Vol 16 Analytical Profiles of Drug Substan (BookSee - Org) V 16

Analytical Profiles
of
Drug Substances
Volume 16
Edited by
Klaus Florey
The Squibb Institute for Medical Research
New Brunswick, New Jersey
Contributing Editors
Abdullah A. Al-Badr
Gerald S. Brenner
1987
ACADEMIC PRESS, INC.

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EDITORIAL BOARD
Abdullah A. Al-Badr
Steven A. Benezra
Gerald S. Brenner
Glenn A. Brewer, Jr.
Nicholas DeAngelis
Lee T. Grady
Eugene Inman
Joseph Mollica
Milton D. Yudis
John Zarembo
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Library of Congress Cataloging in Publication Data

(Revised for volume 16)
Analytical profiles o f drug substances.
Compiled under the auspices o f the Pharmaceutical
Analysis and Control Section, Academy of Pharmaceutical
Sciences.
Includes bibliographical references and indexes.
1. Drugs-Analysis-Collected works. 2. Chemistry,
Pharmaceutical-Collected works. I. Florey, Klaus,
11. Brewer, Glenn A. 111. Academy of Pharmaceutical
Sciences. Pharmaceutical Analysis Control Section.
[DNLM: 1. Drugs-Analysis-Yearbooks.
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ISBN 0-12-260816-X
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AFFILIATIONS OF
EDITORS AND CONTRIBUTORS
Mohammad A . Abounassif, College of Pharmacy, King Saud University,

Riyadh, Saudi Arabia
Abdul Fattah A . Afify, College of Pharmacy, King Saud University,
Abdullah A . Al-Budr, King Saud University, Riyadh, Saudi Arabia
Mohammed A . Al-Yahya, College of Pharmacy, King Saud University,
Salim A . Babhair, College of Pharmacy, King Saud University, Riyadh,
Saudi Arabia
Jos H . Beijnen, Slotervaart Hospital, Amsterdam, The Netherlands
Steven A . Benezra, Wellcome Research Laboratories, Research Triangle
Park, North Carolina
Gerald S. Brenner, Merck Sharp & Dohme Research Laboratories, West
Point, Pennsylvania
Glenn A . Brewer, Jr., The Squibb Institute for Medical Research, New
Brunswick, New Jersey
Auke Bult, Faculty of Pharmacy, State University of Utrecht, Utrecht,
The Netherlands
Nicholas J. DeAngelis, Wyeth Laboratories, Philadelphia, Pennsylvania
Humeida A. El-Obeid, King Saud University, Riyadh, Saudi Arabia
Klaus Florey, The Squibb Institute for Medical Research, New Brunswick, New Jersey
Dennis K . G . Gorecki, College of Pharmacy, University of Saskatchewan,
Saskatoon, Saskatchewan, Canada
Lee T . Grady, The United States Pharmacopeia, Rockville, Maryland
Mahmoud M . A . Hassan, King Saud University, Riyadh, Saudi Arabia
Eugene L. Inman, Lilly Research Laboratories, Indianapolis, Indiana
vii
...
Vlll
AFFILIATIONS
Dominic P . I p , Merck Sharp & Dohme Research Laboratories, West

Point, Pennsylvania
Casimir A . Janicki, McNeil Pharmaceutical, Spring House, Pennsylvania
Vijay K . Kapoor, Department of Pharmaceutical Sciences, Panjab University, Chandigarh, India
Alice E . Loper, Merck Sharp & Dohme Research Laboratories, West
Point, Pennsylvania
David J . Mazzo, Travenol Laboratories, Morton Grove, Illinois
A . G. Mekkawi, College of Pharmacy, King Saud University, Riyadh,
Saudi Arabia
Joseph A . Mollica, Pharmaceuticals, Du Pont, Wilmington, Delaware
Jaber S. Mossa, College of Pharmacy, King Saud University, Riyadh,
Saudi Arabia
Farid J . Muhtadi, King Saud University, Riyadh, Saudi Arabia
Davide Pitrc?, Faculty of Pharmacy, University of Milan, Milan, Italy
James T. Stewart, College of Pharmacy, University of Georgia, Athens,
Georgia
Riccardo Stradi, Faculty of Pharmacy, University of Milan, Milan, Italy
Abdul Hameed U . Kader Taragan, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
Mohammad Tariq, College of Pharmacy, King Saud University, Riyadh,
Saudi Arabia
WillyJ . M . Underberg, Faculty of Pharmacy, State University of Utrecht,
Utrecht, The Netherlands
Roger K . Verbeeck, College of Pharmacy, University of Saskatchewan,
Milton D . Yudis, Schering-Plough, Kenilworth, New Jersey
John E . Zarembo, W. H. Rorer, Inc., Fort Washington, Pennsylvania
PREFACE
Although the official compendia define a drug substance as to identity,

purity, strength, and quality, they normally do not provide other physical
or chemical data, nor do they list methods of synthesis or pathways of
physical or biological degradation and metabolism. Such information is
scattered through the scientific literature and the files of pharmaceutical
laboratories.
I perceived a need to supplement the official compendial standards of
drug substances with a comprehensive review of such infoimation, and
sixteen years ago the first volume of Anulyticul Profiles of Drug Substances was published under the auspices of the Pharmaceutical Analysis and Control Section of the APhA Academy of Phaimaceutical Sciences. That we were able to publish one volume per year is a tribute to
the diligence of the editors to solicit articles and even more so to the
enthusiastic response of our authors, an international group associated
with pharmaceutical firms, academic institutions, and compendial authorities. 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 divg substances of medical value and, therefore, we have welcomed any articles 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.
Klaus Florey
ix
BROMAZEPAM
Mahmoud M.A. H a h a n , PhO6eAhOh 06 PhahmaceuLLcd C h m h L t y ,

Depah;tment od PhahmaceuLLcd C h m h L t y , CoMege 06
Phanmacy, King Saud U n L v m L t q , Riyadh, Saudi Axabia.
Mohammad A. A b u u m n i d , AnbhXant Phodeddon 04 PhamaceuLic d C h m h i x y , Heud 04 Xhe DepdmenX 06 P h a m a c e d i c d
C h m h L t y , College 06 Phahmacy, King Saud U n i v m i , t y ,
Riyadh, Saudi Arrabia.
1. H i s t o r y and T h e r a p e u t i c Category
2.
Description
2.1
Nomenclature
2.2
Formulae
2.3
Molecular Weight
ANALYTICAL PROFILES OF DRUG SUBSTANCES

VOLUME 16
Copyright 0 1987 by Academic Press, Inc.

All rights of reproduction in any form reserved.
MAHMOUD M. A. HASSAN AND MOHAMMAD A. ABOUNASSIF
.3.
4,
Physical Properties
3.1
Appearance, Color, Odor and Taste
3.2
Melting Range
3.3
D i s s o c i a t i o n Constants
3.4
Crystal Structure
Spectral Properties
4.1
U l t r a v i o l e t Spectrum
4.2
I n f r a r e d Spectrum
4.3
Nuclear Magnetic Resonance S p e c t r a
4.4
Mass Spectrum
5.
Synthesis
6.
Metabolism
7.
Pharmacokinetic S t u d i e s
8.
Methods of Analysis
8.1
Elemental Analysis
8.2
I d e n t i f i c a t i o n Tests
8.3
Titrimetric
8.4 Spectrophotometric
8.5 Polarographic
8.6 Atomic Absorption
9.
8.7
Radioimunoassays
8.8
Chromatographic
References
BR 0MAZEPAM
A n a l y t i c a l P r o f i l e of Bromazepam
1. H i s t o r y and Therapeutic Category
The l a s t q u a r t e r c e n t u r y , and i n p a r t i c u l a r t h e decade
1952-1962, has witnessed a break-through i n t h e t r e a t m e n t
and outlook of t h e mentally ill. Two groups of drugs
were developed namely psychotic and psychotropic a g e n t s .
Among t h e l a s t named psychotropic a g e n t s , t h e benzodiazep i n e s . These drugs such as diazepam, chlordiazepoxide
and oxazepam have been shown t o e x e r t a n x i o l y t i c , s l e e p
inducing, muscle r e l a x a n t and a n t i c o n v u l s a n t e f f e c t s i n
g r e a t e r or l e s s e r degree. Search continued f o r making
more d e r i v a t i v e s t o produce s u i t a b l e drug f o r s p e c i f i c
cases or symptoms. I n t h i s way nitrazepam and more
r e c e n t l y flurazepam have been found t o be s p e c i a l l y e f f e c t i v e s l e e p inducers and clonazepam e x e r t s even more
p o t e n t l y t h e a n t i c o n w l s a n t a c t i v i t y known a l r e a d y f o r
diazepam.
Search has continued and i n 1974 bromazepam w a s marketed
as a new psychotropic drug of t h e benzodiazepine s e r i e s
but belonging t o a new c l a s s of pyridyl-benzodiazepines.
It possesses c e r t a i n biochemical f e a t u r e s which d i s t i n guish it from o t h e r benzodiazepines. It has been shown
t o e x e r t a more profound a n x i o l y t i c e f f e c t t h a n o t h e r
similar substances.
2.
Description
2.1
Nomenclature
2.1.1
Chemical Names
7-Brom0-1,3-dihydro-5-(~2-pyridyl)-2H-l,~benzodiazepine-2-one.
2.1.2
Generic Name
Bromazepam.
2.1.3
Trade Names
Lectopam; Lexomil; Lexotan; Lexotanil.
2.2
Formulae
2.2.1
Ehpirical
C14H10BrN
2.2.2
Structural
2.2.3
Research Number (1)

Ro 5-3350.
2.2.4
Chemical A b s t r a c t s R e g i s t r y Number
[ 1812-30-21
2.3
Molecular Weight
316.16
3.
Physical Properties
3.1
Appearance, Color, Odor and T a s t e

A p a l e yellow o d o r l e s s c r y s t a l l i n e s o l i d ( 2 ) .
3.2
Melting Range
BROMAZEPAM
3.3
D i s s o c i a t i o n Constants
Bromazepam h a s t h r e e pKa v a l u e s o f 2.5, 5.2 and
11.8 corresponding t o p r o t o n a t i o n at t h e azomethine
and p y r i d i n e n i t r o g e n atoms, and d e p r o t o n a t i o n a t
t h e n i t r o g e n atom i n p o s i t i o n 1, r e s p e c t i v e l y .
These pKa v a l u e s were determined by s p e c t r a l and
polarographic a n a l y s i s ( 3 ) .
3.4
Crystal Structure
(4)
The c r y s t a l s t r u c t u r e of bromazepam w a s determined

u s i n g c r y s t a l s from amyl a c e t a t e - e t h a n o l o b t a i n e d
by slow e v a p o r a t i o n . The i n t e n s i t y d a t a w e r e
c o l l e c t e d on a n Enraf-Nonius CAD-4 d i f f r a c t o m e t e r ,
c r y s t a l s i z e 0 . 3 x 0.25 x 0.2 mm, c e l l dimentions
from s e t t i n g a n g l e s o f 25 r e f l e c t i o n s and g r a p h i t e
monochromated MoKa r a d i a t i o n . Bromazepam i s monoc l i n i c w i t h space groups P 2 l / c (non-centrosymetric)
The c e l l dimentions are a = 10.304 ( 4 ) , b = 15.897
( 5 ) , c = 8.122 ( 3 ) A', 6 = 106.8 (3)', U = 1273.6 AO,
Z = 4 , Dx = 1.649 Mg m-3, p(MoKa, A = 0.71069 A')
= 3.13 mm-1, F(000) = 6 3 2 , room t e m p e r a t u r e ,
R = 0.040 f o r 1470 observed r e f l e c t i o n s .
I n bromazepam t h e 7-membered r i n g i s i n a b o a t
conformat i o n . The a n g l e between t h e benzo-moeity
o f t h e 1,4-benzodiazepine system and t h e h e t e r o c y c l i c r i n g system i n t h e ? - p o s i t i o n i s 60.3 ( 6 ) ' .
F i n a l atomic parameters f o r bromazepam are l i s t e d i n
Table 1; bond l e n g t h s , bond a n g l e s and s e l e c t e d
t o r s i o n a n g l e s are l i s t e d i n Table 2. The atomic
numbering scheme i s i l l u s t r a t e d i n F i g . 1. Bond
l e n g t h s and a n g l e s g e n e r a l l y a g r e e w e l l w i t h v a l u e s
found i n analogous molecules ( 5 - 7 ) . The N ( l ) - C ( 2 )
formal s i n g l e bond i s s h o r t e n e d t o 1.351 ( 5 ) A' i n
bromazepam and t h e d i s p o s i t i o n o f bonds at N ( 1 ) i s
n e a r p l a n a r s o t h a t t h e geometry of t h e bond
resembles t h a t of a normal double bond ( c f . t o r s i o n
a n g l e s i n Table 2 ) ( 6 ) . The N ( 4 ) - C ( 5 ) l e n g t h i n
bromazepam corresponds t o t h a t o f a C = N bond and
t h e C ( 5 ) - C ( l ' ) and C ( 5 ) - C ( l l ) l e n g t h s are w i t h i n t h e
a c c e p t e d range (1.48-1.50 A') for a C(sp2)-C(sp2)
s i n g l e bond. There i s , t h e r e f o r e , no evidence f o r
any e l e c t r o n d e l o c a l i z a t i o n between t h e 5-pyridyl
r i n g i n bromazepam and t h e 1,4-benzodiazepine
system.
a,
c
0
c,
(ud
BROMAZEPAM
The seven-membered r i n g i s i n a c y c l o h e p t a t r i e n e l i k e b o a t conformation, C ( 1 0 ) and C ( 1 1 ) forming t h e

' s t e r n ' and C(3) t h e 'bow'.
The bow a n g l e s , 60.3
( 8O) i n bromazepam and t h e s t e r n a n g l e 30.8 (8' )
Table 1.
F r a c t i o n a l atomic c o o r d i n a t e s ( x 1 0 ) w i t h e . s . d . ' s
i n p a r e n t h e s e s and e q u i v a l e n t i s o t r o p i c t e m p e r a t u r e
f a c t o r s ( ~ x2 1 0 3 )
617 (1)
-1404 ( 3 )
-528 ( 2 )
-2169 ( 2 )
-1209 ( 2 )
-1298 ( 3 )
-2004 ( 3 )
-1626 ( 5 )
-520 ( 3 )
237 ( 3 )
726 ( 3 )
449 ( 3 )
-324 ( 3 )
-821 ( 2 )
-1841 ( 3 )
-1400 ( 3 )
-2190 ( 3 )
-2820 ( 4 )
-2650 ( 3 )
U
eq
184
177
94
102
95
110
163
86
101
104
108
119
78
75
79
110
126
125
99
The major conformational d i f f e r e n c e between t h i s

compound and o t h e r known 5-phenyl-1,4-benzodiazep i n e s i s i n t h e o r i e n t a t i o n of t h e 5-aryl r i n g . The
a n g l e between t h e mean p l a n e o f t h e 5-aryl r i n g and
t h e 'benzo' p l a n e i s 60.3 (6') i n bromazepam and
75.5 (9') i n o t h e r benzodiazepines ( 8 ) .
Table 2. Molecular dimensions

(b) Bond angles
(")
1.351
1.214
1.453
1.278
1.483
1.394
1 371
119
113
127.1
122.1
114.6
123.3
111.3
1.376
1.362
1 397
1* 395
(31
(3)
(4)
(5)
(4)
(4)
(4)
117.4 ( 4 )
126.1 ( 4 )
116.3 (4)
1.402
117.6 (3)
1.892
121.3
120.3
119.8
119.8
(4)
(4)
(4)
(3)
1.378 ( 6)
C ( 11) -C( 10)-N( 1)

C( 10)-c( 11)-c( 6)
c( 1O)-C( ll)-C( 5)
C( 6 ) -c( 11) -c( 5 )
C( 5 ) -C( 1' ) -N( 2 ' )
c(5 -C(l')-C(2')
c(5 -c(if)-c(6I)
119.5
121.2
119.5
116.9
123.5
118.1
112.8
119.0
117.9
(4)
(4)
(4)
(4)
(4)
(4)
(4)
(4)
(4)
120.7 ( 4 )
c(2 )-c( 1'1-c( 6')

N ( 2 )-c(it)-c(6') 1 2 2 . 3 (4)
C( 2 )-C( 2')-C( 3') 116.8 ( 4 )
C( 1 ) -C( 2')-C( 3')
C( 1 ) - C ( 2')-F
C( 3 ' ) -C( 2 ' ) -F
c( 2 ' )-C(3')-C(4'
N( 2')-C( 3' )-C( 4 ' ) 124.4 (5)
c( 3' )-c( 4 ' )-c( 5 ' ) 117.8 (5)
C( 4 ' )-C( 5')-~(6') 119.2 ( 5 )
C( 5' 1-C( 6 ' 1-C( 1') 1 1 9 . 4 ( 5 )
BROMAZEPAM
(c)
Selected torsion angels
C( l O ) - N ( 1 ) - C ( 2)-C( 3)
N ( l ) - C ( p ) - C ( 3)-N(4)
c(2)-C(3)-N(b)-C( 5 )
C ( 3 ) - N ( 4 ) -C( 5 ) -C( 11)
N ( 4 ) - C ( 5 ) -C( ll)-C( 10)
C ( 5 ) - C ( 11)-C( 10)-N( 1)
4.
(O);
-1.1
-70.3
72.3
-2.7
-37.6
-1.2
e.s.d.'s
ca
0.6'
C(ll)-C(lO)-N(l)-C(2)
39.8
4 ) - C ( 5 ) -C( 1' )-N( 2 ' ) 141.6
4 ) -C( 5 ) -C( 1' ) -C( 2 ' )
C( 11)-C( 5 ) - C ( 1' ) -N( 2 ' ) -39.8
C( I l ) - C ( 5)-C( 1' )-C( 2 ' )
-
N(
Nf
Spectral Properties
4.1
The u l t r a v i o l e t spectrum o f bromazepam i n methanol
i s shown i n F i g . 2. The s p e c t r a of bromazepam i n
1 N NaOH and 1N H C 1 are p r e s e n t e d i n F i g . 3 and Fig.
4 , r e s p e c t i v e l y . The
and maximum wavelengths
cm
are given i n Table 3 ( 9 ) . These v a l u e s a g r e e s w i t h
t h e p u b l i s h e d d a t a (10-12).
Levillain (13), has
s t u d i e d t h e r e l a t i o n s h i p of s t r u c t u r e and t h e W
a b s o r p t i o n c h a r a c t e r i s t i c s of a s e r i e s of 1,4-benzod i a z e p i n e s , i n c l u d i n g bromazepam, c o n s i d e r i n g t h e
electronic distribution of t h e various substituent s
and t h e s t e r e o c h e m i s t r y . The spectrum of bromazepam
i s c o n s i s t e n t w i t h t h a t o f o t h e r benzodiazepines w i t h
s i m i l a r s t r u c t u r e . Als o bromazepam was i d e n t i f i e d
i n s o l i d dosage forms by W a b s o r p t i o n spectrometry
q%
(14).
Table 3.
Solvent
Methanol
1 N NaOH
1N HC1
UV S p e c t r a l c h a r a c t e r i s t i c s
max ( n m )
320
258
1%
cm
54.54
233
398.08
1004.78
350
268
238
493.77
794.25
348
270
238
59.33
39 * 23
386.60
552.15
The above v a l u e s a g r e e s w i t h t h a t of bromazepam.
90
80
70
60
M
40
3a
2G
10
11
z
z
4
.-C
N
1cI
0
E
CI
0,
m
M
.r(
i*
91
81
7(
6t
5(
4
f
200
Fi:;.
4.
250
The U V s p e c t r u m of bromazeparn
in
IN HCI.
4.2
An i n f r a r e d a b s o r p t i o n spectrum o f potassium bromide

d i s p e r s i o n o f bromazepam (Roche - Reference Standard
Lot 0401055) i s shown i n F i g . 5. The s p e c t r a l band
assignments (9,16,17) l i s t e d i n Table 4 .
Table
4.
I n f r a r e d s p e c t r a l assignments o f bromazepam
Wave number (em-')
Assignment
(Vibration m o M
3220,3150 , 3080
N-H
stretch
3000,2940,2860
C-H
stretch
1695
C = 0 stretch
1615
C = N s t r e t c h of both
benzodiazepine and
pyridine.
1600,1590,1570,14~0
Aromatic C = C s t r e t c h
815
Out o f p l a n e CH-deformat i o n of 1,2,4-tris u b s t i t u t e d aromatic

Out o f p l a n e CH deformat i o n of o r t h o - d i s u b s t i t u t e d aromatic
1 , 2 ,h-Trisubst i t u t e d
aromatic and o - d i s u b s t i t u t e d aromatic. A l s o
2-substituted pyridine.
Other c h a r a c t e r i s t i c bands a r e 1440, 1385, 1340,

1320, 1230, 1200, 1090, 1040, 1000, 990, 890, and
790 em-1, I n f r a r e d h a s been used f o r i d e n t i f i c a t i o n
o f bromazepam w i t h o t h e r psychotropic drugs ( 1 8 ) .
Bromazepam was i d e n t i f i e d i n s o l i d dosage forms by
I R a b s o r p t i o n spectrometry (14).
15
BROMAZEPAM
4.3

4.3.1
'H-NMR
Spectrum
A t y p i c a l 'H-NMR
spectrum o f bromazepam i s
shown i n Fig. 6. The sample w a s d i s s o l v e d
i n DMSO d6 and t h e spectrum was recorded on a
400 MHz NMR spectrometer, using TMS as an
i n t e r n a l r e f e r e n c e standard. NMR spectrum
recorded on a Jeol-FX-100 90 MHz s p e c t r o x e t e r
i s a l s o shown i n Fig 6a. The proton assignments are l i s t e d i n Table 5. The spectrum
a f t e r a d d i t i o n of D20 i s shown i n F i g . 7.
H
Table 5.
Proton
p o s i t ion
c-3
c-9
C-6
c-5'
C-8
c-4'
PMR c h a r a c t e r i s t i c s of bromazepam
Group
-CH2
-9H
-6H
-5'H(
-8H
-4'H(
-3'H(
-6'H(
(Benzenoid)
( "
"
)
F'yridine )
(Benzenoid)
Fyridine)
c-3'
Fyridine)
c-6'
Pyridine)
N-1
- l H (Diazepine)
(disappeared a f t e r D20
exchange )
s = singlet
d = doublet
Chemical s h i f t ppm
and split?;irig
4.22
7.23
7.44
7.49
7.41
7.95
8.86
8.57
(s)
(d)
(s)
(m)
(m)
(m)
(d)
(d)
10.66 (s)
rn = m u l t i p l e t
Lr
BROMAZEPAM
F i g . 6s.
'H-NYR
Fig. 7 .
17
spectrum of bromazepam i n DMSO d6 and TMS.
'H-NMR
spectrum of bromazepam i n DMSO d6 and
TYS after DzO t=xchsngr.
18
4.3.2
13C-NMR
Spectra
13C-NMR noise-decoupled and off-resonance

s p e c t r a of bromazepam are shown i n F i g . 8,
and F i g . 9 , r e s p e c t i v e l y . Both were
recorded over 5000 Hz range i n d e u t e r a t e d
dimethyl s u l f o x i d e (DMSO d 6 ) , ( c a n e . 100 mg/
1 m l ) , on J e o l FX-100 90 MHz i n s t r u m e n t .
Sample t u b e 1 0 mm and t e t r a m e t h y l s i l a n e as a n
i n t e r n a l r e f e r e n c e s t a n d a r d a t 2OoC were used.
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 chemical s h i f t t h e o r y and
t h e off-resonance s p l i t t i n g p a t t e r n ( T a b l e 6 ) .
Table 6.
Carbon No.
c-2
5'
Carbon chemical s h i f t s of bromazepam
Chemical s h i f t
6 (ppm)
169.72 ( s )
c-5
56.89 ( t )
167.55 ( s )
C-6
c-7
113.94 ( s f
c-3
C-5a
Carbon No.
127.28 ( s )
133.50 ( d )
C-8
133.79 ( d )
C-la
c-2'
138.49 ( s )
155.63 ( s )
c-9
c-6'
c-5:
c-4
c-3'
s = singlet
d = doublet
Chemical s h i f t
6 (ppm)
123.34 ( d )
148.24 ( d )
122.87 ( a )
136.96 ( d )
124.81 ( s )
t = triplet
19
BROMAZEPAM
Fig. 8 .
C-NMR noise-decoupled Spectrum o f bmmazepam i n OMS0 and TUS.
Pig. 9 .
13C-NMR off-resonance spectrum of bromazepam in DUSO and TUS.
20
4.4
Mass Spectrum
The E l mass spectrum o f bromazepam (Roche Reference

Standard, Lot 0401055) was o b t a i n e d on a Finigan
1020 quadrupole mass spectrometer and shown i n
Fig. 10. I t s GC t r a c e i s shown i n Fig. 11. The
i o n i z i n g e l e c t r o n beam energy w a s at 70 eV. It
shows a molecular i o n !d+ a t m/e 316 ( r e l a t i v e
i n t e n s i t y 34.87) and M+ + 1 a t m / e 317 ( r e l a t i v e
i n t e n s i t y 63.88). Some of t h e most prominent i o n s
a r e given i n Table 7 (9).
Table 7.
The most prominent fragments of

bromaz epam
m/e
Relative i n t e n s i t y
m/e
Relative i n t e n s i t y
317
63.88
236
316
34.87
208
100.00 ( b a s e
Peak)
315
60.70
207
30 37
314
26.84
206
23.33
289
33.24
179
30.89
288
59.95
104
21.09
287
35.95
90
35.54
286
52.84
79
19.69
261
19.28
78
31 90
259
19.36
77
24.08
51
25.67
43.56
F i g . 10.
EI-mass spectrum of bromazeparn.
22
5.
Synthesis
D i f f e r e n t r o u t e s f o r t h e s y n t h e s i s o f bromazepam were
r e p o r t e d (19-21).
Route I
Br
[11
CH NH COOC2H5.HC1
2 2
p y r i d i n e /ref lux
BROMAZEPAM
23
2-(2-Amino-5-bromobenzoylpyridine [ 41 was prepared from

2-phenacylpyridine p-bromophenyl hydrazone [ 11 through
cyclization with concentrated hydrochloric acid to give
5-bromo-2-phenyl-3-( 2-pyridy1)indole [ 21. This compound
was oxidized with chromium trioxide to give 2-(2-benzamido-5-bromobenzoy1)pyridine [3] ( 2 9 , 2 2 ) which on hydrolysis with concentrated hydrochloric acid afforded the
desired compound 2( 2-amino-5-bromobenzoy1)pyridine [4]
This compound is considered to be the starting material
for the synthesis of 1,4-benzodiazepines. A mixture of
[4], glycine ethyl ester hydrochloride and pyridine was
refluxed to give bromazepam [ 5 ] .
Br2
H
I
[5 1
in glacial
acetic acid
\ /
[41
Alternatively, the key intermediate compound [4] was prepared by bromination of 2-(2-aminobenzoyl)pyridine [6]
with bromine and glacial acetic acid (23,24).
24
Route 3
BrCOCH2Br
Br
Heat
-6
[ 51
Bromazepam
I n t h i s r o u t e 2-(2-aminobenzoyl)pyridine [ 41 w a s t r e a t e d
with bromoacetyl bromide in g l a c i a l a c e t i c a c i d . The
crude bromoacetyl d e r i v a t i v e [ 71 w a s converted d i r e c t l y
t o bromazepam [ 5 ] by r e a c t i o n with ammonia through t h e
intermediate aminoacetamidobenzoyl p y r i d i n e
(20).
[a]
BROMAZEPAM
Route
25
CH2
0-
-/
[41
H-N
c o \\
QI
IC
Br
Br
'
4 - a
L
I
CH3COOH
[ 51
An a l t e r n a t i v e method (20)f o r t h e s y n t h e s i s of bromazepam
proceeded v i a t h e carbobenzoxyglyclyl d e r i v a t i v e [ 9 ] .
T h i s compound i s prepared a c c o r d i n g t o t h e method of
Sheehan and Hess d e v i s e d f o r t h e s y n t h e s i s o f p e p t i d e s
( 2 5 , 2 6 ) . A m i x t u r e of t h e 2-(2-amino-5-bromobenzoyl)pyrid i n e and carbobenzoxyglycine i n methylene c h l o r i d e w a s
added. The methylene c h l o r i d e s o l u t i o n a f t e r removal of
N,N'-dicyclohexylurea w a s c o n c e n t r a t e d i n vacuo a t room
t e m p e r a t u r e . The r e s i d u e w a s d i s s o l v e d i n benzene and
chromatographed t o g i v e [ g ] . Cleavage o f t h e carbobenzoxy
group w i t h hydrogen bromide i n g l a c i a l a c e t i c a c i d (27)
gave bromazepam [ 5 ] d i r e c t l y without i s o l a t i o n of t h e
i n t e r m e d i a t e , 2-( 2-aminoacetamido-5-bromobenzoy1)pyridine.
26
Route 5
Clarke e t a1 (1980) (28) have described i n d e t a i l s t h e
p r e p a r a t i o n of bromazepam [ 5 ] from t h e corresponding
benzophenone by t h e use of hexamine and hydrochloric a c i d
as o u t l i n e d below:
Br
/
t
+ 3MeC6H12N4C1
t
NH4C1
3HC02Me
BROMAZEPAM
Three main by-product were i d e n t i f i e d : two i m i d a z o l i d i nones [lo], [ll] and a n aminobenzodiazepine ( 1 2 ) .
27
28
6.
Metabolism
The f i r s t r e p o r t e d m e t a b o l i c work on bromazepam w a s t h a t
published by Sawada i n 1972 ( 2 9 , 3 0 ) . T h i s s t u d y w a s
c a r r i e d o u t b o t h i n dogs, r a b b i t s and r a t s . The i s o l a t e d
u r i n a r y m e t a b o l i t e was assumed t o be a 3-hydroxylated
d e r i v a t i v e o f 2-amino-5-bromobenzoylpyridine [ 1 5 ] . The
s t r u c t u r a l e l u c i d a t i o n w a s based on e l e m e n t a l a n a l y s i s ,
mass spectrometry and n u c l e a r magnetic resonance s t u d i e s .
OH
The b i o t r a n s f o r m a t i o n of bromazepam, was s t u d i e d i n f o u r

s p e c i e s namely human, dog, r a t and mouse ( 3 1 ) . Four
m e t a b o l i t e s w e r e i d e n t i f i e d and t h e i r e x c r e t i o n w a s
q u a n t i t a t i v e l y determined. The major u r i n a r y m e t a b o l i t e s
e x c r e t e d by t h r e e humans given s i n g l e 1 2 mg o r a l doses
of bromazepam-5- 1 4 C were t h e conjugated forms of 3hydroxy bromazepam [13] accounting f o r 13-30% of t h e dose,
and 2-( 2-amino-5-bromo-3-hydroxybenzoyl)pyridine [15]
accounting f o r 4-25% of t h e dose. Conjugated [131 w a s
a l s o t h e major m e t a b o l i t e i n dogs and mice a d m i n i s t e r e d
14C-bromazepam, but i t s e x c r e t i o n by r a t s was v e r y
l i m i t e d . Together w i t h [13], m e t a b o l i t e [ 1 5 ] and 2-( 2amino-5-bromobenzoy1)pyridine [ 4 ] were d e t e c t e d i n t h e
e x c r e t a o f t h e human, dog, r a t and mouse. Although t h e
p y r i d y l N-oxide [I81 d e r i v a t i v e o f bromazepam w a s not
d e t e c t e d as a m e t a b o l i t e i n any s p e i c e s , t h e Nq-oxide [14]
was a minor m e t a b o l i t e i n dog u r i n e . T h i s i s t h e f i r s t
r e p o r t e d i n s t a n c e o f N4-oxidation o f a 1,4-benzodiazepine-2-one.
This s t u d y has shown t h a t t h e metabolism o f
bromazepm i n human, dogs but not i n r a t s f i t s t h e same
p a t t e r n of o t h e r lY4-benzodiazepine-2-one i n a f f o r d i n g
conjugated 3-hydroxylated d e r i v a t i v e (32). The 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 t h e d i f f e r e n t m e t a b o l i t e s
were c a r r i e d o u t by t h i n l a y e r chromatography u s i n g
s e v e r a l s o l v e n t systems.
BROMAZEPAM
29
de Silva et al, 1974 (33) have reported a sensitive and

specific electron-capture GLC assay for bromazepm and
its major metabolites (see under methods of analysis).
Table 8 shows the chemical names and physical properties
of bromazepam and its major metabolites.
Table
8. Chemical names and physical properties of bromazepam

and metabolites
No.
Chemical name
7-Bromo-1,3-dihydro-5-(2pyridyl)-2H-1,4-benzodiazepin-2-one (bromazepam)
Molecular
weight
Melting
point
316.16
237438.50
dec
13
332 2
198-2ooo
14
7-Bromo-1,3-dihydro-5-(2pyridyl)-2H-1,h-benzodiazepin-2-one 4-oxide.
332.2
263O dec.
2-Amino-5-bromobenzoylpyridine.
277.12
97* 5-99O
15
2-Amino-5-brom0-3-hydroxybenzoylpyridine.
293.12
190-196O
16
Bromazepam mercapturic acid

methylester( 7-bromo-1,3dihydro-5- [ 2 ' - ( 6' -N-ac etylL-cystein-S-y1)-pyridyl] -2H1,4-benzodiazepin-2-one.
228
17
18
Met hylthiobromazepam
Pyridyl N-oxide of bromazepam
Taleishi and Shimizu 1976 (34) has recently reported the

isolation of a methylthio-containing metabolite which
was identified as 7-bromo-l,3-dihydro-5-[2'-( 6I-methylthio)pyridyl]-2H-1,4-benzodiazepin - 2-one (methylthiobromazepam) [17]. Also they have shown that the methionine
donor theory (34) is very unlikely in the case of the
formation of methylthiobromazepam. Isolation of the mer-
0
01
Y
M
BROMAZEPAM
31
capturic acid conjugate of bromazepam from bile of rat

given the drug, its identification and subsequent conversion of the mercapturic acid to methylthiobromazepam
in rat liver preparation was reported (35). Scheme 1
shows a proposed pathway for the biotransformation of
bromazepam to methylthiobromazepam in the rat.
II
CH3
[51
' COOH
I
[MeY
14
-SAM]
Br
[I71
: S - @
14CH3
Scheme I. A proposed pathway f o r the biotransformation

of bromazepam to methylthiobromazepam in the
rat.
14
In this study non-radioactive bromazepam, [5- C] bromazepam and bromazepam-N'-oxide ('j'-bromo-1,3-dihydro-5-[2'(1' -ox0 ) -pyridyl 3 -2H-1,h-benzodiazepin - 2-one were
employed. 7-Bromo-l,3-dihydro-5-[ 2 I - ( 6'-methyl ,N-acetylL-cysteinate-5-yl)-pyridyl]-2H-1,4-benzodiazepin-2-one
(bromazepam rnercapturate methylester) was synthesised
from bromazepam "-oxide and methyl N-acetyl-L-cysteinate
in acetic anhydride according to the pyridine-N-oxide
32
n u c l e o p h i l i c s u b s t i t u t i o n d e s c r i b e d by Bauer and
Dickerhofe ( 3 6 ) . The r e s u l t s i n d i c a t e d t h a t 6'-methylthiobromazepam i s o l a t e d p r e v i o u s l y i n t h e r a t u r i n e i s
formed a t l e a s t i n p a r t v i a t h e m e r c a p t u r i c a c i d and t h a t
rat l i v e r c o n t a i n s enzyme(s) capable of c a t a l y s i n g t h e
conversion from t h e mercapturic a c i d t o methylthiobromazepam. Chemical names and p h y s i c a l p r o p e r t i e s of bromazepam and i t s m e t a b o l i t e s are l i s t e d i n Table 8. Chemical
r e a c t i o n s o f bromazepam and i t s known m e t a b o l i t e s are
shown i n Scheme 2.
H
OH
I1
""3
COOH
[I71
Scheme 2. Chemical r e a c t i o n s of bromazepam and i t s known m e t a bolites.
BROMAZEPAM
33
Pharmacokinetic S t u d i e s
D i s t r i b u t i o n and e l i m i n a t i o n of bromazepam h a s been
s t u d i e d i n 4 human s u b j e c t s f o l l o w i n g s i n g l e o r a l and
i n t r a v e n o u s doses o f 6 mg 14C-bromazepam ( 3 7 ) . Absorpt i o n and d i f f u s i o n o f t h e s u b s t a n c e t a k e s p l a c e r a p i d l y ,
peak plasma l e v e l s b e i n g reached about an hour a f t e r
o r a l a d m i n i s t r a t i o n . Bromazepam i s mainly e l i m i n a t e d i n
t h e u r i n e , and, u n l i k e most o t h e r benzodiazepines, excret i o n f o l l o w s a r e g u l a r , r e c t i l i n e a r p a t t e r n which i s
p r a c t i c a l l y completed a f t e r 120 hours. E x c r e t i o n o f t h e
m e t a b o l i t e s c l o s e l y p a r a l l e l s t h a t of t h e unchanged
s u b s t a n c e . H a l f - l i f e f o r t h e e l i m i n a t i o n of bromazepam
from t h e plasma is 2 0 . 1 hours following 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 , compared w i t h 22.5 h o u r s f o r t o t a l r a t i o a c t i v i t y (unchanged bromazepam p l u s m e t a b o l i t e s ) . F i g .
1 2 and F i g . 13 f o r i n t r a v e n o u s and o r a l a d m i n i s t r a t i o n
a r e p r a c t i c a l l y i d e n t i c a l . Binding o f bromazepam by
plasma p r o t e i n s w a s s t u d i e d by e q u i l i b r a t i o n d i a l y s i s ,
which showed t h a t about 70% of t h e substance p r e s e n t i s
bound. T h i s i s c o n s i d e r a b l y l e s s t h a n f o r diazepam and
most o t h e r benzodiazepines, and i s a t t r i b u t e d t o t h e
i n c r e a s e d p o l a r i t y of t h e molecule due t o t h e p r e s e n c e o f
t h e pyridyl radical (38).
Another work d e s c r i b e d t h e a p p l i c a t i o n of gas l i q u i d
chromatography f o r pharmacokinetic s t u d i e s i n man ( 3 9 ) .
The a s s a y can b e used a l s o for r o u t i n e plasma l e v e l
monitoring. The important pharmacokinetic parameters
have been c a l c u l a t e d from t h e plasma c o n c e n t r a t i o n .
= e l i m i n a t i o n h a l f - l i f e = 1 8 . 5 hours, C1 = t o t a l
T 2 (B)
body plasma c l e a r a n c e = 347 m l / m i n and VdR = a p p a r e n t
volume of d i s t r i b u t i o n = O.gl/kgm following s i n g l e o r a l
However, u s i n g m u l t i p l e dosing w i t h
dose o f 6 mg/day.
~
1 ( B ) = 1 9 . 5 hours
3 mg/day, it w a s found t h a t T 2
243 ml/min and Vdg = 071/kg. Kaplan e t a l , 1976 (40)

have r e p o r t e d plasma c o n c e n t r a t i o n s and t i m e p r o f i l e s
which i n agreement w i t h t h e above mentioned work.
34
Fig. 12
-Bromazepam
.....,
Fig. 13
Bromazepam + metabolites
Fig. 12.
14C-Bromazepam and total radioactivity (ug/ml) in

plasma after I.V. injection o f 6.0 mg.
Fig. 13.
14C-Bromazepam in plasma (pg/ml) after oral

administration of 6.0 mg.
BROMAZEPAM
8.
Methods of Analysis
8.1 Elemental h a l y s i s
The elemental composition of bromazepam i s
Element
% Theoretical
53.18
8.2
3-19
Br
25.28
13 * 29
5.06
8.2.1
Color Reactions
Reported c o l o r r e a c t i o n s for bromazepam a r e
as follows ( 4 1 ) :
Diazocoupling
React i o n
with 1,3dinitrobenzene
In
In
1 M N a O H 2 M HC1
I n DMSO
(CH3)bN OH
Bromaze-
Pam
Orange
red
Red v i o l e t
Yellow
Yellow
Orange
brown
8.2.2
Thin-Layer Chromatogram (42)

Layer
: S i l i c a g e l F254, MERCK pre-
coated p l a t e s 0.25 mm.

Mobile phase
- conc. ammonia
(100 + 1) ( p r e p a r e f r e s h l y )
without s a t u r a t i o n o f t h e
chamber atm0sphel.e.
: Ethyl a c e t a t e
MABMOUD M. A. HASSAN AND MOHAMMADA. ABOUNASSIF
36
Application
: 1 0 p l of each s o l u t i o n
(sample and comparison)
Sample s o l u t i o n : Shake 400 mg

of t a b l e t powder w i t h 5 ml
of chloroform for approx.
3 min and f i l t e r .
Bromazepam comparison solut i o n : Dissolve 60 mg of
bromazepam i n 25 m l of
chloroform.
Front d i s t a n c e : 1 2 cm
Detect i o n
: 1. Observe under short-wave
W-light:
decrease o f t h e
fluorescence through bromazepam.
2. Spray t h e d r i e d p l a t e w i t h
a f r e s h l y prepared s o l u t i o n
of f e r r o u s s u l f a t e , d r y f o r
a s h o r t moment and spray
t h e n w i t h an ammonia (1%):
Bromazepam appears as a
v i o l e t spot.
Rf-value
: Bromazepam approx.
0.6
Ferrous s u l f a t e
: Dissolve 1 g of FeS04.
7H20
solution
i n 1 0 0 m l of d i s t . water.
8.2.3
Microcrystal T e s t s
Bromazepam w a s i d e n t i f i e d i n s o l u t i o n by
m i c r o c r y s t a l t e s t s i n microgram q u a n t i t i e s
(43) *
A micro drop of t h e t e s t s o l u t i o n ( i n 50%
e t h a n o l ) w a s placed on t h e g l a s s cover s l i p
and a micro drop of t h e reagent was added.
The drop being s t i r r e d w i t h a s l i g h t s c r a t c h i n g
of t h e g l a s s t o promote t h e formation of
c r y s t a l s . The appearance of t h e c r y s t a l s w a s
recorded as follows:
1. With potassium cadmium i o d i d e , c r y s t a l s
i n t h e form of s m a l l t a b l e t s were obtained
a f t e r one hour ( F i g . 14a).
37
BROMAZEPAM
2.
With potassium mercuric i o d i d e , c r y s t a l s

i n t h e form of round t r i f e r c a t e b l a c k i s h
s t r u c t u r e s , white on black back ground
were formed a f t e r one hour ( F i g . 1 4 b ) .
3.
With gold bromide hydrochloride, c r y s t a l s

i n t h e form of flowers of d e n d r i t e s were
obtained i n s t a n t l y ( F i g . 14c )
The s e n s i t i v i t i e s o f t h e t e s t s were 0 . 2 , 0.5

and 0 . 1 microgram r e s p e c t i v e l y .
8.3
Titrimetric
8.3.1
Non-aqueous T i t r a t i o n
Among s e v e r a l 1,h-benzodiazepine d e r i v a t i v e s ,
bromazepam w a s t i t r a t e d with p e r c h l o r i c a c i d
i n dioxane or tetrabutylammonium hydroxide i n
benzene-methanol ( 4 3 ) .
8.4
Spectrophotometric
Unlike o t h e r 1,b-benzodiazepines, t h e e x i s t e n c e of
t h e a, a ' - d i p y r i d y l t y p e moiety enables bromazepam
t o form complexes w i t h d i v a l e n t metal i o n s . Sabatino
-e t a 1 ( 4 5 ) reported t h a t f e r r o u s i o n s form purple
complexes with p y r i d y l benzodiazepin-2-ones; compounds having a b a s i c d i p y r i d y l t y p e bond s t r u c t u r e
which has e x c e l l e n t metal complexing p r o p e r t i e s .
-N
\\c -
HN-
The method has been a p p l i e d ( 4 6 ) t o t h e determination

o f bromazepam i n b i o l o g i c a l m a t e r i a l s by adding
methanol s o l u t i o n of f e r r o u s c h l o r i d e t o t h e bromazepam obtained a f t e r e x t r a c t i o n from blood o r u r i n e and
measuring t h e absorbance of t h e s o l u t i o n a t 580 nm.
The method proved t o be s p e c i f i c for bromazepam w i t h
no i n t e r f e r e n c e from 2-amino-5-bromobenzoylpyridine,
one of i t s p r i n c i p a l m e t a b o l i t e s .
The procedure w a s a l s o a p p l i e d t o t h e determination
o f bromazepam i n r a t blood ( 4 7 ) .
Fig. 14a
Fig. 14b
Fig. 14c
Fig. 14a. Microcrystals of bromazepam with potassium cadmium iodide.

Fig. 14b.
Microcrystals of bromazepam with potassium mercuric iodide.
Fig. 14c.
Microcrystals of bromazepam with gold bromide hydrochloride.
BROMAZEPAM
39
Smyth e t a1 (48) s t u d i e d t h e c h e l a t i o n o f bromazepam

w i t h F e ( I I ) , C u ( I I ) , and Co(I1) u s i n g s p e c t r o s c o p i c
t e c h n i q u e s such as W - v i s . spectrophotometry.
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 bromazepam, as w e l l as
some o t h e r benzodiazepines, w a s a l s o performed a f t e r
r e a c t i o n w i t h a modified Marquis's r e a g e n t ( 4 9 ) .
pKa v a l u e s and mechanism o f h y d r o l y s i s were a l s o
r e p o r t e d u s i n g W spectrophotometry ( 3 ) .
Another W-spectrophotometric method used by F.
Hoffnann - L a Roche & Co Limited i s as f o l l o w s : ( 5 0 )
Assay: The d e t e r m i n a t i o n should b e c a r r i e d o u t
w i t h i n one hour under subdued d a y - l i g h t .
P u l v e r i z e 20 t a b l e t s and a c c u r e t e l y weigh approximat e l y 1000 mg of t a b l e t powder i n t o a 250 ml-volumetric
f l a s k . Add about 200 m l of 0.1 N methanolic s u l f u r i c
a c i d and shake f o r 1 5 min o r t r e a t w i t h u l t r a s o n i c s .
D i l u t e t o volume w i t h 0 . 1 N methanolic s u l f u r i c a c i d .
F i l t e r a part of t h i s solution (discard t h e first
1 5 m l ) and d i l u t e 10.0 m l o f t h e c l e a r f i l t r a t e t o
100.0 m l w i t h 0 . 1 N methanolic s u l f u r i c a c i d
(= test solution).
Method. o f Analysis: With a spectrophotometer measure
t h e e x t i n c t i o n o f t h e t e s t s o l u t i o n a t 285 nm (max.)
a g a i n s t 0 . 1 N methanolic s u l f u r i c a c i d as a b l a n k
u s i n g 1 em-quartz c e l l s .
Calculation:
mg of bromazepam t a b l e t . =
E. 25000
300
.B
.A
E = e x t i n c t i o n o f t h e sample s o l u t i o n
A = average weight (mg)
B = weight of sample (mg)

300 = E ( 1 % , 1 cm)-value of bromazepam a t 285 nm
(maximum) i n 0.1 N methanolic s u l f u r i c a c i d .
Methanolic s u l f u r i c a c i d : D i l u t e 5.0 g o f 98%

s u l f u r i c a c i d t o 1000 m l w i t h methanol. P r e p a r e
t h i s s o l u t i o n a t l e a s t 24 hours b e f o r e u s e .
40
8.5
Polarographic
The ease of r e d u c t i o n of t h e azomethine ( > C = N4)
group o f bromazepam and i t s 3-hydroxy metabo i t e ,
and t h e ( > C = 0 ) carbonyl group o f i t s benzophenone m e t a b o l i t e s , promotes t h e i r q u a n t i t a t i o n i n t h e
subnanogram range by d i f f e r e n t i a l p u l s e polarography.
D e S i l v a et a1 ( 3 3 ) d e s c r i b e d a d i f f e r e n t i a l p u l s e
polarographic method f o r t h e d e t e r m i n a t i o n o f
bromazepam and i t s m e t a b o l i t e s , 3-hydroxy-bromazepam,
2-amino-3-hydroxy-5-bromobenzoylpyridine and 2-amino5-bromobenzoylpyridine i n u r i n e .
Polarography w a s c a r r i e d o u t u s i n g phosphate b u f f e r
as t h e s u p p o r t i n g e l e c t r o l y t e w i t h pH o f 5.5.
The
peak p o t e n t i a l , Ep, due t o t h e r e d u c t i o n o f t h e
azomethine group o f bromazepam and i t s 3-hydroxym e t a b o l i t e occured a t -0.535 and -0.555 V v e r s
calomel e l e c t r o d e r e s p e c t i v e l y , whereas t h e P e z due
due t o t h e r e d u c t i o n o f t h e c a r b o n y l group o f t h e
m e t a b o l i t e s 2-amino-5-bromobenzoylpyridine and
2-amino-3-hydroxy-5-bromobenzoylpyridine
occured a t -0.630 and -0.635 V v e r s u s calomel e l e c t rode, r e s p e c t i v e l y ( F i g . 15). S e n s i t i v i t y l i m i t s
ranged between 50 and 1 0 0 ng/5 ml u r i n e .
Trace l e v e l s of bromazepam i n blood w a s determined
by d i f f e r e n t i a l p u l s e polarography ( 5 1 ) . The
c u r r e n t r e s u l t i n g from r e d u c t i o n o f t h e 4,5-azomet h i n e bond o f bromazepam w a s measured a t -0.610 V
v e r s u s s i l v e r c h l o r i d e e l e c t r o d e . The method has
a recovery o f 62.1% 7.8 i n t h e r a n g e of 10-1000 ng
bromazepam/l ml o f blood.
Polarography w a s a l s o used t o s t u d y acid-base and

complexing behaviour o f bromazepam ( 3 ) , and f o r t h e
i d e n t i f i c a t i o n o f any one o r more of several 1 , 4 benzodiazepines i n c l u d i n g bromazepam ( 5 2 ) .
8.6 Atomic Absorption

Gonzales-Perez e t a1 ( 5 3 ) d e s c r i b e d a method f o r t h e
d e t e r m i n a t i o n o f bromazepam by atomic a b s o r p t i o n
spectrophotometry. The method i s based on t h e
e x t r a c t i o n o f t h e i o n - p a i r formed by t h e bromazepamn i c k e l (11) c a t i o n i c c h e l a t e and p e r c h l o r a t e from
aqueous t o m e t h y l i s o b u t y l k e t o n e (MIBK) s o l u t i o n s .
BROMAZEPAM
41
I
- 0.350
-0550
-0.750
POTENrlAL VOLTS VERSUSSATURATED
CAZOMELELECTROOE
Cb)
Fig. 15.
Differential pulse polarograms of: (a) I and I 1

(b) I 1 1 and IV in 1.0 M pH 5.5 phosphate buffer as
the supporting electrolyte. Key: A, control urine
blank; B, authentic standard mixture; and C,
authentic recovered from urine.
I.
111.
IV.
Bromazepam
11. 2-Amino-5-bromobenzoylpyridine
3-Hydroxymetabolite of bromazepm
2-Amino-3-hydroxy-5-brombenzoylpyridine
42
The absorbance of n i c k e l i n t h e o r g a n i c phase w a s

determined a t t h e n i c k e l resonance a 232.0 nm l i n e .
The method t h a t showed a d e t e c t i o n l i m i t o f 2 X 10-6M,
was 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 bromazepam i n
drugs.
8.7
Radioimunoassay
Robinson e t a1 (54 ) d e s c r i b e d f o u r radioimmunoassays
f o r t h e d e t e r m i n a t i o n of bromazepam and o t h e r
benzodiazepines. I n t h e most s e n s i t i v e method, t h e
antiserum from t h e Finit-tox serum benzodiazepine
a
a s s a y k i t w a s used w i t h [-H]flunitrazepam and proved
t o be s u i t a b l e f o r detecting sub-therapeutic l e v e l s
o f a l l benzodiazepines t e s t e d except one. The a s s a y
i s p a r t i c u l a r l y a p p l i c a b l e t o blood samples of
f o r e n s i c i n t e r e s t which may b e hemolyzed or decomposing, and r e q u i r e o n l y 75 m l o f blood.
A r a d i o r e c e p t o r a s s a y f o r benzodiazepines i n c l u d i n g
bromazepam i n human blood, plasma, s a l i v a and u r i n e
w a s developed ( 5 5 ) . The method i s based upon competit i o n between [ 3H]flunitrzepam and b i o l o g i c a l l y
a c t i v e benzodiazepines i n b i o l o g i c a l f l u i d s f o r
brain-specific receptors, prepared i n a s t a b l e , dry
form and easy t o handle. The method i s s p e c i f i c f o r
b i o l o g i c a l l y a c t i v e benzodiazepines. Other method,
d e s c r i b e d t h e d e t e r m i n a t i o n o f benzodiazepines i n
serum u s i n g t h e enzyme m u l t i p l i e d immuno assay-single
t e s t (EMIT-ot) drug d e t e c t i o n system ( 5 6 ) . The
method i s u s e f u l t o c l i n i c a l t o x i c o l o g y p r a c t i c e .
8.8
Chromatographic
8.8.1 Paper Chromatography

Bromazepam w a s s e p a r a t e d from o t h e r benzodiazepines by u s i n g a t h i n l a y e r system: Merck
aluminium o x i d e F254 and CHC13 - PhMe - E t O H
(40:60:2) complemented by a Whatman S.G. 81
s i l i c a g e l loaded paper system developed i n
CHCl3-EtOH (49:l). The s p o t s were l o c a t e d by
s h o r t W wavelength and a c i d i f i e d potassium
i o d o p l a t i n a t e ( 57 )
43
BROMAZEPAM
8.8.2
Thin-Layer Chromatography
Haefelfinger (58) has reported a specific
and s e n s i t i v e method for t h e d e t e r m i n a t i o n o f
bromazepam i n plasma by q u a n t i t a t i v e t h i n l a y e r chromatography. S i l i c a g e l p l a t e s were
used w i t h methylacetate-ammonia 33% (1OO:l)
as a s o l v e n t system. The q u a n t i t a t i o n o f t h e
s p o t s w a s performed e i t h e r by measuring t h e
W r e f l e c t a n c e of t h e p l a t e o r by t h e hydrolys i s o f bromazepam t o 2-amino-5-bromobenzoylp y r i d i n e on t h e p l a t e , d i a z o t i z a t i o n and
c o u p l i n g w i t h N-( 1-naphthy1)ethylenediamine
t o form an azo-dye, which was t h e n e v a l u a t e d
d e n s i t o m e t r i c a l l y . The recovery from plasma
was found t o be over 90%. Bromazepam h a s an
Rf v a l u e o f 0.6-0.65.
Thin-layer chromatographic systems and d e t e c t i o n methods d e s c r i b e d f o r t h e 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 bromazepam a r e summarized
i n Table 9 .
Table 9 .
Solvent systems and d e t e c t i o n methods f o r

br oma z epam
Detection
Support
Solvent system
S i l i c a AR
7GF-5 (twc
diment ional
TLC )
Hept a n e / e t hyl a c e t a t e/ethanol,

c o n c e n t r a t e d ammonia
( 50 : 50 :3) and : Heptane/
chloroform/ ethanol/conc e n t r a t e d ammonia (50:50:20:1)
S i l i c a g e l Chloroform/acetone (gO:lO),
G
benzene/isopropanol/ammonia
( 85 :15 :10)
S i l i c a g e l Diisopropylether-toluenee t h y l a c e t a t e methanol
di6o *254
ethylamine (70:15:10:5:2)
W-light
Bratton-Marshall,
d r a g e n d o r f f and
iodoplati n a te
reagents
Ref.
59
60
W - l i g h t (254 nm: 50
10%
w/v ~ $ 0 4
h e a t a t 120' for
1 0 minutes
chamber w i t h
n i t r o u s fumes
Spray w i t h anapht h y l e t hylmediamine R.
44
Thin l a y e r chromatographic d e t e c t i o n and

i d e n t i f i c a t i o n of bromazepam among o t h e r
lY4-benzodiazepines h a s a l s o been r e p o r t e d
(61-64).
8.8.3
Gas Chromatography
Numerous methods f o r t h e g a s chromatographic

a n a l y s i s o f bromazepam i n b i o l o g i c a l f l u i d s
have been r e p o r t e d . Most of t h e s e methods
d e s c r i b e d t h e d e t e r m i n a t i o n o f t h e compound
e i t h e r as t h e i n t a c t 1,b-benzodiazepine-2-0ne
o r as t h e hydrolyzed 2-amino-5-bromobenzoylp y r i d i n e (ABBP) (33, 65-69). Bromazepam h a s
a l s o been assayed as t h e "-methyl
derivative
(70).
The presence o f e l e c t r o n e g a t i v e f u n c t i o n a l
groups ( t h e halogen i n t h e 7 - p o s i t i o n and t h e
2-carbonyl) r e n d e r s bromazepam amenable t o
s e n s i t i v e d e t e r m i n a t i o n by e l e c t r o n - c a p t u r e
g a s - l i q u i d chromatography (EC-GLC); t h e r e f o r e
a l l ( e x c e p t one) t h e above mentioned methods
a p p l i e d (EC-GLC) t o t h e d e t e r m i n a t i o n o f t h e
drug. I n p r i n c i p l e , t h e s e methods are c a p a b l e
of d e t e c t i n g nanogram amounts o f bromazepam
i n b i o l o g i c a l f l u i d s where t h e y compete w i t h
radioimmunoassays. Recently, it h a s been
coupled w i t h mass spectrometry (71). The
experimental c o n d i t i o n s used f o r t h e a n a l y s i s
o f t h e drug i n b i o l o g i c a l f l u i d s a r e
summarized i n Table ( 1 0 ) .
Table 10. Gas liquid chromatographic conditions f o r the determination of bromazepam and its
metabolites
Drug o r metabolite
Column packing
Carrier
2-Amino-5-bromo-benzoylpyridine (ABBP)
2% Carbowax 20M-TPA on silanized

Gas ChromP (100-120 mesh)
2-Amino-5-bromo-benzoylpyridine (ABBP)
3% OV 225 (methyl-phenylcyanopropyl. Ar-cH4

silicone) on Gas Chrom Q ( 6 0 / 8 0 Rest (9O:lO)
Bromazepam
Bromazepam
Column
temp
)etector
. RT
ns
-
20
mi
2e:
-
6-7
EC
65
12.5
EC
66
3% OV-17 on Gas Chrom Q (60/80mesh) Ar-CH4

245 O
(90:lO)
EC
33
3%
240
9.6
EC
67
265"
FI
68
270
3-4
EC
70
3-4
EC
69
OV-17
220
N2
on Gas Chrom Q (60/80mesh) Ar-CH4

( 90-10
214O
Bromazepam
10% UCC-W-98 on Chromosorb W AW DMC:

(80/100mesh)
N2
N'-Methylbromazepam
0.5% OV-17 Chromosorb G HP (100/200

mesh)
N2
4% SE-52 silica capillary column
He
Programmed
130-260'
20% UCC-W on Chromosorb W AW DMCS

(80/100mesh)
He
ProgramMass
71
med
spectro100-310 - meter
-
Ar = Air
46
8.8.4
High P r e s s u r e Liquid Chromatography

A high p r e s s u r e l i q u i d chromatographic procedure f o r t h e d e t e r m i n a t i o n o f bromazepam i n
human plasma has been d e s c r i b e d ( 7 2 ) . A f t e r
e x t r a c t i o n from plasma, t h e e x t r a c t w a s evaporate& and t h e r e s i d u e w a s suspended i n
acetonitrile/tetrabutylammonium hydroxide
(60:40,v / v ) b e f o r e i n j e c t i o n i n t o t h e
chromatograph. A n a l y s i s w a s performed on a
1-1 Bondpack c18 column w i t h mobile phase made
from 20 m l MeOH, 30 m l MeCN, and T O O ml of
s o l u t i o n c o n t a i n i n g 20 ml 10% t e t r a b u t y l ammonium hydroxide i n 1 L water. The i n t e r n a l s t a n d a r d w a s carbamazepine and d e t e c t i o n
w a s by W spectroscopy.
I n o r d e r t o determine t h e lower t h e r a p e u t i c
range of drug l e v e l s i n serum (73 ) , bromazepam,
among o t h e r b a s i c drug, w a s chromatographed
on an ~ ~ Micropack
1 8
MCH 1 0 column u s i n g UV
d e t e c t o r and perchlorate/MeCN s o l u t i o n as
eluent.
Hochmuth et a1 (74) d e s c r i b e d a simple and
r a p i d method f o r t h e t o x i c o l o g i c a l a n a l y s i s
of some benzodiazepines and a n t i d e p r e s s a n t s
i n c l u d i n g bromazepam. A f t e r s o l v e n t e x t r a c t i o n a t pH 9 , t h e a n a l y s i s w a s performed by
HPLC on a reverse-phase column w i t h MeCN 0.6%
KH PO (1:l) a d j u s t e d t o pH 3.
2 4
Heizmann et a1 ( 7 5 ) r e p o r t e d a HPLC method
f o r t h e d e t e r m i n a t i o n o f bromazepam i n plasma
and of i t s main m e t a b o l i t e s i n u r i n e . The
unchanged drug was e x t r a c t e d from plasma w i t h
dichloromethane, and t h e r e s i d u e was subseq u e n t l y analysed by r e v e r s e d phase HPLC w i t h
W d e t e c t i o n (230 n m ) . For t h e d e t e r m i n a t i o n
of t h e m e t a b o l i t e s , t h e u r i n e samples were
incubated t o e f f e c t enzymatic deconjugat i o n
b e f o r e e x t r a c t i o n w i t h dichloromethane. The
column used w a s a 15 cm S u p e l c o s i l LC18 and
t h e mobile phase c o n s i s t e d o f a m i x t u r e o f
methanol/phosphate b u f f e r , pH 7 . 5 , a t a flow
r a t e of 1 ml/min. The r e t e n t i o n times r a n g i n g
from 6 t o 20 minutes.
BROMAZEPAM
9.
41
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Courvoisier, ArzneimittelForsch., 24, 1841 (1974).
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Pharmakol,
39. U.
40.
809 (1978).
and U. W o l l e r t , Naunya-Schmiedeberg's, Arch.
278, 301 (1973).
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222,
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Weissmann and S . C u t l e r , J . Pharmacokin. Biopharm.,
1 (1976).
4,
41. K.A. Kovar and

(1983).
42. F.
D. Linden, Pharm. Acta Helv.,
58, 66
Hoffmann
La Roche & Co. Ltd., C o n t r o l Department,
B a s l e , S w i t z e r l a n d ( P r i v a t e Communication) (1986).
43.
S.N.
T e w a r i and S.K. Shukla, Pharmazie,
44. F.
33, 372 (1978).
Barbato, C . Grieco, C. C i l i p o and A. V i t t o r i a ,

Farmaco, Ed. P r a t &, 187 (1979).
.,
45. J.D.
S a b a t i n o , O.W. Weber, G.R. Padmanobhan, and

Senkowski, Anal. Chem.,
905 (1969).
41,
46. N. G a l l o , V.D.
497 (1980).
Bianco and S. Doronzo, I L Farmaco,
B.Z.
35,
3, 3678 (1980).
47.
M. Fukumoto, Chem. Pharm. B u l l ,
48.
W.F. Smyth, R. S c a n n e l l , T.K. Goggin D. Lucas-Hernandez,

321 (1982).
A n a l y t i c a Chimica Acta,
49.
H.M.
141,
S t e v e n s , J. F o r e s i c S c i . Soc.,
18,69 (1978).
50
50.
S p e c i f i c a t i o n s for Lexotan "Roche" , C o n t r o l Department ,

Hoffhann
La Roche & Co Ltd. , Basle, S w i t z e r l a n d
(1975)
Brooks and M.R. Hackman, Anal. Chem.,
47,
51.
M.A.
52.
W.F. Smyth, M.R. Smyth, J.A.

Analyst , 103,497 (1978).
53.
C. Congalez-Perez, J . Hernandez-Mendez and M . I .

Martin, I L Farmaco, Ed. P r a t . , 3, 383 ( 1 9 8 3 ) .
54.
K. Robinson, M. R u t t e r f o r d , R.N.
Pharmacol. , 32, 773 ( 1 9 8 0 ) .
55.
J. Lund, Scand. J. Clin. Lab. I n v e s t . ,
56.
R.H. D r o s t , T.A. Plomp and R.A.A.

Clin. Toxicol. , 9,
303 (1982).
57.
(1975).
H.M. Stevens and R.W.

11, 183 (1971).
2059
Groves and S.B. Tan,

Gonzalez-
Smith, J. Pharm.
41,
275 (1981).
Maes, J. Toxicol.
J e n k i n s , F o r e n s i c , S c i . SOC., J.
11,1 0
(1978).
58.
P. H a e f e l f i n g e r , Chromatographia,
59.
M.A. Schwartz, W.R. Pool D.L. Hane and E. P o s t n a , Drug

Metabolism and D i s p o s i t i o n , 2 , 31 (1974).
60.
H. Schuetz, F r e s e n i u s ' Z. a n a l . Chem.,
61.
L. L a r i n i , P.E.T. Salgado, T.C. Ralpho and M.G.

An. Farm. Quim. Sao Paulo, 17,37 (1977).
62.
P. F i c a r r a , M.G. V i g o r i t a and A. Tomanapini, B o l l . Chim.

Pharm. ,
720 (1977).
63.
D. Dekker and P.M.C.

( 1978 1
64.
A . Hulshoff and J.H. P e r r i n , J . Chromatogr.,

( 1976 1.
65.
J.A.
66.
J.P.
294,
135 (1979).
Vilela,
116,
, 118,465
D e S i l v a and J. Kaplan, J . Pharm. S c i ,
(1966).
1012
P a r e l , Pharm. Weekbl.
Cano, A.M.
(1975).
129,
55,
263
1278
Baile and A . Viala, Arzneim. Forsch,
25
51
BROMAZEPAM
67.
J . A . D e S i l v a , I. Bekersky, V.C. P u g l i s i , M.A. Brooks

and R.E. W e i n f i e l d , Anal. Chem. , 48, 1 0 (1976).
68.
T. Kaniewska and W. Wejman, J . Chromatogr.,

( 19801
69. J.M.F.
Douse, J. Chromatogr.,
182, 81
301, 137 (1984).
222,
501 (1981).
70.
U. K l o t z , J . Chromatogr.,
71.
H. Maurer and K. P f l e g e r , J. Chromatogr.,
72.
H. Hirayama, Y . Kasuya and T. Suga, J . Chromatogr.,
(1981).
222, 409
414 (1983).
73. H. K a e f e r s t e i n
95 (1983).
74.
75.
and G. S t i c h t , B e i t r . G e r i c h t l . Med.,
277
41,
P. Hochmuth, J.L. R o b e r t , H . Schreck and R. Wennig,

Anal. Chem. Syrup. S e r . , 20, 143 (1984).
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310, 129 (1984).
Acknowledgement
The a u t h o r s would l i k e t o t h a n k M r . Tanvir A. B u t t , College
of Pharmacy, King Saud U n i v e r s i t y , for t y p i n g o f t h i s
manuscript
BUSULPHAN
Muhammad T d q " and AbduReah. A . Al-Ba&**
*U~pumincnX06 P h a c o L o g y and "Vepahtment 06

PhmacclLticd CCZmhfiy, CoReegc 06 P h m a c y , King Saud
UniuemLty, Riyadh, Saudi h u b & .

VOLUME 16
53
A11 rights of reproduction in any form reserved.
MOHAMMADTARIQ AND ABDULLAH A. AL-BADR
54
1.
Foreword
2.
Description
2.1
2.2
2.3
2.4
2.5
3.
Nomenclature
Formulae
Molecular Weight
Elemental Composition
Appearance, Color and Odor
Physical Properties
3.1
3.2
3.3
3.4
Melting Point
Solubility
Storage
Spectral Properties
4.
Synthesis
5.
Pharmacokinetics
6.
Toxicology
7.
Methods of Analysis
7.1
7.2
7.3
7.4
Identification
Titrimetric Methods
Chromatographic Methods
Nuclear Magnetic Resonance Methods
References
BUSULPHAN
1.
55
Foreword
Busulphanis a chemotherapeutic agent, which has been
widely used as antineoplastic drug since its discovery
in 1953 (1). This alkylating agent belongs to the
series of alkanesulfonic acid esters, possessing
significant cytotoxic activity and is the drug of
preference in the treatment of chronic myelocytic or
granulocytic leukaemia. It has recently been proposed
as a drug of choice in polycythaemia and thrombocythemia
(2-13). Busulphanhas also been used for the treatment of
bronchial carcinoma (18-19) , myasthenia gravis (20)
and chronic granulomatous disease in children (21).
The drug is also used as reproductive sterilant for
insect (22-23) and as a general immunosuppressant for
organ transplant in the treatment of autoimmune
disease (24-26).
2.
Description
2.1
Nomenclature
2.1.1
Chemical Names
1,4-Butanediol dimethanesulfonate;
1,4-Bis (methanesulf0noxy)butane;
1,4-di(methanesulfonyloxy)butane;
1,4-di(methylsulfonoxy)butane;
1,4-Butanediylbismethane sulphonate;
Methanesulfonic acid tetramethylene ester;

Tetramethylene bis (methanesulfonate) (27-29)
2.1.2
Generic Name
Busulphan, CB 2041, GT 41, NSC 750, WR 19508
Busulfanum (29).
2.1.3
Trade Names
Myleran, Misulban, Mitosan, Myelosan,
Myeleukon, Myeloleukon, Sulfabutin,
Mielucin (27).
MOHAMMAD TARIQ AND ABDULLAH A. AL-BADR
56
2.2.2
Structural
0
I1
II
H C-S-O-CH2-CH2-CH2-CH2-O-S-CH3
3 It
II
2.2.3
CAS No.
[55-98-17 (27)
2.3
Molecular Weight
246.31
2.4
(31)
C, 29.26%; H, 5.73%;
2.5
0 , 38.98%;
S,
26.03%
(27).

A white almost odorless crystalline powder (29, 32).
3.
Physical Properties
3.1
Melting Point
114-118O,
3.2
119O, 116'.
(27).
Solubility
Soluble, at 20, in 750 parts of water (in which
it is slowly hydrolyzed) and in 25 parts of
acetone; very slightly soluble in alcohol (27, 32).
3.3
Storage
Busulphan should be kept in a well-closed container,
protect from light (30).
BUSULPHAN
3.4
Spectral Properties
3.4.1
Ultraviolet Spectrum
The ultraviolet spectrum of busulphan in
methanol, ethanol and in water was scanned
from 200 to 400 nm using Varian DMS 90
Spectrophotometer. No absorbance has been
noticed
3.4.2
Infrared Spectrum
The infrared spectrum of busulphan as KBr
disc is shown in Figure 1. It was recorded
on a Pye-Unicam SP 1025 infrared
spectrophotometer. The assignments of the
characteristic bands in the infrared
spectrum is shown below:
-1
3.4.3
Frequency cm
Assignments
2960-3060
CH stretch
1430-1415
-CH2-0 vibration
1352
S ( = 0 > 2 stretch (Assymm.)
1180
S ( = 0)2 stretch (Symm.)
1000-770
S-0-C
stretch
1
Proton Nuclear Magnetic Resonance ( H NMR)

Spectrum
The 1H NMR spectrum of Busulphan
Figure 2. The drug is dissolved
and its spectrum determined on a
T60 A NMR spectrometer using TMS
internal standard.
is shown in
in DMSO-db
Varian as the
Assignment of the chemical shifts to the

different protons is shown below:
Wavelenqth urn
3500
3ooo 2500 zoo0
1800
1600
1400
1200
Wavenumber
Figure 1 : Infrared spectrum of Busulphan, KBr disc.
loo0
800
Jo
625
"
'
'
500
400
'
'
300
6 !
5
4
3
2
CH3-~-O-CH2-CHj-CH~CH,-0
0
'
"
1 . "
"
"
"
'
-S- CH3
i;
ch
1
-
TM:
. . . .
. . . .
8.0
7.0
6.O
Figure 2 : Proton nuclear magnetic Resonance spectrum of Busulphan
DMSO -dg
using TMS as reference standard
in
60
Chemical
shift (6)
Multiplicity
Proton
assignment*
1.68-1.90
Multiplet
3 and 4 (Cg2)
3.10
Sing1et
1 and 6 (Cg3)
4.1-4.40
Mu1tiplet
2 and 5 (Cl12)
*Please refer to Figure 2.

3.4.4
Carbon-13 Nuclear Magnetic Resonance Spectra

(C-13 NMR)
The carbon-13 NMR spectra of busulphan in
DMSO-d6 using TMS as an internal reference

are obtained using a Jeol FX 100 MHz
spectrometer at an ambient temperature.
Figure 3 and 4 represent the proton-decoupled
and off-resonance spectra respectively.
0
6 11
5 4 3 2
II
CH3-S-0-CH CH CH CH -0-S
iI
2 2 2 2
11
The
the
the
are
1
- CH3
carbon chemical shifts are assigned on

basis of the chemical shift theory and
off-resonance splitting pattern and
shown below:
Chemical
shift (6)
Multiplicity
Carbon
assignments
24.70
Triplet
3 and 4
36.52
Quartet
1 and 6
69.40
Triplet
2 and 5
Figure 3
: Proton - decoupled carbon - I3 nuclear magnetic resonance spectrum

of Busulphan in DMSO- dgusing TMS as reference standard.
Ill
Figure 4
: Off-
resonance carbon-13 nuclear magnetic resonance spectrum

of Busulphan in DMSO-dG using TMS as reference standard.
BUSULPHAN
63
3.4.5
Mass Spectrum
The electron impact (EI) mass spectrum of
busulphan at 70 eV recorded on Varian
Mat 311 mass spectrometer and the direct
chemical ionization (DCI) mass spectrum
obtained with Finnigan 4000 mass spectrometer
are shown in figures 5 and 6 respectively.
The (EI) spectrum (Figure 5) shows a base
peak at m/e 7 1 , the molecularion peak was
not detected. Other major fragment are at
m/e 175, 122, 111, 109, 97, 79, 55 and 42.
A proposed mechanism of fragmentation of
the EI fragments is shown in scheme 1.
The CI spectrum (Figure 6 ) is simple and
shows a base peak at m/e 151 and at
molecular ion peak at 247 corresponding
to (M + 1) and minor peak at m/e 125.
4. Synthesis
By esterifying 1,4-butanediol [HOCH CH CH CH OH]
2 2 2 2
with methanesulfonyl chloride [CH SO2C1] in the
3
presence of pyridine ( 3 1 , 33) as shown below:
0
II
CH3-S-C1
II
OH-CH2CH2CH2CH2-OH
0
0
II
II
CH3-S-O-CH2CH2CH2CH2-O-E-CH3
11
Pyridine
c
r
64
-29568
175
150
200
2 50
300
Electron impact (El) mass spectrum of Busulphan.
100
Figure 5
100
150
200
250
300
350
Figure 6:Ckmical imitation(CI1 mass spectrum of Busulphan.
+OX
=--a
'c:
0
m
X
V
,m-0
+o- I
c'
o=
0
I
m=o
Xm
1
m
+o=(.=
-51
co
m
I
I
+o= m=
c
re:
..
.rl
CH3
,+
;
OH
iI
(4*7J;-m3
m/e 246
OH
OH
m / e 175
Scheme 1:
Continued.
m/e 71
1.
I
m
+O=T=O
67
T;
I
II
--m=
51
0 -m=o
Im
T;
+O
..
4
68
5.
Pharmacokinetics
Absorption, Metabolism and E x c r e t i o n
Busulphan i s r e a d i l y absorbed from t h e g a s t r o i n t e s t i n a l t r a c t and r a p i d l y d i s a p p e a r s from t h e
blood.
I t i s l a r g e l y e x c r e t e d i n t h e u r i n e as
s u l p h u r - c o n t a i n i n g m e t a b o l i t e ( 2 9 ) . Due t o i t s
a l k y l a t i n g a c t i v i t y , t h e compound might be bound
b o t h r e v e r s i b l y and i r r e v e r s i b l y ( c o v a l e n t l y ) t o
blood component (34). The metabolism t a k e s p l a c e
mainly i n t h e l i v e r . The c l i n i c a l r e s p o n s e
u s u a l l y b e g i n s w i t h i n 1 t o 2 weeks a f t e r i n i t i a t i o n
of t h e r a p y . It a p p e a r s t o be p r a c t i c a l l y c o m p l e t e l y
e x c r e t e d i n t h e u r i n e as methane-sulphonic a c i d
(35)
Nadkarni -e t a1 ( 4 ) u s i n g 14C and 35S-labeled
busulphan found t h a t , a f t e r i n t r a v e n o u s d o s i n g ,
t h e r a d i o a c t i v i t y i n t h e blood r a p i d l y d e c r e a s e d ,
and between 20% and 95% was removed w i t h i n 3 t o
5 minutes. A f t e r o r a l d o s i n g an i n i t i a l l o g
phase of about 0.5 t o 2 h o u r s was observed b e f o r e
measureable blood l e v e l s were achieved. A f t e r
o r a l 3H-labeled busulphan, Vodopick et a l , (36)
found a prompt r i s e i n t h e plasma l e v e l of
r a d i o a c t i v i t y , which i s peaked w i t h i n t h e f i r s t
h o u r , followed by a r a p i d f a l l and t h e n a g r a d u a l
r i s e i n r a d i o a c t i v i t y . Busulphan i s e l i m i n a t e d
w i t h t$ of about 2.5 h o u r , f o r t h e i n i t i a l p h a s e
of r a d i o l a b e l e d d r u g i n plasma. Only about 1%
of a dose of busulphan i s e x c r e t e d as unchanged
drug w i t h i n 2 4 h o u r s . The major p o r t i o n of
busulphan i s probably e l i m i n a t e d by enzymatic
a c t i v i t y r a t h e r t h a n pure chemical d e g r a d a t i o n
(36).
Ehrsson e t a1 (37) have s t u d i e d t h e busulphan
k i n e t i c s and r e p o r t e d t h a t k i n e t i c s were followed
i n p a t i e n t w i t h c h r o n i c m y e l o c y t i c leukemia a f t e r
o r a l dose of 2.4 t o 6 mg. The plasma c o n c e n t r a t i o n t i m e d a t a c o u l d be f i t t e d t o a zero-order a b s o r p t i o n
one-component open model. The e l i m i n a t i o n rate
c o n s t a n t averaged 0.27 ? 0.05 hr-1 (SD). The plasma
AUC w a s l i n e a r l y r e l a t e d t o t h e d o s e . The l o g
t i m e f o r t h e s t a r t of a b s o r p t i o n , t h e t i m e absorpt i o n ends, and t h e a b s o r p t i o n rate c o n s t a n t showed
BUSULPHAN
69
some inter-individual variation. About 1% of

the drug is excreted unchanged in urine over
24 hours.
The fate of the drug in man has been studied
after the administration of radiolabelled drug
followed by measurement of total radioactivity in
plasma and urine (4, 36, 37).
The drug is rapidly eleminated from the plasma and
is reported to be extensively metabolized; twelve
metabolites have been isolated, including
methane-sulphonic acid and 3-hydroxytetrahydrothiophene-1,l-dioxide, most of the metabolites
have not been identified (38).
Wiygul et a1 (39) have studied metabolism of
busulphan in the boll weevil. When one-day male
boll weevils were force-fed 3H- and Ik-labelled
busulphan, 34.34% of total radioactivity was
recovered from the feces, about 1.09% as busulphan,
at 72 hours after injection. Most metabolism of
busulphan took place within 24 hours postingestion.
1,4-Butanediol, 2,3-butanediol, sulfolanes, malic
acid, malonic acid, succinic acid, aminoacids,
and methanessulphonic acid were identified as
principal metabolites.
Jones and Campbell (40) have studied the metabolism,
reactions and biological activities of some
cyclic dimethanesulphonates. Relevance to the
mechanism of action of busulphan (Myleran). The
results obtained suggest that cycloalkylation of
thiol groups by busulphan may represent a normal
detoxification route rather than a mechanism of
action.
Other studies on the metabolism of busulphan in b o l l
weevils, are also reported (41, 42, 43).
6. Toxicity
Recently Bishop and Wassom (44) have published a
detailed review article on the toxicity of busulphan
incorporating around 290 references. The most important
side effect of busulphan in high doses are thrombocytopenia and damage to haemotological tissues (45,53, 2 9 ) .
70
I n t e r s t e t i a l pulmonary f i b r o s i s , known as "Busulphan

lung", i s as w e l l r e c o p i s e d s i d e e f f e c t busulphan
t h e r a p y (54-64).
A w a s t i n g o r Addison - l i k e syndrome
has o c c u r r e d i n some p a t i e n t s r e c e i v i n g l o n g term
t h e r a p y w i t h Busulphan (59, 65, 6 6 ) . Other a d v e r s e
e f f e c t s include endocardia1 f i b r o s i s (67), c a t a r a c t s
( 6 8 ) ) c e l l u l a r d y s p l a s i a of pancreas ( 6 9 ) , a d r e n a l
i n s u f f i c i e n c y (70) and bone marrow damage (71-83)
Laboratory s t u d i e s i n animals have shown s i g n i f i c a n t
immunosuppressive ( 4 8 , 81,) 85, 8 2 , 86) and r e p r o d u c t i v e
t o x i c i t y of busulphan (87 - 1 0 0 ) .
Busulphan, a d m i n i s t e r e d d u r i n g t h e t h i r d trimester of
pregnancy, can a d v e r s e l y a f f e c t f e t a l growth (101).
T e r a t o g e n i c e f f e c t of t h i s drug i n animals h a s been
r e p o r t e d by s e v e r a l workers (102 - 1 0 6 ) . Busulphan i s
p o t e n t i a l l y mutagenic (104
114) and c a r c i n o g e n i c i n
man (115
123).
7.
Methods of A n a l y s i s
7.1
Identification
The USP XX (1980) ( 3 2 ) d e s c r i b e s t h e f o l l o w i n g
i d e n t i f i c a t i o n t e s t s f o r busulphan.
A)
Fuse about 100 mg w i t h about 100 mg of p o t a s s i u m

n i t r a t e and a p e l l e t of potassium hydroxide
weighing approximately 250 mg. Cool, d i s s o l v e
t h e r e s i d u e i n water, a c i d i f y w i t h d i l u t e d
h y d r o c h l o r i c a c i d , and add a few d r o n s of
barium c h l o r i d e TS; a w h i t e p r e c i p i t a t e i s
formed.
B)
To 100 mg add 10 m l of water and 5 m l of 1 N

sodium hydroxide. Heat u n t i l a c l e a r s o l u t i o n
i s o b t a i n e d ; an odor c h a r a c t e r i s t i c of
methanesulfonic acid i s p e r c e p t i b l e .
C)
Cool t h e s o l u t i o n o b t a i n e d i n I d e n t i f i c a t i o n
Test B , and d i v i d e i t i n t o two e q u a l p o r t i o n s .
To one p o r t i o n add 1 d r o p of potassium
permanganate TS; t h e p u r p l e c o l o r changes t o
v i o l e t , t h e n t o b l u e , and f i n a l l y t o emeraldgreen. A c i d i f y t h e second p o r t i o n of t h e s o l u t i o n w i t h d i l u t e d s u l f u r i c a c i d , and add 1 drop
BUSULPHAN
71
of potassium permanganate TS: t h e c o l o r of

t h e permanganate i s n o t d i s c h a r g e d .
B r i t i s h Pharmacopoeia (1980) (30) h a s
a l s o r e p o r t e d similar procedures f o r t h e
i d e n t i f i c a t i o n of busulphan.
7.2
T i t r i m e t r i c Methods
7.2.1
Aqueous
B r i t i s h Pharmacopoeia (1980) (30) d e s c r i b e d
an aqueous t i t r a t i o n procedure f o r t h e
a s s a y of busulphan a s f o l l o w s :
To 0.25 g of t h e drug add 20 m l of w a t e r and
b o i l g e n t l y under a r e f l u x condenser f o r
t h i r t y minutes. Wash t h e condenser w i t h
a small q u a n t i t y of water, c o o l and
t i t r a t e w i t h 0.1 M sodium hydroxide VS,
u s i n g p h e n o l p h t h a l e i n s o l u t i o n as i n d i c a t o r .
Each m l of 0.1 M sodium hydroxide VS i s
e q u i v a l e n t t o 0.01232 g of C6H1406S2.
7.2.2
Gravimetric
Busulphan t a b l e t s are s u g a r c o a t e d and
c o n t a i n o n l y m i l l i g r a m q u a n t i t i e s , hence
t h e f l a s k combustion method i s n o t
a p p l i c a b l e becuase of t h e l a r g e amount of
o r g a n i c matter. It i s n e c e s s a r y t o
e x t r a c t t h e busulphan w i t h a c e t o n e and
decompose i t i n a s e a l e d t u b e w i t h n i t r i c
acid a s follows:
T r i t u r a t e an a c c u r a t e l y weighed q u a n t i t y of
powdered t a b l e t s e q u i v a l e n t t o about 2 mg
of busulphan w i t h 10 m l of h o t a c e t o n e ,
decant t h e l i q u i d through a f i l t e r i n t o a
C a r i u s t u b e and e v a p o r a t e t o s m a l l volume
under a j e t of warm a i r . E x t r a c t i n t h e
same way w i t h two f u r t h e r 10-ml q u a n t i t i e s of
a c e t o n e , d e c a n t i n g and e v a p o r a t i n g a f t e r
each e x t r a c t i o n and e v a p o r a t i n g t o d r y n e s s
a f t e r t h e t h i r d e x t r a c t i o n . Add 0.5 m l of
fuming n i t r i c a c i d , s e a l t h e t u b e and h e a t
a t 300 f o r two hours. A f t e r opening t h e
12
Carius tube transfer its contents to a small

dish with 15 ml of water, evaporate to
dryness, dissolve the residue in water and
acidify with concentrated hydrochloric acid.
Add an excess of 10 per cent barium chloride
solution, heat on a water-bath for three
hours, filter through a platinum filtering
crucible, ignite at 800 for one hour,
cool and weigh.
Each mg of residue =
0.5276 mg of busulphan (124).
7.3
7.3.1
Gas Chromatographic Method

Hassan and Ehrrson (125) developed a gas
chromatographic method for the determination
of busulphan in plasma with electron capture
detection. The method comprise a new
derivatization technique for extraction of
busulphan from plasma in a single step
combined with high separation efficiency
and sensitivity by using capillary column
in combination with electron capture
detection. The derivatization of busulphan
was done directly in plasma by adding sodium
iodide in acetone. Plasma sample (1.0 ml)
was mixed with 0.1 ml of 1.0 ug/ml of
1,5-bis(methanesulfoxy)pentane in acetone
and sodium iodide in water (1 ml acetone
and 8 M Na' iodide). After addition of
0.4 ml n-heptane the reaction was carried
out at 7OoC for 70 minutes under stirring.
The organic phase was separated for analysis
on gas chromatography. 1-2 pl of solution
was injected into Varian 3700 GC equipped
with a constant current 63Ni lectroncapture detector. An OV-I fused silica
cappillary column (25 m x 0.3 mm i.d.) with
a film thickness of 0.52 um (HewlettPackard) was used.
The instrument was operated isothermally
with the oven, detector and injector port
temperatures at 145OC, 25OoC and 2OO0C respectively. The helium was used as carrier gaswith
a flow rate of 2 ml/min,the inlet pressure was
BUSULPHAN
73
0.4 b a r . Nitrogen was used a s make up g a s

and added through t h e hydrogen i n l e t a t a
rate of 30 ml/min i n t h e d e t e c t o r base. A
s p l i t r a t i o of 1 : 10 w a s used. A h i g h
s e p a r a t i o n e f f i c i e n c y and s e n s i t i v i t y w a s
o b t a i n e d u s i n g t h i s method, t h e r e t e n t i o n
t i m e w a s 8 minutes. The s t a n d a r d curve u s i n g
plasma w a s l i n e a r w i t h i n t h e range of
5-300 ng/ml. The p r e c i s i o n of t h i s method
t 3.9% (c.v.) a t t h e 10 ng/ml l e v e l (n = 5)
and ? 2 . 3 % (c.v.) a t t h e 100 ng/ml (n = 5 ) .
7.3.2
Gas Chromatography
Mass Spectrometry
(GC-MS)
Ehrsson and Hassan (126) developed a
chromatographic method f o r t h e determinat i o n of busulphan i n plasma by GC-MS w i t h
s e l e c t e d i o n monitoring. Busulphan
e x t r a c t e d from plasma with methylene c h l o r i d e
and converted t o 1,4-diiodobutane.
1.0 M 1
of plasma was mixed w i t h 0.1 m l of i n t e r n a l
s t a n d a r d (1,5-pentanediol d i m e t h a n e s u l f o n a t e
1.0 pg/ml) i n acetone and e x t r a c t e d w i t h 4 m l
of methylene c h l o r i d e u s i n g a mechanical
shaker (100 s t r o k e s / m i n )
The o r g a n i c phase
w a s s e p a r a t e d and evaporated t o d r y n e s s ,
d e r i v a t i z a t i o n was done by adding 0 . 1 m l
of 1 M sodium i o d i d e i n a c e t o n e w i t h a
r e a c t i o n t i m e of 20 minutes a t 7OoC.
After
a d d i t i o n 0.05 m l of n-hexane and 0.1 m l of
water t h e m i x t u r e was v i g o r o u s l y shaken.
The a l i q u o t (1-2 pg of n-hexane) w a s used
f o r t h e a n a l y s i s by GC-MS;LKB 2091 w i t h an
i o n i z i n g energy of 70 e V was used. The
column(1.5 m x 2 mm i . d . ) w a s packed w i t h
10% SP-2401 on 100-120 Supelcoport and w a s
o p e r a t e d a t 140using a helium flow r a t e of
20 ml/min. The i n j e c t o r and t h e i o n s o u r c e
temperature w e r e 230 and 27OoC, respectively.
The mininam! d e t e c t a b l e c o n c e n t r a t i o n was

5.7 x 10-16 mole/sec. The s t a n d a r d curve
w a s l i n e a r over a range of 10-400 ng/ml.
The r e l a t i v e S.D. was 2.6% a t 100 ng/ml and
? 4.3% a t 10 ng/ml (n = 5 ) .
74
7.4
NMR S p e c t r o m e t r i c Methods
a)
Truczan and Lau-Cm(l27) d e s c r i b e d a s i m p l e

NMR s p e c t r o s c o p i c method f o r t h e a s s a y of
busulphan i n t a b l e t s . 20 G r a m of busulphan
t a b l e t s are f i n e l y powdered, an a c c u r a t e l y
weighed q u a n t i t y of powder e q u i v a l e n t t o 6 mg
of busulphan i s t r a n s f e r r e d t o a 15 m l v i a l .
To t h i s 1 m l of i n t e r n a l s t a n d a r d s o l u t i o n
(11.68 mg of methenamine i n 10 m l of chloroform)
was added, t h e v i a l was c l o s e d w i t h a septum
and crimper s e a l e d w i t h an aluminium seal.
2 M I of chloroform w a s added u s i n g a s y r i n g e by
i n t r o d u c i n g t h e n e e d l e t h r o u g h septum. The
c o n t e n t s were shaked v i g o r o u s l y . The i n s o l u b l e
matter i s allowed t o s e t t l e and 0.5 t o 1 m l
of upper l a y e r i s t r a n s f e r r e d t o NMR t u b e . 1
Drop of r e f e r e n c e s t a n d a r d (50 mg t e t r a m e t h y l s i l a n e (TMS) i n 10 m l chloroform) was added,
caped and shaken. The NMR s p e c t r a were
r e c o r d e d w i t h a 200 MHz, Wise b o r e , F o u r i e r
t r a n s f o r m s p e c t r o m e t e r equipped w i t h 5 mm
p r o t o n probe and computer w i t h 24 K memory.
The s p e c t r o m e t r y c o n d i t i o n s were: ambient
t e m p e r a t u r e = 30OC, frequency 1 h = 200.06 MHz,
Observation frequency range = 1600 H2.
P u l s e width
Pulse r e p e t i t i o n t i m e
FTD d a t a p o i n t s
Observation t i m e
=
=
9.13 sec.
14 sec.
8
2.2 m i n u t e s .
I n t e g r a t e t h e broad t r i p l e t a t about 4.3 ppm

due t o t h e 4-methylene p r o t o n s (-CH2-O-S02-)
of busulphan and t h e s i n g l e t s and about 4.7 ppm
due t o t h e 12 methylene p r o t o n s of t h e i n t e r n a l
s t a n d a r d . The q u a n t i t y of busulphan i n mg i s
o b t a i n e d from:(AU/AS) x (EV./ES) x
Au
As
Ev
Es
=
=
c,
where
I n t e g r a l v a l u e of signal r e p r e s e n t i n g
bulsulphan.
I n t e g r a l v a l u e of signal r e p r e s e n t
methanamine.
Formula weight of busulphan 14, 61.58.
Formula wieght of methenamine/l2, 11.68.
Weight i n mg of m e t h e n m i n e t a k e n f o r t h e
analysis.
BUSULPHAN
b)
15
Feit and Rastrup-Andersen (128) have

investigated the hydrolysis of busulphan by
means of NMR spectroscopy. The final product
was found to be tetrahydrofuran (THF). The
rather unstable intermediate, 4-methanesulphonybutanol, was synthesized and proved
to undergo cyclization to THF by intramolecular
alkylation. The half-life of this first-order
reaction in aqueous solution at 3 7 O was
determined to be approximately 12 minutes at
pH 3 as well as at pH 7.4. From the data
obtained, it is concluded that 4-methansulphonyloxybutanol is unlikely to be responsible for
the biological action of busulphan.
Acknowledgements
The authors would like to thank Mr. Syed Rafatullah,
Assistant Researcher, College of Pharmacy, King Saud
Univeristy, for his technical assistance and to Mr. Tanvir
A. Butt, secretary to the Pharmaceutical Chemistry Dept. for
his secretarial services.
76
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B.M. Nelson and G.A. Andrews, Am. J. Clin. Pathol. 42,

37-44 (1964).
BUSULPHAN
118.
119.
120.
121.
122.
123.
124.
M.L.
83
Feingold and L.G. KOSS, Arch. Intern. Med., 124,
66-71 (1969).
S.
Dahlgren, G. Holm, N. Svanborg and R.
Med. Scand. 192(1),

---
129-135 (1972).
I. Diamond, M.M. Anderson and S . R .

Pediatrics, 25, 85-90 ( 1960).
J.F.
38,
Pezzimenti, H.C. Kim and .T.
2242-2246 (1976).
Watz,
Acts
McCreadie,
Lindenbaum,
Cancer
H. Stott, W. Fox, D.J. Girling, R.J. Stephens and

D.A.G. Galton. Br. Med. J. 2, 1513-1517 (1977).
F.H. Sobles, Pharm. Weckble. 107, 365 (1972).

D.C. Garratt, "The Quantitative Analysis of Drug",
Third Edition, Chapman and Hall Ltd., London, page 801
(1964).
277,
374-
125.
M. Hassan and H. Ehrsson, J. of Chromatogr.,
126.
H. Ehrsson and M. Hassan, J. Pharm. Sci., 7 2 ( 1 0 ) ,
127.
J.Turczan and C. Lau-Cam, Spectrosc. Letters.,
128.
P.W. Feit and N. Rastrup-Andersen, J. Pharm. sci.,
380 (1983).
1203-1205
(1983).
17(6&7), 353-359
1007 (1973).
(1984).
62,
CHLORAMBUCIL
Mohammad T L q * and AbduReah A . ke-Bad&**
*UepcuLtment 06 P h m a c o R o g y , **UepahGncn;t 06 Phacu~laceuLicaI

Chern&in.fhy, College 06 P h m a c y , K i m j Saud LlniLImiXy,
P.O. Box 2 4 5 7 , Riyadh-17451, S a u d i RtLabia.

VOLUME 16
85
Copyright 01987 by Academic Press, Inc.

All rights of reproduction in any form resewed.
86
1.
History
2.
Description
2.1
2.2
2.3
2.4
2.5
2.6
3.
Nomenclature
Formulae
Molecular Weight
Crystal Properties
Physical Properties
3.1
3.2
3.3
3.4
3.5
3.6
3.7
Melting Point
Extraction
Solubility
Moisture Content and Hygroscopicity
Dissociation Constant
Storage
Spectral Properties
4. Synthesis
5. Pharmacokinetics
6. Therapeutic Uses
7.
Toxicity
8. Methods of Analysis
8.1
8.2
8.3
8.4
References
Identification
Titrimetric Methods
Spectrophotometric Methods
87
CHLORAMBUCIL
1. History
This aromatic derivative of mechlorethamine was
synthesized first at the Chester Beatty Research
Institute in England.
It was synthesized
among other aromatic derivatives of the chloroethylmines by the British chemists, Everett, Roberts and Ross
Chlorambucil has shown activity in a variety
(1,2).
of human malignancies (3). These include chronic
lymphocytic leukemia (4,5). Hodgkin's and non-Hodgkin's
lymphoma ( 6 ) , choriocarcinoma, ovarian carcinoma, and
breast carcinoma (7). It is most often employed in the
long-term maintenance management of chronic lymphoet a1 (8) found that a response
cytic leukemia. Moore rate of 19% was achieved in 52 advanced breast cancer
patients.
2. Description
2.1 Nomenclature
2.1.1 Chemical Names

4-[Bis( 2-chloroethyl)amino]benzenebutanoic
acid;
4-[ p- [ Bis ( 2-chloroethyl) amino]phenyl ]
butyric acid;
N,N-di-2-chloroethyl-y-p-aminophenylbutyric
acid;
~-[p-Di(2-chloroethyl)aminophenyl]butyric
acid;
4-[ 4-Di-( 2-chloroethyl ) aminophenyl] butyric
acid;
4-[4-(Bis(2-chloroethyl)amino)phenyl]butanoic
acid;
p-Di(2-chloroethyl)aminophenyl butyric acid
(9 -12).
2.1.2
Generic Names
Chlorambucil, C.B. 1348. NSC 3088 Chlorbuti-
num (10-12).
2.1.3 Trade Names

Chlorambucil , Leukeran, Chloroambucil,
(11)
Amboclorin; Chloraminophene; Linfolysin
(10-12)
88
2.1.4
CAS R e g i s t r y No.
( 305-03-3)
Wiswesser Line N o t a t i o n
2.1.5
(13)
QV3R DN2G2G
2.2
Formulae
2.2.1
Empirical
(14J 5 )
C14H19C12N02
2.2.2
Structural
C 1CH 2CH2
CH CH CH COOH
C1CH2CH2
2.3
Molecular Weight
304.20
2.4
(10,15)
C 55.27%; H 6.30%,
0 10.52% ( 1 0 ) .
2.5
C1 23.31%,
N 4.60%,
A w h i t e or off-white c r y s t a l l i n e or g r a n u l a r
powder with a s l i g h t odor (11).
2.6
Crystal Properties
F l a t t e n e d n e e d l e s from petroleum e t h e r ( 1 0 ) .
3.
Physical.
3.1
Properties
Melting P o i n t
Melts between
64'
and 69'
(12).
89
CHLORAMBUCIL
3.2
Extract ion
Chlorambucil i s e x t r a c t e d by o r g a n i c s o l v e n t s from
aqueous a c i d s o l u t i o n s ( 1 2 ) .
3.3
Solubility
P r a c t i c a l l y i n s o l u b l e i n water (14). S o l u b l e a t
2OoC i n 1 . 5 p a r t s of a l c o h o l , i n 2.5 p a r t s o f
chloroform, and i n 2 p a r t s of a c e t o n e , s o l u b l e i n
e t h e r ( 1 0 ) and i n d i l u t e s o l u t i o n of a l k a l i
hydroxides (11).
3.4
Moisture Content and Hygroscopicity

Not more t h a n O . 5 % , determined by F i s c h e r t i t r a t i o n
(14).
3.5
D i s s o c i a t i o n Constant
pKa v a l u e 5.8
3.6
(16).
Storage
It should be s t o r e d i n a c o o l p l a c e i n a i r t i g h t ,
l i g h t r e s i s t a n t c o n t a i n e r s (11).
3.7
Spectral Properties
3.7.1
The u l t r a v i o l e t spectrum of chlorambucil
i n methanol e x h i b i t s maxima a t 258 nm
( E 1%, 1 cm 650) and 302 nm ( E 1%, 1 cm 8 5 ) ;
and minima a t 225 nm and 281 nm ( 1 2 ) .
The W spectrum o f chlorambucil i n w a t e r
and i n methanol which was scanned from 200
t o 400 nm u s i n g DMS 90 Varian spectrophotometer. It e x h i b i t s a b s o r p t i o n maxima a t
257 nm and a t 302 nm and minima a t 224 and
280 nm. The W s p e c t r a i n w a t e r and i n
methanol a r e shown i n Figs [l] and [ 2 ] ,
respectively.

2
so.
80
90
'
80
. 70
.so
. 50
.LO
40.
.lo
.20
10.
.10
A
2 00
250
Wavelenplh
2 00
IS0
Wardepth
300
300
150
I50
F i g u r e 2 : U l t r a v i o l e t Spectrum o f Chlorambucil i n Methanol.
91
CHLORAMBUCIL
3.7.2
The i n f r a r e d spectrum of chlorambucil i s
shown i n F i g . [ 3 ] . The spectrum w a s
o b t a i n e d a s K B r d i s c and i s recorded i n
Pye-Unicam SP 1025 i n f r a r e d spectrophotometer. The f r e q u e n c i e s and t h e i r s t r u c t u r a l
assignments are as follows:Frequency (em-')
Assignment
1720
O H s t r e t c h and CH
stretch
C = 0 acid
1622
C = C aromatic
1450
CH deformation
3100-29'70
730
C-C1
stretch
Other f i n g e r p r i n t bands a r e 1530, 1465 and

Clarke ( 1 2 ) r e p o r t e d t h e
p r i n c i p a l peak a r e 1695, 1520 and 1229 cm-l.
830 cm-1.
3.7.3
Carbon-13 Nuclear Magnetic Resonance S p e c t r a

( C - 1 3 NMR)
The C-13 NMR n o i s e decoupled and o f f resonance s p e c t r a a r e shown i n Figs. [ 4 ] and
151, r e s p e c t i v e l y . Both were recorded i n
d e u t e r a t e d chloroform on a Jeol-XL-100 MHz
NMR s p e c t r o m e t e r u s i n g t e t r a m e t h y l s i l a n e
as a r e f e r e n c e s t a n d a r d . The s p e c t r a l
assignments are l i s t e d below:-
10 9
H C ~H~
c iC
&@
/
C1CH2CH2
10' 9'
7'
6'
4
3
2
1
CH2-CH2-CH2-COOH
92
Wavenumber
Figure 3 : Infrared Spectrum of Chlorambucil as KBr Disc.
93
,
0
v)
.r
.r
Spectrum o f Chlorambucil in CDC13 (Noise Decoupled).

YO
..
Figure 4 : "C-NMR
Figure 5 : 13C-NMR Spectrum o f Chlorambucil i n CDC13 (Off-Resonance).
95
CHLORAMBUCIL
Chemical s h i f t
( PPm 1
Carbon
No.
180,3
33.8"
33.4"
26.4
130.2
and 6'
129.6
7 and 7'
112.1
144.3
9 and 9'
53.5
10 and 10'
40.4
s = singlet
3.7.4
Mult i p l ic ity
d = doublet
t = triplet
I
Proton Magnetic Resonance S p e c t r a ( H-NMR)
1
The 60 MHz H-NME3 spectrum of chlorambucil
i n d e u t e r a t e d chloroform i s shown i n F i g .
[ 61. The spectrum w a s recorded on
Varian T ~ O - A NMR spectrometer u s i n g
t e t r a m e t h y l s i l a n e (TMS) as t h e i n t e r n a l
standard
The f o l l o w i n g are t h e s t r u c t u r a l assignments:

Chemical s h i f t (ppm)
Assignments
M u l t i p l e t a t 1.8-2.8 -(CH ) -CO p r o t o n s

S i n g l e t a t 3.60
23
-N-CH2CH2 p r o t o n s
M u l t i p l e t a t 6.6-7.08
Aromatic p r o t o n s
S i n g l e t a t 11.5
O H ( a c i d i c ) exchangea b l e w i t h D20,
Fig. [ T I .
500
. - . . .... ,... .. .. "..,".,,',..
l - - - . v
100
200
300
400
--,-
TY
I
[ . . . . I
Qo
7-0
. . . .1
6-0
5-0
.... .... .... .... ....

6
4-0
3-0
2.0
14
Figure 6 : 'H-NMR Spectrum o f Chlorambucil i n Deuterated Chloroform.
200
100
L'
Figure 7 : 'H-NMR Spectrum o f Chlorambucil in Deuterated Chloroform + D20.
98
3.7.5
Mass Spectrum
The e l e c t r o n impact ( E I ) mass spectrum o f
chlorambucil a t 70 e V , r e c o r d e d on Varian
Mat 311 mass s p e c t r o m e t e r u s i n g i o n
source p r e s s u r e of 10-6 T o r r , ion s o u r c e
temperature of 1 8 0 O c and a n emission
c u r r e n t of 300 uA. The spectrum i s shown
i n F i g . [ a ] . The spectrum is dominated by
m / e 254 i o n ( b a s e peak) r e s u l t i n g from t h e
l o s s of CH2C1 and H . A proposed mechanism
of f r a g m e n t a t i o n and t h e m / e o f t h e major
fragments i s given i n Scheme 1.
The chemical i o n i s a t i o n ( C I ) spectrum
( F i g . [ 9 ] ) i s o b t a i n e d on a Finnigan 4000
mass s p e c t r o m e t e r w i t h i o n e l e c t r o n energy
of 1 0 0 e V , i o n source p r e s s u r e o f 0.13 Torr,
i o n s o u r c e t e m p e r a t u r e o f 1 5 0 O c and emission
c u r r e n t o f 300 PA. The spectrum shows a
pronounced peak r e s u l t i n g from t h e loss of
HC1 (& - 36) c o n s t i t u t e t h e base peak a t
m / e 268 and a molecular i o n peak at m / e 304.
A proposed f r a g m e n t a t i o n pathway for t h e
fragment i n t h e E I spectrum i s shown i n
Schemes l a and lb. The mass s p e c t r a l
assignments o f t h e prominant i o n s under C I
conditions a r e given i n t h e following
m/e
Fragment
306
[ M + 2H]+
305
MH+
304
M+
303
[MH
286
[M
268
[M
- HCl]'
270
[M + 2 H
254
[MH
232
[M - 2 H C 1 ] +
H20]+
H20]
(H2 + C H 2 C l ] +
1oo.c
50.0
WIE
50
100
1so
ZOO
250
Figure 8 : Mass Spectrum o f Chlorambucil ( E I ) .
300
1oo.c
5O.C
'r
MI
200
220
240
260
'i
2
a
300
320
Figure 9 : Mass Spectrum of Chlorambucil
340
(CI).
I0
310
100
z
0
N
8
6N
L
W
-N
a,
00
a,
+z
I' Y
z-z
u
+z
Ill
db..
a,
r
.
4
4
rd
+J
.rl
rd
t,
k
k
w
w
.d
s8
a
a,
0
PI
0
m/e 254
H
Scheme l b :
Proposed mechanism of fragmentation of chlorambucil.
103
CHLORAMBUCIL
4.
Synthesis
E v e r e t t et g (1) have s y n t h e s i z e d chlorambucil and
some o t h e r n i t r o g e n mustard. The s y n t h e s i s have been
r e p o r t e d (17,18) as f o l l o w s :
N i tr a t ion
CH2CH2CH2COOH
02N
-(Ot
02N
CH2CH2CH2COOH
CH2CH2CH2COOCH3
CH2CH2CH2COOCH3
HOCH2CH2
a-
/
C1CH2CH2
ClCH CH
\IN
CH2CH2CH2COOCH3
CH2CH2CH2COOH
ClCH ,CH,
Ethylene oxide
CH3COOH
POC 1
CH2CH2CH2COOCH3
HOCH,CH,
C~CH~CH~
2J
Esterification
Reduction Pd/CaCO,
HzN
Hydrolysis
104
5.
Pharmacokinetics
5.1
Absorption, D i s t r i b u t i o n , Metabolism and Excretion
Oral absorption of chlorambucil i s adequate and

r e l i a b l e . The drug has a h a l f - l i f e i n plasma of
approximately 90 minutes, and it i s almost
completely metabolized ( 1 9 ) . Although t h e i n t r a venous r o u t e produced higher c o n c e n t r a t i o n s , absorpt i o n from t h e g a s t r o - i n t e s t i n a l t r a c t w a s consist e n t l y r a p i d and appeared t o be complete; peakplasma concentrations were achieved i n 40 t o 7 0
minutes. A n hour a f t e r a d m i n i s t r a t i o n by e i t h e r
r o u t e t h e r a t e of metabolism w a s s i m i l a r and
s u f f i c i e n t t o make a c o n t r i b u t i o n t o t h e a c t i v i t y
of t h e drug ( 2 0 ) .
There appears t o be extensive metabolic degradation
of t h e drug and a major m e t a b o l i t e , an aminophenyla c e t i c a c i d d e r i v a t i v e which has a h a l f - l i f e of
about 2.5 h r i n man, has been i d e n t i f i e d . The
u l t i m a t e m e t a b o l i t e ( s ) of chlorambucil i n man has
y e t t o be defined and f u r t h e r work i s r e q u i r e d t o
confirm t h e s e i n i t i a l pharmacokinetic observations
(21).
McLean e t a1 ( 2 0 ) reported two m e t a b o l i t e s one w a s
i d e n t i f i e d as t h e @-oxidation yroduct of chloramb u c i l ; 2[ k-N,N-bis( 2-chloroethyl)aminophenyl] a c e t i c
a c i d , a l s o c a l l e d phenylacetic mustard.
This
m e t a b o l i t e i s known t o have an a l k y l a t i n g a c t i o n i n
animals.
Dorr and W i l l i a m ( 2 1 ) e x t e n s i v e l y reviewed t h e
d i s p o s i t i o n of chlorambucil i n human being and
experimental animals. I n r a t s , r a d i o a c t i v e drug
appear t o be c l e a r e d from t h e blood r a p i d l y , and
a t one h r t h e h i g h e s t t i s s u e c o n c e n t r a t i o n i s found
i n t h e l i v e r . However, f a i r l y homogeneous t i s s u e
d i s t r i b u t i o n w a s noted. The drug h a s a l s o been
shown t o d i s t r i b u t e w e l l i n t o a s c i t i c f l u i d a f t e r
subcutaneous administ-ation.
S i x t y percent of drug
r a d i o a c t i v i t y w a s excreted i n t o t h e u r i n e by 24 h r ,
with t h e m a j o r i t y of t h e remainder e x i s t i n g as
t i s s u e as tissue-bound drug ( 2 2 ) . S i g n i f i c a n t drug
binding t o gamma g l o b u l i n p r o t e i n s has been observed.
Apparently no drug i s excreted i n t h e f e c e s .
Because a p o r t i o n of t h e drug molecule has l i p o p h i l i c
CHLORAMBUCIL
105
p r o p e r t i e s , some f a t ( d e p o t ) drug s t o r a g e may occur.

This could e x p l a i n t h e prolonged c l i n i c a l e f f e c t
of chlorambucil o c c a s i o n a l l y observed i n man.
6.
Therapeutic Uses
C l i n i c a l t r i a l s of c h l o r m b u c i l were i n s t i t u t e d i n 1954.
During t h e subsequent 5 y e a r s t h e substance w a s s t u d i e d
i n q u i t e some d e t a i l . The t h e r a p e u t i c uses of
chlorambucil have been discussed by Larionov ( 2 3 ) . The
drug i s given o r a l l y i n t a b l e t form i n doses from 0 . 1
t o 0.4 mg/kg, i . e . 6 t o 25 mg d a i l y . The course of
treatment u s u a l l y l a s t s 3-9 weeks. The t o t a l dose on
average i s about 400 mg per course but i n i n d i v i d u a l
p a t i e n t s it v a r i e s g r e a t l y with t h e form of t h e d i s e a s e ,
t h e s t a g e , t h e condition of t h e organism, previous
t r e a t m e n t , e t c . The t h e r a p e u t i c e f f e c t does not come
a t once; but u s u a l l y i n t h e 2nd t o 3rd week a f t e r s t a r t ing t h e drug. A t t h e s e doses s i d e e f f e c t s i n t h e g a s t r o i n t e s t i n a l t r a c t a r e r a r e l y observed. Towards t h e end
of t h e course , neutropenia and thrombocytopenia may
appear, t h u s n e c e s s i t a t i n g withdrawal of t h e p r e p a r a t i o n .
The maintenance dose of t h e compound i s from 0.03 t o
0 . 1 mg/kg, i . e . 2-6 mg d a i l y .
Chlorambucil g i v e s t h e b e s t e f f e c t i n chronic lymphoid
leumaemia both i n t h e leukaemic and aleukaemic form and,
i n p a r t i c u l a r , i n t h a t v a r i a n t known as f o l l i c u l a r
lymphoma Galton and T i l l ( 2 4 )
Altman e t a1 ( 2 5 ) ,
Israels _
e t -a1 (261 Doan e t a1 ( 2 7 ) . I n chronic lymphoid
leukaemias, chlorambucil i s given i n doses of 0 . 1 0.2 mg/kg, i . e . 6-12 mg d a i l y f o r 4-8 weeks. The e f f e c t
c o n s i s t s remission expressed, i n p a r t i c u l a r , i n r e d u c t i o n
of t h e lymph nodes, s p l e e n and l i v e r , i n c r e a s e d
haemoglobin, f a l l i n lymphocytosis and sometimes i n c r e a s e
i n n e u t r o p h i l s i n t h e blood. The remission l a s t s from
3 months t o 1-3 y e a r s . A f t e r t h e onset of a r e l a p s e ,
remission can again be obtained i n some p a t i e n t s by t h e
same drug ( 2 3 ) .
Moore et a1 ( 8 ) used a continuous d a i l y dose of 0 . 1 0.2 mg/kg. The drug may a l s o be administered on an
i n t e r m i t t e n t b a s i s ( 0 . 4 mg/kg every four weeks ( 2 8 ) .
Both regimens commonly include prednisone. On t h e i n t e r m i t t e n t schedule, monthly pulse doses as high as 1 . 5 2 . 0 mg/kg were w e l l t o l e r a t e d and d i d not produce undue
marrow t o x i c i t y . A n e f f e c t i v e biweekly dosing schedule
106
w i t h l e s s e n e d hematologic t o x i c i t y i s a l s o r e p o r t e d :
0.4 mg/kg every 2 weeks, i n c r e a s i n g by 0 . 1 mg/kg
increments t o t o x i c i t y o r remission ( 5 ) .
7.
Toxicity
According t o Dorr and W i l l i a m ( 2 1 ) chlorambucil i s one
of t h e b e s t t o l e r a t e d o r a l a l k y l a t i n g a g e n t s . Bone
marrow d e p r e s s i o n , i n c l u d i n g n e u t r o p e n i a , thrombocytop e n i a , and lymphopenia, o c c u r s w i t h prolonged u s e .
I r r e v e r s i b l e bone marrow damage, however, may o c c u r .
Thus myelosuppression i s t h e common d o s e - l i m i t i n g
t o x i c i t y with chlorambucil. G a s t r o i n t e s t i n a l d i s t r e s s
w i t h l a r g e r doses o c c u r s but i s u s u a l l y not s e r i o u s l
Rare h e p a t i t i s and d i s t u r b a n c e s o f l i v e r f u n c t i o n have
a l s o been r e p o r t e d .
C e n t r a l nervous system s t i m u l a t i o n h a s occurred w i t h
chlorambucil but i s uncommon u n l e s s l a r g e doses are
used. I n a d v e r t e n t t o x i c i n g e s t i o n s i n c h i l d r e n o f
1.5
5 mg/kg have occurred without f a t a l i t i e s b u t w i t h
moderate t o x i c i t y c h a r a c t e r i z e d by vomiting, a g i t a t i o n ,
i r r i t a b i l i t y , and h y p e r a c t i v i t y followed by l e t h a r g y
and e v e n t u a l m i l d pancytopenia ( 2 9 , 3 0 ) . Chromosomal
damage i s w e l l documented f o r t h i s agent although t h e
e x a c t mechanism i s not w e l l known ( 3 1 ) . Lerner ( 3 2 ) has
f u r t h e r a s s o c i a t e d c h r o n i c chlorambucil t h e r a p y w i t h a
high i n c i d e n c e o f secondary a c u t e myelogenous leukemia.
S i m i l a r t o some o t h e r a l k y l a t i n g a g e n t s , chlorambucil
can a l s o cause a l v e o l a r d y s p l a s i a and pulmonary f i b r o s i s
w i t h long-term u s e ( 3 3 ) . A good c l i n i c a l response t o
drug d i s c o n t i n u a n c e and c o r t i c o s t e r o i d s w a s n o t e d i n
t h i s c a s e . Spermatogenesis i s a l s o depressed w h i l e t h e
p a t i e n t i s on chlorambucil t h e r a p y ( 3 4 ) . LD 0 i n rats
i n a s i n g l e a d m i n i s t r a t i o n i s 21-23 mg/kg ( 3 5 ) .
8.
Methods o f A n a l y s i s
8.1
Identification
B r i t i s h Pharmacopoeia 1980 ( 9 ) d e s c r i b e s t h e
following i d e n t i f i c a t i o n t e s t s :
1) Mix 0 . 4 g of chlorambucil w i t h 1 0 m l o f 2 M
hydrochloric a c i d and a l l o w t o s t a n d f o r t h i r t y
minutes, shaking o c c a s i o n a l l y . F i l t e r , wash t h e
CHLORAMBUCIL
107
r e s i d u e with two q u a n t i t i e s , each o f 1 0 m l , o f

w a t e r , and r e s e r v e t h e mixed f i l t r a t e s and washi n g s for t e s t f o r i d e n t i f i c a t i o n 2. Melting
p o i n t o f t h e r e s i d u e , a f t e r d r y i n g over
phosphorous pentoxide a t a p r e s s u r e not exceedi n g 0.7 KPa (about 5 t o r r ) f o r t h r e e h o u r s ,
about 1 4 6 O .
2)
To 1 0 ml of t h e mixed f i l t r a t e and washings
r e s e r v e d i n t e s t f o r i d e n t i f i c a t i o n , add 0 . 5 m l
of potassium m e r c u r i i o d i d e s o l u t i o n ; a b u f f c o l o r e d p r e c i p i t a t e i s produced. To a f u r t h e r
1 0 m l add 0.5 m l of potassium permanganate
solution t h e purple color i s discharged.
3 ) Mix 0.6 g w i t h 0.2 m l o f phenylhydrazine, h e a t

i n a water b a t h f o r t e n minutes, s t i r r i n g
o c c a s i o n a l l y , add 2 m l of a b s o l u t e e t h a n o l , h e a t
f o r twenty seconds, f i l t e r immediately, and
a l l o w t o stand f o r t h i r t y minutes. C r y s t a l s of
phenylhydrazinium c h l o r i d e are formed, which,
a f t e r washing with two q u a n t i t i e s , each o f 1 m l ,
o f a b s o l u t e e t h a n o l and d r y i n g a t 105O, have
a m e l t i n g p o i n t of about 245' , w i t h decomposition.
U.S. Pharmacopeia XX ( 1 4 ) d e s c r i b e t h e f o l l o w i n g
t e s t s f o r t h e i d e n t i f i c a t i o n o f chlorambucil:
1) The i n f r a r e d a b s o r p t i o n spectrum of a 1 i n
125 s o l u t i o n i n carbon d i s u l f i d e , i n a 1 mm c e l l ,
e x h i b i t s maxima o n l y a t t h e same wavelength as
t h a t o f a s i m i l a r s o l u t i o n o f USP Chlorambucil
Reference Standard.
2 ) D i s s o l v e 50 mg i n 5 ml o f a c e t o n e , and d i l u t e
w i t h water t o 1 0 m l . Add 1 drop of 2 N s u l f u r i c
a c i d , t h e n add 4 drops of s i l v e r n i t r a t e ( t e s t
s o l u t i o n ) : no opalescence i s observed
immediately (absence of c h l o r i d e i o n ) . Warm
t h e s o l u t i o n on a steam b a t h : opalescence
develops ( presecne o f i o n i s a b l e c h l o r i n e )
8.2
U . S . Pharmacopeia XX ( 1 4 ) d e s c r i b e s t h e following
method:
Dissolve about 200 mg o f c h l o r a m b u c i l , a c c u r a t e l y
weighed, i n 1 0 ml o f a c e t o n e , add 1 0 m l o f water,
108
and t i t r a t e w i t h 0 . 1 N sodium hydroxide VS, u s i n g

p h e n o l p h t h a l e i n TS as t h e i n d i c a t o r . Each m l
of 0 . 1 N sodium hydroxide i s e q u i v a l e n t t o 30.42 mg
o f C14HlgC12N02.
B r i t i s h Pharmacopoeia
f o l l o w i n g method:
1980 ( 9 ) d e s c r l b e s t h e
To 0.5 g of chlorambucil, add 20 m l of M sodium

hydroxide and b o i l under a r e f l u x condenser f o r
one hour. Cool, t r a n s f e r t h e m i x t u r e t o a 100-ml
graduated f l a s k w i t h t h e a i d of w a t e r , add 50 m l
o f 0 . 1 M s i l v e r n i t r a t e VS and 5 m l o f n i t r i c a c i d ,
mix, and add s u f f i c i e n t water t o produce 100 m l .
F i l t e r and t i t r a t e t h e excess of s i l v e r n i t r a t e
i n 50 m l o f t h e f i l t r a t e w i t h 0 . 1 M ammonium
t h i o c y a n a t e VS, u s i n g 3 m l o f ammonium i r o n (111)
s u l f a t e s o l u t i o n as i n d i c a t o r . Each m l o f 0.1 M
s i l v e r n i t r a t e VS i s e q u i v a l e n t t o 0.01521 g of
C14H19C12N02*
8.3
8.3.1
C o l o r i m e t r i c Method
P e t e r i n g and Van Giessen ( 3 6 ) d e s c r i b e d a
c o l o r i m e t r i c procedure f o r d e t e r m i n a t i o n o f
n i t r o g e n mustards i n c l u d i n g Chlorambucil ,
Dopan, U r a c i l Mustard and Mustargen i n
plasma. Mix t h e drug (10 t o 70 ug) ,
d i s s o l v e d i n a 5% s o l u t i o n of dimethylacetamide i n e i t h e r e t h a n o l o r 0.9% N a C l
s o l u t i o n , with phthalate buffer s o lu tio n
(pH 4 . 0 ) (1m l ) , 5% b ( p - n i t r o b e n z y 1 ) p y r i d i n e s o l u t i o n i n acetone (1 m l ) and 0.9%
N a C l s o l u t i o n (1m l ) , and d i l u t e w i t h
e t h a n o l t o 4 m l . Heat a t 80' f o r 20 min. ,
c o o l i n i c e , add N-KOH i n 90% e t h a n o l
( 0 . 1 nil), d i l u t e w i t h e t h a n o l a t 600 mp
w i t h i n 2 o r 3 min. Carry o u t a b l a n k
determination.
8.3.2
I n f r a r e d S p e c t r o m e t r i c Method
Kozlov and Sbezhneva (37) r e p o r t e d an
i n f r a r e d s p e c t r o s c o p i c a n a l y s i s of chloramb u c i l i n pharmaceutical p r e p a r a t i o n s .
S p e c i a l l y t o determine t h e amount of a c t i v e
109
CHLORAMBUCIL
11
8.3.3
ingredients i n carcinostatic preparations

c o n t a i n i n g chlorambucil. 0 . 1 g of sample of
t h e p r e p a r a t i o n i s e x t r a c t e d i n 100 m l o f
acetone and t h e absorbance o f a l i q u o t i s
measured a t 770 cm-1. The e r r o r does not
exceed f 1%.
U l t r a v i o l e t Spectrometric )Jethod
El-Tarras e t a 1 (38) developed a simple

method f o r t h e d e t e r m i n a t i o n of chlorambucil
i n pharmaceutical p r e p a r a t i o n s . The
powdered t a b l e t s are e x t r a c t e d i n t o e t h a n o l
(4 X 1 0 ml). The f i l t e r e d e x t r a c t w a s
d i l u t e d t o 50 m l w i t h e t h a n o l . A p o r t i o n of
t h i s s o l u t i o n w a s f u r t h e r d i l u t e d as r e q u i r e d
w i t h e t h a n o l and t h e absorbance measured
a t 250 nm v s e t h a n o l . The c o n c e n t r a t i o n o f
t h e drug i s c a l c u l a t e d u s i n g a c a l i b r a t i o n
curve.
8.3.4
Mass S p e c t r o m e t r i c Methods
e t -a1 (39) developed a s e n s i t i v e and

Chang _
s p e c i f i c method f o r t h e d e t e r m i n a t i o n of
chlorambucil and i t s m e t a b o l i t e s i n b i o l o g i c a l samples. The method i s based on
s e l e c t e d i o n monitoring d e t e c t i o n f o l l o w i n g
simple e x t r a c t i o n of t h e p a r e n t compound,
i t s m e t a b o l i t e and i n t e r n a l s t a n d a r d
(Chlorambucil-d8) from plasma and u r i n e
samples. To determine chlorambucil and
b [ b i s - ( 2 chloroethyl)ar,ino]phenyl a c e t i c
a c i d i n 0.5 m l plasma or u r i n e add 0.5 m l
of 4% HCl04 ( p e r c h l o r i c a c i d ) and [2H8]
chlorambucil as i n t e r n a l s t a n d a r d .
The
mixture i s e x t r a c t e d w i t h e t h y l a c e t a t e and
The o r g a n i c l a y e r w a s d r i e d
h e x m e (1:l).
w i t h p u r i f i e d N2 a t room t e m p e r a t u r e and
s e v e r a l drops o f methylene c h l o r i d e were
added t o d r y r e s i d u e t o form t r i m e t h y l s i l y l
d e r i v a t i v e . T h i s i s submitted t o GLC
column ( 5 0 cm X 2 mm) of 3% of OV-17 on gas
Chrom Q ( 1 0 0 t o 1 2 0 mesh) a f t e r 40 seconds
a t 190C t h e column t e m p e r a t u r e i s r a p i d l y
r a i s e d t o 31OoC; 7 0 eV-m.s
detection i s
u s e d . T r i m e t h y l s i l y l a t e d chlorambucil i s
110
monitored a t m / e 3?6 and 328, t h e d e r i v a t i v e

of t h e m e t a b o l i t e of chlorambucil a t m / e
298, and t h a t of t h e i n t e r n a l s t a n d a r d a t
m / e 383 and 385. The mean recovery and
s t a n d a r d d e v i a t i o n were 94.3
1.3%.
et a1 ( 4 0 ) d e s c r i b e d an a n a l y t i c a l
Jakhammer procedure f o r simultaneous d e t e r m i n a t i o n of
chlorambucil and prednimustine i n plasma.
The method i n v o l v e s e x t r a c t i o n and s e p a r a t i o n followed by d e r i v a t i z a t i o n t o chloramb u c i l methyl e s t e r and mass fragmentography.
The drugs are e x t r a c t e d i n t o chloroform,
hexane ( 3 : 7 ) and s e p a r a t i o n i s done by
p a r t i t i o n a f t e r adding 0 . 1 M phosphate 0 . 1 M b o r a t e b u f f e r (pH 9 . 0 ) . Chlorambucil
i n t h e aqueous phase add predenimustine i n
o r g a n i c phase are each t r a n s e s t e r i f i e d w i t h
methanol-3F3 d i e t h y l e t h e r a t e ( 4 : l ) .
The
compounds are determined u s i n g fragmentography. A Varian MAT mass spectrometer i n
combination w i t h Varian Aerograph 1400 g a s
chromatograph with a column o f 0.5% o f
Carbowax 20 M on Chromosorb W-HP, are used.
Analogues, prepared by i n t r o d u c i n g e i g h t 2H
atoms i n t o t h e b i s - ( 2-chloroethy1)amino group
of b o t h compounds are used as i n t e r n a l
s t a n d a r d s and c a r r i e r s . The d e t e c t i o n l i m i t
i s about 8 ng/ml f o r b o t h d e t e r m i n a t i o n s and
t h e r e l a t i v e s t a n d a r d d e v i a t i o n 3% f o r
chlorambucil and 5% prednimustine a t 30-60
ng/rnl l e v e l .
8.4
8.4.1
Thin Layer Chromatograph (TLC)

Norpoth e t a1 ( 4 1 ) d e s c r i b e d a TLC method
for t h e d e t e r m i n a t i o n o f a l k y l a t i n g cytos t a t i c a g e n t s on a t h i n l a y e r p l a t e w i t h
i s o n i c o t i n a l d e h y d e benzothiazol-2-ylhydrazone. The drug samples are d i s s o l v e d
i n methanol. The s p o t i s a p p l i e d on a s h e e t
o r precoated p l a t e s .
A f t e r developing and
drying t h e chromatogram, it i s sprayed w i t h
3% s o l u t i o n of r e a g e n t i n acetophenone dimethylformamide ( 7 : 3 ) c o n t a i n i n g 0 . 0 1 m l
of c o n c e n t r a t i o n H C 1 p e r 100 m l .
It i s t h e n
111
CHLORAMBUCIL
p l a c e d over a b a t h o f acetophenone and b a t h

and p l a t e are h e a t e d a t 2OO0C for 20
minutes; t h e cooled p l a t e i s t h e n sprayed
w i t h t r i e t h y l a m i n e . Chlorambucil produces
r e d s p o t . For 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
t h e c o l o r i n t e n s i t y o f t h e spot i s measured
u s i n g chromatogram spectrophotometer
8.4.2
Gas Liquid Chromatography ( G L C )

Ehrsson _
et _
a1 (42) d e s c r i b e d a GLC t e c h n i q u e
f o r d e t e r m i n a t i o n of chlorambucil i n plasma
by g a s - l i q u i d chromatography w i t h s e l e c t e d
ion-monitoring.
2 m l o f plasma sample
c o n t a i n i n g 8 l.~g o f chlorambucil p e r m l w a s
mixed w i t h h y d r o c h l o r i c a c i d , phosphoric
a c i d , or phosphate b u f f e r ( 0 . 2 m l ) . The
mixture w a s e x t r a c t e d with methylene
c h l o r i d e ( 2 m l ) f o r 30 minutes. An a l i q u o t
o f o r g a n i c phase was s e p a r a t e d and
evaporated t o dryness with n i t r o g e n g a s .
The r e s i d u e w a s d i s s o l v e d i n methanol-water
0 . 1 M, a c e t i c a c i d (75:15:10) and a n a l y s e d
on g a s chromatography. A l t e r n a t i v e l y t h e
plasma samples w e r e a d j u s t e d t o pH 3 w i t h
1 M phosphoric a c i d and e x t r a c t e d w i t h
e t h y l e n e d i c h l o r i d e f o r 30 minutes. The
organic phase w a s s e p a r a t e d and e x t r a c t e d
with 0 . 5 m l of 0 . 1 M sod. s u l p h i d e f o r 5
minutes. The aqueous phase w a s s e p a r a t e d
and h e a t e d f o r 1 5 minutes a t 80C t o c o n v e r t
chlorambucil i n t o t e t r a h y d r o t h i a z i n e
d e r i v a t i v e , then c o n c e n t r a t e d phosphoric
a c i d (0.91m l ) was added, and H2S removed
w i t h N2 f o r 5 minutes. To t h i s O . 0 5 m l o f
12 M NaOH, 0 . 2 m l of 0.5 M t e t r a b u t y l
ammonium, 0.2 ml methylene c h l o r i d e and
0.05 m l a l l y l b r o m i d e were added and t h e
mixture w a s shaken f o r 30 min a t 2 5 O C .
The
o r g a n i c phase (1-2 mcg) w a s i n j e c t e d i n GLC
( a t Z50C) on a column of 5% of OV-17 on
gas-Chrome-Q o p e r a t e d with 70 eV.m. s .
d e t e c t i o n . The i n t e r n a l s t a n d a r d used w a s
[%8] chlorambucil. The peaks were monitored
a t m / e 305 and 313 f o r chlorambucil and
112
[ H8] chlorambucil r e s p e c t i v e l y .
The
c o e f f i c i e n t of v a r i a t i o n f o r 10ng/ml o f
chlorambucil w a s 5%.
8.4.3
High-pressure Liquid Chromatography (HPLC)

Newel1 et a1 ( 4 3 ) developed a HPLC method
f o r e s t i m a t i o n o f c h l o r a m b u c i l , phenyla c e t i c mustard and predenimustine i n human
plasma. One m l plasma w a s e x t r a c t e d w i t h
2 ml of e t h y l a c e t a t e , t h e emulsion formed
by vigorous a g i t a t i o n on a Whirlimixer was
f r o z e n by immersing t h e t u b e s i n a methanol
carbondioxide b a t h a t - 6 8 O C . The t u b e s
w e r e c e n t r i f u g e d a t 600 pg f o r 10 minutes
a t 4 O C u n t i l t h e thawed aqueous phase c o u l d
be s e p a r a t e d . The procedure w a s r e p e a t e d
with f u r t h e r 2 m l of e th y la c e ta te , t h e
pooled e t h y l a c e t a t e e x t r a c t s were d r i e d
over anhydrous sodium s u l p h a t e f o r 1 hour
a t room t e m p e r a t u r e . 2 m l of d r i e d e t h y l
a c e t a t e e x t r a c t was evaporated t o dryness
i n a stream o f n i t r o g e n at 4 5 O C . The
r e s i d u e w a s d i s s o l v e d i n 100 m l o f e t h y l
a c e t a t e f o r chromatographic a n a l y s i s .
50 1.11of t h e e x t r a c t w a s i n j e c t e d on a
1.1 Bondapak c18
column. L i n e a r g r a d i e n t
o r e l u t i o n w a s done at 2 m l p e r minute w i t h
aqueous 0.175 M - a c e t i c a c i d (from 40 t o
0 % ) i n methanol d u r i n g 1 0 minutes followed
by i s o c r a t i c e l u t i o n w i t h methanol. T h i s
method gave a good s e p a r a t i o n of chloramb u c i l i n l e s s t h a n 1 2 m i n u t e s . The
absorbance o f e l u t e w a s monitored a t 254,
and 280 nm simultaneously. D e t e c t o r response w a s r e c t i l i n e a r over a range o f 5 t o
1000 ng o f t h e compound. The recovery o f
chlorambucil w a s c o n s t a n t over t h e range o f
0.05 t o 10m.
Leff and Bardsley ( 4 4 ) have r e p o r t e d

pharmacokinetic of chlorambucil i n o v a r i a n
carcinoma u s i n g a new HPLC assay. The
a p p a r a t u s c o n s i s t e d o f a Waters A s s o c i a t e s
~ 6 0 0 0pump, a 10 x 0.46 cm s t e e l column
packed w i t h S 5 ODs, a Waters A s s o c i a t e s
Model U6K i n j e c t o r , a me-Unicam LC3 v a r i a b l e
113
CHLORAMBUCIL
wavelength W d e t e c t o r o p e r a t i n g at 254 nm.

The mobile phase was water: methanol 20:80
( v / v ) w i t h 0.1% ammonium a c e t a t e ( w / v )
The
flow r a t e used w a s 1 ml min-1 ( = 500 p s i ) .
Zakaria and Brown ( 4 5 ) r e p o r t e d a r a p i d

a s s a y f o r plasma chlorambucil and i t s
m e t a b o l i t e ( p h e n y l a c e t i c mustard) u s i n g
reversed-phase l i q u i d chromatography.
Plasma ( 2 0 o r 30 ~ 1 i)s i n j e c t e d d i r e c t l y
i n t h e chromatographic system, which
i n c o r p o r a t e s a 5 cm Co: P e l 1 ODS pre-column
f o r sample c l e a n up. A p a r t i s i l PXS-10/25
ODS column ( 2 5 cm X 4.6 mm) o r , f o r f a s t e r
e l u t i o n , a chromegabond MC-18 column
( 1 5 cm X 4.6 mm) i s used for t h e a n a l y s i s .
The mobile phase being methanol - 0.02 M KH2P04 (1:l o r 11:9, r e s p e c t i v e l y ) a t 1 . 5
o r 1 . 0 m l min-l, r e s p e c t i v e l y . C a l i b r a t i o n
graphs are r e c t i l i n e a r over t h e range o f
i n t e r e s t . Simultaneous d e t e c t i o n a t 280
and 254 nm a l l o w s picomole amounts t o be
determined.
Chatterji _
et _
a1 ( 4 6 ) have s t u d i e d k i n e t i c s
of chlorambucil h y d r o l y s i s u s i n g highp r e s s u r e l i q u i d chromatography. The highp r e s s u r e l i q u i d chromatograph w a s equipped
w i t h a fixed-volume l o o p i n j e c t o r and a
f i x e d wavelength d e t e c t o r . A 250 X 4.6 mm
i . d . reversed-phase column w a s used.
S e p a r a t i o n was performed on a reversed-phase
column (Zorbax C-8, 6 pm average p a r t i c l e
s i z e , Dupont I n s t r u m e n t s , W i l l i n g t o n , D e l . )
and m e t h a n o l - a c e t o n i t r i l e - 0 . 0 1 M a c e t a t e
b u f f e r , pH 4.5 ( 6 5 : 5 : 3 0 ) , a t a flow rate
of 1 . 6 ml/min was used as t h e mobile phase.
A 1 0 p1 full l o o p volume w a s q u a n t i t a t i v e l y
i n j e c t e d , and t h e r e c o r d e r w a s set a t 0.32
a u f s (254 nm d e t e c t o r ) .
Ekrsson e t a1 ( 4 7 ) d e s c r i b e d a reverse
phase HPLC method t o s t u d y d e g r a d a t i o n o f
chlorambucil i n aqueous s o l u t i o n . Chloramb u c i l and i t s d e g r a d a t i o n products;4-( 4-( 2-
chloroethyl-2-hydroxyethylamino)-phenyl)
b u t y r i c acid, are determined i n aqueous

methanol. The chlorambucil s o l u t i o n i s
114
MOHAMMAD TARlQ AND ABDULLAH A. AL-BADR
prepared by adding 1 0 mg o f compound i n

0.5 m l methanol and t h e volume i s made
upto 100 m l with d i s t i l l e d water. A t
d i f f e r e n t t i m e i n t e r v a l s e x t r a c t i o n i s done
with e t h y l a c e t a t e , t h e solvent i s
evaporated t o 0 . 2 m l under a n i t r o g e n
atmosphere. Reversed-phase HPLC a n a l y s i s
i s done on a column (15 cm X 4 mm) o f
Li-Chrosorb RP-18 ( 5 urn). Methanollwater
c o n t a i n i n g 0.01 M phosphoric a c i d i s used
as mobile phase. A W d e t e c t o r a d j u s t e d
a t 253.7 nm w a s used f o r d e t e r m i n a t i o n o f t h e
concentration.
Ahmed _
et _
a1 (48) d e s c r i b e d a q u a n t i t a t i v e
method for d e t e r m i n a t i o n of chlorambucil
i n plasma by r e v e r sed-phas e h i gh-pe r f ormanc e
l i q u i d chromatography. The plasma samples
a r e d e p r o t e i n i s e d by adding 4 volumes o f
a c e t o n i t r i l e a t pH 3 . The t u b e s w e r e c e n t r i fuged f o r 2 minutes and t h e s u p e r n a t e n t w a s
r a p i d l y f r o z e n i n s o l i d carbondioxide-acetone
t o complete d e p r o t e i n i s a t i o n . The t u b e s a r e
c e n t r i f u g e d for 2 m i n u t e s . The a l i q u o t s o
o b t a i n e d w a s analysed on Water's Radial-PAK
c18 ( 1 0 u m ) c a r t r i d g e , w i t h a c e t o n i t r i l e 0.2% a c e t i c a c i d (13:7) as mobile phase.
Absorbance was monitored a t 263 nm and
recorded on a Data Module. A s t a n d a r d curve
was developed u s i n g a c e t o n i t r i l e s o l u t i o n
o f chlorambucil s t a n d a r d .
Acknowledgement
The a u t h o r s would l i k e t o t h a n k Mr. Syed A l i Jawad f o r
t h e t e c h n i c a l a s s i s t a n c e and Mr. Tanvir A . B u t t f o r t h e
s e c r e t a r i a l a s s i s t a n c e i n t y p i n g t h e manuscript.
115
CHLORAMBUCIL
References
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E v e r e t t ; J.J. R o b e r t s ; W . C . J .
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Ross.
J. Chem. S O C . ;
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p. C a l a b r e s i ; A.D. Welch. A. R e v . Med.,

and r e f e r e n c e s c i t e d t h e r e i n .
3.
R.B. L i v i n g s t o n ; S.K. Carter, (1970) " S i n g l e Agents i n

Cancer Chemotherapy" , New York, Plenum P u b l i s h i n g
Corp.
4.
T. Han; E.Z. E z d i n l i ; K . Shimaoka and D.V.

Cancer, 31, 502 (1973
5.
W.H. Knospe, V. Loeb, Jr., and C.M.

Cancer, 33, 555 (1974).
6.
E.Z. E z d i n l i and L. Stutzman.
7.
H.A. Freckman; H.L. Fry and F.L. Mendez, e t_

a1 -5JAMA
189. 23 (1964).
8.
G. Moore; I . D . J . Bross; R . Ausman; S. Nadler; R . J o n e s ;

N . S l a c k ; and A.A. R i m , Cancer Chemother. R e p . 52, 661
(1968 )
(1962)
Desai,
Huguley, Jr.,
JAMA 191, 444 (1965).
9.
10.
The B r i t i s h Pharmacopoeia, H e r M a j e s t y ' s S t a t i o n e r y

O f f i c e , Cambridge, Page 94 ( 1 9 8 0 ) .
"The Merck Index, An Encyclopedia of Chemicals and
Drugs'' , E d i t e d by Motha Windhelz , Nineth E d i t i o n ,
Merck and Company I n c , Rahway, N . J . , USA page 260
(1976 1 .
11. M a r t i n d a l e , The Extra Pharmacopoeia 2 8 t h E d i t i o n , The

Pharmaceutical P r e s s , 1 Iambeth High S t r e e t , S.E.I.
London, page 195 ( 1 9 8 2 ) .
12.
E.G.C. C l a r k e , " I s o l a t i o n and I d e n t i f i c a t i o n of Drugs",

The Pharmaceutical P r e s s , London, page 1016 (1971).
13.
C.R.C.;
" A t l a s o f S p e c t r a l Data and P h y s i c a l Constant
for Organic Compounds", E d i t e d by J.G. Grasselli and
W.M. R i t c h e y , Second e d i t i o n , Volume 11, CRC P r e s s ,
Cleveland, Ohio , 1975.
116
14.
"United S t a t e s Pharmacopeia X X , "Twentieth e d i t i o n ,

United S t a t e s Pharmacopoeia1 Convention, I n c . page 1 2 9
(1980)
15
"The Pharmaceutical Codex" , 1 1 t h E d i t i o n , The

Pharmaceutical P r e s s , London, page 161, (1979)
16.
"Textbook of Organic Medicinal and Pharmaceutical

Chemistry" , E d i t e d by Charles 0. Wilson, Ole Gisvold,
Robert F. Doerge, 6 t h E d i t i o n , J . B . L i p p i n c o t t Co. ,
P h i l a d e l p h i a , Toronto page 220 (1971).
17. "Bentley and Driver I s Textbook of Pharmaceutical
Chemistry", 8 t h Ed., E d i t e d by L.M. Atherden, London,

Oxford U n i v e r s i t y P r e s s , page 526 (1969).
, Mack
Page 1076
18. "Remington's Pharmaceutical Sciences", 15th Ed.

P u b l i s h i n g Co., Easton, Pennsylvania, U.S.A.
(1975) *
1 9*
D.S. A l b e r t s ; S.Y. Chang; and H-S.G.

and S.E. J o n e s , Cancer T r e a t . Rev.,
Chen; B.J. Larcom
20.
A. McLean; R . L . Woods; D . Catovsky and P. Farmer,

(Suppl. ) 33 (1979).
Cancer Treat Rev. ,
6 (Suppl.),
9 (1979).
, Robert T. Dorr and

W i l l i a m L. F r i t z , Elsevier Press , N e w York ( 1 9 8 2 ) .
21. "Cancer Chemotherapy Handbook"
22.
B.T. Hill; P.G. Riches.
Eur. J . Cancer,
(1975)
2: 9
23.
"Cancer Chemotherapy", L.F. Larionov, Pergmon P r e s s ,

London. Page 286-288 (1965).
24.
D.A.G.
25.
S . J . Altman; A. Haut, G.E. Cartwright and M.M.

Wintrobe. Cancer, 9,512 (1956).
26.
L.G.
Galton and M. T i l l , Lancet,

-268, 425 (1955).
I s r a e l s ; D.A.G.
Ann. N . Y . Acad. S c i ,
Galton; M. T i l l , E. Wiltshaw,
68,915
(1958).
CH. A. Doan; B.K. Wiseman and B.A. Bouroncle.

N.Y. Acad. S c i . , 6, 979 (1958).
&.
117
CHLORAMBUCIL
28. A.
Sawitsky; K.R. R a i ; 0. Glidewell and R.T.

Blood, 50, 1049 (1977).
29*
A.A.
Silver,
Green; and J . L . Naiman, Am. J. D i s . C h i l d . ,
190 (1968).
30. S.
Wolfson and M.B.
116,
Olney, JAMA,165, 239
(1957).
4,22 (1975).
Treat. Rep. , 62,1135
(1978).
31. B.R. Reeves, Br. Med. J . ,
32.
H.F. L e r n e r , Cancer
Cancer,
33. S.R. Cole; J . J . Myers and A.U. K l a t s k y , _
_ _ -41, 455,
(1978).
34. P. R i c h t e r ; C.J. Calamers and M.C. Morganfeld, Cancer,

25, 1026 (1970).
Acad. S c i . , 68, 826
35.
L.A.
E l s o n , Ann. N . Y .
36.
H.G.
P e t e r i n g and G . J .
37
~ 5 (1963).
9
N.E.
Kozlov and V.G.
29, 33 (1980).
(1958).
Van G i e s s e n , J . Pharm. S c i . ,
52,
Sbezhneva, Farmatsiya (Moscow),
38.
M.F. E l - T a r r a s ; M.M. E l l a i t h y and N.B.

Pharma Fenn. , 2,
31 (1983).
39.
S.Y. Chang; B.J. Larcom; D.S. A l b e r t s ; B. Larsen; P.D.

Walson and I . G . S i p e s , J . Pharm. S c i . ,
80 (1980).
40.
T. Jakhammer; A. Olsson and L. Svensson, Acta Pharma.

Suec. ,
485 (1977).
41.
K. Norpoth, H.W. Addicks and U. W i t t i n g , A r z n e i m i t t e l Forsch. , 2 3 , 1529 (1973).
42.
H. Ehrsson; S. Eksborg; I . Wallin; Y . Marde and B .

Joansson, J . Pharm. S c i . , 6 9 ( 6 ) , 710 (1980).
Tadros,
69,
14,
43. D . R . N e w e l l ; L . I . Heart and K.R. Harrap., J . Chromatogr

164,Biomed. Appl., 6,114 (1979).
44.
P. Leff and W.G.
1289 (1979).
B a r d s l e y , Biochem. Pharmacol.,
28,
118
45.
46.
)
47.
M. Zakaria and P.R. Brown, J. Chromatogr., 230, Biomed.

Appl. , 19, 381 (1982).
D.C.
Sci
Chatterji, R.L. Yeager and J.F. Gallelli; J. Pharm.

71, 50 (1982).
H. Ehrsson; S. Eksborg;Inger Wallin and S.G. Nilsson,

J. Pharm. Sci., @, 1091 (1980).
48. A.E. Ahmed; M. Moenig; and H.H.J. Farrish, J.

Chromatogr. , 233, (1982), Biomed. Appl. , 22, 392
(1982)
CHLORZOXAZONE
James T. Stewart
Department of Medicinal Chemistry and Pharmacognosy
College of Pharmacy
University of Georgia
Athens, Georgia
Casimir A. Janicki
Director, Analytical Control
McNeil Pharmaceutical
Spring House, Pennsylvania
Foreword, History, Therapeutic Category

Description
2.1
Names, Formula, Molecular Weight
2.2
Appearance, Color, Odor, Taste
3. Synthesis
4 . Physical Properties
4.1
Infrared Spectrum
4.2
Ultraviolet Absorption Spectrum
4.3
Nuclear Magnetic Resonance Spectra
4.4
Mass Spectra
4.5
X-Ray Diffraction Pattern
4.6
Thermal Analysis
4.7
Melting Range
4.8
Solubility
4.9
pKa
5 . Methods of Analysis
5.1
Fluorescence Spectrophotometry
5.2
Ultraviolet Spectrophotometry
5.3
Iodometric Titrimetry
5.4
Gas Chromatography
5.5
5.6
Paper Chromatography
5.7
High Performance Liquid Chromatography
5.8
Non-Aqueous Titrimetry
5.9
Colorimetry
5.10
Color Test
5.11
Polarizing Microscopy
5.12
Potentiometry
6 . Stability
7. Drug Metabolic Products, Pharmacokinetics, and Bioavailabi1ity
1.
2.

VOLUME 16
119

JAMES T. STEWART AND CASIMIR A. JANICKI
120
7.1
Drug Metabolic Products

7.11
Urine Metabolism
7.12
In-vitro Metabolism
7.2
Pharmacokinetics and Bioavailability
8. Identification and Determination in Body Fluids and
Tissues
8.1
8.2
8.3
8.4
Gas Chromatography
9. Identification and Determination in Pharmaceuticals
9.1
Colorimetry
9.2
9.3
9.4
Potentiometry
9.5
10. References
CHLORZOXAZOIW
1.
121
Foreword, History, Therapeutic Category

Chlorzoxazone is a centrally active muscle relaxant for
the treatment of painful muscle spasms associated with
muscloskeletal disorders, such as fibrositis, bursitis,
myositis, spondylitis, sprains, and muscle strains. The
skeletal muscle relaxant properties were first
discovered by Marsh at McNeil Laboratories In the late
1950s (1). Onset of therapeutic activity is observed
within one hour and the duration usually lasts up to 6
hours. The drug was reported to be useful in the
treatment of painful spasm of skeletal motor muscles by
acting at spinal levels of the central nervous system
(2). The drug exhibits minimal adverse effects and
almost no gastrointestinal irritation. Chlorzoxazone was
ranked among the top 100 most widely prescribed drugs
for many years ( 3 ) .
2. Description
2.1 Names, formula, molecular weight
Generic name - Chlorzoxazone
Trade name Marketed by McNeil Pharmaceutical
-__.
under the trade name, Paraflex.
Chemical names - 5 Chloro-2-(3H)-benzoxazolone,
5-Chloro-2-benzoxazolol, 5-Chloro-2hydroxybenzoxazole, 2-Hydroxy-5-Chlorobenzoxazole,
5-Chlorbenzoxazolin-2-one, 5-Chlorobenzoxazolidone.
Chemical Abstracts Registry No. 95-25-0
C7H4C1N02
Mol. Wt. 169.58
2.2 Appearance, Color, Odor, Taste - Odorless colorless

crystals or a white to creamy white crystalline
powder with a bitter taste.
122
3. Synthesis
Chlorzoxazone is synthesized by either the acid hydrolysis of 2-amino-5-chlorobenzoxazole (4) or by treating
2-amino-4-chlorophenol with phosgene in ethyl acetate
(5). It can also be prepared from the reaction of
sodium ethoxide with 2-ethoxycarbonylamino4-chlorophenol in tetralin (6).
t
4.
ClCOCl
Physical Properties
4.1 Infrared I
SDectrum - An FTIR sDectrum of chlorzoxazone is shown in Figure 1.
-1
cm
3470
3155
3117
3087
3057
2978
2282
1924
1885
1772
1621
1482
1463
%T
72.6
36.3
39.7
37.3
35.2
46.4
69.1
67.4
70.2
3.2
30.5
13.5
34.3
-1
cm
% T
1367 43.8
1330
1300
1262
1153
1101
1064
964
923
867
845
805
53.6
27.3
26.2
21.1
58.7
54.8
16.4
33.1
46.1
23.7
19.2
-1
% T
cm
732 52.8
713
600
591
552
417
394
382
352
234
216
210
32.2
45.8
41.8
49.0
67.6
70.2
79.1
66.1
8.8
5.0
6.7
11i
-
rI
r
I
rI
I
I
I
I
I
.....
....
i
i
I
i
Fig. 1
FTIR spectrum of chlorzoxazone
124
4.2
Ultraviolet Absorption Spectrum - A solution of the

drug in absolute methanol shows a maximum at 282 ?
2 nm and a m i n i m u m a i 248 _t 2 nm.
There is an
inflection at about 228 GII! ( s e e Figure 2).
--
4.3. Nuclear
lvlagnetic
hpectra
- 13C and proton
- Resenance nuclear magnetic resonance spectra are shown in
Figures 3 and 4.
C
13C NMR
Carbon
6(ppm)
a
39.4 DMSO-dc
J
b
109.8
C
110.8
d
121.4
e
127.7
f
131.7
g
142.1
h
154.2
NH
d
Proton NMR
Proton
fi (DDm)
r
a
2.49 DMSO-d,
b
3.36 HDO
C
7.10
d
7.13
e
7.28
f
11.8
~~
.r
--I
CHLORZOXAZONE
350
220
WAVELENGTH ( n m )
Fig. 2
W spectrum of chlorzoxazone in methanol
Fig. 3
13C-NMR spectrum of chlorzoxazone in deuterated dimethylsulfoxide
127
"-2.1
Fig. 4
- ......7 7
1d.r
8.1
..-.--I---*
.I
7.1
6.1
PPM
5.0
4.1
3.1
2.1
1.0
'H-NMR spectrum of chlorzoxazone in deuterated dimethylsulfoxide
128
Spectra - Electron impact (EI) and chemical

4.4 EM
ionization (CI) mass spectra are shown in Figures 5
and 6.
4.5 X-Ray Diffraction - Figure 7 shows an X-ray
diffraction pattern for chlorzoxazone powder.
4.6 Thermal Analysis - A DSC thermogram of

chlorzoxazone is shown in Figure 8.
4.7 Melting Range - The melting point range is
189-194C.
4.8 Solubility - Chlorzoxazone is very slightly soluble

in water (0.2 - 0.3 mg/ml), sparingly soluble in
ethanol and methanol, soluble in chloroform (1 g in
It is freely
250 ml) and ether (1 g in 60 ml).
soluble in aqueous solutions of ammonia and alkali
hydroxides.
4.9 pKa
The pKa of chlorzoxazone in 8.3.
5.1 Fluorescence Spectrophotometry - Stewart and Chan
have studied the reaction of chlorzoxazone with
dansyl chloride, fluorescamine, 2,4-dihydroxybenzaldehyde, and salicylaldehyde to form fluorophores (7). Fluorescamine gave the highest intensity product and was used to analyze chlorzoxazone
in the 0.27 - 3.4 pg/ml range. The same authors
also reported that chlorzoxazone exhibits native
fluorescence in chloroform using excitation and
emission wavelengths of 286 and 310 nm, respectively (8).
5.2
Ultraviolet Spectrophotometry - Conney et a1

utilized a back-extraction approach to quantitate
chlorzoxazone at 289 nm (9). The drug can also be
directly determined at 282 nm in absolute methanol
(10)
5.3
Iodometric Titrimetr
Beral et a1 titrated
c h l o r z o x a z o d t r e a t m e n t with boiling 10%
sulfuric acid, potassium bromide and potassium
bromate) with 0.1 N sodium thiosulfate after
potassium iodide was added to a cooled solution
(11).
5.4
Gas Chromatography -
Kaempe has chromatographed

chlorzoxazone on a packed 15% Dexsil 300 on HP
11 9
90 80
70 -
60 50 -
113
30 -
20
51
63
d.
I,,,
140
.. .
.. ,
I;
. . ,[ASS
130
200
40
60
Fig. 5
Electron impact (EI) mass spectrum of chlorzoxazone
30
100
120
160
MH+
'01
80 -
60 -
%I
!!
50
Fig. 6
90
I30
m/e
I 70
210
250
Chemical ionization (CI) mass spectrum of chlorzoxazone
90 -
80 -
70 -
60 -
50
W
e
Fig. 7
X-Ray diffraction pattern of chlorzoxazone
5.00
CHLORZOXAZONE.
SCAN RATE:
0.00
'
L O T 2933
1.00 mg
WT:
*. MI
5.00 deg/rnxn
380.M
4M.M
42O.M
4 h no
bu). MI
TEMPERATURE
(K)
Fig. 8 DSC thermogram of chlorzoxazone
460.00
5th 01
DSC
CHLORZOXAZONE
133
Chromosorb W 801100 mesh glass column (6 ft x 6.4

m i.d.).
The retention time was 0.62 relative to
caffeine using a column temperature of 220C (12).
Parker et a1 attempted to chromatograph the drug on
SE-30 (0.05% on glass microbeads 60/80 mesh) at 150
and 165C column temperatures with no success (13).
Desiraju et a1 chromatographed chlorzoxazone on a 6
ft x 4 mm i.d. glass column packed with 3% OV-1 on
60/80 mesh Gas Chrom Q (14). Column temperature
was 130C and retention time was 3.25 minutes using
a column flow rate of 30 ml/minute.
Beckett et a1
- reported that chlorzoxazone can be
chromatographed on a 2.5% SE-30 on 80/100 mesh
Chromosorb W acid-washed HMDS treated 5 ft x 4 mm
i.d. glass column at a column temperature of 225C
(15). Retention time was 0.69 relative to
diphenhydramine. Nitrogen was used as carrier gas
at 50 ml/min a.nd FID detection was utilized.
Ardrey and Moffat also reported that a 2 m x 4 mm
i.d. glass column containing 2.5% SE-30 on 80/100
mesh Chromosorb G acid-washed HMOs treated will
separate chlorzoxazone using a column temperature
of 173C (16). Retention time is given relative to
n-alkanes.
Pedroso and Moraes have chromatographed chlorzoxazone on 2.5% SE-30 and 3% OV-17 at 200 and 220C
column temperatures, respectively (17). They used
the retention data to perform qualitative analysis
of the drug based on Kovat's Retention Indices.
5.5
Stationary Phase
Mobile Phase
Silica Gel HF254
Chloroform-methanol
Chloroform-methanol
Chloroform-methanol
Chloroform-methanol
Chloroform-methanol
Kieselgel 60F254
19:l
9:l
4:l
7:3
3:2
Toluene ethylacetate 85% formic acid (50:45:5)
18
0.32
0.58
0.87
1.00
1.00
0.69
19
134
Kieselgel 60F254
Kieselgel 60F254
Silica Gel 60F254
0.6819
Toluene
Isopropanol concd. ammonium
hydroxide (70:29 :1)
0.55
19
Toluene-dioxane
methanol ammonium hydroxide
(20:50:20:10)
Toluene Acetone
2N Acetic Acid
(30:65:5) -
0.8320
20
Silica Gel 60F254
Toluene
Isopropanol
Ethyl Acetate 2N Acetic Acid
(10:35:35:20)
0.95
Bonded C18F
Methanol 0.5% Phosphoric Acid

3% Sodium Chloride
(60:10:30)
0.3220
Bonded C18F
Isopropanol
0.5% Phosphoric Acid
3% Sodium Chloride
( 4 0 : 10:50)
Bonded C18F
Isopropanol Methanol
0.5% Phosphoric Acid
3% Sodium Chloride
(23:23:10:44)
Bonded C18F
Bonded C18F
Tetrahydrofuran Methanol 0.5% Phosphoric Acid

3% Sodium Chloride
(28:28:10:34)
Methanol 2 N Ammonia 3% Sodium Chloride
(60:10:30)
20
0.35
0.33
20
20
0.32
20
0.60
135
CHLORZOXAZONE
Bonded C18F
Bonded C18F
Bonded C18F
Bonded C18F
Bonded C18F
Bonded C18F
Bonded C18F
Bonded C18F
Bonded C18F
5.6
Isopropanol 2N Ammonia
3% Sodium Chloride
(40:10:50)
0.4720
Isopropanol Methanol
2N Ammonia 3% Sodium Chloride
(23:23:10:44)
0.4320
Tetrahydrofuran Methanol 2N Ammonia 3% Sodium Chloride

(28:28:10:34)
0.5520
Acetonitrile 3% Sodium Chloride

(50:50)
0.4620
Methanol 3 % Sodium Chloride

(60 :40)
0.32
Ace tone
3% Sodium Chloride
(50:50)
0.25
Tetrahydrofuran 3% Sodium Chloride

(43:57)
20
0.18
Isopropanol 3% Sodium Chloride

(40:60)
0.35
Methanol Isopropanol Tetrahydrofuran 3 % Sodium Chloride

(17:17:17:49)
0.36
20
20
20
20
Paper Chromatography - Clarke reported that chlorzoxazone gave an R of 0.93 using a mobile phase of
87 :13 n-butanol-waEer containing 0.48% citric acid
(21)
136
5.7
5.8
5.9
High Performance Liquid Chromatography - Stewart

a1 have chromatographed chlorzoxazone on an octadecylsilane column using varying proportions of
absolute methanol-distilled water (22). Chlorzoxazone was effectively separated from acetaminophen
in a mixture with 50:50 methanol-water at a
2.0 ml/min flow rate using refractive index detection. The drug has also been chromatographed on
an octadecylsilane column using 60:40 watermethanol (23), and a 1:l acetonitrile-water mixture
(10). These methods utilized ultraviolet detection
at 280 nm.
Non-Aqueous Titrimetry - Chlorzoxazone is titrated
with 0.1 N sodium methoxide in dimethvlformamide
using thymol blue as indicator ( 2 4 ) . The drug can
also be titrated in dimethylsulfoxide using O.1N
propanolic potassium hydroxide as titrant and
metanil yellow as visual indicator (25).
Colorimetry - Sanghoi reported a colorimetric
procedure for chlorzoxazone based on base hydrolysis of the drug followed by diazotization of the
product with nitrous acid formed -in situ (26). The
color measured at 405 nm is stable for 60 minutes
and obeys Beer's Law in the 4-32 pg/ml range.
5.10
Color Test - The Koppanyi-Zwikker

-violet color (27).
5.11
Polarizing Microscopy - Watanabe et al., reported

that polarizing microscopy can be used as a qualitative identification tool for chlorzoxazone (28).
5.12
test gives a
Potentiometry A potentiometric method for the

determination of chlorzoxazone, based on the use of
a carbon dioxide gas-sensing electrode, is described by Tagami and Muramoto (29). Upon refluxing the drug with 3N sodium hydroxide, aminophenol
and sodium carbonate are formed.
Acidification of the reaction mixture yields
carbon dioxide which is sensed by the gas-permeable
membrane electrode. A line r calibrati n plot was
obtained within the 3 x 10-8 to 5 x 10-3 M- range of
chlorzoxazone.
Stability
Chlorzoxazone is stable in the solid state for up to 5
years at room temperature. Storage at temperatures for
CHLORZOXAZONE
137
up to 80C and in artificial sunlight conditions (1000

foot candles) for up to 4 weeks did not cause
significant degradation.
Tablets of chlorzoxazone have also been found to be
stable for up to 5 years at room temperature. Temperatures for up to 60"C, humidity conditions of 40"C/80%
relative humidity and artificial sunlight (1000 foot
candles) or up to 4 weeks have not caused degradation
of the product.
An aqueous suspension of the drug in distilled water or
acidic media is generally stable but in solutions with a
pH greater than 7, the solution is generally not stable.
Chlorzoxazone yields 2-amino-4-chlorophenol as its major
degradation product.
7.
Drug Metabolic Products, Pharmacokinetics, and Bioavailabi1ity

7.1 Drug Metabolic Products
7.11 Urine metabolism - The metabolic product of
chlorzoxazone was isolated from urine of human
subjects administered an oral dose of the
drug. Ether extraction of urine yielded a
residue which was identified as 6-hydroxychlorzoxazone (9). Studies showed that the
metabolite is eliminated as the glucuronide
conjugate to the extent of 74% of the dose.
6-Hydroxychlorzoxazone had little or no muscle
relaxant activity when tested in mice and
rats. In 1982, Twele and Spiteller identified
two additional chlorzoxazone metabolites from
human urine, a 5-chloro-2,4-dihydroxyacetanilid produced from 6-hydroxychlorzoxazone by ring cleavage and acetylation of the
amino moiety, and 6-hydroxybenzoxazolone,
produced by substitution of hydrogen for
chlorine and hydroxylation of a neighbor
ring-carbon atom (30).
7.12
In-vitro metabolism - Chlorzoxazone was

incubated with fortified homogenates of mouse
and rat liver. The reaction was stopped by
addition of 3N hydrochloric acid. Spectrophotometric assay of a sample preparation
identified 6-hydroxy-chlorzoxazone as the
major metabolite (31).
It has been established that the liver is the principal site of
metabolism.
138
7.2
Pharmacokinetics and Bioavailability - The

following pharmacokinetic data were obtained
following a 750 mg oral dose of chlorzoxazone to
human subjects ( 1 4 ) :
Peak plasma concentration
Peak time
AUC (0-10 hr)
Apparent Volume of
Distribution (V/F)
Elimination Rate Constant (K)
Absorption Rate Constant (ka)
Elimination Half Life (t )
Apparent Clearance (KV/F)'
36.3 f 2.3 pg/ml

38 f 3.3 minutes
4084 f 284 pg min/ml
13.70 f 5.07 liters
0.73 2 0.29
2.28 2 2.08
1.12 f 0.48
148.0 f 39.9
hrI:
hr
hr
ml/min
There is rapid absorption and elimination of

chlorzoxazone. Chlorzoxazone is widely distributed
in plasma and tissues. Less than 1% of the drug is
excreted unchanged in urine. The drug concentration in fat is twice the plasma concentration,
while liver, muscle, brain and kidney concentrations are one-half or less that found in plasma.
The apparent volume of distribution of approximately 14 liters suggests that the drug is not widely
distributed and that it would be confined to the
circulatory system and possibly the extracellular
fluid
8.
Identification and Determination in Body Fluids and

Tissues
8 . 1 Ultraviolet Spectrophotometry - Conney et a1
reported the extraction and estimation of
chlorzoxazone in urine ( 9 ) . Petroleum ether
containing 1.5% isoamyl alcohol was shaken with the
urine sample for 60 minutes. The organic phase was
separated and the drug was back extracted into 0.5
N sodium hydroxide. Absorbance read at 289 nm
followed Beers Law. Recovery of drug from urine
was 93 f 2%.
8.2
Fluorescence Spectrophotometry - Stewart and Chan

reported the quantitation of chlorzoxazone from
plasma and/or -urine samples ( 8 ) . Chlorzoxazone was
extracted from the biological sample at pH 2.5 with
petroleum ether containing 1.5% isoamyl alcohol.
The organic phase was re-extracted with strong
base, the pH adjusted to pH 6 . 8 with hydrochloric
acid, and the drug extracted into chloroform for
the measurement step. Excitation and emission
wavelengths were 286 and 310 nm, respectively.
CHLORZOXAZONE
139
Drug concentration arid fluorescence were linear

from 0.06 - 3.2 pg/ml and 0.13 - 3.0 vg/ml for
plasma and urine, respectively. Limits of
detection were 6 0 and 130 ng/ml for plasna and
urine, respectively. Percent recoveries of
chlorzoxazone from plasma and urine were 86.63 2
1.66 and 95.37 2 2.42%, respectively.
Stewart and Chan also reported a procedure for the
analysis of chlorzoxazone based upon solvent
extraction of the drug from human plasma or urine
followed by chemical derivatization with fluorescamine (7). Linear detection range was 2-10 pg/ml
using excitation and emission wavelengths of 370
and 505 nm, respectively.
8.3
High Performance Liquid Chromatography - Honigberg,

Stewart, and Coldren reported an HPLC assay for
chlorzoxazone and 6-hydroxy-chlorzoxazone in plasma
based on ether extraction of the compounds from
acidic plasma (22). The separation was achieved on
an octadecylsilane column (30 cm x 3.9 mm, i.d.) at
a flow rate of 2 ml/min with UV detection at 280 nm
and a mobile phase of 40:60 absolute methanoldistilled water. Retention times for the hydroxy
compound and drug were approximately 4 and 8
minutes, respectively. The limit of detection for
each compound was calculated to be 8 0 ng at
signallnoise = 2.
Another HPLC assay for chlorzoxazone in plasma
reported by Ng at McNeil Pharmaceutical (32) used
at 1:l acetonitrile-distilled water mobile phase,
an octadecylsilane column (25 cm x 4..6 mm, i.d.), a
2.0 ml/min flow rate, and UV detection at 280 nm.
Retention time for the drug is 2.9 minutes. Ethyl
5-chloro-2-hydroxycarbanilate was used as internal
standard (retention time of 4.5 minutes).
A plasma
sample was made acidic with hydrochloric acid and
extracted once with ethyl acetate. The ethyl
acetate was evaporated to dryness and the residue
dissolved in methanol prior to injection into the
HPLC
Stewart and Carter reported modifications in the

above mentioned HPLC assay by Honigberg, Stewart
and Coldren in which an octadecylsilane solid-phase
extraction column was used t o separate chlorzoxazone and 6-hydroxychlorzoxazone from human serum
(33). Percent recoveries were 90.24 2 4.28 and
JAMEST.STEWART AND CASIMIRA.JANICKI
140
86.06
It: 3.58% for drug and metabolite, respectively. The separation was achieved on an octadecylsilane column using a mobile phase of 5 0 : 5 0
acetonitrile-aqueous 0 . 0 5 M sodium dihydrogen
phosphate, pH 4 . 5 with phenacetin as internal
standard. The two compounds were detected at the
glassy carbon electrode using a cell potential of +
1300 mV. These modifications halved the original
HPLC analysis time and increased detection for each
compound to 2.5 ng, 30 times the previous limit of
detection using 280 nm.
8.4
Gas Chromatography -
Desiraju et a1 reported a
packed column method for the quantitation of
chlorzoxazone in plasma samples ( 1 4 ) . The drug is
extracted from acidified plasma with ethyl acetate
containing the internal standard, n-hexadecane.
The ethyl acetate is separated andevaporated to
dryness. Pyridine and acetic anhydride are added
to the residue, the tube is capped, and the
reaction allowed to proceed at 42C for 20 minutes.
An aliquot of the mixture is injected into the gas
chromatograph.
The GC parameters were flame ionization detection

(30 and 300 ml/min flow rates for hydrogen and air,
respectively), carrier gas (nitrogen at 30 ml/min),
6 ft x 4 mm i.d. glass column packed with 3% OV-1
on 6 0 / 8 0 mesh Gas Chrom Q, and column temperature
of 130C. The retention times of chlorzoxazone and
internal standard were 3.25 and 4 . 5 minutes,
respectively. The limit of detection was 0 . 5 ug/ml
using 1 ml of plasma. The calibration curve was
linear in the 0.5 - 25 ug/ml range. Extraction
efficiency for chlorzoxazone was 88 It: 8% (n = 6 ) .
9.
Identification and Determination in Pharmaceuticals

Colorimetry - Twenty tablets were weighed and
powdered in a dry mortar and pestle. An aliquot of
the powder equivalent to 40-60 mg of chlorzoxazone
was hydrolyzed with 20% sodium hydroxide solution,
cooled, and reacted with nitrous acid to yield the
diazonium salt, whose absorbance measured at 405 nm
obeyed Beer's Law in the 4-32 ug/ml range ( 2 6 ) .
Acetaminophen and indomethacin were shown to
interfere with the method. Recovery of chlorzoxazone in bulk powder and tablet samples was in the
9 8 . 3 - 99.9% range utilizing the assay procedure.
9.1
CHLORZOXAZONE
9.2
141
Ultraviolet Spectrophotometry - Kirschner of McNeil

Pharmaceutical reported an assay for the drug in
tablets as follows (10):
Standard Preparation: Dissolve a suitable quantity
of USP Chlorzoxazone KS, accurately weighed, in
methanol, and dilute quantitatively and stepwise

with methanol t o obtain a solution having a known
concentration of about 20 pg/mL.
Assay Preparation: Weigh and finely powder not
less than 20 Chlorzoxazone tablets. Transfer an
accurately weighed portion of the powder,
equivalent to about 100 mg of chlorzoxazone, to a
100-ml volumetric flask. Add about 80 ml of warm
methanol, and shake by mechanical means for about
15 minutes. Dilute with methanol to volume, and
mix. Filter, discarding the first 20 mL of the
filtrate, and pipet 2 ml of the filtrate into a
100-ml volumetric flask. Dilute with methanol to
volume, and mix.
Procedure: Concomitantly determine the absorbances
of the Assay and Standard Preparations at 282 nm,
with a suitable spectrophotometer, using methanol
as the blank. Calculate the quantity, in mg, of
chlorzoxazone in the portion of tablets taken by
the formula 5C (Au/As), in which C is the
concentration, in pg per ml, of USP chlorzoxazone
RS in the Standard Preparation and Au and As are
the absorbances of the Assay and Standard Preparations, respectively.
9.3
9.4
Fluorescence Spectrophotometry - Stewart and Chan

used the intrinsic fluorescence of chlorzoxazone to
assay for the drug in commercial tablets containing
acetaminophen ( 8 ) . Recovery of drug was in the
98-101% range. These same authors also
demonstrated that chlorzoxazone could be
successfully analyzed in a tablet dosage form
chemical derivatization with fluorescamine to form
a fluorophore (7).
Potentiometry - Tagami and Muramoto described a
method based on the use of a carbon dioxide
gas-sensing electrode ( 2 9 ) . Twenty tablets were
powdered and a portion equivalent to about 424 mg
of drug was weighed and extracted with 20 ml of
acetone. After stirring and centrifugation, the
supernatant acetone solution was removed and
142
diluted with additional acetone. This extraction

procedure was performed four times. The collected
acetone fractions were evaporated to dryness. The
residue was refluxed for 2 hr with 50 ml of 3N
sodium hydroxide and after acidification, the
carbon dioxide was determined using a gas permeable
electrode. Mean recovery of chlorzoxazone was
99.6% with a standard deviation of 0.24.
9.5
High Performance Liquid Chromatography - The USP

XXI method for Chlorzoxazone with Acetaminophen
tablets is a gradient HPLC method (34). In the
method, m-chloroaniline, p-aminophenol (a degradation prozuct of acetaminophen), p-chlorophenol and
2-amino-4-chlorophenol are separated from
chlorzoxazone, acetaminophen and the internal
standard phenacetin.
An example of the separation is as follows:
Retention time, minutes
Component
2.3
Acetaminophen
6.0
2-Amino-4-chlorophenol
p-Aminophenol
7.1
m-Chloroaniline
7.5
Internal Standard (phenacetin) 8.3
9.2
p-Chlorophenol
10.1
Chlorzoxazone
10. References
1.
2.
3.
4.
5.
6.
7.
8.
9.
Current Therapeutic Research (1975) 17,p 123, Literature Services Section, McNeil Laboratories, Inc., Fort
Washington, PA.
A.P. Roszkowski, J. Pharmacol. Exptl. Therap., 129
(1960) p 75.
Pharmacy Times, 48 (1982) 23-32.
D. Marsh, U S Patent 2, 895, 877 (1959).
J. Sam and J . N . Plampin, J.Pharm. Sci., 53, (1964)
538-544.
K. Harsanyi, F. Toffler, KD. Korbonitis and G.
Leszkovszky, Hung. Patent 150, 577 (1961); thru Chem.
Abstr., 60, 5507b (1964).
J.T. Stewart and C.W. Chan, Anal. Letters, Bl1 (9),
(1978) 667-680.
J.T. Stewart and C.W. Chan, J. Pharm. Sci., 68 (1979)
910-912.
A.H. Conney, N. Trousof and J.J. Burns, J. Pharmacol.
Exptl. Therap., 128 (1960) 333.
CHLORZOXAZONE
143
10. E.L. Kirschner, McNeil Pharmaceutical, Spring House, PA,

(1986), Personal communication.
11. H. Reral, V. Gamentzy and I. Bucur, Rev. Chim.
(Bucharest) 16 (1965) 322.
12. B. Kaempe, Arch. Pharm. Chemi., g . Ed., 1. (1974), 145.
13. K.D. Parker, C.R. Fontan and P.L. Kirk, Anal. Chem., 34
(1962) 757.
14. R.K. Desiraju, N.L. Renzi, R.K. Nayak and K. Ng, J,
Pharm. Sci. 72 (1983) 991-994.
15. A.H. Beckett,G.T.
Tucker and A.C. Moffat, J. Pharm.
Pharmac., 19 (1967) 273.
16. "Clarke's Isolation and Identification of Drugs," 2nd
Edition, A.C. Moffat, Ed., The Pharmaceutical Press,
London, 1986, p 193.
1 7 . R.C. Pedroso and E.D.C.F. Moraes, Rev. Farm. Rioqulm.
Univ. S. Paulo, 18 (1982) 183-195.
J.
18. I. Ullah, D.E. Cadwallader, and I.L. Honigberg, Chromatogr., 46 (1970) 211-216.
19. R.A. Egli andS. Tanner, Fresenius 2. Anal. Chem. 295
(1979) 398-401.
J. Chromatogr., 2 (1984)
20. R.A. Egli and S. Keller, 249-256.
21. E.G.C. Clarke, "Isolation and Identification of Drugs,"
Volume 1, The Pharmaceutical Press, London (1969), p 34.
J.
22 * J.T. Stewart, I.L. Honigberg, and J.W. Coldren, Pharm. Sci., 68, (1979) 32-36.
J.
23. I.L. HonigberE J.T. Stewart, and J.W. Coldren, Pharm. Sci., 68, (1979) 253-255.
24. Japan Pharmacopeia, 9th Ed., Society of Japanese
Pharmacopeia, Japan, p 157.
25. V.J. Schnekenburger and M. Quade-Henkel, Dtsch.
Ap0th.-Ztg., 124 (1984) 1167-1170.
26. N.M. Sanghoi, N.G. Joshi, and K.S. Dubal, Indian Drugs,
18 (1981) 299-302.
27. Tlarke's Isolation and Identification of Drugs," 2nd
Edition, A.C. Moffat, Ed., The Pharmaceutical Press,
London, 1986, p 465.
28. A . Watanabe. Y. Yamaoka. K. Kuroda, T. Yokoyama and T.
Umeda, Yakugaku Zasshi,-l05 (1985) -481-490.29. S. Tagami and Y. MuramotFChem. Pharm. Bull, 32 (1984)
1018-1024.
32 (1982)
30. V.R. Twele and G. Spiteller, Arzneim. Forsch., 759-763.
31. A.H. Conney and J.J. Burns, J. Pharmacol. Exptl. Ther.,
128 (1960) 340.
Ng, McNeil Pharmaceutical, Spring House, PA (1986),
32.
Personal Communication.
33. J.T. Stewart and H.K. Carter, J. Chromatogr., Biomed.
Appl., 380 (1986) 177-183.
m.
144
34.
Second Supplement, U n i t e d S t a t e s P h a r n a c o p e i a , 2 1st

E d i t i o n , U n i t e d S t a t e s Pharmacopeial Convention,
B e t h e s d a , MD, (1985), p . 1 8 2 7 .
L i t e r a t u r e surveyed t h r o u g h December, 1986.
CYCLOSPORINE
*Mahmaud M.A. H c u a n and **Mohammed A . AL-Yahya
06 P h c u u n a c W c d Chmbin.thy, Vep&evLt 06
P h m a c e d c d Che.mhin.thy, CoUege 04 P h m a c y , King Saud
U n i w m L t y , Riyadh, Saudi Atrabiu.
*Pho6ehboh
PhOdeAhotr 06 Phahmacognob y , Ve-ed

06
Phahmacognon y.
VhecZoh 06 ,the MediciMae Akomatic and Pohonoun Plan&
Runahch Cenhn, CoLtege 06 Phahmacy, King Saud U n i w m L t y ,
Riyadh, Saudi k a b i a .
**kshoch~&

VOLUME 16
145

MAHMOUD M. A. HASSAN AND MOHAMMED A. AL-YAHYA
146
1. History and Therapeutic Category

2.
Description
2.1 Nomenclature
2.2 Formulae
2.2.3 Degradation and Structural Elucidation
2.2.4 Conformation
2.3 Molecular Weight
2.4 Appearance, Color, Taste, Odor
3. Physical Properties
3.1
3.2
3.3
3.4
3.5
Crystal Properties
Melting Range
Solubility
Optical Rotation
Spectral Properties
4. Iso lation
5.
Biosynthesis
6. S y nthesis
6.1 Cyclosporine
6.2 Cyclosporine Analogs
6.3 Structure-Activity Relationship
7.
Metabo1ism
8.
Pharmacokinetics
9. Pharmacological Activity and Clinical Applications

10. Pharmaceutical Forms

11.1
11.2
11.3
11.4
Introduction to Analysis
Radioimmunoassay
12. References
13. Acknowledgement
147
CYCLOSPORINE
Cyclosporine
1.
H i s t o r y and T h e r a p e u t i c Category
It w a s from Hardanger Vidda, a b l e a k t r e e l e s s p l a t e a u
i n t h e South o f Norway, t h a t a s o i l sample w a s o b t a i n e d

i n t h e e a r l y s e v e n t i e s . The fungus i s o l a t e d from t h i s
sample w a s c a l l e d Tolypocladium i n f l a t m Gams (1),

formerly ( 2 ) d e s i g n a t e d as Trichoderma p o l y s p o r m
(Link ex P e r s . ) R i f a i or Cylindrocarpon luciderm Booth
( 3 ) . S e v e r a l m e t a b o l i t e s o f t h i s fungus have been
i s o l a t e d of which c y c l o s p o r i n s A , C and G t o have so f a r
been shown t o p o s s e s s s t r o n g immunosuppressive a c t i v i t y .
The main component i n normal f e r m e n t a t i o n b r o t h s w a s
named c y c l o s p o r i n A ( A ) , now known as c y c l o s p o r i n e .
A s r e p o r t e d by Bore1 e t a1 ( 5 - 9 ) , c y c l o s p o r i n e i s a
s e l e c t i v e immunosuppressive drug w i t h a n t i f u n g a l and
antiinflammatory p r o p e r t i e s .
Today c y c l o s p o r i n e can be regarded as t h e m o s t
important r e c e n t i n n o v a t i o n i n t h e f i e l d of organ t r a n s plantation.
2. D e s c r i p t i o n
2.1
Nomenclature
2.1.1
Chemical Names
Cyclosporin A; Cyclosporine.
2.1.2
Generic Names
Cyclosporine (USAN) and i s adopted f o r t h e
b a s i c s t r u c t u r e , meanwhile t h e f o l l o w i n g
names h a s been a c c e p t e d i n o t h e r c o u n t r i e s .
England, c y c l o s p o r i n (BAN name); F r a n c e ,
c i c y l o s p o r i n e ; S w i t z e r l a n d and o t h e r s ,
c i c l o s p o r i n (INN name, WHO).
2.1.3
Trade Names
Sandimmune , Sandimmun
148
2.2
Formulae
2.2.1
Ehpiricd
N O
C 6 2 H l l l 11 1 2
2.2.2
Structural
2.2.3
Cyclosporine ( F i g . 1) i s a n e u t r a l , hydrophobic c y c l i c p e p t i d e composed o f 11 amino

a c i d r e s i d u e s , a l l having t h e S-configurat i o n o f t h e n a t u r a l L-amino a c i d s , except
f o r t h e D-Ala i n p o s i t i o n 8, which has t h e Rc o n f i g u r a t i o n , and S a r i n p o s i t i o n 3. Seven
a c i d s a r e N-methylated. Ten are known a c i d s ,
t h e y are a-Abu i n p o s i t i o n 2 , Sar-in p o s i t i o n
3, N-MeLeu i n p o s i t i o n 4,6,9 and 1 0 , V a l i n
p o s i t i o n 5, Ala i n p o s i t i o n 7 , D-Ala i n p o s i t i o n 8 , N - M e V a l i n p o s i t i o n 11 and MeBmt i n
p o s i t i o n 1.
Degradation and S t r u c t u r a l E l u c i d a t i o n
The s t r u c t u r e o f c y c l o s p o r i n e [l] ( F i g . 1)
was determined by chemical d e g r a d a t i o n (4)
and x-ray c r y s t a l l o g r a p h i c a n a l y s i s (10,ll).
Also with an x-ray c r y s t a l o g r a p h i c a n a l y s i s
of t h e iodo d e r i v a t i v e [ l a ] . It has t h e
molecular formula C62HlllN11012 as deduced
by NMR and mass s p e c t r a . Hydrolytic
cleavage of c y c l o s p o r i n e f u r n i s h e d 11 amino
a c i d s as fragments, among them an a r t i f a c t
o f a new C9-amino a c i d . The amino a c i d
sequence w a s determined by Edman d e g r a d a t i o n
o f i s o c y c l o s p o r i n , a b a s i c rearrangement
product formed from c y c l o s p o r i n e by N,Oa c y l m i g r a t i o n . On t h e b a s i s of t h e
chemical, s p e c t r o s c o p i c and c r y s t a l l o g r a p h i c
e v i d e n c e , t h e s t r u c t u r e of c y c l o s p o r i n e w a s ,
e l u c i d a t e d as t h e n e u t r a l c y c l i c o l i g o p e p t i d e
It i s composed of e l e v e n amino a c i d r e s i d u e s ,
a l l having t h e L-configuration of t h e n a t u r a l
amino a c i d s , except f o r t h e D-alanine i n
p o s i t i o n 8 and t h e non-chiral s a r c o s i n e
(N-methylglycine) i n p o s i t i o n 3. Seven
amino a c i d s
i n p o s i t i o n s 1, 3, 4, 6 , 9 ,
1 0 and 11- are N-methylated.
Ten o f t h e
Fig. I . Struciure of cyclosporine 1 (schematic). McBmt

nyl]-4,N-dimcthyl-~-threoninc
(see text).
(4R)-4-[()-2-bute-
150
J
cyclorporine
not obtoincd
Ib
cyclic derivotivool the
Nmdhyl-C9-amino ocid
cyclosporlnc
IS OCYCLOSWRINE
onhydromrthylthio
Ic
hydontoin
de rivot ive
Edman d e g r a d a t i o n with CHI-NCS under c a r e l u l l y

chosen mild conditions
p e p t i d e hydrolysis w i t h
c h r o m a tog raphy.
6 N HCI followed by ion erchange
n
'F,.I
TlOAcl It
-+
t--Zn I HOAc
cy cloiporlnc
CYCLOSPORINE
151
eleven ring members are derivatives of

known aliphatic amino acids:
a-aminobutyric acid (Abu) in position 2,
Sarcosine (S.ar) in position 3,
N-methylleucine (MeLeu) in positions 4, 6,
9 and 10,
Valine (Val) in position 5,
Alanine (Ala) in position 7,
D-Alanine (D-Ala) in position 8, and
N-methylvaline (Me Val) in position 11.
The amino acids were readily characterised
following acid hydrolysis of cyclosporine.
The novel amino acid was found to have the
composition ( 2S, 3R, 4R, 6E)-3-hydroxy-bmethyl-2-( methylamino)-6-octenoic acid, and
in accord with amino acid nomenclature, is
now designated as (4R)-4-[(E )-2-butenyl]4-N-dimethyl-L-threonine and abbreviated
Thus the novel amino acid has the
MeBmt .
polar features of an N-methyl-L-threonine
which is substituted at the end of the
carbon chain by butenyl and methyl groups.
This amino acid was hitherto not known in
the free form, since only antifacts and
derivatives were obtained from degradation
experiments on cyclosporine(4).
Hydrolysis of cyclosporine followed by ion
exchange chromatography afforded a cyclic
derivative [lb]of the MeBmt
minoacid.
Acidic treatment of cyclosporin [l] in the
absence of water effects an N, O-acylmigration of the methylvalyl moiety and
furnishes isocyclosporine [lc]. A modified
Edman degradation of isocyclosporine [1]
using methyl isothiocyanate produced an
anhydrothiohydantoin-derivative [l] and thus
established MeBmt as the first amino acid
in the peptide sequence. The complete
sequence was established by repetitive
Edman degradation.
2.2.4
Conformation of Cyclosporine
The conformation of cyclosporine in the crystal and in solution was reported (11). The
152
conformational a n a l y s i s was based on X-ray

c r y s t a l l o g r a p h y i n t h e c r y s t a l and on NMR
spectroscopy i n s o l u t i o n u s i n g d i f f e r e n t
l i p o p h i l i c s o l v e n t s . Cyclosporine molecule
i s highly lipophilic, therefore t h e s o l i d
s t a t e conformation and t h a t deduced from
NMR s t u d i e s i n a p o l a r s o l v e n t s a r e c l o s e l y
s i m i l a r . It assumes a r a t h e r r i g i d conformation, b o t h i n t h e c r y s t a l l i n e s t a t e and
i n s o l u t i o n ( F i g . 2 ) . The o n l y d i f f e r e n c e s
a r e t h e side-chain conformation o f MeBmt
( a major change between t h e c h a i n f o l d e d
i n upon t h e molecule i n t h e solid s t a t e ,
and p o i n t i n g o u t i n t o s o l v e n t i n t h e NMR
s t u d y ) , t h e side-chain conformation o f
MeLeu-10,
and t h e d e t a i l e d backbone conformation i n t h e r e g i o n o f D- A l a - 8 ( F i g . 3 ) .
Here t h e X-ray s t r u c t u r e c l e a r l y shows one
H-bond from D-Ala-8 ( N H ) t o MeLeu-6 ( C O ) ,
whereas i n s o l u t i o n a b i f u r c a t e d H-bond t o
b o t h t h e c a r b o n y l 0-atom o f MeLeu-6 and
itself.
t o t h e carbonyl 0-atom of D-&a-8
The conformation change needed t o c o n v e r t
t h e X-ray backbone i n t o t h e NMR backbone
i s small, and can b e brought about by
r e l a t i v e l y s m a l l r o t a t i o n i n t h e backbone
t o r s i o n a n g l e s i n t h e r e g i o n MeLeu-6 t o
MeLeu-9, p r i n c i p a l l y around t h e 7- and 8residues.
The side-chain conformations o f M e V a l and
V a l and t h r e e o f t h e f o u r MeLeu-residues
(MeLeu -4, -6 and -9) are i d e n t i c a l i n
c r y s t a l and i n s o l u t i o n . Only t h a t o f
MeLeu-10 i s r o t a t e d by 120. The side-chain
conformation o f MeBmt
i n s o l u t i o n can be
d e r i v e d from t h e X-ray conformation simply
by means o f 120' r o t a t i o n about t h e cl,B-bond.
T h i s r o t a t i o n which i s e n e r g e t i c a l l y
allowed, could be t h e r e s u l t from t h e
formation of an i n t r a m o l e c u l a r H-bond
between t h e B-OH and t h e carbonyl 0-atom
of MeBmt r e p l a c i n g t h e i n t e r m o l e c u l a r Hbond found i n t h e c r y s t a l . A l a r g e p o r t i o n
o f t h e backbone ( r e s i d u e s 1-6) a d o p t s an
a n t i p a r a l l e l & p l e a t e d s h e e t conformation
which c o n t a i n s t h r e e t r a n s a n n u l a r H-bonds
CYCLOSPORINE
Fig. 2.
153
Stereoview of cyclosporine. a) Solid-state conformation, b) Computer-generated conformation in

apolar solvents (in solution the distal atoms of
Abu-2 and YeBmt-1 have a high flexibility).
Fig. 3.
Superposition of the solid-state conformation (thin line;) and the

computer-generated conformation of cyclosporine in apolar solvent
(thick lines).
155
CY CLOSPORINE
and is markedly twisted. The sarcosine

in position 3 and N-methylleucine in
position 4 participate in a type 11B-turn
(12-14). This means that, in the orientations chosen in Figures l and 2, the CC
group of Abu-2 and the N-CH? group of
MeLeu-4 are up and the CC gEoup of Sar-3
and NH group of Val-5 are down - pro -S
proton of the methylene group of Sar 3 is
axial, and the side-chain of MeLeu-4 is
requatorial. The remaining residues 7-11
form an open loop. This loop contains the
only cis- amide linkgage in the molecule
between the two adjacent N-methylleucine
residues 9 and 10. The remaining H-bond
of a y-type and serves to hold the backbone in a folded L-shaped.
As a consequence of this rather rigid conformation of the cyclosporine skeleton,

six amino acids have their side-chains
directed quasi-perpendicular to the plane
of the peptide ring; in Abu-2, Val-5 and
MeLeu-6 they are pointing upwards, in
MeBmt-1 and
~ e ~ e u -they
6
are pointing
downwards. The side-chains of the remaining
aminoacids lie more or less in the plane
of the peptide ring.
The resulting overall molecular shape in
apolar solution is that of the previously
postulated "butterfly" (10), in which the
MeE3mt
side-chain, known to be important
for immunosuppressive activity, stands out,
probiscis - like, in a manner suggestive of
special function (Fig. 4).
2.2.5
CAS Registery Number

Cyclosporine [79217-60-0]
Cyclosporine A [ 59865-13-31
2.2.6 Clinical Code

CyAIOL 27-400
156
Fig. 4.
Two roughly orthogonal views of a model of native

cyclosporine in the conformation suggested as
probable. The backbone conformation corresponds
to that of iodocyclosporine.
157
CY CLOSPORINE
2.3
Molecular Weight
1202.64
2.4
Appearance, C o l o r , Taste, Odor

White p r i s m a t i c c r y s t a l s .
3. P h y s i c a l P r o p e r t i e s
3.1
Crystal Properties
3.1.1
X-ray D i f f r a c t i o n
C r y s t a l Data
X-ray c r y s t a l s t r u c t u r e s o f c y c l o s p o r i n e
and i t s i o d o d e r i v a t i v e [ I a ] were d e t e r m i n e d
(10,ll). A summary o f t h e c r y s t a l d a t a and
r e f r a c t o m e t r y for c y c l o s p o r i n e i s g i v e n
i n Table 1 (11).
T a b l e 1.
C r y s t a l and D i f f r a c t i o n Data
Molecular f o r mula
N O
C 6 2 H l l l 11 12
Molecules
per c e l l
z = 4
Molecular
weight
1202.5
Diffractometer
CAD4 ( E n r a f Nonius)
Crystallisation
from a c e t o n e
Radiation
C a a ( Graphite
monochromat o r )
C r y s t a l form
colourless,
p r is m a ti c
Intwsity
scans
w/2e = 1.0;
Ow = 1.0+0.3
tan 0
Crystal s i z e
ca. 0.2 x 0.2
a ( T ) / I = 0.02
x 0.3 mm
( tmax
= 120s)
Space group
Sphere of
ref lexion
sine/X 4 0 . 5 1
( 36508 r e f l e x i o n r )
Cell dimen-.
sions
I n t e n s i t ie:
measured
Intensities
significant
5057 ( u n i q u e )
C r y s t a l dens i t y (calc.)
c = 41.242(33
A; V = 7896A
dc = 1 . 0 4 2 g.
cm-3
4272(I
2.50(1);
158
The c r y s t a l d a t a for iodocyclosporine (10),

N 0
+ , M = 1327, c o l o r l e s s
'62'110
11 1 2 prisms from n-heptene, 2 . 5 : l monoclinic,
a = ( 1 0 . 4 7 5 ( 5 ) , b = 1 9 . 6 0 ( 1 ) , c = 21.05(1)A0,
f3 = 99.35 (2'1, U = 4264 A o 3 , Dc = 1.03,
z = 2, space group ~2~ , ( c,; N O . 4). CUKci
r a d i a t i o n , A = 1.54187 A o , g r a p h i t e
monochromator. A c r y s t a l o f approximate
dimensions 0 . 3 x 0.7 x 0.4 mm w a s used on
an Enraf-Nonius CAD4-F automatic d i f f r a c t o meter.
A s t e r e o v i e w o f iodocyclosporine molecule
i s shown i n F i g . 5a, and t h a t of c y c l o s p o r i n e
i n F i g . 5b.
The iodocyclosporine showed t h e a n t i p a r e l l e l
& p l e a t e d s h e e t conformation of r e s i d u e s
1-6, t h e open l o o p of r e s i d u e s 7-11, and
t h e r a t h e r l a r g e t h e r m a l v i b r a t i o n s of some
of t h e side-chain atoms , p a r t i c u l a r l y
t h o s e of MeLeu-9 are a p p a r e n t . The a b s o l u t e
c o n f i g u r a t i o n w a s n o t determined as p a r t of
the structure analysis, since sufficient
of t h e h y d r o l y s i s p r o d u c t s could r e l i a b l y
be i d e n t i f i e d as L-amino-acids.
It became
apparent d u r i n g t h e a n a l y s i s , however , t h a t
A l a - 8 has t h e D-configuration (15).
The conformation of c y c l o s p o r i n e observed
i n t h e c r y s t a l ( F i g . 5b) w i t h 50% p r o b a b i l i t y
v i b r a t i o n a l e l l i p s o i d s of t h e atomic
thermal motion (16) which d i s p l a y t h e
r e l a t i v e l y s m a l l , isotopic v ib r a tio n s of
t h e backbone atoms - i n d i c a t i n g conformat i o n a l r i g i d i t y - i n c o n t r a s t t o t h e more
f l e x i b l e side-chain atoms whose v i b r a t i o n a l
amplitudes i n c r e a s e w i t h t h e d i s t a n c e from
(a).
Fig. 6a shows t h e backbone of t h e c y c l i c
p e p t i d e w i t h t h e 4-, I)-, and w- a n g l e s
and some s t r u c t u r a l f e a t u r e s . Three H-bonds
b r i d g e t h e two s h o r t @- s t r a n d s adding t o
t h e s t a b i l i t y of @-fragment.
/!
Fig. 5a.
A stereoscopic view of the iodocyclosporine molecule showing 50% probability
ellipsoids of thermal vibration. The Cg amino acid is at centre left on the

forward side of the molecule and the conventional sequence then extends upwards
at the front through a-aminobutyric acid, glycine etc. One of the hydrogen bonds
of the 8-pleated sheet is indicated (dotted) in the centre and the interesting
hydrogen bond Ala-8 4 Meleu-6 appears at centre right.
Fig. 5b. ORTEP drawing of the crystal structure of cyclosporine. Anisotropic

atomic vibrations are represented by 50% - probability ellipsoids.
The numbers shown are residue numbers.
loop
Fig. 6 a .
bet a
Backbone conformation of crystalline cyclosporine with indication of the loop
and 6-fragment. The torsion angles 6, $I, and w and in brackets the computermodeled values for the NMR conformation are given. The dashed lines indicate
B-bonds, the dotted lines close contacts from CH3N of MeVal to different atoars
(0 11:3.04 Ao; C 7:3.36 Ao; 0 7:3.26 AO; C 8:3.41 A O ; N 9 : 3 . 5 4 AO; N 10:3.12 Ao).
F i g . 6b.
Stereoview of a space-filling model of

cyclosporine showing the side chain of
between MeLeu-6 and-Meleu-4 into the ply of the &fragment
163
CYCLOSPORINE
The s i d e c h a i n s of t h e Abu, V a l , and MeLeu

r e s i d u e s are i n t h e s t a g g e r e d conformation,
t h e MeLeu s i d e c h a i n s being extended.
Rather unexpected i s t h e conformation o f
t h e Cg-alkylene s i d e chain of MeBmt-1; it i s
n e a t l y f o l d e d i n t o t h e p l y of t h e 6-pleated
s h e e t which a l l o w s t h e molecule t o adopt
a g l o b u l a r compact shape ( F i g . 6 b ) .
3.2
Melting Range
White p r i s m a t i c n e e d l e s from acetone a t - 15',
m.p. 148-151' (17). The s y n t h e t i c c y c l o s p o r i n e
m.p. 149-150 (18).
3.3
Solubility
The compound i s n e u t r a l , r i c h i n hydrophobic amino
a c i d s , i n s o l u b l e i n water and n-hexane, b u t v e r y
s o l u b l e i n a l l o t h e r o r g a n i c s o l v e n t s . It i s
v e r y s o l u b l e i n methanol, e t h a n o l , a c e t o n e , e t h e r
and chloroform.
3.4
Optical Rotation
[a]:'
3.5
(17)
244' ( C = 0.6 i n c h l o r o f o r m ) .
1.89' ( C
= 0.5 i n methanol).
Spectral Properties
3.5.1
The W spectrum o f c y c l o s p o r i n e i n methanol
w a s recorded on a Beck~nann-DK2 spectrophotometer shows only an end a b s o r p t i o n a t
200 nm ( 4 )
3.5.2
The i n f r a r e d c h a r a c t e r i s t i c s i n methylenec h l o r i d e ( C H 2 C 1 2 ) and i n c a r b o n t e t r a c h l o r i d e
( C C l 4 ) were r e p o r t e d ( 4 , l l ) . The I R
spectrum i n methylene c h l o r i d e recorded on
a P e r k i n E l m e r 21.IR spectrophotometer i s
shown i n Fig. 7. It s h o u l d be n o t e d t h a t
s o l u t i o n s t u d i e s , i n d i c a t e d t h e NH groups
Fig. 7.
IR-spectrum of cyclosporine in CH2C12.
165
CYCLOSPORINE
are involved i n H-bonding.

Their absorption
bonds ( 3330, 3290 cm-l i n C C l 4 s o l u t i o n
and 3400-3330 cm-1 i n CH2C12 s o l u t i o n ) are
independent o f t h e c o n c e n t r a t i o n i n t h e
range o f 4.10-2 t o 4.1Oq4 M i n C C 1 4
s o l u t i o n . The spectrum shows a l s o an
i n t e n s i v e amidecarbonyl bond a t
1630-1670 cm-1.
3.5.3
'H-NMR,
13C-NMR
and H-NMR-NOE-difference
s p e c t r a of cyclosporine i n deuterochloroform and deuterobenzene were r e p o r t e d ( 4 ~ 8 ,
11). However, t h e complete assignment of
lH-NMR, 13C-NMR and 15N-NMR s p e c t r a i n
deuterochloroform and deuterobenzene by a
combination of homonuclear and h e t e r o nuclear two d i m e n t i o n a l t e c h n i q u e s were
d e s c r i b e d by Kessler e t a1 ( 1 9 ) .
3.5.3.1
'H-NMR
Spectra
1
The H-NMR spectrum of c y c l o s p o r i n e
( F i g . 8 ) i n CDC13 and TMS w a s
recorded on Bruker HX-gO-E,
90 MHz instrument ( 4 ) . A p a r t o f
1~-NMR
spectrum o f c y c l o s p o r i n e
i n ~ 6 ( F~i g .6 9 ) w a s recorded on
Bruker HX-360, 360 MHz i n s t r u m e n t .
This h a s proved t h e E-configurat i o n o f t h e double bond i n
c y c l o s p o r i n e by u s i n g t h e double
resonance t e c h n i q u e (JE, 5 = 16 Hz)
( 4 ) . From t h e s e s p e c t r a , i d e n t i f i c a t i o n o f s p i n systems were
o b t a i n e d . It i s e v i d e n t from t h e
s p e c t r a t h a t one conformation
s t r o n g l y dominates. Some minor
peaks s p e c i a l l y i n t h e N-methyl
r e g i o n (2.5-3.5 ppm) are v i s i b l e
i n d i c a t i n g t h e presence o f a n o t h e r
conformation i n s l o w exchange. A
completely d i f f e r e n t s i t u a t i o n i s
observed i n DMSO d6 s o l u t i o n ,
where a m i x t u r e o f a t l e a s t seven
conformations l e a d s t o a spectrum
of h i g h complexity. Roughly
Fig. 8 .
'H-NMR-spectrum
0 ??a
of cyclosporine by 90 !Mz in CDcl3, TMS
--
0 ppm.
167
CYCLOSPORINE
speaking, f i v e more o r l e s s
separated s p e c t r a l regions are
distinguishable:
NH protons
- 8.3
-6
P P ~
a-protons as w e l l as
t h e v i n y l i c protons
4.5
N-methyl groups
2.6
C-methyl groups
0.6
A l i p h a t i c f3-, yand 6 protons
1.1 - 2.7 PPRI
pprn
- 3.8 ppm
- 1.8 ppm
The proton chemical shifts for

cyclosporine i n CDC13 and C6D6
are l i s t e d i n Table 2 .
h
C
H-c
F i g . 9.
1
Part of the H-NMR-spectrum of cyclosporlne by 360 llHz in CsDs.
a) Undecoupled spectrum.
b) Decoupled s n e c t r m keeping lock on H-C(n) of Cg As
c ) Decoupled spectrum keeping lock on H-C(6) of Q As
Table 2.
Residue
h- and
chemical shifts of cyclosporine [I] in C D C l
13C-NMR
Amino-acid
residue
Group
MeBmt:
CH3N
~~
co
H-C(a )
H-C(B)
OH
3-52
5.45
3.82
3.87
1.63
2.41
1-73
0.72
5.36
C6D6
3.51
Abu
NH
co
7.93
C6D6
CDC13
3.73
5.47
5.7
4.21
5.72
4.20
3.5
2.07
2.65
33.97
169.65
58.75
74.74
s
d
d
34.33
UO * 73
59.96
74.95
-
35.99 d
35.63 t
36.30
36.04
16.76 q
129.68 d
126.32 d
18.30
126.76
17.96
18.69
2.13
0.71
5.35
1.62
2
and C G D ~ ~ )
l3C-NMR
lH-NMR
CDC13
1.15
5.64
5-52
1-75
7.96
131.38
8.26
173.04 s
174.29
Table 2.
Residue
No.
(Continued)
Amino-acid
Group
CDCl
Sar
MeLeu
32K
5-03
5.12
5.12
Val
32K
H-C(
6)
1.6-1.74
1.79
H-C(
Y)
0.87
0.89
49.54
26.11
9.93 9
39.40 q
10.73
39.58
171.80
50.10
co
3.40
-
H-C( a )
4.76
4.74
4.01
50 37 t.
3.11
2.59
31.32 q
CH N
H-C(
~ 1 )
5.34
H1-C(
B)
2.00
3.08
-
5.60
2.27
1.58
1.42
0.98
0.91
7.46
C6D6
48.86 d
25.06 t
m3N
co
CDCl
C6D6
2K
2K
H-C( a)
13C-NMR
H-NMR
residue
170.50 s
169.35 s
55.51 a
31.39
170.21
56.20
35.99 t
37 * 01
24.90 d
23.49 q
21.18 q
25.79
24.28
22. TOb)
W
RW
c - o r i
wlnmrJ&w
d
ri
* . . .
1 - 3
I A ( u r l 0
170
(u
m
Ln
d
M
cu
m
L n
cu
M
ul
co
t-
co
171
L n d L n L n
? ? ? ?
ti
m t i c o o
c u r - J J
L P
UJ
cu
t-
co
I L n
cu
0
I L n
cs
cu
r-
co
. .
gt-
cucu
5
9
L n L n
Table 2 .
Residue
No.
( Continued)
Amino-acid
residue
CDCl
MeLeu
H-C(CX)
5.10
6)
H-C(y)
CH3(61)
MeVal
13C-NMR
C6D6
2K
m3N
21.86 9
24.81
29.83 q
169.41 s
30.47
170.98
57.54 d
40.73 t
58.36
42.06
1.79
24.55 d
25.62
1.17
23.85 q
24.34
1.16
23.38 q
22. 6bb)
2.85
5.35
2.42
1.29
2.13
1.24
1.49
0.98
0.98
C6D6
CDC13
32K
0.84
2.70
co
CH3(
32K
0.89
CH 3N
H1-C(
11
2K
CH3(62)
10
lH-NMR
Group
2.71
2.42
30.96
174.61
5-27
57.93 d
58.84
2.27
29.05 t
2.98
co
H-C(CX)
5-15
5.14
H-C( 6 )
2.17
29.81
172.85
29 - 99
Table 2.
Group
0
1H-NMR
Pi
3
Amino-acid
residue
k
0
Residue
( Continued)
WDV)
19.38
0.66
20.26 q
20.59
18.75 q
. .
. .
a m
r i m
oco
d o
173
e
4
W
0.96
At 296 K in pprn from internal TMS. The H-NMR data were obtained from a conventional
w
lk
k
Gik
Ld
13c-NMR
CDC1,
32K spectrum or from the cross-sections of a IH,$-COSY
with 2K data points in f2 at
300 MHz
b,
The signals of MeLeu-4(6 ) and MeLe~-9(6~)

as well as those of MeLeu-6(6 ) and MeLeu-lO(6 )
2
1
2
in C D may be exchanged).
66
174
3.5.3.2
13C-NMR
Spectra
I n t h e broadband-decoupled 13C-NMR
s p e c t r a i n CDC13 or C6D6 were
r e p o r t e d ( 1 9 ) . Almost a l l 62 Cs i g n a l s a r e r e s o l v e d and only few
signals overlap, but these s p l i t
when t h e s o l v e n t i s changed. A l l
carbonyl C-atoms r e s o n a t e i n t h e
range between 169 and 175 ppm.
P a r t o f t h e l 3 C - m spectrum ( t h e
carbonyl r e g i o n ) of c y c l o s p o r i n e i n
CDC13 measured on a Bruker-WH-360,
360 MHz instrument i s shown i n
Fig. 1 0 (11). The two o l e f i n i c
C-atoms of MeBmt are a l s o i n a
t y p i c a l range 126-132 ppm, whereas
49 C-signals a r e i n t h e a l i p h a t i c
r e g i o n . The number of a t t a c h e d
p r o t o n s were o b t a i n e d v i a t h e
u s u a l DEPT t e c h n i q u e ( 2 0 , 2 1 ) . A
h e t e r o n u c l e a r 2D-J, 6 spectrum
(22-26) w a s a l s o performed i n CDC13
The chemical s h i f t f o r a l l carbons
and m u t l i p l i c i t i e s i n C X 1 3 and
i n C6D6 are given i n Table 2 .
3.5.3.3
15N-NMR
Spectra
AH-decoupled 15N-NMR spectrum shows

eleven N-signals ( T a b l e 3 ) .
Table 3.
15N-NMR chemical s h i f t s o f 1.
i n an e x t e r n a l c a p i l l a r y .
6 ppm from N H 4 l 5 N O 3
~~
i n C6D6
i n CDC13
248.8 A7
- 255.5 Abu
256.9
257.3 A8
258.0
258.6 Val
255.1 Abu
256.7
- 261.8
248.1 A7
256.9 ~8
- 258.3
-
i n CDCl
259.2
in
C6D6
259.2
260.1
- 261.8
260.4 V a l
264.2
265.2
- 265.1
263.1
263.9
I73
-6
F i g . 10.
[PpnJ
170
spectrum of cyclosporlne: CO region. Those CO groups whose signals

shifted downfield are marked In black. The CO sign81 of residue 8 (shaded)
lowest from those attached to NE groups (residues 1 , 4 , 6 , and 7).
''C-NNFI
176
3.6
Mass Spectrum
3.6.1
EI Spectrum
The e l e c t r o n impact spectrum o f c y c l o s p o r i n e
w a s measured on a CEC-mass spectrometer
21-llOB ( 4 ) . The e l e c t r o n energy w a s 7 0 e V ,
t h e a c c e l e r a t i o n rate 4.6-8 KV, t h e temperat u r e of t h e source 280-300C and a p r e s s u r e
of 10-5 - 10-6 T o m . The molecules w a s
u n s t a b l e g i v i n g a molecular i o n peak a t
m/e 1201 which v a n i s h e s q u i c k l y . A s t h e M+
v a n i s h e s a peak a t m / e 1183 i n c r e a s e s i n
i n t e n s i t y and t h e spectrum remains s t a b l e .
The peak a t m/e 1183 i s formed by eliminat i o n o f a molecule o f water. The e x a c t mass
measurement were made at 1183.831 i 0.003
and 1201.842 f 0.003. The molecular f o r m u l a o b t a i n e d by t h i s measurement w a s
C62HlllN11012
( p h y s i c a l molecular mass
1201.841). T h i s w a s i n agreement w i t h t h a t
deduced from NMR and o t h e r f i n d i n g s . Other
mass s p e c t r a l d a t a w i l l be p r e s e n t e d under
metabolism.
3.6.2
FD Spectrum
The f i e l d d e s o r p t i o n mass spectrum w a s
measured on a MAT 711 mass spectrometer ( 4 ) .
The a c c e l e r a t i o n energy r a t e 6 KV, t h e
temperature of t h e i o n s o u r c e goo and t h e
power f a c t o r f o r m u l t i p l i e r 106. A molec u l a r i o n peak M+ of m / e 1201.8 w a s o b t a i n e d
and m / e 1203.6 f o r t h e d i h y d r o c y c l o s p o r i n e .
Another FD spectrum w a s r e p o r t e d (18). A
molecular i o n peak Mc a t m / e 1202, NH' a t
m / e 1203 and ( M + N a + ) a t m / e 1225.
4.
I s o l a t i o n o f Cyclosporine
Dreyfuss e t a 1 ( 2 ) and o t h e r s ( 4 , 27-30) have r e p o r t e d
t h e i s o l a t i o n of c y c l o s p o r i n e from o t h e r f u n g a l m e t a b o l i t e s of t h e f u n g i Tolypocladium i n f l a t u m Gams. T h i s
fungus w a s p r e v i o u s l y known as Trichoderma polysporum
(Link ex P r e s . R i f a i ( 2 ) .
CYCLOSPORINE
177
The s t r a i n used i s Tolypocladium i n f l a t u m d e s i g n a t e d

No. N RRL 8044. T h i s was grown i n an a e r o b i c submerged
culture.
A r e p o r t e d method of i s o l a t i o n of c y c l o s p o r i n e from
f e r m e n t a t i o n media w a s as f o l l o w s ( 4 ) :
The f e r m e n t a t i o n medium w a s e x t r a c t e d w i t h n-butylacetate.

Then a f t e r t h e s e p a r a t i o n of t h e o r g a n i c l a y e r it w a s
c o n c e n t r a t e d under vacuum. The crude r e s i d u e o b t a i n e d
w a s d e f a t t e d with 90% methanol and petroleum e t h e r . The
product o b t a i n e d was d i s s o l v e d i n chloroform and a p p l i e d
t o a column chromatography of s i l i c a g e l . The p o l a r i t y
of t h e e l u t i n g s o l v e n t w a s g r a d u a l l y i n c r e a s e d by adding
methanol t o chloroform. The f r a c t i o n s o b t a i n e d by
chloroform-methanol ( 98 :5 :1 . 5 ) c o n t a i n e d c y c l o s p o r i n A .
F r a c t i o n s c o n t a i n e d c y c l o s p o r i n -C w a s o b t a i n e d w i t h
chloroform-methanol (97:3). The f r a c t i o n s c o n t a i n i n g
c y c l o s p o r i n A were s u b j e c t e d t o g e l f i l t r a t i o n with
Sephadex LH-20 w i t h methanol. The t o p f r a c t i o n s w e r e
d i s s o l v e d i n t o l u e n e and a p p l i e d t o column chromatography o f aluminium oxide u s i n g t o l u e n e - e t h y l a c e t a t e as
t h e e l u e n t . The f r a c t i o n s o b t a i n e d were evaporated and
d i s s o l v e d i n a l c o h o l , t h e n t r e a t e d w i t h c h a r c o a l . The
f i l t r a t e was evaporated t o g i v e c y c l o s p o r i n A as an
amorphous powder.
Another r e p o r t e d procedure ( 2 7 ) w a s as f o l l o w s :
The f e r m e n t a t i o n b r o t h w a s s e p a r a t e d i n t o t h e m y c e l i a l
cake and t h e c u l t u r e f i l t r a t e . The former w a s homogen i z e d t h r e e t i m e s w i t h methanol-water (9:l) and t h e
combined f i l t r a t e s were c o n c e n t r a t e d under vacuum t o
remove t h e methanol. The water s o l u t i o n w a s e x t r a c t e d
t h r e e times w i t h l , 2 - d i c h l o r o e t h a n e and t h e o r g a n i c
l a y e r s were evaporated t o d r y n e s s . Working-up of t h e
c u l t u r e f i l t r a t e by e x t r a c t i o n w i t h 1 , 2 - d i c h l o r o e t h a n e
r e s u l t e d i n an a d d i t i o n a l p o r t i o n of crude material.
The combined e x t r a c t e d were submitted t o g e l f i l t r a t i o n
on Sephadex LH-20 w i t h methanol. Those f r a c t i o n s which
c o n t a i n t h e c y c l o s p o r i n e ( a s a m i x t u r e ) were pooled
and t h e n s e p a r a t e d i n t o t h e components by s i l i c a g e l
column chromatography (Merck , 0.063-0.2 mm) w i t h e t h y l
a c e t a t e s a t u r a t e d w i t h water. I n accordance t o t h e i r
p o l a r i t y , t h e cyclosporines were e l u t e d i n t h e following
o r d e r : D , G, A , B and C . The crude c y c l o s p o r i n e s were
f u r t h e r p u r i f i e d by c r y s t a l l i z a t i o n ( c y c l o s p o r i n e G
from e t h e r / p e t r o l e u m e t h e r a t room t e m p e r a t u r e ;
c y c l o s p o r i n e s A , C and D f r o m a c e t o n e a t - 15OC). The
178
r e s u l t i n g pure c y c l o s p o r i n e s A, B, C, D and G w e r e
c h a r a c t e r i z e d by a n a l y t i c a l methods ( t h i n l a y e r chromatography, m e l t i n g p o i n t , o p t i c a l r o t a t i o n ) and s p e c t r a l
d a t a ( U , I R , 'H- and l3C-NMR, mass s p e c t r u m ) . They
were found t o be i d e n t i c a l i n a l l r e s p e c t s w i t h
a u t h e n t i c samples i s o l a t e d from f e r m e n t a t i o n i n complex
n u t r i e n t media as p u b l i s h e d by Dreyfuss e t a 1 ( 2 ) ,
Ruegger _
et _
a1 ( 4 ) and Traber e t a1 ( 2 8 , 3 0 ) .
5.
B i o s y n t h e s i s of Cyclosporine
Kobel _
e t -a1 ( 3 1 ) and o t h e r s (27,32,33) have r e p o r t e d t h e
b i o s y n t h e s i s of c y c l o s p o r i n e and o t h e r f u n g a l metab o l i t e s . I n t h e i n i t i a l b i o s y n t h e t i c s t u d i e s o f Kobel
_
et _
a1 (31) , 3H- and 13C-labeled p r e c u r s o r s w e r e f e d t o
t h e c u l t u r e and t h e p o s i t i o n o f i n c o r p o r a t i o n o f t h e
l a b e l i n c y c l o s p o r i n e w a s determined by NMR s p e c t r o s c o p y .
It w a s demonstrated t h a t t h e N-methyl groups of t h e molec u l e and t h e methyl group i n t h e y - p o s i t i o n o f t h e
u n s a t u r a t e d amino a c i d MeBmt a r e i n t r o d u c e d as i n t a c t
methyl groups from methionine and t h a t t h e remaining
carbons of t h e MeBmt moiety a r e d e r i v e d from t h e headt o - t a i l condensation of f o u r a c e t a t e u n i t s . The l3C-NMR
spectrum o f t h e e n r i c h e d c y c l o s p o r i n e d e r i v e d from
[ l-13C]acetate showed f o u r enhanced s i g n a l s corresponding
t o t h e carbon atoms 1, 3, 5 and 7 o f t h e MeBmt u n i t and
no '3C i n c o r p o r a t i o n i n t o any o t h e r amino a c i d .
I n e n n i a t i n , a n analogous example of non-ribosomal

c y c l i c p e p t i d e s y n t h e s i s by f u n g i , Zocher and
Kleinkauf ( 32) demonstrated t h a t a m u l t i f u n c t i o n a l
enzyme c a r r i e s o u t t h e N-methylation o f c o n s t i t u e n t amino
a c i d s following an a c t i v a t i o n s t e p when t h e a c i d s are
bound t o t h e enzyme as t h i o e s t e r s . Supporting evidence
f o r a similar mechanism i n c y c l o s p o r i n e b i o s y n t h e s i s w a s
provided by t h e o b s e r v a t i o n o f Zocher e t a1 ( 3 3 ) t h a t
[14C] s a r c o s i n e w a s n o t ' n c o r p o r a t e d and t h a t t h e
r a d i o a c t i v i t y from [Me-16C]methionine i n c o r p o r a t e d
i n t o each N-methylated amino a c i d w a s d i r e c t l y proport i o n a l t o t h e number o f t h e corresponding amin a c i d
r e s i d u e s i n c y c l o s p o r i n e , t h u s i n d i c a t i n g t h a t Nm e t h y l a t i o n of t h e amino a c i d s o c c u r r e d s i m u l t a n e o u s l y .
From t h e r e s u l t s o f Kobel e t a1 ( 3 1 ) t h e s i t e of
b i o s y n t h e s i s of t h e Bmt u n i t remains u n c l e a r . However,
t h e results o b t a i n e d by t h e Zocher group ( 3 3 ) w i t h
short-term f e e d i n g experiments u s i n g l a b e l e d a c e t a t e
and methionine, i n which probably o n l y i n c o r p o r a t i o n of
179
CYCLOSPORINE
label in the N-methyl group occurred, suggest that the

amino acid Bmt is not biosynthesized on the enzyme
matrix but at some other site before incorporation
into cyclosporine.
6. Synthesis
6.1 Cyclosporine
Winger (18, 34-38) has reported the total synthesis
of cyclosporine and analogues.
The strategy of the total synthesis of cyclosporine
has involved the following steps:-
I. The synthesis of the N-methyl-C-9-amino

acid (Scheme 1) (36, 39-44).
11. Building of the tetrapeptide BOC-D-Ala-MeLeuMeLeu-MeVal-benzylester (37).
111. Preparation of the dipeptide BOC-Abu-Sarbenzylester (18,37,46).
TV.
V.
VI
VII.
Building of the tetrapeptide BOC-MeLeu-ValMeLeu-Ala-benzylester (18).

The synthesis of the hexapeptide BOC-AbuSar-MeLeu-Val-MeLeu-Ala-benzylester (18).
Synthesis of the heptapeptide H-Me-C9-AbuSar-MeLeu-Val-MeLeu-Ala-benzylester (18,47)
Formation of the undecapeptide BOC-Ala-MeLeuMeLeu-MeVal-Me-Cg-Abu-Sar-MeLeu-Val-MeLeuAla-benzylester (18,48).
VIII. Cyclization of the undecapeptide with a free

amino group and free carboxyl group to
produce cyclosporine (48,50,51).
180
tio-
F:,
7 Steps
COOEI
(ZR,3R)-3-methyl-l,2,4,-butanetrio1
6 Steps
(2R,3R,SE)-3-rnethyl-S-heptene1,2-diol
"II_ c y
(2R, 3R, 5E) -2-hydroxy-3-methyl5-heptenal
(2S, 3R, 4R, 6E) -3-hydroxy-4methyl-2-(methylamino)-6-octenoic

acid (MeBmt)
Scheme 1.
Synthesis of MeBmt
CYCLOSPORINE
VIII.
181
T o t a l Synthesis of Cyclosporine (18, 34, 35,
37 1
S t r a t e g y Used f o r t h e Synthesis o f
Cyclosporine
For t h e s y n t h e s i s of cyclosporine [l]( F i g . 1)
t h e p e p t i d e bond between t h e L-alanine i n
p o s i t i o n 7 and t h e D-alanine i n p o s i t i o n
8 w as chosen f o r t h e c y c l i z a t i o n s t e p .
There were two main reasons f o r choosing
t h i s s t r a t e g y : 1. The intramolecular
hydrogen bonds between t h e amide groups of
t h i s l i n e a r p e p t i d e could o p e r a t e so as t o
s t a b i l i z e t h e open chain i n folded conformations approximating t h e c y c l i c s t r u c t u r e
of cyclosporine and t h u s assist c y c l i z a t i o n .
2 . The bond formation between N-methylated
amino a c i d s i s more d i f f i c u l t t h a n between
non-methylated amino a c i d s (37,45) ; t h e r e f o r e , bond formation between t h e only
consecutive p a i r of non-methylated amino
a c i d s i n cyclosporine appeared t h e l o g i c a l
choice f o r t h e c y c l i z a t i o n s t e p .
For t h e s y n t h e s i s of t h e undecapeptide, a
fragment-condensation technique i n t r o d u c i n g
t h e amino a c i d MeBmt a t t h e end of t h e
s y n t h e s i s w a s used. I n t h i s way, t h e
number o f s t e p s a f t e r t h e i n t r o d u c t i o n of
t h i s amino a c i d w a s minimized. Scheme 5
shows t h e s t e p sequence used f o r j o i n i n g
t h e ( p r o t e c t e d ) p e p t i d e fragments. The
carboxy groups were g e n e r a l l y a c t i v a t e d
using a v a r i a t i o n of t h e mixed p i v a l i c
anhydride method r e p o r t e d by Zaoral ( 4 6 )
and adapted f o r t h e
N-methylamino a c i d
d e r i v a t i v e s ( 3 7 ) . T h i s s t r a t e g y allowed
slow anhydride formation i n chloroform a t
- 2OoC with p i v a l o y l c h l o r i d e i n t h e
presence of 2 e q u i v a l e n t s of a t e r t i a r y base
such as N-methylmorpholine before adding
t h e amino a c i d o r p e p t i d e e s t e r s t o be
coupled i n t h e form o f t h e i r f r e e bases.
The f r e e bases were obtained by removal of
t h e Boc-protecting groups with t r i f l u o r o a c e t i c a c i d a t - 2OoC and subsequent
182
n e u t r a l i z a t i o n with NaHC03; t h e benzyloxyp r o t e c t i n g groups of t h e p e p t i d e i n t e r mediates were removed h y d r o g e n o l y t i c a l l y

with palladium/carbon i n ethanol.
The t e t rapept i d e Boc-D-Ala-MeLeu-MeLeuMeVal-OBzl w a s synthesized from t h e l e f t
t o t h e r i g h t by forming bonds 1, 2 and
3 (Scheme 2 ) . Bond 4 was formed on synt h e s i s of t h e d i p e p t i d e Boc-Abu-Sar-OBzl.
The t e t r a p e p t i d e Boc-MeLeu-Val-MeLeu-AlaOBzl w a s synthesized from t h e r i g h t t o t h e
l e f t by forming bonds 5 , 6 and 7 i n t h a t
o r d e r . Subsequent coupling (bond 8 ) w i t h
t h e p r e v i o u s l y synthesized d i p e p t i d e t h e n
furnished t h e hexapeptide Boc-Abu-SarMeLeu-Val-MeLeu-Ala-OBzl. During incorporat i o n of t h e amino a c i d MeBmt, t h e hydroxyand N-methylamino f u n c t i o n s were p r o t e c t e d
i n t h e form of a dimethyloxazolidine derivat i v e . This isopropylidene-protecting group
was r e a d i l y introduced by r e f l u x i n g t h e
amino a c i d i n acetone and has t h e advantage
of avoiding epimerization of t h e amino a c i d
MeBmt during peptide-bond formation. This
means t h a t t h e oxazolidine r i n g r e t a i n s
t h e thermodynamically more s t a b l e t r a n s c o n f i g u r a t i o n of s u b s t i t u e n t s during carboxy
a c t i v a t i o n and peptide formation. On
p r e p a r a t i o n , but before a c t i v a t i o n , t h e
p r o t e c t e d MeBmt amino a c i d w a s s t a b i l i z e d
by a d d i t i o n of N-methylmorpholine (1 e q u i v . ) .
To prepare t h e p r o t e c t e d heptapeptide from
t h e p r o t e c t e d MeBmt amino a c i d and t h e
hexapeptide, t h e dicyclohexylcarbodiimide
( D C C I ) coupling method w a s used i n presence
of N-hydroxybenzotriazole ( 4 7 ) . A f t e r
removal of t h e isopropylidene-protecting
group (1 equiv. of 1 N HCl/MeOH), t h e
f i n a l amide l i n k a g e 1 0 w a s made by coupling
Boc-D-Ala-MeLeu-MeLeu-MeVal-OH with t h e
heptapeptide i n presence of N-methylmorphol i n e i n CH2C12 at room temperature using t h e
reagent (1H-1,2,3-benzotriazol-l-yloxy)tris
(dimethylamino)-phosphonium hexafluorophosphate ( B t O P ( NMe2)'3 PFZ) developed by
Castro et a1 ( 4 8 ) . A f t e r h y d r o l y s i s of t h e
183
CYCLOSPORINE
step sequence for the peptide bond formation
iiiTiiiiii
1
0-Alo
HeLeu HeLeu
10
HeVol
11
H e 6 m t Abu
Sor
MeLeu
Vol
HeLeu
Alo
HeLcu-cHcVol~HeB~t-cAbu~Sar
HeLeu
0 - A lo --A lo t- H rLeu-Vol --HeLeu
Scheme 2.Syniherir olcyclosporinc 1 . Bold arrows: slralrgy lor the synthesis o l the pcptidcs. For delailr see teat. Boc-rrrrbuloxycarbonyl. BzI -bcnzyl. >-OH
isopropylidcne.proleacd MeBml.
184
e s t e r group (NaOH a t OC) and removal of

t h e Boc group (CF3COOH a t - 2OoC) , t h e
unprotected undecapeptide w a s c y c l i z e d a t
room temperature t o cyclosporine 1
(0.0002 M CH2C12, 1 equiv. BtOP(NMe); PFZ
( 4 8 ) , N-methylmorpholine). The y i e l d of
c r y s t a l l i n e cyclosporine w a s 62%. By using
t h e mixed phosphonic anhydride method
described by Wissmann and K l e i n e r ( 4 9 ) o r
t h e pentafluorophenol-DCCI complex of
Kovacs ( 5 0 ) t o e f f e c t c y c l i z a t i o n of t h e
unprotected undecapeptide , t h e y i e l d o f
cyclosporine could be increased t o 65%.
Using t h e fragment-condensation technique
described h e r e it i s now p o s s i b l e t o
synthesize cyclosporine very e f f i c i e n t l y i n
27.5% y i e l d with r e s p e c t t o t h e amino a c i d
MeBmt. Thus, 1 . 6 g of cyclosporine can be
produced s t a r t i n g from 1 g of t h e amino
a c i d MeBmt
7.
Metabolism of Cyclosporine
Maurer e t a1 ( 5 2 ) have r e p o r t e d t h e d i s p o s i t i o n of
cyclosporine i n man and s e v e r a l animal s p e c i e s and a l s o
s t r u c t u r a l e l u c i d a t i o n of i t s m e t a b o l i t e s . The metabol i t e s were i s o l a t e d from dog and human u r i n e and from
rat b i l e and f a e c e s . The s t r u c t u r e s of t h e i d e n t i f i e d
m e t a b o l i t e s were used t o e s t a b l i s h t h e biotransformation
processes. 3H-labeled cyclosporine with s p e c i f i c
a c t i v i t i e s ranging from 25 t o 80 uCi/mg w a s prepared
b i o s y n t h e t i c a l l y by feeding submerged c u l t u r e s of a
s e l e c t e d s t r a i n of t h e fungus
inflatum G a m s with
[methyl-H3] methionine as l a b e l e d precursor ( 3 1 ) .
The radiochemical p u r i t y of t h e l a b e l e d substance w a s
b e t t e r t h a n 95%. 3H-NMR a n a l y s i s revealed t h e
incorporated tritium t o be l o c a t e d i n t h e seven Nmethyl groups and i n t h e methyl group a t p o s i t i o n 4
(C,) of amino a c i d ( F i g . 11).
r.
The s e p a r a t i o n of m e t a b o l i t e s was performed by HPLC.

Nine e t h e r - e x t r a c t a b l e s of cyclosporine were i s o l a t e d
from u r i n e of dog and man and from r a t b i l e and f a e c e s
( F i g . 12).
Site and type of biotransformation in cyclosporine
F i g . 11.
r)
..
1)
186
Structure of cyclosDorine and i t s m e t a b o l i t e s .
F i g . 12.
1.u
..I
&A8
b A I
R ---Ctiz-:ti*.*
CkI,-N
f
cl'I
,,,
O C .CH-NI
CHj
1.i
..I
AI
CO CH- N
I
CH,
...
co
h-R2
.., $ ... I
- C ... C H -NI - iII- CH

- N - C O - CH
1
I
b
0.
......................
CI.C,
I
CI(,/:
'cn,
CII, ,C!l
Cn,
CHI
Cli,
CH,
CH+
*3
-----
tj
CH,
1202 . C 4
ti
cti,
I 2I 0 . 6 ~
On
OH
081
CH,
n
or.
011
Rl
Cyclosporinc
n
011
1!
9
ho.
-
-10
16
I1
-
Ho I ccul a r
ueiqht
Rl
-I
R2
Vet~bo;ite
on
R4
It
CH]
OH
CH)
on
n
-I n
of(
Cn,
oti
ciil
n
n
?!
II
II
II
Other modif i r r t ion
1214.64
/9
C H ch-cnZ
z c r .
ti
1220.62
1234.64
1214.64
1218.64
A ~ I 1218.64
1188.61
CYCLOSPORINE
187
Despite t h e complex s t r u c t u r e o f c y c l o s p o r i n e , t h e
r e a c t i o n s involved i n t h e b i o d e g r a d a t i o n o f t h e molecule
are l i m i t e d and produce o n l y a r e l a t i v e l y s m a l l number
o f m e t a b o l i t e s . The r e s u l t i n g p r o d u c t s a r e l i p o p h i l i c
as i n d i c a t e d by t h e i r occurrence i n t h e e t h e r phase
of the liquid-liquid extraction.
S i t e and t y p e of b i o t r a n s f o r m a t i o n i n c y c l o s p o r i n e are
summarized i n F i g . 11. The c y c l i c o l i g o p e p t i d e s t r u c t u r e
of c y c l o s p o r i n e i s p r e s e r v e d i n a l l i d e n t i f i e d metab o l i t e s . Metabolism mainly c o n s i s t s o f o x i d a t i v e
p r o c e s s e s ( p h a s e I r e a c t i o n s ) . T y p i c a l phase I1
r e a c t i o n s l i k e c o n j u g a t i o n w i t h s u l f u r i c a c i d or g l u c u r o n i c a c i d could n o t be d e t e c t e d . Only a l i m i t e d
number o f t h e c y c l o s p o r i n e s u b u n i t s , namely f o u r of
11 amino a c i d s , undergo r e g i o s p e c i f i c o x i d a t i v e modif i c a t i o n s . Hydroxylation r e a c t i o n s appear t o be
r e s t r i c t e d t o t h e rl-position o f amino a c i d 1 (Cg-amino
a c i d ) and t h e y - p o s i t i o n o f t h e N-methylleucines 4, 6 ,
and 9. Oxidative N-demethylation, so f a r , seems t o
occur o n l y on t h e N-methylleucine 4. The N-methylleuc i n e s 6 and 9 a r e s u b j e c t o f a s i n g l e r e a c t i o n
( h y d r o x y l a t i o n ) whereas amino a c i d 1 and t h e N-methyll e u c i n e 4 are s u b s t r a t e s f o r two t y p e s of transformat i o n : h y d r o x y l a t i o n and i n t r a m o l e c u l a r e t h e r formation
o r h y d r o x y l a t i o n and N-dernethylation.
The proposed pathways f o r t h e b i o t r a n s f o r m a t i o n o f
c y c l o s p o r i n e ( F i g . 13) are based on t h e s t r u c t u r e o f
t h e i s o l a t e d m e t a b o l i t e s . The r e g i o i s o m e r i c monohydroxylated c y c l o s p o r i n e s 1 and 17 and t h e N-demethyl a t e d c y c l o s p o r i n e 2 1 are primary m e t a b o l i t e s .
M e t a b o l i t e s 1 and 17 r e s u l t from h y d r o x y l a t i o n o f
e i t h e r t h e N-methylleucine 9 a t t h e y - p o s i t i o n or t h e
amino a c i d 1 a t t h e q - p o s i t i o n .
Metabolite 21
r e p r e s e n t s the product o f N-demethylation
on t h e N-methylleucine 4. F u r t h e r o x i d a t i o n o f metab o l i t e l on e i t h e r one o f t h e N-methylleucines 4 and 6
o r on t h e Cg-amino a c i d 1, and o f m e t a b o l i t e 17 on t h e
N-methylleucine 9 g e n e r a t e s d i h y d r o x y l a t e d d e r i v a t i v e s
of c y c l o s p o r i n e ( m e t a b o l i t e s 8 , 1 0 , and 1 6 ) . The
i d e n t i f i c a t i o n o f a d i h y d r o x y l a t e d and N-demethylated
d e r i v a t i v e of c y c l o s p o r i n e ( m e t a b o l i t e 9 ) i n d i c a t e s
f u r t h e r o x i d a t i o n t o occur on e i t h e r t h e d i h y d r o x y l a t e d
m e t a b o l i t e 1 6 by N-demethylation o f t h e N-methylleucine
4 o r t h e primary N-demethylcyclosporine 2 1 by hydroxyl a t i o n o f b o t h N-methylleucines 6 and 9.
21
'En,
10
Fig. 13. Proposed pathways for the biotransformation of cyclosporine.
CYCLOSPORINE
189
Metabolite 18, containing a cyclic ether moiety (tetrahydrofuran), may be derived from 17 (monohydroxycyclosporine) by intramolecular addition of the 6-hydroxyl
group to the double bond of the C9-amino acid 1. This
cyclization is suggested to proceed by a nonenzymatic
mechanism. An artifact with a similar cyclic ether
structure has previsouly been observed among the acid
hydrolysis products of cyclosporine.
Three other metabolites possessed the basic cyclic
structure of the parent drug. The spectroscopic data
of two of them were compatible with hydroxylated and
N-demethylated derivatives of cyclosporine, whereas
those of the third indicated a dihydroxylated derivative. Their structures remain incompletely determined.
It should be noted that metabolites 10 and
found in man (53).
16 were not
8. Pharmacokinetics of cyclosporine
The absorption, distribution, metabolism and elimination
of cyclosporine have been investigated by many authors
( 52-57)
The pharmacokinetic data on cyclosporine in man as

follows :
Absorption
Ab solute or a1 bioavailabi1ity
20-50% (mean 34%)
Plasma peak concentration time
1-6 hours
Di stribut ion
Binding to plasma proteins
go% (20OC)
Apparent volume of distribution
3.5 1/Kg
Metabolism
Extent of metabolism
Ca. 99%
Number of components
10-12(9 of which have

been characterised
as metabolites).
Elimination
Terminal e l i m i n a t i o n h a l f - l i f e
14-26 hours
T o t a l body c l e a r a n c e
0.27-0.47
l/h/Kg
because of i t s v e r y low w a t e r - s o l u b i l i t y , t h e o r a l dose

form of c y c l o s p o r i n e i n an o l i v e - o i l - b a s e d s o l u t i o n .
The a b s o l u t e o r a l b i o a v a i l a b i l i t y from t h i s s o l u t i o n
( i n s t e a d y s t a t e ) i s 20-50% (mean 34%).
I n whole blood, about 50% o f c y c l o s p o r i n e i s t a k e n up

by e r y t h r o c y t e s and 10-20% by l e u c o c y t e s . O f t h e t o t a l
amount of drug i n t h e plasma, approximately 90% i s
protein-bound, mostly t o l i p o p r o t e i n s .
I n animal experiments using3H-cyclosporine, t h e h i g h e s t

t i s s u e c o n c e n t r a t i o n s of r a d i o a c t i v i t y were found i n
l i v e r , kidney, a d r e n a l s , p a n c r e a s , thymus, t h y r o i d and
r e n a l f a t . From blood concentration-time d a t a f o l l o w i n g
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 i n man, t h e drug was found
t o be d i s t r i b u t e d according t o a three-compartment
d i s t r i b u t i o n model. The t o t a l a p p a r e n t volume o f
d i s t r i b u t i o n was 3.51/Kgm.
Cyclosporine i s slowly b u t e x t e n s i v e l y metabolised by
l i v e r . I n a d d i t i o n t o t h e p a r e n t d r u g , 10-12 metab o l i t e s have been d e t e c t e d i n human plasma. Nine metab o l i t e s have been i s o l a t e d and i d e n t i f i e d ; a l l c o n t a i n e d
t h e i n t a c t c y c l i c o l i g o p e p t i d e s t r u c t u r e of t h e p a r e n t
molecule. S t r u c t u r a l m o d i f i c a t i o n s c o n s i s t e d of monoand di-hydroxylation and N-demethylation a t v a r i o u s
p o s i t i o n s on t h e c y c l o s p o r i n e .
Terminal e l i m i n a t i o n h a l f - l i v e s from blood o r plasma
o f 14-26 hours have been r e p o r t e d .
The drug and i t s m e t a b o l i t e s are e x c r e t e d mainly v i a
t h e l i v e r and o n l y t o a s l i g h t e x t e n t v i a t h e k i d n e y s .
I n man t h e t o t a l u r i n a r y e x c r e t i o n o f r a d i o a c t i v i t y i n
t h e p e r i o d up t o 96 hours f o l l o w i n g a s i n g l e dose o f
3H-cyclosporine w a s 6% of t h e dose; unchanged cyclos p o r i n e accounted f o r o n l y 0.1% o f t h e o r & dose,
191
CYCLOSPORINE
9 . Pharmacological Activity and Clinical Application

As reported by Bore1 et a1 (5-9), cyclosporine is a
selective immunosuppressive drug with antifungal and
antiinflammatory properties. It has been shown to
strongly suppress the production of haemagglutinating
antibodies against sheep erythrocytes in mice and to
be effective in inhibiting both humoral and cellmediated immune responses. Its effect is reversible,
and specific for T-lymphocytes. The hemopoietic
tissues in immunosuppressed animals are not affected.
Bueding _
et _
a1 (58) demonstrated an antishistosomal
activity and Thommen-Scott ( 59) an antimalarial effect
of cyclosporine, both antiparasitic activities being
independent of immunosuppression. The first transplantation in humans with the aid of cyclosporine were
reported in 1978 by Calne et a1 (60) f o r kidney and by
Fowles et a1 (61) f o r bone marrow. Today cyclosporine
is used successfully to prevent graft rejection following
bone marrow and organ transplantations.
Other possible applications, such as in the treatment
of autoirnune and other diseases, are currently under
investigations.
Cyclosporine ( Sandimmun) is available as :

a)
Drinking Solution: 100 mg/ml cyclosporine dissolved

in olive oil and labrafil.
b)
Intravenous Preparation: Ampoules of 5 m l (250 mg

cyclosporine) and 1 ml (50 mg cyclosporine).
11.Methods of Analysis
11.1 Elemental Composition
Cyclosporine (C H
N 0 ):
62 111 11 12
C
61.90
9-30
12.80
16.00
192
11.2
Introduction to Analysis
Several methods have been developed for the
analysis of cyclosporine in plasma and or blood.
The first method reported was the radioimmunoassay (RIA) of Donatsch (62)which is now available as a kit and has been used extensively
during the clinical development for cyclosporine.
The method was initially developed for plasma,
and as shown later, is also applicable to blood
samples. The main criticism of the RIA method
has been the cross-reactivity of the antisera
with some of the circulatinc metabolites of
cyclosporine. Other RIA procedures were
reported ( 54,55, 63-67).
Subsequent to the introduction of the RIA
method, Niederberger et a1 (68) developed a high
pressure liquid chromatogri:phy ( HPLC) method
based on gradient elution. The technical complexity of this method encouraged several
investigators, (69-75) to reinvestigate the
method. The results of these activities are
shown in Table 4.
11.3
Radioimmunoassay ( RIA)
Several radioimmunoassay procedures have been
developed for both the analysis and immunopharmacological monitoring of cyclosporine ( 54,55,
63-67)
One method described recently by Randall et a1

(64) is as follows:
Sample Preparation: Venous blood was collected
in heparinized tubes from 38 post-transplant
patients receiving cyclosporine, just before
their next dose and 12 h after their last.
Non-temperature-standardized protocol: About

0.5 to 3 h after collection, an aliquot of each
sample f o r whole-blood analysis was set aside
and centrifuged the remaining specimen at room
temperature to remove cells. The plasma and
whole-blood specimens were both then stored at
- 20%.
CYCLOSPORINE
193
Temperature-standardized p r o t o c o l : After c o l l e c t i o n , samples were r e f r i g e r a t e d for no l o n g e r

t h a n 4 h , r e - e q u i l i b r a t e d t o 37OC by i n c u b a t i o n
i n a water b a t h f o r 30 min, t h e n c e n t r i f u g e d
without d e l a y (1100 X g , 1 0 min, room t e m p e r a t u r e )
The r e s u l t i n g plasma w a s s t o r e d f r o z e n a t -2OOC
u n t i l analysis.
Time and t e m p e r a t u r e experiments: A l i q u o t s of
whole blood from p a t i e n t s r e c e i v i n g c y c l o s p o r i n e
were i n c u b a t e d for 0.25, 0.5, 1, 2, 4 , 6, and
24 h a t 4'12, room t e m p e r a t u r e ( a b o u t 22OC), and
37OC. A t t h e s e s p e c i f i e d t i m e s , samples s t o r e d
a t room temperature or 4C were c e n t r i f u g e d a t
room t e m p e r a t u r e ; t h o s e s t o r e d a t 3 7 O C were
c e n t r i f u g e d a t 37OC and t h e plasma was decanted
and f r o z e n .
I n a d d i t i o n , t h e t r e a t e d a l i q u o t s of samples
s t o r e d a t room t e m p e r a t u r e and 4 O C f o r t h e
above t i m e s b y r e - e q u i l i b r a t i n g t o 3 7 O C by
i n c u b a t i n g for 1 5 min i n a 37OC w a t e r b a t h ,
followed by c e n t r i f u g a t i o n a t 37OC and prompt
removal of t h e plasma.
E q u i l i b r a t i o n time experiment: A l i q u o t s o f
whole blood from p a t i e n t s r e c e i v i n g c y c l o s p o r i n e
w e r e maintained a t 4 O C f o r 4 h a f t e r c o l l e c t i o n , t h e n e q u i l i b r a t e d i n a 37OC w a t e r b a t h for
0, 10, 1 5 , 30, 60, 1 2 0 , and 180 min. All
specimens were c e n t r i f u g e d a t 3 7 O C , and t h e
plasma was immediately decanted and stored a t
-2OOC u n t i l a n a l y s i s .
Measurement of c y c l o s p o r i n e : Cyclosporine w a s
measured by radioimmunoassay, u s i n g t r i t i a t e d
c y c l o s p o r i n e as t r a c e r and an a n t i b o d y i n a k i t
s u p p l i e d by Sandoz L t d . , B a s l e , S w i t z e r l a n d .
D u p l i c a t e 5 pL p a t i e n t ' s
samples were mixed
with
1 0 0 pL
of t r a c e r , TOO 1J.L o f T r i s
b u f f e r ( 0 . 5 mol/L, p H 8 . 5 ) , and 100 UL o f sheep
a n t i s e r u m t o c y c l o s p o r i n e , t h e n i n c u b a t i n g for
2 h a t room t e m p e r a t u r e . F r e e and bound f r a c t i o n s
were s e p a r a t e d by e x t r a c t i o n w i t h c h a r c o a l for
min a t 4 O C .
The r a d i o a c t i v i t y was measured i n
each specimen w i t h a l i q u i d s c i n t i l l a t i o n
194
c o u n t e r , u s i n g a quench-corrected c o u n t i n g program. The a n a l y t i c a l recovery o f c y c l o s p o r i n e

i n plasma and whole blood supplemented w i t h
t h e drug w a s about 75%.
C o r r e c t i o n f o r h e m a t o c r i t : Hematocrits of
samples f o r c y c l o s p o r i n e a n a l y s i s were measured
as p a r t o f t h e r o u t i n e b l o o d count on a
Coulter S Plus (Coulter Instruments, Hialeah,
F L ) . The plasma c y c l o s p o r i n e r e s u l t s were
c o r r e c t e d f o r hematocrit by m u l t i p l y i n g t h e
u n c o r r e c t e d plasma c y c l o s p o r i n e
r e s u l t s by t h e
p a t i e n t ' s hematocrit / O .39, t h e n o m a l v a l u e f o r
t h e hematocrit.
1 1 . 4 High Performance Liquid Chromatography

S e v e r a l HPLC procedures were r e p o r t e d for t h e
e s t i m a t i o n o f c y c l o s p o r i n e i n blood and o r
plasma a s w e l l as immunopharmacological monit o r i n g o f c y c l o s p o r i n e ( 5 3 , 5 4 , 67, 69-75).
These methods were developed t o overcome t h e
drawbacks o f t h e R I A methods p a r t i c u l a r l y t h e
overestimation of cyclosporine i n b i o l o g i c a l
f l u i d s . This i s because, t h e a n t i b o d i e s c r o s s
r e a c t w i t h t h e m e t a b o l i t e s . The HPLC c o n d i t i o n s
used f o r t h e a n a l y s i s o f c y c l o s p o r i n e a r e
l i s t e d i n Table 5. A r e c e n t HPLC method w a s
r e p o r t e d by Kates and L a t i n i ( 7 5 ) and i s as
follows :
Chemicals and r e a g e n t s : Cyclosporine and t h e
i n t e r n a l s t a n d a r d , c y c l o s p o r i n D ( F i g . 14).
are o b t a i n e d from Sandoz (Basle, S w i t z e r l a n d ) .
Glass-distilled diethyl ether, a c e t o n i t r i l e ,
q-hexane, and methanol are o b t a i n e d from
Burdick and Jackson Labs. (Muskegon, M I ,
Tris(hydroxymethy1)ainomethane i s
U.S.A.).
purhcased from A l d r i c h (Milwaukee, W I , U.S.A.)
I n s t r u m e n t a t i o n and chromatograghic c o n d i t i o n s :
A Waters Instrument ( M i l f o r d , MA, U.S.A.)
Model 6 0 0 0 ~HPLC pump, Model 480 v a r i a b l e wavelength UV d e t e c t o r , Model 7lOB sample
i n j e c t o r and Model 730 d a t a module a r e employed.
The column used i s a Brown-Lee Labs. ( S a n t a
Clara, CA, U.S.A.) RP-8 MLPC a n a l y t i c a l c a r t r i d g e
195
CYCLOSPORINE
( 1 0 ern X 4.6 mm I.D.; p a r t i c l e s i z e 1 0 m). A

3-em RP-8 guard c a r t r i d g e i s p o s i t i o n e d a t t h e
head o f t h e column. The column i s m a i n t a i n e d
a t 7 O o C with an Eldex (Menlo Park, CAY U . S . A . )
column h e a t e r . The h e a t e r i s l e f t on a t a l l
t i m e s and a c o n s t a n t flow i s m a i n t a i n e d
through t h e column. The flow-rate o f t h e
mobile phase i s 0.6 ml/min which produces a
precolumn p r e s s u r e o f about 34 bars ( 500 p. s i. )
The e f f l u e n t from t h e column i s monitored a t a
wavelength o f 215 nm.
Mobile phase: The mobile phase c o n s i s t s o f a

simple mixture of a c e t o n i t r i l e - w a t e r ( 72:28).
This m i x t u r e i s f i l t e r e d and degassed by
vacuum and s o n i c a t i o n . The mobile phase i s
c o n t i n u o u s l y r e c y c l s e d and r e p l a c e d about
every two weeks.
P r e p a r a t i o n o f e x t r a c t i o n columns: The e x t r a c t i o n o f c y c l o s p o r i n e i n v o l v e s t h e use o f a
Baker-10 SPE e x t r a c t i o n system ( J . T . Baker,
P h i l l i p s b u r g , N J , U.S.A.).
The columns used
are t h e 3 ml cyano d i s p o s a b l e e x t r a c t i o n
columns. These columns a r e p r e p a r e d by washing
w i t h 6 ml of methanol and t h e n 6 ml o f water
under vacuum. They a r e l e f t approximately one
h a l f t o t h r e e q u a r t e r s f u l l of w a t e r u n t i l t h e
sample i s added.
E x t r a c t i o n procedure: One ml o f whole blood
or serum i s added t o PTFE-lined, screw-capped
t u b e s . To t h e blood or serum i s added 450 ng
o f i n t e r n a l s t a n d a r d , 3 ml o f 0 . 1 M T r i s b u f f e r
(pH 9 . 8 ) and 1 0 m l o f d i e t h y l e t h e r . They a r e
rocked f o r 20 min on a labquake r o c k e r (Lab.
I n d u s t r i e s , Berkeley, CA, U.S.A.) and c e n t r i fuged for 20 min t o s e p a r a t e t h e e t h e r and
aqueous l a y e r s . The d i e t h y l e t h e r i s p i p e t t e d
i n t o a c l e a n t u b e and t h e blood or serum residue i s d i s c a r d e d . To t h e d i e t h y l e t h e r i s
added 200 p1 o f 75% methanol i n w a t e r . The
d i e t h y l e t h e r i s t h e n evaporated a t room temperat u r e w i t h a g e n t l e stream of n i t r o g e n . Only
t h e d i e t h y l e t h e r i s e v a p o r a t e d , l e a v i n g 150200 p 1 o f t h e methanol-water remaining.
To
t h i s are added a n o t h e r 100 u1 of methanol. The
196
methanol-water i s t h e n t r a n s f e r r e d t o a 3 m l
Baker e x t r a c t i o n column, The r e s i d u e i s
e l u t e d onto t h e column w i t h water. The column
i s t h e n washed w i t h 3 ml of 25% a c e t o n i t r i l e
i n w a t e r and 6 m l of n-hexane.
The columns a r e
t h e n d r i e d by drawing through a i r for 4 min.
Cyclosporine and c y c l o s p o r i n D are t h e n
d l u t e d off t h e column w i t h t h r e e washings o f
200 p l of methanol. The methanol i s c o l l e c t e d
i n t o d i s p o s a b l e t u b e s and evaporated t o dryness
w i t h a stream of n i t r o g e n a t 5OoC. The
r e s u l t i n g r e s i d u e i s d i s s o l v e d i n 200 p 1 of t h e
mobile phase. An a l i q u o t o f 1 0 0 p 1 i s t h e n
i n j e c t e d o n t o t h e column ( F i g . 1 4 ) .
P r e p a r a t i o n of c a l i b r a t i o n s t a n d a r d s : S t o c k
s o l u t i o n s of c y c l o s p o r i n e and c y c l o s p o r i n D
are prepared i n methanol and s t o r e d i n amber
b o t t l e s a t room t e m p e r a t u r e . A s t o c k s o l u t i o n
of c y c l o s p o r i n D i s prepared i n a c o n c e n t r a t i o n
of 30 n g / u l . The s t o c k s o l u t i o n of c y c l o s p o r i n e
i s prepared i n a c o n c e n t r a t i o n of 10 n g / p l .
T h i s s o l u t i o n i s used t o p r e p a r e s t a n d a r d s for
t h e c a l i b r a t i o n curve. The c a l i b r a t i o n c u r v e
samples are prepared by adding amounts of cyclos p o r i n e (50-800 ng) t o whole blood or serum
from a normal v o l u n t e e r . These are t h e n
t r e a t e d as d e s c r i b e d i n t h e e x t r a c t i o n procedure.
This procedure has a lower l i m i t of s e n s i t i v i t y
below 50 ng/ml.
I
n
71
11
F i g . 14. Chromatograms of extracted whole blood samples. ( A ) Blood from normal healthy
volunteer, not taking cyclosporine; (B) blood to which has been added 200 ng/rnl of cyclosporine and the internal standard; (C) blood from a patient who was taking cyclosporine
( t h e concentration in this patient sample is 304 nglml). Peaks: I = cyclosporine; I1 = cyclosporin D, internal standard.
Table
4. Comparison o f p u b l i s h e d methods o f a n a l y s i s o f c y c l o s p o r i n e
analysis
Sample t y p e
Sample*
preparation
Detect i o n
limit
pg/litre)
Specificity
S t an dardization
Ext
20
High
Int
30
100
Ext
Very l o n g
25
High
Int
Short
20
High
Blood/plasma
HPLC-Grad.
Plasma
Long
Serum
Long
Plasma
Long
100
High
.
Int .
Int .
HPLC-Isocr.
Blood/plasma
Long
100
High
Int
HPLC-Grad.
Serum
Long
30-50
High
Int
HPLC-Isocr.
Plasma
Long
31
High
Int .
ihort
s emiaut oma-
High
Ext
50
High
Int
HPLC-Isocr.
*
1-20
Blood/plasma
HPLC-Col Swit Blood/plasma

HPLC-Isocr
HPLC-Col .Swit 3loodlplasma
HPLC-Isocr
"Short:
**RIA:
ted)
Long
.
.
54955,
63-67
68
5
10
69
70
30
71
20
72
63
45
15
30
73
74
15
67
19
75
p r o t e i n p r e c i p i t a t i o n and i n j e c t i o n ; Long: e x t r a c t i o n & e v a p o r a t i o n ;

a d d i t i o n a l sample m a n i p u l a t i o n .
Radioimmunoassay. Ex:.
=.External; I n t . = Internal;
Col-Swit
= Column s w i t c h i n g .
Ref.
Time( min)
Metabolite
x-react i v i t y
RIA**
HPLC-Isocr
Chromat o-
Very Long:
Grad. = G r a d i e n t ; I s o c r . = I s o c r a t i c ;
Table
5.
HPLC c o n d i t i o n s used f o r t h e a n a l y s i s of c y c l o s p o r i n e
Flow
Sample
Instrument
rate
Mobile phase
Column
m l /min
I
Blood/snrum Waters Model RP-8 YLPC a n a l y t i 6000~
,Blood/
Technicon
Acet o n i t r i l e /
LC-8 S u p e l c o s i l ,
5 urn, s u p e l c o , I n c . , water 55:45 v/v
B e l l e f o n t e , PA.,
a t 75Oc.
A l t e x pumps
AX-110.
UV d e t e c t o r
LCD 725 Uvikon
Recorder
Tarkan 600
W+W A l t e x
m i croproc essor 420.
blood/
plasma
.
.
0.6
Acetonitrilel
w a t e r 72:28
cal cartridge,
10 cm x 4.6 mm
I . D . 3-em RP-8
guard c a r t r i d g e ,
a t 7OoC.
Column I; A l t e x
p e r i s o r b RP-8 ,
40 X 4.6 mm I . D . ,
30-40 urn.
Column 11; Lichrosorb RP-18, 150 X
4.6 mm I . D . , 5 urn,
a t 70Oc.
A l t e r n a t i v e column;
Beckman U l t r a s p h e r e
O D s , 150 X 4.6 mm
I . D . , Guard column;
A l t e x p e r i s o r b RP-2
40 X 3.2 mm I . D . ,
30-40 urn.
Tetentior
time
Detect i o n
9.2
UV a t
215 nm
(mins)
3.0
UV a t
202 nm
1-7
W at
210 nm
C o n s i s t s of 6 solvent s.
1. A c e t o n i t r i l e 1
methanollwat e r
35 :20 :45 v/v/v
2. Acetonitrile1
water 55:45 v/v

3. A c e t o n i t r i l e l
water 72:28 vlv
4.
5-
6.
Tetrahydrofuran
Acetonitrile/
water 90110 v/v
Methanol
Table
5.
Sample
Serum
Serum
( Continued)
1
Instrument
Varian Vist a HPLC

system.
Mobile phase
Beckman u l t r a s p h e r e
ODs, 25 X 0.46 cm,
S t a r t i n g with
t r ifluoroacet i c
acidlacetonitrile
5 um.
Precolumn Vydac C18, 35:65 and increasing proport ions
4 X 0.4 cm.
u n t i l 5:95 a t
Both columns a t
1 5 mins.
7OoC.
I
Waters syst e m cont r o l l e d with
M-660 s o l vent programmer
Beckman u l t r a s p h e r e - A c e t o n i t r i l e /
o c t y l , 25 cm X 4.6
methanol / w a t er
mm, 47 :20 :33 by Vol
5 pm.
Maintained a t 72Oc.
Flow Retention
time
Detect ion 3ef
rate
(mins )
(ml/min
14.1
IUV a t
73
1-5
20.46
and
UV a t
210 nm
74
CYCLOSPORINE
12.
201
References
1. W. G a m s , Persoonia,6,
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M. Dreyf'uss, E. Harri, H. Hofhan, H . Kopel, W. Pache,

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York, E l s e v i e r Biomedical, 1982, p. 5.
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Agents Actions, 6, 468 ( 1 9 7 6 ) .
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J . F . Borel, C . Feurer, C. Magnee, Immunology,
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J.F. Borel, C. F e u r e r , C . Magnee, H . S t a h e l i n ,

32, 1917 (1977).
Immunology, -
8.
J . F . Borel, D. Wiesinger, H.U.

Inflammation,
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T.J.
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T.J.
New
Gubler, Eur. J . RheumatoL
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135
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J . A . Smith, L.G.
316 (1980).
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J . S . Richardson, Adv. P r o t e i n Chem. 34,
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468 (1976).
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(1983), p . 396 No. 2748.
18.
R.M.
19*
H . Kessler, H.R. L o o s l i , H . Oschkinat, Helv. Chim.

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D o d d r e l l , M.R.
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21.
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G . Bodenhausen, R . Freeman, D.L.

65, 839 (1976); J. Magn. Reson.
24.
G. Bodenhausen, R . Freeman, R . Niedermeyer, D.L.

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25 *
63,
T u r n e r , J . Chem. Phys.,
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R . Freeman, G.A. M o r r i s , D.L. T u r n e r , J . Magn. Reson.,

26,
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H. Kessler, D. Ziessow, Nachr. Chem. T e c h . Lab.,
27*
H . Kobel, R. T r a b e r , Eur. J . Appl. Microbiol.

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R . T r a b e r , M. Kuhn, A. Ruegger, H . L i c h t i , H . R .
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488 (1982).
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29
R . T r a b e r , M. Kuhn, H . R . L o o s l i , W . Pache, A . von

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1568 (1977).
30.
R . T r a b e r , H.R. L o o s l i , H . Hoflnan, M. Kuhn, A. von

1655 (1982).
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60,
65,
31. H . Kobel, H . R . L o o s l i , R . Voges, E x p e r i e n t i a 39, 873
(1983
CYCLOSPORINE
32.
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81,
1162 (1978).
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35.
R . Wenger, T r a n s p l a n t a t i o n Proceedings 1 5 (4-Suppl.
36.
R . Wenger, Helv. Chim. Acta, 66, 2308 (1983).
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R.S. Cahn, C . I n g o l d , V. P r e l o g , Angew. Chem.

( 1966 1 ,
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Aarau 1980, p. 93.
41.
42.
43.
g,464
(1982).
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66,2672
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78, 413
M. S c h l o s s e r , K.F. Christman, J u s t u s L i e b i g s Ann. Chem.
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( 1973)
R.W.
92,
H e r r , D.M.
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Johnson, J . Am. Chem. S O C . ,
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K.E.
Pfitzner, J.G.
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J.R.
McDermott, N.L.
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M. Z a o r a l , C o l l e c t . Czech. Chem. Commun.,

( 1962 )
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M o f f a t t , J . Am. Chem. S o c . ,
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3,
51, 2551
3,
1273
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204
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50
H. Wissmann, H . J . K l e i n e r , Angew. Chem., 92, 1 2 9 ( 1 9 8 0 ) ;

Angew. Chem. I n t . Ed. E n g l . , 9,
133 ( 1 9 8 0 ) .
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Amino Acid and P e p t i d e Active E s t e r s " , i n t h e ' P e p t i d e s '
eds. E. Gross and J Meinenhofer, Vol. 2 , Academic
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I n t . J . P e p t . P r o t e i n Res. ,
459 (1981).
52 *
G. Maurer, H.R. L o o s l i , E . S c h r e i e r and B. Keller, Drug

Metab. D i s p o s i t , 12,120 (1984).
53.
A . J . Wood, G. Maurer, W . Miederberger, and T.

Beveridge , T r a n s p l a n t a t i o n Proceedings , Vol. XV , No.
Supp-1, 1983, p . 2409.
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W. Howson, M. Stawecki, J . McMichael, J . Koegler, N.
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Vol. X V , No. 4 , Suppl. 1, (1983) p. 2438.
55 *
J. Newburger and B.D. Kahan, T r a n s p l a n t a t i o n Proceedings,

X V , No. 4, Suppl. 1 (1983), p . 2413.
Vol. -
56.
F. F o l l a t h , M. Wenk, S . Vozeh, e t a l , C l i n . Pharmacol.

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638 (1983).
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18,
4,
2,
B.E.
Bueding, J . Hawkins and Y.N.
11, 380 (1981).
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2, 77 (1981).
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K. Thommen-Scott,
60.
R . J . Calne, D . J . White, S . T h i r u , D.B. Evans, P.

McMaster, D.C. Dunn, G.N. Craddock, D.B. Pentlow, K .
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R.L. Powles, A . J . Barrett, H . C l i n k , H.E.M. Kay, J .

Sloane, T . J . McElwain, Lancet 2 , 1327 (1978).
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CYCLOSPORINE
62,
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Trapp, R . l'oges, J . Immunoassay, 2(1), 19-32 (1981).
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S . J . LeGrue, M. B o i l e a u , W.D. Payne, R . H . Kerman,
Transplantation,
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64.
65.
66.
W.Y. R a n d a l l , D.N. Rush, J . R . J e f f e r y , C l i n . Chem.,
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B.D.
Kahan, M. Ried, J . Newburger, Transp. P r o c e e d . ,
15(1), 446 ( 1 9 8 3 ) .
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W.T. Robinson, H.F. Schran, E.P. B a r r y , Transp.

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W. Niederberger, P. Schaub, J . Beveridge, J .

Chromatogr. Biomed. Appl., 182,454 ( 1 9 8 0 ) -
69.
R.
32
70.
R.J.
71*
K. Nussbaumer, W . Niederberger, H.P. Keller, J. High

Resolut Chromatogr Chromatogr. Comun, 2, 424 ( 1 9 8 2 ) .
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B . Leyland-Jones, A . C l a r k , W. Kreis, Res. Comun.

Chem. P a t h o l Pharmacol., 37, 431 ( 1 9 8 2 ) .
73.
G.C. Yee, D . J .
74.
S.G. C a r r u t h e r s , D . J . Freeman, J . C . Koegler, W.

Howson, P.A. Keown, A. Lanpacis, C.R. S t i l l e r , C l i n .
Chem., 9,
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R . E . Kntes, R . L a t i n i , J . Chromatogr. Biomed. Appl.,

309, 441 (1984).
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( S u p p l ) , lOOP ( 1 9 8 0 ) .
Sawchuk, L.L.
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206
13. Acknowledgement
The authors would like to express their sincere
gratitude to Mr. Abdalla I. Babikir of the Medicinal
Aromatic and Poisonous Plants Research Center, College
of Pharmacy, King Saud University, Riyadh, Saudi
Arabia for his continuous and indispensable assistance
throughout the preparation of this profile.
ENALAPRIL MALEATE
Dominic P. Ip and Gerald S . Brenner
1. History and Therapeutic Properties

2. Description
2 - 1 Nomenclature
2.1.1 Chemical Name
2.1.2 Generic Name
2.1.3 Laboratory Codes
2.1.4 Trade Names
2.1.5 CAS Registry Number
2.2 Formula and Molecular Weight
2.3 Appearance, Color, Odor
3.
Synthesis
4.1 infrared Spectrum
- Nuclear Magnetic Resonance Spectrum
1% - Nuclear Magnetic Resonance Spectrum
4 - 4 Ultraviolet Spectrum
4.5 Mass Spectrum
4.6 Optical Rotation
4 . 7 Thermal Behavior
4.8 Solubility
4.9 Crystal Properties
4.10 Dissociation Constants
4 . 1 1 Partition Behavior
4.12 Confocmational Isomers
::;

VOLUME 16
207

DOMINIC P. IP AND GERALD S. BRENNER
208
5.1
5.2
5.3
5.4
5.5
Elemental Analysis
Colorimetric/Spectrophotometric
Chromatographic
5.3.1 Thin-Layer Chromatography
5.3.2 High Performance Liquid Chromatography
Identification Tests
Titration
6. Stability and Degradation

6.1 Solid State Stability
6.2 Solution Stability
7. Pharmacokinetics and Metabolism

8. Determination in Biological Fluids
9.
References
209
ENALAPRIL MALEATE
1. History and Therapeutic Properties
Enalapril is the ethyl ester of a long-acting angiotensin converting enzyme inhibitor, enalaprilat. Enalapril maleate, discovered and developed by the Merck
Sharp and Dohme Research Laboratories (l), is indicated
f o r the treatment of essential and renovascular hypertension. Enalapril is a pro-drug; following oral administration, it is bioactivated by hydrolysis of the
ethyl ester to enalaprilat, which is the active angiotensin converting enzyme inhibitor.
CH3
e C H 2 C H 2 y H N H C H -CO
-N
COOH
COOCHPCH3
Enalapril
CH3
CHZCHzCHNHCH -CO
-N
COOH
COOH
Enalaprilat
Since the oral absorption of enalapril is superior to

that of enalaprilat, it is the compound that is used in
o r a l dosage forms. Enalaprilat, the deesterified parent diacid of enalapril is the form of the drug that is
administered intravenously. Several review articles
give a detailed account of the history, design, chernistry and pharmacology of the drug. (2-6).
2. Description
2.1 Nomenclature
2.1.1 Chemical Name

( S) -1-[N-[ 1-( ethoxycarbonyl) -3phenylpropyll-Galany11 ->proline,
ixltenedioate(1:l)salt
(2)-2-
210
2.1.2
Generic Name
Enalapril Maleate
2.1.3
Laboratory Codes
G154,739-001D, MK-0421
2.1.4
Trade Names
Vasotec, Renitec, Tnnovace, Pres, Xanef
2.1.5
CAS
Registry Number
76095-16-4
2.2 Formula and Molecular Weight
mol w t 492.53
2.3
Appearance, Color, Odor

Enalapril maleate is a white to off-white,
crystalline, odorless powder
3. Synthesis
Enalapril maleate has been prepared by the scheme outlined in Figure 1 ( 7 ) . Ethyl 2-0x0-4-phenylbutanoate
(1) is condensed with L-alanyl-L-proline (2) to give
Schiff base 3, as a mixture of syn and anti isomers.
Reduction of the imine bond in 3 results in formation
of the diastereomeric mixture 4 (SSS and RSS). The
more biologically active diastereomer (SSS) is isolated
o=
C U
z
+
0,
0
,
0"
l
-
A"
a
f
3'
0"
aJ
m
c1
aJ
rl
I4
.A
I4
m
e
w
0
tn
aJ
C
h
v)
212
by fractional crystallization of the maleate salts to

yield enalapril maleate (5) of greater than 99% purity
in 32 - 34% overall yield. In addition to this synthetic route, others have also been described in the literature (1, 8, 9).
4.1
Infrared Spectrum
The infrared spectrum is shown in Figure 2 (10).
The spectrum was obtained as a potassium bromide
pellet using a Perkin-Elmer Model 281B spectrophotometer. Assignments for the characteristic
bands in the spectrum are listed in Table I.
Table I
Wavenumber, an-
3100-3010
2970 and 2920

2800-2200
(Broad, structured)
1750
1727
1645
1570
1500-1390
1375
1355
1187
750
695
4.2
Assignment
Aromatic C-H Stretch
Methyl and methylene C-H
asymmetric stretch
/
\NH$ Stretch
Acid carbonyl stretch
Ester carbonyl stretch
Amide carbonyl stretch
Monohydrogen maleate
carboxyl stretch
Mainly methylene bending
Methyl symnetric bending
Methylene bending
Ester C-0 stretch
In-phase, out-of-plane
hydrogen bending (5adjacent aromatic
hydrogens)
Phenyl out-of-plane ring
bending
H-Nuclear Magnetic Resonance Spectrum

The proton magnetic resonance spectrum is shown in
Figure 3 (11). The spectrum was obtained using a
Varian Associates Model SC300 spectrometer at a
concentration of 0.55% w/v in deuterium oxide
(D20). Active protons exchange with D20 in
10
N E
Bb
0
8
0
L2
0
0
s
0
Li
0
0
9
0
0
?
0
0
F
0
0
E
0
5:
0
0
0
rr)
5:
lo
-m
-m
Figure 3 .
The P r o t o n Magnetic Resonance Spectrum of E n a l a p r i l Maleate
ENALAPRIL MALEATE
215
solution and appear in the HOD signal at

approximately 4.8-4.9 ppm. Figure 4 shows an
expanded scale spectrum which m r e clearly illustrates the signals pattern.
For several protons in the molecule, slow rotation
about the proline amide C-N bond gives rise to
paired signals. The phenomena has been attributed
to the existence of rotational conformers as in
the case of captopril in aqueous solution ( 1 2 ) . A
tabulation of the chemical shifts and assignments
for the numbered structure below is shown in Table
11. The notations 'major' and 'minor' denote the
relative abundance of the two rotamers relative to
each other in the spectrum.
H\
m
C
II
/COOH
H/'\COOH
Table I1
hemical Shift
(ppm) Multiplicity
'I6,
1.30
1.54
1.59
1.75
2.02
2.02
2.33
2.33
2.80
3.60
3.95
3.97
4.27
4.09
Assignment
CE3CH 0-
Alanyl C%
(minor)
Alanyl C%
(major1
Proline C-4 proton
(minor)
Proline C-4 proton
(major)
Proline C-3 protons
(minor)
Proline C-3 protons
(major)
C-3 ' protons
C-4' protons
Proline C-5 protons
C-2' proton (minor)
C-2' proton (major)
CH3CZ20a
' proton (minor)
--csr
I
6.0
4.0
Figure 4 .
3.0
PPM
2.0
1.2
The Proton Magnetic Resonance Expanded Spectrum of Enalapril Maleate
ENALAPRIL MALEATE
217
hemical Shift
'H, 6 (ppm) Multiplicity
4.3
4.33
6.33
7.37
7.41
m
m
Assignment
a' proton (major)
-H
L'C02H
Aryl protons
Aryl protons
-m
-o&p
13C Nuclear Magnetic Resonance Spectrum

The l3C magnetic resonance spectrum shown in
Figure 5 was run using a Varian Associate Model XL200 spectrometer at a concentration of 8 . 4 % w/v in
deuterated methanol (13). The internal standard
was tetramethylsilane. The pulse width was 4 ps,
acquisition time was 0.9 s.
The assignments for the numbered structure below
are shown in Table 111. As discussed above, the
two rotamers are designated 'major' and 'minor' in
order of their relative abundances.
160
Figure 5.
PPM
50
The Carbon-13 Magnetic Resonance Spectrum of Enalapril Maleate
219
ENALAPRIL MALEATE
Table 111
Chemical
13c, 6 (ppm)
16.2
17.4
18.1
24 -9
27.7
31.8
33.7
35.0
35.2
Assignment
iE:$2H3
Alanyl ?[I3
(major)
(minor)
The signals at 24.9-35.2 p p m

arise from the four carbons C-3', C-4' and C-4, C-3
proline including signals
corresponding to rotamers. An
exact assignment of the signals
in this region was not made.
49.8
Proline C5
57.9
58.4
61.7
61.8
62.3
62.4
65.6
65.7
The signals at 57.9-65.7 ppm

arise from the four carbons Proline C-2', Alanyl C-a, C-2'
and -gH2CH3 including
signaIs attributable to
rotamers. An exact assignmnt
of the signals in this region
was not made.
129.3
Phenyl C-4"
131.2
131.4
Phenyl C-2", C-6", and phenyl

c-3", c-5"
138.0
142.9
Naleate -HcSHPhenyl C-1"
170.5
171.2
171.6
172.0
172.4
176.5
176.7
The signals at 170.5-176.7

arise from alanyl, proline,
ester and maleate carbonyl
carbons. However, definitive assignments were not
made for the carbonyl
carbons.
Carbon-13 NMR may also distinguish enalapril mleate and 'ts RSS diastereomer. Major differences
between
spectra of the two compounds occur
between 31.8 and 34.0 ppm, between 54.7 and 60.8
220
ppm and between 168.6 and 170.7 ppm. However,

this technique is not preferred for accurate
measurement of small quantities of either compound
in the other.
4.4
Enalapril maleate is characterized by inflections
or "shoulders" at about 251 and 263 y%and barely
discernible maxima at abut 21J nm (A1 cm,
about 15) and abut 267 nm (A1 cm, about
8.6). Figure 6 shows the ultraviolet absorption
spectra of enalapril maleate in N/10 methanolic
hydrochloric acid at concentrations of 0.991 mg/ml
and 0.0991 mg/ml.
The low intensity and lack of well-defined maxima
in the ultraviolet absorption spectrum of enalapril maleate, typical of an unconjugated phenyl
moiety, does not make it useful for characterization or quantitation of the compound.
4.5
Mass Spectrum
The direct probe electron impact (EI) mass spectrum of enalapril maleate as shown in Figure 7 was
obtained on the VG MM7035'mass spectrometer employing 70 eV electron energy and a probe temperature
of 16OoC (14).
Under the condition of analysis the maleic acid
moiety is pumped off before the peptide derivative
is vaporized. Molecular ion information for
enalapril base is also absent in the spectrum
obtained by direct probe EI at 70 eV and 20 eV,
respectively. Such information is only possible
when a gross sample overload is employed. Under
field desorption (FD)/MS however, good molecular
ion and pseudomolecular ion [M+H]+ formation
(376, 377) is observed (Figure 8 ) .
The low resolution EI 70 eV spectrum (Figure 7)
shows a base peak at m/e at 91 (benzylic ion), [MH20] atm/e 358 and other major fragment
ions. Accurate mass measurements (high resolution, 70 eV, 16OOC) of the diagnostic fragment
ions at m/e 358, 313, 285, 257, 254, 234, 208,
1
al
0
0
*u1
a
\
220 240 260 280 300 320 340
220 240 260 280 300 320 340
Wavelength ( n m I
F i g u r e 6.
The U l t r a v i o l e t Absorption Spectrum of

E n a l a p r i l Maleate i n ( a ) N / 1 0 Methanolic
H y d r o c h l o r i c Acid; C o n c e n t r a t i o n : 0.991 mg/ml
and (b) N i l 0 Methanolie H y d r o c h l o r i c Acid;
C o n c e n t r a t i o n : 0 . ~ 9 9 1 mg/ml
80 c
.u)
)r
c
0)
60-
0)
50
'T'
150
250
350
Mass
Figure 7.
The Low R e s o l u t i o n (EI) Mass Spectrum of

Enalapril Maleate
223
Figure 8.
The F i e l d D e s o r p t i o n (FD) Mass Spectrum of E n a l a p r i l Maleate
224
169, 168, 160, 126, 9 1 and 70 facilitated a proposed fragmention pattern as shown in Figure 9.
4.6
Optical Rotation
The optical rotation,
of enalapril
maleate (three asymnetric centers) in methanol is,
-42.3' (c=l%).
4.7
Thermal Behavior
Enalapril maleate exhibits a single melting endotherm by differential thermal analysis (DTA) under
a stream of nitrogen. The peak temperature of the
endotherm varies with heating rate, indicating
accompanying decompostion during melting of the
conpound (- 14OOC at 2OC/min. and 149OC at 2OoC/
min.) A DTA curve for enalapril maleate obtained
on a DuPont 1090 equipped with a DTA head is given
in Figure 10.
- 143-
By capillary melting point determination (USP
Class Ia method) enalapril maleate melts at

144OC (15).
4.8
Solubility
The following approximate solubility data (Table
IV) have been obtained at ambient temperature.
Table IV
Solvent
Water
Methanol
Ethanol
Isopropanol
Acetone
Acetonitrile
Dimethylformamide
Chloroform
Diethyl ether
Hexane
Polyethylene glycol 300
Solubility (mg/ml)
25
200
80
1.4
9.1
3.3
>400
0.6
< 0.1
< 0.1
< 0.1
t
3
\"
+5
+*
Figure 9.
A Proposed Fragmentation Pattern to Explain the Mass Spectrum of Enalapril Maleate
r o d - r r ,
(D
0
d-
(0
"
"
N
N
N
E
0
2
0
5
0
2
0
0
(D
0
t
ar
m
ar
'rl
m
m
wG
0
u-i
al
>
3
fi
4
k
al
-4
0
.-(
ar
FL(
M
'rl
221
ENALAPRIL MALEATE
The solubility of enalapril maleate increases with

pH. A pH versus solubility profile constructed to
compare theoretical points to actual data is shown
in Figure 11 (16).
4.9
Crystal Properties
Enalapril maleate is a white to off-white crystalline corrpound. The compound is polymorphic. TWO
nonsolvated polylmrphs have been detected and
characterized by high resolution spec oscopic
NMR)
techniques ( F T I R , Raman, solid state
and solution calorimetry ( 1 7 ) . However, these
plymrphs, referred to as Form I and Form I1 are
very similar in energy, differing only by 0.6
Kcal/ml. The X-ray powder diffraction spectra
for Form I and Form I1 of enalapril maleate are
shown in Figures 12 and 13, respectively. The
spectra were obtained using a Philips Electronics
X-ray diffractometer using CuKa irradiation.
Aqueous acidicfiasic potentiometric titration

yielded pKa values of 2.97 and 5.35 at 25OC for
enalapril (18).
4.11 Partition Behavior
At room temperature, enalapril maleate does not
partition out of the the aqueous media listed
below into mineral oil or n-octanol (19).
Solvent Pairs
n-Octanol/pH 7 buffer
mineral oil/pH 3.5 buffer
4.12 Conformational Isomers
Proton NMR and carbon-13 NMR spectra of enalapril

maleate are characterized by several paired signals attributed to slow rotation about the proline
amide bond, resulting in a slow equilibrium between two rotational conformers (cis and trans).
The two rotamers are distinguishable on the NMR
228
1000000
100000
100
10
PH
-D-
o
Figure 1 1 .
pH
Calculated Pts
Experimental Pts
- Solubility Profile of Enalapril Maleate
F i g u r e 12.
Powder X-Ray D i f f r a c t i o n P a t t e r n of E n a l a p r i l Maleate, Form I
In
In
(u
In
0
O*
H
H
0
F
a
.d
fd
fd
rl
wc
0
E
al
PI
a::
N
23 1
ENALAPRIL MALEATE
time scale. In addition, HPLC has provided firm

evidence for their existence by demonstrating the
chromatographic behavior of enalapril maleate at
various temperatures (20). Figure 14 shows chromtograms obtained by reverse-phase HPLC at column
oven temperatures of 10C and 80C. At 10C, in
addition to the early eluting maleic acid peak ( 1.2 minute), the enalapril base peak is resolved
into two distinct rotamer peaks. At 8OoC, the rotamer peaks have coalesced to yield a single symmetrical peak.
5.
Methods of Analysis
5.1 Elemental Analysis

Analysis of Merck Sharp & Dohme reference l o t ,
L-154,739-001D22 for carbon, hydrogen and nitrogen
was reported as follows:
Element
C
H
N
Calculated
58.53
6.55
5.68
Found
58.53
6.75
5.82
5.2 Colorimetric/Spectrophotometric
Due to the lack of strong W absorption maxima,
m r e sensitive ways for the analysis of enalapril
maleate are needed. One approach has been an autoanalyzer colorimetric assay procedure (21). In
this approach enalapril maleate forms a yellow dye
complex with bromothymol blue in acidic aqueous,
pH 2/CHC13 mixture and is then assayed spectrophotometrically at 415 nm after extraction with
chloroform. This method is suitable for content
uniformity and dissolution assays of enalapril
maleate capsules and tablets.
Analysis of enalapril in pharmaceutical dosage
forms via flow injection analysis has recently
been reported (22). Enalapril is determined by
extraction as an ion-pair with bromthyml blue in
an unsegmented flow system. The procedure is stability indicating: the degradation products of enalapril and excipients in the dosage forms do not
interfere.
232
80"C
10C
a0
Time (Minutes 1
Figure 14.
:
N
Time (Minutes
HPLC Chromatograms of Enalapril Maleate

at 10" and 80C
233
ENALAPRIL MALEATE
5.3
Chromatographic
5.3.1
Three solvent systems (23) for the thinlayer chromatography of enalapril maleate
are given below. The systems separate
enalapril base from its salt forming acid
(maleic acid) and its hydrolysis product
(enalaprilat - see Section 6.1)
Solvent System
Rf (Approximate)
A
(chloroform/methanol/ 0.6 (enalapril
base )
acetic acid
90:10 :1)
0.2 (maleic acid)
0 (enalaprilat)
B
(n-Butanol/water/
acetic acid 3:l:l)
Solvent System
C
(n-Butanol/toluene/
water/acetone/acetic
acid 1:l:l:l:l)
0.7 (enalapril
base and maleic
acid)
0.35 (enalaprilat)
Rf (Approximate)
0.55 (enalapril
base )
0.45 (maleic acid
and enalaprilat)
These solvent systems use a silica gel

adsorbent containing an ultraviolet-fluorescence quench indicator (Analtech GF or Whatman K1F or E Merck Silica Gel G60 type
plates). Detection of components is accomplished by visualization of dried plates
under short wavelength (254 nm) ultraviolet
light. Iodine vapor yields a mre intense
spot for the enalapril base but does not
visualize the maleic acid.
In the systems reported above, the low
sensitivity of the method (detection limit
1 to 2 % ) renders it unsuitable for the
234
detection and quantification of low levels

of impurities in enalapril maleate bulk
substance.
5.3.2

(HpLC)
Several HPLC methods have been developed
which are suitable for: (a) quantification
of enalapril maleate and process impurities
in the bulk substance ( 2 4 ) , (b) analysis of
enalapril mleate and degradates in tablets
(2 5 ), (c) content uniformity assays of
enalapril maleate in tablets (26) and (d)
dissolution of enalapril maleate in tablets
( 2 7 ) . Reverse-phase HPLC conditions are
employed. Detection is by uv at 210 or 215
nm. Flow rate is normally 1.8 or 2.0
ml/min. A mobile phase of aqueous phosphate buffer and organic modifier (acetonitrile or a mixture of acetonitrile and
methanol) and an oven temperature of 7OoC
or 80 provide the selectivity and sensitivity required for satisfactory chromatography.
The significance of specifying a high
column temperature has been discussed in
section 4.12. A summary of these HPLC systems is given in Table V.
5.4
Identification of enalapril maleate can be carried
out by infrared absorption (section 4.1), HPLC
(section 5.3.2) and thin-layer chromatography
(section 5.3.1).
Supportive evidence for identification can be
obtained by differential thermal analysis (section
4 . 7 ) and complexation with bromothyml blue
(section 5.2).
5.5
Titration
Enalapril maleate can be determined by potentiometric titration with aqueous sodium hydroxide and
perchloric acid. With respect to the sodium
&
ru
'9
v
X
cv-l JJ
c
u
0
In
a
. .
0
a3
z
a x
0
OD
9
cv
X
F! 2
4:;
0,
obl
..
obl
o c
a,
mRJ
4J .d
0-
w
0
In
07
DOMINIC P. IP AND GERALD S. BREWER
236
hydroxide titration, 0.1N sodium hydroxide is the

titrant and a combination pH electrode is employed
as the electrode system. The perchloric acid titration employs 0.1N perchloric acid in glacial
acetic acid as the titrant and an anhydrous electrode system, e.g. Metrohm #EA 441/5 silversilver chloride electrode filled with 0.1N lithium
perchlorate in glacial acetic acid (or acetic anhydride), versus a glass electrode is used.
6. Stability and Degradation
6.1 Solid State Stability

There are two major potential modes of degradation
for enalapril maleate: (a) hydrolysis of the
ethyl ester to 11, enalaprilat, the free acid and
(b) cyclization to a diketopiperazine derivative
111. The structure of these compounds are shown
in Figure 15.
Crystalline enalapril maleate is a very stable
solid. The compound stored at room temperature
(amber glass) for four years shows no evidence of
degradation by HPLC analysis. Less than 2% of
degradation can be induced by storage at 8OoC for
3 weeks (in screw-cap vials) and < 1% of decomposition at 4OoC and 75% RH (open vial) for 16 weeks.
Enalapril maleate tablets are stable when protected from elevated temperatures and high humidity. Degradation can be induced, however, if tablets are stored at 4OoC and 75% RH in open containers, resulting in enalapril maleate loss of about
10% or greater for three months. The major degradation product is 11. The mass balance between
loss OF enalapril maleate and formation of I1 and
I11 has been demonstrated.
6.2 Solution Stability
The stability of enalapril maleate in aqueous buffer solutions has been studied as a function of pH
in the range of 2-7 (28). Maximum solution stability occurred around pH 3 . The rate of enalapril
loss and the mode of degradation are dependent
upon the solution pH. At room temperature, tgO
IN
V
In
I,
v)
v)
a
u
m
aJ
crr:
rl
U
C
ri
Ll
.d
a
C
'd
w
238
(time required for 10% loss of starting material)

values are 262 days and 114 days or 0.5 W/ml at
pH 2 and 5, respectively. For up to 20% loss at
pH 2, I11 was the major degradation product while
I1 represented only a minor fraction of the loss.
A t pH 3 , I11 was the major degradate but the fraction of I1 was increased. At pH 5 and ahve, I1
accounted for the bulk of enalapril loss with I11
present only in trace quantities.
7. Pharmacokinetics and Metabolism
The pharmacokinetics and metabolism of enalapril

maleate follming oral administration have been the
subject of a number of investigations and reviews (4,
29-32).
Following oral administration of enalapril maleate to
man ( 3 3 ) peak serum concentrations of enalapril and
enalaprilat occur within 0.5 to 1.5 and 3 to 4 hours
respectively. Based on urinary recovery of the total
drug (enalapril plus its active metabolite, enalaprilat) absorption is at least 61%. The primary route of
excretion of the drug is renal. Approximtely 94% of
the dose is recovered in the urine and feces as enalaprilat or enalapril.
Enalapril, when administered orally, is rapidly
absorbed and bioactivated extensively to enalaprilat.
Bioactivation appears to be largely post-absorptive and
there is no evidence for metabolism of enalapril beyond
bioactivation to enalaprilat in man. The in vitro conversion of enalapril to enalaprilat in human autopsy
tissues has been examined (34). Human cadaver liver
was the only tissue in which significant conversion was
demonstrated.
Enalapril absorption is not influenced by the presence
of food in the gastrointestinal tract (35). The drug
was administered to normal volunteers both in the fasting state and with a normal meal. The area under the
serum concentration-time curves for enalaprilat and urinary recoveries for enalaprilat and the total drug did
not differ significantly between the fed and fasted
conditions.
The pharmacokinetics of enalapril maleate was determined in normal volunteers after repeated single daily
ENALAPRIL MALEATE
239
doses for eight days (36). Serum profiles show little

accumulation of enalprilat over the course of the
study. An average effective half-life for accumulation of approximately 11 hours was calculated from
urine data. An average steady state recovery of enalaprilat is achieved by the third or fourth dose. The
serum concentration profile of enalaprilat is characterized by a prolonged terminal phase, apparently representing a small fraction of the administered dose that
has been bound to angiotensin converting enzyme. The
amount bound does not increase with dose indicating a
saturable site of binding.
8. Determination in Biological Fluids

number of procedures have been developed for the
analysis of enalapril and enalaprilat in biological
Eluids. Those of primary utility include radiometric,
enzyrw inhibition and radioimuno-methods.
The physiological disposition and metabolism of enalapril maleate in laboratory animals has been studied
with both radiolabeled drug and an enzyme-inhibition
assay (37). Following administration of the radioactive drug; urine, feces or plasm samples may be
counted by liquid scintillation techniques for total
radioactivity. Urinary radioactivity may be separated
into enalapril and enalaprilat by thin-layer chromatography *
Enalaprilat can also be determined by its in vitro
inhibition of angiotensin converting enzyme (37).
Plasm, urine and homogenates of feces are placed on
xAD.4 columns, washed and then eluted with methanol to
yield enalapril and enalaprilat. The enalaprilat
component of the mixture is determined by the enzyme
assay which uses as a substrate, the tripeptide carbobenzoxycarbonyl-phenylalanyl-histidyl leucine and converting e n z w isolated from porcine plasm. The product, histidyl-leucine is then quantified by fluorescence after reaction with o-phthalaldehydc ( 3 8 ) . For
the determination of the total drug (enalapril plus
enalaprilat) samples are hydrolyzed at high pH and then
reassayed for enalaprilat. Enalapril levels may then
be calculated as the difference between the total drug
and enalaprilat prior to hydrolysis. An enzyme inhibition assay for enalaprilat has also been developed
240
which uses radiolabeled substrate ( 3 9 ) .thereby eliminating derivatization with o-phthalaldehyde and fluorescence detection of the enzyme roducts. The radiolabeled substrate employed is [flHI hippuryl-kglycylL-glycine. The product, [ 3HI hippuric acid is extracted into a water-immiscible scintillation cocktail
and then quantified by counting in a liquid scintillation spectrometer.
Enalaprilat has also been determined in serum and urine
samples by radioimmunassay procedures. (40,41). In
one such procedure ( 4 0 ) antibodies for the radioimunoassay are raised in rabbits using an immunogen prepared
by coupling the lysine analog of enalaprilat (lisinopril) to albumin with difluoro-dinitrobenzene. The
radiolabeled component is obtained by iodination of the
CH2CH2-c
-N
COOH
Lisinopril
- c -c -N
51
COOH
NH2
amidine derived from the reaction of lisinopril and

methyl p-hydroxybenzimidate. The total drug is
measured as enalaprilat after sample hydrolysis by rat
liver homogenate. Enalapril can then be calculated as
the difference between enalaprilat before and after
hydrolysis.
Acknowledgements
The authors wish to thank Mrs. Betty Moyer or typing
the manuscript and Ms. Florence Berg for performing the
literature search.
ENALAPRIL MALEATE
24 I
9. References
1. A. A. Patchett, E. Harris, E. W. Tristram, M. J.
Wyvratt, M. T. Wu, D. Taub, E. R. Peterson, T. J.
Ikeler, J. temroeke, L. G. Payne, D. L. Ondeyka, E.
D. Thorsett, W. J. Greenlee, N. S. Lohr, R. D.
Hoffsomer, H. Joshua, W. V. Ruyle, J. W. Rothrock, S.

D. Aster, A. L. Maycock, F. M. Robinson, R. Hirschmann,
C. S. Sweet, E. H. Ulm, D. M. Gross, T. C. Vassal and
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Bearn Eds., Raven Press, New York, N.Y., 1984.
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(1984).
4. P. H. Vlasses, G. E. Larijani, D. P. Conner and R.
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K.
5. C. S. Sweet, Fed. Proc. 4 2 , 167 (1983).
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A. A. Morris, D. Weitz, D. L. Bohn, H. C. Wenger, T.
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Lohr, H. Joshua, J. P. Springer, B. H. Arison and A.
A. Patchett, J. Org. Chem. 49, 2816 (1984).
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J. Wyvratt (Merck and Co., Inc.) US 4,374,829.
M.
10. Merck Sharp and Dohme Research Laboratories,

unpublished data.

unpublished data.
12. D. L. Rabenstein and A.
(1982).
A.
Isab, Anal. Chem.,
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242
13. Merck Sharp and Dohrne Research Laboratories,

unpublished data.
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unpublished data.
16. R. G. Bergstcorn, Merck Sharp and Dohme Research
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Lindenbaum, A. W. Douglas, S. D. Klein and J.
McCauley, Int. J. of Pharm., 28, 183 (1986).
A.
18. J. A. McCauley, Merck Sharp and Dohme Research

19. J. P. Draper, Merck Sharp and Dohme Research
20. R. Roman, formerly of Merck Sharp and Dohme Research
Laboratories, unpublished data.
21. J. M. Konieczny, Merck Sharp and Dohme Research
Laboratories, personal comnunication.
22. T. Kato, Anal Chim. Acta,
175,339
(1985).
23. Nerck Sharp and Dohme Research Laboratories,
unpublished data.
unpublished data.
25. J. DeMarcO, Merck Sharp and D o h Research
Laboratories, personal comnunication.
26. R. Roman, formerly of Merck Sharp and Dohme Research

Laboratories and C. Bell, Merck Sharp and Dohm
Research Laboratories, personal comunication.
27. D. J. Mazzo, formerly of Merck Sharp and Dohme
Research Laboratories, unpublished data.
ENALAPRIL MALEATE
243
28. L. L. Ng, Merck Sharp and Dohme Research Laboratories,

personal communication.
29. E. H. U l m , Drug. Metab. Rev.,
14 , 99
(1983).
30. P. A. Todd and R. C. Heel, Drugs 31, 198 (1986).

31. R. S. Perry, M e d . A c t u a l . ,
21,
31 (1985).
32. A. F. Lant, R. W. McNabb and F. H. Noormohamed, J.

Hypertens., 2, 37 (1984).
33. E. H. U l m , M. Hichens, H. J. Gomez, A. E. T i l l , E.
Hand, T. C. V a s s i l , J . Biollaz, H. R. Brunner and J. L.
Schnelling, B r . J. Clin. Pharmacol., 14, 357 (1982).
34. I . Larmour, B. Jackson, R. Cubelal and C. I. Johnston,
B r . J. Clin. Pharmacol., 19,701 (1985).
35. B. N. Swanson, P. H. Vlasses, R. K. Ferguson, P. A.
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36. A. E. T i l l , H. J. Gomez, M. Hichens, J. A. Bolognese,
W. R. McNabb, B. A. B r o o k s , F. Noormhamed and A. F.
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Vassil and E. H. U l m , Drug Metab. Dispos. 10, 1 5
(1982).
38. Y. Piquillaud, A. Reinharz and M. Roth, Biochem.
Biophys. Acta, 206, 136 (1970).
39. B. N. Swanson, K. L. Stauber, W. C. Alpaugh and S. H.
Weinstein, Anal. Biochem. 148, 401 (1985).
40. M. Hirhens, E. L. Hand and W. A. Mulcahy, Ligand Q. 4,
43 (1981).
41. P. J . Worland and B. J a r r o t t , J. Pharm Sci. 75, 512
(1986).
The l i t e r a t u r e w a s reviewed t o J u l y , 1986.
HOMATROPINE HYDROBROMIDE
F a i d J. Muktadi, Mahmoud M.A. H u b a n ,

and
AbduR FaMah A . A d i h y
CoUege
F.J. Muktadi
M.M.A.
Hanun
A.A. A d i d y
06
Phanmacy, King Saud U n i v m L t y ,

Riyadh, Saudi A h a b i a .
Uepan;lmeMA: od Phatunacognony.
M e d i c i d , AmmaZic a n d P o h a n o u n P&m%
R e n u c h Cevtta.
Qep'ahtment 06 Phatunacagnony.

VOLUME 16
245
Copylight 0 1987 by Academic Press. Inc.

FARID J. MUHTADI ETAL.
246
1. Description
1.1 Nomenclature
1.2
Formulae

1.5 Appearance, Color, Odor and Taste
2.
3.
1.6
1.7
pH Range
Physical Properties
2.1
Melting Point
2.2
Solubility
2.3
Crystal Structure
2.4
X-Ray Powder Diffraction
2.5
Spectral Properties
2.5.1
W Spectrum
2.5.2
IR Spectrum
2.5.3
Nuclear Magnetic Resonance Spectra
2.5.4
Mass Spectrum
Preparation of Homatropine Hydrobromide
4. Total Synthesis
4.1 Total Synthesis o f Tropine
4.2 Total Synthesis of Mandelic Acid
4.3 Total Synthesis of Homatropine
5. Therapeutic Uses and Pharmacology
6.
Drug Stability
7.1
Tests of Identification
7.2
Microcrystal Formation
7.3
Titrimetric Methods
7.4
Complexornetry
7.5
Polarographic Methods
7.6
7.7
Acknowledgement
References
247
248
1. D e s c r i p t i o n
1.1 Nomenclature
1.1.1 Chemical Names
1.1.2
a- Hydroxybenzene a c e t i c a c i d 8-methyl-8a z a b i c y c l o [ 3 . 2 . 1 ] oct-3-yl e s t e r , hydrobromide.

l a H , 5aH-tropane-3a-01 mandelate ( e s t e r )
hydrobromide.
Benzeneacetic a c i d , a-hydroxy-, 8-methyl-8a z a b i c y c l o [ 3.2.17 o c t - 3 y l e s t e r hydrobromide , endo-( 2 ) - .
Generic Names
Homatropine Hydrobromide, DL-Hornatropine
Hydrobromide, The hydrobromide of t r o p i n e
mandelate , Tropine mandelate Hydrobromide ,
Mandelyltropine Hydrobromide.
1.1.3
Trade Names
Homatrisol, Bufopto, Homatrocel.
1.2
Formulae
1.2.1
Empirical
CIGH2103N.
1.2.2
HBr
Structural
7
HO OH
II
I
0- C-CH
249
1.2.3
CAS Reg istry No.
[Sl-56-91 C16H2103N.
1.2.4
H Br
Wiswesser Line Notation

T56 A ANTJ A -GOVYGR 6 EH
1. 2. 5
(1)
Stereo ch em istry
B a m i n a t i o n o f t h e M s p e c t r a of some tro p a n e
d e u t e r o h a l i d e s has shown t h a t t h e N-substitue n t i n tro p an es i s predominantly e q u a t o r i a l ( 2 1.
X-ray a n a l y s i s o f t r o p i n e hydrobromide has
shown t h e presence of c h a i r conformation ( 3 ) .
Study of t h e dipole-moment and Kerr-constant
measurements o f a number of tro p a n e d e r i v a t i v es h as shown t h a t t h e p i p e r i d i n e r i n g i s i n
t h e c h a i r form w i t h t h e N-methyl e q u a t o r i a l
( 4 ) . Another study of t h e dipole-moments and
NMR s p e c t r a o f some tr o p a n e d e r i v a t i v e s have
confirmed t h a t t h e p i p e r i d i n e r i n g i s i n t h e
chair conformation w i t h t h e N-methyl group
predominantly e q u a t o r i a l ( 5 ) . I n t r o p i n e ,
however, t h e predominant conformation i s t h e
p i p e r i d i n e r i n g i n a deformed c h a i r form t o gether w i t h a minor amount i n t h e boat form
(6
I*
HO
Tropine
I n a t r o p i n e , t h e a-3 -s u b s titu e n t i s of greet e r bulk th an t h e hydroxyl, and t h e boat form

may w i l l be favored because o f t h e in c re a s e d
i n t e r a c t i o n s i n v o l v i n g t h e dimethylene b rid g e
i n t h e c h a i r conformation ( 7 ).
Hornatropine would be t h e same as a t r o p i n e .
250
II
N -CH3
e /
011
0-c-CH
6HS
A d e t a i l e d review i s a v a i l a b l e f o r t h e boat o r
c h a i r conformation i n t r o p i n e s ( 8 ) .
Other PMR s t u d y suggested a p r e f e r e n c e f o r t h e b o a t

conformation i n s e v e r a l t r o p a n e d e r i v a t i v e s . T h i s
s t u d y showed s t r o n g c r o s s - r i n g i n t r a m o l e c u l a r i n t e r a c t i o n s of t h e t y p e N---C---O
and N---H-0were i n d i c a t e d by t h e broadening of t h e p r o t o n s i g n a l due t o
t h e c o u p l i n g between 1(5)-H and 2(4)-H p r o t o n s i n
t h e boat conformer compared with t h e c h a i r . T h i s
broadening a r i s e s a s a consequence of e c l i p s i n g o f
t h e s e p r o t o n s i n t h e boat conformer ( 9 ) .
1
A p p l i c a t i o n of H-NMR c o n t a c t s h i f t s u s i n g n i c k e l
and c o b a l t d i a c e t y l a c e t o n a t e s , t o t r o p i n e s h a s
demonstrated t h a t t h e six-membered r i n g of t r o p i n e s
is i n a n e a r l y s e m i p l a n e r form. I t i s s u g g e s t e d
t h a t t h e p i p e r i d i n e r i n g of t r o p a n e s is somewhat
d i f f e r e n t i n conformation from t h e normal c h a i r
form ( 1 0 ) .
Carbon-13 magnetic resonance s t u d y has a l s o suggest e d a n o n - c h a i r conformations i n t r o p a n e d e r i.vat i v e s (11).
Me
1.3
Molecular Weight
275.33
356.3
(Homatropine)
(Homat r o p i n e HBr)
25 1
1.4
Elemental Cormosition
C , 69.79%; H, 7.69%; N , 5 . 0 9 % ; 0 , 17.43%
Il-lomatropine)
C , 53.94%; H , 6 . 2 2 % ; B r , 2 2 . 4 4 % ; N , 3.93%; 0 , 13.47%
(Homatropine HBr).
1.5
Appearance, C o l o r , Odor and T a s t e

C o l o r l e s s c r y s t a l s o r a w h i t e c r y s t a l l i n e powder,
o d o r l e s s , with a b i t t e r t a s t e ( 1 2 ) .
Orthorhombic, bipyramidal p r i s m s (from w a t e r ) ,
odorless (13).
1.6
D i s s o c i a t i o n Constant
pKa
1.7
9.9(2O0)
(14)
pH Range
-A 2% s o l u t i o n i n water has a pH 5 . 5 t o 7 (12 ) ,
a 5.67% s o l u t i o n i n water i s i s o - o s m o t i c w i t h serum.
pH (1%aqueous s o l u t i o n ) : 5.4 (13).
2.
i'hysical P r o p e r t i.es
2.1
Melting P o i n t
(12)
214 - 217O (with decomposition)
O t h e r m e l t i n g p o i n t s were a l s o r e p o r t e d :
215 (with decomposition)
f 15)
217-218 (with decomposition)
(1)
2.2
Solubility
One gram d i s s o l v e s i n 6 ml water, 40 m l a l c o h o l ,
1 2 m l a l c o h o l a t 60, 420 m l chlornform. I n s o l u b l e
i n e t h e r . The s o l u b i l i t y i n c r e a s e s r a p i d l y as t h e
t e E p e r a t u r e rises (12 ) .
2.3
Crystal Structure
The c r y s t a l s t r u c t u r e o f b o t h homatropine and homat r o p i n e hydrobromide were r e p o r t e d (16). C r y s t a l s o f
homatropine were o b t a i n e d by r e c r y s t a l l i z a t i o n from
e t h a n o l , whereas t h e c r y s t a l s of homatropine hydrobromide (C16H21N03. H R r ) were o b t a i n e d by r e c r y s t a l -
FARID J. MUHTADIETAL.
252
l i z a t i o n from water.
O s c i l l a t i o n and Weissenberg photographs were used
t o determine t h e space group (Table 1 ) .
In b o t h homatropine and homatropine hydrobromide t h e
n i t r o g e n atom may be o p t i c a l l y a c t i v e , s i n c e t h e
s p a c e groups a r e centrosymmetric, t h e u n i t c e l l s
must be assumed t o c o n t a i n e q u a l numbers of L - a d
D-molecules ( 1 6 ) .
T a b l e 1.
The C r y s t a l S t r u c t u r e o f Homatrop i n e and of Homatropine H B r .
a(A")
b(A")
c(A")
Homatropine
14. 5
15.1
6.97
1.21
Homatropine
HBr
10.3
16.4
19.25
1.48
Compound
2.4
Density (g.cmR3)
observed c a l c u l a t e d
Space
group
1.22
P211/c
1.46
Pcab
X-ray Powder D i f f r a c t i o n
The X-ray d i f f r a c t i o n p a t t e r n o f homatropine hydrobromide was determined with a P h i l i p s X-ray d i f f r a c t i o n s p e c t r o g o n i o m e t e r equipped w i t h PW 1730 generator.
X-ray r a d i a t i o n was provided by a copper t a r g e t (Cuanode 2000 W ) .
High i n t e n s i t y X-ray t u b e o p e r a t e d
a t 40 KV and 20 mA. The monochromator was a s i n g l e
curved c r y s t a l ( y = l .5918A0)
The u n i t was equipped
with P h i l i p s PM 821.0 p r i n t i n g recorded d i g i t a l p r i n t e r . Divergance s l i t and t h e r e c e i v i n g s l i t were 1".
The scanning speed o f t h e goniometer used was 0.02
2 e / s e c . , r e c o r d e r f u l l s c a l e was 10,000 c o u n t s a t
r e c o r d e r speed o f 4 mm/28.
The lower level and t h e
upper l e v e l of t h e s i g n a l c o n t r o l waz 35 E 75 r e s pectively.
The u n i t was a l i g n e d and examined, u s i n g a S o l i c o n
s t a n d a r d , t o a t t a i n maximum o p e r a t i o n c o n d i t i o n s .
The X-ray p a t t e r n of homatropine hydrobromide i s
p r e s e n t e d i n F i g . 1. I n t e r p l a n n a r d i s t a n c e and
r e l a t i v e i n t e n s i t y are t a b u l a t e d i n t a b l e 2 .
253
Table 2 : X-Ray Powder D i f f r a c t i o n P a t t e r n

of Homatropine Hydrobromide
~~
6.98
6.41
6.00
5.15
5.04
4.84
4.64
4.48
4.35
4.28
4.19
3.96
3.57
3.50
3.43
3.30
3.23
3.17
3.12
3.07
3.00
2.97
~
17.8%
17.3%
22.1%
15.7%
21.1%
73.5%
12.6%
14.4%
39.0%
85.9%
100.0%
36.3%
48.7%
46.1%
11.2%
38.5%
21.3%
20.2%
22.7%
20.7%
20.8%
17.8%
2.87
7.83
2.72
2.64
2.61
2.58
2.52
2.50
2.47
2.35
2.32
2.29
2.26
2.15
2.07
2.03
1.98
1.93
1.87
1.80
1.77
11,7%
24.1%
19.5%
10.7%
14.2%
20.0%
22.9%
12.1%
22.0%
13.5%
25.9%
13.7%
13.1%
11.9%
11.3%
16.4%
10.7%
13.0%
11.2%
11.0%
13.5%
~~
d = i n t e r p l a n a r distance, I/Io = relat 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 100).
J
1
lY 1
X-RAY DIFFRACTION PATTEW OF HCMATROPINE HYDROIIRO\IIDL.
254
2.5
Spectral Properties
2.5.1
The UV spectrum of homatropine hydrobromide
i n methanol ( F i g . 2 ) was scanned from 200
t o 350 nm u s i n g DMS 90 Varian S p e c t r o p h o t o meter. I t e x h i b i t s t h e f o l l o w i n g UV d a t a
(Table 3 ) .
Table 3 . UV C h a r a c t e r i s t i c s o f Homatropine
Xmax. a t nm
252
258
264.5
273
(A 1%,lcm)
224.28
26 7
228.5
78.32
6.3
7.5
6.42
2.2
Other r e p o r t e d UV s p e c t r a l d a t a o r homatropine i n 0.1 N s u l f u r i c acid ( 1 7 ) .

Xmax. a t 252 mu ( E 1%, 1 cm 4 . 8 ) , 258 mu
( E 1%, 1 cm 6.1) and 264 mu ( E 1%, 1 cm 5 ) .
Amax. i n 0 . 1 N h y d r o c h l o r i c a c i d a t 254 nm
( E 1%, 1 cm = 3 ) ; a t 259 nm ( E 1% , 1 cm = 5 . 2 )
and 266 nm. ( 1 8 ) .
2.5.2
The I R spectrum of homatropine hydrobromide
as KBr-disc was r e c o r d e d on a Perkin E l m e r
580 B I n f r a r e d S p e c t r o m e t e r t o which i n f r a r e d
d a t a s t a t i o n i s attached (Fig. 3 ) .
The s t r u c t u r a l assignment 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 4 ) .
Table 4. I R C h a r a c t e r i s t i c of Homatropine
Ass i gnment
Frequency cm-
Base
3350
2940,3050
1735
HBr
3270
2965
2 80 0 - 2.585
1755
OH ( s t r e t c h )
CH ( s t r e t c h )
NH
-0-C- ( e s t e r )
255
200
250
300
350
Fig. 2 : THE W SPECTRUM OF HOMATROPINE IIYDROl3KOMIDE IN PEMANOL
3500
3000
2500
2000
19
1600
1400
1200
1000
1800
Fig. 3 : IHE IR SPECTRLM OF HOMATRDPINE HYDROBRDMIDE AS KBr DISC.
800
600
257
HOMATROPINE HY DROBROMIDE
Frequency cm -1
Ease
Assignment
HBr
1600
145 0,1500
1100,1065
755,700
1600
1475
1170,1070
735,700
C=C (aromatic)
C=C (aromatic)
C-0-C (ether)
5 H (monosubstituted
aromatic)
Other characteristic bands are 2750, 2710, 1450,

1420, 1395, 1378, 1328, 1288, 1265, 1195, 1105,
1025, 960, 885, 820 and 615 cm-l.
Other IR data for homatropine and the hydrobromide
salt have been reported (1,14,17).
2.5.3
Nuclear Magnetic Resonance

2.5.3.1
'H-NMR Spectra
The 'H-NMR spectra o f homatropine
hydrobromide in DMSO-D6 and in D20
were recorded on a Varian T-60A, 60
MHz NMR spectrometer iising TMS (tet-
A H 3
ramethylsilane) as an internal reference. These are shown in F i g . 4

and 5 respectively. The following
structural assignment have been made
(Table 5).
76&
>
H
$0
'
" OH
Oi4
12
13
0- C-CH
9
10
1
'6
Table 5. The H-NMR Characterjstics of Homatropine
Group
5 aromatic protons
(12,13,14,15,16 H)
10 H (OH)
10 H
Chemical Shift (pmm)

DMSO-d6
D2
7.33 (s)
7,4 (s)
6.03 (d)
5.13 (d)
5.33 ( s )
J
L
.
.
In
. . . .
In
. I. . .
,
1 ,
l
6 0
so
.
.
.
PPWIII
,
l
,
.
an
,
.
I
.
,
.
1 0
,
.
,
.
.
.
1
*
,
I
Y O
Fig. 4 : THE 'H-NMR SPECTRUM HOMATROPINE HYDROBROMIDE IN D I W - L ) ~
.
.
L
10
Fig. 5 : THE 'H-NMR SPECTRUM OF HOMATROPINE HYDROBROMIDE IN D20.
FARID J. MUWTADIETAL.
260
Group
Chemical S h i f t (pmm)
DMSO- d6
D2
3H
4.70 ( t )
5.03 ( t )
1 H and 5 H
3.71 ( b s )
3.66 ( b s )
8-N-Me
2.63 (s)
2.67 (s)
2,4,6,7 H
2.4-1.27
(m)
2.4-1.8
(m)
s = s i n g l e t , d = d o u b l e t , t = t r i p l e t , bs=broad s i n g l e t
1
Other H-NMR d a t a f o r homatropine hydrobromide was
r e p o r t e d ( 1 ) . Ohashi e t a 1 (10 ) have r e p o r t e d
'H n u c l e a r magnetic resonance c o n t a c t s h i f t s o f some
t r o p a n e s i n c l u d i n g homatropine i n CDC13 a t 220 MHz.
The c o n t a c t s h i f t s used were n i c k e l and c o b a l t d i a cetylacetonates.
2.5.3.2
13C-NMR
The I3C -NMR complete l y decoupled

and o f f resonance s p e c t r a of homat r o p i n e hydrobromide a r e p r e s e n t e d
i n Fig.6 and Fig. 7 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
range i n a mixture o f DMSO-D6 and
CDC13 (conc. 100 mg/ml), on a J o e l
FX-100-90 MHz s p e c t r o m e t e r . A samp l e t u b e ' l 0 mm and TMS a s i n t e r n a l
s t a n d a r d a t 20' were used. 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 o f t h e chemical s h i f t
t h e o r y and t h e o f f - r e s o n a n c e s p l i t t i n g p a t t e r n (Table 6 ) .
12
II
0- C-CH
9 10
13
Fig. 6 : 'IHE l 3 C - I W W L E T E L Y DECOUPLED SPECTRUM OF HOMATROPINE HYDROBNNIDE.
Fig.
THE
'3C-W
OFF-RESONANCE S P E m M OF Wl'p.OPINE HWWBROFlIDE.
263
Table 6 .
Carbon
No.
1
Carbon Chemical S h i f t s of Homatropine
Chemical S h i f t
[PPml
59.18 (d)
32.11 ( t )
c2
70.74
c3
(d)
32.11 ( t )
59.18 (d)
c5
21.19 ( t )
21.19 ( t )
c7
40.21 ( 9 )
Carbon
No.
9
5 0
cll
5 2
13
14
5 5
16
Chemical S h i f t
[PPml
169.38 (s)
62.76 (d)
137.38 (s)
126.34 (d)
124.76 (d)
125.99 (d)
124.76 (d)
126.34 (d)
s = singlet, d = doublet, t = t r i p l e t , q = quartet.
2.5.4
Mass Spectrum
The mass s p e c t r u m o f homatropine 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 a t 70 eV, was
r e c o r d e d on a Finigan-Mat 5100 Mass S p e c t r o meter. The s p e c t r u m ( F i g . 8) shows a molec u l a r i o n peak M+ a t m/e 275. The b a s e peak
i s a t m/e 124.
The most prominent f r a g m e n t s , t h e i r r e l a t i v e
i n t e n s i t i e s and some proposed i o n f r a g m e n t s
are shown i n t a b l e 7.
Table 7 .
m/e
Mass Fragements o f Homatropine

Relative Intensity %
275
4.4
234
1.2
193
1.2
165
1.3
142
6.4
140
4.6
125
10.5
Ions
-
M+
[IT)]
I-
Fig. 8 : 'THE MASS SF'ECIRLN OF ~ l R O P I t E
m/e
-
Relative Intensity %
265
Ions
125-H
124
100
122
1.6
107
7.5
106
3.8
105
6.1
96
14
95
9.3
96 -H
94
21.5
95-H
83
20.3
82
29.2
79
14.3
77
19.2
68
5.9
67
19.1
68 -H
55
6.7
[CH2 =N=CH-CH2]
O t h e r mass s p e c t r a l
r e p o r t e d (19,20).
data
of homatropine have been
266
3.
P r e p a r a t i o n of Homatropine Hydrobromide
Atropine i s hydrolyzed w i t h barium hydroxide s o l u t i o n t o
g i v e t r o p i n e and t r o p i c a c i d .
The t r o p i n e i s h e a t e d w i t h mandelic a c i d i n t h e p r e s e n c e
o f hydrogen c h l o r i d e . Ammonia i s added, and t h e l i b e r a t e d homatropine i s e x t r a c t e d with chloroform. The c h l o roform s o l u t i o n i s e v a p o r a t e d t o d r y n e s s . The r e s i d u e
i s t r e a t e d with hydrobromic a c i d and t h e homatropine
hydrobromide o b t a i n e d is p u r i f i e d by r e c r y s t a l l i z a t i o n
(21, 2 2 ) . The p r e p a r a t i o n i s o u t l i n e d i n Scheme I .
Scheme I .
f) / CH20H
0-C-CH
\C6H
Hydrolysis
eoi
6
+
HOOC -CH-CHZ OH
HOOC\
HocH@
0-E-cii
OH
6HS
Homatropine
267
An alternative method of hydrolyzing atropine was achieved
microbically by the action of Arthrobacter a t r o p i n i (23).
4. Total Synthesis
Since Homatropine is the tropine ester of mandelic acid,
schemes for the total synthesis of tropine and of mandelic
acid were reported.
4.1
Total Synthesis of Tropine

Four schemes for the total synthesis of tropine are
known. Scheme I1 was also modified to give a much
better yield.
Scheme 11: Willstatter's total synthesis of tropine
(24) *
Suberone (cycloheptanone) [l] is reduced to suberol
which is treated with hydrogen iodide to give suberyl
iodide [2]. This is treated with potassium hydroxide
in ethanol to give cycloheptene [ 3 ] . Cycloheptene
is brominated to give 1,Z-dibromocycloheptane [4]
which is treated with dimethylamine to yield dimethylaminocyclohept-2-ene [5]. The latter is converted
to cyclohepta-1,3-diene [6] by exhaustive methylation.
[6] is brnminated at 1,4-positions to give 1,4-dibromocyclohept-2-ene [7]. Elimination of two moles of
the hydrogen bromide of [7] is effected by quinaline
to give cycloheptatriene [ 8 ] .
Substance [8] is treated with hydrogen bromide to
give bromocyclohepta-3,5-diene [ 9 ] which is reacted
with dimethylamine to give dimethyl aminocyclohepta2,4-diene [ l o ] . The latter is treated with sodium in
ethanol followed by bromination to give 1,2-dibromo5-dimethylamino-cycloheptane [ll].
This is warmed in
ether when intramolecular alkylation occurs to give
2-bromotropane methobromide [12]. Hydrogen bromide
is eliminated from [12] by the action of alkali to
yield tropidine methobromide [ 1 3 ] . This is transformed to tropidine methochloride [ 1 4 ] by the action
of potassium iodide followed by the action of silver
chloride. Sub-rtance [14] is pyrolized to give tropidine [15].
Hydrogen bromide is added to an acetic acid solution
of tropidine [15] to yield 3-bromotropane [16! which
is hydrolysed with 10% sulfuric acid at 2@0-21@0 to
give pseudotropine [ 171. JI-tropine [ 171 is oxidized
with chromium trioxide to give tropinone [18]. This
ketone is reduced with zinc and hydriodic acid to
tropine [ 191.
268
Scheme 11 : W i l l s t a t t e r ' s t o t a l s y n t h e s i s o f a t r o p i n e
QBr
exhaust
L
methyln
[4
Br
q u i n o l i ne
1500~
warm
i n Et20
[I21
Br
Br
[111
Br
269
Scheme T I l : Robinson's t o t t i l s y n t h e s i s of atropine
CH-OH
CH3NH2
121
cond.
N -CH 3
CH3
/"="
CH3
CH-OH
I31
[GI
[41
I51
270
Scheme 1 1 1 : Robinson's s y n t h e s i s (25)

Succindialdehyde [ 11 i s condensed with methylamine [ 2 ] t o g i v e t h e condensate b i s c a r b i n olamine [3]. This i n t u r n condensed w i t h acet o n e [ 4 ] t o give t r o p i n o n e [ 5 ] ( T h i s mixture
i s allowed t o s t a n d i n water a t o r d i n a r y t e m p e r a t u r e f o r h a l f an h o u r ) .
Tropinone [ 5 ] i s reduced with z i n c and hydriodic acid t o tropine [6].
The y i e l d can be improved by s u b s t i t u t i o n
of t h e more r e a c t i v e acetone d i c a r b o x y l a t e o r
i t s e s t e r f o r acetone.
Succindialdehyde [l] i s condensed w i t h m t h y l a mine [21 t o g i v e b i s c a r b i n o l m i n e [31. I31
i s condensed w i t h calcium acetonedicarboxyl a t e [4] t o af1'31-d t h e condensate [51. This
i s warmed with hydrochloric a c i d t o g i v e t r o pinone [61. Tropinone [61 i s reduced w i t h
z i n c and hydriodic a c i d t o t r o p i n e [71.
Scheme IV-:
Willstatter I s second s y n t h e s i s ( 2 6 )
S u c c i n y l d i a c e t i c e s t e r [ 11 i s condensed with
methylamine [ 2 ] t o g i v e diethyl-N-methylpyrr o l e d i a c e t a t e [ 31. This i s reduced (H2+Pt)
t o a f f o r d diethyl-N-methylpyrrolidinediacet a t e [41. The c i s form of [41 is c y c l i z e d i n
t h e presence of Na and p-cymene t o g i v e e t h y l tropinone-2-carboxylate [ 51. Hydrolysis of
[5] with 10% s u l f u r i c a c i d g i v e s e t h y l t r o p i none-2-carboxylic a c i d [ 61. The l a t t e r i s
heated t o y i e l d tropinone [TI which i s reduced w i t h z i n c and hydriodic a c i d t o t r o p i n e [a].
Scheme V:
Tropinone can a l s o be s y n t h e s i z e d ( 2 7 ) u s i n g
methylamine hydrochloride a c e t o n d i c a r b o x y l i c
a c i d and g e n e r a t i n g succindialdehyde i n s i t u
by t h e a c t i o n of a c i d on 2,5-dimethoxy t e t r a hydrofuran as follows :
H+
CH3NH2HC.l
CO( CH2COOH)2
"0
CHO
- ';-...
Scheme 111: Robinson's
:li
s y n t h e s i s ( y i e l d improvement)
CH-OH
f NHeCH3
121
I11
cond
CH-OIf
[31
Scheme I&
Cli-
CH2-COOC
CH2COOCa
\ c=o
+ /
\4
H3CT
c=o
I
CH -C-CH2-COOC
2
(51
COOCa
H
2 5
[21
H
2 5
H
.(--------------
CH=
C-
CH2-COOC H
'
A
CH -C1 2 I
III
CR -C-
2 1
H
2 5
CH
- c-
i 2 i
II CN
31
CH2-
cI
H
I3]
CH2-COOC
Cli3
CH2-COOC
CH-COOH
I
c=o
I
&I2
(61
2 5
2 5
[41
CH,-COOC
HI
CH2-COOC
C-
H
CH2- C-
[4
CH=
111
CH2COOCa
W i l l s t a t t e r ' s second s y n t h e s i s
+ H2NCH3
CH=
27 1
II
2 5
H
2 5
212
CH2 - C H
Me-N
CH2
- CH
-CH2
C=O
- CH2
Zn
HI
[71
4.2
CH2
- CH - C H
[81
T o t a l S y n t h e s i s of Mandelic Acid
S e v e r a l schemes f o r t h e t o t a l s y n t h e s i s a r e known
(28-30).
Scheme I
Benzaldehyde [ l ] i s t r e a t e d w i t h a m i x t u r e of a
s a t u r a t e d s o l u t i o n of sodium hydrogen s u l f i t e and
aqueous sodium cyanide t o g i v e m a n d e l o n i t r i l e [ 2 ] .
[ 2 ] i s hydrolyzed w i t h c o l d c o n c e n t r a t e d hydroc h l o r i c a c i d t o y i e l d mandelic a c i d [3] (28).
Scheme I1
Acetophenone [ l ] i s c o n v e r t e d i n t o d i c h l o r o a c e t o phenone [ 2 ] by t h e a c t i o n of c h l o r i n e i n a c e t i c a c i d .
[ 2 ] i s t r e a t e d with sodium hydroxide t o a f f o r d t h e
sodium s a l t o f mandelic a c i d , which i s t r e a t e d w i t h
h y d r o c h l o r i c a c i d t o g i v e mandelic a c i d [ 3 ] ( 2 9 ) .
Scheme I11
Amygdalin [ 13 i s t r e a t e d w i t h fuming h y d r o c h l o r i c
a c i d t o produce m a n d e l o n i t r i l e [ 2 ] which i s t h e n
hydrolyzed w i t h h y d r o c h l o r i c a c i d t o g i v e mandelic
a c i d [ 3 ] (30).
4.3
T o t a l S y n t h e s i s of Homatropine
Tropine i s t h e n e s t e r i f i e d w i t h (+) -mandelic a c i d
i n t h e presence of hydrogen c h l o r i d e ( F i s c h e r S p e i e r e s t e r f i c a t i o n ) t o g i v e ( 2 ) -homatropine.
213
Total Synthesis of Mandelic Acid

Scheme I
NaCN
NaHSO
Scheme I1
@Lo
c12
CH3COOH
Scheme I11
HO
HO
CHC 1 2
[21
i) NaOH
ii) HCI
214
5.
Therapel.itic Uses and Pharmacological E f f e c t s

Homatropine i s used a s a m y d r i a t i c and c y c l o p l e g i c d r u g
i n ophthalmology. I t i s p r e f e r r e d t o a t r o p i n e f o r d i a g n o s t i c purposes because i t s a c t i o n i s more r a p i d , less
prolonged and i s z o r e r e a d i l y c o n t r o l l e d by p h y s o s t i g mine ( 1 2 ) . The e f f e c t of homatropine i s e x e r t e d i n 15 t o
30 minutes and p a s s e s o f f i n 1 2 t o 24 h o u r s . I t j s sornetimes used with c o c a i n e which enhances i t s m y d r i a t i c
action (12).
Homatropine has a l s o l e s s tendency t h a n a t r o p i n e t o i n c r e a s e t h e i n t r a - o c u l a r p r e s s u r e (18), b u t it produces a
l e s s s a t i s f a c t o r y m y d r i a s i s i n c h i l d r e n (18). I t i s
seldom used i n t e r n a l l y (12).
Homatropine i s an a n t i c h o l i n e r g i c d r u g w i t h e f f e c t s s i m i l a r t o h u t much weaker (about o n e - t e n t h ) t h a n t h o s e o f
atropine (12,31l, generally t h e belladonna a l k a l o i d s a r e
absorbed r a p i d l y from t h e g a s t r o i n t e s t i n a l t r a c t . They
a l s o e n t e r t h e c i r c u l a t i o n when a p p l i e d l o c a l l y t o t h e
mucosal s u r f a c e s o f t h e body (31). Some of t h e s e compounds ( a t r o p i n e and homatropine) when a p p l i e d l o c a l l y
t o t h e eye can cause mydriasis and c y c l o p l e g i a (31).
Homatropine i s used a s a 2% s o l u t i o n i n c a s t o r o i l , b u t
more o f t e n i n form of 2 t o 5% hornatropine hydrobromide
ophthalmic s o l u t i o n s (12,31).
I t has been r e p o r t e d (32) t h a t hornatropine i n doses o f
0 . 5 t o 4.0 mg, induces b r a d y c a r d i a i n man, when it i s
a d m i n i s t e r e d subcutaneously
6.
Drug S t a b i l i t y
Homatropine hydrobromide i n t h e d r y s t a t e , i n a i r t i g h t
c o n t a i n e r a t room temperature and p r o t e c t e d from l i g h t ,
showed no decomposition a f t e r f i v e y e a r s when examined
according t o t h e B.P. ( 1 5 ) .
In aqueous s o l u t i o n , homatropine h y d r o l y s e s t o t r o p i n e
andmandelic a c i d . Hydrolysis i s c a t a l y z e d by hydrogen
i o n s and hydroxide i o n s (18). A t 25", t h e r a t e of hydrol y s i s i s a minimum a t pH 3 . 7 .
A s o l u t i o n c o n t a i n i n g homatropine hydrobromide 1%and
b o r i c a c i d 1 . 5 5 % , l o s t 2% of i t s homatropine c o n t e n t a f t e r
a u t o c l a v i n g f o r 20 minutes a t 120" (12).
The pH of t h i s s o l u t i o n f e l l from 4 . 5 t o 3.8 (33,34).
7.
215
7.1
The f o l l o w i n g i d e n t i f i c a t i o n t e s t s a r e mentioned i n
t h e B.P. ( 1 5 ) .
Dissolve 10 mg o f homatropine hydrobromide i n 1 m l

of water, add a s l i g h t e x c e s s o f 10M ammonia and
shake w i t h 5 m l of chloroform. Evaporate t h e c h l o r o form l a y e r t o d r y n e s s on a water b a t h and add 1 . 5 m l
of a 2 p e r c e n t w/v s o l u t i o n of mercury (11) c h l o r i d e
i n e t h a n o l ( 6 0 % ) ; a yellow c o l o r develops which t u r n
t o r e d on warming.
- A 2 p e r c e n t w/v s o l u t i o n y i e l d s t h e r e a c t i o n c h a r a c t e r i s t i c o f a l k a l o i d s and t h e r e a c t i o n s c h a r a c t e r i s t i c o f bromides.
The f o l l o w i n g t e s t i s mentioned i n t h e U.S.P. ( 3 5 ) .

The i n f r a r e d a b s o r p t i o n spectrum of a potassium
bromide d i s p e r s i o n of i t , e x h i b i t s maxima only a t
t h e same wavelengths a s t h a t of a s i m i l a r p r e p a r a t i o n of USP Homatropine Hydrobromide Reference
Standard
A s i m p l e , r a p i d and s e n s i t i v e t e s t or t h e d e t e c t i o n
of homatropine and o t h e r a l k a l o i d s i n p h y s i o l o g i c a l
f l u i d s such a s s a l i v a and u r i n e i s r e p o r t e d (36)
To t h e s o l u t i o n o f a l k a l o i d s , a methanolic bromophenol b l u e i s added t o g i v e b l u e s t a b l e c o l o r .
- Another c o l o r t e s t was r e p o r t e d a s f o l l o w s ( 3 7 ) :
Homatropine i s e v a p o r a t e d t o d r y n e s s w i t h a mixture
of fuming n i t r i c a c i d - a c e t i c anhydride ( 4 : 3 ) and
5% met hano 1i c t e t rame t h y 1ammonium hydroxide so l u t ion
i s added t o a s o l u t i o n of t h e r e s i d u e i n a c e t o n e ,
t h e c o l o r d e v e l o p s and remains c o n s t a n t f o r 6 t o 8
minutes. I t i s p o s s i b l e t o determine t h e a l k a l o i d
q u a n t i t a t i v e l y by measuring t h e c o l o r a t 570 ni.1.
- Other i d e n t i f i c a t i o n t e s t s have been a l s o mentioned

(38) *
7.2
M i c r o c r v s t a l Tests
Homatropine hydrobromide was d i s s o l v e d i n water
(10 mg i n 10 m l ) , 1 t o 2 d r o p s of t h i s s o l u t i o n was
t r e a t e d w i t h t h e r e a g e n t on a m i c r o s c o p i c a l g l a s s
s l i d e . After s p e c i f i c time, t h e c r y s t a l s were
m i c r o s c o p i c a l l y examined (39)
276
7.2.1
Wagner's r e a g e n t was added t o t h e t e s t s o l u t i o n , minute irregular brown b l a d e s were

formed a f t e r 2-3 minutes ( F i g . 9 ) .
7.2.2
P i c r i c a c i d was added t o t h e t e s t s o l u t i o n ,
r a d i a t i n g r o d s and i r r e g u l a r b l a d e s were
formed a f t e r 10 minutes ( F i g . 1 0 ) .
7.2.3
D r a g e n d r o f f ' s r e a g e n t was added t o t h e t e s t

s o l u t i o n , minute i r r e g u l a r orange c r y s t a l s
were formed a f t e r 1 - 3 minutes ( F i g . 1 1 ) .
Other m i c r o c r y s t a l t e s t s have a l s o been
r e p o r t e d (17,40,41).
7.3
T i t r i m e t r i r Determinations
7.3.1
Aqueous
The f o l l o w i n g a s s a y i s mentioned i n U.S.P
(35)
- D i s s o l v e about 400 of Homatropine Hydrohro-
mide, a c c u r a t e l y weighed, i n water t o make

50 m l and mix.
T r a n s f e r 10 m l of t h i s s o l u t i o n t o a b e a k e r ,
add 5 m l of sodium hydroxide TS, and h e a t t h e
s o l u t i o n j u s t t o b o i l i n g . Add 10 m l of d i l u t e
n i t r i c a c i d ( 1 i n 1 5 ) , add water t o make 50 m l ,
and c o o l i n an i c e b a t h . Concomitantly add
5 m l o f d i l u t e n i t r i c a c i d (1 i n 15) t o a
second 10 m l p o r t i o n o f t h e s o l u t i o n of Homat r o p i n e Hydrobromide, add w a t e r t o make 50 m l ,
and c o o l i n an i c e b a t h . Add 1 d r o p o f n i t r o p h e n a n t h r o l i n e TS t o each s o l u t i o n and while
keeping t h e s o l u t i o n s c o l d , t i t r a t e w i t h O.OSN
c e r i c ammonium n i t r a t e u n t i l t h e pink c o l o r i s
discharged.
Each m l o f t h e d i f f e r e n c e i n volumes of 0.05N
c e r i c ammonium n i t r a t e r e q u i r e d i s e q u i v a l e n t
t o 8.907 mg of CI6HZ1NO3.HBr.
Homatropine was determined by t i t r a t i o n w i t h

aqueous 0 . 0 1 M - t e t r a p h e n y l b o r a t e , w i t h t e t r a bromophenolphthalein e t h y l e s t e r as an i n d i c a t o r i n 1,2 - d i c h l o r o e t h a n e (42).
A p o t e n t r i o m e t r i c t i t r a t i o n of homatropine
hydrobromide i n g a l e n i c a l p r e p a r a t i o n s w i t h
sodium t e t r a p h e n y l b o r o n was d e s c r i b e d (43)
277
Fig, 9. Hornatropine HBr with Wagner's reagent
~~
Fig. 10. Hornatropine HBr with picric acid reagent
a-
Fig. 11 Hornatropine HBr with Dragendorff's reagent
218
- Homatropine hydrobromide was t i t r a t e d i n
dimethyl sulphoxide medium w i t h 0.1 M AgN03.

End-point was determined by conductometric,
p o t e n t i o m e t r i c and p o l a r i m e t r i c t e c h n i q u e s ,
and t h e r e s u l t s were s a t i s f a c t o r y by a l l
t h r e e t e c h n i q u e s . Amounts of 0 . 1 mmol (44)
could n o t be determined.
7.3.2
Non-aqueous
- The f o l l o w i n g a s s a y i s mentioned i n t h e B.P.
(15) *
Dissolve 0 . 3 gm i n 20 m l of anhydrous g l a c i a l
a c e t i c a c i d , add 10 m l o f mercury (IT) a c e t a t e
s o l u t i o n and c a r r y o u t Method I f o r non-aqueous
t i t r a t i o n , Appendix V I I I A, d e t e r m i n i n g t h e
end-point p o t e n t i o m e t r i c a l l y . Each m l of
0 . 1 M p e r c h l o r i c a c i d VS i s e q u i v a l e n t t o
0.03563 gm of C16H21N03.HBr.
7.4
-
An a l t e r n a t i v e method i n v o l v e s d i r e c t t i t r a -
t i o n of homatropine hydrobromide w i t h p e r c h l o r i c a c i d i n a c e t i c anhydride medium u s i n g

naphthalbenzein o r Sudan r e d B a s i n d i c a t o r
(45)
Other non-aqueous t i t r a t i o n has been a l s o
r e p o r t e d (46).
Complexometry
P r e c i p i t a t i o n of homatropine a s a t h a l i u m complex
u s i n g one p a r t 0 . 1 N t h a l l i u m s u l f a t e and 3 p a r t s
0 . 1 N i o d i n e i n 0.15 M potassium i o d i d e s o l u t i o n .
The p r e c i p i t a t i o n i n a n e u t r a l medium i s most s e n s i t i v e . The use of t h i s p r e c i p i t a t i o n r e a g e n t f o r an
i n d i r e c t d e t e r m i n a t i o n of s m a l l amounts of homatrop i n e i s based on t h e d e t e r m i n a t i o n of t h a l l i u m i n
the p r e c i p i t a t e (47).
- Another method u t i l i z i n g merceuric-potassium i o d i d e
r e a g e n t i n 0 . 3 sodium hydroxide s o l u t i o n was r e p o r t e d ( 4 8 ) . The d e t e r m i n a t i o n i s based on t h e e x t r a c t i o n of t h e p r e c i p i t a t e formed with chloroform. The

combined e x t r a c t s a r e evaporated and water and 0.1Ms i l v e r n i t r a t e a r e added. Methylthymolblue i s used
a s i n d i c a t o r and s u f f i c i e n t 20% h e x a m e t h y l e n e t e t r a mine s o l u t i o n i s added t o g i v e a b l u e c o l o r . The
mercury i n t h e s o l u t i o n i s t h e n t i t r a t e d w i t h 0.05M
EDTA t o a yellow end p o i n t .
7.5
-
279
Polarographic
The o s c i l l o p o l a r o g r a p h i c behaviour of homatropine
and o t h e r a l k a l o i d s was r e p o r t e d ( 4 9 ) .
A dropping mercury e l e c t r o d e s e r v e d a s a p o l a r i z a b l e
e l e c t r o d e , and a g r a p h i t e one a s a r e f e r e n c e .
The d e p o l a r i z i n g p o t e n t i a l s were r e f e r r e d t o t h e
p o t e n t i a l s of T1 i o n s . S e m i q u a n t i t a t i v e determinat i o n from t h e depth o f t h e c h a r a c t e r i s t i c C u t s - i n
on t h e c u r v e i s p o s s i b l e i n a l k a l i n e s o l u t i o n s .
- Another o s c i l l o p o l a r o g r a p h i c method f o r i d e n t i f i c a t i o n o f homatropine hydrobromide i n a l k a l o i d a l

m i x t u r e s was a l s o d e s c r i b e d (50).
7.6
Spectrophotometric methods
7.6.1
Colorimetry
- 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 homatropine and o t h e r r e l a t e d a l k a l o i d s
was r e p o r t e d ( 5 1 ) . The method i s based on
t h e n i t r a t i o n o f t h e drug w i t h a s o l u t i o n of
20% potassium n i t r a t e i n c o n c e n t r a t e d s u l f u r i c
a c i d a t 50-60' f o r 30 minutes. The product
i s made a l k a l i n e with h o t 20% sodium hydroxide
and t h e c o l o r which develops i s measured.
A s t a b i l i t y i n d i c a t i n g a s s a y f o r t h e determinat i o n of homatropine hydrobromide and methobromide and t h e i r d e g r a d a t i o n p r o d u c t s i n g a l e n i c a l p r e p a r a t i o n s was r e p o r t e d ( 5 2 ) . The method

i s as follows:To 5 ml o f prepared s o l u t i o n ( z = 3 mg o f
a l k a l o i d ) i n a 5Q m l f l a s k p l a c e d i n an i c e w a t e r b a t h , add s a t u r a t e d aqueous hydroxylammonium c h l o r i d e s o l u t i o n ( 1 ml) and 1 0 . 5 M
potasium hydroxide (1 ml) , mix and s e t a s i d e
f o r 1 h r . Add 4 M h y d r o c h l o r i c a c i d ( 2 ml)
t o g i v e pH 1 . 2 t o 1 . 4 , m i x , add 0 . 3 7 M F e r r i c
chloride solution i n 0.1 M hydrochloric acid
(1 ml) and mix a g a i n . Remove t h e f l a s k from
t h e b a t h , a l l o w t h e e v o l u t i o n of gas t o s u b s i d e
and measure t h e e x t i n c t i o n o f t h e s o l u t i o n a t
540 nm.
- Absorpt i o m e t r i c d e t e r m i n a t i o n o f homatropine
hydrobromide i n eye d r o p s ( 5 3 ) ;
D i l u t e 2 . 5 m l of sample t o 2 5 m l and t o 1 m l
of t h e r e s u l t i n g s o l u t i o n [ 0 . 2 5 t o 2 mg of
280
homatropine hydrobromide] add 2.5 m l o f

water, 3 m l of 5 % sodium hydroxide s o l u t i o n and a f t e r 5 minutes, 3 . 5 m l of F o l i n
r e a g e n t . A f t e r 10 m i n u t e s , measure t h e
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 w i t h u s e
of a r e d f i l t e r . The s e n s i t i v i t y of t h e
method i s 0.25 mg i n t h e f i n a l s o l u t i o n
t h e mean e r r o r i s + 3 % .
- Another 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
homatropine hydrobromide has been a l s o

r e p o r t e d (54). The method i s as f o l l o w s :
D i s s o l v e 1 mg of sample i n 100 m l of water
and t o 1 m l of t h e s o l u t i o n add 3 m l o f
mM-bromocresol p u r p l e and 2 m l o f b u f f e r
s o l u t i o n (pH 4 ) . E x t r a c t t h e c o l o r e d 1:l
p r o d u c t i n t o c h l o r o f o r m (2x5 ml) ; d i l u t e
t h e combined e x t r a c t s t o 10 m l w i t h c h l o r o form and measure t h e absorbance a t 405 t o
410 nm. Hydrolysis p r o d u c t s of t h e drug
does n o t i n t e r f e r e u n l e s s p r e s e n t i n t h r e e
f o l d amounts. The r e l a t i v e e r r o r of t h e
method does n o t exceed 2 1%.
Other 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 homat r o p i n e by c i s - a c o n i t i c anhydride h a s been

d e s c r i b e d (55,56).
I t i s as f o l l o w s :
The sample (0.05 gm) was d i s s o l v e d i n 50 m l
t o l u e n e and from t h i s s o l u t i o n , t h e s t a n d a r d s a t 20,50 and 100 y / m l were p r e p a r e d .
Each s t a n d a r d ( 2 m l ) was d i l u t e d w i t h 2 m l
t o l u e n e and 1 m l r e a g e n t p r e p a r e d by d i s s o l v i n g 0.25 gm c i s - a c o n i t i c a n h y d r i d e i n
100 m l r e d i s t i l l e d a c e t i c anhydride. The
s o l u t i o n s were h e a t e d f o r 45 minutes on a
steam b a t h and c o o l e d f o r 15 minutes i n
t h e d a r k . Then were d i l u t e d t o 10 m l .
w i t h 20: 80 a c e t i c a n h y d r i d e - t o l u e n e m i x t u r e
and absorbancy measured a t 535 nm. Aqueous
s o l u t i o n s o f t h e s a l t was b a s i f i e d w i t h
ammonia and e x t r a c t e d w i t h t o l u e n e .
- Homatropine was determined u s i n g t e t r a -
bromophenolphthalein e t h y l e s t e r a t pH
8-10.5 and t h e a d d i t i o n compound formed
was e x t r a c t e d w i t h 1 , 2 - d i c h l o r o e t h a n e and
e x t i n c t i o n i s measured a t 560 nm (57).
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 t h e t h a l i u m
complex of homatropine hydrobromide u s i n g
c r y s t a l v i o l e t was a l s o r e p o r t e d ( 5 8 ) .
7.7
A
7.7.1
Paper chromatography
Homatropine can be d e t e c t e d by paper chromatography.
chromatographic c o n d i t i o n s used or homatropine.
Table 8 i n c l u d e s paper
Table 8 . Paper Chromatography of Hornatropine

Conditions
Detecting reagent
Whatman
No.
1
paper
impreg- 5% aqueous ammonia
s a t u r a t e d w i t h octanol n a t e d w i t h 7 % m e t h a n o l i c
o c t a n o l . Descending t e c h n i que. Drying t h e paper i n
a i r f o r 5 t o 10 minutes.
Solvent system
R
-
value
-
Reference
(59)
- 4 . 8 gm of c i t r i c a c i d Paper Whatman No.1 dipped
U.V. o r Iodoplatinate
0.3
Clark
(17)
- acetate buffer
Location under U . V .
Light (254 mu) o r
Iodoplatinate spray
0.96
Clark
(17)
i n a mixture of 130
water and 870 of
n- But an o 1
(pH 4.58)
i n 5% s o l u t i o n of sodium
dihydrogen c i t r a t e and d r i e d
Whatman No.1 o r No.3 impregnated by d i p p i n g i n 10%
s o l u t i o n of T r i b u t y r i n i n
a c e t o n e and d r y i n g i n a i r .
- BuOH- AC OH- H 2O
(40 : 10 :50)
BUOH - HCO 2H- H2 0
(10: 1 : 10)
Descending t e c h n i q u e
0.2% I o d i n e s o l u t i o n spray
BuOH-C2H5C 02H-H20
(10: 1: 10)
Other paper chromatography h a s a l s o been r e p o r t e d (61).
(60)
7.7.2
Thin l a y e r chromatography (TLC)

Homatropine can b e d e t e c t e d by t h i n l a y e r chromatography. Table 9
t h i n l a y e r chromatographic c o n d i t i o n s used f o r homatropine.
Table 9.
S o l v e n t system
Thin l a y e r chromatography of Eomatropine

Conditions
Detecting reagent
S t r o n g ammonia s o l u t i o n :
methanol (1.5 : 100)
s i l i c a gel G
acidified iodoplatinate
o r under U . V . (254 nm)
Ethanol-pyridine-water
: 6
: 4)
(1
alumina
iodoplatinate spray
Chloroform-acetone-diethylamine
( 5
:
4
:
1)
Acetone - wat e r - 25 % ammonia
solution
:
6)
(90
: 4
K i e s e l g e l GF
254
Kieselgel G
Dragendorf f s p r a y
The upper phase of

But ano 1- a c e t i c ac id-wat e r
( 10
:
1
: 5)
Other T.L.C.
includes
Ce 1l u l ose
f-
value
Reference
0.15
Clark
( 17)
0.87
(65)
Dragendorff e t h y l a c e t a t e
reagent
(64)
Dragendorff s p r a y
(65)
f o r homatropine h a s been a l s o r e p o r t e d ( 6 6 ) .
7.7.3
283
Electrophoresis
- E l e c t r o p h o r e s i s of homatropine was performed
on Whatman No.1 p a p e r (18x46 cm) a t 8 v./cm

f o r 3 h r s . i n t h e p r e s e n c e of u n i v e r s a l buffer
The r e l a t i v e d i s p l a c e m e n t s a t pH
2 . 3 , 4 . 3 , 6 . 4 , 8 . 2 , 10.5 and 11.4 were 6 3 , 5 4 ,
104,105,100,16 r e s p e c t i v e l y ( 6 7 , 6 8 ) .
- Other e l e c t r o p h o r e s i s f o r s e v e r a l a l k a l o i d s
have been r e p o r t e d ( 6 9 ) .
7.7.4
Column chromatography
Chromatographic a s s a y of homatropine hydrobromide and o t h e r r e l a t e d a l k a l o i d s a r e quant i t a t i v e l y e l u t e d w i t h 95% a c e t o n e from a
column packed w i t h b a s i c alumina (70).
The p u r i t y of homatropine hydrobromide can
be determined on alumina column as f o l l o w s
(71) '
Homatropine hydrobromide (0.1 gm) was e l u t e d
o v e r a column packed w i t h b a s i c alumina (5
gm) by u s i n g aqueous a c e t o n e as s o l v e n t .
After e l u t i o n , t h e e l u a t e was d i l u t e d w i t h
w a t e r and homatropine hydrobromide i s t i t r a t e d w i t h 0 . 1 N h y d r o c h l o r i c a c i d u s i n g bromophenol b l u e as an i n d i c a t o r .
7.7.5
Gas chromatography (GC)
The gas chromatography f o r homatropine a r e

l i s t e d i n t a b l e (lo).
Table 1 0 .
The GC of Homatropine
Ref.
-
C a r r i e r gas
2.5% SE-30 on 80-100 mesh chromosorb

W (5 F t x 4mm i n t e r n a l d i a m e t e r ) .
Column t e m p e r a t u r e 225'.
Nitrogen
(SO ml/min.)
F . I . D . hydrogen 0.36 r e l a t i v e
(50 ml/min.)
t o codeine
a i r (300 ml/min.)
Clark
(17)
3% XE-60 s i l i c o n e n i t r i l e polymer
on 100-120 mesh Chromosorb W.
Column t e n p e r a t u r e 225".
Nitrogen
(50 ml/min.)
F . I . D . hydrogen 0.39 r e l a t i v e
(50 ml/min.)
t o codeine
a i r (300 ml/min.)
Clark
(17)
5% SE-30 on 60-80 mesh Chromosorb W

AW (5 f t x 1/8 d i a m e t e r )
Column temperature 230'.
Nitrogen
(30.7 ml/min.)
F.I.D. hydrogen
(22 ml/min.)
0.44 r e l a t i v e
t o codeine
Clark
(17)
3% J X R on s i l a n i s e d G Chrom P on
100-120 mesh (1.8m x 2mm)
Column t e m p e r a t u r e 250".
Nitrogen
(50 ml/min.)
F.I.D.
F.I.D.
5% SE-30 on 60-80 mesh a c i d washed

Chromosorb W a t 190,210,230,
250-270
15 % QF-1 on 60-80 mesh,
Gas Chrom(R)Q. ,
(1.5m x 3 . 2 mm)
column t e m p e r a t u r e 160".
Detector
Retention _
t i_
me
Column c o n d i t i o n
Nitrogen
(50 ml/min)
F.I.D.
Other gas chromatographic a n a l y s i s have been r e p o r t e d (75-78)
4 . 2 (min)
(73)
7.7.6
285
High P r e s s u r e Liquid Chromatography (HPLC)

Fell _
et _
a l . (73) have r e p o r t e d an a n a l y s i s of
homatropine hydrobromide by r e v e r s e d - p h a s e
h i g h - p r e s s u r e l i q u i d chromatography. Homatrop i n e hydrobromide was determined on a column
of H y p e r s i l ODS ( 5 um) with 5 0 mM. Sodium
a c e t a t e i n 10 mM-tetrabutylammonium s u l f a t e
(pH 5 . 5 ) - a c e t o n i t r i l e ( 3 : l ) as t h e mobile
phase and d e t e c t i o n a t 254 nm. The i n t e r n a l
s t a n d a r d was p - t o l u i c a c i d . R e c t i l i n e a r
c a l i b r a t i o n graph was o b t a i n e d f o r homatropine
hydrobromide ( i n eye d r o p s ) i n t h e range 0 t o
4 mg ml-1.
S t u t z and Sass (80) have d e s c r i b e d a high-speed,

high p r e s s u r e l i q u i d chromatography of t r o p a n e
a l k a l o i d s i n c l u d i n g homatropine. The compound
was s e p a r a t e d on a s t a i n l e s s - s t e e l column (1
meter x 4 . 6 mm) packed with sil-X a b s o r b e n t
w i t h 28% aqueous ammonia t e t r a h y d r o f u r a n (1:
100) as t h e s o l v e n t and w i t h a column i n l e t
p r e s s u r e o f 500 l b p e r s q . i n c h . A d i f f e r e n t i a l
r e f r a c t i v e index d e t e c t o r and a UV d e t e c t o r
o p e r a t i n g a t 254 nm were used t o monitor t h e
eluate.
ACKNOWLEDGEMENT
The a u t h o r s would l i k e t o thank Mr. Uday C. Sharma
of Department of Pharmdcognosy, c o l l e g e o f Pharmacy,
King Saud U n i v e r s i t y or h i s s e c r e t a r i a l assistance
i n t h e reproduction of t h e manuscript.
286
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72. V . Quercia, C. Cardini and B. Tucci, Boll. Chim. farm.
107 (12), 737 (1968).
(Ital.), 73.
74.
K.D. Parker, C.R. Fontan, and P.L. Kirk, Anal. Chem.,
35, 356 (1963).
B.F. Gorbowski, B.J. Softly, B.L. Chang, and W.G. Haney,
J . Pharm. Sci., 62, 806 (1973).
75. E. Brochmann - Hanssen and C.R. Fontan, J. Chromatog.,,

19 (2) , 296 (1965).
76.
E. Brochmann-Hanssen and A.B. Svendsen, J. Pharm. Sci.

51, 1095 (1962).
25 ( 2 ) , 122 (1970).
77. V. Quercia, Farmaco, Ed. prat., -
78.
E. Shami, J . Dudzinski, J. Lachman., and J . Tingstand,

62 (81, 1283 (1973).
J. Pharm. Sci., -
79. A.F. Fell, S.J.K. Johnstone, and G. Smith, J. Pharm.

Pharmacol., 31 (Suppl.) 20 (1979).
80.
45 (12), 2134,
M.H. St.utz, and S. Sass; Analyt. Chem., (1973).
MEBENDAZOLE
A . A . AL-Budrz* und M . T d / t i q * *
*Vepu,ahtmeMA: 06 PhatunuzceuLlcal C h m i ~ f i yand VcpahXment**

0 6 Phahmacology, Coflecje ozj Phahmucy, King Saud
UnivehhLty, P.O. Box 2457, Riyudh-11451, Saudi Ahabia.

VOLUME 16
29 1

A. A. AL-BADR AND M. TARIQ
292
1.
Forward, History
2.
Description
2.1
2.2
2.3
2.4
2.5
3.
Nomenclature
Formulae
Molecular Weight
Appearance, Color and Taste
Physical properties
4. Spectral Properties
5.
Synthesis
6. Pharmacokinetics
7.
Toxicity
8.1
8.2
Identification
Titrimetric Methods
8 . 3 Spectrophotometric Methods
8.4 Chromatographic Methods
8.5 Polarographic Method
8.6 Radioimmunoassay Method
References
MEBENDAZOLE
293
Foreword, H i s t o r y
Mebendazole i s a broad spectrum a n t h e l m i n t i c a g e n t
s y n t h e s i z e d and developed by J a n s s e n Pharmaceutica,
Research Laboratody, Beerse, Belgium. A f t e r i t s
i n t r o d u c t i o n t h e r e i n 1972, t h e drug became a v a i l a b l e i n
numerous c o u n t r i e s around t h e world, i n c l u d i n g t h e
United S t a t e s and Canada (1). I t produces high c u r e
rates i n i n f e s t a t i o n s by A s c a r i s , threadworms
( E n t e r o b i u s ) , hookworms (Ancylostoma and Necator S p p . ) ,
and Whipworms ( T r i c h u r i s ) . I t i s a l s o a c t i v e i n some
(about 40%) Dwarf tapeworm (Hymenolepis) i n f e s t a t i o n s
(2-5).
2.
Description
2.1
Nomenclature
2.1.1
Chemical Names
(5-Benzoyl-1H-benzimidazol-2-yl)-carbamic
a c i d methyl ester.
5-Benzoyl-2-benzimidazolecarbamic a c i d methyl
ester.
Methyl 5-benzoyl-2-benzimidozolecarbamate.
Methyl-5-benzoylbenzimidazol-2-ylcarbamate.
Methyl-(5-benzoyl-1H-benzimidazol-2-yl)
carbamat e.
Carbamic acid,5-benzoyl-1H-benzimidzol-2-yl,
methyl e s t e r . (6)
CAS R e g i s t r y Number
[31431-29-71
2.1.2
(6)
Generic N a m e
Mebendazole; R 17,635
2.1.3
(7).
Trade Names
P r o p r e t a r y Names
Bendrax, Besantin, E s a d i r a s e , Equivuarm
p l u s Forverm, Gendal, Granverm, Lomper,
Mebendacin, Mebendozol M K , Mebendazole,
Mebenix, Mebutar, Nemasole, Oxiben, Pantelmin,
Parelmin, S i r b e n , Telmin, T r o t i l , Vermirax,
Vermox, and Zakor (1,6, 8-11).
294
2.2
Formulae
2.2.1
Empirical
2.2.2
Structural
H
2.3
Molecular Weight
295.30 and 295 ( 7 E 9 ) ,
2.4
C , 65.08%, H , 4.44%, N , 14.23%; 0, 16.25%
2.5
Appearance, C o l o r and Taste

Off-white to slightly yellow amorphous powder and
not unpleasant to taste. (10-12)
3.
Physical Properties
3.1
Melting Point
Melts at about 290, 288.5',
decomposition (6-8 6 l o ) .
3.2
above 280' with
Solubility
Almost insoluble in water, ethanol, ether, chloroform, and dilute mineral acids; readily soluble in
formic acid (9 E 11).
3.3
Hygroscopicity
Mebendazole is not hygroscopic and it is stable in
air.
MEBENDAZOLE
3.4
295
Extraction
Mebendazole i s e x t r a c t e d by chloroform from aqueous
a l k a l i n e solutions (11).
4.
Spectral Properties
4.1
The u l t r a v i o l e t a b s o r p t i o n spectrum o f mebendazole
o b t a i n e d from a s o l u t i o n i n n e u t r a l methanol i n t h e
r e g i o n o f 200 t o 400 nm u s i n g DMS 90 s p e c t r o p h o t o meter i s shown i n F i g u r e 1. Three a b s o r p t i o n
maxima a t about 210, 247 and 310 nm and two minima
a t 228 and 273 nm were observed.
C l a r k e (11) r e p o r t e d t h e f o l l o w i n g :
Mebendazole i n 0.05% f o r m i c a c i d i n isopropanol;
maxima a t 248 nm ( E l % , 1 cm 1005) and 313 nm
( E l % , 1 cm 5 1 8 ) , minima a t 230 nm and 275 nm.
Mebendazole i n 0 . 0 1 N sodium hydroxide i n i s o p r o p a n o l , maxima a t 270 nm ( E l % , 1 cm 802) and 355 nm
( E l % , 1 cm 653), minima a t 245 nm and 302 nm.
Mebendazole i n 0 . 1 N h y d r o c h l o r i c a c i d i n i s o p r o p a n o l , maxima a t 234 nm ( E l % , 1 cm 1000) and
288 nm ( E l % , 1 cm 5 2 4 ) , minima a t 215 nm and 266 nm.
4.2
The i n f r a r e d spectrum o f mebendazole i s p r e s e n t e d ;
i n F i g u r e 2. The spectrum was o b t a i n e d from K B r
d i s c u s i n g a Pye Unicam SP 1025 i n f r a r e d
s p e c t r o p h o t o m e t e r . The s p e c t r a l a s s i g n m e n t s are
presented i n t h e following t a b l e .
A. A. AL-BADR AND M.TARIQ
2%
200
250
300Wavelength 350
400
(nm)
FIGURE I : ULTRAVIOLET SPECTRUM OF MEEENDAZOLE IN METHANOL
-E 20.
*20
;"
.-
m
C
0;
FIGURE 2 . INFRARED S P E C T R U M
OF M E B E N D A Z O L E
( KBr d i s c )
298
Frequency cm
-1
Assignment
34 15
NH s t r e t c h
2500-2950
CH s t r e t c h
1720
C = 0 (amide) s t r e t c h
1650
C = 0 stretch
1530
1600
141 0-1460
700- 765
878
900)
C = C stretch
CH3-0 (C-0) s t r e t c h
Monosubstituted benzene
1 , 2 , 4 T r i s u b s t i t u t e d benzene
C l a r k e (11) r e p o r t e d t h e p r i n c i p a l peaks (KBr d i s c )

a r e 705, 1260, 1590, 1635 and 1730 cm-1.
4.3
1
'H Nuclear Magnetic Resonance ( H NMR) Spectrum
1
The H NMR spectrum of mebendazole i s shown i n
F i g u r e 3. The drug i s d i s s o l v e d i n T r i f l u o r o a c e t i c a c i d (TFA) and i t s spectrum determined on a
Varian T60 A NMR s p e c t r o m e t e r u s i n g TMS ( t e t r a m e t h y l s i l a n e ) as t h e i n t e r n a l s t a n d a r d . Assignment
of t h e chemical s h i f t s t o t h e d i f f e r e n t p r o t o n s is
shown be low:
Chemical S h i f t
PPM (6)
7.5 - 8.3
4.08
Multiplicity
P r o t o n Assignment
mu 1t ip 1et
Aromatic
singlet
methyl
FIGURE 3: PROTON MAGNETIC RESONANCE SPECTRUM OF MEBENDAZOLE IN
TRIFLOUROACE T I C
ACID(TFAI
300
4.4
I 3 C Nuclear Magnetic Resonance (13C NMR) Spectrum
The I3C NMR s p e c t r a of mebendazole i n formic a c i d

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
s t a n d a r d a r e o b t a i n e d u s i n g a J o e l FX 100 MHz
s p e c t r o m e t e r a t an ambient t e m p e r a t u r e . F i g u r e s 4
and 5 r e p r e s e n t t h e 1H-decoupled and o f f - r e s o n a n c e
spectra respectively.
/
0
.,
The carbon assignments a r e based on chemical s h i f t

v a l u e s and by comparison with model compounds and
a r e shown below:
Chemical S h i f t
PPM ( 6 )
Multiplicity
C a r bon Ass ignm en t
57.5
quartet
155.8
singlet
148.4
singlet
139.2
s i n g 1e t
134.4
singlet
118.2
doublet
130.5
singlet
130.9
doublet
115.8
doublet
200.5
singlet
136.7
singlet
132.9
doublet
131.3
doublet
13 and 15
136.3
doublet
14
c1
c2
c3
c4
c5
6
c7
c9
clo
5 1
12
and 16
MEBENDAZOLE
301
FIGURE L : PROTON OECOUPLEO CARBON-13 NMR SPECTRUM OF MEBENDAZOLE IN FORMIC ACID
FIGURE 5 : OF-RESONANCE CARB3N-I3 NMR SPECTRUM
OF
MEBENDAZOLE IN FORMIC
ACID
302
4.5
Mass Spectrum and Fragmentometry

F i g u r e 6 shows t h e 70 eV e l e c t r o n impact ( E I ) mass
spectrum o b t a i n e d on Varian MAT 311 mass s p e c t r o meter usi ng ion s o u r c e p r e s s u r e of 10-6 T o r r , i o n
s o u r ce t e mpe ra ture o f 18OoC and an emission c u r r e n t
of 300 PA. The spectrum is dominated by m/e 186
i o n ( ba se peak) r e s u l t i n g from t h e l o s s of CH30H
and C6H5 fragments. A proposed mechanism of
f r ag m enta ti on and t h e mass/charge r a t i o s of t h e
major fragments i s given i n Scheme 1 (a ar,d b ) .
Chemical i o n i s a t i o n (CI) spectrum i s p r e s e n t e d i n
F i g u r e 7 and i s obt a i ne d on a Finnigan 4000 mass
s p e ct r ome t e r wit h ion e l e c t r o n energy of 100 eV,
ion s ourc e p r e s s u r e o f 0 . 3 T o r r , ion s o u r c e
t e m p e r a ture of 15OoC and emission c u r r e n t of 300 PA.
The mass s p e c t r a l assignment of t h e prominent i o n s
under C I c o n d i t i o n s i s gi ve n below:
m/e
Species
296
M+ + 1
2 95
M+
218
M+
C6H5
MEBENDAZOLE
303
lBO(
50.C
324.2
J
2180
208 \
I86
,330
50.0
336.2
235.8 251 6 2Ul
32832.
I05
263
218
2 95
M /E
FIGURE 7
MASS SPECTRUM OF MEBENDAZOLE ( c l )
.+
&@+.
[@!$==
7
m/e 295
N?-NH-c-o
-cH3
'\
0-C'
O+
- co
____)
m/e 77
-+0
NH
- \\C - OCH3
-CH30H
[-
m/e 105
Ill
O 1
IA
-co
*
C-HNy@
-, i ,
m/e 158
m/186
m/e 218
Scheme l a :
Proposed mechanism of fragmentation of mebendazole.
/4
m/e 130
m/e 295
m/e 295
N7rNH-C
0 '&
+
OI
s
W
-CH30H
Figure lb:
m/e 263
= O
-co
4 m/e
Proposed mechanism of f r a g m e n t a t i o n o f mebendazole.
235
5.
Synthesis
1- Mebendazole was prepared (13) from 3,4(NH2)2C,H,CoC6H5
and NHz(CH3S)C = N - COOCH3
according to the following scheme.
II
NH3
HN
' 3
c
II
C-NH-C-0-CH3
.CH3
11
H2N,
,C = N-C-0-CH3
s,
CH3
NH -c-0II
0
11
CH3
MEBENDAZOLE
2-
307
Mebendazole was also prepared in 52% yield by

decomposition of methyl 7-benzoyl-lH-2,1,4benzothiadiazine-3-y1 carbamate in methanol with
2 N HC1. The latter compound is prepared in two
stages from 4 - b e n z o y l - 2 - n i t r o a n i l i n e (14)
Two steps
0
II
CH30H
CH3 2N HC1
H
6.
Pharmacokinetics
6.1 Absorption, Distribution, Metbolism and Extraction
Mebendazole is poorly absorbed by gastrointestinal

tract after oral administration (10). Only
5-10% of the orally administered frug is found
in the blood (1). The poor intestinal absorption
of mebendazole is of therapeutic advantage in the
treatment of intestinal helminthis. However,
for tissue dwelling organism high oral doses are
required ( 1 5 ) .
The absorption of drug in man is markedly enhanced
when the drug is used together with the meals, as
concomitent fat ingestion ehances its intestinal
absorption. Following oral administration peak
plasma level reach in 2-4 hours (16-18).
Munst et a1 (19) reported a significant variation
in plasma mebendazole level in human subjects
following oral ingestion. According to this study
the plasma half-life was ranging between 1 . 5 and
308
5 . 5 h o u r s . The peak c o n c e n t r a t i o n following o r a l

a d m i n i s t r a t i o n were d e t e c t e d a t 30 minutes t o 2
hours of t h e dose. Mebendazole is metabolized
p r i m a r i l y i n l i v e r by d e c a r b o x y l a t i o n (20) , t h e
major m e t a b o l i t e was 2 -amino-5 (6-benzimidazolyl
phenylketone. B r a i t h w a i t e et a1 (21) r e p o r t e d t h a t
mebendazole is more l i p o p h i l i c t h a n any of i t s
m e t a b o l i t e s . The d i s t r i b u t i o n s t u d y by same a u t h o r
showed t h a t 63.3% of mebendazole was d i s t r i b u t e d
i n plasma and 36.7% i n c e l l u l a r f r a c t i o n . Most
of t h e drug i n plasma (95%) was p r e s e n t i n t h e
p r o t e i n bound form. The c o n c e n t r a t i o n of mebendazole
i n l i v e r was c o n s i d e r a b l y h i g h e r as compared t o t h e
f a t t y t i s s u e . G i l b a l d i and Perrier (22) r e p o r t e d
t h a t s t e a d y s t a t e AUC of mebendazole i s independent
of a b s o r p t i o n r a t e i n human s u b j e c t s . Approximately
90% o f t h e o r a l l y a d m i n i s t e r e d dose o f mebendazole
i s e l i m i n a t e d p r i m a r i l y a s unchanged drug i n
faeces. About 5-10% of t h e a d m i n i s t e r e d drug may
be recovered i n t h e form o f i t s c o n j u g a t e s , and
metabolites i n t h e urine (11).
I n r a t s about 85% o f an I . V . dose was e l i m i n a t e d
with t h e b i l e and remainder w i t h t h e u r i n e . The
m a j o r i t y o f t h e dose was recovered a s conjugated
m e t a b o l i t e s (23-24). I n a n o t h e r s t u d y (25), t h r e e
m e t a b o l i t e s of mebendazole were i s o l a t e d from t h e
b i l e o f r a t s and i d e n t i f i e d as : methyl-5(a-hydroxybenzyl)-2-benzimidazole carbamate; 2amino-5- (a-hydroxybenzyl) benzimidazole and 2-amino5-benzoylbenzimidazole.
7.
Toxicity
G a s t r o i n t e s t i n a l symptoms i n c l u d i n g c o n s t i p a t i o n have
been r e p o r t e d i n f r e q u e n t l y f o l l o w i n g use o f mebendazole
(26). High doses may on o c c a s i o l s b e a s s o c i a t e d w i t h
mild
colic
p a i n o r d i a r r h o e a as r e p o r t e d by
C h a v a r r i a ( 2 7 ) u s i n g doses of u p t o 300 mg B I D i n t h e
t r e a t m e n t of t a e n i a s i s
Acute and c h r o n i c t o x i c i t y
s t u d i e s i n animals
i n d i c a t e a wide range between t h e r a p e u t i c and t o x i c
d o s e s . The LD50 (mg/kg o r a l l y ) was more t h a n 640 mg/kg in
r a b b i t s and dogs and more t h a n 1280 mg/kg i n mice and rats
(11). S i g n i f i c a n t haematologic, biochemical, o r pathol o g i c a b n o r m a l i t i e s were n o t found i n animals given
40 mg/kg d a i l y f o r 13 weeks b u t were noted i n r a t s
MEBENDAZOLE
309
given a d a i l y dosage o f 130 mg/kg. Mebendazole i s

c o n t r a i n d i c a t e d i n p r e g n a n t women because i t
h a s shown embryotoxic and t e r a t o g e n i c a c t i v i t y i n
s i n g l e p e r o r a l dose a s low as 10 mg/kg (18, 2 8 , 2 9 ) .
Rare cases o f leukopenia i n human s u b j e c t s f o l l o w i n g
t h e a d m i n i s t r a t i o n o f mebendazole was a l s o reporqed ( 3 0 ) .
I n high d o s e s 40-50 mg/kg/day, t h e drug has been
a s s o c i a t e d w i t h s e v e r e i r r e v e r s i b l e n e u t r o p e n i a . No
s i g n i f i c a n t CNS e f f e c t s i n t h e p a t i e n t s t a k i n g mebendazole
t h e r a p y has been r e p o r t e d e x c e p t s l i g h t headache and
dizziness (31).
8.
8.1
Identification
The i n f r a r e d a b s o r p t i o n spectrum of a
potassium bromide d i s p e r s i o n o f i t , p r e v i o u s l y
d r i e d , e x h i b i t maxima o n l y a t t h e same
wavelength as t h a t o f a s i m i l a r p r e p a r a t i o n of
USP mebendazole RS ( 3 2 ) .
X-ray 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 d a t a f o r i d e n t i f y i n g mebendazole and o t h e r a n t h e l m i n t i c s have
been o b t a i n e d by d i f f r a c t o m e t e r and Debye S c h e r r e r camera t e c h n i q u e s ( 3 3 ) . The d a t a were
t a b u l a t e d i n terms of t h e l a t t i c e s p a c i n g s and
the r e l a t i v e i n t e n s i t i e s of t h e lines.
P a t t e r n s u s i n g t h r e e d i f f e r e n t X-ray wavelengths
with t h e camera method a r e compared w i t h each
o t h e r and w i t h t h e d i f f r a c t o m e t e r p a t t e r n .
8.2
T i t r i m e t r i c Method
USP XX (1980) (32) d e s c r i b e d t h e f o l l o w i n g
t i t r i m e t r i c method f o r t h e a s s a y o f mebendazole:
Dissolve about 225 mg of mebendazole, a c c u r a t e l y

weighed, i n 30 m l o f g l a c i a l a c e t i c a c i d . T i t r a t e
w i t h 0 . 1 N p e r c h l o r i c a c i d VS, d e t e r m i n i n g t h e
end-point p o t e n t i o m e t r i c a l l y , u s i n g a calomelg l a s s e l e c t r o d e system. Perform a blank determinat i o n , and make any n e c e s s a r y c o r r e c t i o n . Each ml
3 10
o f 0 . 1 N p e r c h l o r i c a c i d i s e q u i v a l e n t t o 29.53 mg
Of
C16H13N303'
Wahbi and Onsy, (34) developed t h e two t i t r i m e t r i c

methods t o determine t h e mebendazole i n pharmac e u t i c a l p r e p a r a t i o n s . D i r e c t t i t r a t i o n and back
t i t r a t i o n methods are mentioned a s f o l l o w s :
a)
D i r e c t T i t r a t i o n Method
200 Mg o f mebendazole was t a k e n i n d r y c o n i c a l
f l a s k and d i s s o l v e d i n 2 m l o f 99% formic a c i d
followed by t h e a d d i t i o n o f 60 m l o f g l a c i a l
a c e t i c a c i d . T h i s s o l u t i o n was t i t r a t e d w i t h
0.1 N perchloric acid i n g l a c i a l a c e t i c acid
u s i n g c r y s t a l v i o l e t a s i n d i c a t o r , development
o f a p e r s i s t e n t green c o l o r was d e s i g n a t e d a s
end p o i n t . Blank d e t e r m i n a t i o n was performed
and t h e n e c e s s a r y c o r r e c t i o n s were made. The
c a l c u l a t i o n was done by u s i n g t h e e q u i v a l e n c e
f a c t o r which is 1 m l o f 0 . 1 N p e r c h l o r i c a c i d
i s e q u a l t o 29.53 mg o f mebendazole.
b)
B a c k - T i t r a t i o n Method
200 Mg o f mebendazole was t a k e n i n d r y c o n i c a l
f l a s k , t h e powder was d i s s o l v e d i n 20 m l o f 1 M
p e r c h l o r i c a c i d i n g l a c i a l acetic a c i d . T h i s
s o l u t i o n was t i t r a t e d with 0 . 1 N sodium
acetate i n glacial acetic acid, crystal violet
was used a s i n d i c a t o r , development o f b l u i s h
green a s i n d i c a t o r . These methods a r e
reproducible with standard deviation of 0.3%.
Mebendazole ( i n amounts u p t o 250 mg) i n anhydrous

a c e t i c a c i d medium can be t i t r a t e d w i t h 0 . 1 M p e r c h l o r i c a c i d ( a l s o i n a c e t i c a c i d ) u s i n g 0.5%
c r y s t a l v i o l e t solution a s indicator. Results
a g r e e with t h o s e o b t a i n e d by spectrophotometry a t
313 nm ( 3 5 ) .
8.3
8.3.1
U l t r a v i o l e t S p e c t r o m e t r i c Methods
--
P a t e 1 e t a1 (36) r e p o r t e d two s p e c t r o p h o t o metric methods f o r t h e e s t i m a t i o n o f
MEBENDAZOLE
311
mebendazole i n raw m a t e r i a l . A s o l u t i o n of
t h e sample i n formic a c i d was d i l u t e d w i t h
a mixture o f chloroform : methanol (17:3).
Thin-layer chromatography was run on
s i l i c a g e l i n chloroform : e t h y l a c e t a t e ;
a c e t o n e : formic a c i d (70:80:9.5:0.5),
developed f o r 15 cm and d r i e d f o r 30 minutes
a t 115O. The drug i s t h e n e x t r a c t e d i n t o
chloroform and measured a t 313 nm.
In a n o t h e r methods, a s o l u t i o n o f t h e drug
i n formic a c i d i s d i l u t e d w i t h i s o p r o p a n o l
and t r e a t e d w i t h MeOH and 30% potassium

hydroxide. After 30 min. t h e absorbance was
measured a t 420 nm.
Wahbi and Osny (34) r e p o r t e d a s p e c t r o p h o t o metric method o f a n a l y s i s of mebendazole
i n t a b l e t s with a s t a n d a r d d e v i a t i o n o f
1 . 4 % . Powdered t a b l e t s e q u i v a l e n t t o 50 mg
of mebendazole were t a k e n i n t o 50 m l
s t a n d a r d f l a s k followed by 10 m l o f 70%
p e r c h l o r i c a c i d . The mixture was shaken f o r
10 minutes and f i l t e r e d through a d r y
f i l t e r paper. 1 M 1 of f i l t r a t e taken t o a
100 m l f l a s k and d i l u t e d t o volume w i t h
d i s t i l l e d w a t e r . The absorbance of t h i s
s o l u t i o n was measured i n a 1 cm c e l l a t
288 nm wavelength u s i n g a spectrophotometer.
0 . 2 M 1 o f 70% p e r c h l o r i c a c i d d i l u t e d w i t h
100 m l of d i s t i l l e d w a t e r was used a s b l a n k .
C a l c u l a t i o n o f c o n c e n t r a t i o n was done by
t a k i n g 566 a s t h e (E 1%, 1 cm) v a l u e a t
288 nm o r by u s i n g a s u i t a b l e c a l i b r a t i o n
curve.
8.3.2
Phosphorescence Method
Mebendazole h a s been r e p o r t e d t o show good
phosphorescence a t room temperature when
determined on Whatman 42 f i l t e r paper u s i n g
Pb ( I V ) and T 1 ( I ) a s phosphorescence
enhancing a g e n t s . In t a b l e t s , t h i s method
gave a r e c o v e r y o f 97.6% o f mebendazole
(37) *
312
8.3.3
F l u o r i m e t r i c Method
Abdel F a t t a h et a1 (38) have r e p o r t e d a
f l u o r i m e t r i c d e t e r m i n a t i o n method o f
mebendazole i n p h a r m a c e u t i c a l dosage forms
a f t e r a l k a l i n e h y d r o l y s i s . I n t h i s method
2 t o 5 mg o f mebendazole i s d i s s o l v e d i n
10 m l of 1 M sodium h y d r o x i d e and h e a t e d
f o r 1 h r on a b o i l i n g water b a t h . After
d i l u t i o n w i t h methanol, 1 M sodium h y d r o x i d e
( 4 9 : 1 ) , t h e s o l u t i o n was s p o t t e d on t o
Whatman 42 f i l t e r p a p e r and d r i e d a t 65 f o r
5 t o 10 m i n u t e s . The f l u o r e s c e n c e was
measured a t 460 nm ( e x c i t a t i o n a t 365 nm).
The l i m i t of d e t e c t i o n was 0 . 1 f o r
mebendazole w i t h r e c t i l i n e a r c a l i b r a t i o n
graphs u p t o 10 and 50 ppm ( i n t h e f i n a l
s o l u t i o n ) . For 8 ng o f mebendazole p e r
s p o t , t h e c o e f f i c i e n t o f v a r i a t i o n was
9.5% (n = 1 9 ) . For p h a r m a c e u t i c a l
f o r m u l a t i o n s , r e c o v e r y o f mebendazole ranged
from 99.04 t o 100.56%, w i t h c o e f f i c i e n t o f
v a r i a t i o n of 2.27 t o 2.97%.
8.3.4
C o l o r i m e t r i c Method
Rana et& (39) have d e s c r i b e d a method f o r
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 mebendazole
i n t a b l e t s and s u s p e n s i o n b y adding hydroxyl
amine h y d r o c h l o r i d e , dicyclohexylcarbodiimide
and f e r r i c c h l o r i d e t o a s o l u t i o n o f
t a b l e t s (or a d i l u t i o n o f a s u s p e n s i o n ) i n
isopropanol and formic a c i d and measuring
t h e absorbance a t 520 nm. The c a l i b r a t i o n
graph was l i n e a r f o r 0 . 4 t o 2 . 0 pg/ml of
mebendazole .
8.4
8.4.1
Thin-Layer Chromatography (TLC)

C l a r k e (11) h a s d e s c r i b e d t h e f o l l o w i n g TLC
system f o r t h e i d e n t i f i c a t i o n o f mebendazole.
S o l v e n t system: Chloroform : methanol :
formic a c i d (90:5:5).
Absorbent:
S i l i c a g e l HF 254 (Merck)
MEBENDAZOLE
313
V i s u a l i z i n g a g e n t : I o d i n e vapour;
d r a g e n d o r f f s p r a y (Location under u l t r a v i o l e t
l i g h t ) . Rf : 0.53.
United S t a t e s Pharmacopeia "USPXX 1980 (32)
have used TLC t o determine t h e p u r i t y o f
mebendazole. The method i s d e s c r i b e d as
follows:
D i s s o l v e 50 mg i n 1 . 0 m l o f 98 p e r c e n t
f o r m i c a c i d i n a 10-ml v o l u m e t r i c f l a s k , add
chloroform t o volume, and mix. S i m i l a r l y
p r e p a r e a s o l u t i o n of USP Mebendazole RS
i n t h e same medium having a c o n c e n t r a t i o n o f
5 mg p e r m l . T r a n s f e r 1 . 0 m l o f t h i s
S t a n d a r d s o l u t i o n t o a 200-ml v o l u m e t r i c
f l a s k , add a m i x t u r e o f chloroform and 98
p e r c e n t formic a c i d ( 9 : l ) t o volume, and
mix ( d i l u t e d S t a n d a r d s o l u t i o n ) . On a
s u i t a b l e t h i n - l a y e r chromatographic p l a t e ,
c o a t e d w i t h a 0.25-mm l a y e r o f chromatog r a p h i c s i l i c a g e l m i x t u r e , s p o t 10-1-11
p o r t i o n s of t h e t e s t s o l u t i o n , t h e S t a n d a r d
s o l u t i o n , and t h e d i l u t e d S t a n d a r d s o l u t i o n .
Allow t h e s p o t s t o d r y , and develop t h e
chromatogram i n a s o l v e n t system c o n s i s t i n g
o f chloroform, methanol, and 98 p e r c e n t
formic a c i d (90:5:5) u n t i l t h e s o l v e n t f r o n t
h a s moved about t h r e e - f o u r t h s o f t h e l e n g t h
o f t h e p l a t e . Remove t h e p l a t e from t h e
d e v e l o p i n g chamber, mark t h e s o l v e n t f r o n t ,
a l l o w t h e s o l v e n t t o e v a p o r a t e , and examine
t h e p l a t e under s h o r t - w a v e l e n g t h u l t r a v i o l e t
l i g h t . The R f v a l u e of t h e main s p o t
o b t a i n e d from t h e t e s t s o l u t i o n c o r r e s p o n d s
t o t h a t o b t a i n e d from t h e S t a n d a r d s o l u t i o n ,
and no s p o t , o t h e r t h a n t h e main s p o t , i n
t h e chromatogram o f t h e t e s t s o l u t i o n i s
l a r g e r o r more i n t e n s e t h a n t h e main s p o t
o b t a i n e d from t h e d i l u t e d S t a n d a r d s o l u t i o n .
8.4.2
H i g h - p r e s s u r e Liquid Chromatography (HPLC)

A s e n s i t i v e method f o r s e p a r a t i o n and
s i m u l t a n e o u s d e t e r m i n a t i o n o f mebendazole
and hydroxymebendazole i n human plasma o r
c y s t l i q u i d sample u s i n g f l u b e n d a z o l e as
314
i n t e r n a l s t a n d a r d by high-performance
l i q u i d chromatography w ith e le c tr o c h e m ic a l
detector i s described (40).
The chromatographic c o n d i t i o n s : The
column ( 4 . 6 mm X 5 cm) was packed with
S p h er i s o r b 5 ).rm ODS (Phase Sep. Queensferry
Clwyd, U.K.).
The mobile phase c o n s i s t e d
of 20% 2-propanol i n a pH 7 . 0 phosphate
b u f f e r . The e l u e n t was d e l i v e r e d by Kipp
9208 h i g h - p r e s s u r e l i q u i d chromatography
pump, a t a flow r a t e of 2.5 ml/min. The
e l ec t r o ch e m i c a l d e t e c t o r equipped with a
r o t a t i n g d i s c working e l e c t r o d e was u se d .
The l i m i t of d e t e c t i o n f o r mebendazole and
hydroxymebendazole was
5 and 2.5 ng/ml
r e s p e c t i v e 1y
_-
Allan e t a 1 (41) r e p o r t e d two h ig h performance l i q u i d chromatographic methods

f o r d et er m i n a t i o n o f mebendazole and i t s
m e t a b o l i t e s i n human plasma u sin g a r a p i d
Sep Pak C18 e x t r a c t i o n . The methods elimin a t e t h e need f o r s o l v e n t e x t r a c t i o n a s
such. One method i n c l u d e s i s o c r a t i c e l u t i o n
and o t h e r , g r a d i e n t e l u t i o n . The g r a d i e n t
e l u t i o n system p r o v i d es s u p e r i o r r e s o l u t i o n s
Plasma samples ( 5 ml) was s p i k e w i t h major
m e t a b o l i t e s o f mebendazole, methyl 5fa-hydroxybenzyl) -2-benzimidazole carbamate,
2-amino-5-benzoyl-benzimidazole and 2amino-5- (a-hydroxybenzyl) -benzimidazole
and w i t h 5 . 0 pg o f ethyl-5-benzoylbenzimid a z o l e carbamate as i n t e r n a l s t a n d a r d i n
t h e range o f 10 ng - 30 pg. Samples were
a d j u s t e d t o pH 6 with d i l u t e h y d r o c h l o r i c
a c i d o r sodium c a r b o n a te s o l u t i o n and
e x t r a c t e d by p a s s i n g through a Sep Pak C18
c a r t r i d g e f i t t e d t o a l u e r lock g l a s s
s y r i n g e . A f t e r t h e spikedplasma was p a sse d
through t h e c a r t r i d g e , it was washed with
20 m l o f d i s t i l l e d w a t e r , 0 . 5 m l o f 40%
methanol i n water and 0.4 m l of methanol.
The n e x t 1 . 6 m l of methanol e l u t e d t h e f i v e
compounds from c a r t r i d g e , t h i s 1 . 6 m l o f
methanol was ev a p o r a t ed t o d r y n e ss i n
MEBENDAZOLE
315
p o i n t e d and t h e r e s i d u e was d i s s o l v e d i n
100 1-11 DMSO. A l i q u o t e s of 20 1~1were
i n j e c t e d i n t o HPLC system. The i n s t r u m e n t
used was A l t e x Model 322 MP HPLC ( A l t e x
S c i e n t i f i c , Berkeley, CA, USA) equipped w i t h
f i x e d wavelength d e t e c t o r (254 nm, 8-ul flow
c e l l ) , a Rheodyne (Berkeley, CA, USA) Model
7120 i n j e c t o r and Hewlett-Packard (Avondale,
PA. USA) Model 3380 A i n t e g r a t o r r e c o r d e r .
For mebendazole t h e d e t e r m i n a t i o n limits
a r e 20 ng/ml ( i s o c r a t i c system) and 10 ng/ml
(gradient system).
Chromatographic C o n d i t i o n s :
Column
(250 x 4 . 6 mm I.D.)
LiChrosorb, RP-8 10-ym
reversed-phase packing. A
precolumn (35 x 3 . 2 mm I.D.)
dry packed with C o r a s i l C18
was a l s o used.
Mobile phase :
Methanol-water (55:45 pump A)

and methanol-aqueous
ammonium phosphate 0.05 M
pH 5 . 5 (55:45 pump B)
Flow r a t e
Pressure
1 . 7 ml/min.
1200-1300 p . s . i .
--
K a r l a g a n i s e t a 1 (42) developed an HPLC

method f o r t h e d e t e r m i n a t i o n o f mebendazole
i n b i o l o g i c a l samples. 2 M 1 plasma o f t h e
p a t i e n t s was a l k a l i n i z e d w i t h 8 ml of
sodium c a r b o n a t e b u f f e r (0.05 M, pH 11.3)
and was e x t r a c t e d with 10 m l o f chloroform
by shaking i n a g l a s s t u b e f o r 15 m i n u t e s .
The c o n t e n t s were c e n t r i f u g e d and aqueous
phase was a s p i r a t e d and t h e chloroform
l a y e r was evaporated under reduced p r e s s u r e .
The r e s i d u e was d i s s o l v e d i n 200 1~1mobile
phase, 150 1-11 were i n j e c t e d . A n a l y s i s u s i n g
Waters Model M6000A High-pressure Liquid
Chromatograph equipped w i t h Wisp Model 710
i n j e c t o r and a Perkin-Elmer W d e t e c t o r
Model LC55.
316
Chromatographic Condi tions:

Column
25 cm x 3 mm ( i . d . )
LiChrosorb S1 60 5 m .
Mobile phase :
Acetonitrile/water-saturated
ch 1o r o f orm/2 5% (weight /
volume) ammonia i n water
(75/92.5/0.1, by volume) ,
pH 6 . 5 .
Flow r a t e
0 . 8 ml/min
Pressure
400 p s i
Column
t e mpe ra ture
Ambient
De t e c tor
wavelength
307 nm
Recorder
5 mV (0.02 a b s o r p t i o n u n i t s
full scale).
C a l i b r a t i o n curve was pr epar ed u s i n g 4 0 , 80,

200 and 400 ng of mebendazole. 1 Mg o f
c ic lobe nda z ole was used as a i n t e r n a l
s t a n d a r d , d e t e c t i o n r a t e i n t h e range o f
6.117 ng/ml.
Alton -e t a1 (43) developed a simple, r a p i d
and s p e c i f i c HPLC method or 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 mebendazole i n plasma.
2 M 1 o f plasma was d i l u t e d w i t h 2 n i l o f
0.05 M potassium hydrogen phosphate b u f f e r
(pH 7.0) and e x t r a c t e d w i t h 7 m l of e t h y l
a c e t a t e . The o r g a n i c l a y e r was removed
and d r i e d , t h e r e s i d u e was d i s s o l v e d i n
0.001 N HC1 and r e - e x t r a c t e d w i t h 6 m l o f
petroleum e t h e r and c e n t r i f u g e d , s u p e r n a t e n t
was d i s c a r d e d t h e aqueous f r a c t i o n was
a l k a l i n i z e d w i t h 2 m l 0 . 0 1 N NaOH and
e x t r a c t e d t w i c e with e t h y l acetate. The
s o l v e n t was d r i e d and d i s s o l v e d i n 60 ~1
of e t h y l acetate and 20 p1 was i n j e c t e d i n
chromatograph (Model ALC 202/204 Waters
Associates) f o r analysis.
MEBENDAZOLE
317
Column
Bondapak C-18 300 x 3 . 0 mm

( i .d . ) reversed-phase .
Mobile phase :
0.05 M KH2P04-NaOH b u f f e r
(pH 6 . 0 ) - a c e t o n i t r i l e
( 7 3 : 2 7 v/v).
Flow r a t e
2 . 5 ml/min
Pressure
2500 p s i
Column
temperature
Ambient
Detector
wavelength
UV (313 nm).
Flubendazole was used as i n t e r n a l s t a n d a r d .

i n t h e q u a n t i t y o f 0.15 ug/ml f o r e a c h
sample. T h i s method can be used t o measure
plasma mebendazole l e v e l as low as 10 ng/ml.
A simple and r a p i d method t o d e t e c t t h e presence
of d i f f e r e n t benzimidazole compounds i n drug
p r e p a r a t i o n s have been r e p o r t e d ( 4 4 ) . The
procedure i n v o l v e s two d i f f e r e n t chromatography systems:
i-
Reversed-phase g r a d i e n t .
ii- Normal p a r t i t i o n chromatography w i t h

isocratic elution.
Three d i f f e r e n t m i x t u r e s A , B , and C were
used. The benzimidazoles were d i s s o l v e d i n
p u r e formic a c i d (5 ml) t h e n i n c o n c e n t r a t e d
h y d r o c h l o r i c a c i d (5 ml) and i n 50% (%)
aqueous e t h a n o l (90 m l ) . Mixtures A and C
c o n t a i n mebendazole. The s o l u t i o n s A and B
were s e p a r a t e d by r e v e r s e d - p h a s e g r a d i e n t
e l u t i o n with t h e solvent containing aceton i t r i l e - aqueous 1%HzS04 w i t h t h e
programme: 10% a c e t o n i t r i l e f o r 2 minutes
t h e n from 10% a c e t o n i t r i l e t o 30% a t a r a t e
of 5% p e r minute. Mixture C was e l u t e d
318
i s o c r a t i c a l l y on a amino phase chemically

bonded t o s i l i c a g e l (Type NH2) w i t h t h e
s o l v e n t : methylene c h l o r i d e + i s o p r o p y l
a l c o h o l (90 + 10) - a c e t o n i t r i l e ( 5 0 : 5 0 ) .
I n i t i a l l y f o r d e t e r m i n a t i o n of r e t e n t i o n
time each of t h e benzimidazoleswas s e p a r a t e l y
chromatographed.
Chromatographic Condit ions:
Chromatograph :
Varian LC 8500 HPLC w i t h

UV d e t e c t o r .
Column
LiChrosorb RP 8 - microp a r t i c u l a t e (15 cm 4 . 7 mm

i .d . ) LiChrosorb NH2 (Merck)
(15 cm x 4.7 mm i . d . )
Mobile phase : Methylene c h l o r i d e + i s o propyl a l c o h o l (90 + 10)

a c e t o n i t r i l e (5O:SO).
Flow r a t e
80 m l h - l
De te c t or
sensitivity
0 . 5 AUFS
Column
press ure
De te c t or
wavelength
500 p s i
:
(UV) 254 nm
This method would be u s e f u l i n t h e q u a l i t y

c o n t r o l of raw m a t e r i a l s i n t h e q u a n t i f i c a t i o n o f benzimidazoles i n f o o d s t u f f s of
animal o r i g i n , and i n t h e d e t e c t i o n of
residues.
A simpl e , a c c u r a t e and r e p r o d u c i b l e HPLC
method h a s been d e s c r i b e d (45) f o r deter minat i o n of mebendazole i n t a b l e t s . 20 T a b l e t s

were t a k e n and ground i n f i n e powder,
e q u i v a l e n t t o about 10 - 20 mg of mebendazole
was a c c u r a t e l y measured o u t or each
d e t e r m i n a t i o n . The powder was e x t r a c t e d
wi th 5 m l o f formic a c i d . 5 M 1 o f s a l i c y l a mide s o l u t i o n ( 8 . 5 mg/ml) was added t o each
MEBENDAZOLE
319
e x t r a c t as i n t e r n a l s t a n d a r d . The m i x t u r e
was c e n t r i f u g e d and t h e s u p e r n a t e n t was
used f o r HPLC d e t e r m i n a t i o n .
Chromatograph:
Waters A s s o c i a t e s Model 440
Column
Bondapak C-18
Mobile phase :
T e t r a h y d r o f u r a n - 0 . 5 % formic
acid (30:60).
Flow r a t e
1 . 9 ml/min.
Temperature
25-28OC.
Pressure
1500 p s i .
Detect o r
wavelength
(UV) 254 nm
The r e c o v e r y was between 99.58 - 100.15%.

An HPLC method f o r t h e e s t i m a t i o n mebendazole,
i t s prodrug, 4-arnin0-3-(3~-methoxycarbonyl
2 -thioureido)benzophenone , and t h e i r known
o r expected m e t a b o l i t e s and d e g r a d a t i o n
p r o d u c t s i n aqueous media and r a t blood
was developed ( 4 6 ) . After o r a l a d m i n i s t r a t i o n o f t h e p r o d r u g , t h e prodrug was r a p i d l y
c o n v e r t e d t o mebendazole and t h e area under
t h e blood level vs time c u r v e o f mebendazole,
i n rats dosed w i t h p r o d r u g , was more t h a n
twice t h a t o b t a i n e d a f t e r g i v i n g rats an
equimolar amount o f mebendazole. Only t h e
p r o d r u g , mebendazole and known m e t a b o l i t e s
o f mebendazole were d e t e c t e d i n r a t s g i v e n
t h e prodrug.
Behm et a1 (47) have measured t h e c o n c e n t r a t i o n o f mebendazole and i t s major m e t a b o l i t e s

by HPLC i n s h e e p plasma a t 12.5, 25, 50 o r
100 mg/kg.
A t 1 2 . 5 mg/kg t h e peak plasma
c o n c e n t r a t i o n s occured between n i n e and 24
h o u r s f o r a l l dose r a t e s and d e c l i n e r a p i d l y .
Two major m e t a b o l i t e s were d e t e c t e d ; t h e i r
320
c o n c e n t r a t i o n s exceeded t h a t o f mebendazole
a t a l l dose r a t e s .
8.5
P o l a r o g r ap h i c Method
P i n z a u t i -e t a1 (48) d e s c r i b e d a d i r e c t - c u r r e n t
p o l a r o g r ap h i c r ed u c t i o n method o f mebendazole a t
t h e dropping-mercury e l e c t r o d e , and e s t a b l i s h e d t h e
optimum c o n d i t i o n s f o r t h e d ete r m in a tio n o f t h i s
drug i n dosage form. S i x t a b l e t s o f mebendazole
(about 0 . 3 g) were ground. A q u a n t i t y o f t h e powder
e q u i v a l e n t t o 50 mg o f mebendazole was t r a n s f e r r e d
t o 100 m l v o l u m e t r i c f l a s k . To t h i s powder 8.6 m l
o f 70% p e r c h l o r i c a c i d was added and t h e m ix tu r e
s t i r r e d f o r 10 minutes and d i l u t e d t o volume w i t h
water. The suspension was f i l t e r e d under s u c t i o n
through a f i n e p o r s i t y g l a s s c r u c i b l e . 2 M1
p o r t i o n of t h e f i l t r a t e was t r a n s f e r r e d t o a 50 m l
volumetric f l a s k , 3 m l o f 1 M p e r c h l o r i c a c i d was
added and t h e s o l u t i o n d i l u t e d t o volume w ith t h e
McIlvaine b u f f e r pH 2 . 6 (2.18 v o l . 0.2 M sodium
phosphate with 17.82 v o l . 0 . 1 M c i t r i c a c i d ) .
A 20 m l of t h i s s o l u t i o n (corresponding t o 0 . 4 mg
o f mebendazole) was t r a n s f e r r e d i n t o p o l a r o g r a p h i c
c e l l (Metrohm E506 r e c o r d i n g polarograph equipped
w i t h polarography s t a n d ) .
Deaeration and polarography were performed on u s i n g

Metrohm EA 290 hanging mercury drop system. The
number of e l e c t r o n s involved i n t h e r e d u c t i o n was
c a l c u l a t e d a t a mebendazole c o n c e n t r a t i o n o f
1 x 10-4 M u s i n g a s o l i d s t a t e c o n t r o l p o t e n t i a l
c o u l o m e t r i c a p p a r a t u s . A c a l i b r a t i o n curve was
p r e p a r e d u s i n g 5 t o 50 pg/ml mebendazole s o l u t i o n
p r e p a r ed i n McIlvaine b u f f e r , u s i n g t h i s method
mebendazole a n a l y s i s c o u l d - b e performed with a l i m i t
of d e t e c t i o n 100 ng/ml ( 4 8 ) .
8.6
Radioimmunoassay Method
M i c h i e l s -e t a 1 (49) developed an e x tr e m e ly
h i g h l y s p e c i f i c radioimmunoassay procedure f o r t h e
d e t e r m i n at i o n o f mebendazole i n plasma. Mebendazole
was converted t o methyl [5-[4-(2-aminoethyl)
ben zoy 11-1H-ben zimidazol - 2- y l ] carbamate
T h is
hapten was coupled t o bovine serum albumin u s i n g a
water s o l u b l e carbodiimide as f o llo w s:
MEBEND AZOLE
321
NHH
ICI -0-
C H ~
II
CH3-C-NH-CH2CH2
HC1, 1 N
a!a7
H
H 2 NCH2CH2
It
NH-C-0-CH3
1. DMF/MeOH
carbodi imide /HC1
2 . Bovine Serum
Albumin (BSA).
0
I.
II
BSA-C-HN-CH2-CH2
Synthesis of the hapten and the mebendazole-protein

conjugate used f o r the immunization of the
rabbits (49).
322
The r e s u l t i n g co n j cg a t e was used t o immunize r a b b i t s

according t o co n v e n t i o n a l p r o c e d u r e s. The a n t i serum e l i c i t e d i n t h i s way was t e s t e d f o r i t s
a b i l i t y t o b i n d s p e c i f i c a l l y mebendazole. Blood
was c o l l e c t e d on EDTA and plasma was o b ta in e d
a f t e r c e n t r i f u g a t i o n of t h e blood a t 2200 g f o r
15 minutes. Unmetabolized mebendazole was measured
by d i r e c t radioimmunoassay u s in g a n t i - b o d i e s .
Under t h e s t a n d a r d i z e d a s s a y c o n d i t i o n , t h e r a b b i t
serum bound s p e c i f i c a l l y n e a r l y 50% o f added 0 . 2 ng
H3-Mebendazole a t 1/100 serum d i l u t i o n . Un sp e c if ic
a d s o r p t i o n t o plasma c o n s t i t u e n t s d i d n o t exceed
2 % . The method is q u i t e s e n s i t i v e t o a s s a y
mebendazole i n plasma a t c o n c e n t r a t i o n as low a s
100 picogram/ml. M et ab o l i t es d i d n o t i n t e r f e r e
with t h e binding of parent drug t o antibodies.
Acknowledeements
The a u t h o r s would l i k e t o Thank M r . Syed R a f a t u l l a h ,
A s s i s t a n t Researcher, f o r h i s t e c h n i c a l a s s i s t a n c e and Mr.
T a n v i r A . Butt f o r h i s s e c r e t a r i a l s e r v i c e s .
MEBENDAZOLE
323
References
1.
J . S . Keystone, and J . K . Murdoch, Ann. I n t e r n . Med.,
91, 582 (1979).
2.
W.C. Bowman and M . J . Rand, "Textbook o f Pharmacology",

2nd Ed. Blackwell S c i e n t i f i c P u b l i c a t i o n s , Oxford,
page 374 (1980).
3.
M . J . M i l l e r , I.M. Krupp, M.D.

JAMA,230, 1412 (1974).
-
4.
E.D. Wagner and A.P.

23, 151 (1974).
-
5.
M.S. Wolfe and J . M .
6.
"The Merck Indextr, 9 t h Ed. , Merck 6 Co. I n c . , Rahway,

N . J . U.S.A., page 747 (1976).
7.
" D i c t i o n e r y o f Organic Compoundsrr, 5 t h Ed. , Volume I V Y

Champan and Hall Ltd. , London, page 3644 (1982).
8.
"Remington's Pharmaceutical Sciences", A . Osol , 1 5 t h

Ed. , Mack P u b l i s h i n g Co. , E a s t o n , P e n n s y l v a l n i a , USA,
page 1182 (1980).
9.
"Annual Drug Data Report", Vol. 11, J . R . P u b l i s h e r s ,

Barcelona, Spain, page 137 (1979).
L i t t l e and C . S a n t o s ,
C h a v a r r i a , Am. J . Trop. Med.,

Wershing, JAMA, 230, 1408 (1974).
, The
10.
M a r t i n d a l e , The E x t r a Pharmacopoeia, 28th Ed.

Pharmaceutical Press, London, page 98 (1982).
11*
E.C.G. C l a r k e , " I s o l a t i o n and I d e n t i f i c a t i o n o f Drugs",

Vol. 11, The Pharmaceutical Press, London, page 1056
(1975).
12.
Goodman and G i l l m a n ' s , The Pharmacological B a s i s o f

T h e r a p e u t i c s " , 6 t h Ed. , Mac M i l l a n P u b l i s h i n g Co. , Inc.,
N e w York, page 1018 (1975).
13.
Ger. O f f e n . , 2,029,637 (1971) through CA 74: 1000475

(1971).
14.
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15.
S . De N o l l i n and H . B . Vanden, J . P a r a s i t o l . ,
970 (1973).
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324
16.
M.D. Rawlins, P . C o l i s t e , L. B e r t i l s s o n and L . Palmer,

Eur. J. C l i n . Pharmacol., 8, 91 (1975).
17.
W.A. R i t s c h e l and G . V . Hammer, I n t . J . C l i n . Pharmacol.

Toxicol., 18, 175 (1980).
18.
B.G. Katzung, "Basic and C l i n i c a l Pharmacology", Middle

E a s t E d i t i o n , Lange Medical P u b l i c a t i o n s , C a l i f o r n i a ,
608 (1982).
19.
G . J . Munst, G . Karlaganis and J . B i r c h e r , E u r . , J .

C l i n . Pharmac., l
J
375
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20.
J . P . Brugmans, D . C . Theinpont, I.V. Wijngaarden, O.F.

V a n p a r i j s , V . L . Schuermans and H . L . Lauwers, JAMA,
2
1
7
.
313
(1971).
-
21.
P.A. B r a i t h w a i t e , M.S. Roberts, R . J . Allan and T.R.

Watson, Eur. J . C l i n . Pharmacol., 2 2 , 161 (1982).
22.
M. G i b a l d i and D. P e r r i e r , "Pharmacokinetics" Marcel

Dekker, New York (1975).
23.
R . J . Allan and T.R. Watson, Eur. J . Drug Metab.

Pharmacokinet., 8, 373 (1983).
24.
M. Dawson, R . J . Allan and T.R. Watson, B r . J . C l i n .

Pharmac., 14, 453 (1982).
25.
R . J . Allan and T.R. Watson, Eur. J . Drug Me'tab.

Pharmacokinet., 7, 131 (1982).
26.
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A.P. C h a v a r r i a , Am. J . Tro. Med. Hyg., 26, 118 (1977).
28.
D. L. Morris and S.E. Gould, B r . Med. J . ,

(1982).
29.
O f f i c i a l Product Monograph, Vermox T a b l e t s . R a r i t a n

New J e r s e y , Otho Pharmac. Corp. , 11 J u n e , 8 (1975) .
30.
31.
P.F. Miskovitz and N . R .
29, 1356 (1980).

M.H.
285,
175
J a v i t t , Am. J . Tro. Med. Hz.,
Levin, JAMA,249, 2929 (1983).
325
MEBENDAZOLE
32.
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33.
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O f f . Anal. Chem., 66, 161 (1983).
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A.A.M.
35.
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40, 20 (1978).
I n d i a n J . Pharm., -
Patel,
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I n d i a n J . Pharm. S c i . , 40, 76 (1978).
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37.
F. A b d e l - F a t t a h , W. Baeyens and P . De Moerloose

Biolumin Chemilumin . , [ I n t Symp. Anal. Appl .
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38.
F. Abdel F a t t a h , W . Baeyens, P . De Moerloose, Anal.

Chim. Acta, 154, 351 (1983).
39.
N . G . Rana, R.V. Dave and M.R.

333 (1981).
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B. O o s t e r h u i s , J . C . F.M. Wetsteyn and C . J . V a n B o x t e l ,

Therap. Drug M o n i t o r . , 6 , 215 (1984).
41.
R . J . A l l a n , H.T. Goodman and T.R. Watson, J o u r n a l o f

Chromatography, 183, 311 (1980).
42.
G . K a r l a g a n i s , G . J . Munst and J . B i r c h e r , J . High

Resolu. Chromatogr. and Chromatogr. Commun. 2 , 141
(1979).
43.
K . B . A l t o n , J . E . P a t r i c k and J . L . McGuire, J . Pharm.

S
c i ,68, 880 (1979).
-
44.
Wahbi and S . Onsy, T a l a n t a , 25, 716 (1978).
P a t e l , I n d i a n Drugs, 18
D . Mourot, J . B o i s s e a u and G . Gayot, Anal. Chim. Acta,
99, 371 (1978).
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45.
Da-Peng Wang and Hsin-Shang,

(1984).
46.
M . Dawson, T.R. Watson, E u r . J . Drug Metab.
47.
Pharmacokinet. 8 , 329 (1983).
C.A.
Behm, R.A. C o r n i s h and C . Bryant, Res. V e t . S c i . ,
34, 37 (1983).
326
48.
S . P i n z a u t i , E . La P o r t a and G . Papeschi, J .
Pharmaceuti. Biomed. Anal.,
223 (1983)
49.
M. M i c h i e l s , R . Hendriks, J . Heykants and H.V.D.

Bossche, Arch. Int. Pharmacodyn., 256, 180 (1982).
1,
.--
METOCLOPRAMIDE HYDROCHLORIDE
Davide P i t r g and R i c c a r d o S t r a d i
F a c u l t y o f Pharmacy, U n i v e r s i t y o f Milan
1.
Description
1.1
Nomenclature
1.2
Chemical names
1.3
G e n e r i c names
1.4
Trade names
1.5
Formula and Molecular Weight
1.6
2.
Physical Properties
2.1
2.2

VOLUME 16
321
Copyright 01987 by Academic Press, Inc.

DAVIDE PITRe AND RICCARDO STRADI
328
2.2.1
2.2.2
2.3
2.4
2.5
2.6
2.7
2.8
2.9
2.10
2.11
2.12
3.
'H-NMR
13
C-NMR
Mass Spectrum
Fluorescence Spectrum
X-Ray, Powder Diffraction
Crystal Structure
Melting Range
Solubility
PKa
PH
Partition Coefficient
Manufacturing Procedures
Synthesis
3.1
4.
Stability
5.
Metabolism and Pharmacokinetics

5.1
Metabolism
5.2
Pharmacokinetics
5.3
Acute Toxicity
6.
Methods of Analysis
Elementa1
6.1
6.2
Nonaqueous Titration
6.3
Complexometric Analysis
6.4
Colorimetric Analysis
6.5
Ultraviolet Spectrophotometric Analysis
6.6
Fluorimetric Analysis
6.7
6.8
6.9
High Performance Thin-Layer Chromatography
6.10
Gas
Liquid Chromatography
6.11
6.12
7.
Determination in Body Fluids and Tissues
8.
References
1.
Description
1.1
Nomenclature
1.2
Chemical Names
329
Benzamide, 4-amino-5-chloro-N-[2-(diethylamino)
ethyl 3-2-methoxy,
monohydrochloride, rnonohydrate CAS 54143-57-6
anhydrous
CAS 7831-21-5
4-Amino-5-chloro-N-[2(diethylamino)ethyllo-anisasamide
1.3
Generic Names
Metoclopramide Hydrochloride USAN-BP 1980-F.U.IX
Metoclopramidhydrochloride
DAC 1979
Metoclopramidum hydrochloricum
2.AB-DDR
1.4
Trade Names
Cerucal (Arzneimittelwerk-Dresden)
Elieten (Nippon Kayaku, Tokyo-Japan)
Emperal (Neofarma-Helsinki)
Maxeran ( Delagrange-Paris)
Plasil ( Lepetit-Milan)
Peraprin (Taiyo, Gifuken-Japan)
Primperan (Delagrange-Paris)
Reglan (Robins, Richmond, USA)
DAVIDE PITRh AND RICCARDO STRADI
330
1.5
Formula and Molecular Weight
C0 NH
I
- CH-2
CH2-N
OCH3
.HCI * H20
\
c2
CI
NHp
C
1.6
H C1N 0 . H C l . H 0
1 4 22
3 2
2
Mol.Wt.
= 354.3
Appearance, C o l o r , Odor, Taste
A w h i t e o r a l m o s t w h i t e c r y s t a l l i n e powder, odorless (1)
33 1
2.
Physical Properties
2.1
The i n f r a r e d s p e c t r u m of rnetocloprarnide hydrochlor i d e , i s p r e s e n t e d i n F i g . 1. The spectrum was r e c o r d e d w i t h

a s o l i d sample d i s c composed of 1 m g o f compound/200 m g KBr
on a P e r k i n E l m e r Model 1310.
-1
The f o l l o w i n g bands (cm ) were a s s i g n e d and a r e r e p o r t e d i n
Table 1.
Table 1
Wave Number
Assignments
3200, 3300, 3340, 3400, 3460
VNH
2860, 2950, 2980
VC
H
SP3
3030
VC
H
SP2
2100, 2500, 2660, 2710
v N+H
1600
v c=o
1540
6NH
1270
v C-0-C
700
v c-c1
VOH
(Amide)
(Asymmetric)
Fig. 1
I n f r a r e d Spectrum of Metocloprarnide Hydrochloride
333
2.2
2.2.1
1
H-NMR
1
H-NMR
spectrum o f metoclopramide h y d r o c h l o r i d e ,
shown i n F i g . 2 , was obtained i n DMSO-d with a Varian spec6
trometer EM-390 o p e r a t i n g a t 90 MHz. The chemical s h i f t s a r e
l i s t e d i n t h e Table 2.
Table 2
Moltepli
city
Number of
protons
Assignments
1.33
N-(CH2-CH
-3
3.0-3.2
CH -N-(CH
-2
-2
-CH
3.72
NH-CH
-2
3.95
OCH
6.03
broad s
2 ,exch.
NH
6.60
H~
(arom.)
7.72
(arorn.)
8.44
1,exch.
CONH
10.85
broad s
1,exch.
hH
2
)
3 2
Fig. 2
1
H-NMR (90 MHz) Spectrum of Metocloprarnide
h y d r o c h l o r i d e i n DMSO-d
335
2.2.2
13
C-NMR
13
The
C-NMR spectrum o f metoclopramide hydrochlor i d e i s shown i n F i g . 3. The spectrum was r e c o r d e d i n D 0
2
w i t h Varian XL.200 s p e c t r o m e t e r o p e r a t i n g a t 50.391 MHz. The
chemical s h i f t s a r e l i s t e d i n Table 3.
Table 3
Line
(p.p.m)
Mu1 t i p 1i c i t y
Assignments
N.
N(CH2.CH )
-3 2
8.38
35.15
48.27
N(CH -CH )
-2
3 2
51.40
NH.CH
55.89
OCH
98.00
c3
109.18
110.61
131.43
10
148.57
11
158.07
12
167.46
2.3
CH -N(C2H512
-2
-2
3
(aromatic)
or C
or C
c2
c=o
The u l t r a v i o l e t spectrum of metoclopramide hydro-5

c h l o r i d e i n w a t e r ( c = 5.763 10
m/l) was o b t a i n e d (60)
with a Beckman Mod. 24 s p e c t r o p h o t o m e t e r and i t is shown i n
Fig. 4
. .
'
1
Fig. 3
jl
I
I
h
13
H-NMR
(90 MHz) Spectrum of Metoclopramide

hydrochloride in DMSO-d
6
- i'
Fig. 4
Ultraviolet Spectrum of Metoclopramide

hydrochloride in Water
331
DAVIDE PITRG AND RICCARDO STRADI
338
Mass Spectrum
2.4
The mass spectrum of metoclopramide h y d r o c h l o r i d e

was recorded ( F i g . 5 ) on Finnigan 1020 s p e c t r o m e t e r by
c o w e n t i o n a l e l e c t r o n impact i o n i s a t i o n a t 70 eV. Prominent
fragments and t h e i r r e l a t i v e i n t e n s i t y a r e shown i n Table 4.
Table 4
m/e
Intensity
229
0.23
227
0.90
Attribution
201
7.60
181
6.43
169
0.66
141
1.78
99
27.96
86
100
c1QCH3NH2
4fCH3
J$ro
c1
II
NH2
c1
-0"'
NH2
c1
CH
+/CZHS
-N
2- \C2H5
339
Ell% N.E:
DulR: STRW16 (72
'108 t 2128
RlCi
n.w.02.cL.
96
367616.
173712.
1W.Q
10.
50.1
99
WE
mss S P E C T M
Wrn.'86 9zs7;oo
M n : SIFWI6 172
2128
B P X M'E:
P6
PIC:
167616.
WREi Cll.H22.tI3.02.cL.
188.1
se. I
270
i'
272
I
M
Fig. 5
281
2Qe""'"-
285
~
"
26
"
29s
"
'
Electron impart Mass Spectrum of Monocloprarnide hydrochloride
DAVIDE PITRB AND RICCARDO STRADI
340
Fluorescence Spectrum
2.5
A solution of metoclopramide hydrochloride in

water exhibits fluorescence when excited with ultraviolet
light. When excited at 353 nm, it shows a sharp peak at 353
nm and a second peak at about 273-276 nm.
The emission spectrum of the compound consists of a single
peak at 355 nm. The spectra obtained in water (5 mcg/ml), on
a spectrophotometer Perkin-Elmer MPE 44A,are given in Fig.6.
X-Ray Powder Diffraction
2.6
The X-ray powder diffraction data of metoclopramide hydrochloride was determined (2) by a Philips Powder
Diffractometer PW 1710 with nichel-filtered copper radiation
(1.54051 A) with the following instrumental conditions:
TUBE: BF type, CU/Ni, 40 KV, 40 mA; SLITS: lo-0.1 mm-lo;
DETE TOR:PW1711 proportional counter + discriminator; SCALE:
2x10 cps; TIME CONSTANT: 1"; SCANNING SPEED: 0.008xl'1;
PAPER SPEED 1 cmxlo; SPECIMEN HOLDER: Niskanen.
The X-ray diffraction data are given in Table 5.
Fig. 6
F l u o r e s c e n c e Spectrum of Metoclopramide
hydrochloride i n water
341
342
Table 5
N.
d(A)*
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
9.23
8.46
7.64
6.98
6.64
6.28
6.02
5.26
5.18
4.87
4.71
4.60
4.48
4.27
4.23
4.03
3.95
3.82
3.78
3.71
362
3.50
3.47
3.41
3.36
3.32
I&**
5
25
20
22
13
65
8
20
10
38
45
30
31
10
20
20
6
47
35
17
10
58
30
75
100
11
I&*+
N.
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
50
51
52
3.15
3.07
3.04
2.99
2.95
2.92
2.86
2.82
2.78
2.74
2.69
2.59
2.53
2.51
2.43
2.33
2.31
2.29
2.26
2.16
2.14
2.05
2.01
1.81
1.81
1.71
1.63
12
8
6
11
12
10
20
13
8
21
11
10
9
13
18
10
8
10
11
11
10
8
9
8
8
7
9
PLUS OTHER LINES c5

Interplanar distances = 1.54051/2sin diam.
**
Relative intensities are based on highest intensity of

100
343
Crystal Structure
2.7
The metoclopramide hydrochloride monohydrate is

monoclinic, space group P2 /n with a = 12.138(3),
b =
1
8.553(2), c = 17.120(4) A and p= 98.06(7) Z = 4 . The structure was solved by direct methods and refined to R = 0.055
(3).
The metoclopramide free base ( C A S 364-62-5) is triclinic,
space group P1 with a = 13.310 b = 8.728, c=7.477 A , a =
114.20, p = 81.13 and Y = 101.23O, Z = 2. A study of structure-activity relations shows an analogy between metoclopramide and apomorphine(4)
Melting Range
2.8
Employing the USP method the metoclopramide monohydrochloride monohydrate melts at 182-185O(5) and with terma1 microscopy the melting begins at 181O and completes at
183O(6). The dihydrochloride monohydrate melts in the range
of 148O with decomposition. The melting points f o r metoclopramide free base are about 148O(7) or m.p. 146-148O(8). For
dihydrochloride is also reported an eutectic melting point
of 160 with phenacetin and of 117O with benzanilide(7).
Solubility
2.9
At 25O metoclopramide monohydrochloride is soluble in 0.7 g of water, 3 g of ethanol (96 per cent) and 55 g
of chloroform, whereas it is practically insoluble in ether
(10). Solubility of the base and of dihydrochloride are
reported in Table (6). (11).
Table
Solubility of metocloprarnide g/100 m l at 25O
~~
Solvent
Base
Water
Ethanol 95
Ethanol
Benzene
Chloroform
0.02
2.90
1.90
0.10
6.60
Dihydrochloride
48
9
6
0.10
0.10
DAVIDE PITRfi AND RICCARDO STRADI
344
2.10
Metoclopramide hydrochloride shows two ionisation

costants; pic = 9.71 and pK = 0.42. The determination was
1
carried out spectrometricalfy in aqueous solution and the
values are a mean of 16 determinations with a standard deviation of 0.03 and 0.02 (12).
2.11
pH Range
The pH of a 10% water solution must be between

4.6 and 6.5 according to BP 1980.
2.12
Partition Coefficient
The partition coefficient in octanol/water has

been determined(l3) by reversed-phase HPLC at 20.
The
hydrochloride gives
Experimental
LogP = 2,667
Calculated from R.A. Deckker (14)
LogP = 2.76
3.
Manufacturing Procedures
3.2
Synthesis
345
Among the synthetic methods leading to metoclopramide (I) it seems suitable to report only the ones of
greatest interest that are those using p-aminosalicilic acid
(II), as starting product, a compound whose properties are
economically excellent. The two main processes are listed in
Fig 7.
In Scheme A, referring to the first invention(l5), the acid
(1I)is acetylated to (111) and then treated with methyl sulfate to give the ether-ester (IV). This compound is reacted
with (C H ) N-CH CH -NH to the corresponding amide. The
25
2 2
2
subsequent Zesacetylation leads to metoclopramide ( 1).
In scheme B the first step in the synthesis is the etherester (VI). The protection of the amino aromatic group was
not considered necessary (16). The direct chlorination (18)
of (VI) with sodium ipochloride gives (VII) which is
transformed in excellent yields into (I).
4.
Stabi1ity
Aqueous solution
The maximum stability of metoclopramide, investigated at different pH values, was found at pH = 7.6 while
the maximum degradation was found at pH = 2. One of the degradation products was identified as N,N-diethylethylendiamine(l7).
Scheme
FOOH
FOOCH3
COOCH3
COOCH3
FOOCH3
COOH
60 b
Fig. 7
Synthesis of Metoclopramide
341
5.
5.1
Metabolism
The metabolites identified in the in vitro and in

vivo studies are listed in Table 7, in which metoclopramide
is indicated as (I).
Table 7
Non conjugated metabolites found
CI @ O R 2
NH2
N.
-NHCH CH N(C H )
2 2 252
-CH
-NHCH CH NHC H
2 2
2 5
-C H
-NHCH CH NH
2 2 2
-C Hg
-OH
-CH3
-NHCH CH N(C H )
2 2
2 5 2
-H
VI
-NHCH CH N(C2H512
2 2
-H
-NH
2
-CH
VIII
-NHCH COOH
2
-C H
IX
-NHCH = CH
-CH
-NHCH CH NHCOCH
2 2
3
-CH
I
I1
I11
IV
VII
3
3
3
3
REFERENCES
348
DAVIDE PITRe AND RICCARDO STRADI
By studies of the in vitro metabolism of (I) using hepatic

fractions from several animal species (1) (2) five metabolites (11) (111) (IV) (V) (VI) were detected.
Identification studies of the metabolites in urinary excretes of rat and dog (20) after enzimatic hydrolysis evidenced some compounds already known from the in vitro studies and some new ones corresponding to (VII) (VIII) IX) (X).
In particular in rat urine, were found metabolites (11) (IV)
(V) (VII) (VIII), while in dog urine were present metabolites (V) (VI) (VIII) (IX) (X).
In human urine the excretion
was found to be 60% in the first 24 hours. After enzimatic
hydrolisis was identified metabolite (VIII)(CAS 52702.04.2),
but the main metabolite was found to be unchanged metoclopramide originated from the sulfate and glucuronide. Metabolic products were quantified in rabbit urines (21) (Ii)
4
and (IV) but also unchanged (I),as N -glucoronide and N sulfonate were identified. Another product of N-oxidation of
4
(I) was also found. Metoclopramide N -sulfonate (CAS 2726042.0) is the main metabolite in human urine.
Pharmacokinetic
5.2
14
The studies of pharmacokinetics of

C-metoclopramide in rats, dogs and humans show that the drug is distributed within a few minutes after oral administration and it
is eliminated mainly in the urine of the first 24 hrs. (20).
Table 8
14
Excretion of
C-metoclopramide in rats, dogs, and humans
Dosage
p.0.
m g k
hours
Rats
Dogs
U =
humans
71.9
8.5
1.0
5.7
4.4
1.7
65.3
----
77.8
6.9
1.o
18.3
0.8
8.7
0.8
0-24
24-48
48-72
10
20
100
urinary excr.
dose
F
20
1.0
1.6
fecal excr. % of the administered
349
The pharriiacokinetic of metoclopramide H C 1 was s t u d i e d i n

normal male v o l u n t e e r s a f t e r
i.v.
dosage o f 10 mg.
The
shape o f t h e plasma l e v e l c u r v e i n t h e s e s u b j e c t s f i t s a
two-compartment model w i t h a d i s t r i b u t i o n o f T 1
a = 4.6
p = 165.7 min./2The body
min.
and a n e l i m i n a t i o n o f T
.I/?
plasma c l e a r a n c e was 10.9 ml/min g ( 2 2 ) ( 2 3 ) . G. B e r n e r and
co-workers ( 2 4 ) r e p o r t e d t h e s t u d y o f b i o a v a i l a b i l i t y of
commercial f o r m u l a t i o n s o f metoclopramide i n male h e a l t h y
v o l u n t e e r s . The f o l l o w i n g r e s u l t s were o b t a i n e d : f o r t a b l e t s
57.6-80.3%, f o r s o l u t i o n s 49.7-76.7% and f o r c a p s u l e s 54.8%.
Acute T o x i c i t y
5.3
(LD ) of t h e metoclopraThe a c u t e t o x i c i t y i . v .
50
mide d i h y d r o c h l o r i d e is 85.7 mg/kg f o r t h e r a t and 71 mg/kg
f o r t h e mice ( 2 5 ) .
6.
6.1
E l e m e n t a l Composition
H , 7.12; C 1 , 20.0; N , 11.9; H 0 , 5.1 f o r t h e

2
monohydrate
C , 50.01; H , 6 . 8 9 ; C 1 , 21.08; N, 1 2 . 5 ; 0, 9.50 f o r t h e
a n h y d r o u s form
C, 5 6 . 9 ; H , 7.40; C 1 , 1 1 . 8 3 ; N , 1 4 . 0 2 ; 0, 10.67 f o r t h e
base.
C, 47.4;
6.2
a)
I n f r a r e d Spectroscopic Test
BP 1980 (10) c i t e s t h e u s e of t h e i n f r a r e d abs o r p t i o n spectrum o f a potassium c h l o r i d e d i s p e r s i o n o f t h e rnetoclopramide h y d r o c h l o r i d e i n a c c o r dance w i t h t h e r e f e r e n c e s p e c t r u m r e p r o d u c e d i n
t h e appendix.
DAVIDE PITRk AND RICCARDO STRADI
350
b)
Ultraviolet Spectroscopic Test

The light absorption, in the range 230 to 350 nm
of a 2-cm layer of a 0.001 per cent w/v solution
in 0.01M hydrochloric acid exhibits two maxima, at
273 nm and 303 nm; absorbance at 273 nm about 0.79
and at 309 nm about 0.69. This is another
identification test recommended by B.P.1980 (10).
C)
Color Tests
With a solution of 4-dimethylaminobenzaldehyde yellow-orange(1); ammonium vanadate test - light
brown (sensitivity : 1.0 ug), (7).
/
Vitali's test: pale-yellow/light brown (sensitivity : 1.0 ug) (7).
/
d)
Crystal Tests
Gold Cyanide solution

needles, sometimes in
rosettes (sensitivity: 1 in 1000).
Lead iodide solution - feathery rosettes (sensitivity: 1 in 1000) (7).
6.3
Nonaqueous Titration
The metocloropramide hydrochloride may be titrated
(10) potentiometrically in glacial acetic acid containing
mercuric acetate with perchloric acid in glacial acetic acid

as titrant.
6.4
Complessometric Analysis
The method is ba ed on the use of a column of

5+ form. The released quantity,
amberlite IR 120 in the Zn
corresponding to the absorbed metoclopramide, is titrated
with EDTA using Eriochromo Black T as indicator (26).
6.5
35 1
Colorimetric Analyses
The colorimetric assays have been the most widely

used especially to determine metoclopramide in pharmaceutical preparations. They are listed in the following table
9.
Table 9
Colorimetric assays of Metoclopramide
N.
max
Bear's law Ref.

,ug/ml
--
Citric acid in Ac 0
600
2-14
27
NH4SCN t Co (N03)2
620
--
28
a , P-dinitrostilbene
390
--
29
DiazotizationtN(1-naphthyl)
525
0.4-4
30-11
ethylendiamine
5
HNO
Diazotization
to form the nitrous

2
derivative
375
10-60
31
+ a-Naphthyl-
510
0.1-11
32
500
1-14
33
475
20-120
34
495
1-11
35
NH Sulfamate
4
Diazotization +
440
0.8
36
amine
7
Diazotization
Dragendorff's
Diazotization
10
R-Salt
2,4-dihydroxybenzoic acid
352
6.6
Ultraviolet Spectrophotometric Analysis
B.P 1980 (10) has described a spectrophotometric

assay for the quantitative determination of metoclopramide
tablets. After the extraction of the base in chloroform, the
metoclopramide can be determined by measurement of absorbance at 305 nm. E(1%, lcm) = 265.
6.7
Fluorimetric Analysis
Fluorimetric measurements are carried out in pH=2

buffer solution on the basis of intense emission at 360 nm
that occurs when the sample is exci ed at 310 nm. The me-1
thod, with detection limit of 3x10 ,ug ml , was successfully applied to analyses of pharmaceutical formulations
with a recovery of 100.8-101.8% and a coefficient of variation of 0.9-1.9% (37).
-8
Chromatography
6.8
Several chromatographic assays are summarized in

the following table 10.
Table 10
Solvent system
4.6 g of citric acid
in 130 ml H 0 and
2
870 ml BuOH
Acetate buffer
pH = 4.58
Phosphate buffer
pH = 7.4
Toluene: Methanol :
conc. NH40H
Paper
Detection
U.V.
Iodoplatinate
spray
RfxlOO
45
II
77
II
39
1% of chloranyl in Bz
Ref.
353
Paper:
A
Whatman N 1, s h e e t 11 x 6 cm buffered by dipping i n a 5%
s o l u t i o n o f sodium c i t r a t e , b l o t t i n g and drying a t 25O
f o r 1 hour.
B
Whatman N 1 N 3, s h e e t 17x 19 cm impregnated by dipping
i n a 10% s o l u t i o n of t r i b u t y r i n i n acetone and drying i n
air.
C
Whatman N 1
6.9
Thin-Layer Chromatographx
The methods f o r s e p a r a t i o n and d e t e c t i o n o f metoclopramide a r e summirized i n t a b l e 11.
Table 11
Solvent system
I
I1
III
IV
V
VI
VII
VIII
IX
X
XI
XI1
Plate
A,B
C
C
C
C
C
C
F
C
C
F
R f x 100
40
10
46
16
54
29
55
98
68
47
13
Reference
DAVIDE PITRfi AND RICCARDO STRADI
354
Solvent Sytem
I Strong ammonia solution: methanol (1,5:100)
I1 Methanol-chloroform (1:4)
I11 1,2-Dichloroethane-ethanol-ammonia solution
(sp.gr.0.88) (70: 15:2)
IV n-Butanol-acetic acid-water (4:l:l)
V Isopropanol-ammonia solution (sp.gr. 0.88)
( 80:4:5)
VI
VII
VIII
IX
X
Butanol-acetic acid-water (4:l:l)

Isopropanol-NH OH-water (80:4:5)
4
Dioxan-NH OH-benzene (8:l:l)
4
Chloroform-methanol-28% ammonia (10:4:1)
Chloroform-methanol-dioxane-28% ammonia
(90:14:10:3)
XI Methanol-ammonia (100:1.5)
XI1 Acetone
Detection
Acidified iodoplatinate spray

- Spray solution: 1% solution sodium nitrite in HC1 N and
then with 0.4% of N-(1-naphthy1)-ethylaminediammonium dichloride as coupling agent in methanol.
- Spray solution: 0.2% solution of chloranil in acetonitrile followed by heating the plate at 100-105 for 2 min.
- Spray solution: 2 g of iodic acid dissolved in 10 m l H20
and made up to volume of 100 m l with 90% (w/v) sulfuric
acid (5).
- Ultraviolet light (250 nm).
Plate
A.
B.
C.
D.8
E.
F.
Glassplate, 20 x 20 cm coated with silica gel

Pre-coated silica gel G (Merck)
Silica1 gel 60 F
(Merck)
254
GF Plate. Analtech (6)
Kodak Silice Fluorescente K301
Silica dipped in 0.1N KOH and dried
METOCLOPRAMIDEHYDROCHLORIDE
6.10
355
High Performance Thin-Layer Chromatography
HPTLC was performed on metoclopramide base using

NH OH
HPTLC Fertiplatten Kiegelgel 6OF(Merck)and Me0H:CHCl
3:
4
25% (80:85:0.2) as solvent. The compound may be quantified
by means of excitation fluorimetry at 312 nm and emission at
365 nm integrating the peak area with a videointegrator. The
results are linear from 160 ug to 20 ug with a standard
/
/
deviation of 2-5% (43).
6.11
Gas Liquid Chromatography
T. Daldrup and co-workers (44) described a GLC

assay method on a glass-column (6 ft x 2 inc.) packed with
3% OV-1 on Chromosorb W-HP (100 to 120 mesh) and operated at
-1
15Oo-25O0 (lOC/min), with N as carrier gas (50 ml min )
2
and a flame ionization detector. Internal standard: 2-amino-5-chlorobenzophenone.
Detector and injection temperature
were 350 and 250OC. Retention time: 1.87.
GLC has been used as the method for the determination of the
drug and its 15 metabolites in biological fluids (45) (46)
(47) (48)and Y.K. Tamond, J . E . Axelson (49)have been determined the mctoclopramide in picogram quantities in plasma
63
with a
Ni-electron capture detector. The procedure involved the extraction of the drug from an alkalinized aqueous
solution in benzene and derivatization with heptafluorobutirric anhydride.
The determination of the derivative was done using diazepam
as internal standard. Working conditions: glass column (1.2
m x 2 inc. packed 3% OV-17, coated with 80-160 mesh, chromosorb W. Injection 2 5 8 O ; oven 250 and detection 350.
A mixture of argon-methane (95:5) was employed with a flow
-1
rate of 40 ml/min
The retention times were 3.76 min for
the metoclopramide and 9.1 min for diazepam.
356
6.12
High-pressure Liquid Chromatography
-?
Teng et al.(l) used a column 15 cm x .5 mm packed with silica gel M 171; flow rate 2.0 ml/min and CH OH3
-CHC1 -NH OH conc. (30:70:0.5) at room temperature. Detect4
ion 280 nm. The retention times of metoclopramide is 2.8 min
and 4.5 min for the internal standard (4-amino-5-chloro-ND- ( propylamino ethyl]-Z-me
thoxybenz m i de )
W. Block et a1 ( 5 0 ) determined metoclopramide using reverse-phase HPLC with an RP-18 column under isocratic conditi n
-4.
(CH 0H:H 0:NH OH conc.,75:24.9:0.1). Flow rate 1.2 ml/min
3
2
4
Detection at 308 nm. Elution after 5 min.
7.
Determination of metoclopramide in Body Fluids and

Tissues
The determination of metoclopramide in biological

fluids of man and of various animal species is the object of
many communications as reported below:
Blood (Plasma, Serum)
Colorimetry: (11)
: ( 3 ) (41) (40) (52)
HPLC: (24)
TLC
GC
: (45) (46) (47) (53) (48)
HPLC
: (54) (55) (20) (50) (53) (56) (57)
GC-Mass Spectrometry: (15)
Urine
Colorimetry
: (21) (52)
TLC
: (21) (40) (39)
HPCL
: (20) (56)
GC
: (54) (19)
GLC-Mass Spectrometry: (58)
Saliva
HPLC : ( 59)
Bile
Colorimetry : (21)
Feces
Radioactivity with 14C-metoclopramide : (20)
Microsomal fraction of liver homogenate
HPLC : (39)
357
DAVIDE PITRE AND RICCARDO STRADI
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Y.K. Tam, J.E. Axelson
J .Pharm.Sci. 67, 1073 (1978)
R.P. Kapil, J.E. Axelson, R. Ongley, J.D.E. Price
J.Pharm.Sci. 2, 215 (1983)
C. Bugatti. Faculty of Pharmacy, University of Milan,
Personal Communication
42
MITOMYCIN C
J o s H. Beijnen
Slotervaart HospitalfNetherlands Cancer Institute
Amsterdam, The Netherlands
Auke Bult and Willy J.M. Underberg

Faculty of Pharmacy, Department of Pharmaceutical Analysis
State University of Utrecht
Utrecht, The Netherlands
1.
2.
3.
4.
5.
6.
7.
8.
Historv
Description
2.1 Name, Formula, Molecular Weight
2.2 Appearance, Colour, Odour
Biosynthesis, Chemical Synthesis
Physical Properties
4 . 1 Ultraviolet-Visible Spectrum
4 . 2 Infrared Spectrum
4.3 Nuclear Magnetic Resonance (NMR) Spectrum
4.4 Mass Spectrum
4.5 Circular Dichroism (CD) Spectrum
4.7 Melting Point
4.8 Differential Scanning Calorimetry
4 . 9 Solubility
4.10 Partition Coefficient
4.12 Electrochemistry
Methods of Analysis
5 . 1 Elemental Analysis
5.2 Ultraviolet Spectrophotometry
5 . 3 Thin Layer and Paper Chromatography
5 . 4 Gel Filtration Chromatography
5.5 High Performance Liquid Chromatography
5.6
Identification and Purity Tests in Official
Compendia
Stability, Degradation
6.1 Stability in Aqueous Solutions
6.2 Stability in Pharmaceutical Formulations
6 . 3 Stability in Biological Media
Pharmacology
7 . 1 Mechanism of Action
7 . 2 Clinical Antiturnour Activity
7 . 3 Cljnical Toxicity
7 . 4 Pharmacokinetics
Determination in Body Fluids
References

VOLUME 16
361
Copyright 0 1987 by Academic Press,Inc.

JOSH. BEIJNEN ETAL.
362
1.
HISTORY
In 1956 Hata and coworkers (1) first reported about the

isolation of a new group of antitumour antibiotics which they
named mitomycins. These pigmented compounds were obtained by
extraction and chromatographic purification of the fermentation broth filtrate of Streptomyces caespitosus. In 1958 another Japanese group, led by Wakaki, succeeded in the isolation of a mitomycin derivative, called mitomycin C, from the
same actinomyces strain (2). Several mitomycin derivatives
have since been isolated from Streptomyces species (3-8). Extensive chemical degradation studies, X-ray diffraction crystallography, chromatographic and spectroscopic analysis and
biosynthesis studies have contributed to the elucidation of
the structure and absolute stereochemistry of mitomycin C and
related mitomycins (9-23a). Mitomycin C was selected for further development after it had been established that mitomycin
C possessed profound antineoplastic activity and that the
therapeutic index was more favourable than that of related
mitomycin congeners. The drug has activity against an array
of solid tumours such as adenocarcinomas of the gastrointestinal tract. Unfortunately, clinical experience with mitomycin C has shown that it causes severe toxicity in particular
a delayed, cumulative bone marrow suppression, Mitomycin C is
rarely used as a single agent except for the treatment of superficial cancer of the urinary bladder, intravesically administered. This administration mode has been shown to be very
effective and devoid of serious haematological toxicities. For
more specific information about the clinical usefulness of
mitomycin C combinations in lung, breast and stomach cancer
the reader is referred to review articles and books ( 2 4 - 3 1 ) .
2.
DESCRIPTION
2.1 Name, Formula, Molecular Weight

The generic name is mitomycin C (50-07-7).
The drug is
marketed under the trade names of MutamycinB, Mitomycin-C
The molecular formula for mitomyKyowa@ and Ametycine@
cin C is CI5Hl8N4O5, The molecular weight is 334.13.
MITOMYCIN C
363
2.2 Appearance, Colour, Odour

Blue-vi.olet crystalline powder which is o d o u r l e s s .
3.
BIOSYNTHESIS, CHEMICAL SYNTHESIS
By using 13C, 14C, 3H and 15N-labeled precursors in feeding experiments, investigators have studied the biosynthetic
pathways of mitomycins (20, 21, 32-35). Although considerable
progress has been made in elucidating the biosynthesis routes
the exact pathway remains to be established. Feeding studies
have revealed that D-glucosamine, L-methionine, L-citrulline,
L-arginine, pyruvate and D-erythrose are candidates as biosynthetic precursors of the mitomycines (21). The total chemical synthesis of mitomycin C has been accomplished from
2,6-dimethoxytoluene as starting material by Kishi and collaborators (36). The synthesis route is lengthy (47 steps) and
complex.
4.
PHYSICAL PROPERTIES
4.1 Ultraviolet-Visible Spectrum

The ultraviolet spectrum of mitomycin C ( ~ x ~ O - ~ Min)
water is depicted in Figure 1. The spectrum was recorded by
using a Shimadzu UV-200 double beam spectrophotometer in 1 cm
quartz cell. In the visible region a broad band of low intensity is present. At pH values over 10 the absorption maximum at 360 nm shifts to 295 nm due to keto-enol tautomerization and deprotonation of the 7-amino quinone part of the
mitomyzin C molecule (Figure 2)(37).
Molar absorptivities (E)
and A1 cm values are reported in Table I (9).
Table I
360
555
UV-VIS Spectral Data for Mitomycin C in Methanol(9)
689
6.3
23,000
209
364
JOS H.BEIJNEN ETAL.
400
MMC
01 absorbance
300
250
wavelength I nrn)
350
200
MMC-
Figure 2.
Keto-enol tautomerization and deprotonation of mitomycin C
(MMC)
MITOMYCIN C
365
4.2
Infrared Spectrum
The infrared spectrum of mitomycin C in a potassium bromide pellet is presented in Figure 3 . The spectrum was recorded with a Jouan-Jasco IRA-grating infrared spectrophotometer. Assignments o f a number of prominent bands is listed in
Table 11.
Table I1
Infrared Assignments for Mitomycin C

-1
wavenumber (cm )
assignment
346OS, 331OS, 3260'
V(NH) and
1735'
carbarnate and 7C-NH2

V(C=O) of carbamate function
16OOs, 1558'
V(C=O)
of quinone
(NH2) of aziridine,
'"1
Figure 3 .
Infrared spectrum of mitomycin C
JOSH. BEIJNEN ETAL.
366
4.3 Nuclear Magnetic Resonance fNMR) Spectrum

The proton NMR spectrum was recorded in pyridine-d containing tetramethylsilane as internal reference and wi ?h the
use of a Bruker WM-300 spectrometer a t frequency 300.13 MHz
(37a). The spectrum (detail) is presented in Figure 4 and refers to the situation after proton exchange. The spectral
assignments are presented in Table 111 (in pyridine-d ) and
are derived from L o w and Begleiter ( 3 8 ) . Irrigularitges in
intensity comparison of the signals make the assignments
tentative. Proton exchange with deuterium oxide results in
disappearance of NH, NH and H-1 signals, while the H-3, H-9,
2
H-10 and H-10' signals shift 0.10 to 0.20 to lower ppm
values (spectrum F i g . 4 ) .
Table I11
'H NMR Assignments for Mitomycin C in pyridine-d5

~
~~~~
Chemical shift
6 (ppm)
Multiplicity
Number of
atoms
Assignment
2.00
singlet
CH3 (C-6)
2.10
triplet
NH (Cl-C2)
2.72
quartet
H-2
H-1
3.13
3.19
singlet
OCH3 ( C - 9 )
3.57
doublet
H-3 '
4.02
quartet
H-9
4.53
doublet
H- 3
5.11
triplet
H-10
5.43
quartet
H-10'
7.6
broad
NH2(C-7)
and CONH2
The natural abundance carbon 13 NMR spectrum was recorded under the same experimental conditions except that the
frequency was 75.46 MHz (37a). The proton-noise decoupled
spectrum is presented in Figure 5 and the spectral assignments are summarized in Table IV. The assignments of Keller
and Hornemann are followed ( 3 9 ) .
MITOMYCIN C
367
5.0
4 .O
. . , .1
. . . . , . . . . , . . . .
3.O
chemical shift (ppm)
Figure 4 .
Proton NMR spectrum of mitomycin C (detail)
2.0
368
JOSH.BEIJNEN ETAL.
180
160
140
120
100
80
chemlcal shift (ppm)
Figure 5.
I3c NMR spectrum of mitomycin
60
40
20
MITOMYCIN C
369
13C NMR Assignments for Mitomycin C in pyridine-d 5
Table IV
Chemical shift 6(ppm)

~
Ass-ignment
~~
CH3 (C-6)
8.8
32.6
c-2
36.7
c-1
44.2
c-9
49.5
OCH3 (C-9a)
50.6
c-3
62.5
c-10
104.3
C-6
106.8
C-9a
110.7
C-8a
149.6a
c-7
156.1
C-5a
158.1
C-lOa
176.7
C-8
c-5
178.3
~~~
~~
obscured by solvent
4.4 Mass Spectrum

Electron impact (EI) mass spectroscopy of mitomycins,
including mitomycin C, has been investigated in detail by
Van Lear (40). His observations were confirmed in later studies (41, 42). For this analytical profile electron impact
(EI), chemical ionization (CI) , field desorption (FD) and
fast atom bombardment (FAB) in the positive and negative ion
mode, spectra of mitomycin C (lot nr. 010783) were recorded.
EI mass spectrometry was performed by direct probe analysis
on a Kratos MS-80 mass spectrometer. Source conditions were:
200 OC source temperature, 70 eV electron energy and 100 PA
ionising current. Direct insertion probe CI mass spectra
were obtained by using a Finnigan 3200 quadrupole mass spectrometer combined with a Finnigan 6000 data system. Methane
was used as rhe ionising gas. The emission current was 0.20
mA and the multiplier voltage 1800 V. FD mass spectra were
obtained with a Varian MAT 711 double focussing mass spectrometer equipped with a MAT 100 data acquisition unit. 10 p m
tungsten wire FD emitters containing carbon microneedles
JOSH. BEIJNEN ETAL.
370
Figure 6.
EI mass spectrum of mitomycin C
.360
242
292
3ab
Figure 7.
CI mass spectrum of mitomycin C
MITOMYCIN C
371
with an average length of 30 p m were used. The mitomycin C

sample was dissolved in methanol and then loaded onto the
emitters by the dipping technique. An emission current of
12 mA was used to desorb the sample. The ion source temperature was 70 'C. FAB mass spectrometry was carried out using
a V.G. micromass ZAB-2F mass spectrometer, an instrument with
reverse geometry and fitted with a high field magnet and
coupled to a V.G. 11-250 data system. The sample was loaded
in thioglycerol solution onto a stainless steel probe and
bombarded with Xenon atoms having a 8 keV energy. The recorded spectra are depicted in Figures 6, 7, 8, 9 and 10 and
the major ion assignments are given in Table V.
A high resolution mass spectrum exhibited the molecular ion
at m/z 334.1280 with composition C15H18N405 (calculated mass
334.1277).
Figure 8.
FD mass spectrum of mitomycin
3 72
JOSH. BEIJNEN ETAL.
37
28
18
388
488.
98.
88
18.
68.
2r
58.
18.
38.
21s
I88
288
388
480
Figure 9/10.
FAB(+) mass spectrum (upper spectrum) and FAB(-) mass spectrum (lower spectrum) of mitomycin C
MITOMYCIN C
Table V
373
Mass Spectral Data for Mitomycin C

m/z
assignment
FD
334
335
M+*
(M+H)+
FAB (+)
-
358
(M+H+Na)
357
(M+Na )
336
(M+2 H)+
335
274
(M+H)
( (M+H)-02CNH3)
242
( ( (M+H) -H~COH)
-O~CNH~)
m/z 57, 91 and 215 signals come from the thioglycerol matrix
FAB (-)
334
333
M-'
29 1
(M-CONH) -
(M-H)-
m/z 107, 215 and 321 signals come from the thioglycerol matrix
m/z 139 and 265 signals cannot be assigned with certainty
CI
-
363
335
302
274
EI
-
(M-H3COH)+
242
(M-02CNH2)
( (M-H3COH) -02CNH2)+
334
M+
302
(M-H~COH)+
291
(M-OCWH)
273
259
(M-0
242
( (M-H3COH) -02CNH2)
CNH3)
( (M-02CNH2)-CH3)
JOSH. BEIJNEN ETAL.
374
4.5 Circular Dichroism (CD) Spectrum

The chiral centers at C1, C2, C9a and C9 in the mitomycin C molecule are responsible for the CD Cotton effects at
355 nm and 395 nm. The CD spectrum of mitomycin C in a methanolic solution (c=O.OOl%), determined by using a Jobin Yvon
Dichrograph 111, is shown in Figure 11.
+A c
F i g u r e 11.
CD spectrum of mitomycin C
MITOMYCIN C
375
4 . 6 Optical Rotation
The optical rotation
of mitomycin C in methanol
(c-0.1%) was determined to be + 2 3 2 " . This analysis was performed with a Thorn NPL Automatic Polarimeter Type 243 at 589
nm. The optical rotatory dispersion (Om)spectrum of mitomycin C has been reported by Hornemann et al. ( 4 3 ) .
4.7
Melting Point
In the literature no melting point for mitomycin C is
mentioned except for the statement that it should be over
300 " C (11).
4.8
Differential Scanning Calorimetry

In nitrogen atmosphere no transitions were recorded up
to 330 OC in a closed (air tied) sample holder. In an open
sample holder an undefined large exothermic decomposition
takes place from 215 "C. The applied apparatus was a Feteram
111, the heating rate was 5 K/min and the sample size was 3
mg ( 4 4 ) .
4.9 Solubility
Mitomycin C is readily soluble in methanol, acetonitrile
normale saline and water, but is nearly insoluble in hydrocarbon solutions, The following solubility data have been
reported ( 4 5 ) , determined by suspending an excess amount of
mitomycin C in a solvent, followed by filtration and HPLC
analysis.
Table VI
Solubilities of Mitomycin C
solvent
solubility (mM)
water
sesame oil
isopropylmyristate
hexane
2.73
temperature ("C)
~
0.0180
0.0193
0.0234~10-~
25
25
37
25
JOS H.BEIJNEN ETAL.
376
4.10 Partition Coefficient

The partition coefficient for mitomycin C has been measured in various organic phaseldistilled water systems. The
results have been combined in Table VII.
Table V I I
Partition Coefficients for Mitomycin C
solvent
chloroform
n-octanol
ethyl acetate
1-pent an1
chloroform/2-propano1
chloroform/2-propanol
chloroform/l-pentanol
chloroform/l -pentan01
partition coefficient
0.26; 0.27,
0.41; 0.42
(90+10)
(50+50)
(90+10)
(80+20)
dichloromethane/2-propanol (50+50)
reference
0.51
2.32
0.50
1.52
1.12
1.85
1.04
the aqueous phase was 0.07M phosphate buffer pH
7.4
The organic solvents chloroform (1,2) and ethyl acetate (7,

48) have been used with success or the isolation of mitomycins from the aqueous fermentation broths of Streptomyces
species.

The mitomycin C molecule contains several prototropic
functions. Basic groups are the 7-amino group, the N-4 nitrogen and the aziridine nitrogen. Due to rapid degradation the
pKa values of the conjugated acid concerning the 7-amino
group and N-4 nitrogen can not be deduced from titration experiments with intact mitomycin C. Therefore the acid dissociation constants for these unctions were derived from titrations with stable analogs (37). Results are listed in
Table VIIT. The pKa of the aziridine nitrogen has been determined titrimetrically (11) and kinetically from the pH-rate
re1.ationship.s in degradation studies (49-51) although the
titrimetrical determination also has been influenced by degradation. Mitomycin C also has acidic properties with an
(apparent) pKa 12.44 at room temperature. These acidic properties emerge after keto-enol tautomerization of the 7-amino
quinoid moiety which precedes deprotonation in alkaline medium (Figure 2)(37).
MITOMYCIN C
Table V I I I
377
Prototropic Functions of Mitomycin C

pKa(25"C)
function
determination method
N-4
7-amino
aziridine
-1.2
spectrophotometry
spectrophotometry
-1.3
potentiometric titration 3.2
via pH-rate profile
2.8
2
via pH-rate profile
via pH-rate profile
2.74
via pH-rate profile
2.50(49
spectrophotometry
12.44
7-amino
reference
4 . 1 2 Electrochemistry
Due to the presence of the quinone ring of the mitomycin
C molecule the compound can be reduced and transformed into
its hydroquinone form. This conversion triggers a complicated
pattern of chemical consecutive reactions. Although much progress has been made in the elucidation of the processes occurring during and following electrochemical reduction of mitomycin C the exact complicated mechanisms have not yet been
unravelled (50). This is due to:
rapid degradation of mitomycin C at p H < 3 and p H > 1 2 ;
the extremely high reactivity of the hydroquinone form
of mitomycin C;
the fact that chemical degradation products as well as
electrochemically generated degradation products of mitomycin C can be reduced within the region of reduction
potentials of mitomycin C itself ( 5 0 ) .
Electrochemistry of mitomycin C has been studied in aqueous
solutions (50, 52-54) and aprotic solvents ( 5 5 ) , by using
polarography ( 5 0 , 52-54) and cyclic voltammetry ( 5 0 , 5 2 , 5 3 ,
5 5 ) . Direct current polarographic curves for mitompcin C are
shown in Figure 1 2 . Rapid degradation of mltomycin C within
the time scale of recording a direct current polarographic
curve at p H < 3 and pH > 1 2 strongly influences the shape and
heights of these curves ( 5 0 ) .
378
JOS H.BEIJNENETAL.
PH = S O
-500
-1000
-1500
potential (mV)
Figure 12.
-4
Direct current polarographic curves of mitomycin C (10 M)
MITOMYCIN C
5.
379
METHODS OF ANALYSIS

The elemental analysis of a purified mitomycin C sample
for C, H, N and 0 was reported to be as follows:
Table IX
El-emental Analysis of Mltomycin C (11)
element
% theory
% found
C
H
53.83
5.43
16.76
23.98
53.55
5.57
16.86
24.13
5.2 Ultraviolet Spectrophotometry

Mitomycin C has been determined by using UV spectrophotometry at 360 nm &n methan-qjl solution. This assay is linear
in the range 2x10- to 4x10 M y with a precision of ? 1%.
5.3
Thin Layer and Paper Chromatography
A diverse array of thin layer chromatographic systems
have been described in the literature for analysing mitomycin

C and derivatives (55). Some representative systems have been
summarized in Table X.
The mitomycin C spot can be located by:
irradiation with UV light of 254 nm;
examination under daylight (mitomycin C has a blue-violet colour) ;
spraying with a 1 in 100 solution of ninhydrin in alcohol, followed by heating the plate at 110C for 15 minutes by which mitomycin C appears as a pink spot (56).
Table X
Thin Layer and Paper Chromatography of Mitomycin C
plate
solvent (v/v)
Rf
silicagel
butanol (sat. with water)

ethyl acetate (sat. with water)
methanol-benzene-water (10:5:5)
isopropylalcohol-1ZNH OH (2:l)
4
methano 1
0.57
~_________
1-octanol-acetone-ligroin
(9O-115C) (2:5:5)
chloroform-methanol (9: 1)
cellulose isobutyric acid-0.5M NH40H (10:6)
paper
butanol-water (84:16)
methanol-benzene-water (1:1:2)
ethyl acetate (sat. with water)
reference
0.02
0.88
0.80
(12)
(12)
(12)
(57)
(57)
0.11
0.12
0.75
0.57
0.02
0.56
(58)
(59)
(57)
(60)
(60)
(60)
0.56
380
JOS H. BEIJNENETAL.
5 . 4 Cel Filtration Chromatography

For the separation of mitomycin C from its degradation
products and alkylation adducts Tomasz et a1 (57, 6 1 - 6 4 )
utilized pel filtration chromatography on Sephadex G-25 columns and 0 . 0 2 M NH HCO solution as eluent. By using a 1 . 5 ~
4
3
4 1 . 5 cm Sephadex G-25 (fine) column the elution volume of
mitomycin C is 96 m l ( 5 7 ) .
5.5 High Performance Liquid Chromatography
HPLC procedures reported in the literature have been developed mainly for the analysis of mitomycin C and derivatives in decompositinn mixtures ( 5 1 , 5 9 , 6 5 - 7 1 ) in biological
matrices (plasma, urine, bile, tissues) ( 4 6 , 7 2 - 8 6 ) and other
systems such a s (enzyme containing) culture media ( 6 2 , 6 9 ,
87-90). Detection by W absorbance measurements at 365 nm is
usually preferred ( 5 5 , 7 5 , 7 6 ) because of the high specificity and sensitivity, but detection at 254 nm, which is less
sensitive, is also reported ( 6 6 , 91, 92). The polarographic
electrochemical detection mode is an elegant alternative for
the detection of mitomycin C ( 5 0 , 75, 93), but demands extensive experimental precautions to be taken, such as removal of
oxvgen from the mobile phase and damping of pulsating flow of
the solvent delivery system ( 9 3 ) . Important HPLC systems
have been enumerated in Table XI,
Table XI
High Performance Liquid Chromatography for Mitomycin C
~~
~~
column
mobile phase
p-Bondapak C 1 8 (10 ,pm)

p-Bondapak C18 (10 pm)
Lichrosorb RP18 (10 pm)
Lichrosorh RP8 (5 p'm)
Ultrasphere ODX
Hypersil-MOS (5 pm)
Hewlett-Packard C8 (10 pm)
Radial Pak C18 (10 pm)
Reversed Phase HPLC

methanol-water (35: 65)
methanol-0.01 M phosphate buffer (pH 6,0)(30:7O;w/w)
methanol-water (30:70;w/w) + 0.5% (v/w) phosphate buffer pH 7.0
methanol-water-phosphate buffer pH 7.0 (20:70:10)
acetonitrile-0.03M potassium phosphate buffer (12.5:87.5)
acetonitrile-0.05M phosphate buffer pH 7 (10:90)
acetonitrile-water; gradient from 5% to 41% acetonitrile
methanol-0.01M phosphate buffer pH 7; gradient from 0% to 50% methanol
Lichrosorb RP18 (10 pm)

Lichrosorb RP18 ( l o p )
Hypersil ODS (5 pm)
Hypersil ODS (5 pm)
Resolve C18 (5 pm)
Porasil P (10 pm)

Porasil (10 pm)
Silica Si-60
a
~~
reference
(77-80)
(46)
(67)
(82)
(91)
(75)
(94)
(42)
Ion Pair HPLC

(65)
methanol-water (40:63;w/w) + 0.5X (v/w) glacial acetic acid (~H*=4.3)~
methanol-water (30:70;w/w) + 0.5% (v/w) phosphate buffer pH 7.0
+ 0.5% (v/w) tetrabutylammoniumbromide solution (20%, w/w)
(59)
acetonitrile-water (25:75) + 0.2% sodium laurylsulphate + 0.025M
(66)
sodium acetate
acetonitrile-water (20:80) + 0.2% cetrimide + 0.0125M sodium acetate
(66)
methanol-1 mM octanesulfonicacid sodium salt solution (30:7O;v/v)
(pH* 4.5)a
(85)
Normal Phase HPLC

tetrahydrofuran-methanol-isooctane-dichloro~t~ane (3:7:15:75)
chloroform-methanol (9:1)
ethyl acetate-methanol (95:l)
ethyl acetate-methanol-water-dichloromethane (97:2:1:1)
pH values of organic modifier-water mixtures
382
JOS H.BEIJNEN ETAL.
5.6
Identification and Purity Tests in Official Compendia

In the United States Pharmacopeia XXI (56) the monographs "Mitomycin" and "Mitomycin for Injection" have been
included. In these monographs the identification of mitomycin
C is based on IR and UV spectroscopy and chromatography by
which the sample should exhibit the same spectroscopic and
chromatographic properties as a similar preparation of a USP
Mitamycin Reference Standard. The pH of a solution containing
5 mg/ml should be between 6.0 and 8.0. The water content of
the raw material may not exceed 5%. Furthermore Mltomycin and
Mitomycin for Injection (a dry mixture of mitomycin C and
mannitol) must meet the requirements of specific tests described in the USP XXI such as: Safety Tests, Depressor Substances Tests, Pyrogen Tests and Sterility Tests (56).
6.
STABILITY, DEGRADATION
6.1 Stability in Aqueous Solutions

Mitomycin C and related mitomycins are not stable when
stored as aqueous solutions (11-13, 51, 55, 57, 59, 65-68,
82, 96-108). Degradation is accelerated by acid, alkali,
(buffer) ions and high temperatures (108). At p H < 7 mitomycin
and at
C is converted into l-hydroxy-2,7-diaminomitosenes
pH > 7 it is hydrolysed into 7-hydroxymitosane. The overall
degradation scheme is given in Figure 13. In the mitosene
compounds the C9a methoxy group has been cleaved, a double
bond between C 9 and C9a has been introduced and the aziridine
moiety has been opened. Aziridine ring opening occurs with
configurational retention at C2 and water attachment at C1
with inversion as well. as retention of the C1 stereochemistry
yielding 1,2-trans- and 1,2-cis-l-hgdroxy-2,7-diaminomitosenes, respectively. Under drastic acidic conditions progressive degradation reactions lead to hydrolysis of the 7-amino
group and the C10 carbamate side chain (9-11, 1 3 ) . The mechanism of the initial degradation step of mitomycin C in acid
is shown in Figure 14 (107, 108). In the cascade of reactions
an intermediate is proposed, having a positive charge at C1
(49, 107-110). This electrophilic center serves as target for
attacking (nucleophilic) water molecules resulting in the
formation o f l-hydroxv-2,7-diaminomitosenes. The degree of
protonation of the 2-amino function in the proposed intermediate species (pKa:2.8(65))
determines the C1 stereochemistry
of the resulting mitosenes. A protonated 2-amino function may
direct incoming water molecules to a cis configuration. Whenever the amino function in the intermediate is not protonated
the directing electrostatic power is absent and a reacting
water molecule may approach the C1 carbonium ion from both
MITOMYCIN C
383
sides with roughly equal chances. This explains the remarkable influence of pH on the C 1 stereochemistry of the resulting mitosenes. At pH=l the cisltrans ratio is about 4 while
at pH=6 the ratio approaches 1 (65). When mitomycin C degrades in acidic medium in the presence o f nucleophiles such as
acetate, phosphate or ever! DNA parts, l-nucleophile-2,7-diaminomitosenes are generated (57, 103, 111-114). These are
important observations which lend credibility to the hypothesis that acid activation may play a role in the in vivo mitomycin C DNA alkylation.
In alkaline solution (pH 7 to 13) the 7-amino group of
the mitomycin C molecule is substituted by a hydroxy function
(59, 100-102). Under prolonged severe alkaline treatment the
quinone chromophore is destroyed ( 5 9 ) .
In huffered media the degradation kinetics o f mitomycin
C can be described adequately by (pseudo) first order kinetics. Some kinetic data are summarized in Table X I I . For further detailed kinetic information is referred to the literature (49, 51, 55, 67, 107, 108).
+
i,i \
cis- mitosene
mitomycin C
OH-
7 - hydroxymitosane
Figure 13.
Overall degradation scheme of mitomvcin C
trans -mitosene
NH,
384
JOSH. BEIJNEN ETAL.
.."
L
0
II
c
N-H
Figure 14.
Acid degradation mechanism of rnitomycin C
+ CH.OH
MITOMYCIN C
Table XI1
385
Degradation Kinetics for Mitomycin C
conditions/method
pH 0.87;
perchloric acid solution;

2OoC/ HPLC
pH 2.86; 0.001M acetate buffer;
20C/ HPLC
1.OM phosphate buffer;
pH 3.0;
25'C/ HPLC
pH 4.64; 0.1M phosphate buffer;
40C/ spectrophotometry
pH 7.4;
0.1M phosphate buffer;
37'C/ HPLC
37"C/ spectrophotometry
25OC/ HPLC
kobs ( s - l )
1.M
O-~
reference
(65)
4.Ox
(65)
1.2~10-~
(114)
1.4~10-~
(107)
4.1~1O-~
(105)
1.8~10-~
(104)
5.3~
(67)
6.2 Stability in Pharmaceutical Formulations

Mitomycin C is commercially available in a lyophilized
form containing mannitol (Mutamycin@ : 5 mg mitomycin C +
10 mg Mannitol) or sodium chloride (Mitomycin C-Kyowam :10 mg
mitomycin C + 240 mg sodium chloride; Ametycine lo@ : 10 mg
mitomycin C + 240 mg sodium chloride) as excipients. In this
state the drug is stable for at least two years if kept dry
and in well closed containers at room temperature in the dark
(115). After reconstitution the drug has only limited stability, depending strongly on the pH of the final solution (68).
An overview of stability data of mitomycin C in infusion
fluids is given in Table XIII. More specific and extended
data are described in the references concerned (68, 70, 71,
116-120).
Table XI11
Stability of Mitomycin C in Infusion Fluids at Room Temperature and - 3 O O C
infusion fluid
concentration
stability
reference
5% dextrose
50
pglml
tgO = 3 . 0 h; t50
0.9% sodium chloride
pg/ml
mg/ml
93% remained after 5 days
sterile water
50
2
5% dextrose
50
pg/ml
26% remained after 12 hours
50
pglml
t50
phosphate buffer pH 7.75
50 pg/ml
0.4 mg/ml
~~
5% dextrose
78 h
stable for 7 days

= 12
tgO
=
tgO =
15 days
1 h
0 . 4 mg/ml
0.6 mg/ml
99% remained after 4 weeks storage at -30C
90
=24h
MITOMYCIN C
387
Further research is required to resolve the problem of

conflicting stability data on mitomycin C in 0.9% sodium
chloride solution (68, 70, 116, 119, 120).
Stability in Biological Media
6.3
Chemical instability of mitomycin C in biological fluids
and tissues may be a complicating factor in the bioanalysis
of the drug. Ignorance of the chemical stability of the drug
during sampling and sample pre-treatment procedures may lead
the researcher to misinterpretations. Recently, den Hartigh
et al. (86) published an extehsive study concerning the handling of mitomycin C biological samples. The following recommendations and guidelines were given by these authors (86):
- whole blood samples should be placed in ice immediately
after sampling; separation of plasma and red blood cells
should take place within half an hour after collection
of the samples;
- mitomycin C samples should be protected from long-term
exposure to daylight in order to prevent analyte degradation;
the pH of urine samples should always be checked and, if
necessary, adjusted to neutral values, as a low pH will
induce mitomycin C decomposition and the formation of
precipitates upon which mitomycin C may be adsorbed:
- plasma and urine samples need not necessarily be placed
in the refrigerator or freezer immediately after preparation, provided analysis is carried out within a period
of 2 h;
- plasma and urine samples may be stored in a freezer at
-2OOC; however, analysis should take place within 3 weeks
as longer storage at this temperature may induce considerable reduction of the mitomycin C concentration;
- repeated freezing and thawing of plasma and urine samples does not influence the analytical results of the mitomycin C assay;
- extracts of biological samples may be kept for at least
24 h, in the dark, either dry or dissolved in methanol
at 4 C or 20C prior to analysis.
The in vitro half life of mitomycin C was determined to be
> 14 days under conditions of the clonogenic assay ( 21). Mitomycin C is fairly stable (70-802 of the drug remained) when
incubated for 5 days at 37C in Medium E, RPMI, Fischer's or
MEM culture media, supplemented with serum (69). In Mark's
M-20 culture medium with fetal calf serum, Proctor and Gaulden (90) observed that the amount of mitomycin C was reduced
by 2 9 % after 30 minutes incubation.
388
7.
JOS H. BEIJNENETAL.
PHARMACOLOGY
7.1 Mechanism of Action

Mitomycin C induced cytotoxicity is believed to be mediated by DNA binding. The drug as such, in its oxidized form,
is not cytotoxic although some interaction with nucleic acids
may exist (122). Mitomycin C must be activated before it is
capable of alkylating DNA (123-128). The alkylating capacity
is unmasked by reductive or acidic activation. An outline of
the reductive activation pathway is given in Figure 15. Reduction can be mediated by enzyme systems (87-89, 129-132) or
by chemical reducing agents (58, 126). The conversion of mitomycin C into its reduced form destabilizes the compound
drastically and almost instantaneously the C9a methoxy group
is lost (Figure 15). A subsequent rearrangement in species C
leads to the formation of the quinone methide D which acts as
target for nucleophilic nucleic acid attack, by which a monofu ctional alkylation adduct is formed. An intramolecular
9
SN -displacement of the carbamate moiety leads to the bifunctional cross-linked adduct F (Figure 15) (128). The interaction of mitomycin C with DNA is more than 9OX of the monofunctional type. Chemical structures of monofunctionally linked mitomycin C adducts with DNA, nucleotides and nucleosides
have been reported (62-64, 133-136). Reduction of mitomycin C
may follow either one or two electron reduction steps. The
semiquinone radical is also believed to be capable of producing interstrand cross-links between complimentary strands
of DNA (87, 137). Under aerobic conditions, the semiquinone
radical formed after a one electron reduction can transfer
its electron to oxygen to generate superoxide anion under
regeneration of the oxidized quinone (138-142). Reactive oxygen species can cause DNA strand scission and membrane lipid
peroxidation, leading to cell damage and can contribute in
this manner to mitomycin C induced cytotoxicity. In the absence of any reducing agent or enzymes mitomycin C is also
capable of coupling covalently with DNA or nucleic acid
parts, but then, activation by acid is a requisite (57, 103,
111-113, 143, 144). Acid catalyzed degradation of mitomycin C
(Figure 14) occurs through reaction intermediates possessing
an electrophilic C1 center which is the target for nucleophilic nucleic acid attack.
More studies are necessary in order to elucidate which
of the proposed mechanisms or combination is actually responsible for mitomycin C induced cytotoxicity in vivo.
MITOMYCIN C
389
OH
OH
Figure 15.
Reductive activation mechanism of mitomycin C
390
JOSH. BEIJNEN ETAL.
7.2 Clinical Antitumour Activity

Single agent activity of mitomycin C has been demonstrated against various solid neoplasms in humans such as breast
cancer (29, 145) , stomach cancer (146), pancreatic cancer
(147), colorectal cancer (29), non-small cell lung cancer
(148) , squamous cell carcinoma of the uterine cervix (28)
and head and neck cancer (28). Encouraging results have been
obtained on the treatment of superficial transitional cell
carcinoma of the bladder when mitomycin C is administered
intravesically (28-30, ,l49). Mitomycin C is usually used in
combination with other cytostatics (28-30).
7.3 Clinical Toxicity
Delayed cumulative myelosuppression is the most important toxicity. Others include nausea, vomiting, anorexia,
diarrhea, stomatits, skin rashes and alopecia (24, 28, 150).
Extravasation of mitomycin C causes severe cellulitis accompanied with chronic pain and ulceration (151). Interstitial
pneumonitis, pulmonary fibrosis, hemolytic-uremic syndrome
and renal failure have occasionally also been documented as
due to the drug. Following topical intravesical mitomycin C
therapy for bladder cancer dermatological toxicity has been
encoutered in some cases (154).
7.4 Pharmacokinetics
Following intravenous bolus injection of mitomycin C the
plasma concentration-time curves show a bi-phasic, exponential decline (72, 155). Mitomycin C pharmacokinetics (at clinically relevant doses) is not dose dependent (72, 155). Maj o r routes of elimination are liver metabolism and urinary
excretion. Metabolites have never been identified. Absorption
after oral administration is very irregular(l56). After intravesical instillation, limited absorption of mitomycin C
into the systemic circulation occurs (30, 157). Pharmacokinetic data after intra-arterial administration are available
(73).
MITOMYCIN C
8.
391
DETERMINATION IN BODY FLUIDS
The analysis of mitomycin C in biological samples is

hampered by its instability, low concentrations and the polar
nature of the mitomycin C molecule (55, 7 6 ) . The following
techniques have been used: microbiological assays ( 7 4 , 9 5 ,
1 0 4 , 1 5 8 - 1 6 1 , 1 6 6 ) , bioassay by using a repair deficient mutant of Chinese hamster ovary cells ( 1 6 2 ) , immunological assay ( 1 6 3 , 1 6 4 ) , high-performance differential pulse polarography ( 5 4 ) and HPLC assays ( 4 6 , 7 2 - 8 6 , 93-96, 1 5 5 ) . The HPLC
assay designed by Den Hartigh et al. ( 4 6 , 7 2 ) has shown to be
very suitable for pharmacokinetic studies ( 7 2 - 7 4 ) . The following procedure is followed for plasma samples: 0 . 2 , 0.5 or
1.0 ml samples is mixed with the internal standard porfiromycin (about 500 ng) and with 2 . 0 , 5 . 0 or 10.0 ml, respectively, of chloroform-2-propanol ( l + l , w/w). After shaking
for 1 minute and centrifugating for 5 minutes ( 2 5 0 0 g), the
clear supernatant is transferred into a conical tube and evaporated to dryness at 30-40C under nitrogen. The residue is
dissolved in 50-100 p l of methanol with the aid of a vortextype mixer. Aliquots of 10-20 PI are injected into the chromatograph. HPLC: column: p Bondapak C18 ( 3 0 cm x 3 . 9 mm i.d.,
particle size 10 pm); mobile phase: 0.01 M phosphate buffer
pH 6.0-methanol (70+30, w/w); flow rate 1.0 ml/min; detection: 365 nm.
An overview of published determination methods of mitomycin C in body fluids is given in Table X I V .
Table X I V
Determination Methods for Mitomycin C in Body Fluids
matrix
sample pretreatment
b
determination limit(ng/ml) - test organism reference
microbiological assays
~~~
W
tJ
blood, tissues, urine,

bile
plasma, urine
plasma, urine
serum, urine
no
no
no
no
10
500
60
E.coli R
E.coli B
B.subtilis
E. coli
bioassay with hamster ovary cells

plasma
no
(162)
immunological assays
serum, urine
no
(163)
polarography
plasma, urine
plasma, urine
lI0
1.s
200
25
(54)
(54)
Table XIV (continued)

matrix
high performance liquid chromatography

sample preinternal
chrom to ra hic
determination
a
C
g g p
treatment standard mode limit(ng/rnl) -
serum, urine, ascites

plasma
plasma
plasma, urine, bile,
ascit e s
serum, plasma, urine
1.1.
1.1.
1.1.
no
no
no
1.1.
1.S.
Yes
Yes
plasma, urine
plasma, urine
1.s.
1.s.
yes
no
plasma, urine
1.1.
Yes
plasma, urine
serum
serum
1.1. (plasma)
1.S.
deproteination
with methanol
1.S.
1.1.
no
no
no
plasma, urine
plasma, urine
Yes
Yes
RP
RP
RP
1
5
RP
RP
40 (serum)
5 0 (urine)
4 (plasma)
2 (plasma)
500 (urine)
1 (plasma)
200 (urine)
1 (plasma)
reference
RP (gradient)
RP
NP
10
10
RP
RP
RP
25
10
RP
RP
a
1.1.: sample pre-treatment by liquid-liquid extraction; 1.s.: sample pre-treatment by liquid-solid
extraction.
b
determination limits for 25 p1 ( 8 0 ) , 50 p l ( 8 5 , 1 6 3 ) , 100 p l ( 8 4 ) , 0.2 ml (160),
77, 7 8 , 82, 162), 2.0 ml (54, 7 5 , 9 5 ) and 2.5 ml ( 9 4 ) samples
C
the internal standard is porfiromycin
d
RP: reversed phase chromatography; NP: normal phase chromatography.
1.0 ml ( 4 6 , 72-74,
JOSH. BEIJNEN ETAL.
394
Acknowledgements
The authors wish t o thank Ms C. de Graaf or typing the manuscript and Mr P.W.Y. Chan for preparing the figures.
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399
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47,
132,
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MITOMYCIN C
401
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67,
NEOSTIGMINE
A . A . Al-Ba&*
and Ad. lahiq"
*DepahAnent 06 Phanmacaficak? Chemhin.thy and **Vep&men;t

Phatrmacotogy, College 06 P h m a c y , King Saud UVLive.&Lty,
P.O. Box 2457, Riyadh-77451, (Saudi A m b h l .

VOLUME 16
403
06
Copyright 01987 by Academic Press. Inc.

404
1. History
2.
Description
2.1 Nomenclature
2.2
Formulae
2.3
Molecular Weight

2.5 Appearance, Color, Odor and Taste
3.
Physical Properties
4. Synthesis
5. Pharmacokinetics
6.
Therapeutic and Other Uses
7.
Toxicity
8. Methods o f Analysis
8.1 Identification
8.2
Titrimetric Methods
8.3
Biological Method
8.4 Spectrophotometric Methods

8.6 Ion-Selective Electrodes Method
8.7
Polarographic Method
References
NEOSTIGMINE
1.
405
Historv
As a r e s u l t o f t h e b a s i c r e s e a r c h o f Stedman and
a s s o c i a t e s ( 1 , 2 , 3 ) i n e l u c i d a t i n g t h e chemical b a s i s
o f t h e a c t i v i t y of physostigmine, Aeschlimann and
R e i n e r t (4) s y s t e m a t i c a l l y i n v e s t i g a t e d a s e r i e s o f
s u b s t i t u t e d phenyl esters o f a l l y l c a r b a m i c a c i d s .
Neostigmine ( P r o s t i g m i n ) , a most promising member o f
t h i s series, was i n t r o d u c e d i n t o t h e r a p e u t i c s i n 1931
f o r i t s s t i m u l a n t a c t i o n on t h e i n t e s t i n a l t r a c t . I t
was r e p o r t e d i n d e p e n d e n t l y by Remen (5) and Walker
(6) t o be e f f e c t i v e i n t h e symptomatic t h e r a p y of
m y a s t h e n i a g r a v i s which is due t o d e f e c t i n s y n a p t i c
t r a n s m i s s i o n a t neuromuscular j u n c t i o n . When t h e
patient i s given an a p p r o p r i a t e dose o f n e o s t igmine,
t h e r e s p o n s e t o t i t a n i c s t i m u l a t i o n i s improved a l o n g
w i t h symptomatic improvement i n muscle s t r e n g t h ( 7 ) .
Neostigmine was a l s o found u s e f u l i n p a r a l y t i c i l l e u s ,
a t o n y o f u r i n a r y b l a d d e r and i n glaucoma ( 8 , 9 ) .
2.
Description
2.1
Nomenclature
2.1.1
Chemical Names
3- (Dimethylcarbamoyloxy) -N ,N , N - t r i m e t h y l a n i l i n i u m bromide.
3-(Dimethylcarbamoyloxyphenyl)trimethyl-
ammonium bromide.
Benzenaminium, 3- [ [ (dimethylamino)carbonyl]
oxy] -N ,N,N-trimethyl bromide.
(m-Hydroxypheny1)timethylammonium bromide
dimethyl carbamate.
3- [ [ (Dimethylamino)carbonyl]oxy] - N , N , N ,
trimethylbenzenaminium bromide
3- [ [ (Dimethylamino) carbonyl loxy] -N ,N ,Ntrimethylbenzaminium methyl s u l p h a t e .
(m-Hydroxyphenyl) t r i m e t h y l ammonium methyl
s u l p h a t e dimethylcarbamate.
(3-Dimethylcarbamoxyphenyl)trimethylamonium
methyl s u l p h a t e (10-13).
2.1.2
Generic Names
Neostigmine bromide; Neostigmine DCF; NFN;
Neoeserine; P r o s e r i n e ; Synstigmine;
Eus t igmine ; P h i 10st igmine ; Neos t igmin i
bromidum; N e o s t i g m i n i i bromidum; Neostigmium
bromatum (13,14).
2.1.3
Trade Names
Neost i pmine bromide
P r o s t i g m i n ; J u v a s t i g m i n e ; Konstigmin;
Metastigmin; Normastigmin; P r o s t i g m i n ;
Leostigmin ( 1 3 , 1 4 ) .
Neostigmine methyl s u l p h a t e
I n t r a s t i g m i n a ; J u v a s t i g m i n ; Mestastigmin;
N e o e s s e r i n ; Normastigmin; P r o s t i g m i n ;
Hodostin; S t i g l y n ; Stigmosan (13,14).
2.1.4
CAS R e g i s t r y Number
59-99-4
114-80-7
51-60-5
2.1.5
Neostigmine
Neostigmine bromide
Neostigmine methyl s u l p h a t e (14)
1 N 1 & VOR CK-& E (Neostigmine bromide) (15)
2.2
Formulae
2.2.1
Empirical
C
H BrN202
1 2 19
C13H22N206S
Neostigmine bromide
401
NEOSTIGMINE
2.2.2
Structural
CH I
x0
-
X = Br-
II
c -N
,CH3
CH3
Neostigmine bromide
X = CH3SOi Neostigmine methyl s u l p h a t e
2.3
Molecular Weipht
Neostigmine bromide
303.2
Neostigmine methyl s u l p h a t e 334.4
2.4
C
0
C
0
2.5
47.53%, H 6.32%, N 9.24%, B r 26.36%,

10.55% (bromide).
46.69%, H 6 . 6 3 % ; N 8 . 3 8 % , S
9.59%,
28.71% (methyl s u l p h a t e ) .

Odorless, c o l o r l e s s c r y s t a l o r a white c r y s t a l l i n e ,
s l i g h t l y hygroscopic powder w i t h a b i t t e r t a s t e
(14) *
3.
Physical Properties
3.1
Melting P o i n t
Neost igmine bromide
C r y s t a l s from a l c o h o l and e t h e r melt a t 167 w i t h
decomposition ( 1 3 ) .
C r y s t a l s from a l c o h o l melt between 142OC and 145OC
(13) *
A. A, AL-BADR AND M. TARIQ
408
3.2
Extraction
Neostigmine s a l t s a r e q u a t e r n a r y ammonium compounds
and a r e s o l u b l e i n water. The aqueous s o l u t i o n
i s a c i d i f i e d w i t h d i l u t e a c e t i c a c i d , evaporated
t o dryness and e x t r a c t e d w i t h methanol. The
methanol e x t r a c t w i l l c o n t a i n most o f t h e q u a t e r n a r y
ammonium compound and may b e p u r i f i e d by paper
chromatography ( 1 6 ) .
3.3
Solubility
Neostigmine s a l t s a r e f r e e l y s o l u b l e i n w a t e r .
Moderately s o l u b l e i n chloroform and e t h a n o l .
I n s o l u b l e i n e t h e r (11).
3.4
Acidity
Dissolve 0 . 2 0 g i n 20 m l carbon d i o x i d e - f r e e
w a t e r and t i t r a t e a t pH 7 . 0 w i t h 0 . 0 2 N sodium
hydroxide ( c a r b o n a t e - f r e e ) n o t more than 0 . 2 m l
i s r e q u i r e d (17).
3.5
Moisture Content and H y g r o s c o p i c i t y

Not more than I%, determined by d r y i n g a t 1 0 5 O C
(10)
3.6
Storage
I t should b e s t o r e d i n a i r t i g h t c o n t a i n e r s
p r o t e c t e d from l i g h t ( 1 0 ) .
3.7
Spectral Properties
3.7.1
U l t r a v i o l e t SDectrum
The UV spectrum o f neostigmine bromide i n
e t h a n o l i s given i n Figure 1. I t shows two
maxima a t 260 nm and 266 nm. The spectrum
was recorded on Varian spectrophotometer
model DMS 90.
Clarke (11) r e p o r t e d t h e following:
Neostigmine methyl s u l f a t e i n 1 N H2SO4;
maxima a t 260 nm (E 1 % ,1 cm 20) and 266 nm
(E 1%,1 cm 1 8 ) .
NEOSTIGMINE
lot
2 00
2SOWavelength 300
(nm)
350
400
Figure I: Ultraviolet spectrum of neostigmine bromide in methanol.
410
3.7.2
- (IR)
The IR spectrum of n e o s t i g m i n e bromide i n
K B r d i s c i s p r e s e n t e d i n F i g u r e 2 , and
was recorded on Pye-Unicam I R s p e c t r o p h o t o meter model SP 1025. The f r e q u e n c i e s and
t h e s t r u c t u r a l assignments a s shown below:
Frequency cm-l
3020-
2900 .)
1730
1610
1595)
1490-1450
1030
1070)
780 and 895
As s ignment
CH s t r e t c h
C = 0 (amide) s t r e t c h
C = C s t r e t c h (aromatic)
CH bending v i b r a t i o n s
C - N vibration (aliphatic)
CH (aromatic) bending
vibrations
Clarke (11) r e p o r t e d t h e f o l l o w i n g :
Neostigmine bromide, potassium bromide d i s c
t h e p r i n c i p a l peaks a r e 1711, 1215 and
1154 cm-1.
3.7.3
1
Proton Nuclear Magnetic Resonance ( H NMR)
Spectrum
1
The H NMR spectrum o f neostigmine methyl
s u l p h a t e i s shown i n F i g u r e 3. The drug was
dissolved in
deuterium o x i d e (DzO) and i t s
spectrum was determined on a Varian - T60 A
NMR s p e c t r o m e t e r u s i n g sodium 2,2-dimethyl
2 - s i l a p e n t ane- 5 - s u l p h o n a t e (DSS) a s t h e
i n t e r n a l standard.
--
f
I
8.0
7.0
Figure 3: Proton
6.0
S.OPpm(6k.O
3 -0
2 .o
I .o
NMR spectrum of neostigmine methyl sulphate in D20 with
TMS as internal reference.
NEOSTIGMINE
413
Assignment o f t h e chemical s h i f t s t o t h e
d i f f e r e n t p r o t o n s i s shown below:
Chemical
shift
(ppm)
Multiplicity
3.04)
II
singlets
-C-N(CH
-3 ) 2
3. 72
singlet
4- ( g 3 )
3.80
s i n g 1e t
CH,- SO4
3.12
7.15-7.60
7.66-8.30
3.7.4
Proton assignment
m u l t i p l e t s Aromatic p r o t o n s
Carbon-13 Nuclear Magnetic Resonance

(C-13 NMR) Spectrum
The C-13 NMR s p e c t r a o f neostigmine methyl
s u l p h a t e i n deuterium oxide (D2O) u s i n g
DSS (sodium 2,2-dimethyl 2 - s i l a p e n t a n e 5-sulphonate) as an i n t e r n a l r e f e r e n c e were
o b t a i n e d u s i n g a J e o l FX 100 MHz s p e c t r o m e t e r
a t an ambient t e m p e r a t u r e . F i g u r e s 4 and
5 r e p r e s e n t t h e proton-decoupled and o f f resonance s p e c t r a r e s p e c t i v e l y .
12
CH3
I+
13
CH3 - S O i
-C
0
"
-N
1
CH
.CH3
2
The carbon chemical s h i f t s were a s s i g n e d on

t h e b a s i s o f t h e chemical s h i f t t h e o r y and
t h e o f f - r e s o n a n c e s p l i t t i n g p a t t e r n and a r e
shown below:
414
Figure 4 : Proton -decoupled carbon-I3 NMR spectrum Of n-stigmine
methyl wlphate
in D20 with DSS as internal reference.
Figure 5 : Off -Resonance carbon -13 NMR spectrum of neostigmine methyl sulphate
in 9 0 with DSS a s internal reference.
NEOSTIGMINE
415
Chemical
shift
(ppm)
3.7.5
Multiplicity
Carbon assignment
38.9
quartet
38.8
quartet
158.3
singlet
149.8
s i n gl e t
126.9
doublet
134.0
doub 1e t
119.8
doublet
154.5
singlet
117.1
doublet
59.6
quartet
10, 11 and 12
57.9
quartet
13
Mass Spectrum
The e l e c t r o n impact ( E I ) mass spectrum a t
70 e V was r e c o r d e d on Varian MAT 311 mass
s p e c t r o m e t e r i s shown i n F i g u r e 6 . In t h e
spectrum an ion a t m/e 428 was observed
which most p r o b a b l y a r i s e s from a
recombination o f fragments. The spectrum
shows a b a s e peak a t m/e 72. The
most prominent i o n s a r e shown below:
Figure 6: Electron impld (El)mass spectrum
Of
neostigmine methyl sulphate.
NEOSTIGMINE
417
Fragment
72
208
356
CON ( c H ~+ ) ~
@-
0-CON
(cH~$+
428 - CON (CH,),I+
1'
L-.
42 8
OCON ( CH3)
CH2
The chemical i o n i s a t i o n ( C I ) mass spectrum

was o b t a i n e d on Finnigan 4000 mass
s p e c t r o m e t e r and is shown i n F i g u r e 7 . The
spectrum shows i o n s at m/e 1 2 7 ( t h e b a s e
peak) and a t m/e 303 and both a r e probably
a r i s e d from a recombination o f fragments.
The most prominent i o n s a r e shown below:
Figure 7 : Chemical ionization ( C I mass Spectrum
Of
neostigmine methyl sulphate.
NEOSTIGMINE
419
m/e
127
Fragment
0
II
CH30-S-O-CH3
II
.t
l+
209
303
4.
l+
Synthesis
Neostigmine bromide can b e p r e p a r e d from 3-hydroxydim e t h y l a n i l i n e , which i s made from 3 - n i t r o a n i l i n e by
o r d i n a r y s y n t h e t i c methods. The 3-hydroxydimethyla n i l i n e i s d i s s o l v e d i n an a l c o h o l i c s o l u t i o n o f t h e
c a l c u l a t e d amount of potassium hydroxide, and t h e
s o l u t i o n o f t h e potassium hydroxide, and t h e s o l u t i o n
o f t h e potassium d e r i v a t i v e so formed is t r e a t e d w i t h
dimethylcarbamoyl c h l o r i d e , Me2N.COC1 (made from
dimethylamine and carbonyl c h l o r i d e ) . T h i s g i v e s a
t e r t i a r y b a s e which, by combination w i t h methyl bromide,
y i e l d s t h e required quaternary s a l t - i . e . t h e
dimethylcarbamic e s t e r o f 3-hydroxy-NNN-trimethylanil i n i u m bromide:
(18, 1 9 ) .
420
OCON (CH3)
OCON (CH3)
Neostigmine methylsulphate can be made i n the same

way, except t h a t i n t h e l a s t s t a g e t h e secondary amine
i s combined with methyl sulphate t o form t h e s a l t
[ (R-AMe3)MeSOi]
(18).
OK
OCON (CH3)
CH3S0i
0-C-N,
0
CH3
42 1
NEOSTIGMINE
A modification of t h i s route is t o convert t h e
hydroxydimethylani l i n e i n t o t h e q u a r t e r n a r y s a l t (by
combination w i t h methyl bromide o r methyl s u l p h a t e ) ,
and t h e n t o t r e a t t h e s a l t with dimethylcarbamoyl
c h l o r i d e (18).
N(CH3)2
OK
ClCON (CH3)
P
Neostigmine can be s y n t h e s i z e d e i t h e r d i r e c t l y from

3-hydroxydimethylaniline with phosgene t o g i v e t h e
carbonyl c h l o r i d e and t h e n with dimethylamine t o g i v e
3-dimethylaminophenyldimethylurethane o r t h e l a t t e r
may be prepared from t h e sodium s a l t of t h e s t a r t i n g
m a t e r i a l and dimethylcarbamic c h l o r i d e . In e i t h e r
c a s e t h e product i s converted t o t h e m e t h y l s u l p h a t e
by t r e a t m e n t with dimethyl s u l p h a t e (20).
OCON (CH31
ON a
COC 1 2
N
OH
2-
.CH3S0i
OCOCl
OCON (CH3)
422
5.
Pharmacokinetics
Neostigmine is absorbed p o o r l y a f t e r o r a l a d m i n i s t r a t i o n ,
such t h a t much l a r g e r doses a r e needed t h a n by t h e
p a r e n t e r a l r o u t e . Whereas t h e e f f e c t i v e p a r e n t e r a l
dose o f neostigmine i n man i s 0 . 5 t o 2.0 mg, t h e
e q u i v a l e n t o r a l dose may be 30 mg o r more. Large o r a l
doses may prove t o x i c i f i n t e s t i n a l a b s o r p t i o n i s
enhanced f o r any reason and t h e q u a t e r n a r y a l c o h o l and
p a r e n t compound are e x c r e t e d i n t h e u r i n e . P y r i d o s t i g mine and i t s q u a t e r n a r y a l c o h o l are a l s o t h e predominant
e n t i t i e s found i n u r i n e a f t e r a d m i n i s t r a t i o n o f t h i s
drug t o man (21).
Neostigmine was r a p i d l y e l i m i n a t e d from t h e plasma o f
5 p a t i e n t s t o whom 5 mg o f t h e methylsulphate had been
given t o a n t a g o n i s e r e s i d u a l neuromuscular b l o c k . The
plasma c o n c e n t r a t i o n o f neostigmine d e c l i n e d t o about
8% o f i t s i n i t i a l v a l u e a f t e r 5 minutes with a d i s t r i b u t i o n h a l f - l i f e o f less than one minute. E l i m i n a t i o n
h a l f - l i f e ranged from about 15 t o 30 minutes. Trace
amounts o f neostigmine could be d e t e c t e d i n t h e plasma
a f t e r one hour ( 1 4 ) .
O f neostigmine t h a t reaches t h e l i v e r , 98 p e r c e n t is
metabolized i n 10 minutes. ( 2 2 ) I t s t r a n s f e r from
plasma t o l i v e r c e l l s and then t o b i l e is probably
p a s s i v e i n c h a r a c t e r . Since c e l l u l a r membranes
permit t h e passage o f plasma p r o t e i n s s y n t h e s i z e d i n
l i v e r i n t o t h e blood stream through c a p i l l a r y walls
o r lymphatic v e s s e l s , t h e y may n o t p r e s e n t a b a r r i e r
t o t h e d i f f u s i o n o f q u a t e r n a r y amines such as n e o s t i g mine. P o s s i b l y t h e r a p i d h e p a t i c metabolism o f
neostigmine p r o v i d e s a downhill g r a d i e n t f o r t h e
c o n t i n u a l d i f f u s i o n of t h i s compound ( 2 3 ) . A c e r t a i n
amount may be hydrolyzed slowly by plasma c h o l i n e s t e r a s e . When neostigmine was given by mouth t o 2
p a t i e n t s with myasthenia g r a v i s less t h a n 5 % was found
unchanged i n t h e u r i n e ( 1 4 ) . Following i n t r a m u s c u l a r
a d m i n i s t r a t i o n about 65% o f a dose i.s e x c r e t e d i n t h e
u r i n e unchanged ( 1 0 ) . 20% of an o r a l dose is
e x c r e t e d i n t h e u r i n e and 50% i n t h e faeces; less t h a n
5% o f t h e o r a l dose i s e x c r e t e d i n t h e u r i n e unchanged.
NEOSTIGMINE
6.
423
T h e r a n e u t i c and Ot he r Uses
Neostigmine is a q u a t e r n a r y ammonium a n t i c h o l i n e s t e r a s e .
I t a c t s a t t h e e s t e r a t i c s i t e o f t h e enzyme t o form t h e
i n a c t i v e dimethylcarbamoyl enzyme. Neostigmine h a s
widespread a c t i o n s , b u t f o r t u n a t e l y i t s e f f e c t s a r e
more prominent on c e r t a i n s t r u c t u r e s t h a n on o t h e r s ,
b e i n g p a r t i c u l a r l y e f f e c t i v e on t h e bowel, u r i n a r y
b l a d d e r , and s k e l e t a l muscle, t h e p u p i l , t h e h e a r t ,
blood p r e s s u r e , and s e c r e t i o n s b e i n g a f f e c t e d t o a
much lesser e x t e n t i n doses t h a t are o r d i n a r i l y
e f f e c t i v e on r e l a t e d s t r u c t u r e s (19) .
The drug i n h i b i t s c h o l i n e s t e r a s e a c t i v i t y and p r o l o n g s
and i n t e n s i f i e s t h e m u s c a r i n i c and n i c o t i n i c e f f e c t s
o f a c e t y l c h o l i n e . I t proba bly a l s o has d i r e c t e f f e c t s
on s k e l e t a l muscle f i b r e s . The a n t i c h o l i n e s t e r a s e
a c t i o n s o f ne ost igmi ne are r e v e r s i b l e . I t is used
mainly f o r i t s a c t i o n on s k e l e t a l muscle, and less
f r e q u e n t l y t o i n c r e a s e a c t i v i t y o f smooth muscle ( 1 4 ) .
Neostigmine me t hylsul pha t e i s used i n t h e t r e a t m e n t o f
myasthenia g r a v i s i n u s u a l d o s e s o f 1 t o 2 . 5 mg d a i l y
given i n d i v i d e d dose s by subcutaneous, i n t r a m u s c u l a r ,
o r i n t r a v e n o u s i n j e c t i o n a c c ordi ng t o t h e s e v e r i t y o f
t h e c o n d i t i o n (14).
Neostigmine i s used i n c o n d i t i o n s o f u r i n a r y b l a d d e r
a t o n y due t o p o s t a n e s t h e t i c d e p r e s s i o n o r t o n e u r o l o g i c a l
d i s o r d e r s (19). I t is a l s o used i n t h e t r e a t m e n t o f
p a r a l y t i c i l e u s postoperative urinary retention, i n
d o s es o f 0 . 5 t o 1 mg. For e x p e l l i n g i n t e s t i n a l f l a t u s
p r i o r t o r a di ogra phy o f t h e g a l l - b l a d d e r , kidneys , o r
u r e t e r s , a s i n g l e dose o f 500 ug h a s sometimes been
used ( 1 4 ) .
Neostigmine can b e employed as a d i a g n o s t i c t e s t ,
e s p e c i a l l y a f t e r symptoms have been pur posely
a c c e n t u a t e d by t h e a d m i n i s t r a t i o n o f q u i n i n e . Neostigmine is used as d i a g n o s t i c t e s t a gent i n myotonia
c o n g e n i t a , i n which c o n d i t i o n ne ostigmine a g g r a v a t e s
t h e symptoms. I t may be used as a d i a g n o s t i c agent
f o r e a r l y pregnancy o r t o t r e a t delayed menstr uation:
given i n t r a m u s c u l a r l y on t h r e e s u c c e s s i v e days i t w i l l
i n d u c e m e nst rua ti on w i t h i n 72 hours a f t e r t h e l a s t dose
u n l e s s t h e p a t i e n t i s pre gna nt . Neostigmine i s used
t o p i c a l l y t o t r e a t primary open-angle glaucoma and i n
424
A. A. AL-BADR AND M.TARlQ
t h e emergency t r ea t m e n t o f primary a c u te - a n g le glaucoma.

Miosis lasts 1 2 t o 36 h o u r s . Longer-acting a n t i c h o l i n e s t e r a s e s a r e p r e f e r r e d f o r t h e t r e a t m e n t o f accommod a t i v e e s o t r o p i a (19).
7.
T o x i c i t y and S i d e E f f e c t s
Adverse and s i d e e f f e c t s o f neostigmine i n c l u d e
s a l i v a t i o n , a n o r e x i a, nausea and vomiting , abdominal
cramps and d i a r r h o e a . Symptoms of overdosage i n c l u d e
sweating, lachrymation, watery n a s a l d i s c h a r g e ,
e r u c t a t i o n , i n v o l u n t a r y d e f a e c a t i o n and u r i n a t i o n
f l u s h i n g , m i o s i s , c o n j u n c t i v a l co n g e stio n , c i l i a spasm,
brow ache, nystagmus, r e s t l e s s n e s s , a g i t i o n , f e a r ,
e x c e s s i v e dreaming, i n c r e a s e d b r o c h i a l s e c r e t i o n
combined wi t h b r o n c h o c o n s t r i c t i o n , b r a d y c a r d ia and
hypotension, muscle cramps, s c a t t e r e d f a s i c u l a t i o n s
and e v e n t u a l s e v e r e weakness and p a r a l y s i s , c o n v u lsio n s,
and coma. I t h as a l s o been s t a t e d t h a t p a r a d o x ic a l
e f f e c t could o c c u r due t o i n t e r a c t i o n between n i c o t i n i c
and muscarinic a c t i o n s ; a consequence of t h i s could b e
some evidence o f an a c c e l e r a t i o n p u l s e - r a t e and
e l e v a t i o n o f blood p r e s s u r e . Death may f o llo w due
t o cardiac a r r e s t o r central respiratory paralysis
and pulmonary oedema. The major symptom o f overdosage
i n myasthena g r a v i s i s i n c r e a s e d muscular weakness ( 1 4 ) .
I t i s c o n t r a i n d i c a t e d i n a s t h m a t i c p a t i e n t s . I t should
n o t be employed al o n g with c h o l i n e esters e x c e p t f o r
ophthalmologic u s e. Quinidine i n t e r f e r e s w i t h t h e a c t i o n
o f neostigmine ( 1 9 ) .
Neostigmine methyl s u l p h a t e , by i n c r e a s i n g i n t e s t i n a l
m o t i l i t y , may cause d i s r u p t i o n o f i n t e s t i n a l s u t u r e
l i n e s (10).
8.
Methods o f Analysis
8.1
Identification
a)
To 0 . 1 m l o f a 1 p e r c e n t w/v s o l u t i o n , add
0.5 m l of sodium hydroxide s o l u t i o n and e v a p o r a te
t o d r y n e s s on a water-bath. Heat q u i c k l y on an
o i l - b a t h t o about 250' and m a in ta in a t t h i s
temperature f o r about t h i r t y seconds. Cool,
d i s s o l v e t h e r e s i d u e i n 1 m l o f water, c o o l i n
i c e water, and add 1 m l o f diazoaminobenzene-
425
NEOSTIGMINE
sulphonic acid; a cherry-red colour i s

produced ( 1 5 ) .
C r y s t a l t e s t s . Micro: l e a d i o d i d e s o l u t i o n b r a n c h i n g n e e d l e s ( s e n s i t i v i t y : 1 i n 400) ;
p l a t i n i c i o d i d e s o l u t i o n - bunches o f curved
( s e n s i t i v i t y : 1 i n 400) (11).
The i n f r a r e d a b s o r p t i o n spectrum o f a potassium
bromide d i s p e r s i o n o f i t , p r e v i o u s l y d r i e d a t
1 0 5 O f o r 3 h o u r s , e x h i b i t s maxima o n l y a t t h e
same wavelengths as t h a t of a s i m i l a r
p r e p a r a t i o n o f USP Neostigmine Bromide
Reference Standard (12).
A s o l u t i o n (1 i n 50) responds t o t h e t e s t o f
bromide (neostigmine bromide) (12).
8.2
8.2.1
Aqueous T i t r a t i o n
B r i t i s h Pharmacopoeia (1973) (17) r e p o r t e d
t h e f o l l o w i n g procedure f o r t h e a s s a y of
neostigmine methyl s u l p h a t e :
D i s s o l v e 0.15 g i n 20 m l of w a t e r , t r a n s f e r
t o a semimicro ammonia d i s t i l l a t i o n
a p p a r a t u s , and add 20 m l of a 50 p e r c e n t
w/v s o l u t i o n o f sodium hydroxide. Pass a
c u r r e n t o f steam through t h e m i x t u r e ,
c o l l e c t t h e d i s t i l l a t e i n 50 m l o f 0 . 1 N
s u l p h u r i c a c i d u n t i l t h e t o t a l volume
r e a c h e s about 200 m l , and t i t r a t e t h e e x c e s s
o f a c i d w i t h 0.02 N NaOH u s i n g methyl r e d
s o l u t i o n as i n d i c a t o r . Repeat t h e o p e r a t i o n
w i t h o u t t h e s u b s t a n c e b e i n g examined; t h e
d i f f e r e n c e between t h e t i t r a t i o n s represents
t h e amount o f a c i d r e q u i r e d t o n e u t r a l i s e t h e
dimethylamine formed from t h e n e o s t i g m i n e .
Each m l o f 0.02 N s u l p h u r i c a c i d i s e q u i v a l e n t t o 0.006688 g of Cl3HZ2N2O6S.
Huang -e t a1 (24) d e s c r i b e d a s i m p l e , r a p i d
and accurate method u s i n g t h e a p p l i c a t i o n
of a l t e r n a t i n g - c u r r e n t o s c i l l o p o l a r o g r a p h i c
t i t r a t i o n i n pharmaceutical a n a l y s i s . In
426
t h i s method neostigmine methyl s u l p h a t e i n

i n j e c t i o n s was t i t r a t e d w i t h sodium
t e t r a p h e n y l b o r a t e . To t h e i n j e c t i o n
s o l u t i o n ( 2 0 ml) c o n t a i n i n g 10 mg o f
neostigmine methyl s u l p h a t e were added 10 m l
of 0 . 0 1 N sodium t e t r a p h e n y l b o r a t e and
10 m l o f a c e t a t e b u f f e r s o l u t i o n (pH 5 . 3 ) .
The s o l u t i o n was d i l u t e d t o 50 m l w i t h
w a t e r , a f t e r 5 minutes t h e p r e c i p i t a t e
was formed, t h e c o n t e n t s were f i l t e r e d . A
25 m l p o r t i o n o f f i l t r a t e was t r e a t e d w i t h
8 drops o f 40% sodium hydroxide s o l u t i o n and
t i t r a t e d w i t h 0.01 N TI2S04, t h e d i s appearance o f t h e i n c i s i o n o f sodium
t e t r a p h e n y l b o r a t e on t h e curve of d/E/dt vs
E b e i n g used t o i n d i c a t e t h e end p o i n t . The
r e s u l t s were c o r r e c t e d f o r a blank v a l u e .
The r e c o v e r y range from 9 9 . 3 - 100.2% and
t h e c o e f f i c i e n t o f v a r i a t i o n was 0 . 4 2 % .
Tsubouchi -e t a 1 (25) r e p o r t e d a method o f
a n a l y s i s o f neostigmine u s i n g t h e
a p p l i c a t i o n o f one-phase end-point change
system i n two phase t i t r a t i o n t o amine
drug a n a l y s i s . Neostigmine i n aqueous
s o l u t i o n ( 5 o r 10 mM) was determined by
t i t r a t i o n w i t h aqueous 0 . 0 1 M t e t r a p h e n y l
b o r a t e with tetrabromo p h e n o l p h t h a l i e n
e t h y l ester a s an i n d i c a t o r i n t h e o r g a n i c
phase ( 1 , 2 - d i c h l o r o e t h a n e ) , i n b o r a t e
phosphate b u f f e r medium (pH 5 . 5 . - 7 . 5 ) .
The
end-point was d e t e c t e d by means o f c o l o r
change o f t h e i n d i c a t o r i n t h e o r g a n i c
phase without t r a n s f e r of i n d i c a t o r between
phases. Various common i o n s ( i n c l u d i n g
c a r b o n a t e , a c e t a t e , c i t r a t e , and t a n n a t e )
did not i n t e r f e r e but thiamine , papaverine ,
dodecyle s u l p h a t e and mercury caused
interference.
Diamandis and C h r i s t o p o u l o s ( 2 6 ) developed
a p o t e n t i o m e t r i c t i t r a t i o n o f neostigmine
bromide i n pharmaceutical compounds by
t i t r a t i n g them w i t h sodium t e t r a p h e n y l
b o r a t e . 25 M 1 o f aqueous s o l u t i o n ( 0 . 2 1 mM) o f neostigmine bromide and 5 m l o f
t h e a p p r o p r i a t e b u f f e r s o l u t i o n was added.
NEOSTIGMINE
421
The s o l u t i o n was t i t r a t e d p o t e n t i o m e t r i c a l l y
a t 0.36 m l p e r minute with 0.01 M sodium
t e t r a p h e n y l b o r a t e a t 2 2 O 5 20 w i t h
continuous s t i r r i n g . The end-point was
d e t e c t e d by a t e t r a p h e n y l b o r a t e s e l e c t i v e
e l e c t r o d e . T h i s method was adopted f o r
d e t e r m i n a t i o n o f neostigmine i n powders,
i n j e c t i o n s , drops and s y r u p s .
United S t a t e s Pharmacopeia X I X (1980) (12)
d e s c r i b e d t h e f o l l o w i n g procedure f o r t h e
a s s a y o f neostigmine methyl s u l p h a t e :
Place about 100 mg o f neostigmine methyl
s u l p h a t e , a c c u r a t e l y weighed, i n a 500 m l
Kjeldahl f l a s k , d i s s o l v e i n 150 m l o f w a t e r ,
and add 40 m l of sodium hydroxide s o l u t i o n
(1 i n 1 0 ) . Connect t h e f l a s k by means o f
a d i s t i l l a t i o n t r a p t o a well-cooled
condenser t h a t d i p s i n t o 25 m l o f b o r i c a c i d
s o l u t i o n ( 1 i n 2 5 ) , d i s t i l about 150 m l o f
t h e c o n t e n t s of t h e f l a s k , add methyl
p u r p l e TS t o t h e s o l u t i o n i n t h e r e c e i v e r ,
and t i t r a t e with 0.02 N s u l p h u r i c a c i d .
Perform a blank d e t e r m i n a t i o n , and make t h e
n e c e s s a r y c o r r e c t i o n . Each m l of 0 . 0 2 N
s u l p h u r i c a c i d i s e q u i v a l e n t t o 6.688 mg o f
C13H22N206S'
B r i t i s h Pharmacopoeia (1973) (17) r e p o r t e d
t h e f o l l o w i n g procedure f o r t h e a s s a y of
n e o s t igmine t a b l e t s :
Weigh and powder 2 t a b l e t s . T r a n s f e r a
q u a n t i t y o f t h e powder, e q u i v a l e n t t o 0.15
o f neostigmine bromide, t o a semi-micro
ammonia d i s t i l l a t i o n a p p a r a t u s , add 20 m l
o f a 50% w/v s o l u t i o n o f sodium hydroxide
and 0 . 5 m l o f a 2% s o l u t i o n o f octan-2-01
i n l i q u i d p a r a f f i n and complete t h e a s s a y
d e s c r i b e d under neostigmine methyl s u l p h a t e .
(above) b e g i n i n g a t t h e words "Pass a
c u r r e n t o f steam . . . . I ! .
Each m l o f 0.02 N
s u l p h u r i c a c i d i s e q u i v a l e n t t o 0.006064 g
o f C12H19BrN202.
8.2.2
Non-aqueous T i t r a t i o n
United S t a t e Pharmacopeia X I X (1980) (12)
d e s c r i b e d t h e f o l l o w i n g procedure f o r t h e
a s s a y of neostigmine bromide:
Dissolve about 750 mg of neostigmine bromide
a c c u r a t e l y weighed, i n a m i x t u r e o f 70 m l
of g l a c i a l a c e t i c a c i d and 20 m l o f m e r c u r i c
a c e t a t e TS, add 4 d r o p s o f c r y s t a l v i o l e t TS,
and t i t r a t e with 0 . 1 N p e r c k l o r i c a c i d t o a
b l u e e n d - p o i n t . Perform a blank determinat i o n , and make any n e c e s s a r y c o r r e c t i o n .
Each m l of 0 . 1 N p e r c h l o r i c a c i d i s
e q u i v a l e n t t o 30.32 mg o f C12H19BrN202
(12).
Bayer and Posgay (27) s u g g e s t e d a p e r c h l o r i c
a c i d t i t r a t i o n method f o r t h e d e t e r m i n a t i o n
o f neostigmine i n pharmaceutical p r e p a r a t i o n s .
The neostigmine s o l u t i o n was t i t r a t e d with
0 . 1 N p e r c h l o r i c a c i d which h a s been
s t a n d a r d i z e d with anhydrous potassium
c a r b o n a t e w i t h 0.1% g e n t i a n v i o l e t i n
a c e t i c a c i d c o n t a i n i n g 3% m e r c u r i c a c e t a t e
as indicator.
Surmann et a 1 (28) r e p o r t e d a t i t r a t i o n
method f o r t h e pharmaceutical h y d r o c h l o r i d e s
and hydrobromides i n non-aqueous media.
T h i s method i n v o l v e d i r e c t t i t r a t i o n o f
drug w i t h p e r c h l o r i c a c i d i n a c e t i c
anhydride medium, w i t h n a p h t h o l benzein
o r Sudan Red B an i n d i c a t o r f o r hydroc h l o r i d e s f o r hydrobromides r e s p e c t i v e l y .
Kracmarova and Kracmar (29) r e p o r t e d t h e
f o l l o w i n g non-aqueous tit r a t i o n :
Evaporate t h e sample o f t h e eye d r o p s (5 ml)
on a w a t e r b a t h t o d r y n e s s , d i s s o l v e t h e
r e s i d u e i n anhydrous a c e t i c a c i d (2 x 20 m l ) ,
add c r y s t a l v i o l e t (0.2% i n anhydrous
a c e t i c a c i d ) (2 d r o p s ) a s i n d i c a t o r , and
t i t r a t e with 0.1 N perchloric acid t o a
b l u e e n d - p o i n t , add 5% HgII a c e t a t e s o l u t i o n
NEOSTIGMINE
429
(5 ml), mix and t i t r a t e a g a i n t o a b l u e endp o i n t ; t h e d i f f e r e n c e between t h e t i t r e s

corresponds t o neostigmine bromide.
8.2.3
I o d i m e t r i c Methods
Koka (30) developed an i o d i m e t r i c determinat i o n o f p r o s e r i n e (neostigmine methyl
s u l p h a t e ) i n drug f o r m u l a t i o n s . The drug
sample i s d i s s o l v e d i n water and t h e
solution is t r e a t e d with 2-3 m l of d i l u t e
s u l p h u r i c a c i d , 2-3 m l o f 10% potassium
i o d i d e s o l u t i o n and 5 m l of 0 . 1 N i o d i n e ,
d i l u t e d t o 25 m l with w a t e r and f i l t e r e d ;
10 m l o f f i l t r a t e i s t i t r a t e d with 0 . 1 N
sodium t h i o s u l p h a t e .
Mitchenko and Kirichenko (31) r e p o r t e d t h e
u s e of i o d i m e t r i c method f o r t h e determinat i o n o f neostigmine methyl s u l p h a t e and
p i l o c a r p i n e HC1 i n eye-drops. Neostigmine
methyl s u l p h a t e and p i l o c a r p i n e HC1 a r e
determined by r e a c t i o n with i o d i n e s o l u t i o n
i n d a r k , f i l t e r i n g t h e m i x t u r e and t i t r a t i n g
u n r e a c t e d i o d i n e w i t h sodium t h i o s u l p h a t e
s o l u t i o n . When b o t h a r e p r e s e n t t h e sum
o f t h e two i s determined by above method
and only p i l o c a r p i n e i s determined a l k a l i m e t r i c a l l y , a r g e n t i m e t r i c a l l y o r mercurim e t r i c a l l y . Neostigmine methyl s u l p h a t e
is determined by t h e d i f f e r e n c e . The
r e l a t i v e e r r o r i n determining t h e two d r u g s
i n eye-drops is 3.56 and 2.85 r e s p e c t i v e l y .
8.3
B i o l o g i c a l Method
Buckles and Bullock (32) r e p o r t e d t h e a p p l i c a t i o n

o f enzyme i n h i b i t i o n t o t h e e s t i m a t i o n o f s m a l l
q u a n t i t i e s o f drugs p o s s e s s i n g a n t i c h o l i n e s t r a s e
a c t i v i t y . The method is based on t h e i n h i b i t i o n
of t h e pseudocholinesterase a c t i v i t y of horse
serum. 5 M 1 o f sample c o n t a i n i n g 5 ug of
neostigmine i s mixed w i t h 1 m l o f h o r s e serum,
1 m l o f 0.2% cresol r e d s o l u t i o n and 37 m l o f
w a t e r . The m i x t u r e i s h e a t e d t o 4OoC and pH
a d j u s t e d t o 7 . 9 . After 15 minutes add 5 m l o f
3% a c e t y l c h o l i n e p e r c h l o r a t e s o l u t i o n and r e a d j u s t
430
t h e pH t o 7 . 9 .
Maintain t h e pH between
7 . 8 and 8 . 0 d u r i n g 15 minutes by dropwise
a d d i t i o n o f 0.025 N sodium h y d r o x i d e and r e c o r d
t h e volume of a l k a l i used. C o r r e c t f o r nonenzymic h y d r o l y s i s by c o n d u c t i n g an experiment
w i t h 1 m l b u f f e r i n s t e a d o f h o r s e serum and c a r r y
o u t a b l a n k w i t h 5 m l w a t e r i n s t e a d o f sample.
The p e r c e n t a g e i n h i b i t i o n i s a l i n e a r f u n c t i o n o f
t h e l o g c o n c e n t r a t i o n of n e o s t i g m i n e methyl
sulphate.
8.4
S p e c t r o p h o t o m e t r i c Methods
8.4.1
C o l o r i m e t r i c Methods
Belal et a l , (33) d e s c r i b e d a s p e c t r o photometric determination o f neostigmine
i n t a b l e t s and ampoules. T a b l e t p r e p a r a t i o n s
c o n t a i n i n g n e o s t i g m i n e methyl s u l p h a t e were
powdered and d i s s o l v e d i n 50 m l o f w a t e r
and 1 m l of s o l u t i o n was mixed w i t h 3 m l o f
c i t r a t e - p h o s p h a t e b u f f e r s o l u t i o n (pH 7 .8 )
and 2 m l o f a 0.1% s o l u t i o n o f bromothymol
b l u e i n 0 . 0 1 N sodium hydroxide. The
m i x t u r e was e x t r a c t e d w i t h chloroform b e f o r e
measurement o f i t s absorbance a t 416 nm
vs a r e a g e n t b l a n k . A l t e r n a t i v e l y 1 0 m l
of chloroform e x t r a c t was t r e a t e d w i t h 10 ml
of 0 . 0 1 N sodium hydroxide and t h e aqueous
l a y e r was s e p a r a t e d and made up t o 25 m l
with water, i t s absorbance b e i n g measured
a t 615 nm as a r e a g e n t b l a n k . The c a l i b r a t i o n graph were r e c t i l i n e a r f o r 0 . 2 - 1 . 6 mg.
The r e c o v e r y by t h i s method was 1 0 0 . 5 % .
Belikov -e t a1 (34) have used sulphonepht h a l e i n dyes as e x t r a c t i o n r e a g e n t s f o r

t h e e x t r a c t i o n o f q u a t e r n a r y ammonium
compounds. They r e p o r t e d t h e optimum pH
v a l u e , Xmax, d i s t r i b u t i o n c o e f f i c i e n t and
s e n s i t i v i t y of reactions f o r t h e ion
a s s o c i a t i o n complexes o f n e o s t i g m i n e methyl
s u l p h a t e . I n a g e n e r a l procedure f o r
d e t e r m i n i n g t h e q u a t e r n a r y ammonium
compounds, 0 . 5 m l o f 0.01% s o l u t i o n of
compound is added t o 5 m l o f a b u f f e r s o l u t i o n o f a p p r o p r i a t e pH, 0 . 5 m l of 0.1% dye
43 I
NEOSTIGMINE
s o l u t i o n i s added, t h e complex is e x t r a c t e d
i n t o 5 m l o f chloroform and absorbance o f
o r g a n i c phase i s measured a t 400-434 nm.
Relative e r r o r i s less t h a n 2 % .
Tsubouchi (35) 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
d e t e r m i n a t i o n o f o r g a n i c c a t i o n s by
s o l v e n t e x t r a c t i o n w i t h tetrabromophenolp h t h a l i e n e t h y l e s t e r . 5 1.11 of t h e sample
c o n t a i n i n g less t h a n 0.01 WM of n e o s t i g m i n e
was e x t r a c t e d w i t h 2 m l o f 0.07% e t h a n o l i c
tetrabromophenolphthalien e t h y l e s t e r
(potassium s a l t ) and 5 m l o f b o r a t e phosphate b u f f e r (pH l o ) , d i l u t e t o 25 m l
w i t h water and shake f o r 2 minutes w i t h 1 0 m l
of 1,2,dichloroethane.
S e p a r a t e and f i l t e r
t h e o r g a n i c phase and measure i t s
absorbance a t 615 nm a g a i n s t r e a g e n t b l a n k .
The c o e f f i c i e n t o f v a r i a t i o n was 1.11%.
8.4.2
U l t r a v i o l e t SDectronhotometric Method
Rita (36) developed a u l t r a v i o l e t
spectrophotometric assay o f neostigmine
methyl s u l p h a t e as t h e a l k a l i n e h y d r o l y s i s
p r o d u c t , 3- (dimethy1amino)phenol. An
a l i q u o t of i n j e c t i o n preparation equivalent
t o 5 mg o f n e o s t i g m i n e methyl s u l p h a t e was
a c i d i f i e d and any phenol o r p-hydroxy
b e n z o a t e p r e s e r v a t i v e p r e s e n t was
e x t r a c t e d w i t h chloroform. The r e s i d u a l
aqueous s o l u t i o n was made a l k a l i n e and
h e a t e d on steam b a t h f o r 30 m i n u t e s and
t h e 3- (dimethylamino) phenol formed is
determined by UV-spectrophotometry.
Kracmarova and Kracmar (29) d e s c r i b 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 e v a l u a t i o n of
n e o s t i g m i n e bromide and n e o s t i g m i n e methyl
sulphate with regards t o t h e degradation
p r o d u c t s . To 5 m l o f i n j e c t i o n s o l u t i o n ,
2 m l o f 0 . 0 5 N H2SO4 was added. The m i x t u r e
was d i l u t e d t o 10 m l w i t h water. The
absorbance was r e c o r d e d a t 261 nm f o r
n e o s t i g m i n e bromide o r a t 260 nm f o r
n e o s t i g m i n e methyl s u l p h a t e o r a t 266 nm
f o r b o t h o f t h e compounds.
432
Mytchenko and Kyrychenko (37) r e p o r t e d a

spectrophotometric determination o f
p r o s e r i n e ( n e o s t igmine methyl s u l p h a t e )
i n m e d i c i n a l f o r m u l a t i o n s . The t a b l e t s
are g r i n d e d i n w a t e r and shaked. 0.5%
s o l u t i o n i s f i l t e r e d and t h e absorbance o f
f i l t r a t e i s d i r e c t l y r e c o r d e d a t 260 nm.
T h i s method can a l s o be used f o r a n a l y s i s
o f n e o s t i g m i n e i n eye drops ( c o n t a i n i n g
a l s o p i l o c a r p i n e HC1) a f t e r d i l u t i o n . The
accuracy o f method i s w i t h i n ? 0 . 6 6 % .
8.4.3
I n f r a r e d S p e c t r o p h o t o m e t r i c Methods - I R
Mynka et a1 (38) r e p o r t e d t h e i n f r a r e d
s p e c t r o s c o p y o f drugs o f t h e amide class.
The I R spectrum of n e o s t i g m i n e methyl
s u l p h a t e was determined and i n t e r p r e t e d
by measurement a t 1732 cm-l, A n a l y s i s can
be done w i t h a maximum e r r o r o f 1 . 6 6 % .
Kracmarova and Kracmar (29) r e p o r t e d
s p e c t r o p h o t o m e t r i c a n a l y s i s of n e o s t i g m i n e
i n i n f r a r e d r e g i o n . Grind t h e sample
w i t h l i q u i d p a r a f f i n (12 d r o p s ) and r e c o r d
t h e spectrum i n a sodium c h l o r i d e c e l l i n
t h e range 3-15 1-1.
C h a r a c t e r i s t i c band f o r
neostigmine bromide and n e o s t i g m i n e methyl
s u l p h a t e a r e observed.
8.4.4
Photometric Method
Kracmarova and Kracmar (29) r e p o r t e d a
p h o t o m e t r i c method or t h e e v a l u a t i o n o f
n e o s t i g m i n e bromide and n e o s t i g m i n e
methyl s u l p h a t e as f o l l o w s :
To t h e i n j e c t i o n s o l u t i o n ( 1 . 5 ml) add
b u f f e r s o l u t i o n (pH 4 . 6 ) (5 m l ) , bromoc r e s o l p u r p l e s o l u t i o n (2 g i n 15 m l o f
water and 3 . 2 m l o f 0 . 1 N - potassium
hydroxide d i l u t e d t o 250 m l w i t h w a t e r )
(S ml) and chloroform (25 m l ) . Shake f o r
1 minute i n a s e p a r a t i n g f u n n e l , and f i l t e r
t h e chloroform l a y e r ; r e p e a t t h e e x t r a c t i o n
w i t h 2 m l of chloroform and d i l u t e t h e
combined e x t r a c t s w i t h chloroform t o 50 m l .
NEOSTIGMINE
433
To a 25 m l p o r t i o n add 0 . 1 N potassium
hydroxide (20 m l ) , shake f o r 30 seconds,
f i l t e r t h e aqueous l a y e r , d i l u t e t o 100 m l
w i t h w a t e r and measure t h e e x t i n c t i o n a t
570 nm.
8.5
8.5.1
Paper Chromatographic Method

Giebelmann (39) developed a p a p e r chromatog r a p h i c d e t e c t i o n f o r phenoxy compounds
having a phenyl e s t e r f u n c t i o n a l group
using t h e following s o l v e n t s - butanol-20%
formic a c i d ( 4 : 1 ) , methanol a c e t o n e t r i e t h a n o l a m i n e (100: 100: 3) and b u t a n o l water ( 6 : l ) . As l i t t l e as 2.5-10 1-16 of
n e o s t i g m i n e can be d e t e c t e d w i t h M i l l o n ' s
reagent.
Scott et a l , (40) r e p o r t e d an i n v e s t i g a t i o n
on t h e metabolism o f n e o s t i g m i n e i n
p a t i e n t s w i t h myasthenia g r a v i s . N e o s t i g m i n e an d m -h y d r oxyphen y 1t r i m e t h y 1ammon iurn
bromide are p r e c i p i t a t e d from u r i n e w i t h
aqueous bromine. The p r e c i p i t a t e i s
e x t r a c t e d with 50% methanol. T h i s e x t r a c t
is a p p l i e d t o Amberlite CG-50 r e s i n a t
pH 6.86. Ten b a s e s a r e e l u t e d w i t h 0 . 2 N
h y d r o c h l o r i c a c i d and s u b m i t t e d t o
chromatography on Whatman NO 541 p a p e r
w i t h butanol-ethanol-water-acetic a c i d
(32: 8 : 1 2 : 1) as s o l v e n t . The Rf v a l u e o f
n e o s t i g m i n e was between 0 . 5 - 0 . 3 .
Kracmarova and Kracmar ( 2 9 ) d e s c r i b e d
procedures f o r t h e determination o f
n e o s t i g m i n e bromide and n e o s t igmine methyl
sulphate i n various pharmaceutical preparat i o n s , a s w e l l as f o r t h e chromatographic
control of t h e degradation products;
3 dimethylaminophenol and (3-hydroxyphenyl)
trimethylammonium s a l t s , w i t h t h e u s e o f
Whatman No. 1 p a p e r , butanol-conc-aqueous
ammonia-water (3: 1:4) as s o l v e n t , and
Dragendorff r e a g e n t as t h e d e t e c t i n g a g e n t .
434
Clarke (11) d e s c r i b e d t h e f o l l o w i n g :
(1) Paper: Whatman No. 1, s h e e t 14 x 6 i n ,
b u f f e r e d by d i p p i n g i n a 5% s o l u t i o n o f
sodium hydrogen c i t r a t e , b l o t t i n g , and
d r y i n g a t 250 f o r 1 hour. I t can be
s t o r e d immediately.
Sample: 2 . 5 1-11 of a 1%s o l u t i o n ; i n 2 N

a c e t i c a c i d i f possible, otherwise i n 2 N
h y d r o c h l o r i c a c i d , 2 N sodium hydroxide,
o r ethanol.
S o l v e n t : 4 . 8 g of c i t r i c a c i d i n a m i x t u r e
of 130 m l o f water and 870 m l o f n - b u t a n o l .
This may be used f o r s e v e r a l weeks i f water
i s added from time t o time t o keep t h e
s p e c i f i c g r a v i t y a t 0.843 t o 0.844.
Development: Ascending, i n t a n k
8 x 11 x 15% i n ; 4 s h e e t s b e i n g run a t a
time. Time of run, 5 h o u r s .
Location under u l t r a v i o l e t l i g h t , a b s o r p t i o n ;
locat ion reagents : Iodoplati na te spray ;
weak r e a c t i o n ; bromocresol green s p r a y ,
weak r e a c t i o n . Rf 0.20.
( 2 ) Paper: Whatman No. 1 o r No. 3,
s h e e t 17 x 19 cm, implegnated by d i p p i n g
i n a 10% s o l u t i o n o f t r i b u t y r i n i n
acetone and d r y i n g i n a i r .
Sample: 5 p1 o f 1 t o 5% s o l u t i o n s i n
e t h a n o l o r chloroform.
Solvent:
A c e t a t e b u f f e r (pH 4 . 5 8 ) .
E q u i l i b r a t i o n : The b e a k e r c o n t a i n i n g t h e
solvent is equilibrated i n a thermostatically
c o n t r o l l e d o v e r a t 95O f o r about 1 5
minutes.
Development: Ascending, t h e paper i s f o l d e d
i n t o a c y l i n d e r and c l i p p e d , t h e c y l i n d e r
is i n s e r t e d i n t h e b e a k e r c o n t a i n i n g t h e
s o l v e n t which i s n o t removed from t h e oven.
A p l a t e - g l a s s d i s k t h i c k l y smeared with
s i l i c o n e g r e a s e may s e r v e as a cover. Time
of run, 15 t o 20 minutes.
NEOSTIGMINE
435
Location: Under u l t r a v i o l e t l i g h t ,
a b s o r p t i o n , Rf 1.00.
(3) Paper and sample a s i n 2 above.
S o l v e n t : Phosphate b u f f e r (pH 7 . 4 ) .
E q u i l i b r a t i o n : The peak c o n t a i n i n g t h e
solvent is equilibrated i n a thermostatic a l l y c o n t r o l l e d oven a t 86' f o r about
15 minutes.
Development:
as i n 2 above.
Location: Under u l t r a v i o l e t l i g h t ,
a b s o r p t i o n , Rf 0.66.
8.5.2
Thin-Layer Chromatography (TLC)

Anand (41) r e p o r t e d a t h i n - l a y e r chromatog r a p h i c and s p e c t r o p h o t o m e t r i c i n v e s t i g a t i o n on t h e i d e n t i f i c a t i o n o f a p h e n o l i c
i m p u r i t y i n n e o s t i g m i n e . The samples o f
neostigmine were a p p l i e d on TLC p l a t e s
c o a t e d w i t h alumina-D and were developed
f o r 30 minutes w i t h chloroform-benzenemethanol (5:4:1) t o g i v e a good s e p a r a t i o n .
Neostigmine was d e t e c t e d w i t h i o d i n e
vapours o r w i t h D r a g e n d o r f f ' s r e a g e n t . The
u l t r a v i o l e t spectrophotometric technique
is a l s o used f o r q u a n t i t a t i v e e s t i m a t i o n .
Neostigmine h a s a maxima a t 261 nm.
P o r s t and Kny (42) d e s c r i b e d a n a l y s i s o f
neostigmine eye drops u s i n g TLC system
and a b s o r p t i o n s p e c t r o s c o p y . The drug
i s d e t e c t e d and determined by TLC on
c e l l u l o s e and u l t r a v i o l e t o r v i s i b l e
s p e c t r o p h o t o m e t r y is done a f t e r f o r m a t i o n
o f c o l o r e d compound w i t h 3,S-dichloro-pbenzoquinonechlorimine o r sodium n i t r i t e
o r 4-dimethylaminobenzaldehyde.
Clarke (11) d e s c r i b e d t h e f o l l o w i n g :
P l a t e : G l a s s p l a t e , 20 x 20 cm, c o a t e d w i t h
a s l u r r y c o n s i s t i n g o f 30 g o f s i l i c a g e l G
i n 60 m l o f w a t e r t o g i v e - a l a y e r 0.25 mm
l i k e and d r i e d a t 110 f o r 1 hour.
436
Sample:
acid.
1 u1 o f a 1%s o l u t i o n i n 2 N a c e t i c
S o l v e n t : S t r o n g ammonia s o l u t i o n :
methanol (1.5 : 100). I t should be
changed a f t e r two r u n s .
E q u i l i b r a t i o n : The s o l v e n t i s allowed t o
stand i n t h e tank f o r 1 hour.
Development:
2 1 x 21 x 1 0
covered w i t h
evaporation.
Ascending, i n a t a n k ,
cm, t h e end o f t h e t a n k b e i n g
f i l t e r paper t o assist
Time o f r u n , 30 minutes.
Location r e a g e n t : A c i d i f i e d i o d o p l a t i n a t e
s p r a y , p o s i t i v e r e a c t i o n , R f 0.02,
8.5.3
Gas Liquid Chromatographic Methods

e t a1 (43) developed a s e n s i t i v e and
Chan -s e l e c t i v e a n a l y t i c a l method f o r t h e
measurement o f neostigmine i n human plasma
u s i n g gas chromatography. Plasma
c o n t a i n i n g neostigmine i s washed w i t h
e t h y l e t h e r and b u f f e r e d w i t h potassium
iodide-glycine s o l u t i o n , then iodideg l y c i n e - d r u g complexes are e x t r a c t e d i n t o
dichloromethane. The e x t r a c t i s e v a p o r a t e d
and t h e r e s i d u e is d i s s o l v e d i n methanol.
2 - 5 1-11 o f m e t h a n o l i c s o l u t i o n was i n j e c t e d
i n t o gas chromatographic g l a s s column
(2 m x 0 . 2 5 i n c h e s O.D.) packed w i t h
Diatomite CQ c o a t e d w i t h 3% o f OV-1, 3% o f
OV-17 o r 3 . 8 % of SE-30 and s i l a n i s e d ;
a n i t r o g e n s e l e c t i v e d e t e c t o r was used.
Pyridostigmine bromide was used a s i n t e r n a l
s t a n d a r d . The t e m p e r a t u r e was m a i n t a i n e d
a t 205OC and t h e flow r a t e o f n i t o g e n was
30 m l p e r minute. The d e t e c t i o n l i m i t
was 5 ng/ml and t h e r e was no i n t e r f e r e n c e
with o t h e r b a s i c d r u g s .
Ward et a 1 (44) d e s c r i b e d a simple GLC
a s s a y of neostigmine u s i n g d i e t h y l
analog o f neostigmine a s i n t e r n a l s t a n d a r d .
Neostigmine and i t s analog were d i s s o l v e d
i n 25 1.11 o f chloroform. 2 1.r1o f
chloroform s o l u t i o n was i n j e c t e d i n t o a
437
NEOSTIGMINE
gas chromatograph equiped w i t h a flame

i o n i s a t i o n d e t e c t o r and 1 . 2 m x 2 mm I . D .
g l a s s column packed with 5 % O V - 1 7 on
Gas-Chrom Q i s used a t 210' with helium
(40 ml/minute) a s a carrier gas and flame
i o n i z a t i o n d e t e c t o r . For d e t e r m i n a t i o n o f
neostigmine i n b i o l o g i c a l f l u i d s , t h e drug
i s e x t r a c t e d i n t o chloroform w i t h added
3-diethylcarbamoyloxy-"N-trimethylanil inium
i o d i d e as t h e i n t e r n a l s t a n d a r d . The
r e l a t i v e r e t e n t i o n time of t h e drug and t h e
s t a n d a r d is 1 t o 1 . 3 5 .
De Ruyter et a1 (45) developed a r e v e r s e d phase, i o n - p a i r l i q u i d chromatography o f
q u a r t e r n a r y ammonium compounds or t h e
determination of pyridostigmine,
n e o s t i g m i n e , and edrophonium i n b i o l o g i c a l
f l u i d s . The method i s based on e x t r a c t i o n
of neostigmine i n t o w a t e r - s a t u r a t e d
dichloromethane, t h e n b a c k - e x t r a c t i o n is
done w i t h t e t r a b u t y l ammonium hydrogen
s u l p h a t e t o enhance t h e r e c o v e r y t o more
t h a n 86%. The e x t r a c t s a r e s u b m i t t e d t o
chromatography on a v a r i e t y o f r e v e r s e d phased columns f i t t e d with 214 nm
d e t e c t o r s . For b i o l o g i c a l e x t r a c t s a
column (15 cm x 4 . 6 mm) o f U l t r a s p h e r e
o c t y l ( 5 pm) provided with an RP-2
pre-column was used. For neostigmine
e l u t i o n was c a r r i e d o u t w i t h a s o l u t i o n
c o n t a i n i n g sodium h e p t a n e s u l p h o n a t e
(0.01 M ) , sodium hydrogen phosphate (0.01 M)
and t e t r a m e t h y l ammonium c h l o r i d e (2.5 mM)
i n aqueous 20% a c e t o n i t r i l e . The mobile
phase (2 m l p e r minute) was a d j u s t e d t o
pH 3 with s u l p h u r i c a c i d , edrophonium was
used a s an i n t e r n a l s t a n d a r d . The
c a l i b r a t i o n graphs were r e c t i l i n e a r f o r
u p t o 400 ng per m l . The l i m i t of
d e t e c t i o n was 5 ng p e r m l . The c o e f f i c i e n t
o f v a r i a t i o n was 1 . 5 % .
Davison e t a 1 (46) d e s c r i b e d a method f o r
simultaneous monitoring of plasma l e v e l of
neostigmine and p y r i d o s t i g m i n e i n man.
The a s s a y i n v o l v e p r e l i m i n a r y ion p a i r
438
e x t r a c t i o n o f t h e drugs and i n t e r n a l
s t a n d a r d s (3-diproypl carbamoyloxy) 1-methyl
pyridinium bromide) w i t h t h e u s e o f potassium
i o d i d e and o f g l y c i n e b u f f e r . The e x t r a c t
i s a n a l y s e d by GLC (10% o f OV-17 on
Chromosorb W-AW (100-120 mesh) with a
n i t r o g e n - s e n s i t i v e d e t e c t o r . The calib r a t i o n graphs a r e r e c t i l i n e a r and
reproduced o v e r t h e range 5-100 ng p e r m l
o f e i t h e r drugs i n 3 m l samples.
Pohlmann and Cohan (47) developed a s i m p l i f i e d d e t e c t i o n o f q u a r t e r n a r y ammonium
compounds by gas chromatography. The
method h a s been a p p l i e d t o p y r i d o s t i g m i n e ,
neostigmine and a c e t y l c h o l i n e . The
q u a r t e r n a r y s a l t s i n t h e serum o r u r i n e
a r e first converted w i t h potassium
t r i i o d i d e (K13) i n t o t h e i r i o d i e s which
a r e e x t r a c t e d i n t o chloroform. The
e x t r a c t i s evaporated i n vacuum and t h e
r e s i d u e is d i s s o l v e d i n water o r hexane.
The s o l u t i o n is i n j e c t e d on t o a column
( 1 . 8 m x 2 mm) o f Porapak Q (800-100 mesh),
Chromosorb 101 (80-100 mesh) o r Chromosorb
105 (80-100 mesh), o p e r a t e d a t 14SoC-17O0C
with helium o r n i t r o g e n a s c a r r i e r gas
(20 m l p e r ml). On t h e column t h e
q u a r t e r n a r y ammonium i o d i d e s a r e t h e r m a l l y
decomposed, and t h e methyl i o d i d e r e l e a s e d
i s measured w i t h a 63Ni e l e c t r o n - c a p t u r e
detector. Extraction y i e l d s a r e a t l e a s t
95% and down t o 1 0 f-mol of q u a r t e r n a r y
ammonium compound can be d e t e c t e d w i t h
good r e p r o d u c i b i l i t y .
Chan and Dehghan (48) d e s c r i b e d a GLC
method f o r i s o l a t i o n and d e t e r m i n a t i o n o f
neostigmine, p y r i d o s t i g m i n e and t h e i r
m e t a b o l i t e s i n human b i o l o g i c a l f l u i d s .
The method i n v o l v e s a p r e l i m i n a r y ,
s e l e c t i v e i o n - p a i r e x t r a c t i o n o f t h e drugs
and t h e i r m e t a b o l i t e s i n t o dichloromethane
and i n (dichloromethane-acetone) ,
r e s p e c t i v e l y . The a n a l y s i s o f e x t r a c t was
done by GLC on a column o f 3% (drug) o r
10% ( m e t a b o l i t e ) o f OV-17 on Chromosorb W AW,
439
NEOSTIGMINE
with a n i t r o g e n - s e n s i t i v e d e t e c t o r . For
t h e d e t e r m i n a t i o n o f 3-hydroxytrimethyl
a n i l i n i u m i o n ; a major m e t a b o l i t e o f
neostigmine, 3-hydroxy-N-methylpyridinium
ion was used a s a i n t e r n a l s t a n d a r d . As
l i t t l e as 3 ng/ml o f neostigmine and upto
50 ng/ml o f i t s m e t a b o l i t e can be
determined i n b i o l o g i c a l samples.
8.6
I o n - S e l e c t i v e E l e c t r o d e s Method
Kina et a1 (49) d e s c r i b e d a method of neostigmine
a n a l y s i s based on i o n - s e l e c t i v e e l e c t r o d e s
s e n s i t i v e t o some o r g a n i c compounds used a s drugs.
Liquid membrane e l e c t r o d e s s e n s i t i v e t o n e o s t i g mine have been made by t h e i o n - a s s o c i a t i o n
e x t r a c t i o n method. The exchange components used
were c r y s t a l v i o l e t , d i p i c r y l a m i n e , sodium
t e t r a p e n t y l b o r a t e , l , l O - p h e n a n t h r o l i n e , 4,7diphenyl- 1,lO-phenanthroline . The s o l v e n t s used
f o r t h e s e compounds were 1,Z-dichloroethane and
n i t r o b e n z e n e . The c h o i c e of s o l v e n t s a f f e c t e d
with s e l e c t i v i t i e s o f t h e membranes b u t t h e c h o i c e
o f ion-exchange components d i d n o t . The
s e l e c t i v i t i e s r e l a t i v e t o u n i v a l e n t c a t i o n s were
mostly b e t t e r than 10-4. The u s e f u l c o n c e n t r a t i o n
range o f e l e c t r o d e s was 10 VM t o 0 . 1 M w i t h a
response o f 59 mV p e r decade change i n t h e
c o n c e n t r a t i o n o f t h e drug. The e l e c t r o d e s could
be used i n a n a l y s i s of mixed pharmaceutical
preparations.
Diamandis and C h r i s t o p o u l o s (26) r e p o r t e d a
p o t e n t i o m e t r i c t i t r a t i o n o f neostigmine and o t h e r
pharmaceutical compound i n pharmaceutical formul a t i o n with sodium t e t r a h y d r o b o r a t e . (See
The end p o i n t was d e t e c t e d by a
Section 8.2.1.).
tetraphenylborate-selective e l e c t r o d e .
8.7
P o l a r o g r a p h i c Method
Novotny (50) devised a p o l a r o g r a p h i c method f o r
t h e d e t e r m i n a t i o n o f neostigmine i n pharmaceutical
p r e p a r a t i o n s having a c o n c e n t r a t i o n o f 0 . 5 mg/ml
o f t h e drug. T h i s method i s based on t h e
h y d r o l y s i s of neostigmine and n i t r a t i o n o f
r e s u l t i n g phenol, followed by polarography o f
r e s u l t i n g d e r i v a t i v e . The neostigmine s o l u t i o n
( 0 . 2 t o 0 . 6 m l of 0 . 1 % ) i s e vapor ated t o dr yness
i n a 50 m l be a ke r on a water b a t h . 1 bll o f KOH
s o l u t i o n (20%) i s added and warmed on t h e b a t h
f o r 20 minutes and a ga i n e va por ated t o dryness.
0 . 5 M 1 of wa te r and 2 m l o f conc. HN03 (65%) i s
added and warmed f o r 20 minutes and cooled. 10 M 1
of KOH s o l u t i o n (20%) is added and t h e volume
made upto 1 4 . 5 m l w i t h water. The e s t i m a t i o n i s
c a r r i e d out by pol a rogra phy, w i t h a galvanometer
s e n s i t i v e t o 10-9 amp. To determine t h e
c o n c e n t r a t i o n o f neostigmine i n an unknown
s o l u t i o n , s o l u t i o n o f t h e unknown c o n c e n t r a t i o n o f
neostigmine i s t r e a t e d as d e s c r i b e d above, both
with and wit hout a known amount of neostigmine.
Acknowledgements
The a u t h o r s would l i k e t o thank Mr. Syed A l i Jawad, C o l l e g e
of Pharmacy, King Saud U n i v e r s i t y f o r h i s t e c h n i c a l assist a n c e and Mr. Ta nvir A. Butt or 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 y p i n g t h e manuscript.
441
NEOSTIGMINE
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PIRENZEPINE DIHYDROCHLORIDE
ffumeiida A . ER-Ob&id*,
S a L h A . Rubha,&**,
and A b d u U a h A . M-Badrr*

VOLUME 16
445

HUMEIDA A. EL-OBEID ETAL.
446
Contents
1. Description
1.1 Nomenclature
1.2 Formulae
1 . 3 Molecular Weight
1 . 4 Elemental Composition
1.5 Appearance
2.
Physico-chemical Properties
2.1
2.2
2.3
2.4
2.5
3.
Melting Range
Solubility
Distribution Ratio
Spectral Properties
Crystal Structure
Synthesis
4. Stability
5.1 Spectrophotometric Methods
5 . 3 Radioimmunoassays
6.
Pharmacokinetics
6.1 Absorption
6.2 Distribution
6 . 3 Metabolism
6 . 4 Excretion
7.
Pharmacology
Antisecretory Effects
Efficacy
Adverse React ions
Tissue Selectivity
7 . 5 Mechanism of Action
7.1
7.2
7.3
7.4
Acknowledgement
Re ferences
1.
447
Descrivt ion
1.1 Nomenclature
1.1.1
Chemical Names
5,11-Dihydro-ll-[(4-methyl-l-piperazinyl)
a c e t y l ] -6H-pyrido[ 2,3-b] [ 1 , 4 ] b e n z o d i a z e p i n 6-one d i h y d r o c h l o r i d e monohydrate.
11- [ (4-Methyl-1-piperazinlyl) a c e t y l ] - p y r i d o
[ 2 , 3-b] [ 1 , 4 ] b e n z o d i a z e p i n - 6 (5 H) -one
d i h y d r o c h l o r i d e monohydrate.
1.1.2
G e n e r i c Names
L-S 519, P i r e n z e p i n e
1.1.3
Trade Names
B i s v a n i l , G a s t e r i l , G a s t r o z e p i n , Leblon,
Tabe, Ulcosan.
1.2
Formulae
1.2.1
Empirical
(dihydrochl o r i d e )
C 9H2 3C 12N502
C19H21N502
1.2.2
(base)
Structural
I
CH2-
.2HC1, H 2 0
NwN
CH3
448
1.2.3 CAS No.

[ 29868-97-11 as dihydrochloride salt.
[28797-61-71 as free base.

424.33 as dihydrochloride salt
351.42 as free base.
Dihydrochloride salt: C 53.78%, H 5.46%, N 16.50%,
0 7.52%, C1 16.71% (1).
Free base: C 64.94%, H 6.02%, N 19.93%, 0 9.11% (2).
1.5
Appearance
Pirenzepine dihydrochloride is a white crystalline
substance (2).
2. Physico-chemical Properties
2.1 Melting Range

Pirenzepine dihydrochloride melts in the range
250-252OC (corrected) with decomposition (I).
2.2 Solubility
Pirenzepine dihydrochloride is readily soluble
in water, slightly soluble in methanol and
practically insoluble in ether (1).
2.3 Distribution Ratio
Pirenzepine is a tricyclic compound, but its
physioco-chemical properties differ considerably
from those of other tricyclic compounds which are
used as centrally-acting drugs. Pirenzepine is
unusual because of its pronounced hydrophilic
properties. Comparison of the distribution
coefficient of pirenzepine with those of other
449
t r i c y c l i c drugs a t pH 7.4, has been c a r r i e d out (3).

The r e s u l t s depicted i n Table 1 show t h a t
pirenzepine i s 200-13000 l e s s l i p i d s o l u b l e ,
Table 1:
D i s t r i b u t i o n c o e f f i c i e n t of pirenzepine
and o t h e r t r i c y c l i c drugs.
Drug
-
H 7.4
$Ppe---Chlorpromazine
3160
CNS e f f e c t s
1600
250
Dibenzepine
51
Pirenzepine
0.23
Cyprohept adine
Imipramin
Eberlein -e t a1 (3) analysed t h e pirenzepine

molecule i n t o f i v e s t r u c t u r a l elements. Each o f
t h e f i v e elements can be a l l o c a t e d a hydrophobic
s u b s t i t u e n t consant (TX) and compared with t h o s e
assigned f o r corresponding s t r u c t u r a l elements i n
imipramine molecule (Table 2 ) . A p o s i t i v e T X
value i n d i c a t e s contribution t o t h e l i p o p h i l i c
c h a r a c t e r of t h e molecule and a negative T X
value i n d i c a t e s contribution t o h y d r o p h i l i c i t y of
t h e molecules.
HUMEIDA A. EL-OBEID E T A L .
450
Table 2 :
Contribution o f t h e d i f f e r e n t fragments of
p i r e n z e p i n e and imipramine m o l e c u l e s t o t h e i r
distribution coefficients.
CH2CH2CH2
'
CH,
CH,
fN I
1
4
-
5
-
I m ip r am i n e
Pirenzepine
~~~~
Fragment
No.
Fragment
structure
TX
-CH2-CH2-
+10.02
- 1 .4 9
+ 1.96
+ O . 65
1.96
+ l .96
\
/
N-CH2CH2CH2-
Fragment
structure
+ 0.50
\
/
N-COCH2-
nx
- 0 .9 7
n
~
LogBase ( c a l c u l a t e d )
+ 5.12
(calculated)
LogBase
+O. 010
LogBase(experimental)
+ 4.80
LogBase (experiment a l pH 7 . 4 )
-0.64
45 1
I t can b e shown t h a t t h e s t r u c t u r a l elements 1 and

4, i n p a r t i c u l a r , c o n t r i b u t e l a r g e l y t o t h e
h y d r o p h i l i c i t y of p i r e n z e p i n e . Fragments 1 and
4 i n p i r e n z e p i n e c o n s i s t o f amide groupings each
w i t h two d i p o l e s which can i n t e r a c t s t r o n g l y with
water molecules. The p y r i d y l group of fragment 2
i n p i r e n z e p i n e i s a l s o expected t o c o n t r i b u t e t o
t h e h y d r o p h i l i c i t y of t h e molecule. Adding up t h e
i n d i v i d u a l hydrophobic c o n s t a n t s f o r p i r e n z e p i n e
m o l e cu l ar fragments a l o g P of 0.01 i s o b t a i n e d
f o r t h e whole molecule, which i n d i c a t e s t h a t p i r e n z e p i n e a s t h e uncharged free b a s e w i l l be d i s t r i b u t e d uniformly between t h e o c t a n o l and wa te r .
A t p h y s i o l o g i c a l pH, however, t h e r a t i o s h i f t s
even more towards w at er s o l u b i l i t y due i o n i z a t i o n
o f t h e molecule. Under p h y s i o l o g i c a l c o n d i t i o n s
(pH 7.4) t h e ex p e r i m e n t al l y determined l o g P is
- 0.64 (Table 2 ) , i n d i c a t i n g t h a t about 5 times
more o f t h e compound i s d i s t r i b u t e d i n t h e
aqueous phase t h an i n o c t a n o l . Under t h e same
c o n d i t i o n s imipramine accumulates much more
s t r o n g l y i n t h e o r g a n i c phase. As w i l l b e shown
l a t e r , t h e pronounced h y d r o p h i l i c p r o p e r t i e s o f
p i r e n z e p i n e e x p l a i n many o f t h e s p e c i f i c pharmac o k i n e t i c c h a r a c t e r i s t i c s of t h e drug.
2.4
Spectral Properties
2.4.1
U l t r a v i o l e t Suectrum
The u l t r a v i o l e t a b s o r p t i o n spectrum o f
p i r en z ep i n e i n met h an o 1/HC 1 was ob t a i n ed
on a Cary 219 s p e c t r o ph o to m e te r . The
spectrum, shown i n F i gu r e 1, i s c h a r a c t e r i z e d by a maximum a t 283 nm and a minimum
a t 264 nm. The r e s u l t s a g r e e w i t h t h o s e
r e p o r t e d i n t h e l i t e r a t u r e (4, 5 ) . I t
is a l s o reported t h a t pirenzepine i n
0 . 1 N sodium hydroxide s o l u t i o n shows
a b s o r p t i o n maximum a t 295 nm ( 4 ) .
2.4.2
The i n f r a r e d a b s o r p t i o n spectrum o f
p i r e n z e p i n e o b t a i n ed from a potassium
bromide d i s p e r s i o n was r e c o r d e d on a Pye
452
HUMEIDA A. EL-OBEIDETAL.
Figure 1 : ULTRAVIOLET SPECTRUM OF PIRENZEPINE DItiYDRDQiLORIDE

IN METHANOLIC HCl.
453
Unicam SP 1025 spectrometer and i s shown

i n Figure 2 . The c h a r a c t e r i s t i c bands of
t h e spectrum with t h e assignments a r e
l i s t e d below:
Frequency (cm-l)
Assignment
3520, 3463
Amide N-H s t r e t c h
3240
Water molecule
Aromatic C-H s t r e t c h
3020, 2990
2700
- 2400
stretch
1 7 1 7 , 1678
Amide C = 0
1605, 1585, 1502,)

1479, 1432
1
Aromatic C - C ,
C
N stretch.
1370
Aryl C
800,
2.4,3
t -Amine s a l t N-H
785,
760
-*
N stretch
Aromatic C - H outof-plane bending.
'H Nuclear Magnetic Resonance ('H
NMR)
SDectrum
Pirenzepine s o l u t i o n i n D 2 0 was used t o
obtain t h e proton magnetic spectrum on a
Varian-T60 NMR spectrometer with TMS a s
i n t e r n a l standard. The spectrum i s shown
i n Figure 3, and assignment of t h e
chemical s h i f t s t o t h e d i f f e r e n t protons
is summarized below:
Microns
VI
P
4000
Wavenumber
3000
2500
2000 1800 1600 1400
1000 800
Figure 2 : INFRARED SPECTRUM OF PIRENZEPINE DIHYDROCHLORIDE, KBr DISC.
600 400
200
400
200
100
I7
16
Rl
u
l . . . . l . . . . l . . . . 1 . . . . l . . . . l . . . . 1 . . . . 1 . . . .
7.0
60(ppm)Ei.O ( 6 ) 4-0
3-0
2-0
1.0
0
8.0
Figure 3 : H' SIR SPECIRUM CF PIRENZEPINE DIHYDROCMRIDE IN DZO WITH ?Hs As

IkTERUL REFERENCE.
456
14
= c 1 2
n
N -CH3
13
Multiplicity
18
CH~-
Chemical s h i f t
(6)
15
Proton
Assignment
No. of
protons
3.05
Sing 1e t
-N-CH
-3
3.55
Mu I t i p 1et
-CH -(piperazine)
-2
0
It
4.15
Mult i p l e t
- 3 - C -
7.7
Mu 1t i p l e t
Aromat ic-H
8.4
Mu 1t i p l e t
Aromatic-H
2.4.4
13C Nuclear Magnetic Resonance ( 13C NMR)
Spectrum
The 13C NMR s p e c t r a of pirenzepine i n a
mixture of D20 and formic acid using
dioxane a s i n t e r n a l reference a r e obtained
using a J e o l FX 100 90 MHz spectrometer a t
ambient temperature. Figure 4 and 5 show
t h e IH-decoupled and off-resonance
s p e c t r a , r e s p e c t i v e l y . The carbon
chemical s h i f t s , assigned on b a s i s of
t h e off-resonance s p l i t t i n g p a t t e r n and
t h e t h e o r i e s of chemical s h i f t s , are
presented below:
451
Dioxan
67.4
Figure 4 : 'ti DEaWPLED 13C tW SPEclRuM OF PIRENZEPINE DIHYDROMLORIDE I N DzO/

RIRMIC ACID FlIXlURE W I ' M DIOXANE AS INNWU. REFERNECE.
a'
Figure 5 :
458
IY
10
121
0-c
CH2l3
Chemical S h i f t
(61
18
NWN
CH3
17
16
Multiplicity
44.83
Quartet
50.72
Triplet
51.87
Triplet
57.77
Triplet
122.41
Doublet
128.22
Doub 1e t
128.88
Doub 1e t
130.57
Doublet
131.57
Singlet
132.15
Doublet
132.74
Doub 1e t
135.24
Singlet
139.43
Doublet
143.61
Singlet
146.77
Singlet
165.21
Singlet
165.31
Sing 1et
Carbon
Assignment
18
15
16
14
17
13
clo
8
c7
3
6
c9
c4
c7
c2
c31
c21
12
2.4.5
459
Mass SDectra
The 70 eV e l e c t r o n impact mass spectrum of
pirenzepine, presented i n Figure 6 , was
obtained on Varian MAT 311 mass spectrometer
using ion source pressure of
Torr, ion
source temperature of 18OoC and an emission
current o f 300 PA. The molecular ion i s
d e t e c t a b l e a t m/e 351 and t h e base peak
a t m/e 113. A proposed fragmentation
p a t t e r n and t h e mass/charge r a t i o s o f t h e
major fragments i s shown i n Scheme 1. The
d i r e c t chemical i o n i z a t i o n (DCI) spectrum
(Figure 7) was obtained on a Finnigan 4000
mass spectrometer using methane gas a
reagent with ion e l e c t r o n energy of 100 eV,
ion source pressure o f 0 . 3 Torr, ion
source temperature of 150C and emission
current o f 300 PA. The spectrum is dominated
by a quasi-molecular ion (M + 1 ) . Two
peaks appearing a t m/e 380 and m/e 392 a r e
a t t r i b u t e d t o t h e t r a n s f e r o f carbocations
from t h e c a r r i e r gas. The mass s p e c t r a l
assignments of t h e prominent ions under
DCI conditions a r e shown i n Table 3.
Table 3.
m/e
Species
392
[M + C ~ H ~ I +
380
[M
.__
2.5
Mass s p e c t r a l assignments o f
pirenzepine using DCI with methane
a s reagnet gas.
352
MH+
35 1
M+
CZH51+
Crystal S t r u c t u r e
The c r y s t a l s t r u c t u r e o f pirenzepine
f r e e base was s t u d i e d by Ruzic-Toros
Prodic (6). Pirenzepine monohydrate
monoclinic, P2
a = 13.160 [7], b
1/c
monohydrate
and Kojecwas t o be
= 12.766 [Sly
460
Figure 6 : ELECTRON IMPACT W S SPECTHW! OF PSWZEPINE.
Figure 7 : DIRECT WICAL IONIZATION W S SPECIIW OF PIRENZEPINE.
@%
N
-238
=c I
%lac.
DN -CH,
-N
H,C
L
dh
0
=c I
____t
+p
'd
-CH
-43 3-N = CH2
H2C
m/e 113
5
m/e 351
-42
=N=CHC
2
2
m/e 113
=CH2
m / e 70
N+3
~cH
m/e 71
tH
1
~ A
~ 1N - CHJ
0 = C-CH-N
CH3
- 140
m/e 211
Scheme 1.
-cH3-
Proposed f r a p e n t a t i o n of pirenzepine.
m/e 56
462
c = 12.439 171 A,
B = 113.2 [4]O, U
Z = 4, Dc = 1 . 2 7 7 Mgm-3, p(Mo Ka)
1920.8 Ao3,
-1
= 0.128 mm
=
F i n a l R = 0.045 f o r 2681 observed r e f l e x i o n s .

Bond d i s t a n c e s and a n g l e s (Table 4) i n t h e f u s e d
r i n g s , benzene, d i a z e p i n e and p y r i d i n e , a r e
a f f e c t e d by c o n j u g a t i o n . Deviation from t h e v a l u e s
expected or t h e given atom type and h y b r i d i z a t i o n
could n o t be e x p l a i n e d by t h e thermal v i b r a t i o n s
of t h e atoms (Table 5 ) . The s h o r t e n i n g o f
C[7] - C [ 8 ] , 1.372[4]Ao, i n t h e benzene r i n g could
be due t o i n t e r m o l e c u l a r c o n t a c t s , i n v o l v i n g
t h e s e atoms, with N[1][3.352[3]; 3.470[3]Ao.
The seven-membered d i a z e p i n e r i n g is f o l d e d a t
51 - C[6] and 111 t o form a b o a t conformations.
The p y r i d i n e and benzene r i n g s a r e p l a n a r while
t h e p i p e r a z i n e r i n g i n a c h a i r conformation. A
v a l u e o f 60.9[41 was o b t a i n e d f o r t h e d i h e d r a l
a n g l e between t h e benzene and t h e p y r i d i n e r i n g s .
The w a t e r molecule is involved i n hydrogen bonds
with t h e amide group o f t h e d i a z e p i n e r i n g ,
51 - H[5] .. O[W]I2.800[3])and O(W)-H(W)2
O[ 61 c2.907[ 31 Ao) . N [ 4 ] o f t h e p i p e r a z i n e r i n g
is hydrogen-bonded t o t h e water molecule by
O(W)- H(W)1
N[4](2.821[3]A0).
Molecular
packing i s a l s o i n f l u e n c e d by van d e r Wall
i n t e r a c t i o n s . The r e l a t i v e o r i e n t a t i o n o f t h e
p y r i d i n e and p i p e r a z i n e r i n g , w i t h a d i h e d r a l
a n g l e o f 2 2 . 2 [ 41, e n a b l e s a s h o r t i n t r a m o l e c u l a r
c o n t a c t 11 ... 11 o f 3.087[2]Ao. The
s t r u c t u r a l formula and atomic numbering o f
p i r e n z e p i n e i s given i n F i g u r e 8. The confonnat i o n o f t h e molecule is shown i n F i g u r e 9 and
Table 6.
...
. ..
--
Trummlitz e t a l . ( 7 ) , u s i n g X-ray a n a l y s i s ,
determined t h e c r y s t a l s t r u c t u r e s o f p i r e n z e p i n e
d i h y d r o c h l o r i d e and i t s monoprotonated form,
p i r e n z e p i n e monohydrochloride (Figure 10)
Molecular mechanics (MMPI) and semiempirical
quantum chemical (MNDO) c a l c u l a t i o n s showed t h a t
t h e c a l c u l a t e d minimum energy conformations o f
t h e t r i c y c l e and of t h e e x o c y c l i c amide group are
T a b l e 4.
463
Bond d i s t a n c e s (A) and a n g l e s (O)
C(lO)-C(15)
C(lO)-H(lO)
N(l1)-C(12)
N(l1)-C(15)
N(l1)-C(16)
C(12)-C(13)
1 . 3 4 8 (3)
1.315 (3)
1.364 (4)
0 . 9 9 (2)
1 . 3 7 5 (3)
0.97 (2)
1 . 3 9 5 (3)
0 . 9 7 (2)
1.362 (3)
1 . 4 0 3 (3)
0.86 (2)
1 . 2 3 1 (3)
1 . 4 8 4 (4)
1 . 3 9 7 (4)
0.97 (2)
1.372 (4)
1.377 ( 4 )
1 . 0 0 (3)
1 . 3 8 3 (4)
0.92 ( 2 )
1 . 3 8 1 (3)
0.94 (2)
1 . 4 3 8 (3)
1 . 4 3 3 (3)
1.362 (2)
1.387 (3)
C(Z)-N(l)-C(12)
c (1) -c (2) -c (3)
N (1) -C ( 2 ) -H (2)
C(3)-C(Z)-H(2)
c (2) -c (3) -c (4)
C (2) -C (3) -H( 3)
C (4) -C (3) -H (3)
c (3) -c (4) -c (13)
C(3)-C (4)-H(4)
C ( 1 3) -C (4) -H (4)
C(6)-N(S)-C(13)
C(6)-N(S)-H(5)
C (13) -N (5) -H (5)
N (5) -C (6) -0 (6)
N (5) -C (6) -C (14)
O(6) -C (6) -C (14)
C(8) -C (7) -C (14)
C (8) -C (7) -H (7)
C (14) -C (15)
C(16)-O(16)
C (16) -C ( 1 7)
C (17) -N (1' )
C(17)-H(17)1
C ( 1 7) -H ( 1 7) 2
N(l')-C(Z')
N ( 1 ' ) -C ( 6 ' )
C (2 ' ) -C ( 3 ' )
C (2 ' ) -H(2 ' ) 1
C (2 ' ) -H (2 ' ) 2
C ( 3 ' ) -N (4 ' )
C(3')-H(3')1
C ( 3 ' ) -H ( 3 ' ) 2
N ( 4 ' ) -C ( 5 ' )
N ( 4 ' ) -C ( 7 ' )
C (5 ' ) -C ( 6 ' )
C ( 5 ' ) -1-1 ( 5 ' ) 1
C ( 5 ' ) -H ( 5 ' ) 2
C ( 6 ' ) -H(6 ' ) 1
C (6 ' ) -H ( 6 ' ) 2
C ( 7 ' ) -H ( 7 ' )
C ( 7 ' ) -H( 7 ' ) 2
C (7 I ) -H ( 7 ' ) 3
O(W)-H(W)1
O(W)-H(W)2
116.7 (2)
122.9 (2)
115 (1)
1 2 1 (1)
1 1 9 . 6 (2)
120 ( 1 )
120 (1)
1 1 8 . 7 (2)
123 (1)
118 (1)
129.8 (2)
114 ( 1 )
115 (1)
119.3 (2)
1 1 9 . 3 (2)
121.2 (2)
1 2 0 . 5 (2)
121 (2)
1 .3 8 8 (3)
1.229 (3)
1 .5 1 3 (4)
1.452 (3)
1 . 0 1 (2)
1 . 0 5 (2)
1 .4 6 0 (3)
1.454 (3)
1 .5 0 5 (4)
1 .0 4 (2)
1 .0 4 (2)
1 .4 6 5 (3)
1.05 (2)
1 .0 1 (3)
1.462 (4)
1.468 (5)
1.502 (4)
1.03 (2)
1 .0 1 (2)
1 .0 4 (2)
1 . 0 3 (3)
1.09 (3)
0.97 (4)
1 . 0 5 (3)
0 .9 3 (3)
0 . 7 8 (5)
C (12) -N ( 1 1 ) -C (16)
C (15) -N (11) -C(16)
N ( 1 ) -C ( 12 ) -N ( 1 1 )
N ( l ) -C (12) -C (13)
N (11) -C (12) -C(13)
C(4) -C (13) -N (5)
C (4) -C (13) -C (12)
~ ( s- cj( 1 3 j -c (12)
C (6) -C (14) -C (7)
C(6) -C (14) -C (15)
c (7) -c (14) -c (15)
C ( 10) -C (15) -N ( 1 1)
C(10) -c (15) -c (14)
N (11) -C (15) -C (14)
N (11) -C (16) -0 (16)
N (11) -C (16) -C (17)
0 (16) -C (16) -C (17)
C (16) -C (17) -N ( 1 ' )
1 2 4 .9
120.6
1 1 7 .0
125.0
118.0
119.8
116.8
1 2 3 .1
1 1 8 .1
123.2
1 1 8 .6
1 2 0 .5
1 2 0 .9
1 1 8 .5
120.8
117.9
1 2 1 .3
113.2
464
Table 4.
Contd .....
C (14) -C (7) -H (7)

C (7) -C (8) -C (9)
C (7) -C (8) -H (8)
C (9) -C (8) -H (8)
C (8) -C (9) -C (10)
C (8) -C (9) -H(9)
C(l0)-C(9) -H(9)
C (9)-C (10) -C (15)
C(9)-C(lO)-H(lO)
C(lS)-C(lO)-H(lO)
C( 12) -N(11) -C(15)
Table 5.
118 (2)
120.2 (3)
120 (2)
120 (2)
120.4 (2)
123 (1)
117 (1)
1 1 9 . 4 (2)
1 2 1 (1)
119 (1)
1 1 4 . 3 (1)
C (1 7) -N ( 1 ' ) -C (2 ' )
C(17)-N(lf)-C(6')
C (2 ' ) -N ( 1 ' ) -C (6 ' )
N ( 1 ' ) -C (2 ' ) -C ( 3 ' )
C(2')-C(3')-N(4')
C(3' ) -N ( 4 ' ) -C(S')
C (3 ' ) -N ( 4 ' ) -C ( 7 ' )
C(5 ) -N(4' ) -C ( 7 I )
N (4 ') -C ( 5 ') -C (6' )
N ( 1 ' ) -C(6') -C(S')
H(W) l-O(W) -H(W) 2
Final atomic coordinates (x

5
x 10
lo4
111.0 (2)
111.7 (2)
1 1 0 . 6 (2)
109.9 (2)
110.8 (2)
1 0 9 . 3 (2)
1 1 0 . 9 (2)
110.9 (2)
1 1 0 . 1 (2)
1 1 0 . 6 (2)
110 (4)
f o r C , N and 0;
f o r H) and i s o t r o p i c thermal parameters
(x l o 2 )
U
eq
U
eq
i s derived from t h e a n i s o t r o p i c thermal parameters by

1
= - C . C . U..a* a * . a * . a . . a
3 1 J 1J
J 3 1 j
or
6263
6199
6380
6615
7023
6726
7131
5060
4228
4186
4978
6635
6537
6695
5866
5818
7373
7400
8146
8712
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(2)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
1423
1306
372
-503
-1266
-1540
-2330
-1465
-932
143
698
716
588
-404
-922
162
1361
1426
1997
1375
(1)
(1)
(2)
(1)
(1)
(1)
(1)
(1)
(2)
(2)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
6087
4985
4557
5255
7162
8057
8634
8510
8677
8615
8379
7947
6761
6403
8266
8216
8752
9729
8376
7805
ueq('42)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(I)
(1)
(2)
(1)
4.2
4.6
4.7
4.3
4.3
7.2
6.4
4.8
5.1
5.1
4.5
3.7
3.4
3.5
3.9
3.7
4.2
6.2
4.6
3.8
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(3)
(3)
(3)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(3)
(2)
Table 5.
Contd..
465
..
U
9214
9766
10604
10082
9536
11162
2165
603
632
676
737
509
366
362
496
868
768
857
978
918
1017
951
1068
1012
917
1175
1152
1057
162
223
(1)
(2)
(1)
(2)
(2)
(3)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(3)
(3)
(3)
(3)
(4)
2042
1377
696
28
688
63
2153
195
32
-118
-175
-222
-133
53
143
237
259
248
255
91
184
-46
-44
112
21
-45
55
-38
167
217
(1)
(2)
(1)
(1)
(2)
(3)
(1)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(3)
(3)
(3)
(3)
(4)
7195
6583
7417
8008
8627
6827
8845
451
375
499
696
854
886
871
834
909
780
661
777
594
621
741
857
929
904
747
648
615
843
950
or
Ue9(A2)
(2)
(2)
(1)
(2)
(2)
(3)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(2)
(3)
(3)
(3)
(3)
(4)
4 . 9 (3)
5 . 8 (3)
5 . 0 (2)
5.4 (3)
4 . 7 (2)
8 .4 ( 4 )
7.9 (2)
6 . 1 (7)
5.2 ( 6 )
3.9 ( 6 )
4 .3 (6)
5 . 7 (7)
7.1 (8)
5.2 (6)
4.2 ( 6 )
6 . 3 (7)
5.7 (7)
6 .0 (7)
6 . 6 (8)
6 . 6 (9)
8 . 3 (9)
6 . 6 (7)
7 .0 (8)
5 .1 (6)
7.5 (8)
11 (1)
9 (1)
1 0 (1)
1 0 (1)
1 4 (2)
466
Figure
8 : STRUCTURAL FORMULA OF PIRENZEPINE WITH ATOMIC NUMBERING (6).
Figure 9 :
A VIEW OF THE CRYSTAL STRUCTURE SHOWING

THE PACKING, HYDROGEN BONDS AND CONFOR-MATION OF THE BENZODIAZEPINE AND
PIPERAZINE R I N G S ( 6 ) .
Table 6.
Torsion a n g l e s
C (12) -N (1) -C (2) -C (3)

N ( l ) -C(2) -C(3) -C (4)
C (2) -C (3) -C (4) -C (13)
C(3) -C (4) -C(13)-C (12)
C (3) -C (4) -C (13) -N (5)
C (4)-C (13) -C(12) -N (1)
C(4) -C(13)-C (12) -N(11)
C(13)-C (12) -N(1) -C(2)
C (7) -C (8) -C (9) -C (10)
C(8)-C(S)-C(lO)-C(15)
C (9) -C (10) -C (15) -C (14)
C (9)-C(lO)-C(15) -N(11)
C (10) -C (15) -C (14) -C (7)
C(10) -C(15) -C(14) -C(6)
C(15) -C (14) -C (7) -C (8)
C (14) -C (7) -C(8) -C (9)
N(S)-C(6)-C(14)-C(15)
C(6) -C (14) -C (15)-N (11)
(O)
1.4
1.5
- 2 .0
- 0 .4
-174.7
3.6
-178.9
-4.1
0.3
- 0 .5
1.1
1 7 8 .7
- 1 .4
1 7 7 .8
1.1
-0.6
-42.4
0 .2
(3)
(4)
(3)
(3)
(2)
(3)
(2)
(3)
(3)
(3)
(3)
(2)
(3)
(2)
(3)
(3)
(3)
(3)
C (14) -C (15) -N (11) -C (12)

N (11 ) -C (12) -C (13) -N (5)
C ( 12) -C ( 13) -N (5) -C (6)
C ( 1 3 ) -N (5) -C (6) -C (14)
C (15) -N (11) -C (12) -C (13)
C (13) -N (5) -C (6) - 0 ( 6 )
C(12) -N(11) -C(16) -0(16)
C(12) -N(11) -C(16) -C(17)
N (11) -C (16) -C (17) -N ( 1 ' )
C (16) -C (17) -N (1' ) -C (2 I )
C (17) -N (I ) -C (2 ' ) -C ( 3 ' )
N ( 1 ' ) -C (2 ' ) -C ( 3 ' ) -N ( 4 ' )
C (2 ) -C ( 3 ) -N (4 ' ) -C ( 5 )
C (2 I ) -C ( 3 ) -N (4 ' ) -C ( 7 ' )
C ( 3 ' ) -N (4 I ) -C ( 5 I ) -C ( 6 ' )
N (4 ' ) -C ( 5 I ) -C (6' ) -N (1I )
C(S')-C(6') -N(1 I ) - C ( 2 ' )
6 9 .5
-4.7
3 8 .3
5.3
-64.5
-178.8
179.8
0.2
-50.0
165.2
-178.4
-57.9
58 .7
-178.7
-58.6
5 8 .6
-57.7
( 2)
(3)
( 3)
(3)
(2)
(2)
(2)
(3)
(2)
(1)
(1)
(2)
(3)
(2)
(2)
(2)
(2)
468
Figure 10: STEREO-PAIRS OF DRAWINGS FOR THE MOLECULAR

STRUCTURES OF PIRENZEPINE DIHYDROCHLORIDE(a)AND
PI RENfEPlNE MONOHYDROCHLORIDE(b) OBTAINED
FROM X-RAY ANALYSIS ( 7 ) .
a-
1.491 (3)
1.503(6)
Figure l l
ATOM NUMBERING SCHEME AND CRYSTALLOGRAPHIC BOND

ANGLES
(A,
STANDARD DEVIATONS IN PARENTHESES)
FOR PIRENZEFINE DIWDROCHLORIDE ( FIRST VALUE) AND

PIRENZEPINE MONOHYDROCHLORIDE (SECOND VALUE I( 7).
469
i n agreement w i t h t h e c r y s t a l s t r u c t u r e s . The
t r i c y c l i c r i n g system c o n s i s t s of t h e p l a n a r
r i n g s A and C and nonplanar r i n g B (Figure 1 1 ) .
For b o t h compounds t h e seven-membered r i n g B
h a s a d i s t a r t e d boat shape. This r i n g i s f o l d e d
a l o n g a f o l d i n g l i n e p a s s i n g through N 1 and t h e
middle o f t h e bond N 8 - C9. T h i s s i m i l a r i t y i n
t h e geometry o f t h e t r i c y c l i c systems o f b o t h
compounds does n o t h o l d f o r t h e s i d e c h a i n s
which have t o t a l l y d i f f e r e n t arrangements with
r e s p e c t t o t h e t r i c y c l i c system i n both compounds
( F i g u r e s 10 and 1 2 ) . The s t e r i c s i t u a t i o n is
q u i t e s i m i l a r along t h e bond N 1 - C16, i n t h a t
t h e carbonyl groups are almost i n e c l i p s e d
p o s i t i o n s t o N 1 - C15. S i g n i f i c a n t d i f f e r e n c e s
are observed, however, a l o n g t h e C16 - C 1 7 bond.
For t h e d i h y r o c h l o r i d e N18 i s trans t o
N 1 (T N18-Cl7-Cl6-Nl = 171.3') whereas i n t h e
monohydrochloride N18 and N 1 a r e i n gauche
p o s i t i o n s (T N18-Cl7-Cl6-Nl = 64.1').
In t h i s
arrangement t h e p i p e r a z i n e r i n g w i l l be s i t u a t e d
below t h e t r i c y c l i c r i n g system i n t h e c r y s t a l
s t r u c t u r e o f p i r e n z e p i n e monohydrochloride.
The c r y s t a l s t r u c t u r e s o f b o t h s a l t s showed
s t r o n g N-H .
C 1 - hydrogen bonds f o r t h e p r o t o nated nitrogens of t h e piperazine rings. A
r e l a t i v e l y weak 0
C 1 - hydrogen bonds a l s o
e x i s t f o r t h e d i s t o r t e d w a t e r molecule i n t h e
c r y s t a l l a t t i c e i n t h e dihydrochloride salts.
The amide n i t r o g e n N8 o f t h e monohydrochloride
s a l t shows a f u r t h e r N-H ... C 1 - hydrogen bond
which i s n o t observed i n t h e d i h y d r o c h l o r i d e
structure.
..
...
3.
Synthesis
P i r e n z e p i n e , t o g e t h e r with o t h e r 1 1 - s u b s t i t u t e d 11Hpyrido[ 2 ,3-b] [ 1 , 5 ] benzodiazepin-5 (6H) -ones , had
been prepared by D r . Karl Thomae ( 8 ) . The compounds
are p r e p a r e d by t r e a t m e n t of an 11H-pyrido[ 2 ,3-b] [ 1,51
benzodiazepin-5 (6H) -one with a dihalogen compound,
XCH2COX' i n which X and X I may be a l i k e o r d i f f e r e n t
and d e s i g n a t e an atom o f C 1 , B r and I . The 11h a l o a c e t y l i n t e r m e d i a t e is t h e n t r e a t e d with t h e
a p p r o p r i a t e amine. Thus, a b o i l i n g s o l u t i o n o f 2 1 g
11H-pyrido[ 2 ,3-b] [ 1,51 benzodiazepin-5 (6H) -one) i n 300 m l
470
C16 -NI
C 17- C I6
HI71
N 18-C I7
HI8
HI72
Figure 12 : NEWMAN PROJECTIONS DOWN THE BONDS ClbNl, c r C 1 6 &
v8-Ci7
FOR PIRENZEPINE DIHYDROCHLORIDE ( ABOVE ) AND THE

MONOHYDROCHLORIDE (BELOW) ( 7 1.
47 1
a b s o l u t e dioxane t r e a t e d dropwise sim u lta n e o u sly i n

30 min w i t h 15.8 g ClCH2COC1 i n 40 m l a b s o l u t e dioxane
and 1 4 . 4 g E t 3 N i n 30 m l a b s o l u t e dioxane, t h e m ix tu r e
r e f l u x e d f o r 8 h r and t h e h o t f i l t r a t e e v a p o r a te d i n
vacuo gave ll-chloroacetyl-11H-pyrido[2,3-b][1,5]
benzodiazepin-5 (6H) -one (Scheme 2 ) . The c h l o r o a c e t y l
d e r i v a t i v e and N-methylpiperazine i n a b s o l u t e a l c o h o l
r e f l u x e d f o r 18 h r f i l t e r e d and t h e hot f i l t r a t e
e v a p o r a t e d i n vacuo t o g i v e p i r e n z e p i n e .
C 1CH, COC 1
%
N
H
I
=c
I
CH2C1
H2C -N
wNCH3
Scheme 2 .
Synthesis of pirenzepine
412
4.
Stability
S t a b i l i t y of p i r e n z e p i n e depends on b o t h m o istu r e
c o n t e n t s and pH and t h e e f f e c t o f t h e m o istu r e c o n t e n t s
could be minimized by a d j u s t i n g t o optimum pH ( 9 ) .
P i r e n z e p i n e d i h y d r o c h l o r i d e i n t a b l e t was shown
s t a b l e t o exposure t o l i g h t a t 50 - 5 5 O . No change
i n c o l o r , smell, t a s t e o r g e n e r a l appearance o r
presence o f d eg r a d a t i o n p r o d u ct s was observed (10).
5.
5.1
S p e ct r o p h o t o m et r i c Methods
The d e t er m i n a t i o n of p i r e n z e p i n e d i h y d r o c h l o r i d e
s p e c t r o p h o t o m e t r i c a l l y u s i n g AA method have
r e c e n t l y been r e p o r t e d ( 4 ) . The mean p e r c e n ta g e
r e c o v er y f o r t a b l e t s , each l a b e l l e d t o c o n t a i n
25 mg,was 100.2 -+ 1 . 1 5 . The drug shows maximum
a b s o r p t i o n a t 280 nm i n h y d r o c h l o r i c a c i d and a t
295 nm i n 0 . 1 N sodium hydroxide due t o k e to - e n o l
tautomerism. AA a t 300 nm was found t o be
l i n e a r l y r e l a t e d t o t h e drug c o n c e n t r a t i o n o v e r
a range of 10-80 Ug/ml with a r e l a t i v e s t a n d a r d
d e v i a t i o n of 0 . 4 % .
A r a p i d and d i r e c t method f o r t h e d e te r m in a tio n
o f p i r e n z e p i n e d i h y d r o c h l o r i d e i n t h e p r e se n c e
o f d eg r a d a t i o n p r o d u ct s u s i n g f i r s t d e r i v a t i v e
spectrophotometry h as a l s o been r e p o r t e d ( 5 ) .
The method was a p p l i e d u s i n g s y n t h e t i c m ix tu r e s
o f t h e i n t a c t drug and d e g r a d a tio n p r o d u c ts. The
procedure i s s u i t a b l e f o r m o n ito r in g t h e drug
s t a b i 1i t y
5.2
Chromatographic Eilethods
5.2.1
Thin-Layer Chromatographic (TLC) Methods

Thin l a y e r chromatography have been used
f o r t h e analysis o f pirenzepine (10).
P r e c o a t ed TLC p l a t e s with 0 . 2 mm s i l i c a
g e l FZs4 were used.
5.2.2
413
High-pressure Liquid Chromatography (HPLC)

Daldrup -e t a1 (11) have used reversed-phase
l i q u i d chromatography a s a t e s t f o r
s c r e e n i n g pharmaceuticals , drugs and
insecticides.
High performance r e v e r s e - p h a s e l i q u i d
chromatography r e t e n t i o n d a t a f o r
p i r e n z e p i n e and o t h e r compounds (560
compounds) a r e given. The r e l a t i v e r e t e n t i o n
times were c a l c u l a t e d a s t h e r a t i o s of
r e t n t i o n times of compound and r e f e r e n c e
compound, 5- (p-methylphenyl) -5-phenyl
hydantoin. The UV d e t e c t o r wavelength was
220 nm, where most of t h e compounds gave
response. Two s o l v e n t programs and a
prepacked column C - 1 8 SIL-X-10 were used
f o r the analysis.
Babhair (12) r e p o r t e d a s i m p l e and s e n s i t i v e
method for t h e d e t e r m i n a t i o n o f p i r e n z e p i n e ,
i n dosage forms and i n b i o l o g i c a l f l u i d s ,
u s i n g h i g h - p e r formance 1i q u i d chromatography w i t h U . V . d e t e c t o r . A t a b l e t o f
25 mg drug was ground, suspended i n 1 0 m l
o f w a t e r , shaken and t h e n f i l t e r e d . A
known volume o f t h e f i l t r a t e i s a d j u s t e d
t o a p p r o p r i a t e c o n c e n t r a t i o n . Twenty 1.11
o f t h i s s o l u t i o n were i n j e c t e d . Plasma o r
u r i n e samples were made a l k a l i n e w i t h
ammonia b e f o r e e x t r a c t ion with chloroform
which was evaporated and t h e r e s i d u e was
d i s s o l v e d i n t h e mobile phase, 20 1-1 o f t h i s
s o l u t i o n were i n j e c t e d . The d e t e r m i n a t i o n
l i m i t f o r q u a n t i t a t i o n was about 1 pg/ml o f
p i r e n z e p i n e . Complete s e p a r a t i o n of t h e
drug was achieved i n about 5 . 4 minutes
under t h e p r e s e n t chromatographic c o n d i t i o n s .
A 30 X 3.9 cm i . d. commercially a v a i l a b l e
s t a i n l e s s s t e e l C-18 column was used.
Mobile phase c o n s i s t e d o f a c e t o n i t r i l e ,
methanol and 5% a c e t i c a c i d (70:40:15).
High-performance l i q u i d chromatographic
determination of pirenzepine dihydrochloride
i n i t s pharmaceutical f o r m u l a t i o n have a l s o
474
been r e p o r t e d (13). Normal phase l i q u i d

chromatography has been performed on a
Micropack Si-10 column u s i n g ammonium hydrox i d e (28-30% NH3) i n methanol (0.75 : 99.25%
v/v) mobile phase and a flow r a t e o f 2 ml/min.
Clobazam h as been used a s i n t e r n a l s t a n d a r d
with r e t e n t i o n times o f 1 . 9 and 2.8 minutes
f o r clobazam and p i r e n z e p i n e d i h y d r o c h l o r i d e ,
r e s p e c t i v e l y a t 254 nm. T a b l e t s each l a b e l l e d
t o c o n t ai n 25 mg p i r e n z e p i n e d i h y d r o c h l o r i d e
gave mean p er c e n t ag e result o f 99.98 2 0 . 4 .
The method have a l s o been r e p o r t e d t o be a
s t a b i l i t y i n d i c a t i n g method.
5.3
Radioimmunoassay
A radioimmunoassay system developed f o r p i r e n z e p i n e
was used t o s p e c i f y t h e r e q u i re m e n ts f o r u s i n g such

system i n pharmacokinetics and t o i l l u s t r a t e t h e
general strategy i n establishing s p e c i f i c i t y , sensit i v i t y and r e l i a b i l i t y ( 1 4 ) . The t y p e o f e r r o r i n
t h e f i n a l d a t a was c o n s i d er e d . I t is a widespread
assumption t h a t weak c r o s s - r e a c t i o n s i n m e t a b o l i t e s
r e s u l t i n a r e l a t i v e e r r o r and, t h e r e f o r e , have no
r e l e v a n c e t o t h e d e t e c t i o n o f t h e p a r e n t drug. In
c o n t r a s t t o t h i s a s s u p t i o n , it was shown t h a t such
weak c r o s s - r e a c t i v i t y i n b i o l o g i c a l samples g i v e s
r i s e t o an a b s o l u t e e r r o r which i s independent o f t h e
c o n c e n t r a t i o n and analogous t o t h a t caused by a b la n k
v a l u e i n chemical a n a l y s i s .
Tanswell and Zahn (15) a p p l i e d monoclonal a s s a y s f o r
s t u d y i n g t h e pharmacokinetics o f p i r e n z e p i n e .
6.
Pharmacokinetics
The marked physicochemical p r o p e r t i e s o f p i r e n z e p i n e
r e s u l t i n t h e drug b ei n g h i g h l y h y d r o p h i l i c a t p h y s i o l o g i c a l pH. T h i s p r o p e r t y determines t h e f a t e o f p i r e n z e p i n e
i n t h e organism due t o t h e l i m i t e d a b i l i t y of t h e drug t o
p e n e t r a t e l i p i d membranes. T h i s l i m i t e d p e n e t r a t i o n i s
r e f l e c t e d i n i t s a b s o r p t i o n , d i s t r i b u t i o n , b io tr a n sf o r m a t i o n and e x c r e t i o n .
6.1
Absorption
O r a l l y a d m i n i s t e r e d p i r e n z e p i n e was almost
completely absorbed by r a b b i t s and dogs b u t o n l y
about 11%absorbed by r a t s and t h e blood l e v e l
maxima reached a f t e r 3 h r (16).
475
After o r a l a d m i n i s t r a t i o n o f aqueous s o l u t i o n s o r
t a b l e t s 20-30% o f t h e dose is absorbed ( 1 7 ) . In
an i n t e r n a t i o n a l pharmacokinetic s t u d i e s 20% o f t h e
o r a l dose a d m i n i s t e r e d t o t h e panel o f 87
v o l u n t e e r s was absorbed (18). Uniform plasma
l e v e l s were observed i n t h i s l a r g e panel from
10 d i f f e r e n t c o u n t r i e s a f t e r a s i n g l e o r a l dose
o f 50 mg, with minimal i n t e r p a t i e n t v a r i a t i o n .
Peak serum c o n c e n t r a t i o n were approximately 50 ng/ml
o c c u r r i n g w i t h i n 2 h r s . After o r a l dose o f 20 mg/kg,
t h e drug appeared i n t h e blood a t a c o n c e n t r a t i o n
o f 0 . 1 7 ug/ml o n l y 15 min a f t e r a d m i n i s t r a t i o n .
Afterwards, however, i n c r e a s e i n t h e c o n c e n t r a t i o n
was shown r e a c h i n g t h e maximum of 0 . 4 2 ug/ml
approximately 3 h r a f t e r a d m i n i s t r a t i o n ( 1 9 ) . The
r e s u l t s a r e comparable t o t h o s e o f Hammer e t_a1
(16) who r e p o r t e d t h a t t h e maximum blood
c o n c e n t r a t i o n was a t t a i n e d 3 h r a f t e r a d m i n i s t r a t i o n
a t t h e l e v e l of 0.55 ug/ml.
After o r a l a d m i n i s t r a t i o n o f 25 mg o f t h e drug, t h e
maximum blood l e v e l occurred i n 2 - 3 h r and was
about 24 ng/ml. Computer s i m u l a t i o n was used t o
d e r i v e a s i n g l e dosage scheme f o r p i r e n z e p i n e
c o n s i s t i n g o f an i n i t i a l dose o f 50 mg and
maintenance dose o f 25 mg a t 12 h r - i n t e r v a l s .
T h i s schedule maintained a maximum plasma l e v e l
o f -> 24.1 ng/ml i n normal s u b j e c t s ( 1 7 ) .
The maximum plasma l e v e l appeared 2 h r a f t e r t h e

a d m i n i s t r a t i o n o f 1 2 . 5 - 150 mg p i r e n z e p i n e o r a l l y
t o volunteers (20). After multiple administration
(25 - 50 mg a day) t h e plasma l e v e l was i n c r e a s e d
f o r t h e f i r s t 3 days o f a d m i n i s t r a t i o n and remained
constant t h e r e a f t e r .
Simulated plasma l e v e l s of p i r e n z e p i n e a f t e r an o r a l
t h e r a p e u t i c dosage regimen (50 mg, twice d a i l y ) on
t h e b a s i s o f s i n g l e a d m i n i s t r a t i o n d a t a , showed
s t e a d y s t a t e w i t h i n 2 days o f t r e a t m e n t (18).
P i r e n z e p i n e a d m i n i s t e r e d i . m . was completely
absorbed i n r a b b i t s and dogs. Rats t h a t p o o r l y
absorb o r a l p i r e n z e p i n e showed complete a b s o r p t i o n
o f i . m . dose o f t h e drug.
Using monoclonal a s s a y s (15), plasma p i r e n z e p i n e
l e v e l s and u r i n a r y e x c r e t i o n were measured over time
476
i n 10 h e a l t h y male s u b j e c t s a f t e r 10 mg i . v . or
i . m . dose, i n randomized c r o s s o v e r s t u d y . The
d a t a were f i t t e d t o a 3-compartment open model.
Absorption a f t e r i . m . i n j e c t i o n was r a p i d and
complete (mean b i o a v a i l a b i l i t y 1 0 0 . 3 % ) .
After i . v . b o l u s i n j e c t i o n h i g h plasma l e v e l peaks
are reached which last only f o r a s h o r t time
p e r i o d s i n c e due t o t h e d i s t r i b u t i o n o f p i r e n z e p i n e
i n t o body t i s s u e s t h e plasma l e v e l d e c l i n e s r a p i d l y
with an i n i t i a l h a l f - l i f e o f about 5 min. (17, 2 1 ) .
The peak c o n c e n t r a t i o n a f t e r i . v . a d m i n i s t r a t i o n
(22) was much h i g h e r than t h e t h e r a p e u t i c l e v e l
a f t e r o r a l a d m i n i s t r a t i o n . In t h e f i r s t minutes
a f t e r i . v . i n j e c t i o n o f 10 mg p i r e n z e p i n e ( 0 . 1 5
mg/kg) , t h e plasma c o n c e n t r a t i o n s were almost
1 0 - f o l d h i g h e r than t h e s t e a d y s t a t e l e v e l reached
a f t e r d a i l y doses o f o r a l 100 mg. I n less t h a n
one hour, however, t h e plasma l e v e l o f t h e i . v .
dose i s comparable t o t h e 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 after o r a l a d m i n i s t r a t i o n (18).
The a b s o r p t i o n o f S . C . dose o f p i r e n z e p i n e i s very
swift and comparable t o t h a t o f i . v . (4) and
u n l i k e t h a t o f i . m . r e p o r t e d by Hammer -e t a 1 (16).
6.2
Distribution
Informations provided by whole-animal a u t o r a d i o g r a phy of r a t 10 minutes a f t e r i n t r a v e n o u s i n j e c t i o n
o f 2 mg/kg o f r a d i o a c t i v e p i r e n z e p i n e (15, 2 1 ) ,
showed t h a t t h e drug is widely d i s t r i b u t e d i n t h e
body. The r a d i o a c t i v e drug i s found i n a l l
i n t e r n a l organs and s k e l e t a l muscles. No r a d i o a c t i v i t y was d e t e c t e d i n t h e b r a i n o f t h e r a t o r
t h e f e t u s e s o f g e s t a t i n g mouse s u g g e s t i n g t h a t t h e
blood-brain b a r r i e r r e p r e s e n t s a genuine b a r r i e r
t o p i r e n z e p i n e and t h a t t h e drug does n o t c r o s s
t h e p l a c e n t a l b a r r i e r . Continuous i n t r a v e n o u s
i n f u s i o n o f p i r e n z e p i n e over a p e r i o d o f 2 days a t
a r a t e o f 2 mg/hr/8-9 kg baboon (10 times t h e
human i n f u s i o n d o s e ) , showed t h a t t h e h i g h e s t
l e v e l s a r e i n t h e organs r e s p o n s i b l e f o r t h e
e l i m i n a t i o n of t h e drug i . e . l i v e r and kidneys.
The o t h e r i n t e r n a l organs, t h e s k i n and s k e l e t a l
muscles, however, showed a h i g h e r p i r e n z e p i n e
c o n c e n t r a t i o n p e r g of t i s s u e t h a n t h e plasma.
477
Extremely low drug c o n c e n t r a t i o n was found i n

t h e brain.
The d i s t r i b u t i o n o f p i r e n z e p i n e i n t h e t i s s u e s
a f t e r one-time o r a l a d m i n i s t r a t i o n was i n v e s t i g a t e d
by Kobayashi -e t a 1 (19) a f t e r i s o l a t i o n o f t i s s u e s
and whole-body a u t o r a d i o g r a p h y t e c h n i q u e . After
3 h r , t h e time o f maximum blood c o n c e n t r a t i o n ,
h i g h c o n c e n t r a t i o n of p i r e n z e p i n e was observed i n
t i s s u e s l i k e l i v e r , kidney, p a n c r e a s , lung,
p i t u i t a r y g l a n d , s a l i v a r y g l a n d , a d r e n a l gland e t c .
These r e s u l t s , t h e r e f o r e , agree w i t h t h o s e o f
Hammer and Koss (21) i n t h a t r a d i o a c t i v e p i r e n z e p i n e
was found d i s t r i b u t e d i n a l l t i s s u e s e x c e p t t h e
c e n t r a l nervous system. The r a d i o a c t i v i t y
d i s t r i b u t e d i n t h i s way d e c r e a s e d as time p a s s e d .
Twenty-four hours a f t e r a d m i n i s t r a t i o n , t h e
c o n c e n t r a t i o n was s l i g h t l y h g i h o n l y i n t h e
g a s t r o i n t e s t i n a l t r a c t and l i v e r . R a d i o a c t i v i t y
i n t h e t e s t i s showed a tendency t o d e c r e a s e
g r a d u a l l y d u r i n g 72 h r . Kaubisch et a1 (23)
observed s t r o n g r a d i o a c t i v i t y d i s t r i b u t e d i n t h e
g a s t r o i n t e s t i n a l t r a c t , kidney and s a l i v a r y g l a n d s
i n a whole-body a u t o r a d i o g r a p h y performed a f t e r
i . v. a d m i n i s t r a t i o n of 14C-pirenzepine i n a dose
o f 2 mg/kg. The p r e s e n c e o f r a d i o a c t i v i t y i n t h e
g a s t r o i n t e s t i n a l t r a c t was c o n s i d e r e d t o r e s u l t
from t h e e x c r e t i o n o f t h e r a d i o a c t i v i t y i n t o t h e
i n s i d e o f t h e i n t e s t i n a l t r a c t v i a t h e blood
v e s s e l s and i n t e s t i n a l w a l l . T h i s i s s u p p o r t e d
by t h e f i n d i n g o f Kobayashi e t a 1 (19) who
observed c l e a n p i c t u r e s o f r a d i o a c t i v i t y
d i s t r i b u t i o n a t t h e wall o r mucus o f t h e stomach
and i n t e s t i n e s i n a u t o r a d i o g r a p h y 3 h r and 9 h r
a f t e r a d m i n i s t r a t i o n and c o n s i d e r e d t h a t t h e
r a d i o a c t i v i t y was s e c r e t e d from t h e g a s t r o i n t e s t i n a l
wall o r was d e p o s i t e d t h e r e .
L -
--
After m u l t i p l e o r a l d o s e , Kobayashi e t a 1 ( 1 9 ) ,
observed a s l i g h t tendency towards accumulation
i n t h e l i v e r , kidney and t e s t i s whereas r a d i o a c t i v i t y d e c r e a s e d s l o w l y i n one-day a d m i n i s t r a t i o n .
However, t h e r a d i o a c t i v e c o n c e n t r a t i o n i n each
t i s s u e did not increase i n proportion t o t h e
a d m i n i s t r a t i o n times b u t d e c r e a s e d as time p a s s e d
and l e f t o n l y t r a c e s one week a f t e r t h e t e r m i n a t i o n
o f a d m i n i s t r a t i o n . In m u l t i p l e a d m i n i s t r a t i o n
almost a l l of t h e r a d i o a c t i v i t y was found

excreted i n t o u r i n e and feces 24 h r a f t e r each
administration. I t i s , t h e r e f o r e , considered
t h a t t h e accumulation of t h e drug i n t h e body
is small. Jaup and Blomstrand (24) measured
serum and cerebrospinal concentrations of
pirenzepine i n healthy volunteers a f t e r t h e r a p e u t i c
dosage over 2-5 days. The cerebrospinal f l u i d to-serum r a t i o was 0.095 t o 1, i n d i c a t i n g t h a t
pirenzepine passed t h e blood-brain b a r r i e r , but
only t o a small e x t e n t . Pirenzepine concentrations
i n t h e cerebrospinal f l u i d was about 10% those
of serum.
That t h e penetration of pirenzepine through t h e
blood-brain b a r r i e r i s very limited was a l s o
demonstrated by o t h e r s t u d i e s (18, 25-27).
6.3
Metabolism
Pirenzepine undergoes only s l i g h t metabolism
i r r e s p e c t i v e of the route of administration and
t h e r e i s no s i g n i f i c a n t f i r s t - p a s s e f f e c t . Only
small amounts of metabolites have been detected
(16-18, 21), of which t h e desmethyl d e r i v a t i v e
accounted f o r s i g n i f i c a n t l y less t h a n 10% of t h e
dose. The desmethyl compound has no pharmacological
e f f e c t . The t r i c y c l i c moiety, despiperazinly
pirenzepine was a l s o detected a s a metabolite i n
n e g l i g i b l e amounts ( 1 7 ) . Unlike cimetidine,
pirenzepine does not i n h i b i t t h e h e p a t i c mixed
function oxidase system (28).
6.4
Excretion
In man as well as i n o t h e r animals, pirenzepine i s
excreted unchanged i n u r i n e and feces ( 1 7 , 18, 21).
Due t o t h e s p e c i a l physicochemical p r o p e r t i e s of
pirenzepine only about 10% of t h e dose i s bound
t o plasma p r o t e i n s (17, 20) and hence it i s almost
completely f i l t e r e d i n t o t h e kidneys. The volume
of d i s t r i b u t i o n of pirenzepine i s i d e n t i c a l t o
t h e e x t r a c e l l u l a r space which i n d i c a t e s t h a t
pirenzepine r a p i d l y e q u i l i b r a t e s between plasma
and t h e e x t r a c e l l u l a r f l u i d s of t h e peripheral
t i s s u e s . The t o t a l plasma clearance of
pirenzepine i s 255 ml/min. Because of i t s limited
479
l i p i d s o l u b i l i t y , t h e drug cannot be reabsorbed

from t h e renal tubules s o t h a t t h e renal clearance
of 129 ml/min corresponds t o the glumerular
f i l t r a t i o n r a t e . Hammer -e t a1 (16) reported t h a t
approximately equal amounts of pirenzepine were
excreted v i a t h e b i l e and u r i n e . The h e p a t i c
clearance, mostly b i l i a r y excretion i s about
125 ml/min (17).
Following o r a l administration o f a t h e r a p e u t i c
dose of labeled drug, 82% of t h e substance
excreted i n u r i n e i s unchanged, a s is 92% of t h a t
i n t h e feces. The drug i s eliminated from the
organism f u l l y and almost equally i n u r i n e and
f e c e s , elimination being almost complete a f t e r
4 days (17). Kobayashi -e t a1 (19) reported t h a t
i n s i n g l e a s well as i n m u l t i p l e administration,
almost a l l of t h e dose was excreted i n u r i n e and
feces. In i . v . administration, 44% and 50% of t h e
administered dose were excreted i n t o u r i n e and
f e c e s , r e s p e c t i v e l y 24 h r a f t e r administration and
46% and 53% r e s p e c t i v e l y , 4 days a f t e r administr a t i o n . Similar r e s u l t s were obtained with
subcutaneous administration. Hammer -e t a1 (16)
reported t h a t 36% and 56% of t h e administered
dose were excreted i n u r i n e and f e c e s , r e s p e c t i v e l y ,
following i . v . administration. 42% of a 1 0 mg
dose was a l s o reported (15) eliminated r e n a l l y
with t o t a l clearance o f 253 ml/min and renal
clearance of 107 ml/min. The mean t r a n s i t time
was 9 . 3 h r . A urinary excretion r a t e of 7-12%
was reported by Ohashi -e t a1 (20) within 4 days
a f t e r multiple administration of pirenzepine
(25-50 mg, 3 times a day f o r 4 days). Hammer e t
a1 (16) reported approximately 4% o f the admingt e r e d dose was excreted i n u r i n e a f t e r o r a l
administration. According t o Kobayashi -e t a1 (19)
about 8% was excreted i n u r i n e a f t e r s i n g l e o r a l
administration and about 4.5% i n multiple
administration. The d i f f e r e n c e i n t h e value was
a t t r i b u t e d t o t h e d i f f e r e n c e i n absorption r a t e s
between f a s t e d and nonfasted animals.
Elimination h a l f - l i f e of 10 h r was reported (21)
following i . v . dose o f 8 mg pirenzepine t o humans.
Oral administration o f 25 mg t a b l e t s t o
volunteers r e s u l t e d i n elimination h a l f - l i f e of
480
--
11 h r (21). Ohashi e t a1 (20) reported an

elimination h a l f - l i f e of 13 h r . The long eliminat i o n h a l f - l i f e was a t t r i b u t e d t o slow r e d i s t r i b u t i o n
of pirenzepine from t h e t i s s u e s r a t h e r than t o
slow excretion (21).
Intravenous administration o f 14C-pirenzepine t o

dams i n a dose of 2 mg/kg caused t h e appearance
o f r a d i o a c t i v i t y i n m i l k almost equivalent t o
t h a t i n blood (19).
Pretreatment of healthy volunteers with t h e r a p e u t i c
doses of pirenzepine over 5 days showed no
s i g n i f i c a n t e f f e c t on t h e absorption, d i s t r i b u t i o n
o r elimination of a n t i p y r i n e (28).
7.
Pharmaco 1ogy
7.1
Antisecretory E f f e c t s
Secretory s t u d i e s with pirenzepine c l e a r l y
demonstrate t h a t pirenzepine suppresses both basal
and stimulated acid and pepsin s e c r e t i o n (30-52).
Intravenous pirenzepine reduces b a s a l acid secret i o n by 90% and pentagastrin-stimulated
s e c r e t i o n by approximately 50% ( 3 0 ) . F r i t s c h
-e t a1 (31) reported t h a t o r a l 50 mg of pirenzepine,
i n p a t i e n t s with duodenal u l c e r , i n h i b i t e d t h e
basal acid s e c r e t i o n by 39.9% and peptone-induced
acid output by 21.3%. The percentage i n h i b i t i o n
n e a r l y doubled i f t h e same dose is given a f t e r
a treatment with 50 mg pirenzepine twice d a i l y
f o r 3 t o 7 days. No acute e f f e c t on serum g a s t r i n
l e v e l s can be demonstrated by a s i n g l e dose of
pirenzepine. After pretreatment only small
increase of basal g a s t r i n l e v e l s i s observed,
serum g a s t r i n does not change during stimulation.
Oral pirenzepine (25 mg) reduces acid output by
50% and pentagastrin-stimulated s e c r e t i o n by
30-40%. Doubling t h e dose increases t h e degree
of i n h i b i t i o n of stimulated s e c r e t i o n by
10%
(30). Jaup -e t a1 (32) reported t h a t pirenzepine
50 mg PO b . i . d . reduced b a s a l g a s t r i c a c i d
s e c r e t i o n by 54% a t two hours following t h e l a s t
dose. Pentagastrin-stimulated s e c r e t i o n o f
hydrochloric acid (1 mcg/kg/hr) by continuous i . v .
infusion was reduced by 31%. Secretory s t u d i e s by
48 1
J a u p -e t a1 (33) c l e a r l y demonstrated t h a t p i r e n z e p i n e
s u p p r e s s e s b a s a l , p e n t a g a s t r i n - s t i m u l a t e d and
insulin-stimulated acid secretion in a dose-related
manner. In a d o u b l e - b l i n d , placebo c o n t r o l l e d ,
randomized s t u d i e s on p a t i e n t s w i t h dyspepsia,
Bianchi Porro et a1 (34) r e p o r t e d a 48% d e c r e a s e o f
g a s t r i c s e c r e t i o n i n t h e first hour and 30% i n t h e
second hour a f t e r 50 mg o r a l dose o f p i r e n z e p i n e .
P i r e n z e p i n e caused a r e d u c t i o n t o 20% o f t h e concentr a t i o n o f t h e a c i d produced by vagal s t i m u l a t i o n
(35). The drug i n h i b i t e d t h e v a g a l l y t r a n s m i t t e d
a c i d s e c r e t i o n by about 45% even when t h e v a g a l l y
s t i m u l a t e d a c i d s e c r e t i o n amounts t o more t h a n 50%
o f t h e p e n t a g a s t r i n - s t i m u l a t e d s e c r e t i o n . These
e f f e c t s of pirenzepine a r e simila r t o those of a tro p i n e b u t d i f f e r e n t from t h e e f f e c t s o f c i m e t i d i n e .
Eugenides -e t a1 (36) r e p o r t e d t h a t histamines t i m u l a t e d a c i d o u t p u t p e r two hours i n h e a l t h y
v o l u n t e e r s was reduced with 25 mg p i r e n z e p i n e b . d .
by 12.5% and w i t h 50 mg t . d . s . by 24%. Stockbrugger
et a1 (37) s t u d i e d t h e e f f e c t o f t h r e e d i f f e r e n t
doses o f p i r e n z e p i n e on a c i d s e c r e t i o n s t i m u l a t e d by
modified shamfeeding (MSF) on h e a l t h y v o l u n t e e r s .
Oral p i r e n z e p i n e 25 mg b . i . d . , 50 mg b . i . d . o r 50 mg
t . i . d . reduced b a s a l o u t p u t (0-30 min) by 48%, 59%
and 66% r e s p e c t i v e l y , i n s t i m u l a t e d a c i d o u t p u t (0 120 min) by 45%, 58% and 48% r e s p e c t i v e l y . When t h e
a c i d s e c r e t i o n was c a l c u l a t e d a s volume c o r r e c t e d f o r
d u o d e n a l - g a s t r i c r e f l u x and p y r o l i c l o s s e s , t h e
corresponding f i g u r e s were 33.7%, 3 6 . 3 % and 42.6%.
Mignon e t a1 (38) r e p o r t e d a d o s e - r e l a t e d r e d u c t i o n
o f meal-stimulated a c i d response w i t h i n c r e a s i n g
doses o f i . m . p i r e n z e p i n e . R e s u l t s with 0 . 5 mg/kg
o f p i r e n z e p i n e showed 53% r e d u c t i o n i n a c i d secret i o n . P i r e n z e p i n e 10 mg given t o duodenal u l c e r
p a t i e n t s 45 min and a g a i n 10 min b e f o r e t h e s t a r t of
MSF blocked 48% o f t h e response (39).
--
P i r e n z e p i n e (25 pg/kg) a d m i n i s t e r e d i . m . t o dogs

10 min b e f o r e i n f u s i o n o f bombesin, s i g n i f i c a n t l y
i n h i b i t e d t h e h y p e r s e c r e t i o n induced by t h e
p e p t i d e i n t h e main stomach and i n a dose o f
100 ug/kg p i r e n z e p i n e v e r t u a l l y a b b o l i s h e d t h e a c i d
response t o bombesin ( 4 0 ) . I n Heidenhain pouch
b o t h doses f u l l y prevented a c i d s e c r e t i o n .
Pirenzepine i n j e c t e d during t h e secretory plateau
482
induced by bombesin, produced a mean peak

i n h i b i t i o n of acid output from t h e main stomach
of 76.5% and 80% with doses of 25 and 100 g/kg
respectively. Pirenzepine (25 and 100 ug/kg i . v . ,
10 min before a meal t e s t ) f u l l y prevented a c i d
s e c r e t i o n f o r Hiedenhain pouch. The drug showed
no e f f e c t on bombesin - or meal-stimulated g a s t r i n
r e l e a s e . Acid and pepsinogen s e c r e t i o n induced by
bethanechol i n i s o l a t e d perfused mouse stomach
a r e i n h i b i t e d by pirenzepine (41).
7.2
Efficacy
Pirenzepine i s e f f e c t i v e i n t h e treatment of g a s t r i c
and duodenal u l c e r s . I t i s a l s o e f f e c t i v e i n nonu l c e r dyspepsia and i n preventing u l c e r recurrence.
Pirenzepine i n combination therapy with cimetidine
can be used s u c c e s s f u l l y f o r t h e treatment o f
c i m e t i d i n e - r e s i s t a n t conditions such as duodenal
u l c e r and Zollinger-Ellison syndrome. The
l i t e r a t u r e r e p o r t s a number of s t u d i e s on t h e
e f f i c a c y of pirenzepine i n t h e treatment of g a s t r i c
and duodenal u l c e r s (43, 53-130). The published
world l i t e r a t u r e (from 1977 t o 1981) on t h e e f f i c a c y
of pirenzepine had been t h e s u b j e c t o f a review and
commentary by Bianchi Porro and P e t r i l l o (53). The
review examined t h e d a t a published i n Europe which
deal with pirenzepine i n t h e treatment of g a s t r i c
and duodenal u l c e r s , taking i n t o account only
those r e s u l t s obtained from c o n t r o l l e d endoscopic
t r i a l s . In 12 prospective randomized double-blind
placebo-controlled s t u d i e s pirenzepine was
administered t o 475 duodenal u l c e r p a t i e n t s with
incidence of endoscopically proven healing of
74% when the p a t i e n t s received d a i l y dosage o f
100 o r 150 mg pirenzepine, but only 55% i f t h e
dosage of t h e drug was 75 mg o r less. Similar
healing rates were observed (72% when d a i l y dose
of pirenzepine was 100 o r 150 mg, 55% when t h e
dose was 75 mg o r l e s s ) i n 4 randomized doubleb l i n d placebo -controlled s t u d i e s i n which 84
p a t i e n t s with g a s t r i c u l c e r were admitted.
In another review, Texter and R e i l l y (83)
evaluated the c l i n i c a l evidence concerning t h e
healing e f f e c t of pirenzepine i n g a s t r i c and
duodenal u l c e r s . The reviewers subjected t h e
483
results of t h e previous double-blind, t h e r a p e u t i c

s t u d i e s on u l c e r healing t o mathematical treatments.
The t h e r a p e u t i c s t u d i e s reviewed included a l a r g e
number o f p a t i e n t s i n Europe and Japan. The doseresponse curve f o r duodenal u l c e r incorporating
16 d a t a p o i n t s from 8 t r i a l s i n 530 p a t i e n t s , 343
of whom received pirenzepine, r e s u l t e d i n a
c o r r e l a t i o n c o e f f i c i e n t r = 0.887 with P < 0.005.
Although t h e a n a l s i s is not s t r i c t l y mathematically
c o r r e c t , it suggests t h a t pirenzepine h e a l s
duodenal u l c e r compared t o placebo with a p o s i t i v e
r e l a t i o n s h i p between dose and e f f e c t . The pred i c t e d healing r a t e a t 150 mg was 79%. The
a n a l y s i s a l s o shows t h a t endoscopic healing from
g a s t r i c and duodenal u l c e r s i s l i n e a r with respect t o
time upto 6 weeks ( r = 0.75, P < 0.001) and suggests
t h a t healing r a t e s improve with time on therapy.
This observation on time course is strengthened by
t h e r e s u l t s of a l a r g e Japanese m u l t i c e n t r e t r i a l
on pirenzepine. Therapeutic s t u d i e s on g a s t r i c
u l c e r , showed endoscopic healing r a t e o f 66.7%,
using 75 mg/day pirenzepine f o r 8 weeks, which
increased t o 72% with 150 mg dose f o r 8 weeks.
Brunner (94) reported t h a t pirenzepine is equally
o r a t least n o t e s s e n t i a l l y l e s s e f f e c t i v e than
cimetidine i n t h e treatment o f duodenal u l c e r .
The e f f e c t of pirenzepine and cimetidine on
healing, symptoms and r e l a p s e r a t e of duodenal
u l c e r was s t u d i e d by Eichenberger -e t a1 (95). No
s i g n i f i c a n t d i f f e r e n c e s i n u l c e r healing between
t h e e f f i c a c y o f t h e drugs when 1 gm/day o f cimetid i n e and 75 mg/day pirenzepine were used. The
r e l a p s e r a t e a f t e r treatment with pirenzepine was
lower than a f t e r treatment with cimetidine.
Do -e t a1 (96) reported t h e e f f i c a c y of pirenzepine,

100 mg daily, i n t h e treatment of duodenal u l c e r s
i n a placebo-controlled study, Pirenzepine
produced healing i n 14 out o f 18 p a t i e n t s (78%)
following 6 weeks o f treatment as compared t o 7 of
20 p a t i e n t s (35%) receiving placebo. Relief o f
daytime and nighttime pain was g r e a t e r i n
pirenzepine-treated p a t i e n t s , a s well as lower
consumption of antacid.
484
Cheli -e t a1 (97) reported t h a t pirenzepine i n 100 mg

o r a l l y d a i l y f o r 28 days r e s u l t e d i n c l i n i c a l
cure i n 34 of 44 duodenal u l c e r p a t i e n t s . Sixteen
p a t i e n t s completing treatment, were given
pirenzepine 50 mg d a i l y f o r 6 months, r e s u l t e d i n
u l c e r r e l a p s e i n 18.7%. In 16 o t h e r p a t i e n t s , who
i n t e r r u p t e d treatment completely without
pirenzepine, c l i n i c a l recurrence of duodenal u l c e r
occured i n 68.7%, with endoscopic recurrence i n
62%, i n t h e following 6 months.
--
Pirenzepine was shown by Dal Monte e t a1 (98) t o be

e f f e c t i v e i n t h e maintenance treatment of duodenal
u l c e r s t o prevent u l c e r recurrence when given i n
o r a l doses o f 100 mg d a i l y f o r one year.
Pirenzepine has a l s o been e f f e c t i v e i n doses of
50 mg d a i l y f o r one year (100, 104, 107). However,
o t h e r i n v e s t i g a t o r s have reported no s i g n i f i c a n t
d i f f e r e n c e between pirenzepine 25 mg o r a l l y b . i . d .
and placebo i n t h e prevention o f duodenal u l c e r
r e l a p s e i n a one-year study (102). Hoffenberg
(103) reported pirenzepine i n o r a l dose of 50 mg
b . i . d . f o r 4 weeks, i n i t i a l l y , followed by 50 mg
b . i . d . f o r 4 weeks, was no more e f f e c t i v e than
placebo i n t h e treatment of duodenal u l c e r (48%
versus 40% complete healing). These d a t a suggest
t h a t longer courses of therapy (6-8 weeks) may be
indicated.
Pirenzepine i n o r a l d a i l y doses of 100 - 150 mg
has been e f f e c t i v e i n producing healing of 74%
of p a t i e n t s with duodenal u l c e r s i n c o n t r o l l e d
c l i n i c a l t r i a l s (range 52
90%) (53, 55, 57, 60,
74, 76, 104, 105), whereas lower doses of 75 mg
d a i l y o r l e s s have produced lower response r a t e s
( z 55%) (53, 54, 106). These results a r e supported
by more recent c l i n i c a l s t u d i e s with pirenzepine
i n o r a l d a i l y doses of 100
150 mg and t h i s
appears t o be t h e optimal dose (107 - 113).
Available c l i n i c a l t r i a l s on g a s t r i c u l c e r suggest
t h a t o r a l doses of 100-150 mg d a i l y can produce
healing in approximately 72% of p a t i e n t s with gast r i c u l c e r , s i m i l a r t o duodenal u l c e r , doses of
75 mg d a i l y o r l e s s produce lower healing rates
(53, 89, 114, 115).
485
Oral 50 mg d a i l y dose of pirenzepine was reported

e f f e c t i v e i n t h e treatment o f non-ulcer dyspepsia
i n a double-blind comparative t r i a l i n 59 p a t i e n t s
f o r a period of 4 weeks (116). A d a i l y dose o f
100 mg was a l s o shown t o be e f f e c t i v e (117).
Short-term pirenzepine therapy (100 mg d a i l y f o r
4 weeks) was reported e f f e c t i v e i n improving t h e
endoscopic appearance of acute c o n j e s t i v e and
e r o s i v e g a s t r i t i s and non-ulcer-associated
severe duodenitis (118). The drug was s i m i l a r l y
e f f e c t i v e a s cimetidine 1 gm d a i l y f o r 4 weeks.
Comparative e f f i c a c y of pirenzepine 100 mg PO
d a i l y and cimetidine 1 gm PO d a i l y i n 6-week
treatment o f duodenal u l c e r i n o u t p a t i e n t s has
been indicated by s e v e r a l c o n t r o l led c l i n i c a l
t r i a l s (67, 68, 81, 107, 108). Porro e t a1 (109)
have a l s o reported t h a t pirenzepine 150 mg PO
d a i l y was comparable t o cimetidine 1 gm d a i l y i n
t h e treatment o f a c t i v e duodenal u l c e r i n 90
p a t i e n t s t r e a t e d f o r a period of 4 weeks (72%
versus 75% r e s p e c t i v e l y ) . In most c l i n i c a l s t u d i e s ,
t h e e f f i c a c y of both drugs was approximately 70%.
Both drugs have been equally e f f e c t i v e i n
preventing duodenal u l c e r recurrence following
healing with conventional treatment with e i t h e r
drug (99, 101, 120, 121). Combination therapy with
cimetidine has been e f f e c t i v e i n Zollinger-Ellison
syndrome r e s i s t a n t t o cimetidine alone (121).
--
A s i n g l e 400 mg o r a l dose of cimetidine demonstrated
approximately twice t h e a n t i s e c r e t o r y a c t i v i t y
of 50 mg pirenzepine s i n g l e o r a l dose i n both
basal and stimulated g a s t r i c a c i d s e c r e t i o n
following pentagastrin stimulation. With pirenzepine i n doses o f 50 mg, i n h i b i t i o n of p e n t a g a s t r i n stimulated acid s e c r e t i o n was similar t o t h a t o f
an o r a l 200 mg cimetidine, t h i s cimetidine dose,
however, had a g r e a t e r i n h i b i t o r y e f f e c t on g a s t r i c
basal a c i d s e c r e t i o n (122). The authors i n d i c a t e
t h a t high doses of pirenzepine should be avoided
due t o antimuscarinic e f f e c t s such a s dry mouth
and p a l p i t a t i o n s and t h a t cimetidine i s i n d i c a t e d
as agent of choice f o r reduction of g a s t r i c a c i d
s e c r e t i o n . P a l p i t a t i o n s occurred i n s e v e r a l
p a t i e n t s i n t h i s study with doses of 50 mg. In a
s i n g l e - b l i n d multicenter study Brunner et a1 (108)
evaluated t h e e f f i c a c y of pirenzepine 100 mg d a i l y
486
(50 mg before b r e a k f a s t and dinner) compared with

t h a t of cimetidine 1 gm d a i l y (200 mg t . i . d . with
meals and 400 mg h . s . ) i n t h e treatment of endoscopically-proven duodenal u l c e r i n 254 p a t i e n t s .
Endoscopy was repeated a f t e r 4 weeks of treatment
with both regimens; t h e endoscopist was blinded a s
t o treatment regimens. Healing r a t e s of 64% and
73% occurred with pirenzepine and cimetidine,
r e s p e c t i v e l y , which was not s t a t i s t i c a l l y
s i g n i f i c a n t . Pain r e l i e f was achieved r a p i d l y
with both drugs and t h e incidence of s i d e e f f e c t s
was s i m i l a r .
Pirenzepine i n an o r a l dose of 50 mg b . i . d . was

reported s i m i l a r l y e f f e c t i v e as cimetidine 400 mg
b . i . d . o r a l dose (low-dose therapy) i n t h e
treatment of duodenal u l c e r s i n multicenter t r i a l
(125). Following 4 weeks of therapy, healing
occured i n 27 of 37 pirenzepine-treated p a t i e n t s
(73%) and 29 of 38 cimetidine-treated p a t i e n t s
( 7 6 % ) . Side e f f e c t s were s i m i l a r i n each group,
except f o r more frequently reported antimuscarinic
e f f e c t s i n pirenzepine-treated p a t i e n t s . No
adverse e f f e c t s on i n t r a g a s t r i c milieu were
reported. I n t r a g a s t r i c microbial concentrations
were increased s i g n i f i c a n t l y with both drugs but
remained within normal limits; n i t r i l e concentrat i o n p r i o r t o o r following treatment did not
exceed t h e normal range. Oral 300 mg d a i l y dose
r a n i t i d i n e was reported (104) equally e f f e c t i v e as
o r a l 100 mg d a i l y dose of pirenzepine i n t h e
treatment of duodenal u l c e r i n one c o n t r o l l e d
study (response rates of 87% i n each group).
Pirenzepine was equally e f f e c t i v e as a t r o p i n e as
g a s t r i c a n t i s e c r e t o r y agent, but unlike a t r o p i n e ,
pirenzepine had no e f f e c t s on CNS, i n c r e a s e i n
h e a r t r a t e , mydriasis, urinary bladder c o n t r a c t i o n
o r motor function of g a s t r o e n t e r i c t r a c t (124).
Londong -e t a1 (125) reported t h a t concomitant
pirenzepine and cimetidine therapy r e s u l t i n
s i g n i f i c a n t l y g r e a t e r suppression o f stimulated
acid s e c r e t i o n than e i t h e r drug alone. I t i s
suggested t h a t combination therapy may e x h i b i t
s y n e r g i s t i c e f f e c t s on p a r i e t a l c e l l function and
could be advantageous i n p a t i e n t s w i t h gastrinoma,
487
u l c e r s and h y p e r s e c r e t i o n r e s i s t a n t t o s i n g l e
drug t h e r a p y , and i n p r o p h y l a x i s o f stress u l c e r
bleeding i n c r i t i c a l l y - i l l p a t i e n t s .
Weberg et a1 (126) h a s demonstrated t h a t
p i r e n z e p i n e enhances and prolongs t h e e f f e c t s o f
a n t a c i d s on i n t r a g a s t r i c a c i d i t y . Addition o f
p i r e n z e p i n e t o a n t a c i d t h e r a p y r e s u l t e d i n a more
s u s t a i n e d pH e l e v a t i o n t h a n t h a t observed with
t w i c e t h e amount o f a n t a c i d a d m i n i s t e r e d a l o n e .
Combination t h e r a p y w i t h H2-receptor a n t a g o n i s t s
h a s proven more e f f e c t i v e t h a n H2-antagonist
t h e r a p y a l o n e (118, 1 2 7 ) . The combination o f
p i r e n z e p i n e 50 mg h . s . and r a n i t i d i n e 150 mg b . i . d .
given f o r a p e r i o d o f 6-12 weeks was e f f e c t i v e
i n a l l of 7 p a t i e n t s with a c t i v e recurrent
duodenal u l c e r . Continuous maintenance t h e r a p y
with r a n i t i d i n e 150 mg d a i l y and p i r e n z e p i n e 50 mg
d a i l y r e s u l t e d i n prevention of u l c e r relapse i n
5 p a t i e n t s who, i n t h e p r e v i o u s y e a r , c o n t i n u a l l y
r e l a p s e d while r e c e i v i n g s i n g l e - a g e n t ( c i m e t i d i n e
o r r a n i t i d i n e ) t h e r a p y . These r e s u l t s s u g g e s t t h e
e f f i c a c y of combined H2-antagonist and p i r e n z e p i n e
t h e r a p y i n h i g h l y r e c u r r e n t o r r e s i s t a n t duodenal
u l c e r s (127). Mignon e t a1 (121) r e p o r t e d t h e
e f f e c t i v e n e s s o f combination t h e r a p y o f p i r e n z e p i n e
and c i m e t i d i n e i n 3 of 4 p a t i e n t s w i t h Z o l l i n g e r E l l i s o n syndrome who were unresponsive t o
cimet i d i n e a l o n e .
- L
Unlike c i m e t i d i n e ,
inhibitory effects
o x i d a s e system and
concomitantly w i t h
t h i s system (128).
p i r e n z e p i n e appears t o l a c k
on t h e h e p a t i c mixed f u n c t i o n
it should be safe t o a d m i n i s t e r
drugs t h a t a r e metabolized by
The a v a i l a b l e i n f o r m a t i o n s s u g g e s t t h a t p i r e n z e p i n e
p o s s e s s e s s e l e c t i v e a n t i m u s c a r i n i c e f f e c t s and
minimal CNS effects. S t u d i e s p r e s e n t e d so f a r
do n o t show t h a t t h e drug i s more e f f e c t i v e t h a n
c i m e t i d i n e o r r a n i t i d i n e and no s i g n i f i c a n t
d i f f e r e n c e s i n t o x i c i t y have been observed.
P i r e n z e p i n e i n combination t h e r a p y w i t h H2-receptor
a n t a g o n i s t s , however, s u g g e s t t h a t p i r e n z e p i n e may
be very u s e f u l i n t h e t r e a t m e n t o f r e s i s t a n t duoden a l u l c e r and Z o l l i n g e r - E l l i s o n syndrome.
488
7.3
Adverse Reactions
The major s i d e e f f e c t s of pirenzepine a r e due t o
i t s a n t i c h o l i n e r g i c a c t i v i t y which i n d i c a t e s t h a t
t h e s e l e c t i v i t y of t h e drug is n o t absolute and
t h e a n t i c h o l i n e r g i c e f f e c t s can occur which appear
t o be dose-dependents.
Dry mouth is t h e most common s i d e e f f e c t of
pirenzepine. S a l i v a r y s e c r e t i o n e f f e c t s have been
noted i n many t h e r a p e u t i c t r i a l s . In t h e pharmacological s t u d i e s i n man, pirenzepine i n a dose of
50 mg b . i . d . i n h i b i t e d s a l i v a t i o n compared t o
placebo (131) but not a s much as R-hyoscyamine
i n a dose o f 0.6 mg b . i . d . which was approximately
equipotent i n suppression of g a s t r i c a c i d . Ten of
18 p a t i e n t s i n t h e t r i a l s reported dry mouth with
pirenzepine and 17 of 18 on R-hyoscyamine. Dry
mouth i s reported t o occur i n 13.5% of p a t i e n t s
receiving pirenzepine i n doses o f 150 mg d a i l y .
The incidence is reduced t o 4% with doses of 100 mg
d a i l y and t o 2.5% with doses of 75 mg d a i l y o r l e s s
(54).
Dry mouth does not appear t o be severe
enough t o warrant withdrawal of treatment i n most
cases (106).
Pirenzepine i n high doses i n h i b i t s t h e c o n t r a c t i l e
(132) a c t i v i t y of t h e stomach and colon (132).
Bianchi Porro e t a1 (34), however, reported
pirenzepine (50 mg o r a l l y ) showed no e f f e c t s on
g a s t r i c emptying. Similar r e s u l t s were reported
by Stacher -e t a1 (133) when healthy men received
e i t h e r 50 mg pirenzepine twice d a i l y o r placebo.
Despite t h e considerable e f f e c t s of pirenzepine
on a n t r a l m o t i l i t y , its delaying e f f e c t on g a s t r i c
emptying was i n s i g n i f i c a n t . Constipation has
occurred with pirenzepine i n approximately 2.6%
of p a t i e n t s receiving doses of 150 mg o r a l d a i l y
dose (54). Diarrhea has a l s o occurred with
pirenzepine therapy, however, t h e incidence appears
t o be l e s s than t h a t of constipation (23, 106,
134). Jaup (135) measured esophageal p e r i s t a l t i c
a c t i v i t y a f t e r pirenzepine, 10 mg i . v . o r 50 mg
b . i . d . ( o r a l l y ) i n comparison with a t r o p i n e 0.5 mg
i . v . o r R-hyoscyamine 0.6 mg b . i . d . The l a t t e r
two agents reduced esophageal p e r i s t a l t i c pressure
compared t o placebo while pirenzepine showed
I
489
marked e f f e c t s only s h o r t l y a f t e r i . v . dosing,

when plasma l e v e l s were high. Oral pirenzepine
s l i g h t l y decreased (- 15%) p e r i s t a l t i c amplitude
compared t o placebo while R-hyoscyamine decreased
t h i s parameter over 50%. Other s t u d i e s (136, 137)
have shown decreased amplitude of esophageal
contractions as well as i n h i b i t i o n of lower
esophageal s p i n c t e r pressure with p a r e n t e r a l
administration of pirenzepine. Colonic m o t i l i t y is
reduced by 0 . 3 mg/kg i . m . (138) and g a s t r i c
smooth muscle e f f e c t s a r e s l i g h t a t 0 . 2 mg/kg i . m .
(139) o r 50 mg b . i . d . (131). Despite pirenzepine
e f f e c t s i n a l t e r i n g esophageal and colonic
m o t i l i t y , t h e a v a i l a b l e d a t a does not suggest t h e
drug w i l l increase t h e r i s k of p e p t i c g a s t r o esophageal r e f l u x (140).
The e f f e c t s of intravenous and o r a l pirenzepine
on various g a s t r o i n t e s t i n a l hormones and metab o l i c parameters a r e s i m i l a r t o those observed

f o r antimuscarinic agents except f o r t h e lack of
depression of pancreatic bicarbonate s e c r e t i o n
(141, 142) and p o s s i b l y lack of g a s t r i n (143-145).
Pirenzepine produced no changes i n g a s t r i c i n h i b i t o r y polypeptide, i n s u l i n , glucagon, neurotensin,
vasoactive i n t e s t i n a l polypeptide, somatostatin,
s e c r e t i n , f r e e f a t t y a c i d s , l a c t a t e , pyruvate,
f3-hydroxybutyrate, a c e t o a c e t a t e , p r o l a c t i n and
growth hormone. Pancreatic s e c r e t i o n volume,
amylase, t r y p s i n , chymotrypsin,. l i p a s e , p a n c r e a t i c
polypeptide, enteroglucagon, pepsin and g a s t r i c
a c i d s e c r e t i o n s a r e decreased. One study (146)
noted a s l i g h t prolongation o f g a s t r i n postp r a n d i a l l y , while o t h e r s (143, 145) observed
decreases i n c o n t r a s t t o a t r o p i n e and another
t r i a l (144) showed no change. Masala e t a1 (147)
reported t h a t pirenzepine i n s i n g l e doses o f
75 mg reduced p r o l a c t i n l e v e l s i n females but
produced only minimal e f f e c t s on p r o l a c t i n i n
males. S i n g l e doses o f 75 mg pirenzepine were
reported by Alagna -e t a1 (148) t o produce no
d i r e c t e f f e c t upon t h e adrenal glands. The
responsiveness o f t h e adrenal glands t o exogenous
ACTH was unchanged, However, a reduction i n basal
c o r t i s o l l e v e l s occurred with pirenzepine,
suggesting a reduction i n ACTH s e c r e t i o n . The
drug has not produced adrenal i n s u f f i c i e n c y .
--
490
Visual disturbances have been reported i n 42% o f

s u b j e c t s (149). Brunner (94) however, reported
t h a t only small number of p a t i e n t s t r e a t e d with
pirenzepine complained about impairment o f v i s i o n .
Blurred vision and double vision have occurred
during p i renz ep i n e t r e a t men t i n approximate 1y
6.3% of p a t i e n t s receiving o r a l d a i l y doses of
150 mg (75, 83). Hoffenberg (103) described
amblyopia i n 24% of p a t i e n t s receiving dose of
100 mg d a i l y .
Urinary r e t e n t i o n has been reported i n 0.3% of
p a t i e n t s receiving pirenzepine i n doses o f 100 mg
d a i l y ( 8 3 ) . Kuhn -e t a1 (150) reported t h a t
pirenzepine (8 mg i.m.) decreased t h e pulse r a t e
and s y s t o l i c and d i a s t o l i c blood pressure.
Tachycardia has been observed during pirenzepine
treatment (103). In most s t u d i e s , however, no
e f f e c t s on h e a r t r a t e were observed (83).
Pirenzepine i n dose of 50 mg d a i l y caused
p a l p i t a t i o n s (122). CNS t o x i c i t y has been minimal
i n most s t u d i e s with pirenzepine (83, 151).
This can be explained on t h e bases o f pirenzepine
s e l e c t i v i t y and a l s o t o hydrophilic c h a r a c t e r i s t i c s .
Dizziness, mental confusion, a s t h e n i a and headache
have been reported i n a few p a t i e n t s (152, 153).
Studies i n healthy volanteers showed t h a t no
psychotropic e f f e c t s occurred t h a t a r e commonly
encountered with t r i c y c l i c a n t i d e p r e s s a n t s ,
i n d i c a t i n g t h a t pirenzepine lack appreciable CNS
t o x i c i t y (154). Fink and Erwin (149) a l s o reported
lack of CNS e f f e c t s by pirenzepine with doses
upto 150 mg.
7.4
Tissue S e l e c t i v i t y
Pirenzepine blocks t h e acetylcholine receptors of
t h e p a r i e t a l c e l l s o f the stomach and i s thereby
a marked i n h i b i t o r of g a s t r i c acid s e c r e t i o n both
i n animals and men. Pharmacological and c l i n i c a l
s t u d i e s have shown t h i s a c t i o n of pirenzepine t o
be s e l e c t i v e , s i n c e i n doses which s i g n i f i c a n t l y
i n h i b i t g a s t r i c secretion, s i d e e f f e c t s typical
of a n t i c h o l i n e r g i c agents do not occur o r minimal
(30, 33, 45) and much higher doses a r e needed t o
i n h i b i t s a l i v a t i o n (132) and contraction of
stomach and colon (138) o r t o induce tachycardia
(52, 132).
49 1
Engelhorn (155) has e v a l u a t e d t h e s e l e c t i v i t y o f

p i r e n z e p i n e and a t r o p i n e and h i s experiments
i n d i c a t e d s u b s t a n t i a l d i f f e r e n c e s between t h e
i n f l u e n c e o f e i t h e r a n t i m u s c a r i n i c agent on g a s t r i c
u l c e r and g a s t r i c s e c r e t i o n on one hand and on
smooth muscle organs on t h e o t h e r . Although t h e
a n t i - u l c e r and a n t i s e c r e t o r y e f f e c t s o f p i r e n z e p i n e
approach t h o s e o f a t r o p i n e , s i g n i f i c a n t
d i f f e r e n c e s a r e demonstrated i n i s o l a t e d muscle
preparations a f t e r acetylcholine stimulation as
well a s after transmural stimulation. Pirenzepine
was 16.5 times weaker than a t r o p i n e i n reducing
s a l i v a t i o n i n r a b b i t and is 100 times less
e f f e c t i v e than a t r o p i n e i n doubling p a p i l l a r y
diameter. Atropine caused a dose-dependent
inhibition of t h e gastrointes tin a l tr a n s p o r t
w h i l e p i r e n z e p i n e , even a t h i g h e r d o s e s , d i d n o t
cause a d e c r e a s e b u t r a t h e r an i n c r e a s e . Atropine
caused a dose-dependent i n c r e a s e i n h e a r t r a t e
which, however, could only be o b t a i n e d w i t h
p i r e n z e p i n e i n doses 10 t o 100 times g r e a t e r t h a n
with a t r o p i n e . Jaup et a1 (131, 156) compared t h e
effects o f p i r e n z e p i n e and R-hyoscyamine when
t a k e n i n e q u i p o t e n t doses i n i n h i b i t i n g g a s t r i c
s e c r e t i o n . Both drugs a f f e c t e d t h e s a l i v a r y
s e c r e t i o n b u t t h e i n h i b i t i o n by p i r e n z e p i n e was
s i g n i f i c a n t l y less t h a n t h a t by R-hyoscyamine.
Gastric emptying was s i g n i f i c a n t l y delayed by
R-hyoscyamine compared t o p i r e n z e p i n e . Swallowing
induced eosphageal p e r i s t a l s i s was s i g n i f i c a n t l y
i n h i b i t e d i n 51%. P u p i l s i z e and n e a r p o i n t
d i s t a n c e were s i g n i f i c a n t l y a f f e c t e d by
R-hyoscyamine b u t n o t by p i r e n z e p i n e . The minimal
e f f e c t s o f p i r e n z e p i n e on t h e CNS have been
a t t r i b u t e d t o t h e drug s e l e c t i v i t y and h y d r o p h i l i city.
7.5
Mechanism o f Action
I t i s g e n e r a l l y accepted t h a t p i r e n z e p i n e i s an
a n t i m u s c a r i n i c drug. Among o t h e r examples
demonstrating t h i s , is t h e c o m p e t i t i v e i n h i b i t i o n
by t h e drug of t h e carbachol e f f e c t on t h e
spontaneously b e a t i n g guinea p i g atrium ( 1 5 7 ) ,
t h e s e l e c t i v e antagonism o f c a r b a c h o l - s t i m u l a t e d
a c i d s e c r e t i o n i n t h e p e r f u s e d r a t stomach w h i l e
no such antagonism was n o t i c e d when h i s t a m i n e o r
492
p e n t a g a s t r i n was used as a s t i m u l u s (48), as well

a s t h e s e l e c t i v e antagonism, by p i r e n z e p i n e , o f
t h e m u s ca r i n i c response t o bethanechol on
conscious c a t s with g a s t r i c f i s t u l a (157) and t h e
f i n d i n g t h a t p i r e n z e p i n e , i n g r e a t e r amount t h a t
a t r o p i n e , blocked o n l y t h e m u s c a r in ic e f f e c t s o f
a c e t y l c h o l i n e without a l t e r i n g t h e n i c o t i n i c
component (151). S i m i l a r t o o t h e r a n t i m u s c a r i n i c
a g e n t s , p i r en z e p i n e a f f e c t s o n ly t h e volume and
et a1
a c i d o u t p u t , and n o t g a s t r i c pH (83). B a ld i (158) have demonstrated t h a t p i r e n z e p i n e (0.15 mg/kg,
i . m . ) i n h i b i t s t h e motor response i n t h e human
colon t o a c h o l i n e r g i c s t i m u l u s w i t h p r o stig m in e .
S t u d i e s on r e c e p t o r b i n d i n g , pharmacological
i n v e s t i g a t i o n on i s o l a t e d t i s s u e s and s t u d i e s i n
man have shown t h a t p i r e n z e p i n e d i s t i n g u i s h e s
between d i f f e r e n t s u b c l a s s e s o f m u sc a r in ic r e c e p t o r s
(83, 150, 159-170). Using p i r e n z e p i n e a s novel
t o o l t h a t r e v e a l s h e t e r o g e n e i t y o f m u sc a r in ic
r e c e p t o r s , ev i d en c e s have been p r o v id e d (164-166)
o f t h e e x i s t e n c e o f t h r e e d i s t i n c t i v e t y p e s of
muscarinic receptors c l a s s i f i e d a s types A, B
and C. With p i r e n z e p i n e t h e l o g a f f i n i t y
c o n s t a n t f o r t h e A-type is about 7.7, f o r t h e Bt y p e about 6.6 (10-fold weaker) and 4 - f o l d
f u r t h e r weaker f o r t h e C-type. In t h e same system
t h e l o g a f f i n i t y f o r a t r o p i n e i s about 8.5 t o 9 ,
i n d i c a t i n g t h a t t h e pharmacological r e sp o n se o f
p i r e n z e p i n e as compared t o t h a t o f a t r o p i n e w i l l b e
about 1 0 - f o l d weaker a t t h e A-type, 1 0 0 - f o ld
weaker a t t h e B-type and 400-fold weaker a t t h e
C-type. The pharmacological d a t a a v a i l a b l e on t h e
potency of p i r e n z e p i n e as well a s d a t a from b in d in g
s t u d i e s s u g g e s t t h a t i n t h e calf sy m p a th e tic
g a n g l i a t h e r e seems t o be predominantly Ar e c e p t o r s and i n t h e r a t s y m p a th e tic g a n g l i a more
o f B- r e c e p t o r s . The myocardium have predominantly
C - r e c e p t o r s , t h e conduction t i s s u e s i n t h e h e a r t
c o n t a i n B-type r e c e p t o r s w h i le t h e ileum seems t o
have b o t h t y p e B and C r e c e p t o r s and t h e CNS
b o t h t y p e A and B r e c e p t o r s . In most of t h e cases
t h e r e i s good q u a n t i t a t i v e agreement between t h e
b i n d i n g a n a l y s i s and t h e pharmacological r e s u l t s
(132, 155). Evidence f o r t h e h e t e r o g e n e i t y o f
m u s c ar i n i c r e c e p t o r s i s a l s o p r e s e n t e d (171-173)
using t h e s e l e c t i v e ganglionic receptor agonist
493
McN-A-343 [ 4- (m-chlorophenylcarbamoyloxy) -2butenyltrimethylammonium c h l o r i d e ] . The compound

s e l e c t i v e l y stimulates t h e muscarinic receptors
on ganglionic receptors with minimal influence on
t h e muscarinic receptors i n t h e h e a r t and smooth
muscle. Those receptors stimulated s e l e c t i v e l y
by McN-A-343 have been named as M1-receptors
and those stimulated by conventional muscarinic
drugs are c a l l e d M2-receptors. Further i n v e s t i gations a r e needed on pirenzepine and McN-A-343
t o determine which nomenclature t o follow. I t
appears t h a t MI and M2 receptors correspond t o
t h e A-type and C-type receptors r e s p e c t i v e l y .
Pirenzepine may a l s o have a cytoprotective e f f e c t
i n addition t o i t s a n t i s e c r e t o r y e f f e c t s (174, 175).
Other mechanism f o r pirenzepine ulcer-heating
e f f e c t s were a l s o reported (176).
Acknowledgement
The authors would l i k e t o thank M r . Tanvir A. B u t t f o r
typing t h i s manuscript.
494
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STREPTOMYCIN
J a b e r S . Mossa, Abdul Hameed U . Kader Taragan,

and Mahmoud M.A. Hassan,
J a b e r S . Mossa, A s s o c i a t e P r o f e s s o r o f Pharmacognosy,
C o l l e g e of Pharmacy, King Saud U n i v e r s i t y , Riyadh,
Saudi A r a b i a .
Abdul Hameed U . Kader Taragan, L e c t u r e r o f Pharmacognosy,
Department o f Pharmacognosy, C o l l e g e o f Pharmacy,
King Saud U n i v e r s i t y , Riyadh, S a u d i A r a b i a .
Mahmoud M.A. Hassan, P r o f e s s o r of Medicinal Chemistry,

Department of Pharm. Chem., C o l l e g e o f Pharmacy,
King Saud U N i v e r s i t y , Riyadh, Saudi Arabia.
VOLUME 16
507

All rights of reproductionin any form reserved.
JABER S . MOSSA ETAL.
508
1. History
2. Description
2.1
2.2
2.3
2.4
2.5
Nomenclature
Formulae
Molecular Weight
pH Range.
3.1 Melting Range
3.2 Solubility
3.3 Loss on hying
3.5 Crystal Structure
3.6 Spectral Properties
4. Production of Streptomycin
5. Synthesis of Streptomycin
6. Biosynthesis of Streptomycin
7. Pharmacology and Therapeutic Category
8. Pharmacokinetics
8.1 Absorption
8.2 Distribution
8.3 Excretion
8.4 Half -life
9. Toxicity
10. Dosage
12. Mechanism of Action
STREPTOMYCIN
13.1
13.2
13.3
13.4
13.5
13.6
13.7
13.8
13.9
Identification
Titrimetric Methods
Gasometric
Electrometric
Spectroscopic Methods
Bio-Assay Methods.
Biochemical Methods.
Acknowledgement
References
509
510
1. HISTORY
Streptomycin is a water soluble organic base biosynthesised

by appropriate strain of the actinomycetes,SXkepXornqcU
g h i n m . Prior to the development of Penicillin Waksman and
his collaborators began to study the production of antibiotic substances in the department of microbiology of New
Jersey Agricultural Experiment Station, Rutgers University
in 1939 (1). In the succeeding years they examined some
thousands of actinomycetes, hundreds of fungi and many bacteria and extracted from their culture media a number of
antibacterial substances. Particular attention was devoted to obtaining substances which acted prominently on gram
negative bacteria. As a result of this work, the organism
producing streptomycin was isolated from heavily manured
soil and from a chicken's throat (2,3). The first publication on streptomycin was made by Schatz, Bugie and Waksman
(3) in 1944. A complete bibliography of the work on streptomycin from 1944-1952 was recorded by Waksman in "The Literature on Streptomycin" (4). The cultural descriptions and
the nutritional requirement of ShepXornqcu g h i n w are also
discussed by Waksman ( 5 , 6 ) . The early promising clinical
reports of 1944 based on crude Streptomycin prepared in the
Merck Research Laboratories showed the importance of the
crude drug and started an intensive development program for
its large-scale production. The general line of development of the work on streptomycin have closely followed
those of the work on penicillin. Rapid progress in the
study of streptomycin was greatly helped by the experience
gained with penicillin and by the fact that there already
existed organizations for large-scale manufacture and
exploitation of the latter.
2. DESCRIPTION
2.1 Nomenclature
2 . 1 1 Chemical Names
a) O-2-Deoxy-2-(methylamino)-a-L-glycopyranosyl-
(1 + 2)-0-5-deoxy-3-C-formyl-a-L-lyxofuranosyl-
(1 + 4)-N,N' -bis (aminoiminomethyl)-D-streptamine ( 7 ) .
b) 4-0-(2-0-(2-Deoxy-2-methylamino- a -L-glycopyranosyl)-5-deoxy-3-C-formyl-a-L-lyxofuranosyl)-N
N' -diamidino-D -streptamhe (8).
STREPTOMYCIN
511
c) 2,4-Diguanidino-3,5,6-trihydroxycyclohexyl5-deoxy-2-0-(2-deoxy-2-methylamino-a -L-glucopyranosyl ) -3-formy1-a - L 1yxopentanofuranoside (9).

d) N-Methyl-L-glucosamidinostreptosidostreptidine (10).
e) 2- (N-methyl-a-L-glucopyranosaminido)- 3-Cforinyl-5-desoxy-L-aldopentofuranose (11) .
2.12 Generic Name
Streptomycin A.
(12)
2.13 Trade Names

a)
b)
c)
d)
e)
f)
g)
h)
i)
j)
Estreptomicina
Streptomycinum
Streptomyseen
Streptomycini
Streptovex
Stryzolin
Crystamycin
Dimycin
Oroject NS
Streptopen
2.2 Formulae
2.21 Empirical
a) Streptomycin = C21H39N7012
b) Streptomycin sulphate = (C21H39N,012)23H2S04
c) Streptomycin Hydrochloride =
C21H39N7012.3HC1.
d) Streptomycin calcium chloride =
(C21H39N7012. 3HC1) CaC12
JABER S. MOSSA ETAL.
512
2 . 2 2 Structural
MH
NH- C-NH2
H3C NH
HO
Dl
Streptomycin i s an o p t i c a l l y a c t i v e , t r i a c i d i c
base, C21H39N7012; possessing an aldehydic
carbonyl group. The high proportion of oxygen
molecule i s c h a r a c t e r i s t i c of a carbohydrate.
Streptomycin [ l ] i s made up o f t h r e e components :
S t r e p t i d i n e [ Z ] , S t r e p t o s e [ 3 1 , and N-methylL-glucosamine [ 4 ] , linked together by g l y c o s i d i c
bonds (16).
STREPTOMYCIN
513
NH
Streptomycin
PI
5 14
2.23 Structural elucidation

The difficult task of elucidating the structure
of streptomycin was carried out principally by
four groups of workers led by K. Folkers,
0. Wintersteiner, H.E. Carter and M.L. Wolfrom.
Their work has excellently reviewed f o r several
times (11, 17-21). The structural elucidation of
streptomycin is based on studies o f its various
degradation products.
1) On acid hydrolysis, the weaker glycosidic bond
in streptomycin between streptidine and streptose
splits to give streptidine [21 and streptobiosamine 151 (16,22-26).
2) Mild methanolysis o f streptomycin [ 11 with
methanolic hydrogen chloride gives streptidine
[ 21 and methyl streptobiosaminide dimethyl acetal
hydrochloride [61. Treating [6] with ethyl mercaptan and hydrogen chloride gives ethyl thiostreptobiosaminide diethyl mercaptal hydrochloride [ 7 1 which is also obtained by the action of
ethyl mercaptan and hydrogen chloride on streptomycin. By Raney-nickel and subsequent acid hydrolysis o f [71 , it gives N-methyl-L-glucosamine [4]
and bisdeoxy-streptose 181 (27-28).
3) Hydrolysis of streptomycin xith N1 sodium hydroxideofor three minutes at 100 or eighteen hours
at 40 yields a weakly acid substance, which has
been characterized as maltol [9! (29).
qCH3
OH
The degradation reactions of streptomycin is well

summarized in Scheme I.
516
Streptidine
is a meso Form of 1,3Streptidine 121, ~8~18~604,
diguanido-2,4,5,6-tetrahydrocyclohexane (24,25,
30, 31). The structure of streptidine [21 was
confirmed by synthesis from D-glucosamine (32,33).
Streptobioasamine
The structure of Nitrogen containing disaccharide,
streptobiosamine [51 was determined by a study of
hydrolysis products of the methyl ether and the
ethyl thio-ether formed by the action of hydrogen chloride in methanol and ethyl mercaptan respectively (27, 29, 34, 35). The hydrolysis of
methyl-streptobiosaminide dimethyl acetal gave
N-methylglucosamine 141 (27,28). The structure of
N-methyl-L-glucosamine has been established by
synthesis from L-arabinose (36). Because of its
instability, it is very difficult to isolate streptose [ 31 , the second component of streptobiosamine
[51. The molecular formula CgH1005 was calculated
from the known formulae of streptomycin 111 ,
Streptidine [ 2 ] and N-methyl-L-glucosamine [41
and its structure was deduced by identifying oxidation and reduction products. It was finally
shown to be 3-C-formyl-5-deoxy-L-lyxose, a pentose
containing the reactive aldehyde group associated
with streptomycin molecule (29,37-40). The
structure of streptomycin [I] was finally confirmed by total synthesis (Scheme 11).
2.24 CAS Registry Number
1) Streptomycin
2) Streptomycin sulphate
3) Streptomycin Hydrochloride
(57-92-1)
(3810- 74-0)
(6160-32-3)
2.25 Wiswesser Line Notation

Streptomycin sulphate :
T60TJ BlQ CQ -DQ EM1 FO- DT50TJ B CV H CQ EO
AL6T J BQ CQ DMYZ UM EQ EMYZU M WSQQ 3.
(41)
STREPTOMYCIN
517
2.26 Stereochemistry and Absolute Configuration

The chemistry of the component fragments of streptomycin was thoroughly studied and their structures were established (23-40). The absolute configuration of streptomycin has been elucidated by
chemical (applying Reeve's Copper complexing
method to various degradation products of streptomycin, dihydrostreptomycin and streptobiosamine)
(42,43) and optical correlations (44). The absolute stermch emistry of streptose was established
to be (R) at C-4 and (S) at C-2 (37,40,45). The
streptidine was shown to possess all mrn configuration (46). Although streptidine is a meso
form, the asymmetric attachment of streptobiosamine to it causes each of the ring carbon of
streptidine in streptomycin to be asymmetric. The
asymmetry of streptidine ring in streptomycin is
1(R), 2(R), 3(S), 4(R), 5(R), 61s). The streptobiosamine moiety of streptomycin has been shown to
be attached to position 4 of streptidine in either
R or S absolute configuration (46-48). The three
units of the streptomycin are attached together
by glycosidal bonds which were shown to be formed
by condensations between :
(i) the C-2 hydroxyl o f streptose and C-1 hydroxyl
of N-methyl-L-glucosamine (28,38,39,49-51).
(ii) C-1 hydroxyl of streptose and a hydroxl
group of streptidine that is adjacent to only
one guanidino group (i.e., position 4)
(22,38,39,52-54).
All glycosidic linkages have an a-S configuration
which was confirmed by NMR and crystallographic
studies (44, 55-57). By using the configurational
assignment, the structure of streptomycin in
complete stereochemical details may be written
as indicated below :
JABER S. MOSSAETAL.
518
YH2
C=NH
I
N-H
HOCH2
OH
-I
\,
- 11-
YH2
L-I
II
H NH
HO
2 . 3 Molecular Weight
a)
b)
c)
d)
Streptomycin
Streptomycin
Streptomycin
Streptomycin
sulphate =
hydrochloride =
calcium chloride =
581.6
1457.4
691.0
1492.9 (8)
2 . 4 Appearance, Color, Odor and Taste
Streptomycin occurs as white to slightly pink o r pale

brownish powder or granules; odor: odorless o r with
a slight c d o r ; taste : slightly bitter. Streptomycin
is hygroscopic and may deliquensce on exposure to air
but not affected by air or light ( 1 3 ) .
2 . 5 PH Range
per cent w/v solution of streptomycin has a pH

between 5 and 7.
A 25
519
STREPTOMYCIN
3 . PHYSICAL PROPERTIES
3 . 1 Melting Range
I n d e f i n i t e (12).
3 . 2 So 1u b i 1it y
1)
Streptomycin : I t i s very s o l u b l e i n w a t e r ; almost

i n s o l u b l e i n a l c o h o l ( 9 5 % ) , chloroform, e t h e r and
petroleum e t h e r ( 1 3 ) .
2)
Streptomycin s u l p h a t e : I t i s v e r y s o l u b l e i n
water; p r a c t i c a l l y i n s o l u b l e i n a l c o h o l a c e t o n e ,
chloroform, e t h e r and petroleum e t h e r ( 8 ) .
3)
Streptomycin h y d r o c h l o r i d e : I t i s v e r y s o l u b l e i n
water, p r a c t i c a l l y i n s o l u b l e i n a l c o h o l , c h l o r o form and e t h e r ( 8 ) .
4)
Streptomycin calcium c h l o r i d e : I t is v e r y s o l u b l e
i n water, p r a c t i c a l l y i n s o l u b l e i n a l c o h o l , c h l o r o form and e t h e r ( 8 ) .
3 . 3 Loss on Drying
Strephomycin l o s e s when d r i e d o v e r phosphorus p e n t o x i d e
a t 60 C and a t a p r e s s u r e n o t exceeding 5 mm. o f merc u r y , f o r 3 hours, n o t more t h a n 5 p e r c e n t (13).
3.4 O m i c a l R o t a t i o n
Streptomycin i s o p t i c a l l y a c t i v e w i t h l e v 0 r o t a t i o n .
The f o l l o w i n g o p t i c a l r o t a t i o n s were r e p o r t e d f o r t h e
various s a l t s of streptomycin (7,12) :
25
[a1D
1. Streptomycin h y d r o c h l o r i d e .
-84:
2 . Streptomycin calcium c h l o r i d e . - 7 6
Solvent
Water (C = l . O )
Water (C -1.0)
The s p e c i f i c r o t a t i o n or s t r e p t o m y c i n s u l p h a t e was
determined i n our l a b o r a t o r y
on a P e r k i n E l m e r Polar i m e t e r , Model 241 MC and found t o be [a125 - 75' i n
D
water (C = 1. O ) .
520
3.5 Crystal Structure

Neidle, e t a1 (57) have r e p o r t e d t h e X-ray c r y s t a l l o g r a p h i c a n a l y s i s of s t r e p t o m y c i n oxime s e l e n a t e . S t r e p mycin oxime selenate', C21H40Ng012
[ 1%H2Se04], 4H20
c r y s t a l l i z e s from aqueous methanol as monoclinic
n e e d l e s , s a c e groupoC2, a = 17.10, b = 14.36,
c = 16.13
B = 108 , D = 1 . 5 4 g . ~ m - ~Dc, = 1 . 5 5 g .
cm-3 f o r f o u r formula u n f t s p e r c e l l . The [OOl] p r o j e c t i o n o f one streptomycin oxime i o n i n selenate
c r y s t a l i s shown i n F i g . 1.
B,
3.6 Spectral Properties

3 . 6 1 U l t r a v i o l e t spectrum
The UV spectrum o f s t r e p t o m y c i n s u l p h a t e i n water
was scanned from 200 t o 400 nm on DMS 100 Varian
AG Spectrophotometer (Fig. 2 ) .
Three maxima were
observed a t 241.6, 270 and 328.2 nm. Streptomyc i n i n 0.2N su;phuric a c i d , showed a maxima a t
280 nm w i t h E l 4 = 0.2 ( 9 ) .
1cm
3.62 I n f r a r e d spectrum
The i n f r a r e d spectrum of s t r e p t o m y c i n s u l p h a t e
a s K B r d i s c recorded on a Pye Unicam i n f r a r e d
spectrophotometer i s shown i n F i g . 3.
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 f o l l o w i n g f r e q u e n c i e s (Table 1 ) :
Table 1 : I R C h a r a c t e r i s t i c s of Streptomycin
Sulphate.
Frequency cm
-1
Assignment
3100-3500
NH and OH s t r e t c h (vb).
1630- 1710
1480
1100-1200
-C=O and -C=NH s t r e t c h ( v b ) .

(vb) *
C-0-C-stretch
(vb) *
vb = Very broad.
5.
Other r e p o r t e d I R d a t a 41) a r e 3330, 1670, 1470,

1390, 1110 and 1040 cm-
STREPTOMYCIN
F i g . 1 : [OOlI P r o j e c t i o n o f one Streptomycin oxime

i o n i n t h e Selenate C r y s t a l .
Fig. 2 : UV Spectrum of Streptomycin Sulphate in Water.
521
80
t
1
2000
1800
1600
1400
Fig. 3 : IR S p e c m ~ aof Streptomycin Sdphrte as K k diw.
1200
1000
800
523
STREPTOMYCIN
3 . 6 3 Nuclear Magnetic Resonance Spectra

3.631
1
H-NMR Spectra
1
The H-NMR s p e c t r a of streptomycin i n D 0

( F i g . 4 ) were recorded on a T60A MHz N M i
spectrometer with TMS a s i n t e r n a l reference.
The proton chemical s h i f t s a r e shown i n
Table 2 .
NH
OH
CH
7* 3
Table 2 : PblR C h a r a c t e r i s t i c s o f Stremomycin
Sulphate
Chemical
Group.
shift.
D20
H-1 N-methylglucosamjne
5.53
H-1
Streptose
3 -ti Formyl s t r e p t o s e
5.30
5.06
3.83
3.56
2 - N Me of N-methyl
glucosamine.
- Sec-Me o f S t r e p t o s e .
2.86
1.23
1 1
,
'
I . . . .
8.0
I .
r.0
. I . . . , I . .
. 1 . . . . 1 . . . , 1 .
6.0
5.0
m (4)
4.0
3.0
Z.0
I
Fig. 4
H-NMW Spectrum of Streptomycin Sulphatr iii I)$) and TMS.
1 . . . . 1 ,
1.e
STREPTOMYCIN
525
3.632 "C-NMR
Spectra
The n a t u r a l abundance C-13 NMR n o i s e - d e coupled and s i n g l e frequency o f f - r e s o n a n c e

decoupled (SFORD) s p e c t r a ( F i g . 5 and F i g . 6 )
i n deuterium o x i d e were o b t a i n e d on a J o e l
FX 90 - 90-MHz i n s t r u m e n t . The carbon
chemical s h i f t s were a s s i g n e d on t h e b a s i s
o f t h e t h e o r y of chemical s h i f t and SFORD
s p l i t t i n g p a t t e r n (Table 3 ) .
Table 3 : Carbon Chemical S h i f t s o f Streptomycin
Sulphate.
Carbon No.
1
2
3
4
5
6
7
8
1'
2'
3'
4'
5'
6'
1"
2'7
3"
4"
5"
6"
7''
Chemical S h i f t
6 ppm
57.06
87.13
60.82
108.61 o r 97.11
63.11
61.52
160.33
160.80
108.61 o r 97.11
71.92
84.89
80.67
34.81
186.31
108.61 o r 97.11
64.50
73.33
74.21
80.14
75.97
15.20
Multiplicity
d
d
d
d
d
d
S
S
d
d
S
d
d
d
d
d
d
t
Fig. 5 : 13C-NMR Noisc-decoupkd Spectrum of Streptomycin SuIphate in D20 and TMS.
hli
Fig. 6 : 13C-NMR Off-Resonance Spectrum of Streptomycin Sulphate in D 2 0 and TMS.
528
1 6 0 .3 3 (s)
57.06 ( d )
87.13(d)
160.8 (5)
H2N
60. 82(d)
C
0
- HN
H
0
63. i 1(d)
108.61 o r
97.11 (d)
9 7 .1 1 (d)
80.14 (d)
74.21 (d)
73.33 (d)
64.05 (d)
STREPTOMYCIN
529
4 . PRODUCTION OF STREPTOMYCIN
Streptomycin i s produced by t h e micro-organism b;trreptomyca
g h h w . I t i s a l s o produced by o t h e r s t r a i n s of organisms
l i k e Acfinomyca cj1obhppohu.b ( 5 8 ) , pink v a r i a n t o f bRtreptumqca g h i n e u n (591, bAh.epXumycu g d b u (60). However, f o r
l a r g e s c a l e p r o d u c t i o n , a p p r o p r i a t e s t r a i n o f baheptomyca
g&mb i s used.
SXtepXomycu g & e u
can b i o s y n t h e s i z e s t r e p t o m y c i n i n a
medium c o n t a i n i n g g l u c o s e , sodium c i t r a t e and i n o r g a n i c
s a l t s , but much h i g h e r y i e l d s are o b t a i n e d by u s i n g t h e
media c o n t a i n i n g a l s o an o r g a n i c s o u r c e o f n i t r o g e n such
a s meat e x t r a c t , y e a s t a u t o l y s a t e , Soybean f l o u r , c o r n
s t e e p l i q u o r o r f e r m e n t a t i o n s o l u b l e s (61-88). One o r
more of t h e s e a r e used f o r i n d u s t r i a l b i o s y n t h e s i s . Tremendous amount of work h a s been done on t h e p r o d u c t i o n of
s t r e p t o m y c i n (64 , 89-103) .
The l a r g e - s c a l e p r o d u c t i o n h a s been accomplished by e i t h e r
s u r f a c e c u l t u r e o r deep c u l t u r e p r o c e s s ( 6 4 , 6 5 ) . The deep
c u l t u r e p r o c e s s i s more o f t e n used f o r l a r g e - s c a l e p r o d u c t i o n o f s t r e p t o m y c i n . A b r i e f o u t l i n e of t h e p r o d u c t i o n
o f s t r e p t o m y c i n i s well summarised below (104):
a ) P r o d u c t i o n o f Spore Suspension
The s p o r e s o f h%%eptumqcu g h i n ~ .r e~q ~u i r e d f o r i n o c u l a t i o n i s o b t a i n e d from r a p i d l y growing c u l t u r e on s o l i d
medium (64, 1 0 5 ) . The s p o r e s are e i t h e r suspended i n
s t e r i l e water o r i n skimmed m i l k which i s i n o c u l a t e d w i t h
t h e medium i n t h e shake f l a s k s .
b) P r e p a r a t i o n o f Medium
A l l t h e s o l i d c o n s t i t u e n t s o f t h e c u l t u r e medium a r e
p l a c e d i n t h e ferinenter through t h e c h a r g e h o l e and w a t e r
i s added t o make up t h e volume. The medium i s s t e r i l i z e d
by t h e d i r e c t i n t r o d u c t i o n of steam. S t e r i l i z a t i o n i s
c a r r i e d o u t a t 1 5 l b p r e s s u r e f o r 2 5 minutes. A f t e r s h u t t i n g o f f t h e steam, water i s passed through t h e j a c k e t and
an a i r p r e s s u r e o f 10 l b i s maintained i n t h e f e r m e n t e r .
The s i t r r e r is working d u r i n g s t e r i l i z a t i o n , c o o l i n g and
ferment a t i o n .
c ) P r e p a r a t i o n o f t h e inoculum
The s t e r i l i z e d medium i n t h e s e e d t a n k (115 L . c a p a c i t y ,
provided with s t i r r e r and inoculum measuring t a n k ) i s
530
Flow Sheet 1 : The production of Streptomycin.
STREPTOMYCIN
53 1
i n o c u l a t e d w i t h t h e c o n t e n t o f shake f l a s k . After t h e
i n o c u l a t i o n , t h e niycelium i s grown f o r 36 hours w i t h an
a e r a t i o n r a t e of 100 L p e r minute. After 36 h o u r s , a
heavy growth of mycelium i s o b t a i n e d . About twenty l i t e r s
o f t h i s mycelium suspension i s blown i n t o t h e measuring
t a n k and f i n a l l y t o t h e f e r m e n t e r s through p i p e - l i n e .
d) Fermentation
The f e r m e n t e r s a r e made up o f s t a i n l e s s s t e e l , having t h e
c a p a c i t y of 15000 g a l l o n s and provided w i t h s i t r r e r , a n t i foam n o z z l e , a i r s p a r g e r and a i r o u t l e t . After t h e
inoculum from t h e s e e d tank h a s been added t o t h e ferment e r , a e r a t i o n i s s t a r t e d a t once. Then t h e f e r m e n t a t i o n
0
0
i s c a r r i e d o u t a t an optimum t e m p e r a t u r e of 25 -30 C .
Foaming d u r i n g f e r m e n t a t i o n i s c o n t r o l l e d by t h e a d d i t i o n
o f a n t i f o a m i n g a g e n t s . The f i n a l c o n c e n t r a t i o n o f s t r e p tomycin (50,000 u n i t s p e r l i t r e ) w i l l be reached i n 72
h o u r s . The p r o d u c t i o n of s t r e p t o m y c i n is given i n flow
sheet I .
e) I s o l a t i o n and P u r i f i c a t i o n
The s t r e p t o m y c i n from t h e c u l t u r e f l u i d i s s e p a r a t e d by
a d s o r p t i o n on t h e c h a r c o a l and by subsequent e l u t i o n w i t h
methanolic hydrochloric acid o r other s u i t a b l e solvents
(106-117).
F u r t h e r p u r i f i c a t i o n i s c a r r i e d o u t by chromatography on
alumina (118-122) o r i o n exchange resins (123-164) and by
p r e c i p i t a t i o n w i t h v a r i o u s b a s e p r e c i p i t a n t s (165-179).
S o l v e n t e x t r a c t i o n techniques (180-188), e l e c t r o d i a l y s i s
(189) and e l e c t r o o s m o t i c (190) procedures a r e a l s o
employed.
5 . SYNTHESIS OF STREPTOMYCIN
Although s t r e p t o m y c i n was t h e f i r s t aminoglycoside a n t i b i o t i c t o be d i s c o v e r e d , i t was n o t s y n t h e s i s e d u n t i l some

30 y e a r s l a t e r . The t o t a l s y n t h e s i s of s t r e p t o m y c i n was
achieved by Sumio Umezawa e t a l , i n 1973 (191,192). The
r e a c t i o n s involved a r e i n shown i n Scheme I 1 j summarized
below:
Condensation o f blocked d e r i v a t i v e o f d i h y d r o - s t r e p t o s e
(benzyl a-L-dihydro-streptose)[lO] with t h e glucosamine
d e r i v a t i v e (L-glucopyranosyl b r o m i d e ) [ l l ] i n benzene i n
t h e p r e s e n c e of m e r c u r i c c y a n i d e , followed by hydrol y s i s o f t h e S c h i f f b a s e w i t h 50% a c e t i c a c i d and
532
SCHEME I1
STREPTOMYCIN
533
1111
phcg-J
PhCOO
0
1191
PhCOO
534
phcol&j
PhCOO
0
NH
I1
NHCNH2
1221
tlH
NHCNHz
OH
STREPTOMYCIN
535
reaction with methyl chloroformate (Na2Cog, aq. acetone) gave the protected streptobiosaminide[ 121. NMethvlation of [121 gave [131 Deacetylation followed by
deisopropylidenation (IN HC1, Aqueous MeOH) gave [ 141
which was identical with benzyl a-S-dihydrostreptobioasminide. Hydrolysis of 1141 with 10% Ba(OH)2 affored
benzyl a-L-dihydro-Streptobiosaminide [ 151 . Treatment
of [ 151 with 2,2-dimethoxypropane (p-toluenesulfonic
acid) gave a diisopropylidene derivative and further
blocking by use of p - n i t r o p h en o x y c a r b o n y lc h lo r id e
(NaOH, aqueous acetone) gave [ 161. Partial hydrolysis
of [161 (25% AcOH in MeOH) gave the diol 1171, This
was converted into the dibenzoyl derivative [18].
Partial hydrolysis of [181(75% Acetic acid) removed
the isopropylidene group giving a diol which was transformed (p-N02C6H ococ 1, pyridine) into the carbonate
[19]: Hydrogenofysis of the glycoside linkages
(Palladium Black-dioxane) gave free sugar 1201. The
reaction of [201 with thionyl chloride at room temperature offered the a-glycosyl chloride [211. Direct
treatment of [ 151 with p-nitrophenoxycarbonylchloride
o r phosgene followed by benzolyation also gave [191.
But the yield is very poor.
Finally condensation of [211 with di-N-acety1-di-N-
b e n z y l o x y c a r b o n y l - 0 - c y c l o h e x y l i d e n e s t r e p t i d i n e gave
the 4-0-glycoside [22]. Hydrolysis of 1221 with 0.05 N
Ba(OH2) and dioxane successively removed the carbamate,

carbonate, benzoyl and acetyl groups. Removal of the
remaining protecting groups with 50% acetic acid
followed by hydrogenolysis with Palladium black gave
dihydrostreptomycin [23]. Subsequent mild oxidation
of [23] afforded streptomycin[l] .
6. BIOSYNTHESIS OF STREPTOMYCIN
Biosynthesis of streptomycin has been quite extensively
studied. Streptomycin is formed from glucose as a primary
precursor (193). Biosynthesis of the components of streptomycin, streptidine, streptose and N-methyl-glucosamine
are well summarised by Horner (193) E Snell (194). General
indication of the metabolic relationships of glucose L241
to the various components of streptomycin is shown in
Scheme 111.
536
SCHEME I11
Tranunnular
O<QH
Icarranttemcnt.
on
HO
Mvoinotitol
H,yo3n
on
OH
STREPTOMYCIN
537
Experiments w i t h l a b e l e d compounds demonstrated t h a t t h e

s p e c i f i c a l l y labeled glucose [24] i s converted i n t o s t r e p tomycin [ l ] i n which each of t h e s u b - u n i t s i s l a b e l e d a t
t h e c o r r e s p o n d i n g carbon (Scheme IV). T h i s was e s t a b l i s h e d
by s e v e r a l groups (195-203). Some o f t h e i n t e r m e d i a t e s i n
t h e b i o s y n t h e t i c pathway of s t r e p t o m y c i n have been i s o l a t e d , b u t some o f t h e key i n t e r m e d i a t e s a r e n o t y e t d e t e c t e d .
Recent s t u d i e s e s t a b l i s h e d t h e p r e s e n c e o f s t r e p t i d i n e - 6 phosphate and dihydro-streptose-dTDP, i n t h e s t r e p t o m y c i n
b i o s y n t h e t i c pathway (204). Some i n t e r m e d i a t e s between
g l u c o s e and d i h y d r o s t r e p t o s e a r e known as a r e s u l t o f t h e
work o f Grisebach (205) who demonstrated t h a t [14C-] glucose-dTDP was c o n v e r t e d t o 4-Keto4,6-dideoxyglucose-dTDP
which i n t u r n was c o n v e r t e d t o dihydrostreptose-dTDP.
Nothing i s known y e t r e g a r d i n g t h e i n t e r m e d i a t e between
g l u c o s e and N-methylglucosamine. Recently Kniep and
Grisebach (206) e s t a b l i s h e d t h e r o l e o f d i h y d r o s t r e p t o s y l s t r e p t i d i n e 6-phosphate [ 2 5 ] i n t h e b i o s y n t h e s i s o f s t r e p tomycin. They concluded t h a t t h e nucleotide-bound N-methyl4-glucosamine moiety i n S. g h i n w was c a t a l i z e d and t r a n s f e r r e d t o d i h y d r o s t r e p t o s y l s t r e p t i d i n e 6-phosphate [ 2 5 ]
which i n t u r n s was c o n v e r t e d t o dihydrostreptomycin 6 phosphate [ 2 6 ] . F u r t h e r o x i d a t i o n of [ 2 6 ] gave s t r e p t o mycin-6-phosphate[27] (207) hid h y d r o l y s i s y i e l d s t r e p t o mycin [ l ] (208).
The r e a c t i o n i s summarized i n Scheme V .
7 . PHARMACOLOGY AND THERAPEUTIC CATEGORY
Streptomycin i s an aminoglycoside a n t i b i o t i c which i s

a c t i v e a g a i n s t a wide range of b a c t e r i a , both Gram p o s i t i v e
and Gram n e g a t i v e (209). I t i s a v a l u a b l e t h e r a p e u t i c
a g e n t f o r a l l forms of t u b e r c u l o s i s (210-252). S t r e p t o mycin i s e f f e c t i v e a g a i n s t Tularemia (253,254). I t i s
h i g h l y s p e c i f i c and one o f t h e most e f f e c t i v e a g e n t f o r
t h e t r e a t m e n t o f a l l forms o f p l a u g e (255). I n combinat i o n w i t h P e n i c i l l i n G , it i s c o n s i d e r e d t o be t h e d r u g o f
c h o i c e i n i n f e c t i o n s w i t h SfiepXococcU v h i d a v m and
EvLtmococcu ( 2 5 6 ) . I t i s a l s o e f f e c t i v e a g a i n s t h&phyLococcud uLbw (257).
Streptomycin i s a d r u g o f second c h o i c e i n i n f e c t i o n s due
t o s u s c e p t i b l e s t r a i n s o f a n a e r o b i c bfiCLp,&jCOC&, b a c t e r o i d e s , AmobacXm a e h o g e n a , A . d e c a l h , S h i g e l L a , BhuceRea
and Vibh,Lo carnnin and i n combination w i t h t e t r a c y c l i n e , i n
P o t l ; t e ~ i n f e c t i o n s and i n b r u c e l l o s i s (256,258-264).
Since
s t r e p t o m y c i n i s e f f e c t i v e a g a i n s t H. i n d h e n z a e , E. CULL,
K&bbLelLa pneurnanhe and S&andl?a,
i n combination w i t h
NH
SCHEME I V
II
NH
NHCNH
II
STREPTIDINE
i-koH
HO
-.
OH
~241
#q*
I
H3C
OH
STREPTOSE
HQ
N-METHYL-
CH20H
CH3NH
OH
[11
L-GLUCOSAMINE
STREPTOBIOSAMINE
STREPTOMYCIN
539
SCHEME V
nvo
1251
H$NM
OM
OH
Ill
1261
JABER S.MOSSA ETAL.
540
o t h e r a n t i b i o t i c s it i s useful i n t h e treatment of r e s p i r a t o r y t r a c t i n f e c t i o n s , b a c t e r i a l m e n i n g i t i s and u r i n a r y

t r a c t i n f e c t i o n s (256,265-273).
Streptomycin i s a l s o
e f f e c t i v e f o r typhoid f e v e r (274-277) , whooping cough (278) ,
d i p h t h e r i a (279) and i n murine
l e p r o s y (280-283).
8 . PHARMACOKINETICS
8 . 1 Absorption
Streptomycin is absorbed p o o r l y and i r r e g u l a r l y from
t h e g a s t r o i n t e s t i n a l t r a c t (255,284). T h e r e f o r e , t h e
o r a l route i s not s u i t a b l e f o r administration of s t r e p tomycin f o r s y s t e m i c a c t i o n . However, t h e o r a l r o u t e
h a s been recommended i n t h e t r e a t m e n t o f i n t e s t i n a l
d i s o r d e r s due t o s t r e p t o m y c i n s e n s i t i v e s t r a i n s o f
b a c t e r i a o r t o reduce t h e b a c t e r i a l f l o r a of t h e
i n t e s t i n e p r i o r t o o p e r a t i o n on i n t e s t i n a l t r a c t .
Streptomycin i s r a p i d l y absorbed when a d m i n i s t e r e d by
subcutaneous, i n t r a m u s c u l a r o r i n t r a v e n o u s r o u t e s
(255,256,285). Streptomycin can a l s o be absorbed
through rectum when a d m i n i s t e r e d as cocoa b u t t e r o r
carbowax m i x t u r e (286,287). P a r e n t r a l a d m i n i s t r a t i o n
must be employed t o e n s u r e t h e r a p e u t i c a l l y a d e q u a t e
plasma and t i s s u e l e v e l s of s t r e p t o m y c i n . After an
i n t r a m u s c u l a r dose of 1 g o f streptomycinipeak serum
c o n c e n t r a t i o n s o f 25 t o 50 mg/ml a r e a t t a i n e d i n 0.52 h o u r s , f a l l i n g t o 5 t o 10 mg/ml a t 12 hours (9, 1 5 ) .
The e f f e c t i v e minimum serum c o n c e n t r a t i o n i s about
7 mg/ml ( 1 5 ) .
8.2 Distribution
No m e t a b o l i t e of streptomycin has so f a r been d e t e c t e d .

About one t h i r d t h e c i r c u l a t i n g st-reptomycin i s bound
t o serum p r o t e i n . Streptomycin i s widely d i s t r i b u t e d
t o a l l organs e x c e p t b r a i n .
The drug p e n e t r a t e s r a p i d l y i n t o t h e p e r i t o n e a l , p e r i c a r d i a l , p l e u r a l and s y n o v i a l f l u i d s . Very l i t t l e
enters i n t o cerebrospinal f l u i d except i n meningitis
(256,285,288,289) ,
8.3 E x c r e t i o n
About 50-80% o f s t r e p t o m y c i n i n j e c t e d i s e x c r e t e d i n
u r i n e unchanged i n 24 hours (15,285,290,291).
Most of
t h e o r a l dose i s e l i m i n a t e d i n t h e f a e c e s (15,256).
About 0.5% is e x c r e t e d i n t h e b r e a s t m i l k i n 24 hours
and 1%i s e x c r e t e d i n b i l e (15,286).
STREPTOMYCIN
541
8.4 Half-life
Serum h a l f - l i f e o f s t r e p t o m y c i n i s about 2 . 5 hours i n
young a d u l t s . T h i s w i l l be i n c r e a s e d i n t h e new born
and i n a d u l t s above 40 y e a r s . In r e n a l f u n c t i o n
impairment, t h e h a l f - l i f e may be i n c r e a s e d t o 2-5 days
(15)
9 . TOXICITY
A l l e r g i c r e a c t i o n s a r e r e l a t i v e l y v e r y common w i t h t h e
a d m i n i s t r a t i o n o f s t r e p t o m y c i n . The u s u a l a l l e r g i c symptoms a r e e o s i n o p h i l i a , f e v e r and s k i n r a s h e s . The d r u g
a l s o c a u s e s headache, nausea, a r t h r a l g i a , b l u r r i n g o f
v i s i o n , v e r t i g o , r e n a l i r r i t a t i o n and n e u r o t o x i c i t y . The
most s e r i o u s t o x i c e f f e c t i s damage t o t h e e i g h t h c r a n i a l
n e r v e , c a u s i n g d e a f n e s s which i s permanent. Damage t o
r e n a l t u b u l e s , bone marrow and l i v e r r a r e l y o c c u r s a t t h e
usual t h e r a p e u t i c t i s s u e l e v e l . Application of s t r e p t o mycin d i r e c t l y t o t h e peritoneum o r i t s i n t r a v e n o u s i n j e c t i o n i n l a r g e doses may cause d e a t h by p a r a l y s i s o f t h e
r e s p i r a t i o n (285,286,288, 292-303).
1 0 . DOSAGE
About 0 . 5 t o 1 gram by i n t r a m u s c u l a r i n j e c t i o n d a i l y o r a t
long i n t e r v a l s . A s an i n t e r n a l a n t i s e p t i c , 500 m i l l i g r a m s
by mouth every e i g h t hours (304).
11. PHARMACEUTICAL FORMS
1)
2)
3)
4)
Streptomycin
Streptomycin
Streptomycin
Streptomycin
s u l p h a t e U.S.P.
sulphate i n j e c t i o n B.P.
calcium c h l o r i d e B.P.
hydrochloride B.P.
(305).
(304).
(8) *
(8)
1 2 . MECHANISM OF ACTION
The a n t i b a c t e r i a l a c t i o n of s t r e p t o m y c i n i s a consequence
o f i t s combination w i t h t h e 30s ribosomes o f s u s c e p t i b l e
organisms, t h e r e b y d i s t o r t i n g t h e t r a n s l a t i o n of t h e
g e n e t i c code. As a r e s u l t o f t h i s c o n d i t i o n t h e wrong
amino a c i d s a r e i n c o r p o r a t e d i n t o t h e n a s c e n t p o r t i o n o f
t h e p r o t e i n molecule, t h e p r o t e i n s y n t h e s i s i s d i s r u p t e d
and t h e c e l l d i e s (256, 306, 307).
542
13. METHODS OF ANALYSIS

1 3 . 1 Elemental Composition
c = 43.7%
H = 6.76%
N = 16.86%
0 = 33.01%
(7).
13.2 I d e n t i f i c a t i o n
13.21 Pharmacopeial T e s t s
The f o l l o w i n g t e s t s have been d e s c r i b e d i n
B r i t i s h Pharmacopoeia f o r t h e i d e n t i f i c a t i o n
of s t r e p t o m y c i n (304).
a ) D i s s o l v e about 0 . 2 g o f s t r e p t o m y c i n i n a
m i x t u r e o f 2 m l o f methyl a l c o h o l and 0 . 1 m l
o f s u l p h u r i c a c i d , f i l t e r i f n e c e s s a r y and
allow t o s t a n d a t about 250; c r y s t a l s o f
s t r e p t i d i n e sulphate separate i n t h e course
of 2 o r 3 d a y s . D i s s o l v e t h e c r y s t a l s i n
10 ml of h o t water c o n t a i n i n g 0.1 g o f t r i n i t r o p h e n o l and c o o l . The m e l t i n g p o i n t o f
t h e p r e c i p i t a t e a f t e r r e c r y s t a l l i s a t i o n from
hot water i s about 283%.
b) Boil a small q u a n t i t y of s t r e p t o m y c i n w i t h
1 N sodium hydroxide f o r a few minutes and
add a s l i g h t excess o f h y d r o c h l o r i c a c i d and
a few d r o p s o f f e r r i c c h l o r i d e t e s t s o l u t i o n ;
a b r i l l i a n t v i o l e t c o l o r i s produced.
13.22 Color T e s t s
The f o l l o w i n g c o l o r t e s t s have been d e s c r i b e d
f o r t h e i d e n t i f i c a t i o n of streptomycin :
a ) Glucosamine Test
i ) A 10 mg of s t r e p t o m y c i n i s d i s s o l v e d i n
1 m l o f water, 2 m l o f 1 N h y d r o c h l o r i c
a c i d is added and t h e s o l u t i o n is h e a t e d
on a steam b a t h f o r 10 minutes. Then
2 m l of 2 N sodium hydroxide and 1 m l o f
20% aqueous a c e t y l a c e t o n e a r e added. The
s o l u t i o n i s a g a i n h e a t e d f o r 5 minutes
and cooled. About 1 m l o f e t h a n o l and
25 m l o f c o n c e n t r a t e d h y d r o c h l o r i c a c i d
STREPTOMYCIN
543
a r e t h e n added.
produced (308).
A cherry red color i s
i i ) To about 2 m l o f an aqueous s o l u t i o n o f
s t r e p t o m y c i n , 1 m l of a 2% s o l u t i o n o f
a c e t y l a c e t o n e i n w a t e r and 1 m l o f 1 N
sodium hydroxide a r e added. The m i x t u r e
i s h e a t e d f o r 10 minutes i n a b o i l i n g
water b a t h and t h e n cooled. A pink c o l o r
i s developed on a d d i t i o n o f 2 m l o f a
s o l u t i o n o f E h r l i c h ' s aldehyde (309).
b) Guanido Test
i ) A 1 0 mg o f s t r e p t o m y c i n i s d i s s o l v e d i n
1 m l o f water and 1 m l of o x i d i z e d n i t r o p n r s s i d e r e a g e n t i s added ( t h e r e a g e n t
i s p r e p a r e d by mixing equal volumes of
10% sodium n i t r o p r u s s i d e , 10% potassium
f e r r i c y a n i d e and 10% sodium hydroxide,
i n t h a t o r d e r ; a f t e r t h e c o l o r becomes
b r i g h t green-yellow, 1 m l i s d i l u t e d t o
100 m l w i t h w a t e r ) , a r e d c o l o r devel o p s (310).
i i ) Aqueous s o l u t i o n o f s t r e p t o m y c i n when
t r e a t e d with d i a c e t y l i n t h e presence
o f oxygen g i v e s a pink c o l o r which i s
i n t e n s i f i e d by t h e a d d i t i o n o f l-napht h o l (311).
i i i ) When s t r e p t o m y c i n i s t r e a t e d with l-napht h o l and sodium hypobromite, a r e d c o l o r
i s produced (312,313).
i v ) When s t r e p t o m y c i n i s t r e a t e d w i t h sodium
hypobromite i n s t r o n g a l k a l i n e s o l u t i o n ,
a v o l a t i l e bromo amine i s produced which
turns acidified starch iodide solutions
t o dark b l u e c o l o r (314).
c ) Other c o l o r T e s t s
i ) An a l c o h o l i c s o l u t i o n o f s t r e p t o m y c i n
gives a pink color with sulphuric acid
(315).
JABER S.MOSSA ETAL.
544
ii) An alcoholic solution of streptomycin produces a yellowish-red color with phosphoric acid (315).
iii) With Sakaguchi solution (100 mg o f boric
acid in 10 ml of concentrated sulphuric
acid) streptomycin gives pale-green
after 3-5 minutes (315).
13.23 Microbiological Tests
a) Test with Streptococcus lactis
Streptomycin when treated with milk inoculated with SRhepAococcud ,f!ucLh, prevents
the coagulation of the milk in the usual
way (316) .
b) Triphenyltetrazolium Chloride (TTC) Test
When streptomycin is treated with a culture
of SR/LepAocaccw t h m o p k i e e u d on a sterile
milk medium containing triphenyltetrazolium
chloride, prevents the usual color change
from leucoform to red (317,318).
c) Water-blue Test
A dilute solution of streptomycin prevents
the formation of the blue color when treated

with a culture of KLeebtA.eRea pfiewnoniae containing water blue dye (319).
13.3 Titrimetric Methods
13.31 Aqueous
Delaby and Stephan (320) have reported an iodimetric method for the determination o f streptomycin. The method is as follows :
Dissolve about 0.7 to 1 g of Streptomycin in
15 ml of water, add 15 ml of Nessler's reagent
and 1 ml of 10% potassium iodide. Then add
20 ml of water, neutralize with 2N hydrochloric
acid and add 1 ml in excess. Add 15 ml of
0.1N iodine solution. Shake and titrate the
excess of iodine with 0.05N sodium thiosulphate
solution.
STREPTOMYCIN
545
An i o d o m e t r i c method i n v o l v i n g t h e r e a c t i o n o f
s t r e p t o m y c i n w i t h sodium hyphobromite has been
a l s o r e p o r t e d (321).
The method i s a s
follows :
The sample c o n t a i n i n g s t r e p t o m y c i n s u l p h a t e
i s added t o 5-10 m l b a s i c sodium hyphobrom i t e s o l u t i o n and a i r i s swept s u c c e s s i v e l y
through t h e sample and 2 U-tubes each cont a i n i n g 5 m l o f 0.1N s u l p h u r i c a c i d and
0.1-0.2 g potassium i o d i d e . A f t e r sweeping
a i r (10-15 b u b b l e s / s e c ) through t h e system
or about 15 m i n u t e s , t h e l i b e r a t e d i o d i n e
i s t i t r a t e d w i t h 0.005N sodium t h i o s u l p h a t e .
A n a l y s i s o f 4-8 mg p o r t i o n s o f 90.2% p u r e
s t r e p t o m y c i n s u l p h a t e showed v a l u e s o f 89.2,
88.7 and 8 8 . 4 % .
Huttenrauch and Keiner (322) have d e s c r i b e d a
t i t 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 o f
s t r e p t o m y c i n s u l p h a t e . The method i s a s
follows :
The sample c o n t a i n i n g about 50 t o 150 mg
o f streptomycin sulphate is dissolved i n
25 m l o f w a t e r ; 2 m l o f 1 N ,sodium hydroxide
i s added and t h e m i x t u r e i s h e a t e d a t looo
f o r 5 minutes. A f t e r c o o l i n g , it i s a c i d i f i e d with 1 N sulphuric a c i d , then d i l u t e d
t o 100 m l w i t h water and about 0 . 1 m l o f 2%
f e r r i c c h l o r i d e s o l u t i o n is added. The
s o l u t i o n i s set a s i d e f o r 7 minutes and t h e n
t i t r a t e d w i t h 0.01N c e r i c s u l p h a t e u n t i l t h e
c h a r a c t e r i s t i c red colour i s discharged.
T h i s method can a l s o b e employed f o r t h e
d e t e r m i n a t i o n of s t r e p t o m y c i n i n c u l t u r e
media (323).
M o d i f i c a t i o n of Huttenrauch and Keiner method
by r e p l a c i n g c e r i c s u l p h a t e s o l u t i o n w i t h p o t assium permanganate s o l u t i o n , was r e p o r t e d by
Chandra e t a 1 (324). T h i s modified method
g i v e s r e s u l t s w i t h s h a r p e r end p o i n t .
13 32 Non-aaueous
Penau e t a1 (325) have d e s c r i b e d a nonaquous
method of d e t e r m i n a t i o n o f a n t i b i o t i c s i n c l u d i n g
546
s t r e p t o m y c i n . The s p e c i f i e d amount o f S t r e p tomycin s u l p h a t e is d i s s o l v e d i n p e r c h l o r i c

a c i d and 2 m l of e t h y l e n e g l y c o l . Then about
0.05M b e n z i d i n e s o l u t i o n i n a c e t i c a c i d i s
added. After 10 minutes, 2 d r o p s of i n d i c a t o r
(1% thymolphthalein i n e t h a n o l o r 0 . 5 % t h i a z o l e
yellow i n e t h a n o l ) is added and t h e e x c e s s o f
p e r c h l o r i c a c i d i s t i t r a t e d with a c i d potassium
p h t h a l a t e s o l u t i o n . l m l o f 0.1N H C 1 0 4 a c i d
i s e q u i v a l e n t t o 581.6/30 mg o f s t r e p t o m y c i n
base.
G a u t i e r and P e l l e r i n (326) have r e p o r t e d a nonaqueous method o f e s t i m a t i o n o f s u l p h a t e o f
t h e o r g a n i c b a s e s l i k e s t r e p t o m y c i n , dihydros t r e p t o m y c i n , and o t h e r s by d i r e c t t i t r a t i o n o f
p e r c h l o r i c a c i d i n g l a c i a l a c e t i c a c i d . The
procedure i s a s f o l l o w s :
To a 0.1N s o l u t i o n of t h e sample i n g l a c i a l
a c e t i c a c i d , add s u f f i c i e n t b e n z i d i n e ( a s 0.05M
solution i n g l a c i a l acetic acid) t o p r e c i p i t a t e
95 % of t h e s u l p h a t e ; a l l o w t h e p r e c i p i t a t e t o
s e t t l e and t i t r a t e t h e s u p e r n a t a n t l i q u i d with
0.1 N p e r c h l o r i c acid i n g l a c i a l a c e t i c a c i d .
13.4 Gasometric
Gasometric d e t e r m i n a t i o n o f s t r e p t o m y c i n was r e p o r t e d
by Yamagishi and Yokoo ( 3 2 7 ) and V i a l a (328). Pure
s t r e p t o m y c i n i n s o l u t i o n o r i n complex p r e p a r a t i o n s
o r i n a s s o c i a t i o n w i t h p e n i c i l l i n s , are determined by
measuring t h e volume of n i t r o g e n evolved by s t r e p t i d i n e f r a c t i o n c.f tlie molecule under t h e a c t i o n o f
sodium h y p o c h l o r i t e o r sodium hypobromite and compari n g it with t h a t f r e e d by r e f e r e n c e s o l u t i o n s o f
guanidine hydrochloride o r guanidine a c e t a t e .
13.5 Electrometric
13.51 P o l a r o g r a p h i c
Streptomycin i s r e d u c i b l e a t t h e dropping mercury electrode. A polarographic a n a l y s i s has
been developed ( 3 2 9 ) . The c o n c e n t r a t i o n d i f f u s i o n current curve i s l i n e a r f o r concent r a t i o n s from 0 . 1 t o 1 . 0 mg/ml. The s o l u t i o n
o f s t r e p t o m y c i n i n 3% t e t r a m e t h y l ammonium
STREPTOMYCIN
541
hydroxide is polarographed at 13.6O; the Eh is

1.45 volts vs a mercury electrode. The rate of
decomposition o f streptomycin in alkaline
solution was polarographically studied by
Bricker and Vail ( 3 3 0 ) .
Goodey et a1 (331) has described a polarographic method for the assay of streptomycin in
fermenter broth and associated recovery stages.
A Tinsley Mark 19 pen recording polarograph
is used, with mercury capillaries of drop times
between 2 and 3 seconds at 50 cm height.
Lithium hydroxide is used as base electrolyte.
Doan and Riedel (332) have reported a polarographic method for the estimation of streptomycin sulphate.Solutions containing 100-400 y
/ml were used for the determination. The pH
of the solution was adjusted to 1 2 . 4 by adding
2 ml of 1 N sodium hydroxide. The dissolved
oxygen was removed by bubbling nitrogen through
the sample f o r 5 minutes after that the surface
was flushed with nitrogen. A mercury drop rate
of 4 - 5 seconds was maintained. For all concentrations the half wave potential (E%) was 1 . 2 8 1 . 2 9 volts. Plotting the diffusion current
against concentration resulted in a straight
1ine.
Cunha (333) has published a similar polarographic method f o r determination of streptomycin
sulphate. The limit o f identification of
streptomycin sulphate was 0 . 5 8 - 7 2 . 8 7 y/ml.
Caplis et a1 (334) have reported a method f o r
determination o f antibiotics including streptomycin by alternating-current polarography.
A survey of the applications of polarographic
methods in the production control of antibiotics
including streptomycin was described by
Weissbuch and Unterman ( 3 3 5 , 3 3 6 ) .
1 3 . 5 2 Amperometric
Streptomycin forms water insoluble salt with

various anionic dyes. This can be used or
amperometric estimation of streptomycin ( 3 3 7 ) .
548
The method is based on the precipitation of

streptomycin with a trivalent dye which is synthesised by coupling diazotized para rosaniline
with 1-naphthol-4-sulphonic acid. About 1-3 mg
of streptomycin is dissolved in 5 to 10 ml of
0.02M triethylamine citrate buffer, pH 2.8 and
is titrated with standard solution of the dye.
The potential is maintained at -0.08 volts.
Konopik (338) has described the application of
metal salt solutions to amperometric determination of organic compounds including streptomycin.
13.53 Potentiometric
Machek (339) has carried out a potentiometric
titration for the determination of streptomycin
in mixed preparations. The method is as
follows :
Mix the sample with 15 ml o f water and 20 ml
of butyl acetate and adjust to pH 2.1 by
addition of 2N hydrochloric acid and shake
for five minutes. Extract a 1 ml aliquot of
the filtered upper layer with 10 ml of
phosphate buffer solution (pH 7.2) and determine the penicillin in the extract iodimetrically or microbiologically. Mix the
filtered lower layer with 20 ml of butyl
acetate, adjust to pH 9 . 5 by addition of
2N sodium hydroxide, add Barium chloride
(2ml) and shake for 1 minute; centrifuge,
separate and dilute the aqueous phase to
50 ml, adjust the pH of a 20 ml aliquot to
5.8 by addition of 2N hydrochloric acid
and titrate the streptomycin potentiometrically with 0.1N NaOH to pH 9.6.
A potentiometric microbiological assay for the
antibiotic substances including streptomycin

was proposed (340). The method for the determination of tetracycline hydrochloride with
suspension of E b C h d C k i d cofi and a potentiometric C02 sensor (HNC Model 10-22-00 or an
Orion Model 95-02) has been extended to the
determination of streptomycin. Sample solutions containing 2-4 pg ml-1 of streptomycin
STREPTOMYCIN
549
was used.
The c a l i b r a t i o n graph was r e c t i l i n e a r

w i t h range 0.33 t o 16.67 pg m l - 1 .
13.6 S p e c t r o s c o p i c Methods
1 3 . 6 1 C o l o r i m e t r i c Methods
13.611 The m a l t o l Method
When s t r e p t o m y c i n is h e a t e d i n d i l u t e
a l k a l i n e s o l u t i o n i t forms m a l t o l (29).
Maltol r e a c t s w i t h f e r r i c i o n s t o g i v e
a p u r p l e c o l o r (304) and w i t h t h e
F o l i n - C i o c a l t e u phenol r e a g e n t i t produces a b l u e c o l o r (341). This f a c t
may be used f o r t h e a s s a y o f streptomycin.
A colorimetric assay involving t h e maltol
formation, f o r t h e estimation of s t r e p tomycin i s d e s c r i b e d i n t h e European

Pharmacopoeia (342). The procedure i s
as f o l l o w s :
Dissolve 0 . l g o f s t r e p t o m y c i n i n water
and d i l u t e t o 100.0 m l with t h e same
s o l v e n t . To 5 . 0 m l o f t h i s s o l u t i o n
add 5 . 0 m l o f 0.2N sodium hydroxide and
h e a t f o r e x a c t l y 10 minutes i n a water
b a t h . Cool i n i c e f o r e x a c t l y 5 minutes,
add 3 m l of a 1 . 5 p e r c e n t w/v s o l u t i o n
o f f e r r i c ammonium s u l p h a t e i n 0.5N s u l p h u r i c a c i d and s u f f i c i e n t water t o
produce 25 m l and mix. E x a c t l y 20
minutes a f t e r t h e a d d i t i o n of t h e
f e r r i c ammonium s u l p h a t e , measure t h e
e x t i n c t i o n o f a 2 cm l a y e r a t about
525 nm, u s i n g a s t h e compensation l i q u i d
a s o l u t i o n p r e p a r e d i n t h e same manner,
o m i t t i n g t h e s u b s t a n c e t o be examined.
The e x t i n c t i o n i s n o t less t h a n 90.0 p e r
c e n t o f t h a t o b t a i n e d by c a r r y i n g o u t
t h e operations simultaneously, using
s t r e p t o m y c i n s u l p h a t e CRS i n s t e a d o f t h e
s u b s t a n c e t o be examined. C a l c u l a t e t h e
percentage with reference t o t h e subst a n c e t o be examined and t h e streptomyc i n s u l p h a t e CRS, b o t h d r i e d f o r 4 hours
550
a t 60' o v er phosphorus p e n to x id e a t a
p r e s s u r e n o t exceeding 5 T o r r .
Pharmacopoeia of United S t a t e s , 1955

(343) d e s c r i b e d a similar m a l t o l c o l o r i metric a s s a y f o r d e t e r m i n a t i o n o f s t r e p tomycin i n s t r e p t o d u o c i n or I n j e c t i o n ,
U.S.P.
Boxer e t a1 (344) have r e p o r t e d a s i m p l e and r a p i d c o l o r i m e t r i c a s s a y f o r
streptomycin i n c l i n i c a l p r e p a r a t i o n s ,
u r i n e and b r o t h . The method i s based
on t h e formation o f m a l t o l from s t r e p t o mycin, s e p a r a t i o n of t h e m a lto l
from t h e i n t e r f e r i n g s u b s t a n c e s by ext r a c t i o n with chloroform and subsequent
d e t er m i n a t i o n by e i t h e r phenol r e a g e n t
o r a c i d f e r r i c ammonium s u l p h a t e .
Eisenman and B r i c k e r (345) have d e s c r i bed a c o l o r i m e t r i c method or t h e e s t i mation o f s t r ep t om y c in i n which t h e
m a l t o l i s s e p a r a t e d from t h e r e a c t i o n
m i x t u r e by steam d i s t i l l a t i o n . The
method i s s u c c e s s f u l l y a p p l i e d f o r t h e
d et er m i n a t i o n of str e p to m y c in i n ferment a t i o n b r o t h s , c o n c e n t r a t e s and c l i n i c a l
samples. I f more th a n 500 u n i t s o f
streptomycin a r e p r e s e n t t h e colorimet r i c r e a c t i o n w i t h f e r r i c i o n is employed and t h e c o l o r i s read a t 550 nm.
I f less t h ea n 500 u n i t s a r e p r e s e n t , t h e
phenol r e a g e n t i s used and t h e c o l o r i s
read a t 775 nm. The sta n d a r d c u r v e s
p r e p a r e d from p u r e str e p to m y c in are
linear.
Bagdasarian and Michalska (346) have
r e p o r t e d the m a lto l method f o r d e te r m in a t i o n of s t r e p t om y c in i n u r i n e without
e x t r a c t i n g t h e s tr e p to m y c in p r i o r t o t h e
a s s a y , provided t h a t dosage was moder a t e . This method is recommended f o r
r o u t i n e u s e , b u t cannot be a p p l i e d t o
determining s t r e p to m y c in i n c o n c e n tr a t i o n below 100 y/ml.
STREPTOMYCIN
551
Doery and Mason (347) and Ka r tse v a e t a 1

(348) s e p a r a t e d str e p to m y c in from o t h e r
f e r m e n t a t i o n p r o d u c t s by i o n exchange
chromatography on c a t i o n exchange r e s i n
before convertion i n t o maltol. Final
d e t e r m i n a t i o n of str e p to m y c in was done
by Maltol method.
Bruns e t a 1 ( 3 4 9 ) , have r e p o r t e d a c o lo rimetric method f o r t h e d e t e r m i n a t i o n o f
s t r ep t o m y c i n from t h e b r o t h . The a n t i b i o t i c s are c o l l e c t e d from t h e s o l u t i o n
a t pH 8 . 3 - 8 . 5 on s i l i c a g e l and extract e d i n s u l p h u r i c a c i d . The c o n t e n t i s
determined by m a l t o l method.
Desai e t a1 (350) have d e s c r i b e d a
d i r e c t method f o r e s t i m a t i o n o f s t r e p tomycin i n f e r m en te r b r o t h sample. The
b r o t h sample was d e p r o t e i n i z e d by t h e
a d d i t i o n o f 4 m l of t r i c h l o r o a c e t i c
a c i d , f i l t e r e d c e n t r i f u g e d and d i l u t e d .
After t h e a d d i t i o n o f 2 m l of 2N sodium
hydroxide t o 5 m l o f t h e b r o t h , t h e
m i x t u r e was heated i n a b o i l i n g water
b a t h f o r t h r e e minutes and c o o le d . The
absorbance was r e a d a f t e r a d d i t i o n of
f e r r i c ammonium s u l p h a t e . A blank was
run c o n c u r r e n t l y , c o n s i s t i n g o f t h e
same sample without h e a t .
F e r r a r i e t a1 (351) have adapted t h e
m a l t o l method f o r automated a n a l y s i s
o f s t r ep t o m y c i n i n f e r m e n ta tio n media
u s i n g d i a l y s i s t o s e p a r a t e m a l t o l from
t h e i n t e r f e r i n g s u b s t a n c e s . The m a l t o l
t h u s s e p a r a t e d was a u t o m a t i c a l l y t r e a t e d
w i t h stream o f f e r r i c c h l o r i d e i n 2.2N
h y d r o c h l o r i c a c i d and t h e e x t i n c t i o n o f
t h e r e a c t i o n product a t 530 nm was a u to m a t i c a l l y r e c o r d e d . E r r o r s were claimed
t o be less t h a n with c o n v e n tio n a l manual
methods.
F u r t h e r development o f t h e a u t o - a n a l y z e r
method f o r e v a l u a t i o n of str e p to m y c in
were d e s c r i b e d by Shaw and Fortune ( 3 5 2 ) .
They claimed t h a t h ig h p r e c i s i o n could
552
be maintained even w i t h c o n s i d e r a b l e
s i m p l i f i c a t i o n t o a l l o w f o r h i g h throughp u t o f samples.
Iodimetric-absorptiometric method was

adopted by Sauciuc and Ionescu (353) f o r
micro-determination o f s t r e p t o m y c i n i n
f e r m e n t a t i o n l i q u i d . The method i s
a s follows :
The sample c o n t a i n i n g 25-150 Ug o f
s t r e p t o m y c i n is h e a t e d w i t h 0.1N sodium
hydroxide f o r 4 minutes. The s o l u t i o n
i s t h e n a d j u s t e d t o pH 7.5 by adding
0.055N phosphoric a c i d t h e n t r e a t e d
w i t h f r e s h l y d i l u t e d 0.001N i o d i n e i n
potassium i o d i d e s o l u t i o n . After 5 t o
10 minutes 5 m l of r e a g e n t A (300 m l
water, 200 m l acetic a c i d , 6 g potassium
bromide and l m l h y d r o c h l o r i c a c i d ) is
added followed by a s t r o n g j e t o f 1 m l
of r e a g e n t B (100 m l water, 0.72g Sulphanilamide
0.02 g N-naphthyl-ethylenediamine d i h y d r o c h l o r i d e and 5g
hydroxylammonium c h l o r i d e ) . After 10
minutes t h e e x t i n c t i o n of t h e s o l u t i o n
is measured a g a i n s t t h e blank s o l u t i o n
which is s i m i l a r l y t r e a t e d .
Krystyna (354) h a s r e p o r t e d t h e m a l t o l
c o l o r i m e t r i c method or t h e determinat i o n of streptomycin residues i n milk.
A n t i b i o t i c - f o r t i f i e d milk samples
were cooled, d e f a t t e d by c e n t r i f u g a t i o n ,
d e p r o t e i n i z e d with z i n c s u l p h a t e i n t h e
p r e s e n c e of barium hydroxide, concent r a t e d and a g a i n d e f a t t e d w i t h l i g h t
petroleum. Then t h e sample was t r e a t e d
w i t h sodium hydroxide and h e a t e d on t h e
water b a t h . The m a l t o l formed was
e x t r a c t e d i n t o chloroform from t h e
a c i d i c medium and b a c k - e x t r a c t e d i n t o
w a t e r a t a l k a l i n e pH. The aqueous
e x t r a c t was mixed w i t h F o l i n C i o c a l t e u
phenol r e a g e n t , a f t e r 10 minutes t h e
absorbance was measured a t 660 nm and
r e f e r r e d t o a c a l i b r a t i o n graph.
STREPTOMYCIN
553
The maltol method has been extensively

used in studies of stability (355) and
in the analysis of streptomycin in
pharmaceutical preparations (356), feeds
(357) and urine (358). Minor modifications have been suggested by Borowiecka
(359), Kartseva and Bruns (360) and
Wahbi et a1 (361).
13.6 12 Guanido reaction Method
Both streptomycin and dihydrostreptomycin form orange-red color with oxidized
nitroprusside reagent (310,362). Based
on this principle Valedinskaya (363) has
described a colorimetric method for
estimation o f streptomycin. The procedure is as follows :
10 ml o f oxidized nitroprusside reagent
is added to 10 ml of aqueous sample solution containing approximately 0.1 mg of
streptomycin o r dihydrostreptomycin.
After 3 minutes the absorbance is determined at 490 nm against a reagent blank
prepared by substituting 10 ml of water
for the sample aliquot. A working curve
is prepared with the appropriate compound following the same procedure. The
color is stable for about 4 hour.
Vieira and Pereira (364) have adapted

the nitroprusside method for the determination of streptomycin in urine. The
urine sample is treated with acetone
and centrifuged after 30 minutes. Then
the precipitate is stirred with water
and filtered o r centrifuged. To an aliquot of the clear solution,water is
added to bring to 2.5 ml, then treated
with nitroprusside reagent an3 the absorbance is measured.
Halliday (311) described the application
of the modified Voges-Proskauer reaction to streptomycin, in which a pink
color is generated by the reaction of
the guanidine moiety with 3iacetyl and
554
1-napthol. Quantitative analytical procedure

was developed by Szafir and Bennett (365),
Ashton et a1 (366) and Banerjee (367).
The method is as follows :
Transfer 2 ml of the sample solution to a test
tube, add 15 ml of water and then 1 ml of 0.4
per cent diacetyl solution, 1 ml of 20% potassium hydroxide solution and 1 ml of 10% solution of 1-naphthol in anhydrous ethanol, in
that order. Mix the contents of the tube by
inversion after each addition. Forty minutes
later determine the extinction at 525 MI in
a 1 cm cell against water. Prepare a standard
graph from suitable dilutions of a standard
streptomycin solution, using the same procedure for color development as described for
the sample. Determine the potency of the
diluted sample solution from the standard
graph. A standard graph should be prepared
for each determination to allow for differences in room temperature, reagents, and so on.
Schulz and Posada (368) have developed a colorimetric method involving guanidino group
reaction for determination of streptomycin.
The aqueous sample solution is treated with an
ethanolic reagent containing diacetyl and
naphthalene-2,7-diol; ethanolic sodium hydroxide is added and the mixture is set aside
at room temperature for 45 minutes. The extinction is then measured at 555 nm and the
streptomycin content is calculated with reference to a standard solution containing 50 1.18
of streptomycin sulphate per ml.
The Sakaguchi reaction (312,313) has been
adapted by Buck et a1 (369) to the analysis of
streptomycin and dihydrostreptomycin, using
spectrophotometric determination of the red
color formed by the reaction of 1-naphthol
and sodium hypobromite with the antibiotics.
Natarajan and Tayal (370) have reported a
rapid method of colorimetric estimation of
streptomycin, based on the Sakaguchi reaction.
The procedure i s as follows :
555
STREPTOMYCIN
Mix a n aqueous s o l u t i o n o f t h e sample w i t h 1

m l o f 10% sodium hydroxide, and 2 m l o f 0.02%
e t h a n o l i c 1-naphthol. After 15 m i n u t e s , add
1 m l of sodium hypobramite r e a g e n t and 10 m l
o f c a r b o n t e t r a c h l o r i d e . Shake well, and
add 5 m l o f a b s o l u t e e t h a n o l , shake a g a i n and
measure t h e e x t i n c t i o n o f t h e aqueous p h a s e
a t 530 nm i n a 1 cm c u v e t t e with a r e a g e n t
b l a n k . The r e s u l t s were comparable w i t h t h o s e
from m i c r o b i o l o g i c a l method.
The a p p l i c a t i o n of t h e Sakaguchi r e a c t i o n f o r
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 o f streptomyc i n was a l s o r e p o r t e d by Fontes ( 3 7 1 ) .
M o d i f i c a t i o n of t h e above method by u s i n g 8hydroxyquinoline was d e s c r i b e d by T i b a l d i
(372).
S z i l a g y i and Szabo (373) have r e p o r t e d a simil a r c o l o r i m e t r i c method f o r t h e a n a l y s i s o f
S a k a g u c h i - p o s i t i v e a n t i b i o t i c s , u s i n g N-bromosuccinimide. The method i s as f o l l o w s :
5 m l O f t h e s o l u t i o n o f t h e sample i s t r e a t e d
w i t h 2 m l o f 1-naphthol r e a g e n t . After b e i n g
shaken and set a s i d e f o r 3 minutes, t h e mixt u r e i s r a p i d l y poured i n t o 0.5 m l o f N-bromos u c c i n i m i d e s o l u t i o n , t h e mixture i s shaken
v i g o r o u s l y and 1 m l o f 40% u r e a s o l u t i o n i s
added i n 1 5 second. A v i v i d r e d c o l o u r devel o p s , t h e e x t i n c t i o n o f which is r e a d i n 5
minutes a g a i n s t b l a n k s , w i t h a b l u e f i l t e r ,
r e s u l t s being r e f e r r e d t o standard curves.
The r e l a t i v e e r r o r o f t h e method i s f 4 % .
13.613 Glucosamine r e a c t i o n Method
The glucosamine t e s t f o r t h e i d e n t i f i c a t i o n of
s t r e p t o m y c i n u s i n g a c e t y l a c e t o n e and E h r l i c h ' s
r e a g e n t , can be used q u a n t i t a t i v e l y (309).
Based on t h i s p r i n c i p l e , Kamata e t a1 (374)
have d e s c r i b e d a c o l o r i m e t r i c a s s a y f o r s t r e p tomycin. The p r o c e d u r e of t h e method i s as
follows :
556
To 4 m l of s t r ep t o m y c i n s o l u t i o n , add 1 m l o f
2.5N s u l p h u r i c a c i d . Boil f o r 1 hour, c o o l ,
n e u t r a l i z e and d i l u t e t o 10 m l . To about 2 m l
o f t h i s s o l u t i o n add 5 .5 m l o f r e a g e n t A
(2.225 m l a c e t y l a c e t o n e , 113.75 m l 2 N sodium
c a r b o n a t e 3 4 . 1 m l 1N h y d r o c h l o r i c a c i d and
water) , s t o p p e r , b o i l f o r 20 minutes, c o o l ,
add 10 m l . o f e t h a n o l , f i n a l l y add r e a g e n t
B (0.8 o f Ehrlich'saldehyde, 30 m l o f concent r a t e d h y d r o ch l o r i c a c i d and a b s o l u t e e t h a n o l ) .
Measure t h e i n d e n t s i t y o f t h e c o l o r a t 532 nm.
The method i s r e l i a b l e o n l y f o r samples o f
high p u r i t y .
The a c e t y l acetone-Ehrlich's aldehyde method h a s
been adapted by Masquelier (375) f o r t h e d e t e r m i n a t i o n o f s t r e p t o m y c in i n u r i n e . The u r i n e
sample c o n t a i n i n g s t r e p to m y c in i s t r e a t e d w i t h
l ea d a c e t a t e , t h e e x c e s s o f l e a d is removed by
sodium s u l p h a t e and f i l t e r e d . To about 1 m l
o f t h e f i l t r a t e 1 m l o f a c e t y l a c e to n e r e a g e n t
and 2 d r o p s o f sodium hydroxide a r e added. The
s o l u t i o n i s h ea t ed i n a w a te r h a t h f o r 10 minu t e s and co o l ed . 2 m l o f th e E h r lic h ' s aldehyde
r e a g e n t is t h e n added and t h e red c o l o r developed i s compared w i t h a sta n d a r d s o l u t i o n o f
s t r ep t o m y c i n and w i t h a blank of u r i n e sample.
13.614 Other Methods
The aldehyde group o f str e p to m y c in was expl o i t e d by Marshall e t a1 (358) who developed
a q u a n t i t a t i v e p r o ce d u r e based on t h e format i o n o f a co l o r ed semicarbazone with 4-[4(p-chlorophenazo) -1 -naphthyl ] semicarbazide.
The ex c e s s of t h e r e a g e n t i s e x t r a c t e d w i t h
chloroform and h y d r o c h l o r i c a c i d i s added.
The c o l o r developed i s measured a t 580 nm.
S a v i t s k a y a and Kartseva (376) used 2 , 4 - d i n i t r o p h e n y l h y d r a z i n e which reacts w i t h s t r e p tomycin t o form a yellow hydrazone. The
e x c e s s r e a g e n t is e x t r a c t e d w i t h b u t y l acetate
and t h e c o n c e n t r a t i o n o f t h e str e p to m y c in i s
determined from c a l i b r a t i o n curve p r e p a r e d
with pure streptomycin.
Mattick (377) h a s r e p o r t e d 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 o f a n t i b i o t i c s
STREPTOMYCIN
557
i n c l u d i n g s t r ep t o m y c i n i n m i l k which is based
on t h e i n h i b i t i o n o f t h e r e d u c t i o n of n i t r a t e
t o n i t r o u s o x i d e by Miwiacoccus pqkogcnu.
The n i t r o u s o x i d e produced i s determined color i m e t r i c a l l y a f t e r d i a z o t i s a t i o n w i t h su lp h a n i l i c a c i d and c o u p l i n g with l-naphthylamine.
B a r i l a r i and K a t z (378) have r e p o r t e d a method
u s i n g copper t a r t r a t e and phosphomolybdic a c i d
reagents f o r colorimetric determination o f
s t r ep t o m y c i n . The f r e s h l y p r e p a r e d s o l u t i o n
o f streptomycin s u l p h a t e is heated f o r 8 minu t e s w i t h 2 m l of w a t er and 2 m l of copper
t a r t r a t e s o l u t i o n . After c o o l i n g t h e phosphomolybdic a c i d i s added and t h e b l u e c o l o r
developed i s r e a d p h o t o c o l o r i m e t r i c a l l y .
A p h o t o c o l o r i m e t r i c method f o r t h e determina-
t i o n o f s t r e p t o m y c i n w i t h r e i n e c k a t e s a l t was
d e s c r i b e d by B a r i l a r i e t a1 ( 3 7 9 ) . S t r e p t o mycin i s h ea t ed w i t h w ate r a t 40 and e q u a l
volume o f ammonium r e i n e c k a t e s o l u t i o n i s
added. The p r e c i p i t a t e t h u s formed i s separ a t e d , washed w i t h et h an o l and d i s s o l v e d i n
ac e t o n e water m i x t u r e . Then a c e to n e i s evap o r a t e d . The s o l u t i o n i s th e n t r e a t e d w ith
sodium hydroxide, d i l u t e d w i t h water and neut r a l i z e d with n i t r i c acid. 5 m l of t h i s solut i o n i s t r e a t e d with 5 m l of f e r r i c n i t r a t e
and t h e c o n c e n t r a t i o n o f str e p to m y c in i s d e t e r mined c o l o r i m e t r i c a l l y . The a p p l i c a t i o n o f
t h i s method i n t h e pharmaceutical a n a l y s i s was
r e p o r t e d by Basu and D utta (380).
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 aldehyde and
k e t o n es w i t h o x a l i c a c i d d ih y d r a z id e was adap t e d by Bartos and B u r t i n (381) f o r d e te r m in a t i o n o f s t r ep t o m y c i n . About 1 m l of t h e
sample i s t r e a t e d with 2 m l o f p h o s p h a t e - c i t r a t e - b o r a t e b u f f e r and 2 m l of o x a l i c a c i d
d i h y d r a z i d e r e a g e n t . The m ix tu r e i s allowed
t o s t a n d f o r 4 h o u r s . The b l u e o r v i o l e t c o l o r
r e s u l t i n g i s read colorimetrically.
Using p i c r i c a c i d r e a g e n t , Agrawal and D u tta
(382) have developed a r a p i d c o l o r i m e t r i c
a s s a y method f o r s t r ep t om y c in i n m ix tu r e w i t h
d i h y d r o s t r e p t o m y c i n . The test sample and
558
the blank are treated with 3 ml of 0.1% picric acid,2 ml of 5% sodium hydroxide; heated
for 3 minutes, cooled diluted to 10 ml and
the extinction is measured at 500 nm. Dihydrostreptomycin does not give this color reaction, but can be determined after being converted into streptomycin.
Yavors'kli (383) has reported the application
of dinitrobenzene and their derivatives as
reagent in colorimetric analysis of organic
medicinal substances including streptomycin.
A new spectrophotometric method for the assay
of streptomycin, by salt formation with bromothymol blue was reported by Nour El-Deen et a1
(384).
Barilari et a1 (385) have reported a new photocolorimetric method for the determination o f
streptomycin. Streptomycin solution is treated with potassium iodate solution and sodium
arsenite solution. After the disappearance of
the yellow color, 0 . 0 3 ml of phenol and 4 ml
of sulphuric acid are added, boiled, cooled
and the absorbance is read at 490 nm.
A spectrophotometric method for the estimation
of streptomycin by salt formation with tropaeolin 000, was reported by Nour El-Deen et a1
(384) and Beltagy et a1 (386). The method
is as follows :
To about 3 ml of sample in water add about

1 ml o f cold, dilute sulphuric acid, and 3 ml
of cold aqueous solution Tropaeolin 000. Mix
and cool in ice for one hour, centrifuge,
decant and wash the precipitate with 10 n i l of
cold dilute sulphuric acid. Dissolve the residue in 2 ml of sodium hydroxide, adjust to
100 ml with water. Dilute a mixture of 5 ml
of the solution and 1 ml of dilute sulphuric
acid to 50 ml and measure the extinction at
485 nm.
A new sensitive colorimetric determination of
streptomycin by thiobarbituric acid method
was proposed by Erno (387). The method is
STREPTOMYCIN
559
s p e c i f i c and s e n s i t i v e .
follows :
The procedure i s a s
The s o l u t i o n c o n t a i n i n g s t r e p t o m y c i n i s mixed
w i t h 0.1M potassium i o d a t e i n 0.05 m l o f 0.5M
s u l p h u r i c a c i d , a f t e r 15 minutes, a 2% s o l u t i o n
o f sodium a r s e n i t e i n 5 m l h y d r o c h l o r i c a c i d i s
added. When t h e brown c o l o r i s d i s a p p e a r e d ,
1 m l o f t h i o b a r b i t u r a t e r e a g e n t is added. The
m i x t u r e i s h e a t e d f o r 10 minutes and t h e n t h e
e x t i n c t i o n of t h e s o l u t i o n i s measured a t 415
nm a g a i n s t a b l a n k .
The m o d i f i c a t i o n o f t h i o b a r b i t u r i c a c i d method
was r e p o r t e d by Erno e t a l . (388) and Fukumoto
Hisako (389).
Alykov (390) has d e s c r i b e d an a b s o r p t i o 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 o f amino g l y c o s i d e
a n t i b i o t i c s i n b i o l o g i c a l f l u i d s . Streptomycin
and o t h e r amino g l y c o s i d e s form a complex w i t h
stilbazochrome P i n a c e t a t e o r c i t r a t e b u f f e r
medium. The blood sample is t r e a t e d with 1 m l
o f water and 0 . 5 m l o f t r i c h l o r o a c e t i c a c i d
s o l u t i o n and c e n t r i f u g e d . To t h e s u p e r n a t a n t
l i q u i d 1 m l of s t i l b a z o c h r o m e P r e a g e n t i n
c i t r a t e b u f f e r s o l u t i o n , 1 m l o f 1 mM PrC13
and 6 ml o f e t h a n o l added. After 20 minutes
t h e mixture i s c e n t r i f u g e d and t h e absorbance
o f t h e s u p e r n a t a n t l i q u i d is measured a t 560 nm.
A new and s e l e c t i v e s p e c t r o p h o t o m e t r i c d e t e r mination o f s t r e p t o m y c i n u s i n g O-hydroxyhydroq u i n o n e p h t h a l e i n [2',3',6',7'-tetrahydroxyspiro
(isobenzofuran; 1( 3 H ) , 9 ' - (9H) xanthen) -3-one) ]
and manganese, was r e p o r t e d (391). The method
i n v o l v e s f o r m a t i o n o f a t e r n a r y complex i n a
medium c o n t a i n i n g 40% of methanol and 1%of
p o l y v i n y l p y r r o l i d i n o n e . Measurements a r e made
a t 570 nm a t pH 10 t o 1 0 . 3 .
A p h o t o 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 t r e p t o m y c i n and o t h e r aminoglycoside a n t i b i o t i c s was proposed by Alykov (392). The method
i n v o l v e s t h e measurement of t h e d e c r e a s e i n
t h e absorbance a t 575 nm, due t o Acid b l a c k S
caused by p r e c i p i t a t i o n o f a complex o f C . 1
Acid Black 24 w i t h t h e a n t i b i o t i c s .
560
Eriochrome Slack T was used as a precipitant in

the photometric determination of streptomycin
and other antibiotics in biological fluids (393).
Ninhydrin spectrophotometric method has been
adapted f o r the determination of aminoglycoside
antibiotics in pharmaceutical preparations
(394). About 10 l.11portions of the solutions
of streptomycin in water were applied to precoated Silica gel plate; the chromatogram was
developed in a solution of l g of ninhydrin in
50 ml of 95% ethanol-anhydrous acetic acid
(5:l). After drying the colored bands were
transferred to test tubes and shaken with 5
ml of propan-2-01 containing 5 drops of ammonium hydroxide and the tubes were centrifuged.
The absorbance spectrum o f supernatant solution
was recorded in the range 400 to 800 nm against
water.
Divakar et a1 (395) have reported a spectrophotometric method for the determination of streptomycin, tetracycline and chloramphenicol using
brucine and sodium periodate. About 10 ml of
streptomycin solution was boiled under reflux
f o r 45 minutes with 10 ml o f 2M hydrochloric
acid and the excess of the acid was removed in
vacuo. The residue was dissolved in 20 ml of
warm water and diluted to 50 ml. About 0 . 5 to
3.0 ml of the solution was treated with 3 ml
of SmM-brucine, 1.5 ml of 5m.M.sodium metaperiodate and 2 ml of 2.3M.-sulphuric acid. The
solution is then diluted, boiled for 15 minutes,
cooled and the absorbance was measured at
430-450 nm.
13 62 Ultraviolet Methods
When streptomycin is heated with 0.1N sodium
hydroxide, it produce maltol (L9), which exhibits an absorption maximum at 322 nm in alkaline solution. Therefore the streptomycin can
be assayed by measuring the absorbance at 322
nm before and after hydrolysis in sodium hydroxide solution (396).
Katz (397) has reported an ultraviolet spectroscopic method for the determination of
STREPTOMYCIN
561
s t r e p t o m y c i n i n f e e d s . The s t r e p t o m y c i n i n
f e e d s was e x t r a c t e d w i t h d i l u t e s u l p h u r i c a c i d ,
s e p a r a t e d , n e u t r a l i z e d , f u r t h e r s e p a r a t e d , conc e n t r a t e d and p u r i f i e d by i o n exchange. The
s t r e p t o s e moiety was c o n v e r t e d i n t o m a l t o l and
q u a n t i t a t i v e l y determined by measuring i t s
u l t r a v i o l e t absorbance.
1 3 . 6 3 F l u o r i m e t r i c Methods
Boxer and J e l i n e k (398) have r e p o r t e d a f l u o r i metric method f o r t h e d e t e r m i n a t i o n o f s t r e p tomycin i n blood and s p i n a l f l u i d . The b a s i s
o f t h i s method i s t h e formation o f hydrazone
o f s t r e p t o m y c i n w i t h f l u o r e s c e n t 9-hydrazinoa c r i d i n e h y d r o c h l o r i d e . The e x c e s s of t h e
r e a g e n t t o g e t h e r w i t h hydrazones of a c i d i c ,
n e u t r a l and b a s i c compounds are s e p a r a t e d from
t h e s t r o n g l y b a s i c s t r e p t o m y c i n hydrazone by
e x t r a c t i o n from t h e s o l u t i o n with benzyl a l c o h o l . The aqueous base c o n t a i n i n g s t r e p t o m y c i n
hydrazone i s c e n t r i f u g e d and t h e
intens i t y of t h e f l u o r e s c e n c e o f t h e clear s o l u t i o n
i s measured. The method h a s been extended t o
determine s t r e p t o m y c i n i n u r i n e and t i s s u e s
(399).
Streptomycin and dihydrostreptomycin i n a l k a l i n e s o l u t i o n , produce a s t r o n g f l u o r e s c e n c e
w i t h sodium 1,2-naphthaquinone-4-suIphonate
(400). Based on t h i s p r i n c i p l e , Faure and
Blanquet (401) have developed an a n a l y t i c a l
procedure f o r t h e d e t e r m i n a t i o n of s t r e p t o m y c i n .
Aqueous s o l u t i o n o f s t r e p t o m y c i n was t r e a t e d
w i t h sodium hydroxide and sodium 1,2-naphthaquinone-4-sulphonate s o l u t i o n . Then t h e s o l u t i o n was set a s i d e i n t h e d a r k or 1 hour a t
20 and t h e n t h e f l u o r e s c e n c e was measured. The
d e t a i l e d s t u d i e s o f f l u o r e s c e n c e s p e c t r a were
made i n subsequent p u b l i c a t i o n s (402,403).
A p p l i c a t i o n o f t h e method f o r t h e d e t e r m i n a t i o n
o f s t r e p t o m y c i n i n blood serum o r plasma was
a l s o e x p l o r e d (404-406).
A continuous-flow Auto-Analyzer f l u o r i m e t r i c
procedure f o r t h e d e t e r m i n a t i o n of l a c t i c a c i d
i n m i l k was a p p l i e d t o t h e d e t e c t i o n o f a n t i b i o t i c s ( 4 0 7 ) . The procedure was based on t h e
562
i n h i b i t i o n by a n t i b i o t i c s o f t h e a b i l i t y of
s t a r t e r c u l t u r e s t o produce l a c t i c a c i d .
Alykov (408) has d e s c r i b e d a new f l u o r i m e t r i c
method f o r t h e d et er m i n a tio n o f aminoglycoside
a n t i b i o t i c s . The method i n v o l v e s e x t r a c t i o n
of f l u o r e s c e i n complexan-Pr3+ aminoglycoside
s p e c i e s from an aqueous medium o f pH 5.8 t o
6 . 2 i n t o isoamyl alcohol-benzene (1 :1) r e e x t r a c t i o n o f f l u o r e s c e i n complexan a s s o c i a t e d
w i t h t h e aminoglycoside i n t o aqueous 0.1M sodium f l u o r i d e and F l u o r 2 me tr ic d e t e r m i n a t i o n o f
f l u o r e s c e i n complexan i n t h i s aqueous s o l u t i o n
a t 533 nm. The method i s s u i t a b l e f o r t h e
d e t e r m i n a t i o n o f s t r e p t om y c in and t h e r e l a t e d
a n t i b i o t i c s i n blood, milk and u r i n e . Prot e i n s a r e removed from t h e blood samples by
p r e c i p i t a t i o n with t r i c h l o r o a c e t i c acid.
13. 7 Chromatographic Methods
13.71 Column Chromatography

Column chromatographic (CC) method h a s been
used e x t e n s i v e l y f o r t h e s e p a r a t i o n and p u r i f i c a t i o n of s t r e p t o m y c i n. S e v e r a l r e p o r t s
have been p u b l i s h e d r e g a r d i n g t h e a p p l i c a t i o n
o f c h a r c o a l (lQ6-117), a c i d washed alumina
(118-122) and i o n exchange r e s i n s (123-164)
f o r t h e s e p a r a t i o n and p u r i f i c a t i o n o f s t r e p t o mycin.
Okumura e t a1 (409) have used columns o f a
magnesium a l u m i n o - s i l i c a t e f o r p u r i f i c a t i o n o f
b a s i c a n t i b i o t i c s . R es o lu tio n s such a s s t r e p tomycin and kanamycin from oleandomycin were
d e s c r i b e d . The method a p p e a r s t o have a p p l i c a b i l i t y i n t h e i s o l a t i o n o f t h e a n t i b i o t i c s from
b i o l o g i c a l f l u i d s such a s c u l t u r e media, blood
and u r i n e .
S t . John e t a1 (410) used column o f c a t i o n exchange r e s i n . Amberlite IRC-50 t o s e p a r a t e
streptomycin and s t r ep t om y c in B from fermentat i o n b r o t h s , a n t i b i o t i c being determined c o l o r i m e t r i c a l l y [maltol method), w ith good a g r e e ment w i t h m i c r o b i o l o g i c a l procedure. Applicat i o n o f t h e above method f o r d e te r m in a tio n of
STREPTOMYCIN
563
s t r e p t o m y c i n i n animal f e e d (397) and i n formul a t i o n s (411) were r e p o r t e d . h b e r l i t e CG 120

i o n exchange r e s i n was u t i l i z e d by Conca and
Pazdera (412) f o r s e p a r a t i o n of s t r e p t i d i n e i n
s t r ep t o m y c i n .
Comparative s t u d y o f c a r b o x y l i c c a t i o n exchangersin r e s p e c t s o f t h e s o r p t i o n and d e s o r p t i o n
o f s t r ep t o m y c i n was r e p o r t e d (413).
Other r e p o r t s have d e s c r i b e d i n d e t a i l s t h e u s e
o f p e r m u t i t (414), phenoxy-acetic a c i d c a t i o n
exchanger and p h e n o l i c r e s i n s (415), t h e r e s o l u t i o n o f components of str e p to m y c in complex
(416), t h e s e p a r a t i o n o f str e p to m y c in from
f e r m e n t a t i o n b r o t h s u s i n g continuous p r o c e s s
(417) and e v a l u a t i o n o f v a r i o u s exchangers f o r
s e p a r a t i n g c o l o r e d i m p u r i t i e s (418). The
chromatographic behaviour of str e p to m y c in on
Sephadex G-10 h a s been d e s c r i b e d by S t o r l (419).
13.72 Paper Chromatography
Horne and P o l l a r d ( 4 2 0 ) have d e s c r i b e d a method
f o r i d e n t i f i c a t i o n o f s tr e p to m y c in by u s i n g
p a p e r - s t r i p chromatogram. The p o s i t i o n o f t h e
a n t i b i o t i c were d e t e c t e d by means o f t h e
Sakaguchi r e a g e n t . 3% ammonium c h l o r i d e was
found t o be n e c e s s a r y f o r good s e p a r a t i o n .
Wj-nsten and Eigen (421) were s u c c e s s f u l i n
r e s o l v i n g m i x t u r es o f s tr e p to m y c in and c l o s e l y
r e l a t e d s u b s t a n c e s . B u ta n o l- p ip e r id in e t o l u e n e s u l p h o n i c a c i d m ix tu r e was used as a
d e v e l o p i n g a g e n t . The p o s i t i o n o f t h e a n t i b i o t i c s were r e v e a l e d by bioautography on a g a r .
Solomons and Regna (422), S t o d o l l a e t a1 (423)
Kowezyk (424) have a p p l i e d similar method f o r
t h e s e p a r a t i o n o f s t r ep t om y c in . P e t e r s o n and
Reineke (425) d e s c r i b e d a s i m i l a r method f o r
t h e d e t e r m i n a t i o n o f s t r ep to m y c in i n s a l t - f r e e
p r e p a r a t i o n s and a p p l i c a t i o n s o f t h e method t o
s t u d i e s o f b i o s y n t h e s i s were p u b lish e d by
Hunter e t a1 (199). E f f e c t s o f s a l t s on t h e
movement and zone shape of str e p to m y c in i n
p ap e r chromatography was d i s c u s s e d by So k o lsk i
and Lummis (426).
564

A d e t a i l e d s t u d y by Heding (427,428) produced
a system c a p a b l e of c l e a r l y r e s o l v i n g s t r e p t o mycin, and i t s r e l a t e d compounds w i t h a development time o f 8 hours. The d e v e lo p e r used

was p r ep a r e d by mixing e q u a l volume o f amyl
a l c o h o l c o n t a i n i n g 1%o f b i s - ( 2 - e t h y l h e x y l )
phosphate and 0.5% sodium c h l o r i d e s o l u t i o n
i n b o r a t e b u f f e r . The s p o t s were d e t e c t e d
e i t h e r by u s i n g a s p r a y o f 1 - n a p h t h o l - d i a c e t y l
o r by bioautogrsphy on a n u t r i e n t a g a r p l a t e
seeded w i t h s p o r e s o f B a W bub-.
Other r e p o r t s o f r e s o l u t i o n have been p u b l i shed by Makisumi (429), Kondo e t a1 (430) and
Pant and Agrawal (431). The former used b u t a n o l , a c e t i c a c i d , p y r i d i n e . water (4: 1:1:2)
and b u t a n o l : 6N h y d r o c h l o r i c a c i d ( 7 : 3 ) as
developing r e a g e n t and Sakaguchi's r e a g e n t and
J a f f e ' s r e a g e n t f o r d e t e c t i o n . The Rf v a l u e s
of s t r ep t o m y c i n i n t h e above s o l v e n t systems
were 0.17 and 0 . 0 8 r e s p e c t i v e l y .
Albu-Budai and Unterman (432) r e p o r t e d a quant i t a t i v e procedure based on paper chromatograp h i c r e s o l u t i o n , e l u t i o n and sp e c tr o p h o to m e tr ic
d e t e r m i n a t i o n after c o l o r development w i t h
MN 261 paper s t r i p s
1-naphthol-diacetyl.
p r e v i o u s l y impregnated with phosphate b u f f e r o f
pH 7 were used. The developing s o l v e n t were
pentachlorophenol-butanol-sodium hydroxide-water.
Numerous s p r a y r e a g e n t s have been used i n t h e
d e t e c t i o n o f s t r ep t o m y c in based l a r g e l y on c o lo r i m e t r y . T h i s i n c l u d e s potassium permanganate
(433), i o d i n e (434), sodium p e r i o d a t e w i t h
p i c r i c a c i d (435), t h e a n i l i n e , l a c t i c a c i d ,
p i c r i c a c i d m i x t u r e (436) and bromine-starchpotassium i o d i d e , r e a g e n t (9).
Couling and Goodey (437) have d e s c r i b e d a new
method o f s t r ep t o m y c i n chromatography and i t s
u s e i n t h e examination of t h e r e a c t i o n between
s t r e p t o m y c i n and ammonia, Paper p r e v i o u s l y
impregnated w i t h phosphate b u f f e r a t pH 7 and
sodium hydroxide-pentachlorophenol-butanol as
developing s o l v e n t were U s e d . To d e t e c t t h e
compounds, d i p t h e chromatogram i n p e r i o d a t e
ac e t o n e and t h en i n b e n z i d i n e - a c e to n e t o
STREPTOMYCIN
565
g i v e yellow o r white s p o t i n a b l u e background;

o r d i p i n a mixture o f d i a c e t y l and 1-naphthol
t o g i v e r e d s p o t on a white background.
1 3 . 7 3 Thin-Layer Chromatography
Thin l a y e r chromatographic t e c h n i q u e was ext e n s i v e l y used o r t h e s e p a r a t i o n and i d e n t i f i c a t i o n o f s t r e p t o m y c i n and i t s r e l a t e d compounds by Kondo (430) and Borowiecka (438).
The former used p l a t e s c o a t e d w i t h a c t i v a t e d
carbon whereas t h e latter used p l a t e s c o a t e d
e i t h e r w i t h s i l i c a g e l G o r Kieselguhr G.
VonSchantz e t a1 (439) s t u d i e d t h e a p p l i c a t i o n
of metal p l a t e s f o r t h i n l a y e r d e t e c t i o n o f
pharmaceutical compounds.
S e v e r a l r e p o r t s have appeared on t h e a p p l i c a t i o n o f t h i n l a y e r chromatography i n i d e n t i f y i n g aminoglycoside a n t i b i o t i c s . The s o l v e n t system and t h e Rf v a l u e s are r e p o r t e d
i n t a b l e . 4.
Voigt and Bared (443) and Katayama and Ibeda
(444) have r e p o r t e d two-dimensional TLC method
f o r s e p a r a t i o n and i d e n t i f i c a t i o n of s t r e p t o mycin and i t s r e l a t e d compounds.
S a t 0 and Ikeda (445) were a b l e t o r e s o l v e
streptomycin and i t s dihydro-and dihydrodeoxy
d e r i v a t i v e s by ascending TLC procedure. Simil a r claims were made by Katayama and Ikeda
(446).
Thin l a y e r - b i o a u t o g r a p h i c method h a s been employed f o r t h e s e p a r a t i o n and i d e n t i f i c a t i o n o f
streptomycin (447,448) . The s p o t s o b t a i n e d
from streptomycin by TLC procedure have been
e l u t e d w i t h phosphate b u f f e r and examined
m i c r o b i o l o g i c a l l y w i t h B a W bubLLLi.6 s p o r e s
A s i m i l a r procedure h a s been adapted by Zuidweg
e t a1 (449) and Hamann e t a 1 (450) t o r e s o l v e
streptomycin and o t h e r a n t i b i o t i c s . The former
used t h e p l a t e s c o a t e d with sephadex and t h e
l a t t e r used t h e c e l l u l o s e l a y e r .
A p p l i c a t i o n 4-Dimethylaminobenzaldehyde-antimony t r i c h l o r i d e a s a d e t e c t i n g agent f o r a n t i b i o t i c i n TLC was r e p o r t e d by Mathis and
Table 4 : TLC of Streptomycin
Adsorbent
Solvent System.
Spray Reagent
1. Silica gel G /
Aluminium
oxide.
PropanolEthyl acetate-waterAmmonium hydroxide

(50:10:30:10).
2. Silica gel G
Butanol-watermethanol-tolune
-p-sulphonic acid
(40:20:10:1)
Sodium Nitroprusside, potassium

permanganate and
sodium hydroxide
mixture.
3 . Silica gel G
1) Acetic Acid :
butanol (7 :2)
2) Benzene-acetone
acetic acid
2:2:1.
10% aqueous solution

Copper sulphate
with 2% ammonia.
Ninhydrin.
> J
Rf value
Reference
0.62-07
440
0.4
441
0.32
0.32
442
STREPTOMYCIN
567
Schmitt (451). Paunez (452) described a TLC

method utalizing the layer o f ion-exchange
resin.
More extensive examination of streptomycin
group was reported by Heding (453) and More
recently Borowiecka ( 4 5 4 ) . Forty seven solvent
systems were investigated in deciding on optimum conditions for resolution of streptomycin
and related compounds. Visualization was made
by a spray reagent of sodium nitroprussidepotassium permanganate.
1 3 . 7 4 High Performance Liquid Chromatography
Bagon (455) has developed an assay of antibiotics in pharmaceutical preparations using reversed-phase high-pressure liquid chromatography.
Adapting this method, antibiotics like streptomycin were determined by HPLC on a column
packed with Spherisorb SS-ODs. Aqueous methanol
at a flow rates of upto 1 ml/min. was used as
an eluent. The elutes were monitored by spectrophotometric detection.
Whall (456) has reported a HPLC method for the
determination of streptomycin and dihydrostreptomycin sulphate. The method is as
follows:
Samples were dissolved in water and diluted to
50 ml., 3 ml of each solution is then diluted
to 50 ml with mobile phase containing 0.02 M
sodium hexanesulphonate - 0.025 M sodium phosphate in aqueous acetonitrile, adjusted to
pH 6 . A 25 pl portion was subjected to HPLC
on a column packed with LI Bondapak C18. The
column was fitted with a reversed-phase guard
column and elution was carried out at 1 mljmin.
at 25O. Solutes being detected at 195 nm.
HPLC assay of pyrazinoic acid in human plasma
in the presence o f antituberculosis drugs using
an automatic sampler was proposed by Ratti et
a1 ( 4 5 7 ) . This method could also be used to
determine streptomycin. The chromatographic
conditions are : Mobile phase : 0.01M ammonium
dihydrogen phosphate - acetonitrile ( 3 :7 ) .
568
Column condition : Column of 25 cm length 4.6

mm width packed with Lichrosorb-NH2. Flow rate
at 2 rnl/min. The elutes are detected at 254 nm.
Inchauspe and Samain (458) have described a
ion-pair reversed-phase high performance liquid
chromatographic method for separating aminoglycoside antibiotics using perfluorinated carboxylic acids. The aminoglycoside antibiotics were
separated at ambient temperature on a column
(25 cm x 4.6 mm) packed with ultrasphere c18
(5 pm) with methanol - 50 mM-pentafluoropropionic acid (21:29), methanol - 50 mM-heptafluorobutyric acid (3:2), methanol - 50 mM - camphorsulphonic acid (29:21) o r 0.2 M trifluoroacetic acid as mobile phase and refractive index
detection.
13.75 Electrophoresis
Foster and Ashton (459) described a method for
separation of streptomycin and related compounds
by paper electrophoresis. The test solutions
were applied to the paper which is then wetted
with a buffer solution of pH 5. After passage
o f current for 16 hours the paper was removed
and dried. The spots were revealed by one of
following reagents : 1) diacetyle; 2) naphtharesorcinol spray; 3) Elson Morgans reagent.
Alternatively, the potency was determined microbiologically by laid down the papers on agar
plates seeded with suitable organisms.
Takahashi and Amano (460) employed the paper
electrophoresis method for the identification
o f various antibiotics.
Philippe et a1 (461) have reported a similar
method of electrophoresis f o r the separation of
streptomycin and dihydrostreptomycin. Whatman
3MM paper in borate buffer at pH 9 were used.
The electropherogram was run or 3 hours at
500 V and the spots were developed with
Monastero's alkaline nitroprusside-ferric cyanide reagent.
Other reports o f resolution of streptomycin and
other antibiotics have been published by Paris
569
STREPTOMYCIN
and Theallet (462) Lightbrown and de Rossi (463).

The application of electrophoresis for separation and identification o f the antibiotics in
Pharmaceutical mixtures was reported by
Apreotesei and Teodosiu (464).
Katayama and Ikeda (465) achieved a similar
resolution using electrophoresis layers of
silica gel. They also described a two dimensional technique using a combination of electrophoresis and chromatography on silica gel (466).
Gorge and Monteoliva (467) used two dimensional techniques on paper (chromatography in
one direction and electrophoresis in the second)
or identification of many guanidino compounds
including streptomycin. Application of electrphoresis to the identification of several aminoglycoside antibiotics has been reported by
Garber and Dobrecky (468) and Ochab (469).
Langner et a1 (470) have described a method of
determination of antibiotics including streptomycin in mixtures at ultra-micro levels by
rapid low-voltage electrophoresis. The antibiotics could be separated on microscopic
slides coated with agarose gel. After electrophoresis at 50 V per cm. for 5 to 30 minutes,
the antibiotics were located by Bioautography
on agar seeded with BaCieeUn c5ub;tieid.
13.76 Counter-Current Distribution
Titus and Fried (471) have reported the u s e of
counter-current distribution technique f o r the
analysis of streptomycin preparation. The Craig
technique of counter-current distribution was
employed to show the presence of a related substance in crude streptomycin by distribution
between butanol and water.
Craig countercurrent distribution technique
has been extensively employed for the separation of streptomycin from mannosidostreptomycin
by Plaut and McCormack (472) and O'Keeffe et a1
(473). The former obtained a better resolution
by using the solvent systems of an aqueous
phase containing 0.5% sodium bicarbonate and
1% sodium chloride and solvent phase
570
(Pentasol-amyl a l c o h o l ) c o n t a i n i n g 5% s t e a r i c
a c i d and t h e latter employed a system composed
o f l a u r i c a c i d i n amyl a l c o h o l and aqueous
phospha t e -bora te b u f f e r .
Meyer (474) h a s d i s c u s s e d i n d e t a i l s t h e d i f f e r e n t methods o f c o u n t e r - c u r r e n t d i s t r i b u t i o n
t e c hnique s employed f o r t h e a n a l y s i s of v a r i o u s
compounds i n c l u d i n g s t r e p t o m y c i n .
1 3 .8 Bio-Assay Methods
The p r i n c i p l e o f t h e b i o l o g i c a l a s s a y i s based on t h e
comparison o f t h e i n h i b i t i o n of t h e growth o f b a c t e r i a
produced by known c o n c e n t r a t i o n o f t h e s t a n d a r d p r e p a r a t i o n wi th t h a t produced by measured c o n c e n t r a t i o n
o f t h e sample be ing t e s t e d .
13.81 Agar-Diffusion Method on S o l i d Media
Many a g a r - d i f f u s i o n methods w i t h t h e s u i t a b l e
inoculum o f t h e fol lowing t e s t organisms have
been r e p o r t e d :
Enchenicki cakX (475-477), BacAYw n u b U
(478-483), B a W cd~cf.&ns
(484), B a r n
ficheni~onmn (485), B a W cetrew v a r .
mgcoidu (486-487), SXaphyLocaccw awrew (488490), S.thepRococcub h c ; t i n (491) K L e b n i d h
pneumonkw (492) L a c t o b a c i U u b&atLicw
(493)
a c i d r e s i s t a n t microbacterium V-5(494). The
method h a s been e x t e n s i v e l y used i n determin a t i o n o f stre pt omyc i n i n b i o l o g i c a l body
f l u i d (495-499), u r i n e (500). food and animal
f e e d (501-505) and pha rmaceutical p r e p a r a t i o n s
(506).
M o d i f i c a t i o n s of a g a r d i f f u s i o n method have been

r e p o r t e d . Paper d i s k method h a s been adapted
f o r t h e d e t e r m i n a t i o n o f str eptomycin by Loo
e t a1 (507), Koelzer and Giesen ( 5 0 8 ) , R e i l l y
and Sobe rs (509), and Kanazawa & Kuramata (510).
Resazurin d i s c ha s been employed by Shahani and
Badami (511).
Waksman and R e i l l y (512) a p p l i e d Agar-str eak
method or a s s a y i n g st reptomycin and o t h e r a n t i The potency o f t h e a n t i b i o t i c substances:
b i o t i c s p r e p a r a t i o n s can be determined by
STREPTOMYCIN
571
i n c o r p o r a t i n g graded d i l u t i o n s i n s t a n d a r d n u t r i e n t a g a r media i n p e t r i p l a t e s and s t r e a k i n g

t h e agar surface with selected t e s t b a c t e r i a .
After o v e r n i g h t i n c u b a t i o n , t h e potency is r e a d
and expressed as t h e r e c i p r o c a l o f t h e h i g h e s t
d i l u t i o n i n h i b i t i n g t h e d i f f e r e n t t e s t organisms.
McGuire e t a 1 (513) h as suggested a new l i n e a r
d i f f u s i o n method f o r m i c r o b i o l o g i c a l a ssa y o f
s t r e p t o m y c i n . S l i d e - c e l l method (514) and
modified d r o p - p l a t e method were a l s o r e p o r t e d .
A comparative s t u d y o f cup, p a p e r - d isk , p u lp
d i s k and s u p e r p o s i t i o n a s s a y mthods was r e p o r t e d by I s h i d a e t a1 ( 5 1 6 ) .
Dewart e t a1 (517) and Light Brown e t a 1 (518)
have d e s c r i b e d a d i f f u s i o n a s s a y f o r a n t i b i o t i c s
by an automated procedure. The method i s as
follows :
Disposable p l a s t i c P e t r i d i s h e s loaded with a g a r
p r e p a r e d f o r a s s a y are c a r r i e d a u t o m a t i c a l l y t o
a d e v i c e t h a t punches h o l e s i n t h e a g a r , t h e n
t o a d i s p e n s i n g u n i t t h a t d i s p e n s e s measured
volumes ( e . g . , 50 o r 70 1.11) o f t e s t o r s t a n d a r d
solution i n t o t h e holes; a f t e r incubation, t h e
a r e a o f zones of i n h i b i t i o n a r e measured.
Continuous au t o m a t i c m i c r o b i o l o g i c a l a s s a y o f
a n t i b i o t i c s was r e p o r t e d by Shaw and Duncombe
(519). The method was based on t h e measurement
of r e s p i r a t o r y carbon d i o x i d e .
Improvements o f Agar d i f f u s i o n method f o r a s s a y i n g s t r e p t o m y c i n were a l s o r e p o r t e d (520,521).
B r i t i s h Pharmacopoeia 1958 (304) and Egyptian
Pharmacopoeia 1953 (13) d e s c r i b e a m ic r o b io lo g i c a l Assay method f o r d e t e r m i n a t i o n o f
s t r ep t o m y c i n .
Assay Procedure :
F i l l i n uniform t h i c k n e s s p e t r i - d i s h e s , o r r e c t a n g u l a r t r a y s , t o a d e p th o f 3 t o 4 mm. with
a g a r c u l t u r e medium which h a s been p r e v i o u s l y
inoculated with a s u i t a b l e quantity of a susp en s i o n o f s p o r e s o f a s u i t a b l e s t r a i n o f
BacLUuh h u b U .
572
JABER S. MOSSAETAL.
Allow t h e inoculated p l a t e s t o dry a t room temperature and keep a t 5OC., u n t i l used. Place
on the s u r f a c e of the inoculated medium, small
s t e r i l e cylinders of uniform s i z e approximately
10 mm high o r having an i n t e r n a l diameter of
approximately 5 mm., made of g l a s s , porcelain
o r aluminium, and keep a t about 150OC. Five,
s i x , e i g h t c y l i n d e r s , o r any o t h e r numbers which
can be conveniently adjusted t o t h e s i z e of t h e
agar p l a t e , may be placed on each. Bore i n t h e
medium, i n place of c y l i n d e r s , holes, 5 t o 8
mm i n diameter, by means of a s t e r i l e borer.
Prepare i n s t e r i l e phosphate buffer s o l u t i o n ,
pH 8 . 0 , s o l u t i o n s of t h e standard preparation
o f streptomycin, of known concentrations, as
f o r example 0.5 t o 2 u n i t s per m l and s o l u t i o n s
of t h e sample t o be t e s t e d presumed t o be o f
t h e same order of concentration. F i l l i n t o t h e
cylinders o r holes, t h e s o l u t i o n s of t h e s t a n dard preparation and of t h e sample t o be t e s t e d
i n a l t e r n a t e order by means of a p i p e t t e which
d e l i v e r s a standard amount o f s o l u t i o n s u f f i c i e n t almost t o f i l l t h e holes when they a r e
used,
Place t h e p l a t e s i n a r e f r i g e r a t o r f o r about 2
hours, and then incubate a t about 30 t o 37OC
f o r about 16 hours. Measure, with t h e g r e a t e s t
possible accuracy, t h e diameters of t h e i n h i b i t i o n zones produced by t h e varied concentrations
of t h e standard preparation of Streptomycin and
of t h e sample t o be t e s t e d , and from t h e r e s u l t s
t h e potency of t h e l a t t e r is c a l c u l a t e d .
L i m i t of Error:
The limits of e r r o r (P=O.99)
t h a t can be obtained with t h i s method are 79
and 1 2 1 per c e n t . , where 10 cylinders o r holes
a r e used; 85Oand l l S O , where 20 cylinders o r
holes a r e used; 89.50 and 110.50 where 40 c y l i n d e r s o r holes are used; and 92.50 and 107.50
where 80 cylinders o r holes a r e used f o r t h e
standard preparation of streptomycin and f o r t h e
sample t o be t e s t e d , r e s p e c t i v e l y .
STREPTOMYCIN
513
13.82 Liquid C u l t u r e Method

13.821 T u r b i d i m e t r i c Method
Oswald and Knudsen (522) have r e p o r t e d
a t u r b i d i m e t r i c method f o r a s s a y o f s t r e p tomycin. Two series of t u b e s , one w i t h
graded amounts o f s t a n d a r d s t r e p t o m y c i n
and o t h e r w i t h t h e unknown are i n o c u l a t e d
w i t h t h e same amount o f a s t a n d a r d s u s pension o f t h e t e s t organism, i n c u b a t e d
a t 37O f o r 4 h o u r s . The p e r c e n t a g e o f
transmission read i n a photoelectric
c o l o r i m e t e r . Potency of t h e sample under
t e s t can be r e a d d i r e c t l y from t h e s t a n dard curve.
A p p l i c a t i o n s of t h e t u r b i d i m e t r i c method
o r t h e a n a l y s i s of s t r e p t o m y c i n i n u r i n e ,
serum and c e r e b r o s p i n a l f l u i d were r e p o r t e d by Gibb (523) and Whitlock e t a l ( 5 2 4 ) .
13.822 Other Methods
Bonifas and Chesni (525) have developed
t h e d i l u t i o n method f o r t h e d e t e r m i n a t i o n
o f s t r e p t o m y c i n : K ~ e b p bn m~o n ~
iae
was allowed t o grow a t pH 9-9.5 i n t h e
s y n t h e t i c medium of Monod t o which s u g a r
and i n d i c a t o r c r e s o l r e d were added. The
r e a c t i o n s h i f t e d towards t h e a c i d s i d e
upon s p l i t t i n g t h e s u g a r . Decreasing
amounts o f s t r e p t o m y c i n were added t o t h e
medium. The i n h i b i t i n g amount o f s t r e p tomycin was determined by n o t i n g t h e d i l u t i o n i n t h e t u b e immediately p r e c e d i n g
t h a t i n which t h e s e was t h e f i r s t change
of color of t h e indicator.
Dilution-method was extended t o determine
s t r e p t o m y c i n i n u r i n e and body f l u i d s
(526).
A microbiological assay of a n t i b i o t i c s
based on i n h i b i t i o n of ammonia p r o d u c t i o n
was r e p o r t e d by Kobos (527). The t e s t
s o l u t i o n was i n c u b a t e d f o r 2 hours a t 37O
w i t h a s u s p e n s i o n o f E . cofi i n 6% peptone
medium., t h e pH o f t h e m i x t u r e was
514
a d j u s t e d w i t h sodium hydroxide and t h e

c o n c e n t r a t i o n of ammonia was determined
w i t h u s e of an Orion 95-10 ammonia s e n s o r .
13.9 Biochemical Methods
A radioimmuno a s s a y (528,529) was developed f o r d e t e r mination o f s t r e p t o m y c i n i n plasma and u r i n e . The
method i n v o l v es enzyme-catalysed i n t r o d u c t i o n o f a
l a b e l l e d group i n t o t h e molecule and s e p a r a t i o n o f
t h e l a b e l l e d product by paper chromatography b e f o r e
counting
Schwenzer and Anhalt (530) have r e p o r t e d an automated

f l u o r e s c e n c e p o l a r i z a t i o n immunoassay f o r str e p to m y c in
i n blood serum. I n t h e a s s a y d e s c r i b e d f l u o r e s c e i n l a b e l l e d streptomycin was used a s t h e tracer and a n t i serum a g a i n s t streptomycin was r a i s e d i n r a b b i t s . On
mixing o f t r a c e r , sample and a n tib o d y , t h e p o l a r i z a t i o n o f t r a c e r f l u o r e s c e n c e was measured i n an Abbott
TDX f l u o r i m e t e r .
Acknwo 1edgement
The a u t h o r s s i n c e r e l y thank Mr. A l t a f Hussain Naqvi and
M r . Uday C. Sharm or t h e i r s e c r e t a r i a l h e l p .
STREPTOMYCIN
575
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108. I d e m . ,
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625,
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820 (1949); C.A.,
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43,
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109. P h i l i p D. Coppock, L i l y L. Mulligan and Alan G. White;

Brit, 622, 682 (1949); C.A. 44, 1656 (1950).
110. HarveyE. Alburn and Eric G. Snyder; U.S.,
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2 , 505, 318
111. Robert D. Babson and Max T i s h l e r ; U.S., 2 , 521, 770

(1950); C.A. , 44, 11041 (1950).
582
1 1 2 . Robert L. Peck, U.S. 2 , 540, 284 (1957); C.A.

(1951).
43, 4894
113. Wm. A. B i t t e n b e n d e r and Robert D. Babson; U.S.

238 (1951); C.A., 45, 4414 (1951).
114. Frank J. Wolf; U.S.,

(195 1)
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540,
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C.A. 46, 1220 (1952).
116. David L. Johnson; U.S.,
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2 , 643, 997 (1953), C.A. 47,
117. Arne N. Wick and M i l t o n , J. Vander Brook; U.S.

701, 795 (1955); C . A . , 49, 6550 (1955).
, 2,
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P.S. S k e l l and W.A. S t r o n g ; J. B i o l . Chem; 160, 337
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45, 2158
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125. C h a r l e s H. McBurney; U.S.,
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126. Hideo Kubota;Japan,
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127. Sumio Umezawa and T e t s u o Suami; Japan; 4296 (1954); C.A.

49, 10589 (1955).
583
STREPTOMYCIN
128. Kaneyuki Takagi; Japan; 5649 (1954); C . A . ,

(1955).
49,
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C.A. , 50, 10350 (1956).
130. J o s e f Hoffman, J i r i n a Capkova, Miroslav Hermansky and
50, 10351
Zdenek Severa; Czech; 85, 067 (1955); C.A. (1956).
131. O . B . F a r d i g and I . R . Hooper; U.S. 2 , 717, 892 (1955);
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135. C.R. Bartels; G . Kleiman, D . R . I r i s h and J . N . Korzun,

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(1962), C.A. 58, 5979 (1963).
224,
3,702
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Clinic; 20, 145 (1945); C.A., 39, 4640 (1945).
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498. Gosta Wallmark, Acta Path. Microbiol. Scand.,
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29 ,
16, 331
Antibiotiki; -
500. A . L . O l i t z k i and Z . O l i t z k i ; A n t i b i o t i c Med., 2 , 317

(1956); C . A . , 50, 14854 (1956).
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STREPTOMYCIN
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86
and Haruta. Misao; Shokuhin E i s e i g a k u Z a s s h i ,
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12,
44,
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Anal. Abst., 8 , 3973 (1961).
33(1961);
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Abst., 9, 2550 (1962).
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1 0 , 2877 (1963).
825 (1962)., Anal. A b s t . , 505.
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(1969); C.A.
(1970).
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59682t (1969); Anal. A b s t . , 1 9 , 2678
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2 , 469 (1952); C . A . , 4 7 , 7160(1953).
and Chemotherapy, -
51u.
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I
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c
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609
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THIABENDAZOLE
Vijay X. Kapoor
1.
Description
1.1 Name, Formula and Molecular Weight
1.2
1.3
Salts
2.
Physical Properties
2.1
Infrared Spectrum
2.2
2.3
Optical Rotation
2.4
Melting Range
2.5
Solubility
2.6 Crystal Properties
3.
Synthesis
4.
Stability and Degradation
5.
Metabolism and Phannacokinetics
6.
Methods of Analysis
6.2
Identification Color Test
6.3
Titrimetric Analysis
6.4
Bioassay
6.5
Polarographic Analysis
6.6 Colorimetric Analysis
6.7
Spectrophotometric Analysis
6.71
Ultraviolet
6.72
Infrared
6.8
Spectrofluorometric Analysis
6.9
Chromatographic Analysis
6.91
6.92
6.93
Gas Chromatography
6.94
High Performance Liquid
Chromatography
6.10 Miscellaneous
7.
Limits of Tolerance
8.
References

VOLUME 16
61 1

AU riehts of reproductionin any form reserved.
VIJAY K.KAPOOR
612
Description
1.1 Name, Formula and Molecular Weight
Thiabendazole, an anthelmintic and
antifungal agent, was developed by scientists of
Merck laboratories1 in 1961. Thiabendazole is
2-(4-thiazolyl)-1H_-benzimidazole. The CAS
Registry number is 148-79-8. The most common
proprietory name is Mintezol
Molecular Weight: 201-25

10H7N'3
1.2
White to cream-colored, odorless or
almost odorless powder.
1.3
Salts
Thiabendazole is a weak base. The
hydrochloride (sublimed at 265O) and sulfate
(melted at 262-6O) have been prepared.2 Watersoluble lactate of thiabendazole has also been
prepared along with the citrate, fumarate and
salicylate which had limited solubility in water.
Physical Properties
2.1
Infrared Spectrum
The infrared spectrum4 (KBr disc) of
thiabendazole is presented in Figure 1. The
principal peaks are at 1408, 906 and 74OCm-1.
2.2
The light absorption, in the range 230
to 350 tun, of a 2-cm layer of a 0.0004 per cent
w/v solution in 0.1N hydrochloric acid exhibits
two maxima, at 243 ?h~
and at 302 nm; absorbance
at 243 nm, about 0.47 and at 302 nm, about
0.98.5
2.
THIABENDAZOLE
613
Wavelength microns
6
7
8
9
10 11 12 131415
90
80
70
60
50
40
30
20
l10
o
I
2000
1800
1600
1400
1200
1000
I
I
800 650
Wavenum ber
Figure 1. Infrared Spectrum of Thiabendazole

2.3
Optical Rotation
Thiabendazole exhibits no optical activity.
2.4
Melting Range
The melting range6 of thiabendazole is
between 296O and 303O.
2.5
Solubility
The solubility7 of thiabendazole at
ordinary room temperature is as follows:
Solvent
Water
Alcohol
Chlorofonn
Ether
Acetone
Dilute mineral acids
Solubility, mg/ml
practically insoluble
6.66
3.33
0.5
slightly soluble
soluble
VIJAY K.KAPOOR
614
2.6
Crystal Properties
Crystal structure of thiabendazole is
described.* Crystals of thiabendazole are
orthorhombic, space group p
s, with a =
17.052(7), b = 10.998(4) , and
= 10.030?8) A.
There are erght formula units per cell; observed -3
and calculated densities are 1.414 and 1.421 g cm
Intensity data were collected on an automatic
diffractometert the structural parameters were
refined by full-matrix least-squares to an
index
of 0,066 f o r 1805 reflections, The two ring
systems are approximately planar, but are twisted
by loo with respect to each other. The C-C bond
connecting the two ring systems is 1.442(10) %,
long. Molecules are linked together by N ( 1 ) H(l)---N(14)
hydrogen bonds to form chains
parallel to the
axis,
X-ray powder diffraction data for thiabendazole
is given in Table 1.9 The data were obtained by
diffractometer and Debye-Scherrer camera techniques,
and are tabulated in terms of the lattice spacings
and the relative intensities of the line. Patterns
using three different X-ray wavelengths w i t h the
camera method are given.
3.
Synthesis
Brown et a1.l from Merck laboratories first
reported the synthesis (Scheme I) of thiabendazole
by the reaction of 4-thiazolecarboxamide with
O-phenylenediamine in polyphos horic acid at 250
for three hours. In a patent18 the synthesis of
thiabendazole is reported through another route
(Scheme 11). O-Nitroaniline was condensed with
lactic acid or-the acid halide to give g-lactoyl2-nitroacetanilide which on oxidation followed by
bromination gave ~-(bromopyruvoyl)-2-nitroanilide.
The latter on refluxing overnight with thiofomamide
hydrate followed by zinc dust reduction gave
thiabendazole.
Alternate methods of synthesis havs also been
described.11812 Synthesis of 14C- or 3 S-labeled
thiabendazole i s also described.13
Table 1
X-Ray Diffraction Data f o r Thiabendazole
Diffractometer
CuKa
d (ft)
1/11
6.81
5.64
5056
8
4
7
43*
5 007"
4r23*
3.72
3 39*
3027
2,814
20695
2,525
2 327
-14
loo*
43*
2
-10
-33
--
Camera
corn
CuKa
d (1)
6.76
5057
5oO3*
4021~
4.02
3.71
3 0 38*
3026
3011
2 0803
2.681
20629
2,517
2.419
2.321
2,253
20137
2,055
20022
2,062
2
1
20025
*Most intense lines
1/11
17
I
18
68*
loo*
3
26
51*
5
3
19
6
3
12
1
9
4
5
7
4
CrKa.
d <A)
1/11
d (A)
1/11
5.57
-17
6076
-1113
6.76
5 04"
4.21*
4001
3071
3 38*
3.26
3010
20806
2 0 685
20630
2.515
20418
2.316
2.252
2.132
2.056
2 0 000
14
75*
loof
5
27
5 1*
7
4
5057
5oO3*
4021*
(c.
3.70
3 0 38*
3025
I
--
21
2.808
12
17
21
5
4
2.519
7
7
7
5
20311
63*
loo*
20
43*
-.14.
VIJAY K.KAPOOR
616
$7
PolYphosphoric acid
NHpCO
250/ 3 hr
Scheme I. Synthesis of thiabcndazole
UNo2
CHaCHOHCOR
N-H
R =OH, C I
I
I
H COH
I
c=o
CH3
aNo2
N -H
I
c=o
I
c =o
0r2/0O0
CHI Br
HC
II
Zn/HCl
NH-C
Scheme
n. Alternate
N-H
c=o
I
I
c=o
CHI
'NH2
aNo2
iiya
synthetic pathway t o thiabendazole
THIABENDAZOLE
617
4.
Stability and Degradation

In general thiabendazole is a stable compound.
The USP XXI NF XVI directs that it should be packed
and stored in well-closed containers.6 Thiabendazole, however, has been reported14-17 to be
slightly uv (sunlight) sensitive. Photolytic
degradation products of thiabendazole have been
identified as benzimidazole, benzimidazole-2carboxamide, triazole-4-carboxamide, thiazol-4ylamidine and methyl thiazol-4-carboxylate.14.16 A
recent study involving photooxidation of thiabendazole in methanol in presence and absence of a
photosensitizer, methyleneblue also identified
dimethyl oxalate, thiazole-4-(pcarbomethoxy)carboxamlde, methyl benzimidazole-2-carboxylate,
benzimidazole-2-carboxamide and benzimidazole as the
main products of photolysis.19 Figure 2 gives the
structures of the main degradative products,
Benzi midosole
Benzimidozole-2-corboxomide
CH3OOCNHCO
Methyl benrim~dozole- 2 -corboxylote
1hiozote -4
c;sa
- f N-car bomethoxy) - carboxamide
Figure 2. Moin products of photolysis of thiabendazole
A recent review discusses the different

breakdown products due to environmental factors
of various fungicides including thiabendazole 2o
5,
Thiabendazole is metabolized chiefly to 5hydroxythiabendazole which is excreted in the
urine conjugated either B S glucuronide or as t h e
sulfate (Figure 3) . I 3 Thiabendazole is rapidly
absorbed from the gastrointestinal tract and peak
plasma levels of 13 to 18 pg/ml are found within an
hour of ingestion. The drug and its metabolites
are excreted rapidly, 87% in the urine and 5% in
VIJAY K.KAPOOR
618
Thiabendazole
5 -Hydroxythiabendazole
Hp
mN
Sulfate conjugate
Glucuronide conjugate
Figure 3 . Metabolism o f thiabendazole
the faeces within 48 hours. 21 small amounts of

both unchanged thiabendazole and free 5-hydroxythiabendazole may be found in both the plasma and
urine. Gastrointestinal absorption and secretion
of thiabendazole has been studied.22 Wilson &
a1.23 have reported that 5-hydroxylation of
thiabendazole in rat liver microsomal preparation
is dependent on cytochrome P-450 and has a Km of
3.92 x 10-431 and Vmax of 0.484pole of 5hydroxythiabendazole produced/hr/mg of protein.
5-Hydroxythiabendazole gave a type If binding
spectrum w i t h cytochrome P-450, Desmethylimipramine
and ethoxyquin are reported to inhibit the hepatic
microsomal hydroxylation of thiabendazole.24 A
single o r a l dose of desmethylimipramine (80 mg/kg)
administered to rats inhibited the hydroxylation of
thiabendazole by 45% in vitro at 5 hr after dosage
but did not decrease cytochrome P-450. A single
oral dose of ethoxyquin (200 mg/kg) to rats
inhibited the hydroxylation by 65% in vitro at 1 hr
after dosage, inhibition was less at 5 hr. Oral
ethoxyquin (400 mg/kg) or desmethylimipramine
(80 rng/kg) administered 30 minutes before oral
thiabendazole (100 and 200 mg/kg, respectively)
delayed absorption of thiabendazole and resulted
in its decreased plasma concentrations. Bauer
THIABENDAZOLE
619
et a1.25
have studied the phamacokinetics of

thiabendazole and its metabolites in an anephric
patient undergoing hernodialysis and hemoperfusion.
The half-life, volume of distribution, and
clearance of thiabendazole were 1.17 hr, 2.76 l/kg,
and 27.2 ml/rnin/kg, respectively. While thiabendazole and the 5-hydro= metabolite did not accumulate
during multiple dosing, the glucuronide and sulfate
conjugates accumulated extensively despite hemodialysis and hemoperfusion.
Pharmacokinetics of thiabendazole has been
studied in cattle,268 27 sheep,28 and avian29
species. The findings are consistent with the
literature data that metabolism and excretion of
thiabendazole are more extensive in cattle than
in sheep.
Methods of Analysis
6.1
Elemental Analysis
The elemental analysis of thiabendazole
is as follows:
11
E 1ement
% Calcd.
% Found
C
59.68
59074
H
3.51
3.62
N
20.88
20.57
S
15.93
15.99
6.2
Identification Color Test
The following color reaction can be used
to identify a sample of thiabendazole.586 About
5 mg of the sample are dissolved in 5 ml of 0.12
hydrochloric acid and 3 mg of E-phenylenedimine
dihydrochloride are added f01 lowed by shaking to
dissolve the contents. About 0.1 g of zinc dust
is then mixed and the mixture allowed to stand
f o r 2 minutes. 5 ml of a solution prepared by
dissolving 20 g of ferric ammonium sulphate in
7 5 rnl of water, and 10 ml of 1E sulphuric acid are
then added. When diluted with water to 100 ml a
blue or blue-violet color is developed6.3
The titration with perchloric acid is
the method of choice to assay thiabendazole.586
6.
620
VIJAY K.KAPOOR
The sample is dissolved i n glsclal acetic acid.

After speciEic quantities of acetic snhydride,
mercuric aceta%e and two drops of c r y s t a l violet
soluticn are added, the titration with 0.1E perchloric acid to a hlue-green end point is carried
out. A blank determination is performed and any
necessary correction is made. Each r n l of 0.1N perchloric acid is equivalent to 20.13 mg of CIOH7N3S.
A titration method utilizing the complex
formed by thiabendazole with silver Ions is also
described. 30
6.4
Bioassay
Several microbiological assay methods using
sensitive fungi for the quantitative determination
of thiabendazole have been reported. 31-38 These
methods have been employed f o r determining the
residue of the fungicide on plant materials or in
soil. A simple and rapid bioassay or the direct
determination of thiabendazole residue in soil
involves placing pellets composed of mixtures of
soil (200-500 my> and agar on an agar medium preinoculated with the test organism Penicillium
digitatum.37 After cold pre-incubation followed by
incubatlon at 2 7 O , the size of the inhibition zone
is measured (Figure 4 ) . The lowest de%ectable
concentration of thiabendazole in a sand soil was
In another method 5 extracts
found to be 1.0 ) i g / g .
from citrus fruit and bananas were applied as spots
to silica gel in P e t r i dishes which were then
uniformly inoculated with Penicillium cyclopium
spores. Fungicide concentration was detennined
from comparison of inhibition zones with those of
a standard 48-hr incubation. Detection threshold
was 0.5 p g thiabendazole.
Other microorganisms employed in the bioassa
studies are Penicillium exansum,32 Graphium ulmf3
and Sporobolomyces roseus.38
6.5
Polaroqraphic Analysis
A polarographic method for determining
thiabendazole is described. 39 The electrochemical
behavior of thfabendazole was investigated by
sampled doc. polarography and differential pulse
polarography. Thiabendazole showed one wave peak
in the polarogram when a short drop time (0.4 s )
was used. Detection was most sensitive at pH 8 .
THIABENDAZOLE
c
E 250
c
.-2
.- 200
621
C
0
.-C
2
4
6
8
10
12
Thiabendazole concentration in soil ( p g / g )
Figure 4. Effect of thiabendazole concentration

in-soil on growth inhibition of Penicfllium
di-itatum using the soil-agar pellet technique.
inhibition is expressed a s the square
Grow
The current measured was proportional to the

concentration. The detection limit was 0.5 ppm.
Differential pulse polarography has been used for
quantitative determination of thiabendazole In
citrus fruit peels.
6.6 Colarimetric Analysis
Thiabendazole can be analyzed colorimetrically in feeds.40-43 The method involves:
(a) complete extraction of thiabendazole from Eeed
w i 3 1 C.2N H C 1 in SO% methanol; (h) separation from
other suEstences by ext.raction with chlorof o m from
a l k a l i n e solution; (c) further purif ication by
reextraction into dil.vte H C l ; ( d ) reduction with
622
VIJAY K. KAPOOR
zinc dust in the presence of pphenylenediamine to

form a complex; (el subsequent oxidation with a
ferric solution to form a thiazine dye; and (f)
extraction of the dye into butanol and measurement
at 605 nm against reference standard thiabendazole.
The method gives most accurate and reproducible
he method described above was automated
results.
by !~;hite.~~
In the automated system the solution
was reduced with 0.3% zinc dust in glycerol, and
the products were coupled with 0.17% pphenylenediamine in 2N H2SO4 and 15% Fe NH4 (SOg)2 in 0.1g
H2SO4. The glue complex was measured at 610 nm.
The relative standard deviation was 1.2%, and the
recovery was 95-100% from feed containing 1%
thiabendazole. A modified method was used to
determine the residues of thiabendazole in citrus
fruits-45
A method for estimating thiabendazole in the
milligram and submilligram ranges (0.1-2.0 mg/ml)
has been descrlbed which involves measuring of
absorbance of a copper-thiabendazole-tetramethylguanidine complex at 350-400 nm.46 A modified
method in which absorbance of the complex was
measured at 311 nm permitted estimation of 2 pg/ml
thiabendazole. An improved procedure involves
shaking an alkaline aqueous suspension containing
thiabendazole with solution of cupric acetate and
1-dimethylamino-2-propanol in chloroform to produce
a green color in the chloroform phase.47 The
analysis is completed by spectrophotometric
measurement. Thiabendazole in concentration range
of 0.1-1.0 mg/ml can be estimated by this method.
A simple colorimetric method involving the
reaction of thiabendazole in ch1.oroform with
alkaline buffered bromocresol green is also
6escribed.48
6.7 Spectrophotometric Analysis
6.71 Ultraviolet
Thiabendazole is estimated in
various fruits as a residual fungicide by ultraviolet
spectrophotometry.49-60 The method in general
involves extraction with ethyl acetate-ammonium
hydroxide, purification by acid-base procedure, and
measuring the absorbance of the acid solution at
302-3 nm. In another purification procedure the
THIABENDAZOLE
623
f r u i t p e e l s o r p u l p s are extracted with chloroEorm,

t h e e x t r a c t i s a c i d i f i e d and c h l o r o f o m evaporated.
The a c i d i c c o n c e n t r a t e i s cleaned up with chloroform,
and t h i a b e n d a z o l e e x t r a c t e d a t pH 9.5.
Using t h i s
method t h i a b e n d a z o l e w a s determined spectrophotom e t r i c a l l y a t 302-3 nm a t c o n c e n t r a t i o n s of 0.1 pprn
i n 100 g peel or pulp, o r 0.03 ppm i n whole c i t r u s
f r u i t . Aspect of p r o t e i n i n t e r f e r e n c e i n
t h i a b e n d a z o l e d e t e r m i n a t i o n i s discussed.61
6.72
Infrared
Thiabendazole can a l s o be analyzed
by i n f r a r e d spectrophotometry.62 The method
i n v o l v e s d i s s o l v i n g 0.12 g of t h i a b e n d a z o l e sample
i n 5 m l of l?,~-dimethylformamide and adding 1 g
of anhydrous sodium s u l p h a t e . A p o r t i o n of t h e
s u p e r n a t a n t i s used for i n f r a r e d spectrophotometry,
Absorbance a t
scanning between 2.5 and 16.7 )un.
11.09 )xn is used f o r q u a n t i t a t i o n . The s t a t i s t i c a l
a n a l y s i s of m u l t i p l e d e t e r m i n a t i o n by t h i s method
gave a r e l a t i v e e r r o r o f \ ( 2 % and a c o e f f i c i e n t of
v a r i a t i o n 4 1%.
6.8
Spectrofluorometric A n a l y s i s
Thiabendazole is a n a l zed by spectroThe f u n g i c i d e
f l u o r o m e t r i c method also.54,63-%8
can be detected i n c r o p s by acetone e x t r a c t i o n
followed by p a r t i t i o n i n g i n acetone-water and
e t h y l a c e t a t e and clean-up on magnesium oxidec e l i t e - a l u n f n i u m oxide column. The cornpound i s
t h e n q u a n t i t a t i v e l y measured by d i r e c t fluorometry.
The e x c i t a t i o n and emission wavelengths are 305
and 345 nm, r e s p e c t i v e l y f o r thiabendazole. T h e
method i s r e p o r t e d t o be s e n s i t i v e t o 0.02 ppm
thl sbendazole. 63 An automated f l u o r o 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 t h i a b e n d a z o l e i s a l s o
d e s c r i b e d which gives t h e minirnun l e v e l of
d e t e c t i o n of t h e f u n g i c i d e asw0.05 pg/ml.6*
A
s e n s i t i v e f l u o r o m e t r i c method f o r d e t e r m i n a t i o n
of t h i a z o l e c o n t a i n i n g compound i s r e c e n t l y
described. 68
6.9
described.
Chromatographic A n a l y s i s
6.91 Paper Chromatoqraphy
The f o l l o w i n g system h a s been
4
VIJAY K.KAPOOR
624
Paper:
Solvent:
Equilibration:
Development:
Location:
Rft
(l:6)
Khatrnan Noel, sheet 1 4 x 6

i n , buffered by dipping i n
a 5% s o l u t i o n of sodium
d ihydr ogen c i tret e , b l o t t i n g ,
and drying a t 2 5 O for 1 hour.
4.8 CJ of c i t r i c a c i d in a
mixture of 330 m l of water
and 870 ml of r p b u t a n o l .
None
Ascending. Time of run,
5 hours
a. Examination under
ultraviolet light
b. Bromocresol green spray
C. Fotassium permanganate
spray
0.06
A n a l t e r n a t e system employs acetone-water

i n ascending mode which g i v e s Rf v a l u e of
Location is done by
t h i a b e n d a z o l e as 0.49.69
allowing t h e paper t o react with i o d i n e vapor for
1 minute.
6.92
A v a r i e t y of t h i n - l a y e r chromatog r a p h i c s y s t e m s u s i n g s i l i c a g e l or si"l1ca g e l GF254
p l a t e s f o r i d e n t i f i c a t i o n and e v a l u a t i o n of thiabenda z o l e have been r e p o r t e d . These are t a b u l a t e d below:
S o l v e n t System
Reference
Benzene-acetic acid-acetone-water
70
(10:4: 1:0.4)
71,72
Benzene-acetic acid-acetone-water
(70:20: 10: 2)
Ethyl a c e t a t e - e t h y l methyl ketone65,67
formic acid-water (50: 30: 10: 10)
Ethyl acetate-benzene (50:50)
D i e t h y l e t h e r - g l a c i a l acetic acidmethanol (100:5:2)
74
Acetone
74
73
THIABENDAZOLE
625
Solvent. System
Reference
L i g h t petroleum (60-80)-acetone
(30:lO)
Chloroform-acetone (80: 20)
74
75
Using aluminium o x i d e F254 n e u t r a l p l a t e 74

d i e t h y l ether-methanol i s t h e s y s tern employed.
Cole e t
h a s g i v e n t h e Rf v a l u e s of
t h i a b e n d a z x e i n d i f f e r e n t s o l v e n t systems. These
a r e g i v e n below:
R f Value x 100
S o l v e n t System
E t h y l acetate
55
E t h y l a c e t a t e s a t u r a t e d wit.h
ammonia
E t h y l acetate-toluene-anunonia
(60:40:2)
E t h y l acetate-toluene-ammonia
(40:60: 2)
E t h y l acetate-toluene-anunonia
(20:80:2)
Toluene-ethanol-ammonia (80:20:2)
Toluene-ethyl a c e t a t e - e t h a n o l ammonia ( 60 :10 :30 :2 1
To 1uene- e t k i y 1 ace t a t e- et h a n o 1
ammonia (70: 15: 15:2)
Toluene-ethyl a c e t a t e - e t h a n o l arnmonia (60:10:10:2)
60
41
31
21
43
58
52
38
D e t e c t i o n of s p o t of t h i a b e n d a z o l e on t h e
p l a t e h a s been c a r r i e d o u t by d i f f e r e n t methods
such as v i s u a l i z a t i o n under u l t r a v i o l e t
l i g h t , 7 4 8 7 6 # 7 7 by s p r a y i n g with potassium
iodobismuthate solution followed by exposure t o
bromine vaposs74 o r by exposure t o i o d i n e vapors. 76
A b i o l o g i c a l method f o r d e t e c t i o n and e v a l u a t i o n
of t h i a b e n d a z o l e h a s a l s o been d e s c r i b e d O 7 3 I t
i s done by s p r a y i n g t h e p l a t e s with spores of any
of t h e t h i r t y t w o fungal. species f o l l o w e d b y
i n c u b a t i o n . G u a n t i t a t i v e relations were
e s t a b l i s h e d b e t w e e n spot s u r f a c e a r e a and t h e
amount of f u n g i c i d e . A n enzyme i n h i b i t i o n
VIJAY K.KAPOOR
626
75
technique i s a l s o reported.
using t h i n - 1aye r chsornatoy r aphy want i t a t i v e
determination of thiabendazole has been c a r r i e d
oiit s p e c t r o p h o t o m e t r i c a l l 70,71,72,78,79
or
fluorometrically65,67,8~,~1. Otteneder and
FIezel65 have r e p o r t e d a method by which thiabenda z o l e can be Getermined d i r e c t l y on t h e chromatogram by r e f l e c t a n c e - a b s o r p t i o n photometry with
measurement at 302 nrn o r f l u o r o m e t r i c a l l y a t
355 nm, with e x c i t a t i o n a t 313 nm. Compared with
t h e e l u t i o n and subsequent photometry, direct
e v a l u a t i o n on t h e chromatogram h a s t h e advantage
t h a t it saves t i m e and r e q u i r e s less substance
p e r spot.
6.93
G a s Chroniatoqraphy
Gas chromatographic methods have
been used t o determine r e s i d u e of thiabenda,o1e050,53,67,82-94
An e a r l i e r method t o determine
t h e f u n g i c i d e i n whole c i t r u s f r u i t and i n c i t r u s
and bansna pulp is d e s c r i b e d by Mestres e t al.50
Samples were made a l k a l i n e with ammonia s o l u t i o n
and macerated w i t h e t h y l a c e t a t e and filtered.
T h e f i l t r a t e was cleaned up by e x t r a c t i o n i n t o
0.1g HC1, and making t h e aqueous phase a l k a l i n e
and r e e x t r a c t i n g back i n t o e t h y l a c e t a t e . The
concentrated e x t r a c t was chromatographed on a
lo/, DC-200 s t a t i o n a r y phase using FPD set i n t h e
mode f o r determining s u l f u r . The l i m i t o f s 3
d e t e c t i o n was of t h e o r d e r of 0.1 pj. Hey
used
SE-30 a s s t a t i o n a r y phase with a n i t r o g e n - s e n s i t i v e
Tanaka and Fujimoto83 determined
detection
thiabendazole a s i t s methyl d e r i v a t i v e w i t h flame
i o n i z a t i o n d e t e c t i o n and a column of lo/, Dc-200
on Gas-Chrom Q a t 240O. It p e r m i t s d e t e m l n a t i o n
i n c o n c e n t r a t i o n s down t o 0,l ppm,
Thiabendazole w a s determined by Nose e t al. 8 4
a s g-pentafluorobenzoyl d e r i v a t i v e by e l e c t r o n c a p t u r e g a s chromatography following r e a c t i o n of
thiabendazole with pentafluorobenzoyl c h l o r i d e .
The m i n i m u m amount of thiabendazole detectable
by t h i s method was 0.01 ppm. P r i n c i p a l o p e r a t i n g
c o n d i t i o n s were: 1.5 m x 3 mm I.D. g l a s s column
packed with 5% OV-101 on Gas Chrom G (HP)
(80-100 mesh), i n j e c t i o n p o r t and d e t e c t o r
temperature 270, column temperature 230O. Nitrogen
was used as a c a r r i e r g a s a t a flow-rate of
THIABENDAZOLE
627
40 ml/min.
Combined gas chromatography-mass

spectrometry was operated with column temperature
2 3 0 , separator temperature 260, ion source
temperature 290 and flash heater temperature
270'.
The carrier gas was helium at a flow-rate
of 30 ml/min.
Recently, Bardalaye and Wheeler85
employed this technique to determine thiabendazole
in yams with operating conditions as: 1.83 m x
4 mm I . D . glass column packed with 5% OV-17 on
80-100 mesh Gas-Chrom 0, argon-methane carrier gas
(95 + 5) at a flow-rate of 60 ml/min. Temperatures
of oven, detector and injectors used were 240
300 and 200, respectively.
Tjan and Jansen66
have used pentafluorobenzoyl bromide f o r
derivatization of thiabendazole.
A combined gas Chromatography
mass
spectrometry confirniatory assay for thiabendazole
and 5-hydroxythiabendazole at 0.1 p m in animal
tissue isolates has been developed. 5 On-column
methylation converts these compounds to their Emethyl and E,g-dimethyl derivatives, respectively.
Identification and quantitation are achjeved by
selective ion monitoring of M-1, M and Etl ions
from E-methylthiabendazole and the I4 and M-15
i o n s from IJ,g-dimethyl derivative of fj-hydroxy=
thiabendazole.
6.94 High Performance Liquid
Chromatoqraphy
In recent years high performance
liquid chromatography (HPLC) has become one of
the major analytical tools to determine the
residual thiabendazole alone96-100 or simultaneously
with other addit.iveslOl-lll in fruits. Isshiki
et a l o g 6 have described a simple HPLC method for
eetermining thiabendazole in fruits. Thiabendazole
is extracted with methanol and the extract is
washed with phexane saturated with methanol. The
HPLC determination is carried out u s i n g LiChrosorb
RP-8 as stationary phase and methanol-2.8"A ammonia
wzter (60:40) as mobile phase; 2-methylindole is
added as an internal standard. The detection is
carried out fluorometrically and the limit of
detection is 0.1 pg thiabendazole/
Recoveries
are 92%. Collinge and Nairfalisegg have
determined thiabendazole in curds (artificial
marmalades) and orange and lemon marmalades
--
628
VIJAY K.KAPOOR
by using aqueous solution containing 0,PA of

ammonium nitrate and 0.7% of ammonia (buffered
solution)-methanol (1:1) as mobile phase for
curds, and buffered solution-methanol ( 3 :2) for
marmalades. The flow-rate in column was 1.0
ml/min, the column temperature was 22-26O and the
detection wavelength was 305 nm. Yamada et &.lo0
have described a detailed procedure for the
separation of thiabendazole in fruits in which
the uv-absorbing Contaminants are removed by
fractional separation with an aqueous-ethyl
acetate system. The HPLC conditions for thiabendazole determination reported aret column,
Unisil C-18 10 p 4.6 mm x 300 mmt eluant, methanol0.16 M KH2PO4 water ( 5 0 : 5 0 , v/v % ) I flow rate,
1.0 ml/min; pressure, 60 kg/cm2; detector uv
(298 nm); range 0.01 AUF; and sample size 5 pl.
The detection limit for this method was found to
be 0.08 ppm, and it was shown that 1 ppm of
thiabendazole in the fruits could be estimated
within
10%. An ionggair HPLC has been
described by Belinky.
A simultaneous HPLC determination of
thiabendazole, 0-phenylphenol and diphenyl
residues in cityus fruits without prior clean
up has been described by Kitada et a1.16
A rapid, sensitive and precise HPLC method
using fluorescence detection has been developed
by Watts et a1,112 for the simultaneous
determination of thiabendazole and unconjugated
5-hydroxythiabendazole in human serum. Sample
pretreatment consists only of protein precipitation
with acetonitrile containing the internal standard,
2-methylindole. Detection limits were found to be
0.1 pg/ml serum for thiabendazole and 0.4 pg/ml
serum for 5-hydroxythiabendazole. A microenzymatic
method for the conversion of the glucuronide and
sulfate esters of 5-hydroxythiabendazole was also
developed using P-glucuronidase and sulfatase,
respectively. Thus, quantitation of the separate
metabolites was possible. A special adaptation
of the chromatographic procedure was utilized for
the determination of 5-hydroxythiabendazole
metabolites in the sera of uremic patients, which
can contain large mounts of interfering fluorescent
substances. The method should be of particular
use for monitoring thiabendazole therapy in
THIABENDAZOLE
629
patients to eliminate the potentially toxic

metabolites.
HPIC methods for determining thiabendazole in
waste waters1 138114 and in textile productsll5-117
are also described.
6.10 Miscellaneous
A radioactive indicator method for the
determination of thiabendazole residues has been
described.118
7.
Limits of Tolerance
Tolerances in ppm for the residues of the
fungicide thiabendazole in or on various raw
agricultural commodities established under the
Federal Food, Drug, and Cosmetic Act fixed by the
United States Environmental Protection Agency are:
0.02 for sweet otatoes;llg 0.1 for dried beans,l20
eggs,121 meat; 151 and soyabeanso122 0.4 for banana
pulp119 and rnilki123 1 for hubbard squashr124
3 for bananas119 and rough rice1125 5 f o r
strawberriesol26 6 or su ar beetsol21 8 for rice
hullst127 10 for a les,l 8 carrots,l29 citrus
fruits 130 grapes,P93 pears 128 potatoesl31, rice
straw,$25 sugar beet tops135 and wheat grains; 131
15 for cantaloupes;126 and 40 for rnushro0m.13~
8.
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TIMOLOL MALEATE
DAVID J. MAZZO* AND ALICE E. LOPER#
MANAGER, PARENTERALS RESEARCH AND DEVELOPMENT, TRAVENOL LABORATORIES, MORTON GROVE,

ILLINO1 S
#RESEARCH FELLOW, PHARMACEUTICAL RESEARCH AND

DEVELOPMENT, MERCK SHARP AND DOHME RESEARCH
LABORATORIES, WEST POINT, PENNSYLVANIA
VOLUME 16
641
AU rights of reproduction in any form reserved.
DAVID J. MAZZO AND ALICE E. LOPER
642
1.
2.
Introduction
1.1
Therapeutic Category
1.2
History
Description
2.1
Chemical ..me, Formu a, Molecular

Weight
2.2
Definition
2.3
3.
Synthesis
4.
Physical Properties
4.1
Infrared Spectrum
4.2
Nuclear Magnetic Resonance Spectrum

4.2.1
Proton NMR
4.2.2
Carbon-13 NMR
4.3
4.4
Mass Spectrum
4.5
Optical Rotation
4.6
Electroanalytical Behavior
4.7
Thermoanalytical Behavior
4.7.1
Melting Point
4.7.2
Differential Thermal Analysis

Behavior
TIMOLOL MALEATE
643
Thermogravimetric Analysis
Behavior
4.7.3
4.8
Solubilities
4.9
Crystal Properties
4.10 Hygroscopicity
4.11 Dissociation Constant
5.
Methods of Analysis
5.1
5.1.1
5.1.2
Infrared Spectroscopy
5.1.3
Elemental Analysis
5.2
5.3
5.4
6.
5.3.1
Direct Ultraviolet Spectrophotometry
5.3.2
via Flow Injection Analysis
5.3.3
Specific Rotation
5.4.1
Thin-layer Chromatography
5.4.2

Chromatography
Stability
Degradation
6.1
Solid State Stability
6.2
Solution Stability
644
7.
Biopharmaceutics and Metabolism

7.1
Absorption and Bioavailability
7.2
Metabolism
7.3
Pharmacokinetics
8.
Determination in Biological Matrices
9.
Determination in Pharmaceuticals
10.
9.1
Dissolution Testing
9.2
Potency
9.2.1
Assay
9.2.2
Dosage Uniformity
9.2.3
Stability Testing
References
TIMOLOL MALEATE
1.
645
Introduction
1.1
Therapeutic Category (1)

Timolol maleate is a beta adrenergic
blocker which is non-selective between
beta and beta adrenergic receptors.
It dhes not ha6e significant intrinsic
sympathomimetic, direct myocardial
depressant or local anesthetic
(membrane-stabilizing)activity.
Timolol maleate is effective in
lowering intraocular pressure and is
widely used in patients with openangle glaucoma and aphakic glaucoma.
Timolol maleate is also indicated both
for the treatment of hypertension
(alone or in combination with other
anti-hypertensive agents, especially
thiazide-type diuretics) and to reduce
cardiovascular mortality and the risk
of reinfarction in patients who have
survived the acute phase of myocardial
infarction and who are clinically
stable. Timolol maleate, available for
oral dosing as tablets and for injection and ophthalmic dosing as distinct
sterile aqueous solutions, is usually
well tolerated with most adverse
effects being mild and transient.
However, a number of adverse effects
have been reported with other betaadrenergic blocking agents and should
be considered potential adverse effects
of timolol maleate. A number of adverse reactions considered to be
causally related to timolol maleate
have been reported. For example,
severe respiratory reactions and
cardiac reactions, including death due
to bronchospasm in patients with asthma
and rarely death in association with
cardiac failure, have been reported
(1)
646
1.2
History
Timolol maleate, belonging to the
thiadiazole class of compounds, was
first synthesized in the Merck-Frosst
Laboratories in Montreal, Canada. The
first non-patent literature reference
to timolol maleate appeared in 1972
(2). Timolol maleate, since its introduction in pharmaceutical formulations,
has gained a wide acceptance as an
anti-hypertensive and anti-glaucoma
agent. A search of Chemical Abstracts
(1966 - 1986) and Pharmaceutical
Abstracts (1974 - 1986) produced over
210 unique bibliographic citations for
works dealing with timolol maleate.
2.
Description
2.1
Chemical Name, Formula, Molecular

Weight
The accepted chemical name for timolol
maleate (MK-950) is: (S)-l-[(l,l-
dimethylethyl)-aminol-3-[[4-(4morpholinyl)-1,2,5-thiadiazol-3-
ylloxyl-2-propanol, (2)-butenedioate
(1:l) salt.
The CAS registry number
is 26921-17-5.
Other names which have been used for
timolol maleate include the maleate
salts of (s)-(-)-3-(3-tert-butyl-amino2-hydroxypropoxy)-4-morpholino-l,2,5-
thiazide, (-)-3-morpholino-4-(3-tert-
butylamino-2-hydroxypropoxy)-1,2,5-
thiadiazole and (-)-1-(tert-
butylamino)-3-[(4-morpholino-1,2,5thiadiazol-3-yl)oxy]-2-propanol.
TIMOLOL MALEATE
2.2
647
Definition
Timolol maleate possesses an asymmetric
carbon atom in its chemical structure
and is provided as the levoisomer. It
has tradenames of BLOCADRENs and
TIMOPTICs. Timolol, when referred to,
indicates the free base (CAS Registry
Number = 26839-75-8).
2.3

Timolol maleate is a white, odorless,
crystalline powder.
3.
Synthesis
Timolol maleate has been prepared through a
series of synthetic steps beginning with
D-mannitol and acetone (3). The synthetic
route is presented in Figure 1. The
starting material, I, is reacted with
acetone and the product (11) is washed with
benzene and crystallized from hexane. This
substituted mannitol (I11 is then oxidized
in tetrahydrofuran with lead tetraacetate to
the substituted glyceraldehyde (1111,
hydrogenated with t-butylamine ( I V ) and
treated with benzaldehyde to form a
substituted phenyloxazolidine (V). In a
separate reaction sequence,
C13H24N403S-C4H404 Molecular Weight 432.49 g/mole
H3C..c/0-?H2
HO -CHe
I
HO-C-H
I
HO-C-H
I
H-C-OH
(CH3)zCO
H3C'
'0-C-H I
Pb(0Ach
HO-C-H
I
H-C-OH
I
H-C-0,
H-C-OH
I
CHDH
I
HeC-O/
[""' /o-i:6]
H,C/~'O-CH
(CH3)aCNH2
HOCHzCHCHzNHC(CH3)3
I
OH
6/
rn
Ip
N,
o&N\C(CH3)3
/CH3
'CH3
II
CHO
,N
Ix
Figure 1. Synthetic route to timolol maleate.
649
TIMOLOL MALEATE
aminoacetonitrile hydrochloride (VI) is

reacted in the presence of chlorine with
sulfur monochloride to form a chlorinated
thiadiazole (VII), which, in turn, is
reacted with morpholine to form VIII.
Compounds VI and VIII are reacted to timolol
free base (1x1 which is then converted to
timolol maleate by refluxing with maleic
acid in acetone ( X ) .
4.
Physical Properties
4.1
Infrared Spectrum
The infrared spectrum of timolol
maleate taken in a KBr pellet is shown
in Figure 2 (4). A Digilab Model
FTS-15C Fourier transform infrared
spectrophotometer was used to acquire
the spectrum. Frequency assignments
for some of the characteristic bands
are listed in Table I.
Table I
Infrared Spectral Assignments for
Timolol Maleate
Frequency ( cml)
3250
3000
1700
1200
1100
Assiqnment
0-H
N-H
C=N
c-o(c-OH)
c-0-c
stretch
stretch
stretch
stretch
stretch
The infrared spectrum of timolol maleate taken in

a mineral oil mull is shown in Figure 3 (4).
Both of these spectra are consistent with the
structure of timolol maleate.
4000 3800
3600 3400
3200
3000
2800 2600
2400
2200
2000 1800
1600
V
l
1400
1200
Wavenumbers
Figure 2.
1000 900800 700 600500
Infrared spectrum of timolol maleate taken in a KBr pellet.
;
1
3700
3500
3300
3100
2900
2700
2500
2300
2100
1900
1700
1500
1300
1100
900 800700600
Wavenumbers
Figure 3.
Infrared spectrum of timolol maleate taken in a mineral oil mull.

Mineral oil contributes to the spectrum at ca. 1350-1450, 15001750 and 2875-3000 wavenumbers.
652
4.2
Nuclear Magnetic Resonance Spectrum

4.2.1
Proton NMR Spectrum

The proton magnetic resonance
spectrum of timolol maleate was
obtained using a Nicolet NT 360
spectrometer. The spectrum was
acquired from an approximately
0.02 M solution using d
DMSO
as the solvent. The spgctrum is
shown in Figure 4 and the spectral assignments are listed in
Table I1 ( 4 , s ) .
A
20
ill
LL
10
"
'
"
'
Figure 4.
Proton NMR spectrum of timolol maleate.
654
Table I1
Re lat ive
No. Protons
Assignment
d - DMSO
sglvent effect
- CH- Cg2 -N
2
2*>
3.
3.35
3.47
CH - C H h
/ -2
0
-N
CH2 - CHf
3.72
-N
4.2
residual H20/HOD
,CHpE&
- Cgf
CH
- CHI
4.4
0-C12-
5.97
-0-g
6.03
HC=Cg
8.38
I I
H
-
I +
-N-C(CH3)3
H
20.1
-C=C-COOH
I -
c02
TIMOLOL MALEATE
4.2.2
655
CI3 NMR Spectrum

The CI3 NMR spectrum shown in
Figure 5 was obtained on a
Varian CFT-20 spectrometer using
a 0.5 M timolol maleate fglution
in d - DMSO (5). The C
spectral assignments are listed
in Table 111.
Table I11
CI3 NMR Spectral Assignments for
Timolol Maleate
6
(ppm)
Assignment
167.4
C02H/C02-
153.3
c3
149.8
c4
I
I
HC=CH
135.9
72.0
-0cH2-(Ca)
65.6
O-(CH2-l2 ( a )
65.0
-CHOH-(C@)
-
56.5
-NH-C-
43.7
-NHCH2-(Cy)
24.9
C(CH313
180 170 160 150 145 130 120 110 100 90
80
70
60
50
40
30
20
10
(PPm)
Figure 5.
CI3 NMR spectrum of timolol maleate.
TIMOLOL MALEATE
657
1
a)
The C
and C
carbons are equivalent;
theregore, onfy a single line is
observed for these two carbons.
b)
The C
and C
carbons are equivalent;
there$ore, on?y a single line is
observed for these two carbons.
Both the proton and CI3 NMR spectra are
consistent with the timolol maleate
structure.
4.3
The ultraviolet absorption spectrum of
timolol maleate in 0.1 N aqueous
hydrochloric acid is characterized by
maxima at approximately 210 nm and 294
nm.
The absortivity in absorbance units

drug solution in a 1 cm
is 200 at 294 nm. The W
in Figure 6 was obtained
using a Hewlett-Packard Model HP8451A
diode array spectrophotometer.
4.4
Mass Spectrum
A mass spectrum of timolol is shown in
Figure 7. This spectrum was obtained

using an LKB model 9000 mass spectrometer operated at 70 eV in the direct
probe-electron impact mode. The fragmentation pattern observed is consistent with the chemical structure of
timolol.
The molecular ion is noted at m/e =
316. A strong M-minus-methyl peak is
observed at m/e = 301. The loss of
CIHION leads to m/e = 244. Loss of
C H produces a fragment at m/e = 259
w h e h lacks the intensity to be seen in
a high resolution spectrum. The loss
of C H N(H)-CH gives rise to m/e = 230
with4t8e peak it m/e = 86 representing
this lost group. Principle fragmentations of the morpholine ring are seen
658
I
100
80
60
Relative
Intensity
40
130
57
20
144 154
50
100
150
187 188
I
200
229 230
l
A 250
286
316
300
(mle)
Figure 7.
Low resolution mass spectrum of timolol maleate taken

in the electron impact mode.
at m/e = 286 and m/e = 272. A

MacClafferty rearrangement leads to m/e
= 187 and two ions at m/e = 130 (see
Figure 8). Fragmentation of the heterocyclic nucleus produces ions at m/e =
144 and possible M-144 at m/e = 172.
The peak at m/e = 243 is due to the
loss of C H 0 from the molecular ion.
This fraAe8t also appears as a characteristic doubly charged ion at m/e =
121.5 (due to the same decomposition).
The origin of this peak is apparent
from the metastable peak at m/e = 196
which corresponds to the transition
from m/e = 301 to m/e = 243.
4.5
Optical Rotation
Timolol maleate, having one chiral
carbon, is optically active. The
levorotatory enantiomer is the
biologically active form. The optical
rotation of a 5% (w/v) solution of
timolol maleate in i50 N aqueous HCt5at
25 "C and 405 nm
is -12.2" ( A D =
-4.2 " ) (6).
f7
U
N
0
L
mle 187,0407
(187.0415)
(1 30.1232)
(major)
-(CH,-0-CH-CH,)
154.0616
(154.0625)
CH, = N
@
N\s,
f/
NH
mle 130.0057
(130,0075)
(minor)
Figure 8.
CH,=CH-CH
-H,O
@ CH3
= NKC-CH,
CH,
mle112
Schematic representation of the mass spectral

fragmentation of timolol maleate.
662
4.6
Electroanalytical Behavior
Timolol maleate is electrochemically
active and it undergoes a single
irreversible electro-oxidation. The
E
of an approximately 40 vg/mL
skliition of timolol maleate in 0.01 M
aqueous KC1 is +730 mV (vs Ag/AgCl).
Figure 9 shows the voltammogram of this
solution obtained using a Princeton
Applied Research Model 174A
Polarographic Analyzer with a glassy
carbon working electrode, a
silver/silver chloride reference
electrode and a platinum auxillary
electrode.
4.7
Thermoanalytical Behavior
4.7.1
Melting Point
The melting point of timolol
maleate varies between 201.5 'C
and 2 0 2 . 5 'C as determined by
the USP class Ia capillary
method (6).
4.7.2
Differential Thermal Analysis

Behavior
The differential thermal
analysis (DSC) behavior of
timolol maleate under nitrogen
is shown in Figure 10. This
curve was obtained using a
Perkin-Elmer Series 7 DSC
scanning from 40 "C to 2 5 0 "C at
1 0 "C/minute. A primary
endotherm corresponding to
melting is observed with an
actual peak temperature of 2 0 5 . 8
"C. An additional endotherm
with a peak temperature of ca.
2 1 5 'C is noted corresponding to
compound decomposition.
Figure 9.
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1.2
Potential (Volts vs. AgIAgCI)

Scanning voltammogram of timolol maleate in 0.1 M K C 1 (aq).
100.0
90.0 80.0 -
=3
U
70.0 60.0 -
50.0 -
40.0 30.0 -
20.0 10.0
0.0
-p
'
50.0
75.0
100.0
125.0
150.0
175.0
200.0
225.0
Temperature ("C)
Figure 10.
Differential scanning calorimetry behavior of timolol maleate.
TIMOLOL MALEATE
665
Thermogravimetric Analysis
Behavior
4.7.3
Thermogravimetric analysis of
timolol maleate indicates no
weight loss until 205.8 "C.
From 2 0 5 . 8 " C to ca. 3 5 0 " C
there is an approximately 80%
weight loss due to decomposition/vaporization. A typical
TGA curve for timolol maleate is
shown in Figure 11. The curve
was obtained using a PerkinElmer Series 7 TGA scanning from
30 "C to 4 0 0 "C at 1 0 "C/minute.
A sample of 3.523 mg of timolol
maleate was used.
4.8
Solubilities
The solubilities of timolol maleate in
a variety of solvents at room
temperature ( ~ 2 5"C) is presented in
Table IV (6). Note that these
solubilities are stated in terms of the
current U.S.P. definitions ( 7 ) .
Table IV
Solubility of Timolol Maleate at
Room Temperature
Solvent
Water
Methanol
Ethanol
Chloroform
Propylene Glycol
Ether
Cyclohexane
Isooctane
Solubility
Soluble
Soluble
Soluble
Sparingly Soluble
Sparingly Soluble
Practically Insoluble
8m
8
s:
cu
8rr,
8
cv
8
?
9
m
TIMOLOL MALEATE
4.9
667
crystal Properties ( 8 )
Timolol maleate exists as a crystalline
powder. No polymorphs of timolol
maleate have been reported. Figure 1 2
shows the x-ray powder diffraction
pattern of timolol maleate obtained
with a Phillips Electronics model
APD3720 powder diffractometer using
CuKa radiation. Table V lists the
interplanar distances and the relative
intensities of the major lines in the
x-ray powder diffraction pattern of
timolol maleate.
Table V
X-Ray Powder Diffraction of Timolol Maleate
Peak Angle ( 2 0 " )
7.1
9.9
11.4
14.2
14.8
15.6
16.3
18.1
19.0
19.5
19.8
20.5
20.6
20.9
21.4
22.4
23.0
23.8
24.8
26.0
26.5
27.1
28.3
29.1
29.5
31.6
34.0
38.8
39.3
D Spacinq
12.49
8.95
7.76
6.23
5.96
5.68
5.44
4.90
4.67
4.56
4.47
4.33
4.31
4.24
4.14
3.97
3.86
3.74
3.58
3.42
3.36
3.29
3.16
3.07
3.03
2.82
2.63
2.32
2.29
(I/Imax)(%)
13
13
6
100
27
12
30
43
62
24
18
52
54
67
75
35
9
50
45
7
16
60
32
13
5
26
12
19
14
TIMOLOL MALEATE
669
4.10 Hygroscopicity (9)

The maleate salt of timolol is a stable
anhydrous compound. No hydrates of
timolol maleate have been reported.
The free base of timolol, however,
exists both as a stable crystalline
hemihydrate and anhydrous compound.
Anhydrous timolol maleate is nonhygroscopic when stored under ambient
temperature conditions (-25 " C ) and
relative humidity conditions ranging
from 3 3 % to 76%.
4.11 Dissociation Constant ( 9 )
The pKa of timolol base obtained by
potentiometric titration in water at
The native
25 " C is approximately 9.2.
pH of a saturated aqueous solution of
timolol maleate is 2 4.
5.
Methods of Analysis
5.1
5.1.1
(10)
Ultraviolet spectrophotometry is
used to identify timolol
maleate. A solution in 0.1 N
HC1 (aq) scanned from 200 nm to
400 nm qualitatively exhibits
the same absorbance characteristics at identical wavelengths
as does a similarly prepared and
concomitantly measured solution
of a timolol maleate standard.
Quantitatively, equimolar sample
and standard solutions will
exhibit absorbances at 2 294 nm
(Xmax) whi Q differ by no more
than 3% (AX&= 2 0 0 ) .
5.1.2
Infrared Spectroscopy
Infrared spectroscopy may also
670
be used to identify timolol

maleate. The infrared spectrum
prepared as a mineral oil
dispersion compares
qualitatively (with maxima only
at the same frequencies) to the
spectrum of a similarly prepared
timolol maleate standard.
5.1.3
Elemental Analysis
Timolol maleate may be
identified through the use of
elemental analysis. The results
of an elemental weight percent
determination of carbon,
nitrogen, sulfur, hydrogen and
oxygen are compared to the
respective theoretical values of
49.35%, 17.71%, 10.15%, 7.65%
and 15.17%.
5.2
Titrimetric Analysis (10)

Timolol maleate may be determined by
perchloric acid titration to a visual
or potentiometric endpoint. An
accurately weighed sample ( = 800 mg) is
dissolved in = 90 mL glacial acetic
acid and titrated with 0.1 N perchloric
acid in dioxane to a green endpoint.
Alternatively, the endpoint may be
determined potentiometrically as that
point at which the greatest change in
potential per unit volume change of
titrant is noted. For potentiometric
titrations, the electrode system should
consist of an indicating electrode
prepared by emptying a sleeve type
calomel electrode, drying it and
filling with 0.1 N LiClO in acetic
anhydride. A platinum rfng reference
electrode is to be used.
5.3
5.3.1
Direct Ultraviolet
Spectrophotometry
TIMOLOL MALEATE
67 1
Timolol maleate exhibits an

ultraviolet absorption band near
2 9 4 nm attributed to the
thiadiazole core structure.
This absorption is the basis for
the quantitative determination
of the drug. Assay of the
compound is performed by
comparing the net absorbance at
2 9 4 nm of a sample dissolved in
0.1 N HC1 (as) with the net
absorbance of a standard in 0.1
N HC1 (as) of known concentration. The net absorbance is
calculated by subtracting the
drug free matrix contribution to
absorbance at the wavelength of
determination from the absorbance of the drug solution at
the same wavelength.
where :
= Concentration of the sample
solution
= Concentration of the standard
std
solution
= Absorbance at 2 9 4 nm of the
Asam
sample solution
= Absbrbance at 2 9 4 nm of the
standard solution
=
Absorbance
at 2 9 4 nm of the
Ab
drug free solution matrix
5.3.2
via Flow Injection Analysis
Ultraviolet absorbance is also
used as the detection mode for
timolol maleate determinations
by flow injection analysis (11).
In the case of the flow
injection technique assay,
timolol maleate sample and
672
standard solutions are

periodically injected into a
flowing stream. The resulting
changes in UV absorbance at 294
nm of the stream are measured
relative to the analyte-free
stream. Calculation of the
concentration of timolol is made
in an identical fashion as
direct UV spectrophotometry.
5.3.3
Specific Rotation
The specific rotation of timolol
maleate is determined in 0.1 N
HC1 (aq). Using a solution of =
50 mg/mL, the angle of rotation
at 405 nm, 25 "C, in a 10 cm
cell is determined with a
suitable photoelectric polarimeter. Specific rotation,
calculated on the dried basis,
is between -11.7" and -12.5'.
5.4
5.4.1
Thin Layer Chromatography (10)

Normal-phase thin layer
chromatography on silica gel has
been employed for timolol
maleate. A developing solvent
system consisting of
chloroform/methanol/(28% ammonia
water) [80/20/1 (v/v)l is used
to develop a spot resulting from
10 pL of an approximately 5
mg/mL solution of the drug in
methanol. A reference spot for
impurity purposes applied as 10
pL of = 20 pg/mL solution of
timolol maleate in methanol is
also employed. R for timolol
maleate in this &stem is = 0.7.
Detection is made under visible
light after the developed and
air-dried plate has been exposed
613
TIMOLOL MALEATE
to iodine vapor for 2 hours.

Sensitivity is estimated to be
within an order of magnitude of
the amount of timolol maleate in
the reference spot solution ( =
200 ng drug).
5.4.2

Chromatography (12)
High performance liquid
chromatography may be employed
to analyze timolol maleate. A
chromatographic system
consisting of a column (300 mm x
4.6 mm i.d.) packing with
p-phenyl stationary phase (10 pm
particle size) has been used
with a mobile phase consisting
of 1 part methanol and 2 parts
0.005 M aqueous hexane sulfonic
acid (pH adjusted to 1 with
acetic acid). A column
temperature of 30 'C and a flow
rate of 2.00 mL/min are used.
Detection is by UV absorbance at
280 nm. Typically, 20 pL
injections of a timolol maleate
solution are made. Under these
conditions, timolol is separated
from its known potential process
impurities.
6.
Stability
6.1
- Degradation
Solid State Stability ( 1 3 )

Timolol maleate is an extremely stable
compound at room temperature in the
solid state. In fact, prolonged
exposure to extremes of temperature and
humidity caused minimal degradation of
the solid compound. For example,
timolol maleate stored for = 2 months
at 105 O C in an air atmosphere (ambient
relative humidity) showed no difference
in potency by TLC chromatographic
614
profile when compared to a sample of

unstressed solid. Only heating timolol
maleate to above its melting point and
keeping it molten for 10 minutes
produced discoloration and an
approximate 5% loss in potency. (TLC of
this sample did detect small quantities
of several unknown decomposition
products.) Timolol maleate, when
heated at 95 O C for = 3 weeks in an air
atmosphere (100% relative humidity),
also exhibited a loss in potency of 25%
(by W assay). Two degradates were
isolated from this sample and were
identified as the cis/trans isomer pair
of timolol 0-fumarate ester and timolol
0-maleate ester (Figure 13).
Timolol maleate exhibited surface
discoloration when exposed in an open
dish to intense UV light ( z 20 times
normal sunlight). This sample did not,
however, show any loss of potency by UV
assay nor were any decomposition
products detected by TLC.
6.2
Solution Stability ( 1 3 )
Timolol maleate is extremely stable in
aqueous solutions (protected from
light) with no decomposition being
detected in injectable formulations
stored at room temperature for = 5
years. Timolol maleate can be degraded
in solution if exposed to elevated
temperatures or intense ultraviolet
radiation. The solution decomposition
of the drug is pH dependent with
maximum stability occurring near pH =
4. Solutions of timolol maleate
prepared in each of 0.1 N HC1 (aq) (pH
= 1) and phosphate buffer (pH = 7 . 2 )
and then stored in sealed glass vials
for = 2 months at 105 OC yielded
considerable amounts of degradation
products. A solution prepared in
distilled water and stored identically
OCH,CHCH,NHb-CH,
I
c=o
CH3
HC = CHCOOH
cis and trans

Figure 13.
Potential solid state degradates of timolol maleate

exposed to 105 " C / l O O % relative humidity.
676
was found to be virtually the same as

unstressed control solution. Studies
of timolol aqueous solutions (pH - 5 )
under autoclave conditions indicated
that timolol maleate degrades at the
rate of approximately l%/hour at 120
OC.
Three decomposition products were
isolated from solutions which were
autoclave degraded. Figure 14 depicts
the proposed degradation mechanism and
indicates the three modes of thermally
induced solution degradation; (1)
rearrangement to isotimolol; (2) ether
cleavage to form 4-hydroxy-3morpholino-1,2,5-thiadiazole; and (3)
oxidation followed by ether cleavage to
form 4-hydroxy-3-morpholino-l,2,5thiadiazole 1-oxide.
Aqueous solutions of timolol maleate
(pH 5 ) have also been shown to be
susceptible to degradation when exposed
to intense ultraviolet radiation.
Exposure for 2 hours to U V light
equivalent to = 20 times normal
sunlight produced approximately 2% loss
in potency. Evidence of the three
thermal degradation products mentioned
above as well as several unidentified
degradation products was found in this
solution. Because of the potential for
light-induced degradation, it is
recommended that aqueous timolol
maleate solutions be stored protected
from light.
7.
Biopharmaceutics and Metabolism
7.1
Absorption and Bioavailability

Timolol maleate is rapidly and
completely absorbed after oral
administration (14). Maximum blood
plasma concentrations ranging from 10
ng/mL to 100 ng/mL are attained within
1 to 2.4 hours after either acute or
OH
CH,
I
"\_/",
n IIOCH,CHCH,NHC-CH,
I
I
N\
CH,
Timolol
CH,OH
o=N'rrr(opH
Y SY
lsotimolol
FH3
CH~NHC-CH,
oWNllfoH
N
N
'
'S
CH,
'OH
0
4-hydroxy-3-morpholino1,2,5-thiadiazole l-oxide
4-hydroxy-3-morpholino1,2,5-thiadiazole
Figure 14.
Proposed aqueous solution degradation mechanism

for timolol maleate in solutions exposed to
autoclave conditions.
678
chronic administration of 2.5 mg to 20

mg of timolol maleate twice daily
(15-16). The bioavailability of oral
timolol is reported to be 61% to 75% of
a reference intravenous dose (17-18).
Bioavailability of less than 100% is
attributed to first-pass metabolic
extraction by the liver after oral
administration rather than to
incomplete gastrointestinal absorption
(14). The effect of food on the rate
and extent of oral absorption of
timolol maleate is not significant
(19)
Timolol maleate administered directly

to the eye as an ophthalmic drop
appears to be rapidly absorbed,
producing a decrease in intraocular
pressure within three hours (20).
Systemic absorption of timolol also
occurs after ocular administration,
producing peak blood plasma levels of
no more than 2 to 5 ng/mL at 0.5 hours
to 1.5 hours after instillation of 2
drops of 0.5% timolol maleate
ophthalmic solution in each eye (21).
Some small but statistically
significant cardiovascular effects have
been reported after ophthalmic
administration (20,221. Supporting
studies in rabbits indicate that
timolol reaches peak levels in most
tissues of the eye at ten minutes after
ocular administration with blood serum
concentrations peaking at 30 to 60
minutes (23).
Timolol free base is rapidly absorbed
through intact skin (24).
7.2
Metabolism
After orf4 administration of a 4 mg
C- timolol maleate to man,
dose of
72% of the I4C label is excreted within
84 hours with 66% in the urine and 6%
in the feces. Only 20% of the timolol
TIMOLOL MALEATE
679
dose is recovered unchanged from the

urine. Over 80% of the blood plasma
radioactivity represents timolol
metabolites (14).
Oxidation and hydrolytic cleavage of
!be morpholine ring of an oral dose of
C- timolol maleate produces two major
urinary metabolites in man (25).
HO-CH2- CH
HOOC-CH<
S/
0-CH -CH-CH2-NH-C(CH3)3
bH
Metabolite I
N-{{4-[3-(l,l-dimethylethyl)aminol-2-hydroxypropoxy}-1,2,5- thiadiazol-3-yl}-N-(2-hydroxyethyl)
glycine.
H O - C H ~ - C H ~ - N H - ~ O--C HC -HC H ~ - N H - C ( C H ~ ) ~
N
N
bH
S
Metabolite I1
l-(l,l-dimethylethylamino)-3-{[4-(2-hydroxyethylamino)-1,2,5-thiadiazol-3-ylloxy}-2-propanol.
Metabolite I represents approximately

30% of the total urinary radioactivity
while Metabolite I1 accounts for 10%
(14,251.
Oxidation of the basic oxypropanolamine

side chain of orally administered
timolol maleate results in two other
minor urinary metabolites in man.
Metabolites I11 and IV represent 6% and
3 % , respectively, of administered
radioactivity (26).
680
..
N\s/
OH
Metabolite I11
2-hydroxy-3-[{4-(4-morpholinyl)-l,2,5-thiadiazol-3-yl}oxy]propanoic acid
H-CH2NH-C-CH2-OH
O w N -
\ /
CH3
Metabolite IV
l-~[(1,1-dimethyl-2-hydroxy)-ethyllamino}-3-{[4(4-morpholinyl)-1,2,5-thiadiazol-3-yl]oxy}-2propanol.
Biological testing of synthetic samples

of Metabolites I, I1 and I11 shows only
Metabolite I1 to have significant
6-adrenergic blocking activity.
However, activity was only one-seventh
that of timolol maleate in anesthetized
dogs (26).
These four metabolites are also
produced by other species. Metabolite
I11 represents the major metabolic
pathway for timolol maleate in the dog.
Other metabolites in rodents result
from hydroxylation and oxidation of
either the morpholino ring or the
oxypropanolamine side chain (25).
7.3
Pharmacokinetics
Oral administration of timolol maleate
in doses ranging from 5 mg to 20 mg
TIMOLOL MALEATE
681
results in peak plasma concentrations

of timolol maleate within one to two
hours, followed by an exponential
decline of plasma concentrations with
time ( 1 5 ) . The mean apparent half-life
of elimination of unmetabolized drug
from plasma is approximately 2 . 5 hours
( 1 5 , 1 7 1 , although plasma half-lives as
long as five hours have been reported
in normal volunteers ( 2 7 ) . Half-life
is independent of the administered
dose. Timolol maleate appears to
follow simple linear kinetics over the
dosage range studied; area under the
plasma concentration versus time curve
is proportional to the administered
dose ( 1 5 ) . The mean renal clearance of
unchanged timolol maleate in normal
volunteers was reported as 6 9 . 6
mL/minute ( 2 7 ) . Pharmacokinetic
parameters do not change upon one week
chronic administration of oral timolol
maleate ( 1 5 ) .
Following intravenous administration,
plasma levels decrease biexponentially
with time ( 2 8 ) . Reported half-lives
for timolol maleate elimination from
plasma, 3.3 to 4 . 1 hours, are comparable to the oral half-life.
The pharmacodynamics of oral timolol
maleate have been well characterized
(15,161.
Maximal suppression of
exercise-induced tachycardia, a measure
of @-blockade, occurs when timolol
maleate plasma levels reach 27 to 30
ng/mL. Fifty percent of maximum
inhibition of exercise-induced
tachycardia is produced by timolol
maleate plasma concentrations of 3 to 4
ng/mL.
8.
Determination in Biological Matrices

Timolol maleate may be determined in blood
plasma and urine by isolation and
682
derivatization followed by gas-liquid

chromatography with electron capture
detection (29). Timolol and the internal
standard, desmethyltimolol, are extracted
from either 1.0 mL of alkalinized plasma or
0.1 mL of alkalinized urine into 4% isoamyl
alcohol in heptane. The compounds are then
back-extracted into 0.1 N hydrochloric acid,
the acidic phase alkalinized, and the
compounds extracted into methylene chloride.
After evaporation of the methylene chloride,
timolol maleate and the internal standard
are derivatized by addition of 0.1 mL of
heptaflurobutyrylimidazole-ethyl acetate
reagent and immersion in a boiling water
bath for one hour. The diheptafluorobutyryl
derivatives are recovered in n-heptane,
which is evaporated at 60 "C under a stream
of nitrogen to concentrate the sample.
Typically, 1 to 5 p 1 of sample is
chromatographed on a 1.83 m X 0.64 cm column
packed with 1% OV-17 on 80-100 mesh Gas
Chrom Q. The chromatograph is operated
isothermally with injection port, oven and
detector temperatures of 250 OC, 185 "C and
300 "C, respectively. Helium, the carrier
gas, and 10% methane in argon, the purge
gas,63re maintained at 75 mL per minute.
The
Ni electron-capture detector was
operated in the pulse mode of voltage with a
pulse interval of 5 0 microseconds.
Retention times of derivatized timolol
maleate and internal standard are 7.6 and
6.0 minutes, respectively. Limits of
detection are 2 ng/mL in plasma and 20 ng/mL
in urine.
A capillary column gas-liquid
chromatographic-mass spectrometric assay

monitoring for timolol
with
maleate and
C-timolol maleate in blood
plasma is reported to have a sensitivity of
0.5 ng/mL (30). This method provides the
capability to examine administration of drug
and its stable isotope-labeled counterpart
simultaneously for comparison of two
different routes of administration. The
selectefiion
TIMOLOL MALEATE
683
isolation procedure prior to assay consists

of passing 9.5 to 1.5 mL of plasma,
containing H-timolol maleate as an internal
standard, through a cartridge containing an
octadecyl-silica bonded reversed-phase
packing. The cartridge is flushed and the
compounds are then eluted with acetonitrile0.05 N hydrochloric acid, which is subsequently alkalinized. Back extraction from
cyclohexane-toluene into an aqueous phase
followed by extraction into methylene
chloride precedes evaporation and derivatization with bis(trimethylsily1)-trifluoroacetamidepyridine. One to two ~ J Lare
injected onto a 16 m X 0.25 mm capillary
column coated with SE-30. The splitless
injection port temperature is 260 "C and
oven temperature is 225 " C . Carrier gas
(helium) velocity is 30 cm/second.
The mass spectrometer is operated in the
electron-impact ionization mode at an
ionizing potential of 70 eV, a filament
emission current of 0.8 mamp and electron
multiplier setting of 1900 V. Selected ion
monitoring at m/e 86, 89 and 95, base peaks
corresponqing to the side chains f f timolol
maleate,
C-timolol maleate and H-timolol
maleate, respectively, allows construction
of a standard curve from the ratios
Intensity86/Intensity95 and Intensitygg/
Intensityg5.
~
Other gas-liquid chromatographic methods

employing mass fragmentographic detection
(31) or nitrogen-selective flame ionization
detection (18) have been reported.
High performance liquid chromatography with
electrochemical detection may also be
employed for analysis of timolol maleate in
human blood plasma and breast milk (32). A
25 cm X 4.6 m i.d. column packed with PXS
5/25 Partisil ODS 3 (5 pm) is used with a
mobile phase of methanol :0.2 M sodium
dihydrogen phosphate :88% orthophosphoric
acid :water (500:200:3:297) pumped at a flow
684
rate of 1.0 mL per minute. The applied

potential for oxidative electrochemical
detection is +1.2 V for maximum sensitivity.
A glassy carbon working electrode is
employed with a silver/silver chloride
reference electrode and a platinum auxillary
electrode. Timolol maleate and the internal
standard, propranolol for plasma or serum,
pindolol for breast milk, are extracted from
biological matrices with diethyl ether.
Compounds are extracted from the organic
phase into dilute orthophosphoric acid. For
plasma or serum, a hexane wash of the
organic phase is sufficient to prepare the
acidic aqueous phase for injection of 100 pL
into the chromatographic column. For breast
milk, the acidic aqueous phase must be
alkalinized and the compounds back-extracted
into ether. Timolol maleate and internal
standard are once again extracted into
dilute orthophosphoric acid and the hexane
wash and assay are performed as for plasma.
Calibration curves are linear from the 2
ng/mL limit of detection up to 100 ng/mL.
9.
Determination in Pharmaceuticals
9.1
Dissolution Testing
The dissolution of timolol maleate
tablets is evaluated based on
methodology and apparatus described in
USP XXI(33). The acidic dissolution
medium ( 5 0 0 mL of 0.1 N hydrochloric
acid) is maintained at 37 5 0.5 "C and
basket speed is 100 rpm. After introduction of the tablet, filtered
aliquots of the dissolution medium are
withdrawn for assay for timolol maleate
content against appropriate standards.
The official assay is a high performance liquid chromatographic method
developed for a system with an ultraviolet detector. The wavelength for
detection is 295 nm. A mobile phase
consisting of pH 2.8 phosphate
TIMOLOL MALEATE
685
buffer: methanol (3:2) is pumped at a

rate of 1.8 mL/minute through a 30 cm X
3.9 mm column packed with octadecylbonded silica. About 15 p 1 of
dissolution sample is injected onto the
column. Not less than 80% of the
labeled amount of timolol maleate is
dissolved in 20 minutes. Alternative
assay methods, as outlined in the
previous sections (direct ultraviolet
spectrophotometry, ultraviolet
spectrophotometry via flow injection
analysis and high performance liquid
chromatography), can be used if an
official cornpendial method is not
required.
9.2
Potency Testing
9.2.1
Assay (33)
Official compendial methods are
available for both timolol
maleate tablets and ophthalmic
solution. Tablets are prepared
for assay by weighing and finely
powdering not less than twenty
tablets. An accurately weighed
portion of powder is dispersed
in 0.05 M monobasic sodium
phosphate and sonicated for five
minutes. Acetonitrile is added
followed by an additional five
minutes of sonication. Water is
then added and the dispersion is
shaken for ten minutes. After
dilution to volume with water,
the sample may be centrifuged to
remove dispersed solids from the
timolol maleate solution. The
supernatant liquid may be
assayed with appropriate
standards with the chromatographic system outlined above
(Dissolution, Section 9.1) or by
an alternative assay if an
official cornpendial method is
686
not required. Tablets must

contain not less than 90.0
percent and not more than 110.0
percent of the labeled amount of
timolol maleate.
The official compendia1 assay
for timolol maleate opthalmic
solution requires that an
aliquot of the solution be
diluted to volume with water to
produce a 0.5 mg per mL timolol
maleate solution. Five mL each
of this solution and reference
standard solutions are transferred to separators and made
basic with pH 9.7 carbonate
buffer. Timolol is extracted
into toluene from the alkaline
aqueous phase. The aqueous
phase in each separator is then
transferred to a second set of
separators for an additional
toluene extraction. The aqueous
phase in the second set of
separators is then discarded.
Additional pH 9.7 buffer is
added to the toluene remaining
in the first set of separators
and, after extraction, the
aqueous phase is transferred to
the second set of separators.
After shaking, the aqueous phase
is discarded and the toluene
from both sets of separators is
combined. Timolol in the
combined toluene solutions is
extracted into four portions of
0.1 N sulfuric acid. The
sulfuric acid extracts are
diluted to volume with
additional acid solution. The
extracts are assayed against a
0.1 N sulfuric acid blank at 2 9 4
nm with an ultraviolet
spectrophotometer. The timolol
maleate ophthalmic solution must
TIMOLOL MALEATE
687
contain not less than 90.0

percent nor more than 110.0
percent of label claim.
9.2.2
Dosage Uniformity ( 3 3 )
Tablets must also meet official
compendia1 standards for
uniformity of dosage units. Ten
tablets, assayed individually
must exhibit 85.0 to 115.0
percent of the label claim and
have a relative standard
deviation less than or equal to
6.0 percent. If one unit is
outside the label claim range or
the relative standard deviation
is greater than 6.0 percent, or
both, an additional twenty
tablets must be tested. Of the
thirty tested, not more than one
may be outside the 85.0 to 115.0
percent range, and that must be
within the 7 5 . 0 to 125.0 percent
of label claim range ( 3 4 ) .
Direct ultraviolet spectrophotometry, ultraviolet
spectrophotometry via flow
injection analysis and reversedphase high performance liquid
chromatography with ultraviolet
detection, all as previously
described, are acceptable
techniques for uniformity
determinations. Typically
sample preparation involves the
disintegration and dissolution
of single tablets in solvents
identical to those used for
assay. A set of extractions and
other sample preparation steps
identical to those described in
the potency assay ( 9 . 2 . 1 ) are
then employed.
DAVID J. MAZZO AND ALICE E.LOPER
688
9.2.3
Stability Testing ( 3 3 )
Timolol maleate is tested for
stability using the high
performance liquid chromatographic method described in the
USP XXI. Based upon data
generated in accelerated and
room temperature stability
studies, timolol maleate tablets
carry a five year expiration
dating.
TIMOLOL MALEATE
689
References
Physicians Desk Reference, 41st edition,

Edward R. Barnhart, Publisher, Medical
Economics Company, Inc., Oradell, NJ,
U.S.A., 1987, pp. 1249-1251, 1340-1342.
B. K. Wasson, W. K. Gibson, R. S. Stuart, H.
W. R. Williams, C. H. Yates, J. Med. Chem
15(6), 651-655 (1972).
L. M. Weinstock, United States of America
Patents 3619370, 3655663 and 3657237 (to
Merck and Co., Rahway, NJ, U.S.A.), 1971,
1972 and 1972, respectively.
4)
J. A. Ryan, Department of Pharmaceutical

Research and Development, Merck Sharp and
Dohme Research Laboratories, West Point, PA,
U.S.A., personal communication.
S. M. Pitzenberger and J. S. Murphy,
Department of Medicinal Chemistry, Merck
Sharp and Dohme Research Laboratories, West
Point, PA, U.S.A., personal communication.
6)
The Merck Index, 10th edition, Martha

Windholtz, editor, Merck and Company, Inc.,
Rahway, NJ, U.S.A., 1983, monograph no.
9284, pg. 1353.
The United States Pharmacopeia, 21st
revision, Mack Publishing Company, Easton,
PA, U.S.A., 1985, pg. 7.
J. McCauley, Department of Analytical
Research, Merck Sharp and Dohme Research
Laboratories, Rahway, NJ, U.S.A., personal
communication.
9)
E. Oberholtzer, and Joseph V. Bondi;

Department of Pharmaceutical Research and
Development, Merck Sharp and Dohme Research
Laboratories, West Point, PA, U.S.A.,
690
10 1

PA, U.S.A., 1985, pp. 1063-1064.
111
F. P. Bigley, R. L. Grob, and G. S. Brenner,

Anal. Chim. Acta 181, 241-244 (1986).
12 1
P. Tway, Department of Analytical Research,

Merck Sharp and Dohme Research Laboratories,
West Point, PA, U.S.A., personal
communication.
13
R. Roman, W. Hagerman, Department of

Pharmaceutical Research and Development,
Merck Sharp and Dohme Research Laboratories,
West Point, PA, U.S.A., personal
communication.
14 I
D. J. TOCCO,A. E. W. Duncan, F. A. DeLuna,

H.B. Hucker, V. F. Gruber and W. J. A.
Vandenheuvel, Druq Metab. Disp. 3, 361
( 1975 1.
1 51
A. Bobik, G. L. Jennings, P. Ashley and P.

I. Korner, Eur. J. Clin, Pharmacol. 16, 243
(1979).
16 1
R. K. Ferguson, P. H. Vlasses, J. R. Koplin,

G. I. Holmes, P. Huber, J. Demetriades and
W. B. Abrams, Br. J. Clin. Pharmacol. 14,
719 (1982).
17 1
T. W. Wilson, W. B. Firor, G. E. Johnson, G.

I. Holmes. M C. Tsianco. P. B. Huber and R.
0. Davies; Clin. Pharmacol. Ther. 32, 676
(1982).
18
0. F. Else, H. Sorensen and I. R. Edwards,
19 1
R. Mantyla, P. Mannisto, S. Nykanen, A.

Koponen-and U. Lamminsivu, EuE. J. Clin.
Pharmacol. 24, 227 (1983).
20 1
M. B. Affrime, D. T. Lowenthal, J. A.
Tobert. J. Shirk, B. Eidelson, T. Cook and
G. Onesti, Clin.'Pharmacol. Ther 27, 471
Eur. J. Clin. Pharmacol. 14, 431 (1978).
(19801.
TIMOLOL MALEATE
2 11
G. Alvan, B. Calissendorff. P. Seideman. K.

Widmark and G. Widmark, Clin. Pharmacokin.
5, 9 5 ( 1 9 8 0 ) .
22
C. V. Leier, N. D. Baker and P. A. Weber,

Ann. Int. Med. 104, 1 9 7 ( 1 9 8 6 ) .
23
C. J. Schmitt, V. J. Lotti and J. C.

LeDouarec, Arch. Ophthalmol. 9 8 , 5 4 7 ( 1 9 8 0 ) .
24 1
P. H. Vlasses. L. G. T. Ribeiro, H. H.
Rotmensch, J. V. Bondi, A. E. Loper, M.
Hichens, R. A. Clementi, W. B. Abrams and R.
K. Ferguson, Clin. Pharmacol. Ther. 3 5 , 2 7 9
(1984).
25 1
D. J. TOCCO,A. E. W. Duncan, F. A. DeLuna,

J. L. Smith, R. W. Walker and W. J. A.
Vandenheuvel, Drug Metab. Disp. 8, 2 3 6
(1980).
26 1
B. K. Wasson, J. Scheigetz, C. S. Rooney, R.

A. Hall, N. N. Share, W. J. A. Vandenheuvel,
B. H. Arison, 0. D. Hensens, R. L. Ellsworth
and D. J. TOCCO, J. Med. Chem. 2 3 , 1 1 7 8
(1980).
27 1
D. T. Lowenthal, J. M. Pitone, M. B.
Affrime, J. Shirk, P. Busby, K. E. Kim, J.
Nancarrow, C. D. Swartz and G. Onesti, Clin.
Pharmacol. Ther. 2 3 , 6 0 6 ( 1 9 7 8 ) .
28 1
J. A. Vedin, J. K. Kristianson and C. E.

Wilhelmsson, J. Clin. Pharmacol. 2 3 , 43
( 1 9 8 2 1.
29 1
D. J. TOCCO, F. A. DeLuna and A. E. W.

Duncan, J. Pharm. Sci. 6 4 , 1 8 7 9 ( 1 9 7 5 ) .
30
J. R. Carlin, R. W. Walker, R. 0. Davies, R.

T. Ferguson and W. J. A. Vandenheuvel, J.
Pharm. Sci. 6 9 , 1111 ( 1 9 8 0 ) .
31
J. B. Fourtillan. M. A. Lefebvre. J. Girault

and P. Courtois,'J. Pharm. Sci. 70, 5 7 3
(1981).
69 1
DAVID J. MAZZO AND ALICE E.LOPER
692
32)
M. R. Gregg and D. B. Jack, J . Chromatog.
305,
244 (1984).
33)

PA, U.S.A., 1985, pp. 1063-1064.
34)
Ibid, pp. 1277-1278.
TRAZODONE HYDROCHLORIDE
DeMiS K.J. Gorecki and Roger K. Verbeeck

College of Pharmacy
University of Saskatchewan
1.
Introduction
1.1. History
1.2. Therapeutic Category
2.
Description
2.1.
2.2.
2.3.
2.4.
2.5.
2.6.
3.
Nomenclature
2.1.1. Chemical Name
2.1.2. Generic Name
2.1.3. Proprietary Names
Formulae
2.2.1.
Empirical
2.2.2. Structural
2.2.3. Registry Numbers
Molecular weight
Patent Information
Phvsico-Chemical ProDerties
3.1.
3.2.
3.3.
3.4.
3.5.
3.6.
3.7.
Melting Range
Solubility
Stability and Storage
Crystal structure
Differential Scanning Calorimetry
Spectral Properties
3.7.1. Ultraviolet Spectrum
3.7.2. Fluorescence Spectrophotometry
3.7.3. Infrared Spectrum
3.7.4. Nuclear Magnetic Resonance (NMR) Spectra

VOLUME 16
693

All rights of reproductionin any form reserved.
DENNIS K. G. GORECKI AND ROGER K. VERBEECK
694
3.7.4.1. Proton Magnetic Resonance (PMR)

Spectra
3.7.4.2. Carbon-13 Nuclear Magnetic Resonance
Spectrum (~%-NMEX)
3.7.5. Mass Spectrometry
3.7.5.1. Electron Impact (EI)
3.7.5.2. Chemical Ionization (CI)
4. Synthesis
5.
Pharmacokinetics
5.1. Absorption
5.2. Distribution
5.3. Elimination
6.1. Identification
6.2. Chromatographic Analysis
6.2.1. Thin Layer Chromatography (TLC)
6.2.2. Gas-Liquid Chromatography (GLC)
6.2.3. High Performance Liquid Chromatography (HPLC)
7. References
695
1. Introduction
1.1. History
Trazodone hydrochloride was originally synthesized
in 1966 by Palazzo and Silvestrini (1) in the chemical
laboratories of the firm Francesco Angelini. The
molecule consists of a triazolopyridine ring and a phenyl
piperazinylalkyl moiety. It is the first triazolopyri
-dine derivative to be used clinically, and therefore,
represents a new chemical class of antidepressant drugs.
Its development was a result of an organized approach
rather than fortuitous discovery and was based on the
hypothesis that the mechanisms responsible for physical
and mental pain are the same (2,3).
The history, pharmacology and clinical data on
trazbdone hydrochloride is well documented in a number of
review articles and monographs ( 3 - 8 ) . In addition, an
analytical profile has been described (9).
1.2. Therapeutic Category
Trazodone hydrochloride is an antidepressant drug
indicated in the symptomatic treatment of moderate to
severe depression. Its major advantages include a low
incidence of anticholinergic and cardiovascular side
effects along with minimal stimulatory effects upon
dopamine and norepinephrine receptors and is, therefore,
considered particularly useful in the geriatric
population (5).
2. Description
2.1. Nomenclature
2.1.1.
Chemical Name
2-{3-[4-(3-chlorophenyl)-l-piperazinyll
propyl)-1,2,4-triazolo-[4,3-a]pyridin-3(2H)-one
monohydrochloride
696
2.1.2. Generic Name

Trazodone hydrochloride
2.1.3.
Proprietary Names
Desyrel, Manegan, Molipaxin, Pragmazone,

Thombran, Trittico
2.2. Formulae
2.2.1.
Empirical
C19H22C1N50 HC1
2.2.2.
Structural
2.2.3.
Registry Numbers
Chemical abstracts;
Trazodone:19794-93-5
Trazodone hydrochloride:25332-39-2
2.3. Molecular Weight
408.33
2.4. Elemental Composition
C, 55.89%; HI 5.68%; C1 17.36%; N, 17.15%; 0, 3.92%
2.5. Appearance, Color and W o r
White, odorless crystals (plates)with a bitter

taste.
2.6. Patent Information
U.S. Pat. 3,381,009 (1968 to Angelini Francesco)(l).
Brit. Pat. 1,117,068 (11).
3.
691
Physico-Chemical Properties
3.1. Melting Range
The melting point for trazodone free base is 96OC
(9). The hydrochboride salt melts with decomposition in
the range 222-228 C (grlOr1l). Under vacuum
decomposihion does not occur and a melting range of
231-232.5 C is reported (9).
3.2. Solubility
Trazodone hydrochloride is soluble in chloroform,
sparingly soluble in water, ethanol and methanol (10) and
insoluble in common organic solvents (9).
3.3. Stability and Storage
Trazodone hydrochloride tablets should be stored at
room temperature in tight, light resistant gontainers and
protected from temperatures in excess of 40 C (13).
3.4. Dissociation Constant
The reported pKa for trazodone, in 50% ethanol, is
6.14 (9). This value was obtained potentiometrically
using a glass-calomel electrode.
3.5. Crystal Structure
The crystal and molecular structure for trazodone
hydrochloride has been determined (14). X-ray
diffraction data were obtained using an Oak Ridge
diffractometer employing molybdenum Kolradiation with a
niobium filter.
Trazodone hydrochloride, C H C1N 0-HC1,crystals
are monoclinic with space group182?3c a8d with cell
dimension8 a=16.866[31, b..1O.66[2lr c=11.230[21, A,
=93.04(1) and Z=4 with the final R factor of 0.042 for
3017 reflections. The megsured and calculated densities
are 1.344 and 1.345 mg m- respectively for Z=4.
Figure 1 depicts the 3-dimensional, X-ray derived,
structure of trazodone hydrochloride (58). Bond lengths
and angles are tabulated in Tables 1 and 2.
Figure 1.
Three-Dimensional, X-Ray Derived, Structure of Trazodone Hydrochloride.
Table 1.
Intramolecular Bond Lengths ( A ) of Trazodone Hydrochloride

0
A
-
D i s tances
A
-
1.222
1.349
1.386
1.308
1.384
1.419
1.344
1.421
1.337
1.384
1.394
N(2)-C(10)
C( lO)-C(ll)
C(ll)-C(12)
C( 12)-N( 13)
N( 13)-H(13)
N( 13)-C1(1)
H( 13)-C1(1)
N( 13)-C( 14)
C (14)-C ( 15)
C (15)-N( 16)
N(16)-C(17)
1.451
1.518
1.512
1.498
0.872
3.052
2.180
1.491
1.497
1.455
1.463
Distances
C(17)-C(18)
C(18)-N(13)
N(16)-C(19)
C(19)-C(20)
C(20)-C(21)
C(21)-C(22)
C(22)-C(23)
C(23)-C(24)
C(24)-C(19)
C(22)-C1(2)
A
-
1.506
1.495
1.407
1.394
1.378
1.380
1.363
1.383
1.391
1.747
Table 2.
Bond Angles ( " ) of Trazodone Hydrochloride
Angles
0 (1) -C (1)-N(l)
0 (1)-C( 1)-N(8)
C(l)-N(2)-C(lO)
C (l)-N( 2)-N( 3)
C ( 1)-N (8) -C ( 9)
N(2)-C(l)-N(8)
C( 10)-N( 2)-N(3)
N( 2)-N( 3)-C(9)
N(3)-C(9)-C(4)
N (3) -C (9)-N (8)
C(9) -C(4) -c(5)
C(4)-C(5)-C(6)
C (5)-C( 6)-C ( 7 )
C(6)-C(7)-N(8)
(">
130
128
125
114
108
103
121
104
130
112
119
122
121
118
Angles
C(7)-N(8)-C(9)
N(8)-C(g)-C(4)
C(l)-N(8)-C(7)
N(2)-C(lO)-C(11)
C( lO)-C(ll)-C(
12)
C(ll)-C(l2)-N(13)
C(12)-N(13)-C(14)
C(12)-N(13)-H(13)
N (13) -H (13)-C1(1)
N (13)-C (14)-C( 15)
C ( 14)-C ( 15)-N (16)
C(15)-N(16)-C(17)
N(16)-C(17)-C(18)
C(17)-C(18)-N(13)
(")
123
118
129
113
112
113
109
114
172
112
111
111
111
111
Angles
C( 18)-N( 13) -C ( 1 4 )
C(17)-N(16)-C(19)
C(15)-N(16)-C(19)
N( 16)-C (19)-C(20)
N(16)-C(19)-C(24)
C(19)-C(20)-C(21)
c(20)-c (211-c(221
C(21)-C(22)-C(23)
C(22)-C(23)-C(24)
C( 23)-c(24)-c(
19)
c (24) -c (19) -c (20)
Cl(2) -C( 23)-C(22)
C1(2)-C(23)-C(24)
108
118
117
121
121
120
122
117
118
120
118
119
118
70 1
This study revealed a number of interesting features

about trazodone which may be useful in proposing
structural requirements for neuroleptic activity. The
piperazine ring is in the normal chair conformation with
13) extending upward and N(16) down. The bond lengths
and angles about N(13) resemble that of a protonated2
mine and those associated with N(16) demonstrate sp
character due to the attached chlorophEny1 ring.
Torsiogal angles about N(2)-C(10)(93.2 1 C(ll)-C(12)
(178.2 ) suggest that the propylene side chain is fully
extended. In addition, the triazolopyridine nucleus
(including C(10)) and the chlorophenyl moiety are planar
groups
3.6. Differential Scanning Calorimetry

A differential scanning calorimetry (DSC) curve for
trazodone hydrochloride has been reported (9). Data
obtained using a Perkin Elmer DSC 1B instrument was used
as a purity index for trazodone.
3.7. Spectral Properties

3.7.1. Ultraviolet Spectrum
The ultraviolet spectrum of trazodone
hydrochloride was scanned from 200 to 360 nm with a
Gilford Response spectrophotometer at a concentration of 30 and 75!~moles/Lin methanol and 30
Umoles/L water (Figure 2). Each spectrum is
characterized by two well defined and two less
prominent peaks. The spectra in methanol show
maxima at 213, 256, 278 and 312 nm while that in
water displays maxima at 211, 246, 275 and 312 nm.
The latter are in agreement in previously reported
data (9,101.
The reported A (l%, 1 cm) and molar
absorptivity values for trazodone hydrochloride in
water are s m r i z e d in Table 3 (9).
Table 3. Ultraviolet Characteristics of Trazodone
Hydrochloride
max
(m)
211
246
274
312
A( 106,lcm)
1225
287
94
94
50,100
11,730
3,040
3,840
A
10
Figure 2 .
Ultraviolet Spectra of Trazodone Hydrochloride (-,
methanol;
---- , water).
3.7.2.
703
Trazodone hydrochloride, like most

2-alkyl-substituted-l,2,4-triazolo-[4,3-al pyridin-3
( 2H)-ones , shows an intense fluorescence-when
irradiated with ultraviolet light. The excitation
and emission wavelengths for trazodone are 317 and
455 nm respectively (9).
3.7.3.
Infrared Spectrum
The infrared spectrum of trazodone

hydrochloride is shown in Figure 3. The spectrum
was obtained with a Beckman AccuLab 4 infrared
spectrophotometer from a compressed KBr disc.
Structural assignments of some of the characteristic
absorption bands are presented in Table 4.
Table 4. Infrared Characteristics of Trazodone
Hydrochloride
Frequency (cm-' )
Intensity
Assignment
2975-2840
2535,2460
1705
1640
1595
W
M
S
M
S
Aliphatic CH
+N-H stretching
C=O stretching
triazoline C=N stretch
aromatic C=C stretch
M=Medium; S=Strong; W=Weak
Other characteristic bands appe r at 1540,

1491, 1447, 1350, 1273, 943 and 745 cm-'i Principle
peaks, wavenumbers and structural assignments are in
agreement with previously published results (9,lO).
3.7.4. Nuclear Magnetic Resonance (NMR) Spectra
3.7.4.1.
Proton Magnetic Resonance (PMR)

Spectra
The PMR spectra of trazodone base

and trazodone hydrochloride were recorded on
a Bruker AM 300 NMR spectrometer employing a
frequency of 300.13 MHz. Spectral
assignments were confirmed by homodecoupling, COSY and heteronuclear correlation
experiments.
okooo
4
I
3000
MICRONS
5
I
2000
1600
I800
WAVENUMBER
Figure 3.
7
I
1400
CM
Infrared Spectrum of Trazodone Hydrochloride-KBr Disc.
9
I
1200
10
1
1000
11
I
12
1
800
14
16
10
600
705
The spectrum of trazodone base

obtained in CDCl is presented in Figure 4.
Scale expansion $n the region 6.4 to 7.8 ppm
is illustrated in Figure 5. The chemical
shifts, multiplicities and assignments are
compiled in Table 5. These data are
consistent with the results reported using a
60 MHz instrument ( 9 ) .
Table 5. Proton Magnetic Resonance
Assignments for Trazodone Base
/y\ /-7
9
*\
12
10
13
11
Chemical
Number
Shift
of
6 (ppm) Multiplicities Protons Assignment
7.75
7.11
6.84
6.76
6.46
4.09
3.13
2.56
2.49
2.05
d
m
m
m
m
t
t
t
t
m
3
1
1
2
4
4
2
1
314,6
8
517
9
13
12
11
10
d=doublet; t=triplet; m=multiplet

The PMR spectrum of trazodone
hydrochloride in DMSO-d is shown in Figure
6. Scale expansion in the region 6.5 to 8.0
ppm is depicted in Figure 7. Chemical
shifts and assignments are given in Table 6.
It can be seen that protonation of the
piperzine ring nitrogen nearest the propyl
chain affects the chemical shift and
splitting pattern of adjacent protons in the
piperazine ring.
UCJ
I " " I " " I ~ " ' " ' 1 " ' " " ' ' " I ' " ' I " " I ' " ' I ' ' ~ ' I " " I ' ' "
8.0
Figure 4 .
7.5
7.0
6.5
6.0
5.5
5.0
PPM
4.5
4.0
3.5
3.0
P r o t o n Magnetic Resonance Spectrum of Trazodone Base i n CDC13.
LL
2.5
2.0
1 -
7.8
Figure 5.
'
7.6
7.4
7.2
7.0
PPM
6.6
6.6
6.4
Proton Magnetic Resonance Spectrum o f Trazodone Base in CDCl with Scale

3
Expansion (6.4-7.8 ppm).
I " " " " ' I
8.0
Figure 6.
7.5
'
7.0
6.5
"
'
6.0
5.5
"
'
5.0
PPM
4.5
'
I 1
4.0
3.5
~I
3.0
2.5
2.0
Proton Magnetic Resonance Spectrum of Trazodone Hydrochloride in DMSO-d6.
7.9
Figure 7.
7.8
7.7
7.6
7.5
7.4
7.3
PPM
7.2
7.1
7.0
6.9
6.6
6.7
-6.6
Proton Magnetic Resonance Spectrum of Trazodone Hydrochloride in DMSO-d 6 with

Scale Expansion (6.5-8.0 ppm).
710
Table 6.
Proton Magnetic Resonance Assignments for Trazodone Hydrochloride
Chemical
Number
Shift
of
6 (ppm) Multiplicities Protons Assignment
7.87
7.25
7.04
6.95
6.86
6.64
4.01
3.86
3.53
3.15
2.25
m
m
m
dd
dd
m
t
d
d
m
m
3
1
1
1
1
2
2
2
6
2
1
31416
8
5
7
2
9
1 2 ,13
12,13
11,14,15
10
d=doublet; dd=doublet,doublet; t-triplet

m=multiplet
3.7.4.2.
Carbon-13 Nuclear Magnetic

Resonance Spectrum (IjC-NMR)
The 13C-NMR spectrum of trazodone

hydrochloride base obtained in DMSO-d6 is
given in Figure 8. The spectrum was
obtained at ambient temperature on a Bruker
AM 300 NMR spectrometer at 75.47 MHz with
broad band proton decoupling. Chemical
shifts, and assignments are outlined in
Table 7. Spectral assignments were
confirmed by C-13m-1 heteronuclear
correlation experiments and published
chemical shift data.
d
1
'
160
Figure 8.
'
'
140
'
'
120
'
'
100
'
PPM
'
80
'
'
60
'
40
'
'
"
'
'
20
Carbon-13 Nuclear Magnetic Resonance Spectrum of Trazodone Hydrochloride in DMSO-d 6'
712
Table
7. Carbon-13 Nuclear Mametic
_.
.~~
Resonance Assignments forTrazodone Hydrochloride
Chemical
Shift
6 (ppm)
150.69
147.88
141.06
133.84
130.51
130.47
123.82
119.03
115.12
3.7.5.
Assignment
6
1
7
11
4 , s or 9
4,5 or 9
2
10
12
Chemical
Shift
6 (ppm)
114.92
113.99
110.76
52.79
50.23
44.66
42.54
22.78
Assignment
4,s or 9
8
3
13
17
16
15
14
Mass Spectrometry
3.7.5.1.
Electron Impact (EI)
An electron impact mass spectrum

of trazodone hydrochloride is presented in
Figure 9. This was obtained by direct solid
insertion probe at an electron eneljgy of 70
eV and a source temperature of 180 C using a
VG Analytical MM 16F single focussing mass
spectrometer linked to a VG 2035 data
system+ The spectrum shows a molecular ion
peak M at a mass/charge (m/z) ratio of 371
with a relative intensity of 13.2% and a
base peak at 205. Prominent diagnostic ions
are observed at m/z 70, 136, 166, 176, 209,
231 and 278. The fragmentation pattern
leading to the various ions is outlined
below.
713
20:91J
231
Mass spectra of trazodone and

other related compounds have been reported
elsewhere (9,15,16,17,18).
3.7.5.2.
Chemical Ionization (CI)
The chemical ionization spectrum

of trazodone hydrochloride is shown in
Figure 10. This spectrum was also
determined on a VG Analytical MM 16F mass
spectrometer, however, using ammonia as a
reagent gas. The spectrum shows a pseudo
molecular ion at a mass to charge ratio of
372.
4. Synthesis
A number of synthetic pathways have been described for
the synthesis of trazodone hydrochloride (1,11,19,20). All
of the reported routes employ 1,2,4-triazo-[4,3-a]-pyridin3(2H)-one (I) or 2-substituted derivatives as stzrting
matFria1. The more important methods are outlined in Scheme
1.
The original synthesis reported by Palazzo and Silvestrini (1) consisted of the condensation of l-(3-chloropheny1)-4-(3-~hloropropyl)-piperazine with the sodium salt of
I. Other related patented methods were simultaneously
developed in the same laboratories (11).
More recently, modications of existing pathways were
reported (19). One method involves the condensation of the
usual triazopyridine (I) with bromochloropropane to yield the
2-(3-~hloropropyl)-derivative (11). Subsequent treatment
with N-(3-chlorophenyl)piperazine gives trazodone.
Alternatively, I is alkylated with bromochloropropane and
treated with diethanolamine to form the di-alcohol (111).
This product is then chlorinated with thionyl chloride and
subsequently reacted with 3-chloroaniline to yield trazodone.
714
DENNIS K. G.GORECKI AND ROGER K. VERBEECK

m
100-
80 >
c
cn
-6
c
60 -
>
0
100
50
1-50
250
200
300
350
MASSKHARGE RAT I0
Figure 9.
Mass Spectrum of Trazodone Hydrochloride

Electron Impact.
1s
231
I 1 1
Figure 10.
Mass Spectrum of Trazodone HydrochlorideChemical Ionization.
375
Cl-(CH
) -N
2 3
CH2 CH20H
c1
CH2CH 20H
HC1
I1
.HC1
TRAZODONE
c1
HYDROCHLORIDE
Synthesis of trazodone hydrochloride
Scheme 1.
716
DENNIS K. G . GORECKI AND ROGER K. VERBEECK
The synthesis of 14-C-labelled trazodone hydrochloride has

also been published ( 2 0 ) . This procedure also employs the
classical condensation reaction described in the original
patent.
5. Pharmacokinetics
5.1. Absorption
Following oral administration to man, trazodone is

rapidly absorbed. The time to reach m a x i m plasma
concentrations typically varies from 0.5 to 4.0 hours in
fasting subjects (21-26). Peak plasma concentrations
following oral administration of a single 50 mg dose
range from 0.5 to 1.0 pg/mL (21,24-26).
Following oral
ingestion of a single 100 mg trazodone hydrochloride dose
average m a x i m plasma concentrations of 1.61 Pg/mt and
1.66 ug/mL were reported for a capsule and liquid
formulation respectively ( 2 2 ) .
Oral and intramuscular bioavailability of trazodone
is almost complete as shown by the virtually identical
plasma concentration-time profiles obtained following
oral intramuscular and intravenous administration to
healthy volunteers ( 2 3 ) . Ingestion of food delays the
absorption of trazodone from the gastrointestinal tract
but does not seem to affect its bioavailability (23,27).
Studies in rats and rabbits have shown that food delays
the oral absorption by decreasing the transfer rate of
drug from the stomach to the duodenum ( 2 3 ) .
5.2. Distribution
Trazodone is a weak base with a pKa of 6.14 (91, and

therefore, exists mostly in the unionized form at
physiological pH. The drug distributes in a relatively
large volume: Vd values reported in the literature or
calculated from published data range approximately from
1-2 L/kg for healthy volunteers (21,22,24,25,28).
Trazodone is highly bound to plasma proteins. In vitro
protein binding studies with human plasma have shown that
trazodone is 96, 98 and 97% bound at concentrations of
1.0, 0.1 and O.Olvg/mL respectively ( 2 9 ) .
Studies in laboratory animals show that trazodone
readily crosses the blood brain barrier but is minimally
transferred across the placenta (30,311. Little
information is available in man concerning its
distribution in organs and body fluids. Excretion of
trazodone in human breast milk has been studied and found
to be very small ( 3 2 ) . The average milk/plasma ratio was
0.14, therefore, exposure to babies via breast milk would
be minimal.
717
5.3. Elimination
In general, plasma concentrations of trazodone in
man decline in a biphasic manner with a mean half-life of
approximately 4 hours during the period 3-10 hours
following dosing and a terminal elimination half-life of
6-12 hours (21,22,25,28,32). Total body clearance
(assuming an oral bioavailability of 100%) ranges from
150 mL/min. to 400 mL/min. (21,24,32). Patients treated
with trazodone on a twice daily dosage schedule show
significantly higher plasma levels in the morning as
compared to the levels at night (33). A possible diurnal
variation in clearance may be responsible.
Trazodone is extensively metabolized via
hydroxylation, N-oxidation and hydrolysis (Scheme 2)
(34). Hydroxylation occurs in the benzene ring
(4-hydroxyphenyl metabolite, I) and in the
triazolopyridine ring (dihydrodiol, 11) while N-oxidation
occurs on a piperazine nitrogen (N-oxide, 111). A
hydrolytic reaction results in the formation of
3-0~0-1,2,4-triazo10[4,3-a]-pyridin-2-yl propionic acid
( IV) and 1-m-chlorophenylpiperazine ( V ), a
pharmacologTcally active metabolite ( 35,361. Small
concentrations of 1-m-chlorophenylpiperazine,
approximately 1% of 'frazodone plasma concentrations, have
been reported in man (37). Whether these small
concentrations contribute to the overall effects of
trazodone remains unclear. Approximately 70-75% of an
oral dose of the drug is excreted in urine within 72
hours of administration, most as metabolites. Only less
than 1% of the dose is recovered in urine as unchanged
drug (26,34). The remainder of an oral dose is excreted
in feces, involving biliary excretion, mainly as
trazodone metabolites (23,271.
Results from animal studies indicate that trazodone
does not induce its own metabolites (38).
6.
Methods of Analysis
6.1. Identification
Trazodone hydrochloride reacts with certain reagents
to produce characteristic colors which may be useful for
its identificahion. When treated with Liebermanns Test
reagent at 100 C (sodium nitrite-sulphuric acid)
trazodone gives a transient violet color whereas with
Mandelin's reagent (ammonium vanadate-sulphuric acid) a
grey color develops which later changes to violet (10).
/
QOH
OH
Cl
TRAZODONE
GLUCURONIDE
IV
GLUCURONIDE
Scheme 2.
Biotransformation pathways of trazodone in man
719
An infrared spectroscopic method is described which

is useful for the rapid emergency identification of
certain psychtropic drugs including trazodone (39). The
method employs chloroform extraction of patient's urine
at different pH's and utilizes the infrared finger print
region and spectra of authentic standards to confirm
identity. Infrared spectra are determined as compressed
potassium bromide discs from dried chloroform extracts.
6.2. Chromatographic Analysis

6.2.1. Thin-Layer Chromatography ( T L C )
A number of thin-layer chromatographic
systems have been developed for the determination of
trazodone. These systems have been employed for the
identification (9,10,40,41,42)and quantitation
(43,44,45) of the drug both as pure drug substance
as well as in biological fluids and tissues.
A variety of detection methods have been
used and most systems use silica gel plates as
absorbent. A summary of some of the reported TLC
methods is given below.
System I ( 9 )
Absorbent: Merck Kieselgel-0.25 mm

Mobile Phase:
i)
cyclohexane-benzene-diethylamine (5:4:1)
ii) chloroform-methanol-isopropanol-ether-NH (60:25:25:2)
i ii ) n-butanol :wate? :NH ( 20:10:2 1
iv) abs. ethano1:NH (35:5)
Detection: ultra-viol& light (366 MI),
ninhydrin, rhodanine, ammonium
sulphocyanide and cobaltous chloride or
potassium dichromate, sulphuric acid with
glacial acetic acid.
System I1 (10)
Absorbent: Silica gel G, 0.25 mm treated

with potassium hydroxide in methanol.
Mobile Phase:
i)
methano1:NH (100:1.5)
ii) cyclohexane.toluene:diethyl3
amine (75:15:10)
Detection: ultra-violet light,
Dragendorff's reagent or acidified
iodoplatinate solution.
720
System 111 (40)
Absorbent: Silica gel, F254, 0.25 mm

Mobile Phase:
i)
methano1:NH (100:1.5)
ii) cyclohexane.to1uene:diethyl3
amine (75:15:10)
iii) ch1oroform:methanol (9:l)
iv) acetone
Detection: Dragendorff's reagent
System IV ( 4 2 )
Absorbent: Silica gel 60, F254,

0.25 nun
Mobile Phase:
i)
ethylacetate:methanol:30% NH3
(85:10:5)
ii) cyc1ohexane:toluene:diethylamine (65:25:10)
iii) ethy1acetate:chloroform
(50:50)
iv) acetone
Detection: 10% sulphuric acid followed
by Dragendorff's reagent and acidified
iodoplatinate solution
System V ( 4 5 )
Absorbent: Silica gel, 0.25 nun

Mobile Phase:
i)
methano1:ammonia (100:l.S)
ii) ethy1acetate:methanol:amonia
( 85:10:5)
Detection: 5% sulphuric acid,
iodoplatinate solution followed by
Dragendorff's reagent
6.2.2. Gas-Liquid Chromatography (GLC)

A number of GLC procedures have been
published for the determination of trazodone. The
technique has been used for routine detection of the
drug (9,10,17), biopharmaceutics and pharmacokinetic
studies (16,19,28,47), as well as in therapeutic and
overdose stiuations.(45,46) Chromatography of
trazodone is readily accomplished without
derivatization using a variety of stationary phases
in packed or capillary columns. Detectors employed
include flame ionization (FID), nitrogen-phosphorous
(NPD) and mass spectrometry (MS). The conditions
for the reported GLC methods are as follows:
72 1
System I (9)
Type of sample: drug substance

Column: 6 ft x 1/4" I . D . glass column
packed with 1.5% OV-17 on Chromosorb W HP
80-100
Carrier gas: Nitrogen, &3 lb/inch 2
Temperature: column 268 C, injector 32OoC
Retention time: approximately 3.5 min.
Detector: not stated, presumably F I D
System I1 (10)
Type of sample: general

Column: 2 m x 4 mm I . D . glass packed with
2.5% SE-30 on chromosorb G 80-100
Carrier gas: nitrogen, 45 mt/min.
Temperature: column, retention index - 10
Retention index: 3250
Detector: F I D or NPD
System 111 (16)
Type of sample: plasma ( rat)

Column: 80 cm x 4 mm I . D . glass column
packed with 1% OV-1 on Chromosorb Q
100-120
Carrier gas: nitrogen, 33 mL/min.
Tevrature: column, 240 C; injector,
270 C
Retention time: 2 min.
Detector: F I D and MS
System Iv ( 4 5 )
Type of sample: biological fluids and

tissue
Column: 4 m x 0.25 mm I.D. DB-1
fused-silica capillary (0.25wm film)
Carrier gas: hydrogen, 48 cwsec.
Temperature: column, 265 C
Retention time: 2.87 min.
Detector: F I D (or NPD)
In addition:
i)
Column: 1.8 m column with 3%
SP 2250 on Supegcoport 100-120
Temperape: 10 C/min. from
140-300 C, hold 5 min.
Detector: NJ?D (MS)
ii) Column: 1.8 m column with 3%
SP 2100 on Supegcoport 100-120
Temperape: 13 C/min from
160-320 C, hold 7 min.
Detector: NPD (MS)
DENNISK. G . GORECKI AND ROGER K. VERBEECK
722
System V (28)
of sample: plasma
Column: 1.23 m x 2 mm I.D. glass column
packed with 3% OV-101 on Chromosorb W HP
80-100
Carrier gas: helium, 30 p/min
Tevrature: column, 260 C, injector,
310 C; detector, 275OC
Detector: NPD
System VI (47)
Type of sample: plasma, brain tissue

(rat)
Column: 1 m x 3 mm I.D. glass column
packed with 3% OV-1 on Gas Chrom Q 80-100
Carrier gas: nithogen, 30 ml/min.
Temperature: 270 C
Detector: NPD (MS)
System V I I (17)
Type of sample: urine (rat)

Column: 60 cm x 1 mm I.D. nickel
capillary column packed with 20% UCC-W
(Hewlett Packard) on Chromosorb W AW DMCS
80-100
Carrier gas: helium, 7 p/min.
Temperahure: column, 20 $pin. from
100-300 C; injector, 270 C
Detector: MS
System VIII (18)
'rype of sample: plasma
6.2.3.
"ype
Column: 4 m x 0.25 mm I.D. DB-1

fused-silica capillary (0.25 pm film)
Carrier gas: helium, 2000cm/sec.
Teperature: c o l p , 170 C for 1 gin.,
15 C/min. to 255 CA injector, 250 C;
separator oven 255 C
Detector: MS
3HPLC )
Several HPLC methods have been developed for

the determination of trazodone; most of which have
been employed in analysis of biological fluids and
tissues for unchanged drug and metabolites. The
majority of the procedures utilize reverse-phase
chromatography with mobile phases comprised of an
aqueous buffer with organic solvent modifiers
(21,32,43,48,50-52,54). Others include
723
reversed-phase with ion-pairing agents (49,56,57)

and normal-phase systems (53,551. Detector systems
include ultra-violet ( w ) at various wavelengths,
fluorescence and electrochemical oxidation. A
summary of the reported HPLC systems is presented
below.
System I (48)
Type of sample: serum or plasma
Coluum: 150 x 4.6 mm Supelcosil LC-8 DB

Mobile phase: phosphate buffer 0.01M (pH
4.6): methanol: acetonitrile (47:37:16)
Temperature: ambient
Flow rate: 2.1 mL/min.
Detection: UV at 206 nm
System I1 (32)
Type of sample: breast milk

Column: 150 x 3.9 mm Nova-Pak C18 (4p m )
Mobile phase: methano1:phosphate buffer
0.082 M (PH 6.5): acetonitrile (39:47:14)
Detection: W at 211 mm
System I11 (49)
Type of sample: plasma and tissue

Column: 250 x 4.6 mm trimethyl silyl
(5urn)
Mobile phase: acetonitri1e:phosphate
buffer (pH 3.0) (27:73) with 0.02 M
heptane sulfonic acid and 0.04 M
triethylamine
Detection: W at 214 nm
System Iv (50)
Type of sample: plasma

Column: 300 x 4.6 mm Bondapak C18
(10 V M )
Mobile phase: phosphate buffer 0.05 M (pH
4.7): acetonitrhle (72:8)
Temperature: 60 C
Flow rate: 2.5 mL,/min.
724
DENNIS K. 0 . GORECKI AND ROGER K. VERBEECK
System V (51)
Type of sample: serum

Column: 250 x 4 . 6 mm LiChrosorb l o w 8
Mobile phase: acetonitrile: phosphate
buffer, 0.6%; pH 3.0 (1:1)
Flow rate: 2 mL/min.
Retention time: 3 . 8 min.
System VI ( 5 2 )
Type of sample: biological fluids

Column: 250 x 4 . 6 mm Ultrasphere ODs
(octadecylsilyl) 5 p m)
Mobile phase:
i)
phosphate buffer, 0.1 M (pH
7.0): acetgnitrile (10:3.8); 3.0
ml/min; 65 C; retention time
( 1 0 : 3 . 8 ) ; 8 min.
ii) phosphate buffer 0.1 M (pH 3.0):
acgtonitrile (10:3); 1.5 mL/min.;
60 C; retention
time, 1.25 min.
System VII ( 4 3 )
Type of sample: serum and urine

Column: ODs (C18)
Mobile phase: methanol: 0.011 M phosphate
buffer (pH 7.5): acetonitrile (60:38:2)
System VIII (21)
of sample: plasma
Column: 250 x 4.6 rmn Spherisorb S5 ODs
Mobile phase: acetonitrile: 0.05 M
sulphuric acid ( 1 8 : l )
Retention tinre: 6 . 8 min.
Detection: Uv at 254 nm
System IX (53)
Type of sample: drug substance

Column: 125 mm Spherisorb S5W Silica
"ype
Mobile phase: methanolic ammonium

perchlorate, 10 mM, pH 6 . 7
Relative retention time: 0 . 3 1
Detection: W at 254 nm, electrochemical,
-I- 1 . 2
125
System X ( 5 4 )

Columns: 250 x 4.6 mm MC-18 with 20%
carbon load (5 pm), 300 x 4.6 mm Bondapak C18 (10um)
Mobile phase: phosphate buffer, 0.2 M (pH
4.7): tetrahydrofuran: acetonitrile
(90:5:5)
Temperature: 45'~
Flow rate: 1.5 !nL/min.
System XI (55)
Type of sample: biological fluids

Column: 125 x 5 mm Spherisorb S5W Silica
( 5 pm)
Mobile phase: methanol with 0.02%
perchloric acid
Detection: Fluorescence,A ex=200nm
System XI1 (56)

Column: 150 x 4.6 mm Ultrasphere octyl
(5urn)
Mobile phase: acetonitrile: water (50:50)
with 0.05% of each of
tetramethylammmonium hydroxide and
perchloric acid (70%)
Flow rate: l.O/min.
Detection: Fluorescence, Xex=320 nm;
Aem=440 nm
System XI11 (57)

Column: 250 x 4.6 mm LC-1 (trimethysilyl)
( 5 urn)
Mobile phase: phosphate buffer (0.05 M I
pH 3.0): acetonitrile (9O:lO) with 0.005
M of each of sodium heptane sulfonate and
n-nonylamine
Detection: electrochemical, +1.15 V
126
7.
References
1.
G. Palazzo and B. Silvestrini, U.S. Patent No. 3,381

- 52146W (1968).
009. CA:69,
2.
B. Silvestrini, V. Cioli, S. Burberi and B. Catanese,

Int. J. Neuropharmacol., 1, 587 (1968).
3.
B. Silvestrini. Trazodone. A new approach to the

therapy of depression. In Proceedings of "Depression
and the Role of Trazodone in Antidepressant Therapy",
Eds., G. MOrOZOV, J. Saarma and B. Silvestrini,
Edizioni Luigi Pozzi, Roma, 1978, pp. 1-10.
4.
B. Silvestrini, R. Lisciani and M. De Greqorio, in
Pharmacological and Biochemical Properties of Drug

Substances, Vol. 3, Ed. M.E. Goldberg, Am. Pharm.
Association, Washington, DC, 1981, pp. 94-119.
5.
R.N. Brogden, R.C. Heel, T.M. Speight and G.S. Avery,

Drugs, 2, 401 (1981).
6.
M.M. Al-Yassiri, S.I. Ankier and R.K. Bridges, Life

Sciences, 28, 2449 (1981).
7.
T.A. Ban and B. Silvestrini, Eds., Trazodone in Modern

Problems of Pharmacopsychiatry,Vol 9, S. Karger,
1974.
8.
S. Gershon, K. Rickels and 8. Silvestrini, Eds.,

Trazodone: A New Broad Spectrum Antidepressant,
Amsterdam: Excerpta Medica, 1980.
9.
L. Baiocchi, A. Chiari, A. Frigerio and P. Ridolfi,

Arzneim-Forsch., 23, 400 (1973).
10.
A.C. Moffat, Ed., Clarke's Isolation and Identification

of Drugs, The Pharmaceutical Press, London, 1986, pp.
1035-1036.
11.
Brit. Patent No. 1,117,068. CA:69,

- 67429 q (1968).
12.
The Merck Index, M. Windholz, Ed., 10th edition, Merck

and Co., Inc. Rahway, N.J., U.S.A., 1983 p. 1370.
13.
Desyrel (Trazodone hydrochloride) Product Monograph,

Mead Johnson and Co., Evansville, Indiana.
14.
J.P. Fillers and S.W. Hawkinson, Acta Cryst., B35, 498

(1979).
727
15.
L. Baiocchi, A. Frigerio, M. GiaMangeli and L. Secchi,

Boll. Chim. Farm., 114, 206 (1975).
16.
G. Belvedere, A. Frigerio and C. Pantarotto, J.

Chromatogr., 112, 631 (1975).
17.
H. Maurer and K. Pfleger, J. Chromatogr., 305, 309

( 1984 1.
18.
R.E. Gammans, E.H. Kerns, W.W. Bullen, R.R. Covington

339, 303 (1985).
and J.W. Russell, J. Chromatogr., -
19.
L. Baiocchi and M. Giannangeli, Boll. Chim. Farm.,

152 (1981).
20.
K. Sugiyama, S. Kono, C. Yamato and T. Fujita, J. Lab.

Compd., 9, 723 (1973).
21.
S.I. Ankier, B.K. Martin, M.S. Rogers, P.K. Carpenter

and C. Graham, Br. J. Clin. Pharmacol., 11, 505 (1981).
22.
A.W. Harcus, A.E. Ward, S.I. Ankier and G.R. Kimber, J.

Clin. Hosp. Pharm., 8, 125 (1983).
23.
R. Javch, Z. Zopitar, A. Prox and A. Zimmer,

Arzneim-Forsch., 26, 2084 (1976).
24.
R.E. Gammans, A.V. Mackenthun and J.W. Russell, Br. J.

Clin. Pharmacol., 18, 431 (1984).
25.
A.J. Bayer, M.S.J. Pathy and S.I. Ankier, Br. J. Clin.

Pharmacol., 16, 371 (1983).
26.
C. Yamato, T. Takahashi and T. Fujita, Xenobiotica., 6,
27.
F.W. Koss and U. Bosch, Pharmacokinetics and metabolism

of trazodone in different species. In Proceedings of
"Depression and the Role of Trazodone in Antidepressant
Therapy", as., G. Morozov, J. Saarma and 8.
Silvestrini, Edizioni Luigi Pozzi, Roma, 1978, pp.
11-20.
28.
D.R. Abernethy, D.J. Greenblatt and R.I. Shader,

Pharmacology, 28, 42 (1984).
R.E. Gammans, Personal communication, 1984.
29.
30,
113,
295 (1976).
F.W. Koss and U. Bosch: Trazodone as an example-drug

levels in blood and brain. In "Trazodone--A New
Borad-spectrum Antidepressant". Eds. S. Gershon, K.
Rickels and B. Silvestrini, Excerpta Medica, Amsterdam,
1980.
728
31.
Y. Suzuki: Teratogenicity and placental transfer of

trazodone. In "Modern Problems of Pharmacopsychiatry,
Vol. 9", Eds. T. Ban and B. Silvestrini, Karger, Basel,
1974.
32.
R.K. Verbeeck, S.G. Ross and E.A. McKenna, Br. J. Clin.

Pharmacol., 22, 367 (1986).
33.
L.R. Baxter, J.N. Wilkins and G.B. Smith, J. Clin.

6, 223 (1986).
Psychopharmacol., -
34.
L. Baiocchi, A. Frigerio, M. Giannangeli and G.

Palazzo, Arzneim.-Forsch., 24, 1699 (1974).
35.
A. Adamus, M. Sansone, M. Melzacka and J. Vetulani, J.

Pharm. Pharmacol., 37, 504 (1985).
36.
S. Caccia, M. Ballabio, R. Samanin, M.G. Zanini and S.

Garattini, J. Pharm. Pharmacol., 33, 477 (1981).
37.
S. Caccia, M.H. Fong, S. Garattini and M.G. Zanini, J.

Pharm. Pharmacol., 34, 605 (1982).
38.
C. Yamato, T. Takahashi and T. Fujita, Xenobiotica, 4,

313 (1974).
39.
F. Pala, G. DeCosmo, A.F. Sabato and A. Bondoli,

Resuscitation, 9, 125 (1981).
40.
G. Musumarra, G. Scarlata, G. Romano, S. Clementi and

S. Wold, J. Chromatoyr. Sci., 22, 538 (1984).
41.
T. Daldrup, F. Susanto and P. Michalke, Fresenius Z.

Anal. Chem., 308, 413 (1981). CA:96,
- 29498m (1982).
42.
G. Musumarra. G . Scarlata. G. Cirma, G. Romano, S.

Palazzo, S. Clementi and G . Giulietti, J. Chromatogr.,
350, 151 (1985).
43.
P. Giovanni, C. DiNunzio, A. Scotto di Tella, P.

Schiano di Zenise and P. Ricci, Boll. SOC. Nat. Napoli,
93, 41 (1984). CA: 608, 126695k (1986).
44.
S. Putzolu, J.C. Pecknold, L. Baiocchi,

Psychopharmacol. Bull., 12, 40 (1976).
45.
W.H. Anderson and M.M. Archuleta, J. Anal. Toxicol., 8,

217 (1984).
729
46.
B. Munday, M.J. Kendal, M. Mitchard and T.A. Betts, Br.

J. Clin. Pharmacol., 2, 19 (1975).
47.
S. Caccia, M. Ballabio, R. Fanelli, G. Guiso and M.G.
48.
B. Gerson, S. Chan, F. Bell and K.M. Pappalardo, J.

20, 69 (1986).
Psychiat. Res., -
49.
R.L. Miller and C.L. Devane, J. Chromatogr., 374, 388

( 1986 1.
50.
S.H.Y. Wong, S.W. Waugh, M. Draz and N. Jain, Clin.

, 30, 230 (1984).
Chem.
- -
51.
P. Hochmuth, J.L. Robert, H. Schreck and R. Wennig

Simultaneous determination of benzodiazepines and
antidepressants in emergency toxicology by HPLC. In
"Topics in Forensic and Analytical Toxicology", Ed.
R.A.A. Mais, Elsevir, Amsterdam, 1984, pp. 143-149.
52.
I. Root and G.B. Ohlson, J. Anal. Toxicol., 8, 91

(1984).
53.
Zanini, J. Chromatogr., 210, 311 (1981).
I. Jane, A. McKiMon and R.J. Flanagan, J. Chromatogr.,

191 (1985).
323,
54.
S.H.Y. Wong and N. Marzouk, J. Liquid Chromatogr., 8,

1379 (1985).
55.
R. Caldwell and R.J. Flanagan, J. Chromatogr., 345, 187

(1985).
56.
R.N. Gupta and M. Lew, J. Chromatogr., 342, 442 (1985).
57.
R.F. Suckow, J. Liquid Chromatogr., 6, 2195 (1983).
58.
J.M. Stewart and S.R. Hall. In "The XTAL system of

crystallographic programs, Computer Science Centre,
University of Maryland, College Park, M.D., 1983.
730
8.
Acknowledgements
The authors would like to express sincere appreciation

to Mr. Peter Pavlakidis for performing the literature search
and providing some spectral data, to Mr. D. Leek for
determining and Dr. M. Sardessai, Dr. S . Reid and Dr. J.R.
Dimmock for assistance in analyzing the nuclear magnetic
resonance spectra, to Dr. G. McKay for determining the mass
spectra and to Dr. L. Delbaere, Dr. W. Quail and Mr. G.
Loewen for interpretation of X-ray diffraction data and
providing the three-dimensional structure of trazodone
hydrochloride. Special thanks are extended to Mrs. C.
Shuttleworth for typing this manuscript.
YOHIMBINE
A . G . Mehhawi* and A . A . Al-Ua&**
*vep&eitt
06
Phahmacagnany
**'Depcmhen.t 06 Humnaceu;ticcd ~ h m h h y
C o l l e g e 0 6 P h m a c y , King Saud UVLivmLty, P.U. Box 2 4 5 7 ,
Riyadh-] 1451 (Saudi Ahab,i.ul

VOLUME 16
73 1

All rights of reproduction in any form IeSeNed.
A. G . MEKKAWI AND A, A. AL-BADR
732
1.
Description
1.1
1.2
1.3
1.4
Nomenclature
Formulae
Molecular Weight
2.
Physical Properties
3.
Spectral Properties
4.
Natural Sources and Isolation
5.
Biosynthesis of Yohimbine
6.
Total Synthesis of Yohimbine
7.
Pharmacology and Medicinal Uses
8.
Methods of Analysis
8.1
8.2
Qualitative Analysis
Quantitative Analysis
8.2.1
8.2.2
8.2.3
8.2.4
8.2.5
Acknowledgements
References
Gravimetric Methods
Titrimetric Methods
Spectrofluorimetric Methods
YOHIMBINE
133
From t h e h i s t o r i c a l p o i n t of view yohimbine i s t h e most

i m p o r t a n t of t h e complex i n d o l e a l k a l o i d t y p e s . I t was known
s i n c e a n c i e n t t i m e s , i n t h e c u r d e drug b a r k o r t h e impure
form, as an a p h r o d i s i a c by t h e West A f r i c a n n a t i v e s .
It
o c c u r s i n v a r i o u s t r o p i c a l trees such P a u s i n y s t a l i a yohimbe
P i e r r e and r e l a t e d s p e i c e s , Aspidosperma quebrachoblanco
S c h l e c h t and as a minor a l k a l o i d i n some Rawaolfia
The f i r s t i s o l a t i o n of t h e drug w a s r e p o r t e d by S p i e g e l i n
1896 (1) and Witkop (2) i n 1943 s u g g e s t e d its c o r r e c t
constitution.
-.
1.
Description
1.1
Nomenclature
1.1.1
Chemical Name
17a-Hydroxyyohimban-16a-carboxylic
methyl e s t e r ( 3 , 4 ) .
1.1.2
acid
G e n e r i c Names
Quebrachine, Corynine, Aphrodine ( 3 , 4 , 5 , 6 ) .
1.1.3
a)
Yohimbine b a s e : T F6 D5 C666 E
M ONbTTTTJ T
Q
b)
1.2
.........
(4).
Yohimbine
D5 C666 E
--h y d r o c h l o r i d e .: MT F6
ON&TTTTJ T
Q UVOl &GH . . ( 4 ) .
Formulae
1.2.1
UVOl
Empirical
2 l H 263N2
A. G . MEKKAWI AND A. A. AL-BADR
134
1.2.2
Structural
10
11
1.3
OH
Molecular Weight
354.45
1.4
C 71.16%,
2.
H 7.39%,
N 7.90%,
0 13.54%.
Physical Properties
2.1
Color, Odour and Taste
2.2
Colorless needles when crystallized from ethanol,

colorless or white in bulk. Taste is bitter,
odourless ( 4 , 6 , 7 ) .
Melting Point
240'
2.3
241'.
Sublimation
Under vacuum the base sublimes at 159/0.01 mm (7).
2.4
Solubility
Almost insoluble in water, freely soluble in ethanol,
chloroform and sparingly soluble in diethyl ether
(4,6).
YOHIMBINE
2.5
735
Salts and Their Melting Point

The salts are well crystalline and the hydrochloride
is colorless plates m.p. 3oOo - 302OC , the
nitrate m.p. 269-270C, the tarkarate hexahydrate
m.p. 213OC, followed by solidification and
remelting at 278OC. The thiocyanate is of
rectangular crystals (m.p. 233' - 234OC) and the
colorless methiodide plates from acetone have the
m.p. 249 - 250OC. The melting point for the
0-acetate is 1 3 3 O C and the 0,N-diacetyl derivative
melting point is 1 8 3 O C ( 7 , 8 ) .
2.6
Specific Rotations of Base and Salts

Various specific rotations have been recorded for
the base. [ a ] i O+
EtOH ( 9 ) ;
+
3.
62.2'
107.9'
in pyridine;
56 in
in EtOH ( 7 ) ; the hydrochloride
103.3 (H20)
(7).
Spectral Properties
3.1
The U.V. spectra of yohimbine in methanol and
in 0.1 N H2SO4 were scanned from 200 to 400 nm using
Varian DMS 90 spectrophotometer and the following
maxima and (E 1%, 1 cm) values were found:
In methanol: 3 maxima at 289, 282 and 225 nm
were recorded. The (E 1%, 1 cdfound were : 210
at 289 nm, 247 at 282 nm and 1079 at 225 nm.
In the acidic solution a shift to lower wavelengths
with some hyperchromic effect was observed
(E 1%,1 an) at 280 nm was 225, at 272 nm was 233
and at 221 nm was 1110. Previous reports in 0.1 N
H2S04 (6) were as follows:
(E 1%, 1 an) at 278 nm 204; at 272 nm 208 and 220 nm
1064. Figure 1 illustrates the UV spectrum
behaviour for both the methanolic and the acidic
solutions of yohimbine.
736
YOHIMBINE
3.2
737
The i n f r a r e d spectrum of yohimbine h y d r o c h l o r i d e

i s p r e s e n t e d i n F i g u r e 2. The spectrum i s
o b t a i n e d from K B r d i s c on a Perkin-Elmer I n f r a r e d
Spectrophotometer model 580 B.
I t shows t h e
following :
-1
Frequency (cm )
Assignments
3540
OH
- stretch (free)
3190
NH
- stretch
CH
2900)
2960)
1730
stretch
C = 0
- stretch
C = C
- aromatic
1460)
1472))
1448)
Other c h a r a c t e r i s t i c a b s o r p t i o n bands are :
1172, 1150, 1025, 1000, 970, 750 and 702 cm-.
I R d a t a of yohimbine h y d r o c h l o r i d e have a l s o been
r e p o r t e d (3, 5 , 6 ) .
C l a r k e ( 6 ) r e p o r t e d t h e p r i n c i p l a l peaks a t
1705, 741, 1160 o r 1197 o r 1436 cm-1.
3.3
Nuclear Mangetic Reonance S p e c t r a (NMR)

3.3.1
P r o t o n Nuclear Magnetic Resonance (PMR)

Spectra
The PMR spectrum of yohimbine h y d r o c h l o r i d e
i n DMSO-db and i n DMSO-d6 p l u s D20 were
shown i n F i g u r e s 3a and 3b r e s p e c t i v e l y .
The s p e c t r a were r e c o r d e d on a Varian T60 A
NMR s p e c t r o m e t e r u s i n g t e t r a m e t h y l s i l a n e
Wavelength urn
100.
10 11 12 13 1461qm
90 *
80-
90
80
20
70,
Fac
10.
r- 0,
38003500 3ooo 2500 2000
Wavenumber
-10
1800
1600
1400
1200
Figure 2 : Infrared spectrum of yohimbine, HBr disc.
1000
800
625
139
YOHIMBINE
., . . . . , . .
1500
400
' I . ' . -
300
80
7.0
6.0
S.OPpM(r)L.O
30
2.0
1.0
0
Figure 3a: Proton magnetic resonance spectrum of yohimbine hydrochloride
in D M s 0 - d ~using TMS as reference standard.
200
. . . I . .
'7
I . . . . , .
. . I .
8.0
7.0
60
WPPM(~)L.O
30
2.0
I0
0
Pigurc 3b: Proton magnetic resonance spectrum of yohimbinc hydrochloride
in D M s 0 - d ~ PIUS
@ o using
TMS a5 reference standard.
740
(TMS) as reference standard. The structrual

assignments as follows:
Chemical
shift (PPMJ
Assignments
Multiplets at 0.7-4.60
CH2 and CH protons
Singlet at 3.60
CH3 protons
Multiplet at 6.7-7.4
Aromatic protons
Singlet at 4.05
NH (exchangable
with D20).
Singlet at 10.65
OH (exchangable
with D 2 0 ) .
Mills et a1 (3) have reported the

proton NMR spectrum of yohimbine.
3.4
Carbon-13 Nuclear Magnetic Resonance Spectrum

(C-13 NMR)
The proton-decoupled and off-resonance carbon-13
NMR spectrum of yohimbine have been determined on
Jeol FX 100 MHz spectrometer at an ambient
temperature and are presented in Figures 4 and 5
respectively. The sample was dissolved in
deuterated chloroform and DMSO-dC; using TMS as
reference standard. The carbon assignments are
based on chemical shift values and off-resonance
spectrum and are listed below:
OH
(OHIMBINE
741
sil
TMS
Figure4: Proton- decoupled carbon -13 NMR spectrum of yohimbine in

CDCI3 and D M s 0 - d ~using TMS as reference standard.
TMS
Figure 5 : Off- resonance carbon-13NMR spectrum of yohimbin inCDCi3

and DMSO-ds using TMS a reference standard.
A. 0.MEKKAWI AND A. A. AL-BADR
142
Chemical s h i f t ( 6 )
Multiplicity
Assignment
135.1
Singlet
c2
70.2
Doublet
c3
52.6
Triplet
21.7
Triplet
106.9
S i n gl e t
c7
127.0
Singlet
117.6
Doublet
118.5
Doublet
clo
120.5
Doublet
c11
111.0
Doublet
c12
136.1
S i n g1e t
13
34.7
Triplet
14
36.5
Doublet
15
52.4
Doublet
16
66.8
Doublet
17
31.9
Triplet
18
23.2
Triplet
19
39.8
Doublet
2 0
61.3
Triplet
c2 1
174.8
S i n g 1e t
c22
51.6
Quartet
2
3
YOHIMBINE
3.5
143
Mass S p e c t r a
The e l e c t r o n impact ( E I ) m a s s spectrum a t 70 e V
r e c o r d e d on Varian Mat 311 mass s p e c t r o m e t e r and
t h e chemical i o n i s a t i o n (CI) mass spectrum
o b t a i n e d w i t h F i n n i g a n 4000 mass s p e c t r o m e t e r are
shown i n F i g u r e s 6 and 7 r e s p e c t i v e l y . The E I
spectrum (Fig. 6 ) shows a b a s e peak a t m / e 353
and a n o t h e r peak a t m/e 354 (&) of almost e q u a l
abundance ( 9 8 % ) , o t h e r major fragments a r e a t m / e
184, m / e 169 and a t m / e 156. The C I spectrum
( F i g . 7) shows a b a s e peak a t m / e 355 (M+ + 1 ) .
Other fragments are a t m / e 337 and a t m / e 323.
Other m a s s spectrum d a t a h a s a l s o been r e p o r t e d
(3) *
4.
N a t u r a l Sources and I s o l a t i o n
Yohimbine i s t h e major a l k a l o i d of t h e bark of
P a u s i n y s t a l i a yohimba (Syn. Corynanthe yohimbe) and
a l s o p r e s e n t i n t h e r e l a t e d s p e c i e s p. m a c r o c e r a s ,
P. p a n i c u l a t a and 2. t r i l l e s i i ( f . Rubiaceae) ( 7 , 4 , 8 ) .
S e v e r a l Rauwolfia s p e c i e s w e r e r e p o r t e d t o c o n t a i n
yohimbine ( e . g . &. s e r p e n t i n a ) ( 4 , l O ) . &. c a f f r a ( l l ) ,
R. t e t r a p h y l l a (12) and &. v o m i t o r i a ( l 3 ) ,
( f . Apocynaceae).
The i s o l a t i o n of yohimbine from t h e b a r k of s p e c i e s
c o n t a i n i n g i t as t h e major a l k a l o i d can be done by t h e
method of L e H i r -e t a1 ( 8 ) , where yohimbine i n a
m i x t u r e w i t h t h e o t h e r isomers (a-yohimbine) w a s
b o i l e d w i t h d - t a r t a r i c a c i d , t h e yohimbine t a r t a r a t e
c r y s t a l l i z e d immediately while a-yohimbine t a r t a r a t e
remained i n s o l u t i o n and c r y s t a l l i z e d l a t e r . The
e x t r a c t i o n of t h e a l k a l o i d s from t h e i r n a t u r a l s o u r c e s
i s g e n e r a l l y c a r r i e d o u t by t h e c o n v e n t i o n a l method.
The drug powder i s t r e a t e d w i t h Na2C03, d r i e d and
e x t r a c t e d w i t h a s u i t a b l e o r g a n i c s o l v e n t e.g. benzene
Le H i r -e t a1 (8) a l s o used
o r chloroform (14,15).
column chromatographic s e p a r a t i o n . Court and h i s
s t u d e n t s (16-ZO), working on Rauwolfia s p e c i e s ,used
p r e p a r a t i v e t h i n - l a y e r chromatography as a r o u t i n e
t e c h n i q u e f o r t h e i s o l a t i o n and c h a r a c t e r i s a t i o n of
i n d o l e a l k a l o i d s . Although p r e p a r a t i v e HPLC w a s n o t
used i n t h e r o u t i n e i s o l a t i o n of such compounds b u t
t h e t e c h n i q u e might be of r o u t i n e u s e i n t h e f u t u r e .
A. G. MEKKAWI AND A. A. AL-BADR
744
-9904.
100.0-
50.0169
467'
100.0-
50.0-
18912.
100.
504
383
337
323
M E 300
320
340
360
380
400
420
440
Figure7 : Chemical ionization (CI )mass spectrum of yohimbine
YOHIMBINE
5.
745
Biosynthesis of Yohimbine
The major features of the probable biosynthetic pathway
to yohimbine were summarized by Atta-ur-Rahman and
Basha (21). Scheme 1 illustrates how yohimbine is
biosynthesized from tryptamine and secologanin. The
condensation of tryptamine [l] with secologanin [2] is
catalysed by the enzyme strictosidine synthetase which
controls the C-ring closure mechanism, and results in
the formation of strictosidine [3]. A second glycosidase
enzyme converts strictosidine via a series of reactive
intermediates [4]-[6] to 4,21-dehydrocorynantheine
aldehyde [7] by D-ring closure. This aldehyde can then
isomerize to [8] which cyclizes to yohimbine.
6.
Total Synthesis of Yohimbine

The following total synthesis of yohimbine was described
by Van Tamelen et a1 (22).
p-Benzophenone [l] was condensed with lY4-butadiene to
give the adduct 1,4,5,8,9,1O-cis-hexahydronaphthalene1,4-dione [2]. Selective reduction by zinc metal and
acetic acid gives the octahydronaphthalenedione [3]
which was converted to glycidic acid ester [4],
subsequent hydrolysis to the glycidic acid [5] and then
final decarboxylation to the ketoaldehyde [ 6 ] . The
ketoaldehyde [6] was oxidized to the corresponding ketoacid [7] using silver oxide in the presence of alkali.
The keto acid [7] was then converted to its acid
chloride [8] and the latter was reacted with tryptamine
[9] in the presence of pyridine yielding tryptamide [lo].
The tryptamide [lo] is then converted to the cis-diol
[ll] using solution of osmium tetraoxide in tetrahydrofuran
added to a solution of amide [lo] in pyridineTHF and cooled at dry-ice-acetone temperature. Hydrogenation of the keto-diol [ll] over Adams' catalyst in
ethanol gave a good yield of the desired triol [12].
Intermediate [12] was converted to the dialdehyde [13]
by cleavage of the triol amide [12] in aqueous acetone
and then was cyclized -in situ using phosphoric acid
and heating at 70, where intermediate [14] was obtained.
The lactol [14] was then transformed into the lactollactam methyl ether [15] by means of p-toluenesulfonic
acid catalyzed methanolysis. Lithium aluminium hydride
reduction of [15] in THF afforded the lactol ether base
[16]. Hydrolysis of [16] by means of aqueous hydrochloric
A. G.MEKKAWI AND A. A. AL-BADR
746
,O
c-0-cs3
11
+ H20
- glucose
Glu.
[21
I
+
+ HH 22 00 '
1
I
-Glucose
CB
oc
CH3-0-C
3 \\
II
OH
CH30-C
II
Scheme 1:
Biosynthesis of Yohimbine.
*v
,
I
OH
YOHIMBINE
-@
141
Zn-AcOH
Diels-Alder
t
--.Condn.
--
111
[21
COOEt
Aqueous
NaOH ( h o t )
under
nitrogen
Ethyl chloroacetate
K t-butoxide/benzene
H
[41
[31
COOH
(1) Cu powder i n d i e t h y l g l y c o l a t e 155 - 160'
Silver
oxide/alkali
H
H O
[61
[51
Oxalyl c h l o r i d e
HZ
.
)
i n pyridine
0
Scheme 2:
[91
T o t a l S y n t h e s i s of Yohimbine.
748
HL=O
OH
OH H
LAH
i n THF
OCH3
OH
[I61
Scheme 2:
[I71
Continued.
YOHIMBINE
749
285
[ 181
- 295'
OCOCH3
[ 191
Metaperiodate
Cleavage
o = c
'OCHO
H
Chromic anhydride at Oo/H2 SO4
Scheme 2 :
Continued.
[211
750
i- Chromatography
ii- L-camphorsulphonic
acid
CH3-O-C
'OH
' H
\\
Scheme 2.
[ 23
Continued.
YOHIMBINE
75 1
a c i d gave t h e l a c t o l b a s e 1171. The l a t t e r w a s t h e n

c o n v e r t e d t o t h e l a c t o l acetate [ 1 8 ] ( a s acetate s a l t )
by t r e a t m e n t w i t h a c e t i c a n h y d r i d e - p y r i d i n e .
The
a c e t a t e s a l t [18] w a s subjected t o pyrolysis c o n d i t i o n s ,
where a l i m i t e d amount of a c e t a t e s a l t under 0 . 0 1 mm
p r e s s u r e i n a s u b l i m e r was exposed t o a metal b a t h
p r e v i o u s l y h e a t e d t o 285-295',
where t h e e n o l e t h e r [19]
have been formed, a s t h e a c e t a t e s a l t . The' o l i f i n i c
bond i n t h e c y c l i c e n o l e t h e r system w a s o x i d i z e d u s i n g
osmium t e t r a o x i d e - p e r i o d a t e .
The ozmylation w a s c a r r i e d
i n t h e e n o l e t h e r a t - 7 8 O i n THF-pyridine, where t h e
d i o l [ 2 0 ] was o b t a i n e d . M e t a p e r i o d a t e c l e a v a g e of 1,2g l y c o l [ 2 0 ] gave t h e 0-formate of pseudoyohimbaldehyde
[ 2 1 ] . The c r u d e p e r i o d a t e was d i s s o l v e d i n methanola c e t o n e and t r e a t e d a t Oo w i t h chromic a n h y d r i d e
(2.0-3.5 e q u i v . ) i n s u l f u r i c a c i d f o r 30 m i n u t e s . A f t e r
removal of t h e forrnyl group, pseudoyohimbine [ 2 2 ] was
o b t a i n e d (low y i e l d ) u s i n g alumina chromatography.
The
r e s o l u t i o n of t h e s y n t h e t i c b a s e was accomplished by
means of l-camphor s u l p h o n i c a c i d where pseudoyohimbine
e p i m i e r i z e s t o yohimbine[23]. Scheme 2 i l l u s t r a t e s t h e
sequence of s y n t h e s i s d e s c r i b e d above.
7.
Pharmacology and Medicinal Uses

Yohimbine produces a c o m p e t i t i v e a - a d r e n e r g i c blockade
of l i m i t e d d u r a t i o n and i t i s r e l a t i v e l y s e l e c t i v e f o r
t h e presynaptic a2-receptor.
A t high doses i t a l s o
blocks postsynaptic a-adrenergic receptors.
It a l s o
b l o c k s 5-HT r e c e p t o r s . It h a s a l i t t l e e f f e c t on
smooth muscle and r e a d i l y p e n e t r a t e s t h e c e n t r a l n e r v o u s
system and produces a complex p a t t e r n of r e s p o n s e s i n
d o s e s lower t h a n t h e r e q u i r e d t o produce p e r i p h e r a l aa d r e n e r g i c blockade. These i n c l u d e v a s o p r e s s i n release
and hence m a n i f e s t s an a n t i d i u r e t i c e f f e c t . G e n e r a l
CNS e x c i t a t i o n i n c l u d i n g e l e v a t e d blood p r e s s u r e ,
i n c r e a s e d h e a r t r a t e , i n c r e a s e d motor a c t i v i t y , i r r i t a b i l i t y and t r e m o r s a l s o occured. P a r e n t r a l a d m i n i s t r a t i o n may i n d u c e s w e a t i n g , n a u s e a and vomiting (23 - 3 3 ) .
L e c r u b i e r e t a1 (29) i n v e s t i g a t e d t h e p o s s i b l e u s e of
yohimbine t o improve t h e c o n d i t i o n of o r t h o s t a t i c
hypotension induced by clomipramine. The c o n t r o v e r s i a l
a p h r o d i s i a c p r o p e r t y of yohimbine was r e c e n t l y
i n v e s t i g a t e d by many a u t h o r s . Morales e t a1 (30)
r e p o r t e d t h e a b i l i t y of 6 mg of yohimbine, g i v e n 3 times
a day, t o improve p a r a t h e s i a of t h e lower limbs i n 4
p a t i e n t s o u t of 6 d i a b e t i c p a t i e n t s . S t e e r s e t a1 (31)
152
s t u d i e d t h e pharmacological e f f e c t s of yohimbine i n
p e n i s and
the penis,
yohimbine e x h i b i t s a - a d r e n e r g i c b l o c k i n g p r o p e r t i e s and
does n o t a f f e c t catecholamine l e v e l i n t h i s t i s s u e .
Condra e t a 1 (32) s t u d i e d t h e e f f e c t i v e n e s s of yohimbine
i n t h e t r e a t m e n t of impotence and Nakatau e t a1 ( 3 3 ) have
s t u d i e d t h e pharmacokinetics of yohimbine i n man.
However, t h e p o s s i b l e e f f e c t i v e n e s s of yohimbine as an
a p h r o d i s i a c , a t l e a s t i n r a t s , h a s now r e c e i v e d s u p p o r t
from a s t u d y c a r r i e d o u t b y Clark e t a1 ( 3 4 ) . The
teams of Condra and Nakatau ( 3 2 , 3 3 ) found t h a t i n
p a t i e n t s w i t h c l e a r o r g a n i c impotence, t r e a t m e n t w i t h
yohimbine h y d r o c h l o r i d e e x h i b i t e d a p o s i t i v e r e s p o n s e
r a t e of approximately 30% and p o s t u l a t e d t h a t t h e
f a i l u r e t o improve t h e c o n d i t i o n s of t h e o t h e r two t h i r d s
of p a t i e n t s may be due t o i n t e r i n d i v i d u a l d i f f e r e n c e s
i n t h e pharmacokinetics of t h e drug which a p p e a r s t o be
c l e a r e d i n i n d i v i d u a l s by metabolism.
-i n - v i t r o p r e p a r a t i o n s of human and r a b b i t
i-n v i v o on r a b b i t s and concluded t h a t , i n
8.
8.1
Q u a l i t a t i v e Analysis
8.1.1
Color Tests
Yohimbine when r e a c t e d upon by ammonium
molybdate g i v e s a b l u e c o l o r t h a t changes
slowly t o green ( s e n s i t i v i t y 0.025 pg);
w i t h ammonium vanadate t e s t i t g i v e s a b l u e
changing t o preen and f a d i n g away
( s e n s i t i v i t y i s 0.1 pg). With V i t a l i ' s t e s t
t h e c o l o u r sequence i s y e l l o w / y e l l o w / v i o l e t
( 6 ) . Other n o n - s p e c i f i c t e s t s a r e t h e
x a n t h y d r o l r e a g e n t and 4-dimethylamino
benzaldehyde i n m e t h a n o l i c HC1.
8.1.2
M i c r o c r y s t a l Tests
Wachsmuth (35) determined t h e p r e c i p i t a t i n g
p r o p e r t i e s of f l a v i n i c a c i d ( i n e t h a n o l d i e t h y l e t h e r s o l u t i o n ) on s e v e r a l a l k a l o i d a l
and o t h e r n i t r o g e n c o n t a i n i n g s u b s t a n c e s
and r e p o r t e d t h a t abundant p r e c i p i t a t e s were
o b t a i n e d w i t h yohimbine ( s e n s i t i v i t y 1:lOO).
Habib and Aziz ( 3 6 ) found t h a t yohimbine
g i v e s i r r e g u l a r l a r g e r o s e t t e s of curved
h a i r s w i t h 0.5% aqueous ammonium r e i n e c k a t e
YOHIMBINE
153
which can be i d e n t i f i e d m i c r o s c o p i c a l l y .
With potassium c y a n i d e s o l u t i o n , y o h i m b i n e
g i v e s r o s e t t e s of r o d s and w i t h sodium
c a r b o n a t e s o l u t i o n i t g i v e s r o s e t t e s of r o d s
o r dense r o s e t t e s ( s e n s i t i v i t y i n b o t h
r e a g e n t s 1: 1500) ( 4 ) . C h a r a c t e r i s t i c
c r y s t a l s with d i f f e r e n t reagents obtained i n
o u r l a b o r a t o r y , are i l l u s t r a t e d i n F i g u r e s
8-17.
8.2
Q u a n t i t a t i v e Analysis
8.2.1
G r a v i m e t r i c Methods
Raymond (11) reviewed methods used a t h i s
t i m e i n gravimetric analysis. He extracted
powdered bark of yohimbehe w i t h e t h e r :
chloroform ( 4 : l ) m i x t u r e u s i n g 10 m l of
s o l v e n t f o r each gram of b a r k m a t e r i a l
Feldhoff (15) m o d i f i e d t h e t e c h n i q u e s u s e d
b e f o r e him by l i b e r a t i n g t h e b a s e s i n t h e
w i t h Na2C03 t r e a t m e n t
bark of Yohimbe
and e x t r a c t i n g t h e d r u g i n a s o x h l e t w i t h
d i - o r - t r i c h l o r o e t h y l e n e mixed i n t h e
r e c e i v e r w i t h aqueous o x a l i c a c i d s o l u t i o n .
The o r g a n i c s o l v e n t was e v a p o r a t e d , t h e
aqueous a c i d s o l u t i o n w a s t r e a t e d w i t h NH40H
s o l u t i o n t o l i b e r a t e t h e a l k a l o i d s which
were t h e n e x t r a c t e d w i t h e t h e r . The a l k a l o i d s were p r e c i p i t a t e d w i t h an a l c o h o l i c
s o l u t i o n of H C 1 i n a b e a k e r and t h e aqueous
a l c o h o l i c p a r t was washed twice w i t h e t h e r
and methyl a c e t a t e . The secondary a l k a l o i d s
remained i n s o l u t i o n a f t e r c r y s t a l l i s a t i o n
of yohimbine HC1.
A f t e r b e i n g l e f t 24 h o u r s
i n a r e f r i g e r a t o r , t h e c r y s t a l l i n e yohimbineH C l was f i l t e r e d on a weighed f i l t e r p a p e r ,
washed and d r i e d a t 102% t o c o n s t a n t w e i g h t .
8.2.2
Precipitation Titration
V y t r a s and Riha (37) c a r r i e d o u t p r e c i p i t a t i o n t i t r i m e t r y for 9 alkaloids including
yohimbine u s i n g sodium t e t r a p h e n y l b o r a t e
r e a g e n t and a C r y t u r valinomycin i o n selective electrode.
754
Figure8: Rosettes of circular plates of yohimbine with picric

acide reagent ( a f t e r 3 minutes
1.
Figure91 Radiating pointing plates of phimbine with mercuric

chloride reagent ( a f t e r 14minutes).
Figure 10 : Orange coloured radiating relatively wide crystal

plates of yohimbine with potassium chromate reagent
(after 9 minutes ).
YOHIMBINE
155
Figurell
Tagged plates of crystals of yohirnbine with

Dragendorf f's reagent ( after 5 minutes ),
Figure12. White crystalline minute rods of yohimbine after

reaction w i t h lead iodide reagent (immediately
produced )
Figure 13: Crystal of tetragonal or hexagonal structures

a f t e r addition of Marme's reagent (after 5 minutes).
156
Figure 14
Radiating rod clusters of yohlrnblne and Naf03

solution (after 10 minutes)
Figure IS
Crystal plates, characteristic, to reaction of

yohimbine with NH4S CN aqueous solution
( a f t e r 5 - 7 minutes),
Figure
16:immediate sandy crystals w i t h Wagner's reagent.
Figure 17. Fine clusters of rods with KCN solution (after 15minutes)
YOHIMBINE
b)
M i cr 0- he t e r ome t r i c T i t r a t i o n
I n t h e i r t r i a l t o analyze l a r g e organic
n i t r o g e n - c o n t a i n i n g compounds, Bobtelsky
and B r a z i l y (38) s t u d i e d t h e b e h a v i o u r of
micro amounts of yohimbine t o g e t h e r w i t h
n i t r o n , quinone , s t r y c h n i n e , p a p a v e r i n e ,
n i c o t i n e , a t r o p i n e , morphine o r phenanthrol i n e when t i t r a t e d h e t e r o m e t r i c a l l y w i t h
t u n g s t o s i l i c i c , tungstophosphoric o r
molybdophosphoric a c i d a t pH 1 o r 7. They
found t h a t t h e i n f l u e n c e of t h e a c i d i t y of
t h e s o l u t i o n was d i f f e r e n t i f t h e o r g a n i c
molecule c o n t a i n e d 1 o r 2 h e t e r o c y c l i c N
atoms.
8.2.3
S p e c t r o p h o t o m e t r i c Methods
a)
Colorimetry
C o l o r i m e t r i c a s s a y s of t h e weakly b a s i c
t e r t i a r y a l k a l o i d w e r e f i r s t done on c o l o r
r e a c t i o n s c h a r a c t e r i s t i c of t h e i n d o l e
group ( 8 , 29). I n p r e s e n c e of o t h e r i n d o l e
c o n t a i n i n g compounds, such as t h o s e n a t u r a l l y
present i n t h e n a t u r a l source,such a
method i s of l i t t l e v a l u e . However, Kolsck
(39) used t h e c o l o u r r e a c t i o n of yohimbine
i n d o l e n u c l e u s w i t h p-dimethylaminobenzaldehyde and measured t h e c o l o u r produced
p h o t o m e t r i c a l l y w i t h f i l t e r S 39 o r S 49.
The method i s summarized a s f o l l o w s :
Yohimbine-HC1 w a s d i s s o l v e d i n water, i f
d e s i r e d w i t h a d d i t i o n of l i t t l e e t h a n o l .
I n a 10 m l v o l u m e t r i c f l a s k t o 1 m l of
t h e s o l u t i o n c o n t a i n i n g 0.05 - 3 mp, of t h e
allcaloid s a l t , 2 m l of p-dimethylylaminobenzaldehyde w e r e added and t h e m i x t u r e w a s
l e f t t o c o o l f o r 10 minutes and 0.05 m l
of a 5% H202 ( o r 1%NaN02) s o l u t i o n w a s
added, shaken, and t h e c o l o r w a s allowed
t o develop. To t h i s , d i s t i l l e d water was
added t o make 10 m l of m i x t u r e and d e n s i t y
was measured p h o t o m e t r i c a l l y u s i n g a f i l t e r
S 39 o r S 49.
The mechanism of t h i s
r e a c t i o n w a s s t u d i e d by P i n d u r (40).
758
Jung assayed reserpine and yohimbine

( 4 1 , 4 2 ) photometrically with xanthydrol
reagent in presence of other nitrogen
containing compounds. The indole nucleus of
the two compounds forms a colored compound
with xanthydrol and the extinction is
measured at 515 mu. In this method a
50-150 p g of yohimbine were dissolved in
5 ml of a freshly prepared solution o f 0 . 4 %
xanthydrol in glacial acetic acid Containing
1% v/v HC1 heated on a boiling water bath
for 15 minutes, cooled and the extinction
wasmeasured at 515 mu. The color is stable
for 5 hr. The procedure can be used for
tablets, injections or mixtures after
extracting the alkaloid by a suitable method
The method was also used by Budavaris
a1 ( 4 3 ) .
The yohimbine-methyl orange complex color
as a quantitative method was utilized by
Mohamed and Elsayed ( 4 4 ) for assaying the
base in tablet formulations. A solution of
the powdered tablets ( e 5 0 mg of yohimbine
hydrochloride) in 50 ml of H20 was prepared
A measured volume containing about 300 mg
of tablet base was diluted to 5 0 ml. A 2 m
portion of this solution was transferred to
a separating-funnel containing 2 ml of
buffer (pH 4 . 0 ) and 1 ml of 0.1% methyl
orange solution in 20% ethanol. The
yohimbine-methyl orange complex was extracted
successively with chloroform, diluted to
25 ml with chloroform and the extinction was
measured at 421 nm. For 5 determinations
the recovery was 97.6 - 102.5%.
8.2.4
Spectrofluorimetric Methods
In their spectrophotofluorimetric study
of a vast number of organic compounds of
pharmaceutical interest, Underfried et a1
( 4 5 ) using a Xe arc - source emitting a
continuum from 200-800 mu in their spectrofluorometer,reported that yohimbine at pH 7
had its maximum activation at 270 mu and
its maximum emission of fluorescence at
360 mu.
YOHIMBINE
759
Chiang and Chen (46) assayed yohimbine

after increasing its fluorescence
in solutions by subjecting its heated
solution in presence of H202 to ultraviolet
light. Auterhoff and Schroeder ( 4 7 ) carried
a similar assay and evaluated yohimbine in
Yohimbe bark. They extracted 1 g of the
bark with ethyl ether after alkalinisation
with 10% aqueous ammonia. The ethereal
extract was re-extracted with 0.1 N H2SO4
solution and the acid solution was basified
with NH40H, extracted with chloroform,
concentrated and diluted to 10 ml with
chloroform. The chloroformic extract was
chromatographed on a MN 300 Polygram
cellulose plate impregnated with formamide :
dimethylformamide : acetone (4:3:12) and
developed with heptane : ethylmethyl ketone :
0.25% aqueous ammonia (4:2:1) for 15 cm
and the zone of Rf 0.2, correspondin? to
yohimbine, was extracted with CHC13 : CH3OH
(100 ml).
The extract was filtered, the
solvent removed under reduced pressure at
35OC and the residue dissolved in 10 ml of
30% acetic acid. To 1 ml of this solution,
1 ml of 3% H202 and 4 ml of 30% acetic acid
were added. A similar solution of standard
yohimbine was prepared. Both solutions
were heated for 2 hr at 80C, cooled and
the volume was adjusted to 10 ml with 30%
acetic acid. After excitation at 365 nm
the fluorescence was measured at 496 nm.
Clarke ( 6 ) reported the quantitative
estimation of yohimbine as follows:
The drug when in a mixture consisting of
reserpine, ajmaline and yohimbine may be
separated by thin-layer chromatography and
determined by spectrophotometry or
spectrofluorometry as described previously
for indole alkaloids by some authors (48,49)
760
8.2.5
8.2.5.1
Paper Chromatography (P.L.C.)
Z a f f a r o n i ' s system (50) w a s t r i e d by

Machovicova ( 51 ) on p a p e r chromatography
and was found t o be s u i t a b l e f o r t h e
s e p a r a t i o n of r e s e r p i n e , r e s e r p i c a c i d and
yohimbine.
Q u a n t i t a t i o n of yohimbine u s i n g
P.L.C. by Auterhoff and Schroeder (47) i s
d e s c r i b e d i n s e c t i o n 8.2.4 above
C l a r k e (6) r e p o r t e d t h e f o l l o w i n g paper
chromatography systems f o r t h e s e p a r a t i o n
of yohimbine:
a)
Paper: Whatman No. 1 , s h e e t 16 x 4 i n c h ,

b u f f e r e d by d r i p p i n g i n a 5% s o l u t i o n
of sodium dihydrogen c i t r a t e , b l o t t i n g ,
( I t can
and d r y i n g a t 25' f o r 1 hour.
be s t o r e d i n d e f i n i t e l y ) .
Sample: 2 . 5 ~1 of a 1X s o l u t i o n ; i n 2 N
a c e t i c acid i f possible, otherwise i n
2 N h y d r o c h l o r i c , 2 N sodium h y d r o x i d e ,
or ethanol.
S o l v e n t : 4.8 of c i t r i c a c i d i n a
m i x t u r e of 130 m l of w a t e r and 870 m l
of n-butanol.
T h i s may be used f o r
s e v e r a l weeks i f w a t e r added from t i m e
t o time t o keep t h e s p e c i f i c g r a v i t y
a t 0.843 t o 0.844.
Development: Ascending, i n a t a n k 8 x 11
x 15% i n c h , 4 s h e e t s b e i n g run a t a t i m e .
Time o f r u n , 5 hour.
Location: Under u l t r a v i o l e t l i g h t , p a l e
blue fluorescence; location reagent :
i o d o p l a t i n a t e s p r a y , week r e a c t i o n ;
bromocresol green s p r a y ; weak r e a c t i o n .
b)
Paper: Whatman No. 1 o r No. 3 , s h e e t

1 7 x 19 cm, impregnated by d i p p i n g i n a
10% s o l u t i o n of t r i b u t y r i n i n a c e t o n e and
drying i n a i r .
YOHIMBINE
76 1
Sample: 5 1-11of 1 t o 5% s o l u t i o n i s
e t h a n o l o r chloroform.
Solvent:
Acetate b u f f e r (pH 4.58).
E q u i l i b r a t i o n : The beaker c o n t a i n i n g
the solvent i s equilibrated i n a
t h e r m o s t a t i c a l l y c o n t r o l l e d oven a t 95'
f o r about 15 m i n u t e s .
Development: Ascending. The p a p e r i s
f o l d e d i n t o a c y l i n d e r and c l i p p e d , t h e
c y l i n d e r i s i n s e r t e d i n t h e beaker
c o n t a i n i n g t h e s o l v e n t which i s n o t
removed from t h e oven. A p l a t e - g l a s s
d i s k t h i c k l y smeared w i t h s i l i c o n e
g r e a s e may s e r v e a s a cover. T i m e of
r u n , 15 t o 20 m i n u t e s .
L o c a t i o n : Under u l t r a v i o l e t l i g h t ,
blue fluorescence.
Rf v a l u e 0 . 6 4 .
c)
Paper and Sample: A s above i n ( b )

Solvent:
Phosphate b u f f e r (pH 7 . 4 ) .
E q u i l i b r a t i o n : The beaker c o n t a i n i n g
the solvent i s equilibrated i n a
t h e r m o s t a t i c a l l y c o n t r o l l e d oven a t 86'
f o r about 15 m i n u t e s .
Development:
Location:
0.04.
8.2.5.2
A s above i n (b)
A s above i n ( b ) .
Thin-Layer
.
Rf v a l u e
Chromatography (T.L.C.)
S i l i c a g e l G. p l a t e s p r e a p r e d w i t h 0 . 1 M
NaOH o r 0.1 M KHSO4 developed by 5 s o l v e n t
systems were used by Fike (52) i n t h e s t u d y
of t h e b e h a v i o u r of 140 b a s i c d r u g s
i n c l u d i n g yohimbine and t h e Rf v a l u e s of
t h o s e d r u g s w e r e r e c o r d e d and s u c c e s s f u l
s e p a r a t i o n w a s achieved b u t none of t h e
i n d o l e a l k a l o i d s r e l a t e d t o yohimbine w e r e
i n c l u d e d i n t h e s t u d y . However, Court and
162
A. 0.MEKKAWI AND A. A, AL-BADR
and Habib (16) and Court and Timmins ( 1 7 ) ,

i n t h e i r s t u d i e s on Rauwolfia a l k a l o i d s
and t h e i r behaviour on T.L.C. have piven
v a l u a b l e i n f o r m a t i o n s on t h e c o l o u r r e a c t i o n s
and R f v a l u e s of such r e l a t e d a l k a l o i d s on
s i l i c a g e l l a y e r s employing 10 s o l v e n t
systems and emphasis can b e c o n c e n t r a t e d
on t h e r e l a t e d a l k a l o i d s :
Yohimbine, a-yohimbine, s e r p e n t i n e , s a r p a g i n e
and a j m a l i c i n e which come i n t h e s t r o n g l y
b a s i c f r a c t i o n of Rauwolfia r o o t e x t r a c t .
Q u a n t i t a t i o n p h o t o d e n s i t o m e t r i c a l l y on
s i l i c a g e l l a y e r s of a l k a l o i d s of p l a n t
s o u r c e s w a s c a r r i e d o u t by Massa e t a1 (53)
who found t h a t f l u o r s c e n c e r e f l e c t a n c e
r e a d i n g s of unsprayed s p o t s of f l u o r e s c e n t
a l k a l o i d s were f a v o u r a b l e i n a c t i v a t e d
s i l i c a g e l p l a t e s . They used a double-beam
p h o t o d e n s i t o m e t e r w i t h r e c o r d e r and
i n t e g r a t o r f u n c t i o n i n g i n t h e v i s i b l e and
u l t r a v i o l e t l i g h t s . Nine s o l v e n t s systems were
used and s u i t a b l e c o n c e n t r a t i o n s of
s t a n d a r d s were r u n and t h e s t u d y w a s done
on 25 a l k a l o i d s of which 1 4 a l k a l o i d s
sprayed w i t h Dragendorff-Schuette r e a g e n t ,
were d e t e c t e d a t 400 nm and 11 o t h e r s were
determined by i n h i b i t i o n of f l u o r e s c e n c e
a t 528 nm and 13 a l k a l o i d s e s t i m a t e d by
t h e i r n a t u r a l f l u o r e s c e n c e o r induced by
a s u i t a b l e r e a g e n t such as KMnO4, I o d i n e o r
d i c h l o r o a c e t i c a c i d and h e a t , and e x c i t e d
a t 358 nm. However, Court and Habib (16)
q u a n t i t a t e d 10 Rauwolfia a l k a l o i d s by
combined p r e p a r a t i v e T.L.C. and c o l o r i m e t r y
and found t h a t t h e i r method w a s b e s t
s u i t a b l e f o r B-carboline a l k a l o i d s .
Clarke ( 6 ) d e s c r i b e d t h e f o l l o w i n g TLC
system f o r t h e i s o l a t i o n of yohimbine:
P l a t e : Glass p l a t e s 20 x 20 c m , c o a t e d
w i t h a s l u r r y c o n s i s t i n g of 30 g of s i l i c a
p,el G i n 60 m l of w a t e r t o g i v e a l a y e r
0.25 mm t h i c k and d r i e d a t l l O o f o r 1 h o u r .
YOHIMBINE
163
Sample:
acid.
Solvent:
1 p1 of a 1%s o l u t i o n i n 2 N a c e t i c
S t r o n g ammonia s o l u t i o n : methanol
I t should be changed a f t e r
(1.5 : 1 0 0 ) .
two r u n s .
E q u i l i b r a t i o n : The s o l v e n t i s allowed t o
s t a n d i n t h e t a n k f o r 1 hour.
Development: Ascending, i n a t a n k 2 1 x 2 1 x
10 cm, t h e end of t h e t a n k b e i n g covered
w i t h f i l t e r paper t o a s s i s t e v a p o r a t i o n .
Time of run: 30 m i n u t e s .
Location reagent: Acidified i d o p l a t i n a t e
Rf v a l u e 0.55.
spray, p o s i t i v e reaction.
8.2.5.3
(G.L.C.)
Dusci and Hackett (54) d e s c r i b e d a d i r e c t

p r o c e d u r e f o r a n a l y s i s of yohimbine and
o t h e r n e u t r a l drugs i n t i s s u e s . They used
Hewlett-Packard gas chromatograph model
5700A equipped w i t h a f l a m e i o n i z a t i o n
d e t e c t o r . The column w a s a 4 f t x 4 mm i . d .
g l a s s column pakced w i t h 3% OV-17 on Gas
Chrom Q, 80-100 mesh. The i n s t r u m e n t
s e t t i n g s were a s f o l l o w s :
I n j e c t i o n p o r t t e m p e r a t u r e , 25OoC; d e t e c t o r
t e m p e r a t u r e , 30OoC; n i t r o g e n c a r r i e r g a s
flow r a t e , 60 ml/min.
Oven t e m p e r a t u r e
programmed as 15OOC h e l d f o r 2 m i n u t e s ,
i n c r e a s e d a t 8OC/min t o 29OoC and t h e
f i n a l t e m p e r a t u r e was h e l d f o r 4 m i n u t e s .
Using c h o l e s t e r o l a s a s t a n d a r d , t h e r e l a t i v e
r e t e n t i o n t i m e (RRT) of yohimbine was 0.89.
C l a r k e ( 6 ) r e p o r t e d t h e f o l l o w i n g GC system
f o r t h e s e p a r a t i o n of yohimbine.
5% SE-30 on 60-80 mesh Chromosorb
5 f t x 118 i n c h i n t e r n a l d i a m e t e r
s t a i n l e s s s t e e l column.
Column:
W AW,
Column t e m p e r a t u r e :
Carrier Gas:
23OoC.
Nitrogen.
764
Gas Flow:
30.7 m l p e r minute.
D e t e c t o r : Flame i o n i z a t i o n d e t e c t o r ,
hydrogen 22 m l p e r minute.
Retention t i m e :
8.2.5.4
0.38 r e l a t i v e t o c o d e i n e .
High-speed L i q u i d Chromatography(HPLC)
The advent of HPLC i n t h e f i e l d o f s e p a r a t i o n

and q u a n t i t a t i o n of almost a l l t y p e s of
chemical m i x t u r e s made t h e t a s k s of t h e
a n a l y t i c a l chemists f a r e a s i e r t h a n two
decades ago. HPLC on a l k a l o i d s u s i n g
v a r i o u s t y p e s of chromatographic s e p a r a t i o n
modes w a s c a r r i e d o u t by v a r i o u s a u t h o r s .
Yohimbine s e p a r a t i o n on a d o s r p t i o n HPLC
w a s s u c c e s s f u l and V e r p o o r t e and Svendsen
( 5 5 ) were a b l e t o s e p a r a t e i t from r e s e r p i n e
on Mercksorb S 1 70 ( 5 pm) column u s i n g 6
systems of e l u e n t and a Packard Model 8200
l i q u i d chromatograph equipped w i t h a 254 nm
and 280 nm UV d e t e c t o r w a s used. The
column w a s a s t a i n l e s s s t e e l t u b e (30 cm x
2 mm i.d.). The column t e m p e r a t u r e w a s
maintained a t 2OoC and p r e s s u r e s of 50-250
kg/cm2 were employed t o c o n t r o l flow rates.
However, t h e r e l a t i v e l y high[E 1%,1 cm]of
yohimbine i n U.V. l i g h t a t 226 nm makes t h e
d e t e c t i o n of minute q u a n t i t i e s p o s s i b l e i f
t h e i n s t r u m e n t U.V. wavelength can be
v a r i e d t o t h e optimum. Rodgers ( 5 6 ) w a s
a b l e t o a n a l y s e s e v e r a l classes of t r a n q u i l i z e r drugs i n c l u d i n g Rauwolfia a l k a l o i d s
by t h i s t e c h n i q u e u s i n g a d s o r p t i o n columns of
s i l i c a g e l and c a t i o n exchangers.
Acknowledgements
The a u t h o r would
of Pharmacognosy
and t o T a n v i r A.
Dept. f o r t y p i n g
l i k e t o thank M r . Amir S h e h a t a (B. Pharm.)

Dept. f o r performing t h e m i c r o c r y s t a l t e s t s
B u t t , S e c r e t a r y t o P h a r m a c e u t i c a l Chemistry
t h e manuscript.
YOHIMBINE
765
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54. Dusci, L.J. and Hackett, L . P . ,
353-358 (1977).
Clinical Toxicolopy, g(3)
55. Verpoorte, R. and Svendsen, A . B . ,

227-230 (1974).
J. Chromatog.,
56. Rodgers, D.H., J . Chromatogr. Sci., g ( 1 2 ) ,

(1974).
100,
742-746
CUMULATIVE INDEX
Bold numerals refer to volume numbers
Acetaminophen, 3, I ; 14, 551

Acetohexamide, 1, I ; 2, 573
Allopurinol, 7, I
Alpha-tocopheryl acetate, 3, 1 1 1
Amantadine, 12, 1
Amikacin sulfate, 12, 37
Amiloride hydrochloride, 15, I
Arninoglutethimide. 15, 35
Aminophylline. 11, I
Aminosalicylic acid, 10, I
Amitriptyline hydrochloride, 3, 127
Amoxicillin, 7, 19
Amphotericin B, 6, 1: 7, 502
Ampicillin, 2, I ; 4, 518
Ascorbic acid, 11, 45
Aspirin, 8, I
Atenolol. 13, 1
Atropine, 14, 32
Azathioprine, 10, 29
Bacitracin, 9, 1
Baclofen, 14, 527
Bendroflumethiazide, 5, 1; 6, 597
Benperidol 14, 245
Benzocaine, 12, 73
Benzyl benzoate, 10, 55
Betamethasone dipropionate, 6, 43
Bretylium tosylate, 9, 71
Bromazepam, 16, I
Bromocriptine methanesulfonate, 8, 47
Busulphan, 16, 53
Caffeine, 15, 71
Calcitriol, 8, 83
Camphor, 13, 27
Captopril, 11, 79
Carbamazepine. 9, 87
Cefaclor, 9, 107
Cefamandole nafate, 9, 125; 10, 729
Cefazolin, 4, 1
Cefotaxime, 11, 139
Cefoxitin. sodium, 11, 169
Cephalexin, 4, 21
Cephalothin sodium, 1, 319
Cephradine, 5, 21
Chloral hydrate, 2, 85
Chlorambucil, 16, 85
Chloramphenicol, 4,47, 518; 15, 701
Chlordiazepoxide, I, 15
Chlordiazepoxide hydrochloride, 1, 39; 4, 518
Chloroquine, 13, 95
Chloroquine phosphate, 5,61
Chlorpheniramine maleate, 7, 43
Chlorprothixene, 2, 63
Chlortetracycline hydrochloride. 8, 101
Chlorthalidone, 14, I
Chlorzoxazone, 16, 119
Cholecalciferol. see Vitamin D3

Cimetidine, 13, 127
Cisplatin. 14, 77 : 15, 7%
Clidinium bromide, 2, 145
Clindamycin hydrochloride, 10, 75
Clofibrate, 11, 197
Clonazepam, 6,61
Clorazepate dipotassiurn, 4, 91
Clotrimazole, 11, 225
Cloxacillin sodium, 4, 113
Cocaine hydrochloride, 15, 15 I
Codeine phosphate, 10,93
Colchicine, 10, 139
Cyanocobalamin, 10, 183
Cyclizine, 6, 83; 7, 502
Cycloserine, 1,53
Cyclosporine, 16, 145

Cyclothiazide.1, 66
Cypropheptadine, 9, 155
Dapsone, 5, 87
Dexamethasone, 2, 163; 4, 519
Diatrizoic acid, 4, 137; 5, 556
Diazepam, 1,79: 4, 518
Dibenzepin hydrochloride, 9, 181
Dibucaine and dibucaine hydrochloride, 12, 105
Diflunisal, 14, 491
Digitoxin, 3, 149
Digoxin, 9, 207
Dihydroergotoxine methanesulfonate, 7, 81
769
770
Dioctyl sodium sulfosuccinate, 2, 199: 12, 713
Diperodon, 6 , 9 9
Diphenhydramine hydrochloride, 3, 173
Diphenoxylate hydrochloride, 7, 149
Disopyramide phosphate, 13, 183
Disulfiram, 4, 168
Dobutamine hydrochloride, 8, 139
Dopamine hydrochloride. 11, 257
Doxorubicine. 9, 245
Droperidol, 7, 171
Echothiophate iodide, 3, 233
Emetine hydrochloride, 10, 289
Enalapril maleate, 16, 207
Ephedrine hydrochloride, 15, 233
Epinephrine, 7, 193
Ergonovine maleate, 11, 273
Ergotamine tartrate, 6, I13
Erythromycin. 8, 159
Erythromycin estolate, 1, 101; 2, 573
Estradiol. 15, 283
Estradiol valerate, 4, 192
Estrone, 12, 135
Ethambutol hydrochloride, 7, 231
Ethynodiol diacetate, 3, 253
Etomidate. 12, 191
Fenoprofen calcium 6, 161
Flucytosine, 5, 115
Fludrocortisone acetate, 3, 281
Flufenamic acid, 11, 313
Fluorouracil, 2, 221
Fluoxymesterone, 7, 25 I
Fluphenazine decanoate. 9, 275; 10, 730
Fluphenazine enanthate, 2, 245; 4, 524
Fluphenazine hydrochloride, 2, 263; 4, 519
Flurazepam hydrochloride, 3, 307
Gentamicin sulfate, 9, 295; 10, 731
Glibenclamide, 10, 337
Gluthethimide, 5, 139
Gramicidin, 8, 179
Griseofulvin, 8, 219; 9, 583
Guanabenz acetate. 15, 319
Halcinonide, 8, 251
Haloperidol, 9, 341
Halothane, 1, 119; 2, 573; 14, 597
Heparin sodium, 12, 215
Heroin, 10, 357
Hexestrol, 11, 347
Hexetidine, 7, 277
Homatropine hydrobromide, 16, 245
Hydralazine hydrochloride, 8, 283
Hydrochlorothiazide, 10, 405
Hydrocortisone, 12, 277
Hydroflumethiazide, 7, 297
Hydroxyprogesterone caproate. 4, 209
CUMULATIVE INDEX
Hydroxyzine dihydrochloride, 7, 319
Imipramine hydrochloride, 14, 37
Indomethacin, 13, 21 1
loddmide. 15, 337
lodipamide. 2, 333
lopanoic acid, 14, 181
Isocarboxazid. 2, 295
Isoniazide. 6, 183
Isopropamide. 2, 315; 12, 721
Isoproterenol, 14, 391
lsosorbide dinitrate, 4, 225; 5, 556
Kanamycin sulfate, 6, 259
Ketamine, 6, 297
Ketoprofen, 10, 443
Ketotifen. 13, 239
Khellin. 9, 371
Leucovorin calcium, 8, 315
Levallorphan tartrate, 2, 339
Levarterenol bitartrate, 1, 49; 2, 573; 11, 555
Levodopa, 5, 189
Levothyroxine sodium, 5 , 225
Lidocaine base and hydrochloride, 14, 207; 15, 761
Lithium carbonate. 15, 367
Lorazepam, 9, 397
Maprotiline hydrochloride, 15, 393
Mebendazole, 16, 291
Mefloquine hydrochloride, 14, 157
Melphalan, 13, 265
Meperidine hydrochloride, 1, 175
Meprobamate, 1, 209; 4, 520; 11, 587
6-Mercaptopurine, 7, 343
Mestranol. 11, 375
Methadone hydrochloride, 3, 365; 4, 520; 9, 601
Methaqualone. 4, 245, 520
Methimazole, 8, 351
Methotrexate, 5 , 283
Methoxsalen. 9, 427
Methyclothiazide, 5 , 307
Methylphenidate hydrochloride, 10,473
Methyprylon, 2, 363
Metocloprarnide hydrochloride, 16, 327
Metoprolol tartrate, 12, 325
Metronidazole, 5, 327
Minocycline. 6, 323
Mitomycin C, 16, 361
Moxalactam disodium, 13, 305
Nabilone, 10, 499
Nadolol, 9, 455; 10, 732
Nalidixic acid, 8, 371
Naloxone hydrochloride, 14, 453
Natamycin, 10, 513
Neornycin, 8, 399
Neostigrnine, 16, 403
Nitrazepam, 9, 487
CUMULATIVE INDEX
Nitrofurantoin. 5, 345
Nitroglycerin. 9, 519
Norethindrone, 4, 268
Norgestrel, 4, 294
Nonriptyline hydrochloride. I, 233; 2, 573
Noscapine, 11, 407
Nystatin. 6, 341
Oxazepam. 3,441
Oxyphenbutazone. 13, 333
Oxytocin, 10, 563
Penicillamine. 10, 601
Penicillin-G benzothine, 11, 463
Penicillin C. potassium, IS, 427
Penicillin-V. 1. 249
Pentazocine, 13, 361
Phenazopyridine hydrochloride, 3,465
Phenelzine sulfate, 2, 383
Phenformin hydrochloride, 4, 319; 5 , 429
Phenobarbital. 7, 359
Phenoxymethyl penicillin potassium. I , 249
Phenylhutazone, 11,483
Phenylephrine hydrochloride. 3,483
Phenylpropanolamine hydrochloride, 12, 357; 13,
771
Phenytoin, 13, 417
Pilocarpine, 12, 385
Piperazine estrone sulfate. 5 , 375
Pirenzepine dih ydrochloride, 16, 445
Piroxicam. 15, SO9
Primidone, 2,409
Probenecid. 10, 639
Procainamide hydrochloride, 4, 333
Procarbazine hydrochloride, 5, 403
Promethazine hydrochloride, 5 , 429
Proparacaine hydrochloride, 6, 423
Propiomazine hydrochloride, 2, 439
Propoxyphene hydrochloride, I , 301; 4, 520; 6,
598
Propylthiouracil, 6, 457
Pseudoephedrine hydrochloride, 8,489
Pyrazinamide, 12, 433
Pyridoxine hydrochloride, 13,447
Pyrimethamine, 12, 463
Quinidine sulfate, 12, 483
Quinine hydrochloride, 12, 547
Ranitidine. 15, 533
Reserpine, 4, 384; 5 , 557; 13, 737
Rifampin. 5, 467
Rutin. 12, 623
Saccharin, 13, 487
Salbutamol, 10, 665
Salicylamide. 13, 521
77 1
Secobarbital sodium, 1, 343
Silver sulfadiazine, 13, 553
Sodium nitroprusside, 6 , 487; 15, 781
Spironolactone, 4,431
Streptomycin, 16, 507
Strychnine. 15, 563
Succinycholine chloride, 10, 691
Sulfadiazine, 11, 523
Sulfamethazine. 7, 401
Sulfamethoxazole, 2, 467; 4, 521
Sulfasalazine, 5, 515
Sulfisoxazole, 2, 487
Sulindac. 13, 573
Sulphamerazine. 6, 515
Terpin hydrate, 14, 273
Testolactone, 5, 533
Testosterone enanthate. 4, 452
Tetracycline hydrochloride, 13, 597
Theophylline. 4, 466
Thiabendazole, 16, 61 I
Thiosrrepton, 7, 423
Timolol maleate, 16, 641
Tolbutamide, 3, 513; 5, 557; 13, 719
Trazodone hydrochloride, 16, 693
Triamcinolone, 1, 367; 2, 571; 4, 521. 524: 11,
593
Triamcinolone acetonide, 1, 397, 416; 2, 571; 4,
521; 7, 501; 11, 615
Triamcinolone diacetate. 1, 423: 11, 651
Triamcinolone hexacetonide, 6, 579
Triclobisonium chloride, 2 , 507
Trifluoperazine hydrochloride, 9, 543
Triflupromazine hydrochloride, 2, 523; 4, 521; 5,
557
Trimethaphan camsylate, 3, 545
Trimethobenzamide hydrochloride, 2, 551
Trimethoprim, 7, 445
Trimipramine maleate. 12, 683
Trioxsalen, 10, 705
Tripelennamine hydrochloride. 14, 107
Triprolidine hydrochloride, 8, 509
Tropicamide, 3, 565
Tubocurarine chloride, 7, 477
Tybamate, 4,494
Valproate sodium and valproic acid, 8, 529
Vidarabine. IS, 647
Vinblastine sulfate, 1, 443
Vincristine sulfate. 1, 463
Vitamin Dlr 13, 655
Warfarin, 14, 243
Xylometazoline hydrochloride, 14, 135
Yohimbine, 16, 731
Zomepirac sodium, 15, 673

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Analytical Profiles

ACADEMIC PRESS, INC.

Academic Press Rapid Manuscript Reproduction

COPYRIGHT 0 1987 BY ACADEMIC

ACADEMIC PRESS, INC.

Orlando, Florida 32887

United Kingdom Edition published by

Library of Congress Cataloging in Publication Data

Mohammad A . Abounassif, College of Pharmacy, King Saud University,

Dominic P . I p , Merck Sharp & Dohme Research Laboratories, West

Although the official compendia define a drug substance as to identity,

Mahmoud M.A. H a h a n , PhO6eAhOh 06 PhahmaceuLLcd C h m h L t y ,

ANALYTICAL PROFILES OF DRUG SUBSTANCES

Copyright 0 1987 by Academic Press, Inc.

MAHMOUD M. A. HASSAN AND MOHAMMAD A. ABOUNASSIF

Appearance, Color, Odor and Taste

Nuclear Magnetic Resonance S p e c t r a

MAHMOUD M. A. HASSAN AND MOHAMMAD A. ABOUNASSIF

Research Number (1)

Appearance, Color, Odor and T a s t e

The c r y s t a l s t r u c t u r e of bromazepam w a s determined

The seven-membered r i n g i s i n a c y c l o h e p t a t r i e n e l i k e b o a t conformation, C ( 1 0 ) and C ( 1 1 ) forming t h e

The major conformational d i f f e r e n c e between t h i s

MAHMOUD M. A. HASSAN AND MOHAMMAD A. ABOUNASSIF

Table 2. Molecular dimensions

C ( 11) -C( 10)-N( 1)

c(2 )-c( 1'1-c( 6')

Selected torsion angels

The above v a l u e s a g r e e s w i t h t h a t of bromazepam.

An i n f r a r e d a b s o r p t i o n spectrum o f potassium bromide

Wave number (em-')

Out o f p l a n e CH-deformat i o n of 1,2,4-tris u b s t i t u t e d aromatic

Other c h a r a c t e r i s t i c bands a r e 1440, 1385, 1340,

Nuclear Magnetic Resonance S p e c t r a

spectrum of bromazepam i n DMSO d6 and TMS.

spectrum of bromazepam i n DMSO d6 and

TYS after DzO t=xchsngr.

MAHMOUD M. A. HASSAN AND MOHAMMAD A. ABOUNASSIF

13C-NMR noise-decoupled and off-resonance

C-NMR noise-decoupled Spectrum o f bmmazepam i n OMS0 and TUS.

13C-NMR off-resonance spectrum of bromazepam in DUSO and TUS.

MAHMOUD M. A. HASSAN AND MOHAMMAD A. ABOUNASSIF

The E l mass spectrum o f bromazepam (Roche Reference

The most prominent fragments of

EI-mass spectrum of bromazeparn.

MAHMOUD M. A. HASSAN AND MOHAMMAD A. ABOUNASSIF

2-(2-Amino-5-bromobenzoylpyridine [ 41 was prepared from

MAHMOUD M. A. HASSAN AND MOHAMMAD A. ABOUNASSIF

MAHMOUD M. A. HASSAN AND MOHAMMAD A. ABOUNASSIF

MAHMOUD M. A. HASSAN AND MOHAMMAD A. ABOUNASSIF

The b i o t r a n s f o r m a t i o n of bromazepam, was s t u d i e d i n f o u r

de Silva et al, 1974 (33) have reported a sensitive and

8. Chemical names and physical properties of bromazepam

Bromazepam mercapturic acid

Taleishi and Shimizu 1976 (34) has recently reported the

capturic acid conjugate of bromazepam from bile of rat

Bromazepam mercapturic acid

Scheme I. A proposed pathway f o r the biotransformation

MAHMOUD M. A. HASSAN AND MOHAMMAD A. ABOUNASSIF

Bromazepam mercapturic acid

Scheme 2. Chemical r e a c t i o n s of bromazepam and i t s known m e t a bolites.

243 ml/min and Vdg = 071/kg. Kaplan e t a l , 1976 (40)

MAHMOUD M. A. HASSAN AND MOHAMMAD A. ABOUNASSIF