated ammonia R and 80 volumes of 2-propanol R. Allow the
15-49
plate to dry in air and spray with ninhydrin solution R. Heat at 110C for 5 min. Any spot in the
the spot in the chromatogram obtained with reference solution (b) (0.5 per cent). Disregard any spot
of lighter colour than the background.
Sulphates (2.4.13).15 ml of solution S complies with the limit test for sulphates (100 ppm).
100C to 105C.
ASSAY
potentiometric titration (2.2.20), using 0.1M sodium hydroxide. Read the volume added between the 2
10
H
16
ClNO.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
15-50
Ergocalciferol
1/01
Me
Me
Me
Me
Me
H
H
H
HO
H
H
2
C
C
28
H
44
O 396.7 50-14-6
Ergocalciferol complies with the requirements of the 3rd edition of the European Pharmacopoeia [0082]. These
Action and use Used in treatment of vitamin D deficiency.
Preparations
Ergocalciferol Injection
Ergocalciferol Tablets
When vitamin D
2
is prescribed or demanded, Ergocalciferol shall be dispensed or supplied. When
calciferol or vitamin D is prescribed or demanded, Ergocalciferol or Colecalciferol shall be dispensed
or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ergocalciferol contains not less than 97.0 per cent and not more than the equivalent of 103.0 per
cent of (5Z,7E,22E)-9,10-secoergosta-5,7,10(19),22-tetraen-3-ol.
1 milligram of ergocalciferol is equivalent to 40,000 I.U. of antirachitic activity (vitamin D) in rats.
CHARACTERS
A white or slightly yellowish, crystalline powder or white or almost white crystals, practically insoluble
in water, freely soluble in alcohol, soluble in fatty oils. It is sensitive to air, heat and light. Solutions in
volatile solvents are unstable and are to be used immediately.
A reversible isomerisation to pre-ergocalciferol takes place in solution, depending on temperature and
IDENTIFICATION
with ergocalciferol CRS. Examine the substances prepared as discs.
TESTS
Specific optical rotation (2.2.7). Dissolve 0.200 g rapidly and without heating in aldehyde-free
alcohol R and dilute to 25.0 ml with the same solvent. The specific optical rotation, determined
within 30 min of preparing the solution, is +103 to +107.
Reducing substances Dissolve 0.1 g in aldehyde-free alcohol R and dilute to 10.0 ml with the same
solvent. Add 0.5 ml of a 5 g/l solution of tetrazolium blue R in aldehyde-free alcohol R and 0.5 ml of
dilute tetramethylammonium hydroxide solution R. Allow to stand for exactly 5 min and add 1.0 ml of
glacial acetic acid R. Prepare a reference solution at the same time and in the same manner using
10.0 ml of a solution containing 0.2 g/ml of hydroquinone R in aldehyde-free alcohol R. Measure the
absorbance (2.2.25) of the two solutions at 525 nm using as the compensation liquid 10.0 ml of
aldehyde-free alcohol R treated in the same manner. The absorbance of the test solution is not greater
than that of the reference solution (20 ppm).
Ergosterol Examine by thin-layer chromatography (2.2.27), using a TLC silica gel G plate R.
Test solution. Dissolve 0.25 g of the substance to be examined in ethylene chloride R containing 10 g/l
of squalane R and 0.1 g/l of butylhydroxytoluene R and dilute to 5 ml with the same solvent. Prepare
Reference solution (a). Dissolve 0.10 g of ergocalciferol CRS in ethylene chloride R containing 10 g/l of
15-51
Reference solution (b). Dissolve 5 mg of ergosterol CRS in ethylene chloride R containing 10 g/l of
Reference solution (c). Mix equal volumes of reference solution (a) and reference solution (b). Prepare
Apply to the plate 10 l of the test solution, 10 l of reference solution (a), 10 l of reference solution
(b) and 20 l of reference solution (c). Develop immediately, protected from light, over a path of
containing 0.1 g/l of butylhydroxytoluene R. Allow the plate to dry in air and spray three times with
antimony trichloride solution R1. Examine the chromatograms for 3 min to 4 min after spraying. The
principal spot in the chromatogram obtained with the test solution is initially orange-yellow and then
becomes brown. In the chromatogram obtained with the test solution, any slowly appearing violet
spot (corresponding to ergosterol) immediately below the principal spot is not more intense than the
spot in the chromatogram obtained with reference solution (b) (0.2 per cent). There is no spot in the
chromatogram obtained with the test solution that does not correspond to one of the spots in the
chromatograms obtained with reference solutions (a) and (b). The test is not valid unless the
chromatogram obtained with reference solution (c) shows two clearly separated spots.
ASSAY
Carry out the operations as rapidly as possible, avoiding exposure to actinic light and air.
Test solution. Dissolve 10.0 mg of the substance to be examined without heating in 10.0 ml of
toluene R and dilute to 100.0 ml with the mobile phase.
Reference solution (a). Dissolve 10.0 mg of ergocalciferol CRS without heating in 10.0 ml of toluene R
silica gel (5 m),
of hexane R,
resolution between the peaks corresponding to pre-cholecalciferol and trans-cholecalciferol is not less
than 1.0. If necessary adjust the proportions of the constituents and the flow rate of the mobile phase
to obtain this resolution.
Calculate the percentage content of ergocalciferol from the expression:
100
D
D
S
S
m
m
m =mass of ergocalciferol CRS in reference solution (a), in milligrams,
S
D
=area (or height) of the peak due to ergocalciferol in the chromatogram obtained with the
test solution,
S
D
=area (or height) of the peak due to ergocalciferol in the chromatogram obtained with
STORAGE
Store in an airtight container, under nitrogen, protected from light, at a temperature between 2C
and 8C.
15-52
IMPURITIES
H
Me
H Me
Me
Me
H H
2
C
O
H
H Me
A. (5E,7E,22E)-9,10-secoergosta-5,7,10(19),22-tetraen-3-ol (trans-vitamin D
2
),
H
Me
H Me
Me
Me
H
O
H
Me
H
H Me
B. (22E)-ergosta-5,7,22-trien-3-ol (ergosterol),
H
Me
H Me
Me
Me
H
O
H
Me
H
H Me
C. (9,10,22E)-ergosta-5,7,22-trien-3-ol (lumisterol
2
),
Me
H Me
Me
Me
H
HO
H
Me
H Me
D. (6E,22E)-9,10-secoergosta-5(10),6,8(14),22-tetraen-3-ol (iso-tachysterol
2
),
H
Me
H Me
Me
Me
H
HO
H
Me
H Me
E. (6E,22E)-9,10-secoergosta-5(10),6,8,22-tetraen-3-ol (tachysterol
2
).
__________________________________________________________________________________________________________ Ph Eur
15-53
Ergometrine Maleate
NMe
HN
H
H
N
OH
O
Me H
H
COOH
COOH
,
C
19
H
23
N
3
O
2
,C
4
H
4
O
4
441.5 129-51-1
Ergometrine Maleate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0223].
Action and use Uterine stimulant.
Preparations
Ergometrine Injection
Ergometrine and Oxytocin Injection
Ergometrine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ergometrine maleate contains not less than 98.0 per cent and not more than the equivalent of
101.0 per cent of (6aR,9R)-N-[(S)-2-hydroxy-1-methylethyl]-7-methyl-4,6,6a,7,8,9-hexahydro-
indolo[4,3-fg]quinoline-9-carboxamide maleate, calculated with reference to the dried substance.
CHARACTERS
A white or slightly coloured, crystalline powder, sparingly soluble in water, slightly soluble in alcohol,
IDENTIFICATION
A. Dissolve 30 mg in 0.01M hydrochloric acid and dilute to 100.0 ml with the same acid. Dilute
360 nm (2.2.25), the solution shows an absorption maximum at 311 nm and a minimum at 265 nm
to 272 nm. The specific absorbance at the maximum is 175 to 195.
obtained with ergometrine maleate CRS. Examine the substances prepared as discs.
D. To 0.1 ml of solution S (see Tests) add 1 ml of glacial acetic acid R, 0.05 ml of ferric chloride
solution R1 and 1 ml of phosphoric acid R and heat in a water-bath at 80C. After about 10 min, a blue
or violet colour develops which becomes more intense on standing.
E. Dissolve 0.1 g in a mixture of 0.5 ml of dilute sulphuric acid R and 2.5 ml of water R. Add 5 ml of
ether R and 1 ml of strong sodium hydroxide solution R and shake. Separate the aqueous layer and shake
with two quantities, each of 5 ml, of ether R. To 0.1 ml of the aqueous layer add a solution of 10 mg
of resorcinol R in 3 ml of sulphuric acid R. Heat on a water-bath for 15 min. No colour develops. To
the rest of the aqueous layer add 1 ml of bromine water R. Heat on a water-bath for 10 min, then heat
to boiling and cool. To 0.2 ml of this solution add a solution of 10 mg of resorcinol R in 3 ml of
sulphuric acid R. Heat on a water-bath for 15 min. A pinkish-violet colour develops.
TESTS
Solution S Dissolve 0.100 g, without heating and protected from light, in 9 ml of carbon dioxide-free
water R and dilute to 10.0 ml with the same solvent.
solution Y
5
or BY
5
(Method II, 2.2.2).
15-54
coating substance.
Carry out all operations as rapidly as possible, protected from light. Prepare the test and reference solutions
Test solution (a). Dissolve 50 mg of the substance to be examined in a mixture of 1 volume of
concentrated ammonia R and 9 volumes of alcohol (80 per cent V/V) R and dilute to 5.0 ml with the
Test solution (b). Dilute 1.0 ml of test solution (a) to 10.0 ml with a mixture of 1 volume of
concentrated ammonia R and 9 volumes of alcohol (80 per cent V/V) R.
Reference solution (a). Dissolve 10 mg of ergometrine maleate CRS in a mixture of 1 volume of
concentrated ammonia R and 9 volumes of alcohol (80 per cent V/V) R and dilute to 10.0 ml with the
Reference solution (b). Dilute 5.0 ml of reference solution (a) to 50.0 ml with a mixture of 1 volume of
Reference solution (c). To 2.0 ml of reference solution (b) add 2.0 ml of a mixture of 1 volume of
Apply separately to the plate 5 l of each solution. Develop immediately over a path of 14 cm using a
mixture of 3 volumes of water R, 25 volumes of methanol R and 75 volumes of chloroform R. Dry the
plate in a current of cold air and spray with dimethylaminobenzaldehyde solution R7. Dry the plate in a
current of warm air for about 2 min. Any spot in the chromatogram obtained with test solution (a),
obtained with reference solution (b) (1.0 per cent) and at most one such spot is more intense than
the principal spot in the chromatogram obtained with reference solution (c) (0.5 per cent).
ASSAY
23
H
27
N
3
O
6
.
STORAGE
Store in an airtight, glass container, protected from light, at a temperature of 2C to 8C.
__________________________________________________________________________________________________________ Ph Eur
15-55
Ergotamine Tartrate
1/01
HOOC
COOH
OH H
H OH
,
NMe
HN
H
H NH
O
OH
O
O
H
H CH
2
Ph
O
N
N
Me
(C
33
H
35
N
5
O
5
)
2
,C
4
H
6
O
6
1313 379-79-3
Ergotamine Tartrate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0224].
Preparation
Ergotamine Sublingual Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ergotamine tartrate contains not less than 98.0 per cent and not more than the equivalent of
101.0 per cent of bis[(6aR,9R)-N-[(2R,5S,10aS,10bS)-5-benzyl-10b-hydroxy-2-methyl-3,6-dioxo-
octahydro- 8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-4,6,6a,7,8,9-
hexahydroindolo[4,3-fg]quinoline- 9-carboxamide] tartrate, calculated with reference to the dried
substance. It may contain two molecules of methanol of crystallisation.
CHARACTERS
A white or almost white, crystalline powder or colourless crystals, slightly hygroscopic, slightly soluble
in alcohol. Aqueous solutions slowly become cloudy owing to hydrolysis; this may be prevented by
the addition of tartaric acid.
IDENTIFICATION
A. Dissolve 50 mg in 0.01M hydrochloric acid and dilute to 100.0 ml with the same acid. Dilute
360 nm (2.2.25), the solution shows an absorption maximum at 311 nm to 321 nm and a minimum
at 265 nm to 275 nm. The specific absorbance at the maximum is 118 to 128, calculated with
obtained with ergotamine tartrate CRS. Examine the substances as discs prepared as follows: triturate
the substance to be examined and the reference substance separately with 0.2 ml of methanol R and
then with potassium bromide R as prescribed in the general method.
C. Examine for not more than 1 min in ultraviolet light at 365 nm the chromatograms obtained in
the test for related substances. The principal spot in the chromatogram obtained with test solution
(b) is similar in position and fluorescence to the principal spot in the chromatogram obtained with
reference solution (a). After spraying with dimethylaminobenzaldehyde solution R7, examine in daylight.
The principal spot in the chromatogram obtained with test solution (b) is similar in position, colour
D. To 0.1 ml of solution S (see Tests) add 1 ml of glacial acetic acid R, 0.05 ml of ferric chloride
solution R1 and 1 ml of phosphoric acid R and heat in a water-bath at 80C. After about 10 min, a blue
or violet colour develops which becomes more intense on standing.
E. Dissolve about 10 mg in 1.0 ml of 0.1M sodium hydroxide. Transfer to a separating funnel and
shake with 5 ml of methylene chloride R. Discard the organic layer. Neutralise the aqueous layer with a
few drops of dilute hydrochloric acid R.0.1 ml of this solution gives reaction (b) of tartrates (2.3.1).
Pour the reaction mixture into 1 ml of water R to observe the colour change to red or brownish-red.
TESTS
Carry out all operations as rapidly as possible, protected from light.
Solution S Triturate 30 mg finely with about 15 mg of tartaric acid R and dissolve with shaking in
6 ml of water R.
15-56
solution Y
6
(Method II, 2.2.2).
pH (2.2.3). Shake 10 mg, finely powdered, with 4 ml of carbon dioxide-free water R. The pH of the
Specific optical rotation (2.2.7). Dissolve 0.40 g in 40 ml of a 10 g/l solution of tartaric acid R.
Add 0.5 g of sodium hydrogen carbonate R cautiously in several portions and mix thoroughly. Shake
with four quantities, each of 10 ml, of chloroform R previously washed with five quantities of water R,
each of 50 ml per 100 ml of chloroform R. Combine the organic layers. Filter through a small filter
moistened with chloroform R previously washed as described above. Dilute the filtrate to 50.0 ml with
chloroform R previously washed as described above. Measure the angle of rotation.
Determine the amount of ergotamine base in the chloroformic solution as follows: to 25.0 ml of
the solution add 50 ml of anhydrous acetic acid R and titrate with 0.05M perchloric acid, determining
33
H
35
N
5
O
5
.
The specific optical rotation is 154 to 165, calculated from the angle of rotation and the concen-
tration of ergotamine base.
Related substances Examine by thin-layer chromatography (2.2.27), using a TLC silica gel G
plate R.
Prepare the reference solutions and the test solutions immediately before use and in the order indicated below.
Reference solution (a). Dissolve 10 mg of ergotamine tartrate CRS in a mixture of 1 volume of
methanol R and 9 volumes of methylene chloride R and dilute to 10.0 ml with the same mixture of
solvents.
Reference solution (b). Dilute 7.5 ml of reference solution (a) to 50.0 ml with a mixture of 1 volume of
Reference solution (c). To 2.0 ml of reference solution (b) add 4.0 ml of a mixture of 1 volume of
Test solution (a). Dissolve 50 mg of the substance to be examined in a mixture of 1 volume of
methanol R and 9 volumes of methylene chloride R and dilute to 5.0 ml with the same mixture of
solvents.
Test solution (b). Dilute 1.0 ml of test solution (a) to 10.0 ml with a mixture of 1 volume of
Apply immediately to the plate 5 l of each reference solution and then 5 l of each test solution.
Expose the starting points immediately to ammonia vapour and for exactly 20 s by moving the
starting line from side to side above a beaker 55 mm high and 45 mm in diameter containing about
20 ml of concentrated ammonia R. Dry the starting line in a current of cold air for exactly 20 s.
Develop immediately over a path of 17 cm using a mixture of 5 volumes of ethanol R, 10 volumes of
methylene chloride R, 15 volumes of dimethylformamide R and 70 volumes of ether R. Dry the plate in a
current of cold air for about 2 min. Examine for not more than 1 min in ultraviolet light at 365 nm
for the identification. Spray the plate abundantly with dimethylaminobenzaldehyde solution R7 and dry
in a current of warm air for about 2 min. Any spot in the chromatogram obtained with test solution
(a), apart from the principal spot, is not more intense than the principal spot in the chromatogram
obtained with reference solution (b) (1.5 per cent) and at most one such spot is more intense than
the principal spot in the chromatogram obtained with reference solution (c) (0.5 per cent).
95C for 6 h.
ASSAY
70
H
76
N
10
O
16
.
STORAGE
Store in an airtight, glass container, protected from light, at a temperature of 2C to 8C.
__________________________________________________________________________________________________________ Ph Eur
15-57
Erythromycin
O
HO
Me
Me
O
O
O
Me
O
1
Me
Et
O
Me
Me
OH
Me
Me O
Me
OH
NMe
2
O
R
2
Erythromycin R
1
R
2
A OH OMe
B H OMe
C OH OH
C
37
H
67
NO
13
734 114-07-8
Erythromycin complies with the requirements of the 3rd edition of the European Pharmacopoeia [0179].
Preparation
Erythromycin Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Erythromycin is a mixture of macrolide antibiotics produced by a strain of Streptomyces erythreus, the
main component of the mixture being (3R,4S,5S,6R,7R,9R, 11R,12R,13S,14R)-4-[(2,6-dideoxy-3-
C-methyl-3-O-methyl--L-ribo-hexopyranosyl) oxy]-14-ethyl-7,12,13-trihydroxy-3,5,7,9,11,13-
hexamethyl-6-[(3,4,6-trideoxy-3-dimethylamino--D-xylo-hexopyranosyl)-oxy]oxacyclotetradecane-
2,10-dione (erythromycin A). The sum of the content of erythromycin A, erythromycin B and
erythromycin C is not less than 93.0 per cent and not more than the equivalent of 100.5 per cent,
CHARACTERS
A white or slightly yellow powder or colourless or slightly yellow crystals, slightly hygroscopic, slightly
soluble in water (the solubility decreasing as the temperature rises), freely soluble in alcohol, soluble
in methanol. It dissolves in dilute hydrochloric acid.
IDENTIFICATION
the same solvent.
Reference solution (a). Dissolve 10 mg of erythromycin A CRS in methanol R and dilute to 10 ml with
the same solvent.
Reference solution (b). Dissolve 20 mg of spiramycin CRS in methanol R and dilute to 10 ml with the
same solvent.
layer of a mixture of 4 volumes of 2-propanol R, 9 volumes of ethyl acetate R and 8 volumes of a
150 g/l solution of ammonium acetate R previously adjusted to pH 9.6 with ammonia R. Allow the
plate to dry in air, spray with anisaldehyde solution R1 and heat at 110C for 5 min. The principal spot
principal spot in the chromatogram obtained with reference solution (a) and its position and colour
are different from those of the spots in the chromatogram obtained with reference solution (b).
B. To about 5 mg add 5 ml of a 0.2 g/l solution of xanthydrol R in a mixture of 1 volume of hydro-
chloric acid R and 99 volumes of acetic acid R and heat on a water-bath. A red colour develops.
C. Dissolve about 10 mg in 5 ml of hydrochloric acid R1 and allow to stand for 10 min to 20 min. A
15-58
TESTS
solvent. Measure the angle of rotation at least 30 min after preparing the solution. The specific
Related substances Examine by liquid chromatography (2.2.29), as described under Assay. The
content of erythromycin B and that of erythromycin C is not greater than 5.0 per cent. Inject refer-
ence solution (d). Inject the test solution and continue the chromatography for five times the
retention time of the peak corresponding to erythromycin A. In the chromatogram obtained with the
test solution, the area of any peak other than those corresponding to erythromycin A, erythromycin B
and erythromycin C, is not greater than the area of the principal peak in the chromatogram obtained
with reference solution (d) (3.0 per cent).
mination of water. Use a 100 g/l solution of imidazole R in anhydrous methanol R as the solvent.
ASSAY
Test solution. Dissolve 40.0 mg of the substance to be examined in a mixture of 1 volume of
methanol R and 3 volumes of phosphate buffer solution pH 7.0 R and dilute to 10.0 ml with the same
Reference solution (a). Dissolve 40.0 mg of erythromycin A CRS in a mixture of 1 volume of methanol R
and 3 volumes of phosphate buffer solution pH 7.0 R and dilute to 10.0 ml with the same mixture of
solvents.
Reference solution (b). Dissolve 10.0 mg of erythromycin B CRS and 10.0 mg of erythromycin C CRS in
a mixture of 1 volume of methanol R and 3 volumes of phosphate buffer solution pH 7.0 R and dilute to
50.0 ml with the same mixture of solvents.
Reference solution (c). Dissolve 5 mg of N-demethylerythromycin A CRS in reference solution (b). Add
1.0 ml of reference solution (a) and dilute to 25 ml with reference solution (b).
Reference solution (d). Dilute 3.0 ml of reference solution (a) to 100.0 ml with a mixture of 1 volume
of methanol R and 3 volumes of phosphate buffer solution pH 7.0 R.
The test solution and reference solutions can be used within one day if stored at 5C.
copolymer R (8 m to 10 m) with a pore size of 100 nm,
as mobile phase at a flow rate of 2.0 ml per minute a solution prepared as follows: to 50 ml of a
35 g/l solution of dipotassium hydrogen phosphate R adjusted to pH 9.0 with dilute phosphoric acid R,
add 400 ml of water R, 165 ml of 2-methyl-2-propanol R and 30 ml of acetonitrile R, and dilute to
maintaining the column at 70C, preferably using a water-bath. Inject reference solution (c). Adjust
the sensitivity of the detector so that the height of the peaks is at least 25 per cent of the full scale of
the recorder. The substances are eluted in the following order: N-demethylerythromycin A,
erythromycin C, erythromycin A and erythromycin B. The test is not valid unless the resolution
between the peaks corresponding to N-demethylerythromycin A and erythromycin C is at least 0.8
and the resolution between the peaks corresponding to N-demethylerythromycin A and erythromycin
A is at least 5.5. If necessary, adjust the concentration of 2-methyl-2-propanol in the mobile phase or
reduce the flow rate to 1.5 ml or 1.0 ml per minute. Inject reference solution (a) six times. The test is
not valid unless the relative standard deviation of the peak area for erythromycin A is not greater than
2.0 per cent. Inject alternately the test solution and reference solutions (a) and (b).
Calculate the percentage content of erythromycin A using the chromatogram obtained with refer-
ence solution (a). Calculate the percentage contents of erythromycin B and erythromycin C using the
STORAGE
Store in a well-closed container, protected from light at a temperature not exceeding 30C.
15-59
IMPURITIES
O
HO
Me
O
O
Me
HO
Me
Et
O
Me
OH
Me
Me O
O
O
R
1
Me
OH
NMeR5
R
2
R
3
Me
R4
A. R
1
=

OH, R
2
=

OH, R
3
=

H, R
4
=

OCH
3
, R
5
=

CH
3
: erythromycin F,
B. R
1
=

OH, R
2
=

H, R
3
=

H, R
4
=

OCH
3
, R
5
=

H:
N-demethyl erythromycin A,
O
OH
Me
O
O
Me
HO
Me
Et
O
Me
OH
Me
Me O
O
O
HO
Me
OH
NMe2
O
Me
MeO
C. erythromycin E,
Me
OMe
O
HO
Me
O
O
Me
Et
O
Me
O
Me
Me O
O
Me
OH
NMe2
Me Me
OH
D. anhydroerythromycin A,
O
HO
Me
O
O
Me
HO
Me
Et
O
Me
Me
Me
Me O
O
HO
Me
OH
NMe2
O
Me
OMe
E. erythromycin A enol ether,
O
HO
Me
O
O
Me
Me
Et
O
Me
Me
Me
Me O
O
O
Me
OH
NMe2
O
OH
Me
OMe
F. pseudoerythromycin A enol ether.
__________________________________________________________________________________________________________ Ph Eur
15-60
Erythromycin Estolate
O
HO
Me
Me
O
O
O
Me
O
1
Me
Et
O
Me
Me
OH
Me
Me O
Me
OH
NMe
2
O
R
2
,C
12
H
25
OSO
3
H
Erythromycin R
1
R
2
A OH OMe
B H OMe
C OH OH
C
40
H
71
NO
14
,C
12
H
26
O
4
S1056 3521-62-8
Erythromycin Estolate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Erythromycin Estolate Capsules
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Erythromycin estolate (or erythromycin 2'-propionate dodecyl sulphate) is
(3R,4S,5S,6R,7R,9R,11R,12R,13S, 14R)-4-[(2,6-dideoxy-3-C-methyl-3-O-methyl--L-ribo-
hexopyranosyl)oxy]-14-ethyl-7,12,13-trihydroxy-3,5,7,9, 11,13-hexamethyl-6-[(3,4,6-trideoxy-3-
dimethylamino-2-O-propionyl--D-xylo-hexopyranosyl)oxy]oxacyclotetradecane-2,10-dione dodecyl
sulphate. The potency is not less than 610 I.U. per milligram, calculated with reference to the anhy-
drous substance.
CHARACTERS
A white, crystalline powder, practically insoluble in water, freely soluble in alcohol, soluble in
acetone. It is practically insoluble in dilute hydrochloric acid.
IDENTIFICATION
obtained with erythromycin estolate CRS.
chromatogram obtained with test solution (b) is similar in position and colour to the principal spot in
C. Suspend about 3 mg in 2 ml of dilute sulphuric acid R. Add 0.1 ml of a 0.1 g/l solution of methylene
blue R and 2 ml of chloroform R and shake. The chloroform layer is blue.
D. Dissolve about 10 mg in 5 ml of hydrochloric acid R1 and allow to stand for 10 min to 20 min. A
TESTS
pH (2.2.3). Suspend 0.4 g in 10 ml of carbon dioxide-free water R. Shake for 5 min and allow to stand.
The pH of the clear supernatant liquid is 5.5 to 7.0.
coating substance.
Test solution (a). Dissolve 40 mg of the substance to be examined in acetone R and dilute to 10 ml
15-61
Test solution (b). Dilute 2.5 ml of test solution (a) to 10 ml with acetone R.
Reference solution (a). Dissolve 10 mg of erythromycin estolate CRS in acetone R and dilute to 10 ml with
the same solvent.
Reference solution (b). Dissolve 10 mg of erythromycin estolate CRS and 10 mg of erythromycin
ethylsuccinate CRS in acetone R and dilute to 10 ml with the same solvent.
Reference solution (c). Dissolve 8 mg of erythromycin CRS in acetone R and dilute to 100 ml with the
same solvent.
1 volume of a 150 g/l solution of ammonium acetate R previously adjusted to pH 7.0, 15 volumes of
alcohol R and 85 volumes of chloroform R. Allow the plate to dry in air and spray with anisaldehyde
solution R. Heat at 110C for 5 min and allow to cool. Any spot in the chromatogram obtained with
obtained with reference solution (c) (2.0 per cent).
Dodecyl sulphate 23.0 per cent to 25.5 per cent of C
12
H
26
O
4
S, calculated with reference to the
anhydrous substance. Dissolve 0.500 g of the substance to be examined in 25 ml of
dimethylformamide R. Titrate with 0.1M sodium methoxide using 0.05 ml of a 3 g/l solution of thymol
blue R in methanol R as indicator.
1 ml of 0.1M sodium methoxide is equivalent to 26.64 mg of C
12
H
26
O
4
S.
ASSAY
Dissolve 40.0 mg in 40 ml of methanol R, add 20 ml of phosphate buffer solution pH 7.0 R and dilute to
100.0 ml with water R. Maintain at 60C for 3 h and allow to cool. Carry out the microbiological
assay of antibiotics (2.7.2). Use erythromycin CRS as the reference substance.
STORAGE
Store in an airtight container, protected from light, at a temperature below 30C.
__________________________________________________________________________________________________________ Ph Eur
15-62
Erythromycin Ethyl Succinate
O
HO
Me
Me
O
O
O
Me
O
1
Me
Et
O
Me
Me
OH
Me
Me O
Me
O
NMe
2
O
O
EtOOC
R
2
Erythromycin R
1
R
2
A OH OMe
B H OMe
C OH OH
C
43
H
75
NO
16
862 41342-53-4
Erythromycin Ethyl Succinate complies with the requirements of the 3rd edition of the European
Pharmacopoeia for Erythromycin Ethylsuccinate [0274]. These requirements are reproduced after the heading
Definition below.
Preparations
Erythromycin Ethyl Succinate Oral Suspension
Erythromycin Ethyl Succinate Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Erythromycin ethylsuccinate is (3R,4S,5S,6R,7R,9R, 11R,12R,13S,14R)-4-[(2,6-dideoxy-3-C-
methyl-3-O-methyl--L-ribo-hexopyranosyl)oxy]-14-ethyl-7,12,13-trihydroxy-3,5,7,9,11,13-
hexamethyl-6-[[3,4,6-trideoxy-3-dimethylamino-2-O-[3-(ethoxycarbonyl)propionyl]--D-xylo-
hexopyranosyl]oxy]oxacyclotetradecane-2,10-dione. The potency is not less than 780 I.U. per milli-
gram, calculated with reference to the anhydrous substance.
CHARACTERS
A white, crystalline powder, hygroscopic, practically insoluble in water, freely soluble in acetone, in
ethanol and in methanol.
IDENTIFICATION
obtained with erythromycin ethylsuccinate CRS.
chromatogram obtained with test solution (b) is similar in position and colour to the principal spot in
C. To about 5 mg add 5 ml of a 0.2 g/l solution of xanthydrol R in a mixture of 1 volume of hydro-
chloric acid R and 99 volumes of acetic acid R and heat on a water-bath. A red colour develops.
D. Dissolve about 10 mg in 5 ml of hydrochloric acid R1 and allow to stand for 10 min to 20 min. A
TESTS
Appearance of solution Dissolve 1.0 g in 10 ml of ethanol R. The solution is clear (2.2.1) and not
more intensely coloured than reference solution B
6
(Method II, 2.2.2).
pH (2.2.3). Suspend 1.0 g in 100 ml of carbon dioxide-free water R. The pH of the suspension is 6.0 to
8.5.
15-63
Specific optical rotation (2.2.7). Dissolve 0.100 g in acetone R and dilute to 10.0 ml with the same
solvent. Measure the angle of rotation at least 30 min after preparing the solution. The specific
coating substance.
TESTS solution (a). Dissolve 40 mg of the substance to be examined in acetone R and dilute to 10 ml
Test solution (b). Dilute 2.5 ml of test solution (a) to 10 ml with acetone R.
Reference solution (a). Dissolve 10 mg of erythromycin ethylsuccinate CRS in acetone R and dilute to
Reference solution (b). Dissolve 10 mg of erythromycin ethylsuccinate CRS and 10 mg of erythromycin
estolate CRS in acetone R and dilute to 10 ml with the same solvent.
Reference solution (c). Dissolve 10 mg of erythromycin CRS in acetone R and dilute to 50 ml with the
same solvent.
1 volume of a 150 g/l solution of ammonium acetate R previously adjusted to pH 7.0, 15 volumes of
alcohol R and 85 volumes of chloroform R. Allow the plate to dry in air and spray with anisaldehyde
solution R. Heat at 110C for 5 min and allow to cool. Any spot in the chromatogram obtained with
test solution (a), apart from the principal spot, is not more intense that the spot in the chromatogram
obtained with reference solution (c) (5.0 per cent).
Sulphated ash (2.4.14.). Not more than 0.3 per cent, determined on 1.0 g.
ASSAY
Dissolve 0.100 g in 40 ml of methanol R and dilute to 100.0 ml with buffer solution pH 8.0 R. Allow to
stand for 16 h. Carry out the microbiological assay of antibiotics (2.7.2). Use erythromycin CRS as the
reference substance.
STORAGE
Store in an airtight container, protected from light, at a temperature below 30C.
__________________________________________________________________________________________________________ Ph Eur
15-64
Erythromycin Lactobionate
O
HO
Me
Me
O
O
O
Me
O
1
Me
Et
O
Me
Me
OH
Me
Me O
Me
OH
NMe
2
O
R
2
COOH
HO H
OH H
H
HO H
OH
O
HO
O
OH
OH
OH
,
Erythromycin R
1
R
2
A OH OMe
B H OMe
C OH OH
C
37
H
67
NO
13
,C
12
H
22
O
12
1092 3847-29-8
Erythromycin Lactobionate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Erythromycin Lactobionate Intravenous Infusion
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Erythromycin lactobionate is a mixture of lactobionates of macrolide antibiotics. The main
component is (3R,4S,5S, 6R, 7R,9R,11R,12R,13S,14R)-4-[(2,6-dideoxy-3-C-methyl-3-O-methyl--
L-ribo-hexopyranosyl)oxy]-14-ethyl-7,12,13-trihydroxy-3,5,7,9,11,13-hexamethyl-6-[(3,4,6-trideoxy-
3-dimethylamino--D-xylo-hexopyranosyl)oxy]oxacyclotetradecane-2,10-dione 4-O--D-galactopyran-
osyl-D-gluconate (erythromycin A lactobionate). The sum of the content of erythromycin A
lactobionate, of erythromycin B lactobionate and of erythromycin C lactobionate is not less than
93.0 per cent and not more than the equivalent of 100.5 per cent, calculated with reference to the
CHARACTERS
A white or slightly yellow powder, hygroscopic, soluble in water, freely soluble in ethanol and in
methanol, very slightly soluble in acetone and in methylene chloride, practically insoluble in ether.
IDENTIFICATION
A. Examine by thin-layer chromatography (2.2.27) using silica gel H R as the coating substance.
the same solvent.
the same solvent.
Reference solution (b). Dissolve 10 mg of lactobionic acid R in water R and dilute to 10 ml with the same
solvent.
volumes of glacial acetic acid R, 10 volumes of water R and 90 volumes of methanol R. Allow the plate
to dry in air, spray with a 5 g/l solution of potassium permanganate R in 1M sodium hydroxide and heat at
110C for 5 min. The chromatogram obtained with the test solution shows two spots, one of which
corresponds in position, colour and size to the principal spot in the chromatogram obtained with
15-65
reference solution (a) and the other to the principal spot in the chromatogram obtained with
B. To about 5 mg add 5 ml of a 0.2 g/l solution of xanthydrol R in a mixture of 1 volume of hydro-
chloric acid R and 99 volumes of acetic acid R. A red colour develops.
C. Dissolve about 10 mg in 5 ml of hydrochloric acid R1. A yellowish-green colour develops.
TESTS
Appearance of solution Dissolve 1.0 g in 20 ml of water R. The solution is clear (2.2.1) and
colourless (2.2.2).
Related substances. Examine by liquid chromatography (2.2.29), as described under Assay. The
content of erythromycin B lactobionate and that of erythromycin C lactobionate is not greater than
5.0 per cent. Inject reference solution (d). Inject the test solution and continue the chromatography
for five times the retention time of erythromycin A. In the chromatogram obtained with the test
solution, the area of any peak, apart from the peaks corresponding to erythromycin A, erythromycin
B or erythromycin C, is not greater than the area of the principal peak in the chromatogram obtained
with reference solution (d) (3.0 per cent).
Free lactobionic acid. Not more than 1.0 per cent of C
12
H
22
O
12
anhydrous substance. Dissolve 0.400 g in 50 ml of water R. Titrate with 0.1M sodium hydroxide,
determining the end-point potentiometrically (2.2.20). Calculate the volume of 0.1M sodium hydroxide
required per gram of the substance to be examined (n
1
ml). Dissolve 0.500 g in 40 ml of glacial acetic
acid R and titrate with 0.1M perchloric acid, determining the end-point potentiometrically (2.2.20).
Calculate the volume of 0.1M perchloric acid required per gram of the substance to be examined
(n
2
ml).
12
H
22
O
12
3.580 (n
1
n
2
).
Inject per kilogram of the rabbits mass 1 ml of a solution in water for injections R containing 7.4 mg of
the substance to be examined per millilitre (equivalent to 5 mg of erythromycin).
ASSAY
Test solution. Dissolve 60.0 mg of the substance to be examined in a mixture of 1 volume of
methanol R and 3 volumes of phosphate buffer solution pH 7.0 R and dilute to 10.0 ml with the same
Reference solution (a). Dissolve 40.0 mg of erythromycin A CRS in a mixture of 1 volume of methanol R
and 3 volumes of phosphate buffer solution pH 7.0 R and dilute to 10.0 ml with the same mixture of
solvents.
a mixture of 1 volume of methanol R and 3 volumes of phosphate buffer solution pH 7.0 R and dilute to
50.0 ml with the same mixture of solvents.
Reference solution (d). Dilute 3.0 ml of reference solution (a) to 100.0 ml with a mixture of 1 volume
of methanol R and 3 volumes of phosphate buffer solution pH 7.0 R.
The test solution and the reference solutions can be used within one day if stored at 5C.
a column 0.25 m long and 4.6 mm in internal diameter packed with styrene-divinylbenzene copoly-
mer R (8 m to 10 m) with a pore size of 100 nm,
as mobile phase at a flow rate of 2.0 ml per minute a solution prepared as follows: to 50 ml of a
35 g/l solution of dipotassium hydrogen phosphate R adjusted to pH 9.0 with dilute phosphoric acid R,
add 400 ml of water R, 165 ml of 2-methyl-2-propanol R and 30 ml of acetonitrile R, and dilute to
15-66
a 100 l injector,
maintaining the column at 70C, preferably using a water-bath. Inject reference solution (c). Adjust
the sensitivity of the system so that the height of the peaks is at least 25 per cent of the full scale of the
recorder. The substances are eluted in the following order: N-demethylerythromycin A, erythromycin
C, erythromycin A and erythromycin B. The assay is not valid unless the resolution between the
peaks corresponding to N-demethylerythromycin A and erythromycin C is at least 0.8 and the
resolution between the peaks corresponding to N-demethylerythromycin A and erythromycin A is at
least 5.5. If necessary, adjust the concentration of 2-methyl-2-propanol in the mobile phase or reduce
the flow rate to 1.5 ml or 1.0 ml per minute. Inject reference solution (a) six times. The assay is not
valid unless the relative standard deviation of the area of the peak due to erythromycin A is at most
2.0 per cent. Inject alternately the test solution and reference solutions (a) and (b).
Calculate the percentage content of erythromycin A using the chromatogram obtained with refer-
ence solution (a). Express the result as erythromycin A lactobionate by multiplying the percentage
content of erythromycin A by 1.4877. Calculate the percentage contents of erythromycin B and
erythromycin C using the chromatogram obtained with reference solution (b). Express the result as
erythromycin B lactobionate and as erythromycin C lactobionate by multiplying by 1.4877.
STORAGE
Store in an airtight container at a temperature not exceeding 25C. If the substance is sterile, store in
LABELLING
The label states:
where applicable, that the substance is free from pyrogens.
IMPURITIES
O
HO
Me
O
O
Me
HO
Me
Et
O
Me
OH
Me
Me O
O
O
R
1
Me
OH
NMeR5
R
2
R
3
Me
R4
A. R
1
=

OH, R
2
=

OH, R
3
=

H, R
4
=

OCH
3
, R
5
=

CH
3
: erythromycin F,
B. R
1
=

OH, R
2
=

H, R
3
=

H, R
4
=

OCH
3
, R
5
=

H:
N-demethyl erythromycin A,
O
OH
Me
O
O
Me
HO
Me
Et
O
Me
OH
Me
Me O
O
O
HO
Me
OH
NMe2
O
Me
MeO
C. erythromycin E,
Me
OMe
O
HO
Me
O
O
Me
Et
O
Me
O
Me
Me O
O
Me
OH
NMe2
Me Me
OH
15-67
O
HO
Me
O
O
Me
HO
Me
Et
O
Me
Me
Me
Me O
O
HO
Me
OH
NMe2
O
Me
OMe
O
HO
Me
O
O
Me
Me
Et
O
Me
Me
Me
Me O
O
O
Me
OH
NMe2
O
OH
Me
OMe
__________________________________________________________________________________________________________ Ph Eur
15-68
Erythromycin Stearate
O
HO
Me
Me
O
O
O
Me
O
1
Me
Et
O
Me
Me
OH
Me
Me O
Me
OH
NMe
2
O
R
2
,H
3
C[CH
2
]
16
COOH
Erythromycin R
1
R
2
A OH OMe
B H OMe
C OH OH
C
37
H
67
NO
13
,C
18
H
36
O
2
1018 643-22-1
Erythromycin Stearate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Erythromycin Stearate Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Erythromycin stearate is a mixture of the octadecanoates of erythromycin and stearic acid. The main
component is the octadecanoate of (3R,4S,5S,6R,7R,9R,11R,12R,13S,14R)-6-[[3-(dimethylamino)-
3,4,6-trideoxy--D-xylo- hexopyranosyl]oxy]-14-ethyl-7,12,13-trihydroxy-3,5,7,9,11,13-hexamethyl-
4-[(3-C-methyl- 3-O-methyl-2,6-dideoxy--L-ribo-hexopyranosyl)oxy]oxacyclotetradecane-2,10-
dione (erythromycin A). The sum of the content of erythromycin A, erythromycin B and
erythromycin C, expressed as stearate salts, and of the free stearic acid is not less than 97.0 per cent
and not more than the equivalent of 103.0 per cent, calculated with reference to the anhydrous
substance. The sum of the content of erythromycin A, erythromycin B and erythromycin C is not less
than 60.5 per cent, calculated with reference to the anhydrous substance.
PRODUCTION
Erythromycin stearate complies with the requirements of the monograph on Products of fermentation
(1468).
CHARACTERS
A white, crystalline powder, practically insoluble in water, soluble in acetone and in methanol.
Solutions may be opalescent.
IDENTIFICATION
Second identification: B.
obtained with erythromycin stearate CRS.
B. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel G plate R.
the same solvent.
the same solvent.
15-69
Reference solution (b). Dissolve 10 mg of stearic acid R in methanol R and dilute to 10 ml with the same
solvent.
plate to dry in air and spray with a solution containing 0.2 g/l of dichlorofluorescein R and 0.1 g/l
solution of rhodamine B R in alcohol R. Maintain the plate for a few seconds in the vapour above a
water-bath. Examine in ultraviolet light at 365 nm. The chromatogram obtained with the test
solution shows two spots, one of which corresponds in position to the principal spot in the chromato-
gram obtained with reference solution (a) and the other to the principal spot in the chromatogram
obtained with reference solution (b). Spray the plate with anisaldehyde solution R1. Heat at 110C for
5 min and examine in daylight. The coloured spot in the chromatogram obtained with the test
solution corresponds in position, colour and size to the principal spot in the chromatogram obtained
TESTS
Free stearic acid Not more than 14.0 per cent of C
18
H
36
O
2
anhydrous substance. Dissolve 0.400 g in 50 ml of methanol R. Titrate with 0.1M sodium hydroxide,
determining the end-point potentiometrically (2.2.20). Calculate the volume of 0.1M sodium hydroxide
required per gram of the substance to be examined (n
1
ml). Dissolve 0.500 g in 30 ml of methylene
chloride R. If the solution is opalescent, filter and shake the residue with three quantities, each of
25 ml, of methylene chloride R. Filter, if necessary, and rinse the filter with methylene chloride R. Reduce
the volume of the combined filtrate and rinsings to 30 ml by evaporation on a water-bath. Add 50 ml
of glacial acetic acid R and titrate with 0.1M perchloric acid, determining the end-point potentiometric-
ally (2.2.20). Calculate the volume of 0.1M perchloric acid required per gram of the substance to be
examined (n
2
ml).
18
H
36
O
2
2.845(n
1
n
2
)
Related substances Examine by liquid chromatography (2.2.29) as described under Assay. The
contents of erythromycin B stearate and that of erythromycin C stearate are not greater than 5.0 per
cent. Inject reference solution (d). Inject the test solution and continue the chromatography for five
times the retention time of the peak corresponding to erythromycin A. In the chromatogram obtained
with the test solution, the area of any peak apart from the peaks corresponding to erythromycin A,
erythromycin B and erythromycin C, is not greater than the area of the principal peak in the chroma-
togram obtained with reference solution (d) (3 per cent).
ASSAY
Test solution. Dissolve 55.0 mg of the substance to be examined in 5.0 ml of methanol R and dilute to
10.0 ml with buffer solution pH 8.0 R1. Centrifuge the solution and use the clear solution.
Reference solution (a). Dissolve 40.0 mg of erythromycin A CRS in 5.0 ml of methanol R and dilute to
10.0 ml with buffer solution pH 8.0 R1.
25.0 ml of methanol R and dilute to 50.0 ml with buffer solution pH 8.0 R1.
Reference solution (d). Dilute 3.0 ml of reference solution (a) to 100.0 ml with a mixture of equal
volumes of methanol R and buffer solution pH 8.0 R1.
The test solution and reference solutions can be used within one day if stored at 5C.
copolymer R (8 m to 10 m) with a pore size of 100 nm,
as mobile phase at a flow rate of 2.0 ml/min a solution prepared as follows: to 50 ml of a 35 g/l
solution of dipotassium hydrogen phosphate R adjusted to pH 9.0 with dilute phosphoric acid R, add
400 ml of water R, 165 ml of 2-methyl-2-propanol R and 30 ml of acetonitrile R, and dilute to
maintaining the column and at least a third of the tubing preceding the column at 70C, preferably
by using a water-bath. Inject 100 l of reference solution (c). Adjust the sensitivity of the system so
that the height of the peaks is at least 25 per cent of the full scale of the recorder. The substances are
15-70
eluted in the following order: N-demethylerythromycin A, erythromycin C, erythromycin A and
erythromycin B. The test is not valid unless the resolution between the peaks corresponding to
N-demethylerythromycin A and erythromycin C is at least 0.8 and the resolution between the peaks
corresponding to N-demethylerythromycin A and erythromycin A is at least 5.5. If necessary, adjust
the concentration of 2-methyl-2-propanol in the mobile phase or reduce the flow rate to 1.5 ml/min
or 1.0 ml/min. Inject reference solution (a) 6 times. The test is not valid unless the relative standard
deviation of the peak area for erythromycin A is not greater than 2.0 per cent. Inject alternately the
test solution and reference solutions (a) and (b).
Calculate the percentage content of erythromycin A using the chromatogram obtained with
reference solution (a). Calculate the percentage contents of erythromycin B and erythromycin C
using the chromatogram obtained with reference solution (b). Calculate the content of the corres-
ponding stearate salts by multiplying by 1.387.
IMPURITIES
O
HO
Me
O
O
Me
O
Me
Et
O
Me
OH
Me
Me O
O
O
Me
OH
NMeR5
R
2
R
3
1
Me
R4
A. R
1
= OH, R
2
= OH, R
3
= H, R
4
= OCH
3
, R5 = CH
3
: erythromycin F,
B. R
1
= OH, R
2
= H, R
3
= H, R
4
= OCH
3
, R5 = H: N-demethylerythromycin A,
O
OH
Me
O
O
Me
HO
Me
Et
O
Me
OH
Me
Me O
O
O
HO
Me
OH
NMe2
O
Me
MeO
C. erythromycin E,
Me
OMe
O
HO
Me
O
O
Me
Et
O
Me
O
Me
Me O
O
Me
OH
NMe2
Me Me
OH
O
HO
Me
O
O
Me
HO
Me
Et
O
Me
Me
Me
Me O
O
HO
Me
OH
NMe2
O
Me
OMe
15-71
O
HO
Me
O
O
Me
Me
Et
O
Me
Me
Me
Me O
O
O
Me
OH
NMe2
O
OH
Me
OMe
__________________________________________________________________________________________________________ Ph Eur
15-72
Erythropoietin Concentrated Solution
PPDAASAAPL RTITADTFRK LFRVYSNFLR
LQLHVDKAVS GLRSLTTLLR ALGAQKEAIS
VEVWQGLALL SEAVLRGQAL LVNSSQPWEP
APPRLICDSR VLERYLLEAK EAENITTGCA
EHCSLNENIT VPDTKVNFYA WKRMEVGQQA
GKLKLYTGEA CRTGD
Molecular weight 30,600 (approx)
Erythropoietin Concentrated Solution complies with the requirements of the 3rd edition of the European
The label states (1) the type of erythropoeitin using the appropriate International Nonproprietary
Name (Epoetin Alfa, Epoetin Beta, etc) and (2) the approved code in lower case letters indicative of
the method of production.
Action and use Haematopoietic growth factor.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Erythropoietin concentrated solution is a solution containing a family of closely-related glycoproteins
which are indistinguishable from the naturally occurring human erythropoietin (urinary
erythropoietin) in terms of amino acid sequence (165 amino acids) and average glycosylation pattern,
at a concentration of 0.5 mg/ml to 10 mg/ml. It may also contain buffer salts and other excipients. It
has a potency of not less than 100,000 International Units (I.U.) per milligram of active substance
determined using the conditions described under Assay and under Protein.
Erythropoietin concentrated solution complies with the general monograph on Recombinant DNA
technology, products of (784).
PRODUCTION
Erythropoietin is produced in rodent cells in vitro by a method based on recombinant DNA
technology.
Prior to batch release, the following tests are carried out on each batch of the final product, unless
exemption has been granted by the competent authority.
Host cell-derived proteins Limit to be determined by the competent authority.
Host cell- and vector-derived DNA Limit to be determined by the competent authority.
CHARACTERS
A clear or slightly turbid colourless solution.
IDENTIFICATION
A. It gives the appropriate response when examined using the conditions described under Assay.
B. Examine by isoelectric focusing.
Test solution. Desalt the preparation to be examined by a suitable procedure. For example, dilute in
water R to obtain a concentration of 1 mg/ml. Apply an appropriate volume to a membrane filtration
system suitable for desalting proteins, and desalt the sample according to the manufacturers instru-
ctions. Reconstitute the desalted sample to the original volume with water R.
Reference solution (a). Dissolve erythropoietin BRP in water R to give a concentration of 1 mg/ml.
Desalt the sample as described for the test solution.
Reference solution (b). Use an isoelectric point calibration solution in the pH range of 2.5 to 6.5,
prepared according to the manufacturers instructions.
The isoelectric focusing procedure may be carried out using a 0.5 mm thick polyacrylamide slab gel
containing ampholytes covering the pH range 3 to 5, prepared as follows. In a side-arm flask, mix 9 g
of urea R, 6.0 ml of 30 per cent acrylamide/bisacrylamide (36.5:1) solution R, 1.05 ml of 40 per cent pH
3 to 5 ampholyte, 0.45 ml of 40 per cent pH 3 to 10 ampholyte and 13.5 ml of water R. Degas. Add
15-73
15 l of tetramethylethylenediamine R and 0.3 ml of a 100 g/l freshly prepared solution of ammonium
persulphate R.
Pour into a suitable gel cassette, with approximate dimensions of 15 15 0.05 cm. Insert a
suitable sample-well comb and allow to polymerise.
Use as the anode solution the anolyte for isoelectric focusing pH 3 to 5 R and as the cathode solution
the catholyte for isoelectric focusing pH 3 to 5 R. Allow pre-focusing to take place for 1 h at a constant
power of 10 W, with maximum voltage and current settings of 2000 V and 100 mA respectively.
Apply to the gel 15 l of each solution. Carry out focusing for a further 30 min at the same power
supply settings. Take the gel out of the focusing chamber, place it in 200 ml of a solution containing
35 g of sulphosalicylic acid R and 100 g of trichloroacetic acid R per litre of destaining solution R.
Incubate with gentle shaking at room temperature for 30 min. Drain off the solution and add 200 ml
of destaining solution R. Incubate with shaking for 1 h, drain the gel, and add 200 ml of Coomassie
staining solution R. Incubate for 30 min and then de-stain the gel by passive diffusion with destaining
solution R until the bands are well visualised against a clear background.
The test is not valid unless the distribution of bands in the electrophoretogram obtained with
reference solution (b) corresponds to the technical information supplied with the isoelectric point
calibration reagents, the electrophoretogram obtained with reference solution (a) contains a range of
bands corresponding to the Ph. Eur. reference electrophoretogram of erythropoietin isoforms. If necessary,
the voltage settings and duration may be altered to optimise the separation of the isoforms. Identify
the bands corresponding to isoforms 2 to 7.
In the electrophoretogram obtained with the test solution, the pH range of the bands observed
corresponds to that of the electrophoretogram obtained with reference solution (a). The predominant
bands correspond to isoforms 4, 5 and 6. Additional, fainter bands corresponding to isoforms 2, 3
and 7 may also be present. Other bands are present in no more than trace amounts.
C. Examine by polyacrylamide gel electrophoresis (2.2.31) and immunoblotting (2.7.1).
Carry out the test in 0.75 mm thick polyacrylamide slab gels, using a slab gel electrophoresis
apparatus and gels of approximately 16 cm square. Assemble the gel-cassette in accordance with the
manufacturers instructions.
Separating gel. In a side-arm flask, mix 8.0 ml of 30 per cent acrylamide/bisacrylamide (29:1) solution R,
5.0 ml of 1.5M tris-hydrochloride buffer solution pH 8.8 R, 6.6 ml of water R and 0.2 ml of a 100 g/l
solution of sodium lauryl sulphate R. Degas the solution and add 8 l of tetramethylethylenediamine R
and 0.2 ml of a 100 g/l freshly prepared solution of ammonium persulphate R.
Pour the separating gel mixture into the assembled cassette, to the required depth for the stacking
gel. Overlay the gel solution with 2-propanol R and allow it to polymerise.
Stacking gel. In a side-arm flask, mix 1.0 ml of 30 per cent acrylamide/bisacrylamide (29:1) solution R,
0.75 ml of 1M tris-hydrochloride buffer solution pH 6.8 R, 4.1 ml of water R and 0.06 ml of a 100 g/l
solution of sodium lauryl sulphate R. Degas the solution and add 6 l of tetramethylethylenediamine R
and 0.06 ml of a 100 g/l freshly prepared solution of ammonium persulphate R.
Drain the 2-propanol R from the polymerised resolving gel, pour in the stacking gel solution, insert
the sample-well comb, and allow the stacking gel to polymerise.
Sample buffer. Mix equal volumes of concentrated SDS-PAGE sample buffer R and water R.
Test solution (a). Dilute the preparation to be examined in water R to obtain a concentration of
1.0 mg/ml. To one volume of this solution add the same volume of concentrated SDS-PAGE sample
buffer R.
Test solution (b). Add 0.1 ml of test solution (a) to 0.9 ml of sample buffer.
Reference solution (a). Dissolve the contents of one vial of erythropoietin BRP in 0.25 ml of water R.
Add an equal volume of concentrated SDS-PAGE sample buffer R.
Reference solution (b). Add 0.1 ml of reference solution (a) to 0.9 ml of sample buffer.
Reference solution (c). Use a solution of molecular weight standards suitable for calibrating SDS-
polyacrylamide gel electrophoresis in the range of 10 kD to 70 kD.
Reference solution (d). Use a solution of pre-stained molecular weight standards suitable for calibrating
SDS-polyacrylamide gel electrophoretograms in the range 10 kD to 70 kD and suitable for the
electrotransfer to an appropriate membrane.
Load the prepared gel into the apparatus and add the required volume of SDS-PAGE running
buffer R. Place the test and reference solutions, contained in stoppered test tubes, in a boiling water-
bath for about 2 min. Apply 20 l of each solution to the stacking gel wells, in the following order:
reference solution (c), reference solution (a), test solution (a), empty well, reference solution (b), test
solution (b), reference solution (d).
Carry out the electrophoretic separation using the conditions recommended by the manufacturer of
the equipment. At the end of the separation, remove the gel-cassette from the apparatus, and cut the
gel into two parts: the first part containing reference solution (c), reference solution (a) and test
solution (a); the second part containing reference solution (b), test solution (b) and reference
solution (d).
Place the first part of the gel into Coomassie staining solution R and shake for about 1 h. Place the gel
in destaining solution R and allow to de-stain with shaking until the protein bands are well visible on a
15-74
clear background. The test is not valid unless the molecular weight standards are resolved into
discrete bands, with an approximately linear relationship between distance migrated and log
10
of the
molecular weight. The electrophoretogram obtained with test solution (a) shows a single diffuse band
corresponding in position and intensity to the single band seen in the electrophoretogram obtained
Transfer the second part of the gel onto a membrane suitable for the immobilisation of proteins,
using commercially available electroblotting equipment and following the manufacturers instru-
ctions. After electroblotting, incubate the membrane in a neutral isotonic buffer containing a suitable
blocking agent (for example, 50 g/l of dried milk or 10 per cent V/V foetal calf serum), for 1 h to 2 h,
followed by incubation for 1 h to 14 h in the same blocking solution with a suitable dilution of either
a polyclonal or monoclonal anti-erythropoietin antibody. Detect erythropoietin-bound antibody using
a suitable enzyme- or radio-labelled antibody binding reagent (for example, an alkaline phosphatase-
conjugated second antibody). The precise details of blocking agents, concentrations and incubation
times should be optimised using the principles set out in Immunochemical methods (2.7.1).
The test is not valid unless in the electrophoretogram obtained with Reference solution (d), the
molecular mass standards are resolved into discrete bands, with an approximately linear relationship
between distance migrated and log
10
of the molecular mass.
The lane containing test solution (b) shows a single broad band corresponding in position and
intensity to the single band seen in the lane containing reference solution (b).
D. Examine by peptide mapping.
Test solution. Dilute the preparation to be examined in tris-acetate buffer solution pH 8.5 R to a concen-
tration of 1.0 mg/ml. Equilibrate the solution in tris-acetate buffer solution pH 8.5 R using a suitable
procedure (dialysis against tris-acetate buffer solution pH 8.5 R, or membrane filtration using the
procedure described under Identification B, but reconstituting the desalted sample with tris-acetate
buffer solution pH 8.5 R, are suitable). Transfer the dialysed solution to a polypropylene centrifuge
tube. Freshly prepare a solution of trypsin for peptide mapping R at a concentration of 1 mg/ml in
water R, and add 5 l to 0.25 ml of the test solution. Cap the tube and place in a water-bath at 37C
for 18 h. Remove the sample from the water-bath and stop the reaction immediately by freezing.
Reference solution. Dissolve the contents of one vial of erythropoietin BRP in 0.25 ml of water R.
Prepare as for the test solution, ensuring that all procedures are carried out simultaneously, and
under identical conditions.
Examine the two tryptic digests by liquid chromatography (2.2.29).
a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with butylsilyl silica
gel R (5 m to 10 m)
as mobile phase:
Mobile phase A. A 0.06 per cent V/V solution of trifluoroacetic acid R,
Mobile phase B. To 100 ml of water R add 0.6 ml trifluoroacetic acid R and dilute to 1000 ml with
following the elution conditions as described in the table below (if necessary, the gradient may be
modified to improve the separation of the digest):
Time
(min)
Flow
rate
(ml/min)
Mobile phase
A (per cent
V/V)
Mobile phase
B (per cent
V/V)
Comment
010 0.75 100 0 isocratic
10125 0.75 10039 061 linear gradient
125135 1.25 3917 6183 linear gradient
135145 1.25 170 83100 linear gradient
145150 1.25 100 0 re-equilibration
Equilibrate the column at the initial conditions for at least 15 min. Carry out a blank run using the
above-mentioned gradient.
Inject 50 l of the test solution and 50 l of the reference solution. The test is not valid unless the
chromatogram obtained with each solution is qualitatively similar to the Ph. Eur. reference chromato-
gram of erythropoietin digest. The profile of the chromatogram obtained with the test solution corres-
ponds to that of the chromatogram obtained with the reference solution.
E. Examine by N-terminal sequence analysis.
Perform the Edman degradation using an automated solid-phase sequencer, operated in
accordance with the manufacturers instructions.
Desalt the equivalent of 50 g of erythropoietin. For example, dilute a volume of the substance to
be examined equivalent to 50 g of the active substance in 1 ml of a 0.1 per cent V/V solution of
trifluoroacetic acid R. Pre-wash a C18 reverse-phase sample preparation cartridge according to the
instructions supplied and equilibrate the cartridge in a 0.1 per cent V/V solution of trifluoroacetic
15-75
acid R. Apply the sample to the cartridge, and wash successively with a 0.1 per cent V/V solution of
trifluoroacetic acid R containing 0 per cent, 10 per cent and 50 per cent V/V of acetonitrile R according
to the manufacturers instructions. Lyophilise the 50 per cent acetonitrile eluate.
Redissolve the desalted sample in 50 l of a 0.1 per cent V/V solution of trifluoroacetic acid R and
couple to a sequencing cartridge using the protocol provided by the manufacturer. Run fifteen
sequencing cycles, using the reaction conditions for proline when running the second and third
cycles.
Identify the phenylthiohydantoin (PTH)-amino acids released at each sequencing cycle by reverse-
phase liquid chromatography. The procedure may be carried out using the column and reagents
recommended by the manufacturer of the sequencing equipment for the separation of PTH-amino-
acids.
The separation procedure should be calibrated using:
the mixture of PTH-amino acids provided by the manufacturer, with the gradient conditions
adjusted as indicated to achieve optimum resolution of all amino acids,
a sample obtained from a blank sequencing cycle obtained as recommended by the equipment
manufacturer.
The first fifteen amino acids are:
Alanine-Proline-Proline-Arginine-Leucine-Isoleucine-(no recovered peak)-Aspartic acid-Serine-
Arginine-Valine-Leucine-Glutamic acid-Arginine-Tyrosine.
TESTS
Protein Examine by ultraviolet absorption spectrophotometry (2.2.25).
Test solution. Dilute the preparation to be examined in a 4 g/l solution of ammonium hydrogen
carbonate R to obtain a concentration of 1 mg/ml.
Record the absorbance spectrum between 250 nm and 400 nm. Measure the value at the absorbance
maximum (276 nm to 280 nm), after correction for any light scattering due to turbidity measured at
400 nm. Calculate the concentration of erythropoietin using a specific absorbance value of 7.43. The
concentration of erythropoietin is not less than 80 per cent and not more than 120 per cent of the
stated concentration.
Dimers and higher molecular mass related substances Examine by size-exclusion chromato-
graphy (2.2.30).
Test solution. Dilute the preparation to be examined in the mobile phase to obtain a concentration of
0.2 mg/ml.
Reference solution. To 0.02 ml of the test solution add 0.98 ml of the mobile phase.
a stainless steel column 0.6 m long and 7.5 mm in internal diameter packed with hydrophilic silica
gel for chromatography R, of a grade suitable for fractionation of globular proteins in the
molecular mass range of 20,000 to 200,000,
as mobile phase at a flow rate of 0.5 ml/min a solution prepared as follows: dissolve 1.15 g of
anhydrous disodium hydrogen phosphate R, 0.2 g of potassium dihydrogen phosphate R and 23.4 g of
sodium chloride R in 1 litre of water R (1.5 mM potassium dihydrogen phosphate, 8.1 mM
disodium hydrogen phosphate, 0.4M sodium chloride, pH 7.4); adjust the pH to 7.4 if
necessary,
Inject 100 l of the test solution and 100 l of the reference solution and record the chromato-
grams for a minimum of 1 h. In the chromatogram obtained with the test solution, the sum of the
areas of any peaks eluted in advance of the main peak is not greater than the area of the main peak in
the chromatogram obtained with the reference solution (2 per cent). The test is not valid unless the
area of the main peak obtained with the reference solution is 1.5 to 2.5 per cent of the area of the
main peak obtained with the test solution.
Sialic acid
Test solution (a). Dilute the preparation to be examined in the mobile phase used under Dimers and
higher molecular mass related substances to obtain a concentration of 0.3 mg ml.
Test solution (b). To 0.5 ml of test solution (a) add 0.5 ml of the mobile phase used under Dimers
and higher molecular mass related substances.
Reference solution (a). Dissolve a suitable amount of sialic acid R in water R to obtain a concentration
of 0.1 mg/ml.
Reference solution (b). To 0.8 ml of reference solution (a) add 0.2 ml of water R.
Reference solution (c). To 0.6 ml of reference solution (a) add 0.4 ml of water R.
Reference solution (d). To 0.4 ml of reference solution (a) add 0.6 ml of water R.
Reference solution (e). To 0.2 ml of reference solution (a) add 0.8 ml of water R.
Reference solution (f). Use water R.
Carry out the test in triplicate. Transfer 100 l of each of the test and reference solutions to 10 ml
15-76
glass test tubes. To each tube add 1.0 ml of resorcinol reagent R. Stopper the tubes and incubate at
100C for 30 min. Cool on ice. To each tube, add 2.0 ml of a mixture consisting of 12 volumes of
butanol R and 48 volumes of butyl acetate R. Mix vigorously, and allow the two phases to separate.
Ensuring that the upper phase is completely clear, remove the upper phase, being careful to
completely exclude any of the lower phase. Measure the absorbance (2.2.25) at 580 nm of all
samples. Using the calibration curve generated by the reference solutions, determine the content of
sialic acid in each of the two test solutions, and calculate the mean. Calculate the mol of sialic
acid per mol of erythropoietin, assuming that:
the molecular mass of erythropoietin is 30,600,
the molecular mass of sialic acid is 309.
The preparation to be examined contains not less than 10 mol of sialic acid per mol of erythro-
poietin. The test is not valid unless the individual replicates agree to within 10 per cent and unless
the value obtained from reference solution (a) is between 1.5 and 2.5 times that obtained with test
solution (a).
Bacterial endotoxins (2.6.14). Not more than 20 I.U. of endotoxin in the volume that contains
100,000 I.U. of erythropoietin.
ASSAY
The activity of the preparation is compared with that of erythropoietin BRP and expressed in
International Units (I.U.).
potency. The fiducial limits of error of the estimated potency (P = 0.95) are not less than 64 per cent
Carry out the determination of potency by Method A or B.
A. In polycythaemic mice
The activity of the preparation is estimated by examining, under given conditions, its effect in
stimulating the incorporation of
59
Fe into circulating red blood cells of mice made polycythaemic by
exposure to reduced atmospheric pressure.
The following schedule, using treatment in a hypobaric chamber, has been found to be suitable.
Induce polycythaemia in female mice (of the same strain, weighing 16 g to 18 g). Place female
mice, in a hypoxic chamber and reduce the pressure to 0.6 atmospheres. After three days at 0.6
atmospheres, further reduce the pressure to 0.4 to 0.5 atmospheres and maintain the animals at this
pressure for a further 11 days (the partial vacuum is interrupted daily for a maximum of 1 h at about
11:00, in order to clean the cages and feed the animals). At the end of the specified period, return the
mice to normal atmospheric conditions. Randomly distribute the mice into cages, each containing six
animals, and mark them.
Test solution (a). Dilute the substance to be examined in phosphate-albumin buffered saline pH 7.2 R1 to
obtain a concentration of 0.2 I.U./ml.
Test solution (b). Mix equal volumes of test solution (a) and phosphate-albumin buffered saline pH
7.2 R1.
Test solution (c). Mix equal volumes of test solution (b) and phosphate-albumin buffered saline pH
7.2 R1.
Reference solution (a). Dissolve erythropoietin BRP in phosphate-albumin buffered saline pH 7.2 R1 to
obtain a concentration of 0.2 I.U./ml.
Reference solution (b). Mix equal volumes of reference solution (a) and phosphate-albumin buffered
saline pH 7.2 R1.
Reference solution (c). Mix equal volumes of reference solution (b) and phosphate-albumin buffered
saline pH 7.2 R1.
Radiolabelled ferric chloride solution (concentrated)
59
Fe. Use a commercially available solution of
59
Fe-
ferric chloride (approximate specific activity: 100 - 1000 MBq per milligram of Fe).
Radiolabelled ferric chloride solution
59
Fe. Dilute the concentrated radiolabelled ferric chloride solution
in sodium citrate buffer solution pH 7.8 R to obtain a solution with an activity of 3.7 10
4
Bq/ml.
The concentrations of the test solutions and reference solutions may need to be modified, based on
the response range of the animals used.
Three days after returning the animals to atmospheric pressure, inject each animal subcutaneously
with 0.2 ml of one of the solutions. The six animals in each cage should each receive one of the six
different treatments (three test solutions and three reference solutions), and the order of injection
should be separately randomised for each cage. A minimum of n = eight (eight cages) is
recommended.
Two days after injection of the test or reference solution, inject each animal intraperitoneally with
0.2 ml of radiolabelled ferric chloride solution
59
Fe. The order of the injections should be the same as
that of the erythropoietin injections, and the time interval between administration of the
erythropoietin and the radiolabelled ferric chloride solution must be the same for each animal.
After a further 48 h, anaesthetise each animal by injection of a suitable anaesthetic, record body
weights, and withdraw blood samples (0.65 ml) into haematocrit capillaries from the bifurcation of
15-77
the aorta. After determining the packed cell volume for each sample, measure the radioactivity.
Calculate the response (per cent
59
Fe in total circulating blood) for each mouse using the
expression:
s t
s
5 7
V A
. M A

A
s
=radioactivity in the sample,
A
t
=total radioactivity injected,
7.5=total blood volume as per cent body weight,
M=body weight, in grams,
V
s
=sample volume
Calculate the potency by the usual statistical methods for a parallel line assay. Eliminate from the
calculation any animal where the packed cell volume is less than 54 per cent, or where the body
weight is more than 24 g.
B. In normocythaemic mice
The assay is based on the measurement of stimulation of reticulocyte production in normocythaemic
mice.
The assay may be carried out using the following procedure:
Test solution (a). Dilute the substance to be examined in phosphate-albumin buffered saline pH 7.2 R1 to
obtain a concentration of 80 I.U./ml.
Test solution (b). Mix equal volumes of test solution (a) and phosphate-albumin buffered saline pH
7.2 R1.
Test solution (c). Mix equal volumes of test solution (b) and phosphate-albumin buffered saline pH
7.2 R1.
Reference solution (a). Dissolve the erythropoietin BRP in phosphate-albumin buffered saline pH 7.2 R1 to
give a concentration of 80 I.U./ml.
Reference solution (b). Mix equal volumes of reference solution (a) and phosphate-albumin buffered
saline pH 7.2 R1.
Reference solution (c). Mix equal volumes of reference solution (b) and phosphate-albumin buffered
saline pH 7.2 R1.
The exact concentrations of the test solutions and reference solutions may need to be modified,
based on the response range of the animals used.
At the beginning of the assay procedure, distribute randomly mice of a suitable age and strain (8-
week old B6D2F1 mice are suitable) into six cages. A minimum of n = eight (eight mice per cage) is
recommended. Inject each animal subcutaneously with 0.5 ml of the appropriate treatment (one
solution per cage) and put the animal in a new cage. Combine the mice in such a way that each cage
housing the treated mice contains one mouse out of the six different treatments (three test solutions
and three reference solutions, six mice per cage).
Four days after the injections, collect blood samples from the animals, and determine the number
of reticulocytes using a suitable procedure.
The following method may be employed:
The volume of blood, dilution procedure and fluorescent reagent may need to be modified to ensure maximum
development and stability of fluorescence.
Colorant solution (concentrated). Use a solution of thiazole orange suitable for the determination of
reticulocytes. Prepare at a concentration twice that necessary for the analysis.
Proceed with the following dilution steps. Dilute whole blood five hundred-fold in the buffer used
to prepare the colorant solution. Dilute this solution two-fold in the concentrated colorant solution.
After staining for 3 min to 10 min, determine the reticulocyte count microfluorometrically in a flow
cytometer. The percentage of reticulocytes is determined using a biparametric histogram: number of
cells/red F1 (620 nm).
Calculate the potency by the usual statistical methods for a parallel line assay.
STORAGE
Store in an airtight container at a temperature below 20C. Avoid repeated freezing and thawing.
LABELLING
The label states:
the erythropoietin content in milligrams per millilitre,
the activity in International Units per millilitre,
the name and the concentration of any other excipients.
__________________________________________________________________________________________________________ Ph Eur
16-1
Estradiol Benzoate
H H
PhCOO
OH
Me
H
H
C
25
H
28
O
3
376.5 50-50-0
Estradiol Benzoate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0139].
Preparation
Estradiol Injection
When oestradiol benzoate is prescribed or demanded, Estradiol Benzoate shall be dispensed or
supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Estradiol benzoate contains not less than 97.0 per cent and not more than the equivalent of 103.0 per
cent of oestra-1,3,5(10)-triene-3,17-diol 3-benzoate, calculated with reference to the dried
substance.
CHARACTERS
An almost white, crystalline powder or colourless crystals, practically insoluble in water, sparingly
soluble in acetone, slightly soluble in alcohol and in fatty oils.
IDENTIFICATION
obtained with estradiol benzoate CRS. If the spectra obtained in the solid state for the substance to be
examined and the reference substance show differences, record further spectra using 50 g/l solutions
in chloroform R.
C. Examine the chromatograms obtained in the test for related substances in daylight and in ultra-
violet light at 365 nm. The principal spot in the chromatogram obtained with test solution (b) is
similar in position, colour, fluorescence and size to the spot in the chromatogram obtained with
D. To about 1 mg add 0.5 ml of sulphomolybdic reagent R2. A yellowish-green colour develops which
in ultraviolet light at 365 nm has an intense green fluorescence. Add 1 ml of sulphuric acid R and 9 ml
of water R. The colour becomes pink with a yellowish fluorescence.
TESTS
substance.
coating substance.
Test solution (b). Dilute 2.5 ml of test solution (a) to 50 ml with a mixture of 1 volume of methanol R
and 9 volumes of chloroform R.
Reference solution (a). Dissolve 25 mg of estradiol benzoate CRS in a mixture of 1 volume of methanol R
and 9 volumes of chloroform R and dilute to 25 ml with the same mixture of solvents.
Reference solution (b). Dilute 2 ml of test solution (b) to 10 ml with a mixture of 1 volume of
16-2
10 volumes of alcohol R and 90 volumes of toluene R. Allow the plate to dry in air until the odour of
the solvent is no longer detectable and heat at 110C for 10 min. Spray the hot plate with alcoholic
solution of sulphuric acid R. Heat again at 110C for 10 min. Examine in ultraviolet light at 365 nm.
Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not
more intense than the spot in the chromatogram obtained with reference solution (b) (1.0 per cent).
100C to 105C for 3 h.
ASSAY
Dissolve 25.0 mg of the substance to be examined in alcohol R and dilute to 250.0 ml with the same
solvent. Dilute 10.0 ml of the solution to 100.0 ml with alcohol R. Measure the absorbance (2.2.25)
at the maximum at 231 nm, using alcohol R as the compensation liquid.
25
H
28
O
3
STORAGE
__________________________________________________________________________________________________________ Ph Eur
16-3
Estradiol Hemihydrate
H H
OH
Me
H
H
HO
,H
2
O
C
18
H
24
O
2
,H
2
O 281.4 50-28-2 (anhydrous)
Estradiol Hemihydrate complies with the requirements of the 3rd edition of the European Pharmacopoeia
When oestradiol hemihydrate is prescribed or demanded, Estradiol Hemihydrate shall be dispensed
or supplied
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Estradiol hemihydrate contains not less than 97.0 per cent and not more than the equivalent of
103.0 per cent of estra-1,3,5(10)-triene-3,17-diol, calculated with reference to the anhydrous
substance.
CHARACTERS
A white or almost white, crystalline powder or colourless crystals, practically insoluble in water,
soluble in acetone, sparingly soluble in alcohol, slightly soluble in ether and in methylene chloride.
IDENTIFICATION
obtained with estradiol hemihydrate CRS.
substance.
the same solvent.
Reference solution (a). Dissolve 50 mg of estradiol hemihydrate CRS in methanol R and dilute to 50 ml
Reference solution (b). Dissolve 25 mg of ethinylestradiol CRS in reference solution (a) and dilute to
the solvent is no longer perceptible and heat at 110C for 10 min. Spray the hot plate with alcoholic
solution of sulphuric acid R. Heat again at 110C for 10 min. Allow to cool. Examine in daylight and in
similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to the
the chromatogram obtained with reference solution (b) shows two spots which may however not be
completely separated.
D. To about 1 mg add 0.5 ml of freshly prepared sulphomolybdic reagent R2. A blue colour develops
which in ultraviolet light at 365 nm has an intense green fluorescence. Add 1 ml of sulphuric acid R
and 9 ml of water R. The colour becomes pink with a yellowish fluorescence.
TESTS
Specific optical rotation (2.2.7). Dissolve 0.250 g in alcohol R and dilute to 25.0 ml with the same
substance.
16-4
25.0 ml with methanol R.
Reference solution (b). Dissolve 12.5 mg of 17-estradiol R in 20 ml of acetonitrile R and dilute to
50.0 ml with methanol R. Dissolve 25.0 mg of estradiol hemihydrate CRS in 10 ml of acetonitrile R, add
0.5 ml of the solution of 17-estradiol R and dilute to 25.0 ml with methanol R.
a stainless steel column 0.25 m long and 4 mm in internal diameter packed with octadecylsilyl silica
as mobile phase at a flow rate of 1 ml per minute a mixture prepared as follows: to 400 ml of
acetonitrile R add 50 ml of methanol R and 400 ml of water R; allow to stand for 10 min, dilute to
obtained with 20 l of reference solution (a) is 70 per cent to 90 per cent of the full scale of the
recorder.
conditions, the retention times are: estradiol hemihydrate about 11 min and 17-estradiol about
13 min. Measure the height (A) above the base-line of the peak due to 17-estradiol and the height
(B) above the base-line of the lowest point of the curve separating this peak from the peak due to
estradiol hemihydrate. The test is not valid unless A is greater than three times B. If necessary, adjust
the concentrations of acetonitrile and water in the mobile phase.
Inject separately 20 l of the test solution, 20 l of reference solution (a) and 20 l of reference
solution (b). Continue the chromatography for twice the retention time of the principal peak. In the
chromatogram obtained with the test solution: the area of any peak due to 17-estradiol
(immediately following the principal peak) is not greater than the area of the corresponding peak
obtained in the chromatogram with reference solution (b) (0.5 per cent); the area of any other peak,
apart from the principal peak, is not greater than 0.25 times the area of the principal peak in the
chromatogram obtained with reference solution (a) (0.5 per cent); the sum of the areas of all second-
ary peaks (including 17-estradiol) is not greater than 0.5 times the area of the principal peak in the
chromatogram obtained with reference solution (a) (1.0 per cent). Disregard any peak due to the
solvent and any peak with an area less than 0.01 times that of the principal peak in the chromatogram
mination of water.
ASSAY
solution to 50.0 ml with 0.1M sodium hydroxide solution. Measure the absorbance (2.2.25) of the
solution at the maximum at 238 nm.
18
H
24
O
2
STORAGE
__________________________________________________________________________________________________________ Ph Eur
16-5
Estramustine Sodium Phosphate
Me
H
H
H
H
O P O
ONa
ONa
O N
Cl
O
Cl
C
23
H
30
Cl
2
NNa
2
O
6
P 564.4 52205-73-9
Definition Estramustine Sodium Phosphate is disodium 3-[bis(2-chloroethyl)carbamoyloxy]estra-
1,3,5(10)-trien-17-yl orthophosphate. It contains not less than 97.0% and not more than 103.0% of
C
23
H
30
Cl
2
NNa
2
O
6
P, calculated with reference to the anhydrous substance.
Characteristics A white or almost white powder.
Freely soluble in water and in methanol; very slightly soluble in absolute ethanol and in chloroform.
Identification
A. The light absorption, Appendix II B, in the range 230 to 350 nm of a 0.05% w/v solution exhibits
maxima at 267 nm and 275 nm. The absorbance at 267 nm is about 0.76 and at 275 nm is about
0.71.
estramustine sodium phosphate (RS 128). In preparing the potassium bromide disc precautions
should be taken to exclude moisture and avoid excessive grinding; if necessary heat the prepared disc
at 90 for 2 minutes.
C. A 1% w/v solution yields the reactions characteristic of sodium salts, Appendix VI.
Alkalinity pH of a 0.5% w/v solution, 8.5 to 10.0, Appendix V L.
Clarity and colour of solution A 5.0% w/v solution is not more opalescent than reference suspension
II, Appendix IV A, and is colourless, Appendix IV B, Method I.
Specific optical rotation In a 2% w/v solution, +11 to +13, calculated with reference to the
anhydrous substance, Appendix V F.
Ionisable chlorine Dissolve 0.10 g in 10 ml of water, add carefully, with mixing, 0.1 ml of a mixture
of 10 volumes of silver nitrate solution and 1 volume of nitric acid and examine immediately. Any
opalescence produced is not more intense than that obtained by treating a solution containing
13.4 g of sodium chloride in 10 ml in the same manner (0.1%).
Estradiol 17 -phosphate Dissolve 50 mg in 5 ml of 0.2M sodium hydroxide, add sufficient ethanol
(96%) to produce 10 ml, mix and immediately measure the absorbance at the maxima at 300 nm and
350 nm, Appendix II B. The difference between the two absorbances is not more than 0.34 (1.0%).
Inorganic phosphate Dissolve 25 mg in 10 ml of water, add 4 ml of 1M sulphuric acid, 1 ml of a
10% w/v solution of ammonium molybdate and 2 ml of methylaminophenolsulphite reagent and allow to
stand for 15 minutes. Add sufficient water to produce 25 ml, allow to stand for 15 minutes and filter.
The absorbance of the filtrate at 730 nm, Appendix II B, is not greater than the absorbance at 730 nm
of a solution obtained by repeating the operation using 10 ml of a 0.00180% w/v solution of
potassium dihydrogen orthophosphate and beginning at the words add 4 ml of 1M sulphuric acid .
Volatile matter Carry out the method for gas chromatography, Appendix III B, using solutions in
water containing (1) 0.0040% v/v of pyridine, 0.020% v/v of absolute ethanol and 0.020% v/v of butan-
1-ol (internal standard), (2) 4.0% w/v of the substance being examined and (3) 4.0% w/v of the
substance being examined and 0.020% v/v of the internal standard.
with acid-washed, silanised diatomaceous support (80 to 100 mesh) coated with 20% w/w of
polyethylene glycol (Carbowax 20M is suitable) and maintained at 120.
In the chromatogram obtained with solution (1) the area of the peak due to pyridine is greater than
the area of any corresponding peak in the chromatogram obtained with solution (3). In the
chromatogram obtained with solution (1) the area of the peak due to ethanol is greater than the sum
of the areas of any peaks with a retention time less than that of the peak due to the internal standard
in the chromatogram obtained with solution (3).
gel G as the coating substance and a mixture of equal volumes of butan-2-one, propan-2-ol and
triethylamine hydrogen carbonate solution as the mobile phase. Apply separately to the plate 10 l of
each of four freshly prepared solutions in a mixture of 49 volumes of methanol and 1 volume of
16-6
triethylamine containing (1) 4.0% w/v of the substance being examined, (2) 0.020% w/v of the
substance being examined, (3) 0.080% w/v of 17,17-bis{3-[bis(2-chloroethyl)carbamoyloxy]estra-
1,3,5(10)-trienyl pyrophosphate} BPCRS and (4) 0.040% of estramustine BPCRS. After removal of the
plate, allow it to dry in air, spray with methanolic sulphuric acid (20%) and heat at 110 for 10
minutes. The principal spots in the chromatograms obtained with solutions (3) and (4) are more
intense than any corresponding spots in the chromatogram obtained with solution (1). Any other
secondary spot in the chromatogram obtained with solution (1) is not more intense than the spot in the
Assay To 0.5 g add 40 ml of 1M sodium hydroxide and boil under a reflux condenser for 60 minutes.
Cool and transfer the mixture to a 250-ml graduated flask with the aid of water. Add 100 ml of 0.1M
silver nitrate VS and 10 ml of nitric acid, dilute to 250 ml with water and mix. Filter and titrate the
excess of silver nitrate in 50 ml of the filtrate with 0.1M ammonium thiocyanate VS using 3 ml of
ammonium iron(III) sulphate solution R2 as indicator. Each ml of 0.1M silver nitrate VS is equivalent to
28.22 mg of C
23
H
30
Cl
2
NNa
2
O
6
P.
Storage Estramustine Sodium Phosphate should be kept in a well-closed container and protected
from light.
Action and use Cytotoxic substance.
Preparation
Estramustine Phosphate Capsules
16-7
Estriol
H H
OH
Me
H
H
HO
OH
C
18
H
24
O
3
288.4 50-27-1
Estriol complies with the requirements of the 3rd edition of the European Pharmacopoeia [1203]. These require-
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Estriol contains not less than 97.0 per cent and not more than the equivalent of 103.0 per cent of
estra-1,3,5(10)-triene-3,16,17-triol, calculated with reference to the dried substance.
CHARACTERS
alcohol.
It melts about 282C.
IDENTIFICATION
obtained with estriol CRS.
substance.
the same solvent.
Reference solution (a). Dissolve 10 mg of estriol CRS in methanol R and dilute to 10 ml with the same
solvent.
Reference solution (b). Dissolve 5 mg of estradiol hemihydrate CRS in reference solution (a) and dilute
20 volumes of alcohol R and 80 volumes of toluene R. Allow the plate to dry in air. Spray the plate
with alcoholic solution of sulphuric acid R. Heat the plate at 100C for 10 min or until the spots appear.
Allow to cool. Examine in daylight and ultraviolet light at 365 nm. The principal spot in the chroma-
togram obtained with the test solution is similar in position, colour in daylight, fluorescence in
ultraviolet light at 365 nm and size to the principal spot in the chromatogram obtained with the
TESTS
Specific optical rotation (2.2.7). Dissolve 80 mg in ethanol R and dilute to 10 ml with the same
substance.
Solvent mixture. A mixture of 20 volumes of 2-propanol R1 and 80 volumes of heptane R.
Test solution. Dissolve 20.0 mg of the substance to be examined in 5 ml of 2-propanol R1 and dilute to
Reference solution (a). Dissolve 5 mg of estriol CRS and 2.0 mg of estriol impurity A CRS in 5 ml of
2-propanol R1 and dilute to 10.0 ml with the solvent mixture. Dilute 1.0 ml of the solution to 20.0 ml
with the solvent mixture.
Reference solution (b). Dilute 1.0 ml of the test solution to 10.0 ml with the solvent mixture. Dilute
1.0 ml of the solution to 10.0 ml with the same solvent mixture.
a stainless steel column 0.15 m long and 4.0 mm in internal diameter packed with diol silica gel
16-8
as mobile phase at a flow rate of 1.2 ml/min a linear gradient programme using the following
conditions:
Mobile phase A. Heptane R,
Mobile phase B. 2-propanol R1,
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
1020 88 12 Isocratic
2030 8895 125
Switch to initial
eluent
composition
3035 95 5 Equilibration
Equilibrate the column with a mixture of 20 per cent V/V of 2-propanol R1 in heptane R until a stable
baseline is obtained.
obtained with 20 l of reference solution (b) is about 25 per cent of the full scale of the recorder.
conditions, the retention times are: estriol, about 19 min and estriol impurity A, about 21 min. The
test is not valid unless the resolution between the peaks corresponding to estriol and estriol impurity
A is at least 2.2. If the retention times increase or the resolution decreases, wash the column first with
acetone R and then with heptane R.
Inject separately 20 l of the solvent mixture as a blank, 20 l of the test solution and 20 l of each
of the reference solutions (a) and (b). In the chromatogram obtained with the test solution: the area
of any peak corresponding to estriol impurity A is not greater than half the area of the peak corres-
ponding to estriol impurity A in the chromatogram obtained with reference solution (a) (0.5 per cent)
and any other peak, apart from the principal peak, is not greater than half the area of the principal
all the peaks, apart from the principal peak and the peak corresponding to estriol impurity A, is not
greater than the area of the peak in the chromatogram obtained with reference solution (b) (1 per
cent). Disregard any peak due to the blank and any peak with an area less than 0.05 times the area of
100C to 105C for 3 h.
ASSAY
solution to 50.0 ml with the same solvent. Measure the absorbance (2.2.25) at the maximum at
281 nm.
18
H
24
O
3
taking the specific absorbance to be 72.5.
IMPURITIES
H
HO
Me
H
OH
OH
H
H
A. 9,11-didehydroestriol,
H H
HO
Me
H
O
B. estrone,
16-9
H H
Me
H
OH
OH
H
H
H
3
CO
C. estriol 3-methyl ether,
H H
HO
Me
H
R
4
R
3
R
2
R
1
D. R
1
= R
2
= R
3
= H, R
4
= OH: estradiol,
E. R
1
= R
3
= OH, R
2
= R
4
= H: 17-epi-estriol,
F. R
1
= R
3
= H, R
2
= R
4
= OH: 16-epi-estriol,
G. R
1
= R
4
= H, R
2
= R
3
= OH: 16,17-epi-estriol.
__________________________________________________________________________________________________________ Ph Eur
16-10
Estropipate
H H
Me
H
O
O
S
HO
O O
N
N
H
H
C
18
H
22
O
5
S,C
4
H
10
N
2
436.6 7280-37-7
Definition Estropipate is piperazine 17-oxoestra-1,3,5-(10)-trien-3-yl hydrogen sulphate (1:1). It
18
H
22
O
5
S,C
4
H
10
N
2
, calculated with
Very slightly soluble in water, in ethanol (96%), in chloroform and in ether.
Identification
The infrared absorption spectrum, Appendix II A, is concordant with the reference spectrum of
estropipate (RS 129).
Free oestrone Carry out the method for liquid chromatography, Appendix III D, using two solutions
in methanol containing (1) 0.10% w/v of the substance being examined and (2) 0.0020% w/v of
estrone EPCRS.
3.9 mm) packed with stationary phase C (10 m) (Bondapak C18 is suitable), (b) a mixture of 35
volumes of acetonitrile and 65 volumes of 0.025M potassium dihydrogen orthophosphate as the mobile
phase with a flow rate of 1.5 ml per minute and (c) a detection wavelength of 213 nm. The peak due
to oestrone has a retention time, relative to the peak due to estropipate, of about 5.
In the chromatogram obtained with solution (1), the area of any peak corresponding to oestrone is
not greater than the area of the peak in the chromatogram obtained with solution (2) (2%).
Loss on drying When dried at 105 for 1 hour, loses not more than 1.0% of its weight. Use 1 g.
Assay Carry out the method for liquid chromatography, Appendix III D, using two solutions in
methanol containing (1) 0.01% w/v of the substance being examined and (2) 0.01% w/v of estropipate
BPCRS.
The chromatographic procedure described under Free oestrone may be used.
18
H
22
O
5
S,C
4
H
10
N
2
in the substance being examined using the declared
content of C
18
H
22
O
5
S,C
4
H
10
N
2
in estropipate BPCRS.
Preparation
Estropipate Tablets
When piperazine oestrone sulphate is prescribed or demanded, Estropipate shall be dispensed or
supplied.
IMPURITIES
H H
Me
H
O
HO
A. Oestrone.
16-11
Etacrynic Acid
O COOH
CH
2
O
Cl
Cl
H
3
C
C
13
H
12
Cl
2
O
4
303.1 58-54-8
Etacrynic Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia [0457].
Preparation
Sodium Etacrynate Injection
When ethacrynic acid is prescribed or demanded, Etacrynic Acid shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Etacrynic acid contains not less than 98.0 per cent and not more than the equivalent of 102.0 per
cent of 2-[2,3-dichloro-4-(2-ethylacryloyl)phenoxy]acetic acid, calculated with reference to the dried
substance.
CHARACTERS
A white or almost white, crystalline powder, very slightly soluble in water, freely soluble in alcohol
and in ether. It dissolves in ammonia and in dilute solutions of the alkali hydroxides and carbonates.
IDENTIFICATION
B. Dissolve 50.0 mg in a mixture of 1 volume of 1M hydrochloric acid and 99 volumes of methanol R
and dilute to 100.0 ml with the same mixture of solvents. Dilute 10.0 ml of this solution to 100.0 ml
with a mixture of 1 volume of 1M hydrochloric acid and 99 volumes of methanol R. Examined between
230 nm and 350 nm (2.2.25), the solution shows an absorption maximum at 270 nm and a shoulder
at about 285 nm. The specific absorbance at the maximum is 110 to 120.
obtained with etacrynic acid CRS. Examine the substances prepared as discs.
D. Dissolve about 30 mg in 2 ml of aldehyde-free alcohol R. Dissolve 70 mg of hydroxylamine hydro-
chloride R in 0.1 ml of water R, add 7 ml of alcoholic potassium hydroxide solution R and dilute to 10 ml
with aldehyde-free alcohol R; allow to stand and add 1 ml of the supernatant liquid to the solution of
the substance to be examined. Heat the mixture on a water-bath for 3 min. After cooling, add 3 ml of
water R and 0.15 ml of hydrochloric acid R. Examined in ultraviolet light at 254 nm, the mixture
shows an intense blue fluorescence.
E. Dissolve about 25 mg in 2 ml of 1M sodium hydroxide and heat in a water-bath for 5 min. Cool and
add 0.25 ml of a mixture of equal volumes of water R and sulphuric acid R. Add 0.5 ml of a 100 g/l
solution of chromotropic acid, sodium salt R and, carefully, 2 ml of sulphuric acid R. An intense violet
colour is produced.
TESTS
254
R as the
coating substance.
Test solution. Dissolve 0.2 g of the substance to be examined in alcohol R and dilute to 10 ml with the
same solvent.
Reference solution (a). Dilute 1.5 ml of the test solution to 100 ml with alcohol R.
Reference solution (b). Dilute 0.5 ml of the test solution to 100 ml with alcohol R.
20 volumes of glacial acetic acid R, 50 volumes of ethyl acetate R and 60 volumes of chloroform R.
Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any spot in the chromato-
16-12
in the chromatogram obtained with reference solution (a) (1.5 per cent) and at most one such spot
is more intense than the spot in the chromatogram obtained with reference solution (b) (0.5 per
cent).
diphosphorus pentoxide R at a pressure of 0.1 kPa to 0.5 kPa.
ASSAY
Dissolve 0.250 g in 100 ml of methanol R and add 5 ml of water R. Titrate with 0.1M sodium
13
H
12
Cl
2
O
4.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
16-13
Etamiphylline Camsilate
N
N
N
O
O
Me
NEt
2
MeN
,
Me Me
CH
2
SO
3
H
O
C
13
H
21
N
5
O
2
,C
10
H
16
O
4
S 511.6 19326-29-5
Definition Etamiphylline Camsilate is 7-(2-diethylaminoethyl)theophylline camphorsulphonate. It
13
H
21
N
5
O
2
,C
10
H
16
O
4
S, calculated with
reference to the dried substance, when determined by both methods described under the Assay.
Characteristics A white or almost white powder; odour, faint and camphoraceous.
Very soluble in water; soluble in chloroform and in ethanol (96%); very slightly soluble in ether.
Identification
A. The light absorption, Appendix II B, in the range 230 to 350 nm of a 0.004% w/v solution exhibits
a maximum only at 274 nm. The absorbance at 274 nm is about 0.70.
etamiphylline camsilate (RS 132).
C. Fuse 0.1 g with a pellet of sodium hydroxide, dissolve in water and neutralise with hydrochloric acid.
The resulting solution yields reaction A characteristic of sulphates, Appendix VI.
Free etamiphylline Not more than 2.0% w/w when determined by Method I for non-aqueous
titration, Appendix VIII A, using 2 g dissolved in 75 ml of acetic anhydride and determining the end
point potentiometrically. Each ml of 0.1M perchloric acid VS is equivalent to 27.93 mg of free
etamiphylline.
gel HF
254
as the coating substance and a mixture of 80 volumes of chloroform, 20 volumes of ethanol
(96%) and 1 volume of 13.5M ammonia as the mobile phase. Apply separately to the plate 10 l of
each of two solutions of the substance being examined in water containing (1) 4.0% w/v and (2)
0.0080% w/v. After removal of the plate, allow it to dry in air and examine under ultraviolet light
(254 nm). Any secondary spot in the chromatogram obtained with solution (1) is not more intense
1 g.
Assay
For camphorsulphonic acid Dissolve 1 g in 25 ml of methanol, previously neutralised with 1M
sodium hydroxide VS, and titrate with 0.1M sodium hydroxide VS using thymol blue solution as indicator.
Each ml of 0.1M sodium hydroxide VS is equivalent to 51.16 mg of C
13
H
21
N
5
O
2
,C
10
H
16
O
4
S.
For etamiphylline Dissolve 0.15 g in 20 ml of 2M hydrochloric acid, add 12 ml of a 5% w/v solution
of silicotungstic acid and allow to stand for 5 hours. Filter, wash the residue with 2M hydrochloric acid
until the filtrate yields no precipitate with a 1% w/v solution of quinine hydrochloride and dry at 110.
Each g of residue is equivalent to 0.2830 g of C
13
H
21
N
5
O
2
,C
10
H
16
O
4
S.
Storage Etamiphylline Camsilate should be kept in a well-closed container.
Preparations
Etamiphylline Injection
Etamiphylline Suppositories
When etamiphylline camsylate is prescribed or demanded, Etamiphylline Camsilate shall be
dispensed or supplied
16-14
Etamsylate
OH
HO
SO
3
H
,NHEt
2
C
6
H
6
0
5
S,C
4
H
11
N 263.3 2624-44-4
Etamsylate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1204]. These
Action and use Haemostatic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Etamsylate contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of
diethylamine 2,5-dihydroxybenzenesulphonate, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, very soluble in water, freely soluble in methanol, soluble
in ethanol, practically insoluble in methylene chloride.
IDENTIFICATION
obtained with etamsylate CRS. Examine the substances prepared as discs.
C. Dissolve 0.100 g in water R and dilute to 200.0 ml with the same solvent. Dilute 5.0 ml of the
solution to 100.0 ml with water R. Examined immediately between 210 nm and 350 nm (2.2.25), the
solution shows two absorption, maxima at 221 nm and 301 nm. The specific absorbance at the
maximum at 301 nm is 145 to 151.
D. In a test-tube, introduce 2 ml of freshly prepared solution S (see Tests) and 0.5 g of sodium
hydroxide R. Warm the mixture and place near the open end of the tube a wet strip of red litmus
paper R. The colour of the paper becomes blue.
TESTS
Appearance of solution Solution S, when freshly prepared, is clear (2.2.1) and colourless (Method
II, 2.2.2).
Hydroquinone Examine by thin-layer chromatography (2.2.27), using as the coating substance a
same solvent.
Reference solution. Dissolve 10 mg of hydroquinone R in water R and dilute to 50 ml with the same
solvent.
Apply to the plate 10 l of each solution and dry the starting points in a current of cool air. Develop
over a path of 15 cm using a mixture of 20 volumes of methylene chloride R, 30 volumes of methyl
acetate R and 50 volumes of ethyl acetate R. Dry the plate in a current of hot air and examine in
ultraviolet light at 254 nm. Any spot corresponding to hydroquinone in the chromatogram obtained
with the test solution is not more intense than the principal spot in the chromatogram obtained with
vacuo at 60C.
16-15
ASSAY
Dissolve 0.200 g in a mixture of 10 ml of water R and 40 ml of dilute sulphuric acid R. Titrate with
0.1M cerium sulphate, determining the end-point potentiometrically (2.2.20).
10
H
17
NO
5
S.
STORAGE
IMPURITIES
OH
OH
A. benzene-1,4-diol (hydroquinone).
__________________________________________________________________________________________________________ Ph Eur
16-16
Ethambutol Hydrochloride
H
3
C
N
N
CH
3
CH
2
OH H
H CH
2
OH
H
H
,2HCl
C
10
H
24
N
2
O
2
,2HCl 277.2 1070-11-7
Ethambutol Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Ethambutol Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ethambutol hydrochloride contains not less than 97.0 per cent and not more than the equivalent of
101.0 per cent of 2,2-(ethylenedi-imino)di[(S)-butanol] dihydrochloride, calculated with reference
CHARACTERS
A white, crystalline powder, freely soluble in water, soluble in alcohol, very slightly soluble in ether.
IDENTIFICATION
obtained with ethambutol hydrochloride CRS. Examine the substances prepared as discs.
B. Examine the chromatograms obtained in the test for 2-aminobutanol. The principal spot in the
spot in the chromatogram obtained with reference solution (b).
C. Dissolve 0.1 g in 10 ml of water R and add 0.2 ml of copper sulphate solution R. Add 0.5 ml of dilute
sodium hydroxide solution R. A blue colour is produced.
TESTS
4.0.
2-Aminobutanol Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating
substance.
Reference solution (a). Dissolve 50 mg of 2-aminobutanol R in methanol R and dilute to 10 ml with the
same solvent. Dilute 1 ml of this solution to 10 ml with methanol R.
Reference solution (b). Dissolve 50 mg of ethambutol hydrochloride CRS in methanol R and dilute to
10 volumes of concentrated ammonia R, 15 volumes of water R and 75 volumes of methanol R. Allow
the plate to dry in air, heat at 110C for 10 min, cool and spray with ninhydrin solution R1. Heat the
plate at 110C for 5 min. Any spot corresponding to 2-aminobutanol in the chromatogram obtained
with test solution (a) is not more intense than the spot in the chromatogram obtained with reference
solution (a) (1.0 per cent).
100C to 105C for 3 h.
16-17
ASSAY
To 70 ml of dilute ammonia R2 add 4.0 ml of copper sulphate solution R, mix, add 5.0 ml of dilute sodium
hydroxide solution R and dilute to 100 ml with water R. Dissolve 0.100 g of the substance to be exam-
ined in 20 ml of this solution and dilute to 25.0 ml with the same solution. Prepare a reference
solution in the same manner using 0.100 g of ethambutol hydrochloride CRS. Measure the angle of
optical rotation (2.2.7) of the solutions at 436 nm.
10
H
26
Cl
2
N
2
O
2
from the angles of optical rotation measured and the
concentrations of the solutions.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
16-18
Ethanol
Absolute Alcohol; Dehydrated Alcohol
H
3
C CH
2
OH
C
2
H
6
O 46.07 64-17-5
Ethanol complies with the requirements of the 3rd edition of the European Pharmacopoeia for Anhydrous
Ethanol [1318]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Anhydrous ethanol contains not less than 99.5 per cent V/V of C
2
H
6
O (99.2 per cent m/m), at 20C.
CHARACTERS
A colourless, clear, volatile, flammable liquid, hygroscopic, miscible with water and with methylene
chloride. It burns with a blue, smokeless flame.
IDENTIFICATION
spectrum of anhydrous ethanol.
C. Mix 0.1 ml with 1 ml of a 10 g/l solution of potassium permanganate R and 0.2 ml of dilute sulphuric
acid R in a test-tube. Cover immediately with a filter paper moistened with a freshly prepared solution
containing 0.1 g of sodium nitroprusside R and 0.5 g of piperazine hydrate R in 5 ml of water R. After a
few minutes, an intense blue colour appears on the paper and becomes paler after 10 min to 15 min.
D. To 0.5 ml add 5 ml of water R, 2 ml of dilute sodium hydroxide solution R, then slowly add 2 ml of
0.05M iodine. A yellow precipitate is formed within 30 min.
TESTS
Appearance It is clear (2.2.1) and colourless (Method II, 2.2.2) when compared with water R. Dilute
1.0 ml to 20 ml with water R. After standing for 5 min, the dilution remains clear when compared
with water R (2.2.1).
Acidity or alkalinity To 20 ml add 20 ml of carbon dioxide-free water R and 0.1 ml of phenolphthalein
solution R. The solution is colourless. Add 1.0 ml of 0.01M sodium hydroxide. The solution is pink
(30 ppm, expressed as acetic acid).
Absorbance Examined between 235 nm and 340 nm, the absorbance (2.2.25) measured in a 5 cm
cell using water R as the compensation liquid is not greater than 0.40 at 240 nm, 0.30 between
250 nm and 260 nm, and 0.10 between 270 nm and 340 nm. The absorption curve is smooth.
Volatile impurities Examine by gas chromatography (2.2.28).
Test solution (b). Add 150 l of 4-methylpentan-2-ol R to 500.0 ml of the substance to be examined.
Reference solution (a). Dilute 100 l of anhydrous methanol R to 50.0 ml with the substance to be
examined. Dilute 5.0 ml of the solution to 50.0 ml with the substance to be examined.
Reference solution (b). Dilute 50 l of anhydrous methanol R and 50 l of acetaldehyde R to 50.0 ml with
the substance to be examined. Dilute 100 l of the solution to 10.0 ml with the substance to be
examined.
Reference solution (c). Dilute 150 l of acetal R to 50.0 ml with the substance to be examined. Dilute
100 l of the solution to 10.0 ml with the substance to be examined.
Reference solution (d). Dilute 100 l of benzene R to 100.0 ml with the substance to be examined.
Dilute 100 l of the solution to 50.0 ml with the substance to be examined.
poly[(cyanopropyl)(phenyl)][dimethyl]siloxane R (film thickness 1.8 m),
helium for chromatography R as the carrier gas at a column flow rate of 1.5 ml/min,
with the following programme:
16-19
Time
(min)
Temperature
(C)
Rate
(C per min)
Comment
1232 40240 10 linear
gradient
3242 240 isothermal
Injection
port
280
Detector 280
two peaks eluting before the principal peak, are at least 50 per cent of the full-scale of the recorder.
The test is not valid unless in the chromatogram obtained, the resolution between the first peak
(acetaldehyde) and the second peak (methanol) is at least 2.0. If necessary, decrease the initial
column temperature.
Inject 1 l of each solution. The area of any peak corresponding to methanol in the chromatogram
obtained with test solution (a) is not greater than half the area of the corresponding peak in the
chromatogram obtained with reference solution (a) (200 ppm V/V).
Calculate the sum of the contents (ppm) of acetaldehyde and acetal from the areas of the corres-
ponding peaks in the chromatogram obtained with test solution (a) using the following expression:
E T
E
E T
E
30 10
C C
C
A A
A
A
E
=area of the acetaldehyde peak in the chromatogram obtained with test solution (a),
A
T
=area of the acetaldehyde peak in the chromatogram obtained with reference solution (b),
C
E
=area of the acetal peak in the chromatogram obtained with test solution (a),
C
T
=area of the acetal peak in the chromatogram obtained with reference solution (c).
The sum of the contents of acetaldehyde and acetal is not greater than 10 ppm (V/V), expressed as
acetaldehyde.
Calculate the content of benzene (ppm) from the area of the corresponding peak in the chromato-
gram obtained with test solution (a) using the following expression:
E T
E
2
B B
B
B
E
=area of the benzene peak in the chromatogram obtained with test solution (a),
B
T
=area of the benzene peak in the chromatogram obtained with reference solution (d).
If necessary, the identity of benzene can be confirmed using another suitable chromatographic
system (stationary phase with a different polarity).
It contains not more than 2 ppm (V/V) of benzene.
In the chromatogram obtained with test solution (b), the sum of the areas of any peaks, apart from
the principal peak and any peak due to methanol, acetaldehyde, acetal or benzene, is not greater than
the area of the peak corresponding to 4-methylpentan-2-ol (300 ppm). Disregard any peak with an
area less than 0.03 times that of the peak corresponding to 4-methylpentan-2-ol in the chromatogram
obtained with test solution (b).
for 1 h. The residue weighs not more than 2.5 mg (25 ppm m/V).
STORAGE
IMPURITIES
A. 1,1-diethoxyethane (acetal),
B. acetaldehyde,
C. acetone,
D. benzene,
E. cyclohexane,
F. methanol,
G. butan-2-one (methyl ethyl ketone),
H. 4-methylpentan-2-one (methyl isobutyl ketone),
I. propanol,
J. propan-2-ol,
K. butanol,
16-20
L. butan-2-ol,
M. 2-methylpropanol (isobutanol),
N. furane-2-carbaldehyde (furfural),
O. 2-methylpropan-2-ol (1,1-dimethylethyl alcohol)
P. 2-methylbutan-2-ol,
Q. pentan-2-ol,
R. pentanol,
S. hexanol,
T. heptan-2-ol,
U. hexan-2-ol,
V. hexan-3-ol.
The following type chromatogram is given for information and guidance only; it does not form a mandatory part
of the monograph.
1. Acetaldehyde 6. tert-Butanol 10. Benzene 14. Methyl isobutyl ketone
2. Methanol 7. Methyl ethyl ketone 11. 2-Methyl-1-propanol 15. Pentanol
3. Ethanol 8. 2-Butanol 12. Butanol 16. Furfural
4. Acetone 9. Cyclohexane 13. Acetal 17. Octanol
5. 2-Propanol
__________________________________________________________________________________________________________ Ph Eur
16-21
Ethanol (96 per cent)
Alcohol (96 per cent)
Ethanol (96 per cent) complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ethanol (96 per cent) contains not less than 95.1 per cent V/V (92.6 per cent m/m) and not more
than 96.9 per cent V/V (95.2 per cent m/m) of C
2
H
6
O (M
r
46.07) at 20C, and water.
CHARACTERS
A colourless, clear, volatile, flammable liquid, hygroscopic, miscible with water and with methylene
chloride. It burns with a blue, smokeless flame.
IDENTIFICATION
spectrum of anhydrous ethanol.
C. Mix 0.1 ml with 1 ml of a 10 g/l solution of potassium permanganate R and 0.2 ml of dilute sulphuric
acid R in a test-tube. Cover immediately with a filter paper moistened with a freshly prepared solution
containing 0.1 g of sodium nitroprusside R and 0.5 g of piperazine hydrate R in 5 ml of water R. After a
few minutes, an intense blue colour appears on the paper and becomes paler after 10 min to 15 min.
D. To 0.5 ml add 5 ml of water R, 2 ml of dilute sodium hydroxide solution R, then slowly add 2 ml of
0.05M iodine. A yellow precipitate is formed within 30 min.
TESTS
Appearance It is clear (2.2.1) and colourless (Method II, 2.2.2) when compared with water R. Dilute
1.0 ml to 20 ml with water R. After standing for 5 min, the dilution remains clear when compared
with water R (2.2.1).
Acidity or alkalinity To 20 ml add 20 ml of carbon dioxide-free water R and 0.1 ml of phenolphthalein
solution R. The solution is colourless. Add 1.0 ml of 0.01M sodium hydroxide. The solution is pink
(30 ppm, expressed as acetic acid).
Absorbance Examined between 235 nm and 340 nm, the absorbance (2.2.25) measured in a 5 cm
cell using water R as the compensation liquid is not greater than 0.40 at 240 nm, 0.30 between
250 nm and 260 nm and 0.10 between 270 nm and 340 nm. The absorption curve is smooth.
Volatile impurities Examine by gas chromatography (2.2.28).
Test solution (b). Add 150 l of 4-methylpentan-2-ol R to 500.0 ml of the substance to be examined.
Reference solution (a). Dilute 100 l of anhydrous methanol R to 50.0 ml with the substance to be
examined. Dilute 5.0 ml of the solution to 50.0 ml with the substance to be examined.
Reference solution (b). Dilute 50 l of anhydrous methanol R and 50 l of acetaldehyde R to 50.0 ml with
the substance to be examined. Dilute 100 l of the solution to 10.0 ml with the substance to be
examined.
Reference solution (c). Dilute 150 l of acetal R to 50.0 ml with the substance to be examined. Dilute
100 l of the solution to 10.0 ml with the substance to be examined.
Reference solution (d). Dilute 100 l of benzene R to 100.0 ml with the substance to be examined.
Dilute 100 l of the solution to 50.0 ml with the substance to be examined.
helium for chromatography R as the carrier gas at a column flow rate of 1.5 ml/min,
with the following programme:
16-22
Time
(min)
Temperature
(C)
Rate
(C per min)
Comment
1232 40240 10 linear
gradient
3242 240 isothermal
Injection
port
280
Detector 280
two peaks eluting before the principal peak are at least 50 per cent of the full-scale of the recorder.
The test is not valid unless in the chromatogram obtained, the resolution between the first peak
(acetaldehyde) and the second peak (methanol) is at least 2.0. If necessary, decrease the initial
column temperature.
Inject 1 l of each solution. The area of any peak corresponding to methanol in the chromatogram
obtained with test solution (a) is not greater than half the area of the corresponding peak in the
chromatogram obtained with reference solution (a) (200 ppm V/V).
Calculate the sum of the contents (ppm) of acetaldehyde and acetal from the areas of the corres-
ponding peaks in the chromatogram obtained with test solution (a) using the following expression:
E T
E
E T
E
30 10
C C
C
A A
A
A
E
=area of the acetaldehyde peak in the chromatogram obtained with test solution (a),
A
T
=area of the acetaldehyde peak in the chromatogram obtained with reference solution (b),
C
E
=area of the acetal peak in the chromatogram obtained with test solution (a),
C
T
=area of the acetal peak in the chromatogram obtained with reference solution (c).
The sum of the contents of acetaldehyde and acetal is not greater than 10 ppm (V/V), expressed as
acetaldehyde.
Calculate the content of benzene (ppm) from the area of the corresponding peak in the chromato-
gram obtained with test solution (a) using the following expression:
E T
E
2
B B
B
B
E
=area of the benzene peak in the chromatogram obtained with test solution (a),
B
T
=area of the benzene peak in the chromatogram obtained with reference solution (d).
If necessary, the identity of benzene can be confirmed using another suitable chromatographic
system (stationary phase with a different polarity).
It contains not more than 2 ppm (V/V) of benzene.
In the chromatogram obtained with test solution (b), the sum of the areas of any peaks, apart from
the principal peak and any peak due to methanol, acetaldehyde, acetal or benzene, is not greater than
the area of the peak corresponding to 4-methylpentan-2-ol (300 ppm). Disregard any peak with an
area less than 0.03 times that of the peak corresponding to 4-methylpentan-2-ol in the chromatogram
obtained with test solution (b).
for 1 h. The residue weighs not more than 2.5 mg (25 ppm m/V).
STORAGE
IMPURITIES
A. 1,1-diethoxyethane (acetal),
B. acetaldehyde,
C. acetone,
D. benzene,
E. cyclohexane,
F. methanol,
C. butan-2-one (methyl ethyl ketone),
H. 4-methylpentan-2-one (methyl isobutyl ketone),
I. propanol,
J. propan-2-ol,
16-23
K. butanol,
L. butan-2-ol,
M. 2-methylpropanol (isobutanol),
N. furane-2-carbaldehyde (furfural),
O. 2-methylpropan-2-ol (1,1-dimethylethyl alcohol)
P. 2-methylbutan-2-ol,
Q. pentan-2-ol,
R. pentanol,
S. hexanol,
T. heptan-2-ol,
U. hexan-2-ol,
V. hexan-3-ol.
The following type chromatogram is given for information and guidance only; it does not form a mandatory part
of the monograph.
1. Acetaldehyde 6. tert-Butanol 10. Benzene 14. Methy isobutyl ketone
2. Methanol 7. Methyl ethyl ketone 11. 2-Methyl-1-propanol 15. Pentanol
3. Ethanol 8. 2-Butanol 12. Butanol 16. Furfural
4. Acetone 9. Cyclohexane 13. Acetal 17. Octanol
5. 2-Propanol
__________________________________________________________________________________________________________ Ph Eur
16-24
Dilute Ethanols
Definition The official Dilute Ethanols contain 90, 80, 70, 60, 50, 45, 25 and 20% v/v
respectively of ethanol. They may be prepared as described below, the final adjustment of volume
being made at the same temperature, 20, as that at which the Ethanol (96 per cent) is measured.
NOTE On mixing ethanol and water, contraction of volume and rise of temperature occur.
Acidity or alkalinity; Appearance; Volatile impurities; Residue on evaporation. Comply
with the requirements stated under Ethanol (96 per cent).
ETHANOL (90 PER CENT)
Alcohol (90 per cent); Rectified Spirit
Dilute 934 ml of Ethanol (96 per cent) to 1000 ml with Purified Water.
Content of ethanol 89.6 to 90.5% v/v.
Apparent density 826.4 to 829.4 kg m
3
, Appendix V G.
3
, Appendix V G.
3
, Appendix V G.
3
, Appendix V G.
3
, Appendix V G.
3
, Appendix V G.
3
, Appendix V G.
16-25
3
, Appendix V G.
16-26
Ethanolamine
Monoethanolamine
H
2
N
OH
C
2
H
7
NO 61.08 141-43-5
Definition Ethanolamine is 2-aminoethanol. It contains not less than 98.0% and not more than
100.5% of C
2
H
7
NO.
Characteristics A clear, colourless or pale yellow liquid; odour, slight.
Miscible with water and with ethanol (96%); slightly soluble in ether.
Identification
A. To 0.1 ml add 0.3 g of picric acid and 1 ml of water and evaporate to dryness on a water bath. The
melting point of the residue, after recrystallisation from ethanol (96%) and drying at 105, is about
160, Appendix V A.
B. When freshly distilled the second half of the distillate freezes at about 10.
C. It is alkaline to litmus solution.
Related substances Prepare a 0.1% w/v solution of 3-aminopropan-1-ol (internal standard) in
dichloromethane (solution A). Carry out the method for gas chromatography, Appendix III B, using the
following solutions. For solution (1) prepare a solution containing 0.05% w/v of ethanolamine and
0.1% w/v each of diethanolamine and triethanolamine in solution A. To 0.5 ml of this solution add
0.5 ml of trifluoroacetic anhydride, mix and allow to stand for 10 minutes. For solution (2) prepare a
10% w/v solution of the substance being examined in solution A. To 0.5 ml of this solution add
0.5 ml of trifluoroacetic anhydride, mix and allow to stand for 10 minutes.
The chromatographic procedure may be carried out using (a) a fused silica capillary column (25
m 0.22 mm) bonded with a 0.25-m layer of dimethylpolysiloxane, (b) helium as the carrier gas at a
flow rate of 1.0 ml per minute with a flow rate of the make up gas of 20 ml per minute and (c) a split
injection system with a split ratio of 40 to 1 maintained at 240 and a detector temperature of 250.
Maintain the temperature of the column at 80 for 2 minutes, then increase to 200 at a rate of 8 per
minute and maintain this temperature for 10 minutes.
In the chromatogram obtained with solution (1) the peaks eluting after the solvent peak in order of
emergence are due to (a) ethanolamine, (b) 3-aminopropan-1-ol, (c) diethanolamine and (d)
triethanolamine. In the chromatogram obtained with solution (2) calculate the content of
diethanolamine and triethanolamine by reference to the corresponding peaks in the chromatogram
obtained with solution (1). Calculate the content of any other impurity by reference to the peak due
to ethanolamine in the chromatogram obtained with solution (1). The content of diethanolamine and
triethanolamine is not more than 1.0% w/w of each, the content of any other impurity is not more
than 0.5% w/w and the sum of the contents of all the impurities is not more than 2.0% w/w.
Assay Dissolve 2.5 g in 50 ml of 1M hydrochloric acid VS and titrate the excess of acid with 1M sodium
hydroxide VS using methyl red solution as indicator. Each ml of 1M hydrochloric acid VS is equivalent to
61.08 mg of C
2
H
7
NO.
Action and use Sclerosant.
Preparation
Ethanolamine Oleate Injection
16-27
Ether
H
3
C O CH
3
C
4
H
10
O 74.1 60-29-7
Ether complies with the requirements of the 3rd edition of the European Pharmacopoeia [0650]. These require-
When solvent ether is demanded, Ether shall be supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ether is diethyl ether which may contain a suitable non-volatile antioxidant at a suitable concentra-
tion.
CHARACTERS
A clear, colourless liquid, volatile, highly flammable, soluble in water, miscible with alcohol, with
methylene chloride and with fatty oils.
IDENTIFICATION
B. It complies with the test for distillation range (see Tests).
TESTS
Acidity To 20 ml of alcohol R add 0.25 ml of bromothymol blue solution R1 and, dropwise, 0.02M
sodium hydroxide until a blue colour persists for 30 s. Add 25 ml of the substance to be examined,
shake and add, dropwise, 0.02M sodium hydroxide until the blue colour reappears and persists for 30 s.
Not more than 0.4 ml of 0.02M sodium hydroxide is required.
Distillation range (2.2.11). Do not distil if the substance to be examined does not comply with the test
for peroxides. It distils completely between 34.0C and 35.0C. Carry out the test using a suitable
heating device and taking care to avoid directly heating the flask above the level of the liquid.
Non-volatile matter After ensuring that the substance to be examined complies with the test for peroxides,
evaporate 50 ml to dryness on a water-bath and dry the residue in an oven at 100C to 105C. The
residue weighs not more than 1 mg (20 mg/l).
Substances with a foreign odour Moisten a disc of filter paper 80 mm in diameter with 5 ml of the
substance to be examined and allow to evaporate. No foreign odour is perceptible immediately after
the evaporation.
Aldehydes To 10.0 ml in a ground-glass-stoppered cylinder add 1 ml of alkaline potassium
tetraiodomercurate solution R and shake for 10 s. Allow to stand for 5 min, protected from light. The
lower layer may show yellow or reddish-brown opalescence but not grey or black opalescence.
Peroxides Place 8 ml of potassium iodide and starch solution R in a 12 ml ground-glass-stoppered
cylinder about 15 mm in diameter. Fill completely with the substance to be examined, mix and allow
to stand protected from light for 5 min. No colour develops.
Water (2.5.12). Not more than 2 g/l, determined on 20 ml by the semi-micro determination of
water.
STORAGE
LABELLING
The label states, where appropriate, the name and concentration of any added non-volatile
antioxidant.
__________________________________________________________________________________________________________ Ph Eur
16-28
Anaesthetic Ether
C
4
H
10
O 74.1 60-29-7
Anaesthetic Ether complies with the requirements of the 3rd edition of the European Pharmacopoeia [0367].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Anaesthetic ether is diethyl ether which may contain a suitable non-volatile antioxidant at an
appropriate concentration.
CHARACTERS
A clear, colourless liquid, volatile, very mobile, highly flammable, soluble in 15 parts of water,
miscible with alcohol and with fatty oils.
IDENTIFICATION
B. It complies with the test for distillation range (see Tests).
TESTS
Distillation range (2.2.11). Do not distil if the substance to be examined does not comply with the test
for peroxides. It distils completely between 34.0C and 35.0C. Carry out the test using a suitable
heating device and taking care to avoid directly heating the flask above the level of the liquid.
Non-volatile matter After ensuring that the substance to be examined complies with the test for peroxides,
evaporate 50 ml to dryness on a water-bath and dry the residue at 100C to 105C. The residue
weighs not more than 1 mg (20 mg/l).
Substances with a foreign odour Moisten a disc of filter paper 80 mm in diameter with 5 ml of the
substance to be examined and allow to evaporate. No foreign odour is perceptible immediately after
the evaporation.
Acids To 20 ml of alcohol R add 0.25 ml of bromothymol blue solution R1 and, dropwise, 0.02M sodium
hydroxide until a blue colour persists for 30 s. Add 25 ml of the substance to be examined, shake and
add, dropwise, 0.02M sodium hydroxide until the blue colour reappears and persists for 30 s. Not more
than 0.4 ml of 0.02M sodium hydroxide is required.
Acetone and aldehydes To 10.0 ml in a ground-glass-stoppered cylinder add 1 ml of alkaline
potassium tetra-iodomercurate solution R and shake for 10 s. Allow to stand for 5 min, protected from
light. The lower layer shows only a slight opalescence.
If the substance to be examined does not comply with the test, distil 40 ml, after ensuring that the
substance to be examined complies with the test for peroxides, until only 5 ml remains. Collect the distillate
in a receiver cooled by placing in a bath of iced water and repeat the test described above using
10.0 ml of the distillate.
Peroxides Place 8 ml of potassium iodide and starch solution R in a 12 ml ground-glass-stoppered
cylinder about 15 mm in diameter. Fill completely with the substance to be examined, shake
vigorously and allow to stand protected from light for 30 min. No colour is produced.
Water (2.5.12). Not more than 2 g/l, determined on 20 ml by the semi-micro determination of
water.
STORAGE
Store in an airtight container, protected from light, at a temperature of 8C to 15C. The contents of
a partly filled container may deteriorate rapidly.
LABELLING
The label states, where appropriate, the name and concentration of any added non-volatile
antioxidant.
__________________________________________________________________________________________________________ Ph Eur
16-29
Ethinylestradiol
HO
H H
Me
H
OH
C CH
C
20
H
24
O
2
296.4 57-63-6
Ethinylestradiol complies with the requirements of the 3rd edition of the European Pharmacopoeia [0140].
Preparations
Ethinylestradiol Tablets
Levonorgestrel and Ethinylestradiol Tablets
When ethinyloestradiol is prescribed or demanded, Ethinylestradiol shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ethinylestradiol contains not less than 97.0 per cent and not more than the equivalent of 102.0 per
cent of 19-nor-17-pregna-1,3,5(10)-trien-20-yne-3,17-diol, calculated with reference to the dried
substance.
CHARACTERS
A white or slightly yellowish-white, crystalline powder, practically insoluble in water, freely soluble in
alcohol. It dissolves in dilute alkaline solutions.
IDENTIFICATION
obtained with ethinylestradiol CRS. If the spectra obtained in the solid state show differences, dissolve
the substance to be examined and the reference substance in methanol R, evaporate to dryness and
record new spectra using the residues.
Reference solution. Dissolve 25 mg of ethinylestradiol CRS in a mixture of 1 volume of methanol R and 9
the solvent is no longer detectable and heat at 110C for 10 min. Spray the hot plate with alcoholic
solution of sulphuric acid R. Heat again at 110C for 10 min. Examine in daylight and in ultraviolet
light at 365 nm. The principal spot in the chromatogram obtained with the test solution is similar in
position, colour, fluorescence and size to the principal spot in the chromatogram obtained with the
reference solution.
TESTS
Specific optical rotation (2.2.7). Dissolve 1.25 g in pyridine R and dilute to 25.0 ml with the same
Reference solution (a). Dissolve 5 mg of 17-estradiol R in the mobile phase, add 10.0 ml of the test
solution and dilute to 50.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with
the same solvent.
1.0 ml of this solution to 10.0 ml with the same solvent.
16-30
volumes of water R,
conditions, the retention times are: 17-estradiol about 3.5 min and ethinylestradiol about 4.6 min.
The test is not valid unless the resolution between the peaks due to 17-estradiol and ethinylestradiol
is at least 3.5; if necessary, adjust the concentration of acetonitrile in the mobile phase.
solution: the area of any peak, apart from the principal peak, is not greater than half the area of the
principal peak in the chromatogram obtained with reference solution (b) (1 per cent); the sum of the
areas of all the peaks, apart from the principal peak, is not greater than 0.75 times the area of the
principal peak in the chromatogram obtained with reference solution (b) (1.5 per cent). Inject 20 l
of the mobile phase as a blank. Disregard any peak due to the mobile phase and any peak with an
solution (b).
100C to 105C for 3 h.
ASSAY
Dissolve 0.200 g in 40 ml of tetrahydrofuran R and add 5 ml of a 100 g/l solution of silver nitrate R.
Titrate with 0.1M sodium hydroxide, determining the end-point potentiometrically (2.2.20). Carry out
a blank titration.
20
H
24
O
2
.
STORAGE
IMPURITIES
H H
H
Me
HO
OH
CH
A. 17-ethinylestradiol,
H
H
Me
HO
OH
CH
B. 9,11-didehydroethinylestradiol,
H H
H
Me
HO
O
C. estrone,
H H
H
Me
HO
OH
H
D. -estradiol.
__________________________________________________________________________________________________________ Ph Eur
16-31
Ethionamide
N Et
CSNH
2
C
8
H
10
N
2
S 166.2 536-33-4
Ethionamide complies with the requirements of the 3rd edition of the European Pharmacopoeia [0141]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ethionamide contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent
of 2-ethylpyridine-4-carbothioamide, calculated with reference to the dried substance.
CHARACTERS
A yellow, crystalline powder or small, yellow crystals, practically insoluble in water, soluble in
methanol, sparingly soluble in alcohol, slightly soluble in ether.
IDENTIFICATION
the solution to 100.0 ml with methanol R. Examined between 230 nm and 350 nm (2.2.25), the
solution shows an absorption maximum at 290 nm. The specific absorbance at the maximum is 380
to 440.
obtained with ethionamide CRS.
D. Dissolve about 10 mg in 5 ml of methanol R. Add 5 ml of silver nitrate solution R2. A dark-brown
TESTS
Appearance of solution Dissolve 0.5 g in 10 ml of methanol R, heating to about 50C. Allow to
cool to room temperature. The solution is not more opalescent than reference suspension II (2.2.1).
Acidity Dissolve 2.0 g in 20 ml of methanol R, heating to about 50C, and add 20 ml of water R.
Cool slightly while shaking until crystallisation begins and then allow to cool to room temperature.
Add 60 ml of water R and 0.2 ml of cresol red solution R. Not more than 0.2 ml of 0.1M sodium
hydroxide is required to change the colour of the indicator to red.
254
R as the
coating substance.
same solvent.
Reference solution (a). Dilute 0.5 ml of the test solution to 100 ml with acetone R.
Reference solution (b). Dilute 0.2 ml of the test solution to 100 ml with acetone R.
10 volumes of methanol R and 90 volumes of chloroform R. Allow the plate to dry in air. Examine in
ultraviolet light at 254 nm. Any spot in the chromatogram obtained with the test solution, apart from
solution (a) (0.5 per cent) and at most one such spot is more intense than the spot in the chromato-
100C to 105C for 3 h.
16-32
ASSAY
8
H
10
N
2
S.
__________________________________________________________________________________________________________ Ph Eur
16-33
Ethosuximide
N
O O
Et
Me
H
and enantiomer
C
7
H
11
NO
2
141.2 77-67-8
Ethosuximide complies with the requirements of the 3rd edition of the European Pharmacopoeia [0764]. These
Preparations
Ethosuximide Capsules
Ethosuximide Oral Solution
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ethosuximide contains not less than 98.5 per cent and not more than the equivalent of 101.0 per
cent of (RS)-3-ethyl-3-methylpyrrolidine-2,5-dione, calculated with reference to the anhydrous
substance.
CHARACTERS
A white or almost white powder or waxy solid, freely soluble in water, very soluble in alcohol, in ether
IDENTIFICATION
B. Dissolve 50.0 mg in alcohol R and dilute to 50.0 ml with the same solvent. Examined between
obtained with ethosuximide CRS, prepared in the same manner. Melt a sufficient quantity at 50C and
prepare a film between two previously warmed potassium bromide R plates. Record the spectrum
immediately.
D. Dissolve 0.1 g in 3 ml of methanol R. Add 0.05 ml of a 100 g/l solution of cobalt chloride R and
0.05 ml of a 100 g/l solution of calcium chloride R and add 0.1 ml of dilute sodium hydroxide solution R.
A purple colour develops and no precipitate is formed.
E. To about 10 mg add 10 mg of resorcinol R and 0.2 ml of sulphuric acid R. Heat at 140C for 5 min
and cool. Add 5 ml of water R and 2 ml of concentrated ammonia R1. A brown colour is produced.
Add about 100 ml of water R. A green fluorescence is produced.
TESTS
Cyanide Dissolve 1.0 g in 10.0 ml of alcohol R. Add 0.5 ml of ferrous sulphate solution R2, 1 ml of
dilute sodium hydroxide solution R and 0.1 ml of ferric chloride solution R1. Heat to boiling, cool and
acidify using 3 ml of dilute sulphuric acid R. After 15 min, there is no blue colour and no blue
Related substances Examine by gas chromatography (2.2.28), using anthracene R as the internal
standard.
Internal standard solution. Dissolve 10 mg of anthracene R in chloroform R and dilute to 100 ml with the
same solvent.
Test solution (a). Dissolve 1.00 g of the substance to be examined in chloroform R and dilute to
Test solution (b). Dilute 5.0 ml of test solution (a) to 10.0 ml with the internal standard solution.
16-34
Reference solution (a). Dissolve 10.0 mg of 2-ethyl-2-methylsuccinic acid R in chloroform R and dilute to
Reference solution (b). Dilute 1.0 ml of test solution (a) to 100.0 ml with chloroform R. To 1.0 ml of
this solution add 5.0 ml of the internal standard solution and dilute to 10.0 ml with chloroform R.
Reference solution (c). Dilute 1.0 ml of test solution (b) to 50.0 ml with chloroform R. To 1.0 ml of this
solution add 1.0 ml of reference solution (a) and 5.0 ml of the internal standard solution and dilute
to 10.0 ml with chloroform R.
a glass column 2 m long and 2 mm in internal diameter, packed with silanised diatomaceous earth
for gas chromatography R (150 m to 200 m) impregnated with 3 per cent m/m of
poly(cyanopropyl)methylphenylmethylsiloxane R,
detector at 240C.
Inject 1 l of reference solution (c) and adjust the sensitivity of the detector so that the heights of
the three peaks, apart from the solvent peak, are not less than 70 per cent of the full scale of the
recorder. The substances elute in the following order: 2-ethyl-2-methylsuccinic acid, ethosuximide
and anthracene. The test is not valid unless the resolution between the peaks corresponding to
2-ethyl-2-methylsuccinic acid and ethosuximide is at least 4.
Inject separately 1 l of test solution (b) and reference solution (b). Record the chromatogram for
twice the retention time of the peak corresponding to ethosuximide. From the chromatogram
obtained with reference solution (b), calculate the ratio (R) of the area of the peak due to
ethosuximide to the area of the peak due to the internal standard. From the chromatogram obtained
with test solution (b), calculate the ratio of the sum of the areas of any peaks, apart from the principal
peak, the peak obtained due to the internal standard and the peak due to the solvent, to the area of
the peak due to the internal standard: this ratio is not greater than R (0.1 per cent).
mination of water.
ASSAY
To 20 ml of dimethylformamide R add 0.2 ml of a 5 g/l solution of thymolphthalein R in
dimethylformamide R and titrate with 0.1M tetrabutylammonium hydroxide until a distinct blue colour is
obtained. Immediately add 0.120 g of the substance to be examined and titrate with 0.1M tetrabutyl-
ammonium hydroxide until the distinct blue colour is obtained again. Protect the solution from
atmospheric carbon dioxide throughout the titration.
1 ml of 0.1M tetrabutylammonium hydroxide used in the second titration is equivalent to 14.12 mg of
C
7
H
11
NO
2
.
STORAGE
IMPURITIES
A. 2-ethyl-2-methylsuccinic acid.
__________________________________________________________________________________________________________ Ph Eur
16-35
Ethyl Acetate
H
3
C COOEt
C
4
H
8
O
2
88.1 141-78-6
Ethyl Acetate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0899]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ethyl acetate is ethyl ethanoate.
CHARACTERS
A clear, colourless liquid, volatile, soluble in water, miscible with acetone, with alcohol and with
methylene chloride.
IDENTIFICATION
A. Boiling point (2.2.12): 76C to 78C.
spectrum of ethyl acetate.
C. It gives the reaction of acetyl (2.3.1).
D. It gives the reaction of esters (2.3.1).
TESTS
Appearance of solution A mixture of 1 ml of the substance to be examined and 15 ml of water R is
clear (2.2.1) and colourless (Method II, 2.2.2).
Acidity To 10 ml of alcohol R add 0.1 ml of phenolphthalein solution R and 0.01M sodium hydroxide
until the colour changes to pink. Add 5.5 ml of the substance to be examined and 0.25 ml of 0.02M
sodium hydroxide. The solution remains pink for not less than 15 s.
Reaction with sulphuric acid Carefully add 2 ml to 10 ml of sulphuric acid R. After 15 min, the
interface between the two liquids is not coloured.
a glass column 2 m long and 2 mm in internal diameter, packed with ethylvinylbenzene-
raising the temperature at a rate of 8C per minute from 90C to 240C and maintaining at 240C
for 8 min and maintaining the temperature of the injection port and that of the detector at 240C.
Inject 1 l of the test solution. In the chromatogram obtained with the test solution, the sum of the
areas of any peaks apart from the principal peak is not greater than 0.2 per cent of the area of the
principal peak.
Residue on evaporation Evaporate 100.0 g to dryness on a water-bath and dry in an oven at 100C
to 105C. The residue weighs not more than 3 mg (30 ppm).
Water (2.5.12). Not more than 0.1 per cent, determined on 10.0 ml by the semi-micro deter-
mination of water.
STORAGE
Store in a well-closed container, protected from light, at a temperature not exceeding 30C.
IMPURITIES
A. methyl acetate,
B. ethanol,
C. methanol.
__________________________________________________________________________________________________________ Ph Eur
16-36
Ethyl Chloride
H
3
C CH
2
Cl
C
2
H
5
Cl 64.52 75-00-3
Definition Ethyl Chloride is chloroethane. It may be prepared by the action of hydrogen chloride
on Ethanol or on Industrial Methylated Spirit; in the latter case it may contain a small variable
proportion of methyl chloride.
Characteristics Gaseous at ambient temperatures and pressures, but usually compressed to a
colourless, mobile, flammable and very volatile liquid; odour, ethereal.
Slightly soluble in water; miscible with ethanol (96%) and with ether.
Identification
A. The infrared absorption spectrum, Appendix II A, is concordant with the reference spectrum of ethyl
chloride (RS 135).
B. Burns with a luminous flame with the production of an acid gas.
Acidity or alkalinity Shake 10 ml with 10 ml of iced water and allow the ethyl chloride to evaporate
spontaneously. The residual liquid is neutral to litmus solution.
Ethanol Warm 5 ml of the residual liquid obtained in the test for Acidity or alkalinity with iodinated
potassium iodide solution and sodium carbonate. No yellow crystals of iodoform are produced.
Ionisable chloride 5 ml of the residual liquid obtained in the test for Acidity or alkalinity yields no
turbidity with silver nitrate solution.
Distillation range Fit a dry 100-ml measuring cylinder with a stopper carrying a short exit tube not
less than 6 mm in internal diameter and an accurately standardised short-bulb thermometer covering
the range 20 to +30 and graduated in increments of 0.1. Cover the bulb of the thermometer with
a piece of very fine muslin, free from grease and sizing materials, so that one end hangs down about
10 mm below the bulb. Cool the cylinder in ice, transfer to it 100 ml of the sample, previously cooled
in ice, insert the stopper and adjust the thermometer so that the end of the muslin dips into the liquid
and the bulb is above the surface. Replace the ice with water at 24 to 26 and observe the
temperature when 5 ml of sample has evaporated and again when 5 ml remains. Continually lower
the thermometer to maintain its position relative to the liquid surface throughout the test. Correct the
observed temperature by adding 0.26 for every kPa that the barometric pressure is below 101.3 kPa
or by subtracting 0.26 for every kPa above. The corrected temperature range is 12.0 to 12.5.
Apparent specific gravity (0/15) Cool a sufficient quantity to about 5 by surrounding the
container with a mixture of methanol and solid carbon dioxide. Transfer the cooled substance being
examined to a hydrometer cylinder standing in melting ice and insert a hydrometer and a thermo-
meter. The reading on the hydrometer when the temperature has risen to 0 is 0.921 to 0.926.
Other organic compounds On evaporation, no foreign odour is detectable at any stage.
Non-volatile matter When evaporated and dried at 105, leaves not more than 0.01% w/w of
residue.
Storage Ethyl Chloride should be protected from light and stored at a temperature not exceeding
20.
Action and use Anaesthetic.
16-37
Ethyl Cinnamate
COOEt
C
11
H
12
O
2
176.2 103-36-6
Definition Ethyl Cinnamate is predominantly ethyl (E)-3-phenylprop-2-enoate. It contains not
11
H
12
O
2
Characteristics A clear, colourless or almost colourless liquid; odour, fruity, balsamic.
Practically insoluble in water; miscible with most organic solvents.
Identification
A. The infrared absorption spectrum, Appendix II A, is concordant with the reference spectrum of ethyl
cinnamate (RS 136).
ethanol (96%) exhibits a maximum only at 276 nm. The absorbance at the maximum is about 1.23.
C. To 1 g add 25 ml of 1M sodium hydroxide, boil under a reflux condenser for 1 hour, cool and
acidify with hydrochloric acid. The melting point of the resulting precipitate, after filtration, washing
with water and drying at 60 at a pressure not exceeding 0.7 kPa, is about 133, Appendix V A.
Acidity Mix 30 g with 150 ml of ethanol (96%) previously neutralised to phenolphthalein solution R1.
Not more than 1.0 ml of 0.1M sodium hydroxide VS is required for neutralisation using phenolphthalein
solution R1 as indicator.
following solutions. Solution (1) contains 1.0% w/v of the substance being examined in chloroform.
Solution (2) is the substance being examined.
with acid-washed, silanised diatomaceous support coated with 3% w/w of cyanopropylmethyl phenyl
methyl silicone fluid (OV-225 is suitable) and maintained at 150.
greater than 1% by normalisation.
Water Not more than 0.1% w/w, Appendix IX C. Use 5 g.
Assay In a borosilicate glass flask dissolve 2.5 g of the substance being examined in 5 ml of carbon
dioxide-free ethanol prepared by boiling ethanol (96%) thoroughly and neutralising to phenolphthalein
solution R1. Neutralise the free acid in the solution with 0.1M ethanolic potassium hydroxide VS using
0.2 ml of phenolphthalein solution R1 as indicator. Add 50 ml of 0.5M ethanolic potassium hydroxide VS
and boil under a reflux condenser on a water bath for 1 hour. Add 20 ml of water and titrate the
excess of alkali with 0.5M hydrochloric acid VS using a further 0.2 ml of phenolphthalein solution R1 as
indicator. Repeat the operation without the substance being examined. The difference between the
titrations represents the alkali required to saponify the esters. Each ml of 0.5M ethanolic potassium
11
H
12
O
2
.
16-38
Ethyl Gallate
HO
HO
OH
COOEt
C
9
H
10
O
5
198.2 831-61-8
Definition Ethyl Gallate is ethyl 3,4,5-trihydroxybenzoate.
Characteristics A white to creamy white, crystalline powder; odourless or almost odourless.
Slightly soluble in water; freely soluble in ethanol (96%) and in ether; practically insoluble in
arachis oil.
Identification
methanol exhibits a maximum only at 275 nm. The absorbance at the maximum is about 1.08.
B. Carry out the method for gas chromatography, Appendix III B, using a solution prepared in the
following manner. Boil 0.5 g with 50 ml of 5M sodium hydroxide under a reflux condenser for 10
minutes and distil 5 ml.
with acid-washed, silanised diatomaceous support (80 to 100 mesh) coated with 10% w/w of free fatty
acid phase (Supelco FFAP is suitable) and maintained at 80.
The principal peak in the chromatogram has the same retention time as that of absolute ethanol,
examined under the same conditions.
C. Dissolve 5 mg in a mixture of 25 ml of acetone and 25 ml of water and add 0.05 ml of iron(III)
chloride solution. A purplish black colour is produced which rapidly becomes bluish black.
Acidity Dissolve 0.4 g in 100 ml of warm carbon dioxide-free water, cool and titrate with 0.1M sodium
hydroxide VS using bromocresol green solution as indicator. Not more than 0.1 ml of 0.1M sodium
hydroxide VS is required.
Chloride Shake 0.50 g with 50 ml of water for 5 minutes and filter. 15 ml of the resulting solution
1 g.
Storage Ethyl Gallate should be kept in a well-closed container and protected from light. Contact
with metals should be avoided.
16-39
Ethyl Hydroxybenzoate
Ethylparaben
HO
COOEt
C
9
H
10
O
3
166.2 120-47-8
Ethyl Hydroxybenzoate complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Ethyl Parahydroxybenzoate [0900]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ethyl parahydroxybenzoate contains not less than 99.0 per cent and not more than the equivalent of
100.5 per cent of ethyl 4-hydroxybenzoate.
CHARACTERS
A white or almost white, crystalline powder or colourless crystals, very slightly soluble in water, freely
IDENTIFICATION
obtained with ethyl parahydroxybenzoate CRS.
D. To about 10 mg in a test-tube add 1 ml of sodium carbonate solution R, heat to boiling for 30 s and
cool (solution a). To a further 10 mg in a similar test-tube add 1 ml of sodium carbonate solution R; the
substance partly dissolves (solution b). Add at the same time to solutions (a) and (b) 5 ml of
aminopyrazolone solution R and 1 ml of potassium ferricyanide solution R and mix. Solution (b) is yellow
to orange-brown. Solution (a) is orange to red, the colour being clearly more intense than any similar
colour which may be obtained with solution (b).
TESTS
solution BY
6
(Method II, 2.2.2).
Acidity To 2 ml of solution S add 3 ml of alcohol R, 5 ml of carbon dioxide-free water R and 0.1 ml of
bromocresol green solution R. Not more than 0.1 ml of 0.1M sodium hydroxide is required to change the
a suitable octadecylsilyl silica gel with a fluorescent indicator having an optimal intensity at 254 nm.
Reference solution (a). Dilute 0.5 ml of test solution (a) to 100 ml with acetone R.
Reference solution (b). Dissolve 10 mg of ethyl parahydroxybenzoate CRS in acetone R and dilute to
Reference solution (c). Dissolve 10 mg of methyl parahydroxybenzoate R in 1 ml of test solution (a) and
dilute to 10 ml with acetone R.
glacial acetic acid R, 30 volumes of water R and 70 volumes of methanol R. Allow the plate to dry in air
16-40
ASSAY
Place 2.000 g in a ground-glass-stoppered flask and add 40.0 ml of 1M sodium hydroxide. Boil under
a reflux condenser for 1 h. Allow to cool and rinse the condenser with water R. Titrate the excess
sodium hydroxide with 0.5M sulphuric acid, continuing the titration until the second point of inflexion
and determining the end-point potentiometrically (2.2.20). Carry out a blank titration.
9
H
10
O
3
.
IMPURITIES
HO
COOR
A. R = H: 4-hydroxybenzoic acid,
B. R = CH
3
: methyl 4-hydroxybenzoate,
C. R = CH
2
-CH
2
-CH
3
: propyl 4-hydroxybenzoate,
D. R = CH
2
-CH
2
-CH
2
-CH
3
: butyl 4-hydroxybenzoate.
__________________________________________________________________________________________________________ Ph Eur
16-41
Ethyl Oleate
Ethyl Oleate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1319]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ethyl oleate is a mixture consisting of the ethyl esters of fatty acids, mainly oleic acid. A suitable
antioxidant may be added.
PRODUCTION
CHARACTERS
A clear, pale yellow or colourless liquid, practically insoluble in water, miscible with alcohol, with
methylene chloride and with light petroleum (40C to 60C).
IDENTIFICATION
C. It complies with the test for content of oleic acid (see Tests).
TESTS
Relative density (2.2.5).0.866 to 0.874.
Iodine value (2.5.4).75 to 90.
Content of oleic acid Carry out the test for foreign oils in fatty oils by gas chromatography
(2.4.22, Method A). The fatty acid fraction of the substance contains at least 60.0 per cent of oleic
acid.
of water.
STORAGE
LABELLING
The label states, where applicable, the name and concentration of any added antioxidant.
__________________________________________________________________________________________________________ Ph Eur
16-42
Ethylcellulose
Ethylcellulose complies with the requirements of the 3rd edition of the European Pharmacopoeia [0822]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ethylcellulose is a partly O-ethylated cellulose. It contains not less than 44.0 per cent and not more
than 51.0 per cent of ethoxy (-OC
2
H
5
) groups, calculated with reference to the dried substance.
CHARACTERS
A white or yellowish-white powder or granular powder, odourless or almost odourless, practically
insoluble in water, soluble in methylene chloride and in a mixture of 20 g of alcohol and 80 g of
toluene, slightly soluble in ethyl acetate and in methanol, practically insoluble in glycerol (85 per
cent) and in propylene glycol. The solutions may show a slight opalescence.
IDENTIFICATION
spectrum of ethylcellulose.
B. 0.2 g does not dissolve in 10 ml of water R, but dissolves in 10 ml of toluene R giving a slightly
opalescent solution.
TESTS
Acidity or alkalinity To 0.5 g add 25 ml of carbon dioxide-free water R and shake for 15 min. Filter
through a sintered-glass filter (40). To 10 ml add 0.1 ml of phenolphthalein solution R and 0.5 ml of
0.01M sodium hydroxide. The solution is pink. To 10 ml add 0.1 ml of methyl red solution R and 0.5 ml
of 0.01M hydrochloric acid. The solution is red.
Viscosity (2.2.9). Shake a quantity of the substance to be examined equivalent to 5.00 g of the dried
substance with 95 g of a mixture of 20 g of alcohol R and 80 g of toluene R until the substance is
dissolved. Determine the viscosity using a capillary viscometer. The viscosity, determined at 25C
and expressed in mPas, is not less than 90.0 per cent and not more than 110.0 per cent of that
stated on the label for a nominal viscosity of 10 mPas or more; not less than 80.0 per cent and not
more than 120.0 per cent of that stated on the label for a nominal viscosity of less than 10 mPas but
more than 6 mPas; and not less than 75.0 per cent and not more than 140.0 per cent of that stated
on the label for a nominal viscosity of 6 mPas or less.
Acetaldehyde Introduce 3.0 g into a 250 ml conical flask with a ground-glass stopper, add 10 ml of
water R and stir mechanically for 1 h. Allow to stand for 24 h, filter and dilute the filtrate to 100.0 ml
with water R. Transfer 5.0 ml to a 25 ml volumetric flask, add 5 ml of a 0.5 g/l solution of methyl-
benzothiazolone hydrazone hydrochloride R and heat in a water-bath at 60C for 5 min. Add 2 ml of
ferric chloride-sulphamic acid reagent R and heat again at 60C for 5 min. Cool and dilute to 25.0 ml
with water R. The solution is not more intensely coloured than a standard prepared at the same time
and in the same manner using instead of the 5.0 ml of filtrate, 5.0 ml of a reference solution prepared
by diluting 3.0 ml of acetaldehyde standard solution (100 ppm C
2
H
4
O) R1 to 100.0 ml with water R
(100 ppm).
Chlorides (2.4.4). Disperse 0.250 g in 50 ml of water R, heat to boiling and allow to cool, shaking
occasionally. Filter and discard the first 10 ml of the filtrate. Dilute 10 ml of the filtrate to 15 ml with
water R. The solution complies with the limit test for chlorides (0.1 per cent).
100C to 105C for 2 h.
ASSAY
Internal standard solution. Dilute 120 l of toluene R to 10 ml with o-xylene R.
CAUTION: Hydriodic acid and its reaction by-products are highly toxic. Perform all steps of the test solution
preparation and the reference solution preparation in a properly functioning hood.
Test solution. Transfer 50.0 mg of the substance to be examined, 50.0 mg of adipic acid R and 2.0 ml
16-43
of the internal standard solution into a suitable 5 ml thick-walled reaction vial with a pressure-tight
septum-type closure. Cautiously add 2.0 ml of hydriodic acid R, immediately close the vial tightly and
weigh the contents and the vial accurately. Shake the vial for 30 s, heat to 125C for 10 min, allow to
cool for 2 min, shake again for 30 s and heat to 125C for 10 min. Afterwards allow to cool for 2 min
and repeat shaking and heating for a third time. Allow the vial to cool for 45 min and reweigh. If the
loss is greater than 10 mg, discard the mixture and prepare another. Use the upper layer.
Reference solution. Transfer 100.0 mg of adipic acid R, 4.0 ml of the internal standard solution and
4.0 ml of hydriodic acid R into a suitable 10 ml thick-walled reaction vial with a pressure-tight
septum-type closure. Close the vial tightly and weigh the vial and contents accurately. Afterwards
inject 50 l of iodoethane R through the septum with a syringe, weigh the vial again and calculate the
mass of iodoethane added, by difference. Shake well and allow the layers to separate.
a stainless steel column 5.0 m long and 2 mm in internal diameter packed with diatomaceous
maintaining the temperature of the column at 80C and the temperature of the injection port and
that of the detector at 200C.
Inject 1 l of the upper layer of the test solution and 1 l of the upper layer of the reference
solution.
The relative retention times are as follows: iodoethane 0.6, toluene 1.0 and o-xylene 2.3. Adjust the
sensitivity of the system so that the heights of the two principal peaks are at least 50 per cent of the
full scale of the recorder. The test is not valid unless the resolution between the peaks corresponding
to iodoethane and toluene is at least 2.0.
Calculate the percentage content of ethoxy groups from the following expression:
Q m
Q m d
1 2
2 1
100 100
2 100

45.1
156.0 ( )
Q
1
=ratio of iodoethane peak area to toluene peak area in the chromatogram obtained with the
test solution,
Q
2
=ratio of iodoethane peak area to toluene peak area in the chromatogram obtained with the
reference solution,
m
1
=mass of the substance to be examined used in the test solution in milligrams,
m
2
=mass of iodoethane used in the reference solution in milligrams,
d=loss on drying as a percentage.
STORAGE
LABELLING
The label states the nominal viscosity in millipascal seconds for a 5 per cent m/m solution.
__________________________________________________________________________________________________________ Ph Eur
16-44
Ethylene Glycol Monopalmitostearate
Ethylene Glycol Monostearate
Ethylene Glycol Monopalmitostearate complies with the requirements of the 3rd edition of the European
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ethylene glycol monopalmitostearate is a mixture of ethylene glycol mono- and di-esters of stearic
and palmitic acids. It contains not less than 50.0 per cent of monoesters produced from the
condensation of ethylene glycol and stearic acid 50 of vegetable or animal origin.
PRODUCTION
CHARACTERS
A white or almost white, waxy solid, practically insoluble in water, soluble in acetone and in hot
alcohol.
IDENTIFICATION
C. It complies with the assay (monoesters content).
TESTS
Melting point (2.2.15): 54C to 60C.
(2.4.22, Method A). The fatty acid fraction has the following composition:
sum of contents of palmitic acid and stearic acid: not less than 90.0 per cent.
Free ethylene glycol Not more than 5.0 per cent, determined as prescribed under Assay.
ASSAY
Determine the free ethylene glycol content and monoesters content by size-exclusion chromato-
graphy (2.2.30).
Test solution. Into a 15 ml flask, weigh about 0.2 g (m), to the nearest 0.1 mg. Add 5.0 ml of
tetrahydrofuran R and shake to dissolve. Heat gently, if necessary. Reweigh the flask and calculate the
total mass of solvent and substance (M).
5.0 mg, 10.0 mg and 20.0 mg of ethylene glycol R. Add 5.0 ml of tetrahydrofuran R and shake to
dissolve. Weigh the flasks again and calculate the concentration of ethylene glycol in milligrams per
gram for each reference solution.
divinylbenzene copolymer R (particle diameter 5 m and pore size 10 nm),
the retention times relative to ethylene glycol are about 0.83 for the monoesters and about 0.76 for
the diesters. From the calibration curve obtained with the reference solutions determine the concen-
tration (C) in milligrams per gram of ethylene glycol in the test solution.
Calculate the percentage content of free ethylene glycol in the substance to be examined using the
following expression:
16-45
10
m
M C
From the peak area of the monoesters (A) and the diesters (B), calculate the percentage content of
monoesters using the following expression:
) 100 ( D
B A
A

+
D=the percentage content of free ethylene glycol plus the percentage content of free fatty
acids.
Calculate the percentage content of free fatty acids using the expression:
1 561
270
A
.
I
I
A
=acid value.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
16-46
Ethylenediamine
H
2
N
NH
2
C
2
H
8
N
2
60.1 107-15-3
Ethylenediamine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0716].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ethylenediamine contains not less than 98.0 per cent and not more than the equivalent of 101.0 per
cent of ethane-1,2-diamine.
CHARACTERS
A clear, colourless or slightly yellow liquid, hygroscopic, miscible with water and with ethanol,
slightly soluble in ether. On exposure to air, white fumes are evolved. On heating, it evaporates
completely.
IDENTIFICATION
B. Boiling point (2.2.12): 116C to 118C.
C. To 0.2 ml add 0.5 ml of acetic anhydride R. Boil. A crystalline mass forms after cooling, which
dissolves in 5 ml of 2-propanol R with heating. Cool the solution and add 5 ml of ether R. If necessary,
initiate crystallisation by scratching the walls of the test-tube with a glass rod. Filter through a
sintered-glass filter, wash with several portions of ether R and dry at 100C to 105C. The residue
melts (2.2.14) at 173C to 177C.
TESTS
Solution S Mix 10 g with carbon dioxide-free water R and dilute to 100 ml with the same solvent.
Appearance of solution Solution S is clear (2.2.1) and not more intensely coloured than the refer-
ence solution BY
6
(Method II, 2.2.2).
Carbonate A mixture of 4 ml of solution S and 6 ml of calcium hydroxide solution R is not more
opalescent than reference suspension II (2.2.1).
Chlorides (2.4.4). To 5 ml of solution S add 5 ml of dilute nitric acid R and dilute to 15 ml with
water R. This solution complies with the limit test for chlorides (100 ppm).
Ammonia and other bases Dissolve 1.2 g in 20 ml of alcohol R and add, dropwise with stirring,
4.5 ml of hydrochloric acid R. Evaporate to dryness on a water-bath, breaking up any resulting cake
with a glass rod, and dry at 100C to 105C for 1 h.
1 g of the residue is equivalent to 0.4518 g of C
2
H
8
N
2
. The percentage content of C
2
H
8
N
2
found
in this test does not vary by more than 0.5 from the percentage content determined in the assay.
Residue on evaporation Evaporate 5.00 g to dryness on a water-bath and dry at 100C to 105C
for 1 h. The residue weighs not more than 15 mg (0.3 per cent).
ASSAY
Place 25.0 ml of 1M hydrochloric acid and 0.2 ml of methyl red mixed solution R in a flask. Add 0.600 g
of the substance to be examined. Titrate with 1M sodium hydroxide until the colour changes from
violet-red to green.
1 ml of 1M hydrochloric acid is equivalent to 30.05 mg of C
2
H
8
N
2
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur.
16-47
Ethylmorphine Hydrochloride
O
N
H
H
H
HO
EtO
Me
,HCl
C
19
H
23
NO
3
,HCl,2H
2
O 385.9 125-30-4
Ethylmorphine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ethylmorphine hydrochloride contains not less than 99.0 per cent and not more than the equiva-
lent of 100.5 per cent of (5R,6S)-4,5-epoxy-3-ethoxy-N-methylmorphin-7-en-6-ol hydrochloride,
CHARACTERS
A white or almost white, crystalline powder, soluble in water and in alcohol, practically insoluble in
ether.
IDENTIFICATION
spectrum of ethylmorphine hydrochloride.
B. In a test-tube, dissolve 0.5 g in 6 ml of water R and add 15 ml of 0.1M sodium hydroxide. Scratch
the wall of the tube with a glass rod. A white, crystalline precipitate is formed. Collect the precipitate,
wash and dissolve in 20 ml of water R heated to 80C. Filter and cool in iced water. The crystals,
after drying in vacuo for 12 h, melt (2.2.14) at 85C to 89C.
C. To about 10 mg add 1 ml of sulphuric acid R and 0.05 ml of ferric chloride solution R2. Heat on a
water-bath. A blue colour develops. Add 0.05 ml of nitric acid R. The colour becomes red.
D. Solution S (see Tests) gives reaction (a) of chlorides (2.3.1).
TESTS
solvent.
solution BY
6
(Method II, 2.2.2).
coating substance.
alcohol R and water R and dilute to 10 ml with the same mixture of solvents.
Reference solution. Dilute 0.5 ml of the test solution to 100 ml with a mixture of equal volumes of
Apply separately to the plate 10 l of each solution and develop over a path of 15 cm using a mixture
of 2.5 volumes of concentrated ammonia R, 32.5 volumes of acetone R, 35 volumes of alcohol R and 35
volumes of toluene R. Dry the plate in a current of air and spray with dilute potassium iodobismuthate
(0.5 per cent).
16-48
mination of water.
ASSAY
Dissolve 0.300 g in 30 ml of anhydrous acetic acid R. Add 20 ml of acetic anhydride R and 5 ml of
mercuric acetate solution R. Titrate with 0.1M perchloric acid, determining the end-point potentio-
19
H
24
ClNO
3
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
16-49
Etilefrine Hydrochloride
,HCl
NHEt
OH
H
OH
and enantiomer
C
10
H
15
NO
2
,HCl 217.7 943-17-9
Etilefrine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Sympathomimetic; antihypertensive.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Etilefrine hydrochloride contains not less than 98.0 per cent and not more than the equivalent of
101.0 per cent of (RS)-2-ethylamino-1-(3-hydroxyphenyl)ethanol hydrochloride, calculated with
CHARACTERS
IDENTIFICATION
obtained with etilefrine hydrochloride CRS. Examine the substance as discs prepared using potassium
chloride R.
Prepare the solutions in subdued light and develop the chromatogram protected from light.
the same solvent.
Reference solution (a). Dissolve 25 mg of etilefrine hydrochloride CRS in methanol R and dilute to 5 ml
Reference solution (b). Dissolve 10 mg of phenylephrine hydrochloride CRS in 2 ml of reference solution
(a) and dilute to 10 ml with methanol R.
volumes of concentrated ammonia R, 25 volumes of methanol R and 70 volumes of methylene chloride R.
Dry the plate in a current of warm air and spray with a 10 g/l solution of potassium permanganate R.
Examine in daylight after 15 min. The principal spot in the chromatogram obtained with the test
D. To 0.2 ml of solution S (see Tests), add 1 ml of water R, 0.1 ml of copper sulphate solution R and
1 ml of strong sodium hydroxide solution R. A blue colour is produced. Add 2 ml of ether R and shake.
The upper layer is colourless.
E. Dilute 1 ml of solution S to 10 ml with water R. The solution gives reaction (a) of chlorides
(2.3.1).
TESTS
Solution S. Dissolve 2.50 g in carbon dioxide-free water R prepared from distilled water R and dilute to
Acidity or alkalinity Dilute 4 ml of solution S to 10 ml with carbon dioxide-free water R. Add 0.1 ml
16-50
of methyl red solution R and 0.2 ml of 0.01M sodium hydroxide. The solution is yellow. Not more than
0.4 ml of 0.01M hydrochloric acid is required to change the colour of the indicator to red.
+0.10.
the same solvent.
Reference solution (a). Dilute 1.0 ml of test solution to 10.0 ml with water R. Dilute 1.0 ml to 50.0 ml
Reference solution (b). Dissolve 10.0 mg of etilefrine impurity A CRS in water R and dilute to 50.0 ml
with the same solvent. Dilute 1.0 ml of the solution to 50.0 ml with water R.
Reference solution (c). To 10.0 ml of reference solution (a) add 5.0 ml of reference solution (b) and
volumes of a 1.1 g/l solution of sodium lauryl sulphate R adjusted to pH 2.3 with phosphoric
acid R,
Inject 20 l of reference solution (c). When the chromatogram is recorded in the prescribed
conditions, the retention times are: etilefrine, about 9 min, etilefrine impurity A, about 10 min.
Adjust the sensitivity of the system so that the height of the peak due to etilefrine is at least 50 per
cent of the full scale of the recorder. The test is not valid unless the resolution between the peaks
corresponding to etilefrine and etilefrine impurity A is at least 2.5.
Inject 20 l of the test solution, 20 l of reference solution (a) and 20 l of reference solution (b).
Continue the chromatography of the test solution for three times the retention time of etilefrine. In
the chromatogram obtained with test solution: the area of any peak due to etilefrine impurity A is not
(0.4 per cent); the area of any peak, apart from the principal peak and the peak due to etilefrine
impurity A, is not greater than the area of the principal peak in the chromatogram obtained with
reference solution (a) (0.2 per cent); the sum of the areas of all the peaks apart from the principal
peak and the peak due to etilefrine impurity A, is not greater than five times the area of the principal
peak in the chromatogram obtained with reference solution (a) (1 per cent). Disregard any peak due
to the solvent and any peak with an area less than 0.1 times the area of the principal peak in the
100C to 105C.
ASSAY
10
H
16
ClNO
2
.
STORAGE
IMPURITIES
NHEt
OH
O
A. 2-ethylamino-1-(3-hydroxyphenyl)ethanone (etilefrone),
16-51
H
N
OH
OH
H
R and enantiomer
B. R = CH
3
: (RS)-2-methylamino-1-(3-hydroxyphenyl)ethanol (phenylephrine),
C. R = H: (RS)-2-amino-1-(3-hydroxyphenyl)ethanol (norfenephrine).
__________________________________________________________________________________________________________ Ph Eur
16-52
Etodolac
and enantiomer
H
N
O
Et
COOH
Et
C
17
H
21
NO
3
287.4 41340-25-4
Etodolac complies with the requirements of the 3rd edition of the European Pharmacopoeia [1422]. These
Preparations
Etodolac Capsules
Etodolac Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Etodolac contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent of
2-[(1RS)-1,8-diethyl-1,3,4,9-tetrahydropyranol[3,4-b]indol-1-y1]acetic acid, calculated with
CHARACTERS
and in ethanol.
IDENTIFICATION
obtained with etodolac CRS.
C. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel GF
254
plate R previously
activated by heating at 105C for 1 h.
the same solvent.
Reference solution. Dissolve 10 mg of etodolac CRS in acetone R and dilute to 10 ml with the same
solvent.
Place the plate in an unsaturated chamber containing a mixture of 20 volumes of a 25 g/l solution of
ascorbic acid R and 80 volumes of methanol R. Allow the solution to ascend 1 cm above the line of
application on the plate, remove the plate and allow it to dry for at least 30 min.
of 0.5 volumes of glacial acetic acid R, 30 volumes of ethanol R and 70 volumes of toluene R. Allow the
obtained with the test solution is similar in position and size to the reference solution.
TESTS
Reference solution (b). Dissolve 1.0 mg of etodolac impurity H CRS and 1.0 mg of etodolac CRS in
16-53
Mobile phase A. A mixture of 312 volumes of methanol R and 688 volumes of a solution prepared
as follows: dissolve 13.6 g of potassium dihydrogen phosphate R in 900 ml of water R, adjust the
pH to 7.0 with a 300 g/l solution of potassium hydroxide R and dilute to 1000 ml with water R,
Time
(min)
Mobile
phase A
(per cent V/V)
Mobile
phase B
(per cent V/V)
Comment
conditions
conditions, the retention times are: impurity H about 8 min and etodolac about 9 min. The test is
not valid unless the resolution between the peaks due to impurity H and etodolac is at least 5.0.
Inject separately 10 l of the test solution and 10 l of reference solution (a). In the chromatogram
obtained with the test solution the area of any peak, apart from the principal peak, is not greater than
the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per
cent); the sum of the areas of all the peaks apart from the principal peak is not greater than twice the
area of the principal peak in the chromatogram obtained with reference solution (a) (1 per cent).
Chlorides Not more than 300 ppm. Dissolve 1.0 g of the substance to be examined in 60 ml of
methanol R, add 10 ml of water R and 20 ml of dilute nitric acid R. Titrate with 0.01M silver nitrate,
1 ml of 0.01M silver nitrate is equivalent to 0.3545 mg Cl.
mination of water.
ASSAY
Dissolve 0.250 g in 60 ml of methanol R. Titrate with 0.1M tetrabutylammonium hydroxide determining
the end-point potentiometrically (2.2.20). Carry out a blank titration.
17
H
21
NO
3
.
STORAGE
In a well-closed container.
IMPURITIES
H
N
O
R
1 R
2
COOH
and enantiomer
A. R
1
= H, R
2
= CH
2
-CH
3
: 2-[(1RS)-1-ethyl-1,3,4,9-tetrahydropyrano[3,4-b]indol-1-yl]acetic
acid (8-desethyl etodolac),
B. R
1
= CH
3
, R
2
= CH
2
-CH
3
: 2-[(1RS)-1-ethyl-8-methyl-1,3,4,9-tetrahydropyrano[3,4-b]indol-1-
yl]acetic acid (8-methyl etodolac),
C. R
1
= CH
2
-CH
3
, R
2
= CH
3
: 2-[(1RS)-8-ethyl-1-methyl-1,3,4,9,-tetrahydropyrano[3,4-b]indol-1-
yl]acetic acid (1-methyl etodolac),
D. R
1
= CH(CH
3
)
2
, R
2
= CH
2
-CH
3
: 2-[(1RS)-1-ethyl-8-(1-methylethyl)-1,3,4,9-tetrahydro-
pyrano[3,4-b]indol-1-yl]acetic acid (8-isopropyl etodolac),
E. R
1
= CH
2
-CH
2
-CH
3
, R
2
= CH
2
-CH
3
: 2-[(1RS)-1-ethyl-8-propyl-1,3,4,9-tetrahydropyrano[3,4-
b]indol-1-yl]acetic acid (8-propyl etodolac),
16-54
F. R
1
= CH
2
-CH
3
, R
2
= CH(CH
3
)
2
: 2-[(1RS)-8-ethyl-1-(1-methylethyl)-1,3,4,9-tetrahydro-
pyrano[3,4-b]indol-1-yl]acetic acid (1-isopropyl etodolac),
G. R
1
= CH
2
-CH
3
, R
2
= CH
2
-CH
2
-CH
3
: 2-[(1RS)-8-ethyl-1-propyl-1,3,4,9-tetrahydropyrano[3,4-
b]indol-1-yl]acetic acid (1-propyl etodolac),
H
N
Et
OH
H. 2-(7-ethylindol-3-yl)ethanol,
N
H
Et
H
N
Et
HO
COOH
Et
and enantiomer
I. (3RS)-3-[7-ethyl-3-(2-hydroxyethyl)indol-2-yl]-3-(7-ethylindol-3-yl)pentanoic acid
(etodolac dimer),
H
N
O
Et
Me Et
and enantiomer
J. (1RS)-1,8-diethyl-1-methyl-1,3,4,9-tetrahydropyrano[3,4-b]indol (decarboxy etodolac),
H
N
O
Et
Et
COOMe
and enantiomer
K. methyl 2-[(1RS)-1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indol-1-yl]acetate (etodolac
methyl ester),
H
N
Et
OH
COOH
Me
L. (EZ)-3-[7-ethyl-3-(2-hydroxyethyl)-1H-indol-2-yl]pent-3-enoic acid.
__________________________________________________________________________________________________________ Ph Eur
16-55
Etofenamate
1/01
H
N
O
O
HO O
CF
3
C
18
H
18
F
3
NO
4
369.4 30544-47-9
Etofenamate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1513]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
2-(2-Hydroxyethoxy)ethyl 2-[[3-(trifluoromethyl)phenyl]amino]benzoate.
Content: 98.5 per cent to 101.5 per cent (anhydrous substance).
CHARACTERS
Appearance: yellowish viscous liquid.
Solubility: practically insoluble in water, miscible with alcohol and with ethyl acetate.
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison: etofenamate CRS.
Preparation: films.
TESTS
reference solution GY
1
(2.2.2, Method II).
Impurity F: maximum 0.1 per cent.
Gas chromatography (2.2.28).
Internal standard: tetradecane R.
Solution A. Dissolve 6.0 mg of tetradecane R in hexane R and dilute to 10.0 ml with the same solvent.
Solution B. To 6.0 mg of diethylene glycol R in a 10 ml volumetric flask add 3 ml of N-methyl-
trimethylsilyltrifluoroacetamide R and heat for 30 min at 50C. After cooling dilute to 10.0 ml with
N-methyltrimethylsilyltrifluoroacetamide R.
Test solution. To 0.200 g of the substance to be examined add 10 l of solution A. Add 2 ml of
N-methyltrimethylsilyl-trifluoroacetamide R and heat for 30 min at 50C.
Reference solution. To 2.0 ml of N-methyltrimethylsilyl-trifluoroacetamide R add 10 l of solution A and
10 l of solution B.
Column:
size: l = 25 m, = 0.20 mm,
stationary phase: coated with poly(dimethyl)(diphenyl)siloxane R (film thickness 0.33 m).
Carrier gas: hydrogen for chromatography R.
Temperature:
Time
(min)
Temperature
(C)
Rate
(C per min)
Column 0 13 60 150 7
13 19 150 300 25
19 34 300
Injection port 150
Detector 300
16-56
Detection: flame-ionisation.
Injection: direct; 0.2 l. Inject the test solution and the reference solution.
Reference solution (a). Dissolve 10.0 mg of etofenamate impurity G CRS in methanol R and dilute to
20.0 ml with the same solvent. Dilute 0.2 ml of the solution to 50.0 ml with a mixture of 40 volumes
of water R and 60 volumes of methanol R.
water R and 60 volumes of methanol R.
Reference solution (c). To 5.0 ml of reference solution (a), add 5.0 ml of reference solution (b).
Reference solution (d). Dissolve 10.0 mg of etofenamate for system suitability CRS (contains etofenamate
spiked with about 1 per cent of impurities A, B, C, D and E) in 6.0 ml of methanol R and dilute to
Column:
size: l = 0.10 m, = 4.0 mm,
stationary phase: octadecylsilyl silica gel for chromatography R (3 m),
temperature: 40C.
Mobile phase:
mobile phase A. Dissolve 1.3 g of ammonium phosphate R and 4.0 g of tetrabutylammonium
hydroxide R in 900 ml of water R. Adjust the pH to 8.0 with dilute phosphoric acid R and dilute to
mobile phase B. Methanol R,
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
0 13 40 60
13 20 40 10 60 90
20 25 10 90
25 26 10 40 90 60
26 31 40 60
Injection: 20 l.
System suitability:
retention times: impurity A = 3 min; impurity C = 9 min; impurity G = 11 min; Etofenamate =
13 min; impurity E = 20 min; impurity B = 21 min; impurity D = 22 min,
resolution: minimum of 2.3 between the peaks due to impurity G and etofenamate in the chroma-
togram obtained with reference solution (c).
Limits:
correction factors: correct the areas of the corresponding peaks using the following correction
factors: impurity A = 0.62; impurity C = 0.45; impurity D = 0.77.
impurity A (corrected area): not more than 1.25 times the area of the principal peak in the
chromatogram obtained with reference solution (b) (0.25 per cent),
impurity B: not more than the area of the principal peak in the chromatogram obtained with
reference solution (b) (0.2 per cent),
impurity C (corrected area): not more than the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.2 per cent),
impurity D (corrected area): not more than 2.5 times the area of the principal peak in the chroma-
togram obtained with reference solution (b) (0.5 per cent),
impurity E: not more than the area of the principal peak in the chromatogram obtained with
impurity G: not more than the area of the principal peak in the chromatogram obtained with
any other impurity: not more than half the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.1 per cent),
total: not more than 6 times the area of the principal peak in the chromatogram obtained with
16-57
Pb) R.
Water (2.5.12): maximum 0.5 per cent, determined on 1.000 g.
ASSAY
To 3.000 g add 20 ml of 2-propanol R and 20.0 ml of 1M sodium hydroxide and heat under reflux for
2 h. Add 0.1 ml of bromothymol blue solution R1. Titrate after cooling with 1M hydrochloric acid until
the colour disappears. Carry out a blank titration.
1 ml of 1M sodium hydroxide is equivalent to 0.3694 g of C
18
H
18
F
3
NO
4
.
IMPURITIES
H
N
CF
3
R =
A. R-CO
2
H: 2-[[3-(trifluoromethyl)phenyl]amino]benzoic acid (flufenamic acid),
H
3
C O O
R
B. butyl 2-[[3-(trifluoromethyl)phenyl]amino]benzoate (butyl flufenamate),
C. R-H: N-phenyl-3-(trifluoromethyl)aniline,
O
O O
R
O
R
D. 2,2-oxybis(ethylene) bis[2-[[3-(trifluoromethyl)phenyl]amino]benzoate],
O
O O
R
O
3
C
E. 2-(2-butoxyethoxy)ethyl 2-[[3-(trifluoromethyl)phenyl]amino]benzoate,
O
O
OH
F. 2,2-oxydiethanol,
HO
O O
R
G. 2-hydroxyethyl 2-[[3-(trifluoromethyl)phenyl]amino]benzoate.
16-58
The following chromatogram is given for information and guidance only; it does not form a mandatory part of the
monograph.
5 10 15 20 25
2.5
0
-2.5
5
7.5
10
12.5
15
17.5
20
mAu
2
.
7
4
1
I
m
p
u
r
i
t
y

A
I
m
p
u
r
i
t
y

C
I
m
p
u
r
i
t
y

G
E
t
o
f
e
n
a
m
a
t
e
I
m
p
u
r
i
t
y

E
I
m
p
u
r
i
t
y

B
I
m
p
u
r
i
t
y

D
1
0
.
6
0
9
2
2
.
1
2
1
2
1
.
0
6
9
2
0
.
2
9
5
1
2
.
6
5
2
8
.
9
4
2
Fig. 1513.1 Chromatogram for the test for related substances.
__________________________________________________________________________________________________________ Ph Eur
16-59
Etofylline
N
N
N
O
O
Me
OH
MeN
C
9
H
12
N
4
O
3
224.2 519-37-9
Etofylline complies with the requirements of the 3rd edition of the European Pharmacopoeia [0492]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Etofylline contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of
7-(2-hydroxyethyl)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione, calculated with reference to the
dried substance.
CHARACTERS
A white, crystalline powder, soluble in water, slightly soluble in alcohol.
IDENTIFICATION
obtained with etofylline CRS. Examine the substances as discs prepared using 0.5 mg to 1 mg of the
substance to be examined in 0.3 g of potassium bromide R.
C. Dissolve 1 g in 5 ml of acetic anhydride R and boil under a reflux condenser for 15 min. Allow to
cool and add 100 ml of a mixture of 20 volumes of ether R and 80 volumes of light petroleum R. Cool
in iced water for at least 20 min, shaking from time to time. Filter, wash the precipitate with a
mixture of 20 volumes of ether R and 80 volumes of light petroleum R, recrystallise from alcohol R and
dry in vacuo. The crystals melt (2.2.14) at 101C to 105C.
D. It gives the reaction of xanthines (2.3.1).
TESTS
solution is yellow or green. Not more than 0.4 ml of 0.01M sodium hydroxide is required to change the
254
R as the
coating substance.
and 30 volumes of methanol R and dilute to 10 ml with the same mixture of solvents. Prepare
Reference solution (b). Dilute 0.2 ml of the test solution to 100 ml with methanol R.
Reference solution (c). Dissolve 10 mg of theophylline R in methanol R, add 0.3 ml of the test solution
of concentrated ammonia R, 10 volumes of ethanol R and 90 volumes of chloroform R. Allow the plate
to dry in air and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with
the test solution, apart from the principal spot, is not more intense than the spot in the chromato-
gram obtained with reference solution (a) (1 per cent) and at most one such spot is more intense than
16-60
spots.
100C to 105C.
ASSAY
Dissolve 0.200 g in 3.0 ml of anhydrous formic acid R and add 50.0 ml of acetic anhydride R. Titrate
9
H
12
N
4
O
3
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
16-61
Etomidate
1/01
N
N
H Me
COOEt
C
14
H
16
N
2
O
2
244.3 33125-97-2
Etomidate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1514]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Etomidate contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of
ethyl 1-[(1R)-1-phenylethyl]-1H-imidazole-5-carboxylate, calculated with reference to the dried
substance.
CHARACTERS
A white or almost white powder, very slightly soluble in water, freely soluble in alcohol and in
methylene chloride.
IDENTIFICATION
obtained with etomidate CRS.
TESTS
Solution S Dissolve 0.25 g in ethanol R and dilute to 25.0 ml with the same solvent.
ethanol R and 50 volumes of water R and dilute to 10.0 ml with the same mixture of solvents.
Reference solution (a). Dissolve 5.0 mg of etomidate CRS and 5.0 mg of etomidate impurity B CRS in a
mixture of 50 volumes of ethanol R and 50 volumes of water R and dilute to 250.0 ml with the same
ethanol R and 50 volumes of water R. Dilute 5.0 ml of the solution to 25.0 ml with a mixture of 50
volumes of ethanol R and 50 volumes of water R.
as mobile phase at a flow rate of 2.0 ml/min, the following linear gradient programme:
Mobile phase A. A 5 g/l solution of ammonium carbonate R,
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
6 10 10 90
Isocratic
16-62
Equilibrate the column for at least 30 min with acetonitrile R and then equilibrate at the initial eluent
composition for at least 5 min.
conditions, the retention times are: impurity B about 4.5 min; etomidate about 5.0 min. The test is
not valid unless the resolution between the peaks due to impurity B and etomidate is at least 5.0. If
necessary, adjust the concentration of ammonium carbonate R in the mobile phase or the time
programme of the linear gradient.
Inject 10 l of the test solution and 10 l of reference solution (b). In the chromatogram obtained
of the principal peak in the chromatogram obtained with reference solution (b) (0.2 per cent) and the
sum of the areas of any such peaks is not greater than 1.5 times the area of the principal peak in the
less than 0.25 times the area of the principal peak in the chromatogram obtained with reference
solution (b).
40C for 4 h.
ASSAY
ethyl ketone R and titrate with 0.1M perchloric acid using 0.2 ml of naphtholbenzein solution R as
indicator.
14
H
16
N
2
O
2
.
STORAGE
Protected from light.
IMPURITIES
N
N
H Me
COOR
and enantiomer
A. R = H: 1-[(1RS)-1-phenylethyl]-1H-imidazole-5-carboxylic acid,
B. R = CH
3:
methyl 1-[(1RS)-1-phenylethyl]-1H-imidazole-5-carboxylate (metomidate),
C. R = CH(CH
3
)
2:
1-methylethyl 1-[(1RS)-1-phenylethyl]-1H-imidazole-5-carboxylate.
__________________________________________________________________________________________________________ Ph Eur
16-63
Etoposide
O
O
O
O
H
H
H
H
O H
O
O
OH
OH
O
OH
OMe MeO
Me
C
29
H
32
O
13
588.6 33419-42-0
Etoposide complies with the requirements of the 3rd edition of the European Pharmacopoeia [0823]. These
Preparations
Etoposide Capsules
Etoposide Intravenous Infusion
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Etoposide contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent of
(5R,5aR,8aR,9S)-9-[(4,6-O-(R)-ethylidene--D-glucopyranosyl)oxy]-5-(4-hydroxy-3,5-dimethoxy-
phenyl)-5,8,8a,9-tetrahydroisobenzofuro[5,6-f][1,3]benzodioxol-6(5aH)-one, calculated with
CHARACTERS
A white or almost white crystalline powder, practically insoluble in water, sparingly soluble in
methanol, slightly soluble in alcohol and in methylene chloride.
IDENTIFICATION
obtained with etoposide CRS.
C. Examine by thin-layer chromatography (2.2.27), using silica gel H R as the coating substance.
Reference solution. Dissolve 10 mg of etoposide CRS in a mixture of 1 volume of methanol R and 9
Apply separately to the plate as 10 mm bands 5 l of each solution. Develop immediately over a path
of 17 cm using a mixture of 1.5 volumes of water R, 8 volumes of glacial acetic acid R, 20 volumes of
acetone R and 100 volumes of methylene chloride R. Dry the plate in a current of warm air for 5 min.
Spray with a mixture of 1 volume of sulphuric acid R and 9 volumes of alcohol R and heat at 140C for
15 min. Cover the plate immediately with a glass plate of the same size. Examine in daylight. The
principal band in the chromatogram obtained with the test solution is similar in position, colour and
size to the principal band in the chromatogram obtained with the reference solution.
D. In a test-tube dissolve about 5 mg in 5 ml of glacial acetic acid R and add 0.05 ml of ferric chloride
solution R1. Mix and cautiously add 2 ml of sulphuric acid R. Avoid mixing the two layers. Allow to
stand for about 30 min; a pink to reddish-brown ring develops at the interface and the upper layer is
yellow.
16-64
TESTS
Appearance of solution Dissolve 0.6 g in a mixture of 1 volume of methanol R and 9 volumes of
methylene chloride R and dilute to 20 ml with the same mixture of solvents. The solution is clear
(2.2.1) and not more intensely coloured than reference solution Y
6
or BY
6
(Method II, 2.2.2).
Specific optical rotation (2.2.7). Dissolve 50 mg in a mixture of 1 volume of methanol R and 9
volumes of methylene chloride R and dilute to 10.0 ml with the same mixture of solvents. The specific
optical rotation is 106 to 114, calculated with reference to the dried substance.
Inject separately 10 l of test solution (a), 10 l of reference solution (a) and 10 l of reference
solution (b). When the chromatograms are recorded under the prescribed conditions, the retention
times and the elution order of the peaks are similar to those shown in the type chromatogram (Figure
823-1). In the chromatogram obtained with test solution (a): the area of any peak, apart from the
principal peak, is not greater than the area of the principal peak obtained with reference solution (a)
(0.5 per cent); not more than two peaks have an area greater than the area of the principal peak in the
chromatogram obtained with reference solution (b) (0.2 per cent); and the sum of the areas of any
such peaks is not greater than twice the area of the principal peak in the chromatogram obtained with
reference solution (a) (1 per cent). Disregard any peak due to the solvent and any peak with an area
(a).
100C to 105C under high vacuum for 4 h.
ASSAY
Test solution (a). Dissolve 40 mg of the substance to be examined in a mixture of 50 per cent V/V of
mobile phase A and 50 per cent V/V of mobile phase B and dilute to 10.0 ml with the same mixture.
Test solution (b). Dissolve 50 mg of the substance to be examined in a mixture of 50 per cent V/V of
mobile phase A and 50 per cent V/V of mobile phase B and dilute to 50.0 ml with the same mixture.
Reference solution (a). Dilute 1.0 ml of test solution (a) to 10.0 ml with a mixture of 50 per cent V/V
of mobile phase A and 50 per cent V/V of mobile phase B. Dilute 1.0 ml of this solution to 20.0 ml
with the same mixture.
Reference solution (b). Dilute 4.0 ml of reference solution (a) to 10.0 ml with a mixture of 50 per cent
V/V of mobile phase A and 50 per cent V/V of mobile phase B.
Reference solution (c). Dissolve 50 mg of etoposide CRS in a mixture of 50 per cent V/V of mobile
phase A and 50 per cent V/V of mobile phase B and dilute to 50.0 ml with the same mixture.
Reference solution (d). To 10 ml of test solution (b), add 0.1 ml of a 4 per cent V/V solution of glacial
acetic acid R and 0.1 ml of phenolphthalein solution R. Add 1M sodium hydroxide until the solution
becomes faintly pink (about 0.15 ml). After 15 min, add 0.1 ml of a 4 per cent V/V solution of glacial
acetic acid R.
silica gel for chromatography R (5 m).
Mobile phase A. Mix 1 volume of triethylamine R, 1 volume of anhydrous formic acid R and 998
volumes of water R,
Mobile phase B. Mix 1 volume of triethylamine R, 1 volume of anhydrous formic acid R and 998
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
75 25 equilibration
07 75 25 isocratic
a loop injector,
Inject 10 l of reference solution (d) and continue the chromatography until the peak due to
phenolphthalein is eluted. The test is not valid unless the chromatogram shows two principal peaks
16-65
corresponding respectively to etoposide and cis-etoposide (impurity B) and the resolution between
these peaks is at least 3.0; disregard any peak due to phenolphthalein. If necessary, slightly increase
the proportion of mobile phase A during the isocratic phase of the gradient. When the chromato-
grams are recorded under the prescribed conditions, the retention times of the peaks in the chroma-
togram obtained with reference solution (d) are similar to those shown in the type chromatogram
(Figure 823-2). Inject 10 l of reference solution (c) six times. The test is not valid unless the relative
standard deviation of the peak area for etoposide is less than 1.0 per cent. Inject 10 l of test solution
(b) and 10 l of reference solution (c). Calculate the percentage content of C
29
H
32
O
13
from the areas
of the peaks and the declared content of etoposide CRS.
STORAGE
IMPURITIES
O
O
O
O
H
Me
OH
OH
R =
OMe
OH
MeO
Ar =
O
O
O
O O
O
H
H
OMe MeO
O
H
R
H
A. (5R,5aR,8aR,9S)-9-[(4,6-O-(R)-ethylidene--D-glucopyranosyl)oxy]-5-[4-(benzyloxy-
carbonyl)oxy]-3,5-dimethoxyphenyl]-5,8,8a,9-tetrahydroisobenzofuro[5,6-
f][1,3]benzodioxol-6(5aH)-one (4-carbobenzoyloxyethylidene-lignan P),
O
O
O
Ar
O
H
H H
R
H
B. (5R,5aS,8aR,9S)-9-[(4,6-O-(R)-ethylidene--D-glucopyranosyl)oxy]-5-(4-hydroxy-3,5-
dimethoxyphenyl)-5,8,8a,9-tetrahydroisobenzofuro[5,6-f][1,3]benzodioxol-6(5aH)-one
(picroethylidene-lignan P; cis-etoposide),
OH
H H
H
O
Ar
O
O
O
H
O
Me
H
O
O
O
OH
C. (5R,5aR,8aR,9S)-9-[(4,6-O-(R)-ethylidene--D-glucopyranosyl)oxy]-5-(4-hydroxy-3,5-
dimethoxyphenyl)-5,8,8a,9-tetrahydroisobenzofuro[5,6-f][1,3]benzodioxol-6(5aH)-one
(-etoposide),
16-66
O
O
HO
HO
OH
OH
O
O
O
Ar
O
H
H H
H
D. (5R,5aR,8aR,9S)-9-[(-D-glucopyranosyl)oxy]-5-(4-hydroxy-3,5-dimethoxyphenyl)-
5,8,8a,9-tetrahydroisobenzofuro[5,6-f][1,3]benzodioxol-6(5aH)-one (lignan P),
O
O
O
Ar
O
H
H H
OH
H
E. (5R,5aR,8aR,9S)-9-hydroxy-5-(4-hydroxy-3,5-dimethoxyphenyl)-5,8,8a,9-tetrahydroiso-
benzofuro[5,6-f][1,3]benzodioxol-6(5aH)-one (4demethylepipodophyllotoxin),
O
O
O
O
O
O
H
H
OMe MeO
O
H
R
H
F. (5R,5aR,8aR,9S)-9-[(4,6-O-(R)-ethylidene--D-glucopyranosyl)oxy]-5-[4-[(2-phenoxy-
acetyl)oxy]-3,5-dimethoxyphenyl]-5,8,8a,9-tetrahydroisobenzofuro[5,6-f][1,3]benzodioxol-
6(5aH)-one (phenoxyacetyl-4-etoposide),
O
O
O
O
H
Me
O O
O O
OMe MeO
O
H
H H
H
O
O
O
O
X = OCHO
X
G. (5R,5aR,8aR,9S)-9-[(2,3-diformyl-4,6-O-(R)-ethylidene--D-glucopyranosyl)oxy]-5-[4-
[(benzyloxycarbonyl)oxy]-3,5-dimethoxyphenyl]-5,8,8a,9-tetrahydroisobenzofuro[5,6-
f][1,3]benzodioxol-6(5aH)-one (4-carbobenzoyloxy-diformyl-ethylidene-lignan P),
O
O
O
Ar
O
H
H H
H
OEt
H. (5R,5aR,8aR,9S)-9-ethoxy-5-(4-hydroxy-3,5-dimethoxyphenyl)-5,8,8a,9-tetrahydro-
isobenzofuro[5,6-f][1,3]benzodioxol-6(5aH)-one (4-O-demethyl-1-O-ethyl-
epipodophyllotoxin),
16-67
O
O
O
O
H
H
OMe MeO
H
R
H
OMe
I. (5R,5aR,8aR,9S)-9-[(4,6-O-(R)-ethylidene--D-glucopyranosyl)oxy]-5-(3,4,5-trimethoxy-
phenyl)-5,8,8a,9-tetrahydroisobenzofuro[5,6-f][1,3]benzodioxol-6(5aH)-one (4-O-
methylethylidene-lignan P),
O
O
O
Ar
O
H
H H
H
OMe
J. (5R,5aR,8aR,9S)-9-methoxy-5-(4-hydroxy-3,5-dimethoxyphenyl)-5,8,8a,9-tetrahydroiso-
benzofuro[5,6-f][1,3]benzodioxol-6(5aH)-one (4-O-demethyl-1-O-methyl-
epipodophyllotoxin),
H H
H
O
O
O
O
O
O
O
O
H
H H
H
Ar
Ar
H
O
K. 9,9-oxybis[(5R,5aR,8aR,9S)-5-(4-hydroxy-3,5-dimethoxyphenyl)-5,8,8a,9-tetrahydroiso-
benzofuro[5,6-f][1,3]benzodioxol-6(5aH)-one] (di-4-O-demethylepipodophyllotoxin),
O
O
O
Ar
O
H
H H
OH
H
L. (5R,5aR,8aR,9R)-9-hydroxy-5-(4-hydroxy-3,5-dimethoxyphenyl)-5,8,8a,9-tetrahydroiso-
benzofuro[5,6-f][1,3]benzodioxol-6(5aH)-one (4-O-demethylpodophyllotoxin),
O
O
O
O
H
H
OMe MeO
H
OH
H
OMe
M. (5R,5aR,8aR,9R)-9-hydroxy-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydroiso-
benzofuro[5,6-f][1,3]benzodioxol-6(5aH)-one (podophyllotoxin).
The following type chromatograms are given for information and guidance only; they do not form a
mandatory part of the monograph.
16-68
Fig 823.1 Type chromatogram for the test for related substances
Fig 8232 Type chromatogram for the test for the assay.
__________________________________________________________________________________________________________ Ph Eur
16-69
Etynodiol Diacetate
H H
Me
H H
C CH
OAc
H
AcO
C
24
H
32
O
4
384.5 297-76-7
Definition Etynodiol Diacetate is 19-nor-17-pregn-4-en-20-yne-3,17-diyl diacetate. It contains
24
H
32
O
4
substance.
Very slightly soluble in water; freely soluble in chloroform and in ether; soluble in ethanol (96%).
Identification
A. The light absorption, Appendix II B, in the range 220 to 350 nm of the solution obtained in the test
for Light absorption exhibits a maximum only at 236 nm and shoulders at 229 and 243 nm.
etynodiol diacetate (RS 138).
C. Yields the reaction characteristic of acetyl groups, Appendix VI.
Light absorption Dissolve 50 mg in sufficient methanol to produce 50 ml (solution A). To 10 ml of
solution A add 40 ml of methanol and a mixture of 3 ml of hydrochloric acid and 2 ml of water, mix
and boil on a water bath for exactly 10 minutes. Cool, dilute to 100 ml with methanol and dilute
10 ml of the solution to 100 ml with methanol. The absorbance of the resulting solution at the
maximum at 236 nm, Appendix II B, is 0.47 to 0.50, calculated with reference to the dried
substance, using in the reference cell a solution prepared by diluting 1 ml of solution A to 100 ml
with methanol.
Specific optical rotation In a 1% w/v solution in chloroform, 70 to 76, calculated with reference
to the dried substance, Appendix V F.
Conjugated compounds Absorbance of a 0.050% w/v solution in methanol at 236 nm, not more than
0.47, calculated with reference to the dried substance, Appendix II B.
1 g.
Assay Dissolve 0.2 g in 40 ml of tetrahydrofuran, add 10 ml of a 10% w/v solution of silver nitrate and
titrate with 0.1M sodium hydroxide VS, determining the end point potentiometrically. Each ml of 0.1M
sodium hydroxide VS is equivalent to 38.45 mg of C
24
H
32
O
4
.
Storage Etynodiol Diacetate should be kept in a well-closed container and protected from light.
When ethynodiol diacetate is prescribed or demanded, Etynodiol Diacetate shall be dispensed or
supplied.
16-70
Eucalyptus Leaf
Eucalyptus Leaf complies with the requirements of the 3rd edition of the European Pharmacopoeia [1320].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Eucalyptus leaf consists of the whole or cut dried leaves of older branches of Eucalyptus globulus L.
The whole drug contains not less than 20 ml/kg of essential oil and the cut drug not less than
15 ml/kg of essential oil, both calculated with reference to the anhydrous drug.
CHARACTERS
The drug has an aromatic odour of cineole.
IDENTIFICATION
A. The leaves which are mainly greyish-green and relatively thick are elongated, elliptical and slightly
sickle-shaped and usually up to 25 cm in length, and up to 5 cm in width. The petiole is twisted,
strongly wrinkled and is 2 cm to 3 cm, rarely 5 cm, in length. The coriaceous, stiff leaves are entire
and glabrous and have a yellowish-green mid-rib. Lateral veins anastomose near the margin to a
continuous line. The margin is even and somewhat thickened. On both surfaces are minute,
irregularly distributed, warty dark brown spots. Small oil glands may be seen in transmitted light.
chloral hydrate solution R. The powder shows fragments of glabrous lamina with small thick-walled
epidermal cells bearing a thick cuticle, numerous anomocytic stomata (2.8.3) of more than 80 m in
diameter and occasionally groups of brown cork cells, 300 m in diameter and brownish-black in
their centre; fragments of isobilateral mesophyll with two or three layers of palisade parenchyma on
each side and in the centre several layers of spongy mesophyll with elongated cells with the same
orientation as the palisade cells and containing prisms and cluster crystals of calcium oxalate;
fragments of mesophyll containing large schizogenous oil glands.
substance.
Test solution. Shake 0.5 g of the freshly powdered drug (355) with 5 ml of toluene R for 2 min to 3 min
and filter over about 2 g of anhydrous sodium sulphate R.
Reference solution. Dissolve 50 l of cineole R in toluene R and dilute to 5 ml with the same solvent.
mixture of 10 volumes of ethyl acetate R and 90 volumes of toluene R. Allow the plate to dry in air and
spray the plate with anisaldehyde solution R. Examine in daylight while heating at 100C to 105C for
5 min to 10 min. The chromatogram obtained with the reference solution shows in the middle a zone
corresponding to cineole. The main zone in the chromatogram obtained with the test solution is
similar in position and colour to the zone corresponding to cineole in the chromatogram obtained
with the reference solution, it also shows an intense violet zone (hydrocarbons) near the solvent front
and there may also be other fainter zones.
TESTS
Foreign matter (2.8.2). Not more than 3 per cent of dark and brown leaves, not more than 5 per
cent of stems and not more than 2 per cent of other foreign matter. Cordate or ovate sessile leaves of
young branches, with numerous glands on both sides, visible as points in transmitted light, are not
present. Determine by using 30 g of the drug.
Water (2.2.13). Not more than 10.0 per cent, determined on 20.0 g of powdered drug (355) by
distillation.
ASSAY
Carry out the determination of essential oil in vegetable drugs (2.8.12). Use 10.0 g of the cut drug
immediately before determination, a 500 ml round-bottomed flask, 200 ml of water R and 100 ml of
glycerol R as the distillation liquid and 0.5 ml of xylene R in the graduated tube. Distil at a rate of
2 ml/min to 3 ml/min for 2 h.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
16-71
Eucalyptus Oil
1/01
Eucalyptus Oil complies with the requirements of the 3rd edition of the European Pharmacopoeia [0390].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Eucalyptus oil is obtained by steam distillation and rectification from the fresh leaves or the fresh
terminal branchlets of various species of Eucalyptus rich in 1,8-cineole. The species mainly used are
Eucalyptus globulus Labill., Eucalyptus fructicetorum F. von Mueller (Eucalyptus polybractea R. T.
Baker) and Eucalyptus smithii R. T. Baker.
CHARACTERS
A colourless or pale-yellow liquid with an aromatic and camphoraceous odour and a pungent and
camphoraceous taste.
IDENTIFICATION
Test solution. Dissolve 0.1 g of the substance to be examined in toluene R and dilute to 10 ml with the
same solvent.
Reference solution. Dissolve 50 l of cineole R in toluene R and dilute to 5 ml with the same solvent.
10 volumes of ethyl acetate R and 90 volumes of toluene R. Allow the plate to dry in air and spray with
anisaldehyde solution R and examine in daylight while heating at 100C to 105C for 5 min to 10 min.
The chromatogram obtained with the reference solution shows in the middle a zone due to cineole.
The chromatogram obtained with the test solution shows a main zone similar in position and colour
to the zone due to cineole in the chromatogram obtained with the reference solution. It also shows an
intense violet zone (hydrocarbons) near the solvent front. Other weaker zones may be present.
obtained with the test solution shows five peaks similar in retention time to the five peaks in the
TESTS
Optical rotation (2.2.7). The angle of optical rotation is 0 to +10.
Solubility in alcohol (2.8.10). It is soluble in 5 volumes of alcohol (70 per cent V/V) R.
Aldehydes Place 10 ml in a glass-stoppered tube 25 mm in diameter and 150 mm long and add 5 ml
of toluene R and 4 ml of alcoholic hydroxylamine solution R. Shake vigorously and titrate immediately
with 0.5M potassium hydroxide in alcohol (60 per cent V/V) until the red colour changes to yellow.
Continue the titration with shaking; the end-point is reached when the pure yellow colour of the
indicator is permanent in the lower layer after shaking vigorously for 2 min and allowing separation to
take place. The reaction is complete in about 15 min. Repeat the titration using a further 10 ml of the
substance to be examined and, as a reference solution for the end-point, the titrated liquid from the
first determination to which has been added 0.5 ml of 0.5M potassium hydroxide in alcohol (60 per cent
V/V). Not more than 2.0 ml of 0.5M potassium hydroxide in alcohol (60 per cent V/V) is required in the
second titration.
Reference solution. Dissolve 80 l of -pinene R, 10 l of -pinene R, 10 l of -phellandrene R, 10 l of
limonene R, 0.8 ml of cineole R and 10 ml of camphor R in 10 ml of acetone R.
16-72
of 5C per minute to 200C and maintaining at 200C for 5 min; maintaining the temperature of the
injection port and that of the detector at 220C.
Inject about 0.5 l of the reference solution. When the chromatogram is recorded in the prescribed
conditions the components elute in the order indicated in the composition of the reference solution.
Record the retention times of these substances.
The assay is not valid unless: the number of theoretical plates calculated for the peak due to
limonene at 110C is at least 30,000, the resolution between the peaks corresponding to limonene
and cineole is at least 1.5.
Inject about 0.5 l of the test solution. Using the retention times determined from the chromato-
gram obtained with the reference solution, locate the components of the reference solution on the
chromatogram obtained with the test solution.
Determine the percentage content of each of these components by the normalisation procedure.
-pinene: 2 to 8 per cent,
-pinene: less than 0.5 per cent,
-phellandrene: less than 1.5 per cent,
limonene: 4 to 12 per cent,
1,8-cineole: not less than 70 per cent,
camphor: less than 0.1 per cent.
STORAGE
Store in a well-filled, airtight container, protected from heat.
The following chromatogram is given for information and guidance only; it does not form a mandatory part of
the monograph.
4 8 12 16 20 24 28 32 36 40
1
2
4 5
6
1. -pinene 4. limonene
2. -pinene 5. 1,8-cineole
3. -phellandrene 6. camphor
Fig. 03901 Type chromatogram for eucalyptus oil (chromatographic profile test)
__________________________________________________________________________________________________________ Ph Eur
16-73
Eugenol
OMe
OH
H
2
C
C
10
H
12
O
2
164.2 97-53-0
Eugenol complies with the requirements of the 3rd edition of the European Pharmacopoeia [1100]. These
Action and use Local anaesthetic used in dentistry.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Eugenol is 2-methoxy-4-(prop-2-enyl)phenol.
CHARACTERS
A colourless or pale yellow, clear liquid, darkening on exposure to air, with a strong odour of clove,
practically insoluble in water, freely soluble in alcohol (70 per cent V/V, practically insoluble in
glycerol, miscible with acetic acid, with alcohol, with ether, with fatty oils and with methylene
chloride.
IDENTIFICATION
A. It complies with the test for refractive index (see Tests).
obtained with eugenol CRS.
254
plate R.
Test solution. Dissolve 50 l of the substance to be examined in alcohol R and dilute to 25 ml with the
same solvent.
Reference solution. Dissolve 50 l of eugenol CRS in alcohol R and dilute to 25 ml with the same
solvent.
of ethyl acetate R and 90 volumes of toluene R. Dry the plate in a current of cold air and examine in
solution. Spray with anisaldehyde solution R. Heat at 100C to 105C for 10 min. The principal spot
D. Dissolve 0.05 ml in 2 ml of alcohol R and add 0.1 ml of ferric chloride solution R1. A dark green
colour is produced which changes to yellowish-green within 10 min.
TESTS
Dimeric and oligomeric compounds. Dissolve 0.150 g of the substance to be examined in
ethanol R and dilute to 100.0 ml with the same solvent. The absorbance of the solution (2.2.25) at
330 nm is not greater than 0.25.
Related substances. Examine by gas chromatography (2.2.28).
Test solution. Dissolve 1.00 g of the substance to be examined in ethanol R and dilute to 5.0 ml with
the same solvent.
Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with ethanol R.
Reference solution (b). Dissolve 50 mg of vanillin R in 1 ml of the test solution and dilute to 5 ml with
ethanol R.
a fused-silica capillary column 30 m long and 0.25 mm in internal diameter coated with a film of
polymethylphenylsiloxane R (film thickness 0.25 m).
helium for chromatography R as the carrier gas at a flow rate of 1 ml/min.
16-74
Time
(min)
Temperature
(C)
Rate
(C per min)
Comment
227 80280 8 linear
gradient
2747 280 isothermal
Injection
port
250
Detector 280
Inject 1 l of the test solution and 1 l each of reference solutions (a) and (b). The test is not valid
unless in the chromatogram obtained with reference solution (b), the relative retention time of the
peak due to vanillin is at least 1.1 with respect to the peak due to eugenol. Calculate the percentage
content of related substances from the areas of the peaks in the chromatogram obtained with the test
solution by the normalisation procedure; disregard the solvent peak and any peak with an area less
than 0.05 times that of the principal peak in the chromatogram obtained with reference solution (a).
The content of related substances with a relative retention time greater than 2.0 in respect to the
main peak is not greater than 1.0 per cent; the content of any related substances is not greater than
0.5 per cent; the total content of related substances is not greater than 3.0 per cent.
Hydrocarbons. Dissolve 1 ml in 5 ml of dilute sodium hydroxide solution R and add 30 ml of water R
in a stoppered test-tube. Examined immediately, the solution is yellow and clear (2.2.1).
STORAGE
IMPURITIES
Me
CH
2
H
H
Me
Me
A. (E)-(1R,9S)-4,11,11-trimethyl-8-methylenebicyclo-[7.2.0]undec-4-ene (-caryophyllene),
Me
Me
Me
Me
B. (E,E,E)-2,6,6,9-tetramethylcycloundeca-1,4,8-triene (-humulene, -caryophyllene),
Me
CH
2
H
H
Me
Me
O
H
C. (1R,4R,5S,9S)-4,5-epoxy-4,11,11-trimethyl-8-methylenebicyclo[7.2.0]undecane
(-caryophyllene oxide),
OR
1
R
2
R
3
D. R
1
= H, R
2
= H, R
3
= CH
2
-CH = CH
2
: 4-(prop-2-enyl)phenol,
E. R
1
= CH
3
, R
2
= OCH
3
, R
3
= CH
2
-CH = CH
2
: 1,2-dimethoxy-4-(prop-2-enyl)benzene (eugenol
methylether),
F. R
1
= H, R
2
= OCH
3
, R
3
= CH = CH-CH
3
(cis): 2-methoxy-4-[(Z)-prop-1-enyl]phenol (cis-
isoeugenol),
G. R
1
= H, R
2
= OCH
3
, R
3
= CH = CH-CH
3
(trans): 2-methoxy-4-[(E)-prop-1-enyl]phenol
(trans-isoeugenol),
16-75
H. R
1
= H, R
2
= OCH
3
, R
3
= CHO: 4-hydroxy-3-methoxy-benzaldehyde (vanillin),
I. R
1
= CO-CH
3
, R
2
= OCH
3
, R
3
= CH
2
-CH = CH
2
: 2-methoxy-4-(prop-2-enyl)phenyl acetate
(acetyleugenol),
J. R
1
= H, R
2
= OCH
3
, R
3
= CO-CH = CH
2
: 1-(4-hydroxy-3-methoxyphenyl)prop-2-enone,
K. R1 = H, R
2
= OCH
3
, R
3
= CH = CH-CHO: (E)-3-(4-hydroxy-3-methoxyphenyl)prop-2-enal (trans-
coniferyl aldehyde),
H
2
C
O
Me
OH
OMe
L. 2-methoxy-4-[3-methyl-5-(prop-2-enyl)-2,3-dihydro-1-benzofuran-2-yl]phenol
(dehydrodiisoeugenol),
H
2
C
OH
OMe
HO
MeO CH
2
M. 3,3-dimethoxy-5,5-di(prop-2-enyl)biphenyl-2,2-diol,
N. O. two further unknown dimeric compounds,
Me
P. toluene.
__________________________________________________________________________________________________________ Ph Eur
16-76
Famotidine
S
N
S N
N
NH
2
H
2
N
NH
2
S
NH
2
O O
C
8
H
15
N
7
O
2
S
3
337.4 76824-35-6
Famotidine complies with the requirements of the 3rd edition of the European Pharmacopoeia [1012]. These
2
Preparation
Famotidine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Famotidine contains not less than 98.5 per cent and not more than the equivalent of 101.5 per cent
of N
2
-(aminosulphonyl)-3-[[[2-[(diaminomethylene)amino]thiazol-4-yl]methyl]thio]propanamidine,
CHARACTERS
A white or yellowish-white, crystalline powder or crystals, very slightly soluble in water, freely soluble
in dimethylformamide and in glacial acetic acid, slightly soluble in methanol, very slightly soluble in
ethanol, practically insoluble in ether and in ethyl acetate. It dissolves in dilute mineral acids.
It exists in two forms which melt at about 163C and 169C, with decomposition.
IDENTIFICATION
A. Dissolve 25.0 mg in buffer solution pH 2.5 R1 and dilute to 100.0 ml with the same solution. Dilute
10.0 ml of this solution to 100.0 ml with buffer solution pH 2.5 R1. Examined between 230 nm and
350 nm (2.2.25), the solution shows one absorption maximum, at 265 nm. The specific absorbance
at the maximum is 297 to 315, calculated with reference to the dried substance.
obtained with famotidine CRS. Examine the substances prepared as discs. If the spectra obtained
show differences, suspend 0.10 g of the substance to be examined and the reference substance
separately in 5 ml of water R. Heat to boiling and allow to cool, scratching the wall of the tube with a
glass rod to initiate crystallisation. Filter, wash the crystals with 2 ml of iced water R and dry in an
oven at 80C at a pressure not exceeding 670 Pa for 1 h. Record new spectra using the residues.
the chromatogram obtained with reference solution (f).
D. Dissolve with shaking about 5 mg of the substance to be examined and about 3 mg of p-phenylene-
diamine dihydrochloride R in 5 ml of a 10 g/l solution of hydrochloric acid R. Add about 0.1 g of zinc
powder R, mix and allow to stand for 2 min. Add 5 ml of ferric ammonium sulphate solution R6 and
mix. A blue or violet-blue colour develops.
TESTS
Appearance of solution Dissolve 0.20 g in a 50 g/l solution of hydrochloric acid R, heating to 40C if
necessary, and dilute to 20 ml with the same acid. The solution is clear (2.2.1) and not more
7
(Method II, 2.2.2).
suitable silica gel (5 m) containing a fluorescent indicator having an optimal intensity at 254 nm.
Test solution (a). Dissolve 0.20 g of the substance to be examined in glacial acetic acid R and dilute to
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with glacial acetic acid R.
Reference solution (a). Dilute 3 ml of test solution (b) to 100 ml with glacial acetic acid R.
Reference solution (b). Dilute 1 ml of test solution (b) to 100 ml with glacial acetic acid R.
16-77
Reference solution (c). Dilute 5 ml of reference solution (b) to 10 ml with glacial acetic acid R.
Reference solution (d). Dissolve 4 mg of famotidine impurity A CRS in glacial acetic acid R and dilute to
10 ml with the same acid. Dilute 1 ml of this solution to 10 ml with glacial acetic acid R.
Reference solution (e). Dissolve 4 mg of famotidine impurity B CRS in glacial acetic acid R and dilute to
10 ml with the same acid. Dilute 1 ml of this solution to 10 ml with reference solution (a).
Reference solution (f). Dissolve 20 mg of famotidine CRS in glacial acetic acid R and dilute to 10 ml with
the same acid.
Apply separately to the plate 5 l of each solution in aliquots of 1 l, drying with a current of
nitrogen R after application of each aliquot. Place the plate in a desiccator for 2 h and develop over a
path of 8 cm using a freshly prepared mixture of 2 volumes of concentrated ammonia R, 20 volumes of
toluene R, 25 volumes of methanol R and 40 volumes of ethyl acetate R. Allow the plate to dry in air
until the odour of the solvent is no longer perceptible and examine in ultraviolet light at 254 nm. In
the chromatogram obtained with test solution (a): any spot corresponding to famotidine impurity A is
not more intense than the principal spot in the chromatogram obtained with reference solution (d)
(0.2 per cent); any spot, apart from the principal spot and any spot corresponding to famotidine
impurity A, is not more intense than the principal spot in the chromatogram obtained with reference
solution (a) (0.3 per cent); and not more than three such spots are more intense than the principal
spot in the chromatogram obtained with reference solution (b) (0.1 per cent). The test is not valid
unless the chromatogram obtained with reference solution (e) shows two clearly separated spots and
the chromatogram obtained with reference solution (c) shows a visible spot.
80C at a pressure not exceeding 670 Pa for 5 h.
ASSAY
8
H
15
N
7
O
2
S
3
.
STORAGE
IMPURITIES
S
N
S NH
N
NH
2
H
2
N
NH
2
A. 3-[[[2-[(diaminomethylene)amino]thiazol-4-yl]methyl]thio]propanamidine,
S
N
N
N
H
N
S
O O
S S
N
S
N N
NH
2
NH
2
NH
2
H
2
N
B. 3,5-bis[2-[[[2-[(diaminomethylene)amino]thiazol-4-yl]methyl]thio]-ethyl]-4H-1,2,4,6-
thiatriazine1,1-dioxide,
S S
N
S NH
N
NH
2
H
2
N
O
NH
2
O O
C. N-(aminosulphonyl)-3-[[[2-[(diaminomethylene)amino]thiazol-4-yl]methyl]-
thio]propanamide,
S
N
S NH
2
N
NH
2
H
2
N
O
D. 3-[[[2-[(diaminomethylene)amino]thiazol-4-yl]methyl]thio]propanamide.
__________________________________________________________________________________________________________ Ph Eur
17-1
Hard Fat
Hard Fat complies with the requirements of the 3rd edition of the European Pharmacopoeia [0462]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hard fat consists of a mixture of triglycerides, diglycerides and monoglycerides, which may be
obtained either by esterification of fatty acids of natural origin with glycerol or by transesterification
of natural fats. Each type of hard fat is characterised by its melting point, its hydroxyl value and its
saponification value. It contains no additives.
CHARACTERS
A white or almost white, waxy, brittle mass, practically insoluble in water, freely soluble in ether,
slightly soluble in ethanol. When heated to 50C, it melts giving a colourless or slightly yellowish
liquid.
IDENTIFICATION
Test solution. Dissolve 1.0 g of the substance to be examined in ethylene chloride R and dilute to 10 ml
Apply to the plate 2 l of the test solution. Develop over a path of 12 cm with a mixture of 10
volumes of ether R and 90 volumes of ethylene chloride R. Allow the plate to dry in air and expose to
iodine vapour until the spots appear. Examine in daylight. The chromatogram shows a spot with
an R
f
value of about 0.6, corresponding to triglycerides (R
st
1) and spots corresponding to 1,3-di-
glycerides (R
st
0.5) and to 1,2-di-glycerides (R
st
0.3). A spot corresponding to 1-monoglycerides may
also be visible (R
st
0.05).
TESTS
Alkaline impurities Dissolve 2.00 g in a mixture of 1.5 ml of alcohol R and 3.0 ml of ether R. Add
0.05 ml of bromophenol blue solution R. Not more than 0.15 ml of 0.01M hydrochloric acid is required to
change the colour of the indicator to yellow.
Melting point (2.2.15). 30C to 45C; the melting point does not differ by more than 2C from the
nominal value. Introduce the melted substance into the capillary tube and allow to stand at a
temperature below 10C for 24 h.
Acid value (2.5.1). Not more than 0.5. Dissolve 5.0 g in 50 ml of the prescribed mixture of solvents.
Hydroxyl value (2.5.3, Method A). The hydroxyl value is not more than 50 and does not differ by
more than 5 units from the nominal value. If the nominal value is less than 5, the hydroxyl value is
not more than 5.
Iodine value (2.5.4). Not more than 3.
Saponification value (2.5.6). 210 to 260, determined on 2.0 g. The saponification value does not
differ by more than 5 per cent from the nominal value.
STORAGE
Store protected from light and heat.
LABELLING
The label states:
the nominal melting point,
the nominal hydroxyl value,
the nominal saponification value.
__________________________________________________________________________________________________________ Ph Eur
17-2
Felbinac
COOH
C
14
H
12
O
2
212.3 5728-52-9
Definition Felbinac is biphenyl-4-ylacetic acid. It contains not less than 98.0% and not more than
101.0% of C
14
H
12
O
2
Characteristics A fine white, crystalline powder.
Practically insoluble in water; sparingly soluble in absolute ethanol; freely soluble in
dimethylformamide.
spectrum of felbinac (RS 372).
Clarity and colour of solution A 1.0% w/v solution in acetone is clear, Appendix IV A, and
colourless, Appendix IV B, Method II.
Melting point 163 to 166, Appendix V A, Method I.
Chlorides Dissolve 1.0 g of the substance being examined in 40 ml of acetone, add 6 ml of 10% v/v
nitric acid and sufficient water to produce 50 ml and mix. Pour 15 ml of the mixture as a single
addition into 1 ml of 0.1M silver nitrate solution and allow to stand for 5 minutes protected from light.
When viewed transversely against a black background any opalescence produced is not more intense
than that obtained by treating 15 ml of a mixture of 1.5 ml of 0.002M hydrochloric acid, 40 ml of
acetone, 6 ml of 10% v/v nitric acid and sufficient water to produce 50 ml in the same manner
(0.011%).
Sulphates Dissolve 1.5 g of the substance being examined in 40 ml of dimethylformamide, add 1 ml
of 10% v/v hydrochloric acid and sufficient dimethylformamide to produce 50 ml and mix. To 15 ml of
this solution add 2 ml of a 12% w/v solution of barium chloride solution and allow to stand for 5
minutes. Any opalescence produced is not more intense than that of a standard prepared in the same
manner but using 2 ml of 0.001M sulphuric acid in place of the substance being examined (0.013%).
following solutions. Protect the solutions from light and inject within 20 minutes of preparation.
Solution (1) contains 1.0% w/v of the substance being examined in methanol. For solution (2) dilute
1 volume of solution (1) to 100 volumes with the mobile phase and further dilute 1 volume of this
solution to 10 volumes with the same solvent. Solution (3) contains 0.001% w/v of 4-acetylbiphenyl
and 0.001% w/v of biphenyl in methanol. Solution (4) contains 0.001% w/v of the substance being
examined and 0.001% w/v of o-phenylbenzoic acid in the mobile phase.
(10 cm 4.6 mm) packed with stationary phase C (10 m) (Partisil ODS3 is suitable), (b) as the
mobile phase with a flow rate of 2 ml per minute a mixture of 45 volumes of a 0.1% v/v solution of
glacial acetic acid and 55 volumes of methanol and (c) a detection wavelength of 254 nm.
For solution (1) allow the chromatography to proceed for at least twice the retention time of the
principal peak. In the chromatogram obtained with solution (1) the area of any peaks corresponding
to 4-acetylbiphenyl or biphenyl is not greater than the area of the corresponding peak in the chroma-
togram obtained with solution (3) (0.1% of each) and the area of any other secondary peak is not
greater the area of the principal peak in the chromatogram obtained with solution (2) (0.1%).
Calculate the content of 4-acetylbiphenyl and biphenyl using the peak areas in the chromatogram
obtained with solution (3) and the nominal content of any other impurity from the area of the peak in
the chromatogram obtained with solution (2); the sum of the contents of the impurities is not greater
than 0.5%.
Loss on drying When dried for 3 hours at 105, loses not more than 0.5% of its weight. Use 1 g.
Assay Carry out the method for liquid chromatography, Appendix III D, using solutions in the mobile
phase containing (1) 0.0012% w/v of the substance being examined, (2) 0.0012% w/v of felbinac
BPCRS and (3) 0.0012% w/v of felbinac BPCRS and 0.0012% w/v of o-phenylbenzoic acid.
17-3
(25 cm 4.6 mm) packed with stationary phase C (10 m) (Partisil ODS3 is suitable), (b) as the
mobile phase with a flow rate of 1 ml per minute a mixture of 40 volumes of a 0.1% v/v solution of
glacial acetic acid and 60 volumes of methanol and (c) a detection wavelength of 254 nm.
14
H
12
O
2
in the substance being examined using the declared content of
C
14
H
12
O
2
in felbinac BPCRS.
Preparations
Felbinac Cutaneous Foam
Felbinac Gel
IMPURITIES
O
CH
3
A. 4-acetyl biphenyl,
B. biphenyl,
O
N
S
H
C. (2-(1,1-biphenyl)-4-yl-1-thioxoethyl)morpholine
17-4
Felodipine
N Me Me
COOEt MeOOC
Cl
H
Cl
H
and enantiomer
C
18
H
19
Cl
2
NO
4
384.3 72509-76-3
Felodipine complies with the requirements of the 3rd edition of the European Pharmacopoeia [1013]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Felodipine contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of
ethyl methyl (RS)-4-(2,3-dichlorophenyl)-1,4-dihydro-2,6-dimethylpyridine-3,5-dicarboxylate,
CHARACTERS
A white or light yellow, crystalline powder, practically insoluble in water, freely soluble in acetone, in
ethanol, in methanol and in methylene chloride.
IDENTIFICATION
A. Dissolve 50 mg in methanol R and dilute to 100 ml with the same solvent. Dilute 3 ml to 100 ml
with methanol R. Examined between 220 nm and 400 nm (2.2.25), the solution shows two absorption
maxima, at 238 nm and 361 nm. The ratio of the absorbance measured at the maximum at 361 nm
to that measured at the maximum at 238 nm is 0.34 to 0.36.
obtained with felodipine CRS. Examine the substances prepared as discs.
the same solvent.
Reference solution (a). Dissolve 10 mg of felodipine CRS in methanol R and dilute to 10 ml with the
same solvent.
Reference solution (b). Dissolve 5 mg of nifedipine CRS in reference solution (a) and dilute to 5 ml with
the same solution.
40 volumes of ethyl acetate R and 60 volumes of cyclohexane R. Allow the plate to dry in air and
solution is similar in position, fluorescence and size to the spot in the chromatogram obtained with
D. Dissolve 150 mg in a mixture of 25 ml of 2-methyl-2-propanol R and 25 ml of perchloric acid
solution R. Add 10 ml of 0.1M cerium sulphate, allow to stand for 15 min, add 3.5 ml of strong sodium
hydroxide solution R and neutralise with dilute sodium hydroxide solution R. Shake with 25 ml of
methylene chloride R. Evaporate the lower layer to dryness on a water-bath under nitrogen (the residue
is also used in the test for related substances). Dissolve about 20 mg of the residue in methanol R and
dilute to 50 ml with the same solvent. Dilute 2 ml to 50 ml with methanol R. Examined between
220 nm and 400 nm (2.2.25), the solution shows an absorption maximum at 273 nm.
17-5
TESTS
Absorbance (2.2.25). The absorbance of solution S at 440 nm is not greater than 0.10.
Reference solution (c). Dissolve 50.0 mg of the residue obtained in identification test D (impurity A)
and 25.0 mg of felodipine CRS in the mobile phase and dilute to 50.0 ml with the mobile phase.
Dilute 1.0 ml to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the
mobile phase.
a stainless steel column 0.125 m to 0.15 m long and 4 mm in internal diameter packed with
as mobile phase at a flow rate of 1 ml per minute a mixture of 20 volumes of methanol R, 40
volumes of acetonitrile R and 40 volumes of a phosphate buffer solution pH 3.0, containing
0.8 g/l of phosphoric acid R and 8 g/l of sodium dihydrogen phosphate R,
two peaks are not less than 20 per cent of the full scale of the recorder. The test is not valid unless the
resolution between the first peak (impurity A) and the second peak (felodipine) is at least 2.5. Two
other peaks may be observed corresponding to impurity B and impurity C. The substances elute in
the following order: impurity B, impurity A, felodipine and impurity C.
Continue the chromatography for twice the retention time of felodipine, which is about 12 min. In
the chromatogram obtained with the test solution: the sum of the areas of any peaks corresponding to
impurity B and impurity C is not greater than the area of the principal peak in the chromatogram
obtained with reference solution (a) (1.0 per cent); the area of any peak apart from the principal peak
and any peaks corresponding to impurity B and impurity C is not greater than the area of the prin-
cipal peak in the chromatogram obtained with reference solution (b) (0.1 per cent) and the sum of
the areas of such peaks is not greater than three times the area of the principal peak in the chromato-
gram obtained with reference solution (b) (0.3 per cent). Disregard any peak with an area less than
0.2 times that of the principal peak in the chromatogram obtained with reference solution (b).
100C to 105C for 3 h.
ASSAY
Dissolve 0.160 g in a mixture of 25 ml of 2-methyl-2-propanol R and 25 ml of perchloric acid solution R.
Add 0.05 ml of ferroin R. Titrate with 0.1M cerium sulphate until the pink colour disappears. Titrate
slowly towards the end of the titration.
18
H
19
Cl
2
NO
4
.
STORAGE
IMPURITIES
Cl
Cl
N Me Me
COOEt MeOOC
A. ethyl methyl 4-(2,3-dichlorophenyl)-2,6-dimethylpyridine-3,5-dicarboxylate,
17-6
N Me Me
COOMe MeOOC
Cl
H
Cl
B. dimethyl 4-(2,3-dichlorophenyl)-1,4-dihydro-2,6-dimethylpyridine-3,5-dicarboxylate,
N Me Me
COOEt EtOOC
Cl
H
Cl
C. diethyl 4-(2,3-dichlorophenyl)-1,4-dihydro-2,6-dimethylpyridine-3,5-dicarboxylate.
__________________________________________________________________________________________________________ Ph Eur
17-7
Fenbufen
COOH
O
C
16
H
14
O
3
254.3 36330-85-5
Fenbufen complies with the requirements of the 3rd edition of the European Pharmacopoeia [1209]. These
Preparations
Fenbufen Capsules
Fenbufen Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Fenbufen contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of
4-(biphenyl-4-yl)-4-oxobutanoic acid, calculated with reference to the dried substance.
CHARACTERS
A white, fine, crystalline powder, very slightly soluble in water, slightly soluble in acetone, in alcohol
IDENTIFICATION
obtained with fenbufen CRS.
Reference solution (a). Dissolve 10 mg of fenbufen CRS in methylene chloride R and dilute to 10 ml with
the same solvent.
Reference solution (b). Dissolve 10 mg of ketoprofen CRS in methylene chloride R and dilute to 10 ml
with the same solvent. To 5 ml of the solution, add 5 ml of reference solution (a).
5 volumes of anhydrous acetic acid R, 25 volumes of ethyl acetate R and 75 volumes of hexane R. Allow
the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromato-
togram obtained with reference solution (a). The test is not valid unless the chromatogram obtained
TESTS
dimethylformamide R and 60 volumes of mobile phase A and dilute to 10.0 ml with the same mixture
of solvents.
dimethylformamide R and 60 volumes of mobile phase A. Dilute 1.0 ml of the solution to 10.0 ml with
a mixture of 40 volumes of dimethylformamide R and 60 volumes of mobile phase A.
Reference solution (b). Dissolve 25 mg of fenbufen CRS and 6 mg of ketoprofen CRS in a mixture of 40
volumes of dimethylformamide R and 60 volumes of mobile phase A and dilute to 10 ml with the same
17-8
mixture of solvents. Dilute 1 ml of the solution to 100 ml with a mixture of 40 volumes of
dimethylformamide R and 60 volumes of mobile phase A.
as mobile phase at a flow rate of 2 ml per minute the following solutions:
Mobile phase A. 32 volumes of acetonitrile R and 68 volumes of a mixture of 1 volume of glacial
acetic acid R and 55 volumes of water R,
Mobile phase B. 45 volumes of acetonitrile R and 55 volumes of a mixture of 1 volume of glacial
acetic acid R and 55 volumes of water R,
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
015 100 0 isocratic
(a) is at least 50 per cent of the full scale of the recorder. The test is not valid unless, in the chroma-
togram obtained with reference solution (b), the resolution between the peaks corresponding to
ketoprofen and to fenbufen is at least 5.0.
Inject 20 l of the test solution. In the chromatogram obtained with the test solution: the area of
chromatogram obtained with reference solution (a) (0.1 per cent); the sum of the areas of any peaks,
chromatogram obtained with reference solution (a) (0.5 per cent). Disregard any peak due to the
solvent and any peak with an area less than 0.2 times that of the principal peak in the chromatogram
100C to 105C for 3 h.
ASSAY
Dissolve 0.200 g in 75 ml of acetone R previously neutralised with phenolphthalein solution R1 and add
50 ml of water R. Add 0.2 ml of phenolphthalein solution R1 and titrate with 0.1M sodium hydroxide.
Carry out a blank titration.
16
H
14
O
3
.
STORAGE
IMPURITIES
COOH
O
Cl
A. 3-(4-chlorophenyl)-3-oxopropanoic acid,
R
R'
B. R = CO-CH = CH-CO
2
H, R = H: 4-(biphenyl-4-yl)-4-oxobut-2-enoic acid,
C. R = R = H: biphenyl,
D. R = CO-CH
2
-CH
2
-CO
2
H, R = OH: 4-(4-hydroxybiphenyl-4-yl)-4-oxobutanoic acid.
__________________________________________________________________________________________________________ Ph Eur
17-9
Bitter Fennel
Bitter Fennel complies with the requirements of the 3rd edition of the European Pharmacopoeia [0824]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Bitter fennel consists of the dry, cremocarps and mericarps of Foeniculum vulgare Miller sp. vulgare
var. vulgare. It contains not less than 40 ml/kg of essential oil, calculated with reference to the
anhydrous drug. The oil contains not less than 60.0 per cent of anethole and not less than 15.0 per
cent of fenchone.
CHARACTERS
Bitter fennel is greenish-brown, brown or green.
IDENTIFICATION
A. The fruit of bitter fennel is a cremocarp, of almost cylindrical shape with a rounded base and a
narrower summit crowned with a large stylopod. It is generally 3 mm to 12 mm long and 3 mm to
4 mm wide. The mericarps, usually free, are glabrous. Each bears five prominent slightly carenated
ridges. When cut transversely, four vittae on the dorsal surface and two on the commissural surface
may be seen with a lens.
B. Reduce to a powder (355). The powder is greyish-brown to greyish-yellow. Examine under a
yellow fragments of wide secretory canals, often made up of yellowish-brown-walled polygonal
secretory cells, frequently associated with a layer of thin-walled transversely elongated cells 2 m to
9 m wide, having a parquetry arrangement; reticulate parenchyma of the mesocarp; numerous fibre
bundles from the ridges, often accompanied by narrow spiral vessels; very numerous endosperm
fragments containing aleurone grains and very small calcium oxalate microrosette crystals, as well as
some fibre bundles from the carpophore.
254
Test solution. Shake 0.3 g of the freshly powdered drug (1400) with 5.0 ml of methylene chloride R for
15 min. Filter and carefully evaporate the filtrate to dryness on a water-bath at 60C. Dissolve the
residue in 0.5 ml of toluene R.
Reference solution. Dissolve 50 l of anethole R and 10 l of fenchone R in 5.0 ml of hexane R.
Apply separately to the plate, as bands 20 mm by 3 mm, 10 l of each solution. Develop over a path
of 10 cm using a mixture of 20 volumes of hexane R and 80 volumes of toluene R. Allow the plate to
dry in air and examine in ultraviolet light at 254 nm. The chromatograms show in the central part a
quenching zone corresponding to anethole. Spray the plate with sulphuric acid R and heat at 140C
for 5 min to 10 min until a yellow zone corresponding to fenchone appears in the lower third of the
chromatograms. Anethole appears as a violet band in the central part. The chromatogram obtained
with the test solution also shows a reddish-brown zone in its upper third (terpenes).
TESTS
Estragole The essential oil obtained in the assay contains not more than 5.0 per cent of estragole.
Operate as described in the assay for anethole and fenchone, using the following reference solution.
Reference solution. Dissolve 5 mg of estragole R in 0.5 ml of xylene R.
Determine the content of estragole by normalisation.
Foreign matter (2.8.2). Not more than 1.5 per cent of peduncles and not more than 1.5 per cent of
other foreign matter.
drug (710).
ASSAY
Essential oil Carry out the determination of essential oils in vegetable drugs (2.8.12). Use a 500 ml
round-bottomed flask and 200 ml of water R as the distillation liquid. Reduce the drug to a coarse
powder (1400) and immediately use 5.0 g for the determination. Introduce 0.50 ml of xylene R in the
graduated tube. Distil at a rate of 2 ml/min to 3 ml/min for 2 h.
Anethole and fenchone Examine by gas chromatography (2.2.28).
17-10
Test solution. Dilute the mixture of essential oil and xylene R obtained in the determination of
essential oil to 5.0 ml with xylene R and by rinsing the apparatus.
Reference solution. Dissolve 5 mg of fenchone R and 5 mg of anethole R in 0.5 ml of xylene R.
a capillary column 30 m to 60 m long and 0.3 mm in internal diameter coated with macrogol
20,000 R,
nitrogen for chromatography R as the carrier gas at a flow rate of 0.40 ml/min and split at a ratio of
1 to 200,
maintaining the temperature of the column at 60C for 4 min, then raising the temperature linearly at
a rate of 5C per minute to 170C and maintaining at 170C for 15 min and maintaining the
temperature of the injection port at 220C and that of the detector at 270C.
Inject 1 l of the reference solution. Identify and record the retention times of the components,
which are eluted in the order indicated in the composition of the reference solution.
Inject 1 l of the test solution. Determine the contents of anethole and fenchone by normalisation.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
17-11
Sweet Fennel
Sweet Fennel complies with the requirements of the 3rd edition of the European Pharmacopoeia [0825]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sweet fennel consists of the dry, cremocarps and mericarps of Foeniculum vulgare Miller sp. vulgare
var. dulce (Miller) Thellung. It contains not less than 20 ml/kg of essential oil, calculated with
reference to the anhydrous drug. The oil contains not less than 80.0 per cent of anethole.
CHARACTERS
Sweet fennel is pale green or pale yellowish-brown.
IDENTIFICATION
A. The fruit of sweet fennel is a cremocarp of almost cylindrical shape with a rounded base and a
narrowed summit crowned with a large stylopod. It is generally 3 mm to 12 mm long and 3 mm to
4 mm wide. The mericarps, usually free, are glabrous. Each bears five prominent slightly carenated
ridges. When cut transversely, four vittae on the dorsal surface and two on the commissural surface
may be seen with a lens.
B. Reduce to a powder (355). The powder is greyish-brown to greyish-yellow. Examine under a
yellow fragments of wide secretory canals, often made up of yellowish-brown-walled polygonal
secretory cells, frequently associated with a layer of thin-walled transversely elongated cells 2 m to
9 m wide, having a parquetry arrangement; reticulate parenchyma of the mesocarp; numerous fibre
bundles from the ridges, often accompanied by narrow spiral vessels; very numerous endosperm
fragments containing aleurone grains and very small calcium oxalate microrosette crystals, as well as
some fibre bundles from the carpophore.
254
Test solution. Shake 0.3 g of the freshly powdered drug (1400) with 5.0 ml of methylene chloride R for
15 min. Filter and carefully evaporate the filtrate to dryness on a water-bath at 60C. Dissolve the
residue in 0.5 ml of toluene R.
Reference solution. Dissolve 60 l of anethole R in 5.0 ml of hexane R.
of 10 cm using a mixture of 20 volumes of hexane R and 80 volumes of toluene R. Allow the plate to
dry in air and examine in ultraviolet light at 254 nm. The chromatograms show in the central part a
quenching zone corresponding to anethole. Spray the plate with sulphuric acid R and heat at 140C
for 5 min. Examine in daylight. The chromatograms show in the central part a violet band corres-
ponding to anethole. The chromatogram obtained with the test solution also shows a reddish-brown
zone in its upper third (terpenes).
TESTS
Estragole and fenchone The essential oil obtained in the assay contains not more than 10.0 per
cent of estragole and not more than 7.5 per cent of fenchone.
Proceed as described in the assay for anethole, using the following reference solution.
Reference solution. Dissolve 5 mg of estragole R and 5 mg of fenchone R in 0.5 ml of xylene R.
Determine the contents of estragole and fenchone by normalisation.
Foreign matter (2.8.2). Not more than 1.5 per cent of peduncles and not more than 1.5 per cent of
other foreign matter.
drug (710).
ASSAY
Essential oil Carry out the determination of essential oils in vegetable drugs (2.8.12). Use a 500 ml
round-bottomed flask and 200 ml of water R as the distillation liquid. Reduce the drug to a coarse
powder (1400) and immediately use 10.0 g for the determination. Introduce 0.50 ml of xylene R in
the graduated tube. Distil at a rate of 2 ml/min to 3 ml/min for 2 h.
Anethole Examine by gas chromatography (2.2.28).
17-12
Test solution. Dilute the mixture of essential oil and xylene R obtained in the determination of the
essential oil to 5.0 ml with xylene R and by rinsing the apparatus.
Reference solution. Dissolve 5 mg of anethole R in 0.5 ml of xylene R.
a capillary column 30 m to 60 m long and 0.3 mm in internal diameter coated with macrogol
20,000 R,
nitrogen for chromatography R as the carrier gas at a flow rate of 0.40 ml/min and split at a ratio of
1 to 200,
maintaining the temperature of the column at 60C for 4 min, then raising the temperature linearly at
a rate of 5C per minute to 170C and maintaining at 170C for 15 min and maintaining the
temperature of the injection port at 220C and that of the detector at 270C.
Inject 1 l of each solution. Determine the content of anethole by normalisation.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
17-13
Fenofibrate
Cl
O
O COOPr
i
Me Me
C
20
H
21
ClO
4
360.8 49562-28-9
Fenofibrate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1322]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Fenofibrate contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent
of 1-methylethyl 2-[4-(4-chlorobenzoyl)phenoxy]-2-methylpropanoate, calculated with reference to
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water, very soluble in methylene
chloride, slightly soluble in alcohol.
IDENTIFICATION
obtained with fenofibrate CRS. Examine the substances prepared as discs.
TESTS
Solution S To 5.0 g, add 25 ml of distilled water R and heat at 50C for 10 min. Cool and dilute to
50.0 ml with the same solvent. Filter. The resulting filtrate is solution S.
Appearance of solution Dissolve 0.50 g in acetone R and dilute to 10.0 ml with the same solvent.
6
(Method II,
2.2.2).
Acidity Dissolve 1.0 g in 50 ml of alcohol R previously neutralised using 0.2 ml of phenolphthalein
solution R1. Not more than 0.2 ml of 0.1M sodium hydroxide is required to change the colour of the
indicator to pink.
peaks in the chromatogram obtained is at least 20 per cent of the full scale of the recorder. When the
chromatograms are recorded in the prescribed conditions, the relative retention times are: impurity A
about 0.34, impurity B about 0.36, impurity C about 0.50, impurity D about 0.65, impurity E about
0.80, impurity F about 0.85 and impurity G about 1.35. The test is not valid unless the resolution
between the peaks corresponding to impurity A and impurity B is at least 1.5.
Inject 20 l of reference solution (b) and 20 l of the test solution. Continue the chromatography
of the test solution for twice the retention time of fenofibrate. In the chromatogram obtained with the
test solution, the area of any peak corresponding to impurity A, impurity B or impurity G is not
greater than that of the corresponding peak in the chromatogram obtained with reference solution (b)
(0.1 per cent for impurities A and B and 0.2 per cent for impurity G); the area of any peak, apart
from the principal peak and any peaks corresponding to impurities A, B and G, is not greater than
the area of the peak corresponding to fenofibrate in the chromatogram obtained with reference
solution (b) (0.1 per cent); the sum of the areas of all the peaks, apart from the principal peak, is not
greater than five times the area of the peak corresponding to fenofibrate in the chromatogram
obtained with reference solution (b) (0.5 per cent). Disregard any peak with an area less than 0.1
times that of the peak due to fenofibrate in the chromatogram obtained with reference solution (b).
Halogens (2.4.4). To 5 ml of solution S add 10 ml of distilled water R. The solution complies with
the limit test for chlorides (100 ppm, expressed as chlorides).
17-14
60C.
ASSAY
Reference solution (a). Dissolve 25.0 mg of fenofibrate CRS in the mobile phase and dilute to 25.0 ml
Reference solution (b). Dissolve 10.0 mg of fenofibrate CRS, 10.0 mg of fenofibrate impurity A CRS,
10.0 mg of fenofibrate impurity B CRS and 20.0 mg of fenofibrate impurity G CRS in the mobile phase
and dilute to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 100.0 ml with the
mobile phase.
as mobile phase at a flow rate of 1 ml/min a mixture of 30 volumes of water R acidified to pH 2.5
with concentrated phosphoric acid R and 70 volumes of acetonitrile R,
peaks in the chromatogram is at least 50 per cent of the full scale of the recorder. Inject 5 l of
reference solution (a) six times. The assay is not valid unless the relative standard deviation of the
peak area for fenofibrate is at most 1.0 per cent. Inject 5 l of the test solution and 5 l of reference
solution (a).
STORAGE
IMPURITIES
R
H
O Cl
O
R =
A. (4-chlorophenyl)(4-hydroxyphenyl)methanone,
R COOH
Me Me
B. 2-[4-(4-chlorobenzoyl)phenoxy]-2-methylpropanoic acid (fenofibric acid),
R
H Me
CH
3
O
and enantiomer
C. (3RS)-3-[4-(4-chlorobenzoyl)phenoxy]butan-2-one,
R
Me Me
OCH
3
O
D. methyl 2-[4-(4-chlorobenzoyl)phenoxy]-2-methylpropanoate,
R
Me Me
O
O
CH
3
E. ethyl 2-[4-(4-chlorobenzoyl)phenoxy]-2-methylpropanoate,
R
Me
Me
F. (4-chlorophenyl)[4-[(1-methylethyl)oxy]phenyl]methanone,
17-15
R
Me Me
O
O
O Me
O
Me Me
Me
G. 1-methylethyl 2-[[2-[4-(4-chlorobenzoyl)phenoxy]-2-methylpropanoyl]oxy]-2-
methylpropanoate.
__________________________________________________________________________________________________________ Ph Eur
17-16
Fenoprofen Calcium
and enantiomer
Me H
O
COO
2
Ca
2
+
(C
15
H
13
O
3
)
2
Ca,2H
2
O 558.6 53746-45-5
Definition Fenoprofen Calcium is calcium (RS)-2-(3-phenoxyphenyl)propionate dihydrate. It
contains not less than 97.5% and not more than 101.0% of (C
15
H
13
O
3
)
2
Ca, calculated with refer-
ence to the anhydrous substance.
Slightly soluble in water; soluble in ethanol (96%); slightly soluble in chloroform.
Identification
A. Dissolve 0.1 g in 5 ml of glacial acetic acid and add sufficient methanol to produce 100 ml. Dilute
5 ml of this solution to 50 ml with methanol. The light absorption of the resulting solution, Appendix
II B, in the range 230 to 350 nm exhibits two maxima, at 272 nm and 278 nm, and a shoulder at
266 nm. The absorbance at the maximum at 272 nm is about 0.70 and at the maximum at 278 nm is
about 0.65.
fenoprofen calcium (RS 142).
C. The residue on ignition yields the reactions characteristic of calcium salts, Appendix VI.
0.50% w/v of the substance being examined and (3) 0.04% w/v of fenoprofen calcium and 0.0015%
w/v of 4,4-dimethoxybenzophenone.
(25 cm 4.6 mm) packed with stationary phase C (7 to 8 m) (Zorbax ODS is suitable), (b) a
mixture of 61 volumes of water, 30 volumes of acetonitrile, 7 volumes of tetrahydrofuran and 2 volumes
of glacial acetic acid as the mobile phase with a flow rate of 2 ml per minute and (c) a detection wave-
length of 270 nm. For solution (2) allow the chromatography to proceed for 3 times the retention
time of the peak due to fenoprofen.
The test is not valid unless the resolution factor between the peaks corresponding to fenoprofen and
4,4-dimethoxybenzophenone in the chromatogram obtained with solution (3) is at least 3.0.
twice the area of the peak in the chromatogram obtained with solution (1), not more than one such
peak has an area greater than the area of the peak in the chromatogram obtained with solution (1)
and the sum of the areas of all such peaks is not greater than four times the area of the peak in the
(C
15
H
13
O
3
)
2
Ca.
Storage Fenoprofen Calcium should be kept in a well-closed container.
Preparation
Fenoprofen Tablets
17-17
Fenoterol Hydrobromide
HO N
OH H
Me H
OH
OH
H
,HBr
and enantiomer
C
l7
H
21
NO
4
,HBr 384.3 1944-12-3
Fenoterol Hydrobromide complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Fenoterol Pressurised Inhalation
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Fenoterol hydrobromide contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of (RS)-1-(3,5-dihydroxyphenyl)-2-[(RS)-2-(4-hydroxyphenyl)-1-
methylethylamino]ethanol hydrobromide, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder, soluble in water and in alcohol, practically insoluble in ether.
IDENTIFICATION
A. Dissolve 50.0 mg in dilute hydrochloric acid R1 and dilute to 50.0 ml with the same acid. Dilute
5.0 ml to 50.0 ml with dilute hydrochloric acid R1. Examined between 230 nm and 350 nm (2.2.25),
the solution shows an absorption maximum at 275 nm and a shoulder at about 280 nm. The specific
obtained with fenoterol hydrobromide CRS. Examine the substances prepared as discs.
the same solvent.
Reference solution. Dissolve 10 mg of fenoterol hydrobromide CRS in alcohol R and dilute to 10 ml with
the same solvent.
1.5 volumes of concentrated ammonia R, 10 volumes of water R and 90 volumes of aldehyde-free
methanol R. Allow the plate to dry in air and spray with a 10 g/l solution of potassium permanganate R.
D. Dissolve about 10 mg in a 20 g/l solution of disodium tetraborate R and dilute to 50 ml with the
same solution. Add 1 ml of a 10 g/l solution of aminopyrazolone R, 10 ml of a 2 g/l solution of
potassium ferricyanide R and 10 ml of methylene chloride R. Shake and allow to separate. A reddish-
brown colour develops in the lower layer.
TESTS
solution Y
7
(Method II, 2.2.2).
Phenone The absorbance (2.2.25) of solution S measured at 330 nm is not greater than 0.42
(0.2 per cent).
Diastereoisomers Examine by liquid chromatography (2.2.29).
17-18
the same solvent.
Reference solution. Dissolve 25.0 mg of fenoterol hydrobromide CRS (which contains 4.0 per cent m/m of
the diastereoisomers) in water R and dilute to 10.0 ml with the same solvent.
as mobile phase at a flow rate of 1 ml per minute a solution prepared as follows: to a mixture of
1 volume of a 9 g/l solution of potassium dihydrogen phosphate R and 69 volumes of a 24 g/l
solution of disodium hydrogen phosphate R, previously adjusted to pH 8.5 using phosphoric acid R,
add 30 volumes of methanol R,
a loop injector.
Inject 20 l of the reference solution. When using a recorder, adjust the sensitivity of the system so
that the height of the peak due to the diastereoisomers eluting immediately after the principal peak is
not less than 10 per cent of the full scale of the recorder. The test is not valid unless: the height of the
trough separating the peak due to the diastereoisomers from the principal peak is less than 4 per cent
of full scale of the recorder; the retention time of the principal peak is less than 20 min.
Inject separately 20 l of the test solution and 20 l of the reference solution. In each chromato-
gram measure the height of the peak due to the diastereoisomers by dropping a perpendicular from
the apex of the peak to a line drawn from the trough between the two peaks to the baseline. In the
chromatogram obtained with the test solution, the height of this peak is not greater than the height of
the corresponding peak in the chromatogram obtained with the reference solution (4.0 per cent).
Iron (2.4.9). Dissolve the residue from the test for sulphated ash in 2.5 ml of dilute hydrochloric acid R
and dilute to 10 ml with water R. The solution complies with the limit test for iron (5 ppm).
100C to 105C.
ASSAY
Dissolve 0.600 g in 50 ml of water R and add 5 ml of dilute nitric acid R, 25.0 ml of 0.1M silver nitrate
and 2 ml of ferric ammonium sulphate solution R2. Shake and titrate with 0.1M ammonium thiocyanate
until an orange colour is obtained. Carry out a blank titration.
17
H
22
BrNO
4
.
STORAGE
IMPURITIES
N
OH
HO
OH H
H Me
OH
H
A. (RS)-1-(3,5-dihydroxyphenyl)-2-[(SR)-2-(4-hydroxyphenyl)-1-methylethylamino]ethanol,
N
OH
HO
H Me
OH
O
and enantiomer
,HBr
H
B. 1-(3,5-dihydroxyphenyl)-2-[(RS)-2-(4-hydroxyphenyl)-1-methylethylamino]ethanone
(phenone).
__________________________________________________________________________________________________________ Ph Eur
17-19
Fentanyl
N
N
H
3
C
O
C
22
H
28
N
2
O 336.5 437-38-7
Fentanyl complies with the requirements of the 3rd edition of the European Pharmacopoeia [1210]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Fentanyl contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of
N-phenyl-N-[1-(2-phenylethyl)piperidin-4-yl]propanamide, calculated with reference to the dried
substance.
CHARACTERS
A white or almost white powder, practically insoluble in water, freely soluble in alcohol and in
methanol.
IDENTIFICATION
Examine by infrared absorption spectrophotometry (2.2.24) comparing with the Ph. Eur. reference
spectrum of fentanyl.
If the spectrum obtained in the solid state shows differences, dissolve the substance to be examined
in the minimum volume of ethanol R, evaporate to dryness at room temperature under an air-stream
and record the spectrum again using the residue.
TESTS
Reference solution (a). In order to prepare the in situ degradation compound (fentanyl impurity D),
dissolve 10 mg of the substance to be examined in 10.0 ml of dilute hydrochloric acid R. Heat on a
water-bath under a reflux condenser for 4 h. Neutralise with 10.0 ml of dilute sodium hydroxide R.
Evaporate to dryness on a water-bath. Cool and take up the residue in 10 ml of methanol R. Filter.
as mobile phase at a flow rate of 1.5 ml per minute the following mixtures:
tetrahydrofuran R and 90 volumes of water R,
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
eluent
composition
17-20
conditions, the retention times are: fentanyl, about 10 min and fentanyl impurity D, about 12 min.
The test is not valid unless the resolution between the peaks corresponding to fentanyl and fentanyl
impurity D is at least 8.0. If necessary, adjust the concentration of acetonitrile in the mobile phase or
adjust the time programme for the linear gradient elution.
reference solution (b) (0.5 per cent). Disregard any peak obtained with the blank run and any peak
50C.
ASSAY
ethyl ketone R and titrate with 0.1M perchloric acid, using 0.2 ml of naphtholbenzein solution R as
indicator.
22
H
28
N
2
O.
STORAGE
IMPURITIES
Ar =
H
3
C
O
N
N
Ar
Ar
O
A. N-phenyl-N-[1(2-phenylethyl)piperidin-4-yl]propanamide 1-oxide,
H
3
C
O
N
N
Ar
H
B. N-phenyl-N-(piperidin-4-yl)propanamide,
H
3
C
O
N
N
Ar
Ar
C. N-phenyl-N-[1-(2-phenylethyl)piperidin-4-yl]acetamide,
N
N
Ar
Ar
H
D. N-phenyl-1-(2-phenylethyl)piperidin-4-amine.
__________________________________________________________________________________________________________ Ph Eur
17-21
Fentanyl Citrate
N
N
CH
3
O
,
C COOH HO
CH
2
COOH
CH
2
COOH
C
22
H
28
N
2
O,C
6
H
8
O
7
528.6 990-73-8
Fentanyl Citrate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1103].
Preparation
Fentanyl Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Fentanyl citrate contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of N-phenyl-N-[1-(2-phenylethyl)piperidin-4-yl]propanamide 2-hydroxy-1,2,3-
propanetricarboxylate, calculated with reference to the dried substance.
CHARACTERS
A white or almost white powder, soluble in water, freely soluble in methanol, sparingly soluble in
alcohol.
IDENTIFICATION
spectrum of fentanyl citrate.
TESTS
Appearance of solution Dissolve 0.2 g of the substance to be examined in water R and dilute to
20 ml with the same solvent. The solution is clear (2.2.1) and colourless (Method II, 2.2.2).
Reference solution (a). In order to prepare the in situ degradation compound (impurity D), dissolve
10 mg of the substance to be examined in 10.0 ml of dilute hydrochloric acid R. Heat on a water-bath
under a reflux condenser for 4 h. Neutralise with 10.0 ml of dilute sodium hydroxide R. Evaporate to
dryness on a water-bath. Cool and take up the residue in 10 ml of methanol R. Filter.
conditions:
tetrahydrofuran R and 90 volumes of water R.
Mobile phase B. Acetonitrile R.
17-22
Time
(min)
Mobile
phase A
(per cent V/V)
Mobile
phase B
(per cent V/V)
Comment
eluent composition
conditions, the retention times are: fentanyl, about 10 min; impurity D, about 12 min. The test is not
valid unless the resolution between the peaks corresponding to fentanyl and impurity D is at least 8.0.
If necessary, adjust the concentration of acetonitrile in the mobile phase or adjust the time
reference solution (b) (0.5 per cent). Disregard any peak obtained with the blank run and any peak
with a retention time relative to the principal peak of 0.05 or less and any peak with an area less than
60C.
ASSAY
28
H
36
N
2
O
8
.
STORAGE
IMPURITIES
Ar =
H
3
C
O
N
N
Ar
Ar
O
A. N-phenyl-N-[1(2-phenylethyl)piperidin-4-yl]propanamide 1-oxide,
H
3
C
O
N
N
Ar
H
B. N-phenyl-N-(piperidin-4-yl)propanamide,
H
3
C
O
N
N
Ar
Ar
C. N-phenyl-N-[1-(2-phenylethyl)piperidin-4-yl]acetamide,
17-23
N
N
Ar
Ar
H
D. N-phenyl-1-(2-phenylethyl)piperidin-4-amine
__________________________________________________________________________________________________________ Ph Eur
17-24
Fenticonazole Nitrate
N
N
H
O
Cl Cl S
,HNO
3
and enanti omer
C
24
H
20
Cl
2
N
2
OS,HNO
3
518.4 73151-29-8
Fenticonazole Nitrate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Fenticonazole nitrate contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of (RS)-1-[2-(2,4-dichlorophenyl)-2-[4-(phenylsulphanyl)benzyloxy]ethyl-1H-
imidazole nitrate, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water, freely soluble in methanol
and in dimethylformamide, sparingly soluble in ethanol.
IDENTIFICATION
B. Dissolve 20.0 mg in ethanol R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of this
solution to 10.0 ml with ethanol R. Examined between 230 nm and 350 nm (2.2.25), the solution
shows an absorption maximum at 252 nm, a shoulder at about 270 nm and an absorption minimum
at 236 nm. The specific absorption at the maximum is 260 to 280.
obtained with fenticonazole nitrate CRS.
D. It gives the reaction of nitrates (2.3.1).
TESTS
Reference solution (d). To 5 ml of the test solution add 5.0 mg of fenticonazole impurity D CRS, dissolve
in the mobile phase and dilute to 100.0 ml with the mobile phase. Dilute 2.0 ml of this solution to
30 volumes of a phosphate buffer solution prepared by dissolving 3.40 g of potassium dihydrogen
phosphate R in 900 ml of water R, adjusting to pH 3.0 with phosphoric acid R and diluting to
17-25
peak due to fenticonazole is at least 10 per cent of the full scale of the recorder.
Inject separately 10 l of reference solution (c) and 10 l of reference solution (d). The test is not
valid unless in the chromatogram obtained with reference solution (d), the resolution between the
peak due to fenticonazole impurity D and fenticonazole is at least 2.0. The test is not valid unless in
the chromatogram obtained with reference solution (c) the signal-to-noise ratio is at least five.
Inject separately 10 l of the test solution and 10 l of reference solution (a). Record the chromato-
gram obtained with the test solution for 5.5 times the retention time of the principal peak. In the
chromatogram obtained with the test solution: the area of any peak apart from the principal peak and
the nitric ion peak (which corresponds to the dead volume of the column), is not greater than the
and the sum of the areas of such peaks is not greater than the area of the principal peak in the
chromatogram obtained with reference solution (a) (0.5 per cent). Disregard any peak with an area
less than that of the principal peak in the chromatogram obtained with reference solution (c).
Toluene Not more than 100 ppm, determined by head-space gas chromatography (Method II,
2.2.28).
Test solution. Disperse 0.2 g of the substance to be examined in a 10 ml vial with 5 ml of water R.
Reference solution. Mix 4 mg of toluene R with water R and dilute to 1000 ml. Place 5 ml of this
solution in a 10 ml vial.
a column 25 m long and 0.32 mm in internal diameter coated with a 1.2 m thick layer of
poly(cyanopropyl)(7)phenyl(7)methyl(86)siloxane R,
helium for chromatography R as the carrier gas, split ratio 1:25, column head pressure 40 kPa,
the detector at 220C. Maintain each solution at 90C for 1 h, and transfer onto the column 1 ml of
the vapour phase.
60C.
ASSAY
Dissolve 0.450 g in 50 ml of a mixture of equal volumes of anhydrous acetic acid R and methyl ethyl
ketone R. Titrate with 0.1M perchloric acid, determining the end-point potentiometrically (2.2.20).
24
H
21
Cl
2
N
3
O
4
S.
STORAGE
IMPURITIES
N
N
H
HO
Cl
Cl
and enantiomer
A. (RS)-1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethanol,
N
N
H
O
Cl Cl S
O
and enantiomer
B. (RS)-1-[2-(2,4-dichlorophenyl)-2-[4-(phenylsulphinyl)benzyloxy]ethyl]-1H-imidazole,
17-26
N
N
H
O
Cl Cl
S
O O
and enantiomer
C. (RS)-1-[2-(2,4-dichlorophenyl)-2-[4-(phenylsulphonyl)benzyloxy]ethyl]-1-H-imidazole,
and enantiomer +
NO
3
-
S
N
N
H
HO
Cl
Cl
D. (RS)-1-[2-(2,4-dichlorophenyl)-2-hydroxyethyl]-3-[4-(phenylsulphanyl)benzyl]-
imidazolium nitrate,
and enantiomer
+
NO
3
-
S
N
N
H
Cl
Cl
O
S
E. (RS)-1-[2-(2,4-dichlorophenyl)-2-[4-(phenylsulphanyl)benzyloxy]ethyl]-3-[4-
(phenylsulphonyl)benzyl]imidazolium nitrate.
__________________________________________________________________________________________________________ Ph Eur
17-27
Fenugreek
Fenugreek complies with the requirements of the 3rd edition of the European Pharmacopoeia [1323]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Fenugreek consists of the dried, ripe seeds of Trigonella foenum-graecum L.
CHARACTERS
Fenugreek has a strong characteristic aromatic odour.
IDENTIFICATION
A. The seed is hard, flattened, brown to reddish-brown and more or less rhomboidal with rounded
edges. It is 3 mm to 5 mm long, 2 mm to 3 mm wide and 1.5 mm to 2 mm thick. The widest
surfaces are marked by a groove that divides the seed into two unequal parts. The smaller part
contains the radicle; the larger part contains the cotyledons.
chloral hydrate solution R. The powder shows fragments of the testa in sectional view with thick cuticle
covering lageniform epidermal cells, with an underlying hypodermis of large cells, narrower at the
upper end and constricted in the middle, with bar-like thickenings of the radial walls; yellowish-
brown fragments of the epidermis in surface view, composed of small, polygonal cells with thickened
and pitted walls, frequently associated with the hypodermal cells, circular in outline with thickened
and closely beaded walls; fragments of the hypodermis viewed from below, composed of polygonal
cells whose bar-like thickenings extend to the upper and lower walls; parenchyma of the testa with
elongated, rectangular cells with slightly thickened and beaded walls; fragments of endosperm with
irregularly thickened, sometimes elongated cells, containing mucilage.
254
plate R.
Test solution. Place 1.0 g of the powdered drug (710) in a 25 ml conical flask and add 5.0 ml of
methanol R. Heat in a water-bath at 65C for 5 min. Cool and filter.
Reference solution. Dissolve 3.0 mg of trigonelline hydrochloride R in 1.0 ml of methanol R.
Develop over a path of 10 cm using a mixture of 30 volumes of water R and 70 volumes of
methanol R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The chromato-
gram obtained with the test solution shows in its lower half a quenching zone similar in position and
fluorescence to the zone in the chromatogram obtained with the reference solution. Spray with
potassium iodobismuthate solution R2. The chromatogram obtained with the test solution shows an
intense orange-red zone similar in position and colour to the zone in the chromatogram obtained
with the reference solution. It also shows in its upper half a broad, light brownish-yellow zone
(triglycerides).
TESTS
Swelling index (2.8.4). Not less than 6, determined on the powdered drug (710).
by drying in an oven at 100C to 105C for 2 h.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
17-28
Products of Fermentation
Products of Fermentation comply with the requirements of the 3rd edition of the European Pharmacopoeia
[1468]. These requirements are reproduced below.
Ph Eur ___________________________________________________________________________________________________________
This monograph applies to indirect gene products obtained by fermentation. It is not applicable to:
monographs in the Pharmacopoeia concerning vaccines for human or veterinary use;
products derived from continuous cell lines of human or animal origin;
direct gene products that result from the transcription and translation from nucleic acid to protein,
whether or not subject to post-translational modification;
products obtained by semi-synthesis from a product of fermentation and those obtained by biocatalytic
transformation;
whole broth concentrates or raw fermentation products.
The statements in this monograph apply to individual monographs in the Pharmacopoeia for products
obtained directly by fermentation as defined below. The requirements do not necessarily apply to products of
fermentation that are not the subject of such monographs. However, the requirements may be invoked by
competent authorities for this purpose.
DEFINITION
For the purposes of this monograph, products of fermentation are active or inactive pharmaceutical
substances produced by controlled fermentation as indirect gene products. They are primary or
secondary metabolites of micro-organisms such as bacteria, yeasts, fungi and micro-algae, whether or
not modified by traditional procedures or recombinant DNA (r-DNA) technology. Such metabolites
include vitamins, amino acids, antibiotics, alkaloids and polysaccharides.
They may be obtained by batch or continuous fermentation processes followed by procedures such as
extraction, concentration, purification and isolation.
PRODUCTION
Production is based on a process that has been validated and shown to be suitable. The extent of
validation depends on the critical nature of the respective process step.
CHARACTERISATION OF THE PRODUCER MICRO-ORGANISM
The history of the micro-organism used for production is documented. The micro-organism is
adequately characterised. This may include determination of the phenotype of the micro-organism,
macroscopic and microscopic methods and biochemical tests and, if appropriate, determination of
the genotype of the micro-organism and molecular genetic tests.
When a master cell bank is replaced, the critical steps of the production process must be
revalidated to the extent necessary to demonstrate that no adverse change has occurred in the quality
and safety of the product. Particular attention must be given to possible changes in the impurity
profile of the product if a modified or new micro-organism is introduced into the process.
RAW MATERIALS
The raw materials employed in the fermentation and/or down-stream processing, are of suitable
quality for the intended purpose. They are to be tested to ensure that they comply with written
specifications.
Levels of bioburden in media or in the inlet air for aeration are reduced to an adequately low level
to ensure that if microbiological contamination occurs, it does not adversely affect the quality, purity
and safety of the product. Addition of components such as nutrients, precursors, and substrates
during fermentation takes place aseptically.
IN-PROCESS CONTROLS
In-process controls are in place to ensure the consistency of the conditions during fermentation and
down-stream processing and of the quality of the isolated product. Particular attention must be paid
to ensure that any microbial contamination that adversely affects the quality, purity and safety of the
product is detected by the controls applied.
Production conditions may be monitored, as appropriate, by suitable procedures for example to
control and check:
temperature,
pH,
rate of aeration,
rate of agitation,
pressure,
and to monitor the concentration of the required product.
17-29
DOWN-STREAM PROCESSING
At the end of fermentation, the producer micro-organism is inactivated or removed. Further
processing is designed to reduce residues originating from the culture medium to an acceptable level
and to ensure that the desired product is recovered with consistent quality.
The purification processes employed (for example, charcoal treatment, ultrafiltration, solvent
extraction) are demonstrated to minimise or remove:
residues from the producer micro-organism, culture media, substrates and precursors,
unwanted transformation products of the substrates and precursors.
If necessary, suitable tests are performed either as in-process controls or on the isolated product of
fermentation.
IDENTIFICATION, TESTS AND ASSAY
The requirements with which the product must comply throughout its period of validity, as well as
specific test methods, are stated in the individual monographs.
STORAGE
See the individual monographs.
LABELLING
See the individual monographs.
__________________________________________________________________________________________________________ Ph Eur
17-30
Ferric Chloride Hexahydrate
1/01
FeCl
3
,6H
2
O 270.3 10025-77-1
Ferric Chloride Hexahydrate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ferric chloride hexahydrate contains not less than 98.0 per cent and not more than 102.0 per cent
of FeCl
3
,6H
2
O.
CHARACTERS
Crystalline mass or orange-yellow to brownish-yellow crystals, very hygroscopic, very soluble in
water and in alcohol, freely soluble in glycerol.
IDENTIFICATION
A. It gives reaction (a) of chlorides (2.3.1).
B. It gives reaction (c) of iron (2.3.1).
TESTS
Solution S Dissolve 10 g in distilled water R and dilute to 100 ml with the same solvent.
Acidity In a suitable polyethylene container, dissolve 3.0 g of potassium fluoride R in 15 ml of
water R. Titrate with 0.1M sodium hydroxide using 0.1 ml of phenolphthalein solution R as indicator until
a pink colour is obtained. Add 10 ml of solution S and allow to stand for 3 h. Filter and use 12.5 ml
of the filtrate. Not more than 0.30 ml of 0.1M sodium hydroxide is required to change the colour of
the indicator to pink.
Free chlorine Heat 5 ml of solution S. The vapour does not turn starch iodide paper R blue.
Sulphates (2.4.13). Heat 15 ml of solution S on a water-bath and add 5 ml of strong sodium hydroxide
solution R. Allow to cool and filter. Neutralise the filtrate to blue litmus paper R using hydrochloric
acid R1 and evaporate to 15 ml. The solution complies with the limit test for sulphates (100 ppm).
Ferrous ions To 10 ml of solution S, add 1 ml of water R, 4 ml of phosphoric acid R and 0.05 ml of
potassium ferricyanide solution R. After 10 min, any blue colour in the solution is not more intense than
that in a standard prepared at the same time and in the same manner using 10 ml of water R and 1 ml
of a freshly prepared 0.250 g/l solution of ferrous sulphate R (50 ppm Fe).
Heavy metals (2.4.8). Dissolve 1.0 g in 10 ml of hydrochloric acid R1. Add 2 ml of strong hydrogen
peroxide solution R, then evaporate to 5 ml. Allow to cool and dilute to 20 ml with hydrochloric acid R1
and transfer the solution to a separating funnel. Shake three times, for 3 min each time, with 20 ml of
methyl isobutyl ketone R1. Separate the lower phase, reduce to half its volume by evaporation and
dilute to 25 ml with water R. Neutralise 10 ml of the solution with dilute ammonia R1 to red litmus
paper R and dilute to 20 ml with water R. 12 ml of the solution complies with limit test A for heavy
metals (50 ppm). Prepare the standard using lead standard solution (1 ppm Pb) R.
ASSAY
In a conical flask with a ground-glass stopper, dissolve 0.200 g in 20 ml of water R. Add 10 ml of
dilute hydrochloric acid R and 2 g of potassium iodide R. Allow the stoppered flask to stand for 1 h
protected from light. Titrate with 0.1M sodium thiosulphate, using 5 ml of starch solution R as indicator,
added towards the end of the titration.
1 ml of 0.1M sodium thiosulphate is equivalent to 27.03 mg of FeCl
3
,6H
2
O.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
17-31
Ferrous Fumarate
corrected 1/01
H O
O
O
O
H
_
_
e
2+
C
4
H
2
FeO
4
169.9 141-01-5
Ferrous Fumarate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0902].
Action and use Used in prevention and treatment of anaemias.
Preparations
Ferrous Fumarate Capsules
Ferrous Fumarate Oral Suspension
Ferrous Fumarate Tablets
Ferrous Fumarate and Folic Acid Tablets
Ferrous Fumarate contains in 200 mg about 65 mg of iron.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ferrous fumarate contains not less than 93.0 per cent and not more than the equivalent of 101.0 per
cent of iron(II) (E)-butenedioate, calculated with reference to the dried substance.
CHARACTERS
A fine, reddish-orange or reddish-brown powder, slightly soluble in water, very slightly soluble in
alcohol.
IDENTIFICATION
254
plate R.
Test solution. To 1.0 g add 25 ml of a mixture of equal volumes of hydrochloric acid R and water R and
heat on a water-bath for 15 min. Cool and filter. Use the filtrate for identification test C. Wash the
residue with 50 ml of a mixture of 1 volume of dilute hydrochloric acid R and 9 volumes of water R and
discard the washings. Dry the residue at 100C to 105C. Dissolve 20 mg of the residue in acetone R
Reference solution. Dissolve 20 mg of fumaric acid CRS in acetone R and dilute to 10 ml with the same
solvent.
10 cm using a mixture of 12 volumes of anhydrous formic acid R, 16 volumes of methylene chloride R,
32 volumes of butanol R and 44 volumes of heptane R. Dry the plate at 105C for 15 min and
solution is similar in position and size to the principal spot in the chromatogram obtained with the
reference solution.
B. Mix 0.5 g with 1 g of resorcinol R. To 0.5 g of the mixture in a crucible add 0.15 ml of sulphuric
acid R and heat gently. A dark red semi-solid mass is formed. Add the mass, with care, to 100 ml of
water R. An orange-yellow colour develops and the solution shows no fluorescence.
C. The filtrate obtained for the test solution in identification test A gives reaction (a) of iron (2.3.1).
TESTS
Solution S Dissolve 2.0 g of the substance to be examined in a mixture of 10 ml of lead-free hydro-
chloric acid R and 80 ml of water R, heating slightly if necessary. Allow to cool, filter if necessary and
Sulphates (2.4.13). Heat 0.15 g with 8 ml of dilute hydrochloric acid R and 20 ml of distilled water R.
Cool in iced water, filter and dilute to 30 ml with distilled water R. 15 ml of this solution complies
with the limit test for sulphates (0.2 per cent).
Arsenic (2.4.2). Mix 1.0 g with 15 ml of water R and 15 ml of sulphuric acid R. Warm to precipitate
the fumaric acid completely. Cool and add 30 ml of water R. Filter. Wash the precipitate with
water R. Dilute the combined filtrate and washings to 125 ml with water R. 25 ml of the solution
complies with limit test A for arsenic (5 ppm).
Ferric ion Not more than 2.0 per cent of ferric ion.
17-32
In a flask with a ground-glass stopper, dissolve 3.0 g in a mixture of 10 ml of hydrochloric acid R and
100 ml of water R by heating rapidly to boiling. Boil for 15 s. Cool rapidly, add 3 g of potassium
iodide R, stopper the flask and allow to stand protected from light for 15 min. Add 2 ml of starch
solution R as indicator. Titrate the liberated iodine with 0.1M sodium thiosulphate. Carry out a blank
test. The difference between the volumes used in the two titrations corresponds to the amount of
iodine liberated by ferric ion.
1 ml of 0.1M sodium thiosulphate is equivalent to 5.585 mg of ferric ion.
Cadmium Not more than 10 ppm of Cd, determined by atomic absorption spectrometry (Method I,
2.2.23).
Reference solutions. Prepare the solutions using cadmium standard solution (0.1 per cent Cd) R and
diluting with a 10 per cent V/V solution of lead-free hydrochloric acid R.
Measure the absorbance at 228.8 nm using a cadmium hollow-cathode lamp as source of radiation
Chromium Not more than 200 ppm of Cr, determined by atomic absorption spectrometry (Method
I, 2.2.23).
Reference solutions. Prepare the solutions using chromium standard solution (0.1 per cent Cr) R and
diluting with a 10 per cent V/V solution of lead-free hydrochloric acid R.
Measure the absorbance at 357.9 nm using a chromium hollow-cathode lamp as source of radiation
2.2.23).
Reference solutions. Prepare the solutions using lead standard solution (10 ppm Pb) R and diluting with a
10 per cent V/V solution of lead-free hydrochloric acid R.
Measure the absorbance at 283.3 nm using a lead hollow-cathode lamp as the source of radiation and
Mercury Not more than 1 ppm of Hg, determined by atomic absorption spectrometry (Method I,
2.2.23).
Reference solutions. Prepare the solutions using mercury standard solution (10 ppm Hg) R and diluting
with a 25 per cent V/V solution of lead-free hydrochloric acid R.
Following the recommendations of the manufacturer, introduce 5 ml of solution S or 5 ml of the
reference solutions into the reaction vessel of the cold-vapour mercury assay accessory, add 10 ml of
water R and 1 ml of stannous chloride solution R1.
Measure the absorbance at 253.7 nm using a mercury hollow-cathode lamp as the source of
radiation.
Nickel Not more than 200 ppm of Ni, determined by atomic absorption spectrometry (Method I,
2.2.23).
Reference solutions. Prepare the solutions using nickel standard solution (10 ppm Ni) R and diluting with
a 10 per cent V/V solution of lead-free hydrochloric acid R.
Measure the absorbance at 232 nm using a nickel hollow-cathode lamp as source of radiation and an
Zinc Not more than 500 ppm of Zn, determined by atomic absorption spectrometry (Method I,
2.2.23).
Test solution. Use solution S diluted ten times.
Reference solutions. Prepare the solutions using zinc standard solution (10 ppm Zn) R and diluting with a
1 per cent V/V solution of lead-free hydrochloric acid R.
Measure the absorbance at 213.9 nm using a zinc hollow-cathode lamp as source of radiation and an
100C to 105C.
ASSAY
Dissolve with slight heating 0.150 g in 7.5 ml of dilute sulphuric acid R. Cool and add 25 ml of
water R. Add 0.1 ml of ferroin R. Titrate immediately with 0.1M cerium sulphate until the colour
17-33
changes from orange to light bluish-green.
4
H
2
FeO
4
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
17-34
Ferrous Gluconate
H OH
OH H
H HO
H OH
COO
_
OH
2
Fe
2
+
C
12
H
22
FeO
14
,xH
2
O 446.1(anhydrous) 299-29-6 (anhydrous)
Ferrous Gluconate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0493].
Action and use Used in prevention and treatment of iron deficiency.
Preparation
Ferrous Gluconate Tablets
Ferrous Gluconate contains in 600 mg about 70 mg of iron.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ferrous gluconate is iron(II) di(D-gluconate). It contains not less than 11.8 per cent and not more
than 12.5 per cent of iron(II), calculated with reference to the dried substance.
CHARACTERS
A greenish-yellow to grey powder or granules, freely but slowly soluble in water giving a greenish-
brown solution, more readily soluble in hot water, practically insoluble in alcohol.
IDENTIFICATION
Reference solution. Dissolve 20 mg of ferrous gluconate CRS in 2 ml of water R, heating if necessary in a
water-bath at 60C.
volumes of alcohol R. Dry the plate at 100C to 105C for 20 min, allow to cool and spray with a
50 g/l solution of potassium dichromate R in a 40 per cent m/m solution of sulphuric acid R. After 5 min,
the principal spot in the chromatogram obtained with the test solution is similar in position, colour
B. 1 ml of solution S (see Tests) gives reaction (a) of iron (2.3.1).
TESTS
Solution S Dissolve 5.0 g in carbon dioxide-free water R prepared from distilled water R and heated to
about 60C, cool and dilute to 50 ml with carbon dioxide-free water R prepared from distilled water R.
Appearance of solution Dilute 2 ml of solution S to 10 ml with water R. Examined against the
light, the solution is clear (2.2.1).
pH (2.2.3). The pH of solution S, measured 3 h to 4 h after preparation, is 4.0 to 5.5.
Sucrose and reducing sugars Dissolve 0.5 g in 10 ml of warm water R and add 1 ml of dilute
ammonia R1. Pass hydrogen sulphide R through the solution and allow to stand for 30 min. Filter and
wash the precipitate with two quantities, each of 5 ml, of water R. Acidify the combined filtrate and
washings to blue litmus paper R with dilute hydrochloric acid R and add 2 ml in excess. Boil until the
vapour no longer darkens lead acetate paper R and continue boiling, if necessary, until the volume is
reduced to about 10 ml. Cool, add 15 ml of sodium carbonate solution R, allow to stand for 5 min and
filter. Dilute the filtrate to 100 ml with water R. To 5 ml of this solution add 2 ml of cupri-tartaric
solution R and boil for 1 min. Allow to stand for 1 min. No red precipitate is formed.
Chlorides (2.4.4). 0.8 ml of solution S diluted to 15 ml with water R complies with the limit test for
17-35
Oxalates Dissolve 5.0 g in a mixture of 10 ml of dilute sulphuric acid R and 40 ml of water R. Shake
the solution with 50 ml of ether R for 5 min. Separate the aqueous layer and shake it with 20 ml of
ether R for 5 min. Combine the ether layers, evaporate to dryness and dissolve the residue in 15 ml of
water R. Filter, boil the filtrate until the volume is reduced to 5 ml and add 1 ml of dilute acetic acid R
and 1.5 ml of calcium chloride solution R. Allow to stand for 30 min. No precipitate is formed.
Sulphates (2.4.13). To 3.0 ml of solution S add 3 ml of acetic acid R and dilute to 15 ml with distilled
water R. The solution complies with the limit test for sulphates (500 ppm). Examine the solutions
against the light.
Arsenic (2.4.2). 0.5 g complies with limit test A for arsenic (2 ppm).
Barium Dilute 10 ml of solution S to 50 ml with distilled water R and add 5 ml of dilute sulphuric
acid R. Allow to stand for 5 min. Any opalescence in the solution is not more intense than that in a
mixture of 10 ml of solution S and 45 ml of distilled water R.
Ferric ion In a ground-glass-stoppered flask, dissolve 5.00 g in a mixture of 10 ml of hydrochloric
acid R and 100 ml of carbon dioxide-free water R. Add 3 g of potassium iodide R, close the flask and
allow to stand protected from light for 5 min. Titrate with 0.1M sodium thiosulphate, using 0.5 ml of
starch solution R, added towards the end of the titration, as indicator. Carry out a blank titration. Not
more than 9.0 ml of 0.1M sodium thiosulphate is used in the test (1.0 per cent).
Heavy metals (2.4.8). Thoroughly mix 2.5 g with 0.5 g of magnesium oxide R1 in a silica crucible.
Ignite to dull redness until a homogeneous mass is obtained. Heat at 800C for about 1 h, allow to
cool and take up the residue in 20 ml of hot hydrochloric acid R. Allow to cool. Transfer the liquid to a
separating funnel and shake for 3 min with three quantities, each of 20 ml, of methyl isobutyl ketone
saturated with hydrochloric acid (prepared by shaking 100 ml of freshly distilled methyl isobutyl
ketone R with 1 ml of hydrochloric acid R). Allow to stand, separate the aqueous layer, reduce to half
its volume by boiling, allow to cool and dilute to 25 ml with water R. Neutralise 7.5 ml of this
solution to red litmus paper R using dilute ammonia R1 and dilute to 15 ml with water R. 12 ml of the
solution complies with limit test A for heavy metals (20 ppm). Prepare the standard using lead
at 100C to 105C. for 5 h.
3
micro-
organisms per gram, determined by plate-count.
ASSAY
Dissolve 0.5 g of sodium hydrogen carbonate R in a mixture of 30 ml of dilute sulphuric acid R and 70 ml
of water R. When the effervescence stops, dissolve in the solution 1.00 g of the substance to be exam-
ined with gentle shaking. Using 0.1 ml of ferroin R as indicator, titrate with 0.1M ammonium and
cerium nitrate until the red colour disappears.
1 ml of 0.1M ammonium and cerium nitrate is equivalent to 5.585 mg of iron(II).
STORAGE
__________________________________________________________________________________________________________ Ph Eur
17-36
Ferrous Sulphate
FeSO
4
,7H
2
O 278.0 7720-78-7
Ferrous Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0083].
Preparation
Paediatric Ferrous Sulphate Oral Solution
Ferrous Sulphate contains in 300 mg about 60 mg of iron.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ferrous sulphate contains not less than 98.0 per cent and not more than the equivalent of 105.0 per
cent of FeSO
4
,7H
2
O.
CHARACTERS
A light green, crystalline powder or bluish-green crystals, efflorescent in air, freely soluble in water,
very soluble in boiling water, practically insoluble in alcohol. Ferrous sulphate is oxidised in moist air,
becoming brown.
IDENTIFICATION
A. It gives the reaction of sulphates (2.3.1).
B. It gives reaction (a) of iron (2.3.1).
TESTS
Solution S Dissolve 2.5 g in carbon dioxide-free water R, add 0.5 ml of dilute sulphuric acid R and
Chlorides (2.4.4). Dilute 3.3 ml of solution S to 10 ml with water R and add 5 ml of dilute nitric
acid R. The solution complies with the limit test for chlorides (300 ppm). Prepare the standard using
a mixture of 10 ml of chloride standard solution (5 ppm Cl) R and 5 ml of dilute nitric acid R. Use
0.15 ml of silver nitrate solution R2 in this test.
Ferric ions In a ground-glass-stoppered flask, dissolve 5.00 g in a mixture of 10 ml of hydrochloric
acid R and 100 ml of carbon dioxide-free water R. Add 3 g of potassium iodide R, close the flask and
allow to stand in the dark for 5 min. Titrate the liberated iodine with 0.1M sodium thiosulphate, using
0.5 ml of starch solution R, added towards the end of the titration, as indicator. Carry out a blank test
in the same conditions. Not more than 4.5 ml of 0.1M sodium thiosulphate is used, taking into account
the blank titration (0.5 per cent).
Heavy metals (2.4.8). Dissolve 1.0 g in 10 ml of hydrochloric acid R1, add 2 ml of strong
hydrogen peroxide solution R and boil until the volume is reduced to 5 ml. Allow to cool, dilute to
20 ml with hydrochloric acid R1, transfer the solution to a separating funnel and shake for 3 min with
three quantities, each of 20 ml, of methyl isobutyl ketone saturated with hydrochloric acid (prepared
by shaking 100 ml of freshly distilled methyl isobutyl ketone R with 1 ml of hydrochloric acid R1). Allow
to stand, separate the aqueous layer and reduce to half its volume by boiling, allow to cool and dilute
to 25 ml with water R (solution a). Neutralise 10 ml of solution A to litmus paper R using dilute
ammonia R1 and dilute to 20 ml with water R. 12 ml of the solution complies with limit test A for
Manganese Dissolve 1.0 g in 40 ml of water R, add 10 ml of nitric acid R and boil until red fumes are
evolved. Add 0.5 g of ammonium persulphate R and boil for 10 min. Discharge any pink colour by
adding dropwise a 50 g/l solution of sodium sulphite R and boil until any odour of sulphur dioxide is
eliminated. Add 10 ml of water R, 5 ml of phosphoric acid R and 0.5 g of sodium periodate R, boil for
1 min and allow to cool. The solution is not more intensely coloured than a standard prepared at the
same time in the same manner using 1.0 ml of 0.02M potassium permanganate and adding the same
volumes of the same reagents (0.1 per cent).
Zinc To 5 ml of solution (a) obtained in the test for heavy metals add 1 ml of potassium ferrocyanide
solution R and dilute to 13 ml with water R. After 5 min, any turbidity produced is not more intense
than that of a standard prepared at the same time by mixing 10 ml of zinc standard solution (10 ppm
Zn) R, 2 ml of hydrochloric acid R1 and 1 ml of potassium ferrocyanide solution R (500 ppm).
17-37
ASSAY
Dissolve 2.5 g of sodium hydrogen carbonate R in a mixture of 150 ml of water R and 10 ml of sulphuric
acid R. When the effervescence ceases add to the solution 0.500 g of the substance to be examined
and dissolve with gentle shaking. Add 0.1 ml of ferroin R and titrate with 0.1M ammonium and cerium
nitrate until the red colour disappears.
1 ml of 0.1M ammonium and cerium nitrate is equivalent to 27.80 mg of FeSO
4
,7H
2
O.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
17-38
Dried Ferrous Sulphate
13463-43-9
Definition Dried Ferrous Sulphate is Ferrous Sulphate from which part of the water of
crystallisation has been removed by drying at a temperature of 40. It contains not less than 86.0%
and not more than 90.0% of FeSO
4
.
Characteristics A greyish white powder.
It dissolves slowly, but almost completely, in freshly boiled and cooled water.
Identification Yields reaction A characteristic of iron salts and the reactions characteristic of
sulphates, Appendix VI.
Arsenic Dissolve 1.0 g in 10 ml of water and 15 ml of stannated hydrochloric acid and distil 20 ml. To
the distillate add 0.15 ml of bromine water, remove the excess bromine with 0.15 ml of tin(II) chloride
solution AsT and dilute to 75 ml with water. 25 ml of the resulting solution complies with the limit test
for arsenic, Appendix VII (3 ppm).
Copper Not more than 50 ppm of Cu when determined by atomic absorption spectrophotometry,
Appendix II D, Method II, measuring at 324.7 nm, and using a solution prepared by dissolving 2 g
of the substance being examined in sufficient 5% v/v nitric acid to produce 100 ml. Use copper
standard solution (0.1% Cu), suitably diluted with 5% v/v nitric acid to prepare the standard solutions.
Ferric iron Dissolve 5 g in a mixture of 10 ml of hydrochloric acid and 100 ml of carbon dioxide-free
water in a ground-glass-stoppered flask and add 3 g of potassium iodide. Close the flask and allow to
stand in the dark for 5 minutes. Titrate the liberated iodine with 0.1M sodium thiosulphate VS using
0.5 ml of starch solution added towards the end of the titration as indicator. Repeat the operation
without the substance being examined. The difference between the titrations represents the amount
of iodine liberated by the ferric iron. Not more than 4.5 ml of 0.1M sodium thiosulphate VS is required
(0.5%).
Manganese Dissolve 1.0 g in 40 ml of water, add 10 ml of nitric acid and boil until red fumes cease
to be evolved. Add 0.5 g of ammonium persulphate and boil for 10 minutes. If any pink colour is
produced, decolorise the solution by the dropwise addition of a 5% w/v solution of sodium sulphite
and boil for 2 minutes. Add 10 ml of water, 5 ml of orthophosphoric acid and 0.5 g of sodium periodate,
boil for 1 minute and cool. The colour of the solution is not more intense than that of a solution
prepared at the same time and in the same manner using 1.0 ml of 0.02M potassium permanganate VS
in place of the substance being examined.
Zinc To 5 ml of solution A prepared in the test for Heavy metals add 1 ml of potassium
hexacyanoferrate(II) solution, dilute to 13 ml with water and allow to stand for 5 minutes. Any turbidity
produced is not more intense than that of a solution prepared at the same time by mixing 10 ml of
zinc standard solution (10 ppm Zn), 2 ml of 7M hydrochloric acid and 1 ml of potassium
hexacyanoferrate(II) solution (500 ppm).
Heavy metals Dissolve 1.0 g in 10 ml of 7M hydrochloric acid, add 2 ml of hydrogen peroxide solution
(100 vol) and boil until the volume is reduced to 5 ml. Allow to cool, dilute to 20 ml with 7M hydro-
chloric acid and extract with three successive 20-ml quantities of a solution prepared by shaking
100 ml of freshly distilled 4-methylpentan-2-one with 1 ml of 7M hydrochloric acid. Allow to stand,
separate the aqueous layer and evaporate to half its volume, allow to cool and dilute to 25 ml with
water (solution A). Neutralise 7.5 ml of solution A to litmus paper using 6M ammonia and dilute to
15 ml with water. 12 ml of the resulting solution complies with limit test A for heavy metals, Appendix
VII. Use lead standard solution (1 ppm Pb) to prepare the standard (50 ppm).
Basic sulphate 2.0 g dissolves slowly in a mixture of 7.5 ml of freshly boiled and cooled water and
0.5 ml of 0.5M sulphuric acid, producing a solution that is not more than faintly turbid.
Assay Dissolve 0.5 g in a mixture of 30 ml of water and 20 ml of 1M sulphuric acid and titrate with
0.1M ammonium cerium(IV) sulphate VS using ferroin solution as indicator. Each ml of 0.1M ammonium
cerium(IV) sulphate VS is equivalent to 15.19 mg of FeSO
4
.
Storage Dried Ferrous Sulphate should be kept in a well-closed container.
Preparations
Ferrous Sulphate Tablets
Prolonged-release Ferrous Sulphate Tablets
17-39
Feverfew
1/01
Feverfew complies with the requirements of the 3rd edition of the European Pharmacopoeia [1516]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Feverfew consists of the dried, whole or fragmented aerial parts of Tanacetum parthenium (L.) Schultz
Bip. It contains not less than 0.20 per cent of parthenolide (C
15
H
20
O
3
; M
r
248.3), calculated with
CHARACTERS
Fewerfew has a camphoraceous odour.
IDENTIFICATION
A. The leafy, more or less branched stem has a diameter of up to 5 mm; it is almost quadrangular,
channelled longitudinally and slightly pubescent. The leaves are ovate, 2 cm to 5 cm long, sometimes
up to 10 cm, yellowish-green, petiolate and alternate. They are pinnate or bipinnate, deeply divided
into five to nine segments, each with a coarsely crenate margin and an obtuse apex. Both surfaces are
somewhat pubescent and the midrib is prominent on the lower surface. When present, the flowering
heads are 12 mm to 22 mm in diameter with long pedicels; they are clustered into broad corymbs
consisting of five to thirty flower-heads. The hemispherical involucre is 6 mm to 8 mm wide and
consists of many overlapping bracts, which are rather narrow, are obtuse and scarious and have
membranous margins. The central flowers are yellow, hermaphrodite, tube-shaped with five teeth
and have five stamens inserted in the corolla; the filaments of the stamens are separate from each
other but the anthers are fused into a tube through which passes the style, bearing two stigmatic
branches. The peripheral flowers are female and have a white three-toothed ligule, 2 mm to 7 mm
long. The fruit is an achene, 1.2 mm to 1.5 mm long, brown when ripe, with five to ten white
longitudinal ribs. It is glandular and bears a short, crenate, membranous crown.
B. Reduce to a powder (355). The powder is yellowish-green. Examine under a microscope using
chloral hydrate solution R. The powder shows numerous large, multicellular, uniseriate covering
trichomes consisting of a rhomboidal basal cell, three to five smaller, thick-walled rectangular cells
and a very long, flat, slender terminal cell, often curved at a right angle to the axis of the basal cell;
glandular trichomes with a short, biseriate, two to four celled stalk and a biseriate head of four cells
around which the cuticle forms a bladder-like covering; epidermal cells with a very sinuous anticlinal
walls, a striated cuticle and anomocytic stomata; numerous spirally and annularly thickened vessels;
stratified parenchyma and collenchyma. Fragments of disc florets containing pale yellow amorphous
masses and small rosette crystals of calcium oxalate may be present; spherical pollen grains about
25 m in diameter, with three pores and a spiny exine may be present.
Test solution. To 1 g of the powdered drug (355) add 20 ml of methanol R. Heat in a water-bath at
60C for 15 min. Allow to cool and filter. Evaporate to dryness under reduced pressure and dissolve
the residue in 2 ml of methanol R.
Reference solution. Dissolve 5 mg of parthenolide R in methanol R and dilute to 5 ml with the same
solvent.
Apply to the plates as bands 20 l of each solution. Develop over a path of 10 cm using a mixture of
15 volumes of acetone R and 85 volumes of toluene R. Allow the plate to dry in air. Spray with a 5 g/l
solution of vanillin R in a mixture of 20 volumes of ethanol R and 80 volumes of sulphuric acid R.
After 5 min examine in daylight. The chromatogram obtained with the test solution shows in its
central part a blue principal zone that is similar in position, colour and size to the principal zone in
the chromatogram obtained with the reference solution, and somewhat below the principal zone it
may show a second blue zone. It also shows, one or two blue zones in its lower third. Other violet
zones may be present.
TESTS
Foreign matter (2.8.2). Not more than 10.0 per cent of stem having a diameter exceeding 5 mm,
and not more than 2.0 per cent of other foreign matter.
17-40
ASSAY
Test solution. Completely reduce about 50 g to a powder (355). After homogenisation, introduce
1.00 g of the powdered drug into a flask and add 40 ml of methanol R. Heat in a water-bath at 60C
for 10 min. Allow to cool and filter. Rinse the filter with 15 ml of methanol R. Take up the residue
with 40 ml of methanol R. Repeat the operation. Collect the filtrates and rinsings and evaporate to
dryness under reduced pressure. Take up the residue with methanol R and dilute to 20.0 ml with the
same solvent. Dilute 10.0 ml of this solution to 50.0 ml with the mobile phase. Filter (0.45 m).
Reference solution. Dissolve 5.0 mg of parthenolide R in methanol R and dilute to 10.0 ml with the same
solvent. Dilute 2.0 ml of the solution to 50.0 ml with the mobile phase.
volumes of water R,
Inject 20 l of each solution. The retention time of parthenolide is about 11.5 min.
Calculate the percentage content of parthenolide from the expression:
1 2
2 1
40
m A
m A

A
1
=area corresponding to the principal compound in the chromatogram obtained with the
test solution,
A
2
=area corresponding to the principal compound in the chromatogram obtained with the
reference solution,
m
1
=mass of the drug used in the test solution, in grams,
m
2
=mass of parthenolide in the reference solution, in grams.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
17-41
Fig
Definition Fig is the sun-dried succulent fruit of Ficus carica L.
Characteristics Odour, pleasantly fruity; taste, sweet.
Macroscopical Fruit compound, soft, fleshy, brown or yellowish brown, sometimes covered with a
saccharine efflorescence; at the summit a small opening surrounded by scales and at the base a short,
stalk-like prolongation; fruit up to about 5 cm in length and breadth, consisting of a hollow receptacle
bearing on the inner surface numerous drupelets, each containing a stone about 1.5 to 2.0 mm long;
seed containing endosperm and a curved embryo.
Microscopical Receptacle: epidermal cells polyhedral, stomata raised, trichomes unicellular, thick
walled, of varying length up to about 300 m; hypodermis composed of rounded polyhedral cells,
some containing small rosette crystals of calcium oxalate; aerenchyma made up of large, irregular
cells, forming the greater part of the receptacle, containing large rosette crystals of calcium oxalate
and interspersed with numerous latex tubes, about 30 to 50 m wide, and slender vascular bundles.
Pericarp: epicarp consisting of radially elongated cells with mucilaginous outer walls; mesocarp of
delicate, often disorganised cells; endocarp of radially elongated sclereids with pitted walls.
Endosperm and embryo: small cells containing aleurone grains and fixed oil; starch absent.
Water-soluble extractive Not less than 60.0% when determined by the following method. To 25 g,
minced, add 500 ml of water, boil under a reflux condenser for 1 hour, cool and filter. To 20 ml of
the filtrate add 20 g of washed and ignited sand, evaporate to dryness in a tared, flat bottomed
shallow dish and dry the residue to constant weight at 100. Calculate the water-soluble extractive by
subtracting the weight of sand from the weight of the residue obtained.
Storage Fig should be stored in a dry place.
Action and use Demulcent.
17-42
Flavoxate Hydrochloride
,HCl
N
O O
O
O
Me
C
24
H
25
NO
4
,HCl 427.9 3717-88-2
Definition Flavoxate Hydrochloride is 2-piperidinoethyl 3-methyl-4-oxo-2-phenyl-4H-chromene-
8-carboxylate hydrochloride. It contains not less than 99.0% and not more than 101.0% of
C
24
H
25
NO
4
Slightly soluble in water and in ethanol (96%); sparingly soluble in dichloromethane.
Identification
flavoxate hydrochloride (RS 143).
Heavy metals 2.0 g complies with limit test C for heavy metals, Appendix VII (10 ppm). Prepare the
standard using 2 ml of lead standard solution (10 ppm Pb).
gel GF
254
as the coating substance and a mixture of 1 volume of 18M ammonia, 80 volumes of propan-
2-ol and 200 volumes of ethyl acetate as the mobile phase. Apply separately to the plate the following
four solutions in chloroform: (1) 50 l of a 2.0% w/v solution of the substance being examined, (2)
10 l of a 0.030% w/v solution of 3-methylflavone-8-carboxylic acid BPCRS, (3) 10 l of a 0.015% w/v
solution of 3-methylflavone-8-carboxylic acid ethyl ester BPCRS and (4) 25 l of a 0.0040% w/v solution
of the substance being examined. After removal of the plate, allow it to dry in air and examine under
ultraviolet light (254 nm). Any spot corresponding to 3-methylflavone-8-carboxylic acid ethyl ester in
obtained with solution (3) (0.15%) and any other secondary spot in the chromatogram obtained with
solution (1), other than any spot corresponding to 3-methylflavone-8-carboxylic acid, is not more
intense than the spot in the chromatogram obtained with solution (4) (0.1%).
3-Methylflavone-8-carboxylic acid Carry out the method for thin-layer chromatography, Appendix
III A, using silica gel GF
254
as the coating substance and a mixture of 4 volumes of glacial acetic acid,
25 volumes of ethyl acetate and 70 volumes of cyclohexane as the mobile phase. Apply separately to the
plate the following two solutions in chloroform: (1) 50 l of a 2.0% w/v solution of the substance being
examined and (2) 10 l of a 0.030% w/v solution of 3-methylflavone-8-carboxylic acid BPCRS. After
removal of the plate, allow it to dry in air and spray with dilute potassium iodobismuthate solution. Any
spot corresponding to 3-methylflavone-8-carboxylic acid in the chromatogram obtained with solution
(1) is not more intense than the spot in the chromatogram obtained with solution (2) (0.3%).
1 g.
Assay Dissolve 0.5 g in 40 ml of anhydrous acetic acid, add 10 ml of mercury(II) acetate solution and
titrate with 0.1M perchloric acid VS using oracet blue B solution as indicator until the colour changes to
pink. Repeat the operation without the substance being examined. Each ml of 0.1M perchloric acid VS
24
H
25
NO
4
,HCl.
Storage Flavoxate Hydrochloride should be kept in a well-closed container and protected from light.
Preparation
Flavoxate Tablets
17-43
IMPURITIES
O
O
Me
COOH
A. 3-methylflavone-8-carboxylic acid
O
O
Me
COOEt
B. 3-methylflavone-8-carboxylic acid ethyl ester
17-44
Flecainide Acetate
corrected 1/01
N
HN
O
H
O F
3
C
O
CF
3
H ,H
3
C COOH
and enantiomer
C
17
H
20
F
6
N
2
O
3
,C
2
H
4
O
2
474.4 54143-55-4
Flecainide Acetate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1324].
Preparations
Flecainide Injection
Flecainide Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Flecainide acetate contains not less than 98.0 per cent and not more than the equivalent of 101.0 per
cent of N-[(RS)-(piperidin-2-ylmethyl)]-2,5-bis(2,2,2-trifluoroethoxy)benzamide acetate, calculated
CHARACTERS
A white or almost white, crystalline powder, very hygroscopic, soluble in water and in ethanol. It is
freely soluble in dilute acetic acid and practically insoluble in dilute hydrochloric acid.
IDENTIFICATION
A. Melting point (2.2.14): 146C to 152C. The melting range is not greater than 3C.
B. Dissolve 50 mg in alcohol R and dilute to 50.0 ml with the same solvent. Dilute 5.0 ml of the
solution to 50.0 ml with the same solvent. Examined between 230 nm and 350 nm (2.2.25), the
solution shows an absorption maximum at 298 nm. The specific absorbance at the maximum is 61 to
65.
obtained with flecainide acetate CRS.
D. It gives reaction (b) of acetates (2.3.1).
TESTS
Appearance of solution Dissolve 0.25 g in water R, add 0.05 ml of glacial acetic acid R and dilute to
10 ml with water R. The solution is clear (2.2.1) and colourless (Method II, 2.2.2).
the solution to 10.0 ml with the same solvent.
Reference solution (b). Dissolve 25 mg of flecainide acetate CRS and 25 mg of flecainide impurity A

CRS
17-45
Mobile phase A. Mix 2 ml of concentrated ammonia R, 4 ml of triethylamine R and 985 ml of
water R. Add 6 ml of phosphoric acid R and adjust to pH 2.8 with concentrated ammonia R,
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
90 10 equilibration
a loop injector.
If a suitable baseline cannot be obtained, use another grade of triethylamine. Inject 20 l of
reference solution (b). The test is not valid unless the resolution between the two peaks in the
chromatogram obtained is at least four. Inject 20 l of the test solution and 20 l of reference solution
(a). In the chromatogram obtained with the test solution, the area of any peak apart from the
principal peak is not greater than 0.4 times the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.2 per cent) and the sum of the areas of all the peaks apart
from the principal peak is not greater than the area of the peak in the chromatogram obtained with
reference solution (a) (0.5 per cent). Disregard any peak with an area less than 0.02 times that of the
Impurity B Examine by thin-layer chromatography (2.2.27), using a TLC silica gel F
254
plate R.
the same solvent.
Reference solution. Dissolve 10 mg of flecainide impurity B CRS in methanol R and dilute to 100 ml with
the same solvent (solution A). Dissolve 0.10 g of flecainide acetate CRS in solution A and dilute to
Apply separately to the plate 5 l of each solution. Develop over a path of 10 cm using a freshly
prepared mixture of 5 volumes of concentrated ammonia R and 95 volumes of acetone R. Dry the plate
at 100C to 105C until the odour of ammonia disappears completely and examine in ultraviolet light
at 254 nm to establish the position of the flecainide spot. Then spray with a freshly prepared 2 g/l
solution of ninhydrin R in methanol R and heat at 100C to 110C for 2 min to 5 min and examine in
daylight. In the chromatogram obtained with the test solution, any spot corresponding to impurity B
is not more intense than the spot corresponding to impurity B in the chromatogram obtained with
the reference solution (0.2 per cent). The test is not valid unless the chromatogram obtained with the
reference solution shows two clearly separated spots.
Loss on drying (2.2.32). Not more than 0.5 per cent determined on 1.000 g by drying in an oven at
60C under a pressure not exceeding 0.6 kPa for 2 h.
ASSAY
19
H
24
F
6
N
2
O
5
.
STORAGE
IMPURITIES
N
N
O CF
3
O F
3
C
A. 3-[2,5-bis(2,2,2-trifluoroethoxy)phenyl]-1,5,6,7,8,8a-hexahydroimidazo[1,5-a]pyridine,
17-46
N
H
2
N
H
H
and enantiomer
B. (RS)-(piperidin-2-yl)methanamine,
O CF
3
O F
3
C
N
O
N
H
HO
H
H
and enantiomer
C. (RS)-4-hydroxy-N-(piperidin-2-ylmethyl)-2,5-bis(2,2,2-trifluoroethoxy)benzamide,
O CF
3
O F
3
C COOH
D. 2,5-bis(2,2,2-trifluoroethoxy)benzoic acid,
O CF
3
O F
3
C
N
O
N
H
E. N-(pyridin-2-ylmethyl)-2,5-bis(2,2,2-trifluoroethoxy) benzamide.
__________________________________________________________________________________________________________ Ph Eur
17-47
Flucloxacillin Magnesium
2
Mg
2+
H
N
O
O
N
Me
Cl
F
COO
_
H H
Me
Me
H
O
S
N
(C
19
H
16
ClFN
3
O
5
S)
2
Mg,8H
2
O 1074.2 58486-36-5
Definition Flucloxacillin Magnesium is magnesium (6R)-6-[3-(2-chloro-6-fluorophenyl)-5-
methylisoxazole-4-carboxamido]penicillanate octahydrate. It contains not less than 95.0% and not
more than 100.5% of (C
19
H
16
ClFN
3
O
5
S)
2
Mg, calculated with reference to the anhydrous substance.
Slightly soluble in water and in chloroform; freely soluble in methanol.
Identification
flucloxacillin magnesium (RS 144).
B. Carry out the method for thin-layer chromatography, Appendix III A, using a silanised silica gel F
254
precoated plate (Merck silanised silica gel 60 F
254
plates are suitable) and a mixture of 70 volumes of
a 15.4% w/v solution of ammonium acetate previously adjusted to pH 5.0 with glacial acetic acid and
30 volumes of acetone as the mobile phase. Apply separately to the plate 1 l of each of three solutions
in water containing (1) 0.5% w/v of the substance being examined, (2) 0.5% w/v of flucloxacillin
sodium BPCRS and (3) 0.5% w/v each of cloxacillin sodium BPCRS, dicloxacillin sodium EPCRS and
flucloxacillin sodium BPCRS. After removal of the plate, allow it to dry in air and expose to iodine
vapour until spots appear. The principal spot in the chromatogram obtained with solution (1)
corresponds in colour, position and size to that in the chromatogram obtained with solution (2). The
test is not valid unless the chromatogram obtained with solution (3) shows three clearly separated
principal spots.
C. Yields reaction B characteristic of magnesium salts, Appendix VI.
Specific optical rotation In a 0.5% w/v solution, +163 to +173, calculated with reference to the
Iodine-absorbing substances Not more than 5.0%, calculated with reference to the anhydrous
substance, when determined by the following method. Dissolve 0.125 g in sufficient mixed phosphate
buffer pH 7.0 to produce 25 ml. To 10 ml add 10 ml of mixed phosphate buffer pH 4.0 and 10 ml of
0.01M iodine VS and titrate immediately with 0.01M sodium thiosulphate VS using starch mucilage,
added towards the end of the titration, as indicator. Repeat the operation without the substance
being examined. The difference between the titrations represents the amount of iodine-absorbing
substances present. Each ml of 0.01M sodium thiosulphate VS is equivalent to 0.524 g of iodine-
absorbing substances.
Assay Dissolve 0.1 g in sufficient buffered sodium acetate solution pH 6.0 to produce 500 ml and dilute
25 ml to 100 ml with the same buffer. Place two 2-ml aliquots of the resulting solution in separate
stoppered tubes. To one tube add 10 ml of imidazolemercury reagent, mix, stopper the tube and
immerse in a water bath at 60 for exactly 25 minutes, swirling occasionally. Remove from the water
bath and cool rapidly to 20 (solution A). To the second tube add 10 ml of water and mix (solution
B). Without delay measure the absorbances of solutions A and B at the maximum at 346 nm,
Appendix II B, using in the reference cell a mixture of 2 ml of water and 10 ml of imidazolemercury
reagent for solution A and water for solution B. Calculate the content of (C
19
H
16
ClFN
3
O
5
S)
2
Mg from
the difference between the absorbances of solutions A and B, from the difference obtained by
repeating the procedure using flucloxacillin sodium BPCRS in place of the substance being examined
and from the declared content of C
19
H
17
ClFN
3
O
5
S in flucloxacillin sodium BPCRS. Each mg of
C
19
H
17
ClFN
3
O
5
S is equivalent to 1.025 mg of (C
19
H
16
ClFN
3
O
5
S)
2
Mg.
Storage Flucloxacillin Magnesium should be kept in a well-closed container, protected from
moisture and stored at a temperature not exceeding 25.
Labelling The label states (1) the date after which the material is not intended to be used; (2) the
conditions under which it should be stored.
17-48
Preparations
Flucloxacillin Oral Suspension
Co-fluampicil Oral Suspension
17-49
Flucloxacillin Sodium
H
N
O
O
N
Me
Cl
F
COONa
H H
Me
Me
H
O
S
N
C
19
H
16
ClFN
3
NaO
5
S,H
2
O 493.9 1847-24-1
Flucloxacillin Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparations
Flucloxacillin Capsules
Co-fluampicil Capsules
Flucloxacillin Injection
Flucloxacillin Oral Solution
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Flucloxacillin sodium contains not less than 95.0 per cent and not more than the equivalent of
101.0 per cent of sodium (2S,5R,6R)-6-[[[3-(2-chloro-6-fluorophenyl)-5-methylisoxazol-4-
yl]carbonyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate, calculated
PRODUCTION
(2.2.28) using a suitable, validated method.
CHARACTERS
A white or almost white, crystalline powder, hygroscopic, freely soluble in water and in methanol,
soluble in alcohol.
IDENTIFICATION
obtained with flucloxacillin sodium CRS.
substance.
Reference solution (a). Dissolve 25 mg of flucloxacillin sodium CRS in 5 ml of water R.
Reference solution (b). Dissolve 25 mg of cloxacillin sodium CRS, 25 mg of dicloxacillin sodium CRS and
25 mg of flucloxacillin sodium CRS in 5 ml of water R.
30 volumes of acetone R and 70 volumes of a 154 g/l solution of ammonium acetate R the pH of which
for 1 min; the solution becomes yellow.
17-50
TESTS
solvent. The specific optical rotation is +158 to +168 , calculated with reference to the anhydrous
substance.
test solution (a) and continue the chromatography for five times the retention time of the principal
peak. Inject reference solution (b). In the chromatogram obtained with test solution (a): the area of
chromatogram obtained with reference solution (b) (1 per cent); the sum of the areas of all peaks,
(b).
mination of water.
Inject per kilogram of the rabbits mass 1 ml of a solution in water for injections R containing 20 mg of
ASSAY
Reference solution (a). Dissolve 50.0 mg of flucloxacillin sodium CRS in the mobile phase and dilute to
Reference solution (c). Dissolve 5 mg of flucloxacillin sodium CRS and 5 mg of cloxacillin sodium CRS in
the mobile phase and dilute to 50.0 ml with the mobile phase.
volumes of a 2.7 g/l solution of potassium dihydrogen phosphate R (Adjust to pH 5.0 by addition of
dilute sodium hydroxide solution R),
17-51
peaks in the chromatogram obtained are at least 50 per cent of the full scale of the recorder. The test
is not valid unless the resolution between the first peak (cloxacillin) and the second peak
(flucloxacillin) is at least 2.5. Inject reference solution (a) six times. The test is not valid unless the
relative standard deviation of the peak area for flucloxacillin is at most 1.0 per cent. Inject alternately
test solution (b) and reference solution (a).
STORAGE
LABELLING
The label states:
IMPURITIES
H
HN
S
N
N
O
H COOH
Me
Me
COOH O
Me
Cl
F
*
*
A. (4S)-2-[carboxy[[[3-(2-chloro-6-fluorophenyl)-5-methyl-isoxazol-4-yl]carbonyl]-
amino]methyl]-5,5-dimethyl-thiazolidine-4-carboxylic acid (penicilloic acids of flucloxacillin),
and epimer at C*
HN
S
N
N
O
H COOH
Me
Me
O
Me
Cl
F
H
H
*
B. (2RS,4S)-2-[[[[3-(2-chloro-6-fluorophenyl)-5-methyl-isoxazol-4-yllcarbonyl]amino]-
methyl]-5,5-dimethylthiazolidine-4-carboxylic acid (penilloic acids of flucloxacillin),
N
S
H COOH
Me
Me
O
H H
H
2
N
C. (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic
N
O
Me
COOH
F
Cl
D. 3-(2-chloro-6-fluorophenyl)-5-methylisoxazole-4-carboxylic acid.
__________________________________________________________________________________________________________ Ph Eur
17-52
Flucytosine
H
N O
F
N
NH
2
C
4
H
4
FN
3
O 129.1 2022-85-7
Flucytosine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0766]. These
Preparation
Flucytosine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Flucytosine contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent
of 4-amino-5-fluoropyrimidin-2(1H)-one, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, sparingly soluble in water, slightly soluble in ethanol,
IDENTIFICATION
obtained with flucytosine CRS.
C. Mix about 5 mg with 45 mg of heavy magnesium oxide R and ignite in a crucible until an almost
colourless. Filter and add to the filtrate a freshly prepared mixture of 0.1 ml of alizarin S solution R
and 0.1 ml of zirconyl nitrate solution R. Mix, allow to stand for 5 min and compare the colour of the
solution with that of a blank prepared in the same manner. The colour of the solution changes from
red to yellow.
D. To 5 ml of solution S (see Tests) add 0.15 ml of bromine water R and shake. The colour of the
solution is discharged.
TESTS
solution BY
7
or Y
7
(Method II, 2.2.2).
Test solution (a). Dissolve 50 mg of the substance to be examined in a mixture of 10 volumes of
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with a mixture of 10 volumes of water R and
Reference solution (a). Dissolve 10 mg of flucytosine CRS in a mixture of 10 volumes of water R and 15
Reference solution (b). Dilute 1 ml of test solution (b) to 100 ml with a mixture of 10 volumes of
water R and 15 volumes of methanol R.
Reference solution (c). Dissolve 5 mg of fluorouracil CRS in 5 ml of reference solution (a).
17-53
Apply separately to the plate 10 l of each solution. Develop over a path of 12 cm in an
unsaturated tank using a mixture of 1 volume of anhydrous formic acid R, 15 volumes of water R, 25
volumes of methanol R and 60 volumes of ethyl acetate R. Allow the solvents to evaporate. At the
bottom of a chromatography tank place an evaporating dish containing a mixture of 2 volumes of a
15 g/l solution of potassium permanganate R, 1 volume of hydrochloric acid R1 and 1 volume of water R,
close the tank and allow to stand for 15 min. Place the dried plate in the tank and close the tank.
Leave the plate in contact with the chlorine vapour for 5 min. Withdraw the plate and place it in a
current of cold air until the excess of chlorine is removed and an area of the coating below the
points of application does not give a blue colour with a drop of potassium iodide and starch solution R.
Spray with potassium iodide and starch solution R. Examine the plate in daylight. Any spot in the
than the spot in the chromatogram obtained with reference solution (b) (0.1 per cent). The test is
not valid unless the chromatogram obtained with reference solution (c) shows two clearly
separated spots.
Fluoride Not more than 200 ppm. Carry out a potentiometric determination of fluoride ion, using
a fluoride-selective indicator electrode and a silversilver chloride reference electrode.
Prepare and store all solutions in plastic containers.
Buffer solution. Dissolve 58 g of sodium chloride R in 500 ml of water R. Add 57 ml of glacial acetic
acid R and 200 ml of a 100 g/l solution of cyclohexylenedinitrilotetra-acetic acid R in 1M sodium
hydroxide. Adjust the pH to 5.0 to 5.5 with a 200 g/l solution of sodium hydroxide R and dilute to
the same solvent.
Reference solutions. Dissolve 4.42 g of sodium fluoride R, previously dried at 120C for 2 h, in 300 ml of
water R and dilute to 1000.0 ml with the same solvent (solution (a): 1.9 g/l of fluoride). Prepare three
reference solutions by dilution of solution (a) 1 in 100, 1 in 1000 and 1 in 10,000.
To 20.0 ml of each reference solution, add 10.0 ml of the buffer solution and stir with a magnetic
stirrer. Introduce the electrodes into the solution and allow to stand for 5 min with constant stirring.
Determine the potential difference between the electrodes. Plot on semi-logarithmic graph paper the
potential difference obtained for each solution as a function of concentration of fluoride. Using
exactly the same conditions, determine the potential difference obtained with the test solution and
calculate the content of fluoride.
Heavy metals (2.4.8). 1.0 g complies with limit test C for heavy metals (20 ppm). Use a platinum
100C to 105C.
ASSAY
Dissolve 0.100 g in 40 ml of anhydrous acetic acid R and add 100 ml of acetic anhydride R. Titrate
with 0.1M perchloric acid determining the end-point potentiometrically (2.2.20).
4
H
4
FN
3
O.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
17-54
Fludrocortisone Acetate
H
O
O
F
HO
OH
OAc
Me
Me
H
C
23
H
31
FO
6
422.5 514-36-3
Fludrocortisone Acetate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Fludrocortisone Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Fludrocortisone acetate contains not less than 97.0 per cent and not more than the equivalent of
103.0 per cent of 9-fluoro-11,17,21-trihydroxypregn-4-ene-3,20-dione 21-acetate, calculated
CHARACTERS
ethanol, slightly soluble in ether.
IDENTIFICATION
obtained with fludrocortisone acetate CRS. If the spectra obtained in the solid state show differences,
volume of acetone R, evaporate to dryness and record the spectra again using the residues.
Reference solution (a). Dissolve 10 mg of fludrocortisone acetate CRS in a mixture of 1 volume of
solvents.
Reference solution (b). Dissolve 5 mg of cortisone acetate CRS in 5 ml of reference solution (a).
ence solution (a). Spray the plate with alcoholic solution of sulphuric acid R. Heat at 120C for 10 min
or until the spots appear. Allow to cool. Examine in daylight and in ultraviolet light at 365 nm. The
Test solution (a). Dissolve 25 mg of the substance to be examined in methanol R and dilute to 5 ml
with the same solvent. (This solution is also used to prepare test solution (b)). Dilute 2 ml of the
solution to 10 ml with methylene chloride R.
17-55
saturated methanolic potassium hydrogen carbonate solution R and immediately pass a stream of nitrogen R
through the solution for 5 min. Stopper the tube. Heat on a water-bath at 45C protected from
light for 2 h 30 min. Allow to cool.
Reference solution (a). Dissolve 25 mg of fludrocortisone acetate CRS in methanol R and dilute to 5 ml
with the same solvent. (This solution is also used to prepare reference solution (b)). Dilute 2 ml of
the solution to 10 ml with methylene chloride R.
nitrogen R through the solution for 5 min. Stopper the tube. Heat on a water bath at 45C protected
from light for 2 h 30 min. Allow to cool.
obtained with the corresponding reference solution. Spray with alcoholic solution of sulphuric acid R.
Heat at 120C for 10 min or until the spots appear. Allow to cool. Examine in daylight and in ultra-
violet light at 365 nm. The principal spot in each of the chromatograms obtained with the test
principal spots in the chromatograms obtained with test solution (b) and reference solution (b)
have R
f
values distinctly lower than those of the principal spots in the chromatograms obtained with
test solution (a) and reference solution (a).
red to yellow.
TESTS
substance.
Reference solution (a). Dissolve 2.0 mg of fludrocortisone acetate CRS and 2.0 mg of hydrocortisone
acetate CRS in the mobile phase and dilute to 50.0 ml with the mobile phase.
silica gel for chromatography R,
as mobile phase at a flow rate of 1 ml per minute a mixture of 35 volumes of tetrahydrofuran R
and 65 volumes of water R,
described above the retention times are: hydrocortisone acetate about 8.5 min and fludrocortisone
acetate about 10 min. The test is not valid unless the resolution between the peaks corresponding to
hydrocortisone acetate and fludrocortisone acetate is at least 1.0; if this resolution is not achieved,
adjust slightly the concentration of tetrahydrofuran in the mobile phase. Increasing the concentration
of tetrahydrofuran reduces the retention times.
17-56
and the sum of the areas of all such peaks, is not greater than 0.75 times the area of the principal
due to the solvent and any peak with an area less than 0.025 times the area of the principal peak in
100C to 105C.
ASSAY
Dissolve 10.0 mg of the substance to be examined in alcohol R and dilute to 100.0 ml with the same
solvent. Dilute 5.0 ml of this solution to 50.0 ml with alcohol R. Measure the absorbance (2.2.25) at
the maximum at 238 nm.
23
H
31
FO
6
__________________________________________________________________________________________________________ Ph Eur
17-57
Flumazenil
N
N
N
O
Me
COOEt
F
C
15
H
14
FN
3
O
3
303.3 78755-81-4
Flumazenil complies with the requirements of the 3rd edition of the European Pharmacopoeia [1326]. These
Action and use Benzodiazepine receptor antagonist.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Flumazenil contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of ethyl 8-fluoro-5-methyl-6-oxo-5,6-dihydro-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate,
CHARACTERS
A white or almost white, crystalline powder, very slightly soluble in water, freely soluble in methylene
chloride, sparingly soluble in methanol. It melts between 198C to 202C.
IDENTIFICATION
spectrum of flumazenil.
TESTS
7
(Method II,
2.2.2).
Dimethylformamide diethylacetal Dissolve 0.10 g of the substance to be examined in 0.5 ml of
methylene chloride R and dilute to 10 ml with butanol R. To 5.0 ml of this solution add 2.0 ml of
ninhydrin solution R and heat in a water-bath at 95C for 15 min. Any blue-purple colour in the
solution not more intense than that in a standard prepared at the same time and in the same manner
using 5.0 ml of a 0.1 g/l solution of dimethylformamide diethylacetal R in butanol R (1 per cent).
Reference solution (a). Dissolve 4 mg of chlordiazepoxide R in the mobile phase, add 2.0 ml of the test
solution and dilute to 50.0 ml with the mobile phase. Dilute 0.5 ml of this solution to 10.0 ml with
the mobile phase.
volumes of water R previously adjusted to pH 2.0 with phosphoric acid R,
conditions, the retention times are: chlordiazepoxide, about 6 min and flumazenil about 9 min. The
test is not valid unless the resolution between the peaks due to chlordiazepoxide and flumazenil is at
least 6.0; if necessary, adjust the concentration of acetonitrile in the mobile phase.
17-58
solution: the area of any peak, apart from the principal peak, is not greater than the area of the
any peak due to the solvent and any peak with an area less than 0.25 times the area of the principal
100C to 105C.
ASSAY
Dissolve 0.250 g in 50 ml of a mixture of 2 volumes of acetic anhydride R and 3 volumes of anhydrous
acetic acid R. Titrate with 0.1M perchloric acid, determining the end-point potentiometrically (2.2.20).
15
H
14
FN
3
O
3
.
IMPURITIES
N
N
N
F
O
Me
COOH
A. 8-fluoro-5-methyl-6-oxo-5,6-dihydro-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic
acid,
N
N
N
HO
O
Me
COOEt
B. ethyl 8-hydroxy-5-methyl-6-oxo-5,6-dihydro-4H-imidazo[1,5-a][1,4]benzodiazepine-3-
carboxylate,
Me
2
N OEt
OEt
C. dimethylformamide diethylacetal.
__________________________________________________________________________________________________________ Ph Eur
17-59
Flumequine
1/01
N
F
O
COOH
Me
H
and enantiomer
C
14
H
12
FNO
3
261.3 42835-25-6
Flumequine complies with the requirements of the 3rd edition of the European Pharmacopoeia [1517]. These
Action and use Antimicrobial.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Flumequine contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of (RS)-9-fluoro-5-methyl-1-oxo-6,7-dihydro-1H,5H-benzo[i,j]quinolizine-2-carboxylic acid,
CHARACTERS
A microcrystalline white powder, practically insoluble in water, freely soluble in dilute solutions of
alkali hydroxides, sparingly soluble in methylene chloride, very slightly soluble in methanol.
IDENTIFICATION
obtained with flumequine CRS.
B. It complies with the test for optical rotation (see Tests).
254
plate R.
Test solution. Dissolve 5 mg of the substance to be examined in 10 ml of methylene chloride R.
Reference solution. Dissolve 5 mg of flumequine CRS in 10 ml of methylene chloride R.
of ammonia R, 10 volumes of water R and 90 volumes of alcohol R. Allow the plate to dry in air.
Examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the
the reference solution.
solution with that of a blank prepared in the same manner. The colour of the test solution changes
from red to yellow. The colour of the blank solution remains red.
TESTS
Solution S Dissolve 5.00 g in 0.5M sodium hydroxide and dilute to 50.0 ml with the same solvent.
solution BY
5
(Method II, 2.2.2).
+0.10.
Test solution. Dissolve 35.0 mg of the substance to be examined in dimethylformamide R and dilute to
17-60
Reference solution (a). Dissolve 5.0 mg of flumequine CRS and 5.0 mg of flumequine impurity B CRS in
Reference solution (b). Dilute 1.0 ml of the test solution to 200.0 ml with dimethylformamide R.
volumes of a 1.36 g/l solution of potassium dihydrogen phosphate R,
Inject reference solution (a). When the chromatograms are recorded in the prescribed conditions,
the retention times are: impurity B, about 11 min and flumequine, about 13 min. The test is not
valid unless the resolution between the peaks corresponding to flumequine and impurity B is at least
2.0.
Inject separately dimethylformamide R as a blank, the test solution and reference solution (b).
Continue the chromatography of the test solution for three times the retention time of flumequine.
When the chromatograms are recorded in the prescribed conditions, the relative retention time for
impurity A is about 0.67 with respect to the flumequine peak. In the chromatogram obtained with
principal peak in the chromatogram obtained with reference solution (b) (1 per cent). Disregard any
peak obtained with dimethylformamide and any peak with an area less than 0.1 times the area of the
100C to 105C for 3 h.
ASSAY
14
H
12
FNO
3
.
IMPURITIES
N
COOH
O
H
Me
and enantiomer
A. (RS)-5-methyl-1-oxo-6,7-dihydro-1H,5H-benzo[i,j]quinolizine-2-carboxylic acid
(defluoroflumequine),
N
COOEt
O
H
Me
and enantiomer
B. ethyl (RS)-9-fluoro-5-methyl-1-oxo-6,7-dihydro-1H,5H-benzo[i,j]quinolizine-2-
carboxylate (flumequine ethyl ester).
__________________________________________________________________________________________________________ Ph Eur
17-61
Flumetasone Pivalate
1/01
H
O
F
O
OH
O
Me
Me
Me H
F
HO
Me
O
Me
Me
C
27
H
36
F
2
O
6
494.6 2002-29-1
Flumetasone Pivalate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Flumetasone pivalate contains not less than 97.0 per cent and not more than the equivalent of
103.0 per cent of 6,9-difluoro-11,17-dihydroxy-16-methyl-3,20-dioxopregna-1,4-dien-21-yl 2,2-
dimethylpropanoate, calculated with reference to the dried substance.
CHARACTERS
acetone, slightly soluble in alcohol and in methylene chloride.
IDENTIFICATION
obtained with flumetasone pivalate CRS. If the spectra obtained in the solid state show differences,
dissolve the substance to be examined and the reference substance separately in acetone R, evaporate
to dryness on a water-bath and record new spectra using the residues.
254
plate R.
the same solvent.
Reference solution (a). Dissolve 10 mg of flumetasone pivalate CRS in acetone R and dilute to 10 ml
Reference solution (b). Dissolve 10 mg of desoxycortone acetate CRS in acetone R and dilute to 10 ml
with the same solvent. Dilute 5 ml of the solution to 10 ml with reference solution (a).
Apply to the plate 5 l of each solution. Prepare the mobile phase by adding a mixture of 1.2 volumes
of water R and 8 volumes of methanol R to a mixture of 15 volumes of ether R and 77 volumes of
methylene chloride R. Develop over a path of 15 cm. Allow the plate to dry in air and examine in
solution (a). Spray the plate with alcoholic solution of sulphuric acid R. Heat at 120C for 10 min or
until the spots appear. Allow to cool. Examine in daylight and in ultraviolet light at 365 nm. The
C. Add about 2 mg to 2 ml of a mixture of 0.5 ml of water R and 1.5 ml of sulphuric acid R and shake
to dissolve. Within 5 min, a pink colour develops. Add the solution to 10 ml of water R and mix. The
colour fades and a clear solution remains.
17-62
zirconyl nitrate solution R add 1.0 ml of the filtrate. Mix, allow to stand for 5 min and compare the
TESTS
Solution S Dissolve 0.50 g in acetone R and dilute to 25.0 ml with the same solvent.
solution BY
6
(Method II, 2.2.2).
Reference solution (a). Dissolve 10 mg of dexamethasone pivalate CRS in the mobile phase and dilute to
100.0 ml with the mobile phase. Take 5.0 ml of the solution, add 5.0 ml of the test solution, mix and
as mobile phase at a flow rate of 0.6 ml/min a mixture of 5 volumes of tetrahydrofuran R, 30
volumes of acetonitrile R, 30 volumes of water R and 35 volumes of methanol R,
principal peak in the chromatogram obtained with reference solution (b) is at least 50 per cent of the
full scale of the recorder.
conditions, the relative retention time of dexamethasone pivalate, relative to flumetasone pivalate, is
about 1.1. The test is not valid unless in the chromatogram obtained with reference solution (a) the
resolution between the peaks corresponding to dexamethasone pivalate and flumetasone pivalate is at
least 2.8. If necessary adjust the concentration of tetrahydrofuran in the mobile phase.
Inject 20 l of the test solution. Continue the chromatography for 1.5 times the retention time of
the principal peak. In the chromatogram obtained with the test solution, the area of any peak, apart
gram obtained with reference solution (b) (1.5 per cent); the sum of the areas of any peaks, apart
from the principal peak, is not greater than the area of the principal peak in the chromatogram
obtained with reference solution (b) (2 per cent). Disregard any peak with an area less than 0.025
times the area of the principal peak in the chromatogram obtained with reference solution (b).
100C to 105C for 4 h.
ASSAY
27
H
36
F
2
O
6
STORAGE
IMPURITIES
O
F H
H Me
Me
HO
O
OR
1
OH
Me
R
2
A. R
1
= H, R
2
= F: 6,9-difluoro-11,17,21-trihydroxy-16-methylpregna-1,4-diene-3,20-dione
(flumetasone),
B. R
1
= CO-CH
3
, R
2
= F: 6,9-difluoro-11,17-dihydroxy-16-methyl-3,20-dioxopregna-1,4-dien-
21-yl acetate (flumetasone acetate),
17-63
C. R
1
= CO-C(CH
3
)
3
, R
2
= H: 9-fluoro-11,17-dihydroxy-16-methyl-3,20-dioxopregna-1,4-dien-
21-yl 2,2-dimethylpropanoate (dexamethasone pivalate),
D. R
1
= CO-C(CH
3
)
3
, R
2
= Cl: 6-chloro-9-fluoro-11,17-dihydroxy-16-methyl-3,20-
dioxopregna-1,4-dien-21-yl 2,2-dimethylpropanoate (chlordexamethasone pivalate).
__________________________________________________________________________________________________________ Ph Eur
17-64
Flunitrazepam
N
N
O
Me
O
2
N
F
C
16
H
12
FN
3
O
3
313.3 1622-62-4
Flunitrazepam complies with the requirements of the 3rd edition of the European Pharmacopoeia [0717]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Flunitrazepam contains not less than 98.5 per cent and not more than the equivalent of 101.5 per
cent of 5-(2-fluorophenyl)-1-methyl-7-nitro-1,3-dihydro-2H-1,4-benzodiazepin-2-one, calculated
CHARACTERS
A white or yellowish, crystalline powder, practically insoluble in water, soluble in acetone, slightly
soluble in alcohol and in ether.
IDENTIFICATION
obtained with flunitrazepam CRS.
D. Dissolve about 10 mg in 2 ml of methanol R, heating if necessary. Add 0.05 ml of dilute sodium
hydroxide solution R. An intense yellow colour develops.
red to yellow.
TESTS
Related substances Carry out the test protected from light. Examine by thin-layer chromatography
(2.2.27), using silica gel GF
254
Test solution (a). Dissolve 0.20 g of the substance to be examined in acetone R and dilute to 5 ml with
the same solvent. Prepare the solution immediately before use.
Reference solution (a). Dilute 1 ml of test solution (a) to 20 ml with acetone R. Dilute 3 ml of this
Reference solution (b). Dissolve 8 mg of flunitrazepam CRS in acetone R and dilute to 10 ml with the
same solvent.
Reference solution (c). Dissolve 8 mg of flunitrazepam CRS and 8 mg of nitrazepam CRS in acetone R
17-65
15 volumes of ethyl acetate R and 85 volumes of nitromethane R. Allow the plate to dry in air and
reference solution (a) (0.3 per cent). The test is not valid unless the chromatogram obtained with
reference solution (c) shows two clearly separated principal spots.
100C to 105C.
ASSAY
16
H
12
FN
3
O
3
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
17-66
Fluocinolone Acetonide
1/01
H
O
O
F
O
O
OH
Me
Me
F
HO
H Me
Me
C
24
H
30
F
2
O
6
452.5 67-73-2
Fluocinolone Acetonide complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparations
Fluocinolone Cream
Fluocinolone Ointment
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Fluocinolone acetonide contains not less than 97.0 per cent and not more than the equivalent of
103.0 per cent of 6,9-difluoro-11,21-dihydroxy-16,17-(1-methylethylidenedioxy)pregna-1,4-
diene-3,20-dione, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water, soluble in acetone and in
ethanol.
IDENTIFICATION
obtained with fluocinolone acetonide CRS.
B. Examine the chromatograms obtained in the test for related substances. The retention time of the
principal peak in the chromatogram obtained with reference solution (b) is approximately the same as
that of the peak due to fluocinolone acetonide CRS in the chromatogram obtained with reference
solution (a).
TESTS
substance.
Related substances Examine by liquid chromatography (2.2.29). Carry out the test protected from
light.
Test solution. Dissolve 25.0 mg of the substance to be examined in acetonitrile R and dilute to 10.0 ml
Reference solution (a). Dissolve 2.5 mg of fluocinolone acetonide CRS and 2.5 mg of triamcinolone
acetonide R in acetonitrile R and dilute to 100.0 ml with the same solvent.
Reference solution (b). Dilute 1.0 ml of test solution (a) to 100.0 ml with acetonitrile R.
deactivated end-capped octadecylsilyl silica gel for chromatography R (5 m),
17-67
conditions, the retention times are: triamcinolone acetonide about 8.5 min and fluocinolone
acetonide about 10 min. The test is not valid unless the resolution between the peaks corresponding
to triamcinolone acetonide and fluocinolone acetonide is at least 3.0; if necessary, adjust the concen-
tration of acetonitrile in the mobile phase.
Inject separately 20 l of acetonitrile R as a blank, 20 l of the test solution and 20 l of reference
solution (b). Continue the chromatography of the test solution for four times the retention time of
the principal peak. In the chromatogram obtained with the test solution: the area of any peak, apart
obtained with reference solution (b) (1 per cent) and not more than one such peak has an area
greater than half the area of the principal peak in the chromatogram obtained with reference solution
(b) (0.5 per cent); the sum of the areas of all the peaks, apart from the principal peak, is not greater
(b) (2.5 per cent). Disregard any peak obtained with the blank run and any peak with an area less
(b).
100C to 105C for 3 h.
ASSAY
Protect the solutions from light throughout the assay.
24
H
30
F
2
O
6
STORAGE
IMPURITIES
F H
H Me
Me
HO
O COOH
O
F
O
Me
Me
A. 6,9-difluoro-11-hydroxy-16,17-(1-methylethylidenedioxy)-3,20-dioxopregna-1,4-dien-
21-oic acid,
F H
H Me
Me
HO
O
F
O
Me
Me
COOH
B. 6,9-difluoro-11-hydroxy-16,17-(1-methylethylidenedioxy)-3-oxoandrosta-1,4-diene-
17-carboxylic acid,
F H
H Me
Me
HO
O
OH
F
OH
OH
C. 6,9-difluoro-11,16,17,21-tetrahydroxypregna-1,4-diene-3,20-dione,
17-68
F H
H Me
Me
HO
O CHO
O
F
O
Me
Me
D. 6,9-difluoro-11-hydroxy-16,17-(1-methylethylidenedioxy)-3,20-dioxopregna-1,4-
dien-21-al,
H
Me
Me
H
O
O
F
O
Me
Me
OH
H
O
E. 9,11-epoxy-6-fluoro-21-hydroxy-16,17-(1-methylethylidenedioxy)-9-pregna-1,4-
diene- 3,20-dione,
H H
H Me
Me
O
O
F
O
OH
Me
Me
F. 6-fluoro-21-hydroxy-16,17-(1-methylethylidenedioxy)pregn-4-ene-3,20-dione,
H H
H Me
Me
HO
O
O
F
O
OAc
Me
Me
G. 6-fluoro-11-hydroxy-16,17-(1-methylethylidenedioxy)-3,20-dioxopregn-4-en-21-yl
acetate.
_______________________________________________________________________________________________________________ Ph Eur
17-69
Fluocinolone Acetonide Dihydrate
C
24
H
30
F
2
O
6
,2H
2
O 488.5
Definition Fluocinolone Acetonide Dihydrate is 6,9-difluoro-11,21-dihydroxy-16-,17-
isopropylidenedioxypregna-1,4-diene-3,20-dione dihydrate. It contains not less than 96.0% and not
24
H
30
F
2
O
6
Practically insoluble in water; freely soluble in acetone; soluble in absolute ethanol; sparingly soluble
in dichloromethane and in methanol; practically insoluble in hexane.
Identification
fluocinolone acetonide dihydrate (RS 147).
B. Complies with the test for identification of steroids, Appendix III A, using impregnating solvent I and
mobile phase H. Apply 5 l of each of the three solutions.
C. Complies with the test for identification of steroids, Appendix III A, using the conditions specified in
test B but using solutions prepared in the following manner. For solution (1) dissolve 10 mg in
1.5 ml of glacial acetic acid in a separating funnel, add 0.5 ml of a 2% w/v solution of chromium(VI)
oxide and allow to stand for 30 minutes. Add 5 ml of water and 2 ml of dichloromethane and shake
vigorously for 2 minutes. Allow to separate and use the lower layer. Prepare solution (2) in the same
manner but using 10 mg of fluocinolone acetonide EPCRS.
Light absorption Dissolve 15 mg in sufficient absolute ethanol to produce 100 ml. Dilute 10 ml of
the solution to 100 ml with absolute ethanol. The A(1%, 1 cm) of the resulting solution at the
maximum at 239 nm is 345 to 375, calculated with reference to the anhydrous substance, Appendix
II B.
ence to the anhydrous substance, Appendix V F.
gel GF
254
ether, 8 volumes of methanol and 1.2 volumes of water as the mobile phase. Apply separately to the
plate 5 l of each of three solutions of the substance being examined in chloroform containing (1)
1.0% w/v, (2) 0.020% w/v and (3) 0.010% w/v. After removal of the plate, allow it to dry in air and
examine under ultraviolet light (254 nm). Any secondary spot in the chromatogram obtained with
solution (1) is not more intense than the spot in the chromatogram obtained with solution (2) and
not more than one such spot is more intense than the spot in the chromatogram obtained with
solution (3).
Assay Carry out the tetrazolium assay of steroids, Appendix VIII J, and calculate the content of
C
24
H
30
F
2
O
6
from the absorbance obtained by repeating the operation using fluocinolone acetonide
EPCRS in place of the substance being examined.
Storage Fluocinolone Acetonide Dihydrate should be kept in a well-closed container and protected
from light.
Preparations
Fluocinolone Cream
Fluocinolone Ointment
17-70
Fluocinonide
Me
H
H Me
O
O
O
O
Me
Me
F
HO
OAc
H
H
H F
C
26
H
32
F
2
O
7
494.5 356-12-7
Definition Fluocinonide is 6,9-difluoro-11-hydroxy-16,17-isopropylidenedioxy-3,20-
dioxopregna-1,4-dien-21-yl acetate. It contains not less than 97.0% and not more than 103.0% of
C
26
H
32
F
2
O
7
Practically insoluble in water; slightly soluble in chloroform and in absolute ethanol. It melts at about
220, with decomposition.
Identification
fluocinonide (RS 148).
B. Carry out the method for thin-layer chromatography, Appendix III A, using a silica gel F
254
254
plates are suitable) and a mixture of 12 volumes of water, 80
volumes of methanol, 150 volumes of ether and 770 volumes of dichloromethane as the mobile phase;
mix the water and the methanol before adding to the remaining components of the mobile phase.
Apply separately to the plate 5 l of each of the following solutions. For solution (1) dissolve 25 mg
of the substance being examined in 5 ml of methanol (solution A); dilute 2 ml of solution A to 10 ml
with chloroform. For solution (2) transfer 2 ml of solution A to a 15-ml glass tube with a ground-glass
stopper, add 10 ml of saturated methanolic potassium hydrogen carbonate solution and immediately pass
a current of nitrogen briskly through the solution for 2 minutes. Stopper the tube, heat in a water bath
at 45 protected from light for 2.5 hours and allow to cool. For solution (3) dissolve 25 mg of
fluocinonide BPCRS in 5 ml of methanol (solution B); dilute 2 ml of solution B to 10 ml with chloro-
form. Prepare solution (4) in the same manner as solution (2) but use 2 ml of solution B in place of
2 ml of solution A. After removal of the plate, allow it to dry in air and examine under ultraviolet light
(254 nm). The principal spots in each of the chromatograms obtained with solutions (1) and (2) are
similar in position and size to those in the chromatograms obtained with solutions (3) and (4),
respectively.
Specific optical rotation In a 1.0% w/v solution in chloroform, +81 to +89, calculated with refer-
Related substances Carry out the method for thin-layer chromatography, Appendix III A, using the
precoated plate and the mobile phase specified in test B for Identification. Apply separately to the
plate 5 l of each of four solutions in chloroform containing (1) 0.50% w/v of the substance being
examined, (2) 0.010% w/v of the substance being examined, (3) 0.0050% w/v of the substance being
examined and (4) 0.010% w/v of fluocinolone acetonide BPCRS. After removal of the plate, allow it to
dry in air, heat the plate at 105 for 10 minutes, cool and spray with alkaline tetrazolium blue solution.
Any secondary spot in the chromatogram obtained with solution (1) is not more intense than the spot
in the chromatogram obtained with solution (2) (2%) and not more than one such spot is more
intense than the spot in the chromatogram obtained with solution (3) (1%). The test is not valid
unless the principal spot in the chromatogram obtained with solution (2) has an Rf value relative to
the spot in the chromatogram obtained with solution (4) of at least 1.5.
Loss on drying When dried at 100 to 105 for 3 hours, loses not more than 1.0% of its weight. Use
1 g.
methanol containing (1) 0.012% w/v of the substance being examined and (2) 0.012% w/v of
fluocinonide BPCRS.
mobile phase with a flow rate of 1 ml per minute a mixture of 1 volume of glacial acetic acid, 450
volumes of acetonitrile and 550 volumes of water and (c) a detection wavelength of 238 nm.
17-71
26
H
32
F
2
O
7
26
H
32
F
2
O
7
in fluocinonide
BPCRS.
Storage Fluocinonide should be kept in a well-closed container and protected from light.
Preparations
Fluocinonide Cream
Fluocinonide Ointment
IMPURITIES
H
O
O
F
F
O H Me
Me
O
Me
Me
OAc
O
A. 6,9-difluoro-16,17-isopropylidene-3,11,20-trioxopregna-1,4-dien-21-yl acetate,
H
O
O
F
F
O H Me
Me
O
Me
Me
OH
HO
B. 6,9-difluoro-11,21-dihydroxy-16,17-isopropylidenepregna-1,4-diene-3,20-dione.
17-72
Fluocortolone Hexanoate
H
O
O
HO
H
F
Me Me
Me
H
OCO[CH
2
]
4
CH
3
C
28
H
39
FO
5
474.6 303-40-2
Definition Fluocortolone Hexanoate is 6-fluoro-11-hydroxy-16-methyl-3,20-dioxopregna-1,4-
dien-21-yl hexanoate. It contains not less than 97.0% and not more than 103.0% of C
28
H
39
FO
5
,
Characteristics A white or creamy white, crystalline powder; odourless or almost odourless.
Practically insoluble in water and in ether; sparingly soluble in chloroform; slightly soluble in acetone
and in 1,4 -dioxan; very slightly soluble in ethanol (96%) and in methanol.
It exhibits polymorphism.
Identification
The infrared absorption spectrum, Appendix II A, is concordant with the reference spectrum of fluocortol-
one hexanoate (RS 149).
Light absorption Ratio of the absorbance of the solution prepared as directed in the Assay at the
maximum at 242 nm to that at 263 nm, 2.15 to 2.35, Appendix II B.
Specific optical rotation In a 1% w/v solution in 1,4-dioxan, prepared with the aid of heat, +97 to
+103, calculated with reference to the dried substance, Appendix V F.
Related foreign steroids Carry out the method for thin-layer chromatography, Appendix III A, using
silica gel G as the coating substance and a mixture of 0.2 volume of water, 5 volumes of methanol and
95 volumes of 1,2-dichloroethane as the mobile phase. Apply separately to the plate 3 l of solution (1)
and 1 l of each of solutions (2) and (3). Solution (1) contains 0.50% w/v of the substance being
examined in acetone. Solution (2) contains 1.5% w/v of fluocortolone hexanoate BPCRS in a mixture of
1 volume of methanol and 9 volumes of chloroform. Solution (3) contains 0.030% w/v each of
prednisone EPCRS, prednisolone acetate EPCRS, cortisone acetate EPCRS and deoxycortone acetate
EPCRS in a mixture of 1 volume of methanol and 9 volumes of chloroform. After removal of the plate,
allow it to dry in air until the solvents have evaporated, heat at 105 for 10 minutes, cool and spray
with alkaline tetrazolium blue solution. The principal spot in the chromatogram obtained with solution
(1) corresponds in position, colour and intensity to that in the chromatogram obtained with solution
(2). Any secondary spot in the chromatogram obtained with solution (1) is not more intense than the
proximate spot in the chromatogram obtained with solution (3) (2%).
1 g.
Assay Dissolve 15 mg in sufficient methanol to produce 100 ml, dilute 20 ml to 100 ml with methanol
and measure the absorbance of the resulting solution at the maximum at 242 nm, Appendix II B.
28
H
39
FO
5
242 nm.
Storage Fluocortolone Hexanoate should be protected from light.
Preparation
Fluocortolone Cream
17-73
Fluocortolone Pivalate
H
O
O
HO
H
F
Me H Me
Me
OCOCMe
3
C
27
H
37
FO
5
460.6 29205-06-9
Fluocortolone Pivalate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Fluocortolone Cream
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Fluocortolone pivalate contains not less than 97.0 per cent and not more than the equivalent of
103.0 per cent of 6-fluoro-11,21-dihydroxy-16-methylpregna-1,4-diene-3,20-dione 21-pivalate,
CHARACTERS
chloride and in dioxan, sparingly soluble in alcohol.
IDENTIFICATION
obtained with fluocortolone pivalate CRS.
Reference solution (a). Dissolve 20 mg of fluocortolone pivalate CRS in a mixture of 1 volume of
solvents.
Reference solution (b). Dissolve 10 mg of norethisterone CRS in reference solution (a) and dilute to
C. To about 1 mg add 2 ml of a mixture of 2 volumes of glacial acetic acid R and 3 volumes of
sulphuric acid R and heat for 1 minute on a water-bath. A red colour is produced. Add 5 ml of
water R, the colour changes to violet-red.
17-74
solution with that of a blank prepared in the same manner. The test solution is yellow and the blank
is red.
TESTS
substance.
Test solution. Dissolve 10.0 mg of the substance to be examined in acetonitrile R and dilute to 10.0 ml
Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with acetonitrile R.
Reference solution (b). Dissolve 2 mg of fluocortolone pivalate CRS and 2 mg of prednisolone hexanoate
CRS in acetonitrile R and dilute to 100 ml with the same solvent.
as mobile phase at a flow rate of 1.5 ml per minute a mixture of 25 volumes of methanol R, 30
Inject 20 l of reference solution (b). The test is not valid unless, in the chromatogram obtained, the
resolution between the two principal peaks is at least 5.0. Inject 20 l of the test solution. Continue
the chromatography for twice the retention time of fluocortolone pivalate. In the chromatogram
obtained with the test solution: the area of any peak, apart from the principal peak, is not greater than
the area of the principal peak in the chromatogram obtained with reference solution (a) (1 per cent)
and the sum of the areas of any peaks, apart from the principal peak, is not greater than 2.0 times the
area of the principal peak in the chromatogram obtained with reference solution (a) (2 per cent).
Disregard any peak due to the solvent and any peak with an area less than 0.025 times that of the
100C to 105C.
ASSAY
solution to 100.0 ml with ethanol R. Measure the absorbance (2.2.25) at the maximum at 242 nm.
27
H
37
FO
5
STORAGE
IMPURITIES
H
O
O CH
2
OH
HO
H
F
Me H Me
Me
A. 6-fluoro-11,21-dihydroxy-16-methylpregna-1,4-diene-3,20-dione (fluocortolone),
H
O
O
HO
H
Me H Me
Me
OCOCMe
3
O OH
*
and epimer at C*
B. 6-hydroperoxy-11,21-dihydroxy-16-methylpregna-1,4-diene-3,20-dione 21-
dimethylpropanoate,
17-75
H
O
O
H
F
Me H Me
Me
OCOCMe
3
O
C. 6-fluoro-16-methyl-3,11,20-trioxopregna-1,4-diene-3,20-dione 21-dimethylpropanoate
H
O
O
HO
H
F
Me H Me
Me
OCOCMe
3
D. 6-fluoro-11,21-dihydroxy-16-methylpregn-4-ene-3,20-dione 21-pivalate.
__________________________________________________________________________________________________________ Ph Eur
17-76
Fluorescein Sodium
Soluble Fluorescein
O
COONa
O NaO
C
20
H
10
Na
2
O
5
376.3 518-47-8
Fluorescein Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia [1213].
Action and use Dye used for detection of abrasions of the cornea.
Preparations
Fluorescein Eye Drops
Fluorescein Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Fluorescein sodium contains not less than 95.0 per cent and not more than the equivalent of
103.0 per cent of disodium 2-(3-oxo-6-oxido-3H-xanthen-9-yl)benzoate, calculated with reference to
CHARACTERS
An orange-red, fine powder, hygroscopic, freely soluble in water, soluble in alcohol, practically
insoluble in hexane and in methylene chloride.
IDENTIFICATION
A. Dilute 0.1 ml of solution S (see Tests) to 10 ml with water R. The solution shows yellowish-green
fluorescence. The fluorescence disappears on addition of 0.1 ml of dilute hydrochloric acid R and
reappears on addition of 0.2 ml of dilute sodium hydroxide solution R.
spectrum of fluorescein sodium. Examine the substance prepared as a disc.
C. The absorption by a piece of filter paper of 0.05 ml of the solution prepared for identification A
(before the addition of dilute hydrochloric acid R) colours the paper yellow. On exposing the moist
paper to bromine vapour for 1 min and then to ammonia vapour the colour becomes deep pink.
D. Ignite 0.1 g in a porcelain crucible. Dissolve the residue in 5 ml of water R and filter. 2 ml of the
filtrate gives reaction (a) of sodium (2.3.1).
TESTS
Appearance of solution Solution S is clear (2.2.1) and orange-yellow with yellowish-green
fluorescence.
Related substances and resorcinol Examine by thin-layer chromatography (2.2.27), using as the
coating substance a suitable silica gel.
Test solution (a). Dissolve 0.10 g of the substance to be examined in a 10 g/l solution of hydrochloric
acid R in methanol R and dilute to 10 ml with the same acidic solution.
Test solution (b). Dissolve 0.250 g of the substance to be examined in 5 ml of water R. Transfer to a
separating funnel and wash with 3 ml of water R. Add 2 ml of buffer solution pH 8.0 R and 2.5 g of
sodium chloride R. Shake until the sodium chloride dissolves. Shake the solution obtained with two
quantities, each of 25 ml, of peroxide-free ether R. Dry the ether layers over anhydrous sodium
sulphate R and evaporate to dryness using a rotary evaporator. Dissolve the residue in 10 ml of a
10 g/l solution of hydrochloric acid R in methanol R.
17-77
Reference solution (a). Dilute 0.5 ml of test solution (a) to 100 ml with a 10 g/l solution of hydrochloric
acid R in methanol R.
Reference solution (b). Dilute 1 ml of test solution (a) to 50 ml with a 10 g/l solution of hydrochloric
acid R in methanol R. Dilute 1 ml of the solution to 10 ml with a 10 g/l solution of hydrochloric acid R
in methanol R.
Reference solution (c). Dissolve 25 mg of resorcinol R in methanol R and dilute to 100 ml with the same
solvent.
Reference solution (d). Dilute 10 ml of reference solution (c) to 20 ml with water R.
Reference solution (e). To 5 ml of reference solution (c) add 1 ml of test solution (a) and dilute to
Apply separately to the plate 5 l of test solutions (a) and (b) and 5 l of reference solutions (a), (b),
(d) and (e). Develop over a path of 15 cm using a mixture of 10 volumes of methanol R and 90
volumes of methylene chloride R. Allow the plate to dry in air. Examine in ultraviolet light at 365 nm.
Expose the plate to iodine vapour for 30 min and examine in daylight. In the chromatogram obtained
with test solution (a): any spot, apart from the principal spot, is not more intense than the principal
spot in the chromatogram obtained with reference solution (a) (0.5 per cent) and not more than one
such spot is more intense than the principal spot in the chromatogram obtained with reference
solution (b) (0.2 per cent). In the chromatogram obtained with test solution (b) any spot correspond-
ing to resorcinol is not more intense than the principal spot in the chromatogram obtained with
reference solution (d) (0.5 per cent). The test is not valid unless the chromatogram obtained with
reference solution (e) shows two clearly separated spots. The spots corresponding to resorcinol are
visible only after exposing the plate to iodine vapour.
Dimethylformamide Examine by gas chromatography (2.2.28) using dimethylacetamide R as the
internal standard.
Internal standard solution. Dilute 20 l of dimethylacetamide R to 100 ml with water R.
Test solution (a). Dissolve 1.0 g of the substance to be examined in 10 ml of water R. Add, with gentle
stirring, 10 ml of a 60 g/l solution of hydrochloric acid R. Allow to stand for 15 min and centrifuge.
Dissolve 0.10 g of trisodium phosphate dodecahydrate R in 5 ml of the supernatant.
Test solution (b). Dissolve 1.0 g of the substance to be examined in 10 ml of the internal standard
solution. Add, with gentle stirring, 10 ml of a 60 g/l solution of hydrochloric acid R. Allow to stand for
15 min and centrifuge. Dissolve 0.10 g of trisodium phosphate dodecahydrate R in 5 ml of the super-
natant.
Reference solution. Dilute 20 l of dimethylformamide R to 10 ml with water R. Dilute 1 ml of the
solution to 10 ml with water R. Add 10 ml of the internal standard solution and mix.
macrogol 1000 R,
detector at 170C.
Inject 2 l of each solution. In the chromatogram obtained with test solution (b), the ratio of the
area of any peak corresponding to dimethylformamide to the area of the peak due to the internal
standard is not greater than the corresponding ratio in the chromatogram obtained with the reference
solution (0.2 per cent).
Chlorides (2.4.4). To 10 ml of solution S add 90 ml of water R and 1 ml of dilute nitric acid R, wait
for at least 10 min and filter. 10 ml of the filtrate diluted to 15 ml with water R complies with the
Sulphates (2.4.13). To 5 ml of solution S add 90 ml of distilled water R, 2.5 ml of dilute hydrochloric
acid R, dilute to 100 ml with distilled water R and filter. 15 ml of the filtrate complies with the limit
test for sulphates (1.0 per cent).
Zinc Dilute 5 ml of solution S to 10 ml with water R. Add 2 ml of hydrochloric acid R1, filter and add
0.1 ml of potassium ferrocyanide solution R. No turbidity or precipitate is formed immediately.
at 100C to 105C.
ASSAY
Dissolve 50.0 mg in water R and dilute to 500.0 ml with the same solvent. Dilute 5.0 ml of this
solution to 200.0 ml with buffer solution pH 8.0 R. Measure the absorbance (2.2.25) at the maximum
at 492 nm.
17-78
20
H
10
Na
2
O
5
STORAGE
IMPURITIES
OH
OH
A. benzene-1,3-diol (resorcinol).
__________________________________________________________________________________________________________ Ph Eur
17-79
Fluorometholone
Me
F H
Me
OH
H
HO
O
Me
CH
3
O
C
22
H
29
FO
4
376.5 426-13-1
Definition Fluorometholone is 9-fluoro-11,17-dihydroxy-6-methylpregna-1,4-diene-3,20-
dione. It contains not less than 97.0% and not more than 103.0% of C
22
H
29
FO
4
, calculated with
Characteristics A white to yellowish white, crystalline powder. It melts at about 280, with
decomposition.
Practically insoluble in water; slightly soluble in absolute ethanol and in ether.
Identification
fluorometholone (RS 152).
B. In the Assay, the principal peak in the chromatogram obtained with solution (1) has the same
retention time as the principal peak in the chromatogram obtained with solution (2).
Specific optical rotation In a 1% w/v solution in pyridine, +52 to +60, Appendix V F, calculated
solutions in methanol containing (1) 0.010% w/v of the substance being examined, (2) 0.00005% w/v
of the substance being examined and (3) 0.00005% w/v each of deltamedrane BPCRS and
fluorometholone BPCRS.
mixture of 40 volumes of water and 60 volumes of methanol as the mobile phase with a flow rate of
2 ml per minute and (c) a detection wavelength of 254 nm.
The test is not valid unless in the chromatogram obtained with solution (3), the resolution factor
between the peaks due to deltamedrane and fluorometholone is at least 2.0.
the area of the principal peak in the chromatogram obtained with solution (2) (0.5%) and the sum of
the areas of any secondary peaks is not greater than twice the area of the principal peak in the chroma-
togram obtained with solution (2) (1%).
methanol containing (1) 0.005% w/v of the substance being examined, (2) 0.005% w/v of
fluorometholone BPCRS and (3) 0.00005% w/v each of deltamedrane BPCRS and fluorometholone
BPCRS.
between the peaks due to deltamedrane and fluorometholone is at least 2.0.
22
H
29
FO
4
from the chromatograms obtained and using the declared
content of C
22
H
29
FO
4
in fluorometholone BPCRS.
Preparation
Fluorometholone Eye Drops
17-80
IMPURITIES
H H
Me
Me
O
Me
HO
OH
O CH
3
A. 11,17-dihydroxy-6-methylpregna-1,4-diene-3,20-dione (deltamedrane)
H
Me
Me
O
Me
OH
O CH
3
O
H
B. 9,11-epoxy-17-hydroxy-6-methylpregna-1,4-diene-3,20-dione (epoxymedradiene)
17-81
Fluorouracil
NH
N O
O
F
H
C
4
H
3
FN
2
O
2
130.1 51-21-8
Fluorouracil complies with the requirements of the 3rd edition of the European Pharmacopoeia [0611]. These
Preparations
Fluorouracil Cream
Fluorouracil Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Fluorouracil contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent
of 5-fluoro-1H,3H-pyrimidine-2,4-dione, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, sparingly soluble in water, slightly soluble in alcohol,
IDENTIFICATION
obtained with fluorouracil CRS.
C. In a test-tube, heat 0.5 ml of a saturated solution of chromium trioxide R in sulphuric acid R in a
naked flame until white fumes appear in the upper part of the tube. The solution wets the side of the
tube and there is no appearance of greasiness. Add about 2 mg of the substance to be examined and
heat again in a naked flame until white fumes appear. The solution does not wet the sides of the tube.
TESTS
solution BY
7
or Y
7
(Method II, 2.2.2).
254
R as the
coating substance.
methanol R and water R and dilute to 10.0 ml with the same mixture of solvents.
Reference solution (a). Dissolve 20 mg of fluorouracil CRS in a mixture of equal volumes of methanol R
and water R and dilute to 10 ml with the same mixture of solvents.
Reference solution (b). Dilute 2.5 ml of reference solution (a) to 200 ml with a mixture of equal
volumes of methanol R and water R.
Reference solution (c). Dissolve 5 mg of 5-hydroxyuracil R in a mixture of equal volumes of methanol R
and water R and dilute to 200 ml with the same mixture of solvents.
15 volumes of methanol R, 15 volumes of water R and 70 volumes of ethyl acetate R. Allow the plate to
dry in air and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with
17-82
obtained with reference solution (b) (0.25 per cent). Spray with a freshly prepared 5 g/l solution of
fast blue B salt R and subsequently with 0.1M sodium hydroxide. Any spot corresponding to 5-hydroxy-
uracil in the chromatogram obtained with test solution (a) is not more intense than the spot in the
Heavy metals (2.4.8). Use a platinum crucible. 1.0 g complies with limit test C for heavy metals
(20 ppm). Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R.
80C for 4 h.
Sulphated ash (2.4.14). Use a platinum crucible. Not more than 0.1 per cent, determined on 1.0 g.
ASSAY
Dissolve 0.1000 g in 80 ml of dimethylformamide R, warming gently. Cool and titrate with 0.1M
tetrabutylammonium hydroxide, using 0.25 ml of a 10 g/l solution of thymol blue R in
dimethylformamide R as indicator. Carry out a blank titration.
4
H
3
FN
2
O
2.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
17-83
Fluoxetine Hydrochloride
O NHMe
H
F
3
C
,HCl
and enantiomer
C
17
H
18
F
3
NO,HCl 345.8 59333-67-4
Fluoxetine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparations
Fluoxetine Capsules
Fluoxetine Oral Solution
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Fluoxetine hydrochloride contains not less than 98.0 per cent and not more than the equivalent of
101.5 per cent of (RS)-N-methyl-3-phenyl-3-(4-trifluoromethylphenoxy)propan-1-amine hydro-
chloride, calculated with reference to the anhydrous, acetonitrile-free substance.
CHARACTERS
A white or almost white, crystalline powder, sparingly soluble in water, freely soluble in methanol,
sparingly soluble in methylene chloride.
IDENTIFICATION
obtained with fluoxetine hydrochloride CRS. Examine the substances prepared as discs.
TESTS
Solution S Dissolve 2.0 g in a mixture of 15 volumes of water R and 85 volumes of methanol R and
Optical rotation (2.2.7). 0.05 to +0.05, determined on solution S.
Related substances Examine by liquid chromatography (2.2.29) as prescribed under Assay, setting
the spectrophotometer at 215 nm.
Reference solution. Dissolve 22.0 mg of fluoxetine hydrochloride CRS in 10.0 ml of 0.5M sulphuric acid.
Heat at about 85C for 3 h. Allow to cool. The resulting solution contains considerable quantities of
fluoxetine impurity A and 4-trifluoromethylphenol. To 0.4 ml of the solution, add 28.0 mg of
fluoxetine hydrochloride CRS and about 1 mg of fluoxetine impurity B CRS and of fluoxetine impurity C
CRS and dilute to 25.0 ml with the mobile phase.
When the chromatograms are recorded in the prescribed conditions, the retention times relative to
fluoxetine hydrochloride are: impurity A, about 0.24; impurity B, about 0.27 and impurity C, about
0.94.
Inject 10 l of the reference solution.
The test is not valid unless: in the chromatogram obtained with the reference solution, the
retention time of the peak due to fluoxetine hydrochloride is 10 min to 18 min; the retention time of
the peak due to 4-trifluoromethylphenol is not greater than 35 min; the h/v ratio is not greater than
1.1 (h is the distance between the top of the peak due to impurity C and the base-line; v is the
distance between the top of the peak due to impurity C and the lowest point of the valley defined
17-84
between the peak due to impurity C and the peak due to fluoxetine hydrochloride). If the ratio is
greater than 1.1, reduce the volume of methanol and increase the volume of the solution of
triethylamine in the mobile phase.
Inject 10 l of test solution (a) and 10 l of test solution (b). Continue the chromatography for
three times the retention time of fluoxetine hydrochloride.
In the chromatogram obtained with test solution (b), the area of any peak due to impurity C is not
greater than 0.0015 times the area of the principal peak (0.15 per cent).
In the chromatogram obtained with test solution (a): the areas of any peaks due to impurity A and
B are not greater than 0.0125 times the area of the principal peak in the chromatogram obtained with
test solution (b) (0.25 per cent); none of the peaks, except the principal peak and the peaks due to
impurity A and B, has an area greater than 0.005 times the area of the principal peak in the chroma-
togram obtained with test solution (b) (0.1 per cent); the sum of the areas of all the peaks, apart from
the principal peak, is not greater than 0.025 times the area of the principal peak in the chromatogram
obtained with test solution (b) (0.5 per cent). Disregard any peak with an area less than 0.0025 times
that of the principal peak in the chromatogram obtained with test solution (b).
Acetonitrile Not more than 0.1 per cent, determined by gas chromatography (2.2.28).
Test solution. Dissolve 50 mg of the substance to be examined in dimethylformamide R and dilute to
Reference solution. To 1.0 g of acetonitrile R, add dimethylformamide R, mix and dilute to 100.0 ml with
the same solvent. Dilute 1.0 ml of solution to 1000.0 ml with dimethylformamide R.
a fused-silica capillary column 30 m long and about 0.53 mm in internal diameter coated with
macrogol 20,000 R (1 m thick),
maintaining the temperature of the column at 35C for 2 min then raising the temperature at a rate
of 15C per minute to 220C and maintaining at 220C for 10 min and maintaining the temperature
of the injection port and that of the detector at 250C.
Inject 1 l of the test solution, 1 l of the reference solution and 1 l of the dissolution solvent. In
the chromatogram obtained with the reference solution, note the retention time of acetonitrile.
In the chromatogram obtained with the dissolution solvent, verify that there is no peak with the same
retention time as acetonitrile.
The area of the peak due to acetonitrile in the chromatogram obtained with the test solution is not
greater than that of the peak in the chromatogram obtained with the reference solution.
of water.
ASSAY
Reference solution. Dissolve 55.0 mg of fluoxetine hydrochloride CRS in the mobile phase and dilute to
as mobile phase at a flow rate of 1 ml/min a mixture of 8 volumes of methanol R, 30 volumes of
tetrahydrofuran R and 62 volumes of a solution of triethylamine R prepared as follows: to 10 ml of
triethylamine R, add 980 ml of water R, mix and adjust to pH 6.0 with phosphoric acid R (about
4.5 ml) and dilute to 1000 ml with water R,
When using a recorder, adjust the sensitivity of the system so that the height of the principal peak in
the chromatogram obtained with the reference solution is at least 50 per cent of the full scale of the
recorder.
Adjust the volumes of methanol and the solution of triethylamine in the mobile phase so that the
retention time of fluoxetine hydrochloride is between 10 min and 18 min.
The assay is not valid unless the symmetry factor calculated at 10 per cent of the height of the peak
due to fluoxetine hydrochloride is at most 2.0.
Calculate the content of fluoxetine hydrochloride (C
17
H
19
ClF
3
NO) from the area of the peaks in
17-85
the chromatograms obtained with the test solution and the reference solution using the stated
content of C
17
H
19
ClF
3
NO in fluoxetine hydrochloride CRS and correcting for the content of water and
of acetonitrile in the substance to be examined.
STORAGE
IMPURITIES
NHMe
H OH
and enantiomer
A. (RS)-3-methylamino-1-phenylpropan-1-ol,
NHMe
B. N-methyl-3-phenylpropyl-1-amine,
F
3
C O NHMe
H
and enantiomer
C. (RS)-N-methyl-3-phenyl-3-(3-trifluoromethylphenoxy)propyl-1-amine.
__________________________________________________________________________________________________________ Ph Eur
17-86
Flupentixol Decanoate
S
CF
3
N
N
OCO[CH
2
]
8
CH
3
C
33
H
43
F
3
N
2
O
2
S 588.82 30909-51-4
Definition Flupentixol Decanoate is (Z)-2-{4-[3-(2-trifluoromethylthioxanthen-9-
ylidene)propyl]piperazin-1-yl}ethyl decanoate. It contains not less than 98.0% and not more than
101.0% of C
33
H
43
F
3
N
2
O
2
Characteristics A yellow, viscous oil.
Very slightly soluble in water; soluble in ethanol (96%); freely soluble in chloroform and in ether.
Identification
ethanol (96%) exhibits two maxima at 230 nm and 264 nm. The absorbances at the maxima are about
0.85 and about 0.35, respectively.
flupentixol decanoate (RS 154).
Heavy metals 1.0 g complies with limit test C for heavy metals, Appendix VII (20 ppm). Prepare the
standard using 2 ml of lead standard solution (10 ppm Pb).
Free alcohol Carry out the method for thin-layer chromatography, Appendix III A, using a silica gel
F
254
254
plates are suitable) and a mixture of 10 volumes of
diethylamine, 40 volumes of dichloromethane and 50 volumes of cyclohexane as the mobile phase and an
unsaturated tank. Apply separately to the plate 4 l of each of two solutions of the substance being
examined in dichloromethane containing (1) 0.25% w/v and (2) 0.000625% w/v. After removal of the
plate, allow it to dry in air, spray with a mixture of equal volumes of sulphuric acid and absolute
ethanol, heat at 110 for 5 minutes and examine under ultraviolet light (365 nm) immediately. In the
chromatogram obtained with solution (1) any secondary spot of the same colour as the principal spot is
not more intense than the spot in the chromatogram obtained solution (2) (0.25%).
2-Trifluoromethylthioxanthone Carry out the method for thin-layer chromatography, Appendix
III A, protected from light, using a silica gel F
254
254
plates are
suitable) and a mixture of 3 volumes of diethylamine and 90 volumes of cyclohexane as the mobile
phase and an unsaturated tank. Apply separately to the plate 4 l of each of two solutions in dichloro-
methane containing (1) 0.25% w/v of the substance being examined and (2) 0.000625% w/v of
2-trifluoromethylthioxanthone BPCRS. After removal of the plate, allow it to dry in air, spray with a
mixture of equal volumes of sulphuric acid and absolute ethanol, heat at 110 for 5 minutes and
examine under ultraviolet light (365 nm) immediately. Any secondary spot in the chromatogram
obtained with solution (1) corresponding to 2-trifluoromethylthioxanthone is not more intense than
the spot in the chromatogram obtained with solution (2) (0.25%).
trans-Flupentixol decanoate Carry out the method for liquid chromatography, Appendix III D,
protected from light, using solutions in dichloromethane containing (1) 0.10% w/v of the substance
being examined, (2) 0.001% w/v of trans-flupentixol decanoate dihydrochloride BPCRS and (3) 0.10%
w/v of the substance being examined and 0.001% w/v of trans-flupentixol decanoate dihydrochloride
BPCRS.
(25 cm 4.6 mm) packed with stationary phase A (5 m) (Spherisorb S 5W is suitable), (b) a mixture
of 0.005 volume of 13.5M ammonia, 15 volumes of acetonitrile, 42.5 volumes of dichloromethane and
42.5 volumes of n-heptane as the mobile phase with a flow rate of 2.3 ml per minute and (c) a
The test is not valid unless the chromatogram obtained with solution (3) shows clearly separated
peaks.
In the chromatogram obtained with solution (1) the area of any peak corresponding to trans-
flupentixol decanoate dihydrochloride is not greater than the area of the peak in the chromatogram
Loss on drying When dried at 60 at a pressure of 0.7kPa for 3 hours, loses not more than 1.0% of
its weight. Use 1 g.
17-87
titration, Appendix VIII A, using crystal violet solution as indicator. Each ml of 0.1M perchloric acid VS
33
H
43
F
3
N
2
O
2
S.
Storage Flupentixol Decanoate should be kept in a well-closed container and protected from light at
a temperature below 15.
Preparation
Flupentixol Injection
When flupenthixol decanoate is prescribed or demanded, Flupentixol Decanoate shall be dispensed
or supplied.
IMPURITIES
S
N
N
OH
CF
3
H
A. cis-flupentixol (free alcohol)
S
CF
3
O
B. 2-trifluoromethylthioxanthone
S
CF
3
N
N
H
3
C[CH
2
]
8
COO
H
C. trans-flupentixol decanoate
17-88
Fluphenazine Decanoate
CF
3
S
N
N
N O [CH
2
]
8
CH
3
O
C
32
H
44
F
3
N
3
O
2
S 591.8 5002-47-1
Fluphenazine Decanoate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Fluphenazine Decanoate Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Fluphenazine decanoate contains not less than 98.5 per cent and not more than the equivalent of
101.5 per cent of 2-[4-[3-(2-(trifluoromethyl)phenothiazin-10-yl)propyl]piperazin-1-yl]ethyl
decanoate, calculated with reference to the dried substance.
CHARACTERS
A pale yellow, viscous liquid or a yellow solid, practically insoluble in water, very soluble in ethanol,
in ether and in methylene chloride, freely soluble in methanol.
IDENTIFICATION
A. Dissolve 50.0 mg in methanol R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml to
50.0 ml with methanol R. Examined between 230 nm and 350 nm (2.2.25), the solution shows an
absorption maximum at 260 nm and a broad absorption maximum at about 310 nm. The specific
absorbance at the maximum at 260 nm is 570 to 630.
obtained with fluphenazine decanoate CRS. Examine as discs prepared by placing 50 l of a 25 g/l
solution in methylene chloride R on a disc of potassium bromide R. Dry the discs at 60C for 1 h before
use.
254
Completely impregnate the dry plate in a tank with a 5 per cent V/V solution of tetradecane R in
heptane R. Allow to dry.
the same solvent.
Reference solution (a). Dissolve 20 mg of fluphenazine decanoate CRS in methanol R and dilute to 10 ml
Reference solution (b). Dissolve 10 mg of fluphenazine enantate CRS in reference solution (a) and dilute
10 volumes of water R and 90 volumes of methanol R. Allow the plate to dry in air and examine in
similar in position and size to the spot in the chromatogram obtained with reference solution (a). The
separated spots.
17-89
E. It does not give reaction (a) of chlorides (2.3.1).
TESTS
Related substances Carry out the test protected from light and prepare the solutions immediately before use.
254
the same solvent.
5 volumes of concentrated ammonia R, 30 volumes of cyclohexane R and 80 volumes of acetone R.
Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Spray with a 50 per cent V/V
solution of sulphuric acid R and heat at 100C for 15 min. For both methods of visualisation, any spot
intense than the spot in the chromatogram obtained with reference solution (a) (1.0 per cent) and at
most one such spot is more intense than the spot in the chromatogram obtained with reference
60C at a pressure not exceeding 0.7 kPa for 3 h.
ASSAY
Dissolve 0.250 g in 30 ml of glacial acetic acid R. Using 0.05 ml of crystal violet solution R as indicator,
titrate with 0.1M perchloric acid, until the colour changes from violet to green.
32
H
44
F
3
N
3
O
2
S.
STORAGE
IMPURITIES
A. fluphenazine,
N
S
N
N
OH
CF
3
O
B. fluphenazine S-oxide.
__________________________________________________________________________________________________________ Ph Eur
17-90
Fluphenazine Enantate
CF
3
S
N
N
N O [CH
2
]
5
CH
3
O
C
29
H
38
F
3
N
3
O
2
S 549.7 2746-81-8
Fluphenazine Enantate complies with the requirements of the 3rd edition of the European Pharmacopoeia
When fluphenazine enanthate is prescribed or demanded, Fluphenazine Enantate shall be dispensed
or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Fluphenazine enantate contains not less than 98.5 per cent and not more than the equivalent of
101.5 per cent of 2-[4-[3-(2-(trifluoromethyl)phenothiazin-10-yl)propyl]piperazin-1-yl]ethyl
heptanoate, calculated with reference to the dried substance.
CHARACTERS
A pale yellow, viscous liquid or a yellow solid, practically insoluble in water, very soluble in ethanol,
in ether and in methylene chloride, freely soluble in methanol.
IDENTIFICATION
obtained with fluphenazine enantate CRS. Examine as discs, prepared by placing 50 l of a 25 g/l
solution in methylene chloride R on a disc of potassium bromide R. Dry the discs at 60C for 1 h before
use.
254
Completely impregnate the dry plate in a tank with a 5 per cent V/V solution of tetradecane R in
heptane R. Allow to dry.
the same solvent.
Reference solution (a). Dissolve 20 mg of fluphenazine enantate CRS in methanol R and dilute to 10 ml
Reference solution (b). Dissolve 10 mg of fluphenazine decanoate CRS in reference solution (a) and
dilute to 5 ml with the same solution.
10 volumes of water R and 90 volumes of methanol R. Allow the plate to dry in air and examine in
similar in position and size to the spot in the chromatogram obtained with reference solution (a). The
separated spots.
17-91
E. It does not give reaction (a) of chlorides (2.3.1).
TESTS
Related substances Carry out the test protected from light and prepare the solutions immediately before
use. Examine by thin-layer chromatography (2.2.27), using silica gel GF
254
the same solvent.
5 volumes of concentrated ammonia R, 30 volumes of cyclohexane R and 80 volumes of acetone R.
Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Spray with a 50 per cent V/V
solution of sulphuric acid R and heat at 100C for 15 min. For both methods of visualisation, any spot
intense than the spot in the chromatogram obtained with reference solution (a) (1.0 per cent) and at
most one such spot is more intense than the spot in the chromatogram obtained with reference
60C at a pressure not exceeding 0.7 kPa for 3 h.
ASSAY
Dissolve 0.250 g in 30 ml of glacial acetic acid R. Using 0.05 ml of crystal violet solution R as indicator
titrate with 0.1M perchloric acid, until the colour changes from violet to green.
29
H
38
F
3
N
3
O
2
S.
STORAGE
IMPURITIES
A. fluphenazine,
N
S
N
N
OH
CF
3
O
B. fluphenazine S-oxide.
__________________________________________________________________________________________________________ Ph Eur
17-92
Fluphenazine Hydrochloride
CF
3
S
N
N
N
OH
,2HCl
C
22
H
26
F
3
N
3
OS,2HCl 510.5 146-56-5
Fluphenazine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Fluphenazine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Fluphenazine hydrochloride contains not less than 98.5 per cent and not more than the equivalent
of 101.0 per cent of 2-[4-[3-(2-(trifluoromethyl)phenothiazin-10-yl)propyl]piperazin-1-yl]ethanol
CHARACTERS
A white or almost white, crystalline powder, freely soluble in water, slightly soluble in alcohol and in
IDENTIFICATION
obtained with fluphenazine hydrochloride CRS.
254
the same solvent.
Reference solution (a). Dissolve 20 mg of fluphenazine hydrochloride CRS in methanol R and dilute to
Reference solution (b). Dissolve 10 mg of perphenazine CRS in reference solution (a) and dilute to 5 ml
Apply separately to the plate 2 l of each solution. Develop twice over a path of 15 cm using a
mixture of 1 volume of water R, 4 volumes of diethylamine R and 95 volumes of methyl ethyl ketone R.
gram obtained with reference solution (b) shows two clearly separated principal spots.
17-93
TESTS
2.4.
Related substances Carry out the test protected from light and prepare the solutions immediately before
use. Examine by thin-layer chromatography (2.2.27), using silica gel GF
254
diethylamine R and 95 volumes of methanol R and dilute to 10 ml with the same mixture of solvents.
Reference solution (a). Dilute 1 ml of the test solution to 100 ml with a mixture of 5 volumes of
Reference solution (b). Dilute 5 ml of reference solution (a) to 10 ml with a mixture of 5 volumes of
togram obtained with reference solution (a) (1.0 per cent) and at most one such spot is more intense
65C for 3 h.
ASSAY
Dissolve 0.220 g in a mixture of 10 ml of anhydrous formic acid R and 40 ml of acetic anhydride R.
22
H
28
Cl
2
F
3
N
3
OS.
STORAGE
IMPURITIES
N
S
N
N
OH
CF
3
O
A. fluphenazine S-oxide.
__________________________________________________________________________________________________________ Ph Eur
17-94
Flurazepam Monohydrochloride
N
N
O
F
Cl
Et
2
N
,HCl
C
21
H
23
ClFN
3
O,HCl 424.3 36105-20-1
Flurazepam Monohydrochloride complies with the requirements of the 3rd edition of the European
Preparation
Flurazepam Capsules
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Flurazepam monohydrochloride contains not less than 99.0 per cent and not more than the equiva-
lent of 101.0 per cent of 7-chloro-1-(2-diethylaminoethyl)-5-(2-fluorophenyl)-2,3-dihydro-1H-1,4-
benzodiazepin-2-one monohydrochloride, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, very soluble in water, freely soluble in alcohol, practically
insoluble in ether.
IDENTIFICATION
First identification: C, F.
A. It complies with the test for pH (see Tests).
B. Dissolve 0.10 g in a 2.7 per cent V/V solution of sulphuric acid R in anhydrous methanol R and
dilute to 100 ml with the same acid solution. Dilute 1 ml of this solution to 100 ml with a 2.7 per
cent V/V solution of sulphuric acid R in anhydrous methanol R. Examined between 220 nm and
350 nm (2.2.25), the solution shows two absorption maxima, at 240 nm and 284 nm. The ratio of
the absorbance measured at the maximum at 240 nm to that measured at the maximum at 284 nm is
2.15 to 2.25.
obtained with flurazepam monohydrochloride CRS. Examine the substances prepared as discs.
254
the same solvent.
Reference solution (a). Dissolve 10 mg of flurazepam monohydrochloride CRS in alcohol R and dilute to
Reference solution (b). Dissolve 10 mg of flurazepam monohydrochloride CRS and 10 mg of diazepam
CRS in alcohol R and dilute to 10 ml with the same solvent.
10 volumes of diethylamine R and 90 volumes of ether R. Dry the plate in a current of air and examine
phenolphthalein solution R1 and sufficient dilute hydrochloric acid R (about 1 ml) to render the solution
17-95
colour of the solution with that of a blank prepared in the same manner. The colour of the test
solution is yellow and that of the blank is orange-red.
F. It gives reaction (a) of chlorides (2.3.1).
TESTS
254
R as the
coating substance.
concentrated ammonia R and 98 volumes of methanol R and dilute to 10 ml with the same mixture of
solvents.
Reference solution. Dilute 1 ml of the test solution to 50 ml with a mixture of 2 volumes of concentrated
ammonia R and 98 volumes of methanol R. Dilute 1 ml to 10 ml with a mixture of 2 volumes of
2.5 volumes of diethylamine R and 97.5 volumes of ether R. Dry the plate in a current of air and
examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with the test solution,
100C to 105C for 4 h.
ASSAY
21
H
24
Cl
2
FN
3
O.
STORAGE
IMPURITIES
N
O
F
NEt
2
Cl
A. 5-chloro-2-(2-diethylaminoethylamino)-2-fluorobenzophenone,
H
N
N
Cl
F
O
B. 7-chloro-5-(2-fluorophenyl)-2,3-dihydro-1H-1,4-benzodiazepin-2-one.
__________________________________________________________________________________________________________ Ph Eur
18-1
Flurbiprofen
1/01
COOH
Me H
F
and enantiomer
C
15
H
13
FO
2
244.3 5104-49-4
Flurbiprofen complies with the requirements of the 3rd edition of the European Pharmacopoeia [1519]. These
Preparations
Flurbiprofen Suppositories
Flurbiprofen Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Flurbiprofen contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of (2RS)-2-(2-fluorobiphenyl-4-yl)propanoic acid, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water, freely soluble in alcohol
and in methylene chloride. It dissolves in aqueous solutions of alkali hydroxides and carbonates.
IDENTIFICATION
B. Dissolve 0.10 g in 0.1M sodium hydroxide and dilute to 100.0 ml with the same alkaline solution.
Dilute 1.0 ml of the solution to 100.0 ml with 0.1M sodium hydroxide. Examined between 230 nm and
obtained with flurbiprofen CRS.
zirconyl nitrate solution R add 1.0 ml of the filtrate. Mix, allow to stand for 5 min and compare the
TESTS
The solution is clear (2.2.1) and colourless (Method I, 2.2.2).
Test solution. Dissolve 0.20 g of the substance to be examined in a mixture of 45 volumes of aceto-
acetonitrile R and 55 volumes of water R. Dilute 1.0 ml of the solution to 10.0 ml with a mixture of 45
Reference solution (b). Dissolve 10.0 mg of flurbiprofen impurity A CRS in a mixture of 45 volumes of
18-2
Dilute 10.0 ml of this solution to 100.0 ml with a mixture of 45 volumes of acetonitrile R and 55
volumes of water R.
Reference solution (c). Dissolve 10 mg of the substance to be examined in a mixture of 45 volumes of
Dilute 1.0 ml of the solution to 10.0 ml with reference solution (b).
as mobile phase at a flow rate of 1 ml/min a mixture of 5 volumes of glacial acetic acid R, 35
two principal peaks in the chromatogram obtained are at least 40 per cent of the full scale of the
recorder. The test is not valid unless the resolution between the peak corresponding to impurity A
and the peak corresponding to flurbiprofen is at least 1.5.
Inject 10 l of the test solution and of reference solutions (a) and (b). Continue the chromato-
graphy for twice the retention time of flurbiprofen. In the chromatogram obtained with the test
solution the area of any peak corresponding to impurity A is not greater than the area of the peak in
the chromatogram obtained with reference solution (b) (0.5 per cent); the area of any peak, apart
from the principal peak and the peak due to impurity A, is not greater than the area of the principal
peak in the chromatogram obtained with reference solution (a) (0.2 per cent) and the sum of the
areas of any such peaks is not greater than five times the area of the principal peak in the chromato-
0.1 times that of the principal peak in the chromatogram obtained with reference solution (a)
(0.02 per cent).
ASSAY
Dissolve 0.200 g in 50 ml of alcohol R. Titrate with 0.1M sodium hydroxide, determining the end-point
15
H
13
FO
2
.
IMPURITIES
COOH
H Me
and enantiomer
A. (2RS)-2-(biphenyl-4-yl)propanoic acid,
COOH
Me
COOH
Me *
*
B. 2-(2-fluorobiphenyl-4-yl)-2,3-dimethylbutanedioic acid,
COOH
HO Me
and enantiomer
C. (2RS)-2-(2-fluorobiphenyl-4-yl)-2-hydroxypropanoic acid,
18-3
F R
D. R = CO-CH
3
: 1-(2-fluorobiphenyl-4-yl)ethanone,
E. R = CO
2
H: 2-fluorobiphenyl-4-carboxylic acid.
__________________________________________________________________________________________________________ Ph Eur
18-4
Flurbiprofen Sodium
COONa
Me H
F
and enantiomer
C
15
H
12
FNaO
2
,2H
2
O 302.3 56767-76-1
Definition Flurbiprofen Sodium is sodium (RS)-2-(2-fluorobiphenyl-4-yl)propionate dihydrate. It
15
H
12
FNaO
2
, calculated with reference
Characteristics A white to creamy-white, crystalline powder.
Sparingly soluble in water; soluble in ethanol (96%); practically insoluble in dichloromethane.
Identification
flurbiprofen sodium (RS 157).
B. Heat 0.2 g over a flame until charred and then heat at 600 for 2 hours. The residue yields the
reactions characteristic of sodium salts, Appendix VI.
solutions in a mixture of 25 volumes of water and 50 volumes of methanol containing (1) 0.10% w/v
of the substance being examined, (2) 0.00020% w/v of the substance being examined, (3) 0.00050%
w/v of 2-(biphenyl-4-yl)propionic acid BPCRS and (4) 0.00050% w/v of the substance being examined
and 0.00050% w/v of 2-(biphenyl-4-yl)propionic acid BPCRS.
(15 cm 3.9 mm) packed with stationary phase C (5 m) (Resolve 5 is suitable), (b) a mixture of 5
volumes of glacial acetic acid, 35 volumes of acetonitrile and 60 volumes of water as the mobile phase
with a flow rate of 1 ml per minute and (c) a detection wavelength of 254 nm. Adjust the sensitivity
so that the heights of the principal peaks in the chromatogram obtained with solution (4) are about
40% of full-scale deflection on the chart paper.
In the chromatogram obtained with solution (1) the area of any peak corresponding to 2-(biphenyl-
4-yl)propionic acid is not greater than the area of the peak in the chromatogram obtained with
solution (3) (0.5%), the area of any other secondary peak is not greater than the area of the peak in the
chromatogram obtained with solution (2) (0.2%) and the sum of the areas of any secondary peaks is
not greater than five times the area of the peak in the chromatogram obtained with solution (2) (1%).
Heavy metals 12 ml of a 20% w/v solution in methanol complies with limit test A for heavy metals,
Appendix VII (10 ppm). Use 10 ml of the solution obtained by diluting 10 ml of lead standard solution
(20 ppm Pb) to 100 ml with methanol to prepare the standard and 10 ml of methanol and 2 ml of the
solution of the substance being examined to prepare the reagent blank.
Loss on drying 11.3% to 12.5% when determined by drying over phosphorus pentoxide at 60 at a
pressure of 2 kPa for 18 hours. Use 1 g.
Assay Carry out the method for liquid chromatography, Appendix III D, using solutions in a mixture
of 25 volumes of water and 50 volumes of methanol containing (1) 0.015% w/v of the substance being
examined, (2) 0.015% w/v of flurbiprofen sodium BPCRS and (3) 0.00075% w/v of the substance
being examined and 0.00075% w/v of 2-(biphenyl-4-yl)propionic acid BPCRS.
15
H
12
FNaO
2
in the substance being examined using the declared
content of C
15
H
12
FNaO
2
in flurbiprofen sodium BPCRS.
Preparation
Flurbiprofen Eye Drops
18-5
IMPURITIES
A. 2-(biphenyl-4-yl)propionic acid
18-6
Flutamide
H
N
Me
Me
O
CF
3
O
2
N
C
11
H
11
F
3
N
2
O
3
276.2 13311-84-7
Flutamide complies with the requirements of the 3rd edition of the European Pharmacopoeia [1423]. These
Action and use Anti-androgen.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Flutamide contains not less than 97.0 per cent and not more than the equivalent of 103.0 per cent of
2-methyl-N-[4-nitro-3-(trifluoromethyl)phenyl]propanamide, calculated with reference to the dried
substance.
CHARACTERS
A pale yellow, crystalline powder, practically insoluble in water, freely soluble in acetone and in
alcohol.
IDENTIFICATION
with flutamide CRS.
TESTS
Reference solution (a). Dissolve 2 mg of flutamide CRS and 2 mg of flutamide impurity C CRS in the
mobile phase and dilute to 50.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 20.0 ml
as mobile phase at a flow rate of 0.5 ml/min a mixture of equal volumes of acetonitrile R and
water R,
principal peak in the chromatogram obtained is not less than 50 per cent of the full scale of the
recorder.
conditions, the retention times are: flutamide about 19 min and impurity C about 14 min. The test is
not valid unless the resolution between the peaks corresponding to impurity C and flutamide is at
least 10.5.
solution: the area of the peak corresponding to impurity C, with a retention time of about 0.72
relative to flutamide, is not greater than 1.5 times the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.3 per cent); the area of any other peak apart from the
principal peak and the peak corresponding to impurity C is not greater than the area of the principal
the peaks apart from the principal peak is not greater than 2.5 times the area of the principal peak in
the chromatogram obtained with reference solution (b) (0.5 per cent). Disregard any peak with an
solution (b).
18-7
60C for 3 h.
ASSAY
Dissolve 25.0 mg in methanol R and dilute to 25.0 ml with the same solvent. Dilute 2.0 ml of this
solution to 100.0 ml with methanol R. Measure the absorbance (2.2.25) at the maximum at 295 nm.
11
H
11
F
3
N
2
O
3
STORAGE
IMPURITIES
2
N CF
3
NO
2
A. 4-nitro-3-(trifluoromethyl)aniline,
H
N
CF
3
NO
2
3
C
O
B. N-[4-nitro-3-(trifluoromethyl)phenyl]acetamide,
H
N
CF
3
NO
2
O
3
C
C. N-[4-nitro-3-(trifluoromethyl)phenyl]propanamide,
2
N CF
3
D. 3-(trifluoromethyl)aniline,
H
N
CF
3
O
e
Me
E. 2-methyl-N-[3-(trifluoromethyl)phenyl]propanamide,
H
N
O
e
Me NO
2
CF
3
F. 2-methyl-N-[2-nitro-5-(trifluoromethyl)phenyl]propanamide.
__________________________________________________________________________________________________________ Ph Eur
18-8
Fluticasone Propionate
O
F
F
Me H
H
Me
HO
Me
OCOEt
O S
CH
2
F
C
25
H
31
F
3
O
5
S 500.6 80474-14-2
Definition Fluticasone Propionate is S-fluoromethyl 6,9-difluoro-11-hydroxy-16-methyl-3-
oxo-17-propionyloxyandrosta-1,4-diene-17-carbothioate. It contains not less than 97.0% and
25
H
31
F
3
O
5
Practically insoluble in water; freely soluble in dimethylformamide; sparingly soluble in acetone and
in dichloromethane; slightly soluble in ethanol (96%).
Identification
A. The infrared absorption spectrum, Appendix II B, is concordant with the reference spectrum of
fluticasone propionate (RS 158).
time as the principal peak in the chromatogram obtained with solution (2).
Specific optical rotation In a 0.5% w/v solution in dichloromethane, +32 to +36, calculated with
following solutions. For solution (1) dissolve 5 mg of the substance being examined in 12 ml of a
mixture of 23 volumes of acetonitrile and 77 volumes of methanol with the aid of ultrasound and dilute
to 20 ml with water. For solution (2) dilute 1 volume of solution (1) to 200 volumes with the mobile
phase. Solution (3) contains 0.0008% w/v of fluticasone S-methyl impurity BPCRS and 0.004% w/v of
fluticasone propionate BPCRS in the mobile phase.
The chromatographic conditions described under Assay may be used. Allow the chromatography
to proceed for twice the retention time of the principal peak.
between the peaks due to fluticasone S-methyl impurity and fluticasone propionate is at least 1.5. If
necessary, adjust the ratio of acetonitrile to methanol in the mobile phase.
the areas of any such peaks is not greater than 4 times the area of the principal peak in the chromato-
gram obtained with solution (2) (2%).
Loss on drying When dried to constant weight at 100 to 105, loses not more than 2% of its
weight. Use 1 g.
For solution (1) dilute 1 volume of a 0.04% w/v solution of the substance being examined in aceto-
nitrile, prepared with the aid of ultrasound, to 10 volumes with the mobile phase. Solution (2)
contains 0.004% w/v of fluticasone propionate BPCRS prepared in a similar manner to solution (1).
Solution (3) contains 0.0008% w/v of fluticasone S-methyl impurity BPCRS and 0.004% w/v of
fluticasone propionate BPCRS in the mobile phase.
4.6 mm) packed with stationary phase C (5 m) (Spherisorb ODS 1 is suitable) and maintained at a
temperature of 40, (b) as the mobile phase with a flow rate of 1.5 ml per minute a mixture of 15
volumes of acetonitrile, 35 volumes of 0.01M ammonium dihydrogen orthophosphate adjusted to pH 3.5
and 50 volumes of methanol and (c) a detection wavelength of 239 nm.
The Assay is not valid unless, in the chromatogram obtained with solution (3), the resolution factor
between the peaks due to fluticasone S-methyl impurity and fluticasone propionate is at least 1.5. If
necessary, adjust the ratio of acetonitrile to methanol in the mobile phase.
25
H
31
F
3
O
5
S using the declared content of C
25
H
31
F
3
O
5
S in fluticasone
propionate BPCRS.
18-9
Preparations
Fluticasone Cream
Fluticasone Ointment
IMPURITIES
O
F
F
Me H
H
Me
HO
Me
OCOEt
O SMe
A. S-methyl 6,9-difluoro-11-hydroxy-16-methyl-3-oxo-17-propionylandrosta-1,4-
diene-17-carbothioate (fluticasone S-methyl impurity)
O
F
F
Me H
H
Me
Me
OCOEt
O SCH
2
F
O
B. S-fluoromethyl 6,9-difluoro-16-methyl-3,11-dioxo-17-propionylandrosta-1,4-diene-
17-carbothioate
O
F
F
Me H
H
Me
HO
Me
OCOEt
O SH
C. 6,9-difluoro-11-hydroxy-16-methyl-3-oxo-17-propionylandrosta-1,4-diene-17-
carbothioic acid
O
F
F
Me H
H
Me
Me
O
O SCH
2
F
O
OH
Me
Me
H
H Me
F
F
O
HO
HO
D. 6,9-difluoro-17-[(fluoromethyl)thiocarboxy]-11-hydroxy-16-methyl-3-oxoandrosta-
1,4-dien-17-yl 6,9-difluoro-11,17-dihydroxy-3-oxo-androsta-1,4-diene-17-
carboxylate
O
F
Me H
H
Me
Me
OCOEt
O SCH
2
F
HO
O
E. S-fluoromethyl 9-fluoro-11-hydroxy-16-methyl-3,6-dioxo-17-propionylandrosta-1,4-
diene-17-carbothioate
18-10
O
F
F
Me H
H
Me
HO
Me
OCOEt
O S
2
F. bis(6,9-difluoro-11-hydroxy-16-methyl-3-oxo-17-propionylandrosta-1,4-diene-17-
carbonyl) disulphide
18-11
Flutrimazole
N
N
F
F
and enantiomer
C
22
H
16
F
2
N
2
346.4 119006-77-8
Flutrimazole complies with the requirements of the 3rd edition of the European Pharmacopoeia [1424]. These
Action and use Topical antifungal.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Flutrimazole contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of (RS)-1-[(2-fluorophenyl)(4-fluorophenyl)phenylmethyl]-1H-imidazole, calculated with reference
CHARACTERS
A white or almost white powder, practically insoluble in water, freely soluble in tetrahydrofuran,
soluble in methanol.
IDENTIFICATION
obtained with flutrimazole CRS. Examine the substances prepared as discs.
254
plate R. Heat the plate
at 110C for 1 h.
the same solvent.
Reference solution (a). Dissolve 20 mg of flutrimazole CRS in acetone R and dilute to 10 ml with the
same solvent.
Reference solution (b). Dissolve 20 mg of flutrimazole CRS and 10 mg of metronidazole benzoate CRS in
Apply separately to the plate 10 l of each solution. Develop over a path corresponding to two-thirds
of the height of the plate using a mixture of 10 volumes of 2-propanol R and 90 volumes of ethyl
acetate R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot
TESTS
7
(Method II, 2.2.2).
18-12
+0.05.
Reference solution (a). Dissolve 25.0 mg of imidazole CRS in the mobile phase and dilute to 50.0 ml
with the mobile phase. Dilute 10.0 ml of this solution to 50.0 ml with the mobile phase.
Reference solution (b). Dissolve 30.0 mg of flutrimazole impurity B CRS in the mobile phase and dilute
Reference solution (c). Mix 2.0 ml of reference solution (a) and 2.0 ml of reference solution (b) and
Reference solution (d). Dilute 10.0 ml of reference solution (c) to 50.0 ml with the mobile phase.
Reference solution (e). Mix 2.0 ml of the test solution and 10.0 ml of reference solution (c) and dilute
Reference solution (f). Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute
gel for chromatography (5 m),
as mobile phase at a flow rate of 1.3 ml/min a mixture of 40 volumes of 0.03M phosphate buffer
solution pH 7.0 R and 60 volumes of acetonitrile R,
Inject 20 l of reference solution (e). The test is not valid unless the resolution between the first
peak (imidazole) and the second peak (impurity B) is at least 2.0 and the resolution between the
second peak and the third peak (flutrimazole) is at least 1.5. The symmetry factor of the first peak
and the second peak is not greater than 2.0.
Inject separately 20 l of the test solution, 20 l of reference solution (d) and 20 l of reference
solution (f). Continue the chromatography for 2.5 times the retention time of the principal peak.
In the chromatogram obtained with the test solution:
the area of the peak due to imidazole is not greater than the corresponding area in the chromato-
gram obtained with reference solution (d) (0.1 per cent);
the area of the peak due to impurity B is not greater than the corresponding area in the chroma-
togram obtained with reference solution (d) (0.3 per cent);
the area of any peak, apart from the principal peak and any peak corresponding to impurity B, is
not greater than the area of the principal peak in the chromatogram obtained with reference
solution (f) (0.1 per cent); the sum of the areas of any such peaks is not greater than three times
the area of the principal peak in the chromatogram obtained with reference solution (f) (0.3 per
cent). Disregard any peak with an area less than half the area of the principal peak in the
chromatogram obtained with reference solution (f).
Heavy metals (2.4.8). 2.0 g complies with limit test F for heavy metals (10 ppm). Use a platinum
100C to 105C.
ASSAY
22
H
16
F
2
N
2
.
STORAGE
IMPURITIES
N
H
N
A. imidazole,
18-13
and enantiomer
F
F
OR
B. R = H: (RS)-(2-fluoropheny)(4-fluoropheny)phenylmethanol,
C. R = CH
3
: (RS)-(2-fluorophenyl)(4-fluorophenyl)methoxyphenylmethane.
__________________________________________________________________________________________________________ Ph Eur
18-14
Fluvoxamine Maleate
OMe
F
3
C
N
O
NH
2
H
H COOH
COOH
,
C
15
H
21
F
3
N
2
O
2
,C
4
H
4
O
4
434.4 61718-82-9
Definition Fluvoxamine Maleate is (E)-5-methoxy-4-trifluoromethylvalerophenone O-2-
aminoethyloxime maleate. It contains not less than 99.0% and not more than 101.0% of
C
15
H
21
F
3
N
2
O
2
,C
4
H
4
O
4
Characteristics A white to almost white, crystalline powder.
Freely soluble in ethanol (96%) and in methanol; sparingly soluble in water.
Identification
fluvoxamine maleate (RS 159).
B. Carry out the method for thin-layer chromatography, Appendix III A, using a silica gel HF pre-
coated plate (Analtech Uniplates are suitable) and a mixture of 5 volumes of 13.5M ammonia and 95
volumes of ethanol (96%) as the mobile phase. Apply separately to the plate 5 l of each of the follow-
ing solutions in methanol containing (1) 1.0% w/v of the substance being examined and (2) 1.0% w/v
of fluvoxamine maleate BPCRS. After removal of the plate, allow it to dry in air and examine under
ultraviolet light (254 nm). The two principal spots in the chromatogram obtained with solution (1)
correspond to those in the chromatogram obtained with solution (2).
Acetonitrile Not more than 100 ppm when determined by the test for residual solvents, Appendix
VIII L.
following solutions in the mobile phase. Solutions (1) and (2) contain 0.14% w/v and 0.00028% w/v
respectively of the substance being examined. For solution (3) heat a 0.32% w/v solution of
fluvoxamine maleate impurity standard BPCRS in 0.1M hydrochloric acid on a water bath for 10
minutes.
4.6 mm) packed with stationary phase B (7m) (Zorbax C8 is suitable) and maintained at 35, (b) as
the mobile phase with a flow rate of 2 ml per minute a mixture of 40 volumes of a solution containing
1.25% w/v of diammonium hydrogen orthophosphate and 0.275% w/v of sodium heptanesulphonate
monohydrate and 60 volumes of methanol, adjusting the pH of the mixture to 3.5 with orthophosphoric
acid and (c) a detection wavelength of 254 nm.
When the chromatograms are recorded under the conditions described above, the retention time of
fluvoxamine maleate is 7 to 9 minutes. If necessary, adjust the composition of the mobile phase
(increasing the methanol content decreases the retention time, increasing the water content increases
the retention time).
The test is not valid unless the chromatogram obtained with solution (3) closely resembles the
reference chromatogram supplied with fluvoxamine maleate impurity standard BPCRS and the
resolution factor between the peaks corresponding to fluvoxamine and the Z-isomer is at least 1.0.
In the chromatogram obtained with solution (1) identify the impurities from the chromatogram
obtained with solution (3) and from the reference chromatogram supplied with the reference
material. The area of any peak corresponding to the addition product is not greater than 2.5 times
the area of the principal peak in the chromatogram obtained with solution (2) (0.5%), the area of any
peak corresponding to the Z-isomer of fluvoxamine is not greater than 1.25 times the area of the
principal peak in the chromatogram obtained with solution (2) (0.5%, assuming a response factor of
0.5) and the area of any other secondary peak is not greater than the area of the principal peak in the
chromatogram obtained with solution (2) (0.2%). Calculate the nominal content of any impurity
found. The combined nominal total content of any such impurities is not more than 1.5%. Disregard
the peak corresponding to maleic acid (which elutes immediately after the solvent front) and any peak
solution (2) (0.05%).
Loss on drying When dried over phosphorus pentoxide at 50 at a pressure not exceeding 0.7 kPa for
2 hours, loses not more than 1.0% of its weight. Use 1 g.
C
15
H
21
F
3
N
2
O
2
, C
4
H
4
O
4
.
18-15
Storage Fluvoxamine Maleate should be kept in a well-closed container.
Preparation
Fluvoxamine Tablets
IMPURITIES
CH
3
F
3
C
N
O
NH
2
A. (E)-4-trifluoromethylvalerophenone O-2-aminoethyloxime,
OMe
F
3
C
N
O
H
2
N
B. (Z)-5-methoxy-4-trifluoromethylvalerophenone O-2-aminoethyloxime (Z-isomer),
OMe
F
3
C
N
O
N
COOH
COOH
H
C. (E)-N-[2[[[-(4-methoxybutyl)-4-(trifluoromethyl)benzylidene]amino]oxy]ethyl]aspartic
acid (addition product),
OMe
F
3
C
O
D. 5-methoxy-p-trifluoromethylvalerophenone,
OMe
F
2
HC
N
O
NH
2
E. (E)-5-methoxy-4-difluoromethylvalerophenone O-2-aminoethyloxime,
OMe
F
3
C
N
O
N
NH
2
H
F. (E)-5-methoxy-4-trifluoromethylvalerophenone O-[2-N-(2-aminoethyl)aminoethyl]oxime.
18-16
Folic Acid
N
N N
N
N
N
H
2
N
O
O
COOH H
COOH
H
H
H
C
19
H
19
N
7
O
6
441.4 59-30-3
Folic Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia [0067]. These
Action and use Used in prevention of neural tube defects and treatment and prevention of
megaloblastic anaemias.
Preparations
Folic Acid Tablets
Ferrous Fumarate and Folic Acid Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Folic acid contains not less than 96.0 per cent and not more than the equivalent of 102.0 per cent of
(2S)-2-[4-[[(2-amino-4-hydroxypteridin-6-yl)methyl]amino]benzamido]glutaric acid, calculated with
CHARACTERS
A yellowish or orange, crystalline powder, practically insoluble in water and in most organic solvents.
It dissolves in dilute acids and in alkaline solutions.
IDENTIFICATION
A. Dissolve 0.25 g in 0.1M sodium hydroxide and dilute to 25.0 ml with the same solvent. The specific
optical rotation (2.2.7) is about +20, calculated with reference to the anhydrous substance.
B. Dissolve 10.0 mg in 0.1M sodium hydroxide and dilute to 100.0 ml with the same solvent. Dilute
380 nm (2.2.25), the solution shows three absorption maxima, at 256 nm, 283 nm and 365 nm. The
specific absorbances at these maxima are about 590, 575 and 206, respectively, calculated with
reference to the anhydrous substance. The ratio of the specific absorbance at the maximum at
256 nm to that at the maximum at 365 nm is 2.8 to 3.0.
Test solution. Dissolve 50 mg of the substance to be examined in a mixture of 2 volumes of
solvents.
Reference solution. Dissolve 50 mg of folic acid CRS in a mixture of 2 volumes of concentrated
ammonia R and 9 volumes of methanol R and dilute to 100 ml with the same mixture of solvents.
20 volumes of concentrated ammonia R, 20 volumes of propanol R and 60 volumes of alcohol R. Allow
the plate to dry in air. Examine in ultraviolet light at 365 nm. The principal spot in the chromato-
gram obtained with the test solution is similar in position, fluorescence and size to the principal spot
TESTS
Free amines The absorbance A
2
is not more than one-sixth of the absorbance A
1
, as determined in
the assay.
mination of water.
ASSAY
Dissolve 50.0 mg in 50 ml of 0.1M sodium hydroxide and dilute to 100.0 ml with the same solvent
(solution a).
(1) To 3.0 ml of solution (a) add 20 ml of dilute hydrochloric acid R and dilute to 100.0 ml with water.
18-17
To 50.0 ml of this solution add 0.5 g of zinc powder R, allow to stand protected from light for 20
min with frequent shaking and filter. Discard the first 10 ml of the filtrate, then take 10.0 ml and
dilute to 25.0 ml with water R. Add 5 ml of dilute hydrochloric acid R and 5 ml of a 1 g/l solution of
sodium nitrite R, mix and allow to stand for 2 min. Add 5 ml of a 5 g/l solution of ammonium
sulphamate R, mix and allow to stand for 2 min. Add 5 ml of a 1 g/l solution of
naphthylethylenediamine dihydrochloride R, mix and allow to stand for 10 min. Dilute to 50.0 ml with
water R and measure the absorbance (A
1
) of this reduced solution (2.2.25) at the maximum at
550 nm, using as the compensation liquid a mixture of 25.0 ml of water R, 5 ml of dilute hydrochloric
acid R and 5 ml of a 1 g/l solution of sodium nitrite R, treated as described above.
(2) To 30.0 ml of solution (a) add 20 ml of dilute hydrochloric acid R and dilute to 100.0 ml with
water R. Dilute 10.0 ml of this solution to 25.0 ml with water R, add 5 ml of dilute hydrochloric acid R,
5 ml of a 1 g/l solution of sodium nitrite R, treat as described above, and measure the absorbance (A
2
)
of this non-reduced solution.
Repeat the operation described above using folic acid CRS, the absorbance of the reduced solution
being A
3
and that of the non-reduced solution A
4
.
19
H
19
N
7
O
6
in the substance to be examined using the expression:
A A
A A
C
1 2
3 4
01
01

.
.
C=the declared content of C
19
H
19
N
7
O
6
in folic acid CRS.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
18-18
Formaldehyde Solution
Formalin
NOTE: The name Formalin as a synonym for Formaldehyde Solution may be used freely in many countries,
including the United Kingdom, but in other countries exclusive proprietary rights in this name are claimed.
H
H
O
CH
2
O 30.03 50-00-0
Formaldehyde Solution complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Formaldehyde Solution (35 per cent) [0826]. These requirements are reproduced after the heading
Definition below.
Action and use Used in the treatment of warts.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Formaldehyde solution (35 per cent) contains not less than 34.5 per cent m/m and not more than
38.0 per cent m/m of formaldehyde (CH
2
O; M
r
30.03) with methanol as stabiliser.
CHARACTERS
A clear, colourless liquid, miscible with water and with alcohol. It may be cloudy after storage.
IDENTIFICATION
A. Dilute 1 ml of solution S (see Tests) to 10 ml with water R. To 0.05 ml of the solution add 1 ml of
a 15 g/l solution of chromotropic acid, sodium salt R, 2 ml of water R and 8 ml of sulphuric acid R. A
violet-blue or violet-red colour develops within 5 min.
B. To 0.1 ml of solution S add 10 ml of water R. Add 2 ml of a 10 g/l solution of phenylhydrazine
hydrochloride R, prepared immediately before use, 1 ml of potassium ferricyanide solution R and 5 ml of
hydrochloric acid R. An intense red colour is formed.
C. Mix 0.5 ml with 2 ml of water R and 2 ml of silver nitrate solution R2 in a test-tube. Add dilute
ammonia R2 until slightly alkaline. Heat on a water-bath. A grey precipitate or a silver mirror is
formed.
D. It complies with the limits of the assay.
TESTS
Solution S Dilute 10 ml, filtered if necessary, to 50 ml with carbon dioxide-free water R.
Appearance of solution Solution S is colourless (Method II, 2.2.2).
Acidity To 10 ml of solution S add 1 ml of phenolphthalein solution R. Not more than 0.4 ml of 0.1M
sodium hydroxide is required to change the colour of the indicator to red.
Methanol 9.0 per cent V/V to 15.0 per cent V/V, determined by gas chromatography (2.2.28) using
ethanol R1 as internal standard.
Internal standard solution. Dilute 10 ml of ethanol R1 to 100 ml with water R.
Test solution. To 10.0 ml of the solution to be examined add 10.0 ml of the internal standard solution
and dilute to 100.0 ml with water R.
Reference solution. To 1.0 ml of methanol R add 10.0 ml of the internal standard solution and dilute to
a glass column 1.5 m to 2.0 m long and 2 mm to 4 mm in internal diameter, packed with
ethylvinylbenzene-divinylbenzene copolymer R (150 m to 180 m),
nitrogen for chromatography R as the carrier gas at a flow rate of 30 ml to 40 ml per minute,
detector at 150C.
Inject 1 l of the reference solution. Adjust the sensitivity of the detector so that the heights of the
peaks in the chromatogram obtained are not less than 50 per cent of the full scale of the recorder.
The test is not valid unless the resolution between the peaks corresponding to methanol and ethanol
is at least 2.0. Inject separately 1 l of the test solution and 1 l of the reference solution. Calculate
the percentage content of methanol.
18-19
ASSAY
Into a 100 ml volumetric flask containing 2.5 ml of water R and 1 ml of dilute sodium hydroxide
solution R, introduce 1.000 g of the solution to be examined, shake and dilute to 100.0 ml with
water R. To 10.0 ml of the solution add 30.0 ml of 0.05M iodine. Mix and add 10 ml of dilute sodium
hydroxide solution R. After 15 min, add 25 ml of dilute sulphuric acid R and 2 ml of starch solution R.
Titrate with 0.1M sodium thiosulphate.
1 ml of 0.05M iodine is equivalent to 1.501 mg of CH
2
O.
STORAGE
Store in a well-closed container, protected from light, at a temperature of 15C to 25C.
__________________________________________________________________________________________________________ Ph Eur
18-20
Foscarnet Sodium
1/01
NaO
P ONa
O
O
NaO
CNa
3
O
5
P,6H
2
O 300.0 63585-09-1 (anhydrous)
Foscarnet Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Foscarnet Sodium Hexahydrate [1520]. These requirements are reproduced after the heading Definition
below.
Preparation
Foscarnet Intravenous Infusion
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Foscarnet sodium hexahydrate contains not less than 98.5 per cent and not more than the equivalent
of 101.0 per cent of trisodium phosphonatoformate, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder, soluble in water, practically insoluble in alcohol.
IDENTIFICATION
obtained with foscarnet sodium hexahydrate CRS.
TESTS
Appearance of solution Solution S is not more opalescent than reference suspension I (2.2.1) and
Reference solution (a). Dilute 1.0 ml of the test solution to 50.0 ml with mobile phase. Dilute 1.0 ml of
Reference solution (b). Dissolve 5.0 mg of foscarnet impurity B CRS in the mobile phase, add 2.0 ml of
the test solution and dilute to 50.0 ml with the mobile phase.
sodium sulphate decahydrate R in water R, add 3 ml of glacial acetic acid R, 6 ml of a 44.61 g/l
solution of sodium pyrophosphate R and dilute to 1000 ml with water R (solution A); dissolve
3.22 g of sodium sulphate decahydrate R in water R, add 6.8 g of sodium acetate R and 6 ml of a
44.61 g/l solution of sodium pyrophosphate R and dilute to 1000 ml with water R (solution B).
Mix about 700 ml of solution A and about 300 ml of solution B to obtain a solution of pH 4.4.
To 000 ml of this solution, add 0.25 g of tetrahexylammonium hydrogen sulphate R and 100 ml of
methanol R.
Inject 20 l of reference solution (b). Continue the chromatography for 2.5 times the retention
time of foscarnet. The test is not valid unless the resolution between the peaks due to foscarnet and
impurity B is at least 7.
18-21
sum of the areas of all the peaks, apart from the principal peak, is not greater than twice the area of
the principal peak in the chromatogram obtained with reference solution (a) (0.4 per cent). Disregard
any peak with a relative retention time less than 0.6 and any peak with an area less than 0.2 times
that of the peak in the chromatogram obtained with reference solution (a).
Impurity D Examine by gas chromatography (2.2.28).
Test solution. Dissolve 0.25 g of the substance to be examined in 9.0 ml of 0.1M acetic acid using a
magnetic stirrer. Add 1.0 ml of ethanol R and mix.
Reference solution. Dissolve 25 mg of triethyl phosphonoformate R in ethanol R and dilute to 100 ml with
the same solvent. Dilute 1 ml of the solution to 10 ml with ethanol R.
poly(dimethyl)(diphenyl)(divinyl)siloxane R (film thickness 0.5 m),
raising the temperature of the column from 100C to 180C at a rate of 10C per minute and
maintaining the temperature of the injection port at 200C and that of the detector at 250C.
In the chromatogram obtained with the test solution the area of any peak due to impurity D is not
greater than the area of the peak in the chromatogram obtained with reference solution (0.1 per
cent).
Phosphate and phosphite Examine by liquid chromatography (2.2.29).
the same solvent.
Reference solution (a). Dissolve 28 mg of sodium dihydrogen phosphate monohydrate R in water R and
Reference solution (b). Dissolve 43 mg of sodium phosphite pentahydrate R in water R and dilute to
Reference solution (c). Dilute 1.0 ml of reference solution (a) and 1.0 ml of reference solution (b) to
25 ml with water R.
Reference solution (d). Dilute 3 ml of reference solution (a) and 3 ml of reference solution (b) to 25 ml
with water R.
a stainless steel column 0.05 m long and 4.6 mm in internal diameter packed with an anion
exchange resin for chromatography R,
potassium hydrogen phthalate R in water R, add 2.5 ml of 1M nitric acid and dilute to 1000 ml with
water R,
as detector a spectrophotometer set at 290 nm (indirect detection).
Inject 20 l of reference solution (d). The test is not valid unless: the resolution between the peaks
due to phosphate (first peak) and phosphite is at least 2.0; the principal peak has a signal-to-noise
ratio of at least ten.
Inject 20 l of the test solution and 20 l of reference solution (c). In the chromatogram obtained
with the test solution: the area of any peak due to phosphate and phosphite is not greater than the
area of the corresponding peaks in the chromatogram obtained with reference solution (c) (0.3 per
cent of phosphate and 0.3 per cent of phosphite).
Heavy metals Dissolve 1.25 g in 12.5 ml of 1M hydrochloric acid. Warm on a water-bath for 3 min
and cool to room temperature. Transfer to a beaker and adjust the pH to about 3.5 with dilute
ammonia R1 and dilute to 25 ml with water R (solution A). To 12 ml of solution A, add 2.0 ml of
buffer solution pH 3.5 R. Rapidly pour the mixture into a test tube containing one drop of sodium
sulphide solution R. The solution is not more intensely coloured than a standard prepared
simultaneously and in the same manner pouring a mixture of 5.0 ml of lead standard solution (1 ppm
Pb) R, 5.0 ml of water R, 2.0 ml of solution A and 2.0 ml of buffer solution pH 3.5 R into a test tube
containing one drop of sodium sulphide solution R (10 ppm).
at 150C.
Sterility (2.6.1). If intended for use in the manufacture of parenteral dosage forms without any
without any further appropriate procedure for the removal of bacterial endotoxins, not more than
83.3 I.U. of endotoxin per gram.
18-22
ASSAY
Dissolve 0.200 g in 50 ml of water R. Titrate with 0.05M sulphuric acid, determining the end-point
potentiometrically (2.2.20) at the first inflexion point.
1 ml of 0.05M sulphuric acid is equivalent to 19.20 mg of CNa
3
O
5
P.
STORAGE
IMPURITIES
P
O
COOEt
ONa
NaO
A. disodium (ethoxycarbonyl)phosphonate,
P
O
COONa
OEt
NaO
B. disodium (ethoxyoxydophosphanyl)formate,
P
O
COOEt
OEt
NaO
C. ethyl sodium (ethoxycarbonyl)phosphonate,
P
O
OEt
EtO COOEt
D. ethyl (diethoxyphosphoryl)formate.
__________________________________________________________________________________________________________ Ph Eur
18-23
Fosfestrol Sodium
O
O
P
P
O
O
ONa
ONa
NaO
NaO
Et
Et
C
18
H
18
Na
4
O
8
P
2
,xH
2
O 516.2 (anhydrous) 23519-26-8
Definition Fosfestrol Sodium is a hydrate of tetrasodium (E)-4,4-(1,2-diethylvinylene)bis(phenyl
orthophosphate). It contains not less than 98.0% and not more than 101.0% of C
18
H
18
Na
4
O
8
P
2
,
Freely soluble in water; practically insoluble in absolute ethanol and in ether.
Identification
fosfestrol sodium (RS 161).
B. Yields the reactions characteristic of sodium salts, Appendix VI.
To 20 ml of a 2.5% w/v solution, add 3.0 ml of 0.01M sodium hydroxide VS. The pH of the
resulting solution is not less than 8.8.
Light absorption Measure the absorbance of a 0.0050% w/v solution in 0.1M sodium hydroxide at the
maximum at 242 nm, Appendix II B. The A(1%, 1 cm) at the maximum is 280 to 320, calculated
Chloride Dissolve 1.0 g in 10 ml of water, add 10 ml of 2M nitric acid, filter, wash the precipitate
with 25 ml of water and dilute the combined filtrate and washings to 100 ml with water. 15 ml of the
Inorganic phosphate Dissolve 0.5 g in 20 ml of water, add 5 ml of 2M hydrochloric acid and extract
with four 30-ml quantities of ether. Discard the ether extracts and heat the aqueous solution on a
water bath until any remaining ether has evaporated. Allow to cool, add sufficient water to produce
100 ml (solution A) and then add 4 ml of sulphomolybdic reagent R3, shake, add 0.1 ml of a mixture of
1 volume of freshly prepared tin(II) chloride solution and 9 volumes of 2M hydrochloric acid, shake again
and examine 20 ml. Any colour produced is not more intense than that of 20 ml of a solution
obtained by repeating the operation using a mixture of 5 ml of a 0.01% w/v solution of potassium
dihydrogen orthophosphate and 95 ml of water in place of solution A.
silica gel 60 F
254
254
volumes each of pentan-1-ol, triethylamine and water and 55 volumes of propan-1-ol as the mobile
phase but allowing the solvent front to ascend 10 cm above the line of application. Apply separately
to the plate 10 l of each of three solutions of the substance being examined in methanol (50%)
containing (1) 2.0% w/v (2) 0.030% w/v and (3) 0.010% w/v. After removal of the plate, allow it to
dry in air and examine under ultraviolet light (254 nm). Any secondary spot in the chromatogram
obtained with solution (1) is not more intense than the spot in the chromatogram obtained with
solution (2) (1.5%) and not more than two such spots are more intense than the spot in the chroma-
togram obtained with solution (3) (0.5%).
Free diethylstilbestrol Dissolve a quantity of the substance being examined containing the equiva-
lent of 2 g of anhydrous fosfestrol sodium in 50 ml of water, extract with two 20-ml quantities of
dichloromethane and retain the aqueous phase. Wash the combined dichloromethane extracts with
30 ml of water and filter through anhydrous sodium sulphate. Extract the water wash with 10 ml of
dichloromethane, filter the dichloromethane extract after filtration through anhydrous sodium sulphate,
add to the original extract and add the aqueous extract to the original aqueous phase. Reserve the
aqueous phase for the test for Diethylstilbestrol disodium monophosphate. Evaporate the combined
filtrates to dryness on a water bath and dissolve the residue in 20 ml of absolute ethanol. To 10 ml of
the resulting solution add 10 ml of a solution prepared by dissolving 1 g of dipotassium hydrogen
orthophosphate in 55 ml of water. Transfer a portion of the mixture to a 1-cm closed silica cell, place
the cell 10 cm from a 15-watt, short-wave ultraviolet lamp and irradiate for 30 minutes. The
absorbance of the irradiated solution measured at the maximum at 418 nm, Appendix II B, is not
18-24
greater than that obtained by repeating the operation using 10 ml of a 0.00150% w/v solution of
diethylstilbestrol EPCRS in absolute ethanol, beginning at the words add 10 ml of a solution
(0.015%).
Diethylstilbestrol disodium monophosphate Dilute the combined aqueous phase reserved in the
test for Free diethylstilbestrol to 100 ml with water (solution A). Prepare a solution containing
0.020% w/v of diethylstilbestrol EPCRS in ethanol (96%) (solution B). To 2 ml of each solution add
3 ml of phosphomolybdotungstic reagent, dilute to 50 ml with a 5% w/v solution of anhydrous sodium
carbonate, mix thoroughly and allow to stand for 30 minutes; filter the solutions through a poly-
tetrafluoroethylene filter (0.45 m) and measure the absorbance, Appendix II B, at 660 nm using
water in the reference cell. The absorbance of solution A, multiplied by 1.46, is not greater than that
obtained for solution B (1%).
Assay Heat 0.2 g in a Kjeldahl flask with 2 ml of sulphuric acid and 2.5 ml of nitric acid until brown
fumes cease to be evolved, allow to cool, add 1 ml of nitric acid and heat again. Continue adding nitric
acid and heating until brown fumes are no longer evolved and the solution is colourless when cold.
Heat until dense, white fumes are evolved, cool, transfer the solution to a flask with the aid of 150 ml
of water, add 50 ml of citricmolybdic acid solution and heat slowly to boiling point. Swirling the flask
continuously, add 25 ml of quinoline solution, at first dropwise and then in a steady stream, heat on a
water bath for 5 minutes and cool. Filter, wash the precipitate with water until free from acid, transfer
the precipitate to a flask with the aid of 100 ml of water, add 50 ml of 0.5M sodium hydroxide VS and
shake until dissolved. Titrate the excess of alkali with 0.5M hydrochloric acid VS using phenolphthalein
thymol blue solution as indicator. Each ml of 0.5M sodium hydroxide VS is equivalent to 4.964 mg of
C
18
H
18
Na
4
O
8
P
2
.
Storage Fosfestrol Sodium should be kept in a well-closed container and protected from light.
Preparations
Fosfestrol Injection
Fosfestrol Tablets
18-25
Fosfomycin Calcium
O PO
3
Ca Me
H H
C
3
H
5
CaO
4
P,H
2
O 194.1
Fosfomycin Calcium complies with the requirements of the 3rd edition of the European Pharmacopoeia [1328].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Calcium fosfomycin contains not less than 95.0 per cent and not more than the equivalent of
101.0 per cent of calcium (2R,3S)-(3-methyloxiran-2-yl)phosphonate, calculated with reference to
CHARACTERS
A white or almost white powder, slightly soluble in water, practically insoluble in acetone, in
methanol and in methylene chloride.
IDENTIFICATION
spectrum of fosfomycin calcium. Examine the substance as discs prepared using potassium bromide R.
B. Dissolve about 0.1 g in 3 ml of a 25 per cent V/V solution of perchloric acid R. Add 1 ml of 0.1M
sodium periodate and heat on a water-bath for 30 min. Allow to cool and add 50 ml of water R.
Neutralise with a saturated solution of sodium hydrogen carbonate R and add 1 ml of a freshly prepared
400 g/l solution of potassium iodide R. Prepare a blank at the same time and in the same manner. The
test solution remains colourless and the blank is orange.
C. To about 8 mg of the substance to be examined, add 2 ml of water R, 1 ml of perchloric acid R and
2 ml of 0.1M sodium periodate. Heat on a water-bath for 10 min and add, without cooling, 1 ml of
ammonium molybdate solution R5 and 1 ml of aminohydroxynaphthalenesulphonic acid solution R. Allow
to stand for 30 min. A blue colour develops.
D. It gives reaction (a) of calcium (2.3.1).
TESTS
pH (2.2.3). Dissolve 20 mg in carbon dioxide-free water R and dilute to 20.0 ml with the same solvent.
Specific optical rotation (2.2.7). Dissolve 2.5 g in a 125 g/l solution of sodium edetate R previously
adjusted to pH 8.5 with strong sodium hydroxide solution R, and dilute to 50.0 ml with the same
solution. Measured at 405 nm using a mercury lamp, the specific optical rotation is 11.0 to 13.0,
Calcium 1,2-(dihydroxypropyl)phosphonate Not more than 1.5 per cent. Into a stoppered-glass
flask, dissolve 0.200 g of the substance to be examined in 100.0 ml of water R. Add 50 ml of phthalate
buffer solution pH 6.4 R and 5.0 ml of 0.005M sodium periodate, close and shake. Allow to stand
protected from light for 90 min. Add 10 ml of a freshly prepared 400 g/l solution of potassium
iodide R, close and shake for 2 min. Titrate with 0.0025M sodium arsenite until the yellow colour
almost disappears. Add 2 ml of starch solution R and titrate slowly until the colour is completely
discharged. Carry out a blank test under the same conditions. Calculate the percentage content of
C
3
H
7
CaO
5
P from the expression:
100
) 100 (
97 ) (
2 1

H m
c n n
m=quantity of the substance to be examined in milligrams,
n
1
=volume of 0.0025M sodium arsenite used in the blank titration,
n
2
=volume of 0.0025M sodium arsenite used in the titration of the test solution,
c =molarity of the sodium arsenite solution,
H=percentage content of water.
18-26
Chlorides (2.4.4). Dissolve 0.500 g in water R, add 2 ml of nitric acid R and dilute to 50 ml with the
same acid. To 2.5 ml of the solution add 12.5 ml of water R. The solution complies with the limit test
for chlorides (0.2 per cent).
Heavy metals (2.4.8). Dissolve 2.5 g of the substance to be examined in 6 ml of glacial acetic acid R
and dilute to 25.0 ml with water R. 12 ml of the solution complies with limit test A for heavy metals
(20 ppm). Prepare the standard using lead standard solution (2 ppm Pb) R.
mination of water. Use as the solvent a mixture of 1 volume of pyridine R and 3 volumes of ethylene
glycol R.
ASSAY
Into a stoppered-glass flask, dissolve 0.120 g of the substance to be examined in 20.0 ml of 0.1M
sodium periodate. Add 5 ml of a 50 per cent V/V solution of perchloric acid R and shake. Heat in a
water-bath at 37C for 105 min. Add 50 ml of water R and immediately adjust to pH 6.4 with a
saturated solution of sodium hydrogen carbonate R. Add 10 ml of a freshly prepared 400 g/l solution of
potassium iodide R, close and allow to stand for 2 min. Titrate with 0.1M sodium arsenite until the
yellow colour almost disappears. Add 2 ml of starch solution R and titrate slowly until the colour is
completely discharged. Carry out a blank test under the same conditions.
3
H
5
CaO
4
G
H m
c n n

) 100 (
100 88 ) (
2 1
n
1
n
2
G=percentage content of calcium 1,2-(dihydroxypropyl)phosphonate,
STORAGE
IMPURITIES
PO
3
Ca
H
3
C
OH
OH
*
*
A. calcium 1,2-(dihydroxypropyl)phosphonate
__________________________________________________________________________________________________________ Ph Eur
18-27
Fosfomycin Sodium
O PO
3
Na
2
Me
H H
C
3
H
5
Na
2
O
4
P 182.0 23519-26-8
Fosfomycin Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia [1329].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Fosfomycin sodium contains not less than 95.0 per cent and not more than the equivalent of
101.0 per cent of disodium (2R,3S)-(3-methyloxiran-2-yl)phosphonate, calculated with reference to
CHARACTERS
A white or almost white, very hygroscopic powder, very soluble in water, sparingly soluble in
methanol, practically insoluble in ethanol and in methylene chloride.
IDENTIFICATION
spectrum of fosfomycin sodium. Examine the substance as discs prepared using potassium bromide R.
B. Dissolve about 0.1 g in 3 ml of a 25 per cent V/V solution of perchloric acid R. Add 1 ml of 0.1M
sodium periodate and heat on a water-bath for 30 min. Allow to cool and add 50 ml of water R.
Neutralise with a saturated solution of sodium hydrogen carbonate R and add 1 ml of a freshly prepared
400 g/l solution of potassium iodide R. Prepare a blank at the same time and in the same manner. The
test solution remains colourless and the blank is orange.
C. To about 8 mg of the substance to be examined, add 2 ml of water R, 1 ml of perchloric acid R and
2 ml of 0.1M sodium periodate. Heat on a water-bath for 10 min and add, without cooling, 1 ml of
TEST
solution B
9
(Method II, 2.2.2).
solvent. Measured at 405 nm using a mercury lamp, the specific optical rotation is 13.0 to 15.0,
Disodium 1,2-(dihydroxypropyl)phosphonate Not more than 1.0 per cent. Into a stoppered-
glass flask, dissolve 0.200 g of the substance to be examined in 100.0 ml of water R. Add 50 ml of
phthalate buffer solution pH 6.4 R and 5.0 ml of 0.005M sodium periodate, close and shake. Allow to
stand protected from light for 90 min. Add 10 ml of a freshly prepared 400 g/l solution of potassium
iodide R, close and shake for 2 min. Titrate with 0.0025M sodium arsenite until the yellow colour
almost disappears. Add 2 ml of starch solution R and titrate slowly until the colour is completely
discharged. Carry out a blank test under the same conditions. Calculate the percentage content of
C
3
H
7
Na
2
O
5
100
) 100 (
100 ) (
2 1

H m
c n n
n
1
n
2
18-28
of water. Use as the solvent a mixture of 1 volume of pyridine R and 3 volumes of ethylene glycol R.
without a further appropriate procedure for the removal of bacterial endotoxins, it contains not more
than 0.083 I.U. of bacterial endotoxins per milligram.
ASSAY
Into a stoppered-glass flask, dissolve 0.120 g of the substance to be examined in 20.0 ml of 0.1M
sodium periodate. Add 5 ml of a 50 per cent V/V solution of perchloric acid R and shake. Heat on a
water-bath at 37C for 105 min. Add 50 ml of water R and immediately adjust to pH 6.4 with a
saturated solution of sodium hydrogen carbonate R. Add 10 ml of a freshly prepared 400 g/l solution of
potassium iodide R, close and allow to stand for 2 min. Titrate with 0.1M sodium arsenite until the
yellow colour almost disappears. Add 2 ml of starch solution R and titrate slowly until the colour is
completely discharged. Carry out a blank test under the same conditions.
3
H
5
Na
2
O
4
G
H m
c n n

) 100 (
100 91 ) (
2 1
n
1
n
2
G=percentage content of disodium 1,2-(dihydroxypropyl)phosphonate,
STORAGE
LABELLING
The label states:
IMPURITIES
PO
3
Na
2
H
3
C
OH
OH
*
*
A. disodium 1,2-(dihydroxypropyl)phosphonate.
__________________________________________________________________________________________________________ Ph Eur
18-29
Fosfomycin Trometamol
O PO
3
H
2
Me
H H
, HO
OH
OH
NH
2
C
7
H
18
NO
7
P 259.2 78964-85-9
Fosfomycin Trometamol complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Fosfomycin trometamol contains not less than 98.0 per cent and not more than the equivalent of
102.0 per cent of 1,3-dihydroxy-2-(hydroxymethyl)propan-2-aminium (2R,3S)-(3-methyloxiran-2-
yl)phosphonate, calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white powder, hygroscopic, very soluble in water, slightly soluble in alcohol and in
methanol, practically insoluble in acetone.
IDENTIFICATION
obtained with fosfomycin trometamol CRS.
B. Examine by thin-layer chromatography (2.2.27), using cellulose for chromatography R as the coating
substance.
same solvent.
Reference solution. Dissolve 50 mg of fosfomycin trometamol CRS in water R and dilute to 10 ml with
the same solvent.
volumes of concentrated ammonia R, 20 volumes of water R and 70 volumes of 2-propanol R. Dry the
plate in a current of warm air and expose it to iodine vapour until the spots appear. The principal
C. To about 15 mg of the substance to be examined, add 2 ml of water R, 1 ml of perchloric acid R
and 2 ml of 0.1M sodium periodate. Heat on a water-bath for 10 min and add, without cooling, 1 ml of
TESTS
Specific optical rotation (2.2.7). Measured at 365 nm using a mercury lamp, the specific optical
rotation is 13.5 to 12.5, determined on solution S and calculated with reference to the anhydrous
substance.
Inject 5 l of the test solution, 5 l of reference solution (b) and 5 l of the blank solution.
Continue the chromatography for twice the retention time of the peak due to fosfomycin. In the
chromatogram obtained with the test solution: the area of any peak corresponding to impurity A is
not greater than the area of the corresponding peak in the chromatogram obtained with reference
solution (b) (0.5 per cent, calculated as trometamol salts); the area of any peak, apart from the
principal peak, two peaks corresponding to trometamol and any peak corresponding to impurity A, is
(b) (0.5 per cent, calculated as trometamol salts). The sum of the areas of all the peaks, apart from
the principal peak and the two peaks corresponding to trometamol, is not greater than twice the area
18-30
of the principal peak in the chromatogram obtained with reference solution (b) (1 per cent,
calculated as trometamol salts). Disregard any peak due to the blank and any peak with an area less
(b).
Phosphates Dissolve 0.1 g in 3 ml of dilute nitric acid R and dilute to 10 ml with water R. To 5 ml of
this test solution add 5 ml of water R and 5 ml of molybdovanadic reagent R. Shake vigorously. After
5 min, any colour in the test solution is not more intense that that in a standard prepared at the same
time in the same manner, using 5 ml of phosphate standard solution (5 ppm PO
4
) R (500 ppm).
mination of water.
ASSAY
Examine the substance by liquid chromatography (2.2.29). Prepare the solutions immediately before use.
Reference solution (a). Dissolve 0.60 g of fosfomycin trometamol CRS in the mobile phase and dilute to
Reference solution (b). Dissolve 8.7 mg of fosfomycin trometamol impurity A CRS (disodium salt) in the
Reference solution (c). Dissolve 5 mg of fosfomycin trometamol impurity A CRS (disodium salt) and
10 mg of fosfomycin trometamol CRS in the mobile phase and dilute to 5 ml with the mobile phase.
Blank solution. A 0.3 g/l solution of anhydrous disodium hydrogen phosphate R in the mobile phase.
aminopropylsilyl silica gel for chromatography R (5 m),
as mobile phase at a flow rate of 1 ml/min a 10.89 g/l solution of potassium dihydrogen
phosphate R,
as detector a differential refractometer maintaining the temperature at 35C.
When the chromatograms are recorded in the prescribed conditions, the relative retention times (to
fosfomycin) are about 0.3 for the two peaks corresponding to trometamol and 0.8 for impurity A.
Inject 5 l of reference solution (c). The test is not valid unless the resolution between the peaks
corresponding to fosfomycin and impurity A is at least 1.5. Inject reference solution (a) six times.
The test is not valid unless the relative standard deviation of the peak area for fosfomycin is at most
1.0 per cent. Inject alternately the test solution and reference solution (a).
Calculate the percentage content of fosfomycin trometamol.
STORAGE
Store in an airtight container, protected from moisture.
IMPURITIES
, HO
OH
OH
NH
2
H
3
C
PO
3
H
2
OH
OH
*
*
A. 1,3-dihydroxy-2-(hydroxymethyl)propan-2-aminium (1,2-dihydroxypropyl)phosphonate,
O
PO
3
H
2
Me
OH
O
OH
NH
2
*
*
B. [2-[2-amino-3-hydroxy-2-(hydroxymethyl)propoxy]-1-hydroxypropyl]phosphonic acid,
O
O
OH
NH
2
P
O
OH
OH
C. mono[2-amino-3-hydroxy-2-(hydroxymethyl)propyl] phosphate (trometamol phosphoric
ester),
18-31
*
*
*
*
P
O
OH
O O
PO
3
H
2
Me
OH
O
Me
OH
O
OH
NH
2
D. [2-[[[2-[2-amino-3-hydroxy-2-(hydroxymethyl)propoxy]-1-hydroxypropyl]hydroxy-
phosphoryl]oxy]-1-hydroxypropyl]phosphonic acid (trometamoyloxy fosfomycin dimer).
__________________________________________________________________________________________________________ Ph Eur
18-32
Framycetin Sulphate
, x H
2
SO
4
H
2
N OH
H
2
N
O
OH
HO
H
2
N
CH
2
NH
2
O
O HOCH
2
OH
O
O
O
HO
NH
2
OH
CH
2
NH
2
C
23
H
46
N
6
O
13
,xH
2
SO
4
614.6 (base) 4146-30-9
Framycetin Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0180].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Framycetin sulphate is the sulphate of O-2,6-diamino-2,6-dideoxy--D-glucopyranosyl-(14)-O-
[O-2,6-diamino-2,6-dideoxy--L-idopyranosyl-(13)--D-ribofuranosyl]-(15)-(2-deoxy-D-
streptamine) (neomycin B), a substance produced by the growth of selected strains of Streptomyces
fradiae or Streptomyces decaris or obtained by any other means. The potency is not less than 630 I.U.
of neomycin B per milligram, calculated with reference to the dried substance.
CHARACTERS
A white or yellowish-white powder, hygroscopic, freely soluble in water, very slightly soluble in
alcohol, practically insoluble in acetone and in ether.
IDENTIFICATION
A. It complies with the test for neomycin C (see Tests).
B. It gives reaction (a) of sulphates (2.3.1).
TESTS
solvent. The specific optical rotation is +52.5 to +55.5, calculated with reference to the dried
substance.
Alcohols content Not more than 2 per cent, calculated as methanol. Place 0.200 g (m g) in a small
distillation apparatus. Dissolve in 5 ml of water R and add 0.05 ml of 0.05M sulphuric acid. Distil and
collect about 2.5 ml of the distillate in a 10 ml cylinder. Transfer to a conical flask rinsing the
cylinder with two quantities, each of 1 ml, of water R. Add 25.0 ml of 0.0167M potassium dichromate
containing 40 per cent V/V of sulphuric acid R. Heat on a water-bath for 30 min, cool, transfer to a
750 ml conical flask and dilute to 500 ml with water R. Add 10 ml of a 100 g/l solution of potassium
iodide R. Allow to stand for 5 min and titrate with 0.1M sodium thiosulphate, using starch solution R,
added towards the end of the titration, as indicator, until the dark-blue colour becomes pale green
(n
1
ml of 0.1M sodium thiosulphate). Carry out a blank test using 5 ml of water R (n
2
ml of 0.1M sodium
thiosulphate).
Calculate the percentage content of alcohols as CH
3
OH from the expression:
m
n n ) (
0534 . 0
1 2

18-33
Neamine. Examine by thin-layer chromatography (2.2.27), using silica gel H R as the coating
substance.
the same solvent.
Reference solution (a). Dissolve 0.5 mg of neamine CRS in 2.0 ml of water R.
Reference solution (b). Mix 0.5 ml of the test solution with 0.5 ml of reference solution (a).
Apply separately to the plate as 5 mm bands 5 l of each solution. Dry the bands. Develop over a
path of at least 8 cm with a mixture of 10 volumes of methylene chloride R, 20 volumes of concentrated
ammonia R and 30 volumes of methanol R. Dry the plate at 100C to 105C for 10 min. Spray with
ninhydrin and stannous chloride reagent R and heat the plate at 110C for 15 min. Spray the plate again
with the same reagent and heat at 110C for 15 min. Any spot corresponding to neamine in the
chromatogram obtained with the test solution is not more intense than the spot in the chromatogram
obtained with the reference solution (a) (1 per cent). The test is not valid unless the chromatogram
obtained with reference solution (b) shows two clearly separated principal spots.
Neomycin C Examine by thin-layer chromatography (2.2.27), using a suitable silica gel as the
coating substance.
Test solution. Dissolve 40 mg of the substance to be examined in water R and dilute to 5.0 ml with the
same solvent.
Reference solution (a). Dissolve 40 mg of framycetin sulphate CRS in water R and dilute to 5.0 ml with
the same solvent.
Reference solution (b). Dissolve 30 mg of framycetin sulphate CRS in water R and dilute to 25.0 ml with
the same solvent. Dilute 5.0 ml of this solution to 25.0 ml with water R.
Reference solution (c). Dissolve 40 mg of neomycin sulphate CRS in water R and dilute to 5.0 ml with
the same solvent.
Apply separately to the plate as 5 mm bands 5 l of each solution. Develop over a path of at least
12 cm with a mixture of 20 volumes of methanol R and 80 volumes of a 200 g/l solution of sodium
chloride R. Dry the plate at 100C to 105C for 10 min. Spray with ninhydrin solution R1 and heat the
plate at 100C to 105C for 10 min. In the chromatogram obtained with the test solution: the prin-
cipal spot is similar in position, colour and size to the principal spot in the chromatogram obtained
with reference solution (a); the neomycin C spot, with an R
f
value slightly less than that of the prin-
cipal spot, is not more intense than the spot in the chromatogram obtained with reference solution
(b) (3 per cent). The test is not valid unless in the chromatogram obtained with reference solution (c)
a spot appears with an R
f
value slightly less than that of the principal spot.
4
continuing the titration until the violet-blue colour disappears.
4
).
diphosphorus pentoxide R at a pressure not exceeding 0.7 kPa for 3 h.
Sterility (2.6.1). If intended for introduction into body cavities without a further appropriate
sterilisation procedure, it complies with the test for sterility.
Pyrogens (2.6.8). If intended for introduction into body cavities without a further appropriate
procedure for the removal of pyrogens, it complies with the test for pyrogens. Inject per kilogram of
the rabbits mass 16 mg of the substance to be examined, dissolved in 5 ml of water for injections R.
ASSAY
Carry out the microbiological assay of antibiotics (2.7.2). Use framycetin sulphate CRS as the refer-
ence substance.
STORAGE
Store in an airtight container, protected from light. If the substance is intended for introduction into
body cavities, store in a sterile, tamper-proof container.
LABELLING
The label states:
18-34
IMPURITIES
NH
2
HO
O
O
O
OH
O
O
CH
2
NH
2
H
2
N
HO
OH
O
CH
2
NH
2
OH
OH
H2N
NH2
CH2 HO
A. neomycin C,
H
2
O
CH
2
NH
2
H
2
N
HO
OH
O
OH
H2N
N
OH
B. neamine.
__________________________________________________________________________________________________________ Ph Eur
18-35
Frangula Bark
Frangula Bark complies with the requirements of the 3rd edition of the European Pharmacopoeia [0025]. These
Preparation
Standardised Frangula Bark Dry Extract
When Powdered Frangula Bark is prescribed or demanded, material complying with the require-
ments below with the exception of Identification test A and the test for Foreign matter shall be
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Frangula bark consists of the dried, whole or fragmented bark of the stems and branches of Rhamnus
frangula L. (Frangula alnus Miller). It contains not less than 7.0 per cent of glucofrangulins, expressed
as glucofrangulin A (C
27
H
30
O
14
; M
r
CHARACTERS
It has the macroscopic and microscopic characters described under Identification tests A and B.
IDENTIFICATION
A. The bark occurs in curved, almost flat or rolled fragments or in single or double quilled pieces
usually 0.5 mm to 2 mm thick and variable in length and width. The greyish-brown or dark brown
outer surface is wrinkled longitudinally and covered with numerous greyish, transversely elongated
lenticels; when the outer layers are removed, a dark red layer is exposed. The orange-brown to
reddish-brown inner surface is smooth and bears fine longitudinal striations; it becomes red when
treated with alkali. The fracture is short, fibrous in the inner part.
B. Reduce to a powder (355). The powder is yellowish or reddish-brown. Examine under a
microscope using chloral hydrate solution R. The powdered drug shows: numerous phloem fibres,
partially lignified, in groups with crystal sheaths containing calcium oxalate prisms; reddish-brown
fragments of cork; fragments of parenchyma containing calcium oxalate cluster crystals. Sclereids are
absent.
C. Examine the chromatogram obtained in the test for Other species of Rhamnus; anthrones in
daylight. The chromatogram obtained with the test solution shows two orange brown zones
(glucofrangulins) in the lower third and two to four red zones (frangulins, not always clearly
separated, and above them frangula-emodine) in the upper third.
D. To about 50 mg of the powdered drug (180) add 25 ml of dilute hydrochloric acid R and heat the
mixture on a water-bath for 15 min. Allow to cool, shake with 20 ml of ether R and discard the
aqueous layer Shake the ether layer with 10 ml of dilute ammonia R1. The aqueous layer becomes
reddish-violet.
TESTS
Other species of Rhamnus; anthrones Examine by thin-layer chromatography (2.2.27), using a
Test solution. To 0.5 g of the powdered drug (180) add 5 ml of alcohol (70 per cent V/V) R and heat to
boiling. Cool and centrifuge. Decant the supernatant solution immediately and use within 30 min.
Reference solution. Dissolve 20 mg of barbaloin R in alcohol (70 per cent V/V) R and dilute to 10 ml
Apply separately to the plate, as bands, 10 l of each solution. Develop over a path of 10 cm using a
mixture of 13 volumes of water R, 17 volumes of methanol R and 100 volumes of ethyl acetate R.
Allow the plate to dry for 5 min, spray with a 50 g/l solution of potassium hydroxide R in alcohol (50 per
cent V/V) R, and heat at 100C to 105C for 15 min. Examine in ultraviolet light at 365 nm. The
chromatogram obtained with the reference solution shows a brownish-yellow zone corresponding to
barbaloin in the central part. The chromatogram obtained with the test solution shows no zones of
intense yellow fluorescence and no zone of orange to reddish fluorescence similar in position to the
zone of barbaloin in the chromatogram obtained with the reference solution.
Apply to another plate, as a band, 10 l of the test solution and develop as described above. Allow
the plate to dry for not longer than 5 min and spray immediately with a 5 g/l solution of nitrotetra-
zolium blue R in methanol R. Examine the chromatogram immediately. No violet or greyish-blue zones
appear.
18-36
ASSAY
In a tared, round-bottomed flask with a ground-glass neck, weigh 0.250 g of powdered drug (180).
Add 25.0 ml of a 70 per cent V /V solution of methanol R; mix and weigh again. Heat in a water-bath
under a reflux condenser for 15 min. Allow to cool, weigh and adjust to the original mass with a
70 per cent V /V solution of methanol R. Filter and transfer 5.0 ml of the filtrate to a separating
funnel. Add 50 ml of water R and 0.1 ml of hydrochloric acid R. Shake with five quantities, each of
20 ml, of light petroleum R. Allow the layers to separate and transfer the aqueous layer to a 100 ml
volumetric flask. Combine the light petroleum layers and wash with two quantities, each of 15 ml, of
water R. Use this water for washing the separating funnel and add it to the aqueous solution in the
volumetric flask. Add 5 ml of a 50 g/l solution of sodium carbonate R and dilute to 100.0 ml with
water R. Discard the light petroleum layer. Transfer 40.0 ml of the aqueous solution to a 200 ml
round-bottomed flask with a ground-glass neck. Add 20 ml of a 200 g/l solution of ferric chloride R
and heat under a reflux condenser for 20 min in a water-bath with the water level above that of the
liquid in the flask. Add 2 ml of hydrochloric acid R and continue heating for 20 min, shaking
frequently, until the precipitate is dissolved. Allow to cool, transfer the mixture to a separating funnel
and shake with three quantities, each of 25 ml, of ether R, previously used to rinse the flask. Combine
the ether extracts and wash with two quantities, each of 15 ml, of water R. Transfer the ether layer to
a volumetric flask and dilute to 100.0 ml with ether R. Evaporate 20.0 ml carefully to dryness and
dissolve the residue in 10.0 ml of a 5 g/l solution of magnesium acetate R in methanol R. Measure the
absorbance (2.2.25) at 515 nm using methanol R as the compensation liquid.
Calculate the percentage of glucofrangulins, expressed as glucofrangulin A, from the expression:
A
m
3.06
i.e. taking the specific absorbance of glucofrangulin A to be 204.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
18-37
Fructose
O
OH
OH
HO
HO
CH
2
OH
C
6
H
12
O
6
180.2 57-48-7
Fructose complies with the requirements of the 3rd edition of the European Pharmacopoeia [0188]. These
Preparation
Fructose Intravenous Infusion
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Fructose is D-()-fructopyranose. The substance described in this monograph is not necessarily
suitable for parenteral use.
CHARACTERS
A white, crystalline powder, with a very sweet taste, very soluble in water, soluble in alcohol.
IDENTIFICATION
Test solution. Dissolve 10 mg of the substance to be examined in a mixture of 2 volumes of water R
Reference solution (a). Dissolve 10 mg of fructose CRS in a mixture of 2 volumes of water R and 3
Reference solution (b). Dissolve 10 mg each of fructose CRS, glucose CRS, lactose CRS and sucrose CRS
in a mixture of 2 volumes of water R and 3 volumes of methanol R and dilute to 20 ml with the same
Apply separately to the plate 2 l of each solution and thoroughly dry the starting points. Develop
over a path of 15 cm using a mixture of 10 volumes of water R, 15 volumes of methanol R, 25
volumes of anhydrous acetic acid R and 50 volumes of ethylene chloride R. The solvents should be
measured accurately since a slight excess of water produces cloudiness. Dry the plate in a current of
warm air. Repeat the development immediately, after renewing the mobile phase. Dry the plate in a
current of warm air and spray evenly with a solution of 0.5 g of thymol R in a mixture of 5 ml of
sulphuric acid R and 95 ml of alcohol R. Heat at 130C for 10 min. The principal spot in the chroma-
gram obtained with reference solution (b) shows four clearly separated spots.
B. Dissolve 0.1 g in 10 ml of water R. Add 3 ml of cupri-tartaric solution R and heat. A red precipitate
is formed.
C. To 1 ml of solution S (see Tests) add 9 ml of water R. To 1 ml of the solution add 5 ml of hydro-
chloric acid R and heat to 70C. A brown colour develops.
D. Dissolve 5 g in water R and dilute to 10 ml with the same solvent. To 0.5 ml of the solution add
0.2 g of resorcinol R and 9 ml of dilute hydrochloric acid R and heat on a water-bath for 2 min. A red
colour develops.
TESTS
solution is clear (2.2.1). Add 10 ml of water R. The solution is colourless (Method II, 2.2.2).
Acidity or alkalinity Dissolve 6.0 g in 25 ml of carbon dioxide-free water R and add 0.3 ml of
phenolphthalein solution R. The solution is colourless. Not more than 0.15 ml of 0.1M sodium hydroxide
is required to change the colour of the indicator to pink.
Specific optical rotation (2.2.7). Dissolve 10.0 g in 80 ml of water R, add 0.2 ml of dilute
ammonia R1, allow to stand for 30 min and dilute to 100.0 ml with water R. The specific optical
rotation is 91.0 to 93.5, calculated with reference to the anhydrous substance.
18-38
Foreign sugars Dissolve 5.0 g in water R and dilute to 10 ml with the same solvent. To 1 ml of the
solution add 9 ml of alcohol R. Any opalescence in the solution is not more intense than that in a
mixture of 1 ml of the initial solution and 9 ml of water R.
5-Hydroxymethylfurfural and related compounds To 5 ml of solution S add 5 ml of water R.
The absorbance (2.2.25) measured at 284 nm is not greater than 0.32.
Barium To 10 ml of solution S add 1 ml of dilute sulphuric acid R. When examined immediately and
after 1 h, any opalescence in the solution is not more intense than that in a mixture of 1 ml of distilled
water R and 10 ml of solution S.
Lead in sugars (2.4.10). It complies with the limit test for lead in sugars (0.5 ppm).
of water.
Sulphated ash (2.4.14). Not more than 0.1 per cent. Dissolve 5.0 g in 10 ml of water R, add 2 ml of
sulphuric acid R, evaporate to dryness on a water-bath and ignite to constant mass.
STORAGE
_______________________________________________________________________________________________________________ Ph Eur
18-39
Fucus
Bladderwrack corrected 1/01
Fucus complies with the requirements of the 3rd edition of the European Pharmacopoeia [1426]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Fucus consists of the fragmented or powdered, dried thallus of Fucus vesiculosus L. or F. serratus L. or
Ascophyllum nodosum Le Jolis. It contains not less than 0.03 per cent and not more than 0.2 per cent
of total iodine (A
r
CHARACTERS
Fucus has a salty and mucilaginous taste, and an unpleasant marine odour.
IDENTIFICATION
A. The drug consists of fragments with a corneous consistency, blackish-brown to greenish-brown,
sometimes covered with whitish efflorescence. The thallus consists of a ribbon-like blade, branching
dichotomously with prominent central ribs (pseudoveins). F. vesiculosus typically shows a foliose
blade with smooth edges and bears occasional ovoid, single or paired, air vesicles. The ends of certain
branches are of ovoid shape and a little widened. They bear numerous reproductive organs
(conceptacles). F. serratus has a foliose blade with a serrate margin and no vesicles, the branches
bearing conceptacles are less swollen. The thallus of A. nodosum is irregularly branched, without
pseudo-midrib. It shows single ovoid air vesicles; the falciform conceptacles are located at the end of
small branches.
B. Reduce to a powder (355). The powder is greenish-brown. Examine under a microscope using
chloral hydrate solution R. The powder shows fragments of surface tissue with regular isodiametric cells
with brown contents, and fragments of deep tissue with colourless, elongated cells arranged in long
filaments with large mucilaginous spaces between them. Thick-walled cells in files and in closely
packed groups, from the pseudovein, are sometimes visible.
C. To 5 g of the powdered drug (355) add 20 ml of a 2 per cent V/V solution of hydrochloric acid R.
Shake vigorously and filter. Wash the residue with 10 ml of water R and filter. To the residue add
10 ml of a 200 g/l solution of sodium carbonate R. Shake and centrifuge. Collect the supernatant
liquid. Adjust to pH 1.5 using sulphuric acid R. A white, flocculent precipitate is slowly formed.
TESTS
Heavy metals Not more than 10 ppm for the total content of cadmium, copper, iron, lead, nickel
and zinc and not more than 0.2 ppm of cadmium.
Apparatus. The apparatus consists of the following:
as digestion flasks, tetrafluoroethylene flasks with a volume of about 120 ml fitted with an
airtight closure, which has a valve to adjust the pressure inside the container and a
tetrafluoroethylene tube so that gas can be released. The closure is designed to make the flasks
airtight always, using a given torque,
a microwave oven, with a magnetron frequency of 2450 MHz, with a selectable output from 0 to
630 70 watts in 1 per cent increments, a programmable computer, a tetrafluoroethylene
coated microwave cavity with a variable speed exhaust fan, a rotating turntable drive system and
exhaust tubing to vent fumes,
an atomic absorption spectrometer, equipped with hollow-cathode lamps as source of radiation,
a graphite furnace as atomic generator, and a deuterium lamp as a background corrector.
Method
Clean all the glassware and laboratory equipment with a 10 g/l solution of nitric acid R in water R,
before use.
Test solution. In a digestion flask introduce the prescribed quantity of the substance to be examined
(about 0.50 g of powdered drug (1400)). Add 6 ml of nitric acid R and 4 ml of hydrochloric acid R.
Attach the closure, using a given torque, to make the flask airtight. Prepare six test solutions.
Blank solution. Mix 6 ml of nitric acid R and 4 ml of hydrochloric acid R.
Place the digestion flasks in a microwave oven. Carry out the digestion according to the following
programme in three steps used for six flasks containing the test solution and one containing the blank
solution:
18-40
1. 80 per cent power for 15 min,
2. 100 per cent power for 5 min,
3. 80 per cent power for 20 min.
At the end of the cycle allow the flasks to cool and to each add 4 ml of sulphuric acid R. Repeat the
digestion programme described above. Allow the flasks to cool in air and to each add 1.0 ml of a
10 g/l solution of magnesium nitrate R and 1.0 ml of a 100 g/l solution of ammonium dihydrogen
phosphate R. Dilute quantitatively to 50.0 ml with bidistilled water R.
Measure the content of cadmium, copper, iron, lead, nickel and zinc by the standard additions
method (Method II, 2.2.23), using reference solutions of each heavy metal and the instrumental
settings described in Table 14261.
Table 14261
Cd Cu Fe Ni Pb Zn
Wavelength nm 228.8 324.8 248.3 232 283.3 213.9
Slit width nm 0.5 0.5 0.2 0.2 0.5 0.5
Lamp current mA 6 7 5 10 5 7
Ignition temperature
o
C 800 800 800 800 800 800
Atomisation temperature
o
C 1800 2300 2300 2500 2200 2000
Background on off off off off off
Nitrogen flow 1/min 3 3 3 3 3 3
Arsenic (2.4.2). 1 g of the powdered drug complies with limit test A for arsenic (1 ppm). Prepare the
standard using 1 ml of arsenic standard solution (1 ppm As) R.
Swelling index (2.8.4). Not less than 6.
at 100C to 105C, for 2 h.
Total ash (2.4.16). Not more than 24 per cent.
ASSAY
Total iodine. To 1.000 g of the powdered drug in a tall silica crucible, add 5 ml of water R and 5 g of
potassium hydroxide R. Stir with a glass rod. Heat on a water bath. Add 1 g of potassium carbonate R.
Mix and dry, first on a water-bath then over an open flame. Incinerate raising the temperature
progressively to not more than 600C. Allow to cool. Add 20 ml of water R and heat gently to
boiling, stirring with a glass rod. Filter the hot mixture through an unpleated filter, into a conical
flask. Rinse the residue with four quantities, each of 20 ml, of hot water R. Rinse the filter and the
crucible with 50 ml of hot water R. Combine the solutions. Allow to cool. Neutralise with dilute
sulphuric acid R in the presence of methyl orange solution R. Add 3 ml of dilute sulphuric acid R and 1 ml
of bromine water R. The solution is yellow. After 5 min add 0.6 ml of a 50 g/l solution of phenol R.
The solution is clear. Acidify with 5 ml of phosphoric acid R and add 0.2 g of potassium iodide R. Allow
to stand for 5 min protected from light. Add starch solution R and titrate with 0.01M sodium
thiosulphate.
1 ml of 0.01M sodium thiosulphate is equivalent to 0.2115 mg of iodine.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
18-41
Furazolidone
N
N
O
O
O
2
N
O
C
8
H
7
N
3
O
5
225.2 67-45-8
Definition Furazolidone is 3-(5-nitrofurfurylideneamino)oxazolidin-2-one. It contains not less
8
H
7
N
3
O
5
substance.
Characteristics A yellow, crystalline powder; odourless or almost odourless.
Very slightly soluble in water and in ethanol (96%); slightly soluble in chloroform; practically
insoluble in ether.
Identification A. The infrared absorption spectrum, Appendix II A, is concordant with the reference
spectrum of furazolidone (RS 164).
B. Dissolve 1 mg in 1 ml of dimethylformamide and add 0.05 ml of 1M ethanolic potassium hydroxide. A
deep blue colour is produced.
Acidity or alkalinity Shake 1 g for 15 minutes with
100 ml of carbon dioxide-free water and filter. The pH of the filtrate is 4.5 to 7.0, Appendix V L.
Nitrofurfural diacetate Carry out in subdued light the method for thin-layer chromatography,
Appendix III A, using silica gel G as the coating substance and a mixture of 95 volumes of toluene and
5 volumes of 1,4-dioxan as the mobile phase. Apply separately to the plate 20 l of solution (1) and
10 l of solution (2). For solution (1) dissolve 50 mg of the substance being examined in 5 ml of
dimethylformamide by heating on a water bath for a few minutes, allow to cool and dilute to 10 ml
with acetone. Solution (2) contains 0.010% w/v solution of nitrofurfural diacetate BPCRS in a mixture
of equal volumes of dimethylformamide and acetone. After removal of the plate, heat it at 105 for 5
minutes and spray with a solution prepared by dissolving 0.75 g of phenylhydrazine hydrochloride in
10 ml of ethanol (96%), diluting to 50 ml with water, adding activated charcoal, filtering and then
adding 25 ml of hydrochloric acid and sufficient water to produce 200 ml. Any spot corresponding to
nitrofurfural diacetate in the chromatogram obtained with solution (1) is not more intense than the
spot in the chromatogram obtained with solution (2).
1 g.
Assay Carry out the following procedure protected from light. To 80 mg add 150 ml of
dimethylformamide, swirl to dissolve and add sufficient water to produce 500 ml. Dilute 5 ml to
100 ml with water and mix. Measure the absorbance of the resulting solution at the maximum at
367 nm, Appendix II B. Calculate the content of C
8
H
7
N
3
O
5
taking 750 as the value of A(1%, 1 cm)
at the maximum at 367 nm.
Storage Furazolidone should be protected from light.
Action and use Antibacterial; antifungal; antiprotozoal.
18-42
Furosemide / Frusemide
N
O
COOH
Cl
S
H
2
N
O O
H
C
12
H
11
ClN
2
O
5
S 330.7 54-31-9
Furosemide complies with the requirements of the 3rd edition of the European Pharmacopoeia [0391]. These
Preparations
Co-amilofruse Tablets
Furosemide Injection/Frusemide Injection
Furosemide Tablets/Frusemide Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Furosemide contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent
of 4-chloro-2-(furfurylamino)-5-sulphamoylbenzoic acid, calculated with reference to the dried
substance.
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water, soluble in acetone,
sparingly soluble in alcohol, slightly soluble in ether, practically insoluble in methylene chloride. It
dissolves in dilute solutions of alkali hydroxides.
IDENTIFICATION
A. Dissolve 50 mg in a 4 g/l solution of sodium hydroxide R and dilute to 100 ml with the same
alkaline solution. Dilute 1 ml of the solution to 100 ml with a 4 g/l solution of sodium hydroxide R.
Examined between 220 nm and 350 nm (2.2.25), the solution shows three absorption maxima, at
228 nm, 270 nm and 333 nm. The ratio of the absorbance measured at the maximum at 270 nm to
that measured at the maximum at 228 nm is 0.52 to 0.57.
obtained with furosemide CRS.
C. Dissolve about 25 mg in 10 ml of alcohol R. To 5 ml add 10 ml of water R. To 0.2 ml of this
solution add 10 ml of dilute hydrochloric acid R and heat under a reflux condenser for 15 min. Allow
to cool and add 18 ml of 1M sodium hydroxide and 1 ml of a 5 g/l solution of sodium nitrite R. Allow to
stand for 3 min, add 2 ml of a 25 g/l solution of sulphamic acid R and mix. Add 1 ml of a 5 g/l
solution of naphthylethylenediamine dihydrochloride R. A violet-red colour develops.
TESTS
Reference solution (a). Dissolve 20.0 mg of furosemide impurity A CRS in the mobile phase and dilute
Reference solution (b). Dilute a mixture of 1.0 ml of the test solution and 1.0 ml of reference solution
(a) to 20.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 20.0 ml with the mobile phase.
18-43
as mobile phase at a flow rate of 1 ml per minute a mixture prepared as follows: dissolve 0.2 g of
potassium dihydrogen phosphate R and 0.25 g of cetrimide R in 70 ml of water R; adjust to pH 7.0
with ammonia R and add 30 ml of propanol R,
two peaks in the chromatogram obtained are not less than 20 per cent of the full scale of the
recorder. The test is not valid unless the resolution between the first peak (furosemide impurity A)
and the second peak (furosemide) is at least 4.
Inject 20 l of the test solution. Continue the chromatography for three times the retention time of
from the principal peak, is not greater than the area of the first peak in the chromatogram obtained
principal peak, is not greater than twice the area of the first peak in the chromatogram obtained with
reference solution (b) (0.5 per cent). Disregard any peak with an area less than 0.1 times the area of
the first peak in the chromatogram obtained with reference solution (b).
Chlorides (2.4.4). To 0.5 g add a mixture of 0.2 ml of nitric acid R and 30 ml of water R and shake
for 5 min. Allow to stand for 15 min and filter. 15 ml of the filtrate complies with the limit test for
Sulphates (2.4.13). To 1.0 g add a mixture of 0.2 ml of acetic acid R and 30 ml of distilled water R
and shake for 5 min. Allow to stand for 15 min and filter. 15 ml of the filtrate complies with the limit
100C to 105C.
ASSAY
Dissolve 0.250 g in 20 ml of dimethylformamide R. Titrate with 0.1M sodium hydroxide using 0.2 ml of
bromothymol blue solution R2. Carry out a blank titration.
12
H
11
ClN
2
O
5
S.
STORAGE
IMPURITIES
H
O
N
O O
COOH S
H
2
N
Cl
A. 2-chloro-4-(furfurylamino)-5-sulphamoylbenzoic acid,
H
2
N
S COOH
O O
Cl Cl
B. 2,4-dichloro-5-sulphamoylbenzoic acid,
H
2
N
S COOH
O O
Cl NH
2
C. 2-amino-4-chloro-5-sulphamoylbenzoic acid,
O
H
2
N
S COOH
N
O O
N
O
H
D. 2,4-bis(furfurylamino)-5-sulphamoylbenzoic acid.
__________________________________________________________________________________________________________ Ph Eur
18-44
Fusidic Acid
H
HO
Me
Me
H
Me
H
Me
Me
H
HO
H
COOH
H
OAc
H
H Me
C
31
H
48
O
6
,H
2
O 525.7 6990-06-3
Fusidic Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia [0798]. These
Preparations
Fusidic Acid Cream
Fusidic Acid Eye Drops
Fusidic Acid Oral Suspension
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Fusidic acid is ent-16-acetoxy-3,11-dihydroxy-4,8,14-trimethyl-18-nor-5,10-cholesta-
(17Z)-17(20),24-dien-21-oic acid hemihydrate, an antimicrobial substance produced by the growth
of certain strains of Fusidium coccineum or by any other means. It contains not less than 97.5 per cent
and not more than the equivalent of 101.0 per cent of C
31
H
48
O
6
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water, freely soluble in alcohol.
IDENTIFICATION
obtained with the Ph. Eur. reference spectrum of fusidic acid.
B. Examine by thin-layer chromatography (2.2.27), using silica gel HF
254
the same solvent.
Reference solution. Dissolve 24 mg of diethanolamine fusidate CRS in methanol R and dilute to 10 ml
2.5 volumes of methanol R, 10 volumes of glacial acetic acid R, 10 volumes of cyclohexane R and 80
volumes of chloroform R. Dry the plate in a current of hot air. Examine in ultraviolet light at 254 nm.
The principal spot in the chromatogram obtained with the test solution is similar in position and size
to the principal spot in the chromatogram obtained with the reference solution.
TESTS
Reference solution (a). Dissolve 5 mg of 3-ketofusidic acid CRS in 5 ml of the mobile phase. To 1.0 ml
of this solution add 0.20 ml of the test solution and dilute to 20.0 ml with the mobile phase.
Reference solution (b). Dilute 20 l of the test solution to 100.0 ml with the mobile phase.
a steel column 0.125 m to 0.15 m long and 4 mm to 5 mm in internal diameter packed with
as mobile phase at a flow rate of 2 ml per minute a mixture of 10 volumes of methanol R, 20
18-45
volumes of a 10 g/l solution of phosphoric acid R, 20 volumes of water R and 50 volumes of
acetonitrile R,
Continue the chromatography for at least 3.5 times the retention time of the principal peak. In the
chromatogram obtained with the test solution, the sum of the areas of the peaks, apart from the
principal peak and the solvent peak, is not greater than twice the area of the peak corresponding to
fusidic acid in the chromatogram obtained with reference solution (a) (2.0 per cent). Disregard any
peak with an area less than that of the principal peak in the chromatogram obtained with reference
solution (b). The test is not valid unless: the resolution between the peaks corresponding to 3-keto-
fusidic acid and fusidic acid in the chromatogram obtained with reference solution (a) is at least 2.5;
the principal peak in the chromatogram obtained with reference solution (b) has a signal-to-noise
ratio of at least 3.
of water.
ASSAY
Dissolve 0.500 g in 10 ml of alcohol R. Add 0.5 ml of phenolphthalein solution R. Titrate with 0.1M
sodium hydroxide until a pink colour is obtained.
31
H
48
O
6
.
STORAGE
Store in a well-closed container, protected from light, at a temperature of 2C to 8C.
__________________________________________________________________________________________________________ Ph Eur
18-46
Galactose
*
and epimer at C*
O
OH
OH
OH
HO
CH
2
OH
C
6
H
12
O
16
180.2
Galactose complies with the requirements of the 3rd edition of the European Pharmacopoeia [1215]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Galactose is D-galactopyranose.
CHARACTERS
A white, crystalline or finely granulated powder, freely soluble or soluble in water, very slightly
soluble in alcohol.
IDENTIFICATION
obtained with galactose CRS. Examine the substances prepared as discs.
substance.
Reference solution (a). Dissolve 10 mg of galactose CRS in a mixture of 2 volumes of water R and 3
Reference solution (b). Dissolve 10 mg of galactose CRS, 10 mg of glucose CRS and 10 mg of lactose
CRS in a mixture of 2 volumes of water R and 3 volumes of methanol R and dilute to 20 ml with the
Apply to the plate 2 l of each solution and thoroughly dry the starting points. Develop in an
unsaturated tank over a path of 15 cm using a mixture of 15 volumes of water R and 85 volumes of
propanol R. Dry the plate in a current of warm air. Spray uniformly with a solution of 0.5 g of
thymol R in a mixture of 5 ml of sulphuric acid R and 95 ml of alcohol R. Heat in an oven at 130C for
10 min. The principal spot in the chromatogram obtained with the test solution is similar in position,
colour and size to the principal spot in the chromatogram obtained with reference solution (a). The
test is not valid unless the chromatogram obtained with reference solution (b) shows three clearly
separated spots.
C. Dissolve 0.1 g in 10 ml of water R. Add 3 ml of cupri-tartaric solution R and heat. An orange to red
TESTS
Solution S Dissolve with heating in a water-bath at 50C, 10.0 g in carbon dioxide-free water R
prepared from distilled water R and dilute to 50 ml with the same solvent.
solution B
8
(Method II, 2.2.2).
colourless. Not more than 1.5 ml of 0.01M sodium hydroxide is required to change the colour of the
indicator to pink.
Specific optical rotation (2.2.7). Dissolve 10.00 g in 80 ml of water R and add 0.2 ml of dilute
ammonia R1. Allow to stand for 30 min and dilute to 100.0 ml with water R. The specific optical
rotation is +78.0 to +81.5, calculated with reference to the anhydrous substance.
Barium Dilute 5 ml of solution S to 10 ml with distilled water R. Add 1 ml of dilute sulphuric acid R.
When examined immediately and after 1 h, any opalescence in the solution is not more intense than
that in a mixture of 5 ml of solution S and 6 ml of distilled water R.
18-47
Lead (2.4.10). It complies with the limit test for lead in sugars (0.5 ppm).
of water.
Sulphated ash To 5 ml of solution S add 2 ml of sulphuric acid R, evaporate to dryness on a water-
bath and ignite to constant mass. The residue weighs not more than 1 mg (0.1 per cent).
2
micro-
organisms per gram.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
18-48
Gallamine Triethiodide
OCH
2
CH
2
NEt
3
OCH
2
CH
2
NEt
3
OCH
2
CH
2
NEt
3
3I
_
+
+
+
C
30
H
60
I
3
N
3
O
3
892 65-29-2
Gallamine Triethiodide complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Skeletal muscle relaxant.
Preparation
Gallamine Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Gallamine triethiodide contains not less than 98.0 per cent and not more than the equivalent of
101.0 per cent of 2,2,2-[benzene-1,2,3-triyltris(oxy)]tris(N,N,N-triethylethaninium) tri-iodide,
CHARACTERS
A white or almost white powder, hygroscopic, very soluble in water, slightly soluble in alcohol,
IDENTIFICATION
A. Dissolve 50 mg in 0.01M hydrochloric acid and dilute to 50.0 ml with the same solvent. Dilute
obtained with gallamine triethiodide CRS.
C. To 5 ml of solution S (see Tests) add 1 ml of potassium tetraiodomercurate solution R. A yellow
D. Dilute 0.5 ml of solution S to 2 ml with water R. Add 0.2 ml of dilute nitric acid R. The solution
gives reaction (a) of iodides (2.3.1).
TESTS
Appearance of solution Solution S is clear (2.2.1) and immediately after preparation is not more
7
(Method II, 2.2.2).
Acidity or alkalinity To 50 ml of water R add 0.2 ml of methyl red solution R. Add either 0.01M
sulphuric acid or 0.02M sodium hydroxide until an orange-yellow colour is obtained. Add 1.0 g of the
substance to be examined and dissolve by shaking. Not more than 0.2 ml of 0.01M sulphuric acid or
0.02M sodium hydroxide is required to restore the orange-yellow colour.
as mobile phase at a flow rate of 1 ml/min a solution prepared as follows: dissolve 14 g of
sodium perchlorate R in 850 ml of phosphate buffer pH 3.0 R and add 150 ml of methanol R,
18-49
When the chromatograms are recorded in the prescribed conditions, the retention times relative to
triethylgallamine as perchlorate (about 40 min) are: impurity A, about 0.45; impurity B, about 0.50;
impurity C, about 0.65; impurity D, about 0.75; impurity E, about 0.85 and impurity F, about 0.90
(see Fig. 181-1).
Inject separately 20 l of the test solution and 20 l of the reference solution. Continue the
chromatography for 1.5 times the retention time of triethylgallamine as perchlorate.
In the chromatogram obtained with the test solution: the area of any peak, apart from the principal
peak, is not greater than the area of the principal peak in the chromatogram obtained with the
reference solution (1 per cent) and the sum of the areas of such peaks is not greater than twice the
area of the principal peak in the chromatogram obtained with the reference solution (2 per cent).
Disregard the peak due to iodide with a retention time of zero.
100C to 105C.
ASSAY
Dissolve 0.270 g in a mixture of 5.0 ml of anhydrous formic acid R and 50.0 ml of acetic anhydride R.
30
H
60
I
3
N
3
O
3
.
STORAGE
IMPURITIES
OCH
2
CH
2
NEt
2
OCH
2
CH
2
NEt
2
OCH
2
CH
2
NEt
2
A. 2,2,2-[benzene-1,2,3-triyltris(oxy)]tris(N,N-diethylethanamine),
OCH
2
CH
2
NEt
3

OCH
2
CH
2
NEt
3

CH
2
CH
2
NEt
3

3I
-
+
+
+
B. 2,2-[2-[2-(triethylammonio)ethyl]-1,3-phenylenebis(oxy)]bis(N,N,N-
triethylethanaminium) tri-iodide,
OCH
2
CH
2
NEt
3

OCH
2
CH
2
NEt
3

OCH
2
CH
2
NMeEt
2

3I
-
+
+
+
C. 2,2-[2-[2-(diethylmethylammonio)ethoxy]-1,3-phenylenebis(oxy)]bis(N,N,N-
OCH
2
CH
2
NEt
3

OCH
2
CH
2
NMeEt
2

OCH
2
CH
2
NEt
3

3I
-
+
+
+
D. 2,2-[3-[2-(diethylmethylammonio)ethoxy]-1,2-phenylenebis(oxy)]bis(N,N,N-
OCH
2
CH
2
NEt
3

OCH
2
CH
2
NEt
2

OCH
2
CH
2
NEt
3

2I
-
+
+
E. 2,2-[3-[2-(diethylamino)ethoxy]-1,2-phenylenebis(oxy)]bis(N,N,N-triethylethanaminium)
di-iodide,
18-50
OCH
2
CH
2
NEt
3

OCH
2
CH
2
NEt
3

OCH
2
CH
2
NEt
3

CH
2
CH
2
NEt
3

4I
-
+
+
+
+
F. 2,2,2-[4-[2-(triethylammonio)ethyl]benzene-1,2,3-triyltris(oxy)]tris(N,N,N-
triethylethanaminium) tetraiodide.
The type chromatogram is given for information and guidance only; it is not part of the mandatory require-
ments of the monograph.
Fig. 1811 Type chromatogram
__________________________________________________________________________________________________________ Ph Eur
18-51
Garlic Powder
Garlic Powder complies with the requirements of the 3rd edition of the European Pharmacopoeia [1216]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Garlic powder is produced from the bulbs of Allium sativum L., cut, freeze-dried or dried at a
temperature not exceeding 65C and powdered. It contains not less than 0.45 per cent of allicin
(C
6
H
10
OS
2
; M
r
CHARACTERS
A light yellowish powder.
It shows the microscopic characters described under Identification test A.
IDENTIFICATION
A. Examine under a microscope using chloral hydrate solution R. The powder shows numerous
fragments of parenchyma and groups of spiral or annular vessels accompanied by thin-walled
parenchyma.
substance.
Test solution. To 1.0 g of garlic powder add 5.0 ml of methanol R, shake for 60 s and filter.
Reference solution. Dissolve 5 mg of alanine R in 10 ml of water R and dilute to 20 ml with methanol R.
propanol R, 20 volumes of water R and 40 volumes of ethanol R. Allow the plate to dry in air. Spray
with a 2 g/l solution of ninhydrin R in a mixture of 5 volumes of glacial acetic acid R and 95 volumes of
butanol R and heat at 105C to 110C for 5 min to 10 min. Examine in daylight. The chromatogram
obtained with the reference solution shows a violet zone (alanine) in its central third. The chromato-
gram obtained with the test solution shows a violet or brownish-red zone similar in position to that in
the chromatogram obtained with the reference solution and corresponding to alliin; above and below
this zone are other, generally fainter, violet zones.
TESTS
Starch Examine the powdered drug under a microscope using water R. Add iodine solution R1. No
ASSAY
Examine by liquid chromatography (2.2.29), using butyl parahydroxybenzoate R as the internal
standard. Carry out the assay as quickly as possible.
Internal standard solution. Dissolve 20.0 mg of butyl parahydroxybenzoate R in 100.0 ml of a mixture of
equal volumes of methanol R and water R.
Test solution. To 0.800 g of garlic powder add 20.0 ml of water R and homogenise the mixture in an
ultrasonic bath at 4C for 5 min. Allow to stand at room temperature for 30 min. Then centrifuge for
30 min. Dilute 10.0 ml of the supernatant to 25.0 ml with a mixture of 40 volumes of a 1 per cent
V/V solution of anhydrous formic acid R and 60 volumes of methanol R (stock solution). Shake and
centrifuge for 5 min. Place 0.50 ml of the internal standard solution in a volumetric flask and dilute
to 10.0 ml with the stock solution.
a stainless steel column 0.25 m long and 4 mm in internal diameter packed with silanised
octadecylsilyl silica gel for chromatography R (5 m) combined with a stainless steel pre-column
20 mm long and 4 mm in internal diameter packed with silanised octadecylsilyl silica gel for
as mobile phase at a flow rate of 0.8 ml/min a mixture of 40 volumes of a 1 per cent V/V solution
of anhydrous formic acid R and 60 volumes of methanol R,
a loop injector,
Inject 1 l of the internal standard solution. Inject 10 l of the test solution. Adjust the sensitivity of
the system so that the height of the peak due to butyl parahydroxybenzoate in the chromatogram
obtained with the test solution is about 50 per cent of the full scale of the recorder.
18-52
Calculate the percentage of allicin from the expression:
1 2
2 1
75 . 22
m S
m S

S
1
=area of the peak corresponding to allicin (most prominent peak),
S
2
=area of the peak corresponding to butyl parahydroxybenzoate in the chromatogram
obtained with the test solution,
m
1
=mass of the drug in grams,
m
2
=mass of butylhydroxybenzoate in grams in 100.0 ml of the internal standard solution.
1 mg of butylparaphydroxybenzoate corresponds to 8.65 mg of allicin.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
18-53
Gelatin
1/01
Gelatin complies with the requirements of the 3rd edition of the European Pharmacopoeia [0330]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Gelatin is a purified protein obtained either by partial acid hydrolysis (type A) or partial alkaline
hydrolysis (type B) of animal collagen; it may also be a mixture of the two types. Gelatin described in
this monograph is not necessarily suitable for preparations for parenteral use or for other special
purposes.
PRODUCTION
CHARACTERS
A faintly yellow to light yellowish-brown, tasteless solid, usually occurring as translucent sheets,
shreds, granules or powder. It is practically insoluble in common organic solvents; it swells in cold
water and on heating it gives a colloidal solution which on cooling forms a more or less firm gel. The
isoelectric point of type A gelatin is between pH 6.3 and 9.2 and that of type B gelatin is between pH
4.7 and 5.2.
IDENTIFICATION
A. To 2 ml of solution S (see Tests) add 0.05 ml of copper sulphate solution R. Mix and add 0.5 ml of
dilute sodium hydroxide solution R. A violet colour is produced.
B. To 0.5 g in a test-tube add 10 ml of water R. Allow to stand for 10 min, heat at 60C for 15 min
and keep the tube upright at 0C for 6 h. Invert the tube; the contents do not immediately flow out.
TESTS
Solution S Dissolve 1 g in carbon dioxide-free water R at about 55C, dilute to 100 ml with the same
solvent and keep the solution at this temperature to carry out the tests.
Appearance of solution Solution S is not more intensely coloured than reference solution Y
4
(Method II, 2.2.2) and when prepared from type A or type B gelatin it is not more opalescent than
reference suspension IV (2.2.1). Mixtures of type A and type B gelatin may give opalescent solutions
through formation of coacervates over a wide range of pH, depending on concentration.
Phenolic preservatives Examine by thin-layer chromatography (2.2.27), using a TLC silica gel F
254
plate R.
Test solution. To 1.0 g of the substance to be examined add 20 ml of methanol R and 2 ml of
ammonia R. Mix and allow to stand for 20 h. Decant the clear solution, evaporate to dryness and
dissolve the residue in 0.5 ml of methanol R.
Reference solution (a). Dissolve 2 mg of either ethyl parahydroxybenzoate R or methyl
parahydroxybenzoate R or propyl parahydroxybenzoate R in methanol R and dilute to 10 ml with the
same solvent. Dilute 2 ml of the solution to 10 ml with methanol R.
Reference solution (b). Dissolve 2 mg of benzocaine R in methanol R and dilute to 10 ml with the same
solvent.
Reference solution (c). Dissolve 1 mg of ethyl parahydroxybenzoate R, 1 mg of methyl
parahydroxybenzoate R, 1 mg of propyl parahydroxybenzoate R and 2 mg of benzocaine R in methanol R
Apply to the plate as bands 20 mm by 3 mm 10 l of each solution. To each of the dried starting
bands of the test solution and of reference solution (b) apply 10 l of a 1 g/l solution of dimethyl-
aminonaphthalenesulphonyl chloride R in acetone R. Separately apply as bands 20 mm by 3 mm 10 l of
the dimethylaminonaphthalenesulphonyl chloride solution and 10 l of the test solution (not
derivated). Spray the starting bands with a 50 g/l solution of disodium tetraborate R holding the plate
nearly horizontally. Dry the plate at 60C for 15 min. Carry out the chromatography protected from
light. Develop over a path of 12 cm using a mixture of 8 volumes of ethanol R and 92 volumes of
toluene R. Allow the plate to dry in air and examine immediately in ultraviolet light at 365 nm. The
chromatogram obtained with reference solution (b) shows a zone of reddish fluorescence with an R
f
18-54
value of about 0.5 (dimethylaminonaphthalenesulphonyl derivative of benzocaine) and spots of
dimethylaminonaphthalenesulphonyl chloride. The chromatogram obtained with 10 l of the
dimethylaminonaphthalenesulphonyl chloride solution shows zones of blue fluorescence at the
starting-point and near the solvent front. Apart from the starting-point and the zones of
dimethylaminonaphthalenesulphonyl chloride, the chromatogram obtained with the test solution
does not show zones of fluorescence which are more intense than the zone corresponding to the
benzocaine derivative in the chromatogram obtained with reference solution (b) (for example amino
acids detergents). Examine in ultraviolet light at 254 nm. The chromatogram obtained with reference
solution (a) shows fluorescent quenching zones with R
f
values of 0.3 to 0.5 (esters of parahydroxy-
benzoic acid). The chromatogram obtained with the non-derivated test solution does not show
fluorescent quenching zones which are more intense than the zone obtained with reference solution
(a) (phenolic preservatives, for example esters of parahydroxybenzoic acid). The test is not valid
unless, in the chromatogram obtained with reference solution (c), examined in ultraviolet light at
254 nm, the zones corresponding to benzocaine and the esters of parahydroxybenzoic acid are clearly
separated.
Fig. 03301 Apparatus for the determination of sulphur dioxide
Dimensions in millimetres
Sulphur dioxide Not more than 200 ppm. Introduce 150 ml of water R into the flask (A) (see Fig.
03301) and pass carbon dioxide R through the whole system for 15 min at a rate of 100 ml/min.
Place 10 ml of dilute hydrogen peroxide solution R neutralised to a 1 g/l solution of bromophenol blue R in
alcohol (20 per cent V/V) R in the test-tube (D). Without interrupting the stream of carbon dioxide,
remove the funnel (B) and introduce through the opening into the flask 25.0 g of the substance to be
examined with the aid of 100 ml of water R. Add through the funnel 80 ml of dilute hydrochloric acid R
and boil for 1 h. Open the tap of the funnel and stop the flow of carbon dioxide, the heating, and the
cooling water. Transfer the contents of the test-tube with the aid of a little water R to a 200 ml wide-
necked, conical flask. Heat on a water-bath for 15 min and allow to cool. Add 0.1 ml of a 1 g/l
solution of bromophenol blue R in alcohol (20 per cent V/V) R and titrate with 0.1M sodium hydroxide
until the colour changes from yellow to violet-blue.
Calculate the content of sulphur dioxide in parts per million from the expression: 128a.
a=number of millilitres of 0.1M sodium hydroxide used.
18-55
Peroxides Dissolve 1.0 g with heating in 10 ml of water R and add 2 ml of divanadium pentoxide
solution in sulphuric acid R. Any orange-yellow colour in the solution is not more intense than that in a
standard prepared in the same manner using a mixture of 1 ml of hydrogen peroxide solution (0.1 g/l
H
2
O
2
) and 9 ml of water R (100 ppm of H
2
O
2
).
Gel strength If intended for use in the preparation of pessaries, suppositories or zinc gelatin, it
complies with the following additional requirement.150 g to 250 g. The gel strength is expressed as
the mass in grams necessary to produce the force which, applied to a plunger 12.7 mm in diameter,
makes a depression 4 mm deep in a gel having a concentration of 6.67 per cent m/m and matured at
10C.
Apparatus. The gelometer consists of:
a cylindrical piston 12.7 0.1 mm in diameter with a plane pressure surface with a rounded
edge (0.5 mm radius),
a device for adjusting the height of the bottle containing the gel, so that the surface of the gel can
come in contact with the piston without exerting pressure,
a device whereby the load exerted by the piston can be increased at a constant rate of 40 g per
second,
a device whereby the vertical movement of the piston can be arrested within 1/40 s when the
latter has descended 4 0.1 mm,
a measuring device (balance) whereby the final load can be measured with a precision of 0.5 g,
a bottle 59 1 mm in internal diameter and 85 mm high.
Method. Place 7.5 g of the substance to be examined in the bottle. Add 105 ml of water R, place a
watch-glass over the bottle and allow to stand for 3 h. Heat in a water-bath at 65C for 15 min.
While heating, gently stir with a glass rod. Ensure that the solution is uniform and that any
condensed water on the inner walls of the bottle is incorporated. Allow to cool at room temperature
for 15 min and transfer the bottle to a thermostatically controlled bath at 10.0 0.1C and fitted
with a device to ensure that the platform on which the bottle stands is perfectly horizontal. Close the
bottle with a rubber stopper and allow to stand for 16 h to 18 h. Transfer the bottle without delay to
the gelometer and adjust so that the piston just comes into contact with the surface of the gel without
exerting pressure. Increase the load on the piston at a rate of 40 g per second until it has descended
4 0.1 mm. The load exerted by the piston at that moment, measured in grams, represents the gel
strength. Carry out at least three determinations and take the average.
Arsenic (2.4.2). To 1.0 g in a 100 ml flask add 15 ml of hydrochloric acid R and, while cooling in a
mixture of ice and water, drop 1 ml of strong hydrogen peroxide solution R and 0.1 ml of ferric chloride
solution R1. Allow to stand at room temperature for 30 min, shaking gently occasionally. Add 1 ml of
alcohol R and boil under a reflux condenser for 1 h. Cool the solution and dilute to 25 ml with
water R, rinsing the reflux condenser. The solution complies with limit test A for arsenic (1 ppm).
Prepare the reference solution using water R instead of hydrochloric acid R.
Heavy metals (2.4.8).2.0 g complies with limit test C for heavy metals (50 ppm). Prepare the
Loss on drying (2.2.32). Not more than 15 per cent, determined on 1.000 g by drying in an oven at
100C to 105C.
3
micro-organisms
per gram, determined by plate count. It complies with the tests for Escherichia coli and Salmonella
(2.6.13).
STORAGE
LABELLING
The label states whether the gelatin is intended for the preparation of pessaries, suppositories or zinc
gelatin and, if so, the gel strength.
__________________________________________________________________________________________________________ Ph Eur
18-56
Gemfibrozil
O
COOH
Me Me
Me
Me
C
15
H
22
O
3
250.3 25812-30-0
Definition Gemfibrozil is 2,2-dimethyl-5-(2,5-xylyloxy)valeric acid. It contains not less than
15
H
22
O
3
, calculated with reference to the anhydrous
substance.
Characteristics A white or almost white, slightly waxy, crystalline solid.
Practically insoluble in water; freely soluble in methanol.
spectrum of gemfibrozil (RS 167).
Melting point 58.0 to 61.0, Appendix V A, Method I.
Heavy metals Dissolve the residue from the test for Sulphated ash in 4 ml of 6M hydrochloric acid,
heat on a water bath to complete dissolution, cool and add sufficient water to produce 20 ml. 12 ml
of this solution complies with limit test A for heavy metals, Appendix VII. Use lead standard solution
2,4-Xylyloxy isomer Prepare a 0.05% w/v solution of myristic acid (internal standard) in methanol
(solution A). Carry out the method for gas chromatography, Appendix III B, using the following
solutions. For solution (1) add 1 ml of solution A and 10 ml of boron trifluoride solution to 0.1 g of the
substance being examined, shake, heat the mixture at 60 for 1 hour and allow to cool. Add 5 ml of
water, shake, add 3 ml of hexane to redissolve any white precipitate and shake for 5 minutes. Allow to
separate and use the hexane layer. Prepare solution (2) in the same manner as solution (1) but
without the addition of solution A. For solution (3) add 10 ml of boron trifluoride solution to a mixture
of 1 ml of solution A and 1 ml of a 0.05% w/v solution of 2,2-dimethyl-5-(2,4-xylyloxy)valeric acid
BPCRS in methanol, shake and complete the procedure in the same manner as solution (1) beginning
at the words heat the mixture. For solution (4) add 10 ml of boron trifluoride solution to 1 ml of a
0.05% w/v solution of 2,2-dimethyl-5-(2,4-xylyloxy)valeric acid BPCRS in methanol and use to dissolve
0.1 g of the substance being examined. Shake well and complete the procedure in the same manner
as solution (1) beginning at the words heat the mixture .
The chromatographic procedure may be carried out using (a) a fused silica capillary column
(50 m 0.2 mm) bonded with a 0.25 m layer containing 14% cyanopropylphenyl 86%
dimethylpolysiloxane (BP10 SGE is suitable) at a temperature of 180, a split-injection system with a
split ratio of 30:1 maintained at 250 and a detector temperature of 250 and (b) helium as the carrier
gas at a flow rate of 0.5 ml per minute with a flow rate of the make-up gas of 30 ml per minute.
The test is not valid unless a peak due to 2,2-dimethyl-5-(2,4-xylyloxy)valeric acid appears
immediately following the principal peak in the chromatogram obtained with solution (4) and the
height of the trough separating the two peaks is less than 10% of the height of the peak due to
2,2-dimethyl-5-(2,4-xylyloxy)valeric acid.
In the chromatogram obtained with solution (1) the ratio of the area of any peak corresponding to
2,2-dimethyl-5-(2,4-xylyloxy)valeric acid to the area of the internal standard peak is not greater than
the corresponding ratio in the chromatogram obtained with solution (3) (0.5%).
following solutions. For solution (1) dissolve 0.10 g of the substance being examined in the minimum
volume of methanol and dilute to 25 ml with the mobile phase. For solution (2) dilute 1 volume of
solution (1) to 100 volumes with the mobile phase and further dilute 1 volume of this solution to 5
volumes with the mobile phase. Solution (3) contains 0.0004% w/v of gemfibrozil impurity A BPCRS
in the mobile phase. Solution (4) contains 0.001% w/v of the substance being examined, 0.001% w/v
of gemfibrozil methyl ester BPCRS and 0.0004% w/v of gemfibrozil impurity A BPCRS in the mobile
phase.
The chromatographic procedure described under Assay may be used. For solution (1) allow the
chromatography to proceed for twice the retention time of the principal peak.
between the peaks due to gemfibrozil and gemfibrozil methyl ester is at least 4.0 and the resolution
factor between the peaks due to gemfibrozil methyl ester and gemfibrozil impurity A is at least 1.2.
In the chromatogram obtained with solution (1) the area of any peak corresponding to gemfibrozil
impurity A is not greater than the area of the peak in the chromatogram obtained with solution (3)
(0.1%), the area of any other secondary peak is not greater than the area of the peak in the chromato-
18-57
gram obtained with solution (2) (0.2%) and the sum of the areas of any secondary peaks other
than any peak corresponding to gemfibrozil impurity A is not greater than 2.5 times the area of the
peak in the chromatogram obtained with solution (2) (0.5%).
Sulphated ash Not more than 0.1%, Appendix IX A. To 2 g add 0.1 ml of sulphuric acid, heat
gently at first and then progressively to dull red heat until the residue shows no further changes and
cool.
Solution (1) contains 0.04% w/v of the substance being examined in a mixture of 2 volumes of
methanol and 3 volumes of the mobile phase. Solution (2) contains 0.04% w/v of gemfibrozil BPCRS
in a mixture of 2 volumes of methanol and 3 volumes of the mobile phase. Solution (3) contains
0.01% w/v of the substance being examined and 0.01% w/v of gemfibrozil methyl ester BPCRS in a
mixture of 2 volumes of methanol and 3 volumes of the mobile phase.
(10 cm 4.6 mm) packed with stationary phase C (3 m) (Spherisorb ODS 2 or Regis C18 are
suitable), (b) a mixture of 1 volume of glacial acetic acid, 19 volumes of water and 80 volumes of
methanol as the mobile phase with a flow rate of 1 ml per minute and (c) a detection wavelength of
276 nm.
Inject 20 l of each solution. The test is not valid unless, in the chromatogram obtained with
solution (3), the resolution factor between the peaks due to gemfibrozil and gemfibrozil methyl ester is
at least 4.0.
15
H
22
O
3
in the substance being examined from the declared content of
C
15
H
22
O
3
in gemfibrozil BPCRS.
Storage Gemfibrozil should be protected from light.
Preparations
Gemfibrozil Capsules
Gemfibrozil Tablets
IMPURITIES
O
COOH
Me Me
Me
Me
H
3
CCH
A. 2,2-dimethyl-5-(4-propen-1-yl-2,5-xylyloxy)valeric acid
O
COOMe
Me Me
Me
Me
B. methyl 2,2-dimethyl-5-(2,5-xylyloxy)valerate (gemfibrozil methyl ester)
O
COOBu
i
Me Me
Me
Me
C. isobutyl 2,2-dimethyl-5-(2,5-xylyloxy)valerate (gemfibrozil isobutyl ester)
O
Me
O Me
Me
Me
D. 1,1-propylene-1,3-dioxybis(2,5-xylene) (gemfibrozil bisether)
Me
Me OH
E. 2,5-xylenol
O
COOH
Me Me
Me Me
18-58
F. 2,2-dimethyl-5-(2,4-xylyloxy)valeric acid
Gentamicin Sulphate
NH
2
NH
2
OH
O
O
R
1
R
2
R
3
H
2
N
O
Me
HO
OH
NHMe
O
H
2
SO
4
+
Gentamicin R
1
R
2
R
3
C
1
Me NHMe H
C
2
Me NH
2
H
C
1a
H NH
2
H
C
2a
H NH
2
Me
1405-41-0
Gentamicin Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparations
Gentamicin Cream
Gentamicin Eye Drops
Gentamicin Injection
Gentamicin Ointment
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Gentamicin sulphate is a mixture of the sulphates of antimicrobial substances produced by
Micromonospora purpurea. The potency is not less than 590 I.U. per milligram, calculated with refer-
CHARACTERS
A white or almost white powder, freely soluble in water, practically insoluble in alcohol and in ether.
IDENTIFICATION
A. Dissolve about 10 mg in l ml of water R and add 5 ml of a 400 g/l solution of sulphuric acid R. Heat
on a water-bath for 100 min, cool and dilute to 25 ml with water R. Examined between 240 nm and
330 nm (2.2.25), the solution shows no absorption maximum.
same solvent.
Reference solution. Dissolve 25 mg of gentamicin sulphate CRS in water R and dilute to 5 ml with the
same solvent.
layer from a mixture of equal volumes of chloroform R, methanol R and concentrated ammonia R.
Allow the plate to dry in air, spray with ninhydrin solution R1 and heat at 110C for 5 min. The three
18-59
principal spots in the chromatogram obtained with the test solution are similar in position, colour
and size to the three principal spots in the chromatogram obtained with the reference solution.
C. Examine the chromatograms obtained in the test for composition. The chromatogram obtained
with the test solution shows four principal peaks having the same retention times as the four principal
peaks in the chromatogram obtained with the reference solution.
TESTS
Appearance of solution Solution S is clear (2.2.1) and not more intensely coloured than degree 6
substance.
Methanol Not more than 1.0 per cent m/m, determined by gas chromatography (2.2.28) using
Test solution (a). Dissolve 0.50 g of the substance to be examined in 2.0 ml of water R.
Test solution (b). Dissolve 0.50 g of the substance to be examined in 1.0 ml of the internal standard
solution and add 1.0 ml of water R.
Reference solution. Mix 1.25 ml of methanol R and 1.25 ml of propanol R and dilute to 500.0 ml with
water R.
a column 1.5 m to 2.0 m long and 2 mm to 4 mm in internal diameter packed with
ethylvinylbenzene-divinylbenzene copolymer R (150 m to 180 m),
nitrogen for chromatography R as the carrier gas at a constant flow rate of 30 ml to 40 ml per
minute,
maintaining the column at a constant temperature between 120C and 140C and maintaining the
injector and the detector at a temperature at least 50C higher than that of the column. Inject the
selected volumes of the test solutions and the reference solution. Calculate the content of methanol
taking its density at 20C to be 0.792 g per millilitre.
Composition Examine by liquid chromatography (2.2.29).
the same solvent. To 10.0 ml of this solution add 5 ml of methanol R and 4 ml of phthalaldehyde
reagent R. Mix and dilute to 25.0 ml with methanol R. Heat in a water bath at 60C for 15 min and
cool to room temperature. If the solution is not used immediately, cool to 0C and use within 4 h.
Reference solution. Prepare as prescribed for the test solution using 0.10 g of gentamicin sulphate CRS
a steel column 0.10 m to 0.125 m long and 4.6 mm to 5 mm in internal diameter packed with
a solution of 5.5 g of sodium heptanesulphonate R in a mixture of 50 ml of glacial acetic acid R,
250 ml of water R and 700 ml of methanol R1 as mobile phase at a flow rate of 1.5 ml per
minute,
as detector a spectrophotometer with a flow cell of about 10 l and set at 330 nm (for variable-
wavelength detectors) or 340 nm (for filter detectors).
When the chromatograms are recorded under the operating conditions described above, the
retention time of component C
2
is 10 min to 20 min and there are well separated peaks with relative
retention times of about 0.13 (reagent), 0.27 (component C
1
), 0.65 (component C
1a
), 0.85
(component C
2a
) and 1.00 (component C
2
). Inject 5 l of the reference solution. The system is not
suitable unless the resolution (2.2.29) between the peaks due to components C
2a
and C
2
is at least
1.3. If necessary adjust the methanol content of the mobile phase to achieve this.
Inject 5 l of the reference solution. Adjust the sensitivity of the amplifier and the volume injected
to obtain a deflection for the peak due to component C
1
of about 75 per cent of the recorder chart.
Plot a horizontal baseline on the chromatogram from the level portion of the curve immediately prior
to the reagent peak. Measure the peak height above this baseline for each component.
Calculate the response factor R
x
of each component from the expression:
R
H
F
x
x
x
18-60
F
x
=designated component ratio for component C
x
in gentamicin sulphate CRS,
H
x
=peak height for component C
x
Inject 5 l of test solution. Adjust the sensitivity of the amplifier and the volume injected to obtain
a deflection for the peak corresponding to component C
1
of about 75 per cent of the recorder chart.
Plot a horizontal baseline on the chromatogram from the level portion of the curve immediately prior
to the reagent peak. Measure the peak height above this baseline for each component. Calculate the
relative proportions of components C
1
, C
1a
, C
2
and C
2a
in the substance to be examined from the
expression:
C per cent
H
R
H
R
H
R
H
R
H
R
x
x
x
1a
1a
2a
2a
( )
+

+

+

1
1
2
2
100
H
x
=peak height for component C
x
in the chromatogram obtained with the test solution.
The relative proportions are within the following limits: C
1
25.0 per cent to 50.0 per cent; C
1a
10.0 per cent to 35.0 per cent; C
2
plus C
2a
25.0 per cent to 55.0 per cent.
mination of water.
4
), calculated with reference to the anhy-
drous substance. Dissolve 0.250 g in 100 ml of distilled water R and adjust the solution to pH 11
using concentrated ammonia R. Add 10.0 ml of 0.1M barium chloride and about 0.5 mg of phthalein
purple R. Titrate with 0.1M sodium edetate, adding 50 ml of alcohol R when the colour of the solution
begins to change and continue the titration until the violet-blue colour disappears.
4
).
Sterility (2.6.1). If intended for use in the manufacture of parenteral or ophthalmic dosage forms
without a further appropriate sterilisation procedure, it complies with the test for sterility.
ASSAY
STORAGE
Store in a well-closed container. If the substance is sterile, store in a sterile, airtight, tamper-proof
container.
LABELLING
The label states:
18-61
__________________________________________________________________________________________________________ Ph Eur
Gentian
Gentian complies with the requirements of the 3rd edition of the European Pharmacopoeia for Gentian Root
Action and use Bitter.
Preparation
Compound Gentian Infusion
When Powdered Gentian is prescribed or demanded, material complying with the requirements
below with the exception of Identification test A shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Gentian root consists of the dried, fragmented underground organs of Gentiana lutea L.
CHARACTERS
Gentian root has a characteristic odour and a strong and persistent bitter taste. It occurs as single or
branched subcylindrical pieces of various lengths and usually 10 mm to 40 mm thick but occasionally
up to 80 mm thick at the crown.
IDENTIFICATION
A. The surface is brownish-grey, and the colour of a transverse section is yellowish to reddish-yellow
but not reddish-brown. The root is longitudinally wrinkled and bears occasional rootlet scars. The
branches of the rhizome frequently bear a terminal bud and are always encircled by closely arranged
leaf scars. The rhizome and root are brittle when dry and break with a short fracture but they absorb
moisture readily to become flexible. The smoothed, transversely cut surface shows a bark, occupying
about one-third of the radius, separated by the well-marked cambium from an indistinctly radiate and
mainly parenchymatous xylem.
B. Reduce to a powder (355). The powder is light brown or yellowish-brown. Examine under a
fragments of the subero-phellodermic layer, consisting of thin-walled yellowish-brown cork cells and
thick-walled collenchyma (phelloderm); fragments of cortical and ligneous parenchymatous cells with
moderately thickened walls containing droplets of oil and small prisms and minute needles of calcium
oxalate; fragments of lignified vessels with spiral or reticulate thickening.
Test solution. To 2.0 g of the powdered drug (355) add 50 ml of methanol R, stir constantly for 20 min
and filter without allowing too much solvent to evaporate. Evaporate 25 ml of the filtrate to dryness
under reduced pressure, at a temperature not exceeding 50C. Take up the residue with small
quantities of methanol R so as to obtain 5 ml of a solution, which may contain a sediment.
Reference solution. Dissolve 50 mg of phenazone R in methanol R and dilute to 10.0 ml with the same
solvent.
Apply separately to the plate as bands 30 mm by 8 mm, 50 l of the test solution and 10 l of the
reference solution. Develop over a path of 15 cm using a mixture of 2 volumes of water R, 30
volumes of chloroform R and 70 volumes of acetone R. Allow the plate to dry in air and examine in
ultraviolet light at 254 nm. The chromatogram obtained with the test solution shows quenching
zones in its lower part and usually in its upper part; there is a zone corresponding to amarogentine in
the middle of the chromatogram, at about the same level as the quenching zone due to phenazone in
the chromatogram obtained with the reference solution. Mark the zone due to phenazone. Spray with
a freshly prepared 2 g/l solution of fast red B salt R and allow to stand for 10 min. The zone due to
amarogentine is orange. Expose the plate to ammonia vapour. The zone due to amarogentine
becomes red. Other coloured zones are visible in the lower and upper parts of the chromatogram
obtained with the test solution. In particular, in the upper part there may be a zone with a high R
f
value, usually very intense, with the same colour as the zone due to amarogentine.
TESTS
Chromatography Examine the chromatograms obtained in identification test C. After exposure to
ammonia vapour, the chromatogram obtained with the test solution does not show violet zones
18-62
immediately above the zone due to amarogentine (other species of Gentiana).
Bitterness value Not less than 10,000. The bitterness value is determined by comparison with
quinine hydrochloride, the bitterness value of which is set at 200,000; the bitterness value is the
reciprocal of the dilution that still has a bitter taste.
Quinine hydrochloride stock solution. Dissolve 0.100 g of quinine hydrochloride R in water R and dilute to
100.0 ml with the same solvent. Dilute 1.0 ml of the solution to 100.0 ml with water R.
Gentian root extract. To 1.0 g of powdered drug (710) add 1000 ml of boiling water R. Heat on a
water-bath for 30 min, stirring continuously. Allow to cool and dilute to 1000 ml with water R. Shake
vigorously and filter, discarding the first 20 ml of filtrate.
Prepare a series of dilutions by placing in a first tube 4.2 ml of quinine hydrochloride stock solution
and increasing the volume by 0.2 ml in each subsequent tube to a total of 5.8 ml; dilute the contents
of each tube to 10.0 ml with water R. Determine as follows the dilution with the lowest concentration
that still has a bitter taste. Take 10.0 ml of the weakest solution into the mouth and pass it from side
to side over the back of the tongue for 30 s. If the solution is not found to be bitter, spit it out and
wait for 1 min. Rinse the mouth with water. After 10 min, use the next dilution in order of increasing
concentration.
Calculate the correction factor k from the expression:
500 .
n
n=number of millilitres of quinine hydrochloride stock solution in the dilution of lowest
concentration that is bitter.
Dilute 10/k ml of the gentian root extract to 100.0 ml with water R. 10.0 ml of this dilution has a
bitter taste.
Water-soluble extractive To 5.0 g of powdered drug (710) add 200 ml of boiling water R. Allow to
stand for 10 min, shaking occasionally. Allow to cool, dilute to 200.0 ml with water R and filter.
Evaporate 20.0 ml of the filtrate to dryness on a water-bath. Dry the residue in an oven at 100C to
105C. The residue weighs not less than 0.165 g (33 per cent).
STORAGE
18-63
__________________________________________________________________________________________________________ Ph Eur
Ginger
1/01
Ginger complies with the requirements of the 3rd edition of the European Pharmacopoeia [1522]. These require-
Action and use Flavour
Preparation
Strong Ginger Tincture
Ginger may be known in commerce as unbleached ginger. When Powdered Ginger is prescribed or
demanded, material complying with the appropriate requirements below shall be dispensed or
supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ginger consists of the dried, whole or cut rhizome of Zingiber officinale Roscoe, with the cork
removed, either completely or from the wide flat surfaces only. Whole or cut, it contains not less than
15 ml/kg of essential oil, calculated with reference to the anhydrous drug.
CHARACTERS
Ginger has a characteristic aromatic odour and a spicy and burning taste.
IDENTIFICATION
A. The rhizome is laterally compressed, bearing short, flattened, obovate oblique branches on the
upper side, each sometimes having a depressed scar at the apex; the whole rhizomes are about 5 cm
to 10 cm long, 1.5 cm to 3 cm or 4 cm wide and 1 cm to 1.5 cm thick, sometimes split
longitudinally. The scraped rhizome with a light-brown external surface shows longitudinal striations
and occasional loose fibres; the outer surface of the unscraped rhizome varies from pale to dark
brown and is more or less covered with cork which shows conspicuous, narrow, longitudinal and
transverse ridges; the cork readily exfoliates from the lateral surfaces but persists between the
branches. The fracture is short and starchy with projecting fibres. The smoothed transversely cut
surface exhibits a narrow cortex separated by an endodermis from a much wider stele; it shows
numerous, scattered, fibrovascular bundles and abundant scattered oleoresin cells with yellow
contents. The unscraped rhizome shows, in addition, an outer layer of dark brown cork.
B. Reduce to a powder (355). The powder is pale yellow to brownish. Examine under a microscope
using chloral hydrate solution R. The powder shows groups of large, thin-walled, septate fibres, with
one wall frequently dentate; fairly large vessels with reticulate thickening and often accompanied by
narrow, thin-walled cells containing brown pigment; abundant thin-walled parenchyma of the ground
tissue, some cells containing brown oleoresin; fragments of brown cork, usually seen in surface view.
Examine under a microscope using a 50 per cent V/V solution of glycerol R. The powder shows
abundant starch granules, simple, flattened, oblong to oval or irregular, up to about 50 m long and
25 m wide, with a small point hilum situated at the narrower end; occasional granules show faint,
transverse striations.
Test solution. To 1.0 g of the powdered drug (710) add 5 ml of methanol R. Shake for 15 min and
filter.
Reference solution. Dissolve 10 l of citral R and 10 mg of resorcinol R in 10 ml of methanol R. Prepare
the solution immediately before use.
Apply to the plate as bands, 20 l of each solution. Develop over a path of 15 cm in an unsaturated
chromatographic tank using a mixture of 40 volumes of hexane R and 60 volumes of ether R. Allow
the plate to dry in air. Spray the plate with a 10 g/l solution of vanillin R in sulphuric acid R and
examine in daylight while heating at 100C to 105C for 10 min. The chromatogram obtained with
the reference solution shows in the lower half an intense red zone (resorcinol) and in the upper half
two violet zones (citral). The chromatogram obtained with the test solution shows below the
resorcinol zone in the chromatogram obtained with the reference solution two intense violet zones
(gingerols) and in the middle, between the resorcinol and citral zones in the chromatogram obtained
18-64
with the reference solution, two other less intense violet zones (shogaols). Other zones may be
present.
TESTS
drug (710).
ASSAY
Carry out the determination of essential oils in vegetable drugs (2.8.12). Use 20.0 g of the freshly,
coarsely powdered drug, a 1000 ml round-bottomed flask, 10 drops of liquid paraffin R or other
antifoam, 500 ml of water R as distillation liquid and 0.5 ml of xylene R in the graduated tube. Distil
at a rate of 2 ml/min to 3 ml/min for 4 h.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
18-65
Ginseng
1/01
Ginseng complies with the requirements of the 3rd edition of the European Pharmacopoeia [1523]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ginseng consists of the whole or cut dried root of Panax ginseng C. A. Meyer. It contains not less
than 0.40 per cent of combined ginsenosides Rg1

(C
42
H
72
O
14
,2H
2
O, = 837) and Rb1
(C
54
H
92
O
23
,3H
2
O, = 1163), calculated with reference to the dried drug.
CHARACTERS
IDENTIFICATION
A. The principal root is fusiform or cylindrical, sometimes branched, up to about 20 cm long and
2.5 cm in diameter, and may be curved or markedly re-curved. The surface is pale yellow to cream
and shows longitudinal ridges; stem scars may be seen at the crown. The fracture is short. The
transversely-cut surface shows a wide outer zone with scattered orange-red resin canals and a finely
radiate inner region. The rootlets, numerous in the lower part, are fine with a small diameter.
B. Reduce to a powder (355). Examine under a microscope using chloral hydrate solution R. The light
yellow powder shows abundant fragments of thin-walled parenchymatous cells and fragments of large
secretory canals containing yellowish-brown resin. The powder occasionally shows non-lignified
tracheids and partially-lignified vessels with spiral or reticulate thickening, occurring singly or in small
groups, and scattered cluster crystals of calcium oxalate. Examine under a microscope using a
mixture of equal volumes of glycerol R and water R. The starch granules are very abundant, simple or
two or three compound, and range from 1 m to 10 m in diameter.
Test solution. Boil 1.0 g of the powdered drug (355) under a reflux condenser with 10 ml of a 70 per
cent V/V solution of methanol R for 15 min. Filter after cooling and dilute to 10.0 ml with methanol R.
Reference solution. Dissolve 5.0 mg of aescin R and 5.0 mg of arbutin R in 1 ml of methanol R.
Apply to the plate as bands 20 l of each solution. Develop over a path of 10 cm, in an unsaturated
tank, using the upper layer of a mixture of 25 ml of ethyl acetate R, 50 ml of water R and 100 ml of
butanol R, which has been allowed to separate for 10 min. Allow the plate to dry in air. Spray with
anisaldehyde solution R and heat at 105C to 110C for 5 min to 10 min. Examine in daylight. The
chromatogram obtained with the reference solution shows in the upper third a brown zone corres-
ponding to arbutin, and in the lower third a grey zone corresponding to aescin. Between these two
zones, a little below the zone corresponding to arbutin in the chromatogram obtained with the
reference solution, the chromatogram obtained with the test solution shows two violet-grey zones due
to ginsenoside Rg1 (upper zone) and to ginsenoside Re (lower zone). The violet-grey zone due to
ginsenoside Rb1 is similar in position to the grey zone due to aescin in the chromatogram obtained
with the reference solution. Between the zones corresponding to ginsenoside Rb1 and Re other, less
intense, bands are present; the zone closest to the starting point corresponds to ginsenoside Rc. Other
zones are visible in the lower third of the chromatogram.
TESTS
Panax quinquefolium Examine the chromatograms obtained in the assay. The chromatogram
obtained with the test solution shows the peak corresponding to ginsenoside Rf. In the case of a
substitution by Panax quinquefolium no peak corresponding to ginsenoside Rf is present.
ASSAY
18-66
Test solution. Reduce about 50 g to a powder (355). Place 1.00 g of the powdered drug and 70 ml
of a 50 per cent V/V solution of methanol R in a 250 ml round-bottomed flask. After adding a few
grains of pumice, boil on a water-bath under a reflux condenser for 1 h. After cooling, centrifuge
and collect the supernatant liquid. Treat the residue as described above. Mix the collected liquids
and evaporate to dryness under reduced pressure at a temperature not exceeding 60C. Take up
the residue with 10 ml of a buffer solution containing 3.5 g of sodium dihydrogen phosphate R and
7.2 g of potassium dihydrogen phosphate R in 1000 ml of water R (solution A).
Wash a cartridge containing about 360 mg of a suitable octadecylsilyl silica gel for chromatography R
with 5 ml of methanol R followed by 20 ml of water R. Apply 5 ml of solution A to the cartridge. Elute
with 20 ml of water R, followed by 15 ml of a 30 per cent V/V solution of methanol R. Discard the
eluates. Elute with 20 ml of methanol R. Evaporate the eluate to dryness and take up the residue with
2 ml of methanol R. Discard the eluates after checking that no ginsenosides are present, otherwise
repeat the assay with a cartridge from a different manufacturer.
Reference solution. Dissolve 3.0 mg of ginsenoside Rb1 R, 3.0 mg of ginsenoside Rg1 R and 3.0 mg of
ginsenoside Rf R, accurately weighed, in methanol R and dilute to 10 ml with the same solvent.
aminopropylsilyl silica gel for chromatography R,
Mobile phase A. Acetonitrile R,
Mobile phase B. Water R,
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
0 14 90 10
14 18 90 80 10 20
18 55 80 20
55 60 90 10
Inject the reference solution and adjust the sensitivity of the system so that the heights of the peaks
due to ginsenosides are about 50 per cent of the full scale of the recorder. The test is not valid unless
the resolution between the peaks corresponding to ginsenoside Rf and ginsenoside Rg1 is at least 1.0.
Inject the test solution.
Calculate the percentage content of ginsenosides Rb1 and Rg1 from the expression:
1 g R 1
3 2
Rb1 1
2 1
40 40
A m
m A
A m
m A

A
1
=area of the peak due to ginsenoside Rb1 in the chromatogram obtained with the test
solution,
A
2
=area of the peak due to ginsenoside Rg1 in the chromatogram obtained with the test
solution,
A
Rb1
=area of the peak due to ginsenoside Rb1 in the chromatogram obtained with the reference
solution,
A
Rg1
=area of the peak due to ginsenoside Rg1 in the chromatogram obtained with the reference
solution,
m
1
=mass of the drug to be examined, in grams,
m
2
=mass of ginsenoside Rb1, in grams,
m
3
=mass of ginsenoside Rg1, in grams.
STORAGE
The following chromatogram is given for information only; it does not form a mandatory part of the
monograph.
18-67
0
50
100
150
200
250
300
350
400
mAU
0 5 10 15 20 25 30 min
1
2
3
4
5
6
7
1. ginsenoside Rg1
2. ginsenoside Rf
3. ginsenoside Re
4. ginsenoside Rd
5. ginsenoside Rc
6. ginsenoside Rb2
7. ginsenoside Rb1
Fig. 15231 Chromatogram of the test solution
__________________________________________________________________________________________________________ Ph Eur
18-68
Glibenclamide
O
N N
S
O O
NH
O
OMe
Cl
H H
C
23
H
28
ClN
3
O
5
S 494.0 10238-21-8
Glibenclamide complies with the requirements of the 3rd edition of the European Pharmacopoeia [0718]. These
Preparation
Glibenclamide Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Glibenclamide contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of 1-[4-[2-(5-chloro-2-methoxybenzamido)ethyl]benzenesulphonyl]-3-cyclohexylurea, calcu-
CHARACTERS
methylene chloride, slightly soluble in alcohol and in methanol, practically insoluble in ether. It
dissolves in dilute solutions of alkali hydroxides.
IDENTIFICATION
B. Dissolve 50.0 mg in methanol R, with the aid of ultrasound if necessary, and dilute to 50.0 ml with
the same solvent. To 10.0 ml of the solution add 1.0 ml of 1M hydrochloric acid and dilute to 100.0 ml
with methanol R. Examined between 230 nm and 350 nm (2.2.25), the solution shows a maximum at
300 nm and a less intense maximum at 275 nm. The specific absorbances at the maxima are 61 to 65
and 27 to 32, respectively.
obtained with glibenclamide CRS. Examine the substances as discs prepared using potassium
bromide R. If the spectra obtained show differences, moisten separately the substance to be examined
and the reference substance with methanol R, triturate, dry at 100C to 105C and record the spectra
again.
E. Dissolve 20 mg in 2 ml of sulphuric acid R. The solution is colourless and shows blue fluorescence
in ultraviolet light at 365 nm. Dissolve 0.1 g of chloral hydrate R in the solution. Within about 5 min,
the colour changes to deep yellow and, after about 20 min, develops a brownish tinge.
TESTS
Appearance of solution Dissolve by heating 0.1 g in 10 ml of alcohol R. The solution is clear (2.2.1)
and colourless (Method II, 2.2.2).
254
R as the
coating substance.
Test solution (b). Dilute 0.5 ml of test solution (a) to 10 ml with a mixture of equal volumes of
Reference solution (a). Dissolve 10 mg of glibenclamide CRS in a mixture of equal volumes of
Reference solution (b). Dissolve 8 mg of glibenclamide impurity A CRS in a mixture of equal volumes of
18-69
Reference solution (c). Dilute 5 ml of reference solution (b) to 10 ml with a mixture of equal volumes
Reference solution (d). Dissolve 0.2 g of glibenclamide CRS in 10 ml of methanol R and boil under a
reflux condenser for 10 min.
5 volumes of alcohol R, 5 volumes of glacial acetic acid R, 45 volumes of chloroform R and 45 volumes
of cyclohexane R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. In the
chromatogram obtained with test solution (a): any spot corresponding to glibenclamide impurity A is
cent); any spot, apart from the principal spot and the spot corresponding to glibenclamide impurity
A, is not more intense than the spot in the chromatogram obtained with reference solution (c)
(0.2 per cent). The test is not valid unless the chromatogram obtained with reference solution (d)
100C to 105C.
ASSAY
Dissolve 0.400 g with heating in 100 ml of alcohol R. Titrate with 0.1M sodium hydroxide, using 1.0 ml
of phenolphthalein solution R as indicator, until a red colour is obtained.
23
H
28
ClN
3
O
5
S.
IMPURITIES
NH
2
S
O O
NH
O
OMe
Cl
A. 4-[2-(5-chloro-2-methoxybenzamido)ethyl]benzenesulphonamide.
__________________________________________________________________________________________________________ Ph Eur
18-70
Gliclazide
1/01
N
N N
S
O
O O
Me
H H
C
15
H
21
N
3
O
3
S 323.4 21187-98-4
Gliclazide complies with the requirements of the 3rd edition of the European Pharmacopoeia [1524]. These
Preparation
Gliclazide Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Gliclazide contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of
1-(hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-3-[(4-methylphenyl)sulphonyl]urea, calculated with
CHARACTERS
sparingly soluble in acetone, slightly soluble in alcohol.
IDENTIFICATION
with gliclazide CRS. Examine the substances prepared as discs.
TESTS
acetonitrile R and 55 volumes of water R. Dilute 10.0 ml of the solution to 100.0 ml with a mixture of
45 volumes of acetonitrile R and 55 volumes of water R.
Reference solution (b). Dissolve 5 mg of the substance to be examined and 15 mg of gliclazide impurity
F CRS in 23 ml of acetonitrile R and dilute to 50 ml with water R. Dilute 1 ml of the solution to 20 ml
with a mixture of 45 volumes of acetonitrile R and 55 volumes of water R.
Reference solution (c). Dissolve 10.0 mg of gliclazide impurity F CRS in 45 ml of acetonitrile R and
dilute to 100.0 ml with water R. Dilute 1.0 ml of the solution to 100.0 ml with a mixture of 45
as mobile phase at a flow rate of 0.9 ml/min a mixture of 0.1 volumes of triethylamine R, 0.1
volumes of trifluoroacetic acid R, 45 volumes of acetonitrile R and 55 volumes of water R,
Inject 20 l of reference solution (b). Adjust the sensitivity of the system so that the heights of the 2
principal peaks in the chromatogram obtained with reference solution (b) are at least 50 per cent of
the full scale of the recorder. The test is not valid unless in the chromatogram obtained the resolution
Inject 20 l of the test solution and 20 l each of reference solutions (a) and (c). Continue the
chromatography of the test solution for twice the retention time of gliclazide. In the chromatogram
obtained with the test solution: the area of any peak corresponding to impurity F is not greater than
the area of the corresponding peak in the chromatogram obtained with reference solution (c) (0.1 per
cent); the area of any peaks, apart from the principal peak and the peak due to impurity F, is not
greater than the area of the principal peak in the chromatogram obtained with reference solution (a)
18-71
(0.1 per cent); the sum of the areas of any such peaks is not greater than twice the area of the
Impurity B Examine by liquid chromatography (2.2.29) as described in the test for related
substances.
Test solution. Dissolve 0.400 g in 2.5 ml of dimethyl sulphoxide R and dilute to 10.0 ml with water R.
Stir for 10 min, store at 4C for 30 min and filter.
Reference solution (a). Dissolve 20.0 mg of gliclazide impurity B CRS in dimethyl sulphoxide R and dilute
to 100.0 ml with the same solvent. To 1.0 ml of the solution, add 12 ml of dimethyl sulphoxide R and
Reference solution (b). To 1.0 ml of reference solution (a), add 12 ml of dimethyl sulphoxide R and
with the test solution, the area of any peak corresponding to impurity B is not greater than the area of
the corresponding peak in the chromatogram obtained with reference solution (b) (2 ppm).
ASSAY
15
H
21
N
3
O
3
S.
IMPURITIES
N
H
S
O O
Me
=
A. R-H: 4-methylbenzenesulphonamide,
N
N
O
B. 2-nitroso-octahydrocyclopenta[c]pyrrole,
O CH
3
O
C. ethyl [(4-methylphenyl)sulphonyl]carbamate,
R
O
D. N-[(4-methylphenyl)sulphonyl]hexahydrocyclopenta[c]pyrrol-2(1H)-carboxamide,
N
N
H
O
E. 1-[(4-methylphenyl)sulphonyl]-3-(3,3a,4,6a-tetrahydrocyclopenta[c]pyrrol-2(1H)-yl)urea,
N
H
S
O O
N
N
H
O
Me
F. 1-(hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-3-[(2-methylphenyl)sulphonyl]urea,
18-72
N
N
O
G. N-[(4-methylphenyl)sulphonyl]-1,4a,5,6,7,7a-hexahydro-2H-cyclopenta[d]pyridazine-2-
carboxamide.
__________________________________________________________________________________________________________ Ph Eur
18-73
Glipizide
N
N
HN O
NH
S
NH
O
O O
Me
C
21
H
27
N
5
O
4
S 445.5 29064-61-9
Glipizide complies with the requirements of the 3rd edition of the European Pharmacopoeia [0906]. These
Preparation
Glipizide Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Glipizide contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent of
1-cyclohexyl-3-[4-[2-(5-methylpyrazine-2-carboxamido)ethyl]benzenesulphonyl]urea, calculated with
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water, soluble in methylene
chloride, sparingly soluble in acetone, practically insoluble in alcohol. It dissolves in dilute solutions
of alkali hydroxides.
IDENTIFICATION
A. Dissolve about 2 mg in methanol R and dilute to 100 ml with the same solvent. Examined between
220 nm and 350 nm (2.2.25), the solution shows two absorption maxima, at 226 nm and 274 nm.
The ratio of the absorbance measured at the maximum of 226 nm to that measured at the maximum
at 274 nm is 2.0 to 2.4.
obtained with glipizide CRS. Examine the substances prepared as discs.
D. Dissolve 50 mg in 5 ml of dioxan R. Add 1 ml of a 5 g/l solution of fluorodinitrobenzene R in
dioxan R and boil for 2 min to 3 min. A yellow colour is produced.
TESTS
254
R as the
coating substance.
methanol R and of methylene chloride R and dilute to 10 ml with the same mixture of solvents.
Reference solution (a). Dissolve 10 mg of glipizide CRS in a mixture of equal volumes of methanol R
Reference solution (b). Dissolve 5 mg of glipizide impurity A CRS in a mixture of equal volumes of
Reference solution (c). Dilute 0.5 ml of test solution (a) to 100 ml with a mixture of equal volumes of
18-74
Reference solution (d). Dilute 4 ml of reference solution (c) to 10 ml with a mixture of equal volumes
Reference solution (e). Dilute 5 ml of test solution (a) to 10 ml with reference solution (b).
25 volumes of anhydrous formic acid R, 25 volumes of ethyl acetate R and 50 volumes of methylene
chloride R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. In the chromato-
gram obtained with test solution (a): any spot corresponding to glipizide impurity A is not more
intense than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent); any
spot apart from the principal spot and the spot corresponding to glipizide impurity A is not more
intense than the spot in the chromatogram obtained with reference solution (c) (0.5 per cent) and not
more than two such spots are more intense than the spot in the chromatogram obtained with refer-
ence solution (d) (0.2 per cent). The test is not valid unless the chromatogram obtained with refer-
ence solution (e) shows two clearly separated spots.
Cyclohexylamine Not more than 100 ppm, determined by gas chromatography (2.2.28), using
decane R as internal standard.
Internal standard solution. Dissolve 25 mg of decane R in hexane R and dilute to 100 ml with the same
solvent. Dilute 5 ml of this solution to 50 ml with hexane R.
Test solution (a). Dissolve 3.0 g of the substance to be examined in 50 ml of a 12 g/l solution of
sodium hydroxide R and shake with two quantities, each of 5.0 ml, of hexane R. Use the combined
upper layers.
Test solution (b). Dissolve 3.0 g of the substance to be examined in 50 ml of a 12 g/l solution of
sodium hydroxide R and shake with two quantities, each of 5.0 ml, of the internal standard solution.
Use the combined upper layers.
Reference solution. Dissolve 30.0 mg of cyclohexylamine R in a 17.5 g/l solution of hydrochloric acid R
and dilute to 100.0 ml with the same acid. To 1.0 ml of this solution add 50 ml of a 12 g/l solution of
sodium hydroxide R and shake with two quantities, each of 5.0 ml, of the internal standard solution.
Use the combined upper layers.
earth for gas chromatography R impregnated with 10 per cent m/m of macrogol 20,000 R and 4 per
cent m/m of potassium hydroxide R,
at 120C.
Inject 1 l of the reference solution and adjust the sensitivity of the detector so that the heights of
the first peak (decane) and the second peak (cyclohexylamine) are not less than 50 per cent of the full
scale of the recorder. The test is not valid unless the resolution between these two peaks is at least 2.
same retention time as that of the internal standard. Inject separately 1 l of test solution (b) and 1 l
of the reference solution. From the chromatogram obtained with the reference solution, calculate the
ratio (R) of the area of the peak due to cyclohexylamine to the area of the peak due to the internal
standard. From the chromatogram obtained with test solution (b), calculate the ratio of the area of
any peak corresponding to cyclohexylamine to the area of the peak due to the internal standard: this
ratio is not greater than R.
100C to 105C.
ASSAY
Dissolve 0.400 g in 50 ml of dimethylformamide R. Titrate with 0.1M lithium methoxide using 0.2 ml of
quinaldine red solution R as indicator until the colour changes from red to colourless.
1 ml of 0.1M lithium methoxide is equivalent to 44.55 mg of C
21
H
27
N
5
O
4
S.
STORAGE
18-75
IMPURITIES
N
N
HN
Me
O
S
NH
2
O O
A. 4-[2-(5-methylpyrazine-2-carboxamido)ethyl]benzenesulphonamide,
NH
2
B. cyclohexylamine.
__________________________________________________________________________________________________________ Ph Eur
18-76
Gliquidone
N O
NH
S
NH
O O
O
O
Me Me
MeO
C
27
H
33
N
3
O
6
S 527.6 33342-05-1
Definition Gliquidone is 1-cyclohexyl-3-p-[2-(3,4-dihydro-7-methoxy-4,4-dimethyl-1,3-dioxo-
2(1H)-isoquinolyl)ethyl]phenylsulphonylurea. It contains not less than 98.5% and not more than
101.5% of C
27
H
33
N
3
O
6
Practically insoluble in water; slightly soluble in ethanol (96%) and in methanol; soluble in acetone;
freely soluble in dimethylformamide.
Identification
A. Dissolve 30 mg in 10 ml of methanol. Evaporate the methanol using a rotary evaporator and dry
the residue at a temperature of 50 at a pressure of 2 kPa for 1 hour. The infrared absorption spectrum
of the dried residue, Appendix II A, is concordant with the reference spectrum of gliquidone (RS 170).
B. In the test for Related substances, the principal spot in the chromatogram obtained with solution
(1) corresponds to that in the chromatogram obtained with solution (2).
gel 60F as the coating substance and a mixture of 5 volumes of glacial acetic acid, 5 volumes of ethanol
(96%), 45 volumes of chloroform and 45 volumes of cyclohexane as the mobile phase but allowing the
solvent front to ascend 10 cm above the line of application. Apply separately to the plate 10 l of each
of five solutions in a mixture of 50 volumes of dichloromethane and 50 volumes of methanol containing
(1) 1.0% w/v of the substance being examined, (2) 1.0% w/v of gliquidone BPCRS, (3) 0.0030% w/v
of gliquidone BPCRS, (4) 0.0030% w/v of gliquidone sulphonamide BPCRS and (5) a mixture of 1
volume of solution (3) and 1 volume of solution (4). After removal of the plate, allow it to dry in air
and examine under ultraviolet light
(254 nm). In the chromatogram obtained with solution (1) any spot corresponding to gliquidone
sulphonamide is not more intense than the spot in the chromatogram obtained with solution (4)
(0.3%) and any other secondary spot is not more intense than the spot in the chromatogram obtained
with solution (3) (0.3%). The test is not valid unless the chromatogram obtained with solution (5)
1 g.
Assay Dissolve 0.3 g in 70 ml of dimethylformamide and immediately carry out Method II for non-
aqueous titration, Appendix VIII A, in an atmosphere of nitrogen using 0.1M tetrabutylammonium
hydroxide VS as titrant and a 0.3% w/v solution of thymol blue as indicator. Each ml of 0.1M
tetrabutylammonium hydroxide VS is equivalent to 52.76 mg of C
27
H
33
N
3
O
6
S.
Preparation
Gliquidone Tablets
18-77
IMPURITIES
N
O
O
Me Me
MeO
S
NH
2
O O
A. p-[2-(3,4-dihydro-7-methoxy-4,4-dimethyl-1,3-dioxo-2(1H)-isoquinolyl)ethyl]benzene-
sulphonamide (gliquidone sulphonamide)
18-78
Glucagon
HisSerGlnGlyThrPheThrSerAsp
TyrSerLysTyrLeuAspSerArgArg
AlaGlnAspPheValGlnTrpLeuMet
AsnThr
C
153
H
225
N
43
O
49
S 3483 16941-32-5
Glucagon complies with the requirements of the 3rd edition of the European Pharmacopoeia [0612]. These
Action and use Hyperglycaemic hormone.
Preparation
Glucagon Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Glucagon is a polypeptide hormone obtained from beef or pork pancreas and which increases the
blood-glucose concentration by promoting rapid breakdown of liver glycogen. The potency is not less
than 1 I.U. per milligram, calculated with reference to the dried substance. Glucagon is prepared in
conditions designed to minimise microbial contamination.
CHARACTERS
A white or almost white powder, practically insoluble in water and in most organic solvents. It
dissolves in dilute mineral acids and in dilute solutions of the alkali hydroxides.
IDENTIFICATION
A. It causes a rise of blood-glucose concentration in the test animals when injected as prescribed in
the assay.
B. Examine the electrophoretograms obtained in the test for related substances. The principal band
in the electrophoretogram obtained with test solution (a) corresponds in position to the principal
band in the electrophoretogram obtained with reference solution (a).
TESTS
Absorbance Dissolve 2.5 mg in 0.01M hydrochloric acid and dilute to 10.0 ml with the same acid.
The specific absorbance (2.2.25) determined at the maximum at 276 nm is 21 to 25, calculated with
Related substances Examine by polyacrylamide gel electrophoresis (2.2.31), using rod gels 75 mm
long and 5 mm in diameter and, as buffer, tris-glycine buffer solution pH 8.3 R. The electrode in the
upper reservoir is the cathode and that in the lower reservoir the anode.
Use the following gel mixture: mix 1 volume of a solution containing in 100 ml 36.6 mg of
tris(hydroxymethyl)aminomethane R, 0.23 ml of tetramethylethylenediamine R and 48.0 ml of 1M hydro-
chloric acid and 2 volumes of a solution containing in 100 ml 0.735 g of methylenebisacrylamide R and
30.0 g of acrylamide R. Add sufficient urea R to give a concentration of 480 g/l in the final solution
and dilute to 7 volumes with water R. If necessary, heat to not more than 40C to dissolve the urea.
Degas the solution and add 1 volume of a 5.6 g/l solution of ammonium persulphate R.
Test solution (a). Dissolve 10 mg of the substance to be examined in 0.5 ml of 0.01M sodium hydroxide.
Test solution (b). Dilute 0.25 ml of test solution (a) to 5 ml with 0.01M sodium hydroxide.
Reference solution (a). Dissolve a quantity of glucagon BRP equivalent to 5 I.U. in 0.01M sodium
hydroxide and dilute to 25 ml with the same solvent.
Reference solution (b). Dilute 8 ml of reference solution (a) to 10 ml with 0.01M sodium hydroxide.
Reference solution (c). Dilute 6 ml of reference solution (a) to 10 ml with 0.01M sodium hydroxide.
Reference solution (d). Dilute 4 ml of reference solution (a) to 10 ml with 0.01M sodium hydroxide.
Reference solution (e). Dilute 2 ml of reference solution (a) to 10 ml with 0.01M sodium hydroxide.
Apply 100 l of each solution separately to the surface of a gel. After the addition of the buffer, add
0.2 ml of bromophenol blue solution R. Allow electrophoresis to take place with a constant current of 1
mA per tube for 30 min and then increase the current to 3 mA per tube. Immerse the gels in a 125 g/l
solution of trichloroacetic acid R for at least 1 h. For each 10 ml of trichloroacetic acid solution used
add 0.5 ml of a 2.5 g/l solution of acid blue 90 R and allow to stand for 12 h. Remove the staining
solution and wash twice with a mixture of 1 volume of acetic acid R and 4 volumes of water R, discard
the washings and store the gels in a similar mixture of acetic acid R and water R. Examine the electro-
18-79
phoretograms using a cold-light illuminator. In the electrophoretograms obtained with test solution
(a), any band, other than the bromophenol blue band, that migrates between two and three times the
distance of the principal band is not more intense than the principal band in the electrophoretogram
obtained with reference solution (e). In the electrophoretogram obtained with test solution (b), any
band that migrates immediately in front of the principal band is not more intense than the principal
band in the electrophoretogram obtained with reference solution (a). The test is not valid unless a
band is seen in the electrophoretogram obtained with reference solution (e) and a gradation of
intensity of staining is seen in the electrophoretograms obtained with reference solutions (a) to (e).
Nitrogen 16.0 per cent to 18.5 per cent, determined by the method of sulphuric acid digestion
(2.5.9) and calculated with reference to the dried substance.
Zinc Not more than 0.15 per cent of Zn, determined by atomic absorption spectrometry (Method I,
2.2.23).
to 25.0 ml with the same acid. Dilute if necessary with 0.01M hydrochloric acid to obtain a zinc
concentration of 0.4 g to 1.6 g per millilitre.
Reference solutions. Use solutions containing 0.10 g, 0.40 g, 0.80 g, 1.00 g, 1.20 g and 1.60 g of
Zn per millilitre, freshly prepared by diluting zinc standard solution (5 mg/ml Zn) R with 0.01M hydro-
chloric acid.
Measure the absorbance at 213.9 nm using a zinc hollow-cathode lamp as the source of radiation and
an air-acetylene flame of suitable composition (for example, 2 litres of acetylene and 11 litres of
air per minute).
Loss on drying (2.2.32). Not more than 10.0 per cent, determined on 50.0 mg by drying at 105C
for 24 h.
ASSAY
The potency of glucagon is determined by comparing, in given conditions, the hyperglycaemic effect
it produces with that produced by the International Standard or by a reference preparation calibrated
in International Units.
The International Unit is the specific hyperglycaemic activity contained in a stated amount of the
International Standard which consists of a quantity of freeze-dried glucagon to which lactose and
sodium chloride have been added. The equivalence in International units of the International
Standard is stated by the World Health Organisation.
potency. The fiducial limits or error (P = 0.95) of the estimated potency are not less than 64 per cent
Reference solution. Dissolve the entire contents of an ampoule of the reference preparation in 2 ml of a
9 g/l solution of sodium chloride R adjusted to pH 3.0 with hydrochloric acid R and dilute with the same
solvent to obtain a solution containing, for example, 0.1 I.U. per millilitre. Store the solution at 2C
to 8C and use within 2 days.
Use in the test healthy rabbits, the difference in mass between the heaviest and the lightest being not
greater than 1.2 kg (for example, rabbits with body masses in the range 1.8 kg to 2.8 kg). Keep the
rabbits in the laboratory under uniform conditions on a uniform diet for a least 1 week before use in
the assay. Handle the rabbits with care to avoid undue excitement.
Inject intramuscularly into each rabbit 48 h before the test 1 ml of a 25 g/l solution of cortisone
acetate and withhold all food but not water from 16 h before each test day until after the withdrawal
of the last blood sample on that day. Distribute the rabbits at random into four equal groups of not
fewer than four rabbits.
Prepare two dilutions of the reference solution with a concentration ratio of 1 to 4, for example
0.006 I.U. per millilitre (reference dilution 1) and 0.024 I.U. per millilitre (reference dilution 2). Use
as diluent a 9 g/l solution of sodium chloride R adjusted to pH 3.0 with hydrochloric acid R. Use the
same diluent to dissolve the substance to be examined and to prepare two dilutions, one of which
(test dilution 1) is presumed, on the basis of the stated potency to contain the same number of
units per millilitre as reference dilution 1 and the other (test dilution 2) the same number of units per
millilitre as reference dilution 2. Inject 1.0 ml of each dilution subcutaneously giving the doses in the
order indicated in the following table, the second injection being made approximately 24 h after the
first injection.
18-80
Group of rabbits
1 2 3 4
First
injection
reference
dilution 1
reference
dilution 2
test
dilution 1
test
dilution 2
Second
injection
test
dilution 2
test
dilution 1
reference
dilution 2
reference
dilution 1
Take a suitable blood sample from the marginal ear vein of each rabbit 1 h after each injection
and determine the blood-glucose concentration in each sample. The optimal time depends on the
strain. It is important that for a given assay a definite and exactly identical time interval be
scrupulously respected for each rabbit.
Calculate the potency by the usual statistical methods for the twin cross-over assay.
STORAGE
Store in an airtight container, at a temperature below 8C and preferably at 20C.
LABELLING
The label states:
the number of units per container,
the number of units per milligram,
the storage conditions,
__________________________________________________________________________________________________________ Ph Eur
18-81
Anhydrous Glucose
O
CH
2
OH
OH
OH
OH
HO
C
6
H
12
O
6
180.2 50-99-7
Anhydrous Glucose complies with the requirements of the 3rd edition of the European Pharmacopoeia [0177].
Preparations
Glucose Intravenous Infusion
Glucose Irrigation Solution
Oral Rehydration Salts
Potassium Chloride and Glucose Intravenous Infusion
Potassium Chloride, Sodium Chloride and Glucose Intravenous Infusion
Sodium Chloride and Glucose Intravenous Infusion
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Anhydrous glucose is D-(+)-glucopyranose.
CHARACTERS
A white, crystalline powder, with a sweet taste, freely soluble in water, sparingly soluble in alcohol.
IDENTIFICATION
A. Specific optical rotation (see Tests): +52.5 to +53.3.
Reference solution (a). Dissolve 10 mg of glucose CRS in a mixture of 2 volumes of water R and 3
volumes of anhydrous acetic acid R and 50 volumes of ethylene chloride R. The solvents should be
measured accurately since a slight excess of water produces cloudiness. Dry the plate in a current of
warm air. Repeat the development immediately, after renewing the mobile phase. Dry the plate in a
current of warm air and spray evenly with a solution of 0.5 g of thymol R in a mixture of 5 ml of
sulphuric acid R and 95 ml of alcohol R. Heat at 130C for 10 min. The principal spot in the chroma-
gram obtained with reference solution (b) shows four clearly separated spots.
C. Dissolve 0.1 g in 10 ml of water R. Add 3 ml of cupri-tartaric solution R and heat. A red precipitate
is formed.
TESTS
Appearance of solution Dissolve 10.0 g in 15 ml of water R. The solution is clear (2.2.1),
odourless, and not more intensely coloured than reference solution BY
7
(Method II, 2.2.2).
Acidity or alkalinity Dissolve 6.0 g in 25 ml of carbon dioxide-free water R and add 0.3 ml of
phenolphthalein solution R. The solution is colourless. Not more than 0.15 ml of 0.1M sodium hydroxide
is required to change the colour of the indicator to pink.
Specific optical rotation (2.2.7). Dissolve 10.0 g in 80 ml of water R, add 0.2 ml of dilute
ammonia R1, allow to stand for 30 min and dilute to 100.0 ml with water R. The specific optical
rotation is +52.5 to +53.3, calculated with reference to the anhydrous substance.
18-82
Foreign sugars, soluble starch, dextrins Dissolve 1.0 g by boiling in 30 ml of alcohol (90 per cent
V/V) R. Cool; the appearance of the solution shows no change.
Sulphites Dissolve 5.0 g in 40 ml of water R, add 2.0 ml of 0.1M sodium hydroxide and dilute to
50.0 ml with water R. To 10.0 ml of the solution, add 1 ml of a 310 g/l solution of hydrochloric acid R,
2.0 ml of decolorised fuchsin solution R1 and 2.0 ml of a 0.5 per cent V/V solution of formaldehyde
solution R. Allow to stand for 30 min and measure the absorbance (2.2.25) at the maximum at
583 nm. Prepare a standard as follows. Dissolve 76 mg of sodium metabisulphite R in water R and
dilute to 50.0 ml with the same solvent. Dilute 5.0 ml of this solution to 100.0 ml with water R. To
3.0 ml of this solution add 4.0 ml of 0.1M sodium hydroxide and dilute to 100.0 ml with water R.
Immediately add to 10.0 ml of this solution 1 ml of a 310 g/l solution of hydrochloric acid R, 2.0 ml of
decolorised fuchsin solution R1 and 2.0 ml of a 0.5 per cent V/V solution of formaldehyde solution R.
Allow to stand for 30 min and measure the absorbance at the maximum at 583 nm. Use as
compensation liquid for both measurements a solution prepared in the same manner using 10.0 ml of
water R. The absorbance of the test solution is not greater than that of the standard (15 ppm of SO
2
)
Arsenic (2.4.2). 1.0 g complies with limit test A for arsenic (1 ppm).
Barium To 10 ml of solution S add 1 ml of dilute sulphuric acid R. When examined immediately and
after 1 h, any opalescence in the solution is not more intense than that in a mixture of 1 ml of distilled
water R and 10 ml of solution S.
of water.
Sulphated ash (2.4.14). Not more than 0.1 per cent. Dissolve 5.0 g in 5 ml of water R, add 2 ml of
sulphuric acid R, evaporate to dryness on a water-bath and ignite to constant mass. If necessary, repeat
the heating with sulphuric acid R.
Pyrogens (2.6.8). If intended for use in large-volume preparations for parenteral use, the competent
authority may require that it comply with the test for pyrogens carried out as follows. Inject per
kilogram of the rabbits mass 10 ml of a solution containing 50 mg per millilitre of the substance to
be examined in water for injections R.
STORAGE
LABELLING
The label states where applicable, that the substance is apyrogenic.
__________________________________________________________________________________________________________ Ph Eur
18-83
Glucose
O
CH
2
OH
OH
OH
OH
HO
,H
2
O
C
6
H
12
O
6
,H
2
O 198.2 5996-10-1
Glucose complies with the requirements of the 3rd edition of the European Pharmacopoeia for Glucose
Preparations
Glucose Intravenous Infusion
Glucose Irrigation Solution
Oral Rehydration Salts
Potassium Chloride and Glucose Intravenous Infusion
Potassium Chloride, Sodium Chloride and Glucose Intravenous Infusion
Sodium Chloride and Glucose Intravenous Infusion
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Glucose monohydrate is the monohydrate of D-(+)-glucopyranose.
CHARACTERS
It has the characters described in the monograph Glucose, anhydrous (177).
IDENTIFICATION
It complies with the identification tests prescribed in the monograph Glucose, anhydrous (177).
TESTS
It complies with the tests prescribed in the monograph Glucose, anhydrous (177) with the following
modification:
of water.
Pyrogens (2.6.8). If intended for use in large-volume preparations for parenteral use, the competent
authority may require that it comply with the test for pyrogens carried out as follows. Inject per
kilogram of the rabbits mass 10 ml of a solution containing 55 mg per millilitre of the substance to
be examined in water for injections R.
STORAGE
LABELLING
The label states where applicable, that the substance is apyrogenic.
__________________________________________________________________________________________________________ Ph Eur
18-84
Liquid Glucose
corrected 1/01
Liquid Glucose complies with the requirements of the 3rd edition of the European Pharmacopoeia [1330]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Liquid glucose is an aqueous solution containing a mixture of glucose, di- and polysaccharides
obtained by hydrolysis of starch. It contains not less than 70.0 per cent of dry matter. The degree of
hydrolysis expressed as dextrose equivalent (DE) is not less than 20 and is within 10 per cent of the
value stated on the label.
CHARACTERS
A clear, colourless or brown viscous liquid, miscible with water. The substance may partly or totally
solidify at room temperature and liquefies again when heated to 50C.
IDENTIFICATION
A. Dissolve 0.1 g in 2.5 ml of water R and heat with 2.5 ml of cupri-tartaric solution R. A red
B. Dip a suitable stick with a reactive pad containing glucose-oxidase, peroxidase and a hydrogen-
donating substance such as tetramethylbenzidine in a 5 g/l solution of the substance to be examined
for 1 s. Observe the colour of the reactive pad within 60 s; the colour changes from yellow to green or
blue.
C. It is a clear, colourless or brown viscous liquid, miscible with water. The substance may partly or
totally solidify at room temperature and liquefies again when heated to 50C.
D. It complies with the test for dextrose equivalent (see Tests).
TESTS
Sulphur dioxide (2.5.29). Not more than 20 ppm. Not more than 400 ppm if intended for the
production of hard boiled candies, provided that the final product does not contain more than
50 ppm of sulphur dioxide.
Heavy metals (2.4.8). Dilute 2 ml of solution S to 30 ml with water R. The solution complies with
limit test E for heavy metals (10 ppm). Prepare the standard using 10 ml of lead standard solution
(1 ppm) R.
Loss on drying (2.2.32). Not more than 30.0 per cent, determined on 1.000 g. Triturate the sample
with 3 g of kieselguhr g R, previously dried for 2 h at 80C under reduced pressure, and dry for 2 h at
80C under reduced pressure.
Dextrose equivalent Accurately weigh an amount of the sample to be examined equivalent to
2.85 g to 3.15 g of reducing carbohydrates, calculated as glucose, into a 500 ml volumetric flask.
Dissolve in water R and dilute to 500.0 ml with the same solvent. Transfer the solution to a 50 ml
burette.
Pipette 25.0 ml of cupri-tartaric solution R into a 250 ml flask and add 18.5 ml of the sample solution
from the burette, mix and add a boiling chip. Place the flask on a hot plate, previously adjusted so
that the solution begins to boil within 2 min 15 s. Leave to boil for exactly 120 s, add 1 ml of a
1 g/l solution of methylene blue R and titrate with the sample solution (V
1
) until the blue colour
disappears. Maintain the solution at boiling throughout the titration.
Standardise the cupri-tartaric solution using a 6.00 g/l solution of glucose R (V
0
).
Calculate the dextrose equivalent (DE) from the equation:
D M V
V
DE

1
0
100 300
V
0
=total volume of dextrose standard solution, in millilitres,
V
1
=total volume of sample solution, in millilitres,
M=sample weight, in grams,
D=per cent of dry matter in the substance.
18-85
LABELLING
The label states the dextrose equivalent (DE).
__________________________________________________________________________________________________________ Ph Eur
18-86
Spray-dried Liquid Glucose
1/01
Spray-dried Liquid Glucose complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Spray-dried liquid glucose is a mixture of glucose, disaccharides and polysaccharides, obtained by the
partial hydrolysis of starch. The degree of hydrolysis, expressed as dextrose equivalent (DE), is not
less than 20 and is within 10 per cent of the value stated on the label.
CHARACTERS
A white or almost white, slightly hygroscopic powder or granules, freely soluble in water.
IDENTIFICATION
A. Dissolve 0.1 g in 2.5 ml of water R and heat with 2.5 ml of cupri-tartaric solution R. A red
B. Dip, for 1 s, a suitable stick with a reactive pad containing glucose-oxidase, peroxidase and a
hydrogen-donating substance, such as tetramethylbenzidine, in a 5 g/l solution of the substance to be
examined. Observe the colour of the reactive pad; within 60 s the colour changes from yellow to
green or blue.
C. It is a powder or granules.
D. It complies with the test for dextrose equivalent (see Tests).
TESTS
Sulphur dioxide (2.5.29). Not more than 20 ppm.
Heavy metals (2.4.8). Dilute 4 ml of solution S to 30 ml with water R. The solution complies with
limit test E for heavy metals (10 ppm). Prepare the standard using 10 ml of lead standard solution
(1 ppm) R.
100C to 105C.
Dextrose equivalent Accurately weigh an amount of the sample to be examined equivalent to
2.85 g to 3.15 g of reducing carbohydrates, calculated as dextrose equivalent, into a 500 ml
volumetric flask. Dissolve in water R and dilute to 500.0 ml with the same solvent. Transfer the
solution to a 50 ml burette.
Pipette 25.0 ml of cupri-tartaric solution R into a 250 ml flask and add 18.5 ml of the sample
solution from the burette, mix and add boiling chips. Place the flask on a hot plate, previously
adjusted so that the solution begins to boil within 2 min 15 s. Allow to boil for exactly 120 s, add
1 ml of a 1 g/l solution of methylene blue R and titrate with the sample solution (V
1
) until the blue
colour disappears. Maintain the solution at boiling throughout the titration.
Standardise the cupri-tartaric solution using a 6.00 g/l solution of glucose R (V
0
).
Calculate the dextrose equivalent (DE) from the equation:
D M V
V

1
0
100 300
DE
V
0
=total volume of glucose standard solution, in millilitres,
V
1
=total volume of sample solution, in millilitres,
M=sample weight, in grams,
D=percentage content of dry matter in the substance.
3
bacteria and 10
2
fungi per gram, determined by plate count. It complies with the tests for Escherichia coli and
LABELLING
The label states the dextrose equivalent (DE).
__________________________________________________________________________________________________________ Ph Eur
18-87
Glutamic Acid
COOH
NH
2
H
HOOC
C
5
H
9
NO
4
147.1 56-86-0
Glutamic Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia [0750]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Glutamic acid contains not less than 98.5 per cent and not more than the equivalent of 100.5 per
cent of (S)-2-aminopentane-1,5-dioic acid, calculated with reference to the dried substance.
PRODUCTION
When Glutamic acid is produced by a process involving fermentation steps, it complies with the
requirements of the monograph on Products of Fermentation (1468).
CHARACTERS
A white, crystalline powder or colourless crystals, freely soluble in boiling water, slightly soluble in
cold water, practically insoluble in acetic acid, in acetone, in alcohol and in ether.
IDENTIFICATION
obtained with glutamic acid CRS. Examine the substances prepared as discs. If the spectra obtained
minimum quantity of water R, evaporate to dryness at 60C and record new spectra using the
residues.
D. To 2.0 ml of solution S (see Tests) add 0.1 ml of phenolphthalein solution R and 3.0 ml to 3.5 ml
of 1M sodium hydroxide to change the colour of the indicator to red. Add a mixture of 3 ml of
formaldehyde solution R, 3 ml of carbon dioxide-free water R and 0.1 ml of phenolphthalein solution R, to
which sufficient 1M sodium hydroxide has been added to produce a pink colour. The solution is
decolorised. Add 1M sodium hydroxide until a red colour is produced. The total volume of 1M sodium
hydroxide used is 4.0 ml to 4.7 ml.
TESTS
Solution S Dissolve 5.00 g in 1M hydrochloric acid with gentle heating, and dilute to 50.0 ml with the
same acid.
gel plate R.
Test solution (a). Dissolve 0.10 g of the substance to be examined in 5 ml of dilute ammonia R2 and
Reference solution (a). Dissolve 10 mg of glutamic acid CRS in water R and dilute to 50 ml with the
same solvent.
Reference solution (c). Dissolve 10 mg of glutamic acid CRS and 10 mg of aspartic acid CRS in water R
18-88
Apply separately to the plate 5 l of each solution. Dry the plate in a current of air for 15 min.
water R and 60 volumes of butanol R. Allow the plate to dry in air, spray with ninhydrin solution R and
heat at 100C to 105C for 15 min. Any spot in the chromatogram obtained with test solution (a),
reference solution (b) (0.5 per cent). The test is not valid unless the chromatogram obtained with
reference solution (c) shows two clearly separated spots.
The solution, without the further addition of nitric acid, complies with the limit test for chlorides
(200 ppm).
watch-glass and suspend in 0.5 ml of water R. To the suspension add 0.30 g of heavy magnesium
4
100C to 105C.
ASSAY
Dissolve 0.130 g in 50 ml of carbon dioxide-free water R with gentle heating. Cool. Using 0.1 ml of
bromothymol blue solution R1 as indicator, titrate with 0.1M sodium hydroxide until the colour changes
from yellow to blue.
5
H
9
NO
4
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
Strong Glutaraldehyde Solution
OHC CHO
C
5
H
8
O
2
100.1 111-30-8 (anhydrous)
Definition Strong Glutaraldehyde Solution is an aqueous solution of glutaraldehyde
(pentanedial). It contains not less than 47.0% and not more than 53.0% w/w of glutaraldehyde,
C
5
H
8
O
2
.
Characteristics A colourless or almost colourless solution.
Identification
A. Heat 1 ml with 10 ml of a solution containing 1 g of hydroxylamine hydrochloride and 2 g of sodium
acetate in water on a water bath for 10 minutes, allow to cool and filter. The melting point of the
residue, after washing with water and drying at 105, is about 178, Appendix V A.
B. Add 0.05 ml to 2 ml of ammoniacal silver nitrate solution and mix gently for a few minutes. Silver is
deposited.
Acidity Dilute 10 ml with 10 ml of carbon dioxide-free water and titrate with 0.1M sodium hydroxide VS
using bromothymol blue solution R3 as indicator. Not more than 5.0 ml of 0.1M sodium hydroxide VS is
required to change the colour of the solution.
Clarity and colour of solution Dilute 1 volume with 4 volumes of water. The resulting solution is
clear, Appendix IV A, and not more intensely coloured than reference solution B
6
, Appendix IV B,
Method I.
Assay Dissolve 4 g in 100 ml of a 7% w/v solution of hydroxylamine hydrochloride previously
neutralised to bromophenol blue solution with 1M sodium hydroxide VS and allow to stand for 30
minutes. Add 20 ml of petroleum spirit (boiling range, 40 to 60) and titrate with 1M sodium hydroxide
VS until the colour of the aqueous phase matches that of a 7% w/v solution of hydroxylamine hydro-
chloride previously neutralised to bromophenol blue solution with 1M sodium hydroxide VS. Each ml of 1M
sodium hydroxide VS is equivalent to 50.05 mg of C
5
H
8
O
2
.
Storage Strong Glutaraldehyde Solution should be kept in a well-closed container and stored at a
Action and use Used in treatment of warts.
Preparation
Glutaraldehyde Solution
18-90
Glutethimide
and enantiomer
HN Et
O
O
C
13
H
15
NO
2
217.3 77-21-4
Glutethimide complies with the requirements of the 3rd edition of the European Pharmacopoeia [0613]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Glutethimide contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent
of (RS)-3-ethyl-3-phenylpiperidine-2,6-dione, calculated with reference to the dried substance.
CHARACTERS
A white or almost white powder or colourless crystals, practically insoluble in water, very soluble in
methylene chloride, freely soluble in alcohol, soluble in ether.
IDENTIFICATION
obtained with glutethimide CRS. Examine the substances prepared as discs.
C. Dissolve about 2 mg in 1 ml of methanol R and add 5 ml of a cooled mixture of 1 ml of
formaldehyde R and 9 ml of sulphuric acid R. Heat on a water-bath for 15 min. The solution is reddish
and shows an intense blue fluorescence in ultraviolet light at 365 nm.
TESTS
Solution S Dissolve 1.25 g in a mixture of 10 volumes of water R and 90 volumes of alcohol R and
solution is yellow. Not more than 0.2 ml of 0.01M sodium hydroxide is required to change the colour of
the indicator; the colour change to blue may not be persistent and subsequent change in colour is not
taken into account.
coating substance.
Test solution. Dissolve 0.5 g of the substance to be examined in ethanol R and dilute to 10 ml with the
same solvent.
Reference solution. Dilute 0.5 ml of the test solution to 100 ml with ethanol R.
5 volumes of anhydrous formic acid R, 10 volumes of methanol R and 85 volumes of chloroform R.
Allow the plate to dry in air. Place the plate in a tank containing chlorine prepared by adding hydro-
chloric acid R to a 50 g/l solution of potassium permanganate R and allow to stand for 1 min. Remove
the plate from the tank, allow to stand in air for 2 min and spray with potassium iodide and starch
(0.5 per cent).
Prepare the standard using lead standard solution (1 ppm Pb) prepared by diluting lead standard
solution (100 ppm Pb) R with a mixture of 10 volumes of water R and 90 volumes of alcohol R.
4 h.
18-91
ASSAY
Dissolve 0.150 g in 10 ml of ethanol R. Add 5 ml of silver nitrate solution in pyridine R. Titrate with
0.1M ethanolic sodium hydroxide. Determine the end-point potentiometrically (2.2.20), using a silver
silver chloride reference electrode.
13
H
15
NO
2
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
18-92
Glycerol
Glycerin
OH H
OH O
C
3
H
8
O
3
92.1 56-81-5
Glycerol complies with the requirements of the 3rd edition of the European Pharmacopoeia [0496]. These
Action and use Lubricant; laxative.
Preparation
Glycerol Suppositories
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Glycerol contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent of
propane-1,2,3-triol, calculated with reference to the anhydrous substance.
CHARACTERS
A syrupy liquid, unctuous to the touch, colourless or almost colourless, clear, very hygroscopic,
miscible with water and with alcohol, slightly soluble in acetone, practically insoluble in ether, in fatty
oils and in essential oils.
IDENTIFICATION
B. To 5 ml add 1 ml of water R and mix carefully. Examine the solution by infrared absorption
spectrophotometry (2.2.24), comparing with the Ph. Eur. reference spectrum of glycerol (85 per cent).
C. Mix 1 ml with 0.5 ml of nitric acid R. Superimpose 0.5 ml of potassium dichromate solution R. A
blue ring develops at the interface of the liquids. Within 10 min, the blue colour does not diffuse into
the lower layer.
D. Heat 1 ml with 2 g of potassium hydrogen sulphate R in an evaporating dish. Vapours (acrolein) are
evolved which blacken filter paper impregnated with alkaline potassium tetraiodomercurate solution R.
TESTS
Solution S Dilute 100.0 g to 200.0 ml with carbon dioxide-free water R.
Appearance of solution Solution S is clear (2.2.1). Dilute 10 ml of solution S to 25 ml with
water R. The solution is colourless (Method II, 2.2.2).
indicator to pink.
Aldehydes Place 7.5 ml of solution S in a ground-glass-stoppered flask and add 7.5 ml of water R
and 1.0 ml of decolorised pararosaniline solution R. Close the flask and allow to stand for 1 h at a
temperature of 25 1C. The absorbance of the solution measured at 552 nm (2.2.25) is not greater
than that of a standard prepared at the same time and in the same manner using 7.5 ml of
formaldehyde standard solution (5 ppm CH
2
O) R and 7.5 ml of water R. The test is not valid unless the
standard is pink.
Esters Add 0.1M sodium hydroxide to the final solution obtained in the test for acidity or alkalinity
until a total of 10.0 ml has been added. Boil under a reflux condenser for 5 min. Cool. Add 0.5 ml of
phenolphthalein solution R and titrate with 0.1M hydrochloric acid. Not less than 8.0 ml of 0.1M hydro-
chloric acid is required to change the colour of the indicator.
Impurity A and related substances Examine by gas chromatography (2.2.28).
Test solution. Dilute 10.0 ml of solution S to 100.0 ml with water R.
Reference solution (a). Dissolve 1.000 g of diethylene glycol R in water R and dilute to 100.0 ml with the
same solvent.
Reference solution (b). Dilute 1.0 ml of reference solution (a) to 10.0 ml with the test solution. Dilute
1.0 ml of this solution to 20.0 ml with the test solution.
18-93
Reference solution (c). Mix 1.0 ml of the test solution and 5.0 ml of reference solution (a) and dilute
to 100.0 ml with water R. Dilute 1.0 ml of this solution to 10.0 ml with water R.
a fused-silica column 30 m long and 0.53 mm in internal diameter coated with a mixture of
cross-linked polycyanopropylphenylsiloxane (6 per cent) and polydimethylsiloxane (94 per cent)
(film thickness 3 m),
helium for chromatography R as the carrier gas at a linear velocity of about 38 cm/s and with a split
ratio of about 10:1,
rate of 7.5C per minute to 220C and maintaining at 220C for 4 min; maintaining the temperature
Inject 0.5 l of the reference solution (c). Adjust the sensitivity of the system such that the height of
the peak due to impurity A is at least 50 per cent of the full scale of the recorder. When the chroma-
tograms are recorded in the prescribed conditions, the substances are eluted in the following order:
impurity A and glycerol.
The test is not valid unless: in the chromatogram obtained with reference solution (c), the
resolution between the peaks corresponding to impurity A and to glycerol is at least 7.0.
Inject 0.5 l of the test solution, 0.5 l of reference solution (b) and 0.5 l of reference solution (d).
In the chromatogram obtained with the test solution: the area of any peak corresponding to impurity
A is not greater than half the area of the corresponding peak in the chromatogram obtained with
reference solution (b) (0.1 per cent); the area of any peak, apart from the peak corresponding to
impurity A, and with a retention time less than the retention time of glycerol, is not greater than half
the area of the peak corresponding to impurity A in the chromatogram obtained with reference
solution (b) (0.1 per cent); the sum of the areas of any peaks with retention times greater than the
retention time of glycerol, is not greater than 2.5 times the area of the peak corresponding to impurity
A in the chromatogram obtained with reference solution (b) (0.5 per cent). Disregard any peak with
an area less than 0.05 times the area of the peak corresponding to impurity A in the chromatogram
obtained with reference solution (d).
Halogenated compounds To 10 ml of solution S add 1 ml of dilute sodium hydroxide solution R,
5 ml of water R and 50 mg of halogen-free nickel-aluminium alloy R. Heat on a water-bath for 10 min,
allow to cool and filter. Rinse the flask and the filter with water R until 25 ml of filtrate is obtained.
To 5 ml of the filtrate add 4 ml of alcohol R, 2.5 ml of water R, 0.5 ml of nitric acid R and 0.05 ml of
silver nitrate solution R2 and mix. Allow to stand for 2 min. Any opalescence in the solution is not
more intense than that in a standard prepared at the same time by mixing 7.0 ml of chloride standard
solution (5 ppm Cl) R, 4 ml of alcohol R, 0.5 ml of water R, 0.5 ml of nitric acid R and 0.05 ml of silver
nitrate solution R2 (35 ppm).
Sugars To 10 ml of solution S add 1 ml of dilute sulphuric acid R and heat on a water-bath for 5 min.
Add 3 ml of carbonate-free dilute sodium hydroxide solution R (prepared by the method described for
carbonate-free 1M sodium hydroxide (4.2.2), mix and add dropwise 1 ml of freshly prepared copper
sulphate solution R. The solution is clear and blue. Continue heating on the water-bath for 5 min. The
solution remains blue and no precipitate is formed.
chlorides (10 ppm). Prepare the standard using 1 ml of chloride standard solution (5 ppm Cl) R diluted
complies with limit test A for heavy metals (5 ppm). Prepare the standard using lead standard solution
(1 ppm Pb) R.
mination of water.
Sulphated ash (2.4.14). Not more than 0.01 per cent, determined on 5.0 g after heating to boiling
and ignition.
ASSAY
Thoroughly mix 0.1000 g with 45 ml of water R. Add 25.0 ml of a 21.4 g/l solution of
sodium periodate R. Allow to stand protected from light for 15 min. Add 5.0 ml of a 500 g/l solution of
ethylene glycol R and allow to stand protected from light for 20 min. Using 0.5 ml of phenolphthalein
solution R as indicator, titrate with 0.1M sodium hydroxide. Carry out a blank titration.
3
H
8
O
3
.
STORAGE
18-94
IMPURITIES
OH
O
O
A. diethylene glycol,
HO
OH
B. ethylene glycol,
C. propylene glycol.
__________________________________________________________________________________________________________ Ph Eur
18-95
Glycerol (85 per cent)
Glycerol (85 per cent) complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Glycerol (85 per cent) is an aqueous solution containing not less than 83.5 per cent m/m and not
more than 88.5 per cent m/m of propane-1,2,3-triol (C
3
H
8
O
3
; M
r
92.1).
CHARACTERS
A syrupy liquid, unctuous to the touch, colourless or almost colourless, clear, very hygroscopic,
miscible with water and with alcohol, slightly soluble in acetone, practically insoluble in ether, in fatty
oils and in essential oils.
IDENTIFICATION
B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with Ph. Eur. reference
spectrum of glycerol (85 per cent).
C. Mix 1 ml with 0.5 ml of nitric acid R. Superimpose 0.5 ml of potassium dichromate solution R. A
blue ring develops at the interface of the liquids. Within 10 min, the blue colour does not diffuse into
the lower layer.
D. Heat 1 ml with 2 g of potassium hydrogen sulphate R in an evaporating dish. Vapours (acrolein) are
evolved which blacken filter paper impregnated with alkaline potassium tetraiodomercurate solution R.
TESTS
Solution S Dilute 117.6 g to 200.0 ml with carbon dioxide-free water R.
Apperance of solution Solution S is clear (2.2.1). Dilute 10 ml of solution S to 25 ml with water R.
The solution is colourless (Method II, 2.2.2).
indicator to pink.
Aldehydes Place 7.5 ml of solution S in a ground-glass-stoppered flask and add 7.5 ml of water R
and 1.0 ml of decolorised pararosaniline solution R. Close the flask and allow to stand for 1 h at a
temperature of 25 1C. The absorbance of the solution measured at 552 nm (2.2.25) is not greater
than that of a standard prepared at the same time and at the same manner using 7.5 ml of form-
aldehyde standard solution (5 ppm CH
2
O) R and 7.5 ml of water R. The test is not valid unless the
standard is pink.
Esters Add 0.1M sodium hydroxide to the solution obtained in the test for acidity or alkalinity until a
total of 10.0 ml has been added. Boil under a reflux condenser for 5 min. Cool. Add 0.5 ml of
phenolphthalein solution R and titrate with 0.1M hydrochloric acid. Not less than 8.0 ml of 0.1M hydro-
chloric acid is required to change the colour of the indicator.
Impurity A and related substances Examine by gas chromatography (2.2.28)
Test solution. Dilute 10.0 ml of solution S to 100.0 ml with water R.
Reference solution (a). Dissolve 1.000 g of diethylene glycol R in water R and dilute to 100.0 ml with the
same solvent.
Reference solution (b). Dilute 1.0 ml of reference solution (a) to 10.0 ml with the test solution. Dilute
1.0 ml of this solution to 20.0 ml with the test solution.
Reference solution (c). Mix 1.0 ml of the test solution and 5.0 ml of reference solution (a) and dilute to
100.0 ml with water R. Dilute 1.0 ml of this solution to 10.0 ml with water R.
a fused-silica column 30 m long and 0.53 mm in internal diameter coated with a mixture of
cross-linked polycyanopropylphenylsiloxane (6 per cent) and polydimethylsiloxane (94 per cent )
(film thickness 3 m),
helium for chromatography R as the carrier gas at a linear velocity of about 38 cm/s and with a split
ratio of about 10:1,
18-96
maintaining the temperature of the column at 100C until injection, then raising the temperature at
a rate of 7.5C per minute to 220C and maintaining at 220C for 4 min; maintaining the tempera-
ture of the injection port at 220C and that of the detector at 250C.
Inject 0.5 l of the reference solution (c). Adjust the sensivity of the system so that the height of
the peak due to impurity A is at least 50 per cent of the full scale of the recorder. When the
chromatograms are recorded in the prescribed conditions, the substances are eluted in the follow-
ing order: impurity A and glycerol.
The test is not valid unless: in the chromatogram obtained with reference solution (c), the
resolution between the peaks corresponding to impurity A and to glycerol is at least 7.0.
Inject 0.5 l of the test solution, 0.5 l of reference solution (b) and 0.5 l of reference solution (d).
In the chromatogram obtained with the test solution: the area of any peak corresponding to impurity
A is not greater than half the area of the corresponding peak in the chromatogram obtained with the
reference solution (b) (0.1 per cent); the area of any peak, apart from the peak corresponding to
impurity A, and with a retention time less than the retention time of glycerol, is not greater than the
area of the peak corresponding to impurity A in the chromatogram obtained with reference solution
(b) (0.1 per cent); the sum of the areas of any peaks with retention times greater than the retention
time of glycerol, is not greater than 2.5 times the area of the peak corresponding to the impurity A in
the chromatogram obtained with reference solution (b) (0.5 per cent). Disregard any peak with an
area less than 0.05 times the area of the peak corresponding to impurity A in the chromatogram
obtained with reference solution (d).
Halogenated compounds To 10 ml of solution S add 1 ml of dilute sodium hydroxide solution R,
5 ml of water R and 50 mg of halogen-free nickel-aluminium alloy R. Heat on a water-bath for 10 min,
allow to cool and filter. Rinse the flask and the filter with water R until 25 ml of filtrate is obtained.
To 5 ml of the filtrate add 4 ml of alcohol R, 2.5 ml of water R, 0.5 ml of nitric acid R and 0.05 ml of
silver nitrate solution R2 and mix. Allow to stand for 2 min. Any opalescence in the solution is not
more intense than that in a standard prepared at the same time by mixing 7.0 ml of chloride standard
solution (5 ppm Cl) R, 4 ml alcohol R, 0.5 ml of water R, 0.5 ml of nitric acid R and 0.05 ml of silver
nitrate solution R2 (30 ppm).
Sugars To 10 ml of solution S add 1 ml of dilute sulphuric acid R and heat on a water-bath for 5 min.
Add 3 ml of carbonate-free dilute sodium hydroxide solution R (prepared by the method described for
carbonate-free 1M sodium hydroxide (4.2.2)), mix and add dropwise 1 ml of freshly prepared copper
sulphate solution R. The solution is clear and blue. Continue heating on the water-bath for 5 min. The
solutiion remains blue and no precipitate is formed.
chlorides (10 ppm). Prepare the standard using 1 ml of chloride standard solution (5 ppm Cl) R diluted
complies with limits test A for heavy metals (5 ppm). Prepare the standard using lead standard solution
(1 ppm Pb) R.
mination of water.
Sulphated ash (2.4.14) Not more than 0.01 per cent, determined on 5.0 g after heating to boiling
and ignition.
ASSAY
Thoroughly mix 0.1000 g with 45 ml of water R. Add 25.0 ml of a 21.4 g/l solution of sodium
periodate R. Allow to stand protected from light for 15 min. Add 5.0 ml of a 500 g/l solution of
ethylene glycol R and allow to stand protected from light for 30 min. Using 0.5 ml of phenolphthalein
solution R as indicator, titrate with 0.1M sodium hydroxide. Carry out a blank titration.
3
H
8
O
3
.
STORAGE
IMPURITIES
OH
O
O
A. diethylene glycol,
HO
OH
B. ethylene glycol,
C. propylene glycol.
__________________________________________________________________________________________________________ Ph Eur
18-97
Glycerol Dibehenate
corrected 1/01
Glycerol Dibehenate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1427].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Glycerol dibehenate is a mixture of diacylglycerols, mainly dibehenoylglycerol, together with
variable quantities of mono-and triacylglycerols. It contains 13.0 per cent to 21.0 per cent of
monoacylglycerols, 40.0 per cent to 60.0 per cent of diacylglycerols and 21.0 per cent to 35.0 per
cent of triacylglycerols, obtained by esterification of glycerol with behenic acid.
CHARACTERS
A hard, waxy mass or powder or white or almost white, unctuous flakes, insoluble in water, soluble
in methylene chloride and partly soluble in hot alcohol.
IDENTIFICATION
Test solution. Dissolve 1.0 g of the substance to be examined in toluene R, with gentle heating and
Reference solution. Dissolve 1.0 g of glycerol dibehenate CRS in toluene R, with gentle heating and dilute
solution of rhodamine B R in alcohol R and examine in ultraviolet light at 365 nm. The spots in the
chromatogram obtained with the test solution are similar in position to those in the chromatogram
TESTS
Acid value (2.5.1). Not more than 4.0, determined on 1.0 g, using a mixture of equal volumes of
alcohol R and toluene R as solvent and with gentle heating.
Saponification value (2.5.6). 145 to 165, determined on 2.0 g. Carry out the titration with heating.
Free glycerol Not more than 1.0 per cent, determined as described under Assay.
Composition of fatty acids Examine by gas chromatography (2.4.22, Method C), raising the
temperature of the column up to 240C. The fatty acid fraction of the substance has the following
composition:
behenic acid: not less than 83.0 per cent,
lignoceric acid: not more than 3.0 per cent,
erucic acid: not more than 3.0 per cent.
of water. Use pyridine R as the solvent.
ASSAY
Determine the free glycerol content and the mono-, di-and triacylglycerol contents by size-exclusion
chromatography (2.2.30).
Test solution. Into a 15 ml flask, weigh about 0.2 g (m), to the nearest 0.1 mg. Add 5 ml of
tetrahydrofuran R and shake to dissolve. Reweigh the flask and calculate the total mass of solvent and
substance (M).
Reference solutions. Into four 15 ml flasks, respectively weigh, to the nearest 0.1 mg, about 2 mg,
5 mg, 10 mg and 20 mg of glycerol R. Add 5 ml of tetrahydrofuran R and shake until well mixed.
18-98
Weigh the flasks again and calculate the concentration of glycerol in milligrams per gram for each
reference solution.
divinylbenzene copolymer R (particle diameter 5 m and porosity 10 nm),
the retention times relative to glycerol are about 0.82 for the monoacylglycerols, about 0.76 for the
diacylglycerols and about 0.73 for the triacylglycerols. From the calibration curve obtained with the
reference solutions determine the concentration (C) in milligrams per gram of glycerol in the test
solution.
Calculate the percentage content of free glycerol in the substance to be examined using the
10
m
M C
Calculate the percentage content of mono-, di-and triacylglycerols in the substance to be examined
by the normalisation procedure.
__________________________________________________________________________________________________________ Ph Eur
18-99
Glycerol Distearate
Glycerol Distearate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1428].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Glycerol distearate is a mixture of diacylglycerols, mainly distearoylglycerol, together with variable
quantities of mono- and triacylglycerols. It contains 8.0 per cent to 22.0 per cent of mono-
acylglycerols, 40.0 per cent to 60.0 per cent of diacylglycerols and 25.0 per cent to 35.0 per cent of
triacylglycerols, obtained by partial glycerolysis of vegetable oils containing triacylglycerols of palmitic
or stearic acid or by esterification of glycerol with stearic acid 50 (type I), stearic acid 70 (type II) or
stearic acid 95 (type III) (see Stearic acid (1474)). The fatty acids may be of vegetable or animal
origin.
PRODUCTION
CHARACTERS
A hard, waxy mass or powder or white or almost white, unctuous flakes, insoluble in water, soluble in
methylene chloride, partly soluble in hot alcohol.
IDENTIFICATION
A. Melting point (2.2.14): 50C to 60C (type I and II), 50C to 70C (type III).
Test solution. Dissolve 1.0 g of the substance to be examined in methylene chloride R, with gentle
heating and dilute to 20 ml with the same solvent.
Reference solution. Dissolve 1.0 g of glycerol distearate CRS in methylene chloride R, with gentle heating
C. It complies with the test for composition of fatty acids according to the type stated on the label
(see Tests).
D. It complies with the limits of the assay (diacylglycerol content).
TESTS
alcohol R and toluene R as solvent and with gentle heating.
Saponification value (2.5.6).165 to 195, determined on 2.0 g. Carry out the titration with heating.
Composition of fatty acids Examine by gas chromatography (2.4.22, Method C). The fatty-acid
fraction of the substance to be examined has the following composition:
18-100
Fatty acid used for
production by
esterification
Glycerol
distearate
type I

Stearic acid 50 Stearic acid: 40.0 per cent to
60.0 per cent
Sum of the contents of
palmitic and stearic acids:
not less than 90.0 per cent
Glycerol
distearate
type II
Stearic acid 70 Stearic acid: 60.0 per cent to
80.0 per cent
Sum of palmitic and stearic
acids: not less than 90.0 per
cent
Glycerol
distearate
type III
Stearic acid 95 Stearic acid 90.0 per cent to
99.0 per cent
Sum of the contents of
palmitic and stearic acids:
not less than 96.0 per cent
mination of water. Use pyridine R as the solvent.
ASSAY
Determine the free glycerol content and the mono-, di- and triacylglycerol contents by size-
exclusion chromatography (2.2.30).
tetrahydrofuran R and shake to dissolve. Reweigh the flasks and calculate the total mass of solvent and
substance (M).
Reference solutions. Into four 15 ml flasks, respectively weigh, to the nearest 0.1 mg, about 2 mg,
reference solution.
the retention times relative to glycerol are 0.84 for the monoacylglycerols, 0.78 for the diacylglycerols
and 0.75 for the triacylglycerols. From the calibration curve obtained with the reference solutions
determine the concentration (C) in milligrams per gram of glycerol in the test solution.
10
m
M C
Calculate the percentage content of mono-, di- and triacylglycerols in the substance to be examined
LABELLING
The label states the type of glycerol distearate.
__________________________________________________________________________________________________________ Ph Eur
18-101
Glycerol Monolinoleate
Glycerol Monolinoleate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Glycerol monolinoleate is a mixture of monoacylglycerols, mainly mono-oleyl and monolinoleoyl-
glycerol, together with variable quantities of di- and triacylglycerols. It contains 32.0 per cent to
52.0 per cent of monoacylglycerols, 40.0 per cent to 55.0 per cent of diacylglycerols and 5.0 per cent
to 20.0 per cent of triacylglycerols, obtained by partial glycerolysis of vegetable oils mainly containing
triacylglycerols of linoleic acid. A suitable antioxidant may be added.
CHARACTERS
Amber, oily liquids which may be partially solidified at room temperature, practically insoluble in
water, freely soluble in methylene chloride.
IDENTIFICATION
Reference solution. Dissolve 1.0 g of glycerol monolinoleate CRS in methylene chloride R and dilute to
TESTS
Iodine value (2.5.4). 100 to 140.
Peroxide value (2.5.5, Method A). Not more than 12.0, determined on 2.0 g.
fraction of the substance has the following composition:
oleic acid: 10.0 per cent to 35.0 per cent,
linoleic acid: not less than 50.0 per cent,
linolenic acid: not more than 2.0 per cent,
eicosenoic acid: not more than 1.0 per cent.
of water. Use as the solvent a mixture of equal volumes of anhydrous methanol R and methylene
chloride R.
ASSAY
Determine the free glycerol content and the mono-, di- and triacylglycerol contents by size-exclusion
Test solution. Into a 15 ml flask, weigh about 0.2 g (m), to the nearest 0.1 mg. Add 5 ml of tetrahydro-
furan R and shake to dissolve. Reweigh the flask and calculate the total mass of solvent and substance
(M).
18-102
reference solution.
a gel permeation column 0.6 m long and 7 mm in international diameter packed with styrene-
solution.
10
m
M C
STORAGE
LABELLING
The label states the name and concentration of any added antioxidant.
__________________________________________________________________________________________________________ Ph Eur
18-103
Glycerol Mono-oleates
Glycerol Mono-oleates comply with the requirements of the 3rd edition of the European Pharmacopoeia [1430].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Glycerol mono-oleates are mixtures of monoacylglycerols, mainly mono-oleoylglycerol, together
with variable quantities of di- and triacylglycerols. They are defined by the nominal content of
monoacylglycerols and obtained by partial glycerolysis of vegetable oils mainly containing
triacylglycerols of oleic acid or by esterification of glycerol by oleic acid, this fatty acid being of
vegetable or animal origin.
Nominal content of acylglycerol (per cent)
40 60 90
Monoacylgycerols 32.0 52.0 55.0 65.0 90.0 101.0
Diacylglycerols 30.0 50.0 15.0 35.0 < 10.0
Triacylglycerols 5.0 20.0 2.0 10.0 < 2.0
A suitable antioxidant may be added.
PRODUCTION
CHARACTERS
Amber, oily liquids which may be partially solidified at room temperature, practically insoluble in
water, freely soluble in methylene chloride.
IDENTIFICATION
Reference solution. Dissolve 1.0 g of glycerol mono-oleate CRS in methylene chloride R and dilute to 20 ml
C. It complies with the limits of the assay (monoacylglycerol content).
TESTS
Iodine value (2.5.4).65.0 to 95.0.
fraction of the substance has the following composition:
oleic acid: not less than 60.0 per cent,
linoleic acid: not more than 35.0 per cent,
18-104
mination of water. Use as the solvent a mixture of equal volumes of anhydrous methanol R and
methylene chloride R.
ASSAY
substance (M).
reference solution.
solution.
10
m
M C
STORAGE
LABELLING
The label states:
the nominal content of monoacylglycerol,
__________________________________________________________________________________________________________ Ph Eur
19-1
Glycerol Monostearate 40-55
31566-31-1
Glycerol Monostearate 40-55 complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Glycerol monostearate 40-55 is a mixture of monoacylglycerols, mainly monostearoylglycerol,
together with variable quantities of di- and triacylglycerols. It contains 40.0 per cent to 55.0 per cent
of monoacylglycerols, 30.0 per cent to 45.0 per cent of diacylglycerols and 5.0 per cent to 15.0 per
cent of triacylglycerols, obtained by partial glycerolysis of vegetable oils mainly containing
triacylglycerols of palmitic or stearic acid or by esterification of glycerol with stearic acid 50 (type I),
stearic acid 70 (type II) or stearic acid 95 (type III) (see Stearic acid (1474)). The fatty acids may be
of vegetable or animal origin.
PRODUCTION
CHARACTERS
A hard, waxy mass or unctuous powder or flakes, white or almost white, practically insoluble in
water, soluble in alcohol at 60C.
IDENTIFICATION
A. Melting point (2.2.15): 54C to 64C. Introduce the melted substance into the capillary tubes and
allow to stand for 24 h in a well-closed container.
Test solution. Dissolve 1.0 g of the substance to be examined in methylene chloride R, with gentle
heating, and dilute to 20 ml with the same solvent.
Reference solution. Dissolve 1.0 g of glycerol monostearate 40-55 CRS in methylene chloride R, with gentle
heating, and dilute to 20 ml with the same solvent.
C. It complies with the test for composition of fatty acids according to the type stated on the label
(see Tests).
D. It complies with the limits of the assay (monoacylglycerol content).
TESTS
alcohol R and toluene R as solvent and heating gently.
Saponification value (2.5.6).158 to 177, determined on 2.0 g. Carry out the titration with heating.
fraction of the substance to be examined has the following composition:
19-2
Fatty acid used for
production by
esterification
Composition of fatty
acids
Glycerol mono-
stearate 40 - 55
type I
Stearic acid 50 Stearic acid: 40.0 per
cent to 60.0 per cent
Sum of the contents
of palmitic and stearic
acids: not less than
90.0 per cent
Glycerol mono-
stearate 40 - 55
type II
Sum of palmitic and
stearic acids: not less
than 90.0 per cent
Glycerol mono-
stearate 40 - 55
type III
Sum of the contents
of palmitic and stearic
acids: not less than
96.0 per cent
mination of water. Use pyridine R as the solvent and heat gently.
ASSAY
substance (M).
reference solution.
solution.
10
m
M C
LABELLING
The label states the type of glycerol monostearate 40-55.
__________________________________________________________________________________________________________ Ph Eur
19-3
Self-emulsifying Glyceryl Monostearate
Self-emulsifying Monostearin; Self-emulsifying Mono- and Diglycerides of Food Fatty Acids
Definition Self-emulsifying Glyceryl Monostearate is a mixture consisting principally of mono-, di-
and triglycerides of stearic and palmitic acids and of minor proportions of glycerides of other fatty
acids; it may also contain free glycerol, free fatty acids and soap. It contains not less than 30.0% of
monoglycerides, calculated as C
21
H
42
O
4
, not more than 7.0% of free glycerol, calculated as C
3
H
8
O
3
,
and not more than 6.0% of soap, calculated as sodium oleate, C
18
H
33
NaO
2
, all calculated with
Characteristics A white to cream coloured, hard, waxy solid; odour, faint and fatty.
Dispersible in hot water; soluble in hot absolute ethanol, in hot liquid paraffin and, subject to
turbidity at concentrations below 20%, in hot vegetable oils.
Acid value Not more than 6, Appendix X B.
Iodine value Not more than 3 (iodine monochloride method), Appendix X E.
Alkalinity Shake 1 g with 20 ml of hot carbon dioxide-free water and allow to cool with continuous
shaking. The pH of the aqueous layer is 8.0 to 10.0, Appendix V L.
Water Not more than 2.0% w/w, Appendix IX C. Use 0.5 g and a mixture of 10 ml of anhydrous
methanol and 10 ml of anhydrous chloroform as the solvent.
Assay
For free glycerol Dissolve 0.4 g in 50 ml of dichloromethane in a ground-glass-stoppered separating
funnel, cool if necessary, add 25 ml of water and shake vigorously for 1 minute; add 0.2 ml of glacial
acetic acid, if necessary, to break the emulsion. Repeat the extraction a further three times using 25-,
20- and 20-ml quantities of water and reserve the dichloromethane solution for the Assay for
monoglycerides. Filter the combined aqueous extracts through a filter paper moistened with water,
wash the filter with two 5-ml quantities of water and dilute the combined filtrate and washings to
100 ml with water. To 50 ml of this solution add 25 ml of periodic acetic acid solution, shaking
cautiously, allow to stand at 25 to 30 for 30 minutes and add 100 ml of water and 12 ml of
potassium iodide solution. Titrate with 0.1M sodium thiosulphate VS using 1 ml of starch solution as
indicator. Repeat the determination using 50 ml of water in place of the 50 ml of the solution being
examined. The difference between the titrations represents the amount of sodium thiosulphate
required. Each ml of 0.1M sodium thiosulphate VS is equivalent to 2.3 mg of glycerol.
For monoglycerides Filter the reserved dichloromethane solution obtained in the Assay for free
glycerol through absorbent cotton and wash the separating funnel and the filter with three 5-ml
quantities of dichloromethane. Dilute the combined filtrate and washings to 100 ml with dichloro-
methane and to 50 ml of the solution add 25 ml of periodic acetic acid solution, shaking cautiously.
Allow to stand at 25 to 30 for 30 minutes and add 100 ml of water and 12 ml of potassium iodide
solution. Titrate the liberated iodine with 0.1M sodium thiosulphate VS using 1 ml of starch solution as
indicator. Repeat the determination using 50 ml of dichloromethane in place of the 50 ml of the
solution of the substance being examined. The difference between the titrations represents the
amount of sodium thiosulphate required. Each ml of 0.1M sodium thiosulphate VS is equivalent to
17.9 mg of 1-monoacylglycerols, calculated as C
21
H
42
O
4
. The quantity of 0.1M sodium thiosulphate
VS used in the assay is not less than 85% of the quantity of sodium thiosulphate used in the blank
assay.
For soap Add 10 g to a mixture of 60 ml of acetone and 0.15 ml of a 0.5% w/v solution of bromo-
phenol blue in a mixture of 20 ml of ethanol (20%) and 80 ml of water, the solvent having been
previously neutralised with 0.1M hydrochloric acid VS or 0.1M sodium hydroxide VS. Warm gently on a
water bath until solution is complete and titrate with 0.1M hydrochloric acid VS until the blue colour is
discharged. Allow to stand for 20 minutes, warm until any solidified matter has redissolved and, if the
blue colour reappears, continue the titration. Each ml of 0.1M hydrochloric acid VS is equivalent to
30.45 mg of C
18
H
33
NaO
2
.
19-4
Glyceryl Trinitrate Solution
O
2
N
O O
NO
2
O
NO
2
Glyceryl Trinitrate Solution complies with the requirements of the 3rd edition of the European Pharmacopoeia
When concentrated glyceryl trinitrate solution is demanded, the intention of the purchaser with respect
to the strength expressed in % w/v or g/l should be ascertained.
Preparations
Glyceryl Trinitrate Sublingual Spray
Glyceryl Tinitrate Tablets
Glyceryl Trinitrate Transdermal Patches
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Glyceryl trinitrate solution is an ethanolic solution of glyceryl trinitrate. It contains not less than
9.85 g/l and not more than 101.0 g/l of propane-1,2,3-triol trinitrate and not less than 98.5 per cent
and not more than 101.0 per cent of the declared content of glyceryl trinitrate stated on the label.
CHARACTERS
A clear, colourless or slightly yellow solution, miscible with acetone and with ethanol. Pure glyceryl
trinitrate is a colourless liquid, freely soluble in ethanol, miscible with acetone, not miscible with water.
IDENTIFICATION
Upon diluting glyceryl trinitrate, care should be taken to always use absolute ethanol, otherwise droplets of pure
glyceryl trinitrate may precipitate from the solution.
After examination, the residues and the solutions obtained in both the identification and the test sections should
be heated on a water-bath for 5 min with dilute sodium hydroxide solution R.
A. Place 50 l of a solution diluted, if necessary, with ethanol R, to contain 10 g/l of glyceryl trinitrate,
on a disc of potassium bromide R and evaporate the solvent in a vacuum. Examine by infrared
absorption spectrophotometry (2.2.24), comparing with the Ph. Eur. reference spectrum of glyceryl
trinitrate.
Test solution. Dilute a quantity of the substance to be examined corresponding to 50 mg of glyceryl
trinitrate to 100 ml with acetone R.
Reference solution. Dilute 0.05 ml of glyceryl trinitrate solution CRS to 1 ml with acetone R.
of ethyl acetate R and 80 volumes of toluene R. Allow the plate to dry in air. Spray with freshly prepared
potassium iodide and starch solution R. Expose the plate to ultraviolet light at 254 nm for 15 min.
Examine in daylight. The principal spot in the chromatogram obtained with the test solution is similar
in position, colour and size to the principal spot in the chromatogram obtained with the reference
solution.
C. To a quantity of the substance to be examined corresponding to 10 mg of glyceryl trinitrate, add
10 ml of peroxide-free ether R. Evaporate to dryness in a stream of nitrogen at a temperature not
exceeding 40C. The residue obtained gives the reaction of nitrates (2.3.1).
D. It complies with the limits for the assay.
TESTS
Upon diluting glyceryl trinitrate, care should be taken to always use absolute ethanol, otherwise droplets of pure
glyceryl trinitrate may precipitate from the solution.
After examination, the residues and the solutions obtained in both the identification and the test sections should
be heated on a water-bath for 5 min with dilute sodium hydroxide solution R.
Appearance of solution If necessary dilute the solution to be examined to a concentration of 10 g/l
with ethanol R. The solution is not more intensely coloured than reference solution Y
7
(Method II,
2.2.2).
19-5
Inorganic nitrates Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R.
Test solution. If necessary dilute the solution to be examined to a concentration of 10 g/l with
ethanol R.
Reference solution. Dissolve 5 mg of potassium nitrate R in 1 ml of water R and dilute to 100 ml with
alcohol R.
of glacial acetic acid R, 30 volumes of acetone R and 60 volumes of toluene R. Thoroughly dry the plate
in a current of air until the acetic acid is completely removed. Spray intensively with freshly prepared
potassium iodide and starch solution R. Expose the plate to ultraviolet light at 254 nm for 15 min.
Examine in daylight. Any spot corresponding to the nitrate ion in the chromatogram obtained with
the test solution is not more intense than the spot in the chromatogram obtained with the reference
solution (0.5 per cent of the content of glyceryl trinitrate calculated as potassium nitrate).
Test solution. Dissolve a quantity of the substance to be examined corresponding to 2 mg of glyceryl
trinitrate in the mobile phase and dilute to 20.0 ml with the mobile phase.
Reference solution (a). Dissolve 0.10 g of glyceryl trinitrate solution CRS and a quantity of diluted
pentaerythrityl tetranitrate CRS equivalent to 1.0 mg in the mobile phase and dilute to 100.0 ml with
the mobile phase. Sonicate and filter if necessary.
as mobile phase at a flow rate of 1 ml/min a mixture of equal volumes of acetonitrile R and
water R,
Inject 20 l of reference solution (a) and (b). Adjust the sensitivity of the system so that the height
of the principal peak in the chromatogram obtained with reference solution (b) is at least 70 per cent
the full scale of the recorder. The test is not valid unless, in the chromatogram obtained with
reference solution (a), the resolution between the peaks corresponding to glyceryl trinitrate and
pentaerythrityl tetranitrate is at least 2.0.
Inject 20 l of the test solution. Continue the chromatography for three times the retention time of
obtained with reference solution (b) (1 per cent calculated as glyceryl trinitrate); the sum of the areas
of all the peaks apart from the principal peak is not greater than three times the area of the principal
peak in the chromatogram obtained with reference solution (b) (3 per cent calculated as glyceryl
trinitrate). Disregard any peak with an area less than 0.1 times the area of the principal peak in the
ASSAY
To a quantity of the substance to be examined corresponding to 60.0 mg of glyceryl trinitrate add
80 ml of pyridine R and titrate with 0.1M tetrabutylammonium hydroxide determining the end-point
3
H
5
N
3
O
9
.
STORAGE
Store diluted solutions (10 g/l) protected from light, at a temperature of 2C to 15C. Store more
concentrated solutions protected from light, at a temperature of 15C to 20C.
LABELLING
The label states the declared content of glyceryl trinitrate.
IMPURITIES
A. inorganic nitrates,
R
1
O O
R
3
O
R
2
B. R
1
= NO
2
, R
2
= H, R
3
= H: 1-nitrate of propane-1,2,3-triol,
C. R
1
= H, R
2
= NO
2
, R
3
= H: 2-nitrate of propane-1,2,3-triol,
D. R
1
= NO
2
, R
2
= NO
2
, R
3
= H: 1,2-dinitrate of propane-1,2,3-triol
E. R
1
= NO
2
, R
2
= H, R
3
= NO
2
: 1,3-dinitrate of propane-1,2,3-triol.
__________________________________________________________________________________________________________ Ph Eur
19-6
Glycine
1/01
H
2
N COOH
H H
C
2
H
5
NO
2
75.1 56-40-6
Glycine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0614]. These
Action and use Amino acid used as bladder irrigation during surgery.
Preparation
Glycine Irrigation Solution
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Glycine contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of
2-aminoethanoic acid, calculated with reference to the dried substance.
PRODUCTION
When glycine is produced by a process involving fermentation steps, it complies with the require-
ments of the monograph on Products of fermentation (1468).
CHARACTERS
A white, crystalline powder, freely soluble in water, very slightly soluble in alcohol.
IDENTIFICATION
obtained with glycine CRS. Examine the substances as discs prepared using about 1 mg of the
substance to be examined for 0.4 g of potassium bromide R. If the spectra obtained in the solid state
minimum quantity of alcohol (60 per cent V/V) R, evaporate to dryness and record new spectra using
the residues.
same solvent.
Reference solution. Dissolve 25 mg of glycine CRS in water R and dilute to 10 ml with the same solvent.
of concentrated ammonia R and 70 volumes of propanol R. Dry the plate at 100C to 105C for
10 min. Spray with a 2 g/l solution of ninhydrin R in a mixture of 5 volumes of dilute acetic acid R and
95 volumes of butanol R. Heat the plate at 100C to 105C for 2 min. The principal spot in the
C. Dissolve 50 mg in 5 ml of water R, add 1 ml of strong sodium hypochlorite solution R and boil for
2 min. Add 1 ml of hydrochloric acid R and boil for 4 min to 5 min. Add 2 ml of hydrochloric acid R
and 1 ml of a 20 g/l solution of resorcinol R, boil for 1 min and cool. Add 10 ml of water R and mix.
To 5 ml of the solution add 6 ml of dilute sodium hydroxide solution R. The solution is violet with a
greenish-yellow fluorescence. After a few minutes, the colour becomes orange then yellow and an
intense fluorescence remains.
TESTS
Solution S Dissolve 5 g in carbon dioxide-free water R and dilute to 50 ml with the same solvent.
solution Y
7
(Method II, 2.2.2).
solution complies with the limit test for chlorides (75 ppm).
19-7
100C to 105C for 2 h.
ASSAY
Dissolve 70.0 mg in 3 ml of anhydrous formic acid R and add 30 ml of glacial acetic acid R.
Immediately after dissolution, titrate with 0.1M perchloric acid, determining the end-point potentio-
2
H
5
NO
2
.
__________________________________________________________________________________________________________ Ph Eur
19-8
Gonadorelin Acetate
revised 1/01
5-oxoProHisTrpSerTyrGlyLeuArgProGlyNH
2
,CH
3
COOH
C
55
H
75
N
17
O
13
,xC
2
H
4
O
2
1242 33515-09-2
Gonadorelin Acetate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0827].
Action and use Gonadotrophin-releasing hormone.
Preparation
Gonadorelin Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Gonadorelin acetate is the acetate form of a hypothalamic peptide that stimulates the release of
follicle-stimulating hormone and luteinising hormone from the pituitary gland. It contains not less
than 95.0 per cent and not more than the equivalent of 102.0 per cent of the peptide C
55
H
75
N
17
O
13
,
calculated with reference to the anhydrous, acetic acid-free substance. It is obtained by chemical
synthesis.
CHARACTERS
A white or slightly yellowish powder, soluble in water and in a 1 per cent V/V solution of glacial
acetic acid, sparingly soluble in methanol.
IDENTIFICATION
A. Examine the chromatograms obtained in the assay. The retention time and size of the principal
peak in the chromatogram obtained with the test solution are approximately the same as those of the
Use the test solution and reference solution (a) prepared under Assay.
volumes of glacial acetic acid R, 14 volumes of water R, 45 volumes of methanol R and 60 volumes of
methylene chloride R. Allow the plate to dry in air for 5 min. At the bottom of a chromatography tank,
place an evaporating dish containing a mixture of 10 ml of a 50 g/l solution of potassium permangan-
ate R and 3 ml of hydrochloric acid R, close the tank and allow to stand. Place the dried plate in the
tank and close the tank. Leave the plate in contact with the chlorine vapour for 2 min. Withdraw the
plate and place it in a current of cold air until the excess of chlorine is removed and an area of coating
below the points of application no longer gives a blue colour with 0.05 ml of potassium iodide and
starch solution R. Spray with potassium iodide and starch solution R. The principal spot in the chromato-
gram obtained with the test solution corresponds in position and size to the principal spot in the
TESTS
Appearance of solution A 10 g/l solution is clear (2.2.1) and not more intensely coloured than
5
(Method II, 2.2.2).
Specific optical rotation (2.2.7). Dissolve 10.0 mg in 1.0 ml of a 1 per cent V/V solution of glacial
acetic acid R. The specific optical rotation is 54 to 66, calculated on the basis of the peptide
content as determined in the assay.
The absorbance, determined at the maximum at 278 nm, corrected to a 10 mg/100 ml solution on
the basis of the peptide content determined in the assay, is 0.55 to 0.61.
acids:
19-9
acids, taking one-seventh of the sum of the number of moles of histidine, glutamic acid, leucine,
proline, glycine and arginine as equal to one. The values fall within the following limits: serine 0.7 to
1.05; glutamic acid 0.95 to 1.05; proline 0.95 to 1.05; glycine 1.9 to 2.1; leucine 0.9 to 1.1; tyrosine
0.7 to 1.05; histidine 0.95 to 1.05 and arginine 0.95 to 1.05. Lysine and isoleucine are absent; not
more than traces of other amino acids are present, with the exception of tryptophan.
gonadorelin. In the chromatogram obtained with the test solution: the area of any peak apart from
obtained with reference solution (b) (2 per cent); the sum of the areas of the peaks, apart from the
principal peak, is not greater than 5 times the area of the principal peak in the chromatogram
obtained with reference solution (b) (5 per cent). Disregard any peak with an area less than 0.05
times that of the principal peak in the chromatogram obtained with reference solution (b) (0.05 per
cent).
Acetic acid (2.5.34).4.0 per cent to 7.5 per cent.
mination of water.
70 I.U. of endotoxin per milligram of gonadorelin acetate.
ASSAY
the same solvent.
Reference solution (a). Dissolve the contents of a vial of gonadorelin CRS in water R to obtain a concen-
tration of 0.5 mg/ml.
Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with water R.
Reference solution (c). Dissolve 2.5 mg of the substance to be examined in 1 ml of 0.1M hydrochloric
acid and heat in a water-bath at 65C for 4 h. Add 1 ml of 0.1M sodium hydroxide and dilute to 5.0 ml
with water R.
volumes of a 1.18 per cent V/V solution of phosphoric acid R (adjusted to pH 2.3 with
triethylamine R),
Inject 20 l of reference solution (c). The test is not valid unless the resolution between the first
and second peaks is at least 2.0.
Inject 20 l of the test solution and 20 l of reference solution (a).
Calculate the content of gonadorelin (C
55
H
75
N
17
O
13
) from the peak areas in the chromatograms
obtained with the test solution and reference solution (a) and the declared content of C
55
H
75
N
17
O
13
in gonadorelin CRS.
STORAGE
Store in an airtight container, protected from light at a temperature of 2C to 8C. If the substance is
19-10
sterile, store in a sterile, airtight, tamper-proof container.
LABELLING
The label states:
__________________________________________________________________________________________________________ Ph Eur
19-11
Gonadorelin Hydrochloride
5-oxoProHisTrpSerTyrGlyLeuArgProGlyNH
2
,HCl
C
55
H
75
N
17
O
13,
HCl 1218.5 33515-09-2
Definition Gonadorelin Hydrochloride is the chloride form of a hypothalamic peptide that
stimulates the release of follicle stimulating hormone and luteinising hormone from the pituitary
gland. It contains not less than 88.0% and not more than 98.0% of the peptide C
55
H
75
N
17
O
13
,
calculated with reference to the anhydrous substance. It is obtained by synthesis.
Characteristics A white or slightly yellowish white powder.
Soluble in water and in a 1% v/v solution of glacial acetic acid; sparingly soluble in methanol.
Identification
A. In tests A and B for Related peptides, the principal spot in the chromatogram obtained with
solution (2) is similar in position and size to that in the chromatogram obtained with solution (4).
B. In the Assay, the position of the principal peak in the chromatogram obtained with solution (1)
corresponds to that of the principal peak in the chromatogram obtained with solution (2).
Clarity and colour of solution A 1.0% w/v solution is clear, Appendix IV A, and not more intensely
5
Light absorption Absorbance of a 0.01% w/v solution at the maximum at 278 nm, 0.55 to 0.59,
Appendix II B, calculated with reference to the peptide content as determined in the Assay.
Specific optical rotation In a 1% w/v solution containing 1% v/v of glacial acetic acid, 54 to 63,
calculated with reference to the peptide content as determined in the Assay, Appendix V F.
Chloride 0.83 mg dissolved in 15 ml of water complies with the limit test for chlorides, Appendix VII
(6%).
Amino acids Operate an amino acid analyser according to the manufacturers instructions.
Standardise the apparatus either with the calibration mixture supplied by the manufacturer or with a
acids:
lysine threonine methionine
histidine serine isoleucine
arginine proline leucine
aspartic acid alanine tyrosine
glutamic acid valine phenylalanine
plus half the equimolar amount of L-cystine.
Dissolve 30 mg of DL-norleucine (internal standard) in a mixture of equal volumes of hydrochloric
acid and water and dilute to 100 ml with the same mixture of solvents. Place 1.0 mg of the substance
being examined in a rigorously cleaned hard-glass tube (10 cm 6 mm), add an accurately measured
volume of the internal standard solution containing an amount of DL-norleucine corresponding to
about half the expected number of moles of gonadorelin, immerse the tube in a freezing mixture at
5, evacuate to a pressure not exceeding 0.133 kPa and seal. Heat for 16 hours at 110 to 115,
cool, open the tube, transfer the contents to a 10-ml flask with the aid of five 0.2-ml quantities of
water and evaporate to dryness over potassium hydroxide at a pressure of 2 kPa. Take up the residue in
water and repeat the evaporation; repeat these operations. Dissolve the residue in a suitable buffer
solution (pH 2.2) and dilute to a suitable volume with the same buffer solution. Apply an aliquot of
the solution to the amino acid analyser. Choose conditions such that the peak given by the amino
acid present in the largest amount produces almost maximum deflection on the chart paper.
acids taking one seventh of the sum of the number of moles of histidine, glutamic acid, leucine,
proline, glycine and arginine as equal to one. The values fall within the following limits: serine 0.7 to
1.05; glutamic acid 0.95 to 1.05; proline 0.95 to 1.05; glycine 1.9 to 2.1; leucine 0.9 to 1.1; tyrosine
0.7 to 1.05; histidine 0.95 to 1.05; arginine 0.95 to 1.05. Lysine and isoleucine are absent; not more
than traces of other amino acids are present.
The determination is not valid unless the number of moles of norleucine recovered, corrected for
the volume of test solution applied, is within t 5 per cent of the amount taken for hydrolysis.
Related peptides
A. Carry out the method for thin-layer chromatography, Appendix III A, using silica gel G as the
coating substance and a mixture of 6 volumes of glacial acetic acid, 14 volumes of water, 45 volumes
of methanol and 60 volumes of chloroform as the mobile phase. Apply separately to the plate 10 l of
each of four solutions in water containing (1) 1.0% w/v of the substance being examined, (2) 0.10%
w/v of the substance being examined, (3) 0.020% w/v of the substance being examined and (4)
0.10% w/v of gonadorelin EPCRS. After removal of the plate, allow it to dry in air for 5 minutes. Place
19-12
the plate in a closed tank of chlorine gas, prepared by the addition of 3 ml of hydrochloric acid to
10 ml of a 5% w/v solution of potassium permanganate in an evaporating dish placed at the bottom of
the tank, and allow to stand for 2 minutes. Dry in a current of cold air until a sprayed area of the
plate below the line of application does not give a blue colour with 0.05 ml of potassium iodide and
starch solution; avoid prolonged exposure to air. Spray the plate with a potassium iodide and starch
solution. Any secondary spot in the chromatogram obtained with solution (1) is not more intense than
the spot in the chromatogram obtained with solution (3) (2%).
B. Complies with test A but using a mixture of 20 volumes of 13.5M ammonia, 45 volumes of
methanol and 60 volumes of chloroform as the mobile phase.
Water Not more than 7.0% w/w when determined by the following method. Use dry glassware
throughout; siliconised glassware may be used. Carry out the method for gas chromatography,
Appendix III B, using the following solutions. For solution (1) dilute 50 l of anhydrous methanol
(internal standard) with sufficient propan-2-ol R1 to produce 100 ml. For solution (2) dissolve 4 mg
of the substance being examined in 1 ml of propan-2-ol R1. For solution (3) dissolve 4 mg of the
substance being examined in 1 ml of solution (1). For solution (4) add 10 l of water to 50 l of
solution (1).
The chromatographic procedure may be carried out using (a) a stainless steel column (1 m
2 mm) packed with porous polymer beads (60 to 80 mesh) (Chromosorb 102 is suitable) and
maintained at 120, (b) helium as the carrier gas and (c) a thermal conductivity detector maintained
at 150.
From the chromatograms obtained and taking into account any water detectable in solution (1),
calculate the percentage w/w of water taking 0.9972 g as its weight per ml at 20.
Assay Carry out the method for liquid chromatography, Appendix III D, using two solutions in a
mixture of 85 volumes of a 1% w/v solution of orthophosphoric acid adjusted to pH 3.0 with
triethylamine and 15 volumes of acetonitrile containing (1) 0.01% w/v of the substance being examined
and (2) 0.01% w/v of gonadorelin EPCRS.
mobile phase with a flow rate of 1 ml per minute, a mixture of a 1% w/v solution of orthophosphoric
acid adjusted to pH 3.0 with triethylamine and acetonitrile, the mixture being adjusted so that the
retention time of gonadorelin is about 10 minutes (a mixture of 75 volumes of a 1% w/v solution of
orthophosphoric acid adjusted to pH 3.0 with triethylamine and 25 volumes of acetonitrile is usually
suitable) and (c) a detection wavelength of 220 nm.
The assay is not valid unless the column efficiency is at least 20,000 theoretical plates per metre.
Calculate the content of the peptide, C
55
H
75
N
17
O
13
, using the declared content of C
55
H
75
N
17
O
13
in gonadorelin EPCRS.
Storage Gonadorelin Hydrochloride should be kept in a well-closed container, protected from light
and moisture and stored at a temperature of 2 to 8.
Labelling The label states (1) the weight of the peptide in the container; (2) the date after which the
material is not intended to be used; (3) the conditions under which it should be stored.
Action and use Gonadotrophin-releasing hormone.
Preparation
Gonadorelin Injection
19-13
Gramicidin
corrected 1/01
1405-97-6
Gramicidin complies with the requirements of the 3rd edition of the European Pharmacopoeia [0907]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Gramicidin is a mixture consisting mainly of three pairs of linear polypeptides, usually obtained by
extraction from tyrothricin, the complex isolated from the fermentation broth of Bacillus brevis
Dubos. The main component of gramicidin is the L-valine congener of the L-tryptophan-containing
pair (gramicidin A1). The potency is not less than 900 I.U. per milligram, calculated with reference
CHARACTERS
A white or almost white, crystalline powder, slightly hygroscopic, practically insoluble in water,
sparingly soluble in alcohol, practically insoluble in cyclohexane.
IDENTIFICATION
A. Dissolve 0.100 g in alcohol R and dilute to 100.0 ml with the same solvent. Dilute 5.0 ml of this
shows two absorption maxima, at 282 nm and 290 nm, a shoulder at about 275 nm and an
absorption minimum at 247 nm. The specific absorbance at the maximum at 282 nm is 105 to 125.
B. Examine by thin-layer chromatography (2.2.27), using a plate coated with a suitable silica gel.
Test solution. Dissolve 5 mg of the substance to be examined in 3.0 ml of alcohol R.
Reference solution (a). Dissolve 5 mg of gramicidin CRS in 3.0 ml of alcohol R.
Reference solution (b). Dissolve 5 mg of tyrothricin CRS in 3.0 ml of alcohol R.
Apply to the plate 0.5 l of each solution. Develop over a path of 5 cm using a mixture of 2.5 volumes
of methanol R, 7.5 volumes of butanol R, 12 volumes of water R, 20 volumes of glacial acetic acid R and
40 volumes of butyl acetate R. Dry the plate for 15 min at 125C and dip it into
dimethylaminobenzaldehyde solution R2. Heat the plate at 90C until the spots appear. The principal
spot or group of principal spots in the chromatogram obtained with the test solution is similar in
position, colour and size to the principal spot or group of principal spots in the chromatogram
obtained with reference solution (a) and to the spot or group of spots with the highest R
f
value in the
chromatogram obtained with reference solution (b). The test is not valid unless the chromatogram
obtained with reference solution (b) shows two clearly separated spots or two clearly separated groups
of spots.
TESTS
Absorbance Dissolve 0.100 g in alcohol R and dilute to 100.0 ml with the same solvent. Dilute
5.0 ml of this solution to 100.0 ml with alcohol R. Prepare a similar solution of gramicidin CRS.
Examine both solutions between 240 nm and 320 nm (2.2.25) and measure the absorbances at the
maximum at 282 nm and at the minimum at 247 nm. Calculate for each solution the difference in
absorbance between the maximum and the minimum. Calculated with reference to the dried
substance, the difference obtained for the product to be examined does not differ by more than
4.0 per cent from the difference calculated for gramicidin CRS.
ASSAY
Carry out the microbiological assay of antibiotics (2.7.2), using the turbidimetric method. Use
gramicidin CRS as the reference substance.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
19-14
Griseofulvin
O
MeO
Cl
MeO
H
MeO O
Me
O
C
17
H
17
ClO
6
352.8 126-07-8
Griseofulvin complies with the requirements of the 3rd edition of the European Pharmacopoeia [0182]. These
Preparation
Griseofulvin Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Griseofulvin is (1S,6R)-7-chloro-2,4,6-trimethoxy-6-methylspiro[benzofuran-2(3H),1-
[2]cyclohexene]-3,4-dione, a substance produced by the growth of certain strains of Penicillium
griseofulvum or obtained by any other means. It contains not less than 97.0 per cent and not more
than the equivalent of 102.0 per cent of C
17
H
17
ClO
6
substance.
CHARACTERS
A white or yellowish-white, microfine powder, the particles of which are generally up to 5 m in
maximum dimension although larger particles which may occasionally exceed 30 m may be present,
tasteless, practically insoluble in water, freely soluble in dimethylformamide and in tetrachloroethane,
slightly soluble in ethanol and in methanol.
IDENTIFICATION
obtained with griseofulvin CRS.
B. Dissolve about 5 mg in 1 ml of sulphuric acid R and add about 5 mg of powdered potassium
dichromate R. A wine-red colour develops.
TESTS
Appearance of solution Dissolve 0.75 g in dimethylformamide R and dilute to 10 ml with the same
4
(Method II, 2.2.2).
Acidity Suspend 0.25 g in 20 ml of alcohol R and add 0.1 ml of phenolphthalein solution R. Not more
than 1.0 ml of 0.02M sodium hydroxide is required to change the colour of the indicator.
Specific optical rotation (2.2.7). Dissolve 0.250 g in dimethylformamide R and dilute to 25.0 ml
with the same solvent. The specific optical rotation is +354 to +364, calculated with reference to
Related substances Examine by gas chromatography (2.2.28), using diphenylanthracene R as the
internal standard.
Internal standard solution. Dissolve 0.2 g of diphenylanthracene R in acetone R and dilute to 100.0 ml
Test solution (a). Dissolve 0.10 g of the substance to be examined in acetone R and dilute to 10.0 ml
Test solution (b). Dissolve 0.10 g of the substance to be examined in acetone R, add 1.0 ml of the
internal standard solution and dilute to 10.0 ml with acetone R.
Reference solution. Dissolve 5.0 mg of griseofulvin CRS in acetone R, add 1.0 ml of the internal standard
solution and dilute to 10.0 ml with acetone R.
a glass column 1 m long and 4 mm in internal diameter packed with diatomaceous earth for gas
chromatography R impregnated with 1 per cent m/m of polycyanopropylmethylphenylmethyl-
siloxane R,
19-15
nitrogen for chromatography R as the carrier gas at a flow rate of 50 ml to 60 ml per minute,
Maintain the temperature of the column at 250C, that of the injection port at 270C and that of the
detector at 300C. Continue the chromatography for three times the period of time required for the
appearance of the peak corresponding to griseofulvin which is about 11 min. For the chromatogram
obtained with the reference solution, determine the ratio of the area of the peak corresponding to
griseofulvin to the area of the peak corresponding to the internal standard. For the chromatogram
obtained with test solution (b), determine the ratio of the area of the peak corresponding to dechloro-
griseofulvin (distance t
R
about 0.6 times that of griseofulvin) to the area of the peak corresponding to
the internal standard, determine also the ratio of the area of the peak corresponding to
dehydrogriseofulvin (distance t
R
about 1.4 times that of griseofulvin) to the area of the peak
corresponding to the internal standard.
The ratios calculated from the chromatogram obtained with test solution (b) divided by the ratio
calculated from the chromatogram obtained with the reference solution are less than 0.6 for
dechlorogriseofulvin and less than 0.15 for dehydrogriseofulvin.
Substances soluble in light petroleum Shake 1.0 g with 20 ml of light petroleum R. Boil under a
reflux condenser for 10 min. Cool, filter and wash with three quantities, each of 15 ml, of light
petroleum R. Combine the filtrate and washings, evaporate to dryness on a water-bath and dry at 100C
to 105C for 1 h. The residue weighs not more than 2 mg (0.2 per cent).
100C to 105C.
Abnormal toxicity To each of five healthy mice, each weighing 17 g to 22 g, administer orally a
suspension of 0.1 g of the substance to be examined in 0.5 ml to 1 ml of water R. None of the mice dies
within 48 h.
ASSAY
solution to 100.0 ml with ethanol R. Measure the absorbance (2.2.25) at the maximum at 291 nm.
17
H
17
ClO
6
STORAGE
__________________________________________________________________________________________________________ Ph Eur
19-16
Guaifenesin
O OH
OH H
OMe
and enantiomer
C
10
H
14
O
4
198.2 93-14-1
Guaifenesin complies with the requirements of the 3rd edition of the European Pharmacopoeia [0615]. These
When guaiphenesin is prescribed or demanded, Guaifensein shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Guaifenesin contains not less than 98.0 per cent and not more than the equivalent of 101.5 per cent
of (RS)-3-(2-methoxyphenoxy)propane-1,2-diol, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, sparingly soluble in water, soluble in alcohol and slightly
soluble in ether.
IDENTIFICATION
obtained with guaifenesin CRS.
TESTS
Solution S Dissolve 1.0 g in carbon dioxide-free water R, heating gently if necessary, and dilute to
Acidity or alkalinity To 10 ml of solution S add 0.05 ml of phenolphthalein solution R1. Not more
than 0.1 ml of 0.01M sodium hydroxide is required to change the colour of the indicator. To 10 ml of
solution S add 0.15 ml of methyl red solution R. Not more than 0.1 ml of 0.01M hydrochloric acid is
Related substances Examine by thin-layer chromatography (2.2.27), using a plate with a layer
0.75 mm thick of a coating prepared as follows: mix 0.3 g of carbomer R with 240 ml of water R and
allow to stand with moderate stirring for 1 h; adjust to pH 7 by the gradual addition with stirring of
dilute sodium hydroxide solution R and add 30 g of silica gel H R. Allow to dry in air for 16 h, heat at
100C to 105C for 1 h, allow to cool and use immediately.
Reference solution (a). Dissolve 20 mg of guaifenesin CRS in methylene chloride R and dilute to 10 ml
Reference solution (b). Dilute 0.5 ml of test solution (b) to 10 ml with methylene chloride R.
20 volumes of alcohol R and 80 volumes of carbon tetrachloride R. Allow the plate to dry in air and
spray with a mixture of equal volumes of a 10 g/l solution of potassium ferricyanide R, a 200 g/l
solution of ferric chloride R and alcohol R. In the chromatogram obtained with test solution (a), any
spot apart from the principal spot is not more intense than the spot in the chromatogram obtained
19-17
Guaiacol To 10 ml of solution S, add 0.1 ml of a mixture of equal volumes of ferric chloride solution R1
and water R and allow to stand for 5 min. The solution is not more intensely coloured (Method II,
2.2.2) than a mixture of 0.5 ml of blue primary solution, 1.5 ml of yellow primary solution, 3.5 ml of
red primary solution and 4.5 ml of hydrochloric acid (10 g/l HCl) (500 ppm).
Chlorides and monochlorhydrins To 10 ml of solution S add 2 ml of dilute sodium hydroxide
solution R and heat on a water-bath for 5 min. Cool and add 3 ml of dilute nitric acid R. The resulting
solution complies with the limit test for chlorides (2.4.4) (250 ppm).
Heavy metals (2.4.8). Dissolve 2.0 g in a mixture of 1 volume of water R and 9 volumes of alcohol R
and dilute to 25 ml with the same mixture of solvents. 12 ml of the solution complies with limit test B
for heavy metals (25 ppm). Prepare the standard using lead standard solution (2 ppm Pb) prepared by
diluting lead standard solution (100 ppm Pb) R with a mixture of 1 volume of water R and 9 volumes of
alcohol R.
60C for 3 h.
ASSAY
Dissolve 50.0 mg in 10 ml of water R. Add 20.0 ml of sodium periodate solution R and allow the mixture
to stand for 10 min. Add 25.0 ml of arsenite solution R and 1 ml of potassium iodide solution R. Allow to
stand for 10 min. Titrate with 0.05M iodine, using 2 ml of starch solution R as indicator. Carry out a
blank titration.
10
H
14
O
4
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
19-18
Guanethidine Monosulphate
N
N NH
2
N
H
,H
2
SO
4
H
C
10
H
22
N
4
,H
2
SO
4
296.4 645-43-2
Guanethidine Monosulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Adrenergic neurone blocking agent.
Preparation
Guanethidine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Guanethidine monosulphate contains not less than 99.0 per cent and not more than the equivalent
of 101.0 per cent of 1-(2-perhydroazocin-1-ylethyl)guanidine monosulphate, calculated with
CHARACTERS
A colourless, crystalline powder, freely soluble in water, practically insoluble in alcohol and in
ether.
IDENTIFICATION
A. Dissolve about 25 mg in 25 ml of water R, add 20 ml of picric acid solution R and filter. The
precipitate, washed with water R and dried at 100C to 105C, melts (2.2.14) at about 154C.
B. Dissolve about 25 mg in 5 ml of water R. Add 1 ml of strong sodium hydroxide solution R, 1 ml of
-naphthol solution R and, dropwise with shaking, 0.5 ml of strong sodium hypochlorite solution R. A
bright-pink precipitate is formed and becomes violet-red on standing.
C. It gives the reactions of sulphates (2.3.1).
TESTS
Appearance of solution Solution S is not more intensely coloured than reference solution GY
6
(Method II, 2.2.2).
Oxidisable substances In a conical, ground-glass-stoppered flask, dissolve 1.0 g in 25 ml of water R
and add 25 ml of dilute sodium hydroxide solution R. Allow to stand for 10 min and add 1 g of
potassium bromide R and 1 ml of 0.0083M potassium bromate. Acidify with 30 ml of dilute hydrochloric
acid R. Mix and allow to stand in the dark for 5 min. Add 2 g of potassium iodide R and shake. Allow
to stand for 2 min and titrate the liberated iodine with 0.05M sodium thiosulphate, using starch
solution R as indicator. Not less than 0.3 ml of 0.05M sodium thiosulphate is required to decolorise the
solution.
100C to 105C.
ASSAY
Dissolve 0.250 g, warming if necessary, in 30 ml of anhydrous acetic acid R and add 15 ml of acetic
anhydride R. Titrate with 0.1M perchloric acid (2.2.20), using 0.1 ml of malachite green solution R as
indicator, until a yellowish-green colour is obtained.
10
H
24
N
4
O
4
S.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
19-19
Guar
Guar complies with the requirements of the 3rd edition of the European Pharmacopoeia [1218]. These require-
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Guar is obtained by grinding the endosperms of seeds of Cyamopsis tetragonolobus (L.) Taub. It
consists mainly of guar galactomannan.
CHARACTERS
A white or almost white powder, yielding a mucilage of variable viscosity when dissolved in water,
It has the microscopic characters described under Identification test A.
IDENTIFICATION
A. Examined under a microscope in glycerol R, the substance to be examined (125) shows pyriform to
ovoid cells, usually isolated, having very thick walls around a central somewhat elongated lumen with
granular contents, and smaller polyhedral cells, isolated or in clusters, with thinner walls.
B. In a conical flask place 2 g, add rapidly 45 ml of water R and stir vigorously for 30 s. After 5 min
to 10 min a stiff gel forms which does not flow when the flask is inverted.
C. Mix a suspension of 0.1 g in 10 ml of water R with 1 ml of a 10 g/l solution of sodium borate R; the
mixture soon gels.
D. Examine by thin-layer chromatography (2.2.27), using a suitable silica gel as the coating
substance.
Test solution. To 10 mg in a thick-walled, centrifuge test tube add 2 ml of a 100 g/l solution of
trifluoroacetic acid R, shake vigorously to dissolve the forming gel, stopper the test tube and heat the
mixture at 120C for 1 h. Centrifuge the hydrolysate, transfer the clear supernatant liquid carefully
into a 50 ml flask, add 10 ml of water R and evaporate the solution to dryness under reduced
pressure. To the resulting clear film add 0.1 ml of water R and 0.9 ml of methanol R. Centrifuge to
separate the amorphous precipitate. Dilute the supernatant liquid, if necessary, to 1 ml with
methanol R.
Reference solution. Dissolve 10 mg of galactose R, 10 mg of mannose R in 2 ml of water R and dilute to
mixture of 15 volumes of water R and 85 volumes of acetonitrile R. Spray with aminohippuric acid
reagent R and dry the plate at 120C for 5 min. The chromatogram obtained with the reference
solution shows in the lower part, two clearly separated brownish zones (galactose and mannose in
order of increasing R
f
value). The chromatogram obtained with the test solution shows two zones
corresponding to galactose and mannose.
TESTS
Tragacanth, sterculia gums, agar, alginates, carrageenan. To a small amount of the substance
to be examined add 0.2 ml of freshly prepared ruthenium red solution R. Examined under a
microscope the cell walls do not stain red.
Protein. Not more than 8.0 per cent. Carry out the determination of nitrogen by the method of
sulphuric acid digestion (2.5.9) using 0.170 g. Multiply the result by 6.25.
Apparent viscosity. Moisten 1.00 g calculated with reference to the dried substance with 2.5 ml of
2-propanol R. While stirring, dilute to 100.0 ml with water R. After 1 h, determine the viscosity
(2.2.10) using a rotating viscometer at 20C and a shear rate of 100 s
1
. The apparent viscosity is not
less than 85 per cent and not more than 115 per cent of the value stated on the label.
4
micro-
organisms per gram, determined by plate count. It complies with the tests for Escherichia coli and for
STORAGE
LABELLING
The label states the apparent viscosity in millipascal seconds for a 10 g/l solution.
______________________________________________________________________________________________________________ Ph Eur
19-20
Guar Galactomannan
Guar Galactomannan complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Guar galactomannan is obtained from the seeds of Cyamopsis tetragonolobus (L.) Taub. by grinding of
the endosperms and subsequent partial hydrolysis. The main components are polysaccharides
composed of D-galactose and D-mannose at molecular ratios of 1:1.4 to 1:2. The molecules consist of
a linear main chain of -(14)-glycosidically linked mannopyranoses and single -(16)-
glycosidically linked galacto-pyranoses.
CHARACTERS
A yellowish-white powder, soluble in cold water and in hot water, practically insoluble in organic
solvents.
IDENTIFICATION
A. Mix 5 g of solution S (see Tests) with 0.5 ml of a 10 g/l solution of disodium tetraborate R. A gel
forms within a short time.
B. Heat 20 g of solution S in a water-bath for 10 min. Allow to cool and adjust to the original mass
with water R. The solution does not gel.
Test solution. To 10 mg of the substance to be examined in a thick-walled centrifuge tube add 2 ml of
a 230 g/l solution of trifluoroacetic acid R, shake vigorously to dissolve the forming gel, stopper the test
tube and heat the mixture at 120C for 1 h. Centrifuge the hydrolysate, transfer the clear supernatant
liquid carefully into a 50 ml flask, add 10 ml of water R and evaporate the solution to dryness under
reduced pressure. Take up the residue in 10 ml of water R and evaporate again to dryness under
reduced pressure. To the resulting clear film, which has no odour of acetic acid, add 0.1 ml of
water R and 1 ml of methanol R. Centrifuge to separate the amorphous precipitate. Dilute the super-
natant liquid, if necessary, to 1 ml with methanol R.
Reference solution. Dissolve 10 mg of galactose R and 10 mg of mannose R in 2 ml of water R and dilute
to 10 ml with methanol R.
Apply separately to the plate, as bands 20 mm by 3 mm, 5 l of each solution. Develop over a path of
15 cm using a mixture of 15 volumes of water R and 85 volumes of acetonitrile R. Spray with
aminohippuric acid reagent R and heat at 120C for 5 min. The chromatogram obtained with the
reference solution shows in the lower part two clearly separated brownish zones (galactose and
mannose in order of increasing R
f
value). The chromatogram obtained with the test solution shows
two zones corresponding to galactose and mannose.
TESTS
Solution S Moisten 1.0 g with 2 ml of 2-propanol R. While stirring, dilute with water R to 100 g and
stir until the substance is uniformly dispersed. Allow to stand for at least 1 h. If the apparent viscosity
is below 200 mPas, use 3.0 g of substance instead of 1.0 g.
Apparent viscosity Moisten a quantity of the substance to be examined equivalent to 2.00 g of the
dried substance with 2.5 ml of 2-propanol R and, while stirring, dilute to 100.0 ml with water R. After
1 h, determine the viscosity (2.2.10) using a rotating viscometer at 20C and a shear rate of 100 s
-1
.
The apparent viscosity is not less than 75 per cent and not more than 140 per cent of the value stated
on the label.
Insoluble matter In a 250 ml flask disperse, while stirring, 1.50 g in a mixture of 1.6 ml of sulphuric
acid R and 150 ml of water R and weigh. Immerse the flask in a water-bath and heat under a reflux
condenser for 6 h. Adjust to the original mass with water R. Filter the hot solution through a tared,
sintered-glass filter (160). Rinse the filter with hot water R and dry at 100C to 105C. The residue
Protein Not more than 5.0 per cent. Carry out the determination of nitrogen by sulphuric acid
digestion (2.5.9), using 0.400 g of substance. Multiply the result by 6.25.
Tragacanth, sterculia gum, agar, alginates and carrageenates To a small amount of the
substance to be examined, add 0.2 ml of freshly prepared ruthenium red solution R. Examined under a
microscope, none of the structures are stained red.
19-21
Total ash (2.4.16). Not more than 1.8 per cent, determined on 1.00 g after wetting with 10 ml of
water R.
3
micro-
organisms per gram, determined by plate-count. It complies with the tests for Escherichia coli and for
STORAGE
LABELLING
The label states the apparent viscosity in millipascal seconds for a 20 g/l solution.
__________________________________________________________________________________________________________ Ph Eur
19-22
Halibut-liver Oil
Definition Halibut-liver Oil is the fixed oil extracted from the fresh, or suitably preserved, liver of
the halibut species belonging to the genus Hippoglossus and refined. It contains in 1 g not less than
30,000 IU of vitamin A.
Characteristics A pale to golden yellow liquid; odour and taste, fishy, but not rancid.
Practically insoluble in ethanol (96%); miscible with chloroform, with ether and with petroleum spirit
(boiling range, 40 to 60).
Acid value Not more than 2.0, Appendix X B.
Iodine value Not less than 122 (iodine monochloride method), Appendix X E.
Iodine value of glycerides 112 to 150 when determined by the following method. Isolate the
unsaponifiable matter as described in Appendix X H, but using 1 g of the oil and evaporating the
acetone, drying the residue at 80 in a current of nitrogen and omitting the final titration. Weigh the
residue and immediately determine the iodine value (iodine monochloride method), Appendix X E.
Calculate the iodine value of the glycerides from the expression (100xSy)/ (100S), where x is
the iodine value of the oil, y is the iodine value of the unsaponifiable matter and S is the percentage of
unsaponifiable matter in the oil.
Saponification value Not more than 180, Appendix X G.
Unsaponifiable matter Not less than 7.0%, Appendix X H.
Assay Carry out the procedure as rapidly as possible, avoiding exposure to actinic light and air, oxidising
agents, oxidation catalysts (e.g. copper and iron) and acids.
Carry out Method A (ultraviolet absorption spectrophotometry) but if method A is found not to be
valid, carry out method B (liquid chromatography).
Method A
To 1 g of the oil being examined in a round-bottomed flask, add 3 ml of a freshly prepared 50% w/w
solution of potassium hydroxide and 30 ml of ethanol, boil under a reflux condenser in a current of
nitrogen for 30 minutes and cool rapidly. Add 30 ml of water and extract with four 50-ml quantities of
ether and discard the aqueous layer after complete separation of the final extract. Wash the combined
ethereal layers with four 50-ml quantities of water and evaporate to dryness under a gentle current of
nitrogen at a temperature not exceeding 30 or in a rotary evaporator at a temperature not exceeding
30 under reduced pressure. Dissolve the residue in sufficient propan-2-ol R1 to give an expected
concentration of vitamin A equivalent to 10 to 15 IU per ml. Measure the absorbances of the solution
at 300 nm, 310 nm, 325 nm and 334 nm and at the wavelength of maximum absorption, Appendix
II B, in a suitable spectrophotometer in 1-cm specially matched cells, using propan-2-ol R1 in the
reference cell.
Calculate the content of vitamin A, as all-trans-retinol, in IU per gram from the expression:
V
m
A
100
1830
325
whereA
325
= absorbance at 325 nm,
m=weight of the oil being examined in grams,
V=total volume of solution containing 10 to 15 IU of vitamin A per ml,
325
325,corr
/0.970 where
A
325,corr
A
325, corr
= 6.815A
325
- 2.555A
310
- 4.260A
324
If A
325
325,corr
expression:
V
m
A
100
1830
325,corr
The assay is not valid unless the wavelength of maximum absorption lies between 323 nm and
327 nm and the absorbance at 300 nm relative to that at 325 nm is at most 0.73.
Method B
Carry out the method for liquid chromatography, Appendix III D, using the following solutions.
Prepare solution (1) in the following manner. To 2 g of the oil being examined in a round-bottomed
flask add 5 ml of a freshly prepared 10% w/v solution of ascorbic acid and 10 ml of a freshly prepared
80% w/v solution of potassium hydroxide and 100 ml of ethanol, boil under a reflux condenser on a
water-bath for 15 minutes. Add 100 ml of a 1% w/v solution of sodium chloride and cool. Transfer
19-23
the solution to a 500-ml separating funnel rinsing the round-bottomed flask with about 75 ml of a
1% w/v solution of sodium chloride and then with 150 ml of a mixture of equal volumes of light
petroleum R3 and ether R. Shake for 1 minute and when the layers have separated completely, discard
the lower layer and wash the upper layer with 50 ml of a 3% w/v solution of potassium hydroxide in a
10% v/v solution of ethanol and then with three 50-ml quantities of a 1% w/v solution of sodium
chloride. Filter the upper layer through 5 g of anhydrous sodium sulphate on a fast filter paper into a
250-ml flask suitable for a rotary evaporator. Wash the funnel with 10 ml of fresh extraction mixture,
filter and combine the upper layers. Distil them at a temperature not exceeding 30 under reduced
pressure (water ejector) and fill with nitrogen when evaporation is completed. Alternatively evaporate
the solvent under a gentle current of nitrogen at a temperature not exceeding 30. Dissolve the
residue in propan-2-ol, transfer to a 25-ml graduated flask and dilute to 25 ml with propan-2-ol.
Gentle heating with the aid of ultrasound may be required. (A large fraction of the white residue is
cholesterol.) Solution (2) is a solution of retinyl acetate EPCRS in propan-2-ol R1 containing about
1000 IU per ml of all-trans-retinol. The exact concentration of solution (2) is assessed by ultraviolet
absorption spectrophotometry, Appendix II B. Dilute the solution with propan-2-ol R1 to a presumed
concentration of 10 to 15 IU per ml and measure the absorbance at 326 nm in matched 1-cm cells
using propan-2-ol R1 in the reference cell. Calculate the content of vitamin A in International Units
per millilitre of solution (2) from the following expression, taking into account the assigned content
of retinyl acetate EPCRS:

1
2
326
100
1900
V
V
A
where
A
326
V
2
V
1
1900=conversion factor for the specific absorbance of retinyl acetate EPCRS in International
Units.
Prepare solution (3) in the same manner as solution (1) but using 2 ml of solution (2) in place of the
oil being examined. The exact concentration of solution (3) is assessed by ultraviolet absorption
spectrophotometry, Appendix II B. Dilute solution (3) with propan-2-ol R1 to a presumed
concentration of 10 to 15 IU per ml of all-trans-retinol and measure the absorbance at 325 nm in
matched 1-cm cells using propan-2-ol R1 in the reference cell. Calculate the content of all-trans-
retinol in International Units per millilitre of solution (3) from the expression:

3
4
325
100
1830
V
V
A
where
A
325
V
3
V
4
4.6 mm) packed with octadecylsilyl silica gel for chromatography (5 m to 10 m), (b) as mobile phase
at a flow rate of 1 ml per minute a mixture of 3 volumes of water and 97 volumes of methanol and (c)
Inject in triplicate 10 l of solutions (1) and (3). The retention time of all-trans-retinol is 4 to 6
minutes.
The assay is not valid unless (a) the chromatogram obtained with solution (1) shows a peak
corresponding to that of all-trans-retinol in the chromatogram obtained with solution (3), (b) when
using the method of standard additions to solution (1) there is greater than 95% recovery of the
added retinyl acetate EPCRS and (c) the recovery of all-trans-retinol in solution (3) as assessed by
direct absorption spectrophotometry is greater than 95%.
m A
V C
A
1
2
1

where A
1
solution (1),
A
2
solution (3),
C=concentration of retinyl acetate EPCRS in solution (2) as assessed before the saponification
in IU per ml (= 1000 IU per ml),
V=volume of solution (2) treated (2 ml),
m=weight of the oil being examined in solution (1) (2 g).
19-24
Storage Halibut-liver Oil should be kept in a well-filled, well-closed container and protected from
light.
Action and use Source of vitamin A.
Preparation
Halibut-liver Oil Capsules
Halibut-liver Oil may contain up to about 3000 IU of vitamin D activity per g. When a statement is
made of the vitamin D content, this is determined by an appropriate method. The method described
under Cod-liver Oil (Type A) may be suitable.
19-25
Haloperidol
N
F
O
OH
Cl
C
21
H
23
ClFNO
2
375.9 52-86-8
Haloperidol complies with the requirements of the 3rd edition of the European Pharmacopoeia [0616]. These
Preparations
Haloperidol Capsules
Haloperidol Injection
Haloperidol Oral Solution
Strong Haloperidol Oral Solution
Haloperidol Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Haloperidol contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of 4-[4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl]-1-(4-fluorophenyl)butan-1-one, calculated with
CHARACTERS
A white or almost white, powder, practically insoluble in water, slightly soluble in alcohol, in
methanol and in methylene chloride.
IDENTIFICATION
B. Examine by infrared spectrophotometry (2.2.24), comparing with the spectrum obtained with
haloperidol CRS. Examine the substances prepared as discs.
coating substance.
the same solvent.
Reference solution (a). Dissolve 10 mg of haloperidol CRS in methanol R and dilute to 10 ml with the
same solvent.
Reference solution (b). Dissolve 10 mg of haloperidol CRS and 10 mg of bromperidol CRS in methanol R
15 cm using a mixture of 10 volumes of tetrahydrofuran R, 45 volumes of methanol R and 45 volumes
of a 58 g/l solution of sodium chloride R. Allow the plate to dry in air and examine in ultraviolet light at
not valid unless the chromatogram obtained with reference solution (b) shows two spots which may,
however, not be completely separated.
D. Dissolve about 10 mg in 5 ml of ethanol R. Add 0.5 ml of dinitrobenzene solution R and 0.5 ml of
2M alcoholic potassium hydroxide solution R. A violet colour is produced and becomes brownish-red
after 20 min.
TESTS
Appearance of solution Dissolve 0.2 g in 20 ml of a 1 per cent V/V solution of lactic acid R. The
7
(Method II,
2.2.2).
19-26
Reference solution (a). Dissolve 5.0 mg of haloperidol CRS and 2.5 mg of bromperidol CRS in
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
eluent
composition
Equilibrate the column with acetonitrile R at a flow rate of 1.5 ml/min for at least 30 min and then
with the initial eluent composition for at least 5 min.
conditions, the retention times are: haloperidol, about 5.5 min and bromperidol, about 6 min. The
test is not valid unless the resolution between the peaks due to haloperidol and bromperidol is at least
3.0. If necessary, adjust the concentration of acetonitrile in the mobile phase or adjust the time
programme for the linear-gradient elution.
the principal peak is not greater than the area of the principal peak in the chromatogram obtained
reference solution (b) (1 per cent). Disregard any peak obtained with the blank and any peak with an
solution (b).
100C to 105C.
Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g using a platinum crucible.
ASSAY
ethyl ketone R. Titrate with 0.1M perchloric acid, using 0.2 ml of naphtholbenzein solution R as indicator.
21
H
23
ClFNO
2
.
STORAGE
Store in an well-closed container, protected from light.
IMPURITIES
F
Ar =
N
Cl
OH R =
19-27
Ar
N
O
OH
A. 1-(4-fluorophenyl)-4-(4-hydroxy-4-phenylpiperidin-1-yl)butan-1-one,
F
O
R
B. 4-[4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl]-1-(2-fluorophenyl)butan-1-one,
O
R
F
Et
C. 4-[4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl]-1-(3-ethyl-4-fluorophenyl)butan-1-one,
O
R
R
D. 4-[4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl]-1-[4-[4-(4-chlorophenyl)-4-hydroxy-
piperidin-1-yl]phenyl]butan-1-one,
Ar
N
O
OH
Cl
E. 4-[4-(4-chlorobiphenyl-4-yl)-4-hydroxypiperidin-1-yl]-1-(4-fluorophenyl)butan-1-one,
Ar
N
O
OH
Cl
F 4-[4-(3-chlorobiphenyl-4-yl)-4-hydroxypiperidin-1-yl]-1-(4-fluorophenyl)butan-1-one.
__________________________________________________________________________________________________________ Ph Eur
19-28
Haloperidol Decanoate
N
F
O
OCO[ CH
2
]
8
CH
3
Cl
C
31
H
41
ClFNO
3
530.1 74050-97-8
Haloperidol Decanoate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Haloperidol decanoate contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of 4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]piperidin-4-yl decanoate,
CHARACTERS
A white or almost white powder, practically insoluble in water, very soluble in alcohol, in methanol
IDENTIFICATION
obtained with haloperidol decanoate CRS. Examine the substances prepared as mulls in liquid
paraffin R.
B. To 0.1 g in a porcelain crucible add 0.5 g of anhydrous sodium carbonate R. Heat over an open
TESTS
solvent. The solution is clear (2.2.1) and not more intensely coloured than reference solution B
5
(Method II, 2.2.2).
Reference solution (a). Dissolve 2.5 mg of bromperidol decanoate CRS and 2.5 mg of haloperidol
decanoate CRS in methanol R and dilute to 50.0 ml with the same solvent.
as mobile phase at a flow rate of 1.5 ml/min the following linear gradient programme:
Mobile phase A. A 27 g/1 solution of tetrabutylammonium hydrogen sulphate R,
Time
(min)
Mobile
phase A
(per cent V/V)
Mobile
phase B
(per cent V/V)
Comment
35 40
4080 6020
switch to initial
eluent composition
19-29
conditions the retention times are: haloperidol decanoate about 24 min and bromperidol decanoate
about 24.5 min. The test is not valid unless the resolution between the peaks due to haloperidol
decanoate and bromperidol decanoate is at least 1.5. If necessary, adjust the gradient or the time
solution (b). In the chromatogram obtained with the test solution: the area of any peak apart from
with reference solution (b) (0.5 per cent); the sum of the areas of all the peaks apart from the
principal peak, is not greater than three times the area of the principal peak in the chromatogram
obtained with the reference solution (b) (1.5 per cent). Disregard any peak due to the blank and any
30C.
ASSAY
31
H
41
ClFNO
3
.
STORAGE
Store in a well-closed container at room temperature below 25C, protected from light.
IMPURITIES
F
N
OCO[CH
2
]
8
CH
3
Cl
r = R =
Ar
N
O
OCO[CH
2
]
8
CH
3
A. 1-[4-(4-fluorophenyl)-4-oxobutyl]-4-phenylpiperidin-4-yl decanoate,
O
F
R
B. 4-(4-chlorophenyl)-1-[4-(2-fluorophenyl)-4-oxobutyl]piperidin-4-yl decanoate,
O
R
Et
C. 4-(4-chlorophenyl)-1-[4-(3-ethyl-4-fluorophenyl)-4-oxobutyl]piperidin-4-yl decanoate,
O
R
N
OH
Cl
D. 4-(4-chlorophenyl)-1-[4-[4-[4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl]phenyl]-4-
oxobutyl]piperidin-4-yl decanoate,
19-30
N
O
OCO[CH
2
]
8
CH
3
Cl
r
E. 4-(4-chlorobiphenyl-4-yl)-1-[4-(4-fluorophenyl)-4-oxobutyl]piperidin-4-yl decanoate,
N
O
OCO[CH
2
]
8
CH
3
Ar
Cl
F. 4-(3-chlorobiphenyl-4-yl)-1-[4-(4-fluorophenyl)-4-oxobutyl]piperidin-4-yl decanoate,
G. 4-[4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl]-1-(4-fluorophenyl)butan-1-one
(haloperidol),
r
N
O
OCO[CH
2
]
6
CH
3
Cl
H. 4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]-piperidin-4-yl octanoate,
r
N
O
OCO[CH
2
]
7
C H
3
Cl
I. 4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]piperidin-4-yl nonanoate,
r
N
O
OCO[CH
2
]
9
CH
3
Cl
J. 4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]piperidin-4-yl undecanoate,
r
N
O
OCO[CH
2
]
10
CH
3
Cl
K. 4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]piperidin-4-yl dodecanoate.
Ar
O
CH
3
L. 1-(4-fluorophenyl)ethanone.
__________________________________________________________________________________________________________ Ph Eur
19-31
Halothane
Cl
F
F
F
Br
H
and enantiomer
C
2
HBrClF
3
197.4 151-67-7
Halothane complies with the requirements of the 3rd edition of the European Pharmacopoeia [0393]. These
Action and use General anaesthetic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Halothane is (RS)-2-bromo-2-chloro-1,1,1-trifluoroethane to which 0.01 per cent m/m of thymol has
been added.
CHARACTERS
A clear, colourless, mobile, heavy, non-flammable liquid, slightly soluble in water, miscible with
ethanol, with ether and with trichloroethylene.
IDENTIFICATION
A. It complies with the test for distillation range (see Tests).
spectrum of halothane. Examine the substance in a 0.1 mm cell.
C. Add 0.1 ml of the substance to be examined to 2 ml of 2-methyl-2-propanol R in a test-tube. Add
1 ml of copper edetate solution R, 0.5 ml of concentrated ammonia R and a mixture of 0.4 ml of strong
hydrogen peroxide solution R and 1.6 ml of water R (solution a). Prepare a blank at the same time
(solution b). Place both tubes in a water-bath at 50C for 15 min, cool and add 0.3 ml of glacial acetic
acid R. To 1 ml of each of solutions (a) and (b) add 0.5 ml of a mixture of equal volumes of freshly
prepared alizarin S solution R and zirconyl nitrate solution R. Solution (a) is yellow and solution (b) is
red.
To 1 ml of each of solutions (a) and (b) add 1 ml of buffer solution pH 5.2 R, 1 ml of phenol red
solution R diluted 1 in 10 with water R and 0.1 ml of chloramine solution R. Solution (a) is bluish-violet
and solution (b) is yellow.
To 2 ml of each of solutions (a) and (b) add 0.5 ml of a mixture of 25 volumes of sulphuric acid R and
75 volumes of water R, 0.5 ml of acetone R and 0.2 ml of a 50 g/l solution of potassium bromate R and
shake. Warm the tubes in a water-bath at 50C for 2 min, cool and add 0.5 ml of a mixture of equal
volumes of nitric acid R and water R and 0.5 ml of silver nitrate solution R2. Solution (a) is opalescent
and a white precipitate is formed after a few minutes; solution (b) remains clear.
TESTS
Acidity or alkalinity To 20 ml add 20 ml of carbon dioxide-free water R, shake for 3 min and allow to
stand. Separate the aqueous layer and add 0.2 ml of bromocresol purple solution R. Not more than
0.1 ml of 0.01M sodium hydroxide or 0.6 ml of 0.01M hydrochloric acid is required to change the colour
of the indicator.
Distillation range (2.2.11). It distils completely between 49.0C and 51.0C, 95 per cent distilling
within a range of 1.0C.
Volatile related substances Examine by gas chromatography (2.2.28), using trichlorotrifluoroethane
CRS as the internal standard.
Test solution (a). Use the substance to be examined.
Test solution (b). Dilute 5.0 ml of trichlorotrifluoroethane CRS to 100.0 ml with the substance to be
examined. Dilute 1.0 ml of the solution to 100.0 ml with the substance to be examined. Dilute
1.0 ml of this solution to 10.0 ml with the substance to be examined.
a column 2.75 m long and 5 mm in internal diameter packed with silanised diatomaceous earth for
19-32
gas chromatography R1 (180 m to 250 m), the first 1.8 m being impregnated with 30 per cent
m/m of macrogol 400 R and the remainder with 30 per cent m/m of dinonyl phthalate R,
maintaining the temperature of the column at 50C. Inject 5 l of test solutions (a) and (b).
In the chromatogram obtained with test solution (b), the sum of the areas of the peaks, apart from the
principal peak and the peak due to the internal standard, is not greater than the area of the peak due
to the internal standard, corrected if necessary for any impurity with the same retention time as the
internal standard (0.005 per cent).
Bromides and chlorides To 10 ml add 20 ml of water R and shake for 3 min. To 5 ml of the
aqueous layer add 5 ml of water R, 0.05 ml of nitric acid R and 0.2 ml of silver nitrate solution R1. The
solution is not more opalescent than a mixture of 5 ml of the aqueous layer and 5 ml of water R.
Bromine and chlorine To 10 ml of the aqueous layer obtained in the test for bromides and
chlorides add 1 ml of potassium iodide and starch solution R. No blue colour is produced.
Thymol Examine by gas chromatography (2.2.28), using menthol R as the internal standard.
Internal standard solution. Dissolve 0.10 g of menthol R in methylene chloride R and dilute to 100.0 ml
Test solution. To 20.0 ml of the substance to be examined add 5.0 ml of the internal standard
solution.
Reference solution. Dissolve 20.0 mg of thymol R in methylene chloride R and dilute to 100.0 ml with the
same solvent. To 20.0 ml, add 5.0 ml of the internal standard solution.
a fused-silica capillary column 15 m long and 0.53 mm in internal diameter coated with a 1.5 m
film of polydimethylsiloxane R,
maintaining the temperature of the column at 150C and that of the injection port at 170C and of
Inject separately 1.0 l of the internal standard solution and of the test and reference solutions. In
the chromatogram obtained with the test solution, the area of the peak due to thymol is not less than
75 per cent and not more than 115 per cent of the area of the corresponding peak in the chromato-
gram obtained with the reference solution (0.008 per cent m/m to 0.012 per cent m/m).
Non-volatile matter Evaporate 50 ml to dryness on a water-bath and dry the residue in an oven at
100C to 105C for 2 h. The residue weighs not more than 1 mg (20 mg/l).
STORAGE
Store in an airtight container, protected from light, at a temperature not exceeding 25C. The choice
of material for the container is made taking into account the particular reactivity of halothane with
certain metals.
IMPURITIES
F
3
C
CF
3
A. 1,1,1,4,4,4-hexafluorobut-2-ene,
F
3
C
CF
3
Cl
F
3
C
Cl
CF
3
and
B. 1,1,1,4,4,4-hexafluoro-2-chlorobut-2-ene (cis and trans),
F
3
C
Cl
CF
3
Cl
F
3
C
CF
3
Cl
Cl
and
C. 1,1,1,4,4,4-hexafluoro-2,3-dichlorobut-2-ene (cis and trans),
F
3
C
Br
CF
3
D. 1,1,1,4,4,4-hexafluoro-2-bromobut-2-ene (trans),
Cl CF
3
E. 2-chloro-1,1,1-trifluoroethane,
19-33
Cl
F
F Cl
Cl F
F. 1,1,2-trichloro-1,2,2-trifluoroethane,
Cl
Br CF
2
G. 1-bromo-1-chloro-2,2-difluoroethylene,
Cl
F
H Cl
F F
H. 2,2-dichloro-1,1,1-trifluoroethane,
Cl
F
Br Cl
F F
I. 1-bromo-1,1-dichloro-2,2,2-trifluoroethane,
Cl
F
Cl F
J. 1,2-dichloro-1,1-difluoroethane.
__________________________________________________________________________________________________________ Ph Eur
19-34
Hamamelis Leaf
Hamamelis Leaf complies with the requirements of the 3rd edition of the European Pharmacopoeia [0909].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hamamelis leaf consists of the whole or cut, dried leaf of Hamamelis virginiana L. It contains not less
than 3 per cent of tannins, expressed as pyrogallol (C
6
H
6
O
3
; M
r
126.1), calculated with reference to
the dried drug.
CHARACTERS
IDENTIFICATION
A. The leaf is green or greenish-brown, often broken, crumpled and compressed into more or less
compact masses. The lamina is broadly ovate to obovate; the base is oblique and asymmetric and the
apex is acute or, rarely, obtuse. The margins of the lamina are roughly crenate or dentate. The
venation is pinnate and prominent on the abaxial surface. Usually, four to six pairs of secondary veins
are attached to the main vein, emerging at an acute angle and curving gently to the marginal points
where there are fine veins often at right angles to the secondary veins.
B. Reduce to a powder (355). The powder is brownish-green. Examine under a microscope using
chloral hydrate solution R. The powder shows fragments of adaxial epidermis with wavy anticlinal
walls; abaxial epidermis with stomata mainly paracytic (2.8.3); star-shaped covering trichomes, either
entire or broken, composed of four to twelve unicellular branches which are united by their bases,
elongated, conical and curved, usually up to 250 m long, thick-walled and with a clearly visible
lumen with contents often brown in colour; fibres are lignified and thick-walled, isolated or in groups,
and they are accompanied by a sheath of prismatic calcium oxalate crystals; small, cylindrical
parenchymatous cells of palisade; irregular-shaped cells of spongy mesophyll; sclereids, frequently
enlarged at one or both ends, 150 m to 180 m long, whole or fragmented; fragments of annular or
spiral vessels; isolated prisms of calcium oxalate.
Test solution. To 1.0 g of the powdered drug (355) add 10 ml of alcohol (60 per cent V/V) R, shake for
15 min and filter.
Reference solution (a). Dissolve 30 mg of tannic acid R in 5 ml of alcohol (60 per cent V/V) R.
Reference solution (b). Dissolve 5 mg of gallic acid R in 5 ml of alcohol (60 per cent V/V) R.
10 volumes of anhydrous formic acid R, 10 volumes of water R and 80 volumes of ethyl formate R. Dry
the plate at 100C to 105C for 10 min and allow to cool. Spray with ferric chloride solution R2 until
bluish-grey zones (phenolic compounds) appear. The chromatogram obtained with the test solution
shows in its lower third a principal zone similar in position to the principal zone in the chromatogram
obtained with reference solution (a) and, in its upper part, a narrow zone similar in position to the
principal zone in the chromatogram obtained with reference solution (b). The chromatogram
obtained with the test solution shows in addition several slightly coloured zones in the central part.
TESTS
Foreign matter (2.8.2). Not more than 7 per cent of stem and not more than 2 per cent of other
foreign matter, determined on 50 g.
ASSAY
Carry out the determination of tannins in herbal drugs (2.8.14). Use 0.750 g of the powdered drug
(180).
STORAGE
__________________________________________________________________________________________________________ Ph Eur
19-35
Hawthorn Berries
Hawthorn Berries complies with the requirements of the 3rd edition of the European Pharmacopoeia [1220].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hawthorn berries consist of the dried false fruits of Crataegus monogyna Jacq., (Lindm.), or Crataegus
laevigata (Poir.) D.C. (synonym: Crataegus oxyacantha L.) or their hybrids or a mixture of these false
fruits. They contain not less than 1.0 per cent of procyanidins, calculated as cyanidin chloride
(C
15
H
11
ClO
6
; M
r
CHARACTERS
The false fruit has a sweet mucilaginous taste.
IDENTIFICATION
A. The false fruit of Crataegus monogyna is obovate to globular. It is generally 6 mm to 10 mm long
and 4 mm to 8 mm wide, reddish-brown to dark red. The surface is pitted or, more rarely,
reticulated. The upper end of the fruit is crowned by the remains of five reflexed sepals surrounding a
small sunken disc with a shallow, raised rim. The remains of the style occur in the centre of the disc
with tufts of stiff, colourless hairs at the base. At the lower end of the fruit a short length of pedicel
or, more frequently, a small pale circular scar where the pedicel was attached. The receptacle is fleshy
and encloses a yellowish-brown, ovoid fruit with a hard, thick wall containing a single, elongated,
pale brown, smooth and shiny seed.
The false fruit of Crataegus laevigata is up to 13 mm long. It contains two to three stony fruits,
ventrally flattened, with short hairs at the top. Frequently, in the centre of the disc of the false fruit
occur the remains of the two styles.
B. Reduce to a powder (355). The powder is greyish-red. Examine under a microscope using chloral
hydrate solution R. The powder shows covering trichomes from inside the disc which are long,
unicellular, frequently bent, tapering to a point, with smooth, much thickened and lignified walls,
parenchymatous receptacle fragments the outer layer with red colouring matter, some cells of the
inner layers containing small cluster crystals of calcium oxalate; occasional fragments including
groups of sclereids and vascular strands with associated files of cells containing prisms of calcium
oxalate; pericarp fragments consisting of large thick-walled sclereids with numerous pits, some of
which are conspicuously branched; a few fragments of the testa having an epidermal layer composed
of hexagonal, mucilaginous cells beneath which is a yellowish-brown pigment layer containing
numerous elongated prisms of calcium oxalate; thin-walled parenchyma of the endosperm and
cotyledons containing aleurone grains and globules of fixed oil.
substance.
Test solution. To 1.0 g of the powdered drug (355) add 10 ml of methanol R and heat in a water bath
at 65C for 5 min. Shake frequently. Allow to cool to room temperature and filter. Dilute the filtrate
to 10 ml with methanol R.
Reference solution. Dissolve 2 mg of chlorogenic acid R, 2 mg of caffeic acid R, 5 mg of hyperoside R and
5 mg of rutin R in 20 ml of methanol R.
Develop over a path of 15 cm using as the mobile phase a mixture of 10 volumes of anhydrous formic
acid R, 10 volumes of water R, 30 volumes of methyl ethyl ketone R and 50 volumes of ethyl acetate R.
Dry the plate at 100C to 105C and spray whilst hot with a 10 g/l solution of diphenylboric acid
aminoethyl ester R in methanol R. Subsequently spray the plate with a 50 g/l solution of macrogol 400 R
in methanol R. Allow the plate to dry in air for 30 min and examine in ultraviolet light at 365 nm. The
chromatogram obtained with the reference solution shows in the lower half, in order of increasing R
f
values, the yellowish-brown fluorescent zone of rutin, the light blue fluorescent zone of chlorogenic
acid and the yellowish-brown fluorescent zone of hyperoside; in the upper third appears the light blue
fluorescent zone of caffeic acid. The chromatogram obtained with the test solution shows three zones
similar in position and fluorescence to the zones due to chlorogenic acid, hyperoside and caffeic acid
in the chromatogram obtained with the reference solution and three weak reddish fluorescent zones,
one corresponding to the rutin zone in the chromatogram obtained with the reference solution and
both of the others located above the hyperoside zone. Below and above the caffeic acid zone some
light blue zones appear.
19-36
TESTS
Foreign matter (2.8.2). Not more than 2 per cent. Not more than 5 per cent of deteriorated false
fruit. It does not contain fruits of other Crataegus species (C. nigra Waldst. et Kit., C. pentagyna
Waldst. et Kit. ex Willd. and C. azarolus L.) which are characterised by the presence of more than
three hard stones.
ASSAY
To 2.50 g of the powdered drug (355) add 30 ml of alcohol (70 per cent V/V) R. Heat under a reflux
condenser for 30 min and filter. Wash the residue with 10.0 ml of alcohol (70 per cent V/V) R. Add to
the filtrate 15.0 ml of hydrochloric acid R1 and 10.0 ml of water R. Heat under a reflux condenser for
80 min. Allow to cool, filter and wash the residue with alcohol (70 per cent V/V) R until the filtrate is
colourless. Dilute the filtrate to 250.0 ml with alcohol (70 per cent V/V) R. Evaporate 50.0 ml of this
solution in a round-bottomed flask to about 3 ml and transfer it to a separating funnel. Rinse the
round-bottomed flask sequentially with 10 ml and 5 ml of water R and transfer to the separating
funnel. Shake the combined solution with three quantities, each of 15 ml, of butanol R. Combine the
organic layers and dilute to 100.0 ml with butanol R. Measure the absorbance (2.2.25) of the solution
at 545 nm.
Calculate the percentage content of procyanidins, as cyanidin chloride, from the expression:
A
m
500
75
i.e. taking the specific absorbance of cyanidin chloride to be 75.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
19-37
Hawthorn Leaf and Flower
corrected 1/01
Hawthorn Leaf and Flower comply with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hawthorn leaf and flower consist of the whole or cut, dried flower bearing branches of Crataegus
monogyna Jacq. Lindm.), C. laevigata (Poiret) D.C. (C. oxyacanthoides Thuill.) or their hybrids or,
more rarely, other European Crataegus species including C. pentagyna Waldst. et Kit. ex Willd., C.
nigra Waldst. et Kit., C. azarolus L. It contains not less than 1.5 per cent of flavonoids, calculated as
hyperoside (C
21
H
20
O
12
; M
r
CHARACTERS
IDENTIFICATION
A. The stems are dark brown, woody, ranging from 1 mm to 2.5 mm in diameter, bearing alternate,
petiolate leaves with small, often deciduous stipules and corymbs of numerous small white flowers.
The leaves are more or less deeply lobed with slightly serrate or almost entire margins; those of C.
laevigata are pinnately lobed or pinnatifid with three, five or seven obtuse lobes, those of C. monogyna
pinnatisect with three or five acute lobes; the adaxial surface is dark green to brownish-green, the
abaxial surface is lighter greyish-green and shows a prominent, dense, reticulate venation. The leaves
of C. laevigata, C. monogyna and C. pentagyna are glabrous or bear only isolated trichomes, those of
C. azarolus and C. nigra are densely pubescent. The flowers have a brownish-green tubular calyx
composed of five free, reflexed sepals, a corolla composed of five free, yellowish-white to brownish,
rounded or broadly ovate and shortly unguiculate petals and numerous stamens. The ovary is fused
to the calyx and consists of one to five carpels, each with a long style and containing a single ovule; in
C. monogyna there is one carpel, in C. laevigata two or three, in C. azarolus two or three, or
sometimes only one, in C. pentagyna five or, rarely, four.
B. Reduce to a powder (355). The powder is yellowish-green. Examine under a microscope using
chloral hydrate solution R. The powder shows unicellular covering trichomes, usually with a thick wall
and wide lumen, almost straight to slightly curved, pitted at the base; fragments of leaf epidermis
with cells which have sinuous to polygonal anticlinal walls and with large anomocytic stomata (2.8.3)
surrounded by four to seven subsidiary cells; parenchymatous cells of the mesophyll containing
calcium-oxalate clusters, usually measuring 10 m to 20 m, those associated with the veins contain-
ing groups of small prism crystals; fragments of petals showing rounded polygonal epidermal cells,
strongly papillose, with thick walls, the cuticle of which clearly shows wavy striations; fragments of
anthers showing endothecium with an arched and regularly thickened margin; fragments of stems
containing collenchymatous cells, bordered pitted vessels and groups of lignified sclerenchymatous
fibres with narrow lumina; numerous spherical to elliptical or triangular pollen grains up to 45 m in
diameter, with three germinal pores and a faintly granular exine.
C. Examine by thin-layer chromatography (2.2.27) using a TLC silica gel plate R.
Test solution. Shake 1.0 g of the powdered drug (355) with 10 ml of methanol R in a water-bath at
65C for 5 min. Cool and filter.
Reference solution. Dissolve 1.0 mg of chlorogenic acid R, 2.5 mg of hyperoside R and 2.5 mg of rutin R
in 10 ml of methanol R.
Apply to the plate, as bands, 20 l of the test solution and 10 l of the reference solution. Develop
over a path of 15 cm using a mixture of 10 volumes of anhydrous formic acid R, 10 volumes of
water R, 30 volumes of methyl ethyl ketone R and 50 volumes of ethyl acetate R. Dry the plate at 100C
to 105C and spray the still warm plate with a 10 g/l solution of diphenylboric acid aminoethyl ester R in
methanol R. Then spray the plate with a 50 g/l solution of macrogol 400 R in methanol R. Allow the
plate to dry for about 30 min. Examine in ultraviolet light at 365 nm. The chromatogram obtained
with the reference solution shows in the middle third a yellowish-brown fluorescent zone (rutin),
above it a light blue fluorescent zone (chlorogenic acid) and above it a yellowish-brown fluorescent
zone (hyperoside). The chromatogram obtained with the test solution shows in particular: a
yellowish-brown fluorescent zone corresponding in position to the zone of rutin in the chromatogram
obtained with the reference solution; immediately above it a yellowish-green fluorescent zone
(vitexin-2-rhamnoside); above it a light blue zone, possibly superposed on a yellow fluorescent
zone, similar in position to the zone of chlorogenic acid in the chromatogram obtained with the
reference solution; above it an intense yellowish-brown fluorescent zone similar in position to the
19-38
zone of hyperoside in the chromatogram obtained with the reference solution, immediately above it
a yellowish-brown fluorescent zone which is often not separated from the zone corresponding to
hyperoside; immediately above it a mostly faint yellowish-green fluorescent zone (vitexin) and near
the front a light blue fluorescent zone. Further zones with weaker fluorescence are present.
TESTS
Foreign matter (2.8.2). Not more than 8 per cent of lignified branches with a diameter greater than
2.5 mm and not more than 2 per cent of other foreign matter.
ASSAY
Stock solution. In a 200 ml flask introduce 0.400 g of the powdered drug (250) and 40 ml of alcohol
(60 per cent V/V) R. Heat in a water-bath at 60C for 10 min, shaking frequently. Allow to cool and
filter through a plug of absorbent cotton into a 100 ml volumetric flask. Transfer the absorbent
cotton with the drug residue back into the 200 ml flask, add 40 ml of alcohol (60 per cent V/V) R and
heat again in a water-bath at 60C for 10 min, shaking frequently. Allow to cool and filter into the
same 100 ml volumetric flask as previously. Rinse the 200 ml flask and filter with a further quantity
of alcohol (60 per cent V/V) R and transfer to the same 100 ml volumetric flask. Dilute to volume with
alcohol (60 per cent V/V) R and filter.
Test solution. Introduce 5.0 ml of the stock solution into a round-bottomed flask and evaporate to
dryness under reduced pressure. Take up the residue with 8 ml of a mixture of 10 volumes of
methanol R and 100 volumes of glacial acetic acid R and transfer into a 25 ml volumetric flask. Rinse
the round-bottomed flask with 3 ml of a mixture of 10 volumes of methanol R and 100 volumes of
glacial acetic acid R and transfer into the same 25 ml volumetric flask as previously. Add 10.0 ml of a
solution containing 25.0 g/l of boric acid R and 20.0 g/l of oxalic acid R in anhydrous formic acid R and
dilute to 25.0 ml with anhydrous acetic acid R.
Compensation solution. Introduce 5.0 ml of the stock solution into a round-bottomed flask and
evaporate to dryness under reduced pressure. Take up the residue with 8 ml of a mixture of 10
volumes of methanol R and 100 volumes of glacial acetic acid R and transfer it into a 25 ml volumetric
flask. Rinse the round-bottomed flask with 3 ml of a mixture of 10 volumes of methanol R and 100
volumes of glacial acetic acid R and transfer into the same 25 ml volumetric flask as previously. Add
10.0 ml of anhydrous formic acid R and dilute to 25.0 ml with anhydrous acetic acid R.
Measure the absorbance (2.2.25) of the test solution at 410 nm, after 30 min by comparison with
the compensation solution.
Calculate the percentage content of total flavonoids, calculated as hyperoside, from the expression:
m
A 235 . 1
taking the value of the specific absorbance to be 405.
A=measured absorbance at 410 nm,
m=mass of the drug to be examined, in grams.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
19-39
Heparin Calcium
Heparin Calcium complies with the requirements of the 3rd edition of the European Pharmacopoeia [0332].
Preparation
Heparin Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Heparin calcium is a preparation containing the calcium salt of a sulphated glucosaminoglycan
present in mammalian tissues. On complete hydrolysis, it liberates D-glucosamine, D-glucuronic acid,
L-iduronic acid, acetic acid and sulphuric acid. It has the characteristic property of delaying the
clotting of freshly shed blood. The potency of heparin calcium intended for parenteral administration
is not less than 150 I.U. per milligram, calculated with reference to the dried substance. The potency
of heparin calcium not intended for parenteral administration is not less than 120 I.U. per milligram,
PRODUCTION
It is prepared from the lungs of oxen or from the intestinal mucosae of oxen, pigs or sheep.
It is produced by methods of manufacturing designed to minimise or eliminate microbial
contamination and substances lowering blood pressure.
CHARACTERS
A white or almost white powder, moderately hygroscopic, freely soluble in water.
IDENTIFICATION
A. It delays the clotting of freshly shed blood.
B. Dissolve 0.40 g in water R and dilute to 10.0 ml with the same solvent. The specific optical
rotation (2.2.7) is not less than +35.
C. Examine by zone electrophoresis (2.2.31) using agarose for electrophoresis R as the supporting
medium. To equilibrate the agarose and as electrolyte solution use a mixture of 50 ml of glacial acetic
acid R and 800 ml of water R adjusted to pH 3 by addition of lithium hydroxide R and diluted to
same solvent.
Reference solution. Dilute heparin sodium BRP with an equal volume of water R.
Apply separately to the strip 2 l to 3 l of each solution. Pass a current of 1 mA to 2 mA per
centimetre of strip width at a potential difference of 300 V for about 10 min. Stain the strips using a
1 g/l solution of toluidine blue R and remove the excess by washing. The ratio of the mobility of the
principal band or bands in the electrophoretogram obtained with the test solution to the mobility of
the band in the electrophoretogram obtained with the reference solution is 0.9 to 1.1.
D. It gives the reactions of calcium (2.3.1).
TESTS
Appearance of solution Dissolve a quantity equivalent to 50,000 I.U. in water R and dilute to
10 ml with the same solvent. The solution is clear (2.2.1) and not more intensely coloured than
degree 5 of the range of reference solutions of the most appropriate colour (Method II, 2.2.2).
Protein and nucleotidic impurities Dissolve 40 mg in 10 ml of water R. The absorbance (2.2.25)
measured at 260 nm is not greater than 0.20 and that measured at 280 nm is not greater than 0.15.
Nitrogen Not more than 2.5 per cent, calculated with reference to the dried substance. Carry out the
determination of nitrogen by sulphuric acid digestion (2.5.9), using 0.100 g.
Calcium 9.5 per cent to 11.5 per cent of Ca, calculated with reference to the dried substance.
Determine the calcium by complexometric titration (2.5.11), using 0.200 g
19-40
Sulphated ash (2.4.14). 32 per cent to 40 per cent, determined on 0.20 g and calculated with
0.01 I.U. of endotoxin per International Unit of heparin activity. The addition of divalent cations
may be necessary in order to fulfil the validation criteria.
ASSAY
Carry out the assay of heparin (2.7.5). The estimated potency is not less than 90 per cent and not
more than 111 per cent of the stated potency. The fiducial limits of error of the estimated potency (P
= 0.95) are not less than 80 per cent and not more than 125 per cent of the stated potency.
STORAGE
container.
LABELLING
The label states:
the number of International Units per milligram,
the name and quantity of any added substance,
__________________________________________________________________________________________________________ Ph Eur
19-41
Heparin Sodium
Heparin Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia [0333].
Preparation
Heparin Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Heparin sodium is a preparation containing the sodium salt of a sulphated glucosaminoglycan
present in mammalian tissues. On complete hydrolysis, it liberates D-glucosamine, D-glucuronic acid,
L-iduronic acid, acetic acid and sulphuric acid. It has the characteristic property of delaying the
clotting of freshly shed blood. The potency of heparin sodium intended for parenteral administration
is not less than 150 I.U. per milligram, calculated with reference to the dried substance. The potency
of heparin sodium not intended for parenteral administration is not less than 120 I.U. per milligram,
PRODUCTION
It is prepared from the lungs of oxen or from the intestinal mucosae of oxen, pigs or sheep.
It is produced by methods of manufacturing designed to minimise or eliminate microbial
contamination and substances lowering blood pressure.
CHARACTERS
A white or almost white powder, moderately hygroscopic, freely soluble in water.
IDENTIFICATION
A. It delays the clotting of freshly shed blood.
B. Dissolve 0.40 g in water R and dilute to 10.0 ml with the same solvent. The specific optical
rotation (2.2.7) is not less than +35.
C. Examine by zone electrophoresis (2.2.31) using agarose for electrophoresis R as the supporting
medium. To equilibrate the agarose and as electrolyte solution use a mixture of 50 ml of glacial acetic
acid R and 800 ml of water R adjusted to pH 3 by addition of lithium hydroxide R and diluted to
same solvent.
Reference solution. Dilute heparin sodium BRP with an equal volume of water R.
Apply separately to the strip 2 l to 3 l of each solution. Pass a current of 1 mA to 2 mA per
centimetre of strip width at a potential difference of 300 V for about 10 min. Stain the strips using a
1 g/l solution of toluidine blue R and remove the excess by washing. The ratio of the mobility of the
principal band or bands in the electrophoretogram obtained with the test solution to the mobility of
the band in the electrophoretogram obtained with the reference solution is 0.9 to 1.1.
D. The residue obtained in the test for sulphated ash (see Tests) gives reaction (a) of sodium (2.3.1).
TESTS
Appearance of solution Dissolve a quantity equivalent to 50,000 I.U. in water R and dilute to
10 ml with the same solvent. The solution is clear (2.2.1) and not more intensely coloured than
intensity 5 of the range of reference solutions of the most appropriate colour (Method II, 2.2.2).
Protein and nucleotidic impurities Dissolve 40 mg in 10 ml of water R. The absorbance (2.2.25)
measured at 260 nm is not greater than 0.20 and that measured at 280 nm is not greater than 0.15.
Nitrogen Not more than 2.5 per cent, calculated with reference to the dried substance. Carry out the
determination of nitrogen by sulphuric acid digestion (2.5.9), using 0.100 g.
Sodium 9.5 per cent to 12.5 per cent of Na, calculated with reference to the dried substance and
determined by atomic absorption spectrometry (Method I, 2.2.23).
Test solution. Dissolve 50 mg of the substance to be examined in 0.1M hydrochloric acid containing
1.27 mg of caesium chloride R per millilitre and dilute to 100.0 ml with the same solvent.
Reference solutions. Prepare reference solutions containing 25 ppm, 50 ppm and 75 ppm of Na, using
sodium standard solution (200 ppm Na) R diluted with 0.1M hydrochloric acid containing 1.27 mg of
caesium chloride R per millilitre.
19-42
Measure the absorbance at 330.3 nm using a sodium hollow-cathode lamp as the source of radiation
and a flame of suitable composition (for example 11 litres of air and 2 litres of acetylene per minute).
Sulphated ash (2.4.14). 30 per cent to 43 per cent, determined on 0.20 g and calculated with
0.01 I.U. of endotoxin per International Unit of heparin activity.
ASSAY
Carry out the assay of heparin (2.7.5). The estimated potency is not less than 90 per cent and not
more than 111 per cent of the stated potency. The fiducial limits of error of the estimated potency (P
= 0.95) are not less than 80 per cent and not more than 125 per cent of the stated potency.
STORAGE
container.
LABELLING
The label states:
the number of International Units per milligram,
the name and quantity of any added substance,
__________________________________________________________________________________________________________ Ph Eur
19-43
Low-molecular-weight Heparins
Low-molecular-weight Heparins comply with the requirements of the 3rd edition of the European Pharma-
copoeia for Low-molecular-mass Heparins [0828]. These requirements are reproduced after the heading
Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Low-molecular-mass heparins are salts of sulphated glucosaminoglycans having a mass-average
molecular mass less than 8000 and for which at least 60 per cent of the total mass has a molecular
mass less than 8000. They display different chemical structures at the reducing or the non-reducing
end of the polysaccharide chains.
The potency is not less than 70 I.U. of anti-factor Xa activity per milligram, calculated with refer-
ence to the dried substance. The ratio of anti-factor Xa activity to anti-factor IIa activity, determined
as described under Assay, is not less than 1.5.
PRODUCTION
Low-molecular-mass heparins are produced in conditions designed to minimise microbial
contamination.
Low-molecular-mass heparins are obtained by fractionation or depolymerisation of heparin of
natural origin that complies with the monograph on Heparin sodium (333) or Heparin calcium (332),
whichever is appropriate, for parenteral use, unless otherwise justified and authorised. For each type
of low-molecular-mass heparin the batch- to-batch consistency is ensured by demonstrating, for
example, that the mass-average molecular mass and the mass percentage within defined molecular-
mass ranges lower than 8000 are not less than 75 per cent and not more than 125 per cent of the
mean value stated as type specification; the same limits apply also to the ratio of anti-factor Xa
activity to anti-factor IIa activity.
Nucleotide and protein impurities of the source material Dissolve 40 mg of the source material
before fractionation in 10 ml of water R. The absorbance (2.2.25) measured at 260 nm and 280 nm is
not greater than 0.20 and 0.15, respectively.
CHARACTERS
A white or almost white powder, hygroscopic, freely soluble in water.
IDENTIFICATION
A. Examine by nuclear magnetic resonance spectrometry (2.2.33) using a pulsed (Fourier transform)
spectrometer operating at 75 MHz for
13
C.
Test solution. Dissolve 0.200 g of the substance to be examined in a mixture of 0.2 ml of deuterium
oxide R and 0.8 ml of water R.
Reference solution. Dissolve 0.200 g of the appropriate specific low-molecular-mass heparin CRS in a
mixture of 0.2 ml of deuterium oxide R and 0.8 ml of water R.
Record the spectra at 40C, using cells 5 mm in diameter. Use deuterated methanol R as internal
reference at = 50.0 ppm.
The spectrum obtained with the test solution is similar to that obtained with the reference solution
containing the appropriate specific low-molecular-mass heparin CRS.
B. The ratio of anti-factor Xa activity to anti-factor IIa activity, determined as described under Assay,
is not less than 1.5.
C. Examine by size-exclusion chromatography (2.2.30).
Test solution. Dissolve 20 mg of the substance to be examined in 2 ml of the mobile phase.
Reference solution. Dissolve 20 mg of low-molecular-mass heparins for calibration CRS in 2 ml of the
mobile phase.
a column 0.30 m long and 7.5 mm in internal diameter, packed with porous silica beads (5 m),
having at least 20,000 theoretical plates per metre and having a fractionation range for proteins
of approximately 15,000 to 100,000,
as mobile phase, at a flow rate of 0.5 ml per minute a 28.4 g/l solution of sodium sulphate R
adjusted to pH 5.0 using dilute sulphuric acid R,
as detector a differential refractometer.
Calibration of the system. Inject 25 l of the reference solution. For detection, use a differential
refractometer (RI) detector connected in series to a UV spectrophotometer set at 234 nm such that
19-44
the UV monitor is connected to the column outlet, and the differential refractometer to the UV-
monitor outlet.
It is necessary to measure the time lapse between the two detectors accurately, so that chromato-
grams from them can be aligned correctly. The retention times used in the calibration must be those
from the RI detector.
The normalisation factor used to calculate the molecular weight from the RI/UV ratio is obtained
as follows: calculate the total area under the UV
234
(UV
234
) and the RI (RI) curves by numerical
integration over the range of interest (i.e. excluding salt and solvent peaks at the end of the chromato-
gram). Calculate the ratio r :
RI
UV
234
Calculate the factor f, which when multiplied by r gives M
na
(the known number-average molecular
mass of the calibrant). The value for M
na
is found in the leaflet which is supplied with the calibrant
CRS., i.e.:
M
r
na
Provided the UV
234
and the RI responses are aligned, the molecular mass at any point is calculated
from:
f
RI
UV
234
Inject 25 l of the test solution and record the chromatogram for a period of time ensuring complete
elution of sample and solvent peaks.
The mass-average molecular mass is defined by the expression:
( ) RI M
RI
i i
i
RI
i
=quantity of substance (mass) eluting in the fraction i.
M
i
=molecular mass corresponding to fraction i.
The value for the mass average molecular mass is not greater than 8000 and at least 60 per cent of
the total mass has a molecular mass lower than 8000. In addition, the molecular mass parameters of
a given substance do not differ by more than 25 per cent from those of the corresponding reference
substance.
D. It gives reaction (a) of sodium or the reactions of calcium (as appropriate) (2.3.1).
TESTS
Nitrogen (2.5.9). 1.5 to 2.5 per cent, calculated with reference to the dried substance.
Calcium If prepared from heparin complying with the monograph on Heparin calcium (332), it
contains 9.5 to 11.5 per cent of Ca, calculated with reference to the dried substance. Determine the
calcium by complexometric titration (2.5.11) using 0.200 g.
Sodium If prepared from heparin complying with the monograph on Heparin sodium (333), it
contains 9.5 to 12.5 per cent of Na, calculated with reference to the dried substance and determined
by atomic absorption spectrometry (Method I, 2.2.23).
Test solution. Dissolve 50 mg of the substance to be examined in 0.1M hydrochloric acid containing
1.27 mg of caesium chloride R per millilitre and dilute to 100.0 ml with the same solvent.
Reference solutions. Prepare reference solutions containing 25 ppm, 50 ppm and 75 ppm of Na using
sodium standard solution (200 ppm Na) R diluted with 0.1M hydrochloric acid containing 1.27 mg of
caesium chloride R per millilitre.
Measure the absorbance at 330.3 nm using a sodium hollow-cathode lamp as a source of radiation
and a flame of suitable composition (for example, 11 litres of air and 2 litres of acetylene per minute).
over diphosphorus pentoxide R at a pressure not exceeding 670 Pa for 3 h.
Molar ratio of sulphate ions to carboxylate ions Not less than 1.8, determined by conductivity
(2.2.38).
The sample of heparin used in this titration must be free from ionisable impurities, particularly salts.
Weigh 0.100 g of the substance to be examined taking the necessary measures to avoid the problems
linked to hygroscopicity.
19-45
Take up into about 20 ml of double-glass-distilled water R. Cool to 4C and apply 2.0 ml of this
solution to a pre-cooled column (approximately 10 1 cm), packed with a suitable cation exchange
resin R. Wash through with double-glass-distilled water R into the titration vessel up to a final volume
of about 10 ml to 15 ml. (The titration vessel must be just large enough to hold the electrodes from the
conductivity meter, a small stirrer bar and a fine flexible tube from the outlet of a 2 ml burette). Stir
magnetically. When the conductivity reading is constant, note it and titrate with 0.05M sodium
hydroxide added in approximately 50 l portions. Record the burette level and the conductivity meter
reading a few seconds after each addition until the end-point is reached.
For each measured figure, calculate the number of milliequivalents of sodium hydroxide added
from the volume and the known concentration of the sodium hydroxide solution. Plot on a graph the
figures for conductivity (as y-axis) against the figures of milliequivalent of sodium hydroxide (as x-
axis). The graph will have three approximately linear sections: an initial steep downward slope, a
middle slight rise and a final steep rise. Estimate the best straight lines through these three parts of
the graph. At the points where the first and second lines intersect and where the second and third
lines intersect, draw perpendiculars to the x-axis to estimate the milliequivalents of sodium hydroxide
taken up by the sample at those points. The point where the first and second lines intersect will give
the number of milliequivalents of sodium hydroxide taken up by the sulphate groups and the point
where the second and third lines intersect will give the number of milliequivalents taken up by the
sulphate and carboxylate groups together. The difference between the two will therefore give the
number of milliequivalents taken up by the carboxylate groups.
Bacterial endotoxin (2.6.14). If intended for use in the manufacture of parenteral dosage forms
of endotoxin per International Unit of anti-Xa activity. The addition of divalent cations may be
necessary to fulfil the validation criteria.
ASSAY
The anticoagulant activity of low-molecular-mass heparin is determined in vitro by two assays which
determine its ability to accelerate the inhibition of factor Xa (anti-Xa assay) and thrombin, factor IIa
(anti-IIa assay), by antithrombin III.
The International Units for anti-Xa and anti-IIa activity are the activities contained in a stated
amount of the International Standard for low-molecular-mass heparin.
Low-molecular-mass heparin for assay BRP, calibrated in International Units by comparison with the
International Standard using the two assays given below, is used as reference preparation.
ANTI-FACTOR XA ACTIVITY
Reference and test solutions
Prepare four independent series of four dilutions each of the substance to be examined and of the
reference preparation of low-molecular-mass heparin in tris(hydroxymethyl)aminomethane sodium
chloride buffer solution pH 7.4 R; the concentration range should be within 0.025 I.U. to 0.2 I.U. of
anti-factor Xa activity per millilitre and the dilutions chosen should give a linear response when
results are plotted as absorbance against log concentration.
Procedure
Label sixteen tubes in duplicate: T
1
, T
2
, T
3
, T
4
for the dilutions of the substance to be examined and
S
1
, S
2
, S
3
, S
4
for the dilutions of the reference preparation. To each tube add 50 l of antithrombin III
solution R1 and 50 l of the appropriate dilution of the substance to be examined or the reference
preparation. After each addition, mix but do not allow bubbles to form. Treating the tubes in the
order S
1
, S
2
, S
3
, S
4,
T
1
, T
2
, T
3
, T
4
, T
1
, T
2
, T
3
, T
4
, S
1
, S
2
, S
3
, S
4
, allow to equilibrate at 37C
(water-bath or heating block) for 1 min and add to each tube 100 l of bovine factor Xa solution R.
Incubate for exactly 1 min and add 250 l of chromophore substrate R1. Stop the reaction after exactly
4 min by adding 375 l of acetic acid R. Transfer the mixtures to semi-micro cuvettes and measure
the absorbance (2.2.25) at 405 nm using a suitable reading device. Determine the blank amidolytic
activity at the beginning and at the end of the procedure in a similar manner, using tris(hydroxy-
methyl)aminomethane sodium chloride buffer solution pH 7.4 R instead of the reference and test
solutions; the two blank values do not differ significantly. Calculate the regression of the absorbance
on log concentrations of the solutions of the substance to be examined and of the reference
preparation of low-molecular-mass heparins and calculate the potency of the substance to be exam-
ined in International Units of anti-factor Xa activity per millilitre using the usual statistical methods
for parallel-line assays.
ANTI-FACTOR IIA ACTIVITY
Reference and test solutions
Prepare four independent series of four dilutions each of the substance to be examined and the
reference preparation of low molecular-mass heparin in tris(hydroxymethyl)aminomethane sodium
19-46
chloride buffer solution pH 7.4 R; the concentration range should be within 0.015 I.U. to 0.075 I.U.
of anti-factor IIa activity per millilitre and the dilutions chosen should give a linear response when
results are plotted as absorbance against log concentration.
Procedure
Label sixteen tubes in duplicate: T
1
, T
2
, T
3
, T
4
for the dilutions of the substance to be examined and
S
1
, S
2
, S
3
, S
4
for the dilutions of the reference preparation. To each tube add 50 l of antithrombin III
solution R2 and 50 l of the appropriate dilution of the substance to be examined or the reference
preparation. After each addition, mix but do not allow bubbles to form. Treating the tubes in the
order S
1
, S
2
, S
3
, S
4
, T
1
, T
2
, T
3
, T
4
, T
1
, T
2
, T
3
, T
4
, S
1
, S
2
, S
3
, S
4
, allow to equilibrate at 37C
(water-bath or heating block) for 1 min and add to each tube 100 l of human thrombin solution R.
Incubate for exactly 1 min and add 250 l of chromophore substrate R2. Stop the reaction after exactly
4 min by adding 375 l of acetic acid R. Transfer the mixtures to semi-micro cuvettes and measure
the absorbance (2.2.25) at 405 nm using a suitable reading device. Determine the blank amidolytic
activity at the beginning and at the end of the procedure in a similar manner, using tris(hydroxy-
methyl)aminomethane sodium chloride buffer solution pH 7.4 R instead of the reference and test
solutions; the two blank values do not differ significantly. Calculate the regression of the absorbance
on log concentrations of the solutions of the substance to be examined and of the reference
preparation of low-molecular-mass heparins, and calculate the potency of the substance to be exam-
ined in International Units of anti-factor IIa activity per millilitre using the usual statistical methods
for parallel-line assays.
LABELLING
The label states:
the number of International Units of anti-factor Xa activity per milligram,
the number of International Units of anti-factor IIa activity per milligram,
the mass-average molecular mass and the percentage of molecules within defined molecular mass
ranges,
where applicable, that the contents are the sodium salt,
where applicable, that the contents are the calcium salt.
STORAGE
container.
__________________________________________________________________________________________________________ Ph Eur
19-47
Herbal Drugs
Herbal Drugs comply with the requirements of the 3rd edition of the European Pharmacopoeia [1433]. These
requirements are reproduced below.
Ph Eur ___________________________________________________________________________________________________________
The statements in this monograph are intended to be read in conjunction with the individual monographs in
the Pharmacopoeia. The requirements do not necessarily apply to products that are not the subject of such
monographs.
DEFINITION
Herbal drugs are mainly whole, fragmented or cut, plants, parts of plants, algae, fungi, lichen in an
unprocessed state, usually in dried form but sometimes fresh. Certain exudates that have not been
subjected to a specific treatment are also considered to be herbal drugs. Herbal drugs are precisely
defined by the botanical scientific name according to the binominal system (genus, species, variety
and author).
PRODUCTION
Herbal drugs are obtained from cultivated or wild plants. Suitable collection, cultivation, harvesting,
drying, fragmentation and storage conditions are essential to guarantee the quality of herbal drugs.
Herbal drugs are, as far as possible, free from impurities such as soil, dust, dirt and other contamin-
ants such as fungal, insect and other animal contaminations. They are not rotten.
If a decontaminating treatment has been used, it is necessary to demonstrate that the constituents of
the plant are not affected and that no harmful residues remain. The use of ethylene oxide is
prohibited for the decontamination of herbal drugs.
IDENTIFICATION
Herbal drugs are identified using their macroscopic and microscopic descriptions and any further
tests that may be required (for example, thin-layer chromatography).
TESTS
A test for foreign matter (2.8.2) is carried out, unless otherwise prescribed in the individual mono-
graphs.
A specific appropriate test may apply to herbal drugs liable to be falsified.
If appropriate, the herbal drugs comply with other tests, for example, total ash (2.4.16), ash insoluble
in hydrochloric acid (2.8.1), extractable matter, swelling index (2.8.4) and bitterness value.
The test for loss on drying (2.2.32) is carried out on herbal drugs, unless otherwise prescribed in the
individual monographs. A determination of water (2.2.13) is carried out for herbal drugs with a high
essential oil content.
Herbal drugs comply with the requirements for pesticide residues (2.8.13). The requirements take
into account the nature of the plant, where necessary the preparation in which the plant might be
used, and where available the knowledge of the complete record of treatment of the batch of the
plant. The content of pesticide residues may be determined by the method described in the annex to
the general method.
The risk of contamination of herbal drugs by heavy metals must be considered. If an individual
monograph does not prescribe limits for heavy metals or specific elements such limits may be
required if justified.
Recommendations on the microbiological quality of products consisting solely of one or more herbal
drugs are given in the text on Microbiological quality of pharmaceutical preparations (5.1.4.-Category 4).
Where necessary limits for aflatoxins may be required.
In some specific circumstances, the risk of radioactive contamination is to be considered.
ASSAY
Unless otherwise justified and authorised herbal drugs are assayed by an appropriate method.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
19-48
Hexachlorophene
CH
2
Cl Cl Cl Cl
Cl Cl OH HO
C
13
H
6
Cl
6
O
2
406.9 70-30-4
Definition Hexachlorophene is 2,2-methylenebis(3,4,6-trichlorophenol). It contains not less
13
H
6
Cl
6
O
2
substance.
Characteristics A white or pale buff, crystalline powder; odourless or almost odourless.
Practically insoluble in water; very soluble in acetone and in ether; freely soluble in ethanol (96%). It
dissolves in dilute solutions of the alkali hydroxides.
Identification
The infrared absorption spectrum, Appendix II A, is concordant with the reference spectrum of
hexachlorophene (RS 174).
Chloride Dissolve 0.50 g in 2 ml of ethanol (96%), dilute to 25 ml with water and filter. 5 ml of the
clear filtrate diluted to 15 ml with water complies with the limit test for chlorides, Appendix VII
(500 ppm).
Non-phenolic substances Dissolve 5 g in 38 ml of methanol, add 125 ml of 0.25M sodium hydroxide
and extract with three 15-ml quantities of n-pentane, retaining any foamy interphase with the aqueous
layer. Dry the combined extracts over anhydrous sodium sulphate and evaporate to dryness at a
pressure of 2 kPa. The residue, when dried to constant weight at a pressure of 2 kPa, weighs not
more than 37.5 mg.
2,3,7,8-Tetrachlorodibenzo-p-dioxin Not more than 0.002 ppm when determined by the
following method. Dilute sufficient
13
C-2,3,7,8-tetrachlorodibenzo-p-dioxin solution CRS (internal
standard) with hexane to produce a solution containing about 0.66 ng per ml (1 ml in 100 ml is
usually suitable) (solution A) and dilute sufficient
12
C-2,3,7,8-tetrachlorodibenzo-p-dioxin solution CRS
with hexane to produce a solution containing about 0.66 ng per ml (1 ml in 100 ml is usually suitable
(solution B). Prepare the following solutions for use in the capillary gas chromatographic method
described below. For solution (1) mix 3 ml of solution A with 3 ml of solution B, evaporate the
solvent to dryness in a current of nitrogen and dissolve the residue in 0.5 ml of hexane. For solution
(2) dissolve 1.0 g of the substance being examined in 20 ml of 2.5M sodium hydroxide, extract with
three 10-ml quantities of freshly distilled hexane and wash the combined extracts with 10 ml of 2.5M
sodium hydroxide followed by two 30-ml quantities of water, adding 0.5 ml of methanol if necessary to
break any emulsion. Filter the hexane extract through a layer of 1 g of anhydrous sodium sulphate and
wash the sodium sulphate with two 2-ml quantities of hexane. To the combined filtrate and washings
add 3 ml of solution B and evaporate the solvent in a current of nitrogen in a water bath at 40.
Dissolve the residue as completely as possible in 3 ml of a mixture of 7 volumes of hexane and 3
volumes of ether and pass through a column (20 cm 5 mm) packed with 2 g of basic aluminium oxide
(70 to 230 mesh) (Brockman activated grade I is suitable). Wash the column with 15 ml of a mixture
of 7 volumes of hexane and 3 volumes of ether, discard the washings and elute with 10 ml of a mixture
of equal volumes of hexane and dichloromethane. Evaporate the eluate in a current of nitrogen, dissolve
the residue in 0.5 ml of hexane and pass it through a second column (90 mm 5 mm) packed with
silica gel (70 to 230 mesh) (Merck silica gel 60 for column chromatography is suitable). Elute with
10 ml of hexane, evaporate the eluate to dryness in a stream of nitrogen and dissolve the residue in
0.5 ml of hexane. Prepare solution (3) in the same manner as solution (2) but using 3 ml of solution
A in place of 3 ml of solution B.
Inject 2 l of each of solutions (1), (2) and (3), using a Grob-type splitless injector and a splitless
period of 60 seconds, onto a fused silica capillary column (25 m 0.2 mm), the inner surface of
which is chemically bonded to methyl silicone (CP sil 5 CB is suitable), at an initial temperature of
40 increasing to 275 at a constant rate of 40 per minute with the injector temperature at 300, or
onto another suitable column. Use helium as the carrier gas with a flow rate of 1 ml per minute and,
as the detector, a suitably coupled mass spectrometer of resolution not less than 5000 with an
electron impact ionisation source and equipped with a sequential multiple-ion monitoring facility set
at 320, 322, 324, 332, 334 and 336 mass units.
In the chromatogram obtained with solution (1) the ratio of the heights of the peaks due to
12
C-
2,3,7,8-tetrachlorodibenzo-p-dioxin at 320, 322 and 324 mass units and the ratio of the heights of
the peaks due to
13
C-2,3,7,8-tetrachlorodibenzo-p-dioxin at 332, 334 and 336 mass units are each
about 3:4:2. The method is not valid unless the ratio of the heights of at least two of the three peaks
at 320, 322 and 324 mass units in the chromatogram obtained with solution (2) is approximately the
19-49
same as that of the corresponding peaks in the chromatogram obtained with solution (1).
Calculate the content of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the substance being examined
from the declared content of 2,3,7,8-tetrachlorodibenzo-p-dioxin in
12
C-2,3,7,8-tetrachlorodibenzo-p-
dioxin CRS using the sum of the areas of the peaks due to
12
C-2,3,7,8-tetrachlorodibenzo-p-dioxin
and the sum of the areas of the corresponding peaks due to
13
C-2,3,7-8-tetrachorodibenzo-p-dioxin
in the chromatograms obtained with solution (1) and (3). In the chromatogram obtained with
solution (2) if the areas of the peaks at 320, 322 and 324 mass units are approximately in the ratio
3:4:2 use the sum of the areas of all three peaks in the calculation; otherwise use the sum of the
areas of the two valid peaks.
Depending on the absorption characteristics of the basic aluminium oxide used in the
preparation of solutions (2) and (3), a low recovery of 2,3,7,8-tetrachlorodibenzo-p-dioxin may be
obtained. If, as a result, the detection sensitivity is found to be inadequate, replace the solvent
mixture of 7 volumes of hexane and 3 volumes of ether used to wash the alumina column and to
dissolve the residue with a less polar mixture, for example a mixture of 98 volumes of hexane and 2
volumes of dichloromethane. If too non-polar a mixture is used the isolation of 2,3,7,8-
tetrachlorodibenzo-p-dioxin from other impurities may not be achieved resulting in the presence of
interfering peaks in the chromatogram.
solutions of the substance being examined in methanol containing (1) 0.020% w/v and (2) 1.0% w/v.
(20 cm 5 mm) packed with stationary phase C (10 m) (Spherisorb ODS 1 is suitable), (b) a
mixture of 100 volumes of methanol, 20 volumes of water and 1 volume of glacial acetic acid as the
mobile phase with a flow rate of 2 ml per minute and (c) a detection wavelength of 300 nm.
In the chromatogram obtained with solution (2) the sum of the areas of any secondary peaks with a
retention time not more than 3 times that of the principal peak is not greater than twice the area of
the principal peak in the chromatogram obtained with solution (1) and not more than one such peak
has an area greater than half of the area of the principal peak in the chromatogram obtained with
solution (1).
1 g.
Assay Dissolve 1 g in 25 ml of ethanol (96%) previously adjusted to pH 9.0 and titrate with 0.1M
sodium hydroxide VS to pH 9.0 determining the end point potentiometrically. Each ml of 0.1M sodium
13
H
6
Cl
6
O
2
.
Storage Hexachlorophene should be kept in a well-closed container and protected from light.
Preparation
Hexachlorophene Dusting Powder
When hexachlorophane is prescribed or demanded, Hexachlorophene shall be dispensed or supplied.
Hexamidine Isetionate
O
O
NH
2
N
NH
2
NH
HO
SO
3
H
2
,
C
24
H
38
N
4
O
10
S
2
607 659-40-5
Hexamidine Isetionate complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Hexamidine Diisetionate [1436]. These requirements are reproduced after the heading Definition below.
Action and use Topical antibacterial.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hexamidine diisetionate contains not less than 98.5 per cent and not more than the equivalent of
101.5 per cent of 4,4-(hexane-1,6-diyldioxy)dibenzimidamide bis(2-hydroxyethanesulphonate),
CHARACTERS
A white or slightly yellow powder, hygroscopic, sparingly soluble in water, slightly soluble in alcohol,
IDENTIFICATION
obtained with hexamidine diisetionate CRS.
B. Dissolve about 40 mg in 5 ml of water R and add dropwise with shaking, 1 ml of a 100 g/l solution
of sodium chloride R. Allow to stand for 5 min. An abundant shimmering white precipitate forms
slowly.
TESTS
Appearance of solution Dissolve 0.5 g in carbon dioxide-free water R heating at about 70C and
dilute to 10 ml with the same solvent. Allow to cool to room temperature for 10 min to 15 min. The
solution is not more opalescent than reference suspension II (2.2.1) and not more intensely coloured
than intensity 6 of the range of reference solutions of the most appropriate colour (Method II, 2.2.2).
Acidity or alkalinity Dissolve 2.0 g in water R heating at about 50C and dilute to 20 ml with
water R heating at about 50C. Allow to cool to about 35C, add 0.1 ml of methyl red solution R. Not
more than 0.25 ml of 0.05M hydrochloric acid or 0.05M sodium hydroxide is required to change the
Test solution. Dissolve 20.0 mg of the substance to be examined in mobile phase A and dilute to
Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with mobile phase A.
Reference solution (b). Dissolve 5 mg of the substance to be examined and 5 mg of pentamidine
diisetionate CRS in mobile phase A and dilute to 100 ml with mobile phase A. Dilute 2 ml of the
solution to 5 ml with mobile phase A.
a stainless steel column 0.25 m long and 4.6 mm in internal diameter, packed with styrene-
divinylbenzene copolymer R (8 m),
Mobile phase A. Mix 20 volumes of acetonitrile R and 80 volumes of a 6.8 g/l solution of potassium
dihydrogen phosphate R adjusted to pH 3 using phosphoric acid R,
Mobile phase B. Mix equal volumes of acetonitrile R and of a 6.8 g/l solution of potassium
dihydrogen phosphate R adjusted to pH 3 using phosphoric acid R,
19-51
Time
(min)
Mobile
phase A
(per cent V/V)
Mobile
phase B
(per cent V/V)
Comment
35 40
0100 1000
re-equilibration
Inject 20 l of reference solution (a) and 20 l of reference solution (b). Adjust the sensitivity of
solution (a) is at least 50 per cent of the full scale of the recorder. The test is not valid unless, in the
chromatogram obtained with reference solution (b), the resolution between the peak corresponding
respectively to hexamidine diisetionate and pentamidine diisetionate is at least 5.
Inject 20 l of the test solution. In the chromatogram obtained, the area of any peak, apart from
with reference solution (a) (1 per cent) and not more than one such peak has an area greater than
half the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per
cent); the sum of the areas of all the peaks, apart from the principal peak, is not greater than 1.5
times the area of the principal peak in the chromatogram obtained with reference solution (a)
100C to 105C.
ASSAY
Dissolve 0.250 g in 50 ml of dimethylformamide R. Titrate with 0.1M tetrabutylammonium hydroxide
under a current of nitrogen R, determining the end-point potentiometrically (2.2.20).
24
H
38
N
4
O
10
S
2
.
STORAGE
IMPURITIES
O
O
NH
2
N
NH
2
O
A. 4-[[6-(4-carbamimidoylphenoxy)hexyl]oxy]benzamide.
__________________________________________________________________________________________________________ Ph Eur
19-52
Hexetidine
H
3
C N N CH
3
H
3
C CH
3
NH
2
H
3
C
* *
C
21
H
45
N
3
339.6 141-94-6
Hexetidine complies with the requirements of the 3rd edition of the European Pharmacopoeia [1221]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hexetidine contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent of
1,3-bis(2-ethylhexyl)-5-methylhexahydropyrimidin-5-amine.
CHARACTERS
An oily liquid, colourless or slightly yellow, very slightly soluble in water, very soluble in acetone, in
alcohol and in methylene chloride. It dissolves in dilute mineral acids.
IDENTIFICATION
A. Examine by infrared absorption spectrophotometry (2.2.24) comparing with the spectrum
obtained with hexetidine CRS.
C. To 0.2 ml add 2 ml of sulphuric acid R and 2 mg of chromotropic acid, sodium salt R. Heat in a
water-bath at 60C. A violet colour develops.
D. Dissolve 0.2 ml in 1 ml of methylene chloride R. Add 0.5 ml of copper sulphate solution R, 0.05 ml of
0.25M alcoholic sulphuric acid and 5 ml of water R. Shake, then allow to stand. The lower layer
becomes deep blue.
TESTS
5
or reference solution GY
5
(Method II, 2.2.2).
Optical rotation (2.2.7). Dissolve 1.0 g in ethanol R and dilute to 10.0 ml with the same solvent.
Absorbance (2.2.25). Dissolve 0.50 g in heptane R and dilute to 50.0 ml with the same solvent. At
wavelengths from 270 nm to 350 nm, the absorbance of the solution is not greater than 0.1.
coating substance. Prepare the solutions immediately before use.
Test solution (a). Dissolve 2.0 g of the substance to be examined in heptane R and dilute to 20 ml with
the same solvent.
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with heptane R.
Reference solution (a). Dissolve 20 mg of hexetidine CRS in heptane R and dilute to 2 ml with the same
solvent.
Reference solution (b). Dilute 1 ml of test solution (a) to 100 ml with heptane R.
Reference solution (c). Dilute 5 ml of reference solution (b) to 10 ml with heptane R.
Reference solution (d). Dissolve 10 mg of dehydrohexetidine CRS in test solution (a) and dilute to 10 ml
Apply separately to the plate 1 l of each solution. At the bottom of a chromatography tank, place an
evaporating dish containing concentrated ammonia R1. Place the dried plate in the tank and close the
19-53
tank. Leave the plate in contact with the ammonia vapour for 15 min. Withdraw the plate and
place it in a current of air to remove the ammonia vapour. Develop over a path of 15 cm using a
mixture of 20 volumes of methanol R and 80 volumes of toluene R. Allow the plate to dry in air.
Expose the plate to iodine vapour for 30 min. Any spot in the chromatogram obtained with test
solution (a), apart from the principal spot, is not more intense than the spot in the chromatogram
obtained with reference solution (b) (1 per cent) and at most two such spots are more intense than
the spot in the chromatogram obtained with reference solution (c) (0.5 per cent). The test is not
valid unless the chromatogram obtained with reference solution (d) shows two clearly separated
spots.
acetone R and dilute to 20 ml with the same mixture of solvents. 12 ml of the solution complies with
ASSAY
21
H
45
N
3
.
STORAGE
IMPURITIES
*
*
*
H
3
C
H
3
C
N
N
N
H
CH
3
CH
3
H
3
C
A. 2-ethyl-N-[[1-(2-ethylhexyl)-4-methyl-4,5-dihydro-1H-imidazol-4-yl]methyl]hexan-1-
amine (dehydrohexetidine),
H
3
C NH HN CH
3
H
3
C CH
3
NH
2
H
3
C
* *
B. N,N-bis(2-ethylhexyl)-2-methylpropane-1,2,3-triamine (triamine),
*
N
N N
CH
3
H
3
C
H
3
C
CH
3
CH
3
*
C. 2,6-bis(2-ethylhexyl)-7a-methylhexahydro-1H-imidazo[1,5-c]imidazole (hexedine),
SO
3
H
SO
3
H
D. naphthalene-1,5-disulphonic acid.
__________________________________________________________________________________________________________ Ph Eur
19-54
Hexobarbital
NH
N
O O
O
Me
Me
and enantiomer
C
12
H
16
N
2
O
3
236.3 56-29-1
Hexobarbital complies with the requirements of the 3rd edition of the European Pharmacopoeia [0183]. These
When hexobarbitone is prescribed or demanded, Hexobarbital shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hexobarbital contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of 5-(cyclohex-1-enyl)-1,5-dimethyl-1H, 3H,5H-pyrimidine-2,4,6-trione, calculated with reference to
CHARACTERS
A white, crystalline powder, very slightly soluble in water, sparingly soluble in alcohol and in ether. It
forms water-soluble compounds with alkali hydroxides and carbonates and with ammonia.
IDENTIFICATION
substance to be examined and hexobarbital CRS and determine the melting point of the mixture. The
obtained with hexobarbital CRS.
254
the same solvent.
Reference solution. Dissolve 0.1 g of hexobarbital CRS in chloroform R and dilute to 100 ml with the
same solvent.
chloroform R. Examine immediately in ultraviolet light at 254 nm. The principal spot in the chromato-
togram obtained with the reference solution.
D. To about 10 mg add 1.0 ml of a 10 g/l solution of vanillin R in alcohol R and 2 ml of a cooled
mixture of 1 volume of water R and 2 volumes of sulphuric acid R. Shake and allow to stand for 5 min.
A greenish-yellow colour develops. Heat on a water-bath for 10 min. The colour becomes dark red.
TESTS
Appearance of solution Dissolve 1.0 g in a mixture of 4 ml of dilute sodium hydroxide solution R and
6 ml of water R. The solution is clear (2.2.1) and not more intensely coloured than reference solution
Y
6
(Method II, 2.2.2).
Acidity Boil 1.0 g with 50 ml of water R for 2 min, allow to cool and filter. To 10 ml of the filtrate
add 0.15 ml of methyl red solution R. The solution is orange-yellow. Not more than 0.1 ml of 0.1M
sodium hydroxide is required to produce a pure yellow colour.
254
R as the
coating substance.
the same solvent.
19-55
Reference solution. Dilute 0.5 ml of the test solution to 100 ml with chloroform R.
chloroform R. Examine immediately in ultraviolet light at 254 nm. Any spot in the chromatogram
obtained with the test solution, apart from the principal spot, is not more intense than the spot in the
chromatogram obtained with the reference solution (0.5 per cent).
100C to 105C.
ASSAY
Dissolve 0.200 g in 5 ml of pyridine R. Add 0.5 ml of thymolphthalein solution R and 10 ml of silver
12
H
16
N
2
O
3
.
__________________________________________________________________________________________________________ Ph Eur
19-56
Hexylresorcinol
CH
3
HO
OH
C
12
H
18
O
2
194.3 136-77-6
Hexylresorcinol complies with the requirements of the 3rd edition of the European Pharmacopoeia [1437].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hexylresorcinol contains not less than 98.0 per cent and not more than the equivalent of 101.0 per
cent of 4-hexylbenzene-1,3-diol calculated with reference to the anhydrous substance.
CHARACTERS
A colourless, yellowish or reddish, crystalline powder or needles, turning brownish-pink on exposure
to light or air, very slightly soluble in water, freely soluble in alcohol and in methylene chloride.
IDENTIFICATION
A. Melting point (2.2.14): 66C to 68C. Melting may occur at about 60C, followed by solidifica-
tion and a second melting between 66C and 68C.
obtained with hexylresorcinol CRS. If the spectra obtained in the solid state show differences, dissolve
the substance to be examined and the reference substance separately in methanol R, evaporate to
dryness and record new spectra using the residues.
Test solution. Dilute 0.1 ml of solution S (see Tests) to 10 ml with alcohol R.
Reference solution (a). Dissolve 10 mg of hexylresorcinol CRS in alcohol R and dilute to 10 ml with the
same solvent.
Reference solution (b). Dissolve 10 mg of hexylresorcinol CRS and 10 mg of resorcinol R in alcohol R and
Apply separately to the plate 10 l of each solution. Develop over a path corresponding to two thirds
of the plate height with a mixture of equal volumes of methyl ethyl ketone R and pentane R. Allow the
plate to dry in air for 5 min. Spray the plate with 3 ml of anisaldehyde solution R and heat at 100C to
105C for 5 min. The principal spot in the chromatogram obtained with the test solution is similar in
position, colour and size to the principal spot in the chromatogram obtained with reference solution
clearly separated principal spots.
D. Dissolve 0.1 g in 1 ml of alcohol R. Add one drop of ferric chloride solution R1. A green colour is
produced. Add dilute ammonia R1. The solution becomes brown.
TESTS
Solution S Dissolve 1.0 g in alcohol R and dilute to 10.0 ml with the same solvent.
Acidity Dissolve 0.5 g in a mixture of 25 ml of carbon dioxide-free water R and 25 ml of ether R
previously neutralised to phenolphthalein solution R1 and titrate with 0.1M sodium hydroxide, shaking
vigorously after each addition. Not more than 0.4 ml is required to change the colour of the solution.
Reference solution (b). Dissolve 20.0 mg of phenol R in the mobile phase and dilute to 100.0 ml with
the mobile phase.
19-57
Reference solution (c). Dissolve 20.0 mg of resorcinol R in the mobile phase and dilute to 100.0 ml with
the mobile phase.
Reference solution (d). To 8.0 ml of reference solution (a) add 2.0 ml of reference solution (b), 2.0 ml
of reference solution (c) and dilute to 20.0 ml with the mobile phase.
as mobile phase at a flow rate of 1 ml/min a mixture of 25 volumes of a 3.0 g/1 solution of glacial
acetic acid R, adjusted to pH 5.9 with dilute ammonia R1 and 75 volumes of methanol R,
a loop injector.
Inject 20 l of reference solution (d). Adjust the sensitivity of the system so that the heights of the
three principal peaks in the chromatogram obtained are at least 20 per cent of the full scale of the
recorder. The test is not valid unless the resolution between the second peak (phenol) and the third
peak (hexylresorcinol) is at least 5.0.
the test solution for twice the retention time of hexylresorcinol. In the chromatogram obtained with
the test solution: the areas corresponding to phenol and resorcinol are not greater than the areas of
the corresponding peaks in the chromatograms obtained with reference solution (d) (0.2 per cent);
the area of any peak, apart from the principal peak and the peaks corresponding to phenol and
resorcinol is not greater than the area of the principal peak in the chromatogram obtained with
reference solution (a) (0.5 per cent); the sum of the areas of such peaks is not greater than twice the
area of the principal peak obtained with reference solution (a) (1 per cent). Disregard any peak with
an area less than 0.1 times the area of the principal peak in the chromatogram obtained with reference
solution (a).
Water (2.5.12). Not more than 0.5 per cent determined on 1.000 g by the semi-micro determination
of water.
ASSAY
Dissolve 0.100 g in 10 ml of methanol R in a ground-glass-stoppered flask, add 30.0 ml of 0.0167M
potassium bromate and 2 g of potassium bromide R. Shake to dissolve the substance and add 15 ml of
dilute sulphuric acid R. Stopper the flask, shake and allow to stand in the dark for 15 min, stirring
continuously. Add 5 ml of methylene chloride R and a solution of 1 g of potassium iodide R in 10 ml of
water R, allow to stand in the dark for 15 min, stirring continuously. Titrate with 0.1M sodium
thiosulphate, using 1 ml of starch solution R, shaking thoroughly. Carry out a blank titration under the
same conditions.
12
H
18
O
2
.
STORAGE
Store in a airtight container, protected from light.
IMPURITIES
A. phenol,
B. resorcinol.
__________________________________________________________________________________________________________ Ph Eur
19-58
Histamine Dihydrochloride
1/01
N
HN
NH
2
,2HCl
C
5
H
9
N
3
,2HCl 184.1 56-92-8
Histamine Dihydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Histamine dihydrochloride contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of 4-(2-aminoethyl)imidazole dihydrochloride, calculated with reference to the dried
substance.
CHARACTERS
A white, crystalline powder or colourless crystals, hygroscopic, very soluble in water, soluble in
alcohol.
IDENTIFICATION
obtained with histamine dihydrochloride CRS. Examine as discs prepared using 1 mg of substance.
B. Examine the chromatograms obtained in the test for histidine. The principal spot in the chromato-
C. Dissolve 0.1 g in 7 ml of water R and add 3 ml of a 200 g/l solution of sodium hydroxide R.
Dissolve 50 mg of sulphanilic acid R in a mixture of 0.1 ml of hydrochloric acid R and 10 ml of water R
and add 0.1 ml of sodium nitrite solution R. Add the second solution to the first and mix. A red colour
is produced.
TESTS
solution Y
7
(Method II, 2.2.2).
Histidine Examine by thin-layer chromatography (2.2.27), using a TLC silica gel G plate R.
the same solvent.
Reference solution (a). Dissolve 0.1 g of histamine dihydrochloride CRS in water R and dilute to 10 ml
Reference solution (b). Dissolve 50 mg of histidine monohydrochloride R in water R and dilute to 100 ml
Reference solution (c). Mix 1 ml of test solution (a) and 1 ml of reference solution (b).
Apply to the plate 1 l of test solution (a), 1 l of test solution (b), 1 l of reference solution (a), 1 l
of reference solution (b) and 2 l of reference solution (c). Develop over a path of 15 cm using a
mixture of 5 volumes of concentrated ammonia R, 20 volumes of water R and 75 volumes of aceto-
nitrile R. Dry the plate in a current of air. Repeat the development in the same direction, dry the plate
in a current of air and spray with ninhydrin solution R1. Heat the plate at 110C for 10 min. Any spot
corresponding to histidine in the chromatogram obtained with test solution (a) is not more intense
than the spot in the chromatogram obtained with reference solution (b) (1 per cent). The test is not
spots.
19-59
Sulphates (2.4.13).3 ml of solution S diluted to 15 ml with distilled water R complies with the limit
100-105C.
ASSAY
first and third points of inflexion.
5
H
11
Cl
2
N
3
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
19-60
Histamine Phosphate
N
HN
NH
2
H
3
PO
4
,2
C
5
H
9
N
3
,2H
3
PO
4
,H
2
O 325.2 51-74-1 (anhydrous)
Histamine Phosphate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0144].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Histamine phosphate contains not less than 98.0 per cent and not more than the equivalent of
101.0 per cent of 4-(2-aminoethyl)imidazole diphosphate, calculated with reference to the anhydrous
substance.
CHARACTERS
Colourless, long prismatic crystals, freely soluble in water, slightly soluble in alcohol.
IDENTIFICATION
obtained with histamine phosphate CRS. Examine as discs prepared using 1 mg of substance.
B. Examine the chromatograms obtained in the test for histidine. The principal spot in the chromato-
C. Dissolve 0.1 g in 7 ml of water R and add 3 ml of a 200 g/l solution of sodium hydroxide R.
and add 0.1 ml of sodium nitrite solution R. Add the second solution to the first and mix. A red colour
is produced.
D. It gives reaction (a) of phosphates (2.3.1).
TESTS
solution BY
7
(Method II, 2.2.2).
Histidine Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating
substance.
the same solvent.
Reference solution (a). Dissolve 0.1 g of histamine phosphate CRS in water R and dilute to 10 ml with
the same solvent.
Reference solution (b). Dissolve 50 mg of histidine monohydrochloride R in water R and dilute to 100 ml
Reference solution (c). Mix 1 ml of test solution (a) and 1 ml of reference solution (b).
Apply separately to the plate 1 l of test solution (a), 1 l of test solution (b), 1 l of reference
solution (a), 1 l of reference solution (b) and 2 l of reference solution (c). Develop over a path of
15 cm using a mixture of 5 volumes of concentrated ammonia R, 20 volumes of water R and 75
volumes of acetonitrile R. Dry the plate in a current of air. Repeat the development in the same
direction, dry the plate in a current of air and spray with ninhydrin solution R1. Heat at 110C for
10 min. Any spot corresponding to histidine in the chromatogram obtained with test solution (a) is
not more intense than the spot in the chromatogram obtained with reference solution (b) (1 per
cent). The test is not valid unless the chromatogram obtained with reference solution (c) shows two
clearly separated spots.
19-61
of water.
ASSAY
Dissolve 0.140 g in 5 ml of anhydrous formic acid R and add 20 ml of anhydrous acetic acid R. Titrate
with 0.1M perchloric acid, determining the end-point potentiometrically (2.2.20). Carry out a blank
titration.
5
H
15
N
3
O
8
P
2
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
19-62
Histidine
corrected 1/01
COOH
NH
2
H
HN
N
C
6
H
9
N
3
O
2
155.2 71-00-1
Histidine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0911]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Histidine contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of
(S)-2-amino-3-(imidazol-4-yl)propanoic acid, calculated with reference to the dried substance.
PRODUCTION
When histidine is produced by a process involving fermentation steps, it complies with the require-
CHARACTERS
A white, crystalline powder or colourless crystals, soluble in water, very slightly soluble in alcohol,
IDENTIFICATION
obtained with histidine CRS. Examine the substances prepared as discs. If the spectra obtained show
minimum volume of water R, evaporate to dryness at 60C and record new spectra using the
residues.
D. Dissolve 0.1 g in 7 ml of water R and add 3 ml of a 200 g/l solution of sodium hydroxide R.
and add 0.1 ml of sodium nitrite solution R. Add the second solution to the first and mix. An orange-
red colour develops.
TESTS
Solution S Dissolve 2.5 g in distilled water R, heating in a water-bath and dilute to 50 ml with the
same solvent.
solution BY
7
(Method II, 2.2.2).
Specific optical rotation (2.2.7). Dissolve 2.75 g in 12.0 ml of hydrochloric acid R1 and dilute to
25.0 ml with water R. The specific optical rotation is +11.8 to +12.8, calculated with reference to
gel plate R.
the same solvent.
Reference solution (a). Dissolve 10 mg of histidine CRS in water R and dilute to 50 ml with the same
solvent.
Reference solution (c). Dissolve 10 mg of histidine CRS and 10 mg of proline CRS in water R and dilute
19-63
Apply separately to the plate 5 l of each solution. Dry the plate in a current of air. Develop over
a path of 15 cm using a mixture of 20 volumes of glacial acetic acid R, 20 volumes of water R and 60
volumes of butanol R. Allow the plate to dry in air. Spray with ninhydrin solution R and heat at 100C
solution (b) (0.5 per cent). The test is not valid unless the chromatogram obtained with test solution
(c) shows two clearly separated spots.
Briefly triturate with a glass rod. Immediately close the cell by putting the two watch-glasses together.
Heat at 40C for 15 min. The litmus paper is not more intensely blue coloured than a standard
prepared at the same time and in the same manner using 0.1 ml of ammonium standard solution
(100 ppm NH
4
Heavy metals (2.4.8). Dissolve 2.0 g in a mixture of 3 ml of dilute hydrochloric acid R and 15 ml of
water R, with gentle warming if necessary, and dilute to 20 ml with water R. 12 ml of the solution
100C to 105C.
ASSAY
Dissolve 0.130 g in 50 ml of water R. Titrate with 0.1M hydrochloric acid, determining the end-point
6
H
9
N
3
O
2
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
19-64
Histidine Hydrochloride Monohydrate
COOH
NH
2
H
HN
N
,HCl
C
6
H
9
N
3
O
2
,HCl,H
2
O 209.6 645-35-2 (anhydrous)
Histidine Hydrochloride Monohydrate complies with the requirements of the 3rd edition of the European
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Histidine hydrochloride monohydrate contains not less than 98.5 per cent and not more than the
equivalent of 101.0 per cent of the hydrochloride of (S)-2-amino-3-(imidazol-4-yl)propanoic acid,
PRODUCTION
When Histidine hydrochloride monohydrate is produced by a process involving fermentation steps, it
complies with the requirements of the monograph on Products of fermentation (1468).
CHARACTERS
A white, crystalline powder or colourless crystals, freely soluble in water, slightly soluble in alcohol,
IDENTIFICATION
First identification: A, B, C, F.
B. It complies with the test for the pH (see Tests).
obtained with histidine hydrochloride monohydrate CRS. Examine the substances prepared as discs.
D. Examine the chromatograms obtained in the test for ninhydrin-positive substances. The principal
E. Dissolve 0.1 g in 7 ml of water R and add 3 ml of a 200 g/l solution of sodium hydroxide R. Dissolve
50 mg of sulphanilic acid R in a mixture of 0.1 ml of hydrochloric acid R and 10 ml of water R and add
0.1 ml of sodium nitrite solution R. Add the second solution to the first and mix. A orange-red colour
develops.
F. About 20 mg gives reaction (a) of chlorides (2.3.1).
TESTS
solution BY
6
(Method II, 2.2.2).
Specific optical rotation (2.2.7). Dissolve 2.75 g in 12.0 ml of hydrochloric acid R1 and dilute to
25.0 ml with water R. The specific optical rotation is +9.2 to +10.6, calculated with reference to the
dried substance.
gel plate R.
the same solvent.
Reference solution (a). Dissolve 10 mg of histidine hydrochloride monohydrate CRS in water R and dilute
19-65
Reference solution (c). Dissolve 10 mg of histidine hydrochloride monohydrate CRS and 10 mg of proline
Apply separately to the plate 5 l of each solution. Dry the plate in a current of air. Develop over a
path of 15 cm using a mixture of 20 volumes of glacial acetic acid R, 20 volumes of water R and 60
volumes of butanol R. Allow the plate to dry in air. Spray with ninhydrin solution R and heat at 100C
(100 ppm NH
4
at 145C to 150C.
ASSAY
Dissolve 0.160 g in 50 ml of carbon dioxide-free water R. Titrate with 0.1M sodium hydroxide, deter-
mining the end-point potentiometrically (2.2.20).
6
H
10
ClN
3
O
2
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
19-66
Homatropine Hydrobromide
1/01
N
Me
H
O
O
H
OH
Ph
,HBr
*
C
16
H
21
NO
3
,HBr 356.3 51-56-9
Homatropine Hydrobromide complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Mydriatic; cycloplegic.
Preparation
Homatropine Eye Drops
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Homatropine hydrobromide contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of (1R,3r,5S)-3-[(RS)-2-hydroxy-2-phenylacetoxy]-8-methyl-8-
azabicyclo[3.2.1]octane hydrobromide, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder or colourless crystals, freely soluble in water, sparingly soluble in alcohol.
IDENTIFICATION
obtained with homatropine hydrobromide CRS.
B. Dissolve 50 mg in 1 ml of water R and add 2 ml of dilute acetic acid R. Heat and add 4 ml of picric
acid solution R. Allow to cool, shaking occasionally. Collect the crystals, wash with two quantities,
each of 3 ml, of iced water R and dry at 100C to 105C. The crystals melt (2.2.14) at 182C to
186C.
C. Dissolve about 10 mg in 1 ml of water R, add a slight excess of ammonia R and shake with 5 ml of
chloroform R. Evaporate the chloroform layer to dryness on a water-bath and add 1.5 ml of a 20 g/l
solution of mercuric chloride R in alcohol (60 per cent V/V) R. A yellow colour develops which becomes
red on warming.
D. It gives reaction (a) of bromides (2.3.1).
TESTS
solution B
9
(Method II, 2.2.2).
Apply to the plate 5 l of each solution. Develop over a path of 15 cm using a mixture of 16.5
volumes of anhydrous formic acid R, 16.5 volumes of water R and 67 volumes of ethyl acetate R. Dry
19-67
the plate at 100C to 105C until the odour of the solvent is no longer perceptible, allow to cool
and spray with dilute potassium iodobismuthate solution R until the spots appear. Any spot in the
at 100C to 105C.
ASSAY
16
H
22
BrNO
3
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
19-68
Homatropine Methylbromide
N
Me
H
O
O
H
Me
OH
Ph
+
Br
*
C
17
H
24
BrNO
3
370.3 80-49-9
Homatropine Methylbromide complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Homatropine methylbromide contains not less than 98.5 per cent and not more than the equivalent
of 101.0 per cent of (1R,3r,5S)-3-[(RS)-2-hydroxy-2-phenylacetoxy]-8,8-dimethyl-8-
azoniabicyclo[3.2.1]octane bromide, calculated with reference to the dried substance.
CHARACTERS
IDENTIFICATION
obtained with homatropine methylbromide CRS.
B. Dissolve 50 mg in 1 ml of water R and add 2 ml of dilute acetic acid R. Heat and add 4 ml of picric
acid solution R. Allow to cool, shaking occasionally. The crystals, washed with two quantities, each of
3 ml, of iced water R and dried at 100C to 105C melt (2.2.14) at 132C to 138C.
C. It gives reaction (a) of bromides (2.3.1).
TESTS
coating substance.
16.5 volumes of anhydrous formic acid R, 16.5 volumes of water R and 67 volumes of ethyl acetate R.
Dry the plate at 100C to 105C until the odour of the solvent is no longer perceptible, allow to cool
and spray with dilute potassium iodobismuthate solution R and then with dilute hydrogen peroxide
(0.5 per cent).
100C to 105C.
19-69
ASSAY
Dissolve 0.300 g in 10 ml of water R. Titrate with 0.1M silver nitrate. Determine the end-point
potentiometrically (2.2.20), using a silver indicator electrode and a silversilver chloride reference
electrode.
17
H
24
BrNO
3.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
19-70
Homoeopathic Preparations
1/01
Homoeopathic Preparations comply with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Homoeopathic preparations are prepared from substances, products or preparations called stocks, in
accordance with a homoeopathic manufacturing procedure. A homoeopathic preparation is usually
designated by the Latin name of the stock, followed by an indication of the degree of dilution.
Raw materials
Raw materials for the production of homoeopathic preparations are of botanical, chemical, mineral or
zoological origin.
A raw material of zoological origin must be shown to be free from any pathogenic agent. A raw
material of botanical or zoological origin may be used either in the fresh state or in the dried state.
Where appropriate, fresh material may be kept deep-frozen.
Where justified and authorised for transportation or storage purposes, fresh material may be kept in
alcohol, provided the whole material including the alcohol is used for processing.
Raw materials comply with any requirements of the monographs of the European Pharmacopoeia.
Vehicles
Vehicles are excipients used for the preparation of certain stocks or for the potentisation process.
They may include for example: purified water, alcohol of a suitable concentration, glycerol and
lactose.
Vehicles comply with any requirements of the monographs of the European Pharmacopoeia.
Stocks
Stocks are substances, products or preparations used as starting materials for the production of
homoeopathic preparations. A stock is usually one of the following: a mother tincture or a glycerol
macerate, for raw materials of botanical or zoological origin, or the substance itself, for raw materials
of chemical or mineral origin.
Mother tinctures
Mother tinctures are liquid preparations obtained by the solvent action of a suitable vehicle upon raw
materials of botanical or zoological origin. They may also be obtained from plant juices with or
without the addition of a vehicle.
A mother tincture is designated by the symbols TM or .
Glycerol macerates
Glycerol macerates are liquid preparations obtained from raw materials of botanical or zoological
origin by using glycerol or a mixture of glycerol and either alcohol of a suitable concentration or a
solution of sodium chloride of a suitable concentration.
Potentisation
Dilutions and triturations are obtained from stocks by a process of potentisation in accordance with a
homoeopathic manufacturing procedure: for a liquid preparation this means successive dilutions and
succussions; for a solid preparation it means successive appropriate triturations.
The potentisation steps are usually one of the following:
1 part of the stock plus 9 parts of the vehicle; they may be designated as D, DH or X
(decimal),
1 part of the stock plus 99 parts of the vehicle; they may be designated as C or CH
(centesimal).
The number of potentisation steps defines the degree of dilution; for example, D3, 3 DH or
3X means three decimal potentisation steps, and C3, 3 CH or 3C means three centesimal
potentisation steps.
Dosage forms
A dosage form of a homoeopathic preparation complies with any relevant dosage form monograph in
the European Pharmacopoeia and with the following:
for the purpose of dosage forms for homoeopathic use active substances are considered to be
dilutions or triturations of homoeopathic stocks,
these dosage forms are prepared using appropriate excipients,
19-71
the test for uniformity of content is normally not appropriate. However, in certain
circumstances, it is required.
Homoeopathic dosage form pillule
Pillules for homoeopathic use are preparations of solid consistency obtained from sucrose, lactose or
other suitable excipients. They may be prepared by impregnation of preformed pillules with a
dilution or dilutions of homeopathic stocks or by progressive addition of these excipients and the
addition of a dilution or dilutions of homoeopathic stocks. They are intended for oral or sublingual
use.
Homoeopathic dosage form tablet
Tablets for homeopathic use are preparations from sucrose, lactose or other suitable excipients
according to the monograph on Tablets (0478). They may in addition be prepared by impregnation of
preformed tablets with a dilution or dilutions of homeopathic stocks. The preformed tablets for
impregnation are obtained from sucrose, lactose or other suitable excipients according to the mono-
graph on Tablets (0478). They are intended for oral or sublingual use.
__________________________________________________________________________________________________________ Ph Eur
19-72
Purified Honey
Definition Purified Honey is obtained by purification of the honey from the comb of the bee, Apis
mellifera L. and other species of Apis.
Production The honey is extracted by centrifugation, pressure or other suitable procedure, melted
at not more than 80 and allowed to stand; the impurities that rise to the surface are skimmed off and
Water is added until the product complies with the requirement for Weight per ml.
Characteristics A thick, syrupy, translucent, pale yellow or yellowish brown liquid; odour, pleasant
and characteristic, becoming more pronounced but otherwise unchanged when heated on a water
bath; taste, sweet and characteristic, varying according to the floral origin.
Optical rotation +0.6 to 3.0, Appendix V F, measured in a 20% w/v solution containing 0.2%
v/v of 13.5M ammonia after decolorising with activated charcoal, if necessary.
Chlorides 15 ml of a 1.0% w/v solution complies with the limit test for chlorides, Appendix VII
(350 ppm).
Sulphates 15 ml of a 4.0% w/v solution complies with the limit test for sulphates, Appendix VII
(240 ppm).
19-73
Hop Strobile
Hop Strobile complies with the requirements of the 3rd edition of the European Pharmacopoeia [1222]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hop strobile consists of the dried, generally whole, female inflorescences of Humulus lupulus L.
CHARACTERS
It has a characteristic, aromatic odour.
IDENTIFICATION
A. Hop strobiles are generally isolated and 2 cm to 5 cm long, petiolate, ovoid, made up of many
oval, greenish-yellow, sessile, membranous, overlapping bracts. The external bracts are flattened and
symmetrical. The internal bracts are longer and asymmetrical at the base because of a fold generally
encircling an induviate fruit (achene). The ovary or rarely the fruit, the base of the bracts and
especially the induvial fold, are covered with small orange-yellow glands.
chloral hydrate solution R. The powder shows the following diagnostic characters: fragments of bracts
and bracteoles covered by polygonal, irregular epidermal cells with wavy walls; unicellular, conical,
straight or curved covering trichomes with thin, smooth walls; rare anomocytic stomata; fragments of
mesophyll containing small calcium oxalate cluster crystals; many characteristic orange-yellow
glandular trichomes with short, bicellular biseriate stalks, bearing a part widening into a cup, 150 m
to 250 m in diameter, made up of a hemispherical layer of secretory cells with a cuticle that has been
detached and distended by the accumulation of oleoresinous secretions; fragments of elongated
sclerenchymatous cells of the testa with thick walls showing striations and numerous pits.
Test solution. To 1.0 g of the freshly powdered drug (355), add 10 ml of a mixture of 3 volumes of
water R and 7 volumes of methanol R; shake for 15 min and filter.
Reference solution. Dissolve 1.0 mg of Sudan orange R, 2.0 mg of curcumin R and 2.0 mg of
dimethylaminobenzaldehyde R in 20 ml of methanol R.
Apply separately to the plate as bands, 20 l of each solution. Develop over a path of 15 cm using a
mixture of 2 volumes of anhydrous acetic acid R, 38 volumes of ethyl acetate R and 60 volumes of
cyclohexane R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The chromato-
gram obtained with the reference solution shows three quenching bands; in the lower quarter is the
faint curcumin band, somewhat below the middle is the dimethylaminobenzaldehyde band and above
the band due to Sudan orange. The chromatogram obtained with the test solution shows a number
of quenching bands similar in position to the bands in the chromatogram obtained with the reference
solution; at about the level of curcumin is a faint band due to xanthohumol, near the level of
dimethylaminobenzaldehyde are bands due to humulones, and near the level of Sudan red are bands
due to lupulones. Examine in ultraviolet light at 365 nm. In the chromatogram obtained with the test
solution the bands due to lupulones show blue fluorescence, the bands due to humulones brown
fluorescence and the band due to xanthohumol dark brown fluorescence. Spray with dilute phospho-
molybdotungstic reagent R. Expose the plate to ammonia vapour. Examine in daylight. In the chroma-
togram obtained with the test solution, the bands due to humulones and to lupulones are bluish-grey
and the band due to xanthohumol is greenish-grey. In the chromatogram obtained with the reference
solution, the bands are bluish-grey to brownish-grey.
TESTS
Matter extractable by alcohol (70 per cent V/V) To 10.0 g of the powdered drug (355) add
300 ml of alcohol (70 per cent V/V) R and heat for 10 min on a water bath under a reflux condenser.
Allow to cool, filter and discard the first 10 ml of the filtrate. Evaporate 30.0 ml of the filtrate to
dryness on a water-bath and dry in an oven at 100C to 105C for 2 h. The mass of the residue is not
less than 0.250 g (25.0 per cent).
19-74
STORAGE
__________________________________________________________________________________________________________ Ph Eur
19-75
Hyaluronidase
Hyaluronidase complies with the requirements of the 3rd edition of the European Pharmacopoeia [0912]. These
Action and use Depolymerising enzyme used as a spreading factor.
Preparation
Hyaluronidase Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hyaluronidase is an enzyme extracted from mammalian testes (for example bovine testes) and
capable of hydrolysing mucopolysaccharides of the hyaluronic acid type. It contains not less than
300 I.U. of hyaluronidase activity per milligram, calculated with reference to the dried substance. It
may contain a suitable stabiliser.
PRODUCTION
The animals from which hyaluronidase is derived must fulfil the requirements for the health of
animals suitable for human consumption to the satisfaction of the competent authority.
Hyaluronidase is produced using a validated method of extraction and purification in conditions
designed to minimise the degree of microbial contamination; the method of preparation must have
been shown to reduce any contamination by viruses or other known infectious agents to acceptable
limits.
CHARACTERS
A white or yellowish-white, amorphous powder, soluble in water, practically insoluble in acetone, in
ethanol and in ether.
IDENTIFICATION
A solution containing the equivalent of 100 I.U. of hyaluronidase in 1 ml of a 9 g/l solution of sodium
chloride R depolymerises an equal volume of a 10 g/l solution of sodium hyaluronate BRP in 1 min at
20C as shown by a pronounced decrease in viscosity. This action is destroyed by heating the
hyaluronidase at 100C for 30 min.
TESTS
Appearance of solution Dissolve 0.10 g of the substance to be examined in water R and dilute to
10 ml. The solution is clear (2.2.1).
pH (2.2.3). Dissolve 30 mg in 10 ml of carbon dioxide-free water R. The pH of the solution is 4.5 to
7.5.
Bacterial endotoxins (2.6.14). The endotoxin limit concentration is 0.2 I.U. of endotoxin per
International Unit of hyaluronidase activity.
ASSAY
The activity of hyaluronidase is determined by comparing the rate at which it hydrolyses sodium
hyaluronate BRP with the rate obtained with the International Standard, or a reference preparation
calibrated in International Units, using a slope-ratio assay.
Substrate solution. To 0.10 g of sodium hyaluronate BRP in a 25 ml conical flask add slowly 20.0 ml of
water R at 4C. The rate of addition must be slow enough to allow the substrate particles to swell
(about 5 min). Maintain at 4C and stir for at least 12 h. Store at 4C and use within 4 days.
Test solution. Dissolve a suitable amount of the substance to be examined in hyaluronidase diluent R so
as to obtain a solution containing 0.6 0.3 I.U. of hyaluronidase per millilitre.
Reference solution. Dissolve a suitable amount of hyaluronidase BRP in hyaluronidase diluent R so as to
obtain a solution containing 0.6 I.U. of hyaluronidase per millilitre.
In a reaction vessel, mix 1.50 ml of phosphate buffer solution pH 6.4 R and 1.0 ml of the substrate
solution and equilibrate at 37 0.1C. At time t
1
=

0 (first chronometer) add 0.50 ml of the test
solution containing E
t
mg of the enzyme to be examined, mix, measure the viscosity of the solution
19-76
using a suitable viscometer maintained at 37 0.1C and record the outflow time t
2
using a second
chronometer (graduated in 0.1 second intervals), several times during about 20 min (read on the first
chronometer). The following viscometer has been found suitable: Ubbelohde microviscometer (DIN
51 562, Part 2), capillary type MII, viscometer constant about 0.1 mm
2
/s
2
.
Repeat the procedure using 0.50 ml of the reference solution containing E
r
mg of hyaluronidase
BRP.
Calculate the viscosity ratio from the expression:
6915 0
2
.
t k
r
k=the viscometer constant in mm

2
/s
2
(indicated on the viscometer),
t
2
=the outflow time (in seconds) of the solution.
0.6915=the kinematic viscosity in mm
2
/s

of the buffer solution at 37C.
Since the enzymatic reaction continues during the outflow time measurements, the real reaction time
equals t
1
+t
2
/2, half of the outflow time (t
2
/2) for which a certain measurement is valid being added to
the time t
1
at which the measurement is started. Plot (ln
r
)
1
as a function of the reaction time
(t
1
+t
2
/2) in seconds. A linear relationship is obtained. Calculate the slope for the substance to be
examined (b
t
) and the reference preparation (b
r
).
Calculate the specific activity in International Units per milligram from the expression:
A
E
E
b
b

t
r
r
t
A=the specific activity of hyaluronidase BRP in International Units per milligram.
Carry out the complete procedure at least three times and calculate the average activity of the
STORAGE
Store in an airtight container at a temperature of 2C to 8C. If the substance is sterile, store in a
sterile, tamper-proof container.
LABELLING
The label states:
the activity in International Units per milligram,
__________________________________________________________________________________________________________ Ph Eur
19-77
Hydralazine Hydrochloride
N
N
NHNH
2
,HCl
C
8
H
8
N
4
,HCl 196.6 304-20-1
Hydralazine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparations
Hydralazine Injection
Hydralazine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hydralazine hydrochloride contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of 1-hydrazinophthalazine hydrochloride, calculated with reference to the dried
substance.
CHARACTERS
A white or almost white, crystalline powder, soluble in water, slightly soluble in alcohol, very slightly
IDENTIFICATION
A. Dissolve 50 mg in water R and dilute to 100 ml with the same solvent. Dilute 2 ml of the solution
to 100 ml with water R. Examined between 220 nm and 350 nm (2.2.25), the solution shows four
absorption maxima, at 240 nm, 260 nm, 303 nm and 315 nm. The ratio of the absorbance measured
at the maximum at 240 nm to that measured at the maximum at 303 nm is 2.0 to 2.2.
hydralazine hydrochloride CRS. Examine the substances prepared as discs.
C. Dissolve 0.5 g in a mixture of 8 ml of dilute hydrochloric acid R and 100 ml of water R. Add 2 ml of
sodium nitrite solution R, allow to stand for 10 min and filter. The precipitate, washed with water R
and dried at 100C to 105C, melts (2.2.14) at 209C to 212C.
D. Dissolve about 10 mg in 2 ml of water R. Add 2 ml of a 20 g/l solution of nitrobenzaldehyde R in
alcohol R. An orange precipitate is formed.
TESTS
(2.2.1) and not more intensely coloured than reference solution GY
6
(Method II, 2.2.2).
Reference solution (c). Dissolve 25.0 mg of phthalazine R in the mobile phase and dilute to 50.0 ml
with the mobile phase. Dilute 4.0 ml of this solution to 100.0 ml with the mobile phase.
Reference solution (d). Dilute a mixture of 4.0 ml of the test solution and 10.0 ml of reference solution
(c) to 100.0 ml with the mobile phase.
The solutions must be analysed within one working day. The chromatographic procedure may be
carried out using:
19-78
a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with nitrile silica
gel for chromatographey R1 (10 m),
as mobile phase at a flow rate of 1 ml per minute a solution prepared as follows: to 22 volumes
of acetonitrile R add 78 volumes of a solution containing 1.44 g of sodium lauryl sulphate R and
0.75 g of tetrabutylammonium bromide R per litre and adjust the mixture to pH 3.0 with 0.05M
sulphuric acid,
Inject 20 l of reference solution (a) and adjust the sensitivity of the detector so that the height of
the principal peak in the chromatogram is not less than 70 per cent of the full scale of the recorder.
When the chromatograms are recorded in the prescribed conditions, the retention time of hydralazine
is about 10 min to 12 min. If necessary, adjust the concentration of acetonitrile in the mobile phase.
Inject 20 l of the test solution and continue the chromatography for three times the retention time of
hydralazine. Inject 20 l of reference solution (b). The area of any peak in the chromatogram
obtained with the test solution, apart from the peaks due to the solvent and to hydralazine, is not
greater than the area of the peak in the chromatogram obtained with reference solution (b) (0.2 per
cent).
The test is not valid unless: the chromatogram obtained with reference solution (d) shows two
principal peaks and the resolution between the peaks is at least 2.5; the principal peak in the chroma-
togram obtained with reference solution (b) has a signal- to-noise ratio of at least 3.
Hydrazine Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating
substance.
Test solution. Dissolve 0.12 g of the substance to be examined in 4 ml of water R and add 4 ml of a
150 g/l solution of salicylaldehyde R in methanol R and 0.2 ml of hydrochloric acid R. Mix and keep at a
temperature not exceeding 25C for 2 h to 4 h, until the precipitate formed has sedimented. Add
4 ml of toluene R, shake vigorously and centrifuge. Transfer the clear supernatant liquid to a 100 ml
separating funnel, separate the toluene layer and shake vigorously, each time for 3 min, with two
quantities, each of 20 ml, of a 200 g/l solution of sodium metabisulphite R and with two quantities,
each of 50 ml, of water R. Separate the toluene layer which is the test solution.
Reference solution (a). Dissolve 12 mg of hydrazine sulphate R in dilute hydrochloric acid R and dilute to
100.0 ml with the same acid. Dilute 1.0 ml of this solution to 100.0 ml with dilute hydrochloric acid R
Reference solution (b). Prepare the solution at the same time and in the same manner as described for
the test solution, using 1.0 ml of reference solution (a) and 3 ml of water R.
Apply separately to the plate 20 l of the test solution and 20 l of reference solution (b). Develop
over a path of 10 cm using a mixture of 10 volumes of alcohol R and 90 volumes of toluene R. Allow
the plate to dry in air and examine in ultraviolet light at 365 nm. Any spot showing yellow
fluorescence in the chromatogram obtained with the test solution is not more intense than the
corresponding spot in the chromatogram obtained with reference solution (b) (10 ppm of hydrazine).
ASSAY
Dissolve 80.0 mg in 25 ml of water R. Add 35 ml of hydrochloric acid R and titrate with 0.05M
potassium iodate, determining the end-point potentiometrically (2.2.20), using a calomel reference
electrode and a platinum indicator electrode.
8
H
9
ClN
4
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
20-1
Hydrochloric Acid
1/01
HCl 36.46 7647-01-0
Hydrochloric Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Concentrated Hydrochloric Acid [0002]. These requirements are reproduced after the heading Definition
below.
Preparation
Dilute Hydrochloric Acid
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Concentrated hydrochloric acid contains not less than 35.0 per cent m/m and not more than 39.0 per
cent m/m of HCl.
CHARACTERS
A clear, colourless, fuming liquid, miscible with water.
IDENTIFICATION
A. Dilute with water R. The solution is strongly acid (2.2.4).
B. It gives the reactions of chlorides (2.3.1).
TESTS
(Method II, 2.2.2).
Free chlorine To 15 ml add 100 ml of carbon dioxide-free water R, 1 ml of a 100 g/l solution of
potassium iodide R and 0.5 ml of iodide-free starch solution R. Allow to stand in the dark for 2 min. Any
blue colour disappears on the addition of 0.2 ml of 0.01M sodium thiosulphate (4 ppm).
Sulphates (2.4.13). To 6.4 ml add 10 mg of sodium hydrogen carbonate R and evaporate to dryness
on a water-bath. Dissolve the residue in 15 ml of distilled water R. The solution complies with the
Heavy metals (2.4.8). Dissolve the residue obtained in the test for residue on evaporation in 1 ml of
dilute hydrochloric acid R and dilute to 25 ml with water R. Dilute 5 ml of this solution to 20 ml with
Residue on evaporation Not more than 10 mg (0.01 per cent), determined on 100 g.
ASSAY
Weigh accurately a ground-glass-stoppered flask containing 30 ml of water R. Introduce 1.5 ml of the
acid and weigh again. Titrate with 1M sodium hydroxide, using methyl red solution R as indicator.
1 ml of 1M sodium hydroxide is equivalent to 36.46 mg of HCl.
STORAGE
Store in a stoppered container made of glass or another inert material, at a temperature below 30C.
__________________________________________________________________________________________________________ Ph Eur
20-2
Dilute Hydrochloric Acid
1/01
Dilute Hydrochloric Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Dilute hydrochloric acid contains 9.5 per cent m/m to 10.5 per cent m/m of HCl (36.46).
PREPARATION
To 274 g of concentrated hydrochloric acid add 726 g of water R and mix.
IDENTIFICATION
A. It is strongly acid (2.2.4).
B. It gives the reactions of chlorides (2.3.1).
TESTS
Free chlorine To 60 ml add 50 ml of carbon dioxide-free water R, 1 ml of a 100 g/l solution of
potassium iodide R and 0.5 ml of iodide-free starch solution R. Allow to stand in the dark for 2 min. Any
blue colour disappears on the addition of 0.2 ml of 0.01M sodium thiosulphate (1 ppm).
Sulphates (2.4.13). To 26 ml add 10 mg of sodium hydrogen carbonate R and evaporate to dryness on
a water-bath. Dissolve the residue in 15 ml of distilled water R. The solution complies with the limit
Heavy metals (2.4.8). Dissolve the residue obtained in the test for residue on evaporation in 1 ml of
dilute hydrochloric acid R and dilute to 25 ml with water R. Dilute 5 ml of this solution to 20 ml with
Residue on evaporation Not more than 10 mg (0.01 per cent), determined on 100 g.
ASSAY
To 6.00 g add 30 ml of water R. Titrate with 1M sodium hydroxide, using methyl red solution R as
indicator.
1 ml of 1M sodium hydroxide is equivalent to 36.46 mg of HCl.
__________________________________________________________________________________________________________ Ph Eur
20-3
Hydrochlorothiazide
S
NH
N
O O
S
H
2
N
O O
Cl
H
C
7
H
8
ClN
3
O
4
S
2
297.7 58-93-5
Hydrochlorothiazide complies with the requirements of the 3rd edition of the European Pharmacopoeia [0394].
Preparations
Co-amilozide Oral Solution
Co-amilozide Tablets
Co-triamterzide Tablets
Hydrochlorothiazide Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hydrochlorothiazide contains not less than 98.0 per cent and not more than the equivalent of
102.0 per cent of 6-chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulphonamide 1,1-dioxide,
CHARACTERS
A white or almost white, crystalline powder, very slightly soluble in water, soluble in acetone, spar-
ingly soluble in alcohol. It dissolves in dilute solutions of alkali hydroxides.
IDENTIFICATION
A. Dissolve 50.0 mg in 10 ml of 0.1M sodium hydroxide and dilute to 100.0 ml with water R. Dilute
350 nm (2.2.25), the solution shows absorption maxima at 273 nm and at 323 nm. The ratio of the
absorbance measured at the maximum at 273 nm to that measured at 323 nm is 5.4 to 5.7.
obtained with hydrochlorothiazide CRS.
Test solution. Dissolve 50 mg in acetone R and dilute to 10 ml with the same solvent.
Reference solution (a). Dissolve 50 mg of hydrochlorothiazide CRS in acetone R and dilute to 10 ml with
the same solvent.
Reference solution (b). Dissolve 25 mg of chlorothiazide R in reference solution (a) and dilute to 5 ml
with the same reference solution.
Apply separately to the plate 2 l of each solution. Develop over a path of 10 cm using ethyl acetate R.
Dry the plate in a current of air and examine in ultraviolet light at 254 nm. The principal spot in the
D. Gently heat about 1 mg with 2 ml of a freshly prepared 0.5 g/l solution of chromotropic acid, sodium
salt R in a cooled mixture of 35 volumes of water R and 65 volumes of sulphuric acid R. A violet
colour develops.
TESTS
Acidity or alkalinity Shake 0.5 g of the powdered substance to be examined with 25 ml of water R
for 2 min and filter. To 10 ml of the filtrate, add 0.2 ml of 0.01M sodium hydroxide and 0.15 ml of
methyl red solution R. The solution is yellow. Not more than 0.4 ml of 0.01M hydrochloric acid is
20-4
Test solution. Dissolve 30.0 mg of the substance to be examined in 5 ml of a mixture of equal volumes
of acetonitrile R and methanol R, using sonication if necessary, and dilute to 20.0 ml with phosphate
buffer solution pH 3.2 R1.
Solvent solution. Dilute 50.0 ml of a mixture of equal volumes of acetonitrile R and methanol R to
200.0 ml with phosphate buffer solution pH 3.2 R1.
Reference solution (a). Dissolve 15.0 mg of hydrochlorothiazide CRS and 15.0 mg of chlorothiazide CRS
in 25.0 ml of a mixture of equal volumes of acetonitrile R and methanol R, using sonication if
necessary, and dilute to 100.0 ml with phosphate buffer solution pH 3.2 R1. Dilute 5.0 ml to 100.0 ml
with the solvent solution.
Reference solution (b). Dilute 1.0 ml of the test solution to 50.0 ml with the solvent solution. Dilute
5.0 ml of this solution to 20.0 ml with the solvent solution.
as mobile phase at a flow rate of 0.8 ml per minute a linear gradient programme using the
following conditions:
Mobile phase A. To 940 ml of phosphate buffer solution pH 3.2 R1 add 60.0 ml of methanol R and
10.0 ml of tetrahydrofuran R and mix,
Mobile phase B. To a mixture of 500 ml of methanol R and 500 ml of phosphate buffer solution pH
3.2 R1 add 50.0 ml of tetrahydrofuran R and mix,
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
50=0 100 0 Return to initial
eluent
composition
Equilibrate the column for at least 20 min with mobile phase A. Adjust the sensitivity of the system
so that the height of the principal peak in the chromatogram obtained with 10 l of reference solution
Inject 10 l of reference solution (a). When the chromatogram is recorded in the prescribed condi-
tions, the retention times are: chlorothiazide about 7 min and hydrochlorothiazide about 8 min. The
test is not valid unless the resolution between the peaks corresponding to chlorothiazide and hydro-
chlorothiazide is at least 2.5. If necessary, adjust slightly the composition of the mobile phase or the
time programme of the linear gradient.
Inject separately 10 l of the solvent solution as a blank, 10 l of the test solution and 10 l of
with reference solution (b) (1 per cent). Disregard any peak due to the solvent solution and any peak
Chlorides (2.4.4). Dissolve 1.0 g in 25 ml of acetone R and dilute to 30 ml with water R. 15 ml
complies with the limit test for chlorides (100 ppm). Prepare the standard using 5 ml of acetone R
containing 15 per cent V/V of water R and 10 ml of chloride standard solution (5 ppm Cl) R.
100C to 105C.
ASSAY
Dissolve 0.120 g in 50 ml of dimethyl sulphoxide R. Titrate with 0.1M tetrabutylammonium hydroxide,
determining the end-point potentiometrically (2.2.20) at the second point of inflexion.
7
H
8
ClN
3
O
4
S
2
.
IMPURITIES
A. chlorothiazide,
20-5
S
NH
2
NH
2
O O
S
H
2
N
O O
Cl
B. 4-amino-6-chlorobenzene-1,3-disulphonamide (salamide),
S
NH
N
H
O O
S
O O
Cl
N
H
N Cl
O O
H
2
N
S
O O
S
C. 6-chloro-N-[(6-chloro-7-sulphamoyl-2,3-dihydro-4H-1,2,4-benzothiadiazin-4-
yl 1,1-dioxide)methyl]-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulphonamide
1,1-dioxide.
__________________________________________________________________________________________________________ Ph Eur
20-6
Hydrocortisone
H
O
HO
H
OH
OH
Me
Me
H
O
C
21
H
30
O
5
362.5 50-23-7
Hydrocortisone complies with the requirements of the 3rd edition of the European Pharmacopoeia [0335]. These
Preparations
Hydrocortisone Cream
Hydrocortisone and Clioquinol Cream.
Hydrocortisone and Neomycin Cream
Hydrocortisone Ointment
Hydrocortisone and Clioquinol Ointment
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hydrocortisone contains not less than 97.0 per cent and not more than the equivalent of 103.0 per
cent of 11,17,21-trihydroxypregn-4-ene-3,20-dione, calculated with reference to the dried
substance.
CHARACTERS
acetone and in alcohol, slightly soluble in methylene chloride.
IDENTIFICATION
obtained with hydrocortisone CRS. If the spectra obtained in the solid state show differences, dissolve
the substance to be examined and the reference substance separately in the minimum volume of
acetone R, evaporate to dryness on a water-bath and record new spectra using the residues.
Reference solution (a). Dissolve 20 mg of hydrocortisone CRS in a mixture of 1 volume of methanol R
Reference solution (b). Dissolve 10 mg of prednisolone CRS in reference solution (a) and dilute to 10 ml
volumes of methylene chloride R. Develop over a path of 15 cm. Carry out a second development over
a path of 15 cm using a mixture of 5 volumes of butanol R saturated with water R, 15 volumes of
toluene R and 80 volumes of ether R. Allow the plate to dry in air. Examine in ultraviolet light at
alcoholic solution of sulphuric acid R. Heat at 120C for 10 min or until the spots appear. Allow to cool.
Examine the chromatograms in daylight and in ultraviolet light at 365 nm. The principal spot in the
chromatogram obtained with the test solution is similar in position, colour in daylight, fluorescence in
ultraviolet light at 365 nm and size to the principal spot in the chromatogram obtained with reference
20-7
with the same solvent. This solution is also used to prepare test solution (b). Dilute 2 ml of the
Test solution (b). Transfer 0.4 ml of the solution obtained during preparation of test solution (a) to a
glass tube 100 mm long and 20 mm in diameter and fitted with a ground-glass stopper or a polytetra-
fluoroethylene cap and evaporate the solvent with gentle heating under a stream of nitrogen R. Add
2 ml of a 15 per cent V/V solution of glacial acetic acid R and 50 mg of sodium bismuthate R. Stopper
the tube and shake the suspension for 1 h in a mechanical shaker, protected from light. Add 2 ml of a
15 per cent V/V solution of glacial acetic acid R and filter into a 50 ml separating funnel, washing the
filter with two quantities, each of 5 ml, of water R. Shake the clear filtrate with 10 ml of methylene
chloride R. Wash the organic layer with 5 ml of 1M sodium hydroxide and two quantities, each of 5 ml,
of water R. Dry over anhydrous sodium sulphate R.
Reference solution (a). Dissolve 25 mg of hydrocortisone CRS in methanol R and dilute to 5 ml with the
same solvent. This solution is also used to prepare reference solution (b). Dilute 2 ml of the solution
to 10 ml with methylene chloride R.
Reference solution (b). Transfer 0.4 ml of the solution obtained during preparation of reference
solution (a) to a glass tube 100 mm long and 20 mm in diameter and fitted with a ground-glass
stopper or a polytetrafluoroethylene cap and evaporate the solvent with gentle heating under a stream
of nitrogen R. Add 2 ml of a 15 per cent V/V solution of glacial acetic acid R and 50 mg of sodium
bismuthate R. Stopper the tube and shake the suspension for 1 h in a mechanical shaker protected
from light. Add 2 ml of a 15 per cent V/V solution of glacial acetic acid R and filter into a 50 ml
separating funnel, washing the filter with two quantities, each of 5 ml, of water R. Shake the clear
filtrate with 10 ml of methylene chloride R. Wash the organic layer with 5 ml of 1M sodium hydroxide
and two quantities, each of 5 ml, of water R. Dry over anhydrous sodium sulphate R.
Apply separately to the plate 5 l of test solution (a), 5 l of reference solution (a), 25 l of test
solution (b) and 25 l of reference solution (b), applying the latter two in small quantities to obtain
small spots. Prepare the mobile phase by adding a mixture of 1.2 volumes of water R and 8 volumes
of methanol R to a mixture of 15 volumes of ether R and 77 volumes of methylene chloride R. Develop
over a path of 15 cm. Carry out a second development over a path of 15 cm using a mixture of 5
volumes of butanol R saturated with water R, 15 volumes of toluene R and 80 volumes of ether R.
Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in each of
the chromatograms obtained with the test solutions is similar in position and size to the principal spot
in the chromatogram obtained with the corresponding reference solution. Spray with alcoholic solution
of sulphuric acid R and heat at 120C for 10 min or until the spots appear. Allow to cool. Examine the
plate in daylight and in ultraviolet light at 365 nm. The principal spot in each of the chromatograms
obtained with the test solutions is similar in position, colour in daylight, fluorescence in ultraviolet
light at 365 nm and size to the principal spot in the chromatogram obtained with the corresponding
reference solution. The principal spots in the chromatograms obtained with test solution (b) and
reference solution (b) have an R
f
value distinctly higher than that of the principal spots in the chrom-
atograms obtained with test solution (a) and reference solution (a).
brownish-red colour develops with a green fluorescence which is particularly intense when examined
in ultraviolet light at 365 nm. Add the solution to 10 ml of water R and mix. The colour fades and a
clear solution remains. The fluorescence in ultraviolet light does not disappear.
TESTS
substance.
before use.
Test solution. Dissolve 25.0 mg of the substance to be examined in 2 ml of tetrahydrofuran R and
Reference solution (a). Dissolve 2 mg of hydrocortisone CRS and 2 mg of prednisolone CRS in the mobile
deactivated end-capped octadecylsilyl silica gel for chromatography R (5 m),
volumetric flask mix 220 ml of tetrahydrofuran R with 700 ml of water R and allow to equilibrate;
dilute to 1000 ml with water R and mix again;
20-8
conditions, the retention times are: prednisolone, about 14 min; hydrocortisone, about 15.5 min.
The test is not valid unless the resolution between the peaks due to prednisolone and hydrocortisone
is at least 2.2. If necessary, adjust the concentration of tetrahydrofuran R in the mobile phase.
Inject separately 20 l of the solvent mixture of the test solution as a blank, 20 l of the test
solution and 20 l of reference solution (b). Continue the chromatography of the test solution for
four times the retention time of the principal peak. In the chromatogram obtained with the test
solution: the area of any peak, apart from the principal peak, is not greater than half the area of the
principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent): the sum of
any peak obtained with the blank run and any peak with an area less than 0.05 times the area of the
100C to 105C.
ASSAY
21
H
30
O
5
STORAGE
IMPURITIES
A. prednisolone,
B. cortisone,
C. hydrocortisone acetate,
O
OH
Me
H
H
H
Me
HO
O
OH
OH
D. 6,11,17,21-tetrahydroxypregn-4-ene-3,20-dione (6-hydroxyhydrocortisone),
O
Me
H
H
H
Me
HO
O
OH
OH
E. 11,17,21-trihydroxypregna-4,6-diene-3,20-dione (
6
-hydrocortisone),
O
Me
H
H
H
Me
O
OH
OH
F. 17,21-dihydroxypregn-4-ene-3,20-dione (Reichsteins substance),
O
Me
H
H
H
Me
HO
O
H
OH
O
G. 11,17-dihydroxy-3,20-dioxopregn-4-en-21-al.
__________________________________________________________________________________________________________ Ph Eur
20-9
Hydrocortisone Acetate
H
O
HO
H
OH
Me
Me
H
H
O
OAc
C
23
H
32
O
6
404.5 50-03-3
Hydrocortisone Acetate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparations
Hydrocortisone Acetate Cream
Hydrocortisone Acetate and Neomycin Ear Drops
Hydrocortisone Acetate and Neomycin Eye Drops
Hydrocortisone Acetate and Neomycin Eye Ointment
Hydrocortisone Acetate Injection
Hydrocortisone Acetate Ointment
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hydrocortisone acetate contains not less than 97.0 per cent and not more than the equivalent of
103.0 per cent of 11,17,21-trihydroxypregn-4-ene-3,20-dione 21-acetate, calculated with reference
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water, slightly soluble in ethanol
IDENTIFICATION
obtained with hydrocortisone acetate CRS.
Reference solution (a). Dissolve 20 mg of hydrocortisone acetate CRS in a mixture of 1 volume of
solvents.
10 ml with reference solution (a).
20-10
with the same solvent. This solution is also used to prepare test solution (b). Dilute 2 ml of the
methanolic potassium hydrogen carbonate solution R and immediately pass a stream of nitrogen R briskly
through the solution for 5 min. Stopper the tube. Heat in a water-bath at 45C protected from light
for 2 h 30 min. Allow to cool.
Reference solution (a). Dissolve 25 mg of hydrocortisone acetate CRS in methanol R and dilute to 5 ml
with the same solvent. This solution is also used to prepare reference solution (b). Dilute 2 ml of the
nitrogen R briskly through the solution for 5 min. Stopper the tube. Heat in a water-bath at 45C
obtained with the corresponding reference solution. Spray with alcoholic solution of sulphuric acid R
and heat at 120C for 10 min or until the spots appear. Allow to cool. Examine in daylight and in
ultraviolet light at 365 nm. The principal spot in each of the chromatograms obtained with the test
principal spots in the chromatograms obtained with test solution (b) and reference solution (b) have
an R

value distinctly lower than that of the principal spots in the chromatograms obtained with test
solution (a) and reference solution (a).
D. Add about 2 mg to 2 ml of sulphuric acid R and shake to dissolve. Within 5 min an intense
brownish-red colour develops with a green fluorescence which is particularly intense when viewed in
ultraviolet light at 365 nm. Add the solution to 10 ml of water R and mix. The colour fades and the
fluorescence in ultraviolet light does not disappear.
TESTS
substance.
Reference solution (a). Dissolve 2 mg of hydrocortisone acetate CRS and 2 mg of cortisone acetate R in the
recorder.
conditions the retention times are: hydrocortisone acetate, about 10 min and cortisone acetate, about
12 min. The test is not valid unless the resolution between the peaks due to hydrocortisone acetate
and cortisone acetate is at least 4.2; if necessary, adjust the concentration of acetonitrile in the mobile
phase.
Inject separately 20 l of the test solution and 20 l of reference solution (b). Continue the chrom-
atography for 2.5 times the retention time of the principal peak. In the chromatogram obtained with
20-11
the principal peak in the chromatogram obtained with reference solution (b) (1.0 per cent) and not
more than one such peak has an area greater than half the area of the principal peak in the chromato-
gram obtained with reference solution (b) (0.5 per cent); the sum of the areas of all the peaks, apart
gram obtained with reference solution (b) (1.5 per cent). Disregard any peak due to the solvent and
100C to 105C.
ASSAY
solution to 100.0 ml with alcohol R. Measure the absorbance (2.2.25) at the maximum of 241.5 nm.
23
H
32
O
6
STORAGE
__________________________________________________________________________________________________________ Ph Eur
20-12
Hydrocortisone Hydrogen Succinate
H
O
HO
H
OH
Me
Me
H
H
O
O COOH
O
C
25
H
34
O
8
462.5 2203-97-6
Hydrocortisone Hydrogen Succinate complies with the requirements of the 3rd edition of the European Pharma-
copoeia [0768]. These requirements are reproduced after the heading Definition below.
Preparation
Hydrocortisone Sodium Succinate Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hydrocortisone hydrogen succinate contains not less than 97.0 per cent and not more than the
equivalent of 103.0 per cent of 11,17,21-trihydroxypregn-4-ene-3,20-dione 21-(hydrogen
butanedioate), calculated with reference to the dried substance.
CHARACTERS
A white or almost white powder, hygroscopic, practically insoluble in water, freely soluble in acetone
and in ethanol. It dissolves in dilute solutions of alkali carbonates and alkali hydroxides.
IDENTIFICATION
obtained with hydrocortisone hydrogen succinate CRS. Dry the substances before use at 100C to
105C for 3 h.
Reference solution (a). Dissolve 20 mg of hydrocortisone hydrogen succinate CRS in a mixture of 1
volume of methanol R and 9 volumes of methylene chloride R and dilute to 20 ml with the same
Reference solution (b). Dissolve 10 mg of methylprednisolone hydrogen succinate CRS in reference solu-
tion (a) and dilute to 10 ml with reference solution (a).
0.1 volumes of anhydrous formic acid R, 1 volume of ethanol R and 15 volumes of methylene chloride R.
the chromatogram obtained with reference solution (a). Spray the plate with alcoholic solution of
sulphuric acid R. Heat at 120C for 10 min or until the spots appear. Allow to cool. Examine in
daylight and in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the
test solution is similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and
size to the principal spot in the chromatogram obtained with reference solution (a). The test is not
valid unless the chromatogram obtained with reference solution (b) shows two spots which may
however not be completely separated.
20-13
and dilute to 5 ml with the same solvent. (This solution is also used to prepare test solution (b)).
15 ml glass tube with a ground-glass stopper or a polytetrafluoroethylene cap. Add 10 ml of a 0.8 g/l
solution of sodium hydroxide R in methanol R and immediately pass a stream of nitrogen R briskly
for 30 min. Allow to cool.
Reference solution (a). Dissolve 25 mg of hydrocortisone hydrogen succinate CRS in methanol R with
gentle heating and dilute to 5 ml with the same solvent. (This solution is also used to prepare refer-
ence solution (b)). Dilute 2 ml of the solution to 10 ml with methylene chloride R.
(a) to a 15 ml glass tube with a ground-glass stopper or a polytetrafluoroethylene cap. Add 10 ml of a
0.8 g/l solution of sodium hydroxide R in methanol R and immediately pass a stream of nitrogen R
briskly through the solution for 5 min. Stopper the tube. Heat in a water-bath at 45C, protected
from light, for 30 min. Allow to cool.
obtained with the corresponding reference solution. Spray the plate with alcoholic solution of sulphuric
acid R. Heat at 120C for 10 min or until the spots appear. Allow to cool. Examine in daylight and in
ultraviolet light at 365 nm. The principal spot in the chromatograms obtained with the test solutions
is similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to the
principal spot in the chromatogram obtained with the corresponding reference solution. The princi-
pal spot in each of the chromatograms obtained with test solution (b) and reference solution (b) has
an R
f
value distinctly higher than that of the principal spots in each of the chromatograms obtained
with test solution (a) and reference solution (a).
brownish-red colour develops with a green fluorescence which is particularly intense when viewed in
ultraviolet light at 365 nm. Add the solution to 10 ml of water R and mix. The colour fades and a
clear solution remains. The fluorescence in ultraviolet light does not disappear.
TESTS
Appearance of solution Dissolve 0.10 g in 5 ml of sodium hydrogen carbonate solution R. The solu-
tion is clear (2.2.1).
substance.
acetonitrile R and water R and dilute to 10.0 ml with the same solvent.
Reference solution (a). Dissolve 2 mg of hydrocortisone hydrogen succinate CRS and 2 mg of dexam-
ethasone CRS in 50 ml of acetonitrile R and dilute to 100.0 ml with water R.
Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with a mixture of equal volumes
of acetonitrile R and water R.
volumetric flask mix 330 ml of acetonitrile R with 600 ml of water R and 1.0 ml of phosphoric
acid R and allow to equilibrate; dilute to 1000 ml with water R and mix again,
recorder.
described above the retention times are: dexamethasone, about 12.5 min and hydrocortisone
hydrogen succinate, about 15 min. The test is not valid unless the resolution between the peaks
corresponding to dexamethasone and hydrocortisone hydrogen succinate is a least 5.0; if necessary,
adjust the concentration of acetonitrile in the mobile phase.
20-14
atography for twice the retention time of the principal peak. In the chromatogram obtained with
the test solution: the area of any peak, apart from the principal peak, is not greater than half the
the sum of the areas of all the peaks, apart from the principal peak, is not greater than 0.75 times
cent). Disregard any peak due to the solvent and any peak with an area less than 0.05 times the
area of the principal peak in the chromatogram obtained with reference solution (b).
at 100C to 105C.
ASSAY
Dissolve 0.100 g in alcohol R and dilute to 100.0 ml with the same solvent. Dilute 2.0 ml to 100.0 ml
with alcohol R. Measure the absorbance (2.2.25) at the maximum at 241.5 nm.
25
H
34
O
8
STORAGE
IMPURITIES
A. hydrocortisone,
B. hydrocortisone acetate.
__________________________________________________________________________________________________________ Ph Eur
Hydrocortisone Sodium Phosphate
H
O
HO
H
OH
Me
Me
H
H
O
O P O
ONa
ONa
C
21
H
29
Na
2
O
8
P 486.4 6000-74-4
Definition Hydrocortisone Sodium Phosphate is disodium 11,17-dihydroxy-3,20-dioxopregn-
4-en-21-yl orthophosphate. It contains not less than 96.0% and not more than 103.0% of
C
21
H
29
Na
2
O
8
P, calculated with reference to the anhydrous substance.
Characteristics A white or almost white powder; hygroscopic.
Freely soluble in water; practically insoluble in absolute ethanol and in chloroform.
Identification Test A may be omitted if tests B, C and D are carried out. Tests B and C may be omitted if
tests A and D are carried out.
A. The infrared absorption spectrum, Apppendix II A, is concordant with the reference spectrum of
hydrocortisone sodium phosphate (RS 386).
B. Carry out the method for thin-layer chromatography, Appendix III A, using silica gel G as the coat-
ing substance and a freshly prepared mixture of 60 volumes of butan-1-ol, 20 volumes of acetic
anhydride and 20 volumes of water as the mobile phase. Apply separately to the plate 2 l of each of
the following solutions. Solution (1) contains 0.25% w/v of the substance being examined in
methanol. Solution (2) contains 0.25% w/v of hydrocortisone sodium phosphate BPCRS in methanol.
Solution (3) is a mixture of equal volumes of solutions (1) and (2). Solution (4) is a mixture of equal
volumes of solution (1) and a 0.25% w/v solution of betamethasone sodium phosphate BPCRS in
methanol. After removal of the plate, allow it to dry in air until the solvent has evaporated, spray with
ethanolic sulphuric acid (20%), heat at 120 for 10 minutes and examine under ultraviolet light
(365 nm). The principal spot in the chromatogram obtained with solution (1) corresponds to that in
the chromatogram obtained with solution (2). The principal spot in the chromatogram obtained with
solution (3) appears as a single, compact spot and the chromatogram obtained with solution (4)
shows two principal spots with almost identical Rf values.
C. Dissolve 2 mg in 2 ml of sulphuric acid. A yellowish green fluorescence is produced immediately
(distinction from betamethasone sodium phosphate, dexamethasone sodium phosphate and
prednisolone sodium phosphate).
D. Heat gently 40 mg with 2 ml of sulphuric acid until white fumes are evolved, add nitric acid drop-
wise until oxidation is complete and cool. Add 2 ml of water, heat until white fumes are again
evolved, cool, add 10 ml of water and neutralise to litmus paper with 5M ammonia. The resulting
solution yields reaction A characteristic of sodium salts and reaction B characteristic of phosphates,
Appendix VI.
Alkalinity pH of a 0.5% w/v solution, 7.5 to 9.0, Appendix V L.
Specific optical rotation In a 1% w/v solution, +121 to +129, calculated with reference to the
Inorganic phosphate Dissolve 25 mg in 10 ml of water, add 4 ml of 1M sulphuric acid, 1 ml of a
10% w/v solution of ammonium molybdate and 2 ml of methylaminophenolsulphite reagent and allow to
stand for 15 minutes. Add sufficient water to produce 25 ml and allow to stand for a further 15
minutes. The absorbance of a 4-cm layer of the resulting solution at 730 nm, Appendix II B, is not
more than that of a 4-cm layer of a solution prepared by treating 10 ml of a 0.0036% w/v solution of
potassium dihydrogen orthophosphate in the same manner, beginning at the words add 4 ml .
gel GF
254
ether, 8 volumes of methanol and 1.2 volumes of water as the mobile phase. Apply separately to the
plate 2 l of each of three solutions in methanol containing (1) 1.0% w/v of the substance being
examined, (2) 1.0% w/v of hydrocortisone sodium phosphate BPCRS and (3) 0.020% w/v of hydro-
cortisone EPCRS. After removal of the plate, allow it to dry in air for 5 minutes and examine under
ultraviolet light (254 nm). Any secondary spot in the chromatogram obtained with solution (1) is not
Water Not more than 10.0%, Appendix IX C. Use 0.4 g.
20-16
Assay Dissolve 0.1 g in sufficient water to produce 200 ml. Dilute 5 ml to 100 ml with water and
21
H
29
Na
2
O
8
P taking 333 as the value of A(1%, 1 cm) at the maximum at
248 nm.
Storage Hydrocortisone Sodium Phosphate should be kept in a well-closed container and protected
from light.
Preparation
Hydrocortisone Sodium Phosphate Injection
20-17
Hydroflumethiazide
S
NH
N
O O
S
H
2
N
O O
F
3
C
H
C
8
H
8
F
3
N
3
O
4
S
2
331.3 135-09-1
Definition Hydroflumethiazide is 3,4-dihydro-6-trifluoromethyl-2H-1,2,4-benzothiadiazine-7-
sulphonamide 1,1-dioxide. It contains not less than 98.0% and not more than 102.0% of
C
8
H
8
F
3
N
3
O
4
S
2
Characteristics White or almost white, glistening crystals or crystalline powder; odourless or almost
odourless.
Practically insoluble in water; soluble in ethanol (96%); practically insoluble in chloroform and in
ether.
Identification
hydroflumethiazide (RS 181).
B. Dissolve 10 mg in 10 ml of 0.1M sodium hydroxide, add sufficient water to produce 100 ml and
dilute 10 ml to 50 ml with 0.01M sodium hydroxide. The light absorption of the resulting solution,
Appendix II B, in the range 230 to 350 nm exhibits two maxima, at 274 nm and 333 nm. The
absorbance at the maxima is about 0.92 and about 0.19 respectively.
C. Carry out the method for thin-layer chromatography, Appendix III A, using silica gel GF
254
as the
coating substance and ethyl acetate as the mobile phase. Apply separately to the plate 5 l of each of
two solutions in acetone containing (1) 0.1% w/v of the substance being examined and (2) 0.1% w/v
of hydroflumethiazide BPCRS. After removal of the plate, dry it in a current of air, examine under
ultraviolet light (254 nm) and then reveal the spots by Method I and examine again. By each method of
visualisation the principal spot in the chromatogram obtained with solution (1) corresponds in colour
and intensity to that in the chromatogram obtained with solution (2).
gel G as the coating substance and ethyl acetate as the mobile phase. Apply separately to the plate
10 l of each of two solutions of the substance being examined in acetone containing (1) 1.0% w/v
and (2) 0.010% w/v. After removal of the plate, dry it in a current of air and reveal the spots by
Method I. Any secondary spot in the chromatogram obtained with solution (1) is not more intense than
the spot in the chromatogram obtained with solution (2).
1 g.
of C
8
H
8
F
3
N
3
O
4
S
2
.
Preparation
Hydroflumethiazide Tablets
20-18
Hydrogen Peroxide Solution (30 per cent)
7722-84-1
Hydrogen Peroxide Solution (30 per cent) complies with the requirements of the 3rd edition of the European
Action and use Antiseptic; deodorant.
When hydrogen peroxide is prescribed or demanded, Hydrogen Peroxide Solution (6 per cent) shall
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hydrogen peroxide solution (30 per cent) contains not less than 29.0 per cent m/m and not more
than 31.0 per cent m/m of H
2
O
2
(M
r
34.01). One volume of this solution corresponds to about 110
times its volume of oxygen. A suitable stabiliser may be added.
CHARACTERS
A colourless, clear liquid.
IDENTIFICATION
A. To 1 ml add 0.2 ml of dilute sulphuric acid R and 0.25 ml of 0.02M potassium permanganate solu-
tion. The solution becomes colourless with evolution of gas.
B. To 0.05 ml add 2 ml of dilute sulphuric acid R, 2 ml of ether R and 0.05 ml of potassium chromate
solution R and shake. The ether layer is blue.
C. It complies with the requirement for the content of H
2
O
2
.
TESTS
Acidity To 10 ml add 100 ml of water R and 0.25 ml of methyl red solution R. Not less than 0.05 ml
and not more than 0.5 ml of 0.1M sodium hydroxide is required to change the colour of the indicator.
Organic stabilisers Shake 20 ml with 10 ml of chloroform R and then with two quantities, each
5 ml, of chloroform R. Evaporate the combined chloroform layers under reduced pressure at a
temperature not exceeding 25C and dry the residue in a desiccator. The residue weighs not more
than 10 mg (500 ppm).
Non-volatile residue Allow 10 ml to stand in a platinum dish until all effervescence has ceased,
cooling if necessary. Evaporate the solution to dryness on a water-bath and dry the residue at 100C
to 105C. The residue weighs not more than 20 mg (2 g/l).
ASSAY
Dilute 1.00 g to 100.0 ml with water R. To 10.0 ml of this solution add 20 ml of dilute sulphuric
acid R. Titrate with 0.02M potassium permanganate until a pink colour is obtained.
1 ml of 0.02M potassium permanganate is equivalent to 1.701 mg of H
2
O
2
or 0.56 ml of oxygen.
STORAGE
Store protected from light; if the solution does not contain a stabiliser, store at a temperature below
15C.
LABELLING
If the solution contains a stabiliser, the label states that the contents are stabilised. The competent
authority may require that the name of the stabiliser be stated on the label.
CAUTION
It decomposes vigorously in contact with oxidisable organic matter and with certain metals and if
allowed to become alkaline.
__________________________________________________________________________________________________________ Ph Eur
20-19
Hydrogen Peroxide Solution
Definition Hydrogen Peroxide Solution (6 per cent) is an aqueous solution of hydrogen peroxide
containing not less than 5.0% w/v and not more than 7.0% w/v of H
2
O
2
(34.01), corresponding to
about 20 times its volume of available oxygen. It may contain a suitable stabilising agent.
Characteristics A clear, colourless liquid. It decomposes in contact with oxidisable organic matter
and with certain metals and if allowed to become alkaline.
Identification A. To 1 ml add 0.2 ml of 1M sulphuric acid and 0.25 ml of 0.02M potassium
permanganate. The solution becomes colourless with evolution of gas.
B. Shake 0.05 ml with 2 ml of 1M sulphuric acid, 2 ml of ether and 0.05 ml of potassium chromate
solution. The ether layer is blue.
C. Complies with the requirement for the content of H
2
O
2
.
Acidity Dilute 10 ml with 20 ml of water and add 0.25 ml of methyl red solution. Not less than
0.05 ml and not more than 1.0 ml of 0.1M sodium hydroxide VS is required to change the colour of
the solution.
Organic stabilisers Shake 20 ml with successive quantities of 10, 5 and 5 ml of chloroform.
Evaporate the combined chloroform extracts at a temperature not exceeding 25 at a pressure of
2 kPa and dry in a desiccator. Any residue weighs not more than 5 mg (250 ppm).
Non-volatile matter Place 10 ml in a platinum dish and allow to stand until effervescence has
ceased, cooling if necessary. Evaporate the solution on a water bath. Any residue, when dried at 100
to 105, weighs not more than 20 mg (0.2% w/v).
Assay Dilute 10 ml to 100 ml with water. To 10 ml of the resulting solution add 20 ml of 1M
sulphuric acid and titrate with 0.02M potassium permanganate VS. Each ml of 0.02M potassium
permanganate VS is equivalent to 1.701 mg of H
2
O
2
Storage Hydrogen Peroxide Solution (6 per cent) should be protected from light. If the solution
does not contain a stabilising agent, it should be stored at a temperature not exceeding 15. It should
not be stored for long periods.
Labelling The label states, where applicable, that the solution contains a stabilising agent.
Preparation
Hydrogen Peroxide Mouthwash
20-20
Dilute Hydrogen Peroxide Solution 1/01
Hydrogen Peroxide Solution (3 per cent) complies with the requirements of the 3rd edition of the European
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hydrogen peroxide solution (3 per cent) contains not less than 2.5 per cent m/m and not more than
3.5 per cent m/m of H
2
O
2
(34.01). One volume of this solution corresponds to about ten times its
volume of oxygen. A suitable stabiliser may be added.
CHARACTERS
A colourless, clear liquid.
IDENTIFICATION
A. To 2 ml add 0.2 ml of dilute sulphuric acid R and 0.2 ml of 0.02M potassium permanganate solution.
The solution becomes colourless or slightly pink within 2 min.
B. To 0.5 ml add 1 ml of dilute sulphuric acid R, 2 ml of ether R and 0.1 ml of potassium chromate
solution R and shake. The ether layer is blue.
C. It complies with the requirement for the content of H
2
O
2
.
TESTS
Acidity To 10 ml add 20 ml of water R and 0.25 ml of methyl red solution R. Not less than 0.05 ml
and not more than 1.0 ml of 0.1M sodium hydroxide is required to change the colour of the indicator.
Organic stabilisers Shake 20 ml with 10 ml of chloroform R and then with two quantities, each of
5 ml, of chloroform R. Evaporate the combined chloroform layers under reduced pressure at a
temperature not exceeding 25C and dry the residue in a desiccator. The residue weighs not more
than 5 mg (250 ppm).
Non-volatile residue Allow 10 ml to stand in a platinum dish until all effervescence has ceased.
Evaporate the solution to dryness on a water-bath and dry the residue at 100C to 105C. The
residue weighs not more than 20 mg (2 g/l).
ASSAY
Dilute 10.0 g to 100.0 ml with water R. To 10.0 ml of this solution add 20 ml of dilute sulphuric
acid R. Titrate with 0.02M potassium permanganate until a pink colour is obtained.
1 ml of 0.02M potassium permanganate is equivalent to 1.701 mg of H
2
O
2
STORAGE
Store protected from light; if the solution does not contain a stabiliser, store at a temperature below
15C.
LABELLING
If the solution contains a stabiliser, the label states that the contents are stabilised. The competent
authority may require that the name of the stabiliser be stated on the label.
CAUTION
It decomposes in contact with oxidisable organic matter and with certain metals and if allowed to
become alkaline.
__________________________________________________________________________________________________________ Ph Eur
20-21
Hydrotalcite
Al
2
Mg
6
(OH)
16
CO
3
,4H
2
O 604.0 12304-65-3
Definition Hydrotalcite is a hydrated form of an aluminium magnesium basic carbonate
corresponding to the formula Al
2
Mg
6
(OH)
16
CO
3
,4H
2
O. It contains not less than 15.3% and not
more than 18.7% of Al
2
O
3
and not less than 36.0% and not more than 44.0% of MgO. The ratio
of the content of Al
2
O
3
to the content of MgO is not less than 0.40 and not more than 0.45.
Characteristics A white or almost white, free-flowing, granular powder.
Practically insoluble in water. It dissolves in dilute mineral acids with slight effervescence.
Identification
A. Dissolve 1.0 g in 20 ml of 2M hydrochloric acid. Effervescence occurs. Add 30 ml of water, boil,
add 2M ammonia until just alkaline to methyl red solution, continue boiling for 2 minutes and filter,
reserving the filtrate for test B. Wash the precipitate with 50 ml of a hot 2% w/v solution of
ammonium chloride and dissolve in 15 ml of 2M hydrochloric acid. The resulting solution yields the
reaction characteristic of aluminium salts, Appendix VI.
B. Dilute 1 ml of the filtrate obtained in test A to 10 ml with water. The resulting solution yields the
reactions characteristic of magnesium salts, Appendix VI.
Alkalinity pH of a 4% w/v suspension in carbon dioxide-free water, 8.0 to 10.0, Appendix V L.
Neutralising capacity Mix 0.2 g with a small quantity of water to give a smooth paste and gradually
add sufficient further quantities of water to produce 100 ml. Warm at 37, add 100 ml of 0.1M
hydrochloric acid VS previously heated to 37 and stir continuously for 1 hour using a paddle stirrer at
a rate of about 200 revolutions per minute, maintaining the temperature at 37, and titrate with 0.1M
sodium hydroxide VS to pH 3.5. Subtract the volume of 0.1M sodium hydroxide VS from 100 ml to
obtain the number of ml of 0.1M hydrochloric acid VS required for neutralisation. Not less than
260 ml of 0.1M hydrochloric acid VS is required to neutralise 1 g.
Arsenic Dissolve 0.33 g in 5 ml of 2M hydrochloric acid. The resulting solution complies with the
limit test for arsenic, Appendix VII (3 ppm).
Heavy metals Dissolve 2.7 g in 20 ml of 5M hydrochloric acid and 10 ml of water, add 0.5 ml of nitric
acid and boil for 30 seconds. Cool, add 2 g of ammonium chloride and 2 g of ammonium thiocyanate
and extract with three 10-ml quantities of a mixture of equal volumes of isoamyl alcohol and ether.
Add to the aqueous layer 0.1 ml of phenolphthalein solution and 13.5M ammonia until a pink colour is
produced. Cool, add glacial acetic acid until the solution is decolorised and add a further 5 ml of
glacial acetic acid. Filter, if necessary, and dilute the solution to 40 ml with water. 12 ml of the result-
ing solution complies with limit test A for heavy metals, Appendix VII. Use lead standard solution (2
ppm Pb) to prepare the standard (30 ppm).
Sodium Not more than 0.1% of Na when determined by Method II for atomic emission spectro-
photometry, Appendix II D, measuring at 589 nm. To prepare the test solution dissolve 0.1 g in 4 ml
of 5M hydrochloric acid, dilute to 200 ml with water and use sodium standard solution (200 ppm Na),
diluted if necessary with 0.1M hydrochloric acid, to prepare the standard solutions.
Chloride Dissolve 0.18 g in 10 ml of 2M nitric acid, boil, allow to cool and dilute to 100 ml with
water. To 10 ml add 5 ml of water. The resulting solution complies with the limit test for chlorides,
Appendix VII (0.3%).
Sulphate Dissolve 0.14 g in 15 ml of 1M hydrochloric acid and dilute to 100 ml with water. 15 ml of
the resulting solution complies with the limit test for sulphates, Appendix VII (0.7%).
Loss on ignition When ignited at 800, loses 40.0 to 50.0% of its weight. Use 1 g.
Assay
For Al
2
O
3
Dissolve 0.3 g in 2 ml of 7M hydrochloric acid, add 250 ml of water and 50 ml of 0.05M
disodium edetate VS and neutralise with 1M sodium hydroxide using methyl red solution as indicator.
Heat the solution on a water bath for 30 minutes and allow to cool. Add 3 g of hexamine and titrate
the excess of disodium edetate with 0.05M lead nitrate VS using xylenol orange solution as indicator.
Each ml of 0.05M disodium edetate VS is equivalent to 2.549 mg of Al
2
O
3
.
For MgO Dissolve 0.125 g in the minimum volume of 7M hydrochloric acid, add 30 ml of water, 1 g
of ammonium chloride, 10 ml of triethanolamine, 150 ml of water and 5 ml of ammonia buffer pH 10.9
and titrate immediately with 0.05M disodium edetate VS using mordant black 11 solution as indicator.
Each ml of 0.05M disodium edetate VS is equivalent to 2.015 mg of MgO.
Preparation
Hydrotalcite Tablets
20-22
Hydroxocobalamin Acetate
N
Me
N
Me
Me
N
Me
Me
CH
2
CH
2
CONH
2
N
Me
Me
H
CH
2
CONH
2
CH
2
CH
2
CONH
2
H
2
NCOCH
2
CH
2
H
H
2
NCOCH
2
H
2
NCOCH
2
H
H
H
Me
N
O
H
Me H
N
N
Me
Me
O
O
O
O P O
H CH
2
OH
HO
H
H
H
Co
+

OH
,CH3COOH
C
62
H
89
CoN
13
O
15
P,C
2
H
4
O
2
1406 22465-48-1
Hydroxocobalamin Acetate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Vitamin B
12
analogue used in treatment of vitamin B
12
deficiency.
Preparation
Hydroxocobalamin Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hydroxocobalamin acetate contains not less than 96.0 per cent and not more than the equivalent of
102.0 per cent of Co-[-(5,6-dimethylbenzimidazolyl)]-Co-hydroxocobamide acetate, calculated
CHARACTERS
A dark red, crystalline powder or dark red crystals, soluble in water, very hygroscopic. Some
decomposition may occur on drying.
IDENTIFICATION
A. Dissolve 2.5 mg in a solution containing 0.8 per cent V/V of glacial acetic acid R and 10.9 g/l of
sodium acetate R and dilute to 100 ml with the same solution. Examined between 260 nm and
ratio of the absorbance at the maximum at 274 nm to that at the maximum at 351 nm is 0.75 to
0.83. The ratio of the absorbance at the maximum at 525 nm to that at the maximum at 351 nm is
0.31 to 0.35.
Carry out the test protected from light.
Reference solution. Dissolve 2 mg of hydroxocobalamin CRS in 1 ml of a mixture of equal volumes of
Apply separately to the plate 10 l of each solution. Develop in an unlined tank over a path of 12 cm
using a mixture of 25 volumes of dilute ammonia R1 and 75 volumes of methanol R. Allow the plate to
dry in air. Examine in daylight. The principal spot in the chromatogram obtained with the test solu-
tion is similar in position, colour and size to the principal spot in the chromatogram obtained with the
reference solution.
C. It gives reaction (a) of acetates (2.3.1).
TESTS
Related substances Examine by liquid chromatography (2.2.29). Use freshly prepared solutions and
protect them from bright light.
20-23
Reference solution (c). Dissolve 25 mg of the substance to be examined in 10 ml of water R, warming if
necessary. Allow to cool and add 1 ml of a 20 g/l solution of chloramine R and 0.5 ml of 0.05M hydro-
chloric acid. Dilute to 25 ml with water R. Shake and allow to stand for 5 min. Inject immediately.
as mobile phase at a flow rate of 1.5 ml per minute a mixture prepared as follows: mix 19.5
volumes of methanol R and 80.5 volumes of a solution containing 15 g/l of citric acid R and
8.1 g/l of disodium hydrogen phosphate R,
a loop injector,
Inject separately 20 l of each solution and continue the chromatography for four times the reten-
tion time of the principal peak in the chromatogram obtained with reference solution (a). In the
chromatogram obtained with the test solution, the sum of the areas of any peaks apart from the
reference solution (a) (5 per cent). Disregard any peak whose area is less than that of the principal
peak in the chromatogram obtained with reference solution (b). The test is not valid unless: the
chromatogram obtained with reference solution (c) shows three principal peaks and the resolution
between each pair of adjacent peaks is at least 3.0; the chromatogram obtained with reference solu-
tion (b) shows one principal peak with a signal-to-noise ratio of at least 5.
Loss on drying (2.2.32). 8.0 per cent to 12.0 per cent, determined on 0.400 g by drying at 100C to
ASSAY
Protect the solutions from light throughout the assay. Dissolve 25.0 mg in a solution containing 0.8 per
cent V/V of glacial acetic acid R and 10.9 g/l of sodium acetate R and dilute to 1000.0 ml with the same
solvent. Measure the absorbance of the resulting solution at the maximum at 351 nm (2.2.25).
64
H
93
CoN
13
O
17
STORAGE
__________________________________________________________________________________________________________ Ph Eur
20-24
Hydroxocobalamin Chloride
N
Me
N
Me
Me
N
Me
Me
CH
2
CH
2
CONH
2
N
Me
Me
H
CH
2
CONH
2
CH
2
CH
2
CONH
2
H
2
NCOCH
2
CH
2
H
H
2
NCOCH
2
H
2
NCOCH
2
H
H
H
Me
N
O
H
Me H
N
N
Me
Me
O
O
O
O P O
H CH
2
OH
HO
H
H
H
Co
+

OH
,HCl
C
62
H
89
CoN
13
O
15
P,HCl 1383 58288-50-9
Hydroxocobalamin Chloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
12
12
deficiency.
Preparation
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hydroxocobalamin chloride contains not less than 96.0 per cent and not more than the equivalent of
102.0 per cent of Co-[-(5,6-dimethylbenzimidazolyl)]-Co-hydroxocobamide chloride, calculated
CHARACTERS
IDENTIFICATION
0.31 to 0.35.
reference solution.
TESTS
20-25
a loop injector.
tion (b) shows one principal peak with a signal- to-noise ratio of at least 5.
ASSAY
62
H
90
ClCoN
13
O
15
STORAGE
Store in an airtight container protected from light, at a temperature of 2C to 8C.
__________________________________________________________________________________________________________ Ph Eur
20-26
Hydroxocobalamin Sulphate
N
Me
N
Me
Me
N
Me
Me
CH
2
CH
2
CONH
2
N
Me
Me
H
CH
2
CONH
2
CH
2
CH
2
CONH
2
H
2
NCOCH
2
CH
2
H
H
2
NCOCH
2
H
2
NCOCH
2
H
H
H
Me
N
O
H
Me H
N
N
Me
Me
O
O
O
O P O
H CH
2
OH
HO
H
H
H
Co
+

OH
,H2SO4
2
C
124
H
178
Co
2
N
26
O
30
P
2
,H
2
SO
4
2791
Hydroxocobalamin Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia
12
12
deficiency.
Preparation
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hydroxocobalamin sulphate contains not less than 96.0 per cent and not more than the equivalent
of 102.0 per cent of di-(Co-[-(5,6-dimethylbenzimidazolyl)]-Co-hydroxocobamide) sulphate,
CHARACTERS
IDENTIFICATION
0.31 to 0.35.
reference solution.
C. It gives reaction (a) of sulphates (2.3.1).
TESTS
20-27
a loop injector.
tion (b) shows one principal peak with a signal- to-noise ratio of at least 5.
ASSAY
124
H
180
Co
2
N
26
O
34
P
2
S taking the specific absorbance to be 188.
STORAGE
Store in an airtight container protected from light, at a temperature of 2C to 8C.
__________________________________________________________________________________________________________ Ph Eur
20-28
Hydroxycarbamide / Hydroxyurea
H
2
N N
OH
O
H
CH
4
N
2
O
2
76.05 127-07-1
Definition Hydroxycarbamide contains not less than 98.0% and not more than 100.5% of
CH
4
N
2
O
2
Characteristics A white to off-white crystalline powder; odourless or almost odourless; hygroscopic.
Freely soluble in water and in hot ethanol (96%).
Identification
hydroxycarbamide (RS 182).
time as that of the principal peak in the chromatogram obtained with solution (2).
Heavy metals Moisten 2.0 g in a silica crucible with sulphuric acid, ignite gently and carry out limit
test C for heavy metals, Appendix VII, beginning at the words Allow to cool . Use 3 ml of lead
Urea Carry out the method for thin-layer chromatography, Appendix III A, using a cellulose F
precoated plate (Merck cellulose F plates are suitable), a mixture of 1 volume of glacial acetic acid, 1
volume of water and 4 volumes of butan-1-ol as the mobile phase and developing the chromatograms
for 18 hours. Apply separately to the plate 50 l of each of the following solutions in water, as several
applications, containing (1) 2.0% w/v of the substance being examined and (2) 0.01% w/v of urea.
After removal of the plate, dry it in a current of cold air, spray with a 1% w/v solution of 4-dimethyl-
aminobenzaldehyde in ethanol (96%) containing 2% v/v of hydrochloric acid and heat at 90 for 5
minutes. In the chromatogram obtained with solution (1), any spot corresponding to urea is not
more intense than the spot in the chromatogram obtained with solution (2) (0.5%).
Assay Carry out the method for liquid chromatography, Appendix III D, using three solutions in water
containing (1) 0.10% w/v of the substance being examined, (2) 0.10% w/v of hydroxycarbamide
BPCRS and (3) 0.10% w/v of hydroxycarbamide BPCRS and 0.4% w/v of hydroxylamine hydrochloride.
(25 cm 4.6 mm) packed with stationary phase C (5 m) (Spherisorb ODS 2 is suitable), (b) water as
the mobile phase with a flow rate of 0.5 ml per minute and (c) a detection wavelength of 214 nm.
between the peaks due to hydroxycarbamide and hydroxylamine hydrochloride is at least 1.0.
Calculate the content of CH
4
N
2
O
2
using the declared content of CH
4
N
2
O
2
in hydroxycarbamide
BPCRS.
Storage Hydroxycarbamide should be kept in a well-closed container and protected from moisture.
Preparation
Hydroxycarbamide Capsules/Hydroxyurea Capsules
20-29
Hydroxychloroquine Sulphate
and enantiomer

,H
2
SO
4
N Cl
HN
H Me
N
OH
Et
C
18
H
26
ClN
3
O,H
2
SO
4
434.0 747-36-4
Definition Hydroxychloroquine Sulphate is (RS)-2-{N-[4-(7-chloro-4-quinolylamino)pentyl]-N-
ethylamino}ethanol sulphate. It contains not less than 98.0% and not more than 100.5% of
C
18
H
26
ClN
3
O,H
2
SO
4
Freely soluble in water; practically insoluble in chloroform, in ethanol (96%) and in ether.
Identification
A. Dissolve 0.1 g in 10 ml of water, add 2 ml of 2M sodium hydroxide and extract with two 20-ml
quantities of chloroform. Wash the chloroform extracts with water, dry with anhydrous sodium sulphate,
evaporate to dryness and dissolve the residue in 2 ml of chloroform. The infrared absorption spectrum of
the resulting solution, Appendix II A, is concordant with the reference spectrum of hydroxychloroquine
(RS 183).
B. Yields the reactions characteristic of sulphates, Appendix VI.
Clarity and colour of solution A 10.0% w/v solution is not more than slightly turbid and not more
than slightly yellow.
Lead Not more than 20 ppm when determined by the following method. Carefully heat 2.0 g for 10
minutes with 8 ml of water and 6 ml of nitric acid in a Kjeldahl flask. Cool, add 4 ml of sulphuric acid
and heat until the mixture darkens. Continue heating, with the dropwise addition of nitric acid, until
the liquid becomes colourless and white fumes of sulphur trioxide are produced. Add 3 ml of water,
carefully evaporate until white fumes are again produced, cool and dilute to 18 ml with water. Add
and dissolve 2 g of citric acid, make alkaline with 5M ammonia and add 1 ml of potassium cyanide
solution PbT. Transfer to a separating funnel, add 10 ml of dithizone solution, shake vigorously and
remove the lower layer. Repeat the extraction with two 5-ml quantities of dithizone solution. If, after
the third extraction, the chloroform layer is bright red, continue the extraction with further 5-ml
quantities of dithizone solution until the colour of the reagent no longer changes to bright red. Wash
the combined chloroform solutions by shaking with 10 ml of water and then extract with two 10-ml
quantities of 2M hydrochloric acid. Wash the combined acid solutions with 10 ml of chloroform and
discard the chloroform. Transfer the solution to a Nessler cylinder and make alkaline with 5M
ammonia. In a second Nessler cylinder mix 2 ml of 6M acetic acid with 20 ml of 2M hydrochloric acid,
make alkaline with 5M ammonia and add 4 ml of lead standard solution (10 ppm Pb).
Treat the contents of each cylinder as follows. Add 1 ml of potassium cyanide solution PbT; the
solutions should not be more than faintly opalescent. If the colours of the solutions differ, equalise
them by the addition of a few drops of a highly diluted solution of burnt sugar or other non-reactive
substance. Dilute to 50 ml with water, add 0.1 ml of a solution prepared by dissolving 10 g of sodium
sulphide in sufficient water to produce 100 ml and filtering and mix thoroughly. Compare the colours
of the two solutions by a suitable method, such as by light reflected from a white tile through the
Nessler cylinders. The colour of the solution in the first cylinder is not more intense than that of the
solution in the second cylinder.
Chloride Dissolve 0.50 g in 50 ml of water. 15 ml of the resulting solution complies with the limit test
for chlorides, Appendix VII (350 ppm).
gel GF
254
as the coating substance and a mixture of 72 volumes of methanol, 25 volumes of water and
3 volumes of 13.5M ammonia as the mobile phase. Apply separately to the plate 2 l of each of three
solutions of the substance being examined in water containing (1) 5.0% w/v, (2) 0.050% w/v and (3)
0.025% w/v. After removal of the plate, allow it to dry in air and examine under ultraviolet light
than the spot in the chromatogram obtained with solution (2) and not more than one such spot is
20-30
Use 1 g.
Assay Dissolve 0.5 g in 10 ml of water, add 20 ml of 1M sodium hydroxide and extract with four 25-
ml quantities of chloroform. Combine the chloroform extracts and evaporate to a volume of about
10 ml. Add 40 ml of anhydrous acetic acid and carry out Method I for non-aqueous titration, Appendix
VIII A, using oracet blue B solution as indicator. Each ml of 0.1M perchloric acid VS is equivalent to
21.70 mg of C
18
H
26
ClN
3
O,H
2
SO
4
.
Storage Hydroxychloroquine Sulphate should be protected from light.
Preparation
Hydroxychloroquine Tablets
20-31
Hydroxyethyl Salicylate
O
O
OH
OH
C
9
H
10
O
4
182.2 87-28-5
Hydroxyethyl Salicylate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hydroxyethyl salicylate contains not less than 98.0 per cent and not more than the equivalent of
102.0 per cent of 2-hydroxyethyl 2-hydroxybenzoate.
CHARACTERS
An oily, colourless or almost colourless liquid, sparingly soluble in water, very soluble in acetone, in
ether and in methylene chloride, freely soluble in alcohol.
IDENTIFICATION
obtained with hydroxyethyl salicylate CRS. Examine the substances prepared as thin films.
D. To 1 ml of solution S (see Tests), add 1 ml of water R and 0.2 ml of ferric chloride solution R2. A
violet-red colour appears which disappears immediately after the addition of 2 ml of dilute acetic
acid R. A very faint violet colour may remain.
E. In a test tube 160 mm long, mix 1.0 g with 2.0 g of finely powdered manganese sulphate R. Insert
2 cm into the test-tube a strip of filter paper impregnated with a freshly prepared mixture of 1 volume
of a 20 per cent V/V solution of diethanolamine R and 11 volumes of a 50 g/l solution of sodium
nitroprusside R adjusted to pH 9.8 with 1M hydrochloric acid. Heat the test-tube over a naked flame for
1 min to 2 min. The filter paper becomes blue.
TESTS
Solution S Dissolve 2.5 g in 40 ml of alcohol R and dilute to 50 ml with distilled water R.
sodium hydroxide. The solution is yellow. Add 0.3 ml of 0.01M hydrochloric acid. The solution is red.
Reference solution (a). Dissolve 50.0 mg of hydroxyethyl salicylate CRS in methanol R and dilute to
Reference solution (b). Dilute 2.5 ml of test solution (b) to 10 ml with methanol R.
Reference solution (c). Dissolve 0.10 g of ethylene glycol R in methanol R and dilute to 50 ml with the
same solvent. Dilute 1.25 ml of the solution to 10 ml with methanol R.
20 volumes of glacial acetic acid R, 20 volumes of ethyl acetate R and 60 volumes of cyclohexane R. Dry
20-32
the plate in a current of cold air and examine in ultraviolet light at 254 nm. Any spot in the chrom-
atogram obtained with test solution (a), apart from the principal spot, is not more intense than the
spot in the chromatogram obtained with reference solution (b) (1 per cent). Spray the plate with
ammonium vanadate solution R and heat to 100C for 10 min. Allow to cool for 10 min and examine
in daylight. In the chromatogram obtained with test solution (a): any spot corresponding to ethylene
glycol is not more intense than the spot in the chromatogram obtained with reference solution (c)
(0.5 per cent); any spot, apart from the principal spot and any spot corresponding to ethylene glycol,
is not more intense than the spot in the chromatogram obtained with reference solution (b) (1 per
cent). The test is not valid unless the chromatogram obtained with reference solution (c) shows a
clearly visible spot.
ASSAY
In a flask with a ground-glass stopper, dissolve 0.125 g in 30 ml of glacial acetic acid R. Add 10 ml of
dilute sulphuric acid R, 1.5 g of potassium bromide R and 50.0 ml of 0.02M potassium bromate. Immedi-
ately close the flask and allow to stand protected from light for 15 min. Add 1.5 g of potassium
iodide R immediately after removing the stopper and titrate with 0.1M sodium thiosulphate, adding 1 ml
of starch solution R towards the end of the titration. Carry out a blank titration.
9
H
10
O
4
.
STORAGE
IMPURITIES
A. salicylic acid,
HO
OH
B. ethane-1,2-diol (ethylene glycol).
__________________________________________________________________________________________________________ Ph Eur
20-33
Hydroxyethylcellulose
corrected 1/01
9004-62-0
Hydroxyethylcellulose complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hydroxyethylcellulose is a partly O-(2-hydroxyethylated) cellulose.
CHARACTERS
A white, yellowish-white or greyish-white powder or granules, soluble in hot and cold water giving a
colloidal solution, practically insoluble in acetone, in alcohol and in toluene.
IDENTIFICATION
A. Heat 10 ml of solution S (see Tests) to boiling. The solution remains clear.
B. To 10 ml of solution S add 0.3 ml of dilute acetic acid R and 2.5 ml of a 100 g/l solution of tannic
acid R. A yellowish-white, flocculent precipitate is formed which dissolves in dilute ammonia R1.
C. In a test-tube about 160 mm in length, thoroughly mix 1 g with 2 g of finely powdered manganese
sulphate R. Introduce to a depth of 2 cm into the upper part of the tube a strip of filter paper
impregnated with a freshly prepared mixture of 1 volume of a 200 g/l solution of diethanolamine R
and 11 volumes of a 50 g/l solution of sodium nitroprusside R, adjusted to about pH 9.8 with 1M
hydrochloric acid. Insert the tube 8 cm into a silicone-oil bath and heat at 190C to 200C. The filter
paper becomes blue within 10 min. Carry out a blank test.
D. Dissolve 0.2 g completely, without heating, in 15 ml of a 700 g/l solution of sulphuric acid R. Pour
the solution with stirring into 100 ml of iced water R and dilute to 250 ml with iced water R. In a test-
tube, mix thoroughly while cooling in iced water 1 ml of the solution with 8 ml of sulphuric acid R,
added dropwise. Heat on a water-bath for exactly 3 min and immediately cool in iced water. While
the mixture is cold, carefully add 0.6 ml of ninhydrin solution R2 and mix well. Allow to stand at
25C. A pink colour is produced immediately and does not become violet within 100 min.
TESTS
Solution S Disperse a quantity of the substance to be examined equivalent to 1.0 g of the dried
substance in 50 ml of carbon dioxide-free water R. After 10 min, dilute to 100 ml with carbon dioxide-
free water R and stir until dissolution is complete.
6
(Method II, 2.2.2).
to 2.00 g of the dried substance into 50 g of water R. Dilute to 100.0 g with water R and stir until
dissolution is complete. Determine the viscosity (2.2.10) with a rotating viscometer at 25C at a
shear rate of 100 s
1
for substances with an expected viscosity up to 100 mPas, at a shear rate of
10 s
1
for substances with an expected viscosity between 100 mPas and 20,000 mPas

and at a shear
rate of 1 s
1
for substances with an expected viscosity above 20,000 mPas. If it is impossible to
obtain a shear rate of exactly 1 s
1
, 10 s
1
or 100 s
1
respectively, use a rate slightly higher and a rate
slightly lower and interpolate. The apparent viscosity is not less than 75 per cent and not more than
140 per cent of the value stated on the label.
Chlorides (2.4.4). Dilute 1 ml of solution S to 30 ml with water R. 15 ml of the solution complies
with the limit test for chlorides (1.0 per cent).
Nitrates
If hydroxyethylcellulose has an apparent viscosity of 1000 mPas or less, it complies with test A:
A. Dilute 5 ml of solution S to 10 ml with water R. To 1 ml of the dilution add 19 ml of water R,
2 ml of concentrated ammonia R, 0.5 ml of a 10 g/l solution of manganese sulphate R and 1 ml of a
10 g/l solution of sulphanilamide R. Add 0.1 g of granulated zinc R and allow to stand for 30 min in
iced water, shaking occasionally. Filter by suction through a sintered-glass filter (40). Place 10 ml of
the filtrate in a test-tube, acidify with 2.5 ml of hydrochloric acid R and add 0.5 ml of a 10 g/l solution
of naphthylethylenediamine dihydrochloride R. Allow to stand for 15 min. Any violet-red colour in the
20-34
manner using a mixture of 15 ml of nitrate standard solution (10 ppm NO
3
) R and 5 ml of water R
(3.0 per cent).
If hydroxyethylcellulose has an apparent viscosity of more than 1000 mPas, it complies with test B.
B. Dilute 5 ml of solution S to 10 ml with water R. To 1 ml of the dilution add 19 ml of water R, 2 ml
of concentrated ammonia R, 0.5 ml of a 10 g/l solution of manganese sulphate R and 1 ml of a 10 g/l
solution of sulphanilamide R. Add 0.1 g of granulated zinc R and allow to stand for 30 min in iced
water, shaking occasionally. Filter by suction through a sintered-glass filter (40). Place 10 ml of the
filtrate in a test-tube, acidify with 2.5 ml of hydrochloric acid R and add 0.5 ml of a 10 g/l solution of
naphthylethylenediamine dihydrochloride R. Allow to stand for 15 min. Any violet-red colour in the
manner using a mixture of 1 ml of nitrate standard solution (10 ppm NO
3
) R and 19 ml of water R
(0.2 per cent).
Glyoxal Introduce 1.0 g into a test tube with a ground-glass stopper and add 10.0 ml of ethanol R.
Stopper the tube and stir mechanically for 30 min. Centrifuge. To 2.0 ml of the supernatant liquid
add 5.0 ml of a 4 g/l solution of methylbenzothiazolone hydrazone hydrochloride R in an 80 per cent V/V
solution of glacial acetic acid R in water R. Shake to homogenise. After 2 h, the solution is not more
intensely coloured than a standard prepared at the same time and in the same manner using 2.0 ml of
glyoxal standard solution (20 ppm C
2
H
2
O
2
) R instead of the 2.0 ml of supernatant liquid (200 ppm).
Ethylene oxide Not more than 1 ppm, determined by head-space gas chromatography (2.2.28).
Test preparation. Place 1.00 g (M
T
) of the substance to be examined in a 5 ml vial and add 1 ml of
water R. It swells in water but does not dissolve.
Reference preparation (a). Place 1.00 g (M
R
) of the substance to be examined in a 5 ml vial. Add
0.2 ml (equivalent to 225 mg) of cooled ethylene oxide solution R and 0.8 ml of water R. It swells in
water but does not dissolve.
Reference preparation (b). To 0.1 ml of ethylene oxide solution R in a 5 ml vial add 0.1 ml of a freshly
prepared 10 mg/l solution of acetaldehyde R.
Close the vials immediately with a butyl rubber membrane stopper, coated with aluminium or polytetrafluoro-
ethylene and secured with an aluminium crimped cap.
a glass or fused-silica column 30 m long and 0.32 mm in internal diameter, the inner surface of
which is coated with a 1.0 m layer of polydimethylsiloxane R,
velocity of about 20 cm/s and a split ratio of 1:20,
of 30C per minute to 230C and maintaining at 230C for 5 min; that of the injection port at 150C
and that of the detector at 250C.
Inject 1.0 ml of the gaseous phase of reference solution (b). Adjust the sensitivity of the system so
that the heights of the two principal peaks in the chromatogram obtained are not less than 15 per
cent of the full scale of the recorder. The test is not valid unless the resolution between the peaks
corresponding to acetaldehyde and ethylene oxide is at least 3.5.
Inject separately 1.0 ml of the gaseous phases of the test solution and reference solution (a). In the
chromatogram obtained with the test solution, the area of any peak corresponding to ethylene oxide
is not greater than half the area of the peak due to ethylene oxide in the chromatogram obtained with
Alternatively the content of ethylene oxide in parts per million can be calculated from the
expression:
) ( 25 . 0
R T T R
EO T
M A M A
C M A

10
where
EO
EO
M
C
C
20-35
A
T
the test solution,
A
R
M
EO
=mass of absorbed ethylene oxide (used for preparing ethylene oxide solution R) in grams,
M
T
=mass of the substance to be examined in grams in the test solution,
M
R
=mass of the substance to be examined in grams in the reference solution,
C=correction factor to be determined from the formula,
C
EO
=ethylene oxide content in milligrams per millilitre, determined by titration.
2-Chloroethanol Not more than 10 ppm, determined by head-space gas chromatography (2.2.28).
Test preparation. To 50 mg (M
T
) of the substance to be examined in a 20 ml vial add 2 l of
2-propanol R.
Reference preparation. To 50 mg (M
R
) of the substance to be examined in a 20 ml vial add 2 l of
2-chloroethanol solution R.
Close the vials immediately with a butyl rubber membrane stopper, coated with aluminium or polytetrafluoro-
ethylene and secured with an aluminium crimped cap.
The following head-space injection conditions may be used: the flask is flushed with helium at a flow
rate of 20 ml/min. The vials are heated to 110C for 40 min. The duration of injection is 5 min. The
gas extracts are transferred to a trap consisting of a tube 13.6 cm long and 4 mm in internal diameter
packed with ethylvinylbenzene-divinylbenzene copolymer R (150 m) maintained at 50C.
Rapidly heat the trap to 210C and with a backflush of helium at a flow-rate of 5 ml/min push the
gas towards the analytical column.
a fused-silica column 30 m long and 0.53 mm in internal diameter, the inner surface of which is
coated with a 1.0 m layer of macrogol 20,000 R for chromatography,
helium for chromatography R as the carrier gas with a linear velocity of 60 cm/s,
of 5C per minute to 110C, then raising the temperature at a rate of 8C per minute to 230C and
maintaining at 230C for 5 min; maintain the temperature of the injection port at 150C and that of
Successively record the chromatograms of the test solution and the reference solution.
Calculate the content of 2-chloroethanol in parts per million from the expression:
) ( ) (
R T T R
T
M A M A
C A

A
T
=area of the peak corresponding to 2-chloroethanol in the chromatogram obtained with the
test solution,
A
R
=area of the peak corresponding to 2-chloroethanol in the chromatogram obtained with the
reference solution,
M
T
=mass of the substance to be examined in grams in the test solution,
M
R
=mass of the substance to be examined in grams in the reference solution,
C=content of 2-chloroethanol in micrograms in 2.0 l of 2-chloroethanol solution R.
STORAGE
LABELLING
The label states the apparent viscosity in millipascal seconds for a 2 per cent m/m solution.
__________________________________________________________________________________________________________ Ph Eur
20-36
Hydroxyethylmethylcellulose
Hydroxyethylmethylcellulose complies with the requirements of the 3rd edition of the European Pharmacopoeia
for Methylhydroxyethylcellulose [0046]. These requirements are reproduced after the heading Definition
below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Methylhydroxyethylcellulose is a partly O-methylated and O-(2-hydroxyethylated) cellulose.
CHARACTERS
A white, yellowish-white or greyish-white powder or granules, hygroscopic after drying, practically
insoluble in hot water, in acetone, in ethanol, in ether and in toluene. It dissolves in cold water giving
a colloidal solution.
IDENTIFICATION
A. Heat 10 ml of solution S (see Tests) in a water-bath while stirring. At a temperature above 50C,
the solution becomes cloudy or a flocculent precipitate is formed. The solution becomes clear again
on cooling.
C. In a test-tube about 160 mm long, thoroughly mix 1 g with 2 g of finely powdered manganese
impregnated with a freshly prepared mixture of 1 volume of a 20 per cent V/V solution of
diethanolamine R and 11 volumes of a 50 g/l solution of sodium nitroprusside R, adjusted to about pH
9.8 with 1M hydrochloric acid. Insert the tube 8 cm into a silicone-oil bath at 190C to 200C. The
filter paper becomes blue within 10 min. Carry out a blank test.
D. Dissolve 0.2 g completely, without heating, in 15 ml of a 70 per cent m/m solution of sulphuric
acid R. Pour the solution with stirring into 100 ml of iced water R and dilute to 250 ml with iced
water R. In a test-tube, mix thoroughly, while cooling in iced water, 1 ml of the solution with 8 ml of
sulphuric acid R, added dropwise. Heat in a water-bath for exactly 3 min and immediately cool in iced
water. While the mixture is cold, carefully add 0.6 ml of ninhydrin solution R2 and mix well. Allow to
stand at 25C. A pink colour is produced immediately and does not become violet within 100 min.
E. Place 1 ml of solution S on a glass plate. After evaporation of the water a thin film is formed.
TESTS
Solution S While stirring, introduce a quantity of the substance to be examined equivalent to 1.0 g
of the dried substance into 50 g of carbon dioxide-free water R heated to 90C. Allow to cool, adjust
the mass of the solution to 100 g with carbon dioxide-free water R and stir until dissolution is complete.
6
(Method II, 2.2.2).
to 6.00 g of the dried substance into 150 g of water R heated to 90C. Stir with a propeller-type
stirrer for 10 min, place the flask in a bath of iced water, continue the stirring and allow to remain in
the bath of iced water for 40 min to ensure that solution is complete. Adjust the mass of the solution
to 300 g and centrifuge the solution to expel any entrapped air. Adjust the temperature of the solu-
tion to 20 0.1C. Determine the viscosity (2.2.10) with a rotating viscometer at 20C and a shear
rate of 10 s
-1
. The apparent viscosity is not less than 75 per cent and not more than 140 per cent of
at 100C to 105C.
Sulphated ash (2.4.14). Not more than l.0 per cent, determined on l.000 g.
STORAGE
20-37
LABELLING
__________________________________________________________________________________________________________ Ph Eur
20-38
Hydroxyprogesterone Caproate
H
O
O Me
H
OCO[CH
2
]
4
CH
3
Me
Me
H
C
27
H
40
O
4
428.6 630-56-8
Definition Hydroxyprogesterone Caproate is 3,20-dioxopregn-4-en-17-yl hexanoate. It contains
27
H
40
O
4
substance.
Characteristics A white or almost white, crystalline powder; odourless or almost odourless. It
exhibits polymorphism.
Practically insoluble in water; very soluble in chloroform; freely soluble in ethanol (96%) and in ether.
It dissolves in fixed oils and esters.
Identification
The infrared absorption spectrum, Appendix II A, is concordant with the reference spectrum of hydroxy-
progesterone caproate (RS 184).
Acidity Dissolve 0.2 g in 25 ml of absolute ethanol previously neutralised to bromothymol blue solu-
tion R3 and titrate immediately with 0.01M sodium hydroxide VS until a faint blue colour is produced.
Not more than 1.0 ml of 0.01M sodium hydroxide VS is required.
Related foreign steroids Carry out the method for thin-layer chromatography, Appendix III A, using
silica gel HF
254
as the coating substance and a mixture of equal volumes of cyclohexane and ethyl
acetate as the mobile phase. Apply separately to the plate 10 l of each of two solutions of the
substance being examined in chloroform containing (1) 1.0% w/v and (2) 0.010% w/v. After removal
of the plate, allow it to dry in air and examine under ultraviolet light (254 nm). Any secondary spot in
Assay Dissolve 60 mg in sufficient absolute ethanol to produce 100 ml, dilute 5 ml to 250 ml with
absolute ethanol and measure the absorbance of the resulting solution at the maximum at 240 nm,
Appendix II B. Calculate the content of C
27
H
40
O
4
taking 395 as the value of A(1%, 1 cm) at the
maximum at 240 nm.
Storage Hydroxyprogesterone Caproate should be protected from light.
Preparation
Hydroxyprogesterone Injection
When hydroxyprogesterone hexanoate is prescribed or demanded, Hydroxyprogesterone Caproate
20-39
Hydroxypropylcellulose
9004-64-2
Hydroxypropylcellulose complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hydroxypropylcellulose is a partly O-(2-hydroxypropylated) cellulose. It may contain not more than
0.6 per cent of silica (SiO
2
).
CHARACTERS
A white or yellowish-white powder or granules, hygroscopic after drying, soluble in cold water, in
glacial acetic acid, in ethanol, in methanol and in propylene glycol and in a mixture of 10 parts of
methanol and 90 parts of methylene chloride giving colloidal solutions, sparingly soluble or slightly
soluble in acetone depending on the degree of substitution, practically insoluble in hot water, in
ethylene glycol and in toluene.
IDENTIFICATION
A. Heat 10 ml of solution S (see Tests) in a water-bath while stirring. At a temperature above 40C
the solution becomes cloudy or a flocculent precipitate is formed. The solution becomes clear again
on cooling.
water R. In a test-tube, mix thoroughly while cooling in iced water 1 ml of the solution with 8 ml of
sulphuric acid R, added dropwise. Heat in a water-bath for exactly 3 min and immediately cool in iced
water. While the mixture is cold, carefully add 0.6 ml of ninhydrin solution R2 and mix well. Allow to
stand at 25C. A pink colour is produced immediately and becomes violet within 100 min.
F. 0.2 g does not dissolve in 10 ml of toluene R but dissolves completely in 10 ml of ethanol R.
TESTS
the mass of the solution to 100 g with carbon dioxide-free water R and stir until dissolution is complete.
6
(Method II, 2.2.2).
to 6.00 g of the dried substance into 150 g of water R heated to 90C. Stir with a propeller-type
stirrer for 10 min, place the flask in a bath of iced water, continue the stirring and allow to remain in
the bath of iced water for 40 min to ensure that solution is complete. Adjust the mass of the solution
to 300 g and centrifuge the solution to expel any entrapped air. Adjust the temperature of the solu-
tion to 20 0.1C. Determine the viscosity (2.2.10) with a rotating viscometer at 20C and a shear
rate of 10 s
1
. The apparent viscosity is not less than 75 per cent and not more than 140 per cent of
For a product of low viscosity, use a quantity of the substance to be examined sufficient to prepare
a solution of the concentration stated on the label.
Silica Not more than 0.6 per cent. To the residue obtained in the test for sulphated ash add
sufficient alcohol R to moisten the residue completely. Add 6 ml of hydrofluoric acid R in small por-
tions. Evaporate to dryness at 95C to 105C, taking care to avoid loss from sputtering. Cool and
rinse the wall of the platinum crucible with 6 ml of hydrofluoric acid R. Add 0.5 ml of sulphuric acid R
20-40
and evaporate to dryness. Progressively increase the temperature, ignite at 900C, allow to cool in
a desiccator and weigh. The difference between the mass of the residue obtained in the test for
sulphated ash and the mass of the final residue is equal to the amount of silica in the substance to be
examined.
Chlorides (2.4.4). l ml of solution S diluted to 15 ml with water R complies with the limit test for
100C to 105C.
Sulphated ash (2.4.14). Not more than 1.6 per cent, determined on 1.0 g using a platinum crucible.
STORAGE
LABELLING
The label states:
the apparent viscosity in millipascal seconds for a 2 per cent m/m solution,
for a product of low viscosity, the concentration of the solution to be used and the apparent
viscosity in millipascal seconds.
where applicable, that the substance contains silica.
__________________________________________________________________________________________________________ Ph Eur
20-41
Hydroxyzine Hydrochloride
revised 1/01
and enantiomer
,2HCl
N
N
O
l
OH
H
C
21
H
27
ClN
2
O
2
,2HCl 447.8 2192-20-3
Hydroxyzine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
1
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hydroxyzine hydrochloride contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of (RS)-2-[2-[4-[(4-chlorophenyl)phenylmethyl]piperazin-1-yl]ethoxy]ethanol
CHARACTERS
A white or almost white, crystalline powder, hygroscopic, freely soluble in water and in alcohol,
slightly soluble in acetone.
IDENTIFICATION
obtained with hydroxyzine hydrochloride CRS. Examine the substances prepared as discs.
Reference solution (a). Dissolve 0.50 g of hydroxyzine hydrochloride CRS in a mixture of equal volumes
of methanol R and methylene chloride R and dilute to 10 ml with the same mixture of solvents.
Reference solution (b). Dissolve 0.50 g of meclozine hydrochloride R in a mixture of equal volumes of
methanol R and methylene chloride R and dilute to 10 ml with the same mixture of solvents. Dilute
1 ml to 2 ml with reference solution (a).
concentrated ammonia R, 24 volumes of alcohol R and 75 volumes of toluene R. Allow the plate to dry
in air. Spray with potassium iodobismuthate solution R2. The principal spot in the chromatogram
C. Dissolve 0.1 g in alcohol R and dilute to 15 ml with the same solvent. Add 15 ml of a saturated
solution of picric acid R in alcohol R. Allow to stand for 15 min. A precipitate is formed. Filter.
Recrystallise from alcohol R. Initiate crystallisation, if necessary, by scratching the wall of the tube
with a glass rod. The crystals melt (2.2.14) at 189C to 192C.
TESTS
solution Y
7
(Method II, 2.2.2).
Optical rotation (2.2.7): 0.10 to +0.10, determined on solution S.
20-42
Reference solution (a). Dissolve 10.0 mg of hydroxyzine hydrochloride CRS in the mobile phase and
5.0 ml to 25.0 ml with the mobile phase.
as mobile phase at a flow rate of 1 ml/min a mixture prepared as follows: dissolve 0.5 g of sodium
methanesulphonate R in a mixture of 14 volumes of triethylamine R, 300 volumes of acetonitrile R
and 686 volumes of water R; adjust to pH 2.7 with sulphuric acid R,
In the chromatogram obtained with reference solution (a), measure the height (A) above the
baseline of the peak immediately before the principal peak and the height (B) above the baseline of
the lowest point of the curve (B) separating this peak from the peak due to hydroxyzine. The test is
not valid unless A is greater than ten times B.
solution: the area of any peak, apart from the principal peak, is not greater than one third of the area
of the principal peak in the chromatogram obtained with reference solution (b) (0.1 per cent); the
sum of the areas of all the peaks, apart from the principal peak, is not greater than the area of the
100C to 105C.
ASSAY
Dissolve 0.200 g in 10 ml of anhydrous acetic acid R. Add 40 ml of acetic anhydride R. Titrate with
21
H
29
Cl
3
N
2
O
2
.
STORAGE
IMPURITIES
N
NH
l
H and enantiomer
A. (RS)-1-[(4-chlorophenyl)phenylmethyl]piperazine,
N
N
O
OH
B. 2-[2-[4-(diphenylmethyl)piperazin-1-yl]ethoxy]ethanol (decloxyzine).
__________________________________________________________________________________________________________ Ph Eur
21-1
Hyoscine Butylbromide
N
H Ph
O
O
H
O
OH
Me Bu
n
Br
+
C
21
H
30
BrN0
4
440.4 149-64-4
Hyoscine Butylbromide complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparations
Hyoscine Butylbromide Injection
Hyoscine Butylbromide Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hyoscine butylbromide (scopolamine butylbromide) contains not less than 98.0 per cent and not
more than the equivalent of 101.0 per cent of (1S,3s,5R,6R,7S,8r)-6,7-epoxy-3-[(S)-(3-hydroxy-
2-phenylpropionyl)oxy]-8-butyl-8-methyl-8-azoniabicyclo[3.2.1]octane bromide, calculated with
CHARACTERS
A white or almost white, crystalline powder, freely soluble in water and in methylene chloride,
sparingly soluble in ethanol.
IDENTIFICATION
obtained with hyoscine butylbromide CRS.
D. To about 1 mg add 0.2 ml of nitric acid R and evaporate to dryness on a water-bath. Dissolve the
residue in 2 ml of acetone R and add 0.1 ml of a 30 g/l solution of potassium hydroxide R in methanol R.
A violet colour develops.
E. To 5 ml of solution S (see Tests) add 2 ml of dilute sodium hydroxide solution R. No precipitate is
formed.
F. It gives reaction (a) of bromides (2.3.1).
TESTS
Hyoscine and other related substances Examine by liquid chromatography (2.2.29).
Reference solution (a). Dissolve 10.0 mg of hyoscine hydrobromide CRS in the mobile phase and dilute
to 100.0 ml with the mobile phase. Dilute 10.0 ml of this solution to 50.0 ml with the mobile phase.
Reference solution (c). To 10 ml of reference solution (a) add 20 l of the test solution.
21-2
as the mobile phase at a flow rate of 2 ml per minute a solution of 2.0 g of sodium lauryl
sulphate R in a mixture of 370 ml of 0.001M hydrochloric acid and 680 ml of methanol R,
Inject 20 l of reference solution (c). The chromatographic system is not valid unless in the
chromatogram obtained with reference solution (c) the resolution between the peaks correspond-
ing to hyoscine and butylhyoscine is at least 5.
Inject separately 20 l of each solution. Record the chromatograms for twice the retention time
of the main peak. In the chromatogram obtained with the test solution, the area of any peak
corresponding to hyoscine is not greater than the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.1 per cent) and the area of any peak apart from the
principal peak and the peak corresponding to hyoscine is not greater than the area of the principal
peak in the chromatogram obtained with reference solution (a) (0.2 per cent). Disregard the peak
due to bromide ion which appears close to the solvent peak.
at 100C to 105C.
ASSAY
Dissolve 0.400 g in 50 ml of water R. Titrate with 0.1M silver nitrate, determining the end-point
potentiometrically (2.2.20) using a silver indicator electrode and a silversilver chloride reference
electrode.
21
H
30
BrNO
4
.
__________________________________________________________________________________________________________ Ph Eur
21-3
Hyoscine Hydrobromide
1/01
N
Me
H Ph
O
O
H
O
OH
,HBr
C
17
H
21
NO
4
,HBr,3H
2
O 438.3 6533-68-2
Hyoscine Hydrobromide complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Antiemetic; mydriatic; cycloplegic.
Preparations
Hyoscine Eye Drops
Hyoscine Injection
Hyoscine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hyoscine hydrobromide (scopolamine hydrobromide) contains not less than 99.0 per cent and not
more than the equivalent of 101.0 per cent of (1S,3s,5R,6R,7S)-6,7-epoxy-3-[(S)-(3-hydroxy-2-
phenylpropionyl)oxy]-8-methyl-8-azabicyclo[3.2.1]octane hydrobromide, calculated with refer-
CHARACTERS
A white, crystalline powder or colourless crystals, efflorescent, freely soluble in water, soluble in
alcohol.
It melts at about 197C with decomposition, determined after drying in vacuo for 24 h and then
IDENTIFICATION
obtained with hyoscine hydrobromide CRS. If the spectra recorded in the solid state for the substance
to be examined and the reference substance show differences, proceed as follows: dissolve 3 mg of
the substance to be examined in 1 ml of alcohol R and evaporate to dryness on a water-bath;
dissolve the residue in 0.5 ml of methylene chloride R and add 0.2 g of potassium bromide R and 15 ml
of ether R; allow to stand for 5 min shaking frequently; decant; dry the residue on a water-bath until
the solvent odour is no longer perceptible; using the residue prepare a disc and dry at 100C to
105C for 3 h. Repeat the procedure with hyoscine hydrobromide CRS and record the spectra.
C. Dissolve about 50 mg in 5 ml of water R and add 5 ml of picric acid solution R dropwise and with
shaking. The precipitate, washed with water R and dried at 100C to 105C for 2 h, melts (2.2.14)
at 188C to 193C.
E. It gives the reactions of bromides (2.3.1).
TESTS
21-4
Foreign alkaloids and decomposition products Examine by thin-layer chromatography (2.2.27),
using a TLC silica gel G plate R.
the same solvent.
volumes of concentrated ammonia R, 10 volumes of methanol R, 30 volumes of acetone R and 50
volumes of chloroform R. Dry the plate at 100C to 105C for 15 min. Allow to cool and spray with
dilute potassium iodobismuthate solution R until the spots appear. Any spot in the chromatogram
obtained with the test solution, apart from the principal spot, is not more intense than the spot in
the chromatogram obtained with reference solution (a) (1.0 per cent) and at most one such spot is
more intense than the spot in the chromatogram obtained with reference solution (b) (0.5 per
cent). Disregard any yellow spot at the starting point.
Apohyoscine Dissolve 0.10 g in 0.01M hydrochloric acid and dilute to 100.0 ml with the same acid.
Determine the absorbance (2.2.25) at 245 nm. The specific absorbance is not greater than 3.6,
calculated with reference to the anhydrous substance (about 0.5 per cent).
Water (2.5.12).10.0 per cent to 13.0 per cent, determined on 0.20 g by the semi-micro determina-
tion of water.
ASSAY
17
H
22
BrNO
4
.
STORAGE
Store in a well-filled, airtight container of small capacity, protected from light, at a temperature not
exceeding 15C.
__________________________________________________________________________________________________________ Ph Eur
21-5
Hyoscyamine Sulphate
N
Me
H Ph
O
O
H
OH
2
,H
2
SO
4
(C
17
H
23
NO
3
)
2
,H
2
SO
4
,2H
2
O 713 6835-16-1
Hyoscyamine Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hyoscyamine sulphate contains not less than 98.0 per cent and not more than the equivalent of
100.5 per cent of bis[(1R,3r,5S)-3-[(S)-(3-hydroxy-2-phenylpropionyl)oxy]-8-methyl-8-
azabicyclo[3.2.1]octane] sulphate, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder or colourless needles, very soluble in water, sparingly soluble or
IDENTIFICATION
obtained with hyoscyamine sulphate CRS.
C. To 0.5 ml of solution S (see Tests) add 2 ml of dilute acetic acid R and heat. To the hot solution
add 4 ml of picric acid solution R. Allow to cool, shaking occasionally. Collect the crystals, wash with
two quantities, each of 3 ml, of iced water R and dry at 100C to 105C. The crystals melt (2.2.14)
at 164C to 168C.
E. It gives reaction (a) of sulphates (2.3.1).
TESTS
Appearance of solution Solution S is not more intensely coloured than reference solution BY
6
(Method II, 2.2.2).
coating substance.
Test solution. Dissolve 0.2 g of the substance to be examined in 2 ml of methanol R.
Reference solution. Dilute 0.5 ml of the test solution to 100 ml with methanol R.
Apply separately to the plate 2 l of each solution. Stand a cylinder containing 10 ml of concentrated
ammonia R in the chromatographic tank and around it place the mobile phase consisting of a
mixture of 15 volumes of methanol R and 85 volumes of chloroform R. Allow the tank to saturate and
21-6
develop over a path of 15 cm. Dry the plate at 100C to 105C for 15 min, allow to cool and spray
with dilute potassium iodobismuthate solution R until the spots appear. Any spot in the chromatogram
obtained with the test solution, apart from the principal spot, is not more intense than the spot in
the chromatogram obtained with the reference solution (0.5 per cent).
Apoatropine Dissolve 0.100 g in 0.01M hydrochloric acid and dilute to 100.0 ml with the same acid.
Determine the absorbance (2.2.25) at 245 nm. The specific absorbance is not more than 4.0,
calculated with reference to the dried substance (about 0.5 per cent of apoatropine).
ASSAY
1 ml of 0.1M perchloric acid, is equivalent to 67.7 mg of C
34
H
48
N
2
O
10
S.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
21-7
Hyoscyamus Leaf
Hyoscyamus Leaf complies with the requirements of the 3rd edition of the European Pharmacopoeia [0225].
Preparations
Prepared Hyoscyamus
Hyoscyamus Dry Extract
When Hyoscyamus Leaf or Powdered Hyoscyamus Leaf is prescribed, Prepared Hyoscyamus shall
be dispensed.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hyoscyamus leaf consists of the dried leaf or the dried leaf and flowering, and occasionally fruit-
bearing, tops of Hyoscyamus niger L. It contains not less than 0.05 per cent of total alkaloids,
calculated as hyoscyamine (C
17
H
23
NO
3
; M
r
289.4) with reference to the drug dried at 100C to
105C. The alkaloids consist mainly of hyoscyamine with varying proportions of hyoscine
(scopolamine).
CHARACTERS
Hyoscyamus leaf has a slightly nauseous odour.
IDENTIFICATION
A. The leaves are yellowish-green to brownish-green, brittle and frequently broken. The base of
the lamina is cordate on sessile leaves and acute on petiolate leaves; the apex is acute and the
margin of the lamina lobes is irregularly dentate. The leaves are densely pubescent and sticky on
both surfaces. The midrib is broad and conspicuous with secondary veins arising at a wide angle
and terminating in the apices of the lobes. Stems are hollow and subcylindrical. The flowering tops
are densely pubescent in compact masses, the flowers being crowded together and arising in the
axils of large bracts; the flowers have a gamosepalous calyx which is broadly campanulate and has
five triangular cuspidate lobes, the corolla is five-lobed, shortly funnel-shaped, yellowish with
brown veins. The fruit is a pyxis, about 1.5 cm long when mature, enclosed in an accrescent,
persistent calyx and contains numerous brownish-grey seeds with a wavy, reticulated testa.
B. Reduce to a powder (355). The powder is greyish-green. Examine under a microscope using
chloral hydrate solution R. The powder shows the following diagnostic characters: fragments of leaf
lamina showing epidermal cells with wavy anticlinal walls and smooth cuticle; anisocytic and
anomocytic stomata (2.8.3) are more frequent on the lower epidermis; multicellular uniseriate
covering trichomes and glandular trichomes with thin, smooth walls; glandular trichomes have
long, multicellular stalks or short unicellular stalks and have clavate bicellular or multicellular
heads; dorsiventral mesophyll with a single layer of palisade cells and a spongy parenchyma
containing single or twinned calcium oxalate prisms; annularly and spirally thickened vessels. The
powdered drug may also show the following: fibres and reticulately thickened vessels from stem,
subspherical pollen grains up to 60 m in diameter, with three germinal pores, three furrows and
nearly smooth exine; fragments of the corolla with papillose epidermis; seed fragments containing
yellowish-brown, sinuous, thick-walled sclereids of testa; occasional cluster and microsphenoidal
crystals of calcium oxalate.
C. Examine the chromatograms obtained in the chromatography test. The principal zones in the
chromatogram obtained with the test solution are similar in position, colour and size to the
principal zones in the chromatogram obtained with the same volume of the reference solution.
D. Shake 1 g of the powdered drug (180) with 10 ml of 0.05M sulphuric acid for 2 min. Filter and
add to the filtrate 1 ml of concentrated ammonia R and 5 ml of water R. Shake cautiously with 15 ml of
peroxide-free ether R to avoid formation of an emulsion. Separate the ether layer and dry over
anhydrous sodium sulphate R. Filter and evaporate the ether in a porcelain dish. Add 0.5 ml of nitric
acid R and evaporate to dryness on a water-bath. Add 10 ml of acetone R and, dropwise, a 30 g/l
solution of potassium hydroxide R in alcohol R. A deep violet colour develops.
TESTS
Chromatography Examine by thin-layer chromatography (2.2.27), using silica gel G R as the
coating substance.
Test solution. To 2.0 g of the powdered drug (180) add 20 ml of 0.05M sulphuric acid, shake for 15
min and filter. Wash the filter with 0.05M sulphuric acid until 25 ml of filtrate is obtained. To the
21-8
filtrate add 1 ml of concentrated ammonia R and shake with two quantities, each of 10 ml, of peroxide-
free ether R. If necessary, separate by centrifugation. Dry the combined ether layers over anhydrous
sodium sulphate R, filter and evaporate to dryness on a water-bath. Dissolve the residue in 0.5 ml of
methanol R.
Reference solution. Dissolve 50 mg of hyoscyamine sulphate R in 9 ml of methanol R. Dissolve 15 mg of
hyoscine hydrobromide R in 10 ml of methanol R. Mix 3.8 ml of the hyoscyamine sulphate solution and
4.2 ml of the hyoscine hydrobromide solution and dilute to 10 ml with methanol R.
Apply separately to the plate as bands 20 mm by 3 mm 10 l and 20 l of each solution, leaving
1 cm between the bands. Develop over a path of 10 cm using a mixture of 3 volumes of concentrated
ammonia R, 7 volumes of water R and 90 volumes of acetone R. Dry the plate at 100C to 105C for
15 min, allow to cool and spray with potassium iodobismuthate solution R2, using about 10 ml for a
plate 200 mm square, until the orange or brown zones become visible against a yellow background.
The zones in the chromatograms obtained with the test solution are similar to those in the chrom-
atograms obtained with the reference solution with respect to their position (hyoscyamine in the
lower third, hyoscine in the upper third of the chromatogram) and their colour. The zones in the
chromatograms obtained with the test solution are at least equal in size to the corresponding zones
in the chromatogram obtained with the same volume of the reference solution. Faint secondary
zones may appear, particularly in the middle of the chromatogram obtained with 20 l of the test
solution or near the starting point in the chromatogram obtained with 10 l of the test solution.
Spray the plate with sodium nitrite solution R until the coating is transparent. Examine after 15 min.
The zones corresponding to hyoscyamine in the chromatograms obtained with the test solution and
the reference solution change from brown to reddish-brown but not to greyish-blue (atropine) and
any secondary zones disappear.
Foreign matter (2.8.2). Not more than 2.5 per cent of stem having a diameter exceeding 7 mm.
ASSAY
Take about 100 g of the drug and completely reduce it to a powder (180). Using the powder,
determine the loss on drying and the total alkaloids.
a) Determine the loss on drying (2.2.32) on 2.000 g by drying in an oven at 100C to 105C.
b) Moisten 40.0 g of the powder with a mixture of 8 ml of ammonia R, 10 ml of alcohol R and 30 ml
of peroxide-free ether R and mix thoroughly. Transfer the mixture to a suitable percolator, if
necessary with the aid of the extracting mixture. Allow to macerate for 4 h and percolate with a
mixture of 1 volume of chloroform R and 3 volumes of peroxide-free ether R until the alkaloids are
completely extracted. Evaporate to dryness a few millilitres of the liquid flowing from the
percolator, dissolve the residue in 0.25M sulphuric acid and verify the absence of alkaloids using
potassium tetraiodomercurate solution R. Concentrate the percolate to about 50 ml by distilling on a
water-bath and transfer it to a separating funnel, rinsing with peroxide-free ether R. Add a quantity of
peroxide-free ether R equal to at least 2.1 times the volume of the percolate to produce a liquid of a
density well below that of water. Shake the solution with no fewer than three quantities, each of
20 ml, of 0.25M sulphuric acid, separate the two layers by centrifugation if necessary and transfer the
acid layers to a second separating funnel. Make the acid layer alkaline with ammonia R and shake
with three quantities, each of 30 ml, of chloroform R. Combine the chloroform layers, add 4 g of
anhydrous sodium sulphate R and allow to stand for 30 min with occasional shaking. Decant the
chloroform and wash the sodium sulphate with three quantities, each of 10 ml, of chloroform R. Add
the washings to the chloroform extract, evaporate to dryness on a water-bath and heat in an oven
at 100C to 105C for 15 min. Dissolve the residue in a few millilitres of chloroform R, add 20.0 ml
of 0.01M sulphuric acid and remove the chloroform by evaporation on a water-bath. Titrate the
excess of acid with 0.02M sodium hydroxide, using methyl red mixed solution R as indicator.
Calculate the percentage content of total alkaloids, expressed as hyoscyamine, from the
expression:
m d
n
) 100 (
) 0 . 2 ( 88 . 57

STORAGE
Store protected from light and humidity.
__________________________________________________________________________________________________________ Ph Eur
21-9
Prepared Hyoscyamus
Prepared Hyoscyamus complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Prepared hyoscyamus is hyoscyamus leaf power (180) adjusted, if necessary, by the addition of
powdered lactose or hyoscyamus leaf powder with a lower alkaloidal content to contain 0.05 per
cent to 0.07 per cent of total alkaloids, calculated as hyoscyamine (C
17
H
23
NO
3
; M
r
289.4) with
CHARACTERS
It presents the characters of the powder described in the monograph on Hyoscyamus leaf (225).
IDENTIFICATION
It complies with identification tests B, C and D in the monograph on Hyoscyamus leaf (225).
Examined in glycerol (85 per cent) R, it may be seen to contain lactose crystals.
TESTS
It complies with the tests for chromatography, for total ash and for ash insoluble in hydrochloric
acid prescribed in the monograph on Hyoscyamus leaf (225) and with the following additional test.
at 100C to 105C.
ASSAY
Carry out the assay prescribed in the monograph on Hyoscyamus leaf (225).
Calculate the percentage of total alkaloids, expressed as hyoscyamine, from the expression:
m d
n
) 100 (
) 0 . 2 ( 88 . 57

STORAGE
__________________________________________________________________________________________________________ Ph Eur
21-10
Hypericum
St Johns Wort corrected 1/01
Hypericum complies with the requirements of the 3rd edition of the European Pharmacopoeia [1438]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hypericum consists of the whole or cut, dried flowering tops of Hypericum perforatum L., harvested
during flowering time. It contains not less than 0.08 per cent of total hypericins expressed as
hypericin (C
30
H
16
O
8
; M
r
CHARACTERS
IDENTIFICATION
A. The branched and bare stem shows two more-or-less prominent longitudinal ridges. The leaves
are opposite, sessile, exstipulate, oblong-oval and 15 mm to 30 mm long. The leaf margins show
black glandular trichomes, and many small translucent, oil glands are present on the entire surface
and are visible by transmitted light. The flowers are regular and form corymbose clusters at the
apex of the stem. They have five green, acute sepals, with black secretory trichomes on the
margins; five orange-yellow petals, also with black secretory trichomes on the margins; three
staminal blades, each divided into many orange-yellow stamens and three carpels surmounted by
red styles.
chloral hydrate solution R. The powder shows fragments of polygonal cells of the epidermis with
thickened and beaded walls and paracytic or anomocytic stomata (2.8.3); fragments of the leaf and
sepal with large oil glands and red pigment cells; thin-walled, elongated cells of the petal epidermis
with straight or wavy anticlinal walls; tracheids and tracheidal vessels with pitted walls and groups
of thick-walled fibres; fragments of rectangular, lignified and pitted parenchyma; fibrous layer of
the anther and elongated, thin-walled cells of the filament with a striated cuticle; numerous pollen
grains with three pores and a smooth exine, occur singly or in dense groups, and calcium oxalate
cluster crystals.
Test solution. Stir 0.5 g of the powdered drug (500) in 10 ml of methanol R in a water-bath at 60C
for 10 min and filter.
Reference solution. Dissolve 5 mg of rutin R and 5 mg of hyperoside R in methanol R and dilute to 5 ml
Apply to the plate as 10 mm bands 10 l of the test solution and 5 l of the reference solution.
water R and 90 volumes of ethyl acetate R. Allow the plate to dry at 100C to 105C for 10 min.
Spray the plate with a 10 g/l solution of diphenylboric acid aminoethyl ester R in methanol R and then
with a 50 g/l solution of macrogol 400 R in methanol R. Examine the plate after about 30 min in
ultraviolet light at 365 nm. The chromatogram obtained with the reference solution shows in the
lower third the zone due to rutin and above it the zone due to hyperoside, both with yellow-orange
fluorescence. The chromatogram obtained with the test solution shows in the lower third the
reddish-orange fluorescent zones of rutin and hyperoside and in the lower part of the upper third
the zone of pseudohypericin and above it the zone of hypericin, both with red fluorescence. Other
yellow or blue fluorescent zones are visible.
TESTS
Foreign matter (2.8.2). Not more than 3 per cent of stems with a diameter greater than 5 mm,
and not more than 2 per cent of other foreign matter.
drug (500) by drying in an oven at 100C to 105C for 2 h.
ASSAY
Test solution. In a 100 ml round-bottomed flask, introduce 0.800 g of the powdered drug (500),
60 ml of a mixture of 20 volumes of water R and 80 volumes of tetrahydrofuran R and a magnetic
21-11
stirrer. Boil the mixture in a water-bath at 70C under a reflux condenser for 30 min. Centrifuge (2
min at 700 g) and decant the supernatant into a 250 ml flask. Take up the residue with 60 ml of a
mixture of 20 volumes of water R and 80 volumes of tetrahydrofuran R. Heat again under a reflux
condenser for 30 min. Centrifuge (2 min at 700 g) and decant the supernatant. Combine the
extracts and evaporate to dryness. Take up the residue with 15 ml of methanol R with the help of
ultrasound and transfer to a 25 ml measuring flask. Rinse the 250 ml flask with methanol R and
dilute to 25.0 ml with the same solvent. Centrifuge again, filter 10 ml through a syringe filter
(0.2 m). Discard the first two millilitres of the filtrate. Introduce 5.0 ml of the filtrate into a
measuring flask and dilute to 25.0 ml with methanol R.
Compensation solution. Methanol R.
Measure the absorbance (2.2.25) of the test solution at 590 nm by comparison with the
compensation solution.
Calculate the percentage content of total hypericins, expressed as hypericin, from the
expression:
870
125
m
A
i. e. taking the specific absorbance of hypericin to be 870.
m=mass of the substance to be examined, in grams.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
21-12
Hypromellose
9004-65-3
Hypromellose complies with the requirements of the 3rd edition of the European Pharmacopoeia [0348]. These
Action and use Used in treatment of tear deficiency.
Preparation
Hypromellose Eye Drops
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hypromellose (hydroxypropylmethylcellulose) is a partly O-methylated and O-(2-
hydroxypropylated) cellulose.
CHARACTERS
A white, yellowish-white or greyish-white powder or granules, hygroscopic after drying, practically
insoluble in hot water, in acetone, in ethanol, in ether and in toluene. It dissolves in cold water
giving a colloidal solution.
IDENTIFICATION
A. Heat 10 ml of solution S (see Tests) in a water-bath while stirring. At a temperature above 50C
the solution becomes cloudy or a flocculent precipitate is formed. The solution becomes clear
again on cooling.
water R. In a test-tube, mix thoroughly while cooling in iced water 1 ml of the solution with 8 ml of
sulphuric acid R, added dropwise. Heat in a water-bath for exactly 3 min and immediately cool in
iced water. While the mixture is cold, carefully add 0.6 ml of ninhydrin solution R2 and mix well.
Allow to stand at 25C. A pink colour is produced immediately and becomes violet within 100 min.
F. 0.2 g does not dissolve in 10 ml of toluene R nor in 10 ml of ethanol R.
TESTS
the mass of the solution to 100 g with carbon dioxide-free water R and stir until dissolution is
complete.
6
(Method II. 2.2.2).
Apparent viscosity While stirring, introduce a quantity of the substance to be examined equiva-
lent to 6.00 g of the dried substance into 150 g of water R heated to 90C. Stir with a propeller-type
stirrer for 10 min, place the flask in a bath of iced water, continue the stirring and allow to remain
in the bath of iced water for 40 min to ensure that solution is complete. Adjust the mass of the
solution to 300 g, and centrifuge the solution to expel any entrapped air. Adjust the temperature of
the solution to 20 0.1C. Determine the viscosity (2.2.10) with a rotating viscometer at 20C and
a shear rate of 10 s
1
. The apparent viscosity is not less than 75 per cent and not more than 140 per
cent of the value stated on the label.
21-13
oven at 100C to 105C.
STORAGE
LABELLING
__________________________________________________________________________________________________________ Ph Eur
21-14
Hypromellose Phthalate
Hypromellose Phthalate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Used in treatment of tear deficiency.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hypromellose phthalate (hydroxypropylmethylcellulose phthalate) is a monophthalic acid ester of
hypromellose. It contains methoxy (OCH
3
) and 2-hydroxypropoxy (OCH
2
CHOHCH
3
) groups
and not less than 21.0 per cent and not more than 35.0 per cent of phthalyl (o-carboxybenzoyl
C
8
H
5
O
3
) groups, calculated with reference to the anhydrous substance.
CHARACTERS
White or slightly off-white, free-flowing flakes or a granular powder, practically insoluble in water,
soluble in a mixture of equal volumes of acetone and methanol and in a mixture of equal volumes of
methanol and methylene chloride, very slightly soluble in acetone and in toluene, practically
insoluble in ethanol.
IDENTIFICATION
A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph. Eur.
reference spectrum of hypromellose phthalate.
B. Dissolve about 40 mg in 1 ml of a mixture of equal volumes of acetone R and of methanol R. Allow
the solution to flow over a glass plate and dry. A colourless, transparent film is formed.
TESTS
Free phthalic acid Examine by liquid chromatography (2.2.29).
Test solution. Dissolve 0.20 g of the substance to be examined in about 50 ml of acetonitrile R with
the aid of ultrasound. Add 10 ml of water R, cool to room temperature and dilute to 100.0 ml with
acetonitrile R.
Reference solution. Dissolve 5.0 mg of phthalic acid R in 125 ml of acetonitrile R. Add 25 ml of water R
and dilute to 250.0 ml with acetonitrile R.
a column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilane chemically
bound to porous silica or ceramic micro-particles (5 m 10 m),
as mobile phase at a flow rate of 2 ml per minute a mixture of 15 volumes of acetonitrile R and
85 volumes of an 8.5 g/l solution of cyanoacetic acid R,
Inject 20 l of the reference solution. Adjust the sensitivity of the system so that the height of the
principal peak, apart from the peaks due to the solvent, is at least 50 per cent of the full scale of the
recorder.
Inject 20 l of the test solution. In the chromatogram obtained with the test solution, the area of
any peak corresponding to phthalic acid is not greater than the area of the principal peak in the
chromatogram obtained with the reference solution (1 per cent).
Chlorides Dissolve 1.0 g in 40.0 ml of 0.2M sodium hydroxide, add 0.05 ml of phenolphthalein
solution R and dilute nitric acid R dropwise and with stirring until the red colour disappears. Add an
additional 20.0 ml of dilute nitric acid R with stirring. Heat on a water bath with stirring until the gel-
like precipitate formed becomes granular. Cool and centrifuge. Separate the liquid phase and wash
the residue with three quantities, each of 20 ml, of water R, separating the washings by centrifuga-
tion. Combine the liquid phases, filter, add 5.0 ml of 0.1M silver nitrate, dilute to 200.0 ml with
water R and mix. 50.0 ml of the solution is not more opalescent than a standard prepared by
treating 0.5 ml of 0.01M hydrochloric acid with 10.0 ml of 0.2M sodium hydroxide, adding 7 ml of dilute
nitric acid R, 5.0 ml of 0.1M silver nitrate and diluting to 50.0 ml with water R (0.07 per cent).
tion of water using 50 ml of anhydrous methanol R as the solvent.
ASSAY
Dissolve 1.000 g in 50 ml of a mixture of 1 volume of water R, 2 volumes of acetone R and 2 volumes
21-15
of alcohol R. Add 0.1 ml of phenolphthalein solution R and titrate with 0.1M sodium hydroxide until a
faint pink colour is obtained. Carry out a blank titration.
Calculate the percentage content of phthaloyl groups from the expression:
149
100
n
a m
S
( )
1.795
n=number of millilitres of 0.1M sodium hydroxide used,
S=percentage content of free phthalic acid (see Tests).
STORAGE
__________________________________________________________________________________________________________ Ph Eur
21-16
Ibuprofen
COOH
Me H
Bu
i
and enantiomer
C
13
H
18
O
2
206.3 15687-27-1
Ibuprofen complies with the requirements of the 3rd edition of the European Pharmacopoeia [0721]. These
Preparations
Ibuprofen Cream
Ibuprofen Gel
Ibuprofen Oral Suspension
Ibuprofen Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ibuprofen contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent
of (RS)-2-(4-isobutylphenyl)propionic acid, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder or colourless crystals, practically insoluble in water, freely soluble in
acetone, in ether, in methanol and in methylene chloride. It dissolves in dilute solutions of alkali
hydroxides and carbonates.
IDENTIFICATION
B. Dissolve 50.0 mg in a 4 g/l solution of sodium hydroxide R and dilute to 100.0 ml with the same
alkaline solution. Examined between 240 nm and 300 nm (2.2.25), using a spectrophotometer with
a band width of 1.0 nm and a scan speed of not more than 50 nm per minute, the solution shows a
shoulder at 258 nm and two absorption maxima, at 264 nm and 272 nm. The ratio of the absorb-
ance measured at the maximum at 264 nm to that measured at the shoulder at 258 nm is 1.20 to
1.30. The ratio of the absorbance measured at the maximum at 272 nm to that measured at the
shoulder at 258 nm is 1.00 to 1.10.
obtained with ibuprofen CRS. Examine the substances prepared as discs.
D. Examine by thin-layer chromatography (2.2.27), using silica gel H R as the coating substance.
Reference solution. Dissolve 50 mg of ibuprofen CRS in methylene chloride R and dilute to 10 ml with the
same solvent.
5 volumes of anhydrous acetic acid R, 25 volumes of ethyl acetate R and 75 volumes of hexane R.
Allow the plate to dry at 120C for 30 min. Lightly spray the plate with a 10 g/l solution of potassium
permanganate R in dilute sulphuric acid R and heat at 120C for 20 min. Examine in ultraviolet light at
solution.
TESTS
Solution S Dissolve 2.0 g in methanol R and dilute to 20 ml with the same solvent.
Optical rotation (2.2.7). Dissolve 0.50 g in methanol R and dilute to 20.0 ml with the same
solvent. The angle of optical rotation is 0.05 to +0.05.
21-17
Test solution. Dissolve 20 mg of the substance to be examined in 2 ml of acetonitrile R and dilute to
Reference solution (b). Dissolve 20 mg of ibuprofen CRS in 2 ml of acetonitrile R, add 1 ml of a 0.06 g/l
solution of 2-(4-butylphenyl)propionic acid CRS in acetonitrile R and dilute to 10.0 ml with the mobile
phase.
as mobile phase at a flow rate of 2 ml per minute a mixture of 0.5 volumes of phosphoric acid R,
340 volumes of acetonitrile R and 600 volumes of water R; allow to equilibrate and dilute to
1000 volumes with water R,
obtained with 20 l of reference solution (a) is 70 per cent to 90 per cent of the full scale of the
recorder.
When the chromatogram is recorded in the prescribed conditions, the retention time of
ibuprofen is about 20 min. Inject 20 l of reference solution (b). Measure the height (A) above the
base-line of the peak due to 2-(4-butylphenyl)propionic acid and the height (B) above the base-line
of the lowest point of the curve separating this peak from the peak due to ibuprofen. The test is not
valid unless A is greater than 1.5 times B. If necessary, adjust the concentration of acetonitrile in
the mobile phase in order to obtain the required resolution.
Inject separately 20 l of the test solution and 20 l of reference solution (a). Continue the
chromatography for 1.5 times the retention time of the principal peak. In the chromatogram
obtained with the test solution, the area of any peak corresponding to 2-(4-butylphenyl)propionic
acid is not greater than that of the peak due to 2-(4-butylphenyl)propionic acid in the chromato-
gram obtained with reference solution (b) (0.3 per cent); the area of any peak, apart from the
principal peak and any peak corresponding to 2-(4-butylphenyl)propionic acid, is not greater than
0.3 times the area of the principal peak in the chromatogram obtained with reference solution (a)
(0.3 per cent) and the sum of the areas of any such peaks is not greater than 0.7 times the area of
the principal peak in the chromatogram obtained with reference solution (a) (0.7 per cent).
Disregard any peak due to the solvent and any peak with an area less than 0.1 times the area of the
Prepare the standard using lead standard solution (1 ppm Pb) prepared by diluting lead standard
solution (100 ppm Pb) R with methanol R.
Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in vacuo
over diphosphorus pentoxide R.
ASSAY
Dissolve 0.450 g in 50 ml of methanol R. Add 0.4 ml of phenolphthalein solution R1. Titrate with 0.1M
sodium hydroxide until a red colour is obtained. Carry out a blank titration.
13
H
18
O
2
.
STORAGE
IMPURITIES
COOH
Me H
Bu
i
A. 2-(3-isobutylphenyl)propionic acid,
COOH
Me H
Bu
n
B. 2-(4-butylphenyl)propionic acid,
21-18
CONH
2
Me H
Bu
i
C. 2-(4-isobutylphenyl)propionamide,
COOH
Me H
e
D. 2-(4-methylphenyl)propionic acid,
CH
3
Bu
i
O
E. 4-isobutylacetophenone.
__________________________________________________________________________________________________________ Ph Eur
21-19
Iceland Moss
Iceland Moss complies with the requirements of the 3rd edition of the European Pharmacopoeia [1439]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Iceland moss consists of the whole or cut dried thallus of Cetraria islandica (L.) Acharius s.l.
CHARACTERS
Iceland moss has a bitter, mucilaginous taste.
IDENTIFICATION
A. The thallus, up to 15 cm long, is irregularly dichotomous and consists of glabrous, groove-
shaped or almost flat, stiff, brittle bands, 0.3 cm to 1.5 cm wide and about 0.5 mm thick,
sometimes serrated with the margin appearing ciliated (pycnidia). The upper surface is greenish to
greenish-brown, the lower surface is greyish-white to light brownish and shows whitish, depressed
spots (so-called respiratory cavities). On the apexes of the terminal lobes, very rarely, there are
brown, discoid apothecia.
B. Reduce to a powder (355). The powder is greyish-brown. Examine under a microscope, using
chloral hydrate solution R. The powder shows numerous fragments of the pseudoparenchyma
consisting of narrow-lumened, thick-walled hyphae from the marginal layer and wide-lumened
hyphae from the adjacent layer consisting of loosely entwined hyphae, in which, in the medullary
zone, greenish to brownish algae cells up to 15 m in diameter, are embedded; occasionally
marginal fragments of the thallus with tube-like or cylindrical spermogonia, up to about 160 m
wide and up to about 400 m long.
C. To 1.0 g of the powdered drug (355) add 10 ml of water R and boil for 2 min to 3 min. The
greyish-brown solution forms a gel after cooling.
D. Examine the chromatograms obtained in the test for other lichen species. The chromatogram
obtained with the test solution shows the violet to grey zone of fumaro-protocetraric acid located
slightly below the zone of caffeic acid in the chromatogram obtained with the reference solution.
Other fainter zones are present.
TESTS
Other lichen species Examine by thin-layer chromatography(2.2.27), using a TLC silica gel plate R.
Test solution. To 1.0 g of the powdered drug (355) add 5 ml of acetone R and heat in a water-bath
under a reflux condenser for 2 min to 3 min. Cool and filter.
Reference solution. Dissolve 5 mg of anethole R and 5 mg of caffeic acid R in 2 ml of acetone R.
Develop over a path of 10 cm, using a mixture of 5 volumes of acetone R, 5 volumes of methanol R,
10 volumes of glacial acetic acid R and 80 volumes of toluene R. Allow the plate to dry in air and
spray the plate with anisaldehyde solution R. Examine in daylight while heating at 100C to 105C for
5 min to 10 min. The chromatogram obtained with the reference solution shows in the lower half a
greyish-blue to violet-blue zone (caffeic acid) and in the upper half a blue to blue-violet zone
(anethole). The chromatogram obtained with the test solution shows no red-violet zone a little
below the zone of anethole in the chromatogram obtained with the reference solution.
Swelling value (2.8.4). Not less than 4.5, determined on powdered drug (355).
STORAGE
__________________________________________________________________________________________________________ Ph Eur
21-20
Ichthammol
Ammonium Ichthosulphonate
Ichthammol complies with the requirements of the 3rd edition of the European Pharmacopoeia [0917]. These
Action and use Used in treatment of psoriasis.
Preparation
Zinc and Ichthammol Cream
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ichthammol is obtained by distillation from certain bituminous schists, sulphonation of the distillate
and neutralisation of the product with ammonia. It contains not less than 50.0 per cent m/m and
not more than 56.0 per cent m/m of dry matter, not less than 4.5 per cent m/m and not more than
7.0 per cent m/m of total ammonia (NH
3
; M
r
17.03) and not less than 10.5 per cent m/m of
organically combined sulphur, calculated with reference to the dried substance; not more than
20.0 per cent m/m of the total sulphur is in the form of sulphate.
CHARACTERS
A dense, blackish-brown liquid, miscible with water and with glycerol, slightly soluble in alcohol, in
ether, in fatty oils and in liquid paraffin. It forms homogeneous mixtures with wool fat and soft
paraffin.
IDENTIFICATION
A. Dissolve 1.5 g in 15 ml of water R (solution A). To 2 ml of solution A add 2 ml of hydrochloric
acid R. A resinous precipitate is formed. Decant the supernatant liquid. The precipitate is partly
soluble in ether R.
B. 2 ml of solution A, obtained in identification test A, gives the reaction of ammonium salts and
salts of volatile bases (2.3.1).
C. Evaporate and ignite the mixture of solution A and dilute sodium hydroxide solution R obtained in
identification test B. Take up the residue with 5 ml of dilute hydrochloric acid R. Gas is evolved which
turns lead acetate paper R brown or black. Filter the solution. The filtrate gives reaction (a) of
sulphates (2.3.1).
TESTS
Acidity or alkalinity To 10.0 ml of the clear filtrate obtained in the assay of total ammonia add
0.05 ml of methyl red solution R. Not more than 0.2 ml of 0.02M hydrochloric acid or 0.02M sodium
hydroxide is required to change the colour of the indicator.
Relative density (2.2.5). 1.040 to 1.085, determined on a mixture of equal volumes of the
substance to be examined and water R.
ASSAY
Dry matter Weigh 1.000 g in a tared flask containing 2 g of sand R, previously dried to constant
mass, and a small glass rod. Heat on a water-bath for 2 h with frequent stirring and dry in an oven
at 100C to 105C until two consecutive weighings do not differ by more than 2.0 mg; the second
weighing is carried out after drying again for 1 h.
Total ammonia Dissolve 2.50 g in 25 ml of warm water R. Rinse the solution into a 250 ml
volumetric flask, add 200 ml of sodium chloride solution R and dilute to 250.0 ml with water R. Filter
the solution, discarding the first 20 ml of filtrate. To 100.0 ml of the clear filtrate add 25 ml of
formaldehyde solution R, neutralised to phenolphthalein solution R1. Titrate with 0.1M sodium hydroxide
until a faint pink colour is obtained.
1 ml of 0.1M sodium hydroxide is equivalent to 1.703 mg of NH
3
.
Organically combined sulphur Mix 0.500 g with 4 g of anhydrous sodium carbonate R and 3 ml of
methylene chloride R in a porcelain crucible of about 50 ml capacity, warm and stir until all the
methylene chloride has evaporated. Add 10 g of coarsely powdered copper nitrate R, mix thoroughly
and heat the mixture very gently using a small flame. When the initial reaction has subsided,
increase the temperature slightly until most of the material has blackened. Cool, place the crucible
in a large beaker, add 20 ml of hydrochloric acid R and, when the reaction has ceased, add 100 ml of
water R and boil until all the copper oxide has dissolved. Filter the solution, add 400 ml of water R,
heat to boiling and add 20 ml of barium chloride solution R1. Allow to stand for 2 h, filter, wash with
21-21
water R, dry and ignite at about 600C until two successive weighings do not differ by more than
0.2 per cent of the mass of the residue.
1 g of residue is equivalent to 0.1374 g of total sulphur.
Calculate the percentage of sulphur and subtract the percentage of sulphur in the form of
sulphate.
Sulphur in the form of sulphate Dissolve 2.000 g in 100 ml of water R, add 2 g of cupric chloride R
dissolved in 80 ml of water R and dilute to 200.0 ml with water R. Shake and filter. Heat 100.0 ml of
the filtrate almost to boiling, add 1 ml of hydrochloric acid R and 5 ml of barium chloride solution R1
dropwise and heat on a water-bath. Filter, wash the precipitate with water R, dry and ignite at about
600C until two successive weighings do not differ by more than 0.2 per cent of the mass of the
residue.
1 g of residue is equivalent to 0.1374 g of sulphur present in the form of sulphate.
Calculate the percentage of sulphur in the form of sulphate.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
21-22
Idoxuridine
O
OH
HOCH
2
N
NH
O
O
I
C
9
H
11
IN
2
O
5
354.1 54-42-2
Idoxuridine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0669]. These
Preparation
Idoxuridine Eye Drops
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Idoxuridine contains not less than 98.0 per cent and not more than the equivalent of 101.0 per
cent of 5-iodo-1-(2-deoxy-b-d-erythro-pentofuranosyl)-1H,3H-pyrimidine-2,4-dione, calculated
CHARACTERS
A white or almost white, crystalline powder, slightly soluble in water and in alcohol, practically
insoluble in ether. It dissolves in dilute solutions of alkali hydroxides.
IDENTIFICATION
obtained with idoxuridine CRS. Examine the substances as discs prepared using 1 mg of the
substance to be examined and of the reference substance each in 0.3 g of potassium bromide R.
the chromatogram obtained with test solution (b) is similar in position and size to the principal spot
in the chromatogram obtained with reference solution (c).
C. Heat about 5 mg in a test-tube over a naked flame. Violet vapour is evolved.
D. Disperse about 2 mg in 1 ml of water R and add 2 ml of diphenylamine solution R2. Heat in a water-
bath for 10 min. A persistent light-blue colour develops.
TESTS
Solution S Dissolve 0.500 g in 1M sodium hydroxide and dilute to 50.0 ml with the same solvent.
Related substances Examine by thin-layer chromatography (2.2.27), using as coating substance a
solvents.
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with a mixture of 1 volume of concentrated
21-23
Reference solution (a). Dissolve 20 mg of 5-iodouracil R, 20 mg of 2-deoxyuridine R and 20 mg of
5-bromo-2-deoxyuridine R in a mixture of 1 volume of concentrated ammonia R and 5 volumes of
methanol R and dilute to 100 ml with the same mixture of solvents.
Reference solution (b). Dissolve 0.20 g of the substance to be examined in 5 ml of reference solution
(a).
Reference solution (c). Dissolve 20 mg of idoxuridine CRS in a mixture of 1 volume of concentrated
Reference solution (d). Dilute 1 ml of test solution (b) to 20 ml with a mixture of 1 volume of
Apply separately to the plate 5 l of each solution. Develop twice over a path of 15 cm using a
mixture of 10 volumes of concentrated ammonia R, 40 volumes of chloroform R and 50 volumes of
2-propanol R, drying the plate in a current of cold air after each development. Examine in ultraviolet
light at 254 nm. In the chromatogram obtained with test solution (a): any spots corresponding to
5-iodouracil, 2-deoxyuridine and 5-bromo-2-deoxyuridine are not more intense than the
corresponding spots in the chromatogram obtained with reference solution (a) (0.5 per cent); any
spot, apart from the principal spot and the spots corresponding to 5-iodouracil, 2-deoxyuridine
and 5-bromo-2-deoxyuridine, is not more intense than the spot in the chromatogram obtained
with reference solution (d) (0.5 per cent). The test is not valid unless the chromatogram obtained
with reference solution (b) shows four clearly separated spots.
Iodide Dissolve 0.25 g in 25 ml of 0.1M sodium hydroxide, add 5 ml of dilute hydrochloric acid R and
dilute to 50 ml with water R. Allow to stand for 10 min and filter. To 25 ml of the filtrate add 5 ml of
dilute hydrogen peroxide solution R and 10 ml of chloroform R and shake. Any pink colour in the organic
layer is not more intense than that in a standard prepared at the same time in the same manner
using 1 ml of a 0.33 g/l solution of potassium iodide R instead of the substance to be examined
(0.1 per cent).
60C.
ASSAY
9
H
11
IN
2
O
5
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
21-24
Ifosfamide
1/01
O N
P
O
Cl
H
N
Cl
and enantiomer
C
7
H
15
Cl
2
N
2
O
2
P 261.1 3778-73-2
Ifosfamide complies with the requirements of the 3rd edition of the European Pharmacopoeia [1529]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ifosfamide contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent
of (RS)-N,3-bis(2-chloroethyl)-1,3,2-oxazaphosphinan-2-amine 2-oxide, calculated with reference
CHARACTERS
A white or almost white, fine, crystalline powder, hygroscopic, soluble in water, freely soluble in
methylene chloride.
IDENTIFICATION
spectrum of ifosfamide. Examine the substance prepared as a disc.
TESTS
solution Y
7
(Method II, 2.2.2).
Acidity or alkalinity Dilute 5 ml of solution S to 50 ml with carbon dioxide-free water R. To 10 ml of
this solution add 0.1 ml of methyl red solution R. Not more than 0.1 ml of 0.01M hydrochloric acid is
required to change the colour of the indicator to red. To another 10 ml of the solution add 0.1 ml
of phenolphthalein solution R. Not more than 0.3 ml of 0.01M sodium hydroxide is required to change
the colour of the indicator to pink.
+0.10.
Related substances
Reference solution (a). Dissolve 25 mg of ifosfamide impurity A CRS and 25 mg of chloroethylamine
hydrochloride R (impurity C) in a mixture of equal volumes of methanol R and water R and dilute to
Reference solution (b). Dissolve 15 mg of ifosfamide impurity B CRS in a mixture of equal volumes of
Reference solution (c). Dissolve 5 mg of ethanolamine R (impurity D), 20 mg of ifosfamide impurity A
CRS and 80 mg of chloroethylamine hydrochloride R (impurity C) in a mixture of equal volumes of
volumes of water R, 15 volumes of methanol R, 25 volumes of anhydrous acetic acid R and 50 volumes
of methylene chloride R. Dry the plate at 115C for 45 min. At the bottom of a chromatography tank,
place an evaporating dish containing a 3.2 g/l solution of potassium permanganate R and add an equal
volume of dilute hydrochloric acid R, close the tank and allow to stand for 10 min. Place the plate
whilst still hot in the tank, avoiding contact of the stationary phase with the solution, and close the
21-25
tank. Leave the plate in contact with the chlorine vapour for 20 min. Withdraw the plate and place
it in a current of cold air until the excess of chlorine is removed (about 20 min) and an area of
coating below the points of application does not give a blue colour with a drop of potassium iodide
and starch solution R. Avoid prolonged exposure to cold air. Immerse the plate in a 1 g/l solution of
tetramethylbenzidine R in alcohol R for 5 s. Allow the plate to dry and examine. In the chromatogram
obtained with the test solution: any spot corresponding to impurity A or impurity C is not more
intense than the corresponding spot in the chromatogram obtained with reference solution (a)
(0.25 per cent); any spot corresponding to impurity B is not more intense than the corresponding
spot in the chromatogram obtained with reference solution (b) (0.15 per cent); any other spot is
shows 3 clearly separated spots.
Reference solution (a). Dissolve 5 mg of ifosfamide impurity E CRS and 5 mg of ifosfamide impurity F CRS
in a mixture of equal volumes of methanol R and methylene chloride R and dilute to 100 ml with the
Reference solution (b). Dissolve 10 mg of ifosfamide impurity E CRS and 10 mg of ifosfamide CRS in a
mixture of equal volumes of methanol R and methylene chloride R and dilute to 100 ml with the same
of methylene chloride R and 10 volumes of acetone R. Dry the plate at 115C for 45 min. Proceed as
described in Related substances test A. Any spot corresponding to impurity E or impurity F in the
chromatogram obtained with the test solution is not more intense than the corresponding spot in
the chromatogram obtained with reference solution (a) (0.25 per cent). The test is not valid unless
the chromatogram obtained with reference solution (b) shows 2 clearly separated spots.
Chlorides (2.4.4). Dilute 1 ml of solution S to 100 ml with water R. Dilute 1 ml of this solution to
30 ml with water R. The freshly prepared solution complies with the limit test for chlorides
(100 ppm).
tion of water.
ASSAY
Examine by liquid chromatography (2.2.29), using ethyl parahydroxybenzoate R as the internal
standard. Use the solutions within 24 h.
Internal standard solution. Dissolve 50.0 mg of ethyl parahydroxybenzoate R in 25 ml of alcohol R, dilute
to 100.0 ml with water R and mix.
Test solution. To 0.150 g of the substance to be examined add 10.0 ml of the internal standard
solution and dilute to 250.0 ml with water R.
Reference solution. To 15.0 mg of ifosfamide CRS add 1.0 ml of the internal standard solution and
volumes of water R,
Inject 1 l of the reference solution six times. The assay is not valid unless the resolution between
the peaks due to ifosfamide and the internal standard is not less than 6.0 and the relative standard
deviation of the peak area for ifosfamide is at most 2.0 per cent.
Inject 1 l of the test solution. Calculate the percentage content of C
7
H
15
Cl
2
N
2
O
2
P from the area
of the corresponding peak in the chromatogram obtained and the declared content of ifosfamide
CRS.
STORAGE
21-26
IMPURITIES
Related substances test A
Qualified impurities: A, B, C.
Other detectable impurities: D.
O
H
N
Cl
HO OH
P
O
A. 3-[(2-chloroethyl)amino]propyl dihydrogen phosphate,
O
H
N
Cl
2
O
HO O
P
B. bis[3-[(2-chloroethyl)amino]propyl] dihydrogen diphosphate,
NH
2
Cl
C. 2-chloroethanamine,
NH
2
HO
D. 2-aminoethanol.
Related substances test B
H
N
Cl
l
E. 3-chloro-N-(2-chloroethyl)propan-1-amine,
O N
P
O
Cl
l
and enantiomer
F. (RS)-2-chloro-3-(2-chloroethyl)-1,3,2-oxazaphosphinane 2-oxide.
__________________________________________________________________________________________________________ Ph Eur
21-27
Imipenem
1/01
N
S
O
H H
H
OH
Me
COOH
N NH
H
C
12
H
17
N
3
O
4
S,H
2
O 317.4 64221-86-9
Imipenem complies with the requirements of the 3rd edition of the European Pharmacopoeia [1226]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Imipenem contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent
of (5R,6S)-6-[(R)-1-hydroxyethyl]-3-[[2-[(iminomethyl)amino]ethyl]sulphanyl]-7-oxo-1-
azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid, calculated with reference to the anhydrous
substance.
CHARACTERS
A white to almost white or pale yellow powder, sparingly soluble in water, slightly soluble in
methanol.
IDENTIFICATION
Examine by infrared absorption spectroscopy (2.2.24), comparing with the spectrum obtained with
imipenem CRS.
TESTS
Appearance of solution Dissolve 0.500 g in phosphate buffer solution pH 7.0 R3 and dilute to 50 ml
with the same solution. The solution is not more opalescent than reference suspension II (2.2.1)
and not more intensely coloured than intensity 6 of the range of the reference solutions of the most
appropriate colour (Method II, 2.2.2).
pH (2.2.3). Dissolve 0.500 g in carbon dioxide-free water R and dilute to 100.0 ml with the same
solvent. The pH of the solution is 4.5 to 7.0.
Specific optical rotation (2.2.7). Dissolve 0.125 g in phosphate buffer solution pH 7.0 R3 and dilute
to 25.0 ml with the same solution. The specific optical rotation measured at 25C is +84 to +89,
peak in the chromatogram obtained is at least 50 per cent of the full scale of the recorder. Inject
20 l of the test solution and continue the chromatography for twice the retention time of the
principal peak. In the chromatogram obtained with the test solution: the area of any peak corres-
ponding to thienamycin is not greater than that of the principal peak in the chromatogram obtained
with reference solution (b) (1 per cent); the area of any peak, apart from the principal peak and
any peak corresponding to thienamycin, is not greater than 0.3 times the area of the principal peak
in the chromatogram obtained with reference solution (b) (0.3 per cent); the sum of the areas of all
the peaks, apart from the principal peak and any peak corresponding to thienamycin, is not greater
than the area of the principal peak in the chromatogram obtained with reference solution (b) (1 per
Water (2.5.12). 5.0 per cent to 8.0 per cent, determined on 0.200 g by the semi-micro determina-
tion of water. Use an iodosulphurous reagent containing imidazole instead of pyridine and a clean
container for each determination.
21-28
ASSAY
Keep the solutions in an ice-bath and use within 8 h of preparation.
Test solution. Dissolve 40.0 mg of the substance to be examined in a mixture of 0.7 volumes of
acetonitrile R and 99.3 volumes of a 0.135 g/l solution of dipotassium hydrogen phosphate R adjusted to
pH 6.8 with dilute phosphoric acid R and dilute to 100.0 ml with the same mixture of solvents.
Reference solution (a). Dissolve 40.0 mg of imipenem CRS in a mixture of 0.7 volumes of acetonitrile R
and 99.3 volumes of a 0.135 g/l solution of dipotassium hydrogen phosphate R adjusted to pH 6.8 with
dilute phosphoric acid R and dilute to 100.0 ml with the same mixture of solvents.
Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with a mixture of 0.7 volumes of
acetonitrile R and 99.3 volumes of a 0.135 g/l solution of dipotassium hydrogen phosphate R adjusted to
pH 6.8 with dilute phosphoric acid R.
Reference solution (c). Heat at 80C for 5 min 20 ml of the test solution previously adjusted to pH 10
with sodium hydroxide solution R.
as mobile phase at a flow rate of 1.0 ml/min a mixture of 0.7 volumes of acetonitrile R and 99.3
volumes of a 8.7 g/l solution of dipotassium hydrogen phosphate R adjusted to pH 7.3 with dilute
phosphoric acid R,
Inject 20 l of reference solution (c). When the chromatograms are recorded in the prescribed
conditions, the retention time of imipenem is about 9 min and the relative retention time of
thienamycin relative to imipenem is about 0.8. The assay is not valid unless the resolution between
the peaks corresponding to imipenem and thienamycin is at least 3.5. Inject 20 l of reference
solution (a) six times. The test is not valid unless the relative standard deviation of the peak area for
imipenem is at most 1.0 per cent. Inject alternately the test solution and reference solution (a).
STORAGE
Store in an airtight container, at a temperature of 2C to 8C. If the substance is sterile, store in a
sterile, airtight, tamper-proof container.
LABELLING
The label states:
IMPURITIES
N
S
NH
2
O
H H
H
OH
Me
COOH
A. (5R,6S)-3-[(2-aminoethyl)sulphanyl]-6-[(R)-1-hydroxyethyl]-7-oxo-1-azabicyclo-
[3.2.0]hept-2-ene-2-carboxylic acid (thienamycin).
__________________________________________________________________________________________________________ Ph Eur
21-29
Imipramine Hydrochloride
1/01
,HCl
N
NMe
2
C
19
H
24
N
2
,HCl 316.9 113-52-0
Imipramine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Imipramine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Imipramine hydrochloride contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of 5-(3-dimethylaminopropyl)-10,11-dihydro-5H-dibenz[b,f]azepine hydro-
CHARACTERS
A white or slightly yellow, crystalline powder, freely soluble in water and in alcohol.
IDENTIFICATION
B. Dissolve 20 mg in 0.01M hydrochloric acid and dilute to 100.0 ml with the same acid. Dilute 1.0 ml
of the solution to 10.0 ml with 0.01M hydrochloric acid. Examined between 230 nm and 350 nm, the
solution shows a single absorption maximum (2.2.25), at 251 nm, and a shoulder at 270 nm. The
specific absorbance at the maximum is about 260.
obtained with imipramine hydrochloride CRS. Examine the substances prepared in the form of discs.
D. Dissolve about 5 mg in 2 ml of nitric acid R. An intense blue colour develops.
E. Dissolve about 50 mg in 3 ml of water R and add 0.05 ml of a 25 g/l solution of quinhydrone R in
methanol R. No red colour develops within 15 min.
F. About 20 mg gives reaction (a) of chlorides (2.3.1).
TESTS
Solution S To 3.0 g add 20 ml of carbon dioxide-free water R, dissolve rapidly by shaking and
triturating with a glass rod and dilute to 30 ml with the same solvent.
Appearance of solution Solution S is clear (2.2.1). Immediately after preparation, dilute solution
S with an equal volume of water R. This solution is not more intensely coloured than reference
solution BY
6
(Method II, 2.2.2).
pH (2.2.3). The pH of solution S, measured immediately after preparation, is 3.6 to 5.0.
Related substances Examine by thin-layer chromatography (2.2.27), using a TLC silica gel G
plate R.
Reference solution (a). Dilute 1 ml of the test solution to 10 ml with methanol R. Dilute 1 ml of this
Reference solution (b). Dissolve 5 mg of iminodibenzyl R in methanol R and dilute to 100 ml with the
21-30
volumes of hydrochloric acid R, 5 volumes of water R, 35 volumes of glacial acetic acid R and 55
volumes of ethyl acetate R. Allow the plate to dry in air for 5 min and spray with a 5 g/l solution of
potassium dichromate R in a mixture of 1 volume of sulphuric acid R and 4 volumes of water R. Examine
the plate immediately. The chromatogram obtained with the test solution shows a blue principal
spot. In the chromatogram obtained with the test solution: any spot corresponding to imino-
dibenzyl is not more intense than the spot in the chromatogram obtained with reference solution
(b) (0.2 per cent); any spot apart from the principal spot and any spot corresponding to imino-
dibenzyl, is not more intense than the spot in the chromatogram obtained with reference solution
(a) (0.2 per cent).
at 100C to 105C.
ASSAY
Dissolve 0.250 g in 50 ml of alcohol R and add 5.0 ml of 0.01M hydrochloric acid. Carry out a poten-
tiometric titration (2.2.20), using 0.1M sodium hydroxide. Read the volume added between the 2
19
H
25
ClN
2
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
21-31
Indapamide
N
N
Cl
SO
2
NH
2
O
Me
H
C
16
H
16
ClN
3
O
3
S 365.8 26807-65-8
Indapamide complies with the requirements of the 3rd edition of the European Pharmacopoeia [1108]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Indapamide contains not less than 98.0 per cent and not more than the equivalent of 102.0 per
cent of (RS)-4-chloro-3-sulphamoyl-N-(2-methyl-2,3-dihydro-1H-indol-1-yl)benzamide, calculated
CHARACTERS
A white or almost white powder, practically insoluble in water, soluble in alcohol, slightly soluble in
ether.
IDENTIFICATION
A. Dissolve 50.0 mg in alcohol R and dilute to 100.0 ml with the same solvent. Dilute 2.0 ml of the
shows an absorption maximum at 242 nm and two shoulders, at 279 nm and 287 nm. The specific
obtained with indapamide CRS. Examine the substances prepared as discs using potassium bromide R.
254
the same solvent.
Reference solution (a). Dissolve 20 mg of indapamide CRS in alcohol R and dilute to 10 ml with the
same solvent.
Reference solution (b). Dissolve 10 mg of indometacin R in 5 ml of reference solution (a) and dilute to
10 ml with alcohol R.
of 1 volume of glacial acetic acid R, 20 volumes of acetone R and 79 volumes of toluene R. Allow the
plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromato-
gram obtained with the test solution is similar in position and size to the principal spot in the
TESTS
Optical rotation (2.2.7). Dissolve 0.250 g in ethanol R and dilute to 25.0 ml with the same solvent.
obtained with reference solution (b) is not less than 15 per cent of the full scale of the recorder.
Inject 10 l of each solution and continue the chromatography for 2.5 times the retention time of
the principal peak. The test is not valid unless in the chromatogram obtained with reference
solution (d): the resolution between the peak corresponding to indapamide and the peak corres-
ponding to methylnitrosoindoline is at least 4.0; the principal peak in the chromatogram obtained
with reference solution (b) has a signal-to-noise ratio of at least 6.
21-32
indapamide impurity B is not greater than the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.3 per cent); the area of any peak, apart from the principal
peak and any peak corresponding to indapamide impurity B, is not greater than the area of the
the areas of all the peaks, apart from the principal peak, is not greater than five times the area of the
Methylnitrosoindoline Not more than 5 ppm, determined by liquid chromatography (2.2.29).
10.0 ml with water R. Shake for 15 min. Allow to stand at 4C for 1 h and filter.
Reference solution. Dissolve 25.0 mg of the substance to be examined in 1.0 ml of a 0.125 mg/l
solution of methylnitrosoindoline CRS in acetonitrile R and dilute to 10.0 ml with water R. Shake for
15 min. Allow to stand at 4C for 1 h and filter.
as mobile phase at a flow rate of 1.4 ml per minute a mixture of 7 volumes of acetonitrile R, 20
volumes of tetrahydrofuran R and 73 volumes of a 1.5 g/l solution of triethylamine R adjusted to
pH 2.8 with phosphoric acid R,
Inject 0.1 ml of the solutions.
In the chromatogram obtained with the reference solution, measure from the baseline the height
A of the peak corresponding to methylnitrosoindoline, appearing just before the principal peak,
and the height B corresponding to the lowest point in the curve between this peak and the principal
peak.
The test is not valid unless in the chromatogram obtained with the reference solution: the
difference between A and B is at least 85 per cent of height A, the peak corresponding to methyl-
nitrosoindoline appearing just before the principal peak has a signal-to-noise ratio of at least 3.
methylnitrosoindoline is not greater than the difference between the areas of the peaks correspond-
ing to methylnitrosoindoline in the chromatograms obtained with the reference solution and the
test solution.
tion of water.
ASSAY
Carry out the assay protected from light and prepare the solutions immediately before use or maintain them at
4C.
Test solution. Dissolve 20.0 mg of the substance to be examined in 7 ml of a mixture of equal
volumes of acetonitrile R and methanol R and dilute to 20.0 ml with a 0.2 g/l solution of sodium
edetate R.
Reference solution (a). Dissolve 30.0 mg of indapamide impurity B CRS in 35 ml of a mixture of equal
volumes of acetonitrile R and methanol R and dilute to 100.0 ml with a 0.2 g/l solution of sodium
edetate R. To 1.0 ml of the solution, add 35 ml of a mixture of equal volumes of acetonitrile R and
methanol R and dilute to 100.0 ml with a 0.2 g/l solution of sodium edetate R.
Reference solution (b). Dilute 1.0 ml of the test solution to 50.0 ml with a mixture of 17.5 volumes of
acetonitrile R, 17.5 volumes of methanol R and 65 volumes of a 0.2 g/l solution of sodium edetate R.
Dilute 1.0 ml of the solution to 20.0 ml with a mixture of 17.5 volumes of acetonitrile R, 17.5
volumes of methanol R and 65 volumes of a 0.2 g/l solution of sodium edetate R.
Reference solution (c). Dissolve 20.0 mg of indapamide CRS in 7 ml of a mixture of equal volumes of
acetonitrile R and methanol R and dilute to 20.0 ml with a 0.2 g/l solution of sodium edetate R.
Reference solution (d). Dissolve 25.0 mg of indapamide CRS and 45.0 mg of methylnitrosoindoline CRS in
17.5 ml of a mixture of equal volumes of acetonitrile R and methanol R and dilute to 50.0 ml with a
0.2 g/l solution of sodium edetate R.
21-33
as mobile phase at a flow rate of 2 ml per minute a mixture of 0.1 volumes of glacial acetic
acid R, 17.5 volumes of acetonitrile R, 17.5 volumes of methanol R and 65 volumes of a 0.2 g/l
solution of sodium edetate R,
When the chromatograms are recorded at the prescribed conditions, the retention time of the
principal peak in the chromatogram obtained with reference solution (c) is about 11 min.
Inject reference solution (c) six times. The test is not valid unless the relative standard deviation
of the peak area for indapamide is at most 1.0 per cent. If necessary, adjust the integrator
parameters. Inject alternately the test solution and reference solution (c).
Calculate the per cent m/m content of anhydrous substance.
STORAGE
IMPURITIES
N
NO
Me
A. 2-methyl-1-nitrosoindoline,
N
Me
N
O
Cl
SO
2
NH
2
H
B. 4-chloro-3-sulphamoyl-N-(2-methyl-1H-indol-1-yl)-benzamide.
__________________________________________________________________________________________________________ Ph Eur
21-34
Indometacin
N COOH
O
Cl
OMe
Me
C
19
H
16
ClNO
4
357.8 53-86-1
Indometacin complies with the requirements of the 3rd edition of the European Pharmacopoeia [0092]. These
Preparations
Indometacin Capsules
Indometacin Suppositories
When indomethacin is prescribed or demanded, Indometacin shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Indometacin contains not less than 98.5 per cent and not more than the equivalent of 100.5 per
cent of [1-(4-chlorobenzoyl)-5-methoxy-2-methylindol-3-yl]acetic acid, calculated with reference
CHARACTERS
A white or yellow, crystalline powder, practically insoluble in water, sparingly soluble in alcohol.
IDENTIFICATION
B. Dissolve 25 mg in a mixture of 1 volume of 1M hydrochloric acid and 9 volumes of methanol R and
dilute to 100.0 ml with the same mixture of solvents. Dilute 10.0 ml of the solution to 100.0 ml
with a mixture of 1 volume of 1M hydrochloric acid and 9 volumes of methanol R. Examined between
obtained with indometacin CRS. Examine the substances in the solid state without recrystallisation.
D. Dissolve 0.1 g in 10 ml of alcohol R, heating slightly if necessary. To 0.1 ml of the solution add
2 ml of a freshly prepared mixture of 1 volume of a 250 g/l solution of hydroxylamine hydrochloride R
and 3 volumes of dilute sodium hydroxide solution R. Add 2 ml of dilute hydrochloric acid R and 1 ml of
ferric chloride solution R2 and mix. A violet-pink colour develops.
E. To 0.5 ml of the solution in alcohol prepared in identification test D, add 0.5 ml of
dimethylaminobenzaldehyde solution R2. A precipitate is formed that dissolves on shaking. Heat on a
water-bath. A bluish-green colour is produced. Continue to heat for 5 min and cool in iced water
for 2 min. A precipitate is formed and the colour changes to light greyish-green. Add 3 ml of
alcohol R. The solution is clear and violet-pink in colour.
TESTS
254
R as the
coating substance. Prepare the slurry using a 46.8 g/l solution of sodium dihydrogen phosphate R.
of 30 volumes of light petroleum R and 70 volumes of ether R. Allow the plate to dry in air and
examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with the test
21-35
solution, apart from the principal spot, is not more intense than the spot in the chromatogram
obtained with the reference solution (0.5 per cent).
at 100C to 105C.
ASSAY
Dissolve 0.300 g in 75 ml of acetone R, through which nitrogen R, free from carbon dioxide, has
been passed for 15 min. Maintain a constant stream of nitrogen through the solution. Add 0.1 ml
of phenolphthalein solution R. Titrate with 0.1M sodium hydroxide. Carry out a blank titration.
19
H
16
ClNO
4
.
STORAGE
IMPURITIES
A. 4-chlorobenzoic acid.
__________________________________________________________________________________________________________ Ph Eur
21-36
Indoramin Hydrochloride
N
N
N
O
H
,HCl
H
C
22
H
25
N
3
O,HCl 383.9 33124-53-7
Definition Indoramin Hydrochloride is N-{1-[2-(indol-3-yl)ethyl]-4-piperidyl}benzamide
hydrochloride. It contains not less than 98.5% and not more than 100.5% of C
22
H
25
N
3
O,HCl,
Characteristics A white or almost white powder. It exhibits polymorphism.
Slightly soluble in water; sparingly soluble in ethanol (96%); soluble in methanol; very slightly
soluble in ether.
Identification
ethanol (96%) exhibits three maxima, at 273, 280 and 290 nm. The absorbances at the maxima are
about 0.76, 0.77 and 0.64, respectively.
B. Dissolve 50 mg in 30 ml of water, make the solution alkaline by the addition of 5M ammonia and
shake with 50 ml of dichloromethane. Dry the dichloromethane layer with anhydrous sodium sulphate,
filter and evaporate the filtrate to dryness using a rotary evaporator. The infrared absorption spectrum
of the residue, Appendix II A, is concordant with the reference spectrum of indoramin (RS 188).
C. Yields reaction A characteristic of chlorides, Appendix VI.
Acidity pH of a 2% w/v suspension in water, 4.0 to 5.5, Appendix V L.
silica gel F
254
pre-coated plate (Merck silica gel 60 F
254
volume of 18M ammonia, 20 volumes of absolute ethanol and 79 volumes of toluene as the mobile
phase. Apply separately to the plate 10 l of each of three solutions of the substance being
examined in ethanol (96%) containing (1) 1.0% w/v, (2) 0.0050% w/v and (3) 0.0010% w/v. After
removal of the plate, allow it to dry in a current of warm air and examine under ultraviolet light
than the spot in the chromatogram obtained with solution (2) (0.5%) and not more than one such
spot is more intense than the spot in the chromatogram obtained with solution (3) (0.1%).
Loss on drying When dried at 100 to 105 for 4 hours, loses not more than 0.5% of its weight.
Use 1 g.
Assay Dissolve 0.2 g in 30 ml of anhydrous acetic acid, add 6 ml of acetic anhydride and 6 ml of
mercury(II) acetate solution. Titrate with 0.1M perchloric acid VS determining the end point potentio-
22
H
25
N
3
O,HCl.
Storage Indoramin Hydrochloride should be stored in a well-closed container and protected from
light.
Action and use Alpha-adrenoceptor antagonist.
Preparation
Indoramin Tablets
IMPURITIES
N
Br
H
A. 3-(2-bromoethyl)indole
21-37
H
N
N
O
B. N-(1-benzyl-4-piperidyl)benzamide
H
HN
N
O
C. N-(4-piperidyl)benzamide
21-38
Inositol Nicotinate
OR OR
OR
OR
RO
RO
N
CO
R =
C
42
H
30
N
6
O
12
810.7 6556-11-2
Definition Inositol Nicotinate is myo-inositol hexanicotinate. It contains not less than 98.0% and
42
H
30
N
6
O
12
Practically insoluble in water; sparingly soluble in chloroform; practically insoluble in acetone, in
ethanol (96%) and in ether. It dissolves in dilute mineral acids.
spectrum of inositol nicotinate (RS 190).
Clarity and colour of solution A 5.0% w/v solution in 0.5M sulphuric acid is clear, Appendix IV A,
6
Chloride Dissolve 0.14 g in a sufficient quantity of 2M nitric acid and dilute to 16 ml with water.
The resulting solution complies with the limit test for chlorides, Appendix VII, beginning at the words
pour the mixture as a single addition ... (350 ppm).
Free nicotinic acid To 1 g add 75 ml of water, shake for 15 minutes and titrate with 0.02M sodium
hydroxide VS using phenolphthalein solution R1 as indicator. Not more than 0.8 ml of 0.02M sodium
hydroxide VS is required to produce the first pink colour.
plate 200 mm 200 mm in size and silica gel GF
254
as the coating substance. For the first
development use a mixture of 90 volumes of chloroform and 10 volumes of methanol as the mobile
phase. Apply to the bottom right-hand corner of the plate 5 l of solution (1) containing 5.0% w/v
of the substance being examined in a mixture of 9 volumes of chloroform and 1 volume of methanol
and develop over a path of 12 cm. After removal of the plate, allow it to dry in air and turn the
plate through 90 in a clockwise direction. Apply separately to the bottom right-hand corner of the
plate, and to the right of the solvent front, 5 l of each of two solutions of the substance being
examined in a mixture of 9 volumes of chloroform and 1 volume of methanol containing (2) 0.075%
w/v and (3) 0.050% w/v. For the second development use a mixture of 50 volumes of ethyl acetate
and 5 volumes each of glacial acetic acid, ethanol (96%) and water as the mobile phase. After removal
of the plate, allow it to dry in air and examine under ultraviolet light (254 nm). In the chromatogram
obtained with solution (1) any secondary spot is not more intense than the spot in the chromatogram
obtained with solution (2) (1.5%) and not more than one such spot is more intense than the spot in
the chromatogram obtained with solution (3) (1%).
Acetone Prepare a 0.020% v/v solution of butan-2-one (internal standard) in dimethylformamide
(solution A). Carry out the method for gas chromatography, Appendix III B, using the following
solutions. Solution (1) contains 0.020% v/v of acetone in solution A. For solution (2) add 5 ml of
dimethylformamide to 0.20 g of the substance being examined contained in a suitable vessel, stopper
securely, suspend in a water bath until solution is complete and allow to cool. Prepare solution (3)
in the same manner as solution (2) but using 5 ml of solution A in place of the dimethylformamide.
The chromatographic procedure may be carried out using a glass column (1.5 m 4 mm)
packed with acid-washed, silanised diatomaceous support coated with 10% w/w of polyethylene glycol
1000 and maintained at 60.
In the chromatogram obtained with solution (3) the ratio of the area of any peak corresponding
to acetone to the area of the peak due to the internal standard is not greater than the correspond-
ing ratio in the chromatogram obtained with solution (1).
Use 1 g.
Assay Carry out Method I for non-aqueous titration, Appendix VIII A, using 0.2 g and 1-naphthol-
benzein solution as indicator. Each ml of 0.1M perchloric acid VS is equivalent to 13.51 mg of
C
42
H
30
N
6
O
12
.
21-39
Storage Inositol Nicotinate should be kept in a well-closed container.
Preparation
Inositol Nicotinate Tablets
21-40
Insulin
C
256
H
381
N
65
O
76
S
6
(porcine) 5778 12584-58-6
C
254
H
377
N
65
O
75
S
6
(bovine) 5734 11070-73-8
Insulin complies with the requirements of the 3rd edition of the European Pharmacopoeia [0276]. These
Preparations
Insulin Preparations
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Insulin is the natural antidiabetic principle obtained from beef or pork pancreas and purified. The
content of porcine insulin C
256
H
381
N
65
O
76
S
6
or bovine insulin C
254
H
377
N
65
O
75
S
6
plus the correspond-
ing A21 desamido insulin as appropriate, is not less than 93.0 per cent and not more than
105.0 per cent, calculated with reference to the dried substance
(1)
.
PRODUCTION
The animals from which insulin is derived must fulfil the requirements for the health of animals
CHARACTERS
A white or almost white powder, practically insoluble in water, in ethanol and in ether. It dissolves
in dilute mineral acids and with decomposition in dilute solutions of alkali hydroxides.
IDENTIFICATION
A. Examine the chromatograms obtained in the Assay. The retention time of the principal peak in
the chromatogram obtained with the test solution corresponds to that of the principal peak in the
chromatogram obtained with reference solution (b) or, where appropriate, reference solution (c).
B. Examine by peptide mapping.
Test solution. Prepare a solution of the substance to be examined in 0.01M hydrochloric acid containing
2.0 mg/ml of insulin and transfer 500 l of this solution to a clean tube. Add 2.0 ml of HEPES buffer
solution pH 7.5 R and 400 l of a 1 mg/ml solution of Staphylococcus aureus strain V-8 protease R. Cap
the tube and incubate at 25C for 6 h. Stop the reaction by adding 2.9 ml of sulphate buffer solution
pH 2.0 R.
Reference solution. Prepare at the same time and in the same manner as for the test solution but using
bovine insulin CRS or porcine insulin CRS, as appropriate, instead of the substance to be examined.
Mobile phase A. Mix 100 ml of acetonitrile for chromatography R, 700 ml of water R and 200 ml of
sulphate buffer solution pH 2.0 R; filter and degas,
Mobile phase B. Mix 400 ml of acetonitrile for chromatography R, 400 ml of water R and 200 ml of
sulphate buffer solution pH 2.0 R; filter and degas.
Follow the elution conditions as described in the table below (if necessary, the gradient may be
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
Equilibrate the column at initial conditions for at least 15 min. Carry out a blank run using the
21-41
gram of bovine insulin digest or the Ph. Eur. reference chromatogram of porcine insulin digest, as appropriate.
In the chromatogram obtained with the reference solution, identify the peaks due to digest frag-
ments I, II and III. The symmetry factor of the peaks due to fragments II and III is not greater
than 1.5, and the resolution between the two peaks is at least 1.9.
The profile of the chromatogram obtained with the test solution corresponds to that of the
NOTE: The retention time of fragment I is the same for insulin derived from pork as for human insulin. The
retention time of fragment II is the same for all insulins. The retention time of fragment III is the same for
insulin derived from beef as for insulin derived from pork.
TESTS
Impurities with molecular masses greater than that of insulin Examine by size-exclusion
Resolution solution. Use a solution of insulin (approximately 4 mg/ml), containing more than 0.4 per
cent of high molecular mass proteins. An injectable insulin preparation, whether a solution or a
suspension, that has been clarified with a sufficient amount of 6M hydrochloric acid, containing the
indicated percentage of high molecular mass proteins, or a solution prepared from insulin,
dissolved in 0.01M hydrochloric acid, may be used. Insulin containing the indicated percentage of high
molecular mass proteins may be prepared by allowing insulin powder to stand at room temperature
for about ten days.
Maintain the solutions at 2C to 10C and use within 7 days. If an automatic injector is used,
maintain the temperature at 2C to 10C.
a column 0.3 m long and at least 7.5 mm in internal diameter packed with hydrophilic silica gel for
chromatography R (5 m to 10 m), of a grade suitable for the separation of insulin monomer
from dimer and polymers,
as mobile phase at a flow rate of 0.5 ml/min a mixture consisting of 15 volumes of glacial acetic
acid R, 20 volumes of acetonitrile R and 65 volumes of a 1.0 g/l solution of arginine R; filter and
degas,
Equilibration of the column. Before using a new column for chromatographic analysis, equilibrate by
repeated injections of an insulin solution containing high molecular mass proteins. This can be
done by at least three injections of the resolution solution. The column is equilibrated when
repeatable results are obtained from two subsequent injections.
Inject 100 l of the resolution solution. When the chromatograms are recorded in the prescribed
conditions, the retention times are: polymeric insulin complexes about 13 min to 17 min, covalent
insulin dimer: about 17.5 min, insulin monomer about 20 min, salts about 22 min. The test is not
valid unless the resolution, defined by the ratio of the height of the dimer peak to the height above
the baseline of the valley separating the monomer and dimer peaks, is at least 2.0.
Inject 100 l of the test solution. Record the chromatogram for approximately 35 min. In the
chromatogram obtained, the sum of the areas of any peak with a retention time less than that of the
principal peak is not greater than 1.0 per cent of the total area of the peaks. Disregard any peak
with a retention time greater than that of the insulin peak.
Related proteins Examine by liquid chromatography (2.2.29) as described under Assay, following
the elution conditions as described in the table below:
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
Maintain the solutions at 2C to 10C and use within 24 h. Perform a system suitability check
(resolution, linearity) as described under Assay. If necessary, the relative proportions of the mobile
phases may be adjusted to ensure complete elution of porcine A21 desamido insulin before
commencement of the gradient. The profile of the gradient may also be adjusted to ensure
complete elution of all insulin related impurities.
Inject 20 l of reference solution (b) or, where appropriate, reference solution (c), and 20 l of
the test solution. If necessary, adjust the injection volume to a volume between 10 l and 20 l in
accordance with the results obtained in the test for linearity as described under Assay. Record the
chromatograms for approximately 50 min. In the chromatogram obtained with either reference
solution, A21 desamido insulin appears as a small peak after the principal peak and has a retention
21-42
time of about 1.3 relative to the principal peak. In the chromatogram obtained with the test
solution, the area of the peak due to A21 desamido insulin is not greater than 3.0 per cent of the
total area of the peaks; the sum of the areas of all the peaks, apart from those due to insulin and
A21 desamido insulin, is not greater than 3.0 per cent of the total area of the peaks.
Proinsulin-like immunoreactivity (PLI) Not more than 10 ppm, calculated with reference to
the dried substance and determined by a suitably sensitive immunochemical method (2.7.1) such as
radio-immunoassay, using the International Reference Reagent for porcine proinsulin or bovine
proinsulin, as appropriate, to calibrate the method.
2.2.23), calculated with reference to the dried substance.
to 25.0 ml with the same acid. Dilute if necessary to a suitable concentration (for example, 0.4 g
to 1.6 g of Zn per millilitre) with 0.01M hydrochloric acid.
Reference solutions. Use solutions containing 0.40 g, 0.80 g, 1.00 g, 1.20 g and 1.60 g of Zn
per millilitre, freshly prepared by diluting zinc standard solution (5 mg/ml Zn) R with 0.01M hydrochloric
acid.
Measure the absorbance at 213.9 nm using a zinc hollow-cathode lamp as source of radiation
and an air-acetylene flame of suitable composition (for example, 11 litres of air and 2 litres of
acetylene per minute).
without a further appropriate procedure for the removal of bacterial endotoxins, it contains not
more than 10 I.U. of endotoxin per milligram.
ASSAY
Reference solution (a). Dissolve the contents of a vial of human insulin CRS in 0.01M hydrochloric acid to
Reference solution (b). Dissolve the contents of a vial of porcine insulin CRS in 0.01M hydrochloric acid to
Reference solution (c). If the substance to be examined is bovine insulin, dissolve 40.0 mg of bovine
insulin CRS in 10.0 ml of 0.01M hydrochloric acid.
Reference solution (d). Dilute 1.0 ml of reference solution (b) to 10.0 ml with 0.01M hydrochloric acid.
Reference solution (e). Dilute 1.0 ml of reference solution (c) to 10.0 ml with 0.01M hydrochloric acid.
Resolution solution. Mix 1.0 ml of reference solution (a) and 1.0 ml of reference solution (b).
Maintain the solutions at 2C to 10C and use within 48 h. If an automatic injector is used,
as mobile phase at a flow rate of 1 ml/min the following solutions, prepared and maintained at a
temperature not lower than 20C:
Mobile phase A. Dissolve 28.4 g of anhydrous sodium sulphate R in water R and dilute to 1000 ml
with the same solvent; add 2.7 ml of phosphoric acid R; adjust the pH to 2.3, if necessary, with
ethanolamine R; filter and degas,
Mobile phase B. Mix 550 ml of mobile phase A with 450 ml of acetonitrile R. Warm the solution
to a temperature not lower than 20C in order to avoid precipitation (mixing of mobile phase
A with acetonitrile is endothermic); filter and degas,
Elute with a mixture of 42 volumes of mobile phase A and 58 volumes of mobile phase B,
adjusted if necessary.
Inject 20 l of the resolution solution and 20 l of reference solution (b). Record the chromato-
gram of the resolution solution until the peak corresponding to the principal peak in the chromato-
gram obtained with reference solution (b) is clearly visible. In the chromatogram obtained with the
resolution solution, identify the peaks due to porcine insulin and human insulin. The test is not
21-43
valid unless the resolution between the peaks corresponding to human insulin and porcine insulin is
at least 1.2. If necessary, adjust the concentration of acetonitrile in the mobile phase until this
resolution is achieved.
Inject 20 l of the test solution and, for porcine insulin, 20 l each of reference solutions (b) and
(d) or, for bovine insulin, 20 l each of reference solutions (c) and (e). The test is not valid unless
the area of the principal peak in the chromatogram obtained with reference solution (b) or (c) is
100.5 times the area of the principal peak in the chromatogram obtained with reference solution
(d) or (e). If this test fails, adjust the injection volume between 10 l and 20 l, in order to be in the
linearity range of the detector.
Calculate the content of insulin (C
256
H
381
N
65
O
76
S
6
or C
254
H
377
N
65
O
75
S
6
, as appropriate) plus the
corresponding A21 desamido insulin from the area of the principal peak and the area of the peak
due to A21 desamido insulin in the chromatograms obtained with the test solution and the appro-
priate reference solution, and the declared content of porcine insulin plus A21 desamido porcine
insulin in porcine insulin CRS or bovine insulin plus A21 desamido bovine insulin

in bovine insulin CRS,
as appropriate.
STORAGE
Store in an airtight container, protected from light, at 20C until released by the manufacturer.
When thawed, insulin is stored at 5 3C and used for manufacturing preparations within a short
period of time. To avoid absorption of humidity from the air during weighing, the insulin must be at
room temperature.
LABELLING
The label states:
the animal species of origin,
(1)

One I.U. of insulin is equivalent to 0.0345 mg of porcine insulin and 0.0342 mg of bovine
insulin.
__________________________________________________________________________________________________________ Ph Eur
21-44
Human Insulin
Gly
_
Ile
_
Val
_
Glu
_
Gln
_
Cys
_
Cys
_
Thr
_
Ser
_
Ile
_
Cys
_
Ser
_
Leu
_
Tyr
_
Gln
_
Leu
_
Glu
_
Asn
_
Tyr
_
Cys
_
Asn
Phe
_
Val
_
Asn
_
Gln
_
His
_
Leu
_
Cys
_
Gly
_
Ser
_
His
_
Leu
_
Val
_
Glu
_
Ala
_
Leu
_
Tyr
_
Leu
_
Val
_
Cys
_
Gly
_
Glu
_
Arg
_
Gly
_
Phe
_
Phe
_
Tyr
_
Thr
_
Pro
_
Lys
_
Thr
Chain B
Chain A
C
257
H
383
N
65
O
77
S
6
5808 11061-68-0
Human Insulin complies with the requirements of the 3rd edition of the European Pharmacopoeia [0838]. These
Preparations
Insulin Preparations
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Human insulin is a protein having the structure of the antidiabetic hormone produced by the human
pancreas. The content of human insulin C
257
H
383
N
65
O
77
S
6
plus A21 desamido human insulin is not
less than 95.0 per cent and not more than 105.0 per cent, calculated with reference to the dried
substance
(1)
. It is produced either by enzymatic modification and suitable purification of insulin
obtained from the pancreas of the pig or by a method based on recombinant DNA (rDNA)
technology. Human insulin produced by rDNA technology complies with the requirements of the
general monograph on Recombinant DNA technology, products of (784).
PRODUCTION
Human insulin is produced in conditions designed to minimise the degree of microbial contamina-
tion.
For human insulin produced by enzymatic modification of insulin obtained from the pancreas of the pig, the
manufacturing process is validated to demonstrate removal of any residual proteolytic activity. The competent
authority may require additional tests.
For human insulin produced by a method based on rDNA technology, prior to release the following test is carried
out on each batch of the final bulk product unless exemption has been granted by the competent authority.
Host-cell-derived proteins Not more than 10 ppm determined as described in the general mono-
graph on Recombinant DNA technology, products of (784).
CHARACTERS
A white or almost white powder, practically insoluble in water, in ethanol and in ether. It dissolves in
dilute mineral acids and with decomposition in dilute solutions of alkali hydroxides.
IDENTIFICATION
A. Examine the chromatograms obtained in the Assay. The retention time of the principal peak in
the chromatogram obtained with the test solution corresponds to that of the principal peak in the
B. Examine by peptide mapping.
Test solution. Prepare a solution of the substance to be examined in 0.01M hydrochloric acid containing
2.0 mg/ml of human insulin and transfer 500 l of this solution to a clean tube. Add 2.0 ml of HEPES
buffer solution pH 7.5 R and 400 l of a 1 mg/ml solution of Staphylococcus aureus strain V-8 protease R.
Cap the tube and incubate at 25C for 6 h. Stop the reaction by adding 2.9 ml of sulphate buffer
solution pH 2.0 R.
Reference solution. Prepare at the same time and in the same manner as for the test solution but using
human insulin CRS instead of the substance to be examined.
21-45
silica gel fr chromatography R (3 m),
as mobile phases at a flow rate of 1 ml/min:
Mobile phase A. Mix 100 ml of acetonitrile for chromatography R, 700 ml of water R and 200 ml of
sulphate buffer solution pH 2.0 R; filter and degas,
Mobile phase B. Mix 400 ml of acetonitrile for chromatography R, 400 ml of water R and 200 ml of
sulphate buffer solution pH 2.0 R; filter and degas.
Follow the elution conditions as described in the table below (if necessary, the gradient may be
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
Equilibrate the column at the initial conditions for at least 15 min. Carry out a blank run using the
gram of human insulin digest. In the chromatogram obtained with the reference solution, identify the
peaks due to digest fragments I, II and III. The symmetry factor of the peaks due to fragments II
and III is not greater than 1.5, and the resolution between the two peaks is at least 3.4.
The profile of the chromatogram obtained with the test solution corresponds to that of the chrom-
atogram obtained with the reference solution.
NOTE: The retention time of fragment I is the same for insulin derived from pork as for human insulin. The
retention time of fragment II is the same for all insulins. The retention time of fragment III is the same for
insulin derived from beef as for insulin derived from pork.
TESTS
Impurities with molecular masses greater than that of insulin Examine by size-exclusion
Resolution solution. Use a solution of insulin (approximately 4 mg/ml), containing more than 0.4 per
cent of high molecular mass proteins. An injectable insulin preparation, whether a solution or a
suspension, that has been clarified with a sufficient amount of 6M hydrochloric acid, containing the
indicated percentage of high molecular mass proteins, or a solution prepared from insulin,
dissolved in 0.01M hydrochloric acid may be used. Insulin containing the indicated percentage of high
molecular mass proteins may be prepared by allowing insulin powder to stand at room temperature
for about ten days.
Maintain the solutions at 2C to 10C and use within 7 days. If an automatic injector is used,
a column 0.3 m long and at least 7.5 mm in internal diameter packed with hydrophilic silica gel for
chromatography R (5 m to 10 m), of a grade suitable for the separation of insulin monomer
from dimer and polymers,
as mobile phase at a flow rate of 0.5 ml/min a mixture consisting of 15 volumes of glacial acetic
acid R, 20 volumes of acetonitrile R and 65 volumes of a 1.0 g/l solution of arginine R; filter and
degas,
Equilibration of the column. Before using a new column for chromatographic analysis, equilibrate by
repeated injections of an insulin solution containing high molecular mass proteins. This can be
done by at least three injections of the resolution solution. The column is equilibrated when
repeatable results are obtained from two subsequent injections.
Inject 100 l of the resolution solution. When the chromatograms are recorded in the prescribed
conditions, the retention times are polymeric insulin complexes: 13 min to 17 min, covalent insulin
dimer: about 17.5 min, insulin monomer: about 20 min, salts: about 22 min. The test is not valid
unless the resolution, defined by the ratio of the height of the dimer peak to the height above the
baseline of the valley separating the monomer and dimer peaks, is at least 2.0.
21-46
Inject 100 l of the test solution. Record the chromatogram for approximately 35 min. In the
chromatogram obtained, the sum of the areas of any peak with a retention time less than that of the
principal peak is not greater than 1.0 per cent of the total area of the peaks. Disregard any peak
with a retention time greater than that of the insulin peak.
Related proteins Examine by liquid chromatography (2.2.29) as described under Assay, following
the elution conditions as described in the table below:
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
030 42 58 Isocratic
Maintain the solutions at 2C to 10C and use within 24 h. Perform a system suitability check
(resolution, linearity) as described under Assay. If necessary, the relative proportions of the mobile
phases may be adjusted to ensure complete elution of A21 desamido porcine insulin before
commencement of the gradient. The profile of the gradient may also be adjusted to ensure
complete elution of all insulin related impurities.
Inject 20 l of reference solution (a), 20 l of reference solution (b), 20 l of reference solution
(c) and 20 l of the test solution. If necessary, adjust the injection volume to a volume between
10 l and 20 l in accordance with the results obtained in the test for linearity as described under
Assay. Record the chromatograms for approximately 50 min. In the chromatogram obtained with
reference solution (a), A21 desamido human insulin appears as a small peak after the principal
peak and has a retention time of about 1.3 relative to the principal peak. In the chromatogram
obtained with the test solution, the area of the peak due to A21 desamido human insulin is not
greater than 2.0 per cent of the total area of the peaks; the sum of the areas of all peaks, apart from
those due to human insulin and that due to A21 desamido human insulin, is not greater than
2.0 per cent of the total area of the peaks. For semisynthetic human insulin only: in the chromato-
gram obtained with the test solution, the area of any peak corresponding to the principal peak in
the chromatogram obtained with reference solution (b) is not greater than the area of the corres-
ponding peak in the chromatogram obtained with reference solution (c) (1.0 per cent of porcine
insulin in human insulin).
Proinsulin-like immunoreactivity (PLI) Not more than 10 ppm, calculated with reference to
the dried substance and determined by a suitably sensitive immunochemical method (2.7.1) such as
radio-immunoassay. For human insulin produced by enzymatic modification of porcine insulin use
the International Reference Reagent for porcine proinsulin to calibrate the method. For human
insulin produced by rDNA technology use the International Reference Reagent for human
proinsulin.
2.2.23), calculated with reference to the dried substance.
to 25.0 ml with the same acid. Dilute if necessary to a suitable concentration (for example 0.4 g to
1.6 g of Zn per millilitre) with 0.01M hydrochloric acid.
Reference solutions. Use solutions containing 0.40 g, 0.80 g, 1.00 g, 1.20 g and 1.60 g of Zn
per millilitre, freshly prepared by diluting zinc standard solution (5 mg/ml Zn) R with 0.01M hydrochloric
acid.
Measure the absorbance at 213.9 nm using a zinc hollow-cathode lamp as source of radiation
and an air-acetylene flame of suitable composition (for example 11 litres of air and 2 litres of
acetylene per minute).
without a further appropriate procedure for the removal of bacterial endotoxins, it contains not
more than 10 I.U. of endotoxin per milligram.
ASSAY
Reference solution (a). Dissolve the contents of a vial of human insulin CRS in 0.01M hydrochloric acid to
21-47
Reference solution (b). Dissolve the contents of a vial of porcine insulin CRS in 0.01M hydrochloric acid to
Reference solution (c). Dilute 1.0 ml of reference solution (b) to 50.0 ml with 0.01M hydrochloric acid.
To 1.0 ml of this solution add 1.0 ml of reference solution (a).
Reference solution (d). Dilute 1.0 ml of reference solution (a) to 10.0 ml with 0.01M hydrochloric acid.
Resolution solution. Mix 1.0 ml of reference solution (a) and 1.0 ml of reference solution (b).
Maintain the solutions at 2C to 10C and use within 48 h. If an automatic injector is used,
as mobile phase at a flow rate of 1 ml/min the following solutions, prepared and maintained at a
temperature not lower than 20C:
Mobile phase A. Dissolve 28.4 g of anhydrous sodium sulphate R in water R and dilute to 1000 ml
with the same solvent; add 2.7 ml of phosphoric acid R; adjust the pH to 2.3, if necessary, with
ethanolamine R; filter and degas,
Mobile phase B. Mix 550 ml of mobile phase A with 450 ml of acetonitrile R. Warm the solution
to a temperature not lower than 20C in order to avoid precipitation (mixing of mobile phase
A with acetonitrile is endothermic); filter and degas,
Elute with a mixture of 42 volumes of mobile phase A and 58 volumes of mobile phase B, adjusted
if necessary.
Inject 20 l of the resolution solution and 20 l of reference solution (b). Record the chromato-
gram of the resolution solution until the peak corresponding to the principal peak in the chromato-
gram obtained with reference solution (b) is clearly visible. In the chromatogram obtained with the
resolution solution, identify the peaks due to porcine insulin and human insulin. The test is not
valid unless the resolution between the peaks corresponding to human insulin and porcine insulin is
at least 1.2. If necessary, adjust the concentration of acetonitrile in the mobile phase until this
resolution is achieved.
Inject 20 l of the test solution, 20 l of reference solution (a) and 20 l of reference solution
(d). The test is not valid unless the area of the principal peak in the chromatogram obtained with
reference solution (a) is 10 0.5 times the area of the principal peak in the chromatogram
obtained with reference solution (d). If this test fails, adjust the injection volume between 10 l and
20 l, in order to be in the linearity range of the detector.
Calculate the content of human insulin C
257
H
383
N
65
O
77
S
6
plus A21 desamido human insulin from
the area of the principal peak and the area of the peak corresponding to A21 desamido human
insulin in the chromatograms obtained with the test solution and reference solution (a) and the
declared content of human insulin plus A21 desamido human insulin in human insulin CRS.
STORAGE
Store in an airtight container, protected from light, at 20C until released by the manufacturer.
When thawed, insulin is stored at 5 3C and used for manufacturing preparations within a short
period of time. To avoid absorption of humidity from the air during weighing, the insulin must be at
room temperature.
LABELLING
The label states:
whether the substance is produced by enzymatic modification of porcine insulin or by rDNA
technology,
(1)
One I.U. of insulin is equivalent to 0.0347 mg of human insulin.
__________________________________________________________________________________________________________ Ph Eur
21-48
Interferon Alfa-2 Concentrated Solution
corrected 1/01
(M)CDLPQTHSLG SRRTLMLLAQ MRXISLFSCL
KDRHDFGFPQ EEFGNQFQKA ETIPVLHEMI
QQIFNLFSTK DSSAAWDETL LDKFYTELYQ
QLNDLEACVI QGVGVTETPL MKEDSILAVR
KYFQRITLYL KEKKYSPCAW EVVRAEIMRS
FSLSTNLQES LRSKE
alfa-2a: C
860
H
1353
N
227
O
255
S
9
19,241 76543-88-9
alfa-2b: C
860
H
1353
N
229
O
255
S
9
19,269 99210-65-8
Interferon Alfa-2 Concentrated Solution complies with the requirements of the 3rd edition of the European
Action and use Immunomodulator.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Interferon alfa-2 concentrated solution is a solution of a protein that is produced according to the
information coded by the alfa-2 sub-species of interferon alfa gene and that exerts non-specific
antiviral activity, at least in homologous cells, through cellular metabolic processes involving
synthesis of both ribonucleic acid and protein. Interferon alfa-2 concentrated solution also exerts
antiproliferative activity. Different types of alfa-2 interferon, varying in the amino acid residue at
position 23, are designated by a letter in lower case.
Designation Residue at position 23 (X
1
)
alfa-2a Lys
alfa-2b Arg
This monograph applies to interferon alfa-2a and -2b concentrated solutions.
The potency of interferon alfa-2 concentrated solution is not less than 1.4 10
8
I.U. per milligram
of protein. Interferon alfa-2 concentrated solution contains not less than 2 10
8
I.U. of interferon
alfa-2 per millilitre.
Interferon alfa-2 concentrated solution complies with the monograph on Products of recombinant
DNA technology (0784).
PRODUCTION
Interferon alfa-2 concentrated solution is produced by a method based on recombinant DNA
(rDNA) technology using bacteria as host cells. It is produced under conditions designed to
minimise microbial contamination of the product.
Interferon alfa-2 concentrated solution complies with the following additional requirements.
Host-cell-derived proteins The limit is approved by the competent authority.
Host-cell-or vector-derived DNA The limit is approved by the competent authority.
CHARACTERS
A clear, colourless or slightly yellowish liquid.
IDENTIFICATION
A. It shows the expected biological activity (see Assay).
B. Examine by isoelectric focusing.
Test solution. Dilute the preparation to be examined with water R to a protein concentration of
1 mg/ml.
Reference solution. Prepare a 1 mg/ml solution of the appropriate interferon alfa-2 CRS in water R.
Isoelectric point calibration solution pI range 3.0 to 10.0. Prepare and use according to the
manufacturers instructions.
Use a suitable apparatus connected with a recirculating temperature controlled water-bath set at
10C and gels for isoelectric focusing with a pH gradient from 3.5 to 9.5. Operate the apparatus in
accordance with the manufacturers instructions. Use as the anode solution phosphoric acid R (98 g/l
21-49
H
3
PO
4
) and as the cathode solution 1M sodium hydroxide. Samples are applied to the gel by filter
papers. Place sample application filters on the gel close to the cathode.
Apply 15 l of the test solution and 15 l of the reference solution. Start the isoelectric focusing
at 1500 V and 50 mA. Turn off the power after 30 min, remove the application filters and
reconnect the power supply for 1 h. Keep the power constant during the focusing process. After
focusing, immerse the gel in a suitable volume of a solution containing 115 g/l of trichloroacetic acid R
and 34.5 g/l of sulphosalicylic acid R in water R and agitate the container gently for 60 min. Transfer
the gel to a mixture of 32 volumes of glacial acetic acid R, 100 volumes of ethanol R and 268 volumes
of water R, and soak for 5 min. Immerse the gel for 10 min in a staining solution prewarmed to
60C in which 1.2 g/l of acid blue 83 R has been added to the previous mixture of glacial acetic acid,
ethanol and water. Wash the gel in several containers with the previous mixture of glacial acetic
acid, ethanol and water and keep the gel in this mixture until the background is clear (12 h to 24 h).
After adequate destaining, soak the gel for 1 h in a 10 per cent V/V solution of glycerol R in the
previous mixture of glacial acetic acid, ethanol and water.
The principal bands of the electrophoretogram obtained with the test solution correspond in
position to the principal bands of the electrophoretogram obtained with the reference solution.
Plot the migration distances of the isoelectric point markers versus their isoelectric points and
determine the isoelectric points of the principal components of the test solution and the reference
solution. They do not differ by more than 0.2 pI unit. The test is not valid unless the isoelectric
point markers are distributed along the entire length of the gel and the isoelectric points of the
principal bands in the electrophoretogram obtained with the reference solution are between 5.8
and 6.3.
C. Examine the electrophoretograms obtained under reducing conditions in the test for impurities
of molecular masses differing from that of interferon alfa-2. The principal band in the electro-
phoretogram obtained with test solution (a) corresponds in position to the principal band in the
electrophoretogram obtained with reference solution (a).
D. Examine by peptide mapping.
Test solution. Dilute the preparation to be examined in water R to a protein concentration of
1.5 mg/ml. Transfer 25 l to a polypropylene or glass tube of 1.5 ml capacity. Add 1.6 l of 1M
phosphate buffer solution pH 8.0 R, 2.8 l of a freshly prepared 1.0 mg/ml solution of trypsin for peptide
mapping R in water R and 3.6 l of water R and mix vigorously. Cap the tube and place it in a water-
bath at 37C for 18 h, then add 100 l of a 573 g/l solution of guanidine hydrochloride R and mix well.
Add 7 l of 154.2 g/l solution of dithiothreitol R and mix well. Place the capped tube in boiling water
for 1 min. Cool to room temperature.
Reference solution. Prepare at the same time and in the same manner as for the test solution but use a
1.5 mg ml solution of the appropriate interferon alfa-2 CRS in water R.
silica gel for chromatography R (5 m) with a pore size of 30 nm,
Mobile phase A. Dilute 1 ml of trifluoroacetic acid R to 1000 ml with water R,
Mobile phase B. To 100 ml of water R add 1 ml of trifluoroacetic acid R and dilute to 1000 ml with
Time (min) Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
0 8 100 0 isocratic
Equilibrate the column with mobile phase A for at least 15 min.
Inject 100 l of the test solution and 100 l of the reference solution. The test is not valid unless
the chromatogram obtained with each solution is qualitatively similar to the appropriate Ph. Eur.
reference chromatogram of interferon alfa-2 digest. The profile of the chromatogram obtained with the
test solution corresponds to that of the chromatogram obtained with the reference solution.
TESTS
Impurities of molecular masses differing from that of interferon alfa-2 Examine by SDS
polyacrylamide gel electrophoresis (2.2.31). The test is performed under both reducing and non-
21-50
reducing conditions, using resolving gels of 14 per cent acrylamide and silver staining as the
detection method.
Sample buffer (non-reducing conditions). Use concentrated SDS PAGE sample buffer R.
Sample buffer (reducing conditions). Use concentrated SDS PAGE sample buffer for reducing conditions R
containing 2-mercaptoethanol as the reducing agent.
Test solution (a). Dilute the preparation to be examined in sample buffer to a protein concentration
of 0.5 mg/ml.
Test solution (b). Dilute 0.20 ml of test solution (a) to 1 ml with sample buffer.
Reference solution (a). Prepare a 0.625 mg/ml solution of the appropriate interferon alfa-2 CRS in
sample buffer.
Reference solution (b). Dilute 0.20 ml of reference solution (a) to 1 ml with sample buffer.
Reference solution (c). Dilute 0.20 ml of reference solution (b) to 1 ml with sample buffer.
Reference solution (d). Dilute 0.20 ml of reference solution (c) to 1 ml with sample buffer.
Reference solution (e). Dilute 0.20 ml of reference solution (d) to 1 ml with sample buffer.
Reference solution (f). Use a solution of molecular mass standards suitable for calibrating SDS-PAGE
gels in the range 15 kDa to 67 kDa.
Place test and reference solutions, contained in covered test-tubes, on a water-bath for 2 min.
Apply 10 l of reference solution (f) and 50 l of each of the other solutions to the stacking gel
wells. Perform the electrophoresis under the conditions recommended by the manufacturer of the
equipment. Detect proteins in the gel by silver staining.
The test is not valid unless: the validation criteria are met (2.2.31); a band is seen in the electro-
phoretogram obtained with reference solution (e); and a gradation of intensity of staining is seen in
the electrophoretograms obtained, respectively, with test solution (a) and test solution (b) and with
reference solutions (a) to (e).
The electrophoretogram obtained with test solution (a) under reducing conditions may show, in
addition to the principal band, less intense bands with molecular masses lower than the principal
band. No such band is more intense than the principal band in the electrophoretogram obtained
with reference solution (d) (1.0 per cent) and not more than three such bands are more intense
than the principal band in the electrophoretogram obtained with reference solution (e) (0.2 per
cent).
The electrophoretogram obtained with test solution (a) under non-reducing conditions may
show, in addition to the principal band, less intense bands with molecular masses higher than the
principal band. No such band is more intense than the principal band in the electrophoretogram
obtained with reference solution (d) (1.0 per cent) and not more than three such bands are more
intense than the principal band in the electrophoretogram obtained with reference solution (e)
(0.2 per cent).
Related proteins Examine by liquid chromatography (2.2.29).
1 mg/ml.
0. 25 per cent m/m hydrogen peroxide solution. Dilute dilute hydrogen peroxide solution R in water R in order
to obtain a 0.25 per cent m/m solution.
Reference solution. To a volume of the test solution, add a suitable volume of 0.25 per cent m/m
hydrogen peroxide solution to give a final hydrogen peroxide concentration of 0.005 per cent m/m,
and allow to stand at room temperature for 1 h, or for the length of time that will generate about
5 per cent oxidised interferon. Add 12.5 mg of L-methionine R per millilitre of solution. Allow to
stand at room temperature for 1 h. Store the solutions for not longer than 24 h at a temperature of
2C to 8C.
silica gel for chromatography R (5 m) with a pore size of 30 nm,
Mobile phase A. To 700 ml of water R add 2 ml of trifluoroacetic acid R and 300 ml of acetonitrile
for chromatography R,
Mobile phase B. To 200 ml of water R add 2 ml of trifluoroacetic acid R and 800 ml of acetonitrile for
chromatography R,
21-51
Interval
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
0 1 72 28 isocratic
Equilibrate the column with the mobile phases in the initial gradient ratio for at least 15 min. Inject
50 l of each solution.
In the chromatograms obtained, interferon alfa-2 elutes at a retention time of about 20 min. In
the chromatogram obtained with the reference solution a peak related to oxidised interferon
appears at a retention time of about 0.9 relative to the principal peak. The test is not valid unless
the resolution between the peaks corresponding to oxidised interferon and interferon is at least
1.0. Consider only the peaks whose retention time is 0.7 to 1.4 relative to that of the principal
peak. In the chromatogram obtained with the test solution, the area of any peak, apart from the
principal peak, is not greater than 3.0 per cent of the total area of all of the peaks. The sum of the
areas of any peaks other than the principal peak is not greater than 5.0 per cent of the total area of
all of the peaks.
1.0 mg of protein.
ASSAY
Protein
Test solution. Dilute the preparation to be examined with water R to obtain a concentration of about
0.5 mg/ml of interferon alfa-2.
Reference solutions. Prepare a stock solution of 0.5 mg/ml of bovine albumin R. Prepare eight dilutions
of the stock solution containing between 3 g/ml and 30 g/ml of bovine albumin R.
Prepare 30-fold and 50-fold dilutions of the test solution. Add 1.25 ml of a mixture prepared the
same day by combining 2.0 ml of a 20 g/l solution of copper sulphate R in water R, 2.0 ml of a 40 g/l
solution of sodium tartrate R in water R and 96.0 ml of a 40 g/l solution of sodium carbonate R in 0.2M
sodium hydroxide to test-tubes containing 1.5 ml of water R (blank), 1.5 ml of the different dilutions
of the test solution or 1.5 ml of the reference solutions. Mix after each addition. After approxi-
mately 10 min, add to each test-tube 0.25 ml of phosphomolybdotungstic reagent R prepared the same
day. Mix after each addition. After approximately 30 min, measure the absorbance (2.2.25) of each
solution at 750 nm using the blank as the compensation liquid. Draw a calibration curve from the
absorbances of the eight reference solutions and the corresponding protein contents and read from
the curve the content of protein in the test solution.
Potency
The potency of interferon alfa-2 is estimated by comparing its effect to protect cells against a viral
cytopathic effect with the same effect of the appropriate International Standard of human
recombinant interferon alfa-2 or of a reference preparation calibrated in International Units.
The International Unit is the activity contained in a stated amount of the appropriate Interna-
tional Standard. The equivalence in International Units of the International Standard is stated by
the World Health Organisation.
Carry out the assay by a suitable method, based on the following design.
Use, in standard culture conditions, an established cell line sensitive to the cytopathic effect of a
suitable virus (a human diploid fibroblast cell line, free of microbial contamination, responsive to
interferon and sensitive to encephalomyocarditis virus, is suitable).
The following cell cultures and virus have shown to be suitable: MDBK cells (ATCC No.
CCL22), or Mouse L cells (NCTC clone 929; ATCC No. CCL 1) as the cell culture and vesicular
stomatitis virus VSV, Indiana strain (ATCC No. VR-158) as the infective agent; or human diploid
fibroblast FS-71 cells responsive to interferon as the cell culture, and encephalomyocarditis virus
(ATCC No. VR-129B) as the infective agent.
Incubate in at least four series, cells with three or more different concentrations of the prepara-
tion to be examined and the reference preparation in a microtitre plate and include in each series
appropriate controls of untreated cells. Choose the concentrations of the preparations such that
the lowest concentration produces some protection and the largest concentration produces less
21-52
than maximal protection against the viral cytopathic effect. Add at a suitable time the cytopathic
virus to all wells with the exception of a sufficient number of wells in all series, which are left with
uninfected control cells. Determine the cytopathic effect of virus quantitatively with a suitable
method. Calculate the potency of the preparation to be examined by the usual statistical methods
for a parallel line assay.
potency. The fiducial limits of error of the estimated potency (P = 0.95) are not less than 64 per
cent and not more than 156 per cent of the stated potency.
STORAGE
Store in an airtight container, protected from light, at or below 20C.
LABELLING
The label states:
the type of interferon (alfa-2a or alfa-2b),
the type of production.
__________________________________________________________________________________________________________ Ph Eur
21-53
Interferon Gamma-1b Concentrated Solution
corrected 1/01
C
734
H
1166
N
204
O
216
S
5
16,465 98059-61-1
Interferon Gamma-1b Concentrated Solution complies with the requirements of the 3rd edition of the European
Action and use Immunomodulator.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Interferon gamma-1b concentrated solution is a solution of the N-terminal methionyl form of
interferon gamma, a protein which is produced and secreted by human antigen-stimulated T
lymphocytes in response to viral infections and various other inducers. It has specific
immunomodulatory properties, such as potent phagocyte-activating effects. The protein consists of
non-covalent dimers of two identical monomers. The formula of the monomer is as follows:
M QDPYVKEAEN LKKYFNAGHS DVADNGTLFL
GILKNWKEES DRKIMQSQIV SFYFKLFKNF
KDDQSIQKSV ETIKEDMNVK FFNSNKKKRD
DFEKLTNYSV TDLNVQRKAI HELIQVMAEL
SPAAKTGKRK RSQMLFRGR
The potency of interferon gamma-1b is not less than 20 10
6
I.U. per milligram of protein.
Interferon gamma-1b concentrated solution contains not less than 30 10
6
I.U. of interferon
gamma-1b per millilitre.
Interferon gamma-1b concentrated solution complies with the monograph on Products of
recombinant DNA technology (0784).
PRODUCTION
Interferon gamma-1b concentrated solution is produced by a method based on recombinant DNA
technology, using bacteria as host-cells. It is produced under conditions designed to minimise
microbial contamination.
Interferon gamma-1b concentrated solution complies with the following additional requirements.
Host-cell derived proteins The limit is approved by the competent authority.
Host-cell-and vector-derived DNA The limit is approved by the competent authority.
CHARACTERS
A clear, colourless or slightly yellowish liquid.
IDENTIFICATION
A. It shows the expected biological activity when tested as prescribed in the assay.
B. Examine the electrophoretograms obtained in the test for impurities of molecular masses
differing from that of interferon gamma-1b. The principal bands in the electrophoretogram
obtained with the test solution correspond in position to the principal bands in the electro-
phoretogram obtained with reference solution (a).
C. Examine by peptide mapping.
Solution A. Prepare a solution containing 1.2 g/l of tris(hydroxymethyl)aminomethane R, 8.2 g/l of
anhydrous sodium acetate R, 0.02 g/l of calcium chloride R and adjust to pH 8.3 (2.2.3) with dilute acetic
acid R. Add polysorbate 20 R to a concentration of 0.1 per cent V/V.
Test solution. Desalt a volume of the preparation to be examined containing 1 mg of protein by a
suitable procedure. For example, filter in a microcentrifuge tube and reconstitute with 500 l of
solution A. Add 10 l of a freshly prepared 1 mg/ml solution of trypsin for peptide mapping R in
water R and mix gently by inversion. Incubate at 30C to 37C for 24 h, add 100 l of phosphoric
acid R per millilitre of digested sample and mix by inversion.
Reference solution. Dilute interferon gamma-1b CRS in water R to obtain a concentration of 1 mg/ml.
Prepare as for the test solution, ensuring that all procedures are carried out simultaneously and
under identical conditions.
21-54
a stainless steel column, 0.15 m long and 4.6 mm in internal diameter packed with octadecylsilyl
Mobile phase A (0.05M sodium phosphate buffer solution pH 3.3). Solution I: dissolve 7.80 g of
sodium dihydrogen phosphate R in water R and dilute to 1000.0 ml with the same solvent. Solution
II: dilute 0.33 ml of phosphoric acid R to 100.0 ml with water R. Mix 920 ml of solution I and
80 ml of solution II. Adjust the pH (2.2.3) if necessary,
Mobile phase B. Acetonitrile for chromatography R,
with the following elution conditions (if necessary, the gradient may be modified to improve the
separation of the digest):
Time
(min)
Mobile
phase A
(per cent V/V)
Mobile
phase B
(per cent V/V)
0 30 100 80 0 20
30 50 80 60 20 40
50 51 60 30 40 70
51 59 30 70
59 60 30 100 70 0
Equilibrate the column for at least 15 min at the initial elution composition. Carry out a blank run
using the above-mentioned gradient.
Inject 100 l of the test solution and 100 l of the reference solution. The test is not valid unless
the chromatogram obtained with each solution is qualitatively similar to the Ph. Eur. reference
chromatogram of interferon gamma-1b digest. The profile of the chromatogram obtained with the test
solution corresponds to that of the chromatogram obtained with the reference solution.
D. Examine by N-terminal sequence analysis.
Use an automated solid-phase sequencer, operated in accordance with the manufacturers instruc-
tions.
Equilibrate by a suitable procedure the equivalent of 100 g of interferon gamma-1b in a 10 g/l
solution of ammonium hydrogen carbonate R, pH 9.0.
Identify the phenylthiohydantoin (PTH)-amino acids released at each sequencing cycle by
reverse-phase liquid chromatography. The procedure may be carried out using the column and
reagents recommended by the manufacturer of the sequencing equipment for the separation of
PTH-amino acids.
The separation procedure is calibrated using:
the mixture of PTH-amino acids provided by the manufacturer, with the gradient conditions
adjusted as indicated to achieve optimum resolution of all amino acids,
a sample from a blank sequencing cycle, obtained as recommended by the equipment
manufacturer.
The first fifteen amino acids are:
MetGlnAspProTyrValLysGluAlaGluAsnLeuLysLysTyr.
TESTS
Appearance The preparation to be examined is clear (2.2.1) and not more intensely coloured than
7
(Method II, 2.2.2).
pH (2.2.3). The pH of the preparation to be examined is 4.5 to 5.5.
Covalent dimers and oligomers Not greater than 2 per cent, determined by size-exclusion
Test solution. Dilute the preparation to be examined with the mobile phase to a protein concentra-
tion of 0.1 mg/ml.
Reference solution (a). Dilute interferon gamma-1b CRS with the mobile phase to a protein concentra-
tion of 0.1 mg/ml.
Reference solution (b). Prepare a mixture of the following molecular mass standards: bovine albumin,
ovalbumin, trypsinogen, lysozyme, at a concentration of 0.1 mg/ml to 0.2 mg/ml for each standard.
a stainless steel column 0.3 m long and 7.8 mm in internal diameter packed with hydrophilic
silica gel for chromatography R, of a grade suitable for fractionation of globular proteins in the
molecular weight range of 10,000 to 500,000 (5 m),
21-55
as mobile phase at a flow rate of 1.0 ml/min a mixture prepared as follows (0.2M sodium
phosphate buffer solution pH 6.8). Solution I: dissolve 31.2 g of sodium dihydrogen phosphate R
and 1.0 g of sodium dodecyl sulphate R in water R and dilute to 1000.0 ml with the same solvent.
Solution II: dissolve 28.4 g of anhydrous disodium hydrogen phosphate R and 1.0 g of sodium dodecyl
sulphate R in water R and dilute to 1000.0 ml with the same solvent. Mix 450 ml of solution I
and 550 ml of solution II. Adjust the pH (2.2.3) if necessary,
as detector a spectrophotometer set at 210 nm to 214 nm.
Inject 200 l of each solution. The test is not valid unless: the molecular mass standards in
reference solution (b) are well separated; the retention time of the principal peak in the chromato-
gram obtained with reference solution (a) is between the retention time of trypsinogen and
lysozyme in the chromatogram obtained with reference solution (b).
Compare the chromatograms obtained with the test solution and with reference solution (a).
There are no additional shoulders or peaks in the chromatogram obtained with the test solution
compared with the chromatogram obtained with reference solution (a).
Calculate the percentage content of covalent dimers and oligomers.
Monomer and aggregates Examine by size-exclusion chromatography (2.2.30). The content of
monomer and aggregates is not greater than 2 per cent.
Solution A. Prepare a solution of the following composition: 0.59 g/l of succinic acid R and 40 g/l of
mannitol R, adjusted to pH 5.0 (2.2.3) with sodium hydroxide solution R.
Test solution. Dilute the preparation to be examined with solution A to a protein concentration of
1 mg/ml.
Reference solution. Dilute interferon gamma-1b CRS with solution A to a protein concentration of
1 mg/ml.
Resolution solution. Prepare 500 l of a mixture consisting of 0.04 mg/ml of bovine albumin R and
0.2 mg/ml of interferon gamma-1b CRS in solution A. Use this solution within 24 h of preparation.
a stainless steel column 0.3 m long and 7.8 mm in internal diameter packed with hydrophilic
silica gel for chromatography R, of a grade suitable for fractionation of globular proteins in the
molecular weight range of 10,000300,000 (5 m),
as mobile phase at a flow rate of 0.8 ml/min a 89.5 g/l solution of potassium chloride R (1.2M),
Inject 20 l of the resolution solution. In the chromatogram obtained, the retention time of the
principal peak, corresponding to the native interferon gamma-1b dimer, is about 10 min. bovine
albumin elutes at a relative retention time of about 0.85, relative to the main peak. The test is not
valid unless the resolution between the peaks corresponding to bovine albumin and interferon
gamma-1b is at least 1.5.
Inject 20 l of the test solution and 20 l of the reference solution. The chromatograms obtained
show principal peaks with identical retention times. Calculate the percentage content of monomer
and aggregates from the peak area of the monomer peak and of peaks which elute prior to the
native interferon gamma-1b peak in the chromatogram obtained with the test solution, by the
normalisation procedure, disregarding any peak due to the solvent.
Deamidated and oxidised forms and heterodimers Examine by liquid chromatography (2.2.29).
The content of deamidated and oxidised forms is not greater than 10 per cent. The content of
heterodimers is not greater than 3 per cent.
1 mg/ml.
Reference solution. Dilute interferon gamma-1b CRS with water R to a protein concentration of 1 mg/ml.
Resolution solution. Use interferon gamma-1b validation solution CRS.
a stainless steel column 0.075 m long and 7.5 mm in internal diameter packed with an appro-
priate hydrophilic polymethacrylate, strong cation exchange gel (10 m, 100 nm),
Mobile phase A (0.05M ammonium acetate buffer pH 6.5). A 3.86 g/l solution of ammonium
acetate R, adjusted to pH 6.5 with dilute acetic acid R,
Mobile phase B (1.2M ammonium acetate buffer pH 6.5). A 92.5 g/l solution of ammonium acetate R,
adjusted to pH 6.5 with dilute acetic acid R,
with the following elution conditions (if necessary, the slope of the gradient may be modified to
improve the separation).
21-56
Time
(min)
Mobile
phase A
(per cent V/V)
Mobile
phase B
(per cent V/V)
0 1 100 0
2 30 100 0 0 100
31 35 0 100
35 37 0 100 100 0
38 47 100 0
Inject 25 l of the resolution solution. In the chromatogram obtained, the retention time of the
principal peak is about 26 min. Deamidated and oxidised forms co-elute at a relative retention time
of about 0.95, relative to the principal peak. The test is not valid unless the resolution, defined by
the ratio of the height of the peak corresponding to the deamidated and oxidised forms to the
height above the baseline of the valley separating the two peaks, is at least 1.2.
Inject 25 l of the test solution and 25 l of the reference solution. The chromatograms obtained
show principal peaks with identical retention times. Calculate the percentage content of
deamidated and oxidised interferon gamma-1b as a percentage of the area of the main peak.
Heterodimers have relative retention times of 0.7 and 0.85 relative to the main peak. Calculate the
percentage of heterodimers as a percentage of the sum of the areas of all peaks.
Impurities of molecular masses differing from that of interferon gamma-1b Examine by
polyacrylamide gel electrophoresis (2.2.31). The test is performed under both reducing and non-
reducing conditions, using resolving gels of 15 per cent acrylamide and silver staining as the
detection method.
Sample buffer (non-reducing conditions). Dissolve 3.78 g of tris(hydroxymethyl)aminomethane R, 10.0 g of
sodium dodecyl sulphate R and 0.100 g of bromophenol blue R in water R. Add 50.0 ml of glycerol R and
dilute to 80 ml with water R. Adjust the pH (2.2.3) to 6.8 with hydrochloric acid R and dilute to
100 ml with water R.
Sample buffer (reducing conditions). Dissolve 3.78 g of tris(hydroxymethyl)aminomethane R, 10.0 g of
sodium dodecyl sulphate R and 0.100 g of bromophenol blue R in water R. Add 50.0 ml of glycerol R and
dilute to 80 ml with water R. Adjust the pH (2.2.3) to 6.8 with hydrochloric acid R and dilute to
100 ml with water R. Immediately before use, add dithiothreitol R to a final concentration of
250 mm.
Test solution. Dilute the preparation to be examined in water R to a protein concentration of
1 mg/ml. Dilute 150 l of the solution with 38 l of sample buffer.
Reference solution (a). Prepare in the same manner as for the test solution, but using interferon gamma-
1b CRS instead of the preparation to be examined.
Reference solution (b) (5 ng control). Mix 50 l of a 0.01 mg/ml solution of bovine albumin R with
2000 l of water R and 450 l of sample buffer.
Reference solution (c) (2 ng control). Mix 20 l of a 0.01 mg/ml solution of bovine albumin R with
2000 l of water R and 450 l of sample buffer.
Reference solution (d). Use a solution of molecular mass standards suitable for calibrating SDS-
polyacrylamide gels in the range of 10 kDa to 70 kDa.
Leave each solution, contained in a test tube, at ambient temperature for 15 min, then store on ice.
Apply 25 l of each solution to the stacking gel wells. Perform the electrophoresis under the
conditions recommended by the manufacturer of the equipment. Detect proteins in the gel by
silver staining.
The test is not valid unless: the validation criteria are met (2.2.31); a band is seen in the electro-
phoretograms obtained with reference solutions (b) and (c).
The principal band in the electrophoretogram obtained with the test solution is similar in
intensity to the principal band in the electrophoretogram obtained with reference solution (a). In
the electrophoretogram obtained with the test solution, no significant bands are observed that are
not present in the electrophoretogram obtained with reference solution (a) (0.01 per cent). A
significant band is defined as any band whose intensity is greater than or equal to that of the band
in the electrophoretogram obtained with reference solution (c).
Norleucine Not more than 0.2 mole of norleucine per mole of interferon gamma-1b, determined
by amino acid analysis.
Test solution. Add 2.5 ml of the preparation to be examined onto a column suitable for the desalting
of proteins previously equilibrated with 25 ml of a 10 per cent V/V solution of acetic acid R. Elute
the sample with another 2.5 ml of a 10 per cent V/V solution of acetic acid R. Determine the protein
content by measuring the absorbance of this solution as described under Protein, in the Assay
21-57
section. Pipette a volume containing the equivalent of 100 g of interferon gamma-1b into each of
three reaction vials. Evaporate to dryness under reduced pressure.
Perform the hydrolysis of the three samples as follows. Add to each reaction vial 200 l of a
50 per cent V/V solution of hydrochloric acid R containing 1 per cent V/V of phenol R, evacuate the
samples, purge with nitrogen and hydrolyse in the gas phase. Heat the reaction vials at 110C for
22 h. After hydrolysis evaporate to dryness under reduced pressure.
Perform the derivatisation of the samples as follows. Prepare immediately before use a mixture
consisting of two volumes of ethanol R, one volume of water R and one volume of triethylamine R.
Add 50 l of this solution to each reaction vial and shake lightly. Evaporate to dryness under
reduced pressure. Add to each vial 50 l of a mixture consisting of 7 volumes of ethanol R, one
volume of water R, one volume of triethylamine R and one volume of phenyl isothiocyanate R. Shake
lightly and allow to stand at room temperature for about 15 min. Evaporate to dryness under
reduced pressure. Reconstitute the samples in 250 l of mobile phase A.
Norleucine stock solution. Prepare a 250 nmol/ml solution of DL-norleucine R in 0.01M hydrochloric acid.
This solution may be kept for two months at 4C.
Leucine stock solution. Prepare a 250 nmol/ml solution of leucine R in 0.01M hydrochloric acid. This
solution may be kept at 4C for two months.
Reference solution. Mix 10 l of norleucine stock solution with 100 l of leucine stock solution in each
of the three reaction vials. Evaporate to dryness under reduced pressure. Perform the derivatisa-
tion of the samples as described for the preparation of the test solution.
a stainless steel column 0.15 m long and 3.9 mm in diameter packed with octadecylsilyl silica gel
Mobile phase A. Mix 70 volumes of a 19 g/l solution of sodium acetate R containing 0.05 per cent
V/V of triethylamine R and adjusted to pH 6.4 with dilute acetic acid R and 30 volumes of mobile
phase B,
Mobile phase B. Mix 40 volumes of water R and 60 volumes of acetonitrile R,
Time
(min)
Mobile
phase A
(per cent V/V)
Mobile
phase B
(per cent V/V)
Comment
0 7 100 0 isocratic
7 7.1 100 0 0 100 linear gradient
7.1 10 0 100 washing step
10 10.1 0 100 100 0 linear gradient
10.1 15 100 0 re-equilibration
In the chromatograms obtained with the test solution, identify the peaks corresponding to
leucine and norleucine. The retention time of norleucine is 6.2 min to 7 min.
Calculate the content of norleucine (in moles of norleucine per mole of interferon gamma-1b)
from the peak areas of leucine and norleucine in the chromatograms obtained with the reference
and test solutions, considering that there are 10 moles of leucine per mole of interferon gamma-1b.
20 10
6
I.U. of interferon gamma-1b.
ASSAY
Protein (2.2.25). Dilute the substance to be examined in water R to obtain a concentration of
1 mg/ml. Record the absorbance spectrum between 220 nm and 340 nm. Measure the value at the
absorbance maximum of 280 nm, after correction for any light scattering due to turbidity measured
at 316 nm. Calculate the concentration of interferon gamma-1b using a specific absorbance value
of 7.5.
Potency The potency of interferon gamma-1b is estimated by evaluating the increase of the
expression of human-leukocyte-antigen-DR (HLA-DR) due to the interferon gamma-1b present in
test solutions during cultivation of the cells, and comparing this increase with the same effect of the
appropriate International Standard of human recombinant interferon gamma or of a reference
preparation calibrated in International Units.
The International Unit is the activity contained in a stated amount of the appropriate Interna-
tional Standard. The equivalence in International Units of the International Standard is stated by
the World Health Organisation.
21-58
Carry out the assay by a suitable method, based on the following design.
Use COLO 205 cells under standard culture conditions. Trypsinise a 3- to 5-day-old flask of
COLO 205 cells and prepare a cell suspension at a concentration of 1.0 10
6
cells/ml.
Add 100 l of the dilution medium to all wells of a 96-well microtitre plate. Add an additional 100
l of this solution to the wells designed for the blanks. Add 100 l of each solution to be tested onto
the plate and carry out a series of twofold dilution steps in order to obtain a standard curve. Then
add 100 l of the cell suspension to all wells and incubate the plate under appropriate conditions
for cell cultivation.
After cultivation remove the growth medium and wash and fix cells to the plate. Add an antibody
able to detect HLA-DR expressed due to the presence of interferon gamma-1b and incubate under
appropriate conditions. After washing the plate, incubate with an antibody conjugated to a marker
enzyme which is able to detect the anti-HLA-DR antibody. After this incubation step, wash the
plate and add an appropriate substrate solution. Stop the reaction. Measure the absorbance of the
solution and calculate the potency of the preparation to be examined by the usual statistical
methods.
The estimated specific activity is not less than 80 per cent and not more than 125 per cent of the
stated potency. The confidence interval (P = 0.95) of the estimated potency is not greater than
70 per cent to 140 per cent.
STORAGE
Store in an airtight container, protected from light and at a temperature of 70C.
__________________________________________________________________________________________________________ Ph Eur
21-59
Inulin
Definition Inulin consists of polysaccharide granules obtained from the tubers of Dahlia variabilis,
Helianthus tuberosus and other genera of the family Compositae.
Characteristics A white, amorphous, granular powder; odourless or almost odourless; hygro-
scopic.
Microscopical When mounted in absolute ethanol, appears as large, very irregular, angular masses,
whole or fragmented, with occasional smaller, spherical to ovoid particles.
Slightly soluble in water; freely soluble in hot water; slightly soluble in organic solvents.
Identification
A. Carry out the method for thin-layer chromatography, Appendix III A, using a suspension of silica
gel G in a 0.3% w/v solution of sodium acetate to coat the plate, but spreading a layer 0.5 mm thick.
Use a mixture of 70 volumes of glacial acetic acid, 60 volumes of chloroform and 10 volumes of water
as the mobile phase. Apply separately to the plate 1 l of each of three solutions in water containing
(1) 2.5% w/v of the substance being examined, prepared using heat, (2) 2.5% w/v of the substance
being examined and 10% w/v of oxalic acid, prepared by boiling for 10 minutes and cooling, and (3)
2.5% w/v of D-fructose and 0.1% w/v of D-glucose monohydrate. Develop the chromatograms in a
continuous elution tank for about 4 hours. After removal of the plate, evaporate the solvent in a
current of warm air and spray with a solution in acetone containing 1% v/v of diphenylamine, 1% v/v
of aniline and 1% v/v of orthophosphoric acid and heat for 10 minutes at 130. The two principal spots
in the chromatogram obtained with solution (2) correspond to those in the chromatogram
obtained with solution (3). The spot in the chromatogram obtained with solution (1) remains on
the line of application.
B. Dissolve 10 mg in 2 ml of hot water, add 3 ml of a 0.15% w/v solution of resorcinol in ethanol
(96%) followed by 3 ml of hydrochloric acid, mix and heat at 80. A red colour is produced.
C. Boil 5 ml of a 10% w/v solution for 2 minutes with 0.5 ml of hydrochloric acid, cool and neutralise
to litmus paper with 5M sodium hydroxide. Add 0.5 ml of cupri-tartaric solution R1 and heat. A red
Acidity Dissolve 5 g in 50 ml of freshly boiled water and titrate with 0.1M sodium hydroxide VS using
phenolphthalein solution R1 as indicator. Not more than 0.20 ml is required to change the colour of
the solution to pink.
Clarity and colour of solution A 10.0% w/v solution in hot water is clear, Appendix IV A, and
colourless, Appendix IV B, Method I.
Specific optical rotation In a 2% w/v solution, prepared using heat, 36.5 to 40.5, calculated
with reference to the dried substance, Appendix V F.
Arsenic 1.0 g dissolved in 25 ml of hot water complies with the limit test for arsenic, Appendix VII
(1 ppm).
Calcium Dissolve 1.0 g in 10 ml of hot water, place the solution in a water bath at 40, add 0.5 ml
of a 2.5% w/v solution of ammonium oxalate and allow to stand for 15 minutes. Any turbidity
produced is not greater than that obtained by treating 10 ml of a 0.010% w/v solution of calcium
chloride in the same manner (270 ppm).
Lead Dissolve 2.5 g in 20 ml of hot water and transfer to a separating funnel with the aid of 5 ml of
water. Add 2.5 ml of hydrochloric acid and shake for 5 minutes. Add 0.25 ml of bromothymol blue
solution R3 and sufficient 13.5M ammonia to produce a full blue colour and add 1.5 ml in excess.
Add 1 ml of a 25% w/v solution of citric acid previously adjusted to pH 11 with 13.5M ammonia
(ammonium citrate solution), 1 ml of potassium cyanide solution PbT and sufficient of a 0.002% w/v
solution of dithizone in chloroform until, on shaking, the chloroform layer becomes purple or blue.
Separate the chloroform layer, add to the aqueous layer 2 ml of the dithizone solution, shake and
separate the chloroform layer. To the combined chloroform solutions add 10 ml of a 1% v/v
solution of nitric acid, shake until the chloroform layer becomes green, allow to separate and
discard the chloroform layer. To the aqueous layer add 0.2 ml of the ammonium citrate solution,
0.25 ml of 13.5M ammonia, 0.2 ml of potassium cyanide solution PbT and sufficient of the dithizone
solution until, on shaking, the chloroform layer becomes purple or blue. Transfer the contents of
the separating funnel to a stoppered tube. Repeat the operations without the substance being
examined and using the same quantities of the reagents. To the tube containing the reference
solution add lead standard solution (10 ppm Pb) dropwise until, on shaking, the colour of the
chloroform layer matches that of the chloroform layer obtained from the substance being exam-
ined. Not more than 0.5 ml of lead standard solution (10 ppm Pb) is required (2 ppm).
Chloride Dissolve 2.0 g in hot water, cool and dilute to 100 ml with water. 15 ml of the resulting
solution complies with the limit test for chlorides, Appendix VII (170 ppm).
Oxalate Dissolve 1.0 g in 10 ml of hot water, place the solution in a water bath at 40, add 0.5 ml
21-60
of a 7% w/v solution of calcium chloride and allow to stand for 15 minutes. Any turbidity produced is
not greater than that obtained by treating 10 ml of a 0.003% w/v solution of oxalic acid in the same
manner.
Sulphate Dissolve 2.3 g in 20 ml of hot water, cool, add 2 ml of 2M hydrochloric acid and dilute to
45 ml with water. The solution complies with the limit test for sulphates, Appendix VII (200 ppm).
Reducing sugars Heat 10 ml of cupri-tartaric solution R1 and titrate with a 10% w/v solution of the
substance being examined, adding a few ml at a time and boiling for 10 to 15 seconds between each
addition. When the solution becomes greenish yellow, add 0.25 ml of a 1% w/v solution of
methylene blue and continue the titration until the solution becomes orange. Repeat the operation
using a 0.20% w/v solution of D-fructose in place of the solution of the substance being examined.
The volume of the fructose solution required is not greater than that of the solution of the
substance being examined.
Use 1 g.
Action and use Used for the measurement of glomerular filtration rate.
Preparation
Inulin Injection
21-61
Iodine
1/01
I
2
253.8 7553-56-2
Iodine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0031]. These
Action and use Antiseptic; antithyroid.
Preparations
Alcoholic Iodine Solution
Aqueous Iodine Oral Solution
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Iodine contains not less than 99.5 per cent and not more than the equivalent of 100.5 per cent of
I.
CHARACTERS
Greyish-violet, brittle plates or fine crystals with a metallic sheen, very slightly soluble in water,
soluble in alcohol, slightly soluble in glycerol, very soluble in concentrated solutions of iodides.
Iodine volatilises slowly at room temperature.
IDENTIFICATION
A. Heat a few fragments in a test-tube. Violet vapour is evolved and a bluish-black crystalline
sublimate is formed.
B. To a saturated solution add starch solution R. A blue colour is produced. Heat until decolourised.
On cooling, the colour reappears.
TESTS
Solution S Triturate 3.0 g with 20 ml of water R, filter, wash the filter with water R and dilute the
filtrate to 30 ml with the same solvent. To the solution add 1 g of zinc powder R. When the solution
is decolourised, filter, wash the filter with water R and dilute to 40 ml with the same solvent.
Bromides and chlorides To 10 ml of solution S add 3 ml of ammonia R and 6 ml of silver nitrate
solution R2. Filter, wash the filter with water R and dilute the filtrate to 20 ml with the same solvent.
To 10 ml of the solution add 1.5 ml of nitric acid R. After 1 min, any opalescence in the solution is
not more intense than that in a standard prepared at the same time by mixing 10.75 ml of water R,
0.25 ml of 0.01M hydrochloric acid, 0.2 ml of dilute nitric acid R and 0.3 ml of silver nitrate solution R2
(250 ppm).
Non-volatile substances Heat 1.00 g in a porcelain dish on a water-bath until the iodine has
volatilised. Dry the residue at 100C to 105C. The residue weighs not more than 1 mg (0.1 per
cent).
ASSAY
Introduce 0.200 g into a flask containing 1 g of potassium iodide R and 2 ml of water R and add 1 ml
of dilute acetic acid R. When dissolution is complete, add 50 ml of water R and titrate with 0.1M
sodium thiosulphate, using starch solution R as indicator.
1 ml of 0.1M sodium thiosulphate is equivalent to 12.69 mg of I.
__________________________________________________________________________________________________________ Ph Eur
22-1
Iohexol
I
I
I
N
N OH
HO
O
H OH
Ac
H HO
N OH O
H OH
mi xture of isomers
H
H
*
* *
C
19
H
26
I
3
N
3
O
9
821.2 66108-95-0
Iohexol complies with the requirements of the 3rd edition of the European Pharmacopoeia [1114]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Iohexol contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent of
5-[(acetyl)(2,3-dihydroxypropyl)amino)]-N,N-bis(2,3-dihydroxypropyl)-2,4,6-tri-iodobenzene-1,3-
dicarboxamide, calculated with reference to the anhydrous substance.
CHARACTERS
A white or greyish-white, hygroscopic powder, very soluble in water, freely soluble in methanol,
practically insoluble in ether and in methylene chloride. The substance is a mixture of
diastereoisomers and atropisomers.
IDENTIFICATION
obtained with iohexol CRS. Examine the substances as discs prepared using potassium bromide R.
B. Examine the chromatograms obtained in test A for related substances (see Tests). The retention
time and size of the principal peaks in the chromatogram obtained with reference solution (b) are
approximately the same as those of the peaks due to iohexol in the chromatogram obtained with
TESTS
solution Y
7
(Method II, 2.2.2).
Related substances
A. Examine by liquid chromatography (2.2.29).
NOTE: Iohexol gives rise to two non-resolved peaks in the chromatogram due to endo-exo isomerism. In
addition, a small peak also due to iohexol usually appears at the leading edge of the first main peak. This
small peak has a retention time about 1.2 min less than the first main peak.
Test solution. Dissolve 0.150 g of the substance to be examined in water R and dilute to the 100.0 ml
Reference solution (a). Dissolve 15.0 mg of iohexol CRS and 15.0 mg of iohexol impurity A CRS in a
mixture of 1 to 2 drops of dilute sodium hydroxide solution R and 10 ml of water R and dilute to
100.0 ml with water R. Dilute 1.0 ml of this solution to 10.0 ml with water R.
Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with water R.
as mobile phase at a flow rate of 1 ml/min a mixture of acetonitrile R and water R in a linear
gradient elution passing from 1 volume of acetonitrile R and 99 volumes of water R to 13 volumes
22-2
of acetonitrile R and 87 volumes of water R over 60 min,
Equilibrate the column at the initial eluent composition for at least 10 min. Adjust the sensitivity of
the system so that the height of the principal peak in the chromatogram obtained with 10 l of
reference solution (b) is at least 50 per cent of the full scale of the recorder.
Inject 10 l of water R as a blank. Inject 10 l of reference solution (a). When the chromatogram is
recorded in the prescribed conditions, the retention times are: iohexol impurity A, about 17 min and
for the two peaks due to iohexol (exo-endo isomerism), about 20 min. The test is not valid unless the
resolution between the peaks corresponding to iohexol impurity A, and the second and greater peak
due to iohexol is at least 5; if necessary adjust the final concentration of acetonitrile in the mobile
phase or adjust the time programme for the linear gradient.
Inject separately 10 l of the test solution and 10 l of reference solution (b). In the chromatogram
obtained with the test solution: the area of any peak, apart from the peaks due to iohexol (see above),
is not greater than half the area of the principal iohexol peaks in the chromatogram obtained with
reference solution (b) (0.5 per cent); the sum of the areas of all peaks, apart from the peaks due to
iohexol (see above), is not greater than 1.5 times the area of the principal peaks in the chromatogram
obtained with reference solution (b) (1.5 per cent). Disregard any peak observed with the blank run.
254
Test solution. Dissolve 1.0 g of the substance to be examined in water R and dilute to 10.0 ml with the
same solvent.
Reference solution (a). Dissolve 50 mg of iohexol impurity J CRS and 50 mg of iohexol CRS in water R
Reference solution (b). Dilute 1.0 ml of the test solution to 10.0 ml with water R. Dilute 1.0 ml of this
Wash the plate with the mobile phase, dry for 30 min at room temperature and for 1 h at 90C.
20 volumes of concentrated ammonia R, 20 volumes of methanol R, 35 volumes of 2-propanol R and 50
volumes of acetone R. Examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained
with the test solution, apart from the principal spot, is not more intense than the spot in the chrom-
atogram obtained with reference solution (b) (0.2 per cent). The test is not valid unless the chrom-
atogram obtained with reference solution (a) shows two clearly separated spots.
3-Chloropropane-1,2-diol Not more than 100 ppm, determined by gas chromatography (2.2.28).
Test solution. Dissolve 1.0 g of the substance to be examined in 2.0 ml of water R. Shake with four
quantities, each of 2 ml, of methyl acetate R. Dry the combined upper layers over anhydrous sodium
sulphate R. Filter and concentrate to 2 ml.
Reference solution. Dissolve 0.50 g of 3-chloropropane-1,2-diol R in 100.0 ml of methyl acetate R. Dilute
1.0 ml of this solution with 100.0 ml of methyl acetate R.
a fused-silica column 25 m long and 0.33 mm in internal diameter coated with a 1 m film of
Inject 2 l (splitless for 30 s) of each solution. When the chromatograms are recorded in the
prescribed conditions, the retention time for 3-chloropropane-1,2-diol is about 8 min.
Calculate the content of 3-chloropropane-1,2-diol.
Methanol, ethylene glycol monomethyl ether and 2-propanol Not more than 50 ppm,
100 ppm and 100 ppm, respectively, determined by head-space gas chromatography (2.2.28), using
2-butanol R as the internal standard and using the method of standard additions.
Internal standard solution. Dissolve 0.50 g of 2-butanol R in water R and dilute to 500.0 ml with the
same solvent. Dilute 5.0 ml of this solution to 100.0 ml with water R.
Test solution (a). Dissolve 6.25 g of the substance to be examined in water R, add 5.0 ml of the
internal standard solution and dilute to 25.0 ml with water R.
Test solution (b). Transfer 5.0 ml of test solution (a) and 1.0 ml of water R to a 10 ml vial and cap the
vial immediately.
Test solution (c). Transfer 5.0 ml of test solution (a) and 1.0 ml of reference solution (b) to a 10 ml
vial and cap the vial immediately (spiked with, per gram, 10 g of methanol R, 20 g of 2-propanol R
and 20 g of ethylene glycol monomethyl ether R).
Test solution (d). Transfer 5.0 ml of test solution (a) and 1.0 ml of reference solution (c) to a 10 ml
22-3
Test solution (e). Transfer 5.0 ml of test solution (a) and 1.0 ml of reference solution (d) to a 10 ml
Reference solution (a). Dissolve 0.625 g of methanol R, 1.250 g of 2-propanol R and 1.250 g of ethylene
glycol monomethyl ether R in water R and dilute to 1000.0 ml with the same solvent. Dilute with some
water R between each component added.
Reference solution (b). Dilute 10.0 ml of reference solution (a) to 50.0 ml with water R. Dilute 10.0 ml
of this solution to 100.0 ml with water R.
Reference solution (c). Dilute 5.0 ml of reference solution (a) to 100.0 ml with water R.
Reference solution (e). Add 10.0 ml of reference solution (d) to 10.0 ml of the internal standard
solution and dilute to 50.0 ml with water R. Transfer 6.0 ml to a 10 ml vial and cap the vial immedi-
ately.
The following head-space conditions may be used:
a thermostatically controlled bath adjusted to 95C,
an equilibration time of 15 min,
a transfer temperature of 140C,
a pressurisation time of 30 s,
a loop-filling time of 10 s,
an injection time of 10 s.
a fused-silica column 30 m long and 0.54 mm in internal diameter coated with a 3 m film of
cross-linked poly-[(cyanopropyl)(methyl)][(phenyl)(methyl)]siloxane R,
helium for chromatography R as the carrier gas at a flow rate of about 14 ml/min,
Record the chromatograms obtained with test solutions (b), (c), (d) and (e) and reference solution
(e). When the chromatograms are recorded in the prescribed conditions, the retention times are:
methanol, about 1.2 min; 2-propanol, about 1.8 min; 2-butanol, about 3.5 min and ethylene glycol
monomethyl ether, about 4.5 min. The test is not valid unless the resolution between the peaks
corresponding to methanol and 2-propanol in the chromatogram obtained with reference solution (e)
is at least 2.5.
In the chromatograms obtained with the test solutions, calculate the peak area ratio for each
substance to the internal standard. Using the peak area ratios, perform a linear regression analysis,
and derive from it the concentration of the substance to be determined in the substance to be
examined.
Alternatively, plot the peak area ratios against the added quantity of the substance to be deter-
mined per gram. Extrapolate the line joining the points until it meets the concentration axis. The
distance between this point and the intersection of the axis represents the concentration (micrograms
per gram) of the substance to be determined in the substance to be examined.
Free aromatic amine Not more than 0.05 per cent m/m determined by absorption spectro-
photometry (2.2.25).
Test solution. Transfer 0.200 g of the substance to be examined to a 25 ml volumetric flask and
dissolve in 15.0 ml of water R.
Reference solution. Prepare the reference solution by mixing 10.0 ml of a solution containing 10 g
per ml of iohexol impurity J CRS with 5.0 ml of water R in a 25 ml volumetric flask.
Blank solution. Transfer 15.0 ml of water R to a 25 ml volumetric flask.
NOTE: In conducting the following steps, keep the flasks in iced water and protected as far as possible from
light until all of the reagents have been added.
Place all three of the flasks containing the test solution, the reference solution and the blank solution
in iced water, protected from light, for 5 min. Add 1.5 ml of hydrochloric acid R1 and mix by swirling.
Add 1.0 ml of a 20 g/l solution of sodium nitrite R, mix and allow to stand for 4 min. Add 1.0 ml of a
40 g/l solution of sulphamic acid R, swirl gently until gas liberation has ceased and allow to stand for
1 min. (CAUTION: considerable pressure is produced). Add 1.0 ml of a freshly prepared 3 g/l solution
of naphthylethylenediamine dihydrochloride R in a mixture of 30 volumes of water R and 70 volumes of
propylene glycol R and mix. Remove the flasks from the iced water, dilute to 25.0 ml with water R, mix
and allow to stand for 5 min. Concomitantly determine the absorbance at 495 nm of the solutions
obtained from the test solution and the reference solution in 5 cm cells, using the blank as the
compensation liquid.
Calculate the content of free aromatic amine.
Iodide Not more than 20 ppm, determined by potentiometric titration (2.2.20). Dissolve 6.000 g in
water R and dilute to 20 ml with the same solvent. Add 2.0 ml of 0.001M potassium iodide. Titrate
22-4
with 0.001M silver nitrate using a silver indicator electrode and an appropriate reference electrode.
Subtract the volume of titrant corresponding to the 2.0 ml of 0.001M potassium iodide, determined by
titrating a blank to which is added 2.0 ml of 0.001M potassium iodide and use the residual value to
calculate the iodide content.
1 ml of 0.001M silver nitrate is equivalent to 126.9 g of iodide.
Ionic compounds Not more than 0.05 per cent m/m calculated as sodium chloride and determined
by the measurement of specific conductivity (2.2.38).
Rinse all glassware with distilled water R five times before use.
Test solution. Dissolve 1.0 g of the substance to be examined in water R and dilute to 50.0 ml with the
same solvent.
Reference solution. Dissolve 10.0 mg of sodium chloride R in water R and dilute to 100.0 ml with the
same solvent. Dilute 10.0 ml of this solution to 100.0 ml with water R.
Measure the specific conductivity of the test solution and the reference solution using a suitable
conductivity meter. The specific conductivity of the test solution is not greater than that of the
reference solution.
Prepare the standard using lead standard solution (1 ppm) Pb R.
tion of water.
ASSAY
To 0.500 g in a 250 ml round-bottomed flask add 25 ml of strong sodium hydroxide solution R, 20 ml
of water R, 1 g of zinc powder R and a few glass beads. Boil under a reflux condenser for 30 min.
Allow to cool and rinse the condenser with 20 ml of water R, adding the rinsings to the flask. Filter
through a sintered-glass filter and wash the filter with several quantities of water R. Collect the filtrate
and washings. Add 5 ml of glacial acetic acid R and titrate immediately with 0.1M silver nitrate.
Determine the end-point potentiometrically (2.2.20), using a suitable electrode system such as silver-
mercurous sulphate.
19
H
26
I
3
N
3
O
9
.
STORAGE
Store in a airtight container, protected from light and moisture.
IMPURITIES
N
HO
I O O
I I
N
OH
OH HO
H
* *
H
Ar =
H
3
C NHAr
O
A. 5-acetylamino-N,N-bis(2,3-dihydroxypropyl)-2,4,6-tri-iodobenzene-1,3-dicarboxamide,
H
3
C N O OH
O
Ar OH OH
* *
B. 5-[(acetyl)(2,6,7-trihydroxy-4-oxaheptyl)amino]-N,N-bis(2,3-dihydroxypropyl)-2,4,6-tri-
iodobenzene-1,3-dicarboxamide,
H
3
C N
O OH
O
Ar
OH
OH
*
*
C. 5-[(acetyl)(5,6-dihydroxy-2-hydroxymethyl-3-oxahexyl)-amino]-N,N-bis(2,3-
dihydroxypropyl)-2,4,6-tri-iodobenzene-1,3-dicarboxamide,
22-5
H
*
H
* *
H
3
C N OH
O
OH
OH OH
OH O
HO
N
I I
O O I
HO
N
*
D. 5-[(acetyl)(2,3-dihydroxypropyl)amino]-N-(2,3-dihydroxypropyl)-N-(2,6,7-trihydroxy-4-
oxaheptyl)-2,4,6-tri-iodobenzene-1,3-dicarboxamide,
H
*
H
*
H
3
C N OH
O
OH
OH
HO
O
N
I I
O O I
HO
N
OH
OH
*
*
E. 5-[(acetyl)(2,3-dihydroxypropyl)amino]-N-(5,6-dihydroxy-2-hydroxymethyl-3-oxahexyl)-
N-(2,3-dihydroxypropyl)-2,4,6-tri-iodobenzene-1,3-dicarboxamide,
F. 5-amino-N,N-bis(2,3-dihydroxypropyl)di-iodobenzene-1,3-dicarboxamide,
G. 5-acetylamino-N,N-bis(2,3-dihydroxypropyl)di-iodobenzene-1,3-dicarboxamide,
H. 5-[(acetyl)(2,3-dihydroxypropyl)amino]-N,N-bis(2,3-dihydroxypropyl)di-iodobenzene-1,3-
dicarboxamide,
H
H
I
O I
N
O
N
OH
N HO O
OH
HO
OH
H
*
*
*
I. 2-hydroxymethyl-N,N-bis(2,3-dihydroxypropyl)-5,7-di-iodo-2,3-dihydro-4H-1,4-
benzoxazine-6,8-dicarboxamide,
NH
2
Ar
J. 5-amino-N,N-bis(2,3-dihydroxypropyl)-2,4,6-tri-iodobenzene-1,3-dicarboxamide.
__________________________________________________________________________________________________________ Ph Eur
22-6
Iopamidol
N
OH
N
HO
I O O
HO OH
I I
HN O
CH
3
HO
H
H H
C
17
H
22
I
3
N
3
O
5
777 62883-00-5
Iopamidol complies with the requirements of the 3rd edition of the European Pharmacopoeia [1115]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Iopamidol contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent of
(S)-N,N-bis[2-hydroxy-1-(hydroxymethyl)ethyl]-5-[(2-hydroxypropanoyl)amino]-2,4,6,-tri-
iodobenzene-1,3-dicarboxamide, calculated with reference to the dried substance.
CHARACTERS
A white or almost white powder, freely soluble in water, very slightly soluble in methanol, practically
insoluble in alcohol and in methylene chloride.
IDENTIFICATION
obtained with iopamidol CRS.
C. It complies with the test for specific optical rotation (see Tests).
TESTS
Appearance of solution Dissolve 1 g in water R and dilute to 50 ml with the same solvent. The
Acidity or alkalinity Dissolve 10.0 g in carbon dioxide-free water R and dilute to 100 ml with the
same solvent. Not more than 0.75 ml of 0.01M hydrochloric acid or 1.4 ml of 0.01M sodium hydroxide is
required to adjust the pH (2.2.3) to 7.0.
Specific optical rotation (2.2.7). Dissolve 10.0 g, with heating if necessary, in water R and dilute to
25.0 ml with the same solvent. The specific optical rotation is 4.6 to 5.2, determined at 436 nm
and calculated with reference to the dried substance.
the same solvent.
Reference solution (a). Dilute 1.0 ml of the test solution to 20.0 ml with water R. Dilute 1.0 ml of the
Reference solution (b). Dissolve 25 mg of iopamidol impurity B CRS in water R and dilute to 100 ml
with the same solvent. Dilute 0.5 ml of the solution to 5 ml with reference solution (a).
as mobile phase at a flow rate of 1.5 ml/min a mixture of water R (mobile phase A) and a 25 per
cent V/V solution of methanol R (mobile phase B), with the gradient elution described in the
following table:
22-7
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
0 92.5 7.5
6 92.5 7.5
18 65 35
30 8 92
34 8 92
36 92.5 7.5
two peaks in the chromatogram obtained is at least 50 per cent of the full scale of the recorder. The
test is not valid unless, in the chromatogram obtained, the resolution between the peaks correspond-
ing to iopamidol impurity B and iopamidol is at least 5.0.
with the test solution, the sum of the areas of any peaks apart from the principal peak is not greater
than the area of the principal peak in the chromatogram obtained with reference solution (a)
Free aromatic amines Keep the solutions and reagents in iced water, protected from bright light.
Test solution. In a 25 ml volumetric flask, dissolve 0.500 g of the substance to be examined in 20.0 ml
of water R.
Reference solution. In a 25 ml volumetric flask, mix 4.0 ml of a 25.0 mg/l solution of iopamidol impurity
A CRS with 16.0 ml of water R.
Blank solution. Place 20.0 ml of water R in a 25 ml volumetric flask.
Place the flasks in iced water, protected from light, for 5 min. Add 1.0 ml of hydrochloric acid R to
each flask, mix and allow to stand for 5 min. Add 1.0 ml of a 20 g/l solution of sodium nitrite R
prepared immediately before use, mix and allow to stand for 5 min. Add 1.0 ml of a 120 g/l solution
of ammonium sulphamate R, swirl gently until gas liberation has ceased, and allow to stand for 5 min.
(CAUTION: considerable pressure is produced). Add 1.0 ml of a freshly prepared 1 g/l solution of
naphthylethylenediamine dihydrochloride R and mix. Remove the flasks from the iced water and allow to
stand for 10 min. Dilute to 25.0 ml with water R and mix. Measure immediately the absorbance
(2.2.25) at 500 nm of the solutions obtained from the test solution and the reference solution using,
as the compensation liquid, the solution obtained from the blank solution.
The absorbance of the test solution is not greater than that of the reference solution (200 ppm).
Free iodine Dissolve 2.0 g in 25 ml of water R in a ground-glass stoppered centrifuge tube. Add
5 ml of toluene R and 5 ml of diluted sulphuric acid R. Shake and centrifuge. The upper layer shows no
red colour.
water R and dilute to 20 ml with the same solvent. Add 2.0 ml of 0.001M potassium iodide. Titrate
with 0.001M silver nitrate using a silver indicator electrode and an appropriate reference electrode.
Subtract the volume of titrant corresponding to the 2.0 ml of 0.001M potassium iodide, determined by
titrating a blank to which is added 2.0 ml of 0.001M potassium iodide and use the residual value to
calculate the iodide content.
100C to 105C.
1.4 I.U. of endotoxin per gram.
ASSAY
22-8
washings.
Add 5 ml of glacial acetic acid R and titrate immediately with 0.1M silver nitrate. Determine the end-
point potentiometrically (2.2.20) using a suitable electrode system such as silver-mercurous sulphate.
17
H
22
I
3
N
3
O
8
.
STORAGE
LABELLING
The label states:
IMPURITIES
N
HO
I O O
HO
I I
NHR
2
R
1
H
A. R
1
= NHCH(CH
2
OH)
2
, R
2
= H: 5-amino-N,N-bis[2-hydroxy-1-(hydroxymethyl)ethyl]-2,4,6-
tri-iodobenzene-1,3-dicarboxamide,
B. R
1
= NHCH(CH
2
OH)
2
, R
2
= COCH
2
OH: N,N-bis[2-hydroxy-1-(hydroxymethyl)ethyl]-5-[(2-
hydroxyacetyl)amino]-2,4,6-tri-iodobenzene-1,3-dicarboxamide,
C. R
1
= NHCH(CH
2
OH)
2
, R
2
- COCH
3
: 5-(acetylamino)-N,N-bis[2-hydroxy-1-(hydroxymethyl)-
ethyl]-2,4,6-tri-iodobenzene-1,3-dicarboxamide,
D. R
1
= OH, R
2
= COCHOHCH
3
: 3-[N-[2-hydroxy-1-(hydroxymethyl)ethyl]carbamoyl]-5-[(2-
hydroxypropanoyl)amino]-2,4,6-tri-iodobenzoic acid,
E. R
1
= NHCH(CH
2
OH)
2
, R
2
- COCH(CH
3
)OCOCH
3
: 5-[(2-acetyloxy-propanoyl)amino]-N,N-bis[2-
hydroxy-1-(hydroxymethyl)ethyl]-2,4,6-tri-iodobenzene-1,3-dicarboxamide,
F. R
1
= N(CH
3
)
2
, R
2
= COCHOHCH
3
: N-[2-hydroxy-1-(hydroxymethyl)ethyl]-5-[(2-hydroxy-
propanoyl)amino]-N-dimethyl-2,4,6-tri-iodobenzene-1,3-dicarboxamide,
G. R
1
= NHCH
2
-CHOH-CH
2
OH, R
2
= COCHOHCH
3
: N-[2-hydroxy-1-(hydroxymethyl)ethyl]-N-(2,3-
dihydroxypropyl)-5-[(2-hydroxypropanoyl)amino]-2,4,6-tri-iodobenzene-1,3-dicarboxamide.
__________________________________________________________________________________________________________ Ph Eur
22-9
Iopanoic Acid
CH
3
COOH H
I
I I
NH
2
and enantiomer
C
11
H
12
I
3
NO
2
571 96-83-3
Iopanoic Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia [0700]. These
Action and use Radio-opaque substance used in cholecystography.
Preparation
Iopanoic Acid Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Iopanoic acid contains not less than 98.5 per cent and not more than the equivalent of 101.0 per
cent of (RS)-2-(3-amino-2,4,6-tri-iodobenzyl)butanoic acid, calculated with reference to the dried
substance.
CHARACTERS
A white or yellowish-white powder, practically insoluble in water, soluble in ethanol, in ether and in
methanol. It dissolves in dilute solutions of alkali hydroxides.
IDENTIFICATION
A. Melting point (2.2.14) about 155C, with decomposition.
obtained with iopanoic acid CRS.
C. Examine the chromatograms obtained in the test for related substances (see Tests). Spray the
plate with a 1 g/l solution of dimethylaminocinnamaldehyde R in a mixture of 1 volume of hydrochloric
acid R and 99 volumes of alcohol R. The principal spot in the chromatogram obtained with test
solution (b) is similar in position, colour and size to the principal spot in the chromatogram obtained
D. Heat 50 mg carefully in a small porcelain dish over a flame. Violet vapour is evolved.
TESTS
3
(Method II, 2.2.2).
254
R as the
coating substance.
Test solution (a). Dissolve 1.0 g of the substance to be examined in a mixture of 3 volumes of
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with a mixture of 3 volumes of ammonia R
Reference solution (a). Dissolve 50 mg of iopanoic acid CRS in a mixture of 3 volumes of ammonia R
Reference solution (b). Dilute 1 ml of test solution (b) to 50 ml with a mixture of 3 volumes of
10 volumes of concentrated ammonia R, 20 volumes of methanol R, 20 volumes of toluene R and 50
volumes of dioxan R. Examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained
with test solution (a), apart from the principal spot, is not more intense than the spot in the chrom-
atogram obtained with reference solution (b) (0.2 per cent).
22-10
Halides To 0.46 g add 10 ml of nitric acid R and 15 ml of water R. Shake for 5 min and filter. 15 ml
of the filtrate complies with the limit test for chlorides (2.4.4) (180 ppm, expressed as chloride).
100C to 105C for 1 h.
ASSAY
washings. Add 40 ml of dilute sulphuric acid R and titrate immediately with 0.1M silver nitrate. Deter-
mine the end-point potentiometrically (2.2.20), using a suitable electrode system such as silver-
mercurous sulphate.
1 ml of 0.1M silver nitrate is equivalent to 19.03 mg C
11
H
12
I
3
NO
2
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
22-11
Iotalamic Acid
corrected 1/01
I
I I
OOC
NHAc
CONHMe
C
11
H
9
I
3
N
2
O
4
614 2276-90-6
Iotalamic Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia [0751]. These
Preparations
Meglumine Iotalamate Injection
Sodium Iotalamate Injection
When iothalamic acid is prescribed or demanded, Iotalamic Acid shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Iotalamic acid contains not less than 98.5 per cent and not more than the equivalent of 101.0 per
cent of 3-(acetylamino)-2,4,6-tri-iodo-5-(methylcarbamoyl)benzoic acid, calculated with reference to
CHARACTERS
A white or almost white powder, slightly soluble in water and in alcohol, practically insoluble in
ether. It dissolves in dilute solutions of alkali hydroxides.
IDENTIFICATION
obtained with iotalamic acid CRS.
B. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel GF
254
plate R.
Test solution. Dissolve 50 mg of the substance to be examined in methanol R containing 3 per cent V/V
of ammonia R and dilute to 5 ml with the same solvent.
Reference solution. Dissolve 50 mg of iotalamic acid CRS in methanol R containing 3 per cent V/V of
ammonia R and dilute to 5 ml with the same solvent.
20 volumes of anhydrous formic acid R, 25 volumes of methyl ethyl ketone R and 60 volumes of
toluene R. Allow the plate to dry until the solvents have evaporated and examine in ultraviolet light at
C. Heat 50 mg gently in a small porcelain dish over a flame. Violet vapour is evolved.
TESTS
solvent. The solution is clear (2.2.1) and colourless (Method II, 2.2.2).
254
plate R.
Test solution. Dissolve 1.0 g of the substance to be examined in methanol R containing 3 per cent V/V
of ammonia R and dilute to 10 ml with the same solvent.
Reference solution (a). Dilute 1 ml of the test solution to 50 ml with water R. Dilute 1 ml of the
Reference solution (b). Dissolve 1 mg of iotalamic acid impurity A CRS in 5 ml of reference solution (a).
glacial acetic acid R, 1 volume of anhydrous formic acid R, 1 volume of methanol R, 5 volumes of ether R
and 10 volumes of methylene chloride R. Allow the plate to dry until the solvents have evaporated.
22-12
Examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with the test
solution, apart from the principal spot, is not more intense than the spot in the chromatogram
Halides Dissolve 0.55 g in a mixture of 4 ml of dilute sodium hydroxide solution R and 15 ml of
water R. Add 6 ml of dilute nitric acid R and filter. 15 ml of the filtrate complies with the limit test for
chlorides (2.4.4) (150 ppm, expressed as chloride).
Impurity A Not more than 0.05 per cent m/m determined by absorption spectrophotometry
(2.2.25).
Test solution. Transfer 0.500 g of the substance to be examined to a 50 ml volumetric flask, add
14 ml of water R, shake and add 1 ml of dilute sodium hydroxide solution R.
Reference solution. Prepare the reference solution by mixing 10.0 ml of a 8.5 g/l solution of sodium
hydroxide R containing 25 g/ml of iotalamic acid impurity A CRS with 5 ml of water R in a 50 ml
volumetric flask.
Blank solution. Transfer 14 ml of water R and 1 ml of dilute sodium hydroxide solution R to a 50 ml
volumetric flask.
In conducting the following steps, keep the flasks in iced water and protected as far as possible from light until
all of the reagents have been added.
Place all three of the flasks containing the test solution, the reference solution and the blank
solution in iced water, protected from light. Add 5 ml of a 5 g/l solution of sodium nitrite R and 12 ml
of dilute hydrochloric acid R. Shake gently and allow to stand for exactly 2 min after adding the
hydrochloric acid. Add 10 ml of a 20 g/l solution of ammonium sulphamate R. Allow to stand for
5 min shaking frequently (CAUTION: considerable pressure is produced), and add 0.15 ml of a 100 g/l
solution of -naphthol R in alcohol R. Shake and allow to stand for 5 min. Add 3.5 ml of buffer
solution pH 10.9 R, mix and dilute to 50.0 ml with water R. Concomitantly and within 20 min
determine the absorbance at 485 nm of the solutions obtained from the test solution and the
reference solution in 5 cm cells, using the blank as the compensation liquid.
Calculate the content of impurity A.
20 ml of 1M sodium hydroxide, add 10 ml of water R and adjust to pH 4.5 to 5.5 with acetic acid R.
Add 2.0 ml of 0.001M potassium iodide. Titrate with 0.001M silver nitrate using a silver indicator
electrode and an appropriate reference electrode. Subtract the volume of titrant corresponding to the
2.0 ml of 0.001M potassium iodide, determined by titrating a blank to which is added 2.0 ml of 0.001M
potassium iodide and use the residual value to calculate the iodide content.
Heavy metals (2.4.8). Dissolve 2.0 g in 4 ml of dilute sodium hydroxide solution R and dilute to 20 ml
with water R. 12 ml of this solution complies with limit test A for heavy metals (20 ppm). Prepare
the standard using lead standard solution (2 ppm Pb) R.
at 100C to 105C.
ASSAY
washings. Add 40 ml of dilute sulphuric acid R and titrate immediately with 0.1M silver nitrate.
Determine the end-point potentiometrically (2.2.20), using a suitable electrode system such as silver-
mercurous sulphate.
l ml of 0.1M silver nitrate is equivalent to 20.47 mg of C
11
H
9
I
3
N
2
O
4.
STORAGE
IMPURITIES
COOH
I I
I
NH
2
eHN
O
A. 3-amino-2,4,6-triiodo-5-(methylcarbamoyl)benzoic acid.
__________________________________________________________________________________________________________ Ph Eur
22-13
Ipecacuanha
Ipecacuanha complies with the requirements of the 3rd edition of the European Pharmacopoeia for Ipecacuanha
Root [0094]. These requirements are reproduced after the heading Definition below.
Action and use Expectorant; emetic.
Preparations
Prepared Ipecacuanha
Ipecacuanha Liquid Extract
When Ipecacuanha, Ipecacuanha Root or Powdered Ipecacuanha is prescribed or demanded,
Prepared Ipecacuanha shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ipecacuanha root consists of the fragmented and dried underground organs of Cephaelis ipecacuanha
(Brot.) A. Rich., known as Matto Grosso ipecacuanha, or of Cephaelis acuminata Karsten, known as
Costa Rica ipecacuanha, or of a mixture of both species. It contains not less than 2.0 per cent of total
alkaloids, calculated as emetine (C
29
H
40
N
2
O
4
; M
r
480.7) with reference to the dried drug. The
principal alkaloids are emetine and cephaline.
CHARACTERS
Ipecacuanha root has a slight odour.
IDENTIFICATION
A. C. ipecacuanha. The root occurs as somewhat tortuous pieces, from dark reddish-brown to very
dark brown, seldom more than 15 cm long or 6 mm thick, closely annulated externally, having
rounded ridges completely encircling the root; the fracture is short in the bark and splintery in the
wood. The transversely cut surface shows a wide greyish bark and a small uniformly dense wood. The
rhizome occurs as short lengths usually attached to roots, cylindrical, up to 2 mm in diameter, finely
wrinkled longitudinally and with pith occupying approximately one-sixth of the whole diameter.
C. acuminata. The root in general resembles the root of C. ipecacuanha, but differs in the following
particulars: it is often up to 9 mm thick; the external surface is greyish-brown or reddish-brown with
transverse ridges at intervals of usually 1 mm to 3 mm, the ridges being about 0.5 mm to 1 mm wide,
extending about half-way round the circumference and fading at the extremities into the general
surface level.
B. Reduce to a powder (355). The powder is light grey to yellowish-brown. Examine under a
microscope, using chloral hydrate solution R. The powder shows the following diagnostic characters:
parenchymatous cells, raphides of calcium oxalate up to 80 m in length either in bundles or
scattered throughout the powder; fragments of tracheids and vessels usually 10 m to 20 m in
diameter, with bordered pits; larger vessels and sclereids from the rhizome. Examine under a
microscope using a 50 per cent V/V solution of glycerol R. The powder shows simple or two- to eight-
compound starch granules contained in parenchymatous cells, the simple granules being up to 15 m
in diameter in C. ipecacuanha and up to 22 m in diameter in C. acuminata.
substance.
Test solution. To 0.1 g of the powdered drug (180) in a test-tube add 0.05 ml of concentrated
ammonia R and 5 ml of ether R and stir the mixture vigorously with a glass rod. Allow to stand for
30 min and filter.
Reference solution. Dissolve 2.5 mg of emetine dihydrochloride CRS and 3 mg of cephaline hydrochloride
mixture of 2 volumes of concentrated ammonia R, 15 volumes of methanol R, 18 volumes of ethyl
acetate R and 65 volumes of toluene R. Allow the plate to dry in air. Spray with a 5 g/l solution of
iodine R in alcohol R and heat at 60C for 10 min. Examine in daylight. The chromatograms obtained
with the test solution and with the reference solution show in the lower part a yellow zone corres-
ponding to emetine and below it a light brown zone corresponding to cephaline. Examine in
ultraviolet light at 365 nm. The zone corresponding to emetine shows an intense yellow fluorescence
and that corresponding to cephaline a light blue fluorescence. The chromatogram obtained with the
test solution shows also faint fluorescent zones.
With C. acuminata the principal zones in the chromatogram obtained with the test solution are
similar in position, fluorescence and size to the zones in the chromatogram obtained with the refer-
ence solution.
22-14
With C. ipecacuanha the only difference is that the zone corresponding to cephaline in the chromato-
gram obtained with the test solution is much smaller than the corresponding zone in the chromato-
gram obtained with the reference solution.
TESTS
ASSAY
To 7.5 g of the powdered drug (180) in a dry flask, add 100 ml of ether R and shake for 5 min. Add
5 ml of dilute ammonia R1, shake for 1 h, add 5 ml of water R and shake vigorously. Decant the ether
layer into a flask through a plug of cotton. Wash the residue in the flask with two quantities, each of
25 ml, of ether R, decanting each portion through the same plug of cotton. Combine the ether solu-
tions and eliminate the ether by distillation. Dissolve the residue in 2 ml of alcohol (90 per cent
V/V) R, evaporate the alcohol to dryness and heat at 100C for 5 min. Dissolve the residue in 5 ml of
previously neutralised alcohol (90 per cent V/V) R, warming on a water-bath, add 15.0 ml of 0.1M
hydrochloric acid and titrate the excess acid with 0.1M sodium hydroxide using 0.5 ml of methyl red
mixed solution R as indicator.
1 ml of 0.1M hydrochloric acid is equivalent to 24.03 mg of total alkaloids, calculated as emetine.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
22-15
Prepared Ipecacuanha
Prepared Ipecacuanha complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Expectorant; emetic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Prepared ipecacuanha is ipecacuanha root powder (180) adjusted, if necessary, by the addition of
powdered lactose or ipecacuanha root powder with a lower alkaloidal content to contain 1.9 per cent
to 2.1 per cent of total alkaloids, calculated as emetine (C
29
H
40
N
2
O
4
; M
r
480.7) with reference to the
dried drug.
CHARACTERS
It presents the characters of the powder described in the monograph on Ipecacuanha root (94).
IDENTIFICATION
It complies with identification tests B, C and D in the monograph on Ipecacuanha root (94). Exam-
ined in glycerol (85 per cent) R, it may be seen to contain lactose crystals.
TESTS
It complies with the tests for foreign matter, for total ash and for ash insoluble in hydrochloric acid
prescribed in the monograph on Ipecacuanha root (94) and with the following additional test.
100C to 105C.
ASSAY
Carry out the assay prescribed in the monograph on Ipecacuanha root (94).
1 ml of 0.1M hydrochloric acid is equivalent to 24.03 mg of total alkaloids, calculated as emetine.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
22-16
Ipratropium Bromide
and epimer at C*
N
H Ph
O
OH
O
H
Me
Pr
i
+
Br
*
C
20
H
30
BrNO
3
,H
2
O 430.4 66985-17-9
Ipratropium Bromide complies with the requirements of the 3rd edition of the European Pharmacopoeia [0919].
Action and use Anticholinergic; bronchodilator.
Preparation
Ipratropium Pressurised Inhalation
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ipratropium bromide contains not less than 99.0 per cent and not more than the equivalent of
100.5 per cent of (1R,3r,5S,8r)-3-[(RS)-(3-hydroxy-2-phenylpropanoyl)oxy]-8-methyl-8-(1-
methylethyl)-8-azoniabicyclo[3.2.1]octane bromide, calculated with reference to the anhydrous
substance.
CHARACTERS
A white or almost white, crystalline powder, soluble in water, freely soluble in methanol, slightly
soluble in alcohol.
IDENTIFICATION
obtained with ipratropium bromide CRS.
substance.
Reference solution (a). Dissolve 10 mg of ipratropium bromide CRS in 2 ml of methanol R.
Reference solution (b). Dissolve 5 mg of methylatropine bromide CRS in 1 ml of reference solution (a).
of 2.5 volumes of anhydrous formic acid R, 7.5 volumes of water R, 45 volumes of alcohol R and 45
volumes of methylene chloride R. Allow the plate to dry in air and spray with potassium iodobismuthate
solution R. The principal spot in the chromatogram obtained with the test solution is similar in posi-
tion, colour and size to the principal spot in the chromatogram obtained with reference solution (a).
The test is not valid unless the chromatogram obtained with reference solution (b) shows two clearly
separated principal spots.
C. To 5 ml of solution S (see Tests), add 2 ml of dilute sodium hydroxide solution R. No precipitate is
formed.
D. To about 1 mg add 0.2 ml of nitric acid R and evaporate to dryness on a water-bath. Dissolve the
residue in 2 ml of acetone R and add 0.1 ml of a 30 g/l solution of potassium hydroxide R in methanol R.
A violet colour develops.
22-17
TESTS
solution GY
7
(Method II, 2.2.2).
Optical rotation (2.2.7). The angle of optical rotation, measured on solution S, is 0.10 to +0.10.
Reference solution (a). Dissolve 25 mg of 8s-ipratropium bromide CRS in 200 ml of the mobile phase
(solution A). Dilute 1 ml of the solution to 100 ml with the mobile phase.
Reference solution (b). Mix 1 volume of the test solution and 2 volumes of solution A.
as mobile phase at a flow rate of 2 ml per minute a solution containing 1 g of sodium
methanesulphonate R in a mixture of 120 ml of acetonitrile R and 1000 ml of 0.05M phosphoric
acid,
Inject 20 l of each solution. Continue the chromatography for twice the retention time of the
principal peak in the chromatogram obtained with the test solution. In the chromatogram obtained
with the test solution: the area of any peak corresponding to 8s-ipratropium is not greater than the
area of the peak in the chromatogram obtained with reference solution (a) (0.5 per cent); the area of
any peak, apart from the principal peak and any peak corresponding to 8s-ipratropium, is not greater
than half the area of the peak in the chromatogram obtained with reference solution (a) (0.25 per
cent). The test is not valid unless: in the chromatogram obtained with reference solution (b), the
resolution between the peaks corresponding to ipratropium and 8s-ipratropium is at least 1.5; in the
chromatogram obtained with the test solution, the symmetry factor of the principal peak is less than
2.2; in the chromatogram obtained with reference solution (a), the signal- to-noise ratio of the princi-
pal peak is at least five.
Apo-ipratropium Not more than 0.5 per cent. Dissolve 0.14 g in 0.01M hydrochloric acid and dilute
to 100 ml with the same acid. Measure the absorbance (2.2.25) of the solution at 246 nm (A
246
) and
263 nm (A
263
). Calculate the percentage content of apo-ipratropium from the expression:
A
A
246
263
0 863 100
_
,
.
tion of water.
ASSAY
Dissolve 0.350 g in 50 ml of water R and add 3 ml of dilute nitric acid R. Titrate with 0.1M silver
nitrate, determining the end-point potentiometrically (2.2.20).
20
H
30
BrNO
3
.
IMPURITIES
and epimer at C*
*
N
H Ph
O
OH
O
H
Pr
i
Me
A. (8s)-ipratropium,
22-18
N
O
CH
2
O
H
Ph
Pr
i
Me
B. apo-ipratropium.
__________________________________________________________________________________________________________ Ph Eur
22-19
Isoaminile
NMe
2
NC
H Me
Me Me
mixture of 4 isomers
* *
C
16
H
24
N
2
244.4 77-51-0
Definition Isoaminile is all-rac-4-dimethylamino-2-isopropyl-2-phenylpentanonitrile. It contains
16
H
24
N
2
.
Characteristics A colourless or slightly yellow, oily liquid; odour, characteristic of amines.
Practically insoluble in water; freely soluble in acetone, in chloroform, in ethanol (96%), in ether and
in methanol. It dissolves in dilute acids.
spectrum of isoaminile (RS 194).
Clarity of solution An 11.0% v/v solution in a 9.0% w/v solution of citric acid is not more opalescent
than reference suspension II, Appendix IV A.
Related substances Carry out the method for gas chromatography, Appendix III B, injecting 0.5 l
of the substance being examined warmed to about 50, with stirring.
The chromatographic procedure may be carried out using a glass column (2 m 4 mm) packed
with silanised diatomaceous earth (80 to 100 mesh) (Chromosorb WAW is suitable) coated with 2%
w/w of potassium hydroxide and 10% w/w of silicone grease (Apiezon L is suitable) and maintained at
205 with both the inlet port and the detector at 250. Use helium as the carrier gas at a flow rate of
30 ml per minute.
In the chromatogram obtained the area of any secondary peak is not more than 0.25% and the sum
of the areas of any secondary peaks is not greater than 0.5% by normalisation.
Assay Carry out Method I for non-aqueous titration, Appendix VIII A, using 0.4 g dissolved in 50 ml
of anhydrous acetic acid and determining the end point potentiometrically. Each ml of 0.1M perchloric
16
H
24
N
2
.
Action and use Antitussive.
IMPURITIES
N
A. benzyl cyanide,
H
3
C
NMe
2
Cl
B. 1-dimethylamino-2-chloropropane,
CH
3
CN
C. 3-methyl-2-phenylbutyronitrile,
NMe
Me
Me Me
NH
D. 3-isopropyl-1,5-dimethyl-3-phenylpyrrolidin-2-imine,
22-20
NMe
2
Me Me
Me
E. dimethyl(1,4-dimethyl-3-phenylpentyl)amine.
22-21
Isoconazole
and enantiomer
N
N
O
Cl
Cl
Cl
Cl
H
C
18
H
14
Cl
4
N
2
O 416.1 27523-40-6
Isoconazole complies with the requirements of the 3rd edition of the European Pharmacopoeia [1018]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Isoconazole contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of (RS)-1-[2-(2,6-dichlorobenzyloxy)-2-(2,4-dichlorophenyl)ethyl]-1H-imidazole, calculated with
CHARACTERS
A white or almost white powder, practically insoluble in water, very soluble in methanol, freely
soluble in alcohol.
IDENTIFICATION
obtained with isoconazole CRS. Examine the substances prepared as discs.
coating substance.
the same solvent.
Reference solution (a). Dissolve 30 mg of isoconazole CRS in methanol R and dilute to 5 ml with the
same solvent.
Reference solution (b). Dissolve 30 mg of isoconazole CRS and 30 mg of econazole nitrate CRS in
is similar in position, colour and size to the principal spot in the chromatogram obtained with refer-
D. To about 30 mg in a porcelain crucible add 0.3 g of anhydrous sodium carbonate R. Heat over an
open flame for 10 min. Allow to cool. Take up the residue with 5 ml of dilute nitric acid R and filter.
To 1 ml of the filtrate add 1 ml of water R. The solution gives reaction (a) of chlorides (2.3.1).
TESTS
Appearance of solution Solution S is clear (2.2.1) and is not more intensely coloured than refer-
ence solution Y
6
(Method II, 2.2.2).
+0.10.
22-22
Test solution. Dissolve 0.100 g of the substance to be examined in 3.2 ml of methanol R. Add 3.0 ml
of acetonitrile R and dilute to 10.0 ml with a solution of ammonium acetate R (6.0 g in 380 ml of
water R).
Reference solution (a). Dissolve 2.5 mg of isoconazole CRS and 2.5 mg of econazole nitrate CRS in the
as mobile phase at a flow rate of 2 ml per minute a solution of 6.0 g of ammonium acetate R in a
mixture of 300 ml of acetonitrile R, 320 ml of methanol R and 380 ml of water R,
recorder.
tions, the retention times are: econazole, about 10 min; isoconazole, about 14 min. The test is not
valid unless the resolution between the peaks corresponding to econazole and isoconazole is at least
5.0. If necessary, adjust the composition of the mobile phase.
any peaks due to the solvent and nitrate ion and any peak with an area less than 0.2 times that of the
100C to 105C for 2 h.
ASSAY
ethyl ketone R. Using 0.2 ml of naphtholbenzein solution R as indicator, titrate with 0.1M perchloric acid
until the colour changes from orange-yellow to green.
18
H
14
Cl
4
N
2
O.
STORAGE
IMPURITIES
A. (RS)-1-[2-(2,6-dichlorobenzyloxy)-2-(2,4-dichlorophenyl)ethyl]-1H-imidazole nitrate
(isoconazole nitrate),
and enantiomer
N
N
Cl
Cl
H
HO
,HNO
3
B. (RS)-1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethanol,
and enantiomer
H
2
N
O
Cl
Cl
Cl
Cl
H
C. (RS)-2-(2,6-dichlorobenzyloxy)-2-(2,4-dichlorophenyl)ethylamine,
22-23
and enantiomer
N
N
O
Cl
Cl Cl
H
Cl
D. (RS)-1-[2-(2,4-dichlorobenzyloxy)-2-(2,4-dichlorophenyl)ethyl]-1H-imidazole.
__________________________________________________________________________________________________________ Ph Eur
22-24
Isoconazole Nitrate
and enantiomer
N
N
O
Cl
Cl
Cl
Cl
H
,HNO
3
C
18
H
14
Cl
4
N
2
O,HNO
3
479.1 40036-10-0
Isoconazole Nitrate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1017].
Preparation
Isoconazole Pessaries
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Isoconazole nitrate contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of (RS)-1-[2-(2,6-dichlorobenzyloxy)-2-(2,4-dichlorophenyl)ethyl]-1H-imidazole
nitrate, calculated with reference to the dried substance.
CHARACTERS
A white or almost white powder, very slightly soluble in water, soluble in methanol, slightly soluble in
alcohol.
IDENTIFICATION
obtained with isoconazole nitrate CRS. Examine the substances prepared as discs.
coating substance.
the same solvent.
Reference solution (a). Dissolve 30 mg of isoconazole nitrate CRS in methanol R and dilute to 5 ml with
the same solvent.
Reference solution (b). Dissolve 30 mg of isoconazole nitrate CRS and 30 mg of econazole nitrate CRS in
is similar in position, colour and size to the principal spot in the chromatogram obtained with refer-
D. It gives the reaction of nitrates (2.3.1).
TESTS
Appearance of solution Solution S is clear (2.2.1) and is not more intensely coloured than refer-
ence solution Y
7
(Method II, 2.2.2).
+0.10.
22-25
Reference solution (a). Dissolve 2.5 mg of isoconazole nitrate CRS and 2.5 mg of econazole nitrate CRS
in the mobile phase and dilute to 100.0 ml with the mobile phase.
as mobile phase at a flow rate of 2 ml per minute a solution of 6.0 g of ammonium acetate R in a
mixture of 300 ml of acetonitrile R, 320 ml of methanol R and 380 ml of water R,
tions, the retention times are: econazole, about 10 min and isoconazole, about 14 min. The test is
not valid unless the resolution between the peaks due to econazole and isoconazole is not less than
5.0. If necessary, adjust the composition of the mobile phase.
any peaks due to the solvent and to nitrate ion and any peak with an area less than 0.2 times that of
100C to 105C for 2 h.
ASSAY
ethyl ketone R and titrate with 0.1M perchloric acid. Determine the end-point potentiometrically
(2.2.20).
18
H
15
Cl
4
N
3
O
4
.
STORAGE
IMPURITIES
and enantiomer
N
N
Cl
Cl
H
HO
A. (RS)-1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethanol,
and enantiomer
H
2
N
O
Cl
Cl
Cl
Cl
H
B. (RS)-2-(2,6-dichlorobenzyloxy)-2-(2,4-dichlorophenyl)ethylamine,
and enantiomer
N
N
O
Cl
Cl Cl
H
Cl
D. (RS)-1-[2-(2,4-dichlorobenzyloxy)-2-(2,4-dichlorophenyl)ethyl]-1H-imidazole.
__________________________________________________________________________________________________________ Ph Eur
22-26
Isoleucine
COOH
NH
2
H
Me H
H
3
C
C
6
H
13
NO
2
131.2 73-32-5
Isoleucine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0770]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Isoleucine contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of (2S,3S)-
2-amino-3-methylpentanoic acid, calculated with reference to the dried substance.
PRODUCTION
When Isoleucine is produced by a process involving fermentation steps, it complies with the require-
ments of the monograph on Products of Fermentation (1468).
CHARACTERS
White or almost white, crystalline powder or flakes, sparingly soluble in water, slightly soluble in
alcohol, practically insoluble in ether. It dissolves in dilute mineral acids and in dilute solutions of
alkali hydroxides.
IDENTIFICATION
B. Dissolve 0.5 g in water R and dilute to 25 ml with the same solvent. The solution is
dextrorotatory.
obtained with isoleucine CRS. Examine the substances prepared as discs.
TESTS
6
(Method II, 2.2.2).
substance.
gel plate R.
Test solution (a). Dissolve 0.10 g of the substance to be examined in 0.1M hydrochloric acid and dilute
Reference solution (a). Dissolve 10 mg of isoleucine CRS in 0.1M hydrochloric acid and dilute to 50 ml
with the same acid.
Reference solution (c). Dissolve 10 mg of isoleucine CRS and 10 mg of valine CRS in 0.1M hydrochloric
acid and dilute to 25 ml with the same acid.
20 volumes of glacial acetic acid R, 20 volumes of water R and 60 volumes of butanol R. Allow the
plate to dry in air, spray with ninhydrin solution R and heat at 100C to 105C for 15 min. Any spot in
22-27
the chromatogram obtained with test solution (a), apart from the principal spot, is not more
intense than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent). The
test is not valid unless the chromatogram obtained with reference solution (c) shows two clearly
separated spots.
Chlorides (2.4.4). Dissolve 0.25 g in water R and dilute to 15 ml with the same solvent. The solu-
tion complies with the limit test for chlorides (200 ppm).
watch-glass and dissolve or suspend in 0.5 ml of water R. To the solution or the suspension add
0.30 g of heavy magnesium oxide R. Briefly triturate with a glass rod. Immediately close the cell by
putting the two watch-glasses together. Heat at 40C for 15 min. The litmus paper is not more
intensely blue coloured than a standard prepared at the same time and in the same manner using
0.1 ml of ammonium standard solution (100 ppm NH
4
) R, 0.5 ml of water R and 0.30 g of heavy
magnesium oxide R (200 ppm).
100C to 105C.
ASSAY
Dissolve 0.100 g in 3 ml of anhydrous formic acid R. Add 30 ml of anhydrous acetic acid R. Using
0.1 ml of naphtholbenzein solution R as indicator, titrate with 0.1M perchloric acid until the colour
6
H
13
NO
2
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
22-28
Isomalt
1/01
O
OH
OH
HO O
OH
OH
OH H
H HO
H OH
OH
H
O
OH
OH
HO O
OH
OH
OH H
H HO
HO H
H
OH
+
C
12
H
24
O
11
344.3
C
12
H
24
O
11
, 2H
2
O 380.3
Isomalt complies with the requirements of the 3rd edition of the European Pharmacopoeia [1531]. These
Action and use Sweetning agent.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Isomalt contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent of a
mixture of 6-O--D-glucopyranosyl-D-glucitol (6-O--D-glucopyranosyl-D-sorbitol; 1,6-GPS) and
1-O--D-glucopyranosyl-D-mannitol (1,1-GPM) and neither of the two components is less than
3.0 per cent, calculated with reference to the anhydrous substance. The percentage content of 1,6-
GPS and 1,1-GPM is stated on the label.
CHARACTERS
A white or almost white powder or granules, freely soluble in water, very slightly soluble in ethanol.
IDENTIFICATION
A. It complies with the assay.
B. Examine by thin layer chromatography (2.2.27) using a TLC silica gel F
254
plate R.
same solvent.
Reference solution. Dissolve 50 mg of isomalt CRS in water R and dilute to 10 ml with the same
solvent.
Apply to the plate 1 l of each solution and thoroughly dry the starting points in warm air. Develop
over a path of 10 cm using a mixture of 5 volumes of acetic acid R, 5 volumes of propionic acid R, 10
volumes of water R, 50 volumes of ethyl acetate R and 50 volumes of pyridine R. Dry the plate in a
current of warm air and dip for 3 s in a 1 g/l solution of sodium periodate R. Dry the plate in a current
of hot air. Dip the plate for 3 s in a mixture of 1 volume of acetic acid R, 1 volume of anisaldehyde R, 5
volumes of sulphuric acid R and 90 volumes of ethanol R. Dry the plate in a current of hot air until
coloured spots become visible. The background colour may be brightened in warm steam. Examine
in daylight. The chromatogram obtained with the reference solution shows two blue-grey spots
with R
f
values of about 0.13 (1,6-GPS) and 0.16 (1,1-GPM). The chromatogram obtained with the
test solution show spots similar in position and colour to the spots in the chromatogram obtained
C. To 3 ml of a freshly prepared 100 g/l solution of pyrocatechol R add 6 ml of sulphuric acid R while
cooling in iced water. To 3 ml of the cooled mixture add 0.3 ml of a 100 g/l solution of the substance
to be examined. Heat gently over a naked flame for about 30 s. A pink colour develops.
22-29
TESTS
Conductivity (2.2.38). Not more than 20 Scm
-1
. Dissolve 20.0 g in carbon dioxide-free water R
prepared from distilled water R and dilute to 100.0 ml with the same solvent. Measure the
conductivity of the solution while gently stirring with a magnetic stirrer at a temperature of 20C.
of cupric-citric solution R and a few glass beads. Heat so that the boiling begins after 4 min and
maintain boiling for 3 min. Cool rapidly and add 100 ml of a 2.4 per cent V/V solution of glacial
acetic acid R and 20.0 ml of 0.025M iodine. With continuous shaking, add 25 ml of a mixture of 6
volumes of hydrochloric acid R and 94 volumes of water R and, when the precipitate has dissolved,
titrate the excess of iodine with 0.05M sodium thiosulphate using 1 ml of starch solution R as indicator,
added towards the end of the titration. Not less than 12.8 ml of 0.05M sodium thiosulphate is required
(0.3 per cent, calculated as glucose).
Related substances Examine the chromatograms obtained in the assay. In the chromatogram
obtained with the test solution: the area of any peak corresponding to mannitol or sorbitol is not
greater than half the area of the corresponding peak in the chromatogram obtained with the reference
solution (0.5 per cent); the area of any peak, apart from the two principal peaks corresponding to
1,1-GPM and 1,6-GPS and the peaks corresponding to mannitol and sorbitol, is not greater than
half the area of the peak due to sorbitol in the chromatogram obtained with the reference solution
(0.5 per cent); the sum of the area of all the peaks apart from the peaks corresponding to 1,1-GPM
and 1,6-GPS is not greater than twice the area of the peak due to sorbitol in the chromatogram
obtained with the reference solution (2 per cent). Disregard any peak with an area less than 0.1 times
that of the peak due to sorbitol in the chromatogram obtained with the reference solution (0.1 per
cent).
Nickel (2.4.15). It complies with the limit test for nickel in polyols (1 ppm).
of water. As solvent, use a mixture of 20 ml of anhydrous methanol R and 20 ml of formamide R at
50C 5C.
ASSAY
Test solution. Dissolve 1.00 g of the substance to be examined in 20 ml of water R and dilute to
Reference solution. In a 50.0 ml flask, dissolve 1.00 g of isomalt CRS in 20 ml of water R. In another
flask dissolve 100 mg of sorbitol CRS and 100 mg of mannitol CRS in 5 ml of water R and dilute to
10.0 ml with the same solvent. Mix 1.0 ml of this sorbitol/mannitol solution with the 20 ml solution
of isomalt CRS and dilute to 50.0 ml with water R.
a stainless steel precolumn 30 mm long and 4.6 mm in internal diameter and a stainless steel
column 0.3 m long and 7.8 mm in internal diameter, both packed with a strong cation-exchange
resin (calcium form) R (9 m) and maintained at 80 1C,
as mobile phase at a flow rate of 0.5 ml/min degassed water R,
as detector a differential refractometer maintained at a constant temperature.
Inject 20 l of each solution and continue the chromatography until sorbitol is completely eluted
(about 25 min). When the chromatograms are recorded in the prescribed conditions, the retention
time of 1,1-GPM is about 12.3 min and the relative retentions to 1,1-GPM are: 1,6-GPS about 1.2,
mannitol about 1.6, sorbitol about 2.0 and isomaltulose about 0.8.
Calculate the percentage content of isomalt (1,1-GPM and 1,6-GPS) from the areas of the peaks
of 1,1-GPM and 1,6-GPS and the declared content of 1,1-GPM and 1,6-GPS in isomalt CRS.
LABELLING
The label states the percentage content of 1,6-GPS and 1,1-GPM.
IMPURITIES
O
OH
OH
HO O
OH
HO
O
OH
OH
OH
A. 6-O--D-glucopyranosyl--d-arabino-2-hexulofuranose (isomaltulose),
B. D-mannitol,
22-30
C. sorbitol (D-glucitol),
O
OH
OH
O O
OH
HO
O
OH
OH
OH HO
D. trehalulose.
__________________________________________________________________________________________________________ Ph Eur
22-31
Isometheptene Mucate
Me NHMe
Me
H Me
and enantiomer
,
COOH
OH H
H HO
HO H
H OH
COOH
(C
9
H
19
N)
2
,C
6
H
10
O
8
492.7 7492-31-1
Definition Isometheptene Mucate is (RS)-1,5-dimethylhex-4-enyl(methyl)amine galacto-2,3,4,5-
tetrahydroxyadipate. It contains not less than 98.5% and not more than 100.5% of
(C
9
H
19
N)
2
,C
6
H
10
O
8
Very soluble in water; slightly soluble in absolute ethanol; very slightly soluble in chloroform;
Identification
A. Dissolve 0.2 g in 10 ml of water, make the solution alkaline with 2M sodium hydroxide, extract with
20 ml of dichloromethane, dry the dichloromethane layer over anhydrous sodium sulphate, filter and
evaporate to dryness at a pressure of 2 kPa. The infrared absorption spectrum of the residue, Appendix
II A, is concordant with the reference spectrum of isometheptene (RS 195).
B. B. Prepare a 5.0% w/v solution of the substance being examined in carbon-dioxide free water (solu-
tion A) and acidify 10 ml with 6M hydrochloric acid, scratch the side of the tube with a glass rod to
initiate crystallisation, filter, wash the precipitate with water and dry at 105 for 10 minutes. The
infrared absorption spectrum of the residue, Appendix II A, is concordant with the reference spectrum of
mucic acid (RS 377).
Acidity pH of solution A, 5.4 to 6.6, Appendix V L.
Clarity and colour of solution Solution A is clear, Appendix IV A, and colourless, Appendix IV B,
Method II.
Related substances Dissolve 20 mg of linalool (internal standard) in sufficient dichloromethane to
produce 100 ml (solution B). Carry out the method for gas chromatography, Appendix III B, using the
following solutions. For solution (1) add 1 ml of 5M sodium hydroxide to 5 ml of a 0.050% w/v solu-
tion of the substance being examined, extract with 10 ml of solution B followed by 10 ml of
dichloromethane, combine the dichloromethane layers and filter through anhydrous sodium sulphate.
For solution (2) dissolve 0.5 g of the substance being examined in 5 ml of water, add 1 ml of 5M
sodium hydroxide, extract with two 10-ml quantities of dichloromethane, combine the dichloromethane
layers and filter through anhydrous sodium sulphate. Prepare solution (3) in the same manner as solu-
tion (2) but using 10 ml of solution B and 10 ml of dichloromethane in place of the two 10-ml
quantities of dichloromethane. For solution (4) dissolve 10 mg of 1,5-dimethylhexyl(methyl)amine
BPCRS and 10 mg of 2-methyl-6-methylaminoheptan-2-ol BPCRS in 50 ml of dichloromethane. For
solution (5) dissolve 5 mg of the substance being examined and 5 mg of linalool in 25 ml of solution
(4) and filter, if necessary.
with acid-washed, silanised diatomaceous support (100 to 120 mesh) (Chromosorb W-HP is suitable)
coated with 10% w/w of polyethylene glycol (Carbowax 20M is suitable) and 5% w/w of potassium
hydroxide at a temperature increased from 80 to 140 at a rate of 4 per minute and then maintained
at 140 for 10 minutes or the time required for the emergence of the peak due to 2-methyl-6-methyl-
aminoheptan-2-ol and with the inlet port at 190, the detector at 200 and a flow rate of 40 ml per
minute for the carrier gas.
The test is not valid unless the chromatogram obtained with solution (5) resembles the reference
chromatogram provided with 1,5-dimethylhexyl(methyl)amine BPCRS and 2-methyl-6-
methylaminoheptan-2-ol BPCRS.
Calculate the ratio (a) of the area of the peak due to isometheptene to that of the peak due to the
internal standard in the chromatogram obtained with solution (1). In the chromatogram obtained
with solution (3) the ratio of the area of any peak corresponding to 1,5-dimethylhexyl(methyl)amine
to the area of the peak due to the internal standard is not greater than 2a, the ratio of the area of any
other secondary peak to the area of the peak due to the internal standard is not greater than a and the
ratio of the sum of the areas of any secondary peaks to the area of the peak due to the internal
standard is not greater than 3a.
22-32
Loss on drying When dried to constant weight over phosphorus pentoxide at 60 at a pressure not
Assay Dissolve 0.2 g in 25 ml of water and add 20 ml of 0.0167M potassium bromate VS, 10 ml of a
10% w/v solution of potassium bromide and 8 ml of hydrochloric acid. Allow to stand for 5 minutes in a
stoppered flask, add 10 ml of dilute potassium iodide solution, allow to stand for 5 minutes and titrate
the resulting solution with 0.1M sodium thiosulphate VS using starch solution as indicator. Repeat the
procedure using 25 ml of water in place of the solution of the substance being examined. The differ-
ence between the titrations represents the amount of sodium thiosulphate required. Each ml of
0.0167M potassium bromate VS is equivalent to 12.32 mg of (C
9
H
19
N)
2
, C
6
H
10
O
8
.
Storage Isometheptene Mucate should be kept in an airtight container and protected from light.
IMPURITIES
Me NHMe
Me
A. 1,5-dimethylhexyl(methyl)amine,
Me OH
Me
B. 2-methylheptan-6-ol,
Me OH
Me
C. 2-methylhept-2-en-6-ol,
Me O
Me
D. 2-methylhept-2-en-6-one,
Me NHMe
Me OH
E. 2-methyl-6-methylaminoheptan-2-ol.
22-33
Isoniazid
N
N
NH
2
O
H
C
6
H
7
N
3
O 137.1 54-85-3
Isoniazid complies with the requirements of the 3rd edition of the European Pharmacopoeia [0146]. These
Preparations
Isoniazid Injection
Isoniazid Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Isoniazid contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of
isonicotinonydrazide, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder or colourless crystals, freely soluble in water, sparingly soluble in alcohol,
very slightly soluble in ether.
IDENTIFICATION
obtained with isoniazid CRS.
C. Dissolve 0.1 g in 2 ml of water R and add 10 ml of a warm 10 g/l solution of vanillin R. Allow to
stand and scratch the wall of the test tube with a glass rod. A yellow precipitate is formed, which,
after recrystallisation from 5 ml of alcohol (70 per cent V/V) R and drying at 100C to 105C, melts
(2.2.14) at 226C to 231C.
TESTS
solution BY
7
(Method II, 2.2.2).
Hydrazine and related substances Examine by thin-layer chromatography (2.2.27), using silica gel
GF
254
acetone R and water R and dilute to 10.0 ml with the same mixture of solvents.
Reference solution. Dissolve 50.0 mg of hydrazine sulphate R in 50 ml of water R and dilute to 100.0 ml
with acetone R. To 10.0 ml of this solution add 0.2 ml of the test solution and dilute to 100.0 ml with
a mixture of equal volumes of acetone R and water R.
of 10 volumes of water R, 20 volumes of acetone R, 20 volumes of methanol R and 50 volumes of ethyl
acetate R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any spot in the
the spot in the chromatogram obtained with the reference solution (0.2 per cent). Spray the plate
with dimethylaminobenzaldehyde solution R1. Examine in daylight. An additional spot, corresponding
to hydrazine, appears in the chromatogram obtained with the reference solution. Any corresponding
spot in the chromatogram obtained with the test solution is not more intense than the spot corres-
ponding to hydrazine in the chromatogram obtained with the reference solution (0.05 per cent).
22-34
at 100C to 105C.
ASSAY
Dissolve 0.250 g in water R and dilute to 100.0 ml with the same solvent. To 20.0 ml of the solution
add 100 ml of water R, 20 ml of hydrochloric acid R, 0.2 g of potassium bromide R and 0.05 ml of
methyl red solution R. Titrate dropwise with 0.0167M potassium bromate, shaking continuously, until
the red colour disappears.
6
H
7
N
3
O.
__________________________________________________________________________________________________________ Ph Eur
22-35
Isoprenaline Hydrochloride
OH H
HO
HO
NHPr
i
and enantiomer
,HCl
C
11
H
17
NO
3
,HCl 247.7 51-30-9
Isoprenaline Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Sympathomimetic; beta-adrenoceptor agonist.
Preparation
Isoprenaline Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Isoprenaline hydrochloride contains not less than 98.0 per cent and not more than the equivalent of
101.5 per cent of (RS)-1-(3,4-dihydroxyphenyl)-2-(1-methylethyl)aminoethanol hydrochloride,
CHARACTERS
A white or almost white, crystalline powder, freely soluble in water, sparingly soluble in alcohol,
IDENTIFICATION
A. Melting point (2.2.14): 165C to 170C with decomposition.
obtained with isoprenaline hydrochloride CRS. Examine the substances as discs.
C. It complies with the test for optical rotation (see Tests).
D. To 0.1 ml of solution S (see Tests) add 0.05 ml of ferric chloride solution R1 and 0.9 ml of water R.
A green colour is produced. Add dropwise sodium hydrogen carbonate solution R. The colour becomes
blue then red.
E. To 0.5 ml of solution S add 1.5 ml of water R. The solution gives reaction (a) of chlorides (2.3.1).
TESTS
solution B
7
or BY
7
(Method II, 2.2.2).
pH (2.2.3). The pH of a mixture of 5 ml of solution S and 5 ml of carbon dioxide-free water R is 4.3 to
5.5.
+0.10.
Reference solution (a). Dissolve 2.5 mg of isoprenaline hydrochloride CRS in the mobile phase and dilute
Reference solution (b). Dissolve 2.5 mg of orciprenaline sulphate CRS in the mobile phase and dilute to
Reference solution (c). To 1.0 ml of test solution (b) add 1.0 ml of reference solution (b) and dilute to
22-36
volumes of a 11.5 g/l solution of phosphoric acid R,
a loop injector.
Adjust the retention time of the peak at about 3 min by varying the concentration of methanol in the
mobile phase. Inject 20 l of test solution (a) and 20 l of reference solution (c). Continue the
chromatography of test solution (a) for seven times the retention time of isoprenaline. The test is not
valid unless in the chromatogram obtained with reference solution (c): the resolution between the
two principal peaks is at least 3; the peak due to isoprenaline has a signal-to-noise ratio of at least 3.
In the chromatogram obtained with test solution (a): the area of any peak, apart from the principal
solution (a) (0.5 per cent) and the sum of the areas of such peaks is not greater than twice the area of
the principal peak in the chromatogram obtained with reference solution (a) (1 per cent). Disregard
15C to 25C for 4 h.
ASSAY
Dissolve 0.150 g in 10 ml of anhydrous formic acid R and add 50 ml of acetic anhydride R. Titrate
11
H
18
ClNO
3
.
STORAGE
IMPURITIES
NHPr
i
HO
OH
O
A. 1-(3,4-dihydroxyphenyl)-2-(1-methylethyl)ethanone.
__________________________________________________________________________________________________________ Ph Eur
22-37
Isoprenaline Sulphate
OH H
HO
HO
NHPr
i
and enantiomer
,H
2
SO
4
2
C
11
H
17
NO
3
,H
2
SO
4
,2H
2
O 556.6 6700-39-6
Isoprenaline Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Sympathomimetic; beta-adrenoceptor agonist.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Isoprenaline sulphate contains not less than 98.0 per cent and not more than the equivalent of
102.0 per cent of bis[(RS)-1-(3,4-dihydroxyphenyl)-2-isopropylaminoethanol] sulphate, calculated
CHARACTERS
A white or almost white, crystalline powder, freely soluble in water, very slightly soluble in alcohol.
IDENTIFICATION
A. Dissolve 0.5 g of the substance to be examined in 1.5 ml of water R and add 3.5 ml of
2-propanol R. Scratch the wall of the tube with a glass rod to initiate crystallisation. Collect the
crystals and dry in vacuo at 60C over diphosphorus pentoxide R. Repeat the operations with isoprenaline
sulphate CRS. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the
spectrum obtained with isoprenaline sulphate CRS.
B. To 0.1 ml of solution S (see Tests) add 0.9 ml of water R and 0.05 ml of ferric chloride solution R1.
A green colour is produced. Add dropwise sodium hydrogen carbonate solution R. The colour becomes
blue and then red.
C. Dilute 1 ml of solution S to 10 ml with water R and add 0.25 ml of silver nitrate solution R1. A
shining, grey, fine precipitate is formed within 10 min and the solution becomes pink.
D. Solution S gives reaction (a) of sulphates (2.3.1).
TESTS
Use within 2 h of preparation.
solution Y
6
(Method II, 2.2.2).
pH (2.2.3). Dilute 5 ml of solution S to 10 ml with carbon dioxide-free water R. The pH of the solu-
tion is 4.3 to 5.5.
Isoprenalone Dissolve 0.20 g in 0.005M sulphuric acid and dilute to 100.0 ml with the same solvent.
The absorbance (2.2.25) measured at 310 nm is not greater than 0.20.
tion of water.
ASSAY
Dissolve 0.400 g in 20 ml of anhydrous acetic acid R, warming gently if necessary and add 20 ml of
methyl isobutyl ketone R. Titrate with 0.1M perchloric acid, determining the end-point potentio-
22
H
36
N
2
O
10
S.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
22-38
Isopropyl Alcohol
H
3
C CH
3
OH H
C
3
H
8
O 60.1 67-63-0
Isopropyl Alcohol complies with the requirements of the 3rd edition of the European Pharmacopoeia [0970].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Isopropyl alcohol is propan-2-ol.
CHARACTERS
A clear, colourless liquid, miscible with water and with alcohol.
IDENTIFICATION
C. To 1 ml add 2 ml of potassium dichromate solution R and 1 ml of dilute sulphuric acid R. Boil.
Vapour is produced which changes the colour of a piece of filter paper impregnated with
nitrobenzaldehyde solution R to green. Moisten the filter paper with dilute hydrochloric acid R. The
colour changes to blue.
TESTS
Appearance The substance to be examined is clear (2.2.1) and colourless (Method II, 2.2.2). Dilute
1 ml to 20 ml with water R. After 5 min, the solution is clear (2.2.1).
Acidity or alkalinity Gently boil 25 ml for 5 min. Add 25 ml of carbon dioxide-free water R and allow
to cool protected from carbon dioxide in the air. Add 0.1 ml of phenolphthalein solution R. The
solution is colourless. Not more than 0.6 ml of 0.01M sodium hydroxide is required to change the
colour of the indicator to pale pink.
Benzene and related substances Examine by gas chromatography (2.2.28).
Test solution (b). Dilute 1.0 ml of 2-butanol R1 to 50.0 ml with test solution (a). Dilute 5.0 ml of the
solution to 100.0 ml with test solution (a).
Reference solution (a). Dilute 0.5 ml of 2-butanol R1 and 0.5 ml of propanol R to 50.0 ml with test
solution (a). Dilute 5.0 ml of this solution to 50.0 ml with test solution (a).
Reference solution (b). Dilute 100 l of benzene R to 100.0 ml with test solution (a). Dilute 0.20 ml of
this solution to 100.0 ml with test solution (a).
a fused-silica capillary column 30 m long and 0.32 mm internal diameter, coated with
helium for chromatography R as the carrier gas; split ratio 1:5 with a linear velocity of 35 cm/s,
nitrogen for chromatography R or helium for chromatography R as the auxiliary gas,
Time
(min)
Temperature
(C)
Rate
(C/min)
Comment
12 32 40 240 10 linear
gradient
Injection port 280
Detector 280
Inject 1 l of reference solution (a). Adjust the sensitivity of the system so that the heights of the
two peaks following the principal peak are at least 50 per cent of the full scale of the recorder. The
test is not valid unless the resolution between the first peak (propanol) and the second peak
(2-butanol) is at least 10.
22-39
Inject 1 l of test solution (b). In the chromatogram obtained, the sum of the areas of all the peaks,
apart from the principal peak and the peak due to 2-butanol, is not greater than 3 times the area of
peak due to 2-butanol (0.3 per cent).
peak due to benzene, with a retention time of about 10 min is at least 10 per cent of the full scale of
the recorder.
Inject 1 l of test solution (a). In the chromatogram obtained, the area of any peak due to benzene
is not more than half of the area of the corresponding peak in the chromatogram obtained with
reference solution (b) (2 ppm).
Peroxides In a 12 ml test-tube with a ground-glass stopper and a diameter of about 15 mm,
introduce 8 ml of potassium iodide and starch solution R. Fill completely with the substance to be
examined. Shake vigorously and allow to stand protected from light for 30 min. No colour develops.
Non-volatile substances Evaporate 100 g to dryness on a water-bath after having verified that it
complies with the test for peroxides and dry in an oven at 100C to 105C. The residue weighs not more
than 2 mg (20 ppm).
of water.
STORAGE
IMPURITIES
A. acetone,
B. benzene,
3
C O CH
3
R R
C. R = CH
3
2-(1-methylethoxy)propane (diisopropyl ether),
D. R = H ethoxyethane (diethyl ether),
E. CH
3
-OH methanol,
H
3
C
OH
F. propan-1-ol (n-propanol).
__________________________________________________________________________________________________________ Ph Eur
22-40
Isopropyl Myristate
O Me
O Me
H
CH
3
[CH
2
]
11
CH
2
C
17
H
34
O
2
270.5 110-27-0
Isopropyl Myristate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0725].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Isopropyl myristate contains not less than 90.0 per cent of isopropyl tetradecanoate together with
variable amounts of other fatty acid isopropyl esters.
CHARACTERS
A clear, colourless, oily liquid, immiscible with water, miscible with alcohol, with ether, with
methylene chloride, with fatty oils and with liquid paraffin.
IDENTIFICATION
obtained with the test solution has a retention time similar to that of the principal peak in the chrom-
C. Superpose 2 ml of a 1 g/l solution in alcohol R on a freshly prepared solution of 20 mg of
dimethylaminobenzaldehyde R in 2 ml of sulphuric acid R. After 2 min, a yellowish-red colour appears at
the junction of the two liquids and gradually becomes red.
D. It gives the reaction of esters (2.3.1). After heating to boiling, cool, add 3 ml of alcohol R and
acidify with 1 ml of dilute hydrochloric acid R.
TESTS
7
(Method II,
2.2.2).
Viscosity (2.2.9). 5 mPas to 6 mPas.
of water.
ASSAY
Examine by gas chromatography (2.2.28), using tricosane R as the internal standard.
Internal standard solution. Dissolve 50.0 mg of tricosane R in heptane R and dilute to 250.0 ml with the
same solvent.
dilute to 100.0 ml with the internal standard solution.
Reference solution. Dissolve 20.0 mg of isopropyl tetradecanoate CRS in the internal standard solution
and dilute to 100.0 ml with the internal standard solution.
film of polycyanopropylsiloxane R,
22-41
hydrogen for chromatography R as the carrier gas at a pressure of 0.15 MPa,
a split injector (1/40).
maintaining the temperature of the injection port and that of the detector at 250C.
Inject a suitable volume of the test solution (about 2 l) and the same volume of the reference
solution.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
22-42
Isopropyl Palmitate
H
3
C[CH
2
]
14
O
O
CHMe
2
C
19
H
38
O
2
298.5 142-91-6
Isopropyl Palmitate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0839].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Isopropyl palmitate contains not less than 90.0 per cent of isopropyl hexadecanoate together with
varying amounts of other fatty acid isopropyl esters.
CHARACTERS
A clear, colourless, oily liquid, immiscible with water, miscible with alcohol, with ether, with
methylene chloride, with fatty oils and with liquid paraffin.
IDENTIFICATION
obtained with the test solution has a retention time similar to that of the principal peak in the chrom-
C. Superpose 2 ml of a 1 g/l solution in alcohol R on a freshly prepared solution of 20 mg of
dimethylaminobenzaldehyde R in 2 ml of sulphuric acid R. After 2 min, a yellowish-red colour appears at
the junction of the two liquids which gradually becomes red.
D. It gives the reaction of esters (2.3.1). After heating to boiling, cool, add 3 ml of alcohol R and
acidify with 1 ml of dilute hydrochloric acid R.
TESTS
7
(Method II,
2.2.2).
Viscosity (2.2.9). 5 mPas to 10 mPas.
of water.
ASSAY
Examine by gas chromatography (2.2.28), using tricosane R as the internal standard.
Internal standard solution. Dissolve 50.0 mg of tricosane R in heptane R and dilute to 250.0 ml with the
same solvent.
dilute to 100.0 ml with the internal standard solution.
Reference solution. Dissolve 20.0 mg of isopropyl hexadecanoate CRS in the internal standard solution
and dilute to 100.0 ml with the internal standard solution.
film of polycyanopropylsiloxane R,
hydrogen for chromatography R as the carrier gas at a pressure of 0.15 MPa,
22-43
a split injector (1/40),
maintaining the temperature of the injection port and that of the detector at 250C.
Inject a suitable volume of the test solution (about 2 l) and the same volume of the reference
solution.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
22-44
Diluted Isosorbide Dinitrate
O
O
H
H
H
ONO
2
H
O
2
NO
C
6
H
8
N
2
O
8
236.1 87-33-2
Diluted Isosorbide Dinitrate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Isosorbide Dinitrate Tablets
When diluted sorbide nitrate is prescribed or demanded, Diluted Isosorbide Dinitrate shall be
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Diluted isosorbide dinitrate is a dry mixture of isosorbide dinitrate and Lactose monohydrate (187) or
Mannitol (559). It contains not less than 95.0 per cent m/m and not more than 105.0 per cent m/m of
the content of 1,4:3,6-dianhydro-D-glucitol 2,5-dinitrate stated on the label.
CAUTION: Undiluted isosorbide dinitrate may explode if subjected to percussion or excessive heat. Appro-
priate precautions must be taken and only very small quantities should be handled.
CHARACTERS
Undiluted isosorbide dinitrate is a fine, white, crystalline powder, very slightly soluble in water, very
soluble in acetone, sparingly soluble in alcohol.
The solubility of the diluted product depends on the diluent and its concentration.
IDENTIFICATION
A. Examine by infrared absorption spectrophotometry (2.2.24) the residue obtained in identification
test D, comparing with the spectrum of the residue obtained in the same way with isosorbide dinitrate
CRS. Examine the residues prepared as discs.
Test solution. Shake a quantity of the substance to be examined corresponding to 10 mg of isosorbide
dinitrate with 10 ml of alcohol R for 5 min and filter.
Reference solution. Shake a quantity of isosorbide dinitrate CRS corresponding to 10 mg of isosorbide
dinitrate with 10 ml of alcohol R for 5 min and filter.
5 volumes of methanol R and 95 volumes of methylene chloride R. Dry the plate in a current of air.
Spray with freshly prepared potassium iodide and starch solution R. Expose to ultraviolet light at
254 nm for 15 min. Examine in daylight. The principal spot in the chromatogram obtained with the
test solution is similar in position, colour and size to the principal spot in the chromatogram obtained
Test solution. Shake a quantity of the substance to be examined corresponding to 0.10 g of lactose or
mannitol with 10 ml of water R. Filter if necessary.
Reference solution (a). Dissolve 0.10 g of lactose R in water R and dilute to 10 ml with the same
solvent.
Reference solution (b). Dissolve 0.10 g of mannitol R in water R and dilute to 10 ml with the same
solvent.
Reference solution (c). Mix equal volumes of reference solutions (a) and (b).
volumes of anhydrous acetic acid R and 50 volumes of ethylene chloride R, measured accurately since a
22-45
slight excess of water produces cloudiness. Dry the plate in a current of warm air. Repeat immedi-
ately the development after renewing the mobile phase. Dry the plate in a current of warm air. Spray
with 4-aminobenzoic acid solution R. Dry the plate in a current of cold air until the acetone is removed.
Heat the plate at 100C for 15 min. Allow to cool and spray with a 2 g/l solution of sodium
periodate R. Dry the plate in a current of cold air. Heat the plate at 100C for 15 min. The principal
principal spot in the chromatogram obtained with reference solution (a) for lactose or to the principal
spot in the chromatogram obtained with reference solution (b) for mannitol. The identification is not
spots.
D. Shake a quantity of the substance to be examined corresponding to 25 mg of isosorbide dinitrate
with 10 ml of acetone R for 5 min. Filter, evaporate to dryness at a temperature below 40C and dry
the residue over diphosphorus pentoxide R at a pressure of 0.7 kPa for 16 h. The melting point (2.2.14)
of the residue is 69C to 72C.
TESTS
Inorganic nitrates Examine by thin-layer chromatography (2.2.27), using silica gel H R as the
coating substance.
Test solution. Shake a quantity of the substance to be examined corresponding to 0.10 g of isosorbide
dinitrate with 5 ml of alcohol R and filter.
alcohol R.
15 volumes of glacial acetic acid R, 30 volumes of acetone R and 60 volumes of toluene R. Thoroughly
dry the plate in a current of air until the acetic acid is completely removed. Spray copiously with
freshly prepared potassium iodide and starch solution R. Expose the plate to ultraviolet light at 254 nm
for 15 min. Examine in daylight. Any spot corresponding to nitrate ion in the chromatogram
the reference solution (0.5 per cent, calculated as potassium nitrate).
Isosorbide 5-nitrate and isosorbide 2-nitrate Examine by liquid chromatography (2.2.29) as
described under Assay, changing the detection to 210-215 nm.
When the chromatograms are recorded in the prescribed conditions, the retention times are:
isosorbide dinitrate, about 5 min; isosorbide 2-nitrate, about 8 min; isosorbide 5-nitrate about
11 min.
Inject 10 l of reference solution (c). When a recorder is used, adjust the sensitivity of the system
so that the height of the principal peak in the chromatogram obtained with reference solution (c) is
not less than 20 per cent of the full scale of the recorder.
Inject 10 l of reference solution (e). The test is not valid unless in the chromatogram obtained
with reference solution (e), the resolution between the peaks corresponding to isosorbide dinitrate
and isosorbide 2-nitrate is at least 6.0.
Inject separately 10 l of test solution (a), 10 l of reference solution (c) and 10 l of reference
solution (d). In the chromatogram obtained with test solution (a): the area of any peak corresponding
to isosorbide 2-nitrate is not greater than the area of the principal peak in the chromatogram obtained
with reference solution (c) (0.5 per cent); the area of any peak corresponding to isosorbide 5-nitrate
is not greater than the area of the principal peak in the chromatogram obtained with reference solu-
tion (d) (0.5 per cent).
ASSAY
Test solution (a). Sonicate a quantity of the substance to be examined corresponding to 25.0 mg of
isosorbide dinitrate with 20 ml of the mobile phase for 15 min and dilute to 25.0 ml with the mobile
phase. Filter the solution through a suitable membrane filter.
Reference solution (a). Sonicate a quantity of isosorbide dinitrate CRS corresponding to 25.0 mg of
isosorbide dinitrate with 20 ml of the mobile phase for 15 min and dilute to 25.0 ml with the mobile
phase. Filter the solution through a suitable membrane filter.
Reference solution (c). Dissolve 10.0 mg of isosorbide 2-nitrate CRS in the mobile phase and dilute to
Reference solution (d). Dissolve 10.0 mg of isosorbide mononitrate CRS in the mobile phase and dilute to
Reference solution (e). Dissolve 5 mg of isosorbide 2-nitrate CRS in the mobile phase and dilute to 10 ml
22-46
with the mobile phase. To 1 ml of this solution, add 0.5 ml of reference solution (a) and dilute to
aminopropylmethylsilyl silica gel for chromatography R

(10 m),
volumes of trimethylpentane R,
Inject 20 l of reference solution (b). When a recorder is used, adjust the sensitivity of the system
so that the height of the principal peak in the chromatogram obtained is not less than 50 per cent of
the full scale of the recorder. If the areas of the peaks from two successive injections do not agree to
within 1.0 per cent, then inject a further four times and calculate, for the six injections, the relative
standard deviation. The assay is not valid unless the relative standard deviation for the six injections
is at most 2.0 per cent. Inject test solution (b) and reference solution (b), alternately.
Calculate the content of isosorbide dinitrate as a percentage of the declared content.
STORAGE
LABELLING
The label states the percentage content of isosorbide dinitrate.
IMPURITIES
B. isosorbide 2-nitrate,
C. isosorbide mononitrate (isosorbide 5-nitrate).
__________________________________________________________________________________________________________ Ph Eur
22-47
Diluted Isosorbide Mononitrate
O
O
H
H
H
ONO
2
H
HO
C
6
H
9
NO
6
191.1 16051-77-7
Diluted Isosorbide Mononitrate complies with the requirements of the 3rd edition of the European Pharmaco-
poeia [1118]. These requirements are reproduced after the heading Definition below.
Preparation
Isosorbide Mononitrate Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Diluted isosorbide mononitrate is a dry mixture of isosorbide mononitrate and Lactose monohydrate
(187) or Mannitol (559). It contains not less than 95.0 per cent m/m and not more than 105.0 per
cent m/m of the content of 1,4:3,6-dianhydro-D-glucitol 5-nitrate stated on the label.
CHARACTERS
Undiluted isosorbide mononitrate is a white, crystalline powder, freely soluble in water, in acetone, in
alcohol and in methylene chloride.
The solubility of the diluted product depends on the diluent and its concentration.
IDENTIFICATION
A. Examine by infrared absorption spectrophotometry (2.2.24) the residue obtained in identification
test D, comparing with the spectrum obtained with isosorbide mononitrate CRS. Examine the
substances prepared as discs.
Test solution. Shake a quantity of the substance to be examined corresponding to 10 mg of isosorbide
mononitrate with 10 ml of alcohol R for 5 min and filter.
Reference solution. Dissolve 10 mg of isosorbide mononitrate CRS in alcohol R and dilute to 10 ml with
the same solvent.
5 volumes of methanol R and 95 volumes of methylene chloride R. Dry the plate in a current of air.
Spray with freshly prepared potassium iodide and starch solution R. Expose to ultraviolet light at
254 nm for 15 min. Examine in daylight. The principal spot in the chromatogram obtained with the
test solution is similar in position, colour and size to the principal spot in the chromatogram obtained
Test solution. Shake a quantity of the substance to be examined corresponding to 0.10 g of lactose or
mannitol with 10 ml of water R. Filter if necessary.
Reference solution (a). Dissolve 0.10 g of lactose R in water R and dilute to 10 ml with the same
solvent.
Reference solution (b). Dissolve 0.10 g of mannitol R in water R and dilute to 10 ml with the same
solvent.
Reference solution (c). Mix equal volumes of reference solutions (a) and (b).
slight excess of water produces cloudiness. Dry the plate in a current of warm air. Repeat immedi-
ately the development after renewing the mobile phase. Dry the plate in a current of warm air. Spray
with 4-aminobenzoic acid solution R. Dry the plate in a current of cold air until the acetone is removed.
Heat the plate at 100C for 15 min. Allow to cool and spray with a 2 g/l solution of sodium
22-48
periodate R. Dry the plate in a current of cold air. Heat the plate at 100C for 15 min. The principal
principal spot in the chromatogram obtained with reference solution (a) for lactose or to the principal
spot in the chromatogram obtained with reference solution (b) for mannitol. The identification is not
spots.
D. Shake a quantity of the substance to be examined corresponding to 25 mg of isosorbide mono-
nitrate with 10 ml of acetone R for 5 min. Filter, evaporate to dryness at a temperature below 40C
and dry the residue over diphosphorus pentoxide R at a pressure of 0.7 kPa for 16 h. The melting point
(2.2.14) of the residue is 89C to 91C.
TESTS
Inorganic nitrates Examine by thin-layer chromatography (2.2.27), using silica gel H R as the
coating substance.
Test solution. Shake a quantity of the substance to be examined corresponding to 0.10 g of isosorbide
mononitrate with 5 ml of alcohol R and filter.
alcohol R.
15 volumes of glacial acetic acid R, 30 volumes of acetone R and 60 volumes of toluene R. Thoroughly
dry the plate in a current of air until the acetic acid is completely removed. Spray copiously with
freshly prepared potassium iodide and starch solution R. Expose the plate to ultraviolet light at 254 nm
for 15 min. Examine in daylight. Any spot corresponding to nitrate ion in the chromatogram
the reference solution (0.5 per cent, calculated as potassium nitrate).
Isosorbide dinitrate and isosorbide 2-nitrate Examine by liquid chromatography (2.2.29) as
described under Assay, changing the detection to 210-215 nm.
When the chromatograms are recorded in the prescribed conditions, the retention times are:
isosorbide dinitrate, about 5 min; isosorbide-2-nitrate, about 8 min; isosorbide 5-nitrate about
11 min.
Inject 10 l of reference solution (b). When a recorder is used, adjust the sensitivity of the system
so that the height of the principal peak in the chromatogram obtained with reference solution (b) is
not less than 20 per cent of the full scale of the recorder.
Inject 10 l of reference solution (d). The test is not valid unless, in the chromatogram obtained
with reference solution (d), the resolution between the peaks correspond ing to isosorbide 2-nitrate
and isosorbide 5-nitrate is at least 4.0.
Inject separately 10 l of test solution (a), 10 l of reference solution (b) and 10 l of reference
solution (c). In the chromatogram obtained with test solution (a): the area of any peak corresponding
to isosorbide 2-nitrate is not greater than the area of the principal peak in the chromatogram obtained
with reference solution (b) (0.5 per cent); the area of any peak corresponding to isosorbide dinitrate
is not greater than the area of the principal peak in the chromatogram obtained with reference solu-
tion (c) (0.5 per cent).
ASSAY
Test solution (a). Sonicate a quantity of the substance to be examined corresponding to 25.0 mg of
isosorbide mononitrate with 20 ml of the mobile phase for 15 min and dilute to 25.0 ml with the
mobile phase. Filter the solution through a suitable membrane filter.
Reference solution (a). Dissolve 25.0 mg of isosorbide mononitrate CRS in the mobile phase and dilute to
Reference solution (b). Dissolve 10.0 mg of isosorbide-2-nitrate CRS in the mobile phase and dilute to
Reference solution (c). Sonicate a quantity of isosorbide dinitrate CRS corresponding to 10.0 mg of
isosorbide dinitrate in 15 ml of the mobile phase for 15 min and dilute to 20.0 ml with the mobile
phase. Filter the solution through a suitable membrane filter. Dilute 0.1 ml of the solution to 10.0 ml
Reference solution (d). Dissolve 5 mg of isosorbide mononitrate CRS and 5 mg of isosorbide-2-nitrate CRS
in the mobile phase and dilute to 10 ml with the mobile phase. Dilute 1 ml of the solution to 10 ml
aminopropylmethylsilyl silica gel for chromatography R

(10 m),
22-49
volumes of trimethylpentane R,
Inject 20 l of reference solution (a). When a recorder is used, adjust the sensitivity of the system
so that the height of the principal peak in the chromatogram obtained with reference solution (a) is
not less than 50 per cent of the full scale of the recorder. If the areas of the peaks from two successive
injections do not agree to within 1.0 per cent, then inject a further four times and calculate, for the
six injections, the relative standard deviation. The assay is not valid unless the relative standard
deviation for the six injections is at most 2.0 per cent. Inject test solution (b) and reference solution
(a), alternately.
Calculate the content of isosorbide mononitrate as a percentage of the declared content.
STORAGE
LABELLING
The label states the percentage content of isosorbide mononitrate.
IMPURITIES
O
O
HO
H
ONO
2
H
H
H
B. isosorbide dinitrate,
O
O
H
H
ONO
2
H
H
O
2
NO
C. isosorbide 2-nitrate.
__________________________________________________________________________________________________________ Ph Eur
22-50
Isotretinoin
COOH
Me
Me e
Me Me
H
C
20
H
28
O
2
300.4 4759-48-2
Isotretinoin complies with the requirements of the 3rd edition of the European Pharmacopoeia [1019]. These
Action and use Treatment of acne.
Preparations
Isotretinoin Capsules
Isotretinoin Gel
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Isotretinoin contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent
of (2Z,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-enyl)nona-2,4,6,8-tetraenoic acid,
CHARACTERS
A yellow or light-orange, crystalline powder, practically insoluble in water, soluble in methylene
chloride, slightly soluble in alcohol. It is sensitive to air, heat and light, especially in solution.
Carry out all operations as rapidly as possible and avoid exposure to actinic light; use freshly prepared
solutions.
IDENTIFICATION
A. Dissolve 75.0 mg in 5 ml of methylene chloride R and dilute immediately to 100.0 ml with acidified
2-propanol (prepared by diluting 1 ml of 0.01M hydrochloric acid to 1000 ml with 2-propanol R).
Dilute 5.0 ml of this solution to 100.0 ml with the acidified 2-propanol (solution A). Dilute 5.0 ml of
solution A to 50.0 ml with the acidified 2-propanol. Examined between 300 nm and 400 nm
(2.2.25), the solution shows an absorption maximum at 354 nm. The specific absorbance at the
maximum is 1290 to 1420.
obtained with isotretinoin CRS. Examine the substances prepared as discs.
C. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel GF
254
plate R.
Reference solution (a). Dissolve 10 mg of isotretinoin CRS in methylene chloride R and dilute to 10 ml
Reference solution (b). Dissolve 10 mg of isotretinoin CRS and 10 mg of tretinoin CRS in methylene
volumes of glacial acetic acid R, 4 volumes of acetone R, 40 volumes of peroxide-free ether R and 54
volumes of cyclohexane R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm.
The principal spot in the chromatogram obtained with the test solution is similar in position and size
to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid
unless the chromatogram obtained with reference solution (b) shows two clearly separated principal
spots.
D. Dissolve about 5 mg in 2 ml of antimony trichloride solution R. An intense red colour develops and
later becomes violet.
TESTS
22-51
Reference solution (a). Dissolve 10.0 mg of tretinoin CRS in methanol R and dilute to 10.0 ml with the
same solvent.
Reference solution (c). Mix 1.0 ml of reference solution (a) with 0.5 ml of the test solution and dilute
to 25.0 ml with methanol R.
Reference solution (d). Dilute 0.5 ml of the test solution to 100.0 ml with methanol R.
as mobile phase at a flow rate of 1.0 ml/min a mixture of 5 volumes of glacial acetic acid R, 225
volumes of water R and 770 volumes of methanol R.
Inject separately 10 l of each of reference solutions (b), (c) and (d) and of the test solution. Adjust
the sensitivity of the detector so that the height of the principal peak in the chromatogram obtained
with reference solution (b) is not less than 70 per cent of the full scale of the recorder. The test is not
valid unless the resolution between the peaks due to isotretinoin and tretinoin in the chromatogram
obtained with reference solution (c) is at least 2.0. In the chromatogram obtained with the test
solution: the area of any peak due to tretinoin is not greater than the area of the principal peak in the
chromatogram obtained with reference solution (b) (2.0 per cent); the sum of the areas of any peaks,
apart from the principal peak and any peak due to tretinoin, is not greater than the area of the
principal peak in the chromatogram obtained with reference solution (d) (0.5 per cent).
16 h.
ASSAY
Dissolve 0.200 g in 70 ml of acetone R. Titrate with 0.1M tetrabutylammonium hydroxide determining
20
H
28
0
2
.
STORAGE
It is recommended that the contents of an opened container be used as soon as possible and any
unused part be protected by an atmosphere of an inert gas.
IMPURITIES
A. tretinoin,
Me Me
Me
Me
COOH
Me
B. (2Z,4E,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-enyl)nona-2,4,6,8-tetraenoic
acid (9,13-di-cis-retinoic acid),
Me Me
Me
Me
Me
COOH
C. (2Z,4Z,6E,8E,)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-enyl)nona-2,4,6,8-tetraenoic
acid (11,13-di-cis-retinoic acid),
Me e
Me
Me
Me
COOH
D. (2E,4E,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-enyl)nona-2,4,6,8-tetraenoic
acid (9-cis-retinoic acid),
E. oxidation products of isotretinoin.
__________________________________________________________________________________________________________ Ph Eur
22-52
Isoxsuprine Hydrochloride
N
OH H
Me H H Me
O
HO
H
,HCl
and enantiomer
C
18
H
23
NO
3
,HCl 337.8 579-56-6
Isoxsuprine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Isoxsuprine hydrochloride contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of (1RS,2SR)-1-(4-hydroxyphenyl)-2-[(1SR)-1-methyl-2-
phenoxyethylamino]propan-1-ol hydrochloride, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, sparingly soluble in water and in alcohol, practically
insoluble in ether and in methylene chloride.
IDENTIFICATION
10.0 ml of this solution to 100.0 ml with 0.1M hydrochloric acid. Examined between 230 nm and
350 nm (2.2.25), the solution shows two absorption maxima, at 269 nm and 275 nm. The specific
absorbance at these maxima are 71 to 74 and 70 to 73, respectively. The test is not valid unless, in
the test for resolution (2.2.25), the ratio of the absorbances is at least 1.7.
obtained with isoxsuprine hydrochloride CRS. Examine the substances prepared as discs. If the spectra
obtained show differences, dissolve 50 mg of the substance to be examined and the reference
substance separately in 2 ml of methanol R, add 15 ml of methylene chloride R, evaporate to dryness
and record new spectra using the residues.
the same solvent.
Reference solution. Dissolve 20 mg of isoxsuprine hydrochloride CRS in methanol R and dilute to 10 ml
0.25 volumes of concentrated ammonia R, 15 volumes of methanol R and 85 volumes of methylene
chloride R. Dry the plate in a current of warm air and spray with a 10 g/l solution of potassium
permanganate R. The principal spot in the chromatogram obtained with the test solution is similar in
solution.
D. To 1 ml of solution S (see Tests) add 0.05 ml of copper sulphate solution R and 0.5 ml of strong
sodium hydroxide solution R. The solution becomes blue. Add 1 ml of ether R and shake. Allow to
separate. The upper layer remains colourless.
E. 2 ml of solution S gives reaction (a) of chlorides (2.3.1).
TESTS
Solution S Dissolve 0.50 g, with gentle heating if necessary, in carbon dioxide-free water R, cool and
22-53
Optical rotation (2.2.7). The angle of optical rotation of solution S is 0.05 to +0.05.
Phenones Dissolve 10.0 mg in water R and dilute to 100.0 ml with the same solvent. The absorb-
ance (2.2.25) measured at the maximum at 310 nm is not greater than 0.10 (1.0 per cent, calculated
as impurity B).
Related substances Prepare the solutions immediately before use. Examine by gas chromatography
(2.2.28), using hexacosane R as the internal standard.
Internal standard solution (a). Dissolve 0.1 g of hexacosane R in trimethylpentane R and dilute to 20 ml
Internal standard solution (b). Dilute 1 ml of internal standard solution (a) to 50 ml with
trimethylpentane R.
Test solution. To 10.0 mg of the substance to be examined, add 0.5 ml of N-trimethylsilylimidazole R.
Heat to 65C for 10 min. Allow to cool, then add 2.0 ml of the internal standard solution (b) and
2.0 ml of water R. Shake. Use the upper layer.
Reference solution (a). To 10.0 mg of the substance to be examined, add 0.5 ml of N-trimethylsilyl-
imidazole R. Heat to 65C for 10 min. Allow to cool, then add 2.0 ml of the internal standard solu-
tion (a) and 2.0 ml of water R. Shake. Dilute 1.0 ml of the upper layer to 50.0 ml with
trimethylpentane R.
Reference solution (b). To 10.0 mg of the substance to be examined, add 0.5 ml of N-trimethylsilyl-
imidazole R. Heat to 65C for 10 min. Allow to cool, then add 2.0 ml of trimethylpentane R and
2.0 ml of water R. Shake. Use the upper layer.
a glass column 1.5 m long and 4 mm in internal diameter, packed with silanised diatomaceous
rate of 5C per minute to 215C and maintaining at 215C for 10 min, and maintaining the tempera-
ture of the injection port and that of the detector at 225C.
Inject 1 l of reference solution (a). The substances elute in the following order: isoxsuprine and
hexacosane. Adjust the sensitivity of the detector so that the heights of the two principal peaks are not
less than 50 per cent of the full scale of the recorder. The test is not valid unless, in the chromato-
gram obtained, the resolution between the peaks corresponding to isoxsuprine and hexacosane is at
least 5.0.
Inject 1 l of reference solution (b). In the chromatogram obtained, verify that there is no peak
with the same retention time as the internal standard.
Inject 1 l of the test solution and 1 l of reference solution (a). From the chromatogram obtained
with reference solution (a), calculate the ratio (R) of the area of the peak due to the trimethylsilyl
derivative of isoxsuprine to the area of the peak due to the internal standard. From the chromatogram
obtained with the test solution, calculate the ratio of the sum of the areas of any peaks, apart from the
principal peak, the peak due to the internal standard and the peak due to the solvent, to the area of
the peak due to the internal standard: this ratio is not greater than R (2.0 per cent).
at 100C to 105C.
ASSAY
tiometric titration (2.2.20), using 0.1M sodium hydroxide. Read the volume added between the two
18
H
24
ClNO
3
.
STORAGE
22-54
IMPURITIES
HO
N
O
H OH
Me H
H
Me
H
A. (1RS,2SR)-1-(4-hydroxyphenyl)-2-[(1RS)-1-methyl-2-phenoxyethylamino]propan-1-ol
hydrochloride,
HO
N
O
Me H
H
Me
O
H
B. 1-(4-hydroxyphenyl)-2-(1-methyl-2-phenoxyethylamino)-propan-1-one.
__________________________________________________________________________________________________________ Ph Eur
22-55
Ispaghula Husk
Ispaghula Husk complies with the requirements of the 3rd edition of the European Pharmacopoeia [1334].
Action and use Antidiarrhoeal; bulk-forming laxative.
Preparations
Ispaghula Husk Oral Powder
Ispaghula Husk Granules
Ispaghula Husk Effervescent Granules
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ispaghula husk consists of the episperm and collapsed adjacent layers removed from the seeds of
Plantago ovata Forssk. (P. ispaghula Roxb.).
CHARACTERS
IDENTIFICATION
A. Ispaghula husk consists of pinkish-beige fragments or flakes up to about 2 mm long and 1 mm
wide, some showing a light brown spot corresponding to the location of the embryo before it was
removed from the seed.
B. Reduce to a powder (355). The powder is pale yellow. Examine under a microscope using lactic
acid reagent R. The powder shows mainly fragments of the episperm with polygonal cells filled with
mucilage; fragments of the inner layers of the testa with brownish thin-walled cells often associated
with the outer layers of the endosperm. Examine under a microscope using a 50 per cent V/V
solution of glycerol R. The powder shows occasional starch granules, single or in groups of two to
four, measuring 3 m to 25 m in diameter.
Test solution. To 10 mg of the powdered drug (355) in a thick-walled centrifuge tube add 2 ml of a
230 g/l solution of trifluoroacetic acid R and shake vigorously. Stopper the test tube and heat the
mixture at 120C for 1 h. Centrifuge the hydrolysate, transfer the clear supernatant liquid into a
50 ml flask, add 10 ml of water R and evaporate the solution to dryness under reduced pressure. Take
up the residue in 10 ml of water R and evaporate again to dryness under reduced pressure. Take up
the residue with 2 ml of methanol R.
Reference solution (a). Dissolve 10 mg of arabinose R in a small quantity of water R and dilute to 10 ml
with methanol R.
Reference solution (b). Dissolve 10 mg of xylose R in a small quantity of water R and dilute to 10 ml
with methanol R.
Reference solution (c). Dissolve 10 mg of galactose R in a small quantity of water R and dilute to 10 ml
with methanol R.
Apply to the plate, as bands, 10 l of each solution. Develop over a path of 15 cm using a mixture
of 15 volumes of water R and 85 volumes of acetonitrile R. Spray with aminohippuric acid reagent R and
heat at 120C for 5 min. Examine in daylight. The chromatogram obtained with the test solution
shows two orange-pink zones (arabinose and xylose) and a yellow zone (galactose) similar in position
and colour to the zones in the chromatograms obtained with the reference solutions.
TESTS
Foreign matter (2.8.2). It complies with the test for foreign matter, determined on 5.0 g of the
Swelling index (2.8.4). Not less than 40, determined on 0.1 g of the powdered drug (355).
STORAGE
__________________________________________________________________________________________________________ Ph Eur
22-56
Ispaghula Seed
Ispaghula Seed complies with the requirements of the 3rd edition of the European Pharmacopoeia [1333].
Action and use Bulk-forming laxative.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ispaghula seed consists of the dried ripe seeds of Plantago ovata Forssk. (P. ispaghula Roxb.).
CHARACTERS
IDENTIFICATION
A. Ispaghula seed is pinkish-beige, smooth, boat-shaped and curved. It is 1.5 mm to 3.5 mm long,
1.5 mm to 2 mm wide and 1 mm to 1.5 mm thick. The concave surface shows in the centre a light
coloured spot corresponding to the hilum. The convex surface shows a light brown spot correspond-
ing to the location of the embryo and takes up about one quarter of the length of the seed.
B. Reduce to a powder (355). The powder is pale brown. Examine under a microscope using lactic
acid reagent R. The powder shows mainly fragments of the episperm with polygonal cells filled with
mucilage; fragments of the inner layers of the testa with brownish thin-walled cells often associated
with the outer layers of the endosperm; fragments of the endosperm with cells with thick cellulose
walls containing aleurone grains and oil droplets; a few fragments of embryo with thin-walled cells.
Examine under a microscope using a 50 per cent V/V solution of glycerol R. The powder shows starch
granules, single or in groups of two to four and measuring 3 m to 25 m in diameter.
Test solution. To 50 mg of the powdered drug (355) in a thick-walled centrifuge tube add 2 ml of a
230 g/l solution of trifluoroacetic acid R, and shake vigorously. Stopper the test tube and heat the
mixture at 120C for 1 h. Centrifuge the hydrolysate, transfer the clear supernatant liquid into a
50 ml flask, add 10 ml of water R and evaporate the solution to dryness under reduced pressure. Take
up the residue in 10 ml of water R and evaporate again to dryness under reduced pressure. Take up
the residue in 2 ml of methanol R.
Reference solution (a). Dissolve 10 mg of arabinose R in a small quantity of water R and dilute to 10 ml
with methanol R.
Reference solution (b). Dissolve 10 mg of xylose R in a small quantity of water R and dilute to 10 ml
with methanol R.
Reference solution (c). Dissolve 10 mg of galactose R in a small quantity of water R and dilute to 10 ml
with methanol R.
15 volumes of water R and 85 volumes of acetonitrile R. Spray with aminohippuric acid reagent R and
heat at 120C for 5 min. Examine in daylight. The chromatogram obtained with the test solution
shows two orange-pink zones (arabinose and xylose) and a yellow zone (galactose) similar in position
and colour to the principal zones in the chromatogram obtained with the reference solutions.
TESTS
Foreign matter (2.8.2). It complies with the test for foreign matter, determined on 10.0 g of the
drug.
Swelling index (2.8.4). Not less than 9, determined on the powdered drug (355).
STORAGE
__________________________________________________________________________________________________________ Ph Eur
22-57
Isradipine
N Me Me
MeOOC
O Me
O Me
N
O
N
H
H
and enantiomer
C
19
H
21
N
3
O
5
371.4 75695-93-1
Definition Isradipine is isopropyl methyl (RS)-4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-
dimethylpyridine-3,5-dicarboxylate. It contains not less than 98.0% and not more than 102.0% of
C
19
H
21
N
3
O
5
Characteristics A yellow, crystalline powder.
Freely soluble in acetone; soluble in acetonitrile and in methanol; practically insoluble in n-hexane and
in water.
spectrum of isradipine (RS373).
Related substances Carry out the test protected from light. Carry out the method for liquid chrom-
atography, Appendix III D, using the following solutions. For solution (1) dissolve 50 mg in 1 ml of
methanol with the aid of ultrasound, if necessary, add 25 ml of the mobile phase and mix. For solu-
tion (2) dilute 1 volume of solution (1) to 400 volumes with the mobile phase. Solution (3) contains
0.0018% w/v of isradipine impurity B BPCRS in the mobile phase. Solution (4) contains 0.0020% w/v
each of the substance being examined and isradipine impurity D BPCRS in the mobile phase. For
solution (1) allow the chromatography to proceed for 5 times the retention time of the principal peak.
(10 cm 4.6 mm) packed with stationary phase C (5 m) (Brownlee Spheri ODS 5 is suitable), (b)
as the mobile phase a mixture of 125 volumes of acetonitrile, 270 volumes of tetrahydrofuran and 625
volumes of water with a flow rate of 1.2 ml per minute and (c) a detection wavelength of 230 nm.
In the chromatogram obtained with solution (1) the area of any peak corresponding to isradipine
impurity B is not greater than the area of the corresponding peak in the chromatogram obtained with
solution (3) (0.9%), the area of any other secondary peak is not greater than the area of the peak in the
chromatogram obtained with solution (2) (0.25%) and the sum of the areas of any secondary peaks is
not greater than 6 times the area of the peak in the chromatogram obtained with solution (2) (1.5%).
Assay Carry out the assay protected from light. Carry out the method for liquid chromatography,
Appendix III D, using the following solutions. For solution (1) dissolve 50 mg in 2 ml of methanol
and dilute to 250 ml with the mobile phase. For solution (2) dissolve 50 mg of isradipine BPCRS in
2 ml of methanol and dilute to 250 ml with the mobile phase. Solution (3) is a 0.020% w/v solution of
isradipine impurity D BPCRS in solution (2).
The chromatographic procedure may be carried out using the conditions described under Related
substances but using a detection wavelength of 326 nm.
19
H
21
N
3
O
5
19
H
21
N
3
O
5
in isradipine
BPCRS.
Storage Isradipine should be stored in a well-closed container and protected from light.
Preparation
Isradipine Tablets
22-58
IMPURITIES
N
N
O
N
COOEt MeOOC
Me Me
H
A. ethyl methyl 4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethylpyridine-3,5-
dicarboxylate
N
N
O
N
COOPr
i i
PrOOC
Me Me
H
B. di-isopropyl 4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethylpyridine-3,5-
dicarboxylate
N
N
O
N
COOMe MeOOC
Me Me
H
C. dimethyl 4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethylpyridine-3,5-dicarboxylate
N
N
O
N
COOPr
i
MeOOC
Me Me
D. isopropyl methyl 4-(2,1,3-benzoxadiazol-4-yl)-2,6-dimethylpyridine-3,5-dicarboxylate
N
N
O
N
COOPr
i
MeOOC
CH Me CH
N
O
N
H
E. isopropyl methyl 4-(2,1,3-benzoxadiazol-4-yl)-2-[2-(2,1,3-benzoxadiazol-4-yl)ethenyl]-1,4-
dihydro-6-methylpyridine-3,5-dicarboxylate
O
CH CH Me
MeOOC COOPr
i
N
O
N
N
H
F. isopropyl methyl 4-(2,1,3-benzoxadiazol-4-yl)-6-[2-(2,1,3-benzoxadiazol-4-yl)ethenyl]-1,4-
dihydro-2-methylpyridine-3,5-dicarboxylate
22-59
Itraconazole
corrected 1/01
*
N
N
N
H
N
N
O
Cl
Cl
N
N
N
O
O
H
3
C
H Me
O
and epimer at C*
C
35
H
38
Cl
2
N
8
O
4
706 84625-61-6
Itraconazole complies with the requirements of the 3rd edition of the European Pharmacopoeia [1335]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Itraconazole contains not less than 98.5 per cent and not more than the equivalent of 101.5 per cent
of 4-[4-[4-[4-[[cis-2-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1,3-dioxolan-4-
yl]methoxy]phenyl]piperazin-1-yl]phenyl]-2-[(1RS)-1-methylpropyl]-2,4-dihydro-3H-1,2,4-triazol-3-
one, calculated with reference to the dried substance.
CHARACTERS
sparingly soluble in tetrahydrofuran, very slightly soluble in alcohol.
IDENTIFICATION
itraconazole CRS. Examine the substance prepared as discs.
coating substance.
Reference solution (a). Dissolve 30 mg of itraconazole CRS in a mixture of equal volumes of methanol R
Reference solution (b). Dissolve 30 mg of itraconazole CRS and 30 mg of ketoconazole CRS in a mixture
of equal volumes of methanol R and methylene chloride R and dilute to 5 ml with the same mixture of
solvents.
Apply to the plate 5 l of each solution. Develop in an unsaturated tank over a path of 10 cm using a
mixture of 20 volumes of ammonium acetate solution R, 40 volumes of dioxan R and 40 volumes of
methanol R. Dry the plate in a current of warm air for 15 min and expose it to iodine vapour until the
spots appear. Examine in daylight. The principal spot in the chromatogram obtained with the test
22-60
D. To 30 mg in a porcelain crucible add 0.3 g of anhydrous sodium carbonate R. Heat over an open
TESTS
Solution S Dissolve 2.0 g in methylene chloride R and dilute to 20.0 ml with the same solvent.
solution BY
6
(Method II, 2.2.2).
Optical rotation (2.2.7). The angle of optical rotation is 0.10 to +0.10, determined on solution
S.
methanol R and tetrahydrofuran R and dilute to 10.0 ml with the same mixture of solvents.
Reference solution (a). Dissolve 5.0 mg of itraconazole CRS and 5.0 mg of miconazole CRS in a mixture
of equal volumes of methanol R and tetrahydrofuran R and dilute to 100.0 ml with the same mixture of
solvents.
Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with a mixture of equal volumes
of methanol R and tetrahydrofuran R. Dilute to 5.0 ml of this solution to 10.0 ml with the same
as mobile phase at a flow rate of 1.5 ml/min a gradient programme using the following condi-
tions:
Mobile phase A. A 27.2 g/l solution of tetrabutylammonium hydrogen sulphate R,
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
25 30 80 20 switch to initial
eluent composition
Equilibrate the column for at least 30 min with acetonitrile R at a flow rate of 1.5 ml/min and then
equilibrate at the initial eluent composition for at least 5 min.
conditions, the retention times are: miconazole about 10.5 min and itraconazole about 11 min. The
test is not valid unless the resolution between the peaks corresponding to miconazole and
itraconazole is at least 2.0. If necessary, adjust the concentration of acetonitrile in the mobile phase or
adjust the time programme for the linear gradient elution.
Inject separately 10 l of the mixture of equal volumes of methanol R and tetrahydrofuran R as a
blank, 10 l of the test solution and 10 l of reference solution (b). In the chromatogram obtained
with the test solution: the area of any peak, apart from the principal peak, is not greater than that of
of the areas of all the peaks, apart from the principal peak, is not greater than 2.5 times the area of
Disregard any peak obtained with the blank run and any peak with an area less than 0.1 times the
area of the principal peak in the chromatogram obtained with reference solution (b).
100C to 105C for 4 h.
ASSAY
ethyl ketone R. Titrate with 0.1M perchloric acid, determining the end-point potentiometrically at the
second point of inflexion (2.2.20).
35
H
38
Cl
2
N
8
O
4
.
22-61
STORAGE
IMPURITIES
*
N
N
N
N
N
OMe
H
3
C
H Me
O
and enantiomer
A. 4-[4-[4-(4-methoxyphenyl)piperazin-1-yl]phenyl]-2-[(1RS)-1-methylpropyl]-2,4-dihydro-
3H-1,2,4-triazol-3-one,
N
N
N
H
N
N
O
Cl
Cl N
N
N
O
O
H
3
C
H Me
O
B. 4-[4-[4-[4-[[cis-2-(2,4-dichlorophenyl)-2-(4H-1,2,4-triazol-4-ylmethyl)-1,3-dioxolan-4-
yl]methoxy]phenyl]piperazin-1-yl]phenyl]-2-[(1RS)-1-methylpropyl]-2,4-dihydro-3H-
1,2,4-triazol-3-one,
N
N
N
H
N
N
O
Cl
Cl
N
N
N
O
O
H
3
C
O
and enantiomer
C. 4-[4-[4-[4-[[cis-2-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1,3-dioxolan-4-
yl]methoxy]phenyl]piperazin-1-yl]phenyl]-2-(1-propyl)-2,4-dihydro-3H-1,2,4-triazol-3-
one,
N
N
N
H
N
N
O
Cl
Cl
N
N
N
O
O
H
3
C
O
CH
3
and enantiomer
D. 4-[4-[4-[4-[[cis-2-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1,3-dioxolan-4-
yl]methoxy]phenyl]piperazin-1-yl]phenyl]-2-(1-methylethyl)-2,4-dihydro-3H-1,2,4-triazol-
3-one,
22-62
N
N
N
H
N
N
O
Cl
Cl
N
N
N
O
O
H
3
C
H Me
O
E. 4-[4-[4-[4-[[trans-2-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1,3-dioxolan-4-
yl]methoxy]phenyl]piperazin-1-yl]phenyl]-2-[(1RS)-1-methylpropyl]-2,4-dihydro-3H-
1,2,4-triazol-3-one,
N
N
N
H
N
N
O
Cl
Cl
N
N
N
O
O
O
H
3
C and enantiomer
F. 2-butyl-4-[4-[4-[4-[[cis-2-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1,3-
dioxolan-4-yl]methoxy]phenyl]piperazin-1-yl]phenyl]-2,4-dihydro-3H-1,2,4-triazol-3-one,
N
N
N
H
N
N
O
Cl
Cl
N
N
N
O
O
O
O
O
N
N
N
Cl
Cl
H
and enantiomer
G. 4-[4-[4-[4-[[cis-2-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1,3-dioxolan-4-
yl]methoxy]phenyl]piperazin-1-yl]phenyl]-2-[[cis-2-(2,4-dichlorophenyl)-2-(1H-1,2,4-
triazol-1-ylmethyl)-1,3-dioxolan-4-yl]methyl]-2,4-dihydro-3H-1,2,4-triazol-3-one.
__________________________________________________________________________________________________________ Ph Eur
22-63
Ivermectin
corrected 1/01
Me
OMe
HO
O
O
O
O
Me
OMe
O
O
O
O O
Me
OH
H
H
H
OH
Me
H
R
H
CH
3
Me
Me
Component R Molecular Formula M
r
H
2
B
1a
CH
2
CH
3
C
48
H
74
O
14
875
H
2
B
1b
CH
3
C
47
H
72
O
14
861
70161-11-4 (ivermectin B
1a
) 70209-81-3 (ivermectin B
1b
)
Ivermectin complies with the requirements of the 3rd edition of the European Pharmacopoeia [1336]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ivermectin is a mixture of (2aE,4E,8E)-(5S,6S,6R,7S,11R,13R,15S,17aR,20R,20aR,20bS)-
20,20b-dihydroxy-5,6,8,19-tetramethyl-7-[[(3-O-methyl-4-O-(3-O-methyl-2,6-dideoxy--L-arabino-
hexopyranosyl)-2,6-dideoxy--L-arabino-hexopyranosyl]oxy]-6-[(1S)-1-methylpropyl]-
3,4,5,6,6,7,10,11,14,15,17a,20,20a,20b-tetradecahydrospiro[11,15-methano-2H,13H,17H-
furo[4,3,2-pq][2,6]benzodioxacyclo-octadecene-13,2-[2H]pyran]-17-one (or 5-O-demethyl-22,23-
dihydroavermectin A
1a
) (component H
2
B
1a
) and (2aE,4E,8E)-
(5S,6S,6R,7S,11R,13R,15S,17aR,20R,20aR,20bS)-20,20b-dihydroxy-5,6,8,19-tetramethyl-6-(1-
methylethyl)-7-[[(3-O-methyl-4-O-(3-O-methyl-2,6-dideoxy--L-arabino-hexopyranosyl)-2,6-
dideoxy--L-arabino-hexopyranosyl]oxy]-3,4,5,6,6,7,10,11,14,15,17a,20,20a,20b-
tetradecahydrospiro[11,15-methano-2H,13H,17H-furo[4,3,2-pq][2,6]benzodioxacyclo-octadecene-
13,2-[2H]pyran]-17-one (or 25-(1-methylethyl)-5-O-demethyl-25-de(1-methylpropyl)-22,23-
dihydroavermectin A
1a
) (component H
2
B
1b
). It contains not less than 90.0 per cent of C
48
H
74
O
14
(component H
2
B
1a
) and the sum of the contents of component H
2
B
1a
and component H
2
B
1b
is not
less than 95.0 per cent and not more than the equivalent of 100.5 per cent, both calculated with
reference to the anhydrous and solvent-free substance.
CHARACTERS
A white or yellowish-white, crystalline powder, slightly hygroscopic, practically insoluble in water,
freely soluble in methylene chloride, soluble in alcohol.
IDENTIFICATION
obtained with ivermectin CRS.
B. Examine the chromatograms obtained in the assay. The retention times and sizes of the two
principal peaks in the chromatogram obtained with the test solution are similar to those of the two
principal peaks in the chromatogram obtained with reference solution (a).
TESTS
Appearance of solution Dissolve 1.0 g in 50 ml of toluene R. The solution is clear (2.2.1) and not
more intensely coloured than reference solution BY
7
(Method II, 2.2.2).
22-64
same solvent. The specific optical rotation is 17 to 20, calculated with reference to the anhy-
drous and solvent-free substance.
Inject 20 l of the test solution. Continue the chromatography of the test solution for twice the
retention time of ivermectin. Inject 20 l of reference solution (b) and 20 l of reference solution (c).
In the chromatogram obtained with the test solution: the area of any peak with a retention time
relative to the principal peak of 1.3 to 1.5 is not greater than 2.5 times the area of the principal peak
in the chromatogram obtained with reference solution (b) (2.5 per cent); the area of any other peak,
apart from the two principal peaks, is not greater than the area of the principal peak in the chromato-
gram obtained with reference solution (b) (1 per cent); the sum of the areas of all the peaks, apart
from the two principal peaks, is not greater than five times the area of the principal peak in the
than that of the principal peak in the chromatogram obtained with reference solution (c).
Ethanol and formamide Not more than 5.0 per cent of ethanol and not more than 3.0 per cent of
formamide, determined by gas chromatography (2.2.28), using 2-propanol R as the internal standard.
Internal standard solution. Dilute 0.5 ml of 2-propanol R to 100 ml with water R.
Test solution. In a centrifuge tube, dissolve 0.120 g of the substance to be examined in 2.0 ml of m-
xylene R (if necessary heat in a water-bath at 40C to 50C). Add 2.0 ml of water R, mix thoroughly
and centrifuge. Remove the upper layer and extract it with 2.0 ml of water R. Discard the upper layer
and combine the aqueous layers. Add 1.0 ml of the internal standard solution. Centrifuge and
discard any remaining m-xylene.
Reference solution (a). Dilute 3.0 g of ethanol R to 100.0 ml with water R.
Reference solution (b). Dilute 1.0 g of formamide R to 100.0 ml with water R.
Reference solution (c). Dilute 5.0 ml of reference solution (a) and 5.0 ml of reference solution (b) to
50.0 ml with water R. Introduce 2.0 ml of this solution into a centrifuge tube, add 2.0 ml of
m-xylene R, mix thoroughly and centrifuge. Remove the upper layer and extract it with 2.0 ml of
water R. Discard the upper layer and combine the aqueous layers. Add 1.0 ml of the internal standard
solution. Centrifuge and discard any remaining m-xylene.
Reference solution (d). Dilute 10.0 ml of reference solution (a) and 10.0 ml of reference solution (b) to
50.0 ml with water R. Prepare as prescribed for reference solution (c) (from Introduce 2.0 ml of this
solution...).
a fused-silica capillary or wide-bore column 30 m long and 0.32 mm or 0.53 mm in internal
diameter coated with poly[(cyanopropyl)(phenyl)][dimethyl]siloxane R (film thickness 1.8 m or
3 m),
helium for chromatography R as the carrier gas, with a split ratio 1:5 and a linear velocity of about
35 cm/s,
Time
(min)
Temperature
(C)
Rate
(C per min)
Comment
512 40180 20 linear
gradient
1214 180 isothermal
Injection
port
140
Detector 250
Inject the test solution, reference solution (c) and reference solution (d).
of water.
ASSAY
22-65
Reference solution (a). Dissolve 40.0 mg of ivermectin CRS in methanol R and dilute to 50.0 ml with the
same solvent.
Reference solution (c). Dilute 5.0 ml of reference solution (b) to 100.0 ml with methanol R.
Mobile phase A. Water R,
Mobile phase B. Mix 35 volumes of methanol R and 53 volumes of acetonitrile R,
Equilibrate the column with a mobile phase ratio A:B of 15:85. Inject 20 l of reference solution (a).
The test is not valid unless, in the chromatogram obtained, the resolution between the first peak
(component H
2
B
1b
) and the second peak (component H
2
B
1a
) is at least 3.0 (if necessary, adjust the
ratio A:B of the mobile phase) and the symmetry factor of the principal peak is not greater than 2.5.
Inject 20 l of reference solution (c). Adjust the sensitivity of the system to obtain a principal peak
with a signal-to-noise ratio of at least ten. Inject reference solution (a) six times. The test is not valid
unless the relative standard deviation of the peak area for component H
2
B
1a
is at most 1.0 per cent.
Inject alternately the test solution and reference solution (a). Calculate the percentage contents of
component H
2
B
1a
and ivermectin (sum of components H
2
B
1a
and H
2
B
1b
).
STORAGE
IMPURITIES
OH
OH H
R
H
CH
3
H
Me
H
H
H
Me
Me
O
O
O O
O
Me
Me
OMe
O
O O
O
Me
OMe
HO
25
5
A. R = CH
2
-CH
3
: 5-O-demethylavermectin A
1a
(avermectin B
1a
),
B. R = CH
3
: 25-(1-methylethyl)-5-O-demethyl-25-de(1-methylpropyl)avermectin A
1a
(avermectin
B
1b
),
OH
OH H
R
H
CH
3
H
Me
H
H
H
Me
Me
O
O
O O
O
Me
R
2
R
1
Me
OMe
O
O O
O
Me
OMe
HO
22
28
5
23
C. R
1
= H
2
, R
2
= OH: (23S)-23-hydroxy-5-O-demethyl-22,23-dihydroavermectin A
1a
(avermectin
B
2a
),
D. R
1
= O, R
2
= H: 28-oxo-5-O-demethyl-22,23-dihydroavermectin A
1a
(28-oxo H
2
B
1a
),
22-66
OH
OH H
R
H
CH
3
H
Me
H
H
H
Me
Me
O
O
O O
O
Me
R
2
Me
OMe
O
O O
O
Me
OMe
HO
5
2
25
23
22
E. R = CH
2
-CH
3
: (2S)-5-O-demethyl-22,23-dihydroavermectin A
1a
(2-epimer H
2
B
1a
),
F. R = CH
3
: (2S)-25-(1-methylethyl)-5-O-demethyl-25-de(1-methylpropyl)-22,23-
dihydroavermectin A
1a
(2-epimer H
2
B
1b
),
22
OH
OH H
C
2
H
5
H
CH
3
H
Me
H
H
H
Me
Me
O
O
O O
O
Me
R
1
O
Me
OMe
HO
O
13
osyl =
G. R
1
= H: 5-O-demethyl-22,23-dihydroavermectin A
1a
aglycone (H
2
B
1a
aglycone),
H. R
1
= osyl: 4-O-de(2,6-dideoxy-3-O--L-arabino-hexopyranosyl)-5-O-demethyl-22,23-
dihydroavermectine A
1a
,
OH
OH H
R
H
CH
3
H
Me
H
H
H
Me
Me
O
O
O O
O
Me
Me
OMe
O
O O
O
Me
OMe
HO
23
5
25
2
3
*
and epimer at C*
22
I. R = CH
2
-CH
3
: 2,3-didehydro-5-O-demethyl-3,4,22,23-tetrahydroavermectin A
1a
(
2,3
H
2
B
1a
),
J. R = CH
3
: 25-(1-methylethyl)-2,7-didehydro-5-O-demethyl-25-de(1-methylpropyl)-3,4,22,23-
tetrahydroavermectin A
1a
(
2,3
H
2
B
1b
),
OH
OH H
C
2
H
5
H
CH
3
H
Me
H
H
H
Me
Me
O
O
O O
O
Me
Me
OMe
O
O O
O
Me
OMe
HO
22
*
and epimer at C*
4
1
19
7
K. (4R)-and (4S)-5-O-demethyl-3,4,22,23-tetrahydroavermectin A
1a
(H
4
B
1a
isomers).
__________________________________________________________________________________________________________ Ph Eur
22-67
Java Tea
Java Tea complies with the requirements of the 3rd edition of the European Pharmacopoeia [1129]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Java tea consists of the fragmented, dried leaves and tops of stems of Orthosiphon stamineus Benth.
(O. aristatus Miq.; O. spicatus Bak.).
CHARACTERS
IDENTIFICATION
A. The leaves are friable and may reach 7.5 cm in length and 2.5 cm in width. The petiole is short.
The lamina is oval to lanceolate, the apex acuminate and the base cuneate. The abaxial surface of the
leaves is light greyish-green and the adaxial surface is dark green to brownish-green. The venation is
pinnate with few secondary veins. Examined under a lens (10), the secondary veins, after running
parallel to the midrib, diverge at an acute angle. The margin is irregularly and roughly dentate,
sometimes crenate and the abaxial surface is slightly curved. The petioles are thin, quadrangular,
4 mm to 8 mm long and, like the primary venation, usually violet-coloured. Occasionally,
inflorescences in clusters of bluish-white to violet flowers, not yet opened, are found.
B. Reduce to a powder (355). The powder is dark green. Examine under a microscope using chloral
hydrate solution R. The powder shows fragments of epidermis, with cells with sinuous outlines bearing
unicellular or bicellular conical covering trichomes and articulated uniseriate trichomes up to 450 m
long, consisting of three to eight cells with thick pitted walls; capitate trichomes with unicellular or
bicellular heads; secretory trichomes with unicellular stalks and usually tetracellular heads; diacytic
stomata (2.8.3), which are more numerous on the lower epidermis.
substance.
Test solution. Shake 1 g of the powdered drug (710) for 5 min with 10 ml of methanol R in a water-
bath at 60C and filter the cooled solution.
Reference solution. Dissolve 1 mg of sinensetin R in methanol R and dilute to 10 ml with the same
solvent.
Develop over a path of 10 cm using a mixture of 5 volumes of methanol R, 40 volumes of ethyl
acetate R and 55 volumes of toluene R. Allow the plate to dry in air and examine in ultraviolet light at
365 nm. The chromatogram obtained with the reference solution shows in its middle part a zone of
intense light blue fluorescence due to sinensetin. The chromatogram obtained with the test solution
shows in its middle part a major zone of blue fluorescence corresponding to the sinensetin zone in the
chromatogram obtained with the reference solution. Above this zone one or two zones of more or less
intense blue to violet-blue fluorescence are visible. Two other zones of bluish fluorescence are below
the sinensetin zone. Zones of red fluorescence are visible in the lower third and near the solvent front.
TESTS
Foreign matter (2.8.2). Not more than 5 per cent of stems with a diameter greater than 1 mm and
not more than 2 per cent of other foreign matter.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
22-68
Juniper
1/01
Juniper complies with the requirements of the 3rd edition of the European Pharmacopoeia [1532]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Juniper consists of the dried ripe cone berry of Juniperus communis L. It contains not less than
10 ml/kg of essential oil, calculated with reference to the anhydrous drug.
CHARACTERS
Juniper has a strongly aromatic odour, especially if crushed.
IDENTIFICATION
A. The berry-shaped cone is globular up to 10 mm in diameter, violet-brown to blackish-brown,
frequently with a bluish bloom. It consists of three fleshy scales. The apex has a three-rayed closed
cleft and three not very clearly defined projections. A remnant of peduncle is frequently attached at
the base. The fleshy part is crumbly and brownish. It contains three, seldom two, small, elongated,
extremely hard seeds that have three sharp edges and are slightly rounded at the back, acuminate at
the apex. The seeds are fused with the fleshy part of the cone berry in the lower part on the outside of
their bases. Very large, oval oil glands containing sticky resin lie at the outer surface of the seeds.
B. Reduce to a powder (355). The powder is brown. Examine under a microscope, using chloral
hydrate solution R. The powder shows fragments of epidermis of the cone berry wall containing cells
with thick, pitted colourless walls and brown glandular content, occasionally with anomocytic
stomata (2.8.3); fragments of the three-rayed apical cleft of the cone berry with spaces and epidermal
cells interlocked by papillous outgrowths; fragments of the hypodermis with collenchymatous
thickened cells; fragments of the mesocarp consisting of large thin-walled parenchymatous cells,
usually rounded, with large intercellular spaces and irregular, large, usually scarcely pitted, yellow
idioblasts (barrel cells); fragments of schizogenous oil cells; fragments of the testa with thick-walled,
pitted colourless sclereids containing one or several prism crystals of calcium oxalate; fragments of
the endosperm and embryonic tissue with thin-walled cells containing fatty oil and aleurone grains.
Test solution. Dilute the oil-xylene mixture obtained in the assay to 5.0 ml with hexane R.
Reference solution. Dissolve 4.0 mg of guaiazulene R and 50 l of cineole R in 10 ml of hexane R.
a path of 15 cm using a mixture of 5 volumes of ethyl acetate R and 95 volumes of toluene R. Allow
the plate to dry in air. Spray the plate with anisaldehyde solution R and examine in daylight while
heating at 100C to 105C for 5 min to 10 min. The chromatogram obtained with the reference
solution shows a red zone (guaiazulene) in the upper half and a brownish-violet to greyish-violet zone
(cineole) in the lower half. The chromatogram obtained with the test solution shows a strong violet
zone (mono- and sesquiterpenes) similar in position to the zone due to guaiazulene in the chromato-
gram obtained with the reference solution, a reddish-violet zone a little above the zone corresponding
to cineole in the chromatogram obtained with the reference solution, a greyish-violet zone (terpinen-
4-ol) a little below the zone corresponding to cineole in the chromatogram obtained with the refer-
ence solution and just below it a blue zone. A faint violet zone may be present in a similar position to
the zone corresponding to cineole. Further zones are present.
TESTS
Foreign matter (2.8.2). Not more than 5 per cent of unripe or discoloured cone berries and not
more than 2 per cent of other foreign matter.
Water (2.2.13). Not more than 12.0 per cent, determined by distillation on 20.0 g of the crushed
drug.
ASSAY
tube. Crush the drug and immediately use 20.0 g for the determination. Distil at a rate of 3 ml/min
to 4 ml/min for 90 min.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
22-69
Kanamycin Acid Sulphate
Kanamycin Acid Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Kanamycin acid sulphate is a form of kanamycin sulphate prepared by adding sulphuric acid to a
solution of kanamycin monosulphate and drying by a suitable method. The potency is not less than
670 I.U. per milligram, calculated with reference to the dried substance.
PRODUCTION
It is produced by methods of manufacture designed to eliminate or minimise substances lowering
blood pressure. The method of manufacture is validated to demonstrate that the product if tested
would comply with the following test:
Abnormal toxicity (2.6.9). Inject into each mouse 0.5 ml of a solution containing 2 mg per
millilitre of the substance to be examined.
CHARACTERS
A white or almost white powder, hygroscopic, soluble in about 1 part of water, practically insoluble in
acetone, in alcohol and in ether.
IDENTIFICATION
A. Examine by thin-layer chromatography (2.2.27), using a plate coated with a 0.75 mm layer of the
following mixture: mix 0.3 g of carbomer R with 240 ml of water R and allow to stand, with moderate
shaking, for 1 h; adjust to pH 7 by the gradual addition, with continuous shaking, of dilute sodium
hydroxide solution R and add 30 g of silica gel H R.
Heat the plate at 110C for 1 h, allow to cool and use immediately.
same solvent.
Reference solution (a). Dissolve 10 mg of kanamycin monosulphate CRS in water R and dilute to 10 ml
Reference solution (b). Dissolve 10 mg of kanamycin monosulphate CRS, 10 mg of neomycin sulphate
CRS and 10 mg of streptomycin sulphate CRS in water R and dilute to 10 ml with the same solvent.
Apply separately to the plate 10 l of each solution. Develop over a path of 12 cm using a 70 g/l
solution of potassium dihydrogen phosphate R. Dry the plate in a current of warm air and spray with a
mixture of equal volumes of a 2 g/l solution of dihydroxynaphthalene R in alcohol R and a 460 g/l
solution of sulphuric acid R. Heat at 150C for 5 min to 10 min. The principal spot in the chromato-
gram obtained with the test solution is similar in position, colour and size to the principal spot in the
B. Dissolve 0.5 g in 10 ml of water R. Add 10 ml of picric acid solution R. Initiate crystallisation if
necessary by scratching the wall of the tube with a glass rod and allow to stand. Collect the crystals,
wash with 20 ml of water R and filter. Dry at 100C. The crystals melt (2.2.14) at about 235C, with
decomposition.
C. Dissolve about 50 mg in 2 ml of water R. Add 1 ml of a 10 g/l solution of ninhydrin R and heat for
a few minutes on a water-bath. A violet colour develops.
D. It gives the reactions of sulphates (2.3.1).
TESTS
Kanamycin B Examine by thin-layer chromatography (2.2.27), using a plate prepared as prescribed
under identification test A.
same solvent.
22-70
Reference solution. Dissolve 4 mg of kanamycin B sulphate CRS in water R and dilute to 20 ml with the
same solvent.
Apply separately to the plate 4 l of each solution. Develop over a path of 12 cm using a 70 g/l solu-
tion of potassium dihydrogen phosphate R. Dry the plate in a current of warm air and spray with
ninhydrin and stannous chloride reagent R. Heat the plate at 110C for 15 min. Any spot corresponding
to kanamycin B in the chromatogram obtained with the test solution is not more intense than the
spot in the chromatogram obtained with the reference solution (4.0 per cent).
4
4
).
Inject per kilogram of the rabbits mass 1 ml of a solution in water for injections R containing 10 mg
per millilitre of the substance to be examined.
ASSAY
Carry out the microbiological assay of antibiotics (2.7.2). Use kanamycin monosulphate CRS as the
reference substance.
STORAGE
Store in a well-closed container. If the substance is sterile, store in a sterile, tamper-proof container.
LABELLING
The label states:
__________________________________________________________________________________________________________ Ph Eur
22-71
Kanamycin Sulphate
NH
2
NH
2
OH
O
O
HO
H
2
NCH
2
HO
OH O
OH
O
CH
2
OH
HO
NH
2
,H
2
SO
4
C
18
H
36
N
4
O
11
,H
2
SO
4
,H
2
O 60125389-94-0 (anhydrous)
Kanamycin Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Kanamycin Monosulphate [0032]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Kanamycin monosulphate is O-3-amino-3-deoxy--D-glucopyranosyl-(16)-O-[6-amino-6-deoxy--
D-glucopyranosyl-(14)]-2-deoxy-D-streptamine sulphate, an antimicrobial substance produced by
the growth of certain strains of Streptomyces kanamyceticus. The potency is not less than 750 I.U. per
PRODUCTION
It is produced by methods of manufacture designed to eliminate or minimise substances lowering
blood pressure. The method of manufacture is validated to demonstrate that the product if tested
would comply with the following test:
Abnormal toxicity (2.6.9). Inject into each mouse 0.5 ml of a solution containing 2 mg per
millilitre of the substance to be examined.
CHARACTERS
A white or almost white, crystalline powder, soluble in about 8 parts of water, practically insoluble in
acetone, in alcohol and in ether.
IDENTIFICATION
A. Examine by thin-layer chromatography (2.2.27), using a plate coated with a 0.75 mm layer of the
following mixture: mix 0.3 g of carbomer R with 240 ml of water R and allow to stand, with moderate
shaking, for 1 h; adjust to pH 7 by the gradual addition, with continuous shaking, of dilute sodium
hydroxide solution R and add 30 g of silica gel H R.
same solvent.
Reference solution (a). Dissolve 10 mg of kanamycin monosulphate CRS in water R and dilute to 10 ml
Reference solution (b). Dissolve 10 mg of kanamycin monosulphate CRS, 10 mg of neomycin sulphate
CRS and 10 mg of streptomycin sulphate CRS in water R and dilute to 10 ml with the same solvent.
Apply separately to the plate 10 l of each solution. Develop over a path of 12 cm using a 70 g/l
solution of potassium dihydrogen phosphate R. Dry the plate in a current of warm air and spray with a
mixture of equal volumes of a 2 g/l solution of dihydroxynaphthalene R in alcohol R and a 460 g/l
solution of sulphuric acid R. Heat at 150C for 5 min to 10 min. The principal spot in the chromato-
gram obtained with the test solution is similar in position, colour and size to the principal spot in the
22-72
B. Dissolve 0.5 g in 10 ml of water R. Add 10 ml of picric acid solution R. Initiate crystallisation if
necessary by scratching the wall of the tube with a glass rod and allow to stand. Collect the crystals,
wash with 20 ml of water R and filter. Dry at 100C. The crystals melt (2.2.14) at about 235C, with
decomposition.
C. Dissolve about 50 mg in 2 ml of water R. Add 1 ml of a 10 g/l solution of ninhydrin R and heat for
a few minutes on a water-bath. A violet colour develops.
D. It gives the reactions of sulphates (2.3.1).
TESTS
Kanamycin B Examine by thin-layer chromatography (2.2.27), using a plate prepared as prescribed
under identification test A. Heat the plate at 110C for 1 h, allow to cool and use immediately.
same solvent.
Reference solution. Dissolve 4 mg of kanamycin B sulphate CRS in water R and dilute to 20 ml with the
same solvent.
Apply separately to the plate 4 l of each solution. Develop over a path of 12 cm using a 70 g/l solu-
tion of potassium dihydrogen phosphate R. Dry the plate in a current of warm air and spray with
ninhydrin and stannous chloride reagent R. Heat the plate at 110C for 15 min. Any spot corresponding
to kanamycin B in the chromatogram obtained with the test solution is not more intense than the
4
4
).
Inject per kilogram of the rabbits mass 1 ml of a solution in water for injections R containing 10 mg
per millilitre of the substance to be examined.
ASSAY
STORAGE
Store in a well-closed container. If the substance is sterile, store in a sterile, tamper-proof container.
LABELLING
The label states:
__________________________________________________________________________________________________________ Ph Eur
23-1
Heavy Kaolin
Heavy Kaolin complies with the requirements of the 3rd edition of the European Pharmacopoeia [0503]. These
Preparation
Kaolin Poultice
When kaolin is prescribed or demanded, Light Kaolin shall be dispensed or supplied, unless it is
ascertained that Light Kaolin (Natural) is required.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Heavy kaolin is a purified, natural, hydrated aluminium silicate of variable composition.
CHARACTERS
A fine, white or greyish-white, unctuous powder, practically insoluble in water and in organic
solvents.
IDENTIFICATION
A. To 0.5 g in a metal crucible add 1 g of potassium nitrate R and 3 g of sodium carbonate R and heat
until the mixture melts. Allow to cool. To the residue add 20 ml of boiling water R, mix and filter.
Wash the residue with 50 ml of water R. To the residue add 1 ml of hydrochloric acid R and 5 ml of
water R. Filter. To the filtrate add 1 ml of strong sodium hydroxide solution R and filter. To the filtrate
add 3 ml of ammonium chloride solution R. A gelatinous white precipitate is formed.
B. Add 2.0 g in twenty portions to 100 ml of a 10 g/l solution of sodium lauryl sulphate R in a 100 ml
graduated cylinder about 30 mm in diameter. Allow 2 min between additions for each portion to
settle. Allow to stand for 2 h. The apparent volume of the sediment is not greater than 5 ml.
C. 0.25 g gives the reaction of silicates (2.3.1).
TESTS
Solution S To 4 g add a mixture of 6 ml of acetic acid R and 34 ml of distilled water R, shake for
1 min and filter.
To 10 ml of the filtrate add 0.1 ml of phenolphthalein solution R. The solution is colourless. Not more
than 0.25 ml of 0.01M sodium hydroxide is required to change the colour of the indicator to pink.
Organic impurities Heat 0.3 g to redness in a calcination tube. The residue is only slightly more
coloured than the original substance.
Adsorption power To 1.0 g in a ground-glass-stoppered test-tube add 10.0 ml of a 3.7 g/l solution
of methylene blue R and shake for 2 min. Allow to settle. Centrifuge and dilute the solution 1 in 100
with water R. The solution is not more intensely coloured than a 0.03 g/l solution of methylene blue R.
Swelling power Triturate 2 g with 2 ml of water R. The mixture does not flow.
Substances soluble in mineral acids To 5.0 g add 7.5 ml of dilute hydrochloric acid R and 27.5 ml
of water R and boil for 5 min. Filter, wash the residue on the filter with water R and dilute the
combined filtrate and washings to 50.0 ml with water R. To 10.0 ml of the solution add 1.5 ml of
dilute sulphuric acid R, evaporate to dryness on a water-bath and ignite. The residue weighs not more
than 10 mg (1 per cent).
limit test for sulphates (0.1 per cent).
Heavy metals (2.4.8). To 5 ml of the solution prepared for the test for substances soluble in mineral
acids add 5 ml of water R, 10 ml of hydrochloric acid R and 25 ml of methyl isobutyl ketone R. Shake for
2 min. Separate the layers. Evaporate the aqueous layer to dryness on a water-bath. Dissolve the
residue in 1 ml of acetic acid R and dilute to 25 ml with water R. Filter. 12 ml of the solution complies
with limit test A for heavy metals (50 ppm). Prepare the standard using lead standard solution (1 ppm
Pb) R.
Heavy kaolin intended for internal use complies with the requirements of the monograph modified as shown
below for the test for heavy metals.
23-2
Heavy metals (2.4.8). To 10 ml of the solution prepared for the test for substances soluble
in mineral acids add 10 ml of water R, 20 ml of hydrochloric acid R and 25 ml of methyl isobutyl
ketone R. Shake for 2 min. Separate the layers. Evaporate the aqueous layer to dryness on a
water-bath. Dissolve the residue in 1 ml of acetic acid R and dilute to 25 ml with water R. Filter.
12 ml of the solution complies with limit test A for heavy metals (25 ppm). Prepare the
3
micro-organisms
per gram, determined by plate-count.
LABELLING
The label states, where applicable, that the substance is suitable for internal use.
__________________________________________________________________________________________________________ Ph Eur
23-3
Light Kaolin
Definition Light Kaolin is a native hydrated aluminium silicate, freed from most of its impurities by
elutriation and dried. It contains a suitable dispersing agent.
Characteristics A light, white powder free from gritty particles; odourless or almost odourless;
unctuous.
Practically insoluble in water and in mineral acids.
Identification
A. To 0.5 g in a metal crucible add 1 g of potassium nitrate and 3 g of sodium carbonate and heat until
the mixture melts. Allow to cool. To the residue add 20 ml of boiling water, mix and filter. Wash the
residue with 50 ml of water, add to the residue 1 ml of hydrochloric acid and 5 ml of water, mix and
filter. To the filtrate add 1 ml of strong sodium hydroxide solution, filter and add to the filtrate 3 ml of
ammonium chloride solution. A gelatinous white precipitate is produced.
B. 0.25 g yields the reaction characteristic of silicates, Appendix VI.
C. Triturate 2 g with 2 ml of water. The resulting mixture flows.
Coarse particles Transfer 5 g to a stoppered cylinder (about 16 cm 35 mm), add 60 ml of a 1%
w/v solution of sodium pyrophosphate, shake thoroughly and allow to stand for 5 minutes. Using a
pipette, withdraw 50 ml from a point about 5 cm below the surface of the liquid. To the remaining
liquid add 50 ml of water, shake, allow to stand for 5 minutes and withdraw 50 ml in the same
manner as before. Repeat the operation until a total of 400 ml of suspension has been withdrawn
under the prescribed conditions. Transfer the remainder to an evaporating dish and evaporate to
dryness on a water bath. The residue, after drying at 105, weighs not more than 25 mg.
Fine particles Disperse 5 g in 250 ml of water by shaking vigorously for 2 minutes in a stoppered
flask, pour immediately into a glass cylinder 5 cm in diameter and transfer 20 ml to a glass dish using
a pipette. Evaporate to dryness and dry to constant weight at 105. Allow the remainder of the
suspension to stand for 4 hours at 20 and withdraw a second 20-ml portion using a pipette with its
tip exactly 5 cm below the surface and without disturbing the sediment. Transfer the second portion
to a glass dish, evaporate to dryness and dry to constant weight at 105. The weight of the residue
from the second portion is not less than 70% of the weight of the residue from the first portion.
Arsenic 0.50 g dispersed in 25 ml of water complies with the limit test for arsenic, Appendix VII
(2 ppm).
Heavy metals Heat 6.0 g for 15 minutes under a reflux condenser on a water bath with a mixture of
70 ml of water and 10 ml of hydrochloric acid and filter. To 40 ml of the filtrate add 0.5 ml of nitric
acid and evaporate to low bulk. Add 20 ml of water, 2 g of ammonium chloride and 2 g of ammonium
thiocyanate and extract with two 10-ml quantities of a mixture of equal volumes of isoamyl alcohol and
ether. To the aqueous layer add 2 g of citric acid and sufficient water to produce 60 ml. 12 ml of the
resulting solution complies with limit test A for heavy metals, Appendix VII. Use lead standard solution
Chloride Boil 1.0 g with 80 ml of water and 20 ml of 2M nitric acid under a reflux condenser for 5
minutes, cool and filter. 15 ml of the filtrate complies with the limit test for chlorides, Appendix VII
(330 ppm).
1 g.
Loss on ignition When ignited at 600, loses not more than 15.0% of its weight. Use 1 g.
Soluble matter Boil 2 g with 100 ml of 0.2M hydrochloric acid under a reflux condenser for 5
minutes, cool, filter and evaporate 50 ml of the filtrate to dryness. The residue, after ignition at about
600 for 30 minutes, weighs not more than 10 mg.
Preparations
Kaolin Mixture
Kaolin and Morphine Mixture
When Kaolin or Light Kaolin is prescribed or demanded, Light Kaolin shall be dispensed or supplied
unless it is ascertained that Light Kaolin (Natural) is required.
23-4
Light Kaolin (Natural)
Definition Light Kaolin (Natural) is a native hydrated aluminium silicate, freed from most of its
impurities by elutriation and dried. It does not contain a dispersing agent.
Characteristics A light, white powder free from gritty particles; odourless or almost odourless;
unctuous.
Practically insoluble in water and in mineral acids.
Identification
A. To 0.5 g in a metal crucible add 1 g of potassium nitrate and 3 g of sodium carbonate and heat until
the mixture melts. Allow to cool. To the residue add 20 ml of boiling water, mix and filter. Wash the
residue with 50 ml of water, add to the residue 1 ml of hydrochloric acid and 5 ml of water, mix and
filter. To the filtrate add 1 ml of strong sodium hydroxide solution, filter and add to the filtrate 3 ml of
ammonium chloride solution. A gelatinous white precipitate is produced.
B. 0.25 g yields the reaction characteristic of silicates, Appendix VI.
C. Triturate 2 g with 2 ml of water. The resulting mixture does not flow.
Coarse particles; Arsenic; Heavy metals; Chloride; Loss on drying; Loss on ignition;
Soluble matter Complies with the requirements stated under Light Kaolin.
Fine particles Disperse 5 g in 250 ml of water containing 50 mg of sodium pyrophosphate by shaking
vigorously for 2 minutes in a stoppered flask, pour immediately into a glass cylinder 5 cm in diameter
and transfer 20 ml to a glass dish using a pipette. Evaporate to dryness and dry to constant weight at
105. Allow the remainder of the suspension to stand for 4 hours at 20 and withdraw a second
20-ml portion using a pipette with its tip exactly 5 cm below the surface and without disturbing the
sediment. Transfer the second portion to a glass dish, evaporate to dryness and dry to constant
weight at 105. The weight of the residue from the second portion is not less than 70% of the weight
of the residue from the first portion.
Preparations
Kaolin Mixture
Kaolin and Morphine Mixture
When Kaolin or Light Kaolin is prescribed or demanded, Light Kaolin shall be dispensed or supplied
unless it is ascertained that Light Kaolin (Natural) is required.
23-5
Ketamine Hydrochloride
1/01
O
Cl
NHMe
and enantiomer
,HCl
C
13
H
16
ClNO,HCl 274.2 1867-86-9
Ketamine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Anaesthetic.
Preparation
Ketamine Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ketamine hydrochloride contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of (RS)-2-(2-chlorophenyl)-2-(methylamino)cyclohexanone hydrochloride.
CHARACTERS
A white, crystalline powder, freely soluble in water and in methanol, soluble in alcohol.
IDENTIFICATION
spectrum of ketamine hydrochloride.
TESTS
Reference solution (a). Dissolve 25.0 mg of ketamine impurity A CRS in the mobile phase and dilute to
50.0 ml with the mobile phase (using ultrasound, if necessary). To 1.0 ml of the solution, add 0.5 ml
of the test solution and dilute to 100.0 ml with the mobile phase. Prepare immediately before use.
a stainless steel precolumn 4 mm long and 4.0 mm in internal diameter, followed by a stainless
steel column 0.125 m long and 4.0 mm in internal diameter, both packed with spherical octa-
decylsilyl silica gel for chromatography R (5 m),
of sodium hexanesulphonate R in 1 litre of a mixture of 25 volumes of acetonitrile R and 75
volumes of water R and add 4 ml of acetic acid R,
Inject 20 l of each solution. Continue the chromatography for ten times the retention time of
ketamine. The test is not valid unless in the chromatogram obtained with reference solution (a) the
resolution between the peaks due to impurity A and ketamine is at least 1.5 and unless the retention
time of the peak due to ketamine is 3 min to 4.5 min. If necessary, adjust the concentrations of water
and acetonitrile in the mobile phase. In the chromatogram obtained with the test solution, the sum of
the areas of any peaks apart from the principal peak is not greater than the area of the principal peak
23-6
in the chromatogram obtained with reference solution (b) (0.5 per cent). Disregard any peak with
an area less than 0.2 times that of the peak in the chromatogram obtained with reference solution
(b).
ASSAY
Dissolve 0.200 g in 50 ml of methanol R and add 1.0 ml of 0.1M hydrochloric acid. Carry out a poten-
tiometric titration (2.2.20), using 0.1M sodium hydroxide. Read the volume added between the two
13
H
17
Cl
2
NO.
STORAGE
IMPURITIES
NMe
Cl
OH

A. 1-[(2-chlorophenyl)(methylimino)methyl]cyclopentanol,
Cl
O
OH
B. (RS)-2-(2-chlorophenyl)-2-hydroxycyclohexanone,
O
Cl
OH

C. 2-chlorophenyl-1-hydroxycyclopentyl ketone.
__________________________________________________________________________________________________________ Ph Eur
23-7
Ketoconazole
O
O
N
N
Cl
Cl
O
N
N
Ac
H
and enantiomer
C
26
H
28
Cl
2
N
4
O
4
531.4 65277-42-1
Ketoconazole complies with the requirements of the 3rd edition of the European Pharmacopoeia [0921]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ketoconazole contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of 1-acetyl-4-[4-[[(2RS,4SR)-2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolan-4-
yl]methoxy]phenyl]piperazine, calculated with reference to the dried substance.
CHARACTERS
soluble in methanol, sparingly soluble in alcohol.
IDENTIFICATION
obtained with ketoconazole CRS. Examine the substances prepared as discs.
coating substance.
Test solution. Dissolve 30 mg of the substance to be examined in the mobile phase and dilute to 5 ml
Reference solution (a). Dissolve 30 mg of ketoconazole CRS in the mobile phase and dilute to 5 ml with
the mobile phase.
Reference solution (b). Dissolve 30 mg of ketoconazole CRS and 30 mg of econazole nitrate CRS in the
mobile phase and dilute to 5 ml with the mobile phase.
D. To about 30 mg in a porcelain crucible add 0.3 g of anhydrous sodium carbonate R. Heat over an
open flame for 10 min. Allow to cool. Take up the residue with 5 ml of dilute nitric acid R and filter.
To 1 ml of the filtrate add 1 ml of water R. The solution gives reaction (a) of chlorides (2.3.1).
TESTS
Solution S Dissolve 1.0 g in methylene chloride R and dilute to 10 ml with the same solvent.
solution BY
4
(Method II, 2.2.2).
23-8
+0.10.
Reference solution (a). Dissolve 2.5 mg of ketoconazole CRS and 2.5 mg of loperamide hydrochloride
CRS in methanol R and dilute to 50.0 ml with the same solvent.
as mobile phase at a flow rate of 2 ml per minute a mixture of 0.5 volumes of acetonitrile R and
9.5 volumes of a 3.4 g/l solution of tetrabutylammonium hydrogen sulphate R changing by linear-
gradient elution to a mixture of 5 volumes of acetonitrile R and 5 volumes of a 3.4 g/l solution of
tetrabutylammonium hydrogen sulphate R over 10 min, followed by the final elution mixture for
5 min,
elution composition for at least 5 min.
conditions, the retention times are: ketoconazole, about 6 min; loperamide hydrochloride, about
8 min. The test is not valid unless the resolution between the peaks corresponding to ketoconazole
and loperamide hydrochloride is at least 15. If necessary, adjust the final concentration of acetonitrile
in the mobile phase or adjust the time programme for the linear-gradient elution.
solution (b). In the chromatogram obtained with the test solution, the sum of the areas of all peaks,
obtained with reference solution (b) (0.5 per cent). Disregard any peak obtained with the blank and
100C to 105C.
ASSAY
ethyl ketone R. Titrate with 0.1M perchloric acid, determining the end-point potentiometrically
(2.2.20).
26
H
28
Cl
2
N
4
O
4
.
STORAGE
IMPURITIES
R =
O
O
N
N
Cl
Cl
O
H
23-9
N
N H
3
C
O
R
and enantiomer
A. 1-acetyl-4-[4-[[(2RS,4SR)-2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-
dioxolan-4-yl]methoxy]phenyl]-1,2,3,4-tetrahydropyrazine,
N
N H
3
C
O
R
N
N H
3
C
O
O
and enantiomer
B. 1-acetyl-4-[4-[[(2RS,4SR)-2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-
dioxolan-4-yl]methoxy]-3-[4-(4-acetyl-1-piperazinyl)phenoxy]phenyl]piperazine,
N
N H
3
C
O
O
Cl
Cl
N
N
O
O
H
and enantiomer
C. 1-acetyl-4-[4-[[(2RS,4SR)-2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-
dioxolan-4-yl]methoxy]phenyl]piperazine,
H
N
N
R
and enantiomer
D. 1-[4-[[(2RS,4SR)-2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolan-4-
yl]methoxy]phenyl]piperazine.
__________________________________________________________________________________________________________ Ph Eur
23-10
Ketoprofen
and enantiomer
H Me
COOH
O
C
16
H
14
O
3
254.3 22071-15-4
Ketoprofen complies with the requirements of the 3rd edition of the European Pharmacopoeia [0922]. These
Preparations
Ketoprofen Capsules
Ketoprofen Gel
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ketoprofen contains not less than 99.0 per cent and not more than the equivalent of 100.5 per cent
of (RS)-2-(3-benzoylphenyl)propionic acid, calculated with reference to the dried substance.
CHARACTERS
in alcohol and in methylene chloride.
IDENTIFICATION
B. Dissolve 50.0 mg in alcohol R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml to
50.0 ml with alcohol R. Examined between 230 nm and 350 nm (2.2.25), the solution shows an
absorption maximum at 255 nm. The specific absorbance at the maximum is 615 to 680.
obtained with ketoprofen CRS.
254
the same solvent.
Reference solution (a). Dissolve 10 mg of ketoprofen CRS in acetone R and dilute to 10 ml with the
same solvent.
Reference solution (b). Dissolve 10 mg of indometacin CRS in acetone R and dilute to 10 ml with the
same solvent. To 1 ml add 1 ml of reference solution (a).
1 volume of glacial acetic acid R, 49 volumes of methylene chloride R and 50 volumes of acetone R.
gram obtained with reference solution (b) shows two clearly separated principal spots.
TESTS
Appearance of solution Dissolve 1.0 g in acetone R and dilute to 10 ml with the same solvent. The
6
(Method II,
2.2.2).
Related substances Prepare the solutions immediately before use. Examine by liquid chromatography
(2.2.29).
1.0 ml to 10.0 ml with the mobile phase.
23-11
Reference solution (b). Dissolve 10.0 mg of ketoprofen impurity A CRS in the mobile phase and dilute to
100.0 ml with the mobile phase. Dilute 1.0 ml to 50.0 ml with the mobile phase.
Reference solution (c). Dissolve 10.0 mg of ketoprofen impurity C CRS in the mobile phase and dilute to
100.0 ml with the mobile phase. Dilute 1.0 ml to 50.0 ml with the mobile phase.
Reference solution (d). Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. To 1.0 ml
add 1.0 ml of reference solution (b).
a stainless steel column 0.15 m long and 4.6 mm in internal diameter packed with a spherical
octadecylsilyl silica gel for chromatography R (5 m) with a specific surface of 350 m
2
g
1
and a pore
size of 0.01 m (10 nm),
as mobile phase at a flow rate of 1 ml per minute a mixture of 2 volumes of phosphate buffer
solution pH 3.5 R, freshly prepared, 43 volumes of acetonitrile R and 55 volumes of water R,
a loop injector.
Inject 20 l of reference solution (d). The substances are eluted in the following order: ketoprofen
and ketoprofen impurity A. Adjust the sensitivity of the detector so that the heights of the two
principal peaks in the chromatogram obtained are not less than 50 per cent of the full scale of the
recorder. The test is not valid unless the resolution between the peaks corresponding to ketoprofen
and ketoprofen impurity A is at least 7.0.
Inject 20 l of the test solution, 20 l of reference solution (a), 20 l of reference solution (b) and
20 l of reference solution (c). Continue the chromatography of the test solution for seven times the
retention time of ketoprofen. In the chromatogram obtained with the test solution: the areas of any
peaks corresponding to ketoprofen impurity A and ketoprofen impurity C are not greater than the
areas of the principal peaks in the chromatograms obtained with reference solution (b) and reference
solution (c), respectively (0.2 per cent); the area of any peak, apart from the principal peak and any
peaks corresponding to ketoprofen impurity A and ketoprofen impurity C, is not greater than the area
sum of the areas of all the peaks, apart from the principal peak and any peaks corresponding to the
two named impurities, is not greater than twice the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.4 per cent). Disregard any peak with an area less than 0.1
times the area of the principal peak in the chromatogram obtained with reference solution (a).
Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g at 60C at a pressure
not exceeding 670 Pa.
ASSAY
Dissolve 0.200 g in 25 ml of alcohol R. Add 25 ml of water R. Titrate with 0.1M sodium hydroxide,
16
H
14
O
3
.
STORAGE
IMPURITIES
CH
3
O O
A. 3-acetylbenzophenone,
COOH
O
B. 2-(3-benzoylphenyl)acetic acid,
COOH
Me H
HOOC
C. 2-(3-carboxyphenyl)propionic acid,
23-12
COOH
Me H
Me
O
D. 2-[3-(4-methylbenzoyl)phenyl]propionic acid,
CONH
2
Me H O
E. 2-(3-benzoylphenyl)propionamide,
CN
Me H O
F. 2-(3-benzoylphenyl)propionitrile.
__________________________________________________________________________________________________________ Ph Eur
23-13
Labetalol Hydrochloride
,HCl
N
Me H
OH H
CONH
2
OH
H
mixture of 4 isomers
* *
C
19
H
24
N
2
O
3
,HCl 364.9 32780-64-6
Labetalol Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Alpha- and beta-adrenoceptor antagonist.
Preparations
Labetalol Injection
Labetalol Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Labetalol hydrochloride contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of 2-hydroxy-5-[1-hydroxy-2-[(1-methyl-3-phenylpropyl)amino]ethyl]benzamide
CHARACTERS
A white or almost white powder, sparingly soluble in water and in alcohol, practically insoluble in
ether and in methylene chloride.
IDENTIFICATION
A. Determined on solution S (see Tests), the angle of optical rotation (2.2.7) is +0.05 to 0.05.
B. Dissolve 25.0 mg in 0.1M hydrochloric acid and dilute to 250.0 ml with the same acid. Examined
between 230 nm and 350 nm (2.2.25), the solution shows an absorption maximum at 302 nm. The
obtained with labetalol hydrochloride CRS.
D. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable
Test solution. Dissolve 10 mg of the substance to be examined in 1 ml of alcohol R.
Reference solution (a). Dissolve 10 mg of labetalol hydrochloride CRS in 1 ml of alcohol R.
Reference solution (b). Dissolve 10 mg of labetalol hydrochloride CRS and 10 mg of propranolol hydro-
chloride CRS in alcohol R and dilute to 5 ml with the same solvent.
Apply separately to the plate 2 l of each solution. Place the plate in a chromatographic tank
immediately after the addition of the mobile phase consisting of a mixture of 0.5 volumes of perchloric
acid R, 50 volumes of water R and 80 volumes of methanol R. Close the tank and develop over a path
of 15 cm. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot
TESTS
Solution S must be freshly prepared.
Diastereoisomer ratio Examine by gas chromatography (2.2.28).
23-14
Test solution. Dissolve 2.0 mg of the substance to be examined in 1.0 ml of a 12.0 g/l solution of
butylboronic acid R in anhydrous pyridine R and allow to stand for 20 min.
maintaining the temperature of the column, and that of the injection port and the detector at 300C.
Inject 2 l of the test solution. Two peaks, each corresponding to a pair of diastereoisomers, appear
in the chromatogram. Adjust the sensitivity of the system so that in the chromatogram obtained, the
height of the taller of the diastereoisomer peaks is about 80 per cent of the full scale of the recorder.
The test is not valid unless the height of the trough separating these peaks is less than 5 per cent of
the full scale of the recorder. The area of each peak is not less than 45 per cent and not more than
55 per cent of the total area of the two peaks.
as mobile phase at a flow rate of 1 ml per minute a mixture of 150 ml of tetrahydrofuran R,
300 ml of methanol R, 550 ml of water R, 0.82 g of tetrabutylammonium hydrogen sulphate R, 1 g
of sodium octyl sulphate R and 10 ml of a 10 per cent V/V solution of sulphuric acid R,
obtained with 20 l of the reference solution is at least 50 per cent of the full scale of the recorder.
When the chromatograms are recorded in the prescribed conditions, the retention time of the
principal peak is 10 min to 15 min. If necessary, adjust the water content of the mobile phase
ensuring that the 2:1 ratio of methanol to tetrahydrofuran is maintained.
Inject separately 20 l of each solution. Continue the chromatography for three times the retention
time of the principal peak. In the chromatogram obtained with the test solution: the area of any peak,
apart from the principal peak, is not greater than 0.6 times that of the principal peak in the chromato-
gram obtained with the reference solution (0.3 per cent); the sum of the areas of any such peaks is
not greater than the area of the principal peak in the chromatogram obtained with the reference
solution (0.5 per cent). Disregard any peak due to the solvent and any peak with an area less than 0.1
times the area of the principal peak in the chromatogram obtained with the reference solution.
Heavy metals (2.4.8).1.0 g complies with limit test C for heavy metals (20 ppm). Prepare the
100C to 105C at a pressure not exceeding 0.7 kPa.
ASSAY
Dissolve 0.200 g in a mixture of 10 ml of anhydrous formic acid R and 40 ml of acetic anhydride R.
19
H
25
ClN
2
O
3
.
IMPURITIES
H
N
Me
OH
OH
OR
O
A. R = H: 2-hydroxy-5-[1-hydroxy-2-[(1-methyl-3-phenylpropyl)amino]ethyl]benzoic acid,
B. R = CH
3
: methyl 2-hydroxy-5-[1-hydroxy-2-[(1-methyl-3-phenylpropyl)amino]-
ethyl]benzoate.
__________________________________________________________________________________________________________ Ph Eur
23-15
Lactic Acid
H
3
C COOH
OH H
C
3
H
6
O
3
90.1 50-21-5
Lactic Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia [0458]. These
Preparations
Sodium Lactate Intravenous Infusion
Compound Sodium Lactate Intravenous Infusion
Lactic Acid Pessaries
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Lactic acid consists of a mixture of 2-hydroxypropionic acid, its condensation products, such as
lactoyl-lactic acid and polylactic acids, and water. The equilibrium between lactic acid and polylactic
acids depends on the concentration and temperature. Lactic acid is usually a racemate (RS-lactic
acid), but the (+)-(S)-isomer may predominate. Lactic acid contains the equivalent of not less than
88.0 per cent m/m and not more than 92.0 per cent m/m of C
3
H
6
O
3
.
CHARACTERS
A colourless or slightly yellow, syrupy liquid, miscible with water, with alcohol and with ether.
IDENTIFICATION
B. Relative density (2.2.5): 1.20 to 1.21.
C. It gives the reaction of lactates (2.3.1).
TESTS
Solution S Dissolve 5.0 g in 42 ml of 1M sodium hydroxide and dilute to 50 ml with distilled water R.
Appearance of the substance It is not more intensely coloured than reference solution Y
6
(Method
II, 2.2.2).
Ether-insoluble substances Dissolve 1.0 g in 25 ml of ether R. The solution is not more opalescent
than the solvent used for the test.
Sugars and other reducing substances To 1 ml of solution S add 1 ml of 1M hydrochloric acid,
heat to boiling, allow to cool and add 1.5 ml of 1M sodium hydroxide and 2 ml of cupri-tartaric
solution R. Heat to boiling. No red or greenish precipitate is formed.
Volatile fatty acids Heat 5 g cautiously in a ground-glass-stoppered 100 ml flask at 50C for
10 min. No unpleasant odour resembling that of the lower fatty acids is recognisable immediately
after opening the flask.
Methanol and methyl esters Place 2.0 g in a ground-glass-stoppered, round-bottomed flask and
add 10 ml of water R. Cool in iced water and cautiously add 30 ml of a 300 g/l solution of potassium
hydroxide R. Allow the flask to stand in iced water for 10 min to 15 min. Fit a condenser and steam
distil. Collect the distillate in a 10 ml graduated flask containing 1 ml of ethanol R. Distil to a volume
of at least 9.5 ml and dilute to 10.0 ml with water R. Transfer 1.0 ml of the distillate to a test-tube,
add 5 ml of potassium permanganate and phosphoric acid solution R and mix. Allow to stand for 15 min.
Add 2 ml of oxalic acid and sulphuric acid solution R, stir with a glass rod until the solution is colourless
and add 5 ml of decolorised fuchsin solution R. Allow to stand for 2 h. The solution is not more
intensely coloured than a standard prepared at the same time and in the same manner using, instead
of the distillate, 1.0 ml of a solution containing 100 g of methanol R and 0.1 ml of ethanol R per
millilitre (500 ppm of methanol).
Citric, oxalic and phosphoric acids To 5 ml of solution S add dilute ammonia R1 until slightly
alkaline (2.2.4). Add 1 ml of calcium chloride solution R. Heat on a water-bath for 5 min. Both before
and after heating, any opalescence in the solution is not more intense than that in a mixture of 1 ml
of water R and 5 ml of solution S.
23-16
Pyrogens (2.6.8). If intended for use in the manufacture of large-volume preparations for parenteral
use without a further appropriate procedure for the removal of pyrogens, the competent authority
may require that it comply with the test for pyrogens. Inject per kilogram of the rabbits mass 10 ml
of a solution prepared as follows: to a quantity of the substance to be examined equivalent to 3.46 g
of C
3
H
6
O
3
add 38.4 ml of 1M sodium hydroxide prepared using water for injections R and 100 ml of
water for injections R : boil for 5 min, cool and dilute to 250 ml with water for injections R.
ASSAY
Place 1.000 g in a ground-glass-stoppered flask and add 10 ml of water R and 20.0 ml of 1M sodium
hydroxide. Close the flask and allow to stand for 30 min. Using 0.5 ml of phenolphthalein solution R as
indicator, titrate with 1M hydrochloric acid until the pink colour is discharged.
3
H
6
O
3
.
LABELLING
The label states, where appropriate, that the substance is free from bacterial endotoxins.
__________________________________________________________________________________________________________ Ph Eur
23-17
Lactitol Monohydrate
CH
2
OH
OH H
H HO
H
H OH
CH
2
OH
CH
2
OH
OH
OH
O
O
HO
C
12
H
24
O
11
,H
2
O 362.3 585-86-4 (anhydrous)
Lactitol Monohydrate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Osmotic laxative.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Lactitol monohydrate contains not less than 97.0 per cent of 4-O-(-D-galactopyranosyl)-D-glucitol,
CHARACTERS
A white crystalline powder, very soluble in water, slightly soluble in alcohol, practically insoluble in
methylene chloride.
IDENTIFICATION
obtained with lactitol monohydrate CRS.
C. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel G plate R .
the same solvent.
Reference solution (a). Dissolve 50 mg of lactitol monohydrate CRS in methanol R and dilute to 20 ml
Reference solution (b). Dissolve 5 mg of sorbitol CRS in 2 ml of reference solution (a) and dilute to
volumes of water R and 75 volumes of acetonitrile R. Allow the plate to dry in air and spray with
4-aminobenzoic acid solution R. Dry the plate in a current of cold air until the acetonitrile is removed.
Heat the plate at 100C for 15 min. Allow to cool and spray with a 2 g/l solution of sodium per-
iodate R. Dry the plate in a current of cold air. Heat the plate at 100C for 15 min. The principal spot
the chromatogram of reference solution (b) shows two clearly separated spots.
TESTS
solvent.
Appearance of solution Solution S is clear and not more intensely coloured than reference solution
BY
7
(Method II, 2.2.2).
Acidity or alkalinity To 10 ml of solution S add 10 ml of carbon dioxide-free water R. To 10 ml of
this solution add 0.05 ml of phenolphthalein solution R. Not more than 0.2 ml of 0.01M sodium
hydroxide is required to change the colour of the indicator to pink. To a further 10 ml of the solution
add 0.05 ml of methyl red solution R. Not more than 0.3 ml of 0.01M hydrochloric acid is required to
change the colour of the indicator to red.
Calculate the percentage content of related substances from the areas of the peaks in the chrom-
atogram obtained with the test solution by the normalisation procedure. The content of lactulitol is
not greater than 1.5 per cent and the content of the sum of any other related substances is not greater
than 1.5 per cent.
of cupri-citric solution R and a few glass beads. Heat so that boiling begins after 4 min and maintain
boiling for 3 min. Cool rapidly and add 100 ml of a 2.4 per cent V/V solution of glacial acetic acid R
and 20.0 ml of 0.025M iodine. With continuous shaking, add 25 ml of a mixture of 6 volumes of
hydrochloric acid R and 94 volumes of water R and when the precipitate has dissolved, titrate the
excess of iodine with 0.05M sodium thiosulphate using 1 ml of starch solution R added towards the end
of the titration, as indicator. Not less than 12.8 ml of 0.05M sodium thiosulphate is required (0.2 per
cent).
Nickel (2.4.15). It complies with the limit test for nickel in polyols (1 ppm).
Water (2.5.12). Between 4.5 per cent and 5.5 per cent, determined on 0.30 g by the semi-micro
3
micro-organisms
per gram.
It complies with the tests for Escherichia coli, Salmonella and for Pseudomonas aeruginosa (2.6.13).
ASSAY
the same solvent.
Reference solution (a). Dissolve 10 mg of lactitol monohydrate CRS and 10 mg of glycerol R in water R
Reference solution (b). Dilute 1.0 ml of test solution (a) to 100.0 ml with water R. Dilute 5 ml of this
a stainless steel column 0.30 m long and 7.8 mm in internal diameter packed with strong cation
exchange resin (calcium form) R,
as mobile phase water R at a flow rate of 0.6 ml/min,
as detector a refractive index detector,
maintaining the temperature at 60C.
Inject 100 l of reference solution (a). The test is not valid unless the resolution between the first
peak (lactitol) and the second peak (glycerol) is at least five.
Inject 100 l of the test solution and 100 l of reference solution (b). Continue the chrom-
atography for 2.5 times the retention time of lactitol. The retention time of lactitol is about 13 min
and the relative retention times are: lactose about 0.7, lactulitol about 0.8, glycerol about 1.3,
mannitol about 1.5, dulcitol (galactitol) about 1.8 and sorbitol about 1.9.
12
H
24
O
11
from the areas of the peaks in the chromatogram obtained with
the test solution using the normalisation procedure. Disregard any peaks with an area less than that
of the principal peak in the chromatogram obtained with reference solution (b).
STORAGE
IMPURITIES
A. lactose,
CH
2
OH
H HO
H
H OH
H OH
CH
2
OH
O
O
OH
OH
HO
CH
2
OH
B. lactulitol,
C. mannitol,
23-19
CH
2
OH
OH H
H
HO H
H OH
CH
2
OH
HO
D. dulcitol (galactitol),
E. sorbitol.
__________________________________________________________________________________________________________ Ph Eur
23-20
Lactose
O
OH
HO
HOCH
2
O
OH
O
OH
OH
HOCH
2
OH
,H
2
O
C
12
H
22
O
11
,H
2
O 360.3 10039-26-6
Lactose complies with the requirements of the 3rd edition of the European Pharmacopoeia for Lactose
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Lactose monohydrate is the monohydrate of O--D-galactopyranosyl-(14)--D-glucopyranose. It
may be modified as to its physical characteristics and may contain varying proportions of amorphous
lactose.
CHARACTERS
A white or almost white, crystalline powder, freely but slowly soluble in water, practically insoluble in
alcohol.
IDENTIFICATION
obtained with lactose CRS.
Reference solution (a). Dissolve 10 mg of lactose CRS in a mixture of 2 volumes of water R and 3
slight excess of water produces cloudiness. Dry the plate in a current of warm air. Repeat the
development immediately, after renewing the mobile phase. Dry the plate in a current of warm air
and spray evenly with a solution of 0.5 g of thymol R in a mixture of 5 ml of sulphuric acid R and
95 ml of alcohol R. Heat at 130C for 10 min. The principal spot in the chromatogram obtained with
reference solution (b) shows four clearly separated spots.
C. Dissolve 0.25 g in 5 ml of water R. Add 5 ml of ammonia R and heat in a water-bath at 80C for
10 min. A red colour develops.
D. It complies with the test for water (see tests).
TESTS
Appearance of solution Dissolve 1.0 g in water R, heating to 50C, dilute to 10 ml with the same
solvent and allow to cool. The solution is clear (2.2.1) and not more intensely coloured than
7
(Method II, 2.2.2).
Acidity or alkalinity Dissolve 6.0 g by boiling in 25 ml of carbon dioxide-free water R, cool and add
23-21
0.3 ml of phenolphthalein solution R. The solution is colourless. Not more than 0.4 ml of 0.1M sodium
hydroxide is required to change the colour of the indicator to pink.
Specific optical rotation (2.2.7). Dissolve 10.0 g in 80 ml of water R, heating to 50C. Allow to
cool and add 0.2 ml of dilute ammonia R1. Allow to stand for 30 min and dilute to 100.0 ml with
water R. The specific optical rotation is +54.4 to +55.9, calculated with reference to the anhydrous
substance.
Absorbance (2.2.25). Dissolve 1.0 g in boiling water R and dilute to 10.0 ml with the same solvent
(solution A). The absorbance of the solution measured at 400 nm is not greater than 0.04. Dilute
1.0 ml of solution A to 10.0 ml with water R. Examine the solution from 210 nm to 300 nm. At
wavelengths from 210 nm to 220 nm, the absorbance is not greater than 0.25. At wavelengths from
270 nm to 300 nm, the absorbance is not greater than 0.07.
Heavy metals (2.4.8). Dissolve 4.0 g in water R with warming, add 1 ml of 0.1M hydrochloric acid
and dilute to 20 ml with water R. 12 ml of the solution complies with limit test A for heavy metals
(5 ppm). Prepare the standard using lead standard solution (1 ppm Pb) R.
of water, using a mixture of 1 volume of formamide R and 2 volumes of methanol R as the solvent.
Sulphated ash Not more than 0.1 per cent. To 1.0 g add 1 ml of sulphuric acid R, evaporate to
dryness on a water-bath and ignite to constant mass.
2
micro-organisms
per gram, determined by plate-count. It complies with the test for Escherichia coli (2.6.13).
STORAGE
__________________________________________________________________________________________________________ Ph Eur
23-22
Anhydrous Lactose
C
12
H
22
O
11
342.3
Anhydrous Lactose complies with the requirements of the 3rd edition of the European Pharmacopoeia [1061].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Anhydrous lactose is O--D-galactopyranosyl-(14)--D-glucopyranose or a mixture of O--D-
galactopyranosyl-(14)--D-glucopyranose and O--D-galactopyranosyl-(14)--D-glucopyranose.
CHARACTERS
A white or almost white, crystalline powder, freely but slowly soluble in water, practically insoluble in
alcohol.
IDENTIFICATION
obtained with anhydrous lactose CRS.
Reference solution (a). Dissolve 10 mg of anhydrous lactose CRS in a mixture of 2 volumes of water R
Reference solution (b). Dissolve 10 mg each of glucose CRS, anhydrous lactose CRS, fructose CRS and
sucrose CRS in a mixture of 2 volumes of water R and 3 volumes of methanol R and dilute to 20 ml
slight excess of water produces cloudiness. Dry the plate in a current of warm air. Repeat the
development immediately, after renewing the mobile phase. Dry the plate in a current of warm air
and spray evenly with a solution of 0.5 g of thymol R in a mixture of 5 ml of sulphuric acid R and
95 ml of alcohol R. Heat at 130C for 10 min. The principal spot in the chromatogram obtained with
reference solution (b) shows four clearly separated spots.
C. Dissolve 0.25 g in 5 ml of water R. Add 5 ml of ammonia R and heat in a water-bath at 80C for
D. It complies with the test for water (see tests).
TESTS
Appearance of solution Dissolve 1.0 g in water R, heating to 50C, dilute to 10 ml with the same
solvent and allow to cool. The solution is clear (2.2.1) and not more intensely coloured than
7
(Method II, 2.2.2).
Acidity or alkalinity Dissolve 6.0 g by boiling in 25 ml of carbon dioxide-free water R, cool and add
0.3 ml of phenolphthalein solution R. The solution is colourless. Not more than 0.4 ml of 0.1M sodium
Specific optical rotation (2.2.7). Dissolve 10.0 g in 80 ml of water R, heating to 50C. Allow to
cool and add 0.2 ml of dilute ammonia R1. Allow to stand for 30 min and dilute to 100.0 ml with
water R. The specific optical rotation is +54.4 to +55.9, calculated with reference to the anhydrous
substance.
Absorbance (2.2.25). Dissolve 1.0 g in boiling water R and dilute to 10.0 ml with the same solvent
(solution A). The absorbance of the solution measured at 400 nm is not greater than 0.04. Dilute
1.0 ml of solution A to 10.0 ml with water R. Examine the solution from 210 nm to 300 nm. At
wavelengths from 210 nm to 220 nm, the absorbance is not greater than 0.25. At wavelengths from
270 nm to 300 nm, the absorbance is not greater than 0.07.
23-23
of water, using a mixture of 1 volume of formamide R and 2 volumes of methanol R as the solvent.
Sulphated ash Not more than 0.1 per cent. To 1.0 g add 1 ml of sulphuric acid R, evaporate to
dryness on a water-bath and ignite to constant mass.
2
micro-organisms
per gram, determined by plate-count. It complies with the test for Escherichia coli (2.6.13)
-Lactose and -lactose The following test concerning the pharmaco-technological properties may be
carried out depending on the intended formulations. It is not a mandatory requirement.
Test solution. Dissolve 0.1 mg of the substance to be examined in 225 l of a mixture of 19.5 volumes
of dimethyl sulphoxide R, 22 volumes of N-(trimethylsilyl)imidazole R and of 58.5 volumes of anhydrous
pyridine R. Mix gently and allow to stand for 20 min.
Reference solution. Dissolve 0.50 g of the substance to be examined in water R and dilute to 10.0 ml
with the same solvent. Heat on a water-bath at 75C for 8 min to 10 min, then allow to cool. To 2 l
of the solution add 225 l of a mixture of 19.5 volumes of dimethyl sulphoxide R, 22 volumes of
N-(trimethylsilyl)imidazole R and 58.5 volumes of anhydrous pyridine R. Mix gently and allow to stand
for 20 min.
earth for gas chromatography R impregnated with 3 per cent m/m of poly[(cyanopropyl)methyl-
phenylmethylsiloxane] R,
helium for chromatography R as the carrier gas at a flow rate of 40 ml per minute,
maintaining the temperature of the column at 215C, and that of the injection port and of the
detector at 275C.
Inject 2 l of the reference solution. The test is not valid unless the relative retention times are
about 0.7 for the silyl derivative of -lactose and 1.0 for the silyl derivative of -lactose and the
resolution between the peaks is at least 3.0.
Inject 2 l of the test solution.
Calculate the percentage content of -lactose from the expression:
b a
a
100
S S
S
+
Calculate the percentage content of -lactose from the expression:
b a
b
100
S S
S
+
S
a
=area of the peak corresponding to the silyl derivative of -lactose
S
b
=area of the peak corresponding to the silyl derivative of -lactose
Loss on drying (2.2.32). The following test concerning the pharmaco-technological properties may be
carried out depending on the intended formulations. It is not a mandatory requirement.
Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 80C for 2 h.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
23-24
Lactulose
O
OH
HO
HOCH
2
O
OH
HO
O
OH
HO
OH
and epimer at C*
*
C
12
H
22
O
11
342.3 4618-18-2
Lactulose complies with the requirements of the 3rd edition of the European Pharmacopoeia [1230]. These
Preparation
Lactulose Oral Powder
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Lactulose contains not less than 95.0 per cent and not more than the equivalent of 102.0 per cent of
4-O--D-galactopyranosyl-D-fructofuranose, calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white, crystalline powder, freely soluble in water, sparingly soluble in methanol,
practically insoluble in toluene.
IDENTIFICATION
First identification: B, C, D, E.
the same solvent.
Reference solution. Dissolve 50.0 mg of lactulose CRS in water R and dilute to 10.0 ml with the same
solvent.
10 volumes of glacial acetic acid R, 15 volumes of a 50 g/l solution of boric acid R, 20 volumes of
methanol R and 55 volumes of ethyl acetate R. Dry the plate at 100C to 105C for 5 min and allow to
cool. Spray the plate with a 1.0 g/l solution of 1,3-dihydroxynaphthalene R in a mixture of 10 volumes
of sulphuric acid R and 90 volumes of methanol R. Heat the plate at 110C for 5 min. The principal
B. Examine the chromatograms obtained in the Assay. The principal peak in the chromatogram
obtained with the test solution is similar in position and size to the principal peak in the chromato-
gram obtained with the reference solution (b).
C. Dissolve 0.05 g in 10 ml of water R. Add 3 ml of cupri-tartaric solution R and heat. A red precipi-
tate is formed.
D. Dissolve 0.125 g in 5 ml of water R. Add 5 ml of ammonia R. Heat on a water-bath at 80C for
E. It complies with the test for specific optical rotation (see Tests).
TESTS
solution BY
5
(Method II, 2.2.2).
23-25
pH (2.2.3). To 10 ml of solution S add 0.1 ml of a saturated solution of potassium chloride R. The pH
of the solution is 3.0 to 7.0.
Specific optical rotation (2.2.7). Dissolve 1.25 g of the substance to be examined in water R, add
0.2 ml of concentrated ammonia R and dilute to 25.0 ml with water R. The specific optical rotation is
46.0 to 50.0, calculated with reference to the anhydrous substance.
obtained with the test solution, the sum of the areas of any peaks corresponding to the principal
peaks in the chromatograms obtained with the reference solutions (d, e, f, g and h) (galactose,
lactose, epilactose, tagatose and fructose) is not greater than the area of the peak corresponding to
lactulose in the chromatogram obtained with reference solution (a) (3 per cent).
Methanol Examine by head-space gas chromatography (Method II, 2.2.28).
Internal standard solution. Mix 0.5 ml of propanol R with 100.0 ml of water R. Dilute 1.0 ml to
100.0 ml with water R. Dilute 5.0 ml to 50.0 ml with water R.
Test solution. To 79 mg of the substance to be examined in a 20 ml vial add 1.0 ml of the internal
standard solution and 5 l of a 0.1 per cent V/V solution of methanol R.
Reference solution. To 1.0 ml of the internal standard solution in a 20 ml vial add 5 l of a 0.1 per cent
V/V solution of methanol R.
a column 2 m long and 2 mm in internal diameter, packed with ethylvinylbenzenedivinylbenzene
copolymer R (180 m),
helium for chromatography R as the carrier gas at a flow rate of 30 ml per minute,
the detector at 220C. Maintain each solution at 60C for 1 h, pressurise for 1 min and transfer onto
the column 1 ml of the gaseous phase.
In the chromatogram obtained with the test solution, the ratio of the area of the methanol peak to
that of the internal standard peak is not greater than twice the corresponding ratio for the chromato-
gram obtained with the reference solution (50 ppm, calculated assuming the density of methanol to
be 0.79 g/ml at 20C).
Boron Avoid where possible the use of glassware.
Reference solution. Dissolve 50.0 mg of boric acid R in water R and dilute to 100.0 ml with the same
solvent. Dilute 5.0 ml of this solution to 100.0 ml with water R. Keep in a well-closed polyethylene
container.
In four polyethylene 25 ml flasks, place:
0.50 g of the substance to be examined dissolved in 2.0 ml of water R (solution A),
0.50 g of the substance to be examined dissolved in 1.0 ml of the reference solution and 1.0 ml
of water R (solution B),
1.0 ml of the reference solution and 1.0 ml of water R (solution C),
2.0 ml of water R (solution D).
To each flask, add 4.0 ml of acetate-edetate buffer solution pH 5.5 R. Mix and add 4.0 ml of freshly
prepared azomethine H solution R. Mix and allow to stand for 1 h. Measure the absorbance (2.2.25) of
solutions A, B and C at 420 nm, using solution D as the compensation liquid. The test is not valid
unless the absorbance of solution C is at least 0.25. The absorbance of solution B is not less than
twice that of solution A (9 ppm of boron).
tion of water.
Microbial contamination It complies with a limit for total viable aerobic count of 10
2
micro-
organisms per gram, determined by plate count (2.6.12). It complies with the test for Escherichia coli
(2.6.13).
ASSAY
Test solution. Dissolve 1.00 g of the substance to be examined in 10 ml of water R. Add 12.5 ml of
acetonitrile R with gentle heating and dilute to 25.0 ml with water R.
Reference solution (a). To 3 ml of the test solution add 47.5 ml of acetonitrile R with gentle heating and
Reference solution (b). Dissolve 1.00 g of lactulose CRS in 10 ml of water R. Add 12.5 ml of aceto-
nitrile R with gentle heating and dilute to 25.0 ml with water R.
Reference solution (c). Dissolve 20 mg of lactulose CRS and 20 mg of epilactose CRS in 2.0 ml of
water R. Add 2.5 ml of acetonitrile R with gentle heating and dilute to 5.0 ml with water R.
23-26
Reference solution (d). Dissolve 0.2 g of galactose R in 20 ml of water R. Add 25.0 ml of acetonitrile R
with gentle heating and dilute to 50.0 ml with water R.
Reference solution (e). Dissolve 0.2 g of lactose R in 20 ml of water R. Add 25.0 ml of acetonitrile R with
gentle heating and dilute to 50.0 ml with water R.
Reference solution (f). Dissolve 20 mg of epilactose CRS in 2 ml of water R. Add 2.5 ml of acetonitrile R
Reference solution (g). Dissolve 0.2 g of tagatose R in 20 ml of water R. Add 25.0 ml of acetonitrile R
Reference solution (h). Dissolve 0.2 g of fructose R in 20 ml of water R. Add 25.0 ml of acetonitrile R
a stainless steel column 5 cm long and 4.6 mm in internal diameter followed by a stainless steel
column 0.15 m long and 4.6 mm in internal diameter, both packed with aminopropylsilyl silica gel
for chromatography R (3 m) and maintained at 38C 1C,
sodium dihydrogen phosphate R in 220 ml of water R and add 780 ml of acetonitrile R,
as detector a refractometer maintained at a constant temperature.
When the chromatograms are recorded in the prescribed conditions, the retention time of lactulose
is about 18.3 min and retention times relative to lactulose are about 0.38 for tagatose, 0.42 for
fructose, 0.57 for galactose, 0.90 for epilactose and 1.17 for lactose.
Inject 20 l of reference solution (c). The test is not valid unless the resolution between the peaks
corresponding to lactulose and epilactose is at least 1.3. If necessary, adjust the concentration of
acetonitrile R in the mobile phase to between 75.0 per cent V/V and 82.0 per cent V/V to achieve the
prescribed resolution.
Inject separately 20 l of the test solution and 20 l of reference solution (b) and continue the
chromatography for 2.5 times the retention time of lactulose. Calculate the percentage content of
C
12
H
22
O
11
(lactulose) from the areas of the peaks and the declared content of lactulose CRS.
STORAGE
IMPURITIES
O O
CH
2
OH
OH
HO
O
CH
2
OH
OH
OH
HO HO *
and epimer at C*
A. epilactose,
*
and epimer at C*
O
CH
2
OH
OH
OH
OH
HO
B. galactose,
C. lactose,
D. fructose.
*
and epimer at C*
O
HO
HO
OH
CH
2
OH
OH
E. tagatose.
__________________________________________________________________________________________________________ Ph Eur
23-27
Lactulose Solution
Lactulose Solution complies with the requirements of the 3rd edition of the European Pharmacopoeia for Liquid
Lactulose [0924]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Liquid lactulose is an aqueous solution of lactulose (4-O--D-galactopyranosyl-D-fructofuranose)
normally prepared by alkaline isomerisation of lactose. It may contain lesser amounts of other sugars
including lactose, epilactose, galactose, tagatose and fructose. It contains not less than 620 g/l of
lactulose (C
12
H
22
O
11
; M
r
342.3) and not less than 95.0 per cent and not more than 105.0 per cent of
the declared content of lactulose. It may contain a suitable antimicrobial preservative.
CHARACTERS
A clear, viscous liquid, colourless or pale brownish-yellow, miscible with water. It may be a
supersaturated solution or may contain crystals which disappear on heating.
A 10 per cent V/V solution is laevorotatory.
IDENTIFICATION
Test solution. Dilute 0.50 g of the substance to be examined to 50 ml with water R.
Reference solution. Dissolve 60 mg of lactulose CRS in water R and dilute to 10 ml with the same
solvent.
Apply separately to the plate 2 l of each solution. Develop over a path of 15 cm with a mixture of 10
volumes of glacial acetic acid R, 15 volumes of a 50 g/l solution of boric acid R, 20 volumes of
methanol R and 55 volumes of ethyl acetate R. Dry the plate at 100C to 105C for 5 min and allow to
cool. Spray the plate with a 1.0 g/l solution of 1,3-dihydroxynaphthalene R in a mixture of 10 volumes
of sulphuric acid R and 90 volumes of methanol R. Heat the plate at 110C for 5 min. The principal
B. Examine the chromatograms obtained in the Assay. The retention time of the principal peak in the
chromatogram obtained with the test solution is approximately the same as that of the principal peak
in the chromatogram obtained with reference solution (b).
C. To 0.1 g add 10 ml of water R and 3 ml of cupri-tartaric solution R and heat. A red precipitate is
formed.
D. To 0.25 g add 5 ml of water R and 5 ml of ammonia R. Heat in a water-bath at 80C for 10 min.
A red colour develops.
TESTS
Solution S Mix 10 g with carbon dioxide-free water R and dilute to 100 ml with the same solvent.
solution BY
5
(Method II, 2.2.2).
pH (2.2.3). To 10 ml of solution S, add 0.1 ml of a saturated solution of potassium chloride R. The
pH of the solution is 3.0 to 7.0.
obtained with the test solution: the area of any peak corresponding to the principal peak in the
chromatogram obtained with reference solution (d) (galactose) is not greater than three times the
area of the peak corresponding to lactulose in the chromatogram obtained with reference solution (a)
(15 per cent); the area of any peak corresponding to the principal peak in the chromatogram obtained
with reference solution (e) (lactose) is not greater than twice the area of the peak corresponding to
lactulose in the chromatogram obtained with reference solution (a) (10 per cent); the area of any
peak corresponding to the principal peak in the chromatogram obtained with reference solution (f)
(epilactose) is not greater than twice the area of the peak corresponding to lactulose in the chromato-
gram obtained with reference solution (a) (10 per cent); the area of any peak corresponding to the
principal peak in the chromatogram obtained with reference solution (g) (tagatose) is not greater than
0.8 times the area of the peak corresponding to lactulose in the chromatogram obtained with refer-
ence solution (a) (4 per cent); the area of any peak corresponding to the principal peak in the
chromatogram obtained with reference solution (h) (fructose) is not greater than 0.2 times the area of
23-28
the peak corresponding to lactulose in the chromatogram obtained with reference solution (a)
(1 per cent).
Methanol Examine by head-space gas chromatography (Method II, 2.2.28).
Internal standard solution. Mix 0.5 ml of propanol R with 100.0 ml of water R. Dilute 1.0 ml to
100.0 ml with water R. Dilute 5.0 ml to 50.0 ml with water R.
Test solution. To 0.13 g of the substance to be examined in a 20 ml vial add 1.0 ml of the internal
standard solution. Add 5 l of a 0.1 per cent V/V solution of methanol R.
Reference solution. To 1.0 ml of the internal standard solution in a 20 ml vial add 5 l of a 0.1 per cent
V/V solution of methanol R.
a column 2 m long and 2 mm in internal diameter packed with ethylvinylbenzene-divinylbenzene
copolymer R (180 m),
the detector at 220C. Maintain each solution at 60C for 1 h, pressurise for 1 min and transfer onto
the column 1 ml of the gaseous phase.
In the chromatogram obtained with the test solution, the ratio of the area of the methanol peak to
that of the internal standard peak is not greater than twice the corresponding ratio for the chromato-
gram obtained with the reference solution (30 ppm calculated assuming the density of methanol to be
0.79 g/ml at 20C).
Sulphites Mix 5.0 g with 40 ml of water R, add 2.0 ml of 0.1M sodium hydroxide and dilute to 100 ml
with water R. To 10.0 ml of the solution, add 1.0 ml of hydrochloric acid R1, 2.0 ml of decolorised
fuchsin solution R1 and 2.0 ml of a 0.5 per cent V/V solution of formaldehyde solution R. Allow to stand
for 30 min and measure the absorbance (2.2.25) of the solution at 583 nm using as the compensation
liquid a solution prepared simultaneously and in the same manner using 10.0 ml of water R instead of
the solution of the substance to be examined. The absorbance is not greater than that of a reference
solution prepared simultaneously and in the same manner, using 10.0 ml of sulphite standard solution
(1.5 ppm SO
2
) R instead of the solution of the substance to be examined (30 ppm).
Boron Avoid where possible the use of glassware.
Reference solution. Dissolve 56.0 mg of boric acid R in water R and dilute to 100.0 ml with the same
solvent. Dilute 5.0 ml of this solution to 100.0 ml with water R. Keep in a well-closed polyethylene
container.
In four polyethylene 25 ml flasks, place:
1.00 g of the substance to be examined and 1 ml of water R (solution A),
1.00 g of the substance to be examined and 1 ml of the reference solution (solution B),
1 ml of the reference solution and 1 ml of water R (solution C),
2 ml of water R (solution D).
To each flask, add 4.0 ml of acetate-edetate buffer solution pH 5.5 R. Mix and add 4.0 ml of freshly
prepared azomethine H solution R. Mix and allow to stand for 1 h. Measure the absorbance (2.2.25) of
solutions A, B and C at 420 nm, using solution D as the compensation liquid. The test is not valid
unless the absorbance of solution C is at least 0.25. The absorbance of solution B is not less than
twice that of solution A (5 ppm of B).
Lead (2.4.10). It complies with the limit test for lead in sugars (0.5 ppm, calculated with reference to
the declared content of lactulose).
reference to the declared content of lactulose.
Microbial contamination It complies with a limit for total viable aerobic count of 10
2
micro-
organisms per gram, determined by plate count (2.6.12). It complies with the test for Escherichia coli
(2.6.13).
ASSAY
Test solution. Mix 4.00 g of the substance to be examined with 20 ml of water R. Add 25.0 ml of
acetonitrile R with gentle heating and dilute to 50.0 ml with water R.
Reference solution (a). To 5 ml of the test solution, add 47.5 ml of acetonitrile R with gentle heating
and dilute to 100.0 ml with water R.
Reference solution (b). Dissolve 2.00 g of lactulose CRS in 20 ml of water R. Add 25.0 ml of aceto-
nitrile R with gentle heating and dilute to 50.0 ml with water R.
Reference solution (c). Dissolve 20 mg of lactulose CRS and 20 mg of epilactose CRS in 2.0 ml of
water R. Add 2.5 ml of acetonitrile R with gentle heating and dilute to 5.0 ml with water R.
Reference solution (d). Dissolve 0.2 g of galactose R in 20 ml of water R. Add 25.0 ml of acetonitrile R
23-29
Reference solution (e). Dissolve 0.2 g of lactose R in 20 ml of water R. Add 25.0 ml of acetonitrile R with
gentle heating and dilute to 50.0 ml with water R.
Reference solution (f). Dissolve 20 mg of epilactose CRS in 2 ml of water R. Add 2.5 ml of acetonitrile R
Reference solution (g). Dissolve 0.2 g of tagatose R in 20 ml of water R. Add 25.0 ml of acetonitrile R
Reference solution (h). Dissolve 0.2 g of fructose R in 20 ml of water R. Add 25.0 ml of acetonitrile R
a stainless steel column 0.05 m long and 4.6 mm in internal diameter followed by a stainless
steel column 0.15 m long and 4.6 mm in internal diameter, both packed with aminopropylsilyl
silica gel for chromatography R (3 m) and maintained at 38C 1C,
sodium dihydrogen phosphate R in 220 ml of water R and add 780 ml of acetonitrile R,
as detector a refractometer maintained at a constant temperature.
When the chromatograms are recorded in the prescribed conditions, the retention time of lactulose is
about 18 min and retention times relative to lactulose are about: 0.38 for tagatose, 0.42 for fructose,
0.57 for galactose, 0.90 for epilactose and 1.17 for lactose.
Inject 20 l of the reference solution (c). The test is not valid unless the resolution between the
peaks corresponding to lactulose and epilactose is at least 1.3. If necessary, adjust the concentration
of acetonitrile R in the mobile phase to between 75.0 per cent V/V and 82.0 per cent V/V to achieve
the prescribed resolution.
Inject separately 20 l of the test solution and 20 l of reference solution (b) and continue the
chromatography for 2.5 times the retention time of lactulose. Calculate the percentage content of
C
12
H
22
O
11
(lactulose) from the areas of the peaks and the declared content of lactulose CRS.
LABELLING
The label states:
the declared content of lactulose,
the name and concentration of any added antimicrobial preservative.
IMPURITIES
O O
CH
2
OH
OH
HO
O
CH
2
OH
OH
OH
HO HO *
and epimer at C*
A. epilactose,
*
and epimer at C*
O
CH
2
OH
OH
OH
OH
HO
B. galactose,
C. lactose,
D. fructose,
*
and epimer at C*
O
HO
HO
OH
CH
2
OH
OH
E. tagatose.
__________________________________________________________________________________________________________ Ph Eur
23-30
Lanatoside C
O
Me
OAc
O
O
O
OH
OH
HOCH
2
O
Me
O
O
Me
OH
O
R
OH
HO
R =
OH
H
H
H
H
H
Me
Me
O
O
H
HO
C
49
H
76
O
20
985 17575-22-3
Lanatoside C complies with the requirements of the 3rd edition of the European Pharmacopoeia [0504]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Lanatoside C contains not less than 97.0 per cent and not more than the equivalent of 103.0 per
cent of 3-[(O--D-glucopyranosyl-(14)-O-(O-3-acetyl-2,6-dideoxy--D-ribo-hexopyranosyl)-
(14)-O-2,6-dideoxy--D-ribo-hexo-pyranosyl-(14)-2,6-dideoxy--D-ribo-hexopyranosyl)oxy]-
12,14-dihydroxy-5,14-card-20(22)-enolide, expressed for the dry substance.
CHARACTERS
A white or slightly yellowish, crystalline or finely crystalline powder, hygroscopic, practically
insoluble in water and in ether, soluble in methanol. In an atmosphere of low relative humidity, it
loses water.
IDENTIFICATION
obtained with lanatoside C CRS. When comparing the spectra, special attention should be given to
the presence of a distinct absorption maximum at about 1260 cm
1
and to the intensity of the
absorption maximum at about 1740 cm
1
. Examine the substances as discs prepared by dissolving
1 mg of the substance to be examined or 1 mg of the reference substance in 0.3 ml of methanol R and
triturating with about 0.4 g of dry, finely powdered potassium bromide R until the mixture is uniform
and completely dry.
B. Examine the chromatograms obtained in the test for related substances. The principal band in the
band in the chromatogram obtained with reference solution (a).
D. Dissolve about 5 mg in 5 ml of glacial acetic acid R and add 0.05 ml of ferric chloride solution R1.
Cautiously add 2 ml of sulphuric acid R, avoiding mixing the two liquids. Allow to stand: a brown but
no reddish ring develops at the interface and a greenish-yellow, then bluish-green colour diffuses
from it to the upper layer.
TESTS
solution Y
7
or BY
7
(Method II, 2.2.2).
23-31
coating substance.
Test solution (a). Use solution S.
Reference solution (a). Dissolve 20 mg of lanatoside C CRS in methanol R and dilute to 10 ml with the
same solvent.
Reference solution (b). Dilute 1.5 ml of reference solution (a) to 10 ml with methanol R.
Reference solution (c). Dilute 1 ml of reference solution (a) to 10 ml with methanol R.
Reference solution (d). To 2 ml of reference solution (c) add 2 ml of methanol R.
Apply separately to the plate as 10 mm bands 5 l of each solution. Develop immediately over a path
of 15 cm using a mixture of 1 volume of water R, 20 volumes of methylene chloride R, 30 volumes of
alcohol R and 60 volumes of toluene R. Dry the plate in a current of cool air for 5 min. Carry out a
second development in the same direction. Dry the plate in a current of cool air for 5 min, spray with
a mixture of 5 volumes of sulphuric acid R and 95 volumes of alcohol R and heat at 140C for 15 min.
Examine in daylight. In the chromatogram obtained with test solution (a): any band, apart from the
principal band, is not more intense than the band in the chromatogram obtained with reference
solution (b) (1.5 per cent); at most three such bands are more intense than the band in the chrom-
atogram obtained with reference solution (d) (0.5 per cent); and one of these may be more intense
than the band in the chromatogram obtained with reference solution (c) (1.0 per cent).
100C to 105C.
for loss on drying.
ASSAY
Before carrying out the assay, allow both the substance to be examined and the reference substance to stand in
a desiccator containing a saturated solution of potassium thiocyanate R for 24 h.
solution to 100.0 ml with alcohol R. Prepare a reference solution in the same manner, using 50.0 mg
of lanatoside C CRS. To 5.0 ml of each solution add 3.0 ml of alkaline sodium picrate solution R and
allow to stand protected from bright light in a water-bath at 20 1C for 40 min. Measure the
absorbance (2.2.25) of each solution at the maximum at 484 nm, using as the compensation liquid a
mixture of 3.0 ml of alkaline sodium picrate solution R and 5.0 ml of alcohol R prepared at the same
time.
49
H
76
O
20
solutions, without allowing for the result of the test for loss on drying.
STORAGE
Store in an airtight, well-filled glass container, protected from light, at a temperature below 10C.
__________________________________________________________________________________________________________ Ph Eur
23-32
Lauroyl Macrogolglycerides
Lauroyl Macrogolglycerides comply with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Lauroyl macrogolglycerides are mixtures of monoesters, diesters and triesters of glycerol and mono-
esters and diesters of macrogols with a mean relative molecular mass between 300 and 1500. They
are obtained by partial alcoholysis of saturated oils mainly containing triglycerides of lauric acid using
macrogol or by esterification of glycerol and macrogol with saturated fatty acids or by mixing of
glycerol esters and condensates of ethylene oxide with the fatty acids of these hydrogenated oils.
CHARACTERS
Pale yellow waxy solids, dispersible in hot water, freely soluble in methylene chloride.
IDENTIFICATION
substance.
f
st
st
st
st
0.1) and to
st
0).
C. They comply with the test for fatty acid composition (see Tests).
D. They comply with the test for saponification value (see Tests).
TESTS
Drop point (2.2.17). Introduce into the cup the substance to be examined which has been melted by
heating for 1 h in an oven at 100 2C and allow to stand for 5 h at about 5C. The drop point is
indicated in Table 1231-1.
Table 1231-1
per molecule
(nominal value)
Type of macrogol Drop point
6 300 33 to 38
8 400 36 to 41
12 600 38 to 43
32 1500 42.5 to 47.5
Hydroxyl value (2.5.3). The ranges are presented in Table 1231-2, determined on 1.0 g (Method
A).
Table 12312
per molecule
(nominal value)
Type of macrogol Hydroxyl value
6 300 65 to 85
8 400 60 to 80
12 600 50 to 70
32 1500 36 to 56
Saponification value (2.5.6). The ranges are presented in Table 1231-3, determined on 2.0 g.
23-33
Table 12313
per molecule
(nominal value)
Type of macrogol Saponification
value
6 300 190 to 204
8 400 170 to 190
12 600 150 to 170
32 1500 79 to 93
Alkaline impurities Introduce 5.0 g into a test tube and carefully add a mixture, neutralised if
of bromophenol blue R in alcohol R, 0.3 ml of water R and 10 ml of alcohol R. Shake and allow to stand.
Not more than 1.0 ml of 0.01M hydrochloric acid is required to change the colour of the upper layer to
yellow.
Free glycerol Not more than 3.0 per cent. Dissolve 1.20 g in 25.0 ml of methylene chloride R. Heat if
necessary. After cooling, add 100 ml of water R. Shake and add 25.0 ml of a 6 g/l solution of periodic
acid R. Shake and allow to stand for 30 min. Add 40 ml of a 75 g/l solution of potassium iodide R.
Allow to stand for 1 min. Add 1 ml of starch solution R. Titrate the iodine with 0.1M sodium
caprylic acid: not more than 15.0 per cent,
capric acid: not more than 12.0 per cent,
lauric acid: 30.0 per cent to 50.0 per cent,
myristic acid: 5.0 per cent to 25.0 per cent,
stearic acid: 5.0 per cent to 35.0 per cent.
of water using a mixture of 30 volumes of anhydrous methanol R and 70 volumes of methylene
chloride R as solvent.
STORAGE
Store in an well-closed container.
LABELLING
The label states the type of macrogol used (mean relative molecular mass) or the number of units of
ethylene oxide per molecule (nominal value).
__________________________________________________________________________________________________________ Ph Eur
23-34
Lavender Flower
1/01
Lavender Flower complies with the requirements of the 3rd edition of the European Pharmacopoeia [1534].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Lavender flower consists of the dried flower of Lavandula angustifolia P. Mill.(L. officinalis Chaix). It
contains not less than 13 ml/kg of essential oil, calculated with reference to the anhydrous drug.
CHARACTERS
Lavender flower has a strongly aromatic odour.
IDENTIFICATION
Second identification: A, B, C.
A. The flower has a short peduncle and consists of a bluish-grey tubular calyx divided distally into
four very short teeth and a small rounded lobe, a blue bilabial corolla with the upper lip bifid and the
lower lip trilobate, four didynamous stamens with ovoid anthers.
B. Reduce to a powder (355). The powder is bluish-grey. Examine under a microscope using chloral
hydrate solution R. The powder shows covering trichomes bifurcating at one or more levels; secretory
trichomes with short stalks and eight-celled heads of the Labiatae type; secretory trichomes with
unicellular or multicellular stalks and unicellular heads; secretory trichomes with long uneven stalks
and unicellular heads, separated from the peduncle by an intermediary cell with a smooth cuticle;
certain such trichomes show a crown of small spheroid cells just below the insertion point of the
intermediary cell on the peduncle; fragments of warty epidermis from the inner surface of the petals;
fragments of calyx epidermis with sinuous-walled cells and containing prismatic crystals of calcium
oxalate; spherical pollen grains which have a diameter of about 45 m and an exine with six slit-like
germinal pores and six ribbon-like groins radiating from the poles.
Test solution. To 0.5 g of the powdered drug (355) add 5 ml of hexane R, shake for 5 min and filter.
Reference solution. Dissolve 10 l of linalol R and 10 l of linalyl acetate R in 5 ml of hexane R.
Apply to the plate as bands 10 l of each solution. Develop over a path of 15 cm using a mixture of 5
volumes of ethyl acetate R and 95 volumes of toluene R. Allow the plate to dry in air. Spray the plate
with anisaldehyde solution R and examine in daylight while heating at 100C to 105C for 5 min to
10 min. The chromatogram obtained with the reference solution shows in the lower third a greyish-
blue zone (linalol) and in the middle third a greyish-blue zone (linalyl acetate). The chromatogram
obtained with the test solution shows zones corresponding to linalol and linalyl acetate and in the
middle, between these zones, a reddish-violet zone (epoxydihydrocaryophyllene). Further zones are
also present.
D. Examine the chromatograms obtained in the test for other species and varieties of lavender. The
five principal peaks in the chromatogram obtained with the reference solution are similar in retention
time to peaks in the chromatogram obtained with the test solution. Among them are mainly linalol
and linalyl acetate peaks.
TESTS
Foreign matter (2.8.2). Not more than 3 per cent of stems and not more than 2 per cent of other
foreign matter.
Other species and varieties of lavender Examine by gas chromatography (2.2.28).
Test solution. Dilute 0.2 ml of the essential oil-xylene mixture obtained in the assay to 5 ml with
hexane R, add 1 g of anhydrous sodium sulphate R, shake and use the supernatant liquid.
Reference solution. Dissolve 0.1 g of limonene R, 0.2 g of cineole R, 0.05 g of camphor R, 0.4 g of
linalol R, 0.6 g of linalyl acetate R and 0.2 g of -terpineol R in 100 ml of hexane R.
a fused-silica column about 60 m long and about 0.25 mm in internal diameter, coated with
macrogol 20,000 R,
helium for chromatography R as the carrier gas at a flow rate of 1.5 ml per/min,
23-35
Time
(min)
Temperature
(C)
Rate (C
per min)
Comment
15 70 70 180 2 linear gradient
Injection port 220
Detector 220
Inject the same volume of each solution. When the chromatograms are recorded under the
prescribed conditions, the components are eluted in the order indicated in the composition of the
reference solution. Record the retention times of these substances.
The test is not valid unless: the number of theoretical plates calculated from the limonene peak at
110C is at least 30,000, and the resolution between the peak corresponding to limonene and that
corresponding to cineole is at least 1.5.
Using the retention times determined from the chromatogram obtained with the reference solution,
locate the six components of the reference solution in the chromatogram obtained with the test
solution. Disregard the peaks due to hexane and xylene.
In the chromatogram obtained with the test solution the area of the peak due to camphor is not
greater than 1 per cent of the total area of the peaks.
Water (2.2.13). Not more than 10.0 per cent, determined by distillation using 20.0 g of drug.
ASSAY
Carry out the determination of essential oils in vegetable drugs (2.8.12). Use 20.0 g of the drug, a
1000 ml round-bottomed flask, 500 ml of water R as the distillation liquid and 0.5 ml of xylene R in
the graduated tube. Distil at a rate of 2 ml/min to 3 ml/min for 2 h.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
23-36
Lavender Oil
Lavender Oil complies with the requirements of the 3rd edition of the European Pharmacopoeia [1338]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Lavender oil is obtained by steam distillation from the fresh flowering tops of Lavandula angustifolia
Miller (Lavandula officinalis Chaix).
CHARACTERS
A colourless or pale yellow, clear liquid, with a characteristic odour, miscible with alcohol (90 per
cent V/V), with ether, and with fatty oils.
IDENTIFICATION
substance.
Test solution. Dissolve 20 l of the substance to be examined in 1 ml of toluene R.
Reference solution. Dissolve 10 l of linalol R and 10 l of linalyl acetate R in 1 ml of toluene R.
Apply separately to the plates as bands 10 l of each solution. Develop twice, 5 min apart, over a path
of 10 cm using a mixture of 5 volumes of ethyl acetate R and 95 volumes of toluene R. Allow the plate
to dry in air. Spray with anisaldehyde solution R. Heat at 100C to 105C for 5 min to 10 min while
observing. Examine in daylight. The chromatogram obtained with the reference solution shows two
violet zones, one corresponding to linalol in the lower half and the other corresponding to linalyl
acetate a little above the middle of the chromatogram. Two zones in the chromatogram obtained
with the test solution are similar in position and colour to the zones in the chromatogram obtained
with the reference solution; below the linalol zone are other brownish-green or violet-red zones
(usually two to five), including a brownish-green zone, directly below the linalol zone, that is more
intense; above the linalol zone and even at the solvent front are other violet-red zones. The chromato-
gram obtained with the test solution may also show a weak violet-brown zone (cineole) between the
linalol zone and the violet-red zone above the linalol zone (caryophyllene epoxide).
B. Examine the chromatograms obtained in the test for Chromatographic profile. The retention
times of the principal peaks in the chromatogram obtained with the test solution are similar to those
TESTS
Optical rotation (2.2.7). 12.5 to 7.
Foreign esters (2.8.6). It complies with the test for foreign esters.
essential oils.
Water-soluble portion Introduce 20 ml of saturated sodium chloride solution R into a 50 ml measur-
ing cylinder and carefully add slowly 10 ml of the essential oil. Note the zone of contact of the two
layers, mix and allow to stand. The volume of the essential oil does not change.
Reference solution. Dissolve 0.1 g of limonene R, 0.2 g of cineole R, 0.2 g of 3-octanone R, 0.05 g of
camphor R, 0.4 g of linalol R, 0.6 g of linalyl acetate R, 0.2 g of terpinen-4-ol R, 0.1 g of lavandulyl
acetate R, 0.2 g of lavandulol R, and 0.2 g of -terpineol R in 1 ml of hexane R,
a fused-silica capillary column 60 m long and about 0.25 mm in internal diameter coated with
macrogol 20,000 R,
a split ratio of 1/100,
23-37
rate of 2C per minute to 180C and maintaining the temperature of the injection port at 220C
and that of the detector at 220C.
Inject about 0.2 l of the reference solution. When the chromatograms are recorded under the
prescribed conditions, the components elute in the order indicated in the composition of the refer-
ence solution. Record the retention times of these substances.
The test is not valid unless: the number of theoretical plates calculated from the limonene peak at
110C is at least 30,000; the resolution between the peaks corresponding to limonene and cineole is
at least 1.5.
obtained with the reference solution, locate the components of the reference solution on the chrom-
atogram obtained with the test solution. Disregard the peak due to hexane.
Determine the percentage content of these components by the normalisation procedure.
The percentages are within the following values:
limonene less than 1.0 per cent
cineole less than 2.5 per cent
3-octanone less than 2.5 per cent
camphor less than 1.2 per cent
linalol 20.0 per cent to 45.0 per cent
linalyl acetate 25.0 per cent to 46.0 per cent
terpinen-4-ol 1.2 per cent to 6.0 per cent
lavandulyl acetate more than 1.0 per cent
lavandulol more than 0.1 per cent
-terpineol less than 2.0 per cent
STORAGE
Store in an airtight, well-filled container, protected from light and heat.
Fig. 13381 Type chromatogram for lavender oil
1. limonene 2. cineole
3. 3-octanone 4. camphor
5. linalol 6. linalyl acetate
7. terpinen-4-ol 8. lavandulyl acetate
9. lavandulol 10. a-terpineol
__________________________________________________________________________________________________________ Ph Eur
23-38
Lemon Balm
Lemon Balm complies with the requirements of the 3rd edition of the European Pharmacopoeia for Melissa Leaf
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Melissa leaf consists of the dried leaf of Melissa officinalis L. It contains not less than 4.0 per cent of
total hydroxycinnamic derivatives expressed as rosmarinic acid (C
18
H
16
O
8,
M
r
360.3), calculated
with reference to the dried drug.
CHARACTERS
Melissa leaf has an odour reminiscent of lemon.
IDENTIFICATION
A. Melissa leaf has a petiole varying in length and is oval, cordate and up to about 8 cm long and
5 cm wide. The lamina is thin and the under surface has a conspicuous, raised, reticulate venation;
the margins are roughly dentate or crenate. The upper surface is bright green and the lower surface is
lighter in colour.
B. Reduce to a powder (355). The powder is greenish. Examine under a microscope using chloral
hydrate solution R. The powder shows fragments of the leaf epidermis with sinuous walls; short,
straight, unicellular, conical covering trichomes with a finely striated cuticle; multicellular, uniseriate
covering trichomes with pointed ends and thick, warty cuticles; eight-celled secretory trichomes of
lamiaceous type; secretory trichomes with unicellular to tricellular stalks and unicellular or, more
rarely bicellular, heads; diacytic stomata (2.8.3), on the lower surface only.
Test solution. Place 2.0 g of the powdered drug (355) in a 250 ml round-bottomed flask and add
100 ml of water R. Distil for 1 h using the apparatus for the determination of essential oils in
vegetable drugs (2.8.12) and 0.5 ml of xylene R in the graduated tube. After distillation transfer the
organic phase to a 1 ml volumetric flask rinsing the graduated tube of the apparatus with the aid of a
small portion of xylene R and dilute to 1.0 ml with the same solvent.
Reference solution. Dissolve 1.0 l of citronellal R and 10.0 l of citral R in 25 ml of xylene R.
Apply to the plate as bands 10 l of the reference solution and 20 l of the test solution. Develop over
a path of 15 cm using a mixture of 10 volumes of ethyl acetate R and 90 volumes of hexane R. Allow
the plate to dry in air. Spray the plate with anisaldehyde solution R. Examine in daylight while heating
at 100C to 105C for 10 min to 15 min. The chromatogram obtained with the reference solution
shows in the lower third a greyish-violet to bluish-violet double zone (citral) and above it a grey to
greyish-violet zone (citronellal). The chromatogram obtained with the test solution shows zones
similar in position and colour to the zones in he chromatogram obtained with the reference solution
and between these zones a reddish-violet zone (epoxycaryophyllene). Further zones may be present.
TESTS
Foreign matter (2.8.2). Not more than 10 per cent of stems having a diameter greater than 1 mm
and not more than 2 per cent of other foreign matters, determined on 20 g.
ASSAY
Stock solution. To 0.200 g of the powdered drug (355) add 190 ml of alcohol (50 per cent V/V) R. Boil
in a water-bath under a reflux condenser for 30 min. Allow to cool and filter. Rinse the filter with
10 ml of alcohol (50 per cent V/V) R. Combine the filtrate and the rinsings in a volumetric flask and
dilute to 200.0 ml with alcohol (50 per cent V/V) R.
Test solution. To 1.0 ml of the stock solution in a test-tube add 2 ml of 0.5M hydrochloric acid, 2 ml of
a solution prepared by dissolving 10 g of sodium nitrite R and 10 g of sodium molybdate R in 100 ml of
water R and then add 2 ml of dilute sodium hydroxide solution R and dilute to 10.0 ml with water R and
mix.
Compensation solution. In another test-tube place 1.0 ml of the stock solution, 2 ml of 0.5M hydro-
chloric acid, 2 ml of dilute sodium hydroxide solution R and dilute to 10.0 ml with water R.
Immediately measure the absorbance (2.2.25) of the test solution at 505 nm by comparison with the
compensation solution.
23-39
Calculate the percentage content of total hydroxycinnamic derivatives, expressed as rosmarinic
acid, from the expression:
m
A 5
taking the specific absorbance to be 400 for rosmarinic acid at 505 nm.
A=absorbance of the test solution at 505 nm,
STORAGE
__________________________________________________________________________________________________________ Ph Eur
23-40
Lemon Oil
Lemon Oil complies with the requirements of the 3rd edition of the European Pharmacopoeia [0620]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Lemon oil is the essential oil obtained by suitable mechanical means, without the aid of heat, from
the fresh peel of Citrus limon (L.) Burman fil. Lemon oil contains not less than 2.2 per cent m/m and
not more than 4.5 per cent m/m of carbonyl compounds, calculated as citral (C
10
H
16
O; M
r
152.2).
CHARACTERS
A clear, mobile, pale-yellow to greenish-yellow liquid. The oil may become cloudy at low tempera-
tures. It is miscible with ethanol, with ether and with glacial acetic acid.
IDENTIFICATION
254
Test solution. Mix 1 ml of the substance to be examined with 1 ml of toluene R.
Reference solution (a). Dissolve 10 mg of citropten R and 50 l of citral R in toluene R and dilute to
Reference solution (b). Dissolve 10 mg of citropten R in toluene R and dilute to 100 ml with the same
solvent. To 1 ml of the solution add 10 l of citral R and dilute to 100 ml with toluene R.
of 15 cm using a mixture of 15 volumes of ethyl acetate R and 85 volumes of toluene R. Allow the plate
to dry in air and examine in ultraviolet light at 254 nm. The chromatogram obtained with reference
solution (a) shows in the middle a quenching zone (citral) and in the lower third a bright-blue
fluorescent zone (citropten). The chromatogram obtained with the test solution shows zones
corresponding to those in the chromatogram obtained with reference solution (a) and also, just above
the citral zone, a zone due to bergamotin; below the citral zone, a bright-blue zone (5-geranyloxy-7-
methoxycoumarin); below the citropten zone, a zone due to a psoralen derivative and below this a
zone due to biakangelicin. Examine in ultraviolet light at 365 nm. The zone due to the psoralen
derivative shows greenish-yellow fluorescence, that due to citropten bright violet-blue fluorescence,
that due to 5-geranyloxy-7-methoxycoumarin bright blue fluorescence and that due to bergamotin
yellow fluorescence.
TESTS
Optical rotation (2.2.7). The angle of optical rotation is +57 to +70.
Absorbance (2.2.25). Dissolve 0.250 g in alcohol R, mix and dilute to 100.0 ml with the same
solvent. Measure the absorbance over the range 260 nm to 400 nm. If a manual instrument is used,
measure the absorbance at 5 nm intervals from 260 nm to about 12 nm before the expected
absorption maximum, then at 3 nm intervals for three readings and at 1 nm intervals to about 5 nm
beyond the maximum and finally at 10 nm intervals to 400 nm. Plot a curve representing the
absorption spectrum with the absorbances as ordinates and the wavelengths as abscissae. Draw as a
base-line the tangent between A and B (see Fig. 0620-1). The absorption maximum C is situated at
315 3 nm. From C draw a line perpendicular to the axis of abscissae and intersecting AB at D.
Deduct the absorbance corresponding to point D from that corresponding to point C. The value
obtained is 0.20 to 0.96 and for Italian-type lemon oil it is not less than 0.45.
23-41
Fig. 06201 Typical spectrum of Lemon Oil
Adulterants Examine the chromatograms obtained in the identification test in ultraviolet light at
254 nm. The chromatogram obtained with reference solution (b) shows a quenching zone corres-
ponding to citral. In the chromatogram obtained with the test solution, any quenching zone above
the zone due to bergamotin (methyl anthranilate and menthyl salicylate) or any quenching zone at
about the same level as citropten (chalcones) is not more intense than the quenching zone in the
chromatogram obtained with reference solution (b). Examine in ultraviolet light at 365 nm. The
chromatogram obtained with reference solution (b) shows a bright-blue fluorescent zone correspond-
ing to citropten. In the chromatogram obtained with the test solution, any violet or blue fluorescent
zone above the zone due to bergamotin is not more intense than the fluorescent zone in the chrom-
atogram obtained with reference solution (b). Expose the plate to hydrochloric acid vapour and
examine in daylight. Neither bright red zones (chalcones) nor bright blue or yellow zones (other
adulterants) appear in the lower third or in the middle of the chromatogram obtained with the test
solution, but may appear in the upper third of the chromatogram.
essential oils.
Foreign essential oils Use a distillation apparatus with four bulbs having approximate diameters
from the lowest bulb to the highest of 60 mm, 35 mm, 30 mm and 25 mm respectively, the distance
between the bottom of the flask and the lateral arm being 200 mm. Distil 50 ml of the substance to
be examined at a rate of 1 drop per second until 5 ml of distillate is obtained. The angle of optical
rotation (2.2.7) of the distillate is not more than 6 less than that of the substance to be examined
before distillation. The refractive index (2.2.6) of the distillate is not more than 0.003 less than that
of the substance to be examined before distillation.
Residue on evaporation (2.8.9). 1.8 per cent to 3.6 per cent. Heat on the water-bath for 4 h.
ASSAY
Mix 9.000 g with 20 ml of ethanol R. Add 10.0 ml of hydroxylamine hydrochloride solution R2 and
0.4 ml of bromophenol blue solution R2. Titrate slowly with 0.5M alcoholic potassium hydroxide until the
colour changes from yellow to olive-green. Allow to stand for 5 min and titrate again, if necessary,
until the colour changes from yellow to olive-green.
1 ml of 0.5M alcoholic potassium hydroxide is equivalent to 76.1 mg of carbonyl compounds,
calculated as C
10
H
16
O.
STORAGE
Store in a well-filled, airtight container, protected from light.
LABELLING
The label states, where applicable, that the contents are Italian-type lemon oil.
__________________________________________________________________________________________________________ Ph Eur
23-42
Terpeneless Lemon Oil
Definition Terpeneless Lemon Oil may be prepared by concentrating Lemon Oil under reduced
pressure until most of the terpenes have been removed or by solvent partition. It contains not less
than 40% w/w of aldehydes calculated as citral, C
10
H
16
O.
Characteristics A clear, colourless or pale yellow liquid, visibly free from water; odour and taste,
those of lemon.
Optical rotation 5 to +2, Appendix V F.
Solubility in ethanol Soluble, at 20, in 1 volume of ethanol (80%), Appendix X M.
Assay Carry out the method for determination of aldehydes, Appendix X K, using 1 g, omitting the
toluene and using a volume, not less than 7 ml, of alcoholic hydroxylamine solution that exceeds by 1 to
2 ml the volume of 0.5M potassium hydroxide in ethanol (60%) VS required. Each ml of 0.5M
potassium hydroxide in ethanol (60%) VS is equivalent to 76.73 mg of C
10
H
16
O.
Storage Terpeneless Lemon Oil should be kept in a well-filled, well-closed container, protected from
light and stored at a temperature not exceeding 25.
Preparations
Lemon Spirit
Compound Orange Spirit
23-43
Dried Lemon Peel
Definition Dried Lemon Peel is the dried outer part of the pericarp of the ripe, or nearly ripe,
fruit of Citrus limon (L.) Burm. f.
Characteristics Odour, aromatic; taste, aromatic and bitter.
Macroscopical In strips or pieces; outer surface yellow and somewhat rough from the presence of
numerous minute pits, each corresponding to an oil gland; inner surface with only a small remnant of
white, spongy pericarp. Fracture short.
Microscopical Epidermis of small polyhedral cells. Tissue subjacent to the epidermis parenchymatous,
many of the cells containing prismatic crystals of calcium oxalate. Numerous large oil glands and
small vascular strands embedded in the parenchyma.
Volatile oil Not less than 2.5% v/w, Appendix XI E, using 300 ml of water as the distillation liquid
and no xylene in the graduated tube. Use 20 g, crushed under water, and distil for 3 hours.
23-44
Leucine
COOH
NH
2
H
H
3
C
H H
3
C
C
6
H
13
NO
2
131.2 61-90-5
Leucine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0771]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Leucine contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of
(S)-2-amino-4-methylpentanoic acid, calculated with reference to the dried substance.
PRODUCTION
When Leucine is produced by a process involving fermentation steps, it complies with the require-
CHARACTERS
Shiny flakes or a white or almost white crystalline powder, sparingly soluble in water, practically
insoluble in alcohol and in ether. It dissolves in dilute mineral acids and in dilute solutions of alkali
hydroxides.
IDENTIFICATION
B. Dissolve 0.50 g in water R and dilute to 25 ml with the same solvent. The solution is
laevorotatory.
obtained with leucine CRS. Examine the substances prepared as discs.
TESTS
6
(Method II, 2.2.2).
substance.
gel plate R.
Test solution (a). Dissolve 0.10 g of the substance to be examined in 0.1M hydrochloric acid and dilute
Reference solution (a). Dissolve 10 mg of leucine CRS in 0.1M hydrochloric acid and dilute to 50 ml with
the same acid.
Reference solution (c). Dissolve 10 mg of leucine CRS and 10 mg of valine CRS in 0.1M hydrochloric
acid and dilute to 25 ml with the same acid.
of butanol R. Allow the plate to dry in air, spray with ninhydrin solution R and heat at 100C to 105C
for 15 min. Any spot in the chromatogram obtained with test solution (a), apart from the principal
spot, is not more intense than the spot in the chromatogram obtained with reference solution (b)
23-45
(100 ppm NH
4
100C to 105C.
ASSAY
Dissolve 0.100 g in 3 ml of anhydrous formic acid R. Add 30 ml of anhydrous acetic acid R. Titrate with
0.1M perchloric acid using 0.1 ml of naphtholbenzein solution R as indicator, until the colour changes
from brownish-yellow to green.
6
H
13
NO
2
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
23-46
Leuprorelin
corrected 1/01
H
N
H
HisTrpSerTyrDLeuLeuArgProNHEt
O
C
59
H
84
N
16
O
12
1209 74381-53-6 (base)
53714-56-0 (acetate)
Leuprorelin complies with the requirements of the 3rd edition of the European Pharmacopoeia [1441]. These
Action and use Gonadorelin analogue.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Leuprorelin is a synthetic nonapeptide analogue of hypothalamic peptide, gonadorelin. It is obtained
by chemical synthesis and is available as an acetate. It contains not less than 97.0 per cent and not
more than the equivalent of 103.0 per cent of the peptide C
59
H
84
N
16
O
12
, calculated with reference to
the anhydrous, acetic acid-free substance.
CHARACTERS
A hygroscopic, white or almost white powder.
IDENTIFICATION
spectrum of leuprorelin. Examine as discs prepared using potassium bromide R.
TESTS
Specific optical rotation (2.2.7). Dissolve the substance to be examined in a 1 per cent V/V
solution of glacial acetic acid R to obtain a concentration of 10.0 mg/ml. The specific optical rotation
is 38.0 to 42.0, calculated with reference to the anhydrous, acetic acid-free substance.
acids:
lysine threonine alanine leucine
histidine serine valine tyrosine
arginine glutamic acid methionine phenylalanine
aspartic acid proline isoleucine
together with half the equimolar amount of L-cystine. For the validation of the method, an appro-
priate internal standard, such as DL-norleucine, is used.
Test solution. Place 1.0 mg of the substance to be examined in a rigorously cleaned suitable hard-glass
tube 100 mm long and 6 mm in internal diameter. Add a suitable amount of a 50 per cent V/V
solution of hydrochloric acid R. Immerse the tube in a freezing mixture at 5C, reduce the pressure to
below 133 Pa and seal. Heat at 110C to 115C for 16 h. Cool, open the tube, transfer the contents
to a 10 ml flask with five quantities, each of 0.2 ml of water R and evaporate to dryness over potassium
hydroxide R under reduced pressure. Take up the residue in water R and evaporate to dryness over
potassium hydroxide R under reduced pressure. Take up the residue in a buffer solution suitable for
the amino-acid analyser used and dilute to a suitable volume with the same buffer solution.
Apply to the amino-acid analyser a suitable, accurately measured volume of the test solution such
that the peak height given by the amino acid present in the largest amount is at least 90 per cent of
the full scale of the recorder.
acids taking one seventh of the sum of the number of moles of histidine, glutamic acid, leucine,
proline, tyrosine and arginine as equal to one. The values fall within the following limits: serine
present; glutamic acid 0.85 to 1.1; proline 0.85 to 1.1; leucine 1.8 to 2.2; tyrosine 0.85 to 1.1;
histidine 0.85 to 1.1 and arginine 0.85 to 1.1. Not more than traces of other amino acids are present,
with the exception of tryptophan.
23-47
Related substances Examine by liquid chromatography (2.2.29) as prescribed in the assay.
Inject 20 l of test solution (a) and 20 l of reference solution (c). Record the chromatograms for
90 min. When the chromatograms are recorded in the prescribed conditions, the retention times
relative to the principal peak are: D-Ser-leuprorelin 0.8; D-His-leuprorelin 0.9; L-Leu-leuprorelin 1.2;
O-acetyl-Ser-leuprorelin: 1.5. In the chromatogram obtained with test solution (a), the area of the
peak corresponding to O-acetyl-Ser-leuprorelin is not greater than the area of the principal peak in
the chromatogram obtained with reference solution (c) (1 per cent); the area of any peak, apart from
the principal peak and the peak due to O-acetyl-Ser-leuprorelin, is not greater than half the area of
the principal peak in the chromatogram obtained with reference solution (c) (0.5 per cent) and the
sum of the areas of the peaks, apart from the principal peak, is not greater than two and a half times
the area of the principal peak in the chromatogram obtained with reference solution (c) (2.5 per
cent). Disregard any peak with an area of less than 0.1 times the area of the principal peak in the
chromatogram obtained with reference solution (c).
Acetic acid (2.5.34): 4.7 per cent to 9.0 per cent.
phase B and 95 volumes of mobile phase A and dilute to 10.0 ml with the same mixture of mobile
phases.
16.7 I.U. of endotoxin per milligram. Perform the test using method D (chromogenic peptide kinetic
method).
Sulphated ash (2.4.14). Not more than 0.3 per cent.
ASSAY
Test solution (a). Dissolve the substance to be examined in the mobile phase to obtain a concentration
of 1.0 mg/ml.
Reference solution (a). Dissolve leuprorelin CRS in the mobile phase to obtain a concentration of
1.0 mg/ml.
Resolution solution. Dilute 5.0 ml of reference solution (a) to 50.0 ml with water R. To 5 ml of the
solution, add 100 l of 1M sodium hydroxide and shake vigorously. Heat in an oven at 100C for
60 min, cool immediately and add 50 l of dilute phosphoric acid R. Shake vigorously.
as mobile phase at a flow rate of 1.0 ml/min to 1.5 ml/min a mixture prepared as follows:
dissolve about 15.2 g of triethylamine R in 800 ml of water R, adjust the pH to 3.0 with phosphoric
acid R, and dilute to 1000 ml with water R (solution A). To 150 ml of a mixture of 2 volumes of
propanol R and 3 volumes of acetonitrile R, add 850 ml of solution A,
Inject 20 l of the resolution solution. Record the chromatogram for 60 min. Adjust the flow rate,
so that in the chromatogram obtained, the principal peak elutes at a retention time of approximately
41 min to 49 min. The peak corresponding to D-His-leuprorelin elutes at a relative retention time of
0.9 relative to the principal peak. The test is not valid unless the resolution between the peaks
corresponding to D-His-leuprorelin and leuprorelin is at least 1.5 and the symmetry factor for the
leuprorelin peak is 0.8 to 1.5.
Inject 20 l of test solution (b) and 20 l of reference solution (b). Calculate the content of
leuprorelin (C
59
H
84
N
16
O
12
) from the peak areas in the chromatograms obtained with test solution
(b) and reference solution (b) and the declared content of C
59
H
84
N
16
O
12
in leuprorelin CRS.
STORAGE
If the substance is sterile, store in a sterile, airtight, tamper-proof container.
LABELLING
The label states:
23-48
__________________________________________________________________________________________________________ Ph Eur
23-49
Levamisole Hydrochloride
N
N S
H
,HCl
C
11
H
12
N
2
S,HCl 240.8 16595-80-5
Levamisole Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Levamisole hydrochloride contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of (S)-2,3,5,6-tetrahydro-6-phenylimidazo[2,1-b]thiazole hydrochloride, calculated
CHARACTERS
A white or almost white, crystalline powder, freely soluble in water, soluble in alcohol, slightly soluble
in methylene chloride, practically insoluble in ether.
IDENTIFICATION
First identification: B, D, E.
A. Dissolve 0.5 g in 20 ml of water R. Add 6 ml of 1M sodium hydroxide and 20 ml of methylene
chloride R and shake. Collect the lower layer, wash with two quantities each of 10 ml of water R and
dry over anhydrous sodium sulphate R. Filter and evaporate to dryness in vacuo, at a temperature not
exceeding 40C. The residue melts (2.2.14) at 58C to 61C.
obtained with levamisole hydrochloride CRS. Examine the substances prepared as discs.
E. It complies with the test for specific optical rotation (see Tests).
TESTS
solution Y
7
(Method II, 2.2.2).
Specific optical rotation (2.2.7). 121 to 128 determined on solution S and calculated with
254
R as the
coating substance.
Reference solution (a). Dissolve 25 mg of levamisole hydrochloride CRS in methanol R and dilute to 5 ml
1 volume of concentrated ammonia R, 40 volumes of acetone R and 60 volumes of toluene R. Dry the
plate in an oven at 100C to 105C for 15 min. Examine in ultraviolet light at 254 nm. Any spot in
than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent). Expose the
plate to iodine vapour in an airtight tank for 15 min. Any spot in the chromatogram obtained with
23-50
test solution (a), apart from the principal spot and any spot situated immediately above the starting
point, is not more intense than the spot in the chromatogram obtained with reference solution (b)
(0.5 per cent).
100C to 105C for 4 h.
ASSAY
11
H
13
ClN
2
S.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
23-51
Levobunolol Hydrochloride
O
O
t
BuHN
H HO
,HCl
C
17
H
25
NO
3
,HCl 327.9 27912-14-7
Definition Levobunolol Hydrochloride is (S)-5-(3-tert-butylamino-2-hydroxypropoxy)-1,2,3,4-
tetrahydronaphthalen-1-one hydrochloride. It contains not less than 98.5% and not more than
101.0% of C
17
H
25
NO
3
Characteristics A white or pinkish white, crystalline powder.
Freely soluble in water; sparingly soluble in ethanol (96%).
Identification
levobunolol hydrochloride (RS 200).
Specific optical rotation In a 3% w/v solution in methanol, 19.0 to 20.0, calculated with
0.00050% w/v of the substance being examined, (3) 0.0050% w/v of each of levobunolol hydrochloride
BPCRS and atenolol.
The chromatographic procedure described under Assay may be used.
For solution (1) allow the chromatography to proceed for 3 times the retention time of the
principal peak. The test is not valid unless, in the chromatogram obtained with solution (3), the
resolution factor between the two principal peaks is at least 8.
the areas of any secondary peaks is not greater than twice the area of the principal peak in the
chromatogram obtained with solution (2) (1%).
Loss on drying When dried over phosphorus pentoxide at 110 at a pressure not exceeding 2 kPa for 4
Assay Carry out the method for liquid chromatography, Appendix III D, using solutions in the mobile
phase containing (1) 0.01% w/v of the substance being examined, (2) 0.01% w/v of levobunolol
hydrochloride BPCRS and (3) 0.0050% w/v of each of levobunolol hydrochloride BPCRS and atenolol.
(25 cm 3.9 mm) packed with stationary phase B (10 m) (Lichrosorb RP 8 is suitable), (b) a
solution prepared by mixing 53 volumes of 0.005M sodium heptanesulphonate in methanol with 47
volumes of 0.005M sodium heptanesulphonate in water containing 1 ml of 0.5M sulphuric acid as the
mobile phase with a flow rate of 1 ml per minute and (c) a detection wavelength of 223 nm.
The test is not valid unless in the chromatogram obtained with solution (3) the resolution factor
17
H
25
NO
3
,HCl from the declared content of C
17
H
25
NO
3
,HCl in
levobunolol hydrochloride BPCRS.
Storage Levobunolol Hydrochloride should be kept in a well-closed container and protected from
light.
Preparation
Levobunolol Eye Drops
23-52
IMPURITIES
O
t
BuHN
OH
OH
A. 5-(3-tert-butylamino-2-hydroxypropoxy)-1,2,3,4-tetrahydro-1-naphthol
O
O
NHBu
t
t
BuHN
OH
OH
B. 1,1-(1,2,3,4-tetrahydro-1,5-naphthalenedioxy)bis(3-tert-butylamino)-2-propanol
O N
O
OH OH
O
O
Bu
t
C. meso-5,5-[(3,3-tert-butylamino)bis(2-hydroxypropoxy)]bis-3,4-dihydronaphthalen-
1(2H)-one
23-53
Levocabastine Hydrochloride
1/01
N
F
NC
H
Me
COOH
H
,HCl
C
26
H
30
ClFN
2
O
2
457.0 79547-78-7
Levocabastine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
1
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Levocabastine hydrochloride contains not less than 98.5 per cent and not more than the equivalent
of 101.5 per cent of (3S,4R)-1-[cis-4-cyano-4-(4-fluorophenyl)cyclohexyl]-3-methyl-4-
phenylpiperidine-4-carboxylic acid monohydrochloride, calculated with reference to the dried
substance.
CHARACTERS
A white or almost white powder, practically insoluble in water, sparingly soluble in methanol, slightly
soluble in alcohol and in a 2 g/l solution of sodium hydroxide.
IDENTIFICATION
obtained with levocabastine hydrochloride CRS. Examine the substances prepared as discs.
B. Dissolve 50 mg in a mixture of 0.4 ml of ammonia R and 2 ml of water R. Mix, allow to stand for
5 min and filter. Acidify the filtrate with dilute nitric acid R. It gives reaction (a) of chlorides (2.3.1).
C. It complies with the test for specific optical rotation (see Tests).
TESTS
solution Y
7
(Method II, 2.2.2).
Specific optical rotation (2.2.7). The specific optical rotation is 102 to 106, determined on
solution S, calculated with reference to the dried substance.
Related substances Examine by capillary electrophoresis (2.2.31). Prepare the solutions immediately
before use.
Test solution. Dissolve 25.0 mg of the substance to be examined in a 2 g/l solution of sodium
hydroxide R and dilute to 10.0 ml with the same solvent.
Reference solution (a). Dissolve 2.5 mg of levocabastine hydrochloride CRS and 2.5 mg of levocabastine
impurity D CRS in a 2 g/l solution of sodium hydroxide R and dilute to 200.0 ml with the same
solvent.
Reference solution (b). Dilute 5.0 ml of the test solution to 100.0 ml with a 2 g/l solution of sodium
hydroxide R. Dilute 1.0 ml of the solution to 10.0 ml with a 2 g/l solution of sodium hydroxide R.
The micellar electrokinetic chromatographic procedure may be carried out using:
an uncoated fused-silica capillary with the length to the detector cell of 0.5 m and 75 m in
internal diameter,
an electrolyte solution prepared as follows: dissolve 1.08 g of sodium dodecyl sulphate R and
0.650 g of hydroxypropyl--cyclodextrin R in 5 ml of 2-propanol R and dilute to 50.0 ml with
buffer solution pH 9.0 prepared as follows: dissolve 1.39 g of boric acid R in water R and adjust
to pH 9.0 with 1M sodium hydroxide (about 9 ml). Dilute to 100.0 ml with water R;
an injection of 5 s under a pressure of 3450 Pa,
the following current gradient:
23-54
Time(min) Current (A)
00.17 075
0.1715 75130
1540 130
4060 130170
maintaining the temperature of the capillary at 50C.
Equilibrate the capillary for 2 min with a 2 g/l solution of sodium hydroxide R and then equilibrate
with the electrolyte solution for at least 5 min.
Inject reference solution (a). When the electropherogram is recorded in the prescribed conditions,
the migration times are: levocabastine about 28 min and impurity D about 30 min. The test is not
valid unless the resolution between the peaks due to levocabastine and impurity D is at least 4. If
necessary adjust the current gradient.
Inject a 2 g/l solution of sodium hydroxide R as a blank, the test solution and reference solution (b).
In the electropherogram obtained with the test solution: the area of any peak, apart from the principal
peak, is not greater than the area of the principal peak in the electropherogram obtained with refer-
ence solution (b) (0.5 per cent); the sum of the areas of all peaks, apart from the principal peak, is
not greater than twice the area of the principal peak in the electropherogram obtained with reference
solution (b) (1.0 per cent). Disregard any peak obtained with the blank and any peak with an area
less than 0.1 times the area of the principal peak in the electropherogram obtained with reference
100C to 105C.
ASSAY
first and third point of inflexion.
26
H
30
ClFN
2
O
2
.
STORAGE
IMPURITIES
Qualified impurities: A, B, C, D, E.
Other detectable impurities: F, G, H, I.
N
R
1
H
Me
COOH
H
2
R
3
CN
A. R
1
= R
2
= R
3
= H: (3S,4R)-1-(cis-4-cyano-4-phenylcyclohexyl)-3-methyl-4-phenylpiperidine-
4-carboxylic acid,
B. R
1
= R
2
= H, R
3
= F: (3S,4R)-1-[cis-4-cyano-4-(2-fluorophenyl)cyclohexyl]-3-methyl-4-
phenylpiperidine-4-carboxylic acid,
C. R
1
= H, R
2
= F, R
3
= H: (3S,4R)-1-[cis-4-cyano-4-(3-fluorophenyl)cyclohexyl]-3-methyl-4-
phenylpiperidine-4-carboxylic acid,
N
F
NC
H
COOH
D. 1-[cis-4-cyano-4-(4-fluorophenyl)cyclohexyl]-4-phenylpiperidine-4-carboxylic acid,
N
NC
H
Me
COOH
H
E. (3S,4R)-1-[trans-4-cyano-4-(4-fluorophenyl)cyclohexyl]-3-methyl-4-phenylpiperidine-4-
carboxylic acid,
23-55
N
Me
COOH
H
F (3S,4R)-3-methyl-4-phenylpiperidine-4-carboxylic acid,
N
F
H
Me
COOH
H
H
2
N
O
G. (3S,4R)-1-[cis-4-carbamoyl-4-(4-fluorophenyl)cyclohexyl]-3-methyl-4-phenylpiperidine-
4-carboxylic acid,
F
NC
O
H. 1-(4-fluorophenyl)-4-oxocyclohexanecarbonitrile,
N
F
NC
H
Me
COOH
H
I. (3S,4S)-1-[cis-4-cyano-4-(4-fluorophenyl)cyclohexyl]-3-methyl-4-phenylpiperidine-4-
carboxylic acid.
__________________________________________________________________________________________________________ Ph Eur
23-56
Levocarnitine
Me
3
N COO
_
H OH
+
C
7
H
15
NO
3
161.2 541-15-1
Levocarnitine complies with the requirements of the 3rd edition of the European Pharmacopoeia [1339]. These
Action and use Amino acid derivative.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Levocarnitine contains not less than 98.0 per cent and not more than the equivalent of 102.0 per
cent of (3R)-3-hydroxy-4 -(trimethylammonio)butanoate, calculated with reference to the anhydrous
substance.
CHARACTERS
A white, crystalline powder or colourless crystals, hygroscopic, freely soluble in water, soluble in
warm alcohol, practically insoluble in acetone.
IDENTIFICATION
obtained with levocarnitine CRS. Examine the substances, previously dried in vacuo at 50C for 5 h,
prepared as discs.
C. To 1 ml of solution S (see Tests) add 9 ml of water R, 10 ml of dilute sulphuric acid R and 30 ml of
ammonium reineckate solution R. A pink precipitate is formed. Allow to stand for 30 min. Filter and
wash with water R, with alcohol R and then with acetone R and dry at 80C. The precipitate melts
(2.2.14) at 147C to 150C.
TESTS
Specific optical rotation (2.2.7). 29.0 to 32.0, determined on solution S and calculated with
Reference solution (b). Dissolve 12.5 mg of levocarnitine impurity A CRS in water R and dilute to
50.0 ml with the same solvent. Dilute 2.0 ml of the solution to 20.0 ml with the mobile phase.
Reference solution (c). Dissolve 10.0 mg of levocarnitine impurity A CRS in water R and dilute to
10.0 ml with the same solvent. Dilute 2.0 ml of the solution to 20.0 ml with the mobile phase.
Reference solution (d). Dissolve 0.100 g of levocarnitine CRS in reference solution (c) and dilute to
aminopropylmethylsilyl silica gel for chromatography R (10 m),
potassium dihydrogen phosphate R adjusted to pH 4.7 with dilute sodium hydroxide solution R, and
65 volumes of acetonitrile R,
23-57
When the chromatograms are recorded under the prescribed conditions, the retention times are:
about 9.6 min for levocarnitine and about 10.6 min for levocarnitine impurity A. Adjust the sensiti-
vity of the system so that the height of the principal peak in the chromatogram obtained with refer-
ence solution (b) is at least 20 per cent of the full scale of the recorder.
Inject 25 l of reference solution (d) and continue the chromatography for 15 min. The test is not
valid unless, in the chromatogram obtained with reference solution (d), the resolution between the
peaks corresponding to levocarnitine and to levocarnitine impurity A is at least 0.9.
levocarnitine impurity A is not greater than the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.5 per cent); the area of any peak, apart from the principal
peak and the peak due to levocarnitine impurity A, is not greater than the area of the principal peak
in the chromatogram obtained with reference solution (a) (0.1 per cent).
Chlorides (2.4.4). Dilute 2.5 mlof solution S to 15 ml with water R. The solution complies with the
of water.
ASSAY
Dissolve 0.125 g in a mixture of 3 volumes of anhydrous formic acid R and 50 volumes of anhydrous
acetic acid R. Add 0.2 ml of crystal violet solution R. Titrate with 0.1M perchloric acid until the colour
changes from violet to green.
7
H
15
NO
3
.
STORAGE
IMPURITIES
COO
_
Me
3
N
+
and Z-isomer
A. (E)- or (Z)-4-(trimethylammonio)but-2-enoate,
Me COOH
Me
Me
COOH
H
and enantiomer
B. (1RS,3SR)-1,2,2-trimethylcyclopentane-1,3-dicarboxylic acid (camphoric acid),
CONH
2
Me
3
N
H OH
+
C. [(2R)-3-carbamoyl-2-hydroxypropyl]trimethylammonium (carnitinamide),
CONH
2
Me
3
N
+
and Z-isomer
D. (E)- or (Z)-(3-carbamoylprop-2-enyl)trimethylammonium.
__________________________________________________________________________________________________________ Ph Eur
23-58
Levodopa
HO
HO
COOH
NH
2
H
C
9
H
11
NO
4
197.2 59-92-7
Levodopa complies with the requirements of the 3rd edition of the European Pharmacopoeia [0038]. These
Preparations
Co-beneldopa Capsules
Dispersible Co-beneldopa Tablets
Co-careldopa Tablets
Levodopa Capsules
Levodopa Tablets
When L-dopa is prescribed or demanded, Levodopa shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Levodopa contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of
(S)-2-amino-3-(3,4-dihydroxyphenyl)propionic acid, calculated with reference to the dried
substance.
CHARACTERS
A white or slightly cream-coloured, crystalline powder, slightly soluble in water, practically insoluble
in alcohol and in ether. It is freely soluble in 1M hydrochloric acid and sparingly soluble in 0.1M
hydrochloric acid.
IDENTIFICATION
A. Examine by infrared absorption spectrophotometry (2.2.24) comparing with the spectrum
obtained with levodopa CRS.
B. Dissolve about 2 mg in 2 ml of water R and add 0.2 ml of ferric chloride solution R2. A green colour
develops which changes to bluish-violet on the addition of 0.1 g of hexamethylenetetramine R.
C. Dissolve about 5 mg in a mixture of 5 ml of 1M hydrochloric acid and 5 ml of water R. Add 0.1 ml
of sodium nitrite solution R containing 100 g/l of ammonium molybdate R. A yellow colour develops
which changes to red on the addition of strong sodium hydroxide solution R.
D. To about 5 mg add 1 ml of water R, 1 ml of pyridine R and about 5 mg of nitrobenzoyl chloride R,
mix and allow to stand for 3 min. A violet colour develops which changes to pale yellow on boiling
the mixture. Add. while shaking, 0.2 ml of sodium carbonate solution R. The violet colour reappears.
TESTS
solvent. The solution is not more intensely coloured than reference solution BY
6
(Method II, 2.2.2).
pH (2.2.3). Shake 0.10 g for 15 min with 10 ml of carbon dioxide-free water R. The pH of the
Optical rotation (2.2.7). Dissolve a quantity equivalent to 0.200 g of the dried substance and 5 g of
hexamethylenetetramine R in 10 ml of 1M hydrochloric acid and dilute to 25.0 ml with the same acid.
Allow the solution to stand protected from light for 3 h. The angle of optical rotation is 1.27 to
1.34.
same acid. Dilute 10.0 ml of this solution to 100.0 ml with 0.1M hydrochloric acid. Examined between
230 nm and 350 nm, the solution shows a single absorption maximum, at 280 nm. The specific
absorbance at this maximum is 137 to 147, calculated with reference to the dried substance.
Related substances Examine by thin-layer chromatography (2.2.27), using cellulose for chromato-
graphy R as the coating substance.
23-59
Test solution. Dissolve 0.1 g of the substance to be examined in 5 ml of anhydrous formic acid R and
dilute to 10 ml with methanol R. Prepare immediately before use.
Reference solution (a). Dilute 0.5 ml of the test solution to 100 ml with methanol R.
Reference solution (b). Dissolve 30 mg of tyrosine R in 1 ml of anhydrous formic acid R and dilute to
100 ml with methanol R. Mix 1 ml of this solution with 1 ml of the test solution.
Apply separately to the plate as bands 20 mm long 10 l of the test solution, 10 l of reference
solution (a) and 20 l of reference solution (b). Dry in a current of air. Develop over a path of 15 cm
using a mixture of 25 volumes of glacial acetic acid R, 25 volumes of water R and 50 volumes of
butanol R. Dry the plate in a current of warm air and spray with a freshly prepared mixture of equal
volumes of a 100 g/l solution of ferric chloride R and a 50 g/l solution of potassium ferricyanide R.
Examine the chromatograms immediately. Any spot in the chromatogram obtained with the test
solution, apart from the principal spot is not more intense than the spot in the chromatogram
obtained with reference solution (b) shows, above the principal spot, a distinct spot which is more
intense than the spot in the chromatogram obtained with reference solution (a).
100C to 105C.
ASSAY
Dissolve 0.180 g, heating if necessary in 5 ml of anhydrous formic acid R and add 25 ml of anhydrous
acetic acid R and 25 ml of dioxan R. Titrate with 0.1M perchloric acid, using 0.1 ml of crystal violet
solution R as indicator, until a green colour is obtained.
9
H
11
NO
4
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
23-60
Levodropropizine
1/01
N
N OH
H OH
C
13
H
20
N
2
O
2
236.3 99291-25-5
Levodropropizine complies with the requirements of the 3rd edition of the European Pharmacopoeia [1535].
Action and use Antitussive.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Levodropropizine contains not less than 98.5 per cent and not more than the equivalent of 101.0 per
cent of (2S)-3-(4-phenylpiperazin-1-yl)propane-1,2-diol, calculated with reference to the dried
substance.
CHARACTERS
A white or almost white powder, slightly soluble in water, freely soluble in dilute acetic acid and in
methanol, slightly soluble in alcohol.
IDENTIFICATION
A. Dissolve 1.50 g in a 21 g/l solution of hydrochloric acid R and dilute to 50.0 ml with the same acid.
The specific optical rotation (2.2.7) is 30.0 to 33.5, calculated with reference to the dried
substance.
obtained with levodropropizine CRS.
TESTS
pH (2.2.3). Suspend 2.5 g in carbon dioxide-free water R, heat to dissolve, cool down to room
temperature and dilute to 100 ml with the same solvent. The pH of the solution is 9.2 to 10.2.
Impurity B and related substances Examine by liquid chromatography (2.2.29).
Reference solution (a). Dissolve 12.0 mg of 1-phenylpiperazine R in methanol R and dilute 100.0 ml
Reference solution (b). To 24.0 mg levodropropizine CRS add 1.0 ml of reference solution (a) and
Reference solution (c). Mix 0.5 ml of the test solution with 1 ml of reference solution (a) and dilute to
deactivated octylsilyl silica gel for chromatography (5 m),
as mobile phase, at a flow rate of 1.5 ml/min, a mixture of 12 volumes of methanol R and 88
volumes of a 6.81 g/l solution of potassium dihydrogen phosphate R, adjusted to pH 3.0 with
phosphoric acid R,
Inject 20 l of the test solution, reference solution (b) and reference solution (c). The test is not
valid unless in the chromatogram obtained with reference solution (c), the resolution between the
peaks corresponding to levodropropizine and impurity B is at least 2.0. In the chromatogram
obtained with the test solution, the area of the peak corresponding to impurity B is not greater than
that of the peak due to impurity B in the chromatogram obtained with reference solution (b) (0.5 per
cent); the area of any peak, apart from the principal peak and any peak corresponding to impurity B,
is not greater than 0.2 times the area of the peak due to impurity B in the chromatogram obtained
Disregard any peak with an area less than 0.02 times that of the peak corresponding to impurity B
in the chromatogram obtained with reference solution (b) (0.01 per cent).
23-61
Impurity C Not more than 10 ppm determined by gas chromatography (2.2.28).
Test solution. Dissolve 2.0 g of the substance to be examined in a ground-glass-stoppered tube with
5 ml of methylene chloride R. Stopper the tube and sonicate for 30 min. Add 1.0 ml of water R and
shake vigorously for 1 min. Centrifuge for 5 min at approximately 100 g and use the upper layer.
Reference solution (a). Dissolve 0.20 g of glycidol R in 100.0 ml of methylene chloride R. Dilute 1.0 ml
of this solution to 100.0 ml with methylene chloride R.
Reference solution (b). To 2.0 g of the substance to be examined in a ground-glass-stoppered tube add
1.0 ml of reference solution (a) and 4.0 ml of methylene chloride R. Stopper the tube and sonicate for
30 min. Add 1.0 ml of water R and shake vigorously for 1 min. Centrifuge for 5 min at approxi-
mately 100 g and use the upper layer.
a column 30 m long and 0.53 mm in internal diameter coated with poly[(cyanopropyl)(phenyl)]-
[dimethyl]siloxane R (film thickness 3 m),
helium for chromatography R as the carrier gas at a flow rate of 2 ml/min with a split ratio of 1:12,
detector at 250C.
with the test solution, the area of any peak corresponding to impurity C is not greater than half of the
area of the peak due to impurity C in the chromatogram obtained with reference solution (b)
(10 ppm).
Enantiomeric purity Examine by liquid chromatography (2.2.29).
Test solution. Dissolve 10.0 mg of the substance to be examined in 10.0 ml of a mixture of 40
volumes of ethanol R and 60 volumes of hexane R. Dilute 1.0 ml of the solution to 50.0 ml with a
mixture of 40 volumes of ethanol R and 60 volumes of hexane R.
Reference solution (a). Dissolve 10.0 mg of levodropropizine CRS in 10.0 ml of a mixture of 40
volumes of ethanol R and 60 volumes of hexane R. Dilute 1.0 ml of the solution to 50.0 ml with a
Reference solution (b). Dissolve 10.0 mg of levodropropizine impurity A CRS in 10.0 ml of a mixture of
40 volumes of ethanol R and 60 volumes of hexane R. Dilute 1.0 ml of the solution to 50.0 ml with a
Reference solution (c). Dilute 1.0 ml of reference solution (b) to 50.0 ml with a mixture of 40
volumes of ethanol R and 60 volumes of hexane R.
Reference solution (d). Mix 1 ml of reference solution (a) with 1 ml of reference solution (b).
a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with silica gel OD
for chiral separations R,
as mobile phase at a flow rate of 0.8 ml/min, a mixture of 0.2 volumes of diethylamine R, 5
volumes of ethanol R and 95 volumes of hexane R,
reference solution (d), the resolution between the peaks corresponding to impurity A (first peak) and
levodropropizine is at least 2.0; the retention times of the principal peak in the chromatograms
obtained with the test solution and reference solution (a) are identical.
impurity A is not greater than the area of the principal peak in the chromatogram obtained with
reference solution (c) (2 per cent).
60C over diphosphorus pentoxide R at a pressure of 0.15 kPa to 0.25 kPa for 4 h.
ASSAY
Dissolve 0.100 g in 50 ml of anhydrous acetic acid R. Carry out a potentiometric titration (2.2.20)
using 0.1M perchloric acid. Read the volume added at the second point of inflexion.
13
H
20
N
2
O
2
.
STORAGE
23-62
IMPURITIES
N
N OH
OH H
A. (2R)-3-(4-phenylpiperazin-1-yl)propane-1,2-diol (dextrodropropizine),
N
NH
B. 1-phenylpiperazine,
OH
H
and enantiomer
C. [(2RS)-oxiran-2-yl]methanol (glycidol).
__________________________________________________________________________________________________________ Ph Eur
23-63
Levomenthol
Me Me
H
Me H
OH
H
H
C
10
H
20
O 156.3 2216-51-5
Levomenthol complies with the requirements of the 3rd edition of the European Pharmacopoeia [0619]. These
Action and use Decongestant.
Preparation
Menthol and Benzoin Inhalation
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Levomenthol is (1R,2S,5R)-2-isopropyl-5-methylcyclohexanol.
CHARACTERS
Prismatic or acicular, colourless, shiny crystals, practically insoluble in water, very soluble in alcohol,
in ether and in light petroleum, freely soluble in fatty oils and in liquid paraffin, very slightly soluble
in glycerol.
IDENTIFICATION
Second identification: B, D.
the same solvent.
Reference solution. Dissolve 25 mg of menthol CRS in methanol R and dilute to 5 ml with the same
solvent.
volumes of ethyl acetate R and 95 volumes of toluene R. Allow the plate to dry in air until the odour of
the solvents is no longer perceptible and spray with anisaldehyde solution R. Heat at 100C to 105C
for 5 min to 10 min. The principal spot in the chromatogram obtained with the test solution is
reference solution.
C. Examine the chromatograms obtained in the test for related substances. The principal peak in the
chromatogram obtained with test solution (b) is similar in position and approximate dimensions to
the principal peak in the chromatogram obtained with reference solution (c).
D. Dissolve 0.20 g in 0.5 ml of anhydrous pyridine R. Add 3 ml of a 150 g/l solution of dinitrobenzoyl
chloride R in anhydrous pyridine R. Heat on a water-bath for 10 min. Add 7.0 ml of water R in small
quantities with stirring and allow to stand in iced water for 30 min. A precipitate is formed. Allow to
stand and decant the supernatant liquid. Wash the precipitate with two quantities, each of 5 ml, of
iced water R, recrystallise from 10 ml of acetone R, wash with iced acetone R and dry at 75C at a
pressure not exceeding 2.7 kPa for 30 min. The crystals melt (2.2.14) at 154C to 157C.
TESTS
Acidity or alkalinity Dissolve 1.0 g in alcohol R and dilute to 10 ml with the same solvent. Add
0.1 ml of phenolphthalein solution R; the solution is colourless. Not more than 0.5 ml of 0.01M sodium
Specific optical rotation (2.2.7). 48 to 51, determined on solution S.
23-64
Test solution (b). Dilute 1.0 ml of test solution (a) to 10.0 ml with methylene chloride R.
Reference solution (a). Dissolve 40.0 mg of the substance to be examined and 40.0 mg of isomenthol R
in methylene chloride R and dilute to 100.0 ml with the same solvent.
Reference solution (b). Dilute 0.10 ml of test solution (a) to 100.0 ml with methylene chloride R.
Reference solution (c). Dissolve 40.0 mg of menthol CRS in methylene chloride R and dilute to 100.0 ml
a glass column 2.0 m long and 2 mm internal diameter packed with diatomaceous earth for gas
chromatography R impregnated with 15 per cent m/m of macrogol 1500 R,
Inject separately 1 l of each solution. Record the chromatograms for twice the retention time of
the peak corresponding to menthol. In the chromatogram obtained with test solution (a), the sum of
the areas of the peaks, apart from the principal peak, is not greater than 1 per cent of the area of the
principal peak. Disregard any peak due to the solvent and any peak whose area is less than 0.05 per
cent of the area of the principal peak. The test is not valid unless: in the chromatogram obtained with
reference solution (a) the resolution between the peaks corresponding to menthol and isomenthol is
not less than 1.4 and the principal peak in the chromatogram obtained with reference solution (b) has
a signal- to-noise ratio of not less than 5.
Residue on evaporation Evaporate 2.00 g on a water-bath and heat in an oven at 100C to 105C
for 1 h. The residue weighs not more than 1.0 mg (0.05 per cent).
STORAGE
Store in a well-closed container, in a cool place.
__________________________________________________________________________________________________________ Ph Eur
23-65
Levomepromazine Hydrochloride / Methotrimeprazine
Hydrochloride
S
N
Me H
NMe
2
OMe
,HCl
C
19
H
24
N
2
OS,HCl 364.9 4185-80-2
Levomepromazine Hydrochloride complies with the requirements of the 3rd edition of the European Pharma-
copoeia [0505]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Levomepromazine hydrochloride contains not less than 98.5 per cent and not more than the
equivalent of 101.0 per cent of (R)-10-[3-(dimethylamino)-2-methylpropyl]-2-methoxyphenothiazine
CHARACTERS
A white or very slightly yellow, crystalline powder, slightly hygroscopic, freely soluble in water and in
alcohol, practically insoluble in ether. It deteriorates when exposed to air and light. It exists in two
forms, one melting at about 142C and the other at about 162C.
IDENTIFICATION
A. Prepare the solution protected from bright light and carry out the measurements immediately. Dissolve
50.0 mg in water R and dilute to 500.0 ml with the same solvent. Dilute 10.0 ml of this solution to
100.0 ml with water R. Examined between 230 nm and 340 nm (2.2.25), the solution shows two
absorption maxima, at 250 nm and 302 nm. The specific absorbance at the maximum at 250 nm is
640 to 700.
B. It complies with the identification test for phenothiazines by thin-layer chromatography (2.3.3).
C. Introduce 0.2 g into a 100 ml separating funnel. Add 5 ml of water R and 0.5 ml of strong sodium
hydroxide solution R. Shake vigorously with two quantities, each of 10 ml, of ether R. Combine the
ether layers, dry over anhydrous sodium sulphate R and evaporate to dryness. Keep the residue at
100C to 105C for 15 min and allow to crystallise in iced water. Initiate crystallisation if necessary
by scratching the wall of the flask with a glass rod. Dry the crystals at 60C for 2 h. The crystals melt
(2.2.14) at 122C to 128C.
TESTS
Acidity or alkalinity To 10 ml of solution S add 0.1 ml of bromocresol green solution R. Not more
than 0.5 ml of 0.01M sodium hydroxide or 1.0 ml of 0.01M hydrochloric acid is required to change the
Related substances Carry out the test protected from bright light. Examine by thin-layer chromato-
254
Test solution. Dissolve 0.2 g of the substance to be examined in a mixture of 5 volumes of diethyl-
amine R and 95 volumes of methanol R and dilute to 10 ml with the same mixture of solvents. Prepare
Reference solution. Dilute 0.5 ml of the test solution to 100 ml with a mixture of 5 volumes of
23-66
atogram obtained with the reference solution (0.5 per cent).
100C to 105C for 3 h.
ASSAY
Dissolve 0.300 g in 5 ml of water R and add 50 ml of 2-propanol R. Titrate with 0.1M sodium
19
H
25
ClN
2
OS.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
23-67
Levomepromazine Maleate / Methotrimeprazine
Maleate
S
N
Me H
NMe
2
OMe
H
H COOH
COOH
,
C
19
H
24
N
2
OS,C
4
H
4
O
4
444.6 7104-38-3
Levomepromazine Maleate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Levomepromazine maleate contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of (R)-10-(3-dimethylamino-2-methylpropyl)-2-methoxyphenothiazine (Z)-butene-
dioate, calculated with reference to the dried substance.
CHARACTERS
A white or slightly yellowish, crystalline powder, slightly soluble in water, sparingly soluble in
methylene chloride, slightly soluble in alcohol, practically insoluble in ether. It deteriorates when
exposed to air and light.
IDENTIFICATION
obtained with levomepromazine maleate CRS. Examine the substances prepared as discs.
C. It complies with the identification test for phenothiazines by thin-layer chromatography (2.3.3).
254
Reference solution. Dissolve 50 mg of maleic acid CRS in a mixture of 10 volumes of water R and 90
volumes of acetone R and dilute to 10 ml with the same mixture of solvents.
Apply separately to the plate as bands 10 mm by 2 mm 5 l of each solution. Develop over a path of
12 cm using a mixture of 3 volumes of water R, 7 volumes of anhydrous formic acid R and 90 volumes
of di-isopropyl ether R. Dry the plate at 120C for 10 min and examine in ultraviolet light at 254 nm.
The chromatogram obtained with the test solution shows a band at the starting-point and another
band similar in position and size to the principal band in the chromatogram obtained with the
reference solution.
TESTS
pH (2.2.3). Carry out the test protected from bright light. Introduce 0.50 g into a conical flask and add
25.0 ml of carbon dioxide-free water R. Shake and allow the solids to settle. The pH of the supernatant
Specific optical rotation (2.2.7). Dissolve 1.25 g in dimethylformamide R and dilute to 25.0 ml with
the same solvent. The specific optical rotation is 7.0 to 8.5, calculated with reference to the dried
substance.
Related substances Carry out the test protected from bright light and prepare the solutions immediately
23-68
before use. Examine by thin-layer chromatography (2.2.27), using silica gel GF
254
R as the coating
substance.
Reference solution. Dilute 0.5 ml of the test solution to 100 ml with a mixture of 10 volumes of water R
atogram obtained with the reference solution (0.5 per cent).
100C to 105C for 3 h.
ASSAY
23
H
28
N
2
O
5
S.
STORAGE
IMPURITIES
N
S
OMe
H
A. 2-methoxyphenothiazine,
N
S
OMe
NMe
2
Me H
O
B. levomepromazine 5-oxide.
__________________________________________________________________________________________________________ Ph Eur
23-69
Levonorgestrel
O
H
H H
Et
C
OH
H
CH
C
21
H
28
O
2
312.5 797-63-7
Levonorgestrel complies with the requirements of the 3rd edition of the European Pharmacopoeia [0926]. These
Preparation
Levonorgestrel and Ethinylestradiol Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Levonorgestrel contains not less than 98.0 per cent and not more than the equivalent of 102.0 per
cent of 13-ethyl-17-hydroxy-18,19-dinor-17-pregn-4-en-20-yn-3-one, calculated with reference
CHARACTERS
IDENTIFICATION
obtained with levonorgestrel CRS.
TESTS
Specific optical rotation (2.2.7). Dissolve 0.200 g in chloroform R and dilute to 10.0 ml with the
same solvent. The specific optical rotation is 30 to 35.
coating substance.
the same solvent.
Reference solution (a). Dilute 1 ml of the test solution to 10 ml with methylene chloride R. Dilute 1 ml
of this solution to 20 ml with methylene chloride R.
Reference solution (b). Dilute 4 ml of reference solution (a) to 10 ml with methylene chloride R.
Reference solution (c). Dissolve 5 mg of levonorgestrel CRS and 5 mg of ethinylestradiol CRS in methylene
20 volumes of ethyl acetate R and 80 volumes of methylene chloride R. Allow the plate to dry in air,
spray with a 100 g/l solution of phosphomolybdic acid R in alcohol R, heat at 100C to 105C for
15 min and examine immediately. Any spot in the chromatogram obtained with the test solution,
obtained with reference solution (a) (0.5 per cent) and at most two such spots are more intense than
spots.
100C to 105C.
ASSAY
Dissolve 0.200 g in 45 ml of tetrahydrofuran R. Add 10 ml of a 100 g/l solution of silver nitrate R.
23-70
After 1 min, titrate with 0.1M sodium hydroxide, determining the end-point potentiometrically
(2.2.20). Carry out a blank titration.
1 ml of 0.1M sodium hydroxide is equivalent to 31.25 mg C
21
H
28
O
2
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
23-71
Levothyroxine Sodium / Thyroxine Sodium
O
COONa
NH
2
H
HO
I
I
I
I
C
15
H
10
I
4
NNaO
4
,xH
2
O 799 (anhydrous) 55-03-8 (anhydrous)
Levothyroxine Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Thyroid hormone.
Preparation
Levothyroxine Tablets/Thyroxine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Levothyroxine sodium contains not less than 97.0 per cent and not more than the equivalent of
102.0 per cent of sodium (S)-2-amino-3-[4-(4-hydroxy-3,5-di-iodophenoxy) -3,5-di-
iodophenyl]propanoate, calculated with reference to the dried substance. It contains a variable
amount of water.
CHARACTERS
An almost white or slightly brownish-yellow powder or a fine, crystalline powder, very slightly soluble
in water, slightly soluble in alcohol, practically insoluble in ether. It dissolves in dilute solutions of
alkali hydroxides.
IDENTIFICATION
obtained with levothyroxine sodium CRS.
Test solution. Dissolve 5 mg of the substance to be examined in a mixture of 5 volumes of concentrated
Reference solution (a). Dissolve 5 mg of levothyroxine sodium CRS in a mixture of 5 volumes of
solvents.
Reference solution (b). Dissolve 5 mg of liothyronine sodium CRS in a mixture of 5 volumes of concen-
trated ammonia R and 70 volumes of methanol R and dilute to 5 ml with the same mixture of solvents.
Mix 1 ml of this solution and 1 ml of the test solution.
20 volumes of concentrated ammonia R, 35 volumes of 2-propanol R and 55 volumes of ethyl acetate R.
Allow the plate to dry in air and spray with ninhydrin solution R. Heat at 100C to 105C until the
spots appear. Examine in daylight. The principal spot in the chromatogram obtained with the test
D. To about 50 mg in a porcelain dish add a few drops of sulphuric acid R and heat. Violet vapour is
evolved.
E. To 20 mg add 2 ml of dilute sulphuric acid R. Heat on a water-bath and then carefully over a naked
flame, increasing the temperature gradually up to about 600C. Continue the ignition until most of
23-72
the black particles have disappeared. Dissolve the residue in 2 ml of water R. The solution gives
TESTS
Solution S Dissolve 0.500 g in 23 ml of a gently boiling mixture of 1 volume of 1M hydrochloric acid
and 4 volumes of alcohol R. Cool and dilute to 25.0 ml with the same mixture of solvents.
Appearance of solution Freshly prepared solution S is not more intensely coloured than reference
solution BY
3
(Method II, 2.2.2).
Liothyronine and other related substances Examine the chromatograms obtained in the assay. In
the chromatogram obtained with test solution (a), the area of any peak corresponding to liothyronine
is not greater than that of the principal peak in the chromatogram obtained with reference solution
(b) (1.0 per cent) and the sum of the areas of all the peaks apart from the principal peak and any
peak corresponding to liothyronine is not greater than the area of the principal peak in the chromato-
that of the peak in the chromatogram obtained with reference solution (d).
at 100C to 105C.
ASSAY
Examine by liquid chromatography (2.2.29). Protect the solutions from light throughout the assay.
Test solution (a). Dissolve 20.0 mg of the substance to be examined in methanolic sodium hydroxide
solution R and dilute to 100.0 ml with the same solvent.
Test solution (b). Dilute 2.0 ml of test solution (a) to 200 ml with methanolic sodium hydroxide
solution R.
Reference solution (a). Dissolve 20.0 mg of levothyroxine CRS in methanolic sodium hydroxide solution R
and dilute to 100.0 ml with the same solvent. Dilute 2.0 ml of this solution to 200 ml with methanolic
sodium hydroxide solution R.
Reference solution (b). Dissolve 5 mg of liothyronine sodium CRS in methanolic sodium hydroxide
solution R and dilute to 50.0 ml with the same solvent. Dilute 10.0 ml of this solution to 50 ml with
methanolic sodium hydroxide solution R. Dilute 10.0 ml of this solution to 100 ml with methanolic
sodium hydroxide solution R.
Reference solution (c). Mix equal volumes of reference solution (a) and reference solution (b).
Reference solution (d). Dilute 1 ml of reference solution (a) to 10 ml with methanolic sodium hydroxide
solution R.
a column 0.25 m long and 4 mm in internal diameter packed with nitrile silica gel for chromato-
graphy R (5 m to 10 m),
as mobile phase at a flow rate of 1 ml per minute a mixture of 1 volume of phosphoric acid R, 300
a loop injector.
Inject separately 50 l of each solution. Continue the chromatography for 3.5 times the retention
time of the principal peak. The assay is not valid unless the resolution between the peaks correspond-
ing to levothyroxine and liothyronine in the chromatogram obtained with reference solution (c) is at
least 4 and the principal peak in the chromatogram obtained with reference solution (d) has a signal-
to-noise ratio of at least 5. Calculate the percentage of C
15
H
10
I
4
NNaO
4
r
r
u
972 . 0
C
S
S
S
u
=area of the peak corresponding to levothyroxine in the chromatogram obtained with test
solution (b);
S
r
=area of the peak corresponding to levothyroxine in the chromatogram obtained with
reference solution (a);
C
r
=declared content of C
15
H
11
I
4
NO
4
in levothyroxine CRS.
STORAGE
Store in an airtight container, protected from light, at 2C to 8C.
__________________________________________________________________________________________________________ Ph Eur
23-73
Lidocaine / Lignocaine
H
N
NEt
2
Me
O
Me
C
14
H
22
N
2
O 234.3 137-58-6
Lidocaine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0727]. These
Action and use Local anaesthetic; anti-arrhythmic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Lidocaine contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of
2-diethylamino-2,6-dimethylacetanilide, calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water, very soluble in alcohol and
in methylene chloride, freely soluble in ether.
IDENTIFICATION
A. Melting point (2.2.14): 66C to 70C, determined without previous drying.
obtained with lidocaine CRS.
C. Dissolve 0.20 g in a mixture of 0.5 ml of dilute hydrochloric acid R and 10 ml of water R with
warming and add 10 ml of picric acid solution R. The precipitate, washed with water R and dried,
melts (2.2.14) at about 230C, with decomposition.
D. To about 5 mg add 0.5 ml of fuming nitric acid R. Evaporate to dryness on a water-bath, cool and
dissolve the residue in 5 ml of acetone R. Add 0.2 ml of alcoholic potassium hydroxide solution R. A
green colour is produced.
E. Dissolve about 0.1 g in 1 ml of alcohol R and add 0.5 ml of a 100 g/l solution of cobalt nitrate R. A
bluish-green precipitate is formed.
TESTS
Appearance of solution Dissolve 1.0 g in 3 ml of dilute hydrochloric acid R and dilute to 10 ml with
water R. The solution is clear (2.2.1) and colourless (Method II, 2.2.2).
2,6-Dimethylaniline Dissolve 0.25 g in methanol R and dilute to 10 ml with the same solvent. To
2 ml of the solution add 1 ml of a freshly prepared 10 g/l solution of dimethylaminobenzaldehyde R in
methanol R and 2 ml of glacial acetic acid R and allow to stand for 10 min. Any yellow colour in the
manner using 2 ml of a 2.5 mg/l solution of 2,6-dimethylaniline R in methanol R (100 ppm).
Chlorides (2.4.4). Dissolve 1.4 g in a mixture of 3 ml of dilute nitric acid R and 12 ml of water R.
The solution complies with the limit test for chlorides (35 ppm).
Sulphates (2.4.13). Dissolve 0.2 g in 5 ml of alcohol R and dilute to 20 ml with distilled water R.
15 ml of the solution complies with the limit test for sulphates (0.1 per cent).
tion of water.
23-74
ASSAY
To 0.200 g add 50 ml of anhydrous acetic acid R and stir until dissolution is complete. Titrate with
14
H
22
N
2
O.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
23-75
Lidocaine Hydrochloride / Lignocaine Hydrochloride
1/01
H
N
NEt
2
Me
O
Me
,HCl
C
14
H
22
N
2
O,HCl,H
2
O 288.8 6108-05-0
Lidocaine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Local anaesthetic; anti-arrhythmic.
Preparations
Lidocaine Gel/Lignocaine Gel
Lidocaine and Chlorhexidine Gel/Lignocaine and Chlorhexidine Gel
Lidocaine Injection/Lignocaine Injection
Lidocaine and Adrenaline Injection/Lignocaine and Epinephrine Injection
Sterile Lidocaine Solution/Sterile Lignocaine Solution
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Lidocaine hydrochloride contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of 2-diethylamino-2,6-dimethylacetanilide hydrochloride, calculated with reference
CHARACTERS
A white, crystalline powder, very soluble in water, freely soluble in alcohol.
IDENTIFICATION
First identification: A, B, F.
A. Melting point (2.2.14): 74C to 79C, determined without previous drying.
obtained with lidocaine hydrochloride CRS.
C. Dissolve 0.2 g in 10 ml of water R and add 10 ml of picric acid solution R. The precipitate, washed
with water R and dried, melts (2.2.14) at about 230C.
D. To about 5 mg add 0.5 ml of fuming nitric acid R. Evaporate to dryness on a water-bath, cool and
dissolve the residue in 5 ml of acetone R. Add 0.2 ml of alcoholic potassium hydroxide solution R. A
green colour is produced.
E. To 5 ml of solution S (see Tests) add 5 ml of water R and make alkaline with dilute sodium
hydroxide solution R. Collect the precipitate on a filter and wash with water R. Dissolve half of the
precipitate in 1 ml of alcohol R and add 0.5 ml of a 100 g/l solution of cobalt nitrate R. A bluish-green
F. It gives reaction (a) of chlorides (2.3.1).
TESTS
Impurity A
Solution (a). Dissolve 0.25 g of the substance to be examined in methanol R and dilute to 10 ml with
the same solvent. This solution is used to prepare the test solution.
23-76
Solution (b). Dissolve 50 mg of 2,6-dimethylaniline R in methanol R and dilute to 100 ml with the same
solvent. Dilute 1 ml of the solution to 100 ml with methanol R. This solution is used to prepare the
standard.
Using three flat-bottomed tubes, place in the first 2 ml of solution (a), in the second 1 ml of solution
(b) and 1 ml of methanol R and in the third 2 ml of methanol R (used to prepare a blank). To each
tube add 1 ml of a freshly prepared 10 g/l solution of dimethylaminobenzaldehyde R in methanol R and
2 ml of glacial acetic acid R and allow to stand at room temperature for 10 min. The intensity of the
yellow colour of the test solution is between that of the blank and that of the standard (100 ppm).
Heavy metals (2.4.8). Dissolve 1.0 g in water R and dilute to 25 ml with the same solvent. Carry out
the prefiltration. 10 ml of the prefiltrate complies with limit test E for heavy metals (5 ppm). Prepare
the standard using 2 ml of lead standard solution (1 ppm Pb) R.
of water.
ASSAY
tiometric titration (2.2.20), using 0.1M sodium hydroxide. Read the volume added between the 2
inflexion points.
14
H
23
ClN
2
O.
STORAGE
IMPURITIES
NH
2
Me Me
A. 2,6-dimethylaniline.
__________________________________________________________________________________________________________ Ph Eur
23-77
Lime Flower
Lime Flower complies with the requirements of the 3rd edition of the European Pharmacopoeia [0957]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Lime flower consists of the whole, dried inflorescence of Tilia cordata Miller, of Tilia platyphyllos
Scop., of Tilia vulgaris Heyne or a mixture of these.
CHARACTERS
Lime flower has a faint aromatic odour and a faint, sweet and mucilaginous taste.
IDENTIFICATION
A. The inflorescence is yellowish-green. The main axis of the inflorescence bears a linguiform bract,
membranous, yellowish-green, practically glabrous, the central vein of which is joined for up to about
half of its length with the peduncle. The inflorescence usually consists of two to seven flowers,
occasionally up to sixteen. The sepals are detached easily from the perianth; they are up to 6 mm
long, their abaxial surface is usually glabrous, their adaxial surface and their borders are strongly
pubescent. The five spatulate, thin petals are yellowish-white, up to 8 mm long. They show fine
venation and their borders only are sometimes covered with isolated trichomes. The numerous
stamens are free and usually constitute five groups. The superior ovary has a pistil with a somewhat
5-lobate stigma.
B. Separate the inflorescence into its different parts. Examine under a microscope using chloral
hydrate solution R. The adaxial epidermis of the bract shows cells with straight or slightly sinuous
anticlinal walls; the abaxial epidermis shows cells with wavy-sinuous anticlinal walls and anomocytic
stomata (2.8.3). Isolated cells in the mesophyll contain small calcium oxalate cluster crystals. The
parenchyma of the sepals shows, particularly near the veins, numerous mucilaginous cells and cells
containing small calcium oxalate clusters. The adaxial epidermis of sepals bears bent, thick-walled
covering trichomes, unicellular or stellate with up to five cells. The epidermal cells of the petals show
straight anticlinal walls with a striated cuticle without stomata. The parenchyma of the petals shows
small calcium oxalate clusters and especially in its acuminate part mucilaginous cells. The pollen
grains have a diameter of about 30 m to 40 m and are oval or slightly triangular with three
germinal pores and a finely granulated exine. The ovary is glabrous or densely covered with
trichomes, often very twisted, unicellular or stellate with two to four branches.
substance.
Test solution. Shake 1.0 g of the powdered drug (355) with 10 ml of methanol R in a water-bath at
65C for 5 min. Allow to cool and filter.
Reference solution. Dissolve 2.0 mg of caffeic acid R, 5 mg of hyperoside R and 5 mg of rutin R in 10 ml
of methanol R.
ketone R and 50 volumes of ethyl acetate R. Dry the plate at 100C to 105C and spray the warm plate
with a 10 g/l solution of diphenylboric acid aminoethyl ester R in methanol R. Then spray the plate with a
50 g/l solution of macrogol 400 R in methanol R. Allow the plate to dry for about 30 min and examine
in ultraviolet light at 365 nm. The chromatogram obtained with the reference solution shows in order
of increasing R
f
value zones corresponding to rutin and hyperoside with yellowish-orange to
brownish-orange fluorescence and a zone corresponding to caffeic acid with greenish-blue fluores-
cence. In the chromatogram obtained with the test solution, the main zone shows brownish-yellow to
orange fluorescence. This zone is situated just above the zone of hyperoside in the chromatogram
obtained with the reference solution. In daylight, this zone stands out from the other zones as the
main zone. At the R
f
level of rutin there is also a zone of brownish-yellow fluorescence. Below this
zone, two zones of yellow fluorescence may be present. Between the zones of rutin and hyperoside,
zones of orange and yellow fluorescence are visible. Between the zones of hyperoside and caffeic acid,
up to five zones of yellow to orange fluorescence are present. Immediately below the zone of caffeic
acid is a zone of a blue fluorescence.
TESTS
Foreign matter (2.8.2). Not more than 2 per cent, determined on 30 g. There are no inflorescences
with a bract bearing at the abaxial face stellate, five- to eight-rayed trichomes and flowers having an
23-78
apparent double corolla by transformation of five stamens into petal-like staminoids and having a
pistil which is not lobular nor indented. 6-merous flowers occur only occasionally (Tilia americana L.,
Tilia tomentosa Moench).
STORAGE
__________________________________________________________________________________________________________ Ph Eur
23-79
Lincomycin Hydrochloride
N
H
H
Me
Pr
n
,HCl
CH
3
H HO
H CONH
OH
SMe
HO
O
OH
C
18
H
34
N
2
O
6
S,HCl,H
2
O 461.0 7179-49-9
Lincomycin Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparations
Lincomycin Capsules
Lincomycin Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Lincomycin hydrochloride consists mainly of the monohydrate of methyl 6,8-dideoxy-6-[(2S,4R)-1-
methyl-4-propylpyrrolidine-2-carboxamido]-1-thio-D-erythro--D-galacto-octopyranoside hydro-
chloride, an antimicrobial substance produced by Streptomyces lincolnensis var. lincolnensis or by any
other means. It contains not less than 82.5 per cent and not more than 93.0 per cent of lincomycin
(C
18
H
34
N
2
O
6
S), calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white, crystalline powder, very soluble in water, slightly soluble in alcohol, very
slightly soluble in acetone, practically insoluble in ether.
IDENTIFICATION
obtained with lincomycin hydrochloride CRS.
the same solvent.
Reference solution (a). Dissolve 10 mg of lincomycin hydrochloride CRS in methanol R and dilute to
Reference solution (b). Dissolve 10 mg of lincomycin hydrochloride CRS and 10 mg of clindamycin
plate to dry in air and spray with a 1 g/l solution of potassium permanganate R. The principal spot in
the chromatogram obtained with the test solution is similar in position, colour and size to the
3 ml of sodium carbonate solution R and 1 ml of a 20 g/l solution of sodium nitroprusside R. A violet-red
colour develops.
D. Dissolve 0.1 g in water R and dilute to 10 ml with the same solvent. The solution gives reaction
TESTS
23-80
solution Y
6
(Method II, 2.2.2).
substance.
Lincomycin B Examine the chromatogram obtained in the assay with test solution (a). The area of
the peak due to lincomycin B, which is eluted just before lincomycin, is not more than 5 per cent of
the area of the peak due to lincomycin.
tion of water.
ASSAY
Examine by gas chromatography (2.2.28), using dotriacontane R as the internal standard.
Internal standard solution. Dissolve 0.200 g of dotriacontane R in chloroform R and dilute to 25.0 ml
Test solution (a). Dissolve 0.100 g of the substance to be examined in a 20 g/l solution of imidazole R
in chloroform R and dilute to 100.0 ml with the same solution. Shake until solution is complete. Place
4.0 ml of the solution in a ground-glass-stoppered 15 ml centrifuge tube. Add 1.0 ml of a mixture of
1 volume of chlorotrimethylsilane R and 99 volumes of N,O-bis(trimethylsilyl)acetamide R and swirl
gently. Position the glass stopper loosely in the tube and heat at 65C for 30 min.
Test solution (b). Prepare as described for test solution (a) but add 10.0 ml of the internal standard
solution before dissolution of the substance to be examined.
Reference solution. Prepare as described for test solution (a) using 0.100 g of lincomycin hydrochloride
CRS instead of the substance to be examined and adding 10.0 ml of the internal standard solution
before dissolution of the reference substance.
earth for gas chromatography R impregnated with 3 per cent m/m of poly(methylphenylsiloxane) R,
helium for chromatography R as the carrier gas at a flow rate of about 45 ml per minute,
detector between 260C and 290C. Inject the chosen volume of the test solutions and the reference
solution.
STORAGE
Store in an airtight container at a temperature not exceeding 30C. If the substance is sterile, store in
LABELLING
The label states:
__________________________________________________________________________________________________________ Ph Eur
23-81
Lindane
Cl
Cl Cl
Cl
Cl Cl
C
6
H
6
Cl
6
290.8 58-89-9
Lindane complies with the requirements of the 3rd edition of the European Pharmacopoeia [0772]. These
Action and use Topical parasiticide.
When gamma benzene hexachloride is demanded, Lindane shall be supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Lindane contains not less than 99.0 per cent and not more than the equivalent of 100.5 per cent of
1,2,3,4, 5,6-hexachlorocyclohexane.
CHARACTERS
and in ether, soluble in ethanol.
IDENTIFICATION
obtained with lindane CRS. Examine the substances prepared as discs.
D. Dissolve about 5 mg in 4 ml of alcohol R. Add 1 ml of 0.5M alcoholic potassium hydroxide. Allow to
stand for 10 min. The solution gives reaction (a) of chlorides (2.3.1).
TESTS
Appearance of solution Dissolve 0.50 g in acetone R and dilute to 10 ml with the same solvent. The
7
(Method II,
2.2.2).
coating substance.
Test solution (a). Dissolve 1.0 g of the substance to be examined in chloroform R and dilute to 10 ml
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with chloroform R.
Reference solution (a). Dissolve 0.1 g of lindane CRS in chloroform R and dilute to 10 ml with the same
solvent.
Reference solution (b). Dilute 1 ml of test solution (b) to 10 ml with chloroform R.
Reference solution (c). Dissolve 10 mg of -hexachlorocyclohexane CRS in test solution (a) and dilute to
10 volumes of chloroform R and 90 volumes of cyclohexane R. Dry the plate in a current of air,
irradiate with ultraviolet light at 254 nm for 15 min and spray with a 6 g/l solution of dicarboxidine
hydrochloride R in alcohol (90 per cent V/V) R. Examine in daylight. Any spot in the chromatogram
chromatogram obtained with reference solution (c) shows two clearly separated spots.
Chlorides (2.4.4). To 0.75 g, finely powdered, add 15 ml of water R. Boil for 1 min. Allow to cool,
shaking frequently, and filter. To 10 ml of the filtrate add 3 ml of water R and 2 ml of alcohol R. The
23-82
ASSAY
To 0.200 g add 10 ml of alcohol R and warm on a water-bath until dissolved. Cool, add 20 ml of
0.5M alcoholic potassium hydroxide and allow to stand for 10 min, swirling frequently. Add 50 ml of
water R, 20 ml of dilute nitric acid R, 25.0 ml of 0.1M silver nitrate and 5 ml of ferric ammonium sulphate
solution R2. Titrate with 0.1M ammonium thiocyanate until a reddish-yellow colour is obtained. Carry
out a blank titration.
6
H
6
Cl
6
.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
23-83
Linoleoyl Macrogolglycerides
Linoleoyl Macrogolglycerides comply with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Linoleoyl macrogolglycerides are mixtures of monoesters, diesters and triesters of glycerol and
monoesters and diesters of macrogols. They are obtained by partial alcoholysis of an unsaturated oil
mainly containing triglycerides of linoleic acid using macrogol with a mean relative molecular mass
between 300 and 400 or by esterification of glycerol and macrogol with unsaturated fatty acids or by
mixing of glycerol esters and condensates of ethylene oxide with the fatty acids of this unsaturated oil.
CHARACTERS
Amber, oily liquids which may give rise to a deposit after prolonged periods at 20C, practically
insoluble but dispersible in water, freely soluble in methylene chloride.
The viscosity at 40C is about 35 mPas, the relative density at 20C is about 0.95, the refractive
index at 20C is about 1.47.
IDENTIFICATION
substance.
f
st
st
st
st
0. 1) and to
st
0).
C. They comply with the test for fatty acid composition (see Tests).
D. They comply with the test for saponification value (see Tests).
TESTS
Hydroxyl value (2.5.3). 45 to 65, determined on 1.0 g (Method A).
Iodine value (2.5.4). 90 to 110.
Alkaline impurities Into a test-tube introduce 5.0 g and carefully add a mixture, neutralised if
of bromophenol blue R in alcohol R, 0.3 ml of water R and 10 ml of alcohol R. Shake and allow to
stand. Not more than 1.0 ml of 0.01M hydrochloric acid is required to change the colour of the upper
layer to yellow.
Free glycerol Not more than 3.0 per cent. Dissolve 1.20 g in 25.0 ml of methylene chloride R. Heat if
necessary. After cooling, add 100 ml of water R. Shake and add 25.0 ml of a 6 g/l solution of periodic
acid R. Shake and allow to stand for 30 min. Add 40 ml of a 75 g/l solution of potassium iodide R.
Allow to stand for 1 min. Add 1 ml of starch solution R. Titrate the iodine with 0.1M sodium
oleic acid: 20.0 per cent to 35.0 per cent,
linoleic acid: 50.0 per cent to 65.0 per cent,
23-84
of water, use a mixture of 30 volumes of anhydrous methanol R and 70 volumes of methylene chloride R
as solvent.
STORAGE
Store in a well-closed container, protected from light at room temperature.
LABELLING
The label states the type of macrogol used (mean relative molecular mass) or the number of units of
ethylene oxide per molecule (nominal value).
__________________________________________________________________________________________________________ Ph Eur
23-85
Linseed
Linseed complies with the requirements of the 3rd edition of the European Pharmacopoeia [0095]. These
Action and use Demulcent.
When Powdered Linseed is prescribed or demanded, material complying with the appropriate
requirements below shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Linseed consists of the dried ripe seeds of Linum usitatissimum L.
DESCRIPTION
The seeds have a flattened, elongated ovoid shape and are 4 mm to 6 mm long, 2 mm to 3 mm wide
and 1.5 mm to 2 mm thick; one end is rounded and the other end forms an oblique point near which
the hilum appears as a slight depression. The testa is dark reddish-brown, smooth and glossy but
when viewed with a lens the surface is seen to be minutely pitted. The interior of the testa has a
narrow, whitish endosperm and an embryo composed of two large, flattened, yellowish and oily
cotyledons; the radicle points towards the hilum.
Examined under a microscope, the testa is composed of an epidermis of isodiametric cells with
mucilaginous outer walls and suberised inner walls; within this is an area of collenchymatous cells
followed by a single layer of longitudinally elongated sclereids, each 120 m to 190 m long and
12 m to 15 m wide, with thickened and pitted walls; the testa has a hyaline layer composed of thin-
walled parenchyma and the inner epidermis formed of a layer of flattened polygonal cells each
containing a mass of orange-brown pigment. The endosperm and cotyledons are composed of
polygonal parenchymatous cells with slightly thickened walls, containing aleurone grains up to 20 m
in diameter and globules of fatty oil; starch is absent.
The powder is greasy to the touch; it is yellowish-brown and has a slight characteristic odour and a
mucilaginous and oily taste. It consists of: fragments of the outer epidermal cells of the testa filled
with mucilage; sub-epidermal collenchymatous layer seen in surface view as round cells with distinct
triangular intercellular spaces often in connection with groups of elongated sclereids with pitted walls;
thin-walled pitted cells of the hyaline layer often remaining attached to the elongated sclereids and
crossing them at approximately right angles; pigmented cells of the inner epidermis of the testa;
parenchyma of the endosperm and cotyledons containing aleurone grains and fatty oil. Starch
granules are absent.
TESTS
Odour and taste The drug does not have a rancid odour or taste.
Foreign matter (2.8.2). Not more than 1.5 per cent.
Swelling index (2.8.4). Not less than 4 for the whole drug and not less than 4.5 for the powdered
drug (710).
Sulphated ash (2.4.14). Not more than 6.0 per cent, determined on 1.00 g of powdered drug.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
23-86
Linseed Oil
Definition Linseed Oil is the fixed oil obtained by expression from the ripe seeds of Linum
usitatissimum L.
Characteristics A clear, yellowish brown oil; odour, characteristic. Gradually thickens on exposure
to air forming, when spread in a thin film, a hard transparent varnish.
Slightly soluble in ethanol (96%); miscible with chloroform, with ether and with petroleum spirit
(boiling range, 40 to 60).
Identification Carry out the test for identification of fixed oils by thin-layer chromatography, Appendix
X N. The chromatogram obtained from the oil being examined shows spots corresponding to those
in the typical chromatogram for linseed oil.
Acid value Not more than 4.0, Appendix X B.
Iodine value Not less than 175 (iodine monochloride method), Appendix X E.
Saponification value Not less than 188, Appendix X G.
Unsaponifiable matter Not more than 1.5%, Appendix X H.
Storage Linseed Oil should be kept in suitable, well-filled, well-closed containers.
23-87
Liothyronine Sodium
O
COONa
NH
2
H
HO
I
I
I
C
15
H
11
I
3
NNaO
4
673 55-06-1
Liothyronine Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia [0728].
Action and use Thyroid hormone.
Preparation
Liothyronine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Liothyronine sodium contains not less than 95.0 per cent and not more than the equivalent of
101.0 per cent of sodium (S)-2-amino-3-[4-(4-hydroxy-3-iodophenoxy)-3,5-di-
iodophenyl]propionate, calculated with reference to the dried substance.
CHARACTERS
A white or slightly coloured powder, practically insoluble in water, slightly soluble in alcohol,
practically insoluble in ether. It dissolves in dilute solutions of alkali hydroxides.
IDENTIFICATION
B. Dissolve 10.0 mg in 0.1M sodium hydroxide and dilute to 100.0 ml with the same solvent. Exam-
ined between 230 nm and 350 nm (2.2.25), the solution shows an absorption maximum at 319 nm.
The specific absorbance at the maximum is 63 to 69, calculated with reference to the dried
substance.
obtained with liothyronine sodium CRS.
D. To about 50 mg in a porcelain dish add a few drops of sulphuric acid R and heat. Violet vapour is
evolved.
E. Dissolve the residue obtained in the test for sulphated ash in 2 ml of water R. The solution gives
TESTS
Solution S Dissolve 0.250 g in a mixture of 1 volume of 1M hydrochloric acid and 4 volumes of
alcohol R and dilute to 25.0 ml with the same mixture of solvents.
Appearance of solution Freshly prepared solution S is not more intensely coloured than intensity 5
Levothyroxine and other related substances Examine the chromatograms obtained in the assay.
In the chromatogram obtained with test solution (a), the area of any peak corresponding to
levothyroxine is not greater than that of the principal peak in the chromatogram obtained with refer-
ence solution (c) (5.0 per cent) and the sum of the areas of all the peaks apart from the principal peak
and any peak corresponding to levothyroxine is not greater than half the area of the principal peak in
the chromatogram obtained with refereance solution (c) (2.5 per cent). Disregard any peak with an
area less than that of the peak in the chromatogram obtained with reference solution (e).
Chlorides Not more than 2.0 per cent, expressed as NaCl and calculated with reference to the dried
substance. Dissolve 0.500 g in a 2 g/l solution of sodium hydroxide R and dilute to 100 ml with the
same solvent. Add 15 ml of dilute nitric acid R and titrate with 0.05M silver nitrate, determining the
23-88
at 105C for 2 h.
Sulphated ash (2.4.14). 9.0 per cent to 12.2 per cent, determined on 0.200 g and calculated with
ASSAY
Test solution (a). Dissolve 20.0 mg in methanolic sodium hydroxide solution R and dilute to 100.0 ml
Test solution (b). Dilute 5.0 ml of test solution (a) to 200 ml with methanolic sodium hydroxide solu-
tion R.
Reference solution (a). Dissolve 20.0 mg of liothyronine sodium CRS in methanolic sodium hydroxide
solution R and dilute to 100.0 ml with the same solvent.
Reference solution (b). Dilute 5.0 ml of reference solution (a) to 200 ml with methanolic sodium
hydroxide solution R.
Reference solution (c). Dissolve 5 mg of levothyroxine sodium CRS in methanolic sodium hydroxide solu-
tion R and dilute to 50.0 ml with same solvent. Dilute 10.0 ml of this solution to 100.0 ml with
methanolic sodium hydroxide solution R.
Reference solution (d). Mix equal volumes of reference solution (b) and reference solution (c).
Reference solution (e). Dilute 1.0 ml of reference solution (b) to 25.0 ml with methanolic sodium
hydroxide solution R.
a column 0.25 m long and 4 mm in internal diameter packed with nitrile silica gel for chromato-
graphy R (5 m to 10 m),
as mobile phase at a flow rate of 1 ml per minute a mixture of 5 volumes of phosphoric acid R,
300 volumes of acetonitrile R and 700 volumes of water R,
a loop injector.
Inject separately 50 l of each solution. Record the chromatogram obtained with test solution (a) for
five times the retention time of the principal peak. The test is not valid unless the resolution between
the peaks corresponding to liothyronine and levothyroxine in the chromatogram obtained with refer-
ence solution (d) is at least 4 and the principal peak in the chromatogram obtained with reference
solution (e) has a signal- to-noise ratio of at least 5. Calculate the content of C
15
H
11
I
3
NNaO
4
from
the peak areas in the chromatograms obtained with test solution (b) and reference solution (b) and
the declared content of C
15
H
11
I
3
NNaO
4
in liothyronine sodium CRS.
STORAGE
Store in an airtight container, protected from light, at a temperature between 2C and 8C.
__________________________________________________________________________________________________________ Ph Eur

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1-1

Ester value (2.5.2). 70 to 80.

Ester value (2.5.2). 70 to 80.

i.e. taking the specific absorbance of hyperoside to be 500.

m m A A / cent per 100 /

m m A / A / cent per 100

for the peaks corresponding to methyl ricinoleate and methyl 9,10-dihydroxystearate.

a=percentage content of water,

a=number of millilitres of 0.0167M potassium bromate used for the blank,

c =percentage concentration (g/100 ml) of the substance to be examined, calculated with

does not exceed 0.025 (2.5 per cent).

d=percentage loss on drying.

does not exceed 0.025.

d=percentage loss on drying.

does not exceed 0.025.

does not exceed 0.025.

d=percentage water content.

m=mass of the substance to be examined in grams.

k=the viscometer constant in mm

d=loss on drying as a percentage,

d=loss on drying as a percentage,

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