Analytical Profiles of Drug Substances Volume 7 Edited by
Klaus Florey The Squibb Institute for Medical Research New Br...
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Analytical Profiles of Drug Substances Volume 7 Edited by
Klaus Florey The Squibb Institute for Medical Research New Brunswick, New Jersey
Contributing Editors
Norman W. Atwater Jerome I. Bodin Glenn A. Brewer, Jr. Salvatore A. Fusari
Boen T. Kho Arthur F. Michaelis Gerald J. Papariello Bernard Z. Senkowski
Compiled under the auspices of the Pharmaceutical Analysis and Control Section Academy of Pharmaceutical Sciences
W ACADEMIC PRESS, INC. Harcour! Brace Jovanovich, Publishers
San Diego New York Berkeley Boston London Sydney Tokyo Toronto
EDITORIAL BOARD Norman W. Atwater JeromeI. Bodin Glenn A. Brewer, Jr. Lester Chafetz Edward M. Cohen Jack P. Comer Klaus Florey Salvatore A. Fusari
Erik H. Jensen Boen T. Kho Arthur F. Michaelis Gerald J. Papariello Bruce C. Rudy Bernard Z. Senkowski Frederick Tishler
Academic Press Rapid Manuscript Reproduction
COPYRIGHT 0 1978,. BY ACADEMIC PRESS,INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM O R BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.
ACADEMIC PRESS, INC.
1250 Sixth Avenue, San Diego, California 92101
United Kingdom Edition published by ACADEMlC PRESS, INC. (LONDON) LTD.
24/28 Oval Road, London N W l
7DX
Library of Congress Cataloging in Publication Data Main entry under title:
Analytical profiles of drug substances.
Compiled under the auspices of the Pharmaceutical Analysis and Control Section, Academy of Pharmaceutical Sciences. Includes bibliographicalreferences. I. Drugs-Collected works. 2. Chemistry, Medical and pharmaceutical-Collected works. I. Florey, Klaus, ed. 11. Brewer,Glenn A. 111. Academy of PharmaceuticalSciences. Pharmaceutical Analysis and Control Seetion. 1. Drugs- Analysis-Yearbooks. [DNLM: QV740 AAI A551 RS189. A58 615l.1 70- 187259 ISBN 0-12-260807-0(v.7) PRINTEDINTHE UNITED STATtS OF AMtRlCA
AFFILLATIONS OF EDITORS, CONTRIBUTORS, AND REVIEWERS
K . S. Albert, The Upjohn Co., Kalamazoo, Michigan
N. W .Atwater, E. R. Squibb & Sons, Princeton, New Jersey
L . 2.Benet, University of California School of Pharmacy, San Francisco, California S . A . Beneva, Burroughs Wellcorne Co., Research Triangle Park, North Carolina T. R. Bennetr, Burroughs Wellcorne Co., Research Triangle Park, North Carolina P. K. Bhatracharyya, Hoffman-LaRoche, Inc., Nutley, New Jersey J. I. Bodin, Carter-Wallace, Inc., Cranbury, New Jersey G. A . Brewer, Jr., The Squibb Institute for Medical Research, New Brunswick, New Jersey L. Chaferz, Warner-Larnbert Research Institute, Moms Plains, New Jersey M. K. C. Chao, Parke, Davis and Co., Detroit, Michigan E. M . Cohen, Merck, Sharp and Dohme, West Point, Pennsylvania J . P. Comer, Eli Lilly and Company, Indianapolis, Indiana W. M .Cort, Hoffman-LaRoche, Inc., Nutley, New Jersey R. D.Daley, Ayerst Laboratories, Rouses Point, New York N . DeAngelis, Wyeth Laboratories, Philadelphia, Pennsylvania C. G. Eckhurt, Schering-Plough Research Division, Bloomfield, New Jersey K. Florey, The Squibb Institute for Medical Research, New Brunswick, New Jersey P . R . B . Foss, Burroughs Wellcome Co., Research Triangle Park, North Carolina M. Frantz, The Squibb Institute for Medical Research, New Brunswick, New Jersey S . A . Fusari, Parke, Davis and Company, Detroit, Michigan R. K. Gilpin, McNeil Laboratories, Fort Washington, Pennsylvania W . Herrmann, Godecke A.G., Freiburg, Germany D . D . Hong, Searle Laboratories, Chicago, Illinois W . Hong, Parke, Davis and Company, Detroit, Michigan A . L. Jacobs, Sandoz Pharmaceuticals, East Hanover, New Jersey C. A . Janicki, McNeil Laboratories, Fort Washington, Pennsylvania E. H . Jensen, The Upjohn Co., Kalamazoo, Michigan J . Kirschbaurn, The Squibb Institute for Medical Research, New Brunswick, New Jersey B. T. Kho, Ayerst Laboratories, Rouses Point, New York C.-S. Lee, University of Florida, Gainesville, Florida vii
viii
AFFILIATIONS OF EDITORS, CONTRIBUTORS. AND REVIEWERS
T . McCorkle, Schering-Plough Research Division, Bloomfield, New Jersey G. J . Munius, Hoffman-LaRoche, Inc., Nutley, New Jersey A. F. Michuelis, Sandoz Pharmaceuticals, East Hanover, New Jersey N . G. Nash, Ayerst Laboratories, Rouses Point, New York C . E . Orzech, Ayerst Laboratories, Rouses Point, New York G. J . Papariello, Wyeth Laboratories, Philadelphia, Pennsylvania C. Papustephunou. The Squibb Institute for Medical Research, New Brunswick, New Jersey B. C . Rudy, Burroughs Wellcome Co., Greenville, North Carolina G. Sutzinger, Giidecke A.G., Freiburg, Germany W. D. Schoenleber, Sandoz, Basle, Switzerland B. Z. Senkowski, Alcon Laboratories, Forth Worth, Texas D . H . Szulczewski, Parke, Davis and Company, Detroit, Michigan F. Tishler. Ciba-Geigy, Summit, New Jersey J . Tsuu, Wyeth Laboratories, Philadelphia, Pennsylvania F . Zimmermann, Godecke A.G., Freiburg, Germany
PREFACE
Although the official compendia list tests and limits for drug substances related to identity, purity, and strength, 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. For drug substances important enough to be accorded monographs in the official compendia, such supplemental information should also be made readily available. To this end the Pharmaceutical Analysis and Control Section, Academy of Pharmaceutical Sciences, has undertaken a cooperative venture to compile and publish Analytical Profiles of Drug Substances in a series of volumes of which this is the seventh. The concept of analytical profiles is taking hold not only for compendial drugs but, increasingly, in the industrial research laboratories. Analytical profiles are being prepared and periodically updated to provide physicochemical and analytical information of new drug substances during the consecutive stages of research and development. Hopefully, then, in the not too distant future, the publication of an analytical profile will require a minimum of effort whenever a new drug substance is selected for compendial status. The cooperative spirit of our contributors has made this venture possible. All those who have found the profiles useful are requested to contribute a monograph of their own. The editors stand ready to receive such contributions. Thanks to the dedicated efforts of Dr. Morton E. Goldberg, a long cherished dream has come to fruition with the publication of Volume I of Pharmacological and Biochemical Properties of Drug Substances, M. E. Goldberg, editor, published by APhA Academy of Pharmaceutical Sciences. This new series supplements the comprehensive description of the physical, chemical, and analytical characteristics of drug substances covered in Analytical Profiles of Drug Substances with the equally important description of pharmacological and biochemical properties. Two drug substances, cefazolin and fenoprofen, have the distinction of being the first to be covered by monographs in both series, and beginning with this volume, the cumulative index will cross-reference drug substances appearing in the new series. The goal to cover all drug substances with comprehensive monographs is still a distant one. It is up to our perseverance to make it a reality.
Klaus Florey ix
Analytical Profiles of Drug Substances, 7
ALLOPURINOL
Steven A. Benezra and T. Reney Bennett
1
Copyright @ 1978 by Academic Press, lnc. All rights of reproduction in any form reserved.
ISBN 0- 12-260807-0
STEVEN A. BENEZRA AND T. RENEY BENNETT
2
Table of Contents
1. 2.
3.
4. 5.
6.
Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor Physical Properties 2.1 Infrared Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Ultraviolet Spectrum 2.4 Mass Spectrum 2.5 Melting Point 2.6 Solubility 2 . 7 Dissociation Constant 2.8 Partition Coefficient 2.9 Crystal and Molecular Structure Synthesis Stability Methods of Analysis 5.1 Elemental Analysis 5.2 Spectrophotometric Analysis 5.3 Polarography 5.4 Chromatography 5.41 Thin Layer Chromatography 5.42 Paper Chromatography 5.43 Column Chromatography 5.44 High Performance Liquid Chromatography 5.45 Gas Chromatography 5.5 Mass Spectrometry Metabolism and Pharmacokinetics 6.1 Metabolism 6.2 Tissue Distribution 6 . 3 Pharmacokinetics
3
ALLOPURINOL
1.
Description
1.1 Name, Formula, Molecular Weight Allopurinol is lH-pyrazol0(3,4-d)pyrimidine-4-01
0
136.11
C5H4N40 1.2 Appearance, Color, Odor
Allopurinol is a white to off-white powder with a slight odor
.
2.
Physical Properties 2.1
Infrared Spectrum
The infrared spectrum of allopurinol is shown in Figure 1 . It was taken as a 0.2% dispersion of allopurinol in KBr with a Perkin Elmer model 457 infrared spectrophotometer. Table I gives the infrareg assignments consistent with the structure of allopurinol
.
Table I Infrared Spectral Assignments for Allopurinol Frequency (cm-l)
Assignment
3060
CH stretching vibrations of the pyrimidine ring
1700
CO stretching vibration of the keto form of the 4-hydroxy tautomer
1590
ring vibrations
1245
CH in-plane deformation
Figure 1-
Infrared Spectrum of Allopurinol
ALLOPURINOL
790 2.2
5
CH in-plane deformation
Nuclear Magnetic Resonance (NMR) Spectrum
The NMR spectrum of allopurinol is shown in Figure The spectrum was obtained with a Varian model CFT-20 80 MHz NMR spectrometer. Deuterated DMSO was used as the solvent with tetramethylsilane as an internal standard. Based on the NMR spectrum, the following assignments for allopurinol can be made.
4 2
.
Proton
# of Protons
Chemical Shift (ppm)
b
1
7.95
a
1
8.07
2.3
Ultraviolet Spectrum
The ultraviolet spectrum of allopurinol in 0.1N HC1 was obtained with a Beckman ACTA CIII ultraviolet spectrophoTable I1 summarizes the tometer and is shown in Figure 35 ultraviolet spectral data for allopurinol.
.
Table I1 Ultraviolet Spectral Data for allopurinol Solvent
Amax (n)
E -
0.1N NaOH
257
7200
0.1N HC1
250
7600
Me thano1
252
7600
2
rl .rl
a
1
$4
0
rl
4
rl
0
W
fi 1
Ll
0 a,
u
a tA
a
x (d
111
0
a, d -rl
u a, c
M
s
$4 (d
V
a, rl
I
a,
w b 7
M .rl
h
I, 1
9 03 0
I
I
6
co
I
I
0
Kt
I
I
0
N
I
0 -0 Tt
0 -co co m
0
m
-N N
n 0
-R 0
-8 -
0
OR
E c
0
w
U
a
a,
cn a,
CI
4 0
I
a,
@ l
L.c 1
M *d F
STEVEN A. BENEZRA AND T.RENEY BENNETT
8
2.4
Mass Spectrum
The low resolution ele8 tron impact mass spectrum of allopurinol is shown in Figure 4 It was obtained with a Varian model CH5-DF mass spectrometer. Direct probe at 115OC into the source was used to obtain the mass spectrum. The electron energy was 70 eV. The assignments of the major ions formed in the mass spectrometer is given below.
.
m/e 120 (4.2%)
m/e 136(100%) 2.5
Melting Point Allopurinol melts above 35OOC7
2.6
m/e I09(6.3%)
.
Solubility
The solubility of ajlopurinol in various solvents at 25OC is given in Table 111
.
Table I11 Solubility of Allopurinol at 25OC Solvent
Solubility (mg/ml)
Water n-octanol Chlorofo m Ethanol DMSO 2.7
0.48
urea. 1,2 i 3 i 4 Both t h e t o x i c p r o p e r t i e s and t h e medical use o f c r u d e e r g o t a l k a l o i d s have been known f o r hundreds of year&, b u t t h e first p h y s i o l o g i c a l l y a c t i v e , c r y s t a l l i n e a l k a l o i d , e r g o t o x i n e , was n o t i s o l a t e d b e f o r e 1906.6i7,8i9 I n 1918, S t o l l found a second a c t i v e compound , ergotamine .lo The t h i r d a c t i v e a l k a l o i d , ergonov i n e , was i s o l a t e d i n 1935 by f o u r groups o f workers.ll S t o l l and Hofmann12g13 l a t e r discovered t h a t ergot o x i n e was n o t a s i n g l e compound, b u t c o n s i s t e d o f t h r e e a l k a l o i d s , ergocornine, e r g o c r i s t i n e and e r g o k r y p t i n e , which could b e separated by f r a c t i o n a l c r y s t a l l i z a t i o n of t h e i r di-(4-toluyl)-L-tartrates. R e ~ e n t l y , ~ ~ i tl h5e8 a~p ~p l i c a t i o n o f a s p e c i a l paper chromatographic technique h a s shown t h a t one o f t h e components, e r g o k r y p t i n e , c o n s i s t s of two c l o s e l y r e l a t e d isomers , d e s i g n a t e d as a- and B- isomers , t h e first c o n t a i n i n g L-leucine t h e l a t t e r c o n t a i n i n g Li s o l e u c i n e i n t h e p e p t i d e p a r t of t h e molecule.
,
The n a t u r a l l y o c c u r r i n g a l k a l o i d s cause s t r o n g u t e r i n e c o n t r a c t i o n s and are c i r c u l a t o r y s t i m u l a n t s .17i18 S t o l l and Hofmannlg prepared t h e 9 ,lo-dihydrogenated deriv a t i v e s of e r g o t o x i n e and i t s components. These a l k a l o i d s no longer have a c t i o n on t h e u t e r i n e muscle, b u t possess sympathicolytic-adrenolytic a c t i v i t y accompanied by supp r e s s i o n of t h e vasomotor c e n t e r .20 Dihydroergotoxine, i n t h e form o f i t s methanesulfonic acid s a l t (mesilate), i s now widely used f o r t h e treatment of symptoms o f m i l d t o moderate impairment o f mental f u n c t i o n i n t h e e l d e r l y . 2 l S p e c i a l s t u d i e s have been made of i t s a c t i o n on metabolism i n the brain, i n particular.22-26 T h i s a n a l y t i c a l p r o f i l e h a s been prepared f o r 9,lOdihydroergotoxine, and a d d i t i o n a l l y f o r i t s components 9 ,lo-dihydroergocornine, 9 , l o - d i h y d r o e r g o c r i s t i n e , 9 ,lodihydro-a -ergokryptine , and 9 ,lO-dihydro-fl-ergokryptine i n t h e form of t h e i r mesilates.
DIHY DROERGOTOXINE METHANESULFONATE
1.
85
pescr i D t i o n 1.1 Jame. Def i n i t i o n
Dihydroergotoxine mesilate is t h e s a l t of t h e hydrog e n a t i o n product of t h e n a t u r a l l y o c c u r r i n g ergotoxine. Hence, i t comprises t h e f o u r compounds dihydroergocornine, d i h y d r o e r g o c r i s t i n e , dihydro-a-ergokryptine, and dihydroB-ergokryptine. The drug substance c o n t a i n s e q u a l amounts of dihydroergocornine, - c r i s t i n e and - k r y p t i n e , t h e r a t i o of t h e a- and &isomers of dihydroergokryptine being approximately 2: 1. These p r o p o r t i o n s correspond t o amounts found i n n a t u r a l e r g o t o x i n e i s o l a t e d from s e l e c t e d and c u l t i v a t e d s t r a i n s of ClaviceDs DurDurepl.
1.21
Dihvdroernotoxine mesilat e
R =
,
-CH 2-C 6H 5’
-CH( CH3 1
-CH(CH3)-C 2H 5’
-CH2-CH(CH 3 ) 2
W. DIETER SCHOENLEBER et al.
86
Chemical Abstracts Registry Numbers:
base
:
mesilate:
11 032-41-0
52 655-92-2
Aver age Molecular W e i g h ts : : 584.40
base
mesilate : 680.51
Average Formulas : base
1.22
'
'32. 67'42. 67N505
:
'33.67'45.67
N 5 O8 S
m o n e n t s ~of Dihvdroeraotoxine 1.221
Pihvdroeraocornine mesi l a t e
Dihydroergocornine mesilate i s t h e s a l t of 9,1Oa-dihydr0-12'-hydroxy-2~ ,5'a-bis (1-me t h y l e t h y l 1-ergotaman-3 , 6 ,18trione.
CH3 CHS \ /
CH ,S020H
87
DIHYDROERGOTOXINE METHANESULFONATE
Chemical A b s t r a c t s R e g i s t r y Numbers: base
: 25 447-65-8
mesilate: blolecular Weight:
base
:
563.70
mesilate :
659 81
base
Formula :
29 261-94-7
: C
mesilate:
H 0 N 31 41 5 5
C32H4508N5S
1 . 2 2 2 ~ i h v d r o e r n o c r i s t i n emesi l a t e Dihydroergocristine m e s i l a t e i s t h e s a l t of 9 10ccdihydro-12' -hydroxy-2' -( l-methyle t h y l ) -5 'a-(phenyl-me thy1 -ergotaman-3 , 6',18-trione.
-
HI,
c-
-N
';I
Q$? -CH,
.CH,
88
W. DIETER SCHOENLEBER et al.
Chemical A b s t r a c t s R e g i s t r y Numbers: base
: 17 479-19-5
mesilate:
24 730-10-7 29 261-92-5
Molecular Weight:
: 611.74
base
mesilate: Formula :
707.85
: C
base
mesilate:
H 0N 35 41 5 5
C H 0 N S 36 45 8 5
1 . 2 2 3 DihvdroernokrvDtine mesilata Dihydroergokryptine mesilate i s t h e s a l t o f t h e a- and B-isomers in t h e molar ratio of 2 : l .
0
II
C-
'I
HN
1;1
N
*CH,S020H R = -CH -CH(CH 2
)
3 2
or -CH(CH3)-CH -CH 2 3
DIHYDROERGOTOXINE METHANESULFONATE
Molecular k e i g h t :
89
base
: 577.73
mesilate : 67 3.85 Formula :
base
: C
mesilate:
C
H 0 N 32 43 5 5 H
0 N S
33 47 8 5
1.2231 pihvdro -a-ernokrvD tine mesilate
D i hydro*-ergokr ypt i n e me s i l a te is t h e s a l t of 9,10Ci-dihydr0-12~-hydroxy-2'-(lme thy1 e t h y l 1-5 'a- ( 2-me thylpr o p y l ) -ergotaman-3' ,6' , 1 8 - t r i o n e .
H N-.
/
\
CH3 CH3
90
W. DIETER SCHOENLEBER et al.
Chemical Abstracts Registry Numbers:
base
: 25 447-66-9
mesilate:
Molecular Weight:
base
29 261-93-6 : 577.73
mesilate:
base
Formula :
673.85
: C
mesilate:
C
H
0 N
H
0 N S
32 43 5 5 33 47 8 5
Dihydro-B-ergokryptine mesilate is t h e s a l t of 9,10Cbdihydr0-12'-hydroxy-2~-(1me t hy l e t h y l ) 5 cb ( 1-me t h y l pr opy l ) -ergotaman-3t,6t,18-trione.
-
DMYDROERGOTOXINE METHANESULFONATE
91
Chemical Abstracts R e g i s t r y Numbers: : 1 9 467-62-0
base
mesilate: Molecular Weight:
base
not y e t assigned : 577.73
mesilate:
1.3
673.85
Amearance. Colour
D i hydroergo t ox i n e mesila t e and t h e mesil a t es o f its components are w h i t e t o off-white crystall i n e powders. 2.
P h v s i c a l ProDe r t i e s 2.11
I n f r a r e d SDectra
O f t h e i n f r a r e d s p e c t r a of t h e genated a l k a l o i d s of t h e e r g o t o x i n e t h o s e of t h e dihydro-a-ergokryptine e r g o k r y p t i n e bases have so f a r been
9,10-dihydrogroup, o n l y and d i h y d r o 4 reported.l5
Figure 1 .
Infrared Spectrum of Dihydroergotoxine mesilate KEr p e l l e t , instrument: Perkin Elmer 257
Figure 2 .
Infrared Spectrum of Dihydroergocornine mesilate KBr p e l l e t , instrument: Perkin Elmer 257
Figure 3.
Infrared Spectrum of Dihydroergooristine mesilate KBr pellet, instrument: Perkin Elmer 257
Figure 4 .
Infrared Spectrum of Dihydroergokryptine mesilate KBr p e l l e t , instrument: Perkin E l m e r 257
Figure 5.
Infrared Spectrum of Dihydro-a-ergokryptine mesilate KBr pellet, instrument: Perkin Elmer 257
Figure 6 .
Infrared Spectrum of Dihydroa-ergokryptine mesilate Perkin E l m e r 257
KBr p e l l e t , instrument:
W. DIETER SCHOENLEBER et al.
98
The s p e c t r a o f t h e drug s u b s t a n c e itself and o f i t s c o n s t i t u e n t s are p r e s e n t e d i n f i g u r e s 1 6.
-
Conditions were: p e l l e t made from 1 . 5 mg o f each compound p l u s 400 mg o f KBr. Instrument: Perkinhlmer 257. 2.12 a-
tr
The absorbance o f dihydroergotoxine mesilate was measured i n s e v e r a l s o l v e n t s 2 7 as follows:
solvent
max, nm
95% e t h a n o l
Sh
0.01
0.01
a
275
4 luH40ti i n 95% e t h a n o l
Sh
Sh
275 281
101
291 277 281
0.01
fi t a r t a r i c acid
Sh
cm 94.8 101 85.6 95.1
281 291 H C 1 i n 95% e t h a n o l
1%
292 274 279 288
87.1 93.2 98.6 81.0 98.0 93.3 79.3
The u l t r a v i o l e t spectra o f t h e 3 components, dihydroergocornine, d i h y d r o e r g o c r i s t i n e and dihydroergokrypt i n e , have been r e p o r t e d by M . Prosek and co-workers28 measured on a c e l l u l o s e p l a t e u s i n g a chromatogram spe ct ropho tome ter
.
2.13 F l u o r e s c e n c e Q u i t e i n c o n t r a s t t o t h e i r p a r e n t compounds, d i h y droergotoxine and i t s components do not f l u o r e s c e i n t h e v i s i b l e r e g i o n of t h e ~ p e c t r u m . 5 , ~ 9 ' 3 ~ A yellowish green f l u o r e s c e n c e can however be induced by i n t e n s i v e i r r a d i a t i o n w i t h u l t r a v i o l e t 1ight.30-32 T h i s effect has been u t i l i z e d f o r t h e i d e n t i f i c a t i o n and p a r t l y for t h e q u a n t i t a t i o n of dihydroergotoxine (see a l s o s e c t i o n 6.32).
99
DIHYDROERGOTOXINE METHANESULFONATE
2.14
Proton Nuc l e a r Mame t i c Resonance SDectra
A comprehensive i n v e s t i g a t i o n o f t h e PMR s p e c t r a o f p e p t i d e a l k a l o i d s has so far n o t been r e p o r t e d . A r e c e n t p u b l i c a t i o n by Floss, kenkert and co-workers33 deals p r i m a r i l y w i t h c l a v i n e a l k a l o i d s .
The spectra o f t h e dihydroergotoxine a l k a l o i d m e s i l a t e s i n d e u t e r a t e d dimethyl sulphoxide are presented i n f i g u r e s 7 12.
-
+
The HN-CH3 shows a s i n g l e t f o r GoamQn-fga&yyeg: methyl a t d = 2.3 ppm and a broad, u n c h a r a c t e r i s t i c s i g n a l f o r H a t 10 ppm. The mesilate methyl group shows up a t 6 = 3 ppm. The p a t t e r n of 9,lO-dihydrol y s e r g i c acid, with t h e very c h a r a c t e r i s t i c signals f o r t h e i n d o l e moiety ( p y r r o l i c N t i a t 11, t h e a r o m a t i c p r o t o n s a t about 7 ppm) and t h e a d j a c e n t r i n g s C and D i n t h e a l i p h a t i c r e g i o n , i s p r e s e n t i n t h e s p e c t r a of a l l f o u r components. The amide proton a t p o s i t i o n 19 e x h i b i t s a n e a t s i n g l e t a t 9.3 9.4 ppm. Moreover, t h e p e p t i d e f r a c t i o n leads t o s i g n a l s superimposing t h e already complex a l i p h a t i c p a r t o f t h e spectrum.
-
Indixidual-fgaLu2e.a: The PMR p a t t e r n i s charact e r i s t i c a l l y altered by t h e t y p e o f 5' s u b s t i t u t i o n . Thus, i n t h e s p e c t r a of dihydro-a-ergokryptine and o f d i h y d r o e r g o c r i s t i n e mesilate, t h e p r o t o n s a t C-5' appear as n e a t t r i p l e t s a t 6 = 4.3 and 4.6 ppm, r e s p e c t i v e l y . For dihyaroergocornine and dihydrof3 ergokryptine t h e r e is a d o u b l e t a t 4.3 ppm. The s i g n a l s of t h e i n d i v i d u a l C - 5 ' s u b s t i t u e n t (2-propyl-, l-methylpropyl, 2-methylpropyl, and benzyl) a d d t o t h e corresponding r e g i o n of a l i p h a t i c o r aromatic signals, respectively, yielding a c h a r a c t e r i s t i c p a t t e r n f o r each a l k a l o i d .
-
The conspicuous, a d d i t i o n a l s i n g l e t emerging on t o p o f t h e broad m u l t i p l e t a t 3.35 ppm i n t h e dihydroe r g o c r i s t i n e mesilate spectrum stems from t h e water c o n t e n t of t h e s u b s t a n c e (see a l s o thermogram, s e c t i o n 2.23).
Figure 7 .
PMR Spectrum of Dihydroergotoxine mesilate i n (CD ) SO, instrument: Bruker HX-90
3 2
F ig u re 8.
PMR Spectrum of Dihydroergocornine mesilate i n (CD ) SO, instrument: Bruker HX-90 3 2
Figure 9.
PMR Spectrum of Dihydroergocristine mesilate in ( C D ) SO, instrument: Bruker HX-90 3 2
Figure 10.
PMR Spectrum of Dihydroergokryptine mesilate Bruker HX-90 3 2
i n (CD ) S O , i n s t r u m e n t :
Figure 11.
PMR Spectrum of Dihydro-a-ergokryptine mesilate i n (CD ) SO, instrument: Bruker HX-90 3 2
Figure 1 2 .
PMR Spectrum of Dihydro-B-ergokryptine mesilate in (CD ) SO, instrument: Bruker HX-90 3 2
106
W. DIETER SCHOENLEBER et al.
2.15
13C Nuclear Mannetic
The s p e c t r a o f dihydroergotoxine mesilate and t h e i n d i v i d u a l a l k a l o i d s have been r e c 0 r d e d 3 ~i n dimethylsulphoxide using a Bruker HX-90 MHz NMR spectrophotometer. The chemical s h i f t s o f t h e f o u r c o n s t i t u e n t s are presented i n f i g u r e 13. A s expected, t h e s h i f t d i f f e r e n c e i n t h e s p e c t r a i s a maximum f o r t h e C-5' signal.
2.16
- ,
Although mass s p e o t r a o f non-hydro enated e r g o t a l k a l o i d s and t h a t of dihydroergotamine 5 have been d i s c u s s e d , 36 t 37 dihydroergotoxine h a s so far n o t been i n v e s t i g a t e d . The low r e s o l u t i o n mass spectra of t h i s compound and t h o s e o f i t s components, a s mesilates, are p r e s e n t e d i n f i g u r e s 1 4 - 1 9 .
3
G e n e c a : Due t o t h e low v o l a t i l i t y of t h e e r g o t p e p t i d e a l k a l o i d s i n g e n e r a l , molecular peaks are n o t always p r e s e n t i n t h e s p e c t r a , t h e i r occurrence being s u b j e c t t o c a r e f u l l y optimized instrument parameters and to a c e r t a i n p o r t i o n of good luck. ~ o ~ n ~ ~ ~ Ergot n ~a l k a~l o i d~s n i n g e n e r a l are known t o undergo cleavage between t h e i r p e p t i d e and e r g f n e f r a c t i o n & a t t h e s i t e of t h e 19 2 ' bond accompanied by a hydrogen abstract i o n . T h i s l e a d s t o a common p a t t e r n f o r t h e lysergic acid p a r d 8 f o r a l l ergot alkaloids. In the c a s e of t h e 9 ,lo-dihydrogenated ergotoxine a l k a l o i d s , t h i s species is t h e dihydrolysergamide i o n fragment a t m/e = 269 ( a l o n g w i t h its decay peaks a t m/e = 223, 224, 225 f o r i n d i v i d u a l s w i t h s p l i t - u p ring D >
-
~
~
30.0
12
F i g u r e 1 3 . S t r u c t u r a l P o s i t i o n s ( b o l d numbers) i n t h e d i h y d r o er g o t o x in e a l k a l o i d s ’ s k e l e t o n and l 3 C NMR Chemical S h i f t s of d ih y d ro er g ot o x i n e mesil a t e and of its Components i n DMSO-d 6 (ppm, TMS 0, numbers i n i t a l i c s ) (37-41 = s i g n a l s overlapped by s o l v e n t )
100
1
90 80
/
70 60
Y1
50 55
YO 31
30 20
223
10
3Y3 500
'
1 " " ' . " 1 ' * '
100
Figure 1 4 .
200
300
YO0
500
Low Resolution Mass Spectrum o f Dihydroergotoxine mesilate Instrument: CBC 21-110 B
I
-'.
600
3
' '
"
' 1
M/E
700
4
o o
o m
o m
r
-
o
r
5
o
m
o
o Y
r
o
3
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o -
0
0
r-
0
0 (D
0 0
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0 0 f.
0 0 P)
0 0 N
0
z
9) c)
uSI c
o c
J H
0
~
0
m
0
l
0
m
P
0
(
0
0
m
0
9
0
m
E
\
w
=
2
o 0 cc
0
0 (0
0
0
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9
0 0
0 0 M
0 0
N
0
z
al
(d
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m
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.+
0 O
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al c,
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Id
100
/
90 80 70
60 50 ’10
30 20
553
I
10
100
Figure 1 8 .
200
300
1100
500
600
Low Resolution Mass Spectrum of Dihydrou-ergokryptine m e s i l a t e Instrument: CEC 21-110 B
700
100
/
90 80
70 60
50 YO
30
223 187
209
I.
20
10 97112
100
Figure 19.
138
200
300
Lloo
500
600
Low Resolution Mass Spectrum of Dihydro-R-ergokryptine mesilate Instrument: CEC 21-110 B
700
W.DIETER SCHOENLEBER et al.
114
a t m/e = 167 and 154 f o r the fragments presented be1ow.
HYCONH,
0 1'
m/e = 269
m/e = 167
m/e = 154
I n d i h y d r o e r g o t o x i n e and its c o n s t i t u e n t s , t h e p e p t i d e moiety a l s o leads t o a common fragmentation p a t t e r n i n those pathways where t h e 5 ' - s u b s t i t u e n t is n o t involved, such as t h e f o l l o w i n g fragments:
m/e = 154
m/e = 153 m/e = 70 m/e = 71 base peak
m/e = 125
A further common fragment is t h a t a t m/e = 349 t h a t c o n t a i n s t h e dihydrolysergamide f r a c t i o n p l u s a n 80 mass u n i t s p a r t presumably from t h e dimethyl p y r u v i c acid s e c t i o n of t h e p e p t i d e moiety.
InBiyidul-fzagngat&iQu t u t i o n a t t h e 5' p o s i t i o n leads d i f f e r e n c e s i n the mass s p e c t r a i n q u e s t i o n , expressed not o n l y
The t y p e of s u b s t i t o t h e observed of t h e a l k a l o i d s i n t h e correspond-
DMYDROERGOTOXINE METHANESULFONATE
115
i n g molecular peak, i f o b s e r v a b l e , b u t a l s o i n t h e fragments o f t h e p e p t i d e f r a c t i o n . Typical fragments are l i s t e d below.
Hh ,1 X ‘ O H R
R
I
I11
I1
I
I1
I11
ComDonent
m/ e
m/e
m/ e
dihydroergocornine dihydroergocristine d ihyd r o d - e rgokrypt i n e
196 244
19 5 243
72 1 08
210
209
86
dihydro-B -ergokry p t i n e I n t h e spectrum of d i h y d r o e r g o c r i s t i n e , a n o t h e r fragment shows up which i s o b v i o u s l y s t a b i l i z e d by t h e b e n z y l i c radical a t C-5’. I n analogy t o t h e quoted l i t e r a t u r e , i t s s t r u c t u r e i s l i k e l y t o be t h e following :
m/e = 344 The presence of t h e benzyl group i n dihydroergoc r i s t i n e i s a l s o confirmed by t h e occurence of a fragment a t m/e = 91, corresponding t o t h e tropylium ion.
116
W. DIETER SCHOENLEBER et al.
The o p t i c a l p r o p e r t i e s o f t h e s i n g l e bases have been r e p o r t e d by S t o l l and Hofmann.lg Those of t h e i r mesilates are given below.
O p t i c a l R o t a t i o n o f dihydroergotoxine a l k a l o i d s
[ u ]i ~ n 50 ~ % ethanol, c = l % d i hydroergo t o x i n e
me s i l a t e dihydroergocornine m e s i l a t e d i h y d r o e r g o c r i st i n e mesilate dihydroergokrypt i n e mesilate dihydro-u-ergokryptine mesilate dihydro- b e r g o k r y p t i n e mesilate 2.2
+ + + + + +
13.50 14.00
3.10 21.40 16.60 28.10
Ehy sical ProDe r t i e s of t h e S o l i a
2.21
B e l t i n n Character i s t i c g
As dihydroergotoxine mesilate decomposes a t about 200 degrees, a melting p o i n t cannot be given. The melting o f three corn onents as t h e i r bases have been r e p o r t e d by Hofmann:
8
dihydroergocornine 187 0, decomposition d i h y d r o e r g o c r i s t i n e 160 0 , decomposition dihydroergokryptine 2350, decomposition 2.22
p i f f e r e n t i a 1 Scanninn Ca lorimet r y
The thermogram was obtained w i t h a Perkin Elmer scanning o a l o r i m e t e r a t a h e a t i n g rate o f 10 /min. i n a n i t r o g e n atmosphere i n a l o o s e l y covered v e s s e l . Dihydroergotoxine mesilate and its components e x h i b i t endotherms f o r t h e melting/decomposition between 180° and 200'. Minor and broad endotherms i n d i c a t e t h e l o s s of s o l v e n t and water (see f i g u r e s 20 25).
-
The thermogravimetric a n a l y s e s were carried o u t on a Perkin Elmer TGS-1 thermobalance. The samples were heated a t a r a t e of 1O0/min. under a n i t r o g e n atmosphere. Weight l o s s e s , as i n d i c a t e d i n f i g u r e s 20 - 25, were observed below m e l t i n g which are a t t r i buted t o s o l v e n t and water losses. Massive sample decomposition obviously begins a f t e r melting.
117
DIHYDROERGOTOXINE METHANESULFONATE
5cp
Figure 20.
.
I.
I.
fa?
Y
I.
.. ..
.I
%!w
,,
,. ,,w ,, .. u
D i f f e r e n t i a l Scanning Calorimetric and Thermogravimetric Curves of Dihydroergotoxine mesilate
W. DIETER SCHOENLEBER et
118
sv
Y
u
Figure 21.
a.
.." .. .. rn
.L
U
"
Y
so
1 .
.. .. ..
u
aarr
.."I
2w-
D i f f e r e n t i a l Scanning Calorimetric and Thermogravimetric Curves of Dihydroergocornine mesilate
01.
DIHYDROERGOTOXINE METHANESULFONATE
Figure 22.
D i f f e r e n t i a l Scanning Calorimetric and Thermogravimetr i c Curves of D i hyd ro erg oc r i s t i n e mesilate
119
W.DIETER SCHOENLEBER et al.
120
Figure 23.
Differential Scanning Calorimetric and Thermogravimetric Curves of Dihydroergokryptine mesilate
DIHYDROERGOTOXINE METHANESULFONATE
F i g u r e 24.
Differential Scanning Calorimetric and Thermogravimetric C u r v e s of D i h y d r o u e r g o k r y p t i n e mesilate
121
W.DIETER SMOENLEBER et al.
122
F i g u r e 25.
D i f f e r e n t i a l Scanning Calorimetric and Therrnogravimetr i c Curves of Dihydro-Bergokrypt i n e mesilate
DIHYDROERGOTOXINE METHANESULFONATE
2.24
123
I o n i z a t i o n Constants
Maulding and Zoglio3g have r e p o r t e d t h e i o n i z a t i o n c o n s t a n t s f o r t h e dihydroergotoxine components a t 24O i n water a s 6.91 6.89 and 6.89
0.07 f o r dihydroergocornine 2 &
0.07 f o r d i h y d r o e r g o c r i s t i n e 0.07 f o r dihydroergokryptine
The PKMCS v a l u e s (MCS = methyl c e l l o s o l v e ) accordi n g t o t h e method of Simon40 have been determined41 as
5.78 5.83 5.81 and 5.84 2.25
for for for for
dihydroergocornine, dihydroergocristine, dihydrov-ergokryptine, dihydro-g-ergokryptine.
gart i t i o n C o e f f i c i e n t
The p a r t i t i o n e q u i l i b r i u m of dihydroergotoxine mesilate between water o f pH 1 . 2 and chloroform, on t h e one hand, and water pH 1 . 2 and n-octanol, on t h e o t h e r , have been i n v e s t i g a t e d a t 37.0 0.50.
p a r t i t i o n system
water pH 1.2/CHC13 water pH 1.2/n-octanol 2.26
equilibrium d i s t r i b u t i o n ratio 0.12 : 1 : 22.9
1
Solu b i l i t v
The s o l u b i l i t i e s of dihydroergotoxine mesilate i n d i f f e r e n t s o l v e n t s have been determined a s follows: i n water, 95% e t h a n o l , 50% e t h a n o l , methanol, and acetone: > 2 g/100 m l . , i n chloroform: 0.9 g/100 m l . , i n dichloromethane: < 0 . 1 g/100 ml. , i n ether and petroleum e t h e r : i n s o l u b l e .
3.
Produc t ia
The e r g o t a l k a l o i d s are mainly i s o l a t e d from f i e l d c u l t i v a t e d s c l e r o t i a which a r e produced a f t e r a r t i f i c i a l i n f e c t i o n of r y e ears w i t h c o n i d i a of ClaviceDs pLlrDurea. 5 117 i 43 i 44
W. DIETER SCHOENLEBER et
124
01.
After defatting w i t h benzene, the suspension is made a l k a l i n e w i t h ammonia, t h e a l k a l o i d s are e x t r a c t e d w i t h benzene and subsequently c r y s t a l l i z e d and f r a c t i o n a t e d i n t o t h e d i f f e r e n t a l k a l o i d groups as t h e i r salts. 5
Another more r e c e n t method o f production c o n s i s t s o f the c u l t i v a t i o n of selected s t r a i n s of i n large v e s s e l s under submerged c o n d i t i o n s The s e l e c t i v e c a t a l y t i c hydrogenation of t h e 9,lO-double bond i n t h e e r g o t a l k a l o i d s l e a d i n g t o t h e dihydrogenated species i n q u e s t i o n has been invest i g a t e d and d e s c r i b e d i n d e t a i l . l g ~ 5 3
4.
S t a b i l i t v and Denradation 4.1
Bulk S tabi-
Dihydroergotoxine mesilate h a s been shown t o be s t a b l e a t room temperature f o r up t o t e n years when s t o r e d i n t h e absence of l i g h t . I r r a d i a t i o n by a r t i f i c i a l d a y l i g h t leads t o a s l i g h t l y yellowish a s p e c t o f the powder accompanied by a lowered drug c 0 n t e n t . 5 ~ 4.2
S t a b i u t v i n So l u t i u
Like a l l e r g o t and most i n d o l e a l k a l o i d s , 3 5 dihydroergotoxine mesilate and i t s components a r e prone t o l i g h t enhanced o x i d a t i v e degradation i n aqueous s o l u t i o n s . 55 Therefore, such f o r m u l a t i o n s must be kept i n amber glass v i a l s and i n an i n e r t atmosphere, o r they have t o be s t a b i l i z e d by a n adequate excipien t .56
I n t h e case o f d i h y d r o e r g o c r i s t i n e , t h e main oxid a t i o n products have been i d e n t i f i e d by spectroscopy and s y n t h e s i s 5 7 as N-formyl-B-seco-dihydroergocrist i n e and its deformylation product, B-seco-dihydroergoc r i s t i n e , t h e s t r u c t u r e s o f which are g i v e n i n f i g u r e 26. They f l u o r e s c e very i n t e n s i v e l y b u t do n o t react The pathway w i t h van Urk's r e a g e n t (see s e c t i o n 6 . 2 ) . has been proposed v i a a u t o x i d a t i o n a t t h e 3 p o s i t i o n and subsequent r u p t u r e o f t h e 2-3 bond. The remaini n g components of dihydroergotoxine undergo t h e same type of a u t o x i d a t i v e degradation i n s o l u t i o n , however, t h e s t r u c t u r e s of t h e corresponding products have n o t y e t been e l u c i d a t e d .
DIHYDROERGOTOXINE METHANESULFONATE
125
0 N
R = -CHO = -H
Figure 2 6 .
N-formyl-B-seco-dihydroergocristine B-seco-dihydroergocristine
Autoxidation Products of Dihydroergocristine
W,DETER SCHOENLEBER et 01.
126
4.22
Acid and Base Ca t a l v z e d Isome-r
I n hydroxyl-containing s o l v e n t s , nonhydrogenated e r g o t p e p t i d e a l k a l o i d s are r e a d i l y epimerized a t carbon 8 t o an e q u i l i b r a t e d mixture of t h e l y s e r g i c and i s o l y s e r g i c acid series, called t h e - h e / - i n i n e forms .20 J 58 The double a c t i v a t i o n o f the hydrogen a t C-8 is a p r e r e q u i s i t e f o r t h i s conversion, theref o r e i t i s rendered impossible by t h e 9,lO-dihydrogenation. Consequently, dihydroergotoxine a l k a l o i d s do not e x h i b i t t h i s behaviour. However, a t e l e v a t e d temperatures a n o t h e r type of a c i d c a t a l y z e d rearrangement takes place i n both nonhydrogenated and hydrogenated e r g o t p e p t i d e alkal o i d s : t h e so-called &&isomerization a t C-2' i n t h e p e p t i d e moiety20 ~ 5 *96o l e a d i n g t o more acidic e p i mers. These show almost no pharmacological a c t i v i t y and can r e a d i l y be s e p a r a t e d from t h e i r s t a r t i n g m a t e r i a l due t o t h e i r as t h e i r name r e v e a l s enhanced a c i d i t y .
-
I n a l c o h o l i c s o l u t i o n s and under acid c a t a l y s i s 6 l t h e hydroxy group a t C-12' i n t h e p e p t i d e p o r t i o n is slowly replaced by t h e corresponding alkoxy group.35 I n aqueous s o l u t i o n s dihydrolysergamide and dihyd r o l y s e r g i c acid occur as h y d r o l y s i s products. The two breakdown products can t h e r e f o r e be observed i n small q u a n t i t i e s i n aged l i q u i d pharmaceutical preparations.20 5.
Drua M etabolism and P h a r w o k i n e t i c s
T r i t i u m labelled dihydroergotoxine mesilate (see s e c t i o n 6.4) administered o r a l l y i n man a t 1.0 mg was r a p i d 1 absorbed w i t h an a b s o r p t i o n h a l f - l i f e of 0.52 hours,x2 t h e peak plasma c o n c e n t r a t i o n of 0.5 ngequivalent/ml. was reached after 2.3 hours a f t e r admini strat ion. The e l i m i n a t i o n occurs i n two phases w i t h h a l f l i v e s of 4.1 and 1 3 hours r e s p e c t i v e l y . The main pathway f o r t h e e x c r e t i o n of the dihydroergotoxine a l k a l o i d s is t h e b i l i a r y r o u t e , as shown i n rats f o r
DIHYDROERGOTOXINE METHANESULFONATE
127
d i h y d r o e r g o c r i s t i n e by Nimmerfall and R o s e n t h a l e 1 - . ~ 3 9 ~ ~ Only a very small percentage o f t h e administered dose appears i n t h e u r i n e . Maurer65 succeeded i n i s o l a t i n g and i d e n t i f y i n g 16 m e t a b o l i t e s o f dihydro-B-ergokryptine from r a t b i l e . The major m e t a b o l i t e s stem from t h e o x i d a t i v e b i o t r a n s f o r m a t i o n a t t h e 8' p o s i t i o n i n t h e p r o l i n e p a r t o f t h e p e p t i d e moiety: A series o f s t e r e o i s o meric 8' and 9' as well as 8' and 10' dihydroxylated compounds could be i s o l a t e d , accompanied by p r o d u c t s o f further o x i d a t i o n a t t h e 8' p o s i t i o n such as g l u tamic and hydroxy g l u t a m i c acid d e r i v a t i v e s . As minor m e t a b o l i t e s , g l u c u r o n i d e s o f t h e mono8'-hydroxylated and o f t h e 8' ,g'-dihydroxylated metabolic products were i s o l a t e d t o g e t h e r w i t h a s m a l l amount of dihydrolysergamide and a g'-hydroxylated d e r i v a t i v e conjugated w i t h g l u t a t h i o n e . A similar b i o t r a n s f o r m a t i o n h a s been observed f o r t h e o t h e r components of dihydroergotoxine i n r a t s . 6 6
6.
Methods o f A n d v s i s 6.1 6.11
si
Dihvdroernotox i n e mesilate
Element calculated $ % found 6.12
C
H
N
S
59.4 59.0
6.8 6.8
10.3 10.5
4.7 4.8
ComDonents of Dihvdroe r n o t o x i n e mes i l a t e
The e l e m e n t a l a n a l y s e s of t h e bases were r e p o r t e d by S t o l l and Hofb1ann:15*~9
Element D i hydroergo-
cornine Dihydroergocristine D i h yd r o e r g okrypti n e Dihydro- &ergokryptine
calc. found calc. found oalc. found calc. found
% %
p % % % % %
C
H
N
66.03 66.04 68.70 68.78 66.51 66.36 66.51 66.2
7.33 7.56 6.76 6.86 7.50 7.59 7.50
12.43 12.66 11.46
8.0
11.40
12.13 12.12
12.13 12.0
W. DIETER SCHOENLEBER et oJ.
128
6.21
General Tests
Some g e n e r a l non-specific i d e n t i t y tests f o r e r g o t a l k a l o i d s are Mayer's r e a g e n t (mercuric potassium i o d i d e ) , which g i v e s a p r e c i p i t a t e i n d i l u t i o n s of 1 p a r t p e r m i l l i o n o f t h e a l k a l o i d s . Iodine i n potassium iodide a l s o y i e l d s p r e c i p i t a t e s from d i l u t e s o l u t i o n . Other a l k a l o i d a l r e a g e n t s g i v e p r e c i p i t a t i o n s w i t h e r g o t a l k a l o i d s (phosphomolybdic a c i d , phosphotungstic a c i d , cadmium-potassium i o d i d e , potassium iodobismuthate, e t c . )
.
Since e r g o t alkaloids, i n c l u d i n g t h e i r 9,lOdihydro d e r i v a t i v e s , p o s s e s s a n i n d o l e nucleus, unsubs t i t u t e d a t C-2, t h e y g i v e v a r i o u s i n d o l e c o l o u r t e s t s . 5 Some o f these are g i v e n below:
-
I.
KeJJ&rLs-tgs& The a l k a l o i d is d i s s o l v e d i n glacial acetic a c i d t o which h a s been added a trace o f anhydrous f e r r i c c h l o r i d e . After c a r e f u l l y l a y e r i n g i n concentrated s u l f u r i c acid, an i n t e n s e b l u e - v i o l e t colour i s produced a t t h e i n t e r f a c e .
11.
GlyoxyAia aCid_rmgeRt-- A test based on t h e presence of g l y o x y l i c acid i n acetic acid a s an i m p u r i t y h a s been developed:67 The r e a g e n t g i v e s a b l u e c o l o u r with t h e a l k a l o i d i n t h e presence of concentrated s u l f u r i c acid. T h i s r e a c t i o n h a s been used, f o r example, f o r t h e v i s u a l i z a t i o n of dihydroergotoxine and s e v e r a l o t h e r a l k a l o i d s a f t e r t h i n l a y e r chromatograph on s i l i c a g e l by Puech, Duru and Jacob. 68 The spraying a g e n t contained g l y o x y l i c acid monohydrate ( 0 . 5 g) , f e r r i c c h l o r i d e hexahydrate (0.5 g), acetic acid (60 m l ) and sulfuric acid (30 m l ) p l u s 10 m l o f d i s t i l l e d water. The method i s s e n s i t i v e t o 0.1 microgram of a l k a l o i d .
111. l'anIlrk1s_te9t-- Ergot a l k a l o i d s , includi n g dihydrogenated ones, y i e l d a deep b l u e c o l o u r with 4-dimethylaminobenzaldehyde re-
agent (0.125 g 4-dimethylaminobenza1dehydel
DIHYDROERGOTOXINE METHANESULFONATE
129
0.1 m l 5% FeC13 d i l u t e d i n 65% s u l f u r i c acid t o g i v e 100 m l ) w i t h an a b s o r p t i o n maximum a t about 585 nm. T h i s t e s t h a s been widely used f o r t h e c o l o r i m e t r i c a s s a y o f i n d o l e a l k a l o i d s (see s e c t i o n 6.33). It h a s a l s o found a p p l i c a t i o n i n a f i e l d t e s t f o r halucinogens ,69 A modified van Urk r e a g e n t c o n t a i n i n g e t h a n o l i c hydrochloric acid ( E h r l i c h ' s r e a g e n t ) h a s o f t e n been used f o r t h e
v i s u a l i z a t i o n of a l k a l o i d a l compounds on t h i n l a y e r p l a t e s . 7 0 6.22
SDeci f i c I d e n t i f i c a t i o n 'I 'ests
6.221
I n f rared SDectruql
- see s e c t i o n 2.11 6.222
ChromatoaraD h i c Behavior
R f v a l u e , v i s u a l i z a t i o n i n t h i n l a y e r and k l v a l u e s i n high performance l i q u i d chromatography both f o r t h e dihydroergotoxine group a l k a l o i d s a s a whole and f o r t h e s i n g l e components are g e n e r a l l y cons i d e r e d t o be very s p e c i f i c i d e n t i f i c a t i o n criteria (see s e c t i o n 6.36).
6.223
~3
The amino acids p r e s e n t i n t h e p e p t i d e moiety o f t h e e r g o t a l k a l o i d s can be used f o r i n d i r e c t i d e n t i f i c a t i o n , b u t t h i s method does n o t allow d i s t i n c t i o n between epimers. The a l k a l o i d s are hydrol y z e d w i t h concentrated hydrochloric acid i n a sealed t u b e a t 100' f o r s i x t e e n hours. The genera t e d amino a c i d s are i d e n t i f i e d a f t e r s e p a r a t i o n by t h i n l a y e r , paper o r i o n exchange chromatography according t o familiar methods.
Valine o c c u r s only i n dihydroergocornine, phenylalanine i n d i h y d r o e r g o c r i s t i n e , l e u c i n e results from t h e h y d r o l y s i s o f dihydro-a-ergokrypt i n e , and i s o l e u c i n e from dihydro-B-ergokryptine. Consequently, dihydroergotoxine mesilate w i l l cont a i n these f o u r amino a c i d s , t o g e t h e r w i t h p r o l i n e .
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W. DIETER SCHOENLEBER et al.
T h i s method has been u t i l i z e d f o r t h e quantit a t i o n o f t h e components' r a t i o ( s e c t i o n 6.38).
6.3
A spectrophotometric method used t o determine nonhydrogenated a l k a l o i d s i n dihydroergotoxine h a s been d e s c r i b e d ,T1,T2 measuring t h e unhydrogenated p a r e n t compound a t 318 nm which shows a molar absorpt i v i t y o f 8000 compared t o 80 f o r dihydroergotoxine.
6.32 Fluorometric methods t o measure nonhydrogenated e r g o t a l k a l o i d s as i m p u r i t i e s i n dihydroergotoxine have been d e s c r i b e d 7 l (see a l s o s e c t i o n 2.13). The q u a n t i t a t i v e u - f l u o r o d e n s i t o m e t r i c determination o f t h e hydrogenated a l k a l o i d s o f t h e ergot o x i n e group was r e p o r t e d by Prosek and co-workers .28 The e x c i t a t i o n wavelength was 230 nm, the emission was a maximum between 250 and 320 nm. The u t i l i z a t i o n o f a n adequate cut-off f i l t e r is e s s e n t i a l .
6.33 The van Urk reaction20973-75 has been used by many workers t o determine t h e e r g o t a l k a l o i d s both i n bulk substance and a f t e r chromatographic s e p a r a t i o n (see s e c t i o n 6.21).
For lack of t h e i n t a c t i n d o l e system, t h e oxidat i o n products of dihydroergotoxine mesilate do n o t g i v e t h e van Urk r e a c t i o n so t h a t t h e y w i l l n o t i n t e r f e r e w i t h t h e assay.35 9 5 6 ~ 7 6 6.34 Gravimetric methods f o r a l k a l o i d s are q u i t e nons p e c i f i c b u t can be used t o measure t h e t o t a l a l k a l o i d c o n t e n t o f p r e p a r a t i o n s t h a t do n o t c o n t a i n o t h e r basic substances. I n most methods a p r e c i p i t a n t such as phosphomolybdic acid, potassium i o d i d e , potassium iodobismuthate, p i c r i c acid, phosphotungstic acid, s i l i c o t u n g s t i c acid, p i c r o l o n i c acid, ammonium r e i n e c k a t e and t r i c h l o r o a c e t i c a c i d is u t i l i z e d . Details of some t y p i c a l methods f o r v a r i o u s a l k a l o i d s are d e s c r i b e d by Higuchi and Bodin.77
DIHYDROERGOTOXINE METHANESULFONATE
6.35
131
Yol u m e t r i c Analvs is
S i n c e t h e e r g o t p e p t i d e a l k a l o i d s a r e basic, v a r i o u s ti trimetric methods have been developed f o r t h e i r a s s a y . The methods are non-specific and cannot be used t o measure t h e s t a b i l i t y o f t h e a l k a l o i d s , f o r example. A g e n e r a l view o f volumetric methods has been presented. 77 6.36
ChromatoaraDhv
The h i s t o r y o f t h e e r g o t a l k a l o i d chemistry as a whole and t h a t o f dihydroergotoxine i n p a r t i c u l a r i s t i g h t l y connected t o t h e development o f t h e s e p a r a t i o n techniques. Before 1943, chromatographic methods desc r i b e d dihydroergotoxine mesilate as a "single" compound. Afterwards , three-component s e p a r a t i o n s are r e p o r t e d , and f i n a l l y , t h e four-component s e p a r a t i o n has been achieved. I n t h e following chapter, a l l t h r e e s e p a r a t i o n modes w i l l be quoted.
6.361
PaDer Chr omat op:raDhv
A number o f paper chromatographic systems have been used f o r the s e p a r a t i o n of dihydroergotoxine alkaloids.
S t o l l and Riiegge1-7~ u t i l i z e d a r e v e r s e phase system w i t h d i m e t h y l p h t h a l a t e a s t h e s t a t i o n a r y phase and formamide-water pH 4 (1:4) a s t h e mobile phase. Adamanis and co-workers79 used a m o d i f i c a t i o n of t h e S t o l l system t o a c h i e v e s e p a r a t i o n . The paper was impregnated w i t h 1 0 % d i e t h y l p h t h a l a t e i n e t h e r and developed i n c i r c u l a r , as a time-saving technique, with formamide-water (1:4) a t pH 4.0 c o n t a i n i n g a small amount o f d i e t h y l p h t h a l a t e . The R f o f dihydroe r g ocor n i n e , d ih ydroergo c r ist i n e and d i hyd r o e r g okrypt i n e i n t h e system were 0.66, 0.54 and 0.47, respectively. The a l k a l o i d s of t h e dihydroergotoxine group
( 3 components) have been s e p a r a t e d from each o t h e r 3 l and from t h e i r a u t o x i d a t i o n products56 using paper impregnated w i t h 40% formamide i n ether and developed w i t h benzene-chloroform (1:l) f o r 5 hours. The s p o t s were v i s u a l i z e d by induced f l u o r e s c e n c e v i a U V irradiation.
W. DIETER SCHOENLEBER et al.
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The s e p a r a t i o n of t h e d i h y d r o e r g o t o x i n e a l k a l o i d s from t h e i r l i g h t d e g r a d a t i o n p r o d u c t s has been described80 u s i n g paper impregnated w i t h formamidee t h a n o l (2:3) and development w i t h benzene-chloroform (1:l).
Gawrych and Wilczynska71 have d e s c r i b e d two systems f o r t h e s e p a r a t i o n of e r g o t o x i n e and dihydro-
ergotoxine: 1.
Paper impregnated w i t h formamide, e l u t i o n with formamide-saturated benzene-chloroform (1:1)
2.
Paper impregnated w i t h formamide, e l u t i o n w i t h formamide-saturated chloroform-cyclohexaned i e t h y l a m i n e (2:7:1).
.
s e p a r a t i o n w i t h o u t p r e p a r a t i o n of t h e aper has been r e p o r t e d by Margasinski and c o - ~ o r k e r s ~developl i n g w i t h t h e same mobile phase as Adamanis79 (formamide-water 1 : 4 ) , leading t o R f v a l u e s of 0.14, 0.22 and 0.34 f o r d i h y d r o e r g o c r i s t i n e , d i h y d r o e r g o k r y p t i n e and d i h y d r o e r g o c o r n i n e , r e s p e c t i v e l y . A pc
Reio77 has l i s t e d s i x paper chromatographic systems 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 of d i h y d r o e r g o t o x i n e and v a r i o u s o t h e r a l k a l o i d s , t h e b e s t s u i t e d f o r d i h y d r o e r g o t o x i n e being:
A.
Methyl i s o b u t y l ketone-formic acid-water (10 p a r t s k e t o n e saturated w i t h 1 p a r t 4% formic acid)
F.
Methyl e t h y l ketone-acetone-formic acid-water (40:2:1:6).
The s p o t s were v i s u a l i z e d w i t h van U r k ' s , Ehrlich's,
o r D r a g e n d o r f f ' s r e a g e n t (Potassium bismuth i o d i d e / water). HofInann20 h a s described two pc systems leading t o three-component s e p a r a t i o n s . I.
11.
Dimethylphthalate-impregnated p a p e r , development w i t h 20% formamide/80% pH 4.4 c i t r a t e b u f f e r (Soerensen) and
Paper impregnated w i t h formamide-benzoic a c i d , development w i t h d i e t h y l ether.
DIHYDROERGOTOXINE METHANESULFONATE
133
The van Urk r e a c t i o n served as a means o f d e t e c t i o n
and f o r c o l o r i m e t r i c q u a n t i t a t i o n a f t e r e l u t i o n of t h e a l k a l o i d s from t h e paper. 6.362
Thin Laver ChromatonraD hv
Zinser and Baumg%te183 have used a TLC system t o s e p a r a t e dihydroergotoxine ( a s i t s then-known components) from t h e p a r e n t compounds and s e v e r a l o t h e r Benzene-chloroform-ethanol a l k a l o i d s (see a l s o 8 4 ) (2:4:1) was a p p l i e d on n e u t r a l s i l i c a g e l . Q u a n t i t a t i o n was carried o u t by e l u t i o n with 40% methanol c o n t a i n i n g 1% t a r t a r i c a c i d and subsequent c o l o r i m e t r y a f t e r van Urk-reaction.
.
Wichlinski85 has r e p o r t e d a TLC method w i t h an acetone-benzene-petroleum ether ( 4 : 1:1) system f o r the s e p a r a t i o n o f dihydroergotoxine and some o t h e r e r g o t a l k a l o i d s on s i l i c a g e l G. The same purpose can be accomplished by a p p l y i n g e t h y l acetate-dichloromethane-methanol-conc. ammonia (50:50:3:1) on Merck F 254 s i l i c a g e l . Dihydroergot o x i n e e x h i b i t s a n R f v a l u e o f 0.45 under these cond i t i o n s . 86 S e v e r a l i n d o l e a l k a l o i d s , i n c l u d i n g dihydroergot o x i n e , have been s e p a r a t e d by Puech, Duru and Jacob68 on t h i n l a y e r s i l i c a g e l G p l a t e s w i t h e i t h e r butanolacetic acid-water (3:l:l) o r chloroform-acetonediethylamine (5:4:1). The a l k a l o i d s were v i s u a l i z e d by s p r a y i n g w i t h g l y o x y l i c acid r e a g e n t . D i h y d r o e r g o c r i s t i n e has been included i n a multisupports/mul t i - s o l v e n t s s c r e e n i n g p r o j e c t f o r t h e i d e n t i f i c a t i o n of l y s e r g i c and d i h y d r o l y s e r g i c a c i d d e r i v a t i v e s i e l d i n g a v a r i e t y o f a p p l i c a b l e TLC systems .29 * 8f Hohmann and Rochelmeyer30 used a c e l l u l o s e p l a t e impregnated w i t h dimethylformamide and developed w i t h ethylacetate-heptane-diethylamine (5: 6 :O .02) t o separate t h e dihydroergotoxine a l k a l o i d s i n t o t h e compon e n t s dihydroergocornine, d i h y d r o e r g o c r i s t i n e and dihydroergokryptine. The s p o t s were v i s u a l i z e d by exposing t h e p l a t e t o t h e l i g h t o f a mercury lamp t o form t h e b l u e f l u o r e s c e n t U V d e g r a d a t i o n products of t h e a l k a l o i d s (see a l s o 2.13). A modification of t h i s method was a p p l i e d by Prosek and co-workers28 i n dens i t o m e t r i c s t u d i e s
.
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W. DIETER SCHOENLEBER et a].
Zarchska and Ozarowsky88 have used talc as t h e adsorbent i n a TLC system. The formamide-impregnated p l a t e was d r i e d f o r 1 hour a t 25O, a mixture of n-heptane-tetrahydrofuran-toluene (5:4:1) was used f o r development. The t h r e e a l k a l o i d s were v i s u a l i z e d w i t h dimethylaminobenzaldehyde (modified van Urk r e a g e n t ) ,
Rsder , Mutschler and Rochelmeyer89 r e p o r t e d two TLC systems w i t h homogeneous a z e o t r o p i c s o l v e n t mixt u r e s f o r t h e s e p a r a t i o n of dihydroergocornine and
d i h y d r o e r g o c r i s t i n e , namely, dichloromethane-methanol (92.7:7.3), and chloroform-ethanol (92:8), respectively. Reichelt and Kudrnacgo used s i l i c a g e l impregnated w i t h formamide as a support. The TLC p l a t e s we re developed w i t h f ormamide- sa t ur a t ed d i i s o p r o p y l ether-tetrahydrofuran-diethylamine (80:20:0.2) o r w i t h d i b u t y l ether-dichloromethane-diethylamine (60:20:0.2), each y i e l d i n g a three-component separation.
Q u i t e r e c e n t l y , Reicheltgl r e p o r t e d on t h e three-component s e p a r a t i o n of dihydroergotoxine mesil a t e from bulk and pharmaceutical form samples on formamide-saturated t h i n l a y e r p r e f a b r i c a t e d p l a t e s ( S i l u f o l "Kavalier", Merck o r Schleicher + S c h u e l l ) w i t h d i iso propy l ether- t e t r a h y d ro f uran-die t h y l amine (90:10:0.7) a s a mobile phase. The dihydrogenated a l k a l o i d s were detected w i t h van U r k ' s a g e n t . Dihydroergocornine, - c r i s t i n e , and - k r y p t i n e s e p a r a t e r e a d i l y i n t h e s y s tea e ther-dime t h y 1formamide-oonc. ammonia (85:15:2) on Merck s i l i c a g e l F 254 prefab p l a t e s r e s u l t i n g i n o timal R f v a l u e s of 0.50, 0.44 and 0.55, r e s p e c t i v e l y . 66
-
Another n e a t three-component s e p a r a t i o n v i a reversed phase - c a n be accom l i s h e d on dimethyl p h t h a l a t e impregnated AVICEL Qp/ s i l i c a g e l ( 9 : l ) p l a t e s w i t h dimethyl p h t h a l a t e - e q u i l i b r a t e d pB 2 c i t r a t e b u f f e r .86 The components are e l u t e d w i t h ethanol-water-acetic a c i d ( 9 :9:2) and allowed t o react w i t h van Urk r e a g e n t . Spectrophotometry a t 585 nm y i e l d s q u a n t i t a t i v e r e s u l t s .
135
DIHYDROERGOTOXINE METHANESULFONATE
6.363
Gas Chromatonraohv
Not being v o l a t i l e without decomposition, t h e dihydroergotoxine a l k a l o i d m e s i l a t e s a r e n o t suscept i b l e t o GLC. However, r e c e n t l y an i n t e r e s t i n g GLC approach h a s been r e p o r t e d by Szepesi and Gazdag.92 The mesilates are converted t o bases by i o n exchange. Subsequently, t h o s e a r e decomposed a t 235O i n t h e s t a i n l e s s s t e e l i n j e c t i o n p o r t of a g a s chromatograph i n a s u r p r i s i n g l y r e p r o d u c i b l e manner. Hence t h e method l e n d s i t s e l f not only t o t h e i d e n t i f i c a t i o n b u t also t o t h e q u a n t i t a t i o n o f dihydroergocornine, d i h y d r o e r g o c r i s t i n e and dihydroergokryptine. The n a t u r e of t h e chromatographing species, d e r i v e d from t h e a l k a l o i d s ' p e p t i d e f r a c t i o n , i s s t i l l under investigation. GLC Conditions :
Column:
2% D e x s i l 300 on 80/100 mesh Gas Chrom Q i n a 1 m x 3.2 mm s t a i n l e s s s t e e l tube.
Carrier gas:
Nitrogen, 11.5 ml/min.
I n j e c t i o n p o r t temperature: Column temperature: 6.364
-
235O
180 2800C/heating r a t e 50 /min
.
. .
Hiah Perf0 rmance J,iauiQ
Chromatoaraohv biittwer and Kluckhohn93 have u t i l i z e d two HPLC systems i n t h e s e p a r a t i o n of v a r i o u s alkaloids.Dihyd r o e r g o c r i s t i n e served as an example f o r t h e 9,lOdihydrogena ted e r g o t p e p t i d e a1kaloid s. Its r e t e n t i o n r e l a t i v e t o ergotamine was r e p o r t e d as 0.81 i n t h e system a c e t o n i t r i l e - i s o p r o p y l ether (40:60) on .Sil-X (Perkin-Elmer) a s t h e s t a t i o n a r y phase and a s 0.73 i n t h e system a c e t o n i t r i l e - i s o p r o p y l ether (25:75) on Corasil-11 (Waters).
Jane and bhealsg4 have r e p o r t e d three d i f f e r e n t r e l a t i v e r e t e n t i o n times f o r dihydroergocornine, d i h y d r o e r g o c r i s t i n e and dihydroergokryptine, as t h e i n d i v i d u a l a l k a l o i d s on C o r a s i l C 18 (Waters). The method has been improved by V i v i l e c c h i a and co-workers95 u s i n g
W.DIETER SCHOENLEBER et al.
136
Figure 27. Three-component S e p a r a t i o n of d i hyd r o e r g o t o x i n e mesilate. Conditions ind icated i n s e c t i o n 6.364
DIHYDROERGOTOXINE METHANESULFONATE
137
t h e corresponding m i c r o p a r t i c l e s , P-hndapak-C 18 (Waters), s e p a r a t i n g these t h r e e a l k a l o i d components of d i hyd r o e r g o t o x i ne
.
I n a l l t h e above cases, t h e d e t e c t i o n was c a r r i e d o u t a t 254 nm w i t h a f i x e d wavelength U V detector. Q u i t e r e c e n t l y , Hartmann, RSdiger , A b l e i d i n g e r and bethke96 r e p o r t e d the HPLC s e p a r a t i o n of dihyd r o e r g o t o x i n e mesilate. The s p l i t t i n g - u p o f dihydroe r g o t o x i n e mesilate i n t o dihydroergocornine, dihydroe r g o c r i s t i n e and d i h y d r o e r g o k r y p t i n e ( 3 component s e p a r a t i o n ) was accomplished u s i n g prepacked as well a s self-made columns w i t h 5 o r 1 0 p C-8 o r C-18 reversed phase material. The mobile phase was acet o n i t r i l e - 0 . 0 2 & ammonium c a r b o n a t e i n water ( 0 . 5 4 : l ) or a c e t o n i t r i l e - 1 / 3 0 & phosphate b u f f e r , pH a d j u s t e d t o 7.5 ( 0 . 5 4 : l ) (see f i g . 2 7 ) . According t o t h e same a u t h o r s , t h e base l i n e s e p a r a t i o n of d i h y d r o e r g o k r y p t i n e I n t o i t s two isomers , r e s u l t i n g i n a f o u r component s e p a r a t i o n of dihydroe r g o t o x i n e , c a n be accomplished by t h e a p p l i c a t i o n of t h e f o l l o w i n g HPLC systems: I.
11.
111.
LiChrosorb RP 18 (Merck) 5 MI (12.5 cm, 4 mm 1 . D . ) or 1 0 IJm (25 cm, 4 mm I . D . ) / water-acetonitrile-triethyl amine ( 32 :8 :1) (v /v/v) LiChrosorb RP 8 (Merck) 7 pm (25 cm, 3 mm I.D. ) / w a t e r - a c e t o n i t r i l e t r i e t h y l amine (75:40:1) (v/v/v) bBondapak C-18 ( h a t e r s ) 1 0 P m (30 cm, 4 mm I.D.)/water-acetonitrile-triethyl amine (22.8:11.4:1) (v/v/v)
See f i g u r e 28. A spectrophotometer set a t the a b s o r p t i o n maximum of
about 280 nm was used as a d e t e c t o r . T h i s method has been u t i l i z e d f o r 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 purposes a s well as f o r t h e determ i n a t i o n of t h e r e l a t i v e ratios of t h e f o u r dihydroe r g o t o x i n e components.
W.DIETER SMOENLEBER et al.
138
INXCT
Figure 28.
Four-component HPLC Separation of dihydroergotoxine mesilate. Conditions indicated i n section 6 . 3 6 4
DIHYDROERGOTOXINE METHANESULFONATE
6.37
139
13C-Nuclear Magnetic Resonan c e
Q u i t e r e c e n t l y Ernie and L 0 0 s l i 3 ~have shown t h a t t h i s powerful technique can be used t o determine t h e p r o p o r t i o n s of t h e f o u r components o f dihydroergotoxine m e s i l a t e . Dissolved i n d e u t e r a t e d dimethylsulphoxide , t h e four a l k a l o i d s show discrete resonance s i g n a l s f o r C-5' t h u s e n a b l i n g convenient ( b u t a d m i t t e d l y time-consuming) component r a t i o q u a n t i t a t i o n , f i g u r e 29 (see a l s o s e c t i o n 2.15).
Figure 29.
50 t o 70 ppm S e c t i o n o f t h e 1 3 C NMR
Spectrum o f dihydroergotoxine me si l a t e (500 mg i n 1.5 m l dimethylsulfoxide-d6). C-5' S i g n a l s i n b l a c k .
W.DIETER SCHOENLEBER et 01.
140
6-38 Amino acid a n a l y s i s (see a l s o s e c t i o n 6.223) h a s been u t i l i z e d f o r t h e d e t e r m i n a t i o n of the p r o p o r t i o n s o f t h e f o u r dihydroergotoxine components. The drug substance is s u b j e c t e d t o h y d r o l y s i s w i t h h y d r o c h l o r i c acid leading t o t h e breakdown o f t h e p e p t i d e f r a c t i o n . The r e s u l t i n g amino acids are t h e n chromatographed by i o n exchange and detected after r e a c t i o n w i t h ninhyd r i n e according t o common procedures. From the peak areas o f l e u c i n e , i s o l e u c i n e , phenylalanine and v a l i n e , t h e r e l a t i v e p r o p o r t i o n s of the four compon e n t s can be c a l c u l a t e d . 6.4
Countercurrent DistributiQn
Galeffi and Delle Monache97 have u t i l i z e d 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 o s e p a r a t e dihydroergot o x i n e i n t o 3 components. A s o l v e n t system o f 1 p a r t of chloroform and 1 part o f carbon t e t r a c h l o r i d e was e q u i l i b r a t e d w i t h phosphate b u f f e r s o f v a r i o u s pH values. After 270 t r a n s f e r s a t pH 4.8 and 250 a t pH 4.5, dihydroergocornine could be separated from d i h y d r o e r g o c r i s t i n e and dihydroergokryptine. The l a t t e r two could be separated a f t e r more t h a n 750 a d d i t i o n a l t r a n s f e r s a t pH 4.5.
6.5
-
D e t e r U . w t i o n i n Bodv F l u i d s
Due t o t h e i r s t r o n g biopotency, t h e dihydroergotoxine a l k a l o i d s are administered i n very small dosages of a few milligrams per day, leading t o minute c o n c e n t r a t i o n s i n body f l u i d s and t i s s u e s . A s e n s i t i v e fluorome tr i c der i v a t i za t i o n method h a s been developed,g8 b u t i t is s t i l l n o t s e n s i t i v e enough t o cover t h e extremely low c o n c e n t r a t i o n range f o r t h e a l k a l o i d s . As mentioned i n s e c t i o n 6.363, gas chromatography and GC-MS c o u p l i n g , i n c l u s i v e l y , cannot be u t i l i z e d r e a d i l y because of t h e low v o l a t i l i t y and t h e thermal i n s t a b i l i t y o f t h e compounds i n question. Therefore, t h e o n l y methods p r e s e n t l y a v a i l a b l e for biopharmaceutical s t u d i e s are t h e r a d i o t r a c e r (3H, 1%)and t h e radio immuno a s s a y techniques.63 These methods on t h e o t h e r hand r e q u i r e a very high l e v e l o f s o p h i s t i c a t i o n concerning s t a n d a r d s t a b i l i t y and
DIHYDROERGOTOXINE METHANESULFONATE
141
p u r i t y f o r t h e first one, high s p e c i f i c r a d i o a c t i v i t y and a c c u r a t e l y e s t a b l i s h e d t e s t s p e c i f i c i t y f o r t h e l a t t e r . It is due t o these c r u c i a l experimental cond i t i o n s t h a t s u c c e s s f u l biopharmaceutical s t u d i e s have only r e c e n t l y been reported.63'65 6.6
Determi n a t i o n i n Pharmaceutical Forglg
Dihydroergotoxine mesilate can b e i d e n t i f i e d i n t h e v a r i o u s pharmaceutical forms by spectrophotometry, photometry and c o l o r i m e t r y following t h e van Urk r e a c t i o n , o r by i t s paper, t h i n l a y e r o r high p e r f o r mance l i q u i d chromatographic behaviour. Q u a n t i t a t i o n i n dosage forms h a s been accomp l i s h e d by c o l o r i m e t r y ,31,56,75 t 76 t h i n l a y e r chromatography p l u s c o l o r i m e t r y , 3 0 , 8 6 densitometry28 and h i g h performance l i q u i d chromatography. 96
W. DIETER SCHOENLEBER et
142
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a,
a,
a,
m,
DIHYDROERGOTOXINE METHANESULFONATE
A c k n o w l e m e nts The a u t h o r s are Indebted t o many c o l l e a g u e s a t Sandoz L t d . f o r t h e i r v a l u a b l e h e l p , p a r t i c u l a r l y t o H. Bethke, F. E r n i , Mrs. D. C i r o n , J.-R. Kiechel, P. Koci, and h.-R. L o o s l i . The t e c h n i c a l a s s i s t a n c e for t h e proper present a t i o n of t h e mass s p e c t r a by J . Clerc a t t h e Swiss F e d e r a l I n s t i t u t e of Technology i n Zurich i s gratef u l l y acknowledged.
147
Analytical Profiles of Drug Substances, 7
DIPHENOXYLATE HYDROCHLORIDE
Donald D . Hong
149
Copyright @ 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-260807-0
DONALD D. HONG
150
CONTENTS 1.
Description 1.1 Name, Formula, Molecular Wei ght 1.2 Appearance, Color, Odor
2.
Physical Properties 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10 2.11 2.12 2.13
Bulk Density and Specific Gravity Differential Scanning Calorimetry Di ssociation Constant F1 uorescence Infrared Spectrum Mass Spectrum Nuclear Magnetic Resonance Spectrum Me1 ti ng Range Optical Rotation Partition Coefficient PH Sol ubi I i ty U1 traviolet Spectrum
3.
Synthesis
4.
Drug Metabolism 4.1 4.2
Biotransformation Products Pharmacokinetics
5. Stability 6.
Methods o f Analysis Elemental Analysis Microchemical Identification Chromatographic Analysis 6.31 Thin Layer Chromatography 6.32 Gas Liquid Chromatography 6.4 Colorimetric Analysis 6.5 Spectrophotometric (UV) Analysis 6.6 Titrimetric Analysis 6.61 Non-aqueous Titration 6.62 Semi-microtitrimetry Using Sodium Lauryl Sulfate
6.1 6.2 6.3
7. Acknowledgments 8. References
DIPHENOXYLATE HYDROCHLORIDE
1.
151
Description
1.1 Name, Formula, Molecular Weight The chemical name o f diphenoxylate h y d r o c h l o r i d e i n Chemical Abstracts i s found under I s o n i p e c o t i c acid, designated as I s o n i p e c o t i c acid, 1-(3-cyano-3,3d i phenyl propyl )-4-phenyl- , e t h y l e s t e r , monohydroc h l o r i d e (3810-80-8)
- HCI C30H32N202 HC1 1.2
MW 489.06
Appearance, Color, Odor Diphenoxylate h y d r o c h l o r i d e i s a white, odorless, m i c r o c r y s t a l l i n e powder having a b i t t e r t a s t e . A s a t u r a t e d aqueous s o l u t i o n o f t h e chemical i s c o l o r 1ess
.
2.
Physical P r o p e r t i e s
2.1
Bulk Density and S p e c i f i c G r a v i t y The b u l k d e n s i t y of t h e compound i s about 0.2 g/ml w i t h a s p e c i f i c g r a v i t y o f 0.832 0.836 ( 1 ) .
-
2.2
D i f f e r e n t i a l Scanning Calorimetry The DSC thermogram o f diphenoxyl a t e h y d r o c h l o r i d e obtaiged on a Perkin-Elmer DSC-2 a t a h e a t i n g r a t e o f 10 C/minute i s shown i n Figure 1. The endo228' C thermic range corresponds t o 219.2 - 223.5 (onset of m e l t - p o i n t of maximum m e l t i n g r a t e end o f m e l t ) .
-
DONALD D. HONG
152
210
Tunpnrun OC FIQURE 1:
DSC THERMOORAM OF DIPHENOXYLATL HYDROCHLORIDE (Cm RER CWSA\
DIPHENOXYLATE HYDROCHLORIDE
2.3
153
D i s s o c i a t i on Constant The pKa o f diphenoxylate h y d r o c h l o r i d e by t h e titrim e t r i c method was found t o be 7.1 (1).
2.4
F1uoresence Diphenoxylate h y d r o c h l o r i d e e x h i b i t s no n a t u r a l fluorescence when observed as 0.001 0.100% w/v s o l u t i o n s i n MeOH ( 1 ) .
-
2.5
I n f r a r e d Spectrum The i n f r a r e d absorption spectrum o f diphenoxylate h y d r o c h l o r i d e i n a K B r d i s p e r s i o n i s shown i n F i g u r e 2. Table 1 l i s t s t h e major absorption band assignments (2). Table 1.
Band (cm-l) 2800-3 140 2320 1770-2000 1730 1605,1450-1500 1025 855 2.6
I n f r a r e d Band Assignments Ass ignment CHs, t r e t c h R3N-H, CzN s t r e t c h Aromatic overtone bands CcO s t r e t c h 0 Aromatic C=C s t r e t c h O-CH CH (0-C) s t r e t c h C h a r i c t i h s t i c o f ethyl ester
Mass Spectrum The mass spectrum o f diphenoxylate i s shown i n F i g u r e 3. The peaks o f i n t e r e s t a t m/e 246, 377 and 452 a r e as i n d i c a t e d .
2.7
Nuclear Magnetic Resonance (NMR) The spectrum i n Figure 4 was obtained w i t h a Varian A60 NMR Spectrometer u s i n g a 10.6% s o l u t i o n o f diphenoxylate h y d r o c h l o r i d e i n deuterated c h l o r o form. Band assignments are r e l a t i v e t o TMS i n t e r n a l standard and summarized i n Table 2 ( 2 ) .
PI
a
b
a
a,
LI)
I II 36
m xm
I43
155
I
x5-
1
Figure 3. Mass spectrum (electron impact) of diphenoxylate (After brim and Heykants, r e f . 3)
-.
d o
DONALD D. HONG
156
Table 2.
NMR Spectral Assignments E
CN
D
C
C HC1
PPM Chemical S h i f t
Protons
1.19 4.21 7.4 2.7 2.5 - 3.8
A B C D E
2.8
Multiplicity Triplet Quartet Mu1 t i pl e t Broad Sing1 e t Broad Bands
Me1 t i ng Range
The me1t i n g range of diphenoxyldte hydrgchloride given i n USP X I X , p. 158, is 220 t o 226 C. 2.9
Optical Rotation Diphenoxylate hydrochloride exhibits no optical a c t i v i t y (1).
2.10
P a r t i t i o n Coefficient
K =
‘CHCl
= 473
‘H20 (pH 6.8)
There i s l i t t l e , i f any, variation i n K between CHC13 and water as a function o f pH ( 1 ) .
I . . a0
I
.
.
.
.
l
,
.
.
.
I
.
.
l
.
.
.
.
l
.
.
.
.
l
~
.
.
.
I
~
.
.
,
I
I . . . . ~ . . . . I . . . . I . . . . I . . . . I . . . . I . . . . I . . . . I . . . . ~ . . . . l . . . . ~ . . . . l , . , . , . . . , I . . ,
7.0
6.0
5.0
PPM( 6 )
4.0
C l O w L 4. WUAn U Q f T l C
20
lluLyyyf
mEcTnu oc D l M u u u v u l E
WDI*a(LOnIDT
I
I
.
DONALD D. HONG
158
2.11
pH A saturated solution of the compound i n water has a pH of about 3.3 ( 4 ) .
2.12
Solubility S o l u b i l i t i e s i n various solvents a t 25' C a r e tabulated i n Table 3.
Table 3.
Solubility Data f o r Diphenoxylate HC1
Sol vent
Solubility, mg/ml
Methanol Chloroform
) 50 ) 50
Acetone Benzene Ethanol Ethyl Acetate
Water
Heptane Ethyl Ether Simulated gastric f l u i d
2.13
5
6 3 3 2.3 0.8 (0.1 (0.1
(0.1
Acetic Acid 500 Dimet hy 1f o rmami de 500 Chloroform* 360 Dioxane 46 Tetrahydrof uran 21 i-Propanol 2.5 Carbon Tetrachloride 1 Hexane 0.5 0.1N HC1 (0.1 0.1N NaOH (0.1 0.1N NH40H loo.
308
CHESTER E. ORZECH et a].
Kobinger and Lund report that hydroflumethiazide is e a s i l y soluble i n the lower alcohols and ketoner, tetrahydrofuran, and pyridine. The p r r t i t i o n coefficient between e t h y l acetate or methyl isobutyl ketone and water i r about 30. It is also e a s i l y soluble i n b86eS (15). Hydroflumethiazide i8 soluble i n a mixture of polyethylene glycol 400, N-ma thyl-2-pyrrolidinoac, and water (40:5:55), or i n a mixture of polyethylene glycol 400, d i methylfonnamide, and water (40:5:55); the s o l u b i l i t i e s a r e 11.2 and 15.8 mg/ml, respectively (16). 2.8 Ionization Constant Kobinger and Lund report a pK1 of 8.9 and a pK2 of 10.7 a t room temperature (15). Smith e t a1 report a pK1 of 8.73, determined by rpectrophotomctry a t 273 nm, and of 8.79, determined by fluoronetry with e x c i t a t i o n a t 333 nm and emission a t 393 nm (17).
.
2 9 Fluorescence Spectra Hydroflumethiazide fluorescence ha6 an excitation maximum a t 333 nm and an emiosioa maximum a t 393 nm. Fluorercence is much more intenre a t pH 8 or les8 (17).
2.10 Melting Point8 Reported melting points range from about 200' t o 275OC (18-36, 38). The m l t i n g point i r specified as 270' t o 275'C i n NF XIV. 3.
SYNTHESIS
Hydrof lumethiazide ha8 been prepared by vrrious procedurer from t r i f luoroma thyl-5-aminobenzene-2 ,I-di8olforumide (18-28, 30, 32, 34-38), tr i f luoromethyl-5-8minobentene-2,4disulfonyl chloride (31, 33), or 6-(trifluoromcthyl)-7-~ulfamyl-l,2,4-benzothiadiatinc 1,l-dioxide (29). A few of these procedures including ryntheser of the s t a r t i n g material6 a r e outlined i n Figure 6. 4
.
STABILITY-DEGRADATION
Kobinger and Lund (15) reported 1 percent loss i n 24 hours in 0.1E hydrochloric acid a t 38*C, and 0.5 percent i n 24 hours i n 0 . E rodium hydroxide a t room temperature. However, boiling in 3E sodium hydroxide for 2 hour q w a t i t a t i v e l y conver tad hydrof lumethiazide t o 2,4-dirulfamyl-5-trif luoromc thylani 1ine
.
r
Figure 6. Synthesis of Hydrof lume thiazide
.
/.
clso/ 2. NQOH
1
,
1. (HCHOJn,
CHESTER E. ORZECH et al.
310
Fazzari (8) reported that less than 0.04 percent of the disulfonamide formed when hydroflumethiaside was stored for 5 hours i n 0.2E sodium hydroxide a t room temperature. 5.
METABOLISM
No reports of metabolic studies on hydroflunrethiazide were found. 6.
METHODS OF ANALYSIS 6 . 1 Identification Tests
Hydroflumethiazide is e a s i l y identified by the prope r t i e s described i n section 2 above. Where i d e n t i f i c a t i o n of hydroflumethiazide i n s o l i d formulations is necessary, i t can be extracted with a small volume of sodium hydroxide solution; the e x t r a c t is made s l i g h t l y acid t o precipitate the hydroflumethiazide, which can be washed with water and dried before further testing. Hydroflumethiazide can be extracted from a c i d i c aqueous solution with diethyl ether and recovered by evaporating the ether extract; the recovered material can be identified from i t s infrared spectrum, a s reported by Fazzari (8) and as directed i n NF XIV (39). Ethyl acetate and mcthyl isobutyl ketone a r e a l s o s u i t a b l e for extraction of hydroflumethiazide ( 1 5 ) . If the i d e n t i f i c a t i o n of 6 m P L l amounts of hydroflume thiaz ide is necessary, thin layer chromatography procedures are q u i t e specific. Several procedures for identifying thiazide diuretics, using t h i n layer chromatography ( 4 0 , 41, 4 2 , 4 3 , 44, 4 5 ) or paper chromatography (4), with various detection methods, have been reported. Ultraviolet absorption spectra i n acid and a l k a l i n e solution show comparatively small s h i f t s i n wavelength maxima in the case of hydroflumethiazide ( 4 ) , but may help t o d i f f e r e n t i a t e i t from somc other thiazides. Microscopic t e s t s for hydroflumethiazide a r e described by Croenewegen ( 1 4 ) and by Kuhnert-Brandstitter e t a 1 (13). 6.2 Elemental Analysis
The elemental composition of hydroflume thiazide is a8 follass :
311
HYDROFLUMETHIAZIDE Element
7. Theory
Carbon Hydrogen F luor ine N i tr ogen Oxygen Sulfur
29.00 2 -43 17.21 12.68 19.32 19.36
6.3 Colorimetric Method Bermlo (46) determined hydroflumethiazide by hydrolyzing i t to ~,4-disulfamyl-5-trifluoromet h y l a n i l i n e , diazot i z i n g the hydrolysis product and coupling to chromotropic a c i d or t o N-(1-naphthy1)e thylenediamine dihydrochloride. Kobinger and Lund (15) a l s o indicate that t h i s technique can be used f o r q u a n t i t a t i o n of hydroflumethiazide. 6.4 T i t r a t i o n Methods DePaulis and Diuietromaria (7) t i t r a t e d hvdroflumethiazide i n anhydrous eihylenediamine-solutionwiih sodium me thoxide i n benzene -methanol (85 :15), using a s a t u r a t e d solut i o n of azo v i o l e t i n benzene as indicator. Chiang (47) t i t r a t e d hydroflumethiazide i n dimethylformamide with sodium methoxide i n benzene-methanol s o l u t i o n , using a s a t u r a t e d s o l u t i o n of p-nitrobenzene-azo-resorcinol i n benzene a s i n d i c a t o r ; thymol blue indicator gave u n s a t i s f a c t o r y endpoints. Sugar and lactose i n t e r f e r e d , however. 6.5 Paper Chromatography
Adam and Lapiere (40) used two systems for paper chromatography of hydroflumethiazide: ( a ) Whatman No. 1 paper, with 1-pentanol, 12E a m o n i a , and water (80:20:60) a s mobile phase; (b) Whatman No. 1 paper impregnated w i t h formamide, with chloroform and 1-pentanol (80:20) a s mobile phase. The spots were detected using s h o r t wavelength u l t r a v i o l e t r a d i a t ion. Pilsbury and Jackson (4) a l s o used two s y s t e m t o d i f f e r e n t i a t e hydroflume thiazide from other thiazide diure tics: ( a ) Whatman No. 3 paper impregnated with t r i b u t y r i n , developing f o r 20 minutes a t 9OoC with pH 7.4 phosphate buffe r ; (b) Whatman No. 1 paper, with amyl alcohol and a m o n i a (9:l) a s mobile phase. The spots were located using s h o r t wavelength u l t r a v i o l e t r a d i a t i o n , and a l s o by spraying with 0.1E sodium hydroxide s o l u t i o n followed by a s a t u r a t e d solut i o n of sodium 1,2-naphthoquinone-4-sulfonate i n ethanol and water (1:l); the l a t t e r treatment develops a n orange-red color
CHESTER E. ORZECH et
312
with thiazides
.
01.
.
6 6 Column Chromatography Fazzari ( 8 9 48) used a &sic celitt@ column to purify
t a b l e t e x t r a c t s prior t o u l t r a v i o l e t quantitation. So& e t a1 ( 4 3 ) used non-ionic resin colunms t o recover and clean up thiazides i n urine prior t o thin layer chromatography.
Hydroflumethiazide can be separated and quantitated by columa chromatography with a gravity fed eluant as follows (49 1: Sephade@ LH-20, 23 cm x 1.2 em I.D. Me thanol-cyclohexane-chloroform-ace t i c acid (40r30t30:l vol/vol) Elution Volum: 72 t o 117 m l Column: Eluant:
-
Fluorescence 333 nm excitation, 380 nm emission About 100 u g of hydroflumethiazide
Quantitation: Sample:
Pressurized liquid chromatography can a l s o be used t o determine hydroflumethiazide. The following system is s u i t able (49): W t e r s y-Boadapal@ 30 cm x 4 m I.D. pre-packed coltrmn Solvent: Methanol 0.02g pH 7.4 phosphate buffer (2:8 vol/vol) Pressure: 1500 p s i Plow Ratet 1 m l h i n u t e sample: About 1 u g Detection: Schoeffel Variable Wavelength Ultrav i o l e t Detector, 280 nm Column:
-
6.7 Ultraviolet Me thods
Identification of hydroflrwethiazide by u l t r a v i o l e t absorption was discussed ihsections 2.3 8nd 6;l. Fazzari (8) developed a p a r t i t i o n chromatographyu l t r a v i o l e t method for hydroflumcthiazide i n drug formulations. The compound i r h e l d on an alkaline C e l i t 8 colunm, which is washed with chloroform and ether, then eluted with a c e t i c acid in ether. The eluate is then extracted with 0.21 sodium hydroxide and the strength of the sample is determined by the absorptivity a t 273 fun. This method is now the NF assay procedure for hydroflumethiazide tablets (39).
HYDROFLUMETHIAZIDE
313
Other i n v e s t i g a t o r s (4, 5, 6, 7) have described the use of u l t r a v i o l e t absorption measurements t o determine hydro-
flume t h i a z ide. 6.8 Polarography
Polarographic s tudies on the reduction of hydroflumet h i a z i d e have been reported (50, 51, 52, 53). These are p r i marily s t u d i e s of the reduction of the trifluoromethyl group or sulfonamide group, although Lund (51) r e p o r t s the a s s a y of hydroflume t h i a z i d e i n urine by polarography. 6.9 Thin Layer Chroma tographr
Table I1 lists t h i n layer chromatography systems for hydroflumethiazide. Hydroflu& thiazide can be iocated on the p l a t e by examination under an u l t r a v i o l e t lamp, or by any of the s p r a y reagents described (42, 43, 44, 45, 54). Garceau e t a1 (55) reported a q u a n t i t a t i v e t h i n l a y e r chromatography procedure with fluorometric d e t e c t i o n , for the determination of hydroflumethiazide i n urine and plasma. The l i m i t of d e t e c t i o n was reported t o be 10 ng of drug per m l of plasma. ACKNOWLEDGMENTS The writers wish t o thank Dr. B. T. Kho f o r h i s review of the manuscript, Dr. G. S c h i l l i n g of Ayerst Research Laboratories f o r h i s mass s p e c t r a l data and i n t e r p r e t a t i o n , the l i b r a r y s t a f f f o r t h e i r literature search, and Mrs. B. Juneau f o r typing the p r o f i l e .
314
CHESTER E. ORZECH et al.
TABLE XI THIN LAYER CHROMATOGRAPHY SYSTEMS FOR HYDROFLUMETHIAZIDE
Absorbent
Solvent Sys tern
Alumina G S i l i c a Gel G
Ethyl Acetate E t h y l Ace ta teBenzene (80: 20) Ethyl Acetate Ethyl Acetate-ater (98: 2 E t h y l Ace tate-Wa ter (197: 3) Toluene-Xylene-l,4Dioxane-2-Propanol25% Amnonia (1:1:3:
S i l i c a Gel G Alumina GF254 S i l i c a Gel DSFS S i l i c a Gel GF
S i l i c a Gel G S i l i c a Gel G S i l i c a Gel G S f l i c a Gel F254 S i l i c a C e l F254 Silica G e l
G25-22
*Not Recorded
3:2)
Benzene-B thyl Acetate (2:8) Ethyl AcetateMe thanol- Anmonia (85:10:5) Ethyl AcetateBenzene (8:2) Ethyl Acetate Ethyl Acetate E t h y l AcetateMethanol-0. !l Annnonia (96:2:2)
u
Reference
0.36
40
0.41 0.46
40 40
0.52
41
Oa
53
41
0.43
42
NR*
43
NR*
43
0.78 0.45 0.45
54 44 45
0.52
55
315
HYDROFLUMETHIAZIDE
REFERENCES 1.
2. 3. 4. 5. 6.
7. 8. 9. 10.
11.
12. 13. 14. 15. 16. 17. 18. 19. 20.
21. 22.
0. R. Samnul, W. L. Brannon, and A. L. Hayden, J. A s s . Offic. A n a l . Chem. 47, 918-991 (1964). E. G. C. Clarke ( e d i t o r ) , " 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 Press, London, 1969, page 743. L. J. Bellamy, "The Infrared Spectra of Complex Molecules", t h i r d e d i t i o n , John Wiley & Sons, New York, 1975, pp. 372 and 406-7. V. B. P i l s b u r y and J. V. Jackson, J. Pharm. Pharmacol. 18, 713-20 (1966). J. Kracmar and M. Lastovkova, Pharmazie 25, 464-70 (1970). J. Kracmar and M. Lastovkova, Cesk. Farm. 20, 287-98 (1971); C.A. 76, 1448785. D. DePaulis and C. Dipietromaria, Boll. chim. farm. 99, 15-19 (1960); C.A. 54, 9 2 0 7 ~ . F. R. Fazzari, J. Ass. Offic. Anal. Chem 53, 582-4 (1970) I. Sunshine ( e d i t o r ) , "Handbook of Analytical Toxicology", The Chemical Rubber CO., Cleveland, Ohio, 1969, page 222. " B r i t i s h Pharmacopoeia 1973", Her Majesty's S t a t i o n e r y Office, London, 1973, page 232, G. S c h i l l i n g , A y e r s t Research Laboratories personal comnunica tion. 0. I. Corrigan and R. F. Timoney, J. Pharm. Pharmacol. 26, 838-40 (1974). M. Kuhnert-Brandstgtter, A. Kofler, R. Hoffman, and H.-C Rhi, Sci. Pharm, 33, 205-30 (1965); C.A. 64, 7966g. H. Groenewegen, Pharm.Teekblad 95, 345-50 (1960); C.A. 55, 87670. W. Kobinger and F. J. Lund, Acta Pharmacol. Toxicol. l5, 265-74 (1959); C.A. 53, 14332e. S. Ueda, Japan. Patent No. 25,692 (1963); C.A. 60, 9107f. R. B. Smith, R. V. Smith, and C. J. Yakatan, J. Pharm. Sci. 65, 1208-11 (1976). CIBA Ltd., B r i t . Patent No. 861,367 (1961); C.A. 55, 199691. C, T. Holdrege, R. B. Babel, and L. C. Cheney, J. Am. Chem. SOC. 81, 4807-10 (1959). F. Lund and W. 0. Godtfredsen, Brit. Patent No. 863,474 (1961); C.A. 55, 19971b. R, S e l l e r i a n 7 0 . Caldini, Ann. Chim. (Rome) 5 0 , 170-6 (1960); C.A. 54, 22676~. H. L. Yale, K. Losee, and J. Bernstein, J. Am. Chem. Soc. 82, 2042-6 (1960).
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-
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,
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24. 25. 26. 27. 28
c. Novello, S. C. b e l l , E. L. A. Abrams, C. Ziegler, and J. M. Sprague, J. Org. Chem. 25, 970-81 (1960). L. H. Werner, A. Halamandaris, S. Ricca, Jr., L. Dorfman, and G. DeStevens, J. Am. Chem. SOC. 82, 1161-6 (1960), W. J. Close, L. R. Swett, L. E. Brady, J. H. Short, and M. Vernsten, J. Am. Chern. SOC. 82, 1132-5 (1960). G. DeStevens and L. H. Werner, Swiss Patent No. 385,869 (1965); C.A. 63, 8383g. H. Hoehn, Ger. Patent No. 1,118,788 (1961); C.A. 57, 2236a. G. DeStevens and L. H. Werner, Ger. Patent No. 1,147,233 (1963); C.A. 60, 1778b. A. Kongsted, Dan. Patent No. 92,966 (1962); C.A. 58, 9108a. Z. Foldi, D. Heidt, and R. Koenig, Hung. Patent No. 143,398 (1962); C.A. 58, 68470. R. B. Margerison, A. C. Shabica, and J. B. Z i r g l e r , Ger. Patent No. 1,093,370 (1962); C.A. 56, 5987a. H. L. Yale and J. Bernstein, Fr. Patent No. 1,330,713 (1963); C.A. 60, 1777g. L. C. Cheney and C. T. Holdredge, Fr. Patent No. 1,368,708 (1964); C.A. 62, 9157c. J. Klosa and H. Voigt, J. Prakt. Chem. l6, 264-76 (1962); C.A. 2,5689c. D. A. Johnson, U.S. Patent No. 3,007,948 (1962); C.A. 56, 5889e. CIBA Ltd., Brit. Patent No. 880,652 (1962); C.A. 56, 7340a. F. C. N w e l l o , Fr. Patent No. 1,393,953 (1965); C.A. 63, 8383c. Merck and Co., Inc., B r i t . Patent No. 948,069 (1964); C.A. 60, 15895d. "The National Formulary", 14th ed., Mack Publishing Co., Easton, Pa., 1975, page 329ff. R. Adam and C. L. Lapiere, J. Pharm. Belg. l9, 79-89 8134f. (1964); C.A. M. Duchene and C. L. Lapiere, J. Pharm. Belg. 20, 275-84 (1965); C.A. 64, 79701. R. Neidlein, H. K r u l l , and M. Meyl, Deut. Apoth.-Ztg. 105, 481-2 (1965); C.A. 66, 4075511. 0. Soh, J. Simoa, M. A. Hanna, G. G h a l i , and R. Tolba, J. Chromatogr. 87, 570-5 (1973). M. I. Walash and S. P. Agarwal, J. Drug Res. 2, 217-25 (1973); C.A. 8 l , 68646t. S. P. Agarwal, M. I. Blake, Indian J. Pharm. 35, 181-3 (1973); C.A. 80, 63904~. J. Bermjo, Galenica Acta (Madrid) l 4 ., 255-64 (1961); C.A. 56, 10286e. H. C. Chiang, J. Pharm. Sci. 5 0 , 885-6 (1961).
F.
23
.
29. 30. 31, 32. 33 34 35
. . .-
36 37. 38
.
39. 40 41. 42. 43
.
44. 45. 46. 47.
fi,
317
HYDROFLUMETHIAZIDE
48
.
49.
51. 52 53 54 55
.
. ..
F. R. Fazzari, J. Ass. Offic. Anal. Chem. 55, 161-2 (1972). G. J. Krol and R. E. Pickering, Ayerst Laboratories Inc., personal comunication. H. Lund, Acta Chem. Scand. l3, 192-4 (1959); C.A. 54, 22676a. H. Lund, Abhandl. Deut. Akad. Wiss. Berlin, K1. Chem., Ceol., Biol. 1964 ( l ) , 434-42; C.A. 62, 8674d. A. I. Cohen, B. T. Keeler, N. H. Coy, and H. L. Yale, Anal. Chem. 34, 216-19 (1962). 0. Menousek, 0. Exner, and P. Zurnan, Collect. Czech. Chem. Coamun. 33, 4000-7 (1968); C.A. 70, 33765b. B. G. Osborne, J. Crhometogr. 70, 190-3 (1972). Y. Garceau, I. Davis, and J. Hasegawa, J. Pharm. Sci. 63, 1793-5 (1974).
-
-
L i t e r a t u r e surveyed through March, 1976.
Analytical Profiles of Drug Substances, 7
HYDROXYZINE DIHYDROCHLORIDE Josef Tsau and Nicholas DeAngelis
319
Copyright @ 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN n-12-260807-o
320
JOSEFTSAU AND NICHOLAS DEANGELIS Contents
Introduction Description 2.1 Name, Formula, Molecular Weight 2.2 Appearance, Color, Odor 3. Physical Properties 3.1 Infrared Spectrum 3.2 Nuclear Magnetic Resonance Spectrum 3.3 Ultraviolet Spectra 3.4 Acidity Constant 3.5 Melting Range 3.6 Thermal Analyses 3 . 7 Crystal Properties and X-Ray Powder Diffraction 3.8 Solubility 3.9 Mass Spectrum 4. Synthesis 5 . Stability 6. Distribution, Excretion, and Metabolism 7. Identification and Elemental Analysis 8. Methods of Analysis 8.1 Non-aqueous Titration 8.2 Formation of Color Derivatives 8.3 Conductometric Titration 8.4 Chromatographic Methods 8.4 a. Gas Liquid Chromatography (GLC) 8.4 b. High Performance Liquid Chromatography (HPLC) 8.4 c. Paper Chromatography 8.4 d. Thin Layer Chromatography (TLC)
1. 2.
9. References
HYDROXYZINE DIHYDROCHLORIDE 1.
321
Introduction
Hydroxyzine is classified as an antihistaminic drug which has several pharmacological effects:sedation, relaxation of muscles, and hypnotic properties. It is comparatively non-toxic and chemically unrelated to phenothiazine, reserpine, and meprobamate drugs.192 Hydroxyzine is mainly used to reduce anxiety and tension, for skeletal muscular relaxation or as an antiemetic drug. Its antispasmodic effect is probably due to its interference with the mechanism that responds to spasmogenic agents such as serotonin, acetylcholine, and histamines.3 2.
Description 2.1
Name, Formula, Molecular Weight Hydroxyzine dihydrochloride is designated as ethanol, 2-D-[4-[(4-chlorophenyl) phenylmethyl3-1-piperazinyll ethoxyl-, dihydrochloride in Chemical Abstracts and NF XIV4. The commonly used trade names are Atarax, Ataraxoid, Cartrax, Enarax, Marax, and Vistaril.
OH.2HCl
C21H27ClN202 2.2
2HC1
Mol. Wt. = 447.83
Appearance, Color, Odor Hydroxyzine dihydrochloride is a white, odorless crystalline powder.
3.
Physical Properties 3.1
Infrared Spectrum The infrared (I.R.) spectrum of hydroxyzine dihydrochloride is given in Figure 1, and is comparable to that published by Hayden et al.5 The spectrum was
.
0
z
..
OF4
5
d
ri rn
HYDROXYZINE DIHYDROCHLORIDE
323
recorded with a Perkin-Elmer 467 Grating I . R . Spectrophotometer and was obtained in a 1% KBr pellet. Structural assignments of some of the significant bands are given in Table I. Table I Infrared Assignments for Hydroxyzine Dihydrochloride Frequency range (cm-l)
Assignment
3100-3700
0-H stretching
2800-3100
C-H stretching
2410
tertiary amine salt6
1596, 1495, 1455
aromatic C=C in plane skeletal vibrations
1090
C-0-C stretching
700, 760, 805
aromatic CH out-of-plane bending
3.2
Nuclear Magnetic Resonance Spectrum The nuclear magnetic resonance (NMR) spectrum of hydroxyzine dihydrochloride in deuterated dimethylsulfoxide (d6) containing tetramethylsilane as the internal standard is shown in Figure 2. This spectrum was obtained on a Varian A60 NMR spectrometer. The spectral peak assigments are given be low: Chemical Shift (ppm) 3-0-4-4
6.0-6.4 7.2-8.3
protons all methylene protons (16) methine proton (1) all aromatic protons (9)
The NMR spectrum after D,O exchange indicates that there are 3 exchangeable protons (1 from OH and 2 from HC1 groups) which are likely broad and weak peaks hidden under aromatic proton peaks and methylene proton peaks,
Y
. . . I . . U
..
I d . . . , . .I .. .. . . ,
. I . . . 2 . . I . . . . % . . . 1 . . . . 1 . . . . 1 . . . . 1 . . . . 1 . . . . 1 . . l . . . . I . . . . l . .. . 1 . . . . 1 . . . .
?a
u
u
11181
u
U
I
.
I
1
.
I
1
1
1
u
0
a
Figure 2-NMR Spectrum of Hydroxyzine Dihydrochloride, N.F. Reference Standard LOT NO. 6818. Solvent: Deuterated Dimethylsulfoxide, Internal Standard: tetramethylsilane. Instrument: Varian A 60.
6
HYDROXYZINE DIHYDROCHLORIDE
3.3
325
U l t r a v i o l e t Spectra The u l t r a v i o l e t ( W )absorption s p e c t r a of hydroxyzine dihydrochloride i n acetate buffer a t pH 4.5, i n d i l u t e NaOH s o l u t i o n a t pH 10.3 and pH 1.8 i n d i l u t e HC1 s o l u t i o n are given i n Figure 3. The s p e c t r a were recorded on a Cary 14 Spectrophotometer. The UV s p e c t r a a l l have a max a t 230 run with andamof 33.0 a t pH 4.50, 33.5 a t pH These values agree 10.3 and 35.2 a t pH 1.83. reasonably well with t h a t previously reported7 b"=35.0). The o v e r a l l W spectrum of hydroxyzine dihydrochloride i s c h a r a c t e r i s t i c of disubs t i t u t e d benzenes and some biphenylmethyl derivatives.8 We found t h a t t h a t p a r t of t h e spectrum due t o the benzenoid groups is pH dependent, Figure 4. The UV spectrum of hydroxyzine dihydroc h l o r i d e i n ethanol i s similar t o those shown i n Figure 35.
3.4
I o n i z a t i o n Constant The f i r s t stage i o n i z a t i o n constant of hydroxyzine dihydrochlor i d e w a s determined potentiometr i c a l l y g t o be pKa = 1.834
+
[1.018 n / ( 1
+
0.517
fi)]+
0.160 I
where I i s t h e i o n i c s t r e n g t h adjusted with NaC1. The authors a l s o determined t h e PKa spectrophotom e t r i c a l l y t o be 2.0. 3.5
Melting Range Hydroxyz ine dihydrochlor i d e m e 1t s a t about 200 C with d e c o m p o ~ i t i o n . ~I t m 1 ing range i s reported El elsewhere t o be 192-3OC.
3,18,
3.6
Thermal Analyses The DSC thermogram of hydroxyzine dihydrochloride obtained a t a scan r a t e of 10°C/min. exhibited a wide range endotherm from 190°C t o 225"C, where melting w a s accompanied by decomposition. The TGA thennogram obtained a t a scan r a t e of 10"C/min. showed no weight l o s s from room temperature t o l50"q about 10% l o s s from 150°C t o 200°C and over 80% l o s s above 25OOC.
326
JOSEF TSAU AND NICHOLAS DEANGELIS
Figure 3-UV Spectra of Hydroxyzine Dihydrochloride, Instrument: Cary 14
HYDROXYZINE DIHYDROCHLORIDE
F'i
I:e
4-UV Spectra of Hydroxyzine DihydI-ochlor i.de Instrument: Cary 14
327
JOSEFTSAU AND NICHOLAS DEANGELIS
328
3.7
Crystal Properties and X-Ray Powder Diffraction The crystal habit of hydroxyzine obtained from both sublimation and recrystallization from 95% alcohol is needle-like crystals.12 The X-ray powder diffraction pattern of hydroxyzine dihydrochloride obtained with a Norelco Phillips diffractometer using CuQ radiation is shown in Figure 5. The calculated d-spacings are given in Table 11, and agree well with those previously pub1ished 38
.
Table I1 X-Ray Powder Diffraction Pattern For Hydroxyzine Dihydrochloride
2 0 (degree)
d
I/I0 -
6.2 8.2 11.8 12.4 13.4 14.5 15.3 16.8 17.2 19.5 20.2 21.4 22.3 23.5 24.7 26.9 27.6 29.1 30.0
14.28 10.79 7.49 7.13 6.59 6.09 5.78 5.26 5.13 4.53 4.37 4.13 3.96 3.76 3.58 3.28 3.20 3.04 2.94
19 14 6
d (interplanar distance)
=
1/10 = relative intensity
3.8
6
45 43
46
50 100 18 56 30
46
39 64 89
54
31 26
L sin
Solubility The authors determined the solubility of hydroxyzine dihydrochloride to be 7700 mg/ml in water, 60 mg/ml in chloroform, 2 mg/ml in acetone andL0.1 mg/ml in ether.
Figure 5-X-Ray Diffraction Pattern of Hydroxyzine Dihydrochloride, NF Reference Standard Lot No. 6818. Radiation-CuKg Instrument: Norelco Philips Diffractometer.
,
JOSEF TSAU AND NICHOLAS DEANGELIS
330
3.9
Mass Spectrum The electronic impact (E.I.) mass spectrum of hydroxyzine dihydrochloride shown in Figure 6 (spectrum is of the hydroxyzine base) was obtained with a MS-902 double focusing, high resolution mass spectrometer. The probe temperature was 200°C and the ionization electron beam energy was 70 eV. Structural assignments of the major peaks are given in Table I11 for E.1, and chemical ionization (C.1.). Table I11 High Resolution Mass Spectrum of Hydroxyzine Assignment
% Relative Intensity
375 (c.1.)
MH+
90
374.1732 (E.I.) (Calc. 374.17611
M+
6
299
M+- CH, OCH, CH, OH 18
201
Cl-(4-c-g+
100
173
d-LCH,+
10
4.
B
u
Synthesis
ud:w
The synthesis of piperazine ethers including h zine dihydrochloride were first reported by Morren. There are five major synthetlc methods depending on the availability of starting material and intermediates.Il A typical reaction scheme is given in Figure 7.13 5.
Stability Hydroxyzine dihydrochloride solutions are unstable when irradiated with intense UV light. 14915 The authors obtained the results given below on hydroxyzine dihydrochloride solutions (10 mg/ml) stored in sealed ampules under N2 atmosphere for 15.5 months,
ii
a
Lo
W
5
k CI
a
U QI
Lo
Figure 7
A Synthetic Route €or Hydroxyzine Dihydrochloride
p-chlorobenzhydryl chloride
piperazine
p-chlorobenzhydryl p iperaz ine
+
" ' An
wcH-NW
C 1CH2CH, OCH, CH, OH
N-CHZ CHZ OCH2 CH, OH
CHCl, ref Zuxing acid-base extract ion
I
hydroxyz ine base HCl/THF hydroxyzine dihydrochloride
1
HYDROXYZINE DIHYDROCHLORIDE
333
pH
Percent of initial concentration
3
10 1
51
9
5
100
93
72
7
94
51
42
This study indicates that hydroxyzine solution is most stable at a pH of approximately 5.
6. Distribution, Excretion, and Metabolism In rats after intraperitoneal administration, hydroxyzine distributes rapidly to the organs with the highest concentration in lungs, followed by fat, liver, spleen and kidneys. l6 Hydroxyzine is also rapidly absorbed in the gastrointestinal tract.3 Hydroxyzine is excreted mainly within the first day, in feces via the bile. It is completely metabolized in bile and urine principally to the glucuronides of the diphenylmethane derivatives l6
.
The following metabolites were identified in rats163 17918; p-chloro-p -hydroxybenzophenone,p-chlorobenzhydrol and its glucuronide, p-chlorobenzophenone, piperazine, 2-c2- ( 1-piperaziny1) ethoxy1 ethano1, norchlorcyclizine and hydroxyzine N-oxide. 7.
Identification and Elemental Analysis Hydroxyzine dihydrochloride can be identified by means of its characteristic I.R. and U.V. spectra or by means of the many chromatographic methods given in Section 8.
The theory elemental analysis is: C=56.3%, H=6.53%,
N=6.26%,
CL23.7570, 0=7.14"h.
JOSEF TSAU AND NICHOLAS DEANGELIS
334
8. Methods of Analysis 8.1
Non-Aqueous Titration The NF X I V 4 describes non-aqueous titration methods to determine hydroxyzine dihydrochloride drug substance as well as injection, tablet, and syrup dosage forms, The procedure involves dissolving about 150 mg of hydroxyzine dihydrochloride drug substance, or for dosage forms chloroform extraction of hydroxyzine base from alkaline solution. Glacial acetic acid and mercuric acetate are added and the solution is titrated with 0.1N perchloric acid using quinaldine red as the indicator. Two procedures which are very similar to the above NF method were described by Ciaccio et a1,19 and Pasich and Stasiewska.20
In another method21 hydroxyzine was precipitated by sodium tetraphenylborate from acidic solution. This precipitate was redissolved in 50% acetone, acidified with acetic acid and potentiometrically titrated with AgNO, solution. 8.2
Formation of Color Derivatives With concentrated % SO, hydroxyzine develops a yellow color with a maximum absorbance at 460 ,.22,23 Hydroxyzine dihydrochloride when treated with cobalt thiocyanate reagent and extracted with CHC1, saturated with ammonium thiocyanate produces a color which has a maximum absorbance near 600 nm. 24
8.3
Conductometric Titration Hydroxyzine dihydrochloride drug substance and tablet dosage forms were analyzed by titrating conductometrically using picric acid as titrant.25
8.4 Chromatographic Methods 8.4a.
Gas Liquid Chromatography (GLC) The following GLC methods have been reported. Method 1- For biological fluids26
HYDROXYZINE DIHYDROCHLORIDE
335
column: 1/8" x 7 1 stainless steel packed with 3% OV17 on Gas Chrom. Q, 100-120 mesh. carrier gas: N,, 60 ml/min temperatures: column 275"C, detector 300"C, inlet block 300°C detector: FID retention time: 5 minutes sample preparation: two extractions from pH 7.2 buffer into ether. Evaporate to dryness, Reconstitute in 100 pl acetone containing internal standard griseofulvin comment: GLC-mass spectrometry recommended by the authors to increase sensitivity.
--
Method 2-Cardini et a127 showed that by using 2% GE-SE30 on Aeropak (Varian Aerograph), 80-100 mesh, hydroxyzine was well separated from other tranquilizers, such as glutethimide, methaqualone and carLsoprod01. Method 3-Method 1 was adapted to determine hydroxyzine in an injection formulation, and was proven to be stability indicating.28 column: 2 mm i.d. x 70 cm glass column packed with 3% OV-17 on Gas Chrom Q, 100-120 mesh. carrier gas: N, temperatures: column 275"C, detector, 300"C, injection port 300°C detector: FID retention time: 3 minutes sample preparation: 3X extraction of the equivalent of about 100 mg of hydroxyzine from NaCl saturated alkaline solution into chloroform and then add internal standard (n-dotriacontane). 8.4b,
High Performance Liquid Chromatography (HPLC) The authors have developed a HPLC method to determine hydroxyzine dihydrochloride in injection formulations. This method is stability indicating. The chromatographic conditions are given below. column: Bondapak phenyl/corasil, 35-50 Fun, 4 mm i.d. x 50 cm (Waters Assoc.) acetonitrile mobile phase: 0.25% (NH, ),C03 in water
(5:3) flow rate:
1.5 ml/min
JOSEFTSAU AND NICHOLAS DEANGELIS
336
d e t e c t o r wavelength: 230 nm sample and standard s o l u t i o n s : prepare and properly d i l u t e with d i s t i l l e d water t o about 0.1 mglml.
8.4~.
Paper Chromatography Sybirska and G a j k ~ i n s k areported ~~ a c a t i o n exchange paper chromatographic method t o quantify hydroxyzine. Several o t h e r paper chromatographic methods have been developed t o systematically i d e n t i f y t r a n q u i l i z i n g drugs including hydroxyzine. 30, 31
8.4d.
Thin Layer Chromatography (TLC) TLC was used t o study hydroxyzine dihydrochloride and i t s metabolites i n b i o l o g i c a l f l u i d s and The chromatographic behavior of hydroxyzine dihydrochloride i n s e v e r a l TLC systems i s given i n Table I V and various d e t e c t i o n methods are given i n Table V.
Table IV TLC Methods for Hydroxyzine Dihydrochloride
Eluent methanol:12N NH,, OH(100:1.5) benzene:ethano1 :12N NH,, OH (95: 15:s)
Ad sorbent
Impregnated with
Silica Gel G II
cyc1ohexane:diethylamine: benzene ( 75 :20:15)
0.1N NaOH
acetone ch1oroform:methanol (90:10)
I1 11
cyc1ohexane:benzene:diethyl-
m i n e (75:15:10) methano1 acetone methano1 95% ethanol chloroform:acetone:NH,, OH (80:20 :1) methano 1 :acetone( 12:88) ethano1:carbon tetrachloride (16:84) methano1:benzyl alcoho1(31.7:68.3) ethano1:toluene (68:32)
Silica Gel
0.1M KOH
11
11
II
11
II
0.1M NaHSOG II
II
Silica GelgYPsm Silica Gel G W254 Silica Gel G W254
Rf
Ref -
0.68
32
0.4
I1
0.22 0.33 0.59
0.08 0.59 0.39
II
11
0.25
0.40
I1
34 I1
II
Na,CO,
II
11
I1
0.45
II
II
0.56
0.1N Na, CO, 0.1N
It
0.48 0.53
35 33 11
I1 II
Table IV TLC Methods for Hydroxyzine Dihydrochloride Eluent methano1:cyclohexane:methyl acetate (17.8 :33.6: 48.6)
Adsorbent Silica Gel G W 2 54
acetone:methanol: ammonium hydroxide (50:50 :1) Silica Gel G cyc1ohexane:benzene: diethylamine (75: 15: 10) Silica Gel G 11 methanol:acetone(l: 1) I1 95% ethanol n-butano1:ethanol:water I1 (5:2:2) isoamyl alcohol (water saturated): acetic acid (85:15) ch1oroform:cyclohexane (1:l)
Impregnated with 0.1N
Na, CO,
9
Ref -
0.49
33
0.64
36
0.41 0.8
16 It
0.68
It
0.65
11
0.30
II
0
1
HYDROXYZINE DIHYDROCHLORIDE
339
Table V D e t e c t i o n of Hydroxyzine Dihydrochloride on Thin-Layer P l a t e s Detecting reagent
A.
B.
-
Color Room temp 100°C
A
white
bluish
B
-
ye 1low
C
-
D
Fluorescence 100°C Room temp
-
-
32
flesh
-
violet
yellow
-
-
-
37
E
brown
-
-
-
II
F
violet
-
-
G
brown
-
-
-
-
-
-
green-yellow
gold
-
II
32
F o l i n - C i o c a l t e a u r e a g e n t ( F i s c h e r S c i e n t i f i c Co., C a t . NO. SO-P-24) d i l u t e d 1:l w i t h d i s t i l l e d water p r i o r t o use. FPN r e a g e n t
5% Feel3 s o l u t i o n : 70% p e r c h l o r i c acid-water (1:5) : c o n c e n t r a t e d HN03 -water (1:l)-1:9:10 C.
Ref
Mandelin r e a g e n t
10 mg/ml of ammonium vanadate i n c o n c e n t r a t e d H, SO,, D.
%Mn04 s o l u t i o n (1%)
E.
Dragendorff r e a g e n t
F.
Iodop l a t i n a t e r e a g e n t
G.
I o d i n e chamber
JOSEF TSAU AND NICHOLAS DEANGELIS
340
References 1.
J. H. Moyer, K. Pevey, and V. Kinross-Wright, J. Am. Acad. Gen. Pract., No. 6, 97 (1957). Merck Index, 9th Ed., Merck and Co., Inc. (1976). Physician's Desk Reference, 31St Ed., 1341-2 and 1247-8 (1977). N.F. XIV, Mack Printing Co., 339 (l975). "Infrared and Ultraviolet Spectra of Some Compounds of Pharmaceutical InterestYff J.A.O.A.C., Washington, D.C. , 1972. W. E. Thompson. R. J. Warren, I.B. Eisdorfer. and J. E. Zarembo,*J. Pharm. Scii, 54, 1819 (1965). E. C. G. Clarke, IIIsolation and Identification of DrugsYtt Part 2, The Pharmaceutical Press, London, 1969. T. J. Siek, J. Forensic Sci., 19, 193 (1974). S. Lukkari, Farm. Aikak., 80, 161 (1971); C.A. -975 41145 t H. G. Morren, Synthesis-Belgium, 523, 899 (1954); 53, 18072 d. C.A. United States Patent Office, H. Morren, Pat. No. 2,899,436, Aug. 11, 1959. P. Rajeswaran and P. L. Kirk, Bull. Narcotics, XIII (3), Part 11, 21 (1961). Chem. -'Week 79 (5), 70(August 4, 1956). J. Pawlaczyk, P. T. P. Nauk and Wyd. Lek., Pr. Kom. Farm 5, 117(1966); C.A. 67, 5679 w. -0s J. Pawlaczyk, Ann. Pharm. I , 127 (1969); C.A. 72, 103676 r. S. F. Pong and C. L. Huang, J. Pharm. Sci., 63, 1527 (1974). G. Cannizaro, Boll. Chim. Farm. 104, 39 (1965); C.A. 63, 2264 g. J. A. Close, J. G. Gobert and L. A. M. Rodriguez, Proc. Eur. Soc. Study Drug Toxicity, 9, 144 (1968). L. L. Ciaccio, S. R. Missan, W. H. McMullen, and T. C. Grenfell, Anal. Chem., 9,1670 (1957). J. Pasich and K. Stasiewska, Farm Polska, 18, 261 (1962); C.A. 58, 1303 h. B. Sawicki and B. Janik, Acta Pol. Pharm., 23, 573 (1966); C.A. 66, 108296 c. J. Hoyois, J. Pharm. Belg., 2, 200 (1959). H. Auterhoff and J. Kuehn, Arch. Pharm., 306, 241 (1973); C.A. 79, 49044 n. I. M. Roushdi, S.A. Soliman, and M. H. Sadek, U.A.R.J. Pharm. Sci., 12, 353 (1971). K. Nikolic and 2. Cupic, Acta Pharm. Jugoslav., 21, 159 (1971); C.A. 76, 103816 U.
z,
2. 3.
4. 5.
6.
7.
8. 9.
.
10
11. 12. 13. 14. 15. 16. 17. 18. 19
.
20. 21.
22. 23.
24. 25.
.
-
-
-
-
--
-
-
--
--
--
-
-
-
341
HYDROXYZINE DIHYDROCHLORIDE
26. 27. 28. 29. 30. 31, 32. 33. 34. 35. 36. 37. 38.
E. Van Der Kleyn, G. C. Beelen, and M. A. F r e d e r i c k , 345 (1971). C l i n i c a Chemica A c t a . , C, C a r d i n i , V. Q u e r c i a , and A. Calo, J. Chromatog. 37, 190 (1968). L. S i v i e r i , Wyeth L a b o r a t o r i e s Inc. P e r s o n a l Communication. H S y b i r s k a and H. Gajkzinska, Bromatol Chem. Toksykol., 1, 189 (1974); C.A. 81, 164121 p. J. Vecerkova, M. Sulcova, and K. Kacl, J. Chromatog., 7 , 527 (1962). -. A. Viala and R. Monnet, Trav. SOC. Pharm. M o n t p e l l i e r L l8, 79 (1958). I. Zingales, J. Chromatog., 2, 405 (1967). E. Rb'der, E. Mutschler, and H. Rochclmeyer, J. Chromatog., 131 (1969). W. W. F i k e , Anal. Chem., 38, 1697 (1966). T. Fuwa, T. Kido, and H. Tanaka, Yakuzaigaku, 25, 138 (1965); -C.A. 64, 6405 h. J. J. Thomas and L. Dryon, J. Pharm. Belg., 481 (1964). A. N o i r f a l i s e , J. Chromatog., 20, 61 (1965). J. C. C o l l e t e r , M. G a d r e t and J. Roy, Bull. SOC. Pharm Bordeaux, 8 7 (1961).
2,
-
g,
2,
100,
Analytical Profiles of Drug Substances, 7
6-MERCAPTOPURINE
Steven A . Benezra and Penelope R . B. Foss
343
Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 012-260807-0
STEVEN A. BENEZRA AND PENELOPE R. B. FOSS
344
Table of Contents
1.
Description
1.1 Name, Formula, Molecular Weight 1 . 2 Appearance, Color, Odor 2.
Physical Properties Infrared Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Ultraviolet Spectrum 2.4 Mass Spectrum 2.5 Melting Point 2.6 Solubility 2.7 Dissociation Constant
2.1
3.
Synthesis
4.
Stability
5.
Methods of Analysis 5.1 5.2 5.3 5.4 5.5 5.6
6.
Elemental Analysis Nonaqueous Titrhetric Analysis Spectrophotometric Analysis Polarography Mass Spectrometry Chromatography 5.61 High Performance Liquid Chromatography 5.62 Column Chromatography 5.63 Gas Chromatography 5.64 Thin Layer Chromatography
Pharmacokinetics and Metabolism 6.1 Excretion Rate 6.2 Tissue Distribution 6.3 Metabolism
345
6-MERCAPTOPURINE
1. Description 1.1 Name, Formula, Molecular Weight Mercaptopurine is purine-6-thiol monohydrate
SH
170.19
C5H4N4S * H 2 0
1.2 Appearance, Color, Odor Mercaptopurine is a yellow, practically odorless, crystalline powder'. 2.
Physical Properties
2.1 Infrared Spectrum The infrared2spectrum of mercaptopurine (anhydrous) is shown in Figure 1 It was taken as a 0.2% dispersion of mercaptopurine in KBr with a Perkin Elmer model 457 infrared spectrophotometer. Table I gives the infrared gssignments consistent with the structure of mercaptopurine
.
.
Table I Infrared Spectral Assignments for Mercaptopurine Band (cm-l) 3420, 3490 3120, 3040, 2780 1200 930
Assignment Aromatic NH stretch Aromatic CH stretch C=S stretch CH bend
2.2 Nuclear Magnetic Resonance (NMR) Spectrum The NMR spectrum of mercaptopurine is shown in Figure 24 It was obtained with a Varian CFT-20 80 MHz NMR spectrometer. Deuterated DMSO was used as the solvent with tetramethylsilane as an internal standard. Based on the NMR spectrum, the following proton assignments for mercaptopurine can be made.
.
Figure 1-
Infrared Spectrum of 6-Mercaptopurine
2
k 7
*rl
a
0
a
U 0
cd
k a,
7
\D
0
w
5
k
al
u
U
a b)
rn
STEVEN A. BENEZRA AND PENELOPE R. B. FOSS
348
Chemical Shift (ppm) 2.75-3.75 broad singlet 8.15 singlet 8.35 singlet 11.0-12.0 broad singlet
Proton S-H C-H (aromatic) C-H (aromatic) N-H
2.3 Ultraviolet (W)Spectrum The W spectra of mercaptopurine in 0.1N NaOH, 0.1N HC1, and methanol were taken with a B e c p n ACTA CIII W spectrophotometer and are shown in Figure 3 Table I1 summarizes the W data.
.
Table I1 UV Spectral Data €or Mercaptopurine Solvent 0.1N NaOH
)i
0.1N HC1 Methanol
max (nm) 230 312 222 327 216 329
(3) 14000(5) 19600 924012; 21300(3) 8940(3) 19300
2.4 Mass Spectrum The low r solution mass spectrum of mercaptopurine is shown in Figure 4% It was obtained with a Varian-MAT model 731 mass spectrometer. Direct probe at 145OC into the electron impact source was used. The electron energy was 70eV. The assignment of fragment ions is given below.
.
m /e I25 ( 18%)
H
H2NXLJ7+
y>> ;
H2N
N
m/e 119 ( 16%)
m /e 98 ( 10%)
CzNSH'
(CN?
+
m/e71 ( 1 1 % )
m/e 26 (20%)
349
6-MERCAPTOPURINE
I .4 I .2
I .o 0.8 0.6 0.4
0.2
0 I.4
W 0
7
I .2 I .o
a m
0.8
0
0.6
m
0.4
02
rn
a
0.2 0 I .2 r
I .o
0.8 0.6 0.4
0.2 0
-
-
:P i-1
1
I
1
1
1
1
1
1
1
1
1
nm Figure 3- Ultraviolet Spectra of 6-Mercaptopurine Top-O.1N HC1, Middle-O.1N NaOH, Bottom-Methanol
1
100
> Icn I-
80
60
z -
w
> -I- 40 a
A
w
20
0 SO
I00 m /e
Figure 4-
Mass Spectrum of 6-Mercaptopurine
351
6-MERCAPTOPURINE
2.5 Melting Point Mercaptopurine decomposes above 308°C1
.
2.6 Solubility Mercaptopurine is insoluble in water, acetone, and ether. It is soluble in hot ethanol and dilute alkalilsolutions. It is slightly soluble in dilute sulfuric acid
.
2.7 Dissociation Constant The pK and pKa of mercaptopurine qetermined poten1 7.77 ani 11.17 respectively tiometrically fs
.
Synthesis Mercap@yyrine has been prepared by a variety of synthetic procedures These procedures are outlined in Figure 5. Process 1 involves treatment of 4-amino-5-formamido-6-hydroxypyrimidine with phosphorous pentasulfide, decomposition of excess phosphorous pentasulfide with base and acidification to pH 4-5. Mercaptopurine precipitates from the acidic solution. Process 2 treats hypoxanthine with phosphorous pentasulfide. The solid formed is treated with NH40H to pH 5. Mercaptopurine precipitates from the solution. Process 3 utilizes 4amino-6-chloro-5-nitropyrimidine as the starting material. Treatment of this compound with potassium hydrosulfide gives 4,5-diamino-6-mercaptopyrimidine. The 4,5-diamino-6-mercaptopyrimidine is heated in concentrated formic acid to yield 7amino-thiazolo(5,4-d)pyrFmidine. Mercaptopurine is precipitated after treatment of 7-amino-thiazolo(5,4-d)pyrimidine with NaOH and adjusted to pH 5 with acetic acid. Process 4 is a varient of process 3. The 4,5-diamino-6-mercaptopyrimidine is treated with 50% formic acid to give 4-amino-5-formamido-6-mercaptopyrFmidine, which in turn can be treated as the 7-amino-thiazolo(5,4-d)pyrimidine in process 3 to give mercaptopurine. Process 5 treats 4-aminoimidazole-5-carboxamide with phosphorous pentasulfide. The product, 4-aminoimidazole-5-thiocarboxamide, is heated with formamide to form the six-membered ring. Removal of the formamide and recrystallization of the residue gives mercaptopurine. 3.
.
Stability The decomposition of mercaptopurine in 0.1N NaOH, 0.1y2 HC1, distilled water, and photolytically has been studied When mercaptopurine is refluxed for 6 days in 0.1N NaOH, 4aminoimidazole-5-thiocarboxamide and 4-amino-5-cyanoimidazole are the major products formed. When refluxed for 4 days in 0.1N HC1 and 7 days as an aqueous suspension in distilled water, mercaptopurine decomposes primarily to 4-aminoimidazole5-thiocarboxamide. Mercaptopurine in 0.1N NaOH and as an 4.
.
c
I z
?=J
I*&
0
N
d-
353
6-MERCAPTOPURINE
aqueous suspension in distilled water forms hypoxanthine when irradiated for 72 hours with a medium pressure mercury lamp. 5. Methods of Analysis 5.1 Elemental Analysis (As the Hydrate)
C -
Element X calculated
H -
35.28
3.55
N -
-S
32.92
18.83
5.2 Nonaqueous Titrimetric Analysis Nonaqueous titration is t e official method of analysis in the USP for mercaptopurine . An accurately weighed sample of mercaptopurine is dissolved in dimethylformamide. The solution is titrated with standardized 0.1N sodium methcrxide using thymol blue as an indicator. Precautions must be taken against absorption of atmospheric carbon dioxide.
P
5.3 Spectrophotometric Analysis in tabThe official USP analysis of mercaptopurine . lets is a spectrophotometric analysis. A portion of powdered tablets is weighed. Twenty ml distilled water, and 1.5 m l NaOH TS is added to the powder in a 100 m l volumetric flask. The flask is brought to volume with distilled water, filtered, and a portion of the filtrate diluted with dilute hydrochloric acid. The solution is compared against a Reference Standard prepared in a similar manner at 325 nm in 1 cm cells. 5.4 Polarography Alternating current polarography has beep3used to determine decomposition kinetics of mercaptopurine A catalytic wave with Q=0.45 was observed for mercaptopurine in 1N H2S04.
.
5.5 Mass Spectrometry Quantitative analysif40f mercaptopurine in plasma has been accomplished with GCfMS Mercaptopurine was extracted from plasma, derivatized with methyl iodide, and separated by gas chromatography using a 1.83 M, 3% OV-225 column at 2OO0C, and detected with a mass spectrometer equipped with a peak monitor. Limit of detection was 20 ng/ml of mercaptopurine in plasma.
.
5.6 Chromatography 5.61 High Performance Liquid Chromatography High performance liquid chromatography was used to determine mercapSopurine and metabolites in cultured cells A 0.18 x 100 cm colunm packed with and animal tissues Beckman M71 strong cation exchange resin was eluted with 0.4M
.
STEVEN A. BENEZRA AND PENELOPE R. B. FOSS
354
ammonium formate (pH 4.6) at 8 ml/hr. The column was kept at 50°C. The retention time of mercaptopurine under these conditions was 39 minutes. Detection was accomplished with a UV detector at 322 nm. 5.62 Column Chromatography Column chromatography has been yEed to separate mercaptopurine from its metabolites in urine , A cation exchange resin, Zeo-carb 225, eluted with 20% (v/v) ammonium hydroxide separated mercaptopurine and other 6-thiopurines from a concentrated urine sample. 5.63 Gas Chromatography A gas chrornatog7aphic analysis of mercaptopurine A 1.5M x 6.3 mm 0.d. column in serum has been described packed with 10% w/w SE-30 maintained at 135OC was used. Mercaptopurine derivatized with trimethylanilinium hydroxide had a retention time of 22 minutes. 5.64 Thin Layer Chromatography The separation of mercavtovurine from mixtures of . purines and pyrimidines has been accomplished by thin layer chroma graphy using anion exchange ECTEOLA cellulose plates The plates were developed in acetone:O.lM H SO : ethyl acetate (45:10:45). A second development was doie In deionized water:acetone (8:2). The R value of mercaptopurine was 0.36. Cellulose plates developedf in 0.1N HC1, H20, and isopropano1:methanol:H 0:NH OH (60:22:20:1) gave R valyes for mercaptopurine of 8 . 4 3 , 40.26, and 0.55 respectfvely
.
ie .
.
6. Pharmacokinetics and Metabolism 6.1 Excretion Rate In the rat, by the end of 48 hours after injection, 55% of mercaptopurine was excreted in the uri28. The largest proportion was excreted in the first 24 hours 3Jor an intraperitoneal injection in the mouse of 1 mg of S -6-mercaptopurine, 43.5% of the radioactive material was excreted in the urin in the first four hours. At the end of 2 days over 55 was excreted. Approximately the same urinary ex60% of S cretion rate was found with oral dofps, but some radioactive C02 was detected in the expired air
.
.
6.2 Tissue Distribution In the mouse the concentration of radioactive mercaptopurine was highest in the gut, almost twice as high as in the blood, and lowest in the brain. Mercaptopurine has some difficulty passing the blood-brain barrier. The brain2foncentration is one-tenth the concentration in the blood The presence of a tumor in different sites in rats, as well as in mice, causes lower blood levels of mercaptopurine when compared to non-tumor bearing animals. The apparent volume of
.
355
6-MERCAPTOPURINE
distr tumor
$9ution .
is markedly increased in the presence of a
6.3 Metabolism The pathway of metabolism of droxylation v i a the enzymes xanthine dase. Mercaptopurine is transformed side, 6-tltf~ypf2~acid,sulfates, and the liver
mercaptopurine is by hyoxidase and aldehyde oxiinto mercaptopurine ribonucleotide metabolites in
356
STEVEN A. BENEZRA AND PENELOPE R.B. FOSS References
1. USP XIX, p.307 (1975) 2.
J. Lagasca, Burroughs Wellcome, personal communication
3. P.R.B. Foss, Burroughs Wellcome, unpublished data
4. R. Crouch, Burroughs Wellcome, personal communication 5. The Merck Index, 8th Ed. p.658 (1968)
6. D. Brent, Burroughs Wellcome, personal communication 7. J.J. Fox, I. Wempen, A. Hampton, J. her. Chem. SOC., 1669 (1958)
So,
8. U.S. Patent 2,697,709
9. U.S. Patent 2,721,866 10. U.S. Patent 2,724,711 11. U.S. Patent 2,756,828
12. B.J. Carthy, C.B. Lines, Burroughs Wellcome, personal communication 13. V. Parrak, M.M. Tuckerman, J. Pharm. Sci.,
63,
622 (1974)
14. J.M. Rosenfeld, V.Y. Taguchi, B.L. Hellcoat, M. Kowai, Anal. Chem., 49, 725 (1977) 15. H.-J. Breter, Anal. Biochem.,
80, 9
16. A.H. Chalmers, Biochem. Med.,
12, 234
(1977) (1975)
17. D.G. Bailey, T.W. Wilson, G.E. Johnson, J. Chrom., 305 (1975) 18. M.J. Harber, J . L . Maddocks, J. Chrom.,
101,231
111,
(1974)
19. J.L . Maddocks, G.S. Davidson, Brit. J. Clin. Pharm., 359 (1975) 20. E.J. Sarcione, L. Stulzman, Can. Res.,
2,
20, 387 (1960)
6-MERCAPTOPURINE
357
21.
G.B. Elion, S. Bieber, G.H. Hitchings, Ann. N . Y . Acad. Sci., 60, 297 (1954)
22.
M.G. Donelli, T. Columbo, A. Porgione, S. Garattini, Pharmacol., 2, 311 (1972)
23.
R.H. Adamson, Ann. N.Y. Acad. Sci.,
179, 432
(1971)
Analytical Profiles of Drug Substances, 7
PHENOBARBITAL
Marcus K . C . Chao. Kenneth 5’. Albert, und Salvatore A . Fusari
3 59
Copyright @ 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-260807-0
MARCUS K. C. CHAO et al.
360
Contents 1. General Information
1.1 Nomenclature 1.11 Chemical Names 1.12 Generic Names 1.13 Trade Names 1.2 Formulas and Molecular Weight 1.3 Description 1.4 Forms in Official Compendia 2. Physical Properties 2.1
Spectra 2.11 Infrared 2.12 Raman 2.13 Nuclear Magnetic Resonance 2.14 Mass 2.15 Ultraviolet
2.2
Crystal Properties 2.21 2.22 2.23
Crystal Morphology X-Ray Diffraction Melting Point
2.3 Solubility 2.4 Dissociation Constants 2.5 Distribution Coefficients 3.
Synthesis
4. Stability and Degradation 5. Metabolism and Pharmacokinetics 6.
Identification
- Microchemical Tests
7. Methods of Analysis 7.1 7.2 7.3 7.4
Differential Thermal Elemental Colorimetric Spectrophotometric
361
PHENOBARBITAL
7.41 7.42 7.43 7.5 7.6 7.7 7.8
Ultraviolet Phosphorimetric Fluorometric
Polarographic Titrimetric Gravimetric Chromatographic 7.81 7.82 7.83 7.84 7.85
Paper Thin-Layer Open-Column Gas Modern Liquid
7 . 9 Ion Selective Electrode 7 . 1 0 Immunoassay 8.
Automated Analytical System
9.
References
362
MARCUS K. C. CHAO et al.
1. General Information 1.1 Nomenclature
1.11 Chemical Names1 5-ethyl-5-phenyl-2,4,6(lH,3H,5H)pyrimidinetrione; 5-ethyl-5-phenylbarbituricacid. The compound is listed in Chemical Abstracts under the heading pyrimidinetrione. CAS registry number2 f o r this compound is 50-06-6 and for the sodium salt is 57-30-7. 1.12 Generic Names1
Phenobarbital, phenobarbitone, phenylethylmalonylurea. 1.13 Trade Names1s3 Luminal, Gardenal, Barbenyl, Barbiphenyl, Dormiral, Euneryl, Neurobarb, Barbipil, Lubrokal, Lubergal, Phenyral, Cratecil, Nunol, Phenonyl, Noptil, Phenobal, Agrypnal, Eskabarb, Etilfen, Gardepanyl, Somonal. 1.2 Formulas and Molecular Weight' 1' 2H12N203 0
Molecular Weight 232.24
1.3 Description White, odorless, glistening, small crystals or white crystalline powder which exhibits polymorphism2 .
PHENOBARBITAL
363
1.4 Forms in Official Compendia The USP2 contains Phenobarbital as the free acid, the sodium salt and the following formulations: Phenobarbital Elixir Phenobarbital Tablets Phenobarbital Sodium Injection Sterile Phenobarbital Sodium Phenobarbital Sodium Tablets and the USP reference standard for phenobarbital is available. Physical Properties
2.
2.1
Spectra 2.11
Infrared
The infrared spectrum4 of phenobarbital is presented in Figure l. The band assignments are swarized in Table 1. Table 1 Infrared Band Assignments for Phenobarbital4 Wave
NO.
(cm-1)
3080-3220 1710, 1770 1500, 1590
1250-1450
830 720, 770
Assignment Inter bonded N-H stretching. C=O stretching. Aromatic ring skeletal vibration, h i d e 111 Bond (mainly C-N stretching). N-H Wagging. C-H out of plane deformation of mono-substituted phenyl.
Two other spectra of phenobarbital were obtained from the same lot of sample but using different preparation techniques. Figure 2 represents the spectrum of which the sample is dissolved in a small amount of chloroform and mixed with KBr, after it is vacuut dried the resulting powder is pressed to a disc . Figure 3 is the spectrum obtained from the similar procedure except using methanol as the solvent. The literature5p6 presents other spectra obtained from different disc
Figuro 1. Infrarod Spoctrum of Phonobarbitol (Lot 597821) in Kbr. Instruront: Digilab FtS14
Figuro 2. Infrarod Spectrum of Phonobarbital (Lot 597821) from chloroform i n K l r . Inrtrumont: D i g i l a b FtSlS
Figuro 3.
Infrarod Spoctrum of Phonoborbital (Lot597821) from mothanol i n Kbr. Inrtrumont: Porkin-Elmor 6 2 1
PHENOBARBITAL
367
preparation methods such as ( C ~ H G - C ~ H ~ ~ and KB~) (C6H6-CH30H-KBr). The obvious different appearance of these spectra confirms that phenobarbital forms different crystals depending on the solvent used. Mesley and Clements7 evaluated the infrared identification of phenobarbital with particular reference to the occurrence of polymorphism. 2.12 Raman The Raman spectrum8 is shown in Figure 4. The band assignments are listed in Table 2. Table 2 Raman Band Assignments for Phenobarbital8 Wave
NO.
(cm-1)
1690, 1740 630 620, 1000, 1040, 1590
Assignment C=O stretching. Ring "breathing" vibration. Mono-substituted phenyl group.
2.13 Nuclear Magnetic Resonance The NMR spectrum9 of phenobarbital in DMSO at 90 Meg. Hz. is shown in Figure 5 and the spectral assignments are in Table 3. Table 3 Nuclear Magnetic Resonance Spectral Assignments for Phenobarbital Chem ica1 Shift
m 0.8
9.2
2.2 7.8 7.3 2.7 11.6 -1.6
Relative Intensity 3 2 5
2
Mu1tip1icity triplet quartet singlet singlet
Assignment
- CH3 - CH2 -
2.14 Mass The mass spectrum6 of phenobarbital is shown in Figure 6 . The parent peak is at m/e 232 and the base peak at m/e 204 where the pattern is
Figuro 4.
Raman Spoctrua of P h o n o b a r b i t a l . Courtory of A n a l y t i c a l C h o m i r t r y 44, 1229, 1972.
Figure 5.
Nuclear M a g n o t i c Resonance Spoctrum of Phenobarbital ( l o t 597821) in DMSO.
.,k
Instrument: Bruker WH-90
i t
i
Figure 6.
Moss Spoctrun of Phenobarbital (Lot 597821) Instrument: Finigan 1015
PHENOBARBITAL
371
consistent with a progressive l o s s of CO. The structural assignments are listed in Table 4 . Table 4 Mass Spectrum Assignments for Phenobarbital Nominal Mass
Re1ative Intensity
232
16.2 4.0 100.0
217
204 189 174
161 146 117 77
4.0
6.9 13.5 10.8
23.2 8.4
Structure Assignment M+
M-(CH ) M- (COY M- (HNCO) M- (HNCONH) M- (CONHCO) M- (CONHCONH) M- (C H5) - (CONHCONH) C6H5
3
2.15 Ultraviolet Spectrum The UV spectra of phenobarbital in 0.01 N HC1 (unionized form), pH 10 buffer (singly ionized form) and 0.1 N NaOH (doubly ionized form) are presented in Figure 7 . The spectra are in agreement with those reported in the literatureB o d The absorption maximum of pH 10 and pH 13 solutions are at 240 nm and 256 nm, a(l%, 1 cm) values are 431 and 314 respectively. 2.2 Crystal Properties 2.21 Crystal Morphology Phenobarbital exhibits polymorphismll~l2. The stable form13 exists as rhomboids with angles of 61° and a leveling plane at 94'. A metastable phase forms long acicular crystals which are monoclinic needles, Another metastable phase is monoclinic hemimorphic. The refrafgive indexes of phenobarbital have been reported a=1.447, B=1.620 and y=l. 667.
MARCUS K. C. CHAO et a].
372
Figuro 7. Ultraviolot Abaorbonco Spoctrum of Phonobarbital (Lot 597821) in aolutiona with difforont pH. Inatrumont: Porkin- Elmor 124
0.6
0.5
y1
U
0.4
z
a
a
2a
0.3
0.2
0.1
220
240
260
280
Wavolongth (nm)
300
320
340
373
PHENOBARBITAL
2.22 X-Ray Diffraction Williams15 presented the x-ray diffraction patterns of phenobarbital wtich is crystallized from diethyl ether. Huang reported five polymorphic forms of phenobarbital with characteristic x-ray diffraction patterns. More recently, Mesley and co-workers12 have isolated thirteen crystal forms and documented the diffraction data in the literature. 2.23 Melting Range Phenobarbital1 reportedly melts at 174-178OC. Williams15 determined the melting points of three crystal phases of phenobarbital; the stable phase at 174OC; one metastable phase at 156157OC; and another metastable phase at 166-167OC. Mesleyl2 summarized the melting points for eleven possible crystal forms of phenobarbital. 2.3 Solubility One gram of phenobarbital dissolves in about one liter of water, 8 ml of alcohol, 40 ml of chloroform, 13 ml of ether and about 700 ml benzene. The compound is soluble in alkaline hydroxides and carbonates. A satu ated aqueous solution is acid to litmus (about pH 5) .
5
The solubility of phenobarbital in water at various te eratures have been determined: /l) . (0.97 g / l ) p ; 250C141.2 g/1)18; 37OC (1.42 g200E1 Aldrich and Watson studied the solubility of phenobarbital at different pH values in aqueous solution: pH 5.2 (1.1 g/l); pH 7.1 (1.75 g/l); pH 8.1 (9.57 g/l); pH 8.5 (16.4 g/l); pH 9.1 (55.3 g / l ) . Also, the effect of pH upon the solubility of phenobarbital in alcohol-a eous solution has been investigated by LJrdangy8. A study of the solubility of phenobarbital in alcoholglycerin-water ystems at various composition has been presented18 . 2.4
Dissociation Constants
The first and second ionization constants, pK1 and pK2, of phenobarbital have been determined
MARCUS K. C. CHAO et 01.
3 74
to be 7.3 and hi.8 by Krahl2I and Butlerz2 respec-
tively. Biggs confirmed the pK1 value by ultraviolet spectrophotometry and it is in good agreement with that reported by Krahl. 2.5 Distribution Coefficients Bush24 determined a number of distribution coefficients (K) for phenobarbital between a few selected organic solvents and aqueous solution. The results are tabulated as follows:
Where
Organic Solvents
K -
1-Chlorobutane Benzene Dichloromethane Diethyl Ether
0.40 1.0 3.0 50
=
Concentration in organic phase Concentration in aqueous phase
More information of the distribution coefficients (K) of phenobarbital between a few organic solvents and aqueous solutions in different pH values ranged from to 12 have been reported. Riedel and co-workers23 measured the K values for three organic solvents and the results are presented below: K Values in Various Solvents at Different pH
PH
CH2C12
4
19.0 11.5 4.9 3.0 1.10
5
6 7 8 9 10
0.35
0.01 DCE
=
CHC13
DCE -
3.6
10.1 8.1
4.0
3.6 2.7 0.54 0.14 0.08
5.7
4.0 1.13 0.18 0.04
1,2-dichloroethane
Meijer26 obtained additional data on K values of phenobarbital as listed below:
PHENOBARBITAL
375
K Values in Various Solvents at Different pH Solvent
pH 3.4
Hexane 0.006 Toluene 0.538 Chloroform 4.16 1,2-Dichloroethane 5.26 Ether 16.4 Ethyl Acetate 57.3 Acetone
-
3.
pH 7 . 4 0.005 0.552 2.35 2.85
17.4 52.0
762
pH 12.4 0.0004 0.0006 0.004
0.0014 0.003
7
Synthesis
Two synthetic routes of general applicabilit have been used for phenobarbital. One method27 ~ 5 8 is based on the condensation of a-ethylbenzenepropanedioic ester (I) with urea (11) in the presence of sodium ethoxide. The synthetic route is shown in Figure 8, scheme 1.
The other synthetic method29 includes the following steps: (1) condensing benzeneacetonitrile (111) with diethyl carbonate (IV) in ether solution to form a-cyanobenzeneacetic ester (V); (2) ethylating this ester to a-cyano-a-ethylbenzeneacetid ester (VI) which is further condensed with urea yielding the iminobarbituric acid (VII) ; ( 3 ) hydrolyzing compound (VII) to phenobarbital. The complete reaction is presented in Figure 8, scheme 2. 4.
Stability and Degradation
Phenobarbital is quite stable in aqueous solutions of low pH, but is readily hydrolyzed at high pH. Figure 9 shows graphically the essential features of the hydrolytic decomposition of phenobarbital in alkaline solutions30,31. The barbituric acid ring is ruptured between the 1-6 (or 3 - 4 ) bond yielding the intermediate compound which loses carbon dioxide to form N-(aminocarbony1)-a-ethylbenzeneacetamide. Under more severe conditions, especially on heating, the second peptide bond is broken with the formation of the a-ethylbenzeneacetic acid and urea as major products. Kapadia and ~o-workers3~ separated and identified these decomposed products by paper chromatography. The kinetics and mechanism of phenobarbital degradation
MARCUS K. C. CHAO et 01.
376
Scheme I
HI 0
Figure 8. Synthetic Procedure for phenobarbital
377
PHENOBARBITAL
H
cbnl
\? cans / N.(aninocorbonyl)
-
yo
‘$-yf O H a-ethylbenrenracetride
(phenylethylacetyl urea)
a -ethylbenzeneacetic ocid (a-phrnyl-n-butyric acid)
(urea)
Figure 9. Hydrolytic decomposition of phenobarbital in alkaline solution
MARCUS K. C. CHAO et 01.
378
have been studied by Goyan33 and Tishler34. Bush24 measured the half-life of phen barbital in 0.1 N KOH at 25OC as 46 hours. Mauldingg5 obtained the apparent first order rate constants for decomposition of phenobarbital at 8OoC in 0.1 N NaOH (pH 11.5) as 3 x 10-4 s c-l and in 0.033 N NaOH (pH 10.99) as 1.5 x 10-z sec-1. Schmitz and Hi1136 reported the stability of phenobarbital in hydroalcoholic and glycero-hydro-alcoholic solutions. Phenobarbital is stable to strong oxidizing reagents such as permanganate and dichromate24. This provides a simple "clean-up" procedure to separate phenobarbital from other, easily oxidized, compounds. 5. Metabolism and Pharmacokinetics Phenobarbital is partially metabolized and partially excreted unchange in the urine. Raun-Jansen and co-workers39 reported that about 11 to 25% of the dose of phenobarbital was found in the patient's urine in the form of unchanged drug. Butler38 found that the p -hydroxyphenobarbital
(I) is the major metabolite of phenobarbital. On
this evidence, Maynert39 indicated that the primary pathway for the metabolism of phenobarbital is hydroxylation of the phenyl ring. The possible metabolic scheme is shown in Figure 10, scheme 1. It is reported that some other possible metabolites such as ortho- or meta-hydroxyphenobarbital and dihydrodiol derivatives have not been found in the biotransformation product6 of phenobarbital, However, Harvey, et. al. isolated and identified 5-(3,4-dihydroxy-1,5-cyclohexadin-l-yl)-5-ethylbarbituric acid (11), 5-(l-hydroxyethyl)-5-phenylbarbituric acid (111) and 5-(3,4-dihydroxyphenyl)5-ethylbarbituric acid (IV) as the metabolites in the urine of humans using gas chromatography-mass spectrometry. Figure 10, scheme 2 suggests the other possible metabolic pathway with the formation of metabolites (11), (111) and (IV) from phenobarbital41. Maynert39 studied the absorption, distribution and excretion of phenobarbital in man and determined
PHENOBARBITAL
379
Scheme
I
Scheme
2
O Y
'\
GH.5
J
C
$-1/ O H
-0 =O
Figure 10. Metabolic Pathways
380
MARCUS
K. C. CHAO et al.
the half-life for the disappearance of the drug from the plasma ranges from 53 to 140 hours. Studies on the bi ogical half-life of phenobarbital in human babiesg3 and children43 have been reported. The value of blood and plasma level assays for determining the dosage of phen i 1 has been the subject of recent reviews €8 . Vajda and co-workers47 measured the phenobarbital concentrations in hum n brain, cerebrospinal fluid and plasma. Lint8 studied the pharmacokinetic aspects of elimination from plasma and distribution to brain and liver of phenobarbital in rats.
zB:r!,
McArthurLC9and co-workers studied the binding of phenobarbital to human whole blood in vitro. A study of the application of two dimensional immunoelectrophoresis to investigate the binding of phenobarbital to human serum proteins has been reported50. 6. Identification
-
Microchemical Tests
Phenobarbital reacts with iodide-potassium iodide reagent solution to form characteristic crystals in little dark grains with a few red . Also blades and dark splinter-rods in clusters5iarge mentioned is a method using ammoniacal nickel acetate as the reagent with which phenobarbi forms in single rectangular crystals. Davis developed a procedure for the identification and differentiation of twenty barbiturates using a silver reagent which is composed of 10% aqueous silver nitrate and ethylenediamine (85:15, v/v).
fP
7. Methods of Analysis 7.1 Differential Thermal As phenobarbital exhibits polymorphism, a recrystallization step is recommended prior to a differential thermal analysis in order to ensure the presence of a single crystalline phase. Figure 11 shows a thermogram of a recrystallized sample which contains 0.17% impurity. 7.2 Elemental The data9 as shown in the following table
PHENOBARBITAL
381
Figuro 11. Thornogron: Difforontial Thormal Analysis of Phonobarbital ( l o t 597821) Instrumont: Mottlor TA 2000
Tomp or at uro (OC)
173
174
175
176
177
170
I
MARCUS K. C. CHAO et 01.
382
is obtained for Phenobarbital Lot 597821.
7.3
Element
% Found
7, Theory
C H N
62.54 5.30 11.97
62.06
5.21
12.06
Colorimetric
Colorimetric methods based on the formation of a complex of phenobarbital with cobalt or copper salts i the presence of a base h e been well s tudiedfl ,53. Pfiel and Goldbach!): determined phenobarbital by precipitating it with mercury and y complex spectrophotometrically measuring th modified with dithizone. A few laboratories55?fg:y5 the reaction of dithizone with Hg (11)-phenobarbital complex and applied the method to determine phenobarbital in biological fluids. 7.4
Spectrophotometric 7.41 Ultraviolet
Curry58 reviewed the use of ultraviolet spectrophotometric methods for assaying phenobarbital either by directly measuring the absorbance at 250-255 nm in an alkaline solution at pH 13 or by determining the difference in absorption at 240 m in solutions of pH 10 and pH 2. Jain and Cravey58 also indicated that the concentration of phenobarbital is proportional to the difference in absorbance readings between an alkaline solution in pH 13 at 240 nm and a buffer solution in pH 10 at 260 nm. The potential interference with the differential ultraviolet spectrophotometric6tnalysis of phenobarbital has been studied by Jatlow . Phenobarbital in elixir and tablet products is assayed by extraction and subsequent measurement of ultrav olet absorbance at 240 nm in pH 9.6 borate buffer .
3
7.42 Phosphorimetric Weinfordner and TinG1 developed a procedure for the phosphorimetric measurement of phenobarbital. The excitation and emission maximum
PHENOBARBITAL
383
wavelengths are at 240 and 380 nm; the detection limit is 0.1 vg/ml. 7.43
Fluorometric
A fluorometric method62 is also available for the determination of phenobarbital. The excitation and emission maximum wavelengths are at 265 nm and 400 nm in a buffer at pH 13. The sensitivity is reported to be 0.5 parts per million in the final solution.
7.5 Polarographic A differential pulse polarographic procedure63 was developed for the determination of phenobarbital. The sample is nitrated with potassium nitrate in sulfuric acid and the nitroderivative is analyzed by differential pulse polarography *
7.6 Titrimetric Phenobarbital can be determined by potentiometric titration2 with 0.1 N NaOH. Cheronis and Ma64 listed titrimetric methods for phenobarbital determination including an acidimetric titration in non-aqueous media. argentiometry, use of precipitation agents. iodimetry and the Kjeldahl method. A review of non-aqueous titrimetric ethods for phenobarbital as presented by Kucharsky!6! , In addition, Agarwal6t described a spectrophotometric titration of phenobarbital in acetonitrile using triethyl-n-butylammoniumhydroxide as a titrant. 7.7 Gravimetric Gravimetric determination was one of the main methods used in earlier days for assaying phenobarbital in different dosage forms67 and tissues68. The procedure involves extraction with chloroform followed by evaporating the solvent and dissolving the residue in ethyl ether. The ether solution is again evaporated to dryness and the amount of phenobarbital is measured gravimetrically.
MARCUS K. C.CHAO et al.
384
7.8 Chromatographic 7.81 Paper Paper chromatography was one of the early methods use for the identification of phenobarbital. Curry5 t documented different detection methods for henobarbital on the developed chromatogram. Clarktg summarized a number of chromatographic systems and Rf values for phenobarbital. The identification of barbiturates from biological specimens by paper hromatograph have been reviewed by Jain and Cravey76 and deZeeuwTl. 7.82 Thin-Layer Thin-layer chromatography has been widely used as a rapit simple screening method for phenoqybital. Clark 9 deZeeuw71 Kirchner72 and Stahl reported many systems for phenobarbital and its corres nding Rf values. Gardner-Thorpe and co-workersTz evaluated a number of procedures and found that a system using chloroform-acetone (9O:lO) as the mobile phase and ultraviolet absorption at 254 nm as the detection method would be suitable for routine clinical work, R value for phenobarbital is 0 . 5 . Itiaba, et. al.75 used a fiberglass sheet impregnated with silica gel as the stationary phase, Rhodamine B dye as the spray reagent, and detected phenobarbital under ultraviolet light. By this method Rf values determined for three different mobile phases are: petroleum ether-ethyl acetate (100:14),Rf = 0.41; benzene-acetone (100:2), Rf = 0.28; cazbon tetrachloride-acetone (100:5), Rf = 0.28. Dunges and Peter76 described a new procedure in which a derivative formed from the reaction of phenobarbital with a dimethylaminonaphthalenesulphonic acid is applied directly to thin-layer plates and the amount of phenobarbi a1 is determined fluorometrically. Jain and Cravey76 presented a review article including more than ten recently developed TLC systems for the determination of phenobarbital. Atwe1177 reported a list of Rf values for phenobarbital on silicic acid gel thin layer plate using 17 various mobile phases. A quantitative thin-layer chromatography using scanning of remission technique was applied to phenobarbital determination in capsules78.
PHENOBARBITAL
385
7.83 Liquid Phenobarbital is separated from phenytoin on a reversed phase (silanized celite) column using 25% amyl alcohol in chloroform as the stationary phase and 0.05 M borate buffer pH 9.5 as the mobile phase. After the separation, phenobarbital is determined from its ultraviolet absorption in 0.1 N NaOH at 253 nm79. Another system14 employing a column packed with a mixture of acid washed celite 535 (2 g ) and 12% BaC12 solution (1 ml) and using a water-saturated solution of 15% arnyl alcohol in chloroform as the mobile phase was also presented for the separation of phenobarbital from phenytoin. Kullberg and co-workers80 described a procedure using Amberlite XAD-2 as the adsorbent resin to separate phenobarbital from urine samples, using a mixture of ethyl acetate1,2-dichloroethane (3:2) as the mobile phase. A similar rocedure was presented by Hetland, et. al.8y where a mixture of ethyl acetate-methanolammonium hydroxide (170:20:10) was used as the mobile phase. 7.84 Gas Brochrnann-Hanss en8 reviewed some early studies on gas chromatographic s aration and identification of barbiturates. Clarkg8 and Gudzinowicz83 summarized a number of systems for phenobarbital and its corresponding retention time, A number of gas chromatographic studies of phenobarbital determination were collected in the b ok edited by Meijer, et. al.84. Jain and Cravey78 thoroughly reviewed the development and application of gas chromatography to determine phenobarbital in biological samples. The article includes detailed information about derivatization, column packing materifls, stationary phases and internal standards. Berry critically examined twelve gas chromatographic systems for phenobarbital measurement in plasma and urine samples. Recently, Ehrsson86 suggested an extract5ve methylation procedure in carbon disulfide for barbiturate determination and compared this procedure with other methylation methods. Various detectors have been used for the determination of
MARCUS K. C.CHAO et
386
01.
phenobarbital. The application of a nitrogenspecific detector has been studied87$88. Gyllenhaal89 and Wallego have developed similar procedures converting phenobarbital to its bispentafluorobenzyl derivative and measuring it with an electron-capture detector. Phenobarbital in the range of nanograms to picograms from a sample size of 0.1-1ml of plasma or 1-5 ml of urine can be detected by a mass spectroscopy-computer systemg1 where an internal standard labeled with stable isotopes is used. The more recent gas chromatographic work on the determination of phenobarbital is summarized in Table 5 . 7.85 Modern Liquid Modern liquid chromatography provides a new technique for the separation, identification and quantitative determination of phenobarbital in pharmaceutical dosages or in biological samples. The method is accurate, simple, rapid and sensitive. Results and working conditions reported from different laboratories are sumarized in Table 6 . A high-speed molecular-size exclusion techniquelll has been applied to analyze phenobarbital in blood plasma without any clean-up preparation. The column used is a 2.6 mm, i.d. x 1 ml stainless steel tube packed with Vit-X-328 (PerkinElmer); the mobile phase is double distilled water; flow rate at 0.5 ml/min. and UV detection is used,
7.9 Ion Selective Electrode Carmark and Freiser112 developed a coatedwire selective electrode for the assay of phenobarbital based on the ion pair complex between phenobarbital anion and the quaternary armnonium cation, tricaprylylmethylammoniun ion. The electrode is sensitive for the concentration range from 0.1 M to 10-5 M of phenobarbital at pH 9 . 6 , and the results obtained are in agreement with the standard USP method. The assay using the ion selective electrode can be accomplished within twenty minutes where the USP method needs four hours. The useful lifetime of the electrode is about three months.
TABLE 5 DETERMINATION OF PHENOBARBITAL BY GAS CHROMATOGRAPHY Derivatization
Column Length Base
Stationary Phase
N.M.
2 m
3.175 mm
Chromosorb W 4% SE-30 8 0 / 100 6% QF-1 on acid washed DMCS
TMAH
200 cm
3mm
2% QF-1
TAAH (Flash Heater)
6 ft,
0.25 in.
TMAH
5 ft.
0.25 in.
(Flash Heater)
(On Column)
Support
Detector
Ref.
F.I.
92
Chromosorb W
F.I. E.C.D.
93
3% QV-17
Gas Chrom Q 100/120
F,I.
94
5% OV-17
Chromosorb W
F.I.
95
60/80
60/80
TMAH
N.M.
N.M.
3% XE-60
Gas Chrom Q l o o / 120
N.M.
96
TMPAH
200 cm
2 m
3% SP2250
Supelcoport methyl-phenyl (50 : 50) 100/120
F.I.
97
(Pre Column)
TABLE 5 (Continued) Derivatization
Column Length Base
Stationary Phase
N.M.
6 ft.
3% OV-17 or SP2250
2mm
Supp ort
Detector
Deactivated E.C.D. Chromosorb 750
Ref. 98
100/120
TBAH (on Column)
1.8 m
2mm
3% ov-101
Chrom G HP 100/120
F.I.M.
TMAH
6 ft.
1/8 in.
10% UV-W98
Chromosorb W HP/80/100
-
100
N.M.
4 ft.
4mm
1%OV-17
High Performance Chromosorb G
F.I.
101
N.M.
213 cm
4 mm
3% OV-17
Celite 545
F.I.
102
100/120
TAAH = Tetraalkylammonium Hydroxide TMAH = Trimethylanilinium Hydroxide F.I. = Flame Ionization Detector E.C.D. = Electrolytic Conductivity Detector N.M. = None Mentioned TBAH = Tetrabutylammonium Hydroxide TMPAH = Trimethylphenylammonium Hydroxide
99
TABLE 6 DETERMINATION OF PHENOBARBITAL BY MODERN LIQUID CHROMATOGRAPHY*
Column
Mobile Phase
Flow Rate (ml/hr)
Retention Volume (m1) 11
Remark
Anion Exchange, Pellicular Type LSF (0.04 in. i.d. x 120 in. 1)
NaCl
26
SAX (Du Pont) ( 2 . 1 mm i.d. x
0.01 M Na3B03 0.01 M NaN03
28.8
9.36(c)
(b)
0.01 M Na3B03 0 . 0 5 M NaN03
28.8
2.25(')
(b)
0.01 M Citric Acid
21.6
4.59(c)
(b)
d.d. Water
24
1.17(C)
(b)
1 m 1)
ETH Permaphase (Du Pont) ( 2 . 1 mm i.d. x 1 m 1)
8OoC
(a)
*UV detector ( 2 5 4 nm) is used for the measurement unless otherwise mentioned in the remark.
Ref. 103
104
104
TABLE 6 (Continued) Flow Rate (ml/hr)
Retention Volume (ml)
Remark
Ref.
Column
Mobile Phase
Micro Pak 10 p (Varian) (2 mm i.d. x 50 cm 1)
CH2C12-CH30HNHbOH ( 9 2 : 7 : 1
45
3
VBondapak c18 (Waters) (4 mm i.d. x 30 cm 1)
CHgOH-Buffer pH 3 (60:40)
120
4.4
Silica Gel (h)
d.d. Water
50
3
(f)
107
Bondapak C18 on Corasil I1 (1/8 in. i.d. x 2 ft. 1)
CH OH-HzOHOic (25:75: 0.1)
120
4.4
3OoC
108
Corasil c18 (Waters) (h)
Absolute Methanol 1% (NH4)H2PO4 (40: 60)
84
2.15
(el
109
Corasil Phenyl (Waters) (h)
Absolute Methanol1% (NH4)H2P04 (40:60)
84
1.6
(e)
109
-
(d)
105
(e)
106
W detection
at 230 nm
(g)
TABLE 6 (Continued)
Column
Mobile Phase
Flow Rate (ml/hr 1
Retention Volume (ml)
1~.
Porasil (Waters) ( 4 mm i.d. x 30 cm 1)
Chloroformdioxane 2-propanolacetic acid (310:9.7 :l.0: 0.1)
-
90
4.5
SAX (Permaphase AAX) (2.1 m i.d. x 50 cm 1)
4 mM (NH412HP04 pH 6.2
24
1.7
Remark (i)
Ref.
77
110
(a) Separation of four barbiturates and their metabolites. (b) Separation of phenobarbital from fifteen other barbiturates. (c)The retention volume has been corrected for the column dead volume. (dl Simultaneous measurement of phenobarbital and phenytoin in serum. (e)Data obtained from mobile phases with different mixture composition have also been reported. (f)The system has been applied to the separation of phenobarbital from a mixture of six antiepileptic drugs and to the determination of phenobarbital in serum extract as well. (dSimultaneous measurement of phenobarbital and phenytoin in dosage form. (h)Column size is not mentioned. Simultaneous determination of phenobarbital and phenytoin in plasma.
MARCUS K. C. CHAO et al.
392
7.10 Immunoassay Recently, a homogeneous immunoassay methodll3 (Enzyme Multiplied Inmunoassay Technique, EMIT) has been applied to the quantitative determination of phenobarbital in biological samples, The method employs three reagents antibody, enzyme and enzyme substrate. The drug to be analyzed combines with the enzyme to form a drug-enzyme conjugate. When the antibody complexes with the conjugate, the active site of the enzyme is blocked and the enzyme is inactive. However, in the presence of the free drug, the drug conjugate competes with the free drug for the antibody binding site. If the free drug is bound to the antibody, the conjugate would be uncomplexed and the enzyme active site is available for the enzymatic reaction with the enzyme substrate. The level of enzyme activity is directly proportional to the concentration o r e drug present in the sample. Pippenger€18 f15 and Schneiderll6 evaluated the method for the quantitation of phenobarbital in serum and plasma. 9
Another immunoassay technique, radioimmunoassay, was also performed on the m surement of phenobarbital in saliva and plasmaff7. The principle of the technique is that the drug present in the sample equilibrates and displaces a proportional quantity of radioactive-labeled drug which is bound to the specific antibody, the uncomplexed radioactive-labeled drug is then estimated in a liquid scintillation spectrophotometer or a gamma counter. The method is reported to be sensitive to saliva and plasma levels of phenobarbital and it on requires 10 u l sample for the measurement. ChungiY8 and Satoh119 described a highly specific procedure using phenobarbital at picomole levels.
8. Automated Analytical System Jain and Cravey70 reviewed the automated analysis o arbiturates. Blackmore and co-workersf2k described an automated method for screening large numbers of urine samples for drugs using the Technicon Auto Analyzer. The method utilizes differential ultraviolet spectrophotom for the determination of phenobarbital. Adamsl W Y
PHENOBARBITAL
3 93
reported an automated gas chromatographic technique for assaying phenobarbital in biological samples. The method employs an automated capsule system of injection, the Perkin-Elmer MS-41 and an automatic data processor, Perkin-Elmer PEP-1. An auto analyzer method has been used to determine ph nobarbital in compressed tablets and elixir152. In this method, the sample solution is dialyzed into a recipient stream of pH 9 . 5 borate buffer followed by ultraviolet spectrophotometric measurement at 240 nm. Literature surveyed through September, 1977. Acknowledgments: The authors are indebted to Dr. Ira J. Holcomb for his invaluable suggestions and comments. The expert secretarial assistance of Ms. Kay McLeod is gratefully acknowledged.
MARCUS K. C. CHAO et a].
394
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PHENOBARBITAL
3 99
106. Twitchett, P.J. and Moffat, A.C., J. Chromatogr. 11, 149 (1975). 107. Eedel, E.; Klocke and Bayer, H., "Methods of Analysis of Anti-Epileptic Drugs", Meijer, J.W.A.;Meinardi, H.; Gardner-Thorpe, C. and van der Kleijn, E. (Ed.), American Elsevier Co., Inc., New York, 1972, p. 194. 108. Holcomb, I.J., Parke, Davis & Co., Personal Communication, 1976. 109. Honigberg, I.L.;Stewart, J.T.;Smith, A.P.; Plunkett, R.D. and Justice, E.L., J. Pharm. Sci. 64, 1389 (1975). 110. Etazawa, S . and Komuro, T., Clin. Chim. Acta. 73, 31 (1976). 111. Telepchak, M.J., J. Chromatogr. 83, 125 (1973). 112. Carmark, G.D. and Freiser, H., A E l . Chem, 49, 1577 (1977). 113. Rubenstein, K.E.; Schneider, R.S. and Ullman, E.F., Biochemical and Biophysical Research Communications 47, 846 (1972). 114. Pippenger. C.E.; Bastiani, R.J. and Schneider, R.S., "Clinical Pharmacology of Anti-Epileptic Drugs", Schneider, H.; Janz, D.; Gardner-Thorpe, C.; Meinardi, H. and Sherwin, A.L. (Ed.), Springer-Verlag, New York, 1975, p. 3 3 1 . 115, Pippenger, C.E., "Quantitative Analytic Studies in Epilepsy", Kellaway, P. and Petersen, I. (Ed.), Raven Press, New York, 1976, p. 59. 116. Schneider, R.S.; Bastiani, R.J.; Rubenstein, K.E.; Ullman, E.F. and James, K.R., Clin. Chem. 20, 869 (1974). 117. Cook, C.E.; Amerson, E . ; Poole, W.K.; Lesser, P. and O'Tuama, L., Clin. Pharmacol. Ther. 18, 742 (1975). 118. Chung, A.; Kim, S.Y.; Cheng, L.T. and Castro, A., Experientia 3, 820 (1973). 119. Satoh, H.; Karoiwa, Y.; Hamada, A. and Uematsu, T., J. Biochem. 75, 1301 (1974). 120. Blackmore, D.J.; Curry, A.S.; Hayes, T.S. and Rutter, E.R., Clin. Chem. 17,896 (1971). 121 * Adams, R.F., Clin. Chem. Newsletter 4 , 15 (1972); 4, 22 (1972). 122. Perrizo, C.H., Parke, Davis & C o . , Personal Communication. Literature surveyed through September, 1977.
Analytical Profiles of Drug Substances, 7
SULFAMETHAZINE
Constantin Papastephanou and May Frantz
401
Copyright @ I978 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISBN 0-12-260807-0
402
CONSTANTIN PAPASTEPHANOU AND MAY FRANTZ
SULFAMETHAZINE Contents 1.
2.
3. 4,
5. 6.
7,
Description 1.1 Name, synonyms, formula, molecular weight 1.2 Appearance, color, odor Physical Properties 2.1 Infrared spectrum 2.2 Nuclear magnetic resonance Spectrum 2.3 Mass spectrum 2.4 Ultraviolet spectrum 2.5 Optical Rotation 2.6 Luminescence Properties 2 . 7 Solubility 2.8 Melting Range 2.9 Differential Thermal Analysis 2.10 Powder X-ray Diffraction 2.11 Dissociation Constants Synthesis Stability-Degradation Drug metabolism Methods of analysis 6 . 1 Elemental analysis 6.2 Volumetric methods 6.3 Colorimetric analysis 6.4 Column chromatography 6.5 High pressure liquid chromatography 6.6 Gas chromatography 6.7 Paper chromatography 6.8 Thin-layer chromatography 6.9 Microbiological assay 6.10 Assay in body fluids and tissues References
403
SULFAMETHAZINE
SULFAMETHAZINE 1. Description 1.1 Name, synonyms, formula, molecular weiqht: 4-Amino-N-(4,6-dimethyl-2-pyrimidinyl) benzenesulfonamide: N4-(4-6-dimethyl-2-pyrimidyl)sulfanilamide: 4,6-Dimethyl-2-sulfanilamidopyrimidine; Sulfamezathine, sulfadimerazine, sulfadimidine, sulfamidine, sulfadimethylpyrimidine.
H
2
N
O SO2NH
fy3 N-
CH3 Molecular weight: 278.33 1.2 Appearance, color, odor: White to yellowish white powder, which may darken on exposure to light. Has a slightly bitter taste and is odorless. 2. Physical Properties 2.1 Infrared spectrum: The infrared spectrum of a USP reference standard sulfamethazine is shown in Figure 1. The spectrum, obtained on a Perkin Elmer Model 621 spectrometer as a KBr pellet, indicates the presence of the absorptions shown in Table I (1). Table I Infrared Assiqnments for Sulfamethazine Frequency (cm-1) Characteristic of 3420 3320 N-H stretch (NH2 + NH) 3220 1635 Aromatic C = C and 1590 C = N stretch 1505 1500 1475 1300 1140 s02 860 p-substituted phenyl ring
a,
c,
h
a
a,
rl rl
a,
C
m
5
w
U
0 E 7 k c, a,
a k
aa, m k
H
c
+I
a,
rl
k
7
tP F
-I4
SULFAMETHAZINE
405
2.2
Nuclear Maqnetic Resonance Spectrum: The 60 MHz NMR spectrum ( F i g u r e 2 ) o b t a i n e d i n dimethyl s u l f o x i d e - d g c o n t a i n i n g TMS a s i n t e r n a l r e f e r e n c e , u t i l i z i n g t h e Perkin-Elmer R12B spectrometer, i n d i c a t e s t h e presence o f resonances a t s6.57 (d, J = 9 . 0 Hz, 2H o r t h o t o NH2) and 7.67 (a, J = 9.0 Hz, 2H o r t h o t o S 0 2 ) a s s i g n e d t o p - s u b s t i t u t e d benzene r i n g p r o t o n s , 6.70 s ( 1 H ) a s s i g n e d t o t h e h e t e r o c y c l i c p r o t o n , t h e amine 5.90, t h e S02NH p r o t o n p r o t o n s resonance a t resonance a t $11.05 and t h e methyl p r o t o n s resonance a s a s i n g l e t a t s 2 . 1 5 . The data a r e c o n s i s t e n t w i t h t h e a s s i g n e d s t r u c t u r e 1.
s
s
The comparison of NMR d a t a o f s u l f a methazine, L w i t h t h a t o f s u l f a m e r a z i n e , 2 i n d i c a t e s t h e absence o f two v i c i n a l l y coupled h e t e r o c y c l i c p r o t o n s resonance a s t h e r e s u l t o f methyl s u b s t i t u t i o n i n t h e former compound. The p r e s e n c e of methyl resonance a t t 2 . 1 5 (6H) i s i n d i c a t i v e o f a symmetrical s u b s t i t u t i o n p a t t e r n c o n s i s t e n t w i t h s t r u c t u r e 1. The chemical s h i f t o f t h e remaining proton o f t h e h e t e r o c y c l i c r i n g i s o n l y mildly a f f e c t e d by t h e p r e s e n c e o f t h e a d d i t i o n a l methyl group (66.70 (L) YS 6 6 . 8 6 ( 2 J ) , ( 2 ) .
1-
Sulfamethazine:
R = CH3
2-
Sulfamerazine:
R = H
1-n G d
Figure 2.
I
I
a
7
4
NMR spectrum of sulf amethaz i n e .
5
6
I
I,
P
iom
407
SULF AMETHAZINE
2.3
Mass S p e c t r u m : The mass s p e c t r u m ( F i g u r e 3 ) w a s o b t a i n e d on a n AEI-MS902 mass s p e c t r o m e t e r b y d i r e c t sample i n t r o d u c t i o n i n t o t h e s o u r c e (165OC). The s p e c t r a w e r e r e c o r d e d o n m a g n e t i c a n a l o g tape a n d p r o c e s s e d b y i n - h o u s e programs u s i n g a PDP-11 computer. The m a s s spectrum shows no m o l e c u l a r i o n
b u t a n i n t e r e s t i n g rearrangement i o n a t m / e 214 a r i s i n g from t h e l o s s of SO2 from t h e m o l e c u l a r ion. O t h e r fragment i o n s consistent w i t h the s t r u c t u r e a r e d e p i c t e d below, ( 4 ) .
as02/mf1
m/e
H2N
92
(333
m/e
m/e 1 2 3
156
+
H ?
2.4
U l t r a v i o l e t Spectrum F i g u r e 4 shows t h e UV s p e c t r u m o f a q u e o u s s o l u t i o n s o f s u l f a m e t h a z i n e i n w a t e r (pH 6 . 6 ) , 0.01N- H C 1 a n d 0.01g NaOH. A t pH 6.6,
(El'
1c m
o n e peak a t 2 4 1 nm i s o b s e r v e d a t 255 nm.
= 670) w i t h a s h o u l d e r
S u l f a m e t h a z i n e i n 0.01N NaOH shows 2 p e a k s a t 243 a n d 257 nm w i t h El%- o f 765 a n d 776 1c m respectively. When t h e d r u g i s d i s s o l v e d i n 0.01g HC1, t w o p e a k s a r e o b s e r v e d a t 2 4 1 a n d 297 nm w i t h E1% of 5 6 1 a n d 266 r e s p e c t i v e l y . 1c m 2.5
Optical Rotation S u l f a m e t h a z i n e e x h i b i t s no o p t i c a l a c t i v i t y s i n c e i t h a s no c e n t e r of asymmetry.
CONSTANTIN PAPASTEPHANOU AND MAY FRANTZ
408
4 1 5 7 S U L F R M E T H R Z I N E 165 DEG 2 9 - O C T -76
MF12043
108
90 80
70 60
50
40 30 20 10 0
Q
8
CU
8
3-
8
CD
8
03
0
0
0
8
9
0
0
8 cU 3 CD 03 8 CLJ . - i 7 4 . - l c l F l c c l ~
MRSSiCHRRGE
INTENSITY SUM = 2 0 5 7 0 Figure 3.
BRSE PERK
Z
=22.9r!
Mass s p e c t r u m of s u l f a m e t h a z i n e .
SULFAMETHAZINE
409
Figure 4. W s p e c t r u m of aqueous solutions of s u l f a m e t h a z i n e () in w a t e r , (, ) O . 01N H C 1 ,
...
(---)
0.01N NaOH.
410
CONSTANTIN PAPASTEPHANOU AND MAY FRANTZ
Luminescence P r o p e r t i e s : S u l f a m e t h a z i n e h a s no f l u o r e s c e n c e I t does, a c t i v i t y i n aqueous s o l u t i o n s a t any pH. however, show a phosphorescence peak a t 410 nm when e x c i t e d a t 310 nm a t 77OK. The l i f e t i m e i s 0.8 seconds, ( 5 ) . 2.6
Solubility: Sulfamethazine i s very s l i g h t l y s o l u b l e i n w a t e r and chloroform: s l i g h t l y s o l u b l e i n e t h a n o l , a c e t o n e and 0.1g H C l ; s p a r i n g l y s o l u b l e i n 0.1EJ NaOH and i n s o l u b l e i n benzene ( 7 ) . 2.7
M e l t i n q Ranqe: The m e l t i n g range r e p o r t e d i n t h e USP X I X f o r s u l f a m e t h a z i n e i s between 197O and 200° u s i n g t h e C l a s s I procedure ( 6 ) . 2.8
D i f f e r e n t i a l Thermal A n a l y s i s
2.9
The o n l y t h e r m a l e v e n t i n t h e d i f f e r e n t i a l
t h e r m a l a n a l y s i s curve o f t h i s compound i s a s h a r p endotherm a t 197OC ( 8 ) . Sunwoo and E i s s e n ( 9 ) h a v e c a l c u l a t e d t h e h e a t of f u s i o n t o be 7438 170 ca l/mol e
.
2.10 Powder 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 p a t t e r n o f s u l f a m e t h a z i n e i s shown i n T a b l e I1 and F i g u r e 5 (10) There h a s been c o n s i d e r a b l e s p e c u l a t i o n a s t o t h e p r e s e n c e o f 2 o r 4 forms o f sulfamethaz i n e . Shiang Yang and G u i l l o r y (11) and Mesley and Houghton ( 1 2 ) r e p o r t t h e p r e s e n c e of two forms o f s u l f a m e t h a z i n e . The d a t a shown i n t h e s e two papers, however, i n d i c a t e s v e r y small d i f f e r e n c e s i n f u s i o n t e m p e r a t u r e , I R s p e c t r a and x-ray powder d i f f r a c t i c n data Kuhner-BrandstCdtter and Wunsch (13) r e p a r t e d t h a t t h e y were a b l e t o d e t e c t f o u r forms o f s u l f a m e t h a z i n e by thermomicroscopic methods, N o I R s p e c t r a o f t h e s e forms w e r e p u b l i s h e d i n subsequent r e p o r t s .
.
SULFAMETHAZINE
411
Table I1
X-ray powder diffraction pattern of sulfamethazine 2
9.68 10.70 15.21 17.16 18.44 19.71 21.33 24.64 25.92 27.70 28.81 29.23 30.25
0
d
(g)
9.14 8.27 5.83 5.17 4.81 4.50 4.17 3.61 3.44 3.22 3.10 3.06 2.95
Relative Intensity
1.000 0.045 0.101 0.075 0.083 0.571 0.088 0.338 0.103 0.049 0.303 0.052 0.046
100
so
80
70
ba
so
40
ao
i
h”Y-3-
....--
Figure 5 .
2.y
a
L
L
7
-
---_I--
X-ray d i f f r a c t i o n pattern o f sulfamethaeine.
F m.-
u
413
SULFAMETHAZINE
2.11
Dissociation Constants The pK was found to have a value of 7'.42 0.2 for equilibrium I and a value of + 2.65 - 0.2 for equilibrium I1 (14).
-
I. R 1 S O Z N H R e RlS02NR
+
11. R N H 2 a R N H 3 Synthesis Sulfamethazine is prepared by reacting acetylsulfanilyl chloride with 2-amino-4,6dimethylpyrimidine suspended in dry pyridine or in acetone and pyridine, followed by alkaline hydrolysis of the 2-(N4-acetylsulfanilamido)-4,6dimethylpyrimidine; the resulting salt being neutralized with S02. The 2-amino-4,6-dimethylpyrimidine is prepared by condensing acetylacetone with guanidine carbonate in toluene (15). Alternatively it can be prepared by condensation of equimolar amounts of sulfanilylguanidine and acetylacetone directly, It is precipitated in the presence of water in the form of very pale yellow crystals (16) Figure 6. Figure 6 Synthesis of Su Ifamethazine 3.
NH H2N o c ) $ f H !
-
CO
I
+
NH2
Sulfanilylguanidine
-
CH3 -2 H20
(332 A0
-
CH3
Acetylacetone
Sulfamethazine
CONSTANTIN PAPASTEPHANOU AND MAY FRANTZ
414
4.
Stability and Deqradation Venturella (17) reports that refluxing 10 g of sulfamethazine in 135 ml of 2g HC1 for 6 hours yielded sulfanilic acid (I) and 2-amino-4,6dimethyl-pyrimidine (11)
.
H
2
N O SO3H
H2N
I
(D
.-t
I
2
0
f N
ai a
.rl
k 0
d
E
al
c
.rl
k IU k
w
2
0 E
a,
U
4J
k
a,
m
bl
k 7
F
.rl
k
al 4J
c
-d
al
a
al
r:
k
.d
m k
CONSTANTIN PAPASTEPHANOU
488
2.5
O p t i c a l Rotation The U. S . P. X I X g i v e s a s p e c i f i c
r o t a t i o n v a l u e o f + 210° t o + 224O f o r t u b o c u r a r i n e c h l o r i d e , c a l c u l a t e d on an anhydrous b a s i s , and determined i n a s o l u t i o n c o n t a i n i n g .lo0 mg i n 1 0 m l o f water ( 4 ) . 2.6
Luminescence P r o p e r t i e s The u n c o r r e c t e d e x c i t a t i o n and emission s p e c t r a f o r t u b o c u r a r i n e c h l o r i d e (10 mg/ m l water) are shown i n F i g u r e 5. The maximum e x c i t a t i o n wavelength i s 375 nm and t h e maximum emission wavelength is 425 nm ( 5 ) . 2.7
Solubility Tubocurarine c h l o r i d e i s s o l u b l e i n w a t e r : s p a r i n g l y s o l u b l e i n e t h a n o l : and a l m o s t i n s o l u b l e i n e t h e r and chloroform ( 6 ) . 2.8
Melting Range The m e l t i n g p o i n t r e p o r t e d i n t h e U . S . P . X I X for t u b o c u r a r i n e c h l o r i d e i s a b o u t 270° w i t h decomposition ( 4 ) . 2.9
D i f f e r e n t i a l Thermal A n a l y s i s The p r i n c i p a l f e a t u r e s of t h e d i f f e r e n t i a l t h e r m a l a n a l y s i s a r e a s h a r p endotherm a t 125O and a decomposition endotherm a t 273O ( 7 ) . The endotherm a t 1 2 5 O r e p r e s e n t s t h e loss of w a t e r of h y d r a t i o n . 2.10
Powder X - r a y D i f f r a c t i o n The powder x-ray d i f f r a c t i o n p a t t e r n o f t u b o c u r a r i n e c h l o r i d e i s shown i n Table I and F i g u r e 6 ( 8 ) .
I 0 .
W 0 y1 0
u 0
" 0
N 0
A l I S N 3 1 N I 33N33S380flld
0
-
0
0 CI 0
0 N o
0 0
0 CI W
0
o m
0 0 u
c
-
I
I
o r
f
4 >
Y
2
w
N = u m
O u 0
0 0 W
0
m
u
0 I 0 n
0 y1 N
k a, c, C
a,
.d
a
.rl
k 0
rl
E
:
.d
k rd k
7u
+I
4 0
W
a
h
k k
a, -9 U a,
8c
7
Y
u
E 7 k c, a,
% a, U
2U u) 0)
k 0 7
b.4
rl
a,
m 1 trr
k
b.4
.A
CONSTANTIN PAPASTEPHANOU
490
Table I Powder X-ray Diffraction Pattern of d-tubocurarine 2 9 4.58 11.80 12.31 13.25 14.52 16.90 17.33 17.84 19.37 19.96 20.73 21.07 21.58 22.51 23.28 24.81 25.74 26.00 27.02 27.70 28.80 30.84 33.73
0
d (A)
19.29 7.50 7.19 6.68 6.10 5.25 5.12 4.97 4.58 4.45 4.28 4.22 4.12 3.95 3.82 3.59 3.46 3.43 3.30 3.22 3.10 2.90 2.66
Relative Intensity 1.000 0.224 0.125 0.197 0.194 0.368 0.101 0.267 0.205 0.506 0.215 0.088 0.325 0.353 0.395 0.312 0.139 0.179 0.498 0.141 0.202 0.278 0.155
s
Q)
C k m k
8
0
2 U
0
W
crc
a
x
CONSTANTIN PAPASTEPHANOU
492
2.11
D i s s o c i a t i o n Constant The pK w a s found t o be 7 . 4
(6).
3.
I s o l a t i o n and P u r i f i c a t i o n The South American arrow poisons known a s c u r a r e were f i r s t d e s c r i b e d i n w r i t i n g a t t h e beginning of t h e s i x t e e n t h century. The first attempt t o i s o l a t e t h e a c t i v e components of c u r a r e was made i n t h e e a r l y n i n e t e e n t h c e n t u r y by Boussingault and Roulin (9). I t was recognized e a r l y t h a t t h e poisons were of s e v e r a l v a r i e t i e s and t h a t , i n g e n e r a l , t h e c o n t a i n e r s used by n a t i v e groups f o r t h e preparat i o n o f the poisons w e r e f a i r l y d i a g n o s t i c of t h e c u r a r e i n them. Curares were t h e r e f o r e c l a s s i f i e d i n t o t h r e e c a t e g o r i e s : calabash- o r gourd-curare, pot-curare, and tube-curare ( t u b o c u r a r i n e ) . d-Tubocurarine chloride was f i r s t i s o l a t e d from a specimen o f tube c u r a r e i n 1935 by K i n g ( l 0 ) and l a t e r i s o l a t e d from Chondodendron tomentosum ( a l s o s p e l l e d Chondrodendron) Ruiz and Pavon by D u t c h e r i n 1946 (11). I t was l a t e r o b t a i n e d i n pure c r y s t a l l i n e form a l s o by Dutcher i n 1952 ( 1 2 ) by r e c r y s t a l l i z a t i o n from 0.1g HC1. S t a b i l i t y and Deqradation Pure t u b o c u r a r i n e c h l o r i d e appears t o be v e r y s t a b l e when s t o r e d i n a t i g h t c o n t a i n e r a t 5OC. A Squibb House Standard of d-tubocurarine c h l o r i d e was reassayed a f t e r t e n y e a r s of s t o r a g e and t h e potency of t h i s s t a n d a r d w a s found t o be i d e n t i c a l t o t h a t of t h e o r i g i n a l s t a n d a r d (13). One r e p o r t i n t h e l i t e r a t u r e claims t h a t pharmaceu ti ca 1 p r e p a r a t i o n s of tubocura r i n e c h l o r i d e may degrade a f t e r s e v e r a l y e a r s o f s t o r a g e a t room temperature (14). 4.
Druq Metabolism I n man, about 50% of t h e i n j e c t e d dose of t u b o c u r a r i n e is e x c r e t e d unchanged i n t h e u r i n e i n t h e f i r s t 1 0 hours (15, 16). There i s a s p e c i e s v a r i a t i o n i n t h e u r i n a r y e x c r e t i o n of the d r u g ( l 7 ) . The e l i m i n a t i o n of t u b o c u r a r i n e i n feces or by 5.
TUBOCURARINE CHLORIDE
493
other routes is negligible. Cohen e t a 1 (18) have shown i n a s t u d y of t h e metabolism of tubocurarine-H3 i n dogs t h a t t h i s drug does n o t undergo s i g n i f i c a n t d e g r e e s of b i o transformation. The kidneys normally p r o v i d e t h e major avenue o f e l i m i n a t i o n , w i t h t h e l i v e r contributing an a l t e r n a t e route. I n the absence of normal r e n a l f u n c t i o n , the a b i l i t y o f the l i v e r t o e l i m i n a t e t u b o c u r a r i n e v i a t h e b i l e is g r e a t l y increased. I n v e s t i g a t i o n of t h e serum p r o t e i n p a t t e r n of 52 p a t i e n t s undergoing upper abdominal s u r g e r y showed a h i g h l y s i g n i f i c a n t c o r r e l a t i o n between t h e l e v e l of gamma g l o b u l i n i n serum and t h e dose of t u b o c u r a r i n e r e q u i r e d t o m a i n t a i n adequate muscular relaxation. Baraka and G a b a l i (19) a t t r i b u t e d t h i s t o t h e a b i l i t y o f gamma g l o b u l i n t o b i n d tubocurarine. Olsen et a1 ( 2 0 ) s t u d i e d t h e b i n d i n g of ether i o d i d e (TCE) tubocurarine dirmethyl-14CJ and o t h e r r a d i f ~l a b e l l e d amines t o bovine n a s a l septum, c h o n d r o i t i n s u l f a t e and human plasma protein. They found t h a t 30-40"/, TCE was bound t o plasma p r o t e i n and t h a t each gram o f c a r t i l a g e could b i n d about 10-7 mol, TCE. They concluded t h a t c a r t i l a g e may r e p r e s e n t a measurable d i s t r i b u t i o n pool f o r t u b o c u r a r i n e . 6.
Methods o f A n a l y s i s 6.1 I d e n t i t y T e s t s To 20 m l of a s o l u t i o n of t u b o c u r a r i n e h y d r o c h l o r i d e (1 i n 2,000 i n water) are added 0.02 m l s u l f u r i c acid and 2 m l potassium i o d a t e s o l u t i o n (1%in w a t e r ) . A f t e r mixing and warming on a steam b a t h f o r 30 minutes, a yellow color i s produced (4,211 When 5 m l of m e r c u r i c n i t r a t e s o l u t i o n (3 m l Hg i n 27 m l HNO3) a r e added t o 0 . 5 m l of a s o l u t i o n of t u b o c u r a r i n e h y d r o c h l o r i d e (1%w / v ) , a c h e r r y red c o l o r s l o w l y a p p e a r s (21,22,23). When a few drops o f f e r r i c chloride s o l u t i o n are added t o 1 m l of a s o l u t i o n of tuboc u r a r i n e h y d r o c h l o r i d e (1%w/v) a g r e e n c o l o r i s
CONSTANTIN PAPASTEPHANOU
4 94
produced which t h e n changes t o brown a f t e r warming t h e m i x t u r e i n a water b a t h ( 2 3 ) . 6.2 Elemental Analysis The r e s u l t s from t h e e l e m e n t a l a n a l y s i s of t u b o c u r a r i n e a r e l i s t e d i n Table I I ( 2 ) . T a b l e I1 Elemental A n a l y s i s of d-Tubocurarine Chloride % Found
Element
58.08 6.65 3.62 8.99
C H N
c1
% Calculated 57.58 6.79 3.63
9.19
U l t r a v i o l e t Absorption According t o t h e B r i t i s h Pharmacopoeia ( 2 2 ) p u r e t u b o c u r a r i n e can be measured by i t s a b s o r p t i o n a t a b o u t 280 nm. The c o n c e n t r a t i o n s of d - t u b o c u r a r i n e i n a s o l u t i o n may be c a l c u l a t e d by a t t h e maximum o f t a k i n g 105 as t h e v a l u e of E l % 1c m a b o u t 280 nrn. The European Pharmacopoeia l i s t s t h e of tubocurarine a t same a s s a y b u t g i v e s t h e E l % l c m 2 8 0 nm a s 118. Cohen e t a1 have r e p o r t e d a method b a s e d on W a b s o r p t i o n f o r t h e d e t e r m i n a t i o n o f t u b o c u r a r i n e i n plasma ( 2 4 , 2 5 ) . E x t r a c t i o n o f t h e c u r a r e from the plasma i s based on i t s s o l u b i l i t y i n e t h y l e n e d i c h l o r i d e a t a n a l k a l i n e pH, and t h e n t h e s a l t i s reformed i n 0.01g HC1. Satisfactory measurements can be made t o c o n c e n t r a t i o n s of less t h a n 1 pg/ml o f plasma. 6.3
Colorimetric Analysis Momot ( 2 6 ) d e s c r i b e s a c o l o r i m e t r i c 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 d - t u b o c u r a r i n e i n human blood. Plasma, 3 m l , i s shaken for 5 minutes w i t h 10 m l e t h y l e n e d i c h l o r i d e and 0 . 5 m l g l y c i n e buffer. The m i x t u r e is c e n t r i f u g e d , t h e aqueous phase is d i s c a r d e d and t h e s o l v e n t phase is t r a n s 6.4
TUBOCURARINE CHLORIDE
495
f e r r e d t o a n o t h e r t e s t t u b e c o n t a i n i n g 3 d r o p s of c i t r a t e b u f f e r a n d some m e t h y l o r a n g e c r y s t a l s . The s o l u t i o n i s mixed, c e n t r i f u g e d , a n d a f t e r r e m o v a l o f t h e excess d y e , 0 . 5 m l e t h a n o l i s a d d e d a n d t h e s a m p l e i s r e a d i n a colorimeter. 6.5
Fluorescence Assay T u b o c u r a r i n e c a n be e x t r a c t e d from body f l u i d s and t i s s u e s w i t h e t h y l e n e d i c h l o r i d e a t a l k a l i n e pH i n t h e p r e s e n c e o f e x c e s s p o t a s s i u m i o d i d e and t h e n b a c k - e x t r a c t e d i n t o h y d r o c h l o r i c a c i d . The d r u g i s t h e n a n a l y z e d b y c o m p l e x i n g w i t h r o s e b e n g a l i n p o t a s s i u m h y d r o g e n p h o s p h a t e and t h e r e s u l t i n g t u b o c u r a r i n e - r o s e b e n g a l complex i s e x t r a c t e d i n a chloroform-phenol mixture(25% phenol i n c h l o r o f o r m ) . The c h l o r o f o r m l a y e r i s t h e n c a r e f u l l y removed, mixed t h o r o u g h l y w i t h a c e t o n e and measured f l u o r o m e t r i c a l l y (27,28). 6.6
T h i n - L a y e r Chromatoqraphy A r a p i d TLC method f o r t h e d e t e r m i n a -
t i o n o f t u b o c u r a r i n e i s d e s c r i b e d b y Crone and Smith(29) u s i n g microscope s l i d e s c o a t e d w i t h S i l i c a G e l H and a d r u g s a m p l e o f 5pg i n 1 I J - ~ 1g HC1. The chromatograms a r e d e v e l o p e d u s i n g 1N_ HC1 f o r a b o u t 5 m i n u t e s ( S o l v e n t I ) , o r 1g HC1: e t h a n o l (1:l) f o r 15-20 m i n u t e s ( S o l v e n t 11). The s l i d e s a r e t h e n d r i e d w i t h a h a i r - d r y e r and s p r a y e d w i t h D r a g e n d o r f f s r e a g e n t (30) Tubocurarine has a n Rf o f 0 . 1 2 a n d 0.54 i n s o l v e n t s I a n d I1 respectively. d-Tubocurarine c h l o r i d e can b e s e p a r a t e d from d - c h o n d r o c u r a r i n e c h l o r i d e ( a n a t u r a l l y o c c u r i n g compound,similar i n s t r u c t u r e to tubocurarine) using S i l i c a G e l GF p l a t e s i n a s o l v e n t s y s t e m c o n s i s t i n g o f a 80:lO:lO m i x t u r e o f m e t h y l e t h y l k e t o n e , w a t e r a n d f o r m i c a c i d . The p l a t e is sprayed with i o d o p l a t i n a t e spray reagent (30). U s i n g t h i s s y s t e m d - t u b o c u r a r i n e c h l o r i d e h a s a n Rf o f 0.30 a n d d - c h o n d r o c u r a r i n e c h l o r i d e h a s a n R f of 0.17(32). T u b o c u r a r i n e and o t h e r s k e l e t a l m u s c l e r e l a x a n t s h a v e b e e n e x t r a c t e d from b i o l o g i c a l m a t e r i a l and a n a l y z e d q u a l i t a t i v e l y and q u a n t i t a f
.
CONSTANTIN PAPASTEPHANOU
496
t i v e l y by TLC (33). Extracts have been a n a l y z e d u s i n g t h r e e systems: (I), alumina Woelm, acid (chloroform:methanol, 80:20) ; (11), alumina Woelm, b a s i c (methanol:chloroform, 75:25) and (111), alumina G Merk (methano1:acetic acid:water,92:3:3). Q u a l i t a t i v e a n a l y s i s o f t h e t e s t drugs on developed chromatograms w a s a f f e c t e d by examination under W l i g h t and b y s p r a y i n g w i t h i o d o p l a t i n a t e s o l u t i o n ( 3 1 ) . Q u a n t i t a t i o n of d r u g s was a l s o accomplished by t r a n s f e r r i n g t h e a r e a containing t h e spot t o a centrifuge tube containing phosphate b u f f e r pH 6.5. A f t e r shaking and c e n t r i f u g i n g , an a l i q u o t was removed and mixed w i t h methylene d i c h l o r i d e and sodium 9,lO dimethoxyanthracene-2-sulfonate s o l u t i o n . T h i s m i x t u r e was a g a i n shaken, c e n t r i f u g e d and a n a l i q u o t of t h e e x t r a c t removed. To t h i s was added t e t r a b u t y l ammonium hydroxide and t h e r e s u l t i n g f l u o r e s c e n c e determined (ex. 368 nm, e m . 448 nm). Tubocurarine gave Rf v a l u e s o f 0.83, 0 . 6 5 and 0.72 i n systems I, 11, and 111, r e s p e c t i v e l y . F i o r i and Marigo (34) have d e s c r i b e d a method f o r t h e d e t e c t i o n of d-tubocurarine, gallamine, decamethonium, and s u c c i n y l c h o l i n e i n b i o l o g i c a l material. The method i s b a s e d on t h e p u r i f i c a t i o n of e x t r a c t s from body f l u i d s and o r g a n s by i o n exchange s e p a r a t i o n on Amberlite I R C 50 r e s i n . The c u r a r e s a r e e l u t e d and p r e c i p i t a t e d a s r e i n e c k a t e s , which a r e t h e n r e g e n e r a t e d t o a l l o w t h e i r i d e n t i f i c a t i o n by means o f paper and t h i n l a y e r chromatography. Thin-layer chromatography w i t h a c i d alumina l a y e r s and ch1oroform:methanol (80:20) as the s o l v e n t system g i v e s a good r e s o l u t i o n (Rf = 0.22 f o r t u b o c u r a r i n e ) . The s p o t s a r e r e v e a l e d by t h e i o d o p l a t i n a t e r e a g e n t ( 3 1 ) . The e l u a t e s o f u n t r e a t e d spots are examined s p e c t r o p h o t o m e t r i c a l l y i n t h e W t o detect t u b o c u r a r i n e and f i n a l l y are i n j e c t e d into t h e tail v e i n o f mice f o r a biological test. Hiqh-Pressure Liuuid Chromatoqraphy T w i t c h e t t and Moffat (35) have shown t h a t t u b o c u r a r i n e can be e f f e c t i v e l y s e p a r a t e d from o t h e r 6.7
TUBOCURARINE CHLORIDE
497
drugs and quantitatively measured using octadecylsilane as the stationary phase with aqueous methanol eluents. Gas Chromatography A paper by Vidic (36) described a gas chromatographic method for the determination of choline and its esters, neostigmine, tubocurarine methylcurarine and scopolamine Bu bromide. 6.8
Electrophoresis Marini-Bettolo and Coch Frugoni (37) have studied the electrophoretic separation of some 70 alkaloids on paper at pH 2-12. Mazzei and Lederer ( 3 8 ) have also studied the paper electrophoretic behavior of tubocurarine and other quaternary ammonium compounds, Wan (39) has described a procedure for the thin-layer electrophoresis of alkaloids on glass plates coated with cellulose powder. Electrophoresis was carried out in acid and alkaline electrolytes at 500 V and 3,000 V. 6.9
6.10 Bioassay The only assay procedure described in the U . S . P . XIX (4) is the rabbit "head drop" method (40). In the assay the reference standard and test sample of tubocurare are each injected intravenously into a number of rabbits in a crossover pattern. The endpoint of the test,"head-drop", is relaxation of the neck muscles to such a degree that the animal's head cannot be raised or turned in response to a physical stimulus.
6.11 Radioimmunoassay Horowitz and Spector (41)have developed a radioimmunoassay that depends upon a competition between unlabelled d-tubocurarine and a standard of labelled 3H-d-tubocurarine for the specific antibodies present in rabbit antisera. The assay is extremely sensitive measuring as little as 5 ng/ml and requires only 10 pl of serum or
498
CONSTANTIN PAPASTEPHANOU
urine sample. The advantage of this type of assay is the fact that no extraction from biological materials is required. 6.12 Assays in Bodv Fluids and Tissues The following methods have been used for the assay of tubocurarine in body fluids and tissues: Method Reference Ultraviolet absorption 24,25 colorimetric 26 Fluorescence 27,28 Thin-layer chromatography 33,34 High-pressure liquid chromatography 35 Radioimmunoassay 41
TUBOCURARINE CHLORIDE
499
References
1. 2. 3. 4. 5. 6.
7. 8. 9.
10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
20. 21. 22.
Toeplitz, B.: The Squibb Institute, Personal communication. Funke, P. T. & Puar, M.S. ; The Squibb Institute Personal communication. Whigan, D.: The Squibb Institute, Personal communication. Anon; U. S. Pharmacopeia XIX, p. 530 (1975). Papastephanou, C.; The Squibb Institute, Unpublished data. Perlman, S.: The Squibb Institute, Personal communication. Jacobson, H.; The Squibb Institute, Personal communication. Ochs, Q.; The Squibb Institute, Personal communication. Boussingault & Roulin ; Ann. de Chim. et de phvs. : 39,29 (1828). King, H. : J. Chem.Soc., 1381(1935). Dutcher, J.D.: J.Am. Chem.Soc. ,@, 419(1946). Dutcher, J.D. : ibid, 74, 2221 (1952). Brewer, G.A.: The Squibb Institute, Personal communication. Tyfezynska, J. : Diss. Pharm. Pharmacol., 19,585 (1967)C.A. 68,89889W. Kalow, W. : Anesthesioloqy, 20. 505 (1959). Stovner, J. & Lund, I. ; Br. J.Anaesth. , a 2 3 5 (1970). March, D.F. : J. Pharmacol. Exp.Ther., 105,299 (1952). Cohen, E.N. & Brewer, H.W. : Anesthesioloqy,28, 309 (1967). Baraka, A. & Gabali, F.: Br. J. Anaesth., 40, 89 (1968). Olsen, G.D., Chan, E.M. & Riker, W.K. : . J. Pharmacol. Exp. Ther., =,242(1970). Anon,; Pharmacopeie Francaise. Codex Medicamentarius Galicus, p. 1248,1965, supp. 1968. Anon., The British Pharmacopoeia, H.M. Stationery Office, London p.488 (1973).
CONSTANTIN PAPASTEPHANOU
500
23. 24. 25. 26. 27. 28. 29. 30. 31. 320 33.
34. 35. 36. 37. 38. 39. 40.
41
Anon. ;The European Pharmacopoeia, Maisonneuve S.A. p. 379 (1969). Elert, B.: Am. J. Med. Techno1.,=331(1956). Cohen, E.N. in Curare and Curare-like Aqents, Bovet, Bovet-Nitti and Marini-Bettolo (Editors), Elsevier, (1959) p. 377. Momot, G.A. ; Tr. Tsent. Inst. Usoversh.Vrachei, 95. 237(1966). Cohen, E.N. ; J. Lab. & Clin. Med., 61, 338 (1963). Cohen, E . N . ; ibid. 979 (1963). Crone, H.D. & Smith, E.M. : J.Chromatogr. ,77, 234 (1973). Jackson, J.V. in Isolation and Identification of Druqs, Clarke, G. C. (Editor), Pharmaceutical Press, London, (1969) p. 799. Stahl, E. : Thin-Layer Chromatoqraphy, Springer-Verlag, (1965) p. 493. Williamson, D. E. ; Chromatoqraphia, 281 (1973). Wittmer, D., Atwell, S. & Haney, W.G. : J. Forensic Sci., 20,86(1975). Fiori, A. & Marigo, M.; J. ChrOmatOqr.,3l, 171 (1967). Twitchett, P. J. & Moffat, A. C. : J. Chromatoqr., 111, 149 (1975). Vidic, H.J.: Dross, H. & Kewitz, H.; Z.Klin.Chem.Klin.Biochem. ,lO,l56(1972). Marini-Bettolo, G.B. & Coch Frugoni,J.A. : Gass. chim. ital., 86,1324 (1956). Mazzei, M. & Lederer, M. : J.Chromatoqr.,Z 292 (1968). Wan, A. S. C, : ibid,60.371 (1971) Varney, R.F., Linegar, C.R. & Holaday, H.A. t J. Pharmacol. Exp. Ther., 97.72 (1949). Horowitz, P.E. & Spector, S. : J. Pharmacol. Exp.Ther., 185.94 (1973).
,a,
Analytical Profiles of Drug Substances, 7
ADDENDAANDERRATA
501
Copyright 0 1978 by Academic Press, h e . All rights of reproduction in any form reserved.
ISBN 0-12-2m7-0
ADDENDA AND ERRATA
502
Affiliations of Editors and Contributors Volume 6, p . vii Correct affiliation: S . A. Benezra, Burroughs Wellcome Co., Research Triangle Park, North Carolina Amphotericin B Volume 6, p. 26 4 . 3 Mass Spectrometry (TMS = trimethyl-silane) Cy clizine
Volume 6, p. 83 The name of the co-author was inadvertently omitted Correct title: Cyclizine Steven A. Benezra and Chen-Hwa Yang
.
Triamcinolone Acetonide Volume 1, p . 416 7. Determination in Body Fluids and Tissues. A radioimmunoassay to determine plasma levels after topical application has been developed by M. Ponec, M. Frolich, A . DeLiister, A . J. Moolenaar, Arch; Dermatol. Res., 259; 63 (1977).
-
CUMULATIVE INDEX Italic numerals refer to volume numbers Acetaminophen, 3,1 Acetohexamide, I, I ; 2, 573 Allopurinol, 7, 1 Alpha-tocopheryl acetate,.?, 11 1 Amitriptyline hydrochloride, 3, 127 Amoxicillin, 7, 19 Amphotericin B, 6, I ; 7, 502 Ampicillin,2, 1;4,517 Bendroflumethiazide, 5 , I ; 6,597 Betamethasone dipropionate, 6,43 Cefazolin*; 4 , 1 Cephalexin,l, 21 Cephalothin sodium, I , 319 Cephradine, 5 , 2 I Chloral hydrate,2,85 Chloramphenicol,4, 47,517 Chlordiazepoxide, I , 15 Chlordiazepoxide hydrochloride, I , 39;4, 517 Chloroquine phosphate,5,61 Chlorpheniramine maleate, 7, 43 Chloroprothixene, 2,63 Clidinium bromide, 2, 145 Clonazepam, 6,61 Clorazepate dipotassium,4,91 Cloxacillin sodium,l, I13 Cyclizine, 6 , 83; 7, 502 Cycloserine, I , 53 Cyclothiazide, 1 , 6 6 Dapsone, 5.87 Dexamethasone, 2, 163;4,518 Diatrizoic acid,4, 137;5,556 Diazepam, I , 79;4,517 Digitoxin.3, 149 Dihydroergotoxine methane sulfonate, 7, 81 Dioctyl sodium sulfosuccinate, 2, 199 Diperodon, 6.99 Diphenhydramine hydrochloride, 3, 173 Diphenoxylate hydrochloride, 7, 149 Disulfiram.4, 168
Droperidol, 7, 171 Echothiophate iodide, 3,233 Epinephrine, 7, 193 Ergotamine tartrate, 6 , 113 Erthromycin estolate, I , 101;2,573 Estradiol valerate.4, 192 Ethambutol hydrochloride, 7,23 I Ethynodiol diacetate,3,253 Fenoprofen calcium*, 6 , 161 Flucytosine,S, I15 Fludrocortisone acetate,3,281 Fluorouracil, 2,221 Fluoxymesterone, 7,251 Fluphenazine enanthate,2,245;4,523 Fluphenazine hydrochloride, 2,263; 4 , 5 18 Gluthethimide.5, 139 Halothane, I , 119;2,573 Hexetidine, 7,277 Hydroflumethiazide, 7,297 Hydroxyprogesterone caproate, 4,209 Hydroxyzine dihydrochloride, 7.3 19 Iodipamide,3,333 Isocarboxazid ,2,295 Isoniazide,6, 183 Isopropamide,2,315 Isosorbide dinitrate, 4,225; 5,556 Kanamycin sulfate, 6,259 Ketamine,6,297 Levarterenol bitartrate, I , 49; 2,573 Levallorphan tartrate, 2,339 Levodopa,S, 189 Levothyroxine sodium, 5,225 Meperidine hydrochloride, I , 175 Meprobamate, I , 209;4,519 6-Mercaptopurine, 7,343 Methadone hydrochloride,3,365;4,519 Methaqualone,l, 245,519 Methotrexate.5.283 Methyclothiazide, 5,307 Methyprylon,Z, 363
* Monographs in “Pharmacological and Biochemical Properties of Drug Substances, Volume 1” M. E. Goldberg, D. Sc., Editor American Pharmaceutical Association, 1977. Cefazolin, 155 Fenoprofen, 183 503
504
Metronidazole,S, 327 Minocycline,6,323 Nitrofurantoin, 5,345 Norethindrone, 4,268 Norgestrel, 4,294 Nortriptyline hydrochloride, I , 233;2,573 Nystatin,6,341 Oxazepam,3,44 I Phenazopyridine hydrochloride, 3,465 Phenelzine sulfate, 2,383 Phenformin hydrochloride, 4.3 19; 5,429 Phenobarbital, 7,359 Phenoxymethyl penicillin potassium, 1, 249 Phenylephrine hydrochloride,3,483 Piperazine estrone suIfate,5,375 Primidone, 2,409 Procainamide hydrochloride, 4,333 Procarbazine hydrochloride, 5,403 Promethazine hydrochloride, 5,429 Proparacaine hydrochloride, 6,423 Propiomazine hydrochloride, 2,439 Propoxyphene hydrochloride, I, 301;4,519; 6,598 Propylthiouracil, 6,457 Reserpine,4, 384; 5,557 Rifampin,5,467 Secobarbital sodium, 1,343
CUMULATIVE INDEX
Spironolactone, 4,43 1 Sodium nitroprusside, 6,487 Sulphamerdne, 6, 515 Sulfamethazine, 7,401 Sulfamethoxazole,2,467; 4,520 Sulfasalazine,5, 515 Sulfisoxazole,2,487 Testolactone,S, 533 Testosterone enanthate,4, 452 Theophylline,Q. 466 Thiostrepton, 7,423 Tolbutamide,3,513;5,557 Triamcinolone,1,367;2,571;4,520.523 Triamcinolone acetonide. 1, 397,416;2,571; 4, 520; 7 Triamcinolone diacetate, I , 423 Triamcinolone hexacetonide, 6,579 Triclobisonium chloride, 2,507 Triflupromazine hydrochloride,2,523; 4, 520; 5,557 Trimethaphan camsylate, 3,545 Trimethobenzamide hydrochloride, 2,551 Trimethoprim, 7,445 Tropicamide. 3,565 Tubocurarine chloride, 7,477 Tybamate,4, 494 Vinblastine sulfate, 1,443 Vincristine sulfate, 1,463