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Analytical Profiles

of
Drug Substances
Volume 11

Edited by

Klaus Florey
The Squibb Institute for Medical Research
New Brunswick, New Jersey

Contributing Editors
Gerald S. Brenner Lee T. Grady
Glenn A. Brewer, Jr. Hans-Georg Leemann
Lester Chafetz Joseph Mollica
Nicholas DeAngelis Milton D. Yudis
Compiled under the auspices of the
Pharmaceutical Analysis and Control Section
Academy of Pharmaceutical Sciences

ACADEMIC PRESS 1982


A Subsidiary of Harcourt Brace Jovanovich,Publishers
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EDITORIAL BOARD

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Rafik Bishara Lee T. Grady
Gerald S . Brenner Boen T. Kho
Glenn A. Brewer, Jr. Hans-Georg Leemann
Lester Chafetz Joseph A. Mollica
Nicholas DeAngelis Bruce C. Rudy
John E. Fairbrother Milton D. Yudis
Klaus Florey
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AFFILIATIONS OF EDITORS,
CONTRIBUTORS, AND REVIEWERS

E . Abignente, University of Naples, Naples, Italy


H . Y. Aboul-Enein, King Saud University, Riyadh, Saudi Arabia
A. A . AZ-Badr, King Saud University, Riyadh, Saudi Arabia
I . A . Al-Meshal, King Saud University, Riyadh, Saudi Arabia
M . A . Al-Yahyu, King Saud University, Riyadh, Saudi Arabia
S. L. Ali, Zentrallaboratorium Deutscher Apotheker e. V., Eschborn, Germany
N. W. Atwater, E. R. Squibb and Sons, Princeton, New Jersey
D. M. Baaske, American Critical Care, Chicago, Illinois
R. Bishara, Eli Lilly and Company, Indianapolis, Indiana
G. S. Brenner, Merck Sharp & Dohme Research Laboratories, West Point,
Pennsylvania
G. A. Brewer, Jr., The Squibb Institute for Medical Research, New Brunswick,
New Jersey
P. de Caprariis, University of Naples, Naples, Italy
J. E . Carter, Ortho Pharmaceuticals, S o m e d e , New Jersey
L. Chajetz, Warner-Lambert Research Institute, Morris Plains, New Jersey
N. DeAngelis, Wyeth Laboratories, Philadelphia, Pennsylvania
W. DeWitte, Ciba-Geigy, Suffern, New York
H . A . El-Obeid, King Saud University, Riyadh, Saudi Arabia
A. A. Elazzouny, National Research Center, Dokki, Cairo, Egvpt
J. Fairbroth, Stiefel Laboratories Ltd., Sligo, Ireland
K. Florey, The Squibb Institute for Medical Research, New Brunswick, New
Jersey
S . A. Fwari, Parke-Davis, Inc., Detroit, Michigan
L. T. Grudy, The United States Pharmacopeia, Rockville, Maryland
M . M. A. Hassan, King Saud University, Riyadh, Saudi Arabia
J. G. Hoogerhde, Schering-Plough Corporation, Bloomfield, New Jersey

vii
viii AFFILIATIONS OF EDITORS, CONTRIBUTORS, A N D REVIEWERS

J. H . Johnson,American Critical Care, Chicago, Illinois


H . Kudin, The Squibb Institute for Medical Research, New Brunswick, New
Jersey
B. T. K b , Ayerst Laboratories, Rouses Point, New York
F. Kreuzig, Biochemie GmbH, Kundl, Austria
H. G. Leemunn, Sandoz, Basel, Switzerland
E. A. Lo@, King Saud University, Riyadh, Saudi Arabia
M. E. Mohamed, King Saud University, Riyadh, Saudi Arabia
1. MoZZica, Ciba-Geigy Corporation, Summit, New Jersey
F. I. Muhtadi, King Saud University, Riyadh, Saudi Arabia
V. D. Reij, Wyeth Laboratories, Philadelphia, Pennsylvania
B. C. Rudy, Mary Kay Cosmetics, Dallas,Texas
C. M . S h r m , Wyeth Laboratories, Philadelphia, Pennsylvania
D. H . Sieh, The Squibb Institute for Medical Research, New Brunswick, New
Jersey
H. Stober, Ciba-Geigy, Suffern, New York
K. D. Thkker, The United States Pharrnacopeia, Rockville, Maryland
T. D. Wilson, Sterling Winthrop Research Institute, Rensselaer, New York
B. E . Wyka, Schering- Plough Corporation, Bloomfield, New Jersey
M. D. Yudis, Schering- Plough Corporation, Bloomfield, New Jersey
PREFACE

It is now well over a decade that I perceived the need to supplement the official
compendial standards of drug substances with a comprehensive review of perti-
nent physical, chemical, and analytical data and methods. Ten years ago the first
volume of Analytical Projiles of Drug Substanceswas published under the auspices
of the Pharmaceutical Analysis and Control Section of the APhA Academy of Phar-
maceutical Sciences. That we were able to publish one volume per year is a tribute
to the diligence of the editors to solicit monographs and even more so to the en-
thusiastic response of our authors, an international group associated with phar-
maceutical firms,academic institutions, and compendial authorities. I would like
to express my sincere gratitude to them for making this venture possible.
I am pleased to report that five years ago a companion series entitled Phur-
macological and Biochemical Properties of Drug Substances was initiated by Mor-
ton E. Goldberg under the auspices of the section on Pharmacology and Tox-
icology, APhA Academy of Pharmaceutical Sciences. So far, three volumes have
been published.
Over the years, we have had queries concerning our publication policy. Our
goal is to cover all drug substances of medical value and, therefore, we have
welcomed any monographs of interest to an individual contributor. We also have
endeavored to solicit profiles of the most useful and used medicines, but many in
this category still need to be profiled.
Starting with this, the eleventh volume, we shall also supplement previously
published profiles with new data as we can find volunteers to write such s u p
plements. In this volume, five of the original profiles in Volume 1have been up-
dated.
The goal to cover and update all drug substances of medical value with com-
prehensive monographsis still a distant one. I estimatethat only about a quarter of
such compounds have been profiled so far. We would very much like to accelerate

ix
X PREFACE

the rate of publication and hope that even more authors can be encouraged to
write profiles. All those who have found these profiles useful are requested to con-
tribute monographs of their own. We, the editors, stand ready to receive such con-
tributions.

Klaus Florey
AMINOPHYLLINE
Kailus D.Thakker and Lee T.Grady

1. Description 2
1 . 1 Nomenclature 2
1.2 Formula, Molecular Weight, and Composition 2
1.3 Appearance, Color, and Odor 2
2. Physical Properties 3
2.1 Spectral 3
2.2 Other Properties 3
3. Methods of Preparation 9
4. Stability-Degradation 9
4.1 Stability in Solution 9
4.2 Stability in Solid State 9
5. Methods of Analysis 10
5.1 Identification Tests 10
5.2 Gravimetric Methods 11
5.3 Titrimetric Methods 11
5.4 Spectroscopic Methods 13
5.5 Chromatographic Methods 13
5.6 Immunoassays 26
6. Metabolism 31
I. Biopharmaceutics and Pharmacokinetics 31
8. Toxicity 33
9. References 34

Analytical Profilcs of Drug Substances Copyright 01982 by The American


Volume I I 1 Pharmaceutical Association
ISBN 0-12-260811-9
2 KAILAS D. THAKKER AND LEE T. GRADY

1. Description

1.1 Nomenclature

1.11 Chemical Name

Aminophylline is chemically known as 1H-


P
purine-2,6-dione, 3,7-dih dro-l,3-dimethyl-, compound wTth
1,2-ethanediamine (2:l).

1.12 Adopted Names

Aminophylline was also known as theophylline


ethylenediamine and euphylline. 1

1.13 Trade Names

Aminophylline is known as carena;


inophylline; metaphylline; theophyldine; aminocardol;
ammophylline; cardiocilina; cardophyllin; phylcardin;
tefamin; cardiomin; grifomin; minaphil; peterphylline;
stenovasan; the drox; diophylline; genophylline; phyllindon
and theolamine. P
1.2 Formula, Molecular Weight, and Composition:

11

Anhydrous: C16H24N1004 420.44

Dihydrate: C16H28N1006 456.44

Theophylline 85-87%
Ethylenediamine 12-15%

1.3 Appearance, Color and Odor

Aminophylline is available in the anhydrous form


or as the dihydrate. The dihydrate occurs as white or
slightly yellowish granules or powd r. It has a faint
ammoniacal odor and a bitter taste. f
AMINOPHYLLINE 3

2. Physical Properties

2.1 Spectral

2.11 Infrared

The infrared spectrum of aminophylline in


mineral oil mull, obtained on a B ckman 4250 spectro-
photometer, is shown in Figure l.5 It is generally
consistent wi h the reported infrared spectrum of
theophylline.5,4 The stretches for -NH2 in ethylenediamine
and -NH in theophylline appear as a broad band (combined
with the mineral oil signal) in the region of 3.0-4.0 P M .
Other signals are in the same vicinity as those for
theophylline. The fingerprint region beyond 8.5 IJM is
distinctive and can be used for identification.

2.12 Ultraviolet

Spectral characteristics of aminophylline

''
solutions in the ultraviolet region were reported by Andrade
an Inacio.'
LE1cm
Absorption max ma occurred at 243-5 nm
= 1701 and at 273-5 [E:im= 5001 in pH 9.5 borate buffer.

Figure 2 shows the ultraviolet spectrum of


aminophylline in water obtained on a Beckman 5260 recording
spectrophotometer.

2.13 Nuclear Magnetic Resonance

2.13.1 Proton NMR

An 80 MHz proton magnetic resonance


spectrum of aminoph lline in d6-dimethyl sulfoxide, obtained
on a Varian FT-80A,' containing tetramethylsilane as an
internal reference, is shown in Figure 3 . It is jimilar to
the reported (60 MHz) proton NMR of theophylline. The
assignments, based on assignments of theophylline protons,
are shown in Table I.

2.13.2 Carbon-13 NMR

The 20 MHz proton-noise decoupled


spectrum of aminophylline in d6-dimethyl sulfoxide,
obtained on a Varian FT-80A is shown in Figure 4.6 The
assignments are shown in Table 11. These are based on
assignments of dimethyluracil and 1-methylhypoxanthine.7
t
P

WAVELE W T H UM

Fig. 1. IR Spectrum of Aminophylline


0.8

0.7

a.6

y Q!5
U
z
4
m
g *1

v)
m
a
0. :

0 .i

.1

240 260 280 300 320 340 36


WAVELENGTH (nms)

Fig. 2. W Spectrum of Aminophylline

5
l ' l ~ l ' l ~ l ~~ l l ' t \ l ' l r l ~ l ~ l ' l ' l ~
10 9 a I 6 5 4 3 2 1 0
PPm

Fig. 3 . H' NMR of Aminophylline


Solvent
I I

Fig. 4. I 3 C NMR of Aminophylline


8 KAILAS D. THAKKER AND LEE T. GRADY

Table I

Proton Position
Proton Assignment (see structure) Chemical Shift (ppm)

-CH2- 10,ll 2.75


-N-CH3- 1 3.23
3 3.42
N-C%-
N-H 7 5.67
C-H 8 7.71

aIntensity of signal is proportional to the concentration.


This proton may be delocalized in the ring.

Table I1

Carbon Position
Carbon Assignment (see structure) Chemical Shift

N-CH3 29.7
N-CH3 27.5
c=c 109.6
c=c 148.5
N-C-N 142.7
-c=o 151.4
N-C=O-N 155.6

-CH2 on ethylenediamine is buried in the solvent signal as


shown in Figure 4.

2.2 Other Properties

2.21 Differential Scanning Calorimetry and


Melting Point

The thermogram of aminophylline2 shows two


endothermic transitions, one at 120" and another at 272°C.
The first transition reflects the melting point of
aminophylline; the second transition reflects the melting
point of theophylline. Theophylline is known to sublime on
melting. 2

2.22 Solubility

Aminophylline is soluble in water


AMINOPHYLLINE 9

(1 g/5 ml) .l It is insoluble in dehydrated alcohol and in


ether.8

3. Methods of Preparation:

Aminophylline was first prepared by Gruter' by


dissolving theophylline in aqueous solutions of ethylene-
diamine in stoichiometric proportions and evaporating in
vacuum over sodium hydroxide. Alternate methods include
treating anhydrous or h drated crystals of theophylline with
ethylenediamine vapor," and treating a 3 M solution of
theophylline in a weak organic base (pyridine, quinoline or
a-picoline) with a 2 -
M aqueous solution of ethylene-
diamine.11

4. Stabilitv-Degradation

4.1 Stability in Solution

Solutions of aminophylline become turbid on


standing due to absorption of carbon dioxide, with
subsequent precipitation of theophylline.' S 8 During the
preparation of aminophylline injection, excess
ethylenediamine is necessary to keep aminophylline from
decomposing.12

4.2 Stabilitv in Solid State

Aminophylline crystals, in the presence of


moisture, can absorb carbon dioxide fr m air and decompose
into theophylline and ethylenediamine.8 This accounts for
its characteristic odor.

Mixtures containing aminophylline and ephedrine


hydrochloride were found to be discolored13 due to an
exchange reaction between the two drugs. The ethylene-
diamine in aminophylline is presumed to liberate ephedrine
base which decomposes rapidly. The color change was
accelerated by temperature and humidity. 13.
Numerous reports are found in the literature on the
stability of aminophylline in suppository bases,14-21
especially those containing fatty acids. Dissolution of
suppositories made with cocoa butter base was markedly lower
than those ma e with macrogol base after storage at 22" for
up to a yearaP5 Other physical properties of cocoa butter
suppositories such as melting point (Tm) and melting time
have been known to increase within weeks of storage at 22"
10 KAILAS D. THAKKER AND LEE T. GRADY

and 30" as a result of decomposition16 . Increase in the in


vitro melting point (above 37" in some casf$) was correlated
with poor rate of release of theophylline.

Decomposition of aminophylline suppositories is


presumed to be due to the formation of insoluble amides
ethylenediamine and fatty acids of the suppository
base.
b
etwe'' our decomposition products that have been
isolated" were identified as mixturf6 of amides of oleic,
palmitic, lauric and myristic acids. Further
characterization of the decomposition products by IR
GLC, and TLC confirmed the presence of alkyl amides. 20""
Stabili rs such as hydroxylamine hydrochloride have been
useful. %f

5. Methods of Analysis

5.1 Identification Tests

Spectral identification tests include IR, W,Mass


and N M R spectroscopy. The melting point of theophylline
liberated from aminophylline is the basis of one of the
compendia1 identification tests. 22

The following color reactions are also useful


identification tests; many are used for dosage forms and for
mixtures of pharmaceuticals.

(i) Ethylenediamine present in aminophylline


reacts with sodium rhodizon85e to form a water-soluble,
violet-colored precipitate. Ethylenediamine must be
released from aminophylline and converted to acetate or
hydrochloride.
0

0
+2 a+
(ii) Ethylenediamine also foryz a yellow
precipitate with 2,4-dinitrochlorobenzene. Primary
aliphatic and aromatic amines interfere with the test.
AMINOPHYLLINE 11

(iii) Aminophylline develops an orange color


with ferric chloride and a yellow color with Ehrlich’s
reagent. Color de elopment is rapid. This test was
designed by Cooper” €or identification of pharmaceuticals
in tablets.

(iv) Reaction between dimethylglyoxime and


oxidized solutions of purines to form a colored product is
the basis of the identification test developed by Kido. 26

(V) Reaction of theophylline with potassium


chlorate in hydrochloric acid, followed by exposure to
ammonia vapors, produces a purple residue. This reaction is
the basis of on of the compendia1 identification tests for
aminophylline.2 5

(vi) Bratton-Marshall: Aminophylline in acid


solution, when treated with diazotized p-aminobenzene-sul-
fonic acid or p-aminonitrobenzene produces a yellow or a red
color, respectively.27

(vii) Addition of 1-2 drops of a 5% solution of


sodium nitroprusside to a solutig of aminophylline in
acetone produces a violet color.

(viii) Aminophylline powder, on mixing and


heatin with copper sulfate powder, also produces a violet
color.99

5.2 Gravimetric Methods

Earlier methods of analysis of aminophylline were


based on extraction of theophylline with organic solvents
such as chloroform and 2-propg~;4~ followed by gravimetric
determination of the residue.

5.3 Titrimetric Methods

5.31 Alkalimet ric

The weakly acidic nature of theophylline


lends itself to titrations with alkali. Titrations with
alkali have be59 carried out t o Jgsay aminophylline using a
potentiometric or colorimetric end-point
(thymolphthalein as indicator) in mixtures of
pha rmaceutica1s.
12 KAILAS D. THAKKER AND LEE T. GRADY

5.32 Acidimetric

Ethylenediamine's basic nature was used in


many early methods of analysi
strong acid were carried out.
ethylenediamin by solvent extraction31 or ion-exchange
~ h r o m a t o g r a p h 3was
~ followed by titrations with
hydrochloric acid using a colorimetric end-point (methyl
orange as indicator).

5.33 Argentometric

When theophylline is treated with solutions


of silver nitrate, it forms a silver salt that is insoluble
in water and soluble in nitric acid solutions. Basej6gtlthis
property, several assay methods have been develop
including the compendia1 assay for aminophylline.
general, silver theophyllate is precipitated, filtered,
washed, re-dissolved in nitric acid and titrated with
ammonium thio cy Aminophylline resent in mixtures of
pharmaceuticals'8:5p;39 and xanthines" can be analyzed.
Some presence of amm nia is necessary when precipitating
silver theophyllate." It has been suggested that
argentometric titrations give higher values due to
adsorption of silver ions by the very voluminous silver
theophyllate precipitate. 31

In an unusual application, silver (lloAg)


nitrate was used and radi metric titration was carried out
to analyze aminophylline. 41

5.34 Complexometric

Bosly developed a titrimetric method for


aminophylline by Precipitating Hg-(the~phylline)~with
mercuric acetate, itrating excess H g H ions with
ammonium thiocyana:zd4' Similarly, the Cu salt of
theophylline is also precipitated excess copper titrated
with ethylenediaminetetraacetate.4y94

5.35 Non-Aqueous

Non-aqueous titrations of aminophylline


using sodium methoxide as the titrant have been carried out
with dirnethylf~rmamide~~or ben~ene-methanol~~ as the
solvent for aminophylline. A differential titrimetric
method for aminophylline was dexgloped using acetous
perchloric acid as the titrant. This method allows
AMINOPHY LLINE 13

determination of ethylenediamine and theophylline content in


a single titration, and is useful for aminophylline tablets,
injections and suppositories.

5.4 Spectroscopic Methods

Aminophylline solutions obey the Beer-Lambert Law


for a zqncentration range of 0.5-1.2 mg%.5 Schack and
Waxler developed a potentiometric assay for aminophylline
in biological fluids using chloroform/2-propano1 extraction
to isolate theophylline, followed by W-absorbance
measurement at 275 nm. The method of Schack and Waxler was
used most extensively for analysis of aminophylline in early
pharmacokinetics research.a Several other
spectrophotometric methods have been developed for
aminophylline in mixtures of pharmaceuticals, each one using
an extraction t organic solvent to isolate
aminophylline. ''*kg In one case, graphical correction was
applied to the W absorbances of mixtures of aminophylline
and phen barbital, measured at two wavelengths of
maxima.58 In another application, extraction of serum
samples with a salt-solvent pair of ammonium sulfate and
ch1oroform:hexane (7:3 v/v) was carried out followed b back
extraction of theophyllinea into aqueous borate buffer ( PH
9 . 0 ) an measurement of UV absorbance at 275 nm. 51
PlavsicgZ used charcoal extraction to isolate theophyl ine
from other interfering substances. Since theophylline was
reversibly adsorbed on charcoal, elution with organic
solvent was followed by measurement of W absorbance.

Determination of aminophylline in the blood of


patients was carried out after gfidation with potassium
dichromate in an acidic medium, separation of the
oxidation product by steam distillation and measurement of
W absorbance at 257 nm.

5.5 Chromatographic Methods

5.51 Thin-Layer Chromatography

In addition to systems developed for theo-


~ h y l l i n e ,several
~ TLC systems have been developed for

"Aminophylline and theophylline are indistinguishable at


biological pH's. Therefore assays of theophylline in
biological fluids are also included here due to their
obvious applicability.
Table I11
Thin-Layer Chromatographic Systems for Aminophylline

No. Solvent Stationary Developing Solvent Detection Application Ref.


for Drug Phase Methods

1. chloroform Silica gel ch1oroform:acetic acid W,Dragen- Analysis of 54


F254 (100 :20) dorff ' s suppositories
reagent

2. water aluminum benzene:ethanol (9:l W, iodine, Mixture of 55


oxide or 9:1.5) modified pharmaceuticals
Dragendorff ' s
reagent

Silica gel (i) acetone:chloro- ferric Analysis of 56


F254 form:l-butanol:25% chloride, mixtures of
ammonium hydroxide iodine xanthines
(30:30:40:10)
(ii) ch1oroform:ethyl
ether (9O:lO)
(iii) ch1oroform:eth-
anol (9O:lO)

4. -----a Silica gel H ch1oroform:acetone: lJv Analysis of 18


methanol (8:l:l) suppositories
Table 111 (contd.)

No. Solvent St at ionary Developing Solvent Detect ion Application Ref.


for Drug Phase Methods

5. -----a Silica g e l methylene chloride: uv Analysis of tab- 57


F254 methano1:acetic acid lets ampuls,
(90:10:3) suppositories

anot mentioned.
16 KAILAS D. THAKKER AND LEE T. GRADY

analysis of aminophylline and its dosage forms. Table 111


lists the methods developed for aminophylline.

Zorka et alS8 separated and identified


several pharmaceuticals in a mixture including amino-
phylline using silica gel G plates and neutral, acidic, and
alkaline mobile phases. Detection was done by UV.

Riechert5' developed a "micro"-TLC method


for analysis of theophylline in biological fluids.
Kieselgel 60 F-254 DL-"Fertigplatten" thin-layer plates were
used and developed with ch1oroform:methanol (9O:lO) for a
mixture of caffeine and theophylline and with ethyl
acetate:methanol:25% ammonia (80:ZO:lO) for a mixture of
theobromine and theophylline present in saliva, plasma or
urine. Sensitivity of detection claimed was 25 ng/lO ~ 1 .
None of the dietary xanthines or other commonly co-
administered drugs appear to interfere.

5.52 PaDer ElectroDhoresis60


Whatman Paper No. 1 was used with a
potential gradient of 20 V/cm. Several xanthines were
separated. Among these, theophylline and aminophylline were
best chromatographed in Britton-Robinson buffer at pH 10.
Detection agent was 1% solution of disodium-2-hydroxy-3,6-
naphthalenedisulfonate.

5.53 Pressurized Liquid Chromatography

Numerous liquid chromatographic methods for


determinations of theophy 1 ne n biological fluids are
listed in the literature.4,f1-g' The maj ri of the

'',"
methods use reverse-phase chromatog p
li9
'l-" few are
listed that use i er normal-phase p b z ' or ion-exchange
chromatography. In many cases, chromatographic
conditions h e e n developed for a specific
application,QY,8B~'9 since adaptation of reported methods
may introduce some inttgference from co-adm nistered drugs
such as acetazo&mide, trisulfapyrimidinebl or
cephalosporins.

Table IV lists some of the recent, most


cited methods of analysis of theophylline in biological
fluids by liquid chromatography.
Table IV

No. Stationary Mobile Phase Pre-treatment of sample Comments Ref.


phase No.

1. Reverse-phase methano1:sodium Extraction of plasma with Separation of 61


C-18 acetate (pH 4 . 2 ) chloroform:2-propanol theophylline from
(ion-pair) containing 10 mM (50:50) paraxanthine.
tet rabutylammonTum
chloride (10:90)

2. Reverse-phase acetonitri1e:water Urine: pH adjusted before Analysis of theo- 62


C-18 containing 10 mM chromatography phylline in urine
(ion-pair) tet rabutylammonium Serum: ultrafiltration and serum; separation
chloride (2.5:97.5) from other xanthines
and metabolites.

3. Reverse-phase ethano1:water Assay of theophylline 6 3


C-18 (20:80) in plasma; separation
of theophylline from
sulfisoxazole and
ampic i11in.

4. Reverse-phase acetonitrile:O.Ol Molecular ultrafiltration Direct injection. 64


C-18 - sodium acetate
M to remove plasma proteins
(p-Bondapak (pH 4 . 0 ) (1:9);
C-18) 2 . 0 ml/min flow
Table IV (contd.)

No. Stationary Mobile Phase Pre-treatment of sample Comments Ref.


phase No.

5. Reverse-phase acetonitri1e:water De-proteinization with Micromethod--only 65


C-18 ( 6 : 9 4 ) ; 3.0 ml/min 2.5 volumes of acetoni- 10-1.11sample is
flow trile required.

6. Reverse-phase acetonitri1e:ace- De-proteinization with Microscale method-- 66


C-18 tate buffer aqueous acetonitrile- only 3 0 p 1 plasma
(p-Bondapak (pH 4 . 0 ) ( 7 : 9 3 ) s olution needed for analysis;
-
Q)
7.
C-18)

Reverse-phase methanol:l% pro- Extraction with chloroform


direct injection.

50-1.1
1 sample needed. 67
C-18 pionic acid (20:80) evaporation; sample
u-Bondapak redissolved in methanol
C-18

8. Reverse-phase methano1:tetrahy- Same as Ref. 62 p-Hydroxyethyltheo-


- 68
C-18 drofuran:water con- phylline as inter-
(p-Bondapak taining 10 d/liter nal standard; sep-
C-18) sodium acetate aration from other
(40:10:50) xanthines and commonly
administered antibiotics.
Table IV (contd.)

No. Stationary Mobile Phase Pre-treatment of sample Comments Ref.


phase NO

9. Reverse-phase methano1:sodium Deproteinization of serum B-Hydroxyethyltheo- 69


acetate (15:85) samples with two volumes phylline used as
of methanol internal standard;
no interference
from antibiotics
or metabolites.

10. Reverse-phase methano1:amonium Extraction with organic 100 u1 of serum 70


phosphate solvent before analysis sample can be
analyzed; theo-
phylline can be
analyzed in pres-
ence of anticon-
vulsants.

11. R verse-phase acet nitrile : Extraction with chloro- 8-Hydroxyethyltheo- 71


C-18 acetate buffer form:2-propanol (95:5) phylline used as
(pH 4.0) (9O:lO) internal standard;
theophylline re-
covery was found to
be between 71 and 75%.
Table IV (contd.)

No. Stationary Mobile Phase Pre-treatment of sample Comments Ref.


phase NO

12. Reverse-phase acetonitrile: Extraction of 0.5-0.2 ml Separation of xan- 72


C-18 acetate buffer of acidified plasma thines; sensitivity
(pH 4 . 0 ) (12:88) with dichloromethane of detection 0.1 mg/
liter

13. Reverse-phase methanol:0.05 M Molecular filtration of Method applicable 73


C-18 phosphate buffer serum to separate proteins to human serum,
(pH 4 . 7 ) (12:88 v/v) urine and saliva
h)
0 samples, also sep-
aration and quan-
titation of theo-
phylline and its
metabolites.

14. Reverse-phase 2 0 ~1 of blood are 74


sufficient for
analysis; detection
by direct-curren t
pulse and differ-
ential pulse am-
perometry.
Table IV (contd.)

No. Stationary Mobile Phase Pre-treatment of sample Comments Ref.


phase No.

15. Reverse-phase methano1:buffer Extraction with Completely automat- 75


C-8 (14:86 v/v) 2-propanol:chloroform ed analysis. De-
(25 :75) veloped on Technicon
"Fast-LC" .200-p 1
sample required. No
interference was
observed.

16. Reverse-phase sodium acetate: Adjustment of pH to 5.5 Theophylline, sul- 76


RP-8 0.02 M methanol famethoxazole,
(2:l)- ampicillin and
caffeine well
separated.

17. Reverse-phase acetonitrile: Same as Ref. 85 Authors found that 77


C-18 phosphate buffer the retention
(pH 4.8) (1:l); time of internal
3.0 ml/min flow; standard varied
50" from run t o run
when using the
conditions described
in Ref. #5; column
temp. was therefore
maintained at 50°C.
Table IV (contd.)

No. Stationary Mobile Phase Pre-treatment of sample Comments Ref.


phase No.

18. Reverse-phase acetonitrile: Protein denaturation by No pre-column nec- 78


C-18 Whatman 10 mM phosphate acetonitrile essary; no inter-
Partisi1 buffer (10:90) ference observed.
10-ODS

19. Reverse-phase methano1:pH 2.0 Precipitation of proteins Theophylline ana- 79


C-18 hydrochloric acid by 50% v/v trichloroace- lyzed in presence
p-Bondapak solution (containing tic acid of methyl xanthines
N
N
0.02 M potassium and caffeine.
chloride)

20 * Reverse-phase acetonitri1e:ace- Plasma samples extracted Theophylline well 80


C-18 tate buffer with chloroform:2-propanol separated from
(8:92) (95:s); solvent removed paracetamol and
and samples re-dissolved and xanthines.
dissolved in mobile phase

21. Reverse-phase Solvent A: water Ion-pair extraction using Simultaneous quan- 81


C-18 5 p containing .01 - M tetrabutylammonium sul- titation of theo-
sodium acetate fate and ethyl acetate: phylline and its
and 0.005 M te- chloroform:2-propanol, major metabolites.
t rabu ty1ammonium (45:45:10 v/v) after ad-
hydrogen sulfate. justment of urine pH to
6.0-6.5
Table IV (contd.)

No. Stationary Mobile Phase Pre-treatment of sample Comments Ref.


phase No.

21. Solvent B: ( 5 0 : 5 0 )
contd. methano1:solvent A
gradient elution
with 9% solvent B at
start, 46% solvent B
at end of run (for
program see Ref. 81).

g 22. Reverse phase methanol:10 mM Proteins precipitated 50 111 of serum are 82


p-Bondapak monobasic sodium by perchloric acid, sufficient; theophy-
C-18 phosphate (1:4); supernatant neutralized lline is well separ-
0.8 ml/min flow and injected ated from dietary
xanthines, caffeine,
theobromine and
theophylline meta-
bolites.
Table IV (contd.)

No. Stationary Mobile Phase Pre-treatment of sample Comments Ref.


phase NO

23. Normal phase ch1orform:dioxane: Equal volume of saturated 100 u 1 of plasma 83


column formic acid ammonium sulfate is added are sufficient; theo-
(silica) (95.5:4.5:0.01 to plasma, and then phylline well sep-
v/v) extracted with chloro- arated from the
f orm:2-propanol (95 :5 metabolites.
v/v); solvent is evapor-
ated and residue
redissolved in mobile phase

*
K
3
.
24. Normal phase (i) chloroform: Extraction from plasma Mass spectrometry 84
Lichrosorb 2-propanol: with chloroform 2- used for identi-
Si-60 glacial acetic acid propanol (95:5) fication.
(92:7:1) with 40%
hexane
(ii) Ethylene chloride:
methanolic ammonium
formate (98:2)

25. Cation-exchange 0.66% acetic acid 0.1-ml sample extracted Applicable to plasma 85
Partis i1 with ethyl acetate or saliva samples;
SCx column no interference.
temp. at 5OoC
Table IV (contd.)

No. Stationary Mobile Phase Pre-treatment of sample Comments Ref.


phase NO

26. Strongly basic acetate buffer; Sample filtered to remove General method for 86
anion ex- linear gradient particulate matter W-absorbing com-
change resin from 0-6.0 M, pounds in urine;
(35% cross- flow 0.72 a / m i n 1 30 compounds
linkage) tested.
26 KAILAS D. THAKKER AND LEE T. GRADY

5.54 Gas Chromatography

Earlier attempts to analyze blood levels of


theophylline resulted in evelopment of various gas
chromatographic methods. g5h_f0' Most of these methods
require extraction and derivatization before
chromatography. Table V lists some of the more recent
methods.

5.6 Immunoassays

5.61 Enzyme Immunoassay (EMIT)

The enzyme immunoassay (EMIT) developed by


Syva Corporation (Palo Alto, California) is the most widely
nSs
used method for the assay of the0 lline (in biological
fluids) in clinical laboratories.

In principle, theophylline antibodies are


preparedlo4 by injecting a solution of bovine gamma globulin
linked to theophylline ( o r a chemically similar derivative)
into sheep. Theophylline (or a derivative) is also linked
to an enzyme, in this case, glucose-6-phosphate
dehydrogenase. When a patient's serum containing free
theophylline is mixed with a solution of antibodies and
enzyme-labelled theophylline, the free drug and the enzyme-
labelled drug compete for the binding sites on antibodies.
The reduction in enzyme activity when bound to antibodies
can be monitored by using the proper substrate; in this case
NADH. This assa s rapid, specific, and requires small
sample size. s easily adaptable to ommercial
kinetic analyzers, and can be modified"' to suit the
application.
-_ Comparison of the EMIT assay with
chromat~~t;;~~#cmethods shows good agreement between the two
assays.

Vinet et all2' developed another enzyme


immunoassay. In this method, the sample is extracted with
chloroform/2-propano1, and back-extracted into aqueous
sodium hydroxide. Inhibition of beef liver phosphatase by
theophylline is determined at 25" C, using p-nitrophenyl
acetate as the substrate in a pH 9.4 2-amino-2-methyl-l-
propanol buffer system.

In another application nephelometric,


competitive immunoassay was developed. '21 A precipitate was
obtained by combining theophylline-antibody complex with a
macromolecule, and scattering of light by the precipitate
Table V

No. Stationary Conditions Pre-treatment of sample Comments Ref.


phase NO

1. 3% OV-17 on Column at 230°C; Sample extracted with 20 p 1 of plasma are 93


Gas Chrom Q nitrogen-phospho- chloroform:2-propanol sufficient; de-
rus detector ( 5 0 : 5 0 ) , evaporation and tection sensitivity
redissolution of residue 100 pmol/liter; theo-
in 0.02 M tetrabutyl- phylline well sep-
ammonium-h yd ro xid e arated from other
xanthines and co-ad-
ministered drugs.

2. Silicone sta- F.I.D. Extraction of sample with 94


tionary phase, salt-solvent pair of
2% SP 2510 DA ammonium sulfate and
methylene chloride: hex-
ane:acetic acid (80:20:0.1)

3. 5% OV-225 on Electron-capture Sample extracted with 100 p 1 of serum are 95


80/100 Gas detector; column ethyl acetate, and then sufficient; detec-
Chrom Q at 250°C treated with pentafluor- tion sensitivity
obenzyl bromide for 0.1 ug/ml.
derivatization
Table V (contd.)

No. Stationary Conditions Pre-treatment of sample Comments Ref.


phase No.

4. 3% XE-60 on Electron-capture Derivatization by penta- Sensitivity of de- 96


80/100 Gas detector; injector fluorobenzyl bromide tection 5 ng/ml.
Chrom Q and column at followed by column chrom-
220"C, detector atography prior to in-
at 225°C jection

5. 3% OV-17 on Nitrogen detector; Extraction with tetrahexyl- 25-pl sample is 97


100/120 Gas column at 240°C ammonium hydrogen sulfate sufficient.
Chrom Q in aqueous sodium hydrox-
ide

6. 3% OV-17 on Nitrogen-phospho- Off-column derivatization No interference 98


100/120 Gas rus detector; as follows: Sample is ex- from drugs or
Chrom Q column at 240°C tracted with dichloro- metabolites.
methane, dried and
reacted with --
N,N-dimethyl-
acetamide, tetramethyl-
ammonium hydroxide and 1-
iodopentane. It is then
transferred into cyclohexane:
pentane mixture (95: S), the
solvent evaporated, and it
is redissolved in methanol.
Table V

No. Stationary Conditions Pre-treatment of sample Comments Ref.


phase No.

7. 3% OV-17 on Flame-ionization Extraction of sample with 99


Gas Chrom Q detector; column ether:dichloromethane:
at 190°C 2-propanol ( 6 : 4 : 1 ) , re-
extraction of the organic
layer with 1 N sodium
hydroxide, acidification
with phosphoric acid,
re-extraction with organic
h)
solvent, evaporation and
\o redissolution of residue
in tetrapropylammonium
hydroxide

8. 3% SP 2250 on Flame-ionization Sample extracted with di- 100


100/120 detector; column chloromethane, evaporated,
Supelcoport temperature pro- and dissolved in toluene,
grammed from 160°C butylating agent is
to 240°C at 8" then added.
C/min
Table V (contd.)

No. Stationary Conditions Pre-treatment of sample Comments Ref.


phase No.

9. 3% OV-17 on Column temperature Extraction with chloro- Mass spectrometry 101


100/120 programmed from form, butylation with using probability-
Chrom W HP 180°C to 280°C; tetramethylammonium based matching.
column well detector at hydroxide and --
N,N-di-
conditioned 280"C, mass methylacetamide
spectral source
at 150°C

10. 3% SP2250 on Column temperature Samples are introduced in No interference. 102


100/120 programmed at a flash heater for
Supelcoport 190°C to 3OO0C at ethylation
type 50-50 10"/min
methylphenyl
silicone
AMINOPHYLLINE 31

was used to quantitate theophylline content.

5.62 Radioimmunoassay (RIA)

The principle of radioimmunoassay is similar


to EMIT, except that in this case decrease in radioactivity
is measured. Ra ioimmunoassays f r th ophylline have been
developed using 'H-theophylline. lY2 ,123 8-Carboxy-
theophylline was used to prepare antibodies. There is no
interference from endogenous purines or known metabolites of
theophylline at the concentrations studied. 122

6. Metabolism

UsinglJZ4C] aminophylline injection, Caldwell,


Monks and Smith determined that the metabolites of
aminophylline are the same as those of theophylline. 125,126
These are (i) 3-methylxanthine (ii) 1,3-dimethyluric acid
and (iii) 1-methyluric acid. However, the - rate and extent
of conversion to 1,3-dimethyluric acid and 3-methylxanthine
were higher or aminophylline than for theophylline.126
Therefore, "C recovery in urine (0-24 hours) was higher for
aminophylline (87%) than for theophylline ( 7 6 % ) . The
formation of 3-methylxanthine follows saturation kinetics;
therefore, the presence of circulating methylxan ines from
foods affects the elimination of aminophylline. '" The
appearance of the other wo metabolites follows first order
kinetics. Jenne et all2' determined that 1-demethylation of
theophylline to 3-methylxanthine is the dominant reaction
determining theophylline concentration in serum. Presence
of ethylenediaminelyyst affect this conversion, but how it
does is not known.

7. Biopharmaceutics and Pharmacokinetics

Pharmacokinetics of aminophylline has been studied


extensively. Since aminophylline and theophylline are
indistinguishable in biological fluids, pharmacokineticists
d o not differentiate between the two. lthough the pharma-
cokinetics of theophylline was reported' earlier, the advent
of newer analytical techniques has since led to an extensive
amount of work.
Aminophyl line is administered orally 129-133 as a
sustained-re1
lwe
dose capsulg- ordosagetablet,
fo
1329133,134,135 Or as a single-
15',130 intravenously (or by
infusion) "' ntramuscularly, or rectally as suppositories
or enema. 1389131 Among different aminophylline dosage
32 KAILAS D. THAKKER AND LEE T. GRADY

forms, rect 1 positories give the widest


variation.lf8,fYf

Absorption of aminophylline from tablets or capsules is


rapid, plasma levels reaching therapeutic range within 1-1 l/2
hours. Sustained-release preparations of aminophylline are
used often to maintain theophylline levels within
for about 12 hours in treatment of
as .
theraPey51'15'~f39
thma The rectal route is used often with
infants and children.

Plasma theophylline levels of 5-15 pg/ml (after


administrati of aminophylline) are considered safe and
O''
therapeutic. Although in most ca toxic symptoms do
not appear at levels above 25 ug/ml,Bz8 the patient-to-
patient variation is fglhi@ that individualization of the
therapy is necessary. In some case intra-patient
variation in doselblood levels is observedfh6 during
continuous long-term therapy. Saliva and bre ft5
milk levels
of theophylline are lower than plasma levels,
but1 &75PZ$
correlation between plasma/saliva ratio is obtained.

.'
The pharmacokinetic behavior of aminophylline once it
appears in the blood can be described by the same ode1 that
is used to describe theophylline pharmacokinetics The
volume of distri u i n at steady-state for all ages is
0.45 literlkg'158-E5P except in neonates where it is
slightly larger.152 Elimination is rapid, with a half-life
of about 6 hours in normal, healthy, non-smoking adults.
Among the factors that affect the ation of
theophylline fr sma are age,1f'i'P'9 physi lo and
disease stat 1'B-p89 co-administer 163-18 diet ,165
time of day,fb6 and smoking habits.fg7di'8s'In a case when
any of these factors is operating, the half-life of
elimination varies from 5 to 30 hours.

Very recently, Monks et have compared the


disposition and elimination of theophylline and
aminophylline. Elimination of aminophylline was faster than
theophylline in the same subjects. Although qualitatively
the disposition (and metabolism) of theophylline and
aminophylline were similar, the authors claimed there were
small but significant differences in rates of elimination
and the extent of dose eliminated within 48 hours.
AMINOPHYLLINE 33

8. Toxicitv

Toxicity of aminophylline results when bl d levels


exceed the level of 20 pg/ml of theophylline.lY8 The
severity of toxicity is directly related to plasma levels of
theophylli In mild toxicity, symptoms are nausea and
vomiting ,1981171 diarrhea, abdominal pain, nervousness,
insomnia, tachycardia and headache.
serious tachycardia, grand ma1 seizures,
cardiac arrythmias may occur In some cases, death may
result from acute toxicity. ij4
34 KAILAS D. THAKKER AND LEE T. GRADY

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AMINOPHYLLINE 35

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AMINOPHYLLINE 37

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38 KAILAS D. THAKKER AND LEE T. GRADY

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--
78. J. W. Nelson, A. L. Cordry, C. G. Aron, and R . A.
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79. F. Nielsen-kudsk and A. K. P e d e r s e n , Acta Pharmacol.


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80. P. J. Naish, M . Cooke, and R. E. Chambers, -


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81. K. T. Muir, J. H. G. Jonkman, D. Tong, M . K u n i t o n i , and


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AMINOPHY LLINE 39

83. P. L. von Aerde, E. Moerman, R. van S e v e r e n , and P.


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89. S. A. McKenzie, A. T. Edmunds, E. B a i l l i e , and J. H.


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91. L. W. Bond and D. L. Thornton, C l i n . Chem., --


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95. S. Sun, J. Pharm. S c i . , --


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40 KAILAS D. THAKKER AND LEE T. GRADY

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101. M. Sheehan, R. H. Hertel, and C. T. K e l l y , C l i n .


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102. C. V. Abraham, 0. A tk in so n , and D. Gresham, Am. J.


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--
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109. A. C a s t r o , J. I b a n e z , W. V o ig h t, T. Noto, and H.


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110. D. N. D i e t z l e r , N. Weidner, V. L. T i e b e r , J. M.
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111. N. Weidner, J. M. McDonald, V. L. T i e b e r , C. H. Smith,


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Biochem., --
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AMINOPHYLLINE 41

115. D. A. L a c h e r , J . A. S i n n , J. S a v o r y , and M. R. Wills,


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117. J. P. Long, C l i n . Chem., --

118. F. D. Lasky, J. A 1 R a z i , and A. Kramen, C l i n . Chem.,


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--
119. R. L. Boeckx, E. M. F r i t h , and F. E. Simons, "her.
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Drug Monit., _-

120. B. V i n e t and L. Z i z i a n , C l i n . Chem., --


25 (8), 1370
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121. T. Nishikawa, H. Kubo, and M. S a i t o , C l i n . Chim. Acta,


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122. A. L. Neese and L. F. Soyka, C l i n . Pharmacol.
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123. C. E . Cook, M. E. Twine, M. Myers, E. Amerson, J. A.


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Pharm.,
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124. J. C a l d w e l l , T. J. Monks, and R . L. S m i t h , B r . J .
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126. J. W. J e n n e , T. W. C h i c k , B. A. Miller, and R. D.


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127. T. J . Monks, R. L. S m i t h , and J. C a l d w e l l , J. Pharm.


Pharmacol., -33, 93 (1981).

128. J. W. J e n n e , H. T. Nagasawa, and R. D. Thompson, C l i n .


Pharm. and Therap., -- 19 (3), 375 (1976).

129. L. H e n d e l e s , M. Weinberger, and L. B i g h l e y , Am. J.


Hosp. Pharm., - 34, 525 (1977).

130. M. L. S l o t f e l d t , C. E. Johnson, G. Grambau, and J. G.


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42 KAILAS D. THAKKER AND LEE T. GRADY

131. H. Lamont, E. Moermann, M. B o g a r t , M. Van Der


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132. G. E. M a r l i n , M. A. B u t c h e r , J. A. Klumpp, and P. J.
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133. P. W. Trembath and S. W. Boobis, C l i n . Pharmacol.


Ther.,
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134. S. McKenzie and E. B a i l l i e , J. I n t . Med. Res., 7


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Suppl. 1, 22 (1979).

135. M. C. Meyer, A. B. S t r a u g h n , and P. Lieberman, C h e s t ,


78 ( 2 ) , 300 (1980).
--
136. L. J. Lesko, A. T. Canada, G. Eastwood, D. Walker, and
D. R. Brousseau, J. Pharm. S c i . , --
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137. M. W. Weinberger, R. A. Matthay, E. J. Ginchansky, C.


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138. E. B. T r u i t t , J r . , V. A. McKusick, and J. C. K r a n t z ,


J r . , J. Pharmacol. Exp. Therap., 1 6 0 , 309 (1950).

139. J. Ahrens, Dtsch. Med. Wochenschr., 102 (13), 482


--
(1977).

140. M. W. Weinberger and E. A. Bronsky, J. P e d i a t r . , -


84,
421 (1974).

141. E. Ginchansky and M. Weinberger, J . P e d i a t r . , --


91 (4),
655 (1977).

142. P. R a n g s i t h i e n c h a i and R. W. Newcomb, J. P e d i a t r . ,


91 ( 2 ) , 325 (1977).
--
143. F. Nielsen-Kudsk, I. Magnussen, T. S . J e n s e n , and K.
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144. F. E . Simons, K. J. Simons, G. G. S h a p i r o , W. E .


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(1976).
AMINOPHYLLINE 43

145. M. H. J a c o b s , R. M. S e n i o r , and G. Kessler, J. Am.


Med. Assoc., 235, 1983 (1976).

146. P. D. Watson, R. C. S t r u n k , and L. M. T a u s s i g , -


J.
91 (2), 321 (1977).
P e d i a t r . , --

147. N. N. Khanna, H. S. Bada, and S. M. Somani, -


J.
96, 494 (1980).
Pediatr., -

148. S. P. G a l a n t , S. A. Gillman, L. H. Cummins, P. P.


131 (9),
Kozak, and J. J. O r c u t t , Am. J. D i s . C h i l d . , --
970 (1977).

149. R. Koysooko, E . F. E l l i s , and G. Levy, C l i n .


15 (5), 454 (1974).
Pharmacol. Therap., --

150. P. A. Mitenko and R. I. O g i l v i e , C l i n . Pharmacol.


14, 509 (1973).
Therap., -

151. L. H e n d e l e s , M. Weinberger, and L. B i g h l e y , Am. Rev.


R e s p i r . D i s . , 118, 97 (1978).

152. 3. V. Aranda, D. S. S i t a r , W. E. P a r s o n s , P. M.
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413 (1976).

153. G. G i a c o i a , W. J. J u s k o , J. Menke, and J. R. Koup, -


J.
Pediatr., - 89, 829 (1976).

154. J. P. Rosen, M. D a n i s h , M. C. Ragni.


-~ C. L. S a c c a r , S.
64, 248
J. Y a f f e , and H. I. Lecks, P e d i a t r i c s , -
(1979).

155. F. E. R. Simons and K. J. Simons, J. C l i n . Pharmacol.,


18, 472 (1978).
-

156. P. M. Loughnan, D. S. S i t a r , R. I. O g i l v i e , A. E i s e n ,
88, 874 (1976).
2. Fox, and A. H. N e i m s , J. P e d i a t r . , -

58,
157. E. F. E l l i s , R. Koysooko, and G. Levy, P e d i a t r i c s , -
542 (1976).

158. K. M. P i a f s k y , D. S. S i t a r , R. E. Rangno, and R. I.


O g i l v i e , N. Engl. J. Med., 296, 1495 (1977).

159. K. M. P i a f s k y , D. S. S i t a r , R. E. Rangno, and R. I.


O g i l v i e , C l i n . Pharmacol. Therap., -21, 310 (1977).
44 KAILAS D. THAKKER AND LEE T. GRADY

160. A. Mangione, T. E. I m h o f f , R. V. L e e , L. Y. Shum, and


W. J u s k o , - s t , 73, 616 (1978).
C h e-

161. N. Vicuna, J. L. McNay, T. M. Ludden, and H.


7, 33 (1979).
S c h w e r t n e r , B r . J. C l i n . Pharamcol., -

162. K. C. Chang, T. D. B e l l , B. A. L a u e r , and H. C h a i ,


n c e t , 1, 1132 (1978).
-a -
L

163. R. A. Landay, M. A. G o n z a l e z , and J. C. T a y l o r , -


J.
A l l e r g y C l i n . Immunol., 62, 27 (1978).

164. M. Weinberger, D. Hudgel, S. S p e c t o r , and C. C h i d s e y ,


J. A l l e r g y C l i n . Immunol., 59, 228 (1977).

165. A. Kappas, K. E. Anderson, A. H. Conney, and A. P.


23, 445 (1978).
Alvares, C l i n . Pharmacol. Ther., -

166. L. J. Lesko, D. B r o u s s e a u , A. T. Canada, and G .


Eastwood, J. Pharm. S c i . , -- 69 (3), 358 (1980).

167. J. J e n n e , M. Nagasawa, R. McMugh, F. McDonald, and E .


17, 195 (1975).
Wyse, L i f e S c i . , -

168. J. R. P o w e l l , J. T h i e r c e l i n , S. Vozeh, L. Sansom, and


S. Reigelman, Am. Rev. Resp. D i s . , 116, 17 (1977).

169. W. J. J u s k o , J. J. S c h e n t a g , J. H. C l a r k , M. G a r d e n e r
a n d A. M. Yurchak, C l i n . Pharmacol. T h e r a p . , 24,
406 (1978).

170. L. H e n d e l e s , L. B i g h l e y , R. H. R i c h a r d s o n , C. D.
H e p l e r , a n d J. C a r m i c h a e l , Drug I n t e l l . C l i n . Pharm.,
11, 12 (1977).
-

171. T. R. Kordash, R. G. van D e l l e n , and J. T. M c C a l l , -


J.
Am. Med. Assoc., 238, 139 (1977).

172. L. W. Z w i l l i c h , F. D. S u t t o n , J r . , T. A. N e f f , W. M.
Cohn, R. A. Matthay, and M. W. W e i n b e r g e r , Ann.
I n t e r n . Med., - 82, 784 (1975).

173. M. S. Schwartz and D. F. S c o t t , E p i l e p s i a , -


15, 501
(1974).

174. C. L. Winek, J. D. B r i c k e r , W. D. Collom, and F. W.


Fochtman, F o r e n s i c S c i . I n t . , 15 (3), 233 (1980).
ASCORBIC ACID
Ibrahim A . Al-Mesh1 and Mahmoud M . A. Hassan

1. Description 46
1.1 Nomenclature 46
1.2 Formulae 46
1.3 Molecular Weight 47
I .4 Elemental Composition 47
1.5 Appearance, Color, Odor, and Taste 47
2. Physical Properties 47
2.1 Crystal Properties 47
2.2 Solubility 48
2.3 Specific Rotation 48
2.4 Dissociation Constant 49
2.5 Identification 49
2.6 Spectral Properties 51
3. Preparation 61
3.1 Isolation 61
3.2 Synthesis 63
4. Biosynthesis of Ascorbic Acid 63
5. Metabolism 66
6. Daily Requirement 66
7. Mode of Action 66
8. Vitamin Deficiency 67
9. Methods of Analysis 67
9.1 Titrimetric Methods 67
9.2 Spectrophotometric Methods 70
9.3 Turbidimetric Method 73
9.4 Chromatographic Methods 73
9.5 Enzymatic Method 75
9.6 Polarographic Method 15
9.7 Chronometric Method 75
10. References 76

Copyrighl 0 1982 by The Amenan


Analytical Profiles of Drug Substances phrrm.ceuUcd Awduion
Volume II 45
ISBN 0-12-260811-9
46 IBRAHIM A. AL-MESHAL AND MAHMOUD M. A. HASSAN

1, Description

1.1. Nomenclature

1.1.1 Chemical Names

a) L-Ascorbic a c i d
b) L-Xyloascorbic a c i d
c) 3-0x0-L-gluofuranolactone (enol form).
d) L-3-Ketothreohexuronic a c i d l a c t o n e

1.1.2 Generic Names

Vitamin C ; A s c o r b i c a c i d

1.1.3 Trade Names

Adenex; A l l e r c o r b ; A n t i s c o r b u t i c Vitamin;
Ascorbicap; Ascorbajen; A s c o r i l ; A sc o ri n ;
A s c o r t e a l ; A s c o r v i t ; Cantan; C a n t a x i n ; Cata-
v i n C ; Ce b i c u r e ; Cebid; Cebione; Cecon;
Cegiol an ; Cell i n ; Cenetone; Cereon; Cergona;
Ce s c o r b a t ; Cetamid; Cetan; Cetemican; Ceva-
l i n ; Ce v a t i n e ; Cevex; Cevimin; Cevi-Bid;
Ce-Vi-Sol; Cevitamin; C e v i t a n ; Cimin; C e v i t a -
mic Acid; C e v i t e x ; Ciamin; C i p c a ; C o l a s c o r ;
Concemin; C-vimin; Davitamon C ; E r i v i t C ;
Hybrin; L a r o s c o r b i n e ; Lemascorb; Megascorb;
P l a n a v i t C ; Pr o s c o r b i n C ; Redoxon; Ribena;
Sc o r b a c i d ; Scorbu-C; T e s t a s c o r b i c ; V i c e l a t ;
V i t a c e ; V i t a c i mi n ; V i t a c i n ; V i t a s c o r b o l ;
Vitix.

1.2 Formulae

1.2.1 Empirical

‘gH8’6
1.2.2 Structural CtI*OtI
I

HO
HR=o
HOCH

OH
ASCORBIC ACID 41

1.2.3 CAS No.

(50-81-7)

1.2.4 (Wiswesser Line Notation

TOSV EHJ CQ DQ EYQ IQL (1)


1.2.5 Stereochemistry

The nature o f the ring system in ascorbic


acid was determined by a study of the methy-
lated derivatives of the acid. By this means
complete confirmation was obtained of the
accuracy o f the views advanced, concerning
the stereochemical configuration of the
molecule and the nature of the reactive
enolic groups(2). The furanose structure for
ascorbic acid shown above (1.2.2) was put
forward by Lerbert el a1 ( 2 ) on the basis
o f its chemical behaviour as well as its
oxidation products.

1.3 Molecular Weight

176.12

1.4 Elemental Composition


C, 40.91%; H, 4.58%; 0 , 54.51%.

1.5 Qpearance, Color, Odor and Taste

White or slightly yellow crystals or powder. Odor-


less or almost odorless; pleasant sharp acidic
taste(3).
2. Physical Properties

2.1 Crystal Properties

Usually plates, sometimes needles, monoclinic


system (4).

2.1.1 X-ray Diffraction

The available data (2) of the X-ray, reveals


that the total of 12 carbon and oxygen atoms
48 IBRAHIM A. AL-MESHAL AND MAHMOUD M. A. HASSAN

all but one can be accomodated in one plane


without appreciable valency strain whilst
the remaining carbon (C5) lies less than 1A"
above the plane as shown in the following
model :

2.1.2 Melting Range


Ascorbic acid melts at 190-192" with decom-
position ( 4 ) .

2.2 So lubi1ity

Ascorbic acid is soluble at 20", in 3.5 parts of


water and 25 parts of alcohol (95 per cent); 50
parts of absolute alcohol, 100 ml of glycerol, 20 ml
of propylene glycol. Solubility in hot water 40.0%
at 40°, 80% at 100". Insoluble in ether, chloroform,
benzene and light petroleum (boiling range 40-60").
2.3 Specific Rotation

[a] is+ 20.5" to 21.5" (C = 1, water)

[a] i3+ 48 (C = 1, methanol) (4).

l9 + 24 (water) (2).
['I 5780
l8 + 116 (sodium salt in neutral solution) ( 4 )
5780
l8
+ 130 (N/20 NaoH) ( 2 ) .
5780

5780 + 149 (N/l NaoH)


l8 (2).

I'[ l8
5780
+ 155 (N/2 NaoH) (2)

l8 + 161 (2N NaoH) (2)


5780
ASCORBIC ACID 49

2.4 Dissociation Constant

Ascorbic acid is a moderately strong organic acid,


two ionization constants:

pK1 4 . 1 7 and pK 11.57. pH = 3 (5mg/ml), pH = 2


2
(50 mg/ml) (4).
2.5 Identification

i) Solution of ascorbic acid decolorises, 2,6-


dicholorophenol-indophenol solution (5).

ii) Solution and ascorbic acid reduces silver


nitrate solution immediately in the cold
producing a black precipitate (5).

iii) Dissolve 0.1 g of ascorbic acid in sufficient


w a t e r to produce 100 ml and dilute 1 ml to
100 ml with 0.01M h y d r o c h l o r i c a c i d . The
light absorption of the resulting solution
exhibits a maximum only at 244 nm; A (1 per
cent, 1 cm) at 244 nm, about 560 (6).

iv) To 2 ml of a 5 per cent w/v solution add 0.5


g of s o d i u m h y d r o g e n c a r b o n a t e ; carbon
dioxide is evolved ( 6 ) .

v) To 1 ml of a 5 per cent w/v solution add


about 0.2 ml of ZM n i t r i c a c i d and 0.2 ml of
0.1M silver n i t r a t e ; a grey precipitate is
produced (6).

vi) To 5 ml of a 1 per cent w/v solution add


0.05 ml of a freshly-prepared 5 per cent w/v
solution of s o d i u m n i t r o p r u s s i d e and 2 ml of
2M s o d i u m h y d r o x i d e followed by 0.6 to 0.7
ml of h y d r o c h l o r i c a c i d , added dropwise with
stirring; the yellow color turns blue (6).

vii) Specific optical rotation, in a 10 per cent


w/v solution, +20.5' to +21.5O (6).

viii) A solution (1 in 50) reduces alkaline cupric


tartrate TS slowly at room temperature but
more readily upon heating ( 7 ) .
50 IBRAHIM A. AL-MESHAL AND MAHMOUD M. A. HASSAN

ix) To 2 ml of a solution (1 in 50) add 4 drops


of methylene blue TS, and warm to 40": the
deep blue color becomes appreciably lighter
or is completely discharged within 3 minutes
(7).

XI Dissolve 15 mg in 15 ml of a solution of
trichloroacetic acid (1 in 201, add about
200 mg of activated charcoal, shake the
mixture vigorously for 1 minute, and filter
through a small fluted filter, returning the
filtrate, if necessary, until clear. To 5 ml
of the filtrate add 1 drop of pyrrole, and
agitate gently until dissolved, then heat
in a bath at 50": a blue color develops ( 7 ) .

xi) For the examination of vegetable infusions


for the presence of vitamin C, the ascending
- descending paper-chromatographic method of
Block was used on the dinitrosazones. Many
combinations of solvent were used, but a
mixture of xylene and nitrobenzene (95:5)
were the most satisfactory. Of these, the
latter solvent furnished compact spots of
such definition that treatment with alcoho-
lic potash to reveal them was unnecessary
(8)

xii) Amounts of vitamin C down to 3 pg can be


detected as dark areas on the layer exposed
to short-wave UV light; brief heating to
120°C renders it fluorescent in radiation of
365 nm ( 9 ) .

xiii) The customary identification o f free ascor-


bic acid depends on its strong reducing pro-
perties and any of the reactions known from
paper chromatography may be utilized. The
limit of detection with indophenol reagent
(blue) is around 0.1 pg dipyridly-iron
(red) and molybdophosphoric acid (blue) are
almost as sensitive; after brief heating,
derivatives and decomposition products
yield the colors also. Amounts of 3-5 pg
can be visualized with iodoplatinate reagent
(yellow) and with alkaline silver nitrate
reagent (9).
ASCORBIC ACID 51

2.6 Spectral Properties

2.6.1 Ultraviolet Spectrum

The UV spectrum of ascorbic acid (0.002%) in


aqueous, acidic methanolic, ethanolic and
alkaline solution was scanned from 200 to 400
nm using Varian Carry 119 Spectrophotometer
(Fig. I ) . The UV maxima are as follows:

'max (nm)

Aqueous solution 263


Acidic solution 243
Methanol 244
Ethano 1 245

Other reported data (2) are as foJlows:

'max (nm>

Aqueous solution 260-265


Acidic solution (pH3) 245
Ethano 1 245
Methanol 263
Sodium salt in aqueous solution 265

2.6.2 Infrared Spectrum

The IR spectrum of ascorb,icacid as KBr-disc


was recorded on a Perkin-Elmer 580B FT-
spectrometer (Fig. 2 ) . The structural assign-
ments have been correlated with the following
band frequencies (Table 1).

Table - 1: IR Characteristics of Ascorbic


Acid,
Frequency Cm-I Assignment
3510
3405 OH
3306
1755 c=o
1670
1110
c-0-c
1025
52 IBRAHIM A. AL-MESHAL AND MAHMOUD M. A . HASSAN

. . .
200 210 225 230 2 4 250 260 270 284 2YO 300 310 2 0 3% 3u) 350 3m 380 390

Fig. 1. UV Spectrum of Ascorbic Acid. Ascorbic Acid


i n Water; --- Ascorbic Acid i n Ethanol; +scorbic Acid i n
-
Methanol; ....
Ascorbic Acid i n Acid Solution.
1
4w mnmlmbr(tlDm am fWe mr f4a c w I a o c o o 4 m 2 I

Fig. 2. IR Spectrum of A s c o r b i c A c i d as KBr d i s c .


54 IBRAHIM A. AL-MESHAL AND MAHMOUD M. A. HASSAN

Other characteristic absorption bands are:

3208, 1500, 1390, 1372, 1320, 1275, 1250,


1222, 1200, 1140, 1075, 1068, 1045, 1025,
990, 870, 820, 755, 720, 680.

2.6.3 Nuclear Magnetic Resonance Spectra

2.6.3.1 Proton Spectra

The PMR spectra of ascorbic acid in


dueterium oxide,in pyridine and in
pyridine D5 were recorded on a
Varian-T-60-A, 60 MHz spectrometer
using sodium-2,2-dimethyl-2-sila-
pentane-5-sulphonate and tetramethyl-
silane as reference standard respec-
tively (Fig.3, Fig. 4 and Fig. 5).
The following structural assignments
have been made (Table 2):

Table - 2: PMR Characteristics o f Ascorbic


Acid.

Chemical Shift (ppm)

D20 Pyridine Pyridine D5 Assignment

4.87(d) 5.43(d) 5.36 (d) 5-H


4.10(m) 4.68(m) 4.33 (m) 6-H
3.77(s)
3.68(d)
4.40(s)
4.30(s)
4.36 (s)
4.23 (d) ' 7-CH2

s=singlet, d=doublet, m=multiplet.


400

Fig. 3. PMR Spectrum of A s c o r b i c A c i d in D20.


I
L
0
z
L
1
I
0
m
0
0
0
m
;I
56
m
4

Fig. 5. PMR Spectrum of Ascorbic Acid i n Pyridine D5.


58 IBRAHIM A. AL-MESHAL AND MAHMOUD M. A. HASSAN

PMR data in D20 and in a mixture of


DMSO D6 and CDC13 were also reported
(10,11,12).
13
2.6.3.2 C-NMR

The 13C-NMR completely decoupled and


off-resonance spectra are shown in
Fig. 6 and Fig. 7 respectively. Both
were recorded over 5000 Hz range in
dimethylsulfoxide on Jeol FX-100, 100
MHz spectrometer. Using 10 mm sample
tube and tetramethylsilane as refe-
rence standard, at ambient tempera-
ture. The carbon chemical shifts are
assigned on the basis of the additi-
vity principles and the proton-
coupled spectrum (Table 3).

11
I
HO - c 6-
I

Table - 3: Carbon Chemical Shifts of Ascorbic


Acid.

Carbon No. Chemical Shift ppm


c-1 170.31(s)
c-2 117.93 ( s )
c-3 152.62 (s)
c-4 74.56 (d)
c-5 68.42 (d)
C -6 61.93(t)

s=singlet; d=doublet; t=triplet

13C-NMR data in Water have been also


reported (1 3) ,
Fig. 6. 13C-NMR Spectrum of Ascorbic Acid (Completely de-
coupled).
~~

Fig. 7 . 13C-NMR Spectrum of Ascorbic Acid (off-Resonance) .


61
ASCORBIC ACID

2.6.4 Mass Spectrum


The mass spectrum o f ascorbic acid obtained
by electron impact ionization, was recorded
on a Ribermag R-10-10 mas spectrometer equip-
ped with direct inlet probe. The spectrum
(Fig. 8) shows a molecular ion peak M+ at m/e
176 with a relative intensity o f 5.9%. The
most prominent fragments and their relative
intensities are shown in Table 4:

Table - 4: Prominent Mass Fragments of Ascorbic


Acid.

M/e Relative Intensity % Fragment

177 1.0 M + l
176 5.9 M +
1

116 100.0

85 36.0

71

70
24.7

23.1
HF
H
H o g

61 29.7
a'
HO-
'i'
C
PH
- 0
C
I I
H H
3. Preparation

3.1 Isolation

Many methods were reported €or the isolation o f ascor


bic acid from plants. However, the most popular is
by using freshly prepared solution of 5-6% methaphos-
phoric acid (14). This solution is a good extractant
as well as stabilizing agent f o r a limited period by
complexing metal ions and minimizing the rate of oxi-
dation. It has also been claimed that ascorbic acid
can be stablized by diluted perchloric acid solution
ASCORBIC ACID 63

o r 2,3-dimercapto-l-propanol (15). An alternative


method of extracting ascorbic acid from foods is by
forming a slurry of the frozen material with absolute
ethanol has been found to be as effective as extrac-
tion with metabphosphoric acid (16).Also a mixture
of 8% acetic acid and 0.5% oxalic acid was used (17)
3.2 Synthesis

L-ascorbic acid is conventionally synthesized (18,19)


by hydrogenating D-glucose to D-sorbitol. The latter
is made to yields L-sorbitol by oxidation with Aceto-
bacter suboxydan, this followed by introducing carbo-
xyl group at C 1 while the L-sorbose is in the form of
its diacetone derivative. The resulting diacetone-2-
keto-L-gluconic acid is then heated with hydrochloric
acid t o give ascorbic acid (Scheme 1 , p . 11).
Alternative route from sorbose by oxidation with
nitrogen peroxide.
Another method for synthesizing L-ascorbic acid was
reported ( 2 0 ) , involving a one-step oxidation of 1,2-
0-isopropyhene-a-D-glucofuranose to 1,2-0-isopropyli-
dene-a-D-xylo-hexofuranurono-6,3-Lactone-S-ulose and
acid treatment of the later followed by reduction.
4. Biosynthesis of Ascorbic Acid

In both plants and animals ascorbic acid is formed from D-


glucuronic acid. UDP-glucuronic acid is first converted to
D-glucuronic acid lactone via D-glucuronic acid-l-phos-
phate. This compound is then reduced at carbon atom 1 t o
form L-gulonic acid. (Since in the numbering of the carbon
atoms of carbohydrates the most highly oxidized carbon
atom is given the lowest possible number, the original
carbon atom 6 of glucuronic acid becomes carbon atom 1 of
gulonic acid). After the conversion of the gulonic acid
to the corresponding y-lactone, the hydroxyl group at
carbon atom 2 is oxidized to a keto group. The 2-keto-L-
gulonic acid lactone formed is subsequently converted t o
L-ascrobic acid by enolization (21). Direct conversion of
D-glucuronic acid to L-gulonic acid by isomerization at
carbon atom 5 has not yet been conclusively established
(scheme 2,~.12).
64 IBRAHIM A. AL-MESHAL AND MAHMOUD M. A . HASSAN

CH20H
CH20H CH20H
I I I
HO - C -
I
H IIO - C - H co
HO - C - H I I
HO- C- H HO- C--B
I H? I Acetobactv I
H -C-O€i B- C- OH H- C- OH
I cat. I suboxydans I
HO -c - B HO - C - I-; HO - c - H
I I I
CHO
Ct120H CH20€i
D-glucose
G-sorbitol L-sorbose
COOH
I
co
I
HO - c-
I
acetone + (1)*<Mn04 ~

ki - C - OH

I
H+ ( 2 ) H 2 0 (H+) I
9:CMe2 H HO - C
I
-E
CH OII
2,3,4,6-diacetone sorbose 2
2-keto-L-gulonic acid

(CH20H
C -OH

bo
HO-
II
C
11 I
eno-
lize
I lactoniz
H - C-OH
I H
HO- C-H OH O€i
HO-C-H
I I
I
CH20H
CH20H
L-ascorbic acid
(Vitamin C)
Scheme 1: Synthesis of ascorbic acid.
ASCORBIC ACID 65

-
0
II
..
6

Q"Dp-
COOH COOH C

HO Ho@o-p ,@"20H
OH OH OH
UDP-D-Glycuronic D-Glucuronic acid- D-Glucuronic
acid 1-P acid lactone

CH20H CH20H
L-Gulonic acid L-Gulonic acid
lactone

HO-C-H
I I
CH20H CH20H

2-Keto-L-gulonic L-Ascorbic
acid lactone. acid.

Scheme 2: The biosynthesis of L-ascorbic acid.


66 IBRAHIM A. AL-MESHAL AND MAHMOUD M. A . HASSAN

5. Metabolism
Ascorbic acid is readily absorbed and metabolised. However,
after oral administration of large quantities, only small
amounts are excreted in the urine while there is a steady
rise in the level of ascorbic acid in the plasma. If the
oral ingestion is continued for a sufficient period, the
plasma concentration rises to a maximum, after which a
rapid urinary excretion of a large part of the ingested
ascorbic acid occurs (22). Ascorbic acid (ASA) and dehydro-
ascorbic acid (DAsA) are metabolized by humans, and the
levels of AsA in blood were maximum at the 4th hr after
AsA administration but at 2nd hr after DAsA administration.
The amount of AsA and DAsA excreted in urine in 6 hr was
respectively. 60 and 70% of the amount excreted in 24 hr
after administration, About 30 and 40% of administered
dose were excreted by men and women respectively in 24 hr.
Of the vitamin excreted after administration of either
AsA or DAsA, about 90 and 10% were in the form of AsA and
DAsA, respectively (23).
The amino acids phenylalanine and tyrosine are not meta-
bolized completely in vitamin C-deficient individuals,
Under these conditions they are metabolized only partly
and are excreted in the urine as homogentisic, p-hydroxy-
phenylpyruvic and p-hydroxyphenyllactic acids. It appears
that vitamin C plays the role of a coenzyme in the meta-
bolism of tyrosine through its deaminated product, because
scorbutic liver slices cannot metabolize this amino acid
in the absence of this vitamin. Vitamin C in adequate
amounts delays the oxidation of epinephrine by the body
(24 1.
6. Daily Requirement!
Ascorbic acid is needed in daily quantities of about 70 mg
to sustain full stamina, and is an essential nutrient for
human beings. In the case of insufficiency the symptoms
of scurvy appears (21).

7. Mode o f Action
We have as yet no complete understanding o f the mode of
action of ascorbic acid. It is, however, known that this
substance is involved in certain hydroxylation reactions
which are catalysed by mixed function oxygenases in the
reduction o f folic acid to tetrahydrofolic acid as well
as well in the regulation of the redox equilibrium between
Fez+ and Fe3+ and Cu" and C U ~ +(21).
ASCORBIC ACID 61

8. Vitamin Defficiency

Patients placed on a diet deficient in vitamin C


exhibited the following: 1) 10 days, plasma fell to
a low level; 2) 30 days plasma level was zero; 3) 13
weeks, first clinical evidence of scurvy; 4) 132 days,
hyperkeratotic papules developed; 5) 141 days, wounds
failed to heal and 6) 162 days, perifollicular hemor-
rhages of scurvy developed; ascorbic acid value of
white cell platelets fell to zero. Loss of weight
occurred, accompanied by lowered blood pressure (24).

9. Methods of Analysis

9.1 Titrimetric Methods

British Pharmacopoeia 1973

The B.P. 1975 has described the assay


as follows:

Weigh and powder 20 tablets. Dissolve a


quantity of the powder equivalent to 0.15 g
of ascorbic acid as completely as possible in
a mixture of 30 ml of water and 20 ml of
d i l u t e sulphuric acid and titrate with 0.lN
anunonium c e r i c sulphate, using f e r r o i n sulphate
solution as indicator. Each ml of 0 . 1 1 m o -
niwn c e ri c sulphate is equivalent to 0.008806 g
of C6H806.

British Pharmacopoeia 1980

The B.P. 1980 has described the assay as


follows :

Dissolve 0.2 g in a mixture o f 80 ml of freshly


boiled and cooled water and 10 ml of M sulfuric
acid. Titrate with 0 . 0 5 M iodine VS using 1 ml
of starch solution as indicator until a persis-
tent blue color is obtained. Each ml of 0.05M
iodine VS is equivalent to 0.00881 g of C H 0
6 8 6'

United States Pharmacopoeia 1980

a) Ascorbic Acid: Dissolve about 400 mg of


ascorbic acid, accurately weighed, in a
68 IBRAHIM A. AL-MESHAL AND MAHMOUD M. A. HASSAN

mixture of 100 ml of carbon dioxide-free


water and 25 ml of 2N s u l f u r i c acid. Titrate
the solution at once with 0.1 N iodine VS,
adding 3 ml o f starch TS as the end-point is
approached. Each ml of 0.1 N iodine is
equivalent to 8.806 mg of C6H806.

b) Ascorbic acid injections: Transfer to a 100-ml


volumetric flask an accurately measured volume
of ascorbic acid injection, equivalent to about
50 mg of ascorbic acid and previously diluted
with water if necessary. Add 20 ml of metaphos-
phoric-acetic acids TS, dilute with water to
volume, and mix. Accurately measure a volume
of the dilution, equivalent to about 2 mg of
ascorbic acid, into a 50-ml conical flask, add
5 ml of metaphosphoric-acetic acids TS, and
titrate with standard d i c h l o r o p h e n o l - i n d o p h e n o l
solution until a rose-pink color persists f o r
at least 5 seconds. Correct for the volume of
the dichlorophenol-indophenol solution consumed
by a mixture of 5.5 ml of metaphosphoric-
acetic acids TS and 15 ml of water. From the
ascorbic acid equivalent o f the standard
d i c h l o r o p h e n o l - i n d o p h e n o l solution calculate
the ascorbic acid content in each ml o f the
injection.

Various other titrimetric methods of assay


of vitamin C in vegetable tissues, whether as
ascorbic acid or as total vitamin C (ascorbic
plus dehydroascorbic acids), were studied and
compared. For the extraction of the vitamin,
an aqueous mixture of 8 per cent, acetic and
0.5 per cent oxalic acids was used instead of
metaphosphoric acid. Hot and cold extractions
gave practically the same results, except with
hard, dry tissues, which required hot extraction.
To effect removal of colloidal matter, which
interferes with filtration and clarification
of the extract, it is considered advisable to
add 25 to 50 per cent o f ethanol. In the
presence of ethanol causes an error o f at least
2 to 12 per cent. To establish (visually) the
titration end-point in the indophenol method,
it is recommended that a standard having the
same composition as the sample under titration
ASCORBIC ACID 69

b u t p r e v i o u s l y o x i d i s e d w i t h i o d i n e and
mercuric a c e t a t e be employed. T h i s g e n e r a l l y
e n a b l e s 93 t o 100 p e r c e n t of v i t a m i n C e x p e r i -
m e n t a l l y added t o be determined. I n t h e method
of d e t e r m i n a t i o n with methlene b l u e , t h e r a t i o
o f methylene b l u e used t o v i t a m i n C c o n t e n t
d e c r e a s e s as t h e l a t t e r v a l u e i n c r e a s e s . T h i s
d e c r e a s e of t h e r a t i o i s small; t o o b t a i n s a t i s -
f a c t o r y r e s u l t s , t h e a l i q u o t t i t r a t e d should
n o t c o n t a i n >0.04 mg o f v i t a m i n C . V i s u a l
d e t e r m i n a t i o n o f t h e end-point w i t h t h e a i d
o f a s t a n d a r d i s a g a i n recommended. The i o d i -
m e t r i c method, with potassium i o d a t e was a l s o
s t u d i e d . The c o n c l u s i o n s drawn a r e : 1) t h a t
t h e indophenol method i s s u f f i c i e n t l y a c c u r a t e ,
i s t h e most simple method and h a s t h e w i d e s t
s p h e r e of a p p l i c a t i o n ; 2) t h a t t h e methylene b l u e
method g i v e s r e s u l t s 3 t o 10 p e r c e n t lower
t h a n t h e indophenol method and 3) t h a t t h e
i o d i m e t r i c method g i v e s r ? s u l t s 20 t o 40 p e r
cmt higher (17).

Reduction i s e f f e c t e d by adding M sodium


s u l p h i d e s o l u t i o n a c i d i f i e d with HC1, and
removing t h e e x c e s s o f s u l p h i d e w i t h M
e t h a n o l i c mercuric c h l o r i d e s o l u t i o n . Reduc-
t i o n i s complete i n 10 t o 15 min. C l e a r
f i l t r a t e s a r e r e a d i l y o b t a i n a b l e €or t i t r a t i o n
w i t h dichlorophenol-indophenol ( 2 5 ) .

Simple methods o f d e t e r m i n i n g a s c o r b i c
a c i d , t h i a m i n e and n i c o t i n i c a c i d a r e
applied t o mixtures containing t h e s e
vitamins with various pharmaceutical
p r e p a r a t i o n s . Ascorbic a c i d can b e
determined i o d i m e t r i c a l l y i n t h e p r e s e n c e
o f calcium l a c t a t e , p h y t i n g l u c o s e calcium
g l y c e r o p h o s p h a t e , c a f f e i n e sodium b e n z o a t e ,
n i c o t i n i c a c i d , amidopyrine o r t h i a m i n e .
For m i x t u r e c o n t a i n i n g a s c o r b i c a c i d and
nicotinic acid, the ascorbic acid i s deter-
mined i o d i m e t r i c a l l y and t h e t o t a l a c i d i s
70 IBRAHIM A. AL-MESHAL AND MAHMOUD M. A. HASSAN

then titrated against 0.1 N solution of


hydrochloric acid with phenol red as indi-
cator. Thiamine i s determined in the
presence of ascorbic acid by a modifi-
cation of the U.S.S.R. Pharmacopoeia
VIII argentimetric method ( 2 6 ) .
The platinum - tungsten bimetallic elec-
trode system is applicable to the
quantitative iodimetric determination
of ascorbic acid according to the B.P.
1953. The equivalence point is given
by the very sharp break in the titration
curve. The error, = f 0.0002 on samples
of 0.05 to 0.15 g is much less than for
the usual volumetric procedure (27).
Other Sitrimetric method for the deter-
mination of ascorbic acid is by involv-
ing two stage oxidation by potasium
iodate ( 2 8 ) .

9.2 Spectrophotometric Method


9.2.1 Colorimetric
An assay method described for ascorbic acid
involves the reaction with diazotised 4-
methoxy-2-nitroaniline in acid medium, and
subsequent development of a blue color in
alkaline solution. This color, with a maximum
absorbancy at 570 nm, i s compared with stan-
dards in suitable photo-electric colorimeter.
It can be carried out directly, e.g., in the
presence of dehydro-ascorbic acid and all
other vitamins. Its sensitivity permits the
determination of quantities down to 0.5 mg
with a low limit of 10 pg per ml, when a 50-
ml sample aliquot is used (29a).
9.2.2 Ultraviolet
In aqueous solution ascorbic acid is charac-
terised by a single very intense band with
its head at 260-265.nm. The molecular extinc-
tion coefficient is approximately 7000 for
solutions containing about 2 mg/100 cc. In
stronger solutions (ca.50 mg per 100 c.c.)
wide deviations from Beer's law are encoun-
ASCORBIC ACID 71

tered. But for concentrations ranging between


0.5 and 2.5 mg per 100 C.C. Beer's law holds
with sufficient exactitude to permit of the
use of spectrophotometric measurements for
quantitative estimations of concentrations.
The intensity of the band diminishes rapidly,
falling to half value in a few hours (decom-
position of ascorbic acid by oxidation (2).
Other color reactions have been proposed as a
basis for measuring ascorbic acid: reaction
with diazotised p-aminobenzoic acid to produce
a pink color and the interaction of ascorbic
acid with dimethoxydiquinone to form a reddish
-violet color which is measured spectrophoto-
metrically at 510 nm (29b).
Ascorbic acid, cystine, thioglycollic acid,
a-mercaptopropionic acid and sodium mercapto-
butane sulphonate are determined by the fluore-
scence produce by the reduction of sodium 1 : Z -
naphthaquinone-4-sulphonate (Folin's reagent)
in U.V. The use of p-chloromercuribenzoic
acid in the determination of ascorbic acid
with 2,6-dichlorophenol-indophenol below pH3-5
is recommended because of the extent of spon-
taneous fading of indophenol that occurs. p -
Chloromercuribenzoic acid does not affect the
reaction between indophenol and ascorbic acid,
but it inhibits almost entirely the decolori-
sation of indophenol by various interfering
substances (31).
9.2.3 Spectrofluorimetric

The reaction of dehydroascorbic acid with o-


phenylenediamine to give a fluorescent quinox-
aline was used as the basis of an assay to
determine y quantities of ascorbic and dehydro-
ascorbic acids, The development of the fluore-
scent derivative of the vitamin is prevented
by forming a boric acid-dehydroascorbic acid
complex prior to addition of the diamine
solution. This provides a means of differen-
tiating between the fluorescence from the
vitamin and that from possible interfering
substances. When applied to pharmaceutical pre-
parations, beverages and special dietary foods,
72 IBRAHIM A. AL-MESHAL AND MAHMOUD M. A . HASSAN

the method shows a high degree of specificity.


No interfering substances were found (32).

A rapid colorimetric determination of ascorbic


acid (10 to 200 mg per 100 g) with the sodium
salt of 2,6-dichlorophenol-indophenol was
studied. An amyl acetate extract of 2,6-
dichlorophenol-indophenol has max. absorption
at 525 nm the extinction of which remains
unchanged for 4 hrs. The sample is extracted
with 2 per cent metaphosphoric acid and
diluted to produce a 0 . 4 to 1.0 mg per cent
solution of ascorbic acid. 2,6-dichlorophenol-
indophenol solution (5 ml in water) is added
to the sample solution which is rapidly ex-
tracted with amyl acetate for the extinction
to be measured. Another 5-ml portion of the
2,6-dichlorophenol-indophenol solution is
added to metaphosphoric acid and similarly
treated. The amount of ascorbic acid is
determined from the difference in the extinc-
tion of the two extracts, which is propor-.
tional to the concentration of ascorbic acid
up to 4 mg per 100 ml ( 3 3 ) .
Other method depends on the reduction of
ferric chloride by ascorbic acid and the
colorimetric determination of the ferric
chloride by means of reaction with aa-dipy-
ridly; the reaction is carried out in the
presence of phosphoric acid to eliminate in-
terference by reduction. A solution (1 ml)
containing N 0.02 mg of ascorbic acid is tre-
ted with 0.3 ml of 85 per cent. Phosphoric
acid to give a pH of 1 to 2, 5 ml of 0.5 per
cent aqueous aa-dipyridly solution and 1 ml of
1 per cent ferric chloride. The method has
been applied to orange juice, honey and urine;
it is sensitive to 5 lJg of ascorbic acid per
ml ( 3 4 ) .
A direct method for the determination of vita-
min C without removal of the reagent, 2,4-
dinitrophenylhydrazine, is applied to blood
and urine. The ascorbic acid content i s calcu-
lated from a chart (35).
Ascorbic acid can be semi-quantitatively
estimated with the comparison method after
color reaction with molybdophosphoric acid.
ASCORBIC ACID 73

The red zone of dehydroascorbic acid-DNP is


scraped off, eluted with 85% sulphuric acid,
filtered or, better, centrifuged, and the
light absorption of the solution measured at
520 to 525 nm against water as blank. The
analysis result is worked out with the help of
a standard solution which is treated identi-
cally ( 9 ) .

9.3 Turbidimetric Method


This method (36) is used €or the determination o f
ascorbic acid in foods, by the reaction of selenious
acid with ascorbic acid and stannous ions at low pH
and room temperature.

9.4 Chromatographic Methods


9.4.1 Paper Chromatography

Methods used €or the separation of ascorbic


acid by paper chromatography are shown in
Table 5 ( p . 21).

9.4.2 Gas-Liquid Chromatography

Gerstl and Ranf€t (42) extracted food with


metaphosphoric acid, separted the ascorbic
acid on a column of cellulose and formed the
trimethysilyl ether derivative by reaction
with N.0-bis-(trimethylsily) acetamide before
chromatographing on a column of Gas Q contain-
ing 3% SE-30.
9.4.3 High Pressure Liquid Chromatography

Packla and Kissinger ( 4 3 ) , used a strong anion


resin and elution with pH 4.75 buffer solution,
has been applied to the determination of
ascorbic acid in milk products, baby foods,
fruit juice concentrates, whole fruits and
fortified cereals. The procedure was not
directly suitable for measuring dehydroascorbic
acid. However, sood et a1 (44) used HPLC in
TABLE - 5 Methods used for separating ascorbic acid by paper chromatography
P
- -- -~ ~ ~~~ ~ ~

Chromatogram Solvent System Rf Value Reagent Application Reference

Paper Chro- 1) n-butanol saturated 2,6-dichlorophenol- Plant and 37


matography. with water + oxalic indophenol with sub- animal
sequent colorimetry. tissues.
2) Phenol saturated with
water + oxalic acid.
Paper (Sch- 1) 50% Methanol. 0.7-0.8 1) 1% silver-nitrate Lemon juice 38
leicher and solution in 10% white,red and
Schull No. Aqueous ammonia. black-currents.
602). 2) Wat er-n-butano1- 0.6-0.7 2 ) 0.005N iodine solu- tomatoes and
glacial acetic acid tion in 0,04% starch green pappers.
(50:40:14:5) so 1ut ion.
3) 0.04% aqueous solu-
tion of 2,6-dichloro-
phenol-indophenol.
4) U.V.
Paper Chro- butanol-acetic acid- 0.36 ammonium-molybdate solu- 39
matography. water (4:1 :5) tion.

Paper Chro- n-butanol-acetic acid- Alkaline tetrazolium Method f o r de- 40


matography. water (4:l:S) salts. tection of lfpg
quantity.
ASCORBIC ACID 75

the reverse phase ion-pairing mode to deter-


mine ascorbic acid in food with tridecyl ammo-
nium formate as counter-ion.

9.5 Enzymatic Method

A new enzymatic method based on the oxidation of


the ascorbic acid to dehydroascorbic acid with
ascorbic oxidase permit assaying for ascorbic acid
and dehydroascorbic acids in vegetable extracts (45).

9.6 Polarographic Method


Ascorbic acid can be analysed in a variety of mix-
tures and multivitamins preparations by cathode ray
reverse sweep polarography (46).

9.7 Chronometric Method

The reduction oxidation couple between ascorbic acid


oxidation and reduction of the semiquinoid form p-
phenylenediaimine is the basis of a new chronometric
assay of vitamin C.
Ascorbic acid interrupts formation of the colored
product by coupling its oxidation to reduction of
the semiquirioid proceeds to dye form (47).
76 IBRAHIM A. AL-MESHAL AND MAHMOUD M. A. HASSAN

10. References

1. G.C.G. Grasselli and N.M. Ritchey "Atlas of Spectral Data


and Physical Constants for Organic Compounds" 2nd Ed. ,
Vol. 2, CRC Press Inc., Cleveland, Ohio, 1975.
2. R.W. Herbert, E.L. Hirst, E.G.U. Percival, R.J.W. Reynolds
and Smith, J. Chem. SOC., 1270 (1933).
3. J.E. Hoover "Remington's Pharmaceutical Sciences", - 15th
Ed., Mack Publishing Company, U.S.A., (1975).
4. M. Windholz "The Merck Index" 9th Ed. Merck and Co., Inc.,
Rahaway, U.S.A. (1976).
5. "The British Pharmacopeia", Her Majesty's Stationary
Office, Cambridge, (1973).
6. "The British Pharmacopeia", Her Majesty's Stationary
Office, Cambridge, (1980).
7. "The United States Pharmacopeia", XX, Mack Publishing
Co., Easton, Pa (1980).
8. R.C.R. Barreto, Rev. Quim. Ind., Rio de Janeiro, - 24, 13,
(1955). Through, Anal. Abstract , 3, 567 (1956).
9. E. Stahl , "Thin Layer ChromatograpFy", 2nd Ed. , Springer-
Verlag., p. 304, (1966).
10. C.J. Pouchert and J.R. Campbell "The Aldrich Library of NMR
.
and Spectra" Vol XI (1974).
11. "High Resolution NMR Spectra Catalog", Varian Analytical
Instrument Division, Vol. 2, The National Press, U.S.A.
(1963) .
12. D.T. Sawyer and J.R. Brannan, Anal. Chem., 38 (2), 192,
(1966).
13. S . Berger, Tetrahedron, 33, 1587 (1977).
14. R.D. King "Developments in Food Analysis Technique-1"
Applied Science Publishers, 18 (1978).
15. C.B. Bourgeois, P.R. Mainuy, R. George and A.M. Czornomaz,
Analusis, 2, 556 (1973). Through Reference 14.
16. V.G. Randall, E.L., Pippen, A.L. Potter and R.M. McCready,
J. Fd. Sci., 40, 894. Through Reference 14.
17. G, Enachescu,%al. Inst. Cerc. Agron. Romn., 22, 463,
(1955) . Through Anal. Abstract , 3, 3474 (1956):
18. T.A. Geissman, "Principles of Organic Chemistry", 3rd. Ed.,
W.H. Freeman and Company, p. 497, (1968).
19. T. Reichstein and A. Grussner, tlelv. Chim. Acta., -17, 311,
(1934).
20. J. Bakke and 0. Theander Chemical Comm. , 175 (1971).
21. M. Luckner "Secondary Metabolism in Plants and Animals",
Science Paperbacks, p. 74, (1977).
22. E.G.C. Clarke "Isolation and Identification of Drugs",
The Pharmaceutical Press, London, (1971).
ASCORBIC ACID 77

23. T. Masaru, W. Sanae, M. Katsuko, T. Sachiko, F. Akji


(Biochem. Lab. Kagawa Nutr. Coll., Tokyo, Japan). Vila-
mins 45(3), 136, 1974, fJapan). Through
24. C.D. Wilson, 0. Gisvold and R . F . Doerge "Textbook o f Orga-
nic Medicinal and Pharmaceutical Chemistry", 7th Ed.,
J.B. Lippincott Co., Philadelphia, (1977).
25. E . Piyanowski, Prezem. Rony Spozywezy, 11,410 (1954).
26. G.A. Vaisman and S.G. Rozhntskaya Aptechnoc Delo. (3),
16. Through: Anal. Abstract, 2, 563 (1955).
27. S.R. Mohanty, K.R.K. Rao and L.V. Kannan, Anal. Chim.
Acta, 14 (6) 587 (1956). Through: Anal. Abstract, 2, 347,
(1956);
28. G.S. Deshmukh and M.G. Bapat, Z. Anal. Chem. 145(4), 256
(1955).
29. (a) M. Schmall, C.W. Pifer and E.G. Wollish, Anal. Chem.
25(10), 1486 (1953). Through Anal. Abstract, 1,370,
-
(1954).
(b) M.H. Hashmi, M.A. Shahid, M.A. Akhtar and N.A. Chugtani,
Mikrochim Acta, 5, 901 (1973).
30. H.. Freytag, Z. anal. Chem., 139(4), 263, 1953. Through.
Anal. Abstract, 1, 371, (1954).
31. J.A. Owen and B.Iggo, Biochem. J., 62(4), 675 (1956).
32. C. Mike, J. Deutch and C.E. Weeks, J. Assoc. Office Agr.
Chemists, 48(6), 1248 (1965). Through Chem. Abstract, 64,
72840 (1966).
33* H. Imai and T. Fugitani J. Chem. SOC., Japan, Pure Chem.
Sect., s(11), 1212 (1955). Through: Anal. Abstract 2,
2567 (1956).
34. M.X. Sullivan and H.G.N. Clarke, J. Ass. Off. Agric. Chem.,
38(2), 514 (1955). Through: Anal. Abstract, 2, 258 (1956).
-
35. J.H. Roc and C.A. Kuether, J. Biol. Chem. 147, 399 (1943).
Through: Chem. Abstract, 37, 31185 (1943).
36. W.J. Ralls, J. Agric. Food Chem. 23(3), 609, (1975).
Through: Chem. Abstract, 83, 41610 F (1975).
37. Yu-Tuan Cheng., F.A. Isherwood and L.W. Mapson, Biochem.
J., 55(5), 821 (1953). Through: Anal. Abstract, I, 372,
(1954).
38. V. Sanda Ceskosl. Farmac., 2(3), 79 (1954). Through: Anal.
Abstract, 2, 2563, (1955).
39. W. Hermann, R. Strohecker and F. Matt., Z. Lebensmitt. Un-
tersuch. U. Forsch., 97, 263, (1953). Through: Anal.
Abstract, 1, 369, 1954.
40. 2. Padre, E. Smid and V. Sicho, Naturwissenschaften, 42(8) ,
210, (1955). Through: Anal. Abstract, 2, 853 (1956).
41. J.E. Schlack, J. Ass. Office, Analyt. Chemist., 57, 1346,
(1974). Through: Reference 14.
42. R. Gerstl and K. Ranfft, Z. Lebensmitt-elunters. U. Forsch.,
154, 12, (1974). Through Reference 14.
-
78 IBRAHIM A. AL-MESHAL AND MAHMOUD M. A. HASSAN

43. L . A . Packla and P.T. Kissinger, Anal. Chem., 48, 3 6 4 ,


( 1 9 7 6 ) . Through Reference 1 4 .
4 4 . S.P. Sood, L.E. Sartori, D.P. Wittmer and W.G. Haney,
Anal., Chin., 4 8 , 796, ( 1 9 7 6 ) . Through Reference 1 4 .
4 5 . A. Marchesini, F . , Montauori, D. Muffats, D. Maestri, J.
Food Sci., 3 9 ( 3 ) , 5 6 8 , ( 1 9 7 4 ) . Through Chem. Abstract,
8 2 , 2717 F ( 1 9 7 5 ) .
4 6 . K S . Owen, F.W. Franklin, J. Food Technol., E ( 3 ) , 2 6 3 ,
( 1 9 7 5 ) . Through Chem. Abstract, 83, 1 3 0 1 0 2 J ( 1 9 7 5 ) .
4 7 . B. Roe, J . H . Bruemmer, Proc. Fla. State Hortic. SOC.,
87, 210, ( 1 9 7 5 ) . Through Chem. Abstract, 83, 130100 G
-
(1975).
CAPTOPRIL
Harold Kadin

1. Description 80
1.1 Name, Formula, Molecular Weight 80
1.2 Appearance, Color, Odor 80
2. History 80
3. Synthesis 84
4. Physical Properties 84
4.1 Spectral Properties 84
4.2 Solid State Properties 97
4.3 Solution Data 106
5. Stability 107
5.1 Solid State Stability 107
5.2 Solution Stability 108
6. Analytical Tests and Methods 112
6.1 Elemental Analysis 112
6.2 Spectrophotometric Methods 112
6.3 Chromatographic Methods 113
6.4 Titrimetric Methods 120
7 Analysis in Biologic Fluids and Tissues and in Animal Rations 120
7.1 Thin Layer Radiochromatography (TLRC) 120
7.2 Gas Chromatography-Mass Spectroscopy (GC-MS) 122
7.3 Gas Chromatography-Flame Photometric Detection (GF-FPD) 123
7.4 High Performance Liquid Chromatography with UV Detection (HPLC-UVD) 124
7.5 Spectrofluorometry 124
7.6 Radioimmunoassay (RIA) 124
7.7 Semiautomated Ellman Colorimetry 125
7.8 High Performance Liquid Chromatography with 126
Electrochemical Detection (HPLC-ECD)
7.9 High Performance Liquid Chromatography with 128
Fluorescence Detection (HPLC-FD)
8. Drug Metabolism-Pharmacokinetics 128
8.1 Blood Level Studies 128
8.2 Urinary Excretion Studies 129
8.3 Miscellaneous Distribution Studies 134
9. Acknowledgments 131
10. References 131
11. Review Coverage Dates 137

Analylical Profilca of Drug Substances Copynghi 0 1982 by The American


Volume 11 79 Pharmaceutical Association
ISBN 0-12-260811-9
80 HAROLD KADIN

1. Description
1.1 Name, Formula, Molecular Weight
Captopril, Capoten@, or Lopirins
is l-(3-mercapto-2-D-methyl-l-oxopropyl)-L-proline
(S,S) with -
Chem. -
Abstr. Registry Number
62571-86-2.

The asterisks indicate the two S,S optically


active centers.
1.2 Appearance, Color, Odor
Captopril is a white to off-white crystalline
powder with a slight mercaptan odor.
2. History
The captopril story began in 1971 with a
report (1) on the isolation and synthesis of
teprotide, an antihypertensive nonapeptide from
the venom of a Brazilian Pit Viper. This venom
peptide was hypotensive through inhibition of an
exopeptidase, known as the angiotensin converting
enzyme (ACE). The latter performs dipeptide
scission at the carboxyl end of the decapeptide,
angiotensin I to yield angiotensin 11, the most
powerful natural vasoconstrictor known. ACE
further potentiates hypertension through
scission-inactivation of the nonapeptide
vasodilator, bradykinin. Clinical investigations
with both teprotide (2) and captopril ( 3 ) have
implicated ACE as the key enzyme in human
hypertensive diseases.
The first ACE inhibitor shown to be
clinically efficacious against hypertension was
the synthetic venom peptide, teprotide ( 2 ) .
CAPTOPRlL 81

However teprotide was expensive and only effective


parenterally. A simpler, orally effective ACE
inhibitor was desired.
Analogs of the snake venom hypotensive
peptides were quantitatively rated for their in
vitro inhibition of ACE (4) and for their effect
o n e contractile properties of guinea pig ileum
( 5 ) . The ratings aided construction of a
hypothetical active site for ACE (Figure 1). This
model was based primarily on the similarity of the
enzymatic properties of ACE to those of
carboxypeptidase A (CASE A) even though the latter
yields amino acids rather than the dipeptides of
the former. Further, the active site of CASE A,
like ACE contains zinc but unlike that of ACE had
been structurally characterized, in the
crystalline state, by x-ray diffraction (6). Thus
the elaboration of the hypothetical ACE active
site and the possibility of a simple ACE inhibitor
were stimulated by a 1973 report (7) on,
D-2-benzylsuccinic acid, a simple inhibitor, of
CASE A.
Specific points of inhibitor attachment
within the real CASE A site were extrapolated to
that within the hypothetical ACE site ( 8 ) . On the
basis of these extrapolations, synthesis of simple
ACE inhibitors were initiated in 1974. As each of
a great number of candidates was quantitatively
rated in the aforementioned --
in vitro screens
(previously developed for the venom peptide
studies), the hypothetical site was verified and
refined. In the refined model schematically
represented in Figure 1, three dimensional amino
acid configurations within the catalytic site
provided suitably spaced subsites or multiple
points of attachment to substrate or inhibitor.
The semi-circular clefts in the figure represent
hydrophobic subsites which may interact with the
lipophilic side chains of inhibitor or substrate.
The latter are also putatively bound to the ACE
site via the X-H group at their nonscissile
terminal peptide bond. As indicated, one of the
first simple ACE inhibitors was patterned after
all of the venom peptide inhibitors in sharing a
terminal proline (succinyl-L-proline). The choice
of proline was also influenced by the superior
biological stability exhibited by teprotide with
82 HAROLD KADIN

RELATIVE
ENZYME INHIBITOR IN VlTRO INHIBITION

CAABOXYPEPTIDASE A

D-2-EENZYLSUCCINIC ACID

-7 FH2 ?-
0 = CCH,-CH-C -C = 0

ANGIOTENSIN-CONVERTING ENZYME

SUCC1NYL.L-PROLINE 1

D-2-METHYLSUCCINYL.L-PAOLI" 15

CAPTOPRIL 14000

F i g . 1. Key steps i n t h e d e s i g n o f a s p e c i f i c i n h i b i t o r of
t h e a n g i o t e n s i n c o n v e r t i n g enzyme.
CAPTOPRIL 83

its two terminal prolines (9). In an emulation of


D-2-benzylsuccinic acid, but with appropriate
lengthening of the chain, the nitrogen and
carbonyl of the penultimate "venom peptide" amide
linkage were substituted, respectively, with a
peptidase-inhibitory methylene and a highly
anionic, zinc binding carboxyl. As indicated in
Figure 2 the analogy was then extended to the
better snake venom inhibitors bearing a
penultimate alanine in addition to the terminal
proline (D-2-methylsuccinyl-L-proline) . The
largest increase in inhibitory activity (about 14
thousand fold over succinyl-L-proline) was
obtained when the zinc binding carboxyl was
replaced with a thiol which has considerably
greater affinity for zinc. The snake venom
peptide, teprotide, does not have comparable
affinity for zinc, however its binding is enhanced
by additional interactions of its amino acid side
chains with "clefts" on the enzyme beyond the
active peptidase site (10).
In short, the orally active antihypertensive
drug, captopril, was announced to the scientific
community in 1 9 7 7 (11). It was made generally
available for treatment of hypertensive diseases
in 1 9 8 1 (12,841. Clinical investigations (13)
suggest it is also highly efficacious in the
treatment of congestive heart failure. Its
uniquely designed highly specific affinity for the
active site of ACE has resulted in a high ratio of
clinical success with a relatively low index of
side effects. It has been effective where
conventional antihypertensive therapies fail or
have an untoward number of side effects.
84 HAROLD KADIN

3. Synthesis
A process (14) is presented (Figure 2) in a
chemical reaction sequence which follows this
brief description.
Methacrylic acid (I) is condensed with thio-
lacetic acid (11) to give racemic 2-methyl-3-
acetylthiopropionic acid (111). L-proline is then
acylated with the acid chloride (IV) of the thio-
ester (111). The resulting proline thioester (VI)
is resolved from its - - isomer by aqueous cry-
R,S
stallization. Saponification of compound VI with
sodium hydroxide affords the sodium salt of
captopril which after acidification yields
captopril (VII) .
4. Physical Properties
4.1 Spectral Properties
4.11 Infrared Spectra
The infrared spectrum of captopril in
chloroform is presented in Figure 3 and as a KBr
pellet in Figure 4. The infrared spectrum in the
latter indicates the presence of the following
frequencies and their structural assignments (15).
cm-1 Assignment
1750 C = 0 (COOH group)
1725
1640 C = 0 (amide)
2560 S - H
DiffereQfes in the fingerprint regions
(1350-900 cm ) of the mineral oil mull infrared
spectra of batches 3 and 4 (Figure 5 and 6,
respectively) indicate that the low melting batch
3 and the high melting batch 4 are polymorphs (see
Section 4.21) .
CAPTOPRIL 85

Figure 2

Chemical Reaction Schematic Diagram


I + I1 I11

CH2 = CCOOH
MW = 86.01
AcSH
MW 76.11
+ AcSCH2CHCOOH
MW = 162.20
-+
Methacrylic Thiolacetic Acetylthioisobutyric

IV
AcSCH2CHC
\ COOH
c1 COOH
MW = 180.65 MW = 115.13 MW = 259.32
Acid Chloride" L-Proline Proline Thioester

VI VI I

"'?
H3C 0
AcSCH2CHCN
COOH
d)

COOH
Proline Thioester MW = 217.28
Captopril
0

Ac = CH3C-
II
(a) Reflux (b) SOCl , DMF, Distillation
(c) H 0 + NaHCO Ci C12 Wash, HC1,
ci!ystallizaiG.on 41.
(d) H20 + NaOH, HC1, CH2C12 extraction
0
Ll
0
rl
c
u
c
.ti
a
k
ld
a
c
fd
4J
ul
a,
ro
5
0
X
d
.ti
Ll
a
0
4J
a
Id
u
w
0
5
Ll
LI
i,
a,
m
a
a c
a 0
4J
c
m
Q)
k
4J
ro
c
H
86
4J
a,
rl
r(
a,
PI
k
m
M
c
-4
ak
ld
a
c
ld
&
a,
[o
7
0
X
Li
a
0
4J
PI
ld
u
u-l a,
0 a
2
k 5
4J k
0
a,
a
2
rl
v) wI
a c
a, 4
k 2
id k
k a,
44 PI
G
H
..
lJ
k
&
(I)
c
H
WAVELENGTH (MICRONS)

3500 2500 2Ooo lsbo 1600 1400 1200 200


FREQUENCY (CM-’)

F i g u r e 5. I n f r a r e d S p e c t r u m of C a p t o p r i l , B a t c h 3 , Mineral O i l M u l l

Instrument: Perkin-Elmer, Model 6 2 1


WAVELENGTH (MKRONS)

F R E w € w (CM-')

Figure 6. Infrared Spectrum of Captopril, Batch 4, Mineral Oil Mull

Instrument: Perkin-Elmer, Model 621


90 HAROLD KADIN

4.12 Nuclear Magnetic Resonance Spectra


1
The 270 MHZ H-NMR spectrum of captopril in
CDCl is shown in Figure 7. The spectrum was
obtained from the University of Chicago courtesy
of Professor Josef Fried. Spectral assignments
are shown in Table 1.
The 13C-NMR of captopril in CD OD is shown in
Figure 8. The spectrum was obtained on a Varian
Associates XL-100 NMR spectrometer equipped with a
Nicolet TT-100 data system. Major peaks are
assigned in Table 2 . Minor peaks arise from the
presence of cis-trans isomerism at the amide bond
(16).

Table 1
Proton-NMR Data for Captopril
Proton Chemical Shift ( 6 )'PPM from TMS (ext.
COOH 9.81 (s)
CL-CH 4.60 (m)
B-CH2 2.03 (m)i 2.25 (m)
Y-CH,L 2.07 (m)
6 -CH2 3.63 (m)
9
-CH-C 2.44 (d,q) J=6,9
-STHA 2.82 (m)
-CH3 L 1.17 (d) J=6
SH 1.53 (ad) J=9,8
' multiplicities: d=doublet; q=quartet;
m=multiplet. J=proton-proton coupling
constants in Hertz.
0
F
CJ
I
X
5:
k
a,
x
ZJ
..k
m
4J
c
$
k
4J
(II
c
H
4 i
f *
0
0
4
I
I 4
X
c
a
-d
L!
rd
3
..
c,
d
$
k
4J
m
c
H
92
CAPTOPRIL 93

Table 2

Carbon-13 NMR Data f o r C a p t o p r i l i n CD30D.

Carbon # Chemical S h i f t ( 6 )
1 ppm from TMS

175.69
59.84
30.03
25.49
48.24
174.91
43.1
17.07
28.1

R e f e r e n c e d from c e n t e r peak o f t h e C D 3 0 D
m u l t i p l e t a t 4 9 . 0 ppm

4.13 Ultraviolet Spectra

S p e c t r a of c a p t o p r i l i n a q u e o u s m e t h a n o l ,
( F i g . 9 ) w a t e r , 0.1M sodium h y d r o x i d e and 0 . 1 M
h y d r o c h l o r i c a c i d ( F i g . 101, a r e p r e s e n t e d (17).
These s p e c t r a d e p i c t a n end a b s o r p t i o n s l o p e
w i t h o u t peak o r s h o u l d e r . The s l o p e s p e c t r u m i n
0 . 1 M sodium h y d r o x i d e was s h i f t e d c o n s i d e r a b l y
towards h i g h e r wavelengths. S i n c e weak s u l f h y d r y l
a b s o r p t i o n i s r e p o r t e d ( 1 8 ) i n t h e 220-230 nm
r e g i o n , t h i s a b s o r p t i o n s h i f t may b e d u e t o
i o n i z a t i o n o f t h e s u l f h y d r y l f u n c t i o n by t h e
a l k a l i . This s h i f t towards higher wavelengths
w i t h i n c r e a s e i n pH h a s been u s e d by O n d e t t i ( 1 9 )
t o d e t e r m i n e t h e pK o f t h e s u l f h y d r y l i n
captopril (Section 8.32).
HAROLD KADIN

. . . . . .

I ; 0 1 . . . . . . . . . . .0,

. !. !. ..
I '

. . ./ i ! .a,* , , , 4 , -

Fig. 9. U l t r a v i o l e t absorption s p e c t r a of C a p t o p r i l i n
10%aqueous methanol s o l u t i o n .
Instrument: Cary 15.

Fig. 10. U l t r a v i o l e t absorption s p e c t r a of C a p t o p r i l i n


H20, 0.1E H C 1 and 0.1N NaOH
Instrument: Cary 15
CAPTOPRIL 95

The spectra suggest that there is a maximum


at about 200 nm, attributable to the thiol
function. However, precise determination of the
peak absorbance was difficult because the
extremely large blank absorbances prevented
maintenance of a stable balance at this
wavelength. Consequently the peak maximum is
uncertain in Figure 9. However the peak
absorbance is valid in Figure 15 in which the
solvent was an HPLC mobile phase (System 4 - Table
8 - Section 6.32).

4.14 Mass Spectrum


The mass spectral pattern indicated in Figure
12 was obtained (20) on an AEI MS-9 Mass
Spectrometer.
The fragmentation, responsible for the spec-
trum in Figure 12+ is depicted schematically in
Figure 11. The M of m/z 217 and the other frag-
ments are consistent with a sulfur-containing
compound of the composition C 9 H1 5NO 3 S (Section
1.1).
Fiaure 11
Mass Spectral Fragmentation Schematic Captopril

170 103
J
172+m/z
H
m/z 70
m/z 199 + H20

M+ F
m/z 2 1 7 , 1 m / z 1{3
m/z 202 + CH3
+ C02 r m / z 140 + SH
b m / z 126 + CH2SH
I
L m / z 171 + S C H 2 4 m/z 127 + C02
96 HAROLD KADlN

6640 SQ14225 BFlTCH ttNNQ23NB

90.-

80-
>
F
U

II)
78.-

7
Z
W
t-
Z
H

50
W
>
H

t-
a
_I
w
L l

Figure 12. Mass Spectrum of Captopril

Instrument: AEI MS-902 Spectrometer Equipped with


Frequency Modulated Tape Recorder,
Spectrum Processed o n Digital
Equipment Corporation PDP-11 Computer
CAPTOPRIL 97

4.2 Solid State Properties


4.21 Polymorphism
An unstable, low (86OC) melting and a stable,
high (106OC) melting form of captopril have been
observed. These forms exhibited different unit
cells (Section 4.26) on single crystal X-ray
examination, differences in their powder X-ray
(Section 4.27), and differences in the solid state
infrared spectra (Figures 5 and 6). Agreement of
their optical rotations, infrared in solution and
bioassays established them as polymorphs.
4.22 Differential Thermal Analysis
(D.T.A. 1
DTA of the high melting polymorph ( 2 2 )
yielded a sharp, well-defined endotherm at 106OC
whereas the low melting polymorph produced a sharp
endotherm at 86OC. When the low melting polymorph
was allowed to resolidify and the DTA repeated,
the endotherm at 86OC had disappeared and an
endotherm at 106OC appeared. The latter suggests
that the high melting form is the stable
polymorph. A DuPont 900 Thermoanalyzer programmed
for a temperature rise of 15' per min was utilized
for these thermograms.
4.23 Melting Range
The U.S.P. (Class 1) melting range for the
high melting polymorph was 105.2 - 105.9' ( 2 1 ) .
This agrees well with its D.T.A. endotherm of
106OC. The low melting polymorph has a melting
range of 87-88', in agreement with its DTA
endotherm of 86O.
4.24 Differential Scanning Colorimetry
(D.S.C.)
Use of DSC as a purity index for captopril is
supported by titrimetric assays (17) of the
carboxyl function (alkalimetry) and of the
sulfhydryl function (iodimetry). For instance for
batch 4 these yielded 99.6% for the carboxyl and
99.2% for the sulfhydryl in very good agreement
with the DSC of 99.7% mole 8 ( 2 2 ) .
98 HAROLD KADIN

4.25 Hygroscopidity
Under ordinary conditions captopril is not
hygroscopic. Equilibrium moisture studies (23)
indicate no moisture pickup by captopril up to 50%
relative humidity at room temperature. Above 50%
R.H. it shows a tendency to cake after one to two
days.
Captopril did not exhibit any visual physical
changes and remained dry from 0 to 67% R.H. on
exposure for 14 days. Samples exposed to 81% R.H.
for 14 days appeared moist (24).
4.26 Single Crystal X-ray Diffraction
Single crystal X-ray analyses have been
completed (25) for both the low (melting range
86-87OC) and high (melting range 105-106O) melting
polymorphs. Both forms are orthorhombic with the
following crystal data:
A. High melting polymorph -
a = 6.834(2), -b =
0
" 3 space group
8.821(2), c = 17.982(4)A; V = 1084A,
P2 2 2 wiFh four molecules3per unit cell; cal-
culakeh density = 1.33 gcm- .Refined to R = 0.04
for the 745 observed single crystal intensities.
B. Low melting polymorph - -=
a = 9.496(3), b
0
03 space group
12.304(3), c = 19.282(5)A; V = 2253A;
P2 2121 witF eight molecules egr unit cell;
calculated density = 1.28 gcm .
Refined to R =
0.06 for the 1093 observed single crystal inten-
sities.
The structure in both has the S,S absolute
configuration with a 2 ( T r a n s ) conformation about
the N-C(0) amide bond (the O-C-N-C(2) dihedral
angles vary from -4 to +6O). The molecular
conformation differ in detail, most notably in the
conformation about the (S)C-C(C0) bond.
Atomic coordinates relative to orthogonal
axes for the high melting form are:
CAPTOPRIL 99

S -7.010 1.595 -5.766


N1 -6.499 -1.729 -1.938
c2 -5.799 -2.755 -1.152
c3 -6.274 -2.492 0.282
c4 -7.067 -1.228 0.252
c5 -7.511 -1.024 -1.144
C6 -6.188 -4.132 -1.626
06A -7.165 -4.364 -2.286
06B -5.394 -5.106 -1.204
c7 -6.215 -1.576 -3.227
07 -5.3 08 -2.253 -3.754
C8 -7.041 -0.624 -4.050
c9 -6.163 0.238 -4.947
c10 -8.052 -1.432 -4.821
It was predicted that salt formation with
resultant dissociation to a carboxylate anion
would influence Capoten to crystallize in its less
common E ( c i s ) conformation. This prediction was
tested (25) by performing single crystal analysis
on the dicyclohexylamine salt of captopril. The
analysis indicated that the salt was indeed in the
E conformation, i.e. the carbonyl groups of the
amide and carboxyl functions are cis to each
other.
4.27 Powder X-ray Diffraction
The stable, higher melting polymorph and the
lower melting, metastable polymorph are shown in
the powder X-ray patterns Figures 13 and 14
respectively (26). The values given in the
patterns are also listed in Tables 3 and 4 for the
high and low melters respectively. The tables
also show the relative intensities (based on peak
areas) of the various peaks.
A powder X-ray pattern taken on the low
melting polymorph after it was heated to about
95OC showed conversion to the stable form.
The X-ray pattern was taken with copper Ka,
nickel filtered X-radiation.
a k
a a,
u 4J
a,
w E
0 0
100
0
PI
P
I
L '
I
!
I
i
i
101
Table 3
Powder X-Ray Diffraction Data f o r Figure 13 (High Melting Polymorph)
2-13 (DEG.) D(ANGSTROMS) PEAK REL. PEAK AREA REL. AREA
9.99 8.85 22.7 0.208 88.1 0.270
11.35 7.80 37.9 0.347 138.4 0.424
14.24 6.22 15.2 0.139 122.3 0.374
16.45 5.39 21.0 0.192 77.5 0.237
17.21 5.15 57.1 0.523 169.6 0.519
17.98 4.93 47.5 0.435 166.8 0.511
19.25 4.61 34.4 0.315 146.3 0.448
19.85 4.47 109.1 1.000 324.5 0.994
20.78 4.27 37.7 0.346 130.1 0.398
22.23 4.00 49.8 0.456 161.7 0.495
24.52 3.63 24.1 0.221 86.4 0.265
25.03 3.56 15.3 0.140 60.2 0.184
25.97 3.43 62.6 0.574 326.6 1.000
26.56 3.36 13.7 0.126 68.2 0.209
28.26 3.16 68.7 0.630 243.1 0.744
29.79 3.00 10.6 0.097 102.0 0.312
30.81 2.90 8.4 0.077 42.5 0.130
31.66 2.83 9.1 0.083 43.9 0.134
33.45 2.68 8.3 0.076 47.9 0.147
34.28 2.61 17.9 0.164 98.2 0.301
Table 3 (Continued)
~ - ~ ( D E G . ) D(ANGSTROMS) PEAK REL. PEAK AREA REL. AREA

36.17 2.48 14.8 0.136 62.3 0.191


36.34 2.47 13.3 0.122 37.5 0.115
37.53 2.40 9.3 0.085 64.9 0.199
38.63 2.33 15.3 0.140 73.3 0.224
Sorted Data (Highest Peak First)
z-O(DEG.) D(ANGSTR0MS) PEAK REL. PEAK AREA REL. AREA

19.85 4.47 109.1 1.000 324.5 0.994


28.26 3.16 68.7 0.630 243.1 0.744
25.97 3.43 62.6 0.574 326.6 1.000
17.21 5.15 57.1 0.523 169.6 0.519
22.23 4.00 49.8 0.456 161.7 0.495
17.98 4.93 47.5 0.435 166.8 0.511
11.35 7.80 37.9 0.347 138.4 0.424
20.78 4.27 37.7 0.346 130.1 0.398
19.25 4.61 34.4 0.315 146.3 0.448
24.52 3.63 24.1 0.221 86.4 0.265
9.99 8.85 22.7 0.208 88.1 0.270
Table 4

Powder X-Ray Diffraction Data f o r Figure 1 4 (Low Melting Polymorph)

2 - 8 (DEG.) D (ANGSTROMS) PEAK REL. PEAK AREA REL. AREA

8.71 10.15 24.5 0.392 68.7 0.259


9.31 9.50 13.7 0.219 26.3 0.099
11.77 7.52 14.3 0.229 89.1 0.336
12.71 6.96 14.0 0.224 43.0 0.162
13.13 6.74 16.6 0.266 69.4 0.261
15.00 5.91 31.8 0.509 142.4 0.536
17.21 5.15 14.7 0.235 43.2 0.163
17.89 4.96 14.0 0.224 36.8 0.138
18.23 4.87 39.5 0.632 101.0 0.380
18.40 4.82 34.8 0.557 77.2 0.291
19.51 4.55 16.9 0.270 91.2 0.343
20.10 4.42 13.1 0.210 57.3 0.216
20.61 4.31 30.6 0.490 112.0 0.422
20.87 4.26 13.1 0.210 34.8 0.131
21.38 4.16 13.0 0.208 33.8 0.127
22.14 4.02 62.5 1.000 265.5 1.000
23.25 3.83 13.9 0.222 69.1 0.260
23.50 3.79 12.1 0.194 34.4 0.130
24.10 3.69 16.8 0.269 113.4 0.427
24.86 3.58 12.5 0.200 43.4 0.163
25.20 3.53 17.4 0.278 83.3 0.314
Table 4 (Continued)

~-WDEG.) D(ANGSTROMS) PEAK REL. PEAK AREA FEL. AREA

25.88 3.44 17.8 0.285 105.0 0.395


27.33 3.26 14.6 0.234 90.5 0.341
28.35 3.15 12.8 0.205 33.3 0.125
28.94 3.09 14.4 0.230 104.1 0.392
30.05 2.97 14.0 0.224 72.8 0.274
34.47 2.60 16.6 0.266 99.3 0.374

Sorted data (Highest Peak First)

2-B(DEG.) D(ANGSTR0MS) PEAK REL. PEAK AREA REL. AREA

22.14 4.02 62.5 1.000 265.5 1.000


18.23 4.87 39.5 0.632 101.0 0.380
18.40 4.82 34.8 0.557 77.2 0.291
15.00 5.91 31.8 0.509 142.4 0.536
20.61 4.31 30.6 0.490 112.0 0.422
8.71 10.15 24.5 0.392 68.7 0.259
25.88 3.44 17.8 0.285 105.0 0.395
25.20 3.53 17.4 0.278 83.3 0.314
19.51 4.55 16.9 0.270 91.2 0.343
24.10 3.69 16.8 0.269 113.4 0.427
34.47 2.60 16.6 0.266 99.3 0.374
13.13 6.74 16.6 0.266 69.4 0.261
17.21 5.15 14.7 0.235 43.2 0.163
106 HAROLD KADlN

4.3 Solution Data


4.31 Solubility
Captopril at 25OC is freely soluble (1 to 10
parts solvent to 1 part solute) in water,
methanol, ethanol (SD3A), isopropanol, chloroform,
or methylene chloride. However, it is only
soluble (10-30 parts solvent to 1 part solute) in
ethyl acetate (27). The solubility of captopril
in water, at 250CI is 160 mg/ml (28). A
solubility-temperature profile of captopril in
water obeyed a linear equation up to 4OoC (28).
Beyond this temperature captopril showed extra-
ordinarily high water solubility.
Solubility in sesame and corn oils was less
than 1 mg/ml at 25OC, whereas the solubility in
the synthetic oil triacetin (glyceryl triacetate),
at 25OC, was greater than 20 mg/ml (29).
4.32 pKa
-
The pK of the carboxyl of captopril (pK ) is
reported (23) to be 3.7. Whereas a carboxyl break
was readily observed with alkali potentiometry,
the sulfhydryl break could not be detected (17).
Therefore, the pK of the sulfhydryl in captopril
(pK ) was not estfmated by classical potentio-
metgy. It was, however, estimated at 9.8 (pK2) by
Ondetti (19) and Weiss (30) using sulfhydryl U.V.
shifts to higher wavelengths with increase in pH
(Section 4.13). The method utilized was adapted
from Benesch and Benesch (31).
4.33 Metal Complex Formation
Captopril was modelled (11) as a selective
and competitive inhibitor of the angiotensin
converting enzyme (Section 2 ) . Part of this
inhibition resides in the binding of the zinc
cofactor within the enzyme's active site by
captopril's thiol function. Constrained within
the active site by the multiple interactions of
site and inhibitor, captopril bars entry of
angiotensin I and thus prevents its conversion to
the most powerful natural pressor, angiotensin 11.
Since captopril lacks an amino group, it does not
CAPTOPRIL 107

complex metals in solution with the well-docu-


mented avidity (32,33) of amino group bearing
thiols like cysteine, glutathione, and in vivo
metal depletors like 2 - m e r c a p t o p r o p i o n y ~ g l y c i n e
and penicillamine. Indeed, Weiss (30) reported
that an alkali potentiometric study of the extent
of zinc ion complexation with cysteine (I), 2-
mercaptopropionyl glycine (11), and captopril
(111) indicates the order of binding, at pH 7.4,
to be I > I1 > 111.
Captopril binds mercuric ion (Section 7 ) to
block its colorimetric reaction with Ellman's
reagent, thus allowing a measurement of
non-sulfhydryl colorimetric interferences.
4.34 Optical Rotation
The optical rotation of the captopril in
absolute ethanol (34), using the Perkin-Elmer 155
Automatic Polarimeter, was determined to be: a
= -127.8'. The -
R,S
- isomer rotates at about +5'?
4.35 Partition Coefficients
A partition ratio (solvent/aqueous) after shaking
equal volumes of cosaturated aqueous (pH 2) and
methylene chloride was 1.39 ( 2 1 ) . A comparably
determined partition ratio between equal volumes
of cosaturated 0.1M HC1 and octanol was 1.9 (30).
When utilizing salcing out partition from aqueous
acid into methylene chloride at the captopril
concentrations prevalent in the urine analysis
(about 25-50 mcg/ml for a 100 mg dose) NaCl but
not Na2S04 was found to enhance captopril
oxidation (Section 7). This has been attributed
to trace chlorine generation from acidic chloride
plus oxygen.
5. Stability
5.1 Solid State Stabilitv
No significant decomposition was detected
(35) in SQ 14,225 bulk samples, stored at +5'c,
+33'C and +5OoC f o r up to 6 months or exposed to
900 foot-candles in a light box f o r 30 days, when
compared to -2O'C samples which served as the
108 HAROLD KADIN

c o n t r o l . Samples were examined f o r a p p e a r a n c e ,


c o l o r , o d o r , LD s a f e t y and by q u a n t i t a t i v e TLC
and IlPLC , i o d i m 2 p r i c t i t r a t i o n , i n f r a r e d , and
optical rotation.
5.2 Solution S t a b i l i t y

C a p t o p r i l i n aqueous s o l u t i o n undergoes an
oxygen f a c i l i t a t e d , f i r s t o r d e r , f r e e r a d i c a l
oxidation a t its t h i o l to yield captopril
d i s u l f i d e ( 2 8 ) . H y d r o l y s i s a t t h e amide l i n k a g e
o c c u r s o n l y u n d e r f o r c i n g c o n d i t i o n s (see S e c t i o n
5 . 2 5 ) . O x i d a t i o n w a s d e l a y e d by a d j u s t m e n t t o
lower pH, a d d i t i o n o f c h e l a t i n g a g e n t s , i n c r e a s i n g
captopril concentration, u t i l i z a t i o n of nitrogen
o r l o w oxygen h e a d s p a c e , and i n c o r p o r a t i o n o f
a n t i o x i d a n t s . O x i d a t i o n seems t o o c c u r l e s s
r e a d i l y i n methanol ( 3 6 ) . N o d e g r a d a t i o n o f
c a p t o p r i l w a s o b s e r v e d ( 4 0 mcg/ml) i n t h i s s o l v e n t
f o r up t o 2 weeks a t 5OC.
5.21 S t a b i l i t y and S o l u t i o n pH

O x i d a t i o n r a t e c o n s t a n t s a t v a r i o u s pli v a l u e s
( 2 8 ) i n Table 5 , s u g g e s t t h a t c a p t o p r i l i s
o p t i m a l l y s t a b l e below pH 3.5, t h e o x i d a t i o n r a t e
b e i n g e s s e n t i a l l y c o n s t a n t from pH 2 t o 3. The
r a t e c o n s t a n t s i n c r e a s e r a p i d l y above pH 4 . Using
HPLC and c o l o r i m e t r y ( 3 8 ) , c a p t o p r i l a q u e o u s
s t a b i l i t y w a s s t u d i e d , a t 50 mcg/ml, i n a r o t a t i n g
b a s k e t d i s s o l u t i o n a p p a r a t u s f o r up t o 1 8 0 m i n u t e s
a t 37OC i n d i s t i l l e d water, and a t pH 1, 2 and 3 .
E x c e l l e n t s t a b i l i t y a t pH 1 and 2 b u t a p p r e c i a b l e
d e g r a d a t i o n a t pH 3 , and i n d i s t i l l e d w a t e r w a s
observed. S u r p r i s i n g l y , t h e r a t e of d e g r a d a t i o n
a t pH 3 exceeded t h a t i n d i s t i l l e d water. The
more r a p i d o x i d a t i o n a t pH 3 w a s a t t r i b u t e d t o
c a t a l y s i s v i a g r e a t e r t r a c e m e t a l s o l u t i o n from
the dissolution baskets.
CAPTOPRIL 109

Table 5
Oxidation Rate Constant for Captopril (5 mg/ml)
in Citrate-Phosphate Buffers at Various pH
Values at 5OoC

PH
(day ) x 10f
Rate-Eonstan

2.13 8.38
2.59 9.01
2.89 8.22
3.13 8.31
3.53 9.92
3.88 9.13
4.23 12.94
4.67 19.43
5.16 28.93
5.63 42.03
5.22 Solution Stability, Metal Ions
and Chelating Agents
Transition metal ions most effectively cata-
lyze oxidation of captopril through a recycling of
oxygen free radicals (28). The most effective of
these catalysts are ubiquitous copper and iron, in
given order. As little as 1 ppm of copper has
been observed to catalyze captopril oxidation in
solution (28).
As has been demonstrated with cysteine (39)
lower levels of disodium edetate (EDTA Na2) may
enhance metal ion catalyzed thiol oxidation,
whereas higher levels retard oxidation.
Disodium edetate 0.1% (Na2EDTA 0.1%) best
stabilized 0.5 mg captopril per ml (of
citrate-phosphate buffer at pH 4, p = 0.5) in
Teflon-faced rubber sealed vials (37).
Analysis of urinary captopril was necessary
for dosage form bioavailability and dose titration
studies. The necessity for long term storage of
samples prior to analysis required development of
an acid-chelate stabilization (40). This
stabilization utilized diethylenetriamine
pentaacetic acid (DTPA) reputed (40) to be a more
effective metal chelator than Na2EDTA. A
110 HAROLD KADIN

relatively large amount of DTPA (about 4 0 0 mg in 5


ml) was mixed into the periodic urine voiding.
This was followed by an acidification to about pH
2 with a mixture of citric and oxalic acids and a
rapid refrigeration. DTPA, citric and oxalic have
been reported ( 4 1 ) to be effective sequestering
agents in the pH range 2 to 5 for Al, Cu, Fe, Ni
and Zn. Analysis of captopril in urine samples
stabilized in this manner, before and after 90-120
days of refrigeration, agreed remarkably well(40).
5.23 Concentration and Solution
Stability
The greater the captopril concentration, the
slower the oxidation ( 2 8 ) . For example, no signi-
ficant degradation (within + 3 % ) could be detected
by automated Ellman colorimgtry ( 4 2 ) at a concen-
tration of 2 5 0 mg captopril per ml of solution at
pH 1 2 . 5 - 1 4 after overnight room temperature
storage in uncovered beakers. At the opposite
extreme, captopril at an analysis concentration of
2 5 - 5 0 micrograms per ml of solution at pH 1 3 . 5
stored overnight at room temperature in open tubes
lost 8 4 % of its sulfhydryl activity ( 2 1 ) .
5.24 Oxygen Tension and Solution
Stability
The following 2 1 / 2 hour accelerated
stability at 4OoC on 2 5 ml of 1 mg captopril per
ml solutions in unstoppered 100 ml volumetric
flasks using air, oxygen, or nitrogen purging,
where indicated, was carried out ( 2 8 ) .
Table 6
Oxygen Tension and Solution Stability
M PPm 8 Captopril
Code Na2HP04 PH Cu Purge Recovered (HPLC)
1 0.08 7.9 2 None 73
2 0.08 7.9 2 Air 58
3 0.16 7.5 10 None 88
4 0.16 7.5 10 Air 62
5 0.08 7.8 2 N2 96
6 0.04 7.8 1 O2 0
(KH2P041
CAPTOPRIL 111

Under these conditions it is apparent that


oxygen causes rapid, complete degradation, air
facilitates degradation, and nitrogen protects
captopril in solution.
5.25 Amide Hydrolysis and Solution
Stability
Captopril solutions at 5 mg per ml in 0 . 5 M
-
HC1, containing 0.1 mg EDTANa /ml to minimize
oxidation , were heated at elegated temperatures.
The rate of hydrolysis was monitored by HPLC. The
data yielded first order linear plots from which
Wang (43) calculated rate constants (Table 7). An
Arrhenius plot yielded a heat of activation for
amide hydrolysis of 21.4 kcal/mole, comparable to
other amides.
Table 7
Rate Constants for Captopril Hydrolysis
-
k t90%
5.5 x lo,*-7 hr-l
-1 10 years
4OoC PH 3
4OoC PH 4 5.5 x hrml 10 years
22oc PH 3 6.7 x 10-lOhr -1 86 years
22oc PH 4 6.7 x 10 hr 86 years
It is clear from these t ' s (times for 90% amide
hydro1ysis1 , that hydro188f s contributes insigni-
ficantly to degradation.
112 HAROLD KADIN

6. Analytical Tests and Methods


6.1 Elemental Analysis
The following results were obtained:
Element Calculated Found for Batch 5
C 49.78 49.97
H 6.96 6.84
N 6.45 6.50
S 14.77 14.63

6.2 Spectrophotometric Methods


6.21 Colorimetry
Spectrophotometry for captopril has included
the widely utilized Ellman's reaction (44) in
which sulfhydryl reduction of 5,5'-dithiobis-2-
nitrobenzoic acid yields a mole of intensely
yellow 2-nitro-5-thiobenzoate anion per mole of
captopril. Manual (40,451 and automated colori-
metries have been utilized for captopril analysis
in chows (Section 7.71), urine (Section 7.72) and
in pharmaceutical formulations by TLC (Section
6.31).
Kinetic colorimetry vs pH (from pH 5 to 10)
studies (46) of a manual version of Ellman's
colorimetry of captopril established that the pH
optimum was at pH 7 (1 M phosphate, 0.05 M EDTA) .
Maximum color was attained within 2 min. and
stability was maintained for at least 45 min.
The S-nitroso-Bratton-Marshall colorimetry
(47) has been applied to analysis for captopril in
various formulations (48). It was adapted from a
method (47) for cysteine in biological materials.
In this method the thiol reacts with nitrous acid
to form a relatively stable S-nitroso derivative.
Excess nitrous acid is destroyed by sulfamic acid.
The S-nitroso derivative is then hydrolyzed by
mercuric ions to release nitrous acid. The latter
diazotizes sulfanilamide, presumably at a faster
rate than destruction of the nitrous acid by
CAPTOPRIL 113

excess sulfamic acid. The diazotized


sulfanilamide is then coupled to
N-(1-naphthy1)ethylenediamine to yield a stable
measureable red azo dye. The method was found to
be less suitable for analysis of captopril in
urine than Ellman's Reaction (Section 7.72).
Five simple, spectrophotometric identity
tests for captopril reported by Valatin (49)
include the observation of an evanescent red when
captopril reacts with nitrous acid. In addition
captopril color tests were described yielding
purple with nitroprusside, blue with ferric
chloride, red on addition of neutral N-ethylmale-
imide followed by strong alkali, and finally
orange-yellow (specific for proline) on acid
hydrolysis followed by neutralization then reac-
tion with ninhydrin.
6.22 Fluorometry
Captopril was reacted with
N-[p-(2-benzoxazoyl)- phenyl] maleimide in pH 6.85
buffer and the fluorescence of the
captopril-maleimide adduct was then measured at
310 nm excitation and 365 nm emission (49).

For coupling of captopril to


N-(7-dimethylamino-4-methyl coumarinyl) maleimide
to yield a highly fluorescent derivative see
Section 7.5.
6.3 Chromatosraphic Methods
6.31 Thin Layer Chromatography (TLC)

For quantitation of captopril (purity assay)


standard and sample solutions are each
chromatographed at 100 pg and 200 pg levels on
Analtech silica gel G plates using the solvent
system benzene-acetic acid (75:25). The captopril
zones on the plate are located by spraying a
guide-channel with a basic methanolic solution of
5,5'-dithiobis-2-nitrobenzoic acid. Captopril on
the guide-channel appears as a yellow zone. The
silica gel containing captopril zones on the
untreated portion of the plate are removed from
the plate, eluted with 5% aqueous trichloroacetic
acid, and reacted with a methanolic solution of
5,5'-dithiobis-2-nitrobenzoic acid, at an alkaline
114 HAROLD KADIN

pH, to form an intensely yellow 2-nitro-5-


thiobenzoate anion that is measured at 412 nm in a
spectrophotometer (50).
In a thin-layer semi-quantitative procedure
for measuring the individual impurities in
captopril, samples are chromatographed at 100 pg
and 200 pg levels and standards are
chromatographed at concentrations ranging from 0.5
pg to 4.5 pg. TLC separation is carried out on
Analtech Silica Gel G plates, developed in
conventional and in continuous development
chambers, using the solvent system benzene-acetic
acid (75:25). After development, the plates are
air dried and placed in a chamber saturated with
iodine vapors. A semi-quantitative estimate of
the concentration of each impurity in the sample
is made based on a visual comparison of the size
and intensity of each impurity zone with the
appropriate standard zones (50).
These TLC procedures, after appropriate
initial extractions, have been adapted for
formulations. They have also been applied to
semi-quantitative analysis of captopril disulfide
in tablets and as a TLC identity test for
captopril in tablets (50).
Streaked or spotted plates should be
introduced into the developing chamber (2 per
chamber) immediately after applying the solutions
to the plates. This minimizes the conversion of
captopril to its disulfide.
Placebo powders were spiked with known
amounts of captopril and assayed using the TLC
procedure. The results of 15 assays gave a
recovery of 100.6%, a standard deviation ( s ) of
1.39 and a coefficient of variation (C.V.) of 1.38
(50)
6.32 High-Performance Liquid Chroma-
tography ( HPLC)
Three HPLC systems were investigated (51) for
selective separation of captopril from pharmaceu-
tical excipients, synthetic intermediates,
degradation products and impurities. These
included anion exchange, amino, and
CAPTOPRIL 115

octadecylsilane (ODS) systems. The first and


second appeared not to be the systems of choice
when they did not achieve necessary separations of
pharmaceutical excipients from captopril. A
heavily loaded version of the third system ( 1 5 %
O D s , monolayer bonded on silica, Partisil ODS-2)
was selected as optimum for bulk and tablet
analysis. The third system was clearly superior.
Nevertheless, the first two systems had very
occasional usage when formulations contained
excipients which interfered with captopril or its
disulfide analysis in the heavily loaded C18
system.
The characteristics of these systems and of
several others investigated are summarized in
Table 8 below.
Since captopril has a U.V. absorption maximum
at about 2 0 0 nm with broad end absorption (see
Figure 1 5 ) U.V. detection was at 230 nm or lower,
compatible with a good baseline and low solvent
interference inherent in a good signal to noise
ratio. An alternative to U.V. detection, for
captopril per se (see Section 7.4 for captopril
U.V. derivative HPLC) is the electrochemical
detector (systems 8 and 9) introduced for sul-
fhydryl analysis by Saetre and Rabenstein ( 5 2 ) .
The electrochemical detector's near neutral
detection potential makes it especially thiol
selective. It has been reported (53) to have a
sensitivity at least 200 fold over that of U.V.
detection.
Fig. 15. U.V. Spectrum of C a p t o p r i l in Mobile Phase -
System 4 (Table 8)
Instrument: Beckman Acta C I11

116
Table 8

C a p t o p r i l HPLC S y s t e m s

System Flow Loop Remarks


No. Rate Injection and
ml/min Volume p 1 S t a t i o n a r y Phase Mobile Phase Detection Reference

1 0.6 20 250 x 4 . 6 mm 5 2 5 mg c i t r i c U.V. 51


P a r t i s i l SAX acid. H20 220 nm
10um S t r o n g +37.7mg N H 4 C I T
a n i o n Whatman to 1 liter with
CH30H a d j u s t t o
p H 3.30 w i t h U.V. 51
0.1 N HC1 220 nm

2 1.0 20 300 x 3 . 9 mm 0 . 0 1 M N a EDTA


u Bondapak NH2 i n 0.05% SAC- U.V. 51
1 0 um a m i n o CH3CN, 9 5 : 5 2 2 0 nm
Waters

3 1.0 20 250 x 4 . 6 mm CH,OH-H,O-85% U.V. 51


P a r t i s i l ODS 5% ~ ~ $ 20 7 : 7~
5: ' 2 2 0 nm
10um ODS mono- 0.1
l a y e r Whatman
System Flow Loop Remarks
No. Rate Injection and
ml/min Volume p 1 Stationary Phase Mobile Phase Detection Reference

4 1.0 20 250 x 4 . 6 mm CH OH-H 0-85% U.V. 51


Partisil ODS 15% H3$04, 3 0 :50: 220 nm Optimum
lOpm ODS mono 0.05 resolution
layer Whatman system
5 1.0 20 200 mm Hypersil U.V. 54
ODS +
TSIM 5um H go4, 47.5: 230 nm
ODS + trimethyl- 52.5:l. 0
silylimidazole
monolayers
Shandon
6 1.0 1.0 200 mm Hypersil CH OH-H 0 - 1 8 U.V. 54
(ml) ODS + TSIM 5pm H3$04, $3 :57 : 230 nm As say
ODS + trimethyl- 1.0 disulfide
silylimidazole in low
monolayers potency
Shandon captopril
formula-
tions
7 1.0 20 2 5 0 x 4 . 6 mm CH30H-(0.1 M Electrochem., 53
Partisil ODS 5 % KH PO + + 0.10 V , Hg
10pm ODS mono 152mMfi2P04) , Pool vs
layer Whatman 55% :456 Ag/AgCl
8 0.5 20 300 x 4.6 mm MC CH30H-(0.05 M Electrochem., 55
Chromegabond 209 KH PO + 30mM + O.lOV, Adapted
10um heavily H $0 f, Hg film for
loaded ODS 53% :15% vs. Ag/AgCl "total"
ES Industries urinary
captopril
analysis
9 1.0 20 250 x 4.6 mm CH OH-H 0-85% U.V. 56
Partisil ODS 5% H3d04, ?0:80: 214 nm
10pm ODS 0.1
Whatman
10 1-2 Waters 300 x 3 . 9 mm p CH C1 : HAc, U.V. 57
-
c
Variable Porasil 1Opm 9 T l2 240 nm Qua1 .
W
Volume silica gel nor- Sepn. of
mal phase Waters captopril
from its
R,S isomer
11 0.5-1 Waters 250 x 4.6 mm H O(to pH 3.0 U.V. 58
Variable Lichrosorb RP18 z6.5 M H3P04) 220 nm
Volume 5pm "capped" ODS - CH3CN,
Merck 65 : 35
12 100 -- 30 x 5.7 cm Prep- CH OH-H20-0.05 RI 59
PAK C18 ODS M dPO 45 : Prep.
Waters 55 ? 0?65 Sepn.
captopril
impurities
ODS = Octadecylsilyl
120 HAROLD KADIN

6.33 Gas Liquid Chromatography (GLC)


A c a p t ~ p r i l - ~synthesis
~c separated captopril
from its R,S optical isomer by GLC of its methyl
ester (after CH2N2 treatment) on 1.5% OV-17 at
18OOC (60). The gas chromatogram of the
pre-resolution isomer mixture showed two peaks of
almost the same peak area. After a dicyclohexy-
lamine resolution, GLC of the desired captopril
(S,S fraction) indicated a single peak at the
retention time of the second peak in the chromato-
gram of the mixture. The method is recommended,
over the more conventional, appreciably less
sensitive, measurement of optical rotations (Sec-
tion 4.34), for process control of the isomer
separation.
G.C.-mass spectrometry (Section 7.2) and
G.C.-flame photometry (Section 7.3) of the N-ethyl
maleimide adduct (Section 7 ) of captopril as
methyl and heptafluoroisopropyl esters, respec-
tively, have also been reported.
6.4 Titrimetric Methods
Acid iodate (Iodine) titration, before and
after chromatography through a Jones Reductor
Column was used to measure disulfide in captopril
(61). For small amounts of disulfide this
technique measures, relatively inaccurately, small
differences between two large numbers. Therefore
the subsequently developed, more accurate TLC
(Section 6.31) and HPLC (Section 6.32) direct
assays are preferred. However the iodine
titration, with starch indicator, is useful as a
thiol purity assay (61). A "Dead Stop" end point
indicator (62) makes the titration more amenable
to automation.
7. Analysis in Biological Fluids and Tissues
and in Animal Rations
Extreme instability of captopril in biologi-
cal media necessitated development (63) of an
immediate, quantitative thiol derivatization with
N- ethylmaleimide (NEM) followed by freezing.
Studies (64) with radioactive captopril in whole
blood had indicated that a 5 min delay before
addition of NEM resulted in a 10% l o s s of
CAPTOPRIL 121

captopril whereas a 30 min delay yielded a 65%


loss. The NEM derivative was found (65) to be
stable in frozen whole blood or in blood stored at
5OC for at least 3 months: it was unstable in a
frozen or 5OC phosphate buffer (pH 6 . 8 5 ) when
stored for more than 4 weeks.
Antioxidants and metal chelators were inef-
fective (63) as captopril stabilizers in whole
blood.
Long term stabilization, at the relatively
high concentrations of 1 to 100 mcg per ml pre-
valent in urine after captopril dosage, has been
achieved (40) with metal chelators, acid pH, and
5 O C storage (see Section 5.22).

Stability and homogeneity analysis of capto-


pril in animal feeds was required for multiple
long range toxicological studies. Despite a high
proportion of thiol reactive air, inorganics, and
proteins in the feeds, stabilization during the
analysis was achieved (46) by an isopropanolic
extractant fortified with trichloracetic acid and
metal chelators (see Section 7.71).
7.1 Thin Layer Radiochromatography (TLRC)
NEM stabilization allowed TLRC of captopril
in whole blood (63). Aliquots of whole blood were
analyzed for total radioactivity and NEM- treated
aliquots were extracted with methanol.
Reconstituted residues of the extracts were
applied to silica gel GF plates, developed with
chloroform/ethyl acetate/glacial acetic acid
(4:5:3) and analyzed for radioactivity associated
with captopril and its disulfide by zonal analy-
sis. The limit of detection was about 10 ng
captopril/ml of blood.
In TLRC analysis of the highly radioactive
captopril preparations used for drug metabolism
studies the thiol of captopril is particularly
susceptible to free radical degradation
(Section 4.2). Reaction of thiol across the
highly active carbonyl double bond of formaldehyde
(incorporated in the mobile phase) protects the
radioactive captopril during the chromatography
(66).
122 HAROLD KADIN

When dilute aqueous solutions of captopril


and an excess of formaldehyde are used the product
of the reaction has been shown by NMR and IR to be
the captopril hemithioacetal.

cp COOH

The hemithioacetal can be reverted to captopril,


for sulfhydryl analysis, by acid, base, or
bisulfite.
7.2 Gas Chromatography-Mass Spectroscopy
(GC-MS)
NEM-Captopril has been determined by GC-MS in
the selected ion mode (67) in whole blood, urine
(68), amniotic fluid (691, milk (69), and tissues
(68) (liver, kidney, lung, and placenta). For
determination in whole blood, proteins are
precipitated from vortexed whole blood plus
internal standard with freshly prepared 10%
metaphosphoric acid. The NEM-captopril is
absorbed from 0.45 pM membrane filtrates on
purified suction-dried Amberlite XAD-2 resin and
eluted with a freshly purified (neutral alumina
chromatography) ethyl acetate. The drug is
extracted from the ethyl acetate into 5% sodium
bicarbonate. After acidification and saturation
of the aqueous layer with sodium chloride, the NEM
derivative is extracted with ethyl acetate. After
evaporation of the solvent extract, samples are
methylated for GC-MS.
The G.C. was carried out with dried helium
gas at 6 ml/min on a 3 % OV-101 column with injec-
tor at 28OOC and column temperature programming at
200-280°C at 20°C/min. All glass surfaces had
been previously silanized with Sylon-CT. The
spectrometer was a modified Electronic Associates
Quad 300 quadropole. The samples (1 p l ) were
coinjected with 0.5 p 1 of N,O-bis(trimethylsily1)
trifluoroacetamide. The latter was used to
prolong column life. The peak height intensity
CAPTOPRIL 123

data of the m/z 230 and 248 ions was collected by


selected ion monitoring. The limit of detection
and the practical detection limit with a 90%
confidence limit are 5.5 and 16.5 ng per ml of
blood, respectively.
The internal standard, an NEM blocked,
deuterated or fluorinated derivative of captopril,
compensates for extraction and other possible
losses.
The procedure described (67) could be applied
to most of the biological media mentioned with but
slight revision. However milk required a greater
modification. The high fat content of milk made
XAD-2 chromatography unfeasible. Therefore
NEM-captopril was instead extracted from sodium
chloride saturated milk samples with ethyl
acetate.
7.3 Gas Chromatography-Flame Photometric
Detection (GC-FPD)
A GC method for captopril in blood and urine
utilizing a sulfur selective dedicated FPD has
been reported (70). Blood is treated with a s o h .
of N-ethylma1eimi.de in phosphate buffer s o h (pH
7.4) and with metaphosphoric acid (10% soln);
after centrifugation, the supernatant soln. is
extracted with ethyl acetate, and, after further
purification, the extract is evaporated, the
captopril-N-hexylmaleimide adduct is added as
internal standard, and the mixture is treated to
convert the two adducts into their
hexafluoroisopropyl esters for analysis by g.1.c.
at 215O on a glass column (1 m x 3 mm) packed with
2 % of OV-210-yn Gas-Chrom Q (80 to 100 mesh), with
N (50 ml min ) as carrier gas and
flame-photometric detection. Urine is analysed
similarly, but without deproteinisation. The
calibration graph (peak-height ratio vs. concn.)
is rectilinear for 1 to 5 ug ml- of I.
The sensitivity though not specified appears
to be in the microgram range. It is therefore not
satisfactory for human blood studies which are in
the nanogram range.
124 HAROLD KADIN

7.4 High Performance Liquid Chromatography


with U.V. Detection (HPLC-UVD)
An HPLC procedure for captopril in whole
blood and urine has been reported (71). Captopril
is thiol-blocked with p-bromophenacyl bromide
(~BPAB)or N-(4-dimethylamino-3,5-dinitrophenyl)
maleimide (DDPM), then the addition products were
separated and determined by HPLC on a reversed
phase column. Captopril in blood or urine could
be derivatized with pBPAB then excess pBPAB
removed by hexane extraction. The captopril
disulfides in the same samples were then reduced
by tributylphosphine to captopril which in turn
were thiol-blocked with DDPM. HPLC of the adduct
mixture thus allowed a separate analysis of
captopril or disulfides, before and after
reduction. The method is reported to be
sensitive, and precise down to 5 ng per ml of
whole blood and 0.1 mcg per ml of urine.
7.5 Spectrofluorometry
The drug, stabilized with dithioerythritol is
absorbed on a Brinkmann XAD-2 column from an
acidified diluted blood and eluted with pH 6.9
phosphate-30% dimethylformamide buffer. Captopril
in the eluate is reacted with
N-(7-dimethylamino-4-methyl coumarinyl) maleimide
(DACM) to form a fluorescent derivative (Section
6.22). After acidification, the derivative is
extracted into toluene. The fluorescence is
measured at 375 nm/425 nm-excitation/fluorescence.
The analysis was validated by the GC-MS procedure
described above in the range of 0.5 mcg to 10 mcg
per ml of blood (72).
7.6 Radioimmunoassay (RIA)
Development and application of a simple,
highly sensitive RIA for NEM-captopril in blood
plasma has been described (89). The lower
detection limit is 2 ng per ml of plasma.
CAPTOPRIL 125

7.7 Semiautomated Ellman Colorimetry,


(Figure 16)
7.71 Semiautomated Sulfhydryl Analysis
in Laboratory Animal Rations
Feeds were extracted with isopropanol
fortified with trichloroacetic, ethylenediamine
tetraacetic, and diethylenetetramine pentaacetic
acids. Pigments in the initial extract and
turbidity on aqueous dilution necessitated a
cleanup using isooctane after aqueous dilution of
the extract. Further aqueous dilution of the
isopropanol-water extract insolubilized trace
isooctane and more polar lipids. Finally
centrifugation in open top tubes removed the last
traces of isooctane. In the design of the
colorimetry from manual colorimetry ( 7 3 ) the
sequence of adding Ellman's reagent before the
alkali was reversed to maintain solubility of
Ellman's within the autoanalyzer lines. In
addition, methanol and aqueous buffer were added
to Ellman's reagent to enhance both stability and
solubility. Finally the pH of Ellman's reaction
was maintained at pH 8.5 rather than at its
optimum pH 7 (Section 6 . 2 1 ) to dissolve trace
protein haze. Due to the limited selectivity of
the colorimetry it was necessary to analyze for
and subtract the response of drug- unfortified
(blank) feed. The cleanup served to minimize this
blank such that captopril could be analyzed down
to 0.03% in a variety of animal feeds with good
accuracy.
7.72 Semiautomated Ellman Colorimetry
for Urinary Captopril and Its
Disulfides
The necessity for long term storage of
samples prior to analysis and the presence of an
oxidation-prone thiol of captopril required
development of an acid-chelate stabilization
method for urinary captopril (Section 5.22). Thin-
layer radiochromatography (Section 7 . 1 ) indicated
that human urinary captopril was primarily free
(unchanged) and, in almost equal proportion,
126 HAROLD KADIN

disulfide-conjugated with cysteine ( 7 4 ) . Rela-


tively small amounts of captopril disulfide were
observed. An electrochemical reduction (52) was
utilized to release disulfide-conjugated captopril
for thiol colorimetry. Of several rugged reduc-
tion cells evaluated, one with a VycorB Disc
separating the anode and the mercury pool cathode
was preferred. Methylene chloride partitions from
acidified salt-saturated urines, before and after
reduction, allowed the measurement of free and
disulfide-conjugated captopril. The drug
partitioned into the solvent whereas the aqueous
phase retained acid protonated, amino
group-bearing thiols like cysteine. Subsequent
solvent evaporation volatilized other potential
colorimetric interferences. An automated colori-
metry of 25 samples per hour was developed by
utilizing relatively thiol selective Ellman's
Reagent, 5,5'-Dithio-Bis-(2-Nitrobenzoic Acid)
(Sections 6.21 and 7.71). Results were confirmed
by an HPLC method with electrochemical detection,
developed while this method was in use (42).
7.8 High Performance Liquid Chromatography
with Electrochemical Detection (HPLC-
-
ECD)
Captopril in urine has been determined by an
HPLC-ECD procedure in which the drug is separated
from interferences on a monolayer bonded, heavily
loaded (about 20% carbon) octadecylsilane reverse-
phase column with a mobile phase of 55% methanol
and 45% aqueous phosphate buffer (55). A commer-
cially available thin layer mercury film detector
was used (Section 6.32). The anodic current,
resulting from the oxidation of Hg to Hg-captopril
complex, was a linear function of captopril con-
centration. The peak height was reproducible with
a relative standard deviation of 0.54% (n=5).
Captopril concentration as low as 0.5 mcg per ml
of urine can be quantitated. Sample treatment
involved simply centrifugation, filtration, and
deoxygenation with nitrogen. Recovery of capto-
pril from urine spiked at 2.0 mcg per ml was
better than 95%.
CAPTOPRIL 127

TECHNICON AUTOANALYZER
FOR ELLMAN COLORIMETRY
WASTE

P r
IFLOW CELL I
COLOR I M ETER
RECORDER

WASTE

NOMINAL
- FUNCTION
ML/MIN
~~

YELLOW HCI WASH 1.2


2 MIN
MIXING YELLOW' CELL 1.2
c 01LS
YELLoW' ,TEA-EDTA 1.2
J GREY
4 , AIR 1.o

HCI DIL 0.8

ELLMANS 0.8

WASTE
* = SOLVAFLEX SOLVENT TECHNICON
RESISTANT TUBING, SAMPLER I I
OTHER TUBING TYGON WITH 50 2/1 CAM
ELLMAN'S =
TEA-EDTA 0.08% ELLMAN'S
= 1.86% EDTA Nao 2H20 + IN 50% MEOH (COLD)
2Ooh TRIETHANOLAMINE + + 0.01 M
0.02% TWEEN 80 ACETATE pH 4.7

Figure 16
128 HAROLD KADIN

7.9 High-Performance Liquid Chromatography


with Fluorescence Detection (MPLC-FD)
Jarrott et a1 (75) describe an assay for
quantitating plasma captopril levels. Blood from
patients taking this drug was collected into tubes
containing edetate and ascorbic acid, and the
plasma was separated by centrifugation. After
addition of an internal standard, the plasma was
deproteinized and the supernate was adjusted to pII
6.5. N-(l-Pyrene)-maleimide was added to
derivatize captopril and an internal standard to
fluorescent adducts. These derivatives then were
extracted into ethyl acetate-benzene (1:l) and
separated from other derivatized thiols by high-
performance liquid chromatography. The sensi-
tivity of the assay was 150 pmoles/ml. The HPLC-
FD uses a mobile phase of methanol-potassium
phosphate buffer (5 mM, pH 6.5) (52:48) and the
flow rate was 2 ml/min through a radially com-
pressed octadecylsilane 10 cm x 8 mm (i.d.1 column
and a spectrofluorometer with a 20 p 1 flow cell.
Excitation was at 340 nm with emission taken at
390 nm.
8. Drug Metabolism - Pharmacokinetics
Two features salient to the metabolism and
pharmacokinetics of captopril include a) the
reversible transformation of its thiol function to
dimer and mixed disulfide (74,76-79) and b) the
biological stability of its amide linkage (80,
Section 3). The following will emphasize these
aspects.
8.1 Blood Level Studies
Captopril is absorbed rapidly as indicated by
measurable blood levels of the drug 15 min after
ingestion (81). These findings are compatible
with reports of the onset of antihypertensive
activity as soon a s 15 min after a single oral
dose (3) in hypertensive patients and with the
rapid onset of blockade of angiotensin I-induced
increases in blood pressure in healthy subjects
(91). Other studies revealed that the captopril
metabolites included its dimer disulfide and the
mixed disulfides with glutathione, cysteine, and
serum albumin (78,811 .
CAPTOPRIL 129

After single oral dosage with 100 mg


radioactive captopril, ten healthy subjects
attained a mean maximal concentration ( C ) in
blood of 800 + 76 ng/ml at a mean time (Fax) of
0.93 + 0.08 hours (8l,82). Methanol extr%%.on,
ultrariltration, and dithiothreitol reduction
studies (77) coupled with TLRC (Section 7.1)
established that about 5 0 % of the total blood
radioactivity was unchanged captopril, about 10%
dimer disulfide and the remainder other polar
metabolites including protein and non-protein
mixed disulfides (81,82). Thereafter the
captopril fraction of total radioactivity declined
rapidly with time whereas the mixed disulfide
fraction increased. The curvilinearity of
semilogarithmic plots of these captopril blood
levels with time indicated that half-life (t 1 ,
values could not be accurately calculated. #or
unchanged captopril levels, curvilinearity may be
due to the mixed disulfide fraction acting
reversibly to replenish captopril, possibly
extending the duration of pharmacologic activity
(34,76-79). However plots of total radioactivity
allowed calculation of t of 4.3 + 0.2 hours for
the 4-12 hour time inter$al and 1g.4 + 2.6 hours
for the 12-48 hour time interval (81): Mean
concentrations of total radioactivity in blood
(expressed as captopril equivalents) were
41 + 7 ng/ml at 24 hours and 20 + 5 ng/ml at
48 fiours after drug administration.

Blood level and urinary excretion studies in


6 hypertensive patients, dosed over a 10 day
perigd, indicated that the metabolic disposition
of C-captopril was the same at the beginning
and end of the study, and was comparable to the
disposition in healthy subjects (83).
Radiolabeled captopril was administered on days 1
and 10, and blood and urine samples were analyzed
for captopril and its metabolites by radiometric
assay procedures. Non-radioactive drug (100 mg
t.i.d.1 was used for dosage on Days 3 to 9.
8.2 Urinary Excretion Studies
In the fasting state, absorption averaged
7 0 - 7 5 % of an oral dose, as evidenced by human
urinary excretion studies (81,82,84). The
presence of food in the gastointestinal tract
130 HAROLD KADIN

reduces absorption of an oral dose by about 35 to


40% (84).
At least 95% of the radioactivity recovered
in urine after a 100 mg oral dose of radiolabelled
captopril to mildly hypertensive patients was
accounted for as captopril and specific urinary
metabolites. Captopril and captopril-cysteine
mixed disulfide each accounted for about 45% of
the urinary radioactivity (about 3 3 % of the dose)
and the disulfide dimer accounted for about 5% of
the radioactivity in urine (about 4% of the dose)
(74) .Additional minor urinary metabolites, in
rats and dogs, are S-methyl captopril,
captopril-S-methyl sulfoxide, N-acetyl
cysteine-captopril and glutathione-captopril mixed
disulfides (80). These minor metabolites have
not, as yet, been reported in man, but appear to
be present in monkeys (90).
Renal dysfunction, as measured by creatinine
clearance, decreased the excretion rate of
captopril after a single 100 mg oral dose (85).
The authors suggest a method of dosage reduction,
based on creatinine clearance measurements, in
cases of moderate to severe renal dysfunction.
8.3 Miscellaneous Distribution Studies
Biological stability of the amide function
was confirmed by studies (80) on rat dermal
collagen; uptake of radioactive proline was
considerable, whereas insignificant uptake of
captopril labelled in the proline moiety was
observed.
Whole body radioautography of rats indicated
that captopril ( 5 0 mg/kg-intravenously) did not
readily enter the central nervous system whereas
it readily entered the placenta of pregnant rats
(86)
Captopril did not readily enter the breast
milk of lactating women dosed with captopril at
100 mg t.i.d. for 7 days (87,88). Relatively
insignificant milk peak levels were found (4.7
ng/ml)
CAPTOPRIL 131

9. Acknowledgments
I would like to express my appreciation to
individuals who have been very helpful for the
contributions indicated: Drs. J. Fried and M.
Porubcan - NMR, Drs. Y.J. Wang and T. Prusik -
Stability, Mr. A. Restivo and Mr. D. Domina -
Synthesis and Solubility, Drs. A. Cohen and P.
Funke - MS and GC-MS, Ms. M. Malley and Dr. J.
Gougatas - Single Crystal X-Ray, Mr. F. Dondzila,
Mr. S. Perlman and Dr. J. Kirschbaum - HPLC, Mr.
H. Roberts - TLC, Mr. R. Poet and Dr. G. Brewer -
Proof Reading and Manuscript Clarity, Ms. D.
Walker - Word Processing, Dr. D. Cushman -
History, Drs. B. Migdalof and D. McKinstry - Drug
Metabolism - Pharmacokinetics.
10. References
1. Ondetti, M.A., Williams, N.J., Sabo, E.F.,
Pluscec, J., Weaver, E.R.; and Kocy, O.,
Biochemistry, -
10, 4033 (1971).
2. Gavras, H., Brunner, H.R., Laragh, J.H.,
Sealey, J.E. Gavras, I., and Vukovich, R.A.,
New Eng. J. Med., 291, 817 (1974).
3. Case, D.B., Atlas, S.A. Laragh, J.H., Sealey,
J.E., Sullivan, P.A., and McKinstry, D . N . ,
Progr. Cardiovasc. Dis.; 21, 195 (1978).
4. Cushman, D.W. and Cheung, H.S., Biochem.
Pharmac., -
20, 1637 (1971).

5. Rubin, B., Laffan, R.J. Kotler, D.G.,


O'Keefe, E.H., DeMaio, D.A., and Goldberg,
M.E., J. Pharmacol. Exp. Therap., -
204, 271
(1978).

6. Quiocho, F.A. and Lipscomb, W.N., Adv.


-
Protein Chem., 25, 1 (1971).
7. Byers, L.D. and Wolfenden, R., Biochem., 12,
2070 (1973).
8. Cushman, D.W., Cheung, H.S., Sabo, E.F. and
Ondetti, M.A., Biochemistry, 16, 5484 (1977).
132 HAROLD KADIN

9. Cushman, D.W., Cheung, H.S., Sabo, E.F., and


Ondetti, M.A., Fed. Proc., -38, 2778 (1979).

10. Cushman, D.W., Pluscec, J., Williams, N.J.


Weaver, E . R . , Sabo, E.F., Kocy, O., Chueng,
H.S., and Ondetti, M.A., Experientia, - 29,
1031 (1973).

11. Ondetti, M.A., Rubin, B., and Cushman, D.W.,


Science, -
196, 441 (1977).

12. New Drug Application No. 18-343 Submitted by


E . R . Squibb & Sons Inc., Approved April 6,
1981.

13. Levine, T.B., Franciosa, J.A., Cohn, J.N.,


Circulation, -
62, 35 (1980).

14. Ondetti, M.A. and Cushman, D.W., United


States Patent 4,105,776, August 8, 1978.
15. Toeplitz, B., Personal Communication, April,
1976.

16. Porubcan, M.A., Personal Communication, June,


1981.
17. Whigan, D.B., Personal Communication, April,
1976.

18. .
Karchmer , J H. , "Treatise on Anal. Chem. ' I ,
Part 11; Volume 13, Page 410 (1966), Edited
By Kolthoff, I.M. and Elving, P.J., John
Wiley Publishers.
19. Ondetti, M., Personal Communication, February
1976.
20. Puar, M.S. and Funke, P.T., Personal
Communication, February, 1976.
21. Kadin, H., Personal Communication, March,
1976.

22. Valenti, V., Personal Communication, March,


1976.

23. Wadke, D.W., Personal Communication, March,


1976.
CAPTOPRIL 133

24. Karten, J. and Kabadi, B., Personal


Communication, July, 1980.
25. Malley, M. and Gougatas, J.Z., Personal
Communication, May, 1976.
26. Jacobson, H. and Ochs, Q . , Personal
Communication, May, 1976.
27. Weiss, A.L. and Wang, Y.J., Personal
Communication, February, 1980.
28. Wang, Y.J., Personal Communication, December,
1980.
29. Weiss, A.L., Personal Communication, March,
1980.
30. Weiss, A.L. Personal Communication, May,
1976.
31. Benesch, R. and Benesch, R., J. Am. Chem.
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SOC. - 5877 (1955).
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T., Ogura, T., Nakamura, Y., and Kawaguchi,
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Chem. Pharm. Bull., -
33. Jocelyn, P.C. , "Biochemistry of the
Sulfhydryl Group", Page 344 (19721, Academic
Press.
34. Dondzila, F., Personal Communication, May,
1981.
35. Shipkowski, E.R., Personal Communication,
October, 1980.
36. Roberts, H.R., Personal Communication, May,
1977.
37. Timmins, P., Personal Communication, January,
1980.
38. Poet, R., Personal Communication, May, 1980.
39. Hanaki, A. and Kamide, H., Chem. Pharm.
Bull., 26, 325 (1978).
134 HAROLD KADIN

40. Kadin, H., Poet, R.B., "Sequential


Electrochemical Reduction, Solvent Partition
and Automated Thiol Colorimetry for Urinary
Captopril and its Disulfides", J. Pharm.
Sci., In Press.
41. Chas Pfizer and Company, New York, "Chelating
Agent Usage Calculator".
42. Kadin, H., Personal Communication, March,
1979.
43. Wang, Y.J., Personal Communication,
September, 1979.
44. Ellman, G.L. and Lysko, H., J. Lab. and Clin.
Med., 70, 518 (1967).
45. Poet, R., Personal Communication, February,
1978, July 1979.
46. Kadin, H., Personal Communication, October,
1978.
47. Lidell, H. and Saville, B., Analyst, -
84, 188
(1959).
48. Whigan, D.B., Personal Communication,
January, 197 9.
49. Valatin, P., Personal Communication, July,
1980, Sept., 1980.

50. Roberts, H., Personal Communication, January,


1978, August, 1979.
51. Perlman, S. and Kirschbaum, J., J. Chromat.,
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206, 311 (1981).
52. Saetre, R. and Rabenstein, D.L., Anal. Chem.,
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50, 276 (1978).
53. Yeh, P., Abstracts, No. 60, Page 54, Eastern
Analytical Symposium, New York, N.Y.,
November, 198 0.
54. Tenneson, M.E., Personal Communication,
February-March, 1980.
CAPTOPRIL 135

55. Yeh, P., Personal Communication, October,


1980.
56. Perlman, S., Personal Communication, April,
1980.
57. Dondzila, F., Personal Communication, May,
1979.
58. Dondzila, F., Personal Communication, May,
1981.

59. Dondzila, F., Personal Communication,


December-January, 1980.
60. Hasegawa, M., Ohyabu, S., Ueda, T., and
Yamaguchi, M., J. of Labelled Compounds and
Radiopharmaceuticals, -
18 I 643 ( 1981).
61. Whigan, D.B., Willis, S.L., and Jenkins,
E.J., Personal Communication, October, 1976.
62. Rohmer, M., Personal Communication, February,
1980.
63. Migdalof, B.H., Singhvi, S.M., and Kripalani,
K.J., J. Liquid Chromat., 3(6), 857 (1980).
64. Yeh, P. and Roberts H., Personal
Communication, July, 1979.
65. Ivashkiv, E. and Roberts, H., Personal
Communication, July, 1978.
66. Egli, P., Personal Communication, October,
1979.
67. Funke, P.T., Ivashkiv, E., Malley, M.F., and
Cohen, A.I., Anal. Chem., -
52, 1086 (1980).
68. Ivashkiv, E., Personal Communication,
March-April, 1980.
69. Ivashkiv, E., Personal Communication, August,
1979.
136 HAROLD KADIN

70. Matsuki, Y., Fukuhara, K., Ito, T., Ono, H.,


Ohara, N . , and Yui, T., J. Chromat., -
188, 177
(1980).
71. Kawahara, Y., Hisaoka, M., Yamazaki, Y.,
Inage, A., and Morioka, T., Chem. Pharm.
-
Bull., - 150 (1981).
- 29(1),
72. Ivashkiv, E., Personal Communication, August,
1980.
73. Kadin, H., Personal Communication, January,
1977.
74. Kripalani, K.J., Meeker, F.S., Dean, A.V.,
McKinstry, D . N . , and Migdalof, B.H., Fed.
Proceedings, - 39, 307 (1980).

75. Jarrott, B., Anderson, A., Hooper, R. and


Louis, W.J., J. Pharm. Sci., -
70, 665 (1981).

76. Komai, T., Ikeda, T., Kawai, K., Kameyama,


E., Shindo, H., J. Pharm. Dyn., -
4, 677
(1981).
77. Wong, K.K., Lan, S.J., and Migdalof, B.H.,
Biochem. Pharmacology, -
30, 2643 (1981).

78. Migdalof, B.H., Wong, K.K., Lan, S.J.,


Kripalani, K.J. and Singhvi, S.M., Fed.
Proc.,
- --39, 757 (1980).
79. Shindo, H., Komai, T., Ikeda, T., Kawai, K.,
Kawamata, W., and Kameyama, E., J.
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Pharmacobiodyn., -
30, S-9 (1980).

80. Ikeda, T., Komai, T., Kawai, K., and Shindo,


H., Chem. Pharm. Bull., -
29(5),
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81. Kripalani, K.J., McKinstry, D . N . , Singhvi,


S.M., Willard D . A . , Vukovich. R.A. and
Migdalof , B. H. , Clin. Pharmacol. and Therap. ,
27,
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82. McKinstry, D . N . , Singhvi, S.M., Kripalani,
K.J., Willard, D.A., and Migdalof, B.H.,
"Recent Advances in Hypertension Therapy:
Captopril", Excerpta Medica, Page 3, (1981),
Amsterdam-Oxford-Princeton.
CAPTOPRIL 137

83. McKinstry, D.N., Kripalani, K.J., Migdaloff,


B.H., Willard, D.A., Clin. Pharmacol. &
Ther.. 2 7 . 2 7 0 ( 1 9 8 0 ) .
84. Singhvi, S.M., McKinstry, D.N., Shaw, J.M.,
Willard, D.A., and Migdalof, B.H., J. Clin.
Pharmacol. (in press).
85. Rommel, A. J. Pierides, A.M., and Heald, A.
Clin. Pharmacol. Ther., -
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86. Heald, A.F. and Ita, C.E., Pharmacologist,


19,
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Pharmacol. & Ther., -
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88. Devlin, R.G. and Fleiss, P.M., J. Clin


Pharmacol. , -
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89. Pearson, F.M., Martin, V.I., Aldujaili,


E.A.S., Williams, B.C., and Edwards, C.R.W.,
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90. Migdalof, B.H., Personal Communication,


January, 1 9 8 2 .
91. Ferguson, R.K., Brunner, H.R., Turini, G.A.,
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I,
775 I (1977) .
11. Review Coverage Dates
This review summarizes communications up to
June 10, 1 9 8 1 . However Sections 2 and 8 are'
updated to about January, 1 9 8 2 .
CEFOTAXIME

Farid J . Muhtudi and Mahmoud M . A. Hassan

1 Description 140
1.1 Nomenclature 140
1.2 Formulae 140
1.3 Molecular Weight 142
1.4 ElementalComposition 142
1.5 Appearance, Color, Odor, and Taste 142
2. Physical Properties 142
2.1 Solubility 142
2.2 Moisture Content 142
2.3 pH Range 142
2.4 Optical Rotation 142
2.5 Spectral Properties 142
3. Synthesis of Cefotaxime 152
4. Metabolism 152
5. Pharmacokinetics 156
6. Microbiological Activity 156
7. Methods of Analysis 159
7.1 Identification Tests 159
7.2 Non-Aqueous Titration 159
7.3 Chromatography 159
7.4 Spectrophotometry 165
7.5 Microbiological Assay 166
8. References 167

Analytical Profiles of Drug Substances Copyright 0 1982 by The American


Volume I I 139 Pharmaceutical Association
ISBN &12-260811-9
140 FARID J . MUHTADI AND MAHMOUD M. A. HASSAN

1. Description

1.1. Nomenclature

1.1.1 Chemical Names

(a) Sodium 7-[2-(2-amino-4-thiazolyl)-2-


m e t hox y iminoa c e tam i d o ] c epha 1o s po r a n a t e .
(b) Sodium 3-acetoxymethyl-7-[ 2-(2-amino-
4- t h i a z o 1y 1) -2- m e t hox y i m i n o ] a c e t am id0 ]
-3-cephem-4-carboxyla te .
(c) (6 R-trans)-3-[ (Acetyloxy) methyl]-7-[ [
(2-amino-4-thiazolyl) (methoxy- imino)
a c e t y 11 amino]- 8-0xo-5 - t h ia-1 -azab i c yclo
[ 4 . 2 . 0 ] oct-2-ene-2-carboxylic acid
monosodium s a l t .
(d) (6-R, 7R)-7-[ 2-(2-Amino-4-thiazolyl)
glyoxylamido] -3-(hydroxy methyl) -8-0x0-
5-thia-1-azabicyclo [ 4 , 2 , 0 ] oc t-2-ene-
2-carboxylic a c i d a-(O-methyloxime),
a c e t a t e ( e s t e r ) monosodium s a l t .
(e) 5-Thia-1-azabicyclo [ 4.2.01 oc t-2-ene
2-2 c a r b o x y l i c a c i d , 3 - [ ( a c e t y l o x y )
methyl] -7-- [ [ (2-amino-4-thiazolyl)
(me thox y i m i n o ) y a c e t y l amino ] -8 -0xo- ,
[6R-[6a,78(Z)II-

1.1.2 Generic Name

Cefotaxime sodium; HR 756; RU-24,662;


RU-24,756

1.1.3 P r o p r i e t a r y Names

C l a f o r a n ; Primafen; Z a r i v i z ; T a r i v i d .

1.2. Formula

1 . 2 . 1 Empirical C16H16N507S2Na
1.2.2 Structural
m
?2
V
I
0 =. v
I
0
I
hl
X
V
142 FARID J . MUHTADI AND MAHMOUD M. A. HASSAN

1 . 2 . 3 CAS no.

[ 611846-23-31 (1) C16H16N507S2Na


[ 60846-21-11 (1) C H N 0 S (as f r e e acid)
16 1 7 5 7 2
[63527-52-61 (2)

1.3. M o l e c u l a r Weight
477.23
1.4. E l e m e n t a l Composition
C, 40.23;; H , 3.38%; N , 14.67%;
0, 23.47%; S , 13.44%; Ma 4.82%.
1.5. Appearance, C o l o r , Odor and T a s t e :
White t o creamy w h i t e c r y s t a l l i n e powder,
o d o r l e s s and h a s a s a l t y t a s t e a t the b e g i n n i n g ,
f o l l o w e d by b i t t e r n e s s.
2. Physical Properties
2.1. Solubility
F r e e l y s o l u b l e i n water ( 0 . 5 g s o l u b l e i n 5 ml) ( 3 ) ,
s l i g h t l y s o l u b l e i n alcohol ( a b s o l u t e , 95%),
i n s o l u b l e i n chloroform (4)
2.2. M o i s t u r e C o n t e n t
Not more t h a n 6 p e r c e n t , d e t e r m i n e d by t h e Karl
F i s c h e r method, u s i n g a 0.2 g sample d i s s o l v e d
i n 2 m l methanol ( 3 ) .
0
Loss on d r y i n g a t 60 C u n d e r vacuum i n t h e
p r e s e n c e o f phosphorus p e n t o x i d e f o r 4 h r .
s h o u l d n o t exceed 6 % ( 3 ) .
2.3. pH r a n g e
The pH of c e f o t a x i m e a s a 10%a q u e o u s s o l u t i o n
i s 4 . 5 t o 6.5 d e t e r m i n e d p o t e n t i o m e t r i c a l l y ( 3 ) .
2.4. O p t i c a l R o t a t i o n
0
[ a]D ( c = l % aqueous s o l u t i o n ) + 58 t o + 64
(on d r i e d b ases) (3).
The o p t i c a l r o t a t i o n of c e f o t a x i m e ( c = l %
aqueous s o l u t i o n ) was d e t e r m i n e d u s i n g a
P e r k i n E l m e r 2 4 1 MC P o l a r m a t i c and found t o be:

[ a~D240 + 59.30.
CEFOTAXIME 143

2.5. Spectral Properties


2.5.1 Ultraviolet Spectra
The UV s p e c t r a o f c e f o t a x h e i n a n aqueous
s o l u t i o n and i n 0 . 1 N H C 1 a r e p r e s e n t e d i n
Fig. 1 and F i g . 2 r e s p e c t i v e l y . These were
scanned from 200 t o 400 nm u s i n g a Pye-
UnicumSP 8-100 U l t r a v i o l e t s p e c t r o -
photometer. The f o l l o w i n g t a b l e 1 shows
t h e UV d a t a .
Table 1. UV c h a r a c t e r i s t i c s of cefotaxime

solvent c onc en t r a t i o n max nm. E (lX, lcm)

water 0.5 mg/ml 234


0.1N R C l 0.05 mg/ml 205,262
0.1N HC1 0.01 mg/ml 263 420 (5)

2.5.2 Infrared Spectra


The I R s p e c t r a of c e f o t a x i m e a s KBr-disc
and a s n u j o l mull a r e shown i n F i g . 3 and
Fig. 4 r e s p e c t i v e l y . The KBr-disc w a s
recorded on a P e r k i n E l m e r 58OB i n f r a r e d
s p e c t r o m e t e r . The s t r u c t u r a l a s s i g n m e n t s
have been c o r r e l a t e d w i t h t h e f o l l o w i n g band
f r e q u e n c i e s (Table 2).
Table 2. I R c h a r a c t e r i s t i c s of c e f o t a x i m e
-1 Assignment
Frequency Cm

3420 -NH2
3340 (broad) -NH, -NH2
2940 -S-CH2

1 7 60 -C=O lactam 0
II
1730 -C=O e a r b o x y l i c , O-C-CH3
0
11
1650 -C-NH
0
1620 -C-NH,-C=N-,-C=C-
0
1540 -C-N-
1385-13 55 -0-CO-CH3

1180 C=O i n e s t e r
1050 C-0 stretching
230
240
250

304

350

400

Fig.1 The UV spectrum of Cefotaxtme in water

200
210

250
2 60
270

300

350

400

Fig.2 The UV spectrum o f C e f o t a x i m e in O . I N H C I .

144
3.0 40 5.0 MICRONS 6.0 70 8.0 9.0 10 12 14

I
4000 3500 3000 2500 2000 1800 1600 1400 1200 1000 1)oo
8
CD
0
0
a0
0
0
0
4
0
0
2
0
0
$
0
0
'0,
0
0
2
0
0
0
cu
0
0
v)
cy
a
0
0
c3
0
0
In
rn
CEFOTAXIME 147

2.5.3 Nuclear Magnetic Resonance S p e c t r a

2.5.3.1 Proton SDectra

The PMR s p e c t r a of c e f o t a x i m e i n
d e u t e r a t e d d i m e t h y l s u l f o x i d e (DMSOD6) and
d e u t e r i u m o x i d e w e r e r e c o r d e d on a v a r i a n
T-60A, 60-MHZ NMR s p e c t r o m e t e r u s i n g sodium -2,
2-dimethyl-2-silapentane-5-sulfonate (DSS) a s a n
i n t e r n a l r e f e r e n c e . These a r e shown i n F i g . 5
and F i g . 6 r e s p e c t i v e l y .
The f o l l o w i n g s t r u c t u r e a s s i g n m e n t s h a v e been
made ( T a b l e 3 ) ( 6 ) .

T a b l e 3. PMR c h a r a c t e r i s t i c s of c e f o t a x i m e

Group Chemical Sh Et ( P P )
DMSQD6
D2°

-0co CH, 2.00(s) 2.12(s)


2-G2 3 . 3 3 (9) 3 . 5 3 (9)
N - E 3 3 . 8 3 (s) 4.00(s)
0-CH
-3
AC . 4.97(9) 4.83 ( d )
6-H 4.97(q) 5.20(d)
7-H 5.60(2d) 5.82 ( d )
5-H 6.70(s) 6.97 (s)
2-Nl12 7.22 (bs) -
CONH
- 9.47 ( d )

s = s i n g l e t , d=doublet, q = q u a r t e t , 2d=doublet
of d o u b l e t s , bs=broad s i n g l e t .
13
2.5.3.2 C-NMR
13
C-NMR c o m p l e t e l y d e c o u p l e d and o f f r e s o n a n c e
s p e c t r a a r e p r e s e n t e d i n F i g . 7 and F i g . 8
respectively. Both were r e c o r d e d o v e r 5000 H,
r a n g e i n d e u t e r i u m o x i d e ( c o n c . 100 m g / l m l D20)
148
0
149
150 FARID J. MUHTADI AND MAHMOUD M . A. HASSAN

4 a
1
I

-TTJ_LLl- -J ' I ' -I


'_J _ ' ' '-11 I rI ' I '

F i g . 7. The 1 3 C NMR of Cefotaxime, completely decoupled


spectrum .

'I
I I II

Fig. 8 . The 1 3 C NMR of Cefotaxime, o f f resonance (Proton


coupled) spectrum
CEFOTAXIME 151

on FT-80A-8OMHz NMF. s p e c t r o m e t e r , u s i n g 1 0 mm sample


t u b e and d i o x a n e a s a r e f e r e n c e s t a n d a r d a t 2OoC.
T h e c a r b o n chemical s h i f t a r e a s s i g n e d on t h e b a s i s
of t h e a d d i t i v i t y p r i n c i p a l s and t h e o f f r e s o n a n c e
s p l i t t i n g p a t t e r n ( T a b l e 4) ( 6 ) .

12
NOCH3

H2N !?1 6
OCCH3
15

COONa
13

T a b l e 4. Carbon Chemical s h i f t s o f c e f o t a x i m e

Carbon no. Chemical s h i f t Carbon Chemical s h i f t


[ PPm 1 no. [ PPm 1
8-C=0 174.69(s) C-3 117.37 ( S )
1o-c=0 171.25(s) C-5 113.64 (d)
13-C=0 168.78 ( s ) c-7 65.07 (d)
15-C=0 165.08( s) C-6 59.59(d)
,
c-2 164.65(s) C-14 58.23 ( t )
c-11 148.62 (s) c-2 26.35( t )
,
c-4 141.40( s) C-12 63.55 ( 4 )
c-4 132.42 (s) C-16 21.22 ( 4 )

s=singlet, d=doublet, t = t r i p l e t , q=quartet.


152 FARID J. MUHTADI AND MAHMOUD M. A. HASSAN

3. S y n t h e s i s of Cefotaxime

Cefotaxime i s a s e m i - s y n t h e t i c c e p h a l o s p o r i n . The
s t a r t i n g material f o r s u c h c e p h a l o s p o r i n s i s 7-aminoceph-
a l o s p o r a n i c a c i d (7-ACA) which o b t a i n e d e i t h e r by mild
a c i d o r enzymatic h y d r o l y s i s of c e p h a l o s p o r i n C . ( 7 , 8 , 9 , 1 0 )

P r e p a r a t i o n of t h e S i d e Chain: (5)

The s t a r t i n g m a t e r i a l e t h y l a c e t o a c e t a t e [ l ] w a s o x i -
mated t o produce oximinoethylacetoacetate [ 21. Methyla-
t i o n of [ 21 followed by bromination y i e l d e d syn-methoxy-
iminobromoketone [ 3 ] . Condensation of [ 3 ] w i t h t h i o u r e a
[ 4 ] i n aqueous medium and a t a low t e m p e r a t u r e gave syn-
a m i n o t h i a z o l y l d e r i v a t i v e [ 51. The a m i n o t h i a z o l y l r i n g
w a s p r o t e c t e d by t r i t y l a t i o n t o g i v e t h e N - t r i t y l d e r i v a -
t i v e [6]. S a p o n i f i c a t i o n of t h e l a t t e r by b o i l i n g w i t h
NaOH s o l u t i o n a f f o r d e d t h e c o r r e s p o n d i n g a c i d [ 7 ] . Acy-
l a t i o n of t h e amino group of 7-ACA w i t h t h e r e s u l t i n g
a c i d [ 7 ] proved d i f f i c u l t . T h i s h a s been overcome by t h e
u s e of symmetrical a n h y d r i d e [ 8 ] , which w a s o b t a i n e d by
condensing two m o l e c u l e s of [ 7 ] i n t h e p r e s e n c e of d i c y -
c l o h e x y l c a r b o d i i m i d e [ a ] . 7-ACA w a s a c y l a t e d by compound
[8] t o g i v e [ 91. Removal of t h e t r i t y l r e s i d u e under
a c i d i c c o n d i t i o n gave t h e f r e e a c i d form (R=H) of c e f o -
taxime [ l o ] .

P r e p a r a t i o n of a S t a b l e Sodium S a l t :

T h i s w a s prepared by adding t h e s o l i d f r e e a c i d t o
a n aqueous a l c o h o l i c s o l u t i o n of a n o r g a n i c sodium s a l t
t o g i v e t h e s t a b l e a c t i v e D-form. (5)

The s y n t h e s i s of c e f o t a x i m e i s p r e s e n t e d i n Scheme 1.

I t i s i n t e r e s t i n g t o n o t e t h a t t h e syn-isomer of
cefotaxime i s u p t o 100 times more a c t i v e a g a i n s t c e r t a i n
organisms t h a n t h e a n t i - i s o m e r .

4. Metabolism

The metabolism of c e f o t a x i m e i n r a t , dog and man


u s i n g 14C-labelled c e f o t a x i m e h a s been s t u d i e d by
Chamberlain e t a l . (11). Cefotaxime i s w e l l absorbed i n
t h e t h r e e species a f t e r intramuscular administration. It
i s e l i m i n a t e d mainly v i a t h e u r i n e . The major m e t a b o l i t e
being d e s a c e t y l c e f o t a x i m e . The amount of unchanged c e f o -
CEFOTAXIME 153

OH
Scheme 1: S y n t h e s i s of Cefotaxime N’

methylation

bromination

then I
N,0CH3

H2N<NH2 + ‘OC2H5

S CH*
/
[41 Br [31

condensation

i
H2N -(syl:*5
s

[ 51

tr i t y l a t ion
154 FARID J . MUHTADI AND MAHMOUD M. A. HASSAN

condensation
C 6H 1 1 N=C=NC6H1

[a1

/
N
H C-0
3
+ symmetrical anhydride
[81

COOH
7- A M

1 Acylation
155
CEFOTAXIME

Tr,n

H /
<N),/)[;'j
S
0-CH
3

0'
(4 CH 20Ac

C02H

1
acidification

0 -CH
3
N'

H 2N ~R=H acid
C02R ~
cH20Ac

R=Na Cefotaxime
[lo1
156 FARID J . MUHTADI AND MAHMOUD M. A. HASSAN

taxime and t h e major m e t a b o l i t e e l i m i n a t e d i n t h e u r i n e


i s s i m i l a r f o r each s p e c i e s . Two f u r t h e r m e t a b o l i t e s ,
UP1 and UP2 a r e d e t e c t e d i n dog and human u r i n e b u t n o t
i n r a t urine.

The f o r m a t i o n of b o t h UP m e t a b o l i t e s i s r e l a t e d t o
t h e r a t e of r e n a l e l i m i n a t i o n of d e s a c e t y l c e f o t a x i m e
( 11) *

The m e t a b o l i c pathway f o l l o w s t h e r o u t e i n dog and


human which may occur i n t h e l i v e r i s p r e s e n t e d i n Scheme
?
L.

5. P harmacokine t i c s

The p h a r m a c o k i n e t i c s of c e f o t a x i m e w a s r e p o r t e d t o
be d o s e independent f o r d o s e s up t o 2.0 g ( 1 2 ) . Following
t h e i n t r a m u s c u l a r (i.m.) i n j e c t i o n of a 0 . 5 g (13) and
1 . 0 g d o s e s ( 1 2 ) , t h e mean peak serum l e v e l of 11.7 \ig/ml
and 22 ug/ml a t 0.5 h were o b t a i n e d r e s p e c t i v e l y . A f t e r
i n t r a v e n o u s ( i . v . ) p o l u s i n j e c t i o n of 1 . 0 g c e f o t a x i m e ,
t h e mean peak l e v e l w a s 105 ug/ml and an a p p a r e n t volume
of d i s t r i b u t i o n of 25 l i t r e s w a s e s t i m a t e d ( 1 2 ) . Values
of 32 and 37 l i t r e s f o r t h e volume of d i s t r i b u t i o n were
a l s o r e p o r t e d ( 1 3 ) . Cefotaxime i s mainly e l i m i n a t e d by
r e n a l e x c r e t i o n and s e r u m - h a l f - l i f e ranged from 0.92 t o
1.35 h a f t e r 1 g -i.m. i n j e c t i o n , and from 0.84 t o 1 . 2 5 h
a f t e r i . v . a d m i n i s t r a t i o n ( 1 2 ) . The m a j o r i t y of t h e d o s e
being e x c r e t e d unchanged i n t h e u r i n e (-51% w i t h i n 8 h)
(12). I n a n o t h e r s t u d y (13) t h e serum c l e a r a n c e of 341
mlfmin. per 1.73 m2 and r e n a l c l e a r a n c e of 130 mlfmin p e r
1.73 m2 were o b t a i n e d . P r o t e i n b i n d i n g of c e f o t a x i m e
ranged from 35 t o 45%.

6. Microbiological Activity

Cefotaxime p o s s e s s e s a n e x t r e m e l y broad a n t i b a c t e r i a l
spectrum and i s v e r y a c t i v e a g a i n s t C positive bacteria
e s p e c i a l l y on E n t e r o b a c t e r i a c e a e , Haemophilus i n f l u e n z a e
o r Neisseria gonorrhoeae. I t is a l s o a c t i v e on Bacteroides
f r a g i l i s and Pseudomonas a e r a g i n o s a ( 1 4 ) . Cefotaxime
p e n e t r a t e s w e l l through t h e c e l l w a l l s , showing h i g h 6-
l a c t a m a s e s t a b i l i t y and s t r o n g a f f i n i t y t o t h e i m p o r t a n t
t a r g e t enzymes ( 1 4 ) .
CEFOTAXIME 157

Scheme 2 : Metabolism of Cefotaxime.


OCH3
N'

co-
Cefotaxime

Deacetylation

OCH3
'N

2
-<: D;k"oE(A
Desacetyl
NH

cefotaxime
co- CH OH

Desacetyl
cef o t a x ime l a c t o n e
ELo
09 0
158 FARID J. MUHTADI AND MAHMOUD M. A. HASSAN

OCH3
N’ H H

UP 2
CEFOTAXIME 159

7. Methods of A n a l y s i s

7 . 1 . I d e n t i f i c a t i o n Tests

The f o l l o w i n g i d e n t i f i c a t i o n t e s t s are
mentioned under c e p h a l e x i n i n t h e B.P. 1980
(15) :-

A) The i n f r a - r e d a b s o r p t i o n spectrum determined


i n t h e s o l i d s t a t e , i s concordant w i t h t h e
r e f e r e n c e spectrum of c e f o t a x i m e .

B) Mix 20 mg w i t h a few d r o p s of an 8 0 % V/V


s o l u t i o n of s u l f u r i c a c i d c o n t a i n i n g 1 % V/V
of n i t r i c a c i d ; a yellow c o l o r is produced.

C) Mix 20 mg w i t h 5 d r o p s of a 1 % V/V s o l u t i o n of
g l a c i a l a c e t i c a c i d and add 2 d r o p s of a 1 % W/V
s o l u t i o n of copper s u l f a t e and d r o p of 2M sodium
hydroxide; an o l i v e - g r e e n c o l o r i s produced.

7.2. Non-Aqueous T i t r a t i o n Assay

Weigh a c c u r a t e l y a b o u t 0.15 g of c e f o t a x i m e and


d i s s o l v e i n 50 m l g l a c i a l a c e t i c a c i d . Assay
potentiometrically with 0.1 N perchloric acid.

1 m l of 0.1 N p e r c h l o r i c a c i d c o r r e s p o n d s t o
23.87 mg of c e f o t a x i m e ( 3 ) .
7.3. Chromatography

7.3.1 Thin Layer Chromatography (TIX)

TLC of c e f o t a x i m e i s perforlned on s i l i c a
g e l c o a t e d , s e l f - i n d i c a t i n g chroma t o g r a p h i c
p l a t e s (60 F 2 5 4 , Merck).

The developing s o l v e n t system is:-

Ethylacetate/Ethanol/Water/Formic a c i d
(60 : 25 : 1 5 : 1) by volume.
Development i s c a r r i e d o u t i n a l i n e d t a n k ,
and t h e s o l v e n t i s allowed t o m i g r a t e a b o u t
1 5 cm from t h e s t a r t i n g p o i n t s .
The p l a t e s are v i s u l i z e d by examining under
u l t r a v i o l e t l i g h t a t 254 nm ( 3 ) .
160 FARID J. MUHTADI AND MAHMOUD M. A. HASSAN

The above method c a n be adopted f o r t h e


d e t e r m i n a t i o n of t o t a l i m p u r i t y c o n t e n t of
cefotaxime as f o l l o w s ( 3 ) :-

A 2 % s o l u t i o n of t h e sample t o be
determined i s d i s s o l v e d i n a m i x t u r e of
8 v o l . a c e t o n e and 2 v o l . water.
Cefotaxime r e f e r e n c e s u b s t a n c e (HR 756
s t a n d a r d ) i s d i s s o l v e d i n t h e same m i x t u r e t o
produce 0.02 %, 0.04 % and 0.08 % s o l u t i o n s .

Two s p o t s 0.01 and .0.02 m l of t h e t e s t e d


s o l u t i o n s are a p p l i e d t o t h e c h r o m a t o p l a t e
t h e s e r e p r e s e n t i n g 200 and 400 pg r e s p e c t i v e l y .

A 0.01 m l of e a c h s t a n d a r d s o l u t i o n is
a l s o applied t o t h e chromatoplate represent-
ing 2 , 4 , 8 p g r e s p e c t i v e l y .
The p l a t e i s developed a s above.
The t o t a l i m p u r i t y c o n t e n t should n o t
exceed 5 %.

Another TLC system i s recommended f o r


cefotaxime a s f o l l o w s : -

A s o l u t i o n of c e f o t a x i m e i s a p p l i e d i n t o
a s i l i c a g e l G c h r o m a t o p l a t e . The p l a t e i s
developed i n t h e s o l v e n t Methanol : Ammonia
(100 : 1 . 5 ) . A f t e r development, t h e p l a t e
i s a i r d r i e d , sprayed w i t h 0.5 % i o d i n e i n
chloroform. Cefotaxime developed a d a r k
brown s p o t which has Rf v a l u e of 0.83 ( 4 )

7.3.2 Paper Chromatography

Descending paper chromatography w a s


accomplished u s i n g Whatman no. 1 paper.
The s o l v e n t system c o n s i s t e d of i s o p r o p a n o l -
p y r i d i n e - w a t e r (65 : 5 : 30, V / V ) . The
chromatogram was s u b j e c t e d over-night
f o r development. Examination was conducted
under t h e UV l i g h t a t 254 nm.

7.3.3 Gas Liquid Chromatography (GLC)

A GLC method f o r t h e d e t e r m i n a t i o n of
cefotaxime h a s been adopted i n our l a b o r a t o r y
CEFOTAXIME 161

u s i n g a Varian GC - 3700 g a s chromatograph


equipped w i t h Varian CDS 111 i n t e g r a t o r .
Column c o n d i t i o n s : 3 Z OV-17on chromosorb WHP
(80 - 100 mesh) g l a s s column (2 m x 2 mm)
The column w a s c o n d i t i o n e d i s o t h e r m a l l y
a t 120° f o r 1 0 min. and t h e n t h e temp-
e r a t u r e w a s programmed a t 10°/min up t o
2100.
C a r r i e r g a s : Helium, f l o w r a t e was a d j u s t e d
t o 20 m l / m i n .
D e t e c t o r : FID, hydrogen and a i r f l o w rates
were a d j u s t e d t o 40 ml/min. and 300 ml/min.
respectively .
Ethanol w a s used a s t h e s o l v e n t and t h e c h a r t
speed w a s a d j u s t e d t o g i v e 0.5 cm./min.
The r e t e n t i o n t i m e = 8.15 min.
The GLC of cefotaxime i s p r e s e n t e d i n F i g . 9.

Another GLC method h a s been d e s c r i b e d € o r


t h e d e t e r m i n a t i o n of r e s i d u a l s o l v e n t s i n
cefotaxime ( 3 ) .

Apparatus : A 1 . 5 m PORAPAK Q column programed a t 150'


with a flame i o n i z a t i o n d e t e c t o r was used.
I s o p r o p a n o l w a s used a s t h e i n t e r n a l s t a n d a r d .

Solution I : 70 mg cefotaxime s t a n d a r d (HR 756) and 0.08 mg


i s o p r o p a n o l were d i s s o l v e d i n water t o produce
1ml.

S o l u t i o n I1 : 0.05 mg methanol, 0.13 mg e t h a n o l and 0.08 mg


i s o p r o p a n o l were mixed w i t h water t o produce
1ml.

Procedure : S o l u t i o n s I and I1 w e r e i n j e c t e d and t h e


s o l v e n t l e v e l s w e r e determined from t h e
peak h e i g h t s c o r r e c t e d a g a i n s t t h e i n t e r n a l
standard.

Results : Methanol should n o t exceed 0.2 % e t h a n o l o r


any o t h e r o r g a n i c s o l v e n t should n o t exceed 1 % .
162 FARID J. MUHTADI AND MAHMOUD M. A. HASSAN

F i g 9. GLC of c e f o t a x i m e
A = cefotaxime

7.3.4 High Performance L i q u i d Chromatography (HPLC)

S e v e r a l HPLC s y s t e m s f o r t h e i d e n t i f i c a t i o n
and s e p a r a t i o n of c e f o t a x i m e and i t s metabo-
l i t e s have been r e p o r t e d . These s y s t e m s a r e
g i v e n i n T a b l e 5.

HPLC o f c e f o t a x i m e and i t s m e t a b o l i t e s
u s i n g system 1 (11) i s p r e s e n t e d i n F i g . 10.
T a b l e 5. HPLC Systems of Cefotaxime

System Column Mobile P h a s e Retention Time Detect ion Ref.


no. (min) ( nm)

1. A n u c l e o s ill Acetonitrile- 13.0 W region


ODS column wa t e r - a c e t i c a c i d
( 8 : 9 1 : 1 by v o l )

2. A Microbondapak, wa t er-me t h a n o l
C18 corasil (39 : 7 ) c o n t a i n i n g 30 .O 254 nm
0.005 M h e p t a n e
guard column
sulfonic acid

3. Si-C18, 10 Methanol-water- - 235 nm


(1 : 5)
microns
KH2 P O 0.06 %
4
N a 2 HPO 2H20
4
t o r e a c h pH 7 . 8

4. Li ch r os o r b Phosphoric acid- 17.0 310 nm cL7 1


RP-8 methanol
Table 5 contd.

System Column Mobile Phase R e t e n t i o n Time Detection Ref.


no. (min) (nm)
5. A Rever sed-phase 20 m m o l sodium 4.8 254 nm
Hibar RT 250-4 d i h y d r o g enp ho s pha t e
L i c h r o s o r b RP i n water-methanol-
18-7 um, c o r a s i l acetonitr ile
C18 37-50 Vm (83:7:10, V / V / V )
guard column

6. A 10-cm octade- 1 4 % methanol, 12.0 254 nm (19)


c y l s i l a n e column 1% acetic acid, ( s e t a t 0.005
a n d a 4-cm pre- in distilled absorbance
column c o n t a i n i n g water unit)
copellicular
oc t a d e c y l s i l a n e
CEFOTAXIME 165

A
I D

13
R e t e n t i o n time (min)

F i g . 1 0 HPLC of C e f o t a x i m e and i t s M e t a b o l i t e s
A , D e s a c e t y l c e f o t a x i m e ; B , UP1; C , UP2;
D, Desacetyl cefotaxime l a c t o n e ; E , cefotaxime.

7.4 Spectrophotometry

A PMR method f o r q u a n t i t a t i v e d e t e r m i n a t i o n
of c e f o t a x i m e and o t h e r c e p h a l o s p o r i n s i n b u l k
d r u g s and v a r i o u s d o s a g e f o r m s i s r e p o r t e d ( 2 0 ) .
The d e t e r m i n a t i o n i s based on t h e i n t e g r a t i o n
of t h e 6-H and 1 o r 7-H of t h e @-lactam r i n g
system r e l a t i v e t o t h a t of t h e n i n e p r o t o n s of
t-butanol. The method i s r a p i d , a c c u r a t e and
precise, with an average standard deviations
of 2 1 . 5 1 i n s t a n d a r d m i x t u r e s and k 1 . 1 5 i n
p h a r m a c e u t i c a l f o r m u l a t i o n s . The p r o c e d u r e
f u r n i s h e s a s p e c i f i c means o f i d e n t i f i c a t i o n
of e a c h i n d i v i d u a l c e p h a l o s p o r i n a s w e l l a s
d e t e c t i o n of t h e commonly e n c o u n t e r e d i m p u r i t i e s .

Procedure:

Weigh a c c u r a t e l y a p o r t i o n of t h e powder
e q u i v a l e n t t o 100 mg of t h e c e p h a l o s p o r i n i n t o
a g l a s s s t o p p e r e d sample t u b e . Add 1.0 m l
a c c u r a t e l y measured D20 c o n t a i n i n g a n a c c u r a t e
w e i g h t of t - b u t a n o l , s t o p p e r and s h a k e f o r 3 min.
T r a n s f e r a b o u t 0 . 5 m l of t h e r e s u l t i n g s o l u t i o n
i n t o a NMR t u b e and r e c o r d t h e s p e c t r u m , a d j u s t i n g
t h e s p i n r a t e t o r e d u c e t h e s p i n n i n g s i d e bands a s
166 FARID J . MUHTADI AND MAHMOUD M. A. HASSAN

much a s p o s s i b l e . I n t e g r a t e t h e peaks of i n t e r e s t
(The 6- o r 7-H of t h e B-lactam r i n g a p p e a r i n g a t
4.86 - 5.80 ppm and t h e 9 p r o t o n s of t - b u t a n o l
a p p e a r i n g a t 1.23 ppm) a t l e a s t t h r e e times and
determine t h e average i n t e g r a l s .

The amount of c e p h a l o s p o r i n i s t h e n c a l c u l a t e d
as follows:-

Where Ac i s t h e i n t e g r a l v a l u e of t h e c e p h a l o s p o r i n
signal, % t h a t of t h e t - b u t a n o l s f g n a l , E.Wc is t h e
m o l e c u l a r weight of c e p h a l o s p o r i n and E.W is one
b
n i n t h of t h e m o l e c u l a r weight of t - b u t a n o l (= 8 . 2 4 ) .

7.5 M i c r o b i o l o g i c a l Assay

C e f otaxime i s m i c r o b i o l o g i c a l l y assayed by t h e
a g a r w e l l d i f f u s i o n method of Grove and R a n d a l l (21)
a s modified by Chabbert and Boulinger ( 2 2 ) .

The f o l l o w i n g t a b l e 6 summarises t h e media and


t h e t e s t organisms a s r e p o r t e d .

T a b l e 6. Nedia and Test Organisms Used

Med iurn Test Organism -


Ref.

1. Agar a n t i b i o t i c Spores of B a c i l l u s s u b t i l i s (23)


no. 2 (Difco)
II 11
2. B a c i l l u s s u b t i l i s (ATCC 6633) (24)
II 11
3. S a r c i n a l e u t e a (ATCC 9341) (24)
4. II II
Escherichia coli 3989 (13)
It
5. 11
E. c o l i (ATCC 9637) (26)
I1 II
6. Proteus morganii (19)
7 . A n t i b i o t i c no. 3 Staphylococcus epidermid i s (25)
(Oxoid) Q 305
8. Mueller-Hinton P r o t e u s m i r a b i l i s (ATCC 14273) (18)
agar
Pseudomonas a e r u g i n o s a ( K 1118)
CEFOTAXIME 167

8. References

1. "Annual Drug Data Report", Ed. J . R . P r o u s , V 0 1 . 1 1 1 ,


J . R . P r o u s S . A . , B a r c e l o n a , S p a i n , p. 5 3 , ( 1 9 8 1 ) .

2. Chemical A b s t r a c t , I n d e x Guide S u p p l . , 1977-1979


c u m u l a t i v e , The American Chemical S o c i e t y , U.S.A.

3. C l a f o r a n " Q u a n t i t a t i v e and Q u a l i t a t i v e methods of


analysis" Roussel Uclaf, P a r i s , France.

4. S . Khawaja, U n i v e r s i t y of Riyadh, S a u d i A r a b i a
" P e r s o n a l Communication''.

5. R. Bucourt, D. Bormann, R. Heymes and ?I. P e r r o n n e t


6 , S u p p l . A , p. 6 3 , (1980).
J . A n t i m i c r o b i a l Chemotherapy, -

6. M.M.A. Hassan and F . J . Muhtadi, "Unpublished Data".

7. B. Loder, G.G.F. Newton and E.P. Abraham, Biochem.


J., 2,4 0 8 , (1961).
8. R.B. Morin, B . G . J a c k s o n , E . H . F l y n n , and R.W. Roeske,
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9. B. F e c h t i g , H. P e t e r , H. B i c k e l and E . V i s c h e r ,
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10. E . H . F l y n n , Ed., C e p h a l o s p o r i n s and P e n i c i l l i n s , C h e m i s t r y ,


B i o l o g y , Academic P r e s s , New York, p. 27, ( 1 9 7 2 ) .

11. J . Chamberlain, J . D . Coombes, D. D e l l , J . M . Fromson,


R . J . I n g s , C.M. Macdonald and J . McEwen, J . A n t i -
m i c r o b i a l Chemotherapy, 6, Suppl. A , p. 6 9 , ( 1 9 8 0 ) .

1 2 . F. Esmieu, J. G u i b e r t , H . C . R o s e n k i l d e , I. Ho and A.
L e Go, J . A n t i m i c r o b i a l Chemotherapy, - 6 , Suppl. A,
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13. K. P . Fu, P . Aswapokee, I . H o , C . M a t t h i j s s e n and


H . C . Neu, J . A n t i m i c r o b . Agents Chemother, -1 6 , 592,(1979).

1 4 . E. S c h r i n n e r , M. L i m b e r t , L. P e n a s s e and A. Lutz
J. A n t i m i c r o b i a l Chemotherapy, -
6 , Suppl. A , p. 25, (1980).

15. B.P. (1980) "The B r i t i s h Pharmacopeia" Her M a j e s t y


S t a t i o n e r y O f f i c e , Cambridge ( 1 9 8 0 ) .
168 FARID J. MUHTADI AND MAHMOUD M. A. HASSAN

16. D.S. Reeves, L.O. White, H.A. H o l t , D . B a h a r i ,


M. J . Bywater and R . P . Bax, J. A n t i m i c r o b i a l Chemotherapy,
-
6, Suppl. A , p. 93, ( 1 9 8 0 ) .

1 7 . T. Bergan, R. S o l b e r g , Chemotherapy ( B a s e l ) , 27 (3),


155, (1981).

18. F. Kees, E. S t r e h l , K. S e e g e r , G . S e i d e l , P. Dominiak


and H. G r o b e c k e r , Arzneim. F o r s c h . , 31 (l), 3 6 2 , ( 1 9 8 1 ) .

19. R. Wise, N . Wright and P . J . W i l l s , J . A n t i m i c r o b .


Agents and Chemother., 9, 526, ( 1 9 8 1 ) .

20. F . J . Muhtadi, M.M.A. Hassan and M.M. Tawakkol,


Spectroscop. l e t t . I n P r e s s (1982).

21. D.C. Grove and W.A. R a n d a l l , Assay Methods of


A n t i b i o t i c s , A n t i b i o t i c Monograph, Vol. 2. M e d i c a l
E n c y c l o p e d i a , N e w York, p. 3 4 , ( 1 9 5 5 ) .

22. Y . Chabbert and H. B o u l i n g e r , Rev. F r a n c . E t . C l i n .


B i o l . (-
2 ) 636, (1957).

23. N . Lambert-Zechovsky, C . A u f r a n t , E . Bingen, C. Blunn,


M.C. Proux and H . M a t h i e u , J. A n t i m i c r o b i a l Chemo-
therapy, -6 , S u p p l . A, p. 235, ( 1 9 8 0 ) .

24. H. Lode, B. Kemmerich, G . G r u h l k e , G. D z w i l l o ,


P. Koeppe and I . Wagner, J . A n t i m i c r o b i a l Chemotherapy,
6, Suppl. A, p. 193, ( 1 9 8 0 ) .
-

25. W.M. Glb'ckner, U. H o f f l e r , J. K i n d l e r , G. P e t e r s and


H.G. S i e b e r t h , J . A n t i m i c r o b i a l Chemotherapy, 5,
Suppl. A , p. 219, ( 1 9 8 0 ) .

26. J . P . F i l l a s t r e , A. L e r o y , G. Humbert and M. Godin,


J . A n t i m i c r o b i a l Chemotherapy, 6, Suppl. A , p. 1 0 3 ,
(1980).

Acknowledgment

The a u t h o r s would l i k e t o t h a n k R o u s s e l - U c l a f ,
P a r i s , France f o r kindly providing cefotaxime
sample.
CEFOXITIN, SODIUM
Gerald S. Brenner

I . Introduction 170
1.1 History 170
1.2 Name, Formula, Molecular Weight 170
1.3 Definition 170
1.4 Appearance, Color, Odor 170
2. Physical Properties 171
2.1 Infrared Spectrum 171
2.2 Magnetic Resonance Spectra 171
2.3 Ultraviolet Spectrum 179
2.4 Mass Spectrum 179
2.5 Optical Rotation 182
2.6 Solubility 182
2.7 Crystal Properties 183
2.8 Hygroscopicity 183
2.9 Acid Dissociation Constant 183
3. Synthesis 183
4. Stability-Degradation Products 185
4.1 Bulk Stability 185
4.2 Solution Stability 186
5 . Pharmacokinetics and Metabolism 187
5.1 Pharmacokinetics 187
5.2 Metabolism 187
5.3 Intramuscular Absorption and Bioavailability 187
5.4 Effect of Probenecid 188
6. Methods of Analysis 188
6.1 Identification Tests 188
6.2 Ultraviolet Spectrophotometric Analysis 189
6.3 Chromatographic Analysis 189
6.4 Perchloric Acid Titration 190
6.5 lodometric Assay 190
6.6 Microbiological Assay 190
6.7 Hydroxylamine Assay 190
7. Determination in Biological Fluids 191
8. References 192

Analytical Profiles of Drug Substances Copyriaht Q 1982 by The American


Volumc I I I69 Pharmaceutical Association
ISBN &12-260(111-9
170 GERALD S. BRENNER

1. Introduction
1.1 Historv
Sodium cefoxitin, a semi-synthetic cephamycin anti-
biotic was synthesized, tested and developed by the Merck
Sharp and Dohme Research Laboratories. Cefoxitin is active
against Gram-positive and Gram-negative bacteria ( 1 ) and is
therapeutically important because of its resistance to de-
struction by bacterial 6-lactamase (2,3,4).
1.2 Name, Formula, Molecular Weight
The Chemical Abstracts name for sodium cefoxitin
(MK-306) is 3-[: [Aminocarbonyl)oxylmethyl I-7-methoxy-8-0x0-
7~~2-thienylacetyl)aminol-5-thia-l-azabicyclo~4.2.Oloct-2-
ene-2-carboxylic acid sodium salt. The CAS registry no. is
33564-30-6.
Other names include 3-carbamoyloxymethyl-7a-methoxy-
7~-(2-thienylacetamido)-3-cephem-4-carboxylic acid sodium
salt, sodium 3-carbamoyloxymethyl-7-methoxy-7-[2-(2-thienyl~
acetamidol-3-cephem-4-carboxylate and sodium (6R,7R)-3-(hy-
droxymethyl)-7- -methoxy-8-oxo-7-[2-(2-thienyl)acetamidol-5-
thia-l-azabicyclo[4.2.Oloct-2-ene-2 carboxylate carbarnate
(ester) .

‘1 6H16N3Na07S2 Molecular Weight: 449.44


1.3 Definition
Sodium cefoxitin, unless specified otherwise, is
defined as the crystalline, non-solvated salt form of the
compound. Cefoxitin when specified refers to the free acid.
1 .4
Appearance, Color, Odor
White to off-white granules or powder having a
slight characteristic odor.
CEFOXITIN, SODIUM 171

2. Physical Properties
2.1 Infrared Spectrum
The infrared spectrum of c e f o x i t i n presented i n
Figure 1 was taken i n a KBr p e l l e t using a P e r k i n Elmer
Model 621 infrared spectrophotometer. A list of t h e
assignments made f o r some of t h e c h a r a c t e r i s t i c bands is
given i n Table I ( 5 ) .

Table I

IR Spectral Assignments f o r Cefoxitin

Frequency (ern-') Assignment

3200-3500 various N-H s t r e t c h


3000 (broad) OH s t r e t c h
1780 B-lactam C=O s t r e t c h
1715 carboxyl C=O s t r e t c h
1680 carbamate C=O s t r e t c h
1660 amide C=O stretch
1420 various -CH2 bends
1080 carbamate C-0 s t r e t c h

The infrared spectrum of sodium c e f o x i t i n taken a s


a mineral o i l m u l l is shown i n Figure 2.

2.2 Magnetic Resonance Spectra


2.21 Proton Spectrum
The proton magnetic resonance spectrum of
c e f o x i t i n presented i n Figure 3 was obtained using a Varian
A-60A spectrometer with DMSO-d6 a s t h e solvent. The
s p e c t r a l assignments a r e given i n Table I1 ( 6 ) . The proton
magnetic resonance spectrum f o r sodium c e f o x i t i n i n
DMSO-d6 is shown i n Figure 4 .

Table I1

Proton Magnetic Resonance Assignments f o r Cefoxitin

ppm ( 6 ) Relative I n t e n s i t y Multiplicity Assignment

9.38 1 Singlet 0
II
-C-NH-
7.34 1 Mu 1ti p l et thienyl-a
G -85-7.05 2 Mu1t i p l e t thienyl-B
0
-% u
a,
0
-0 c
a0 .rl
U
x
C
0 'z
-O 0
9 -
ir
V
m
!-I
w
C
H
I I 1 I
0 0 0 0 0 0
g c o W c t N
O/o 3 3N V l1I W S NV t l l
172
0
0
P-
O
0
al
H
8
0
r
I
0
0
0
2
0
0
::
0
0
0
w
I73
174
A
1m

FIGURE 4

I
A
I

I . J . I .
I
. .
,
.
,
I
, ,
. . . . I ,
,
. .
,
. I
,
.
,
, .
,. 1
,
,
,
. .
,
.
,
1 .
,
. .
,

.
.
1 .
,

. . . I

8.0 7.0 6.0 5.0 4.0 3.0 2.0 10 0


PPM

Figure 4 . Proton Magnetic Resonance Spectrum of Sodium Cefoxitfn in DMSO-d 6 '


176 GERALD S. BRENNER

Table I1 (cont'd)
ppm (6 1 Relative Intensity Multiplicity Assignment
6.52 2 Singlet 0
I1
0-c-NH2
5.13 1 Singlet 6-H
4.49-5.0 2 Mu1tiplet 0
II
-CH20C-
0

3'83
3.08-3.80
3.40
} 6-7
Singlet
Mu1tiplet
Singlet
thien yl-CH2-C
b-CH
7-OC~;~
II

2.22 Carbon-13 Spectrum


The carbon-13 magnetic resonance spectra of
cefoxitin presented in Figures 5 and 6 were obtained using a
Varian CFT-20 spectrometer with DMSO-d as the solvent at
a concentration of 0.5 -
M. The spectra assignments are P
given in Table I11 (7).
Table I11
Carbon-13 Magnetic Resonance Assignments for Cefoxitin

ppm( 6 ) Assignment
170.4 amide C=O
162.5 carboxyl C=O
160.3 C8, lactam C=O
156.3 carbamate C=O
136.5 c21
126.5
126.4} C31 and C4l
c a r boxy I
\

onide
C
\

c
corbomok
7 ow3
I

J1
I c,

175 I60 PPM I25

Figure 6. C-13 WiA Spectrum of Cefoxitin in DMSO-d


6'
CEFOXITIN, SODIUM 179

Table I11 (cont'd)

ppm ( 6 ) Assignment

125.8
125.5
124.9 c j1
95.1 c7
62.8 c6
61.9 C
52.5 OEB, -I
0
II
35.9 thien yl-CH2-C-
-
25.7 c4
2.3 U l t r a v i o l e t Spectrum
The u l t r a v i o l e t absorDtion sDectrum o f c e f o x i t i n i n
pH 7.0 phosphate buffer is chabacterized by a peak a t about
2 5 nm and a shoulder a t about 262 nm (Figure 7) with
A?$m values of approximately 330 and 209 respec-
t i v e l y . The shoulder a t 262 nm is d u e t o t h e 3-cephem
chromophore and t h e maximum a t 235 nm is due to thienyl-
a c e t y l s i d e chain.

2.4 Mass Spectrum


The mass spectrum o f c e f o x i t i n was obtained u s i n g a
Finnigan 3200 mass spectrometer i n t h e e l e c t r o n impact moie
w i t h an ionizing energy of 70 eV and a probe temperature of
175OC. The output from t h e mass spectrometer was analyzed
(8) and presented i n t h e form of a bar graph shown i n Figure
8.
The ion of highest mass seen i n t h e spectrum o f
c e f o x i t i n f r e e acid (molecular weight 427) is m/e 366, which

0
corresponds t o t h e lactone formed from t h e

C H 2-CO N H OCH3 ~cr''


N N
0 CH2

0
d
Cefoxit i n 1ac tone
\
\ \

I
250 nm 300 nm

Figure 7. UV Spectrum of Cefoxitin in pH 7 Phosphate Buffer.


Concentration: 2.75 mg/100 ml.

180
“1
D
Ln
(3
c
.d
U
*r(
x
0
u
a
D CJ
D
(3 w
0
D
u)
N
c
0
.rl
U
D
D 3
N 4
0
rn
a
d
D
2
c 0
0, 0
+
D
Q D
d In
AlISN31NI 3 A l l V l 3 Y
181
182 GERALD S. BRENNER

carboxylic acid and carbamate group. The n e x t ion (m/e 322)


corresponds t o l o s s o f C02 from the l a c t o n e . Scission o f
t h e s i d e chain amide bond of t h e l a c t o n e g i v e s m/e 241,
which l o s e s CH20
J
to give m/e 210. A t lower mass one s e e s

m /e 124,
@CH=C=O +

m/e 112,

m/e 97,

2.5 Optical Rotation


The s p e c i f i c r o t a t i o n o f a 1% (w/v) sodium
c e f o x i t i n s o l u t i o n i n methanol determined a t 509 nm and
25OC i s +210° + 4' calculated on an anhydrous and
solvent f r e e b a s i s .

2.6 Solubility
The s o l u b i l i t y o f sodium c e f o x i t i n i n t h e following
s o l v e n t s a t room temperature is s t a t e d i n terms of t h e
U.S.P. d e f i n i t i o n s : v e r y s o l u b l e i n water, s p a r i n g l y
soluble i n methanol and dimethylformamide, s l i g h t l y s o l u b l e
i n ethanol and i n s o l u b l e i n e t h e r , chloroform, acetone
aromatic and a l i p h a t i c hydrocarbons.

The s o l u b i l i t y of c e f o x i t i n i n water is t y p i c a l of
an organic acid with l i m i t e d aqueous s o l u b i l i t y and
increases with increasing pH. A t pH 1 , t h e measured
s o l u b i l i t y is about 0.3 mg/ml and a t pH 4 , t h e observed
value is about 25 mg/ml ( 9 ) .
CEFOXITIN, SODIUM 183

2.7 Crvstal ProDerties


2.571 C r y s t a l l i n i t y
Sodium c e f o x i t i n exists i n s e v e r a l solvated
and i n a desolvated c r y s t a l l i n e form. An amorphous form has
a l s o been i d e n t i f i e d . X-ray powder d i f f r a c t i o n p a t t e r n s and
polarized l i g h t microscopy can d i s t i n g u i s h c r y s t a l l i n e from
amorphous forms ( 1 0 ) .

2.72 X-Ray Powder Diffraction


The x-ray powder d i f f r a c t i o n p a t t e r n o f
c r y s t a l l i n e desolvated sodium c e f o x i t i n was obtained with a
Philips-Norelco d i f f r a c t o m e t e r , using copper Kcc r a d i a t i o n .
Samples of t h i s form show broad d i f f r a c t i o n l i n e s suggesting
t h a t t h e s o l i d is m i c r o c r y s t a l l i n e and composed of extremely
small c r y s t a l l i t e s ( 1 1 ) . The amorphous form shows no
d i s t i n c t X-ray powder d i f f r a c t i o n p a t t e r n .

2.73 D i f f e r e n t i a l Scanning Calorimetry


C r v s t a l l i n e sodium c e f o x i t i n e x h i b i t s a
decomposition exotherm a t approximately 19O0-2OO0C by
d i f f e r e n t i a l scanning calorimetry (DSC). The amorphous form
does not e x h i b i t any well-defined thermal t r a n s i t i o n s by DSC
below 35OoC. Gradual decomposition of t h i s s o l i d is
observed by DSC a s temperatures increase over 15OoC ( 1 1 ) .

2.8 Hygroscopicity
The water uptake o f sodium c e f o x i t i n a s a function
of r e l a t i v e humidity was s t u d i e d a t ambient temperature
( 1 1 ) . Equilibrium water levels were approximately I-2% a t
35% RH, 4-6% a t 47% RH and 15-17% a t 76% RH.

2.9 Acid Dissociation Constant


The d i s s o c i a t i o n constant o f c e f o x i t i n derived from
aqueous t i t r a t i o n (12) and s o l u b i l i t y d a t a (9) a r e i n good
agreement and i n d i c a t e t h a t c e f o x i t i n i s a r e l a t i v e l y s t r o n g
organic acid with a pKa of approximately 2.2.

3. Synthesis
Cefoxitin has been prepared by chemical modification of
cephamycin C , a n a t u r a l l y occuring a n t i b i o t i c produced by
Streptomyces lactamdurans (13, 1 4 ) . This r o u t e i s presented
i n Figure 9. Cepharnycin C (I) is tosylated t o t h e N-tosyl
d e r i v a t i v e (11) and then e s t e r i f i e d w i t h methyl chloromethyl
ether t o y i e l d t h e methoxymethyl ester (111). The ester i s
t h e n t r e a t e d with 2-thienylacetyl c h l o r i d e t o exchange t h e
aminoadipoyl s i d e chain f o r t h i e n y l a c e t y l . The ester
protecting group i s then removed w i t h acid t o y i e l d
c e f o x i t i n (IV).
r II:
I
1 CICH20C H3

Figure 9
S y n t h e s i s of C e f o x i t i n from Cephamycin C
CEFOXITIN, SODIUM 185

C e f o x i t i n h a s a l s o been p r e p a r e d b y t o t a l s y n t h e s i s (15)
and v i a t h e a c y l a t i o n and m e t h o x y l a t i o n of 7-amino
c e p h a l o s p o r a n i c a c i d ( 1 6 , 17, 18, 19).

4. Stability-Degradation Products
4.1 Bulk S t a b i l i t v
A t room t e m p e k a t u r e sodium c e f o x i t i n i s s t a b l e for
a t l e a s t t h r e e y e a r s when p r o t e c t e d from m o i s t u r e . A t ele-
v a t e d t e m p e r a t u r e , t h e s o l i d e x h i b i t s a b i p h a s i c decomposi-
t i o n p a t t e r n t y p i f i e d by an i n i t i a l more r a p i d d e c o m p o s i t i o n
p e r i o d f o l l o w e d by a slower d e c a y p e r i o d ( 2 0 ) . T h i s phenome-
non may b e related t o d e g r a d a t i o n of c e f o x i t i n b y low l e v e l s
of w a t e r i n t h e sample. S i n c e 6 - l a c t a m c l e a v a g e is water-
consuming, t h e e x t e n t of t h i s d e g r a d a t i o n pathway is l i m i t e d
by a v a i l a b l e water i n t h e s o l i d . Amorphous sodium c e f o x i t i n
h a s been shown t o be c o n s i d e r a b l y less s t a b l e t h a n i t s
c o r r e s p o n d i n g c r y s t a l l i n e form ( 2 0 ) .

A t e m p e r a t u r e d e p e n d e n t d i s c o l o r a t i o n of t h e s o l i d
h a s been n o t e d . The d i s c o l o r a t i o n i s n e g l i b l e a t 5OC and
becomes greater a t e l e v a t e d t e m p e r a t u r e . I t h a s been shown
t h a t an i n e r t a t m o s p h e r e ( a r g o n or n i t r o g e n ) m a r k e d l y
d e c r e a s e s t h i s change. T h i s development of color is n o t
d i r e c t l y r e l a t e d t o l o s s of p o t e n c y , i . e . c o n s i d e r a b l e
d i s c o l o r a t i o n c a n o c c u r w i t h no m e a s u r a b l e loss of p o t e n c y .

S e v e r a l d e g r a d a t i o n p r o d u c t s of s o l i d sodium
c e f o x i t i n h a v e been i d e n t i f i e d . From material s t o r e d a t
6OoC for t h r e e d a y s , two compounds h a v e been i s o l a t e d and
identified.

I,p
186 GERALD S. BRENNER

4.2 Solution S t a b i l i t y
The s o l u t i o n s t a b i l i t y o f sodium c e f o x i t i n has been
studied i n aqueous b u f f e r s i n t h e pH range 3 t o 9 ( 2 0 ) . The
degradation of sodium c e f o x i t i n i n t h i s pH range follows
apparent f i r s t - o r d e r k i n e t i c s . Maximum s t a b i l i t y i n water
i s i n t h e pH range o f 5-7. Under these pH c o n d i t i o n s ,
sodium c e f o x i t i n undergoes 10% chemical l o s s i n two days a t
25OC. Ten percent l o s s a t pH 3 occurs i n about 40 hours
and a t pH 9 i n about 14 hours. TLC s t u d i e s were c a r r i e d o u t
during k i n e t i c r u n s . P a t t e r n s become complex suggesting
t h a t t h e i n i t i a l B-lactam hydrolysis product i s unstable and
s u s c e p t i b l e t o transformation t o a considerable number of
secondary products (20).

The s o l u t i o n s t a b i l i t y o f sodium c e f o x i t i n was a l s o


studied a f t e r c o n s t i t u t i o n with frequently used I . V .
infusions and admixture w i t h comnonly used I . V . and I.M.
a d d i t i v e s (21 1. S t a b i l i t y i n t h e s e systems was e s s e n t i a l l y
t h e same a s t h a t observed f o r unbuffered s o l u t i o n s . I n
t h e s e s t u d i e s , sodium c e f o x i t i n was shown t o maintain
potency i n s o l u t i o n f o r a t l e a s t 30 days a t 5OC and f o r 30
weeks when stored i n t h e frozen s t a t e .

I n an attempt t o i s o l a t e degradation products


formed i n s o l u t i o n , a 10% aqueous s o l u t i o n of sodium
c e f o x i t i n was heated f o r four days a t 8OoC and then
subjected t o preparative TLC. F r a c t i o n s i s o l a t e d were
examined by NMR, mass spectrometry and i n f r a r e d . The
following compounds were i d e n t i f i e d .

Thiophene-2-acetic acid Thiophene -2- acetamidr

CON+
N- ( 2'-mothoxyacotamido) thiophene-
2- ace t am ido
CEFOXITIN, SODIUM 187

5. Pharmacokinetics and Metabolism


The pharmacokinetics and metabolism of sodium c e f o x i t i n
i n humans following parenteral administration have been t h e
subject of a number of i n v e s t i g a t i o n s and reviews
(22,31,32).

5.1 Pharmacokinetics
Followina intravenous administration (bolus o r
i n f u s i o n ) , c e f o x i t i n i s d i s t r i b u t e d r a p i d l y between serum
and t i s s u e and e x h i b i t s a terminal serum h a l f - l i f e of 30 t o
50 m i n u t e s . Total body clearance of c e f o x i t i n ranges from
approximately 250 m l t o 350 m l / m i n while r e n a l clearance i s
approximately 200 t o 300 m l / m i n . Urine contains a t l e a s t
90% of t h e dose a s unchanged drug and less than 5% of t h e
dose i s eliminated by metabolism and b i l i a r y clearance
(23,24,25). The d i s p o s i t i o n k i n e t i c s are f i r s t o r d e r ,
showing no e f f e c t of dose (0.25 t o 3 g.) or infusion r a t e .
Multiple dose regimens i n t h i s range given every f o u r hours
do not cause accumulation i n healthy volunteers. The volume
of d i s t r i b u t i o n i n t h e vascular compartment is about 8
l i t e r s (26-34). These d a t a a r e adequately described by a
two-compartment open model w i t h elimination occurring from
t h e c e n t r a l compartment.

5.2 Metabolism
Sodium c e f o x i t i n i s not metabolized appreciably i n
man. Urine samples from s e v e r a l human s t u d i e s were sepa-
rated by HPLC and TLC techniques. These s t u d i e s show t h a t
more than 90% of t h e c e f o x i t i n administered by e i t h e r t h e
intravenous o r intramuscular route i s recovered i n t h e u r i n e
a s i n t a c t drug. A microbiologically i n a c t i v e metabolite,
descarbamoyl c e f o x i t i n , was found t o t h e extent of 1-6% i n
some i n d i v i d u a l s , 2 t o 4 hours post dosing (26,271. This
metabolite was not found i n t h e u r i n e o f a l l s u b j e c t s .

Descarbarnoyl c e f o x i t i n
188 GERALD S . BRENNER

5.3 Intramuscular Absorption and B i o a v a i l a b i l i t y


Sodium c e f o x i t i n is raDidly and completely absorbed
following intramuscular administration. Peak serum levels
a r e a t t a i n e d i n 30 m i n u t e s o r less and 85 t o 95% of t h e
i . m . dose is recovered i n u r i n e within 12 hours of
administration.

Intramuscular administration o f sodium c e f o x i t i n


with either 0.5 o r 1.0% l i d o c a i n e hydrochloride a s a d i l u e n t
has no apparent e f f e c t on t h e b i o a v a i l a b i l i t y o f t h e
an tibiotic (32,35>.

5.4 E f f e c t o f Probenecid
The concurrent o r a l o r intravenous administration
of probenecid with i . m . o r i . v . i n j e c t i o n s of c e f o x i t i n has
a l a r g e influence on t h e time course o f t h e a n t i b i o t i c i n
serum (26,36). Probenecid administered intravenously
concurrent with c e f o x i t i n i n c r e a s e s t h e serum h a l f - l i f e o f
c e f o x i t i n from approximately 40 minutes t o 80 m i n u t e s and
reduces t h e r e n a l clearance of t h e drug from 200-300 ml/min
t o less than 100 m l / m i n .

6. Methods o f Analysis
6 . 1 I d e n t i f i c a t i o n Tests
U l t r a v i o l e t spectrophotometry i s used t o i d e n t i f y
sodium c e f o x i t i n . The spectrum o f a sample dissolved i n pH
6.0 phosphate buffer scanned from 220 t o 310 nm compares
q u a l i t a t i v e l y t o t h a t of a c e f o x i t i n standard s i m i l a r l y
tested.

The i d e n t i t y o f sodium c e f o x i t i n is a l s o
established by i n f r a r e d spectroscopy. The i n f r a r e d
absorbance spectrum of a s o l i d sample prepared e i t h e r a s a
potassium bromide d i s c o r mineral o i l m u l l is compared t o a
standard sample prepared i n an i d e n t i c a l manner.

A color test has been employed f o r t h e d e t e c t i o n of


c e f o x i t i n i n s o l u t i o n (37). To t h e r e s i d u e obtained by
drying a s o l u t i o n containing 10-50 mcg of c e f o x i t i n i s added
1-2 m l of 0.01% ninhydrin i n concentrated sulfuric acid and
t h e color is allowed t o develop a t room temperature. A
vivid blue c o l o r appears i n a few m i n u t e s . Other
cephalosporin a n t i b i o t i c s do not give t h e same c o l o r
response.
CEFOXITIN, SODIUM 189

Cefoxitin and f i f t e e n other cephalosporins have


been i d e n t i f i e d by thin-layer chromatography coupled with
color r e a c t i o n s (38) and by spectroscopic methods (39).
6.2 U l t r a v i o l e t Spectrophotometric Analysis
I n t a c t sodium c e f o x i t i n e x h i b i t s a UV absorption
band near 262 nm a t t r i b u t e d t o t h e OX-N-C=C linkage i n t h e
molecule. Beta-lactam r i n g opening l e a d s t o disappearance
of t h i s absorption band. This observation is t h e b a s i s of a
q u a n t i t a t i v e assay f o r c e f o x i t i n which i s s t a b i l i t y
indicating. Calculation of i n t a c t compound i s based on t h e
net absorbance a t 262 nm of t h e sample and of t h e standard
i n pH 6.0 phosphate buffer a s determined by s u b t r a c t i n g a
base l i n e correction a t 262 nm from t h e maximum a t t h e same
wavelength. The correction i s found by extending t o 262 nm
t h e s t r a i g h t portion of t h e UV curve between 340 and 300 nm.

6.3 Chromatographic Analysis


6.31 Thin Layer Chromatography
Thin layer chromatography using t h e system
chloroform/acetone/formic acid (10:9: 1 ) w i t h 0.25 rnm s i l i c a
gel p l a t e s has been employed f o r both sodium c e f o x i t i n and
the f r e e acid. The a i r dried p l a t e is sprayed w i t h 0.2% p-
dimethylaminocinnamaldehyde i n methanol/conc. s u l f u r i c acid
( 4 : l ) and heated a t 105' f o r f i v e m i n u t e s . The Rf f o r
c e f o x i t i n i n t h i s sytem is approximately 0.45.

Cefoxitin has a l s o been chromatographed on


s i l i c a w i t h developing s o l v e n t s of n-butanol/water/acetic
acid ( 4 : l : l ) and benzene/methanol/acetic acid (50:10:6)
giving approximate Rf values of 0.7 and 0.2 r e s p e c t i v e l y .
Detection can be accomplished by fluorescence quenching or
iodine s t a i n i n g .

6.32 High Performance Liquid Chromatography


Several HPLC procedures have been developed
t o s e p a r a t e c e f o x i t i n from process i m p u r i t i e s and
degradates. A system frequently used is described below.

Mobile Phase: 20% a c e t o n i t r i l e i n water


containing 1% a c e t i c acid.

Column : Ten micron, microporous


oct,adecylsilane bonded reversed phase packing i n a 3.9 mn X
30 cm column. Column temperature and pressure a t 25OC
( o r ambient) and 1000-1500 p s i g , respectively. Flow r a t e of
I .O-l.3 ml/min.

Detection: U.V. a t 254 nm


190 GERALD S. BRENNER

Procedure: Aliquots (10 mcl) o f a sample


s o l u t i o n containing 0.25 m g h l i n 0.02 M pH 7 phosphate
buffer a r e i n j e c t e d . The r e t e n t i o n time
f o r c e f o x i t i n is
approximately 10-15 m i n u t e s .

6.4 Perchloric Acid T i t r a t i o n


Sodium c e f o x i t i n can be determined by non-aqueous
t i t r a t i o n . An accurately weighed sample of about 800 mg i s
dissolved i n about 100 m l of g l a c i a l a c e t i c acid and t i t r a -
t e d potentiometrically w i t h standardized 0.1N p e r c h l o r i c
acid i n s p e c t r a l grade dioxane. An e l e c t r o d z p a i r i s
employed c o n s i s t i n g of a calomel e l e c t r o d e which has been
r e f i l l e d w i t h 0.1N LiC104 i n a c e t i c anhydride a s t h e
i n d i c a t i n g e l e c t r o d e , and a platinum r i n g a s t h e r e f e r e n c e
electrode.

6.5 Iodometric Assay


Sodium c e f o x i t i n bulk chemical and formulations can
be determined by iodometric assay. I n t h e assay, a l i q u o t s
of t h e sample and o f a s u i t a b l e c e f o x i t i n standard s o l u t i o n
a r e hydrolyzed f o r 20 minutes with 1 N NaOH and then
a c i d i f i e d t o pH 3.0 with a c e t a t e bufTer and I N H C 1 . Then,
0.01N iodine s o l u t i o n is added and, a f t e r a 2 h i n u t e w a i t ,
t h e excess iodine is t i t r a t e d with 0.01N sodium t h i o s u l f a t e
s o l u t i o n t o a s t a r c h end p o i n t . A blank determination i s
made a t t h e same time on a l i q u o t s o f both sample and
standard s o l u t i o n s t r e a t e d only with buffer and 0.01N iodine
and allowed t o stand f o r 20 minutes.

An automated iodometric assay has been developed


based on the manual method described above w i t h t h e
exception t h a t excess iodine is measured c o l o r i m e t r i c a l l y
r a t h e r than by t h i o s u l f a t e t i t r a t i o n ( 4 0 ) .

6.6 Microbiological Assay


For bulk and formulated products an agar d i f f u s i o n
( p l a t e ) assay can be conveniently c a r r i e d out using
Staphylococcus aureua a s t h e t e s t organism ( 4 1 ) .
6.7 Hydroxylamine Assay
Sodium c e f o x i t i n can be determined by means of t h e
colored complex formed between f e r r i c ion and t h e hydroxamic
acid formed by t h e a c t i o n o f hydroxylamine on t h e beta-
lactam ( 4 2 ) . This method has been s u c c e s s f u l l y automated
CEFOXITIN, SODIUM 191

and has been specified by t h e FDA a s t h e d e f i n i t i v e assay


method f o r c e r t i f i c a t i o n of t h e a n t i b i o t i c .

7. Determination i n Biological F l u i d s
High performance l i q u i d chromatography has been u t i l i z e d
f o r t h e determination of c e f o x i t i n i n b i o l o g i c a l f l u i d s .
Cefoxitin and i t s descarbamoyl metabolite have been
q u a n t i t a t i v e l y analyzed i n human urine employing an anion-
exchange column w i t h U.V. detection ( 2 3 ) . More r e c e n t l y
Wheeler, e t a l . (43) have employed HPLC u t i l i z i n g a C-18
reversed phase packing and a solvent system of
a c e t o n i t r i l e / a c e t i c acid/O. 005M potassium dihydrogen
phosphate (25/0.5/74.5, v / v / v ) f o r the q u a n t i t a t i o n of
c e f o x i t i n i n serum and s a l i v a .

An HPLC method has been developed f o r t h e determination


of c e f o x i t i n i n serum which l e n d s i t s e l f t o automation
( 4 4 ) . The system is described below:
Mobile Phase: 11% methanol i n pH 6.86 buffer
containing 1% a c e t o n i t r i l e .

Column: Ten micron C-8 reversed phase packed i n a


3.9 mn x 25 cm column with a disposable precolumn i n l i n e
and a precolumn i n the i n j e c t i o n loop. A flow r a t e o f 3.0
m l / m i n is used.

Detection: U.V. a t 254 nm.

Procedure: Serum samples (100 1.111 a r e t r e a t e d with


100 1 of i n t e r n a l standard (aqueous cefmetazole, 15 u l / m l
and 75 u 1 10% t r i c h l o r o a c e t i c a c i d . SarnDles a r e m i x e d ,
allowed t o stand for 15 m i n u t e s and centkifuged t o remove
p r e c i p i t a t e d protein. For automated a n a l y s i s , c l e a r
supernatant is t r a n s f e r r e d t o microcentrifuge tubes. A
standard curve i s generated by spiking blank serum with
appropriate levels of c e f o x i t i n and then processing a s
described above.

Cefoxitin i n b i o l o g i c a l f l u i d s has a l s o been determined


microbiologically by t h e cup-plate diffusion-technique using
e i t h e r Staphylococcus aureus MB2876 (26,27) o r B a c i l l u s
s u b t i l i s MB36 (35) a s t h e t e s t organism.
192 GERALD S.BRENNER

8. References

1 . H. R . Onishi, D. R. Daoust, S. B. Zimnerman, D. Hendlin


and E. 0. Stapley, "Abstracts, XI1 I n t e r s c i e n c e Confer-
ence on Antimicrobial Agents and Chemotherapy", A t l a n t i c
C i t y , N.J., 1972, p. 77 and preceding two a b s t r a c t s .

2. G. Darland and J . Birnbaum, Antimicrob. Agents Chemo-


ther. -
1 1 , 725 (1977).

3. H. R . Onishi, D. R . Daoust, S. B. Zimerman, D. Hendlin


and E. 0. Stapley, Antirnicrob. Agents Chemother., -
5 , 38
(1974).

4 . H. C. Neu, Antimicrob. Agents Chemother., 6, 170 (1974).

5. J . A. Ryan and J . Stevenson, Merck Sharp and Dohme


Research Laboratories, personal communication.

6. A. Douglas, Merck Sharp and Dohme Research Laboratories,


personal comnunication.

7. A. Douglas, Merck Sharp and Dohme Research Laboratories,


personal communication.

8. W. J . A. Vandenheuvel, Merck Sharp and Dohme Research


Laboratories, personal communication.

9. J . A. McCauley and A. Shah, Merck Sharp and Dohme


Research Laboratories, personal communication.

10. E. R. Oberholtzer and B. Singleton, Merck Sharp and


Dohme Research Laboratories, personal communication.

1 1 . E. R. Oberholtzer, Merck Sharp and Dohme Research Labora-


t o r i e s , personal communication.

12. G . Bicker, Merck Sharp and Dohme Research Laboratories,


personal communication.

13. S. Karady, S. H. P i n e s , L. M. Weinstock. F. E. Roberts,


G. S. Brenner, A. M. Hoinowski, T. Y. Cheng and M.
S l e t z i n g e r , J . Am. Chem. SOC. -
94, 1410 (1972).
14. L. M. Weinstock (Merck & Co., I n c . ) Fr 2,253,022; Ger
Offen 2,456,528; Jap. K75,105,687, Neth 7,414,820
CEFOXITIN, SODIUM 193

15. R. W. R a t c l i f f e and B. G. Christensen, Tetrahedron Lett


4 6 , 4645 (1973); -
- 46, 4649(1973); 46, 4653 (1973).
16. G. G. Hazen (Merck and Co., Inc.) US 3,780,033.
17. B. C. Christensen and L. D. Cama (Merck & Co., Inc.) Fr
2,163,144; Ger Offen 2,258,278.
18. B. C. Christensen and R. A. Firestone (Merck & Co.,
I n c . ) US 3,775,410.

19. B. G . C h r i s t e n s e n e t . a].. (Merck & Co., Inc.) Brit


1,348,984; Ger Offen 2,143,331.
20. E. R . Oberholtzer and G . S. Brenner, J. Pharm. S c i . , -
68,
863 ( 1979).
21. M. J . O'Brien, J . B. Portnoff, and E. M. Cohen, Am. J .
Hosp. Pharm 36, 33 (1979).

22. K . C. Kwan and J . D. Rogers i n 6-lactam A n t i b i o t i c s


(Handbook o f Experimental Pharmacology) ed. A. L.
Demain. Springer-Verlag, Heidelberg, i n press.

23. R. P. Buhs, T. E. Maxim, N. Allen, T. A. Jacob and F.


J . Wolf, J . Chromatog -
99, 609 (1974).
24. A. M. Geddes, L. P. Schnurr, A. P. B a l l , D. McChie, G .
R. Brookes, R . Wise and J . Andrews, Br. Med. J . -
1 , 1126
( 1977).

25. M. N . Logan, R . Wise and R. P. Grimley, J . Antimicrob.


Chemother. -5 , 620 (1979).
26. C. S. Goodwin, E. B. Raffery, A. D. Goldberg, H. Skeggs,
A. E. T i l l , and C. M. Martin, Antimicrob. Agents
Chemother. - 6, 338 (1979).
27. P. F. Sonneville, R. R . Kartodirdjo, H. Skeggs, A. E.
T i l l and C. M. Martin, Europ. J . C l i n . Pharmacol. -
9,
397 (1976).
28,
28. C. Simon, E. Meyer and V. Malerizyk, Arm, Forsch. -
1541 (1978).
194 GERALD S. BRENNER

29. G . J . Pazin, S. N. Schwartz, M. Ho, J . A. Lyon and A.


W. Pasculle, Rev. I n f e c t . D.S. -
1 . 189 (1979).

30. J.J. Schrogie, R . 0. Davies, K . C. Yeh, D. Rogers, G .


I . Holmes, H. Skeggs and C. M. Martin, J . Antimicrob.
Chemother. -4 , :Suppl. B, 69 (1978).

31. J . J . Schrogie, J . D. Rogers, K . C. Yeh, R. 0. Davies,


C. I . Holmes, H. Skeggs and C. M. Martin, Rev. I n f e c t .
Disc. -1 , 90 (1979).

32. W. Brumfitt, J . Kosmidis, J . M. T. Hamilton-Miller and


J . N. G . G i l c h r i s t , Antimicrob. Agents Chemother. -
6 , 290
(1974).

33. A. M. Brisson, J.B. F o u r t i l l a n , D. Barthes, P. Courtois


and B. Bezq-Giraudon, Therapie 35, 209 (1980).

34. R . Wise, B. Cadge, A. P. Gillett and A. Bhamjee, J .


Infection 1 : Suppl. 1 , 49 (1979).

35. P. F. Sonneville, K . S. Albert, H. Skeggs, H. Centner,


K . C. Kwan and C. M. Martin, Europ. J . C l i n . Pharmacol.
-
12, 273 (1977).

36. P. H. Vlasses, A. M. Holbrook, J . J . Schrogie, J . D.


Rogers, R. K . Ferguson and W. B. Abrams, Antimicrob
Agents Chemo. Ther. 1, 847 (1980).

37. J . R. Carlin and T. A. Jacob, Merck Sharp and D o h e


Research Laboratories, personal comnunications.

40. J . M. Konieczny, Merck Sharp and Dohme Research Labora-


t o r i e s , personal communications.

41. Code of Federal Regulations, Sections 4 4 2 . 1 4 a ( b ) ( l ) ( i )


and 436.105.

42. Code o f Federal Regulations, Sections 4 4 2 , 1 4 a ( b ) ( l ) ( i i )


and 4 4 2 . 4 0 ( b ) ( l ) ( i i ) .

43. L. A. Wheeler, M. de Meo, B. D. Kirby, R. S. Jerauld and


S. M. Finegold, J . Chromatog. -
183, 357 ( 1980).

44. R. S h a f f e r , Merck Sharp and Dohme Research Laboratories,


personal comnunication.

L i t e r a t u r e Reviewed t o June 1981


CEFOXITIN, SODIUM 195

Acknowledgement

The author wishes t o thank Mrs. E. Moyer f o r typing t h e


manuscript, Ms. F. R. Berg and Ms. A. M. Hendrick f o r
l i t e r a t u r e r e t r i e v a l work and Mr. C. Dillman and Ms. N .
G i l b e r t f o r preparation of the f i g u r e s .
CLOFIBRATE
Mahmoud M . A. Hassan and Aida A. Elaxxouny

1. Description 198
1.1 Nomenclature 198
1.2 Formulae 198
1.3 Molecular Weight 199
1.4 Appearance, Color, Taste, Odor 199
2. Physical Properties 199
2.1 Boiling Range 199
2.2 Density 199
2.3 Refractive Index 199
2.4 Solubility 199
2.5 Spectral Properties 199
3. Synthesis 207
4. Metabolism 209
5. Pharmacology 209
6. Methods of Analysis 21 1
6.1 Elemental Analysis 21 1
6.2 Identification Tests 21 1
6.3 Purity Tests 21 1
6.4 Official Methods 212
6.5 Ultraviolet Spectrophotometry 213
6.6 Thin Layer Chromatography 214
6.7 Thin Layer-Gas Liquid Chromatography 214
6.8 Gas Liquid Chromatography 214
6.9 Gas Chromatography-Mass Spectrometry 217
6.10 High-Performance Liquid Chromatography 218
7. Proton Magnetic Resonance Spectrometry 219
8. References 22 1

Analytical Profiles of Drug Substances Copyrilht 0 1982 by The American


Volume I I 197 Phlmuceuliul h o c u t i o n
ISBN & 1 2 - ~ l l - 9
198 MAHMOUD M. A. HASSAN AND AIDA A. ELAZZOUNY

1. Description

1.1 Nomenclature

1.11 Chemical Names

a. 2-(4-chlorophenoxy) -2-methylpropanoic acid


e t h y l ester.

b. E t h y l 2-p-chlorophenoxy isobutyrate

c. E t h y l 2-(p-chlorophenoxy)-2-methyl
propionate.

d. Propanoic a c i d , 2-(4-chlorophenoxy)-2-
methyl-, e t h y l ester.

1.12 Generic Name

Clof i b r a t e

1.13 Trade Names

Amotril ; Anparton ; Ateculon ; Atheropront ;


A t e r i o s a n ; Atromidin ; Atromid-s ; C l a r i p e x ;
Clobren-SF ; CPIB ; H y c l o r a t e ; Lipavion ;
Neo-Atromid ; Normet ; R e c o l i p ; Regelan ;
S e r o t i n e x ; Skleromexe ( 1 ) .

1.2 Formulae

1.21 Empirical

'1 2H15'3'
1.22 Structural
CLOFIBRATE 199

1.23 CAS R e g i s t r y No.

(637-07-0)

1.24 Wiswesser L i n e N o t a t i o n (2)

GR DOXVO 2

1.3 M o l e c u l a r Weight

242.71

1.4 Appearance, C o l o r , Taste, Odor

S t a b l e , c o l o r l e s s t o pale-yellow l i q u i d w i t h a f a i n t ,
c h a r a c t e r i s t i c o d o r and a c h a r a c t e r i s t i c t a s t e .

2. Physical Properties

2.1 B o i l i n g Range

158-1 60
20
148-150

2.2 Density

d25 : 1.138 - 1.144


2.3 R e f r a c t i v e Index

n 2o
D
1.500 - 1.505

2.4 Solubility

Insoluble i n water, s o l u b l e i n acetone, a l c o h o l ,


benzene, and c h l o r o f o r m .

2.5 Spectral Properties

2.51 I n f r a r e d Spectrum

The I n f r a r e d Spectrum of C l o f i b r a t e i s r e c o r d -
ed a s a f i l m on a Unicam Sp-1025 S p e c t r o p h o t o -
meter and i s shown i n F i g . 1 . The a s s i g n m e n t s
f o r t h e c h a r a c t e r i s t i c bands i n t h e I n f r a r e d
Spectrum a r e l i s t e d i n T a b l e 1.
Fig. 1. I R Spectrum of Clofibrate as F i l m .
CLOFIBRATE 20 1

Table I

I R C h a r a c t e r i s t i c s of Clof i b r a t e

-1
Frequency Cm Assignments (3)

1750 C = 0 (ester).

1605
1590 C = C (aromatic).
1500

1150 c-0-c
1100 (C-0 s t r e t c h i n g ) .

1390
1375
(Symmetrical de-
format i o n ) .
830 a r o m a t i c para-
d i s u b s it u t i o n

770
68 0
c-c1

Other bands c h a r a c t e r i s t i c of C l o f i b r a t e are


3020, 2970, 1290, 1250, 1190, 1030, 1020, 980
and 920 Cm-l.

2.52 U l t r a v i o l e t Spectrum (UV)

The UV spectrum of c l o f i b r a t e i n methanol w a s


scanned from 400-200 nm u s i n g V a r i a n Cary 219
Spectrophotometer and i s shown i n F i g . 2 .
The spectrum e x h i b i t s t h r e e maxima a t 288
(708), 280 (10233) and 226 (8511).

2.53 Nuclear Magnetic Resonance Spectrum

2.531 Proton Spectrum

The p r o t o n NMR S p e c t r a of C l o f i b r a t e i n
d e u t e r a t e d chloroform and i n Acetone-&,
were recorded on a Varian T-60 A, 60 MHz
NMR S p e c t r o m e t e r , u s i n g t e t r a m e t h y l s i l a n e
202 MAHMOUD M. A. HASSAN AND AIDA A. ELAZZOUNY

as an i n t e r n a l reference. The PMR spec-


trum i n d e u t e r a t e d chloroform i s shown
i n F i g . 3. The PMR s p e c t r a l assignment
of c l o f i b r a t e a r e g i v e n i n T a b l e 2 ( 4 ) .

Table 2

PMR C h a r a c t e r i s t i c s of C l o f i b r a t e

Protons Chemical S h i f t s
C0Cl3 Acet one-D
6

-CH2CH3 1.20 1.23 t


-C-(CH ) 1.56 1.57 s
3 2
-CH CH 4.18 4.20 q
-
Four a r o m a t i c 6.96 7.03 g
pro t o n s

s = singlet; t - triplet; q = quartet

2.532 S N M R Spectrum

13C NMR Spectrum of c l o f i b r a t e i n deu-


t e r a t e d chloroform u s i n g t e t r a m e t h y l s i -
l a n e as a n i n t e r n a l r e f e r e n c e w a s r e c o r -
ded on a J e o l FX 100, 100 MHz i n s t r u m e n t
a t ambient t e m p e r a t u r e and u s i n g 10 mm
sample t u b e . The d a t a c o n s i s t of 8192
d a t a p o i n t s over a 5000 Hz S p e c t r a l Width.
The c o m p l e t e l y decoupled spectrum is
shown i n F i g . 4. The carbon c h e m i c a l
s h i f t v a l u e s , shown i n T a b l e 3 a r e d e r i v -
ed from b o t h a d d i t i v i t y p r i n c i p l e s and
t h e off-Resonance Spectrum F i g . 5 ( 5 ) .
CLOFI BRATE 203

Fig. 2. W Spectrum of Clofibrate in Methanol.

Fig. 3. PMR Spectrum of Clofibrate and Tetramethylsilane


in CDC13
204 MAHMOUD M. A. HASSAN AND AIDA A. ELAZZOUNY

F i g . 4. I3C-NMR Spectrum of C l o f i b r a t e i n CDC13

Fig. 5. 13C-NMR Proton-Coupled S p e c t r u m of C l o f i b r a t e i n


CDC13
CLOFIBRATE 205

Table 3

13C NMR C h a r a c t e r i s t i c s of C l o f i b r a t e

Carbon No. Chemical S h i f t Carbon No. Chemical S h i f t


p pm ppm

154.18 7 79.62

120.85 8 25.34

129.04 9 25.34

121.43 10 173.67

129.04 11 61.30

120.85 12 14.03

2.54 Mass Spectrum

The mass spectrum of c l o f i b r a t e ( F i g . 6 )


o b t a i n e d by e l e c t r o n impact i o n i z a t i o n shows
a molecular i o n M+ a t m / e 242 ( r e l a t i v e i n t e n -
s i t y 41.1%) and a b a s e peak a t 128. The pro-
posed f r a g m e n t a t i o n p a t t e r n i s p r e s e n t e d i n
T a b l e 4.
- -

180 190 200 210 220 230 240 250 260 270 280 290 300

Fig. 6. Mass Spectrum of C l o f i b r a t e .


CLOFIBRATE 207

Table 4

Mass F r a g m e n t a t i o n P a t t e r n of C l o f i b r a t e .
a

CB2 -

m/e -
RI% Ion
- m/e
- R I% Ion
-
243 100 M.H
+ 242 41.1 M+

169 27 a 169 33.1 a

129 10 - 130 33.0 b+2H

115 78 - 128 100.0 b

87 21.2 c+H

R I = Relative Intensity

Mass s p e c t r a l d a t a , b o t h by e l e c t r o n i m -
p a c t and by m e t h a n e c h e m i c a l i o n i z a t i o n have
a l s o been r e p o r t e d . ( 2 , 6, 7 ) .

3. Synthesis:

The s y n t h e s i s of C l o f i b r a t e h a s b e e n a c h i e v e d by
two main methods (8-15).

Method I : T h i s i n v o l v e s c o n d e n s a t i o n of p a r a - c h l o r o -
p h e n o l w i t h a c e t o n e and c h l o r o f o r m i n t h e p r e s e n c e o f
sodium h y d r o x i d e f o l l o w e d by e s t e r i f i c a t i o n of t h e
resulting acid, afforded clof ibrate.
208 MAHMOUD M. A. HASSAN AND AIDA A. ELAZZOUNY

Method I1 : By condensing phenol w i t h e t h y l 2-chloro-2-


methyl-propionate i n t h e p r e s e n c e of a s u i t a b l e dehydro-
c h l o r i n a t i n g a g e n t and t h e n c h l o r i n a t i o n of t h e r e s u l t a n t
product, afforded c l o f i b r a t e .

C~G OH + CH3COCH 3 + CHC13

,or +
dehydrochlor i n a t i n g
agent
OH
Clof i b r a t e

F-C00C2R5
cl- CH3
- CH3
I

c12
COOC2H5

Clof i b r a t e
CLOFIBRATE 209

4. Metabolism:
The metabolism of c l o f i b r a t e and d i f f e r e n t c l o f i b r i c
a c i d d e r i v a t i v e s i n s e v e r a l a n i m a l s p c e c i e s and i n man were
reported (16-22). I t h a s been shown t h a t c l o f i b r a t e i s
c o m p l e t e l y h y d r o l y s e d t o c l o f i b r i c a c i d (para-chlorophenoxy
i s o b u t y r i c a c i d ) which is t h e n c o n j u g a t e d and e x c r e t e d .
I n man t h e two c o n j u g a t e s of c l o f i b r i c a c i d found i n u r i n e
were p r e s e n t i n p l a s m a . The h i g h e s t plasma c o n c e n t r a t i o n s
of c l o f i b r i c a c i d m e t a b o l i t e s were g e n e r a l l y found i n pa-
t i e n t s w i t h r e n a l d i s e a s e . S i n c e c l o f i b r a t e undergoes
r a p i d h y d r o l y s i s i n v i v o and i n v i t r o , t h e r e s u l t i n g c l o -
f i b r i c a c i d i s persumed t o b e t h e a c t i v e d r u g . S t u d i e s i n
v i v o and i n v i t r o w i t h c l o f i b r a t e and c l o f i b r i c a c i d i n d i -
c a t e t h a t t h e l a t t e r may e x e r t i t s e f f e c t by m u l t i p l e modes
of a c t i o n ( 2 3 ) .

5. Pharmacolopy: (24-32)
Clof i b r a t e r e d u c e s e l e v a t e d t r i g l y c e r i d e and c h o l e s -
t e r o l c o n c e n t r a t i o n s i n serum; t h e e f f e c t on serum l i p o -
p r o t e i n s i s p a r t i c u l a r l y e v i d e n t on t h e v e r y l o w - d e n s i t y
f r a c t i o n . When a d m i n i s t e r e d t o r a t s , i t c a u s e d a d e c r e a s e
i n serum t r i g l y c e r i d e and a l t e r a t i o n s i n a d i p o s e t i s s u e
u p t a k e and release of L i p i d s ( 3 3 ) . The b i o c h e m i c a l changes
produced i n c l u d e a d e c r e a s e i n a d e n y l c y c l a s e a c t i v i t y ,
i n h i b i t i o n of acetyl-coenzyme. A carboxylase, i n h i b i t i o n
of c h o l e s t e r o l and t r i g l y c e r i d e b i o s y n t h e s i s . The i n h i b i -
t i o n of h e p a t i c t r i g l y c e r i d e f o r m a t i o n i s a n e a r l y meta-
b o l i c consequence of c l o f i b r a t e a d m i n i s t r a t i o n and p r e -
c e d e s t h e f a l l i n serum t r i g l y c e r i d e . I t s e f f e c t s on
blood c o a g u l a b i l i t y s u g g e s t t h a t i t may r e d u c e t h e hyper-
coagulability frequently associated with a t h e r o s c l e r o s i s .
Uric a c i d c o n c e n t r a t i o n s , where e l e v a t e d , h a v e f r e q u e n t l y
shown a t r a n s i e n t r e d u c t i o n , and t h e s h o r t e n i n g of t h e
r e c a l c i f i e d C l o t t i n g - t i m e , which o c c u r s d u r i n g post-pran-
d i a l lipacmia, i s prevented.

C l o f i b r a t e w a s f i r s t used i n c o n j u n c t i o n w i t h a n d e r o -
s t e r o n e b u t i t became e v i d e n t t h a t t h e e f f e c t s produced
were n o t enhanced by t h e s t e r o i d . I t i s used i n a t h e r o
s c l e r o t i c conditions manifested i n coronary h e a r t d i s e a s e
and i n c e r e b r a l and v a s c u l a r d i s e a s e s , i n f a m i l i a l hyper-
c h o l e s t e r o l em i a and i n x a n thoma t o u s c o n d i t i o n s . E x u d a t i v e
d i a b e t i c r e t i n o p a t h y h a s been improved by c l o f i b r a t e .
Samuel e t a 1 ( 3 4 ) r e p o r t e d t h e s i g n i f i c a n t r e d u c t i o n
of c h o l e s t e r o l l e v e l s by t h e combined o r a l a d m i n i s t r a t i o n
of neomycin and c l o f i b r a t e . Musa e t a 1 ( 3 5 ) s t u d i e d t h e
e f f e c t s of c l o f i b r a t e upon t h e d i s t r i b u t i o n , m e t a b o l i s m ,
210 MAHMOUD M. A. HASSAN AND AlDA A. ELAZZOUNY

t r a n s p o r t and plasma b i n d i n g of 1 3 1 1 - t h y r o x i n e i n e n t h y -
roid individuals. I t had no c o n s i s t e n t e f f e c t upon t h e
b i n d i n g c a p a c i t i e s of t h y r o x i n e - b i n d i n g g l o b u l i n o r
t h y r o x i n e b i n d i n g pre-albumin. The h e p a t i c d i s t r i b u t i o n
s p a c e and c o n t e n t of t h y r o x i n e - i o d i n e were lower a f t e r
c l o f i b r a t e . The h e p a t i c t h y r o x i n e c l e a r a n c e and plasma-
t o - l i v e r t h y r o x i n e f l u x were unchanged. C l o f i b r a t e d i d
n o t a l t e r t h e plasma p y r o x i n e i o d i n e , t h e d a i l y t h y r o -
x i n e degradation r a t e o r t h e t o t a l thyroxine d i s t r i b u t i o n
s p a c e . These f i n d i n g s f a i l e d t o s u p p o r t t h e h y p o t h e s i s
t h a t c l o f i b r a t e p r o d u c e s i t s h y p o l i p i d e m i c e f f e c t by
d i s p l a c i n g t h y r o x i n e from i t s b i n d i n g p r o t e i n s and s h u n t -
ing it i n t o t h e l i v e r .

H a r r i s o n and Harden ( 3 6 ) s t u d i e d t h e e f f e c t of- c l o -


f i b r a t e on 6 p a t i e n t s w i t h h y p o t h y r o i d i s m and i s c h e m i c
h e a r t d i s e a s e . The p a t i e n t s were m a i n t a i n e d on t h e maxi-
mum d o s e of t h y r o x i n e which t h e y c o u l d t o l e r a t e b u t a l l
s t i l l had e v i d e n c e o f h y p o t h y r o i d i s m and l e v e l s of cho-
l e s t e r o l i n t h e serum were e l e v a t e d . C l o f i b r a t e produced
a r a p i d f a l l i n serum c h o l e s t e r o l a v e r a g i n g 37% and w a s
m a i n t a i n e d up t o t h e two y e a r s t h e d r u g was c o n t i n u e d .
I n t h r e e p a t i e n t s i t was p o s s i b l e t o i n c r e a s e t h e d o s e of
t h y r o x i n e d u r i n g t h e r a p y w i t h c l o f i b r a t e ; i n two p a t i e n t s
w i t h u n t r e a t e d s e v e r e h y p o t h y r o i d i s m , c l o f i b r a t e was
w i t h o u t e f f e c t on serum c h o l e s t e r o l u n t i l t h y r o x i n e w a s
added .

Clof i b r a t e I n vivo
Hydrolysis

-
Clof i b r i c a c i d Glucuron i d e
Metabolism of Clof i b r a t e
CLOFIBRATE 21 1

6. Methods of A n a l y s i s

6.1 Elemental Analvsis

c, 59.38% ; H, 6.232 ;

C 1 , 14.61:; ; 0 , 19.78%.

6.2 I d e n t i f i c a t i o n Tests

Those mentioned i n t h e B.P. (1980) ( 3 7 ) .

A. The i n f r a - r e d a b s o r p t i o n s p e c t r u m , Appendix I1
A , is c o n c o r d a n t w i t h t h e r e f e r e n c e s p e c t r u m of
clof ibrate.

B. The l i g h t a b s o r p t i o n , i n t h e r a n g e 220 t o 250nm,


of a 2-cm l a y e r of a 0.001 per c e n t w/v s o l u t i o n
i n a b s o l u t e e t h a n o l e x h i b i t s a maximum o n l y a t
226 nm; a b s o r b a n c e a t 226 nm, a b o u t 0.91, Appen-
d i x I1 B.

C. The l i g h t a b s o r p t i o n , i n t h e r a n g e 250 t o 350 nm,


of a 2-cm l a y e r of a 0.01 p e r c e n t w/v s o l u t i o n ,
i n a b s o l u t e e t h a n o l e x h i b i t s two maxima, a t 280
nm; and 238 nm a b s o r b a n c e a t 280 nm, aboilt 0.87,
and a t 283 nm, a b o u t 0.62, Appendix I1 R .

D. To 0.05 m l of a 1 0 per c e n t w/v s o l u t i o n i n


e t h e r add 0.05 m l of a s a t u r a t e d s o l u t i o n of
hydroxylammonium c h l o r i d e i n e t h a n o l (96 p e r
c e n t ) and 0.05 m l of a s a t u r a t e d s o l u t i o n of
potassium hydroxide i n e t h a n o l (96 p e r c e n t ) .
Heat f o r two m i n u t e s on a w a t e r - b a t h , c o o l ,
a c i d i f y w i t h 0 . 5 M h y d r o c h l o r i c a c i d , and add
0.05 m l of a 1 p e r c e n t w/v s o l u t i o n of i r o n
(111) c h l o r i d e ; a v i o l e t c o l o u r is produced.

6.3 P u r i t y Tests

(a) Water Content : n o t more t h a n 0.2%.

(b) Acidity: Hix 1 0 . 0 g w i t h 1 0 0 m l o f n e u t r a l i z e d


212 MAHMOUD M. A. HASSAN AND AIDA A. ELAZZOUNY

a l c o h o l , add 3 d r o p s of p h e n o l p h t h a l e i n T.S.
and t i t r a t e w i t h 0.1 N sodium h y d r o x i d e : n o t
more t h a n 0 . 9 m l is r e q u i r e d f o r n e u t r a l i z a t i o n

(c) Para-Chlorophenol:

The USP (XX) d e s c r i b e s a spectrophotomet-


r i c method f o r t h e d e t e c t i o n of p-chlorophenol.
The p e r c e n t a g e of p-chlorophenol i s n o t allowed
t o exceed 0.003%, w h i l e t h e B.P. (1980) d e s -
c r i b e s a g a s chromatographic p r o c e d u r e .

6.4 O f f i c i a l Methods: U.S.P. XX (38)

To a beaker c o n t a i n i n g 7 5 m l of 1 N sodium
hydroxide add a b o u t 3 g of a s t r o n g l y b a s i c
p o l y s t y r e n e anion-exchange r e s i n , and a l l o w t h e
mixture t o stand f o r about 15 minutes, with
o c c a s i o n a l s t i r r i n g . Wash t h e r e s i n w i t h water
u n t i l t h e l a s t washing i s n e u t r a l t o l i t m u s
paper, t h e n wash w i t h t h r e e 50 m l p o r t i o n s of
methanol. Place a plug of g l a s s wool i n t h e
b a s e of a 1-X 15 c m ion-exchange t u b e , and
t r a n s f e r t o t h e t u b e a s u f f i c i e n t amount of
Ion-exchange r e s i n , s l u r r i e d i n methanol, t o
produce a column bed h e i g h t of from6-cm t o
8-cm.

T r a n s f e r a b o u t 200 mg of C l o f i b r a t e , accu-
r a t e l y weighed, t o a 100 m l v o l u m e t r i c f l a s k ,
add methanol t o volume, and mix. T r a n s f e r 10.0
m l of t h i s s o l u t i o n t o t h e Ion-exchange column,
and c o l l e c t t h e e l u a t e i n a 100 m l v o l u m e t r i c
f l a s k . R i n s e t h e column w i t h 25 m l of methanol,
c o l l e c t t h e r i n s i n g i n t h e volumetric f l a s k ,
d i l u t e w i t h methanol t o volume, and mix. Trans-
f e r 5 . 0 m l of t h i s s o l u t i o n t o a 50 m l volume-
t r i c f l a s k , d i l u t e w i t h methanol t o volume,and
mix.

D i s s o l v e a n a c c u r a t e l y weighed q u a n t i t y
of USP C l o f i b r a t e RS i n methanol, and d i l u t e
q u a n t i t a t i v e l y and s t e p w i s e w i t h methanol t o
o b t a i n a s o l u t i o n having a known c o n c e n t r a t i o n
of about 2 0 pg p e r m l .

Concomitantly d e t e r m i n e t h e a b s o r h a n c e s of
t h e Standard p r e p a r a t i o n and t h e Assay p r e p a r a -
CLOFIBRATE 213

t i o n i n 1-cm c e l l s a t t h e wavelength of maximum


absorbance a t about 226 nm, w i t h a s u i t a b l e
s p e c t r o p h o t o m e t e r , u s i n g methanol a s t h e b l a n k .
C a l c u l a t e t h e q u a n t i t y , i n mg, of C12H15C103 i n
t h e p o r t i o n of C l o f i b r a t e t a k e n by t h e formula
lOC(Au/As), i n which C i s t h e c o n c e n t r a t i o n , i n
ug per m l , of USP C l o f i b r a t e RS i n t h e Standard
p r e p a r a t i o n , and Au and AS a r e t h e a b s o r b a n c e s
of t h e Assay p r e p a r a t i o n and t h e Standard pre-
.
para t i o n , r e s p e c t i v e l y

6. 5 U l t r a v i o l e t Spectrophotometry

A f t a l i o n e t a1 ( 3 9 ) r e p o r t e d t h a t e t h a n o l could
n o t be used a s a s o l v e n t f o r t h e u l t r a v i o l e t s p e c t r o -
photometry of c l o f i b r a t e i n g e l a t i n o u s c a p s u l e s w i t h
v e g e t a b l e o i l s because t h e l a t t e r i n t e r f e r e s t r o n g l y
a t t h e Xmax of c l o f i b r a t e ( 2 2 7 nm). However, by us-
ing dioxane, t h e a b s o r p t i o n c o e f f i c i e n t of v e g e t a b l e
o i l w a s found c o n s t a n t from 255 t o 280 nm w h i l e t h a t
of c l o f i b r a t e i n c r e a s e d from 0 . 2 4 a t 265 nm t o 0.48
a t 280 nm ( u s i n g a 0.01% s o l u t i o n ) . I n t h e a s s a y ,
t h e c o n t e n t of 5 c a p s u l e s w a s homogenized, 0 . 5 g d i s -
solved i n dioxane t o g i v e 25 m l s o l u t i o n , 1 m l d i l u t -
ed f u r t h e r w i t h d i o x a n e t o 100 m l , and t h e a b s o r p t i o n
w a s determined a t 280 nm and 265 nm w i t h r e s p e c t t o
a s t a n d a r d s o l u t i o n of 0.01 g v e g e t a b l e o i l i n 100 m l
dioxane measured a t 280 nm. The t r u e a b s o r p t i o n w a s
taken a s t w i c e t h e d i f f e r e n c e a t t h e two wave l e n g t h s
E r r o r s of ?4% were o b t a i n e d when a p p l i e d t o s y n t h e t i c
m i x t u r e of 1 :1.

A spectrophotometr i c a s s a y had been d e s c r i b e d


( 4 0 ) f o r t h e q u a n t i t a t i o n of c l o f i b r i c a c i d i n
plasma and u r i n e . T h i s i n v o l v e s s o l v e n t e x t r a c t i o n
of c l o f i b r i c a c i d from a c i d i f i e d plasma o r u r i n e and
subsequent measurement of t h e UV absorbance a t 2 2 6 n m .

The U.S.P. X I X ( 4 1 ) d e s c r i b e s an a s s a y procedure


f o r c l o f i b r a t e based on t h e p a s s a g e of t h e s o l u t i o n
of c l o f i b r a t e i n methanol on a column of s t r o n g l y
b a s i c p o l y s t y r e n e anion-exchange r e s i n . The a b s o r -
bances of t h e a s s a y p r e p a r a t i o n and a s t a n d a r d c l o -
f i b r a t e p r e p a r a t i o n are c o n c o m i t a n t l y determined i n
1-cm c e l l s a t 226 nm u s i n g methanol a s t h e blank.
214 MAHMOUD M. A. HASSAN AND AIDA A. ELAZZOUNY

6.6 Thin Layer Chromatography (42)

M i x t u r e s of c l o f i b r a t e and x a n t h i n o l n i c o t i n a t e
were , s e p a r a t e d on s i l i c a g e l l a y e r s . Using c h l o r o -
form a s s o l v e n t , t h e hRf of c l o f i b r a t e and x a n t h i n o l
n i c o t i n a t e a r e 82 and 3 r e s p e c t i v e l y . Using c h l o r o -
form a c e t i c a c i d 95:5, t h e hRf v a l u e s a r e :
Clof i b r a t e ( 7 8 ) , x a n t h i n o l (0) and n i c o t i n i c a c i d
(15) *

6.7 Thin Layer - Gas L i q u i d Chromatography:


A s p e c i f i c and s e n s i t i v e method f o r t h e d e t e r m i -
n a t i o n of c l o f i b r a t e i n b i o l o g i c a l f l u i d s w a s d e s c r i -
bed ( 43 ) . C l o f i b r a t e w a s s e p a r a t e d from a s s o c i a t e d
f a t t y a c i d s by TLC and t h e m e t h y l ester was q u a n t i -
f i e d by GLC.

6.8 Gas L i q u i d Chromatography:

Gas l i q u i d c h r o m a t o g r a p h i c methods occupy a pro-


minent p o s i t i o n i n t h e q u a n t i t a t i o n of c l o f i b r a t e i n
d r u g s , t i s s u e s and b i o l o g i c a l f l u i d s . I n t h e method
of S i l v e s t r i ( 4 4 ) , c l o f i b r a t e i s e x t r a c t e d from t a b -
l e t s w i t h e t h y l e t h e r and from t i s s u e s , blood o r u r i n e
by homogenization w i t h t h e a d d i t i o n of N a C l and 7%
HClO4 s o l u t i o n w i t h e t h e r - l i g h t p e t r o l e u m (1 :1).
The e t h e r e x t r a c t is washed w i t h water, d r i e d o v e r
anhydrous sodium s u l p h a t e and e v a p o r a t e d t o s m a l l
volume i n a stream of n i t r o g e n . GLC performed a t
19OoC on a column ( 6 f t . x 1 / 8 i n . ) of 5% b u t a n e - d i o l
s u c c i n a t e on Chromsorb P(100 t o 120 mesh), w i t h n i t -
r o g e n as c a r r i e r g a s and a f l a m e i o n i z a t i o n d e t e c t o r ,
methyl h e p t a d e c a n o a t e is used a s i n t e r n a l s t a n d a r d .
Clof i b r i c a c i d i s s i m i l a r l y d e t e r m i n e d a f t e r a d s o r p -
t i o n on A m b e r l i t e IRA-400 and e s t e r i f i c a t i o n by
treatment with methanolic HC1. Overall recoveries
were 98X ( + 5 % ) f o r t h e ester and 92% ( + l O Z ) f o r t h e
f r e e a c i d . The l i m i t of d e t e c t i o n i s 0 . 5 ug of t h e
ester o r a c i d p e r m l of u r i n e o r g . of t i s s u e .

Karmen and Haut ( 4 5 ) d e s c r i b e d a method f o r


a s s a y i n g c l o f i b r a t e i n serum based on GLC of t h e
methyl e s t e r . Two i n t e r n a l s t a n d a r d s similar i n
c h e m i c a l s t r u c t u r e t o c l o f i b r a t e (chlorophenoxy a c e -
t i c and chlorophenoxy p r o p i o n i c a c i d s ) a r e added; t h e
compounds a r e e x t r a c t e d , c o n v e r t e d t o m e t h y l e s t e r s
and s u b j e c t e d t o GLC u s i n g a n a l k a l i f l a m e i o n i z a t i o n
CLOFIBRATE 215

d e t e c t o r s e l e c t i v e l y s e n s i t i v e t o halogen.

Knuechel and Ochs ( 4 6 ) d e s c r i b e d a g a s chromato-


g r a p h i c method f o r t h e d e t e c t i o n of c l o f i b r a t e i n t h e
serum of t r e a t e d p a t i e n t s . The r e t e n t i o n time of c l o -
f i b r a t e w a s found between m e t h y l p a l m i t a t e and m e t h y l
stearate. C l o f i b r a t e was d e t e c t e d i n v e r y s m a l l
amounts i n serum g l o b u l i n s which were s e p a r a t e d by
f r a c t i o n a l p r e c i p i t a t i o n , w h e r e a s t h e amounts e x t r a c t e d
from albumin c o r r e l a t e d w e l l w i t h t h o s e of t h e serum.
F r a c t i o n s o f c h o l e s t e r o l ester from serum of c l o f i b -
r a t e - t r e a t e d p a t i e n t s , c o n t a i n e d s m a l l amounts of c l o -
f i b r a t e i n s p i t e of r e p e a t e d f r a c t i o n a t i o n on s i l i c a
g e l column, d u e t o e s t e r i f i c a t i o n of c l o f i b r a t e by
cholesterol.

Berlin ( 4 7 ) reported a q u a n t i t a t i v e gas c h r o m a t e


g r a p h i c method of c l o f i b r i c a c i d i n plasma u s i n g 2-(4-
chloro-3-methylphenoxy)-2-methylpropionic a c i d as in-
t e r n a l s t a n d a r d . To blood plasma (200 p l ) i s added
H3PO4 a c i d 2M (50 p l ) and i n t e r n a l s t a n d a r d s o l u t i o n in
t o l u e n e (0.5 m l ) . A f t e r s h a k i n g (10 min) t h e o r g a n i c
phase i s t r a n s f e r r e d t o a 3 m l t a p e r e d g l a s s t u b e and
t h e plasma i s r e - e x t r a c t e d w i t h 0 . 5 m l t o l u e n e . Diso-
dium hydrogen p h o s p h a t e 0.5M ( 0 . 5 ml) i s added t o t h e
combined o r g a n i c p h a s e s and t h e m i x t u r e i s s h a k e n f o r
1 0 min. The o r g a n i c p h a s e i s removed and t h e aqueous
phase i s made a c i d i c w i t h H3PO4 a c i d 5 M (50 p l ) CH2C12
(100 1-11>i s added and t h e m i x t u r e i s e x t r a c t e d on a
whirl-mixer f o r 30 s e c o n d s . A f t e r removal of t h e
aqueous p h a s e , diazomethane i n e t h e r (50 1-11>is added
and t h e s o l u t i o n i s e v a p o r a t e d under n i t r o g e n t o a b o u t
1 / 5 of i t s volume and one p l i s i n j e c t e d . Blank plasma
samples showed no p e a k s i n t e r f e r i n g w i t h t h e c l o f i b r i c
a c i d o r i n t e r n a l s t a n d a r d m e t h y l ester p e a k s . The
method i s l i m i t e d t o d e t e r m i n a t i o n of plasma c o n c e n t r a -
t i o n s down t o 1 pg ml-I d u e t o t h e c o n c e n t r a t i o n
chosen f o r t h e i n t e r n a l s t a n d a r d .

Another method f o r t h e q u a n t i t a t i v e d e t e r m i n a t i o n
of c l o f i b r i c a c i d i n blood p l a s m a had been d e s c r i b e d
( 45 ) . The s u b s t a n c e is e x t r a c t e d from a c i d i f i e d
plasma i n t o benzene, t h e e x t r a c t i s e v a p o r a t e d t o
d r y n e s s and t h e r e s i d u e i s m e t h y l a t e d and s u b m i t t e d
t o chromatography on a g l a s s column packed w i t h 3%O V 4 7
on chromosorb WHP, 100-120 mesh. The c o n d i t i o n s a r e
a s f o l l o w s : i n j e c t o r t e m p e r a t u r e , 18OoC; d e t e c t o r
216 MAHMOUD M. A. HASSAN AND AIDA A. ELAZZOUNY

t e m p e r a t u r e , 210°; column i n i t i a l t e m p e r a t u r e , 150°


f o r 6 min. t h e n programmed a t 195' a t 10°/min. and
h e l d a t t h e f i n a l t e m p e r a t u r e f o r 5 min. b e f o r e re-
c y c l e . The f l o w r a t e s were: helium c a r r i e r g a s , 25
ml/min. helium a u x i l i a r y g a s , 35 ml/min; a i r , 400 m l /
min. hydrogen, 40 m l / m i n . The r e t e n t i o n t i m e s of
c l o f i b r i c a c i d m e t h y l ester and of a c t a d e c a n e ( e x t e r -
n a l s t a n d a r d ) were 3 and 5 min. r e s p e c t i v e l y .

A r a p i d g a s c h r o m a t o g r a p h i c method i s d e s c r i b e d
( 4 9 ) f o r t h e d e t e r m i n a t i o n of c l o f i b r i c a c i d i n
p l a s m a and u r i n e . The a s s a y i n v o l v e s a n e x t r a c t i o n
i n t o t o l u e n e and b a c k - e x t r a c t i o n of c l o f i b r i c a c i d
and t h e i n t e r n a l s t a n d a r d ( 2 - n a p h t h o i c a c i d ) i n t o t h e
methylating agent (trimethylanilinium hydroxide).
The s i l a n i z e d g l a s s column ( 6 f t . x 4 mm i . d . ) was
packed w i t h 3% of SE-30 on 80-100 mesh Gas Chrom Q
0
and was o p e r a t e d a t 150 C w i t h a c a r r i e r g a s ( n i t r o -
gen) f l o w - r a t e o f 25 ml/min ; t h e i n j e c t i o n p o r t tem-
p e r a t u r e w a s 29OoC. The f l a m e i o n i z a t i o n d e t e c t o r
was o p e r a t e d a t 27OoC w i t h a hydrogen f l o w - r a t e of
20 mllmin. and a n oxygen f l o w - r a t e of 200 mllmin.
Under t h e s e c o n d i t i o n s , t h e r e t e n t i o n times were 1 . 5
min. f o r c l o f i b r i c a c i d and 2 . 5 min. f o r t h e i n t e r n a l
s t a n d a r d . The blood samples a r e drawn i n t o h e p a r i n i -
zed t u b e s and t h e plasma w a s s e p a r a t e d by c e n t r i f u g a -
t i o n . To 1 . 0 m l of plasma i n a 1 5 m l g l a s s t u b e were
added 1 m l of 0.4 M h y d r o c h l o r i c a c i d and 6 m l o f
t o l u e n e c o n t a i n i n g 120 pg of t h e i n t e r n a l s t a n d a r d .
The t u b e w a s shaken f o r 5 min. and c e n t r i f u g e d f o r
3 min. a t 4000 g . A 5 m l p o r t i o n of t h e o r g a n i c
phase i s t r a n s f e r r e d t o a p o i n t e d c e n t r i f u g e t u b e .
T r i m e t h y l a n i l i n i u m h y d r o x i d e ( 5 0 p l ) i s added and
t h e m i x t u r e i s e x t r a c t e d on a Vortex-mixer f o r 1 min.
A f t e r b r i e f c e n t r i f u g a t i o n , 1 p 1 o f . t h e aqueous
layer is injected directly. The d e t e r m i n a t i o n of
u r i n a r y c l o f i b r i c a c i d is c a r r i e d o u t e s s e n t i a l l y a s
d e s c r i b e d f o r plasma. For a n a l y s i s of g l u c u r o n i d e
m e t a b o l i t e of c l o f i b r i c a c i d i n u r i n e , t h e sample i s
d i l u t e d 1 : l O w i t h 0.2 M sodium a c e t a t e b u f f e r of pH
5.0 and 4 ml of t h e d i l u t e d sample are i n c u b a t e d o v e r
n i g h t w i t h 2000 Fishman u n i t s of a g l u c u r o n i d a s e -
a r y l - s u l p h a t a s e p r e p a r a t i o n from R e l i x Pomatia.

Wolf and Zimmerman ( 5 0 ) d e s c r i b e d a sumultaneous


GLC d e t e r m i n a t i o n of C l o f i b r a t e and i t s m e t a b o l i t e
c l o f i b r i c a c i d i n human plasma. T h i s h a s been ach-
i e v e d by u s i n g a g a s c h r o m a t o g r a p h i c column packed
CLOFIBRATE 217

w i t h Gas Chrom Q c o a t e d w i t h 10% S i l a r 1 0 C and n i t -


r o g e n a s c a r r i e r g a s . The method is r a p i d and do n o t
require a derivatization step, it is sensitive to
1 u g / d of e i t h e r compound i n b i o l o g i c a l s a m p l e s and
c o u l d b e used t o c h a r a c t e r i z e t h e i n v i v o c o n v e r s i o n
of c l o f i b r a t e ester t o t h e f r e e a c i d .

A c o m p a r a t i v e s t u d y of g a s - l i q u i d c h r o m a t o g r a -
p h i c b e h a v i o u r of t h e p e n t a f l u o r o b e n z y l esters and
t h e m e t h y l esters of t e n c h l o r o p h e n o x y a l k y l a c i d s
i n c l u d i n g c l o f i b r i c a c i d was a l s o r e p o r t e d ( 5 1 )
P a r a - c h l o r o p h e n o l and para-hydroxy b e n z o i c a c i d esters
which are added a s c a p s u l e p r e s e r v a t i v e s were d e t e r -
mined by g a s chromatography ( 52 ) . T h e s e were re-
a c t e d w i t h (EtO)2 P ( 0 ) C l and MeONa i n h e x a n e a t 50°
f o r 3 0 min. t o g i v e t h e d i e t h y l p h o s p h a t e esters.
These esters and c l o f i b r a t e were s e p a r a t e d and d e t e r -
mined on a column packed w i t h 3 . 5 % s i l i c o n e J X R on
Chromosorb G and HP a t 190' u n d e r N2 c a r r i e r . Flame
p h o t o m e t r i c and Flame t h e r m i o n i c d e t e c t o r s were u s e d .
The c o e f f i c i e n t s of v a r i a t i o n f o r 3 . 8 7 , 7.74 pg p a r a -
c h l o r o p h e n o l i n 1 0 m l were 0 . 8 7 , 0.56% w i t h FTB and
1.5, 0.56% w i t h FPD r e s p e c t i v e l y . P a r a c h l o r o p h e n o l
i n commercial p r e p a r a t i o n s was 1.6-5.1 ppm.

6.9 Gas Chromatography - Mass S p e c t r o m e t r y :


I m p u r i t i e s i n c l o f i b r a t e h a v e been s t u d i e d GC-MS
( 53 ) . T h r e e main i m p u r i t i e s were f o u n d , t h e m e t h y l
ester a n a l o g u e of c l o f i b r a t e , i t s d e s c h l o r o a n a l o g u e
and t h e d i c h l o r o a n a l o g u e .

J o h a n s s o n and Ryhage ( 5 4 ) have i d e n t i f i e d o t h e r


i m p u r i t i e s i n t h r e e c l o f i b r a t e p r e p a r a t i o n s . Samples
were o b t a i n e d by d i s s o l v i n g 0 . 5 m l of c a p s u l e - con-
t e n t i n 0.5 m l c h l o r o f o r m 5 p 1 of t h e s a m p l e w a s
i n j e c t e d i n t o t h e combined g a s c h r o m a t o g r a p h - mass
s p e c t r o m e t e r LKB 2091. The G . C . column u s e d w a s 3%
SE-30 g l a s s column 2.7 M x 2 mm ( i . d . ) . A constant
t e m p e r a t u r e of 16OoC f o r t h e f i r s t 8 min. w a s u s e d .
The c a r r i e r g a s f l o w - r a t e was 25 m l h e l i u m l m i n . The
mass s p e c t r a were o b t a i n e d w i t h a c o n s t a n t accelerat-
i n g v o l t a g e of 3 . 5 KV, a n e l e c t r o n e n e r g y of 70 eV
and a n i o n i z i n g c u r r e n t of 100 PA. R e p e t i t i v e scan-
n i n g of t h e m a s s r a n g e 1 0 t o 500 i n 25 w a s u s e d .
218 MAHMOUD M. A. HASSAN AND AIDA A. ELAZZOUNY

6.10 High-Performance Liquid Chromatography:

Bjornsson e t a 1 (55 ) developed a r a p i d , s e n s i -


t i v e and s p e c i f i c h i g h p r e s s u r e l i q u i d chromatogra-
p h i c method f o r t h e q u a n t i t a t i v e a n a l y s i s of c l o f i b -
r i c a c i d i n plasma, s a l i v a and u r i n e . Plasma (0.1 -
-1 m l ) , s a l i v a (1 ml) o r u r i n e d i l u t e d 1 : l O O w i t h
d i s t i l l e d water ( 1 . 0 ml) is p l a c e d i n a screw-capped
t u b e , and 100 p 1 of i n t e r n a l s t a n d a r d s o l u t i o n (con-
t a i n i n g 6.7 pg of t h e i n t e r n a l s t a n d a r d ) , 0.5 m l of
0 . 5 N s u l p h u r i c a c i d and 5 m l of t o l u e n e a r e added.
The samples a r e e x t r a c t e d by mixing f o r 10 min. f o l -
lowed by c e n t r i f u g a t i o n a t 1200 g f o r 10 min. t o
s e p a r a t e t h e o r g a n i c and aqueous p h a s e s . The lower
aqueous phase i s f r o z e n by immersing t h e t u b e i n a
d r y - i c e a c e t o n e b a t h , and t h e o r g a n i c p h a s e i s poured
i n t o a n o t h e r t u b e , which h a s a n e l o n g a t e d cone a t i t s
b a s e . Then 50 p 1 of 0 . 2 N NaOH are added, and t h e
m i x t u r e i s e x t r a c t e d on a Vortex-mixer f o r 2 rnin.
A f t e r b r i e f c e n t r i f u g a t i o n , t h e aqueous phase i s
drawn i n t o a s y r i n g e t h a t a l r e a d y c o n t a i n s 10 p 1 of
a s o l u t i o n of 5% g l a c i a l a c e t i c a c i d i n water and
t h i s m i x t u r e i s i n j e c t e d i n t o t h e chromatograph. For
t h e a n a l y s i s of t h e g l u c u r o n i d e c o n j u g a t e of c l o f i b -
r i c a c i d i n u r i n e , 5 m l of 6N h y d r o c h l o r i c a c i d were
added t o each sample, and t h e s o l u t i o n s were h e a t e d
a t 98OC f o r 30 min. b e f o r e t h e e x t r a c t i o n . The
samples were t h e n cooled and a n a l y s e d as d e s c r i b e d
above, e x c e p t t h a t t h e a d d i t i o n of d i l u t e s u l p h u r i c
a c i d i n t h e f i r s t s t e p w a s o m i t t e d . A V a r i a n Micro-
pak CH-10 r e v e r s e - p h a s e column ( 2 5 c m x 6 . 3 mm 0.d.
x 2.2 mm i . d . ) was used. The a b s o r b a n c e w a s measured
a t 235 11111. One pump of t h e dual-pump g r a d i e n t - e l u -
t i o n chromatograph c o n t a i n e d a c e t o n i t r i l e and t h e
o t h e r 0.5% a c e t i c a c i d i n d i s t i l l e d water; an i s o c r a -
t i c 42% a c e t o n i t r i t e m i x t u r e of t h e two s o l v e n t s was
used. The f l o w - r a t e of t h e s o l v e n t m i x t u r e w a s 70
me/hr w i t h a column i n p u t p r e s s u r e of 197 a t m (2900
p.s.i.). C o n c e n t r a t i o n s between 1 . 0 and 25 pg p e r
sample could be measured w i t h a c o e f f i c i e n t of v a r i -
a t i o n from 1-6%. 40-50 samples c a n e a s i l y be a s s a y e d
i n a day. The method d o e s n o t r e q u i r e p r i o r chroma-
tographic preparation o r m u l t i p l e extractions.

Woodhouse e t a1 ( 5 6 ) d e s c r i b e d a high-perfor-
mance l i q u i d chromatographic method f o r measuring
plasma c o n c e n t r a t i o n s of c l o f i b r i c a c i d a f t e r adminis-
t r a t i o n of c l o f i b r a t e t o humans. 50 pg of t h e i n t e r -
CLOFIBRATE 219

n a l s t a n d a r d (4-chloro-2-methylphenoxyacetic a c i d ) i n
methanol and 3M H C 1 (0.5 ml) were added t o plasma
(1 ml) i n a g l a s s s t o p p e r e d c e n t r i f u g e t u b e , s h a k e n ,
allowed t o s t a n d f o r 5 min. and t h e n e x t r a c t e d w i t h
6 m l e t h e r . Ether w a s evaporated t o d r y n e s s under
n i t r o g e n and t h e r e s i d u e d i s s o l v e d i n m e t h a n o l . 10
1~1p o r t i o n s of t h i s s o l u t i o n were i n j e c t e d i n t o t h e
chromatograph u s i n g a s t o p - f l o w i n j e c t i o n t e c h n i q u e .
The s t a i n l e s s s t e e l column (25 x 0.46 c m i . d . ) w a s
packed w i t h c18 P a r t i s i l (10 pm). The m o b i l e p h a s e
w a s 27% a c e t o n i t r i l e c o n t a i n i n g 0.4% o r t h o p h o s p h a t e
b u f f e r ( t o m a i n t a i n t h e pH a t 4.2) a t a f l o w - r a t e
of 2 mlfmin and a b a c k p r e s s u r e of 50 b a r . R e t e n t i o n
times of i n t e r n a l s t a n d a r d and c l o f i b r i c a c i d were
6 and 7 min. r e s p e c t i v e l y .

7, P r o t o n Magnetic Resonance S p e c t r o m e t r y :

Hassan and L o u t f y ( 4 ) h a v e d e v e l o p e d PMR


a n a l y t i c a l method f o r t h e q u a n t i t a t i o n of c l o f i b r a t e
a s a d r u g e n t i t y and i n c a p s u l e d o s a g e form. The
f o u r a r o m a t i c p r o t o n s q u a r t e t c e n t r e d a t 7 . 0 3 ppm
( F i g . 3) was chosen f o r q u a n t i t a t i o n of c l o f i b r a t e .
Malonic a c i d w a s employed a s a n i n t e r n a l s t a n d a r d ,
s i n c e i t e x h i b i t s two p r o t o n s m e t h y l e n e s i n g l e t a t
3.36 ppm ( F i g . 7) which i s w i d e l y s e p a r a t e d from
t h o s e of c l o f i b r a t e . Acetone was used a s t h e s o l v e n t ,
s i n c e c l o f i b r a t e and m a l o n i c a c i d a r e s o l u b l e i n i t
and i t s m e t h y l p r o t o n s s i n g l e t a t 2.07 ppm ( F i g . 7 )
d o e s n o t i n t e r f e r e w i t h t h e d o w n f i e l d p r o t o n s of
b o t h compounds. The p r o c e d u r e proved t o b e s i m p l e ,
r a p i d , a c c u r a t e and p r e c i s e . S t a n d a r d d e v i a t i o n s of
*1.07% and +1.34% were o b t a i n e d f o r p u r e d r u g and
c a p s u l e s r e s p e c t i v e l y . The PMR spectrum i n a d d i t i o n ,
p r o v i d e s a s p e c i f i c means of i d e n t i f i c a t i o n of c l o -
f i b r a t e , and d e t e c t i o n of i m p u r i t i e s .

Another PMR p r o c e d u r e have been a l s o r e p o r t e d


(57 1 f o r t h e d e t e r m i n a t i o n of c l o f i b r a t e . For t h e
b u l k d r u g , 100 t o 150 mg of sample i s shaken w i t h
5 m l of a s o l n . (10 mg ml-I of t h e i n t e r n a l s t a n d a r d
(hexamethylcyclotrisilazane) i n CCl4 and t h e m i x t u r e
is t r a n s f e r r e d t o a NMR t u b e . For c a p s u l e s , t h e
c o n t e n t s a r e e x t r a c t e d w i t h C C l 4 ( 4 x 5 m l ) , t h e com-
bined e x t r a c t s a r e made up t o 25 m l w i t h CCl4 and
5 m l of t h i s s o l n . i s p l a c e d i n t h e n.m.r. t u b e ,
t o g e t h e r w i t h 1 m l of t h e i n t e r n a l s t a n d a r d s o l n .
(40 t o 50 mg m l - I ) . The NMR spectrum i s t h e n
220 MAHMOUD M. A. HASSAN AND AIDA A. ELAZZOUNY
5 . . . . . l . . . . , . . . . , . . . . l . , . . , . . .
I ' I I I '
600 400 3OQ 2M

I
,JyI.11- 8.0 70 6.0 5.0 P H ( 6 ) 4 0

PARTS RFU MILLION, 6

F i g . 7 . PMR Spectrum of C l o f i b r a t e , Malonic a c i d and


T e t r a m e t h y l s i l a n e i n Acetone.

r e c o r d e d , and t h e c l o f i b r a t e concn. i s c a l c u l a t e d
by comparing t h e i n t e g r a l of t h e s i n g l e t peak a t
1 . 4 7 p.p.m. f o r c l o f i b r a t e w i t h t h a t f o r t h e i n t e r n a l
s t a n d a r d . The p r o c e d u r e i s s i m p l e , r a p i d and accu-
r a t e , and t h e r e c o v e r y i s 9 9 . 6 ? 3 . 2 0 X .

Acknowledgement :

The a u t h o r s would l i k e t o t h a n k Elr. T a n v i r A . B u t t o f t h e


Department of P h a r m a c e u t i c a l C h e m i s t r y , C o l l e g e of Pharmacy,
King Saud U n i v e r s i t y , f o r t y p i n g t h e m a n u s c r i p t .
CLOFIBRATE 22 1

9. References:

1. The Merck I n d e x , An E n c y c l o p e d i a of Chemicals and Drugs,


Martha Windholz -- e t a l , IX E d i t i o n , Merck and Co. I n c . ,
Rahway, N . J . , U.S.A. , P. 305 ( 1 9 7 6 ) .

2. Ateas o f S p e c t r a l Data and P h y s i c a l C o n s t a n t s of O r g a n i c


Compounds, e d i t e d by J . G . G r a s s e l l i and W . M . R i t c h e y ,
Vol. 3 , CRC P r e s s , Cranwood Parkway - C l e v e l a n d , Ohio,
P . 247 (P 2403) (1975).

3. K . N a k a n i s h i and P.H. Solomon, " I n f r a r e d A b s o r p t i o n Spec-


t r o s c o p y " Second E d i t i o n , Holden-Day, I n c , S a n f r a n c i c s c o ,
(1977).

4. M.M.A. Hassan and M.A. L o u t f y , Spec. L e t t . , 12 ( 3 ) , 177


(1979).

5. M.M.A. Hassan, Unpublished r e s u l t s .

6. B.S. F i n k l e , R.L. F o l t z and D.M. T y l o r , J . Chromat. S c i ,


-
1 2 , 304 ( 1 9 7 4 ) .

7. E . S t e n h a g e n , S . Abrahamsson and F.W. M c L a f f e r t y , " Re-


g i s t e r y of Mass S p e c t r a l Data" J o h n Wiley and S o n s ,
London, 2, 1447 ( 1 9 7 4 ) .

8. W.G.M. J o n e s , J . M . Thorp, and F7.S. Waring, B r i t . P a t .


860, 303 ( 1 9 6 1 ) .

9. J.M. Thorp, B r i t . a t . 898, 596 (1962).

10. M. J u l i a , M. B a i l l a r g e and G. T s c h e r n o f f , B u l l . SOC.


Chem. F r a n c e , 776 (1956).

11. G . B a r g e l l i n i , Gazz. Chem. I t a l . , 36, 334 ( 1 9 0 6 ) , Chem.


Z e n t r . 2, 326 ( 1 9 0 6 ) .

12. C.A. B i s h o f f , Ber., 33, 933 ( 1 9 0 0 ) .

13. D.T. W i t i a k , T . C-L HO, R.E. Hackney and W.E. Connor,


J . Med. Chem., 11,
1086 ( 1 9 6 8 ) .

14. V . N . Fedoseeva and B.M. Klebanov., K i m . Farm. Zh., -


3
( 1 0 ) 27 ( 1 9 6 9 ) .

15. J . E . Hoover , "Remington's P h a r m a c e u t i c a l S c i e n c e s "


F i f t e e n t h E d i t i o n , Mack P u b l i s h i n g Company, E a s t o n ,
222 MAHMOUD M. A. HASSAN AND AIDA A. ELAZZOUNY

P e n n s y l v a n i a , P. 796 ( 1 9 7 5 ) .

16. L. A l m i r a n t e , L. B r u s e g h i n i and L. F a z i , B u l l . Chim.


Farm, 108,292 (1969).

17. M.E. Laker and P.A. Mayes, Biorned. E x p r e s s . 2 ( 3 ) , 88


(1978).

18. M.E. Foed and E . G . Mcqueen, C l i n . Exp. P h a r m a m l . P h y s i o l ,


6 , ( 3 ) , 267 ( 1 9 7 9 ) .
-

19. B . J . Kudchodkar, H.S. S o d h i , L . H o r l i c k and D.T. Mason,


C l i n . Pharmacol. T h e r . , 22 ( 2 ) , 154 ( 1 9 7 7 ) .

20. D.T. W i t i a k and M.W. W h i t e h o u s e , Biomed. P h a r m a c o l . ,


-
18, 971 (1969).

21. D.T. W i t i a k , R . E . Hackney, and M.bJ, W h i t e h o u s e , J . Med.


Chem. __
1 3 , 607 ( 1 9 6 9 ) .

22. J.M. Thorp, L a n c e t , 1,1323 (1962).

23. D.T. W i t i a k , D.R. F e l l e r , E.S. S t r a t f o r d , R . E . Hackney ,


R. N a z a r e t h and G . Wagner, J . bled. Chem. 14,
1971) and
references therein cited.

24. J.M. Thorp, and W.S. \*Jaring, N a t u r e , 194 , 948 (1962).

25. M.F. O l i v e r , N . Engl. J. Med., 299, 1360 (1978).

26. R . I . Levy; D.S. F r e d r i c k s o n ; R. Shulman; D.W. B i l h e i m e r ;


J . L . Breslow; N . J . S t o n e , S . E . Lax; H . R . S l o a n ; R.M.
K r a u s s , and P.N. H e r b e r t , 77,
267 ( 1 9 7 2 ) .

27. S.M. Grundy; E.H. Ahrens, J r . G . S a l e n ; and E. O u i n t a o ;


J. C l i n . I n v e s t . , 48, 33a ( 1 9 6 9 ) .

28. P. S e g a l ; P.S. Roheim and H.A. E d e r ; J. C l i n . I n v e s t .


-
51, 1632 (1972).

29. B.M. Wolfe; J . P . Kane; R . J . Havel; and H.P. Brewster;


J. C l i n . I n v e s t . , 52, 2146 ( 1 9 7 3 ) .

30. N . A . D’Costa, F.C. Smigura, K. Kulhay and A . A n g e l ; J .


Lab. C l i n . Med., 90, 823 ( 1 9 7 7 ) .
CLOFIBRATE 223

31. M. Berman; M. Hall, R . I . Levy, S . E i s e n b e r g ; D.W.


B i l h e i m e r ; D , P h a i r ; and R.H. G r o e b e l . .J. L i p i d Res.,
1 9 , 38 (1978).
-

32. Goodman and Gilman's,"The P h a r m a c o l o g i c a l Basis of Thera-


p e u t i c s " , A . G . Gilman, W:S. Goodman, and A. Gilman, S i x t h
E d i t i o n , Macmillan P u b l i s h i n g Co., I n c . Mew York, P . 840
(1980).

33. H.J. F a l l o n , L.L. Adams and R . G . Lamb, L i p i d s , 1(2),


106 (1972).

34. P. Samuel, e t a l , C i r c u l a t i o n , 41, 109 (1970).

35. B.U. Musa, J . T . O g i l v i e , and J . T . Dowling, Metabolism


-
1 7 , 909 (1968).

36. M.T. 1 1 , 213


H a r r i s o n and R. McG. Harden, S c o t . Med. J . -
(1 966) .
37. B r i t i s h Pharmacopoeia, Vol. 1 ,
P r i n t e d i n England f o r Her M a j e s t y ' s S t a t i o n a r y O f f i c e
a t t h e U n i v e r s i t y Press, Cambridge, P . 1 1 6 , ( 1 9 8 0 ) .
38. The United S t a t e s Pharmacopoeia 2 0 t h R e v i s i o n , The
N a t i o n a l F o r m u l a r y , 1 5 t h E d i t i o n , U n i t e d S t a t e s Pharma-
c o p o i a l Convention, I n c . , R o c k v i l l , Md. , P . 157 (1980).

39. H. A f t a l i o n , R. S i m i n o v i c i and F.M. A l b e r t , Rev. Roum.


Chim., 5 ( 9 ) , 1195 (1968).

40. A.M. B a r r e t t and J . M . T h o r p . , B r . J . Pharmacol. Chem-


other. -3 2 , 381 (1968).

41. The United S t a t e s Pharmacopoeia, 1 9 t h R e v . , U n i t e d S t a t e s


Pharmacopoeia1 Convention, I n c . , R o c k v i l l e , Md. P . 9 6 ,
(1979).

42. A. d e J u a r e z , M. E s t r e l l a and G . C a r l o s , P r o - a n a l i s i s ,
2 ( 5 ) , 125 (1969).
-

43. A. S e d a r h a t , H . Nakamura and E.H. Ahrens, J . L i p i d R e s . ,


1 5 ( 4 ) , 352 (1974).

44. S. S i l v e s t v i , Famaco, Ed. P r v t . , g ( 3 ) , 197 (1970).

45. A. Karmen and H . Haut. , Biochem. Med. , 12 ( 2 ) , 154 (1975).


224 MAHMOUD M. A. HASSAN AND AIDA A. ELAZZOUNY

46. F. Knuechel and H . Ochs, Arzneim. Forsch. , 24 ( 4 ) , 576


(1974).

47, A. B e r l i n , J. Pharm. P h a r m a c o l . , 27, 54 ( 1 9 7 5 ) .

48. C.C. Tuong and A. Tuong, J . Chromat. , 106,97 (1975).

49. R. Gugler and C. J e n s e n , J . Chromat., 117,1 7 5 (1976).

50. M.S. Wolf and J . J . Zimmerman, J . Pharm. S c i -


6 9 ( I ) , 92
(1 9 8 0 ).
51. J. De Beer, C . V . Peteghem and A . H e y n d r i c k x , J . Chromato-
g r . , 157, 97 ( 1 9 7 8 ) .

52. M. K o i b u c h i and A. E j i m a , E i s e i Kagaku, 2 (6) 301


(1979).

53. E. D i d i n g , H. Sandstr'dm. H. O s t e l i u s , and R. K a r l e n ,


Acta Pharm. S u e c i c a , 13,
55 ( 1 9 7 6 ) .

54. E. J o h a n s s o n and R. Ryhage, J . Pharm. P h a r m a c o l . , 28,


927 ( 1 9 7 6 ) .

55. T.D. B j o r n s s o n and T.F. B l a s c h k e , J . C h r o m a t o g r . , 137,


145 ( 1 9 7 7 ) .

56. R.N. Woodhouse, D . G . Cresswell, L.F. Chasseaud and R.R.


B r o d i e , J . Chromatogr. 138,
218 ( 1 9 7 7 ) .

57. H.M. E l - F a t a t r y and H . Y . Aboul-Enein, Spec. L e t t . , 11


( 1 2 ) , 921 ( 1 9 7 8 ) .
John G . Hoogerhekk and Bruce E . Wyka

1. Description 226
1.1 Names, Formula, Molecular Weight, and Structure 226
1.2 Drug Properties 226
1.3 Appearance, Color, Odor, and Taste 226
2. Physical Properties 227
2.1 Nuclear Magnetic Resonance Spectra 227
2.2 Mass Spectrum 229
2.3 Infrared Spectrum 232
2.4 Ultraviolet Spectrum 232
2.5 Melting Range 235
2.6 Thermal Properties 235
2.7 Crystal Properties 236
2.8 Solubility 241
2.9 Dissociation Constant 241
3. Synthesis 242
4. Stability 242
5 . Drug Metabolism and Pharmacokinetics 244
5.1 Drug Metabolism 244
5.2 Pharmacokinetics 244
6. Methods of Analysis 246
6.1 Elemental Analysis 246
6.2 Identification 247
6.3 Spectrophotometric Analysis 247
6.4 Titrimetric Analysis 248
6.5 Chromatographic Analysis 248
6.6 Radiochemical Analysis 25 1
6.7 Microcalorimetric Analysis 252
6.8 Microbiological Analysis 252
7. Acknowledgements 252
8. References 253

Analytical Profiles of Drug Substances Copyright 0 1982 by The American


Volume 11 225 Pharmaceutical Association
ISBN 0-12-260811-9
226 JOHN G. HOOGERHEIDEAND BRUCE E. WYKA
1. Description

1.1 Names, Formula, M o l e c u l a r Weight and S t r u c t u r e

C l o t r i m a z o l e was f i r s t s y n t h e s i z e d i n 1969 b y
Plempel e t a l . (1) and t e s t e d under t h e name Bay b5097.
The compound-iias been marketed w i d e l y under t h e t r a d e names
o f Canesten, L o t r i m i n , Gyne-Lotr imin, and Mycelex.

The chemical name o f c l o t r i m a z o l e i s 1 - ( 2 - c h l o r o -


pheny1)diphenylmethyl-1H-
- imidazole.

The m o l e c u l a r f o r m u l a o f c l o t r i m a z o l e i s
C 2 2 H i 7 C l N z and t h e m o l e c u l a r weight i s 344.8 g/mol.
The s t r u c t u r e i s g i v e n i n F i g u r e 1.

1.2 Drug P r o p e r t i e s

C l o t r i m a z o l e i s a broad-spectrum a n t i m y c o t i c agent
e f f e c t i v e a g a i n s t pathogenic dermatophytes, yeasts, and
s e v e r a l species o f Candida, T r i c h o hyton, Microsporum,
Epidermophyton, and Maa-i. & reparations o f the drug
are used b o t h i n t h e t o p i c a l t r e a t m e n t o f dermal i n f e c t i o n s
and t o combat v u l v o v a g i n a l c a n d i d i a s i s .

Results o f i n i t i a l c l i n i c a l studies w i t h t h i s
compound p u b l i s h e d i n 1969 ( 2 ) were f o l l o w e d b y more
d e t a i l e d r e p o r t s o u t l i n i n g t h e spectrum and mechanisms o f
clotrimazole activity (1,3,4). At therapeutic
c o n c e n t r a t i o n s d r u g a c t i o n i s f u n g i s t a t i c ; however, a t h i g h
c o n c e n t r a t i o n s ( 2 0 p g / m l ) some - in- vitro fungicidical
a c t i v i t y has been observed (1,4).

1.3 Appearance, C o l o r , Odor and T a s t e

Clotrimazole i s a colorless, odorless, tasteless,


c r y s t a l 1i n e s o l i d .
CLOTRIMAZOLE 227

2. Physical Properties

2.1 Nuclear Magnetic Resonance S p e c t r a

2.1.1 P r o t o n Magnetic Resonance

The p r o t o n NMR spectrum ( F i g u r e 2) o f a 10% ( w / v )


solution o f clotrimazole i n deuterated chloroform a t
ambient temperature was o b t a i n e d b y u s i n g a V a r i a n CFT-20
spectrometer o p e r a t i n g a t a frequency o f 79.5 MHz. The
chemical s h i f t s g i v e n i n Table I a r e d o w n f i e l d from t h e
internal reference tetramethylsilane. P r o t o n assignments
are as i n d i c a t e d i n F i g u r e 1.

A 220 MHz p r o t o n NMR spectrum o f a s o l u t i o n o f


c l o t r i m a z o l e i n C 6 D 6 has a l s o been r e p o r t e d i n t h e
1i t e r a t u r e ( 5 ) .
Table I
PMR S p e c m s i g n r n e n t s

Protons Chemical S h i f t s (6) Intensity Multiplicity

5-H 6.75 1H triplet,


J=1.5 Hz
4-H 7.00 1H triplet,
J=1.5 Hz
2-H 7.40 1H triplet,
J=1.5 Hz
3'-H t o 6 ' - H
2"-H t o 6"-H 6.95-7.40 14H broad
8"-H t o 12"-H mu1t i p l e t s
228 JOHN G . HOOGERHEIDE AND BRUCE E. WYKA

10"

F i g u r e 1. S t r u c t u r e of C l o t r i m a z o l e .

I I 1 I I I I I I L
l " 1 " ' I " I I
9 8 7 6 s 5 4 3 2 1 0
8H 79.5 MHz
F i g u r e 2. Proton N u c l e a r Magnetic R e s o n a n c e S p e c t r u m of
C l o t r i m a z o l e in CDC13.
CLOTRIMAZOLE 229

2.1.2 Carbon-13 Magnetic Resonance

The carbon-13 p r o t o n decoupled NMR spectrum ( F i g u r e


3 ) o f a 20% ( w / v ) s o l u t i o n o f c l o t r i m a z o l e i n d e u t e r a t e d
c h l o r o f o r m a t ambient t e m p e r a t u r e was o b t a i n e d b y u s i n g a
V a r i a n XL-100 s p e c t r o m e t e r o p e r a t i n g a t a f r e q u e n c y o f 25.2
MHz. The c h e m i c a l s h i f t s a r e i n ppm ( 6 ) w i t h r e f e r e n c e t o
internal tetramethylsilane. The carbon-13 NMR spectrum
i n d i c a t e s t h e presence o f f i v e q u a r t e r n a r y carbons a t
6 = 75.0, 135.6, 139.1, 140.4, and 140.9, and seventeen
o l e f i n i c carbons a t 6 = 121.5, 127.0, 128.0 (4 e q u i v a l e n t
c a r b o n s ) , 128.1 ( 3 e q u i v a l e n t c a r b o n s ) , 128.5, 129.8, 130.1
( 4 e q u i v a l e n t c a r b o n s ) , 130.4, and 132.2 ppm.

2.2 Mass Soectrum

The mass spectrum ( F i g u r e 4 ) o f c l o t r i m a z o l e was


o b t a i n e d b y u s i n g a V a r i a n MAT CH5 medium r e s o l u t i o n s i n g l e
focusing instrument. The e l e c t r o n e n e r g y was 70 eJ, and
t h e probe and source t e m p e r a t u r e s used were 140 C and
25OoC, r e s p e c t i v e l y . The r e s u l t s a r e g i v e n i n T a b l e 11.

Table I1

m/e Ions
-
344 M'

309 (M-35)'

277 (M-67)'

242 (M-102)'

241 (M-103)'

239 (M-105)'

199

c)
t
165 H5C6CC6H 4

*C3H3N2 =
1111
' '~ " ' " " ' '
IIY)
!
I40
;':
'3-0 I , , , ,
190
I 2 , , I , # I
00
# I , , I I I 1
,,,,,
I I , , , / I ,
, , * , oI , , , ' .
20
P
4r"' '
' I ' 8 a I ' " " ' ' , l ' ' ' 1 1 ; ' ' , ~ ' ~ , ' ' , , , , , , . l , , , , , , , , . I . i l , I . I I

18. v v m l

Figure 3 . Carbon-13 Nuclear Magnetic Resonance Spectrum o f Clotrimazole in CDC13.


0
VI
m
0
O
m
0
In
(u
v)
0 0 :
O W
I
3
z
v)
v)
:a
- I
7
0
z
A 1 SN31NI 3AllWl3U
23 1
232 JOHN G . HOOGERHEIDE AND BRUCE E. WYKA

2.3 I n f r a r e d Spectrum

The i n f r a r e d spectrum ( F i g u r e 5 ) o f c l o t r i m a z o l e
as a d i s p e r s i o n i n m i n e r a l o i l was o b t a i n e d b y u s i n g a
P e r k i n Elmer Model 180 i n f r a r e d spectrophotometer . The
major a b s o r p t i o n bands a r e g i v e n i n Table 111.

The i n f r a r e d s p e c t r u m o f c l o t r i m a z o l e as a
d i s p e r s i o n i n potassium bromide has been r e p o r t e d i n t h e
1i t e r a t u r e ( 5 ) .

Table I 1 1
I n f r a r e d Band Assignments

Wavenumber ( c m - l ) Assignment

3170, 3115, 3085, 3075 (w) aromatic C-H ( s t r e t c h )

1585, 1570 (w) aromatic C=C, C=N


(stretch)

1510, 1500, 1450 (m) a r o m a t i c C=C, C=N


(stretch)

770, 760, 750 ( v s ) aromatic C-H o u t - o f - p l ane


bend

720, 700, 680 ( S - V S ) aromatic C-H o u t - o f - p l ane


bend

Notations: vs = very strong; s = strong; m = medium;


w = weak

2.4 U l t r a v i o l e t Spectrum

The u l t r a v i o l e t s p e c t r a o f c l o t r i m a z o l e i n
methanol and methanol i c 0.1N h y d r o c h l o r i c a c i d ( F i g u r e 6 )
were o b t a i n e d b y u s i n g a C a G Model 118 spectrophotometer.
The maxima, m i n i m a , s h o u l d e r s and r e s p e c t i v e m o l a r
a b s o r p t i v i t i e s are g i v e n i n Table I V .
WAVELENGTH MICRONS
2.5 3 4 5 6 7 8 9 10 12 14 1822 3550

Figure 5. I n f r a r e d Spectrum o f C l o t r i m a z o l e i n Mineral O i l .


234 JOHN G . HOOGERHEIDE AND BRUCE E. WYKA

250 300 350


WAVELENGTH (nm)

Figure 6 . Ultraviolet Spectra o f Clotrimazole.


CLOTRIMAZOLE 235

Table I V

U 1t r av io 1e t Spectra 1 Character is t ics o f C 1o t r imazo 1e


2
Solvent A (nm) E x 10-

Methanol 274 (shoulder) 1.03


269 (shoulder) 2.40
265 (shoulder) 3.32
263 (shoulder) 3.21
259 (maximum) 3.40
253 (maximum) 2.94
249 (minimum) 2.64

Methanol i c 274 (shoulder) 1.75


0.1N 269 (shoulder) 3.28
hydrochloric 263 (maximum) 3.96
acid 261 (minimum) 3.84
259 (maximum) 3.93
254 (shoulder) 3.12
246 (minimum) 2.38

2.5 M e l t i n g Range

The f o l l o w i n g me1 t i n g ranges have been r e p o r t e d :

M e l t i n g Range Reference

143 t o 144OC 596


144 t o 145OC 7

141 t o 145OC a

2.6 Thermal P r o o e r t i e s

2.6.1 D i f f e r e n t ia1 Scann i n g C a l o r i m e t r y

The d i f f e r e n t i a l scanning c a l o r i m e t r y c u r v e
( F i g u r e 7 ) o f c l o t r i m a z o l e was o b t a i n e d b y u s i n g oa DuPont
Model 990 Thermal Analyzer a t a h e a t i n g r a t e o f 1 0 C/minute
under a n i t r o g e n atmosphere. A s i n g l e sharp endotherm was
observed w i t h an e x t r a p o l a t e d onset temperature o f 143OC.
236 JOHN G. HOOGERHEIDEAND BRUCE E. WYKA

P u r i t y a n a l y s i s b y d i f f e r e n t i a l scanning c a l o r i -
mgtry ( F i g u r e 8 ) was p e r f o r m e d a t a h e a t i n g r a t e o f
1 C/minute under a n i t r o g e n atmosphere. The p u r i t y o f t h e
sample was d e t e r m i n e d t o b e 99.5 m o l e p e r c e n t . The l a t e n t
h e a t o f f u s i o n AH^) was c a l c u l a t e d t o be 7540 c a l / m o l .

2.6.2 Thermograv i m e t r y

Thermogravimetric a n a l y s i s ( F i g u r e 9 ) o f c l o t r i -
mazole was p e r f o r m e d b y u s i n g a DuPont Modelo950 Thermo-
g r a v i m e t r i c A n a l y z e r a t a h e a t i n g r a t e o f 10 C/minute under
a n i t r o g e n atmosphere. No weighto loss was observed f r o m
ambient temper,ature t o about 180 C. The g r a d u a l w e i g h t
loss above 180 C i s due t o v a p o r i z a t i o n o f t h e m e l t .
2.7 Crystal Properties

2.7.1 X-Rav D i f f r a c t i o n

The X-ray powder d i f f r a c t i o n p a t t e r n ( F i g u r e 10)


o f c l o t r i m a z o l e was o b t a i n e d b y u s i n g a P h i l l i p s DP-3500
X - r a y D i f f r a c t o m e t e r and Cu K, r a d i a t i o n (1.5148 if
) . The
d a t a a r e g i v e n i n T a b l e V.

2.7.2 P o l ymorph ism

Borka e t a l . ( 6 ) have r e p o r t e d t h e f o r m a t i o n o f a
metastable f o f i o f clotrimazole from a cr%stal f i l m
p r e p a r a t i o n . They r e p o r t a m e l t i n g p o i n t o f 106 C f o r t h i s
m e t a s t a b l e f o r m and an i n f r a r e d spectrum t h a t i s d i f f e r e n t
from t h e s t a b l e form.
CLOTRIMAZOLE !37

'0
TEMPERATURE. "c
F i g . 7. D i f f e r e n t i a l S c a n n i n g C a l o r i m e t r y Curve of
Clotrimazole.

TEMPERATURE, *K
F i g . 8. D i f f e r e n t i a l S c a n n i n g C a l o r i m e t r y Curve of C l o t r i -
mazole f o r P u r i t y A n a l y s i s .
W
7
0
N
a
E
.r
L
+J
0
c
0
le
0
0)
w
L
3
0
1,
L
c,
a,
E
.I-
5
l-0
L
rn
0
E
L
a,
L:
I-
0,
W
L
S
m
*I-
L L
238
1
00

01 I I 1 1 i 1 1 i
40 36 32 20 24 20 16 12 8
28

Figure 10. Powder X-ray D i f f r a c t i o n P a t t e r n o f Clotrimazole.


JOHN G. HOOGERHEIDE AND BRUCE E. WYKA

Table V
X-Ray Powder D i f f r a c t m t e r n o f C l o t r i m e r o l e

-
28 d(!lr -
wb
9.172 9.641 80
9.398 9.410 10
9.817 9.009 47
10.203 8.669 10
12.361 7.161 100
14.178 6.247 19
15.159 5.345 7
15.641 5.665 7
16.645 5.326 32
l a . 517 4.791 a2
15.415 4.572 68
19.830 4.477 37
20.647 4.302 61
22.502 3.951 29
22.983 3.870 43
24.101 3.693 36
24.200 3.678 36
24.266 3.668 36
25.086 3.550 41
25.519 3.490 22
26.177 3.404 14
27.474 3.246 32
28.132 3.172 46
28.354 3.148 26
28.810 3.099 14
30.923 2.892 11
30.999 2.885 11
31.318 2.856 9
31.461 2.843 7
31.783 2.815 16
32.655 2.742 23
34.182 2.623 11
34.503 2.599 19
34.577 2.594 19
36.980 2.431 10
37.080 2.424 10
37.196 2.417 9
37.374 2.406 8
37.598 2.392 11
37.680 2.387 11
37.778 2.381 10
37.057 2.376 10
37.989 2.372 8
37.998 2.368 8
38.513 2.337 9

a d ( i n t e r p l a n a r distance) = n A/2 s i n
1/11 = r e l a t i v e i n t e n s t t y
CLOTRIMAZOLE 241

2.8 Solubilitv

The s o l u b i l i t y o f c l o t r i m a z o l e has been d e t e r m i n e d


i n solvents by using v i s u a l , g r a v i m e t r i c o r spectrophoto-
m e t r i c analysis (Table V I ) .

2.9 D i s s o c i a t i o n Constant

The pKa o f c l o t r i m a z o l e i n 50% aqueous e t h a n o l i s


r e p o r t e d t o be 4.7 ( 5 ) .

Table V I

S o l u b i l i t y o f C l o t r i m a z o l e i n Common S o l v e n t s

Measured

Solvent (m m ° C Met hod

Acetone 50 grav i m e t r i c
Benzene >loo visual
Chloroform >loo visual
Diethyl ether 14 g r av ime t r ic
Dimet h y l formamide >loo visual
Dimet h y l s u l f o x i d e 45 spec t r o p h o t omet r ic
E t h a n o l USP 95 grav imetr i c
Ethyl Acetate 45 g r av i m e t r i c
Met h ano 1 >loo visual
Mineral o i l 0.8 spec t r o p h o t omet r ic
Petroleum ether 1.1 spec t r o p h o t o m e t r ic
P o l y e t h y l e n e g l y c o l 400 60 spec t r o p h o t omet r ic
P r o p y l ene g l yco 1 35 spectrophotometr i c
Water <0.03 spectrophotometric
242 JOHN G . HOOGERHEIDE AND BRUCE E. WYKA

3. Synthesis

Clotrimazole i s synthesized by t h e r e a c t i o n o f
o - c h l o r o t r i t y l c h l o r i d e w i t h i m i d a z o l e i n t h e presence o f a
t e r t i a r y amine, as d e s c r i b e d b y Buechel, e t a l . ( 5 ) as
shown i n F i g u r e 11. The y i e l d i n this-syythesis is
solvent-dependent; reactions i n solvents w i t h h i g h
d i e l e c t r i c constants g i v e the higher y i e l d s .

Maul and S c h e r l i n g ( 9 ) used barium { 4C) c a r b o n a t e


as s t a r t i n g m a t e r i a l t o s y n t h e s i z e ' ' C - c l o t r i m a z o l e . They
made t h e i n t e r m e d i a t e s 2 - c t j l o r o - { c a r b o x y l - C ) benzoic
a c i d , 2-ch\ojo- { c a r b o x y l - C 1 benzoylchloride, 2-chloro-
C 1 benzophenone, ( 2 - c h l o r o p h e n y l d i p h e n y l -
{carboxyl-
{ 1 4 C ) methanol and (2-chloropheny1)diphenyl - { ' 1 C)-
methylchloride en route tq radiolabelled clotrimazole,
1-(2-~hlorophenyl)diphenyl-{ k1- methyl-1H-imidazole.
-

4. Stability

C l o t r i m a z o l e i s s t a b l e i n t h e s o l i d s t a t e unger
normal s t o r a g e c o n d i t i o n s . It i s u n a f f e c t e d b y h e a t (70 C)
and exposure t o d a y l i g h t f o r up t o two weeks (10).

I n s o l u t i o n , t h e s t a b i l i t y o f c l o t r i m a z o l e i s pH
dependent. I n an a l k a l i n e medium i t i s s t a b l e , b u t
h y d r o l y z e s i n an a c i d i c medium t o ( 0 - c h l o r o p h e n y 1 ) -
diphenylmethanol p l u s i m i d a z o l e . Buechel e t a l . r e p o r t on,
the relative hydrolytic stability of notrirnazole i n
s o l u t i o n i n e t h a n o l - w a t e r and i s o p r o p a n o l - w a t e r m i x t u r e s
under a c i d i c , n e u t r a l , and a l k a l i n e c o n d i t i o n s ( 4 ) .

Thermal and 1 i g h t pH-stabi 1 i t y s t u d i e s have been


performed. Known amounts o f c l o t r i m a z o l e were s e a l e d i n
g l a s s ampuls a f t e r t h e a d d i t i o n o f 10 m l o f an aqueous
b u f f e r . The pH range s t u d i e d was 1 t o 13. Table V I I g i v e s
t h e r e s u l t s f o r samples s t o r e d a t 7 5 0 , 850, and 95OC f o r
one week. R e s u l t s are g i v e n i n Table V I I I f o r samples
s t o r e d i n t h e d a r k and u n d e r 350 f o o t - c a n d l e s o f
f l u o r e s c e n t l i g h t f o r t h r e e months. Analyses were
performed by u s i n g t h i n - l a y e r chromatoqraphy and e l u t i o n
f o l l o w e d b y UV s p e c t r a l a n a l y s i s (10).
CLOTRIMAZOLE 243

Clp / PCI 3
CH2CI UV-light

CI CI
(1) (I[)

cm 1

J
(rn)
F i g u r e 11 S y n t h e t i c Pathway t o C l o t r i m a z o l e
I . 2-chlorobenzylchloride
11. 2-chlorobenzotrichloride
I1 I . 2-chlorotri tyl chloride
I V . Clotrirnazole

Table V I I

pH-Thermal S t a b i l i t y P r o f i l e

Recovery o f
C l o t r i r n a z o l e i n Percent*

pH -
- Buffer State -
75OC 85OC
-- 95OC
-

1 Hydroch 1o r i c a c i d solution 0 0 0
2 C it r a t e part solution 0 0 0
4 Citrate suspension 84 55 4
4 Acetate suspension 84 65 6
6 Phosphate suspension 101 98 90
7 Phosphate suspension 105 101 94
8 Phosphate suspension 105 101 105
10 Borate suspension 102 102 100
13 Sodium h y d r o x i d e suspension 101 105 102

* A f t e r one week.
244 JOHN G . HOOGERHEIDE AND BRUCE E. WYKA

5. Drug Metabolism and Pharmacokinetics

5.1 Drua Metabolism

After oral or topical administration clotrimazole


undergoes r a p i d b i o t r a n s f o r m a t i o n i n t o i n a c t i v e met a-
bolites. Duhm and c o - w o r k e r s (11) i s o l a t e d f i v e
c l o t r i m a z o l e m e t a b o l i t e s f r o m r a t u r i n e and b i l e b u t found
no c l o t r i m a z o l e . S t r u c t u r e s o f t h e metabol i t e s a r e shown
i n F i g u r e 12.

Human u r i n e and serum t e s t e d a f t e r o r a l doses


o f c l o t r i m a z o l e c o n t a i n o n l y t r a c e amounts o f t h e d r u g
substance. The two m a j o r m e t a b o l i t e s found i n u r i n e ,
serum, and b i l e a r e ( 2 - c h l o r o p h e n y l ) (4-hydroxypheny1)-
phenylmethane and ( 2 - c h l o r o p h e n y l ) bis-phenylmethane; a
s m a l l e r amount o f (2-chloropheny1)bis-phenylmethanol is
a1 so p r e s e n t .

5.2 Pharmacokinetics

E a r l y c l i n i c a l s t u d i e s employing o r a l adminis-
t r a t i o n o f c l o t r i m a z o l e p r o v i d e d d a t a on t h e pharmaco-
k i n e t i c s o f s y s t e m i c a l l y d i s t r i b u t e d drug substance i n
humans. As discussed above, metabolism of c l o t r i m a z o l e i s
e x t r e m e l y r a p i d , and r e 1 i a b l e pharmacokinetics d a t a have
been o b t a i n e d o n l y b y u s i n g a n a l y t i c a l m e t h o d s w h i c h
determine b o t h $tle d r u g substance and m e t a b o l i t e s . In
experiments w i t h C - l a b e l l e d drug, Duhm - et -a l . (11) found
t h a t no c l o t r i m a z o l e was d e t e c t e d i n serum f o r a 20 m i n u t e
i n t e r v a l a f t e r dosing. Peak serum l e v e l s o f up t o 4 u g h 1
were confirmed b y a number o f workers (1,12-15); these
l e v e l s were reached between two and f o u r h o u r s a f t e r d o s i n g
i n a d u l t s and a t about s i x hours a f t e r a d m i n i s t r a t i o n t o
children. Rosenkrantz and P u e t t e r (16) e s t i m a t e d t h a t up
t o 98% o f c l o t r i m a z o l e i n serum i s bound t o serum p r o t e i n s .

Several workers have f o l l o w e d systemic c l o t r imazole


c l e a r a n c e b y d e t e r m i n i n g t h e d r u g and m e t a b o l i t e s e x c r e t e d
i n urine. W e i n g a e r t n e r , e t a l . ( 1 7 ) f o u n d no d r u g
substance i n u r i n e u n t i l b e t w e e n 3 0 and 60 minutes a f t e r
a d m i n i s t r a t i o n ; t h e y r e p o r t e d peak u r i n e c o n c e n t r a t i o n s a t
between 6 and 1 2 h o u r s . Plempel and co-workers (1) have
r e p o r t e d peak u r i n e c o n c e n t r a t i o n s o f 30 t o 60 u g h 1 1 2 t o
14 hours a f t e r d o s i n g .
CLOTRIMAZOLE 245

Table V I I I

pH-L i g h t S t a b i l i t y P r o f i l e

Recovery o f
C l o t r i m a z o l e i n Percent**
pH -
- Buffer State Light Dark
-

1 Hydrochloric acid sol ut ion 9 12


2 C it r a t e part solution 20 41
4 Citrate suspension 98 102
6 Phosphate suspension 97 103
7 Phosphate suspension 99 102
8 Phosphate suspension 101 103
10 Borate suspension 99 103
13 Sod iurn h y d r o x i d e suspension 103 99

**Stored i n t h e dark and under 350 f o o t - c a n d l e s o f f l u o r e s c e n t


1 i g h t for 3 months.

I I
OH OH

CI

F i g u r e 12. C l o t r i m a z o l e M e t a b o l i t e s :
I . (2-chlorophenyl )diphenylmethanol
I I . ( 2-chl orophenyl ) d i phenylmethane
III. (2-chlorophenyl ) , (4-hydroxyphenyl ) phenyl-
methane
IV. (2-chlorophenyl ) , (4-hydroxyphenyl ) p h e n y l -
me t h a no1
V. 2-chlorobenzophenone
246 JOHN G. HOOGERHEIDE AND BRUCE E. WYKA

Pharmacokinetics o f t o p i c a l l y appl i e d c l o t r i m a z o l e
has been s t u d i e d i n s e v e r a l l a b o r a t o r i e s . Duhm a n d
co-workers ( 1 8 ) found t h a t w h i l e C - c l o t r i m a z o l e appl i e d
as a cream p e n e t r a t e d t h e s k i n t o a d e p t h o f 2000 bm, no
d r u g s u b s t a n c e o r m e t a b o l i t e s were d e t e c t e d i n t h e serum.
A s m a l l q u a n t i t y o f t h e m a t e r i a l ( u p t o 0.4%) was d e t e c t e d
i n t h e u r i n e over a f i v e - d a y p e r i o d . In a s i m i l a r study,
H o l t ( 1 9 ) used a m i c r o b i o l o g i c a l a s s a y w i t h a d e t e c t i o n
l i m i t o f 0.01 pg/ml; over a t h i r t y - d a y p e r i o d o f
c l o t r i m a z o l e c r e a m a p p l i c a t i o n h e o b s e r v e d no d r u g
s u b s t a n c e i n e i t h e r serum o r u r i n e . Wallace, - et -al. (20)
n o t e d t h a t 24 h o u r s a f t e r a t e n - d a y t r e a t m e n t w i t h
clotrimazole t o p i c a l preparations, drug l e v e l s i n t h e skin
were as h i g h as 2 pg/mg; t h i s v a l u e d e c r e a s e d t o a b o u t
0.4 vg/mg a f t e r f o u r d a y s .

In studies using c l o t r i m a z o l e vaginal t a b l e t s ,


100 mg, Duhm e t a l . ( 1 8 ) f o u n d peak serum l e v e l s o f
0.03 pg/ml a f t e r 2 4 h o u r s .

P h a r m a c o k i n e t i c s and p h a r m a c o l o g y o f c l o t r i m a z o l e
a r e f u r t h e r c o v e r e d i n a c o m p r e h e n s i v e summary b y Sawyer,
.
e t a1 ( 2 1 ) as we1 1 as i n r e v i e w s b y Meade ( 2 2 ) and Seneca
v3r
6. Methods o f A n a l y s i s

6.1 Elemental A n a l y s i s

Conventional procedures f o r t h e d e t e r m i n a t i o n o f
C,H,N, and C1 y i e l d e d t h e f o l l o w i n g r e s u l t s f o r a sample
c o n f o r m i n g t o USP XX s p e c i f i c a t i o n s .

%
-
E 1ement Found Theory

C 76.63 76.63
H 4.93 4.97
N 8.12 8.12
c1 10.18 10.28
CLOTRIMAZOLE 247

6.2 Identification

S e v e r a l methods have been proposed f o r t h e


identification o f clotrimazole. Kuhnert-Brandstaetter, e t
-
a l . ( 8 ) observed t h a t t h e d r u g s u b s t a n c e f o r m s e u t g c t i c s
w i t h p h e n a c e t i n and b e n z a n i l i d e w h i c h m e l t a t 110 and
115 C, r e s p e c t i v e l y . D a t a on e u t e c t i c m e l t i n g p o i n t s i s
used o to c o n f i r m t h e i d e n t i t y o f c l o t r i m a z o l e , w h i c h m e l t s
a t 143 C .

K r i h a r , e t a l . ( 2 4 ) have c h a r a c t e r i z e d t h e
u l t r a v i o l e t s p e c t r a o f T o t r i m a z o l e i n m e t h a n o l and i n 0.1N
HC1 as an a i d t o t h e i d e n t i f i c a t i o n o f t h i s d r u g substance,

The USP X X i d e n t i f i c a t i o n t e s t s f o r c l o t r i m a z o l e
( 2 5 ) i n c l u d e b o t h t h i n - l a y e r chromatography, i n w h i c h t h e
sample s p o t must appear a t t h e same R f v a l u e as r e f e r e n c e
s t a n d a r d m a t e r i a l , and i n f r a r e d s p e c t r o p h o t o m e t r y .

6.3 Spectrophotometric Analysis

L i m i t e d u s e h a s b e e n made o f u l t r a v i o l e t
s p e c t r o p h o t o m e t r y i n t h e a n a l y s i s o f c l o t r i m a z o l e due t o
i t s low a b s o r p t i v i t y . Kr6&nar, e t a l . ( 2 4 ) suggested t h a t
such a n a l y s e s s h o u l d be p o s s i b l e ; S z a b o l c s ( 2 6 ) used
m e a s u r e m e n t s a t 2 6 1 nm t o a s s a y c l o t r i m a z o l e i n
formulations.

Upon h e a t i n g w i t h t r i c h l o r o a c e t i c o r p e r c h l o r i c
a c i d s , s o l u t i o n s o f c l o t r i m a z o l e become b r i g h t y e l l o w ; t h e
c o l o r fades r a p i d l y w i t h t r i c h l o r o a c e t i c a c i d b u t p e r s i s t s
with perchloric acid (1). T h i s r e a c t i o n , which forms t h e
b a s i s f o r a c o l o r i m e t r i c assay o f t h e d r u g s u b s t a n c e , g i v e s
s o l u t i o n s which obey B e e r ' s l a w up t o c o n c e n t r a t i o n s o f 1 0
pg/ml when r e a d a t 436 nm ( 2 7 ) . T h i s method has been used
f o r t h e e s t i m a t i o n o f c l o t r i m a z o l e i n b i o l o g i c a l f l u i d s and
t i s s u e s a f t e r e i t h e r s o l v e n t e x t r a c t i o n (16,27) o r
extraction followed by thin-layer chromatography
(1,13,17,28).
248 JOHN G.HOOGERHEIDE AND BRUCE E. WYKA

6.4 T i tr i m e t r i c An a1y s is

Assay o f c l o t r i m a z o l e b u l k d r u g s u b s t a n c e i s
p e r f o r m e d b y nonaqueous t i t r a t i o n ( 2 5 ) . The s a m p l e
d i s s o l v e d i n g l a c i a l a c e t i c a c i d i s t i t r a t e d w i t h 0.1M
perchloric acid i n glacial acetic acid t o a green
e n d - p o i n t ; p - n a p h t h o l b e n z e i n i s used as i n d i c a t o r . Each
m i l l i l i t e r o f 0.1M p e r c h l o r i c a c i d i s e q u i v a l e n t t o 34.48
m i l 1i g r a m s o f c l o t T i m a z o l e .

A f t e r e x t r a c t i o n with c h l o r o f o r m , Szabolcs ( 2 6 )
assayed c l o t r i m a z o l e i n p h a r m a c e u t i c a l f o r m u l a t i o n s b y
t i t r a t i o n w i t h 0.1M - p e r c h l o r i c a c i d ; g e n t i a n v i o l e t was
used as i n d i c a t o r .

A two-phase t i t r a t i o n method developed b y


P e l l e r i n , e t a l . ( 2 9 - 3 1 ) s e r v e s as a b a s i s f o r t h e
t i t r a t i o n o f c T o t r i m a z o l e w i t h sodium l a u r y l s u l f a t e .
Samples o f c l o t r i m a z o l e f o r m u l a t i o n s a r e p a r t i t i o n e d
between c h l o r o f o r m and 2N s u l f u r i c a c i d . M e t h y l y e l l o w i s
a d d e d as i n d i c a t o r a n d t h e m i x t u r e t i t r a t e d w i t h
s t a n d a r d i z e d sodium l a u r y l s u l f a t e i n w a t e r . The end p o i n t
i s r e a c h e d when t h e c h l o r o f o r m l a y e r t u r n s g o l d - o r a n g e
( 3 2 ) . T h i s s t a b i l i t y - i n d i c a t i n g t i t r a t i o n has been used t o
assay c l o t r i m a z o l e i n f o r m u l a t i o n s (32,33) and t o f o l l o w
h y d r o l y s i s o f t h e drug substance ( 5 ) .

6.5 Chromatograph ic A n a l y s i s

6.5.1 Paper Chromatography

C l o t r i m a z o l e c a n be s e p a r a t e d f r o m i t s i m p u r i t i e s
and d e g r a d a t i o n p r o d u c t s b y d e s c e n d i n g p a p e r c h r o m a t o g r a p h y
(10). The method uses paper i m p r e g n a t e d w i t h p r o p y l e n e
g l y c o l and a m o b i l e phase o f p r o p y l e n e g l y c o l - s a t u r a t e d
l i g r o i n . C l o t r i m a z o l e i s d e t e c t e d a t R f = 0.4 b y s p r a y i n g
w i t h Dragendorff reagent,

6.5.2 Th i n - L a y e r Chromatography

T h i n - l a y e r c h r o m a t o g r a p h y (TLC) o n s i l i c a g e l h a s
been used e x t e n s i v e l y t o s e p a r a t e c l o t r i m a z o l e f r o m
f o r m u l a t i o n and b i o l o g i c a l m a t r i c e s p r i o r t o q u a n t i t a t i o n .
A summary o f t h i n - l a y e r a d s o r b e n t s and m o b i l e phases used
i n these separations i s g i v e n i n Table I X .
Table I X
Th in-L ayer Chromatography Systems f o r C 1o t r imazol e

P l a t e Medium So 1vent Detect i o n R f Value Reference


( see be1ow) (see below) (see below)

1 s t Dimension : 0.57
2nd Dimens i o n : I 0.64 9

1 s t Development:
2nd Development: I1 - 17

1 s t Development:
2nd Development: I1 - 13

1 s t Development:
2nd Development: TI1 0.4 27

F I11 0.55 27

C I1 0.3 1,28

G IV 0.65 25

H IV,V 0.4 25

J IV,V 0.85 10
250 JOHN G. HOOGERHEIDEAND BRUCE E. W Y K A

Table IX (continued)
Plate Medium
a. Silica gel 60.
b. Silica gel foil (Polygram Sil NH.R, Machery-
Nagel ) .
c. Silica gel 60 F .
Solvent System
A. Benzene:methanol (4:l).
B. Methanol.
C. Chloroform.
D. Petroleum ether (40-60°):acetone:
benzene:ethanol :pyr id ine
(70:12:10:7:1).
E. Benzene.
F. Petroleum ether (40-60') :ethyl-
acetate:acetone:ethanol :
ammon i a ( 25%) (45:25 :25 : 5 :0.5 ) .
G. Xy1ene:n-propanol :ammonia (180:20:1).
H. Ether equilibrated with ammonia vapor.
J. Ethyl acetate:ammonia (25%) (98:2).
Detection Method
I. Autoradiography.
11. Trichloroacetic acid in n-butylacetate.
111. Spray sequentially with ethanolic iodine, sodium
carbonate, and sulfanil ic acid/sodium nitrite.
IV. Fluorescence quenching under short-wave
ultraviolet light.
V. Spray with Dragendorff reagent.
CLOTRIMAZOLE 25 1

Q u a n t i t a t i v e a n a l y s e s e m p l o y i n g TLC h a v e b e e n
c a r r i e d o u t i n s e v e r a l ways. R i t t e r , e t a l . (27) used
densitometry t o determine c l o t r i m a z o l e t h e TLC p l a t e
a f t e r formation o f a red s u l f a n i l i c acid derivative. Their
c a l i b r a t i o n c u r v e s were l i n e a r between 0.5 and 4 p g c l o t r i -
mazole per spot. O t h e r i n v e s t i g a t o r s have d e t e r m i n e d t h e
d r u g substance s p e c t r o p h o t o m e t r i c a l l y b y s c r a p i n g t h e bands
o f f t h e TLC p l a t e and r e a c t i n g e i t h e r w i t h b u t y l a c e t a t e
and p e r c h l o r i c a c i d (1,13,17,28) o r w i t h bromphenol b l u e
(10).
6.5.3 Gas Chromatography

C l o t r i m a z o l e i n human s k i n s a m p l e s h a s b e e n
d e t e r m i n e d b y gas c h r o m a t o g r a p h y w i t h e l e c t r o n - c a p t u r e
detection (20,34). Samples were e x t r a c t e d w i t h e t h e r ,
d r i e d , r e d i s s o l v e d i n benzene and c h r o m a t o g r a p h e d on 6',
1/8" g l a s s columns packed wdth 3% OV-17 on Gas Chrom Q.
Column t e m p e r a t u r e was 250 C and t h e c a r r i e r gas was
argon-methane a t 9 ml/min. S t a n d a r d c u r v e s were l i n e a r
o v e r t h e r a n g e o f 1-25 ng i n j e c t e d .

6.5.4 H i g h P e r f o r m a n c e L i q u i d Chromatography

S t a b i l i t y - i n d i c a t i n g assays o f c l o t r i m a z o l e i n t h e
b u l k d r u g s u b s t a n c e and i n f o r m u l a t i o n s have been c a r r i e d
o u t b y h i g h performance 1 i q u i d c h r o m a t o g r a p h y ( 3 5 ) . The
a n a l y s e s were p e r f o r m e d on a IA Bondapak C i a column w i t h a
m o b i l e phase o f methanol:O.O25M K 2 H P h ( 3 : l ) a t 1 . 0 m l / m i n .
Chromatographic r e s p o n s e was l i n e a r f r o m 2 t o 40 p g c l o t r i -
mazole i n j e c t e d . Average r e c o v e r y o f d r u g s u b s t a n c e f r o m
f o r m u l a t e d m a t e r i a l s r a n g e d f r o m 99.5 t o 100.0 p e r c e n t .
A n a l y s e s were r e p r o d u c i b l e , w i t h between-day r e l a t i v e
s t a n d a r d d e v i a t i o n s between 0.6 and 1.8 p e r c e n t .

6.6 Radiochemical A n a l y s i s
14
Duhm, e t a l . (18,36) used C-labelled c l o t r i -
mazole t o detEmTne drug d i s t r i b u t i o n s i n b i o l o g i c a l
samples. R a d i o a c t i v i t y was d e t e r m i n e d i n serum and u r i n e
b y t h e use o f l i q u i d s c i n t i l l a t o r s c o n t a i n i n g 8 g / 1 o f
b u t y l PBD i n t o l u e n e : d i o x a n e * e t h a n o l ( 1 : l : l ) . S k i n samples
were burned; t h e r e s u l t i n g I4C02 was absorbed i n base and
c o u n t e d b y means o f s c i n t i11 a t o r s .
252 JOHN G. HOOGERHEIDE AND BRUCE E. WYKA

6.7 Microcalorimetr i c Analysis

I n h i b i t i o n o f t h e r e s p i r a t i o n o f Saccharomyces
c e r e v i s i a e b y a n t i f u n g a l agents p r o v i d e s t h e b a s i s f o r a
s e n s i t i v e c l o t r i m a z o l e assay. Beezer and c o - w o r k e r s ( 3 7 )
m o n i t o r e d r e s p i r a t i o n m i c r o c a l o r i m e t r i c a l l y b e f o r e and
a f t e r a d d i t i o n o f c l o t r i m a z o l e ; degree o f i n h i b i t i o n o f
r e s p i r a t i o n was r e l a t e d t o t h e c o n c e n t r a t i o n of
c l o t r i m a z o l e . The minimum d r u g c o n c e n t r a t i o n d e t e c t a b l e b y
t h i s method was 3 x 10’5M.-

6.8 M i c r o b io 1og i c a l A n a l y s i s

M i c r o b i o l o g i c a l assays o f c l o t r i m a z o l e a r e a g a r
d i f f u s i o n assays based on a c o m p a r i s o n between t h e g r o w t h
i n h i b i t i o n zones p r o d u c e d b y s t a n d a r d s o l u t i o n s and t h o s e
produced b y t e s t samples. The t e s t o r g a n i s m C a n d i d a
p s e u d o t r o i c a l i s v a r . c a r s h a l t o n has been used b y several
*12,27,38) t o a s s a y c l o t r i m a z o l e i n serum,
u r i n e , and f e c e s . When t e s t i n g t h e s t a b i l i t y o f

++
c l o t r i m a z o l e i n c u l t u r e medium, H o e p r i c h and Huston ( 3 9 )
employed K l u v e r o m c e s f r a i l i s as t e s t o r g a n i s m .
s t u d y o f c o t r i m a z o l e s t a i i t v on samDle d i s c s . S a u b o l l e
and i o e p r i c h (40) used Candida” a l b i c a n s as t e s t ’ o r g a n i s m .
H o l t ( 4 1 ) has d i s c u s s e d s e o f s e v e r a l o r g a n i s m s and
In a

assay t y p e s i n t h e a n a l y s i s o f a n t i f u n g a l d r u g s , i n c l u d i n g
c l o t r i m a z o l e.

Two p a p e r s h a v e compared m i c r o b i o l o g i c a l a s s a y
r e s u l t s t o t h i n - l a y e r chromatographic determinations o f
c l o t r i m a z o l e (27,42).

7. Acknowledaements

The a u t h o r s w i s h t o t h a n k t h e S c h e r i n g C o r p o r a t i o n
Research L i b r a r y and P h y s i c a l and A n a l y t i c a l C h e m i s t r y
S t a f f s , i n p a r t i c u l a r Ms. Jean Nocka, Ms. M i c a e l a K a t z ,
D r . Henry S u r p r e n a n t , D r . Mohindar S. Puar, Ms. Jane
L i m p e r t , and Ms. L a u r e l Andersen, f o r t h e i r a s s i s t a n c e i n
the preparation o f t h i s analytical p r o f i l e .
CLOTRIMAZOLE 253

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E., Deutsch. Med. Wschr. 94, 1356 ( 1 9 6 9 ) .

2. Oberste-Lehn, H., Baggesen, I., and Plempel, M.,


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3. Marget, W. and Adam, D., -


Med.- n . 64, 1235 ( 1 9 6 9 ) .
K l i-

4. Buechel, K. H., Plempel, M., and Bartmann, K., E.


-
B e r . , 39 (1973).

5. Buechel, K. H., D r a b e r , W., Regel, E., and Plempel,


M., Arzneim. -
F o r s-
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6. Borka, L., and V a l d i m a r s d o t t i r , S., --


A c t a Pharm. -
Suec.
-
12, 479 ( 1 9 7 5 ) .

7. Schering-Plough L a b o r a t o r i e s , Qua1 i t y C o n t r o l Data.

8. K u h n e r t - B r a n d s t a e t t e r , M., Boesch, L., and E c k s t e i n ,


G., S c i . Pharm. 46, 54 ( 1 9 7 8 ) .

9. Maul, W. and S c h e r l i n g , D., -


J. Label. Comp.
Radiopharm. -
14, 403 ( 1 9 7 8 ) .

10. Schering-Plough Corporation, unpublished r e s u l t s .

11. Duhm, B., Medenwald, H., P u e t t e r , J., Maul, W.,


Med.- J .
Patzschke, K., and Wegner, L.A., P o s t g r a d . -
-
50, 13 (1974) S u p p l . 1.

12. Burgess, M. A. and Bodey, G. P., Antimicrob. Agents


Chemother. 2. 423 (19721.

13. W e i n g a e r t n e r , L . , G r u e n d i g , C., P a t s c h , R., and


S i t k a , U., Z e n t r a l b l . -
Pharm.- 111, 465 (1972).

14. Plempel, M., Postgrad. -


Med.- J-
. 55, 662 ( 1 9 7 9 ) .
15. Weuta, H., "Proc. 9 t h I n t e r n a t . Cong. Chemother.",
London, J u l y , 1975, i n Chemotherapy - 6, 179 ( 1 9 7 5 ) .
254 JOHN G . HOOGERHEIDE AND BRUCE E. WYKA

16. Rosenkranz, H. and P u e t t e r , J . , E.


2. D r u g . Metab.
Pharmaco. -
2, 73 (1976).

17. Weingaertner, L., Sitka, U., and G r u e n d i g , C., E.


-
J . -C l i n . Pharmacol. -
8, 131 (1973).

18. Duhm, B., Maul, W., Medenwald, H., P a t z s c h k e , K . ,


Wegner, L. A., and Oberste-Lehn, H., A r z n e i m . F o r s c h .
22, 1276 (1972).
-

19. J . -Cutan.
H o l t , R. J . , - - - P a t h o l . 3, 45 (1976
20. W a l l a c e , S. M . , Shah, V . P., E p s t e n, W. L.
Greenberg, J . , and Riegelman, S., 3. Dermatol,
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113, 1539 (1977).

21. S a w y e r , P. R . , Brogden, R. N . , Pinder, R. M.,


S p e i g h t , T. M., and Avery, G. S., Drugs -
9, 424
(1975).

22. Meade, R. H., Am. -J -


- - - Pharrn. 36, 1326 (1979).
. Hosp.
23. Seneca, H., B i o l o g i c a l B a s i s o f Chemotherapy o f
I n f e c t i o n s and I n f e s t a t i o n s , P h i l a d e l p h i a , D a v i s , pp.
921-929 (1971).

24. KrsEmar, J . , S o t o l o n g o , M. A., and Kr6Emarov6, J.,


Cesk.
-- Farm. - 26, 345 (1977).

25. U n i t e d S t a t e s Pharmacopeia X X , p . 159.

26. Szabolcs, L., --


A c t a Pharm. Hung. -
46, 43 (1976).

27. Ritter, W., Plempel, M., and P u e t t e r , J., Arzneim.


F o r_
_ h . -24,
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28. M a r g e t , W. and Adam, D., Acta Pediatr. - - 60, 341


Scand.
(1971).

29. P e l l e r i n , F., G a u t i e r , J . A., and Demay, D., Ann.


- - F-
Pharm. r a n c . 20, 97 (1962).

30. P e l l e r i n , F., G a u t i e r , J . A., and Demay, D., Ann.


Pharm. F r a n c . 22, 495 (1964).
CLOTRIMAZOLE 255

31. P e l l e r i n , F., G a u t i e r , J. A., and Demay, D., Talanta


12,
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32. U n i t e d S t a t e s Pharmacopeia XX, p . 160.

33. Kudo, A., Jap. -J. -C l i n . Rep. -


6, 85 (1972).

34. Wallace, S. M., Shah, V. P., Riegelman, S., and


E p s t e i n , W. L., -
Anal. - 611, 461 (1978).
Lett. -

35. Hoogerheide, J. G . , S t r u s i a k , S. H., Taddei, C. R.,


Townley, E. R., and Wyka, B. E., J. - Assoc.-O f f-
. Anal.
Chem. ( t o be p u b l i s h e d J u l y , 1 9 8 l r .

36. Duhm, B., Maul, W., Medenwald, H., P a t z s c h k e , K.,


Wegner, L . A. and P u e t t e r , J., P o s t g r a d . Med. J. 50,
13 (1974), Suppl 1. .
37. Beezer, A., Chowdhry, 6. Z., N e w e l l , R. D., and
T y r r e l l , H. J . V., -
A n a l-
. Chem.
- 49, 1781 ( 1 9 7 7 ) .
38. H o l t , R. J. and Neman, R . L., J. C l i n . P a t h o l . 25,
1089 (1972).
39. Hoeprich, P. D. and Huston, A. C., A b s t r a c t 104,
Proc. -
- 1 4 t h I n t e r s c i e n c e Conf. Antimicrob A X s
Chemother., San F r a n c i s c o , CA, S e p t . 11-13 (i97*

40. S a u b o l l e , M. A. and H o e p r i c h , P. D., A b s t r a c t 102,


Proc. -
- 14th I n t e r s c i e n c e Conf. A n t i m i c r o b . A q X s
Chemother., San F r a n c i s c o , CA, S e p t . 11-13 ( 1 9 7 4 ) .

41. H o l t , R. J . , J . C l i n . P a t h o l . 28, 767 ( 1 9 7 5 ) .

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.
M i c r o b i o l , Mexico, p . m , A C 9 - l m .

L i t e r a t u r e s u r v e y t e r m i n a t e d December, 1980.
DOPAMINE HYDROCHLORIDE
James E . Carter, John H . Johnson,
and David M. Bamke

1. Description 258
1.1 Chemical and Proprietary Names 258
1.2 Empirical Formula 258
1.3 Appearance, Color, and Odor 258
2. Physical Properties 258
2.1 Melting Range 258
2.2 Solubility Profile 259
2.3 Infrared Spectrum 259
2.4 Ultraviolet Spectrum 259
2.5 Proton Magnetic Resonance Spectrum 259
2.6 "C Magnetic Resonance Spectrum 263
2.7 Mass Spectra 265
2.8 Differential Scanning Calorimetry 266
2.9 Crystal Properties 266
3. Synthesis 268
4. Analysis 269
4.1 Elemental Analysis 269
4.2 Colorimetric Assay 269
4.3 Nonaqueous Titration 269
4.4 Thin-Layer Chromatography (TLC) 269
4.5 Gas Chromatography 270
4.6 High Performance Liquid Chromatography (HPLC) 270
5. Stability 270
6. Analysis of Biological Samples 27 1
7. Metabolism and Excretion 27 1
8. Acknowledgement 27 1
References 272

Copyright 0 1982 by The American


Analytical Profiles of Drug Substances
Volume I I 257 Pharmaceutical Association
ISBN 012-260811-9
258 JAMES E. CARTER E T A L .

1. Description
1.1 chemical and Proprietary Names
Dopamine hydrochloride is the mn-proprietary
name for 4-(2-aminoethyl)-1,2-benzenediol hydrochloride.
The free base (dopamine) has also been known in the
chemical literature as 3-hydroxytyramine and 3,4
dihydroxyphen~thylamine. The drug is available generically
for the correction of hamdynamic imbalances present in
the shock syndmm due to myocardial infarction, t r a m ,
endotoxic septicemia, open heart surgery, renal failure
and chronic cardiac decanpensation as in congestive heart
failure (1). Proprietary names include Cardiosteril, Docard,
Dopamine Fabre, Dopamine Gullini, Dopastat, Dynatra,
Intropin, Intropinject, Intropine and Orion.
1.2 Empirical F o d a

C8H12Cm2
Wlecular Weight
189.64
Structure

1.3 Appearance, Color & Odor


Dopamine hydrochloride is a white to off white
odorless crystalline pcxder.
2. Physical Properties
2.1 Melting range
The Merck Index reports the mlting point of the
hydrochloride salt as 241OC with decanposition (2). The
DOPAMINE HYDROCHLORIDE 259

equilibrium melting range ( i n the absence of a i r ) has been


established a s 245OC - 246OC with deccenposition ( 3 ) .

2.2 Solubility Profile

Dopamine hydrochloride is f r e e l y soluble i n water:


soluble i n mthanol and hot ethanol: p r a c t i c a l l y insoluble
in ether, chloroform, benzene and toluene. Dopamine
hydrochloride is soluble in aqueous solutions of a l k a l i
hydroxides.

2.3 Infrared Spectrum

The KBr pellet infrared spectrum of 0.5% d o w e


hydrochloride obtained with a Perkin-Elmer 283 Infrared
Spectrophotomter is contained i n Figgie 1. The b r q d strong
absorption bands appearing a t 3350 cm and 3230 cm are due
to the O-H and N-H stretching vibrations. The i n t e m l e c u l a r
and i n t r a m l e c u l a r hydrogen bonding s h i f t s these absorption
bands to-lower frequencies than a f r e e h y d r o q l group (3700 -
3500 an ) and a f r e e a q n e (3500 - 3300 an ) . The -1
aromatic (3100 - 3000 cm ) and a l i p h a t i c (3000 - 2900 cm )
C-H stretching bands a r e prevent as expected. The N-H
bending vibrations 25 tile NH group shaw medium absorption
centered a t 1610 cm . The 2-H out of plane bending of
the 1 , 2 , 4 t r i s u b s t i t u t e d F e n e ring i indicated by
the sharp bands a t 876 cm and 812 cm . -B
2.4 Ul'zaviolet spectrum
Dopamine hydrochloride exhibits a single absorption
peak centered a t 280 nm w i t h a mlar absorptivity (E) of 2707
(Figure 2 ) . The spectrum was obtained with a Beclanan Acta
I11 double beam spectro@otometer.

2.5 Proton Maanetic Resonance S m t r u m

The 60 MHz proton magnetic resonance spectrum was


obtained with a Varian Associates T-60A spectrcneter. The
spectrum i n CD OD with tetramethylsilane (TMS) as internal
reference is d n t a h e d i n Figure 3. The s p l i t i n g patterns
are not simple. Howsver, t h e integration and qeneral
locations of psaks are consistent w i t h the structure. The
peaks a t 0.8 and 3.35 are due to mall amounts of CH30H
in the deuterated solvent.
Fig. 1 KBr Infrared Spectrum of Dopamjne Hydrochloride.
Instmnent: Perkin-Elmer MAel 283
Oopamfne H y d r o c h l o r i d e
UV Spectrum

Scan Speed 0 . 5 nmlscc


Chart Expansion 20 nmlinch
C o n c e n t r r t l o n Span 1.0
deuterium
280 nm
Concentration 2.109 x 10-4 n o l e / L
Absorbance 0.571
Nolar AbsorDtivlty 2707
Reference Cell 1% Sodium B i r u l f i t e
Operator U.UrekSv;&
Date t i - i 2 - # /

I I
240 240 200 SbO $0 nm

Fig. 2 Ultraviolet spectrum of IXpamhe Hydrochloride


Instrument: BeclaMn Acta 111

261
I

r :
I I I I I!

f--- I

li

I I I I I
1 . . . . 1 . . . . 1 . . . . 1 . . . . 1 . . . . . I . . . . I . . . . I . 1 . . . I
80 7 0 60 50 PPM:d: 4 0 30 20 10 0

Fig. 3 Proton Magnetic Resonance Spectnnn of Dopamine Hydrochloride in CD3OD


Instrument: Varian T-60A
DOPAMINE HYDROCHLORIDE 263

Chemical s h i f t s (b) i n ppn r e l a t i v e to TMS are:

H O G C H ,-CH ,-NH ,.HCI

Proton # of Chemical
Assigment Protons S h i f t (b) Mu1t i p l i c i t y

a 4 3.0 multiplet
b 5 4.95 multiplet
C 3 6.75 multiplet

2.6 I3C Magnetic Resoname Spectrum

The "C analysis of dopamine hydrochloride was


taken on a JEOL FX-270 superconductiny NMR operating a t a
frequency of 67.83 IWz using a 45" (9 usec) pulse, a 3.6
second r e p e t i t i o n rate and 16384 data points ( 4 ) .

The sample was dissolved i n D20; P-pfoxane was


added. as a reference. Spectra of the e n t i r e C range were
obtained with ccmplete decoupling and gated decoupling with
Nuclear Overhauser Effect (NOE) to obtain the chemical s h i f t s
and coupling constants. The ccmpletely decoupled sFectrum is
sham i n F j w e 4.
The resolution of a l l a r m t i c carbons (not cham
in Figure 4 ) was obtained using a 2.5 kHz width. The
assignrrent of t h e a r m t i c carbon l i n e s was aided by
canparison to 3,4 dihydroxybenzene.
Fig. 4 l 3 C Magnetic Resonance Spectnnn of Dopamine Hydrochloride w i t h p-dioxane as reference.
Instrurtlent: JExlL FX 270
DOPAMINE HYDROCHLORIDE 265

With the carbons n-red as indicated below the


chemical shifts and coupling constants are as s h m in
T a b l e I.

r
2
HO

Table I. I3C assignments and C-H


coupling constants for
Dopamine Hydrochloride

Carbon Number Shift (ppn) Jm(Hz)


129.1 -
116.3 159.3
143.9 -
142.7 -
116.4 156.7
121.1 160.0
32.0 128.3
4c.7 143.9
2.7 Mass Spectra

The electron inpact (EI) mass spectnrm at 70 eV


and the methane derived chemical ionization (CI) mass
spectrum were obtained with a Hewlett-Packard 5985 quadraple
mass spectrcaneter (5).
The CI spectrum contains t w prcaninent ions at n4e
137.0 and m/e 154.0. The m/e 154.0 corresponds to C H NO
which is protonated dopamine. Loss of m n i a leave8 &gg8;
which is the base peak i n the spec=tnrm at m/e 137.0.
266 JAMES E. CARTER ETAL.

The E I spectrum i s surprisingly simple. The base


peak a t m/e 124.1 corresponds to C H 0 The intact
dopamine a t m/e 153.0 i s seen in &e8s&ctrum. The m/e
77.0 and m/e 78.l+are due $0 the a m t i c r i n g and
correspond t o C H and C F respectively. The
hydrochloride s812 is no$ geen i n e i t h e r the E I o r C I
spectrum which js as expected.

2.8 Dif ferenti a1 Scanning Calorink=tq

Dopamine hydrochloride was heated a t a rate of 10'


per minute i n a Perkin-Elmr DSC-2 d i f f e r e n t i a l scanning
calorimeter frcan 460'K t o 530'K. A single sharp endothem
w a s observed with an onset temperature of 516'K (243OC) and
with the endothem maximum a t 519.8'K (246.8'C). An accurate
heat of t r a n s i t i o n bH) could m t be calculated because of
deccanposition upon rwlting.

2.9 C r y s t a l Properties

Crystals f r m a representative l o t of dopamine


hydrochloride are predaninantly triangular p l a t e s ( 3 ) . The
o p t i c a l crystallographic examination indicated t h a t the
crystals are triclinic or Fossibly m n o c l i n i c .

X-ray powder patterns w e r e obtained w i t h copper


radiation and a nickel f i l t e r . The d-spacings and r e l a t i v e
k t e n s i t i e s (I/I ) are listed i n T a b l e 11. The f i r s t column
shws the p a t t e r 8 f o r the drug a f t e r grinding to break up t h e
p l a t e s and therefore the preferred o r i e n t a t i o n of the
c r y s t a l s . Column 2 shows the d i f f r a c t i o n pattern of the
crystals without p r i o r grinding. The absence of several
key d i f f r a c t i o n l i n e s and the reduced influence of o t h e r s
i n this orientation is c l e a r l y d m n s t r a t e d by canparing
t h e two columns.
DOPAMINE HYDROCHLORIDE 267

TABLE I1
X-ray Diffraction M e r Patterns of Dopamine Hydrochloride

Ground Sample Unground Sample


d-spcing I/Io d-spacing IDo

6.37 5
5.56 30 5.56 10
5.50 60
5.24 5 5.24 20
4.57 75 4.58 15
4.18 95 4.19 30
4.07 30 4.07 15
3.98 100 3.97 15
3.81 65 3.82 100
3.72 60 3.71 15
3.53 70 3.53 30
3.49 45 3.49 65
3.43 70 3.43 20
3.23 65 3.23 20
3.19 45
2.96 10 2.97 5
2.83 10 2.83 5
2.77 55 2.77 15
2.75 45
2.69 10
2.63 60
2.62 60 2.62 80
2.55 60
2.41 10 2.55 15
2.27 10
2.17 20 2.17 10
1.90 10 2.09 5
1.99 5
1.86 10 1.96 5
1.92 5
1.90 5
JAMES E. CARTER E T A L .

3. Synthesis

Dopamine hydrochloride i s available from a variety


of ccprmercial sources. Details of the synthetic process are
considered proprietary information. However, one workab1.e
process using known reactions and readily available materials
is shown in Figure 5 ( 6 ) .

CH30~cHoCH3N02

CH3O
-
cH30D
CH,O
CH=CHNOp
H2

-
cH30x3
CH 3O
CH2CH2NH2
H Br
HO

Fig. 5 Synthetic Scheme for Dopamine Hydrochloride

The d h t h o q p h e n e t h v l a r h e (knma s
homveratrylamine) has been converted to dopamine
hydrochloride in one step with w i d b e hydrochloride ( 7 ) .
DOPAMINE HYDROCHLORIDE 269

4. Analysis

4.1 E l m t a l Analysis

Elerwntal analysis of a typical dopamine


hydrochloride sample is as follaws:

Element % Theoretical % Found

C 50.67 50.51
H 6.38 6.55
c1 18.70 I-

N 7.39 7.37
0 16.87 ---
4.2 Colorimetric Assay

A colorimetric assay f o r the analysis of d0pami.m


has been adapted f m The United S t a t e s Phaxmampeia
Epinephrine Assay ( 8 ) . "The Photarnetric Detection of
Adrenaline in P h a r m e u t i c a l Products" was f i r s t described by
Jhty in 1948 ( 9 ) .

4.3 Non-aqueous T i t r a t i o n

The purity of dopamine hydrochloride may be


assessed by non-aqueous t i t r a t i o n with crystal violet as
the indicator. The hydrochloride salt is dissolved in g l a c i a l
acetic acid, mercuric acetate is added to remove the chloride
as unionized mercuric chloride, crystal violet is added and
the amine is titrated to a green end-point with 0.1 N -
p r c h l o r i c acid.

4.4 Thin-layer Chromatoqraphy (TLC)

Thin-layer chrormtography on silica g e l is


p a r t i c u l a r l y helpful in assessing the purity of the r a w
drug. I n a solvent system of ethyl acetate-methanol-
m n i n u n hydrGxide (85:10 :5) a l l p o t e n t i a l
wthoxy-phenethylamine impurities are readily separated
and differentiated. The 3-hydroxy-4-methoxyphenethylamine and
dopamine fluoresce under s h o r t wave ultraviolet l i g h t .
After spraying w i t h ninhydrin and developing a t 110' f o r
5 minutes dopamine appears as a brown spot (R N 0.07) , the
f
3-hYdr0~-4-~thO analog
~ appears as a yellaw spot
(Rf N 0 . 2 2 ) , the 3-methoxy-4-hydroxy analog appears as
a pink spot (Rf N 0.26) and the dimethoxy compound a p p a r s
as a pink spot (Rf N 0.30).
270 JAMES E. CARTER ETAL.

I n a solvent system of n-buitanol-glacial acetic


acid-water (12:3:5), doparnine has an R of approximately 0.6.
f
The canpounds may also be visualized by spraying t h e silica
p l a t e with 0.5% iodine i n chloroform.

4.5 Gas ChmnatouraDhv

Because of the high-boilirLg, polar and oxidizable


nature of dopamine, it i s not readily amenable
to gas chrmtography.

4.6 High Performance Liquid Chrmtography (HFTC)

N o work has been reported for t h e HPLC analysis of


dopamine hydroFhloride i n pharmaceutical dosage forms as
might be expected f o r a non-patented product. One relevant
papr reports t h e chrmatographic behavior of dopamine and
related catecholamines i n a reverse phase system on t h e
ubiquitous octadecylsilane column (10) . The authors found
dopamine to be insensitive t o pH changes b e b e e n 2 and 5
w i t h n i t r i c acid but to be retained longer as t h e pH
increased in acetic acid. Doparnine appeared to form ion
pairs being retained f o r longer times with t r i c h l o r o a c e t i c
acid and octylsulfonic acid than with mineral acids.

5. Stability

Dopamine hydrochloride ( l i k e other catecholamines) i s


r e l a t i v e l y unstable t o heat, l i g h t and oxygen. The presence
of oxidizing agents or trace mtals such as copper or iron
also increases t h e rate of degradation. Properly stored i n
g l a s s containers t h e raw drug i s stable f o r three to f i v e
years.

Dopamine hydrochloride is generally f o m l a t e d with 1%


sodium b i s u l f i t e as an antioxidant. The r e s u l t a n t solution
has a pH of about 4 and when properly protected f r m a i r , heat
and l i g h t i s s t a b l e f o r three to f i v e years.

The s t a b i l i t y and compatibility of dopamine solutions


with c m n intravenous f l u i d s (11)various antibioiics (12)
and miscellaneous drugs and additives w i t h which it could be
potentially mixed (13) has been reported. Dopamine i s
unstable a t an aklaline pH. Thus dopamine solutions are not
stable i n sodium bicarbonate but are stable i n dextrose,
sodium chloride, sodium lactate, l a c t a t e d Ringer's solution,
and mannitol.
DOPAMINE HYDROCHLORIDE 27 1

6. Analysis of Biological Samples

Because of its rapid metabolism, methods f o r the


routine analysis of dopamine i n blood and urine have not
been developed. Methods which have been reported are
generally concerned with endogenous dopamine and its
mtabolism rather than the clinical pharmacology and
analysis of infused dopamine.

The analysis of l 4 C - d o m e and its radiolabelled


metabolities following an infusion i n human subjects has
been reported ( 1 4 ) .

A novel approach which may be applicable to analysis


following a dopamine infusion has recently been developed
(15). This method converts dopamine (and other catechols)
t o i t s mthoxy derivative i n the presence of catechol-c-
methyltransferase and S-adenosylmthionine- H methyl. The
t r i t i a t d amines are extracted from plasma with d i e t h y l
ether, separated by TLC and measured i n a s c i n t i l l a t i o n
counter. The method i s reported sensitive t o 0.1 mle/L.

7. Metabolism and Excretion

Dopamine metabolism and T g r e t i o n was studied follawing


intravenous administration of C material to six healthy
males ( 1 4 ) . Dopamine and its metabolites are excreted
primarily in the urine: the rsdioactive dose was
quantitatively recovered a f t e r 5 days. Approximately 75% of
the dose was converted i n t o dopamine-related lnetabolites.
The principal product was 3-methoxy-4-hydroxyphenylacetic
acid. Other p r h e n t metabolites were 3-methoxydopamine,
3,4-dihyroxyphenylacetic acid, 3,4-dihydroxyphenylethanol
and 3-methoxy-4-hydroxyphenylethanol. The remainbg 25%
of the infused dopamine w a s transformed i n t o norepinephrine
and a p a r e d i n the urine principally as metabolites of
norepinephrine.

The conjugates of doparnine arid its metabolites were


studied in cultured human skin fibroblastc and rat hepatam
c e l l s ( 1 6 ) . These studies along with references c i t e d
therein indicate the principal dopamine conjugates a s the
3-0 sulphate, and the 3+glucuronide.

8. Ackncwledgemnt

The manuscript was expertly typed by M s . Martine Bunting.


212 JAMES E. CARTER E T A L .

References

1. Anon., I n t r o p i n product literature, Awrican Critical


C a r e , WGaw Park, IL 60085.

2. The Merck I d a , 9 t h Edition, 3422, Merck & Co., Inc.,


F?ahway, N J , 1976.

3. S. P a l e d , Walter C. Mccrone Associates, Inc., Chicago,


I L , 60616. Personal Comnunication.

4. W i l l i a m J. McGranahan, Northwestern University,


D e p r t m n t of Chemistry, Evanston, I L 60201. Personal
Cammication.
5. Hoying L. Hung, Northvestem University, Departrent of
Chemistry, Evanston, I L 60201. Personal C m i c a t i o n .

6. Paul W. Erhardt, American Critical C a r e , McGaw Park, IL,


60085. Personal C a m m i c a t i o n .

7. P. Fabre, -
French P a t e n t 2332748-y36, (1977).

8. Anon., United States Pharmacopeia, XX Revision, 919,


Mack Publishing Co., Easton, PA, 1980.

9. J.R. Doty, Anal.


-- Chem., -
20, 1166 (1948).

10. P.A. A s r m s and C.R. meed, -


J. Chromatogr., 169, 303
(1979).

11. L.A. Gardella, J.F. Zaroslinski and L.H. Possley, Am.


-
J.
- Hosp. - -
Phann., 32, 575 (1975).

12. L.A. Gardella, H. Kesler, J.E. C a r t e r and J.F.


- -J. Hosp. Phann., 33, 537 (1976).
Zaroslinski, Am.

13. L.A. Gardella, H. Kesler, A.H. Amann and J.E. Carter,


- -J. Hosp. Pharm.,
Am. 35, 581 (1978).
14. M. Goodall and H. Alton, Biochem. Pharmacol., -
17, 905
(1968).

15. G. Koch, U. Johansson and E. Arvidsson, - -- l h . C


J. C -hm.
C l h Biochem., -
18, 367 !1980).

16. P.A. Crooks, X.O. Breakefield, C.H. Sulens, C.M.


C a s t i g l i o n e and J . K . Caward, Biochem. -
J., __ - 187
176,
(1978).
ERGONOVINE MALEATE
Van D.Rdj

1. Description 274
1.1 General Classification 274
1.2 Name, Formula, Molecular Weight 274
1.3 Appearance, Color, Odor 274
2. Physical Properties 275
2.1 Infrared Spectra 275
2.2 Ultraviolet Spectra 275
2.3 Fluroescence and Phosphorescence Spectra 275
2.4 Nuclear Magnetic Resonance Spectra 279
2.5 Mass Spectrum 283
2.6 Differential Scanning Calorimetry 283
2.7 Melting Range 283
2.8 Crystal Properties 283
2.9 Solubility 283
2.10 Optical Rotation 289
2.11 Circular Dichroism Configuration 289
3. Synthesis 289
3.1 Biosynthesis 289
3.2 Chemical Synthesis 290
4. Stability 290
5. Identification 293
6. Methods of Analysis 293
6.1 Elemental Analysis 293
6.2 Direct Spectrophotometric Analysis 293
6.3 Colorimetric Analysis 293
6.4 Titrimetric Analysis 293
6.5 Automated Analysis 295
6.6 Fluorometric Analysis 295
6.7 Chromatographic Analysis 295
6.8 Radiochemical Procedures 296
I. Metabolism 296
8. References 308

Copyright 0 1982 by The American


Analytical Profiles of Drug Substances
Volume 11 273 Pharmaceutical Aamiation
ISBN 012-260811-9
274 VAN D.REIF

Description
1.
1.1 General Classification
Ergonovine is a naturally occurring alkaloid
found in ergot (Claviceps purpurea). It is classed as one
of the water-soluble, amine ergot alkaloids, and is an
orally-active oxytocic (1,2). The maleate salt exhibits
greater stability than the free base and is the usual form
in which the alkaloid is utilized (3).
1.2 Name, Formula, Molecular Weight
The name used by Chemical Abstracts for ergono-
vine maleate is [8,&(S)]-9,10-didehydro-N-(2-hydroxy-l-
methylethyl)-6-methylergoline-8-carboxam~deI (Z)-2-bute-
nedioate (1:l) salt. The Chemical Abstracts Registry
Number is 129-51-1. Ergometrine, ergostetrine, ergoba-
sine, ergotocine, and D-lysergic acid L-2-propanolamide
are other names that have been used for this alkaloid
(2,4).

H
I

HC-COOH
It
HC-COOH

c 1gH23N302 ' CqH404 Mol. Wt. = 441.48


1.3 Appearance, Color, Odor
Ergonovine maleate is a white to greyish white,
odorless, crystalline powder.
ERGONOVINE MALEATE 275

2. Physical Properties
2.1 Infrared Spectra
The infrared spectra of ergonovine maleate in
potassium bromide and in mineral oil are given in Figures
1 and 2 , respectively. Both were recorded on a Perkin-
Elmer 467 Grating Spectrophotometer. The KBr spectrum is
similar to that previously published (5). The KBr proce-
dure is also used for an official identity test (6).
Structural assignments (7,8) of some of the significant
bands are given in Table I. As reported (71, a carbonyl
band for the maleate moiety is not distinguished in the
KBr dispersion, and is seen only as a shoulder at 1690
cm-l in the mull.
Table I
Infrared Assignments for Ergonovine Maleate
(0.5% KBr Dispersion)

Frequency range (cm-l) Assignment


3260-3500 N-H, 0-H stretch
1650 amide C=O stretch
1570 carboxylate annion
1055 primary hydroxyl
750,775 indole C-H

2.2 Ultraviolet Spectra


The ultraviolet spectrum of ergonovine maleate in
ethanol is shown in Figure 3 . An absorptivity o f 20.5
(E=9160) was determined for the maximum at 311 nm in alco-
hol. A similar spectrum is obtained using water as sol-
vent; the absorptivity at the maximum at 311 nm was found
to be 18.7 (E=8260). The absorptivity values agree with
those previously published (9,101.
2.3 Fluorescence and Phosphorescence Spectra
A fluorescence emission spectrum in ethanol is
shown in Figure 4 . Excitation was at 3 2 5 nm. It was
obtained on a Perkin-Elmer Model 512 fluorescence spectro-
photometer. A similar spectrum was reported by Bowd
et al. (10) for ergonovine, and phosphorescence maxima
were found at 514,554, and 611 nm at 77OK in ethanol.
WAVELENGTH (Microns)

2.5 3.0 4.0 5.0


I
6.0
1
8.0 10.0 12.0 16.0 20.0 30.0 40.0 1.0
I

I I I I I I I I i i i i i
4OoO 3500 m 2500 2Ooo 1800 1600 1400 1200 loo0 800 600 400 200
WAVE NUMBER cm-4

Figure 1 - Infrared Spectrum of Ergonovine Maleate, USP Reference Standard,


Lot L(KBr Pellet)
WAVELENGM (Microns)
2.5 3.0 4.0 5.0 6.0 7.0 8.0
I
9.0
I
10
,
12
I
14
1
16 18 20
I I I
M 40
I
I 1 I I 1
1

w
V
z
U
E
h)
21
5,
z
21 U
e
I
-z
c

7
800 600 400
WAVE NUMBER(Crn-4

Figure 2 - I n f r a r e d S p e c t r u m o f E r g o n o v i n e Maleate, USP R e f e r e n c e S t a n d a r d ,


Lot L (Mineral O i l Mull)
278 VAN D.REIF

0.7

0.6

0.5

y 0.4
z
a
m
ac
0
Ln
$ 0.3

0.2

0.1 Fig. 3. Ultraviolet


Spectrum of Ergonovine
Maleate (USP Reference
0 1 1 1 1 1 1 1 Standard, Lot L ) . Sol-
210 235 260 285 310 335 360 vent-Alcohol.
WAVE LENGTH IomJ

Fig. 4. Fluorescence
E m i s s i o n Spectrum of Ergo-
I I 1 I 1 novine Maleate, 0 . 1 mg/ml
560 500 440 380 320 (USP Reference Standard,
NANOMETERS Lot L)
ERGONOVINE MALEATE 279

2.4 Nuclear Magnetic Resonance Spectra


The proton NMR spectrum of ergonovine maleate
(USP Reference Standard, Lot L ) in deuterated dimethyl-
sulfoxide (d6) with tetramethylsilane as internal stand-
ard is shown in Figure 5. The spectrum was obtained with
a 100 MHz Varian XL-100 spectrometer (11). Spectral
assignments are given in Table 11. Bands corresponding
to side chain hydrogens were identified by irradiation of
the side-chain methine hydrogen (s=3.9). This resulted
in conversion of doublets at 1.12, 3.44, and 8.25 ppm to
singlets. The alcohol hydrogen was assigned to the shift
at 3.68 ppm because resonance was lost at this shift
after D20 exchange. The shift at 10.98 ppm, assigned to
the indole-NH, also showed a partial loss of resonance
after D20. Similarly, the only resonances above 7.5 ppm
for related indoles, 8.5 ppm (CDC13) for lysergic acid
dimethylamide and 11.4 ppm (deuteropyridine) for penni-
clavine, were attributed to the indole-NH (12,13). The
6-NCH3,H8,H9, and aromatic proton assignments are also
similar to those reported for penniclavine and lysergic
ac id dime thy1amide .

Table I1
Proton NMR Spectral Assignments for Ergonovine Maleate
Chemical Shift ( S ) Multiplicity Assignment
1.12 doublet -CH3 (side chain)
3.10 singlet 6-NCH3
3.44 doublet C-CH20
3.68 singlet C-OH
3.9 mu1 t ip let NCH-C
4.30 mu1 t ip let c(H-8
6.14 singlet maleate C-H
6.57 sharp multiplet H-9
7.6-7.4 mu 1tip le t H-12,H-13,H-14,H-2
8.25 doublet 8CNH
10.98 singlet indole-NH
ERGONOVINE MALEATE 28 1

The 13C NMR spectrum of ergonovine maleate (USP


Reference Standard, Lot L) in deuterated dimethylsulfoxide
is shown in Figure 6. It was recorded on a Varian
FT-80-A spectrometer at ambient temperature (11). The
spectral assignments are given in Table 111. The chemi-
cal shifts are similar to those reported by Bach et al.
(14) for ergonovine.

Table 111
13C NMR Assignments for Ergonovine Maleate
Carbon E
!
-C02H 169.3
-CON 168.1
HC=CH 136.2
c-10 134.6
C-15 131.8
C-16 126.0
c-11 125.7
C-13 123.3
c-9 121.0
c-2 120.5
C-14 112.5
c-12 111.6
c-3 106.8
OCH2 65 .O
c-5 61.8
c-7 53.6
HN-CH 47.7
N-CH3 41.7
C-8 40.8
c-4 24.7
-CH3( side chain) 17.7
ERGONOVINE MALEATE 283

2.5 Mass Spectrum


The mass spectrum of ergonovine maleate was
obtained with a Kratos DS-50s Data System coupled with a
MS-902 double focusing, high resolution mass spectrometer
(15). The ionizing electron beam energy was at 70eV and
the probe temperature was 250°. A line graph of the mass
spectrum is shown in Figure 7 and identification of the
pertinent masses are presented in Figure 8. The retro-
Diels-Alder reaction fragments at m/e 282 and 196 and the
fragments at m/e 220-223 were postulated by Bellman (16)
for other lysergic acid analogs. The identity of frag-
ments at m/e 167 and 154 have been postulated for other
ergot alkaloids (17,181.
2.6 Differential Scanning Calorimetry
The DSC thermogram (19) of ergonovine maleate
(USP Reference Standard, Lot L) is shown in Figure 9.
The thermogram was obtained at a heating rate of loo/
min in a nitrogen atmosphere using a Perkin-Elmer DSC-2.
No endotherms or exotherms were observed other than that
associated with the decomposition melt.
2.7 Melting Range
The melting range must be taken under a pro-
tective environment to limit decomposition. The follow-
ing melting point temperatures have been reported for
ergonovine maleate:
OC
- Reference
167 (with decomposition) 4
180-190 (in paraffin) 20
187-191 (silicone oil) 21
186-196 (under nitrogen) 21
185-190 (sealed capillary) 21
2.8 Crystal Properties
The X-ray diffraction pattern of ergonovine
maleate (USP Reference Standard, Lot L) as obtained with
a Philips diffractometer using C u K d radiation (191, is
presented in Figure 10. The calculated "d" spacings are
listed in Table IV.
2.9 Solubility
The following solubilities have been reported for
ergonovine maleate (4,221:
53111SNllNI 3hllVl3tl
-
m/e ASSIGNMENT RELATIVE INTENSITY
325 m+ 100
310 mt -CH3 0.4
307 mt-H$ 2.2
294 m t -CHflH 2 .o

282

223 25.5

222

221 6.8

196

167 [ &NH]' 7.3

154 [ & 1' 6.7

Figure 8 - Mass Spectrum Fragments

285
286 VAN D.REIF

t
0
c3
z
w

50 75 100 125 150 175 200 225


OC

F i g . 9. D i f f e r e n t i a l Thermal A n a l y s i s Spectrum of Ergo-


n o v i n e Maleate (USP R e f e r e n c e S t a n d a r d , L o t L)
loo -
90 -

80-

70 -

60-
c 50-
B
I-
z
40-

29
Figure 10 - X-ray Diffraction Pattern of Ergonovine Maleate (USP Reference Standard,
Lot L)
288 VAN D.REIF

Table I V
X-Ray Powder Diffraction Pattern

2 0 d (Ao) 1/10
39.6 2.28 4
38.7 2.33 4
36.5 2.46 11
34.4 2.61 4
32.8 2.73 4
29.6 3.02 7
28.9 3.09 13
26.4 3.38 26
24.8 3.59 19
23.9 3.72 24
22.9 3.88 20
22.1 4.02 32
21.6 4.11 98
19.9 4.46 16
18.6 4.77 18
17.7 5.01 13
16.2 5.47 64
15.1 5.87 40
14.2 6.24 89
8.8 10.00 100
7.5 12.00 36
ERGONOVINE MALEATE 289

Solvent Solub i 1ity (mg/ml)


water 28
alcohol 8
chloroform nearly insoluble ( <0.1)
ether nearly insoluble ( < 0.1)
2.10 Optical Rotation
The following optical rotations were reported
(23) for ergonovine maleate:
[d]i0= +53 (cz1.0 in water)
20
[a1578 = +57 (Cz1.O in water)
20
["(I546 = +74 (C=l.O in water)
20
[HI436 = +252 (C=l.O in water)
USP material ( 6 ) meets the specification,
[O( Ii5" = +51 to +56 ( G 0 . 5 in water).
2.11 Circular Dichroism-Configuration
A circular dichroism spectrum was reported (24)
for ergonovine maleate. Maxima were at approximately 218
and 318 nm, and a minimum was at 248 nm. Other isomers
containing the D-lysergic acid moiety gave similar spec-
tra; a compound containing the L-lysergic acid moiety
showed a reversed effect. This is consistent with the
positive Cotton above 300 nm reported for D-lysergic acid
(25). The rotatory dispersive effects are due to the
configuration at C-5; the configuration at C-8 has only
an additive or subtractive effect. In the case of D-
lysergic acid, and therefore, ergonovine, the hydrogen
at C-5 is in t h e 4 position (25) and the C-5 center is
in the S configuration (26).
3. Synthesis
3.1 Biosynthesis
Ergonovine was originally isolated from ergot
extracts (2,27-29). It has since been biosynthesized in

+
cultures of Claviceps species (30-351, and has been iso-
lated from seeds of I omoea violacea (36,371 and
Argyreia nervosa (38 The following pathway has been
constructed (39-44) for biosynthesis of the ergoline
skeleton by Claviceps species:
290 VAN D.REIF

mevalonic acid + tryptophan )


- 4-dimethyl-
allotryptophan

elymoclavine + agroclavine ,<- chanoclavine-I


5.
6-methyl-Ll 8,9-ergoline-8-carboxylic acid
\1
lysergic acid
The final step to produce ergonovine was shown to involve
incorporation of alanine with lysergic acid (44).
3.2 Chemical Synthes.is
Various semi-synthetic procedures for ergonovine
maleate involve addition o f the alkyl side chain to lyser-
gic acid. The D-lysergic acid starting material is
obtained by alkaline hydrolysis of an ergot alkaloid frac-
tion of biosynthetic origin (45). A relatively high-yield
sequence is shown in Figure 11. Lithium lysergate is
reacted with a sulfur trioxide-dimethylformamide complex
at O°C. L-2-amino-1-propanol is added, and the product is
isolated after addition of water and treatment with maleic
acid. Procedures for formation of the amide bond using
lysergic acid chloride ( 4 6 ) or phosgene (47) have been
patented. A complete fiftegn-step synthesis o f the
D-lysergic acid starting material from 3b-carboxyethylin-
dole has also been reported (48).

4. Stability
The maleate salt shows improved stability over ergono-
vine base, but the salt also oxidizes and darkens in the
presence of oxygen (3). The stability of ergonovine s o l u -
tions has been improved by the use of antioxidants such as
ascorbic acid (491, methionine, and d-mercaptopropionyl-
glycine (50). The known degradation products are shown in
Figure 12. Heat treatment of ergonovine (a dextrorotatory
isomer) under acid or alkaline conditions causes a reversi-
ble isomerization at the 8-position to form the dextrorota-
tory ergonovinine, also known as isoergonovine. More
stringent acid or base treatment eventually results in
hydrolysis to lysergic acid, isolysergic acid, and 2-amino-
1-propanol (51,521. When ergonovine is refluxed in the
LiOH-HZO
D-LYSERGIC ACID w
CH30H

CO NH -c -CH3

- DMF
CH3CHNH2
I
CH20H

I MALEIC A C I D

ERGONOV I NE MALEATE

Figure 11 - Synthesis of Ergonovine Maleate from Lysergic Acid


292 VAN D.REIF

y
CONH-C-CH~
A,

-
H+ or OH^

OH{ ERGONOVlNlNE
ERGONOVINE
(Iso-ergonovine)

d3 HN
OH *
._

LYSERGIC ACID ISO-LY SERG IC ACID

& &
HN
y
CONH-C-CH3

H;HzoH +

HN
y
CONH-C-CH3

H;HpH

LUMIERGONOVINE I LUMIERGONOVINE II

Figure 12 - Degradation of E r g o n o v i n e
ERGONOVINE MALEATE 293

presence of amines (53), the centers at C-8 and C-5 are


both racemized to give mixtures of ergonovine, ergonovi-
nine, L-lysergic acid L-2-propanolamide and L-isolysergic
acid L-2-propanolamide. When acidic solutions of ergono-
vine are irradiated, the 9,lO double bond is hydrated to
form lumiergonovine I and small quantities of lumiergono-
vine I1 (54-56). Degradation of the ergonovine indole
ring has been indicated by reports (57) of unidentified
fluorescent degradation products which do not react with
p-dimethylaminobenzaldehyde.

5. Identification
Alternately known as Van Urk's (58) or Erhlich's (59)
reagent, 1-dimethylaminobenzaldehyde has had widespread use
in ergonovine identity tests (59-61) and chromatographic
detection sprays (6,60,62). Indoles give a blue color with
this reagent (61); phenols and amines may also react,
usually to give other colors (58,591. Color reactions may
also be produced with aldehydes such as vanillin, piper-
onal, and paraldehyde (63). Iodinated potassium iodide
(brown flocculent) and ferric chloride in phosphoric acid
at 80° (blue-violet color) are two additional identity
test reagents (60).

6. Methods of Analysis
6.1 Elemental Analysis
The elemental analysis of ergonovine maleate
(USP Reference Standard, Lot L) is presented below.
Element % Calculated % Reported (64)
C 62.57 62.55
H 6.17 6.03
N 9.52 9.38
6.2 Direct Spectrophotometric Analysis
Bayer (65) described an ultraviolet spectrophoto-
metric method for ergonovine maleate tablets. The tablets
are extracted with 1% tartaric acid and the absorbance is
measured at 317 nm.
294 VAN D.REIF

Colorimetric Analysis
6.3
Official assays for ergonovine maleate drug sub-
stance, tablets, and injection (6,601 employ the p-di-
methylaminobenzaldehyde reagent. Drug substance or
injection samples are dissolved in water; tablet samples
are dissolved in aqueous tartaric ac id-benzalkonium chlor-
ide. The reagent is dissolved in aqueous sulfuric acid-
ferric chloride. After color development, absorbances are
read at 555 nm. Light or hydrogen peroxide (63,66,67) have
also been used in place of ferric chloride to catalyze
color development for quantitation. A similar quantitative
procedure using sodium nitrite t o catalyze color formation
was shown to have the advantages of speed, sensitivity, and
color stability (62).
The specificity of the reaction for indoles under
several conditions was studied by Kupfer (61). The speci-
ficity was very dependent on the particular conditions
employed and the development times. In general, for
indoles to be reactive, an unsubstituted 2- or 3- position
was required. The mechanism proposed by Pohm (68) involved
attachment of one aldehyde molecule at the 2-positions of
two indole molecules with release o f water.
An assay for ergonovine maleate tablets using
metaldehyde to produce a color at 550 nm has been published
(69). A colorimetric assay for tablets using ion-pairing
with bromocresol purple was reported (70).
6.4 Titrimetric Analysis
Ergonovine maleate can be determined titri-
metrically in glacial acetic acid with a 0 . 0 5 N perchloric
acid titrant and crystal violet indicator (71,721. The NF
XIV drug substance assay (73) is similar, except that ace-
tic anhydride is added to the system. Ergonovine was
determined in mixtures after thin-layer chromatography
with chloroform-methanol (97:3) on alumina. Spots were
eluted with chloroform, acetone was added, and the mixture
was titrated with 0.005 N perchloric acid in methanol (74).
The maleate moiety has been determined with 0.01 potas-
sium methoxide titrant, thymol blue indicator, and pyri-
dine as solvent (71).
ERGONOVINE MALEATE 295

6.5 Automated Analysis


Kirchhoefer and Wells (75) developed automated
methods for ergonovine maleate tablets and injections.
Samples were dissolved in pH 6.0 tartrate buffer. For
colorimetric quantitation, the sample stream was made
basic and extracted with n-butanol. The extract was mixed
with p-dimethylaminobenzaldehyde reagent, and after react-
ion, the stream was mixed with sodium nitrite for determin-
ation at 550 nm. For fluorometric detection the sample
stream was diluted with tartrate buffer. Excitation was
at 325 nm and the emission was read at 432 nm. The methods
were tested for interference from common tablet and injec-
tion excipients.
Robertson et al. (76) reported a general procedure
for basic drugs in which ergonovine was included. Brom-
thymol blue solution was mixed with an aqueous buffered
sample stream. The ergonovine-dye complex was extracted
into chloroform and determined at 410 nm.
6.6 Fluorometric Analysis
Fluorometry has been used to determine ergonovine
maleate in tablets and injections in an automated procedure
(75) (see section 6.51, and after column chromatography
(77) (see section 6.73). Samples were read in 0.1 tar-
tartic acid buffer with excitation and emission at 325 and
432 nm, respectively.
6.7 Chromatographic Analysis
6.71 Thin-Layer Chromatography
The thin-layer chromatographic systems used
to analyze ergonovine and ergonovine maleate are listed in
Table V. The applications, means of quantitation or
detection,and procedure specificity are listed when this
information was supplied in the literature sources. The
last five systems employ ion-exchange layers.
6.72 Paper Chromatography
Paper chromatographic systems for ergonovine
and ergonovine maleate are given in Table VI. Applications
and detection agents are also listed. Sprince (100) des-
cribed a variation of the Ehrlich reagent which gave rapid,
long-lasting color development for paper chromatography.
A p-dimethylaminobenzaldehyde in HCl spray was followed by
a 1% sodium nitrite spray.
296 VAN D.REIF

6.73 Column Chromatography


The column chromatographic
- . methods that have
been used to analyze ergonovine maleate or ergonovine base
are described in Table VII.
6.74 Gas Chromatography
Sondack (103) analyzed ergonovine maleate in
tablets and injectables after derivatization with N-tri-
methylsilyldiethylamine and N-trimethylsilylimidazole. A
1% OV-1 on Gas Chrom Q column at 260° was used. The sep-
aration of ergonovinine and lumiergonovine I from ergono-
vine was demonstrated.
6.75 High-Performance Liquid Chromatography
High-Performance Liquid Chromatographic
systems for ergonovine and ergonovine maleate are listed in
Table VIII, along with applications and specificity infor-
mat ion.
6.76 Electrophoresis
The electrophoresis of ergonovine on cellu-
lose thin layers at 500V and 3000V was reported (112).
The following electrolytes were found to be the most suit-
able: formic acid-acetic acid-water (26:120:1000),
ammonia-water (2:98), and ammonia-water (1:9). A high
voltage paper electrophoretic separation of ergonovine
from its diasteromer, D-lysergic acid D-propanolamide, was
reported (113).
6.8 Radiochemical Procedures
A radioimmuno assay for ergonovine and other
ergot alkaloids in plasma was reported (114). Detection
was at the picomole level from 4 ml of plasma. The anti-
sera, produced by a conjugate of lysergic acid with human
serum albumin, reacted with lysergic acid derivatives, but
not with tryptophan. An antibody produced by a mixed
anhydride reaction of lysergic acid with bovine serum
albumin reacted with simple lysergic acid derivatives and
some clavines (115). A similar coupling using a Mannich
reaction yielded an antibody specific for lysergic acid.

7. Metabolism
Two metabolites of ergonovine, identified as the
6-glucuronides of 12-hydroxyergonovine and 12-hydroxy-
ergonovinine, were excreted in the bile of rats (99). Two
other metabolites were tentatively identified as glucur-
onides of ergonovine and ergonovinine. An earlier report
(116) demonstrated the formation of two unidentified polar
metabolites of ergonovine in rats.
Table V

Thin-Layer Chromatography Systems

Appl icat ion Separation of Ref.


-
(Quantita t ion/De tec tion Ergonovine from:

Toluene-Butanol Quantitation of ergono- Ergonovinine , 78


(4N HC1 saturated) vine in ergot mixtures ergot alkaloids
(6:4)/silica gel (colorimetric)
Chloroform-Methanol Quantitation of ergono- - 79
(3:l)/silica gel vine in tablets,injections
(in situ fluorometric)
--
Ch 1oro form-Methano1 Quantitation of ergot Ergotamine, 74
(97:3)/alumina mixtures (titrimetric) ergocryptine,
ergocrystine
Ethyl Acetate-n-Heptane- - Quantitation of ergot 8 Ergot alkaloids 80
Diethylamine (7:6:O .0005 ) / mixture s
Cellulose-formamide (in
-- situ fluorometric)
Chloroform-Ethano1- 23 Ergot alkaloid quanti- Ergonovinine , 81
Ace tone tation (colorimetric) 10 ergot alkaloids
(6:4:4)/silica gel G
Chloroform-E thanol 5 Ergot alkaloid quanti- Ergonovinine, 81
(9:l)/silica gel G tation (colorimetric) 10 ergot alkaloids
Table V (continued)

Solvent/Plate Rf x 100 Application Separation of Ref.


-
(Ouantitation/Detection) Ergonovine from:
Ethyl Acetate-DMF- 17 Ergot alkaloid quanti- 11 Ergot alkaloids 82
Ethanol tation (colorimetric)
(13:1.9:0.l)/silica gel
Benzene-DMF (13:2)/ 12 Ergot alkaloid quanti- 11 Ergot alkaloids 82
silica gel tation (colorimetric)
Methanol-Acetic Acid- 30 Ergot alkaloid quanti- Ergotamine, agrocla- 83
Water (2:1:2)/silica tation (colorimetric) vine, chanoclavin
ge 1
Methanol-Water-Tartaric 41 Ergot alkaloid quanti- Ergotamine, agrocla- 83
Acid (40:60:l)/silica tation (colorimetric) vine, chanoclavin
ge 1
Benzene-Ch lor0form- 11 Ergot extract quanti- 8 Ergot alkaloids, 84
Ethanol (2:4:1)/ tation (colorimetric) ergonovinine
silica gel
Chloroform-Methanol- - Purity determination
Water (75:25:3)/ and Identity for Ergo-
silica gel novine maleate (visual
after p-dimethylamino-
benzaldehyde)
Chloroform-Acetone- 45 Identification of Impuri- Ergonovinine,related 24
Methanol-Triethylamine ties in Ergonovine a 1kalo id s
15 :10:5 : 1 / s i 1ica ge 1 maleate (UV)
Table V (continued)
Application Separation of Ref.
(Quantitationstection) Ergonovine from:
~

6 Systems/silica - Ergot Alkaloid Identifi- Ergonovinine, 85


gel, alumina, cation ( 10% CuSO4-2% 5 ergot alkaloids
cellulose ammonium hydroxide, 5:l)
34 Systems /silica 0 to 80 Alkaloid Identification Ergonovinine, 86
gel, alumina in I. violacea extracts LSD, iso-LSD
(UV,p-dimethylamino-
benzaldehyde + sodium
nitrite
17 Systems/silica 0 to 66 Ergot Alkaloid Identifi- 14 ergot alkaloids 87
gel, alumina, cellulose cation ( W ,Ehrlich's
Reagent)
Chloroform-Methanol 10 LSD Identification Ergonovinine, 20 88
(9:l)/silica gel GF (Ehrlich's Reagent) ergot alkaloids
Chloroform (NH3 sat- 13 LSD Identification Ergonovinine, 20 88
uratedl-Methanol (Ehrlich's Reagent) ergot alkaloids
(18:l)/silica gel GF
Trichloroethane- 21 LSD Identification 9 alkaloids 89
Methanol (9:1)/ (Ehrlich's Reagent)
silica gel
Diisopropyl Ether- 0 Ergot alkaloid sepa- 15 ergot alkaloids 90
THF-Toluene-Diethyl- ration (w)
amine ( 7 0 : 15 :15:O. 1 ) /
si1ica ge1- formamide
Table V (continued)
Solvent/Plate Rf x 100 Application Ref.
-
(Quantitation/Detection) Ergonovine from:
Acetone/O.lM 78 Determination of Ergotoxine Ergonovinine, 13 91
Ammonium Carbonate/ Alkaloids (vani11in/H2SOq ) ergot a1kaloids
Ethanol (32.5:
67.5:l)/silica gel G
11 Systems/silica 0-83 methylsergide, methylsergide, 92
gel, alumina methergine detection methergine
Chloroform-Benzene- - Ergot alkaloid identifica- 4 Ergot alkaloids 93
Methanol (forma- tion (0.5% chlorimino-2,6-
mide saturated,50 :4: dichloroquinone/methanol;
lO)/silica gel G 0.3% Congo Red, pH 7; 0.5%
quinone/aq. HC1)
Acetic Acid-HC1 36-4 Alkaloid identificat ion 30 alkaloids 94
(pH 0.6-2.35)/ (6M acetic acid + UV,
alginic acid-cellu- Dragendor ff ' s )
lose, Rexyn 102-
cellulose
1M Acetic Acid in 15 Alkaloid identification 30 alkaloids 94
Water-E thanol (6M acetic acid + UV,
(7:3)/Rexyn 102- Dragendorf f ' s )
cellulose
1M HC1 in Water- 21 Alkaloid identification 30 alkaloids 94
EFhanol ( 1 : 4 ) / (6M acetic acid + UV,
Dowex 50-X4-cellu lose Dragendor ff ' s )
Table V (concluded)
Solvent/Plate Rf x 100 Application
-- Separation of Ref.
-
(Quantitation/Detection) Ergonovine from:
0.5M Acetate/AG 1- 21 Alkaloid identification 30 alkaloids 95
~4 Tacetatel- (6E acetic acid + UV,
cellulose Dragendor f f ' s
0.5M Ammonium Ace- 49 Alkaloid identification 30 alkaloids 95
ta te/DEAE ce 1 lu 1o se (6M acetic acid + UV,
Dragendor f f ' s
Table VI
Paper Chromatography Systems for Ergonovine
Stationag Mobile
- - Application Separations Detection Reference
Phase
n-Butanol-Acetic
Whatman No. 1 - Estimation of Water-insoluble UV light 96
(descending) Acid-Water ergonovine from water-sol-
(4:1:5) maleate in ble ergot alka-
ergot ex- loids, ergometrine
tracts and from ergometrinine
injections
Whatman No. 3 Ace tone-5% determination - p-d imethyl- 97
5% octanol Aqueous Ammonia of ergonovine aminobenzaldehyde
(ascending) (2:3) in ergot extracts
A.H. Thomas n-Butylacetate- determination ergonovine from uv 98
NO. 3677- Nitromethane- of ergonovine ergonovinine
pH 6 sodium Ethyleneglycol- in water-sol-
hydroxide- monoethylether ble ergot
pot assium Ace tate-Pyr id ine extracts
pho spha te Water (100 :50: 5 0 :
8:lO)
Paper loaded Formamide-O.1N
- limit test for - p-d imethylamino- 60
with 10% Pota s s ium impurities in benzaldehyde
d imethy 1 Hydroxide (1:4) ergonovine maleate
phthalate in
chloroform
Table V I (continued)

Stationary Mobile Application


-- Separations
- Detection
~- Reference
Phase

Whatman No. 1 Butanol (water metabolite iden- Ergonovine from uv 9 99


or No. 3 saturated) t i f ication 12-hydroxy- Ehrlich ' s
ergonovine, Reagent
metabolites

Whatman No. 1 Butanol metabolite iden- Ergonovine from uv , 99


or No. 3 (ammonia, tification 12-hydroxy- Ehrl ich ' s
water saturated) ergonovine, Reagent
metabolites
W
E:
Table VII
Column Chromatography for Ergonovine

Packing Eluent App 1icat-


ion Separates Ergonovine Detec t ion Ref.
-
from
-
Silica gel Chloroform- Impurity Ergometrinine, D-LSA- TLC 24
Acetone-Meths- Isolation D-2-propranolamide
no 1-Tr iethyl- male ate
amine ( 15 : 10:
5:l)
Sephadex 96% Ethanol Plant Extracts 5 Ergot Alkaloids Van Urk 101
LH-20 or DMF or Reagent
Ace tone
Sephadex Water Plant Extracts Lysergic Acid Van Urk 101
G 25 Re agent
Ce 1ite I Ether Assay Tablets Fluorescence 77
Sodium Injections
Bicarbonate
Celitel Butanol-Ben- Degraded Lumiergonov ine I Van Urk 56
Water zene-20% Ace- Extracts and I1 Reagent
tic Acid
(1:1:2)
Cel itel (1)Chloroform Powdered Ergot Water-Insoluble p-Dimethyl- 98
Citric Acid (2)Sodium Bi- Extracts Ergot Alkaloids amino benzal-
carbonate dehyde
( 3 )Chloroform
Table VII ( c o n t i n u e d )
--
Packing Eluent Application
-- -S e_
parates Ergonovine Detection
~- Re€.
-
from
-

Cellulose/ (110.1% p y r i - Ergot Alkaloid Water I n s o l u b l e E r g o t p-Dimethyl- 102


pH 3 . 0 dinelether Mixtures A l k a l o i d s , Ergonovinine amino b e n z a l -
Citrate- (2110% d i e t h y l - dehyde
Phosphate amine / e t h e r
(3)ether

W
E:
Table VIII
High-Performance Liquid Chromatography Systems for Ergonovine
Co 1umn Mobile Application Ergonovine Separates Detection -
Ref.
from
-
microBondapak Acetonitrile- Injection, Ergonovinine,Lysergic 312 nm 104
C18 1% Acetic Acid Tablet Assay Acid
(1 :4)
LiChrosorb Acetonitrile- Identification Ergonovinine, 11 Ergot 320 nm 105
RP-2, Rp-8 0.01M Ammonium of Components of Alkaloids
and RP-18 Carbonate (2:3) Plant Extracts,
Fermentation
Mixtures
LiChrosorb n-Hexane-
- Identification Ergonovinine, 11 Ergot 320 nm 105
SI-60 Chloroform- of Components of A1 kaloid s
Ethanol(4:4:1) Plant Extracts,
Fermentat ion
Mixtures
Corasil C18 Methanol-0.1% Assay of Illicit Ergot Alkaloids Fluorometric 107
Ammonium Car- LSD Preparations
bonate (3:2)
Corasil 11 Acetonitrile- Assay of Illicit Ergot Alkaloids 254 nm 108
Isopropyl LSD Preparations
Ether (1:3)
microBondapak Methanol-Ace- Forensic Mixture Ergot Alkaloids 254 nm 109
C18 tic Acid-0.005 Identity
M Heptane Sul-
-
fonic Acid (40:1:59)
Table VIII (continued)

High-Performance Liquid Chromatography Systems for Ergonovine


Co 1umn Mobile App 1ic at ion
- Ergonovine Separates Detection Ref.
microPak NH2 Diethyl Ether- Separation o f Ergonovinine, 15 Ergot 310 nm 110
E thano1 Ergot Mixtures Alkaloids
(84:12)
Diethyl Ether- Separation of Ergonovinine, 15 Ergot 310 nm 110
Isopropanol Ergot Mixtures A1 ka loid s
(3:2)
Chloroform-Iso- Separation of Ergonovinine, 15 Ergot 310 nm 110
propano1 Ergot Mixtures Alkaloids
(9:l)
LiChrosorb Diethyl Ether- Separation of Ergonovinine, 15 Ergot 310 nm 110
NH2 Ethano 1 Ergot Mixtures Alkaloids
(93:7)
LiChrosorb Hexane-Chloro- Separation o f Ergonovinine, 10 Ergot 320 nm 111
SI-60 f o rm-Ac e to- Ergot Mixtures Alkaloids
ni tr ile-Methanol
(55 :20:25 :3)
308 VAN D. REIF

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ERGONOVINE MALEATE 309

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~~

80. M. Prosek, E. Kucan, M. Katic, an=. Bano, Chromato-


graphia, 9 , 273 (1976).
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312 VAN D.REIF

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95. Ibid., 116, 131 (1976).
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.
Pharmacol. .~ 1 .SO2 (1949).
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graphia, 11,23 (1978).
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I - _ -
, I

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through --Chem. Abstr. 933173798q (1980).
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Experientia, 15, 111 (1959).
FLUFENAMIC ACID
Enrico Abignente and Paolo de Caprarh

1. Description 314
1.1 Name 314
I .2 Formula, Molecular Weight 314
1.3 Elemental Composition 314
1.4 Appearance, Color, Odor, Taste 314
2. Physical Properties 314
2.1 Infrared Spectrum 314
2.2 Nuclear Magnetic Resonance Spectrum 314
2.3 Ultraviolet Spectrum 316
2.4 Mass Spectrum 320
2.5 Fluorescence Spectrum 320
2.6 Melting Range 322
2.7 Solubility, Partition Coefficient 323
2.8 Crystal Properties, Polymorphism 325
2.9 Dissociation Constant 326
3. Synthesis 327
4. Drug Metabolism and Pharmacokinetics 328
4.1 Metabolism 328
4.2 Absorption and Excretion 328
4.3 Protein Binding 33 1
5. Methods of Analysis 332
5.1 Identification Tests 332
5.2 Titrimetric Analysis 333
5.3 Colorimetric Analysis 333
5.4 Spectrophotometric Analysis 333
5.5 Fluorometric Analysis 334
5.6 Indirect Atomic Absorption Analysis 335
5.7 Chromatographic Analysis 336
6. Determination in Body Fluids and Tissues 342
7. Acknowledgement 343
8. References 343

Analytical Protilcs of Drug Subsurncea Copyright 0 1982 by The American


Volumc I I 313 PharmaceuticalAssociation
ISBN 0-12-260811-9
314 ENRICO ABIGNENTE AND PAOLO DE CAPRARIIS

1. D E S C R I P T I O N
1. 1 . N a m e
F l u f e n a m i c a c i d is d e s i g n a t e d b y C h e m i c a l A b -
stracts s i n c e 1972 a s 2 - [ [ 3-(trifluoromethyl)phenyl]ami-
n o l b e n z o i c a c i d , w h e r e a s b e f o r e 19 7 2 i t w a s n a m e d N-
(a,a , a-trifluoro-m-toly1)anthranilic a c i d . O t h e r n a m e s
a r e : N - ( 3-trifluoromethylphenyl)anthranilic a c i d , a n d
3'-trifluoromethyldiphenylamine-2-carboxylic acid ( 1 ) .
T h e CAS R e g i s t r y N u m b e r is 530 - 7 8 - 9 .
1. 2. F o r m u l a , M o l e c u l a r W ei ght

COOH

4JF3
C H F NO2 M o l e c u l a r Weight: 281. 2 4
1 4 10 3
1. 3 . E l e m e n t a l C o m p o s i t i o n
C 59. 79%; €3 3 . 58%; F 20. 27%; N 4. 98%; 0 11. 3 2 %
1. 4. A p p e a r a n c e , C o l o r , O d o r , Taste
Pale y e l l o w n e e d l e s , p r a c t i c a l l y o d o r l e s s w i t h a
slight bitter t a s t e ,

2 . P HYS IC AL P R O P E R T I E S
2 . 1. I n f r a r e d S p e c t r u m
T h e IR s p e c t r u m of f l u f e n a m i c a c i d s h o w n in Fi-
g u r e 1 w a s obt ai ned w i t h a B e c k m a n M y c r o l a b 620MX
s p e c t r o p h o t o m e t e r i n a K B r p e l l e t c o n t a i n i n g 0. 4 m g of
f l u f e n a m i c a c i d / 1 0 0 m g of K B r . S o m e s p e c t r a l a s s i g n -
m e n t s a r e g i v e n i n T a b l e I.
T h i s s p e c t r u m is f n good a c c o r d a n c e w i t h t h e IR s p e c -
t r u m (1 8 0 0 - 600 c m - ) r e p o r t e d b y K u h n e r t - B r a n d s t ' a t t e r
e t al. ( 2 6 ) f o r the p o l y m o r p h i c m o d i f i c a t i o n I11 of f l u f e n a -
mic a c i d (see S e c t i o n 2. 8).
2. 2. N u c l e a r M a g n e t i c R e s o n a n c e S p e c t r u m
T h e 1 H NMR s p e c t r u m of f l u f e n a m i c a c i d is p r e -
92

80

70

50

30

10

10

C
1
316 ENRICO ABIGNENTE AND PAOLO DE CAPRARIIS

TABLE I
IR Spectral Assignments
of Flufenamic Acid

Wavenumber, c m - ' Vibration Mode


1670 C=O stretching
1612, 1592, 1535 aromatic C=C stretching
1190, 1125 CF3 a s y m m . s t r e t c h i n g
803, 768, 758, 703 C-H out of plane bending

sented in F i g u r e 2. The s p e c t r u m was r e c o r d e d on a V a -


r i a n E M - 3 6 0 60 MIlz s p e c t r o m e t e r using a CDC13 solution
containing tetramethylsilane a s an i n t e r n a l standard. The
s p e c t r a l assignments are given in Table 11, and a r e in
accordance with the NMR s p e c t r u m of etofenamate, which
is the 2-(2-hydroxyethoxy)ethyl e s t e r of flufenamic acid,
reported by Boltze and Kreisfeld (2) a s r e g a r d s the a r o -
matic protons of the benzene rings.

TABLE I1
NMR S p e c t r a l Assignments
of Flufenamic Acid

Protons Chemical Shift, d Multiplicity


a 6.60-6.96 multiple t
b 7.05-7. 53 multiple t
C 8. 05 doublet

2. 3 . Ultraviolet Spectrum
The UV s p e c t r u m of flufenamic acid ( F i g u r e 3 )
w a s scanned f r o m 400 t o 210 nm on a C a r y 219 s p e c t r o -
START OF SWEEP EN0 OF SWEEP

Fig. 2. NMR Spectrum of Flufenamic Acid


318 ENRICO ABIGNENTE AND PAOLO DE CAPRARIIS

F i g . 3. U l t r a v i o l e t Spectrum of Flufenamic Acid


FLUFENAMIC ACID 319

p h o t o m e t e r , u s i n g a solution of 9. 28 p g of f l u f e n a m i c a c i d
/ m l of 95% ethanol. T h e a b s o r p t i o n d a t a a r e l i s t e d in Ta-
ble 111.

T A B L E I11
UV Absorption Data of F l u f e n a m i c Acid in 95%
ethanol solution

L max' nm
1c m
219 4. 32 745
238 s houlde r -
2 88 4. 2 2 5 90
337 3. 8 8 269

The s p e c t r a l f e a t u r e s observed are substantially in a c -


c o r d a n c e with t h o s e p r e v i o u s l y d e s c r i b e d in the l i t e r a t u r e
(3, 4, 5, 6, 7, 8 ) . T h e log & vs. wavelength plot of a m e t h a -
nolic solution of flufenamic a c i d w a s r e p o r t e d by U n t e r -
halt ( 3 ) , w h e r e a s Ikeda e t al. ( 5 ) p r e s e n t e d the s p e c t r u m
of the d r u g in 0. 1 M phosphate buffer (pH 7. 0 ) . D e l l e t
al. (8) r e p o r t e d Amax v a l u e s of flufenamic a c i d and i t s
phenolic m e t a b o l i t e s in n e u t r a l , a c i d i c and a l k a l i n e m e -
thanol. In T a b l e l V are listed s o m e absorption data re-
p o r t e d in the l i t e r a t u r e .
~~

T A B L E IV
UV Absorption D a t a of F l u f e n a m i c Acid
1q"
Solvent Amax. n m log E
cm
Ref. -
Methanol 2 88 4.22 590 3
340.5 3. 8 9 276
0 . 0 1 N NaOH 289 4.10 448 3
0 . 1 NNaOH 288 4. 1 8 545 4
0 . 01 N HC1 287 4. 2 2 5 90 3
(in CH30H) 345 3. 94 310
320 ENRICO ABIGNENTE AND PAOLO DE CAPRARIIS

2. 4. M a s s S p e c t r u m
T h e mass s p e c t r u m of f l u f e n a m i c a c i d is s h o w n
i n F i g u r e 4. T h e s p e c t r u m w a s r u n o n a H e w l e t t - P a c k a r d
Mod. 5982A s p e c t r o m e t e r w i t h a n i o n i z i n g e n e r g y of 70
e V , i n t e r f a c e d with a H e w l e t t - P a c k a r d Mod. 5934A d a t a -
h a n d l i n g s y s t e m . T h e c o m p u t e r c a l c u l a t e d i o n masses a n d
compared their peak intensities to the b a s e peak (m/e =
265). T h i s i n f o r m a t i o n w a s t h e n a u t o m a t i c a l l y p l o t t e d a s
a series of l i n e s w h o s e h e i g h t s a r e p r o p o r t i o n a l t o t h e
peak intensities. T h e molecular ion peak w a s observed
a t m / e = 281. S o m e c h a r a c t e r i s t i c p e a k s o b s e r v e d a r e
l i s t e d in Table V . T h e f r a g m e n t a t i o n p a t t e r n o b s e r v e d is

TABLE V
M a s s S D e c t r u m of F l u f e n a m i c A c i d
Mass (m/e) Species A b u n d a n c e 01,
281 M+ 44. 8
263 M+-H20 100.0
235 263-CO 22. 8
216 235-F 12. 3
166 216-CF2 14. 9

c o n s i s t e n t with mass s p e c t r a l d a t a p u b l i s h e d b y B o l t z e
a n d K r e i s f e l d ( 2 ) f o r e t o f e n a m a t e a n d C o t e l l e s s a et al.
( 43 ) for t h e m e t h y l e s t e r of f l u f e n a m i c a c i d .
2. 5 . F l u o r e s c e n c e S p e c t r u m
F l u f e n a m i c a c i d s h o w s n a t i v e f l u o r e s c e n c e i n so-
me o r g a n i c s o l v e n t s , e . g. d i o x a n a n d c h l o r o f o r m , w h e r e -
a s i n e t h a n o l f l u o r e s c e n c e is t o o w e a k t o be a n a l i t i c a l l y
u s e f u l (8. 9). M i l l e r et al. (10) r e p o r t e d t h a t f l u f e n a m i c
a c i d s h o w e d n o s i g n i f i c a n t f l u o r e s c e n c e at room t e m p e r a -
t u r e i n a c i d i c , n e u t r a l or a l k a l i n e e t h a n o l s o l u t i o n , b u t
w a s s t r o n g l y f l u o r e s c e n t at l o w t e m p e r a t u r e ( 7 7 0 K ) , p r e -
s u m a b l y b e c a u s e of t h e v i r t u a l a b o l i t i o n of b i m o l e c u l a r
q u e n c h i n g i n t h e latter c o n d i t i o n s . D e l l a n d K u t s c h b a c h
(11) i n v e s t i g a t e d t h e i n f l u e n c e on f l u o r e s c e n c e i n t e n s i t y of
t h e s o l v e n t a n d t h e a d d i t i o n of a h a l o g e n o a c e t i c a c i d , a s
00.

80.,

60,,

40.,

E!O#,

W
0,
c!

F i g . 4. Mass Spectrum of F l u f e n a m i c A c i d
322 ENRICO ABIGNENTE AND PAOLO DE CAPRARIIS

well a s the influence of a t r e a t m e n t with a l k a l i a n d / o r a n


oxidant. T o obtain a f l u o r e s c e n t solution s o l v e n t s with a
d i e l e c t r i c c o n s t a n t = 0 a r e r e q u i r e d , and f l u o r e s c e n c e i n -
t e n s i t y is s t r o n g l y i n c r e a s e d by t h e addition of a haloge-
n o a c e t i c a c i d with a pKa value < 1 (e. g. t r i c h l o r o a c e t i c
a c i d ) . T h e s e findings a r e the b a s i s f o r t h e m o s t c o m m o n
m e t h o d of f l u o r o m e t r i c d e t e r m i n a t i o n of f l u f e n a m i c a c i d
(see Section 5. 5).
Dell e t al. (8) published the f l u o r e s c e n c e s p e c t r u m of
flufenamic a c i d in a C C 1 4 / t r i c h l o r o a c e t i c a c i d solution:
i n t h i s m e d i u m flufenamic a c i d s h o w s the e x c i t a t i o n m a x i -
m u m a t 372 n m with a s u b s i d i a r y m a x i m u m a t 296 n m ,
and t h e e m i s s i o n m a x i m u m a t 445 n m . T h e r e is a l i n e a r
r e l a t i o n s h i p between the f l u o r e s c e n c e i n t e n s i t y and the
c o n c e n t r a t i o n of f l u f e n a m i c a c i d u p t o 1 0 p g / m l .
S o m e f l u o r e s c e n c e d a t a r e p o r t e d in the l i t e r a t u r e f o r
flufenamic a c i d a r e l i s t e d in T a b l e VI.

T A B L E VI
F l u o r e s c e n c e Maxima of F l u f e n a m i c Acid
in v a ri o u s solvents

Solvent Excitation Emission Ref.


maximum, n m m a x i m u m , nm -
Methanol 305 400 8
Ethanola 335 510(420)b 10
Dioxan 3 34 410 9
Chloroform 340 430 9
CCl,/TCA 372 (296)b 445 8
a : a t 77OK.
b : subsidiary maximum.

2. 6. Melting r a n g e
In T a b l e VII a r e l i s t e d s o m e m e l t i n g point v a l u e s
r e p o r t e d by v a r i o u s a u t h o r s f o r flufenamic a c i d ,
T h e d i s c r e p a n c i e s a m o n g t h e s e v a l u e s a r e due t o t h e
d i f f e r e n t p o l y m o r p h i c m o d i f i c a t i o n s which c a n be p r e s e n t
i n c o m m e r c i a l p r o d u c t s (see Section 2 . 8).
FLUFENAMIC ACID 323

TABLE VII
M e l t i n g R a n g e of F l u f e n a m i c A c i d
M. P . , OC Crystallization Ref.
solvent
125 50% e t h a n o l 12
1 2 4 - 125, with r e s o l i d i f i c a -
tion a n d r e m e l t i n g a t 134-
136 ligroine 13
127-128 NR 3
132-133 cyclohexane 14,15
133 NR 16
1 3 3 - 134 NR 2
1 3 3 -1 3 4 o r 1 25- 126, r e s o -
l id ify in g a n d r e m e l t i n g at
1 3 4 -1 3 6 NR 17
134. 3 ethanol 18
1 3 4 - 136 cyclohexane 19

NR : not r e p o r t e d

2 . 7. S o lu b i l i t y, P a r t i t i o n Coef fi ci e n t
Flufenamic acid was reported to be soluble a t
r o o m t e m p e r a t u r e in m e t h a n o l , e t h a n o l , d i e t h y l e t h e r ,
c h l o r o f o r m , a c e t o n e , D M F , and p e a n u t oil ( 3 , 4 , 1 3 ) . T h e
s o l u b i l i t y in w a t e r a t 22OC is gi ven b y R o l t z e a n d K r e i s -
f e ld ( 2 ) a s 0 . 0067 mg/ml. In T a b l e VIII a r e l i s t e d s o m e
v a l u e s of s o l u b i l i t y in w a t e r a t v a r i o u s p H v a l u e s .
A s t u d y b y G h a n e m et al. ( 2 1 ) i n d i c a t e s t h a t t h e s o l u b i -
l i t y of f l u f e n a m i c a c i d is i n c r e a s e d b y n o n i o n i c s u r f a c t a n -
t s , u r e a a n d s o d i u m citrate. T h e e f f i c i e n c y of t h e s u r -
f a c t a n t s t o w a r d s s o l u b i l i z a t i o n is i n t h i s o r d e r : T w e e n
80 > R r i j 9 9 > T w e e n 40 > M y r j 53. T h e e f f e c t of u r e a ,
a m i d o p y r i n e , p h e n a z o n e a n d p a r a c e t a m o l on t h e s o l u b i l i t y
of f l u f e n a m i c a c i d a n d o t h e r a n t i r h e u m a t i c d r u g s w a s s t u -
d i e d b y D a a b i s e t al. ( 2 2 ) .
1- O c t a n o l / w a t e r p a r t i t i o n c o e f f i c i e n t w a s e s t i m a t e d b y
Dunn (2 3 ) t a k i n g a d v a n t a g e of t h e a d d i t i v e - c o n s t i t u t i v e n a -
324 ENRICO ABIGNENTE AND PAOLO DE CAPRARIIS

TABLE VIII
Solubility of F l u f e n a m i c Acid in
Water a t v a r i o u s pH V a l u e s
PH Solubility , Ref,
mg/ml
3 0. 003a 13
7 1. 8a 13
7 1 20
8 4. oa 13

a : a t 37OC

t u r e of l o g P, as follows:

= log 'anthranilic acid + nphenyl


+ n3-cF3 =
= 1. 21 + 2 . 60 + 1 . 0 7 = 4. 88
T e r a d a e t al. ( 2 4 ) have d e t e r m i n e d the t r u e p a r t i t i o n
coefficient, P, a n d the a p p a r e n t p a r t i t i o n coefficient, P',
of f l u f e n a m i c acid. P w a s m e a s u r e d by e q u i l i b r a t i n g a
1-octanol solution of the d r u g , the i n i t i a l c o n c e n t r a t i o n ,
C,, of which w a s 1 0 - 3 - 1 0 - 2 m o l / l , with 0. 01 N HC1:
u n d e r t h i s condition, d r u g m o l e c u l e s e x i s t e x c l u s i v e l y a s
the unionized f o r m . A f t e r e q u i l i b r i u m w a s a t t a i n e d , c o n -
c e n t r a t i o n i n the a q u e o u s p h a s e , Cw, was m e a s u r e d s p e -
c t r o p h o t o m e t r i c a l l y and P w a s c a l c u l a t e d b y the equation:
P = C,/Cw, s i n c e t h e c o n c e n t r a t i o n change in t h e o r g a -
nic p h a s e c a n b e n e g l e c t e d f o r s u c h a highly hydrophobic
compound, obtaining l o g P = 5 . 62.
PI w a s d e t e r m i n e d with the 1- o c t a n o l / p h o s p h a t e b u f f e r
(pH 8. 0) s y s t e m , u s i n g the equation:

w h e r e C a n d V a r e t h e e q u i l i b r i u m c o n c e n t r a t i o n and v o
l u m e of a q u e o u s ( s u b s c r i p t w) and o r g a n i c ( s u b s c r i p t 0 )
p h a s e s , r e s p e c t i v e l y . Ci is t h e i n i t i a l c o n c e n t r a t i o n i n
the a q u e o u s p h a s e . L o g PI w a s found t o b e 1 . 7 4 .
FLUFENAMIC ACID 325

Lombardino et al. (25) have a l s o determined the p a r t i -


tion coefficient of flufenamic acid with some 1-octanol/
buffer s y s t e m s .
2. 8. Crystal P r o p e r t i e s , Polymorphism
Flufenamic acid can exist a s s e v e r a l crystalline
modifications. Kuhnert-Brandstatter et al. have d e s c r i -
bed five different modifications and have reported t h e i r
melting points and infrared s p e c t r a , a s well a s the t h e r -
mogram obtained by differential scanning c a l o r i m e t r y f o r
the f i r s t four f o r m s (16, 26).
According to K r c flufenamic acid can exist a s at l e a s t
seven crystalline modifications with different melting po-
ints (27). K r c has reported the f r e e energy vs. t e m p e r a -
t u r e plot of seven crystalline f o r m s of flufenamic acid.
Modifications I, I1 and I11 were described in detail in
t e r m s of c r y s t a l morphology, optical p r o p e r t i e s , X - r a y
diffraction powder data and infrared s p e c t r a .
Other authors a l s o studied the polymorphism of flufena-
mic acid. Galdecki et al. (28) investigated the c r y s t a l l i -
zation of the d r u g f r o m boiling solvents, B u r g e r and
Ramberger ( 2 9 ) examined the applicability of s o m e t h e r -
modynamic r u l e s to the polymorphic s y s t e m of flufenamic
acid. These r u l e s c o r r e l a t e the heats of transition o r
fusion, IR s p e c t r a and densities of the modifications with
t h e i r stability behavior. In this study flufenamic acid w a s
investigated mainly by quantitative DSC and qualitative s o -
lubility determination (by thermomicroscopy) a s well a s
by IR spectroscopy to differentiate eight crystalline modi-
fications (Table IX). It w a s pointed out ( 2 9 ) that modifi-
cations I, I1 and I11 investigated by the various authors
a r e identical, whereas modification IV studied by Kuhnert
( 2 6 ) coincides with modification V by K r c ( 2 7 ) , who did
not describe Kuhnert's modification V. Since the l a t t e r
f o r m had the lowest melting point of all eight modifica-
tions, it was indicated by B u r g e r and R a m b e r g e r a s modi-
fication VIII.
F r o m the practical point of view, modifications I and
I11 a r e the most important, because they can be present
in the c o m m e r c i a l product. The transition point of these
two f o r m s is a t 42°C: modification I11 i s the stable f o r m
326 ENRICO ABIGNENTE AND PAOLO DE CAPRARIIS

at room t e m p e r a t u r e ( b e l o w 42" C) , w h e r e a s m o d i f i c a t i o n
I is t h e s t a b l e form a b o v e 42OC ( 2 7 ) . M o d i f i c a t i o n I11
w a s o b t a i n e d by B u r g e r a n d R a m b e r g e r by s t i r r i n g f o r 1 2
h o u r s a t 2OoC a x y l e n e s u s p e n s i o n of a commercial p r o -
d u c t f o r m e d by m o d i f i c a t i o n I ( 2 9 ) .

T A B L E IX
M e l t i n g P o i n t s of c r y s t a l l i n e M o d i f i -
c a t i o n s of F l u f e n a m i c A c i d
Mo d ific a t i on M. P . , O C Ref.
I 133 16,26
134 27, 2 9
I1 128 1 6 , 26, 27, 2 9
I11 125 16,26
126 29
126. 5 27
IV 124 27
Va 122 1 6 , 26, 29
122.5 27
VI 120 27
VII 118 27
VIIIb 100-110 26
108 2 5 29

a : T h i s m o d i f i c a t i o n w a s i n d i c a t e d a s IV i n t h e p a -
p e r s 1 6 a n d 26.
b : T h i s modification was indicated as V in the p a p e r s
1 6 a n d 26.

2. 9. D i s s o c i a t i o n C o n s t a n t
T h e pKa of f l u f e n a m i c a c i d w a s r e p o r t e d to b e
3 . 9 by A g u i a r a n d F i f e l s k i ( 2 0 ) a n d 4 . 5 by F r e y a n d El-
S a y e d (30). Terada et al. ( 2 4 ) h a v e found a v a l u e of
3 . 8 5 u s i n g t h e pH -dependent s o l u b i l i t y m e t h o d ( 3 1 ) ; t h i s
value is c o n s i d e r a b l y d i f f e r e n t from t h e c o r r e s p o n d i n g
FLUFENAMIC ACID 327

value obtained by p o t e n t i o m e t r i c t i t r a t i o n i n 5 - 10% a q u e o u s


acetone ( 3 2 ) . In T a b l e X a r e l i s t e d s o m e pKa v a l u e s ob-
tained by p o t e n t i o m e t r i c t i t r a t i o n in v a r i o u s a q u e o u s s o l -
vent s y s t e m s .

TABLE X
pKa V a l u e s of F l u f e n a m i c Acid
obtained bv P o t e n t i o m e t r i c T i t r a t i o n
Solvent pKa Ref.
Water 7. 5 33
75% Aqueous methanol 5.75 3
50Vn Aqueous ethanol 5. 94 33
80% Aqueous 2-methoxy-
ethanol 6. 0 33
Dioxane: w a t e r ( 2 : 1) 6. 8 25

3 . SYNTHESIS
Wilkinson and F i n a r ( 1 2 ) f i r s t s y n t h e s i z e d flufenamic
a c i d by r e a c t i n g o-iodobenzoic a c i d with m - t r i f l u o r o m e -
thylaniline in p o t a s s i u m c a r b o n a t e a q u e o u s solution, i n t h e
p r e s e n c e of c o p p e r b r o n z e . T h e c r u d e p r o d u c t w a s p u r i -
fied via i t s a m m o n i u m s a l t .
Moffett and A s p e r g r e n (19) p r e p a r e d f l u f e n a m i c a c i d
s t a r t i n g f r o m o - c h l o r o b e n z o i c a c i d which w a s r e a c t e d
with m-trifluoromethylaniline i n 85070 aqueous p o t a s s i u m
hydroxide and a m y l alcohol with c o p p e r p o w d e r . Some
patented synthetic m e t h o d s follow the l a t t e r s c h e m e , as
i l l u s t r a t e d in F i g u r e 5.

Figure 5
Synthesis of F l u f e n a m i c Acid
F l u f e n a m i c a c i d w a s a l s o obtained via the r e a c t i o n b e t -
ween o-iodobenzoic a c i d and m-trifluoromethylphenylhydro-
xylamine ( 3 4 ) . Another method involves the r e a c t i o n of
328 ENRICO ABIGNENTE AND PAOLO DE CAPRARIIS

m e t h y l o - c h l o r o b e n z o a t e with N-(3-trifluoromethylphenyl)-
f o r m a m i d e (35). F l u f e n a m i c a c i d w a s a l s o p r e p a r e d by
photolysis of the c o r r e s p o n d i n g b e n z o t r i a z i n o n e ( 3 6 ) .

4. DRUG METABOLISM AND PHARMACOKINETICS


4. 1. M e t a b o l i s m
T h e m e t a b o l i c t r a n s f o r m a t i o n s which f l u f e n a m i c
a c i d u n d e r g o e s in m a n and a n i m a l s a r e d e p i c t e d in F i g u r e
6 , a c c o r d i n g t o Glazko ( 3 7 ) and O b e r e t al. (38), which
d e m o n s t r a t e d that t h e d r u g is e x c r e t e d m a i n l y i n f o r m of
i t s m e t a b o l i t e s . T h e i r s t u d i e s w e r e c a r r i e d out by t r a c e r
m e t h o d s u s i n g [14C] - c a r b o x y l - l a b e l l e d f l u f e n a m i c acid.
T h e 4'-hydroxy- and 5 - h y d r o x y - d e r i v a t i v e and f l u f e n a m i c
a c i d itself a r e e l i m i n a t e d in u r i n e chiefly i n conjugated
f o r m , w h e r e a s t h e 4', 5 - d i h y d r o x y - d e r i v a t i v e is not conju-
gated. A l l f o u r compounds h a v e b e e n found i n t h e s t o o l
i n unconjugated f o r m .
4'-Hydroxy- and 5-hydroxyflufenamic a c i d s w e r e s y n t h e -
s i z e d by Bowman et al. (39).
4. 2. Absorption and E x c r e t i o n
4. 21. In A n i m a l s
T h e r a t e of p e r m e a t i o n of f l u f e n a m i c a c i d
t h r o u g h the gut m e m b r a n e and the amount of d r u g a b s o r -
bed w e r e studied i n v i t r o on s e g m e n t s of t h e small i n t e -
s t i n e of golden h a m s t e r s ( 2 0 ) . T h e p e r m e a t i o n of flufena-
m i c a c i d w a s pH-dependent, a c c o r d i n g t o t h e postulation
t h a t i t is only the unionized m o l e c u l e of t h e d r u g t h a t pas-
s e s through a cell, due t o i t s lipoid solubility. T h i s s t u -
d y showed t h a t a t pH 2. 5 the p e r m e a t i o n of f l u f e n a m i c
acid, which is a p p r o x i m a t e l y 96% unionized, is 20 t i m e s
f a s t e r t h a n a t pH 7. 2, at which only 0. 05% of t h e d r u g is
i n the unionized f o r m .
T h e a b s o r p t i o n and t h e u r i n a r y e x c r e t i o n of f l u f e n a m i c
a c i d w a s s t u d i e d i n r a b b i t s following both c u t a n e o u s and
o r a l application ( 4 0 ) of t h e same d o s e ( 3 0 m g / k g ) . A f t e r
4 8 h o u r s from cutaneous application, 5. 9% of t h e applied
d o s e w a s found i n the u r i n e ; t h e blood level of flufena-
m i c a c i d r e m a i n e d c o n s t a n t o v e r the f i r s t s i x h o u r s at
about 3 pg/ml, o v e r c o m i n g t h e blood l e v e l obtained o r a l -
l y a s f r o m the 4th hour.
m
u.
q
R
Erc
&
0
I
u3
m f
0
r
CD
4 c
R a, a,
lil
rcl
Erc I
rl
.rl~
Erc
R +I
I 0
E
(I)
0 d
.rl
/
0
I e
lu
Y
\ 4 c
0
k
I
V
3 30 ENRICO ABIGNENTE AND PAOLO DE CAPRARIIS

Rosenberg and Bates ( 4 1 ) compared the blood concentra-


tions produced in r a t s following o r a l administration of f l u -
fenamic acid alone o r together with cholestyramine. F l u -
fenamic acid strongly bound to the r e s i n , s o that a 6 0 -
70% d e c r e a s e in both the r a t e and the extent of absorption
of the d r u g was observed: following an o r a l dose of 5 0
m g / k g a peak plasma concentration of 8 9 . 2 p g / m l w a s
reached at 1 hour, whereas when cholestyramine and the
d r u g were coadministered the peak l e v e l dropped to 3 2 . 3
pg/ml. F u r t h e r studies on pharmacokinetics of flufena-
m i c acid in the r a t a r e reported. F r e y and El-Sayed ( 3 0 )
determined the flufenamic acid concentrations in s e r u m
and g a s t r i c mucosa a f t e r o r a l and subcutaneous admini-
stration. Lin et al. ( 4 2 ) determined p l a s m a l e v e l s a f t e r
intravenous administration, Cotellessa e t al. ( 4 3 ) d e t e r -
mined plasma and u t e r u s levels a f t e r intravenous and o r a l
administration.
A s r e g a r d s the excretion, Glazko ( 3 7 ) showed that dogs
eliminated only 2-770 of an o r a l dose in the urine and 53-
7 9 % in the f e c e s , w h e r e a s the corresponding values f o r
monkeys were 45-8070 and 12-2170, respectively, S i m i l a r
r e s u l t s were obtained by Ober et al. ( 3 8 ) in e x p e r i m e n t s
with dogs. Lombardino et al. ( 2 5 ) r e p o r t e d that the s e -
r u m half life a f t e r o r a l administration of the aluminium
salt of flufenamic acid w a s 3 h o u r s f o r rats and dogs,
and 4 hours f o r rabbits.
4 . 2 2 . In Humans
In addition to the r e s u l t s obtained in r a b -
bits ( 4 0 ) , P a n s e e t al. have a l s o studied the absorption
of flufenamic acid through the human skin ( 4 4 ) .
Glazko ( 3 7 ) reported that n e a r l y 100% of an o r a l dose
of flufenamic acid w a s absorbed; the r e n a l elimination of
the drug and i t s metabolites was 5170, of which only 2 . 6%
w a s unaltered drug. Such r e s u l t s were m o r e r e c e n t l y
confirmed by Dell e t al. ( 4 5 , 4 6 ) with two different meth-
ods for the determination of all fluorine-containing c o m -
pounds a s a group in the urine, which showed that the r e -
nal elimination of flufenamic acid and i t s metabolites w a s
4 9 . 470 within t h r e e days a f t e r o r a l administration. The
peak p l a s m a level w a s reached a f t e r two hours, and the
p l a s m a elimination half life w a s found t o be approximate-
FLUFENAMIC ACID 331

l y 3 h o u r s . D e l l e t al. ( 4 5 ) a l s o r e p o r t e d t h a t 3. 670 of
a n o r a l d o s e of flufenamic a c i d was e x c r e t e d unconjugated
into the u r i n e within 6 d a y s : however, f e m a l e s u b j e c t s
e l i m i n a t e d only 1. 9% and the m a l e o n e s 5. 370. On the
c o n t r a r y , no difference w a s o b s e r v e d between m e n and
women in the t o t a l amount of all m e t a b o l i t e s e x c r e t e d by
the r e n a l route. Another study on t h e sex-dependence of
the r e n a l e x c r e t i o n of flufenamic a c i d and o t h e r f e n a m a t e s
in m a n and a n i m a l s was r e p o r t e d by L o r e n z a n d D e l l (47).
T h e bioavailability of o r a l p h a r m a c e u t i c a l f o r m u l a t i o n s
of flufenamic a c i d w a s investigated by A r i a s a n d Cadorni-
ga ( 4 8 ) and Angelucci e t al. (49).
4. 3 . P r o t e i n binding
T h e bovine and human s e r u m a l b u m i n (BSA and
HSA, r e s p e c t i v e l y ) binding affinity of flufenamic a c i d w a s
investigated by Chignell b y c i r c u l a r d i c h r o i s m s t u d i e s
(50-52). T h e r o l e of hydrophobicity f o r the binding affini-
t y w a s investigated by Dunn on HSA (23) and by T e r a d a e t
al. on BSA (24). T h e hydrophobicity a s well a s the with-
d r a w i n g ability of the - C F 3 substituent c o n t r i b u t e signifi-
cantly t o the binding affinity, which w a s d e t e r m i n e d f o r
BSA by m e a s u r i n g the ability of flufenamic a c i d t o d i s p l a -
c e 2 - (4' - hydroxypheny1azo)benzoic a c i d c o m p e t i t i v e l y u n d e r
conditions of pH 7 . 0 and 25OC (24). T h e binding constant,
K, w a s d e t e r m i n e d : the value obtained f o r BSA by T e r a -
d a e t a l . , 6 . 5 l~o 5 1. m o l - l , s e e m s t o c o n f o r m t o a v a -
lue, 1. 3 x l o 6 1. m o l - l , obtained by Chignell with HSA at
pH 7 . 4 (51).
T h e i n t e r a c t i o n between BSA and s e v e r a l c a t i o n i c and
anionic d r u g s including flufenamic a c i d w a s studied by
B l a n c h a r d e t al. (53) u s i n g the e l e c t r o n s p i n r e s o n a n c e
s p i n labeling technique.
T h e binding of flufenamic a c i d t o HSA w a s s t u d i e d by
O t a g i r i e t al. ( 5 4 ) v i a m i c r o c a l o r i m e t r i c i n v e s t i g a t i o n s .
T h e h e a t flux g e n e r a t e d by the binding is p r o p o r t i o n a l t o
the amount of the d r u g bound t o the p r o t e i n . If only one
binding s i t e on t h e d r u g m o l e c u l e c o n t r i b u t e s t o the h e a t
flux, then the d a t a can r e a d i l y be i n t e r p r e t e d i n t e r m s of
the binding constant, AG, AH, and A S f o r binding t o that
site. If m a n y s i t e s a r e involved having d i f f e r e n t e n t h a l -
332 ENRICO ABIGNENTE AND PAOLO DE CAPRARIIS

pies of binding, then unambiguous interpretation of the


data may be impossible. T h i s is the c a s e of flufenamic
acid, which h a s been reported t o have t h r e e v e r y high
affinity s i t e s f o r HSA and other s i t e s of lower affinity(51).
Sudlow et al. (55, 56) have c h a r a c t e r i z e d two distinct
binding s i t e s ( I and 11) f o r anionic d r u g s on HSA by the
u s e of fluorescent probes. Flufenamic acid binds s e l e c t i -
vely to s i t e 11, a s well a s ibuprofen, flurbiprofen and
ethacrynic acid , whereas phenylbutazone and warfarin
bind to s i t e I. Drugs which bind t o s i t e I1 a r e all a r o m a -
t i c carboxylic acids, which would be l a r g e l y ionized a t
physiological pH.
Kaneo et al. (57) examined the binding t o BSA of s i x
nonsteroidal antiinflammatory d r u g s including flufenamic
acid by the u s e of dialysis a t pH 7 . 4 and 37OC. It was
found that flufenamic acid strongly binds t o BSA and the
f r e e fraction of t h i s d r u g e x i s t s within 1% over the t h e r a -
peutic range.

5. METHODS OF ANALYSIS
5. 1. Identification T e s t s
Flufenamic acid can be identified by virtue of i t s
U V , IR, N M R , m a s s and fluorescence s p e c t r a (see Sec-
tion 2). Various chromatographic methods a r e a l s o s u i -
table f o r purposes of identification ( s e e Section 5 . 7 ) .
Devaux e t al. (58) described two color r e a c t i o n s and
one fluorescent reaction. The color r e a c t i o n s a r e due t o
the diphenylamine s t r u c t u r e , w h e r e a s the fluorescent r e a -
ction was explained by the formation of substituted a c r i -
dones ( s e e Section 5.5). T h e s e reactions can be c a r r i e d
out a s follows:
a ) Flufenamic acid (at l e a s t 1 m g ) and about 0. 5 g of
oxalic acid a r e heated into an oil bath at 180-200°C 4-5
minutes. After cooling the r e s i d u e is dissolved in 95%
ethanol t o obtain a stable, intense blue color. The a b s o r -
ption maximum is at 585-590 nm.
b ) Flufenamic acid ( a t l e a s t 100 p g ) is added in a mix-
t u r e of 1 m l CH3COOH:H2S04 (d.1.83)(98:2), 5 rnl CH3-
CO0H:HCl (d. 1.18)(50:50), and 1 m l 0. 10% aqueous levu-
lose. The mixture is heated a t 100°C 25 minutes t o ob-
FLUFENAMIC ACID 333

tain a violet color. The absorption maximum is at 597nm.


c ) Flufenamic acid is dissolved in conc. H2SO4 and
heated 10 minutes at 100°C: the solution exhibits an in-
tense green fluorescence when excited by white light, and
blue when excited by UV light.
5. 2. T i t r i m e t r i c Analysis
Flufenamic acid can be titrated in acetone with
0 . 1 N aqueous potassium hydroxide in the p r e s e n c e of
phenolphtaleine (3). A nonaqueous t i t r i m e t r i c method w a s
described by Walash and Rizk (59), which used 0 . 1 N s o -
dium methoxide as the t i t r a n t and DMF o r tetramethyl-
u r e a as the solvent, m e a s u r i n g the end point e i t h e r with
a thymol blue indicator o r potentiometrically. Various
potentiometric methods in aqueous solvent s y s t e m s a r e
cited in Section 2. 9.
5. 3. Colorimetric Analysis
Flufenamic acid and i t s metabolites w e r e d e t e r -
mined a s a group colorimetrically in urine a f t e r alkaline
hydrolysis and fusion with sodium peroxide: fluorine was
then distilled a s H2SiF6 in the p r e s e n c e of H2S04 and
Si02. A. solution of alizarine-3-methylamino-N, N-diace-
tato-cerium(II1) was added to the distillate to obtain a co-
l o r reaction, and a c o l o r i m e t r i c determination w a s effec-
ted at 617 nm. Measurements in the range h l p,g a r e
possible (46).
The color reactions cited in Section 5. 1 can a l s o be
used for quantitation.
5.4. Spectrophotometric Analysis
The f i r s t UV spectrophotometric method f o r fena-
m a t e s analysis w a s described by Carey (60). The W ab-
sorbance of flufenamic acid ( s e e Section 2. 3 ) e i t h e r in
methanol a t 288 nm (3,40,44) o r in 0. 1 N NaOH at 287-
290 nm (4, 30,411 can be used f o r quantitative analysis.
Beltagy (61) described a spectrophotometric method f o r
the determination of s e v e r a l acidic d r u g s including flufe-
namic acid which w a s determined obtaining the ion-pair
association complex of the d r u g and safranine in a pH 7. 4
buffer, then extracting the complex with chloroform and
measuring the absorbance of the e x t r a c t .
334 ENRICO ABIGNENTE AND PAOLO DE CAPRARIIS

5. 5. F l u o r o m e t r i c A n a l y s i s
Mehta and Schulman ( 9 ) a f f i r m e d t h a t t h e native
f l u o r e s c e n c e exhibited by f l u f e n a m i c a c i d i n o r g a n i c s o l -
v e n t s (see Section 2. 5) could b e u s e f u l f o r i t s d e t e c t i o n
and d e t e r m i n a t i o n , N e v e r t h e l e s s the m e t h o d m o s t corn -
monly u s e d involves the f l u o r o m e t r i c d e t e r m i n a t i o n of flu-
f e n a m i c a c i d i n c a r b o n t e t r a c h l o r i d e a f t e r addition of a
CC14 solution of t r i c h l o r o a c e t i c a c i d which s t r o n g l y i n c r e -
ases the f l u o r e s c e n c e i n t e n s i t y (11). T h e f l u o r e s c e n c e
m a x i m a u n d e r t h i s condition a r e r e p o r t e d i n Section 2. 5.
T h i s method w a s u s e d by a n u m b e r of a u t h o r s t o a s s a y
flufenamic a c i d i n body f l u i d s and t i s s u e s (8, 37, 40, 49, 62,
63, 64).
T h e r e a c t i o n of f l u f e n a m i c a c i d with f o r m a l d e h y d e gives
1-(m-trifluoromethylphenyl)-4-oxo-1,2 - d i h y d r o - 3, 1- b e n z o -
xazine (4), which is s u i t a b l e f o r f l u o r o m e t r i c d e t e r m i n a -
tion of the p a r e n t d r u g (63). T h e r e a c t i o n s c h e m e is d e -
picted in F i g u r e 7. T h e m e t h a n o l i c s o l u t i o n of t h e b e n z o -
xazine d e r i v a t i v e s h o w s two e x c i t a t i o n m a x i m a at 278 and
0
COOH

NH
I
- HCHO

Figure 7
Reaction of F l u f e n a m i c Acid with F o r m a l d e h y d e
342 n m and a n e m i s s i o n m a x i m u m a t 440-450 n m ( 4 ) . T h e
c o r r e s p o n d i n g v a l u e s r e p o r t e d by D e l l e t al. ( 6 3 ) a r e 346
and 458 nm.
Another fluorogenic r e a c t i o n of f l u f e n a m i c a c i d , a l r e a d y
c i t e d i n Section 5. 1, w a s s t u d i e d by D e l l and K a m p ( 4 ) .
F l u f e n a m i c a c i d w a s h e a t e d with c o n c e n t r a t e d s u l f u r i c
a c i d to give a m i x t u r e of two i s o m e r i c a c r i d o n e s , I and
11, as i l l u s t r a t e d i n F i g u r e 8. T h e f l u o r e s c e n c e f e a t u r e s
of t h e s e compounds, which had b e e n p r e v i o u s l y s y n t h e s i -
zed by Wilkinson and F i n a r (12), a r e v e r y s i m i l a r s o that
FLUFENAMIC ACID 335

Reaction of F l u f e n a m i c Acid with conc. H2S04


the quantitative d e t e r m i n a t i o n c a n b e m a d e on t h e mixtu-
r e . In T a b l e XI a r e l i s t e d the wavelengths of excitation
and e m i s s i o n m a x i m a i n n e u t r a l , a c i d i c and a l k a l i n e m e -
thanol ( 4 ) .

T A B L E XI
F l u o r e s c e n c e Data of Trifluoromethylacridones
Solvent E x c i t a t i o n / e m i s s i o n m a x i m a , n m , of:
system 4 - C F 3 - a c r i d o n e (I) 2 - C F 3 - a c r i d o n e (11)

Methanol 400 / 420 400 / 4 2 1


Methanol- H C1 400/440 400/440
Methanol - NaOH 400 / 4 5 5 - 4 6 2 400/460

H a t t o r i e t al. ( 6 5 ) p r o p o s e d a f l u o r o m e t r i c method
which involves the t r e a t m e n t of a n ethanolic solution of
flufenamic a c i d with 0. 5% A1C13 solution i n a b s o l u t e e t h a -
nol t o obtain a n a l u m i n i u m c h e l a t e , which f l u o r e s c e s a t
440 n m following activation a t 358 n m . T h e m a x i m u m
f l u o r o m e t r i c s e n s i t i v i t y of flufenamic a c i d c l a i m e d f o r
t h i s method is 4 n g / m l .
5 . 6. I n d i r e c t Atomic Absorption A n a l y s i s
It w a s found that flufenamic acid, c o p p e r and
2-(2-hydroxyethyl)pyridine combined i n the r a t i o 1:1:1 t o
f o r m a c h e l a t e complex (18). T o obtain t h i s r e s u l t the
s a m p l e containing the d r u g w a s t r e a t e d with a r e a g e n t
p r e p a r e d adding 9. 0 m l of 0. 1% c u p r i c s u l f a t e solution t o
336 ENRICO ABIGNENTE AND PAOLO DE CAPRARIIS

1.5 m l of 2-(2-hydroxyethyl)pyridine. The complex w a s


then extracted with propyl acetate, and the amount of flu-
fenamic acid p r e s e n t in the s a m p l e was obtained i n d i r e c -
tly f r o m the amount of copper determined in the organic
solvent by atomic absorption analysis.
5. 7. Chromatographic Analysis
5.71. P a p e r Chromatography
Flufenamic acid can be detected by p a p e r
chromatography (66) using the following solvent s y s t e m s :
a ) Methyl isobutyl ketone : f o r m i c acid : w a t e r (10 p a r t s
of ketone s a t u r a t e d with 1 p a r t of 470 f o r m i c acid); Rf =
0.95.
b ) Chloroform : methanol : f o r m i c acid : w a t e r (a mixtu-
r e of 1 p a r t of methanol and 1 p a r t of 470 f o r m i c acid
used t o s a t u r a t e 10 p a r t s of chloroform); Rf = 0. 95.
c ) Benzene : methyl ethyl ketone : f o r m i c acid : water
( a mixture of 9 p a r t s of benzene and 1 p a r t of ketone sa-
turated with 1 p a r t of 2’70 f o r m i c acid); Rf = 0. 94.
d ) Benzene : f o r m i c acid : water (10 p a r t s of benzene
s a t u r a t e d with 1 p a r t of 270 f o r m i c acid); Rf = 0. 91.
e ) Methyl ethyl ketone : diethylamine : w a t e r (921:2:77);
Rf = 0. 83.
f ) Methyl ethyl ketone : acetone : f o r m i c acid : water
(40:2:1:6); Rf = 0. 95.
Flufenamic acid can be visualized by UV light, or s p r -
aying the p a p e r with 0.470 p-nitrobenzenediazonium fluo-
borate solution in 1:2 dioxane:water o r with 2’70 aqueous
phosphomolybdic acid solution.
Schmollack and Wenzel (67) developed a method f o r the
detection and quantitative determination of flufenamic acid
using a c h a m b e r p a p e r a n a l y s i s apparatus. Flufenamic
acid w a s then determined fluorometrically a f t e r t r e a t m e n t
with formaldehyde vapor t o obtain the strongly fluorescent
benzoxazine derivative.
5.72. Thin L a y e r Chromatography
Several thin l a y e r chromatographic methods
have been developed f o r identification and quantitative de-
termination of flufenamic acid. Some d e t a i l s of these
methods a r e s u m m a r i z e d in Table XII. It h a s t o point
out that in all c a s e s s i l i c a g e l plates w e r e used.
T A B L E XI1
T h i n L a y e r C h r o m a t o g r a p h y of F l u f e n a m i c Acid
Ref. Solvent S y s t e m Plates Rf D e t e c t ion
3 Cyclohexane:CHC13:CH30H:CH3COOH (60:30:5:5) S. G. G F 0. 54 W(254nm)
3 B e n z e n e : e t h e r : CH3 COOH: CH3 OH ( 120 : 60: 18: 1) id. 0. 7 8 id.
4 Benzene:C2H50H:CH3COOH (20:2:1) S. G. H F NR UV following h e a -
t i n g with HCHO
8 Cyclohexane :e t h y l acetate : CH3 COOH ( 2 0 :30 :2 ) S. G. F 6 0 0.58 W ( 3 5 6 nm)
8 Cyclohexane: CHC13:CH3COOH (40:50: 10) S. G . H F NR id.
40 id. S.G. H F F 0.43 W
44 id. S. G. H F NR UV
63 id. id. 0. 58 U V ; heat. HCHO
+ U V ; iodine
63 B e n z e n e : m e t h a n o l (9: 1) id. , 0. 23 id.
63 Cyc1ohexane:ethyl acetate (1:1 0 ) id. 0. 23 id.
41 1sopropanol:ammonia:water (20: 1 : 2 ) NR 0. 64 NR
68 To1uene:acetic a c i d ( 9 : l ) S. G. G 0.73 H N 0 2 spray
68 To1uene:acetic a c i d (97. 5:2. 5 ) id. 0. 60 id.
69 Chloroform:methanol(7:3) i n NH3 atm. S.G. 60 0. 37 HCHO/HCOOH at
loooc + w
S. G . : silica gel. NR : not r e p o r t e d .
338 ENRICO ABIGNENTE AND PAOLO DE CAPRARIIS

Unterhalt ( 3 ) h a s identified f l u f e n a m i c a c i d i n a m i x t u r e
with o t h e r n o n s t e r o i d a l a n t i i n f l a m m a t o r y d r u g s : the s p o t
v i s u a l i z e d by UV light w a s e l u t e d with m e t h a n o l and flufe-
n a m i c a c i d w a s d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y a t 288
n m with a m e a n r e c o v e r y of 88%.
Dell and K a m p ( 4 ) a s s a y e d f l u f e n a m i c a c i d in u r i n e and
s e r u m : t h e i r method involved a n e t h e r e a l e x t r a c t i o n f r o m
t h e biological fluids followed by T L C . T h e p l a t e s w e r e
then exposed t o f o r m a l d e h y d e v a p o r ( 2 h o u r s at 8OoC) and
t h e UV-fluorescent spot w a s eluted with m e t h a n o l and f l u -
f e n a m i c a c i d d e t e r m i n e d f l u o r o m e t r i c a l l y . An a l t e r n a t i v e
method p r o p o s e d by the s a m e a u t h o r s involved t h e v i s u a -
lization of flufenamic a c i d by UV light and a t r e a t m e n t of
the s c r a p e d off s i l i c a g e l with conc. H2SO4 followed by
f l u o r o m e t r i c d e t e r mi nat ion.
T h e method d e s c r i b e d by P a n s e e t a l . ( 4 0 ) f o r t h e d e -
t e r m i n a t i o n of flufenamic a c i d in u r i n e and p l a s m a w a s
s i m i l a r , involving e x t r a c t i o n f r o m b i o l o g i c a l m a t e r i a l ,
T L C and elution of the d r u g , followed by q u a n t i t a t i v e d e -
termination ei t h e r spectrophotometrically in methanolic
solution o r f l u o r o m e t r i c a l l y in a CC14 solution i n the p r e -
s e n c e of t r i c h l o r o a c e t i c a c i d . T h e l a t t e r m e t h o d w a s a p -
plied by s e v e r a l a u t h o r s ( a l r e a d y c i t e d in Section 5. 5) f o r
t h e d e t e r m i n a t i o n of f l u f e n a m i c a c i d in b i o l o g i c a l fluids
and t i s s u e s . P a n s e e t al. ( 4 4 ) d e s c r i b e d a l s o a method
f o r t h e d i r e c t d e n s i t o m e t r i c d e t e r m i n a t i o n of f l u f e n a m i c
a c i d on the s i l i c a gel thin l a y e r s .
F l u f e n a m i c a c i d w a s d e t e r m i n e d d i r e c t l y in p l a s m a by
G e i s s l e r e t a l . ( 6 9 ) by addition of m e t h a n o l t o p r e c i p i t a t e
p r o t e i n s , T L C , t r e a t m e n t of t h e d r i e d p l a t e with f o r m a l -
dehyde v a p o r in t h e p r e s e n c e of f o r m i c a c i d a t 100°C f o r
45 m i n u t e s t o f o r m t h e benzoxazine d e r i v a t i v e , and d i r e c t
f l u o r i m e t r y of t h e plate. Use of f o r m i c a c i d s h o r t e n s t h e
r e a c t i o n t i m e and e n h a n c e s t h e f l u o r e s c e n c e i n t e n s i t y and
the sensitivity (quantities I 2 ng/spot m a y be detected).
A method involving r e v e r s e d - p h a s e thin l a y e r c h r o m a t o -
g r a p h y w a s r e p o r t e d by B o l t z e and K r e i s f e l d (2), which
u s e d s i l i c a gel p l a t e s i m p r e g n a t e d with a 10'70 solution of
Dow-Corning 200 i n e t h e r . F l u f e n a m i c a c i d w a s c h r o m a -
t o g r a p h e d u s i n g buffer : dioxane : a c e t o n e (2: 1 : l ) a s the s o l -
vent s y s t e m , in which t h e buffer w a s a t pH 5. 2 , 6. 2, and
FLUFENAMIC ACID 339

7. 2, r e s p e c t i v e l y . In T a b l e XI11 a r e l i s t e d Rf and R M v a -
l u e s found f o r flufenamic a c i d .

T A B L E XI11
Reversed-Dhase T L C of F l u f e n a m i c Acid
pH of the buffer Rf RM= l o g ( l / R f - l )
-
5. 2 0. 78 - 0.55
6. 2 0. 76 - 0.5
7. 2 0. 69 - 0. 350

5.73. Gas Chromatography


Roseboom and Hulshoff ( 7 0 ) h a v e developed
a r a p i d and s i m p l e c l e a n - u p and d e r i v a t i z a t i o n p r o c e d u r e
that c a n be g e n e r a l l y applied t o the g a s c h r o m a t o g r a p h i c
d e t e r m i n a t i o n of a c i d i c d r u g s including f l u f e n a m i c a c i d i n
p l a s m a s a m p l e s . T h e d r u g w a s e x t r a c t e d f r o m acidified
p l a s m a with c h l o r o f o r m : i s o p r o p a n o l (95:5), which w a s
then e v a p o r a t e d . T h e r e s i d u e w a s d i s s o l v e d i n toluene,
then the d r u g w a s b a c k - e x t r a c t e d with a small v o l u m e of
a methanolic 20% t e t r a m e t h y l a m m o n i u m hydroxide solution
(TMAH). T h e solution obtained w a s added t o N , N - d i m e -
t h y l a c e t a m i d e . A f t e r t r e a t m e n t with n-butyl iodide the
d r u g w a s c h r o m a t o g r a p h e d a s i t s n-butyl ester. A gas
c h r o m a t o g r a p h equipped with a flame ionization d e t e c t o r
w a s u s e d . T h e g l a s s c o l u m n s (150 c m x 2 mm I. D. ) w e r e
packed with 3% OV-1, 3% OV-17 o r 3y0 SP-1000, all on
100-120 m e s h C h r o m o s o r b W HP. T h e c a r r i e r gas ( n i -
t r o g e n ) f l o w - r a t e w a s mantained a t 20 m l / m i n . T h e r e c o -
v e r y of flufenamic a c i d in the first e x t r a c t i o n s t e p with
ch1oroform:isopropanol w a s 69%, but with toluene, which
c a n a l s o be u s e d f o r the e x t r a c t i o n f r o m p l a s m a , a r e c o -
v e r y of 95% w a s achieved. Toluene h a s the advantage
that no e v a p o r a t i o n of the e x t r a c t is n e c e s s a r y , and it
can b e e x t r a c t e d d i r e c t l y with the TMAH solution. In t h e
b a c k - e x t r a c t i o n with TMAH a r e c o v e r y of 62Y0 w a s obtai-
ned. T h e r e t e n t i o n t i m e s of the n-butyl e s t e r of flufena-
m i c a c i d with v a r i o u s s t a t i o n a r y p h a s e s a r e l i s t e d i n Ta-
ble XIV.
340 ENRICO ABIGNENTE AND PAOLO DE CAPRARIIS

TABLE XIV
G a s ChromatoaraDhic Data f o r Flufenamic Acid (701
Stationary Column Re tent ion
phase temperature, time,
OC sec
370 ov-1 2 00 2 34
370OV-17 210 271
370 sP-1000 230 218

Another g a s chromatographic method f o r quantitative de-


termination of flufenamic acid in p l a s m a w a s reported by
Cotellessa et al. (43). Flufenamic acid w a s extracted
f r o m plasma with benzene, a f t e r dilution of p l a s m a s a m -
ple with an equal volume of 0. 25 M acetate buffer (pH
4.35). The benzene e x t r a c t w a s evaporated to d r y n e s s
under vacuum. The r e s i d u e w a s dissolved in methanol
and methylated with diazomethane. The s a m e procedure
w a s a l s o applied t o r a t u t e r u s homogenates. The methy-
lated sample w a s dissolved in a n acetone solution of the
i n t e r n a l standard 3-chloro-6-aminobenzophenone. G a s
chromatographic analysis w a s c a r r i e d out using a gas
chromatograph equipped with a flame ionization detector.
The stationary phase w a s 370 OV-17 on Gas-Chrom Q
(100-120 m e s h ) packed into a g l a s s column ( 3 m x 2 m m
I. D. ). The column t e m p e r a t u r e w a s 23OoC and the c a r -
r i e r g a s (nitrogen) flow-rate w a s 40 m l / m i n . F I D s e n s i -
tivity was 1 p g / m l p l a s m a and 5 ,ug/g u t e r u s .
A m a s s s p e c t r o m e t e r coupled with the g a s chromato-
graph was employed to a s c e r t a i n the identity of the m e -
thyl e s t e r of flufenamic acid with the GC peak. A m a s s
s p e c t r u m of the methyl e s t e r is presented. The recove-
r i e s f r o m plasma in the range 10-100 ,ug/ml and f r o m
u t e r u s homogenates were 98. 270 and 9070, respectively.
A g a s chromatographic method f o r the detection of non-
s t e r o i d a l antiinflammatory d r u g s including flufenamic acid
in urine collected f r o m h o r s e s that had received these
compounds orally h a s been developed by Hunt et al. (71).
T h i s procedure involves the isolation of the d r u g s f r o m
FLUFENAMIC ACID 341

urine by solvent extraction and on-column methylation of


the carboxylic acid group.
5. 74. High P e r f o r m a n c e Liquid Chromatography
A method f o r the separation and d e t e r m i n a -
tion of some nonsteroidal antiinflammatory d r u g s including
flufenamic acid w a s described by Dusci and Hackett ( 7 2 ) .
This procedure, which can be applied to s e r u m s a m p l e s
of s m a l l volume (100 pl), involved the extraction of the
drugs with acetonitrile. The e x t r a c t w a s taken to d r y n e s s
at 5OoC under a s t r e a m of nitrogen. The r e s i d u e w a s re-
dissolved in 1 0 0 pl of the elution solvent (60% acetonitrile
in 45 mM KH2P04 adjusted to pH 3. 0 with H 3 P 0 4 ) . An
aliquot of 10-20 pl was injected in a high performance li-
quid chromatograph equipped with a variable wavelength
U V detector. The column (30 c m x 3. 9 m m I. D. ) w a s
packed with pBondapak c18. The conditions f o r individual
analysis of flufenamic acid were as follows: flow-rate of
the elution solvent 2. 0 ml/min, wavelength 282 nm. F o r
the separation of flufenamic acid f r o m the mixture of an-
tiinflammatory drugs (flufenamic and mefenamic acid, na-
proxen , ibuprofen , indomethacin, phenylbutazone, oxyphen-
butazone) a flow-rate of 0 . 8 m l / m i n and a wavelength of
225 nm was used: under these conditions the elution t i m e
of flufenamic acid w a s 10. 5 min.
Using the above -mentioned elution solvent , flufenamic
and mefenamic acid were not separated. A modified elu-
tion solvent (3570 acetonitrile in 0. 7% NHqC1 buffered to
pH 7. 8 with ammonia) allowed to obtain the separation of
all the drugs investigated. Using a flow-rate of 1. 0 m l /
min, the elution time of flufenamic acid w a s 10. 2 min
(mefenamic acid 7. 8 min). The r e c o v e r y of flufenamic
acid was 92t370 in a s e r i e s of ten plasma s a m p l e s e x a m i -
ned, in the range 1. 0-20 p g / m l .
Lin et al. ( 4 2 ) have developed a H P L C procedure f o r
the determination of flufenamic acid and mefenamic acid
in plasma, A single extraction s t e p i s followed by r e v e r -
sed-phase chromatography. Flufenamic acid and mefena-
mic acid can be internal standards f o r each other during
e i t h e r assay. The extraction of flufenamic acid f r o m a c i -
dified plasma s a m p l e s (1 m l ) , t o which 4 p g of mefenamic
acid had been added, w a s accomplished with carbon t e t r a -
342 ENRICO ABIGNENTE AND PAOLO DE CAPRARIIS

chloride. T h e e x t r a c t w a s e v a p o r a t e d t o d r y n e s s u n d e r a
s t r e a m of nitrogen and t h e r e s i d u e w a s r e d i s s o l v e d i n t o
0. 5 m l of methanol, and a n aliquot w a s i n j e c t e d in t h e
c h r o m a t o g r a p h , which w a s equipped with a s t a i n l e s s steel
column ( 3 0 c m x 4 m m I. D. ) packed with a s t a b l e r e v e r -
s e d - p h a s e s t a t i o n a r y p h a s e of p o r o u s silica b e a d s c o a t e d
with c h e m i c a l l y bonded cyanopropylsilane m o n o l a y e r s . T h e
elution solvent w a s w a t e r : a c e t o n i t r i l e : a c e t i c a c i d (60:30:
10). T h e f l o w - r a t e w a s 1 m l / m i n with a n o p e r a t i n g p r e s -
s u r e of 1000 p s i at r o o m t e m p e r a t u r e . T h e effluent w a s
m o n i t o r e d continuously at 254 n m . Under t h e s e conditions
flufenamic a c i d had an elution t i m e of 10. 4 min, with a
m e a n r e c o v e r y f r o m p l a s m a of 100.7?3.4% in t h e 1-10 p g
r a n g e . T h e s e n s i t i v i t y l i m i t w a s 1 p g / m l of p l a s m a .
A method f o r t h e d e t e r m i n a t i o n of flufenamic a c i d a n d
i t s m a j o r m e t a b o l i t e s , 4 ' - h y d r o x y - and 5-hydroxyflufena-
m i c a c i d , w a s d e s c r i b e d by Kubo e t al. ( 7 3 ) : t h e a c i d i -
fied s e r u m w a s e x t r a c t e d with ethyl a c e t a t e . T h e o r g a n i c
e x t r a c t was e v a p o r a t e d and the r e s i d u e w a s r e d i s s o l v e d
i n ethanol and c h r o m a t o g r a p h e d , u s i n g a c o l u m n packed
with Bondapack c 1 8 . T h e m o b i l e p h a s e c o n s i s t e d of wa-
t e r : ethanol (52:48) containing 0. 1% N a 2 H P 0 4 and 0. 5%
t e t r a b u t y l a m m o n i u m b r o m i d e , a d j u s t e d t o pH 7. 81. T h e
r e c o v e r i e s f r o m p l a s m a w e r e 98. 870 f o r f l u f e n a m i c a c i d ,
97.0% f o r 4 ' - h y d r o x y - d e r i v a t i v e J a n d 98.070 f o r 5-hydro-
xy-derivative.

6. DETERMINATION IN BODY FLUIDS AND TISSUES


Many m e t h o d s a m o n g t h o s e outlined in t h i s a n a l y t i c a l
p r o f i l e have been applied t o t h e detection a n d quantitative
d e t e r m i n a t i o n of flufenamic a c i d in biological s a m p l e s of
a n i m a l o r human origin. Such a p p l i c a t i o n s w e r e r e p o r t e d
i n the p a p e r s l i s t e d below :
Colorimetry 46
Spe c t rophot o m e t r y 4, 30, 40,41, 44, 60, 61
Fluorimetry 4, 8, 11, 37, 40, 45, 47, 49, 62,
63, 64, 65, 67, 69
Indirect A A A n a l y s i s 18
PC 67
TLC 4, 8,40, 41, 44,45, 63, 64, 68, 6 9
FLUFENAMIC ACID 343

GC 43, 70, 7 1
HPLC 42, 72, 7 3

7. AC KNOWL ED G EM EN T
T h e a u t h o r s would l i k e t o t h a n k D r . M i c h e l e L i g u o r i
and M r . Vincenzo Migliaro, Ente F a r m a c o l o g i c o Italiano,
N a p l e s , f o r p r o v i d i n g IR, NMR a n d UV s p e c t r a of f l u f e -
namic acid.

8. R E F E R E N C E S
1. M e r c k Index, 9th e d . , 1976, M e r c k a n d C o . , R a h w a y ,
N. J . ; p. 4028.
-
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-
3. B. U n t e r h a l t , A r c h . P h a r m . 303, 4 4 5 (1970).
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P h a r m . Sci. 63, 1168 ( 1 9 7 4 ) .
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-
H. J a c o b i a n d R. K a m p , A r z n e i m . - F o r s c h . 31, 9
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-
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344 ENRICO ABIGNENTE AND PAOLO DE CAPRARIIS

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m a and N. Y a m a g i s h i , C h e m . P h a r m . B u l 1 . 21, 1 6 3 2
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20. -
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-
m a n , A r z n e i m . - F o r s c h . 25, 1 6 2 9 ( 1 9 7 5 ) .
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-
- S a n d e r , A r c h . P h a r m . 307, 8 4 5 (1974).
27. J. K r c , J r . , M i c r o s c o p e 25, 31 ( 1 9 7 7 ) .
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(1980).
30. H. H. F r e y a n d M. A . E l - S a y e d , A r c h . I n t . P h a r m a -
-
codyn. T h e r . 230, 300 (1977).
31. A . A l b e r t a n d E . P. S e r j e a n t i n "The D e t e r m i n a t i o n
of Ionization Constants", C h a p m a n a n d Hall, London,
1971, p. 72.
32. H. T e r a d a a n d S. M u r a o k a , M o l . P h a r m a c o 1 . - 8, 9 5
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33. U. Jahn and T. W a g n e r - J a u r e g g , A r z n e i m . - F o r s c h .
24, 4 9 4 (1974).
34. H. M o r i y a m a , H. N a g a t a and T. T a m a k i , S u m i t o m o
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35. S. K o m u r a , K. K k a m u r a and M. T a k e n a k a , O t s u k a
FLUFENAMIC ACID 345

C h e m i c a l D r u g s C o . , L t d . , J a p a n . P a t e n t 7 3 26, 744,
A p r . 9, 1973. C.A. - 79, 31680 ( 1 9 7 3 ) .
36. G. R . Allen, J r . , and F. R . R . Church, A m e r i c a n
Cyanamid Co., S. A f r i c a n P a t e n t 71 00, 512, Sep. 3,
1971. C.A. 76, 140237 ( 1 9 7 2 ) .
37. A . J. Glazko, Ann. P h y s . Med. , Suppl. 9, 24 ( 1 9 6 7 ) .
38. R . E. O b e r , K. Ritchie and S. F. Chang, F e d . P r o c .
24, 547 ( 1 9 6 5 ) .
39. R. E. Bowman, K. D. Brunt, K. E. Godfrey, L.
K r u s z y n s k a , A . A . Reynolds, R . I. T h r i f t , D. Waite
and W. R . N. Williamson, J. Chem.Soc. P e r k i n I, 1
(1973).
40. P. P a n s e , P. Z e i l l e r and K. H. Sensch, A r z n e i m . -
F o r s c h . 21, 1 6 0 5 ( 1 9 7 1 ) .
41. H. A . R o s e n b e r g and T. R . B a t e s , P r o c . S o c . E x p .
-
Riol. Med. 145, 93 ( 1 9 7 4 ) .
42. C. K. Lin, C. S. Lee and .J. H. P e r r i n , J . P h a r m .
Sci. 69, 95 ( 1 9 8 0 ) .
43. L. C z e l l e s s a , R . Riva, P. Salva, F. M a r c u c c i and
E. Mussini, J. C h r o m a t o g r . 192, 4 4 1 ( 1 9 8 0 ) .
-
44. p. P a n s e , P. Z e i l l e r and K. H. Sensch, A r z n e i m . -
F o r s c h . 24, 1 2 9 8 ( 1 9 7 4 ) .
45. H. D. Dell, J. F i e d l e r , H. J a c o b i and B. Wasche,
A r z n e i m . - F o r s c h . 27, 1 3 2 2 (1977).
46. H. D. D e l l and J. F i e d l e r , F r e s e n i u s ' Z.Ana1. Chem.
-
270, 278 ( 1 9 7 4 ) .
47. D. L o r e n z and H. D. Dell, Naunyn-Schmiedeberg's
-
A r c h . P h a r m a c o l . 277, Suppl. , R 4 4 (1973).
48. J. A r i a s and R . Cadorniga, Boll. Chim. F a r m . - 112,
804 (1973).
49. L. Angelucci, €3. P i e t r a n g e l i , P. C e l l e t t i and S. F a -
villi, .J. P h a r m . Sci. 65, 455 ( 1 976).
50. C. F. Chignell, 1 , i f e S c i . 7, 1181 (1968).
51. -
C. F. Chignell, Mol. P h a r m a c o l . 5, 4 5 5 ( 1 9 6 9 ) .
52. C. F. Chignell and D. K. S t a r k w e a t h e r , M o l . P h a r -
m a c o l . 7, 229 ( 1 9 7 1 ) .
53. J. B l a n c h a r d , T . N. T o z e r , D. L. S o r b y and L. D.
-
Tuck, Mol. P h a r m a c o l . 11, 1 3 3 ( 1 9 7 5 ) .
54. M. Otagiri, G. E. H a r d e e and J. H. P e r r i n , Bio-
c h e m . P h a r m a c o l . 27, 1 4 0 1 (1978).
55. G. Sudlow, D. J. B i r k e t t and D. N. Wade, Mol.
346 ENRICO ABIGNENTE AND PAOLO DE CAPRARIIS

P h a r m a c o l . 11, 824 (1975).


56. G. Sudlow, T J . B i r k e t t a n d D. N. W a d e , Mol.
-
P h a r m a c o l . 1 2 , 1052 ( 1976).
57. Y. Ka n e o, A. K ai , S. K i r y u a n d S. I g u c h i , Y a k u g a k u
Z a s s h i 96, 1412 (1976). C.A. - 86, 1 0 0 7 4 7 ( 1 9 7 7 ) .
58. G. D e v a G , P. M e s n a r d a n d A . M. B r i s s o n , Ann.
P h a r m . F r . 27, 239 (1969).
59. M. I. W a l a s r a n d M. R i z k , I nd i a n J . P h a r m . 39, 8 2 -
(1 9 7 7 ). C . A . - 87, 157253 (1977).
60. -
J. B. C a r e y , J. C l i n . I n v e s t . 40, 1 0 2 8 ( 1 9 6 1 ) .
61. Y. A . B e l t a g y , Z e n t r a l b l . P h a r m . , P h a r m a k o t h e r . La-
- -
b o r a t o r i u m s d i a g n . 116, 925 (1977). C. A , 88, 1 5 8 5 4 8
(1978).
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63. H. D. Del l , J. F i e d l e r a n T B . W z s c h e , A r z n e i m . -
-
F o r s c h . 27, 1312 ( 1977).
64. H. D. Del l , H. Jacobi, R. K a m p a n d J. K o l l e , A r z -
n e i m . - F o r s c h . 31, 2 1 ( 1981).
65. Y. H a t t o r i , T. Arai, T. M o r i a n d E. F u j i h i r a ,
-
C h e m . P h a r m . Bull. 18, 1 0 6 3 (19 7 0 ) .
66. L. R e io , J. C h r o m a t o g r . 68, 1 8 3 ( 1 9 7 2 ) .
67. -
W. S c h m o l l a c k a n d U. W e n z e l , P h a r m a z i e 29, 5 8 3
(1 9 7 4 ).
68. B. D e m e t r i o u a n d B. G. O s b o r n e , J. C h r o m a t o g r . - 90,
4 0 5 (1 9 7 4) .
6 9. H. E. G e i s s l e r , E. M u t s c h l e r a n d A . S c h u m a c h e r ,
J. C h r o m a t o g r . - 146, 1 6 9 ( 1978).
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6 5 (1 9 7 9 ) .
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J. D u s c i a n d L. P. H a c k e t t , J. C h r o m a t o g r . 1 7 2 ,
5 1 6 (1 9 7 9) .
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-
t o g r . 174, 254 (1979).

L i t e r a t u r e surveyed through October, 1981.


HEXESTROL
Hassan X Aboul-Enein,
Essam A. Lo@, and
Mohumed E . Mohumed

1. Description 348
1 . 1 Nomenclature 348
1.2 Formulae 348
1.3 Molecular Weight 348
I .4 Elemental Composition 349
1.5 Appearance, Color, Odor 349
2. Physical Properties 349
2.1 Crystal Properties 349
2.2 Melting Point 349
2.3 Solubility 350
2.4 Identification 350
2.5 Spectral Properties 350
3. Synthesis 358
4. Stability and Decomposition Products 361
5 . Metabolism 361
6. Methods of Analysis 362
6.1 Titrimetry 362
6.2 Colorimetry 362
6.3 Ultraviolet Spectrophotometry (Uv) 363
6.4 Chromatographic Analysis 364
6.5 Mass Fragmentography 370
6.6 Biological Assays 370
References 372

Copyright 0 1982 by The American


Analytical Profiles of Drug Substances Pharmaceutical hsociation
Volume I I 347 ISBN 0-12-Mo811-9
348 HASSAN Y. ABOUL-ENEIN ET AL.

ANALYTICAL PROFILE-HEXESTROL

1. Descr i p t ton

1.1 Nomenclature

1.11 - Chemical N a m e s

(?) 3 , 4 - Di(p-hydroxy phenyl) n-hexane.


4 , 4 ' - (1, 2 d i e t h y l - 1, 2 - e t h a n e d i y l )
bis-phenol.
4 , 4 ' - (1, 2 d i e t h y l e t h y l e n e ) d i p h e n o l .
4 , 4 ' - dihydroxy - y,b-diphenyl hexane.
4 , 4 ' - dihydroxy - a ,B-diethyldiphenylethane.
p, p l - dihydroxy - d i p h e n y l hexane.
meso - 3 , 4 - b i s (p-hydroxyphenyl) -n-
hexane .
1 . 1 2 - Generic Name

H e x e s t r o l , H e x o e s t r o l , Dihydrodiethyl-
s t i l b e s t r o l , Hexanoestrol, Cycloestrol ,
Hormoestrol, S y n e s t r o l .

1.13 - Trade Name

S yn t r o g h e , Fo 11i p l e x , S yn t hovo .
1.2 Formula

1 . 2 1 - Emprical

'18 H22 '2

1.22 - Structural

C2R5

1.3 Molecular Weight

270.4
HEXESTROL 349

1.4 Elemental Composition

C , 79.96%; H, 8.2%; 0,11.84%

1.5 Appearance, Color, odor

w h i t e , o d o r l e s s , c o l o r l e s s c r y s t a l s or
c r y s t a l l i n e powder (1).

2. Physical properties

2.1 Crystal properties

The f o l l o w i n g t a b l e shows t h e c r y s t a l form of


h e x e s t r o l d e r i v a t i v e s and t h e i r m e l t i n g p o i n t s .
These d e r i v a t i v e s c a n be used f o r i d e n t i f i c a t i o n
purposes too ( 2 ) .
0
Derivative Crystalization Crystal M.P., C
solvent form

Meso Form

Di-Me e t h e r Me2CO-MeOH plates 146


Dipropionyl Pet. ether crystals 127-128
Dibutyryl Pet. ether crystals 106-107
Dicaproyl Pet. e t h e r crystals 96-97
Disuccinyl CHC13-pet. e t h e r crystals 150-153

DL(+) Form

Di-Me ether C&-pet. ether 56

2.2 Melting p o i n t

The f o l l o w i n g are t h e m e l t i n g p o i n t s f o r h e x e s t r o l
i n i t s meso, DL-forms and t h e i r a n t i p o d e s .
0
Form M.P., C Ref

Meso
-
185-188 3
184-185 4
186 5

DL (+)Form 128 2
350 HASSAN Y. ABOUL-ENEIN ET AL.

L(-) Form 80 2
D(+) Form 80 2

2.3 Solubility

Freely soluble i n ether, soluble i n acetone,


a l c o h o l , methanol, s o l u b l e i n v e g e t a b l e o i l s
upon warmming a l s o s o l u b l e i n d i l u t e s o l u t i o n
of a l k a l i h y d r o x i d e . S l i g h t l y s o l u b l e i n
c h l o r o f o r m , benzene; i n s o l u b l e i n water (1, 3 ) .

2.4 Identification

The f o l l o w i n g i d e n t i f i c a t i o n tests a r e d e s c r i b e d
i n t h e P h a r m a c e u t i c a l Codex 1979 ( 5 ) .

1) To a b o u t 25Omg add l m l of a c e t i c a n h y d r i d e and


2 m l of d e h y d r a t e d p y r i d i n e , b o i l u n d e r a r e f l u x
c o n d e n s e r f o r 1 5 m i n u t e s , c o o l , add 5Oml of water
and s h a k e t h o r o u g h l y u n t i l a p r e c i p i t a t e is
produced which, a f t e r washing w i t h water and
d r y i n g , m e l t s a t a b o u t 138OC.

2) D i s s o l v e a b o u t lOmg i n 5 m l of H2SO4; t h e
s o l u t i o n i s c o l o r l e s s ( d i s t i n c t i o n from
s t i l b o e s t r o l , which g i v e s a g o l d e n - y e l l o w
color).

2.5 Spectral properties

2.51 Ultraviolet spectra

H e x e s t r o l i n e t h a n o l shows maxima a t 230nm


( E l % , l c m , 775) and 280 nm ( E l % , lcm, 1 4 0 ) ;
0.11NaOH h e x e s t r o l g i v e s maxima a t 242 nm
( E l % , l c m , 965) and 295 nm (El%, l c m , 1 7 5 ) .
A s shown i n f i g u r e ( 1 ) and i n agreement t o
t h e f i g u r e s p u b l i s h e d by C l a r k e ( 3 ) .

2.52 Infrared Spectra

The i n f r a r e d spectrum of h e x e s t r o l i n K B r
d i s c i s g i v e n i n f i g u r e ( 2 ) . Major band
a s s i g n m e n t s are a s follows:
-1
Frequency Cm Assignment
3400 P h e n o l i c OH
1610, 1600 Aromatic r i n g
C=C s t r e t c h
HEXESTROL 35 1

L
200 nm 250 nm 300 nm 350 nm

F i g . 1. The u l t r a v i o l e t a b s o r p t i o n spectrum of H e x e s t r o l
i n ethanol.
I n s t r u m e n t : Pye Unicam SP8-100

Wavenumber

Figure 2. I R s p e c t r u m of H e x e s t r o l i n K B r .
Instrument: P e r k i n Elmer 567
352 HASSAN Y. ABOUL-ENEIN ET AL.

Other f i n g e r p r i n t bands c h a r a c t e r i s t i c to-l


h e x e s t r o l a r e 1530, 1450, 1300 and 1110 cm .
2.53 Nuclear Mametic Resonance Spectrum

a) PMR
A t y p i c a l PMR spectrum of h e x e s t r o l i s shown
i n Figure (3).

The sample was d i s s o l v e d i n d e u t r a t e d CDC13


and a drop of d e u t r a t e d d i m e t h y l s u l f o x i d e
(DMSO-db) u s i n g TMS a s t h e i n t e r n a l s t a n d a r d .

The spectrum was determined on a V a r i a n


T60-A s p e c t r o m e t e r .

The f o l l o w i n g s t r u c t u r a l a s s i g n m e n t s have
been e l i c i t e d from F i g u r e ( 3 ) .

Chemical S h i f t (6) Assignment

T r i p l e t a t 0.50 CH CH -
-3 2
Multiplet centered a t 1.3 C H e 2

M u l t i p l e t c e n t e r e d a t 1.90 -CH-CH-

Doublet of d o u b l e t eight aromatic


c e n t e r e d a t 6.86 protons
character i s t i c
f o r para
substitution
of t h e r i n g .

Broad s i n g l e t exchangable p h e n o l i c OH
w i t h D20 a t 7.67 group.

b) 13C-NMR
*
The I3C-NMR of h e x e s t r o l has been d e t e r m i n e d ,
t h e off-resonance spectrum ( F i g . 4 ) shows
seven s i n g l e t s . The complete spectrum i s
shown i n F i g . 5. The spectrum was determined
on a Varian FT-80A i n DMSO-db a s s o l v e n t ,
t u b e d i a m e t e r 10 mm, s p e c t r a l width 5000 Hz

*H.Y. Aboul-Enein, unpublished d a t a


I . I I I . 1
1 . . . . 1 . . . . 1 . . . . 1 . . . . ~ . . . . ~ . . . . ’ ~ ” . ” ” ~
ao ZO 6.0 5.0 PPM( b) 4.0 3.0 2.0 1.0

F i g u r e 3. PMR s p e c t r u m of H e x e s t r o l i n C D C l -DMSO-d w i t h IT% a s


i n t e r n a l standard. 3 6

Instrument: V a r i a n T60-A
354
355
356 HASSAN Y. ABOUL-ENEIN ET AL.

a c q u i s a t i o n t i m e : 1.638 sec.; p u l s e w i d t h :
4 Usec; number of d a t a p r i n t : 16384.

S p e c t r a l a s s i g n m e n t s a r e l i s t e d below:

Chemical S h i f t Assignments
( i n ppm r e l a t i v e t o TMS)

12.06 CH3
26.97 CH2
52.77 -
CH-
134. 4 2 c 1- c.1
115.05
c 2-c 6 02- c 6
128.82 c3-c 5 c--c
3 5
155.39 c4-c*4
2.54 Mass Spectrum

The mass spectrum of h e x e s t r o l , o b t a i n e d by


chemical i o n i z a t i o n w i t h i s o b u t a n e g a s , i s
shown i n P i g . 6. The spectrum w a s determined
by d i r e c t i n l e t t o Ribermag 10-10R m a s s
s p e c t r o m e t e r and e x h i b i t s c o m p a r a t i v e l y
l i t t l e fragmentation.

The f o l l o w i n g t a b l e g i v e s t h e most prominent


i o n s and t h e i r r e l a t i v e i n t e n s i t i e s .

Mass ( d e ) Relative Intensity %

27 0 4.6 (M+)
219 10.5
178 11.8
1 77 93
1 36 9.9
.
I

80 90 14011012Q 130 W 1 5 0 1601?01801902002102PO2303+0250260270

F i g u r e 6. Mass Spectrum of H e x e s t r o l ( C I - i s o b u t a n e ) determined


by d i r e c t i n l e t i n s e r t i o n .
358 HASSAN Y. ABOUL-ENEIN ET AL.

135 100 ( b a s e peak)


134 36.9
107 42.6

+. +'
1 0 -@ CH CH
r - C Hl 2

CH2CH3
CH-CH I
m/e 177 mfe 178
+*

O
D
+*
CH2CH3 CH2CH3

H@ @
H! LH

m f e 135 m f e 134
3. Synthesis

S e v e r a l methods have been p u b l i s h e d and p a t e n t e d


f o r t h e s y n t h e s i s of h e x e s t r o l . I n 1938 Campbell
-
et-a1- (6) i s o l a t e d h e x e s t r o l i n p o o r y i e l d , from
t h e p r o d u c t s of d e m e t h y l a t i o n of a n e t h o l e .

Some of t h e s y n t h e t i c a p p r o a c h e s f o r h e x e s t r o l
a r e summerized a s f o l l o w s : -

1) C a t a l y t i c h y d r o g e n a t i o n of p s e u d o d i e t h y l -
s t i l b e s t r o l g i v e s h e x e s t r o l a l o n g w i t h some
d i e t h y l s t i l b e s t r o l (7).

=:
EtEt
H O G @OH

I
catalytic
hydrogenat i o n

0
Et
I
HO&- ,CH @H+HO c = c
I
I Et
Et
HEXESTROL 359

Hydrogenation of t h e d i m e t h y l e t h e r s of d i e t h y l s -
t i l b e s t r o l and pseudodiethylstilbestrol w i t h
subsequent d e m e t h y l a t i o n a f f o r d s t h e meso-isomer
of h e x e s t r o l which i s more p o t e n t t h a n t h e
DL-isomer ( 7 ) .
H

CH CH

HO

meso-f orm

2) Hydrogenation of 4 , 4 ' dihydroxy y-6 d i p h e n y l y-6


hexadiene g i v e s h e x e s t r o l i n q u a n t i t a t i v e y i e l d ( 7 ) .

HOO'L!!
CH CH

0 OH
Fd Icharcoa 1
>- . Hexestrol

3) H e x e s t r o l may be prepared by t h e a c t i o n of e t h y l
magnesium bromide on a n i s a l d a z i n e w i t h subsequent
d e m e t h y l a t i o n (8).

Hexes t r o l
The above methods f o r t h e s y n t h e s i s of h e x e s t r o l
i n v o l v e t h e u s e of i n t e r m e d i a t e s which a r e d i f f i c u l t
t o prepare.
360 HASSAN Y. ABOULENEIN ET AL.

4) B e r n s t e i n and Wallis d e s c r i b e d ( 4 ) a n a l t e r n a t e
i n e x p e n s i v e method f o r t h e s y n t h e s i s o f h e x e s t r o l
s t a r t i n g from p-hydroxypropiophenone a s shown
i n scheme ( 1 )

I I1
I1 -b
,CH30 N a -@ YHCH

alcohol OH2

I11

I11 F
HBr
C H 3 @'
0 CH-CH
Br 2 5
IV

Scheme 1 VI
5) Kharasch and Kleiman(9) p r e p a r e d h e x e s t r o l d i m e t h y l -
e t h e r from a n e t h o l e hydrobromide and G r i g n a r d r e a g e n t
i n t h e p r e s e n c e of a h a l i d e of c o b a l t , n i c k e l o r i r o n ,
t h e f r e e r a d i c l e g e n e r a t e d from t h i s r e a c t i o n
dimerizes t o give hexestrol dimethylether i n y i e l d s
r a n g i n g from 14-41%. The h i g h e r m e l t i n g p o i n t f o r
t h e meso-form a l l o w s i t s s e p a r a t i o n from t h e DL-
by-product formed i n t h i s r e a c t i o n .
r 1
HEXESTROL 361

4. S t a b i l i t y and Decomposition p r o d u c t s

H e x e s t r o l is a r e l a t i v e l y s t a b l e compound a t room
t e m p e r a t u r e ; however i t i s recommended t o b e k e p t
i n a w e l l c l o s e d c o n t a i n e r p r o t e c t e d from l i g h t .

5. Yetabolism

I l e x e s t r o l i s m e t a b o l i z e d i n a s i m i l a r way a s d i e t h y l -
s t i l b e s t r o l , i t i s excreted c h i e f l y as a glucuronide
conjugate ( 3 ) . This glucuronide i s mostly excreted
i n t o t h e b i l e which i s s u b j e c t e d t o h y d r o l y s i s by
i n t e n t i n a l g l u c u r o n i d a s e enzyme d u r i n g i t s p a s s a g e
i n t o t h e g u t . T h i s a l l o w s t h e d r u g be r eab so r b ed ,
r e c o n j uga t e d and r e - e x e r e t e d ( h e p a t i c c i r c u l a t i o n )
( 1 0 ) . O t h e r p o s s i b l e m e t a b o l i t e i n t e r m e d i a t e s which
should b e i n v e s t i g a t e d a r e shown in scheme ( 2 ) . This
i s i n a n a l o g y t o t h e p o s s i b l e m e t a b o l i t e s of d i e t h y l
s t i l b e s t r o l which h a s r e c e n t l y a t t r a c t e d t h e a t t e n t i o n
by b e i n g l i n k e d t o t h e o c c u r a n c e o f v a g i n a l a d e n o c a r c i -
noma i n a d o l e s c e n t d a u g h t e r s whose m o t h e r s had r e c e i v e d
d i e t h y l s t i l b e s t e r a l d u r i n g p r e g n a n c e (11, 1 2 , 13).

HO
HO * H o e o H

OH

Me0 Me0

HO Me0

Scheme 2. Expected M e t a b o l i t e s of H e x e s t r o l
HASSAN Y. ABOUL-ENEIN ET AL.

6. Methods of A n a l y s i s

6.1 Titrimetry

- Aqueous (Bromometry)
The f o l l o w i n g method h a s been d e s c r i b e d f o r t h e
q u a n t i t a t i v e d e t e r m i n a t i o n of h e x e s t r o l d i a c e t a t e (14) :
To 70 mg of sample add 1 0 m l of 0.5N m e t h a n o l i c KOH,
and h e a t under r e f l u x f o r 30 m i n u t e s on a water-bath.
Cool, add 5Oml of a c e t i c a n h y d r i d e , shake u n t i l t h e
h y d r o l y s a t e i s d i s s o l v e d , t h e n add 2 m l of 30% K B r
s o l u t i o n , 2 ml of conc. H2SO4 and 20 m l of 0.1N-KBr03,
and set a s i d e i n t h e d a r k f o r 1 0 min. Add lgm of K I
and 100 m l of H 2 0 , and t i t r a t e w i t h O.lN-Na2S203 u s i n g
s t a r c h a s i n d i c a t o r . The e r r o r i s - +1% over t h e r a n g e
7 0 t o 90 mg of h e x e s t r o l d i a c e t a t e .

6.2 Colorimetry

H e x e s t r o l has been determined i n o i l y s o l u t i o n and


t a b l e t form a s f o l l o w s ( 1 5 ) :

For t a b l e t : e x t r a c t a sample c o n t a i n i n g 2 t o 3 mg of
h e x e s t r o l w i t h methanol, d i l u t e t h e e x t r a c t w i t h water
t o 100 m l , and f i l t e r . To a 20 m l a l i q u o t add b o r a t e
b u f f e r s o l u t i o n (pH 1 1 ) .

For o i l y i n j e c t i o n s : Mix a sample c o n t a i n i n g 0.4 t o


0.6 mg of h e x e s t r o l w i t h l i g h t petroleum (5ml) , shake
t h e s o l u t i o n w i t h methanol (6ml) , add b u f f e r s o l u t i o n
(13ml), shake, and c o l l e c t t h e aq. methanol phase i n
a lOOml f l a s h . Twice r e p e a t t h e e x t r a c t i o n w i t h methanol
(6ml) and b u f f e r s o l u t i o n (13ml) and t o t h e combined aq.
e x t r a c t s add methanol (2ml).

For d e t e r m i n a t i o n of h e x e s t r o l : h e a t t h e prepared
s o i u t i o n on a water b a t h f o r 30 min., c o o l i t t o room
temperature, add d i a z o t i z e d s u l p h a n i l i c a c i d s o l u t i o n
(12ml) and mix. A f t e r 40 minutes d i l u t e w i t h b u f f e r
s o l u t i o n t o 100 m l , and measure t h e e x t i n c t i o n a t 495 nm
a g a i n s t water. Beer's law i s obeyed f o r 0.5 t o 4 mg of
h e x e s t r o l p e r 1 O O m l . R e s u l t s a g r e e to. w i t h i n +lo% w i t h
t h e o r e t i c a l values.
HEXESTROL 363

B e l i k o v , d e s c r i b e d a s i m i l a r p r o c e d u r e (16) which
depends on d i a z o d i z a t i o n of h e x e s t r o l w i t h d i a z o -
s u l p h a n i l i c a c i d . However, t h e azo-dye formed i s
measured i n a l k a l i n e medium a t 420 nm.

Another c o l o r i m e t r i c method f o r d e t e r m i n a t i o n of
hexestrol i n feeds i s reported a s follows (17):

The ground f e e d i n g s t u f f (40gm) mixed w i t h lOgm of


sand i s s e t a s i d e w i t h c h l o r o f o r m o v e r n i g h t , t h e n
e x t r a c t e d w i t h CHC13 f o r 6 h o u r s . The e x t r a c t i s made
up t o 2 O O m l of CHC13. The r e s i d u e from e v a p o r a t i o n
of t h e f i n a l CHC13 e x t r a c t i s d i s s o l v e d i n t r i e t h y l a m i n e -
t e t r a h y d r o f u r a n - w a t e r (1:5 : 1 4 ) , t h e s o l u t i o n is r e t a i n e d
i n an o l e a t e d c e l l u l o s e column f o r 1 h o u r , t h e n t h e
impurities a r e eluted with triethylamine - tetrahydro-
f u r a n - water m i x t u r e . The column i s a c i d i f i e d w i t h
N-H2SO4 and e x t r a c t e d w i t h e t h y l e t h e r , t h e e x t r a c t
i s evaporated, the r e s i d u e i s dissolved i n ethanol,
and t o t h i s s o l u t i o n a r e added water, dil.HC1, a
molybdotungstophosphate r e a g e n t and t h e n Na2C03 s o l u t i o n .

The e x t i n c t i o n of t h e c e n t r i f u g e d s o l u t i o n i s measured
a t 750 run a g a i n s t a r e a g e n t b l a n k and compared w i t h
t h a t of s t a n d a r d s o l u t i o n of h e x e s t r o l t r e a t e d s i m i l a r l y .

6.3 U l t r a v o i l e t S p e c t r o p h o t o m e t r y (U.V.)

H e x e s t r o l w a s q u a n t i t a t i v e l y determined i n tablet
s p e c t r o p h o t o m e t r i c a l l y ( 1 8 ) . The d r u g was e x t r a c t e d
w i t h CHC13 from a n a c i d i f i e d powdered sample ( c o n t a i n i n g
a b o u t 5mg of h e x e s t r o l ) . The CHC13 e x t r a c t w a s con-
c e n t r a t e d and 2 , 2 , 4 t r i m e t h y l p e n t a n e w a s added and
h e x e s t r o l w a s e x t r a c t e d w i t h 0.1N NaOH. The combined
a l k a l i n e s o l u t i o n w a s a c i d i f i e d and r e - e x t r a c t e d w i t h
CHC13. The CHC13 s o l u t i o n was washed w i t h water and
d r i e d o v e r Na2S04 and e v a p o r a t e d t o d r y n e s s .

The r e s i d u e w a s d i s s o l v e d i n e t h a n o l and measured a t


280nm. The p e r c e n t a g e r e c o v e r y ranged from 94.4-98.9%.

Kovalenko r e p o r t e d a q u a n t i t a t i v e method f o r t h e a s s a y
of h e x e s t r o l ( 1 9 ) . About 25mg of h e x e s t r o l w a s weighed
and d i s s o l v e d i n 25-3Om1 of 0.1N NaOH i n a 5Oml v o l u m e t r i c
f l a s k . The volume w a s a d j u s t e d t o 5Oml w i t h 0.1N NaOH
s o l u t i o n . Then p i p e t t e 5ml of t h i s s o l u t i o n i n t o a
second 5Oml v o l u m e t r i c f l a s k and t h e volume a d j u s t e d
364 HASSAN Y. ABOUL-ENEIN ET AL.

t o 5Oml w i t h 0.1N NaOH s o l u t i o n . The same s e r i a l d i l u t i o n


was r e p e a t e d a n d t h e c o n c e n t r a t i o n of h e x e s t r o l i n t h e
l a s t s o l u t i o n w a s d e t e r m i n e d by m e a s u r i n g t h e a b s o r p t i o n
a t 241 nm u s i n g 0.1N NaOH s o h t i o n f o r t h e b l a n k . S i n c e
t h e s e s o l u t i o n s f o l l o w t h e Lambert-Beer law 1.8 pg m l ,
t h e d e t e r m i n a t i o n c a n b e made from a s t a n d a r d c u r v e .

6.4 Chromatographic A n a l y s i s

6 . 4 1 P a p e r Chromatography

Tompsett h a s d e s c r i b e d a method f o r d e t e c t i o n
of h e x e s t r o l and o t h e r s t i l b e s t r o l d e r i v a t i v e s
a l o n g w i t h t h e p - h y d r o x y m e t a b o l i t e s of
p h e n o b a r b i t o n e and p h e n y t o i n i n u r i n e u s i n g
t h e 2-dimensional paper chromatography ( 2 0 , 2 1 ) .

The two s y s t e m s u s e d were i s o p r o p a n o l :


NH3 (0.99) :H20 (8 :1:1) and C6H6 :E t O A c :H 2 0 ( 2 :1: 1).

The d e t e c t i n g a g e n t s u s e d were P a u l y ' s r e a g e n t


(red-brown) , d i a z o t i z e d p - n i t r o - a n i l i n e (brown) ,
d i a z o t i z e d d i e t h y l a m i n o e t h y l p-aminophenyl
s u l p h o n e (brown) and 1 - n i t r o s o - 2 - n a p h t h o l
n i t r i c a c i d m i x t u r e (+ve). The s e n s i t i v i t y
r a n g e f o r t h i s method i s 5-80ug.

6.42 Column Chromatography

Column chromatography h a s b e e n a p p l i e d t o
p u r i f y the c a t t l e feed e x t r a c t s containing
h e x e s t r o l and o t h e r s t i l b e s t r o l s t o remove
i n t e r f e r i n g substances b e f o r e i t s determina-
t i o n . An example of t h e columns u s e d f o r t h e
p u r i f i c a t i o n of t h e e x t r a c t s i s A 1 0 column
2 3
(22).

Verbeke ( 2 3 ) used columns c o n t a i n i n g XAD-2,


C e l i t e , o r n e u t r a l A1203 (Brockman A c t i v i t y I )
f o r p u r i f i c a t i o n o f t i s s u e e x t r a c t s and u r i n e
u s i n g d i s t i l l e d water; 1 5 m l water-washed
e t h e r f o l l o w e d b y , lhl CgHg t h e n benzene-
i s o o c t a n e ( 1 : l ) ; and b e n z e n e : i s o o c t a n e (1:l)
a s e l u e n t s r e s p e c t i v e l y f o r d e t e c t i o n of
h e x e s t r o l and o t h e r a n a b o l i c s .
HEXESTROL 365

6 . 4 3 Thin Layer Chromatography

S e v e r a l r e p o r t s have been published on t h e


d e t e c t i o n , q u a n t i t a t i v e d e t e r m i n a t i o n of
h e x e s t r o l and o t h e r c h e m i c a l l y r e l a t e d d r u g s
f o r example d i e t h y l s t i b e s t r o l i n f e e d s (meat,
milk) and i n b i o l o g i c a l f l u i d s .

Table (1) summerizes t h e s o l v e n t systems


a n d t h e d e t e c t i n g a g e n t s used i n t h e
cited references.

6.44 Gas-Liquid Chromatography

Various g a s - l i q u i d chromatographic methods


have been developed f o r d e t e c t i o n of h e x e s t r o l
i n meat and o t h e r a g r i c u l t u r a l p r o d u c t s . Gain
and S c h o l l ( 3 1 ) d e s c r i b e d a method f o r
d e t e r m i n a t i o n of h e x e s t r o l i n m o l a s s e s - based
l i q u i d feed supplements, a f t e r t h e p r e p a r a t i o n
of t h e b i s ( t r i m e t h y l s i l y l ) acetamide d e r i v a t i v e .
However t h e method showed i n t e r f e r i n g peaks o r
low recovery due t o emulsion f o r m a t i o n . Most
of t h e g a s chromatographic d e t e r m i n a t i o n
r e q u i r e s d e r i v a t i z a t i o n of h e x e s t r o l b e f o r e
i n j e c t i n g i n t o t h e g a s chromatograph.

Table ( 2 ) summerizes t h e d a t a o b t a i n e d from


t h e l i t r a t u r e s t i l l 1980.
T a b l e 1. (contd.)

S o l v e n t system Stationary Detect i n p R-emarks Ref.


phase agent

2 d i m e n s i o n a l TLC 5%H2SO4- s e n s i t i v i t y 0.5- 23


a) CHC13:EtOH:C H induced f l u o r e lOppb
6 6
(36:1:4) scence a t
n-C H :Et20:CH2C12 366m
6 14
W
m (4:3: 2)
m
petroleum e t h e r Silica gel v a n i l li n a p p l i e d t o animal 27
(40-65OC) : ( a c t i v a t e d a t 120 OC reagent) feeding s t u f f
2 d i m e n s i o n a l TLC
using: Silica gel H 28
a) CHC13:EtOH(9:1)
b) C 6H :E t OAc ( 3 :1 )
t.1.c
CH2 C12:Me2C0(4 :1) K i e s e l g e l or ultraviolet a t s e n s i t i v i t y range
Kieselgel F 254m 0 . 2 . 2 up, 29
254
( t h e h i g h performance
t l c r a n g e 10-200ng
T a b l e 1.

S o l v e n t system Stationary Detecting Remarks Ref.


phase agent

Toleune: EtOAc ( 1 9 :1 ) A l u m i n ium Ultraviolet S e n s i t i v i t y 5,lOppb 24


ox i d e a t 254 nm

C6H6 :E t O A c (20: 1) ( i n f r e s h l i v e r and


kidney)

CH2C12:Me2C0(4: 1) Silica gel U 1 tr av i o l e t recommended € o r 25


a t 254 nm r o u t i n e test of
CHC13:EtOAc(4:1)
e s t r o g e n s i n food
2 d i m e n s i o n a l TLC w i t h

n C6H14:Et20:CH2C12
(4:3:2) i n b o t h d i r e c t i o n
or
EtOAc:C6H6(l:3)
a f t e r s o l v e n t system ( a )

n-C H :Et20:CH2C12 Silica gel G ultraviolet s e n s i t i v i t y of t h e 26


6 14
( 4 :3 :2) 254m. por 20' test i s 0 . 5 y g
T a b l e 1. (contd.)

S o l v e n t system Stationary Detecting Remarks


Ref.
phase agent

h.p. t.1.c.
CH C1 :Ple2C0(4:1) Kieselgel F d e n s itome- (t.1.c. range
2 2 254
W
trically at 200-2000ng) .
91
W 287 (h.p.t.1.c range
10-2 Ong )

C6H14:Me2C0(3 :2 ) o r Po 1yamid e t h e i n f l u e n c e s of
C6H6:Me2C0(3:1) chemical s t r u c t u r e
and m i g r a t i o n r a t e 30
are discussed.
T a b l e 2.

Column Carrier G a s Detector Remarks Ref.

3% OV-1 on Chromosorb Ar-CH electron d er i v a t i z e d by 32


4
hep t a f l u o r o b u t y r i c
WHP (80 t o 100 mesh) a t (19:l) capture a n hyd r i d e
var ious temperature
He electrolytic s e n s i t i v i t y €or electron
conductor c a p t u r e (40-400 p i c o g ) ; 32
and f o r e l e c t r o l y t i c
c o n d u c t o r (1-5 ng)

2% OV-17, 3%0V-1,3%0V-1 electron d e r i v a t i z e d by hepta-


N2
5% of n e o p e n t y l g l y c o l capture fluorobutyric anhydride; 33
s e b a c a t e o r 2% Carbowax s e n s i t i v i t y range
20M on s i l a n i s e d Chromosorb 6 t o 262 ng/ml
W (80 t o 100 m e s t ) a t
var i o u s temperatures .
3% QF-1 o n 100 t o 200 flame ioniza- i n j e c t e d as a dipropionate
mesh Gas. Chrom Q a t 170'. t ion d e t e c to r derivative; sensitivity 34
0.05 mg/ml

Ap i e z o n L-Ep i k o t e Electron- d er i v a t i z ed by
1001(5 :1) on s i l a n i s e d N2 capture trifluoroacetic
Chromosorb G a t 160Oc. anhydr iod e .
3 70 HASSAN Y. ABOUL-ENEIN ET AL.

6.45 High Performance L i q u i d Chromatography

H e x e s t r o l has been determined among o t h e r


a n a b o l i c s y n t h e t i c and n a t u r a l hormones i n meat
by HPLC. The columns used were Rp-2 (10 v m ) ,
Rp-18 ( 5 pm) o r Zorbax CN ( 5 pm) by g r a d i e n t
e l u t i o n w i t h a c e t o n i t r i l e : H 2 0 (1:9) p l u s 25%,
i n c r e a s i n g r e c t i l i n e a r l y i n 5 m i n u t e t o 45%, of
a c e t o n i t r i l e : H20 ( 9 : l ) . The f l o w r a t e w a s 2 m l
min.-l, and 100 mg e a c h of L i C l and LiC104 were
added t o each 1 0 h l of e l u e n t .

The e l u a t e w a s examined by voltammetry w i t h


v i t r e o u s carbon e l e c t r o d e s and on silver-AgC1
(3M-KC1) r e f e r e n c e e l e c t r o d e ; t h e p o t e n t i a l
sweep r a t e was 5 m ~ s - 1 .

Other columns used f o r t h e d e t e r m i n a t i o n of


h e x e s t r o l and o t h e r r e l a t e d d r u g s i s L i c h r o s o r b
RP-8 w i t h m i x t u r e s (35:13, t o 25:23) of methanol
and water ( c o n t a i n i n g i n a l l i n s t a n c e s 2 p a r t s of
a c e t o n i t r i l e ) as a mobile phase (0.8 t o 1.6 m l
min-l). The v o l t a m m e t r i c c u r v e of t h e e l u a t e was
recorded by t h e u s e of a v i t r e o u s - c a r b o n e l e c t r o d e ,
a p l a t i n u m c o u n t e r - e l e c t r o d e and a silver-AgC1
r e f e r e n c e e l e c t r o d e a t pH 3 , t h e peak p o t e n t i a l ( V )
f o r h e x e s t r o l w a s +.9. Amounts r a n g e from 1-4 ng
g-l of h e x e s t r o l i n meat could be determined by
HPLC .
6.5 Mass Pragmentography

H e x e s t r o l among o t h e r s t i l b e s t r o l w a s q u a n t i t a t i v e l y
determined by combined GC/MS ( 3 8 ) .
The s e n s i t i v i t y r a n g e i s 40 p a r t p e r 1 09 , t h e method
is s u c c e s s f u l l y used f o r d e t e c t i o n of e s t r o g e n s and
i n meat p r o d u c t s . I t i n v o l v e s e x t r a c t i o n of l i v e r ,
kidney and muscle t i s s u e i n a c e t o n i t i l e : water ( 9 : l ) .
The drug w a s c o n v e r t e d t o i t s t r i m e t h y l s i l y l d e r i v a -
t i v e f o r a n a l y s i s on a column packed w i t h 2%0V-17
on Chromosorb G and coupled t h r o u g h a Watson-Biemann
H e s e p a r a t o r t o t h e mass s p e c t r o m e t e r .

6.6 B i o l o g i c a l Assays

Heinert, i d e n t i f i e d hexestrol i n milk using the


mouse u t e r i n e weight b i o a s s a y method ( 3 9 ) . Rennet
HEXESTROL 371

c o a g u l a t i o n of t h e m i l k p e r m i t t e d d e t e c t i o n of
0.001 ppm of h e x e s t r o l which could a l s o be d e t e c t e d
i n c h e e s e a f t e r c o a g u l a t i o n of m i l k . L i e m - e t-
a1 (40)
d e s c r i b e d a b i o l o g i c a l a s s a y of e s t r o g e n i c s u b s t a n c e s
i n cosmetic i n c l u d i n g h e x e s t r o l , based on a p p l i c a t i o n
of t h e cosmetic p r o d u c t s t o t h e shaven s k i n of
c a s t r a t e d female mice; v a g i n a l smears were t a k e n
subsequently f o r a n a l y s i s .

Acknowledgements

The a u t h o r s wish t o t h a n k M r . Khalid N.K. Lodhi


f o r h i s technical a s s i s t a n c e i n determining t h e
NMR s p e c t r a of h e x e s t r o l and M r . Uday C. Sharma
f o r t y p i n g t h e manuscript.
372 HASSAN Y. ABOUL-ENEIN ET AL.

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