(Analytical Profiles of Drug Substances 12) Klaus Florey (Eds.) - Academic Press (1983) PDF

Analytical Profiles
of
Drug Substances
Volume 12
Edited by
Klaus Florey
The Squibb Institute for Medical Research
New Brunswick, New Jersey
Contributing Editors
Abdullah A. Al-Badr Glenn A. Brewer, Jr.
Norman W. Atwater Hans-Georg Leemann
Steven A. Benezra Joseph A. Mollica
Compiled under the auspices of the
Pharmaceutical Analysis and Control Section
APhA Academy of Pharmaceutical Sciences
ACADEMIC PRESS 1983

A Subsidiary of Harcourt Brace Jovanovich, Publishers
New York London
Paris San Diego San Francisco Silo Paulo Sydney Tokyo Toronto
EDITORIAL BOARD
Abdullah A. Al-Badr Klaus Florey

Norman W. Atwater Salvatore A. Fusari
Steven A. Benezra Lee T. Grady
Rafik Bishara Boen T. Kho
Gerald S. Brenner Hans-Georg Leemann
Glenn A. Brewer, Jr. Joseph A. Mollica
Nicholas DeAngelis James W. Munson
John E. Fairbrother Milton D. Yudis
Academic Press Rapid Manwrcript Reproduction

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AFFILIATIONS OF EDITORS,
CONTRIBUTORS, AND REVIEWERS
H. Y. Aboul-Enein, King Saud University, Riyadh, Saudi Arabia

S. Ahuju, Ciba-Geigy Corporation, Summit, New Jersey
A. A. Al-Budr, King Saud University, Riyadh, Saudi Arabia
S. L. Ali, Zentrallaboratorium Deutscher Apotheker e.V., Eschborn Germany
N. Atwuter, E. R. Squibb & Sons, Princeton, New Jersey
G. Atzl, Sandoz Ltd., Basel, Switzerland
S. A. Benezru, Wellcome Research Laboratories, Research Triangle Park,
North Carolina
R. Bishuru, Lilly Research Laboratories, Indianapolis, Indiana
D. Both, The Squibb Institute for Medical Research, New Brunswick, New Jersey
G. Brenner, Merck Sharp & Dohme Research Laboratories, West Point,
Pennsylvania
G. A. Brewer, The Squibb Institute for Medical Research, New Brunswick, New
Jersey
R. D. Brown, Bristol Laboratories, Syracuse, New York
2. L. Chung, Abbott Laboratories, North Chicago, Illinois
J. Cohen, Ciba-Geigy Corporation, Summit, New Jersey
N. DeAngelis, Wyeth Laboratories, Philadelphia, Pennsylvania
R. Dowse, Rhodes University, South Africa
J. Fuirbrother, Stiefel Laboratories Ltd., Sligo, Ireland
E. Felder, Bracco Industria Chimica S.p.a., Milan, Italy
K. Florey, The Squibb Institute for Medical Research, New Brunswick, New Jersey
S. A. Fusuri, Warner-Lambert Research Institute, Morris Plains, New Jersey
L. T. Grady, The United States Pharmacopeia, Rockville, Maryland
J. M. Huigh, Rhodes University, South Africa
S.A. Hunnu, Bristol Laboratories, Syracuse, New York
M. M.A. Hassun, King Saud University, Riyadh, Saudi Arabia
I. Kunfer, Rhodes University, South Africa
vii
viii AFFILIATIONS OF EDITORS, CONTRIBUTORS, AND REVIEWERS
T. I . Khalifu, King Saud University, Riyadh, Saudi Arabia

B. T. Kho, Ayerst Laboratories, Rouses Point, New York
J . Kirschbuum, The Squibb Institute for Medical Research, New Brunswick,
New Jersey
H . G . Leemann, Sandoz Ltd., Basel, Switzerland
M. A. Loutfy, King Saud University, Riyadh, Saudi Arabia
J . R. Luch, Ciba-Geigy, Suffern, New York
J . B. Martin, Abbott Laboratories, North Chicago, Illinois
J . P. McGrot-y, Bristol Laboratories, Syracuse, New York
J . Mollicu, Ciba-Geigy Corporation, Summit, New Jersey
P. M. Monteleone, Bristol Laboratories, Syracuse, New York
N . Muhammed, Bristol Laboratories, Syracuse, New York
F. J . Miihtudi, King Saud University, Riyadh, Saudi Arabia
J . W. Munson, The Upjohn Company, Kalamazoo, Michigan
F. Nuchtmann, Sandoz Ltd., Bade, Switzerland
G. R. Padmanabhan, Ciba-Geigy Corporation, Suffern, New York
D. Pitre, Bracco Industria Chimca S.p.a., Milan, Italy
A. Post, Smith Kline & French Laboratories, Philadelphia, Pennsylvania
W. D. Roth, Sandoz Ltd., Basel, Switzerland
R. S. Suntoro, * Smith Kline & French Laboratories, Philadelphia, Pennsylvania
M. D. Yudis, Schering-Plough, Inc., Bloomfield, New Jersey
*Deceased
PREFACE
The compilationofAnalytica1Profiles of Drug Substancesto supplementthe infor-

mation contained in the official compendia is now a well-established activity.
That we are 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 enthusiastic response of our
authors, an internationalgroup associated with pharmaceutical firms, academic insti-
tutions, and compendia1authorities. I would like to express my sincere gratitude to
them for making this venture possible.
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.
In the preface to the eleventh volume. I announced that we would try to supplement
previously published profiles with new data. Unfartunately, most of the original
contributors are no longer available to undertake this task, and it has proven to be
difficult to find other volunteers. We shall continue to pursue the updating program,
but it will not be as comprehensive as originally envisioned.
Again, I would like to request of all those who have found these profiles useful to
contribute monographs of their own. We, the editors, stand ready to receive such
contributions.
ix
AMANTADINE
Joel Kirschbaum
1. Introduction 2
1.1 History, Therapeutic Use, and Mechanism of Action 2
1.2 Nomenclature, Molecular Weight, and Structure 2
1.3 Appearance, Color, Odor, and Precautions 2
1.4 Synthesis 4
1.5 Reactions, Stability, and Metabolism 5
2. Physical Properties of Crystalline Amantadine 6
2.1 Single Crystal X-Ray Diffraction 6
2.2 X-Ray Powder Diffraction 7
2.3 Mass Spectrometry 9
2.4 Infrared Spectrometry 9
2.5 Electron Tunnelling and Photoelectron Spectrometry 11
2.6 Thermal Analysis 13
2.7 Microscopy 13
2.8 Surface Area 13
2.9 Hydration 13
2.10 Polymorphism 13
3. Spectrometryof Amantadine in Solution 14
3.1 Nuclear Magnetic Resonance Spectrometry (NMR) 14
3.2 Ultraviolet Spectrometry 16
1. Bulk Solution Properties 16
4.1 Solubilitiesin Aqueous and Nonaqueous Solvents 16
4.2 Ionization 18
4.3 Dipole Moments 19
4.4 Hydrodynamic Properties 19
5. Methods of Analysis 20
5.1 Compositional Analysis 20
5.2 Identity and Colorimetric Methods 20
5.3 Titration 21
5.4 Spectrometry 21
5.5 Gas-Liquid Chromatography 22
5.6 Thin-Layer Chromatography 22
5.7 High-Performance Liquid Chromatography 22
5.8 Electrochemistry 22
5.9 Fluorescence Spectrometry 29
5.10 Tissue Culture 30
5.11 Comparison of Methods 30
References 31
ANALYTICAL PROFILES OF DRUG SUBSTANCES 1 Copyright by the American Pharmaceutical Arwriation.

VOLUME 12 ISBN 0-12-260812-7
JOEL KIRSCHBAUM
1. Introduction
1.1 History, Therapeutic Use and Mechanism of Action
Amantadine is an orally active antiviral agent

(1,2). It was discovered by workers at DuPont via an
empiric screening program (3). Other than
vaccination, it is the only prophylactic drug
presently useful against many viral infections,
especially influenza A and C. Once administered, its
effect is immediate to reduce signs of infection
among 50% to 70% of individuals exposed to the virus.
A use panel recommended ( 4 ) it for individuals with a
high risk of serious morbidity or mortality due to
cardiovascular, immunodeficiency, metabolic, neuro-
muscular or pulmonary diseases, the elderly, and the
unvaccinated and the important (5). It is 91%
effective in preventing influenza. The antiviral
activity of amantadine hydrochloride appears at an
early phase of the infection (6). The mode of action
appears to be the inhibition of the uncoating of the
virus ( 7 ) once it has penetrated the host cell. Such
a failure prevents replication. Gene 7 , coding for
the virus matrix protein, carries the property of
amantadine resistance ( 8 ) , and can be transferred by
recombination between influenza viruses. It was
conjectured (9) that other highly symmetrical
hydrocarbons, perhaps in the shapes of the Platonic
solids like cubane and dodecahedrane, when
derivatized like amantadine, might have similar
properties to pass through the membrane of a cell and
destroy virus particles inside it (10).
Amantadine is also useful in treating
Parkinson's disease (2). This use was found by a
chance observation of a significant improvement in
such a patient taking 200 mg of amantadine daily for
flu prophylaxis. It also appears clinically
effective in the treatment of drug-induced
extrapyramidal symptoms (11).
Amantadine relieves Parkinson's disease
(including drug-induced Parkinsonism by
neuroleptics) , apparently by a mechanism involving
dopamine (12); indeed, amantadine enhances L-dopa
activation (2).
As expected, various investigators found
amantadine to have other uses; not only against other
AMANTADINE 3
viruses (12), but also in treating cancer (13),

aiding priapus (14), and inhibiting rust (15).
Rimantadine, an amino group analogue
[1-(1-aminoethyladamantane)] is also active against
virus (2,16). Rimantadine is 4-8 times more
effective than amantadine hydrochloride to protect
against influenza A virus infection, but it is more
toxic (17).
1.2 Nomenclature, Molecular Weight and Structure
Amantadine hydrochloride is the United States

adopted name (18). The preferred chemical name is
tricyclo r3.3.1. 13'7]decan-l-amine, hydrochloride.
Other names include 1-adamantanamine hydrochloride,
1-aminotricylo [ 3 . 3 . 1 . I. "1 decane hydrochloride,
1-adamantylamine hydrochloride, adamantylamine
hydrochloride, and 1-aminoadamantane hydrochloride,
and, less correctly, midantane and dimantane
hydrochloride (19). Its molecular weight is 187.71
daltons. Amantadine hydrochloride was given the
chemical abstracts service systematic number
665-66-7 ; the free base, amantadine , was numbered
CAS-768-94-5. It is currently marketed under the
name Symmetrel (Endo Laboratories). Other names
include EXP-105-1, Mantadix, Matadan, Mydantan and
Virafral. In Wiswesser notation it is L66 B6 A B- C
1B ITJ BZ &GH.
Amantadine hydrochloride can be represented a
variety of ways, as shown below:
Amantadine hydrochloride possesses a unique,
rigid, relatively unstrained ring system that is
composed of three fused cyclohexane rings in the
chair conformation (20). Amantadine is considered to
be the smallest repeating unit of the diamond lattice
(21). The symmetrical cage structure causes the
infrared, nuclear magnetic resonance and mass spectra
to be comparatively simple, 2s will be illustrated
later. As expected from this lack of asymmetry,
there is no observable optical rotation (22) using
the D lines of sodium, at a concentration of 1% in
water.
1.3 Appearance, Color, Odor and Precautions
Amantadine hydrochloride is a white, odorless,

free-fl owing crystalline powder. No precautions are
given for this relatively non-toxic compound.
4 JOEL KIRSCHBAUM
ia
HCH
5
4
NH2
7 HCI 3
5 10
1.4 Synthesis
Adamantane is found naturally at low

concentrations (approximately 0.02%) in various
petroleum fractions (23). However, it may be
synthesized by isomerization of ten carbon cyclic
hydrocarbons, the probable basis of the naturally
formed adamantane. A convenient starting material,
dicyclopentadiene (I) was hydrogenated quantitatively
to endo-trimethylnorbornane (11,
endo-tetrahydrodicyclopentadiene). After refluxing
overnight with such Lewis acids as aluminum
trichloride or tribromide, adamantane (111) was
found. The possible mechanism (24) is shown below.
Bromination to 1-bromoadamantane, an ionic
process, can he followed by a sequence of reactions
with either ammonia, methylcyanide, urea or thiourea
as sources of the amino group, to give amantadine
(25-30). More complicated reactions of the l-bromo-
compound involve dehalogenation, reaction with
methylcyanide and saponification (31,32). Other
syntheses utilize the 1-carboxylic acid (33) and the
1-nitrate (34).
AMANTADINE 5
Direct amination (35) of adamantane or

introduction of a source of an amino group during the
rearrangement of I1 (also known as
tricyclo[5.2. 1.02,6]decane) gives a yield of 75%
amantadine (36)
bridgehead carbon via >
The reaction precedes (37)
+ ~j
NC12 )NC12 *
-Cl+
+H+
Various other combinations of isomerization and
pH*.
the
conversion to amantadine have been described (38,39).

Amantadine can also be synthesized by the
photochemical reaction of chloramine with adamantane
(40).
I II
&- &+-&+
F(
+
"RH & m
1.5 Reactions, Stability and Metabolism
Possible reactions are substitution at the amino

group of amantadine, replacement of the amino group,
rearrangement of the cage structure or replacement of
the cage hydrogens, and have been discussed elsewhere
(20). A vast number of derivatives of the amino
group have been prepared (41). The amino group can
undergo all of the typical reactions of primary
amines, such as Schiff base formation (42),
alkylation (43,44), halogenation (45) and amination
(46). Deamination with sodium nitrite and acetic
6 JOEL KIRSCHBAUM
acid or nitrous acid gives 1-hydroxyadamantane in 97%

yield (20). The in situ reaction with
trichloroacetyl isocyanate in NMR tubes was used to
analyze for the amino function ( 4 7 ) . As expected,
various compounds like acid chlorides were reacted
with amantadine ( 4 8 , 4 9 ) to create potential drugs
with new properties. The relative stability of the
1-adamantyl cation ( 5 0 ) permits conversion of the
amino group to nitro, and then to a large series of
derivatives ( 5 1 ) .
The cage structure can be rearranged ( 5 2 ) in a
reversal of the synthesis. The cage hydrogens can be
replaced by fluorine, as induced by light ( 5 3 ) or by
perfluoridation ( 5 4 ; 19F-NMR, infrared and mass
spectra discussed), as well as by tritium ( 5 5 ) .
The amantadine structure has been characterized
as being extremely stable, as predicted from the
equatorial position of the amino group and the facile
rearrangement of ten carbon hydrocarbons to
adamantane ( 2 0 ).
After oral administration, amantadine was found
in the heart, kidney, liver and lungs ( 5 6 ) .
Concentration of the drug in the lungs may be part of
its prophylactic action. After an oral dose of 2.5
mgfkg, maximum concentration of 0 . 3 ug/mL was reached
in 1-4 hours ( 5 7 ) , with a plasma half-life of 9-15
hours ( 5 8 ) . The rate of excretion depends of the pH
of the urine; i . e . , at pH 5 . 0 , 5-7% per hour of body
content was excreted, but at pH 8 the excretion rate
was 4% per hour ( 5 9 ) . As expected, with patients
having negligible renal function, excretion was
impaired, with plasma concentrations reaching 4 . 4
UgfmL, and accompanied by toxic manifestations of the
drug (60).
In hepatic micro soma1 preparations,
N-hydroxy-1-aminoadamantane and 1-nitrosoadamantane
were identified as metabolites (61). Approximately
0.1% of the administered amantadine was found in
urine in the form of 1-amino-3-hydroxyadamantane
(62).
2. Physical Properties of Crystalline Amantadine
2.1 Single Crystal X-Ray Diffraction
Although the x-ray structure of amantadine

hydrochloride was not determined, the structure of
the parent compound adamantane was elucidated (63).
AMANTADINE I
The three-dimensional representation below is

reproduced with the permission of the
C r y s t a l l o g r a p h i c Data C e n t r e , Cambridge ( 6 4 ) .
E l e c t r o n d i f f r a c t i o n d a t a (65) f o r adamantane a g r e e d
w i t h t h e x-ray c r y s t a l l o g r a p h y . The band l e n g t h of
C-H and C-C (1.54 t 0.01A) a p p e a r normal, and t h e
C-C-C a n g l e s a r e t e t r a h e d r a l (109.5 +- 1.5O).
2.2 X-Ray Powder D i f f r a c t i o n
To observe x-ray d i f f r a c t i o n p a t t e r n s , a P h i l i p s
powder d i f f r a c t i o n u n i t e m i t t i n g CuKa r a d i a t i o n a t
1.54A was used w i t h a s c i n t i l l a t i o n c o u n t e r d e t e c t o r
(66). The r e l a t i v e l a c k of peaks s e e n i n F i g u r e 1
w a s e x p e c t e d from t h e h i g h l y symmetrical s t r u c t u r e of
amantadine h y d r o c h l o r i d e . Below a r e t h e s o r t e d d a t a
based on h i g h e s t i n t e n s i t y of 1.00 u s i n g CuKa
radiation.
20(Degrees) ' d' (Angstroms) R e l a t i v e Area
18.2 4.88 1.000

15.9 5.58 0.739
27.4 3.26 0.499
14.4 6.17 0.405
23.9 3.72 0.344
Figure 1. Powder X-Ray Diffraction Pattern of Amantadine Hydrochloride. See Text for Details
AMANTADINE 9
18.6 4.78 0.298

9.0 9.81 0.235
2.3 Mass Spectrometry
The mass spectrum (67) of amantadine

hydrochloride (Figure 2) shows that the amino
substituent was present as a major ionic species
(68). The molecular peak was at m/e 151, with an
intensity as great as 60% (69). Below is the
suggested fragmentation pathway (62).
Secondary ion mas? spectrometry $70) using

silver showed (M + H) and (Ag + M) adducts.
Protonated amfntadine gave rise to the fragment ion
( M + H - NH3)
Mass
.
spectrometry combined with gas
chromatography has been used to determine amantadine
in biological tissues and fluids, cf. section 5.5.
2.4 Infrared Spectrometry
Figure 3 shows the infrared spectra of a

commercial preparation of amantadine hydrochloride
using mineral oil and potassium bromide (71). The
instrument used was a Perkin-Elmer Model 983 Fourier
transform infrared spectrometer. The minor
differences in band intensities of the two spectra
could be due to either pressure effects in the
preparation of the potassium bromide pellet or
10 JOEL KIRSCHBAUM
5765 Q D Q M R N T R N R M I NE . H C L ( FlLClR I Cli 1 205C
90-
80-
>
t-
v)
70-
Z
60-
Z
H
50--
W
>
+ 48.-
a
30-
DL
20-
10-
0- T F V
3
D
d r l d r - i d
M F1 SS1'C H FIR C;E

INTENSITY SUM =56750 BFISE PERK % = 1 3 . 4 4
Figure 2: Mass Spectrum of Amantadine Hydrochloride,
Instrument AEI-MS 902.

AMANTADINE 11
polymorphism. Below are the interpretations (71, 72) aof

the absorbances of these relatively featureless specra.
Ab sorption ( cm- I ) Assignment
3000
+
NH3 stretching (broad)
2923 CH stretching (antisymmet ric)
2855 CH2 stretching (symmetric)
2700-2250 2+ stretching
NH3+
2000 NH3+ overtones
1600 NH3+ deformation
1500 NH3 deformation
1452 CH deformation
2
1365 NH deformation
1307 CH3 wag
1300 and below 2
Fingerprint region
a
zp absorbances at 3000-2800, 1460, 1377 and 123
cm were du to mineral oil. The absorbance at
3500-3400 cm-F was due to water from the KBr in the
pellet.
A diagnostic test for the presence of the
adamantane skeleton is the l.ow-intensity absorption
in the region 1017-1038 cm-l.
The far-in rared spectrum was determined from
- to 100 cm
6501 -5 .
The torsional vibration at 230
cm , which was identical in both the solid and in
cyclohexane solution, was assigned (73) to the amino
group. A barrier height of 2.00 kcal/molel was
calculated. The +band at approximately 490 cm- was
assigned to a NH torsional vibration.
3
2.5 Electron Tunnelling and Photoelectron
Spectrometry .
Inelastic electron tunnelling spectroscopy is a
non-optical vibrational spectroscopy used to study
the adsorption of adsorbates on barrier oxide films
grown on metals. The interpretation of the spectrum
(74) assigns peaks to C-C, -CH2, -CH and C-C-C to be
caused by scissoring, bending, twisting, wagging and
rocking. Vibrations associated with the amine
substituent are almost completely absent, probably
due t o interaction with the adsorbing oxide surface.
Photoelectron spectroscopy is used to determine
ionization potentials, which can test the theoretical
procedures used to predict orbital energies. The
12 JOEL KIRSCHBAUM
F i g u r e 3 . I n f r a r e d s p e c t r a of Amantadine h y d r o c h l o r i d e .
Upper p o r t i o n ; m i n e r a l o i l m u l l : Lower p o r t i o n , potassium
bromide p e l l e t , See t e x t f o r d e t a i l s .
li-
I
AMANTADINE 13
ionization potentials, 11, for a series of adamantane

derivatives are similar (75), 9.22 to 9.25 eV,
indicating that substituents have little effect.
2.6 Thermal Analysis
Thermal gravimetric analysis (76) of a

commercial preparation of amantadine hydrochloride,
using a heating rate of 20°/min., showed no loss in
weight until 190°, indicating a lack of volatile
solvents. Sublimation occurred at about 190" since
the inside of the apparatus was covered with powder,
These results are in good agreement with a melting
range (19) of 180-192'.
Differential thermal analysis and differential
scanning calorimetry (76) gave a series of endotherms
which may be due to sublimation. The parent
compound, adamantane, also sublimes. This unusual
(21) property for a hydrocarbon is considered due to
a face-centered cubic lattice with only forces
between the four molec 15s in a unit cell being
Y
effective (space group TdF43m, a = 9.426 2 0.008A).
2.7 Microscopy
A commercial preparation of amantadine

hydrochloride was found to contain irregularly shaped
crystals ranging from approximately 18 x 25 pm to
35 x 50 pm (76). There was no visual evidence for
polymorphism.
2.8 Surface Area
As measured by nitrogen gas adsorption (76), the

surface ar a of one lot of amantadine hydrochloride
9
was 0.73 m Ig.
2.9 Hydration
The crystals are not solvated with water, based

on the thermal gravimetric and differential thermal
analyses previously described, and the elemental
analysis (cf. section 5.1).
2.10 Polymorphism
There is weak evidence for polymorphism based on

infrared spectrometry (section 3.3) but none by
microscopy.
14 JOEL KIRSCHBAUM
3. Spectrometry of Amantadine in Solution
3.1 Nuclear Magnetic Resonance Spectrometry (NMR)

1
3.11 H-NMR
Figure 4 is the 100 MHz proton NMR spectrum

(77) of amantadine hydrochloride in
deuterochloroform, as obtained on a Varian
XL-100-15 spectrometer equipped to perform
Fourier transform spectrometry. Proton chemical
shifts were referenced to internal
tetramethylsilane (TMS) at 0 ppm. The high
degree of symmetry resulted in the simple
spectrum which was interpreted as follows:
Chemica1 Relative
Shift (ppm) Area Assignment
1.35 2H -NH
1.55 6H 8-Ci
1.62 6H 6-CH
2.05 3H y-CH
Chemical shifts of 1-substituted adamantane (78)

show large variations due to both the substituent
( 7 9 ) and the solvent.
3.12 13C-NMR
The exceptional structure of amantadine has

prompted many carbpj~l-13 NMR structural studies.
Figure 5 shows the C-NMR spectrum of a commercial
preparation of amantadine hydrochloride in
deuterochloroform run at 15 MHz on a Jeol FX 60Q NMR
system ( 7 7 ) . The natural abundance carbon shifts
were referenced to the center line of the CDCl
multiplet at 77.0 ppm fion tetramethylsilane, an3
interpreted as follows. The results are in excellent
agreement with values reported previously (80).
Chemical Shift (ppm) Assignment
29.7 6-C
36.2 B -C
46.2 Y-C
47.2 a-C
In addition, the calculated shift values agree
Figure 4 : Proton Magnetic Resonance Spectrum of Amantadine Hydrochloride
in Deuterochloroform, as Recorded at 100 MHz.

16 JOEL KIRSCHBAUM
with the experimental results, showing that no large

steric interactions ,ayd strain exist between the
carbon atoms (81). C Chemical shifts induced by
protonation of the amine showed the effects of charge
to be transmitted along the carbon skeleton (82) and
involve the next-nearest neighbor (6-ef fect) (83).
Substituent effects three bonds away (y-effect) have
also been studied (84).
Comparisons have been made of shifts in 1- and
2-substituted adamantanes (85) , and relaxation times
(86). Relaxation times are summarized in section
4.4, Hydrodynamic properties, lanthanide and other
shift reagents were used to study the structure (87,
88), donor strength (89), and 8- and y-effects (90).
3.13 15N-NMR
The natural abundance 15N nuclear magnetic

resonance shift was measured (91) for amantadine
P3drochlorideY and found to be 317.1 ppm relative to
N-nitric acid. In methanol, the shift of the free
base was 317.5, and in benzene the shift was 316.3.
The three y carbons of 1-aminoadamantane (see the
NMR section for designations of the various carbons
in the skeleton) appear to have no influence on the
shifts of the free amine or of the hydrochloride.
3.2 Ultraviolet Spectrometry
Below is the ultraviolet spectrum of a

commercial preparation of amantadine hydrochloride at
a concentration of 100 mg/mL method, obtained with
the aid of a Perkin-Elmer Model 320 Spectrophotometer
(92). At 226 nm, the molar absorptivity, E , was
0.128; at 222 nm, E was 0.351, and at 205 nv, E was
0.835. Using traditional nomenclature, the Eli
values are 0.0068, 0.0187 and 0.044, respective'fy.
4. Bulk Solution Properties
4.1 Solubilities in Aqueous and Nonaqueous Solvents.

Solubilities of a commercial preparation of
amantadine hydrochloride were determined (93) at room
temperature in various solvents with about one minute
of mixing.
Figure 5 : 13C-Magnetic Resonance Spectrum of Amantadine Hydrochloride in
Deuterochloroform, as Recorded at 15 MHz.

18 JOEL KIRSCHBAUM
So lvent Solubility (mg/mL)
Acetonitrile 4
Chloroform 75
Ethano1 200
Hexanes <1
Isopropanol 35
Methanol 250
Methylene chloride 10
Water-Methanol (1 :1) >250
Water >250
Hydrochloric acid solution 0.1M 350
Aqueous buffer, pH 2 75
Aqueous buffer, pH 4 50
Aqueous buffer, pH 7 -2
Aqueous buffer, pH 10 <1
Sodium hydroxide solution 0.1M <1
The high solubility in acidic aqueous solvents

precluded measuring an intrinsic dissolution rate,
since tablets compressed under 6000 p.s.i virtually
exploded into aqueous solution (94).
4.2 Ionization
Solutions of recrystallized amantadine

hydrochloride were titrated potentiometrically with
0.1M carbonate-free potassium hydroxide at various
temperatures and concentrations (95). Extrapolation
AMANTADINE 19
t o z e r o i o n i c s t r e n g t h gave pK v a l u e s of 10.71 f
0.01 a t 20', 10.58 f 0.07 a t 250aand 10.14 f 0.02 a t
37". A l i n e a r p l o t gave -d(pK ) / d T = 0.034. These
pKa r e s u l t s are similar t o s u c ! a l k y l a n a l o g u e s a s
2-amino-2-methylpropane (pKa = 10.68 a t 25') and
3-amino-3-ethylpentane (pKa = 10.59) , and s u p p o r t t h e
c o n c l u s i o n (96) t h a t t h e r e i s l i t t l e s t r a i n i n t h i s
a l i c y c l i c molecule.
4.3 Dipole Moments
Dipole moments were measured at various

c o n c e n t r a t i o n s i n benzene (97) f o r a series of
1 - s u b s t i t u t e d adamantanes. Amantadine h a s a d i p o l e
moment, of 1.38 Debyes. T h i s h i g h d i p o l e moment may
r e s u l t (98) from an enhanced p o s i t i v e i n d u c t i v e
e f f e c t of t h e adamantyl s k e l e t o n . I n solvents
forming hydrogen bonds w i t h t h e amino group,
anomalous v a l u e s a r e found ( 9 9 ) .
4.4 Hydrodynamic P r o p e r t i e s
R e l a x a t i o n times (TI) were ermined f o r

amantadine i n v a r i o u s s o l v e n t s usingdp'C-NMR (100).
V i s c o s i t i e s of t h e s o l u t i o n a r e a l s o t a b u l a t e d below.
1 3 ~ - ~(set)
1
Solvent Viscosity -
B 1 6
CD OD
cI 0.76 3.9 6.5 279
CDdlg 0.67 5.1 9.4 3.7
CC14 0.89 9.1 16.4 8.2
T v a l u e s a p p e a r t o be determined by t h e tumbling of
1
a monomer; any s o l u t e - s o l u t e o r s o l v e n t - s o l u t e p a i r s
t h a t are formed a r e t o o s h o r t - l i v e d t o b e measured by
t h i s technique.
I n deuteromethanol t h e moment o f i n e r t i a i s I
II'
300, I , 419 and p , which i s 1 1 / 1 , 1.4.
T t e d i f f u s i o n c o n s t a n t s a r e ' Liven below, where
R1 d e s c r i b e d t h e r o t a t i o n about t h e p r e f e r r e d C
a x i g , anClR2 i s t h e d i f f u s i o n c o n s t a n t , i n u n i t s 02
10 sec . The d a t a i n d i c a t e l i t t l e motion about
t h e C-NH2 bond.
Solvent
R1
-- y
CD OD 5.1 1.7
CD?13 7.2 2.2
20 JOEL KIRSCHBAUM
CC14 8.4 5.7

-0
The partial molar volume, V , in water at 25"
was determined (101) to be 138.9 2 0.3 cm3 mo1-l:
The discrepancy with the calculated value of 141.7
was assumed to be caused by interaction of the amino
group with water via hydrogen bonds.
5. Methods of Analysis
5.1 Compositional Analysis
The results of elemental analyses (102) of two

preparations of amantadine hydrochloride agreed with
theoretical results of carbon, 63.40%; chlorine,
18.89%; hydrogen, 9.13%, and nitrogen 7.46%.
Water content was measured by vapor phase
chromotography. A portion of a commercial
preparation of amantadine hydrochloride was dissolved
in methanol and, after retention on a precolumn,
water content of 0.07% was determined by comparison
with an external standard (103).
Emission spectrochemical analysis of a
commercial preparation (66) gave metallic impurity
contents of copper, 7 pg/g and aluminum, 45 pg/g.
5.2 Identity and Colorimetric Methods
A compendia1 method (104) involves reacting

amantadine hydrochloride in pyridine with acetic
anhydride to produce 1-acetamidoadamantane, which
should have a melting range between 147" and 150"
using the U . S . P procedure for class I compounds,
Alternatively, the characteristic infrared system can
be easily obtained for the solid.
Amantadine hydrochloride undergoes the
characteristic reactions of a primary amine. To 2 mL
of amantadine hydrochloride dissolved in
rnethanolic-acetonitrile (less than 0.3% water) add 1
mL each of 30% trichloroacetic acid and 0.2%
dimethylaminobenzaldehyde solution. The Schiff-base
condensation product forms within 10 min standing at
room temperature to give a bright yellow color (105).
Amantadine hydrochloride undergoes reaction with
l-fluoro-2,4-dinitrobenzene in acetonitrile solution
at an apparent pH of 9, at either 65" or on standing
overnight at room temperature, to give a yellow color
(106).
It also reacts with ninhydrin (107) to form the
AMANTADINE 21
iminoindandione, with acetic anhydride (104, 108) to

form 1-acetamidoadamantane (described in the above
identity test) and with isothiocyanate (log), as well
as forming the derivatives discussed in Section 1.5,
reactions, stability and metabolism; 5.4,
spectrometry, and 5.9, fluorescence.
Reactions of amantadine hydrochloride with
reagents on a microscope slide gave characteristic
crystals. A 1M solution of 1-adamantanamine
hydrochloride in 1M HC1 reacted with either palladium
chloride , gold chloride, iridium chloride, ruthenium
chloride, platinum chloride or potassium osmate to
give various shapes, colors and polarization colors
(109). Complexes were formed when one drop of
amantadine hydrochloride was reacted with one drop of
solutions of either 5-nitrobarbituric acid, HAuCl ,
HAuCl plus NaBr , chloroplatinic acid, chloroplatinfc
4
acid plus NaBr, mercuric chloride and platinum iodide
plus chloroplatinic acid with sodium iodide. Under
the microscope different colors and shapes were
visible (110). The original papers contain sketches
of the crystals.
5.3 Titration
The compendial method (104) uses perchloric acid

and either crystal violet indicator solution or
titrat ion to an endpoint determined
potentiometrically. The accuracy was found to be
within approximately 0.1% in various solvents (111).
Using the Kjeldahl procedure , amantadine was
distilled from amantadine hydrochloride, collected in
0.1 hydrochloric acid and determined by difference
after titration with base (112).
5.4 Spectrometry
Nuclear magnetic resonance (113) was used to

quantitate amantadine hydrochloride. Capsules were
dissolved in water, 30% sodium hydroxide was added
and, after extraction into benzene, acid was added to
a portion, then deuterium oxide and, finally, several
drops of hydrochloric acid. The characteristic
proton NMR spectrum (cf. section 3.1) of 0.4 mL of
purified compound was determined using succinimide as
NMR reference standard. The NMR and compendial
titration method, described in the previous section,
gave similar results. The relative standard
22 JOEL KIRSCHBAUM
deviation of the NMR procedure is 0.8%, it is,

however, an order of magnitude higher than
titrimetry.
Amantadine hydrochloride in biological fluids
was reacted with p-nitrobenzaldehyde (114) to give
1-(p-nitrobenzylidenamino)adamantane, which has
maximal absorption at aqproximately 292 nm (molar
absorptivity is 1.89 x 10 ) . The limit of detection
was approximately 1 pg per mL of blood and plasma.
5.5 Gas-Liquid Chromatography
The method preferred by the pharmaceutical

manufacturer for quantitat ing amantadine
hydrochloride is gas-liquid chromatography (115).
The various GLC systems are summarized in Table 1.
Figure 6 exemplifies gas chromatographic assays for
amantadine in plasma and urine, and were redrawn from
reference (120) with the permission of the Elsevier
Scientific Publishing Company.
5.6 Thin-Layer Chromatography
Table I1 summarizes TLC systems developed for

amantadine.
5.7 High-Performance Liquid Chromatography
The lack of an HPLC method in the literature

prompted development of an assay by the author. Both
reverse and normal phase systems gave multiple
irreproducible peaks for commercial amantadine
hydrochloride known to be of high purity. This
phenomenon was indicative of non-covalent
interactions. Derivatization similar to the U.S.P.
identity test (Section 5.2) but using phthalic
anhydride, gave a compound absorbing at 254 nm. An
octadecylsilane column was used with a mobile phase
of methanol-water-85% phosphoric acid (60:40:0.1)
flowing at 1 mL/min to obtain the chromatogram shown
on next page. Amantadine has negligible absorbance
at 254 nm. Reproducibility and linearity of the
amantadine peak were excellent. Details will be
published subsequently (126).
5.8 Electrochemistry
Oxidation (127) of amantadine in trifluoroacetic

acid gave several anodic waves. The voltammetric
AMANTADINE 23
1 2
- 1
0 4 8 0 4 8 12
Figure 6 .
0 4
1 2
8
Rerention
Gas-Liquid Chromatograms:
J
0
time
4
(min)
I
8
Upper P o r t i o n , A ,
c o n t r o l plasma; B , plasma e x t r a c t c o n t a i n i n g 457 ng
amantadine h y d r o c h l o r i d e (1) p e r mL: Lower P o r t i o n , C ,
C o n t r o l u r i n e ; D , Urine e x t r a c t c o n t a i n i n g 62.4 ug/mL drug
(1). Chlorphentermine i s t h e i n t e r n a l s t a n d a r d ( 2 ) .
Table I. Gas Liquid Chromatography of Amantadine
Bulk Amantadine
Column Carrier T(Co1umn) Detector Reference
5% by weight SE-30 on Chromaton N-AW-DMCS 125" Flame Ionization 116

(particle size 0.16-0.20 mm) N2
10% by weight 0s-124 on Chromaton-N-AW-HMDS 190" Flame Ionization 116

(particle size 0.16-0.20 mm) N2
5% by weight polyethylene glycol (M.W. 1SOO) N2

160" Flame Ionization 116
and 1% KOH on Chromaton N (particle size
0.20-0.25 IIIUI)
3% OV-17 on Chromosorb W He 180" Flame Ionization 117

20% Carbowax on Chromosorb W He 98" Scintillation - 57
Gas Flame
3% OV-1 on Chromosorb W-HP (100-120 mesh) N2,H2,Air 150-250" N-P Detector 118
3% OV-17 on Chromosorb W-HP (100-120 mesh) N 2 ,H2 ,Air 150-250" N-P Detector 118
Amantadine in Blood and Plasma

1.5% SE-30 on Anakrom AS (90-100 mesh) 220" Electron Capture 114
(claim linear response of 0.03-0.15 pg) N2
Table I GLC, continued
co l
m Carrier T(Co1umn) Detector Reference
2% Dexsil 300 on Gas Chrom P or 0 180-260" Mass Spectrometry 62

N2
(80-100 mesh)
Amantadine in Various Tissues and Body Fluids
20% Carbowax 20 M on base-treated Chromosorb W He, H

2'
95" Flame Ionization 123, 124
Air
3% OV-17 on Gas Chrom Q (100-120 mesh) Air, €? 0 150" Flame Ionization 61

2
N2
N
vI 7.5% PEG 20,000 on Gas Chrom Q (100-120 mesh) N2, He 170" Flame Ionization 61
5% Apiezon L o r Gas Chrom 0 (100-120 mesh) H2, Air 160" Flame Ionization 61
2% OV-101 on Chromosorb W (100-120 mesh) Air, H
2 130" Flame Ionization 61
N2
After conversion to isothiocyanate, 3% OV- He 170"or190" Mass Spectrometer 108
225 on Gas Chrom Q
After conversion to isothiocyanate He 180" Mass Spectrometer 108

3% OV-17 on Chromosorb W-HP
After conversion to isothiocyanate He 160" Ma ss Spectrometer 108

OV-101 or Gas Chrom Q
Table I. GLC, continued
COZUmn Carrier T(Colunm1 Detector Reference
After conversion t o N-trichloroacetyl N2

220" Electron Capture 119
derivative, 3% OV-17 on Chromosorb Q (100-
120 mesh). Claim linear response 25ng-ll~g/ml,
determined by MS.
Amantdine in Plasma and Urine
5% Apiezon on Gas-Chrom Q (100-120 mesh) 150" Flame Ionization 120

H2
N After conversion t o trichloroacetyl Ar-CH 200" Electron Capture 121
ch
derivative 5% SE-30 on Chromosorb W HP (90: 16)
(80-100 mesh)
Amantdine i n Urine
10% Apiezon L and 2% KOH on 80/100 145" Flame Ionization 122
N2
Chromosorb W AW
20% Carbowax 20 M on Gas Chrom P or Q 180-260" Mass Spectrometry 62

N2
20% Emulphor ON-870 on Gas Chrom P or Q 180-260" Mass Spectrometry 62
N2
1%SE 30 on Gas Chrom P or Q 180-260" Mass Spectrometry 62
N2
1%OV-17 on Gas Chrom P or Q 180-260" Mass Spectrometry 62
NH2
AMANTADINE 21
Pig 7. HPLC of Amantadine Hydrochloride.
data is E 1 / V = 2.30 and E 122/V=2.67.

Coulgkztry with bromge (128) was used to
quantitate amantadine hydrochloride in aqueous
solution at pH 8 using concentrations as low as 0.5 u
mole per liter.
The interaction of amantadine with lecithin
bilayers used as a membrane model was studied using
polarography (129). Amantadine affects permeability.
5.9 Fluorescence Spectrometry
The lack of natural fluorescence required

fluorescence of amantadine to be induced. Reaction
with fluorescamine (130) at a concentration of 1 pM,
followed by extraction at pH 5 into ethyl acetate,
gave a product with a maximum at 395ex/475em nm. The
limit of sensitivity is 1 ug/mL.
Reaction of 9 - i s o t h i o c y a n a t o a c r i d i n e ( 1 3 1) with
amantadine hydrochloride in ethanol, with heating,
yielded a product that was quantitated from 0.5-2
;g/mL using- 295ex/490em nm. Thin layer fluorimetry
was used.
Table 11. Thin-Layer Chromatography of Amantadine
Support Mobile Phase Detection Re ference
Silica gel Methanol-ammonia (100:1.5) Plate sprayed sequentially 125

with ninhydrin solution, Iron
(111) chloride-perchloric acid,
Dragendorff reagent (Bismuth
subnitrate in acetic acid then
aq. KI), and finally iodoplati-
Silica gel Cyclohexane-toluene-diethylamine nate solution. Second plate 125
(75: 15: 10) had Marquis reagent (formalde-
hyde in H SO ) poured over it.
2 4
Silica gel Chloroform-methanol ( 9 : 1) 125
0
Silica gel Acetone 125
Silica gel Butanol-acetic acid-water (4:1:5) Ninhydrin 57
Silica gel F254 Methanol-25% NH3 (100:1.5) Dragendorff 118
Silica gel F254 Dichloromethane-acetone (4:l) Chlorinate in C12; spray 118

with o-tolidine in ethanol
Silica gel G CHCl -methanol-benzene-25% NH3 Dragendorff 112

(50:20:40:2)
Silica gel G Lower phase HAc-CC1 -CHCl -H 0 61
3 2 3’
( 100 :60: 9 6 : 50) 5%; sulfanilic acid (0.5%)-1-
naphthylamine (0.1%)
Silica gel G n-Butanol-toluene-HAc-H 0 61
2
ninhydrin in n-butanol (98%)
Silica gel G n-Hep tane and HAc (2%) ; triphenyl- 61
tetrazolium reagent; or after
30 JOEL KIRSCHBAUM
5.10 Tissue Culture
Tissue culture and human trial methods for

measuring inhibition of viruses were reviewed
previously (1,2,132). Viral growth inhibition by
amantadine hydrochloride was determined by plaque
formation in Petrie plates with a gradient thickness
of agar plus compound (133). As for most
microbiological assays, analytical errors were
relatively large, typically averaging 22% (134).
5.11 Comparison of Methods
Infrared spectrometry is recommended for an

identity test. Gas-liquid chromatography was the
most frequently used procedure €or quantitating bulk
amantadine hydrochloride and amantadine in tissues
and body fluids. Although titrimetric methods are
more accurate, possible impurities may also be
titrated, limiting the usefulness of the simple
technique. No high-performance liquid
chromatographic assay was found in a search of the
literature, through written requests to several
investigators who published HPLC methods for
adamantanes or by contacting representatives of the
manufacturing pharmaceutical company. Accordingly,
an HPLC method was developed in this laboratory.
6. Acknowledgements
The author gratefully acknowledges the

assistance of the many contributors cited as
"personal communications". With one exception, this
information was especially obtained for this review
by investigators at the Squibb Institute for Medical
Research. The Crystallographic Data Centre,
Cambridge, gave permission t o reproduce the x-ray
derived structure. The Elsevier Scientific
Publishing Co., Amsterdam, gave permission to redraw
the gas-liquid chromatography figure. The figures
utilized the illustrative skills of Jose Alcantara
and the photographic talents of Charlotte Raymond.
The preparation of camera-ready copy was by
Lynn Mendenko. Eugene Ivashkiv provided translations
from Russian. The author appreciates the critical
reading and thoughtful comments of Dr. J. Adamovics,
Dr. G. Brewer and Mr. S . Perlman. Credit for
obtaining many of the references belongs to
Muriel George, Phyllis Gottstine, Letitia Fenwick and
AMANTADINE 31
Michael M a s i e l l o . The Chemical A b s t r a c t s c i t a t i o n s

were appended t o a r t i c l e s t h a t were d i f f i c u l t t o
o b t a i n o r where o n l y t h e a b s t r a c t was read.
L i t e r a t u r e was surveyed t o J a n u a r y , 1983.
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32 JOEL KIRSCHBAUM
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AMANTADINE 33
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34 JOEL KIRSCHBAUM
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87. D.J. Chadwick and D.H. Williams, J . Chem. SOC.,
AMANTADINE 35
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92. D. Sieh, Personal Communication.
93. L. Kerr and J. Kirschbaum, Personal Commun-
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94. E. Rudnic, Personal Communication.
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96. B.A. Korolev, A.P. Khardin, S.S. Radchenko, I . A .
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1632 (1978); CA, 89, 196776n (1978).
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101. F. Shahidi, P.G. Farrell and J.T. Edward, J .
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102. G. Conroy, Personal Communication.
103. M. Jemal and R. Mark, Personal Communication.
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105. P. Stear, Personal Communication.
106. J. Kirschbaum, Personal Communication.
107. P.A. Crooks, Chem. Ind. (London), 1980, 467.
108. N. Narasimhachari, E. Helgeson and U. Prakash,
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36 JOEL KIRSCHBAUM
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Knqaku, 1 9 , 9 5 ( 1 9 7 0 ) ; CA, 73, 12723h ( 1 9 7 0 ) .
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Conyn:$ation.
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(1979). .
AMIKACIN SULFATE
Peter M . Monteleone, Naseem Muhammad,
Robert D . Brown, John P . McGrory,
and Samir A. Hanna
1, Historical and Description 38

1.1 Chemical Origin 38
1.2 Chemical Structure 39
1.3 Name, Formula, and Molecular Weight 39
1.4 Appearance 40
1.5 USP Standard 40
2. Physical Properties 40
2.1 Melting Range 40
2.2 Specific Rotation 40
2.3 Solubility 40
2.4 Electrometric Titration Curve and Apparent pK. Value 41
2.5 Spectra 41
2.6 X-Ray Powder Diffraction 49
2.7 Thermal Analysis (DSCand TGA) 49
3. Synthesis 56
4. Drug Metabolism and Pharmacokinetics 56
5. Stability-Degradation 58
6. Methods of Analysis for Bulk and Injection 59
6.1 Identification 59
6.2 Moisture Content 59
6.4 Microbiological Assay 59
6.5 Other Compendial Tests 60
6.6 Assay for Sulfate 60
7. Chromatographic Methods 60
7.2 High-Pressures Liquid Chromatography (HPLC) 60
7.3 Gas-Liquid Chromatography (GLC) 60
8. Determination in Body Fluids 62
8.1 Microbiological Assay 62
8.2 Fluorescenceimmunoassay(FIA) 63
8.3 Radioimmunoassay (RIA) 63
8.4 Radioenzymatic Assay (REA) 63
8.5 Spectrophotometric-EnzymaticAssay 65
8.6 High-pressure Liquid Chromatography (HPLC) 66
8.7 Gas-Liquid Chromatography (GLC) 66
References 67
ANALYTICAL PROFILES OF DRUG SUBSTANCES 37 Copyright by [he American Pharmaceulical Association

VOLUME 12 ISBN 0-12-260812-7
38 PETER M . MONTELEONE E T A .
1.0 Historical and Description:

Amikacin is a semisynthetic aminoglycoside
produced by the strategic chemical alteration of
kanamycin A. The discovery of kanamycin was re-
ported in 1957 by Dr. Hamao Umezawa’. It is made
by fermentation and has been used to treat many
serious infections caused by gram-negative
bacteria. A s the use of Kanamycin A and other
aminoglycosides became widespread, resistant
strains of bacteria began to emerge. The insight
gained into the enzymatic mechanisms by which
resistant strains inactivated aminoglycosides and
the understanding of structural features related
to microbiological activity formed the basis for
synthesizing better aminoglycosides2. In 1972,
Dr. Hiroshi Kawaguchi at the Bristol-Banyu
Research Institute in Tokoyo described the dis-
covery of BB-K8 (amikacin) and demonstrated its
improved effectiveness3.
In 1976 the F . D . A . approved the marketing of
amikacin. Since then it has found worldwide use
in the treatment of serious gram-negative infec-
tions. The usual marketed forms are 50 and 250
mg/ml solutions designated Amikacin Sulfate
~njection~.
Several articles review the general micro-
biological5, pharmacological, and biochemical
properties6 of amikacin, as well as therapeutic
experience7, with Amikacin Sulfate Injection.
1.1 Chemical Origin:
-
Amikacin is prepared by acylation of
kanamycin A at the C-1 amino group of the
2-deoxystreptamine moiety with the I,(-1-y-
amino-a-hydroxybutyrl group. Acylation of
each of the four amino groups in kanamycin A
with the L(-)-y-amino-a-hydroxybutyrl sub-
stituent gives 4 different derivatives. A s
mentioned, amikacin, originally named BB-K8,
is the C-1 amino derivative. The other three
amino derivatives are BB-K6 (C-6’1, BB-K11
(C-3”) and BB-K29 (C-3) ’.
AMIKACIN SULFATE 39
1.2 Chemical Structure:

-
The structure of am kacin is shown in
Figure 1. The chemistry and proof of struc-
ture have been reported by H. Kawaguchi3.
L (-1
H-C0-C H-C
1
OH
OH
HO
FIGURE 1 STRUCTURE O F AMIKACIN
1.3 Name, Formula and Molecular Weight:

The USAN and USP Dictionary of Drug
Names lists the following names for amikacing:
o-Streptamine, 0-3-amino-3-deoxy-a-
o-glucopyranosyl (1-t6) -0- [ 6-amino-
6-deoxy-a-~-glucopyranosyl (1+4)1-
N1-(4-amino-2-hydroxy-l-oxobutyl)-
2-deoxy, ( S ) - ;
O-3-Amino-3-deoxy-a-D-glucopyranosyl
(1-+4)-0-[6-amino-6-deoxy-a-~-gluco-
pyranosyl (1+6)l-N3-(4-amino-~-2-
hydroxybutyryl)-2-deoxy-~-strept-
amine.
Cz2H43N5013 M.W. = 585.61
Amikacin sulfate is a 1:2 sulfate salt of
amikacin. The following are its formula and
formula weight.
C22H43N5013*2H2S04 F.W. = 781.75
40 PETER M. MONTELEONE E T A .
Amikacin h a s b e e n r e p o r t e d t o e x i s t a s a
f r e e b a s e (CAS Reg. N o . 37517-28-51, a 1 : 2
s u l f a t e (CAS Reg. N o . 39831-55-5), a 1:1
s u l f a t e (CAS Reg. N o . 56086-43-2) and a 1:1
c a r b o n a t e (CAS Reg. N o . 75282-58-5).
1.4 Appearance :
Amikacin i s a w h i t e , c r y s t a l l i n e p o w d e r 3 .
The u s u a l m a r k e t e d form i s a s t e r i l e , color-
less t o s t r a w yellow s o l u t i o n f o r i n j e c t i o n
p r e p a r e d by d i s s o l v i n g a m i k a c i n w i t h d i l u t e
s u l f u r i c a c i d a t a 1 : 2 r a t i o o f a m i k a c i n base
t o H2S04. The s o l u t i o n may c o n t a i n p r e s e r -
v a t i v e s , such a s b i s u l f i t e s and b u f f e r s , s u c h
a s c i t r a t e . The amount o f a m i k a c i n i s e i t h e r
50 o r 250 mg p e r m 1 4 r l o ,
1.5 USP S t a n d a r d :
The c u r r e n t USP s t a n d a r d f o r a m i k a c i n ,
L o t G-1, i s t h e f r e e base a n d h a s a n a s s i g n e d
p o t e n c y of 9 1 4 mcg/mg on a n " a s i s " b a s i s .
A n a l y s i s o f t h a t material g a v e a m o i s t u r e
c o n t e n t of 7 . 3 % 1 2 .
2.0 -
Physical Properties:
2.1 M e l t i n a Ranae:
H . Kawaguchi r e p o r t e d a m e l t i n g r a n g e
203-2040C3. commercial m a t e r i a l r a n g e s f r o m
201-204°C'
2.2 -
Specific Rotation:
The s p e c i f i c r o t a t i o n of a m i k a c i n h a s
been r e p o r t e d a s = +99O, (C= 1 . 0 , H 2 0 ) '.
The CFR r e q u i r e m e n t f o r t h e d r i e d s u b s t a n c e i s
+ 9 7 O t o +1O5Ol4
2.3 Solubilitv:
The e q u i l i b r i u m s o l u b i l i t y i n w a t e r a t
25OC h a s been r e p o r t e d a s 1 8 5 m g / m l 1 3 . Dosage
AMIKACIN SULFATE 41
solutions,prepared as the sulfate salt, con-

tain up to 250 mg/ml as amikacin.
2.4 Electrometric Titration Curve and
-
Apparent pKa Value:
Figure 2 is the electrometric titration
curve for amikacin. It was determined by
conditions which are similar to those reported
for k a n a m y ~ i n ’ ~ .Amikacin (0.1 m mole) was
dissolved in water, and 0.5 m mole of potas-
sium hydroxide was added. The solution was
titrated with 0.5N hydrochloric acid using
SCE/glass electrozes. Instrumentation in-
cluded a Radiometer recording titration
system with PM-64 p H meter TTTGO titrator,
REC-61 servograph and an ABU-13 autoburet16.
If the four amine groups in amikacin are
considered to be equivalent, then an apparent
pKa value of 8.1 may be estimated from the
half neutralization point’’.
2.5 Spectra:
2.5.1 Infrared
- Spectrum
Figure 3 is the infrared spectrum
for amikacin in a potassium bromide
pellet. Band assignments are listed in
Table 1”.
Ma’or bands reported by H.
Kawaguchi’ occurryd at 3350 , 2930 , 1640,
1580 and 1350 cm- .
2.5.2 Proton Nuclear Magnetic Resonance
-
SDectrum
The 90 MHz proton NMR spectrum for
amikacin in D20 is shown in Figure 4.
Table 2 lists the shift assignments”.
H. Kawaguchi has reported the 100 MHz
spectrum for amikacin3.
42 PETER M. MONTELEONE ETAL.
13
12
11
10
8
P"
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
FIGURE 2 ELECTROMETRIC TITRATION CURVE OF AMlKAClN

FIGURE 3 INFRARED SPECTRUM O F AMlKAClN
F I G U R E 4 P R O T O N NMR S P E C T R U M O F A M l K A C l N
d
.rl
0
Id
GI
m
u
&
0
c
ra
4-1 a,c
zrd
h CI)
0 n c,
ICC X c
z a,
[I)
-I-' n 0 !2
c
uI
N I
3: o=u
u I
a
2
rd 0
A c*l
d
0 o r l
co Lo
a3 L O -
O N d o
0
a,
Lo
m n
b rc,
n o @a,
PI m o 0 4 rlrl
rn N f A A
m a - 2 3
a cv r l o 0 0
LO nn
0
rd rl
F-l
ui
c
H
m LO
..
c
u
N
I
f
I
CDN
..
rlf
rl CD Lnm
a,
I+ ri N
A
rd
E-1
45
2.5.3 Carbon-13 NMR Spectrum

The Carbon-13 NMR spectrum for
amikacin is shown in Figure 5. It was
obtained on a Bruker WM-360 WB instru-
ment at the following conditions: 90.6
MHz; spectral width, 15152 Hz; repetition
time, 0.6 sec.; solvent, D20; number of
scans, 2496.
The chemical shifts and spectral
assignments are listed in Table 3”.
These are in accord with the values
originally reported by S . Toda, who
studied the C-13 NMR spectra of amikacin,
its three positional isomers and four
related compounds2’.
2.5.4 Ultraviolet Spectrum
-
Amikacin in water at a concentra-
tion of 1.0 mg/ml shows only end absorp-
tion beginning at about 240 nmI6.
2.5.5 -
Mass Spectrum
The positive ion fast atom bom-
bardment (FAB) mass spectrum of amikacin
is presented in Figure 6. It was obtain-
ed on a VG-ZAB-2F mass spectrometer using
a solution prepared in thioglycero122.
The spectrum shows a strong protonated
molecular ion [M+H]+ which agreed with
the molecular ion with hydrogen loss
observed by negative ion FAB. Spectra
were intense and easy to observe with
most characteristic ions greater than
mass 210. Electron impact (EI) and
positive chemical ionization (PCI) mass
spectrometry failed to yield spectra
which included the molecular ion23.
Amikacin is quite labile in part
because the L-y-amino-a-hydroxybutyric
acid (L-HABA) side chain can fragment or
rearrange very readily. Structures are
given for some of the most prominent
FIGURE 5 CARBON - 13 NMR SPECTRUM OF AMlKAClN
Table 3: Carbon-13 chemical s h i f t s for amikacin.
CHEMICAL SHIFT
CARBON (pm) *
c-1 50.4
c-2 35.0
c-3 49.4
c-4 87.5
c-5 75.4
C-6 81.1
c-1’ 99.1
c-2’ 72.6
c-3’ 73.8
c-4’ 71.8
c-5’ 73.7
C-6’ 42.3
c-1- 100.2
c-2- 72.4
c-3- 54.9
c -4& 70.0
c-5- 72.8
C-6- 61.1
c= 0 177.2
Ca 70.7
C B 36.9
C Y 38.1
*Chemical shifts of free base i n ppm downfield from TMS (in D20)
AMIKACIN SULFATE 49
fragments present in the spectrumz3.

Most include some reaction of the L-HABA
moiety. The structures, given in Table 4
and Figure 7, are consistent with the
ions formed and result from very simple
cleavages. Other structures are also
consistent with some of these ions,
particularly because the two sugars are
structural isomers23.
2.6 X-Ray Powder Diffraction:
The x-ray powder diffraction pattern for
amikacin was obtained using a Rigaku Powder
Diffractometer. Copper Ka radiation ( A =
1.54051) was used with a nickel filter
(Figure 8). The results are listed in
Table 5'l.
2.7 Thermal Analysis (DSC & TGA)
-:
A thermogravimetric analysis curve was
obtained using a Perkin-Elmer TGS-2 thermo-
gravimetric analyzerz1. The analysis was
performed at a heating rate of 10°C/minute
from 4OoC to 300OC.
The TGA curve (Figure 9) shows a weight
loss of 8% from 5OoC to 100°C, due to loss of
water which is probably loosely bound and
evolved slowly. A second weight loss begins
at 22OOC and continues beyond the end of the
scan at 300OC. This is due to decomposition
and is apparently related to the last two
transitions observed by DSC.
A differential scanning calorimetry curve
was obtained (Figure 10) on a Perkin-Elmer
DSC-2 differential scanning calorimeter.
Using nitrogen as the purge gas, the scan was
performed at a rate of 10°C/minute from 4OoC
to 300OC. A broad endotherm was obtained from
119OC to 147OC, which was shown to be revers-
ible and may be due to a crystal phase transi-
tion or melting. Subsequent irreversible
endotherms were obtained at 187OC. 222OC and
260OC. These are attributed to decomposi-
tion2
a
I-
0
w
P
rn
rn
CI)
a
I
m
a
u.
(0
W
a
50
Table 4: F A B mass spectral ions and structural components for amikacin.
Ions
- Structure Formation
586 [M+H1 + Protonated molecular ion.
556 [M+H minus ( c H ~ - N H I~+) Cleavage from the L-HABA side

chain or from Ring A.
542 [M+H minus ( C H ~ - C H , - N H ~ ) I + Cleavage from the L-HABA side

chain.
512 [M+H minus ( C H O H - C H ~ - C H ~ - NH~)]+ Loss of all but the carbonyl of the
L-HABA side chain.
498 [M+H minus CHO-~OH-CH~-NH~) I+ Loss of C 4 ’ - C 5 - - C 6 ’ linked in Ring A.
440 [M+H minus L-HABA) minus (CH~=CH-OH)

I+ L o s s of the L-HABA side chain and
,.
elimination of c S ” - C ~ in Ring c.
429 [M+H minus 2 ( ~ ~ minus

0 ) ( N H ~ - C H ~ - C H O H - C H O H - C H ~ IO+H ) Loss of 2H20 and C S ’ - C / - C ~ ”o
-f
CK
Ring A or C with the ring oxygen and
H rearrangement.
382 [M+H minus (Ring A minus H) minus (CH2=CHNH2)]* Loss of Ring A with H rearrangement
and part of the L-HABA side chain.
324 [M+H minus (Ring A minus H) minus L-HABA + HI+ Simple cleavaqes with H rearrangement
leaving 2 rings.
217 [M+H minus (Ring A+H) minus (Ring C+H) minus Cleavage of both sugars and part of
(CHz=CH-NHz) 1’ the side chain.
215 [M+H minus (Ring A+H) minus (Ring C+H) minus A s above, yet H rearrangment is
(CH3-CHz-NHz) I+ reversed at one point.
OH
$4
I
HO
bH
0
m l z 382 m l z 440
8 RING B
AMIKACIN
m l z 324 mfz 215
FIGURE 7 AMlKAClN FRAGMENTS

F I G U R E 8 X - R A Y D I F F R A C T I O N P A T T E R N OF AMlKAClN
Table 5: X-ray powder diffraction of amikacin.
d* (i) 1/ 109<$9 d (i) 1/10

-- d (i) 1/10
--
6.701 52 4.149 20 3.114 5
6.188 36 4.022 9 3.023 8
6.078 16 3.994 8 2.996 8
5.719 14 3.883 25 2.915 5
5.618 19 3.742 38 2.870 9
5.548 5 3.696 13 2.803 5
5.291 11 3.630 30 2.754 5
5.023 63 3.531 17 2.728 9
4.935 20 3.504 19 2.657 6
4.766 100 3.310 16 2.567 6
4.667 13 3.259 2 2.330 8
4.485 48 3.229 6 2.312 8
4.287 34 3.184 11
ftd = nX interplanar distance

2 sin 8
i'cf:I/Io- relative intensity (based on highest
intensity of 100)
F I G U R E 0 TGA C U R V E S F O R A M l K A C l N
3.0 Synthesis:
Amikacin (BB-K8) is a semisynthetic derivative
of kanamycin acylated with L(-)-y-amino-a-hydroxy-
butyric acid (L-HABA) at the C-1 amino group of the
2-deoxystreptamine moiety.
Amikacin (BB-K8) has been prepared by Hiroshi
Kawaguchi et a1.3 starting from kanamycin, I. The
carbobenzoxylation of kanamycin at the 6’-amino
group was achieved by the activated ester method
using N- (benzylcarbonyloxy)succinimide (CBZ-NOS),
11. The reaction product was purified by ion-
exchange chromatography on Amberlite CG-50 to give
6’-N-benzyloxycarbonyl kanamycin, 111, as shown in
Figure 11. The acylating agent was prepared by
protecting the y-amino group of L-HABA, IV, with
carbobenzoxy chloride, followed by reaction with
N-hydroxysuccinimide to yield active ester of
N-CBZ-protected-L-HABA, VI.
The acylating of 6’-N-Benzyloxycarbonyl kana-
mycin, 111, at C-1 amino group of the 2-deoxy-
streptamine moiety was carried out by an equimolar
reaction with Compound VI at room temperature. The
reaction mixture was subjected to hydrogenolysis
to remove protecting groups at both the 6’-amino
group of the 6’-amino-6’-deoxy-~-glucose moiety of
kanamycin and the y-amino group in the acyl side
chain. The crude amikacin (BB-K8) obtained was
purified by column chromatography on Amberlite
CG-50 with aqueous ammonia. The active fractions
were combined, evaporated in vacuo and the residue
was crystallized from methanol-isopropanol to yield
needle crystals of amikacin base, VII. Synthesis
of amikacin is also claimed in three U . S .
patents2 5, 6. The first of these is the one
claiming the product, while the latter two claim
alternative processes for its preparation.
4.0 Drug Metabolism and Pharmacokinetics:
Amikacin is usually given by intramuscular
injection or intravenous infusion. Protein
binding is minimal. Almost all of the dose is
excreted unmetabolized in the urine6f2’.
AMIKACIN SULFATE 57
L I-)
wM+ticy- $H-COOH
on
CbX-CJ
u-3
HHCO-CH-CH2-CHz
bn hz
VII Amikacin ( B B - K 8 )
Figure 11. Synthesis of Amikacin

58 PETER M . MONTELEONE ET AL.
The study published by H. Lode et a1.28 illus-

trates the pharmacokinetic properties of amikacin.
They reported a mean half-life of 114 2 1 6 . 7
minutes after a 1-hour continuous intravenous in-
fusion of amikacin at a dose of 7.5 mg/kg. Peak
serum levels during infusion reached 37.5 ? 4.9
mcg/ml. Eight hours later they dropped to 1.3 ?
0.5 mcg/ml. In the 24-hour period after dosing,
93.5% of the dose was recovered in the urine
(12 subjects). Using a single intramuscular
injection of 5 mg/kg, peak serum levels reached
21.4 f 5.4 mcg/ml, then declined to 2.4 f 0.9 mcg/
ml by 8 hours ( 3 0 patients).
Based on studies in beagle dogs and humans
after intravenous and intramuscular dosing,
B. C. Cabana and J. G. Taggart” concluded that
the pharmacokinetic profiles of amikacin and
kanamycin are similar.
Evaluating data from a crossover study in
humans, J. T. Clarke et a1.30 also reached the
conclusion that amikacin and kanamycin have similar
1
pharmacokinetics.
D. Zaske and K. CrossleyZ7, as well as K. A.
Kerridge6 have published articles which include
reviews for the pharmacokinetics of amikacin.
5.0 Stability - Degradation:
The stability of amikacin and amikacin sulfate
is reported in a series of papers by Kaplan
et al. 2, 3, These authors provide data on
the stability of amikacin base powder and solution,
aqueous amikacin sulfate solutions, and solutions
of amikacin sulfate admixed with various intra-
venous solutions with and without other therapeutic
agents present. Data given in these papers demon-
strate the chemical and thermal stability of
amikacin. Different lots of the free base powder
subjected to 56OC for four months showed an average
potency loss of 7.2 percent. At 25OC for 24 months
an average loss of 3.9 percent was observed. A
solution of the free base in distilled water at
approximately 185 mg per ml was autoclaved in a
closed vial for 30 minutes at 1 5 lb. pressure and
AMIKACIN SULFATE 59
1 2 1 O C ; no l o s s i n p o t e n c y was o b s e r v e d , a c o l o r
c h a n g e t o l i g h t y e l l o w was o b s e r v e d .
The s t a b i l i t y o f a q u e o u s s o l u t i o n s o f a m i k a c i n
s u l f a t e w a s summarized a s f o l l o w s : The a m i k a c i n
a c t i v i t y w a s maintained a t g r e a t e r than 90 percent
o f t h e o r i g i n a l l y p r e s e n t amount a f t e r e l e v a t e d
t e m p e r a t u r e s t o r a g e a t 56OC a n d 45OC f o r 4 m o n t h s ,
37OC f o r 1 2 months, a n d 25OC f o r up t o 36 months.
The p H o f t h e s o l u t i o n s w e r e 4 . 5 t o 6.8 f o r v a r i o u s
c o n c e n t r a t i o n s o f amikacin s u l f a t e w i t h and w i t h o u t
t h e presence of paraben p r e s e r v a t i v e s .
6.0 Methods o f A n a l y s i s f o r Bulk and I n j e c t i o n :
R e q u i r e m e n t s and methods a r e d e s c r i b e d i n t h e
1982 CFR, T i t l e 2 1 , p a r t s 444.6 and 444.206.
6.1 Identification:
I d e n t i f i c a t i o n i s p e r f o r m e d by c o n t i n u o u s
f l o w TLC ( 2 1 CFR 4 3 6 . 3 1 8 ) .
6.2 - oisture Content:

M
M o i s t u r e i s d e t e r m i n e d by K a r l F i s c h e r
t i t r a t i o n ( 2 1 CFR 4 3 6 . 2 0 1 ) . I t must n o t b e
more t h a n 8 . 5 % .
6.3 -
Specific Rotation:
The CFR r e q u i r e m e n t f o r s p e c i f i c r o t a t i o n
f o r a m i k a c i n b u l k i s n o t less t h a n + 9 7 O a n d
n o t more t h a n +105O ( a n h y d r o u s ) . The d e t e r -
m i n a t i o n i s made by t h e p r o c e d u r e d e s c r i b e d i n
436.20 u s i n g a n a q u e o u s s o l u t i o n c o n t a i n i n g
a m i k a c i n a t 2 0 mg/ml and a 1 d e c i m e t e r p o l a r i -
meter t u b e .
6.4 Microbiolosical Assav:
P o t e n c y i s d e t e r m i n e d by t h e m i c r o b i o -
l o g i c a l a s s a y f o r b o t h t h e b u l k and i n j e c t i o n .
I t is a t u r b i d i m e t r i c assay using Staphylo-
c o c c u s a u r e u s ATCC 6538P ( 2 1 CFR 4 3 6 . 1 0 6 ) .
Ti minimum p o t e n c y o f 9 0 0 mcg/mg, ( a n h y d r o u s )
i s r e q u i r e d f o r t h e b u l k and 9 0 % t o 1 2 0 % o f
t h e claimed value f o r t h e i n j e c t i o n .
6.5 Other Compendia1 Tests:

-
Amikacin bulk must additionally meet the
CFR requirements for safety, pH, residue on
ignition and crystallinity.
Amikacin injection must also pass the
test for safety, pyrogens, and sterility,
and have a pH of not less than 3 . 5 and not
more than 5.5.
6.6 Assay for Sulfate:
Although assay for sulfate in amikacin
injection is not a compendia1 requirement,
it may be determined by the gravimetric
method using barium sulfate p r e ~ i p i t a t i o n ~ ~ .
The procedure is very similar to that de-
scribed for gentamicin sulfate36.
7.0 Chromatographic Methods:
7.1 Thin-Layer Chromatography (TLC)
-
Table 6 summarizes TLC methods for
amikacin and related compounds.
7.2 High Pressure Liquid Chromatography
-
(HPLC):
Since amikacin has poor UV absorbance, it
is derivatized as part of the HPLC procedure.
S. M. Maitra et a1.42 separated amikacin
from its isomers by first forming o-phthal-
dehyde derivatives. They reported the
separation of amikacin from BB-K29, BB-K6,
and BB-K11 by this technique. Separation was
carried out on a C-18 column using a mobile
phase of methanol/water ( 7 0 : 3 0 ) containing
tripotassium EDTA. Retention times (minutes)
for the derivatives were amikacin, 6.6;
BB-K29, 7.1; BB-K6, 8 . 3 ; and BB-K11, 1 1 . 7 .
7.3 Gas-Liquid Chromatography (GLC):

The GLC procedure by J. W. Mayhew and
S. L. Gorbach for determination of amikacin
AMIKACIN SULFATE 61
Table 6: TLC conditions f o r amikacin and related compounds.
Rf Value
Plate Developer Detection F o r Amikacin Reference
Silica Gel A - 0.16 (a) 2
Silica Gel F2511 A - 0.16 ( b ) 37
Silica Gel F 2 5 4 B Ninhydrin 0.23 (c) 38
Silica Gel C Ninhydrin - (d) 39
Silica Gel D Bioautography .06 (el 40
Developers
A. Chloroform/methanol/28% ammonium hydroxide/water (1:4:2:1).

B. Tetrahydrofuran/methanol/ammonium hydroxide/water (1:l:l:l)
C. Methanol/amrnonium hydroxide/chloroform (60:30:25).
D. Chloroform/methanol/25% ammonium hydroxide (1:7:4).
NOTES
(a) Related compounds and Rf values reported as follows: BB-K6,

0.29; BB-K29, 0.211; BB-K11, 0.15; BB-K19, 0.17, and BB-K31,
0.16.
(b) System used to separate amikacin and w-amino-a-hydroxyalkanoic
acid derivatives homologous to amikacin.
(c) Related compounds chromatographed: BB-K11, 0 . 2 0 ; BB-K29, 0.36,
kanamycin A , 0.68, and y-amino-a-hydroxybutyric acid, 0.85.
(d) Continuous f l o w f o r 5% hours. Spots from Standard and Test
Solutions must correspond f o r Identification. Arnikacin
moves about 1.5 cm from the origin4* .
(e) In this system kanamycin A had an R f = 0.30.
in serum is described in section 8.7 of this

profile.
8.0 Determination in Body Fluids:
-
Aminoglycosides, such as amikacin, have the
potential to produce ototoxicity and nephrotox-
icity, if certain serum levels are exceeded for a
prolonged period of time4. However, minimum serum
concentrations are needed for these antibiotics to
be effective. By knowing the serum levels during
the course of treatment, the physician can provide
the safest and most effective therapy for the
patient. To meet this need, many rapid, sensitive
and precise assays have been developed. Some of
those for amikacin are described in this section.
8.1 Microbiological
- Assay:
P. Marengo and his c o - ~ o r k e r shave
~ ~ re-
ported a rapid, highly specific agar diffusion
method for the measurement of amikacin in body
fluids. The assay method uses a strain of
Pr0videncj-a stuartii resistant to multiple
antibiotics with the exception of amikacin.
D. Stevens -
et
- a1.44 described the stan-
dardization of a four-hour assay for amikacin,
gentamicin and tobramycin using Enterobacter
cloacae as the test organism. The method
-
performs well in the presence of other anti-
biotics including cefamandole and cefoxitin;
however, tetracycline and chloramphenicol will
interfere.
D. Buda et al.45 patented a microbiologi-
cal radioassay method to measure the serum and
plasma levels of amikacin and other amino-
qlycosides. The method measures the amount of
'CO2 produced during the incubation of an
aliquot of test sample in a "C urea test
medium containing Proteus rettgeri ATCC 31168
as the urease producing microorganism.
K. Price -
et
- a1.5 determined amikacin in
blood and urine samples by a 16-18 hour agar
diffusion plate method using B. subtilis
ATCC 6633 as the test organism.
AMIKACIN SULFATE 63
S. C. Edberg and A. M i ~ k i nused

~ ~ growth
curve analysis based on continuous measurement
of the turbidity of bacterial suspensions of
S. aureus MHMC386 grown at 37OC. A special
spectrophotometer with a two compartment cell
monitored the turbidity.
8.2 Fluorescenceimmunoassay (FIA):
The procedure reported by S. G . Thompson
d ~an~example of a homogene-
and J. F. B ~ r is
ous substrate-labeled FIA for amikacin. It
uses amikacin labeled by the fluorogenic
enzyme substrate 6-galactosylumbelliferone.
The labeled amikacin is non-fluorescent;
however, when it is hydrolyzed by B-galacto-
sidase, a fluorescent product results. As in
other immunoassays, labeled and unlabeled
amikacin compete for the antibody binding
sites. When labeled amikacin is bound to the
antibody, the hydrolysis of its fluorescent
label is inhibited. Enzymatic hydrolysis of
the unbound labeled drug releases a fluores-
cent product which can be related to the
original concentration of amikacin in the
sample.
The procedure requires two microliters
of serum. Good precision and recovery data
were shown over the range of 5 to 30 mcg of
amikacin per ml, and a comparison was made
with data from a radioimmumoassay. Specifi-
city in the presence of other aminoglycosides
and other antibiotics was discussed.
An example of a fluorescence polarization
immumoassay for certain aminoglycosides, in-
cludin amikacin, was reported by M. E. Jolley
et al. The fluorescent labeled aminoglyco-
side was prepared by reaction with 5-[4,6-
dichlorotriazin-2-yl)-amino] fluorescein.
Binding of the labeled compound to the anti-
bodies resulted in changes in its fluorescence
polarization thereby forming the basis for
quantitation. Good recovery data for amikacin
was obtained over the range of 3 to 50 mcg/ml
of serum. Specificity in the presence of
other drugs and antibiotics was discussed.
8.3 Radioimmunoassay (RIA)

J. E. Lewis -
et
- a1.49 described an RIA for
amikacin requiring as little as 5 microliters
of serum, urine or cerebrospinal fluid. Assay
time was about 2 hours. A sensitivity of 5 ng
of amikacin was reported. Antisera were pro-
duced by immunizing rabbits with amikacin
conjugated to porcine thyroglobulin. Selec-
tivity in the presence of other aminoglyco-
sides was discussed, and a comparison was
made with results obtained by microbiological
and radioenzymatic assays.
C. Ashby et a1.” reported an RIA using
gentamicin. ’‘
three antiserum systems making possible the
specific assa of amikacin, tobramycin and
I-labeled aminoglycosides were
used. Serum samples ranging from 2 to 50 mcg
of amikacin per ml were used to demonstrate
accuracy.
8.4 - -:
Radioenzymatic Assay (REA)
J. 14. Broughhall and D. S. Reevess1 de-
scribed the 14C-acetyltransferase technique
for assay of aminoglycosides. The enzyme
transfers a 14C-labeled acetyl group from the
coenzyme substrate to the aminoglycoside.
Separation of the 14C-labeled aminoglycoside
is carried out by absor tion onto 9hospho-
cellulose paper. The “C-acetyl coenzyme
is removed by washing, and the 14C-labeled
aminoglycoside remaining on the paper is
counted. Assays can be completed within an
hour.
P. Botha -
et
- a1.52 reported that 2 years
experience in assaying serum for aminoglyco-
sides, including amikacin, had shown the
acetyltransferase method to be rapid and
accurate. The method uses 6’-N-transacetylase
prepared from E. coli CH15 and 14C-acetyl
coenzyme A to Tabel the amikacin prior to
isolating and counting. Standard curves,
shown for 3-13 mcg of aminoglycoside per ml,
were linear.
AMIKACIN SULFATE 65
P. Santanam and F. H. Kaysers3 described

the preparation of an aminoglycoside 6’-N-
acetyltransferase from E. coli A21218. The
improved enzyme extract acetylated amikacin
within 5 minutes at 35OC in human serum
buffered at pH 7.5 with trismaleate buffer.
Aminoglycoside calibration curves for concen-
trations of 2 to 20 mcg/ml of serum were shown
in their report. Phosphocellulose papers used
in the isolation step were pre-treated to
eliminate non-specific binding of 14C-acetyl
coenzyme A.
C. Manuel -
et
- a1.54 evaluated a REA to
show that accurate and precise assays could
be performed on capillary as well as venous
blood. Twenty microliters of plasma was re-
quired.
R. P. McNight and J. E. W i l l i ~de-
~~
scribed a procedure using kanamycin 6’-acetyl-
transferase (EC2.3.1.55) to transfer the
l4c-acety1 group from 14c acetyl coenzyme A
to the 6’ nitrogen of the aminoglycoside. The
reaction takes 10 minutes at 37’C to complete.
As little as 2 ng of amikacin could be
measured. Detailed data was presented to
demonstrate precision, accuracy and specifi-
city.
J. Bhattacharya et a1.56 described a way
to remove 67Ga interference in the radio-
enzymatic assay of amikacin and other amino-
glycosides.
8.5 Spectrophotometric-Enzymatic Assay:
-
E. Scarbrough et a1.57 reported a spec-
trophotometric-enzymatic assay for amikacin.
The method uses a purified kanamycin acetyl-
transferase. When the acetylation takes place,
a coenzyme A is produced. This is reacted
with 5,5’-dithiobis-(2,2’)-nitrobenzoic acid
(DNTB) to produce a disulfide and thionitro-
benzoic acid which absorbs at 412 nm. The
assay was reported to be linear from 0 to 60
mcg of amikacin with the lowest amikacin
standard shown to be about 2 - 3 mcg/ml of
serum. Reaction time is approximately

7 minutes.

-(HPLC)
- :
S. K. Maitra et a1.58 detailed the HPLC

-1
procedure for amikacin in serum by the
o-phthaldehyde derivatization technique.
Amikacin was separated from serum on a small
silica column where it was reacted with
o-phthaldehyde to produce a fluorescent
derivative which was quantitated by HPLC.
Sensitivity for amikacin was about 1 mcg/ml
of serum.
J. P. Anhalt and S. D. Brown5' described
the procedure whereby the fluorescent o-
phthaldehyde derivative was formed using post
HPLC column continuous flow derivatization.
A CM-Sephadex (C-25) was used to extract the
aminoglycosides from serum.
8.7 Gas-Liquid Chromatography (GLC):
J. S. Mayhew and S . L. Gorbach6' publish-
ed a detailed description of a GLC method for
amikacin in serum. Interferences were removed
by acid precipitation. The amikacin was de-
rivatized in two steps using N-trimethylsilyl-
imidazole followed by N-heptafluorobutyryl-
imidazole. The derivative was extracted into
hexane before chromatographing. A Nickel-63
detector was used. Sensitivity for amikacin
was about 0.6 mcg/ml of serum. Time required
per assay was approximately 50 minutes.
9.0 Acknowledsements:
The authors thank Marie Foulks for typing the
manuscript, and Vilmars Sprancmanis and Dr. Ken A.
Kerridge for help in obtaining a literature search.
We gratefully acknowledge the work of Dave F.
Whitehead, Tim R. Marr, Dr. George R. Dubay, and
Dr. M. S. Balakrishnan in providing data on the
spectral and thermal properties of amikacin.
AMIKACIN SULFATE 67
10.0 References :
H. Umezawa, M. Ueda, K. Maeda,

K. Yagishita, S. Kondo, Y. Okami,
R. Utahara, Y. Osata, K. Nitta, and
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-
10, 181-189 (1957).
H. Kawaguchi, J. Infect. Dis., 134,
S242-S248 (1976).
H. Kawaguchi, T. Naito, S. Nakagawa and
K. Fujisawa, -
J. Antibiot., -
25, 695-708
(1972).
Amiki@, Physicians Desk Reference,
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K. E. Price, D. R. Chisholm, M. Misiek,

F. Leitner and Y. M. Tsai, 2. Antibiot.,
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-
K. A. Kerridge, Pharmcol. Biochem. Prop.
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Drug Subst., -
R. D. Meyer, Ann. Intern. Med., -
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-
USAN and USP Dictionary Drug Names, 31 c
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P. J. Cloes, M. Dubost and H.
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AMIKACIN SULFATE 69
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61, 123-236 (1978).
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H. Langmaak, Journal of InEectious
-
Diseases, -
134, S316-22 (19/6).
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70 PETER M . MONTELEONE ETAL.
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communication.
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& .
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ther.,
-- 6 , 498-500 ( 1 9 7 4 ) .
-
D. L. Stevens, B. M. Page and C.
Adeniyi - Jones, Am. J. Chem. Pathol.,
-
70, 808-15 (1978):
D. Buda, R. L. Broman and J. R.

Walters, U.S. Patent No. 4 , 0 7 3 , 6 9 4 ,
Feb. 14, i 9 7 8 .
S. C. Edberg and A. Miskin, 2. Pharm.
Sci.,
- -6 9 , 1442-43 (1980).
S. G . Thompson and J. F. Burd, Anti- _-

microb. Agents Chemother., -18, 2 6 4 - 6 8
71980).
M. E. Jolley, S. D. Stroupe, C-H.J.

Wang, H. N. Panas, C. L. Keegan,
R. L. Schmidt. and K. S. Schwenzer,
Clin.
-- Chem. (Winston-Salem, N.C.)
1190-97 (1981).
,-c,
J. E. Lewis, J. C. Nelson and H. A.
Elder, Antimicrob. Agents Chemother.,
7, 4 2 - 4 5 ( 1 9 7 5 ) .
-
C. D. Ashbv, J. E. Lewis and J. C.
Nelson, Clin. Chem. (Winston-Salem,
2z, 1734-1737 71978) .
-- ) , -
N.C.
J. M. Broughall and D. S. Reeves,

Chemother. Proc. Int. Congr. Chemo-
ther.,
-- -
2, 159-164 ( 1 9 7 6 ) .
AMIKACIN SULFATE 71
(52) P. Botha, P. A. Berman, G. Elisha,

S. Afr. Med. J., 56,
J. J. Callanan, -
211-13 (1979).
(53) P. Santanam, F. H. Kayser, J. Anti-

microb. Chemother., -
5 , 477-79 ( 1 9 7 9 ) .
(54) C. Manuel, A. Rancononi and B. Pangon,

J. Antimicrob. Chemother., -
- 6, 560-561
(1980)
(55) R. P. McKnight and J. E. Willis, -

Clin.
-
Chem. (Winston-Salem, N.C.), -
27, 1256-
1261 (1981).
(56) I. Bhattacharya, R. Seligsohn and

S. A. Lerner, Antimicrob. Agents
Chemother., -
- 14; 448-53 ( 1 9 7 8 ) .
(57) E. Scarbrough, J . W. Williams and

D. B. Northrop, Antimicrob. Agents
Chemother., -
1 6 , 221-24 ( 1 9 7 9 ) .
(58) S . K. Maitra, T. T. Yoshikawa, C. M.

Steyn, L. B. Guze and M. C. Schotz
Antimicrob. Agents Chemother., -14,
880-85 (1978).
(59)
Chem. (Winston-Salem, N. C. 1
1940-7 (1978).
zF
J . P. Anhalt and S. D. Brown, Clin.
(60) J . W. Mayhew and S. L. Gorbach, J.

Chromatography, 1 5 1 , 133-146 (1978).
BENZOCAINE
Syed Laik Ali
1. History 14
2. Description 74
2.1 Name, Formula, Molecular Weight 74
2.2 Appearance, Colour, Odour 14
3. Synthesis 14
4.2 Solubility 15
4.3 Dissociation Constant 15
4 . 4 Loss on Drying 76
4.5 Ultraviolet Spectrum 16
4.6 Infrared Spectrum 16
4.1 Nuclear Magnetic Resonance Spectrum 79
4.8 Mass Spectrum 19
4.9 Differential Thermal Analysis (DTA) 79
5 . Identification and Colour Reactions 82
6. Degradation and Stability 83
7. Dissolution, Liberation and Diffusion 87
9 . Drug Metabolism and Pharmacokinetics 97
References 100
ANALYTICAL PROFILES OF DRUG SUBSTANCES 73 Copyright by the American PhamLlceuticd Aasocvatiun.

VOLUME 12 ISBN 0-12-2Ml812-7
74 SYED LAIK ALI
1. H i s t o r y . E t h y l p - a m i n o b e n z o a t e was f i r s t s y t h e -
s i s e d by a german chemist R i t s e r t i n 1 8 9 0 and d u e
t o i t s low t o x i c i t y and good l o c a l a n e s t h e t i c pro-
p e r t i e s was g i v e n t h e t r a d e name o f A n e s t h e s i n (1).
I n 1 9 0 1 t h e s u b s t a n c e was t e s t e d p h a r m a c o l o g i c a l l y
by K o b e r t - R o s t o d k ( 2 ) and was f o u n d t o p o s s e s s
remarkable a n e s t h e t i c p r o p e r t i e s . I n e a r l y 1902
d i f f e r e n t f o r m u l a t i o n s of e t h y l p-aminobenzoa t e ,
powder, p a s t e , l o z e n g e s , d r a g e e s and o i n t m e n t , were
b r o u g h t i n t h e m a r k e t u n d e r t h e t r a d e - n a m e o f Ane-
s t h e s i n ( 3 ) . L a t e r on i n 1 9 0 4 F. B u c k a ' s "Kopf-Phar-
macy" i n F r a n k f u r t am Main was f u r t h e r i n v o l v e d i n
marketing of A n e s t h e s i n p r o d u c t s .
2. Description
2.1. Name, Formula, M o l e c u l a r Weight.
E t h y l p-aminobenzoae; B e n z o c a i n e , b e n z o i c a c i d ,
4-amino e t h y l e s t e r ; E t h o f o r m ;
C9H1N02 165.19
NH2
2.2. A p p e a r a n c e , C o l o u r , Odour. White o r c o l o u r -
l e s s c r y s t a l s o r c r y s t a l l i n e powder w i t h a b i t t e r
t a s t e , f o l l o w e d by l o c a l a n e s t h e s i a o f t h e t o n g u e ,
almost odourless.
3 . S y n t h e s i s B e n z o c a i n e c o u l d be s y n t h e s i s e d i n
number o f ways. p - N i t r o b e n z o i c a c i d i s e s t e r i f i e d
and t h e r e s u l t i n g p r o d u c t i s r e d u c e d w i t h m e t a l l i c
t i n and h y d r o c h l o r i c a c i d ( 4 ) o r h y d r o g e n i n p r e -
s e n c e o f p l a t i n u m o x i d e ( 5 ) t o e t h y l p-aminoben-
z o a t e . O t h e r methods a r e t h e e s t e r i f i c a t i o n o f
p-aminobenzoic a c i d ( 6 ) o r t h e r e d u c t i o n o f e t h y l
p - n i t r o b e n z o a t e w i t h ammonium s u l p h i d e (7) .
S t a r t i n g w i t h n i t r o t o l u e n e benzocaine can a l s o be
s y n t h e s i s e d a s shown i n F i g . 1 ( 8 ) .
BENZOCAINE 75
Fig. 1
V a r i o u s s a l t s o f b e n z o c a i n e w i t h b e n z e n e - , naph-
t h a l i n a n d phenolsulphonic a c i d s have been synthe-
s i s e d and f o u n d t o b e o f t h e r a p e u t i c v a l u e ( 9 ) .
4. Physical Properties
4.1. M e l t i n g Range. B e n z o c a i n e melts b e t w e e n
88-92OCI b u t t h e r a n g e b e t w e e n b e g i n n i n g and end
of m e l t i n g d o e s n o t exceed 2O ( 1 0 ) .
4.2. S o l u b i l i t y (11) The s o l u b i l i t y o f b e n z o c a i n e
i n v a r i o u s s o l v e n t s a t 2OoC i s g i v e n i n Table 1
as 1 p a r t per s p e c i f i e d p a r t s - s o l v e n t .
Table 1
S o l u b i l i t y o f b e n z o c a i n e a t 20’C
Solvent Solubility
Water 2500
A 1c o h o 1 5
Chloroform 2
Ether 4
Almond o i l o r o l i v e o i l 30-50
Mineral a c i d s s o l u b l e under s a l t
f o r ma t i o n
4.3. D i s s o c i a t i o n C o n s t a n t . A c i d d i s s o c i a t i o n c o n -
s t a n t o f b e n z o c a i n e h a s b e e n g i v e n a s pK, 2.5
( 1 2 ) . E s t i m a t i o n o f m i c r o q u i l i b r i u m c o n s t a n t of
e t h y l p - a m i n o b e n z o a t e was d o n e s p e c t r o h o t o -
m e t r i c a l l y and a K v a l u e o f 4.17 x 10-0 was
o b t a i n e d . S p e c t r o p h o t o m e t r i c method a p p l i e d f o r
e s t i m a t i n g m i c r o e q u i l i b i r i u m c o n s t a n t s is simp-
l e r , f a s t e r and more a c c u r a t e t h a n t h e c o n v e n -
76 SYED LAIK ALI
t i o n a l method e m p l o y i n g t h e d i s s o c i a t i o n c o n s t a n t
o f an a l k y l a t e d d e r i v a t i v e ( 1 3 ) .
4.4. Loss on D r y i n q . When d r i e d t o c o n s t a n t w e i g h t
a t a p r e s s u r e n o t e x c e e d i n g 20 cm Hg l o o s e s n o t
more t h a n 0.5 p e r c e n t o f i t s w e i g h t , (11).
4.5. U l t r a v i o l e t S p e c t r u m ( 1 4 ) . B e n z o c a i n e i n
s o l u t i o n absorbs u l t r a v i o l e t r a d i a t i o n t o produce
s p e c t r u m w i t h d i f f e r e n t maxima i n d i f f e r e n t s o l u -
t i o n s . I n methanol o r e t h a n o l , 0 . 1 N H C l and 0 . 1 N
NaOH i t h a s a b s o r p t i o n maxima a t 220, 292, 270,
226 and 284 nm r e s p e c t i v e l y . The c o r r e s o n d i n g
m o l e c u l a r e x t i n c t i o n c o e f f i c i e n t s and APb v a l u e s
a r e reported as following ( 1 4 ) . 1C H
Methanol 0.1N H C l 0.1N NaOH

Absorption
maxima 292 nm 270 nm 284 nm
220 nm 226 nm
A 1b 1246 79
f Clm 538 770 1002
€ 20580
8890
1310
12720
16550
The UV s p e c t r a a r e g i v e n i n F i g . 2.
4.6. I n f r a r e d Spectrum. The i n f r a r e d s p e c t r u m of
b e n z o c a i n e is g i v e n i n F i g . 3. The s p e c t r u m was
o b t a i n e d w i t h P e r k i n - E l m e r 257 s p e c t r o p h o t o m e t e r
from a KBr p e l l e t . The s t r u c t u r a l a s s i g n m e n t s may
b e 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
e n c v ( cm - 1 1
Assignment
3200-3500 c h a r a c t e ristic s t r e t c h i n g
v i b r a t i o n s o f primary
amino g r o u p
1680 characteristic stretching
v i b r a t i o n s o f C=O g r o u p i n
ester
1 6 0 0 and 1 5 1 0 c h a r a c t e ristic skeletal
s t r e t c h i n g v i b r a t i o n s of
the aromatic ring
1250-1310; 1100-1150 c h a r a c t e r i s t i c C-0 s t r e t -
ching v i b r a t i o n s
BENZOCAINE 77
A i n nm-
Fig. 2 UV spectrum of benzocaine in

(methanol)
------ ( 0 . 1 N NaOH)
- - - -(0.1N H C 1 )
Fig. 3 IR Spectrum of benzocaine,

KBr pellet, Perkin-Elmer 257
Spectrophotometer
78 SYED LAIK ALI
Fig. 4 N M R Spectrum o f benzocaine,

Varian T 60 Spectrometer
Fig. 5 Mass Spectrum of benzocaine

BENZOCAINE 79
4.7. Nuclear M a g n e t i c R e s o n a n c e Spectrum.

The 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 of b e n z o -
c a i n e a s shown i n F i g . 4 was o b t a i n e d on a V a r i a n
T-60 NMR s p e c t r o m e t e r i n d e u t e r a t e d c h l o r o f o r m
c o n t a i n i n g 1%t e t r a m e t h y l s i l a n e a s t h e i n t e r n a l
s t a n d a r d . The f o l l o w i n g s p e c t r a l a s s i g n m e n t s a r e
made f o r F i g . 4.
Chemical S h i f t ( ) Assignment
1 . 3 2 ppm m e t h y l p r o t o n s of e t h y l g r o u p
4.28 ppm p r o t o n s o f amino g r o u p
4.31 ppm m t h y l e n e p r o t o n s of e t h y l g r o u p
6.62 ppm two a r o m a t i c p r o t o n s a d j a c e n t
t o ester group
7.88 ppm two a r o m a t i c p r o t o n s a d j a c e n t
t o amino g r o u p
4.8. Mass S p e c t r u m .
Mass S p e c t r u m is g i v e n i n F i g . 5
I n s t r u m e n t : V a r i a n MAT 4 4
Sample t e m p e r a t u r e ( d i r e c t i n l e t ) : 8OoC
Source temperature: 2 0 0 0 ~
E l e c t r o n e n e r g y : 70 e V
The p r o m i n e n t i o n s of t h i s s p e c t r u m c a n be
c o r r e l a t e d t o t h e structure a s following:
m/e 1 6 5 = M ; 1 3 6 = M
120 = M -
- C2H5
C2H5O; 92 = M - COOC2H5
4.9. D i f f e r e n t i a l Thermal A n a l y s i s ( 1 5 , 1 6 , 1 7 )
The two-component s y s t e m b e n z o c a i n e - p i c r i c a c i d h a s
b e e n s t u d i e d i n o r d e r t o e l u c i d a t e some d i s c r e p a n -
c i e s c o n c e r n i n g t h e m e l t i n g p o i n t s of benzocaine
p i c r a t e . Two m o d i f i c a t i o n s , a m e t a s t a b l e o n e w i t h
m.p. 129O and a s t a b l e o n e w i t h m.p. 162O a r e
known. I t was shown by B o r k a ( 1 6 ) t h a t t h e e q u i -
molar p i c r a t e y i e l d s f o u r p o l y m o r p h i c f o r m s , and &
d i m o r p h i c i n h o m o g e n e o u s l y me1 t i n g p i c r a t e w i t h t h e
c o m p o s i t i o n o f 2 mole b e n z o c a i n e : 1 mole p i c r i c
a c i d . The s o l i d - s o l i d t r a n s f o r m a t i o n of t h e l o w
melting modification i n t o t h e high-melting stable
form i s r e p o r t e d . The s t a b l e m o d i f i c a t i o n c a n be
o b t a i n e d by e x c e s s i v e d r y i n g of t h e i s o l a t e d sub-
s t a n c e a t 105O. The t h e r m a l e n e r g y i n t r o d u c e d i s
SYED LAIK ALI
.-V
€ ................... .....................
2E .
Y
... ...
8 ...
...
w
AT
U
13 1 I
i
5 8b 120 160
Temperature "C
Fig. 6 Differential thermal analysis of

benzocaine picrate (Form I)
1st heating
------ reheating, after cooling to
room temperature
.-U
E
L
B
Y
2 ........................................
.... ::
w *.
AT ..
..
.-V ....
E
b, 5
50
'13
C
w
Fig. 7 Differential thermal analysis of

benzocaine picrate (Form 11) heated
to melting point
-- 1st heating (to 135OC)
------ reheating, after cooling to
room temperature
BENZOCAINE 81
c
0
u I
C 1 . 1
, . I I
W 100 130 160

Temperature "c
Fig.8 Differential thermal analysis of
benzocaine picrate ( F o r m 11) heated
above melting point.
1st heating (to 160°C)
------ reheating after cooling to

room temperature
Temperature "C
Fig. g Differential thermal analysis of
benzocaine picrate (Form 11) showing
crystal transformation to form I
82 SYED LAIK ALI
consumed by t h e c r y s t a l s which i n t u r n u n d e r g o
s o l i d - s o l i d t r a n s f o r m a t i o n from t h e l o w - m e l t i n g
metastable m o d i f i c a t i o n t o t h e high-me1 t i n g s t a b l e
form. The i n f r a r e d s p e c t r a of t h e two m o d i f i c a t i o n s
e x h i b i t some d i f f e r e n c e s , m a i n l y i n t h e 3500 cm'l
and t h e 1 5 0 0 - 1 7 0 0 cm'l r e g i o n .
D i f f e r e n t i a l t h e r m a l a n a l y s i s u s i n g a Mettler TA
2000 a p p a r a t u s was a p p l i e d f o r t h e i n v e s t i g a t i o n o f
b e n z o c a i n e p i c r a t e polymorphs. A p p r o x i m a t e l y 2 mg
o f t h e polymorphs were u s e d f o r e a c h a n a l y s i s . I n
F i g . 6 - 9 d i f f e r e n t i a l thermograms o f two polymor-
p h i c forms, o b t a i n e d under d i f f e r e n t c o n d i t i o n s a r e
g i v e n . The e n t h a l p h y c h a n g e 4 H f o r s t a b l e f o r m
( f o r m I ) was c a l c u l a t e d t o be 47.10 Cal g'l and
f o r t h e m e t a s t a b l e f o r m ( f o r m 11) 30.77 C a l 9-1.
5. I d e n t i f i c a t i o n and C o l o u r R e a c t i o n s .
B e n z o c a i n e g- i v e s c h a r a c t e r i s t i c r e a c t i o n s o f a n
a r o m a t i c amine. A f t e r d i s s o l v i n g i t i n water and
a d d i t i o n o f few d r o p s 3 N h y d r o c h l o r i c a c i d and
1 0 b sodium n i t r i t e f o l l o w e d b y 2 m l o f a s o l u -
t i o n o f 1 0 0 mg 2 - n a p h t o l i n 5 m l 1 N sodium hy-
d r o x i d e a n o r a n g e - r e d p r e c i p i t a t e i s formed ( 1 0 ) .
B e n z o c a i n e s o l u t i o n i n d i l u t e HC1 ( 2 0 mg i n 3 d r o p s
o f 2 M HC1) s p e a r a t e s on a d d i t i o n o f 1 m l water
and 5 d r o p s o f i o d i n e d a r k brown o i l d r o p s o f
per i o d i d e ( 1 8 ) . An i n t e n s e r e d - v i o l e t c o l o u r a t i o n
i s o b t a i n e d when 1 m l p h e n o l (1%) and 2 d r o p s o f
p o t a s s i u m b r o m a t e s o l u t i o n (0.1N) a r e added t o
b e n z o c a i n e s o l u t i o n i n d i l u t e HC1 ( 5 mg i n 2 m l
2 M HC1) ( 1 8 ) . B e n z o c a i n e g i v e s w i t h p i c r i c a c i d
s o l u t i o n a y e l l o w c r i s t a l l i n e p r e c i p i t a t e which
a f t e r washing w i t h water and d r y i n g a t 105OC
melts b e t w e e n 124-13OoC ( 1 9 ) . S a l t s o f b e n z o c a i n e
with v a r i o u s s u l f o n i c acids with d i f f e r e n t melting
p o i n t s a r e known. S a l t s w i t h p - t o l o u e n e s u l f o n i c
a c i d (m.P. : 185-187OC) , w i t h w - t o l u e n e s u l f o n i c
acid ( d e c o m p o s e s a t 235OC), w i t h b e n z e n e d i s u l -
f o n i c a c i d (decomposes a t 235OC), w i t h 2 and
4-phenol s u l f o n i c a c i d s r e s p e c t i v e l y (m.p. :
201-203O and 1 9 6 - 198OC), w i t h a n i s o l e s u l -
f o n i c a c i d (m.p. : 188OC) w i t h p h e n o l d i s u l f o n i c
a c i d (m.p. : 220 - 221OC) , w i t h 2 - o x y n a p h t h a l i n
s u l f o n i c a c i d and g u a j a c o l s u l f o n i c a c i d (m.p.
175OC) a r e known ( 2 0 ) .
BENZOCAINE 83
6. D e g r a d a t i o n and S t a b i l i t y . E s t e r h y d r o l i s e s of
b e n z o c a i n e i n a u u e o u s media i s well known ( 2 1 1 .
A t t e m p t s h a v e b e e n made t o decrease i t s d e g r a d a t i o n
t h r o u g h t h e use of c o m p l e x i n g a g e n t s and s u r f a c -
t a n t s . T h e a p p l i c a t i o n of m o l e c u l a r complex forma-
t i o n a s a means of d r u g s o l u b i l i z a t i o n and s t a b i l i -
s a t i o n h a s been s t u d i e d . C a f f e i n e i n i n c r e a s i n g
c o n c e n t r a t i o n s h a s b e e n used as complexing a g e n t t o
i n h i b i t the benzocaine d e g r a d a t i o n i n barium
h y d r o x i d e s o l u t i o n ( F i g . 1 0 ) (22,231. The i n f l u e n c e
of b e t a - c y c l o d e x t r i n on t h e base c a t a l y z e d d e g r a -
d a t i o n of b e n z o c a i n e h a s b e e n i n v e s t i g a t e d . K i n e t i c
d a t a p r e s e n t e d s u b s t a n t i a t e s t h e p o s t u l a t e d 1:l
s t o i c h i o m e t r i e r a t i o f o r t h e benzocaine-cyclodex-
t r i n i n t e r a c t i o n . T h i s shows t h a t t h e d e g r e e of
r e t a r d a t i o n of b e n z o c a i n e h y d r o l y s i s i s c o n s i d e -
r a b l e g r e a t e r than t h a t observed f o r t h e c a f f e i n e -
t y p e complexes i n d i c a t i n g a n i n t e r a c t i o n i n v o l v i n g
t h e i n c l u s i o n f o r m a t i o n and o t h e r a t t r a c t i v e f o r c e s
(24) ( F i g . 11 and 1 2 ) . C a t a l y s i s and i n h i b i t i o n of
t h e a l k a l i n e h y d r o l y s i s of b e n z o c a i n e and a n a l o g s
by c a t i o n i c s u r f a c t a n t s h a s a l s o b e e n r e p o r t e d (25).
S t a b i l i t y c h a r a c t e r i s t i c s of b e n z o c a i n e i n c l u d i n g
t h e e f f e c t s o f pH and p h o s p h a t e i o n s have b e e n
i n v e s t i g a t e d (26). B e n z o c a i n e d e g r a d a t i o n i s b o t h
a c i d and base c a t a l y z e d b u t is much s l o w e r i n t h e
p r e s e n c e of p h o s p h a t e i o n . S t a b i l i t y s t u d i e s were
p e r f o r m e d a t 25, 4 0 , 60 and 7OoC and a t pH v a l u e s
2, 7 and 11 (26). A t a l l t h r e e p H v a l u e s t h e d e g r a -
d a t i o n r a t e was s l o w e r i n 0.11 M p h o s p h a t e b u f f e r
t h a n i n water. T h u s b e n z o c a i n e was a p p r o x i m a t e l y
1 . 3 ( a t pH 7) 7.3 ( a t pH 2) and 8.0 ( a t pH 11)
times more s t a b l e i n p h o s p h a t e b u f f e r t h a n i n
n o n b u f f e r e d s o l u t i o n s of e q u i v a l e n t pH. E x t r a p o l a -
t i o n of t h e e l e v a t e d t e m p e r a t u r e d a t a t o room
temperature resul t e d i n a p r e d i c t e d f i r s t - o r d e r
r a t e c o n s t a n t of 0.0057 h r ' l , w h i c h compares
f a v o u r a b l y w i t h t h e r a t e c o n s t a n t o f 0.0051 h r - 1
o b s e r v e d a t room temperature. p-Aminobenzoic a c i d
was found t o be t h e o n l y d e c o m p o s i t i o n product.
E s s e n t i a l l y a l l t h e benzocaine l o s t due to degrada-
t i o n was a c c o u n t e d f o r b y t h e a p p e a r a n c e of p-amino-
b e n z o i c a c i d (26). B e n z o c a i n e i n a t h r o a t l o s z e n g e
f o r m u l a t i o n was f o u n d t o be u n s t a b l e w i t h v a r i o u s
e x c i p i e n t s . F r a c t i o n a l f a c t o r i a l experiments iden-
t i f i e d t h e e x c i p i e n t s c i t r i c a c i d , c o r n s y r u p and
SYED LAIK ALI
84
Fig. 10 I n f l u e n c e of i n c r e a s j n y c a f f e i n e -
c o n c e n t r a t i o n , 0 . 2 5 % (-0-1 ,
0.5% (-o-), 1.0% (-o-), 1.5%
(-m-), 2 . 0 % (-A-) and 2.5%
(-A-) o n t h e h y d r o l y s i s of
b e n z o c a i n e i n 0 . 0 4 n bar&um
h y d r o x i d e s o l u t i o n a t 30 C
L
c
C
2 4 6 8 10 2 4 6 0 10
Time Ch3 d l i m e Chi+
Fig. 1 1 Influence of beta Fig. 12 Influence of temperatures
cyclodextrin on benzocaine on benzocaine hydrolysks in 0.04N
hydro&ysis in 0.01N Ba(0H) Ba(OH)2, Key: A, with betacyclo-
at 30 C dex tr in :B, without bet acyc lodext P in
86 SYED LAIK ALI
n a t u r a l c h e r r y f l a v o u r a s t h e c a u s e s o f t h e incom-
p a t i b i l i t y . The p r i m a r y aromatic amine g r o u p i n g
i n s t e a d o f t h e e s t e r l i n k a g e o f b e n z o c a i n e was
involved i n t h e s t a b i l i t y problem ( 2 7 ) . I n a n o t h e r
i n v e s t i g a t i o n of 23 d i f f e r e n t b e n z o c a i n e f o r m u l a -
t i o n s some were 'found t o c o n t a i n n o t t h e l a b e l l e d
amount o f b e n z o c a i n e . p-Aminobenzoic a c i d c o u l d n o t
b e d e t e c t e d i n any o f t h e s u b s t a n d a r d b e n z o c a i n e
f o r m u l a t i o n s ( 2 8 ) . I n o n e of t h e s e f o r m u l a t i o n s a
compound h a v i n g t h e s t r u c t u r a l f o r m u l a
-CH=N COOC~HS
0
was i d e n t i f i e d t h r o u g h mass s p e c t r a a f t e r g a s
chromatographic s e p a r a t i o n (29) .
The i n f l u e n c e of a p r o t e c t i v e c o a t i n g o f b e n z o c a i n e
a s s u n s c r e e n i n g a g e n t upon t h e p h o t o s t a b i l i t y o f
dyes h a s been s t u d i e d . Colour s t a b i l i t y of t a b l e t s
c o a t e d w i t h c e r t i f i e d d y e s were t e s t e d . The p r o t e c -
t i v e e f f e c t o f t h e UV a b s o r b e r b e n z o c a i n e a p p e a r d
most e f f e c t i v e f o r F D and C y e l l o w no. 6 when
a p p l i e d a s t h e m o d i f i e d s u g a r c o a t or t h e f i l m c o a t
(30)
The e f f e c t o f p l a s t i c i z e r s on t h e i n t e r a c t i o n o f
PVC w i t h b e n z o c a i n e h a s b e e n i n v e s t i g a t e d (31).
R e s u l t s show t h a t d r u g p e r m e a b i l i t y i n c r e a s e s w i t h
p l a t i c i z e r c o n c e n t r a t i o n and t e m p e r a t u r e , w h i l s t
s o r p t i o n i n c r e a s e s with p l a s t i c i z e r c o n c e n t r a t i o n
and d e c r e a s e s w i t h t e m p e r a t u r e . The e f f e c t o f ATBC
( a c e t y l t r i - n - b u t y l c i t r a t e ) i s more p r o n o u n c e d
t h a n t h a t o f DEHP ( d i - 2 - e t h y l h e x y l p h t h a l a t e ) f o r
b o t h s o r p t i o n and p e r m e a t i o n p r o c e s s e s . The i n t e r -
a c t i o n o f PolyHEMA, a polymer w i d e l y u s e d i n t h e
m a n u f a c t u r e o f c o n t a c t l e n s e s , w i t h b e n z o c a i n e was
s t u d i e d a t 3OoC. b e n z o c a i n e was s o r p e d i n a r e v e r -
s i b l e manner and g a v e l i n e a r u p t a k e i s o t h e r m s (32).
The t r a n s p o r t of b e n z o c a i n e t h r o u g h a n y l o n 6
membrane was i n v e s t i g a t e d a s a f u n c t i o n of tempera-
t u r e ( 1 0 - 7OoC), c o n c e n t r a t i o n ( 0 . 6 , 1.1 and
6.0 x 10'3M), membrane a r e a and pH i n a s i m p l e
a l l - g l a s s p e r m e a t i o n c e l l . F i g . 1 3 shows t h e i n f l u -
e n c e of pH on t h e a p p a r e n t p e r m e a b i l i t y c o e f f i c i e n t
BENZOCAINE 87
c
t
n
I
ul
N
E
c!
I
Q
X
Y
PH -
Fig. 1 3 Influence of p H on t h e apparent
permeabflity coefficients for
6 x 1 0 M benzocaine trassfer across
a nylon 6 membrane at 50 C
f o r 6 x 10’3M b e n z o c a i n e t r a n s f e r a c r o s s a n y l o n
6 membrane ( 3 3 ) . The i n f l u e n c e o f p h a s e t r a n s i t i o n s
on t h e s o r p t i o n of b e n z o c a i n e b y p o l y a m i d e membra-
n e s h a s a l s o b e e n i n v e s t i g a t e d . The s o r p t i o n of
b e n z o c a i n e by non-or i e n t e d p o l y a m i d e membranes h a s
b e e n s t u d i e d a s ‘a f u n c t i o n o f temperature o v e r t h e
r a n g e 1 0 - 8OoC ( 3 4 ) .
7. D i s s o l u t i o n , L i b e r a t i o n and D i f f u s i o n
T h e i n c r e a s e d s o l u b i l i t y o f b e n z o c a i n e i n t h e pre-
s e n c e of v a r i o u s c o m p l e x i n g a g e n t s was a t t r i b u t e d
t o t h e f o r m a t i o n o f s o l u b l e c o m p l e x e s ( 3 5 ) . The
p o s s i b l e i n t e r a c t i o n s i n a mu1 t i c o m p o n e n t s y s t e m
c o n t a i n i n g b e n z o c a i n e , s o d i u m s a l i c y l a t e and PEG
300 were i n v e s t i g a t e d by d i s s o l u t i o n methods. D i s s o -
l u t i o n s t u d i e s i n d i c a t e t h e e x i s t e n c e of s y n e r g i s t i c
e f f e c t b e t w e e n sodium s a l i c y l a t e and PEG 300 on t h e
d i s s o l u t i o n r a t e of b e n z o c a i n e powder. The e f f e c t
is c o r r e l a t e d t o t h e s u r f a c e t e n s i o n measurement o f
sodium s a l i c y l a t e and PEG 300 s o l u t i o n s . D i s s o l u -
t i o n medium was s t i r r e d a t 7 5 rpm u s i n g a PTFE
20 40 60 20 40 60
Time L m i n J d Time Lminl-
Fig. 14 Effect of sodium salicylate on Fig. 15 Effect of PEG 300 o n

the dissolu&ion of benzocaine in the dissolution of bgnzo-
water at 37 C, 00.0% ; 0 1 . 0 % ; caine in water at 37 C,
A 2.0%; 0 5.0%;~10.0~/~. Key : see Fig. 14
BENZOCAINE 89
r e c t a n g u l a r b l a d e ( 4 . 5 cm by 1 . 7 cm) c o n n e c t e d by a
g l a s s rod ( d i a m e t e r : 6 mm) t o a c o n s t a n t s p e e d
motor. The r a t e o f d i s s o l u t i o n o f b e n z o c a i n e powder
is g r e a t l y i n c r e a s e d i n t h e p r e s e n c e o f e i t h e r
s o d i u m s a l i c y l a t e o r PEG 300. F i g . 1 4 and 1 5 i l l u -
s t r a t e t h e d i s s o l u t i o n p a t t e r n of b e n z o c a i n e ( 3 6 ) .
Spectral c h a n g e s observed i n b e n z o c a i n e - sodium
s a l i c y l a t e system have a l s o been r e p o r t e d ( 3 7 ) .
The i n v i t r o s t u d y o f d r u g r e l e a s e t h r o u g h s i l i -
c o n e r u b b e r membranes of b e n z o c a i n e s u s p e n d e d i n
carbomer h y d r o g e l s c o n t a i n i n g d i f f e r e n t concen-
t r a t i o n s o f low m o l e c u l a r w e i g h t p o l y o l s ( e t h y -
l e n e g l y c o l , p r o p y l e n e g l y c o l , g l y c e r o l and s o r -
b i t o l ) was s t u d i e d t o e s t a b l i s h g e n e r a l p r i n c i p -
l e s and p r o c e d u r e s f o r c o n t r o l o f t h e e f f e c t s o n
p e r c u t a n e o u s a b s o r p t i o n c a u s e d by c h a n g e s i n d r u g
s o l u b i l i t y and or d i f f u s i v i t y i n t h e v e h i c l e . Benzo-
c a i n e was s e l e c t e d a s a model o f n e u t r a l d r u g w i t h
low water s o l u b i l i t y . The e f f e c t o f t h e a d d i t i v e s
on t h e r e l e a s e is e x p r e s s e d i n terms o f t h e r e l a -
t i v e r e l e a s e d amount, i.e. t h e r a t i o of t h e amount
of drug r e l e a s e d from each a d d i t i v e - c o n t a i n i n g g e l
t o t h e amount released f r o m e a c h a d d i t i v e - f r e e g e l .
The e x p e r i m e n t a l v a l u e s a r e c o r r e l a t e d w i t h v a l u e s
c a l c u l a t e d by a s i m p l e e q u a t i o n i n v o l v i n g known
measurable parameters such a s t h e drug concentra-
t i o n i n t h e g e l , t h e d r u g s o l u b i l i t y i n t h e pure
l i q u i d p h a s e and t h e v i s c o s i t y o f t h i s p h a s e ( 3 8 ) .
The release of b e n z o c a i n e f r o m s u p p o s i t o r i e s h a s
b e e n i n v e s t i g a t e d u s i n g a c o n t i n u o u s Flow Bead-Bed
D i s s o l u t i o n A p p a r a t u s . B e n z o c a i n e was s e l e c t e d a s a
model compound w i t h b o t h a low m e l t i n g (33.5 - 35.5%)
and a h i g h m e l t i n g ( 3 7 - 39%) g l y c e r i d e - t y p e
s u p p o s i t o r y b a s e . I n v i t r o release of b e n z o c a i n e
d e c r e a s e d as t h e m e l t i n g p o i n t o f t h e s u p p o s i t o r y
i n c r e a s e d . The a p p a r a t u s c o n s i s t e d o f a g l a s s b e a d -
bed c o n t a i n i n g t h e s u p p o s i t o r y . A c o n t i n o u s f l o w o f
l i q u i d was p a s s e d t h r o u g h t h e bead-bed a t a c o n -
s t a n t r a t e . Direct c o n t a c t o f t h e s u p p o s i t o r y was
m a i n t a i n e d w i t h t h e d i s s o l u t i o n medium, c o n f i n i n g
t h e s u p p o s i t o r y w i t h i n t h e b e a d s . Drug release was
a f f e c t e d by t h e t e m p e r a t u r e o f t h e d i s s o l u t i o n
media, i n c r e a s i n g , d e c r e a s i n g a n d i n c r e a s i n g a g a i n
a t c e r t a i n t e m p e r a t u r e s . Dependence o f b e n z o c a i n e
release f r o m s u p p o s i t o r y b a s e a s a f u n c t i o n o f
90 SYED LAIK ALI
t e m p e r a t u r e was d e m o n s t r a t e d ( 3 9 ) ( F i g . 1 6 ) .
The r e l e a s e of b e n z o c a i n e from o i n t m e n t b a s e s and
t h e i n f l u e n c e of e m u l g a t i n g a g e n t s s u c h a s Span,
Tween and o t h e r l i p o p h i l i c a g e n t s , s u c h a s propy-
l e n e g l y c o l m o n o l a u r a t e and s o r b i t a n m o n o l a u r a t e
h a s been s t u d i e d ( 4 0 , 4 1 , 4 2 , 4 3 , 4 4 ) . D i s s o l u t i o n
s t u d i e s c o n d u c t e d i n m i c e l l a r s o l u t i o n c l e a r l y show
t h a t d i s s o l u t i o n r a t e is n o t p r o p o r t i o n a l t o t h e
a p p a r e n t s o l u b i l i t y of t h e d r u g . T h i s was f i r s t
d e m o n s t r a t e d by H i g u c h i ( 4 5 ) who found t h a t t h e
r a t i o of d i s s o l u t i o n of b e n z o c a i n e i n p o l y s o r b a t e
80 s o l u t i o n t o t h a t w i t h o u t t h e s u r f a c t a n t was
s u b s t a n t i a l l y lower t h a n t h e r a t i o p r e d i c t e d by t h e
Noyes-Whitney t h e o r y . Mechanism of s u r f a c t a n t
e f f e c t s on t h e drug a b s o r p t i o n h a s been r e p r o t e d i n
a review a r t i c l e ( 4 6 ) .
The d i f f u s i o n , p e n e t r a t i o n and s u r f a c e e f f e c t s o f
benzocaine i n c o r p o r a t e d i n v a r i o u s polyethylene
g l y c o l o i n t m e n t b a s e s t h r o u g h human s t r a t u m corneum
h a s been s t u d i e d . R e s u l t s showed a d e c r e a s e i n d r u g
d i f f u s i o n i n t h e p r e s e n c e of r e l a t i v e l y h i g h amounts
of t h e l o w e r m o l e c u l a r weight p o r t i o n s of p o l y e t h y -
Fig. 16 Dependence of benzocaine release from

suppositories as a funchion of
tegperatur8, Key : A 45,; 40°;
039 ; 37 ; and A 33 ;
BENZOCAINE 91
l e n e g l y c o l . Thermal a n a l y s i s i n d i c a t e d t h a t benzo-
c a i n e d i s s o l v e s i n p o l y e t h y l e n e g l y c o l . T h i s would
imply an a l t e r a t i o n of t h e r e l e a s e - p a t t e r n i f t h e
p o l y e t h y l e n e g l y c o l c o m p o s i t i o n is a1 tered ( 4 7 ) .
The i n f l u e n c e of f o r m u l a t i o n , m a n u f a c t u r i n g v a r i -
a b l e s and t e m p e r a t u r e on i n v i t r o r e l e a s e of benzo-
c a i n e d i s s o l v e d i n g e l - t y p e o i l y v e h i c l e s h a s been
repor ted (48) .
The t r a n s p o r t of benzocaine along w i t h o t h e r
p-aminobenzoate e s t e r s t h r o u g h a t u b u l a r d i m e t h y l
p o l y s i l o x a n e membrane i n t o a f l o w i n g l i q u i d was
examined. T h e o b s e r v e d t r a n s p o r t b e h a v i o u r r a n g e d
from c o m p l e t e c o n v e c t i v e d i f f u s i o n c o n t r o l f o r t h e
h e x y l ester t o c o m p l e t e membrane c o n t r o l f o r benzo-
c a i n e . The i m p l i c a t i o n s of convectiv e d i f f u s i o n a l
c o n s i d e r a t i o n s t o i n t e s t i n a l a b s o r p t i o n and d i s s o -
l u t i o n s t u d i e s have been d i s c u s s e d ( 4 9 ) .
The i n v i t r o r e l e a s e of b e n z o c a i n e from o l e a g i n o u s ,
a b s o r p t i o n , e m u l s i o n and w a t e r s o l u b l e o i n t m e n t
v e h i c l e s t h r o u g h a c e l l u l o s e membrane t o a n a q u e o u s
s i n k was s t u d i e d a t 37.5OC and was d e m o n s t r a t e d
t h a t v e h i c l e c o m p o s i t i o n markedly a f f e c t e d t h e r a t e
of r e l e a s e of t h e a c t i v e i n g r e d i e n t ( S O ) . The
e f f e c t of s u p p o s i t o r y v e h i c l e , drug c o n c e n t r a t i o n
and n o n i o n i c s u r f a c t a n t s on i n v i t r o d i a l y s i s
t h r o u g h a c e l l u l o s e membrane were s t u d i e d . I n v i t r o
d i a l y s i s correlated w e l l with i n vivo absorption
and d r u g r e l e a s e was g r e a t e r from w a t e r - s o l u b l e
v e h i c l e s t h a n from o l e a g n i o u s v e h i c l e s . I n c l u s i o n
of a n o n i o n i c h y d r o p h i l i c o r l i p o p h i l i c s u r f a c t a n t
( s o r b i t a n m o n o l a u r a t e , p o l y s o r b a t e 80) i n c o c a b u t -
ter resulted i n a s t a t i s t i c a l l y s i g n i f i c a n t i n c r e a s e
f o r i n v i t r o drug r e l e a s e , while a l i p o p h i l i c sur-
f a c t a n t showed l i t t l e e f f e c t i n v i v o and a hydro-
p h i l i c s u r f a c t a n t depressed r e l e a s e i n v i v o . Both
t y p e s of s u r f a c t a n t s had s m a l l e f f e c t s on r e l e a s e
from p o l y e t h y l e n e g y l c o l . Some f o r m u l a t i o n s f a c t o r
o t h e r t h a n t h e c o n c e n t r a t i o n of b e n z o c a i n e a f f e c t e d
t h e r e l a t i v e amounts of b e n z o c a i n e r e l e a s e d i n
v i t r o from t h e c o m m e r c i a l l y a v a i l a b l e p r o d u c t s
examined. The d i a l y s i s method is u s e f u l f o r e v a l u -
a t i n g t h e e f f e c t s of f o r m u l a t i o n on d r u g r e l e a s e
from s u p p o s i t o r i e s (51).
92 SYED LAIK ALI
8. Method o f A n a l y s i s
Titrimetry. T i t r a t i o n of benzocaine d i s s o l v e d
i n h y d r o c h l o r i c a c i d w i t h sodium n i t r i t e s o l u t i o n
using t h e s t a r c h - i o d i d e paper a s an e x t e r n a l i n d i -
c a t o r is t h e a s s a y method used i n U n i t e d S t a t e s
Pharmacopea (10)'. E n d p o i n t c a n a l s o be d e t e r m i n e d
p o t e n t i o me t r i c a l l y a p p l y i ng a c a l o me1 - p l a t i n u m
e l e c t r o d e system. I n assay methods o f BP 8 0 (52)
and e u r o p e a n pharmacopoea (53) b e n z o c a i n e is d i s s o l -
ved i n d i l u t e H C l , a b o u t 3 g p o t a s s i u m bromide
a d d e d , s o l u t i o n c o o l e d w i t h ice and t h e n t i t r a t e d
a m p e r o m e t r i c a l l y w i t h 0.1 M sodium n i t r i t e s o l u t i o n
u s i n g a d o u b l e p l a t i n u m wire e l e c t r o d e . B r o m i n a t i o n
o f b e n z o c a i n e i n a c e t i c a c i d s o l u t i o n u s i n g a mix-
t u r e of p o t a s s i u m bromide and 1 N potassium b r o m a t e
i n d i l u t e HC1 i s p e r f o r m e d a s a n i n d i r e c t a s s a y
method o f b e n z o c a i n e . On a d d i t i o n o f p o t a s s i u m
i o d i d e t h e e x c e s s of l i b e r a t e d bromine r e a c t s tr,
g i v e f r e e i o d i n e w h i c h is b a c k t i t r a t e d w i t h a s t a n -
d a r d t h i o s u l p h a t e s o l u t i o n . B e n z o c a i n e c a n be t h u s
e v a l u a t e d i n d i r e c t l y t h r o u g h t h e amount of s t a n d a r d
p o t a s s i u m bromate consumed (54). B e n z o c a i n e c a n
d i r e c t l y be t i t r a t e d w i t h p o t a s s i u m b r o m a t e a f t e r
d i s s o l v i n g i t i n 25 % H C l , a d d i n g potassium b r o m i d e
and u s i n g methyl red a s a n i n d i c a t o r (55) o r ampero-
m e t r i c a l l y u s i n g a d o u b l e p l a t i n u m e l e c t r o d e (56).
A non-aqueous t i t r a t i o n of b e n z o c a i n e w i t h
p e r c h l o r i c a c i d u s i n g c r y s t a l v i o l e t a s an i n d i -
c a t o r is a l s o known (57).
C o l o r i m e t r i c A n a l y s i s . A c o l o r i m e t r i c method f o r
t h e d e t e r m i n a t i o n of s u b s t a n c e s c o n t a i n i n g a
p r i m a r y aromatic m i n e s u c h a s b e n z o c a i n e i s based
upon d i a z o t i s a t i o n , c o u p l i n g w i t h N ( l - n a p h t y l )
e t h y l e n e d i a m i n e h y d r o c h l o r i d e and measuring a t 545
nm (58,59,60,61). O t h e r c o u p l i n g a g e n t s r e p o r t e d i n
l i t e r a t u r e a r e o( - n a p h t y l a m i n e thymol i n a l k a l i n e
s o l u t i o n (63) and 2 - n a p h t o l (61). C o l o r i m e t r i c
d e t e r m i n a t i o n of b e n z o c a i n e is a l s o p e r f o r m e d a f t e r
r e a c t i o n w i t h p-dimethylaminobenzaldehyde (65) A
c o l o r i m e t r i c d e t e r m i n a t i o n of benzocaine between
.
505-520 nm a f t e r r e a c t i n g w i t h h y d r o x y l a m i n e and
i r o n (111) s a l t s i s a l s o known (66). R e a c t i o n w i t h
sodium 1,2-naphthochinon-4-sulfonate and c o l o r i -
metric d e t e r m i n a t i o n b e t w e e n 460-480 nm is a l s o
r e p o r t e d (67,68). C o l o r i m e t r i c d e t e r m i n a t i o n s o f
BENZOCAINE 93
b e n z o c a i n e a f t e r r e a c t i o n w i t h : v a n i l l i n e and measu-
r i n g b e t w e e n 380-410 nm ( 6 9 1 , w i t h 9 - c h l o r o a c r i d i n e
and m e a s u r i n g a t 435 ( 7 0 1 , w i t h n i t r o u s a c i d and
m e a s u r i n g b e t w e e n 390-430 nm a r e known ( 7 1 , 7 2 ) . Use
o f Bindon 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 o f
benzocaine has a l s o been r e p o r t e d ( 7 3 ) .
Fluorimetry. Local a n e s t h e t i c s c o n t a i n i n g a
p r i m a r y a r o m a t i c amino g r o u p react w i t h 9 - c h l o r o c r i -
d i n e t o y i e l d a m i n o a c r i d i n e h y d r o c h l o r i d e s . The f o r -
mation of t h e s e d e r i v a t i v e s r e s u l t i n quenching of
fluorescence of the 9-chloracridine reagent solu-
t i o n . M o n i t o r i n g t h e f l u o r e s c e n c e a t a c t i v a t i o n and
e m i s s i o n w a v e l e n g t h s o f 385 and 4 2 0 nm r e s p e c t i v e l y
e n a b l e s to a n a l y s e drug i n t h e - 10-6M
r a n g e . An e t h a n o l i c s o l u t i o n o f b e n z o c a i n e i s
a d j u s t e d t o pH4.0 and acridine-tetrahydrofurane
s o l u t i o n e q u i v a l e n t t o a n a p p r o x i m a t e one t o f o u r -
f o l d molar e x c e s s is a d d e d . The m i x t u r e is h e a t e d
a t 600 f o r 1 0 min, c o o l e d and t h e d e c r e a s e i n t h e
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 r e a c t i o n m i x t u r e i s
compared t o t h e r e a g e n t b l a n k . The method d i f f e r s
from o t h e r w i d e l y used t e c h n i q u e s i n t h a t i t i n v o l -
v e s quenching of f l u o r e s c e n c e rather than increased
fluorescence i n t e n s i t y as t h e b a s i s f o r t h e f l u o r i -
.
metric m e a s u r e m e n t s ( 7 4 ) Dambrowski and P r a t t ( 7 5 )
i n t r o d u c e d t h e u s e o f 2 , 6 - d i a m i n o p y r i d i n e a s a new
f l u o r e s c e n c e r e a g e n t and showed t h a t t h e s e n s i t i -
v i t y was i n t h e r a n g e of - 10'7M f o r benzo-
caine (75).
Phosphorimetry. Room-temperature phosphoresence
has been r e p o r t e d t o be u s e f u l f o r a n a l y t i c a l d e t e r -
m i n a t i o n o f v a r i o u s d r u g s . B e n z o c a i n e d i d n o t show
r o o m - t e m p e r a t u r e p h o s p h o r e s c e n c e when a d s o r b e d on
s o d i u m a c e t a t e . However i t was f o u n d t h a t b e n z o c a i n e
c o u l d be r e a d i l y h y d r o l y z e d by r e f l u x i n g i n d i l u t e
a c i d . The h y d r o l y s e d s o l u t i o n was s u b s e q u e n t l y
n e u t r a l i s e d w i t h aqueous a l k a l i s . Small a m o u n t s o f
t h i s s o l u t i o n when a p p l i e d to sodium a c e t a t e and
e v a p o r a t e d t o d r y n e s s showed t h e c h a r a c t e r i s t i c
p - a m i n o b e n z o i c a c i d p h o s p h o r e s c e n c e on e x c i t a t i o n
w i t h a UV lamp. Q u a n t i t a t i v e d a t a h a s n o t b e e n
gathered (76).
94 SYED LAIK ALI
Chroma t o g r a ph i c Method s
Sample P r e p a r a t i o n . Aqueous s o l u t i o n s c a n b e
a p p l i e d d i r e c t l y ( 7 7 ) o r t h e s o l u t i o n is f i r s t o f
a l l made a l k a l i n e w i t h NaOH and t h e l o c a l a n e s t h e t i c
t h e n e x t r a c t e d w i t h ether ( 7 8 ) . O i l s o l u t i o n s ,
s u p p o s i t o r i e s or o i n t m e n t a r e d i s s o l v e d f i r s t o f
a l l i n l i g h t p e t r o l e u m and t h e n e x t r a c t e d w i t h 1 N
HC1. The HC1 e x t r a c t is made a l k a l i n e and t h e n reex-
t r a c t e d w i t h c h l o r o f o r m . With some o i n t m e n t s i t i s
more a d v a n t a g e o u s t o d i s s o l v e t h e o i n t m e n t i n
b e n z e n e ( 7 2 ) . T a b l e t s c a n be e x t r a c t e d d i r e c t l y
w i t h m e t h a n o l a f t e r c r u s h i n g ( 7 7 ) . Drug f o r m u l a -
t i o n s a r e d i s p e r s e d i n 1%HC1, f i l t e r e d , f i l t r a t e
made a l k a l i n e w i t h ammonia, s o l u t i o n e x t r a c t e d w i t h
c h l o r o f o r m and t h e n r e e x t r a c t e d w i t h HC1 ( 7 9 ) .
P a p e r Chromatography. B e n z o c a i n e c a n be s e p a r a t e d
o n p a p e r , i m p r e g n a t e d w i t h formamide, u s i n g b e n z e n e
as m o b i l e p h a s e ( 7 7 ) . The s o l v e n t s y s t e m n - b u t a n o l -
a c e t i c a c i d - w a t e r , 4:1:5 ( 8 0 ) is a l s o w i d e l y u s e d .
Better results, p a r t i c u l a r l y f o r t h e s e p a r a t i o n of
decomposition products can be achieved i n s o l v e n t
s y s t e m c o n t a i n i n g n - b u t a n o l - HC1-water, 60:10:75
( 7 9 ) . I n p a p e r c h r o m a t o g r a p h y w i t h fromamide a s
s t a t i o n a r y p h a s e a l l s u b s t a n c e s w i t h a f r e e amino
group f l u o r e s c e i n UV-light a f t e r t h e chromatogram
h a s b e e n d r i e d a t 105OC.
T h i n l a y e r Chromatography. S i l i c a g e l G t h i n l a y e r
p l a t e s p r e p a r e d i n 0 . 1 N NaOH (811, s i l i c a g e l G F
254 p l a t e s ( 2 8 ) and a l u m i n a p l a t e s ( 8 2 ) h a v e b e e n
used f o r b e n z o c a i n e s e p a r a t i o n . The b e h a v i o u r o f
b e n z o c a i n e and i t s f u r t h e r homologues h a s a l s o b e e n
i n v e s t i g a t e d through p a r t i t i o n chromatography using
c e l l u s l o s e powder i m p r e g n a t e d w i t h o l e y l a l c o l h o l
and a q u e o u s b u f f e r s o l u t i o n s a s m o b i l e p h a s e s ( 8 3 ) .
Under t h e s e c o n d i t i o n s a n i n t e r e s t i n g r e l a t i o n s h i p
b e t w e e n t h e pH v a l u e o f t h e m o b i l e p h a s e and t h e
m o b i l i t y was d e r i v e d . Cyclohexane-benzene-diethyl-
amine, 75:15:10 ( 8 1 ) , b e n z e n e - e t h a n o l , 95:5 ( 8 0 )
and t o l u e n e - e t h a n o l , 95:s ( 2 8 ) h a v e b e e n u s e d a s
m o b i l e p h a s e s f o r t h e s e p a r a t i o n of b e n z o c a i n e .
Roeder and c o w o r k e r s ( 8 4 ) h a v e used mixtures o f
d i f f e r e n t homogeneous a z e o t r o p i c s o l v e n t s f o r t h e
t h i n - l a y e r chromatographic s e p a r a t i o n of benzocaine
from o t h e r l o c a l a n e s t h e t i c s . Chloroform-methanol,
BENZOCAINE 95
80 : 1 0 and e t h a n o l a s m o b i l e p h a s e s and s i l i c a g e l
t h i n l a y e r p l a t e s t r e a t e d w i t h 0.1 N NaOH h a v e b e e n
used f o r t h e s e p a r a t i o n o f b e n z o c a i n e f r o m o t h e r
l o c a l a n e s t h e t i c s ( 4 5 ) . Fuwa ( 8 8 ) r e p o r t e d t h e u s e
o f t h e s i l i c a g e l TLC p l a t e s and b e n z e n e - a c e t o n e -
ammonia, 80:20:T a s m o b i l e p h a s e f o r TLC s e p a r a -
t i o n . A TLC s y s t e m t h a t s e p a r a t e s b e n z o c a i n e f r o m
its h y d r o l y s i s product p-aminobenzoic acid h a s been
r e p o r t e d ( 8 7 ) . S i l i c a g e l TLC p l a t e s and a s o l v e n t
s y s t e m o f b e n z e n e - d i o x a n e - a c e t i c a c i d , 90:75:8
were a p p l i e d ( 8 7 ) .
D e t e c t i o n c a n b e made by q u e n c h i n g i n s h o r t wave-
l e n g t h UV l i g h t , a f t e r s p r a y i n g w i t h E h r l i c h ' s
r e a g e n t which g i v e s y e l l o w s p o t s ( 7 7 ) , d i a z o t i -
s a t i o n f o l l o w e d by c o u p l i n g w i t h & - n a p h t o l ( 8 0 )
and s p r a y i n g w i t h i o d i n e - p o t a s s i u m i o d i d e s o l u -
tion (82).
Q u a n t i t a t i v e 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 benzo-
c a i n e a t 285 nm a f t e r s e p a r a t i o n on t h i n l a y e r
p a l t e s h a s been r e p o r t e d ( 2 8 ) . Benzocaine h a s a l s o
b e e n d e t e r m i n e d a f t e r TLC s e p a r a t i o n by r e a c t i n g i t
w i t h d a n s y l c h l o r i d e r e a g e n t and t h e s u b s e q u e n t
d e t e r m i n a t i o n of f l u o r e s c e n c e i n t e n s i t y of a d d u c t s
t h r o u g h T L C - F l u o r i m e t r y ( 8 8 ) . Microgram q u a n t i t i e s
o f b e n z o c a i n e were d e t e r m i n e d t h r o u g h t h i s method.
R f - v a l u e s o f f l u o r e s c i n g a d d u c t s on d i f f e r e n t TLC
p l a t e s a r e r e p o r t e d ( 8 8 ) . TLC s e p a r a t i o n and s p e c -
t r o p h o t o m e t r i c d e t e r m i n a t i o n o f p r o c a i n e and b e n z o -
c a i n e i n v a r i o u s pharmaceutical p r e p a r a t i o n s h a s
a l s o been performed ( 8 9 , 9 0 ) .
High P e r f o r m a n c e L i q u i d C h r o m a t o g r a p h y .
B e n z o c a i n e and a n t i p y r i n e i n e a r d r o p s h a v e b e e n
s e p a r a t e d and d e t e r m i n e d t h r o u g h HPLC on /((-Ban-
d a p a c k C 1 8 column (Waters) u s i n g a m o b i l e p h a s e o f
0 . 0 2 M KH2P04 i n water c o n t a i n i n g 408 m e t h a n o l .
D e t e c t i o n was d o n e a t 254 nm ( 9 1 ) . HPLC h a s a l s o
been a p p l i e d f o r t h e s i m u l t a n e o u s a s s a y of benzo-
c a i n e and i t s h y d r o l y s i s p r o d u c t p - a m i n o b e n z o i c
a c i d . A C 1 8 m i c r o p a r t i c u l a t e column w i t h a m o b i l e
phase of methanol - a c e t i c a c i d - water, 33:4:63
were u s e d a t a m b i e n t t e m p e r a t u r e w i t h a f l o w r a t e
o f 2 ml/min and d e t e c t i o n w a v e l e n g t h 254 nm. The
r e t e n t i o n times f o r p - a m i n o b e n z o i c a c i d and b e n z o -
96 SYED LAIK ALI
c a i n e were r e p o r t e d t o be 2 min 28 sec and 7 min 1 0

sec r e s p e c t i v e l y . The s e n s i t i v i t y i s g i v e n a s
6-'
7 g/ml f o r b e n z o c a i n e and 0.3 g/ml f o r p-amino-
b"
b n z o i c a c i d f o r an i n j e c t e d v l u m e o f 1 0 1. The
s e n s i t i v e t y c a n be i n c r e a s e d f o u r f o l d i f 6"
detec-
t i o n wavelength o f 294 nm i s used ( 2 6 ) . A l i (28)
h a s g i v e n two modes of l i q u i d chromatography, ad-
s o r p t i o n and r e s e r v e d - p h a s e systems f o r t h e HPLC
a n a l y s i s of b e n z o c a i n e . I n a d s o r p t i o n mode a S i
60,25 cm column w i t h a mobiel p h a s e n-hexane-
c h l o r o f o r m , 30:70 a t a wavelength of 254 nm were
used. A RP-18, l o p column (Knauer) and a m o b i l e
phase of methanol-water-0.1N HC1, 54:36:10 were
used i n r e v e r s e d - p h a s e s y s t e m a t a d e t e c t i o n wave-
l e n g t h of 285 nm. F l o w - r a t e i n b o t h s y s t e m s was
k e p t a t 1 . 5 ml/min. Numerous b e n z o c a i n e f o r m u l a -
t i o n s i n d i f f e r e n t d o s a g e forms were assayed
through t h e s e two HPLC s y s t e m s ( 2 8 ) .
Gas-Liquid -Chr oma toq raphy
Ebel ( 9 2 ) h a s g i v e n a l i s t of d i f f e r e n t s t a t i o n a r y
p h a s e s and g a s - c h r o m a t o g r a p h i c c o n d i t i o n s f o r t h e -
s e p a r a t i o n o f s e v e r a l l o c a l a n e s t h e t i c compounds.
A l i (28) h a s d e t e r m i n e d b e n z o c a i n e t h r o u g h g a s
chromatography on a 3% SE 30 on V a r a p o r t column
w i t h a FID d e t e c t o r . 15OOC oven t e m p e r a t u r e i s o -
therm, 240 and 27OOC f o r i n j e c t i o n p o r t and
d e t e c t o r b l o c k t e m p e r a t u r e s r e s p e c t i v e l y were
used. G a s - l i q u i d chromatography of l o c a l a n e s t h e -
t i c s and r e l a t e d compounds on F l e x o l Chromosorb W
and S E 30 on g l a s s bead columns is a l s o r e p o r t e d
( 9 3 ) . F l e x o l on Chromosorb W column was p r e f e r r e d
f o r t h e l o w e r m e l t i n g compounds s u c h a s t h e amino-
c a l c o h o l s , w h i l e a low l o a d SE 30 on g l a s s - b e a d
column was p e r f e r r e d f o r h i g h e r m e l t i n g l o c a l
a n e s t h e t i c b a s e s and s a l t s . I d e n t i f i c a t i o n and
d e t e r m i n a t i o n of b e n z o c a i n e i n cosmetics h a s a l s o
been r e p o r t e d ( 9 3 a ) . A column packed w i t h 1 . 2 5 b
OV 1 7 + 1 . 2 5 b OV 25 on v a r a p o r t 30, 100-120 mesh
was used. The oven t e m p e r a t u r e programme p r o c e e d e d
from 16OoC t o 23OoC, a t r a t e of 8O/min; t h e
i n j e c t i o n and d e t e c t o r t e m p e r a t u r e s were a d j u s t e d
t o 25OoC and 3OO0C r e s p e c t i v l y ( 9 3 a ) .
BENZOCAINE 97
9. Drug Me t ab o l i s m and P h a r m a c o k i n e t i c s
An i n s i t u c o r n e a l p e r f u s i o n s y s t e m which e n a b l e s
q u a n t i t a t i o n of c o r n e a l d r u g u p t a k e h a s been u s e d
t o e v a l u a t e t h e u p t a k e o f b e n z o c a i n e i n r a b b i t s . By
determining t h e d e c l i n e i n drug c o n c e n t r a t i o n i n
t h e p e r f u s i o n a p p a r a t u s a s a f u n c t i o n o f time i t
was p o s s i b l e t o measure d r u g c l e a r a n c e from t h e
s y s t e m , t h e r e b y o b t a i n i n g a measure o f c o r n e a l d r u g
u p t a k e . Using t h i s method t h e c l e a r a n c e o f benzo-
c a i n e from t h e s y s t e m by r a b b i t c o r n e a s was exami-
ned. T h e i n s i t u p e r f u s i o n s y s t e m p r o v i d e s r e p r o -
d u c i b l e r e s u l t s and o b v i a t e s many o f t h e p r o b l e m s
associated w i t h isolated i n v i t r o corneal studies.
T h i s method c a n be u t i l i z e d t o s t u d y t h e e f f e c t of
p h y s i c o c h e m i c a l d r u g p r o p e r t i e s on c o r n e a l u p t a k e
a s well a s i n o c u l a r p h a r m a c o k i n e t i c modeling and
dosage form e v a l u a t i o n ( 9 4 ) .
E f f e c t s o f a n e s t h e t i c s l i k e b e n z o c a i n e etc. on
s u l f a t e t r a n s p o r t i n t h e red c e l l have been i n v e s t i -
gated. Experiments suggested t h a t a n e s t h e t i c s a c t
t h r o u g h a g e n e r a l mechanism and t h a t t h e s u l f a t e
t r a n s p o r t i n t h e red b l o o d c e l l i s a good model f o r
s t u d y i n g t h e mechanism of a c t i o n of a n e s t h e t i c s
(95)
Transport of benzocaine a c r o s s chromatophore has
b e e n s t u d i e d . T r a n s p o r t of u n p r o t o n a t e d l o c a l
a n e s t h e t i c , although electrically n e u t r a l , requires
t h e p r e s e n c e o f a membrane p o t e n t i a l ( 9 6 ) . Absorp-
t i o n of 3 H - l a b e l l e d b e n z o c a i n e from o i n t m e n t s
f o l l o w i n g a d m i n i s t r a t i o n i n r a t s h a s been s t u d i e d .
T o t a l r a d i o a c t i v i t y i n t h e blood of r a t s f o r f i v e
h o u r s f o l l o w i n g r e c t a l a d m i n i s t r a t i o n of 3H-ben-
z o c a i n e i n o l e a g e n o u s , a b s o r p t i o n e m u l s i o n (water
i n o i l and o i l - i n - w a t e r ) a n d water s o l u b l e o i n t m e n t
v e h i c l e s was measured. The release was g r e a t e s t
from t h e w a t e r - s o l u b l e v e h i c l e and f o l l o w e d t h e
same r e l a t i v e o r d e r a s i n a n i n v i t r o e x p e r i m e n t
( F i g . 1 7 ) ( 9 7 ) . No i n t a c t b e n z o c a i n e was found i n
t h e blood using radiochromatography. I n v i t r o hydro-
l y s i s o f b e n z o c a i n e by r a t b l o o d d i d n o t o c c u r . T h e
r a t e o f a p p e a r a n c e and amount of d r u g i n t h e blood
a f t e r r e c t a l a p p l i c a t i o n s h o u l d be a u s e f u l measure
of t h e r a t e and amount of d r u g t r a n s f e r from a
dosage form t o t h e s e n s o r y nerve e n d i n g s i n t h e
r e c t a l mucosa ( 9 7 ) . The a b s o r p t i o n of d e t e c t a b l e
SYED LAIK ALI
98
1 a 3 4 6
HOURS
Fig. 17 Blood radioactivity3after the

application of 20% H-benzocaine in
different ointment vehicles. Key :
1, white petroleum; 2, absorption
vehicle; 3, water-in-oil emulsion;
4, oil-in-water emulsion; 5, poly-
ethylene glycol. The standard error
of the mean is shown on one side of
each point.
BENZOCAINE 99
amounts of b e n z o c a i n e a f t e r a p p l i c a t i o n t o abroded
abdominal s k i n i n dogs and t h e a b i l i t y o f v a r i o u s
b e n z o c a i n e c o n t a i n i n g p r e p a r a t i o n s t o obtuned t h e
i t c h and b u r n i n g e l e c t r i c a l s t i m u l a t i o n i n humans
h a v e been r e p o r t e d ( 9 8 , 9 9 ) . A b s o r p t i o n of some
l o c a l a n e s t h e t i c s h a s been r e p o r t e d t o b e d e p e n d e n t
on t h e mucous s u r f a c e ( 9 8 ) . B e n z o c a i n e is c l e a r l y
degraded i n v i v o , however t h e d e g r a d a t i o n occurs
p r o b a b l y i n t h e l i v e r ( 1 0 0 ) . Rectal a d m i n i s t r a t i o n
of t h e same c o n c e n t r a t i o n of 3H-benzocaine i n t h e
same v e h i c l e t o male and f e m a l e r a t s r e s u l t s i n a
lower blood r a d i o a c t i v i t y v e r s u s time c u r v e s f o r
t h e male r a t s (51). No m e t a b o l i c s t u d i e s o f benzo-
c a i n e have been r e p o r t e d . M e t a b o l i c s t u d i e s of amino
a c i d d e r i v a t i v e s of b e n z o c a i n e r e v e a l e d t h e p o s s i b i -
l i t y of r a p i d h y d r o l y s i s o f t h e d e r i v a t i v e s t o
b e n z o c a i n e by t i s s u e s and plasma h y d r o l y z i n g
enzymes. They a r e c o n s i d e r e d a s p r o d r u g s (101).
C l i n i c a l p h a r m a c o k i n e t i c s of s e v e r a l l o c a l a n e s t h e -
t i c s h a s been examined by T u c k e r ( 1 0 2 ) . Blood con-
c e n t r a t i o n s of l o c a l a n e s t h e t i c s a f t e r p e r i n e u r a l
i n j e c t i o n s a r e n o t c l o s e l y r e l a t e d t o age, weight
or pregnancy b u t may be i n f l u e n c e d by d i s e a s e s
a s s o c i a t e d w i t h haemodynamic c h a n g e s and by o t h e r
d r u g s g i v e n a t o r around t h e time o f r e g i o n a l
b l o c k a d e ( 1 0 2 ) . The a b s o r p t i o n and u r i n a r y excre-
t i o n of b e n z o c a i n e were i n v e s t i g a t e d i n r a t s by t h e
method of mass fragmentography. The i n t a c t benzo-
c a i n e was found s l i g h t l y i n t h e serum a f t e r o r a l
d o s e . p-Aminobenzoic a c i d , h a y d r o l y z e d p r o d u c t of
b e n z o c a i n e r a p i d l y r e a c h e d t o t h e maximum c o n c e n -
t r a t i o n a t 1 0 min, t h e n soon d i s a p p e a r e d from t h e
serum ( 1 0 3 ) .
A c know1edg emen t
The a u t h o r t h a n k s D r . Hausser and D r . H.W. D i b b e r n ,
Hoechst, F r a n k f u r t , F e d e r a l R e p u b l i c o f Germany f o r
p r o v i d i n g some u s e f u l i n f o r m a t i o n .
100 SYED LAIK ALI
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DIBUCAINE AND DIBUCAINE
HYDROCHLORIDE
Gandharva R . Padmanabhan
1. Description 106
1.2 Appearance, Color, Odor 106
1.3 Therapeutic Category 106
2. Physical and Chemical Properties 106
2.1 Infrared Absorption Spectra 106
2.2 Nuclear Magnetic Resonance Spectra (NMR) 110
2.3 Ultraviolet Absorption Spectra 110
2.4 Mass Spectra 114
2.5 Fluorescence Spectrum 117
2.7 Differential Scanning Calorimetry (DSC) 118
2.8 ThermogravimetricAnalysis (TGA) 118
2.9 Solubility 118
2.10 X-Ray Diffraction 120
2.1 1 Polymorphism 120
2.12 Partition Coefficient 120
3. Synthesis 122
6. Toxicity 125
7.2 Elemental Analysis 125
7.3 Nonaqueous Titration 126
7.4 Phase Solubility Analysis 126
7.5 Thin-layer Chromatography 126
7.6 High-PerformanceThin-layer Chromatography 129
7.7 High-pressure Liquid Chromatography 129
7.8 Gas Chromatography 130
7.9 Gas Chromatography-MassSpectrometry 130
7.10 Paper Chromatography 132
7.11 Polarography 132
7.12 Spectrophotometry 132
8. Miscellaneous 133
8.1 Dibucaine Number 133
References 133
ANALYTICAL PROFILES OF DRUG SUBSTANCES 105 Copyrightby the American Pharmaceutical Assuciatmn.
ISBN 0-12-260812-7
VOLUME 12
106 GANDHARVA R. PADMANABHAN
1. Description
1.1 Name, Formula, Molecular Weight
Dibucaine is 2-butoxy-N-[2-(diethylamino)-
ethyl]-4-quinolinecarboxamide
&'- 0CH2CH2CH2CH3
CONHCH~CH~N(C~HC,)
2
c2 OH29N302 Molecular Weight: 343.47

Other Names: Nupercaine, Percain, Cinchocain,
Cincain and Sovcain.
Dibucaine hydrochloride is 2-butoxy-N-[2-(diethyl-
amino)ethyl]-4-quinolinecarboxamide monohydro-
chloride
J
/
,J OCH2CH2CH2CH3
\ #
HC1
C20H2gN302'HCl Molecular Weight: 379.93

1.2 Appearance, Color, Odor
Dibucaine occurs as a white to off-white
powder, having a slight characteristic odor. It
darkens on exposure to light. Dibucaine hydro-
chloride occurs as colorless or white to off-
white crystals or as a white to off-white crystal-
line powder. It is odorless, is somewhat hygro-
scopic and darkens on exposure to light.
1.3 Therapeutic Category

Local Anesthetic
2. Physical and Chemical Properties

2.1 Infrared Absorption Spectra
The infrared spectra of mineral oil suspen-
sions of dibucaine and dibucaine hydrochloride are
shown in Figures 1 and 2. The spectral assignments
are listed in Tables 1 and 2.
DIBUCAINE AND DIBUCAINE HYDROCHLORIDE 107
TABLE I
I R Spectrum of Dibucaine
-1
Wavenumber, c m Assignment
3300 -NH
0
II
1665 -NH-C -
1605
1 550 Amide I1
1458)
Nujol
1378
1250 Aromatic e t h e r
7 80 0 - D i s u b s t i t u t e d phenyl
TABLE 2
I R Spectrum of Dibucaine HC1
-1
Wavenumber, c m Assignment
3205 -NH
2620
2500 Salt
H O
1670
I II
-N-C-
1605
1555 Amide I1
1458) Nuj 01
1378
1254 Aromatic e t h e r
765 0 - D i s u b s t i t u t e d phenyl
F i g u r e 1. I n f r a r e d Absorption Spectrum Dibucaine
Figure 2. I n f r a r e d Absorption Spectrum of Dibucaine Hydrochloride
2.2 Nuclear Magnetic Resonanace S p e c t r a (NMR)

The p r o t o n NMR s p e c t r a of d i b u c a i n e and
d i b u c a i n e h y d r o c h l o r i d e are shown i n F i g u r e s 3
and 4 . The s p e c t r a were determined on a Perkin-
E l m e r R-24B 60 MHz spectrometer a t ambient
temperature. The samples were d i s s o l v e d i n
d e u t e r a t e d chloroform c o n t a i n i n g t e t r a m e t h y l -
s i l a n e a s a n i n t e r n a l s t a n d a r d . The s p e c t r a l
assignment f o r d i b u c a i n e h y d r o c h l o r i d e i s shown
i n Table 3 .
TABLE 3
Chemical
No. of
Shift Multiplicity Assignment
Protons
6 (ppm)
8.7-9.1 Broad 1
0
-C-NH-CH 2 -
-
7.2-8.3 Multiplet 5 Aromatic E's
4.3-4.7 Triplet 2 -0-CE2-CH2-
9
3.6-4.3 Broad 2 -C-NH-CE2-CH2
, CE2
2.8-3.6 Multiplet 6 -CE2-N,
CE2-
/ cH2-c!!3
0.8-2.3 M u l t i p l et 13 N and
'CHz-CB3
13
The C- NMR spectra of d i b u c a i n e and d i b u c a i n e
h y d r o c h l o r i d e doterairled i n d e u t e r a t e d chloro-
form as s o l v e n t a r e shown i n F i g u r e s 5 and 6 .
2.3 U l t r a v i o l e t Absorption S p e c t r a
The UV s p e c t r a of d i b u c a i n e and d i b u c a i n e
hydrochloride (Figure 7 ) i n 1E H C 1 e x h i b i t
maxima and minima as shown i n Table 4 .
I
80
I
70
I
60
1
50
I
40
1
30
I
20
,
10
1
0
PPM
Figure 4. NMR Spectrum of Dibucaine Hydrochloride
f-
I 1 I T I I I I
90 80 70 60 50 40 30 20 10
PPM
Figure 3. NMR Spectrum of Dibucaine

I I I I I
175 150 125 100 75 50 25 0
PPM
l.3
Figure 5. C-NMR Spectrum of Dibucaine
I
I
150
1 I
100
PPM
I
50
nw
0
I
Figure 6. 13C-NMR Spectrum of Dibucaine Hydrochloride

10
09
08
07
06
Q)
0
C
0
05
2
0
a
04
03
0 2.
01
0 0- I 1 I I
250 300 350 400
Wavelength, Nanometers
Figure 7 . U l t r a v i o l e t Absorption Spectrum of Dibucaine

Hydrochloride
Dibucaine Dibucaine H C 1
'max, nm A ( 1% ,lcm) E A ( 1% ,lcm) E
247 7 30 25,070 650 24,700

320 260 8,930 232 8,810
2.4 Mass S p e c t r a
The low r e s o l u t i o n mass s p e c t r a of d i b u c a i n e
and d i b u c a i n e h y d r o c h l o r i d e o b t a i n e d
u s i n g a s o l i d probe i n s e r t i o n are shown i n
F i g u r e s 8 and 9. The s p e c t r a were run on a
Kratos MS 25 spectrometer i n t e r f a c e d w i t h a d a t a
handling system. Tables 5-6 i l l u s t r a t e t h e
fragments and t h e i r mass/charge r a t i o s .
TABLE 5
Fragmentation P a t t e r n of Dibucaine
100
90
80
70
60
50
40
30
20
10
0 1,
I 11 dl
L,
350
mfe
Figure 8
Low Resolution Mass Spectrum of Dibucaine
90 -
80 -
70-
60 -
50-
40 -
30-
20 -
10-
50
m/e
Figure 9
Low Resolution Mass Spectrum of Dibucaine Hydrochloride
TABLE 6
Fragmentation Pattern of Dibucaine Hydrochloride
m/e I Fragment
343
271
215
+
172
144
2.5 Fluorescence Spectrum

The following UV absorbance (Aa) and fluo-
rescence maxima (Af) of dibucaine in various
solvents have been reported (1-2)
Medium pH or Ho Species A ,nm
a
A f ,nm
Present ~~
H2S04 Ho=- 7 .9 325 508

2+
H2S04 H0 =-0.7 BH2 320 445
1+
Water pH=6.2 BH 328 409
1+
Ethanol - BH 328 400
1+
Chloroform - BH 329 392
Water pH=ll.5 B 328 403
Ethanol - B 327 390
Chloroform - B 328 388
Triply protonated cation
Doubly protonated cation
Mono protonated cation

Base
2.6 Melting Range

Dibucaine m e l t s between 64°C and 65°C when
t e s t e d according t o t h e USP XX, Class I a procedure.
Dibucaine h y d r o c h l o r i d e m e l t s between 95°C and
97.5"C when t e s t e d according t o t h e same procedure.
2.7 D i f f e r e n t i a l Scanning Calorimetry (DSC)

The DSC thermogram of d i b u c a i n e showed one
endotherm between 61" and 70°C w i t h a m e l t i n g
p o i n t of 64°C and a p u r i t y v a l u e of 98.9 mole
p e r c e n t when t h e thermogram w a s followed i n a
DuPont Model 900 instrument a t a scan r a t e of
20°C/minute (Figure 1 0 ) . S i m i l a r l y , t h e DSC
thermogram of d i b u c a i n e h y d r o c h l o r i d e showed one
endotherm between 94" and 104°C w i t h a m e l t i n g
p o i n t of 97°C. The p u r i t y of t h e h y d r o c h l o r i d e
was determined t o be 99.4 mole % (Figure 11).
2.8 Thermogravimetric Analysis (TGA)

The TGA of d i b u c a i n e e x h i b i t e d a weight
l o s s of 0.17% between 40°C and 130°C.
The TGA of t h e h y d r o c h l o r i d e s a l t e x h i b i t e d
a weight l o s s of 0.81% between room temperature
and 75"C, a loss of 0.14% between 75°C and 150°C
and a r a p i d l o s s above 150°C.
2.9 Solubility
Approximate s o l u b i l i t i e s i n d i f f e r e n t
s o l v e n i s were determined a f t e r e q u i l i b r a t i n g 10
mg (more, i f n e c e s s a r y , t o o b t a i n a s a t u r a t e d
s o l u t i o n ) of t h e drugs a t room temperature w i t h 1
mL of s o l v e n t .
S o l u b i l i t y , mg/mL
Solvent
Dibucaine Dibucaine H C 1
Water <0.1 >loo0
0.1z HC1 >33l >loo0
0.1E NaOH <0.1 KO.1
Methanol >loo0 >loo0
Ethanol, Absolute >loo0 >loo0
95% Ethanol >loo0 >loo0
Chloroform >loo0 >loo0
Ethyl Acetate 100- 1000 10-33
Ether 100-1000 0.1-1
Petroleum Ether 10-33 <0.1
Acetone 100-1000 100-1000
n-Hexane 10-33 <0.1
.1 g d i s s o l v e s i n 10-30 1
F i g u r e 10.DSC Scan of Dibucaine
F i g u r e 11. DSC Scan of Dibucaine H y d r o c h l o r i d e

2.10 X-Ray Diffraction

The x-ray powder diffraction pattern obtained
for dibucaine base is shown in Figure 12. The
data were collected on a GE Model XRD-spectrogonio-
meter using CuK, (1.542A') with a Ni filter as a
radiation source. The diffraction pattern obtained
for dibucaine hydrochloride is shown in Figure
13.
2.11 Polymorphism
No polymorphism has been reported for
dibucaine base and dibucaine hydrochloride.
2.12 Partition Coefficient

The following partition coefficient data
were obtained when 50 mL of 0.1 and 1.0 mg/mL
of dibucaine hydrochloride in 0.1N hydrochloric
acid solutions were partitioned individually
with 50 mL of ether and chloroform and when 50 mL
of 0.1 and 1.0 mg/mL of dibucaine in chloroform
and ether solutions were partitioned individually
at room temperature with 50 mL of pH 7 phosphate
buffer and 0.1N sodium hydroxide.
Aqueous Phase Organic Phase Partition Coefficient?
0.1N HC1 Chloroform 0.8-1.1

0.1N HC1 Ether -to
pH 7 Buffer Chloroform >15
pH 7 Buffer Ether >14
0.1N NaOH Chloroform >20
0.1N NaOH Ether >14
2.13 Dissociation Constant

The following pKa values have been reported (1):
pKBH+ = 8.31 (potentiometric titration)
pKBH;+ = 1.6 (absorption spectrophotometry)

psH;+ = - 5 . 3 (absorption spectrophotometry)
3+ 2+ +
BH3 , BH2 and BH are respectively triply pro-
tonated, doubly protonated and mono protonated
species.
I I I I
5 15 25 35
Degrees Two Theta
F i g u r e 1 2 . X-Ray Powder D i f f r a c t i o n P a t t e r n of Dibucaine

3* Synthesis
Dibucaine and dibucaine hydrochloride are prepared
by the following sequence of reactions (Figure 14)
starting with isatin (3). Isatin (I) is reacted with
malonic acid in the presence of an acid to form carbo-
styrilic acid (11). The acid is then treated with
phosphorous oxychloride to yield 2-chlorocinchoninic
acid chloride (111) in solution. The solution is then
reacted with diethylaminoethylamine to form 2-chloro-
N(2-diethylaminoethyl) cinchoninamide (IV) in solution.
The cinchoninamide solution is then treated with
sodium n-butylate to form dibucaine base(V). The base
is purified and then converted to the hydrochloride
salt (VI) by reacting with hydrogen chloride.
4. Stability-Degradation
Dibucaine hydrochloride (I) (Figure 1 5 ) , when
boiled for 4 hours in 2N hydrochloric acid, resulted
in complete hydrolysis to 2-hydroxyquinoline-4-carboxy-
lic acid diethylaminoethylamide (11) and 2-hydroxy-
quinoline-4-carboxylic acid (III)(4). Hydrolysis of
dibucaine hydrochloride, with pH = 5.45 and at a
temperature of 134°C for 40 hours resulted in the
formation of Compound I1 only Autoclaving of a solution
of dibucaine hydrochloride in a mixture of 10% sodium
hydroxide and ethanol at 120°C for 2 hours resulted in
the formation of 2-butoxyquinoline-4-carboxylic acid
(IV). Under the influence of an oxidizing agent such
as m-chloroperbenzoic acid, dibucaine can be oxidized
to its N-oxide analog (V). The N-oxide can further
react with reagents such as ferrous sulfate to yield
the desethyl analog (VI) of dibucaine and dibucaine
(5).
5. Drug Metabolism and Pharmacokinetics

An apparent half-life of approximately 11 hours
with a peak serum concentration at 2 hours after
administration was obtained following the administration
of single 5 mg dibucaine hydrochloride oral dose (15)
to human volunteers. Serum levels of dibucaine in
monkeys and dogs after intravenous administration
indicated an apparent elimination half-life of approxi-
mately one hour. Serum peak concentrations of dibucaine
were found after 1-6 hours in monkeys, dogs and humans
after rectal administration of an ointment formulation.
Rectal administration of the ointment formulation to
human volunteers at 0.2-0.6 mg/kg level, t.i.d. for 3
days resulted in peak serum level after the third or
fourth dose and declined to base-line within 48 hours
I I
5 1; 2; 35
Degrees Two Theta
Figure 1 3 . X-Ray Powder D i f f r a c t i o n P a t t e r n of

Dibucaine Hydrochloride
COOH
IV 111
NaOCgHg
\ II
0
0
t
IV
VI
Figure 15
Chemistry of Dibucaine and Dibucaine Hydrochloride
of t h e l a s t dose. D i s p o s i t i o n of d i b u c a i n e f o l l o w i n g
m u l t i p l e r e c t a l a d m i n i s t r a t i o n of 0.1% s u p p o s i t o r i e s
i s comparable t o ointment a d m i n i s t e r e d s i m i l a r l y
(6-7).
6. Toxicity
The a c u t e i n t r a p e r i t o n e a l LD50 v a l u e s of d i b u c a i n e
h y d r o c h l o r i d e i n male mice and female mice observed
over a p e r i o d of 15 days were found t o b e r e s p e c t i v e l y
7 1 mg/kg and 74 mg/kg. The a c u t e o r a l LD50 v a l u e s of
d i b u c a i n e h y d r o c h l o r i d e i n m a l e rats and female r a t s
observed o v e r a p e r i o d of 1 5 days w e r e found t o be
r e s p e c t i v e l y 371 mgfkg and 395 mg/kg (8).
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
Two i d e n t i f i c a t i o n tests are g i v e n i n t h e
USP XX f o r d i b u c a i n e , one a n i n f r a r e d a b s o r p t i o n
t e s t and t h e o t h e r a n u l t r a v i o l e t a b s o r p t i o n
t e s t . For d i b u c a i n e h y d r o c h l o r i d e , f o u r i d e n t i -
f i c a t i o n tests are g i v e n i n USP XX. The t e s t s
i n c l u d e d are i n f r a r e d a b s o r p t i o n , u l t r a v i o l e t
a b s o r p t i o n , m e l t i n g p o i n t of i s o l a t e d f r e e b a s e
and a t e s t f o r c h l o r i d e .
Methods t o i d e n t i f y and d i f f e r e n t i a t e
d i b u c a i n e from n i n e o t h e r l o c a l a n e s t h e t i c s have
been r e p o r t e d i n t h e l i t e r a t u r e ( 9 ) . The methods
are based o r t h e m e l t i n g , i n f r a r e d and photomicro-
g r a p h i c p r o p e r t i e s of t h e d e r i v a t i v e s o b t a i n e d
with styphnic acid, p i c r i c acid, c h l o r o p l a t i n i c
a c i d , p i c r o l o n i c a c i d , ammonium r e i n e c k a t e and
methyl i o d i d e .
7.2 Elemental A n a l y s i s
The f o l l o w i n g e l e m e n t a l compositions were
o b t a i n e d f o r d i b u c a i n e and d i b u c a i n e h y d r o c h l o r i d e
when 2 mg samples were employed f o r a n a l y s i s
w i t h a Perkin-Elmer Model 240 CHN Analyzer.
DIBUCAINE
Element Theory, % Found, %
Carbon 69.94 69.67
Hydrogen 8.51 8.60
Nitrogen 12.23 12.07
DIBUCAINE HYDROCHLORIDE
Element Theory, % Found, %
Carbon 63.22 62.99
Hydrogen 7.96 8.19
7.3 Nonaqueous Titration

Dibucaine may be titrated in glacial acetic
acid with perchloric acid in glacial acetic acid
as titrant. The titration can be carried out
potentiometrically or with crystal violet as
indicator. Dibucaine hydrochloride may be titrated
similarly in glacial acetic acid containing
mercuric acetate with perchloric acid
in glacial acetic acid as titrant. Two equivalents
of acid are consumed in the titration of dibucaine
and dibucaine hydrochloride. The titration is not
specific for the drug in presence of some of
their degradation compounds.
7.4 Phase Solubility Analysis

Phase solubility analysis of dibucaine
hydrochloride has been carried out using the
following system (10):
Dibucaine Hydrochloride
Solvent Temperature Approx. Solubility
mdg
Ethyl acetate 25 15.4
7.5 Thin-layer Chromatography

A number of thin-layer chromatographic
systems have been developed for the identification
of the drug and for the determination of the
compounds related to the drug.
System I - The following system may be employed
particularly to control the impurities
likely to be present from the synthesis
of the drug.
Adsorbent : Silica Gel G plate, 20 cm x 20 cm
coated to a thickness of 250 microns
Mobile Phase: A mixture containing 30 mL of
acetone, 50 mL of toluene, 5 m L of
methanol and 1 m L of concentrated
ammonium hydroxide.
System I Continued
Detection
Systems: 1. Spray with 0.5% potassium
dichromate in 20% sulfuric acid
followed by heating at 140°C
for 10 minutes and viewing
under shortwave UV
2. Irradiate with high intensity
UV for 10 minutes followed by
visualization under long wave UV
System I1 - The following system may be employed

particularly when 3-chloro dibucaine
content in the drug has to be deter-
mined.
Adsorbent: Silica G plate, 20 cm x 20 cm,
coated to a thickness of 250
microns
Mobile Phase: A mixture of 35 mL of glacial
acetic acid, 55 mL of ethyl acetate,
5 mL of concentrated hydrochloric
acid and 5 mL of water
Detection
Systems : 1. Shortwave UV
2. Irradiation with high intensity
UV for 10 minutes followed by
visualization under longwave UV
System I11 - The following system may be employed

for the estimation of transformation
products in formulations
Adsorbent: Silica Gel G plate coated to a
thickness of 250 microns
Mobile Phase : A mixture of 80 mL of chloroform,
20 mL of methanol, 1 mL of
ammonium hydroxide and 1 mL of
water
Detection
System : Irradiate with high intensity
W for 10 minutes followed by
visualization under long-wave
w
Other Systems: The following systems have also

been employed for the analysis
of dibucaine or dibucaine hydro-
chloride.
System IV - Chloroform/Acetone/Diethylamine
(5:4:1); Silica Gel GF; Dragendorff
Spray, Iodoplatinic Acid Spray and
UV Detection Systems (11)
System V - Chloroform/Diethylamine ( 9 : l ) ;
Silica Gel GF; Detection Systems
same as in System IV
System VI - Methanol/Ammonium Xydroxide (100:
1.5); Silica Gel GF; Detection
Systems same as in System IV
System VII - n-Butanol/Acetic Acid/Water (5:3:2);
same as in System IV
. System VIII - Chloroform/Methanol (9:l); Silica
Gel GF; Detection systems same as
in System IV
System IX - Dioxane/Water ( 9 : l ) ; Silica Gel GF;
Shortwave W,Longwave UV, 0.5%
Iodine in Chloroform Spray,
Acidified Potassium Iodoplatinate
Spray and 40% Sulfuric Acid Spray
Followed by Heating and Longwave
UV Detection Systems (11)
System X - Dioxane/Water/Chloroform (8:l: 1);
same as in System IX
System XI - Dioxane/Water/Toluene (8:l:1);
same as in System IX
System XI1 - Acetone/Benzene/Methanol/Concentra-
ted Ammonium Hydroxide (30:50:5:1);
Silica Gel G; Longwave UV After 10
minute Irradiation with High
Intensity W
System XI11 - Chloroform/Methanol/Water (80:20:2);
Silica Gel G; Dichromate in 20%
Sulfuric Acid spray followed by
heating and visualization under
short-wave UV
7.6 High Performance Thin-layer Chromatography

The following system has been reported for
the quantitation of dibucaine in injectable
solutions and in plasma and serum samples (12).
Developing Solvent: Ether/Benzene/Cyclo-
hexane/Diethylamine
(20:12.5:10:3.5)
Adsorbent: HPTLC Silica Gel 6 0
F254 (Merck)
Chamber Saturation: 15 minutes
Development Distance: 4 cm
Time of Development: 5 minutes
Sample Volume 200 nL
Detection Mode: Reflectance (240 nm)

The following systems have been reported for
the quantitation of impurities in dibucaine and
dibucaine hydrochloride samples (13).
System I
Column : 25 cm x 4.6 mm i.d. Zorbax C-8
stainless steel column with 6.5 cm
x 2.1 mm i.d. Whatman CO-Pel1 ODS
guard column
Detection: W-313 nm
Temperature: Ambient
Flow Rate: 1 mL/minute
Mobile Phase: Linear gradient from 100% A
to 95% B in 20 minutes. A = 1:l
methanol-water; B=0.2% ammonium
hydroxide in 2:8 methanol-aceto-
nitrile
System 11 (11)
Column : 25 cm x 4.6 mm Lichrosorb RP 8
column
Detection: UV 254 nm
Temperature: 25 C
Flow Rate: 2 mIJminute
Mobile Phase: A. Methanol-Water-Diethylamine
(90:10:0.02)
B. Methanol-Water-Diethylamine
(80:20:0.02)
C. Methanol-Water-Diethylamine
(75: 25:0.02)
System I1 Continued
Approximate
Retention A = 4.5 minutes
Time of B = 7.4 minutes
Dibucaine C = 10.9 minutes
System I11 (14)

Column: 50 cm x 2.1 mm (i.d.) stainless
steel column packed with Permaphase
ODS
Detection: W-254 nm
Fluorescence: Excitation-325 nm and
Emission-390 nm
Temperature: 40O C
Flow Rate: 0.85 mL/minute
Mobile Phase: 50% Isopropanol, 45% Methanol and
5% 0.001N NaOH
7.8 Gas Chromatography

The following system has been employed for
the analysis of dibucaine in the drug substance
and in a suppository formulation.
Column: 6 ft x 4 mm (i.d.) column with
3% OV-17 on Gas Chrom Q (100 x 120
mesh)
Temperature: -
Column 25OoC; Injector - 27OOC;
Detector - 300°C
Detector: Flame Ionization Detector
Carrier: Helium 60 cc/minute
Sample: Inject 2.0 pL of a 10 mg sample
in 1 mL of tetrahydrofuran
7.9 Gas Chromatography-Mass Spectrometry (GC-MS)

Sensitive methods €or the analysis of
dibucaine in serum samples have been reported
using GC-MS with selected ion-monitoring for
separation and detection. The following experi-
mental conditions were used for the analysis of
the drug in biological fluids.
Method I (15)
Column : 2.5 ft x 2 mm i . d . silanized glass
column packed with 1.5% OV-17 on
80/100 mesh Chemosorb W-HP
Detection: GC-MS selected ion monitoring
at m/e = 228 and at m/e = 237
Method I Continued
Temperature: I n j e c t o r - 260°C; Column - 250°C:
GC-MS I n t e r f a c e -250°C
Carrier: Helium
MS E I Source: 37 ev
Internal
Standard: Nonadeuterated d i b u c a i n e
Method I1 (15)
Column : 2.5 f t x 2 mm i . d . s i l a n i z e d g l a s s
column w i t h 1 . 5 % OV-1 on Chromasorb
W-HP 80/100 mesh
Detection: GC-MS (CI) s e l e c t e d i o n m o n i t o r i n g
a t m / e = 344 and m / e = 353
Temperature: Column - 215°C; I n j e c t o r - 260°C;
GC-MS I n t e r f a c e - 250°C
Carrier: Methane
MS C I Source: 55-85 ev
C I Reagent
Gas: Methane
Internal
Standard : Nonadeuterated d i b u c a i n e
Method I11 (16)

Column: 0.5 m x 3 mm i . d . g l a s s column
packed w i t h 3%Poly 1-110 on Gas
Chrom Q 80-100 mesh
Detection: E . I . S e l e c t e d ion-monitoring a t
m/ e=86
Temperature: Column - 260°C; I n j e c t o r - 350°C;
S e p a r a t o r - 3OO0C, I o n Source -
310 C
Carrier: Helium, 30 mL/min
In t e r n a 1
Standard : Chloropromazine H C 1 (m/e=86)
Method I V (17)
Column : 2 m x 2 mm g l a s s column packed
w i t h 3% OV-17 on 80-100 mesh Gas-
Chrorn Q
Detection : GC-MS s e l e c t e d i o n monitoring a t
m / e = 326 and a t m / e = 335 and 336
Method IV Continued
Temperature: Column: Programmed at G°C/minute
from 260-300°C; Injector - 300°C;
Ion Source - 300°C
Carrier: Not indicated; 30 mL/minute
MS-EI Source: 75 ev; ionizing current - 300 PA
Internal
Standard : Deuterium-labeled dibucaine (Dg
and Dlo)
7.10 Paper Chromatography
Stationary
Phase: Whatman #1 paper impregnated with
a 1:l solution of acetone and
.
formamide Formamide was adjusted
to pH 5.6 with benzoic acid before
mixing. Remove the excess of the
impregnated solution by blotting
between dry filter papers
Mobile Phase: 2% pyridine in 1:l benzene-
chloroform
Detection: Dragendorff spray reagent
Sample
Solution: Spot 10 pL of 1% dibucaine
hydrochloride in 1:l methanol-
chloroform (18)
R of
f
Dibucaine HC1: -0.75
7.11 Polarography
Polarography has been employed for the
analysis of dibucaine in soLutions and for the
identification of dibucaine (19-20). Polarography
was carried out using a borate-biphosphate buffer
with pH of 5 to 7.5 and measuring the reduction
current at -0.6 V vs calomel electrode. The method
was linear between 5 and 150 mg/100 mL.
7.12 Spectrophotometry
Dibucaine and dibucaine hydrochloride in
formulations can be analyzed by spectrophotometry
(21) by taking advantage of the maxima at 247 nm
and 320 nm in acidic solutions. The technique
when preceded by acid-ether and base-ether
extraction steps is selective for all products
discussed under Section 4 , Stability-Degradation,
except for compound VI.
8. Miscellaneous
8.1 Dibucaine Number
When succinylcholine, which is a neuromuscular
agent, was introduced for anesthetic procedures,
it was observed that certain individuals failed to
recover from the paralytic effects and this poor
recovery was attributed to the low activity of
the enzyme cholinesterase in plasma. The identi-
fication of the atypical enzyme activity has been
carried out by the selective inhibition of the
plasma esterase by dibucaine with benzoylcholine
as substrate. A quantitative measure of this
selective inhibition, expressed as a percent of
inhibition, is called the dibucaine number (22-25).
9. References
1. Martucci, J . D. and Schulman, S. G., Anal. Chim.
Acta, 77, 317 (1975)
2. Hrdy, 0. and Slouf, A., Cs. Pharm., 1,7 1 ( 1 9 5 2 ) ;
and Die Pharmaczie, 8, 1 5 9 ( 1 9 5 3 )
3. CIBA-GEIGY, Personal Communication
4 . Morch, J . , Dansk. Tidsskr. Farm., 27, 1 7 5 (1953)
5. Senn, H. and Kathriner, A., CIBA-GEIGY, Personal
Communication
6 . Alkalay, D., Carlsen, S., Khemani, L., Wagner
Jr., W. E . , and Le Sher, A., CIBA-GEIGY, Personal
Communication
7. Bartlett, M. F. and Egger, H., CIBA-GEIGY, Personal
Communication
8. Thomann, P. and Pericin, C., CIBA-GEIGY, Personal
Communication
9 . Rich, N. W. and Chatten, L. G., J . Pharm. Sci.,
-
5 4 , 9 9 5 (1965)
10. Grady, L. T., Pharmacopeial Formum, United States
Pharmacopeial Convention, Inc., p. 1 4 3 6 , Sept.-
Oct. 1 9 8 1
11 Grady, L. T., USP-NF Reference Standards Committee,
United States Pharmacopeia, Letter 9 9 , p. 432-
438, dated February 2 5 , 1 9 8 1
1 2 . Giibitz, G. and Wintersteiger, R., Sci. Pharm.,
4 6 , 275 (1978) (German)
-
13. Liu, R., CIBA-GEIGY, Personal Communication
1 4 . Takeoka, T., Kojima, T. and Kobayashi, H., Nippon
Hoigaku Zasshi, s, 20 (1979) (Japan)
15. Alkalay, D., Carlsen, S. and Wagner, W. E., Anal.
Letters, 14(B20), 1 7 4 5 (1981)
1 6 . Kageura, M., Totoki, K. and Nagata, T., Nippon
Hoigaku Zasshi, 2, 188 ( 1 9 7 8 ) (Jap. J . Legal
Med.) (English)
17. Shinka, T., Kuhara, T. and Matsumoto, T., Quant

Mass Spectrom. Life Sci., 2, 315 (1978)
18. Korzun, B. P., CIBA-GEIGY, Personal Communication
19. Dusinsky, G., Pharmazie, 9, 27 (1954)
20. Pech, J., Collection Czechosl. Chem. Communications,
6,
- 132 (1934)
21. United States Pharmacoepia, Twentieth Revision,
Mack Printing Company, Easton, PA, 1980, pages
226-228
22. Kalow, W. and Genest, K., Canad. J . Biochem.
Physiol., 35, 339 (1957)
23. Harris, H. and Whittaker, M., Nature, 191,496
(1961)
24. Brody, I. A., Resnick, J. S. and Engel, W. K.,
Arch. Neurol., 13,126 (1965)
25. Irwin, R. L. and Hein, M. M., Biochem. Pharmacol.,
-
15, 145 (1966)
10. Acknowledgement
The author expresses appreciation to Ingrid
Becue, Richard Brown, James B. Smith and Jane Johnson
f o r help in preparation of this manuscript.
ESTRONE
Douglas Both
I . Introduction 136
1.1 History 136
1.2 Structure, Nomenclature, and Molecular Weight 137
1.3 Biosynthesis and Metabolism 137
1.4 Synthesis and Commercial Production 142
2.1 Crystal Structure 144
2.2 Powder X-Ray Diffraction 145
2.3 Melting Point 145
2.4 Thermal Analysis 145
2.5 Magnetic Susceptibility 149
3. Spectrometry 149
3.1 Proton Nuclear Magnetic Resonance 149
3.2 Carbon-I3 NMR Spectra 149
3.4 Infrared Spectrometry 154
3.5 Ultraviolet and Visible Spectrophotometry 156
3.6 Optical Rotatory Dispersion and Specific Rotation 156
3.7 Fluorescence and Phosphorescence 157
4. Solution Properties 158
4.1 Solubility 158
4.2 Partition Coefficients 158
4.3 Molecular Volume 158
4.4 Heat of Formation and Combustion 160
4.5 Acid Ionization Constant 160
4.6 Stability 160
5. Chromatographic and Other Separation-based Analysis 161
5.1 Column Chromatography 161
5.2 Thin Layer Chromatography 163
5.3 High-Performance Liquid Chromatography 165
5.4 Gas-Liquid Chromatography 167
5.5 Gas Chromatography-Mass Spectrometry 169
6. Radioassay 170
7. Colorimetric Analysis 172
8. Titrimetric Analysis 174
References 174
ANALYTICAL PROFILES OF DRUG SUBSTANCES 135 Copyrightby the American Pharmaceutical Association.
VOLUME 12 ISBN 0-12-260812-7
136 DOUGLAS BOTH
1.0 INTRODUCTION
1.1 HISTORY
In 1 8 9 6 l Y 2 , Dr. Emil Knauer, using ovarian transplants

into immature female animals, first demonstrated the
existence of sex hormones. In 1909, Henry H. Dale
developed a posterior pituitary gland extract which
stimulated uterine contraction that found great use during
complicated labor. This was the beginning of the steroid
era.
In 19273, two Berlin gynecologists, Dr. Selmar

Aschheim and Dr. Bernard Zondek, while developing a method
for the early diagnosis of pregnancy, established that the
urine of pregnant women contained a high concentration of
estrogen. Before this discovery, the isolation of
estrogens had been unsuccessfully pursued from placental
extracts.
Dr. E.A. Doisy and Dr. E. Allen, were working towards

the isolation, purification and crystallization of estrone
from pregnancy urine. Aldolf Butenandt, under the
direction of nobel prize winner Adolf Windaus of Gijttingen
University, was also independently working towards this
goal. After nearly two years, in August of 1929, Doisy4
and Allen reported the first account of the purification
and crystallization. A few months later in Octob%r 192g5,
Butenandt also reported its isolation. Butenandt claimed
that Doisy and Allen preempted him by virtue of an annual
cleaning of the Gijttingen Laboratory. The laboratory was
shut down for several weeks, just as he had obtained a
potent extract and was 10-14 days from purification.
At7 first, only the physiological effects and some

chemical properties were known. The first physico-chemical
characterization of the crystal and the correct empirical
formula were reported by Thayer in 1930. The name estrone
was not adopted until 1935. The first partial
of estrone was performed by Inhoffen in 1 9 4 1 and the first
total synthesis in 1948 by Anner.
The isolation of estrone was of great importance, for

it was the first estrogenic steroid hormone isolated. The
great interest generated by this compound was responsible
for the isolation of later steroid hormones. It was these
later steroids that revolutionized the treatment of many
different illnesses,
ESTRONE 137
1.2 STRUCTURE, NOMENCLATURE AND MOLECULAR WEIGHT
The basic skeletal structure of estrone is the

cyclopentanoperhydrophenanthrene nucleus. This nucleus is
composed of the four rings designated A,B,C and D. Each
carbon atom of the ring is numbered in rotation. The
methyl group is found in the beta position in naturally
occuring estrone. See figures 1 and 2.
Estrone is a white crystalline powder or a colorless

flat plate-like crystal. The powder is virtually odorless.
The name estrone is the offically adopted name, other
common synonymous names include: Oestrone,
3-hydroxyestra-1,3,5(10) trien-17-one, folliculin,
ketohydroxyestrin, menformon, theelin and follicular
hormone.
The empirical formula is C H O2 with a molecular

weight of 270.37. Estrone is gl!en2$he chemical abstracts
systematic number [53-16-71,
1.3 BIOSYNTHESIS AND METABOLISM
Estrone is resent in plants and animals. It has been

found in plantslg-14 such as the date, apple, pomegranate,
oat, apricot and in the oils of corn and olive. In
the major source is the ovary and to a lesser
extent, the adrenal cortex, feto-placental unit and Leydig
cells of the testis.
In Vitro s t u d i e ~ l ~
-- have
’ ~ ~shown that estrone arises
from acetate. After several Nicotinamide-Adenine
Dinucleotide Phosphate (NADPH) dependent condensation
reactions, acetate forms squalene, which subsequently
undergoes elimination and cyclization to yield cholesterol.
Cleavage of the side chain of cholesterol and several
rearrangements yield pregnenolone. Oxidation and
isomerization produce progesterone which leads to
4-androstene-3, 17-dione and finally to estrone. An
overview of the pathway can be seen in figure 3 .
Questionsz0 have been raised as to whether cholesterol

is a required intermediate in the synthesis. It seems that
several experiments have shown that the precursers of
cholesterol are incorporated into certain cells much more
readily. B. R. Bhavnani2 discusses other nonclassical
approaches to the biosynthesis of steroids which bypass
cholesterol as an intermediate.
138 DOUGLAS BOTH
ESTRONE
F i g u r e 1. The S t r u c t u r e of E s t r o n e
F i g u r e 2. Conformation Diagram o f E s t r o n e .
(Redrawn from R e f e r e n c e 82.)
ESTRONE 139
Figure 3 . Summary of the biosynthesis of estrone and other steroids.

(Adapted from references 18, 19.)
140 DOUGLAS BOTH
During the synthesis and metabolism, addition or

removal of hydrogen is catalyzed by enzymes found in the
ovary. Addition of oxygen is catalyzed by hydroxylases,
usually taking place in the liver and kidney, which are
important sites for the inactivation of steroids.
Hydroxylation reactions usually occur at the 2, 6 , 8 , 11,
16 or 18 carbon positions, and require molecular oxygen
while utilizing NADPH.
Omura22 worked out the electron transport system for

the 11-8 hydroxylation which occurs in the adrenal
mitochondria. Hydroxylation involves the transport of
hydrogen from NADPH to flavoprotein. The reduced
flavoprotein transfers electrons to a non-haem protein.
The reduced non-haem protein will transfer electrons to
cytochrome P-450. It is believed that this type of pigment
system exists for other biosynthesizing tissue such as the
feto-placental unit.
Estrone exists in bound and unbound forms. It is this

binding with protein that plays a role in the metabolism of
estrone. E ~ t r o n e binds
~ ~ ’ ~strongly
~ to red blood cells
which may increase its solubility, and thus, function to
regulate its distribution and availability to target
organs. ConjugationZ5 of estrone in the liver works to
inactivate the steroid while secretion of a sulfate
conjugate by the adrenal cortex serves as a reservoir for
later activation in target organs. Hydroxylation26 and
methylation appear to be major modes of estrone metabolism.
Both metabolites, as well as estrone, in the form of
glucuronic acid or sulphate conjugates, appear in
significant quantities in urine. Estrone can be readily
converted into a number of products as shown in figure 4.
During the metabolismz7 of estrone, a rapid

equilibrium is established between estrone, estriol and
178-estradiol. This equilibrium has been studied in vitro
in the liver and kidney by Rayan28 and Velle29. The
equilibrium ratios of estrone, estriol and 17B-estradiol in
urine are approximately 45:45:10. The rate constant for
the conversion of estrone to 176-estradiol in the uterus,
however, is 10-20 times larger than the rate constant for
the reverse reaction. MuseyZ6 discusses the pathways for
the metabolism of estrone sulfate and other conjugates in
greater depth; also discussed are conjugate transport
dynamics and hydrolysis.
ESTR 0NE 141
'2. HVOROXVESTRONE '2. YETHOXVESTRONE
-
Flgure 4 Summery of the metabollrm of emone.
(Adapted from reference 18, 10.)
142 DOUGLAS BOTH
Plasma 30’35 levels of estrone for men range form

0.1-0.42 ug/L. For women, levels range from 0.2-0.7 pg/L,
depending on the phase of the menstrual cycle. Urinary
excretion levels for men are 3-8.2 pg/24 hr, and for women
are 0.3-2.4 pg/24 hr.
1.4 SYNTHESIS AND COMMERCIAL PRODUCTION

The first partial synthesis36 of estrone was
accomplished by Inhoffen in 1941. The first total
synthesis was reported by Anner37 and Miescher in 1948.
Since that time, a multitude of starting products,
intermediates and routes have been reported. Estrone is an
important synthetic product, for it is a key intermediate
in the synthesis of many complex 19-nor steroids.
As early as 192838, Parke-Davis and Co. produced an

ovarian extract of estrone. Following the early crude
products were purer forms extracted from urine, as reported
by Schering-Kahlbaum and later Roussel and N.V. Organon.
After the discovery of the levels of estrone in the urine
of stallions and pregnant mares, Zondek, and later American
firms, produced estrone extracted from urine. While the
extraction process initially proved economically feasible,
improved partial or total synthesis became more cost
effective.
The in production of synthetic estrone was

Roussel who converted dehydroisoandrosterone to estrone.
Dehydroisoandrosterone can be produced from cholesterol
which, in turn, can be extracted from plant and animal
oils, grease, wool and waxes. Other methods of production
included: pyrolysis, microbiological reduction or
transformation. For a review of fermentation in industrial
applications see Reference 41, there are several papers
that review the general synthesis of estrone See References
42-46. Table (1) gives a listing of selected syntheses.
Table 1 - Selected Syntheses
REFERENCE METHOD
47 Regiospecific fuctionalization of 2,3,

BIS(trimethylsily1)estratrien -17-one using
cobalt catalyst
48 Regiospecific Diels-Alder using, 6-
methoxy-l-vinyl-3,4-dihydronapthalene
ESTRONE 143
49 Biomimetric polyene cyclization
50 From ethyl-l-carbethoxy-2-oxo-l-
cyclohexaneacetate
51 From methyl-l-keto-2-methyl-7-
methoxy-1,2,3,4,4A,9,10A-octahy-
dro-2-phenanthrenecarboxylate
52 From 1,4-androstandiene-l9-01-3,20-
dione-19-benzoate
53 From a mixture of androsta-1,4-

diene-3,17-dione-l7-(cyclic ethylene acetal)
and 20-(hydroxymethyl) pregna-lY4-diene-
3-one.
54 By irradiation of 3,3,17,17-bis
(ethylenedioxy)-19-hydroxy-5-androstene
55 From 4-pregnene-17aYl9,21-trio~-3,
20-dione-19,21-diacetate
56 From androsta-1,4-diene-3,17-dione-17-
ethylene acetal
57 From 3,17-dioxestra-4,9-diene by isomerizing

in the presence of an acylhalide
58 By fermentation using brevibacterium (ATCC

19653)
59 By asymmetric conversion of
2-methyl-2-carbethoxyethyl cyclopentane-
1 ,3-dione
60 By systematic degradation from

106-hydroxy-19-norperlplogenin
61 From 19-nor-4-androstene-3,17-dione with

p-cymene catalyzed by lead on carbon
62 An expeditious synthesis
63 From 5-androstene-3f3,19-diol-l7-one by
microbiological action of mycobacterium
phlei strain w
144 DOUGLAS BOTH
64 By oxidation of 19-nor-1(10),5-androstadiene
36-01-17-one
65 By steroselective intramolecular
cycloadditon of olefinic-o-quinodmethane
66 From andro-1,4-diene-3,17-dione by pyrolysis
67 From 17B-estradiol by enzymatic oxidation

and reduction using Pseudomonas testosteroni
68 Microbiological transformation with the use

of cholest-4-ene-3-one as a steroid inducer
69 By microbiological conversion of
19-hydroxy-androst-4-ene-3,17-dione
70 From m-CH OC H CH CH:CH2

3 6 4 2
71 By cationic olefinic cyclization
72 From 2B-hydroxy-3,17-dioxoandrost-
4-ene-19-al
73 From 19-hydroxycholesterol acetate by

mycobacterium conversion
74 By microbiologica~dehydrogenation of
3-keto-4-steroids
75 From l-hydroxy-4-androstene-3,17-dione by
fermentation with Penicillium Sp (ATCC
12,556)
76 From andro-1,4-diene-3,17-dione by
pyrolyzing with hot kerosine.
2.0 PHYSICAL PROPERTIES
2.1 CRYSTAL STRUCTURE
Estrone exists in three polymorphic crystalline forms.

The polymorphic form obtained depends on the mode of
crystallization. Two forms are orthorhombic; Form I is
stable and Form I1 is a metastable state. The third form,
Form 111, is monoclinic and metastable.
ESTRONE 145
Forms I and I11 are usually obtained by sublimation,

while Form I1 can be obtained by evaporation from a
solution of acetone or methanol. The crystalline cohesion
of the three forms are different. Forms I and 11177 have
layers of parallel molecules linked by hydrogen bonds and
Form I1 has a herring bone arrangement with weaker hydrogen
bonds and stronger dispersion bonds. Figure 5 7 8 is a
diagram of the two crystalline forms. Bernard B u ~ e t t a ' ~ - ~ ~
has studied the crystalline form of estrone quite
extensively. Table 2 is a summary of the parameters of the
three crystalline forms as measured by Busettair7.
2.2 POWDER X-RAY DIFFRACTION
Figure 6 is the x-ray powder diffractionB2 spectrum of

the U.S.P reference standard estrone. The spectrum was
obtained using a Philips powder diffraction unit utilizing
the Ka emission of copper at 1.54A. The sample was scanned
"as is" from 8 to 48 degrees ( 2 0 ) . The d spacing in
angstroms is given above each major peak in the diffraction
spectrum. The d spacings of the major peaks appear to be
consistant with published valuesM3.
2.3 MELTING POINT
AtH4 1 8 0 " C , unstable crystals in the shapes of rods

and prisms begin to sublime. At 22OoC, stable rectangular
shaped crystals sublime. At about 2 3 0 " C , some of Form I11
is converted into form I. The remaining portion of the
original substance melts at 254°C (Form 111) or at 256°C
(Form 11) while Form I melts at 259°C.
2.4 THERMAL ANALYSIS
Differential thermal a n a l y ~ i s ~of~ 'estrone ~~ shows a

small broad endotherm at about 230°C which seems to
indicate a crystalline transition described in section 2 . 3 .
A sharp strong endotherm at 258°C probably corresponding to
the melting process is also seen. The derivatogram shows
weight loss of 1 9 . 4 , 2 3 . 8 , 3 4 . 4 and 5 1 . 2 % at 3 5 0 , 3 6 5 , 395
and 4 2 5 " C , respectively.
The differential thermal analysis of the U.S.P.

reference standard estroneE7, scanned at 2"C/min, shows a
sharp endotherm at 264°C and a broad minor endotherm
between 2 3 1 and 237°C.
The U. S.P. reference standard estrone", dried at

105°C for 3 hours, lost 0.13% of its weight.
146 DOUGLAS BOTH
",
-
ORTHORHOMBIC FORM
Figure 5 - The two crystalline forms of estrone.

(Redrawn from reference 78.)
TABLE 2 - CRYSTAL DATA
FORM I FORM I1 FORM I11
Evaporation from
Mode of Crystallinzation Sublimation Acetone or Methanol Sublimation
State Stable Metastable Metastable
Crystal form Or thorhombic Orthorhombic Monoclinic
Space Group PZ1' zl, 2, PZ1' Z1' z1 p2 1

Molecules per Two independent
unit cell (Z) 4 4 (4) molecules
" 3 1481 1440 1461
Cell volume (A)
Cell dimensions:
0
+ 0.005 A
a - 12.188 10.043 9.271
0
+ 0.005 A
b - 16.301 18.424 22.285
0
c -
+ 0.005 A 7.463 7.787 7.610
+ 0.2"
a - 90 90 90
B 5 0.2" 90 90 111.45
y 0.2" 90 90 90
Fig. 6. Powder X-Ray Diffraction Spectrum of Estrone.
ESTRONE 149
2.5 MAGNETIC SUSCEPTIBILITY
The mean molar magnetic susceptibility88 of estrone

re r stallized from methanol was found to be -176.47 x 106
s y
cm /mole, when measured according to the Faraday method.
Values calculated according to the Pascal empirical
systematic, the revised Pascal systematic and the empirical
systematic f r bonds were rep0 ted to be -170.74 x 10 ,
8 6 3
-180.95 x 10 and -180.10 x 10 cm /mole respectively.
3.0 SPECTROMETRY
3.1 PROTON NUCLEAR MAGNETIC RESONANCE
The proton nuclear magnetic resonance spectrum of the

U.S.P. reference standard estrone is shown in figure 7 .
The spectrum was obtained on a Varian XL-100 at 100.1 MHZ
in deuterochloroform (CDC1 ) using tetramethylsilane (TMS)
as a internal reference. 3
Because of the similar magnitude of the coupling

constants and chemical shifts of the protons of the
aliphatic rings, normal splitting patterns are distorted.
Virtual coupling of these protons make spectral assignment
difficult. The spectrum shows only one singlet at 0.9 ppm
from TMS belonging to the axial methyl protons.
3.2 CARBON-13 NMR SPECTRA
The proton decoupled Carbon-13 nuclear magnetic

resonance spectrum89 of the U. S.P. Reference Standard
estrone is shown in figure 8. The spectrum was obtained on
a Varian XL-100 at 15.4 MHZ. The resonance assignments of
the carbons of estrone are shown in Table 3. The resonance
chemical shifts of the carbons from TMS are shown to range
from 13.3 to 219.4 ppm.
The Carbon-13 spectra of estrone and other Eteroid

hormones were studied in several recent papersg0 93. The
tritium nuclear magnetic resonance spectra of estrone and
estrone sulphate have also been studied, yielding
information concerning the distribution of tritium between
labeled sites on the steroidg4,
L
u
0,
Fig. 7. Proton NMR Spectrum of Estrone: Instrument: Varian XL-100

L
9
Fig. 8. Carbon-13 NMR Spectrum of Estrone. Instrument: Varian XL-100

152 DOUGLAS BOTH
TABLE 3
CARBON-13 RESONANCE ASSIGNMENTS
CARBON PPM DOWNFIELD FROM TMS
1 125.5
2 112.5
3 154.7
4 114.8
5 136.8
6 37.9
7 26.0
8 28.9
9 43.3
10 129.7
11 25.4
12 31.1
13 47.2
14 49.7
15 20.9
16 35.2
17 219.4
18 13.3
3.3 MASS SPECTROMETRY
Figure 9 gives the low-resolylion mass spectrum of the
U.S.P. reference standard estrone .
The high-resolution
spectrum is given in Table 4. The molecular ion was found
at m/z 270.1585 while the anticipated molecular ion is m/z
270.1620. The fragmentation seen in Table 4 appears to be
consistant with the structure and mass spectrum of estrone.
TABLE 4
HIGH RESOLUTION MASS SPECTRUM
Mass Found Mass Calc. Formula Compositional Loss
270.1585 270.1626 C18H2202

242.1298 242.1307 16H1802 C2H4
237.1306 237.1279 C17H170 CH50(CH3+H20)
226.1388 226.1357 16H180 C2H40
213.1232 213.1279
15H17' C3H50
UJ
ul
a
3
(3
154 DOUGLAS BOTH
211.1115 211.1123 C3H70 (H20+C3H5)

C15H150
199.1072 199.1123
1qH1'5 C4H70
185.0943 185.0966
13'1 3' C5H90
172.0913 172.0888 C6H100
C12H120
159.0776 159.0810 C7H110
c1lH1lo
146.0702 146.0732 'gH12'
CIOHIOo
144.0542 144.0524 'gH14'
1OH8O
133.0654 133.0653 'gH13'
C9H90
131.051 1 131.0497 'gHISO
C9H70
120.0603 120.0575
'sH8' 10H14'
107.0537 107.0497 'llH15'
C7H70
3.4 INFRARED SPECTROMETRY
The infrared spectrum of the U.S.P. reference standard
estrone is shown in figure 10. The spectrumg7 was obtained
as a solid sample disc composed of 1 mg of estronel300 mg
KBr. Table 5 gives a possible interpretation of the given
spectrum. The interpretation appears to be consistent with
a previous published paperg8.
TABLE 5
IR SPECTRAL INTERPRETATION
FREQUENCY (an-') Assignment
3325 0-H Stretch
3050-3000 C-H Aromatic Stretch

2990-2800 C-H Aliphatic Stretching
1725 C=O Stretch
1620-1580 A dpublet straddling 1600

cm-l and a single peak at 1500
cm indicative of C-C aromatic
stretching and endocyclic bonding
Fig. 10. Infrared Spectrum of Estrone.
156 DOUGLAS BOTH
1475-1350 CH3-C Bending
1285-1250 -
A dpublet straddling 1275
cm - OH bonding and C-0
stretching indicative of aromatic
OH
1200-850 C-C Stretching region

quite complex, showing large
numbers of H-present.
3.5 ULTRAVIOLET AND VISIBLE SPECTROPHOTOMETRY
The ultravioletg9 spectrum of estrone 3n p-dioxane

shows maxima a about 282 nm ( E = 2.37 x 10 ) and 296 nm
5
(E = 2.13 x 10 ). An ethanolic solution of the U.S.P.
reference standard8’ estrone at a concentration of 1 in
25,000 w/v exhibits a maxmium at about 280 nm with a
absorptivity of 2.72. In concentrated sulfuric acid, l o o
strong absorption occurs at about 300 and 450 nm, and in
0.1g sodium h droxide at about 239 and 293 nm. The far
ultravioletluY spectrum of estrone in n-hexane exhibits a
strong absorption at about 200 nm, a shoulder at about 225
nm and a broad band at 275 nm.
The visible spectrum’O 2 of estrone treated with

Engelbrecht-Mori-Anderson cholesterol reagent (E.M.A.) (1.0
g FeCl 6 H 0, 40 mL 85% H PO in 500 ml AcOH, 500 ml H2S04),
scannea a$ 2OoC, showed Lxfma at about 392 and 484 nm.
3.6 OPTICAL ROTATORY DISPERSION AND SPECIFIC

ROTATION
Several optical rotator dispersion studieslo3’ll4

have been reported. EstroneroS exhibits a maximum
absorption at 310 nm with molecular rotations of 157,000 in
a solution of methanol. Estrone shows a negative cotton
effect at 275 nm and a weak positive cotton effect at 225
nm with a molecular rotation of 33,750. The specific
rotation of a 1%solution of the U.S.P. reference standard
estrone determined in a solution of dioxane was +163O.
Estrone has been determined by differential-

spectropolarimetry.l o 6 This method is based on the
difference in the optical activity of estrone and sodium
borohydride reduced estrone (Estradiol). Estrone can be
determined in the range of 30-1200 ug/ml.
ESTRONE 157
3.7 FLUORESCENCE AND PHOSPHORESCENCE
The f l u o r e s c e n ~ eof~ estrone

~ ~ ~ ~ ~in
~ ethanol, when
excited at 280 nm, shows a sharp fluorescence emission peak
at 307 nm and a second, broad peak at approximately 410 nm
with a decay time of 3.8 ns. A broad, emission band at
410 nm is the fluorescence of the carbonyl group, while the
phenolic chromophore emission is at the shorter wavelength.
Most of the excitation energy that is absorbed is due

to the phenolic chromophore. A very efficient energy
transfer to the carbonyl group allows for its weak
fluorescence. In solid film, estrone exhibits no carbonyl
fluorescence. This is believed to be a result of hydrogen
bonding between the phenol hydroxyl and the carbonyl group
in the crystalline film.
The phosphorescence1O9 emission spectrum of estrone

consists of a single broad peak at 410 nm. The quantum
yield of the phosphorescence is 0.025 with a decay time of
2.5 S. The phosphorescence is due to only the phenolic
chromophore.
The fluorescence of estrone may be used to detect its

presence at higher concentrations. However, the intensity
of the fluorescence is not great enough for use in most
cases where estrone is in trace amounts.
Estrogens can produce fluorophores when placed in

solutions of strong acids such as sulfuric and
phosphoric1l0'lZ1. The reaction of estrogens with
1-dimethylaminonaphthalene-5-sulfonyl chloride (Dansyl
Chloride) usually results in a substitution at the carbon
3-position of most estrogens to yield a fluorescent
d e r i v a t i ~ e ' ~ ~ ' ~ This
~ ~ . is best carried out in
acetone-water mixtures at pn 11-12. Under these
conditions, most estrogens show maximum fluorescence
intensity in about 30 minutes at room temperature, with a
limit of detection of about 0.5 pg/ml. A semi-automated
fluorometric method for detecting the total estrogen
content of plasma during late pregnancy has been
reported124.
158 DOUGLAS BOTH
4.0 SOLUTION PROPERTIES
4.1 SOLUBILITY
Table 6 gives the ~ o l u b i l i t y ~

of~ estrone
~ ’ ~ ~ ~in
various solutions and solvents. E p ~ h t e i n lhas
~ ~ derived
equations that demonstrate the correlation between aqueous
solubility and Van der Waals molecular volume. The
solubilization128’129 of estrone in aqueous solutions of
different association colloids has been studied. The
solubilization of estrone in sodium dodecyl sulphate at
40°C and in Tween 20 at 2OoC were reported to be 0.014 and
0.0068 moles of estronel mole of micellar substrate
respectively.
TABLE 6
SOLUBILITY OF ESTRONE (BY G.L.C.)
Solvent Temp OC Solubility mg/mL

tetrahydrofuran 30 48.336
p-dioxane 30 19.200
acetone 30 11.535
chloroform 30 15.680
methylene chloride 30 6.384
absolute ethanol 30 3.516
95% ethanol 40 6.227
methano1 30 4.041
ethyl ether 30 2.127
toluene 30 1.011
cyclohexane 30 0.023
hexane 30 0.004
water 25 0.0008
4.2 PARTITION COEFFICIENTS
Table 7 gives selectedlUo partition coefficients for

estrone in various solvent systems. The partition
coefficient (K) is defined here as the concentration of
solute in the upper phase/concentration of solute in the
lower phase.
4.3 MOLECULAR VOLUME
The average apparent molecular volume130 of estrone in

a solution of methanol at a concentration of 0.0207
lpgles/1000 g of solvent was determined to be 372.5
A /molecule with a precision of 2.5%. It has been shown in
ESTRONE 159
steroids that the average molecular volume per atom of

carbon is constant and that the molecular volume decreases
as the number of substituted hydroxyl groups increases.
TABLE 7
PARTITON COEFFICIENTS
Partition
Solvent System Coefficient(K)
etherll.5M sulfuric acid 100

etherjwater 90
ether/pH 10 carbonate buffer 28
ether/l.OM sodium hydroxide 0.5-0.6
n-hexanejwater 6.8
petroleum etherjwater 3.34
petroleum ether/50% water-
50% methanol 0.06
petroleum ether/30% water-
70% methanol 0.56
40% ethyl acetate-60% n-hexanej
50% ethanol-50% water 2.2
10% ethyl acetate-90% cyclohexanej
50% ethyl acetate-50% cyclohexanel
10% methanol-90% waterjcarbon
tetrachloride 0.01
tetrachloride 0.04
tetrachloride 0.07
40% methanol-60% waterlcarbon
tetrachloride 0.15
tetrachloride 0.33
tetrachloride 1.3
tetrachloride 2.8
70% ethanol-30% waterjcarbon
tetrachloride 0.67
70% ethanol-30% water/5% chloroform-
95% carbon tetrachloride 0.40
70% ethanol-30% water/lO% chloroform-
160 DOUGLAS BOTH

70% ethanol-30% water/20% chloroform-
4.4 HEAT OF FORMATION AND COMBUSTION

The heat of combustion131 for estrone, using a
microbomb calorimeter, was determined to be AHo= 2355.3 2
3.1 Kcal/mole. Using this information the heaf of
formation was calculated to be -88.0 Kcal/mole.
4.5 THE ACID IONIZATION CONSTANT
The reported acid ionization constant (pK ) of estrone

shows great variation ranging from 9.36 to 11.8.
Previous132’1 3 3 methods of measurement included: back
titration, conductimetric and most recently U.V.
spectrophotometric methods. Recent workg9’1 3 4 places the
pKa between 10.34 and 10.914. The most recent
spectrophotometric determination135 reports the K to be
10.77 2 0.02 with seven determinations. Egorova1%a
discussed the correlation between structure and the
dissociation constant.
4.6 STABILITY
Estrone in most cases is a relatively stable compound.
A 0.1% wlw solution of estrone in chloroform was shown to
be stable for approximate1 3 years by TLC, using ten
different solvent s sternslg7. Estrone dissolved in sesame
oil and in showed little change in
physiological activity after six months of storage at room
temperature.
Four ml of blood140 were mixed with 1 ml ACD

stabilizer (2.13 g sodium citrate, 0.74 g citric acid, 2.0
g glucose1100 ml water), 1 ml of this mixture was then
incubated for 10 hours at 37’C with 1 pg of estrone and
estradiol and 10 mg glucose. The degradation of estrone
was twice that of estradiol with fresh blood and half that
of estradiolwith 42 day old blood. In the absence of
glucose degradation of estrone was half that of estradiol
with fresh as well as stored erythrocytes.
Estrone141 is not reduced by enzyme extracts of hog
ovaries, beef suprarenal glands or bull testes.
ESTRONE 161
Estrone dissolved in absolute alcohol decomposes when

exposed to ultraviolet radiation. When estrone in
d i o ~ a n e lis
~ ~irradiated with ultra-violet light at 313 nm
it forms 13a-estrone (lumiestrone), which is reversible
when unfiltered ultraviolet light is used. Creepage143 of
estrogens on glassware occurs only in uncovered vessels in
the presence of salts and absence of proteins. Larger
amounts of creepage occurs in freshly cleaned vessels and
when small volumes of concentrated solutions are placed in
larger vessels. No sorption onto glass from buffered
aqueous solution or decomposition in tightly closed
containers in the absence of proteins have been found.
Silanization of glassware can drastically reduce creepage.
The ~ t a b i l i t y ’ ~ ~of
’ ’estrone
~~ on TLC plates has been
studied. Estrone decomposes as a result of contaminants
present in air and not a result of the oxadative effect of
oxygen to any great extent.
5.0 CHROMATOGRAPHIC AND OTHER SEPARATION BASED

ANALYSIS
5.1 COLUMN CHROMATOGRAPHY
Estrone has been separated from other estrogens in a

variety of matrices using column chromatography. Column
chromatography has also been used to concentrate or
separate prior to analysis by HPLC, G.C., RIA or TLC.
Thus, column chromatography serves to remove interfering
compounds and to concentrate prior to detection by a more
sensitive method. Table 8 gives selected examples of
column chromatographic procedures.
TABLE 8
SELECTED COLUMN CHROMATOGRAPHIC PROCEDURES
Reference Description
145 Separation prior to G.C., TLC,

using anion exchange resin (AGl-X2 cl),
elution with a methanollwater solution.
146 Separation of estrogens prior to G.C.

on AGlX2 column, methanol water
elution.
147 Purification after hydrolysis on

Merckogel 6000, followed by separation
on Sephadex LH 20, elution with
162 DOUGLAS BOTH
heptane-chloroform-ethanol-water
mixture.
148 Separation of conjugated urinary

estrogens on Sephadex G-15, elution
with 0.01M ammonium formate.
149 Separation of estrogen conjugates on

DEAE-Sephadex using gradient elution
(0-0.4g) sodium chloride.
150 Separation of androgens, estrogens, and
progestrogens on Lipidex elution with
hexane-chloroform mixture.
151 Separation of Sephadex LH-20 using
methylene chloride elution.
152 Separation of 14 testicular steroids

using celite column prior to HPLC.
153 Analysis of steroid mixtures using

silicic acid column eluted with
gradient of ethyl ether.
154 Extraction of estrogen conjugates from

pregnancy urine using amberlite XAD-2
resin and elution with 30% ethyl
alcohol.
155 Separation of free estrogens on

Sephadex LH-20 eluting with
cyclohexane-benzene-methanol mixture,
156 Purification of urine for

quantification of complete estrogen
profile, using Sephadex G-25,
DEAE-Sephadex A-25, Sephadex LH-20 and
DEAE Sephadex A-25 followed by G.C. or
selective ion monitoring.
157 Separation of C CI9 and C steroids
using Sephadex 2A120 and eluied with
n-hexane-ethyl acetate-methanol
mixture.
ESTRONE 163
158 Separation of U.V. absorbing

constituents in urine on Diaion CDR-10
using a linear gradient of ammonium
acetate (0-6M).
159 A two step anion exchange separation

for the purification of estrogens using
DEAE-Sephadex A-25 prior to capillary
gas chromatography.
160 Separation of radioactive steroids and

steroid conjugates from urine using
Amberlite XAD-2 and DEAE- Sephadex A-25
with NaCl gradient elution.
161 Separation of conjugated estrogens in

urine using Sephadex G-25,
DEAE-Sephadex and Sephadex G-15.
162 Separation of estrone, estradiol, and

estriol on florisil 60 mesh column,
elution with methyl chloride- ethanol
solution.
163 Separation of steroid hormones from

plasma using a Merck extrelut column.
Elution with ethyl ether.
5.2 THIN LAYER CHROMATOGRAPHY
Thin layer chromatography (TLC) can be used to

separate complex mixtures or provide inital sample
purification for further more sensitive separation and
detection. Reviews of TLC steroid methods are found in
r e f e r e n ~ e s l ~ ~ ’Sander166
~~~. describes the theory and
applications of reverse phase TLC. Hais167 has studied the
relationship between chemical structure and TLC sequence in
single and multicomponent systems for the separation of a
group of 6-estrane and 10-androstane derivatives. Table 9
summarizes selected TLC separation methods.
TABLE 9
TLC SEPARATION METHODS
168 Separation and purification of urinary

estrogens on silica gel H-ascorbic
164 DOUGLAS BOTH
acid, developed in benzene containing

5 % ethanol, detection by gas
chromatography.
169 Silica gel containing ethanolic

ammonium bisulfate, development in
benzene-ethanol (85:15) using a
spectrodensitometer for detection.
170 Detection using an automatic

conductivity detector.
171 Separation of steroid mixtures on

silica gel G or aluminum oxide G with
phosphor 6-115 added to coating.
172 Separation of steroids on starch bound

silica gel using phosphomolybdic acid
visualization.
173 Separation of free estrogens and

estrogen acetates on silica g e l
containing dichlorofluoroscein, using a
15% ethanol in benzene and 10%
isopropyl ether in benzene system.
174 Separation of a variety of steroid

hormones on silica gel containing 5%
gypsum with development in a variety of
solvent systems.
175 Separation of free steroids and steroid

heptafluorobutyrates on silica gel G
and GF using benzene: ethyl acetate
(60:40), (9O:lO) and chloroform:
acetone (95:s).
176 Separation of steroids on Gelman sheets

in a chloroform: acetone (30:l) system
using silicotungstic acid
visualization.
177 Detection of estrogens on silica gel by

coupling the estrogens to the diazonium
compound fast dark blue R salt.
Development twice in diethyl
ether-cyclohexane (80:ZO).
ESTRONE 165
178 Simultaneoug separation of common

mammalian A -3-oxosteroids and
estrogens of adrenal, testicular,
ovarian and placental origin.
179 Separation of estrogens on silica gel G

using one and two dimensional develop-
ment, in five different solvent
systems.
180 Separation of antithyroid drugs and

estrogens from animal tissue extracts
by HPTLC with a detection limit of
10-200 ng.
181 Determination of separated estrogens on

silica gel by measurement of their
dansyl derivative fluorscence.
182 Identification of nine major components

of conjugated estrogens on Kieselguhr G
plates containing sodium hydroxide and
impregnated with formamide.
183 Separation of estrogens, androgens,

gestogens and corticoids on dimethyl-,
octyl- and octadecylsilyl silica gel
using a methanol-water solvent system.
5.3 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
Estrone and many steroids exhibit rather strong U.V.

absorption. However, U.V. detection of these compounds in
physiological media is often limited because they are
present in low concentration, and the media often contain
other compounds which interfere with simple U.V. detection.
Thus for many other methods the sample must be pure or
highly concentrated. It is for these reasons the HPLC
analysis of steroid hormones is a preferred method. HPLC
analysis allows for sensitive detection on relatively
"dirty" samples. A simple preparative HPLC column
connected to a fraction collector can clean and concentrate
even the "dirtiest" of samples.
Derivatization can greatly enhance the sensitivity for

detection by resulting in improved U . V . absorption or
fluorescence, or altered retention time or oxidation
potential to allow for electrochemical detection. The
166 DOUGLAS BOTH
great number of combinations that can be achieved by

varying the parameters of mobile phase, column and detector
allow for the separation and detection gf complex mixtures
of steroid hormones. Several papersla4 186 review the
analysis of estrogens by HPLC. Table 10 gives selected
HPLC methods for the analysis of estrone and steroid
mixtures containing estrone.
TABLE 10
SELECTED HPLC METHODS
187 Analysis of estrogens in tablet and

injectable forms by measurement of
their dansyl derivatives.
188 Separation of esterified estrogens in

bulk mixtures and combination drug
preparations by reverse phase HPLC.
189 Analysis of estrogens in pregnancy

urine .
190 Separation of estrogen conjugates using
strong anion exchanger column.
191 Separation of 14 testicular steroids

including estrone by reverse phase
HPLC.
192 Determination of trace estrogenic
hormones using voltammetric detection.
193 Separation of various estrogen mixtures

by reverse and normal phase.
194 Separation of estrogen mixtures using a

mobile phase containing silver nitrate.
195 Separation of catechol estrogens and

detection with electrochemical
detector.
196 High speed L.C. separation of equine

estrogens including estrone.
ESTRONE 167
197 Purification of 19 hormonal steroids

prior to immunoassay.
198 Retention behavior for 43 steroids on

bonded reverse phase systems.
199 Reverse phase determination of

estrogens.
200 Separation of estrogens in primate

urine.
201 Determination of unconjugated estrogens

in amniotic fluid using an amperometric
detector.
5.4 GAS-LIQUID CHROMATOGRAPHY
Numerous analytical methods for the gas

chromatographic analysis of estrone and estrogen mixtures
containing estrone have been reported. Most methods
utilize a glass column typically 1-3 meters in length or a
glass capillary column 20-30 meters long. Estrone is
considered to be thermally stable, therefore instability on
the column appears to be of little concern. It is usually
the stability of other compounds of interest that quite
often limit the column operating temperature. Most
analyses are run at column temperatures of 140-260°C.
Estrone has been chromatographed in derivatized and

free forms. The derivatized forms offer improved thermal
stability, lower detection limits and decreased chance of
irreversible absorptivity losses. Therefore they are quite
suited for use with selective detectors such as electron
capture. Estrone has been frequently detected as
heptafluorobutyrate, chloroacetate, trimethylsilyl ethers,
trifluoroacetates and methoxamine-trimethylsilyl
derivatives.
Each derivative is useful in specific applications,

but not all derivatives are of equal value in a given
separation and quantitation. These same comments apply to
the many types of column supports and stationary phases.
In general, the high performance, acid washed, silanized
supports with particle sizes of 80-120 mesh having 2500 or
more theoretical plates appear to work well. Many
stationary phases which appear useful generally are those
168 DOUGLAS BOTH
of medium olarity and higher thermal stability. Several

papers202'506 review solid supports for steroid analysis.
Several papers185'207-214 review the separation and

identification of steroids. Papers which review retention
times of various steroid derivatives and derivative
applications include references 215-226. Table 11 gives
selected methods of analysis of various estrone containing
samples.
TABLE 11
SELECTED GAS-LIQUID CHROMATOGRAPHY METHODS
227 Estrogen separation from human urine as

-
0-methyloxime-heptafluorobutyrate
derivatives.
228 Separation of submicrogram amounts of

steroid hormones using non-selective
stationary phase.
229 Separation of estrogen stereoisomers as

heptafluorobutyric derivatives using a
completely automated splitless glass
capillary system.
230 Separation of estrogens using a solid

injection system.
23 1 Plasma estrogen measurement as

heptafluorobutyrate derivatives.
232 Urinary estrogen determination on

non-pregnancy urine. Extraction and
enzymatic hydrolysis followed by
silylation of concentrate.
233 Quantitation of urinary estrogens

throughout pregnancy. Enzymatic
hydrolysis and ion-exchange followed by
derivatization as heptafluorobutyric
anhydride derivatives.
234 Analysis of estrone in dermatological

products using internal-external
standard ratioing. Cream or lotion
ESTR0NE 169
analysis by hydroxide extraction and

filtration prior to G.C.
235 Analysis of conjugated estrogens using

dual derivatization and dual injection.
Enzyme hydrolysis and derivatization as
trimethylsilyl and methoxamine-
trimethylsilyl steroids.
236 Resolution of equine estrogens using a

short glass capillary column coated
with Silar 1OC. Detection of
oxime-trimethylsilyl ether derivatives
using a flame ionization detector.
5.5 GAS CHROMATOGRAPHY-MASS SPECTROMETRY
-
Combined gas chromatography mass spectrometry
(GC-MS) using the technique of selective ion monitoring or
fragmentography has been applied for the detection of
estrogens in the lower picogram range.
A typical method involves extraction and

derivatization of the estrogens followed by separation by
gas chromatography and detection using the mass
spectrometer as a detector by monitoring selected peaks of
the mass spectrum.
It was shown237 that the methyl and trimethylsilyl

ether estrogen derivatives of fully trimethylsilylated
estrogen derivatives are more suitable than methyl ether,
acetate or trifluoroacetate estrogen derivatives for use in
combined GC-MS. Several selected methods are listed in
Table 12.
TABLE 12
SELECTED COMBINED G.C.- M.S. METHODS
238 Determination of estrogens in the lower

picogram range using isotopically
labeled internal standards.
239 Comparison of a radio-gas

chromatography method for estrogens in
tissue to G.C. -
M.S. method.
170 DOUGLAS BOTH
240 Determination of steroid hormones in

plasma and urine.
241 Determination of unconjugated estrone,

178-estradiol and estriol in blood.
242 Identification of estrogens isolated

from pregnant mares' urine.
243 Determination of interfering estrogenic

compounds in tablet
preparations of conjugated and
esterified estrogens.
244 Determination of estrone and

178-estradiol in seminal plasma of man,
bull and boar.
6.0 RADIOASSAY
Radioimmunoassay (RIA) methods for the determination

of estrone in biological samples are quite sensitive.
Detection limits for estrone are in the picogram range,
with the use of high affinityfspecific antisera. There are
a great number of methods for the assay of estrone and
other steroid hormones in the literature, Table 13 lists
selected methods. Presently radioimmunoassay is the most
widely used technique for the quantitation of picogram
quantities of steroid hormones. However, because of the
inconveniences associated with the use and disposal of
radioactive compounds and the high per assay costs,
enzymeimmunoassay and fluorescence immunoassay methods have
been increasing in popularity. For recent reviews on
fluorescence-, enzyme- and radio-, immunoassay methods see
references (245-253).
TABLE 13
SELECTED RADIOASSAY METHODS
Referenre Comment
254 Non-chromatographic RIA for total

estrone in plasma.

178-estradiol in pregnant and
non-pregnant plasma.
ESTRONE 171

178-estradiol in urine.
257 Determination of total estrogens in

urine.
258 Non-chromatographic R I A for estrone and

17B-estradiol in plasma.
259 Solid phase R I A for estrone and

178-estradiol in plasma.
260 Total estrone in non-pregnant

peripheral plasma.
261 Determination of estrone in male

saliva.
262 Determination of estrone and equilin in

plasma after administration of a
conjugated equine estrogen preparation.
263 Determination of estrone and estradiol

in plasma.
264 Determination of estrone 178-estradio1,

estriol, testosterone,
5a-dihydrotestosterone and
androstenedione in plasma after
extraction and separation.
265 Sequential R I A for unconjugated and

conjugated estrone, 178-estradiol and
estriol in male plasma.
266 Determination of total urinary estriol.
267 Simultaneous R I A for progestins,

androgens and estrogens in rat testis.
268 Determination of oestrone in the plasma

of rhesus monkey.
269 Determination of estrone, equilin and

dehydroepiandrosterone in peripheral
plasma of pregnant pony mares.
172 DOUOLAS BOTH
270 Enzyme immunoassay for total estrogens

in pregnancy plasma.
271 Determination of estrone and estradiol

in bovine peripheral plasma.
272 RIA of estrone and 17B-estradiol

comparison of method with fluorimetry.
273 RIA of estrone in plasma comparison of

different methods.
274 Simultaneous determination of six

steroids in plasma.
275 Separation and extraction prior to RIA

for estrone, 17B-estradiol and
17a-estradiol in plasma.
276 Determination nf estrone, 17a-estradiol

or 176-estradiol in human and ruminant
plasma.
277 Simultaneous measurement of five

steroids in avian plasma.
278 Immunoenzymological assay of steroids

in plasma.
279 Non-chromatographic determination of

unconjugated estrone, 17B-estradiol and
estriol in plasma.
280 Determination of steroids in plasma of

the monkey.
281 Determination of conjugated estrogens

in plasma of cows.
7.0 COLORIMETRIC ANALYSIS
Colorimetric methods of analysis were once very

popular, but with the advent of more sensitive separative
and instrumential methods, colorimetric analysis today is
of less importance.
Table 14 gives s e l e ~ t e d ~colorimetric

~ ~ ’ ~ ~ ~ reactions.
It should be noted that these colorimetric reactions
ESTRONE 173
generally are not selective to estrone, and reaction with

other steroids is quite possible.
TABLE 14
SELECTED COLORIMETRIC REACTIONS
Reaction with 2,4-dinitrophehylhydrazine and nitromethane

at 100°C for 15 min, Xmax = 565 nm, 13 pg gives 0.3A.
Reaction with 4-nitrophenylhydrazine and

benzyltrimethylammonium hydroxide to give a pink color.
Xmax - 530 nm, 245 ug gives 0.3A.
Reaction of 2,4-dinitrophenylhydrazine and sodium

hydroxide. Amax = 420 nm, 64 pg gives 0.3A.
Reaction with 1:l mixture of 0.5% 1-nitroso-2-napthol in

ethanol and 0.05% sodium nitrate in 3.5 g nitric acid.
Xmax = 450 nm, 11Opg gives 0.3A.
Reaction of diazobenzene-p-sulfonyl chloride in alkaline

solution. Xmax = 510 nm.
Reaction with a 1:l mixture of 0.6% aqueous potassium

ferricyanide and 0.9% aqueous ferric chloride hexahydrate.
Xmax = 720 nm, 11 ug gives 0.3A.
Zimmerman reaction - 1% solution of 3,5-dinitrobenzoic acid

in 40% aqueous solution of benzyltrimethylammonium
hydroxide. Xmax = 530 nm, 175 pg gives 0.3A.
Reaction with salicyloylhydrazide to yield blue

fluorescence, excitation at 340 nm, emission at 420 nm.
Reaction with 1,3,5-trinitrobenzene Xmax = 475 nm, 110 Vg
gives 0.3A.
Reaction with 3,5-dinitrobenzoic acid in aqueous

benzyltrimethylammonium hydroxide solution, Xmax = 530 nm.
Reaction at 100°C in a 10% solution of potassium

guaiacolsulphonate in concentrated sulphuric acid. Xmax =
500 nm.
Reaction when heated in the presence of a 2% soluton of

hydroquinone in 66% sulphuric acid. Xmax = 500-550 nm.
174 DOUGLAS BOTH
8.0 TITRIMETRIC ANALYSIS
Estrone was determined285 in pharuceutical

preperations by titration after extraction with ethanol and
alkalinizaiton with sodium hydroxide to pH 11. It was
first complexed with lead citrate and then determined by
back titration with EDTA to a eriochrome black end point.
Estrone was also determined286 in the parts per

million range by titration with bromine in a methanol-water
solution containing hydrochloric acid and sodium bromide
using biamperometric end point detection.
9.0 ACKNOWLEDGEMENTS
I wish to thank Dr. Mira Szyper for her in-depth

critical reading and comments. The comments and reading of
Dr. Glenn Brewer, Mr. Ray Poet, Dr. Joel Kirschbaum,
Mr. Solomon Perlman, Dr. Jack Isidor, Dr. Mohammed Jemal
and Mr. Richard Koski are greatly appreciated.
Special thanks to Quentin Ochs for the x-ray

diffraction work, Dr. Michael Porubcan for the IR, Gloria
Jennings for the NMR and Dr. Phillip Funk for the mass
spectroscopy work. Special thanks to Dr. Sy-Rong Sun,
Director of the USP Drug Research and Testing Laboratory,
for suppling information concerning the USP Standard
Estrone.
Special thanks to Arminda Rubial for her translation

of several important papers. Thanks to Phyllis Gottstine
and Muriel George for getting those hard to get references.
10.0 References
1. A.Q. Maisel, "Hormone Quest," Random House, N.Y.

(1965)
2. E. Allen, J. h e r . Med. Assoc. 81, 819 (1923)
3. S . Aschheim et al., Klin. Woschr., 5, 1322 (1927)
4. E.A. Doisy et al., her. J. Physiol., 90, 2 (1929)
5. A. Butenandt, Naturwissenschaften, 17,879 (1929)
6. A. Butenandt, Trends Biochem. Sci., A, 215 (1979)

ESTRONE 175
7. S.A. Thayer et al., Proc. SOC. Exp. Biol. A. Medi,

735 (1930)
8. H. Inhoffen, Ber., 74, 1911 (1941)
9. G. Anner et al., Experientia., 4, 25 (1948)
10. El S . Amin, Phytochemistry, 8(1), 295 (1969)
11. A. Gawienowski et al., w.,80,685 (1969)
12. E.P. Haussler, Festschrift E.C. Barell, 327 (1936)
13. W. Velle et al., Acta Endocr., 33, 277 (1960)
14. EL S. Amin et al., Phytochemistry, 18(2), 344 (1979)
15. D. Beall, Nature, -

144, 79 (1939)
16. W. Westerfield et al., J. Biol. Chem., 126, 195

(1938)
17. D. Beall, Biochem. J., 34, 1293 (1940)
18. M.H. Briggs, "Steroid Biochemistry and Pharmacology",

Academic Press, N.Y. (1970)
19. W.R. Butt, "Hormone Chemistry", 2nd ed, Wiley, N.Y.

(1975)
20. D. Greenberg, "Metabolic Pathways" Vol. 2, 3rd ed,

Academic Press, N.Y. (1968)
!1. B.R. Bhavnani et al., "Steroid Biochemistry," CRC

Press, Boca Raton, Florida (19791, pp 1-51
22. T. Omura et al., Federation Proc., 24, 1181 (1965)
23. E.D. Plotka et al., Clin. Chem. Acta, 3, 287 (1973)
24. A.A. Sandberg, Recent Prog. Horomone Res., 13,209

(1957)
25. A. Sneddon, Biochem. J., 86, 385 (1963)
26. P.I. Mussey. "Formation and Metabolism of Steroid

Conjugates", CRC Press, Boca Raton, Florida (1979)
176 DOUGLAS BOTH
27. K.D. Manchester, "Scientific Tables," 6th ed,

CIBA-Geigy Limited, Arsley, N.Y. (1962)
28. K.J. Ryan et al., Endocr., 52, 277 (1953)
29. W. Velle et al., Acta Endocr. Copenh., 33, 277 (1960)
30. S. Ichii et al., Anal. Biochem., 5, 422 (1963)
31. Pochi et al, J. Clin. Endocr., 25, 1660 (1965)
32. A.A. Sandberg et al., 18,253

B., (1958)
33. K. Lisse, Endokeinologie., 55 (3-4), 145 (1969)
34. J.B. Brown, Mem. SOC. Endocr., 2, 1 (1955)
35. J.B. Brown, et.al., Recent Prog. Hormone Res., 2,

337 (1962)
36. H. Inhoffen, -
Ber., 74, 1911 (1941)
37. G. Anner et al., Experientia., A, 25 (1948)
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(1980)
ETOMIDATE
Zui L. Chang and Joseph €3. Martin
1. Description 192
I , 1 Nomenclature 192
1.2 Formulas and Molecular Weight 192
2.2 Proton Magnetic Resonance Spectrum (PMR) 194
2.4 Raman Spectrum 198
2.5 Ultraviolet Spectrum (UV) 20 1
2.6 Solubility 20 1
2.7 X-Ray Powder Diffraction 20 1
2.9 Differential Thermal Analysis 203
2.10 Specific Optical Rotation 205
3. Synthesis 205
5. Method of Analysis 208
5.3 Chromatographic Analysis 209
5.4 Titrimetry 210
6. Analysis of Pharmaceutical Formulations 21 1
6.1 Spectrophotometric Method 21 1
6.2 High Performance Liquid Chromatography (HPLC) 21 1
8. Determination of Etomidate in Biological Fluids 212
References 213
ANALYTICAL PROFILESOF DRUG SUBSTANCES 191 Copyright hy the American Pharmaccurlcal Associatian.
VOLUME 12 ISBN 0-12-260812-7
192 ZUI L. CHANG AND JOSEPH B. MARTIN
1. Description
1.1 Nomenclature
1.11 Chemical Name
(K )- (+)e t h y l 1- (l-phenyle t h y l )-lH-imidazole-5-

car box y l a t e
1.12 Generic Name
E t omida t e .
1.2 Formulas and Molecular Weight
CHCH3
I
C14H16N202 M.W. 244.29
1.3 Appearance, C o l o r , Odor
Etomidate is a f i n e w h i t e powder w i t h no d i s c e r n i b l e
odor.
2.1 I n f r a r e d Spectrum
The i n f r a r e d spectrum of Etomidate i s p r e s e n t e d i n

Figure 1. The spectrum was measured i n t h e s o l i d
s t a t e as a potassium brani.de d i s p e r s i o n . The fol-
lowing bands (cm'l) have been a s s i g n e d f o r F i g u r e
1 (1).
a. 3128 and 3100 cm'l Two bands due t o t h e C-H
t h e imidazole r i n g .
0
B
8
Y
0
0
0 -
C I -
-'s*
U
U
51s
0 s
* E
0
8
m
0
5:
m
0
0
0
9
0
e
0
t
0
9
0
094q*
0 - -
b. 3028 and 3062 cm-1 Two weak bands due t o t h e

C-H s t r e t c h i n g v i b r a t i o n s
of t h e benzene r i n g .
C. Between 3000 and Complex of weak t o medium

2850 cm-1 bands due t o t h e C-H
t h e methylene and methyl
g rou p s .
d. 1700 cm-1 Strong band due t o t h e C=O
s t r e t c h i n g v i b r a t i o n of
t h e a,$ u n s a t u r a t e d e s t e r
group.
e. 1597 and 1580 cm-1 Two week bands due t o t h e

skeletal s t r e t c h i n g v i b r a -
t i o n s of t h e benzene r i n g .
f. 1518 cm-1 Probably due t o t h e skel-

eta1 stretching vibration
of t h e imidazole r i n g .
g* 1205 cm-1 Strong band due t o t h e C-0

t h e a,$ u n s a t u r a t e d ester.
h. 765 and 710 cm'l Two bands due t o t h e C-H

o u t of p l a n e bending v i -
b r a t i o n s of t h e monosub-
s t i t u t e d benzene r i n g .
2.2 P r o t o n Magnetic Resonance Spectrum (PMR)
The proton magnetic resonance spectrum of e t o m i d a t e

as shown i n F i g u r e 2 w a s o b t a i n e d on a Varian As-
s o c i a t e s HA-100 PMR Spectrometer as a 10% w/v s o l u -
t i o n i n a s o l v e n t of d e u t e r a t e d chloroform. The
s p e c t r a l peak assignments ( 2 ) a r e presented i n Table
I.
Figure 2. Proton Magnetic Resonance Spectrum of Etomidate
Table I
PMR S p e c t r a l Assignments f o r Etomidate
Proton
Assignment Chemical S h i f t (ppm) Multiplicity
7.74 Doublet
7.78
7.1-7.4 Mu1t i p l e t
N-CH-Ar 6.37 Quartet
OCH2 4.23 Quartet
1.83 Doublet
1.27 Triplet
2.3 Mass Spectrum
The mass spectrum of etomidate as shown i n F i g u r e 3

was o b t a i n e d u s i n g a n Associated E l e c t r i c a l I n d u s t r i e s
Model MS-902 Mass Spectrometer w i t h t h e i o n i z a t i o n
e l e c t r o n beam energy of 70 eV. High r e s o l u t i o n data
were compiled and t a b u l a t e d w i t h t h e a i d of a n on-
l i n e PDP-11 Computer.
The mass spectrum assignments of t h e prominent i o n s

and subsequent fragments a r e shown i n Table I1 and
F i g u r e 4 (3).
8
cy
In
1
0
ul
0
AllSN3lNI 3hllVl311
T a b l e I1
High R e s o l u t i o n Mass Spectrum of Etomidate
Measured Mass (m/e) C a l c u l a t e d Mass
77.0392 7 7.0391
78.0467 78.0470
79.0545 7 9.0548
95.0242 95.0246
103.0543 103.0547
104.0621 104.0626
105.0699 105.0704
199.0884 199.0872
244.1218 244.1212
2.4 Raman Spectrum
The Raman s p e c t r u m of e t o m i d a t e as shown i n F i g u r e 5

was o b t a i n e d i n t h e s o l i d s t a t e on a Cary Model 83
S p e c t r o m e t e r . The f o l l o w i n g bands (cm-l) have been
a s s i g n e d f o r F i g u r e 5 (1).
a. 3135 and 3103 cm-1 Two bands due t o t h e C-H

t h e imidazole ring.
b. 3018 and 3070 cm-1 Two bands due t o t h e C-H

t h e benzene r i n g .
c. 3000 a n d 2850 cm-1 Complex of bands due t o

the stretching vibrations
of t h e methylene and
methyl groups.
d. 1710 cm-1 S t r o n g band due t o t h e C=O

t h e a,B u n s a t u r a t e d e s t e r
group.
Figure 4. Fragmentation Pathways of Etomidate
0 0 0 0
0
0 a 9 0 N 0
0
N
0
0
0
0
0
9
0
0
m
0
0
P
0
0
2
0 -
0
-cr
0
0
9
0
!i!
0
0
0
N
0
0
t
N
0
0
a
N
0
0
N
W
0
0
9
W
0
# 1 1 I 1 0
0
0 0 0 0 0 P
2 m 9 0 N
AlISN31NI
ETOMIDATE 201
e. 1610 and 1590 cm'l Two bands due t o t h e skel-

etal stretching vibrations
of t h e benzene r i n g .
f. 1528 cm'l Probably due t o t h e s k e l -

etal s t r e t c h i n g v i b r a t i o n s
of t h e imidazole r i n g .
g. 1009 and 992 cm'l Strong d o u b l e t c h a r a c t e r -

i s t i c of a m o n o s u b s t i t u t e d
benzene r i n g .
2.5 U l t r a v i o l e t Spectrum (UV)
When t h e UV spectrum of 0.001% s o l u t i o n of e t o m i d a t e

i n i s o p r o p a n o l s o l u t i o n was scanned from 400 t o 200
nm, one maximum a t 240 nm (€= 12,200) w a s observed
( F i g u r e 6).
The spectrum was o b t a i n e d w i t h a Beckman Acta V

Spectrophotometer.
2.6 Solubility
The f o l l o w i n g s o l u b i l i t y d a t a have been determined

f o r e t o m i d a t e a t room t e m p e r a t u r e (4):
Solvent mg Etomidate/100 m l S o l v e n t
Water 0.0045
0.01 N H C 1 0.30
0.1N-HC~ 2.09
Hexane 1.29
Chloroform Greater t h a n 100
Methanol Greater than 100
Ethanol Greater t h a n 100
I s o p r opanol 90-100
Propylene Glycol Greater t h a n 100
Diethyl Ether 23.8
Ace tone Greater t h a n 100
E t h y l Acetate 90-100
Benzene 90-100
2.7 X-Ray Powder 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 of e t o m i d a t e

w a s determined by v i s u a l o b s e r v a t i o n of a f i l m
0.6
0.5
0A
b
'
z
b
Y)
m
0.3
0.2
0.1
200 250 300 350

WAVELENGTH (nm)
Figure 6. Ultraviolet Spectrum of Etomidate

ETOMIDATE 203
o b t a i n e d w i t h a 143.2 mi Debye-Scherrer Powder Cam-

e r a (Table I V ) . An Enraf-Nonius D i f r a c t i s 601 Gen-
e r a t o r ; 38 KV and 18 MA w i t h n i c k e l f i l t e r e d copper
r a d i a t i o n ; X = 1.5418, w a s employed ( 5 ) .
Table I11
X-Ray Powder D i f f r a c t i o n P a t t e r n of Etomidate

d-Spacings and I n t e n s i t i e s
0
d(A>* do* a**

a**
9.3 15 2.87 5
7.0 50 2.80 20
6.1 40 2.63 10
5.65 100 2.54 3
5.25 40 2.42 5
4.85 25 2.39 3
4.63 80 2.32 2
4.30 25 2.27 2
4.08 30 2.21 2
3.99 40 2.16 2
3.90 35 2.13 1
3.80 10 2.07 2
3.72 10 1.99 5
3.64 40 1.92 4
3.50 5 1.89 5
3.37 4 1.82 2
3.28 10 1.80B 2
3.20 5 1.74 2
3.03 20 1.71 2
*d = I n t e r p l a n a r d i s t a n c e .
**1/11 = R e l a t i v e i n t e n s i t y (based on t h e h i g h e s t
i n t e n s i t y of 1 0 0 ) .
2.8 M e l t i n g Range
Etomidate melts i n t h e range of 67.0 and 69.0"C.
2.9 D i f f e r e n t i a l Thermal A n a l v s i s
A s h a r p endothermic peak a t 66.5"C i s i n d i c a t i v e of

t h e m e l t i n g p o i n t of e t o m i d a t e ( F i g u r e 7 ) .
I I I I I
0 20 40 60 80 100 120 140 160 180 200
1 "C (CORRECTED FOR CHROME1 ALUMEL THERMOCOUPLES)
Figure 7. Differential Thermal Analysis C u r v e of Etomidate

ETOMIDATE 205
2.10 Specific Optical Rotation
The o p t i c a l r o t a t i o n s of 1%etomidate i n 12
s o l v e n t s measured w i t h a sodium lamp a t 589 nm
and with a mercury lamp a t 570, 546, 436 and 365
nm are summarized i n Table I V ( 4 ) .
Table I V
O p t i c a l Rotations* of Etomidate i n Various

S o l v e n t s a t Various Wavelengths
589 nm 578 nm 546 nm 436 m 365 nm

N a H g H g H g Hg
0.1 N HC1 +33.61 +3 5.31 + 40.56 + 73.17 +123.88
c kl0’;rofo nn +51.81 +54.55 + 62.89 +117.16 +208* 25
i so p r opanol +64.52 +67.77 + 78.21 +146* 9 8 +2 64.64
ethanol +69.49 +72.96 + 84.07 +15 7.63 +283.29
me t hanol +69.44 +72.94 + 84.23 +15 8.2 2 +284.75
hexane +7 3.04 +7 6.69 + 88.43 +163.37 +286.50
MIK +78.85 +82.80 + 95.64 +179.43 +320.69
ethylacetate +80* 42 +84.56 + 97.76 +183.32 +328.31
dime thyl- +80.83 +84.98 + 98.18 +185.86 +337.62
ace t amide
acetone +82.37 +86.52 + 99.74 +186.87 +334.74
d i e th y l e t h e r +84.89 +89.09 +102.49 +189.38 +332.57
benzene +90.01 +94.50 +108.94 +201.69 +3 54.29
*Mean value of 2 measurements.
3. Synthesis
Etoniidate may be s y n t h e s i z e d by t h e r e a c t i o n scheme shown

i n Figure 8 ( 4 ) .
4. Stability-Degradation
Etomidate has been r e p o r t e d t o hydrolyze i n s o l u t i o n t o

t h e f r e e a c i d ( 4 ) a s shown i n F i g u r e 9.
N , N-Dimethyformamide (C2H5)3N
0 ; H - N . - CH?- COOC2H5
CH3 R-(+)
Xylene
II HCOOH
t
0-- 7H
I
-
I
N CH2 COOC2H5
CH, R-(+)
Na O C 2H5 H C O O C 2H.j
H ‘c40
~ ~ H - N - C HI - C O O C ~ H S
1
CH3 o// C \ H R-(+l
Figure 8. Synthetic Pathway of Etomidate

ETOMIDATE 207
FIGURE 8 (Continued)
-N-I CH-
I
COOC~HS
iK.*H
CH3 CH2 0 C
I
H-C-CH3 R-(t)
I
$ -CH3
0
H- R-(t)
Etomidote
Figure 9 - Hydrolysis of Etomidate
CH 3CH 2OC q-JHt HOC y-3 I

I
CHCH, CHCH
I
R-(+) e t h y l 1-( l-phenylethyl- R- (+) 1-(1-phenylethy1)-

1H-imidazole-5-carboxylate 1H-imidazole-5-carboxylic
(E tomida t e ) acid
(Etomidate F r e e Acid)
Under mre d r a s t i c c o n d i t i o n s of r e f l u x i n a c i d systems,

t h e h y d r o l y s i s i s a c c e l e r a t e d . Almost t o t a l d e g r a d a t i o n
t o o t h e r products o c c u r s i n s t r o n g base r e f l u x . I n neu-
t r a l r e f l u x , a s w e l l as under exposure t o h e a t and l i g h t ,
very l i t t l e d e g r a d a t i o n (<O. 5%) occurs.
5. Method of A n a l y s i s
5.1 Identification
Etomidate may b e i d e n t i f i e d by i n f r a r e d spectropho-

tonretry and u l t r a v i o l e t spectroscopy. The charac-
t e r i s t i c bands i n t h e i n f r a r e d spectrum ( F i g u r e 1)
of etomidate are given i n S e c t i o n 2.1. The u l t r a -
v i o l e t spectrum of etomidate i s shown i n F i g u r e 6.
ETOMIDATE 209
A t y p i c a l elemental a n a l y s i s of a sample of etomi-

d a t e i s p r e s e n t e d i n Table V I I .
Table V I I
Elemental A n a l y s i s of Etomidate
Element Theory Found
Carbon 68.83% 68.70%

Hydrogen 6.60 6.63
Oxygen 13.10 13.23
5.3 Chromatographic A n a l y s i s
A chloroform:methanol:ammonium hydroxide s y s -
t e m (100:100:2) h a s been used t o induce t h e
m i g r a t i o n of t h e compound and shortwave ul-
t r a v i o l e t l i g h t was used t o d e t e c t t h e etomi-
d a t e and d e g r a d a t i o n product. The Rf f o r
e t o m i d a t e was 0.74. The major d e g r a d a t i o n
product, e t o m i d a t e a c i d , i f p r e s e n t has a n Rf
of 0.45.
5.32 High Performance L i q u i d Chromatography
High performance l i q u i d chromatography (HPLC)

h a s been used t o q u a n t i t a t e t h e amount of ma-
j o r d e g r a d a t i o n p r o d u c t , etomidate a c i d i n
etomidate. The following a r e t y p i c a l chroma-
tographic conditions f o r analysis.
Column : Micro-Bondapak@/C18 ( o c t y l d e c y l
c h e m i c a l l y bonded t o s i l i c a )
Mobile Phase : A c e t o n i t r i l e :methanol :wa ter

(11:5: 8 4 ) c o n t a i n i n g 0.02 - M
c i t r a t e buffer.
Flow Rate: 2.0 m l / m i n
Detector: 254 nm
I n t e r n a l Standard: 3,4-Dimethoxybenzoic acid
5.33 Gas-Liquid Chromatography (GLC)
The p u r i t y of e t o m i d a t e can be determined by

d i r e c t i n j e c t i o n of e t o m i d a t e d i l u t e d i n a
s u i t a b l e s o l v e n t i n t o a chromatograph. Sev-
eral columns have been used t o determine t h e
p u r i t y of e t o m i d a t e (4).
The f o l l o w i n g are t y p i c a l chroma t o g r a p h i c

c o n d i t i o n s f o r t h e GLC d e t e r m i n a t i o n of
etomidate (4):
Column A: 3% OV-17 on Supelcoport 80/100

mesh, 1 m x 2 mm ( i - d . ) x 6 mm
(0.d.). U-shaped pyrex g l a s s
column.
Carrier Gas N2: 25 ml/min
Column B: 3% OV-17 on Supelcoport 80/100

mesh, 1 m x 3 mm ( i - d . ) g l a s s
column.
Carrier Gas N2: 70 ml/min
Column C: 3% SE-30 on Supelcoport 80/100

mesh, 2 m x 2 mm (i.d.) g l a s s
column.
Carrier Gas N 2 : 25 ml/min
Detect i o n : Flame i o n i z a t i o n
Temperature: I n j e c t o r : 290°C
D e t e c t o r : 290°C
Column: I s o t h e r m a l 190°C o r
programmed a t 20°C/min
I n i t i a l Temp.: 50°C
F i n a l Temp: 300°C
5.4 Titrimetrv
Etomidate e x h i b i t s b a s i c p r o p e r t i e s . I t can be po-

t e n t i o m e t r i c a l l y t i t r a t e d with s t a n d a r d i z e d 0. l N
p e r c h l o r i c a c i d u s i n g a modified glass-calomel eiec-
t r o d e system, i n which 0.1 N l i t h i u m p e r c h l o r a t e i n
a c e t i c a c i d h a s been s u b s t i T u t e d f o r potassium
c h l o r i d e , employing g l a c i a l a c e t i c a c i d as t h e sam-
p l e solvent.
ETOMIDATE 211
6. A n a l y s i s of P h a r m a c e u t i c a l Formulations
6.1 S p e c t r o p h o t o m e t r i c Method
Etomidate [ (R)- (+) e t h y l 1-(1-phenylethy1)-1H-imida-

zole-5-carboxylate] i s known t o hydrolyze i n t h e
p r e s e n c e of water, y i e l d i n g R-(+) 1-(1-phenylethy1)-
1H-imidazole-5-carboxylic a c i d . In s o l u t i o n t h e hy-
d r o l y s i s r e a c t i o n i s c a t a l y z e d by hydroxyl as w e l l
as by hydrogen ions. A n a l y t i c a l methodology i s
based on a n e x t r a c t i o n w i t h d i e t h y l e t h e r , f o l -
lowed by UV-measurement of t h e o r g a n i c l a y e r con-
t a i n i n g e t o m i d a t e and of t h e aqueous l a y e r contain-
i n g t h e h y d r o l y s i s product.
During t h e e x t r a c t i o n , t h e a c i d i c f u n c t i o n a l group
of t h e h y d r o l y s i s product, R-(+) 1-(1-phenylethy1)-
1H-imidazole-5-carboxylic a c i d , i s i o n i z e d by t h e
a d d i t i o n of t h e NaHCOg-solution and t h e r e f o r e re-
mains i n t h e aqueous l a y e r . On t h e o t h e r hand,
e t o m i d a t e i s n o t i o n i z e d and passes i n t o t h e o r -
g a n i c l a y e r , where i t i s measured spectrophoto-
.
m e t r i c a l 1y
6.2 High Performance L i q u i d Chromatography (HPLC)
Etomidate i n j e c t a b l e s o l u t i o n can be analyzed by a n

HPLC procedure using 2 -amino-5-chl o r 0benzo phenone as
t h e i n t e r n a l s t a n d a r d . The method uses a C 1 8 micro-
Bondapak column and 0.005 M sodium phosphate mono-
b a s i c : a c e t o n i t r i l e (60:40)-as t h e e l u e n t ( 5 ) .
Etomidate f r e e a c i d , t h e primary d e g r a d a t i o n product

of etomidate c a n be s p e c i f i c a l l y q u a n t i t a t e d i n
etomidate f o r m u l a t i o n u s i n g a n HPLC a n a l y s i s proce-
dure. The HPLC procedure u s e s a c18 microbondapak
column u s i n g acetonitri1e:methanol:water (11:5:84),
0.02 -
M i n c i t r a t e buffer, a s the eluent (6).
7. Drug Metabolism and Pharmacokinetics
The major m e t a b o l i c pathway of etomidate, (R)-(+)ethyl

l-(l-phenylethyl)-1H-imidazole-5-carboxylate, i s hydrolysis
of t h e ester t o produce p r i m a r i l y R-(+) 1-(1-phenylethy1)-
1H-imidazole-5-carboxylic a c i d . Oxidative N-dealkylation
occurs t o a s l i g h t e x t e n t , producing mandelic and benzoic
a c i d s , which a r e e x c r e t e d i n t h e urine. A l l t h e metabolic
p r o d u c t s a r e pharmacologically i n e r t (7).
Etomidate has a s h o r t d u r a t i o n of a c t i o n , presumably be-

cause of r a p i d h y d r o l y s i s i n t h e plasma (8).
L e w i ( 9 ) h a s r e p o r t e d t h a t t h e metabolism of e t o m i d a t e
i n t h e l i v e r i s a c a p a c i t y - l i m i t e d Michaelia Menten pro-
cess.
It has been suggested t h a t etomidate i s hydrolyzed a t

least i n p a r t i n plasma. The e a r l i e s t work on t h e a s s a y
of t h e drug advocated t h e a d d i t i o n of f l u o r i d e i o n t o
plasma s a m p l e s c o n t a i n i n g e t o m i d a t e t o i n h i b i t t h e p l a s -
ma esterases and t h u s , h y d r o l y s i s of t h e drug. Quick re-
covery with absence of hangover e f f e c t s and t h e absence
of cumulation w i t h r e p e a t e d doses of etomidate have been
explained by r a p i d h y d r o l y s i s i n t h e serum and l i v e r (10).
Meuldermans, e t . a l . (11) have s t u d i e d t h e i n - v i t r o meta-

bolism of e t o m i d a t e i n r a t l i v e r f r a c t i o n s . The main
metabolic pathway f o r t h e i n - v i t r o metabolism was t h e
h y d r o l y s i s of t h e e t h y l ester. Decarboxylation and oxi-
d a t i v e N-dealkylation were a l s o observed. The plasma
p r o t e i n binding and d i s t r i b u t i o n of etomidate i n dog, r a t
and human blood were a l s o reported by Meuldermans, et.
a l . (12).
The s i t e of h y d r o l y s i s of etomidate and e f f e c t s of b i s -

(p-nitrophenyl) phosphate (BNPP), a s p e c i f i c i n h i b i t o r
of t h e h e p a t i c rnicrosomal hydrolase, on t h e d u r a t i o n of
a c t i o n and e l i m i n a t i o n of e t o m i d a t e were r e p o r t e d by
Ghoneim and Van Hamme ( 1 3 ) .
The phannacokinetics of etomidate have been r e p o r t e d by

s e v e r a l i n v e s t i g a t o r s ( 9 , 13-18).
8. Determination of Etomidate i n B i o l o g i c a l F l u i d s
Etomidate i n blood plasma h a s been determined by mass

fragmentography (combined GLC-mass spectrometry w i t h
s i n g l e i o n monitoring). A 1.5 m x 2 mm ( i . d . ) g l a s s
column was packed w i t h 5% 08-225 on 100-120 mesh Supel-
coport. The column and i n j e c t i o n p o r t t e m p e r a t u r e were
230' and 250', r e s p e c t i v e l y . The method has a s e n s i t i -
v i t y of 1 n g / d and a c o e f f i c i e n t of v a r i a t i o n of 2.4%
(19).
A h i g h performance l i q u i d chromatography 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 etornidate i n serum h a s
been r e p o r t e d by Uges and Bouma (20).
ETOMIDATE 213
Assay of e t o m i d a t e i n plasma by c a p i l l a r y g a s chromato-

graphy w i t h n i t r o g e n - s e l e c t i v e d e t e c t i o n h a s been re-
p o r t e d by DeBoer, A.G., e t . a l . (21).
References
1. W W
tion.
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a shbur n, Abbot t Lab o r a t o r i e s , P e r s o n a l Commun i c a-
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Communicat ion.
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48, 213-216, 1976.
11. W.E.G. Meuldermans, e t . a l . : I n - v i t r o metabolism of

e t o m i d a t e by r a t l i v e r f r a c t i o n s . Arch I n t Pharma-
codyn Ther 221, 140-149, 1976.
12. W.E.G. Meuldermans, e t . al.: The plasma p r o t e i n

binding and d i s t r i b u t i o n of e t o m i d a t e i n dog, r a t
and human blood. Arch I n t Pharmacodyn Ther, 221,
150-162, 1976.
13. M.M. Ghoneim and M.J. Van Hamme: H y d r o l y s i s of

Etomidate. A n e s t h e s i o l o g y , 50, 227-229, 1979.
14. R.S. Reneman: E t o m i d a t e , a new, s a f e , s h o r t - a c t i n g

i n t r a v e n o u s h y p n o t i c - e x p e r i m e n t a l pharmacology a n d
interactions. Boerhaave S e r P o s t g r a d Med Educ, 1 2 ,
145-151, 197 6.
15. R. S. Reneman, e t. a l . : The e x p e r i m e n t a l pharmacol-

ogy of e t o m i d a t e , a new p o t e n t , s h o r t - a c t i n g i n t r a -
venous h y p n o t i c . A n a e s t h e s i o l Resusc, 106, 1-5,
1977.
16. J. J. Ambre, e t . a l . : P h a r m a c o k i n e t i c s of e t o m i d a t e ,
a new i n t r a v e n o u s a n e s t h e t i c . Fed P r o c 3 6 , 997,
1977.
17. M.M. Ghoneim, e t . a l . : P h a r m a c o k i n e t i c s of i n t r a -

venous a n a e s t h e t i c s : I m p l i c a t i o n s f o r c l i n i c a l use.
C l i n Pharmacokinet, 2, 344-372, 1977.
18. M.J. Van Hamme, e t . a l . : P h a r m a c o k i n e t i c s of e t o m i -

d a t e , a new i n t r a v e n o u s a n e s t h e t i c . Anesthesiology,
49, 274-277, 1978.
19. M.J. Van Hamme, e t . a l . : Mass f r a g m e n t o g r a p h i c de-

t e r m i n a t i o n of plasma e t o m i d a t e c o n c e n t r a t i o n s . J.
Pharm S c i , 66, 1344-1366, 1977.
20. D.R.A. Uges a n d P. Bourn: D e t e r m i n a t i o n of e t o m i -

d a t e i n seruni. Pharm Weekbl. Sci. Ed. 1, 459-460,
197 9.
21. A.G. DeBoer, e t . a l . : Assay o f e t o m i d a t e i n plasma

by c a p i l l a r y gas chromatography w i t h n t i r o g e n - s e l e c -
t i v e detection. J. Chromatogr. 1 6 2 ( 4 ) , 591-595,
1979.
Acknowlednements
The a u t h o r s wish t o thank Miss Diane Penza € o r t y p i n g

t h e manuscript.
HEPARIN SODIUM
Friedrich Nachtmann, Gunter Atxl,
and Wolf Dieter Roth
1. Description 216
1.1 Nomenclature 216
1.2 Occurrence 216
1.3 Structure 216
1.4 Molecular Weight 217
1.5 Elemental Composition 218
1.6 Appearance, Colour, Odour 218
2.2 Ultraviolet Absorption 220
2.3 Circular Dichroism 22 1
2.4 'H-NMR 222
2.5 'T-NMR 224
2.6 Solubility 226
2.7 Viscosity 226
2.8 Optical Rotation 229
2.10 Titratable Groups 230
2.12 Elemmlal Analysis 232
2.13 Crystal Properties 232
3. Production 233
4. Stability 234
4.1 Heparin Sodium 234
4.2 Aqueous Sotutions of Heparin ,Sodium (5000nJ/ml) 236
4.3 Combination of Heparin Sodium with Other Substances 237
5. Metabolism and Pharmacokinetics 238
6.1 IdentificationTests 240
6.2 Colorimetric Analysis 240
6.3 Fluorimetric Analysis 242
6.4 Infrared Spectroscopy 242
6.5 Titrimetric Methods 242
6.6 Turbidimetric and Nephelometric Analysis 243
6.7 Polarographic Analysis 243
6.8 Electrophoretic Analysis 243
6.9 Chromatographic Methods 250
6.10 Amidolytic Methods 256
6.1 1 Esterolytic Methods 258
6.12 Anticoagulant and Allied Tests 259
References 263
ANALYTICAL PROFILES OF DRUG SUBSTANCES 215 Copyright by the American Pharmaceutical Association
VOLUME I2 ISBN 0-12-260812-7
216 FRIEDRICH NACHTMANN ETAL.
1. Description
H e p a r i n Sodium is the ccmrnercial sodium salt of the in-
stable heparinic acid, a n acidic rmcopolysaccharide. The name
is derived from the f i r s t isolation fran dog l i v e r by W a n
i n 1916 (1).
Besides the ccmrnercial sodium salt of heparin, calcium,
barium, lithium, potassium, amrmnium, zinc and lead salts are
also ham, of which, hawever, only heparin calcium is of can-
n-ercial importance. With organic nitrogen bases, heparin forms
s l i g h t l y soluble salts, which are of technological importance
as intennediate products i n the isolation of heparin.
1.1. Nomenclature
Generic name:
Heparin Sodium [9041-08-11
Brand names:
Lip-Hepinette, Liquemin, Pularin, 'Ihrcmbocid,
Thrantoliquine, Vetren
1.2. Occurrence
Heparin occurs in the body tissues of mmnals and could
be detected in the lung, l i v e r , spleen, heart, mcosa, skin,
plasm and lynph.
H e p a r i n is synthesized while bound to protein i n the gra-
nulae of the mast cells ( 2 ) . According to Jeanloz, heparin is
bound to O-L-serin-protein by mean of galactose and xylose ( 3 ) .
I n vim, the sugar is separated fran protein by means of pro-
tease. The release n-echanism of heparin is not ham.
On an industrial level, heparin is mainly isolated fran
t h e lung and nuccsa of the ox and pig, whereby that cbtained
fran porcine mccsa is of greatest importance.
1.3. Structure
For a long time, it was not possible to elucidate t h e
structure of heparin on account of the different origins and
due t o various acccmpanying substances such as heparan sul-
phate, dermatan sulphate and hyaluronic acid being present.
Heparin is a biopolymr belonging to the class of mco-
polysacdmrides. More than 70 % of the structure of "conventio-
nal" heparins can be accounted f o r by repeating disaccharide
units consisting of 1,4-linked L-idmnic acid and D-glucos-
amine. !The iduronic acid residues are O-sulphated a t position
2, and the glucosamine residues are N-sulphated and O-sulpha-
ted a t position 6 ( 4 ) .
HEPARIN SODIUM 217
a - L -I d A a-D-ClcAm
OSOj
The above regular blocks can be intemqted or extended

by residues of R-D-glucumnic acid and 6-0-sulphated N-acetyl-
a-D-glucosamine:
p-D-GlcA a - D - G l c kin
Heparin A, isolated fran porcine intestinal mcosa, is

characterised by a content of such residues that is several
times that of B-type heparin, which is isolated fran beef lung
(5). The location of these minor residues w i t h i n the p l y -
saccharide chain is not knm, ht it is clear that they co-
occur i n a t l e a s t some of the mlecules bonded glycosidically
t o residues of the m j o r , sulphated hexosamine and iduronic
acid, constibents.
Purified heparinase degrades heparin to a mixture of di-,
tetra-, hexa- and oligosaccharides ( 6 ) . The relative anrlunts
of di- and oligosaccharides abtained vary w i t h the source of
heparin used. The tetra- and higher oligosaccharides represent
areas which are strructurally different fran those degradable
iduronic acid
D-glucuronic acid
-
t o disaccharides.
Rosenberg and Lam suggest that a tetrasaccharide unit, L-
N-acetylated D-glucosamine-6-sulphate 4
N-sulphate D-glucosamine-6-sulphate
my represent the structural basis of heparin's anticoagulant
activity (7) .
As possible tertiary structure of hewin, Tabisch des-
cribes a helix, similar to that of peptides, with a pitch of
21 (8).
1.4. Molecular Weiqht

Heparin is a mixture of plyanions in a relatively wide
range of mlecular e i g h t s .
By mans of electqhoresis, 21 fractions =re detected in the
range 3,000 - 37,500 Daltons. (See chapter 2.9.).
1.5. Elemental Compos i t i o n

Different results are obtained fran the e l m n t a l analy-
sis of heparin sodium depending on the origin and m u f a c t u -
ring procedure. A survey is given i n chapter 2.12.
1.6. Appearance, colour, odour

Heparin sodium is a vhite to creamy-white, mderately
hygroscopic powder. It is practically odourless .
2.1. -
Infrared Spectrum
The infrared spectrum of heparin sodium isolated fran
porcine rmcosa w s recorded between 4000 and 600 cm-l using a
E3eckn-m Acculab apparatus. ?he spectrum of a KBr disc of the
product is given in figure 2.1. The d i s c was prepared with 1.5
mg of heparin sodium and 300 mg of potassium branide.
The spectm is i n good accordance with the l i t e r a t u r e
(9-11). Analogous spectra are obtained i n an aqueous solution
(D20). The following frequencies are given for the characte-
ristic bands (12,13)
' AS 1605 ? 5 cm-l % = 11.6)

cm
u s coo- 1420 ? 5 cm-l % = 3.8)
cm
p AmideI 1623 ? 3 cm-l
P Amide I1 1480 2 2 cm-I
AS s=o 1245 an-', 1222 cm-l

-1
u s=o
u Asc4-s
v s c-0-s
--
-1060
980 an-'
820 cm
QTI
-1
A-type heparins (isolated fran porcine mucosa) exhibit an -1

appreciable extra absorption-band in the region of 1130 cm ,
otherwise, the spectra of A- and B-type (or lung) heparins
shm little differences.
NOISSI!4SNVUl lN33t13d
. i0
o)
0
. (0
o
0
- 0
P
0
s
0
f
0
0
0 7
g z
[r
W
m
o x
0 3
$ 5
5
a
z
0
0
8
0
0
0
P
220 FRIEDRICH NACHTMANN E T A .
2.2. Ultraviolet Absorption

Bell and Krantz already examined the W absorption of
heparin sodium i n 1950 (14). They Cbtained different spectra
f o r low-, medium- and high-potency heparins. ?he spectra ex-
hibited absorption mirtla a t 245 nm and 292 nm and minima
between 240 and 260 nm. According to the authors, t h i s ab-
sorption behaviour is due to impurities. Further investiga-
tions later on by Awe and Stildemann (15) confirmed this hypo-
thesis: a minimurn a t 235 nm and a maximum a t 255 nm were found
f o r r a w heparins; these bands disappear a f t e r purification of
the product.
1.00
0.75
a
0
K
m
2
sf
2 0.50
0.25
Figure 2.2 ___* Wavelength [nm]
W-spectrum of heparin sodium (A type)

concentration: 0.8 mg/rnl H20; S p e c t m t e r : Zeiss DM 4
HEPARIN SODIUM 221
Ferrari and D e Ambrosi &ned 43 samples of cmm?rcial

.
heparin sodium (16) Different W spectra =re found depending
on the origin and activity. A correlation between the W spec-
trum and the a c t i v i t y could not been proven.
The W spectrum of heparin sodium, isolated f r m porcine
muma, is given i n figure 2.2. In conformity with the fin-
dings of Awe and Sti.Memann, the product exhibits no W rnaximurn
or mininaun. Between 300 - 230 nm, the W absorbance is very
poor, it is only frm this wavelength onwards that the absor-
bance increases strongly.
2.3. Circular D i c h r o i s m
Studies on the circular dichroism of heparin are descri-
bed by various authors i n the literature (17 - 22). The spec-
tra cbtained by V i l l i e r s e t al. (22) are given i n figure 2.3.
+20
+15
+ 10
+5
I
2
X
I
I1
L E I 0
-5
-10
- 15
Figure 2.3
CD spectrum of heparinic acid -( 1 , sodium heparinate
(-. - .-) , magnesium heparinate ( ...
.) and calcium heparhate
(----) in salt-free aqueous solution (TN = 26 mequiv/l) ; pub-
lished by V i l l i e r s e t al. (22)
-
Instrument : Jasco J-40 B d i c h r m t e r ; Temperature: 22 23OC
The characteristic range of wavelengths is bebeen 180

260 nm. The CD data of plymers are usually stated as nmo-
-
mlar e l l i p t i c i t y [el = e x m.r.w/l x c, where:
m.r.w. = mean residue mlecular w i g h t
1 =path length (am)
c = concentration (g/lOO rnl) .
On account of the differing data for the mean residue
mlecular weight in the literature, [G] = 10 x 0 / (1 x TN) was
used in figure 2.3 whereby Th is the normality of the solution,
determined fran titration curves.
The CD spectra of heparinic acid, and the sodium, calci-
um, and magnesium salts show two bands of opposite signs. The
first band (at 210 nm) is negative and its magnitude depends
on the counter-ion. "he second band is locaw a t lower nm
values. ?he corresponding maximum (at 195 nm) depends on the
counter-ion. The rtlaximum of the second CD band (below 185 nm)
was not reached for heparinic acid.
Unsubstituted polysaccharides do not shew any CD bands in
-
the 185 250 nm region of the spectrum. Therefore the bands
i n figure 2.3 result form the interaction of circularly pola-
rised electranagnetic waves w i t h chranophores of the substi-
tuent groups bund to the polysaccharide backbne. Heparin CD
bands are described as "amide - Cotton effects" (17) and
"amide-like Cotton effects" (18). The CD bands wre found to
be slightly dependent on temperature.
-
In the range of 20 6OoC, the changes are linear and in-
dependent of the munter-ion ( 2 2 ) .
2.4. 'H-NMR
The spectrum of heparin sodium, isolated fran porcine
mucosa, was recorded a t 360 MHz using a Bruker WH-360 spectro-
mter (figure 2 . 4 ) .
40 mg of the sample wre dissolved i n 0.25 m l of DS. Ihe
masuring temperature was 3 5 V , the pD value 6.7. Ihe spectrum
confinns the fact t h a t heparin sodium isolated fran mmsa or
ox lung is mainly canposed of repeating disaccharide sequences
in bhich a-L-idopyranuronic acid-2-sulphate and 2-deoxy-2-
s u l p ~ ~ - ~ g l u c q ? y r a n o s e - 6 - s u l p are
h a ~linked a t positi-
ons 1 and 4 (23, 24). Generally, this sequence accounts for
> 85 % of ox lung heparins and > 70 % of porcine maxal hep-
rins ( 2 3 ) .
of heparin sodium (A-type) in D S ; Instrument: B r u k e r WH-360
The clearly detectable signal a t 2 . 1 p p i s due to acet-

&do-deoxy-hexose residues by means of which the mmsal A-
type heparin is characterised ( 2 5 , 2 6 ) . Unlike B-type heparin
(€ran ox lung), approx. 30 per cent of A-type heparin is can-
posed of residues of 2-acetamido-2-deoxy~-Pglucose and &&
glucurOnic acid ( 2 4 ) . For further interpretation of the spec-
t n n n , the reader should r e f e r to the literature (23-26).
2.5. 13C-NMR
The spectrum (figure 2 . 5 ) was recorded a t 90.5 MHz using
a B r u k e r WH-360 spectraneter.
190 mg of heparin sodium were dissolwd i n 2 ml of D20.
A t a pD value of 4.9, the temperature was 7OoC when recording
the spectrum. D i m , 8'= 67.8 ppn, was used f o r standardi-
sation. The signal a t 23 p p is due to a m t h y l carbohydrate
which is fran the 2-acetamido-2-deoxy-a-D-glumse i n the A-
type heparin. As regards the identification of the signals or
the interpretation of the spectrum, the reader should r e f e r to
the l i t e r a t u r e (24, 25, 27, 2 8 ) .
+) we are grateful to ~ r M.. wli, sand02 LU., f o r recor-

ding the spectrum.
Figure 2.5 +)
l3C-bM3 spectrum of heparin sodium (A-type) in D20
A : spectmm without decoupling
B : spectnrm w i t h decoupling
Instrument: Bruker -360
2.6. Solubility
According to the Merck Index, 5 % of heparin sodium is
mluble in water (29). According to Martindale, The ]Extra
Pharmacopoeia, 1 part of heparir5 sodium dissolves i n 2.5
parts of water (30). Saturated solutions of heparin sodium,
i m l a t e d fran porcine numa, wre prepared in 4 d i f f e r e n t
solvents a t 2OoC. The concentrations of t h e solutions were
determined quantitatively by a coagulation mthod. The re-
s u l t s are given i n table 2.1.
Table 2.1
Solubility of heparin sodium (2OOC)
Acetone
2.7. Viscositv
For polymlecular substances such as heparin sodium,
there is a correlation between m l e c u l a r w i g h t and viscosi-
ty. The interdependence was examined f o r bovine heparin (31).
A graphical representation of the r e s u l t s is given in figure
2.6.
The two large peaks present i n the d i f f e r e n t i a l curve
suggest the p o s s i b i l i t y of two discrete species i n the unfrac-
tionated product. The detectable discontinuities in the inte-
gral d i s t r i b u t i o n curve indicate heterogeneity within a rela-
t i v e l y circwnscr- d i s t r i b u t i o n of molecular weights. The
discontinuities are of the order of a tetra-saccharide.
The viscanetric t i t r a t i o n of heparin with sodium hydro-
xide and calcium hydroxide was examined by V i l l i e r s e t al.
(22). The t i t r a t i o n curves are given in f i g u r e 2.7.
HEPARIN SODIUM 227
100 100
90 90
80 80
70 70
60 ' 60
50 50 5
v
X
40 ,40 U
?
30 30 Q
20 20
10 10
0 0
70 90 110 130 150 170 190 210
[TI x103
Figure 2.6
.Integral dispersion curve for heparin sodium iractionated
with alcohol. Abscissa: i n t r i n s i c viscosity, ordinate: m l a -
t i v e e i g h t recovered during fractionation; published by
Lasker (31).
+
For N a , the specific viscosity ( j l s p ) rises slightly as E
increases fran 0 to 0.6, i.e., when the strongly acidic groups
are neutralised. A t B -0.6 the carboxylic groups start to be
ionised. The new electric charges produce a further extension
of the already partially extended m a c m l e c u l e s . Beyond 5 = 1,
the specific viscosity decreases because of the salt effect of
the excess of alkaline reagent. me ca2+ viscmetric curve is
canpletely different. The specific viscosity decreases Mi-
ately as a increases fran 0. This trend indicates a strong
interaction that 61lows m c r m l e c u l e s to contract.
F
w
Figure 2.7
V i s c a n e t r i c t i t r a t i o n curve of heparinic acid (TN 5 . 2 mequiv/
1) neutralised with sodium hydroxide (-.-.-#--- ) and calcium
hydroxide (---- ) a t 25OC i n salt-free w a t e r solution; pub-
lished by V i l l i e r s e t al. ( 2 2 ) .
Investigations rtFLde by Chung and Ellerton proved that the
viscosity of heparin salts is dependent on the size and &arge
of the ca ion 1 9 ) . ?hereby the following order was found:
5; 1+
Na+<K+<Mg<Sr <E3a2+<Ca2+. The viscosity of heparin is also
.
lowxed by periodate oxidation (19) The f l e x i b i l i t y of hepa-
r i n is increased by cleaving the vicinal hydmxyl groups.
Furthermore, a t constant ionic strength, the viscosity of
heparin in solution is dependent on the pH value. The viscosi-
t y increases between pH 2 and 5. This rise is due to the in-
crease in hydrodynamic wlum of the mlecule (19). The type
of solvent has a large influence on the viscosity of the
heparin solution. The viscosity increases linearly w i t h in-
crease in dielectric constant ( 3 2 ) .
Moreuver it was proved that the viscosity of heparin is
dependent on the shear stress and the ionic strength of the
solutions (19). The viscosity decreases w i t h increase in shear
stress and increase in ionic strength. Desulphatation also
leads to a decrease i n viscosity ( 3 2 ) .
HEPARIN SODIUM 229
2.8. Optical R o t a t i o n
O p t i c a l rotation values for heparin-sodium frm the li-
terature are given in Table 2.2.
Origin of sample Reference

+47.6O Bovine lung 9
+45.6O Bovine intestine 9
+46.3O P o r c i n e intestine 9
+61.6O Whale lung 9
+59.40 Whale intestine 9
+47.0° +) Not defined 10
+46.5O 2 7' V a r i o u s origin 15
+34.4O to +54.1° Various origin 33
+40.9O t o +49.5' Various origin 34
+48.7O Bovine 35
+65.4O Pihale 35
+48.4O 2 3.2' P o r c i n e intestine 36
2.9. Molecular Weight

On account of the varying origin and different procedures
for the m u f a c t u r e of heparin sodium, various values are
given in the literature for the w i g h t average mlecular
weight (table 2.3) .
The mlecular wights are distributed over a relatively
w i d e range. By means of electrofocusing on plyacrylamide gel
slabs, heparin-sodium was s p l i t up into 2 1 fractions ( 4 3 ) . The
mlecular weights of these fractions ranges between 3,000 and
37,500.
230 FXIEDRICH NACHTMANN E T A .
Table 2.3
Weight average mlecular weight of heparin sodium
Weight A v e r a g e Origin &thod of eference

lvblecular Weight Determination
16600 N o t defined Sedimentation 37

Diffusion
14200 Not defined Sedimentation 10
Diffusion
14400 W i n e intestine Sedimentation 9
13600 Porcine intestine Sedimentation 9
11600 Wale lung Sedimentation 9
10800 Wale intestine Sedimentation 9
11900 ? 200 Porcine intestine Sedimentation 38
Diffusion
15000 4 500 Porcine intestine L i q u i d chrana- 39
Ww?hY
22000 4 2600 +) Porcine intestine Liquid chrana- 40
togr@Y
17900 ? 1300 +) Wine lung Liquid chrana- 40
tOSraPhy
10600 4 400 Porcine intestine Liquid chrana- 41
tOSraPhY
Laser l i g h t
scattering
10500 ? 300 Wine lung Liquid chrana- 41
tographY
Laser l i g h t
scattering
11000 Porcine intestine Liquid chrana- 42
tWW?hY
10500 2 600 Wine lung Liquid chrana-
tosraphY
+) Dextrans =re used f o r calibration. The differences i n

mlecular shape of dextrans and heparin sodium can appear
as differences i n mlecular weight.
2.10. Titratable Groups
Heparinic acid originating f r a n porcine intestinal rmcosa
was t i t s a t e d with ptassium hydroxide (38), sodium hydroxide
and calcium hydroxide (22). A typical t i t r a t i o n curve w i t h
potassium hydroxide is given i n figure 2.8.
HEPARIN SODIUM 231
.-
-0
2 20
.-0
c
.-L
a
Q
a,
I
-
a,
8. Et OSO3
8 *O 0 0 0 0 0 O D 0
e
c
F
u)
Q
s2
0
Z 60
NotThrou h '.
I R A - ~ O O ?OH) '.- .-. - . -. -_
-
I I 1 1 1 1
0 2 4 6 8 1 0 1 2
PH
F i g u r e 2.8
Titration curves of heparinic acid before and after passage
through IRA-400 i n the hydroxyl fonn. Titration curve f o r the
amount of ethyl s u l p h r i c acid (EtOSO3H) equivalent to sul-
phate groups estimated to be present in one mle of heparinic
acid; pblished by Helbert and Marini ( 3 8 ) .
Frcm the curves one can identify 40 ionisable sulphate
g r q s , 20 ionisable &xyl groups (S-shaped part of the
t i t r a t i o n curve) and 1 ionisable amino group for the product
examined (38). Furthermore, 6 to 7 equivalents of sodium sul-
phate per mle of heparin s o d i u m were detected, which do not
represent an integral ccmponent of the mlecule and which
could be removed w i t h an anion exchanger (e.g.: IRA-400, - '
H
0
form, see figure 2.8.) .
By means of conductanetric tests, Casu
and Gennaro cbtained values fran 1.94 - 2.40 f o r the ratio of
sulphate to car33oxyl groups in heparin (44).
2.11. Dissociation Constant

The dissociation constants (%) of various heparin salts
w e r e determined by Herwats e t al. (45). A dissociation con-
stant of 10 k 5 mM was found f o r heparin sodium. Thereby one
presumed that sodium is only bound t o the carboxyl grou~+. I n
canparison, the bond between s o d i u m and citrate (Q = 300 nM)
is a least ten times maker. Py means of competition experi-
ments, the dissociation constant could be determined for
various other cations. The following order was found:
Of all the cations examined, calcium w a s the one rrost

strongly bound.
2.12. Elemental Analysis
Various data is given i n the literature, depending on the
origin, p l r i t y and mnufacturing procedure. A sumnary is given
i n table 2.4.
Table 2.4
Elemental analysis of heparin sodium
% C % H % N %S
2.2 13.3 15.1 Bovine 35
2.5 9.0 13.1 Whale 35
2.1 14.2 12.5 Bovine lung 9
2.6 11.3 11.5 Bovine intestine 9
2.1 12.4 11.7 Porcine intestin1 9
2.3 7.3 9.2 Whale lung 9
2.3 8.2 9.0 Whale intestine 9
23.4 3.3 2.2 11.6 12.4 Porcine intestinc 38
22.2 3.5 2.1 11.3 11.6 Porcine intestin( 36
18.2 - 3.6 - 2.3 - 9.3 - 11.6 - Various origin 34
21.7 4.4 3.6 10.4 16.8
2.13. C r y s t a l Properties
X-ray fibre diffraction d a t a wre phlished by Nieduszyn-
ski and Atkins (46,47). Orientated f i l m of heparin sodium
(origin: porcine intestinal rmcosa) c r y s t a l l i s e in a triclinic
unit cell with parameters a = 1.30 4 0.01 nm, b = 1.02 4 0.01
run, c (fibre axis) = 1.59 4 0.02 nm, a = 104 4 lo, R = 96 4 lo,
HEPARIN SODIUM 233
r= 116 i 1' and with volume = 1.79 m3.The density of a

crystalline sample was determined by flotation to be 1.72 g/
cm3. Heparin sodium mlecules require nearly their own volume
of w a t e r i n order t o crystallise. The crystallographic perio-
dicity ranges fran 1.65 to 1.73 m, which is consistent w i t h
a tetrasaccharide repeating unit.
3. Production
Heparin sodium is cbtained by extraction fran animal or-
gans. According to Scott and Charles, the lung, muscle and
liver are particularly suitable. Lately, it has also to an in-
creasing extent been abtained fran nucosa, whereby higher ac-
t i v i t i e s are attained (48). The mst recent development is to
use the k i n e obtained when pickling intestines as the r a w
mterial f o r the mufacture of heparin.
The prduction of heparin sodium takes place principally
i n two steps: isolation of a mre o r less p r e crude heparin
and subsequent p r i f i c a t i o n of the crude heparin t o obtain the
parenterally administrable p r d u c t .
The various procedures for the preparation of crude
heparin are based on the principle that heparin is withdrawn
fran the autolysed or digested organs by means of alkali and
a f t e r separation of the protein-lib acccmpanying substances,
precipita- with alcohols o r acetone as a barium or a l k a l i
salt.
Scott and Charles autolyse the frozen, d u t e d tissue
and extract the heparin with d i l u t e alkali (48). After pres-
sing out the protein mss coagulated on heating, the heparin
i s precipitated w i t h sulphuric acid a t pH 2.5. After removal
of f a t by means of alcohol and digestion with trypsin, the
crude heparin is then precipitated with t w i c e the munt of
alcohol.
Taylor imprwed t h i s mthod by using digestive enzyrnes
and carrying out several extractions w i t h alkali ( 4 9 ) . Taka-
m s a describes a deproteinisation of the mmsa extract w i t h
picric acid and subsequent precipitation with hydrochloric
acid a t pH 2.5 followed by precipitation of the heparin sodium
salt with n-ethanol (50). Thanpson precipitates the heparin out
of the filtered autolysate by means of cetylpyridinium chlo-
ride (51). Wins rmves the f a t fran the tempered, fermented
tissue azeotrqically by mans of dichloroethylene (52). Gede-
on Richter enriches the heparin i n a canplex w i t h protein by
means of heat coagulation and extracts i n a countercurrent of
saline solution (53). Riker uses proteolysis and the addition
of salt to release the heparin fran the tissue. It i s then ad-
234 FMEDRICH NACHTMANN ETAL.
soon a strongly basic anion exchanger, eluted with sali-

ne solution and precipitated w i t h an organic solvent (54). As
starting m t e r i a l schering uses the brine produced when pre-
serving and dehydrating animal intestines w i t h sodium chlo-
ride (55). Heparin is c b t a i n d fran this brine, without pro-
teolytic digestion, by means of precipitation with methanol
and purification on a basic ion exchanger.
The crude heparin mnufactured fran other raw materials
than brine, as described above, must undergo deproteinisation,
decoloration and depyrogenisation processes. Further p r i f i -
cation is cbtained by dissolving and reprecipitating with al-
cohols or acetone or by precipitation of heparin as a slightly
soluble salt (barium, o q a n i c nitrogen bases) and reconverting
t o the sodium salt with soda or sodium chloride. Decoloration
of the product can be carried out by means of f i l t r a t i o n
through activated d~arcoalo r by oxidation with potassium per-
manganate, hydrogen peroxide o r -chlorite.
4. Stability
4.1. Heparin Sodium
Lyophilised, dry heparin sodium can be stored f o r several
years a t roan temperawe and 3OoC, without loss of activity,
i f kept in tightly closed containers (56).
Figure 4.1 shaws the biological activity (AFTT activity)
of heparin sodium (origin: mcosa) detennined over a period of
4 years as a percentage of the declaration.
The product is stable a t room temperature (RT, 15 - 25OC)
and 3OOC. The other quality characteristics (pH value after
dissolution, colour index, colour stability) and the content
of active ingredient remained unchanged throughout the storage
period.
HEPARIN SODIUM 235
I
110
- - - - - - -0- _ _ - - - - - -- _ _ _ _ -_- -_- -- - -

0
6
._ 100
c
---L---.
0
---___
0
E
a
-
_ - - - - - - 8- - - - - -
0
a,
n --- - - - _ _ _
c
0 - - - - _ _--
_
8 90
0 12 24 36 48
Months
Figure 4.1
S t a b i l i t y of heparin sodium (A type) a t roan temperature (RT)
and 3OoC
4.2. Aqueous Solutions of Heparin Sodium (5000 I U / m l )

Figure 4.2 skm a graphical representation of the re-
sults.
L
0
$ 90 -
RT
110
100
c
0
s 90
80
0 12 24 36 48
Months
Fiqure 4.2
S t a b i l i t y of sterile aqueous solutions of heparin sodium
(5000 I U / m l ) a t roan temperature (RT) and 3OOC.
HEPARIN SODIUM 237
lhroughout the period of 36 mnths, no changes regarding

the efficacy, the pH value and the appearance of the solution
could be found to occur i n the ampule solution of heparin so-
dium (origin rmcosa) ready f o r administration (56).
Experimnts carried out by Goodall e t al. showed that
hqxtrin sodium undergoes no loss in quality i n the pH range
f r a n 4 to 9 (57). This has been confinned by other experiments
(56). According to other tests, injections of heparin sodium
are s t a b l e f o r mre than 7 years a t rcun temprature and for
6 - 8 years a t 37OC (30). ?he activities determined immdia-
t e l y after s t e r i l i s a t i o n tests (12loC, 5 or 10 minutes) shaved
no s i g n i f i c a n t changes in canparison w i t h t h e untreated solu-
tions so that any negative influence on the heparin sodium
solutions due to heating my be neglected (56).
4.3. Canbination of Heparin Sodium w i t h Other Substances

S t a b i l i t y tests carried out with heparin sodium in can-
bination with dihydrcergotamine rresylate both in the lyophili-
sed and dissolved form, produced equally good results (56). A t
roan temperature and 3OoC, heparin sodium does not undergo
s t a t i s t i c a l l y detectable changes in a c t i v i t y over a period of
4 years.
Many authors (57 -
64) conducted experiments on the e f f i -
cacy of heparin sodium infusions a f t e r various standing times.
Altbugh in the course of extensive studies, some authors
-
(57 62) did not ascertain any loss i n a c t i v i t y of heparin
sodium (20 -1000 I U / m l ) i n 5 per cent dextrose solution, 0.9
per cent NaCl solution or in g l a s s b o t t l e s within a period of
1 2 t o 24 hours, and Bowie e t al. (58) even speaks of stability
periods of up to 1 2 months, i n canparison with the initial
values, Jacobs (63) and Okuno (64) find a c t i v i t y losses of up
t o 50 per cent within 24 hours f o r heparin sodium in NaCl,
dextrose, Ringer's and Hartmann's solution.
5. Metabolism and Pharmaoakinetics

The heparin activities found in the plasm of noml per-
sons, a f t e r hydrolytic destruction of the native heparin-pro-
tein ca~lplex,amount to 10 - 24 u n i t s / m l of plasm (65).
According to Heilbrunn, t h i s endogenous heparin serves to
mintain the protqlasmic flow equilibrium between the sol
and gel state, a fundamental function i n the cellular haneo-
stasis mechanism (66). Heparin, the mst effective anticoagu-
lant h m a t present, intervenes a t various pints in the
cascade of the oncping coagulation mechanism. Ihe aim of hepa-
rin therapy is, by activation of antithranbin 111, to fom an
inhibitor camlex hich circulates for a lonu tinre i n the
The heparin preparations on the market can be classified

into fractions of different affinities to antithranbin 111,
depending on their origin (mu-, lung) ; accOrding to Thanas
e t al., heparin procured fran the rmcosa possesses a higher i n
vitro activity than that abtained fran the lung (68). The same
authors found the contrary situation in vim (69). According
t o HLiLjk et al., the activities ik+&ned could be cansidered
equal to the biological efficacy (70). According to Rmen-
berg's model, the activity of heparin correlates with the pro-
portion of the linkage on the cofactor antithranbin I11 (71).
Y i n e t al. s h e d that this cofactor is lllaximally saturated
w i t h a p p o x . 0.3 U n i t s / m l although individual doses of mre
than 1 Unit/ml plasma are usually necessary in order to re-
main above the &sired time unit inhibition (72).
After i.v. application, heparin distributes i t s e l f wry
rapidly in the plasm mqxwtment and reacts as a carrier of
negative charge with nwnemus proteins - only 15 20 per cent-
of a i c h form a canplex with antithranbin I11 (73). Further-
mre, within approx. 40 minutes of injecting, 80 90 per cent-
of the injected heparin is m v e d frcm the system and metabo-
lised by the reticuloendothelial tissue of the liver and
spleen. The man half-life dxtm? in practically a l l human
cases m m t s to apprax. 1.5 hous (74 - 76) whereas the la-
test investigations carried out by Ciplle e t al. yielded a
man value of only 0.9 hours (77). Ihe ~ l u m eof distribution
m u n t s to apprax. 5.5 per cent of the body wight and there-
fore corresponds t o the plasm volume (74 - 77).
Many recent plblications prove that small subcutaneously
applied doses of heparin considerably decrease the danger of
-
deep-win thranbsis and p l m n a r y embolism (78 80). This
prophylactic effect of heparin is enhanced by the sinultaneous
administration of dihydergotamine (81 - 83). It is assum3d
that t h i s effect is based on an interaction between dihydro-
HEPARIN SODIUM 239
ergotamine and heparin, which leads to a higher and longer

lasting heparin plasma concentration (83). A study carried
out by Beennann and Lahnborg shows that the heparin plasma
level is the same f o r the canbination as f o r the pure heparin
i n normal persons, but that i n persons who are w e m i g h t ,
the levels are clearly elevated and the half-life increases
fran 1.4 to 2.1 hours (84).
There is no lack of investigations and clinical documen-
tation proving that heparin fran topical preparations pene-
trates to the blood circulation via the skin (85). One should
h a e v e r not expect any permeation rates for heparin which ex-
ceed an approx. threshold value of 1 per cent of the mterial
applied externally. I n a p b l i c a t i o n by Schaefer and Zech it
is state3 that, under suitable conditions (e.g. lanolin alco-
hol ointmnt), a penetration of up to 2 per cent of heparin
into the deeper layers of the skin takes place within 10 mi-
nutes (86). A l s o Kidron e t al. here able to detect absorption
of heparin from the gas-intestinal tract in connection with
a tenside (Cetanacrogol 1000) i n the case of rats (87). I n
s p i t e of t h i s success, one cannot imagine heparin therapy
without the parenteral form of administration.
Investigation of the metabolism of heparin have hitherto
only been carried out using radioactively marked substances or
by determination of the biological activities. chemical detec-
tion has not been carried out up till ncw since the different
mlar relationships of E-glucosamine, glucuronic acid, L-idu-
ronic acid and sulphide groups do not permit the use of the-
cal analysis or radioimnunological mthcds (88). U n t i l now,
exact statements about the mtabolism have only been possible
in a few points:
- R-dogenous heparin:
Rtiogenous h q a r i n released fran the roast cells is
immdiately taken up by macrophages and catabolysed. As a re-
sult, it loses its coagulation-inhibiting effect (89).
- &ogenous heparin:
Bparinase I and heparinitase I1 could be detected i n
the liver (90).
A heparin sulphamidase (91) and three different depoly-
merising enzymes (92) where also characterised. The degrada-
tion of heparin either takes place a t the heparin-antithrmbin
I11 canplex or a t the free substance i n the plasm.
Under the influence of disease ( l i v e r cyrrhosis), the
decrease in coagulation-inhibiting effect of i.v. administered
heparin is significantly slaver, since in such cases, the
c l a r i f y i n g function of the liver cells f o r heparin is dis-

tu&ed (93). I n the case of p l m n a r y embolism and severe
p h l e b o t h r h s i s , the decrease in a c t i v i t y is about 30 per
cent quicker than i n the healthy body (94), whereas in the
case of chronic renal insufficiency, an approximtely 20 per
cent slower decrease in a c t i v i t y was found (95).
6.1. I d e n t i f i c a t i o n %sts
Various tests can be used for the i d e n t i f i c a t i o n of
heparin:
6.1.1. Infrared Spectrwn

See chapters 2.1. and 6.4.
6.1.2. Chranatqraphic Methods

Paper chranatqraphy, thin-layer c h r m t q r a p h y and gel
chranatography (chapter 6.9.) are suitable.
6.1.3. Electrcphoresis
See chapter 6.8.
6.1.4. B i . o c h d c a l and B i o l o g i c a l Tests
The biochemical and biological rrethods given in chapters
6.10., 6.11. and 6.12. are selective f o r heparin and therefore
m y be used as i d e n t i t y tests.
6.2. C o l o r h t r i c Analysis
There are a large n h r of dyes which form a mtachro-
mtic colour a f t e r addition of heparin. Tests carried out by
Jaques e t al. shaved that BislMrCk hrown, azure A, b r i l l i a n t
cresol blue, cresol violet, brmcresol blue, neutral red,
basic fuchsin, pyronine and a c r i f l a v i n e a l l possess t h i s pro-
perty (96). Methylene blue (971, n i l e blue (98) or acridine
orange (99) are equally suitable. For the colorimetric deter-
mination of heparin there are three dyes which are mst often
used: toluidine blue (loo), alcian blue (101 - 103) and azure
A (103 - 106). ?he detection l i m i t f o r the determination of
heparin w i t h the dyes mentioned lies around 1 pg (103). I n the
presence of heparin i n d i l u t e , aqueous solution, t o l u i d i n e
blue e x h i b i t s 3 absorption bands (107). The a-band (620 nm)
corresponds t o t h e mnaneric dye, the &band (540 nm) t o the
dimeric dye and t h e p-band (500 nm) is produced by the binding
HEPARIN SODIUM 241
of the p l y a n i o n . Analogous relationships are found for the

s t r u c t u r a l l y very similar azure A; the canplex w i t h toluidine
blue is hawever mre i n t e n s e l y coloured than the ccanplex w i t h
a z u r e A. F'ractions of heparin possess differing properties:
mnosaccharide and disaccharide fragments only react slowly
w i t h o u t appearance of metachranasis whereas the f u l l meta-
chrcmatic activity i s produced by hexasaccharide units of he-
parin. The t o l u i d i n e blue mthod was described for the deter-
mination of heparin i n plasm (108 - 110), urine (111), mast
cells and granulocytes (112) and tissue (103, 113). The dyes
mentioned can also be used for the colouration of heparin
after separation by TLI= or electrophoresis.
Investigations carried o u t by Awe and StiElemann shcw
t h a t the values found by spectrophotanetry g r e a t l y depend on
t h e plrity of the active ingredient (114).
Anderson e t al. investigated the best measuring range,
l i n e a r i t y and standard d e v i a t i o n for the mst important dyes
(115). A ,summy is given in table 6.1.
Table 6.1.
Characteristic data for the determination of heparin - with
t o l u i d i n e blue ( A ) , azure A (B) and alcian blue (C) .
Method Heparin Useful
range of
heparin I Regression Standard Correla-
equation
Y =
deviatioi tion coef-
of ficient
I
scatter
about re
pg gression
A Porcine rmcous 0.042 0.995
Bovine lung 0.045 0.995
B Porcinermcous 0.016 0.966
W i n e lung 0.065~-0.011 0.024 0.971
C Porcine rmcous 0.030 0.993
Bovine lung 0.008 0.999
A canparison of Wo spectrophotanetric methods ( t o l u i d i n e

blue and m t h y l e n e blue) with the biological a c t i v i t y of hepa-
rin preparations was described by Marvola e t al. (116).
The spectrophotawtric methods cannot be used t o deter-
mine the biological activity of various preparations. ?he
correlation c o e f f i c i e n t s found m u n t to 0.386 - 0.702 only.
The colour reactions of sugars can also be used for the guan-
titative detennination of heparin. The mst significant is the
r e a c t i o n of m n i c a c i d s with carbazole. The mthod described
by Dishe (117, 118) was improved by B i t t e r and Muir (119) and

by Gauthier and Kenyon (120). The absorption mimum is
530 nm, linear c a l i b r a t i o n curves were found between 4 and
40 pg/ml of uronic acid. The reaction time a t 75OC m u n t s to
15 minutes. B i a n c h h i and Osima found no connection between
anticoagulant a c t i v i t y and content of uronic acid i n heparin
(121).
6.3. Fluorimetric Analysis

Phosphatd and sulphated p l y a n i o n s are bound by be&er-
i n e sulphate. The fluorescence of the r e s u l t i n g canplex can
be used f o r a quantitative determination of heparin (122).
Another dye which forms a fluorescent canplex with heparin is
acridine orange. The reaction conditions wre studied by Ca-
t i n i e t al. (123, 124). The emission wavelength of the com-
plex a t pH 2 to 10 is 640 nm. Linear c a l i b r a t i o n curves were
found f o r 7.8 - 303 pg of heparin/ml.
Fluorescent reactions of sugars can also be used for the
determination of heparin. The product of deamination of hepa-
rin with HNO2 reacts with 3,5-diami&nzoic acid and under
mild conditions (pH 3.0, 37OC), gives a stable derivative
(125). Less than 0.1 pg of 2-amin0-2-de~exose can be deter-
mined using standard cells.
6.4. Infrared Spectroscopy

The characteristic, a-tric S=O vibration (see chapter
2.1) can be used f o r quantitative sta-nts. For the measure-
mt of aqueous solutions of heparin sodium, cell w i n d m of
IRTRAN-2 or KSR-5 are reccrnmnded (12). Another msuring
tedmique consists of taking an internal standard such as po-
tassium thioqanate. The r e l a t i o n between the S=O vibrations
of heparin sodium (1240 m-l) and the CaN vibration of the in-
ternal standard (2065 m-l) is determined (126).
US' g D20 as the s o l w n t , spectra in the range 1500 -
1800 an3 can be recorded. &is permits t h e quantitative eva-
luation of the c a h x y l a t e band of heparin sodium (13).
6.5. T i t r b t r i c Methods
6.5.1. Potenticmetric t i t r a t i o n
The procedure was described by klbert and Marini ( 3 8 ) .
Heparin sodium is passed through a column of Dowex-50 in the
hydrogen form. An a l i q u o t of this is titrated for acid groups.
Another aliquot is first passed through a column of IRA-400 i n
the hydraxyl form to ratwlve inorganic anions, then titrated.
The difference is due to the p i o n acid groups.
HEPARIN SODIUM 243
6.5.2. C o n d u c t h t r i c t i t r a t i o n
For determination of the carboxyl content, the dialyzed
sample of heparin sodium is t i t r a t e d with 0.1M Hc1 with a con-
ductimeter and e s t h t e d by the m u n t of standard acid needed
up to t h e f i r s t i n f l e c t i o n p i n t ( 4 4 ) . For sulphate plus car-
boxyl groups, t h e dialyzed sodium salt is converted to the
f r e e acid w i t h A m b e r l i t e IR-120 ( H
'
) and titrated with 0.1M
NaOH. !the f i r s t pint of i n f l e c t i o n is equivalent t o the sul-
&ate content, t h e second t o the sulphate and carboxyl con-
tent.
6.5.3. Colorimtric t i t r a t i o n
A colorimetric t i t r a t i o n is described by Jaques (107).
Heparin sodium is added t o a toluidine blue solution u n t i l the
colour changes f r a n blue to plrple. The colour abtained is
m t c h d with that abtained with reference heparin. The proce-
dure is a f a s t , semiquantitative test and applicable f o r both
crude and F r i f i e d preparations.
6.5.4. W i d i n e t r i c titration
Some plysaccharides react with quaternary alkylammnium
halides resulting i n a precipitate. The reaction of heparin
and other plysaccharides w i t h cetyltrimethylammnium bromide
was studied by recording the t u r b i d i t y by means of an automa-
tic t i t r a t i o n device (127).
6.5.5. Colloid t i t r a t i o n
The plyrrrer p l y d i a l lyldimethylamrronium chloride (Cat-
Floc) binds heparin stochianetrically. After addition of an
excess of Cat-Floc, back t i t r a t i o n is carried out w i t h p-
tassium plyvinylsulphate. Wluidine blue is used as the indi-
cator (128).
6.6. Turbidimetric and N e p h e l m t r i c Analysis
Heparin sodium forms s l i g h t l y soluble canplexes with
various reagents and these can be used f o r a quantitative de-
termination. M a r b e t and Winterstein describe the canplex with
2-(dimethylaminanethyl)dibenzofuran (129). The method was used
t o determine heparin in urine. me t u r b i d i t y of t h e heparin-
protamine canplex i n an excess of protamine was suggested by
Fekete f o r the determination of heparin (130). Another pssi-
b i l i t y is t h e use of the canplex of heparin with cetylpyridi-
nium chloride (131, 132). High t u r b i d i t y values =re found i n
the presence of a 0.4 M solution of sodium chloride. !he
method can be autanated (132).
6.7. Polarograpkic Analysis

An oscillopolarographic detennination of heparin and
other plyanions is described by Bohacek and Singh (133). The
optimal d i m f o r heparin was found to be 1M citric acid.
6.8. Electrophoretic Analysis

This analytical technique is mainly used f o r qualitive
detexnination of heparin. It is also possible to use it f o r
quantitative analyses.
6.8.1. Paper electrcphoresis

A determination of heparin in the presence of serum prc-
teins by mans of paper e l e c t q h o r e s i s is described by Ca-
selli (134). Basic dyes (basic fuchsin, gentian violet, mthy-
lene blue, toluidine blue) were used f o r staining. The mthod
m y he used i n the concentration range fran 10 - 100 p g of
plysaccharide.
Conti e t al. report on a separation of heparin on What-
man No. 1 electrqhoresis paper i n the presence of veronal
buffer pH 8.6 (135). Selective staining of heparin is possible
with auramine 0, malachite green or b r i l l i a n t green. These
dyes do not stain chondroitin sulphuric acid, hyaluronic acid
or nucleic acids.
A canbination of paper electrophoresis and gel electm-
phoresis has been used to identify heparin and other a c i d i c
mcqolysaccharides i n mammalian tissues (136).
6.8.2. Agarose gel e l e c t q h o r e s i s

The separation is based on the differences of the net
charge and a b i l i t y of acid mccplysaccharides to form cun-
plexes With different cations and 0c - amines. A g a r o s e
electrcphoresis i s mst useful for the characterisation of
sulphated mccplysaccharides. It accepts heavily contamina-
ted mixtures without change of pattern of fractionation (107).
A micrc-electqhoretic mthod on agarose f o r heparin and
relatsd canpounds was developed by Jaques e t 61. (107, 137,
138). Most of the samples of camercial heparin examined were
separated i n t o 2 fractions. 2 p l samples containing 0.1 t o 0.5
pg of heparin are applied t o the agarose gel on a microsccpe
s l i d e i n parallel with 0.8 pg of reference heparin. The elec-
t q h o r e s i s is conducted i n a Lucite chamber with three can-
partrnents and a rretal tray as l i d and cold finger (6.3 V/cm
for 10 t o 25 min a t 2OC). The nuccplysaccharide i n the gel is
fixed by immrsing the s l i d e i n 0.1 per cent cetyltrimethyl-
HEPARIN SODIUM 245
m n i u m branide or 0.1 per cent cetylpyridinium chloride so-

lution. The slide is stained with toluidine blue. The back-
ground i s cleared by washing w i t h 1 per cent acetic acid, and
t h e s l i d e is dried i n t h e air. ?he procedure separates heparin
f r a n rnmy other mterials present in tissue extracts. Optical
density of the spts is canpared w i t h that of s l i d e s prepared
with a range of concentrations of standard heparin, either
with a suitable d e n s i t c m t e r o r visually.
The e f f e c t s of d i f f e r e n t buffers on the r e l a t i v e electro-
phoretic migration of sulphated mccplysaccharides i n agarose
are summrised in T a b l e 6.2.
An e s s e n t i a l l y linear r e l a t i o n is found between log area
of t h e s p t and log of quantity of heparin applied up to 0.4
units. About 0.1 unit of heparin applied is q t i m a l f o r ma-
surmnt .
A r e l a t i v e standard deviation of 6.2 per cent f o r densi-
tanetric masurements could be achieved (139). The microelec-
trcphoresis procedure is mre sensitive than the colour tests
(107). It is 30 times as sensitive as the carbazole and 50
times as sensitive as the aqueous mtachranic test f o r h e p -
rin.
Kupchella and Curran separated heparin and a c i d i c glycos-
amino-glycans via cross-aver electrophoresis with t h e dyes
acridine orange and toluidine blue (140). Glycosaminoglycans
were applied to agarose gel films as 0.01 m l of 5 mg/ml
aqueous solutions and electrophoresis was carried o u t a t 200 V
(approximately 8.5 m A ) . Five minutes a f t e r starting, a dye-
soaked wick was applied t o the anode s i d e of the g e l , and
0.9 m l of dye was applied t o the wick. Electrophoresis was
continued u n t i l t h e dye-front passed t h e o r i g i n (about 15 min) .
The individual glycosaminoglycans are d i f f e r e n t i a l l y pigmen-
ted.
A discontinuous electrophoretic mthd f o r fractionation
of heparin into two or three d i f f e r e n t cayonents and of these
fran other a c i d i c mcopolysaccharides =re described by Bian-
c h i n i et al. (141). The mthod consists of electrophoresis
f i r s t i n agarose in barium acetate and then i n diaminopropane
h f f e r i n the sarne direction.
Table 6.2
Effects of Wfer on relative electropbretic migration of sulphated rmcapolysaccharides i n aga-
rose (values relative to migration of reference ox lung heparin), according to Jaques et al. (107)
Heparha
ox Forcine Heparitins Chonkoitins
Buffer M pH lung RUC. A B C D A B C KS
~~ ~~
Ba&ital 0.12 8.6 0.99 0.90 (0.81) 0.83 0.86/ 1.04 0.88 0.88 0.88 0.67
0.97 0.93
'&is 0.12 7.5 0.98 (0.76) 0.90/ 0.91/ 0.93 0.93 0.93 0.69
0.95 0.98
KCl-Hcl 0.02 2.0 1.00 0.87- 0.66 0.71- 0.61- 0.70
1.03 0.90 0.92 0.79 0.82
(2)
HAC-LiOH 0.35 3.0 1.00 0.93 0.72 0.76 0.88 1.04 0.84 0.82 0.84
1.00
Ethylene- 0.1 8.0 1.00 1.00 0.92 0.92 0.89 1.04 1.16 1.05 1.16
diamine
Prcpyl- 0.025 8.5- 1.00 0.98 1.11 1.12 0.92 1.08 1.35 1.25 1.35 1.20
diamine 10.0
Percentage rerrraining a f t e r fixation w i t h CPC (M NaC1)
100 100 39 70 0 0 0 0 0 ?
%ference: Ieo Heparin No. 4439 and Upjohn Heparin Lot No. ZX320. Muc. = intestinal mcosa; KS =
keratomlphate. / indicates alternative values depending on samples or runs; two values unconnec-
ted indicates two separate bands. Forcine intestinal nucosa heparin usually gives bm bands in
barbital and acid M f e r s in varying proportions. cpc = cetylpyridinium chloride
HEPARIN SODIUM 247
6.8.3. Cellulose acetate electruphoresis

The separation principle is the same as agarose electro-
phoresis. A disadvantadge is the small load capacity of the
cellulose acetate d r a n e s . The n-ethod is most useful f o r
identification of mll quantities of acid mcopolysaccharides
i n a relatively plre state (107). Cellulose acetate mmbranes
were used by Herd to identify mcopolysaccharides (142). Seven
ccnm?rcial heparin preparations were examined on cellulose
acetate d r a n e s by means of electrophoresis (143). The elec-
trolyte systems eXamined were: calcium acetate 0.1M and 0.3M,
zinc acetate 0. l M , El 0.1M and tris-ba&)iturate M f e r pH
8.8, I = 0.05. Heparin fractions were stained w i t h 0.5 per
cent alcian blue in 0.01M El. The use of the tris-barbiturate
buffer system allowed a l l samples except one to be resolved
i n t o two wll-defined zones a t 300 V f o r 50 min. The separa-
t i o n my he carried out using less than 1 pg of heparin.
A c o l o r k t r i c n-ethd for the quantitative determination
of heparin and other glycosaminoglycans based on the solubi-
l i s a t i o n of toluidine blue-polyanionk glycosamiraOglycans com-
plexes fran cellulose acetate membranes was described by Hsu
e t al. (144). The glycosaminoglycans were separated employing
three t y p s of electrolyte: 0.1N El, 0 . B htylamine and
0.05M calcium acetate containing 40 per cent of diethylene
qlycol .
A l l h a m glycosaminoglycans could be separated i n a
single mnodimensional electrophoresis on cellulose acetate by
Cappelletti e t al. (145). A very narrow s t a r t i n g band was ab-
tained by formation of a discontinuity in the electric f i e l d
and a l l ccmpounds wre then selectively resolved by combining
their migration properties i n a barium acetate buffer and
t h e i r differential sensitivity to precipitation by ethanol.
By wrking a t l o w current and temperature for longer
times, e l e c t r q h e r a y a n s are abtaincJ shawing well-defined
.
bands for heparin preparations (146) The electxphoreses were
perfomd i n barium acetate (0.1M) , adjusted to pH 5.8 w i t h
acetic acid, on cellulose acetate s t r i p s a t a voltage corres-
ponding to 0.4 mA/cm a t 4OC for 16 h. After staining w i t h
alcian blue, the densitanetric traces were recorded w i t h a
spectrqhotaneter a t 380 nm. Heparins w i t h similar profiles on
gel f i l t r a t i o n gave different electrophoretic patterns. It is
concluded that high-mlecular-weight species canplex Ba2+-ions
stronger than do low-mlecular-weight species.
6.8.4. Polyacrylamide gel electrophoresis
Polyacrylamide gel electrophoresis is a mthod which se-
parates acid nuccpolysaccharides as a function of their mle-
248 FEUEDRICH NACHTMANN ETAL.
cular sizes. The electrophoresis is performed in gel 0.2 an

thick on 5.0 x 7.5 or 10 can s l i d e s (107). After separation
(2 V / a n f o r 50 min with electrodes in b a r b i t a l l x f f e r ) , the
g e l is stained with 0.1 per cent toluidine blue in 1 per cent
acetic acid f o r 30 m i n and the stain reimved with 1 per cent
acetic acid. A p l o t of mlecular w i g h t x l o 3 on a log scale
versus relative distance f r a n the o r i g i n gives a s t r a i g h t
line. The mthod is very useful f o r the determination of t h e
mlecular sizes and degree of p l y d i s p e r s i t y of pure sulphated
muqlysaccharides.
6.8.5. Electrofocushg
A considerably greater heterogeneity of heparin is de-
tectable by mans of electrofocusing. Substances which show
2 bands in conventional electrophoretic systems can be resol-
ved into 21 bands by electrofocusing with ampblyte mixtures
(147). Polyacrylamide gel slabs (5.0 x 15 an), 0.2 an thick
containing 2 per cent of ampholyte (pH 3.0 to 5.0) f r a n LKB
(Sweden) were used. 100 t o 200 pg of heparin was applied to
the gel i n 2 p l volunrts a t 2 an f r a n t h e neyative end and
subjected to a p t e n t i a l of 15 V/cm f o r 18 hours. The 91 slab
was stained with toluidine blue. The 21 bands are d i s t r i b u t e d
between pH 4.2 and 3.2.
For 15 cannercia1 heparin samples tested, 21 f r a c t i o n s
were found by electrofocusing (148). These fractions shm a
mlecular-weight range from 3,000 t o 37,500 with a constant
i n t e r v a l between mlecular w i g h t s . me technique is s p e c i f i c
and unequivocally distinguishes heparin f r a n other acid mco-
polysaccharides. Results by f i g h e t t i and G i a n a z z a d m n s t r a t e
that t h e charge heterogeneity of heparin absented i n electro-
focusing is e l i c i t e d by strong interaction of t h i s p l y -
saccharide with Ampholine, the mixture of amphoteric sub-
stances used to generate and s t a b i l i z e the pH-gradient (149).
6.8.6. and lung o r i g i n
Ccinparison of heparin of mcosal
The differences wre summrised recently by W f i e and
Cuwie (150).
When electrofocused fractions are eluted f r a n the gel and
subsequently exposed to plyacrylamide electrcphoresis, a
range of mlecular w i g h t s is abtained. ?his is true of hepa-
rin of mcosal or lung origin. An important difference exists,
hawever, between lung and mcosal heparins. This is shown i n
figure 6.1. Unfractionated lung heparin y i e l d s a s i n g l e ccm-
ponent i n agarose, a d i f f u s e band i n plyacrylamide and 22
c c n p n e n t s upon electrofocusing. Unfractionated mcosal hepa-
rin yields two canponents in agarose, a d i f f u s e band in p l y -
HEPARIN SODIUM 249
acry1ami.de and 22 canponents upon electrofocusing. Lung hepa-

rin fractions derived fran the electrofucusing prccedure
yield a single band i n agarose, plyacrylamide and electro-
focusing. Mucosal heparin fractions derived fran the electro-
focusing procedure yield two bands i n agarose, two bands i n
polyacrylamide and a variable number of bands upon electro-
focusing.
-
ELECTROPHORETIC MIGRATION PATTERNS IN:
a b C
-
Heparin Agarose Polyacrylamide LKB Ampholyte
1(beef lung) I 11111111111111111111ll

(22)
2 (pork intestine) II 11111111111111111111l

(22)
Single Fraction
from l c I I I
Single Fraction
from 2c II II Ill
Separation on charge molecular size pH and chain len-

the basis of: density gth producing an
insoluble complex
with ampholyte.
Cathode and point of application considerably to left of each pattern;

anode to right.
Figure 6.1
Sumnary diagram of electrophoretic patterns abtained for dif-
ferent heparinS prior t o and following e l e c t r o f m i n g , accor-
ding to McDuffie and Cawie (150).
Mhen mlecular wights of the lung heparin fractions are
determined i n plyacrylamide gels, a family of different chain
lengths is &served. Molecular w i g h t determinations of mu-
ccsal heparin fractions, containing 2 canponents, yield 2
f d l i e s of differing &ain lengths w i t h a high degree of
parallelism.
6.9. Chranatographic Methods

6.9.1. Paper chromatography
Paper chrmtography is mostly used for the identifica-
t i o n of heparin.
Molho and Molho-Lacrout described a one-dimensional paper
partition chranatography of canmercial heparin with propanol/
water (1:1.5) by using toluidine blue as a detecting agent
(151). A very intense spot, Rf = 0.57 and a weaker spot = 0
w e r e found. Ihe former had strong anticoagulant activity,
whereas the latter had about 1/25 of the total activity.
Awe and Stiidemann reported the separation of heparin and
heparin-like canpounds by paper chranatography with aqueous
-
propano1 (30 45 per cent) as the solvent (152). The method
permits one to detect the degree of purity of heparin and dif-
ferentiation between heparin and synthetic sulphonated poly-
saccharides.
Another paper ChraMtographic system was developed f o r
t h e resolution of mixtures of sulphated mcoplysaccharides
by Spolter and Mam (153). The effect on t h e +
values of he-
parin and chondroitin sulphate of varying the ammnium forma-
te buffer/isoprqanol ratio i n the m b i l e phase was studied.
One of these solvent mixtures, amrmnium f o m t e buffer/isopro-
pan01 (65: 35) resolved ccnurercial bovine heparin into two
principal canponents.
Cenik and Kronberger used pper chrcmatography f o r the
quantitative and qualitative determination of heparin i n blood
(154). The chranatographic separation is performed i n propa-
nol/phosphate h f f e r of pH 6.4 (75:25). The Rf value f o r hepa-
rin was 0.90 - 0.93. For quantitative determination the CQ-
lour reaction w i t h toluidine blue was used.
Bayer described the separation of heparin and acidic
polysaccharides w i t h propano1 85 per cent/acetic acid/water
(30:10:40) as the m b i l e phase (155). The chranatcgram was
developed by the ascending technique f o r 6 -
16 hours and
sprayed with 0.05 per cent aqueous toluidine blue. Heparin
appears as a v i o l e t spot on a blue background, Rf = 0.45.
Castor and D o r s t e w i t z investigated the following solvents
f o r the paper chranatographic identification of heparin and
other acid mxopolysaccharides (156): 0.4M AlC13, 0.5M FeCl3,
0.1, 0.5, 1.OM MgC12 and 0.04M aqueous an-mnium acetate solu-
t i o n in methanol (45:55). Most of the k n m manutalian acid
muccplysaccharides could be identified. Protein contamina-
t i o n of significant degree did not interfere w i t h the method.
HEPARIN SODIUM 251
An improvement of the staining of plysaccharide sul-

phates was developed by Benaim (157). The mobile phase con-
sists of 5 m l of a 20 per cent solution of Cetavlon (hexa-
decyltrimethylamrrpnium branide) i n alcohol and 95 m l of a
0.2 per cent solution of toluidine blue or azure A. The paper
is dipped f o r 15 - 30 seconds, washed and dried.
6.9.2. Thin-layer chranatcgraphy
Like paper c h r a n a t q q h y , the i d e n t i f i c a t i o n of heparin
is the main use of thin-layer chranatography.
Giiven prepared thin-layers f r a n a mixture of 10 g of hy-
drated MgO, 1.5 g of CaC12 and 60 g of H20 and dried f o r 24
hours a t roan temperature (158). Heparin samples (about 50 I U )
were applied and developed f o r 3 hours i n mthanol/pyridine/
water (30:20:20). After spraying with 2 per cent sodium n i t r i -
t e and then with 0.5 per cent p n i t r o a n i l i n e in c o n c e n t r a t d
acetic acid, heparin appeared as a red s p t with an Rf value
of 0.7. The same author also used thin-layers prepared with
.
c e l l u l o s e (159) The developing systems armronia (25 per cent) /
pyridine/water (60:35:5) and butanol/acetic acid/water (3:l:l)
i n canbination w i t h t h e following colour reagents =re stu-
died: diazotized p-nitroaniline, ninhydrin, azure A, p d i -
methyl-aminchnzaldehyde, copper sulphate/mnia, eriochrcme
black T and water blue. The colour produced by h q a r i n on cel-
lulose thin-layer plates with azocarmine G, acridine orange
and brmcresol green w a s also investigated by Giiven and Er-
tan (160).
Thin-layer chranatography was applied by Bischel e t al.
f o r separating sulphated acid polysaccharide molecules (161).
Both cellulose and Sephadex 6 5 0 w e r e used as mdia f o r chro-
matography. Separation on cellulose p l a t e s is achieved with a
citrate bdfer p H 3.1.
mixture of n - p r o p a n o l / i ~ r ~ l / O . 0 3 7 M
For Sephadex plates, a mixture of n-propanol/isopropaml/etha-
no1/0.037 M citrate buffer pH 3.1 (1:4:2:13) is used. Deve-
loping time is approximately 18 - 36 h f o r Sephadex and 13 h
f o r cellulose plates. A f t e r staining with toluidine blue, as
little as 0.1 p g of plysaccharides can be detected.
6.9.3. Ion-exchange chranatoqraphy

Manley plblished a method f o r rapid separation of mco-
plysaccharides by salt-gradient, ion-exchange paper chranato-
graphy (162). The samples here separated on A m b e r l i t e SB-2
ion-exchange paper using ascending chranatography with a so-
dium chloride solution of s t e a d i l y increasing mlarity and &
tection of the nuccplysaccharides with alcian blue. h g e r t z
and &ichard fractionated
P
aluronic acid, chondroitin A and
heparin by e l u t i o n with NaC -El mixtures f r a n ECTEOLA-cellu-
lose columns (163). Similar methods using DEAEicellulose (164,

165) and D0we.x 1 X 2 (166) have been pblished.
A canbined chranatcgraphic fractionation for a canplete
separation of acid mcopolysaccharides using cetylpyridinium
chloride cellulose, DE-52 cellulose and Dohex 1 X 2 successi-
vely as column supports was described by Bohn and Kalbhen
(167).
Ion-exchange chranatography on DEAEiSephadex o r prota-
mine-Sephamse separated cmmrcial heparin along a continuum
of anticoagulant activities (168). 95 per cent of the applied
heparin was recovered from the DEAeSephadex column between
0.75 and 1 . 1 M NaCl. ?he specific anticoagulant activity of the
fractions rose steadily from less than 50 IU/mg to high levels
consistently greater than 250 IU/mg in fractions eluting close
t o 1.OM NaCl.
W r i m e n t s using DEAEiSephacel (DEAEicellulose i n bead
form) columns w i t h sodium chloride gradient eluent wen
carried out by Sache e t al. (169). ?he mst active anticoagu-
lant fraction was always associated with a heparin canponent
eluted a t high NaCl mlarity (approximately 0.8M).
6.9.4. G e l chranatography
G e l chranatography has been used f o r mny pars f o r frac-
tionating heparin. During the last few years an improvemnt
was made by introducing high pressure liquid chrcmtography
(WE)
Many authors describe separations using Sephadex-gels.
Skalka used Sephaaex G l O O and 200 (170). It was found that in
low ionicstrength eluents the mlecules of heparin are alte-
red to such an extent that they cannot penetrate into the
accessible pores of the gels. In solution of a higher ionic
strength, they are retarded even on 6 1 0 0 .
Porcine heparin was fractionated into 10 fractions of
varying mlecular w i g h t by gel f i l t r a t i o n on Sephadex G l O O
by Laurent et al. (171). Anticoagulant activity of the frac-
tions increased w i t h mlecular weight up to 18,000 and then
was f a i r l y constant.
Many ccnurercial heparin sodium injections (5 000 I U / m l )
wre fractional on a 2.5 x 45 cm column of Sephadex 6 5 0 , me-
dium grade (172). ?he heparin was eluted with d i s t i l l e d w a t e r
a t a flow rate of 36 ml/h. Each one m l fraction was mnitored
mtachranatically for heparin content by mixing 50 p l of the
fraction with 3 m l of an aqueous solution of 0.0025 per cent
toluidine blue and n-easuring the transmittance a t 500 m. X-k-
parin preparations of nucosal and lung origin gave similar re-
HEPARIN SODIUM 253
s u l t s in v i t r o except that the anti-Xa activation a c t i v i t y of

the lung preparation did not appear to increase w i t h decrea-
sing mlecular s i z e as was found with the mcosal heparins.
Rosenberg e t al. fractionated porcine heparin by cetylpy-
ridinium chloride precipitation and gel f i l t r a t i o n on Sephadex
G l O O (173). Additionnally, heparin fractions sere mixed with
antithranbin and fractionated by gel f i l t r a t i o n .
Recently, lrosito e t al. investigated a column chranato-
graphic technique f o r the fractionation of heparin using vari-
ous diameters of bead fonn cmss-linked dextran gels and an
autcmated apparatus (174). It was &served t h a t Sephadex 6 5 0
resulted i n the separation of three sell-formed peaks and pro-
vided superior resolution canpared to other gels. The s i z e of
the columns was 1 x 60 cm, the eluent 0.086M Tris-El, pH 8.0
containing lM sodium chloride a t 25OC.
Another gel type -
plyacrylamide agarose gel was used-
.
by Johnson and Mu1loy (175) Commrcially available heparins
were fractionated to give fractions whose mlecular w i g h t s
sere estimated by viscosimetry. A similar system was used by
O g m e t al. (176). !they mde experiments W i t h Ultrogel (AcA
44) agarose-acrylamide gel which was found to be favourable
f o r the separation of the higher mlecular w i g h t region of
heparin.
Gel f i l t r a t i o n experinlents w i t h Ultrogel (AcA 54) were
described by Sache et al. (169). Eorcine heparin sodium was
dissolved in 0.3M NaCl solution pH 6.8, applied to the column
(2.6 x 100 cm) and eluted w i t h the same solution. The anti-
thranbin specific a c t i v i t y was m w h a t syrmnetrically d i s t r i -
buted along the whole profile of the elution curve of heparin.
Gel f i l t r a t i o n w i t h ~ ~ particles -
1 1 (5 10 pn) becomes
increasingly important f o r the determination of m l e c u l a r
e i g h t distribution of heparin sodium. Rodriguez used 5 - 10
pn p a r t i c l e size controlled porous glass of different pore
diameters (40 A, 100 and 250 d) and simultaneous detection
with a d i f f e r e n t i a l r e f r a c t m t e r and ultraviolet detector a t
254 nm ( 4 2 ) . The column length was 10 - 60 cm, with 2.3 mrn in-
ternal diameter. The mobile phase was 0.1M sodium acetate
(0.01 per cent in sodium a i d e t o i n h i b i t bacterial qrowth) i n
b i d i s t i l l e d water. The columns were calibrated with different
molecular weight fractions of heparin sodium and baseline reso-
lution obtained between 6.5 k Daltons differences i n molecular
weight. Analysis time was 25 minutes per sample. Most effecti-
ve w a s the 250 8, pore diameter s i z e mterial.
A m d i f i c a t i o n of this technique was described by the
same author ( 4 1 ) . Instead of columns self-packed w i t h control-
led porous glass, comnercially available columns consistinq of

30 an x 3.9 mn internal diameter stainless steel tubing packed
with micrqarticles (10 p) of a fully porous silica-ether
[si(W),CH3] could be used. Molecular w i g h t averages could be
determined accurately to about k 500 Daltons. A difference
between hovine lung and porcine intestinal mcosal heparin was
noticed i n this study: bovine lung heparin sodium gave a
f a i r l y syrrmtrical peak about the maxima whexeas mst mcosal
heparin gave a peak that was skewed taward low molecular
weight.
The calibration mthod proved to be wry critical. Dif-
ferences i n mlecular shape of ccrnpounds can appear as diffe-
rences i n mlecular weight in -1 chranatqraphy. Different
results are cbtained i f the columns are calibra- w i t h dex-
trans (40) instead of heparin sodium.
An HPK-system with W-detection a t 205 m, a differen-
tial refractaneter or a mving w i r e flame ionization detector
was described by Sugisaka and Petxacek (40). Columns of 2.3 mn
internal diameter, 1 m long, packed with controlled porous
glass and columns of 4.0 nun internal d i e t e r , 30 cm long,
packed with porous silica (particle size 10 prn) were studied.
The rmbile phase was sodium sulphate, 0.5M, pwnped a t a flow
rate of 1.0 rnl/min. The columns were calibrated by injecting
dextran solutions. Prabably due t o the calibration method, the
results for the fractionation of canmrcial heparins are dif-
ferent fran those of other investigations (41, 42). A similar
system f o r mlecular weight estimations of heparin fractions
was applied by Sache e t al. (169). 7Xm. stainless steel columns
(300 x 4.7 rmn internal diameter) packed with porous silica,
10 pn particle size, =re connected. The m b i l e phase was
0.02M sodium sulphate a t a flow rate of 1 rnl/min.
6.9.5. Affinity CsUraMtcgraphy

Affinity dwamtography is used for separation of high-
activity and low-activity heparin species. HLiijk e t al. coupled
p l r i f i d bovine antithranbin via amino groups to cyanogen bro-
mide-activated Sepharose (70). sarrp?les of heparin sodium,
dissolwd i n 0.2M NaCl - 0.lM Tris-HC1, pH 7.4 were applied to
a column containing 3 rnl of antithranbin-Sephamse gel. A f t e r
equilibration and mshing w i t h the same h f f e r at 4OC, the
sample was eluted with a linear salt gradient (sodium chloride
0.2M to 3 M i n 0.1M Tris-El, pH 7.4). The effluent MS analy-
sed by the carbazole reaction. A separation into a high-affi-
n i t y and a low-affinity heparin was achieved.
Bleyl and Raka determined the affinity between heparin
and a n t i t h r h i n by carrpetitive affinity chranatography (177).
HEPARIN SODIUM 255
They conclude that the chemical modification caused by cova-

l e n t linkage of antithrombin to a matrix alters t h e binding
characteristics between antithrombin and heparin.
Schrrrer patented an a f f i n i t y chranatography method for the
separation of high-activity heparin (178). The mthod employs
an a f f i n i t y column of protamine coupled to a water-insoluble
solid support such as agarose, and a series of sodium chloride-
imidazole e l u t i o n M f e r s (pH 6.5 - 7.5) varying in sodium
chloride m l a r i t y fran about 1.3 to about 2.0.
The fractionation of heparin by chranatography on imnnbi-
lized thranbin was plblished by Nordenman and Bjork (179). Bo-
vine a-thranbin was coupled to cyanogen branide-activated Se-
pharose in the presence of an excess of acetylated heparin.
The material was packed into a column, 1.6 x 3.4 cm. Elution
and detection were similar to techniques described by H W k e t
al. (70). The authors suggest that the separation of heparin
on matrix-linked thranbin into fractions with d i f f e r e n t anti-
coagulant activities is due m i n l y to ion-exchange chmmato-
graphy, the inmobilized thranbin a c t i n g as a weak anion ex-
changer-
A n w technique involves the binding of antithranbin I11
t o a concanavalin A-sepharose column, r e s u l t i n g i n a higher
capacity f o r high-activity heparin (180). Antithranbin I3 is
bound w i t h its &hydrate side chains to concanavalin A.
Chemical rrodification of the glycoprotein is not required and
active antithrcmbin m y be recovered. 40 per cemt of heparin
samples =re bound to t h e column with binding capacities of
15 mg/100 mg antithrcmbin. The peak of bound heparin e l u t e s a t
a sodium chloride concentration of 0.6M. The anticcagulant po-
tency was 270 unitdmg in the APTT assay.
6.9.6. HyJrophobic interaction chrunatoqraphy

Ogam et al. separated porcine nucosal heparin by hydro-
phobic i n t e r a c t i o n cfiramatography on phenyl-sepharose CG4B
i n t o two groups, one w i t h high a f f i n i t y and one w i t h low a f f i -
n i t y f o r t h e cpls (176). The former group i a s further separa-
t e d i n t o three fractions d i f f e r i n g i n hydmphcbicity. While
the N-acetyl content of the f r a c t i o n with the lowest hydro-
phabicity were 0.12 ml/ml of hexosamine, those of the frac-
tions with higher h y d q h o b i c i t y =re 0.15 - 0.17 ml/ml. ?he
anticoagulant activities of the fractions w i t h higher hydro-
phobicity ranged fran 210 to 254 units/mg, whereas that of the
f r a c t i o n with lower hydrophobicity was 100 units/mg. The
samples were loaded on a 2 x 43 cm column and eluted stepwise
w i t h mixtures of ammnium sulphate/hydrochloric acid a t roan
tgnperaixre.
6.9.7. Countercurrent d’lranatography

This mthcd was described by Wst e t al. for continuous
flow fractionation of glycosaminoglycans by partition i n two-
phase systems (181). In the 1-butanol/aqueous N a C l two-phase
system containing hexadecylpyridinium chloride, fractionation
i s dependent primarily upon chemical canposition, o r charge
density, of the glycosaminoglycans and is relatively Mepen-
dent of molecular weight. Chondroitin 4-sulphate was fractio-
nated according to degree of sulphation and could be comple-
t e l y resolved frcm heparin. A heparin sample was shcrwn to con-
tain three discrete ccmponents differinq with respect to
sulphaminohexose and sulphate substitution.
6.9.8. Gas chranatography
Gas chranatcqraphy is used for the determination of dif-
ferent parts of the heparin mlecule. A differentiation bet-
ween mucous, lung and whale heparin is possible by gas chroma-
tographic determination of acetic acid obtained a f t e r hydro-
l y s i s of N-acetyl groups in heparin (182). The content of ace-
tic acid i n mcous, lung and whale heparin wre 1.52, 0.33 and
5.69 per cent.
The quantitative determination of hexuronic acids i n he-
parin using gas liquid chranatography was described (183). N-
Desulphation of heparin was achieved using 0.06N methanolic
HC1. The resulting N-desulphated heparin rrethyl ester was sa-
ponified to free acid w i t h 0.1N NaOH. It was then N-acetylated
with acetic anhydride. Trimethylsilylation of hexuronic acids
was carried out with hexamethyldisilazane and trimethylchlom-
silane. For gas chranatographic separation and quantification
a packed column f i l l e d w i t h Apiezon M on Gas-chran CLZ a t
190°C coupled t o a flame ionisation detector was used.
Another possibility was investigated by Inoue (184). He-
parin was d e a m i ~ t e dw i t h n-butyl n i t r i t e and rrethanolised.
The mnaneric silylated derivatives of uronic acids w e r e quan-
t i f i e d by gas chromatography. The separation was performed
with a packed column (200 an x 0.4 an internal diameter)
packed with 3.5 per cent SE30 on Gas-Chrm Q a t a l i n e a r tem-
perature programme fran 140 t o 2OOOC (l°C/min). A species-
specific difference i n the iduronic acid to glucwonic acid
r a t i o of heparin was noted.
Gas chranatography can also be applied for the quantifi-
cation on sulphate i n glycosaminglycans (185).
6.10. Amidolytic Methods
In standard procedures f o r the synthetic substrate rre-
thods, heparin is analysed as a heparin/antithranbin I11 ( A F
HEPARIN SODIUM 257
111) canplex. The pesence of AT-I11 in the test plasm is

crucial, either nonnal plasma or plrified AFIII can be used
to assure prcjpr canplexation. These assays can be adapted
for both end-point and kinetic mthods. Recently, test k i t s
containing the substrate, factor Xa, AT-111, Mfer, and nor-
ml human plasm in lyophilised fonn have becane available.
The procedure is as follows (186):
Heparin + AT-I11 d heparin/AFIII
Heparin/AFIII + factor Xa -~heparin/AFIII/Xa+residual Xa
Residual Xa + R-pNA _c,R-COOH + pNA
pNA = p-nitroaniline
Because the plrified Xa preparations are difficult to cb-
Residual thranbin + R-pm

-
tain, heparin assays involving thranbin were developed (186):
Heparin + AS111 4heparin/A?LIII
Heparin/AFIII + thranbin heparin/AFIII/thrallbhrcmbin t
residual thranbin
R-COOH + pNA
?he first chr-nic tripeptide, Bz-Phe-Val-Arg-pNA (S-
2160) was intrcduced by mndsen et al. as a specific substra-
te for thranbin (187). Many other similar peptides have been
described until nw. All of these substrates are coupled with
p-nitroaniline (pm) as a chrumphore at the terminal carboxyl
grow of the peptide and wntain arginine or lysine. The ab-
-
sorbance of F€W is measured at 400 410 nm. @lA bound to the
synthetic peptide does not absorb significantly at 400 - 410
nm, whereas the free form absorbs strongly here.
Nowadays fluorcgenic substrates are available for the de-
termination of serine protease enzymes such as thranbin or
factor Xa. An example is Pphenylalanine-proline-arqinine-5-
amido-isophthalic acid-dimethylester-diacetate (H-D-Phe-Pro-
Arg-AIE) the structure of which is given belw (188):
H-BPhe-PreArg .+N%<=’=3
thrallbin
5-43
0
?he excitation wavelength is 335 nm, the enission wave-
length 430 nm. The method is used for the determination of
heparin in humn plasm.
A surmrary of the mst important synthetic substrates for

the determination of heparin is given i n table 6.3.
Table 6.3
Synthetic substrates for amidolytic determination of he-
parin
Substrate Detec- Literature
tion
S 2160 Bz-Phe-Val-Arg-pNA Thranbin Absorbance 187,189
S-2238 H-D-Phe-PipArg-pNA l%ranbin Absorbance 189 ,190,
191
ChrCfIlOZym Tos-Gly-Pro-Arg-pNA l % r d i n Absorbance 192,193,
TH 194
H-D-Phe-Pro-Arg-AIE Thranbin Fluores- 188
cence
t-Boc-Val-Pro-Arg- Thrdin Fluores- 195
MCA cence
s-2222 Bz-Ile-Glu-Gly-Arg- Factor Xi Absorbance 189,194,
pNA 197,198,
199
t-Boc-Ile-Glu-Gly- Factor Xi Fluores- 195
Arg-McA Cence
t-Boc-Ser-Gly-Arg- Factor Xi Fluores- 196
MCA CenW
AIE = amidoisophthalic acid-dimthylester-diacetate
MCA = methylcoumaryl-7-amide
t-m = tert,-butyloxycarbonyl
Synthetic substrate mthods will play an increasingly im-

portant part in the determination of heparin in clinical che-
mistry laboratories. k z p analysers can be used for these
analyses. Chranogenic and fluorogenic methods give canparable
results (186). The advantage of the fluorimetric mthods is
their higher sensitivity.
6.11. Esterolytic Methods

This assay of heparin is based on the heparin-accelerated
rate of a-thrcanbin inactivation by antithrcmbin I11 (200, 201).
After incubation, a-N-benzoyl-L-arginine-ethylester (BAEE) is
added, which canpletely inhibits further a - t h r d i n inactiva-
tion. The residual a-thranbin hydrolyses -+to produce etha-
nol, which is measured i n the presence of NAD and alcohol de-
hydrogenase by mnitoring NADH-formation a t 340 nm. ?he mthcd
was used for the determination of heparin in lmmn plasma.
There was found to be good correlation with an amidolytic
method.
HEPARIN SODIUM 259
6.12. Anticoagulant and Allied Tests

Reviews on these techniques W E written by Jaques (107)
and Utes (75). There are two applications of anticoagulant
tests: mnitoring drug dosage in heparin theram and standar-
dization of the drug f o r anticoagulant therapy. In a l l coagu-
lation tests involving heparin, extra attention rmst be qiven
to the special significance of pH and surfaces.
Values for clotting tines w i t h mst coagulation systems
are essentially the same between pH 5.5 and 7.6, but the anti-
coagulant effect of heparin is mrkedly pH dependent and is
minimal a t pH 6.4. A decrease i n the coagulation system of
only 0.1 pH u n i t can significantly decrease the anticoagulant
action of heparin. Lyophilisation gmerally results in a pH
shift, since b i c e n a t e , citrate, and ammnium salts decan-
pose. (31 reconstituting lyophilised plasm, a l l the material
must be redissolved, and the pH measured and adjusted. A l l
solutions must be ad-~ustedt o pH 7.0 t o 7.2. A t pH 7.0 t o 7.2,
heparin solutions can be sterilised by autoclaving.
Blood coagulation is activated by surface contact. The
surfaces w i t h which blood canes into contact thanselves af-
fect profoundly the inhibitory effectiveness of heparin on
blood coagulation. Therefore, spcial procedures f o r preser-
vation of test tubes used for the detemination of whole blood
clottinq t h e , Howell whole blood assay etc. are necessary
(107).
6.12.1. Hematological procedures

The hematological procedures are chiefly coagulation
tests carried out in the clinical laboratory for the investi-
gation of coagulation disorders. 'Ihe tests used are sunmrised
i n table 6.4.
Withhi ;Ihe range of measurable clotting times there is a
linear relation between log clotting time and heparin concen-
tration (107). This mans that phannacOaynamic parameters
(e.g., half-life) calculated from coagulation values W i l l not
agree w i t h those calculated fran concentrations of the drug i n
the blood. Clotting t i m s can be recorded by Semi-autanated
instruments. One possibility is the nephelometric masurement
of f i b r i n f o m t i o n which WIS described for the assay of
plasma heparin concentration by means of a thranbin time
methcd by Noren e t al. (220).
Table 6.4
D i r e c t hematolcgical heparin tests on blood or plasma
Test Matrix Reference
Whole blood clotting time Fresh blood 202,203,107

S i l i c o n e c l o t t i n g tirre Fresh blood taken 203
i n silicone
P a r t i a l thranbplastin time Plasm 202,203
(PTT)
Activated PTT (APTT) Plasma 203,204,107
Thranbin t i m e Plasma 203,205,206
T i t r a t i o n with thranbin Plasm 207
Protamine titre Blood , plasma 203,208,209
210
Tbluidine blue titre Plasma 208,211
Factor Xa inhibitor Plasma 107,203,212,
213,214
‘Ihranbin inhibitor Plasma 219
Polyhrene n e u t r a l i s a t i o n Plasma 215 I 216 I 217
(titration)
P a r t i a l p l y b r e n e neutrali- Plasm 218
sation
Many s t u d i e s dealing w i t h a canparison of the different

tests were plblishea. ’kien and L i e canpared five mthcds
(221). The lowest detectable heparin concentration i n nonnal
plasma was: Factor Xa i n h i b i t i o n 0.012, calcium thranbin time
0.02, p l y b r e n e t i t r a t i o n 0.07 and APTT 0.08 I U / m l . The accu-
racy was lower in pathological than in n o m l plasm. A t a
heparin concentration of 0.5 I U / m l plasma, the coefficients
of v a r i a t i o n of eleven pathological plasmas were: Polybrene
-
t i t r a t i o n 3 per cent, factor Xa i n h i b i t i o n 15 20 per cent,
calcium thranbin time 35 per cent. W i t h the APTT method the
found heparin concentrations ranged frm 0.05 t o above 0.55
I U / m l plasma.
4 methods were canpared by Vinazzer (222). The lowest
s e n s i t i v i t y wis found for the thranbin time, s l i g h t l y better
r e s u l t s were abtained by the APTWr&hod. Addition of puri-
f i e d anthithranbin I11 t o the r e a c t i o n mixtures improved the
s e n s i t i v i t y of b t h mthods. l+henfactor Xa was used, the re-
s u l t s were canparable t o the APTCtest. The s e n s i t i v i t y was,
hawever, about 10 times higher h e n the remaining factor Xa
was d i r e c t l y measured w i t h a ckranogenic substrate.
Recently Phillips found, that plasm heparin levels mea-
sured using d i f f e r e n t mthods &wed a wide v a r i a t i o n (223).
HEPARIN SODIUM 261
I n particular, an anti-factor Xa clotting technique qave very

much higher results than thranbin t h e and partial thranbin
t k techniques. This was less marked when a synthetic ckrano-
genic substrate assay was used.
Whole blood coagulation t i m e , automated whole blood coa-
gulation time and APTT w x e cunpared by Sckriever et al. ( 2 2 4 ) .
Log APTT m s linearly correlated with blood heparin level and
with the 2 whole blood coagulation tests.
6.12.2. Phamacological procedures

Tests canparing the activity of two or more heparin pre-
parations i n a test system are designated "pharmacological
procedures". A review was given by Jaques (107), the most im-
portant coagulation tests are surrPMrised in table 6.5.
Tests of heparin preparations fran different sources with
different test systems give different anticoagulant a c t i v i t i e s
(107). Walton e t al. reported that when preparations of hepa-
rin from the lungs and mcosa of ox, pig, and sheep were
tested f o r potencies by the BP and USP assay prcedures, a
discrepancy of I+ to 4 0 per cent wis absenred &tween estima-
tes obtained by the two procedures on canparison of samples
of heparin derived fran lung and intestinal rmcosa (240) .
Shen and Barlow canpared human and sheep plasma ( 2 2 8 ) .
The specific activity of certain heparin fractions assayed i n
human plasma differ& fran that assayed i n sheep plasma. It
is concluded that the USP heparin assay does not necessarily
reflect the true activity in human blood clotting system.
The BP assay of heparin was canpared with som other
methods ( 2 4 1 ) . ?he APTT/BP potency ratios varied with the
preparations, lxlt was not dependent on the tissue source of
heparin. For rmcosal heparins, the anti-Xa/BP potency r a t i o s
were close t o 1, ht for heparin of lung origin the anti-Xa
potency was,1/4 of the BP potency. Heparin fractions prepa-
red by column gel chranatography showed a wide divergence bet-
wen the BP potency and those abtained with other lnethods.
Tests on camercial mcous heparin shaved that there is
no correlation between the potency found by the USP method and
an amidolytic method using Chranozym-TH as substrate ( 2 4 2 ) .
"he explanation of these results my be found i n the f a c t that
heparin preparations inhibit a number of steps i n the coagu-
lation sequence (107). Camxcial heparins are a mixture of
anticoagulant and nonanticoagulant heparins, with differing
relative proportions of the different &ain length heparins.
It appears most unlikely that the same specific heparin chain-
length provides the mst effective inhibitor for each step i n
the coagulation sequence. I n order t o investigate the problems
of canparing different preparations of heparin, a collabora-

tive study was performed (243). Porcine nucosal heparin tested
versus i t s e l f by different methods proved a good accuracy of
the methods. However, when different heparin preparations were
canpared, estimates for the same method by different labora-
tories o r for different substrate batches varied by as much
as 40 t o 50 per cent.
Wle 6.5
Pharmacological coagulation tests
Coagulation tests Substrate Reference

i n vitro
Modified Howell Fresh whole blood 208,107

Fischer Whole chicken plasm and 208
thranboplastin
Jorpes e t al. Ox blood 208
USP &calcified citrated sheep 208,225,226,
plasm 227
Modified USP Recalcified citrated h m n 228
plasm
&calcified citrated p o r c h 229
plasm
Recalcified bovine plasm 230,231,232,
and thranbokinase 233,234
BP Sulfated whole blood and 208,235
thranbokinase
Antithranbin Oxalated ox plasm and 208,236
thranbin
Antilkranbin C i t r a t e d ox blood and 208
thranbin
APTT Different plasms 237
Protamine neutrali- 238,239
sation
Tests in vivo
Jorpes e t al. Sheep with i n 4 l l i n g 208
catheters
Kuo e t al. Blood samples fran 240,107
conscious, trained dogs
The mnplexity of heparin leads to the conclusion of

Jaques that anticoagulant activity cannot be equated w i t h
heparin, nor heparin defined by anticoagulant activity (107).
HEPARIN SODIUM 263
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HYDROCORTISONE
Klaus Florey
1. Introduction 278
1.1 Foreword 278
1.2 Histoory 278
2. Description 278
3. Isolation and Synthesis 279
3.1 Isolation 279
3.2 Chemical Synthesis 279
3.3 Biosynthesis 280
4. Physical Properties 28 I
4.1 Spectra 28 1
4.2 Solid Properties 289
4.3 Solution Properties 293
5.1 Historical Synopsis 294
5.3 Ultraviolet 296
5.4 Colorimetric 297
5.5 Fluorescence 299
5.6 Polarographic 300
5.7 Isotope Dilution 300
5.9 Electrophoretic 306
5.10 Bioassay-Enzymatic 308
5. I 1 Saturation Analysis 308
7. Metabolic Products-Pharmacokinetics 310
8. Determination in Biological Fluids and Tissues 313
9. Determination in Pharmaceutical Formulation 3 14
References 315
ANALYTICAL PROFILES OF DRUG SUBSTANCES 277 Copyright by the American PharmaccuticalAssociation.

ISBN 0-12-260812-7
VOLUME 12
278 KLAUS FLOREY
1. Introduction
1.1Foreword
The compilation of an analytical profile
for hydrocortisone, also known as cortisol, turned
out to be a more ambitious undertaking than I had
expected. When scanning through Chemical Abstracts,
the literature is simply staggering. Fortunately,
there are quite a few very good and easily acces-
sible books and reviews available which will lead
the reader to secondary sources. First and fore-
most, there is the classic book on steroids by
Fieser and Fieser.' Helpful are also the more
modern books,on steroid analysis by Heftman* and
Gordg and Szaz.3 As in the case of aspirin, I have
endeavored to cover the newer literature as compre-
hensively as possible and have included only those
older references which are of historical interest.
Again, my apologies to all those scientists whose
publications in this field are not included here.
1.2History
Althouqh hydrocortisone is the major
hormone secreted by-the human adrenal cortex , it
was by no means the first adrenocortical hormone to
be isolated by the early investigators.
Reichstein14 the first to publish its isolation
from adrenal glands, gave it the letter M (see also
Section 3 ) . When Kendall and Hench made their
momentous discovery of the relief of the symptom of
rheumatoid arthritis5 in 1948, cortisone was the
first hormone to be made available by Merck and
Company. Only in the early fifties was hydrocorti-
sone also introduced into therapy, but was soon
eclipsed in potency and effectiveness by analogs
such as the A 1 - and 9a-halo compounds. Yet, it
still is much prescribed, since it is relatively
inexpensive. It is still the ultimate standard by
which the efficacy of other corticosteroids is
measured.
2. Description
2.1 Name, Formula, Molecular Weight
Hydrocortisone is pregn-4-3,20 dione,
11,17,21-trihydroxy-, ( 1 1 B l - i also 4-pregnene-118,
1701, 21-triol-3,20-dioneI 17-hydroxycorticosterone;
Reichstein's compound M; Kendall's compound F;
HYDROCORTISONE 219
cortisol Chem. Abstracts Registry Number [50-23-71.
21 CH20H
I
c=o
‘21H3005 M.W. 362.46
2.2 Appearance, Color, Odor, Taste

Bitter tasting, white to practically
white, odorless, crystalline powder.
3. Isolation and Synthesis
3.1 Isolation
In the late thirties, the isolation of
adrenocortical hormones was actively persued by
Pfiffner, Vars and Wintersteiner at Columbia, Mason,
Myers and Kendall at the Mayo Foundation, Cortland
and Kuizenga at the Upjohn Co. and Reichstein at
the University of Basle, Switzerland. It was
Reichstein4 who first described the isolation of
hydrocortisone and elucidated its structure. He
called it Substance M; but for several years, it
was better known as Kendall’s F. For details
relating to structure elucidation, I refer to
Fieser and Fieserl and also to Reichstein and
Shoppee.
3.2 Chemical Synthesis
As already mentioned (Section 1,2) ,
cortisone was the first corticosteroid introduced
into therapy, since the 11-keto group is more
readily accessible to synthesis. Hydrocortisone
was first synthesized in the Merck laboratories by
Wendler, Graber, Jones and Tishler7 from 20-cyano-
A17-pregnene-21-ol-3,11 dione, which in turn had
been prepared by Sarett8 in connection with his
cortisone synthesis. Introduction of oxygen at the
11 position has been crucial for the commercial
280 KLAUS FLOREY
production of corticosteroids from abundant natural

sources. The microbiological hydroxylation of
steroids at position 11, therefore, has been a
major breakthrough, pioneered in the Upjohn, Squibb
and Pfizer laboratories (for details, see
references 9 and 10) .The Pharmaceutical Manufac-
turinq Encyclopedia lists the microbiological
118 hvdroxvlation of Reichstein's compound S (11-
desox;- 17-hydroxy-corticosterone) to hydrocortisone
as a mode of manufacturing.
CH2OH CH2OH
I I
c=o C=O
However, I am not aware that this route is really

used on an industrial scale. The main commercial
routes to hydrocortisone are either based on
naturally occuring steroids, having an oxygen
function at carbon 12, or on llu-hydroxylation of
a suitable intermediate. In the first category are
the Merck process using bile acids and the process
starting with hecogenin, a constituent of sisal.
In the second category is the Upjohn process
starting with progesterone, derived from diosgenin,
a constituent of dioscorea or stigmasterol derived
from soybeans. Syntex played a leading role in the
development of suitable intermediates for the
diosgenin and hecogenin derived processes.
3.3Biosynthesis
The mammalian biosynthesis of hydrocortisone
and the other corticosteroids occurs in the adrenal
gland. The common precursor is cholesterol. The
immediate precursors of hydrocortisone are primarily
ll-desoxy-cortisol (Reichstein's compound S),which
is 118 hydroxylated enzymatically, and cortico-
sterone, which is hydroxylated at carbon 17.12 It
is curious to note that no mammalian lla-hydroxy-
lating enzymes are known which are the prevalent
HYDROCORTISONE 281
enzymes in microorganisms (see 3.2). The elucida-

tion of biosynthetic pathways was pioneered by
Hechter and Pincus and their collaborators at the
Worcester Foundation in the late forties.13 For
details on the biosynthesis of adrenal steroids,
I refer to an excellent review by Samuels and
Uchikawa14 and the book on steroid biochemistry by
Heftmann.15
4.1 Spectra
4.11 Infared
The first spectrum of hydrocortisone,
prepared in a nujol mull was published by Collings-
worth, et a1 in 1953.16 A sDectrum of hvdrocorti-
.
sone dispersed in a potassium bromide disk was
recorded by Hayden17 in 1954. The classical atlas
of infrared spectra of steroids by Dobriner,
Katzenellenbogen and Jones18 contains only the
spectrum of hydrocortisone acetate. Mesleyl’
described infrared spectra of hydrocortisone
acetone, had no absorption near 760 cm-B.
polymorphs. Form A, evaporated from eth no1 or
Form B,
evaporated from chloroform, gave a double peak at
761 and 752 due to chloroform.
The spectrum of the USP reference
standard of hydrocortisone in a mineral oil
dispersion,218presented in Figure 1, essentially
agrees with the spectra in reference 16.
4.12 Ultraviolet Spectrum
Hydrocortisone exhibits the ultra-
violet absorption spectrum typical of a,B-unsatu-
rated ketones. Such a spectrum, with a maximum at
241 nm, was recorded already in 1936 for Reichstein4
by Mehler of Zurich, Switzerland on a Hilger quartz
spectrograph. The Merck Index gives a X maximum
242 nm Ei 445.20 The chapter on steroids by
A.A. Forist and J.L. Johnson in Pharmaceutical
Analysis2I gives a X maximum 242 nm, E maximum
16,200. See a l s o the review article on ultra-
violet absorption of steroids by L. Dorfman.22 A
A maximum 242 nm, E maximum 15,800 has also been
reported.23 It also has a weak maximum at 290 nm
(log E 2.O1Iz4 and inflections at:
WAVELENGTH (MICRONS)
0.2
0.4
0.6
0.8
1.o
Figure 1. Infrared spectrum of hydrocortisone (USP reference standard) in a

mineral oil dispersion (Instrument: Perkin-Elmer 621).
HYDROCORTISONE 283
x (nm)
- log E
315 1.82
324 1.75
332 1.73
338 1.65
346 1.56
357 1.23
4.13Fluorescence
Hydrocortisone does not have any
native fluorescence, but it can be induced by
reaction with concentrated acids (see Section 5.5).
4.14Phosphorescence
The singlet + triplet transitions by
phosphogescence excitation spectroscopy at both 77
1
and 4.2 K were studied for several steroids.25
The following data pertain to hydrocortisone:
Phosphorescence emission: 3880 origin; 4350 8
maximum; So+T absorption: 3826 ; S-tS (rl,II) dire t
absorption: 3,600, 3,440, 3,310 ; A E: 1600 cm E .
4.15 Nuclear Magnetic Resonance26
The 100 MHz proton NMR spectrum of
hydrocortisone in DMs0-d~is shown in Figure 2.
The spectrum contains characteristic resonances at
6 0.76 s (l8-CH3), 61.37 s (19-CH3), 65.55 s (C4-H),
64.26 M ((211-H) and a coupled A B quartet at 64.50,
4.05 (21-CH2). Three peaks that exchange with D20
were detected at 65.14, 4.64 and 4.26 (11, 17 and
21-OH). All proton chemical shifts are referenced
to internal TMS at 0.0 PPM.
The carbon-13 NMR spectrum of hydro-
cortisone (Figure 3) is summarized in Table 1.
Assignments of all 21 carbons are listed and are
consistent with those of Blunt and Stothers.27 The
reference peak in the carbon spectrum was the DMSO
multiplet which was assigned as 39.5 PPM from TMS.
The spectra shown in Figures 2 and 3
were obtained on a Varian XL-100-15 NMR spectro-
meter equipped with a Nicolet TT-100 data system.
4.16Mass28
The low-resolution mass spectrum of
hydrocortisone (see Figure 4) shows the expected M+
at m/z 362. Corticosteroids generally show frag-
Figure 2. Proton NMR spectrum of hydrocortisone. Instrument: Varian XL-100-15.
N
oo
01
Fig. 3. Carbon-13 NMR spectrum of hydrocortisone. Instrument: Varian XL-100-15

6394 SQ9318 B R T C H *NX001 200 DEG
01-MRY-80 MB0764
1001
90-
80.-
>
I-
II)
70.-
Z
I
z
U
W
>
U
l-
a
J
w
CY
MRSS/CHRRGE
I N T E N S I T Y SUM = 4 0 8 2 5 BRSE PERK Z = 3 . 4 3
Figure 4. Low Resolution Mass Spectrum of Hydrocortisone. Instrument: MS-9

HYDROCORTISONE 287
TABLE 1. CARBON-13 NMR DATA FOR HYDROCORTISONE

CARBON # CHEMICAL SHIFT ( 6 )
1 34.0
2 33.5
3 198.0
4 121.5
5 172.3
6 32.8
7 31.4*
8 31.2"
9 55.6
10 38.9
11 66.5
12 39.1
13 46.3
14 51.6
15 23.3
16 33.0
17 88.5
18 16.9
19 20.4
20 211.5
21 65.8
6 in PPM from TMS (ext): Reference peak DMSO at
39.5 PPM.
* These assignments may be interchanged.
mentation patterns resulting from the l o s s of

D-ring substituents combined with the loss of small
neutral molecules such as water. The principle
hicrh mass ions are thus formed from the loss of
these groups, as depicted below. *
a
m/z 313
M+ m/z 362
m/z 302 m/z 344 +H
m/z 329
\r m/z 267
* Scheme is intended to show losses rather than to
explicitly show fragmentation pathways.
** Rearrangement
288 KLAUS FLOREY
Ions of mass less than m/z 267 (CigH230) occur

throucrh the loss of carbons (and attached hydrogens)
progressing from the D-ring. .
L m/z 267 -
In contrast to 1,4-dien-3-ones, the 4-en-3-ones

yield less intense diagnostic A-ring ions of m/z123
and 124. The analogous 1,4-dien-3-one would have
had a much more intense A-ring m/z 121, 122 ions.
The mass spectrum and fragmentation
pattern presented here is in essential agreement
with data reported earlier.29
A comparison of field desorption,
chemical ionization and electron impact mass spectra
of some steroids30 revealed that, both in the field
desorption and chemical ionization mode, the
molecular ions ( (M)+ and (M+H)+, respectively) are
the base peaks for hydrocortisone, while m/e 302 is
the base peak in the electron impact mode.
The field desorption kinetics in the
mass spectrum of hydrocortisone indicate that the
(M-C2H302)+ ions are formed by the gas phase
decomposition of the hydrocortisone molecular ion
at times >10-9-8 s.31
HYDROCORTISONE 289
4.2 Solid Properties

4.21 Melting Range
The following melting ranges have
been reported:
OC . Reference
217-220 with some decomposition 20
from ethanol and isopropanol
212-213 (commercial samples) 20
About 214 with decomposition 32,33
207- 214 34
215-218 24
201 (from ethanol, micro rn.p.1 35
4.22Differential Thermal Analysis
Hydrocortisone (USP reference
standard) when heated at 15O/min. (Instrument:
Dupont 900) gave a single endotherm at 221.50.36
4.23Thermogravimetric Analysis
Hydrocortisone (USP reference
standard) when heated at 15O/min. (Instrument:
Dupont 900) had a 0% weight l o s s to a temperature
of 222O. 36
4.24Calorimetry
The heat of combustion of hydro-
cortisone and other steroids was determined in a
microcalorimeter.37
4.25Crystal Properties
Microscopic pictures of hydro-
cortisone crystals were already presented by
Reichstein4 in 1936. As most steroids, hydro-
cortisone crystallizes in polymorphic and solvated
modifications. These can be summarized as follows:
Solvent of
Modification Crystallization I R Characteristics
I Ethano1,acetone OH C=O and C=C Other :

3430 1715,1708,1645 902 (strong band) 19,38
3310 (sh) 1631,1611 887 (sh)
I1 Desolvation from 3435

chloroform 3350(sh) 1712,1643, 38
1630,1611
I11 Methanol Solvate Met hano1 3515 1722,1643 38

3405 1628,1607
3225
IV Chloroform Solvate Chlorofo m 3435 1712,1643 761 ,752 19,38,39

3350(sh) 1630,1609
The optical crystallographic properties of hydrocortisone crystal-

lized from ethanol were determined as follows:35
Crystal System and Habit; monoclinic plates;
Optic sign: +; Optic axial angle: 56O (58O calc.)
Dispersion: R>V; Refractive Indices: Nx: 1.540; Ny: 1.562; Nz: 1.638
Solvents, other than ethanol, gave hexagonal (dichloromethane) and
orthorhombic (methanol) crystals.42
Hydrocortisone was subjected to single crystal x-ray analysis and
HYDROCORTISONE 291
the established structure compared to that of 9a-

fluorohydrocortisone and other related steroids.41
Hydrocortisone methanol solvate (Cambridge Crystal
Library Code: (ORTPIS), when subjected to single
crystal x-ray analysis, was found to crystallize in
the orthorhombic space group P212121 with a=14.372
(4); b=18.400(5); c=7.706(2); 8,2=4. Ring A is a
distorted "sofa", rings B and C are in the "chair"
configuration, and ring D is close to a C(13)
envelope, with a pseudorotational parameter A of
26.1O. The ring junctions B/C and C/D are both
trans. The molecule as a whole is slighgly convex
towards the 8-side, with an angle of 10.9 between
the C(lO)-C(19) and C(13)-C(18) vectors.42 These
findings have been compared with energy calculations
according to the Westheimer The electronic
structures of hydrocortisone and other related
steroids was examined by extended Hueckel molecular
orbital calculations. Total and orbital energies,
Mulliken population, analytical data and dipole
moments were reported.44 The crystal structure of
the pyridine solvate (Cambridge Crystal Library
Code: CORTPY) has also been determined.45
The x-ray powder diffraction data
for hydrocortisone are presented in Table 2 and
Figure 5.46 An early x-ray powder attern was
presented by Collingsworth, et al. 1g Low micron
particles of hydrocortisone were obtained by ultra-
sonic irradiation of supersaturated solutions.47
TABLE 2
Powder X-Ray Diffraction

Pattern of Hydrocortisone (USP Reference Standard)
20 d (8) I 1/10
5.99 14.72 4.5 0.52
9.40 9.40 0.5 0.006
10.60 8.34 0.5 0.006
11.90 7.34 2.0 0.023
12.89 6.86 1.0 0.012
14.70 6.02 86.0 1.000
15.56 5.69 1.0 0.012
16.37 5.41 8.0 0.093
17.20 5.15 4.0 0.047
17.69 5.01 22.0 0.256
19.07 4.65 4.3 0.050
Figure 5. Powder X-Ray Diffraction Pattern of Hydrocortisone (USP Reference
Standard). Instrument: Norelco
HYDROCORTISONE 293
20 d (8) I
- 1/10
19. 80 4.48 4.5 0.052
20.49 4.33 2.0 0.023
20.98 4.23 1.0 0.012
23.52 3.78 2.5 0.029
24.50 3.63 0.5 0. 006
27.59 3.23 1.5 0.017
4.3 Solution Properties

4.31 Solubilitv
According to Clarke,48 hydrocortisone
has the following solubilities:
%
Water 0.029 (1 in 3 , 5 0 0 )
Ethano1 2.5 (1 in 4 0 )
Acetone 1.25 (1 in 8 0 )
Propylene glycol 1.0 (1 in 100)
Slightly soluble in chloroform, almost
insoluble in ether.
The solubility/tem erature profile
in ethanol ranges from 6.3% at 65' to 1 . 5 % at 25°.49
The solubility in water is increased by the addition
of surfactants such as Tween 20 ( 0 . 0 2 7 mol. of
steroid/mol. of Tween 20) .50 The solubility in
tris-buffered KC1 was found to be 161.0 1-1 molesfi
and,in egg lecithin liquid crystals, 1 5 . 5 n moles/
P mol. lipid.51 The solubilization of hydro-
cortisone and other steroids by long-chain polyoxy-
ethylene surfactantsS2 as well as in polyethylene-
glycol fatty acid esters53 has been studied.
Calculations on the dissolution of
hydrocortisone have been presented in connection
with comparing the measurement of the interfacial
energy between solid particles and a dissolving
solvent to the film theory of dissolution in which
diffusion is the principal mechanism.54 A non-sink
dissolution rate equation has also been applied to
hydrocortisone.55
4.32 Partition Coefficients, Diffusion
An extensive list of partition co-
294 KLAUS FLOREY
efficients (Table 3) were presented by Engel and

Carter. 5
For partition coefficients in
surfactants and solubilizers, see references 52
and 53 (Section 4.31). An estimation of hydro-
cortisone partition coefficients in ether:water
based upon molecular constitution has been
attempted.57 The absorption kinetics of hydro-
cortisone at the water-neutral oil interface has
been studiedI5*using the Gibbs equation59 and
verifying the Szyskowski law.60 The effect of
surfactants on the diffusion of hydrocortisone
across a cellulose acetate membrane has been
investigated.
Optical Rotation
4.33
The following values have been
reported:
Reference
( a )Do temp.
+167 (abs. ethanol) 22 20
+16 3O (methanol) 22 20
+150-15 6O (dioxane) 20 ,25 32,33 ,48
+162 24
Optical rotatory dispersion studies
were conducted by Foltz, Lippman and D J e r a ~ s i . ~ ~
The following values were obtained:
(a)700 + 113O; (a)589 + 162O; (a)300 +
12020; "max" (a)385 + 4560; "min" (a)367,5 +
312O; Ilmaxll(a1357.5 + 460°; 'lmin" (a!352,5 +
428O; "max" (a)315 + 3506O; c = 0.10 in dioxane;
temp. 23-25. Drude equation: (MI = 172/A2-
0.0551; A, 235 nm; % deviation (M) Obsd -
(M) calcd: -+ 1.0%; 620-410 nm; -+ 3.7%, 700-400
nm.
5.1Historical Synopsis
As with all hormones which are present in
biological systems only in minute quantities, an
assay system, usually a biological one, is needed
as a guide for their detection and isolation. In
the early work leading to isolation of hydro-
cortisone and other corticosteroids from adrenal
HYDROCORTISONE 295
TABLE 3
Partition Coefficients for Hydrocortisone
(from reference 56)
1. Benzene/water 0.36
2. Benzene/50% methanol in water 0.14
3. Benzene/50% methanol in water 0.31
4. Petroleum ether/35% ethanol in water <o. 02
5. 25% Sec. butanol in n-hexane/water 0.24
6. 35% Sec. butanol in n-hexane/water 0.84
7. 25% Ethyl acetate in n-hexane/water 0.04
8. 50% Ethyl acetate in n-hexane/water 1.00
9. 30% Ethyl acetate in cyclohexane/30% ethanol
in water 0.31
in water 0.08
in water 0.04
in water 0.96
in water 0.39
in water 0.16
in water 2.7
in water 1.2
in water 0.02
18. Ethyl acetate/water 12.2
19. Water/carbon tetrachloride 0.16
20. 30% Methanol in water/70% chloroform in carbon
tetrachloride 0.70
tetrachloride 2.6
tetrachloride 5.9
23. 30% Ethanol in water/carbon tetrachloride 0.06
24. 0.05% NaCl in water/50% n-hexane in chloroform 2.57
25. 20% Ethanol in water/50% n-hexane in chloroform 2.57
26. 30% Ethanol in water/chloroform 0.06
29. 0.1N Acetate buffer/methylene chloride 0.12
30. 20% Methanol in water/50% n-hexane in chloroform 4.0
31. 80% Methanol in water/l.2-dichlvroethane 1.26
2% KLAUS FLOREY
tissue, the response of adrenalectomized animals

was used. Later on, corticosteroids were classi-
fied according to their relative activity in
promoting deposition of glycogen in the liver and
in controlling electrolyte balance in body fluids.
Hydrocortisone was found to have the highest gluco-
corticoid activitv and to be rather inactive
in controlling the electrolyte balance (cf. ref. 1,
p. 604).
When cortisone and hydrocortisone found
therapeutic applications, the Porter-Silber method62
permitted insight in the concentration of steroids
with the dihydroxyacetone side chain in biological
fluids. The blue tetrazolium method of blader and
provided a convenient and stability indica-
ting control procedure of these steroids in pharma-
ceutical preparations. Z a f f a r ~ n iapplied
~~ the
well-known color reaction of steroids with sulfuric
acid to hydrocortisone.
Paper chromatography, as an analytical
tool for steroids, was introduced in the fifties by
Zaffaroni and Bush, TLC and GLC followed in the
sixties, and HPLC in the seventies. For background
and discussion of methods, three excellent recent
books on steroid analysis are available. 2 ~ 3 ~ 6An
5
older book on pharmaceutical analysis also includes
a chapter on steroid analysis.21
5.2 Elemental Analysis
The elemental composition is as follows:20
C:69.59%; H:8.34%; 0:22.07%
5.3 Ultraviolet
The stronq abscmption band in the ultra-
violet ( A max 242 nm; Ei 445, see section 4.12) has
been used for quantitation of hydrocortisone itself
(cf. 21), but due to the interference of excipients,
it rarely can be used in the quantitative determi-
nation of dosage forms without difficulties. The
sodium borohydride method of GorZjg66 overcomes this
obstacle by using the reduced solution as a blank
in the reference cell. Kir~chbaurn~~ found that
addition of propylene glycol assures complete
reduction.
HYDROCORTISONE 297
5.4 Colorimetric
5.41 Sulfuric Acid
It was already known in the thirties
that steroids give color reactions with concentrated
sulfuric acid (cf. 3, p. 1 8 2 ) . A s already mentioned,
Z a f f a r ~ n iwas
~ ~ the first to apply it to hydro-
cortisone. He found absorption maxima at 2 8 0 , 3 9 5
and 4 7 5 mp. My personal prejudice concerning this
method coincides with that of Gorog and S z z z , who
....
state (cf. 3, p. 1 8 2 ) : I' spectra are not
reproducible as well as in the common solvents: the
positions and intensities of the maxima depend on
the time elapsed between dissolution of the steroid
and the recording of the spectrum, the temperature,
the quality of the sulfuric acid, the purity of the
sample, etc." The method today seems to be only of
historical interest, and I refer the reader to the
classical paper by Bernstein and Lenhard6* and the
chapter by Smith and Bernstein.69
They present the following data for
hydrocortisone:
1%
max (nm) Elcm
237 420
2 82 462
391 325
475 1 53
Sulfuric acid has also been used

diluted with other solvents, but rarely water. A
mixture of glacial acetic acid and concentrated
sulfuric acid ( 4 : 6 ) was found to give an absorption
maximum at 4 7 0 nm with hydrocortisone but not
cortisone.7 0
When hydrocortisone was heated in
sulfuric acid and treated with phosphorus
pentoxide, it gave an absorption maximum at 5 4 8 nm
(optical density 0 . 6 2 5 ) . 7 1
5.42 Other Acids
Hydrocortisone in orthophosphoric
acid (cf. 3, p. 1 8 6 ) gives spectra very similar to
sulfuric acid. The U.V. absorption maximum of
hydrocortisone in 7 2 % perchloric acid has been
given as 2 8 3 nm.72 Concentrated hydrochloric acid
has great specificity for the 116-hydroxyl group.
298 KLAUS FLOREY
Hydrocortisone gives a sharp, intense maximum at

465 nm in contrast to lla-h droxy, 11-keto, or
11-unsubstituted steroids.7 3
5.43 Porter-Silber
The principle of the Porter-Silber
method62 is the reaction of the dihydroxyacetone
side chain with phenylhydrazine in methanolic
sulfuric acid to produce a stable yellow color with
a maximum at 410 nm. This reaction is specific for
17-hydroxy steroids, and the sensitivity of the
method permitted its use to determine glucocorti-
costeroids in biological fluids. It was first
applied in 1952 to determine h y d r o c ~ r t i s o n ein
~~
urine and in 1957 to plasma75. With the advent of
chromatographic and RIA techniques, the method
may no longer have the importance it once had, but
it still is described in a modern (1975) book on
the determination of hormones.76
5.44Tetrazolium
The tetrazolium methods still belonq-
to the most important analytical techniques to
determine corticosteroids as such and in pharma-
ceutical preparations. For an excellent discussion
of mechanism and history, see reference 3 , p. 331.
It was first used for hydrocortisone as a spray
reagent for paper chromatography by Burton,
Zaffaroni and Keutman.77 Mader and Buck are
credited with extending the use of tetrazolium to
pharmaceutical steroid analysis but did not
describe the application to hydrocortisone in
their original paper.63 Hydrocortisone was first
quantitated by Henley,78 using 2-iodophenyl-3-
nitrophenyl-phenyl tetrazolium chloride. The USP33
favors the use of blue tetrazolium (B.T.; diani-
sole-bis-diphenyl tetrazolium chloride), the B.P.32
the use of triphenyl tetrazolium chloride (TZ) for
the determination of hydrocortisone and its dosage
forms. Absorbance maxima for the two variants are
at 525 nm and at 485 nm, respectively. The tetra-
zolium method is stability indicating, albeit an
indirect one measuring the reducing capacity of
the steroid side chain to form formazans. The
blue tetrazolium method has also been automated
for the assay of hydrocortisone in tablet^.^^^,^^
HYDROCORTISONE 299
5.45 Miscellaneous
For the determination of the free
21-hydroxy group of hydrocortisone in the presence
of its acetate,oxidation of the side chain with
cupric acetate to the glyoxal and condensation
with 0-phenylenediamine, (A max 331 nm, E 10,200) or
its 4,5-dimethyl derivative ( A max 351 nm, E 12,700)
have been used.80 Condensation with phenylhydrazine
gives an absorption maximum at 364 nm ( E = 19,000)
which has been used for the determination of hydro-
cortisone in tablets.81 For the determination of
residual hydrocortisone in prednisolone, the rate of
thiosemicarbazone formation was determined at
3 0 2 nm.82 A l s o used were reactions with Dische 83
reagent (diphenylamine in acetic and sulfuric acid),
bismuth oxidation,84 condensation with isonicotinic
acid hydrazide (370 nmIa5 also automated for
tabletsa6 and reaction with ammonium molybdate
(A max 655 nm) . 8 7
5.5 Fluorescence
Although hydrocortisone has no native
fluorescence, it can be induced by treatment with
concentrated sulfuric acid. This was already
observed by Wintersteiner and Pfiffners8 in 1936.
The various conditions of acid concentration and
temperature leading to slight variations of the
excitation wavelength (470 nm) and absorption
maximum (530 nm) as well as intensity of fluores-
cence are described by Goldzieher.89 The great
sensitivity of the reaction made it an ideal tool
to study the concentration of hydrocortisone in
biological fluids and tissues in a quantitative
fashion. It was first explored by Sweatgo in 1954.
He was able to determine as little as 5 nanograms
of hydrocortisone in 1 ml of ethanolic sulfuric
acid. The early work on the fluorimetric analysis
in several laboratories has been reviewed by
Silber.gl Phosphoric acid has also been used to
induce fluorescence,92r93as have been acetic acid-
antimony t r i ~ h l o r i d e ,aluminum
~~ salt and isonico-
tinylhydrazine (Ex.: 380 nm; Em: 495 nrnlg4 and
lithium hydroxide pellets (Ex.: 396/Em: 510) .95
A fluorescing derivative, amenable to TLC,96 has
been obtained by reactions with 4(6-methylbenzo-
thiazol-2yl) phenyl isocyanate.
300 KLAUS FLOREY
5.6Polarographic
Rased on the reduction of the 3-carbonyl
-
function, the polarographic reduction of hydro-
cortisone has been studied in well-buffered 50%
ethanol solutions.97 The halfwave potential ( E % )
was found to be dependent on pH, ranging from -1.26
for pH 2.8 to -1.74 for pH 10.8. A halfwave
potential of -1.50 was found in 90% ethanol at
pH 8.5.98 In DMF buffered at pH 9.15, a halfwave
potential of -1.64V was found.99 In acetonitrile-
tetrabutylammonium perchlorate, a halfwave potential
of -1.58 was found. The approximate detection limit
was determined as 3~10'~moles/liter . Polar-
ography of hydrocortisone has also been performed
with prior derivatization with Girard's reagent T
exhibiting a halfwave potential of 1.12lo1 and
betainyl hydrazine (E$ -1.42).98 Polarography has
been used for the determination of hydrocortisone
in ointments,99r102human bloodlol (see sections 8
and 9), and as a microbial conversion product after
TLC separations.lo3
5.7Isotope Dilution
Preparation of tritiated hydrocortisone
and its detection in fermentation brothlo4 and
adrenal sliceslo5 has been described.
5.8Chromatographic Methods
5.81 Paper
With the advent of TLC and HPLC, the
importance of paper chromatographic methods to
determine hydrocortisone declined steeply. It now
is only of historical interest. Yet, in the fifties
it was successfully used to enlarge the knowledge of
the distribution of hydrocortisone in body fluids
and tissues and to assay its purity, as well as
stability in dosage forms.
Both the Bushlo6 and Z a f f a r ~ n i ~ ~
type of chromatographic systems have been used for
hydrocortisone,and a typical sam ling is presented
in Table 4 according to Neher. o y Additional
systems can be found in references 108, 109, and
110. The use of fully acetylated paper,lll paper
impregnated with stearato chromic chloride for
reverse phase as well as centri-
fugal acceleration113has been applied to hydro-
cortisone.
HYDROCORTISONE 301
TABLE 4
Paper Chromatographic Systemslo7
Zaffaroni Type Systems:
Propylene/toluene 0.01
Formamide/chloroform 0.24
Formamide/ethyl acetate-butyl acetate-
water (15:85:1) 0.40
Formamide/butyl acetate:water (100:5) 0.42
Formamide/n-butanol-butyl acetate-
water (15:85:5) 0.60
Bush Type Systems:
Hexane-tert. butanol-methanol-water 0.19
Benzene-methanol-water (100:50:50) 0.25
Cyclohexane-dioxane-methanol-water
(4:4:2:1) 0.25
Toluene-ethyl acetate-methanol-
water (9O:lO: 50: 50) 0.37
For detection and quantitation,

ultraviolet light,ll1,ll4 blue tetrazolium,77
diphenylstyryl phenyltetrazolium chloride,l16 and
alkaline fluorescence117 have been used. Quantita-
tion of hydrocortisone by direct photometry of
paper chromatograms, using an adapter in a Beckman
DU spectrophotometer has been reported.ll* For
preparative paper chromatography to separate hydro-
cortisone from other adrenal constituents, see
reference 119.
5.82 Thin-Layer
Thin-layer chromatography started to
be applied to hydrocortisone in 1960. The two
earliest publications specifically mentionin hydro-
cortisone seem to be those b Carr and ReddyqPo and
by Cerny, Joska and Labler. 1 x 1 TLC is still an
important method for hydrocortisone. Until 1981,
the USP assay32 for hydrocortisone was based on
quantitative TLC,and the BP33 uses TLC for the
related steroid test. For general references, also
concerning quantitation, see references 3 , 107 and
110. Some typical systems have been summarized in
Table 5. While ultraviolet, blue tetrazolium and
sulfuric acid are employed as detection agents in
TABLE 5
SUPPORT SOLVENT SYSTEM Rf DETECTION REF.

-
Cellulose acetate Water-tert.butano1-pet.ether (2:1:3) - Fluorometric 120
Silica Gel Benzene-ethyl acetate (1:l) 0.22-0.36 Sulfuric acid 121
Celite Formamide/benzene-chloroform 0.13 Sulfuric acid, 125
(1:l) blue tetrazolium,
isonicotinic acid,
hydrazide
Silica Gel G Chloroform-methanol-water (188:12 :1) Sulfuric acid 126
Ethyl acetate-chloroform-water Ultraviolet
(90 :10 :1)
Silica Gel G Hexane-ethyl acetate (1:1) 0.06 Phosphomo1ybdic 127
(starch bound) Ethyl acetate 0.38 acid (10%in
Benzene-isopropanol (4:1) 0.55 ethanol)
Alumina
(acetic acid treated) Benzene-dioxane ( 2 : l ) 0.25 Ultraviolet 128
Benzene-dimethylformamide (9:1) 0.53
Chloroform-ethanol (96:4) 0.35
Diisopropylether 0.25
Silica Gel G Dichloroethane-methyl acetate- Diphenyl (styryl- 129
water (2:l:l) phenyl)-tetrazolium
Silica Gel G Chloroform 0.43 Toluene sulfonic acid 130
(impregnated with Methylene chloride-toluene (1:1! 0.10
formamide
TABLE 5 (Cont'd)
SUPPORT SOLVENT SYSTEM Rf DETECTION REF.

-
-
polyamide Resin Chloroform-ethanol (97:3) 0.50 Blue tetrazolium 131
Methylene chloride 0.23
Silica Gel G Cyclohexane-ethyl acetate- 0.23 Potassium periodate 132

ethanol (4.5 :4.5 :1) formaldehydog enic "
I'

Ethyl acetate-n-hexane-acetic
acid-ethanol (72:13.5:10:4.5) 0.44
Benzene-ethanol (4:1) 0.37
Silica Gel Butyl acetate-methanol (99:l) 0.48 Ultraviolet 107
W
W
0 Chloroform-methanol (9:1) 0.42 Ultraviolet
304 KLAUS FLOREY
most cases, Lisboa122 has described quite a number

of other "in situ" color reactions. Attention has
also been paid to the elution and uantitation of
steroids after chromatography. I g2 I Nanogram
amounts have been determined, and best recovery
was achieved by elution with methan01.l~~
A system for the separation of
hydrocortisone from its 17-keto oxidation product
has been described.133 Densitometry has also been
used for quantitation.134,135r136
5.83 Column
In the early years, column and
partition chromatography was used extensively for
the separation of hydrocortisone from adrenal gland
mixtures138 and the determination in blood.lol For
general reviews, see references 2, 3 and 119.
Diatomaceous earth (celite, superce1)101r138r139
silica ge1,140-143sephadex LH-20,144 G-50145 and
diatomaceous earth treated with a ~ e t o n i t r i l e l ~ ~
have been used as adsorbents with various solvent
mixtures.
5.84 High Performance Liquid
Chromatography (HPLC)
HPLC has swept the field of
pharmaceutical analysis. Shall wonder then that
HPLC is also rapidly replacing older chromato-
graphic methods for the control of the purity of
hydrocortisone and for its determination in form-
ulation and biological fluids (see section 8 and 9).
General reviews can be found in reviews by
Vestergaard,2 Di,! Smith,147 O'Hare and Nice,147
and Gorog and Szasz.3 The first reported use of
HPLC for h drocortisone seems to be by Siggia and
Dishman. 1 4 g Some typical data are summarized in
Table 6. A proposed selected method for the
analysis of hydrocortisone in serum has been
described.149
A description of early attempts on
gas chromatography of hydrocortisone-can be-found ,
in the book b Wotiz and Clark.163 VandenHeuvel
and Horning16< already found in 1960 that gas
chromatography of underivatized hydrocortisone and
other steroids with the dihydroxy acetone side
TABLE 6
HPLC SYSTEMS
MOBILE PHASE COLUMN DETECTION -

REF.
Water and methanol, water (3:7) Trifluoroethylene beads coated Ultraviolet 148
with Amberlite LA-1
Ethyl acetate ( 5 % ), acetonitrile Zipax coated with oxydipropionitrile Ultraviolet 150
(0.2%) in hexane
1%Methanol in water Cyano ethyl silicone Ultraviolet 151
Chloroform, dioxane (20:l) Silica gel Ultraviolet 152
Ethylene chloride, ethanol, water Zorbax-Sil Ultraviolet 153
Aqueous ethanol with tetrapentyl Silica coated with octadecyl silane Ultraviolet 154
ammonium chloride
1.5% Methanol and 0.2% water Silica gel Ultraviolet 155
in chloroform
Methanol, water, acetic acid c18 Bondapak Ultraviolet 156
(53:42: 5)
Methylene chloride, methanol, Silica gel Ultraviolet 157
tetrahydrofuran, acetic acid
Acetonitrile, water, glacial ODS Ultraviolet 158
acetic acid (38:60:2)
Methanol-O.OlM ammonium acetate c18 Bondapak (gradient elution) Ultraviolet 159
pH 6.9 (10:90)
60% aqueous methanol c18 Lichrosorb Ultraviolet 160
TABLE 6 (Cont'd)
MOBILE PHASE COLUMN DETECTION REF.

-
*Ethylene dichloride, butanol, water Fluorescence 161
(91: 8 . 5 : 0.5)
Butyl chloride, tetrahydrofuran, s lica gel Ultraviolet 33
methanol, glacial acetic acid
(190:14:7:6)
Acetonitrile, methanol, water Silica coated with octadecylsilane Ultraviolet 33
(2:2:6)
Acetonitrile (20%) in 0.01M Lichrosorb RP-8 Ultraviolet 162
phosphate buffer pH 6 containing
0.01M tetrabutyl ammonium bromide
w
0
ch
dansyl hydrazine derivative
chain eliminated the side chain during vaporization and that the retention time
was that of the corresponding 17-keto compound. The main use of gas chromato-
graphy has been found in the determination of hydrocortisone in biological
fluids where it is competing with radioimmunoassays. Recently, it has been
coupled with mass spectrometry.165 GC/MS and RIA methods have been
Some typical data are presented in Table 7.
5.9 Electrophoretic
Hydrocortisone, being neutral, should not be moved by electrophoresis.
However, there is a report that hydrocortisone migrated in pH 7.0 phosphate buff-
er and pH 10 bicarbonate buffer when subjected to high voltage electroph~resis?~~
TABLE 7
GAS CHROMATOGRAPHIC SYSTEM
COLUMN
DERIVATIZATION COLUMN PACKINGS TEMP. DETECTOR REF.
-
- Silicone SE-30 on 222 Flame ionization 164
Chromosorb W
Trimethylsilyl ether 3% dimethylpolysiloxane - Flame ionization 167
on Gas Chrom. P
Methoxime-trimethylsilyl ether 1% SE-30 on Gas Chrom. P 250 Flame ionization 168
Methoxime-trimethylsilyl ether 3% OV-1 Diatomite CQ 240 Flame ionization 169
Periodate oxidation to SE-30 250 Flame ionization 170
w
eticholenic acid
0
rl llfksilylether and 17,21 1% OV-1 on Chromosorb G 240 Flame ionization 171
cyclic dimethyl
s iliconide
Trimethylsilyl ether-enol- 1% OV-1 on Gas Chrom. P 250 Flame ionization 172
trimethylsilyl ether
Chromium trioxi.de oxidation to 3% OV-17 on Chromosorb W 230 Electron capture 173
androstenetrione and forma-
tion of heptofluorobutyrate
3.20 Dimethoxime-11,17,21 tri- 3% SE-30 - Mass fragmentography 165
methylsilyl ether at m/e 636; m/e 638
(14C-hydrocortisone)
308 KLAUS FLOREY
5.10Bioassay-Enzymatic
The original workers, isolating hydro-
cortisone and other adrenal hormones from adrenal
tissue, used adrenalectomized animals and later on
such tests as the liver glycogen, e ~ s i n o p h i l , ~ ~ ~
thymus involution' and electrolyte-excret-
ing177 (cf. 1, 600ff). The enzyme 20-B-hydroxy
steroid dehydrogenase coupled with DPNH has also
been used to determine hydrocortisone in blood
plasma.178
5.11Saturation Analysis
The great importance of the accurate
determination of hydrocortisone body fluid levels
in normal and diseased states has led to the
development of the following techniques which can
be classified as saturation analysis.' Each of the
techniques has its proponents and pitfalls,179 but
at present, radioimmunoassay seems to be the most
useful.
5.111
Competitive Protein Binding
A review of this technique can be
found in the chapter by W. R. Slaunwhite, Jr. in
reference 2. Murphy first employed this technique
to study hydrocortisone levels in plasma and other
body fluids.lso,lB1 The competition of hydro-
cortisone in the plasma sample with added hydro-
cortisone-4-C14 for corticosteroid-binding globulin
(a1 fraction) is the basis of the method. The
method was extended to urine and the purification
prior to assay refined.l*' TLC has been used for
prepurification.183 Tritiated instead of carbon-14
labelled hydrocortisone has also been used.178
Horse transcortin was used to measure hydrocort-
isone in umbilical cord serum and amniotic f 1 ~ i d . l ~ ~
A commercial kit has been described.186 Ultra-
centrifugation was used to determine unbound
hydrocortisone.187
5.112 Double Isotope Derivative Assay
The double isotope derivative
assay is a modification of competitive protein
binding analysis where a trace amount of tritiated
hydrocortisone is added to determine recovery
values durin extraction. In a comparison of the
two methods,q 8 8 it was found that generally there
is good agreement; however, a large discrepancy
HYDROCORTISONE 309
was found in the values obtained in newborns and

patients with adrenogenital syndrome, where
competitive protein binding gives much higher
values, probably due to the presence of related
steroids competing with hydrocortisone.
5.113Radioimmunoassa
Ruder, Guy and ;ipsettlsg are
credited with the first description of a radio-
immunoassay for hydrocortisone in 1972, but others
were also hard at work. That this technique
offered advantages as to higher sensitivity and
greater specificity over protein-binding methods is
attested by the mushrooming literature which here
is reviewed very selectively. Brief reviews are
those by G. E. Abraham in references 2 and 192; a
detailed review is that of P. Vecsei in reference
193. Antigens used for immunization were the 21-
hemisuccinate, the 3-oxime-21-acetate, the 3-oxime,
the 3,20-dioxime and the 6a- or 66-hemisuccinoxy
derivatives to obtain antibody titers in sheep and
rabbits. These antibodies had varying percenta es
of cross reactivity to related steroids. 3H, 7zSe
and 125I-ligands are used, but in one comparison
study,lq4 tritiated hydrocortisone was found to
have the best sensitivity. Most laboratories use
dextran coated charcoal to separate protein-bound
and unbound radioactivity. The usual solvents for
extraction of plasma hydrocortisone are alcohol
and dichloromethane. Comparison of the radioimmuno-
assay with chemical ionization/mass spectrometry166
and with HPLCIq5 have been reported. An inter-
laboratory evaluation of four RIA kits has been
made.lg6 An automated assay method has been
evaluated.
5.114Other
A chemiluminescent immunoassaylg8
has been described. Enzyme-labelled immunoassays,
using alkaline phosphataselg9 and E. coli B-galac-
tosidase200 have also been developed.
6. Stability - Degradation
Hydrocortisone is very stable as a solid. In
aqueous and alcoholic solution, the dihydroxy-
acetone side chain, as in all such corticosteroids,
is prone to oxidative rearrangement and degradation
at very acid and particularly also alkaline pH's.
310 KLAUS FLOREY
For instance, it was reported in 1967201 that

hydro~ortisone-4-~~C in aqueous or methanolic
solution decomposed to products of lesser and high-
er polarity. The main decomposition product was
identified as 118-h droxyandrost-4-ene-3,17 dione.
In another study,20Y it was found that aqueous
solutions of hydrocortisone were spontaneously
oxidized to 21-dehydrocortisol and other products.
In pH 9.1 carbonate buffer, the conversion rate was
1.6-2.8%/hr. Addition of EDTA or high concentra-
tions of plasma or plasma protein fractions slowed
the degradation.
Recently, Hansen and Bundgaard162 have studied
the degradation of hydrocortisone in aqueous
solution in detail. They identified degradation
products by HPLC (see Section 5.84) and treated the
data kinetically. They confirmed results of
previous investigators, partially obtained with
other corticosteroids with a dihydroxyacetone side
chain. They identified the glycolic acid V, not
previously reported. Generally, they found
degradation patterns to be more complex in basic
solutions. Their results are summarized in
Figure 6.
The photolytic degradation of hydrocortisone in
alcoholic solutions when exposed to fluorescent
lighting was found to be first order. The half-
life of such a solution at room temperature was
found to be 160 days. The degradation involved the
A-ring of the molecule, as measured by a decrease
in U.V. absorption, but not the side chain as
determined by blue tetrazolium. No degradation
products were described.203
7. Metabolic Products - Pharmacokinetics
The metabolism of hydrocortisone is complicated
since it can be different both qualitatively and
quantitatively in health or disease. Much pioneer-
ing work has been done with and without the use of
14C-labeled hydrocortisone in isolated liver204 and
kidney205 in intact rats,206 and in man.207,208
However, the definitive quantitation study in
normal man is that of Fukushima, Bradlow, Hellman,
Zumoff and Gallagher,209 which is summarized in
Figure 7. It was found that 9 0 % of the radio-
activity in the neutral steroid extract would be
HYDROCORTISONE 311
HC=O c=o
I I
C-OH c=o
YT- IX
(minor)
IV
(major)
21 C H 2 0 H
IV
I (minor)
20 c=o
c=o
I
c=o
b'"
I11
I11
COOH
I (intermediate)
I
I
0 CHOH
+ y1 + others
C CHOH
VIII VI . ..OH
(major) (major) (minor) + others
(major) (major) (minor)
Fig. 6. Degradation pathways of hydrocortisone in aqueous

solution. The numbering is that of reference 162.
All oxidative pathways in neutral and alkaline
solution are metal (copper) catalyzed and can be
blocked by EDTA.
312 KLAUS FLOREY
CH20H CH20H
I I
c-0 c-0
68-Hydroxyhydrocortisone Cortisone
CH20H
l
Hydrocortisone
HO”
8
Hd
R-OH Tetrahydrocortisol; Allotetrahydrocortisol
R-0 Tetrahydrocortisonei Rllotetrahydrocortisona
CH20H
I OH HO&.
CHOH
’ __+
o*
HO‘ H HO’
li
20 o+R - Dihydrocortisol R-OH 20 o+B -- cortol 20 o+E -- allocortol
J R=O 20 a+B cortolone 20 a+B allocortolone
1
HO‘ @
116-Hydroxyetiocholanolone
+ HA .(y.p
;I
118-hydroxy androsterone
Gluconurides
Figure 7. Metabolic Pathways of Hydrocortisone.

HYDROCORTISONE 313
accounted for by the compounds depicted in Fig. 7,

and 70% were recovered in urine as the glucuronides.
By far, the largest fractions were the tetrahydro
derivatives both of hydrocortisone and cortisone
with the side chain intact, followed by the
cortolones and cortols. The formation of 6B-
hydroxycortisone and 17-keto compounds was found
to be minor.
Recently, the pharmacokinetics of orall
The
administered hydrocortisone was described. l X 0
mean elimination half-life of hydrocortisone from
plasma was found to be about 9 0 minutes.
Detailed reviews on the subject are available
available. I 210 I 2 2 0
8. Determination in Biological Fluids and Tissues
Since hydrocortisone is a hormone, determina-
tion in bioiogical fluids and tissues-has been of
particular importance, starting with the animal
assays leading to its isolation (cf. ref. 1,
600ff). A perusal of the general reviews,147
I I I mentioned in the preceding pages
may be useful. The following is a compilation of
references, most of them grouped according to
fluid or tissue:
Blood (Plasma, Serum) :
Porter-Silber: 7 5 , 7 6
Fluorometric: 91, 1 4 2 , 2 1 1 , 212
Paper Chromatography: 1 0 6 , 117
Polarographic: 1 0 1
HPLC: 1 6 1 , 1 4 9 , 1 5 7 , 1 5 6 , 1 5 3
Gas Chromatography: 173
Gas Chromatography/Mass Spectrometry:
165, 198
Competetive Protein Binding: 1 7 9 , 180,
181, 183, 186, 187, 219
Radioimmunoassay: 1 6 0 , 1 6 6 , 1 8 9 , 197
Chemiluminescent 1 A : 1 9 8
Enzyme 1 A : 1 9 9 , 2 0 0
Enzymatic: 1 7 8
Urine :
Porter-Silber: 74
Fluorometric: 213
314 KLAUS FLOREY
Paper Chromatography: 109, 207, 209, 214

TLC: 215, 216
HPLC: 155
Compet. Protein Binding: 181, 182, 184
Radioimmunoassay: 195
Reverse Isotope Dilution: 209
Adrenals :
Biological assays: cf. 1, p. 600ff
Paper Chromatography: 118, 217
Amniotic Fluid:
Compet. Protein Binding: 185
Liquid Chromatography: 40
Milk :
TLC: 137
Cerebral Spinal Fluid:
Compet. Protein Binding: 181
9. Determination in Pharmaceutical Formulation
The following tabulation highlights the methods
referenced previously which have been used in the
assay of pharmaceutical formulations:
Tablets:
Spectrophotometric (isonicotinic acid
hydrazide) : 78
Automated colorimetric: 79, 86, 176
Spectrophotometric (ammonium molybdate); 87
Tetrazolium: 33, 115
Paper Chromatography: 115
TLC: 33
Column: 146
HPLC: 33
Topicals:
Blue Tetrazolium: 33
Spectrophotometric (cupric acid ox.): 81
Polarography: 99, 102
TLC: 124, 136
Column Partition: 138
HPLC: 33, 147, 150
HYDROCORTISONE 315
Parenteral:
Blue Tetrazolium: 33
HPLC: 1 5 8
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METOPROLOL TARTRATE
James R. Luch
1. Description 326
1.1 Introduction 326
1.2 Formula, Name, Formula Weight 326
2.1 Ultraviolet Absorption Spectrum 326
2.2 Infrared Absorption Spectrum 328
2.3 Proton Nuclear Magnetic Resonance Spectrum 330
2.4 Carbon-13 Nuclear Magnetic Resonance Spectrum 330
2.6 Fluorescence Spectrum 334
2.9 Differential Scanning Calorimetry 337
2.10 Thermogravimetric Analysis 337
2.13 Solubility 339
2.14 Water Absorption Isotherm 339
2.15 Distribution Ratio 34 1
3. Synthesis 34 1
4. Stability 342
4.1 Solid State Stability 342
4.2 Solution Stability 342
5. Pharmacokineticsand Metabolism 342
6. Analytical Methods 343
6.2 Nonaqueous Titration 343
6.4 Gas Chromatography 346
6.5 Gas Chromatography-MassSpectrometry 349
6.6 High Pressure Liquid Chromatography 350
6.7 Ultraviolet Spectrophotometry 353
References 354
ANALYTICAL PROFILESOF DRUG SUBSTANCES 325 Copyright by ihe American Pharmaceutical AsSwafion.
VOLUME 12 ISBN 0- 12-260812-7
326 JAMES R.LUCH
1. Description
1.1 Introduction
Metoprolol tartrate is a synthetic, selective
beta 1-adrenoreceptor blocking agent ? - 3
1.2 Formula, Name, Formula Weight
OH
I
OCH2CHCH2NHCH(CH& COOH
I
H-C-OH
I
HO-C-H
I
COOH
2
Metoprolol tartrate
Formula Weight: 684.82 ( C I ~ H * ~ N O ~CqH6O6
)~
Chemical Abstracts Number: 56392-17-7
Metoprolol tartrate is a 2:1 salt consisting
of a racemic mixture of optical isomers of the
base and naturally occurring dex&o-tartaric acid.
The compound has been described by the following
chemical names.
i. 2-Propanol, 1-[ 4-(2-methoxyethyl)phenoxy]-3-
[ (1-methylethyl)amino]- , (+)- , [ R- (R* ,Rn) ] -
2,3-dihydroxybutanedioate (2:l)(salt)
ii. (+)-l-(Isopropylamino)-3-[p-(2-methoxyethyl)-
phenoxyl-2-propanol L-(+)-tartrate (2:l)(salt)
iii. 1-(1sopropylamino)-3- [p-(2-methoxyethyl)-
phenoxyl-2-propanol (2:l) dexho-tartrate salt
Trade Names: Betaloc, Lopresor, Lopressor, Seloken
1.3 Appearance, Color, Odor
Metoprolol tartrate is a white, virtually
odorless crystalline powder.
2.1 Ultraviolet Absorption Spectrum
The ultraviolet absorption wavelength maxima
(Amax) and molar absorptivities ( E ) of metoprolol
tartrate in several solvents are listed in Table I.
A typical ultraviolet absorption spectrum of meto-
prolol tartrate in 0.1g HC1 obtained on a Hewlett-
Packard Model 8450A spectrophotometer is given in
Figure 1.
METOPROLOL TARTRATE 327
Figure 1: Ultraviolet Absorption Spectrum

of Metoprolol Tartrate in 0.1 N HCI
Wavelength (nm)
328 JAMES R. LUCH
TABLE I
Solvent
0.1g HC1 221 19.5

274 2.83
281 shoulder 2.31
4
Water 223 23.4
2 74 3.60
280 shoulder 2.94
4
- NaOH
0.01N 223 24.0
274 3.66
280 shoulder 3.00
Methanol 223 21.5
276 3.11
282 2.62
Chloroform 277 3.36
2 83 2.86
2.2 Infrared Absorption Spectrum

The infrared absorption spectrum of metoprolol
tartrate obtained as a Nujol mull on a Perkin-Elmer
Model 281B grating infrared spectrophotometer is
presented in Figure 2 . Spectral assignments €or
major absorption bands provided in Table I1 are
consistent with the structure of metoprolol
tartrate.
TABLE I1
~~~ ~~ ~~
-1
Wavenumber (cm ) Assignment ( s )
3600-2300 -fH2, -OH, Aliphatic and

Aromatic CH
1580 Carboxylic Acid Salt
1580, 1515 Aromatic Ring
1250, 1015 Aromatic Ether
1180 Isopropyl Group
1100 Aliphatic Ether, Secondary
A1coho 1
820 1,4-Disu~tituted Benzene
Figure 2: Infrared Absorption Spectrum of Metoprolol Tartrate
330 JAMES R.LUCH
2.3 Proton Nuclear Magnetic Resonance Spectrum

The 80 MHz proton nuclear magnetic resonance
(NMR) spectra of metoprolol tartrate5 obtained in
CDC13 at ambient temperature (lower trace) and 60°C
(upper trace) are given in Figure 3. The spectra
have been obtained on a Varian CFT-20 NMR instrument
using tetramethylsilane as an internal standard. The
chemical shifts, multiplicities and spectral assign-
ments are provided in Table 111.
TABLE I11
oioo
OCH2CHCH2NHCH(CH&
mo o COOH
@
0 1 63
H-C-OH
* @
HO-C-H
I 0
0 0 @ I @
COOH
2
Proton Chemical Shift Number of Multiplicity
Positi o n 6 (ppm) Protons
1 1.33 12 Doublet
2 2.80 4 Triplet
3 3.13-3.31 6 Broad
4 3.32 6 Singlet
5 3.53 4 Triplet
6 3.95 4 Broad
7 4.40 4 Singlet & Broad
8 6.77 4 p-Substituted
Rromatic
9 7.07 4 p-Substituted
Aromatic
10 7.25 8 Broad,
Exchangeables
Figure 3: 80 MHz 'H Nuclear Magnetic Resonance Spectra of Metoprolol Tartrate
at Ambient Temperature (lower trace) and 60°C (upper trace)
332 JAMES R.LUCH
2.4 Carbon-13 Nuclear Magnetic Resonance Spectrum

The 13C nuclear magnetic resonance (NMR)
spectra of metoprolol tartra e5 obtained in CDC13
at ambient temperature with FH-decoupling (lower
trace) and without '8-decoupling (upper trace)
are presented in Figure 4 . The spectra have been
recorded at 2 5 . 2 MHz on a Varian CFT-20 NMR
instrument. The chemical shifts, multiplicities
and spectral assignments are given in Table IV..
TABLE IV
OH
0 I0 @ O 63
OCH2CHCH2NHCH(CH3)2 COOH
10
H-C-OH
I@
HO-C-H
@@ @ I
COOH
@CH2CH20CH3
63
Carbon Multi licity Without
Position 'H-decoupling
1 18.67, 19.01 Quartet for each

2 35.09 Triplet
3 48.12 Triplet
4 50.37 Doublet
5 53.36 Quartet
6 65.50 Doublet
7 70.08 Triplet
8 73.60 Triplet
9 73.86 Doublet
10 114.37 Doublet
11 129.57 Doublet
12 131.35 Singlet
13 156.85 Singlet
14 178.35 Singlet
gure 4: I3C Nuclear Magnetic Resonance Spectra of Metoprolol Tartrate
ith 'H-decoupling (lower trace) and Without 'H-decoupling (upper trace)
Without 'H-decoupling
With 'H-decoupling
1 1 1 I I I I 1 I I 1 1 1 I I I 1 1 I 1
180 160 140 120 100 80 60 40 20 0
PPM
334 JAMES R.LUCH
2.5 Mass Spectrum

The low r e s o l u t i o n mass spectrum of m e t o p r o l o l
t a r t r a t e o b t a i n e d on a Kratos MS 25 s p e c t r o m e t e r
a t 7 0 ev u s i n g a s o l i d i n s e r t i o n probe a t a probe
temperature of 100°C is shown i n F i g u r e 5 . The
mass spectrum of metoprolol t a r t r a t e i s t h e
spectrum of i t s f r e e base r e s u l t i n g from thermal
d i s s o c i a t i o n when t h e compound i s v a p o r i z e d .
Prominent .fragments and t h e i r mass t o c h a r g e
r a t i o (m/e) a r e l i s t e d i n Table V .
TABLE V
m/ e
268, 267 [M + H]+, M + ( f r e e base)
252 [M - CH3]+
152
[ HO-o-CH2CH20CH3 ] +
OH
I
116 [CH2CHCH,NHCH(CH,) '
1
2
107
102 [HOCHCH*NHCH(CH3)2]+
77 C6H5+
72
+
CH2=NHCH(CH3)2
45 CH3-6=CH2
30 CH2=hH2
2.6 Fluorescence Spectrum

A s o l u t i o n of metoprolol t a r t r a t e i n w a t e r
o r methanol e x h i b i t s f l u o r e s c e n c e when e x c i t e d
w i t h u l t r a v i o l e t l i g h t . The c o r r e c t e d e m i s s i o n
s p e c t r a 6 of m e t o p r o l o l t a r t r a t e i n water ( s o l i d
t r a c e ) and i n methanol (dashed t r a c e ) , g i v e n i n
F i g u r e 6 , show a n emission maximum a t 298 nm and
a quantum y i e l d of approximately 0.3. These
s p e c t r a have been o b t a i n e d u s i n g a n e x c i t a t i o n
wavelength of 275 nm which corresponds t o t h e
maximum of t h e a b s o r p t i o n spectrum.
Figure 5: Low Resolution Mass Spectrum of Metoprolol Tartrate
100
80
=.
.-
c
6o
v)
c
al
c
-
C
.-9
c
-mal
r-
40
20
MassKharge Ratio
1 250
Figure 6: Fluorescence Spectra of Metoprolol Tartrate
2 . 3 ~-. .-.
'O0I ---------- Water
Methanol 3.2~1O-~M I
80-
60-
40-
20-
-
I I I I 1 I I
270 290 310 330 350 370
Wavelength (nrn)
2.7 Optical Rotation

Metoprolol tartrate contains three asymmetric
carbon atoms; one at the 2-propanol position of
the base and the other two in the tartaric acid
portion of the molecule. Optically active, natural
tartaric acid (dexZto-tartaric acid) is used to
prepare metoprolol tartrate and thus solutions of
the compound exhibit optical rotation even though
the base portion of the molecule is racemic. The
specific rotation determined at 20°C on a 2%
aqueous solution of metogrolol tartrate using
the sodium D line, [,]go c, is within the range
of + 6 . 5 " and +10.5". A value for [a]200c of
+8.5" has been reported €or a typica!? sample of
metoprolo1 tartrate. 5
2.8 Me1ting Range

Metoprolol tartrate has been observed to
melt over a 1-2 degree range between approximately
120-123°C when tested according to the USP XX
Class Ia procedure. For example, a sample of
metoprolol tartrate has been determined to melt
between 121.1 and 122.3"C by the USP Class Ia
procedure. 7
2.9 Differential Scanning Calorimetry
The differential scanning calorimetry curve
of metoprolol tartrate obtained7 on a DuPont Model
900 instrument at a scan rate of 10°C/minute
exhibits a sharp melting endotherm with an onset
temperature of 115°C and a peak temperature of
122.3OC as shown in Figure 7. A heat of fusion
value of 18,700 cal/mole has been obtained for a
metoprolol tartrate sample having a purity of 98.9
mole percent as determined by differential
scanning calorimetry.
2.10 Thermogravimetric Analysis
Thermogravimetric analysis of metoprolol
tartrate typically exhibits a weight l o s s of less
than 0 . 5 %between room temperature and 135°C. For
example, thermogravimetric data obtained7 for a
metoprolol tartrate sample on a Perkin-Elmer TGS-1
thermobalance at a scan rate of 10°C/minute showed
a weight l o s s of less than 0.2%.
2 . 1 1 X-ray Diffraction
The X-ray powder diffraction pattern obtained7
for metoprolol tartrate on a Diano Model 8235
diffractometer using the Cu K, line (1.542A) as
the radiation source with a Ni filter is shown in
Figure 8 .
JAMES R.LUCH
I I 1 I 1 I I
20 40 60 a0 100 120 140
1200
600
0 I ' I ' I ' I ' I ' I ' I ' I

7 11 15 19 23 27 31 35
Degrees Two Theta
Figure 8: X-ray Powder Diffraction Pattern of Metoprolol Tartrate
2.12 D i s s o c i a t i o n Constant
Dissociation constant data obtained f o r t h e
secondary amine of metoprolol t a r t r a t e by p o t e n t i o -
m e t r i c t i t r a t i o n a r e given i n Table V I . The pKa
v a l u e s r e p o r t e d i n The Merck Index8 f o r t a r t a r i c
a c i d a r e 2.93 and 4.23 a t 25OC.
TABLE V I
PG Conditions Reference
8.9 ? 0.2 - i n water a t 25OC

8 x 10-4M 4
9.68 ? 0.02 ionic strength, 0.1 9
9.5 ? 0.2 10
2.13 Solubility
The approximate s o l u b i l i t y of m e t o p r o l o l
t a r t r a t e i n s e v e r a l s o l v e n t s a t 25°C is g i v e n i n
Table V I I . The s o l u b i l i t y h a s been determined
a f t e r shaking a s a t u r a t e d suspension of t h e d r u g
f o r 2 hours.
Solvent S o l u b i l i t y (mg/ml)
Water > 1000

Methanol > 500
Chloroform 496
Acetone 1.1
Acetonitrile 0.89
Hexane 0.001
I
2 . 1 4 Water Adsorption Isotherm

Metoprolol t a r t r a t e i s a hygroscopic compound
a t high h u m i d i t i e s . The w a t e r a d s o r p t i o n i s o t h e r m 4
a t 25OC, given i n F i g u r e 9 , i n d i c a t e s t h e m a t e r i a l
r a p i d l y absorbs water a t r e l a t i v e humidities
g r e a t e r t h a n 70% and c o n v e r s e l y d e s o r b s w a t e r as
t h e r e l a t i v e humidity i s decreased. No h y d r a t e o r
change i n c r v s t a l form h a s been observed.
Figure 9: Water Adsorption Isotherm of Metoprolol Tartrate at 25°C
2.15 Distribution Ratio

Distribution ratio data, expressed as the
organic phase concentration divided by the aqueous
phase concentration, are summarized in Table VIII.
TABLE V I I I
Organic
Phase
Aqueous
Phase
Distribution
Ratio
I
Reference
1-Octanol 0.067M Phosphate 0.587 20.007

Buffer, pH 7 . 4
1-Oc tanol 0.0675 Phosphate 0.665 20.008
Buffer, pH 7.4
with 0.9% NaCl
Hexane 0.0675 Phosphate 0.0040 50.0005
Buffer, pH 7 . 4
Hexane 0.0675 Phosphate 0.0047 +0.0001
Buffer, pH 7.4
with 0.9% NaCl
Chloroform 0 . 1 5 NaOH 542 +16
Chloroform 0.15 HC1 0.0040 +0.0003
3. Synthesis
/O\
OCHZCH-CH~
?H
HzNCH(CHs)z
342 JAMES R. LUCH
4. Stability
4.1 Solid State Stability
Metoprolol tartrate stored at room tempera-
ture and at 3 5 O C for five years is physically and
chemically stable. After storage at 5 O o C for up to
thirty months, no degradation has been observed--
the only change has been that the material became
slightly off-white; at lower temperatures and at
shorter time intervals at 5 O o C , it has been
completely unchanged in color. 9 Under high
humidity metoprolol tartrate is hygroscopic and
rapidly adsorbs water at relative humidities
greater than 7 0 % ; however, upon drying and
reanalysis, the material is found to have retained
its chemical and physical integrity.
4.2 Solution Stability

No chemical change has been observed for
solutions of metoprolol tartrate buffered at pH
values of 4 , 7 and 9 which have been stored at
6 0 ° C for 10 days. l4 lletoprolol tartrate solutions
prepared in 0 . 1 E H C 1 , pH 7 phosphate buffer and
O . l N NaOH have been refluxed for 20 hours with no
evizence of chemical change. Ampuls containing an
aqueous solution of metoprolo1 tartrate, 1 mg/mI,
and 0.9% sodium chloride have not shown any
evidence of chemical change after storage for 7 7
months at room temperature and at 5 0 ° C . 9
5. Pharmacokinetics and Metabolism

An extensive review of the pharmacological pro-
perties and therapeutic efficacy of metoprolol tartrate
in hypertension and angina pectoris has been given by
Brogden &. l 5 Studies in rats and dogs indicate that
metoprolol is almost completely absorbed from the
gastrointestinal tract. l 6 Distribution is typical of
that of a moderately lipophilic, basic drug. Studies in
man show that metoprolol is readily and rapidly absorbed
after oral administration and is rapidly distributed to
body tissues. 3 Plasma levels vary considerably
between individuals, due partly to significant hepatic
first-pass elimination, which results in 50% of the
administered oral dose reaching the systemic circula-
tion. 18-20 Metoprolol is only slightly (12-14%) bound
to human serum protein, namely albumin, which is
reflected in its large volume of distribution (5.6
L/kg). 17,21,22
The elimination half-life of metoprolol is about

3 to 7 hours in most patients and is independent of
dose and duration of therapy. 1 8 * 2 0 * 2 3 The drug undergoes
extensive biotransformation and is excreted principally
via the kidneys; only about 3% of a dose is excreted
as unchanged drug after oral administration and about
10% after an intravenous dose. l 7 Three main metabolites
of metoprolol have been isolated in man as well as in
the rat and the dog. These three metabolites account
for 85% of the total urinary excretion in man.24
Arfwidsson d25 have reported an additional 4
urinary metabolites in the rat.
6. Analytical Methods
6.1 Elemental Analysis
The elemental composition determined for a
typical' sample of metoprolo1 tartrate on a
Perkin-Elmer Model 240 CHN Analyzer is given
below.
Element Theory (%) Observed ( X )
Carbon 59.63 59.90

Hydrogen
Nitrogen
6.2 Nonaqueous Titration

Metoprolol tartrate can be titrated in
glacial acetic acid with 0.11 perchloric acid in
glacial acetic acid or dioxane. The end point is
determined potentiometrically using a glass
electrode and a calomel electrode containing
glacial acetic acid saturated with lithium
chloride. Each ml of 0.lE perchloric acid is
equivalent to 3 4 . 2 4 mg of metoprolol tartrate.
A potentiometric titration with 1M_ sodium
hydroxide in a two phase system of water and 0 . e
pentachlorophenol in methylene chloride has been
used to determine metoprolol tartrate.26
6.3 Thin-layer Chromatography

Thin-layer chromatographic systems developed
for the identification and the determination of
metoprolol tartrate and related compounds are
summarized below.
344 JAMES R. LUCH
System 113 The following system is given as a

test for chromatographic purity in
the metoprolol tartrate monograph in
USP xx.27
Adsorbent: Silica Gel glass plate, 250 pm

layer
Solvent Chloroform (under NH3 atmosphere)
System :
Chamber : Saturate a filter paper lined
chamber containing the solvent
system and several beakers, each
containing 45 ml of concentrated
ammonium hydroxide, for 1.5 hours.
Detection Chlorine Gas-Starch: Dry the

System : developed plate until the odor of
ammonia is no longer perceptible and
place in a chamber of chlorine gas
(add 5 ml of 5N HC1 to a beaker
containing 0.5-g of potassium
permanganate) for 1 minute. Allow
the plate to stand in air for
several minutes and spray with
detecting reagent (mix 3 ml ethanol
with 10 ml of potassium iodide
solution, 1 g in 100 ml water, and
10 ml starch solution, 3 g soluble
starch triturated in 10 ml cold
water and then added to 90 ml
boiling water with constant
stirring).
Alternate Chromate-H?SOQ-UV 366 nm: 2 8 Dry the
Detection developed plate with warm air for
Systems: 10 minutes and spray with detecting
reagent (add 20 ml concentrated
sulfuric acid to 90 ml of water
containing 0.5 g of potassium
dichromate). Dry at 12OoC for 10
minutes and visualize the cooled
plate under long wave ultraviolet
light at 366 nm.
Anisaldehyde: Dry the developed
plate with warm air for 2-3 minutes
and spray with freshly prepared
anisaldehyde reagent (0.5 ml anisal-
dehyde in 10 ml glacial acetic
acid, 85 ml methanol and 5 ml concen-

trated sulfuric acid). Dry at 100°C
for 20 minutes and visualize under
daylight or under long wave ultra-
violet light, 366 nm.
Quantitative Determination: 2 a (The
following procedure has been used as
a dosage form stability method.)
Metoprolol tartrate is extracted from
the dosage form with chloroform-
methanol (1:l) and a portion of the
solution equivalent to 2 mg of
metoprolol tartrate is applied as a
band to a silica gel plate containing
a fluorescent indicator concomitantly
with a metoprolol tartrate standard
solution band. After development the
plate is dried and the silica gel
zones containing metoprolol from the
sample and standard are removed from
the plate after visualizing under
ultraviolet light (254 nm).
Metoprolol from the sample is eluted
from the silica gel with 0.15
ethanolic ammonia and quantitated by
ultraviolet absorption spectroscopy
vb a metoprolol standard solution
similarly prepared.
A number of additional systems have been
used for the analysis of metoprolol
tartrate.
System 1112,l 3 Chloroform-Methanol ( 9 5 : 5 ) , saturated
chamber with solvent and ammonia
atmosphere, Silica Gel P254 (Merck) ,
anisaldehyde or chl~rine-tolidine~~
detection
System 1 1 1 ~ 2 Methanol-Ethyl Acetate ( 4 0 : 2 0 ) ,
sandwich chamber, Silica Gel F254
(Merck), anisaldehyde detection
System 1v4 1-Butanol-Glacial Acetic Acid-Water
(71:7:22), Silica Gel with fluores-
cent indicator, iodine vapors or
ultraviolet light (254 nm) detection
346 JAMES R. LUCH
System v4 Methanol-Ethyl Acetate-Diethylamine

(60:35:5), Silica Gel with fluores-
cent indicator, iodine vapors or
System V130 Chlorofom-Methanol-Ammonium Hydrox-
ide (80:15:2), Silica Gel 60 F254
(Merck), chlorine gas-starch detec-
tion
System VI19 Benzene-Methanol (1:1), sandwich
chamber, Silica Gel F254 (Merck),
anisaldehyde detection
System VIII Benzene-Methanol-Glacial Acetic Acid
(30:30:5), sandwich chamber, Silica
Gel F254 (Merck), anisaldehyde
detection
System IX9 Benzene-Methanol (45 :5), sandwich
chamber, Silica Gel F25q (Merck),
anisaldehyde detection
System ~9 Benzene-Methanol-Glacial Acetic
Acid (40:20:5), sandwich chamber,
Silica Gel F254 (Merck), anisal-
dehyde detection
System X131 0.1% Ammonium Acetate in Methanol-
Water (56:44), reverse phase KC18F
plates (Whatman), ultraviolet light
(254 nm) or iodine vapor followed by

Gas chromatographic systems reported for the
determination of metoprolol tartrate are summarized
below.
System 132 The following system has been used
for the determination of metoprolol
in human plasma.
Column : 6 ft x 4 mm i.d. glass column
packed with 3% OV-101 on Chromosorb
WHP (80-100 mesh)
Temperatures: For Hewlett Packard Model 76208
chromatograph; injector (25OoC),
column (195OC), detector (31OOC)
For Varian Model 3700 chromatograph;
injector (23OoC), column (206OC),
detector (31OOC)
Carrier: For Hewlett-Packard; helium at

approximately 60 ml/minute
For Varian; nitrogen at 28 ml/minute
Detection: Electron capture detection of the
pentafluoropropionyl derivative of
metoprolol
33
System I1 The following system has been
employed for the determination of
metoprolol in plasma and urine.
Column: 195 cm x 2 mm i.d. glass column
with 3% JXR on Gas Chrom Q (100-120
mesh)
Temperatures: Injector (not reported), column
(16OoC), detector (300°C)
Carrier: Argon-Methane (95:5) at 50 ml/minute
trifluoroacetyl derivative of
metoprolol
34
System I11 The following system has been used
to determine metoprolol in plasma.
Column : Glass column packed with 1% OV-17
on Gas Chrom Q (80-100 mesh),
column dimensions not reported
Temperatures: Injector (25OoC), column (185"C),
detector (300°C)
Carrier: Argon-Methane (95:5) at 60 ml/minute
pentafluoropropionyl derivative of
metoprolol
35
System IV The following system has been
reported for the determination of
metoprolol in blood or plasma.
packed with 3% JXR on Chromosorb G
(80-100 mesh)
Temperatures: Injector (220°C), column (2OO"C),
detector (350°C)
Carrier: Nitrogen at 30 ml/minute
348 JAMES R. LUCH

metoprolol
36
System V The following system has been used
in plasma and cerebrospinal fluid.
packed with 4% OV-101 on Chromosorb
W (100-120 mesh)
Temperatures: Injector (210°C) , column (195OC) ,
detector (255°C)
Carrier: Nitrogen at 25 mllminute
metoprolol
30
System VI The following system has been used
in pharmaceutical dosage forms.
packed with 3% J X R on Gas Chrom Q
(100-120 mesh)
Temperatures: Injector (25OoC), column (210OC) ,
detector (25OOC)
Carrier: Nitrogen at 45 ml/minute
Detection: Flame ionization detection of
trimethylsilyl derivative of meto-
prolol prepared using bis(trimethy1-
sily1)-trifluoroacetamide
In addition to the systems listed above,
metoprolol has been used as an internal
standard for the gas chromatographic deter-
mination of atenolol in plasma and urine
using electron capture detection of the
pentafluoropropionyl derivatives. 3 7 Alprenolol
and other 8-adrenorceptor blocking agents,
including metoprolol, have been derivatized
to form the 2,4-dichlorobenzeneboronate
adducts for gas chromatographic d ermination
using electron capture detection.'' Two
metabolites of metoprolol have been determined
in plasma and urine by gas chromatography
using electron capture detection. 39
6.5 Gas Chromatography-Mass Spectrometry

Sensitive methods reported for determining
metoprolol and its major metabolites in plasma and
urine using gas chromatography-mass spectrometry
(GC-MS) are given below.
40
System I The following GC-MS system used
selected ion monitoring €or the
quantitation of metoprolol and two
of its metabolites in plasma and
urine.
packed with 3% OV-17 on Gas Chrom Q
(100-120 mesh)
Detection: MS selected ion monitoring at m/e =
266, 270 and 2 7 1
Temperatures: Injector (250°C) , column ( 2 0 0 O C ) ,
separator and ion source (250OC)
Carrier: Helium at 15 ml/minute
MS Source: Electron impact at 60 eV
Sample: Trifluoroacetyl derivatives pre-
pared by using N-methyl-bis-(tri-
fluoroacetamide)
24
System I1 The fallowing GC-MS system has been
used for the determination of
urinary metabolites of metoprolol
tartrate.
Column: 6 ft x 2 mm i.d. glass column
packed with a mixture of 5% OV-1
and 5% OV-17 on Chromosorb W (80-
100 mesh)
Detection: MS using electron impact at 70 eV
Temperatures: Injector (24OoC), column and ion
source (not reported), separator
(210°C)
Carrier: Helium at 54 ml/minute
Sample: Trimethylsilyl derivatives prepared
by using bis-(trimethylsily1)-
acetamide
350 JAMES R.LUCH

High pressure liquid chromatography (HPLC)
systems that have been used to determine metoprolol
tartrate and related compounds in various samples
are summarized below.
System I The following HPLC system is given
as an assay procedure in the mono-
graph for metoprolol tartrate tablets
in USP m.27
Column: pBondapak c18 column (30 cm x
3.9 mu i.d., Waters Associates), USP
column packing L1
Mobile Phase: A mixture of 550 ml methanol and
470 ml water containing 961 mg 1-
pentanesulfonic acid sodium salt
(monohydrate), 82 mg anhydrous
sodium acetate and 0.57 ml glacial
acetic acid
Flow Rate: 1 ml/minute
Detection: Ultraviolet absorption (254 nm)
Internal Oxprenolol hydrochloride
Standard :
The following two HPLC systems have been used
to determine metoprolol in animal feed samples.
System 1 1 ~ 1
Column: pBondapak c18 (30 cm x 3.9 mm
i.d. , Waters Associates)
Mobile Phase: A mixture of 520 ml methanol
and 480 ml water containing 1.10 g
1-heptanesulfonic acid sodium salt
(monohydrate) and 5.0 ml glacial
acetic acid
Flow Rate: 1.2 ml/minute
Principle: Metoprolol tartrate is extracted
from feed samples with 0.1N HC1 con-
taining oxprenolol hydrochioride
(internal standard). Interfering
feed extractables are removed by
alkalizing the aqueous solution and
partitioning metoprolol into hexane-
methylene chloride ( 8 5 : 1 5 ) prior to
HPLC analysis.
42
System I11
Column: 15 cm x 4.5 m i.d. stainless steel
column packed with Partisil 10
(Whatman)
Mobile Phase: Methylene Chloride-Methanol-e
Diethylamine in Methanol (89:lO:l)
Flow Rate: 1 d/minute
Principle: Metoprolol tartrate is extracted
from feed samples with 0.01E HC1,
the aqueous extract is made alkaline
and metoprolol is partitioned into
methylene chloride prior to HPLC
analysis.
43
System IV Metoprolol and one of its metabo-
lites (a-hydroxymetoprolol) have
been determined in plasma by the
following HPLC system.
Column: 25 cm x 4.6 nun i.d. stainless steel
column packed with 5 pm silica B/5
(Perkin-Elmer)
Mobile Phase: Hexane-Isopropanol-Methano1-
Concentrated Ammonium Hydroxide
(850:100:50: 1)
Flow Rate: 3 rdminute
Detection: Fluorescence, excitation wavelength
at 224 nm and no emission filter
Internal (+)-Ethyl-2- (4-(3-isopropylamino-
Standard : 2-hydroxypropoxy)phenyl)ethyl
carbamate
44
System V The following system based on ion-
pairing with a chiral counter ion,
(+)-10-camphorsulfonate, has been
used for the separation of meto-
prolol enantiomers.
Column: 10 cm x 3.2 mm i.d. or 15 cm x
3.2 mm i.d. stainless steel column
packed with 10 pm LiChrosorb-DIOL
(Merck)
352 JAMES R. LUCH
Mobile Phase: A mixture of methylene chloride-

l-p?ntanol (199:l) containing 2 . 2 x
10- -M (+)-10-camphorsulfonate
Detection: Ultraviolet absorption ( 2 5 4 nm)
System v19 The following system has been used

to investigate the purity of meto-
prolol tartrate.
Column: 15 cm x 4 mm i.d. stainless steel
column packed with LiChrosorb RP 8,
5 um or 10 pm particles (Merck)
Mobile Phase : A mixture of 100 ml of water
containing 1.8 ml perchloric acid
and 1 1 . 2 g sodium perchlorate
(monohydrate) with 200 ml aceto-
nitrile diluted to 1000 ml with
water,
Flow Rate: About 0.8-0.9 ml/minute
Detection: Ultraviolet absorption ( 2 7 0 nm)
System V I I ~ The
~ following HPLC system has been
reported for the determination of
several beta-blocking agents,
including metoprolol, in plasma and
urine.
Column: pBondapak c18 column (30 cm x
3.9 mm i.d., Waters Associates)
Mobile Phase: Methanol-Water-Acetic Acid (50: 4 9 :1)
containing 1-heptanesulfonic acid,
prepared by adding one bottle of PIC
B-7 reagent (Waters Associates) per
liter of mobile phase
Flow Rate: 1.3 mllminute
Detection: Fluorescence, excitation wavelength
at 2 2 2 nm and no emission filter
6.7 Ultraviolet Spectrophotometry

An ultraviolet spectrophotometric procedure
is used to determine content uniformity of
metoprolol tartrate tablets in the USP monograph
for this item.27 In this procedure metoprolol is
extracted from the dosage form with 0.1E H C 1 and
after alkalizing the aqueous extract metoprolol is
partitioned into chloroform for quantitation by
ultraviolet spectrophotometry. Assay and content
uniformity data for metoprolol tartrate tablets
also have been obtained using 0.1E H C 1 for
extraction and subsequent quantitation by ultra-
violet spectrophotometry.
The determination of amines and quaternary

ammonium ions by ion pair extraction with picrate
has been described and investigated by Gustavii
and S ~ h i 1 1 .
A ~stability
~ assay for metoprolol
tartrate active substance and dosage forms has been
developed using this approach.47 The active sub-
stance is dissolved in water and diluted with pH
6.5 phosphate buffer. Metoprolol is then extracted
into methylene chloride as a picrate ion pair which
is measured spectrophotometrically at approximately
347 nm. A similar procedure is used for the tablet
assay except 95% ethanol-water (30:ZO) is used to
extract the drug from the tablets.
Acknowledgment
The author expresses appreciation to Rebecca Yang, Donald
Kender, Jane Johnson and Richard Brown for their help in
preparing this manuscript and to Jijrgen Vessman for
reviewing the manuscript.
354 JAMES R. LUCH
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35th Edition, 906-908 ( 1 9 8 1 ) .
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1321-1322 (1977).
3. M. Windholz, Ed., "The Merck Index", 9th Edition,
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5 . R. K. Rodebaugh, CIBA-GEIGY, Personal Communication.
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0
19. C. Bengtsson, G. Johnsson and C. G. Regardh,
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20. G . Johnsson, A c h p h m a c o l . & taxied!. , 36,
suppl V, 59-68 ( 1 9 7 5 ) .
21. K. A. Johansson, C. Appelgren, K. 0. Borg and R.
Elofsson, A& p h m . duct., 11,333-346 ( 1 9 7 4 ) .
22. C. Appelgren, K. 0. Borg, R. Elofsson and K. A.

Johansson, A& phatun. huec., 11,325-332 (1974).
23. S. Zak, CIBA-GEIGY, Personal Communication.
24. K. 0. Borg, E. Carlsson, K-J. Hoffmann, T-E.
Jijnsson, H. Thorin and B. Wallin, AcAa p h m a c o l .
e.A h t U x i c O l . , 36, suppl V, 125-135 (1975).
25. A. Arfwidsson, K. 0. Borg, K-J. Hoffmann and I.
Skgnberg, Xenobiofica, 5, 691-711 (1976).
26. K. Gustavii, P-A. Johansson and A. Briindstrijm,
A& phahm,. hk&c., 13,391-406 (1976).
27. USP XX, Third Supplement, p. 192 (1982).
28. Analytical Department (Pharmaceuticals Division,
Switzerland), CIBA-GEIGY, Ltd., Personal Communi-
cation.
29. C . G. Greig and D. H. Leaback, NUdhte, 188,310
(1960).
30. R. J. Bisknell, B. N. Clewes and C. D. Ratcliffe,
CIBA-GEIGY (England), Personal Communication.
31. G. Szbkely, CIBA-GEIGY, Ltd., Personal Communi-
cation.
32. S . Zak, F. Honc and T. G. Gilleran, A d . L a . ,
13, 1359-1371 (1980).
-
33. M. Ervik, A o t a p h m a c u l . QA: Zoaxicol., 36, s u p p l
V, 136-144 (1975).
34. F. M. Williams, B. N. Singh, P. K. Ambler and R.
Dorrington, Cfin. Ctp. phU4mUcOl. phyAiOl., 3,
473-482 (1976).
35. P. H. Degen and W. Riess, J. Chrtomatogh., ,&I
72-75 (1976).
36. A. J. Wood, Bk. J. cLLn. p h m a c . , 4, 240-241
(1977).
37. S. H. Wan, R. F. Maronde and S. B. Matin, J .
P h m . S c i . , 67,1340-1342 (1978).
38. C . F. Poole, L. Johansson and J. Vessman, J.
Chrtomatogn., 194, 365-377 (1980).
39. C. P. Quarterman, M. J. Kendall and D. B. Jack,
J. Chhomatogn., 183, 92-98 (1980).
40. M. Ervik, K-J. Hoffmann and K. Kylberg-Hanssen,
BLomed. M a n SpecaXom., 8, 322-326 (1981).
41. J. Luch and J. Pensabene, CIBA-GEIGY, Personal
Communication.
42. G. Kildgn, P-0. Lagerstrbm and B-A. Persson, AB
Hiissle, Personal Communication.
43. D. B. Pautler and W. J. Jusko, J. Chrtomatogh,
Biomed. Appl., submitted for publication (1982).
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179-183 (1981).
356 JAMES R. LUCH
45. M. A . Lefebvre, J . G i r a u l t and J. B. F o u r t i l l a n ,

J. Liq. Ckrturnatugn., 4, 483-500 (1981).
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241-258 (1966).
47. M. Schrtider-Nielsen, AB H B s s l e , P e r s o n a l Communi-
cation
L i t e r a t u r e s e a r c h e d through March, 1982.

PHENYLPROPANOLAMINE
HYDROCHLORIDE
Isadore Kanfer, John M . Haigh, and Roslind Dowse
1. Introduction 358
2. Description 358
2.1 Name, Formula, Molecular Mass 358
2.2 Trade Names 358
2.3 Appearance, Odour and Colour 359
3. Synthesis 359
4.1 Solubility 359
4.2 Melting range 359
4.7 Differential Scanning Calorimetry 362
4.8 Proton Magnetic Resonance Spectrum 362
4.9 Ultraviolet Spectrum 364
5.2 Ultraviolet Spectrophotometric Analysis 365
5.3 Colorimetric Analysis 367
5.4 Spectrofluorimetric Analysis 367
5.5 Titrimetric Analysis 367
5.6 Chromatographic Analysis 368
6. Stability 37 1
7. Absorption, Disposition, and Pharmacokinetics 377
8. Identification and Determination in Biological Fluids 377
References 380
ANALYTiCAL PROFILES O F DRUG SUBSTANCES 357 Copyright by the Anierican PharnYlceutical Association.
VOLUME 12 ISBN 0-12-260812-7
358 ISADORE KANFER E T A .
1. Introduction
Phenylpropanolamine hydrochloride belongs to the sympath-

omimetic mine class of drugs and is structurally related to
ephedrine hydrochloride. Its synthesis was first reported in
1910 (1 1 and the first American patent was registered in 1939.
The effects of phenylpropanolamine hydrochloride are largely
the result of alpha-adrenergic agonist activity resulting
from both direct stimulation of adrenergic receptors and
release of neuronal norepinephrine. The principal adverse
effect of phenylpropanolamine hydrochloride is dose-related
hypertension and ventricular arrhythmia has been described
(2). Phenylpropanolamine hydrochloride is widely used as a
decongestant and it has been used as an anorectic agent for
over 40 years ( 3 ) . A report in 1939 ( 4 ) described its effect
as an hypertensive agent when administered parenterally.
2. Description
2.1 Name, Formula, Elolecular Mass
Phenylpropanolamine hydrochloride, sometimes

referred to as dZ-norephedrine can also be named in a number
of ways :
(a) a-(1-aminoethy1)benzenemethanol hydrochloride
(b) a-(1-aminoethy1)benzyl alcohol hydrochloride
+
(c) (-)-2-amino-phenylpropan-l-ol hydrochloride
(d) 2-amino-1-phenyl-1-propanol hydrochloride
(e) a-hydroxy-B-aminopropylbenzene hydrochloride
(f) I-phenyl-2-amino-1-propanol hydrochloride
H CH3
MM 187.67
C9H13No
2.2 Trade Names
Propadrine, Control, Obestat and Dietac. Numerous

products containing phenylpropanolamine hydrochloride in
combination with other active ingredients are commercially
PHENYLPROPANOLAMINEHYDROCHLORIDE 359
a v a i l a b l e as a p p e t i t e s u p p r e s a n t s and c o l d and i n f l u e n z a
remedies.
2.3 Appe a r a n c e , mour and Colour
The compound i s a w h i t e c r y s t a l l i n e powder w i t h an

odour resembling t h a t o f crude benzoic acid.
3. Synthesis
Phenylpropanolamine h y d r o c h l o r i d e i s prepared by r e a c t i n g
benzaldehyde with n i t r o e t h a n e i n 95% e t h a n o l i n t h e p r e s e n c e
o f sodium hydroxide t o form a - ( 1 - n i t r o e t h y l l b e n z y l a l c o h o l
and t h e n r e d u c i n g t h i s n i t r o - a l c o h o l t o t h e corresponding
amino compound. A stream of hydrogen c h l o r i d e passed i n t o a
s u i t a b l e s o l u t i o n o f t h e base y i e l d s t h e h y d r o c h l o r i d e ( 5 ) .
4.1 Solubility
The sample i s s o n i c a t e d f o r one minute a t ambient

temperature.
Solvent mg/ml Solubility
Water )50-<I 000 Soluble
Methanol 2 5 0 4 1 000 Soluble
Isopropanol 21 0-<33.3 Sparingly soluble
D i et h yl e t h e r <0.5 Practically insoluble
Ethyl acetate <0.5 Practically insoluble
Chloroform <0.5 Practically insoluble
Benzene <O .5 Practically insoluble
Carbon t e t r a c h l o r i d e <0.5 Practically insoluble
Acetonitrile <0.5 Practically insoluble
Acetone (0.5 Practically insoluble
Cyclohexane <0.5 Practically insoluble
4 -2 Melting r a n g e
Phenylpropanolamine h y d r o c h l o r i d e c r y s t a l s m e l t a t
190-194OC. The f r e e base m e l t s a t 101-101.5°C (6).
4.3 S p e c i f i c Rotation
The s p e c i f i c r o t a t i o n , [a] , of phenylpropanolamine

h y d r o c h l o r i d e i n water i s +32" ( 7 ) .
4.4 Crystal Structure
The crystal structure was determined using single

crystals of phenylpropanolamine hydrochloride obtained by
slow evaporation of an aqueous solution at room temperature.
The intensity data were collected on an automatic four-circle
diffractometer using Ni-filtered R l K , radiation. Phenylpro-
panolamine hydrochloride has a monoclinic crystal system with
possible space groups of p2 (non-centrosymmetric) or 'P21 /m
(centrosymmetric). ~ h : cell dimensions are a = 7.44~4,
b = 9.4611, c = 14.595AJ B = 103.4' with each asymmetric unit
containing two molecules (8).
4 .S Dissociation Constant
The pK of phenylpropanolamine hydrochloride deter-

mined potentiom&rically at 20'C is 9.44
+ 0.04 (9).
-
4.6 Infrared Spectrum
The infrared spectrum of phenylpropanolamine hydrochloride

is shown in Figure 1. It was obtained from a Nujol mull
between KBr plates using a Perkin-Elmer 180 Infrared Spectro-
photometer. Characteristic band assignments are listed
below.
Frequency (cm-l) Assignment

3368 0-H stretching
3303 NH3f stretching
2800-2400 NH3+
1990 NH3+
1623 NH3 out of plane deformation
1598 C=C aromatic stretching
1581 NH + out of plane deformation
1508, 1491 C=$ aromatic stretching
1450 0-H out of plane deformation
1329 NH3+ in plane deformation
1241 1208 0-H in plane deformation
1128, 1088, 1054 C-H in plane deformation,
monosubstituted benzene
1031 C-0 stretching
816, 802 NH3 + rocking
747, 703 C-H out of plane deformation,
monosubstituted benzene
Figure 1 . I n f r a r e d spectrum of phenylpropanolamine h y d r o c h l o r i d e i n Nujol mull.
4.7 Differential Scanning Calorimetry
Phenylpropanolamine hydrochloride was heated from

320 to 520°K at a rate of 20°/minute under an atmosphere of
nitrogen in a Perkin-Elmer Model DSC-2 Differential Scanning
Calorimeter. The thermogram is depicted in Figure 2. A
single endotherm was observed with an onset temperature of
194.5OC which corresponds to the melting point. The heat of
transition (AH melting) calculated in relation to an indium
standard is 168 + 6 .
- '
g
J
4.8 Proton Magnetic Resonance Spectrum
m e 60 MHz proton magnetic resonance spectrum of

phenylpropanolamine base was obtained with a Perkin-Elmer
Model R-12 Spectrometer. The spectrum in CDC13 with
tetramethylsilane (TMS) as the internal standard is depicted
in Figure 3. The integration and multiplicities are consist-
I8
z
W
ii
Figure 3. Proton magnetic resonance spectrum of phenylpropanolamine in CDC13.
364 ISADORE KANFER ETAL.
e n t w i t h t h e p r o t o n assignments. Chemical s h i f t s ( 6 ) i n ppm
r e l a t i v e t o TMS are :
Proton Mmber Chemical
Wltiplicity
assignment of p r o t o n s J(Hz) S h i f t (6 )
- CH3 3 9.0 0.94 Lbublet
-NH2> -OH 3 - 2.07 Singlet
N-C-H 1 9.0 3.12 Quintuplet
0-C-H 1 9.0 4.51 Lbublet
Aromatic 5 - 7.41 Singlet
An i n s p e c t i o n of t h e D 0 exchange spectrum shows d i s a p p e a r -
2
ance of t h e resonance a t 2.07 ppm. This c o r r e s p o n d s t o
t h r e e p r o t o n s , one hydroxyl p r o t o n and two amino p r o t o n s .
4.9 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 of phenylpropanolamine
h y d r o c h l o r i d e i n methanol and 0.1M HC1 a t a c o n c e n t r a t i o n of
1 m g / m l w e r e o b t a i n e d w i t h a Eieckman Acta M V I u l t r a v i o l e t
spectrophotometer. "be spectrum i n methanol i s d e p i c t e d i n
Figure 4. The spectrum o b t a i n e d i n methanol shows s h o u l d e r s
1
I, .
Figure 4. U l t r a v i o l e t spectrum of phenylpropanolamine

h y d r o c h l o r i d e in methanol.
PHENYLPROPANOLAMINE HYDROCHLORIDE 365
a t 267, 248 and 243 nm w h i l s t i n 0.1M HC1 s h o u l d e r s o c c u r a t

267, 247 and 242 nm.
Absorption
Solution E
M a x i m a (nm) -
Methanol 264.0 133.63
258.0 179.04
252.0 145.44
0.1M HC1 262.8 144.32
257.0 185.04
251 .O 148.82
4.10 Mass Spectrum
The l o w r e s o l u t i o n m a s s spectrum of p h e n y l p r o p a n o l -
amine h y d r o c h l o r i d e i s shown i n F i g u r e 5. It w a s obtained
w i t h a V a r i a n MAT CH5-DF mass s p e c t r o m e t e r . Direct p r o b e a t
80°C i n t o t h e i o n s o u r c e w a s u s e d t o o b t a i n t h e mass s p e c t r u m
The m o l e c u l a r i o n i s n o t o b s e r v e d . The a s s i g n m e n t s of some
of t h e major i o n s formed are :
m/e -
r
-
Ion
+
132 17 Ph-CH=C ( CH ) NH
2 2
105 29 Ph-C=O+
91 26 Ph-CH2+
77 100 Ph+
Theory ( % )
Element Hydrochloride -
Base
C 57.62 70.61
H 7.47 9.80
N 18.92 9.15
0 7 -46 10.44
c1 8.52 -
5.2 U l t r a v i o l e t Spectrophotometric Analysis
P e r i o d a t e o x i d a t i o n of phenylpropanolamine hydro-
c h l o r i d e h a s been used. The sample t o be d e t e r m i n e d i s
p l a c e d i n a s e p a r a t o r y f u n n e l , sodium b i c a r b o n a t e and sodium
m e t a p e r i o d a t e are added and a f t e r s t a n d i n g fsr a b o u t 1 5 min-
u t e s t h e s o l u t i o n i s e x t r a c t e d w i t h hexane a f t e r t h e a d d i t i o n
of 1 M h y d r o c h l o r i c acid. The e x t r a c t i s f i l t e r e d and t h e
Figure 5. Mass spectrum of phenylpropanolamine hydrochloride.
absorbance determined a t 242nm i n lcm c u v e t t e s u s i n g hexane

as t h e r e f e r e n c e . The amount of t h e o x i d a t i o n product o f
phenylpropanolamine h y d r o c h l o r i d e i s determined by comparison
of t h e sample absorbance a g a i n s t t h e absorbance of a Phenyl-
propanolamine Hydrochloride Reference Standard t r e a t e d i n t h e
same manner (1 0). Ether (11 1 and chloroform ( 1 2 ) have a l s o
been used f o r t h e e x t r a c t i o n of t h e d e r i v a t i v e . Wallace ( 1 3 )
used a l k a l i n e p e r i o d a t e o x i d a t i o n t o form benzaldehyde which
w a s subsequently converted t o t h e semicarbazone d e r i v a t i v e ,
t h u s enhancing t h e s p e c i f i c i t y and s e n s i t i v i t y of t h e proce-
dure. Phenylpropanolamine h y d r o c h l o r i d e h a s also been
determined a f t e r a l k a l i n e e x t r a c t i o n i n t o an o r g a n i c s o l v e n t
followed by b a c k - e x t r a c t i o n under aqueous a c i d i c c o n d i t i o n s
(14).
5.3 C o l o r i m e t r i c Analysis
Phenylpropanolamine h y d r o c h l o r i d e can be r e a c t e d
w i t h ninhydrin i n a c i t r a t e b u f f e r a t an e l e v a t e d tempera-
t u r e and determined c o l o r i m e t r i c a l l y a t 570nm ( 1 5 ) . This
r e a c t i o n h a s been a p p l i e d t o t h e d e t e r m i n a t i o n of phenylpro-
panolamine h y d r o c h l o r i d e i n a multicomponent m i x t u r e by an
automated system which, a f t e r phase s e p a r a t i o n , u t i l i z e s t h e
stream s p l i t t i n g t e c h n i q u e t o d i v i d e t h e c h l o r o f o m stream
i n t o segments ( 1 6 ) . An i o n - p a i r e x t r a c t i o n t e c h n i q u e u s i n g
an a c i d i c d y e , bromothymol b l u e , has been u t i l i z e d and t h e
r e s u l t i n g chloroform e x t r a c t determined a t 420x1111 ( 1 7 ) .
5.4 S p e c t r o f l u o r i m e t r i c Analysis
Phenylpropanolamine h y d r o c h l o r i d e has been d e t e r -

mined by measuring i t s fluorescamine d e r i v a t i v e , 4-phenyl-
spiro[furan-2(3H) ,1 '-phthalan)-3,3'-dione a t 480nm w h i l s t
e x c i t i n g a t 398nm ( 1 8 ) . The r e a c t i o n f a v o u r s a pH o f 9 f o r
o p t i m a l r e a c t i v i t y (1 9 1.
5.5 T i t r i m e t r i c Analysis
After e x t r a c t i o n of phenylpropanolamine hydrochlor-

i d e from an a l k a l i n e aqueous s o l u t i o n with chloroform,
shaking with s a t u r a t e d sodium c h l o r i d e s o l u t i o n and back-
e x t r a c t i n g w i t h an e x c e s s of s u l p h u r i c a c i d , t h e e x c e s s a c i d
i s t i t r a t e d with a standard sodium hydroxide s o l u t i o n u s i n g
methyl r e d as i n d i c a t o r ( 2 0 ) .
5.6 Chromatographic Analysis
5.6.1 Column Chromatography

A weakly basic anion exchange resin,
Amberlite IR-45, was found to be suitable for the separation
of phenylpropanolamine hydrochloride from various dosage forms
and yielded a 99.6% recovery of the drug which was then deter-
mined titrimetrically (21 ). Being a nitrogenous base, the
drug is retained on a sulphonated polystyrene cation exchange
resin. Determination is then effected by measuring the
ultraviolet absorption after elution with hydrochloric acid
(22, 23). Phenylpropanolamine hydrochloride has been deter-
mined by mixing with ammonium hydroxide, the base eluted with
chloroform from a Celite column and the absorbance measured
at 258.5nm (24). Separation of phenylpropanolamine hydro-
chloride from mixtures of drugs in various dosage forms has
been described. The method involves the retention of
phenylpropanolamine on the first of four Celite columns
followed by elution, addition of sodium hydroxide, extraction
of the free base with chloroform and subsequent determination
at 257nm using sulphuric acid as the reference solution (25).
An on-column periodate oxidation of phenylpropanolamine to
benzaldehyde on a weakly basic Celite column has also been
described and the derivative determined at 267nm (26).
5.6.2 Paper Chromatography

A descending paper chromatographic technique
using Whatman No. 1 paper has been reported. The solvent
system consisted of a 1:l mixture of butanol (saturated with
1 M hydrochloric acid and methanol. After spraying with
Dragendorff's reagent, the resulting orange-red spots were
quantitatively determined by densitometry (27).
5.6.3 Thin Layer Chromatography

A number of thin layer chromatographic
systems have been described for phenylpropanolamine hydro-
chloride and these are listed in Table 1.
5.6.4 Gas Chromatography

Gas chromatography has been extensively used
as a method for determining phenylpropanolamine in pharma-
ceutical preparations and, to a lesser extent, for the
determination of the drug in body fluids. The drug has been
chromatographed directly without derivatization and also as
the silyl, pentafluorophenyloxazolidine, acetone, butanone,
trifluoroacetyltrimethylsilyl, heptafluorobutyryl and
TABLE 1
T H I N LAYER CHROMATOGRAPHIC SYSTEMS FOR PHENYLPROPANOLAMINE
S o l v e n t System AdsoFbant Detection -

Rf Reference
Chloroform l a y e r from a S i l i c a Gel G Sprayed w i t h 0.3% p - n i t r o a n - Not 28

m i x t u r e of c h l o r o f o r m / (F254) ( 6 0 ~ ) i l i n e p l u s 5% s o d i u m n i t r i t e , reported
a c e t i c acid/methanol/ h e a t e d a t 70°C for 15 m i n u t e s
w a t e r (85 :20 :8 :20 1 t h e n s p r a y e d w i t h 20% sodium
carbonate
Not reported S i l i c a Gel G Sprayed w i t h b u f f e r (pH 9.31, 0.50 19
(F254) ( 6 0 ~ ) oversprayed with f l u o r e s c -
amine/acetone s o l u t i o n and
re-sprayed with b u f f e r
Benzene/ethyl acetate Gelman I T L C p-nitrobenzoyl c h l o r i d e 29
(70 :30) f i b r e SAF a p p l i e d and h e a t e d (lOO°C) 0 -79
% e n z e n e / e t h y l acetate
(30 :70) 0 -90
Hexane/ethyl acetate
(50 : 5 0 ) 0.76
Hexane/ethyl acetate
(30:70) 0.90
Ethyl acetate/methanol/ HPTLC S i l i c a Dipped i n t o o - p h t h a l a l d e h y d e 0-40 30

f o r m i c a c i d (69:30 :1 ) Gel 60 s o l u t i o n f o l l o w e d by 20%
polyethylene g l y c o l i n
methanol
TABLE 1 ( c o n t i n u e d )
S o l v e n t Svstem Adsorbant Detection -
Rf Reference
Heptane/methanol (60 :40) HPTLC S i l i c a Fluorescamine s o l u t i o n 0.45 31
Benzene/methanol ( 8 3 :1 7 G e l 60 0.37
Benzene/acetone/methanol/
dioxane (40 :40 :4 : 5 ) 0.28
Ethyl acetate/methanol/ Silica Gel Sprayed w i t h 0 . 3 % n i n h y d r i n Not 32
water/ammonia (85 :10 :3 :l ) acid, h e a t e d a t 100°C f o r reported
5 minutes, sprayed with 5 %
H2S04, h e a t e d w i t h h o t a i r
f o r 5 minutes, sprayed with
iodoplatinate reagent then
p-nitroaniline reagent,
h e a v i l y s p r a y e d w i t h 25%
a l c o h o l i c NaOH s o l u t i o n
Chloroform/methanol ( 4 :1 Silica Gel G I o d i n e vapour 0.17 33
Chloroform/methanol (4:l) Silica G e l G I o d i n e vapour 0.14
(HF254)
n-butanol/acetic acid/ DC-cellulose Ninhydrin r e a g e n t Not 34
w a t e r ( 4 : l :5) reported
2,6-dinitro-4-trif~uoromethy~benzenesu~phonicacid derivat-
ives. The gas chromatographic conditions are listed in Table
2.
5.6.5 High Performance Liquid Chromatography

High performance liquid chromatography has
been used for the determination of phenylpropanolamine alone
and in pharmaceutical formulations but relatively little
information has been published on the high performance liquid
chromatographic analysis of phenylpropanolamine in biological
fluids. Table 3 lists the various methods and associated
conditions which have been reported. A technical report
issued by Waters Associates describes the following condit-
ions for the determination of phenylpropanolamine : 0.3m x 4 m
(i.d. 1 pbndapak C18 column, methanol/water (50:50 including
PIC reagent B-7, detected at 254nm with a retention time of
3.1 minutes for the drug. A similar technical bulletin
issued by the Ix1 Pont Company lists the following conditions :
0.25m x 4.6mm (i.d.1 Zorbax TMS column, hexanesulphonic acid/
acetic acid/water/methanol (0.1:1:68.9:30), detected at 254nm
at a flow rate of 2.5 ml/min and retention time of 2.4
minutes.
6. Stabilitv
Phenylpropanolamine hydrochloride is a relatively stable

compound. A solid state mixture of phenylpropanolamine
hydrochloride, phenylephrine hydrochloride and aspirin in the
presence of starch, sugar and talc was stressed at 70°C for
42 days. Phenylpropanolamine hydrochloride was found to be
quite stable under these conditions, showing a loss of about
10% when assayed by a thin layer chromatographic method (28).
A recent study on phenylpropanolamine hydrochloride in a
film-sealed tablet, however, indicated that the molecule is
subject to decomposition in pharmaceutical formulations with
time at normal and elevated temperatures (52). A significant
decrease in phenylpropanolamine hydrochloride concentration
was observed in stressed samples when assayed by the perio-
date oxidation method (11). Stability studies carried out on
phenylpropanolamine hydrochloride in a decongestant syrup
formulation containing sucrose indicated a reduction in
concentration of the drug. Fbrther studies showed decompo-
sition of phenylpropanolamine hydrochloride in the presence
of fructose, dextrose and 5-(hydroxymethyl)-2-furaldehydeJ
but not with sorbitol or levulinic acid. It was speculated
that Schiff base formation probably occurred under the test
conditions (59 .
TABLE 2
GAS CHROMATOGRAPHIC SYSTEMS FOR PHENYLPROPANOLAMINE
mug Carrier column Injected

R (mins) Detector as Ref
Source Gas Temp(OC) T
Tablets, 2.0m x 4.0mm (i.d.1 glass He 200 2.80 FID Silyl 35
sP U P 0.1 % s i l i c o n e o i l (DC- derivative
710) on 60-80 mesh
dimethyldichlorosilane
treated glass beads
2.4m x 3 . 2 m (o.d.1 He 180 1.80 FID Phenylpropan- 36
Pyrex glass olamine
2% SE-30 o n Chromo-
sorb W (HP)
Plasma 2.0m x 2.0mm (i.d.1 190 5.00 FID Pentafluoro- 37
N2 phenyloxazol-
glass
1.25% OV-17 on G a s idine deriv-
Chrom Q ative
Tablets 1 . 8 m x 4mm glass He 230 0.85 FID Phenylpropan- 38
3% OV-17 on Gas olamine
Chrom Q
Urine 2.0m x 2.2mm s t a i n - He 230 0.30 Nitrogen Phenylpropan- 39
less steel olamine
3% OV-1 on Gas
Chrom Q
mu9 Carrier Column Injected

COlUmn R (mins) Detector as Ref
Source Gas Temp('C) T
Tablets, 1 .8m x 6 . 4 m (i.d.1 220 2 -40 FID Phenylpropan- 40

N2
capsules, glass olamine
liquids 1 % HI-EFF-8BP and 1 0 % hydrochloride
SE52 o n Gas Chrom Q
4 % HI-EFF-8BP on Gas 220 1.25
Chrom Q
Raw 1.8-2.4m x 3m ( i . d . ) Ar 104 9.10 EC Phenylpropan- 41
Material glass olamine
1.15% SE-30 on Gas 13.40 Acetone d e r i v -
Chrom P ative
4
W
123.50 Butanone d e r i v -
W
ative
Raw 1.4m x 4mm (i.d.1 He Prog- 28.00 FID Trif l u o r o a c e t y l - 42
Mater i.al glass rammed trimethylsilyl
3% P o l y A 1 0 3 on 70-250 derivative
Gas Chrom Q
Serum 1.4m x 3 . 2 m (o.d.1 140 3.70 EC Heptaf l u o r o - 43

s t a i n l e s s steel N2 butyryl deriv-
5 % EGS on AW-DMCS ative
Chromosorb W
Raw l.lm x 2.5m g l a s s 180 2.55 FID Phenylpropan- 44
N2
Material 18.8% Apiezon N o n 138 2.77 olamine
Diatoport S 101 3.18
TABLE 2 (continued)
mug Injected
COlUmn Carrier R (mins) Detector as Ref
Source Gas Tkn~p(~C) T
Raw 1.8m x 2mm (i-d.1
N2
250 1.38 EC ,,
2 64 in i t r o - 45
Material glass 4 - t r i f luoro-
3% OV-1 on Supelcoport methyl-benzene-
0.9m x 2mm (i.d.1 220 1.38 sulphonic acid
glass derivative
3% SP-2250 on Supel- 230 0.98
coport
Raw 1.2m x 4mm (i.d.1 glass 185 1.60 FID Phenylpropan- 46
N2
Materia1 2% SE-30 and 2% o lamine hydro-
Carbowax 20M on chloride
-a
W Anachrom ABS
P
Raw 1.8m x 2 m g l a s s He -0- 8.36 Nitrogen Phenylpropan- 47
Material 3% OV-17 on Anachrom grammed olamine
ABS 100-250
Bio- 1 .Om x 6mm (o.d.1 FID Phenylpropan- 33
N2
logical glass olamine
Material 7.5% Carbowax 20M on 165 4.10
Chromosorb W
2.0% Carbowax 20M on 120 1-10
Chromosorb G
Wine 1 .lm x 3mm (i.d. g l a s s He 170 Not Nitrogen Phenylpropan- 34
12.5% Apiezon L on reported olamine
Chromosorb W impregnat-
ed with 2 % Igepal CO 880
TABLE 3
HIGH PERFORMANCE L I Q U I D CHROMATOGRAPHIC SYSTEMS FOR PHENYLPROPANOLAMINE
mug Flow rate

While Phase Detection
Source (ml/min 1
RT(mhs)
-
Ref
Tablets, 0.5m x 2 . l m (i.d.1 0.02M ( NH4 2HP04 1 .o 2.2 Stated 48

syrup DU Pont Zipax SCX i n 36% dioxane/ o n l y as
U.V.
water
0.3m x 4mm (i.d.1 0.05M KH2P04 i n 2 .o 3 .O 254nm 49
Waters pBondapak Cl8 w a t e r containing
1 3 % (v/v) methanol
T a b l et S n 0.3m x 4mm (i.d.1 Water/methanol/acetic 2 .o Not 254nm 50
capsules, Waters Wondapak acid (55:44:1) con- Stated
w t a i n i n g 0.005M sodium
2 liquid phenyl
heptane s u l p h o n a t e
0.3m x 4mm (i.d.1 13% a c e t o n i t r i l e i n Gradient 7.5 2 54nm 51
Waters PEbndapak CN w a t e r containing 1.8% elution
acetic acid
13%a c e t o n i t r i l e i n 8.8
w a t e r c o n t a i n i n g 1.8%
acetic acid and
0.005M sodium h e p t a n e
sulphonate
Tablets 0.25m x 4.6m (i.d.1 2.85 x 10-3M e t h y l e n e - 3.8 3.3 216.5nm 52
Whatman P a r t i s i l - l O - diamine buffer ( p H
ODs 7.44)/acetonitrile
(1 :I 1
mug Flow r a t e
Mobile Phase R (mins) Detection Ref
Source
COlUmn
(ml/min) T -
Urine 0.3m x 3.9mm (i.d.1 Methanol/water/acetic 2 .o 8.2 254nm 53
Waters p b n d a p a k C18 acid (45 :54 :1
0.3m x 3,9m (i.d.1 Methanol/water (60 :40 1 1 .o 7.4 254nrn 54
Waters UEbndapak w i t h 0.004M sodium
phenyl h e p t a n e s u l p h o n a t e and
Waters VBondapak (38 1 % acetic acid 1 .o 11.7
Urine 0.25111 x 2mm (i.d.1 Chloroform/ethyl 1.2 9.9 450nm 55
Lichrosorb S I 100 acetate/ethanol/
and Wakogel LC 5H n-hexane ( 2 5 :10 :1 :50)
Plasma LDC 51J O D s 40% a c e t o n i t r i l e i n 1.8 8.2 Fluorescence 56
w a t e r containing detection
ammonium acetate and
acetic acid
Serum a 0.3m x 3.9mm (i.d.1 Acetonit r i l e / w a t e r 1.3 4.8 220nm 57
urine Waters pEbndapak C18 (25:75) w i t h 0.005M
sodium h e p t a n e s u l -
p h o n a t e and 0 - 2 % 1 M
HC1
0.15m x 4.6mm (i.d.1 0.1M KH PO i n 1 0 % 1 .o 5.4 198nm 58
2 4
S p h e r i s o r b S5W aqueous e t h a n o l
PHENY LPROPANOLAMINE HYDROCHLORIDE 377
7. Absorption, Disposition and Pharmacokinetics
Relatively little information has been reported on the

metabolism of phenylpropanolamine hydrochloride. In urine
studies with human subjects it was found that approximately
9 0 % of the drug was excreted in 24 hours, predominantly
unchanged (1 1 , 60, 61 1. Sinsheimer et aZ. (60) found only
4% as transformation products, the major biotransformations
being parahydroxylation to 4-hydroxynorephedrine and oxida-
tive deamination of the side chain, resulting in hippuric
acid. Phenylpropanolamine is also metabolised by phenyl-
ethanolamine-N-methyltransferase to the corresponding N-
methylated metabolite (62, 63). In the rat and rabbit,
80-90% of 14C of small oral doses of [14C]norephedrine is
excreted in the urine within 24 hours, with 1-2% of the dose
being excreted in the faeces. In the rat, 60% occurs as the
unchanged drug and 35% as 4-hydroxynorephedrine. Small
amounts of 4-hydroxyhippuric acid and lJ2-dihydroxy-l-phenyl-
propane were also detected. In the rabbit however, 76% is
excreted as deamination products consisting of 31% 1J2-
dihydroxy-I-phenylpropane, 27% 1-hydroxy-2-oxo-I -phenylpro-
pane and 24% benzoic acid (60). In a study with human
subjects under controlled conditions of urinary pHj acidif-
ication of the urine resulted in the excretion of largely
unchanged phenylpropanolamine hydrochloride, whereas basic
urine conditions resulted in reabsorption of the drug with
little influence on metabolsim (64).
The mean elimination half-life of phenylpropanolamine

hydrochloride in man has been reported as 3.9 hours and the
elimination rate constant as 0.18 hr-' (11 1. After oral
administration of phenylpropanolamine hydrochloride as an
aqueous solution, absorption is rapid, being completed in
less than 2.5 hours (64). The tissue distribution of the
drug in dogs 2 hours after administration is kidney > lung >
liver > spleen > brain > heart > muscle > plasma > fat >
cerebrospinal fluid (651.
8. Identification and Determination in Biological Fluids
The isolation and quantitative determination of

phenylpropanolamine from serum/plasma and urine has been
accomplished by gas chromatography (34, 37, 39, 43) and high
performance liquid chromatography (53, 55-57). Typical serum
phenylpropanolamine concentrations after a single dose of
25 mg of the hydrochloride salt range from about 5-80 ng/ml
over a period of 24 hours (56). In a more recent study using
378 ISADORE KANFER ETAL..
a 150 mg s u s t a i n e d - r e l e a s e t a b l e t , serum and u r i n e c o n c e n t r a -

t i o n s ranged from about 60-275 ng/ml and 0.02-100 pg/ml
r e s p e c t i v e l y (57). Typical chromatograms d e p i c t i n g t h e high
performance l i q u i d chromatographic s e p a r a t i o n of t h e d r u g i n
serum and u r i n e are shown i n Figures 6 and 7 ( 5 7 ) .
ACKNOWLEDGEMENTS
The a u t h o r s would l i k e t o thank P r o f e s s o r M.E. Brown,

Chemistry Department , Rhodes U n i v e r s i t y , Grahamstown ,
South Africa f o r t h e DSC d a t a , M r A. Sonneman of t h e
Chemistry Department, %odes U n i v e r s i t y f o r running t h e
NMR spectrum and P r o f e s s o r H.K.L. Hundt and D r J. Steyn,
Pharmacology Department, F a c u l t y o f Medicine, L n i v e r s i t y
of t h e Orange Free State, Bloemfontein, South A f r i c a f o r
t h e mass spectrum.
M - e
INJECT
INJECT
INJECT
f
9p'
5:
( a ) HPLC chromatogram of blank serum e x t r a c t . (a) HPLC chromatogram of blank u r i n e e x t r a c t .
'd
8
n
r
x
r
P,
2a
(b) HPLC chromatogram of serum e x t r a c t ( b ) HPLC chromatogram of u r i n e e x t r a c t
c o n t a i n i n g phenylpropanolamine ( 1 1 c o n t a i n i n g phenylpropanolamine ( 1 )
and ephedrine (2). and ephedrine ( 2 ) .
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47, 51 (1982).
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62, 1174
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PHENY LPROPANOLAMINE HYDROCHLORIDE 38 1
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62, 802
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18. L.L. S h a n k l e , J. P h a r m . Sci., -

67, 1635 (1978).
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(1 975 ) .
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37, 683 ( 1 9 5 4 1
21. M.C. V i n c e n t , E. Krupski and L. F i s c h e r , J. Am. P h a r m .

ASSOC., 46, 85 ( 1 9 5 7 ) .
22. D.J. S m i t h , J. A s s o c . Off. Anal. Chem., 53, 1 1 6 (1970).
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49, 536 (1966).
26. C.C. C l a r k , J. Assoc. O f f . Anal. Chem., 56, 100 ( 1 9 7 3 ) .
27. H. S c h r i f t m a n , J. P h a r m . Sci., 55, 985 (1966).
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59, 1416 ( 1 9 7 6 ) .
30. G. CXibitz, C h r o m a t o g r a p h i a , 12, 779 (1979).
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C h r o m a t o g r a p h i a , 13, 291 ( 1 980 ) .
32. M.L. Bastos, D. J u k o f s k y and S.J. k l 6 , J. C h r o m a t o g . ,
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81, 93 (19 7 3 ) .
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P h a r m a c . , 26, 945 ( 1 9 7 4 ) .
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and M. Donike, Europ. J. C l i n . Pharmacol., 8, - 161 (1975X
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382 ISADORE KANFER ETAL..
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65, 740 (1976).
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51 j 938 (1962).
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49, 541 (1966).
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895 (1977).
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68, 1135 (1979).
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(1 978).
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(1980).
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(1980).
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J. Chromatog., 196,
334 (1980).
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(1981 1.
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(1983) i n p r e s s .
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9 2 , 345 (1977).
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J. P h m . S c i . , 71, 116 ( 1 9 8 2 ) .
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1 0 7 s (1965).
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M. E. Wolfe "Burger I s Medicinal Chemistry", John Wiley
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Biochemical Aepects", Marcel. Dekker I n c . (New York) ,
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This p r o f i l e attempts t o c o v e r t h e p u b l i s h e d l i t e r a t u r e o n
phenylpropanolamine h y d r o c h l o r i d e u p t o J u l y 1982.
PILOCARPINE
Abdullah A . Al-Badr and Hassan Y. Aboul-Enein
1. Description 386
I . 1 Nomenclature 386
1.2 Formulae 386
1.5 Appearance, Odor, and Color 387
2.1 Melting points 387
2.2 pK.value 388
2.6 Stability 392
2.7 Spectral Properties 394
3. Synthesis 402
4. Biosynthesis 408
5. Metabolism 41 1
6. Methods of Analysis 41 I
6.1 Titrimetric Methods 41 1
6.3 Gravimetric Analysis 414
6.4 Polarimetric Analysis 414
6.5 Phase-Solubility Analysis 414
6.6 Fluorescence Analysis 415
6.7 Spectrophotometric Analysis 415
6.8 Chromatographic Analysis 42 1
6.9 IT-NMR Quantitative Analysis 425
6.10 Thermofractographic Analysis 426
References 427
ANALYTICALPROFILESOF DRUG SUBSTANCES 385 Copyrighi by the Arnericnn Pharmceulical Aimcialion.

VOLUME 12 ISBN 0-12-260812-7
386 ABDULLAH A. AL-BADR AND HASSAN Y . ABOUL-ENEIN
PILOCARPINE
1. Description
1.1 Nomenclature
1.11 Chemical names
-
2-Ethy1-3 [ 1-methy l-5- imidazo1y1) methy1] -4-buta-
nolide.
(3s-cis)-3-Ethyldihydro-4- [ (-methyl-1H-imidazole-
5-yl) methyl]-2-( 3H)-furanone.
(3s ,4R)-3-Ethyl dihydro-4-[ (1-methyl-1H-imidazol-
5-yl) methyl] furan-2 (3H)-one.
1.12 Generic names
Pilocarpine.
Pilocarpine hydrochloride; pilocarpine muriate;
almocarpine .
Pilocarpine nitrate; Licarpin.
1.13 Trade names
For base: OCUSERT Pilo.

For nitrate: PV Carpine.
1.14 Chemical Abstract Registry Number
[ 92-13-71 (base).
[54-71-71 (HC1 salt).
[ 148-7241 (nitrate salt).
1.2 Formulae
1.21 Empirical
C11H16N202 (base).
C H C1N202 (HC1 salt).
11 17
C11H17N305 (nitrate salt).
PILOCARPINE 387
1.22 Structural
1.3 Molecular Weight
208.25 (base).
244.72 (HC1 salt).
271.30 (nitrate salt).
1.4 Elemental Composition

C, 63.44%; H, 7.74%; N, 13.45%; 0, 15.37% (base).
C, 53.99%; H, 7.00%; C1,14.49%; N, 11.44%;
0, 13.08% (HC1 salt).
C, 48.70%; H, 6.32%; N , 15.49%; 0, 29.49%(Ilitrate salt).
1.5 Appearance, Odor and Color

a) Pilocarpine base
Colorless oil or crystal.
b) Pilocarpine hydrochloride
Colorless crystals or a white, crystalline powder;

odorless or almost odorless. Hygroscopic.
c) rilocarpine nitrate
Colorless crystals or a white, crystalline powder;

odorless.
2.1 Melting Point
Pilocarpine base. 34O (1)

Pilocarpine hydrochloride. 195-19800 (1)
Pilocarpine nitrate. 173.5-174 (dec.) (1).
388 ABDULLAH A . AL-BADR AND HASSAN Y. ABOUL-ENEIN
2.2 pKa Value
Pilocarpine base shows two ionisation constants, PK1 -

-
7.15 and pK2 = 12.57 at 20°.
2.3 Specific Rotation
The specific rotation for pilocarpine and its official

salts are shown on the following table and this constant
is used to differentiate and identify pilocarpine, the
cis-isomer and the pharmacologically active one, from its
-
trans-isomer, isopilocarpine.
Pilocarpine base.
[n]k8 + 106' (C = 2) (1)
Pilocarpine hydrochloride.
[a]A8 + 88.5 to 91' (C. = 2) (2)
Pilocarpine nitrate.
[a]i8+ 77' to 83' (C = 10) (1)
2.4 C r y s t a l Structure
The crystal structure of pilocarpine-trichlorogermanate

C H N 0 GeCl ,1/2 H 0 has been determined (3).
11 17 2 2' 3 2 0
The space group is P (No.4) with a = 14.24 A #
0 21 C
b = 17.61 A , c = 6.83 A , $ = 97.25'. There are four
formula units per unit cell as shown in Figure 1.
The clinogrophic projection of pilocarpine molecule
is shown in Figure 2. The conventional planar projec-
tion of pilocarpine molecule showing bond distance in
r( unit is shown in Figure 3.
2.5 Identification
2.51 Color Test

British Pharmacopoeia 1980(48) described the following:
a) Pilocarpine hydrochloride
Dissolve 10 mg into 5 ml of water, add

0.1 ml of M Sulphuric Acid, 0.1 ml
PILOCARPINE 389
Figure 1. XZ P r o j e c t i o n of h a l f a u n i t c e l l i n d i c a t i n g
packing and hydrogen bond scheme. (3)
Figure 2. Clinographic p r o j e c t i o n of a P i l o c a r p i n e Molecule.

(3)
Figure 3. Conventional planar projection of a pilocarpine molecule
showing bond distances in 2 units. (3)
PILOCARPINE 391
of H 0 s o l u t i o n (20 v o l . ) , 1 m l t o l u e n e and
2 2
0.5 m l of potassium chromate s o l u t i o n . Shake
w e l l and a l l o w t o s e p a r a t e ; The t o l u e n e l a y e r
i s c o l o r e d b l u i s h v i o l e t and t h e aqueous l a y e r
remains yellow.
b) Pilocarpine n i t r a t e .
1) D i s s o l v e 10 mg i n t o 2 m l H20, add 0.1 m l of

5% w/v s o l u t i o n of potassium d i c h r o m a t e ,
1 m l of H202 s o l u t i o n (10 v o l . ) and 2 m l of
chloroform and s h a k e ; t h e chloroform l a y e r
turns violet.
2) It y i e l d s - the reactions characteristic for
n i t r a t e s and a l k a l o i d s .
The USP XX(2) d e s c r i b e s similar c o l o r t e s t s men-

t i o n e d under p i l o c a r p i n e HC1 b u t u s i n g benzene
i n s t e a d of t o l u e n e . For p i l o c a r p i n e n i t r a t e ,
t h e USP XX (2) c i t e s t h e u s e of i n f r a r e d ad-
s o r p t i o n spectrum of p o t a s s i u m bromide d i s p e r -
s i o n of p i l o c a r p i n e , p r e v i o u s l y d r i e d and s h o u l d
e x h i b i t maxima a t t h e same wavelength a s of a
s i m i l a r p r e p a r a t i o n of USPXX p i l o c a r p i n e
n i t r a t e r e f e r e n c e s u b s t a n c e . Furthermore,
p i l o c a r p i n e n i t r a t e responds t o t h e same c o l o r
t e s t d e s c r i b e d under p i l o c a r p i n e h y d r o c h l o r i d e .
Another c o l o r test d e s c r i b e d i n t h e p i l o c a r p i n e
n i t r a t e monograph i n t h e USP XX (2) i s as
follows :
Mix a s o l u t i o n (1 i n lo) i n an e q u a l volume
of f e r r o u s s u l p h a t e and supperimpose t h e m i x t u r e
upon 5 m l of H2S04 c o n t a i n e d i n a t e s t t u b e .
The zone of c o n t a c t becomes brown.
2.52 C r y s t a l t e s t
P i l o c a r p i n e can b e i d e n t i f i e d ( 4 a ) by forming cha-

r a c t e r i s t i c c r y s t a l s , with t h e following reagents:
a ) Gold bromide s o l u t i o n ; f e a t h e r i n g r o s e t t e s
( s e n s i t i v i t y 1 i n 1500).
b ) P l a t e n i c c h l o r i d e solution;' p l a t e s i n c l u s t e r .
( s e n s i t i v i t y 1 i n 1500).
392 ABDULLAH A. AL-BADR AND HASSAN Y. ABOUL-ENEIN
2.53 Degradation Test
The following tests were cited by the WHO (4b) in

the International Pharmacopea. If the substance
does not pass this test, this indicates that gross
degradation of both pilocarpine hydrochloride and
pilocarpine nitrate had occurred.
Dissolve 20 mg of the substance in 20 ml of H20,
add 3 drops of bromocresol green solution (to 0.20
gm of bromocresol green add 7.4 ml sod. hydroxide
2 gm/l), dilute to 1000 ml H20. A blue color
develops immediately.
2.6 Stabilitv
Pilocarpine possesses several pharmacological properties,

e.g., it possesses a miotic action and lowers the intra-
occular pressure. Its chief clinical application in
optholmology has been for the treatment of glucoma in
buffered isotonic solution ranging from 0.5 to 6% as
pilocarpine nitrate or hydrochloride. However, pilo-
carpine in aq. solution decomposes through two major
pathways which are both base catalysed as shown in
scheme (la).
Pilocarpine is relatively stable in acidic pH (5,6, 12),

as the pH increases pilocarpine progressively becomes
unstable(5, 6 , 7 , 8)especially at elevated temperature.
The constituents of the buffer affects its stability
e.g., phosphate and carbonate catalyse degradation of
pilocarpine whereas borate buffer does not (8,9).
Addition of 0.5% of methyl cellulose slightly increases
the stability of pilocarpine solution (10). The effect
of sterilization and long sotrage of pilocarpine solu-
tion has been studied by Zelikson (11). Generally
the degradation of pilocarpine is accompanied by marked
drop of pH (12, 8 , 9, 10).
Chung -
et -
a1 (13) published a detailed study on the
kinetics of pilocarpine in aqueous solution. They pro-
posed a cyclic mechanism for the hydrolysis in which
is catalysed by both acid and base ions. The appropri-
ate constant, equilibrium constant and the energy of
activation for the hydroxyl ion catalyse hydrolysis
were calculated. The hydrolysis in high alkali pH was
found to be accompanied by some epimerisation (schemelb).
PILOCARPINE 393
OH-/H~O
3OoC
pH:> 8 H H
H20
R
i 300c
H5C2 H
C 2 H 5 X o H / y R
’O 0
28% 28%
OH- pH > 8
C2H5
I
eR 44%
v
1
u
0-
72% 28%
Scheme (la) : Hydrolysis and epimerization mechanism of

pilocarpine in aqueous solution.
Pilocarpic acid. Isopilocarpic I sopi locarpine

acid.
Scheme (lb): Mechanism of cyclization of pilacarpic acid
to isopilocarpine (13).
They suggested that pilocarpine solution be prepared at

a pH4to5 for acceptable stability and biological
activity .
Baechlin and Etter ( 1 4 ) published a study on the stabi-
lity of pilocarpine in various buffer media e.g., ace-
tate, phosphate, and showed that, the behavior of pilo-
carpine as a function of pH was comparable to that of
lactones which are stable in acid medium but are appen-
ded almost instantly in alkaline media. Its equilib-
rium position and stability was dependant, in large, on
the pH e.g. at pH 4, 87% of intact pilocarpine was
found, at pH 6 only 49% of pilocarpine was found. The
author also reported the effect of presence of the pre-
servative e.g., benazlkonium chloride and phenyl mercury
borate on the hydrolysis of pilocarpine solution, These
preservatives slowly increase rate of hydrolysis with-
out modifying the equilibrium position.
Several papers were published on the analysis of pilo-

carpine and its degradation products by various tech-
niques, e.g. HPLC, LC, 13C NMR, among other method,
which will be discussed in its respective section.
Furthermore, a review on the analysis of pilocarpine

eye drops was published by Ermer (15).
2.7 Spectral properties:
2.71 Ultraviolet Spectrum:
Pilocarpine HC1 in H20 exhibitsa maximum at 215

nm as shown in Figure 4 . Clarke (4a), reported the
UV absorption spectrum of pilocarpine in 0.2 N
H SO t o show a maximum at 215 nm (El%, 1 Cm, 250).
2 4
Ben Bassat and Lavie (16) reported the ultravio-
let spectroscopic properties of pilocarpine and
the effect of quaternization of this drug among
other characteristic changes in the NMR and IR
properties.
2.72 Infrared Spectrum:
The IR spectrum of pilocarpine HC1 in KBr disc is

shown in Figure 5. The major band assignments are
as follows:
f
a,
k
%
.d
a
F i g u r e 5. I n f r a r e d Spectrum of P i l o c a r p i n e ; KBr d i s c .
PILOCARPINE 397
-1
cm
Frequency Assignment
3080, 3020 N-CH quaternary and aromatic

CH szretch.
1770 C = 0 (five membered y-lactone
ring).
1620 C = C aromatic stretch.
0th r finger print bands are found at 1180, 1030,

-f
cm and that is in agreement with previously
reported data ( k , 16a, 16b).
2.73 Proton Magnetic Resonance Spectrum
A typical PMR spectrum of pilocarpine HC1 is shown

in Figure 6. The spectrum was determined on Varia-
T60 A spectrometer. The sample was dissolved in
D20 with 3-[ trimethylsilyll-propionic acid sodium
salt as an internal standard.
The following structural assignments have been

made for Figure 6.
Chemical shift (6) Assignment
Triplet at 1.1 CH CH2-

-3
Quartet at 1.7 CH$H2 -
Complex multiplets centered
at 3.00 -CH2 at C8 and CHat C4
Singlet at 3.9 'N+-CH -3

4
Complex multiplet between CH2 at C5 andCHatC3
4 and 4.6
Singlet at 7.4 aromatic proton at C'4

Singlet at 9.4 aromatic proton at C'
2
The data is in agreement with the reported Spectrum(l7).
The characteristic changes in the NMR spectrosco-
pic properties of pilocarpine and its quaternary
derivatives were studied by Ben Bassat and Lavie
(16a) and substantiated by Aboul-Enein (18).
F i g u r e 6 . PMR Spectrum of Pilocarpine in D20
PILOCARPINE 399
2.74 I3C-NMR Spectrum

Simeral and Maciel (19a) reported the 13C NMR shifts
for several cholinergic neural transmission agents
among which was pilocarpine and they reported that
these shifts did not correlate with the potencies
of the cholinergic activity. A typical I3C NMR
of pilocarpine (19b) is shown in Figure 7.
Neville --
et a1 (20) studied the stereoselective em-
pimerization of pilocarpine to isopilocarpine as
well as the hydrolysis of the parent compound(s) t o
form the o en chain pilocarpinate (isopilocarpi-
nate) by NMR spectroscopy. The mechanism for
the empimerization of pilocarpine and the non-
epimerization of isopilocarpine was discussed and
rationalized. The basis for the assay of the de-
gradation products for aqueous pilocarpine solution
was based on the use of the differences for the CH2
group adjacent to the oxygen lactone ether.
The I3C NMR chemical shift differences between

pilocarpine and isopilocarpine as well as the
effect of quaternization on these alkaloids have
been recently published (21).
2.75 Mass Spectrum
The mass spectrum of pilocarpine hydrochloride

obtained by electron ionization (EI) is shown in
Figure 8.
F i g u r e 7. Carbon-13, NMR Spectrum of P i l o c a r p i n e Hydrochloride i n Water
PILOCARPINE 401
Figure 9. Standard Curve of Absorbance Ratios
100.0
50.0
.-
Figure 8. Mass Spectrum of P i l o c a r p i n e

Hydrochloride (EI) By D i r e c t I n s e r t i o n
402 ABDULLAH A. AL-BADR AND HASSAN Y.ABOUL-ENEIN
The mass spectrum was obtained from Finnigan 3100

model by direct insertion. The molecular ion, M'+'
is at m/e 208 and the base peak is at m/e 95. The
proposed fragementation pattern is presented
in Table 1. Other peaks are shown at mfe 163,
149, 133, 121 and 109.
Table 1
ml e Ion
208 M+
193 M-15 (a)
179 M-29 (b)
95 (C)
3. Synthesis
Several syntheses of pilocarpine have been reported in the
literature. The total synthesis of pilocarpine was first
reported by Preobrashenski et a1 (22-31).
The synthesis can be divided into four steps as follows:
I) The synthesis of (+)-pilopic acid 2.

11) Chain homologation of pilopic acid to (+)-homopilopic
acid 8.
111) The cznversion of (+)-homopilopic acid to (+)-
pilocarpidine 15.
IV) The conversionaf (+)-pilocarpidine - 15 to (+) pilo-
carpine 16 as outlined in scheme 2.
Dey has introduced improvements to the synthesis of pilo-

carpine (32) and was first to resolve racemic pilocarpine
through its tartrate. Scheme 3 summarises Dey's route
in which racemic homopilopic acid was an intermediate for
the synthesis of pilocarpine.
Chumachenko et a1 (33, 34, 35) reported a synthesis start-

ing from furfural as shown inscheme 4 .
Few years later, DeGraw ( 3 6 , 37) essentially reported the
synthesis of racemic homopilopic acid and racemic pilocar-
pine without referring to the work of Chumachenko which was
essentially similar to it as shown in scheme 5.
PILOCARPINE 403
404 ABDULLAH A . AL-BADR AND HASSAN Y . ABOUL-ENEIN
Scheme 2. Preobrashenski et a1 Synthesis (22-31).

PILOCARPINE 405
COOEt
I 1 ) D i e t h y l malonate
COOEt
CH
Michael a d d i t i o n
-
I - 1
-
II b Et C CHCOOEt
CH 2)Ethylation. I I
EtOOC CH20Et
I
CH20Et
X
0
0
4-Ethoxy C r o t o n a t e . V
zN
t 2)
1) Hydrolysis
Decarboxylat i o n . *
HOO 0
8
-
Cis-conf i g u r a t i o n used. Racemic
mixture.
Scheme 3 . Dey's S y n t h e s i s of racemic homopilopic

acid (32).
O C H O I____t EtO
-
17 18
-
fOOEt H
4 CH2COOEt
I c
I--, COOEt
Et
19
COOEt
1 ) Catalytic hydrogena-
CH
1
- co *HZ '@ 2) Hydrdysis
Racemic 8
t ion
0
I
I 21
hyd z ly s i s
(+>
J- g
Prebradhenski et a1
route.
(+>
J
- Pilocarpine.
I
-
22
23
- 16
-
Scheme 4 . Chumachenko et a1 route f o r the Synthesis
of pilocarpine (33-35).
PILOCARPINE 407
HBr 'fi
0
Goo%
1) Catalytic
kydrogenatiof
of mellglester
Racemic
g+
0 2) Hydrolysis.
C = H g
-
R = CH3 25
CH2COC1
Resolution
*(+I -8 Sod. salt of
'.
ditert-butyl 3
acetamido-
U
malonat e.
COOC (CH3)
I
E H C H 2 -CO - C - NHCOCH3
* 0
I
COOC (CH3) 1) Hydrolys
4
2) Decarboxy-
lat ion.
26
-
Preobrashenski route
)L (+) - pilocarpine.
Scheme 5. De Graw Synthesis of pilocarpine (36-37).

All the above synthesps have, in common, the following

points :
1) They are multiple step synthesis starting with a chiral

compound which gives rise to a low overall yield which
render the methods difficult for the commercial synthe-
sis of pilocarpine in large amounts.
2) The first stage includes the synthesis of the lactone

part of the molecule followed by the imidazole ring.
A different approach was developed by Link and Bernauer (38)

in which the imidazole nucleus was used as the starting
materials (scheme 6). However, the overall yield is very
low.
Noordam et al, recently reported novel synthesis of (+) pilo-

carpine in high yield (39),(40).
The method reported by these authors are efficient and much

less laborious than the previously mentioned methods. It
depends on stereoseletive synthesis starting from a commer-
cially available L-histidine amino acid as outlined in
scheme 7.
The strategy of this synthesis depends on the conversion of

the amino group of L-histidine into the bromo-derivative
with conversion of configuration. The bromo-derivative
27 was reacted with dibenzyl ethyl malonate to give 2-
mzhyl-2 ,3-dibenzyl-R-l(l-methyl-5-imidazolyl) -2,3,3 pen-
tane tricarboxylate 28 again with the conversion of con-
figuration rendering the synthesis stereoselective.
Compound 28 was subjected to catalytic hydrogenation and

hydrogenolysis followed by lactonization to yield a mixture
o f (+)-pilocarpine and (+)-isopilocarpine which was separa-
ted by fractional crystallization as nitrates from a mix-
ture. The overall yield of the method is high.
The full account of the structure activitv relationship of

pilocarpine and analogs have been recently published (41).
4. Biosynthesis
Biot (42) assumed that, pilocarpine might arise from 2-0x0-
3-(5-imidazolyl) propanol (its phosphate is a precursor in
the biosynthesis of histidine) and either two molecules of
acetic acid or four carbon compound such as butyric acid
PILOCARPINE 409
R EtOOC, H
Diethyl succinate
KOC (CH3)3
Stobbe condensation.
COO
- K+
R = COO CH
3
= CHO
1. Reduction
2 . Hydrolysis Catalytic
3. Hydrogenation
Resolved and (+)-used
0
(*)-Pilosfnine
H H
CH OC
1) Reduction
2 ) Acetylation 3
3) Elimination
(+) - P i l o c a r p i n e and
C a t a l y t i c (+)-Isopilocarpine
0
Reduct ion' (97 : 3 )
N
Scheme 6. Link and Brenauer S y n t h e s i s of p i l o c a r p i n e

and i s o p i l o c a r p i n e ( 3 8 ) .
__
H
N2H- t: AH Walden inversion
I n >
COOH through several steps
L. Histidine
0CH3 Walden inversion

Br-
0
alkylation with dibenzylt
ethyl malonate.
COOH3
27
-
H
CgHgCH2OOC I
I
Et- c-
I
CgHgCH2OOC
(+) -Pilocarpine
1) Reduction (+) -1sopilocarpine
w
2) Hydrogeration ( 4 5 : 55)
3) Lactonigation.
Scheme 7. Noordam et a1 Synthesis (39-40).

PILOCARPINE 411
o r acetoacetic acid is incorporated to forn; the alkaloid as

shown in scheme 8.
Pilosine, the L-hydroxy benzyl analoge of pilocarpine is a
naturally occuring imidazole alkaloid. This leads to the
assumption that threonine might serve as a 4-carbon unit
( 4 3 ) . The condensation of 2-0x0-butyric acid (a metabo-
lite of threonine)with urocanic acid (metabolite of histi-
dine) result in the biosynthesis of pilocarpine as illus-
trated in scheme 9.
Nunes (44) studied the biosynthesis of pilocarpine by

feeding Pilocarpus pennatifolius with several specifically
labelled potential precursor. Only L-methionine (S-methyl
14C) showed significant incorporation in the methyl group
attached to the imidazole nucleus. He assumed that pilo-
carpidine is biosynthesized in the roots and then transpor-
ted to the leaves where nitrogen methylation occurs.
5. Metabolism
The corneal metabolism of pilocarpine in pigmented rabbits

have been studied by Lee - a1 (45). It was found that
et -
extensive metabolism of pilocarpine occur in the cornea
of pigmented rabbit and the major metabolite, is pilocar-
pic acid. This finding contrasts with studies done in the
albino rabbits where low level of pilocarpic acid was
observed.
The author also studied the low occular bioavailability
( 4 6 ) of topically applied pilocarpine which was mainly
attributed to preconeal drug loss in conjunction to the
resistance to corneal penetration. Among other factors
e.g., drainage, lacrimation, vasodilation which influences
drug loss at the absorption site. Mathematical model were
formulated f o r these findings.
Friedman and Patton (47) showed that, after adminis-
tration of pilocarpine; 25 ml of 1 x lO-*M into the eye of
the rabbits of different ages, pilocarpine concentration was
higher in the aq. humor of 20 days old rabbits than the 6 0
days old rabbit. This shows that, less pilocarpine is
needed to achieve the same effect in younger rabbits.
Methods of Analysis
6. -
--
6.1 Titrimetric methods
CH3-
I/
c
\
7 2 + Pathway 1
'i
HO-
HO
H
1
Scheme 8. Biosynthesis of pilocarpine according to

Biot ( 4 2 ) .
CH3-CHZ\
c = o +
HC
o = cI
\
=\ CNN> Pathway 2 1
/ OH
HO- C
CH3CH2\
t-+"O--"
' I
"0
c=c --.CH
CH20H CN6 = NH
pilocarpine
Scheme 9. Biosynthesis of pilocarpine according to

Brochmann-Hansen and Nunes ( 4 3 ) .
PILOCARPINE 413
6.11 Non-Aqueous Titration:

a) Pilocarpine hydrochloride.
This salt is assayed in USP XX (2) and B.P.
1980 (48) by non-aqueous titration with 0.1N
perchloric acid using crystal violet in glacial
acetic acid containing mercuric acetate.
b) Pilocarpine iiitrate:
This salt is assyed in USP XX (2) and B.P.
1980 (48) by non aqueous titration with 0.1N
perchloric acid. The end point is determined
potentiometrically.
6.12 Conductimetric titration:
Jarzebinski and Suchocki (49) reported a method
for determination of pilocarpine hydrochloride
among other hydrochlorides of several alkaloids
by direct-conductimetric titration with 0.01N or
0.005N NaOH in aqueous ethanol medium.
Another conductimetric method was reported (50)

for determination of pilocarpine hydrochloride in
eye drops, whereby the sample was made alkaline
and extracted with chloroform. The combined ex-
tracts were evaporated, the residue dissolved in
aqueous ethanol and titrated conductimetrically
with O.01N HCl.
6.13 Ion-Selective Electrode
Crytur valenomycin ion-selective electrode was
used (51) for the end point determination in the
precipitation titration of pilocarpine with sodium
tetraphenyl borate.
6.2 Polarographic Analysis
Clark --
et a1 (52) determined pilocarpine and some other
related imidazole derivatives using the polarographic
behaviour of the copper complexes of these compounds.
In one molar citric acid, one molar sodium citrate -
one molar copper sulphate medium at 25'C. The E of
I/ 2
the Cu++ - Cu+ citrate system is controlled by the
concentration of pilocarpine o r imidazole compounds.

Thus pilocarpine and related imidazoles are determined
by differential pulse polarography with dropping mer-
cury electrode. The E of the first wave was used for
1/2
the calculation. When the hanging mercury electrode
was used, the peak height was proportional to piloc-
arpine concentration from 0.4 to 1.6 mM.
6.3 Gravimetric Analysis
Pilocarpine among various alkaloids form insoluble meta-
llic complexes with several inorganic salts such as
HgC12, SbC13, Hg12 in KI, BiI3' Sb13, MnC12 in KI, U 0 2
(N03)2 and the [CO (CNS)4]-- group. Rejnecke's salt
picric acid and flavianic acid also
precipitate many alkaloids. These ppts can be
used for the gravemetric determination of pilocarpine
specially the precipitate with ilavianic acid. (53).
Another method was reported by Ganescu et a1 (54) based
on the formation of chromium-thiocyanate complex, with
pilocarpine, whkch has the formula PHCr (SCN)4- (A)2.
where P = Pilocarpine, A = Amine preferably aniline o r
morpholine. This complex can be determined gravemetri-
cally, spectrophotometrically o r by oxidation with
KMn04.
6.4 Polarimetric Analysis
The content of pilocarpine and its inactive degradation

products, pilocarpic acid, can be determined by a
polarimetric method (55), as follows:
The solution was made alkaline with aqueous ammonia,
extracted with chloroform (in which pilocarpic acid
interferes in the spectrophotometric procedure and is
insoluble). The solution was diluted with chloroform
and the specific rotation was measured. The content of
pilocarpine can be determined from calibration graph.
6.5 Phase-Solubility Analysis
-
Pilocarpine among other substances can be determined by
the phase-solubility analysis (56), employing solubility-
product relationship technique. Known and increasing
weights of the samples and of picric acid are shaken
together for 48 hours with fixed volume of acetate
PILOCARPINE 415
buffer solution of pH 3.9 in a series of stoppered

flasks. The picrate ion concentration is determined
in each supernatant liquid by measurement of the ex-
tinction at 357 nm. The concentration of the principal
component in the sample can be calculated whether o r
not the impurity forms picrate o r is insoluble in the
buffer solution. The accuracy is comparable with that
of conventional phase-solubility analysis.
6.6 Fluorescence Analysis
Pilocarpine among several drugs react with 9-bromo-

methylacridine in acetonitrile media (57) to give
quarternary ammonium salts which on subsequent photo-
lysis yield fluorescent products. The amine concen-
tration-fluorescence correlation is linear and can be
used for the assay of various tertiary amines (includ-
ing pilocarpine) in aqueous solutions and biological
fluids, for example, human blood.
6.7 Spectrophotometric Analysis

6.71 Infra-red Spectrophotometric Analysis
Pilocarpine, free base o r liberated from official

salts,hydrochloride and nitrate,has been deter-
mined by infra-red spectrometry (58). The calcu-
lation of pilocarpine content was measured from
extinction at 9 um with correction for the base
line from 8.9 to 9.3 pm and by comparison with
correlated extinction of a similarly treated
standard solution of pilocarpine. The method is
specific and the recoveries were 99.0 to 102%.
Another infra-redanalysis of pilocarpine among
other drugs was also reported (59).
Ryan (60) reported a sensitive and convenient

method of infra-red measurement of pilocarpine-
isopilocarpine isomerisation. Pilocarpine base
is dissolved directly o r extracted from salts in
basic buffer with chloroform. After evaporatioli,
the residue dissolved in CS2 and examined by
IR spectroscopy at the 3 mm cell. The ratio of
extinction at 1100 cm-l and 1082 cm-l i s used to
calculate the isopilocarpine content (the trans,
less pharmacologically active isomer) from a
standard curve (Figure9). Drug formulation excepi-

ents do not interfere (they are insoluble in CS2).
However, non-polar substances that cannot be sepa-
rated by prior extraction do interfere. The co-
efficient of variation for a 50 : 50 isomer mix-
ture was f 1.83% (seven determination).
6.72 Ultraviolet Spectrometric Analysis
Belikov et a1 ( 6 1 ) reported a spectrophotometric

analysis for pilocarpine and other imidazole deri-
vativesand the optimum pH range required for their
determination. Pilocarpine was measured at 230 nm
at an optimum pH range of 4.0 to 4 . 1 9 .
Another method was published ( 6 2 ) based on heating

pilocarpine hydrochloride with 10% malonic acid in
acetic anhydride at 8OoC for 15 minutes. After
dilution with ethanol, the extinction is measured
at 333 nm. The limit of dilution was reported to
be 10 to 30 ng/ml. Dosage forms require prelimi-
nary extraction of the base. For pilocarpine
hydrochloride, the coefficient of variation was
2 t o 6.5%.
6.73 Spectrofluorimetric Analysis
Pilocarpine among some alkaloids containing a ter-

tiary amino groups ( 6 3 ) were determined spectro-
fluorometrically by converting the non-fluorescent
alkaloid into fluorescent compound. This was
achieved by base-catalysed condensation with the
mixed anhydride of organic acid. A solution con-
taining 10% malonic acid in acetic anhydride is
heated with alkaloid at 8OoC for 1 5 minutes. The
solution was then diluted to a specific volume with
ethanol. A suitable aliquate ( 5 0 111) was diluted
to 10-25 ml of ethanol and set aside for 10 minu-
tes. The fluorescence produced was measured for
pilocarpine HC1 at 450 nm (Extinction at 395 nm).
The limit of determination of this method was
reported to be 2.8 Pg/ml.
6 . 7 4 Colorimetric Analysis
Several colorimetric methods have been published

on the determination of pilocarpine and its salts
in various pharmaceutical preparations. The com-
pilations of the methods are summarized in
Table 2 .
Table 2
Reagent used to produce color. Wavelength or Comment. Ref.

color produced.
1% NaOH + 1% sod. Nitroprusside, Calibration curve should

addition of 1% HC1 in water after - be established. (64)
3 minutes.
Aminone at pH 4 . 213 orange complex at Applied for eye drops. (65)
pH 4 .
10% acetic acid, 5% P o t chromate + 560 nm. Standard curve is (66)
3% aq. H202. Shake for 20 minutes. required.
Extract the color produced with Applied to eye ointment.
chloroform.
Sulphuric acid + bismuth Iodate. 1:l complex Pilocarpine The color is stable for
B i 1 4 soluble in acetone 30 minutes. Beer's (67)
490 nm. law is obeyed for 10-60
dml.
0.75 M Hydroxylamine +
2.4 M 515 nm. - (68)
sod. hydroxide, heat at 40' for
5 minutes. Mix with ferric
chlorate.
1 M hydroxylamine HC1 + 7% 480 nm. Remove interfering (69)
sod. sulphate + 3.5M NaOH. Set substances if necessary. (70)
aside f o r 10 min. Add 5.25 M
HC1 + 0.3 M ferric chloride in
0.1 M HC1. Set aside for 10 min. contd.......
Wavelength or Comment. Ref.
Reagent used to produce color. color produced.
Acid form of Rosebengal, the color 550 rim. Excess saturated aq. lithium carbo- (71)
extracted with chloroform. nate solution i s added to affect
displacement o f pilocarpine from
thesolution during the color forma-
tion and extraction of the complex.
Methyl orange, extracting the 420 nm. The assay is unaffected by the anti- ( 7 2 )
complex at pH 4.5 with chloroform. oxidants, preservatives of non-
alkaloid component. The method is
applied to pilocarpine, physostigmine
P
r
and mixture of the two alkaloids in
m eye drops and ointments.
Citrate buffer pH 6 . 8 t 0 . 3 % sodium 520 nm. Calibration curve is required, the ( 7 3 )
aqua or aminopentacyanoferrate coefficient of variation 1.8%.
solution, heat at 45OC for 3 The color intensity is maximum at
hours, cool. pH 5-7 and borate buffer solution
can be used but not acetate OK
phosphate buffer solutions.
0.6 M NaOH+O.lM sod. pentacyano- 520 nm. Coeficient variation is 7.3%. (74)
nitrosylferrate, set aside for one Boric acid present in ophthalmic
hour in the dark, add 0.6M acetic solution does not interfere.
acid (the pH should be about 7 ) .
Set aside for 50 minutes.
contd.......
Wavelength or Ref.
Reagent used to produce color. Comment.
color produced.
The formaldehyde produced by heat- 545 nm. The method is recommended where (75)
ing pilocarpine with benzyl per- ingredients interfere in the
oxide is determined colorimetri- hydroxylamine HC1 method.
cally with chromotropic acid. Eserine must be separated by
TLC prior to the analysis.
3% Ammonium reineckate solution Pink violet comp- The composition of the complex (76)
in acetone. lex measured at has been verified.
533 nm.
Can be applied to several (77)
5
\o
Molybdophosphoric acid. The con- 750 nm. organic bases mainly
tent of Movl in the ppt formed
alkaloids.
is determined colorimetrically
after dissolution and reduction
to molybdenium blue and excess
reagent is masked with tartrate.
2,4-dinitrophenyl acetate method. 357 nm at The base should be extracted with (78)
This depends on catalytic action pH 7.45. CHC13 (to exclude pilocarpic acid
of the imidazole portion of pilo- which interfere with the determi-
carpine on the hydrolysis 2,4- nation). The presence of NaCl
dinitrophenyl acetate. increases the rate of hydrolysis
of the reagent, peroxide, hydro-
xylamine and other nucleophiles
do interfere with the assay.
contd.....
Wavelength or Comment. Ref.
Reagent used to produce color. color produced.
Reaction should be standardized

bv the use of a blank.
Catalytic determination by 2,4- 366 nm at pH 7.4 The velocity of the reaction is

dinitrophenyl acetate. directly proportional to pilocar-
pine concentration. Calibration (79)
graph should be established. The
authors studied the fixed time
and the fixed concentration to
establish equations for construct-
ing the calibration graph in each
case.
The differential acid dye 420 nm. Applied to pilocarpine and other
method. drugs. Blank solution is
Pot. acid phthalate buffer, needed. The author claims the (80)
pH 4 . 2 , 0.05% methyl orange use of this method to assay
solution in aqueous ethanol. pilocarpine in biological fluids.
The color produced extrac-
ted with chloroform.
PILOCARPINE 42 1
Clarke (4a) described a solvent system used for

the paper chromatography of pilocarpine consisting
of citric acid : water ; n-butanol (4.8 gm:130 m l :
8.70 m l ) . The drug can bedetected by several
agents such as bromocresol green spray or iodo-
platinate spray.
Sun (80) reported a method of separation of seve-

ral alkaloids including pilocarpine using the
Whattman paper no. F, moistened with calcium acid
phosphate solution (0.375 M), eluting the paper
with either sec. butanol or propanol : water (3:l)
using the descending technique. Rfvalue wasreported.
Niezgodzki --
et a1 (81) described an analytical
method for the determination of several alkaloids
including pilocarpine in injections by means of
cationic paper (0.3 inch thick) and measured the
spot areas planimetrically. The method can
detect 0.2 mg of pilocarpine HC1 with i 2.06%
standard deviation.
Several methods have been published for the detec-

tion and semiquantitative determination of pilo-
carpine using thin-layer chromatography which are
summarized in Table 3.
et -
Bayne - a1 (86) and Dziedzic et a1 (87) reported
similar methods for the determination of pilocar-
pine in biological fluids (aq. humour of rabbit
cornea) in sub u g quantities. Both methods had
derivatized pilocarpine (after extraction from the
biological fluid) with heptafluorobutric anhydride.
The derivatized pilocarpine was then introduced
to the gas chromatogram under the following con-
ditions:-
a) Silanized glass column (6ft x 2mm) containing
3% OV-17 Chromosob W (100-120 mesh) at 190°,
with N2 as gas carrier (25 ml/min) and 3H-elec-
tron-capture detector (86).
Table 3
Solvent system. Absorbant. Localizing agent. Ref.
Strong ammonia solution : Silica Gel G. Acidified iodoplatinate spray. (4a)

methanol(1.5 : 100)
Should be changed after
two hours.
Chloroform : acetone : diethyl- Kiesel Gel Dragendorff reagent. (82)

amine,(5 : 4 : 1.) GF 254
Chloroform : acetone : Silica gel Iodoplatinate spray or

diethylamine(5:5 :1)or 60. Dragendorff reagent.
Cyc1ohexane:diethyl-
amine,(g : 1.)
Butanol : anhydrous AcOH: Hydrolysed cotton W light.

dater,(4 : 1 : 5.) wool prepared
specially for this
purpose + CaS04
Butanol : acetic acid : Silufvl Iodine or Dragendorff reagent. (85)

water(4 : 1 : 5.) W Sheets.
Table 4
Column Mobile phase. Flow Comment. Ref.

Detector
pressure
Aminex A-1 0.2 M-Tromethamine 200 p s i UV a t S e n s i t i v i t y 0 . 1 mg of i s o - (90)

Cation exchange b u f f e r s o l u t i o n 5% 217 nm. pilocarpine i n the pressure
resin. s o l u t i o n of i s o p r o - of 100 mg of p i l o c a r p i n e o r
panol i n 0.2M T r i s - v i c e v e r s a , no i n t e r f e r e n c e
b u f f e r o f pH 9 by p i l o c a r p i c a c i d .
(0.4 ml/min).
UBondapak c18 Borate b u f f e r 1 ml/ W at The method w a s a p p l i e d f o r (91)

and VBondapak CN s o l u t i o n pH 9.2: min . 254 nm. ophthalmic p r e p a r a t i o n .
!2 THF ( 7 : 3 ) .
LiChrosorb wc18 Water : methanol 1.5 ml/ Differen- P i l o c a r p i n e and i t s degrada-

(10 pm). (97:3) c o n t a i n i n g min. tial ref- t i o n p r o d u c t s can b e separa- (92)
5% KH2P04( pH 2 - 5 ) racto- t e d i n about 30 min. w i t h
meter. d e t e c t i o n l i m i t of 6.6 pg.
L i ChrosorbRPCp, Water : methanol 1.5 m l / W at D e t e c t i o n l i m i t 300 ng (93)

(10 Um). (97 :3) c o n t a i n i n g min . 216 nm.
5% M,PO,, pH 2.5
5 pM Silica Hexane-2% ammonium 2 ml/ UV a t The method can b e a p p l i e d f o r (94)

hydroxide i n min . 220 nm. r o u t i n e a n a l y s i s of p i l o c a r -
isopropanol p i n e and i s o p i l o c a r p i n e and
c70 :30 1) t h e i r degradation products
i n t h e p r e s e n c e of each o t h e r
i n ophthalmic s o l u t i o n .
contd.....
Column. Mobile Phase. Flow
Pressure. Detector. Comment. Ref.
VBondapack c18 40% methanol with 0.005 1 ml/min. W 235 nm. Applied for analysis of (95)
M heptane sulfonic pilocarpine physostig-
acid solution pH 3.6 mine, rubreserine, de-
gradation products and
preservatives in ophth-
almic solution detection
limit 0.003 ug.
VBondapack c18 1M mole sodium octane- 1.6 m l / W 254 nm. Detection limit less than (96)
1-sulphonate in min . 3.8 ng in the presence of
ethanol: water, (4:l). isopilocarpine.
Applied for analysis in
aq. humour.
Derivatization by quater-
ization with a-bromo-4-nitro-
toluene is required for
analysis.
PILOCARPINE 425
b) A column (1.2 m x 4 mm) containing 2% OV-105 on

Gas-Chrom Q (80-100mesh) at 200° with N2 as
gas carriers (70 ml/min) and 3H-electron-cap-
ture detector (89).
The following systmes have been employed for the

analysis of pilocarpine (4a); (a) 5 feet x 4 mm
internal diameter glass column containing 2.5%
SE-30 on 80-100 mesh Chromosob WAW H MDS, column
temperature 2250, carrier gas, nitrogen (50 ml/
min); detector, flame ionization detector. Rt
value is 0.36 relative to codeine; @ ) 5 feet x
118 inch internal diameter stainless steel column
containing 5% SE-30 on 60-80 mesh Chromosob W AW,
column temperature 230°, carrier gas, nitrogen
(30.7 ml/min), detector, flame ionisation detector,
Rt value is 0.73 relative to codeine.
6.84 Ion-Exchange Chromatog-

Jarzebinski (88) used column chromatography for
the quantitative analysis of several alkaloids
including pilocarpine. The solution of the alka-
loid passed through a column, 14 cm x 1 cm, packed
with Wofatit KPS (Zinc+2 form). The zinc+2 in the
eluent was determined with 5 mM EDTA in the pre-
sence of Erichrome black T in a buffer medium at
pH 10.4.
Ion-exchange resin has been used for the purifi-

cation of pilocarpine (89).
6.85 High-pressure Liquid Chromatography
Several procedures have been published by several

authors for the analysis of pilocarpine, isopilo-
carpine and their degradation products by HPLC
technique, both in pharmaceutical dosage forms
and biological fluids.Table 4 , summarise the
HPLC systems used for pilocarpine analysis.
6.9 13C NMR Quantitative Analysis
Neville et a1 (97) have reported a method for analysis

of pilocarpine and its degradation products in ophth-
almic formulation using 1% NMR spectroscopy. The
method depends on the determination of the 1% NMR
of the freeze-dried preparation in D20 to give a
solution of about 20% concentration of pilocarpine.

The spectrum was determined at 30° by the Fourier
Transform technique using 2M tetramethyl ammonium
bromide solution in D20 as external standard. The
concentration of pilocarpine, degradation products and
isopilocarpine was calculated from the internal c8 peak
in the total alkaloid concentration from the integral
C14 peak. The accuracy is stated to be within f 5%.
6.10Thermofractographic Analysis
Stahl and Schmitt (98) reported a method of analysis

for several alkaloids including pilocarpine, using the
thermofractogram. The samples 5-10 mg was submitted
to 9 temperature gradient in the range of 50-450° in
an oven for 2 minutes; and the use of 50 mg of mole-
cular seive 4A containing 20% water as propellant.
Pilocarpine was transferred without decomposition to
the TLC plate under conditions of carrier-gas dis-
tillation for further identification.
PILOCARPINE 427
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Acknowledgement
The authors wish to thank Mr. Altaf Hussain Naqvi for

typing the manuscript and School of Pharmacy, University of
Mississippi, University, Mississippi, U.S.A. for determining
the mass spectrum of pilocarpine.
PYRAZINAMIDE
Ernst Felder and Davide Pitre
1. Foreword, History and Therapeutic Category 434

2. Description 434
2.2 Formula, Molecular Weight 435
2.3 Appearance, Color, Odor and Taste 435
3.1 Spectra 435
3.2 Physical Properties of Solid State 442
3.3 Solubility 445
4. Metal Complex Salts 441
5. Synthesis and Manufacturing 448
6. Stability 450
7.1 Metabolism 450
7.2 Pharmacokinetics 45 1
7.3 Protein Binding 452
7.4 Acute Toxicity 452
8. MicrobiologicalAssay 452
8.1 Biological Method for Pyrazinamide Determination 452
9.1 Elemental 453
9.2 Identification Tests 453
9.3 Official Methods 453
9.4 Nonaqueous Titrimetric Analysis 453
9.5 Colorimetry 453
9.6 Ultraviolet SpectrophotometricMethod 454
9.7 ComplexometricAnalysis 454
9.8 Chromatography 454
9.9 Counter Current Distribution 456
10. Determination of Pyrazinamide in Body Fluids and Tissues 457
References 458
ANALYTICAL PROFILES OF DRUG SUBSTANCES 433 Copyright by the American Pharmaceutical Associalinn.
VOLUME 12 ISBN 0-12-260812-7
434 ERNST FELDER AND DAVIDE PITRE
Foreword, History and Therapeutic Category
Pyrazinamide is together with Isoniazid and Rifampicin a key

drug in the short-course chemotherapy o f pulmonary tuberculosis and is
included in several multidrug regimens recommended by the IUAT and WHO
(192).
In 1936 flalmer and Walter (3) first described Pyrazinamide together
with N-a1 kylsubstituted Pyrazinamides that were reported to be useful
as analeptics.
Only in 1952 Kuschner et a1.(4) and Malone (5), starting from the ob-
servation that Nicotinamide showed some antitubercular activity, re-
cognized the antitubercular activity of the isosteric Pyrazinamide in
experimental mouse tuberculosis. Subsequently it was found to be clini-
cally active in humans (Minutes of the llthe Conf. on Chemoth. o f Tu-
berc.). The drug was Introduced in primary chemotherapy of tuberculosis
in the early 1950's.
Whereas toxicity was a major problem with high daily dosages given for
long periods this was not the case with moderate daily dosage.
In the last decade the introduction o f short-course chemotherapy of tu-

berculosis, based on the experimental work in mice (6,7,8) has fully re-
valuated Pyrazinamide. Pyrazinamide containing regimens produce a high
rate culture negativation at two months and a low incidence of bacterio-
logical relapses after stopping chemotherapy (9,10,11,12,13).
2. Description
2.1 Nomenclature
2.1.1 Chemical names
Pyrazinecarboxamide; Pyrazine-2-carboxamide
CAS: 98-96-4
2.1.2 Generic Name
Pyrazi namide
2.1.3 Trade names
Aldinamid; Zinamid (Merck, Sharp & Dohme)

Eprazin (Krugman)
Piraldina (Bracco)
Pirilene (Lepetit, France)
Pyrafat (Saarstickstoff-Falot)
Tebrazid (Continental Pharma)
Tisamid (Orion)
PYRAZINAMIDE 435
2.2 Formula, Molecular weight
Mol. w t . = 123.11
C5H5N30
Wiswesser l i n e notation: T6N DNJ BVZ
2.3 Appearence, color, odor and t a s t e
White o r almost white, c r y s t a l l i n e powder; odorless o r a l -

most odorless (14) o f s l i g h t l y b i t t e r t a s t e (15,16).
3.1 Spectra
3.1.1 I n f r a r e d Spectrum
The i n f r a r e d spectrum o f Pyrazinamide Bracco Reference Stan-

dard i s shown i n f i g . 1. The spectrum was obtained as a 0.3% disper-
sion o f Pyrazinamide i n KBr w i t h a mod. 257 Perkin-Elmer spectropho-
tometer. The wave numbers and the assignments o f the p r i n c i p a l ab-
sorption bands o f the spectrum f i g . 1 are given i n t a b l e I. (19)
Table I
Wave number (cm-’) assignment
3425, 3290, 3160 J NH

1716 UC=O (Amide I)
1614 sNH+VCN (Amide 11)
1585, 1528 vC=C and YC=N (py r i n g )
1382 v i b r a t i o n o f py ring,
1183-782 6CH out o f plane, NH2 rock
The r e l a t i o n between nitrogen-hydrogen s t r e t c h i n g frequencies and

- 7
N... 0 distances o f c r y s t a l s containing NH... 0 hydrogen- ands was
-
given (20). Usefulness o f the v i b r a t i o n a t 1000 1050 an i n assi-
gnment o f the p o s i t i o n o f mono and d i substituted pyrazines was r e p o r t -
t e d (21). The differences i n spectral features o f d . 6 , v a n d b f o r m s
o f pyrazinamide was correlated w i t h differences i n c r y s t a l structures
(17 ,la).
iot
W
Fig. 1 - Infrared Spectrum of Pyrazinamide ( K B r pellet).

PYRAZINAMIDE 437
3.1.2 Nuclear Magnetic Resonance Spectra
3.1.2.1 'H NMR

1
Several authors (22,23,24,25,26) r e p o r t about the H-NMR spectra
o f pyrazi ne d e r i v a t i v e s.
I n f i g . 2 t h e proton NMR spectrum o f Pyrazinamide, obtained i n DMSD so-
l u t i o n w i t h a Varian XLlOO spectrometer operating a t 100 MHz, i s given
(27)*
The i n t e r p r e t a t i o n o f the spectrum i s given i n t a b l e 2.
Table 2
Chemical s h i f t Mu1t i p 1 i c i t y Intensity Assignment

bH (PPm) TMS
9.21 doublet 1H H-3 (53.5 1.5Hz)
8.85 doublet 1H H-6 ( 5 5 . 6 ~2.4Hz)
8.71 quartet 1H H-5 (55.6 * 2.4Hz)
(53.5 C 1.5Hz)
8.25 and 7.88 singlets, broad 2H -CONH2
3.4 - water
2.5 - DMSO
0.00 - TMS
The signals a t 8.25 and 7.88 ppm disappear a f t e r exchange w i t h D20.
3.1.2.2 "C-NMR
The 13C-NMR spectrum shown i n f i g . 3 was obtained i n DMSO solu-

t i o n w i t h a Varian XL-100 spectrometer operating a t 25.2 MHz (28).
The i n t e r p r e t a t i o n o f the spectrum i s given i n t a b l e 3.
Table 3
Sc (Ppm) TMS -
Line Intensity Assignment
165.0 1 40 c=o
147.3 2 129 C-6
145.0 3 35 c-2
143.6 4 99 c-3
143.2 5 107 c-5
40 6-12 - DMSO
0.0 13 TMS
The assignments o f t h e three carbons C-3, C-4, C-5 are made on the basis
o f s e l e c t i v e decoupling.
The 13C-NMR spectrum o f Pyrazinamide i s n o t reported i n l i t e r a t u r e .

438
Fig. 3 - 13C-NMR (25.2. MHz) Spectrum of Pyrazinamide in DMSO-d6.
440 ERNST FELDER AND DAVIDE PITRB
3.1.3 Mass spectrum
The mass spectrum o f pyrazinamide was obtained w i t h a s i n g l e

focus spectrometer Hitachi mod. RMU-60 (70eV, 80 uA) w i t h source a t
250". The sample was i n s e r t e d a t 180" w i t h a g l i s s i n l e t system (29).
The mass spectrum i s presented i n f i g . 4
The fragmentation pathways are as follows:
CONH~+
(CH~NO)+
m/e 44( 23%)
T
(C5 H5 N5 O)+
Mt m/e 123 (96%)
I
m/e 80 (100%)
-HCN
m/e 79 (25%)
1
. .
-HCN
m/e 53 (70%) m/e 52 (48%)
The spectrum indicates the presence of metastable ions a t m/e" 35.1

(532) and m/en
80
52 (d)
123
Fig. 4 - Low Resolution Mass Spectrum of Pyrazinamide.
3.1.4 UV Spectra
The UV spectrum o f pyrazinamide was determined in water, me-

thanol, chloroform, dilute acid and dilute alkali, with a Cary mod. 219
spectrophotometer (30).
The UV spectrum of Pyrazinamide in water is presented in fig. 5. The
molar absorptivities and corresponding wavelenghts are given in table 4.
Solvent 2 max E (max) 2 min g min

(nm) (nm)
Water 209 8765 2 22 238.5 2000

(n=5) 269 8036 2 20 294 480
31 0 611 2 10
Methanol 269 7900 235 1600

320 530 296.5 280
Chloroform 322 530 269 7500

298 250
0.1N NaOH 268.5 7950 238.5 1500

310 640 295 540
0.1N HCl 209 8000 238.5 2000

269 8200 295 51 0
310.5 620
These values agree with published data (31,32)
3.2 Physical Properties of the Solid State
3.2.1 Crystal Morphology
Pyrazinamide may occur in four polymorphic forms (33) namely: d -

Pyrazinamide, obtained from ethanol at room temperature (33) or from
hydroalcoholic solution (34).$P razinamide, from ethanol at ;'0 8 Py-
razinamide, by fusion (33) and iPyrazinamide, from pouring a solution
in nitromethane at 80'-140" into tetrachloromethane at room temperatu-
re (33) or from a mixture of hexane-ethyl alcohol (34,35).
Crystallographic data of these forms are listed in table 5.

Fig. 5 - U l t r a v i o l e t Spectrum of Pyrazinamide in Water.
Table 5
Crystal lographic Form Form Form Form

Data 06 6 'd b
188-193 187-189 185-1 89 188'
23.07 10.70 10.84 5.728
6.63 3.73 3.75 5.221
3.73 14.38 7.20 9.948
101 .o 101.7 106.9 97.27
567.5 561.9 280.0 279.58
5
P21/a-C2h Pz1/a Pa pi
4 4 2 2
1.44 1.45 1.46 1.46
1.44 1.45 1.46 1.46
Letter (2) (4) (11 (3)
The c r y s t a l structure ofd-Pyrazinamide (34) shows a planar pyrazine

r i n g w i t h distances o f C-H 1.348 A and C-C = 1.383 A. The carboxa-
mide group forms an angle of about 5' w i t h respect t o the nucleus.
6-Pyrazinamide has a planar structure o f the nucleus w i t h the carbo-

xamide group deviated 2.3'. The product e x i s t s as a dimer by hydro-
gen bonding between the carboxamide groups.
The c r y s t a l l i n e structure o f \I-Pyrazinamide and & -Pyrazinamide has

also been reported (35).
3.2.2 X-Ray Powder 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 o f Pyrazinamide Bracco

(Reference Standard), was determined by a P h i l i p s Powder Diffractometer
w i t h n i c k e l - f i l t e r e d copper r a d i a t i o n (36).
- .......................
Instrumental conditions
TUBE: Cu 50 KV, 30 mA; FILTER Ni;3SLITS lo-0.1-lo; DETECTOR Proportio-
nal t Discriminator; SCALE 1 x 10 cps; SCANNING SPEED 1/4O 20 X min;
PAPER SPEED 300 mm/h; TIME CONSTANT 2 sec.; SPECIMEN HOLDER: Niskanen +
I n t e r n a l Standard.
PYRAZINAMIDE 445
Table 6
I/I, xlOOxx
11.20 80 3.02 3
6.42 17 2.89 3
5.76 53 2.83 4
5.64 82 2.79 4
5.01 100 2.51 16
4.32 7 2.43 3
3.76 9 2.35 4
3.65 13 2.26 15
3.37 40 2.16 2
3.28 12 2.14 3
3.25 65
3.21 30
plus other l i n e s 1
3.07 6
x i n t e r p l a n a r distances = nA / 2 s i n 8 = 1.54051 A
xx Relative i n t e n s i t y based on highest i n t e n s i t y o f 100
3.2.3 M e l t i n g Point
UPS XX (14) and B r i t . Ph. 1980 (16) r e p o r t a melting p o i n t

o f 188-189°C. The e u t e c t i c mp. w i t h Benzamide (33) i s 141'C.
3.2.4 D.T.A.
The d i f f e r e n t i a l thermal analysis curves were recorded on a

M e t t l e r TA 2000 thermal analyzer a t a heating r a t e o f 5°C per minute
(39). The thermogram thus obtained, presented i n f i g . 6, shows an endo-
therm of the s o l i d - s o l i d type a t 146' (AH = 1600 J/mole) and a second
endotherm a t 188.1' ( A H = 25900 J/mole) i n d i c a t i n g melting o f the sub-
stance.
On cooling, an exotherm of c r y s t a l l i z a t i o n i s observed a t 178°C. A se-

cond thermal analysis on the same sample shows a s i n g l e melting endo-
therm a t 188.1"C.
3.3 Solubi 1i t y
Homogeneous s o l u b i l i t y data are l i s t e d i n t a b l e 7 (37).

I
!
I
Fig. 6 - DTA Curve of Pyrazinamide.

PYRAZINAMIDE 447
Table 7
Solubility of Pyrazinamide ir, g/lOO g o f solution
Sol vent T =O°C T = 38OC I

Water 0.64 2.65
Methyl alcohol 0.84 1.63
Ethyl I' 0.29 0.74
n- Propy 1 'I 0.19 0.65
n-Butyl I' 0.14 0.65
Methyl acetate 0.59 1.18
Ethyl 'I 0.31 0.70
n - Propyl I' 0.23
n-Butyl I' 0.14 0.40
Chloroform 0.28
I sobutane 0.0004
3.4 Partition coefficient
The partition coefficients i n n-butanol/water and n-octanol/

water have been determined at 3OOC.
-
n-butanol/water 1:l P = 1.05 5 0.01
-
n-octanol/water 1:0.2 P = 0.330 0.003 (38)
4. Metal Complex Salts
Various complex salts of Pyrazinamide with multivalent metals

have been prepared and are listed in table 8.
Table 8
(C6H5N30)2
M = CO x = 2c1
M = Co X = 26r
M = co x = 21
M Co x = 2C1O4
M = co X = 2N03 H20
M = CO X = 2SCN
M = CO x = so4
M = Cu x = 2C1O4
ERNST FELDER AND DAVIDE PIT&
M = CU X = 2CH3CO0 (47)
M = Hg X = 2N03 (48)
M = Hg X = ZCH3CO0 (47)
M = Fe x = so4 (52)
M = Pd x = 2c1 (49)
M = Pt x = 2c1 (49)
M = Mn X = ZCH3CO0 (47)
M = Ni X = 2CH3CO0 (47)
M = Ni x = 2c1 (40)
M = Ni X = 2Br (40)
M = Ni x = 21 (40)
M = Ni x = 2C1O4 (40)
M = Ni X = 2SCN (40)
M = Zn x = 2c1 (45)
Under different experimental conditions adducts of the type M X4

(C H N30) and M X (C6H5N30) -where M = Ti, Zr, Sn X = C1, Br- (50)
an$ fe (C H N 07 Sd .
H20 (5?) were also obtained. Some physical pro-
perties OF $o$t of ?hese complexes, as visible and IR spectra, or X-ray
structural analysis, magnetic moment, vibrational spectra, etc. were
given (40,41,42,43,44,45,46,47,48,49,50,51).
5. Synthesis and Manufacturing
In 1936 Dalmer and Walter (30) first described Pyrazinamide

together with N-a1 kylsubstituted Pyrazinamides that were reported to be
useful as analeptics. The compounds were prepared by standard methods
for the preparation of acid amides, preferably by reaction of the acid
chloride or of a lower alkylester of the acid with ammonia.
The basic method of Pyrazinamide manufacturing is shown in fi9. 7 (53,54)
Pyrazine-2,3-dicarboxylic acid I1 can be prepared by potassium perman-

ganate oxidation of quinoxaline I (56) and converted to the anhydride
V I (57) with the method of Gabriel and Sonn. Refluxing of the anhydride
V I with methanol yields pyrazine-2,3-dicarboxylic acid monomethyl ester
which is decarboxylated to methylpyrazinoate IV and converted without
previous isolation to Pyrazinamide.
A1 ternatively pyrazine-2.3-dicarboxylic acid (11) is decarboxylated to

pyrazine-2-carboxylic acid (111) esterified with methanol and the so ob-
tained methylpyrarinoate (IV) purified by distillation under reduced
pressure.
N
I
z
0
z
111
w
n
5
u
A
I
0
m
L
V
I
0
0
u
0
0
-T
I1
0
11
CL
I =
u- u
+ ma=
v)
N I N
N T u v
E S Z
O Z N II II-
O I I
u-u-u p : p :
pyrazinoic acid 111, the key intermediate, can also be prepared by con-
densation of 2,3-diaminopropionic acid VII with glyoxal, followed by
oxidation with air (59). Oxidation of methylpyrazine VIIIa with sele-
nious acid in pyridine or of ethylpyrazine VIIIb with potassium perman-
ganate are further alternatives for preparation of I11 (60).
Ammonoxidation of methylpyrazine VIIIa yields 2-cyanpyrazine IX (61),
which is easily converted to Pyrazinamide (62).
6. Stabi 1 i ty
Pyrazinamide exhibits good stability in the solid state.

There is no apparent degradation of bulk sample, either in wet or in
dry atmosphere.
Pyrazinamide is also stable when exposed to natural daylight (63).
7. Metabol ism and Pharmacokineti cs
7.1 Metabol i sm
The metabolism of Pyrazinamide has been studied in humans,

(64,65,67), dogs and rhesus monkeys (65,66) .
Pyrazine-2-carboxylic acid (11) and 5-Hydroxy-pyrazine-2-carboxyl ic
acid have been reported as the most important metabolites.
In human urine two other metabolites are also excreted in minor quanti-
ties; one of them was "tentatively assigned" as pyrazinuric acid (66).
The following table outlines the metabolism scheme and the related en-
zymes
Table 9
/N
___,
Pyrazinamide Xantine
deamidase
I Oxidase
Glyci ne conjugation
+
Tentatively assigned
PYRAZINAMIDE 451
The in vitro biotransformation o f Pyrazinamide (68) in rat liver homo-

genate allowed the identification o f the amide of 5-hydroxypyrazine-2-
-carboxylic acid.
This metabolite is formed in the "cytosol fraction" through xantine
oxidase and is an alternative to the above scheme.
A Pyrazinamide deamidase was demonstrated in the tissues of mouse, rat,

pig and rabbit. This enzyme is mainly localized in the microsomes (69).
7.2 Pharmacokinetics
Pyrazinamide, administered orally, is well absorbed in the

gastro-intestinal tract (70,64).
Serum and urine concentrations o f Pyrazinamide and its metabolites after

oral administration have been determined using the methods listed i n pa-
ragraph 10.
Results obtained vary according to the methods. They can, however, be

summarized as follows: in the blood high concentrations of Pyrazinamide
are present, but pyrazinoic acid and 5-hydroxy-pyrazinoic acid can also
be detected, while in urine little Pyrazinamide i s excreted, but pyrazi-
noic acid and 5-hydroxy-pyrazinoic acid concentrations are several times
those of unchanged Pyrazinamide. The latter, filtered by the kidneys,
is reabsorbed, while pyrazinoic acid is not reabsorbed. Peak blood le-
vels obtained by different authors using different methods are report-
ed in table 10.
Table 10
Hours after Concentration

Dose administration (g/ml)x (%) Note Reference
1 g 2 45 15 y/ml 70
after 15 h
1 g 1 - 3 50 - 60 30-40 /ml 71
after 12 h
1.5 g 2 32 half life 64
9-10 h
3 g 2 65 - 64
3 9 1 - 2 134-125 - 67
20 mg/kg 1 - 4 65 5-20 x/ml 72
after 12 h
25 mg/kg 2 47 - 75
35 mg/kg 1 - 3 40 - 80 half life 74
9 h
-
452 ERNST FELDER AND DAVIDE PITRk
The cumulative u r i n a r y excretion reaches o n l y 40% i n 24 hours o f the

administered dose and no Pyrazinamide can be detected i n aqueous aceto-
ne extracts o f feces c o l l e c t e d a f t e r o r a l administration.
7.3 Protein b i n d i n g
No binding o f Pyrazinamide t o plasma proteins was observed i n

humans, r a b b i t s and dogs, w i t h t h e e q u i l i b r i u m d i a l y s i s method (75).
7.4 Acute t o x i c i t y
The acute t o x i c i t y (DL50) o f Pyrazinamide was found t o be:
i n the mouse 2500 mg/kg i.p. and 2730 mg/kg p.0.

i n the r a t 2350 rng/kg i . v . and 3800 mg/kg p.0. (76)
8. M i crobi o l ogical Assay
8.1 B i o l ogi c a l Method f o r Pyrazi namide Determination
A b i o l o g i c a l method f o r the assay o f Pyrazinamide i n body

f l u i d s (serum, blood, urine) has been developed (72).
A f t e r p r e c i p i t a t i o n and removal o f proteins the pH o f the f l u i d was ad-

justed t o pH 5.5. The medium f o r t e s t i n g was made according t o the F i t z -
simons formula o f the Middlebrook 7H10 agar, changing the r a t i o o f the
b u f f e r i n g phosphate s a l t s i n order t o adjust the pH t o 5.5. The surfa-
ce o f the agar s l a n t was inoculated w i t h a 7H9 broth c u l t u r e o f a Pyra-
-
zinamide susceptible s t r a i n o f M. tuberculosis, which was r e s i s t a n t t o
streptomycin and isoniazid. Deproteinized t e s t f l u i d (0.5 m l ) was add-
ed t o the bottom o f the inoculated tube and the tubes incubated a t 37OC
f o r two t o three weeks i n an u p r i g h t position.
A Pyrazinamide content o f 20 t o 60 mcg per 0.5 m l o f body f l u i d r e s u l t e d

i n an i n h i b i t i o n o f mycobacterial growt due t o v e r t i c a l d i f f u s i o n o f the
drug.
The acid pH o f the medium i s a deterrent t o a precise q u a n t i t a t i v e de-

termination by t h i s test. The b i o l o g i c a l t e s t may however serve as a
qua1i t a t i v e control on the antimycobacterial a c t i v i t y o f Pyrazinamide
as determined by the chemical methods.
453
PYRAZINAMIDE
9.1 Elemental
The elemental composition of Pyrazinamide is:

Element % Theoretical
-
48.78
4.09
34.14
12.99
9.2 Identification Tests
Compendia1 identification tests involve comparing either IR

or UV absorption of Pyrazinamide with its reference standard (14.16).
The identification reactions reported are:
a) Perception of ammonia smell from a boiling solution of 20 mg of

Pyrazinamide in 5 ml of 5N NaOH.
b) Development of an orange-red color upon dissolution of 0.1 g of

Pyrazinamide in 10 ml of H20, and addition of 1 ml of FeSO TS.
The color changes to blue on addition of 1 ml o f NaOH TS; (f5).
9.3 Offi ci a1 Methods
These methods are based on the alkaline hydrolysis of Pyrazi-

namide and titration of the liberated ammonia (14,16).
9.4 Non-aqueous Ti trimetric Analysis
Pyrazinamide can be titrated in glacial acetic acid containing

mercuric acetate, with percloric acid in glacial acetic acid as titrant.
The ti tration can be carried out potentiometrically (77).
9.5 Col or i me try
Colorimetric methods for Pyrazinamide determination are report-

ed :
a) hydrolysis by dilute alkali to pyrazinoic acid, which is quantita-

tively determined by the orange-red color developed with ferrous
ammonium sulfate (78).
b) reaction with a1 kaline nitroprusside (nitropentacyanoferroate) to

give a red-orange color with an absorption maximum at 490-500 nm
(64,70). For separate determination of both pyrazinamide and Py-
razinoic acid a preliminary differential solvent extraction (i .e.
benzene-n-butanol ) is described (64).
c) For determination, also i n presence o f pyrazinemonocarboxyl i c acid,

n i c o t i n i c acid, i t s amide, etc., a s o l u t i o n o f the drug i s mixed
w i t h 0.5 M CoC12, glycerol and 2 N KOH; a f t e r standing f o r f i v e
minutes, 3% H202 i s addedd. The yellow c o l o r developped i s measur-
ed c o l o r i m e t r i c a l l y (79).
9.6 U1t r a v i o l e t Spectrophotometric Method
Pyrazinamide can be determined by measurement o f the absorban-

ce a t 269 nm i n aqueous solution. E (l%, 1 cm), a t 269 nm = 655 (80).
9.7 Compl exometric Analysis
The method i s based on the formation o f the complex Pyrazinami-

de-HgC1 (m.p. 245°C) from a c i t r i c acid s o l u t i o n containing an excess o f
HgC12. 2
A f t e r f i l t r a t i o n o f the complex, the excess o f reagent i s t i t r a t e d w i t h

a s o l u t i o n o f Complexon 111 using Eriochrome I black (pH = 10) as indica-
t o r (81).
9.8 Chromatography
9.8.1 Paper chromatography
Pyrazinamide paper chromatography has been reported i n d i c a t i n g

f i v e systems and a v a r i e t y o f v i s u a l i s i n g reagents (32,82) as suitable.
The solvent systems used were:
a) 4.8 g o f c i t r i c acid i n a mixture o f 130 m l water and 870 m l o f

n-butanol,
b) Acetate b u f f e r (pH = 4.58)
c) Phosphate b u f f e r (pH = 7.4)
d) Butyl acetate - acid a c e t i c - water (5:l:l)
e) n-Butanol - HC1 1N ( 1 : l )
Using Whatman N 1 f i l t e r paper the f o l l o w i n g R f values were obtained:

0.56 (a), 0.83 (b), 0.80 (c), 0.57 (d), 0.58 (e).
The spots were visualized using e i t h e r :
1) UV l i g h t
2) Potassium permanganate spray reagent (1% aqueous sol .)
3) Iodoplatinate spray reagent
4) Water s o l u t i o n o f 4% nitroprusside and NaOH 4N (1:l)
PYRAZINAMIDE 455
9.8.2 Thin layer chromatography
TLC methods for the separation and detection of Pyrazinamide

are summarized in the table 1 1
Table 1 1
Solvent system -
Plate Rf
- Reference
I A 0.63 19
I1 B 0.26 73
I11 B 0.44 73
IV B 0.79 73
V B 0.76 73
VI B 0.48 57
VII C -- 70
Solvent System:
I Conc. ammonia : methanol (1.5 : 100)

I1 Toluene : Ethyl acetate : 85% formic acid (50:45:5)
I11 Toluene : isopropanol : conc. ammonia (70:29:1)
IV Toluene : ethyl acetate : isopropanol : acetic acid
(10:35 :35:20)
V Toluene : dioxane : methanol : conc. ammonia (20:50:20:10)
V I Chloroform : methanol : conc. ammonia (20:20:1)
VII Benzene : chloroform : acetic acid (8:l:l)
Plate:
A Silica gel G
B Kieselgel 60F 254 (Merck)
C A1203 60F 254 (Merck)
Detection systems:
Iodine - carbontetrachloride spray reagent

UV light at 254 nm
-
Sodium nitroprusside 4% NaOH 4N (1:l)
Picryl chloride 1.5%
A direct detection with diphenylamine 0.1% is reported (81).
9.8.3 Gas chromatography
A gas-chromatography method has been described for the deter-

mination of Pyrazinamide, using a glass column (1.8 m) of Versamide 900
on Chromosorb W silanized (100-120 mesh), operated at 165" with N2(50 ml/
m) as carrier gas and a FID detector (83).
-
GLC of Pyrazinamide with lithium iodide containing poly-(ethylene gly-
col) as stationary phase was reported (84).
456 ERNST FELDER AND DAVIDE PITRB
GC/MS of TMS derivatives o f Pyrazinamide was done for methabolic studies

operating between 120’ and 180’ with a column packed with 3% OV-101 on
Chromosorb W 80-100 mesh (67).
9.8.4 High pressure liquid chromatography
A HPLC method for quantitative determination of Pyrazinamide

and separation from pyrazinoic acid has been developed (85).
The operating conditions are as follows:
- Apparatus: Hewlett-Packard 10
- Column : 25 cm X 4 mm’!abiH column packed with Lichrosorb RP8
(7
- Injection: 20 I1 o f aqueous solution (about 10 mg/ml o f Pyrazinamide)
- Eluent A : iqueous 0.005 M tetrabutylammonium phosphate (pH 7.5)
- Eluent B : 0.005 M tetrabutylammonium phosphate in than01 - water
8:2 (v/v)
- Flow rate : 1.5 ml/min
- Gradient profile:
Minutes % Eluent 8
0 5
2 5
10 45
11 45
13 5 (reconditioning step)
18 stop
- Column temperature: 25’C
- Detector wavelenght: 254 nm
- Relative retention time: pyrazinoic acid: 1.9 min.
Pyrazinamide: 1 min.
9.9 Counter Current Distribution
A counter current distribution of Pyrazinamide in an automatic

Craig machine by partitioning into 29 tubes using the solvent system
-
ethyl acetate 0.01N sodium hydroxide (K = 0.49 5 0.01) has been per-
formed (64).
- -
For pyrazinoic acid the distribution is possible with the solvent system
ethyl acetate 0.01N sulfuric acid ( K 0.44 5 0.01).
9.10 Pol arography
Quantitative determinations by polarography o f Pyrazinamide

and its metabolites in human tissues and pla- * - have been reported
(86,87,67).
PYRAZINAMIDE 451
JO. Determination of pyrazinamide in body fluids and Tissues
Most of the methods referred in this section are similar to

other general analytical methods, a1 ready described with differences
only in the extraction procedures.
At first colorimetric methods, based on reaction of Pyrarinamide, or o f

its hydrolysis products, with Ferrous Ammonium Sulphate (78), Alkaline
Nitroprusside (64,70), Cobalt Chloride (79) were used.
Later, spectrophotometric measurements (80) or polarographic methods

(65) without prior separation from a biological fluid (86,87) were pre-
ferred.
A combined gas chromatographic-mass spectrometric technique (67) for s i -

mu1 taneous identification and quantitative determination of Pyrazinamide
and its main metabolites in serum and urine of human subjects was des-
cri bed.
A microbiological assay using a Pyrazinamide sensitive Mycobacterium

strain (72) is also reported,
11. REFERENCES
1. Annual meeting o f the IUAT, Prague, June 1-8 (1980)
2. Report o f a J o i n t IUAT/WHD Study Group, Technical Report

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Kenzie and Y. Subba Row, J.Org.Chem., 13, 834 (1948)
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Rev.Tuber. 65, 511 (1952)
6. R.M. McCune, F.M. Feldmann, H.P. Lambert, W. McDermott,

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13. Hong Kong Chest Service/British Medical Research Councils,

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14. U.S.P. XX O f f i c i a l Monographs: Pyrazinamide Tablets, pag. 692
15. Jap. P. I X
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17. C. Tamura, Nippon Kagaku Eashi, 3, 971 (1962)
18. Shigom Ioshida, Chem. Pharm. B u l l . z,628 (1963)

19. M. Grandi, Bracco S.p.A., Milan, personal comunication
20. A. Lautie, F. Froment, A. Novar, Spectrosc. L e t t . 9, 289 (1976)

PYRAZINAMIDE 459
21. J. Bus, Th.J. Liefken, W. Sehwaiger, Rec.Trav.Chim., Pays Bas,

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92, 123 (1973)
22. R.H. Cox, A.A. Bothner-By, J. Phys. Chem. 11,1645 (1968)
23. G.G. Dvoryantseva, V.P. Lezina, V. Bystrv, T.N. Ulyanova, G.P.

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( 1968)
24. G.S. Marx, P.E. Spoerri, J.Org.Chem. 2, 1 1 1 (1972)

25. G.P. Syrova, Yu.N.Sheiker, I.S. Musatova, A.S. Elina,
Chim.Geterosik1. Soedin. 266 (1972)
26. G.P. Syrova, Yu.N.Sheiker, Chim.Gererosik1 .Soedin. 345 (1972)
27. Bracco S.p.A. Milan, unpublished results
31. The Merck Index IX Ed., 7740
32. E. Felder, D. Pitre, U. Tiepolo, Min.Med. 2, 1699 (1962)
33. C. Tamura, H. Kowano, Acta Cryst. 2, 693 (1961)
34. Y. Takaki, Y. Sasada, T. Watanabe, Acta Cryst. 13,693 (1960)

35. G. Ro, H. Sorum, Acta Cryst. B,
1677 (1972)
G. Ro, H. Sorum, Acta Cryst. Q, 991 (1972)
36. 6. Liborio, University o f Milan, Institute o f Mineralogy,

personal comnunication
37. H. Negoro, Takamine Kenkyusho Nempo, lJ, 66 (1959)
38. M. Grandi, Bracco S.p.A., personal communication
39. M. Grandi, Bracco S.p.A. , personal comnunication
40. P:P. Singh, J.N. Seth, J.Inorg.Nucl.Chem., 2, 593 (1975)
41. A. Tenhunen, Suom.Kemistilehti, B 42, 361 (1969)
42. A. Tenhunen, Suom.Kemistilehti, B 43, 506 (1970)

ERNST FELDER AND DAVIDE PITRE
43. A. Tenhunen, Suom.Kemistilehti, 6 2, 97 (1970)

44. A. Tenhunen, Ann.Acad.Sci.Fen., Ser.A 2, 161 (1971)
45. A. Tehnunen, Suom.Kemistilehti, B 45, '81 (1972)
46. M. Sekizaki, Acta Crystallogr. Sect. B 29, 327 (1973
47. T.A.Azizov, O.F. Khodzhaev, N.A. Perpiev, Koord.Khim. Q, 1234,

( 1978)
48. K. Brodersen, N. Hacke, B 107, 3260 (1974)
49. P.P. Singh, J.N. Seth, S.A. Khan, 1norg.Nucl.Chem.Lett. 11,

525 (1975)
50. S.C. Jain, M.S. Gill, G.S. Rao, J.Indlan.Chem.Soc., 2, 537

( 1976)
51. O.F.Khodzhaev, E.K. Khudaiberdiev, Kh. Kh. Khatimov,

Koord.Khim. 5, 689 (1979)
52. Kh. Kh. Khakimov, E. Khudaiberdiev, M.A. Atizov, Mater. Yubi-

leinoi Resp.Nauchn.Konf .Farm., Posuyashch. 50-Letiyu Obrat.
SSR, 138 (1972)
53. S. Kushner et al., J.Am.Chem.Soc. 74,3617-3621 (1952)
54. U.S. Patent 2.627.641 (1954 to Am. Cyanamid)

55. R.L. Yeager et al., Am.Rev.Tuber. 65, 523 (1952)
56. J .A. Solomons, P. E. Spoery, J.Am.Chem.Soc. 75, 679 (1953)

57. S.Gabrie1, A. Sonn, B 40, 4851 (1907)
58. Brit.pat. 566653 (1945 to CA 41, 1251)

59. Swiss pat. 415645 (1967 to Eprova A.G.)
60. H. Gainer, J.Org.Chem. 24, 691 (1959)
61. Fr.pat. 1,331.102 (1963 to Merck & Co.)
62, H. Foks et al., Acta Pol.Pharm. 49-54 (1976)
63. U. Tiepolo, Bracco S.p.A., Milan, unpublished data

64. G.A. Ellard, Tubercle 3, 144 (1969)
PYRAZINAMIDE 461
65. I.M. Weiner, J. Tinker, J.Pharm. & Exper.Therap. 180, 411 (1972)
66. G.M. Fanelli, I.M. Weiner, J.Clin.Invest. -

52, 1946 (1973)
67. 3. Roboz, R. Suzuki, Ts'ai-Fayu, J.Chrom. 147, 337 (1978)
68. D.Pitre, R. Maffei Facino, M. Carini, G. Avarone, Pharm. Res.

Commun. 13, 951 (1981)
69. I. Troida, Amer.Reu.Resp.Dis., 107, 630 (1973)

70. P.A. Caccia , Amer.Rev. of Tub.and Pulm.Dis. 75, 105 (1957)
71. G.Grassi, 6. Tansini, F. Leidi, G. Perna, Atti Soc.Lomb.

Sc.Med.-Biol. 14,10 (1959)
72. K.D. Stottmeier, R.E. Beam, G.P. Kubica, Am.Rev. of Res.Dis.

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98, 70 (1968)
73. F. Boulahbal, S. Khaled, H. Bouhassen, D. Larbaoui, Le Pyrari-

namide 25 ans apres, Symp.Alger, 35 (1979)
74. J. Grosset, Tubercle 2, 519 (1978)
75. P.G. Venosta, Bracco S.p.A., Milan, personal communication
76. E.Boldrini, Bracco S.p.A., Milan, personal communication
77. G. Mascellani, C. Casalini, Anal.Chem. 47, 2468 (1975)
78. W.S. Allen, S.M. Aronovic, L.M. Biancone, J.H. Williams,

Anal. Chem. 25, 895 (1953)
79. D. Aoki, Y. Ivoyama, K. Furusumi, Yakuzaigaku E,9 (1956)

80. S. Chiba, Takamine Kenkyujo Nempo 8, 132 (1956)
81. V. Ignat, T. Costantinescu, Farmacia 16, 151 (1968)
82. E.G.C. Clarke, Isolation and Identification of drugs, The Phar-

maceutical Press - London, 1969
83. A. Cal7, C. Cardini, V. Quercia, Boll.Chim.Farm. 108,175 (1969)
84. N. Amaguchi, T. Nakagawa, T. Uno, J.Chrom.170, 81 (1979)
85. M.Grandi, Bracco S.p.A., Milan, personal communication

86. ti.K. Svoboda, J.A.P.A. 45, 504 (1956)
87. P.O. Kane, Nature 183, 1674 (1959)

PYRIMETHAMINE
Mohammed A . Loutfy and Hassan Y. Aboul-Enein
1. Description 464
1 . 1 Nomenclature 464
1.2 Formulae 464
1.5 Appearance, Colour, Odour, and Taste 465
2.2 Solubility 465
3. Synthesis 47 1
5.1 Gravimetric Method 474
5.2 Titrirnetric Method 475
5.4 Spectrophotometric Analysis 477
5.5 Nuclear Magnetic Resonance 478
References 479
ANALYTICAL PROFILES OF DRUG SUBSTANCES 463 Capynghl by [he American Pharmaceutical Association
VOLUME 12 lSRN0-12~260812-7
464 MOHAMMED A. LOUTFY AND HASSAN Y. ABOUL-ENEIN
1. Description
1.1. Nomenclature
1.1.1. Chemical Names
5- (4-Chlorophenyl)-6-ethyl-2,4-pyrimidine-
diamine (1);
2,4-Diamino-5-(p-chlorophenyl)-6-ethylpyrimi-
dine ( 2 ) ;
5-(4-Chlorophenyl)-6-ethylp yrimidine-2,4-
diamine (3,4) ;
2,4-Pyrimidinediamine, 5-(4-chlorophenyl)-6-
ethyl (5).
1.1.2. Generic Names
Pyrimethamine; Pyrimethaminum; BW 50-63;

RP 4753.
1.1.3. Trade Names
Daraprim; Chloridin; Darapram; Malocide;

Supacox (with amprolium hydrochloride,
ethopabate, and sulphaquinoxaline); Maloprim
(with dapsone); Fansidar (with sulphadoxine
available only in certain overseas countries
(4)) ; Whitsyn S (with sulphaquinoxaline) .
1.2. Formulae
1.2.1. Empirical
C12H13C1N4 '
1.2.2. Structural
c1 (0
NHZ
PYRIMETHAMINE 465
1.2.3. Wiswesser Line Notation
T6NCNJ BZ D
Z ER DC and F2 (6).
1.2.4. Chemical Abstract Registry Number
58-14-0 (6).
248.71
1.4. Elemental Composition

C, 57.94%; H, 5.27%; C1, 14.25%; N, 22.53%.
1.5. Appearance, Colour, Odour, and Taste
A white, crystalline powder, odourless and tasteless.

pKa 7 at 2
0' (4).
2.1. Melting Point

233-234' (capillary) ; 240-242' (copper block) (1);
239-242' (3,7); 235-236' (8); 237-238' (9).
2.2. Solubility
Practically insoluble in water; slightly soluble in

ethanol (about 9 gm/litre), in dilute HC1 (about 5
gm/litre), in acetone and in chloroform (1 gm/125ml).
Very sparingly soluble in propylene glycol and
dimethylacetamide at 70°. Soluble in boiling
ethanol (25 gm/litre), and in warm dilute mineral
acids.
2.3. Tdentffication
The following identification tests have been

described (2-5) :
a. Dissolve 0.05 gm in 5 ml dilute sulphuric acid

and add 0.2 ml of alkaline potassio-mercuric
iodide; a creamy-white precipitate is formed.
466 MOHAMMED A. LOUTFY AND HASSAN Y.ABOUL-ENEIN
b. Ignite 0.1 gm with 0.5 gm of anhydrous sodium

carbonate, extract the residue with water, and
filter. The filtrate, after neutralisation with
nitric acid, yields the reactions characteristic
of chlorides.
c. Identity tests for pyrimethamine have been

reported ( l o ) , utilising alkaloidal precipitants
and preparation of the 2,4-diacetyl derivative,
m.p. 172O (from 50 X ethanol).
Microcrystal Tests
Gold bromide - hydrochloric acid solution

produces serrated needles (sensitivity:lin 1000);
Potassium chromate solution forms irregular
blades (sensitivity: 1 in 1000) (11).
2.4. Spectral Properties
2.4.1. Ultraviolet Spectrum
The UV spectrum of pyrimethamine, in aqueous

acidic solution, was scanned in the region of
400-200 nm using a Pye-Unicam SP 8-100 Ultra-
violet spectrometer, and is shown in Figure 1.
The UV spectrum of a solution in 0.005 N HC1
exhibits a maximum at about 272 nm (El%,
1 cm = 320), and a minimum at about 260 nm.
In alcohol (95%), maximum at 287 nm (El%,
1 cm = 365) (2,4,11).
The UV spectrum of pyrimethamine in alcoholic
NaOH shows a maximum at 286 nm as shown in
Figure 2.
2.4.2. Infrared Spectrum
The IR spectrum of pyrimethamine in KBr-disc

is shown in Figure 3. The IR spectrum was
recorded on a Perkin Elmer 580B Infrared
spectrometer. The structural assignments
have been correlated with the band frequencies
represented in Table 1.
PYRIMETHAMINE 467
90. -90
80. - 80
70. - 70
60. - 60
50. - 50
40. -40
30. .30
2 0. - 20
10- - 10
1 .
W a v e Leng t h
Fig. 1. W SPECTRUM OF PYRIMETHAMINE IN 0.005 N HC1.
90. - 90
80. - 80
70. - 70
60. - 60
50- - 50
40- - 40
30- . 30
20- .20
10- . 10
Table 1. IR Characteristics of Pyrimethatnine
Frequency Cm-1 Assignment
34403
3310
NH asymmetric and symmet-
2 stretch.
ric
3130 C-H aromatic stretch.
1650 C=N aromatic stretch.
C=C aromatic stretch and

1630}
1560 substituted aromatic ring.
1470 CH3 and CH2 bending

1430) stretch
1410
C-H deformation of chloro-
1090 substituted aromatic ring.
990 Chloro-substituted aromatic

ring.
830 C-H deformation of p-

substituted aromatic ring.
8101
750
C-H out of plane bending
vibration.
The above assignments ,re in agreement with

published data (6).
2.4.3. Nuclear Magnetic Resonance Spectrum

2.4.3.1. Proton Spectrum
The PMR spectrum of pyrimethamine in

deuterated dimethyl sulfoxide (DMSO-d6)
was recorded on a Varian T-60A, 60-MHz
NMR spectrometer using TMS as an
internal standard. The spectrum is
shown in Figure 4 .
The structural assignments have been

made in Table 2.
Fr
0
\ m
b0
.rl
Fr
Fig. 4 . PMR SPECTRUM OF PYRIMETHAMINE IN DMSO-d6, USING TMS AS AN INTERNAL STANDARD.
PYRIMETHAMINE 471
Table 2. PMR Characteristics of Pyrimethamine
Proton Assignments Chemical Shift (6)
CH3CH2- 0.97 (t)
-
CH3CH2- 2-00 (9)
-
NH2 at C4 5.5 (bs)
-
NH2 at C2 5.8 (bs)
-
Exchangable with D 2 0
Four aromatic protons of
p-chlorosubstituted phenyl
G n g---------------------------------------
7.3 (m) ---
t=triplet; q*quartet; bssbroad singlet;
momultiplet .
2.4.3.2. 15N-NMR
Stadeli et al. (12) have reported the
15N-NMR spectra for several aminopyri-
dines and aminopyrimidines including
pyr imethamine.
The assignments of "N-NMR for pyrime-
thamine is represented in Table 3.
Table 3. "N-NMR of Pyrimethamine
-
N(1) N(3) -
N(2) N(4) Solvent
-176.3 -176.3 -300.9 -299.4 a
-247.5 -247.5 -294.5 -274.9 b
-249.9 -250.7 -295.8 -274.5
-----------------c--__c_________________-------
C
a=dimethyl sulphoxide at 6
0
'.
b=trifluoroacetic acid.
c-fluorosulphonic acid.
2.4.4. Mass Spectrum
The mass spectrum of pyrimethanmine is shown

in Figure 5. The most prominent ions and
their abundances are shown in Table 4 ( 1 3 ) .
Table 4. Mass Spectrum of Pyrimethamine
Mass (m/e) Fragment Abundance %
247 M-1 due to 100 (base peak)

C1-35 isotope.
248 M+ 50
249 M+1 due to 36
C1-37 isotope.
250 - 15
219 M-C2H5 12
Figure 5 shows a molecular ion peak at m/e =

248, and peaks at 247, 249 and 250 which
represent the natural isotopic abundance for
chlorine and M+1 peak ( 1 3 ) . Other peaks
appear at m/e = 212 (10%); m/e = 232 (5%); and
m/e = 106 (5%).
The fragmentation pattern of pyrimethamine

follows the general fragmentation patterns of
2-aminopyrimidines (14-16) .
3. Synthesis
Various synthetic procedures have been described for

pyrimethamine, starting from open-chain compounds and
different reactions of cyclization.
a. Ethyl propionate is condensed with p-chlorophenyl

acetonitrile, in the presence of sodium methoxide
(17). The resulting a-propionyl-p-chlorophenyl
acetonitrile I is converted into the hemiketal by
reaction with isoamyl alcohol, ethyl orthopropionate
(18-20) or with dimethyl sulphate (8). The hemiketal
undergoes dehydration to a-(p-chloropheny1)-6-ethyl-
$-alkoxyacrylonitrile. The later is then
reacted with guanidine or guanidine HC1 whereupon
cyclization occurs due to liberation of alcohol and
an addition reaction involving an amino group of
guanidine and the nitrile group (Scheme 1).
100-
80.
60 -
40.
Fig. 5. MASS SPECTRUM OF PYRIMETHAMINE.

CH30Na
NCCH2C6H4Cl(p) + CH3CH2COOEt >
I R=iso-C H
5 11;
=C2H5 ;
=CH3
/ NH2 :2H5
HN=C-
‘ NH,
p-ClC H
H2‘@ANH2
Scheme 1
b. Logemann e t a l . ( 9 ) have p r e p a r e d t h e drug by h e a t i n g
a-propionyl-p-chlorophenyl a c e t o n i t r i l e (I) w i t h
a n i l i n e and t r e a t i n g t h e p r o d u c t w i t h t h e c a l c u l a t e d
amount of N a / C H OH and c y c l i z i n g w i t h g u a n i d i n e
2 5
(Scheme 2 ) .
PhNH2
I T> NC-HC-C6H4C1
I
(p-) Enolisation ,
I \
PhN=C-C2H5
NC-C-C6H4C1 (p->
II
Ph-HN-C-C 2H5
HN=C (NH2)
Pyrimethamine
Scheme 2
PYRIMETHAMINE 475
c. Jacob (21) has patented the synthesis of pyrimetha-

mine as shown below (Scheme 3). Et
L
1) Chlorination
2) NH3
> Pyrimethamine
Scheme 3
Several reports and patents are listed in the

literature for the synthesis of the drug (22-31).
4. Metabolism and Pharmacokinetics

After an oral dose, a peak plasma concentration is
reached in about 2 hours. During therapy with 50 mglday,
plasma concentration of 0.3 - 0.6 pg/ml is attained. The
plasma half-life for pyrimethamine is about 90 hours (4).
Several authors (32-33) have studied the pharmacokinetics

of the drug in combination with dapsone and sulfalene in
human volunteers.
Recently, Yamaoka et a1.(34) have studied the tissue

distribution of orally administered pyrimethamine to pigs.
Pyrimethamine affects the nucleoprotein metabolism of the
malarial parasites by interference in the folic-folinic
acid systems and its action is exerted mainly at the time
when the nucleus divides. It has little effect upon
immature schizonts in the red corpuscles and therefore it
is slow to control a malarial attack; its chief value is
as a suppressant. It has been observed (11) that about
1. mg still remains in the body 30 days after a single
dose of 100 mg. Pyrimethamine is excreted in the milk of
nursing mothers. Several metabolites of pyrimethamine
appear in the urine; little is known of their structure
or antimalarial activity (11).
5.1. Gravimetric Method
Drey (10) has reported the assay and identification

of pyrimethamine and its preparations. In this
method, pyrimethamine is determined gravimetrically
by precipitation from 5% H2S04 solution with
phosphotungstic acid, washing with 2% H2S04, and

drying at 500 for 2 hours or over P2O5 in vacuo for
less than 4 hours, then heating at atmospheric
pressure at 110' for 1.5 hours. Each gm is equiva-
lent to 0.2040 gm of C12H13N4C1.
5.2. Titrimetric Method
Non-aqueous
Most pharmacopoeias (2,3,5) recommend the quantita-

tive determination of pyrimethamine by non-aqueous
titration, using quinaldine red as an indicator and
0.1 N acetous perchloric acid as a titrant.
5.3. Chromatography
5.3.1. Paper Chromatography
Clarke (11) has described several solvent

systems for identification of the drug as
shown in Table 5.
Table 5. Solvent System used in Paper chromatography

of pyrimethamine.
Solvent System Visualizing Agent -

Rf
1. Citric acid-water-n- W , iodoplatinate 0.42

butanol (4.8 gm: spray
130 ml: 870 ml).
2. Acetate buffer w 0.42

(pH=4.58) .
3 . Phosphate buffer uv 0.08
(pHe7.4) .
5.3.2. Thin-Layer Chromatography
Serfontein et al. (35) have reported a rapid

and comprehensive system for the routine
identification of pyrimethamine, and other
drugs, in biological materials. The method
is based on the separation of the drugs from
2-propanol extracts of serum, urine and
tissue homogenates at different pH values
PYRIMETHAMINE 477
using microphase extraction techniques

followed by examination with two-dimensional
thin-layer chromatography. The drugs are
visualized after spraying with various chrome
genic and fluorogenic reagents.
Identification of pyrimethamine has been also

carried out (11) by using strong ammonia-
methanol (1.5:lOO) as solvent system and the
drug is visualized after spraying with
acidified iodoplatinate.
De Angelis et al. (36) have determined the

drug, in biological fluids (plasma and urine),
by thin-layer chromatography.
5 . 3 . 3 . Gas-Liquid ChromatograDhy
Jones -et - a l . ( 3 7 ) have described an assay of

pyrimethamine in human plasma by GLC. In
this method, samples, prepared by extraction
of plasma containing pyrimethamine and the
internal standard BW197U toluene, were
analyzed using a column of 10% OV-17 on Chro-
mosorb W HP with N as the carrier gas. The
injection port, column, and detector were at
300, 2 3 5 , and 3500, respectively. After the
internal standard was eluted the column
temperature was increased at 160/minute to
2800 for 4 minutes. Pyrimethamine and the
internal standard had retention times of 7 . 3
and 10.8 minutes, respectively. Quadruplicate
samples of pyrimethamine gave a coefficient
of variation of 5 . 5 % for pyrimethamine plasma
concentrations of 5-400 ng/ml. Pyrimethamine
and the internal standard gave recoveries of
75.6 and 7 4 . 7 % , respectively, which also were
independent of concentration. The minimum
detectable amount of pyrimethamine was 5 0 pg;
plasma concentrations of 5-400 ng/ml are
comEortably assayed. In contrast to other
more sensitive methods, this method has
smaller errors and allows replicate injections
of a sample; pyrimethamine plasma levels can
be monitored in human volunteers for several
weeks after administration of a single 25 mg
dose of pyrimethamine.
Bonini et al. ( 3 8 ) have described a gas

chromatographic determination of four anti-
malarials, including pyrimethamine, singly
and in a mixture in biological media. In
this method, pyrimethamine has been deter-
mined in blood and urine using a column packed
with 2% OV-17 on Chromosorb W AW DMCS 100-120
mesh with N gas as the carrier gas and the
column temperature programmed to increase
from 250 to 3500 at 8Olminute. The limit of
detection was 0.191 ng for pyrimethamine.
The recoveries in blood and urine were 82.3
and 89.5%, respectively for 0.75 ug pyrimetha-
minelml.
Other gas-liquid chromatographic methods for

pyrimethamine has been reported (39-41).
5.3.4. High Performance Liquid Chromatography
Yamazaki et al. (42) have reported an HPLC

assay for pyrimethamine and several antibio-
tics, in food samples. The drugs were simul-
taneously extracted from food samples with
acetonitrile. The extract was then subjected
to HPLC with a Zorbax sil column, using W
detector at 284 nm. The detection limit for
pyrimethamine was 0.4 m m .
Jones and Ovenell ( 4 3 ) have devoloped a high-

performance liquid chromatographic method for
simultaneous determination of pyrimethamine
and dapsone in plasma. The solvent system,
consisting of di-isopropyl ether-methanol-21%
aqueous NH40H (96 : 4 : 0.1) at a flow-rate
of 2 ml/minute, passed through column packed
with 5-um spherical silica (Spherisorb S5W) .
Metoprine, an analogue of pyrimethamine, was
used as an internal standard. The limit of
detection was about 5 ng injected for pyrime-
thamine .
5-4. Spectrophotometric Analysis
5.4.1. Colorimetric
Recently, Sane and Dhamankar (44) have deve-

loped an extractive colorimetric determina-
tion of pyrimethamine in pharmaceutical
preparations. In this method, pyrimethamine
PYRIMETHAMINE 479
is determined by formation of a chloroform-

extractable colored species (maximum absor-
bance at 415-420 nm) by reaction with bromo-
cresol purple, bromophenol blue, methyl
orange, picric acid, or bromothymol blue, in
a medium of pH 1-4.5. The recovery ranges
from 99.4 to 101.17%.
Other colorimetric methods for the assay and

identification,ofpyrimethamine have been
reported (5, 41, 45). The determination is
based on an acid dye ion-pair extraction of
the color formed with bromocresol green and
measuring at 622 nm. One of the methods (41)
is suitable for determination of the drug at
5 mg/kg level of feed.
5.4.2. Ultraviolet
The method officially adopted by U . S . P . XIX

(6) for the quantitative determination of
pyrimethamine tablets involves spectrophoto-
metric assay. The absorbance of the test
solution and standard preparation is measured
in 1 cm cells at 273 nm.
5.5. Nuclear Magnetic Resonance

Girgis and Askam (46) have described determination
of pyrimethamine by NMR spectroscopy. The 60-MHz
NMR spectrum of the drug, in trifluoroacetic acid
solvent and using anhydrous caffeine as an internal
standard, is recorded. The method is based on the
integration of selected peaks of the characteristic
resonance pattern of pyrimethamine-and those of
caffeine. The coefficient of variation was 1.96%.
Acknowledgement
The authors wish to thank F. Hoffman-La Roche & Co. Limited,

Basle, Switzerland, for donating a sample of Pyrimethamine
Lot No: 654351.
480 MOHAMMED A. LOUTFY AND HASSAN Y . ABOUL-ENEIN
6. References
1. M. Windholz, "The Merck Index", 9th Ed., Merck & Co.

Inc., Rahway, N.J., U.S.A., 1976, p. 1036.
2. "Specifications for the Quality Control of Pharma-

ceutical preparations", 2nd Ed., World Health
Organization, Geneva, 1967, p. 507.
3. "British Pharmacopoeia", Vol. 1, Her Majesty's

Stationary Office, London, 1980, p. 381.
4. "The Pharmaceutical Codex", 11th Ed., The Pharma-

ceutical Press, London, 1979, p. 767.
5. "The United States Pharmacopoeia'', XIX, Mack

Publishing Co., Easton Pa., 1965, p. 430.
6. G.C.G. Grasselli and N.M. Ritchey, "Atlas of

Spectral Data and Physical Constants for Organic
4, CRC Press Inc.,
Compounds", 2nd Ed., Vol. -
Cleveland, Ohio, 1975, p. 422.
7. Martindale, The Extra Pharmacopoeia, 27th Ed.,

Pharmaceutical Press, London, 1977, p. 354.
8. M. Furukawa, Y. Set0 and S. Toyoshima, Chem. Pharm.

Bull. (Tokyo), 2, 914 (1961).
9. W. Logemann, L. Almirante, and L. Caprio, Chem. Ber.

87, 435 (1954);
- Chem. Abstr. 9, 4660 (1955).
10. R.E.A. Drey, J. Pharm. and Pharmacol. -

9, 739 (1957).
11. E.G.C. Clarke, "Isolation and Identification of

Drugs", Vol. 1,Pharmaceutical Press, London, 1978,
p . 528.
12. W. Stadeli, W.V. Philipsborn, A. Wick, and I.Kompis,

Helv. Chim. Acta, 63, 504 (1980).
13. E. Stenhagen, S. Abrahamsson, and F.W. McLafferty,

"Registry of Mass Spectral Data", Vol. -
2, John
Wiley & Sons Inc., 1974, p. 1172.
14. J.M. Rice, G.O. Dudek, and M. Barber, J. Amer.

Chem. SOC., 87,4569 (1965).
15. T. Nishiwaki. Tetrahedron 22. 3117 (1966).

PYRIMETHAMINE 481
16. Ibid, 23, 1153 (1967).
17. "Remington's Pharmaceutical Sciences", 15th Ed.,

Mack Publishing Co., Easton, Pennsylvania, 1975,
p . 1157.
18. "Bentley and Driver's Textbook of Pharmaceutical

Chemistry", 8th Ed., L.M. Atherden, Oxford Univer-
sity Press, London, 1969, p . 656.
19. P.B. Russell and G.H. Hitchings, J. Amer.Chem.Soc.,

73, 3763 (1951).
-
20. P.B. Russell and N. Whittaker, Ibid, -
74, 1310(1952).
21. R.M. Jacob, U.S. 2, 680, 740, June 8, 1954; through

Chem. Abstr. 49, 7007 (1955).
22. J.W. Mentha, J.V. Shaffner, and R.M. Cresswell,

Patent U . S . 3 , 939, 181,Feb. 17, 1976; through
Chem. Abstr.-E, 46734 (1976).
23. R. Baltzly and P.B. Russell, J. Org. Chem. 21, 912,

(1956).
24. G.H. Hitchings, P.B. Russell, and E.A. Falco, U.S.

-2, -576,
- 939, Dec.4, 1951; through Chem. Abstr. 46,
6162 (1952).
25. Ibid, U.S. 2, 602, 794, July 8, 1952; through Chem.
Abstr. 47,4921 (1953).
26. Burroughs, Wellcome and Co. Inc., Brit. 749, 051,

May 16, 1956; through Chem. Abstr. 51, 16568 (1957).
27. N. Whittaker, Brit. 750, 017,June 6, 1956; through

51, 1272 (1957).
Chem. Abstr. -
28. N. Whittaker, Brit. 743, 221, June 11, 1956;through
Chem. Abstr. 51, 2038 (1957).
29. G.H. Hitchings, P.B. Russell, and N. Whittaker, J.

Chem. SOC., 1019 (1956).
30. R.M. Jacob, Fr, 1, 070, 420, July 26, 1954; through
Chem. Abstr. 53,-43=(1=).
I
31. G.H. Hitchings and P.B. Russell, Ger. 934, 947,

Nov. 10, 1955; through Chem. Abstr. 53, 7213 (1959).
32. L. Donno, G. Vocaturo, C. Pollini, J.O. Ekanem,

Curr. Ther. Res. 27, 346 (1980).
33. R.A. Ahmad and J.H. Rogers, Br. J. Clin. Pharmacol.
10, 519 (1980).
-
34. R. Yamaoka, H. Yamamoto, and M. Kohanawa, Annu. Rep.
Natl. Vet. Assay Lab., 16,63 (1980); through
Chem. Abstr. 94, 24665 (1981).
35. W.J. Serfontein, D. Botha, and L.S. DeVilliers, J.
Chromatogr. 115,507 (1975).
36. R.L. De Angelis et al., J. Chromatogr. 106,41
(1975).
37. C.R. Jones, P.R. Ryle, and B.C. Weatherley, J.

Chromatogr. 224, 492 (1981).
38. M. Bonini, F.Mokofio, and S. Barazi, J. Chromatogr.

224,
- 332 (1981).
39. P.C. Cala, N.R. Trenner, R.P. Buhs, G.V. Downing,

J.L. Smith, and W.J.A. VandenHewel, J. Agr. Food
20, 337 (1972).
Chem. -
40. J.R. Harris, P.G. Baker, and J.W. Munday, Analyst,

102, 873 (1977).
-
41. Analytical Methods Comittee, Analyst, 106,1208
(1981).
42. T. Yamazaki, H. Hironaka, K. Kindo, and Y. Yamamoto,

Fukuoka-shi Eisei Shikenshoho, 5, 96 (1979); Chem.
95, 5231 (1981).
Abstr. -
43. C.R. Jones and S.M. Ovenell, J. Chromatogr.,w,

179 (1979).
44. R.T. Sane and A.Y. Dhamankar, Indian Drugs, 19,80

(1981); Anal. Abstr. -
43, 75 (1982).
45. Analytical Methods Committee, Analyst, =,764(l977).
46. P. Girgis and V. Askam, J. Ass. Publ. Analyst, 12,

55 (1974).
QUINIDINE SULFATE
Mohammed A. Loutfy, Mahmoud M.A. Hassan,
and Farid]. Muhtadi
1. Description 484
1.2 Formulae 484
1.5 Appearance, Color, Odor, and Taste 489
2.2 Eutectic Temperature 489
2.3 Solubility 489
2.6 Loss on Drying 490
2.7 pHRange 490
3. Preparation of Quinidine Sulfate 50 1
3.1 Isolation of Quinidine 50 1
3.2 Quinidine Sulfate 503
4. Synthesisof Quinidine 503
4. I Partial Synthesis 503
4.2 Total Synthesis 503
5. Biosynthesis of Quinidine 512
6. Metabolism 515
7. Phannacokinetics 516
8. Routes of Administration, Dosage, and Preparations 517
9.2 Gravimetric Method 5 19
9.3 Titrimetric Methods 5 19
9.5 Spectrophotometry 531
References 536
ANALYTICAL PROFILES OF DRUG SUBSTANCES 483 Copyrightby the American Phamcevrical Association.
VOLUME I2 ISBN 0-12-260812-7
484 MOHAMMED A. LOUTFY ETAL..
1. Description
1.1. Nomenclature
1.1.1 Chemical Names
Cinchonan-9-01, 6' -methoxy, (9s)- ,

sulfate (2:l) (salt), dihydrate.
(8s, gS)-6'-methoxy cinchonan-9-01,

sulfate (2:l) (salt), dihydrate.
a - ( 6-methoxy-4-quinolyl)-5-vinyl-2-
quinuclidine methanol , sulfate (2:l)
salt, dihydrate.
6-methoxy-a- (5-vinyl-2-quinucli-
dinyl) -4-quinolinemethanol , sulfate
(2:l) salt, dihydrate.
( S ) - CL -(6-methoxy-quinolin-4-yl)-a -
[ (2R,4S,5R)-( 5-vinylquinuclidin-2-yl)l-
methanol, sulfate (2:l) salt , dihy-
drate.
1.1.2 Generic Names
Quinidine sulfate; Quinidine sulfate (2:l)

( salt ) dihydrate.
1.1.3 Trade Names
Quinidex; Quinicardine; Quinora; Kiditard;

Kinidin; Cin-Quin.
1.2. Formulae
1.2.1 Fmpirical
(C H N 0 ) H2S04 2H20
20 24 2 2 2'
C40H54N4010S
QUINIDINE SULFATE
1.2.2 Structural
1.2.3 CAS registry number
[ 6591-63-51 Quinidine sulfate

(dihydrate)
[ 50-54-41 Quinidine sulfate

( anhydrous )
1.2.4 Wiswesser Line Notation
T66 BNJ H O l E YQ-DT66

A B CNTJ AlUl & GH &
QH & H2-S-04 DX (1)
486 MOHAMMED A. LOUTFY E T A .
1.2.5 Stereochemistry
The stereochemistry of quinidine and o t h e r

r e l a t e d a l k a l o i d s i s w e l l summarised by
F i n a r ( 2 ) and Turner and Woodward ( 3 ) .
I f Q r e p r e s e n t s t h e quinoline h a l f ,
t h e s t r u c t u r e of quinidine may be
w r i t t e n a s follows:-
The above formula c o n t a i n s f i v e c h i r a l

c e n t e r s : 1 , 3 , 4 , 8 and 9. Since t h e
bridge must be a c i s f u s i o n , c e n t e r s 1
and 4 behave as "one c h i r a l u n i t " , t h e r e -
f o r e , t h e number of o p t i c a l l y a c t i v e
forms would be t h e same a s obtained from
f o u r c h i r a l c e n t e r s . When t h e 1-8 bond
i s broken, t h e c h i r a l i t y of t h e n i t r o g e n
is l o s t .
Quinine, q u i n i d i n e , cinchonine and

cinchonidine give on degradation t h e
o p t i c a l l y i d e n t i c a l 8-oximino-3-vinyl-
q u i n u c l i d i n e , meroquinene and cincholo-
iponic acid. It t h e r e f o r e follows t h a t
t h e c o n f i g u r a t i o n s of C3 and C 4 a r e t h e
same f o r a l l t h r e e compounds.
Conclusive evidence f o r t h e c i s
arrangement a t C3 and C 4 w a s provided
by Prelog and Zalan ( 4 ) . They reduced
cinchonine t o dihydrocinchonine and
converted t h e product i n t o cinchlopoin
e t h y l e s t e r i n which C 3 and C4 r e t a i n
t h e o r i g i n a l c o n f i g u r a t i o n of cinchonine.
The l a t t e r w a s converted i n t o t h e dib-
romide which,by means of a s e r i e s of
r e a c t i o n s , a l l of which proceeded under
mild c o n d i t i o n s and d i d not involve t h e
c h i r a l c e n t e r s , w a s converted i n t o 1,2-
diethylcyclohexane [l]. This w a s shown
QUINIDINE SULFATE 487
t o be o p t i c a l l y i n a c t i v e (it could not be

resolved).
C2H5
I
[13
The o p t i c a l r e s u l t s provide conclusive

evidence f o r a c i s arrangement of t h e two
e t h y l groups i n t h e diethyl-cyclohexane [1]
and s i n c e none of t h e s t e p s employed i n t h e
conversion of cinchonine t o [l]involves
t h e c h i r a l c e n t e r s a t C3 and C4, t h e v i n y l
group of t h e n a t u r a l cinchona bases must be
c i s t o t h e C7-c8 bond i n all a l k a l o i d s .
The 9-deoxy d e r i v a t i v e s ( l : e , CH2 has

replaced CHOH) of cinchonine and cinchonidine
have d i f f e r e n t s p e c i f i c r o t a t i o n s , a t +
179.3' and - 29.9', respectively. Since t h e
c o n f i g u r a t i o n s of C3 and C4 a r e t h e same i n
both bases and s i n c e C9 i s no l o n g e r
o p t i c a l l y a c t i v e , t h e d i f f e r e n c e between
t h e two must be a t C8, and t h i s i s t h e r e f o r e
a l s o t h e case for cinchonine and cinchonidine.
S i m i l a r l y s i n c e [ a ] of ~ deoxyquinine i s -
97.7' and t h a t of deoxyquinidine i s + 211.1',
t h e n quinine and q u i n i d i n e d i f f e r a t Cg.
The assignment of c o n f i g u r a t i o n s a t C8 may be

deduced from t h e f a c t t h a t quinidine and cin-
chonine a r e both d e x t r o r o t a t o r y and both can
be converted i n t o t h e i r c y c l i c e t h e r s [2].
On t h e o t h e r handyquinine and cinchonidine
a r e both l e v o r o t a t o r y and do not form c y c l i c
ethers.
488 MOHAMMED A. LOUTFY ETAL.
The c y c l i c e t h e r s t r u c t u r e i s only p o s s i b l e
i f t h e group a t t a c h e d t o C3 and C8 a r e i n t h e
endo-position [ 31. Thus i n cinchonine and
q u i n i d i n e , t h e hydrogen atoms a t C3 and C8
a r e c i s with r e s p e c t t o each o t h e r . Also
because c4 and c8 a r e c i s - o r i e n t e d , it
follows t h a t t h e hydrogen atoms a t C3, C4
and c8 a r e a l l c i s - o r i e n t e d i n cinchonine
and quinidine whereas i n cinchonidine and
quinine t h e hydrogens a t C3 and C 4 are c i s ,
but t h e hydrogen a t C3 and c8 a r e t r a n s .
For each c o n f i g u r a t i o n a t C8, two isomers
a r e p o s s i b l e which d i f f e r i n o r i e n t a t i o n a t
Cg. Since a l l a l k a l o i d s a r e i d e n t i c a l i n
c o n f i g u r a t i o n except at c8 and Cg, f o u r
isomeric substances are p o s s i b l e i n each
s e r i e s . For example, two of t h e s e substances
a r e presented by quinine and quinidine , t h e
o t h e r two members are epiquinine and e p i -
quinidine. The o t h e r ' members a r e
cinchonine, cinchonidine, epicinchonine and
epicinchonidine.
I n most r e s p e c t quinine and quinidine p a r a l l e l

one another c l o s e l y i n t h e i r chemical behavior
and d i f f e r q u a l i t a t i v e l y from t h e isomeric
p a i r , epiquinine and e p i q u i n i d i n e . Since
quinine and quinidine d i f f e r i n configura-
t i o n a t c8, t h e s e f a c t s suggest t h a t t h e two
alkaloids d i f f e r a l s o i n configuration at
Cg. I f t h e s e c o n f i g u r a t i o n s a r e accepted,
then t h e r e l a t i v e c o n f i g u r a t i o n s a t C3, C4,
C8 and Cg a r e now known. It i s now p o s s i b l e
t o w r i t e t h e a b s o l u t e c o n f i g u r a t i o n s of
quinine and quinidine. Other s t u d i e s showed
t h a t both a l k a l o i d s are of t h e e r y t h r o con-
f i g u r a t i o n ( 5 ).
) quinine ( + ) quinidine
782.95 (dihydrate)
746.92 ( anhydrous)
C , 61.36%; H, 6.95%; N , 7.16%,
0, 20.44%; S, 4.09% (dihydrate)
C, 64.32%; H, 6.75%; N, 7.50%

0, 17.14%; S, 4.29% (anhydrous)
1.5. Appearance, Color, Odor and Taste
Fine, needle-like white crystals, frequently

cohering in masses, odorless, has a very bitter
taste, darkens on exposure to light.
2. Physica1 Properties.
2.1. Melting Range (6)

205 - 2100
216' by hot bar method
2.2. Eutectic Temperature (6)

Sal. 161O
Dic. 147" (Both by hot stage method)
Sal. 164'
Dic. 147' (Both by hot bar method)
Sal = acetaminosalol Dic = dicyandiamide
2.3. Solubility
1 g is dissolved in about 100 ml water,

10 ml. alcohol, in 3.0 ml methanol, in 5.0 ml
boiling water and in 15 m l chloroform, insoluble
in ether.
Quinidine sulfate has two pKa values,

the quinoline nitrogen at 20' is 5.4, whereas
the pKa value of the quinuclidine nitrogen at
20' is 10 (7).
pKa values : 4.2, 8.8 at 25' (8).
490 MOHAMMED A . LOUTFY ETAL.
2.5. Specific Rotation
[ a ID2'' + 275' t o + 290' (3%w / v i n 0 . 1 M

hydrochloric a c i d ) ( 9 )
[a about + 212' ( i n 95% e t h a n o l ) ( 1 )
[ a I D + 184.17' (CHC13) (10)
The s p e c i f i c r o t a t i o n was determined a s

1 mg/l m l e t h a n o l u s i n g a P e r k i n E l m e r
25
Polarmatic model 241 MC and found [ c1 I D + 215.5
2.6. Loss on d r y i n g
When d r i e d t o c o n s t a n t weight a t 130' l o s e s n o t

l e s s t h a n 3.0% and n o t more t h a n 5.0% of i t s
weight ( 8 ) .
2.7. pH range
The pH of 1% w/v aqueous q u i n i d i n e s u l f a t e

s o l u t i o n i s 6 . 0 t o 6.8 ( 9 ) .
2.8.1 U l t r a v i o l e t . Spectrum
The W spectrum o f q u i n i d i n e s u l f a t e i n
e t h a n o l ( F i g . 1) w a s scanned from 200 t o
400 nm u s i n g DMS 90 Varian Spectrophotometer.
It e x h i b i t e d t h e f o l l o w i n g W d a t a (Table 1).
Table 1 UV c h a r a c t e r i s t i c s of q u i n i d i n e
sulfate
X max. a t nm -
E
205 5 -
231 -
273 31320
317.5 5089.5
331 5481
Other r e p o r t e d W s p e c t r a l d a t a for
q u i n i d i n e s u l f a t e i n methanol (1):-
Amax. a t 208 nm, 236 nm (34900), 280 nm
(3740) and 334 nm ( 5 8 7 0 ) .
F i g u r e 1. The W Spectrum o f Q u i n i d i n e S u l f a t e i n Ethanol
0.
and f o r q u i n i d i n e i n e t h a n o l (11):-
Amax. a t 236 nm ( E 1%, 1 cm 1110), 278 nm
( E 1%, 1 cm 1 3 2 ) and 332 nm ( E l%, 1 cm 163).
2.8.2 I n f , r a r e d Spectrum
The I R spectrum of q u i n i d i n e s u l f a t e as KBr-

d i s c w a s recorded on a P e r k i n Elmer 580 B
I n f r a r e d Spectrophotometer t o which I n f r a r e d
Data S t a t i o n i s a t t a c h e d ( F i g . 2 ) . The
s t r u c t u r a l assignments 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 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 q u i n i d i n e
sulfate
-1
Frequency ,cm As si gnment
3340 OH bonded
2300 NH+( q u i n u c l i d i n e )
2950 CH s t r e t c h
1620 CN
[ C=C ( a l k e n e )
1600,1510,1475 C=C (aromatic)
1245,1230,1100 ,1050 e t h e r linkage
860,835,800,720 T r i s u b s t i t u t e d benzene
The I R e x h i b i t e d t h e f o l l o w i n g o t h e r
c h a r a c t e r i s t i c bands:
1435, 1360, 1310, 1150, 940, 920, 765, 625
and 610 cm-l.
Other I R d a t a f o r q u i n i d i n e s u l f a t e (1)and
f o r q u i n i d i n e (11)have been a l s o r e p o r t e d .
Hayden and Sammul ( 1 2 ) d e s c r i b e d t h e I R

s p e c t r a of dimorphous and amorphous forms
of q u i n i d i n e .
2.8.3 Nuclear Magnetic Resonance S p e c t r a
2.8.3.1 Proton S p e c t r a
The PMR s p e c t r a of b o t h q u i n i d i n e
s u l f a t e i n DMSO D6 and q u i n i d i n e
i n C D C l 3 were r e c o r d e d on a Varian
T - ~ O A , 60 MHz NMR Spectrometer

u s i n g TMS ( T e t r a m e t h y l s i l a n e ) as an
i n t e r n a l r e f e r e n c e . These are shown
i n F i g . 3 and Fig. 4 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 a l assignments
have been made (Table 3).
Table
Group
2H
3H
5H
7-H, 8-H
vinylic
0-CH3
quinuclidine
s = s i n g l e t , d=doublet q = q u a r t e t , m=
m u l t i p l e t bs=broad s i n g l e t , b d =broad doublet.
Other PMR d a t a of q u i n i d i n e have been r e p o r t e d (1).
I , , 1 1 1 , I . . I .... ... .
l " " I " " 1 '
500
I ' 1 1
400 *
I I
iw
. I
lk
'
iI
I
. -
I
*
.
1 . . . . 1 -
I
... 1 . - .. 1 I . 1
1 . . . . 1 . . . . 1 . . . . 1 . . . . l . * . . ~ ,
. I
FIG. 3 MhR SPECTRUM OF Q U I N I D I N E SULFATE I N OMSO Dg

500
FIG. 4 KHR SPECTRUM OF QUINIDINE IN CDCL~

2.8.3.2 13C-NMR
The l3C-NMR n o l s e decoupled and o f f

resonance s p e c t r a are p r e s e n t e d i n
F i g . 5 and Fig. 6 r e s p e c t i v e l y .
Both were r e c o r d e d over 5000 Hz
width i n DMSO D6 ( c o n c e n t r a t i o n
72.4 mg/l m l ) on J o e l FX-100 NMR
Spectrometer, u s i n g a 1 0 mm sample
t u b e and TMS as a r e f e r e n c e s t a n -
d a r d a t 20’. The carbon chemical
s h i f t s a r e a s s i g n e d on t h e b a s i s o f
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 resonance s p l i t t i n g p a t t e r n
( T a b l e 4).
Table 4. Carbon Chemical S h i f t s of

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

[ PPm 1 PPml
125.82( s )
‘6 157.50(s ) c4
147.17 ( d) C 121.53( d )
7
145.90( s ) C 118.71(t)
c3 19
C
9
143.56( s ) c2 115.98(d)
139.40(d) C 101.94(d )
‘18 5
‘8 130*99(d) 5 0
66.86( a )
Figure 5. The 13C-NMR Noise Decoupled Spectrum of

Quinidine Sulfate
I
Figure 6. The 13C-NMR O f f Resonance Spectrum of
Quinidine Sulfate

[.PPml PPml
51 58.97(d)
'17
37.72 (d)
'16 56.33(t 1 '13

26.99 ( d )
c20
48.34 ( 9 ) 23.19(t)
C 18.22(t)
15 47.56(t) '14
s = s i n g l e t , d=doublet , t e r i p l e t , q = q u a r t e t .
Other 13C-NMR d a t a f o r q u i n i d i n e ( 1 3 , 1 4 ) ,
q u i n i d i n e maleate ( 1 3 ) , e p i q u i n i d i n e (14 ) and
dihydroquinidine (14) have a l s o been r e p o r t e d .
2.8.4 Mass Spectrum
The mass spectrum of q u i n i n e h y d r o c h l o r i d e

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
which w a s recorded on a Finnigan Model 3000
D GC-MS-system. The spectrum scanned t o
mass 500 w i t h u n i t r e s o l u t i o n . E l e c t r o n
energy w a s 7Oev. The chemical i o n i z a t i o n
mass s p e c t r a l d a t a were o b t a i n e d on a
Finnigan Model l O l 5 D GC Mass Spectrometer.
Methane w a s used as GC c a r r i e r gas and a l s o
served as t h e C I r e a c t a n t g a s i n t h e i o n
source. The i o n s o u r c e t e m p e r a t u r e was
1 8 0 O c and e l e c t r o n energy 100 ev., i o n
r e p e l l e r , 3V.
E l e c t r o n impact mass s p e c t r a l d a t a i s pre-
s e n t e d i n Fig. 7 ( 1 5 ~ 6 )
Base Peak 136
42, 55, 67, 81, 95, 117, 122, 136, 158, 173,
174, 189, 214, 226, 240, 253, 269, 283, 295,
309, M'324
The fragmentation p a t t e r n is p r e s e n t e d below
(16)
C I mass s p e c t r a l d a t a and prominent fragment
i o n s are ( 1 5 )
M.H+ 325(100), 3 0 7 ( 1 2 ) , 1 3 6 W , 2 9 5 ( 4 ) ,
323(4)*
'?in
r-
0 t
7
u) tn
0
M
z
H3CO
q u i n i d i n e m / e 324
3. P r e p a r a t i o n of q u i n i d i n e s u l f a t e
Quinidine i s t h e dextrorotatory diastereoisomer of

q u i n i n e which i s o b t a i n e d from v a r i o u s s p e c i e s of
cinchona or t h e i r h y b r i d s and from Remijia pendunculata
a l l of t h e f a m i l y Rubiaceae. It i s found i n cinchona
b a r k s t o t h e e x t e n t of 0.25-3.0% (7,17,18 ) .
3.1. I s o l a t i o n of q u i n i d i n e
S e v e r a l methods have been r e p o r t e d f o r t h e i s o l a -

t i o n o f q u i n i d i n e from cinchona b a r k , t.he most
important method i s as follows ( 19):-
Powdered cinchona (25Og) i s w e l l mixed w i t h C a O

( 6 0 g ) , water (600 m l ) and 30% NaOH s o l u t i o n (301111)
and l e f t f o r 24 hours. The mixture i s t h e n exhaus-
t i v e l y e x t r a c t e d under r e f l u x w i t h benzene. The
benzene e x t r a c t i s f i l t e r e d w h i l e hot i n t o a
separating funnel containing concentrated H SO
2. 4
( 7 g ) i n water (500 m l ) t o convert t h e a l k a l o i d s
t o t h e i r b i s u l f a t e s a l t s . The mixture i s s e p a r a t e d
and t h e a c i d aqueous l a y e r i s h e a t e d t o 90' and
n e u t r a l i s e d w i t h 5% Na2C03 s o l u t i o n u s i n g l i t m u s
as an i n d i c a t o r (The b i s u l f a t e s a l t s a r e now
converted i n t o t h e s u l f a t e s a l t s ) . The c l e a r
orange s o l u t i o n becomes t u r b i d due t o t h e separa-
t i o n of some r e s i n o u s m a t e r i a l . D i l u t e H2S04
( 2 d r o p s ) and animal c h a r c o a l ( 2 g ) a r e added and
t h e r e s u l t i n g mixture i s h e a t e d a g a i n a t 90' f o r
1 5 minutes and f i l t e r e d . The f i l t e r a t e i s allowed
t o cool down when q u i n i n e s u l f a t e s e p a r a t e s o u t
and c o l l e c t e d by f i l t e r a t i o n , The c o l d f i l t e r a t e
i s rendered a l k a l i n e w i t h NaOH s o l u t i o n and shaken
s u c c e s s i v e l y w i t h e t h e r . The e t h e r e x t r a c t which
c o n t a i n s q u i n i d i n e and c i n c h o n i d i n e i s evaporated
t o dryness and t h e r c s i d u e i s d i s s o l v e d i n d i l u t e
h y d r o c h l o r i c a c i d . Few drops of haematoxylin
i n d i c a t o r are added and t h e r e s u l t i n g m i x t u r e i s
n e u t r a l i z e d w i t h ammonia s o l u t i o n t o f a i n t yellow
c o l o r . Sodium potassium t a r t a r a t e ( R c c h e l l e s a l t )
i s now added and t h e mixture k e p t f o r a while upon
which c i n c h o n i d i n e t a r t a r a t e i s p r e c i p i t a t e d and
removed by f i l t e r a t i o n . The f i l t e r a t e c o n t a i n i n g
soluble quinidine tartarate i s t r e a t e d with potas-
sium i o d i d e s o l u t i o n where q u i n i d i n e hydrogen
i o d i d e i s p r e c i p i t a t e d , c o l l e c t e d and decomposed
w i t h ammonia when f r e e q u i n i d i n e p r e c i p i t a t e s o u t .
Schematic method f o r t h e i s o l a t i o n of major c i n -
chona a l k a l o i d s i s p r e s e n t e d i n F i g . 8.
Powder cinchona + C a O + NaOH + Water

r e f l u x with benzene
J7
hot f i l t r a t e
4
e x t r a c t with d i l . H2S04
Alkaloids b i s u l f a t e
Alkaloids s u l f a t e , b o i l with charcoal

4
Cool f i l t r a t e
1 1
filtrate
Quinidine, Cinchonine, Cinchonidine sulfate
Add NaOH and e x t r a c t with e t h e r
.c I
4
+
Add b o i l i n g water+Na2C03
Q.uinine
aqueous ether
Cinchonine Quinidine, Cinchonidine
evaporate t o dryness e x t r a c t with d i l . H C 1
e x t r a c t with alcoholt
decolorise with charcoal
and leave t o c r y s t a l l i s e
I
neutralize acid solution,
Cinchonine add Na k tartarate
PA
I
4
filtrate
(Cinchonidine t a r t . ) (Quinidine t a r t . )
Add H C 1 Ada IU
Alkaloid H C 1
+ NH40H
1
ppt. (Quinidine H 1 )
1
Cinchonidine
Add + NHhOH
Quinidine
J
Fig.8 Schematic Method for t h e Isolation of Cinch-

ona Alkaloids.
3.2. Quinidine s u l f a t e
Q u i n i d i n e s u l f a t e i s o b t a i n e d by n e u t r a l i z i n g t h e
a l k a l o i d q u i n i d i n e w i t h d i l u t e s u l f u r i c a c i d and
r e c r y s t a l l i s i n g from b o i l i n g water t o g i v e f i n e
n e e d l e l i k e c r y s t a l s of q u i n i d i n e s u l f a t e ( 2 0 ).
4. S y n t h e s i s of Q u i n i d i n e
4.1. P a r t i a l S y n t h e s i s
Rabe and Kindler i n 1918 ( 2 1 ) achieved t h e f i r s t
p a r t i a l s y n t h e s i s of q u i n i n e and q u i n i d i n e from
quinotoxine .
Quinotoxine w a s converted by t h e a c t i o n of sodium
hypobromite i n t o N-bromoquinotoxine which w a s
c y c l i z e d by a l k a l i w i t h t h e l o s s of hydrogen
bromide t o g i v e quininone. Reduction of t h e k e t o n e
w i t h aluminium powder and e t h a n o l i n t h e p r e s e n c e
o f e t h o x i d e gave a mixture of s t e r e o i s o m e r i c a l c o -
h o l s from which q u i n i n e and q u i n i d i n e were i s o l a t e d .
G u t z w i l l e r and Uskokovic i n 1973 ( 2 2 ) developed

a s l i g h t l y d i f f e r e n t scheme f o r t h e p a r t i a l synth-
e s i s of q u i n i n e and q u i n i d i n e from quinotoxine.
Q u i n i t o x i n e w a s d i s s o l v e d i n dichloromethane and
t r e a t e d w i t h sodium h y p o c h l o r i t e s o l u t i o n t o g i v e
N-chloroquinotoxine. This was c y c l i z e d w i t h phos-
p h o r i c a c i d t o g i v e a mixture of q u i n h o n e and
quinidinone. The r e s u l t i n g m i x t u r e was d i s s o l v e d
i n benzene and t r e a t e d with a s o l u t i o n of d i i s o -
butylaluminium h y d r i d e i n t o l u e n e t o g i v e a mixtu.re
of q u i n i n e and q u i n i d i n e which w a s s e p a r a t e d by
crystallization.
Q u i n i d i n e can a l s o be prepared by p a r t i a l racemiza-
t i o n of q u i n i n e ( 2 3 ) . Q u i n i n e i s t r e a t e d w i t h a
meta.lic a l k o x i d e where p a r t i a l r a c e m i z a t i o n o c c u r s
t o give quinidine.
4.2. Total Synthesis
S e v e r a l methods f o r t h e t o t a l s y n t h e s i s o f
q u i n i d i n e have been r e p o r t e d @+-29,22).Two of
t h e s e methods are p r e s e n t e d i n scheme 1 and 2.
Other methods are i n c l u d e d i n q u i n i n e hydroch-
l o r i d e ( 30).
Method I Total Synthesis according to Uskokovic

-25,26).
N-benzoylhexahydroisoquinolone [ 11 is hydrogenated
with rhodium on alumina catalyst to give predomi-
nantly cis-isoquinolone [ 21 which is treated with
sodium azide in poly-phosphoric acid to give a
mixture of the seven-membered lactams which is
separated by fractional crystallisation to give [ 31.
Lactam [3] is treated with dinitrogen tetroxide to
give the N-nitrosolactam [4] which is rearranged
upon heating to the diazolactone [5] and fragmented
with extrusion of nitrogen to give a mixture of
racemic N-benzoylmeroquinene [ 61 and the seven
membered lactone [gal (in 50 and 30% yield, respec-
tively). The latter [?a] can be converted into [6]
which upon esterification gives N-benzoylmero-
quinene methyl ester [6al. This ester is treated
with 6-methoxylepidyllithium [ 71 in tetrahydrofuran
to give the racemic N-benzoylketone [ 8 ] . This is
treated with diisobutylaluminium hydride in toluene
at - 78' [route a] to remove the benzoyl group with
concomitant reduction of the ketone function to
give the aminoalcohol [9]. The aminoalcohol [ 91 is
first acetylated with acetic acid containing 10%
boron trifluoride etherate and treated with boiling
benzene - acetic acid - sodium acetate where cycliza-
tion proceeds to give a mixture of desoxyquinine
and desoxyquinidine [ 1 2 ] . This can also be achieved
without acetylation. The aminoalcohol [9] is reflu-
xed with benzene - acetic acid mixture ( 4 : 1 ) for
4-5 days when cyclization proceeds via dehydration
[ 113 to give both desoxyquinine and desoxyquinidine
[121.
Upon stirring a solution of [12] in dimethylsulfo-
xide-t-butylalcohol ( 4 : l ) containing potassium
t-butoxide in an atmosphere of oxygen affords a
mixture of quinine [13] and quinidine [ 1 4 ] . Separa-
tion can be effected by a combination of crystalliza-
tion and chromatography.
An alternative synthetic route [b] via the amino

epoxide [lo] is as follows:-
N-benzoylketone [8] is converted into a mixture

of diastereomeric N-benzoyl epoxides [ 10a] by
bromination followed by sodium borohydride
Scheme 1: Total Synthesis of Quinidine (Method I)

-
[a1 Acetylation
[91
MOHAMMED A . LOUTFY ETAL.
reduction. Reductive debenzoylation of [loa] with

diisobutylaluminium hydride in toluene at - 78'
furnished a mixture of diastereomeric amino-
epoxides [ 101. Treatment of [ 103 with toluene-
ethanol (19:l) at reflux for 12 hr. give quinine
[13] and quinidine [14]. Separation is effected
by preparative thin layer chromatography.
Method I1 Total Synthesis of Quinidine ( 29,22)

3-[ 3(R)-ethyl-4(R)-piperidyl]-propionic acid
ethyl ester [l] is N-chlorinated with N-chloro-
succinimide in a two phase system (waterlether)
to give N-chloramine [2]. This is in trifluoro-
acetic acid is subjected to photolysis by a 200 W
Hanovia medium pressure mercury lamp below 15' to
give 3-[3(R)-( 2-~hloroethyl)-b(R)-piperidyl]-
propionic acid ethylester [3]. Compound [3] is
condensed with benzoyl chloride [4] to give the
condensate 3-[ l-Benzoyl-3(R)-( 2-chloroethy1)-
4(R)-piperidyll-propionic acid ethylester [ 51.
Compound [ 5 ] is saponified to give 3-[ 1-benzoyl-3
( R ) - ( 2-chlorethyl)-b (R ) -piperidyl]-propionic acid
[6]. Dehydrochlorination of compound [6] with
potassium t-butoxide in DMSO gives N-benzoyl-
homomeroquinene [ 7 ] which is esterified to give
N-benzoyl-homomeroquinene ethylester [ 81. Compound
[8]can be proceeded to quinotoxine via two routes.
Route [a] Claisen condensation of [8]with ethyl
quininate [9] to give the 6-keto ester [lo] which
by hydrolysis and decarboxylation gives quinotoxine
[ 111. Route [b] Condensation of [8]with 6-methoxy-
4-quinolyllithium [ 121 affords N-benzoylquinotoxine
[ 131 which upon hydrolysis yields quinotoxine [ 111.
Quinotoxine [ll] is dissolved in dichloromethane
and treated with sodium hypochlorite solution to
give N-chlorotoxine [14]. This is cyclized by
treatment with phosphoric acid to give a mixture
(1:l) of quininone [15] and quinidinone [16]. The
resulting mixture is dissolved in benzene and redu-
ced with diisobutylaluminium hydride (DIBAL-H) to
give a mixture of quinine [17] and quinidine [18]
which is separated by crystallization.
Scheme 2: Total Synthesis of Quinidine (Method 11)
I
COOC2Hg COOC2H5
I
COOC2H5
C 00 C 2 H 5
& H
+
[31
c1
2 COCl
[41
0 c6H5
0
0 c6H5 C6H5
[6 1
R R
I
I
C151
R=OCH3
5. Biosynthesis of Quinidine
Postulation of the biosynthetic pathway of cinchona

alkaloids started in 1950 with the suggestion o f Goutarel
et al. ( 31) that cinchona alkaloids are derived from
indolic precursors since cinchonamine (indole alkaloid)
occurs as a minor alkaloid in cinchona. This w a s proved
when Kowanko and Leete (32) have isolated labelled quinine
upon feeding try~tophan-2-~~C into cinchona plants. They
have shown that the quinoline ring and Cg unit of quinine
originated from tryptophan. Further studies have proved
that quinine is biosynthesized by a combination of indolic
and monoterpenoid units which leads to the corynanthe type
indole alkaloids. Thus tryptophan (32) geraniol (33-35)
and loganin (36 ) were incorporated into quinine. Tracer
experiments on Cinchona ledgeriana carried out by Batter-
sby and. Parry ( 37) have established the biosynthetic path-
way of quinine and quinidine as presented in scheme 111.
Scheme 111: Biosynthesis of quinine and quinidine.

__T
corynantheal
I
H
I
H A
I
Quininone
Quinidinone
H
Quinine
L.
I
H?CO
Quinidine
6. Metabolism
The metabolism of quinidine has been extensively studied
in human and rat urines. The metabolic products of
quinidine found in man are:-
(3s)-3-hydroxyquinidine (13,16,38) $-quinidinone (13,16)
¶
O-desmethylquinidine (39,40), and quinidine-N-oxide

( 41,421.
Brodie et al. (43) reported the presence of monohydroxy-
non-phenolic metabolite of quinidine, quinidine carbosty-
ril and dihydroxy non-phenolic metabolite of quinidine.
Palmer et al. (16)have suggested the existence of other
unidentified hydroxylated metabolites as well as conju-
gated compounds.
Barrow et al. (44) have separated nine metabolites of
quinidine (Fig. 11) in the urine of male Sprague-Dawley
rats after a single dose of quinidine ( 5 0 mg/Kg). Five
of these metabolites have been identified as:
O-desmethylquinidine, 3-hydroxyquinidine, unchanged
quinidine and the two diastereoisomers of quinidine-
10, ll-dihydrodiols. They concluded that the urinary
profiles of the rat and man are different. 2'-Quini-
dinone, a major metabolite in man was not detected in the
rat and the two diastereoisomers of quinidine 10, 11-
dihydrodiols were only reported in the rat urine (44).
The major metabolites in man are:-
( 3s )-3-hydroxyquinidine
Ho\
H H
HO
2-quinidinone 0-Desmethylquinidine
7. Pharmacokinetics
When a d m i n i s t e r e d o r a l l y , q u i n i d i n e s u l f a t e i s absorbed
r a p i d l y and peak c o n c e n t r a t i o n s i n plasma are a t t a i n e d
i n 60 t o 90 minutes. The a b s o r p t i o n of q u i n i d i n e glu-
conate i s slower and maximal c o n c e n t r a t i o n s a r e not
observed u n t i l 3 t o 4 hours a f t e r an o r a l dose ( 4 5 ) .
Q u i n i d i n e accumulates r a p i d l y i n most t i s s u e s except
b r a i n , and t h e apparent volume of d i s t r i b u t i o n i s 2 t o
3 l i t e r s p e r kilogram ( 4 5 ) . Following o r a l a d m i n i s t r a t i o n ,
t h e a b s o l u t e b i o a v a i l a b i l i t y of q u i n i d i n e i s about 70%
o f t h e i n g e s t e d dose b u t may v a r y widely between p a t i e n t s
(46,47,48).
Plasma q u i n i d i n e c o n c e n t r a t i o n s are g e n e r a l l y h i g h e r and
appear e a r l i e r when t h e drug i s a d m i n i s t e r e d on an empty
stomach (49,50). About 70 t o 80% of q u i n i d i n e i n plasma
i s bound t o plasma albumin. The drug e n t e r s e r y t h r o c y t e s
and a p p a r e n t l y b i n d s t o hemoglobin; a t a s t e a d y s t a t e
c o n c e n t r a t i o n s of q u i n i d i n e i n plasma and e r y t h r o c y t e s
are approximately e q u a l s ( 5 1 ) .
Q u i n i d i n e i s metabolized by t h e l i v e r and e x c r e t e d i n
t h e u r i n e . The mean v a l u e f o r t h e e l i m i n a t i o n h a l f - l i f e
of q u i n i d i n e i s 6 t o 7 hours (45,52-54). Ochs e t a l .
( 5 4 ) have r e p o r t e d t h a t t h e e l i m i n a t i o n h a l f - l i f e of
q u i n i d i n e i s g r e a t e r i n t h e e l d e r l y persons ( o v e r 60 y e a r s )
when compared t o t h e younger p e r s o n s (less t h a n 35 y e a r s ) .
T o t a l body q u i n i d i n e c l e a r a n c e i s about 4.5 ml/min/Kg
w i t h wide p a t i e n t t o p a t i e n t v a r i a t i o n s (48, 53).
Since q u i n i d i n e i s a weak b a s e , e x c r e t i o n i s enhanced i f

t h e u r i n e i s a c i d i c . When t h e u r i n a r y pH i s i n c r e a s e d
from t h e 6-7 range t o t h e 7-8 r a n g e , r e n a l c l e a r a n c e of
q u i n i d i n e d e c r e a s e s as much a s 50% and c o n c e n t r a t i o n i n
t h e plasma i n c r e a s e s ( 5 5 ) .
8. Routes of A d m i n i s t r a t i o n , Dosage and P r e p a r a t i o n s

Q u i n i d i n e i s u s u a l l y given o r a l l y , although it can be
administered e i t h e r intramuscularly o r intravenously
under s p e c i a l circumstances.
The u s u a l o r a l dose o f q u i n i d i n e s u l f a t e i s 300 t o 500 mg
f o u r t i m e s a day. I n most p a t i e n t s q u i n i d i n e w i l l r e a c h
a s t e a d y s t a . t e on such a schedule i n about 24 hours and
i t s c o n c e n t r a t i o n i n plasma w i l l f l u c t u a t e l e s s t h a n 50%
between doses ( 4 5 ) . Because of t h e l a r g e i n t e r i n d i v i d u a l
v a r i a t i o n , d r u g i n t e r a c t i o n s , and o t h e r causes of v a r i -
a b i l i t y , it i s w i s e t o examine t h e ECG c a r e f u l l y a f t e r
t h e i n i t i a l dose of q u i n i d i n e and t o measure t h e plasma
c o n c e n t r a t i o n of t h e drug a t s t e a d y s t a t e . Adjustment
o f dosage i s o f t e n necessary. I f an e f f e c t i v e concentra-
t i o n must be achieved r a p i d l y , a l o a d i n g dose of 600-
1000 mg can be given ( 4 5 ) .
Q u i n i d i n e s u l f a t e , U.S.P.,
T a b l e t s and c a p s u l e s c o n t a i n 1 0 0 , 200 o r 300 mg of t h e
drug. P r e p a r a t i o n s f o r slow a b s o r p t i o n a r e a l s o a v a i l a b l e ,
t h e s e i n c l u d e a 300-mg extended-release t a b l e t of q u i n i -
dine sulfate (Quinidex) .
Q u i n i d i n e s u l f a t e i s a l s o a v a i l a b l e a s an i n j e c t i o n i n
1 m l a m p o u l s c o n t a i n i n g 200 mg/ml. The n e c e s s a r y dose i s
d i l u t e d t o 800 mg/50 m l i n 5% glucose s o l u t i o n and i s
i n j e c t e d i n t h e r a t e of 1 6 mg p e r minute, w i t h continuous
o b s e r v a t i o n of t h e p a t i e n t and of t h e ECG. It i s impor-
t a n t t o record t h e a r t e r i a l pressure a t frequent i n t e r v a l s
( 45 1.
9.1 Identification
9.1.1 Color Tests
The following c o l o r t e s t s have been

reported (8,11,56).
a. Thalleioquin t e s t : The a d d i t i o n of
2 drops of bromine s o l u t i o n t o 5 m l of
a s a t u r a t e d s o l u t i o n of quinidine or
quinine or a 1:lOOO s o l u t i o n of t h e i r
s a l t s , followed by 1 ml of ammonia solu-
t i o n produces an emerald-green c o l o r due
t o t h e formation of t h a l l e i o q u i n .
Quinidine and i t s diastereoisomer quinine
a r e d i f f e r e n t i a t e d by ( i )t h e i r o p t i c a l
r o t a t i o n s (quinidine i s dextrorotatory
while quinine i s l e v o r o t a t o r y ) , and by
( i i )t h e i r behavior toward a l k a l i t a r t a -
r a t e (18). I n n e u t r a l or s l i g h t l y a c i d
s o l u t i o n s quinine i s p r e c i p i t a t e d by
a l k a l i t a r t a r a t e , while quinidine i s not.
( i i i )On t h e o t h e r hand, quinidine i n
moderately d i l u t e s o l u t i o n i s p r e c i p i t a t e d
by soluble iodides but quinine i s not
a f f e c t e d (18).
b. To a 0.5% w/v s o l u t i o n add an equal
volume of M sulphuric a c i d ; an i n t e n s e
blue fluorescence i s produced ( 9 ) .
c. A 1% s o l u t i o n gives a yellow but not
a blue c o l o r with bromothymol blue ( 6 ) .
d. To 5 m l o f a 1% s o l u t i o n add 1 m l
of s i l v e r n i t r a t e s o l u t i o n and s t i r with
a g l a s s rnd; a f t e r a s h o r t i n t e r v a l a
white p r e c i p i t a t e , s o l u b l e i n n i t r i c a c i d ,
i s produced ( d i s t i n c t i o n from many o t h e r
alkaloids ) ( 6) .
e. Quinidine sulphate y i e l d s t h e reac-
t i o n s c h a r a c t e r i s t i c of sulphates.
A study of color changes of
quinidine and o t h e r a l k a l o i d s , i n r e l a t i o n
t o time has been described (57). The
c o l o r t e s t s with concentrated sulphuric
a c i d , Erdmann's , Froehde's, Mandelin's,

and Marquis's r e a g e n t a p p l i e d t o a l a r g e
number of a l k a l o i d s , i n c l u d i n g q u i n i d i n e ,
have been r e p o r t e d ( 5 7 ) .
9.1.2 Micro-Crystal Tests
Photomicrographs o f t h e c r y s t a l s formed
are i l l u s t r a t . e d i n Fig.9 (58).
The following m i c r o - c r y s t a l t e s t s are a l s o
useful i d e n t i f i c a t i o n tests:
i ) Potassium i o d i d e s o l u t i o n g i v e s
irregular crystals, often triangular
( s e n s i t i v i t y : 1 i n 3000) (11).
i i ) Sodium c a r b o n a t e s o l u t i o n produces
dense r o s e t t e s , forming o v e r n i g h t ( s e n s i -
t i v i t y : 1 i n 1000) (11).
iii) Dissolve t h e sample (1mg) i n water
( 2 ml), a c i d i f y w i t h d i l u t e s u l p h u r i c
a c i d (1 d r o p ) and add a few drops o f an
aqueous s o l u t i o n c o n t a i n i n g 5% o f cadmium
i o d i d e and 10% o f potassium i o d i d e .
Q u i n i n e g i v e s c o l o r l e s s c r y s t a l s and
t h e s o l u t i o n becomes t u r b i d ; q u i n i d i n e
g i v e s pale-yellow c r y s t a l s more s l o w l y ,
but t h e c o l o r change o f t h e s o l u t i o n i s
e a s i l y d e t e c t e d . This r e a c t i o n h a s been
u t i l i s e d f o r t h e d i s t i n c t i o n between
q u i n i n e and q u i n i d i n e ( 5 9 ) .
9.2 Gravimetric Method
Vignoli e t a l . ( 6 0 ) have d e s c r i b e d d e t e r m i n a t i o n
o f a l k a l o i d c o n t e n t of cinchona powder by e x t r a c -
t i o n w i t h aqueous s o l u t i o n s of v a r y i n g pH. The
extract w a s evaporated t o a sludge on a steam
b a t h and t h e n oven-dried a t 95'. The d r y e x t r a c t
was weighed and t h e a l k a l o i d c o n t e n t determined.
9.3 T i t r i m e t r i c Methods
9.3.1 Aqueous
Schneider ( 6 1 ) h a s d e s c r i b e d t h e determina-
t i o n of some m i n e s a l t s , i n c l u d i n g quini-
d i n e s u l f a t e . The salt ( 0 . 1 g m ) i s d i s -
s o l v e d i n 90% e t h a n o l ( 1 0 m l ) , and t h e
FIG, 9. l ? I C R O C H E M I C A L C R Y S T A L S OF O U l N l D l N E
SULFATE WITH KI.
s o l u t i o n is p a s s e d , a t 2 t o 3 m l p e r
minute, through a n anion-exchange r e s i n .
The combined p e r c o l a t e s are d i l u t e d w i t h
d i s t i l l e d water and t h e l i b e r a t e d q u i n i -
d i n e i s t i t r a t e d w i t h 0 . 1 N HC1 u s i n g
T a s h i r o ' s i n d i c a t o r , u n t i l t h e green-blue
c o l o r changed t o v i o l e t ( 6 2 ) .
Thomis and K o t i o n i s ( 6 3 ) have p u b l i s h e d
t h e i n f l u e n c e of o r g a n i c b a s e s on t h e
p a r t i t i o n o f i n d i c a t o r a c i d s (and v i c e
v e r s a ) i n a water-chloroform system. T h i s
a f f o r d s a method f o r t i t r a t i n g q u i n i d i n e
and o t h e r b a s e s . A s o l u t i o n o f t h e sample
( 0 . 2 m l ) , mixed w i t h 2 ml of b u f f e r solu-
t i o n (pH 5.5) and 15 m l of chloroform,
i s t i t r a t e d w i t h 0.001 M bromothymol b l u e ,
vigorous shaking b e i n g a p p l i e d between
a d d i t i o n s . The end p o i n t i s marked by
a yellow c o l o r i n t h e aqueous l a y e r . This
t i t r a t i o n i s used as a n approximate d e t e r -
mination. An a c c u r a t e d e t e r m i n a t i o n i s
t h e n made by adding an excess of 0.0004M
bromothymol b l u e t o t h e s o l u t i o n o f t h e
b a s e , b u f f e r e d a t pH 7.5, e x t r a c t i n g w i t h
chloroform and determining t h e bromothymol
bl.ue i n t h e chloroform l a y e r by e x t r a c t i n g
it w i t h aqueous sodium hydroxide and com-
paring t h e color of t h e a l k a l i n e e x t r a c t
w i t h s t a n d a r d s . These p r i n c i p l e s are used
i n t h e determination of a l k a l o i d s , includ-
i n g q u i n i d i n e , and o t h e r b a s i c drugs i n
some pharmaceutical p r e p a r a t i o n s .
9.3 2 Non-Aqueous
Non-aqueous t i t r a t i o n s have been p u b l i s h e d
f o r t h e q u a n t i t a t i o n of q u i n i d i n e a l k a l o i d
and s a l t . The drug i s t i t r a t e d by per-
c h l o r i c a c i d i n a c e t i c a c i d and t h e end-
p o i n t i s determined p o t e n t i o m e t r i c a l l y .
The method w a s used f o r t h e d e t e r m i n a t i o n
of q u i n i d i n e s u l f a t e , u s i n g b r i l l i a n t
green and F e t t b l a u B as i n d i c a t o r s ( 6 4 ) .
Non-aqueous t i t r a t i o n o f s m a l l amount of
a l k a l o i d i n t h e p r e s e n c e of a n a d s o r p t i o n
e l e c t r o d e h a s been r e p o r t e d ( 6 5 ) .
Determination of q u i n i d i n e , and o t h e r
a l k a l o i d s , by means of t h e hydrochloric
a c i d complex of chloroaluminium isopro-
poxide i n non-aqueous media, has been rep-
o r t e d ( 6 6 ) . The d e v i a t i o n i s ? 1%in the
range 38 t o 245 mg of t h e a l k a l o i d .
9.3.3 Complexometric
Rolski e t a l . (67) have r e p o r t e d complexo-

metric determination of a l k a l o i d s using
copper p i c r a t e . The method i s based on
t h e f a c t t h a t a l k a l o i d s give constant
complexes w i t h c u p r i c p i c r a t e ; t h e i r
compositions depend only on t h e t y p e of
a l k a l o i d , These complexes were p r e c i p i -
t a t e d from 0.1 gm a l k a l o i d s o l u t i o n with
0.02 M c u p r i c p i c r a t e . The p r e c i p i t a t e
w a s f i l t e r e d and washed, then 0.02 M
sodium v e r s e n a t e , ammonium b u f f e r (pH 10.4)
and murexide were added t o t h e f i l t r a t e .
The excess versenate w a s t i t r a t e d with
0.02 M zinc s u l f a t e t o t h e green color.
The method gave t h e error of ? 0.3%.
The a p p l i c a t i o n of volume-colorimetry
t o t h e micro-determination of a l k a l o i d s
has been described (68). I n t h i s method,
t h e a l k a l o i d i s p r e c i p i t a t e d with Schei-
b l e r ' s phosphotungstic a c i d r e a g e n t , t h e
p r e c i p i t a t e i s t r e a t e d with sodium amal-
gam, and t h e blue c o l o r of t u n g s t i c
anhydride i s tit r a t e d v o l u m e t r i c a l l y
with potassium permanganate t o a d i s -
appearance of t h e b l u e c o l o r .
Several o t h e r complexometric determina-
t i o n s of a l k a l o i d s , including q u i n i d i n e ,
have been a l s o r e p o r t e d (69-72).
9.3.4 Amperometric
The e l e c t r o g e n e r a t i o n of bromine has been

used i n t h e assay of alkenes (73-75).
The coulometric generation of bromine
as a t i t r a n t w a s used as a b a s i s of a
coulometric method of a n a l y s i s of quini-
dine s u l f a t e and o t h e r medicinals. The
r e a c t i o n between bromine and t h e s e drugs
i s t o o slow t o permit t h e use of a
conventional amperometric end-point

detection technique. A residual method
combining a standarised arsenite solution
with amperometry was found to give excel-
lent results ( 7 6 ) . Quinidine sulfate
gave relative standard deviation of less
than 2% and relative error of less than
0.5% using the arseno-amperometric end-
point device. The method was applied to
the analysis of quinidine sulfate powder
and quinidine sulfate tablets ( 7 6 ) .
9.3.5 Polarographic
Polarographic analysis of cinchona

alkaloids has been reported(77-79). The
oscillopolarographic behaviour of these
alkaloids was studied and the oscillo-
grams of quinidine in N LiC1, N LiOH,
N NaOH, and N H2SO4 were obtained with
the use of dropping and streaming mercury
electrodes ( 7 7 ) . The depolarisation
potentials were reported and various
possibilities of the differentiation of
similar derivatives were investigated.
Even 0.0001 M solution of quinidine ,
and other cinchona alkaloids, can be
detected with the use of oscillopola-
rographic methods.
Molnar (78) has published quantitative
polarographic determination of cinchona
alkaloids, including quinidine. These
alkaloids give characteristic oscillo-
grams which can be used for their determi-
nation with an accuracy of 24%.
9.4 Chromatography
9.4.1. Paper Chromatography
Clarke(l1) has described several solvent
systems for identification of quinidine,
as shown in Table 5.
524 MOHAMMED A . LOUTFY ETAL..
Table 5. Paper Chromatography of Quinidine
Solvent System Visualizing agent
1. Citric acid-n-butanol UV, iodoplatinate

-water (4.8gm:870 ml: spray
130 ml)
2. Acetate buffer
( pH=4.58 )
3. Phosfate buffer
(pH=7.4
Several paper chromatographic methods (80-85)have been

described for the separation, identification and quanti-
tative determination of cinchona alkaloids including
quinidine. Alwas et al. have described
application of paper ionophoresis for the separation of
alkaloid mixture including cinchona alkaloids (86). The
electrophoresis is carried out on Whatman No. 1 paper
at 1 mA per Cm and 300 V for 3 hours. The solution of
alkaloids (10 to 30 g) are applied to the paper near the
anode, and the strips are moistened with 1% aqueous
ammonium carbonate (pH 7-8). The separated alkaloids can
be determined quantitatively after elution from the paper.
Quenzer and Hardy (87)have reported a death investigation
involving quinidine. Chromatographic procedures, per-
formed on samples of brain, kidney, liver, blood, and
gastric content, indicated that the cause of death was an
overdose of quinidine. The drug extraction recovery from
spiked whole blood sample was 89%.
9.4.2 Thin-Layer Chromatography
About 35 cinchona alkaloids are known, of

which quinidine and quinine are the pharma-
ceutically most important, quinidine because
of its cardiac depressant and quinine beca-
use of its antimalarial properties. These
two alkaloids have been studied by TLC more
extensively than the other cinchona alkal-
oids such as the stereoisomers cinchonine
and cinchonidine (both of which lack the
methoxy group at C6, which is present in
Table 6. TLC Techniques Used for Quinidine
h Rf
Solvent System (Rf x 100) Ref.
1. Chloroform-diethylamine (9:l) 28 140

2. Chloroform-methanol - 25% ammonia (85:14:1) 44 130
3. Chloroform-acetone - diethylamine (5:4:1) 26 97,141
4. Chloroform-acetone - (3 m l 25% ammonia + 17 m l 26 142
zbsolute ethanol) (5:4:1)
5. Chloroform-acetone-methanol - 25% ammonia (60:20:20:1) 41 142
6. Chloroform-ethyl acetate - isopropanol-diethylamine 21 141
(20:70:4 :6)
7. Chloroform-dichloromethane-diethylamine (20:15:5 ) 34 -
8. Dichloromethane-diethyl ether-diethylamine (20:15:5) 35 -
9. Kerosene-acetone-diethylamine (23:9:9) 41 141
10. Acetone - 25% ammonia (58:2) 37 143
11. Ethyl acetate - isopropanol - 25% ammonia (45:35:5) 55 143
12. Toluene - ethyl acetate - diethylamine (7:2:1) 20 143
13. Toluene - ethyl acetate - diethylamine (10:10:3) 28 108
14. Toluene - diethyl ether - diethylamine (20:12:5) 26 108
15. Toluene - diethyl ether - dichloromethane-diethylamine 29 127
(20:20:20 :8)
contd.. ..
Solvent System h Rf
(Rf x 100) Ref.
16. Carbon tetrachloride - n - butanol - methanol - 10% ammonia 71 144

(12:9:9 :1)
17. Cyclohexanol-cyclohexane-n-hexane (1:l:l)+ 5% 60 144
diet hylamine
18. Methanol - 25% ammonia (1OO:l) 46 128
19. Strong ammonia - methanol (1.5:lOO) 55 11
20. Chloroform-acetone-diethylamine (20:20:1) 145
21. Benzene-diethyl ether-diethylamine (20:12:5 ) 101
22. Methanol - chloroform-diethylamine (50:50 :1) 45 137
Table 7. TLC Detection of Q u i n i d i n e
Reagent Background Color Ref.

Color
1. Quenching, 254 nm - -
2. Fluorescence, 366 nm (formic a c i d or Light b l u e 88
sulphuric a c i d s p r a y )
3. Dragendorf f ' s modification :
Munier - Macheboeuf Yellow Orange-red -
Munier Light yellow Orange-red -
Light yellow Brown 130
s Munier, sodium n i t r i t e
white
Vaguj f a l v i Light yellow Orange -
Bregoff-Delwiche Light yellow Orange 88
4. Iodine vapour Yellow white Brown 88
5. Iodine i n potassium i o d i d e White Brown -
6. Iodine i n methanol Light yellow Brown 146
7. Iodine vapour, p r r o l e vapour Yellow Brown 147
8. Iodine i n potassium i o d i d e and s i l v e r - - 135
acetate
9. F e r r i c c h l o r i d e , i o d i n e i n potassium i o d i d e Light green Brown 148
yellow
contd.. .
Color
- ~~~ ~
10. Iodoplatinate Violet Violet -

11. I o d o p l a t i n a t e , a c i d i f i e d Dark v i o l e t Violet 149
12. F e r r i c hexacyanoferrete (111) Light green Dark green 138
blue blue
13. F e r r i c chloride-perchloric a c i d Yellow white Violet -
14. Methyl orange Light orange Orange 134
15. Tetraphenylborate, q u e r c e t i n - InUV: b l u e 150
16. Phenothiazine, i o d i n e vapour Violet Brown 147
17. Phenothiazine, bromine vapour Violet Green 147
(ammonia vapour)
q u i n i d i n e and q u i n i n e ) , and t h e i r c o r r e s -
ponding dihydro d e r i v a t i v e s d i h y d r o q u i n i d i n e
and dihydroquinine ( i n which t h e v i n y l group
a t C 3 i s r e p l a c e d by an e t h y l g r o u p ) .
TL chromatographic s e p a r a t i o n of cinchona
a l k a l o i d s has been reviewed by Verpoorte,
e t al. ( 8 8 ) , w i t h emphasis on t h e mobile
phases used, and t h e s e n s i t i v i t i e s of
v a r i o u s d e t e c t i o n methods. Some g e n e r a l
conclusions are drawn on t h e e s t a b l i s h m e n t
of optimum c o n d i t i o n s f o r s p e c i f i c separa-
t i o n s of cinchona a l k a l o i d s . TLC s e p a r a t i o n
(89-106) and TLC i d e n t i f i c a t i o n of cinch-
ona a l k a l o i d s i n food(107-108) and i n b i o l o -
g i c a l material (109-123) have been r e p o r t e d .
S e p a r a t i o n , i d e n t i f i c a t i o n , and 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 q u i n i d i n e , and o t h e r cin-
,
chona a l k a l o i d s o f a b u s e have been r e p o r t e d
(124-136) S e p a r a t i o n o f q u i n i d i n e and
q u i n i n e from d i h y d r o q u i n i d i n e and dihydro-
q u i n i n e has been d e s c r i b e d (88).
Table 6 l i s t s s o l v e n t s used f o r t h e separa-
t i o n o f q u i n i d i n e . The TLC d e t e c t i o n methods
are given i n Table 7.
D e t e c t i o n of dihydro-alkaloids i n commercial
q u i n i d i n e and q u i n i n e has been p u b l i s h e d
(137).
Hashmi e t al. (138)have r e p o r t e d semi-
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 cinchona
a l k a l o i d s by c i r c u l a r TLC.
Robles and Wient j e s ( 139 ) have s t u d i e d , by
TLC, t h e decomposition on s t e r i l i z a t i o n ,
of q u i n i d i n e hydrogen s u l f a t e .
9.4.3
- G
A g a s - l i q u i d chromatographic d e t e r m i n a t i o n
of q u i n i d i n e s u l f a t e has been adopted i n our
l a b o r a t o r y , u s i n g a Varion GC-3700 gas
chromatograph equipped w i t h a flame i o n i s a -
t i o n d e t e c t o r . The g l a s s column ( 2 m x 2 mm
w a s packed w i t h 3% OV-17 on 80-100 mesh
Chromosorb W HP. The c a r r i e r gas ( h e l i u m )
flow-rate w a s maintained a t 25 ml/minute.
Ethanol w a s used a s 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 1 cm/minute.
530 MOHAMMEDA. LOUTFY E T A .
Fig. 10 GLC of Quinidine Sulfate
0-Desmethylquini.dine
3-Hydroxy-quinidine
Quinidine
Quinidine-lO.11-
dihydrodiols
I &
~~
L , 1 1 I I
0 10 20 30 40 50 60 70 min
Fig. 11. HPLC Separation of Quinidine
Metabolites
The retention time = 13.5 minutes. The

GLC of quinidine sulfate is illustrated
in Fig. 10.
Separation and quantitative determination
of cinchona alkaloids, among which quinidine
is included, by TLC and GLC, has been des-
cribed (97). In this method, the column
was packed with 2% OV-17 on AW Gas-Chrom p.
A GLC determination of dihydro impurities
in quinidine salts has been developed by
Smith et al. (95).
Midha and Charette (151)have described a
gas-liquid chromatographic determination of
quinidine from plasma and whole blood.
Metabolites of quinidine do not interfere
and the limit of detection is 50 ng of
quinidine per m l .
Other gas-liquid chromatographic assays of
quinidine has been also described (152-153).
9.4.4 High-Performance Liquid Chromatography
HPLC separation of quinidine metabolites is
given in Fig. 11 (44).
High-performance liquid chromatographic
systems for quinidine and its metabolites
are listed in Table 8 , along with speci-
ficity information and cited references.
Reece and Peikert (153) have described
a comparison of HPLC and GLC assays.
HPLC analyses of quinidine and dihydroquini-
dine in plasma samples have been carried
out using reversed-phase sy~tems(159~161-162).
An HPLC assay has been described (163),
employing extraction and post-column acidi-
fication with fluorescence detection, and
without use of internal standardisation.
Other HPLC procedures for quinidine and
other alkaloids have been reported (164-165).
9.5 Spectrophotometry
9.5.1 Colorimetric
A micro-determination of quinidine and

Table 8. HPLC Systems of Q u i n i d i n e
Column Mobile phase Retention t i m e Detection Ref.

(minute )
1. Merckosorb S i Chloroform-methanol W, 254 nm 154

60 (5 v m ) (8:2) 6.5 and 280 nm.
(7:3) 6.2
D i e t h y l ether-methanol
(8:2) 3.7
(7:3) 2.9
(6:4) 2.4
2. A reversed-phase , Methanol-water-acetic acid W , 254 nm 155
Bondapak
t
n
t4
W (25:75:1)
(80 :20 :1)
water-acetic a c i d ( 9 9 : l ) + U V , 254 nm. 44
w a ter-acet o n i trile -a c e t i c acid
(40 :59 :1)
1 . 5 mM phosphoric a c i d - fluorescence, 153
a c e t o n i t r i l e (90 :lo) 418 nm
0.05 M phosphate b u f f e r (pH=3)- UV, 254 nm 156
a c e t o n i t r i l e (73:27) and 325 nm.
3. A S i l i c a gel tetrahydrofuran-ammonium - 106
hydroxide
D i e t h y l ether-water-
diethylamine
~~ ~~ ~~
Mobile phase Retentlon time Detection Ref.

(minute)
4. Bondapak 0.05 M phosphate W,230 nm or 158

alkyl phenyl buffer (pH 4.75)- fluorescence
acetonitrile-tetrahyd-
rofuran (80:15:5)
0.75 M-acetate buffer (pH=3.6)- - W , 330 nm. 159

acetonitrile (3:2)
5. Li Chrosorb Si 60 Chloroform-isopropyl alcohol- UV, 312 nm 160
diethylamine-water
(940:57:1:2.65)
quinine in serum has been described (166).

The method is suitable for determining
concentration up to 1 mg per 100 ml, The
c o l o r , formed with Rose bengal, is measured
at 550 nm.
Vukcevic and Bozin( 167)have published a
quantitative analysis of alkaloids, includ-
ing quinidine, in cinchona tincture by
chromatography and spectrophotometry,
Schmitz and Menges (168) have determined
cinchona alkaloids in galenical preparations
with Tropaeolin 00.
Graham and Thomas (169) have reported
a quantitative assay of quinidine, and
other alkaloids, by color reaction with
dichromate-sulphuric acid.
9.5.2 Ultraviqlet
Kamath et. al. (170) have described the

W absorption spectra of quinidine, and
other cinchona alkaloids, in 11 aliphatic
alcohols. The extinction coefficient of
cinchonine and cinchonidine at 332 nm are
c! 2 to 4% of those of quinine and quinidine
at the same wavelength. Thus, quinidine,

or quinine, can be determined in the pres-
ence of cinchonine and or cinchonidine
to within 1 to 4% in aliphatic alcohol
medium.
Kracmar and Kracmarova (171) have studied
the influence of structure and solvent on
the spectrophotometric behaviour of quini-
dine, and other drugs containing quinoline
and isoquinoline chromophores.
9.5.3 Infrared
Hayden and Sammul (12) have reported a

study of the cinchona alkaloids in potassium
bromide discs and applied KBr -
disc tech-
nique to the analysis of these alkaloids.
Under certain experimental conditions,
anomalous IR spectra are obtained for
quinidine and quinine. Cinchonine and
cinchonidine do not exhibit significant
variations in spectra.
9.5.4 Nuclear Magnetic Resonance

In the development of a method for control
of dihydro impurities in preparations con-
taining quinidine and quinine salts considera-
tion was given to the use of NMR, following
Huynh-Ngoc and Sirois (172).
9.5.5 Atomic Absorption Spectrometry

Recently, an indirect determination of quini-
dine , and other alkaloids , by atomic absorp-
tion spectrometry, has been developed (173).
9.5.6 Spectrofluorimetric
Gelfman and Seligson (174)have described the

determination of quinidine in serum by pre-
cipitation-fluorescence method. In this
method, trichloroacetic acid is shown to be
a satisfactory substitute for metaphosphoric
acid as a protein precipitant in the fluori-
metric assay of quinidine in serum. The
original protein precipitation - fluorescr-
once method (175) is still widely used
together with extraction - fluorescence
methods( 176-177)to determine quinidine and
its metabolites.
Other fluorimetric methods for quantitation
of quinidine have been reported (118,178-179).
Ivanenko (180)has published fluorimetric
determination of quinidine in bile and blood.
The method is based on extraction technique.
Alekseichik et al. (181) has described
qualitative analysis of quinidine, and other
drugs, by determining their fluorescence
spectra in ethanol , concentrated HC1, 10%
NaOH, and aqueous solutions at 0.01-0.001
.
mg /ml
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ACKNOWLEDGEMENT
The a u t h o r s would l i k e t o t h a n k M r . Uday C .

Sharma?for h i s s e c r e t a r i a l a s s i s t a n c e i n t h e
r e p r o d u c t i o n of t h e manuscript.
(*of College of Pharmacy, Department of
Pharmacognosy , King Saud U n i v e r s i t y ) .
QUININE HYDROCHLORIDE
Farid J . Muhtadi, Mohammed A . Loutfy,
and Mahmoud M.A. Hassan
1. Description 548
1. I Nomenclature 548
1.2 Formulae 548
1.5 Appearance, Color, Odor, and Taste 554
2.2 Eutectic Temperature 554
2.3 Solubility 554
2.4 Loss on Drying 554
2.5 pH Range 555
3. Preparation of Quinine Hydrochloride 567
3.1 Synthesis of Quinine 567
3.2 Quinine Hydrochloride 567
4. Synthesis of Quinine 569
4.1 Partial Synthesis 569
4.2 Total Synthesis 569
5. Biosynthesis of Quinine 583
6. Metabolism 586
7. Pharmacokinetics 587
8. Indications and Dosages 587
8.1 For The Treatment of Malaria 587
8.2 For The Relief of Nocturnal Leg Cramps 588
9. Toxicity 588
10.2 Gravimetric Methods 59 1
10.5 Spectroscopic Methods 606
References 612
ANALYTlCAL PROFILES OF DRUG SUBSTANCES 547 Copyrighi by ihe American Pharmaceutical Assacration.
VOLUME 12 ISBN 0-12-260812-7
548 FARID J. MUHTADI ETAL.
1. Description
1.1. Nomenclature
1.1.1 Chemical Names
a) Cinchonan-9-01, 6'-methoxy, (8a, 9R)

hydrochloride (1:l) salt, dihydrate.
b) (8S, 9R)-6'-methoxy cinchonan-9-01,

hydrochloride (1:1) salt, dihydrate.
c) a-(6-methoxy-4-quinolyl)-5-vinyl-2-
quinuclidinemethanol, hydrochloride
d) -
6-methoxy- a ( 5-vinyl-2-quinuclidinyl)
-4-quinolinemethanol, hydrochloride
e) ( a s ) - ~.-(6-methoxy-quinolin-h y1)- a
-[ (2R, 4S, 5R)-( 5-vinylquinuclidin-
2 yl)] methanol, hydrochloride (1:l)
salt, dihydrate.
1.1.2 Generic Names
Quinine hydrochloride j
Quinine chloride;
Quinine monohydrochloride;
Quinine muriate.
1.2. Formulae
1.2.1 Empirical
QUININE HYDROCHLORIDE 549
1.2.2 Structural
The structure of quinine was finally postulated

by Rabe (1)and was confirmed by the total
synthesis of quinine which was achieved by
several authors (2-7).
1.2.3 CAS Registry Number
[ 130-89-2I
1.2.4 Wiswesser Line Notation
T66 BNJ HOlE YQ-DT66

A B CNTJ AlUl & EH & QH (8)
1.2.5 Stereochemistry
The stereochemistry of quinine and

other related cinchona alkaloids is
well summarised by Finar (9)and Turner
and Woodward (10).
550 FARID J . MUHTADI ETAL.
If Q represents t h e quinoline h a l f , t h e
s t r u c t u r e of q u i n i n e may be w r i t t e n as
f o l l o w s :-
The above formula c o n t a i n s f i v e c h i r a l c e n t e r s :

1,3,4,8 and 9 . Since t h e b r i d g e must b e a c i s
f u s i o n , c e n t e r s 1 and 4 behave as "one c h i r a l
u n i t " , t h e r e f o r e , t h e number of o p t i c a l l y a c t i v e
forms would be t h e same as o b t a i n e d from f o u r
c h i r a l c e n t e r s . When t h e 1-8 bond i s broken,
t h e c h i r a l i t y of t h e n i t r o g e n i s l o s t .
Q u i n i n e , q u i n i d i n e , cinchonine and cinchonidine

g i v e on d e g r a d a t i o n t h e o p t i c a l l y i d e n t i c a l 8-
oximino-3-vinylquinuclidine [ 1] , meroquinene [ 21
and c i n c h o l o i p o n i c a c i d [ 31. It t h e r e f o r e f o l l o w s
t h a t t h e c o n f i g u r a t i o n s o f C and C4 are t h e same
f o r a l l t h r e e compounds.
3
Conclusive evidence f o r t h e c i s arrangement a t

C3 and C 4 w a s provided by P r e l o g and Zalan (11).
They reduced cinchonine t o dihydrocinchonine and
converted t h e product i n t o c i n c h l o p i o n e t h y l e s t e r
[ 4 1 i n which C3 and C 4 r e t a i n t h e o r i g i n a l con-
f i g u r a t i o n o f cinchonine. [ 4 ] w a s converted i n t o
t h e dibromide [ 5 ] which by means o f a s e r i e s of
r e a c t i o n s , a l l of which proceeded under mild
c o n d i t i o n s and d i d not i n v o l v e t h e c h i r a l c e n t e r s ,
was converted i n t o 1,2-diethylcyclohexane [6].
This was shown t o be o p t i c a l l y i n a c t i v e ( i t
could not be r e s o l v e d ) .
QUININE HYDROCHLORIDE 55 1
The o p t i c a l r e s u l t s provide c o n c l u s i v e evidence

f o r a c i s arrangement of t h e two e t h y l groups
i n t h e diethyl-cyclohexane [ 6 ] and s i n c e none
of t h e s t e p s employed i n t h e conversion of c i n -
chonine t o [ 6 ] i n v o l v e s t h e c h i r a l c e n t e r s a t
C3 and C4, t h e v i n y l group of t h e n a t u r a l
cinchona b a s e s must be c i s t o t h e c7-c8 bond i n
a l l alkaloids.
The 9-deoxy d e r i v a t i v e s ( i : e , CH2 h a s r e p l a c e d

CHOH) of cinchonine and c i n c h o n i d i n e have
d i f f e r e n t s p e c i f i c r o t a t i o n s , a t + 179.3' and
- 29.9', r e s p e c t i v e l y . Since t h e c o n f i g u r a t i o n s
of C3 and C4 a r e t h e same i n b o t h b a s s e s and
s i n c e C9 i s no l o n g e r o p t i c a l l y a c t i v e , t h e
d i f f e r e n c e between t h e two must be a t c 8 , and
t h i s i s therefore, a l s o t h e c a s e f o r cinchonine
and cinchonidine. S i m i l a r l y s i n c e [ a ] o~f
deoxyquinine i s - 97.7' and t h a t of deoxy-
q u i n i d i n e i s + 211.1', t h e n q u i n i n e and q u i n i d i n e
d i f f e r a t c8.
The assignment of c o n f i g u r a t i o n s a t c8 may be

deduced from t h e f a c t t h a t q u i n i d i n e and c i n -
chonine a r e b o t h d e x t r o r o t a t o r y and b o t h can be
converted i n t o t h e i r c y c l i c e t h e r s [ T I . On t h e
o t h e r hand q u i n i n e and c i n c h o n i d i n e are b o t h
l e v o r o t a t o r y and do not form c y c l i c e t h e r s .
The c y c l i c e t h e r s t r u c t u r e i s only p o s s i b l e if t h e
group a t t a c h e d t o C 3 and C 8 are i n t h e endo-
p o s i t i o n [ 81. Thus i n cinchonine and q u i n i d i n e ,
t h e hydrogen atoms a t C 3 and c8 a r e c i s with
r e s p e c t t o each o t h e r . Also,because C 4 and C 8
a r e c i s - o r i e n t e d , it follows t h a t t h e hydrogen
atoms a t C 3 , C 4 and C 8 a r e a l l c i s - o r i e n t e d i n
cinchonine and quinidine whereas i n cinchonidine
and quinine t h e hydrogens a t C 3 and C 4 are c i s ,
but t h e hydrogen a t C 3 and c8 are t r a n s .
For each c o n f i g u r a t i o n a t c8, two isomers a r e
p o s s i b l e which d i f f e r i n o r i e n t a t i o n a t Cg.
Since a l l a l k a l o i d s a r e i d e n t i c a l i n c o n f i g u r a t i o n
except a t c8 and Cg, four isomeric substances
a r e p o s s i b l e i n each s e r i e s . For example, two
of t h e s e substances are presented by quinine and
q u i n i d i n e , t h e o t h e r two members a r e epiquinine
and epiquinidine. The o t h e r two members a r e
cinchonine, cinchonidine, epicinchonine and
epicinchonidine.
I n most r e s p e c t quinine and quinidine p a r a l l e l
one another c l o s e l y i n t h e i r chemical behavior
and d i f f e r q u a l i t a t i v e l y from t h e isomeric p a i r ,
epiquinine and epiquinidine. Since quinine and
quinidine d i f f e r i n c o n f i g u r a t i o n a t c8, t h e s e
f a c t suggest t h a t t h e two a l k a l o i d s d i f f e r a l s o
i n configuration a t C
z. It i s p o s s i b l e t o deduce
t h e c o n f i g u r a t i o n a t 9 by comparing t h e b a s i c i -
t i e s of quinine and i t s Cg-epimer with t h e
b a s i c i t i e s of (-)-ephedrine and (+)-$-ephedrine.
:$-CH2 Hl*N CH2 -

H$UH:I3 k
l
.H3 H
Ph Ph Q Q
( - )ephedrine ( +)-%ephedrine ( - )quinine (+) epiquinine
( pKt3 9.14 9.22 7.73 8.40
The c o n f i g u r a t i o n of ephedrine (erythro-

c o n f i g u r a t i o n ) and $-ephedrine ( t h r e o -
c o n f i g u r a t i o n and t h e s t r u c t u r e of quinine
and epiquinine have been drawn ( a s above) so
t h a t comparison can be made for c8 and C 9 .
Inspection of t h e pKa values shows t h a t JI-
ephedrine i s a s t r o n g e r base t h a n ephedrine and
t h a t epiquinine i s a s t r o n g e r base t h a n q u i n i n e ,
by a n a l o m , (+)-epiquinine i s t h e r e f o r e probably
r e l a t e d t o (+)-$-ephedrine i n c o n f i g u r a t i o n and
(-)-quinine t o (-)-ephedrine. Thus, t h e con-
f i g u r a t i o n s a t C 8 and C9 i n (-)-quinine and ( + ) -
epiquinine a r e probably t h o s e shown i n t h e above
formulae. If t h e s e c o n f i g u r a t i o n s are accepted,
then t h e r e l a t i v e c o n f i g u r a t i o n s a t C 3 , C 4 , c8
and Cg a r e now known. It i s now p o s s i b l e t o w r i t e
t h e a b s o l u t e c o n f i g u r a t i o n s of t h e s e a l k a l o i d s .
Lyle and Keefer ( l l a ) have confirmed t h a t t h e
n a t u r a l cinchona a l k a l o i d s a r e a l l of t h e erythro-
c o n f i g u r a t i o n with r e s p e c t t o t h e i r C8 and C
systems.
9
I
HO --C
Q/H ' H
( - )quinine (+)quinidine
H--d ,OH
Q
'
epiquinine epiquinidine
554 FARID J. MUHTADI E T A .
396.88 ( d i h y d r a t e )
360.88 (anhydrous )
C , 60.53%; H, 7.36%; N, 7.06%;

0 , 16.13%; C1, 8.92% ( d i h y d r a t e )
C, 66.57%; H, 6.98%; N, 7.76%;

0, 8.87%; C 1 , 9.81% (anhydrous)
1.5. Appearance, C o l o r , Odor and Taste
Fine c o l o r l e s s o r white s i l k y n e e d l e - l i k e
c r y s t a l s , o f t e n grouped i n c l u s t e r s , o d o r l e s s ,
and has a v e r y b i t t e r t a s t e .
2.1. Melting Range
Quinine hydrochloride m e l t s a t : -
145 - 153' ( 1 2 ) by hot s t a g e method
162O ( 1 2 ) by h o t b a r method
158 - 160° (13)
156 - woo (8)
2.2. Eut ec t i c Tempera t u r e
Phenacetin 100'
Benzanilide 114' ( 1 2 ) by h o t s t a g e method
Phenacetin 106"
Benzanilide 118' ( 1 2 ) by hot b a r method
2.3. Solubility
One gram d i s s o l v e s i n 23 m l o f w a t e r a t 20°,

i n 1 . 0 m l of a l c o h o l ( 9 6 % ), i n about 1 . 0 m l
chloroform, i n about 7 . 0 m l g l y c e r o l and i n
about 350 m l e t h e r .
2.4. Loss on Drying
When d r i e d t o c o n s t a n t weight a t 105O, l o s s e s

6.0 t o 10% of i t s weight ( u s i n g 2 . 0 g ) ( 1 4 ) .
2.5. pH Range
1% aqueous solution of quinine hydrochloride

has a pH of 6.0 - 7.0.
2.6. Optical Rotation

The following optical rotations were reported.
[ a ,I Solvent Ref.
- 57.1 chloroform (15)

- 133.7 water (15)
- 149.8 1.3% in water (8)
- 145.5 97% ethanol (15)
- 240 to - 258 2% solution in 0.1 N
hydrochloric acid (13,14)
The specific rotation of quinine hydrochloride
as 2% ethanolic solution has been determined by
using a Parkin Elmer Polarmatic model 241 MC and
found to be
[ cy,] -144.25'
2.7.1 Ultraviolet Spectrum
The UV spectrum of quinine hydrochloride in

ethanol was scanned from 190 to 400 nm using
DMS 90 Varian Spectrophotometer. It exhibi-
ted the following UV characteristics (Fig.1).
Table 1 UV characteristics of quinine
hydrochloride.
X h x . at -
E
205
232 -
277 2381
321 2858
332 3334
Figure 1. The W Spectrum of Quinine Hydrochloride i n Ethanol
Other r e p o r t e d U.V. s p e c t r a l d a t a f o r
quinine hydrochloride i n a l c o h o l ( 8 ) :-
Xmax. a t 278 nm (2512) and 331 nm (3236)
and for quinine i n ethanol ( 1 6 ) :-
Xmax. a t 236 nm ( E 1%, 1 cm 1110), 278 nm
( E 1%, 1 cm 133) and 332 ( E 1%, 1 cm 1 6 3 ) .
The UV absorption s p e c t r a of quinine and i t s
hydrochloride s a l t i n o t h e r s o l v e n t s were
also reported (16-18).
2.7.2 I n f r a r e d Spectrum
The I R spectrum of quinine hydrochloride a s

KBr-disc was recorded on a Perkin Elmer
580B I n f r a r e d Spectrophotometer t o which
I n f r a r e d Data S t a t i o n i s a t t a c h e d (Fig. 2 ) .
The s t r u c t u r a l assignments have been c o r r e l -
a t e d with t h e following frequencies (Table 2 ) .
Table 2. I R c h a r a c t e r i s t i c s of quinine
hydrochloride.
-1
Frequency cm Assignment
3300 OH bonded
+
NH ( q u i n u c l i d i n e )
2580
1615 CN
[ C=C ( a l k e n e )
1600,1512,1480 C=C (aromatic )
1248,1230,1100,1030 e t h e r linkage
860,838,808,725 T r i subst it u t ed
benzene
Other c h a r a c t e r i s t i c absorption bands a r e :
1438,1365,1345,1320,1140,1130,1010,990 ,
935, cm-1.
Other I R d a t a f o r quinine hydrochloride ( 8 )
and f o r quinine (16) have been a l s o reported.
Hayden and Sammul (19) described t h e I R
s p e c t r a of dimorphous and amorphous forms of
quinine.
FIG. 2 THE IR SPECTRUM OF Q U I N I K E HYDROCHLORIDE A S KBR DISC.
2.7.3 Nuclear Magnetic Rosonance S p e c t r a
2.7.3.1 Proton S p e c t r a
The PMR s p e c t r a of q u i n i n e hydro-

c h l o r i d e i n d e u t e r a t e d chloroform
and 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
were recorded on a Varian T - ~ O A ,
60-MH, NMR Spectrometer u s i n g TMS
( T e t r a m e t h y l s i l a n e ) as an i n t e r n a l
r e f e r e n c e . These a r e shown i n
Fig. 3 and Fig. 4 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 assignments
have been made (Table 3).
Table 3 PMR c h a r a c t e r i s t i c s of
quinine hydrochloride
Group Chemical S h i f t (ppm)

CDC13 DMSO D6
s = s i n g l e t , d = doublet, q = quartet
m = m u l t i p l e t b s = broad s i n g l e t
bd = broad d o u b l e t , 2d = doublet of d o u b l e t s ,
b t = broad t r i p l e t .
Other PMR s p e c t r a l d a t a of q u i n i n e and i t s hydro-

c h l o r i d e d i h y d r a t e s a l t i n C D C l have been r e p o r t e d
3
(8, 2 0 ) .
4
m
J
V
n
u
-z
W
ff
a
0
J
I
V
0
e
n
>
Y
--.-
W
L'
c
3
0
U
0
E
3
e
l-
u
W
a
VY
of
E
M
2
LL
560
FIG. 4 Hf'% SPECTRUM OF k J l N l N E HYDROCHLORIDE I N Dr;sO-Dg
562 FARlD J . MUHTADI ETAL.
13
2.7.3.2 C-NMR
13
C-NMR n o i s e decoupled and o f f
resonance s p e c t r a are p r e s e n t e d i n
F i g . 5 and Fig. 6 r e s p e c t i v e l y .
Both were recorded over 5000 Hz
width i n C D C l 3 ( c o n c e n t r a t i o n
52.9 mg/l ml) on J e o l FX-100 NMR
Spectrometer, u s i n g a 1 0 mm sample
t u b e and TMS as a r e f e r e n c e s t a n -
dard a t 20'.
The carbon chemical s h i f t s are
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
resonance s p l i t t i n g p a t t e r n
(Table 4 ) .
Table 4. Carbon Chemical S h i f t s of

q u i n i n e hydrochloride.

[ PPm 1 [ PPm 1
c6 158.12(s) c5 99.79 ( d )
146.81( d ) c10 65.99 ( d )
c3 144.34(s) c11 60.14 ( d )
c9 143.42(s) c16 56.99 ( t )
r
w
El
a
0
-1
I
U
0
a
CI
t
I
W
-z
-z
2
Q
LL
0
z
3
a
I-
U
W
n.
v)
n
W
-I
n.
3
0
U
W
P
W
v)
L
0
z
4
oc
r
T
V
M
4
In
2
LL
FIG. 6 13C-NMR OFF RESONANCE SPECTRUM OF 3 U I N I N E HYDROCHLORIDE.
‘18 137.37 ( d c20 54.70 ( 9 )

131.03( d ) 44.11( t )
‘8 ‘1 5
C4 125.25(s) 37.12( d )
‘17
C 122.17(d) 26.91( a )
7 ‘13
118.81(d) 24.29( t )
c2 52
C 18.13( t )
19 117 15( t ‘14
s = s i n g l e t , d = doublet, t = t r i p l e t , q = q u a r t e t .
Other 13C-NMR s p e c t r a l d a t a of qninine have been

r e p o r t e d (21, 22).
2.7.4 Mass Spectrum
The mass spectrum of quinine hydrochloride obtained

by e l e c t r o n impact i o n i z a t i o n which w a s recorded
on a Finnigan Model 3000 D GC-MS-system. The
spectrum scanned t o mass 500 with u n i t r e s o l u t i o n .
Electron energy was 7Oev. The chemical i o n i z a t i o n
mass s p e c t r a l d a t a were obtained on a Finnigan
Model 1015D GC Mass Spectrometer. Methane w a s
used as GC c a r r i e r gas and a l s o served as t h e C I
r e a c t a n t gas i n t h e i o n source. The i o n source
temperature w a s 180Oc and e l e c t r o n energy 100 ev.,
i o n r e p e l l e r , 3V.
E l e c t r o n impact mass s p e c t r a l d a t a : Fig. 7 (23).

Base Peak 136
42, 55, 67, 81, 95, 117, 128, 136, 158, 172, 1.89,
202, 222, 251, 269, 295, 309, M+ 324
C I mass s p e c t r a l d a t a and prominent fragment i o n s

are
M.H+ 325 (1001, 136 (371, 307 (101,and 323 ( 7 ) .
Fig. 7 The Mass Spectrum of Quinine
3. P r e p a r a t i o n of Quinine h y d r o c h l o r i d e
3.1. I s o l a t i o n of Q u i n i n e
Quinine i s t h e p r i n c i p a l a l k a l o i d o f cinchona
b a r k of which s e v e r a l s p e c i e s are known. Cinchona
o f f i c i n a l i s L. ( C . l e d g e r i a n a Moens) , Family
Rubiaceae i s t h e most important. It c o n t a i n s about
8% q u i n i n e ( 2 4 ) . Q u i n i n e w a s i s o l a t e d from cinchona
b a r k by P e l l e t i e r and Caventou i n 1820 ( 2 5 ) .
S e v e r a l methods have been r e p o r t e d f o r t h e

i s o l a t i o n of q u i n i n e from cinchona b a r k , t h e most
important method i s as f o l l o w s ( 2 6 ) : -
Powdered cinchona (250g) i s w e l l mixed w i t h

C a O ( 6 0 g ) , water (600 m l ) and 30% NaOH s o l u t i o n
( 3 0 m l ) and l e f t f o r 24 hours. The mixture i s t h e n
e x h a u s t i v e l y e x t r a c t e d under r e f l u x w i t h benzene.
The benzene e x t r a c t i s f i l t e r e d while h o t i n t o a
s e p a r a t i n g f u n n e l c o n t a i n i n g c o n c e n t r a t e d H2S04
( 7 g ) i n water (500 m l ) t o convert t h e a l k a l o i d s
t o t h e i r b i s u l f a t e s a l t s . The mixture i s s e p a r a t e d
and t h e a c i d aqueous l a y e r i s h e a t e d t o 90' and
n e u t r a l i s e d w i t h 5% Na2C03 s o l u t i o n u s i n g l i t m u s
as an i n d i c a t o r (The b i s u l f a t e s a l t s are now
converted i n t o t h e s u l f a t e s a l t s ) . The c l e a r
orange s o l u t i o n becomes t u r b i d due t o t h e s e p a r a -
t i o n of some r e s i n o u s m a t e r i a l . D i l u t e H2SO4
( 2 d r o p s ) and animal c h a r c o a l ( 2 g ) a r e added and
t h e r e s u l t i n g mixture i s h e a t e d a g a i n a t 90' f o r
1 5 minutes and f i l t e r e d . The f i l t e r a t e i s allowed
t o c o o l down when q u i n i n e s u l f a t e s e p a r a t e s o u t
and c o l l e c t e d by f i l t e r a t i o n . The q u i n i n e s u l f a t e
s o c o l l e c t e d i s d i s s o l v e d i n b o i l i n g w a t e r and
t r e a t e d w i t h Na2C03 s o l u t i o n t o p r e c i p i t a t e
q u i n i n e which i s c o l l e c t e d and c r y s t a l l i z e d .
The procedure o u t l i n e i s p r e s e n t e d i n F i g . 8.
3.2. Quinine Hydrochloride
Q u i n i n e h y d r o c h l o r i d e i s o b t a i n e d by n e u t r a l i z -
i n g t h e a l k a l o i d quinine with d i l u t e hydrochloric
a c i d and r e c r y s t a l l i s i n g from b o i l i n g w a t e r t o g i v e
f i n e c o l o r l e s s c r y s t a l s of q u i n i n e h y d r o c h l o r i d e .
568 FARID J. MUHTADl ETAL.
Powdered cinchona + CaO + NaOH + Water

reflUX with C6H6
1 filter
Hot f ilt erat e
Extract with d i l . H2S04
I
f
Alkaloids b i s u l f a t e s
Heating a + Na2C03
90 O (PH 6 . 5 )
Alkaloids s u l f a t e s
Purify + charcoal b o i l
and f i l t e r
Cool f i l t e r a t e
+
F i l t e r a t e of
*
P r e c i p i t a t e of
quinine s u l f a t e
Quinidine cinchonine
sulfates etc.
Na2S03
1
Quinine
solution
Fig. 8. Procedure Outline f o r t h e I s o l a t i o n of

Quinine.
4. Synthesis of Quinine
4.1. Partial Synthesis
Rabe and Kindler in 1918 (3) achieved the first

partial synthesis of quinine from quinotoxine.
Quinotoxine was converted by the action of sodium

hypobromite into N-bromoquinotoxine which was
cyclized by alkali with the l o s s of hydrogen
bromide to give quininone. Reduction of the
ketone with aluminium powder and ethanol in the
presence of ethoxide gave a mixture of stereo-
isomeric alcohols from which quinine and quinidine
were isolated.
Gutzwiller and Uskokovic in 1973 (7) developed

a slightly different scheme for the partial syn-
thesis of quinine from quinotoxine.
Quinitoxine was dissolved in dichloromethane and

treated with sodium hypochlorite solution to give
N-chloroquinotoxine. This was cyclized with phos-
phoric acid to give a mixture of quinone and
quinidinone. The resulting mixture was dissolved
in benzene and treated with a solution of diiso-
butylaluminium hydride in toluene to give a mixture
of quinine and quinidine which was separated by
crystallization.
4.2. Total Synthesis
Several schemes (I to IV) for the total

synthesis of quinine have been reported. The
first total synthesis of quinine was completed
in 1944 by Woodward and Doering (2). Rabe and
Kindler (3) carried out a partial synthesis of
quinine starting from quinotoxine. Woodward
and Doering (2) completed the total synthesis
by synthesizing (+)-quinotoxine.
The first total synthesis of quinine is
presented in Scheme I.
m-Hydroxybenzaldehyde [l] is condensed with
aminoacetal [2] to give 7-hydroxyisoquinoline [ 3 ]
which is treated with formaldehyde in methanol
containing piperidine to give 7-hydroxy-8-piper-
idinmethylisoquinoline [ 41. This by heating with
methanolic sodium methoxide at 220° is converted
into ~-hydroxy-8-methylisoquinoline[ 5 1.
Compound [ 51 on catalytic reduction, followed by
acetylation gives N-acetyl-7-hydroxy-8-methyl-
1,2,3,4-tetrahydroisoquinoline [6], which on
further catalytic reduction by heating with a
Raney nickel catalyst under pressure and then
followed by oxidation with C r O 3 is converted into
N-acetyl-~-keto-8-methyldecahydroisoquinoline[ T I .
This compound is a mixture of cis-and trans-iso-
mers, these are separated and the cis-isomer is
treated with ethylnitrite in the presence of
sodium ethoxide to give the homomeroquinene
derivative [8]. This on reduction gives the
corresponding aminocompound [g], which may now be
written more conveniently as shown. Exhaustive
methylation of [ 91 followed by hydrolysis gives
t cis homomeroquinene [lo] which after esterifica-
tion and benzoylation gives N-benzoylhomomero-
quinene ethylester [ll].
On condensation of [ 111 with excess ethylquininate
[lg] using sodium ethoxide produces the inter-
mediate B-ketoester [20]. This on heating with
hydrochloric acid is hydrolysed and decarboxylated
to (+)-quinotoxine [21]. This is resolved via its
dibenzoyltartrate. (+)-quinotoxine [ 221 which is
converted into quininone [23] upon N-bromination
and cyclization. Reduction of [23] with aluminium
powder and ethanol gives a mixture of stereoiso-
meric quinine and quinidine [24]which are separated.
Quinic acid required for the synthesis of
quinine was prepared by Rabe et al. ( 27). p-
anisidine [121 is condensed with acetoacetic
ester [ 1 3 ] to give the condensate [14]. This is
treated with sulfuric acid where ring closure
occurs to give 2-hydroxy-4-methyl-6-methoxy-
quinoline [15]. The phenolic hydroxyl group of [15]
is eliminated upon treatment with a mixture of
phosphorous pentachloride and phosphorous oxy-
chloride to give 2-chloro-4-methyl-6-methoxyquinol-
ine [16] which upon hydrogenation gives 4-methyl-
6- methoxyquinoline [ 171. Knoevenagel condensation
of the latter followed by oxidation gives quinic
[l81 which upon esterification gives ethylquininate
[191
Scheme 11: Total synthesis of quinine and
quinidine according to Uskokovic et al. (4).
Scheme I: T o t a l S y n t h e s i s of Q u i n i n e (Woodward and

Doering).
i) H p R a n e y N i
i i ) ~r03
-G
0
- IJCOCH3
CH3
[71 Ii ) C H ON0
2 5
i i ) C H ONa
2 5
Q c g L T
H5c$2c H2N-CH ‘ QN=C C O C H 3
11 [81
CH3 [91 HO C H 3
( f )-Quinine Resoln. ( - )-Quinine
1241 [241
( f ) Quinidine (+)Quinidine
H F O COOC2H5
d ' 3 " " 3 P'ii)KMnOq
C6H5CH0
ester.
L
[191 [181
N-benzoylhexahydroisoquinolone [ 11 is hydrogen-
ated with rhodium on alumina catalyst to give
predominantly cis-isoquinolone [2] which is
treated with sodium azide in poly-phosphoric acid
to give a mixture of the seven-membered lactams
which is separated by fractional crystallisation
to give [ 31. Lactam [3] is treated with dinit-
rogen tetroxide to give the N-nitrosolactam [4]
which is rearranged upon heating to the diazo-
lactone [5] and fragmented with extrusion of
nitrogen to give a mixture of racemic N-benzoyl-
meroquinene [6] and the seven membered lactone [?a]
(in 50 and 30% yield, respectively). The latter
[5a] can be converted into [6] which upon esterifi-
cation gives N-benzoylmeroquinene methyl ester
[6a]. This ester is treated with 6-methoxyle-
pidyllithium [7] in tetrahydrofuran to give the
racemic N-benzoylketone [8]. This is treated with
diisobutylaluminium hydride in toluene at - 78O
[route a] to remove the benzoyl group with con-
commitant reduction of the ketone function to give
the aminoalcohol [g] . The aminoalcohol [9] is
first acetylated with acetic acid containing 10%
boron trifluoride etherate and treated with boiling
benzene - acetic acid - sodium acetate where
cyclization proceeds to give a mixture of desoxy-
quinine and desoxyquinidine [12]. This can also
be achieved without acetylation. The aminoalcohol
[9] is refluxed with benzene - acetic acid mixture
(4:l) for 4-5 days when cyclization proceeds via
dehydration [ 111 to give both desoxyquinine and
desoxyquinidine [12].
Upon stirring a solution of [12] in dimethyl-
s u l f o x i d e - t - b u t y l a l c o h o l ( 4 :1) containing potas-
sium t-butoxide in an atmosphere of oxygen affords
a mixture of quinine [13] and quinidine [14].
Separation can be effected by a combination of
crystallization and chromatography.
An alternative synthetic route [b] via the amino
epoxide [lo] is as follows:-
N-benzoylketone [8] is converted into a mixture
of diastereomeric N-benzoyl epoxides [lo a] by
bromination followed by sodium borohydride reduc-
tion. Reductive debenzoylation of [lo a] with
diisobutylaluminium hydride in toluene at - 78'
furnished a mixture of diastereomeric amino-
576 FARID J. MUHTADI ETAL
Scheme 11: T o t a l Synthesis (Uskokovic etal. 1
EtOH HC1
[21
N-0
[3
i;' H
' c
0
c6H5
L41
I)-
I
R=H [61 Ac6H5
R=CH3 [ 6a 1
CHeLi
r
[6al + conden. .
4
"71
0
A c6H5
H3C0
[81
H CO
3
191
578 FARID I. MUHTADI ETAL.
[91 [a1 L a c e t ylation

epoxides [lo]. Treatment of [lo] with toluene-

ethanol (19:l) at reflux for 12 hr. give quinine
[13] and quinidine [14]. Separation is effected
by preparative thin layer chromatography.
Scheme 1II:Total synthesis according to Gates et

al. ( 5 )
6-methoxylepidine [ 11 is condensed with N-acetyl-
3-vinyl-4-piperidineacetic acid ester [ 21 to give
the ketone [ 31. Reduction of [ 31 followed by
dehydration of the resulting alcohol with acetic
anhydride gives the intermediate [ 41. Saponifica-
tion of the latter affords the secondary m i n e [5]
Alkaline hydrolysis of [ 51 in aqueous alcohol
under reflux, cyclization occurrs to give a
mixture of desoxyquinine and its c8 epimer
desoxyquinidine [ 61.
Oxidation of the mixture [6] with oxygen in the
presence of potassium t-butoxide and triphenyl-
phosphine in dimethylformamide -t.-butyl alcohol
introduces of the hydroxyl group at C9 giving the
diasteromeric quinine and quinidine [ 73.
The intermediate [4] can also be prepared by
the Wittig reaction of quininaldehyde [8]with the
quaternary phosphonium compound derived from the
corresponding bromide obtained from meroquinene
alcohol [g].
Scheme IV: Total synthesis according to Taylor
and Martin (6) :
4-chloro-6-methoxyquinoline [l] is treated with
2 equiv. of methylenetriphenylphosphorane [ 21 to
give [3]. This is condensed with N-acetyl-3(R)-
vinyl-4 (S)-piperidineacetaldehyde [ 41 to give the
olefin [5]. Removal of the N-acetyl group in situ
by hydrolysis followed by spontaneous intramole-
cular Michael addition to give a mixture of
desoxyquinine and desoxyquinidine [6] which is
converted by base-catalyzed hydroxylation to
quinine [ 71 and quinidine [ 81.
Other scheme for the total synthesis of quinine
through the synthesis of homomeroquinene and
quinotoxine is reported by Uskokovic et al. ( 7 ) .
Scheme 111: Total Synthesis (Gates e t a l . )

Scheme I V : T o t a l S y n t h e s i s of Q u i n i n e (Taylor and M a r t i n )

c1
H 3 c 0+ y 3
CHO
CH=PPh3
I
H3c000 [31 H
..
+ COCH3
[41
conden.
-
\
H3C0
5. Biosynthesis of Quinine
Postulation of the biosynthetic pathway of cinchona
alkaloids started in 1950 with the suggestion of Goutarel
et al. ( 2 8 ) that quinine and other cinchona alkaloids
are derived from indolic precursors, since cinchonamine
(indole alkaloid) occurs as a minor alkaloid in cinchona.
This was proved when Kowanko and Leete ( 2 9 ) have isolated
labelled quinine upon feeding trypt0phan-2-~~C into
cinchona plants. They have shown that the quinoline
ring and Cg unit of quinine originated from tryptophan.
Further studies have proved that quinine is biosynthesi-
zed by a combination of indolic and monoterpenoid units
which leads to the corynanthe type indole alkaloids.
Thus tryptophan ( 2 9 ) , geraniol (30-32) and loganin ( 3 3 )
were incorporated into quinine. Tracer experiments on
Cinchona ledgeriana carried out by Battersby and Parry
( 3 4 ) have established the biosynthetic pathway of
quinine as presented in scheme V.
Scheme V: Biosynthesis of Quinine
Loganin Secologanin
p)-JyOH
\ H
Tryptophan
Vincoside -
584 FARID J. MUHTADI ETAI;.
Corynantheal
LHO
Cine honaminal
I
-
H
H k
Quinidine
6. Metabolism
The cinchona alkaloids are extensively metabolized in
the body, especially in the liver, so that less than 5%
of an administered dose is excreted unaltered in the
urine (35-37).
The metabolism of quinine has been studied both in
human and rat urines. The major urinary metabolites in
man are hydroxyderivatives of quinine (37). The route
of quinine metabolism in man proposed by Brodie et al.
(37)involves two parallel pathways as presented in the
f o l l o w i n g scheme.
Scheme VI: Metabolism of quininein Man
H3CO
dihydroxy derivative quinine carbostyri1

(non-phenolic)
Barrow et al. (38) have separated eight metabolites of

quinine in the urine of male Sprague-Dawley rats after
a single dose of quinine (50 mg/Lg). Six of which have
been identified as O-desmethylquinine, hydroxyquinine,
quinine, quinine carbostyril and the two diastereoismers
of quinine-10, ll-dihydrodiol. These were separated by
reversed-phase HPLC on a semi-preparative column by
gradient elution. Separation of these metabolites is
presented in Fig. 12.
7. Pharmacokinetics
Quinine is readily absorbed after oral administration.

Absorption occurs mainly from the upper small intestine,
and is almost complete even in patients with marked
diarrhea. Rectally administered doses are poorly
absorbed and intramuscular or subcutaneous doses of
quinine salts are slowly absorbed (13, 35).
Peak plasma concentration of quinine occurs within
1 to 3 hours after a single oral dose (35). Therapeutic
plasma concentrations appear to be in the range 3 to 7
ug/ml during therapy with oral doses of 500 to 650 mg
thrice daily (13). Malarial infection inhibits hepatic
metabolism and thus plasma concentrations resulting from
a given dose will vary according to the severity of the
infection (391.
Plasma half-life 6-9 hours, which is increased up to
1 5 hours in malarial infection and decreased to about
3 to 4 hours in patients being treated with antiepileptic
drugs (39).
After termination of quinine therapy, the plasma level
falls rapidly and only a negligible concentration is
detectable after 24 hours. A large fraction (approxim-
ately 70%) of the plasma quinine is bound to proteins (35).
Quinine is excreted mainly in the urine, but small
amounts also appear in the feces, gastric juice, bile
and saliva. Renal excretion of quinine is twice as
rapid when the urine is acidic as when it is alkaline (35).
8. Indications and Dosages

8.1. For the Treatment of Malaria
The usual oral dose of quinine or its salts is
325 mg four times daily for 7 days. The drug is
given after meals, preferably in capsules, to
588 FARID J. MUHTADI E T A .
minimize gastric irritation (35).

Intravenous injections of quinine are to be
reserved for certain emergencies such as pernicious
or cerebral malaria. The dihydrochloride is emplo-
yed and I.v. injection should be given very slowly,
preferably by the drip method (35).
8.2. For the relief of Nocturnal Leg Cramps

Recumbency leg muscle cramps (night cramps) are
quickly and effectively relieved by quinine in
most cases. The dose is 200 to 300 mg before
retiring (35).
9. Toxicity
Poisoning by quinine is usually due to clinical over-

dosage or to hypersensitivity. The fatal oral dose of
quinine for adults is approximately 8 g (35).
When quinine is repeatedly given in full doses a typical
cluster of symptoms occurs to which the term cinchonism
has been applied. In its mildest form it consists in
ringing in the ears, headache, nausea, and slightly
disturbed vision. When medication is continued or
after large single doses, symptoms also involve the
gastrointestinal tract, the nervous and cardiovascular
systems and the skin (35).
10.1. Identification
10.1.1 Color Tests
The following c o l o r t e s t s have been

described f o r t h e i d e n t i f i c a t i o n of quinine ,
H C 1 (12-14, 40 -44 ) :
a. Dissolve 1 0 mg i n s u f f i c i e n t water t o
produce 10 ml and t o 5 m l of t h e s o l u t i o n
add 0.2 m l of bromine water and t h e n 1 m l
of 2 M ammonia, an emeraldgreen c o l o r i s
produced.
b . Dissolve 5 mg i n 5 ml of water, add

1 ml of 2 M ammonia and 5 ml of e t h e r , shake
and acidif'y with 2 M n i t r i c a c i d , t h e aqueous
l a y e r y i e l d s r e a c t i o n s c h a r a c t e r i s t i c of
chlorides.
c . Quinine a l k a l o i d can be i d e n t i f i e d w i t h
ammonium t e t r a k i s ( t h i o c y a n a t o ) z i n c a t e ( 4 2 ) .
d. I n t o x i c o l o g i c a l work, it i s o f t e n d e s i -
r e d t o d e t e c t very s m a l l q u a n t i t y of quinine.
A very s e n s i t i v e t e s t has been r e p o r t e d (45).
10.1.2 Micro-Crystal T e s t s
The following micro-crystal t e s t s a r e a l s o

u s e f u l i d e n t i f i c a t i o n t e s t s , photomicro-
graphs of t h e c r y s t a l s have been described
(16, 46-49) (Fig. 9 and 1 0 ) .
i ) Platinic iodide s o l u t i o n g i v e s curved
s e r r a t e d needles ( s e n s i t i v i t y 1 i n 1 5 0 0 ) .
i i ) Sodium phosphate s o l u t i o n forms needles

( s e n s i t i v i t y 1 i n 1000).
iii) Dissolve 1 mg i n 2 ml of water, a c i d i f y

with d i l u t e s u l p h u r i c a c i d (1 drop) and add
a few drops of an aqueous s o l u t i o n c o n t a i n i n g
5% of cadmium i o d i d e and 10% of potassium
i o d i d e . Colorless c r y s t a l s a r e produced
and t h e s o l u t i o n becomes t u r b i d .
F i G , 9, MICROCHEMICAL CRYSTALS OF Q U I N I N E HYDRCCHLORIDE

WITH DISODIUM HYDROGEN PHOSPHATE,
F I G . 13. K I C R C C H E M I C A LCRYSTALS OF ~ U I P J I NWEI T P

EETHYL I O D I D E .
i v ) A drop of t h e drug i s p u t on a micro-

scope s l i d e next a drop of an almost
s a t u r a t e d s o l u t i o n of p i c r i c a c i d o r of 3 $?
g o l d c h l o r i d e , covered and observed f o r 1 5
minutes or more. If t h e p i r r a t - e ( o r a u r a t e )
does n o t c r y s t a l l i s e spontaneously, a s m a l l
amount o f a suspension of f i n e s e e d c r y s t a l s
i s added t o one edge of t h e drop and i t s
e f f e c t observed.
v) I d e n t i f i c a t i o n of q u i n i n e i n human u r i n e
h a s been d e s c r i b e d (48) by t h e f o l l o w i n g
method :
A f t e r a c i d h y d r o l y s i s t o f r e e t h e b a s e from
i t s g l u c u r o n i d e , q u i n i n e i s e x t r a c t e d from
a l k a l i n e s o l u t i o n by chloroform. The s o l v e n t
i s evaporated and t h e r e s i d u e , d i s s o l v e d i n
methanol, i s s u b j e c t e d t o TLC. The p l a t e
i s sprayed w i t h i o d o p l a t i n a t e , and t h e
methanolic e x t r a c t o f t h e s p o t i s t h e n
subjected t o s p e c i f i e d microscopical tests.
The product i s i d e n t i f i e d by t h e c r y s t a l
formed.
10.2. Gravimetric Method
B e r l i n and Robinson (50) have d e s c r i b e d a thermo-

g r a v i m e t r i c d e t e r m i n a t i o n of q u i n i n e w i t h d i l i t u r i c
a c i d . S o l u t i o n of q u i n i n e i n 50% e t h a n o l w a s t r e -
a t e d w i t h 0.02 M d i l i t u r i c a c i d i n 40% methanol s o
t h a t t h e molar r a t i o of d i l i t u r i c a c i d t o q u i n i n e
was approximately 7 : l . The p r e c i p i t a t e w a s washed
w i t h c o l d 50% Ethanol s a t u r a t e d w i t h d i l i t u r i c
a c i d followed by c o l d 95% e t h a n o l and t h e n d r i e d
i n t h e thermobalance between 140° and 195'. A
c o n s t a n t weight w a s o b t a i n e d i n 1 5 t o 20 m i n u t e s ,
corresponding t o anhydrous q u i n i n e d i l i t u r a t e .
Amounts of q u i n i n e between 2 and 40 mg have been
determined w i t h a maximum d e v i a t i o n of 3%.
lo.3. T i t r i m e t r i c Methods
10.3.1 Aqueous
Schneider ( 5 1 ) has p u b l i s h e d t h e a p p l i c a -
b i l i t y o f t h e s t r o n g l y b a s i c anion-exchangers
t o t h e d e t e r m i n a t i o n of q u i n i n e , H C 1 and
quinine ,H2SO4. In this method , the salt

(about 0.1 gm) is dissolved in 90% ethanol
(10 ml) , pass the solution through an anion-
exchange resin. Pass 90% ethanol (10 ml)
through the column, then rapidly pass a
further 20 ml. Dilute the combined perco-
lates with freshly boiled, cooled water (50
to 70 m l ) and titrate the liberated mine with
0.1 N HC1 in the presence of Tashiro’s
indicator, until the green-blue colour
changed to violet (52).
10.3.2 Non-Aqueous
Non-aqueous titration methods have been

described for the analysis of quinine alkaloid
or salt ( 53-59). The drug is titrated by
perchloric acid in acetic acid and the end
point is determined potentiometrically. The
method is applied for the determination of
small amounts of the alkaloid in the presence
of an adsorption electrode ( 60). Quantita-
tion of quinine in media of nitromethane and
of chloroform-dioxane has been also reported
(61)
10.3.3 Complexometric
Application of volume-colorimetry to the

micro-determination of alkaloids, including
quinine, has been described (62). The assay
method depends on the precipitation of the
alkaloid with phosphotungstic acid reagent,
the precipitate is treated with a reducing
agent, the blue color of tungstic anhydride
is titrated volumetrically with an oxidising
agent.
MaJlat and Bayer (63) have also reported

the determination of quinine salts
by titration with tungstosilicic acid.
Complexometric determinations of alkaloids,

including quinine, have been also described,
by the use of EDTA (disodium salt) (64)
or copper picrate (65).
Budesinsky and Vanickova (66) have published

a complexometric titration of quinine. The
drug i s p r e c i p i t a t e d a t pH 1 . 2 t o 1.5
w i t h bismuth potassium i o d i d e , and t h e
excess of t h e reagent i s determined as b i s -
muth complexometrically.
Yeh and Tsang (67) have described t h e

q u a n t i t a t i o n of q u i n i n e , H SO4 by c a t i o n -
2
exchange followed by complexometric t i t r a t i o n .
The e r r o r i s 0.5-2%.
10.3.4 Conductimetric
High-frequency t i t r a t i o n of q u i n i n e , HC1,qui-
nine,H2S04, a n d o t h e r s a l t s of organic
compounds , has been r e p o r t e d (68). Graphs
i l l u s t r a t i n g t h e high-frequency conducti-
metric t i t r a t i o n of t h e s e s a l t s have been
described. For quinine, H C 1 , t h e t i t r a n t
i s 0.01 N s i l v e r n i t r a t e ; for quinine,H$O4,
t h e t i t r a n t i s 0 . 0 1 M barium c h l o r i d e , 0.01 M
barium a c e t a t e , or 0.008 N potassium hydroxide.
I n t h e t i t r a t i o n of q u i n i n e , H2S@4with potas-
sium hydroxide, e x t r a p o l a t i o n i s necessary
t o determine t h e p o i n t of i n f l e c t i o n of t h e
graph.
10.3.5 Amperometric
Lemahieu e t a l . ( 6 9 ) have r e p o r t e d an
aniperometric determination of q u i n i n e , H C 1
i n dimethyl sulphoxide. The method i s based
on t i t r a t i o n w i t h s i l v e r n i t r a t e i n dimethyl
sulphoxide t o give an end-point corresponding
t o t h e formation of AgC1; and a less sharp
end-point f o r t h e p r e c i p i t a t i o n of s i l v e r
c h l o r i d e . U s e of t h e f i r s t end-point and
two platinum i n d i c a t o r e l e c t r o d e s with a
p o t e n t i a l d i f f e r e n c e of 100 mV allows
t i t r a t i o n down t o a 0.2 m M c o n c e n t r a t i o n of
q u i n i n e , HCI.. Use of one i n d i c a t o r e l e c t r o d e
and a s i l v e r - Ag+ r e f e r e n c e e l e c t r o d e
prodcces a l e s s sharp end-poir,t, not obtain-
a b l e below a m M concentration.
Gengrinovich e t i z l . ( 7 0 ) have described

t h e use of i o d i n e ch1ori.de and a r o t a t i n g
platinum e l e c t r o d e for t h e amperometric
t i t r a t i o n c f quint.ne, HC1. The t i t r a t i o n
i s based on t h e a d d i t i o n o f I C 1 t o t h e
v i n y l group of q u i n i n e . The r e a c t i o n i s
complete i n 20-25 minutes.
Charles and Knevel (71) have p u b l i s h e d

coulometric a s s a y of q u i n i n e s u l p h a t e u s i n g
an arseno-amperometric end-point d e t e c t i o n
t e c h n i q u e . T h i s i s based on t h e f a c t t h a t
t h e r e a c t i o n of bromine w i t h q u i n i n e s u l p h a t e
i s t o o slow t o allow d i r e c t coulometric
t i t r a t i o n of t h e drug. The c o e f f i c i e n t of
v a r i a t i o n i s 0.5%.
10.3.6 Polarographic
Souckova and Zyka(72,73) have r e p o r t e d a
polarographic d e t e r m i n a t i o n of q u i n i n e , H C l
by t i t r a t i o n with t u n g s t o s i l i c i c a c i d ,
tungstophosphoric, and molybdophosphoric
acids (73). The a p p a r a t u s h a s a dropping -
mercury cathode and S.C.E. anode, t h e v o l t a g e
b e i n g 0.65V. The pH o f t h e s o l u t i o n i s
a d j u s t e d w i t h HC1. The m a x i m u m e r r o r i s f
1% w i t h t u n g s t o s i l i c i c a c i d and 22% w i t h t h e
two o t h e r a c i d s .
Molnar and Molnarwa (74) have d e s c r i b e d

o s c i l l o p o l a r o g r a p h i c d e t e r m i n a t i o n of q u i n i n e
a l k a l o i d . The o s c i l l o p o l a r o g r a p h i c behaviour
of q u i n i n e d e r i v a t i v e s has been s t u d i e d and
t h e i r oscillograms i n N LiC1, N LiCH, N
NaOH, and N H$O4 were o b t a i n e d w i t h t h e u s e
of dropping and streaming mercury e l e c t r o d e s .
Concentration of 10-4 M s o l u t i o n of q u i n i n e
a l k a l o i d s can b e d e t e c t e d with t h e use of
o s c i l l o p o l a r o g r a p h i c methods.
Q u a n t i t a t i v e o s c i l l o g r a p h i c polarography of
c e r t a i n a l k a l o i d s , including quinine alka-
l o i d , have been a l s o r e p o r t e d
Cinchona a l k a l o i d s g i v e c h a r a c t e r i s t i c
o s c i l l o g r a m s which can b e used f o r t h e i r
d e t e r m i n a t i o n with an accuracy of f 4%.
Girard and R o u s s e l e t ( 7 6 ) have p u b l i s h e d a

polarographic and c o l o r i m e t r i c d e t e r m i n a t i o n
o f q u i n i c i n e i n t h e presence G f l a r g e amount
of q u i n i n e .
1 0 . 4 . Chromatographic Methods
Chromatographic methods have been d e s c r i b e d f o r

t h e s e p a r a t i o n , i d e n t i f i c a t i o n and q u a n t i t a t i o n of
q u i n i n e i n pharmaceutical dosage forms and i n
mixtures (77-93).
10.4.1 Paper Chromatography
The f o l l o w i n g s c r e e n i n g procedure h a s been

reported ( 9 4 ) f o r detecting quinine i n
chemical-toxicological analysis of b io lo g ic a l
m a t e r i a l (blood):
To t h e blood ( 5 m l ) add water ( 3 1 . 5 m l ) and

10% Na2W04 s o l u t i o n (10 m l ) , mix, and add
s l o w l y , w i t h s t i r r i n g , 10% H2S04 (3-5 m l ) .
Heat t h e mixture i n a b o i l i n g - w a t e r b a t h
for 10 minutes, f i l t e r , shake t h e cooled
f i l t r a t e w i t h e t h e r (30 ml) , and d i s c a r d t h e
e t h e r . Adjust t h e aqueous phase t o pH 9
w i t h c o n c e n t r a t e d aqueous ammonia, shake t h e
s o l u t i o n with e t h e r ( 3 0 m l ) , d r y t h e e t h e r
w i t h anhydrous sodium s u l p h a t e , e v a p o r a t e
a t 37' i n a stream o f n i t r o g e n . D i s s o l v e
t h e r e s i d u e i n one o r two drops of chloroform,
apply t h e s o l u t i o n t o Whatman CT30 anion-
exchange paper, and c a r r y o u t ascending
chromatography w i t h 0 . 1 M EDTA f o r 1 4 minutes.
Examine t h e w e t paper i n UV l i g h t a t 254 nm.
R a m a and Singh (83) have p u b l i s h e d a r a p i d

s e p a r a t i o n of q u i n i n e , and o t h e r a l k a l o i d s ,
by ascending paper chromatography on s t r i p s
impregnated w i t h 0 . 1 M zirconium o x y c h l o r i d e
by u s i n g aqueous s o l v e n t s c o n t a i n i n g 0.001 N
H C 1 o r 0.001 N NaOH. The i n d i v i d u a l a l k a l o i d ,
as w e l l as mixtures o f 4 a l k a l o i d s , were
s e p a r a t e d w i t h i n 15-20 minutes.
Q u i n i n e and s e v e r a l o t h e r a l k a l o i d s have been

s e p a r a t e d by u s i n g Whatman No. 1 paper which
h a s been soaked i n S o r e n s e n ' s phosphate
b u f f e r s o l u t i o n ( M / l 5 , pH 5 ) and d r i e d , w i t h
b u t a n o l as t h e mobile phase. The s p o t s a r e
l o c a t e d by s p r a y i n g w i t h i o d o p l a t i n a t e
r e a g e n t ( 91 ) .
5% FARID J. MUHTADI ETAL.
S t r e e t and Niyogi (95) have r e p o r t e d a

new technique of chromatography and iono-
phoresis on ion-exchange paper. This has
been a p p l i e d t o s e p a r a t i o n of a mixture of
compounds including quinine. The mixture
i s subjected t o ascending chromatography i n
0 . 1 M - EDTA on diethylaminoethylcellulose
paper f o r 20 minutes and s u b j e c t t o iono-
phoresis a t a constant c u r r e n t of 1 0 m A
f o r 30 minutes.
Steger and Storz (96) have described micro-

a n a l y t i c a l determination of a l k a l o i d s ,
including quinine, with paper chromatogram
soaked with molybdosilicic a c i d . A f t e r
drying with w a r m a i r , e x t r a c t with a
reducing q u i n o l , sodium carbonate, and
sodium s u l p h i t e . The e x t i n c t i o n of t h e
b l u e s o l u t i o n i s measured c o l o r i m e t r i c a l l y
a t 660 nm.
A l w a s e t a l . (88) have r e p o r t e d t h e
s e p a r a t i o n and q u a n t i t a t i v e a n a l y s i s of
quinine, and o t h e r a l k a l o i d s , by a p p l i c a t i o n
of paper ionophoresis. Jakube e t a l .
have described t h e use of paper chromatogra-
phy i n t h e assay o f m i x t u r e s of pharmaceuticals,
including quinine s a l t s . I n t h i s method,
mixtures of water, low-boiling a l c o h o l
(methanol, ethanol , o r isopropanol) , and
ammonia have been found t o be t h e b e s t
s o l v e n t s , and a mixture of FeC13 w i t h
K 3 Fe (CN)6, t h e b e s t d e t e c t i n g agent.
Quinine (0.005 t o 0.015 mg) , and some

o t h e r a l k a l o i d s , have been d e t e c t e d i n
s e v e r a l foods by using a paper-chromato-
graphic method (98). I n t h i s method,
e t h a n o l i c e x t r a c t i s s u b j e c t e d t o descending
technique on Whatman N o d paperand develop-
ment i s achieved by chloroform. A f t e r drying,
t h e chromatogram i s sprayed with potassium
iodoplatinate.
10.4.2 Thin-Layer Chromatography
The TLC analysis of cinchona alkaloids has been

thoroughly reviewed by Verpoorte , et a1 ( 7 9 ) .
From the TLC systems described in the review,
18 solvent systems were found to be the most
suitable for the separation of the 24 Cinchona
alkaloids.
Table 5 shows the 18 solvent systems used

f o r quinine.
Table 6 describes TLC detection techniques for

quinine.
The sensitivity of a number of separation methods

has been described. Some general conclusions
concerning the optimal conditions for specific
separations are also described
Oswald and Fluck (86) have reported separate

determination of cincho.naalkaloids by TLC.
Quinine, quinidine, and cinchonine are separated
on silica gel G. , with benzene-ethyl ether -
diethylamine (20:12:5) as solvent; cinchonidine
migrates with quinidine. The alkaloids are
identified with Dragendorff reagent. The
spot area, measured by planimeter, is directly
proportional to the amount of alkaloid in the
range of 3 to 4 p g. The limit of error is ?: 8%.
Bralinova ( 7 7 ) has described 6 eluent systems

in the separation of quinine and quinidine on
silica gel, using chloroform-acetone-diethylamine
(20:20:1) as solvent.
Schwarz and Sarsunova (87) have published thin -

layer chromatographic data for 27 alkaloids,
including quinine , on aluminium oxide. The most
useful solvents are: benzene-ethanol; chloroform-
ethanol; and ethyl ether-ethanol.
Sarsunova and Hrivnak (114) have reported the

separation and evaluation of cinchona alkaloids by
TLC, on silica gel -254 with chloroform-acetone-
diethylamine (5:4:1) as solvent.
Table 5 TLC Techniques Used for Quinine
Ref.
Solvent System RF
( RFxlOO )
1. Chloroform-diethylamine ( 9 :1) 17 59
2. Chloroform-methanol - 25% ammonia (85 :14:1) 44 60
3. Chloroform-acetone-diethylamine (5:4:1) 17 61
4. Chloroform-acetone - ( 3 m l 25% ammonia + 17 m l absolute 21 39
ethanol) (5:4:1)
5. Chloroform-acetone-methanol -25% ammonia (60 :20 :20 :1) 37 61
-
fn
00
6.
7.
8.
Chloroform-ethylacetate-isopropanol-diethylamine ( 2 0 :70:4 :6 )
Chloroform-dichloromethane-diethylamine (20:15:5)
Dichloromethaqe-diethyl ether -diethylamine (20:15:5)
11
22
23
39
39
39
9. Kerosene-acetone-diethylamine (23:9 :9) 32 62
10. Acetone - 25% ammonia (58:2) 32 39
11. Ethyl acetate-isopropanol -
25% ammonia (45:35:5) 49 39
12. Toluene-ethyl acetate - diethylamine (7:2:1) 12 63
13. Toluene-ethyl acetate-diethylamine (10:10:3) 18 39
1 4 . Toluene-diethyl ether-diethylamine (20:12:5) 18 64
15. Toluene-diethyl ether-dichloromethane-diethylam-ine 20 65
(20 :20 :20 :8)
16. Carbon tetrachloride-n-butanol-methanol-10% ammonia (12 :9:9:1) - 39
Solvent System
17. Cyclohexanol - cyclohexane-n-hexane (1:l:l) + 5% 41 66

diethylamine
18. Methanol-25% ammonia (100:1 ) 45 61
Conditions :
Silica gel plates Si 60 F 254 pre-coated aluminum sheets,
20x20 cm (Merck);
temperature, 2422';
relative humidity, 2525%;
normal chromatography chamber, saturated for 30 minutes before use
Table 6 TLC Detection of Quinine

Color
1. Quenching, 254 nm 39
2. Fluorescence 360 nm (formic a c i d o r s u l p h u r i c
a c i d spray) Light b l u e 39
3. Dragendorff's modification:
Munier - Macheboeuf Yellow Orange-red 39
Munier Light-yellow Orange-red 67
Munier-sodium n i t r i t e Light -yellow- Brown 67
white
Vaguj f a l v i Light -yellow Orange 39
Bregoff-Delwiche Light -yellow Orange 39
4. Iodine vapour Yellow-white Brown 39
5. Iodine i n K I White Brown 68,69
6. Iodine i n methanol Light -yellow Brown 60
7. Iodine i n K I and s i l v e r a c e t a t e - - 70
8. F e r r i c c h l o r i d e , iodine i n K I Light green- Brown 71
yellow
9. Iodoplat i n a t e Dark v i o l e t Violet 39
10. Iodoplatinate, a c i d i f i e d Dark v i o l e t Violet 72
11. F e r r i c hexacyanoferrate Light green- Dark green 45
blue blue
Background
Reagent Color Color Ref.
~ ~~~
12. F e r r i c chloride-perchloric acid Yellow-white Violet 39

13. Methyl orange Light orange Orange 73
14. Tetraphenylborate quercetin - i n UV:blue 74
15. Phenothiazine, i o d i n e vapor Violet Brown 60
16. Phenothiazine bromine vapor (ammonia
vapor ) Violet Light brown 60
602 FAFUD J. MUHTADI ETAL.
Suchocki e t a l . (115) have r e p o r t e d determination

of quinine, H C 1 and o t h e r compounds, by TLC, on
a s i l i c a g e l with one of s e v e r a l solvent systems.
The s p o t s a r e l o c a t e d with conventional reagents
and are evaluated d e n s i t o m e t r i c a l l y .
Hashmi e t a l . (85) have described semi-quantitative
determination of quinine by c i r c u l a r TLC. Thirteen
a l k a l o i d s , i n c l u d i n g quinine , have been separated
i n t o groups by an e x t r a c t i o n scheme with t h e use of
water, chloroform and ethanol solvent systems.
Aliquots of t h e various e x t r a c t s ( c o n t a i n i n g
0 . 1 t o 7.0 pg of t h e a l k a l o i d ) a r e a p p l i e d t o a
l a y e r of s i l i c a g e l f o r chromatographic a n a l y s i s .
The following method has been described f o r d e t e c t -
i n g quinine i n t o x i c o l o g i c a l a n a l y s i s of b i o l o g i c a l
materials.
The sample of u r i n e ( 1 0 m l ) , b u f f e r e d a t pH 9.5 i s
e x t r a c t e d with chloroform-isopropyl alcohol (24:l).
A c o n t r o l sample of u r i n e c o n t a i n i n g quinine
( 1 0 Ug/10 m l ) i s a l s o e x t r a c t e d . The solvent l a y e r
i s f i l t e r e d , b o i l e d t o remove ammonia, and a c i d i f i e d
with 0.1 N HC1. The solvent i s evaporated and t h e
r e s i d u e i s d i l u t e d with methanol. The s o l u t i o n i s
then a p p l i e d , i n p o r t i o n s , t o s i l i c a g e l G and t h e
chromatograms a r e developed w i t h ethanol-methanol-
concentrated ammonia (17:2:1) as t h e solvent. The
a i r - d r i e d p l a t e i s heated a t 75' f o r 10 minutes,
t h e n sprayed w i t h i o d o p l a t i n a t e and Dragendorff
reagents .
D i f f e r e n t i a t i o n of quinine from i t s oxidation
products has been i n v e s t i g a t e d ( 9 2 ) . A s e n s i t i v e
d e t e c t i o n reagent f o r quinine on t h i n - l a y e r chroma-
togram has been a l s o described ( 9 3 ) . Small amounts
of quinine can be separated from impure samples and
b i o l o g i c a l materials by chromatographic procedures.
Fig. 11 GLC of Quinine Hydrochloride

10.4.3 Gas-Liquid Chromatography
A GLC method f o r t h e determination of

quinine, H C 1 has been c a r r i e d out i n our
l a b o r a t o r y , using a Varian GC-3700 gas
chromatograph equipped with Varian CDS 111
integrator.
Column conditions:
3%OV-17 on chromosorb W-Hp (80-100mesh
s i z e ) ; g l a s s column ( 2 m x 2 mm). The
column run isothermally a t 280' f o r 1 0
minutes and then t h e temperature was
programmed a t 10°/minute. Carrier gas :
helium, flow r a t e w a s a d j u s t e d t o 25 m l /
minute. Detector : FID, hydrogen and a i r
flow r a t e s were a d j u s t e d t o 30/minute and
300 ml/minute, r e s p e c t i v e l y . Ethanol was
used as t h e solvent and t h e c h a r t speed w a s
a d j u s t e d t o give 1 cm/minute. The r e t e n t i o n
t i m e = 13.4 minutes.
The GLC of quinine, H C 1 i s presented i n
Figure 11.
Sarsunova and Hrivnak ( 1 1 4 ) have described
q u a n t i t a t i v e determination of quinine , and
o t h e r cinchona a l k a l o i d s , by e x t r a c t i o n of
t h e s p o t s from t h e TLC p l a t e s followed by
GLC. The a l c o h o l i c s o l u t i o n , containing
codeine as an i n t e r n a l standard, i s analysed
by GLC on a column of 2% of OV-17 on AW
Gas-Chrom p. The method i s s p e c i f i c enough
f o r r o u t i n e drug a n a l y s i s .
A gas-liquid chromatographic procedure has
been a l s o r e p o r t e d (120) f o r t h e determina-
t i o n of s e v e r a l b a s i c drugs, i n c l u d i n g
quinine, i n s m a l l blood samples.
Bonini, e t . a1 (121) have described gas-
phase chromatographic determination of
four a n t i m a l a r i a l s , including quinine,
s i n g l y and i n a mixture i n blood and u r i n e .
I n t h i s method, a column packed w i t h . 2%
OV-17 on Chromosorb W AW DMCS 100-120
mesh with N gas as t h e c a r r i e r gas and t h e
column temperature programmed t o i n c r e a s e
from 250 t o 350' a t 8O/minute. The l i m i t
of d e t e c t i o n was 0.52 ng f o r quinine. The
r e c o v e r i e s i n blood and u r i n e were 84.3 and
92.8% f o r 1 i.lg q u i n i n e / l m l .
10.4.4 Hioh Performance Liquid Chromatography
Quinine has been determined among other

cinchona alkaloids by HPLC. A number of
HPLC assay procedures for quinine and its
impurities have been described (82).
Pound and Sears (122) have used a silica
gel column with a tetrahydrofuran-ammonium
hydroxide mobile phase to analyse quinine
in commercial formulations.
Low and Kennedy (82,123) have described
ion-pair reversed-phase chromatography in
surveying the quinine products available
in Australia.
Table 7 sununarises the solvent systems
used in the cited references.
Barrow et al. (38) have reported HPLC
separation and isolation of quinine metabo-
lites in rat urine. The extract was
evaporated under N, and a solution of the
residue in methanol was analysed on a
column of IJ Bondapak Cl8 and detection at
254 nm. Eight metabolites of quinine were
separated and only 6 were identified.
These were separated on either an analytical
or a semi-preparative reversed-phase column
by gradient elution (Fig. 12).
HYDROXYQUININE+
0-DESMETHYL-
I QUININE-10,ll-
DIHYDRODIOLS
J
QUININE
lPiNINE
-CARBOSTYRIL
I
1 I , 1
0 10 20 30 40 50 60 70 min.
Fig. 12 HPLC Separation of Quinine Metabolites
Table 7. HPLC Systems of quinine
System Column Mobile phase Retention Time Detect ion Ref.

No. (minute)
Merckosorb Si Chloroform-methanol 6.4 W , 254 80

60 (8:2) and 280 nm.
(7:3) 5.9
Diethyl ether-methanol
(8:2) 3.9
(7:3) 2.7
(6:4) 2.2
A reversed- Methanol-water-acetic - W, 254 nm. a2
phase, acid (25:75:1)
u Bondapak
‘18
4 Silica gel Diethylether-water- - - 124
diethylamine
Li Chrosorb Chloroform-isopropyl - W, 312 nm. 125
Si 60 alcohol-diethylamine-wat r
(940:57 :1:2.62)
Li Chrosorb Water-acetonitrile - 250 nm 126
Rp-8 (1:3); or
adjusted to pH 3 with 435 nm.
perchloric acid
A study of t h e r e t e n t i o n behavior of
quinine, and some o t h e r b a s i c drug sub-
stances, by ion-pair HPLC has been described
(127). By appropriate adjustment of expe-
rimental parameters, complex separations
can be achieved.
Jeuring e t a l . (126) have reported a r a p i d
determination of quinine and i t s hydro-
chloride i n s o f t drinks and t o n i c water by
reversed-phase ion-pair chromatography. I n
t h i s assay method, quinine i s determined i n
t h e concentration range of 20-100 mg and
recoveries ranged from 97 t o 103%.
10.5. Spectroscopic Methods
10.5.1 Colorimetric
Hasselmann (128) has reported a micro-

determination of quinine i n serum by means
of t h e colored compound formed with Rose
bengal. The method i s s u i t a b l e f o r determi-
ning concentration up t o 1 mg per 100 ml.
The reported method i s as follows:
To serum (10 m l ) add 20% t r i c h l o r o a c e t i c
a c i d ( 5 ml), shake and centrifuge. Adjust
an a l i q u o t (10 ml) of f i l t r a t e t o pH 11.7
with N NaOH, add 2% Rose bengal s o l u t i o n
(1 n i ~ )and chloroform (3.5 d).AUOW t o
stand for several hours, shaking 5 o r 6
times during t h i s period. Measure t h e c o l o r
i n t h e chloroform l a y e r a t 550 nm.
A blank i s necessary.
Drey (129) has described spectrophotometric
assay of quinine, 2 H C 1 i n t a b l e t s .
Malat (130) has reported an e x t r a c t i o n -
spectrophotometric determination of organic
bases, including quinine, with some metallo-
chromic i n d i c a t o r s . The procedure involves
t h e addition of t h e i n d i c a t o r t o a s o l u t i o n
of quinine,H$O4, a d j u s t i n g t h e pH a t 1 . 4
t o 6.8 and e x t r a c t i o n with chloroform.
The absorption spectrum i s then recorded
from 400 t o 700 nm. Eriochrome red B i s
t h e most s u i t a b l e f o r determination of quin-
i n e ; t h e absorbance being measured a t 475nm.
Schmitz and Menges (131) have a l s o reported

a colorimetric determination of quinine i n
t i n c t u r e s with Tropaeolin 00. I n t h i s
method, about 0.5 gm of t i n c t u r e of cinchona,
i s d i l u t e d t o 250 m l with water, and a 5-ml
portion i s mixed with 1 0 ml of an a c e t a t e
b u f f e r s o l u t i o n of pH 4.6 and 3 m l of a
saturated aqueous Tropaeolin 00 s o l u t i o n .
The well-shaken mixture i s then e x t r a c t e d
with chloroform and t h e combined e x t r a c t s
are a c i d i f i e d with 3 ml of an a c i d reagent
(1 ml of concentrated ~ 2 ~ and 0 4 99 ml of
methanol), and made up t o 50 m l with chloro-
form. The e x t i n c t i o n i s then determined
and quinine content i s estimated by compari-
son with standard curves. Blank determina-
t i o n i s necessary.
Graham and Thomas (132) have described a
q u a n t i t a t i v e determination of a l k a l o i d s ,
including quinine, using dichromate -
sulphuric a c i d . The s o l u t i o n containing
t h e a l k a l o i d ( 0 . 1 - 4 P moles) i s mixed
with 5% aqueous potassium dichromate
s o l u t i o n (1 m l ) , heat a t 30° f o r 5 minutes ,
add concentrated ~ 2 S 0 4( 8 m l ) , mix, cool
i n i c e f o r 20 minutes and measure t h e
e x t i n c t i o n a t 650 nm. Subtract a reagent
blank. The c h a r a c t e r i s t i c green c o l o r i s
given by 25 a l k a l o i d s . Methanol, ethanol,
u r e a , and many s a l t s i n t e r f e r e , but ethanol
may be used a s a solvent i f an equal amount
i s included i n the blank s o l u t i o n .
10.5.2 Ultraviolet
Volkova and Getman (133) have described an

extraction-photometric determination of
quinine as t h e t e r n a r y complex with t i t a -
nium and s a l i c y l a t e . In t h i s method, t h e
t e s t s o l u t i o n (10ml), containing 1ODg
of quinine, i s mixed with 1 m l of 0.01 M -
Tic14 i n 0.8 N H C 1 , 1ml of 0.05 M sod. -
s a l i c y l a t e and 1 0 ml of CHC13. The pH
of t h e s o l u t i o n i s adjusted t o 3 with
0.1 N NaOH and t h e yellow t e r n a r y complex
i s completely e x t r a c t e d i n t o t h e C H C l 3
by vigorous shaking f o r 2 minutes.
The e x t r a c t is f i l t e r e d , and t h e e x t i n c t i o n
of t h e f i l t r a t e i s measured a t 380 o r 400nm.

Chloride , s u l p h a t e , n i t r a t e , and a c e t a t e
do n o t i n t e r f e r e . A t low c o n c e n t r a t i o n o f
q u i n i n e , metal i o n s do n o t i n t e r f e r e ; i r o n
can b e masked w i t h a s c o r b i c a c i d o r sodium
.
t h i o s u l p h a te
Prudhomme (134) has r e p o r t e d a c o l o r i m e t r i c
a s s a y of q u i n i n e i n b i o l o g i c a l f l u i d s and
i n organs. I n t h i s method, q u i n i n e i s
determined by adding 2% e o s i n t o t h e l i q u i d ,
b u f f e r e d t o pH 7 , and e x t r a c t i n g t h e r e d
product w i t h chloroform and comparing
the solution colorimetrically with standard
s e r i e s . Liquids c o n t a i n i n g l e s s t h a n 0.01
mg of q u i n i n e are e x t r a c t e d f o r 24 hours.
Urine i s f i r s t t r e a t e d w i t h l e a d a c e t a t e
and H2SO4, and blood w i t h potassium o x a l a t e
followed by s a t u r a t e d sodium s u l p h a t e and
N H2SO4 a t 45-50'. Compounds of e o s i n w i t h
q u i n i n e can b e d i f f e r e n t i a t e d by UV absorp-
t i o n s p e c t r a . The r a t e o f e l i m i n a t i o n o f
q u i n i n e i n u r i n e , and i t s d i s t r i b u t i o n
among organs have been s t u d i e d .
P r o e t a l . (135) have r e p o r t e d s p e c t r o -
photometric d e t e r m i n a t i o n of q u i n i n e and
.
h e r o i n (diamorphine ) I n t h i s method ,
equimolar c o n c e n t r a t i o n of t h e two a l k a l o i d s
have almost t h e same a b s o r p t i o n a t 297.5 nm;
w h i l s t a t 330 nm t h e a b s o r p t i o n i s due e n t i -
r e l y t o quinine. A 100 mg of t h e mixed
a l k a l o i d s w i t h 10 m l of anhydrous methanol
i s f i l t e r e d through a s b e s t o s , t h e f i l t r a t e
and methanol washings b e i n g d i l u t e d w i t h
0 . 1 N NaOH, and of t h i s s o l u t i o n 10 m l are
a g a i n d i l u t e d w i t h NaOH - methanol s o l u t i o n .
The e x t i n c t i o n i s measured a t 297.5 and
330 nm. The s t a n d a r d d e v i a t i o n f o r b o t h
c o n s t i t u e n t s i s 4 1 . 5 p.p.m. on m i x t u r e s
c o n t a i n i n g 30 t o 90 p.p.m., and 6 t o 30 p.p.m
of q u i n i n e and diamorphine, r e s p e c t i v e l y .
Hadorn and Z u r c h e r ( 1 3 6 ) have p u b l i s h e d
t h e a n a l y s i s and composition of beverages
c o n t a i n i n g q u i n i n e and i t s decomposition
p r o d u c t s . I n t h i s method, 6 samples were
found t o contain ( p e r l i t r e ) 25-57 mg of

quinine t o g e t h e r with o t h e r c o n s t i t u e n t s .
The quinine e x t r a c t e d from t h e beverages
with e t h y l e t h e r w a s pure, but t h e quinine
e x t r a c t e d with chloroform or carbon t e t r a -
c h l o r i d e contained decomposition products
t h a t could be d e t e c t e d by TLC; t h e s e d i d not
i n t e r f e r e with t h e spectrophotometric
determination of quinine i n d i l u t e H2SO4
medium a t 346 nm. The s p e c t r a of quinine
i n e t h a n o l , chloroform, and carbon t e t r a -
c h l o r i d e d i f f e r e d only s l i g h t l y from one
a n o t h e r , but widely from t h e spectrum of
quinine i n d i l u t e H2SO4.
Kamath e t a l . (137) have s t u d i e d t h e W
absorption of cinchona a l k a l o i d s , i n c l u d i n g
q u i n i n e , i n 11 a l i p h a t i c a l c o h o l s . Quinine
can be determined i n t h e presence of o t h e r
alkaloids t o within 14% i n a l i p h a t i c
a l c o h o l medium.
Schmitt (138) has described t h e q u a n t i t a t i v e
and q u a l i t a t i v e determination of W-absorb-
i n g compounds i n substances t o o dark or t o o
t u r b i d f o r d i r e c t a n a l y s i s . The f i r s t or
second d e r i v a t i v e s of t h e W s p e c t r a a r e
used. They show much more d e t a i l than t h e
d i r e c t s p e c t r a . The determination of
quinine i n a t u r b i d beverage by t h i s
technique has been r e p o r t e d
A q u a n t i t a t i v e spectrophotometric determina-
t i o n of quinine and o t h e r cinchona a l k a l o i d s
has been a l s o described
10.5.3 Atomic Absorption Spectrometry

Recently , Minami e t a l . (140) have developed
a q u a n t i t a t i v e a n a l y s i s of a l k a l o i d s , includ-
i n g q u i n i n e , by t h e atomic absorption s p e c t -
rometry. I n t h i s method, 1 ml of a s o l u t i o n
of Reinecke s a l t ( 6 mg/l m l ) and 5-10 m l of
nitrobenzene a r e added t o 5-20 m l s o l u t i o n
of t h e a l k a l o i d ( c o n t a i n i n g 1.5-100 11 g/ml)
i n 0 . 1 M HC1. The mixture i s shaken and
t h e nitrobenzene l a y e r i s removed, d r i e d ,
and C r i s determined t h e r e i n by conventional
flame a.a.s. at 357.87 nm. By t h i s
procedure, quinine, and several other

alkaloids, can be indirectly determined with
good precision. There is no interference
from up to 37-fold molar amounts of 15
common inorganic ions.
10.5.4 Spectrofluorimetric
Ragazzi and Veronese (141) have published
a fluorimetric determination of quinine and
some other alkaloids, after separation by
TLC on magnesium oxide. Quinine is separa-
ted from natural materials or from mixed
pharmaceutical preparations on a layer
prepared from a suspension of hydrated
magnesium oxide in 2.5% aqueous CaC12, using
ethyl acetate - acetone (4:l) as the develop-
ing solvent. After locating the spots of
quinine under W light, they are removed
from the plate and dissolved in acid and the
solution is used for the fluorimetric
determination. Quinine emits fluorescence
at 450 nm when excited at 350 nm.
Brzezinska and Dzeidzianowicz (142) have
reported a fluorimetric assay of quinine
in the presence of aspirin and phenacetin.
Quinine is extracted from the mixture by a
known volume of 0.1 N H2SO4 and the intensity
of fluorescence of quinine sulphate extract
is then measured and referred to a calibra-
tion graph. Beer's law is obeyed for 1 to
50 p.p.m. of quinine in the extract.
Schmollack and Wenzel (143) have described
a fluorimetric determination of quinine in
the nanogram range with use of a chamber
paper-analysis (KAPA) apparatus. Fluori-
metric measurements of quinine sulphate
solution is carried out at 366 nm, showing
a rectilinear calibration between 5 and 50
pg/ml. For 48 measurements at 30 l.lg/ml, the
coefficient of variation is 7.69%.
Other fluorescence analysis of quinine
as well as various natural and synthetic
drugs has been reported (144).
Fluorescence spectra have been determined
in ethanol, concentrated HC1, 10% NaOH, and
aqueous solutions at 0.01-0.001 mg/d

( 145). The following identification test
has been described (146).
Dissolve 1 gm in 50 m l of water; the solu-
tion is not fluorescent. Dilute with lOOml
of water and add M H2S04, an intense blue
fluorescence is produced.
McCloskey et al. (147) have developed a
spectrofluorimetric determination of quinine
in the blood and urine, following the consum-
ption of tonic preparations.
10.5.5 Phosphorimetric
Harbaugh et al. (148)have reported pulsed-

source time-resolved phosphorimetric method
for the quantitative determination of quin-
ine and other drugs. The phosphorescence
emission spectra, life times, and relative
signals (peak emissions) have been determ-
ined with the use of the apparatus and
procedures previously describe& by Fischer
and Winefordner
Time-resolved phosphorimetry provides a
useful means for identifying quinine and
other drugs even in some of their mixtures.
For a multi-component mixture, the paramet-
ers cited indicate which of the drugs can
be separated spectrally or temporarily or
by P conobination of the two techniques.
Acknowledgement
The authors wish to thank M r . Uday C. Sharma

of the Pharmacognosy Dept., College of
Pharmacy, King Saud University, Riyadh,
for his secretarial assistance in the
reproduction of the manuscripts.
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117. K. Wahl and T. R e j e n t , J. Anal. Toxicol., 3, 216 (1979).
118. O.A. Akopyan, B . I . S h v y d i k i i , S . I . Baik, D.Y. Rogovskii,
Z.S. Rokach, A . I . Shkadova, and O.M. Shcherbina, Farm.
Zh., 49 (1979); Anal. A b s t r . , 3,667 (1980).
k,
119. C.L. Brown and P.L. K i r k , Mikrochim. Acta, 5, 720
(1957).
120. A.W. Missen, Rep. N.Z., Dep. S c i . Ind. Res., Chem.
Div., C.D. 2282, 36 (1979); Chem. Abstr., 92, 103928
(19801
121. M. Bonini, F. Mokofio, S. B a r a z i , J. Chromatogr.,
-
224, 332 (1981).
122. N . J . Pound and R.W. S e a r s , Can. J. Pharm. S c i . , 10,
122 (1975).
123. J.K.C. Low and J.M. Kennedy, Unpublished results.
124. R. G i m e t and A. F i l l o u z , J. Chromatogr.,

333 (1979).
125. M. Bauer and G. Untz , J. Chromatogr. , 192, 479 (1980).
126. H . J . J e u r i n g , V.H. W i l l e m , V.D. P i e t e r , and T.B.
R e i n i e r , Z. Lebensm. - U n t e r s . Forsch. , 169, 281
(1979); Anal. A b s t r . , 38, 570 (1980).
127, R.G. Achari and J.T. J a c o b , J. Liq. Chromatogr., 3,
81 (1980).
128. M. Hasselmann, Compt. Rend., 244, 2860 (1957); Anal.
Abstr. , 5, 2316 (1958).
129. R.E.A. Drey, J. Pharm. and Pharmacol., 2, 739 (1957).

130. M. Malat, Anal. Chim. Acta, log, 191 (1979).
131. B. Schmitz and W. Menges, Dtsch. Apothztg., a,

747 (1957); Anal. Abstr. , 2, 1332 (1958).
132. H.D. Graham and L.B. Thomas, J. Pharm. Sci., 2,901
(19611.
133. A.I. Volkova and T.E. Get'man, UKr. Khim. Zh., 2,
1320 (1965); Anal. Abstr., L4 2187 (1967).
134. R.O. Prudhomme, J. Pharm. Chim. , 2, 8 (1940).
135. M.J. Pro, W.P. Butler, and A.P. Mathers, J. Ass.

Off. Agri. Chem., 3, 849 (1955); Anal. Abstr.,
-3 , 1845 (1956).
136. H. Hadorn and K. Zurcher, Mitt. Lebensmitt. Hyg.,
Bern, 55, 194 (1964); Anal. Abstr. , l2, 4856 (1965) .
137. B.R. Kamath, C.V. Bhat, and S.L. Bafna, Indian J.
Chem. , 6 , 510 (1968); Anal. Abstr. , l8, 1204 (1970)
138. A. Schmitt , Chem. Abstr. , 92, 37108 (1980).
139. V.K. Vera and B. Zora, Acta Pharm. Jugosl., Id, 111
(1968); Anal. Abstr. , 2,4337 (1970).
140. Y. Minami, T. Mitsui, and Y. Fujimura, Bunseki

Kagaku, 30, 811 (1981); Anal. Abstr., 43,70 (1982).
141. E. Ragazzi and G. Veronese, Mikrochim. ichnoanalyst.

-
Acta, 5-6, 966 (1965); Anal. Abstr., lb,2188 (1967).
142. D. Brzezinska and W. Dziedzianowicz, Farmacja Pol.,

-
22 , 416 (1966);Anal. Abstr., 14,
7092 (1967).
143. W. Schmollack and U. Wenzel, Pharmazie, 29, 583 (1974).
144. K. Nikolic, D. Malesev, and K. Velasevic, Acta Pharm.

Jugosl., l8, 209 (1968); Anal. Abstr. a,4330 (1970).
145. R.N. Alekseichik, V.P. Korolyuk, E.A. Tukalo, and
E.D. Safonova, Chem. Abstr., 92, 99616 (1980).
146. J.M. Meola and M. Vanko, Clin. Chem., 20, 184 (1974).
147. K.L. McCloskey, J . C . G a r r i o t t , and S.M. R o b e r t s , J.

Anal. Toxicol., 2, 110 (1978).
148. K.F. Harbaugh, C.M. 0' Donnell, and J . D . Winefordner,

Analyt. Chem. , 4 6 , 1206 (1974).
149. L. F i s h e r and J . D . Winefordner, Anal. A b s t r . , 23,

5073 (1972).
RUTIN
Taha I . Khalifa, Farid J . Muhtadi,
and Mahrnoud M.A. Hassan
1. Description 624
1.2 Formulae 625
1.5 Appearance, Color, Taste, and Odour 626
2.1 Crystal Properties 626
2.3 Solubility 626
3. Stability and Incompatibility 639
4. Isolation 639
4.1 Industrialition 639
5. Synthesisof Rutin 642
6. Biosynthesis of Rutin 65 1
7. Biological Properties 65 1
7.1 Phannacological Activity 65 1
7.2 MicrobiologicalActivity 654
7.3 Therapeutic Uses 656
7.4 Metabolism of Rutin 656
8.1 IdentificationTests 658
8.2 Quantitative Determination 660
8.3 UV Spectrophotometry 664
8.4 PMR Spectrometry 664
8.5 Fluorimetry 666
8.7 Densitometry 667
8.8 Gravimetry 668
8.9 Other Analytical Uses 668
References 675
ANALYTlCAL PROFILES OF DRUG SUBSTANCES 623 Copyright by the American Pharmaceutical Associalion.
ISBN 0-12-260812-7
VOLUME I2
624 TAHA I. KHALIFA ETAL.
1. Description
1.1. Nomenclature
1.1.1. Chemical Names
3-[[6-0-(6-Deoxy- -L.mannopyranosyl)
-~-D-glucopyranosyl]oxy]-2-(3,4-di-
hydroxyphenyl)-5,7-dihydroxy-4H-I-
benz op yran-4 -one.
/
Flavone, 3, 5
, 4 , 5-7-pentahydroxy,
3(6(0(6-deoxy- -L-mannopyranosyl))
$-D-glucopyranoside.
2-(3, 4-Dihydroxyphenyl)-3, 5 , 7 tri-

hydroxy 4-ox0-4H-chromen-3-yl rutino-
side.
/ /
3, 3 , 4 , 5-7-pentahydroxyflavone-
3-rutinoside.
Quercet in-3-rut i n o s i d e .
1.1.2. Generic Names (1-8)
Rutin; R u t i n o s i d e ; Rutoside; Vitamin P,

Melin; Phytomelin, E l d r i n ; E l i x a t h i n ;
Sophorin; G l o b u l a r i c i t r i n ; P a l i u r o s i d e ;
O s y r i t r i n ; O s y r i t i n ; M y r t i c o l o r i n ; Viola-
q u e r c i t r i n ; B i r u t a n ; Rutabion; Rutozyd;
Tanrutin.
1.1.3. Pharmacopoeias
Rutin i s o f f i c i a l i n t h e f o l l o w i n g phar-
macopoeias (8) :
Hungarian; Japanese; P o l i s h ; Roumanian;

Russian and S w i s s .
1.1.4. Pharmacopoeia1 P r e p a r a t i o n s
Th& f o l l o w i n g p r e p a r a t i o n s are o f f i c i a l
i n t h e r e s p e c t i v e pharmacopoeias ( 9 ) :
(a) R u t i n I n j e c t i o n (Japanese Pharmaco-

p o e i a ) , A s t e r i l e aqueous s o l u t i o n
of r u t i n . No s t r e n g t h s p e c i f i e d .
I t should be p r o t e c t e d from l i g h t .
RUTIN 625
(b) T a b u l e t t a e R u t i n i ( R u s s i a n and
German pharmacopoeias), Each t a b l e t
c o n t a i n s 20 mg (Russian) o r 50 mg
(German) of r u t i n .
1.1.5. P r o p r i e t a r y N a m e s (8, 10).
B i r u t a n (E. Merck, Germany) ; R u t i n o n

(Rheinpharma, Germany) ; R u t a c i d (CID,
Egypt). I n j t e n s ( D a g r a , H o l l a n d , Rutaminal
Schenely, USA).
1.2. Formulae
1.2.1. Empirical
'27 H30 '16

1.2.2. Structural
OH
R u t i n o s e is: 6-0-(6-Deoxy-'DC-L-mannopyra-
nosy1)-D-glucose w i t h e m p i r i -
c a l f o r m u l a C12H22010 mol.
wt.326.30 and s t r u c t u r a l
f o r m u l a as f o l l o w s :
H
H OH
T h i s s t r u c t u r e w a s proposed by Zemplen
and Gerecs (11) and confirmed by t h e
t o t a l s y n t h e s i s of r u t i n achieved by
Shakhova e t a 1 ( 1 2 ) .
1.2.3. Chemical A b s t r a c t R e g i s t r y Number[CAS No]

( 1 ) [153-18-41
1.2.4. Wiswesser Line Notation
T 66 BO EVJ CR-CQ DQ & DO A GQ IQ

610.51
C , 53.11%; H,4.95%; 0,41.93%
1.5. Appearance, Color, T a s t e , and Odour
P a l e yellow n e e d l e s from water which g r a d u a l l y

darkens on exposure t o l i g h t , tasteless and
odourless.
2.1. Crystal Properties
2.1.1. Water of C r y s t a l i z a t i o n
The c r y s t a l s from water c o n t a i n 3 H20,

and become anhydrous a t l l O O C and 10 mm
Hg .
2.2. Melting P o i n t
Anhydrous r u t i n browns a t 125OC, m e l t s a t 188.7'C,

becomes p l a s t i c a t 195-197OC, and decomposes w i t h
e f f e r v e s c e n c e a t 214-215OC ( 1 )
2.3. Solubility
One gram r u t i n d i s s o l v e s i n about 8 l i t e r s water,

about 200 m l b o i l i n g water, and 7 m l b o i l i n g
methanol. It i s s o l u b l e i n p y r i d i n e , formamide
and a l k a l i n e s o l u t i o n s ; s l i g h t l y s o l u b l e i n a l -
cohol, a c e t s n e , e t h y l acetate; p r a c t i c a l l y inso-
l u b l e i n chloroform, carbon b i s u l f i d e , e t h e r ,
benzene and petroleum s o l v e n t s ( 2 , 3 ) .
RUTIN 627
2.4. Optical Rotation
[ d l i 3+ 13.82' (ethanol); [dli3 - 39.43' (pyri-

d i n e ) ; Deca-methyl d e r i v a t i v e [=I1'
D
- 33' (etha-
nol.
2.5.1. U l t r a v i o l e t Spectrum
The UV spectrum of a u t h e n t i c r u t i n i n
95% methanol was scanned u s i n g Pye Unicam
SP 800; from 200-500 nm. 2-4 d r o p s of
2 M NaOH s o l u t i o n were added t o t h e c e l l
s o l u t i o n and t h e spectrum w a s measured i n
presence of a l k a l i . Other s p e c t r a l
s h i f t s were recorded by scanning
d i f f e r e n t 95% e t h a n o l s o l u t i o n s of r u t i n
t o which w a s added s u c c e s s i v e l y powdered
sodium a c e t a t e and b o r i c a c i d and by
adding two d r o p s 5% a l c o h o l i c aluminium
c h l o r i d e s o l u t i o n and t h e n dil.HCl(13-13.
The W and V i s i b l e S p e c t r a l maxima and
s h i f t s f o r r u t i n are shown i n Table I and
F i g . 1.
Table I! W and V i s i b l e S p e c t r a l Maxima and S h i f t s f o r

Rutin.
bH 0
628
Ethanol S o l u t i o n S p e c t r a l Maxima (nm) Spectral effect Structural diagnosis
Band Band Rand

I I1 111
Alone 259 266,S, 363 1 2 nm hypsochromic 3 -

OH
299,s s h i f t (band 111) substituted
/
Plus 2 drops 2 M 272 327 415 52 nm bathochromic 4 - OH f r e e
NaOH s o l u t i o n s h i f t (band 111)
P l u s 2 d r o p s 5% 275 303,s 433 7 0 nm bathochromic 5 - OH f r e e

A 1 C1 s o l u t i o n s h i f t (band 111)
3
P l u s powdered 271 325 393 1 2 nm 7 - OH f r e e
*
h)
NaOAc s h i f t (band I)
/ /
P l u s NaOAc and 262 298 387 2 0 nm bathochromic 3 ,4 , di OH f r e e
s h i f t (band 111)
H3B03
S = Shouder
These f i n d i n g s are i n agreement w i t h t h e r e p o r t e d d a t a (14, 15 and 16).

2.5.2. I n t r a r e d Spectrum
The I R spectrum of r u t i n as K B r d i s c has

been determined on a Perkin-Elmer 580 B
I n f r a r e d Spectrophotometer (Fig. 2 ) . The
s t r u c t u r a l assignments have been correla-
t e d f o r t h e c h a r a c t e r i s t i c bands as lis-
t e d i n Table 2.
Table 2. I n f r a r e d band f r e q u e n c i e s of Rutin and i t s

c o r r e l a t i o n t o s t r u c t u r a l assignment.
Frequency (Cm'l) Assignment
3330 OH (bonded)
1660 c=o
1620 c=c
1600 Aromatic s t r u c t u r e
1510 C = C aromatic
1460
1360 c - 0 - c
1295 c-0-c
1200 c-0-c
1060 c-0-c
8 10 S u b s t i t u t e d aromatics
These f i n d i n g s are i n agreement w i t h r e p o r t e d d a t a
(16). 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 t o r u t i n
are: 970, 880, 730 and 700.
2.5.3. Nuclear Magnetic Resonance S p e c t r a
2.5.3.1. Proton Spectra
The p r o t o n NMR S p e c t r a of f l a -
von i d s have been e x t e n s i v e1y
s t u d i e d (13). A t y p i c a l PMR
s p e c t r a of r u t i n are shown i n
Fig. 3 & 4. The sample w a s
d i s s o l v e d i n DMSO-D6 and TFA
r e s p e c t i v e l y , and run on a
Varian T 60A, 60-MHz NMR Spect-
rometer. All chemical s h i f t s
fig. 3 NMR Spectrum o f R u t h in DMSO-D,
I I I I I I I 1 I +
I 1 - I
500 400 300 200 100
I . 1 I . I
I I I I I I I I 1
80 TO 6.0 54 4.0 3.0 2.0 in 01

PPM (6)
fig. 4 NMR Spectrum of Rutr'n in TFA.

RUTIN 633
reported a r e i n reference t o
t e t r a m e t h y l s i l a n e (TMS) a t 0
ppm. The PMR s p e c t r a l a s s i g n -
ments of r u t i n are given i n
T a b l e 3.
Table 3. Chemical S h i f t s of R u t i n i n DMSO-D6 and TFA
Group Position Chemical S h i f t s (6)
DMSO-D5 -
TFA
CHM4 Rhamnosyl Me 103(d) 1.45 (bS)
Rhamnoglucosyl 3.20;3.40 3.90;4.20

(bS) (bS)
H-1 Rhamnosyl 4.40 ( S ) 5.00 (S)
5.30 (bS) 5.00 (S)
H- 6 Aromatic 6.17 (SL) 6.72 ( S )
H-8 Aromatic 6.36 (SL) 6.83 (SC)

/
H- 5 Aromatic 6.80 (d) 7.10 (d)
/
H- 6 Aromatic 7.50 (bS) 7.85 (d)
/
H-2 Aromatic 7.50 (S) 8.00 (S)
(S) = S i n g l e t ; (d) = d o u b l e t ; (bS) = broad s i n g l e t ;

(SL) = S i n g l e t showing long range c o u p l i n g .
2.5.3.2. Carbon-13 Magnetic Resonance

Spectroscopy
Proton-noise and o f f - r e s o n a n c e
decoupled 13C-NMR s p e c t r a were
measured on a V a r i a n FT-80 A-
80 MHz F o u r i e r t r a n s f o r m NMR
Spectrometer o p e r a t i n g a t 23.5
m z . Samples were p r e p a r e d i n
1 0 mm 0.d. t u b e s i n approxtmate-
ly 10% s o l u t i o n i n D?'fSO-D6 w i t h
tetramethylsilane a s i n t e r n a l
r e f e r e n c e . S p e c t r a were r e c o r -
ded w i t h 8 K d a t a p o i n t s a t a
probe t e m p e r a t u r e of 23OC. For
an average s p e c t r a l width of
5000 Hz., a 1 0 ps p u l s e w i d t h
corresponding t o a t i l t angle
of 30° w a s employed w i t h 2 s
i n t e r v a l between p u l s e s . 13C-
NMR c o m p l e t e l y decoupled and
o f f - r e s o n a n c e of r u t i n a r e p r e -
s e n t e d i n F i g s . 5 and 6 and t h e
carbon chemical s h i f t s 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 e l y
p r i n c i p a l s and t h e o f f r e s o -
nance s p l i t t i n g p a t t e r n are
shown i n T a b l e 4 .
R e c e n t l y 1 3 C NMR d a t a of f l a v o -
n o i d s w e r e r e p o r t e d ( 17-24) .
Chang ( 2 5 ) determined 1% NMR
of t h e aglycone of r u t i n amongst
o t h e r f a l v o n o i d s by t h e g a t e d
decoupling t e c h n i q u e . The
s p e c t r a l i n t e r p r e t a t ion s we re
based on t h e i n f o r m a t i o n from
13C-lH c o u p l i n g p a t t e r n s .
OH
Rutinose
635
4600 H z
2doo
13
Fig. 5 C NM R OP Rutin, Noise Decoupled S p c t f U m .
4
1
i
I 3h
N
fig.6 C NMR OF Rutin, off Resonance Spectrum.
n n n n n n n
z z z w- z z z
m r l m o N r l e e
. . . . . . .
m b m * m o b
m r l r l U ) m e o
m
rl
m N N r ( r ( O 0 b
r l r l r l r l r l r l r l
n
a,
v)
0
U
1
cd rl
W\U)\d\N\Ul e U) M
W
U)
I
u
3 N O 3 A 1 3 V 8 V 3ns
a
*d
cd
U
I-r
aJ
U
G
0
n n n n
m m c n m m
w w v w .d
m h
. . . . .
r l U ) U l m *
e
rl
U
.d
a,
U ) m U ) e e 0 0
r l r l r l r l r l rl a
Ll
(d
M
:
cd w
Ul 03 N \* \m 0
G
0
P
Ll
cd
3 N 0 3 1 1 3 V 8 V 3 f l S U
.K
RUTIN 637
2.5.4. Mass Spectrum
The mass spectrum of r u t i n o b t a i n e d by

d e s o r p t i o n chemical i o n i z a t i o n (DCI)
u s i n g ammonia as a r e a c t a n t g a s shows a
molecular ion a t m / e 611 amu. The
prominent fragments 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 are shown i n Table 5 .
OH
A B C
Table 5 . Mass Spectrum of R u t i n

-
m/e Relative i n t e n s i t y (2’) Fragment
303 100.00 A + 2 H
611 28.88 M+
628 2.22 M+ + NH3
164 57.77 -
180 66.66 C
3 04 44.44 A + 3 H
308 22.22 -
320 24.44
32 6 42.22
449 11.11
465 33.33
J !
164 180 197 212 431
- -
,430 ,4[0
449
,
463472482
290
I . -
;I0
1 . 1
530 550 570 590
611 628
Fig. 7 Mass Spectrum of Rutin .

RUTIN 639
3. S t a b i l i t y and I n c o m p a t i b i l i t y
Rutin is more s t a b l e than q u e r c e t i n i n t h e presence of

low c o n c e n t r a t i o n s of a l k a l i . I n a c i d s o l u t i o n s , r u t i n
is hydrolysed t o q u e r c e t i n which is r e l a t i v e l y s t a b l e
under t h e s e c o n d i t i o n s ( 2 6 ) . It should be p r o t e c t e d
from l i g h t . Rutin i s incompatible w i t h a c i d s and s a l t s
of heavy metals ( 8 ) .
4. Isolation
R u t h i s undoubtedly t h e most widespread of a l l querce-

t i n g l y c o s i d e s and probably o c c u r s i n up t o 25% of any
given l o c a l f l o r a ( 1 4 ) . It h a s been found t o occur i n
many p l a n t s , e s p e c i a l l y t h e buckwheat p l a n t (Fagopyrum
esculentum Moench., Polygonaceae) up t o about 3% (1)
l e a v e s of tobacco ( N i c o t i a n a tabacum L . , Solanaceae) (1)
-
Ruta g c a v e o l e n s Rutaceae, flower buds of Sophora
japonica ; teguminosae up t o 18% (27) t h e
s t a l k s of tomato,Solanum persicum s o l a n a c e a e ,
l e a v e s of Eucalyptus spp., f l o w e r s of c e r t a i n Mangolia
( 28 ) and many o t h e r p l a n t s .
A g e n e r a l procedure f o r t h e i s o l a t i o n of r u t i n comprises
drying of p l a n t material, followed by e x t r a c t i o n wtth
a l c o h o l , t h e s o l u t i o n c o n c e n t r a t e d and t h e g l y c o s i d e is
l e f t f o r c r y s t a l i z a t i o n (29).
A d e t a i l e d procedure f o r i s o l a t i n g r u t i n from small quan-
t i t i e s of p l a n t m a t e r i a l i s o u t l i n e d i n Fig. 8 ( 30 ) .
4.1. Industrialization
Due t o t h e h i g h p e r c e n t a g e s of r u t i n c o n t a i n e d i n
t h e f a m i l y of Eucalyptus known as Myrtaceal [4-
?4% ( 2 7 ) ] ; thesc. s p e c i e s a r e processed now i n
A u s t r a l i a f o r t h e commercial p r o d u c t i o n of r u t i n
(27 6 31). Although many s o l v e n t s f o r t h e e x t r a c -
t i o n s t a g e have been i n v e s t i g a t e d i n c l u d i n g 95%
e t h a n o l , and hot d i l u t e i s o p r o p y l a l c o h o l ( 3 2 ) , a
s i n g l e - s t a g e b a t c h e x t r a c t i o n w i t h b o i l i n g water
is recommended. (Fig. 9) r e p r e s e n t s a flowsheet
diagram f o r t h e production of 50.000 l b l r u t i n p.a
from Eucalyptus l e a v e s a c c o r d i n g t o Humphrey's
method (31).
TAHA I. KHALIFA ETAL..
Ground p l a n t
mate f i a l
I Extract with boiling

80% Ethanol (2x200 m l )
I Alcoholic
Extract I
Discard Ether Aqueous

4 Extract Solution
S o l i d mate-
r,+
I 1
G ,
Cry st a l l i n e
Solid
Wash w i t h
water,followed iscard
by e t h e r
Crude
Rut i n
P u r i f i c a t i o n on Magnesium
s i l i c a t e column
Pure
Rut i n
. 8.
F i g~. Procedure O u t l i n e f o r I s o l a t i o n of Rutin.
RUTIN 641
I ,
H a r v e s t e d Leaves
of E u c a l y p t u s
1 Moi;;ye.
Comminution
U s i n g hammer
aS3<44 mesh
Ffill
I
Extract ion
Retention t i m e
usinE b o i l i n g
1 hour
water
I
Filtration
>0.7.5% R u t i n -
C o n s t r u c t i o n Material
(wood o r ceramic)
Crystalization
4 hours I
temp. 4n0c
R u t i n r e c o v e r y 95-97%
F i l t e r cake
n r v i n p:
Crush i n g Rutin p u r i t y
I Packing
P r o t e c t from l i g h t ,
F i g . 9: Flow s h e e t f o r t h e commercial p r o d u c t i o n of R u t i n .
642 TAHA I. KHALIFA ETAL..
5. Synthesis of Rutin
The synthesis of rutin can be achieved according to the

following three schemes. These schemes differ in the
synthesis of ouercetin (the aglycone moiety of rutin).
Scheme 1: Kostanecki --
et al. 1904 (33 ) .
Based upon the Claisen reaction between 2-hydroxy-

4, 6-dimethoxyacetophenone [l] and 3, 4-dimethoxybenzal-
dehyde [2] to give the intermediate [3] which upon
treatment with HC1, cyclization occurs to give 5, 7, 3 ,
,
4
' -tetramethoxyflavonone [4]. Oximination affords [5]
which upon treatment with F2SO4 enolisation occurs to
give 5, 7, 1'34', -tetramethoxyflavonol [6]. Demethyla-
tion with HI affords quercetin [7].
Scheme 2: Robinson -
et -
al. 1926 (34 ) .
Condensation ofw-methoxypholoroacetophenone [I]

with veratric acid anhydride [2] in the presence of the
potassium salt of veratric acid to give the diarylester
[3]. On hydrolysis with alcoholic KOH affords 5, 7-di-
hydroxy-3,/3 ,4' -trimethoxyflavone [ 41 , which on deme-
thylation with HI gives quercetin [5].
Scheme 3: Shakhova et al. 1962 (35), complete synthe-
sis of rutin.
W-methoxyphloroacetophenone [2] was condensed with

0-benzylvanillinic acid, anhydride [ 13 in triethylamine
to give 5 , 7-dihydroxy-4 -benzyloxy-3, /3 -dimethoxyfla-
vone [3]. On treatment with AcOH-HC1 mixture gave 5,
7,4' -trihydroxy-3,'3 -dimethoxyflavone [4]. Demethy-
lation of the latter with HI yielded (about 802) quer-
cetin [5]. Ouercetin potassium salt [6] was produced
upon treating [5] with AcOK in ethanol.
Levoglucosan [7] was acetylated with Ac20 in the
presence of AcONa to give 2, 3, 4-triacetyllevoglucosan
[8] which with TIC14 gave 1-chloro-2, 3, 4-triacetyl D-
glucose [9].
L-rhamnose tetraacetate [lo] treated with TiBr4 in
CHC13 gave 1-bromo-2, 3, I-triacetyl-L-rhamnose [ll].
[lo] + [11] heated with Hg (OAC)~in C6H6 gave (53x) CC -
acetochloro-f3-l-L-rhamnosido-6-D-glucose [12]. [12]
was treated with AgOAc and acetylated with Ac20 to pro-
dilce (68.703 B-heptaacetyl-f3-1-L-rhamnosido-6-D-glu-
cose [13]. This with 33% HBr in AcOH gave (61%) d -
acetobromo-~-l-L-rhamnosido-6-D-glucose [14]. [14] and
quercetin potassium salt [6] were dissolved in NH40H
which was evaporated and treated with methanol and
RUTIN 643
Scheme 1 : Synthesis of Quercetin By Kostanecki e t al.

644 TAHAI. KHALIFA E T A .
H3C0
OCH3 0
HO
I HI
Querc etin
RUTIN 645
Scheme 2 : Synthesis of Quercetin By Robinson bt al.

0 0
I1 II
c-0 -c
1 1
COCH20CH3
OH
+
[11
ArCOO
0ch3
ArCOO 0
[31
KOH
EtOH
646 TAHAI. KHALIFA ETAL..
HO
OH 0
[41
HO
OH
OH 0
[51
RUTIN 647
Scheme 3 : Synthesis of Rutin

- co OH
O t OH O O H
COCH20CH3
HO FOCH2
OCH3
I
OH 0
[31
HO
OH
[51
RUTIN 649
CH2 -0
H OH
[71
-4 Tic14
CH20H
0 0
-
0
+ TiBr4
Ho(o$l
OH OH
AgOAc
Ace0 I OH
[i21
0cch3
II
0
HO
0
RUTIN 651
p u r i f i e d o v e r a chromatographic column packed w i t h

polycaprolactum r e s i n t o g i v e r u t i n [151.
6. B i o s y n t h e s i s of R u t i n
P o s t u l a t i o n of t h e b i o s y n t h e t i c pathway of f l a v o n o i d s
s t a r t e d i n 1936 w i t h t h e s u g g e s t i o n o f Rohinson ( 3 6 )
t h a t t h e C15 s k e l e t o n of f l a v o n o i d s t o b e composed of
two p a r t s c 6 and Cg as f o l l o w s :
'6 c-c I cq ( cg+c3 )

B i r c h e t a l . (37 ) proposed t h a t r i n g A of t h e f l a v o -
noid s t r u c t u r e i s produced by t h e a c e t a t e pathway i . e .
3 a c e t a t e u n i t s condensed h e a d - t o - t a i l .
Grisebach ( 38) f e d l 4 C H 3 COOH and CH314COOH and proved
t h a t r i n g A i s b i o s y n t h e s i z e d from a c e t a t e .
Neish and o t h e r s proved t h a t r i n g B i s formed from
d i f f e r e n t r o u t e ( 3 9 ) i . e . cinnamic acid.The b i o s y n t h e s i s
of r u t i n i s p r e s e n t e d i n Scheme 4 .
7. Biological Properties
7.1. Pharmacological A c t i v i t y
R u t i n a s w e l l as i t s aglycone, q u e r c e t i n , have
a d i r e c t c o n s t r u c t o r a c t i o n on t h e c a p i l l a r y bed
and d e c r e a s e t h e p e r m e a b i l i t y and f r a g i l i t y of
t h e v e s s e l s (40). It h a s been s u g g e s t e d t h a t
t h e s e s u b s t a n c e s could b e c l a s s e d a s v i t a m i n s ,
p a r t i c u l a r l y of t h e "Vitamin P" t y p e . R u t i n h a s
been found t o relax t h e i s o l a t e d i n t e s t i n e (41).
Administered i n t r a v e n o u s l y t o t h e dog and r a b b i t ,
i n d o s e s of 5, 20 and 100 mg/kg r e s p e c t i v e l y ,
r u t i n i n v a r i a b l y produces a lowering of t h e blood
pressure (42). Experimentally r u t i n p r o t e c t s
a g a i n s t c a p i l l a r y i n j u r y (43-44) and d e c r e a s e s
t h e erythematous r e s p o n s e t o l o c a l chloroform
i r r i t a t i o n (43 & 4 4 ) . T h i s action may be
due t o t h e a n t i a c i d a n t a c t i o n of r u t i n on adrena-
l i n e , thus r e s u l t i n g i n a s l i g h t increase i n its
l e v e l and so i n c r e a s i n g t h e t o x i c i t y o f t h e p r e -
c a p i l l a r y s p h i n c t e r s and d e c r e a s i n g t h e t o t a l
number of t r u e c a p i l l a r i e s f i t t e d w i t h f l o w i n g
blood (43 & 4 4 ) . An i n t e r e s t i n g e x t e n s i o n
Scheme 4 : Biosynthesis of Rutin
Shikimic a c i d k
- Prephenic a c b-.
id
RUTIN 653
OH
HO
OH
OH 0
Quercetin
UDP-D-glucose
OH
Quercetin 3-glucoside
UDP-L-rhamnose
HO
Rutin OH OH OH
654 TAHAI.KHALIFA ETAL.
of t h i s h a s been t h e r e s u l t s o b t a i n e d from t h e
a d m i n i s t r a t i o n of r u t i n t o r a b b i t s s u f f e r i n g from
s t a n d a r d i z e d experimental p r o s t h i h e . A dose of
50 t o 100 mp;/kg p e r day by stomach t u b e produces
a marked acminution i n t h e loss of t i s s u e ganga-
r e n e following p r o s t b i t e of r a b b i t f e e t , b u t is
i n e f f e c t i v e i n p r e v e n t i n g loss of t i s s u e f o l l o -
wing p r o s t b i t e of r a b b i t ears (45) Quercetin
is less e f f e c t i v e i n t h i s regard t h a n r u t i n ( 4 6 )
Rutin p r o t e c t s a g a i n s t h i s t a m i n e shock i n an in-
d i r e c t way h u t i s n o t a t r u e a n t i h i s t a m i n i c (47).
7.1.1. LD 0
-5
LD50 determined i n mice by i n t r a v e n o u s
a d m i n i s t r a t i o n of propylene g l y c o l s o l u -
t i o n is 950 mg/kp hody weight (48).
7.2. Microbiologlcal Activity
Naghski, Copley and Couch(49-50) r e p o r t e d i n 1947

t h a t q u e r c e t i n , t h e aglycone of r u t i n , e x c r e t e d
some i n h i b i t o r y e f f e c t on t h e growth of S t a o h y l o -
--
coccus aureus and o t h e r organisms, w h i l e r u t i n
and q u e r c e t r i n w a a inacrive t5l),
A recent a n t i m i c r o b i a l s c r e e n i n n of r u t i n and
a u e r c e t i n was Derformed bv c u v - d a t e agar d i f f u -
s i o n method a g a i n a t Gram-positive, Gram-negative
b a c t e r i a and y e a s t - l i k e fungus (52). The results
of a c t i v i t v are p r e s c r i b e d i n Table 6 .
The minimum i n h i b i t o r v c o n c e n t r a t i o n of r u t i n
a g a i n s t Pseudomonas a e r u n i n o s a and P r o t e u s vulga-
-
ris were 10 mg and 10 mg/ml r e s p e c t i v e l y . Quer-
c e t i n minimum i n h i b i t o r y c o n c e n t r a t i o n a g a i n s t
tively.
-
0 0
QI hl
d
0
0
l-i
0 c
Kl
0 cr)
r-l
c
*rl
u
PI
U
&
PI
7
0
656 TAHA I. KHALIFA ETAL..
7.3. T h e r a p e u t i c Uses
C o n s i d e r a b l e i n t e r e s t h a s been evinced i n t h e
p o s s i b l e c l i n i c a l a p p l i c a t i o n of r u t i n t o t h e
medical t r e a t m e n t of t h e s i c k (53 - 5 7 ) .
R u t i n w a s f o r m e r l y used i n t r e a t m e n t of d i s e a s e
s t a t e s c h a r a c t e r i s e d by c a p i l l a r y f r a g i l i t y , b u t
evidence of i t s v a l u e i s i n c o n c l u s i v e . It h a s
been claimed t o b e e s p e c i a l l y of v a l u e i n t r e a t -
ment of r e t i n a l haemorrhage. Though t h e r e i s no
evidence t h a t c a p i l l a r y s t r e n g t h i s s p e c i f i c a l l y
a s s o c i a t e d w i t h v i t a m i n C , some workers have
claimed b e t t e r r e s u l t s from t h e u s e of r u t i n and
a s c o r b i c a c i d s t h a n from r u t i n a l o n e ( 8 ) .
R u t i n from tobacco l e a v e s w a s found e f f e c t i v e i n
t r e a t i n g p a t i e n t s w i t h h y p e r t e n s i o n complicated
by i n c r e a s e d c a p i l l a r y f r a g i l i t y ( 2 7 T 5 8 ) .
R u t i n can i n h i b i t t h e a c t i o n of h y a l u r o n i d a s e ,
p a r t i c u l a r l y when combined w i t h a s c o r b i c a c i d .
T h i s l e d t o t h e t e s t i n g of r u t i n w i t h a s c o r b i c
a c i d as an o r a l c o n t r a c e p t i v e b u t i t s e f f i c a c y i n
t h i s r e s p e c t h a s n o t y e t been confirmed. R u t i n
h a s been used w i t h s u c c e s s i n t r e a t i n g some t y p e s
of h e r e d i t a r y haemorrhagic d i s o r d e r s , s u c h as
haemophilia, and a l s o b l e e d i n g gums, m i g r a i n e
headaches, toxaemia i n pregnancy, e t c . ( 27 >.
I n t h e US, r u t i n i s o f t e n i n c o r p o r a t e d i n v i t a m i n
p r e p a r a t i o n s because of i t s e f f e c t i v e "vitamin P"
f u n c t i o n ( 27 ) .
Reports t h a t r u t i n a s w e l l as o t h e r "vitamin P"
l i k e flavonoids decreased m o r t a l i t y o r hastened
t h e r e c o v e r y of Roentgen-ray i r r i d i a t e d a n i m a l s
were c i t e d (59,60).
R u t i n and g e n e r a l l y b i f l a v o n o i d s s t i m u l a t e t h e
p r o d u c t i o n of blood p l a t e l e t s , which a r e impor-
t a n t i n c o a g u l a t i o n , and a r e recommended i n
t r e a t m e n t of thrombopenia ( 61 >.
7.3.1. T h e r a p e u t i c Dose (8)
50 - 300 mg d a i l y .
7. 4 . Metabolism of R u t i n
R u t i n and o t h e r f l a v o n o i d s are r e p o r t e d (62-67)

t o be r a p i d l y absorbed f o l l o w i n g o r a l a d m i n i s t r a -
t i o n and c o n v e r t e d i n t o a v a r i e t y of hydroxy a r o -
m a t i c a c i d s which are r a p i d l y e l i m i n a t e d i n t h e
u r i n e . The metabolism of r u t i n i s p r e s e n t e d i n
Scheme 5.
RUTIN 657
Scheme 5 : Metabolism of Rutin
m-hydroxy phenyl 3-rnethoxy, 4-hydroxy-

acetic acid phenyl a c e t i c a c i d
658 TAHA I. KHALIFA ET AL.
8.1. I d e n t i f i c a t i o n Tests
8.1.1. Spot Appearance
Daylight : grenish yellow

w : deep p u r p l e
U V / N H ~ : yellow
8.1.2. Chemical Tests
a. D i l u t e s o l u t i o n of r u t i n g i v e s g r e e n
c o l o u r w i t h f e r r i c c h l o r i d e T.S. ( 1 ) .
b. R u t i n i s coloured brown by t o b a c c o en-
zyme under e x p e r i m e n t a l c o n d i t i o n s ( 2 ) .
c. On a p p l i c a t i o n of t h e modified indophe-
n o 1 method f o r t h e d e t e c t i o n of phenols
( 68 ) , r u t i n (> 10 pg) changes f i r s t
t o b l u e then t o green. The c o l o u r
f a d e s on t h e a d d i t i o n of 2 d r o p s 0.02%
aqueous s o l u t i o n of N a C l O ( 6 9 ) .
Among r e l a t e d compounds, h e s p e r i d i n
( > l o p g ) , h e s p e r t i n ( > 2 u g ) , and
a c a c e t i n (> 5 pg) g i v e a similar
c o l o r a t i o n which becomes more i n t e n s e
on a d d i t i o n of NaOCI.
8.1.3. M i c r o c r y s t a l Tests
R u t i n as w e l l a s i t s a g l y c o n e s q u e r c e t i n
(0.2% m e t h a n o l i c s o l u t i o n ) gave c h a r a c t e -
r i s t i c golden c r y s t a l s ( F i g . l o ) , w i t h 2 ,
4-dinitrophenylhydrazine H C 1 r e a g e n t ( l g
i n 30 m l methanol + 2 m l H2S04) ( 7 0 ) .
T h i s t e s t could b e u t i l i z e d f o r t h e r a p i d
d i f f e r e n t i a t i o n of r u t i n and q u e r c e t i n .
Fly, 10 Micfocf’sfals of Rutin A 1 and Quercetin ( 0 ) with 2,4 Dinitrophenylhydraline
TAHA I. KHALIFA E T A .
8.2. Q u a n t i t a t i v e Determination
8.2.1. Colorimetry
Use i s made of t h e c o l o u r e d d e r i v a t i v e s
having h i g h molar a b s o r p t i v i t y i n UV and
v i s i b l e r e g i o n , formed e i t h e r by c h e l a t i o n
w i t h metals o r by r e a c t i o n w i t h s u b s t i t u -
t i o n reagents.
8.2.1.1. Che l a t ion
i. With ALC13
A c c u r a t e l y weighed 5 g p l a n t
sample& of t h e material were
transferred into extraction
t h i m b l e s and e x t r a c t e d w i t h ab-
s o l u t e alcohol f o r 8 hours i n a
Soxhlet a p p a r a t u s . The a l c o h o l
e x t r a c t i o n w a s allowed t o c o o l to
room t e m p e r a t u r e and made up t o
250 m l volume u s i n g a b s o l u t e a l -
cohol. A 25 m l p o r t i o n of t h i s
s o l u t i o n w a s made up t o 100 m l
volume w i t h isoamyl a l c o h o l and
thoroughly mixed; a 20 m l a l i -
quot w a s t h e n t r a n s f e r r e d t o a
s e p a r a t i n g f u n n e l and shaken
w i t h f i v e s u c c e s s i v e p o r t i o n s of
25 m l of 0 . 1 M AlCl3 s o l u t i o n ,
a f t e r each s h a k i n g t h e s e t t l e d
aqueous l a y e r b e i n g r u n o f f i n t o
1000 m l v o l u m e t r i c f l a s k and
mixed t h o r o u g h l y , p l a c e d i n 1 cm
c e l l and t h e a b s o r p t i o n a t 416nm
n o t e d . The p e r c e n t a g e r u t i n w a s
t h e n c a l c u l a t e d from a s t a n d a r d
curve p r e p a r e d w i t h p u r e r u t i n
(71).
A s t h i s method does n o t
measure t h e rutin-AlC13 complex
alone but everything i n the
s o l u t i o n d e r i v e d i n t h i s way
which a b s o r b s a t 416nm, t h e p r e -
sence of o t h e r s u b s t a n c e s must
be checked by chromatography and
an examination of t h e a b s o r p t i o n
curve i n t h e r a n g e 35Onm-5OOnm.
Other flavonoid-AlC13 complexes
u s u a l l y have a b s o r p t i o n m a x i m a
RUTIN 661
which d i f f e r from t h e r u t i n -
A l C 1 3 complex.
An a l t e r n a t i v e p r o c e d u r e
( 72 ) f o r t h e d e t e r m i n a t i o n
of r u t i n overcoming t h e f o r e -
mentioned d i s a d v a n t a g e s i s t o pa-
p e r chromatograph t h e m e t h a n o l i c
e x t r a c t of t h e p l a n t m a t e r i a l
w i t h e t h y l acetate-anhydrous
a c e t i c acid-water (50 :15 :18) a s
developer. After drying t h e
r u t i n s p o t was c u t o u t and ex-
t r a c t e d w i t h nleefianol (2 m l ) ,
t h e n w i t h anhydrous a c e t i c a c i d
(0.6 ml) and 20% aqueous p y r i -
d i n e (10 ml) and 12% m e t h a n o l i c
A1C13 r e a g e n t (2.5 m l ) were
added. The r e s u l t i n g s o l u t i o n
was d i l u t e d t o 25 m l and t h e ab-
s o r p t i o n was measured a t 42Onm
(5-cm c e l l s ) a g a i n s t water.
Beer's l a w was obeyed w i t h up t o
250 pg of r u t i n .
ii. With Beryllium N i t r a t e
To a sample c o n t a i n i n g 0.1 +
1 . 2 u moles of r u t i n ( o r i t s ag-
lycone q u e r c e t i n ) , an e q u a l
amount of B e (NO3)2 d i s s o l v e d i n
methanol was added, t h e n 2 N Na
a c e t a t e s o l u t i o n (1.5 ml) w a s
added, and t h e m i x t u r e was d i l u -
t e d t o 25 m l w i t h methanol.
A f t e r 10 minutes t h e absorbance
a t 465 nm was measured and t h e
r e s u l t s were r e f e r r e d t o a s t a n -
d a r d curve ( 7 3 ) .
R u t i n a s w e l l as i t s aglycone
q u e r c e t i n , r e a c t w i t h Be2+
g i v i n g r e s p e c t i v e l y , yellow and
orange I:1 complexes ( 7 3 ) .
i i i . W i t h Quadrivalent Titanium
Salts
The c o n c e n t r a t i o n s of r u t i n
an a l c o h o l i c e x t r a c t could be
measured by t h e i n t e n s i t y of
c o l o r of an orange yellow complex
formed w i t h Ti0 S04. The pH of

r u t i n containing solution was
a d j u s t e d t o 5.8 w i t h 3 N Na
a c e t a t e . The c o l o u r , which i s
s t a b l e f o r 30 minutes, w a s mea-
s u r e d and t h e r e s u l t s were r e a d
from a graph o b t a i n e d from
r e a d i n g s of s t a n d a r d s o l u t i o n s
( 7 4 1. T h i s method e n a b l e s low
c o n c e n t r a t i o n s (17 ppm) of r u t i n
t o be determined i n pharmaceuti-
c a l p r o d u c t s as well as i n p l a n t
m a t e r i a l ( 7 4 1.
iv. With Uranyl S a l t s
The c o n c e n t r a t i o n of r u t i n
i n a l c o h o l i c e x t r a c t s was d e t e r -
mined a b s o r p t i o m e t r i c a l l y by
measuring t h e i n t e n s i t y of t h e
colour of an orange complex
formed by r u t i n and Uranyl ace-
t a t e . The maximum a b s o r p t i o n
was o b t a i n e d w i t h equimolar s o l u -
t i o n s i n a 1:l r a t i o (75 ) . Con-
c e n t r a t i o n s i n ppm range can be
determined by t h i s method ( 75 ) .
v. With Cupric S a l t s
Rutin (10-M) was determined

by t h e absorbance measurement of
r u t i n CU (IT) complex i n s l i g h t -
l y a l k a l i n e methanol medium
( 76 & 77 1-
Other i n o r g a n i c i o n s produ-
c i n g coloured complexes w i t h
r u t i n e.g. Gallium (111) ( 78
and Antimony I11 ( 78 ) were
a l s o used f o r i t s 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 In p l a n t material
and pharmaceutical p r e p a r a t i o n s .
8 . 2 1.2. Electrophilic Substitution
i. With p-aminobenzoic a c i d
A methanolic s o l u t i o n of
r u t i n ( 4 ug) w a s a p p l i e d t o a
Whatman No. 1 paper, and deve-
loped f o r 10 hours by t h e
RUTIN 663
a s c e n d i n g t e c h n i q u e w i t h n-buta-
n o l - a c e t i c acid-water (20:5:11).
The r u t i n zone w a s l o c a t e d on
d r y chromatogram under UV o r by
t r e a t m e n t w i t h ammonia fumes.
The chromatogram w a s s e c t i o n e d
and e l u t e d i n a t e s t t u b e by
shaking w i t h 5 m l of acid-metha-
no1 ( 1 : l ) . For t h e c o l o r i m e t r i c
d e t e r m i n a t i o n 0.5% p-aminoben-
z o i c a c i d (0.4 m l ) , 10% H 2 S O 4
( 0 . 4 m l ) , 0.2% NaN02 s o l u t i o n
( 2 ml) and 10% NaOH s o l u t i o n
(5 ml) were added and t h e m i x t u r e
w a s shaken. The a b s o r p t i o n a t
420 nm, was measured immediately
( 79 ) . T h i s method c o u l d be
a p p l i e d t o o t h e r f l a v o n o i d com-
pounds 6 i s claimed c o n v e n i e n t f o r
samples w i t h a low r u t i n c o n t e n t
( 79 >.
ii. With E-aminocaproic a c i d
Rutin condensation product

w i t h <-aminocaproic a c i d w a s de-
termined c o l o r i m e t r i c a l l y a t 410
nm u s i n g Al2(SO4)3 a s chromoge-
n i c a g e n t ( 8 0 ) . No i n t e r f e r e n c e
w a s shown by u n r e a c t e d r u t i n
(80 >.
Other e l e c t r o p h i l i c s u b s t i -
t u t i o n r e a g e n t s used f o r t h e
e s t i m a t i o n of r u t i n and o t h e r
flavonoids i n plant e x t r a c t s
i n c l u d e d i a z o t i s e d amines, 4-
aminophenazone, 2 , 6 dibromoqui-
none c h l o r i m i d e , n i t r o u s a c i d
( 81 ) , and b o r i c a c i d i n d r y
a c e t o n e ( 82 ) .
Generally t h e s u b s t i t u t i o n
methods s u f f e r from s e v e r a l d r a w
backs. Not o n l y are t h e m a j o r i t y
of t h e r e a g e n t s r a t h e r u n s t a b l e ,
b u t t h e r e a c t i o n s are c a r r i e d o u t
i n a l k a l i n e s o l u t i o n , where many
p h e n o l s are r a p i d l y o x i d i z e d .
Furthermore, t h e p r e s e n c e of
c a r b o n y l groups i n s e v e r a l f l a v o -
noids reduces t h e i r a c t i v i t y .
Again, t h e p r e s e n c e of c o l o r l e s s
p h e n o l i c compounds makes i t a l -
most i m p o s s i b l e t o u s e such
methods f o r t h e d e t e r m i n a t i o n of
t h e f l a v o n o i d components i n crude
plant extracts.
8.3. W Spectrophotometry
R u t i n h a s been determined i n pharmaceutical prepa-

r a t i o n s by measuring i t s a b s o r p t i o n a t 256 nm
a f t e r being e l u t e d w i t h a 1:l m i x t u r e of 0.1 N
h y d r o c h l o r i c a c i d and methanol from s i l i c a g e l
p l a t e s ( 83 ).
R u t i n (Ca. 15 mg) i n compound p r e p a r a t i o n c o n t a i n -
i n g a e s c u l i n ( C a . 5 mg) i n a d d i t i o n was d e t e r -
mined by a p p l y i n g t h e drug sample t o t h e t o p of a
column of 3 g of polyamide. Aesculin w a s e l u t e d
w i t h water, then r u t i n w i t h methanol. The r u t i n
c o n t e n t w a s determined s p e c t r o p b o t o m e t r i c a l l y a t
360 nm ( 8 4 ) . The recovery of r u t i n is s a i d t o be
95% ( 84 ) .
The q u a n t i t y of r u t i n and o t h e r f l a v o n o i d of
orange j u i c e of high pulp c o n t e n t determined by
e x k r a c t i n g w i t h e t h y l a c e t a t e , and t h e f l a v o n o i d s
of t h e e x t r a c t were s e p a r a t e d on a polyamide l a y e r
w i t h ljenam~-dioxan-form~ca c i d ( 4 : 5 : 1) as develo-
ping s o l v e n t . The chromatogram was i r r i d i a t e d
w i t h l i g h t of wavelength 254 nm where r u t i n zone
was i d e n t i f i e d by i t s d a r k brown f l u o r e s c e n c e .
R u t i n zone w a s e x t r a c t e d w i t h methanol and t h e ab-
s o r p t i o n measured a t 358 nm ( 85 ) .
8.4. PMR Spectrometry
A r a p i d and simple PMR procedure f o r t h e e s t i m a -

t i o n of r u t i n i n b u l k d r u g s and i n p h a r m a c e u t i c a l
p r e p a r a t i o n s h a s been r e c e n t l y r e p o r t e d ( 86 1.
The peaks a t 1.03 ppm and 7 . 5 0 ppm a s s i g n e d t o t h e
t h r e e p o r t i o n s of rhamnose methyl group and t h e
two 2 , 6 p r o t o n s of t h e a r o m a t i c r i n g of t h e
aglycone moiety were chosen 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 r u t i n .
Acetamide, e x h i b i t i n g t h r e e methyl p r o t o n s s i n g l e t
a t 2 . 0 0 w a s used as an i n t e r n a l s t a n d a r d ( F i g . 1 1 ).
DMSO-D6 h a s been used a s a s o l v e n t i n t h e a s s a y .
The method proved t o be r e l i a b l e and a c c u r a t e be-
s i d e s i t a l s o f u r n i s h e s a s p e c i f i c means of iden-
t i f i c a t i o n of r u t i n a s w e l l as simultaneous d e t e c -
t i o n of any h y d r o l y t i c p r o d u c t s of t h e assayed
r u t i n v i z . q u e r c e t i n , rhamnose and g l u c o s e . T h i s
f i n d i n g h a s c o n t r i b u t e d g r e a t l y t o t h e method.
8.5. Fluorimetry
A method based on measuring t h e i n t e n s i t y of r u t i n
aglycone, q u e r c e t i n , as w e l l as o t h e r f l a v o n o i d s
complexes w i t h A 1 d i r e c t l y on p a p e r chromatograms
w a s d e s c r i b e d by Tyukavkina e t a 1 ( 87 ) . R u t i n
and o t h e r f l a v o n o i d s were s e p a r a t e d by a s c e n d i n g
chromatography on slow paper w i t h CHCl3-acetic
a c i d (1:2) a s a s o l v e n t . The chromatogram w a s
t r e a t e d w i t h 0.02 M A1C13 - 0 . 1 M Na acetate i n
50% e t h a n o l . Pieces of t h e chromatogram (3x4 cm)
were a t t a c h e d t o t h e w a l l of a c e l l i n a f l u o r i -
meter, and t h e f l u o r e s c e n c e of t h e complexes were
e x c i t e d w i t h a mercury lamp through a USF-3 f i l t e r
a t an a n g l e of 45O ( 8 7 ) . The r e c t i l i n e a r p a r t o f
t h e c a l i b r a t i o n graph l i e s i n t h e range of 1 . 6 -
3 iJg f o r q u e r c e t i n . The method w a s proved f o r
various flavonoids i n prepared mixture with a
d e t e c t i o n l i m i t of 0.05 I.rg and 0.8 I.rg f o r querce-
t i n and d i h y d r o q u e r c e t i n r e s p e c t i v e l y .
Another f l u o r i m e t r i c - p l a n i m e t r i c method f o r t h e
e s t i m a t i o n of r u t i n and f l a v o n o i d compounds w a s
d e s c r i b e d by J e r z y e t a 1 ( 88 ) . The f l a v o n o i d
compounds were s e p a r a t e d by two-dimensional p a p e r
chromatography a s d e s c r i b e d by Glotzbach and
Rimpler ( 89 ) and t h e n t h e c o n t e n t s of conpo-
n e n t f l a v o n o i d s were determined by p l a n i m e t r y
a f t e r d i r e c t measurement of t h e f l u o r e s c e n c e a t
365 nm ( 89 ) .
8.6. Polarography
A method based on t h e d e t e r m i n a t i o n of n i t r o d e r i -
v a t i v e s of r u t i n (and q u e r c e t i n ) i n p h a r m a c e u t i c a l
p r e p a r a t i o n s w a s d e s c r i b e d by Davidek and Manousek
( go .) The drug ( 0 . 1 g) w a s d i s s o l v e d i n me-
t h a n o l (25 m l ) , and an a l i q u o t of t h i s s o l u t i o n
(0.5 ml) w a s mixed w i t h methanol (0.5 m l ) , 0.2 N
H2SO4 (5 ml) and 3 M KN02 (2 ml) i n a p o l a r o g r a -
p h i c v e s s e l . A f t e r bubbling t h e mixed s o l u t i o n
w i t h n i t r o g e n f o r 2 . 5 minutes, 2.5 M N a a c e t a t e
(2 ml) was added and t h e b u b b l i n g w a s c o n t i n u e d
f o r a f u r t h e r 4 minutes. The p o l a r o g r a p h i c c u r v e
w a s recorded and t h e f i r s t s t e p measured. The
h e i g h t of t h e s t e p w a s e v a l u a t e d by means of a
c a l i b r a t i o n curve o r by s t a n d a r d a d d i t i o n . It i s
r e p o r t e d t h a t even 10-6M s o l u t i o n of r u t i n may b e
analyzed; t h e h e i g h t of t h e waves are independent
of t i m e and t h e e r r o r is f 4% ( g o ) . A s c o r b i c
a c i d and o t h e r compounds l i k e l y t o be p r e s e n t i n
p h a r m a c e u t i c a l s are s a i d t o have no i n t e r f e r e n c e
( 90 1.
RUTIN 667
Another p o l a r o g r a p h i c d e t e r m i n a t i o n of r u t i n and
q u e r c e t i n i n c o n c e n t r a t i o n of Ca 1 0 - 6 ~a f t e r n i t r o -
s a t i o n by means of t h e f o u r - e l e c t r o n wave produced
by t h e r e d u c t i o n of t h e n i t r o s o group w a s a l s o
mentioned by t h e same a u t h o r s ( 91 ).
8.7. Densitometry
Cine e t a 1 ( 92 ) d e s c r i b e d a d e n s i t o m e t r i c method
€ o r t h e d e t e r m i n a t i o n of r u t i n and q u e r c e t i n i n
m i x t u r e s . S e p a r a t i o n of r u t i n from q u e r c e t i n w a s
done on s i l i c a g e l G p l a t e s u s i n g a 72:18:10
benzene-pyridine a c e t i c a c i d system. The two
compounds were determined a t 370 nm and 400 nm
respectively. The d e t e r m i n a t i o n e r r o r was 5%.
8.8. Gravimetry
The g r a v i m e t r i c methods by Rodwell ( 9 3 ) , Naghski

-
et -
a1 ( 9 4 ) , based on Sando and B a r t l e t t ' s ( 95 )
method of i s o l a t i n g r u t i n were used i n t h e p r e l i -
minary work. An estimate of t h e v a r i a t i o n i n t h e
r e s u l t s o b t a i n e d by t h e s e methods w a s made, t h e
s t a n d a r d d e v i a t i o n b e i n g about 0.5% f o r r e s u l t s
v a r y i n g from 5-20% r u t i n .
8.9. Other A n a l y t i c a l Uses
8.9.1. A s Chrcmogenic Reagent
R u t i n and i t s aglycone, q u e r c e t i n , were

found t o b e s e n s i t i v e r e a g e n t s € o r d e t e c -
t i n g i n o r g a n i c c a t i o n s on paper chromato-
g r a p h s ( 9 6 ) . The t e s t e d c a t i o n s i n c l u d e
Ag, Hg, Cu, B i , Sb, Sn, Fe, A l , N i , Co,
Mg, L i , Mo, Be, Ga, G e , I n , P r , N e , Sm,
U, V , W, T i , L a , Th, Z r , and a r s e n a t e
( 96 & 97 1 -
From t h e a b s o r p t i o n c u r v e s i t w a s conclu-
ded t h a t e a c h atom of a b i , t e r and
q u a d r i v a l e n t metal combines w i t h 2 , 3 and
4 m o l e c u l e s of r u t i n o r q u e r c e t i n r e s p e c -
t i v e l y ( 9 6 & 92).
8.9.2. A s A n A n a l y t i c a l Reagent
Oka and Matsuo ( 98 ) r e p o r t e d a

s p e c t r o p h o t o m e t r i c method f o r t h e determi-
n a t i o n of microgram q u a n t i t i e s of Germa-
nium u s i n g q u e r c e t i n , t h e aglycone of
668 TAHAI. KHALIFA ETAL.
r u t i n . Q u e r c e t i n w a s allowed t o react with

G e i n n e u t r a l s o l u t i o n (PH 6.4 - 7 . 1 ,
phosphate b u f f e r ) t o g i v e a y e l l o w i s h comp-
l e x (A max 410 nm) which i s s o l u b l e i n
water c o n t a i n i n g > 40 % methanol. The ab-
s o r p t i o n spectrum of q u e r c e t i n i t s e l f h a s
X max of 258 nm and 375 nm and h a s l i t t l e
i n f l u e n c e on t h e l i g h t a b s o r p t i o n a t 410,
420, 430 and 440 nm f o l l o w s Beer's l a w f o r
0 . 5 pg of G e p e r m l i n t h e p r e s e n c e of
an e x c e s s q u e r c e t i n (> 1 4 t i m e s t h e equi-
v a l e n c e of Ge) ( 98 ) .
Other n e t h o d s were r e p o r t e d u s i n g r u t i n
o r i t s aglycone q u e r c e t i n f o r t h e d e t e r -
m i n a t i o n of c a t i o n s e . g . Sn ( 99 ) , Z r
( gg 1, B ( gg 1, and V ( 100 ).
8.10. Chromatography
8.10.1.. Paper Chromatography
8.10.1.1. One-Dimensional Descending PC
The chromatographic d a t a of r u t i n
u s i n g one-dimensional descending
PC under d i f f e r e n t c o n d i t i o n s i s
g i v e n i n Table 7 .
8.18.1.2. Two-Dimensional Descending PC
R u t i n i s r o u t i n e l y used as a
s t a n d a r d marker i n s c r e e n i n g a l -
coholic plant e x t r a c t s f o r t h e i r
flavonoid p a t t e r n s using t h e
s o l v e n t s n-butanol-acetic acid-
water (4: 1:5; t o p l a y e r ) BAW,
and 5% a c e t i c a c i d ( 1 4 ) .
R u t i n i s u s e f u l s i n c e i t occu-
p i e s a p o s i t i o n approximately i n
t h e middle of t h e chromatogram
and a l s o i s , i t s e l f , v e r y
common i n p l a n t s and t h u s one of
t h e most l i k e l y compounds t o be
found d u r i n g s u r v e y work.
Table 7. Paper Chromatography of Rutin
Technique One-dimensional descending PC
Paper Whatman No. 2.
Solvent
sl s2 s3 s4 s5 ‘6 s7
Detection W; Brownish Yellow Fluorescent s?ot
I
hRf 45 46 15 a3 45 51 23
Reference (101-1l92X( 101-102) (1031 (lo$) 0 0 3 ) (103) (102 )

S1 BAW (4:1:5, upper phase).
S2 Acetic acid-conc. HC1-water (30:3:10).
S3 Ethyl acetate-water (saturated).
S4 150-propanol-water (6: 4 ) .
S5 n-Heptane-n-Butanol-water (29:14:57).
s6 Acetic acid-water (15:85).
S7 Water.
8.10.1.3. P r e p a r a t i v e PC
P r e p a r a t i v e PC i s such a w e l l
known t e h c n i q u e and i t s u s e i n
t h e f l a v o n o i d f i e l d h a s been s o
well-reviewed r e c e n t l y (13, 1 4 ,
& 104 ) t h a t a b a r e o u t l i n e of
recommended t e c h n i q u e s should b e
sufficient .
The u s u a l p a p e r used f o r l a r g e
s c a l e s e p a r a t i o n (1-100 mg) i s
Whatman No. 3 o r i t s e q u i v a l e n t .
The s o l u t i o n t o b e s e p a r a t e d
(Ca. 1 0 ml) i s a p p l i e d as a con-
t i n u o u s even narrow s t r e a k o r
band a l o n g t h e s t a r t l i n e by suc-
c e s s i v e a p p l i c a t i o n s . For r u t i n
and t h e m a j o r i t y of f l a v o n o i d s
s e p a r a t i o n i s f i r s t e f f e c t e d by
t h e use of BAW m i x t u r e s ( e . g .
6:1:2). It i s convenient t o
l o c a t e t h e s p o r t s by t h e i r f l u o -
r e s c e n c e i n W. A f t e r l o c a t i o n ,
t h e bands are c u t o u t , t h e com-
pounds e l u t e d s e p a r a t e l y , u s u a l l y
w i t h 70% aqueous methanol, and
he s o l u t i o n s c o n c e n t r a t e d f o r
r e p u r i f i c a t i o n i n a second s o l -
vent.
8.10.2. L i q u i d Column Chromatography (LC)
Tomas e t a1 ( 1 0 5 ) s e p a r a t e d r u t i n , quer-
c e t i n and o t h e r f l a v o n o i d s on sephadex G-
25. Glyzosides were r e a d i l y e l u t e d w i t h
water, w i t h good s e p a r a t i o n of r u t i n and
q u e r c e t r i n : t h e accompanying aglycones
w e r e r e t a i n e d a t t h e t o p of t h e column and
could b e s u b s e q u e n t l y e l u t e d w i t h 0.1%
aqueous ammonia s o l u t i o n .
A 16~0.9cm column of Amberlite XAD-2
(200-400 mesh) maitltained a t 95OC w a s used
f o r t h e s e p a r a t i o n of many f l a v o n o i d s
( 1 0 6 ) . The column w a s f i r s t e q u i l i b r a -
t e d w i t h 20% e t h a n o l a t a f l o w r a t e of
60 ml/hour and a s o l u t i o n o r s u s p e n s i o n of
f l a v o n o i d s i n 20% e t h a n o l (each c o n t a i n i n g
l e s s t h a n 500 pg/ml) w a s placed on t h e t o p
of t h e column. A 100 m l volume of 20%
e t h a n o l i s run f i r s t , followed by l i n e a r
g r a d i e n t e l u t i o n w i t h a t o t a l volume of
RUTIN 671
1000 m l , t h e e t h a n o l c o n c e n t r a t i o n i n c r e a -
si-ng from 20 t o 9Oc. The r u t i n group
f l a v o n o i d s w e r e e l u t e d from t h e column i n
t h e o r d e r r u t i n , q u e r c i t r i n , and t h e n quer-
c e t i n (106 ) . T h i s p r o c e d u r e w a s a l s o
a p p l i e d t o t h e d e t e r m i n a t i o n of f l a v o n o i d s
i n c r u d e m e t h a n o l i c e x t r a c t s from p l a n t s
( 106 ) .
The chromatographic behaviour of r u t i n
u s i n g LC under d i f f e r e n t p a r a m e t e r s i s
summarized i n T a b l e 8.
8.10.3. Thin Layer Chromatography (TLC)
Although a n a l y t i c a l TLC of r u t i n and o t h e r

f l a v o n o i d compounds on m i c r o c r y s t a l l i n e
c e l l u l o s e , s i l i c a g e l o r polyamide i s con-
s i d e r e d a r a p i d method of i n i t i a l s c r e e n -
i n g o r checking t h e p u r i t y of i s o l a t e d
compounds ( 1 3 , 1 4 and 81 ) , t h e t e c h -
n i q u e is n o t f a v o u r i t e l y used f o r i n i t i a l
examination of c r u d e p l a n t e x t r a c t s
because t h e r e s o l v i n g power i s g e n e r a l l y
i n s u f f i c i e n t . However, TLC is o f t e n t h e
method of c h o i c e f o r f i n a l p u r i f i c a t i o n
of r u t i n and o t h e r f l a v o n o i d s , e s p e c i a l l y
using s i l i c a g e l s i n c e here contamination
i s less t h a n on p a p e r ( 109 ) .
The chromatographic d a t a of r u t i n u s i n g
d i f f e r e n t TLC t e c h n i q u e s a r e d e p i c t e d i n
T a b l e . 9.
The chromatographic behaviour of f l a v o -
n o i d s on t h i n l a y e r s i s sometimes m i s -
l e a d i n g s i n c e some of them w i l l g i v e t h e
same Rf v a l u e s even w i t h more t h a n one
s o l v e n t system. T h i s l e d H u r s t and
Harborne t o develop a method based on
r e d u c t i v e c l e a v a g e of t h e s e compounds t o
give rise t o phenols, phenolic alcohols
and p h e n o l i c a c i d s which c o u l d b e more
r e a d i l y i d e n t i f i e d (110). Q u e r c e t i n , t h e
aglycone of r u t i n , g i v e s r i s e t o Dhloro-
$ u c i n o l (A-ring fragment) and 3,4-dihyd-
roxyphenylpropionic a c i d and 3,4-dihydroxy-
phenylpropanol (B-ring f r a g m e n t s ) (110).
Table 8 . Column Chromatography of Rutin
Packing Sephadex G-25, medium particle size grade Sephadex LH-20

Column 35 cm long X 2.5 cm diameter 45 cmx2.5 cm
Material
Solvent
H20 I 0.05 M NaCl 0.1 M NH40H 0.01 M Sod. Molybdate Methanol
Flow rate 25mlllw1.r 25 ml/hour 25 ml/hour 25 ml/hour 3-5 ml/min.

Temperature
Detect ion
Kd 5.60 6.50 3.90 0.26 Ve/Vo* 4.00

9
N
(107) (107) (107) (107) ( 108)
Kd = Dsstribution coefficient
* Under these conditions Ve/Vo = 2.2 Kd + 1; Ve = elution volume, Vo = intersitial volume.
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8.10.4. Gas L i q u i d Chromatography (GLC)
The a g l y c o n e s of r u t i n and o t h e r f l a v o n o i d s
could be s e p a r a t e d as t r i m e t h y l s i l y l (TMS)
e t h e r s by GLC ( 1 1 2 ), b u t l i t t l e u s e h a s
been made of t h i s t e c h n i q u e c o n s i d e r i n g
i t s s e n s i t i v i t y ( t o t h e nanogram l e v e l ) ,
r a p i d i t y and t h e f a c t t h a t t h e compounds
can be r e a d i l y e s t i m a t e d q u a n t i t a t i v e l y
(113). I n almost r e p o r t e d c a s e s f l a v o n o i d
e t h e r s have been s e p a r a t e d on columns con-
t a i n i n g t h e s i l i c o n e t h e r polar phases
e . g . OV 1, OV 1 7 o r SE 30 ( 1 1 2 ) .
8.10.5. Electrophoresis
E l e c t r o p h o r e s i s on e i t h e r paper o r t h i n
l a y e r s i,s a r e p o r t e d t e h c n i q u e (:114) which
h a s been s c a r c e l y used i n t h e f l a v o n o i d
f i e l d . R u t i n , and q u e r c e t i n amongst o t h e r
f l a v o n o i d g l y c o s i d e s and a g l y c o n e s could
be r e a d i l y s e p a r a t e d u s i n g b o r a t e b u f f e r s
on TLC c e l l u l o s e l a y e r s ( 114 ) . However,
e l e c t r o p h i l i c examination of e x t r a c t s con-
t a i n i n g charged flavonoid-compounds of a l l
t y p e s , which may be more widely d i s t r i b u -
t e d t h a n h i t h e r t o b e l i e v e d , may pay hand-
some d i v i d e n d s ( 8 1 and 115).
RUTIN 675
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RUTIN 681
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ron. 24, 1407, (1968).
TRIMIPRAMINE MALEATE
Abdullah A . Al-Badr
1 . Description 684
1 . 1 Nomenclature 684
1.2 Formulae 684
1.5 Chemical Abstract Registry Number 685
1.6 Appearance, Color, and Odor 685
2. I Melting Point 685
2.2 Solubility 685
3. Synthesis 696
4. Metabolism and Excretion 699
5.2 Spectrophotometric Methods 700
5.3 Polarographic Method 702
References 710
ANALYTICAL PROFILESOF DRUG SUBSTANCES 683 Copyright by thc American Pharmaceutical Association
VOLUME 12 ISBN 0-12-269812-7
684 ABDULLAH A. AL-BADR
1. Description
1.1 Nomenclature
1.11 Chemical Names
- lO,ll-Dihydro-N,N,B-trirnethyl-5H-dibenz[b,f]
azepine-5-propanamine hydrogen maleate
- 5-[3-(Dirnethylamino)-2-methylpropyl]-l0,11-
dihydro-5H-dibenz[b,f]azepine hydrogen
maleate
- 5-(3-Dimethylamino-2-methylpropy)iminodiben-
zyl hydrogen maleate
- 3-(lO-ll-Dihydro-5H-dibenz[b,f]azepine-5-y1)
-2-methyl-propyl-N,N-dimethyl ammonium hydro
-gen maleate
- 5-(3-Dimethylamino-2-methylpropy)-lO,ll-di-
hydro-5H-dibenz[b,f]azepine hydrogen maleate
- Trimipramine acid maleate

- Trimipramine hydrogen maleate
1.12 Generic Names
Trimepramine,Trimeprimine, Trimeproprimine
RP 7162, Sapilent.
1.12 Trade Names
Stangyl, Surmontil
1.2 Formulae
1.21 Impirical
2OH26N2 (base)
(maleate salt)
C20H26N2y ‘qH4’4
TRIMIPRAMINE MALEATE 685
1.22 Structural
lo 11
CH COOH
t CH3
CH COOH
CH3
1.3 Molecular Weieht
Trimipramine 294.42
Trimipramine Maleate 410.5
1.4 Elemental Composition
C 81.58% H 8.90% N 9.52% (base)
C 70.22% H 7.36% N 6.82% 0 15.60% (maleate)
1.5 Chemical Abstract Reeistw Number
[ 739-71-91 base
[521-78-81 maleate salt
1.6 Appearance, Color and Odor
A white crystalline, powder, odorless or almost

odorless (1).
2.1 Meltine Point
Trimipramine 45O (21

Trimipramine maleate 140-144' (1)
2.2 Solubility
Trimipramine maleate is slightly soluble in water

and in ethanol (96%); freely soluble in chloroform;
practically insoluble in ether (1).
686 ABDULLAH A . AL-BADR
2.3 Identification
Clarke (2) described the following tests:
I) Trimipramine can be identified by forming crys-

tals with platinic chloride solution where need-
les, often serrated are formed (sensitivity: 1
in 1000). Trimipramine also forms dense rosettes
with trinitrobenzoic acid solution (sensitivity
1 in 1000).
11) Trimipramine can be identified by the following

color tests:
- Ammonium molybdate test; blue color is pro-

duced (sensitivity 0.1 ug) .
- Ammonium vanadate test; blue color is pro-

duced (sensitivity 0.1 pg) .
- Vitalits test; deep greenlyellowlyellow

(sensitivity 0.1 pg) .
The following tests are cited in both European

Pharmacopoeia 1975 (3) and the B.P. 1980 (1) for
the identification of trimipramine maleate:
a) To 0.1 g add 2 ml of alcohol, heat to boiling

and add 1 ml of a standard solution of picric
acid in alcohol. Scratch the walls of the
tube with a glass rod until crystallization
begins. Allow to stand for 15 minutes, filter,
wash the precipitate with alcohol and dry at
100" to 105". The picrate has a melting point
of 131'.
b) Triturate 0.1 g with 3 ml of water and 1 ml

of strong sodium hydroxide solution and
extract with three quantities, each of 5 ml
of ether. To the aqueous solution add 2 ml o f
bromine solution. Heat in a water bath for 10
minutes, then heat to boiling and cool. Add
to 0.1 ml of this solution, a solution of 10
mg of resorcinol in 3 ml of sulphuric acid
and heat on a water bath for 2 minutes, then
cool. A deep blue color develops.
c) A 0.002 per cent w/v solution in 0.1 N hydro-

chloric acid, examined between 230 nm and
350 nm, shows a single absorption maximum at
about 250 nm. The extinction at the maximum
at about 250 nm in a 1 cm cell is about 0.42.
The following test is cited in the European

Pharmacopoeia 1975 (3).
Dissolve about 5 mg in 2 ml of nitric acid; an

intense blue color is produced whichturns green
on standing.
2.4 Spectral Properties
2.41 Ultraviolet Spectrum
The ultraviolet spectrum of trimipramine maleate

in neutral methanol solution in the region of
200 to 350 nm exhibits a maximum at 247 nm and
a minimum at 233 nm. The spectrum is shown in
Figure 1.
Trimipramine in 0.1 N sulphuric acid exhibited

a maximum at 250 nm (E 1 cm 300) and an in-
1%’
flexion at about 268 nm (E 1 cm 250) (2).
1%’
According to B . P . 1980 (2) the UV spectrum of
trimipramine maleate of a 1 cm layer of 0.002
percent w/v solution in 0.1 M HC1 exhibits a
maximum only at 250 nm and an absorbance at
250 nm, about 0.42.
2.42 Infrared Spectrum
The infrared spectrum of trimipramine maleate is

shown in Figure 2. The spectrum was obtained as
KBr disc. The structural assignments have been
correlated with the following band frequencies:
-1
Frequency(cm 1 Assignment
3430 N-CH3
3057 Aromatic CH stretch
2949, 2825 Asymmetric and Symmetric

CH stretch
688
1 1 L
0
c
O
B
0
m s 0
N
0
689
2921, 2852 Asymmetric and Symmetric

CH2 stretch.
1620, 1570 Aromatic C=C stretch.
Other finger print band characteristics to

trimipramine (determined in KBr disc) are 1351,
1488 and 1580 cm-' (2).
2.43 Proton Nuclear Magnetic Resonance Spectrum (PMR)

The 60 MHz spectrum of trimipramine maleate in
deuterated dimethylsulphoxide is shown in
Figure 3. The spectrum was determined in Varian
T60 A NMR Spectrometer with tetramethylsilane
(TMS) as reference standard. Assignment of the
bands are as follows:
Chemical Multiplicity Assignment

shift ppm
0.98 d CH2-CH(CH
-3 ) -CH2-
/CH?
2.8 S -N
\
CH,
3.1-3.9 m Methylene protons.
(unresolved)
6.15 S CH=CH (ma1eate)
7.05 m Aromatic protons.
s: singlet; d: doublet; m:multiplet.
Proton magnetic resonance was reported t o be

useful for the identification of trimipramine
and some other tricyclic psychotropic drugs.
The drug can be characterized by examining the
signal given by the protons of the ring and
side chain. It is also reported that NMR spectro-
metry is a technique of choice for the rapid
identification of the drug (4).
s
U
.d
3
Q)
U
m
01
4
ld
E
..
r-7
Q)
L
3
M
2.44 13C-Nuclear Magnetic Resonance Spectrum(”C NMR)
The I3C-NMR spectrum of trimipramine maleate in

deutrated chloroform using tetramethylsilane as
an internal reference is obtained on a Jeol FX
100-100 MHz at an ambient temperature using
10 mm sample tube. The spectrum is shown on
Figure 4 and the carbon chemical shift values,
shown in Table 1 are derived from the off-reso-
nance spectrum.
.7 i a
CH COOH
II
CH COOH
Table (1) 13C-NMR characteristics of trimipramine iiialeate
Carbon No. Chemical Shift Carbon No. Chemical Shift

(PPI (PPm)
1 126.96 11 32.06
9 126.96 ’‘a 133.81

2 119.48 133.81
‘la
8 119.48 12 54.38
3 130.12 13 27.68
7 130.12 14 16.76
4 135.56 15 62.47
6 135.56 16 43.27
4a 147.45 17 123.19
6a 147.45 18 169.27
10 32.06
I TMS
I
PPM (6)
Figure 4 : Carbon-13 NMR Spectrum of Trimipramine maleate in CDC13
with TMS as internal reference.
2.45 Mass SDectrum and F r a m e n t o m e t r y
The mass spectrum of t r i m i p r a m i n e m a l e a t e

(Figure 5) o b t a i n e d by e l e c t r o n impact i o n i s a -
t i o n , u s i n g Finnigan mass s p e c t r o m e t e r shows
a molecular i o n M+'at m / e 294 ( r e l a t i v e i n t e n -
s i t y 2 5 % ) . Table (2) shows t h e proposed f r a g -
mentation of t r i m i p r a m i n e .
Table (2) Proposed fragmentation of trimipramine ( E I )

m le Relative ion
intensity % -
294 25 l+* p 3
kH2CH-CH2
I -N\
CH3
CH3
249 100
CH-CH= CH2
I
CH3
234 35
CH-CH=CH2
208 50
193 70
m'
Fig. 5 . Mass Spectrum of Trimipramine maleate (EI).
Re1ative ion
intensity %
65
+ CH3
CH2-CH-CH=N
0 ,
t \
CH3 CH3
+ ,CH3
84 63 CH2=CH-CH=N
\
+ HCH3
72 32 CH-N
l \
CH3 CH3
CH =N
+ NCH3
58 82
2 \
CH3
Cailleux and Allain (5) reported that chemical

ionisation was superior to electron impact for
identification of four drugs including trimi-
pramine, by gas-chromatography-mass spectro-
metry. The drugs cannot be separated by GLC at
220' on an SE 30-Chromosob column, nor can they
be unequivocally distinguished by electron-
impact mass spectrometry. Spectra are repro-
duced to show that these drugs can be clearly
distinguished by chemical ionisation-mass
spectrometry with CH4 as reagent gas.
3. Svnthesis
Trimipramine can be synthesized by the following methods:
a) Condensation of lO,ll-dihydro-5H-dibenz[b,f] azepine
and (CH3) 2NCHZCH(CH3)CH2C1 in toluene with sodamide
(6) ( 7 )
@& /CH3
*C1CH2-CH-CH2 -N\
toluene* Trimipramine
NaNH2
A CH3
TRIMIPMMINE MALEATE 6?7
b) Decarboxylatlon of the 5- [ [CH3) 2NCH2CH(CH3)CH20CO]

derivative of l0,ll-dihydro-5H-dibenz [b,f]azepine(6)
I CH -co2
COOCH~CH-CH~-N( :Trimipramine
I
CH3
CH3
c) Reaction of the 5- [ C H J S O ~CH2CH(CH3)CH2] derivative
of lO,ll-dihydro-5H-dibenz[b,f] azepine with dimethy-
lamine (6).
I x CH
CH2CH-CH2-S-OCH3+ H-N( 3, Trimipramine
I I1
CH3 0 CH3
d) By the general method described by Budai -

et -
a1 (8)
according to the following scheme:
CH3 absolute benzene

C1-COOC H
2 5
+ C1-CH2-CH-CH2N\
I
CH3
CH3
:1CH, -CH-CH, -N,

,COOC2H5
r,,
~H~CH-CH~N,
,
COOC2H5
CH2CH-CH N
I CH3 1 'CH3
CH3
CH3
CH3+ 1-
0
0
OCH3
I Toluene, K2C03
-+
CH2CH-CH-N,CH3
I 0 CH3
1 2
CH3
e) l0,ll-Dihydro 5H-dibenz [ b , f ] azepine was converted

to its 5-COC1 derivative by the reaction with phos-
gene. This product was allowed to react with HOCH2CH
(CH )CH2-N(CH ) to form 5 - (3-dimethylamino-2-
metgylcarboxy?a$e) intermediate. The latter is dicarbo-
xylated to give trimipramine ( 9 ) .
m I
H
CH
ClCOCl
- -4
t0Cl
I
1 3 / CH3
HO-CH2-CH-CH2 -N
\
I CH3 c
~ @Q-+
I JH3
COOCH2CH-CH2N,
I
CH3
CH3
ICH2CH-CH2N,0 CH3
I c*3
CH3
Trimipramine
4. Metabolism and Excretion
Studies on rabbits and dogs ..ave shown that trimipramine

is extinsively metabolized (2). Populaire -et -
a1 (10)
reported that, after oral ingestion of trimipramine, both
the drug and its monodemethylated derivative were found in
the circulating blood of dogs and rabbits; the concentra-
tion in the blood were low, and maximum concentrations
were reached within the first 6 hours. Within 72 hours,
dogs excreted 1.5-8% of the ingested dose,(50-70% in the
conjugated form), in the urine and 2-25% in the feces;
the corresponding values in rabbit were 10-20% (90%) and
~ 2 respectively.
% At least 26 metabolites were detected
in the excreta. The metabolism of trimipramine in humans
and animals appeared to be similar.
5.1 Titrimetric Methods
a) Aqueous Titration
Potassium hexathiocyanatochromate K3Cr(SCN)6 was

used (11) in the determination of trimipramine.
The reagent precipitated trimipramine base (HB) as
Cr(SCN)6 H3-3B and the excess reagent was titrated
with KBr03. The method was suitable €or analysing
6-20mgsamples with relative deviation of 0.2-0.8%.
b) Non-Aqueous Titration
B.P. 1980 (1) and European Pharmacopoeia 1975 (3)

determined trimipramine by the non-aqueous titra-
tion with 0.1 N perchloric acid using crystal vio-
let solution as an indicator.
c) Oscillometric Titration
Pomazanska - Kolodziejska (12) reported the use of

an oscillometric method for titration of trimipra-
mine among other related pharmaceutical compounds
with HC1 in acetone/ethanol solution.
5.2 Spectrophotometric Methods

5.21 Nuclear Magnetic Resonance Spectroscopy
A new method was described (13) for the assay
of trimipramine maleate and its base using 1H
NMR technique. The method employed is precise,
accurate, rapid and helpful in qualitative
identification and purity of the drug.
5.22 Fluorescence-Phosphoresence
Trimipramine, among other dibenzazepines, gave
with KMnO4, a green fluoresence which can be
used f o r analytical purpose (14) with a sensi-
tivity of >0.06-0.1 y/ml.
Gifford --
et a1 (15) studied the luminesence
characteristics of trimipramine and several
classes of drugs affecting the central nervous
system. The compound was studied in ethanol at
at 77K. The characteristics for trimipramine
are :
Excitation maxima 300 nm, the phosphoresence
maxima 450-470 nm and the phosphoresence life
time 0.70 sec.
5.23 Colorimetric Methods
a) French -
et -
a1 (16) reported a colorimetric
analysis of some dibenzazepines including
trimipramine. The determination of these
drugs has been studied by:
1. Treatment in acid solution with HN02 and
measurement of the extinction of the
reaction mixture at 390 nm.
2. Addition of bromothymol blue to solution
buffered at pH 7 and extraction with
benzene, with measurement of the extinc-
tion of the benzene extract at 410 nm.
3. Direct measurement of the extinction of
the acid solution at 251 nm. The analysis
TIUMIPRAMINE MALEATE 701
by the three methods was used for the

assays of tablets and injections. Although
all the three methods have essentially
the same accuracy and precision for bulk
drugs, the colorimetric procedures are
less subject to interfere by other mate-
rial (e.g. U.V. absorbers) that may be
present ill pharmaceutical preparation.
b) Slunjski and Turkovic (17) reported that the

reaction between 32% HNO3 and trimipramine
produces a blue color which, after several
minutes, changes to yellow. After evapora-
ting off the solution to dryness on a water
bath, dissolution of the residue in alcoholic
KOH produces a stable red violet color exhi-
biting maximum absorption at 560 nm. The
reaction can be used to determine 0.3 to 1.2
mg of drug in dragees or down to 1 ppm of the
drug or its metabolites in urine, and is
specific for compounds of this structure
(e.g . imipramine, desipramine and trimipra-
mine).
c) Klinge and Beyer (18) reported a simple
method for detection and determination of
trimipramine and its derivatives in chemical
toxicology. The drug is extracted from blood,
urine or body tissue extract into chloroform
in the presence of Na2C03. After evaporation
of chloroform, the residue is dissolved in
warm 80% acetic acid (5 ml) and the solution
is diluted to 10 ml with 80% acetic acid. The
drug is determined by heating 5 ml of the
solution with one drop of fresh 2% NaN02
solution in a boiling water-bath for 10 min
and measuring the extinction of the stable
yellow color at 415 to 420 nm.
5.24 Atomic Absorption
Trimipramine, among other azepine bases, has
been microdetermined by atomic absorption (19).
The sensitivity of the method is 1-4 X 10-4M.
The method involves the formation of an ionic
complex between the drug and sodium dioctyl sul-
fosuccinate (DOSS). After the pH of the reaction
medium is adjusted to protonate the drug, known
amount of DOSS is added to form an ionic com-

plex with the drug. If the complex is suffi-
ciently stable, the excess DOSS is complexed
with Cu o-phenathroline. The latter complex is
extracted with methyl isobutyl ketone and the
Cu concentration is determined by atomic absor-
ption, if DOSS-drug complex has low stability,
it may be removed by extracting with CCl4 and
the excess DOSS is then determined as described
above.
5.3 Polarographic Method
Volke -
et -
a1 (20) used a 3-electrode polarograph,
with a rotating-dlsc indicator electrode (~1300rpm)
and s.c.e., f o r the attempted anodic oxidation, of
trimipramine and other related compounds. Acetoni-
trile media were used, with 0.1 M tetrabutylammonium
perchlorate as supporting electrolyte. At both pla-
tinum and gold indicator electrodes,
5.4 Chromatographic Methods
5.41 Gas-Chromatography
Clarke (2) reported the retention time of
trimipramine to be 0.64 relative to codeine
using 2.5% SE-30 on 80-100 mesh chromosob
W A WHMDS, 5 feet X 4 mm id glass column.
Clarke (2) also reported a retention times of
trimipramine to be 0.30 (0.15) relative to
codeine using 3% XE-60 silicon nitrile polymer
on 100-120 mesh chromosob W.
Viala -et -
a1 (21) described a gas chromatogra-
phic technique f o r the identification of trimi-
pramine using two types of columns, XE-60/
Igapal o r Aeropack and UNCON polar o r pre-
alkalanized varport-30. The latter has the base
o r the salt o f the compound in methanolic
solution.
A rapid method is given (22) f o r the extraction

and identification of trimipramine and some
other basic drugs as well as their metabolites
in urine. Gas-Chromatography,(glass coil
packed with 3% OV-17 on gas-chrom Q 100-120
mesh, N carrier, flame ionization detector), was

used as the primary source of identification.
5.42 Gas-Liquid Chromatography
Trimipramine was determined, among other tri-

cyclic antidepressants., in biological fluids and
tissues, by gas-liquid chromatography:
a) Reite (23) published a gas-liquid chromato-

graphy method for the determination o f trimi-
pramine and its main metabolite (monodes-
methyltrimipramine) in human serum using
nitrogen detection. The drug and its main
metabolite were extracted from the serum
with hexane and the metabolite wasderivatized
with trifluoroacetic anhydride.
b) Dawling and Braithwaite (24) reported a

simplified method f o r monitoring trimipramine
among some tricyclic antidepressant therapy
using gas-liquid chromatography with nitro-
gen detection. The column was silanized glass
packed with 3% SP 2250 on supelcoport,
tarrier gas was Ar, and the internal standard
was maprotiline-HC1.
c ) The pharmacokinetic characteristics, o f two
different formulations (capsule and tablets)
of trimipramine, were determined with a new
gas-liquid chromatographic method ( 2 5 ) .
Plasma plus amidopyrine (internal standard)
is mixed with 10 M NaOH and extracted with
hexane; the separated organic phase is dried
and evaporated at 60" under nitrogen. A
solution of the residue in ethyl acetate is
analyzed by GLC on a solumn ( 6 ft X 2 mm)
of Gas Chrom-Q(80-100 mesh) supporting 3% of
OV-17, with nitrogen ionisation detection.
After 9.5 min at 225' the column temperature
is increased to 275' (maintained f o r 5 . 5 min)
in 2 min; retention times are 4.05 min for
amidopyrine and 8.15 min for trimipramine.
5.43 Column Liquid Chromatography
Van den Berg (26) described a column liquid

chromatography system for the analysis of tri-
cyclic antidepressants including trimipramine.

A high separation efficiency can be obtained
with a mixture of ethyl acetate, n-hexane, and
methylamine as eluent on a silica gel column.
The retention is easily regulated by varying
the concentration of n-hexane, the modifier
methylamine, and the H20 content of the ethyl
acetate. Ultraviolet detection permits determi-
nation down to the 10-ng level in the serum.
Clarke (2) described a solvent system consisting
of citric acid: H20 :n-butanol (4.8 gm: 130 ml :
870 ml). The system may be used for several
weeks, if water is added from time to time to
keep the specificgravity at 0.843 to 0.844.
Trimipramine gives an Rf value of 0.73. The
spots can be located under ultraviolet light,
blue fluorescence. Iodoplatinate spray and
bromocresol green spray were used as strong
and weak location reagents respectively.
5.45 High-pressure Liquid Chromatography
Trimipramine, among other tricyclic anti-
depressant was separated by high performance
liquid chromatography on silica gel column using
an eluting solvent of dichloromethane n-hexane
(1:l) to which 0.2% of isopropyl alcohol and
0.13% of propylamine were added ( 2 7 ) .
et a1 (28) described a relatively

De Zeeuw --
simple and rapid procedures for the toxicologi-
cal analysis of some commonly used tricyclic
antidepressants including trimipramine. The
methodology consisted of a single extraction
from aqueous media at pH 10 with hexane followed
by high-performance liquid chromatography (HPLC)
on silica gel using straight phase system.
Uges and Bouma (29) determined trimipramine and
its metabolites in serum by straight phase HPLC.
The drug was separated from solution or from
serum by HPLC on a column of silica gel, using
CH2C12:CH30H: buffer pH 3.2 (90:10:0.15) as the
mobile phase. The internal standard was proma-
zine-HC1 and the compound was extracted from

serum with CHZC12 under basic condition.
Alary and Villet (30) separated trimipramine by
dichotomic extraction as a function of solvent
polarity and pH and identified it by high-per-
formance thin-layer chromatography and liquid
chromatograph trimipramine was isolated by ex-
traction with hexane in an alkaline medium
(pH > 1 2 ) . Cyclohexane:ethanol:butanol-25%
NH40H (80:20:10:1) was used as the solvent in
TLC. Cyc1ohexane:ethanol:diethylamine (80:ZO:lO
:0.25) was used as the solvent in high pressure
liquid chromatography.
et -
Villet - a1 (31) reported a chromatographic
method for separation of trimipramine among
some psychotropic agents.The drug was separated
by a micro thin-layer chromatography method and
a high-performance liquid chromatography method
(Si 60 column, cyc1ohexane:ethanol:butanol:NHOH
25% (80:20:10:0.4) at 1.75 ml/mm transposed
from the first method, The high performance
liquid chromatography method has the advantage
of simultaneous separation, identification and
quantitation.
Table (3) shows various high pressure chromato-
graphy systems used for the analysis of trimi-
pramine in biological fluids.
Several thin-layer chromatography methods for
the separation and analysis of trimipramine
have been described in the literature.
Macek and Vecerkova (35) reported a new method
for identifying 161 medicinals including trimi-
pramine. The method involves separation of sub-
stances into groups by extraction first at a
low pH, and at a high pH, and then using an ion
exchanger. The further separation is then done
with paper chromatography, with thin-layer
chromatography also serving for identification
of the individual compounds.
Table (3) High-pressure Chromatography Systems
- -
Column Mobile Phase Flow rate Detector Remarks Ref.
20 cm X 4.6 mm Methanol:2M-NHg: W 254 nm can be applied to the (32)

Partisil 5 1 M-NH4N03(27: 2 :1) analysis of the drug and
(mean Particle amitryptyline and to their
size 6 pm) metabolites in gastric
fluids, blood and 1iver .
30 cm X 4.4 mm 35% acetonitrile UV 235 nm Retention time of the (33)

column packed in 45 mm KH2PO4 drug 11.8 min.
with p Bonda- adjusted to pH 3.0 The method is described
pack c18 with phosphoric to determine subtherapeu-
acid. tic to overdose level
of the drug.
Applied to other tricyc-
lic antidepressant.
Octadecylsilane Methanol : H20 1 ml/min-l Fluore- The retention volume from (34)
-coated spheri- (35 : 65) scence a point of injection for i
sorb. spectro- 25 cm column with 3.7 1.
meter. The Xf (fluorescence wave-
length) = 412 mm
TRIMIPRAMINE MALEATE I01
Clarke (21 described a solvent system consisting

of strong ammonia solution; methanol (1.5:lOO).
The system should be changed after two runs.
Trimipramine gives an Rf value of 0.58. The
chromatogram is visualized by acidified iodo-
platinate spray.
Groningsson and Schill (36) described a thin-

layer chromatography of trimipramine as an ion
pair with C1-, Br-, SCN- and ClO4- as the coun-
ter ionsin the aqueous phase. The stationary
phase was a cellulose powder impegnated with
the solution containing the counter ion. The
mobile phases are chloroform, cyclohexane + 1-
pentanol (7:3), cyclohexane + 1, pentanol (1:l).
The visualization was made using Rhodamine B
and Iodine.
Table (4) show other thin-layer chromatography

systems.
5.47 Thin-LayeT Chromatography-Mass Spectrometry
Combined thin-layer chromatography-mass spectro-

metry technique was applied for the analysis of
trimipramine (37). Thin-layer chromatography was
carried out on glass plates coated with GF254
silica gel. The drug was applied to the plates
from the stand.solvent. The solvent system con-
sists of acetic acid:ethanol:water (30:50:20).
The silica gel containing the drug was removed
from the plate and inserted into the mass spec-
trometer. The highest m/e in mass spectrum of
TLC sample was m/e 294.
Table (4) Thin-layer chromatographv systems for trimipramine analysis
I
I
Stationary Developing Solvent Detecting Remarks Rf Ref.
Phase Agent value
~ ~~
Silica gel Acetone: methanol: NH40H Alc. FINO3 0.54

50: 50 : 1
Silica gel Dehydrated peroxide-free Spraying with Fluorescence can 0.170 (39)
(activated) ether: acet0ne:diethylamine dil. iodo- be performed after
(9O:lO:l) platinate 24 hours.
Benzene:acetone (100: 20) Teagent in Positive results 0.154
shaken with 10 ml of 5% N-HC1 follow- are obtained with
aq. NH solution. ed by 50% 100 mg of the pro-
3 H2SO4 and duct.
examlning
under U.V.
radiation.
~~~~ ~
Kieselgel G 1) Methano1:acetone [12:88) W light (254 0.4

2) Ethano1:tetrachloroetfiane mm after spray
(16:84) with Dragen- 0.8
3) Methanol :benzene dorff's
(31.7:68.3) reagent). 0.65
4) Ethanol :toluene (68:32) 0.5
5) Methano1:cyclohexane:ethyl
acetate (17.8:33.6:48.6) 0.64
I
Contd.. . .
Stationary Developing Solvent Detecting Remarks Rf Ref.
Agent valut
Silica gel G Hexane:anhydrous diethylamine 55% H3PO3 It is possible to - (41)
(93:7) saturated identify a psycho-
wl’th KC104 tropic drug in urine
and heat. in the event of toxi-
cological emergency.
Kieselgel -Ether:acetone:ethyl acetate: Iodine Used for rapid - (42)
GF254 diethylamine (85 :11 :2 :2) vapour identification.
-Benzene:acetone:diethylamine Bromlne Used for rapid
(50 : 10 : 5) vapour identification.
-Methanol:cyclohexane:Methyl Used for the -
acetate (17.8 :33.6:48.6] identification of
-Butanol:toluene:methanal: the tertiary amine.
H20 : acetic acid
(22:48:18:7:5)
Kieselge
- 1 Cyc1ohexane:ethanol:butanol: - 0.61
60F254 25% NH40H (80:20:10:0.4)
Kieselgel Methanol:25% aq.NH3 -W radi- -

(100 : 1) ation
GF 254 -Spray witl
5% NaN02
soln. in
80% metha-
nol.
6. References
1. British Pharmacopoeia 1980, London Her Majesty's

Stationary Office, 1980, p.466.
2. E.G.C. Clarke, "Isolation and Identification of Drugs"

The Pharmaceutical Press, London, 1975, p.587.
3. "European Pharmacopoeia", V01.111, Maisonneuve, S.A.

Saint - Ruffine, France, 1975, p.357.
4. J. Poirot-Lagubeau, P. Mesnard, P. Gerval, E. Frainnet,

and M. Petraud; -
Ann.-Pharm.
- FT., - 33, 279 (1975).
5. A. Cailleux and P. Allain, - Anal. Toxicol., -

J. - 3 , 39
(1979).
6. R.M. Jacob and M. Messer, Compt. Rend., -

252, 2117
(1961).
7. H. Wunderlich, E. Carstens, A. Stark, H.J. Heidrich

and S. Henker, Ger. [East) 130, 712 (1978), through
Chemical Abstractgo,
- 103863W (1979).
8. Z. Budai,P.Benko and L . Pallos, Ger. Offen. 1, 920
170, 29 Jan. 1970, through Chemical A m c t - 72 (1972).
9. Societe des usines chimiques Rhone-Poulene, Fr.1,172,

014 Feb.,4,1959 through Chemical Abstract -
54, 19730i
(1960).
10. P. Populaire, B. Terlain, S. Pascal, G. Lebreton and

B. Decouvelaere, ---
Prod.Probl.Pharm. -
25, 632 (1970).
11. A. Olech, -
Acta -
Pol.-
Pharm., -
32, 73 (1975).
12. T. Pomazanska-Kolodziejska, --
Farm. Pol., -
30, 1005
(1974).
13. A.A. Al-Badr and S.E. Ibrahim, Spectrosc. -

Lett.
-12,
419 (1979).
Can. J. -
14. E. Adonai Martin, -- Chem., -
45, 75 (1967).
15. L.A. Gifford, J.N. Miller, J.W. Bridges and D.T. Burns,
Talanta,24,
- 273 (1977).
16. W . N . French, F . Matsui and J . F . T r u e l o v e , -

J. _
Pharm.
_ _ -S c i
3 , 33 (1968).
-
J . Pharm. B e l g . , -
17. M. S l u n j s k i and I . Turkovic, -- 25, 400
(1970).
1 8 . D . Klinge and K . H . Beyer, -

D t . ApothZig., -
108, 780, (1968).
19. J . A l a r y , A. V i l l e t and A. Coeur, -

Ann.- - Fr., -
Pharm. 34,
419, (1976).
20. J. Volke, M . M . E l - L a i t h y and V . Volkova, J.E l e c t r o -

analyst. -
Chem.,-60, 239 (1975).
2 1 . A. V i a l a , J.P. Cano, C . Gola and F . Gouezo, -

J . Chromato-
g r , 59, 297, (1971).
-
2 2 . L.J. Dusci and L . P . Hackett, Clin. Toxicol., 14,587
(1979).
23. S . F . R e i t e , -
Medd. - Farm.
Nor. - - Selsk., -
37, 148 (1975).
2 4 . S. Dawling and R . A . J . Chromatogr, -

Braithwaite, - 146, 499
(1978).
25. G . C a i l l e , J . G . Besner, Y . Lacasse and M . Vesina, -

Bio-
pharm.
-- Drug Dispos. 1, 187 (1980).
26. J . H . M . Van den Berg, H . J . J . M . De Ruwe, R.S. Deelder and

T.A. Plomp; - J. Chromatogr., 138, 431 (3977).
J,
2 7 . M . R . D e t a e v e r n i e r , L . Dryon and D . L . Massart, -
Chromatogr., - 128, 204 (1976).
28. R . A . De Zeeuw and H . G . M . Westenberg, _

J . -Anal. T o x i c o l . ,
2 , 229 (1978).
-
29. D . R . A . Uges and P. Bouma, -

Pham. -
Weekbl., S c i . Ed. 1,
417 (1979).
30. J . A l a r y and A . V i l l e t , J . Pharm. Belg., -

35, 408 (1980).
31. A V i l l e t , J. Alary and A. Coeur, T a l a n t a , -

27, 659 (1980).
32. W . M . Hoskins, A . Richardson and D . G . -

Sanger;
- J. F o r e n s i c
S O ~ . 17,
, 185 (1977).
- 55 (1979).
33. L.P. Hackett and L.J. Dusci, Clln. Toxicol.,l5,
J. Chromatogr., -
34. L.A. King, - 208, 113 (1981).
35. K.Macek and J . Vecerkova, Pharmazie, -

20, 605 (1965).
36. K. Groningsson and G. Schill, -

Acta. -
Pharm. Suecica, -
6,
447 (1969).
37. G.J. Down and S . A . Gwyn, -

J. Chromatogr., 103,208 (1975).
3 8 . J.J. Thomas and L . Dryon, 9. Pharm. Belg., 19, 481 (1964).
39. A.Viala, F. Gouezo and C. Gola, -

9. Chromatogr., -
45, 94
(1969).
40. E. Roeder, E. Mutschler and H. Rochelmeyer, -

J. Chrnmatogr.
-
42, 131 (1969).
41. J.J. Kebler, - SOC. Pharm.

Bull. - - Strasbourg., -
13, 41 (1970).
42. J.A. Marca and H. Muehlemann, -

Pharm.-
Acta.-
Helv., -
46, 558
(1971).
ACKNOWLEDGEMENT
The author would like to thank M r . Mohammad Salim

Feroze f o r typing the manuscript.
DIOCTYL SODIUM
SULFOSUCCINATE
Satinder Ahuja and Jerold Cohen
1. Description 714
1 . 1 Name, Formula, Molecular Weight, Elemental Composition 7 14
2.8 Solubilization 717
2.9 Effect On Surface Tension Of Liquids 718
6.1 Titrimetric Analysis 718
6 . 2 Colorimetric Analysis 718
6.4 Turbidimetric Analysis 718
6.8 Miscellaneous 719
References 719
ANALYTICAL PROFILES OF DRUG SUBSTANCES 713 Copyright by the American Pharmaceutlaal Asswiatmn
VOLUME 12 ISBN 0-12-260812-7
714 SATINDER AHUJA AND JEROLD COHEN
The following supplement contains updated information

pertaining to the analytical chemistry of dioctyl sodium
sulfosuccinate. A literature survey was conducted and is
complete up to June, 1982. The numbering system for topics
discussed is the same as that in the original profile
(Volume 2, pp.199-219).
1. DESCRIPTION
1.1 Name, Formula, Molecular Weight,

Elemental Composition
Dioctyl sodium sulfosuccinate is known as docusate
sodium (1). It is also known as sulfobutanedioic acid 1 , 4 -
bis(2-ethylhexyl) ester sodium salt, sulfosuccinic acid 1 , 4 -
bis(2-ethylhexyl) ester S-sodium salt, Comfolax, Molcer,
Soliwax and Valsol OT ( 2 ) . It has a molecular weight of
444.56 (C20H37NaO7S).
C2Il5
I
COOCH2CH(CH2) 3CH3
I
CH2
I
CH-SO3Na
I
COOCH2CH(CH2) 3CH3
I
C2H5
2. PHYSICAL PROPERTIES
2.3 Mass Spectrometry

A chemical ionization mass spectrum (Kratos MS 25
with isobutane as reagent gas) was run on the acid form of
dioctyl sodium sulfosuccinate prepared by acidification of a
methanolic solution with HC1 gas ( 3 ) . The interpretation of
major fragmentation ions (Figure 1) is as follows ( 4 ) :
DIOCTYL SODIUM SULFOSUCCINATE 715
157 113
- r
H @
‘
0
m/z 4 2 3
HO-! CH3
It0 0: 1~ 2 ~ 5
L-
229 12999
I
1- S02H
t l@
‘ZH5
O/\I/\/\CH3
m/z 358
3-0
HO 0 ‘ZH5
c.
211
H - -OH
‘ZH5
/\J/\/\CH 3 m/z 341

O W ‘ H 3
8 0 ‘ZH5
100 -
w -
m -
m -
0 0 -
w -
4 0 -
5 0 -
Figure 1. Chemical Ionization Mass Spectrum of the

Acid Form of Dioctyl Sodium Sulfosuccinate.
(Drawn to show major fragments)
DIOCTYL SODIUM SULFOSUCCINATE 717
2.8 Solubilization
The critical micelle concentration value of 3.0
nmoles/l was determined by plotting desorption potential (d.c.
polarography without electrolyte) vs. log concentration (5).
The solution states of dioctyl sodium sulfosuccinate were ex-
amined by lH NMR (6). Two hydrocarbon chains of its molecules,
in the monomeric state, aggregate with each other in water.
Addition of aqueous sodium chloride solution to the Aerosol
OT-n-octane system showed a peak corresponding to micellar-
solubilized water and another peak corresponding to separated
water (7). Systems containing aluminum chloride differed from
those containing mono or divalent electrolytes. In 0.27M
AlC13, the two peaks merged into a single peak, indicating
breakdown of the micellar system. The magnitude of cation
effect was in the order Na<Ca<Al. The dynamic behavior of
Aerosol OT in the monomeric state in methanol and micellar
states in water, chloroform and benzene was studied by mea-
surements of 13C-NMR spin lattice relaxation time and the
effect of a lanthanide shift reagent (tris(6,6,7,7,8,8,8-
heptafluoro-2,2-dimethyl-3,5 octane dionate) ytterbium in
CHC13 (8,9). The results show that Aerosol OT molecules in
the micellar state associate with each other by the polar
head groups; their hydrocarbon chains are flexible and the
mobility of side chain is restricted to a greater extent than
that of the main chain.
The rotational isomerism of Aerosol OT was studied

by IH NMR spectroscopy in three different solvents (methanol,
chloroform, isooctane) from -10 to +6OoC t o determine its
effect on stability of waterloil microemulsion (10). The
temperature dependance of various proton-proton spin-coupling
constants was investigated and relative energies of the
rotamers were determined. In methanol solution, the solute-
solvent interactions with the polar head group are totally
isotropic and the composition of the equilibrium mixtures of
the rotamers depends on the steric interaction between the two
bulky ester groups of the molecule. In solvents like chloro-
form or isooctane, the energetically favored rotamer has all
polar groups spatially confined in such a way as to lead to a
more pronounced amphilic character of the molecule; this gives
rise to maximum hydrogen bond formation with water molecules
and a strong intra-molecular ion-dipole interaction with the
counterions. This property is believed to enhance its surface
activity and, hence its aggregational and solubilizing ten-
dency in apolar systems.
The solubilization of imidazole, pyrazole and methanol by

Aerosol OT in carbon tetrachloride was studied by following
the chemical shifts of solubilizate H as a function of sur-
factant concentration (11). It affected the self-aggregation
of the solubilizates as illustrated by the larger equilibrium

constants for the binding of 3-H and 5-H of imidazole than
for the 2-H.
2.9 Effect On Surface Tension Of Liquids

A method to determine the surface compressional mod-
ulus by the Fourier transformation of the surface tension re-
laxation function has been reported. The method can provide
rapid measurements for very dilute solutions, in the low fre-
quency range (12).
6. METHODS OF ANALYSIS
6.1 Titrimetric Analysis

The two-phase mixed indicator titration method for
the determination of anionic surfactants was extended to in-
clude dioctyl sodium sulfosuccinate by using 2:3 (v/v) chlo-
roform: 1-nitropropane as the organic phase and a multiple
extraction-titration technique (13).
DSS can also be determined by extractive titration

with carbethopendecinium bromide (14).
6.2 Colorimetric Analysis

Several methods have been proposed for the analysis
of anionic surfactants in trace quantities in water: extrac-
tion into toluene from aqueous solution with ethyl violet and
spectrophotometric determination at 615 nm (15); extraction
into benzene from aqueous solution with bis[2-(2-pyridylazo)-
5-diethylamino phenolato] cobalt (111) ion as the counter-ion
and spectrophotometric determination at 550 nm (16); extract-
ion into chlorobenzene from aqueous solution with 1-(4-nitro-
benzyl)-4-(4-diethylamino pheny1azo)-pyridinium bromide and
spectrophotometric determination at 573 nm (17); extraction
into benzene from aqueous solution with bis[2-(5-chloro-2-
pyridylazo)-5-diethylamino phenolato] cobalt (111) chloride
and spectrophotometric determination at 560 nm (18).
6.4 Turbidimetric Analysis

Dioctyl sodium sulfosuccinate was determined after
formation and stabilization of a dispersion with barium sul-
fate in water-alcohol mixture and measurement of turbidity
at 650 nm (19). The limit of detection was 100-200 pg/g and
reproducibility was %5%.
DIOCTYL SODlUM SULFOSUCCINATE 719
6.7 Polarographic Analysis

Dioctyl sodium sulfosuccinate was determined by
polarographic desorption potential measurement ( 2 0 ) . It
showed a well defined adsorption-desorption wave and a linear
relationship was observed between the desorption potential and
the logarithm of surfactant concentration. The determination
was limited by the critical micelle concentration (CMC), as
the desorption potential remained constant above the CMC of
the surfactant. The method can be applied for the determina-
tion of a few ppm of the surfactant.
6.8 Miscellaneous
A nitrogen blowing technique allowed quantitative
recovery of surfactants present in solution and can be applied
to analysis of surface waters and waste waters (21).
Dioctyl sodium sulfosuccinate can be used to form

ion pairs with organic bases which are extractable with immis-
cible organic solvents. This technique has been used for
identification and assay of the organic bases by ultraviolet
absorption spectrophotometry in dosage forms and biological
fluids ( 2 2 ) .
REFERENCES
1. "The United States Pharmacopeia - National Formu-

lary" XX/XV Ed., Mack Publishing Co., Easton,
Pa. 1 9 8 0 , p. 261.
2. "Merck Index", 9th Ed. , Merck and Go. , Inc. ,
Rahway, New Jersey, 1 9 7 6 , p. 438-439.
3 . S. Ahiija and G. Thompson, Ciba-Geigy Corp.
Suffern, N.Y., l'eKSOna1 Communication, 1 9 8 2 .
4 . M. Stogniew and R. Schiesswohl, Ciba-Geigy Corp.
Suffern, N . Y . , Personal Communication, 1 9 8 3 .
5. N. Shinozuka, H. Suzuki and S. Hayano, Kolloid-Z.Z.
Polym., 248 ( 1 & 2 ) , 959 ( 1 9 7 1 ) .
6 . M. Ueno, H. Kishimoto and Y. Kyogoku, Chem. Lett.,
# 6 , 599-602 (1977).
7. S.G. Frank, Y.H. Shaw and N.C. Li, J. Phys. Chem.,
77 ( Z ) , 238 ( 1 9 7 3 ) .
8. MTUeno, H. Kishimoto and Y. Kyogoku, J. Colloid
Interface Sci., 63 (l), 113 ( 1 9 7 8 ) .
9. M. Ueno, H. Kishimoto and Y. Kyogoku, Bull. Chem.
SOC. *., 3 ( 7 ) , 1776 (1976).
10. A . N . Maitra and H.F. Eicke, J. Phys. Chem. 8 5 ( 1 8 ) ,
2687 ( 1 9 8 1 ) .
11. A. El Seoud and J. H. Fendler, J. Chem. SOC.

Farraday Trans. 1, 7 1 ( 3 ) , 452 ( 1 9 7 5 ) .
12. G. Loglio, U. Tesei a n d R. Cini., Ber. Bunsenges.
Phys. Chem., 81 (11), 1154 ( 1 9 7 7 ) .
13. Z. Li and M. J. Rosen, Anal. Chem., 53 (9), 1 5 1 6
(1981).
14. J . Blazek, A. Dymes and M. Travnickova, Cesk. Farm.
27 ( 9 ) , 379 ( 1 9 7 8 ) .
15. STMotomizu, S. Fujiwara, A. Fujiwara and K. Toei,
Anal. Chem., 54 ( 3 ) , 392 ( 1 9 8 2 ) .
16. S. Taguchi and K. Goto, Talanta, 27 ( 3 ) , 289 (1980)
17. K. Higuchi, Y. Shimoishi, H. Miyata, K. Toei and
T. Hayami, Analyst, 1 0 5 , 768 ( 1 9 8 0 ) .
18. S. Taguchi, I. K a s a h a x Y. Fukushima and K. Goto,
Talanta, 28 ( 8 ) , 616 ( 1 9 8 1 ) .
19. S.M.F. Tavernier and R. Gijbels, Talanta, 28 (4),
221 (1981).
20. N. Shinozuka, H. Suzuki and S. Hayano,
Bunseki Kagaku, 2 ( 4 ) , 517 ( 1 9 7 2 ) .
21. C. Divo, S. Gafa, T. La Noce, A. Paris, C. Ruff0
and M. Sanna, Riv. Ital. Sostanze Grasse, 57 ( 7 ) ,
329 ( 1 9 8 0 ) .
22. F. Pellerin, D. Mancheron and D. Demay, Ann. Pharm.
Fr. 30 ( 6 ) , 429 ( 1 9 7 2 ) .
--
ISOPROPAMIDE
Alex Post and Ralph S . Santoro'
1 . Physical Properties 722

1 . 1 Mass Spectrum-Field Desorption (FD) 722
1.2 Carbon-13 NMR Spectrum 722
2.1 Isopropamide Iodide Chemical 728
3 . Metabolism 73 1
References 73 1
'Deceased
ANALYTICAL PROFILES OF DRUG SUBSTANCES 721 Copyrighiby the American Phamdceutical Assmatirn
VOLUME 12 ISBN 0-12-260812-1
722 ALEX POST AND RALPH S. SANTORO
1.1 Mass Spectrum - Field D e s o r p t i o n (FD) ( l )
The FD mass s p e c t r u m o f i s o p r o p a m i d e i o d i d e i s
shown i n F i g u r e 1 and t h e f r a g m e n t a t i o n i o n s a r e
t a b u l a t e d i n T a b l e 1. The s p e c t r u m was o b t a i n e d
o n a V a r i a n MAT CH-5 DF mass s p e c t r o m e t e r . The
emitter was l o a d e d by t h e d i p p i n g t e c h n i q u e from
a m e t h a n o l s o l u t i o n . An emitter c u r r e n t o f 2 3
m i l l i a m p e r e s was u s e d t o o b t a i n t h e spectrum.
The s p e c t r u m shows a n i n t e n s e p e a k f o r t h e c a t i o n
a t M/Z 353. Minor p e a k s a r e o b s e r v e d a t b o t h
lower and h i g h e r M/Z v a l u e s .
1.2 carbon-13 NMR S p e c t r u m ( 2 )
The d e c o u p l e d carbon-13 NMR s p e c t r u m of i s o p r o p a -

mide i o d i d e was o b t a i n e d u s i n g a s o l u t i o n of a p -
p r o x i m a t e l y 1 0 0 mg/ml i n d i m e t h y l s u l f o x i d e - d 6 .
The d e u t e r i u m s i g n a l o f d i m e t h y l s u l f o x i d e was
u s e d a s t h e i n t e r n a l r e f e r e n c e . The s p e c t r u m was
o b t a i n e d o n a V a r i a n FT-80 F o u r i e r t r a n s f o r m NMR
s p e c t r o m e t e r . The c h e m i c a l s h i f t a s s i g n m e n t s a r e
shown i n T a b l e 2.
F i g u r e 1: Field Desorption Mass Spectrum of Isopropamide Iodide
724 ALEX POST AND RALPH S . SANTORO
T a b l e 1: F i e l d D e s o r p t i o n Mass Spectrum of Isopropamide I o d i d e
Peak I n t e n s i t y ~ a t a
MASS X RA MASS X RA MASS X RA
40 00 0 01 328 00 0 42 383 00 0 36
43 00 0 04 329 00 0 15 384 00 0 44
58 00 8 52 330 00 0 56 385 00 0 12
59 00 0 40 331 00 6 42 386 00 0 42
60 00 0 02 332 00 8 78 387 00 0 43
78 00 0 02 333 00 0 75 388 00 0 49
115 00 0 11 335 00 0 02 389 00 0 43
127 00 0 04 336 00 0 02 390 00 0 84
128 00 0 02 337 00 0 02 391 00 0 59
155 00 0 01 338 00 1 68 392 00 0 21
159 00 0 02 339 00 0 73 393 00 2 27
161 00 0 01 340 00 0 07 394 00 0 82
185 00 0 02 341 00 0 03 395 00 3 90
228 00 0 01 343 00 0 02 396 00 3 27
235 00 0 01 344 00 0 03 397 00 5 21
236 00 0 06 345 00 0 06 398 00 4 03
237 00 0 81 346 00 0 10 399 00 1 12
238 00 0 45 347 00 0 03 400 00 0 22
239 00 0 04 350 00 0 04 401 00 0 17
265 00 0 04 351 00 0 82 402 00 0 14
267 00 0 02 352 00 1 16 403 00 0 15
277 00 0 02 353 00 100 00 404 00 0 02
291 00 0 01 354 00 66 03 406 00 0 02
293 00 0 04 355 00 10 05 407 00 0 06
294 00 0 09 356 00 0 84 409 00 0 14
295 00 0 04 357 00 0 03 410 00 0 16
296 00 0 04 358 00 0 01 411 00 0 05
297 00 0 02 359 00 0 17 412 00 0 03
298 00 0 04 360 00 1 56 413 00 0 07
303 00 0 05 361 00 1 03 414 00 0 09
304 00 0 02 362 00 0 63 415 00 0 07
308 00 0 24 363 00 0 23 416 00 0 05
309 00 0 31 364 00 0 14 417 00 0 17
310 00 11 04 365 00 4 88 418 00 0 08
311 00 3 74 366 00 2 30 419 00 0 04
312 00 0 79 367 00 12 44 421 00 0 09
313 00 0 20 368 00 7 41 422 00 0 02
314 00 0 30 369 00 7 20 424 00 0 02
315 00 0 45 370 00 2 14 425 00 0 02
316 00 0 55 371 00 0 50 429 00 0 11
317 00 0 47 372 00 0 76 430 00 0 04
318 00 0 19 373 00 1 30 431 00 0 02
319 00 0 15 374 00 0 89 434 00 0 02
320 00 0 20 375 00 1 42 436 00 0 07
321 00 0 83 376 00 1 44 437 00 0 11
322 00 0 83 377 00 1 22 438 00 0 05
323 00 0 33 378 00 0 35 474 00 0 01
324 00 1 15 379 00 1 54 476 00 0 01
325 00 8 20 380 00 0 75 478 00 0 02
326 00 0 13 381 00 1 61
327 00 0 91 3 8 1 00 1 07
a,
5
.rl
5
0
H
a,
5
3
P
0
k
P
::
H
ICI
0
5
k
T a b l e 2: Isopropamide I o d i d e - Carbon 1 3 NMR S p e c t r u m
8
CH2
Carbon Chem. S h i f t Multiplicity, (C-H)

(PPm)
1 173.9 singlet
2 142.3 singlet
3 128.7 doublet
4 128.2 doublet
5 127.1 doublet
6 62.9 doublet
7 59.2 singlet
8 54.4 triplet
9 42.7 quartet
15 32.1 triplet
16(a) 30.1 quartet
17 16.9 quartet
18 16.5 quartet
(a) P o s s i b l e Methyliodide
1.3 X-Ray D i f f r a c t i o n
The s t r u c t u r e of i s o p r o p a m i d e i o d i d e , d e t e r m i n e d
by s i n g l e c r y s t a l x - r a y d i f f r a c t i o n , was shown t o
be orthorhombic w i t h u n i t c e l l dimensions, a =
17.548(5), b -14.564(4), c = 8.993(3)A0.(3)
Datta, e t a ~ ( i s~ i n) a g r e e m e n t w i t h t h e a b o v e .
They a l s o r e p o r t e d t h a t t h e a n g l e between t h e two
p h e n y l g r o u p s i s 82O and t h e amide g r o u p is p l a n -
a r , making a n g l e s of 84 a n d 89O w i t h t h e two
p h e n y l g roups.
ISOPKOPAMIDE 727
1.4 P a r t i t i o n Coefficient
1.41 True P a r t i t i o n Coefficient of Isopropamide

Ion P a i r s
The t r u e p a r t i t i o n c o e f f i c i e n t f o r isopro-
pamide ion p a i r s w i t h benzoate, p-toluene-
sulfonate, and s a l i c y l a t e (molar r a t i o ;
1:l) between n-octanol and 0 . 1 pH 7.4
phosphate buffer have been reported by
Shim, e t a ~ ( and ~ a) r e l i s t e d i n Table 3.
Table 3 : True P a r t i t i o n Coefficients

of Isopropamide Ion P a i r s
Anion P a r t i t i o n coe f f i c i e n t ( a )
Benzoate 2.6 (0.1)

p-Toluenesulfonate 8.7 (0.7)
Salicylate 11.7 (1.1)
( a ) Mean of three determinations a t 25OC w i t h S.D.

i n parenthesis
1.42 Apparent P a r t i t i o n Coefficient
The apparent p a r t i t i o n c o e f f i c i e n t s i n
n-octanol and pH 7.4 aqueous phosphate
buffer (0.1%) were determined f o r iso-
propamide-bile s a l t ion p a i r s . The p a r t i -
t i o n c o e f f i c i e n t s varied w i t h t h e concen-
t r a t i o n of t h e b i l e s a l t and indicated t h a t
w i t h increasingly l a r g e r molar amounts of
bile s a l t , the partition coefficient i n -
creased.
The apparent p a r t i t i o n c o e f f i c i e n t s f o r the

isopropamide-bile s a l t ion p a i r s a t equi-
molar concentrations a r e l i s t e d i n Table 4.
Table 4: Apparent Partition Coefficient for

Isopropamide-Bile Salt Ion Pairs
Apparent Partition
Bile Salt Coefficient (a)
Sodium Cholate 0.29

Sodium Deoxycholate 0.63
Sodium Dehydrocholate 0.38
Sodium Taurocholate 0.44
Sodium G1 ycocholate 0.43
Sodium Taurodeoxycholate 0.60
(a) At 22 2 l0C
2.1 Isopropamide Iodide Chemical
2.11 Identification
Chatten et al. ('1 showed that isopropa-

mide iodide could be differentiated from
other medicinal agents containing one or
more quaternary nitrogen functions. Deri-
vatives, prepared by reacting these com-
pounds with ammonium reineckate, picric and
chloroplatinic acids, were characterized by
their melting points and crystalline mor-
phology (Table 5). Differentiation from
related compounds was effected.
Table 5: Derivatives of Isopropamide Iodide
Derivative Melting Point (OC) M icrocrvstallosraPhv
Reineckate 154-155 oil globules

Picrate 140-140.5 oil globules
Chloroplatinate Black mass oil globules
ISOPROPAMIDE 729
2.12 Spectrophotometric Analysis in Presence of

Inte r fe r e nc e s
Infrared spectrophotometry was used t o

rapidly determine small amounts of isopro-
pamide in pharmaceutical preparations con-
taining aminopyrine, phenacetin, and caf-
feine. (8) With acetone a s the solvent
and a compensation solution containing
aminopyrine and phenacetin, linear re-
sponses were obtained at a major absorption
band, 702 cm-1. The method showed recov-
eries of 98-102% 2 1.67% ( n = 6) for solu-
tions of 0.5-2.5 mg isopropamide per milli-
liter.
2.13 Colorimetric Analysis
The methyl orange dye-ion pairing procedure

was modified by Chatten and O k a m ~ r a ( ~ in
)
their essay o f isopropamide iodide tab-
lets. Ion pairing was effected at pH 8.0
and after extraction into methylene chlo-
ride, they obtained absorbance readings at
415 nm.
2.141 Thin-layer chromatography
Th in-layer chromatography was used

by Mozsik and Toth(l0) t o separate
isopropamide from other parasympa-
tholytics and quantified each by
densitometry. Both silica gel G
impregnated with 1.ON NaOH and
aluminum oxide G adsorbents were
used with two mobile phases. Detec-
tion was with the conventional iodc-
platinate reagent. (Table 6)
Table 6: Thin-laver Chromatography of Isopropamide
Adsorbent Mobile Phase Rf

-
.
S i1ica/l ON NaOH Chloroform-ethanol ( 5 : 5 ) 0.48
Aluminum Oxide Chloroform-ethanol ( 5 : 5 ) 0.70

2.142 Ion-exchange chromatography
The USPXX describes a n anion exchange

chromatographic-spectrophotometric
read-out procedure for the isolation
and quantitation of isopropramide in
tablets. (11)
2.143 High pressure liquid chromatography
Antispasmodic agents, including iso-

propamide iodide, were assa ed by
HPLC. Honigberg, et a1.(l2T found
that superior separations were ob-
tained o n a phenyl (diphenyldi-
c hlorosilane) column when compared
t o that on an octadecyl (octadecyl-
trichlorosilane) column. Pertinent
data for the phenyl column are listed
in Table 7. An earlier report by
this group(13) ; in which acetoni-
trile was used, yielded less satis-
factory results.
ISOPROPAMIDE 731
T a b l e 7. High P r e s s u r e L i q u i d Chromatoqraphy
of I s o p r o p a m i d e
R e t e n t i o n Time
Mobile Phase pH (Seconds)
C H 3 0 H ( a ) - 1 % (NH4 ) H2P04 ( 6 0 :40) 5.85 90

CH3OH-l%(NHq)H2P04(50:50) 5.50 122
CH30H-1% ( N H 4 ) H2PO4 (40:60) 5.50 150
CH30H-1% (NH4) H2PO4-
1% (NH4 ) 2 HP04 ( 6 0 :20 :20) 8.20 112
CH$H-1% ( N H 4 ) H2PO4-
1% (NH4) 2HP04 ( 50 :25 :25) 7.90 118
CH30H-1% ( N H 4 ) H2PO4-
l%(NH4)2HP04(40:30:30) 7.60 110
CH30H-O.5% (NH4 2CO3 ( 6 0 :40) 8.65 146
CH30H-O.5% (NH4) 2CO3 (50:50) 8.70 150
CH30H-0.5% (NH4)2CO3(40:60) 8.80 162
A b s o l u t e methanol used a l l mobile p h a s e m i x t u r e s

Flow r a t e : a b o u t 1 . 4 m l per m i n u t e a t room t e m p e r a t u r e
Met a b o l i s m
Kinetic s t u d i e s o n t h e intravenous administration of

[ l 4 C ] i s o p r o p a m i d e t o r a t s showed t h a t i n s i g n i f i c a n t
amounts were p r e s e n t i n b l o o d , muscle, f a t , g a s t r o i n -
t e s t i n a l t i s s u e s , and e y e a n d t h a t l a r g e amounts were
p r e s e n t i n t h e l i v e r a n d k i d n e y s . As t h e m y d r i a t i c
response i s prolonged, suggested t h a t t h e l i v e r acts
a s a s t o r a g e d e p o t or i t i s slow1 m e t a b o l i z e d t o a
p h a r m a c o l o g i c a l l y a c t i v e form. ( 1 4 7
References
1. G . R o b e r t s , p e r s o n a l communication.
2. D. S t a i g e r , p e r s o n a l communication.
3. Chawdhury, S.A. a n d Koch, M . H . J . , Acta
C r y s t a l l o g r . , S e c t . B , 1 2 8 6 (1976) .
4. D a t t a , N. , B r e e n , P., a n d P a u l i n g , P., J.C.S.,
P e r k i n 11, 7 8 1 ( 1 9 7 7 ) .
5. Shim, C.K., N i s h i g a k i , R. , I g a , T., a n d Hanano,
M . , I n t . J. Pharm., &, 1 4 3 ( 1 9 8 1 ) .
6. G a g i n e l l a , T.S., B a s s , P., P e r r i n , J . H . , and
,
V a l l n e r , J.J., J. Pharm. S c i . 62, 1 1 2 1 ( 1 9 7 3 ) .
7. C h a t t e n , L.G., Napper, A.C., a n d B a r r y , P.J. ,
ibid. , 56, 834 ( 1 9 6 7 ) .
8. O i , N. and M i y a z a k i , K. , J. Pharm. SOC. Jap. I 87,

739 (1967).
9. C h a t t e n , L.G.and Okamura, K.O., J. Pharm. S c i . ,
62 1328 (1973).
-I
10. M o z s i k , G. and T o t h , E., J. Chromatog., 45, 478

(1969).
11. United S t a t e s Pharrnacopeiafiational Formulary,
1980.
12. H o n i g b e r g , I.L. , Stewart, J.T., S m i t h , A.P. I
P l u n k e t t , R.D., a n d J u s t i c e , E.L., J. Pharm.
S c i . , 64, 1389 (1975).
13. H o n i g b e r g , I.L., S t e w a r t , J.T., a n d S m i t h , A.P.,
i b i d , 63, 766 (1974).
14. G a g i n e l l a , T.S., B a s s , P., P e r r i n , J.H., and
V a l l n e r , J.J. , J. Pharm. Pharmac., 25, 271 (1973).
CUMULATIVE INDEX
Bold numerals refer to volume numbers
Acetaminophen, 3, 1 Cephalothin sodium, 1,319

Acetohexamide, 1, 1; 2,573 Cephradine, 5,21
Allopurinol, 7, 1 Chloral hydrate, 2,85
Alpha-tocopheryl acetate, 3, 111 Chloramphenicol, 4,47,518
Amantadine, 12, 1 Chlordiazepoxide, 1, 15
Amikacin sulfate, 12, 37 Chlordiazepoxide hydrochloride, 1,39; 4,518
Aminophylline, 11, 1 Chloroquine phosphate, 5,61
Aminosalicylic acid, 10, 1 Chlorpheniramine maleate, 7,43
Amitriptyline hydrochloride, 3, 127 Chloroprothixene, 2,63
Amoxicillin, 7, 19 Chlortetracycline hydrochloride, 8, 101
Amphotericin B, 6, 1; 7,502 Clidinium bromide, 2,145
Ampicillin, 2, 1; 4,518 Clindamycin hydrochloride, 10,75
Ascorbic acid, 11,45 Clofibrate, 11, 197
Aspirin, 8, 1 Clonazepam, 6,61
Azathioprine, 10,29 Clorazepate dipotassiurn, 4,91
Bacitracin, 9, 1 Clotrimazole, 11,225
Bendroflumethiazide, 5, 1; 6,597 Cloxacillin sodium, 4, 113
Benzocaine, 12,73 Codeine phosphate, 10,93
Benzyl benzoate, 10,55 Colchicine, 10, 139
Betamethasone dipropionate, 6,43 Cyanocobalamin, 10,183
Bretylium tosylate, 9,71 Cyclizine, 6,83; 7,502
Brornocriptine methanesulfonate, 8,47 Cycloserine, 1,53
Calcitriol, 8, 83 Cyclothiazide, 1,66
Captopril, 11, 79 Cypropheptadine, 9, 155
Carbamazepine ,9,87 Dapsone, 5,87
Cefaclor, 9, 107 Dexamethasone, 2,163; 4,519
Cefamandole nafate, 9, 125; 10,729 Diatrizoic acid, 4, 137; 5, 556
Cefazolin, 4, 1 Diazepam, 1,79; 4,518
Cefotaxime, 11, 139 Dibenzepin hydrochloride, 9, 181
Cefoxitin, sodium, 11, 169 Dibucaine and dibucaine hydrochloride, 12, 105
Cephalexin, 4,21 Digitoxin, 3, 149
733
734 CUMULATIVE INDEX
Digoxin, 9,207 Isoniazide, 6, 183

Dihydroergotoxine methanesulfonate, 7 , 8 1 Isopropamide, 2,315; 12,721
Dioctyl sodium sulfosuccinate,2, 199; 12,713 Isosorbide dinitrate, 4,225; 5,556
Diperodon, 6,99 Kanamycin sulfate, 6,259
Diphenhydramine hydrochloride, 3,173 Ketamine, 6,297
Diphenoxylate hydrochloride, 7, 149 Ketoprofen, 10,443
Disulfiram, 4, 168 Khellin, 9,371
Dobutamine hydrochloride, 8, 139 Leucovorin calcium, 8 , 3 15
Dopamine hydrochloride, 11,257 Levarterenol bitartrate, 1,49; 2,573; 11,555
Doxorubicine, 9,245 Levallorphan tartrate, 2,339
Droperidol, 7, 171 Levodopa, 5, 189
Echothiophate iodide, 3,233 Levothyroxinesodium, 5,225
Emetine hydrochloride, 10,289 Lorazepam, 9,397
Epinephrine, 7, 193 Meperidine hydrochloride, 1, 175
Ergonovine maleate, 11,273 Meprobamate, 1,209; 4,520; 11,587
Ergotamine tartrate, 6, 113 6-Mercaptopurine, 7,343
Erythromycin, 8, 159 Mestranol, 11,375
Erythromycinestolate, 1, 101; 2,573 Methadone hydrochloride, 3,365; 4,520;
Estradiol valerate, 4, 192 9,601
Estrone, 12, 135 Methaqualone,4,245,520
Ethambutol hydrochloride, 7,23 1 Methimazole, 8,35 1
Ethynodiol diacetate, 3,253 Methotrexate, 5, 283
Etomidate, 12, 191 Methoxsalen, 9,427
Fenoprofen calcium, 6, 161 Methyclothiazide,5,307
Flucytosine, 5, 115 Methylphenidate hydrochloride, 10,473
Fludrocortisoneacetate, 3,281 Methyprylon, 2,363
Flufenamic acid, 11,313 Metoprolol tartrate, 12,325
Fluorouracil, 2,221 Metronidazole, 5,327
Fluoxymesterone,7,25 1 Minocycline, 6,323
Fluphenazine decanoate, 9,275; 10,730 Nabilone, 10,499
Fluphenazine enanthate, 2,245; 4,524 Nadolol, 9,455; 10,732
Fluphenazine hydrochloride, 2,263; 4,519 Nalidixic acid, 8,371
Flurazepam hydrochloride, 3,307 Natamycin, 10,5 13
Gentamicin sulfate, 9,295; 10,731 Neomycin, 8,399
Glibenclamide, 10,337 Nitrazepam, 9,487
Gluthethimide,5, 139 Nitrofurantoin, 5,345
Gramicidin, 8, 179 Nitroglycerin, 9,519
Griseofulvin, 8,219; 9,583 Norethindrone, 4,268
Halcinonide,8,251 Norgestrel, 4,294
Haloperidol, 9, 341 Nortriptyline hydrochloride, 1,233; 2,573
Halothane, 1, 119; 2,573 Noscapine, 11,407
Heparin sodium, 12,215 Nystatin, 6, 341
Heroin, 10,357 Oxazepam, 3,441
Hexestrol, 11,347 Oxytocin, 10,563
Hexetidine, 7,277 Penicillamine, 10,601
Hydralazine hydrochloride, 8,283 Penicillin-G benzothine, 11,463
Hydrochlorothiazide,10,405 Penicillin-V, 1,249
Hydrocortisone, 12,277 Phenazopyridine hydrochloride, 3,465
Hydroflurnethiazide,7,297 Phenelzine sulfate, 2,383
Hydroxyprogesteronecaproate, 4,209 Phenformin hydrochloride, 4,319; 5,429
Hydroxyzine dihydrochloride, 7,3 19 Phenobarbital, 7,359
Iodipamide, 3,333 Phenoxymethyl penicillin potassium, 1,249
Isocarboxazid,2,295 Phenylbutazone, 11,483
CUMULATIVE INDEX 735
Phenylephrine hydrochloride, 3,483 Sulfamethoxazole,2,467; 4,521

Phenylpropanolamine hydrochloride, 12,357 Sulfasalazine, 5,515
Pilocarpine, 12,385 Sulfisoxazole, 2,487
Piperazine estrone sulfate, 5,375 Testolactone, 5, 533
Primidone, 2,409 Testosterone enanthate, 4,452
Probenecid, 10, 639 Theophylline, 4,466
Procainamide hydrochloride, 4,333 Thiostrepton, 7,423
Procarbazine hydrochloride, 5,403 Tolbutamide, 3,513; 5,557
Promethazine hydrochloride, 5,429 Triamcinolone, 1,367; 2,571; 4,521,524;
Proparacaine hydrochloride, 6,423 11,593
Propiomazine hydrochloride, 2,439 Triamcinoloneacetonide, 1,397, 416; 2, 571;
Propoxyphenehydrochloride, 1,301; 4,520; 4,521,7,501; 11,615
6,598 Triamcinolonediacetate, 1,423; 11,651
Propylthiouracil, 6,457 Triamcinolone hexacetonide, 6,579
Pseudoephedrine hydrochloride, 8,489 Triclobisoniumchloride, 2,507
Pyrazinamide, 12,433 Trifluoperazine hydrochloride, 9,543
Pyrimethamine, 12,463 Triflupromazine hydrochloride, 2,523; 4,521;
Quinidine sulfate, 12,483 5,557
Quinine hydrochloride, 12,547 Trimethaphan camsylate, 3,545
Reserpine, 4, 384; 5,557 Trimethobenzamidehydrochloride, 2,55 1
Rifampin, 5,467 Trimethoprim, 7,445
Rutin, 12,623 Trimipramine maleate, 12,683
Salbutamol, 10,665 Trioxsalen, 10, 705
Secobarbital sodium, 1,343 Triprolidine hydrochloride, 8,509
Spironolactone, 4,431 Tropicamide, 3,565
Sodium nitroprusside, 6,487 Tubocurarine chloride, 7,477
Succinylcholinechloride, 10,691 Tybamate, 4,494
Sulfadiazine, 11,523 Valproate sodium and valproic acid, 8,529
Sulphamerazine, 6,515 Vinblastine sulfate, 1,443
Sulfamethazine, 7,401 Vincristine sulfate, 1,463

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Analytical Profiles

ACADEMIC PRESS 1983

Abdullah A. Al-Badr Klaus Florey

Academic Press Rapid Manwrcript Reproduction

United Kingdom Edirion published by

PRINTED IN THE UNITED STATES OF AMERICA

H. Y. Aboul-Enein, King Saud University, Riyadh, Saudi Arabia

T. I . Khalifu, King Saud University, Riyadh, Saudi Arabia

The compilationofAnalytica1Profiles of Drug Substancesto supplementthe infor-

ANALYTICAL PROFILES OF DRUG SUBSTANCES 1 Copyright by the American Pharmaceutical Arwriation.

1.1 History, Therapeutic Use and Mechanism of Action

Amantadine is an orally active antiviral agent

viruses (12), but also in treating cancer (13),

1.2 Nomenclature, Molecular Weight and Structure

Amantadine hydrochloride is the United States

1.3 Appearance, Color, Odor and Precautions

Amantadine hydrochloride is a white, odorless,

Adamantane is found naturally at low

Direct amination (35) of adamantane or

conversion to amantadine have been described (38,39).

1.5 Reactions, Stability and Metabolism

Possible reactions are substitution at the amino

acid or nitrous acid gives 1-hydroxyadamantane in 97%

2. Physical Properties of Crystalline Amantadine

2.1 Single Crystal X-Ray Diffraction

Although the x-ray structure of amantadine

The three-dimensional representation below is

2.2 X-Ray Powder D i f f r a c t i o n

20(Degrees) ' d' (Angstroms) R e l a t i v e Area

18.2 4.88 1.000

18.6 4.78 0.298

2.3 Mass Spectrometry

The mass spectrum (67) of amantadine

Secondary ion mas? spectrometry $70) using

2.4 Infrared Spectrometry

Figure 3 shows the infrared spectra of a

5765 Q D Q M R N T R N R M I NE . H C L ( FlLClR I Cli 1 205C

M F1 SS1'C H FIR C;E

Figure 2: Mass Spectrum of Amantadine Hydrochloride,

Instrument AEI-MS 902.

polymorphism. Below are the interpretations (71, 72) aof

Ab sorption ( cm- I ) Assignment

ionization potentials, 11, for a series of adamantane

2.6 Thermal Analysis

Thermal gravimetric analysis (76) of a

A commercial preparation of amantadine

2.8 Surface Area

As measured by nitrogen gas adsorption (76), the

The crystals are not solvated with water, based

There is weak evidence for polymorphism based on

3. Spectrometry of Amantadine in Solution

3.1 Nuclear Magnetic Resonance Spectrometry (NMR)

Figure 4 is the 100 MHz proton NMR spectrum

Chemical shifts of 1-substituted adamantane (78)

The exceptional structure of amantadine has

Chemical Shift (ppm) Assignment

in Deuterochloroform, as Recorded at 100 MHz.

with the experimental results, showing that no large

The natural abundance 15N nuclear magnetic

3.2 Ultraviolet Spectrometry

Below is the ultraviolet spectrum of a

4.1 Solubilities in Aqueous and Nonaqueous Solvents.

Deuterochloroform, as Recorded at 15 MHz.