Profiles of Drug Substances Vol 07

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
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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
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7DX
Library o Congress Cataloging in Publication Data f

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
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
N. W .Atwater, E. R. Squibb & Sons, Princeton, New Jersey
K . S. Albert, The Upjohn Co., Kalamazoo, Michigan
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 o Drug Substances in a series of f 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 o Drug Substances f 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
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
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
ALLOPURINOL
1.
Description
1 1 Name, Formula, Molecular Weight .

Allopurinol is lH-pyrazol0(3,4-d)pyrimidine-4-01
C5H4N40 1.2 Appearance, Color, Odor
136.11
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)

3060
Assignment CH stretching vibrations of the pyrimidine ring CO stretching vibration of the keto form of the 4-hydroxy tautomer ring vibrations CH in-plane deformation
1700 1590 1245
Figure 1-
Infrared Spectrum of Allopurinol
ALLOPURINOL
790 2.2
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 M z NMR spectrometer. Deuterated DMSO was used as the solvent H with tetramethylsilane as an internal standard. Based on the NMR spectrum, the following assignments for allopurinol can be made.
4 2
Proton
# of Protons
1 1
Chemical Shift (ppm)

7.95
8.07
b
a
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 0.1N NaOH 0.1N HC1 Me thano1
Amax (n)
E -
257 250 252
7200 7600 7600
rl .rl
$4
rl
rl
u
0 a,
L l
fi 1
tA
(d
x
a
a, d
0
-rl
111
u a, c
a, rl
V
$4 (d
w
b 7
a,
.rl
0 -0 Tt
0 -co co m m
w
-N N
m m
E
c
a,
U
-R
-
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cn
CI
4 0
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-8 -8
I
I
@ l
I,
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I
0 0
0 0
OR OR
0 0
M *d F
L.c 1
a,
STEVEN A. BENEZRA AND T.RENEY BENNETT
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 136(100%)
2.5
m/e 120 (4.2%)
m/e I09(6.3%)
Melting Point Allopurinol melts above 35OOC7
2.6
Solubility
The solubility of ajlopurinol in various solvents at 25OC is given in Table 111
Table I11 Solubility of Allopurinol at 25OC Solvent Water n-octanol Chlorofo m Ethanol DMSO
2.7
Solubility (mg/ml)
<0,01 0.60 0.30

4.6
0.48
Dissociation Constant
The pka 9f allopurinol determined spectrophotometrically is 10.2
w
0
AlISN31NI 3 A I l V l 3 U
10
2.8
Partition Coefficient
The partition coefficients of allopurinol at 2' 5C in n-octanol pH 1.2 and n-octanol/pH 6.0 are 0.24 and 0.33 respectively
#.
2.9
Crystal and Molecular Structure
Allopurinol crystals obtained by slow evaporation from acetone: water (1:l) and analyzed with a Picker FACS-1 x-ray diffractometer were found to be in the monoclinic space group P2 Ic. The cell constants were a=3.683A0, b=14.685AD, c=10.318k0, and .B=97.47". The bases are hydrogen bonded in sheets parallel to the 102 plane. Layers of the sheets are stacked with a staggered overlap of the base rings, All ring nitrogensS atoms, oxygen, and C 2 are involved in hydrogen bonding () 3. Synthesis
Allopurinol can be prepared from a variety of synthetic procedures. Three of tQelfynthetic routes to allopurinol are shown in Figure 5 Synthetic route 1 uses malononitrile and triethylorthoformate to form ethoxymethylene malononitrile which reacts with hydrazine to give 3-amino-4-cyanopyrazole. The pyrazole is hydrolyzed to form 3-aminopyrazole-4-carboxamide. The carboxamide is reacted with formamide to give allopurinol. Synthetic route 2 uses formamidine hydrochloride and cyanoacetamide to form 3-amino2-cyanoacrylamide. The 3-amino-2-cyanoacrylamide is reacted with hydrazine to give 3-aminopyrazole-4-carboxa1nide which is converted to allopurinol as in route 1 The third synthetic . route uses cyanoacetamide, triethylorthoformate, and morpholine to prepare 3-morpholino-2-cyanoacrylamide. The 3-morpholino-2-cyanoacrylamide i s reacted with hydrazine as in route 2 to give the carboxamide which is converted to allopurinol by reaction with formamide.
4.
Stability
The primary decomposition product in acidic and basic solutions is 3-aminopyrazole-4-carboxamide. In basic solutions the 3-aminopyrazole-4-carboxamide decomposes to 3-aminopyrazole-4-carboxylic acid and 3-aminopyrazole. In acidic solutions the 3-aminopyrazole-4-carboxylic acid is not formed
--.
( u
Izz
, "
I Z 0
cu
I1
tj P
w
0
( 9
I
I vl
+cu
0
+ I
0*"3
I
u I u
cu
12
STEVEN A. BENEZRA AND T. MNEY BENNETT
At 105C the maximum stability of allopurinol in solution occurs at pH 3.1-3.4. Rapid decomposition occurs at high pH. At 25OC the shelf life of a 2% suspenfion of allopurinol at pH 9.5 is predicted to be 5.4 years
5.
Methods of Analysis 5.1 Elemental Analysis Element C H

% Calculated
4.3 41
2.96 41.17 11.76
5.2 Spectrophotometric Analysis The official USP analysis of allopurinol and alloA purinol in tablet form is a W spectrophometric analysis portion of the drug or powdered tablets is dissolved in sodium hydroxide solution and diluted stepwise with dilute hydrochloric acid to a concentration of approximately 10 ug/ml. The sample solution is compared against a standard solution at 250 nm to determine the purity of allopurinol or the labelled strength of the tablets.
5.3
Polarography
Dryhurst and De have analyzed filopurinol in the presence of uric acid by DC polarography The useful analytical pH range for the analysis of allopurinol was found to be pH 0-6. At pH values above 6 the height of the polarographic wave decreases. The half-wave potential for the reduction of allopurinol was found to be a function of pH and = -1.118-0.067pH. At low pH followed the equation E values of supporting el!d$rolyte the polarographic wave is due to the 4 electron reduction of allopurinol to 2,3,6,7tetrahydroallopurinol. At higher pH values of supporting electrolyte (pH 4.7-6.0) the reduction involves 2 electrons to give 6,7-dihydroallopurinol.
5.4 Chromatography 5.41 Thin Layer Chromatography Various thin layer chromatographic systems or allopurinol are given in Table IV.
ALLOPURINOL
13
Table IV Thin Layer Chromatography Systems for Alloprinol Mobile Phase 2-methoxyethano1:methyl ethyl ketone:amonia 3:6:2 n-butanol:5% aqueous ammonia 2:l ch1oroform:ethanol 3:2 ch1oroform:ethanol 3:l acetone Stationary Phase Rf Cellulose Cellulose Silica gel Silica gel Silica gel 5.42 Paper Chromatography Paper chromatography has been used to separate allopuffnol from metabolites and chemically related compounds The systems used for the separations are given in Table V. 0.36 0.21 0.64 Ref. 5
14
14 14
0.14
0.57
Table V Paper Chromatography Systems for Allopurinol Mobile Phase

5% Na2HP0 :iso-amyl alcohol 4 2: 1
Paper Whatman 3MM Whatman 3MM Whatman 3MM

S & S 597
Rf
0.55
1-butano1:water 84:16 1-p ropanol:water 7 :3 1-butano1:water 84:16(NH3 atmosphere)
0.44
0.62 0.27
5.43 Column Chromatography Column chromatography has been used i g separate allopurinol from 3-aminopyrazole-4-carboxamide A Dowex lxs (chloride form) anion exchange resin was used. The allopurinol was retained on the column using a mobile phase of pH 10 phosphate buffer. Phosphate buffer at pH 4 was used to quantitat4vely elute the allopurinol from the column. A Dowex-5O(H ) column has been used to separate allopurinol
14
STEVEN A . BENEZRA AND T. RENEY BENNETT
from xanthine and uric acid16. with 1N hydrochloric acid.
Allopurinol was eluted
5.44 High Performance Liquid Chromatography

High performance liquid chromatography has been utilized to separate allopurinol from various metabolites and analogues. Table VI gives the conditions and retentions times used for the separations. Table VI HPLC Conditions for Allopurinol Separations Column Mobile Phase Flow Rate(ml/min)
10.0
Rt(min> 0.7
Ref -
Spherisorb CH CN:H20: 10 &OH I 1060: 4 80: Aminex A-27 Whatman PA-3 8
17
NH OAC(pH 8.71,
4. oc
10 .
0.25
18
19
Potassium Phosphate O.O15M, pH 5.7, 4OoC

5.45 Gas Chromatography
10
Allopurinol in tablets has been analyzed by gas chromatography. The allopurinol was extracted with 1.5N NaOH. The trimethylsilyl derivative was formed with BSTFA and injected onto a 2M, 3% OV-la column maintained at 1 5 O O C . The retention index was 1723.6
5.5
Mass Spectrometry
Multiple peak integrated current mass spec5fometry has been used to analyze allopurinol in blood plasma Samples of plasma were dehydrated and 2-3 mg portions of the dehydrated plasma admitted directly into the mass spectrometer. The m/e 52.0187 peak of allopurinol was integrated over the lifetime of the peak. Allopurinol was determined in the 10-100 ppm level with an accuracy of 5 20%.
ALLOPURINOL
15
6.
Metabolism and Pharmacokinetics
6.1 Metabolism
Allopurinol acts as a substrate for, and inhibitor of xanthine oxidase. It is converted primarily to 4,6-dihydroxypyrazolo(3,4-d)pyrimidine (oxypurinol). Allopurinol-lriboside and oxypurinol-7-riboside are also metabolic products, although to a lesser extent than oxypurinol. Allopurinol-1-ribonucleotide, oxypurinol-1-ribonucleotide, and oxypuri~gl-7~~ibonucleotide have been found in concentrations of 10 -10 M in rat liver and kidney. The amounts and relative proportions of the nucleotides y y e dependent upon dose, route of administration, and time
6.2
Tissue Distribution
After 2 hours dose in mice, allopurinol is approximately equally distributed in the blood, liver, spleen, heart, and intestine. The brain and lung contain approximately6half the concentration of allopurinol found in the blood
6.3
Pharmacokinetics
Allopurinol has a half-clearance time in plasma of about 2 hours in man. This is due predominately to the rapid conversion of allopurinol to oxypurinol. Oxypurinol has a more prolonged half-clearance time than allopurinol, on the order of 28 hours. The rate of excretion of allopurinol was approximately 4 hours for half-clearance and on the order of 28 hours for oxypurinol. The half-clearance values vary, depending on whether the allopurinol waa6given as a single dose or over a prolonged administration
16
1.
2.
USP XIX, p. 18 (1975)

J. Lagasca, Burroughs Wellcome, personal communication
3.
D.H. Mitchell, Burroughs Wellcome, personal cmunication
4 .
5.
R Crouch, Burroughs Wellcome, personal communication .

T. Bennett, unpublished data
6.
7 .
8.
D. Brent, Burroughs Wellcome, personal communication

J. Spence and A Jones, Burroughs Wellcome, personal .
communication
P. Prusiner and M. Sundaralingam, Acta Cryst., 2148 (1972)

U.S. Patent 2,759,949
m,
9.
1 . U.S. Patent 3,694,484 0 11. U . S . Patent 3,864,341
.. 12. T.G. Beaumont, T S Comber, A.P. Conroy, R.P. Gandhi, and G.T. Hill, Burroughs Wellcome, personal communication 13. G. Dryhurst and P.K. De, Anal. Chem. Acta, (1972)
58, 183
57
335
14. P.D. Gressel and J.F. Gallelli, J. Pharm. Sci., (1968)
15. T.A. Krenitsky, G.B. Elion, R.A. Strelitz, and G.H. Hitchings, J. Biol. Chem., 242, 2675 (1967) 16. G.B. Elion, A. Kovensky, G.H. Hftchings, E Metz, and . R.W. Rundles, Biochem. Pharmacol., 2, 863 (1966) . 17. G Hill, Burroughs Wellcome, personal communication
. . 18. M Brown and A Bye, J. Chrom.,
1 3 195 4,
(1977)
19. D.J. Nelson, C.J.L. Bugge, H.C. Krasny, and G.B. Elion, Biochem. Pharmacol.. 22. 2003 (1973)
ALLOPURINOL
17
20.
V. M i l l e r , V . Pac6kov6, and E. Smolkovb, J . Chrom., 216 (1976) W. Snedden and E.B.

Parker, Anal. Chem.,
123,
21.
43,
1651 (1971)
AMOXICILLIN
Pranah K . Bhnttacharyya and Winifred M . Cort
19
Copyright @ 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.
ISBN 0-12-260807-0
20
PRANAB K. BHATTACHARYYA AND WINIFRED M. CORT
TABLE OF CONTENTS
1. Description 1.1 Name 1.2 Formula and Molecular Weight 1.3 Isomers 1.4 Hydrates 1.5 Salts 1.6 Appearance, Color, Odor
2. Physical Properties 2.1 Ultraviolet Absorption 2 . 2 ORD Spectrum 2.3 CD Spectrum 2 . 4 IR Spectrum 2.5 Raman Spectrum 2.6 NMR Spectrum 2.7 X-Ray Diffraction Data
3.
4.
Amoxicillin Stability Solubility
5. Manufacturing Procedure
6.
7.
Methods of Analysis Pharmacological Evidence
8, References
AMOXICILLIN
1 Description .
1.1 Name: Amoxicillin Chemical abstracts designates it by its complete chemical name; that is 4-thia-1-azabicyclo p. 2 . heptane-2-carboxylic acid, 6- [ a i o (mn 4-hydroxyphenyl acetyl) amin4 -3,3-dimethyl-7-oxo-pS- pa,5a,6B(S*)) 26787-78-0
Amoxicillin is generally referred to as 6 @ - -() a-amino-p-hydroxyphenyl acetamid4 penicillanic acid trihydrate and in early studies was called hydroxyampicillin.
12 .
Formula and Molecular Weight
O H
C16H1gN30@. 3H20
1.3 Isomers
COOH MW. 419.46
In penicillins, the absolute configurations of the carbons 2,5,6, are S , R, R respectively. In addition the carbon at the 10 position adjacent to the aromatic ring has the R configuration formerly called D() --. As in ampicillin, the S epimer at C10 was also synthesized and had inferior antibacterial activity. The synthesis was further extended to include isomers ( in all) at the C10, with ortho, 6 meta, and para-hydroxy on the aromatic ring, and the two most active ones in vitro had the R configuration on C10 and were meta and para-hydroxy substituted compounds. In vivo the R p-hydroxy isomer resulted in the highest blood levels, and, as a result, this has been the active amoxicillin ( ) 1.
1 4 Hydrates .
Unlike ampicillin, the anhydrous forms have not been studied extensively, although produced ( ) 2.
22
1.5
Salts
Salts consisting of the hydrochloride, sulfate, 8-napthalene sulfonate, sodium and potassium have been synthesized and identified ( 2 ) . Similar to ampicillin the hetacillin type derivative, that is 6-(4-p-hydroxyphenyl-2,2-dimethyl-5-oxoimidazolidin-l-yl)penicillanic acid was synthesized () 1. However, the form currently in use is the trihydrate.
1.6 Appearance, Color and Odor

Amoxicillin trihydrate is a white to off-white free flowing crystalline powder. One of the tests required for certification is the recognition of the crystals and observation of birefringence, typical of all penicillins ( ) 3 . Amoxicillin similar to other penicillins also has the typical penicillin odor described as a sulfurous type. 2 . Physical Properties 2.1 Ultraviolet Absorption: The ultraviolet spectra ( 4 ) of amoxicillin trihydrate, which were recorded on a Cary Model 14 Spectrophotometer, are given in Fig. 1 & 2. The spectra represent a phenolic type chromophore. Table I:
Ethano1
W spectral data
Wavelength,nm:
E
230(max. ) 10850
274(max.) 1400 272(max.) 1080 291(max.) 3000
*281(Sh.) 1160 %278(Sh.) 920
0 1 N HC1 .
Wavelength,nm:
E
229(max. ) 9500
248(max.) 2200
0.1 N KOH Wavelength,nm:

E
~325(Sh.) 750
AMOXICILLIN
23
I(
EX
10-3
5
I N HCL 2. In 0.1 N KOH
FIGURE I
The UV Spectra of omoxicillin trihydrote in O.IN HCI and OlN K r ~ c t i i o l y . W
24
PRANAB K . BHATTACHARYYA AND WINIFRED M. CORT
I In ETHANOL Exlo%
1 6
b
I
1 4
12 -
1 ' 0
8.
6.
4,
0 2c
0 3
FIGURE 2
The UV Spectrum of amoxicillin trihydrate in othanol.
AMOXICILLIN
25
2.2
ORD Spectrum:-
The optical rotatory dispersion spectrum was obtained on a JASCO Model 5-20 Automatic Recording Spectropolarimeter. The ORD curve (see Fig.3) is in agreement with the structure of the compound. Table 11: Experimental data (4) from Extrema Start NaD Hg Hg Hg Shoulder Peak Intersection Trough Intersection Peak Last
2.3
Spectrum
Wavelength (nm) 650 589 546 436 365 290 248 232133 220 212 199/200 195
Rotation ($) +880 +1y 090 +1I 320 +2 520 +4 240 +10 y 000 +38,400
0
-23 200
0
+130 0 0 0
0
CD Spectrum:-
The CD spectrum was obtained on a JASCO Model 5-20 Automatic Recording Spectropolarimeter. The optical circular dichroism (see Fig.4) shows Cotton effects typical or a phenolic type chromophore. Table 111: Experimental data (4) from CD Spectrum Extrema Start Inflection Maximum Inflection Intersection Maximum Intersection Maximum Intersection Last Wavelength (nm)
292 280 273174 268 263 236/37 217 207109 203 200
Ellipticity ( ) 0
0
-2 $000 -2,560 -2 y 000

0
+45,200
0
-31,200
0
+32,000
nm
FIGURE 3
T)M
OR0 curve of omaricillim trihydmte in water at loom
.-
AMOXICILLIN
27
2 00
250
300
A nm
350
FIGURE 4
The circular dichroism of amoxicillin trihydrate in water at room temperature.
28
2.4 IR Spectrum:
The IR spectrum (5) (see Fig.5) of amoxicillin trihydrate was obtained on the Perkin Elmer Model 621 Double-Beam Spectrophotometer as a potassium bromide pellet. The frequencies and assignments are given in the following table. Table IV Assignment
-1 Frequency (cm )
OH stretching
NH3+
8-lactam C=O stretching Amide I C-0 stretching COO- asymmetric stretching Aromatic ring 2.5 Raman Spectrum:
3550-3400 3200-2500 1776 1688 1582 1519
The Raman spectrum (6) (see Fig.6) of the compound in solid state was obtained on a S ex Model 1401 Double Spectrometer using the argon 4880 excitation derived from Model 164 Spectra-Physics Laser. A fluorescence background was also observed.
Table V:
Raman spectral assigments Frequency(cm %3500
Assignment
-1
OH stretching
NII stretching
3410
1780
IIN-
1695 Aromatic C
= C
1620, 1610 (sh)

635
-s-
..
FIGURE 5
The infrared Spectrum of amoaicillin trihydmte.
FIGURE 6
The Ramon Spectrum of amoxicillin trihydrate.
AMOXICILLIN
31
2.6
N M R Spectrum:
The NMR spectrum ( 7 ) (see Fig.7) was obtained on a Varian XL-100 Spectrometer. The sample (about 50 mg) was dissolved into 0.5 ml of D 2 0 with about 2 mg of DSS (sodium 2,2 - dimethyl -2- silapentane-5 - sulfonate) added as an internal reference. Approximately 5 microlitres of DCL were also added for catalysing the rate of substitution of the exchangeable protons by deuterium nuclei.
Interpretation of the NMR spectrum of amoxicillin trihydrate has been reported ( 8 ) . Table VI: Proton Assignment a b
C
Proton NMR assignments Chemical Shift(G),ppm 1.34,l.36 4.40

5.14 5.46 6.91
Multiplicity Singlet
I1
I1
II
d e* f*
7.37
Doublet
I1
*Coupling constant JHH = 9 Hz
I
I
AMOXICILLIN
33
2.7
X-ray Diffraction Data:
The X-ray powder diffraction data are given in the following table () 9. Instrument Conditions Instrument: GE Model XRD-6-Diffractometer operated at 50 KV, 12.5 mA. Target: Copper (CuKa = 1.54181). Beam slit: . ' 3 Detector Slit: 0.05. Soller: M.R. Filter: Ni. Detector: 1.75 KV and 0.95 KV. Pulse height selection: 5 volts. Window: out. At cps full scale: 1K. Time constant: 2.5. Focus: ligo. Take off angle: 4 ' . Scan and chart speeds: 1 /inch. Sample treatment: as is. Table VII: X-ray Diffraction Results
12.20 14.72 *15.13 *16.25 18.05 19.34 19.77 x20.20 k25.76 *26.68 *28.71 *Broad lines
7.26 6.02 5.86 5.45 4.91 4.59 4.49 4.40 34 .6 3.34 3.11
0.63 0.24 0.54 0.16 1.00 0.75 0.24 0.12 0.42 0.51 0.55
1/11 = Relative Intensity
34
3.
Amoxicillin Stability Amoxicillin degradation is typical of all penicillin hydrolyses as reviewed by Ivashkiv ( 0 . In alkali 1) it decomposes first by opening the lactame to its penicilloic acid. This is the basis for the iodine 1) absorption assay ( 1 as well as the hydroxylamine reaction. Penicilloic acid ultimately loses C02 and forms penilloic acid. Amoxicillin in acid, again similar to other penicillins, hydrolyzes to form first amoxicillin penicillenic acid which absorbs at 320 nm. This hydrolysis has been the basis for determination of ampicillin (12) and amoxicillin ( 3 . Neither 1) 6-aminopenicillanic acid nor penicilloic acid forms penicillenic acid. Additional acid breakdown products are penillic acid, penaldic acid, penicillamine and ultimately penilloaldehyde. Although the semi-synthetic penicillins are made from 6-amino penicillanic acid, (6-APA), it is not found as a degradation product. In fact, there are numerous patents on making (6-APA) from penicillins 1) using penicillin amidases ( 4 . Amoxicillin in oral dosage forms is an insoluble emulsion at 125 and 250 mg/5 ml. These have at least 90% retention of activity after 2 weeks at room temperature (RT) or at 4OC. However, solubility at pH 6 is only 4.2 mg/ml (21 mg/5ml); of the solutions kept for 7 days at RT, only 50% of the amoxicillin remains intact although 100% is retained at 4OC. Since the insoluble amoxicillin is more stable than dissolved material, the stability of the oral dosage form is dependent on its lack of solubility. Dry bulk amoxicillin, capsules, and dry oral dosage powders have retained full activity for as long as five years. However, expiration dates are usually three years.
4 Solubility .
Solubility data is somewhat meagre and is as follows ( 5 . 1)
AMOXICILLIN
35
Solvent Water Methano1 Ethanol (Absolute) Acetone Dioxane Ethyl Acetate Hexane Acetonitrile Benzene
Q4
7.5
3.4
1.3
0.8 Insoluble
Insoluble Insoluble Insoluble However, at pH
4-8 this varies from 4 . 2 to 9 . 0 mg/ml.
Solubility in water depends on pH.
5. Manufacturing Procedure
A number of synthetic procedures have been described ( 2 ) . The method of choice (1) involves preparation of 6-(D-a-amino-p-hydroxyphenylacetamido) penicillanic acid by protecting the amino group as an enamine function (the hydroxyl did not have to be protected). The sodium salt of D-(-)-a-amino-phydroxyphenyl acetic acid 1 was heated with methyl . acetoacetate in methanol to give the enamine This was crystallized by distilling off the methanol and replacing it with dry toluene. This was treated with ethyl chloroformate in cold dry acetone containing catalytic quantity of N-methylmorpholine to form the mixed Anhydride =which was then treated with the potassium salt of 6-APA in aqueous acetone for 30 minutes, then concentrated under reduced pressure. The aqueous concentrate was mixed with isobutyl methyl ketone pH 1.9 adjusted, solvent removed, aqueous phase adjusted to pH 5.1, concentrated and crystallized. Crystals are washed with acetone to dry. Summary of synthesis is presented in Figure 8.
z .
36
SYNTH ESlS
+CH3 CH0 - C - O C H 3 -Ch M0
_) i
HOaC,H-COONa
CHjC
II
I
k ~ 3 MIEK extr. pH adj.
OCH 3
-10%
COO ET CL ACETONE
CATALYST
ET
Amoxicillin P
FIGURE 8
Synthesis of amoxlcillin .
AMOXICILLIN
37
6 Methods of Analysis .
The standard specifications in the Code of Federal Regulations requires ( ) that the bulk drug be tested 3 and certified. Testing includes potency 900-1050 mcg/mg on anhydrous basis, moisture 11.5-14.5%, pH 3.5-6.0, it must pass safety (436.331, it must be crystalline, give a positive IR identification, acid and base (lithium-methoxide and perchloric acid) titrations should not be less than 90% on an anhydrous basis. Assays may be performed iodometrically (436.204), colorimetrically by hydroxylamine, or microbiologically (436.105) using S. lutea in a horizontal agar diffusion procedure. Results of the iodometric procedure shall be conclusive. After the above tests, all bulk amoxicillin in the U.S. must be submitted to the FDA for certification. The marketed dosage forms, capsules and oral suspensions must use certified bulk amoxicillin, and all lots must be tested and submitted for certification. The tests for the capsules (440.103a) include moisture, assay and TLC and for the oral suspensions (440.1036) they include the same tests plus pH. There have been other analytical procedures proposed for amoxicillin similar to published methods for other penicillins. These include measurement of U.V. absorption at 320 nm of the amoxicillin penicil1) lenic acid formed in the presence of acid and Cu2+ ( 3 . A spectrofluorometric method has also been proposed (16) using the reaction with formaldehyde and measuring at 430 nm after excitation at 366 nm. A fluorescamine assay (17) has been proposed for ampicillin which will determine 14 nanograms/ml at pH 6.5-7.5 and will also be applicable to amoxicillin. A method using high pressure liquid chromatography (HPLC) has been published for ampicillin ( 8 . HPLC appears to be an excellent 1) quantitative chromatographic procedure for amoxicillin (19,20) since it requires no heat (no destruction) and is readily quantitated by measurement of UV peak intensities. It is hoped that automated HPLC, eventually, will be an official procedure.
38
PRANAB K . BHATTACHARYYA AND WINIFRED M. CORT
7. Pharmacological Evidence Amoxicillin was synthesized and covered in a patent (21) several years after ampicillin. In the meantime many investigations (CF reference 10) were undertaken on ampicillin which became one of the major current penicillins. A series o f papers presented in 1970 and published in 1971, focused attention on amoxicillin. Neu and Winshell ( 2 2 ) showed first that amoxicillin and ampicillin had equivalent antibacterial spectra. All four references ( 2 2 , 2 3 , 2 4 , 2 5 ) reported that amoxicillin was better absorbed and produced higher serum levels than ampicillin. This information 1 apparently stimulated Long, et. al. ( ) to publish their work on the synthesis. Sutherland et. al. ( 2 6 ) further expanded on comparisons of ampicillin and amoxicillin and showed equivalent activity against Staph. aureus 6-haemalytic streptococci, Strep pneumoniae S. faecalis, H. influenzae, N. gonorrhoeae, E. coli, Klebsiella, salmonellae, and S. marcescens. MIC values against 34 organisms were calculated. The degree of protein binding was 17% and thus equal to ampicillin and better than benzyl penicillin which binds to 50-60%. Serum levels 2 hours after oral administration were approximately twice as high as ampicillin. The blood level data were confirmed in clinical volunteers ( 7 . 2) Blood levels of 6-10 mcg of penicillin immediately stimulated many clinicians to study amoxicillin. This resulted in three symposia, one in London (28), another held in New Orleans ( 2 9 ) and a third in Tokyo ( 0 . Increased penicillin blood levels were reported 3) by many investigators. May and Ingold ( 2 9 ) showed that amoxicillin penetrated deeper than ampicillin and all other penicillins into cell walls and was more effective against gram negative infections. They also reported that amoxicillin penetrated deeper into respiratory secretions and speculated that higher tissue levels were obtained. In another review (31) 147 references on amoxicillin were quoted. Peak serum levels were twice those of ampicillin and generally more prolonged. Amoxicillin was more stable in gastric acid than ampicillin. Levels in sputum, sinus mucosa and interstitual fluid are higher than ampicillin. High levels were found in the liver of rats and kidneys of mice which confirmed higher tissue level speculations.
AMOXICILLIN
39
Further studies in healthy adults resulted in amoxicillin serum levels of 7 . 3 4 mcg/ml and in ampicillin 3 . 6 4 mcg/ml following administration of 500 mg oral dose. In patients with impaired renal function, levels as high as 20 mcg/ml were obtained. Amoxicillin was also reported to be successfully used in various other infections (31) including biliary, gynaecological, and bone infections and in septic abortions. In hundreds of patients it was an effective antibacterial agent in urinary tract infections (70-93% cure), upper and lower respiratory infections, skin and soft tissue and G . I . tract infections. Eleven studies on uncomplicated gonorrhoeae resulted in 92% cure (31).
40
References
1 .
Long,A.A.W., Nayler, J.H.C., Smith,H., Taylor,T., and Ward N., J. Chem. SOC. (C) 1920 (1971).
2. Long,A.A.W., and Nayler,J.H.C., U.S. Patent 3,674,776 (1972). 3 . Code Federal Regulations Title 21, 440.3 and 436.203 (1976). Communication.
4. Toome, V., Hoffmann-La Roche Inc., Private

5. Waysek, E., Hoffmann-La Roche Inc., Private Communication.
6 Chang, F., Hoffmann-La Roche Inc., Private .

Communication. 7. Go, M.V., and Johnson, J.H., Hoffmann-La Roche Inc. Private Communication.
8 Wilson, W.L., Avdovich, H.W., and Hughes, D.W., . J. Assoc. of Anal. Chem. 57 ( ) 1300 (1974). 6,
9. Chiu, A.M., Hoffmann-La Roche Inc., Private

Communication. 10. Ivashkiv, E., Analytical Profiles 2 1, edited
by Klaus Florey,Acad. Press N.Y. (1973).
1 . Code Federal Regulations Title 21, 436.204 ( 9 6 . 1 17)

12.
- 247 (1967). 92,
Smith,J.W.G., DeGrey,G.E., and Patel,V.J., Analyst
13. Marini,D. (1976).
, Rass Chim. 28, 35 (1976) CA 85 83296e
14. U . S . Patents 3,008,955; 3,008,956; 3,014,845;

3,014,846. 15. Bass E., Hoffmann-La Roche, Inc., Unpublished Data. 16. Davidson,D.F., Clin. Chim. Acta
69,
67 (1976).
AMOXICILLIN
41
17. Kusnir, J., and Barna, K., 2. Anal. Chem. Band Heft A, 288 (1974).
18. Tsuji, K., and Robertson, J.H., Pharmaceutical Science - 1542 (1975). 64,
271
19. Emery, M., and Debesis, E., High Pressure Liquid Chromatographic Assay of Amoxicillin, Hoffmann-La Roche Inc., Private Communication.
20.
Blythe, R., Beechman, Private Communication.
21. Nayler, J.H.C., and Smith, H., British Patent 981, 178 (1964). 22. 23. 24. 25. 26. 27. 28. Neu, H.C., and Winshell, E.B., 1970 Antimicrobial Agents And Chemotherapy, p.407 (1971). Sutherland, R., and Rolinson, G.M., Ibid. p411 (1971). Croydon, E.A.P., and Sutherland, R., Ibid. p427 (1971). Acred, P., Hunter, P.A., Mizen, L., and Rolinson, G. N., m d . p416 (1971). Sutherland, R., Croyden, E.A.P., and Rolinson, G.N., British Med. Journ. 73,July 1 (1972). Bodey, G.P., and Nance, J., Antimicrobial Agents And Chemotherapy 1,358 (1972). Int. Symposium on Amoxicillin, London Sept. 1973, Publ. by Excerpta Elsevier Publ. Co. Inc., Amsterdam (1974). Symposium on Amoxicillin Perspectives, College of Physicians and Surgeons, Columbia Univ. held in New Orleans, Oct. (1973). Chemotherapy 21 ( ) 1355-1844 (1973) edited by 8 Japanese Society Chemotherapy, Tokyo Japan.
29.
30.
31.
- 88 (1975). 9,
Brogen, R.N., Speight, T.M., and Avery, G.S., Drugs
CHLORPHENIRAMINE MALEATE
Churles G . Eckhurt find Thonius McCorkle
43
Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-260807-0
44
CHARLES G. ECKHART AND THOMAS McCORKLE
TABLE OF CONTENTS
1.
Description
1.1 1.2
Formula, N a m e , Molecular Weight Appearance, Color, Odor
2.
Physical Properties Elemental A n a l y s i s Mass Spectrum 2.3 P r o t o n Magnetic Resonance Spectrum 2.4 Carbon-13 Magnetic Resonance Spectrum 2.5 I n f r a r e d Spectrum 2.6 E q u i l i b r i u m Data 2.7 U l t r a v i o l e t Spectrum 2.8 C r y s t a l P r o p e r t i e s 2.9 Thermal Analyses 2.10 X-Ray D i f f r a c t i o n 2.11 S o l u b i l i t y and P a r t i t i o n i n g S t u d i e s
2.1 2.2
3.
Synthesis
4, S t a b i l i t y
4.1
4.2
4.3
5.
pH-Thermal S t a b i l i t y pH-Light S t a b i l i t y Phase S o l u b i l i t y A n a l y s i s
Drug Metabolic P r o d u c t s
5.1 5.2
The Metabolism of Chlorpheniramine Maleate i n Man The Metabolism of Chlorpheniramine Maleate i n t h e Dog and R a t
6 .
A n a l y t i c a l Methods
6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8
6.9
Acid-Base T i t r a t i o n C o l o r i m e t r i c Methods S p e c t r o p h o t o m e t r i c and F l u o r o m e t r i c Methods Thin-Layer Chromatography Pape r Chromatography S e p a r a t i o n by 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 Gas-Liquid Chromatography High Performance, P a r t i t i o n and IonExchange L i q u i d Chromatography Polarography
45
1.
Description
1.1
Formula, Name, Molecular Weight
CHCOOH
It
CHCOOH
C16H19Cm2
C4H404
Chlorpheniramlne maleate is a racemic mixture of optical isomers and i s known by the following chemical names (1).
i
ii
dlmethyl-(Z)-2-butenedioate (1:l) 2-[p-chloro- a-(2-dimethylaminoethyl)benzyl] pyridine maleate ( 1 : l )
2-pyridinepropanamine,
-(4-chlorophenyl)-N,N-
Molecular Weight: 1.2
390.87
Appearance, Color, Odor
Chlorpheniramine maleate is an odorless, white crystalline solid. 2 . Physical Properties 2.1 Elemental Analysis
Analysis of chlorpheniramine maleate (Batch CM-6G1001) was determined for carbon, hydrogen, nitrogen and chlorine. The carbon, hydrogen and nitrogen analysis was performed on a Perkin Elmer Model 240 instrument. Analysis for chlorine was performed by combustion of the sample and coulometric titration using an American Instrument Co. Chloride Titrator.
46
CHARLES G . ECKHART AND THOMAS McCORKLE
2OH 23N4 2' ' Theoretical Percentages
C: N:
61.45%, H: 7.17%, C 1 : 61.34%, H: 7.18%, C 1 :
5.93%, 9.07% 6.02%, 9.11%
Experimental P e r c e n t a g e s 2.2
Mass Spectrum
- C:
N:
The mass spectrum of c h l o r p h e n i r a m i n e maleate (Batch CM-6G-1001) was o b t a i n e d on a V a r i a n MAT CH5 medium r e s o l u t i o n s i n g l e f o c u s i n g magnetic s e c t o r i n s t r u m e n t a t 70 e w i t h V 0 a probe t e m p e r a t u r e of 110 C. Mass spectrum c a l i b r a t i o n w a s The r e s u l t s are t a b u l a t e d performed w i t h perfluoroalkane-225. i n T a b l e 1 and are i n f u l l s u p p o r t of t h e s t r u c t u r e given.
TABLE 1
MAS s
274
230
ION M+
M -44
M -58
+ +
2 16
203 167
78 72
CH2N(CH3)
CHCH2N(CH3) CH2CH2N(CH3)
M -71
M -107
+ +
@
N
CH CH N (CH3)2
+ c1
58 44 The mass spectrum i s shown i n Fig. 1.
.i r
c
Q,
c a
k
,I -
E
6.I 0
48
Pattern recognition methods were used to differentiate drug classes (e.g. sedatives, tranquilizers) and to predict their pharmacologic activity based on their mass spectral fragmentation pattern (2). Chlorpheniramine maleate was used as an example. 2.3 Proton Magnetic Resonance Spectrum (PMR)
The 100 MHz proton nmr spectrum of chlorpheniramine maleate (Batch CM-6G-1001) was obtained as a 5% (WlV) solution in hexadeuteriodimethylsulfoxide (d6-DMSO) with tetramethylsilane (TMS) as an internal reference. The Spectrum was obtained on a Varian XL-100-15 Fourier Transform Spectrometer at ambient temperature. The assignments,chemical shifts and multiplicities are given in Table 2 and are in full support of the structure shown. TABLE 2
PMR Spectral Assignments
4
HOOC \
,
6
COOH
* / c = c \H 6
49
Table 2 (con't.) Position of Proton

1
Chemical Shift ( 6 ) 10.5 8.7 78 . 74 . 7.2-7.5 6.2 4.2 2.5-3.1 28 . 25 .
Intensity 2 H
Multiplicity Singlet Mu1t iplet Mu1t iplet Singlet Mu 1t iplets Singlet Triplet Mu1t iplet Singlet
2
3
1H
1 H 4 H 2 H 2 H 1 H 4 H 6 H
4
5
6
7
8 9
10 DMSO/d5H
The PMR spectrum is shown in Fig. 2 . 24 . Carbon-13 Magnetic Resonance Spectrum (CMR)
The carbon-13 magnetic resonance spectrum was run on chlorpheniramine maleate (Batch CM-6G-1001). The spectrum was run on a Varian Associates XL-100-15, equipped with a 620-L100 disc-based com uter. The spectrum was run in a The field was locked to the saturated solution of d F -DMSO. deuterium resonance of the solvent. The data was zero filled to 16K prior to transformation; aquisition time was 0 8 sec. . with no post delay used. The spectrum (Fig. 3) represents 25.159 MHz with a 9 0 pulse width in two micro seconds. The shifts shown in ' Table 3 are downfield of an internal TMS reference.
-/
Fig. 2
1 0 0 M z P r o t o n Magnetic Resonance Spectrum of H
Chlorpheniramine Maleate i n d6- DMSO ( 5 % w/v) w i t h TMS as an I n t e r n a l Reference.
JWO
,ow
,o l
I
I
-
1 .
, , , , . ,
180
160
140
120
100
80
60
40
20
PPM(8,)
C-13 Magnetic Resonance Spectrum of Chlorpheniramine Maleate in Saturated d6- D M S O Solution with TMS as an Internal
Fig.
Standard.
52
TABLE 3
CMR Spectral Assignments
10
HOOC,
12
11
c=c
,OH CO
11
12 -~
Position of Carbon 8 10 9
Chemical S h i f t ( 6 ) 28.76 42.27 48.94 55.51 122.00 123.02 128.49 129.61 131.42 135.99 136.97 141.56 149.20 161.20 167.55
7
5 3 3, 5 2, 6 4
11
1
6 2 12
53
2.5
I n f r a r e d Spectrum
The i n f r a r e d spectrum of chlorpheniramine maleate (Batch CM-6G-1001) was o b t a i n e d a s a Nujol mull on a P e r k i n E l m e r Model 180 g r a t i n g i n f r a r e d spectrophotometer. The major a b s o r p t i o n bands are t a b u l a t e d below i n T a b l e 4. These assignments are i n f u l l s u p p o r t of t h e g i v e n s t r u c t u r e .
TABLE 4
I n f r a r e d Assignments -1 Wavenumber (cm 1 Assignment Aromatic and u n s a t u r a t e d C-H s t r e t c h

N-H
3090-3010 (w)
2700-2400 (m, v b r . ) 1705 (m) 1620 (m), 1570 ( s )
stretch
C-0 s t r e t c h a r o m a t i c C=C, C=N s t r e t c h a r o m a t i c C-C, C=N, and c a r b o x y l a t e C-0-stretch c a r b o x y l a t e C-0(5)
1584 ( s )
1359 (s)
stretch
886 (m), 868

Notations:
maleate m-medium, s-strong, vbr-very broad
w-weak,
The i n f r a r e d spectrum i s shown i n Fig. 4. 2.6 E q u i l i b r i u m Data The pKa's of c h l o r p h e n i r a m i n e maleate are as pKal = 9.2 pKa2 = 4.0 The s o u r c e of t h e pKal d a t a i s r e f e r e n c e ( 3 ) . pKa2 has been determined s p e c t r o p h o t o m e t r i c a l l y by Higuchi, e t . a l . (4) and Doye, e t . a l ( 5 ) .
f 0 llows :
4J
a , cd
a,
a ,
.rl
k
r(
c
a
0
a,
55
An interesting application of ionization constants was made by Doyle and Proctor (6) in their study of selective elution parameters f o r several amine-containing drugs by K' partition chromatography. A graphical method using p a s and extraction constants was used.
2.7
Ultraviolet Spectrum (UV)
The ultraviolet spectrum (Fig. 5) of chlorpheniramine maleate (Batch CM-6G-1001) was obtained in methanol on a Cary 118CX spectrophotometer. The spectral data is listed in Table 5. TABLE 5 Ultraviolet Spectrum of Chlorpheniramine Maleate in Methanol Xrnax, nm
250 shoulder 255 26 1 267 275 shoulder 4.27 5.05 5.38 3.86 1.17
Table 6 lists the spectral data of chlorpheniramine maleate obtained in various solvents. TABLE 6 The UV Maximum and Molar Absorptivities in Various Solvents Solvent CH30H
0.1N NaOH (CH30H)
0.1N HC1 (CH30H)
H2
AUlaX
261 nm 261 nm 265 nm 261 nm
5.38 5.63 8.48 5.76
56
20 2
260
300
340
Fig. 5 . U l t r a - v i o l e t Absorption Spectrum of Chlorphenlramine Maleate I n Methanol.
CHLORF'HENIRAMINE MALFATE
57
TABLE 6 (con't.)
Solvent 1 N NaOH (aq. )

1 N HC1 (as.)
CHC l3
1 max
262 n m 265 nm 263 n m 5.77 8.39 5.77
It is found t h a t t h e band maximum i s r e l a t i v e l y i n s e n s i t i v e t o s o l v e n t e f f e c t s , b u t t h e f i n e s t r u c t u r e is l o s t i n a c i d i c media.
2.8
Crystal Properties
According t o B r a n d s t a t t e r - H u h n e r t , et.al. ( 7 ) u s i n g t h e thermo-microscopic t e c h n i q u e , c h l o r p h e n i r a m i n e maleate c r y s t a l i z e s n e a r i t s m e l t i n g p o i n t (129-134OC) t o g i v e p l a t e s , rhombs, g r a i n s and prisms. The s u p e r c o o l e d m e l t w i l l s o l i d i f y t o a g l a s s on t h e microscope s t a g e . A t t h e p r e s e n t t i m e no o p t i c a l c r y s t a l l o g r a p h y h a s been done s i n c e X-ray d i f f r a c t i o n d a t a is a v a i l a b l e . There i s o p t i c a l c r y s t a l l o g r a p h y on dexchlorpheniramine m a l e a t e ( t h e (+) o p t i c a l isomer). The N a t i o n a l Formulary X I 1 (1965) h a s g i v e n t h e f o l l o w i n g d a t a ( 8 ) : na 1.509 O p t i c sign:
nB
1.564
ny
1.683 2V = 70' (calc.)
A x i a l Angle:
The e x t i n c t i o n is p a r a l l e l t o t h e c r y s t a l edge. These d a t a are a l s o r e p o r t e d i n (9). 2.9 Thermal Analyses
Thermal G r a v i m e t r i c A n a l y s i s (TGA)oindicates no weight loss from ambient t e m p e r a t u r e t o 140 C f o r c h l o r pheniramine maleate. The D i f f e r e n t i a l Scanning C a l o r i m e t r y (DSC) Curve (Fig. 6 ) e x h i b i t s a s i n g l e s h a r p m e l t i n g endotherm w i t h t h e o n s e t t e m p e r a t u r e of 1 3 3 O C . Grady, et.al. (10) r e p o r t a HF 11,400 cal./mole on a 99.55 mole % p u r e sample of chlorpheniramine maleate.
1C (CORRECTED FOR CHROMEL ALUMEL THERMOCOUPLES1 .'
Fig. 6 D S C Curve of Chlorpheniramine Maleate Onset Temperature 1 3 C Peak Temperature 1 6 C 3' ; 3' .
59
2.10 X-Ray Diffraction

The X-ray powder diffraction pattern (Table 7) was obtained on chlorpheniramine maleate (Batch CM-6G-1001) with a Phillips ADP 3500 X-Ray Diffractometer utilizing CuK c1 radiation (1.5418 8). TABLE 7 The X-ray Powder Diffraction Pattern of Chlorpheniramine Maleate
20 12.214 12.957 16.943 17.011 18.262 18.543 18.613 18.805 19.214 19.536 19.661 20.215 20.561 20.777 20.965 21.368 21.836 24.058 24.636 24.959 25.599 26.214 28.059 29.960 30.618 32.161 33.622 33.697 33.918 34.722
d&)*
I d *
9 25 6 6 7 14 13 14
100
7.246 6.832 5.233 5.212 4.857 4.784 4.766 4.718 4.619 4.543 4.515 4.392 4.319 4.275 4.237 4.158 4.070 3.699 3.613 3.568 3.480 3.399 3.180 2.982 2.920 2.783 2.666 2.660 2.643 2.583
5 7 90 11 19 14 23 25 59 29 21 17 29 20 15 7 20 8 8 9
11
*d (interplanar distance) = nX / 2 sin 0
**I11 = relative intensity (based on highest intensity of 100)
60
2.11
S o l u b i l i t y and P a r t i t i o n S t u d i e s
The s o l u b i l i t y of c h l o r p h e n i r a m i n e maleate i n e i g h t e e n s o l v e n t s was s t u d i e d by g r a v i m e t r i c o r u l t r a v i o l e t s p e c t r a l d e t e c t i o n . The d a t a is summarized i n T a b l e 8. TABLE 8 S o l u b i l i t y , mg/ml Solvent Acetone Benzene Carbon t e t r a c h l o r i d e Chloroform
1, 2 D i c h l o r o e t h a n e
a t 25OC
50.
Measurement
G r av irne t r i c
G r avime t r i c
0.7
4. x
lo-2
Gravimetric
G r av irnet r i c
240.
47.
6.0
4.0
330. 21.
8. x
Gravimetric
G r av irnet r i c
Dioxane E t h y l acetate Ethyl alcohol Glycerine Hep t a n e 0.1N H C 1 Methyl a l c o h o l Methylene c h l o r i d e Petroleum e t h e r P a r e n t e r a l phosphate buffer s o h . Saturated salt solution 0.1N NaOH
G r av irnet r ic
G r av irnet r i c
Ultraviolet
LO
-3
G r av irnet r i c
G r av irnet r i c
47.
130.
G r av irne t r i c
G r av irne t r i c
190.
0.1
G r av irne t 2: i c
190.
28. 180.
Ultraviolet
U 1 r av i o l e t t
Ultraviolet
Water
160.
U 1 r av i o l e t t
61
In order t o f a c i l i t a t e extraction and chromatographic studies, l i q u i d l i q u i d partitioning data were accumulated and are tabulated i n Table 9.
TABLE 9
Liquid-Liquid Partitioning Data Percent i n Organic Phase Aqueous pH

0.1
n-BuOH
CHC13
0.6
n-Hep t ane
1.3 1.7 2.7 2.6 3.5 3.6 4.3 5.8 6.2 6.7 7.1 7.7 8.0 8.7 9.4 10.8 11.9 12.5
08 .
1.0
44.0 70.0 91.2 99.0 99.6 99.7 99.6 99.6
8.5 7.1 74.7 81.5 94.9 97.4 99.4
99.8
62
3.0
Synthesis
The f o l l o w i n g s y n t h e s i s has been used t o produce chlorpheniramine maleate (11).
c1
C1
4.
Stability
The s t a b i l i t y of chlorpheniramine maleate has been s t u d i e d i n our l a b o r a t o r i e s using a c c e l e r a t e d h e a t and l i g h t c o n d i t i o n s on s o l u t i o n s of various p v a l u e s (Batch CM-6GH 1001). The experimental c o n d i t i o n s as w e l l as t h e r e s u l t s are g i v e n i n the f o l l o w i n g two paragraphs.
63
4.1
pH-Thermal S t a b i l i t y
T h i s s t u d y was done u s i n g 30 mg of c h l o r p h e n i r a m i n e maleate i n 10 m l of aqueous b u f f e r o v e r a wide pH range i n s e a l e d g l a s s ampules. The samples were s t o r e d f o r 1 week a t 95OC. They were t h e n assayed by q u a n t i t a t i v e paper chromatography. R e s u l t s are summarized as f o l l o w s :
pH
2
B u f f e r Component 0.1 0.1 0.1 0.1 0.1 0.1 01 . pH-Light
P e r c e n t Recovery 99 95 100 97 96 96 99
4 6
7 8 10 13 4.2
citric sodium sodium sodium M sodium M sodium M sodium

M M M M
acid citrate phosphate phosphate phosphate borate hydroxide
Stability
T h i s s t u d y was done w i t h 1 5 m of c h l o r p h e n i r a m i n e g maleate i n 5 m l of aqueous b u f f e r , o v e r t h e pH 2 t o 8 r a n g e , i n s e a l e d g l a s s ampules. Two sets of S a m les w e r e p r e p a r e d . 8 One s e t w a s s t o r e d f o r t h r e e months a t 25 C i n a l i g h t box under a f l u o r e s c e n t l i g h t of about 350 c a n d l e power. The 0 o t h e r set w a s s t o r e d f o r t h r e e months a t 25 C i n t o t a l darkn e s s . The samples were t h e n a s s a y e d by q u a n t i t a t i v e p a p e r chromatography. R e s u l t s of t h e s e a s s a y s a r e summarized a s f 0llows : P e r c e n t Recovery pH B u f f e r Component Light Dark 2 4 6 8
01 . 01 . 01 . 01 .
M citric M sodium M sodium M sodium
acid citrate phosphate phosphate
101 103 102 102
101 101 101 100
4.3
Phase S o l u b i l i t y A n a l y s i s
Grady, e t . a l . (10) s t u d i e d t h e p u r i t y of 115 d r u g s by s e v e r a l t e c h n i q u e s i n c l u d i n g p h a s e s o l u b i l i t y a n a l y s i s . S o l u b i l i t y and i m p u r i t y d a t a on c h l o r p h e n i r a m i n e maleate and dexchlorpheniramine m a l e a t e were i n c l u d e d i n t h e study. 5. Drug M e t a b o l i c P r o d u c t s 5.1 Metabolism of Chlorpheniramine Maleate i n Man (12)
The pharmacokinetics and metabolism of 3H c h l o r pheniramine maleate have been s t u d i e d i n man. A f t e r a p.0.
64
dose ( 1 2 mg), 3H appeared r a p i d l y i n plasma and a t two h o u r s was e q u i v a l e n t t o 32.48 ng of c h l o r p h e n i r a m i n e p e r m l ; r a d i o a c t i v i t y p e r s i s t e d i n plasma throug) 48 hours. A t f i v e minutes a f t e r a n i.v. d o s e (4 mg), H3in plasma w a s e q u i v a l e n t t o 20.88 ng of d r u g p e r m l a d H a g a i n p e r s i s t e d 9 i n plasma. T h i s p e r s i s t a n c e 9f plasma H w a s most probably due t o t i s s u e d e p o s i t i o n of H-drug ( o r m e t a b o l i t e s ) , d i r e c t l y i n d i c a t e d by i t s l a r g e volume of d i s t r i b u t i o n , 250% of body weight. The drug was 7 2 % bound t o plasma p r o t e i n a t a c o n c e n t r a t i o n of 280 ng/ml. Unchanged drug a s w e l l as i t s m e t a b o l i t e s were p r e s e n t i n p l sma, however, 3H i n plasma, and i n c o n t r a s 5 t o t h e p e r s i s t a n t l e v e l s of t h e l e v e l s of H-chlorpheniramine d e c l i n e d s t e a d i l y , a l t h o u g h slowly, a f t e r t h e i.v. dose and a f t e r t h e two-hour peak a f t e r a d m i n i s t r a t i o n p.0. The plasma h a l f - l i f e of a p.0. dose was e s t i m a t e d a t 1 2 t o 15 hourg and t h a t of an i.v. dose, 28 hours. E x c r e t i o n of t h e H-drug w a s slow and o n l y o n e - t h i r d of a p.0. o r i.v. d o s e was recovered from t h e e x c r e t a d u r i n g 48 h o u r s a f t e r a d m i n i s t r a t i o n . The p.0. dose was c m p l e t e l y absorbed, however, and only small ! I amounts of t h e H-drug were e x c r e t e d i n t o f e c e s a f t e r i t s 3 a d m i n i s t r a t i o n . F e c a l e x c r e t i o n of H of an i.v. dose 3 i n d i c a t e d t h a t H-chlorpheniramine underwent a n e n t e r o h e p a t i c c i r c u l a t i o n . Chlorpheniramine was e x t e n s i v e l y metabolized and e x c r e t e d i n t h e u r i n e as mono- and didesmethyl c h l o r pheniramine, two u n i d e n t i f i e d m e t a b o l i t e s and small amounts of chlorpheniramine. The g r e a t e s t p o r t i o n of t h e drug was e x c r e t e d as u n i d e n t i f i e d p o l a r m e t a b o l i t e ( s 1 .
5.2
Metabolism of Chlorpheniramine Maleate i n t h e Dog and R a t ( 1 3 )
S t u d i e s of t h e pharmacokinetics and metabolism of 3 14C and H-chlorpheniramine maleate i n t h e r a t and dog have a l s o been conducted. An o r a l dose of t h e drug w a s r a p i d l y and e x t e n s i v e l y absorbed from t h e g a s t r o i n t e s t i n a l t r a c t . I n t h e dog 75 p e r c e n t of t h e r a d i o a c t i v i t y of an o r a l d o s e was e x c r e t e d i n t h e u r i n e and f e c e s i n 11 days and 86 p e r c e n t of a n i.v. d o s e i n 16 days. Most of t h e r a d i o a c t i v i t y was e x c r e t e d i n t h e u r i n e i n e i t h e r case. I n t h e rat 93 p e r c e n t of an o r a l d o s e was r e c o v e r e d from t h e excreta i n 5 days; e x c r e t i o n of t h e drug was almost e q u a l l y g i v i d e d between u r i n e and f e c e s . R a d i o a c t i v i t y of t h e H d r u g d i s appeared q u i t e slowly from t h e plasma of t h e dog, and t h e plasma h a l f - l i f e of r a d i o a c t i v i t y of a n i.v. d o s e w a s approximately 10 days. Plasma r a d i o a c t i v i t y y was composed of chlorpheniramine m e t a b o l i t e s as w e l l as unchanged drug, however, and chlorpheniramine i n c o n t r a s t d i s a p p e a r e d
65
rapidly from plasma having a half-life of 3 hrs. The drug was completely metabolized in the dog and rat and was excreted into the urine as relatively polar and non-polar products. N-dealkylation represented one pathway of metabolism and didesmethyl chlorpheniramine was the main N-dealkylated metabolite. Only 1 to 2 per cent of the dose was excreted unchanged.
6. Analytical Methods
61 .
Acid-Base Titration
Acid-base titrations were performed potentiometrically using non-aqueous solvents, with 0.1M HC104 as the titrant. A Beckman Model K automatic titrator was used to obtain the data which is tabulated in Table 1 . 0 TABLE 10 Neutralization Equivalents of Chlorpheniramine Maleate By Non-Aqueous Titration Neutralization Equivalent Theoretical: Experimental: Solvent 191 ( S ) * 189 (m) 182 ( 8 ) 192 (m) 195 415 (w) 385 (m) 195.4 390.8
Acetic Anhydride Acetonitrile m-Cresol chloroform Nitromethane Acetic Acid
394 (m) -
Symbol denoting relative magnitude of potentiometric inflection point:

s
m w
- sharp - medium
- weak
Titration as an assay method is cited in the USP (1) where chlorpheniramine maleate is titrated with perchloric acid in an acetic acid media. An aqueous titration assay (14) has also been proposed that involves the back-titration of excess H C 1 that was not taken up by the free base.
66
6.2
C o l o r i m e t r i c Methods
S e v e r a l c o l o r i m e t r i c p r o c e d u r e s f o r t h e a s s a y of c h l o r p h e n i r a m i n e maleate have been proposed. Among t h e e a r l i e s t (15, 16) was t h e f o r m a t i o n of t h e Reinecke s a l t (ammonium tetrathiocynatodiamminechromate 111) which was d i s s o l v e d i n a c e t o n e and the absorbance w a s read. The d i s a d v a n t a g e s of t h i s method a r e t h a t i t l a c k s s p e c i f i c i t y , and t h a t t h e c o l o r e d complex can be decomposed by t h e p r e s e n c e of water. S e v e r a l v a r i a t i o n s of t h e J o n e s and Brady r e a c t i o n ( 1 7 ) have been s t u d i e d (18-22). The g e n e r a l r e a c t i o n i n v o l v e s f o r m a t i o n of a n adduct through a t t a c k on t h e p y r i d i n e r i n g by cyanogen bromide, b u t f u r t h e r complexation w i t h a n i l i n e is p o s s i b l e w i t h many N-(2-pyridyl) a n t i h i s t a m i n e s . The rea c t i o n is a n a d a p t a t i o n of t h e Koenig r e a c t i o n (23, 24). Procedures f o r t h e complexation of chlorpheniramine maleate w i t h s u l f o n e p h t h a l e i n dyes such as bromcresol g r e e n (25) bromcresol p u r p l e (26) o r bromthymol b l u e (27) have been d e s c r i b e d . An automated p r o c e d u r e ( 2 8 ) t h a t compared e t h y l orange t o bromphenol b l u e as complexing dyes h a s been developed u s i n g t h e Technicon a u t o m a t i c a n a l y z e r . E t h y l orange w a s t h e p r e f e r r e d dye s i n c e a n orange r e s i d u e developed on t h e g l a s s j u n c t i o n s of t h e a p p a r a t u s when bromphenol b l u e was used. Another automated t e c h n i q u e (29) was developed t h a t used bromthymol b l u e and bromcresol p u r p l e t o s e l e c t i v e l y determine p o l a r and non-polar c l a s s e s of pharmac e u t i c a l amines i n a m i x t u r e of t h e two classes. A s i n g l e a p p a r a t u s was used w i t h two sets of dye r e a g e n t s , depending on t h e c l a s s of compound t o be analyzed. 6.3 S p e c t r o p h o t o m e t r i c and F l u o r o m e t r i c Methods
A n a l y t i c a l f l u o r e s c e n c e t e c h n i q u e s have been used f o r t h e a n a l y s i s of chlorpheniramine maleate (30-31). One t e c h n i q u e (31) d e s c r i b e s t h e complexation of r o s e b e n g a l dye. The second r e f e r e n c e d e s c r i b e s a comparison of two methods, o x i d a t i o n by hydrogen p e r o x i d e and a t t a c k by cyanogen bromide. Both procedures show low s e n s i t i v i t y , and t h e r o s e b e n g a l dye method shows s e n s i t i v i t y t o s o l v e n t i m p u r i t i e s (32-33). U l t r a v i o l e t s p e c t r o s c o p y h a s been used f o r t h e d e t e r m i n a t i o n of chlorpheniramine maleate i n i n j e c t a b l e 8 (34) and i n s y r u p (35). One of t h e drawbacks of t h e s e proc e d u r e s is t h a t they i n v o l v e extraction of t h e f r e e b a s e from t h e sample and subsequent comparison of t h e UV spectrum
67
to that of the maleate. According to Hamilton, et.al. (36) this gives rise to a 2-3% negative bias, since the maleate salt gives a higher absorbance than the free base alone. An interesting proposal for the use of orthogonal functions in deconvoluting U V spectra of chlorpheniramine maleate and its excipients is given by Abdine, et.al. (37).
6.4
Thin-Layer Chromatography (TLC)
Table 11 summarizes the TLC systems used, showing the type of adsorbant and the developing solvent used. The Rf values are also given. (R = Z solute / Z mobile phase), where f Z is equal to the distance travelled on the plate. TABLE 11
A Summary of Several Thin Layer Chromatography
(TLC) Systems for Chlorpheniramine Maleate Solvent System cyclohexane:benzene: 0.38 diethylamine (75: 15: 10) methano1
0.19
Adsorbant Silica gel dipped or prepared with 0.1M KOH Silica gel dipped or prepared with 0.1M KOH Silica gel dipped or prepared with 0.1M KOH Silica gel dipped or prepared with
0.1M WS04
Reference
38, 39
38
acetone
0.06
38
methanol
0.08
38, 39
Silica gel (0.5 mm. )Merck GF Silica gel Merck GF
ammonia:methanol (1.5: 100) benzene:methanol: conc. aq. ammonia ( 4 0 : 10: 1)
0.60
43
40
68
CHARLES G. ECKHART AND THOMAS MCCORKLE
TABLE 11 (con't.) Adsorbant Silica G e l G S o l v e n t System e t h y l acetate: methanol :conc. aq. ammonia (170:20: 10) e t h y l acetate: methanol : a ter : w conc. aq. ammonia (85:10: 3: 1) chloroform: methanol: conc. aq. ammonia
Rf
0.88
Reference 41a
0.25mm S i l i c a G e l F254 Merck
0.33
42b
0.25 mm Polygram S i l i c a G e l Sheets Machery , Nagel

&
0.67
42
co.
(90:10: 1)
ammonia (8.8. = 0.91) methanol (1.5:lOO) ethyl acetate: cyclohexane: p-dioxane: methanol: w a t e r :ammonium hydroxide
0.5 mm S i l i c a G e l GFZs4 Merck

0.25 mm S i l i c a G e l Glass Microf i b e r s h e e t I.T.L.C. Type SA Gelman 0.25 mm S i l i c a G e l Glass Microf i b e r s h e e t I.T.L.C. Type SA Gelman 0.25 mm S i l i c a G e l Glass Microf i b e r s h e e t I. T.L.C. Type SA Gelman 0.25 mm S i l i c a G e l G l a s s Microf i b e r s h e e t I. T.L.C. Type SA Gelman
0.44
43c 44
(50:SO: 10: 10: 1.5:0.5)
e t h y l acetate: cyclohexane: p-dioxane: methanol: water: ammonium hydroxide (50:SO: 10: 10:0.5: 1.5) ethyl acetate: cyclohexane: ammonium hydroxide: w methanol : a ter
0.58
44
0.70
44
(70:15:2:8:0.5)
e t h y l acetate: cyclohexane: methanol: ammonium hydroxide (70:15: 10:s)
0.91
44
69
Notes:
a . b .
c .
This reference gives TLC systems for 90 drug substances. This reference shows how drugs of abuse may be identified by way of their different R f well as their reaction to different spray as reagents. The author of this reference directly scrapes the compound of interest from the TLC plate and introduces it into a mass spectrometer.
6.5
Paper Chromatography (PC)
Table 12 summarizes paper chromatographic systems for chlorpheniramine maleate. TABLE 1 2 Paper Chromatographic Systems for Chlorpheniramine Maleate Support Whatman No. 1 paper dipped in 5% dihydrogen citrate Whatman No. 1 paper dipped in 10% tributyrin in acetone Whatman No. 1 paper dipped in 10% tributyrin in acetone Whatman No. 4 paper dipped in 0.1 N HC1 in acetone Whatman 3 M paper M impregnated with 2% (NH4) SO4 (pH 5 . 3 f Solvent System butano1:water: citric acid 870:130:4.8g. acetate buffer pH 4.58, 95OC
Rf
0.45
Reference 38
0.69
38
phospate buffer pH 7.4, 86OC
0.13
38
n-bu ta n o l saturated with 0.5 N HC1 isobutanol: acetic acid :water

(100: 10: 24)
0.35
40
0.52
67*
70
TABLE 12 ( o ' . cnt) Support Solvent System
Rf
0.87
Reference 6 7*
Whatman 3 M paper M isobutanol: impregnated with acetic acid 0.5M KNa2P04(pH 4 . 2 ) :water (100: 10: 24) Whatman No. 1 paper n-butanol: impregnated with 5% water:citric monosodium citrate acid (50:50:3g) Whatman No. 4 paper n-BuOH saturated with 1.0 N HC1
0.78
46
0.65
47
Note: This reference contains R data on 270 drug f substances, using two paper chromatographic systems. 6.6 Separation by Countercurrent Distribution
Onishi, et.al. (48) separated chlorpheniramine maleate quantitatively from several other drug substances using Craig-type counter-current distribution. Fifty transfers were needed to a 100% recovery of chlorpheniramine maleate from a mixture.
6.7
Gas-Liquid Chromatography (GLC)
The following table summarizes the recent GLC systems showing the liquid phase used, the column length and the oven temperature. TABLE 13 Gas-Liquid Chromatographic Systems for Chlorpheniramine Maleate Column SE-30 Lenpth 2 m 2 m l m Temperature 350C
3OO0C
Reference 49
Ap iezon
L/KOH
OV17
49
49
350C
71
TABLE 13 (con't.)
Column Carbowax 2OM/KOH Carbowax 20M

CDMS DEGSI KOH
Length
l m
Temperature
Reference 49 49 49 49 49 50 51 52 52 53 53 53
23OoC
23OoC 23OoC 190c 190c 25OoC 23OoC
l m 2 m l m
DEGS Chromosorb W
2m 2 m
3% OV-17 on Gas Chrom Q

1.2% Carbowax 20x4 on Gas Chrom Q
1.2X Carbowax 20M on chromosorb WHP
2 m
1.3 m
2 15OC
20oOc 1300- 15OoC (10 /min) 1300- 15OoC (10 /min) 18OoC 200
4% Carbowax 20M on Anakrom ABS 0.25% Uncon oil 50-HB-5100 on g l a s s beads
0.76 m 2 m
3% Carbowax 20M on Anakrom HBS

2% SE-30 on Chromosorb W (HP) a c i d washed and silanized
me t h y l v i n y l s i l i c o n e gum rubber
0.76 m
2.44 m
54 55
2 m
250'
56
2% Carbowax 20M on 60180 chromosorb W

2% SE-30 and 2% Carbowax 20M on 80/90 Anakrom
5 ft.
190c
45
4 ft.
185OC
57
ABS
72
TABLE 13 (con't.)
Column
5% s i l i c o n e gum rubber (SE-30) on Chromosorb G 80/100 a c i d washed amd HMDS t r e a t e d
Length
l m
Temperature 215OC
Reference 58
2.5% SE 30 on 80/100 Chromosorb G
2 ft.
1.8 m
20oOc 200- 25 OC
5ga
14; cyclohexanedimethanol auccinate and 10% SE-5S ( s i l i c o n e gum rubber) on 80/100 mesh Gas Chrom Q
0.07% SE-30 on 120/170 Glass Beads 0.08% PDEAS on 120/170 Glass Beads 1.07% XF-1150 on Chromosorb w-HMDS 100/120 mesh 1.08% Carbowax 20M on 100/120 Gas Chrom P
60
6 ft.
175OC
61b
6 ft.
175OC
61
6 ft.
175OC
61
6 ft.
175OC
61
3%OV-17 on 60/80 Gas Chrom Q

10% D e x s i l 300 on 80/100 Chromosorb W (HP)
6 ft.
195OC 220, 258OC
62
6 ft.
63
17% Dexsil 300 on 80/ 100 Chromosorb
8 ft.
222OC
63
W (HP)
1%Carbowax 20 M on Gas Chrom Q
3 ft.
205OC
64
73
TABLE 13 ( o ' . cnt) Column Length
Temverature
215OC
Reference
1.2% Carbowax 20 M 6 ft. on KOH treated Gas Chrom Q Notes: a .

b.
64
This reference gives gas chromatographic data on 600 drug substances. This system was judged by the authors to give the best peak shapes and shortest retention times for the 16 antihistamines studied on the four systems.
6.8
High Performance Partition and Ion-Exchange Liquid Chromatography (HPLC)
Several high performance liquid chromatographic procedures have been developed in recent years for the purpose of assaying chlorpheniramine maleate dosage forms and separating the antihistimine from excipients or other drugs. Table 14 summarizes the HPLC systems used along with some earlier open column ion-exchange work. TABLE 14 Liquid Chromatographic Systems for Chlorpheniramine Maleate Column Permaphase ODS 37-44 microns pellicular DuPont Mobile Phase 0.2 M KCI 0.2M boric acid 0.2 ml conc. ammonia in 390 ml water 610 ml ethanol pH 8.8
50-90% acetonitrile 50-10% 0.1% ammonium carbonate - pH 8.5-8.9
Reference
65
Corasil/C-l8 37-50 microns pellicular packing Waters Corasil/pheny1 37-50 microns pellicular packing Waters
66
6040% acetonitrile 40-20% 1.0% ammonium acetate pH 7.4-7.6
66
74
TABLE 14 ( c o n ' t - )
Column -Coras i l l p h e n y l 37-50 microns p e l l i c u l a r packing Waters CorsilIC-18 37-50 microns p e l l i c u l a r packing Waters Zipax SCX 25-37 microns p e l l i c u l a r packing DuPont UBondapak Phenyl ca. 10 microns porous packing Waters pBondapak Phenyl ca. 10 microns porous packing Waters UBondapak CN ca. 10 microns porous packing Waters Zorbax CN ca. 5 microns porous s p h e r i c a l packing Dupont 75% a c e t o n i t r i l e
25% 0.01 M KH PO
Mobile Phase 50-90% a c e t o n i t r i l e 50-10% 0.1% ammonium c a r b o n a t e pH 8.5-8.9 60-80% a c e t o n i t r i l e
Reference 66
66
40-20% 1.0% ammonium

a c e t a t e pH 7-4-7.6
0.01 M 0-04M (NH ) H P 0 4 w i t h
68
32%4dZoxane i n water
45% methanol 55% water 0.005 M h e p t a n e s u l f o n a t e (sodium . s a l t ) 1%acetic a c i d

33%methanol 67% 0-01M sodium a c e t a t e
69
69
(PH 4-01
69
70
75
P a r t i t i o n chromatography u s i n g Celite 545 (JohnsManville Corp.) and s e v e r a l 25 x 200 mm columns have been used f o r t h e s e l e c t i v e e l u t i o n of chlorpheniramine (71-73). The Hudanick (18) c o l o r i m e t r i c t e c h n i q u e is used.
An ion-exchange e l u t i o n technique has been developed and e v a l u a t e d by Smith (74, 75) who u s e s a s u l f onated p o l y s t y r e n e r e s i n column t o hold chlorpheniramine maleate p r i o r t o e l u t i o n . Chlorpheniramine was s u b s e q u e n t l y determined c o l o r i m e t r i c a l l y , by t h e method of Hudanick (18). A good review of t h e ion-exchange chromatography of n i t r o g e n - c o n t a i n i n g b a s e s is g i v e n by Jandera and Churacek (76); 171 r e f e r e n c e s a r e given.
6.9
Polarography
Chlorpheniramine Maleate e x h i b i t e d (77) a 2 - e l e c t r o n i r r e v e r s i b l e r e d u c t i o n t h a t r e q u i r e s two p r o t o n s be consumed. The procedure recommends t h a t t h e a s s a y b e run i n 0 . 2 M s u l u r i c ac d (pH 0.7) and a sample c o n c e n t r a t i o n from 10- t o 10 M. A d.c. polarogram is run from -0.4V t o -0.8V. The d i f f u s i o n c u r r e n t is r e a d a t -0.7V and t h e c o n c e n t r a t i o n is o b t a i n e d from a p l o t of t h e d i f f u s i o n c u r r e n t vs. v a r i o u s c o n c e n t r a t i o n s of s t a n d a r d s .
-3
76
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M.V. T a g g a r t Schering-Plough Corp. P e r s o n a l Communication (1968). F.J. Pruyn N o r t h e a s t e r n U. t o M.V. T a g g a r t Plough Corp. P e r s o n a l Communication (1968).
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N a t i o n a l Formulary (1970) 1 3 t h Ed. American Pharmaceut i c a l A s s o c i a t i o n , Washington, D.C. pg. 164. I b i d . pg. 242.
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S.J. Mule', M.L. B a s t o s , D. Jukofsky and E. S a f f e r J. Chromatogr. 63, 289 (1971).

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CHARLES G. ECKHART AND THOMAS McCORKLE C G Farmilo and K Genest, Progress in Chemnical .. . . Toxicology, Vol. 1, A Stolman, Ed., Academic Press, . . New York, N Y (1963) p 264.
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Doyle and J. Levine, JAOAC, 51(1), 191-9 (1968).
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E Jacobensen and K Hogberg, Anal. Chim Acta, 7 l , 157 . . (1974).
Acknowledgment The authors wish to thank Dr. M.D. Yudis and the Physical. Analytical Chemical Research and Development staff of the Schering Research Division or their assistance in the preparation of this monograph.
DIHYDROERGOTOXINE METHANESULFONATE
W . Dieter Schoenleber, Allen L . Jacobs, and Glenn A . Brewer, Jr.
81
Copyright @ 1978 by Academic Press, Inc. All rights of repraluction in any form reserved.
ISBN 0-12-260807-0
82
W.DETER SCHOENLEBER et 01.
Contents
Ergot A l k a l o i d s General I n t r o d u c t i o n
1. D e s c r i p t i o n
1.1 Name, D e f i n i t i o n 1 . 2 Formula, Molecular Weight 1.3 Appearance, Colour
2.
Physical Properties 2.1 Spectra 2.11 2.12 2.13 2.14 2.15 2.16 2.17 2.2 Infrared Ultraviolet Fluorescence Proton Nuclear Magnetic l 3 C Nuclear Magnetic M s Spectrum as Optical Rotation
P h y s i c a l P r o p e r t i e s of t h e S o l i d 2.21 2.22 2.23 2.24 2.25 2.26
klting C h a r a c t e r i s t i c s Differential Scanning C a l o r i m e t r y Thermogravimetry I o n i z a t i o n Constant P a r t i t i o n Coefficient Solubility
3.
4.
Production S t a b i l i t y and Degradation
4.1
4.2
Bulk S t a b i l i t y S t a b i l i t y i n Solution
5.
Drug Metabolism and Pharmacokinetics
83
6.
Methods o f Analysis 6.1 6.2 6.3 Elemental Analysis I d e n t i t y Tests Q u a n t i t a t i v e Methods 6.31 6.32 6.33 6.34 6.35 6.36 U l t r a v i o l e t Spectroscopy Fluorometry Colorimetry Gravime t r y Volumet r y Chromatography 6.361 6.362 6.363 6.364 6.37 6.38 6.4 6.5 6.6 Paper Thin Layer Gas Liquid High Performance Liquid
1% Nuclear Magnetic Resonance
Amino Acid Analysis
Counter Current D i s t r i b u t i o n Determination i n Body F l u i d s and T i s s u e s Determination i n Pharmaceutical Forms
7. Heferences
W.DIETER SCHOENLEBER et al.
F r n o t Alkaloids General IntrOdUCtiOQ Ergot is t h e s c l e r o t i a l stage of fungi o f t h e genus ClaviceDs t h a t grow p a r a s i t i c a l l y i n t h e p i s t i l s of d i f f e r e n t g r a s s e s . These s c l e r o t i a produce a l k a l o i d s o f t h e e r g o t group, t h e most important being Secale C o r a , e r g o t of r y e , which i s produced by t h e s t r a i n ClaviceDs gurr>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 t h e8 a~ ~ l i c a t i o n o f a s p e c i a l l 5 pp
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
85
1.
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)-C 2H 5
-CH( CH3 1
-CH2-CH(CH 3 ) 2
86
W. DIETER SCHOENLEBER et al.
Chemical Abstracts Registry Numbers:
base
1 032-41-0 1
mesilate:
52 655-92-2
Aver age Molecular W i g h ts : e

base
: 584.40
mesilate : 680.51
Average Formulas :
base
'
:
'32. 67'42. 67N505 '33.67'45.67

N 5 O8 S
1.22
m o n e n t s ~ Dihvdroeraotoxine of
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
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:
29 261-94-7
:
base
563.70 659 81
mesilate :
Formula : base
: C
H 0 N 31 41 5 5
mesilate:
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.
Q$?
-CH,
HI,
c-
-N
'I ;
.CH,
88
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:
base
: 611.74
mesilate:
Formula : base
707.85 H 0N 35 41 5 5
: C
mesilate:
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 .
C -
II
1;1
HN
'I
*CH,S020H
R = -CH -CH(CH
2 3 2
)
or -CH(CH3)-CH -CH 2 3
89
Molecular k e i g h t :
base
: 577.73
mesilate : 67 3.85
Formula :
base
: C C H 0 N 32 43 5 5
mesilate:
33 47 8 5
0 N S
1.2231 pihvdro -a-ernokrvD tine mesilate
D i hydro*-ergokr ypt i n e m s i l a te is t h e e s a l t of 9,10Ci-dihydr0-12~-hydroxy-2'-(lm thy1 e t h y l 1-5 'a- ( 2-me thylpr o p y l ) -ergoe taman-3' ,6' , 1 8 - t r i o n e .
H
N-.
/ \
CH3 CH3
90
Chemical Abstracts Registry Numbers:
base
: 25 447-66-9
mesilate:
29 261-93-6
: 577.73
Molecular Weight:
base
mesilate:
673.85 32 43 5 5 33 47 8 5
H H
Formula :
base
: C
0 N
mesilate:
0 N S
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:

base
: 1 9 467-62-0
mesilate: Molecular Weight: base
not y e t assigned
: 577.73
mesilate:
673.85
1.3
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 9,10-dihydrogroup, o n l y and d i h y d r o 4 reported.l5
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
Figure 1 .
Infrared Spectrum of Dihydroergotoxine mesilate K r p e l l e t , instrument: Perkin Elmer 257 E
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 K r pellet, instrument: Perkin Elmer 257 B
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 .
KBr p e l l e t , instrument:
Infrared Spectrum of Dihydroa-ergokryptine mesilate Perkin E l m e r 257
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 95% e t h a n o l
0.01
max, n m
Sh Sh
1%
c m
94.8 101 85.6 95.1
101
275
281 291
H C 1 i n 95% e t h a n o l
275
281
0.01
0.01
4 luH40ti i n 95% e t h a n o l fi t a r t a r i c acid
Sh
291 277
281
Sh
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
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.
i n (CD ) S O , i n s t r u m e n t :
PMR Spectrum of Dihydroergokryptine mesilate Bruker HX-90 3 2
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
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 dimethylH sulphoxide using a Bruker HX-90 M z 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 .
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 ~a l k a l o i d~ n ~ 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
90
80
70
60
Y1
/
55
31
50
YO
30
20
10
223
3Y3
500
1 " " ' . " 1 ' * '
'
-'.
' '
"
' 1
M/E
100
200
300
YO0
500
600
700
Figure 1 4 .
Low Resolution M s Spectrum o f Dihydroergotoxine mesilate as Instrument: CBC 21-110 B
r-
0
(D
9) c)
I n
0 0
0 0 f .
u S I c
0 0
P)
0 0 N
J H
o c
o
o
4
o
-
o 0 cc
0
(0
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4
-6-l
al
(d
0)
ln
* I
ii
0 0
9
cr
0 0
M
0 0
\
a l c,
d .rl
rd
r n
s
Q)
4 c,
3
M
L
0 h
Q)
5 P
G-4
E v ) o c J H
I d
Q)
c,
0
O
. +
0
n
0
-
0
)
0
.
100
90 80
70
/
553
60
50
10
30
20 10
100
200
300
1100
500
600
700
Figure 1 8 .
Low Resolution Mass Spectrum of Dihydrou-ergokryptine m e s i l a t e Instrument: CEC 21-110 B
100
90
80
70
60
/
223
187
50
YO
30
20
209
I.
10
97112
138
100
200
300
Lloo
500
600
700
Figure 19.
Low Resolution Mass Spectrum of Dihydro-R-ergokryptine mesilate Instrument: CEC 21-110 B
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
I
I
R
I1
I11
I1
I11
ComDonent dihydroergocornine dihydroergocristine d ihyd r o d - e rgokrypt i n e dihydro-B -ergokry p t i n e
m/ e
196 244
210
m/e
19 5 243
209
m/ e
72 1 08
86
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
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 ~ 50 % e t h a n o l , c = l % n ~
d i hydroergo t o x i n e
m silate 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:
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
5cp
I.
I.
I.
fa?
.. ..
.I
%!w
, ,
,. ,,w .. , ,
u
Figure 20.
D i f f e r e n t i a l Scanning Calorimetric and Thermogravimetric Curves of Dihydroergotoxine mesilate
118
W. DIETER SCHOENLEBER et
01.
s v
a.
.." .. .. rn
.L
so
"
1 .
.. .. ..
aarr
.."I
2 w
Figure 21.
D i f f e r e n t i a l Scanning Calorimetric and Thermogravimetric Curves of Dihydroergocornine mesilate
119
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
120
Figure 23.
Differential Scanning Calorimetric and Thermogravimetric Curves of Dihydroergokryptine mesilate
121
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
122
W.DIETER SMOENLEBER et al.
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
123
2.24
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 p 1 . 2 and chloroform, on H H t h e one hand, and water p 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
equilibrium d i s t r i b u t i o n ratio
0.12 : 1 : 22.9
water pH 1.2/CHC13 water pH 1.2/n-octanol 2.26

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 i a
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
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 .
125
0
N
R = -CHO
= -H
N-formyl-B-seco-dihydroergocristine
B-seco-dihydroergocristine
Figure 2 6 .
Autoxidation Products of Dihydroergocristine
126
W,DETER SCHOENLEBER et 01.
4.22
Acid and Base Ca t a l v z e d Isomer
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 *9 l e a d i n g t o more acidic e p i 6o 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 m was g 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
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
C
H
Element calculated $ % found 6.12
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-
N
12.43 12.66 11.46
11.40
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
% % % % %
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

8.0
12.13
12.12
12.13 12.0
128
W. DIETER SCHOENLEBER et oJ.
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 . 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 .
11.
1 1 l'anIlrk1s_te9t-- Ergot a l k a l o i d s , includ1. i 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
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 6.221 SDeci f i c I d e n t i f i c a t i o n ''ests I 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 .
130
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 ws a maximum between 250 and 320 nm. The u t i l i z a 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
131
6.35
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.
132
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. 2.
Paper impregnated w i t h formamide, e l u t i o n with formamide-saturated benzene-chloroform (1:1)
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.
F.
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) 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.
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
11.
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.
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
134
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
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.
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
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
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. 235O
I n j e c t i o n p o r t temperature: Column temperature: 6.364
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
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
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.
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)
11.
111.
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.
138
W.DIETER SMOENLEBER et al.
INXCT
Figure 28.
Four-component HPLC Separation of dihydroergotoxine mesilate. Conditions indicated i n section 6 . 3 6 4
139
6.37
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 m si l a t e e (500 m i n 1.5 m l dimethylsulfoxide-d6). g C-5' S i g n a l s i n b l a c k .
140
W.DIETER SCHOENLEBER et 01.
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 p 4.5, dihydroergocornine could be separated from H 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
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 For l gg
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
142
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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.
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
150
DONALD D. HONG
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 Drug Metabolism

4.1 4.2
4.
Biotransformation Products Pharmacokinetics
5. Stability
6.
Methods o f Analysis
6.1 6.2 6.3
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
7. Acknowledgments
8. References
151
1.
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 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
MW 489.06
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 ) .
152
DONALD D. HONG
210
Tunpnrun OC
FIQURE 1:
DSC THERMOORAM OF DIPHENOXYLATL HYDROCHLORIDE (Cm RER CWSA\
153
2.3
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. I n f r a r e d Band Assignments Ass ignment
Cs t r e t c h 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
Band (cm-l) 2800-3 140 2320 1770-2000 1730 1605,1450-1500 1025 855 2.6 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 ,
L) I
m xm
I II
36
I43
155
x5-
-.
d o
Figure 3. Mass spectrum (electron impact) of diphenoxylate (After brim and Heykants, r e f . 3)
156
DONALD D. HONG
Table 2.
NMR Spectral Assignments

E D
CN
C
H1 C
Protons A B C D
E
PPM Chemical S h i f t 1.19 4.21 7.4 2.7 2.5 - 3.8
Multiplicity Triplet Quartet M 1 t i pl e t u Broad Sing1 e t Broad Bands
2.8
M1 t i ng Range e
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 H20 (pH 6.8)
= 473
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
7.0
I . . . . ~ . . . . I . . . . I . . . . I . . . . I . . . . I . . . . I . . . . I . . . . ~ . . . . l . . . . ~ . . . . l , . , . , . . . , I . . ,
60 .
5.0
PPM( 6 )
4 . 0
20
C l O w L 4. W A U Q f T l C U n
lluLyyyf
mEcTnu oc D l M u u u v u l E
WDI*a(LOnIDT
158
DONALD D. HONG
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 H 1 C Solubility, mg/ml

) 50 ) 50
Sol vent
Methanol Chloroform
Reference
5
Acetone
Benzene Ethanol Ethyl Acetate
6 3 3
2.3 0.8 (0.1 (0.1
Water
Heptane Ethyl Ether Simulated gastric f l u i d
(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 NO aH (0.1 0.1N NH40H <o. 1 *The f r e e base i n CHC 2.13
3 has a s o l u b i l i t y of -420 mg/m
U1 t r a v i o l e t Spectrum
The UV spectrum of diphenoxylate hydrochloride i n methanol i s shown i n Figure 5. The spectrum i s an aromatic multiplet having maxima a t 264 nm (a = 1.06), 258 nm (a = 1.32) and 252 nm (a = 1.09).
159
FIQURE 6 ULTRAVIOLET SPECTRUM OF DIPHENOXYLATE HYDROCHLORIDE IN METHANOL
160
DONALD D. HONG
3.
Synthesis
W. Van Bever, e t a1 ( 6 ) have reviewed t h e chemistry o f s y n t h e t i c a n t i d i a r r h e a l agents as p e r t a i n e d t o diphenoxyl ate, d i fenoxin, 1operamide and re1ated compounds. S p e c i f i c a l l y diphenoxylate h y d r o c h l o r i d e can be synthesized ( 7 ) as presented i n Figure 6. Diethanolamine i s condensed w i t h p-toluenesulfonyl c h l o r i d e i n aqueous sodium carbonate s o l u t i o n and NYN-bis-(2-hydroxyethyl)-p-toluenesulfonamide (I)s i s o l a t e d . The comi pound i s reacted w i t h t h i o n y l c h l o r i d e t o form N,N-bis(2-chloroethyl)-p-toluenesulfonamide (11). The amide i n t u r n i s t r e a t e d w i t h sodium h y d r i d e and benzyl cyanide i n to1 uene t o g i v e N- (p-to1 uenesul fonyl)-4-cyano-4p h e n y l p i p e r i d i n e (111). The p i p e r i d i n e i s then hydrol y z e d i n aqueous s u l f u r i c a c i d t o form 4-carboxy-4phenyl p i p e r i d i n e t o s y l a t e s a l t ( I V ) which c r y s t a l l i z e s upon d i l u t i n g and c o o l i n g t h e m i x t u r e w i t h water. The t o s y l a t e i s i s o l a t e d and t r e a t e d w i t h e t h y l alcohol t o form 4-carbethoxy-4-phenyl p i p e r i d i n e (V). The r e a c t i o n m i x t u r e i s made a l k a l i n e and t h e p i p e r i d i n e i s separated from t h e sodium t o s y l a t e by s o l v e n t e x t r a c t i o n . The s o l v e n t i s then s t r i p p e d . D i phenyl acetoni tri 1e , ethylene bromide and sodi um hydride a r e reacted i n toluene t o g i v e 2,2-diphenyl-4b r o m o b u t y r o n i t r i l e ( V I ) and c r y s t a l 1ized. A m i x t u r e c o n t a i n i n g (V) and ( V I ) along w i t h sodium carbonate, potassium i o d i d e and methyl i s o b u t y l ketone i s reacted. The m i x t u r e i s then t r e a t e d w i t h HC1/ toluene t o g i v e t h e f i n a l product, 2,2-diphenyl-4(4carbethoxy-4-phenyl ) p i p e r i d i n e l b u t y r o n i t r i l e hydroc h l o r i d e ( V I I ) which i s r e c r y s t a l l i z e d from e t h y l alcohol.
4. Drug Metabolism 4.1 B i o t r a n s f o r m a t i o n Products
Karim, et a1 (8, 9 ) , u s i n g 14C-labelled diphenoxyl a t e h y d r o c h l o r n e , r e p o r t e d t h a t t h e major p o r t i o n o f t h e administered l a b e l i n man was found i n t h e feces, followed by u r i n e and plasma. A f t e r o r a l a d m i n i s t r a t i o n approximately 10% o f t h e dose was found i n t h e u r i n e and about 40% i n t h e feces. Even a f t e r intravenous admini s t r a t i o n about 70% o f t h e dose was recovered i n t h e feces. I n a l l t h r e e r o u t e s o f e l i m i n a t i o n t h e major metabol i c product o f d i phenoxyl a t e hydrochloride was diphenoxyl i c acid. Hydroxydiphenoxyl i c a c i d and
161
Figure 6. Synthesis o f Diphenoxylate Hydrochloride
,CH2CH20H
HN
\
CH2CH20H
+@
sop
Na2C03
)
so2c1 ,CH2CH20H S02N CH2CH20H

3
H2S04
6
H
Ts
EtOH
(j
0
COOEt
COOH
(IV)
BrCH2CH2Br
0
I
NaH
___$
BC CH C-CN rH
0, / H
C
0
(VI)
2) HC1
0 CN
CN I 0 -C- CH2CH2
1
W I )
HC1
162
DONALD D. HONG
3-cyano-3,3-di phenyl propionic a c i d have a1 so been i d e n t i f i e d (10). I n t h e u r i n e both f r e e and conjugated metabol i t e s were i s o l a t e d . Some unchanged drug was recovered i n the plasma along w i t h a compound t h a t was more p o l a r than the acid. A summary o f t h e metabolites o f diphenoxylate hydroc h l o r i d e , t h e i r mass spectral data and the species from which they were found i s shown i n Figure 7. I n man t h e i d e n t i t y o f t h e Tf$tabolite from feces f o l l o w i n g o r a l a d m i n i s t r a t i o n o f C-labelled diphenoxyl a t e HC1 i s dependent on the dosage form o f the drug. Karim, e t a1 (11) found t h a t when the drug was administ e r e d i n ethanol i c s o l u t i o n , t h e major metabol i t e s were diphenoxylic a c i d and compounds more p o l a r than the acid. When the drug was given as a t a b l e t o r as a caps u l e more than 50% o f the metabolite was as t h e unchanged drug. These studies suggest t h a t t h e drug i s b e t t e r absorbed i n ethanol s o l u t i o n than as a s o l i d dosage form. The reason f o r t h i s i s t h a t diphenoxylate i s n o t very s o l u b l e i n water and t h e d i s s o l u t i o n r a t e o f t h e drug i n the tablet/capsule would be a rate-1 i m i t i n g step.
4.2
Pharmacoki n e t i cs
I n a study i n v o l v i n g three normal male subjects, Karl$, e t a1 (9) reported the pharmacokinetic analysis of C-diphenoxylate hydrochloride using a one-compartment open model showed a r a p i d absorption ( t b = 19.7 minutes) and a r a p i d e l i m i n a t i o n ( t b = 2.5 hdurs). The plasma h a l f - l i f e (tb= 4.4 hours) o f t h e major metabolite, diphenoxyl i c acid, $as higher than the diphenoxylate. I n t h e same study these i n v e s t i g a t o r s showed t h a t w i t h i n 4 days approximately 49% o f t h e administrated l a b e l was recovered i n t h e feces. The f e c a l radioa c t i v i t y i n the 0 t o 24 hour sample was very low. Most o f the a c t i v i t y was recorded i n t h e 24 t o 48 and 48 t o 72 hour samples. Approximately 14% o f the administered l a b e l was recovered i n u r i n e w i t h i n 3 days, w i t h about 11% being found i n the f i r s t 24 hours.
5.
Stability
D i phenoxyl a t e hydrochloride , under o r d i n a r y condi t i o n s o f a i r and l i g h t , i s an extremely s t a b l e compound. I t s major degradation product i s diphenoxylic acid. To study the degradation p a t t e r n o f d i phenoxyl a t e hydrochloride (l), t h e compound was subjected t o the f o l l o w i n g conditions, e x t r a c t e d w i t h chloroform and compared w i t h known degradation products by TLC. The
Figure 7: Mass Spectral Data o f Diphenoxylate and Its Metabolites Compound

@-C-CH2CH2-N CN
M+
452
Molecular Formul a
30H3ZNZ02
Base Peak
246
Species Man Man Dog Rat Man Dog Rat
X
! :H
@
~ - Y C H ~ C H ~ - N
6
CN
XZH5
498
3 lH 34N204
2 46
HO- @ - C - C H ~ C H ~ - N
@
\Q-EIICH~CH~-N% -
32 5 ; H
COOC2H5 486
468
30H3ZN23
246
C30H34N204
246
@ ) - C -CCNH 2 C H 2 - N 3 @ )
COOH
424
COOH
CN HO- @ C - C H 2 C H 2 - N 3 b
6
CN
28H28N202
218
Man Dog Rat Man Dog Rat
16H13N02
@- C-CH~COOH
Man
164
DONALD D. HONG
Table 4. Degradation Products of D i phenoxylate Hydrochloride
Diphenoxylate: R1=CN; R2=C2H5 Condition Degradation Product Diphenoxylic Acid (0.5%) R1=CN; R2=H Diphenoxylic Acid (major) Amide (1%) and Acid (0.2%) o f D i phenoxyl ic Aci d Amide: Rl=CONH2i R2fH Acid: R1=COOH , R2-H No degradation found No degradation found
1. 24 h r r e f l u x i n 1M H2S04/ MeOH ( 1 : l )
2. 24 h r r e f l u x i n 1M NaOH/ MeOH 1: 1)
3. 24 h r r e f l u x i n H20/MeOH (1: 1) f o r 4 weeks 4. 105' 5. 24 h r r e f l u x i n 1M H2S04/ Peroxide
4-Phenyl-4-piperi d i necarboxyl ic a c i d (B) 2,2-Diphenylbutyrolactone (C) 4-Phenylpiperid-3-ene (D)
3-Cyano-3,3-diphenylpropanol (A)
CN
I
Jb-C- CH2CH2- N
0
CN I 0-C-CH2CH20H
I
D i phenoxylate
3
a
COOC2H5
I
H N 3 ( (A)
YOH
(6)
165
r e s u l t s a r e summarized i n Table 4. 6. Methods o f Analysis 6.1 Elemental Analysis Table 5 shows t h e r e s u l t s o f an elemental a n a l y s i s o f diphenoxylate h y d r o c h l o r i d e ( L o t RER 4-103-A) (12). Table 5. Elemental Analysis o f Diphenoxylate Hydrochloride
E l emen t C H c1 N 0
6.2
Theory 73.68 6.80 7.25 5.73 6.54
Found 73.99 6.79 7.20 5.73 6.33
Microchemi c a l I d e n t i f i c a t i o n E.G.C. Clarke (13) has summarized t h e i d e n t i f i c a t i o n t e s t s and Rf values o f diphenoxylate along w i t h various o t h e r drugs. They a r e c l a s s i f i e d under t y p e o f p r e c i p i t a t i o n w i t h 27 a l k a l o i d a l reagents, type o f c r y s t a l s , and s e n s i t i v i t y w i t h s e l e c t e d reagents. 6.3 Chromatographic Analysis 6.31 Thin Layer Chromatography. Most o f t h e development work on diphenoxylate h y d r o c h l o r i d e using TLC evolved around one b a s i c system. The systems a r e summarized i n Table 6. 6.32 Gas L i q u i d Chromatography. A GLC procedure has been developed t o measure d i p h e n o x y l i c a c i d , t h e major m e t a b o l i t e o f diphenoxylate h y d r o c h l o r i d e i n u r i n e (16). The procedure i n v o l v e s base h y d r o l y s i s t o l i b e r a t e d i p h e n o x y l i c a c i d from compounds conjugated i n u r i n e and then removal o f i n t e r f e r i n g substances by a1 umi na c o l umn chromatography. Q u a n t it a t i o n was c a r r i e d o u t using p-chlorodiphenoxylic a c i d as an i n t e r n a l standard. To increase v o l a t i l i t y , i t was necessary t o methylate d i p h e n o x y l i c a c i d and i t s d e r i v a t i v e s w i t h diazomethane. S e n s i t i v i t y i s about 200 ng/ml. To f u r t h e r increase t h e s e n s i t i v i t y o f t h e method, the authors (17) used G U M S t o d e t e c t t h e d i p h e n o x y l i c a c i d i n plasma using a deuterium l a b e l l e d i n t e r n a l standard and m o n i t o r i n g t h e base peak o f the m e t a b o l i t e .
Table 6. Sol vent Ch1or0 f o r m :Methano1 : Acetic Acid (92:3:5) Ch1oroform:Methanol: Acetic Acid (92:3:5) Ch1oroform:Methanol : Acetic Acid (94:5:1) n-Butanol :Acetic Acid: Water (10:2:8) Adsorbent Adsorbent
TLC Systems Detection

I
Dosage Form Chemical Tablets Chemical Chemi c a l
R -f
Ref 1
Note a b
0.7
A
A A
I1
I11
0.63
0.8
0.73
14 15
5
IV,V
Detection S i l i c a Gel 60 F254 (250 u)
A.
I.
11. 111. IV. V.
Potassium iodobismuthate I o d i n e vapors Zonal scraping ( r a d i o a c t i v e counting) Dragendorf f t-Butylhypochlorite/KI starch
Note:
a.
b.
This system i s capable o f r e s o l v i n g diphenoxylate hydrochloride from i t s possible degradation products and i m p u r i t i e s . These i n c l u d e b r o m o b u t y r o n i t r i l e (R 0.90), tetraphenyldicyanobutane ( R 0.82) , amide o f diphenoxylate ( R 0.42), dipffenoxyl i c a c i d ( R 0.38), amide o f dibhenoxylic a c i d (Rf 0.23), dipethagine (Rf 0.13) and p i p e r i d f n e e s t e r (R 0.03). Active e x t r a c t e d w i f h chloroform.
167
A second GLC method has been developed t h a t i s capable of measuring diphenoxylate hydrochloride chemical d i r e c t l y (18). Cholesteryl propionate i s used as the internal standard. T h e method i s described b r i e f l y as follows:
Instrumental Conditions Instrument: Gas chromatograph w i t h FID detector Column: 1%Dexsil 300 GC on 80-100 Chromosorb Q , 2 f t x 4 mm i d , glass Carrier Gas: Nitrogen, 62 ml/min. (Hydrogen and a i r flow rates should be optimized f o r maximum s e n s i t i v i t y and s t a b i l i ty Temperatures : Col umn 260:C Injector 2900C Detector 290 C Retention time: Cholesteryl propionate 6.2 min. D i phenoxylate hydrochloride 8.0 m i n . 6.4 Colorimetric Analysis
Diphenoxylate hydrochloride can be analyzed by the dye-sal t procedure (19). T h i s involves extraction of the compound from a basic aqueous solution w i t h chloroform. A p o r t i o n of the organic extract i s transferred t o a centrifuge tube t o which bromocresol green and an aqueous phase buffered t o pH 5.6 are added. The res u l t i n g yellow colored complex i n the organic phase is back extracted t o a basic aqueous phase. The absorbance of the resulting blue aqueous phase i s then read.
6.5
Spectrophotometric (UV) Analysis
Di phenoxylate hydrochloride may be determined spectrophotometrically i n 0.01N HC1 i n methanol. The UV absorption maximum i s a t about 258 nm.
6.6
Titrimetric Analysis
6.61 Non-Aqueous Titration. Being an amine, diphenoxylate hydrochloride can be t i t r a t e d w i t h acetous 0.1N perchloric acid. The t i t r a t i o n may be performed manually using crystal violet as indicator o r determined potentiometrically. Mercuric a c e t a t e C T.S. i s added t o overcome the a c i d i t y of the H 1 which causes an early end-point. A blank determination
168
DONALD D. HONG
should be c a r r i e d o u t and any c o r r e c t i o n s made. Each m l o f 0.1N p e r c h l o r i c a c i d i s equivalent t o 48.91 mg o f diphenoxylate hydrochloride (20). 6.62 Semi-microtitrimetry Using Sodium Lauryl Sulfate. F. P e l l e r i n e t a1 (21,22) have described t h e t i t r a t i o n o f diverse nitrogenous -compounds i n c l u d i n g diphenoxylate w i t h sodium l a u r y l s u l f a t e i n a 2-phase system o f water/chloroform. The method depends upon t h e r e a c t i o n o f t h e quaternary ammonium cation, i n 1:l r a t i o , w i t h the sodium l a u r y l s u l f a t e which i s an anionic detergent. The s a l t t h a t i s formed i s i n s o l u b l e i n water b u t s o l u b l e i n chloroform and methyl y e l l o w i s used as t h e i n d i c a t o r .
7.
Acknowledgments The author wishes t o express h i s a p p r e c i a t i o n t o Dr. G. Anthony f o r reviewing t h e manuscript; t o Ms. P. Finnegan f o r the I R and NMR data; t o D r . A. Karim f o r the i n f o r m a t i o n on t h e mass spectrum and f o r the s e c t i o n on drug metabolism; t o M r . G. Lee f o r the data on GC; t o Corporate Information Service f o r the 1it e r a t u r e search; t o Ms. M. LLorente for technical assistance; and t o Ms. A. Fenton f o r t y p i n g the monograph.
169
8. References
1.
2.
I . Mead, G . D. Searle & Co. ( H i g h Wycombe), personal
3.
4. 5. 6.
P. Finnegan, Searle Laboratories , personal commun i c a t i on. A. Karim and J. Heykants i n Synthetic Antidiarrheal Drugs , (Modern Pharmacology -Toxicology Series , Vol. 7), W . Van Bever and H . La1 , eds. , Marcel Dekker, e Inc., Nw York, June 1976, p. 148, 152. USP XIX, p. 157. M. LLorente, Searle Laboratories , personal communication. W. Van Bever, R. Stokbroeckx, J . Vandenberk and M. Wouters i n Synthetic Antidiarrheal Drugs,
D.
commun i c a t i on.
8.
7. J . Witt, Searle Laboratories, personal communication. A..Karim and J . Heykants i n Synthetic Antidiarrheal Drugs, p. 1 3 2 f f . 9. A. Karim, R. E. Ranney, K. L. Evensen and M. L .
Clark, Clin. Pharma. Therap., 2, 407 (1972). J . Heykants, J . Brugmans and H. Verhaegen, Arzneimittel Forsch , 22 , 529 (1972). A. Karim and J . Heykazs i n Synthetic Antidiarrheal p. 151. Drugs, E. Ziel inski , Searle Laboratories , personal communi cation. E. G. C . Clarke, Bull. Narcotics, U.N., Dept. Social 13, 57, Affairs, - No. 4, 17-20 (1961); C . A . , - 6025b
23.
m,
10.
11.
12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
x,
(1962)
V. T. Rice, G. D. Searle & Co. ( H i g h Wycombe), personal communication. A. Karim and J . Heykants i n Synthetic Antidiarrheal Drugs, Ibid, p. 135. R. D. A. Brownsill, R. F. Palmer and M. J . Tidd, Chromatographi a, 9, 127 (1976). G. C. Ford, N. J . Haskins, R. F. Palmer, M. J . Tidd and P. H . Buckley, Biomed. Mass Spec., 3, 45 (1976). G. D. Lee, Searle Laboratories, personal communication. M. Baier and M. Aingorn, Searle Laboratories, personal communication. USP XIX, p. 158. F. P e l l e r i n , J . A. Gautier and D. Demay, Ann. Pharm. -Franc., 20, 97 (1962); C.A., 57, 15243 c (1962). -i d-4 Ib , 9 2 5 , (1964); C . A . , -, 12979 f (1965). 62
DROPERIDOL
Cusimir A . Junicki and Roger K . Ciipin
171
Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-260807-0
172
CASIMIR A. JANICKI AND ROGER K. GILPIN
TABLE OF CONTENTS
1.
Description
1.2
1.1
Name, Formula, Molecular Weight Appearance, Color, Odor Infrared Spectrum Nuclear Magnetic Resonance Spectrum Ultraviolet Spectrum Mass Spectrum Melting Range Differential Scanning Calorimetry Solubility PKa
2.
Physical Properties
2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8
3. 4. 5. 6.
Synthesis Stability-Degradation Drug Metabolic Products and Pharmacokinetics Methods of Analysis

6.2 6.3 6.4 6.5 6.6
6.1
Elemental Analysis Nonaqueous Titrimetric Analysis Colorimetric Analysis Spectrophotometric Analysis Thin Layer Chromatographic Analysis Gas Chromatographic Analysis
7.
8.
Determination in Biological Fluids Determination in Pharmaceuticals References
9.
DROPERIDOL
173
1. Description
1.1 Name, Formula, Molecular Weight
Droperidol is l-El-[4-(4-Fluorophenyl)-4oxobutyl]-1,2,3,6-tetrahydro-4-pyridinyl~-l,3-dihydro-2H-benzimidazol-2-one. The parenteral product is known as INAPSINEB injection. It is also a component of INNOVARB and THALAMONALB injection.
1.2 powder.
2.
Appearance, Color, Odor White to light tan, odorless crystalline
Physical Properties 2.1 Infrared Spectrum
The infrared spectrum is presented in Figure 1. The spectrum was obtained from a potassium bromide dispersion using a Perkin-Elmer 521 grating IR spectrophotometer. A list of the assignments made for some of the characteristic bands is given in Table I (1)
Table 1 IR Spectral Assignment for Droperidol

3060
-CH-
2950 2895
-CH2-
MICRONS
2.5 100
w
3
I
4
I
5
I
6
I
7
I
8
I
9
I
1 0
I
12
I
15
I
20
I
3040
I I
80
6040-
*
I -
20
I I
1
0 4000
3500
3000
2500
2000
1800
1600 CM'l
1400
1200 1000
800
600
400
200
Figure 1.
The Infrared Spectrum of Droperidol, KBr Dispersion. Instrument; Perkin-Elmer 521
DROPERIDOL
175
1700 1685 1595 1500 1485 1235 828 745 2.2
C=O ring C=O ring aromatic substitution p-substitution p-substitution o-substitution
Nuclear Magnetic Resonance Spectrum
The 90 mHZ spectrum of droperidol presented in Figure 2 was obtained in DMSO d6 at an approximate concentration of 98 mg/ml with tetramethylsilane as the internal standard. Spectral assignments are listed in Table I1 (1).
Table I1
NMR Spectral Assignments for Droperidol Characteristic of Chemical Shift (ppm) Multiplicity Proton 8 .O 7.25 6.8-7.2
5.9
multiplet mu1tiplet mu 1tip1et mu1tip l et mu1tiplet
2.4-3.1
alb Cld ( rm 1 nl P k elf {glh
Figure 2.
The NMR Spectrum of D r o p e r i d o l i n CDC13 w i t h T S a s M I n t e r n a l Standard. I n s t r u m e n t ; Perkin-Elmer R-32
DROPERIDOL
177
3.25 1.8-2.2 10.5

2.3
mu1tiplet multiplet singlet
i r
j
Ultraviolet Spectrum
The ultraviolet absorption spectrum of dxoperidol obtained from a 9:1, 0.1 M hydrochloric acid: methanol solution is shown in Figure 3 . Droperidol exhibits two maxima, one about 245 nm (E: = 15,600) due to the butyrophenone part of the molecule and the second about 280 nm ( E = 7500) due to the benzimidazolinone part of the molecule. The W spectrum of droperidol obtained from a 9:1, 0.1 M Citric acid: methanol solution is very similar to the spectrum in Figure 3 . 2.4 Mass Spectrum
The mass spectrum obtained on a Finnigan model 10151, EI mass spectrometer is presented in Figure 4. The fragmentation patterns are discussed in a paper by Blessington ( 3 ) . 2.5 Melting Range*
A dried droperidol sample melts between 144O and 148OC, NF X I V Class 1 procedure. The existence of polymorphic forms plus a hydrated form is partially demonstrated by a melting point determination. The most stable polymorphic form melts between 146.5O - 148.5OC, with the least stable 142.5OC. The hydrate melts form between 139.8O at approximately 115OC followed by recrystallization and then remelting at approximately 14OOC (4).
2.6
A
Differential Scanning Calorimetry (DSC)
The DSC of droperidol is shown in Figure 5. melting endotherm is observed at 421K (148OC) using a temperature program of 10/minute. The DSC
*Data given as uncorrected values appearing in listed references.
1.0
0.8
0 2
0.6
2 m
4
0.4
0.2
200
225
250
WAVELENGTH
275
300
325
I nm I
Figure 3.
The W Absorption Spectrum of Droperidol in 9:l 0 1 Hydrochloric Acid:Methanol. .a
4J
u a
c
4J
0 k
rl
a,
180
,430
420
410
TEMPERATURE I"KI
Figure 5.
The,DSC of Droperidol. Instrument; Perkin-Elmer DSC-2. 0 Heating Rate: 1 ' per min. ATM.: Nitrogen
DROPERIDOL
181
shows different metling peaks for the other polymorphic forms. The least stable polymorph has one peak about 416OK and the hydrate has two peaks at about 394OK and 418OK (4).
2.7
Solubility
The approximate solubilities obtained at room temperature are listed in Table I11 (1). Table I11 Approximate Solubilities of Droperidol Solvent Solubility (g/lOO ml) 1.3 1.5 40 17 0.24 0.34 0.15 0.41 11 . 1.2
2.4
Acetic acid (0.1 M) Acetone Benzene Chloroform Dimethylformamide Ether Ethanol Hydrochloric Acid (0.1 M) Lactic Acid (0.1 M) Methanol 2-Propanol Propylene Glycol Tartaric Acid (0.1 Water
2.8
0.55
2.3
<0.001
&
The PKa of droperidol is 7.64 determined titrimetrically (1)
3.
Synthesis
Droperidol is synthesized by refluxing a mixture of 4-Chloro-l-(4-fluorophenyl)-l-butanone, sodium carbonate, 1-[1,2,3,6-tetrahydro-4-pyridinyl]-1,3-dihydro-2H-benzimidazol-2-one, and potassium iodide in a solvent of 4-methyl-2-pentanone. The droperidol is recrystallized from 1:l ethyl acetate: diisopropyl ether or alcohol ( 5 ) .
182
""8"
\ /
Fn i - C H 2 - C H 2 - C H z -
4.
Stability
Degradation
Although droperidol is sensitive to heat and light (6), it is stable for up to 5 years when stored in the dark at room temperature (1). Droperidol is slightly hygroscopic (1). When droperidol was refluxed overnight in 1M hydrochloric acid, it was found to undergo compiete hydrolysis (2). The hydrolysis is given in Figure 6. The major products are 1-[4-(4-fluorophenyl)-4-oxobutyll-4-piperidinone (I) and 1,3dihydro-2H-benzinidazolin-2-one (11). A solution of droperidol in monoglyme was refluxed overnight in 2M sodium hydroxide, and droperidol was recovered-unchanged. Droperidol solutions between p H 2 . 5 and 4 . 0 in Type I glass ampuls (tartaric acid of lactic acid) are very stable and have shown no loss of potency after a 5 year period at room temperature. These solutions were stable even after 7 months at 6OoC
DROPERI DOL
II
HN
YNH 0
Figure 6 .
The Acid H y d r o l y s i s of D r o p e r i d o l .
184
and even when exposed to sunlight. Solutions of droperidol containing fentanyl citrate in a 50:l ratio, with or without parabens, have been found to be stable for up to three years at room temperature when stored in Type I glass. In addition, solutions of droperidol containing fentanyl citrate in a 50:l ratio have been found to be compatible for up to 3 months with 1 mg/ml atropine sulfate ( 6 ) .
5 .
Drug Metabolic Products and Pharmacokinetics
The butyrophenones used clinically are neuroleptics in the sense of Delay and Deniker ( 7 ) . Droperidol, an important member of the butyrophenones series of compounds, is characterized by its short onset of activity ( 8 , 9 ) and its rapid metabolism.* The metabolism of droperidol has been studied by a number of investigators in several species. These studies are catagorized below: Data presented Species Rat, Dog & Man Sheep, Goat & Rat Mice, Dog & Cat Rat Dog & Rat Rat Man Man References 10 10 11 12 13
8
9
A = Metabolic B = Pharmacokinetic C = Distribution D = Other (pharmacological, toxicological, etc.)
*Although it is rapidly metabolized, droperidol has a relatively long duration of activity.
DROPERIDOL
185
The most commonly reported pathway for metabolism of droperidol is oxidative N-dealkylation giving rise to 4-fluoro-y-oxobenzenebutanoic acid and 1-(1,2,3,6-tetrahydro-4-pyridiny1)-1,3-dihydro2H-benzimidazol-2-one, followed by rapid metabolism of 4-fluoro-oxobenzenebutanoic acid to 4-fluorobenzeneacetic acid and its glycine conjugate, N-(4fluorobenzoyl) glycine (7,8,11,12), the most important of the urinary metabolites (7,121. These proposed metabolic pathways are summarized in Figures 7 and 8 .
6.
Methods of Analysis 6.1 Elemental Analvsis
E1ement
Carbon Hydrogen Nitrogen Oxygen Fluorine 6.2
Theory 69.64 5.84 11.08 8.43 5.01
Nonaqueous Titrimetric Analysis
The nonaqueous titration procedure of analysis is the official method listed in the National Formulary XIV (14). An accurately weighed 300 mg sample of droperidol is dissolved in 30 to 40 ml of glacial acetic acid. After addition of anaphtholbenzeine indicator, the solution is titrated with a previously standardized 0.1M HC104 solution to an end point. 6.3 Colorimetric Analysis
Haemers and Van Den Bossche (15) have described a general procedure for the quantitative determination of several butyrophenones, one of which was droperidol. Their procedure involves the reaction of butyrophenones with 3,5-dinitrobenzoic acid in an alkaline medium, resulting in the formation of a red complex. Using this procedure, the amount of droperidol in an injectable (2.5 mg/ml) form was determined.
DROPERIDOL
4 f Iuoro-y -ox0 benzenebut anoic acid
I
Figure 7.
1- 11,2.3, 6-tetrahydro-
4-pyridinyl l -1 .3-dihydro5-step degradation 2H-benzirnidazol-2-one
4-fluorobenzeneacetic
acid
Pathway in the Metabolic Disposition of Droperidol.
DROPERIDOL
187
8
Fe!-CH2-CH2-C-OH
4- f iuoro-y -oxobenzenobutrnoic acid
4-f iuorobenzoneacetic acid
Figure 8.
Pathway in the Metabolic Disposition of Droperidol.
188
The method was found to be useful for both aqueous and alcoholic solution.
6.4
Spectrophotometric Analysis
Spectrophotometric determination of droperidol by a non-specific procedure involving the extraction of the compound into 0.1M hydrochloric acid and reading it at 246 nm has been reported (16). The effect of the amount of ether and extraction time on the purified drug was reported.
A specific assay method for determining droperidol by itself or in the presence of fentanyl citrate (2) involves a double extraction procedure prior to recording the W spectrum. The initial droperidol solution is diluted with water to contain 0.15 mg/ml droperidol. Fifteen ml of the diluted droperidol solution is added to 10 ml of a pH 6 phosphate buffer, 10 ml of a 1% sodium bisulfite solution, and extracted with 15 ml of chloroform. Five ml of the chloroform layer are re-extracted into 50 ml of 0.1M citric acid and the W spectrum recorded from 235 to 300 nm.
6.5 Solvent System
Thin Layer Chromatographic Analysis Absorbent Silica Silica Silica Silica Silica Silica Silica Silica Silica Detection Technique Rf
3, 4 3, 4 3, 4 31 4
Ref 17 17 17 17 1 1 6 18
2
Gela Gela Gela Gela Gel 60Fb Gel Gb Gel GC Gel GC Gel Q4Fd
1, 2
3 2
.80
.75 .44 .45 .46 .78

.58 .25 .59
11 2 4
Solvent Systems A B Acetone (100) Benzene: acetone: petroleum ether: ammonium hydroxide (10:10:10:2)
DROPERIDOL
189
F
G
H
I
Acetone: petroleum ether (7:3) n-Butanol: isopropanol: acetic acid: water (3:3:2:4) Ethyl acetate: chloroform: methanol: 0.1M sodium acetate buffer, pH=4.7 (54:23:18:5) Chloroform: methanol: ammonium hydroxide (91:8 : 1) Methanol: 0.1M sodium acetate buffer, pH=4.7 (95:5) Chloroform: methanol (95:s) Ethyl acetate: methanol: ammonium hydroxide ( 8 5 : 10:3 ) Detection Techniques
1 2 3 4
Ultraviolet light Iodine staining Dragendorff Reagent Iodoplatinate Reagent Adsorbents
b
C
Unspecified Merck Analtech Quantum Industries

6.6
Gas Chromatographic Analysis
Droperidol has been reported to be unsuited for gas chromatographic analysis due to oncolumn decomposition (17). 7. Determination in Biological Fluids
Presently, there are no published chemical methods for determining droperidol in blood or urine. However, a radiochemical assay method has been published (13). Tritium-labeled droperidol is synthesized according to the method of Soudijn - - (19). Total radioactivity in urine or et a1 plasma samples containing tritium-labeled droperido1 is determined by counting directly in a 20% ethanol-toluene scintillator or a 20% Beckman BBS3-toluene scintillator, respectively. The intact droperidol from blood or urine is obtained by
190
extracting an alkaline solution of the urine or plasma with freshly seeded chloroform ( 2 5 pg of unlabeled droperidol per ml of chloroform). A portion of the chloroform layer is transferred to a scintillator vial, evaporated to dryness, and the radioactivity determined using a 20% ethanol-toluene scintillator. The remainder of the chloroform layer is evaporated to dryness, and the residue redissolved in a small volume of chloroform. The entire chloroform sample is spotted onto a Silica G plate and developed with 91:8:1 chloroform: methanol: ammonium hydroxide. The droperidol spot (Rf between 0.6-0.7) is scraped and placed into a scintillation vial. Twenty percent ethanol-toluene scintillator is added and the radioactivity determined. The counting efficiency is not affected by the variation in the amounts of silica gel present in the counting vials.
8.
Determination in Pharmaceuticals
Chekova proposed a method for solutions containing parabens, which involved the dilution of the dosage form with 0.1M hydrochloric acid and removing the parabens with-an ether extraction. The W spectrum of the hydrochloric acid solution is recorded. Haemers and Van den Bossche reacted droperidol with 3,5-dinitrobenzene to form a colored complex, which is used for the quantitative determination of droperidol in dosage forms. Both these methods suffer from the fact that they are not specific for intact droperidol. The specific ultraviolet assay method for droperidol, described in section 6.4, is the preferred assay. The assay is modified slightly when the dosage form contains parabens. After the droperido1 is extracted into citric acid, the citric acid is washed with diethyl ether. This removes any W interferences in the citric acid due to the parabens (6).
DROPERIDOL
191
References 1.
2.
Demoen, Paul, Janssen Pharmaceutica, Beerse, Belgium, unpublished data. Janicki, C.A., Brenner, R.J.,Schwartz, 57, B.E., J. Pharm. Sci., 1968, - 451. Blessington, B., Org. Mass Spectrom, 1971, 5, 1113.
c
3. 4.
5.
Van Rompay, J., Janssen Pharmaceutica, Beerse, Belgium, unpublished data. Janssen, P.A.J., and Gardocki, J.F.,U.S. Patent 3,141,823, (1964). Janicki, Casimir,A., McNeil Laboratories, unpublished data. Janssen, P.A.J., Proc. Eur. SOC. Study 9, Drug Tox., 1967, - 107. DiMascio, A., Butyrophenones in Psychiatry, A. DiMascio and R . I . Shader Eds., Raven Press, New York, N . Y . , 1972, 11-23. Janssen, P.A.J., in "Psychopharmacological Agents", Vol.11, M. Gordon, Ed., Academic Press, New York, N.Y., 1967, 199-248. in "The Application of Shephard, N.W., Neuroleptanalgesia in Anesthetic and Other Practice", N.W. Shephard, Ed., Pergamon Press, London, G.B., 1965, 1-27. Soudijn, W., Van Wijngaarden, I., Janssen, P.A.J., Int. Anesthesiol. Clin., 12, 1974, - 145. Soudijn, W., Van Wijngaarden, I., 1, Allewijn, F., Eur. Pharmacol., 1967, 47.
6.
7.
8.
9.
10.
11.
12.
192
13.
Cressman, W.A., Plostnieks, J., Johnson, P.C., Anesthesiology, 1973, 3 8 , 363.
14. National Formulary XIV, Mack Printing Co., Easton, Pa., 1975, 245. 15. 16. Haemers, A . , Van Den Bossche, W., J. 21, Pharm. Pharmacol., 1969, - 245. Chekova, L.P., Shemyakin, F.M., Molnar, L.N., Farmatsiya (Moscow) 1972, 21, - 41. Quaglio, M . P . , Cavicchi, G . S . , Murglia, M., Boll. Chin. Farm., 1971, 110, - 385. Gilpin, R.K., McNeil Laboratories, unpublished data. Soudijn, W., Van Wijngaarden, I., J. Labelled Compds., 1968, - 159. 4,
17.
18. 19.
EPINEPHRINE
Dale H . Szulczewski und Wen-hui Hong
193
Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN O-I2-260807-0
194
DALE H. SZULCZEWSKI AND WEN-HA1 HONG
CONTENTS
1. General Information 1.1 Nomenclature 1 1 Chemical Names .1 1.12 Generic Name 1.13 Trade Names 1.2 Formula 1.21 Empirical 1.22 Structural and Stereochemical 1.3 Molecular Weight 1.31 Free Base 1.32 Bitartrate 1.4 Elemental Composition (base) 1.5 Description 1 6 Forms of Epinephrine Recognized in Official Compendia . 2 . Physical Properties 2.1 Associated with the Solid State 2.11 Crystallinity 2.12 X-ray Diffraction 2.13 Melting Point 2.2 Solubility 2.3 Spectral 2.31 Ultraviolet 2.32 Infrared 2.33 Mass 2.34 Optical Rotation and Dispersion 2.35 NMR 2.4 Distribution 3 . Ionization in Aqueous Solution 4. Biochemical Considerations 5. Synthesis 6 . Stability and Behavioral Chemistry 6.1 Solid State 6.2 Aqueous Solutions 6.21 Racemization 6.22 Reaction with Bisulfite 6.23 Oxidation 6.24 Miscellaneous Reactions 6.25 Dosage Form Stability
EPINEPHRINE
195
7.
8.
9.
Analytical Methods 7.1 Fluorometric 7.2 Colorimetric 7.3 Polarimetric 7.4 Titrimetric 7.5 Chromatographic 7.51 Thin-layer and Paper 7.52 Gas Chromatography 7.53 Liquid C o l u m n Chromatography 7.6 Spectrophotometric Acknowledgments References
196
DALE H. SZULCZEWSKI AND WEN-HAI HONG
1.
General Information 1.1 Nomenclature 1.11
Chemical Names l-~-3,4-dihydroxyphenyl-~-methylarninoethanol; 1-1-(3,4-dihydroxyphenyl)-2-methylaminoethanol; 3,4-dihydroxy (1-hydroxy-2-methylaminoethy1)benzene; l-methylaminoethanolcatechol; 3,4-dihydroxy-a-(methylaminomethyl)benzyl alcohol; (R) (-) -3,4-Dihydroxy-a1 (methylamino)methyllbenzyl alcohol; 4-[l-Hydroxy-2-(methylamino)ethyl]-1,2-benzendiol. 1.12 Generic Name Epinephrine
Trade Names Many trade names (1) exist for this drug. Some of the more comonly used trade names in the United States are Adrenalin (Parke, Davis), Suprarenalin (Armour Co.) , and Suprarenin (Sterling Drug, Inc.). 1.2 Formula 1.21 1.22 Empirical CgH13N03; (OH)2C6H3CHOHCH2NHCH3 Structural and Stereochemical
1.13
&
1.3
Molecular Weight 1.31 1.32 Free Base 183.21 Bitartrate (1:l salt) 333.30 (C,H1,N03'C,H606)
1.4
Elemental Composition (base) C 59%, H 7.1%, N 7.7%, 0 26.2%
EPINEPHRINE
197
Description The USP XIX (2) describes epinephrine as "white to nearly white, odorless, microcrystalline powder or granules, gradually darkening on exposure to light and air. With acids, it forms salts that are readily soluble in water, and the base may be recovered by the addition of ammonia water or alkali carbonates. Its solutions are alkaline to litmus."
1.5
Forms of Epinephrine Recognized in Official Compendia. The USP XIX ( 3 ) contains e:pinephrine as the free base, the bitartrate salt, and the following formulations:
16 .
Formulation Epinephrine Epinephrine Injection Epinephrine Nasal Solution Sterile Epinephrine Oil Susp. Epinephrine Bitartrate Ophthalmic Solution Epinephrine Bitartrate for Ophthalmic Solution
2.
Category Bronchodilator Adrenergic Adrenergic Bronchodilator Adrenergic Adrenergic
Physical Properties
2.1
Associated with the Solid State 2.11
Crystallinity Although dpinephrine was the first hormone to be obtained in crystalline farm, crystallographic information with regard to crystalline structure is not available. Winchell ( 4 ) describes epinephrine as colorless or light brown crystals or powder which darkens on exposure to air and quotes refractive indices as determined by Keenan ( 5 ) as being N1 = 1.555, N2 = 1.733 X-ray Diffraction Powder X-ray diffraction data ( 6 ) for epinephrine are given in Table 1.
2.12
Melting Point Kirk-Othmer ( 7 ) report the melting point of epinephrine as 211 212OC (215OC when heated rapidly).
2.13
TABLE I.
Powder X-ray Diffraction Data
d
4-0227 5.13 7.84 4.23 7.84
C9H1.p03
I/I
4-0232
CH -NH-CH2-CH
100
70
70
70
Adrenaline
O H
Rad., CuKa D i a . 1 9 cm 1/11, V i s u a l Ref., See below

~ ~~
X = 1,5418 c u t o f f , 9.3
F i l t e r ,Ni
con.
d corr. abs.? N o
d2
7.84 5.13 4.86 4.58 4.23 3.91 3.75 3.60 3.38 3.27 3.13 2.99 2.85 2.69 2.57 2.51 2.42 *2.29 2 .1 8 2.12
1/11
hkl
B
2.05 2.00 1.95 1.91 1.67 1.82 1.77 1.70
1/11
hkl
70 y b 50 70
40
sys
a0
a
Ref.
b0
CO
S.G. A
S a 2v
nu6 C Y Sign mp-216d. C o l o r White
Ref.
Taken for t h e I n s t i t u t e of P h y s i c s a t U n i v e r s i t y College, Cardiff. Average t e m p e r a t u r e - 17.4OC. M o l . Wt., 183.2
40 5 70 20 70 10 30 10 30 30
40 5 40 10 5 10 20
1.55
lob
10 5 10
1.49 1.41
20
:P
3%
*Probably mre than one line.
EPINEPHRINE
199
2.2
Solubility Temperature 2O0C 25OC Solubility 1 part in 3000 1 part in 2000 insoluble insoluble Reference
7
Solvent Water Ethano1 Ethyl Ether Chloroform
---
i
2 2
The solubility of epinephrine in water is dependent on pH. It has minimal solubility at pH 9.4 and its solubility increases as pH deviates from this value due to formation of water soluble species bearing net positive or negative charges. (See section on ionization in aqueous solution.) Aqueous alkaline systems are seldom employed because of stability considerations whereas at pH's of pharmaceutical or therapeutic importance the intrinsic solubility of epinephrine in water is adequate so as to not cause complications. 2.3 Spectral 2.31
Ultraviolet Epinephrine is reported (8,g) to have the f o l lowing ultraviolet absorption characteristics in aqueous systems:
(net charge-1) 295 (4400)
7
243 (7000)
Sh285 (2500) 280(2750)
e 224 (6000)
(1
232(4900)
3
cation (net charge ' ) l t
Isobestic points
A, n m
281 (2750) 2.32
f
267(1500)
Infrared - The infrared spectrum of epinepharine is given in Fig. 1 . 2.33 Mass The mass spectrum of epinephrine(l0) as obtained on a mass spectrometer with an electron beam energy of 70 eV using the direct heated inlet system is shown in Figure 2 As discussed by the authors, phenylalkylamines . such as epinephrine, noradrenalin, and isoprenaline which posess 2 phenolic hydroxyl groups give an observable
Figure 1
Infrared Spectrum of Epinephrine, USP.Synthetic,KBr Pellet
FiWe 2
Mass spectrum of epinephrine
2 02
DALE H. SZULCZEWSKI AND WEN-HAIHONG
molecular ion. The 100% peak (m/e = 44) arises from B cleavage with production of the CH2NHCH3+ ion. For a thorough discussion on the topic of the mass spectra of phenylalkylmine derivatives, the original article should be consulted. Further information pertinent to the identification of microquantities of epinephrine can be obtained by mass spectroscopy of the 1-dimethylamino-5-naphthalenesulfonyl (Dansyl) derivative (11). Characteristics of the mass spectrum obtained for the tridansyl derivative of epinephrine are shown in Table 11. Optical Rotation and Dispersion The USP XIX ( 2 ) uses optical rotation as a criterion of acceptability and specifies it as follows: "The optical rotation of e'pinephrineas determined at 25OC on a2% solution in 0.5N HC1 using sodium D light should not be less than - 5 O O nor more than -53.5O." The optical rotatory dispersion curve of epinephrine was determined by Lyle (12) and also by Manna and Ghislandi (13) who extended rotatory dispersion studies to a series of epinephrine derivatives. The dispersion spectrum is a plain curve over the region 700 to approximately 325 n m. Dispersion follows the Drude equation (14,15). 2.34
with the constant A having a value of -32.3 (absolute ethanol, 0.5N with respect to HC1 as solvent and temperature of 22OC). The dispersion curve obtained under these conditions is shown as Figure 3 . These dispersion studies indicate that levorotatory epinephrine (that which is used medicinally and is contained in the USP XIX [2])has the D configuration. The nuclear magnetic resonance spectrum of epinephrine is shown in Figure 4 (16). The peak assignments as per Jardetsky follow: Chemical shifts (in cps) of 0.1M e.pinephrine in D20 with respect to benzene as external standard ring -24.4 CH 94.2 CH2 195.1 CH3 224.5 2.35
NMEt
EPINEPHRINE
203
3OO 350 400 450 SOU 550 600 650 7 0 m y

Figure 3
- Optical rotary dispersion
curve of epinephrine
Table I1 Molecular weight and relative abundance of peaks for the dansyl derivative of Epinephrine. The relative abundance of the peaks was established by comparison to m/e 170 (or 171) = 100%. Instrument CH5, Varian Mat, electron beam energy 70 eV.
Empirical Formula
No. of Danyl Groups
M+
882
Mw
Relative Abundance of Peaks
Other Characteristic Peaks Ew (% Relative Abundance)
Temperature of Inlet system

OC
M+ 234 250 263 ----3
277 30 868(0.5); 864(0.5); 648 (2) ; 630 (0.5) ; 605(3); 441.5(0.3); 398 (4); 384 (2); 371 (2); 350 (2). 340
40
Figure 4
- NMR
spectrum of 0.3MJ-epinephrine in D20.
Temperature 27'
pH 5 6 .
206
The NMR spectrum of epinephrine and other biogenic phenylalkamines was studied by Reisch et a1 (17) and found to be of value for their characterization. Distribution Epinephrine is not easily extracted from aqueous solution. Changes in pH do not significantly benefit extraction since epinephrine free base, like other catecholamines, is quite hydrophillic (18). Polar water immiscible solvents (n-butanol, etc.) have been used to extrace epinephrine from an aqueous phase.
2.4
3.
Ionization in Aqueous Solution
Ionization characteristics of epinephrine and related compounds have been the subject of numerous studies (19-26). Among these investigations that of Martin's ( 2 4 ) is most definitive since complications which arise from the presence of substituted ammonium and phenolic moieties having comparable acidities are considered. The first deprotdnation reaction of fully protonated epinephrine involves proton loss from both ammonium and phenolic groups and generates a mixture of zwitterionic and neutral molecules. This reaction is associated with a macroscopic pKa of 8.69 (25OC, )I = 0.1). The second deprotonation, associated with proton loss from the zwitterionic and neutral forms previously generated results in formation of a single specie bearing a net charge of -1 and is associated with a macroscopic pKa of 9.90 (conditions as before). The ionization sequence for epinephrine on a molecular level with corresponding microconstants follows:
EPINEPHRINE
207
The ratio of tautomeric molecules having no net charge, i.e., zwitterion/nonzwitterion, is approximately 4 at 25OC. This then means that the first deprotonation primarily involves proton loss from one of the phenolic hydroxyls, whereas the second is mainly loss from the amino group present in the zwitterion. The distribution of various species present in an aqueous solution of epinephrine as a function of pH is given in Figure 5. The ionization constants used in calculating this distribution were taken from Martin's work ( 2 4 ) as tabulated in Table 111. The isoelectric point of epinephrine is 9.4.
4. Biochemical Considerations
Epinephrine is not active orally due to destruction in the gastrointestinal tract and conjugation and oxidation which occur in the liver ( 2 7 ) . Normally the drug is administered parenterally, although preparations suitable for inhalation are available to provide largely for a local effect in the lungs. Epinephrine is metabolized as in the following scheme ( 2 7 ) .
3.4-dehvdroxvmandelic acid
I - l13;mqy
cm
D -OH T
MA0
NHCH3 matanephrine
6
/
3-methoxy-4-hydroxy rnandelic acid
,.'d=mO
#'
H3",",Q-~:$H
H3cm::y H NHCH3 3-methoxy-4-hydroxypheny lg lycol metanephr ine sulfate or glucuronide
4'
conjugate
* Catechol-0-methyltransferase
Wonoarnine oxidase
208
1.00
0.80
+ 1 churge
-1 charge
0.60
C 0
*d L1
I 4 CI,
$ 0 40
W
no net charge
10
0.20
0.00
11
12
13
I4
I Pkl
Figure 5
Pa2
PH
- Species distribution diagram for epinephrine
T a b l e I11
PK1 Epinephrine 8.79 8.66 8.71
PK2
Pkl 8.88 8.72 8.81
pk2 9.51 9.57 9.39
R = kl/k2
4.3
pk21
pkl
T OC
P
- 0
10.10
9.95 9.90
0.50 0.32 0.41
25
7.1 3.8
25 20
0.10 0.10
210
5.
Synthesis
A crystalline compound was first isolated from suprarenal medulla extracts by Takamine ( 2 8 ) and Aldrich (29) which showed the same pharmacological action as extracts fromthis gland. This compound was later characterized as 1+-3,4dihydroxyphenyl-$-methyl-aminoethanol primarily through the work of Pauly (30)and Jowett (31). Resolution of racemic epinephrine as first synthesized by Stolz and Dakin (32,331 was effected by Flacher (34) and it was found that the synthetic l-isomer was identical with that isolated from the natural source.
Many catechol sympathomimetic amiiies are synthesized on a commercial scale by essentially the same process. This process ( 3 5 ) can be divided into 4 stages, i.e.,
1 production of chloracetocatechol, . 2. conversion of chloracetocatechol to adrenalone hydrchloride by reaction with methylamine, 3 . purification of the adrenalone as produced in 2 and reduction of the salt to racemic epinephrine, 4 resolution o f the resulting racemate to yield the . desired lev0 isomer which is generally accomplished by crystallization from methanol as the tartrate.
A flow diagram for this process (35) follows:

C 1CH2COOH
POC 13
HO
CC 14
0
H0QE-CH2C1 HO
catecho1
chloroacetocatechol
IKO
racemic epinephrine catal y s t
HO
CH3*2
E-CH~NHCH~
adrenalone
EPINEPHRINE
211
6. Stability and Behavioral Chemistry
Solid State Epinephrine is a relatively unstable compound and is susceptible to decomposition in the solid state. Special precautions must be observed during the synthesis of the free base, and material so obtained should be stored under regulated conditions, i.e., N2 atmosphere, amber bottles, etc. (35). In regard to the stability of the solid base, moisture content appears to be a significant factor since thoroughly dried material is more stable than that which has a significant moisture content. Aqueous Solutions 6.21 Racemization Epinephrine racemizes at an appreciable rate even at near ambient temperature. Data obtained by Kisbye and Schroeter (36,37,38) indicate that racemization of epinephrine is acid catalyzed. This is an important consideration in dosage form design since an acidic environment is desirable in regard to prevention of discoloration. Estimates of the rate of racemization at near ambient temperatures are given in Table IV (38). At pH 3.5 or less, racemization occurs by an SN type reaction. This acid catalyzed reaction is thought to occur by rapid protonation of the secondary alcoholic oxygen atom followed by rate determining elimination of this hydroxyl as water. Attack of a second molecule of water on the shielded incipient carbonium ion then results in production of the optical isomer. Since the same reaction occurs on the 2-isomer, a racemic mixture results. The &isomer of epinephrine has little pharmacological activity as compared to the 1isomer. Reaction with Bisulfite Closely allied with epinephrine's racemization reaction is its reaction with anions of sulfurous acid. Bisulfite or metabisulfite salts are frequently used as antioxidants in aqueous formulations of epinephrine where they function to retard color formation. Initial studies (39,413) demonstrated that the potency of solutions of epinephrine could be lost without discoloration or true racemization being the cause. Further, it was shown that at pH 4.7 or higher, epinephrine was lost more rapidly in the presence of bisulfite than in its absence. 6.22 6.2
61 .
TABLE I V
P r e d i c t e d R a t e s a n d T i m e s of M a i n t e n a n c e of a Minimum 95 a n d 90 P e r C e n t O p t i c a l A c t i vi t y
Predicted Rate -1 C o n s t a n t , Min.
T i m e of Minimum 95% O p t i c a i A c t i vi t y
~
T i m of Minimum 90% O p t i c a l A c t i vi t yb
~~~ ~
pH 2.5, 25O pH 2.5, 35O pH 3.0, 2 5 ' pH 3.0, 35O pH 3.5, 2 5 ' pH 3.5, 35O
2.63 9.34 2.45 1.91
x x
x x
3 1.9 X 105 min. (ca. 45 mo.) 54 .

104 5 7.3 X 10 5 2.0 x 10 6 2.6 X 10 5 7.5 x 10
4.0 X 105 min.
(ca. 9 mo.)
min. (ca. 1 mo.)

min. (ca. 17 mo.)
6.92 X
min. (ca. 5 mo.)

min.
(ca. 60 mo.)
6.77 X
min. (ca. 17 mo.)
1.1 X 105 min. (ca. 2 mo.) 1.5 X 106 min. (ca. 35 mo.) 4.3 X 105 min. (ca. 10 mo.) 5.5 X 106 min. (ca. 120 m.) 1 6 X 106 min. (ca. 36 mo.) .
EPINEPHRINE
213
Subsequent investigation (41) demonstrated that anions of sulfurous acid actually react with epinephrine to produce 1(3,4-dihydroxyphenyl)-2-methylaminoethane sulfonic acid according to the following equation: e Q 703~ @ R-CHOHCH2NH2CH3 + SO3 = R-CH-CH NH CH t O H ' 2 2 3
H d
The kinetic behavior of epinephrine's reaction with bisulfite is explicable on the basis of the following reaction 4) scheme ( 1 .
LEp
+ Ep' +d I-Ep
102,
Pi
so?I
EpBi (racemic product)
Rate measurements were consistent with and indicative of the following expression which describes the rate of loss of epinephrine from bisulfite containing aqueous solution. (41)
The preceding studies showed that since sulfite is so much more reactive than water, epinephrine does not racemize when bisulfite is present. In the presence of bisulfite, optical and physiological activity is lost through formation of the sulfonate. Oxidation Since epinephrine is an o-diphenol containing a hydroxyl group in the position, it is a strong reducing agent (42). As such, it is easily oxidized by such oxidizing agents as molecular oxygen, iodine, potassium ferricyanide, potassium persulfate, and manganese dioxide. Oxidation of epinephrine is thought to occur through the transient formation of epinephrine quinone with formation, under proper conditions, of adrenochrone (43,44,45,46). Oxidation of epinephrine by molecular oxygen can also result in formation of a brownish insoluble material of indefinite structure. An investigation of the oxidation of epinephrine by molecular oxygen (47) indicated that the reaction involved is extremely complex. Data obtained suggests that oxidation
6.23
214
of epinephrine in aqueous solution can occur in the absence of heavy metal ions and likely involves free radical sequences. Oxidative discoloration of epinephrine solutions occurs more rapidly as pH is increased. Miscellaneous Reactions In addition to the preceding reactions which are relevant to dosage form design, manufacture, and storage, epinephrine can undergo a variety of other reactions which have relevance to other areas. Reaction products of these reactions and conditions are summarized in table 5.
6.24
Dosage Form Stability Available information indicates that oxidation of epinephrine in aqueous dosage forms is an extremely important consideration in regard to dosage form design, manufacture, and storage. This is the situation because oxidation produces both inactivation and discoloration. Discolored solutions of epinephrine are considered to be unusable ( 2 regardless of potency.
6.25
Published reports (51,52,53) indicate that the kind of container (ampoule vs. multiple dose vial) significantly influenced shelf life. Epinephrine solutions formulated with metabisulfite contained in ampoules did not show significant formation of 1-(3,4-dihydroxyphenyl)-2-methylaminoethane sulfonic acid even after 7 years storage at 15OC. Racemization of the epinephrine contained in these ampoules was likewise insignificant. Results of stability studies of epinephrine ampoules using various analytical probes are given in Table VI ( 5 2 ) .
7.
Analytical Methods
7.1
Fluorometric Measurement of native or induced fluorescence provides a useful means of assaying epinephrine. While the scope of this analytical approach is limited, in that stereoselectivity is not attainable, sensitivity and a fair degree of analytical specificity are provided. Naturally occurring catecholamines such as epinephrine, norepinephrine, dopa, and dopamine possess native fluorescence (54,55). All of these compounds are maximally excited at 285 nm and fluoresce at 325 nm. Since several catecholamines which occur in living tissue possess essentially the same fluorescent characteristics, native fluorescence is not
EPINEPHRINE
Tabla V Products ?armed fruo Epinepheine
215
Reaction Product
Reaction Conditions
Reference
wild conditiona of pH and t-rature. Praaance of HS03-or SO3-.
41
1-(3,4-dihydroxyphenyl)Z-luthylamlnoethMe SUltonic acid
Strong acid and high telqperature. 3,4-dihydroryphenyl acetaldahyd. t r t h y l

.rlW
40
Uild conditions, oxidation
(I2, 0 etc)
4 3- 46
Uild temperaturn Conditions. oxidation in basic environment Adrenolutine &isomer of epinephrine Variable pH, reaction occurs mare rapidly at l o w pH, relatively pild conditions
43-46
36,37,39
Elevated temperature, strong acid
49
Diadrenalina ether
Elevated tomperatwe, strong acid
49
Adndne
NH2CHJ
Elevated temperature, strong baae
50
HO
3,4-dihydroxyacetophenone, m t h y l amine
Complex iona containing epinephrine and boron
Uild conditions. Presence of anioni of boric acid
106,101
216
TA8L.E V I The S t a b i l i t y of Adrenaline I n j e c t i o n s a (pH a d j u s t e d t o 3 - 0 by a d d i t i o n of h y d r o c h l o r i c a c i d ) Adrenaline recovery i n per c e n t of o r i g i n a l content
Heat treatment
Unheated
Stordge period (mnthsl

0
pH
3.0 3.0 3.0
Color
Colorimetric method
Fluorimetric method
R d t b. p . method
Rdt U t e r u s
method
6 20
colorless light r e d light red colorless light brown light bmm
100 99 99 101 99 98
102 100 106 96 97

100 95 95
98 (96-104)' 94 (95-1061 99 (98-103) 99 (97-103) 97 (94-106) 92 (95-105) 93 197-104) 92 (91-110)
102 (86-116) 90 (92-1091 99 (87-115) 98 (89-112) 104 (92-109) 94 (94-106) 88 (90-111) 83 (88-113)
1000 20 min.
0 6 20 0 6 20
3.0 3.0 30 . 3.0

3.0 30 .
1200 20 nin.
light r e d 99 brown, d a r k p p t . 9 9 brown, dark ppt. 96
The S t a b i l i t y of Adrenaline I n j e c t i o n s (pH a d j u s t e d to 3.6 by adding sodium m e t a b i s u l p h i t e l Adrenaline recovery i n per c e n t of o r i g i n a l c o n t e n t
treatment Unheated
period (mnthsl
0
prr
36 .
3.4 3.4
Color colorless
Colorimetric method
Fluorimetric mthod
Rat b. p .
nethod
Rat u t e r u s method
6 20 1000 20 min.
0 6
99 100 100 99 100
102 97 100 101 96
100 (95-105)' 100 (93-108) 96 (94-106) 100 (97-103)
101 (92-109) 87 (89-113) 97 (93-108) 96 (93-108)
34 .
3.3
colorless
20 min.
120
0 6 20
34 . 3.3 3.3
colorless
98 99 99
94 95 98
97 (93-108) 98 (94-106) 97 (95-106)
100 (84-120) 98 (89-113) 94 (83-121)

p 3.0, Watel H
PH 3 6 ..
'Adrenaline b i t a r t r a t e 1 . 8 2 gm, sodium c h l o r i d e 8.0 gm, hydrochloric a c i d q . B . , for i n j e c t i o n 1000 m l .
'As dbove except h y d r o c h l o r i c a c i d omitted and 0.5 gln sodium m e t d b i s u l f i t e ddded.
c . Flducial limits, i n p a r e n t h e s i s , are expresaed a s percentages f P
0.051.
EPINEPHRINE
217
conunonly employed in the assay of a particular hormone or compound in body fluids or tissue. Application of this phenomenon, however, has been made to dosage form analysis (56). Primarily because epinephrine's native fluorescence occurs in the ultraviolet range, need existed for an analytical approach which would produce fluorescence in the visible region. Two general schemes evolved to provide this. The first of these involves production of a highly fluorescing hydroxyindole by oxidative cyclization of epinephrine under basic conditions. The second involves production of a fluorescent quinoxaline. was observed by Loew ( 5 7 ) , epinephrine produces a highly fluorescent material when oxidized in a basic aqueous environment. Initial investigations (58-61) indicated that assay of epinephrine on this basis was feasible. The nature of the reaction involved in producing the fluorescing material was studied, and it was determined that the fluorescing material produced is a hydroxyindole formed as in the following scheme
As
(43-46).
clh
Epinephrine
CH,
Epinephrlne-quinone
Adrewchromt
The chemistry of adrenochrome has been reviewed (62). The fluorometric analysis of epinephrine, based on production o f the trihydroxyindole derivative, has undergone extensive modification and is still being modified (63) to provide for a more reliable or selective procedure. The method has been used extensively for analysis of catecholamines in body
218
fluids ( 5 4 ) as well as for analysis of epinephrine in dosage forms (63). For a thorough review of this procedure in regard to analysis of epinephrine in body fluids or tissues, a basic text should be consulted (54).
The second approach used in fluorimetric assay of epinephrine and other catechol amines involves condensation with ethylenediamine. While this has been applied quite extensively to analysis of epinephrine and norepinephrine in biological systems, no examples of applications to dosage form analysis have appeared. Investigations reported thus far (64-69) suggest that epinephrine reacts with ethylenediamine in the f o l lowing manner.
I
CHa
Adrenaline Adrenochrome
A different product is produced from norepinephrine's reaction, i.e.,
Noradrenaline
EPINEPHRINE
219
The quinoxaline produced from norepinephrine has different fluorescent characteristics than the hydroxyindole produced by epinephrine permitting simultaneous determination of both compounds in biological samples. Details of this procedure can be found in a basic text (70). Colorimetric Colorimetric procedures which have been applied to analysis of epinephrine are based on the presence of the catechol or phenolic groups and hence have a variable degree of selectivity. Some of the more recently developed colorimetric procedures are reasonably selective; however, since none are stereoselective, their application is limited to those situations where enantiomorphic composition is determined independently or the much less potent d-isomer is known to be absent. Other common sources of analytical interference include biogenic catecholamines (norepinephrine) and the sulfonated product which results from reaction of bisulfite and epinephrine. Barker et a1 (71) in a critical survey of colorimetric procedures for epinephrine indicated that a colored material could be produced from this hormone by oxidation with potassium persulfate. The procedure was confirmed by Rees (72) and shown to give results which were in accord with those obtained pharmacologically. Details of this procedure are given in Garratt's text on the quantitative analysis of drugs ( 7 3 ) . Other oxidizing agents such as iodine (74) have been used to generate a colored product and provide differentiation between epinephrine and norepinephrine in biological samples. One complication which arises in analytical colorimetric schemes involving oxidation is that bisulfite is known to interfere. Doty's (75) method which employs ferrous ion to generate color copes with this problem in the sense that interference from anions arising from sulfurous acid is no longer apparent; however, epinephrine sulfonate may interfere. A more recent approach to colorimetric determination of epinephrine (76) involves reaction of thiosemicarbazide with epinephrine under basic conditions. Under the conditions of this procedure, apparently only catechols yield a colored product. Other color producing reactions which involve epinephrine are known. Some of these are useful f o r identification of the hormone ( S O ) , while others serve as means of detection in paper and thin layer chromatography. (42,83)
7.2
220
DALE H. SZULCZEWSKI AND WEN-HAI HONG
7.3 Polarimetric Methods which involve measurement of optical rotation have been proposed and are used to assay epinephrine in dosage forms. Several factors restrict the direct application of polarimetric methods and require that they be used in conjunction with other procedures. A primary consideration in this regard is that loss of optical acticity can be associated with either complete or only partial loss of pharmacological activity. Unless an ancillary method is used which assays total (d + 1) epinephrine content, correlation of observed rotation and 1-epinephrine content is not possible.
This problem was recognized by Rosenblum, Goldman, and Feldman ( 7 7 ) as well as Hellberg (78) who used concurrent polarimetric and colorimetric measurements to determine epinephrine content. Unfortunately the colorimetric procedures used are not selective and interference from other chromogenic compounds associated with epinephrine is possible. Nevertheless their approach is valid and if a more selective procedure (e.g., Prasad's fluorometric method 1631) is used to estimate total (d + - epinephrine, accurate results may be expected. - 1) Another analytical approach based on independent determinations of total epinephrine and enantiomorphic distribution is that proposed by Welsh (79) and modified by Higuchi (80). This scheme involves acetylating the mixture to be analyzed, isolating the acetyl derivative produced, and determining total epinephrine by gravimetric or spectrophotometric means. Enantiomorphic composition is then determined by polarimetric measurement of the isolated acetyl derivative. This is the procedure which has received official recognition by the USP. 7.4 Titrimetric Epinephrine can be titrated using a nonaqueous system. Usually the titration is carried out in glacial acetic acid using perchloric acid as titrant and crystal violet as indicator. The purity of bulk epinephrine and epinephrine tartrate U.S.P. is established in this manner ( 2). Photometric microtitration procedures have been developed and applied (81). Chromatography 7.51 Thin-layer and Paper Thin layer and paper chromatography have been used extensively for analysis of epinephrine and other adrenergic compounds contained in body fluids or pharmaceutical formulations. This subject is reviewed in several books
7.5
EPINEPHRINE
221
devoted to the subject of paper and thin layer chromatography (82,831. Summaries of chromatographic behavior and detection reagents are given in Tables VII. discussed by Macek (83), multiple spot formation is a considerable problem in both paper and thin layer chromatographic analysis of epinephrine. Additional spots may be as the result of formation and differential migration of free base and various complex salts formed during isolation or the chromatographic process or, as indicated by Roberts and Broadley (49), may be products of the reaction of epinephrine in the presence of hydrochloric acid.
As
Gas Chromatography Since epinephrine is a polar, nonvolatile compound, derivitization is a requirement for analysis by gas chromatography. Information relevant to derivatization and the gas chromatographic behavior of the derivatives has been summarized in several review articles and one text (89, 90,
91 ) .
7.52
Silylation has been found to be a useful means of converting epinephrine to a derivative with suitable gas chromatographic characteristics. Sen and McGeer (92) found that such a derivative could be produced by treating epinephrine with hexamethyldisilazane (HMDS). This silylated product was characterized by Horning (93) as the N-trimethylsilyl derivative of epinephrine. Horning (94) further demonstrated that preferential 0-silylation could be accomplished using trimethylsilylimidazole (TMSI) in acetonitrile. Under these conditions, N-silylation does not occur. The chromatogram shown as Fig. 6 was obtained after derivatization in this manner
(95).
When desired the basic secondary amino moiety can be blocked by N-acylation of the 0-trimethylsilyl derivative using Nacetyl or N-heptafluorobutyrylimidazole reagents (96).
222
Table VII
PC and TLC
Values for Epinephrine (82) PC
Systems:
S1 S2
S3
TLC
54
S5
S6
n-Butanol-acetic acid-water 4:1:5 (84) Isobutanol-formic acid-water 100:12:10 ( 8 5 ) Paper impregnated with citrate buffer p H 4 ; n-butanol saturated with water (86) Silica G e l G (pH 4 buffer)/n-butanol saturated with water (treated with SO2) (87) as above except pH 4 buffer + .01 M borax/ n-butanol saturated with water ( 8 7 ) Cellulose/phenol-O.lN HC1 (85:15) (88)
Epinephrine Adrenalone
38
09
15
27 38
15
07
62
44
--
--
--
Acetone S B
900'
110'
120.
130.
140. 150' Temperature
160.
170.
580.
Fig. 6 Chromatogrim of a mixture of catecholamines after their trimethylsilyfation and conversion into cnamines". PE = @-Phenylcthylamine; NEP = norcphedrine; @OH = fi-hydmxy-/l-phcnylethylamine; TY R = tyramine; 3.4-DMPE = /?-(3&diniethoxyphenyl)ethylarnine; MN = metanephrine; DO = dopanline; E = epinephrine; N M N = nonnetmephrine; NE r- norepinephrine. Conditions: loo/, F-60, temperature programming at 1.S"/niin.
224
Various chromatographic conditions have been employed for the chromatography of epinephrine and related compounds. Columns packed with such liquid phases as siloxane polymer SE-30 or methysiloxane polymer F-60 are useful. Column temperatures in the region 170-220C appear to be appropriate. Liquid Column Chromatography Liquid column chromatography has been applied to the analysis of epinephrine and related compounds. Since the underivatized hormone does not easily partition into water immiscible liquids, liquid column chromatography as associated with epinephrine is usually conducted using ion exchange resins or adsorbents.
7.53
Starch columns were employed to separate epinephrine, norepinephrine, and related catechol amines (97, 98), however, lengthy elution times were involved. Good separation of epinephrine, norepinephrine, and hydroxytyramine was achieved more quickly by using columns packed with Amberlite IRC-50 in the ammonium form and 0.4 molar ammonium acetate buffer, pH 5.0 as eluent (99). Masuoka and others used chromatography on alumina to accomplish group separations of noncatechols, catechol amines, and catechol acids as contained in biological samples (100,101). Mattock and Wilson (102-104) made use of complex formation which occurs between epinephrine and borate ions to accomplish group separation of methylated catecho1 amine metabolites, i.e., metanephrine etc., from the catechol amines epinephrine and norepinephrine. Group separation of epinephrine and norepinephrine from DOPA was found by McDonald and Murphy (105) to be easily accomplished using columns containing Sephadex G-10. Liquid column ion exchange chromatography was applied to analysis of epinephrine containing pharmaceuticals by Smith (106) who used a sulfonated polystyrene resin to adsorb the hormone and aqueous HC1 for elution. Higuchi (107) used partition chromatography to analyze epinephrine as part of work done on the reaction of epinephrine and bisulfite. In this case the hormone was acetylated to render it more lipophillic. The procedure separated intact d, - epinephrine from degradation products. - 1 Fu and Sibley (108) developed a high efficiency liquid chromatographic method for epinephrine in pharmaceutical formulations, using a cationic exchange resin column and an aqueous phosphate buffer as the mobile phase. The method allows separation of epinephrine from its oxidative and other
EPINEPHRINE
225
degradation products and hence is stability-indicating. Recently, Holcomb (112) developed a method for the analysis of Epinephrp Injection using paired-ion chromatography on C18 Bondapak column. The mobile phase used was methano1:water (2:3) containing EDTA, acetic acid, and dioctyl sodium succinate. Although high efficiency liquid chromatography has become widely used, very little data for epinephrine are found in the literature. Perhaps as the technique of paired-ion chromatography (109) becomes better known, applications will appear. Spectrophotometric Analysis of epinephrine through direct spectrophotometric measurement is subject to many of the same considerations as is colorimetric analysis of the hormone. Epinephrine absorbs ultraviolet radiation due to the presence of the catechol chromophore. Other related biogenic catechol amines (norepinephrine) and inactive compounds which are associated with epinephrine (l-(3,4-dihydroxyphenyl)-2-methylaminoethane sulfonic acid, d-epinephrine, etc.) likewide contain this chromophore andhence have similar spectra. Further complications, arising from spectral interference due to the presence of colored material, are possible. These factors limit the application of direct ultraviolet measurement in the analysis of epinephrine to those cases in which supplemental information, regarding absence of interfering materials, is avail76 .
able.
In 1N mineral acid (HC1 or H2S04), epinephrine has an absorption maximum near 280 nm. The British Pharmacopia (110) specifies that the ultraviolet spectrum of a given sample show a maximum at only this wavelength. Adrenalone, a possible process-related impurity, is detected and monitored by W measurement at 310 nm (49,110). At this wavelength epinephrine does not have significant absorption but adrenelone absorbs maximally (111)
8 Acknowledgments .
The authors wish to express special thanks to Mrs. Pohl for her secretarial assistance in preparing this manuscript.
226
References 1.
2. 3. 4.
5.
6 . 7.
The Merck Index, 9th ed, Merck & Co Inc, Rahway NJ, p 472, 1976 United States Pharmacopeia, Nineteenth Revision, Mack Printing Company, Easton, Pa, p 170, 1975 Ibid, 170-172, 1975 Wenchell A, The Optical Properties of Organic Compounds, 2nd ed, Academic Press Inc, New York, NY, p 160, 1954 Keenan G, J Assoc Off Agri Chem, 27:154, 1944 ASTM Powder Diffraction Data, #4-0232 Kirk RE and Othmer DF, Encyc Chem Tech, 2nd ed, 8:231, Kappe T and Armstrong M, J Med Chem 8:368, 1965 Rozum YS, Biokhimiya 17:476, 1952 Reisch J, Pagnucco R, Alfes H, Jantos N, Mollmann H, J Pharm Pharmacol 20:81, 1968 Seiler N, Schneider H, and Sonnenberg K, Z Anal Chem, Lyle GG, J Org Chem, 25:1779, 1960 LaManna A and Ghislandi V, Farmaco (Sci) 19:377, 1964 Drude P, Lehrbuch der Optik, 2nd ed, S Herzel Verlag, Leipzig Heller W, J Phys Chem 62:1569, 1958 Weiner N and Jardetsky 0, Naunyn Schmiedebergs Arch Pharmacol 248:308, 1964 Reisch J, A l f e s H, and Mollmann H, 2 Anal Chem 238(1):
29 , 1968 252:127, 1970 1963
8. 9. 10.
11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.
Higuchi T and Brochmann-Hansen E, Pharmaceutical Analysis, Interscience, NY, p 491, 1961 Sinistri C and Villa L, Farmaco (Sci) 17:949, 1962 Kappe T and Armstrong M, J Med Chem 8:368, 1965 Tuckerman M and Mayer J, J Am Chem SOC 81:92, 1959 Leffler E, Spencer H, and Burger A, J Am Chem SOC,
73:2611, 1951
Lewis G, Brit J Pharmacol 9:488, 1954 Martin B, J Phys Chem 75:2657, 1971 Jameson R and Nelli W, J Chem SOC 2391, 1965 Edsall JT, Martin RBI and Hollingworth BR, Proc Nat Acad Sci 44:505, 1958 Goodman LS and Gilman A, The Pharmacological Basis of Therapeutics, 4th ed, The Macmillan Co, New York, 1970 Takamine J, J SOC Chem Ind 20:746, 1901 Aldrich TB, Amer J Physiol 5:457, 1901 PauIy H, Ber 36:2944, 1903 Jowett HAD, J Chem SOC 85:192, 1904 Stolz F, Ber 37:4149, 1904
EPINEPHRINE 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57 * 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68.
227
Dakin HD, J P h y s i o l (Lond) 32:34P, 1905 F l a c h e r F, Z P h y s i o l Chem 58:581, 1908 Payne KR, I n d Chemist 37:523, 1 9 6 1 Kisbye J a n d Schon SA, Dansk T i d s s k r Farm 25:185, 1 9 5 1 Kisbye J , Dansk T i d s s k r Farm S u p p l 2:156, 1956 S c h r o e t e r LC and H i g u c h i T, J Am Pharm ASSOC, S c i Ed 47: 426, 1 9 5 8 S c h r o e t e r LC, H i g u c h i T, and S c h u l e r EE, J Am Pharm ASSOC, S c i Ed 47:723, 1958 Somers G a n d West GB, Q u a r t J Pharm 17:308, 1944 F H i g u c h i T and S c h r o e t e r LC, J Am Chem SOC 82:1904, 1960 F e i g l F , S p o t T e s t s i n Organic A n a l y s i s , E l s e v i e r Publ i s h i n g C o , New York, p.132, 188, 202, 273, 460, 1960 E h r l e n I , Farm Revy 47:242, 1948 Lund A , A c t a Pharrnacol T o x i c 0 1 5 : 7 5 , 1 2 1 8 , 1949 F i s h e r P, B u l l SOC Chim B e l g 58:205, 1949 Harley-Mason J , J Chem S O C , 1 2 7 6 , 1950 S o k o l o s k i T a n d H i g u c h i T , J Pharm S c i 51:172, 1962 F e l l m a n , JH , Nature 1 8 2 :311 , 1958 Roberts DJ a n d B r o a d l e y K J , J Chromatogr 27:407, 1967 I b i d 40:460, 1969 Backe-Hansen K a n d H o l s t G , A c t a Pharm S u e c i c a 3:269, 1966 Lundgren P and Strom S , Acta Pharm S u e c i c a 3:273, 1966 Backe-Hansen K , D r o t t n i n g A , Vennerod AM, a n d B r i s e i d J e n s e n K , J Pharm Pharmacol 15:804, 1 9 6 3 U d e n f r i e n d S , F l u o r e s c e n c e Assay i n B i o l o g y a n d Medicine, Academic P r e s s , New York, 1 4 0 , 1962 Duggan DE, Bowman RL, B r o d i e BB, a n d U d e n f r i e n d S , Arch Biochem B i o p h y s 68:l, 1957 J a m e s T, J Pharm S c i 62:669, 1 9 7 3 Loew 0, Biochem Z 85:295, 1918 P a g e t M, B u l l S c i Pharmacol 37:537, 1930 Gaddum J H and S c h i l d H, J P h y s i o l (Lond) 80:9P, 1934 Hueber EF, K l i n Wochenschr 1 9 : 6 6 4 , 1940 Heller JH, S e t l o w RB, and Mylon E, Am J P h y s i o l (Lond) 161:268, 1949 Heacock RA, Chem Rev 59:181, 1959 P r a s a d VK, R i c i RA, Nunning BC, a n d G r a n a t e k AP, J Pharm S c i 62:1130, 1 9 7 3 W a l l e r s t e i n J S , A l b a RT, a n d H a l e MG, Biochem Biophys A c t a 1 : 1 7 5 , 1947 Natedson S , Lugovoy J K , and P i n k u s J B , Arch Biochem 23: 1 5 7 , 1949 Weil-Malherbe H a n d Bone AD, Biochem J 51:311, 1952 Harley-Mason J a n d L a i r d AH, Biochem J 69:59P, 1 9 5 8 Weil-Malherbe H I Pharmacol Rev 11:278, 1959
228
Weil-Malherbe H, Biochim Biophys Acta 40:351, 1960 Udenfriend S , Fluorescence Assay in Biology and Medicine, Academic Press, New York, 153-7, 1962 71. Barker JH, Eastland CJ, and Evers N, Biochem J 26:2129, 1932 72. Rees HG, Quart J P h a m 9:659, 1936 73. Garratt DC, The Quantitative Analysis of Drugs, Chapman and Hall, London, 19, 1964 74. Von Euler US and Hamberg U, Science 110:561, 1949 75. Doty JR, Anal Chem 20:1166, 1948 76. Salama RB and Wahba Khalil SKI J Pharm Sci 63:1301, 1974 77. Rosenblum H, Goldman R, and Feldman H, J Amer Pharm ASSOC, Sci Ed. 39:255, 1949 78. Hellberg HJ, Pharm Pharmacol 7:191, 1955 79. Welsh L, J Amer Pharm Assoc Sci Ed 44:507, 1955 80. Higuchi T, Sokoloski TD, and Schroeter LC, J Amer Pharm ASSOC, Sci Ed. 48: 553, 1959 81. Tuckerman, M, J Pharm Sci 51:700, 1962 82. Stahl E, Thin Layer Chromatography, Springer-Verlag, New York Inc, p 499, 520, 589, 1969 83. Macek K, Pharmaceutical Applications of Thin Layer and Paper Chromatography, Elsevier Publishing Co, New York, NY, 1972 84. Pohloudek-Fabini R and Konig K, Pharmazie 13:131, 1958 85. Skaliks HC, Arzneimittel-Forsch 7:386, 1957 86. Wagner G, Sci Pharm 23:148, 1955 87. Halmekaski J, Suom Kernisti1 B36:58, 1963, CA 59:2589h, 1963 88. Choulis NH, J Pharm Sci 56:904, 1968 89. Drozd J, J Chromatogr 113:303, 1975 90. Ahiya S, J Pharm Sci 65:163, 1976 Pierce A, Silylation of Organic Compounds, Pierce Chemi91 cal Co, Rockford, Ill, 192, 1968 92. Sen NP and McGeer PL, Biochem Biophys Res Commun 13:390, 1963 93. Capella P and Horning EC, Anal Chem 38:316, 1966 94. Horning MG, M s AM, and Horning EC, Biochim Biophys os Acta 148:597, 1967 95. Capella P and Horning EC, Anal Chem 38:316, 1966 96. Horning MG, Moss AM, Boucher EA, and Horning EC, Anal Lett 1:311, 1968 97. Kirshner N and Goodall M, Federation Proc 15:110, 1956 98. Hamberg U and Von Euler US, Acta Chem Scand 4:1185, 1950 99. Kirshner N and Goodall McC, J Biol Chem 226:207, 1957
69. 70.
EPINEPHRINE
100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 1 1 1 .
229
112.
Masuoka DT, D r e l l W, S c h o t t HF, Alcaraz AF, and J a m e s E C Anal Biochem 5:426, 1963 D r e l l W, F e d e r a t i o n Proc 16:174, 1957 Mattock GL and Wilson DL, Anal Biochem 11:575, 1965 T r a u t n e r EM and Messer M, Nature 169:31, 1952 Riegelman S and F i s c h e r EZ, J Pharm S c i 51:210, 1962 McDonald T J and Murphy BEP, J C l i n P a t h 26:37, 1973 Smith D J , J Ass Off Anal Chem 55:596, 1972 Higuchi T , Sokoloski TD, and S c h r o e t e r LC, J Am Phann A s s o c 48:553, 1959 Fu CC and S i b l e y M J , J Pharm S c i 66:425, 1977 W i t t m e r DP, Nuessle NO, and Haney WG, Anal Chem 47:1422, 1975 B r i t i s h Phannacopeia 14, 1973 Moed HD, Van Dijk J, and Niewind H, R e c Trav Chim 74: 919, 1955 P e r s o n a l communication w i t h D r . I . J. Holcomb, Parke, Davis & C o . , D e t r o i t , Michigan.
L i t e r a t u r e r e f e r e n c e s are complete through e a r l y 1977.
ETHAMBUTOL HYDROCHLORIDE
Ching-San Lrr and Leslie 2. Bcnet
231
Copyright 0 197R by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-260807-0
232
CHING-SAN LEE AND
LESLIE Z. BENET
CONTENTS
1. Description 1.1 Name, Formula, Molecular Weight 1 . 2 Appearance, Color, Odor 2 . 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 Melting Range 2.7 Solubility 2.8 Dissociation Constant 3 . Synthesis 4 , Pharmacokinetics 5. Drug Metabolism 6 . Methods of Analysis * 6 . 1 Elemental Analysis 6 . 2 Microbiological Analysis 6 . 3 Colorimetric Analysis 6 . 4 Reineckate Analysis 6.5 Gas Chromatographic Analysis 6 . 5 1 Electron Capture Detection 6 . 5 2 Flame Ionization Detection 6 . 6 GC-CIMS Analysis 7. Acknowledgments 8 . References
233
1. D e s c r i p t i o n
1.1 N a m e , Formula, Molecular Weight While s c r e e n i n g s e l e c t e d compounds, Thomas and cqworkers' found t h a t N , N'-di-isopropylethylenediamine w a s e f f e c t i v e i n t h e treatment of e x p e r i m e n t a l t u b e r c u l o u s i n f e c t i o n s i n mice. A number of congeners of t h i s compound were examined; t h e one t h a t e v e n t u a l l y proved t o be t h e most t u b e r c u l o s t a t i c w a s d-2, 2'-(ethylenediimino)-di-l-butanol. The summary formula f o r t h e d i h y d r o c h l o r i d e s a l t of ethambut o 1 i s C10H2402N2(2HC1), w i t h a c o r r e s p o n d i n g m o l e c u l a r weight of 277.5. The commonly used t r a d e name i s Myambutol (Lederle). HOH2C
HC- N - (CH2) 2 -
H N
CH20H / CH \ 2H5
M.W.
= 277.5
2HC1
Appearance, C o l o r , Odor Ethambutol i s a w h i t e , s c e n t l e s s , b i t t e r t a s t i n g , t h e r m o s t a b l e c r y s t a l l i n e powder.
1.2
2.
Physical Properties 2.1.
I n f r a r e d Spectrum According t o Wilkinson e t a1.2, t h e p r i n c i p a l i n f r a r e d bands, i d e n t i c a l f o r t h e (+) and (-) b a s e s , are: 3310, 3180, 2990, 2900, 1475, 1385, 1374, 1360, 1228, 1148, i 0 9 5 , 1068, 1055, 1015, 990, 885, 846, 839, 816, and 768 cmAn I R spectrum of ethambutol h y d r o c h l o r i d e w a s a l s o o b t a i n e d on a K B r d i s c and i s p r e s e n t e d i n F i g . 1. The I R s p e c t r a f o r b o t h f r e e b a s e and h y d r o c h l o r i d e s a l t i n d i c a t e t h e absence of f r e e -OH i n t h e ethambutol m o l e c u l a r hydrogen bonding. Assignment of some of t h e a b s o r p t i o n bands i s g i v e n i n Table I.
WAVCLEIIGTH (MICRONS)
2.5
3.n
3.5
4.0
5.n
6.0
o.n
3.0
1o.n
15.0 15. 0 1on
80
60
40
20 0
1.5 1.3
1.2
1.1
1.0
0.9
0.8
0.7
Fig. 1
Infrared spectrum of ethambutol hydrochloride in KBr pellet. Instrument: Perkin-Elmer Model 337.
235
TABLE I
IR Spectral Assignments for Ethambutol HCl Wavelength of Absorption (cm-l)
3450-3200
Vibration Mode intermolecular H-bonded, polymeric association combination of intramolecular H-bonded, -OH in chelate form and -CH2, -CH3 stretching the fingerprint region
3100-2550
1500-780
2.2
Nuclear Magnetic Resonance Spectrum The nuclear magnetic resonance (NMR) spectrum (Fig.2) was obtained by preparing a 2 mg/ml solution of ethambutol free base in deutero chloroform containing tetramethylsilane as the internal reference. A Varian XL-100 NMR Spectrometer was used. The NMR proton spectral assignments are given in Table 11.
H-F-0-H
Mass Spectrum Chemical ionization mass spectra of ethambutol were obtained using a quadrupole mass spectrometer. Source pressures were maintained at 15 torr (isobutane). The source temperature was 150-170". Samples (1 & .5 mcg) were placed into a glass capillary by a direct-insertion probe, which was then gradually heated above 200' t o effect volatilization. The major peak at m/e 205 in Fig. 3 corresponds to the M+1 molecular ion of the ethambutol free base.
2.3
IPY( T )
6.n
7.n
I
i:.n
I
9n .
I
1n.n
I
w
HZ
!d c
5.0
4.n
1.0
70 .
10 .
Pm Fig. 2
(6)
NMR spectrum of ethambutol in deuterated chloroform. Instrument: Varian XL-100.
237
TABLE I1
NMR Spectral Assignments for Ethambutol
Chemical shift (PPd
Protons
Splittings triplet quintet Bextet quartet singlet
3.56
- 1.56 - 2.60 2.70 - 2.78 2.91 3.24 - 3.44

3.70
.83 1.29 2.48
.98
--
--
2.4 Ultraviolet Spectrum The ethanolic solution of ethambutol shows only end absorption in the W region starting at 220 run. No absbrption maximum is observable in the visable light field. The W spectrum was scanned using a Cary 15 Spectrophotometer. 2.5 Optical Rotation The specific rotations of the optically active isomers in water2 are (+)-base [a]65 + 13.7", (+)-dihydrochloride rC(1;5 + 7.6" 2.6 Melting Range The following melting point temperatures ("C) have been reported: M.P. Reference 2 3 2
(+I -C10H24N202
(+)-CloH24N202*2HC1
87.5 82.0 201.8 198.5 200.0
88.8 83.5 202.6 200.3 202.0
4
3
Solubility The dihydrochloride salt of ethambutol is easily soluble in water and dimethylsulfoxide; soluble in glycol; sparingly soluble in ethanol and very difficulflysoluble in acetone and chloroform 5 On the contrary, the free base of ethambutol is very soluble in chloroform, methylenechloride, and ethylenedichloride; less soluble in benzene and carbon tetrachloride; and sparingly soluble in water 6.
2.7
205
H0H2C\
/CH20H
w u
5 2
HC-
N -(CH2I2-
N -CH
2
H5
2 200 150
m/e
Fig. 3 Chemical ionization mass spectrum of ethambutol using isobutane as the reagent gas. Instrument: Finnigan quadrupole mass spectrometer - Model 2700.
239
Dissociation Constant Ethambutol is a weak base with reported pKa values 7 of 6 . 6 and 9.5. The first pKa value of 6 . 6 was confirmed using an aqueous titration technique 3. Synthesis The highly active d-isomer was prepared by brief heating of ethylene dichloride with excess d-2-amino-1-butanol 8. A 42% yield of purified d-2,2- (ethy1enediimino)-di-1-butanol was obtained after removal of the less soluble meso isomer corresponding to approximately the amount of levo impurity in the d-2-amino-1-butanol. Ethylene bromide or ditosyl glycol may be used as substitutes for ethylene chloride. An interesting alternative synthesis 9 , reductive alkylation of 2-aminobutanol with glyoxal using sodium borohydride as the reducing agent, gave good yields. Bernardi et al. lo have reported a new synthesis of ethambutol based on the key intermediate, 2-chloro-3-buten-l-ylchloroformate, easily obtainable in high yield by the reaction of phosgene with 3 , 4 epoxy-1-butene.
3.
28 .
Pharmacokinetics Large differences in pharmacokinetic parameters have been reported in the literature. This is best exemplified by the variation found in peak plasma levels reported following oral and intravenous dosing of comparable dose levels as shown in Table 111. For example following oral dosing of 25-30 mg/kg, reported peak values range from 1 to 6 mcg/ml. Likewise plasma concentrations following a 2 hr intravenous infusion of a 20 mg/kg dose are reported to range between 1 and 45 mcg/ml. Similar differences have been noted in pharmacokinetic parameters as shown in Table IV. The various studies have utilized microbiological, colorimetric, radiochemical and most recently gas liquid chromatographic assays, some of which may be measuring metabolites of ethambutol as well as unchanged drug. The majority of the studies reported in Tables I11 and IV were done on tuberculous patients with normal kidney function. The large variations in kinetic parameters among tuberculous patients may be attributed to their disease histories and age scatterings in addition to the insensitive and nonspecific assays use 98 with well controlled normal volunteers A recent study indicates only minor variation among individuals. The peak values from this oral study tend to be higher than those reported by others. The data over the first 12 hrs post dosing favor longer half-life values than those previously reported. Twenty-four hr plasma samples and 24 to 72 hr urine samples indicate that ethambutol possesses an even longer terminal half-life of about 10 hrs. Another study 21 in the same group 4.
240
CHING-SAN LEE AND LESLIE Z. BENET
TABLE I11 V a r i a t i o n i n Reported Peak Plasma L e v e l s of Ethambutol Following Oral and I n t r a v e n o u s Dosing Reference Dose (mg/kg> 4 8 12.5 25 50 25 25 25 25 15 20 25 50 20 15
Oral Dosing Peak Conc. (mcg /ml)
Peak T i m e (hr) 2-4
Assay Method
11
.7
1.3 2.0 4.2 8.6 2 -4 2-5 2
microbiol.
12 13 14 15 16
2-4 3 2 2 2-4
microbiol., colorimet. microbiol. colorimet. microbiol.
1-6
2-2.5 3.1-3.5 4-5 8.6 3.4 3.3-6.0
I . V . Dosing Peak Conc. (mcg/ml)
17
18
3 1.5-4.0
microbiol.
GLC
Re erence
Dose (mg/kg) 15-20 15-25 20-25 20 15
Infusion Time (hr) 2
Assay Methol
19 20 16
35-45 5-23
microbiol. colorimet. microbiol microbiol. GLC
1.5
2 2 1
1-10
2.4-26 11.6-15.4
17
21
TABLE IV Summary of Ethambutol Pharmacokinetic Parameters Reported in Man Elimination Half-Lives Fraction Renal Failure Volume of Plasma Reference in Normals Excreted Half-Lives Distribution Clearance (hrs) Unchanged (hrs) L/kg (ml/min) Renal Clearance Assay (ml/min) microbiol. 7.2 12-15 7.4
. 8
11
4
4.2 12-15
3 Oa . 1.5b
14
15 22
0.6-0.8 (oral) 0.46 (oral)
microbiol. variablef colorimet. 532
08 .4 (iv>
446
GFR 412
radiochem.
23 18 21
~~ ~
2.5
3.6-5.5 2.5-3.6 0.54-0.67 (oral) 0.75-0.80 ( iv) 6.9-9. 6e
.3
100
microbiol. GLC GLC
1.65
~ ~ ~~ ~~
530
415
Estimated from Fig. 3 in reference 22. Calculated from clearance data in reference 22.
Two compartment model was used for data analysis.
Probably corresponds only to the central compartment value. Reference 3.
No correlation with inulin clearance.
242
of human subjects with ethambutol injection indicates very minor variation in peak serum levels following intravenous infusion over a one hour period. A much larger volume of distribution, which approximates 165% of body weight, was estimated for ethambutol in the intravenous study. 5, Drug Metabolism Initial metabolism of ethambutol results from the oxidation of the primary alcohol groups to an aldehyde, this is followed by a further oxidation t o a dicarboxylic acid to prodyce the end metabolite 24. The catalytic enzyme responsible for ethambutol metabolism is alcohol dehydrogenase. These extremely polar metabolites are pharmacologi'cally inactive and excreted by the kidneys readily without further conjugation. The biotransformation of ethambutol is shown below: H5C2
- CH - CH20H
r"
f2
CH2 NH H5C2
H5C2
- CH - CHO I NH
H5C2 - CH
--+
H5C2
r2
y 2
NH
COOH
I
CH2
- CH - CH20H
1 - CH - CHO
NH
NH H5C2 - CH - COOH
The total urinary metabolic pattern over 24 hrs following ethambutol administration was obtained using countercurrent distributi n of radioactive material in the urine of four patients 22. Following an intravenous dose of 14C-ethambutol in one subject, 5% of the dose was excreted as dialdehyde, 3% as diacid, and 65% as unaltered 14C-ethambutol. Similarly, of the 54% of administered 14C excreted into the urine in the 24 hrs following oral dosing, 8% of the dose was excreted as dialdehyde, 7% as diacid and 39% as ethambutol. The diacid end metabolite was also identified using ion exchange liquid chromatography 3 . The reaction of ninhydrin with the diamino dicarboxylic acid metabolite yields a purple color that absorbs maximally near 570 nm. An automatic amino acid analyzer (Beckman Model 116C) was used for separation and identification, The ion exchange resin used was Beckman UR-30. Usingnorleucineas an internal standard, it is possible to quantitate the metabolite in the urine 3 . Figure 4 shows the liquid chromatogram of O.lml of human urine spiked with the diacid metabolite andnorleucineas the internal standard. Table V lists the conditions applied for the metabolite analysis.
B
._
Y Y
50
70
RETENTION TIME
90
(MINI
U O
130
Fig. 4
Chromatogram of 0.1 ml of human urine spiked with 0.25 U M of ethambutol diacid metabolite and 0.15 yM of internal standard (norleucine). Instrument: Beckman Amino Acid Analyzer - Model 116 C.
244
CHING-SAN LEE AND LESLIE Z. BEN'ET
TABLE V Conditions Applied for the Analysis of Ethambutol Metabolite Methodology column size resin type height of resin column column flow rate column back pressure first buffer second buffer buffer change time column temperature
6. Methods of Analysis
2 hour physiological fluid
on the model 116C
0.9 X 69 cm Beckman UR-30 56.0 cm 68 ml/min 250 psi 3.25 f .01 (0.2N) : 4.25 5 .O2 (0.2N) 55 min 55.5" c
6.1 Elemental Analysis (as C H 0 N 10 24 2 2

Element
C
2HC1) Reported 4 43.5

9.7
% Theory
43.3 9.5
H
N
c1
1. 01 25.6
10.4 25.6
6.2 Microbiological Analysis This technique consists of measuring the diameter of the zone of inhibition of disks impregnated with the sample to be tested, or of cylinders containing the sample to be tested, on a homogeneous culture of Mycobacterium sme Mycobacterium aurum 26, M cobacterium parafortuitum Mycobacterium dierhoferi 1 7
Colorimetric Analysis This method is based on the formation of a complex of bromothymol blue and ethambutol, a complex whose yellow color may be quantitatively measured by means of spectrophotometr ;, The technique was first described by Kelly and de la Huerga11 modified by Strauss and Erhardt 13 and by Froseth 28. According to Strauss and Erhardt 13, the sensitivity for the determination of ethambutol in serum is 0.5 mcg/ml.
6.3
245
6.4 Reineckate Analysis The Reineckate assay procedure 29 is a gravimetric determination. The prepared test sample solution of ethambuto1 is precipitated by adding saturated ammonium reineckate solution. Each gram of the precipitate dried at 105" is equito 0.3292 gm of ethambutol hydrochloride. The washed precipitate can be further dissolved in methanol and the absorbance read at 525 nm. The chelation of ethambutol with reineckate produces a stable color with low absorptivity, and small amounts can not be determined with sufficient accuracy.
6.5
Gas-Liquid Chromatographic Analysis 6.51
Electron Capture Detection Developed by Lee and Benet 30, this method involves the use of an internal standard, dextro-2,2'-(ethylenediimino)di-1-propanol and derivatization with trifluoroacetic anhydride (TFA). The method allows micro determination of ethambutol in biologic fluids such as plasma, blood, dialysate and urine. Briefly described: 0.2 ml samples, to which 5 mcg of internal standard [dextro-2,2'-(ethylenediimino)-di-lpropanol] has been added, are extracted with 8 ml of chloroform under alkaline conditions (3-4 drops 4N NaOH). Two to three drops of 1 N HC1 are added before chloroform aliquots are evaporated to dryness under nitrogen. Methylene chloride (0.5 ml) is added and evaporated to dryness. Residues are dissolved in 1 ml of benzene and made alkaline by addition of 3-4 drops of diluted pyridine (1:4 in benzene). Derivatization with 20 p 1 trifluoracetic anhydride added to a closed tube is completed in 2 hr at room temperature. Excessive derivatization agent is washed into an aqueous phase with 3 ml of 0.01 M HC1. Appropriate aliquots of the benzene layer (23 1~1)are injected into a GC equipped with a scandium tritide electron capture detector under the following conditions: glass column, 6 ft x 1/8 in, 3% OV-17 on Gas-Chrom Q, 100-200 mesh; carrier gas (nitrogen) flow-rate, 20 ml/min; injector temperature, 210"; oven temperature, 157"; detector temperature, 230'. Under the chromatographic conditions described above, the internal standard and ethambutol have retention times of 2.5 and 4 min, respectively. The electron capture detector response is linear between 0.1 and 1 mcg where therapeutic concentrations can be conveniently interpolated. Frozen ethambutol samples and the TFA derivatives of ethambuto1 and internal standard have been shown to be stable up to seven days 30.
246
Flame Ionization Detection In 1968, Calo et al.3L published a gas chromatographic method for the separation of three antitubercular drugs, isoniazid, iproniazid and ethambutol. The benzene SOlution of trimethylsilyl ethambutol, injected along with an alcohol solution of isoniazid and an ethereal solution of iproniazid was chromatographed using a programmed temperature system. The retention time for the trimethylsilyl ethambutol was 12 min. In 1970, Richard et al.32 reported a gas chromatographic determination of ethambutol involving derivatization with N-trimethylsilylimidazole. The method made use of a single step derivatization, isothermal temperature and a multipurpose column. The retention time for the trimethylsilyl ethambutol was 4 min. No internal standard was used. Lee and Benet 33 modified rhis method by the inclusion of an internal standard 1,lO-decanediol and the use of a different derivatizing agent, bis(trimethylsily1)trifluoroacetamide. The use of an internal standard presents an improvement over the other published chromatographic techniques 31932. The flame ionization method is suitable for urine samples or for dosage form analysis in which high levels of ethambutol are anticipated. The flame ionization method avoids the tedious and possibly erroneous dilution of the samples necessary for the GLC assay using an electron capture detector. Briefly described 33: appropriate aliquots of sample, to which 10 mcg of decanediol had been added, were extracted with 8 m l of chloroform for 10 min under alkaline conditions. Portions of the chloroform were transferred to another tube. Two to three drops of 1 N H U were added before the chloroform aliquots were evaporated to dryness under nitrogen. Residues were dissolved in 0.1 ml of spectroquality chloroform. Derivatization was initiated by adding 10 p 1 of b i s ( t r i m e t h y l s i l y 1 ) t r i f l u o r o R c e t a m i d e and was completed at of room temperature in 30 min. Aliquots (2-3~1) the reaction mixture were injected into the gas chromatograph. Nitrogen was used as the carrier gas at a flow rate of 6 6 . 6 ml/min measured at the flame ionization detector base. The air flow rate was 300 ml/min and hydrogen flow 27 ml/min. Other chromatographic conditions are the same as those described in 6 . 5 1 . The trimethylsilyl derivative of ethambutol exhibited a retention time of 6 min, and the 1,lOdecanediol internal standard had a retention time of 4 min. The response of the flame ionization detector is linear in the 10-100 mcg range.
6.52
24 7
Using a dual column and dual detector gas liquid chromatograph, simultaneous micro and macro determinations of ethambutol become possible using both the electron capture and flame ionization procedures described. Details such as derivative formation, assay recoveries and choice of internal standard were discussed 33. GC-CIMS Analysis The method 34 involves the use of a tetradeuterated ethambutol standard and derivatization with trimethylsilyimidazole prior to injection onto a GC-CIMS. Mass fragmentography was monitored at m/e 349 and 353, the M+l ions for the di-TMS derivatives of ethambutol and internal standard respectively, and at m/e 333 and 337, corresponding to the M-CH3 ions for ethambutol and ethambutol-dq. The nominal sensitivity on a 0-20 mcg/ml range is 50 ng/ml.
6.6
7. Acknowledgments The authors express there appreciation to Lederle Laboratories for their aid and support in our ongoing ethambutol studies. Special'thanks to Dr. Raymond G. Wilkinson for supplying us withsamples of dextro 2,2'-(ethy1enediimino)di1-propanol, Dr. Robert G. Kelly for radiolabelled ethambutol and John B. Hill for his continuing help in obtaining oral and intravenous dosage forms. The authors thank M s . Irene Beck for her help in preparation of the final typed form of this monograph. Studies in the authors laboratories at the University of California were funded in part by NIH grant GM 1 6 4 9 6 . During the course of this work Dr. Lee received support as a NIH Predoctoral Fellow through TrainingGrant GM 00728.
248
References
1.
2. 3.
4. 5. 6. 7.
8.
9. 10.
J . P . Thomas, C.O. Baughn, R.G. W i l k i n s o n , and R.G. Shepherd, Am. Rev. Resp. D i s . 83, 8 9 1 (1961). R.G. W i l k i n s o n , M.B. C a n t r a l l , and R.G. Shepherd, J . Med. Pharm. Chem. 5, 835 (1962). C. S . Lee, T h e P h a r m a c o k i n e t i c s o f Ethambutol. A d i s s e r t a t i o n s u b m i t t e d i n p a r t i a l s a t i s f a c t i o n of t h e r e q u i r e ments f o r t h e d e g r e e of Doctor o f P h i l o s o p h y (Pharmaceut i c a l Chemistry) t o t h e Graduate D i v i s i o n , U n i v e r s i t y of C a l i f o r n i a , San F r a n c i s c o , 1977. R.G. W i l k i n s o n , R.G. Shepherd, J . P . Thomas, and C. Baughn J. Am. Chem. SOC. 83, 2212 (1961). R. K a n t h e r , A n t i b i o t i c a e t Chemotherapia 1 ,203 (1970). 6 E.H. G i r g i s , Z . Gad, and M. Maram, J . Pharm. S c i . 63, 1764 (1974). R.G. Shepherd, C. Baughn, M.L. C a n t r a l l , B. Goodstein, J.P. Thomas, and R.G. W i l k i n s o n , Ann. N.Y. Acad. S c i . - 686 (1966). 135, R.G. W i l k i n s o n , R.G. Shepherd, J . P . Thomas, and C. Baughn J. Am. Chem. SOC. 83, 2212 (1961). J . H . B i l l i a n and A X . D i e s i n g , J. Org. Chem. 2, 1068 (1957). L. B e r n a r d i , M. F o g l i o , and A. T e m p e r i l l i , J. Med. Chem. -, 555 (1974). 17
V.A. P l a c e , E.A. P e e t s , D.A. Buyske, and R.R. L i t t l e , Ann. N.Y. Acad. S c i . 135, 775 (1966). A. Tacquet and B. Devulder, Rev. Tub. e t Pneumo.32, 729 (1968). I. S t r a u s s and F. E r h a r d t , Chemotherapy 15,1 4 8 (1970). T. Dume, C . Wagner, and E. W e t z e l s , Dtsch Med. Wschr. - 1430 (1971). 96, L. Anex, G. Favez, and C. Willa, Schweiz. Med. Wschr. - 126 (1972). 102, A. Delaude and J.L. A l b a r e d e , G a z e t t e Medicale d e F r a n c e (Supplement) 79, 7301 (1972). J . C . P u j e t and C. P u j e t , G a z e t t e Medicale de F r a n c e (Supplement) 79,7312 (1972). C.S. Lee, J . G . Gambertoglio, D.C. Brater, and L.Z. B e n e t , The P h a r m a c o k i n e t i c s o f Ethambutol F o l l o w i n g Oral Dosing i n Normal V o l u n t e e r s . C l i n . Pharmacol. T h e r . , I n p r e s s (1977). A. R o s s i , G.C. Morandini, and G. PiacenzO, B u l l . SOC. Med. C h i r . P a v i a 82, 792 (1968). G. Arraud, M. Gavend, A . R o u l e t , B. P a r a m e l l e , and G. B e s s a r d , G a z e t t e Medicale de F r a n c e (Supplement) 3, 7321 (1972).
11.
12. 13. 14.
15.
16. 17. 18.
19.
20.
249
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22. 23. 24. 25. 26. 27.
28.
29.
30. 31.
32.
33.
C.S. Lee, D.C. Brater, J . G . Gambertoglio, and L . Z . B e n e t , D i s p o s i t i o n K i n e t i c s of Ethambubol. P r e s e n t e d t o t h e B a s i c P h a r m a c e u t i c s S e c t i o n , APhA Academy of Pharmaceutic a l S c i e n c e s , Phoenix Meeting, November 1977. T.G. C h r i s t o p h e r , A. B l a i r , A. F o r r e y , and R.E. C u t l e r , P r o c . D i a l y s i s T r a n s p l a n t Forum 2, 96 (1973). J . W. J e n n e and W.H. Beggs, Am. Rev. Resp. D i s . 107,1013 (1973). E.A. P e e t s , W.M. Sweeney, V.A. P l a c e , and D.A. Buyske, Am. Rev. Resp. D i s . 91, 51 (1965). V.A. P l a c e and J . P . Thomas, C l i n i c a l Pharmacology of Ethambutol. Am. Rev. Resp. D i s . 87,901 (1963). J . C . P u j e t and C. P u j e t , Bordeaux Medical 2, 1215 (1972). J . K e l l y and J . de l a Huerga, Myambutol Monograph, L e d e r l e L a b o r a t o r i e s , New York, 1967. B. F r o s e t h , Scand. J . Resp. D i s . 3, (1969). 81 P. T a r l i , F. L a t t a n z i , and S . Benocci, I1 Farmaco 2 4 , 7 0 7 (1969). C.S. Lee and L . Z . B e n e t , J. Chromatog. 128,188 (1976). A. C a l o , C. C a r d i n i , and V. Q u e r c i a , J . Chromatog. 37, 194 (1968). B.M. R i c h a r d , J . E . Manno, and B.R. Manno, J . Chromatog. 8 9 , 80-83 (1974). C . Lee and L . Z . B e n e t , Micro and Macro D e t e r m i n a t i o n x of Ethambutol i n B i o l o g i c a l F l u i d s by Gas-Liquid Chromat o g r a p h y . J . Pharm. S c i . I n p r e s s .
A.D.
34.
B l a i r , A.W. F o r r e y , T.G. C h r i s t o p h e r , B. Maxwell, R . E . C u t l e r , and R.G. J e n k i n s , P r o c . 2nd I n t e r n a t . Conf. on S t a b l e I s o t o p e s , U.S. Energy R and D Admin., Oakbrook, I l l . , 1975, p. 208.
L i t e r a t u r e s u r v e y e d through J u l y , 1977.
FLUOXYMESTERONE
Joel Kirschhnum
251
Copyright @ 1978 by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN 0-12-260807-0
252
JOEL KIRSCHBAUM
CONTENTS
1.
H i s t o r y , D e s c r i p t i o n , P r e c a u t i o n s and S y n t h e s i s 1.1 H i s t o r y 1 . 2 Name, Formula and Molecular Weight 1 . 3 Appearance, Color and Odor 1 . 4 Precautions 1 . 5 S y n t h e s i s and S t e r e o c h e m i s t r y Physical Properties 2 . 1 I n f r a r e d Spectrometry 2 . 2 Nuclear Magnetic Resonance Spectrometry (NMR) 2 . 3 Mass Spectrometry 2 . 4 X-ray Powder D i f f r a c t i o n 2 . 5 U l t r a v i o l e t Spectrometry 2 . 6 O p t i c a l R o t a t o r y D i s p e r s i o n and O p t i c a l R o t a t i o n 2 . 7 F l u o r e s c e n c e Spectrometry 2 . 8 M e l t i n g Range 2 . 9 D i f f e r e n t i a l Thermal A n a l y s i s 2.10 Thermal G r a v i m e t r i c A n a l y s i s 2 . 1 1 Microscopy and C r y s t a l Type 2 . 1 2 Polymorphism 2 . 1 3 Hydration 2 . 1 4 S u r f a c e Area Solution Properties 3 . 1 I n t r i n s i c D i s s o l u t i o n Rate 3 . 2 S o l u b i l i t i e s i n Aqueous and Nonaqueous S o l v e n t s 3 . 3 Partition Coefficients 3 . 4 Phase S o l u b i l i t y A n a l y s i s Methods of A n a l y s i s 4 . 1 Elemental and I n o r g a n i c Analyses 4 . 2 I d e n t i f i c a t i o n , U l t r a v i o l e t and C o l o r i m e t r i c Analyses 4 . 3 Chromatographic A n a l y s i s 4 . 3 1 Gas Chromatography 4 . 3 2 High P r e s s u r e Liquid Chromatography 4 . 3 3 Thin Layer Chromatography 4 . 3 4 Paper Chromatography 4 . 3 5 Column Chromatography 4 . 4 Assays f o r Fluoxymesterone i n T i s s u e s and Body Fluids Stability: E f f e c t s of Temperature, T i m e and L i g h t
2.
3.
4.
5.
6.
i
Acknowledgements References
-.
FLUOXYMESTERONE
253
History, Description, Precautions and Synthesis 1 1 History . Fluoxymesterone is an androgen used in replacement therapy and for inoperable mammary cancer.' It originated as a derivative of cortisone and hydrocortisone in which the 9ahydrogen atom is replaced by halogen2, leading to greatly increased glucocorticoid activity. 1.2 Names, Formula and Molecular Weight Fluoxymesterone is the United States adopted name3 (USAN). The preferred chemical name4 is 9-fluoro-118, 178d ihydroxy-17-methylandrost-4-en- 3-one, Other chemical names are: 11B, 17$-dihydroxy-9a-fluoro-17a-methyl-4-androsten- 3one, 9a-fluoro-11f3-hydroxy-l7a-methyltestosterone, 9a-fluoro118, 178-dih droxy-17a-methyl-4-androsten-3-one, 9a-fluoro17a-methyl-A -androstene-llf3-17f3-dio1-3-one, and 9 r f c - luoro-11, 17-dihydroxy-17-methyl-(ll~-l7~)-androst-4-en-3-one. The chemical abstracts systematic number is 76-43-7. The National Service Center (National Cancer Institute) number is 12165.
1.
OH
Fluoxymesterone has also been called, Androsterolo, Androfluorene, Androfluorone, Fluotestin, Halotestin, Oratestin, OraTestryl, Testoral and Ultandren. Appearance, Color and Odor Fluoxymesterone is a white or practically white, odorless powder consisting of free flowing crystals or crystalline material.
1.3
1.4 Precautions Since the human dose is 1 to 30 mg daily, care should be taken to avoid inhaling the powder. 1.5 Synthesis and Stereochemistry The synthesis596 of fluoxymesterone is summarized in Figure 1. Microbiological oxidation7 of androstenedione ( ) yields lla-hydroxyandrostenedione (11) , which may be I
Figure 1:
Synthesis of Fluoxymesterone
FLUOXYMESTERONE
255
o x i d i z e d w i t h chromium t r i o x i d e t o t h e 11-keto d e r i v a t i v e a d r e n o s t e r o n e (111). S c i s s i o n of t h e dihydroxyacetone s i d e c h a i n of c o r t i s o n e (IV) w i t h sodium b i s m u t h a t e a l s o y i e l d s 1 1 C a r e f u l r e a c t i o n of I11 w i t h p y r r o l i d i n e g i v e s eneamine 1 . formation8 (V) a t t h e r e a d i l y a c c e s s i b l e 3-ketone p o s i t i o n , w i t h o u t d e r i v a t i z a t i o n of t h e 11-ketone. Grignard r e a c t i o n w i t h methylmagnesium bromide, followed by removal of t h e eneamine, l e a d s t o t h e 17a-methyl; 178-hydroxyl d e r i v a t i v e (VI). Reconversion of t h e 3-ketone t o t h e eneamine, followed by r e d u c t i o n w i t h l i t h i u m aluminum h y d r i d e , and t h e n hydrolysis of t h e eneamine, g i v e s t h e 118, 17B-dihydroxy-3-ketone der i v a t i v e ( V I I ) . The secondary ll-hydroxy i s s e l e c t i v e l y conv e r t e d t o t h e t o l u e n e s u l f o n a t e ester, which, on b a s i c hydroly s i s , g i v e s t h e 9(11) o l e f i n ( V I I I ) . R e a c t i o n of V I I I w i t h aqueous N-bromoacetamide g i v e s t h e 9a-bromo-118-hydroxy d e r i v E s s e n t i a l l y t h i s is t h e a d d i t i o n t o t h e double ative (IX). bond of hypobromous a c i d , w i t h t h e i n i t i a l bromonium i o n forming on t h e less hindered s i d e . Bromine i s s u b s e q u e n t l y d i s p l a c e d by a l k o x i d e i o n t o g i v e t h e 9 , 11-epoxide ( X ) . The s y n t h e s i s of fluoxymesterone i s completed by opening t h e o x i r a n e w i t h hydrogen f l u o r i d e 9 t o g i v e t h e 9a-fluoro-llBhydroxy d e r i v a t i v e (XI). R e f e r e n c e s 1 0 t o 1 4 are a d d i t i o n a l sources. Although t h e x-ray s t r u c t u r e of fluoxymesterone w a s n o t determined, t h e s t r u c t u r e of the related s t e r o i d 9a-bromo178- hydroxy-17a-4-androstene- 3, 11-dione was e l u c i d a t e d . 1 5 The o p t i c a l r o t a t o r y d i s p e r s i o n s p e c t r a are similar. The 3k e t o , 9a-fluoro and 17-methyl s u b s t i t u e n t s a r e on one s i d e of t h e molecule. The 11-keto, 17-hydroxy and 18- and 19-dimethyl groups are on t h e o t h e r s i d e : Physical Properties 2.1 I n f r a r e d Spectrometry F i g u r e 2 shows t h e i n f r a r e d spectrum of fluoxymester o n e , r u n as a m i n e r a l o i l m u l l , u s i n g a D i g i l a b Model 14D F o u r i e r t r a n s f o r m spectrophotometer. Below are t h e i n t e r p r e t a t i o n s of v a r i o u s absorbances.16 Absorption (cm-l) 3580 3520 3370 3050 3010 Structural Feature alcohols 2.
A s s ignmen t
0-H s t r e t c h
alkene
C-H s t r e t c h
-0
0 -0
( P
0 -0 00
0 -0
0 -0
.o
.o
.o
0 0 ' 0
(Y
0 0 ' 0 0
,z
c3 -
FLUOXYMESTERONE
257
Absorption (cm-1) 1650 1626 1349, 1232, 1245, 1219, 1175 1146, 1085 1035, 953, 933, 926 903
Structural Feature A4-3-one A4-3-one skeletal modes alcohols and a1iphatic fluorine
Assignment C=O stretch C=C stretch C-C stretch and C-H deformation C-0 stretch and C-F stretch
1457, and 1370 em-1 are due, in part, to the aliphatic C-H vibrations of mineral oil.
at 1146, 953, 933 and 926 cm-1, and the C11 alcohol for the bands at 1035 and 1035 cm-l. Peaks at 2960, 2920, 2870, 2850,
The C17 alcohol may be responsible for absorptions
Infrared spectra were previously determined in liquid paraffin and examinedl7-19 during a futile search for polymorphism
Nuclear Magnetic Resonance Spectrometry (NMR) Figure 3 is the 100 M z NMR spectrum of fluoxyH mesterone in deuterodimethylsulfoxide containing tetramethylsilane as internal reference at 0 Hz. The instrument was a Varian Associates, Inc., Model XL-100 NHR spectrometer equipped to perform Fourier transform spectrometry. Below is an interpretation of the various resonances.20
2.2
Figure 3:
Fourier Transform, Nuclear Magnetic Resonance of Fluoxymesterone in DeuterodimethylSulfoxide, as Recorded on a Modified Varian XL-100 NMR Spectrometer.
Chemical S h i f t (PPd 1.00 1.04 1.3-1.8
R el at i v e Area
Multiplicity Singlet Singlet Overlapping Mu1t i p l e t s Singlet
Type
CH3 group CH
Assignment
H , a s 20B-CH3
3 3 13
group
H,as 17B-CH3
A l i p h a t i c CH2's
CH3 group
1 , 7 , 1 2 , 1 5 , 16-H's
H , a s 19i3-CH3
1.5
1.8-2.4 4 . 05a 4.10 4. 90a 5.65
a
Overlapping Multiplets Singlet Doublet of quartets Doublet of doublets Singlet
A l l y l i c CH2's and m e t h i n e s Exchangeable H (u+H = 20 Hz) Carbinol H Exchangeable H V i n y l hydrogen
2,6,8,14-H' 17-OH
11-H
1 1
1
11-OH
4-H
C o n c e n t r a t i o n dependent.
260
JOEL KIRSCHBAUM
Changing s o l v e n t s t o such a r o m a t i c o n e s as d e u t e r o p y r i d i n e produces changes i n t h e chemical s h i f t s of p r o t o n s on t h e t e r t i a r y - s u b s t i t u t e d methyl groups. T h i s shows t h e i n f l u e n c e of n - e l e c t r o n bonding to hydroxyl groups.21
Mass Spectrometry F i g u r e 4 i s t h e low r e s o l u t i o n mass spectrum of fluoxymesterone as o b t a i n e d on a n A E I S c i e n t i f i c Apparatus, L t d . , Model MS902 mass spectrometer. The sample w a s i n s e r t e d i n t o t h e s o u r c e a t a temperature of approximately 180". The molecular i o n of m / e 336 i s observed, a l o n g w i t h several mass fragment i o n s r e p r e s e n t i n g t h e l o s s of small molecules, hydrogen f l u o r i d e and water i n several pathways.22
2.3
-H
m / e 279 % 2 I0
-HF
M+
1-HF m / e 316
-H20
p m / e 318
l-HF -H20 ) m / e 298
The weak i o n s a t m f e 123 and 124 are r e p r e s e n t a t i v e o f A4-3-ones (CgH110, C8H120). A1 ,4-3-0nes u s u a l l y have v e r y i n t e n s e i o n s a t m / e 1 2 1 and m l e 122. Although t h e s e appear t o be r e a s o n a b l e and t y p i c a l l y s t e r o i d a l assignments, t h e y should b e considered t e n t a t i v e s i n c e t h e assignments a r e n o t confirmed by h i g h - r e s o l u t i o n m a s s spectrometry .23 X-ray Powder D i f f r a c t i o n To o b s e r v e x-ray d i f f r a c t i o n p a t t e r n s , a P h i l i p s powder d i f f r a c t i o n u n i t e m i t t i n g CuKa r a d i a t i o n a t 1 . 5 4 1 w a s used w i t h a s c i n t i l l a t i o n counter d e t e c t o r . Four samples from d i f f e r e n t l o t s were scanned and recorded from -2 to 40 d e g r e e s , (20). The t a b l e below i s t h e s o r t e d d a t a corresponding t o t h e x-ra d i f f r a c t i o n p a t t e r n of fluoxymesterone, F i g u r e 5 . The data94 a g r e e w e l l w i t h p r i o r powder data.25 Lack of s i g n i f i c a n t d i f f e r e n c e s i n d i c a t e s no a p p a r e n t polymorphism. 2.4
Figure 4 :
Mass Spectrum of Fluoxymesterone. Instrument: AEI-MS902. See Text For Details.
4156 FLUOXYMESTERONE SQ9752 180 DEG
2 8 - 0 C-7 6 T-
IIR2 0 3 5
I -
lio1
10
I
INTENSITY
MRSS/CHRRGE SUM = 3 5 6 6 4 BRSE PERK % = 8 . 1 3
Figure 5 :
100
Powder X-ray Diffraction Pattern of Fluoxymesterone. See t e x t for d e t a i l s .

100
50
40
FLUOXYMESTERONE
263
28 (Degrees) 15.71 11.29 15.12 18.94 17.50 20.90 20.22 26.25 29.40 23.45 16.05 16.22 29.91 22.51 30.25 27.19 8.15 32.46 14.27 18.09 30.84 26.76 27.95 33.14 35.94 25.49 34.75 38.07 28.55 23.96 21.92 27.53 23.11 21.32
' d ' (Angstroms)

5.66 7.84 5.86 4.69 5.07 4.25 4.39 3.39 3.04 3.79 5.52 5.46 2.99 3.95 2.95 3.28 10.85 2.76 6.21 4.90 2.90 3.33 3.19 2.70 2.50 3.49 2.58 2.36 3.13 3.71 4.05 3.24 3.85
R l a t ive Area e
1.000 0.995 0.961 0.758 0.581 0.425 0.353 0.330 0.291 0.268 0.262 0.256 0.233 0.225 0.218 0.209 0.199 0.195 0.188 0.187 0.181 0.174 0.173 0.172 0.167 0.162 0.148 0.135 0.121 0.121 0.104 0.087 0.079 0.077
~-
4.17
'Inter planar distance. 'Relative area, or intensity, i s based on highest intensity of 1.00 using CuKa radiation.
264
JOEL KIRSCHBAUM
U l t r a v i o l e t Spectrometr I n e t h a n o l , t h e ,A , w a i r e p o r t e d 1 4 t o be 240 nm S p e c t r a l s h i f t s occur depending on t h e (E = 16,700). s o l v e n t , A t a Amax of 239 nm, u s i n g t r a d i t i o n a l nomenclat u r e , t h e E i E m v a l u e f o r fluoxymesterong, i s 495 ( r e f e r e n c e .M 26). I n 0 l h y d r o c h l o r i c a c i d , t h e Ei& v a l u e is 430. O p t i c a l R o t a t o r y D i s p e r s i o n and O p t i c a l R o t a t i o n The r o t a t o r y d i s p e r s i o n c u r v e s of t h e v a r i o u s 116hydroxy-17a-methyl t e s t o s t e r o n e s are similar t o t h a t of t e s t o s t e r o n e . For fluoxymesterone, specific all^,^^ [a1700 +77", [a1589 +114', [a1275 +3410", %ax" [a]410-430 +199", "min" [ a ] 367.5 -165", "max" [ a ] 360 -79' , "min" [a]353 -198" , " i n f l e c t i o n " [a1340 +484", " i n f l e c t i o n " [a1325 +1450; c = 0.11. 2.6 The s p e c i f i c r o t a t i o n s i n mined29 t o be as f o l l o w s , Wavelength (nm) 589 576 546 436
were d e t e r -
2.5
Specific Rotation
+noo
+116' +131 +211
Fluorescence Spectrometry Fluoxymesterone w a s r e p o r t e d t o g i v e a yellow-green f l u o r e s c e n c e 3 0 a t a c o n c e n t r a t i o n of 100 pg/mL i n 37% hydroc h l o r i c a c i d . T h i s thousand-fold h i g h e r c o n c e n t r a t i o n t h a n i s u s u a l f o r f l u o r o m e t r y i n d i c a t e s t h a t e i t h e r an u n s t a b l e species or an impurity is responsible f o r fluorescence, Melting Range Following t h e U.S.P. procedure31 f o r class LA compounds, t h e m e l t i n g r a n g e of fluoxymesterone i s 262.7-263.7", c o r r e c t e d . 2 6 This v a l u e i s i n f a i r agreement w i t h t h e r e s u l t s of d i f f e r e n t i a l thermal a n a l y s i s (cf. S e c t i o n 2.9, below).
2.8
2.7
D i f f e r e n t i a l Thermal A n a l y s i s A Dupont Model 900 D i f f e r e n t i a l Thermal Analyzer shows fluoxymesterone t o have a s h a r p endotherm3' a t 269". Decomposition on m e l t i n g p r e c l u d e s d i f f e r e n t i a l scanning calorimetry studies for purity,
2.9
FLUOXYMESTERONE
265
2.10 Thermal G r a v i m e t r i c A n a l y s i s Fluox e s t e r o n e , c o n t a i n s less t h a n 0.1% t o t a l volat i l e material. 3yA Perkin-Elmer Model TGS-2 thermogravimetric a n a l y z e r w a s used. The temperature was i n c r e a s e d by 2O0/min. up t o 260', under a n i t r o g e n atmosphere, 2.11 Microscopy and C r y s t a l Type One l o t of fluoxymesterone c o n s i s t s of r e c t a n g u l a r c r y s t a l s of d i f f e r e n t s i z e s . 3 3 T y p i c a l small c r y s t a l s are 100 by 7 p , medium c r y s t a l s are 325 by 20p and t y p i c a l l a r g e c r y s t a l s are 750 by 6 0 p . Hot s t a g e microscopy w a s performed a t a r a t e of 3'/min., u s i n g a Mettler FP 52 t e m p e r a t u r e cont r o l l e r . T h i s l o t showed some c r y s t a l s e x h i b i t i n g t h e f i r s t s i g n s of m e l t i n g a t 253.1", and a l l c r y s t a l s w e r e completely melted a t 280.8'. Another l o t c o n s i s t e d of small, f i n e b i r e f r i n g e n t c r y s t a l s 1 t o 211 s q u a r e . O c c a s i o n a l l a r g e r s l a b s 25 t o 35p s q u a r e are found. T h i s l a t t e r l o t began m e l t i n g a t 273.4' and w a s completely melted a t 285.1'. These s t u d i e s show e v i d e n c e of p o s s i b l e polymorphism. A d d i t i o n a l e v i d e n c e i s from a n o t h e r h o t s t a g e microscopy study34 i n which r o d s , g r a i n s , p r i s m s and stems were v i s i b l e , a g a i n i n d i c a t i n g mult i p l e c r y s t a l forms. 2.12 Polymorphism There i s no e v i d e n c e f o r polymorphism from i n f r a r e d s p e c t r o s c o p y , powder x-ray d i f f r a c t i o n , d i f f e r e n t i a l thermal a n a l y s i s , and o n l y weak d a t a from microscopy. However, i t i s a n t i c i p a t e d t h a t , l i k e many o t h e r compounds, f u r t h e r i n v e s t i g a t i o n w i l l d i s c l o s e d i s t i n c t c r y s t a l forms w i t h markedly d i f f e r e n t p h y s i c a l and s p e c t r o s c o p i c p r o p e r t i e s . 2.13 Hydration The c r y s t a l s are n o t s o l v a t e d w i t h water, based on a water c o n t e n t of less t h a n 0.1%, (cf., thermal g r a v i m e t r i c a n a l y s i s S e c t i o n 2.10 above)
2.14 S u r f a c e Area A s measured by g a s a d ~ o r p t i o n ,t ~ e s u r f a c e area of h~ t h r e e l o t s of fluoxymesterone, were 0.7, 1.06 and 1 . 4 rn2/g. Solution Properties 3.1 I n t r i n s i c D i s s o l u t i o n R a t e The i n t r i n s i c d i s s o l u t i o n r a t e w a s determined a f t e r compressing powder under a 1500 P.S.I.G. p r e s s u r e u s i n g 318" d i a m e t e r disc-shaped d i e s . I n one l i t e r of 0.W h y d r o c h l o r i c a c i d a t 37', a g i t a t e d a t a r a t e of 50 rpm, t h e i n t r i n s i c d i s s o l u t i o n rate of fluoxymesterone i s 2325 x mg min.-l cm-2, u s i n g u l t r a v i o l e t spectrometry.
3.
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S o l u b i l i t i e s i n Aqueous and Nonaqueous S o l v e n t s S o l u b i l i t i e s of f luoxymesterone w e r e determined i n v a r i o u s solvents.33 R e s u l t s are r e p o r t e d u s i n g t h e U.S.P. d e f i n i t i o n s .37
3.2
Solvent D i s t i l l e d water 0. I Hydrochloric a c i d M 0. lM Sodium hydroxide Methanol Acetone Acetonitrile Chloroform Diethylether Benzene Hexane s Py r i d i n e
Solubility Practically insoluble Practically insoluble Practically insoluble Sparingly soluble Slightly soluble Slightly soluble Slightly soluble Practically insoluble Practically insoluble Practically insoluble Soluble
P a r t i t i o n Coefficients Fluoxymesterone w a s p a r t i t i o n e d between water and e q u a l volumes of e i t h e r hexanes, d i e t h y l e t h e r o r chloroform. A f t e r one hour of mixing, t h e s t e r o i d c o n t e n t was determined by u l t r a v i o l e t spectrometry of both phases, wherever p o s s i b l e . The following p a r t i t i o n r a t i o s 3 3 were found f o r t h e compound i n [aqueous p h a s e l o r g a n i c phase]; hexanes, 7.1; d i e t h y l e t h e r , 0.27; and chloroform, 0.016. Phase S o l u b i l i t y A n a l y s i s Using p r e v i o u s l y d e s c r i b e d methodology, 38 f luoxymesterone a t a c o n c e n t r a t i o n of 1 4 . 4 mg/g i n 95% e t h a n o l w a s determined29 g r a p h i c a l l y t o b e 99.7% pure.
4.
Methods of Analysis
3.3
3.4
Elemental and I n o r g a n i c Analyses The elemental a n a l y s i s of fluoxymesterone is: carbon, 71.68% (71.4%, t h e o r e t i c a l ) ; hydrogen, 8.71% (8.69% t h e o r e t i c a l ) , and f l u o r i n e , 5.23% (5.65%, t h e o r e t i c a l ) .34 Emission Spectrochemical a n a l y s i s f o r metals w a s performed40 u s i n g a Spec I n d u s t r i e s carbon arc A.C. u n i t w i t h a Bausch and Lomb d u a l g r a t i n g s p e c t r o g r a p h . Data, recorded o n g l a s s p l a t e s , were i n t e r p r e t e d by means of a microphotometer. Fluoxymesterone contained t h e f o l l o w i n g metallic imp u r i t i e s , i n ug/g; i r o n , 2; manganese, 0.3; calcium, 5 ; magnesium, 95; copper, 0.4; aluminum, 3, and sodium, 68. The
4.1
FLUOXYMESTERONE
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magnesium may be from the Grignard reaction in the synthesis. Residue-on-ignition41 of the same lot42 is 0.13%. Heavy metals content (ref. 43, method 11) is less than 0.001%. After 3 hours at 105" in vacuum, the loss-on-drying31 value42 is 0.07%. A portion of the same lot of fluoxymesterone was dissolved in pyridine and, after retention on a precolumn, water content was determined by vapor phase (gas) chromatography by comparison with authentic standards. The value44 of 0.1% agrees well with the loss-on-drying data. 4.2 Identification, Ultraviolet and Colorimetric Ana1 ses y Compendia1 identification tests45 involve comparing either the infrared or ultraviolet absorption spectra of sample fluoxymesterone with that of an authentic sample. To this author, a chromatographic method is preferable since elution time depends on much of the molecule interacting, via weak bonding forces, with the solid and mobile phases. Ultraviolet absorption (cf. Section 2.5) depends principally only on the 3-one-4-ene, A and B ring region being intact. Fluoxymesterone has been quantitated by a differential borohydride reduction assay.46 This differential assay involves measuring the ultraviolet absorbance of an aliquot of methanolic steroid solution, containing sodium borohydride decomposed prior to the addition of steroid. Its absorbance is determined against a methanolic reference solution of steroid reduced by sodium borohydride. The utility of this procedure is that many interferences from excipients and other unconjugated steroids can be eliminated in the assay of a formulation. The addition of fluoxymesterone to various colorimetric reagents ives results typical of steroids with its s u b s t i t ~ e n t s . ~ ~A s~ tested in our laboratories33, fluoxy. ~ mesterone reacts with acidic ethanolic 4-nitrophenylhydra~ i n e after heating, cooling and the addition of sodium ~ ~ , hydroxide to give a brilliant violet color. With methanolic isoniazid50, fluoxymesterone gives a yellow-green color. Fluoxymesterone added to 4-aminoantipyrine51 in methanolic hydrochloric acid gives a pale green color. Flu0 ymesterone added to ethanolic tetramethylammonium hydroxide55 , and heated, gives a cloudy amber color. If added to ethanolic tetramethylammonium hydroxide53 and picric acid, an orangered (tea colored) solution results with fluoxymesterone. In concentrated sulfuric acid54 fluoxymesterone gives a deep yellow color. Adding water slowly causes a deep violet color
268
JOEL KIRSCHBAUM
t o form a t t h e i n t e r f a c e . Fluox e s t e r o n e g i v e s a r o y a l purp l e c o l o r w i t h b l u e t e t r a z o l i u m , g and a yellow-brown c o l o r w i t h aluminum c h l o r i d e i n nitromethane. 55 Chromatographic A n a l y s i s 4.31 Gas Chromatography Bulk fluoxymesterone, fluoxymesterone i n t a b l e t s and fluoxymesterone i n u r i n e can be q ~ a n t i t a t e d ~ ~ usi n g g l a s s columns packed w i t h 3% o r 4% XE-60 phase on Gas Chrom Q s u p p o r t . The temperature w a s approximately 225" and t h e n i t r o g e n p r e s s u r e from 1 0 t o 24 P.S.I.G. Typically, fluoxymesterone ( r e t e n t i o n t i m e 36.8 min.) can b e s e p a r a t e d from t e s t o s t e r o n e (9.4 m i n . ) , e p i t e s t o s t e r o n e (8.5 m i n . ) , 9a-fluoro-17~-hydroxy-l7-methyl-4-androstene-3,ll-dione (16.8 m i n . ) , and s e v e r a l o t h e r s t e r o i d s . The peaks are Gaussian. R e s i d u a l s o l v e n t s can a l s o be determined by g a s chromatography44 (cf. S e c t i o n 4.1). Methanol c o n t e n t of f luoxymesterone w a s less t h a n 0.1%. 4.32 High P r e s s u r e Liquid Chromatography Reverse phase h i g h p r e s s u r e l i q u i d chromatog r a p h i c methods have been developed t o s e p a r a t e and q u a n t i t a t e b u l k and formulated fluoxymesterone, u s i n absorbance measurements a t 254 mu. A compendia1 procedure45 u t i l i z e s a n o c t a d e c y l s i l a n e column and aqueous methanol mobile phase (4 :1) t o s e p a r a t e and q u a n t i t a t e fluoxymesterone ( r e t e n t i o n t i m e 4-7 min.) w i t h n o r e t h i n d r o n e as t h e i n t e r n a l s t a n d a r d ( e l u t i o n i n 8-14 min.). F i g u r e 6, c o u r t e s y of W. Beyer, Upjohn Co., I n c . , shows t h e r e s u l t s of t h i s procedure u s i n g a flow rate of 0.4 mL/min. The r e s p o n s e i s l i n e a r , e l e c t r o n i c a l l y measuring peak areas of fluoxymesterone and n o r e t h i n d r o n e , from 0.05 t o 0.50 mg fluoxymestrone p e r IUL. The c o e f f i c i e n t of v a r i a t i o n i s 1.4%.
A UBondapak, prepared, o c t a d e c y l s i l a n e column, w i t h aqueous a c e t o n i t r i l e ( 3 : l ) flowing a t 3 mL,/min., gave a r e t e n t i o n t i m e f o r fluoxymesterone of 3 t o 7 min. and f o r n o r e t h i n d r o n e of 6 t o 1 0 min.57 The c o e f f i c i e n t of v a r i a t i o n i s 0.32%. Another assay58 u t i l i z i n g aqueous a c e t o n i t r i l e (3:2) u s e s a reverse-phase o c t a d e c y l s i l a n e prepared P a r t i s i l column. With a flow r a t e of 0.8 mL/min., t h e r e t e n t i o n t i m e f o r fluoxymesterone i s 1 2 min. R e p e t i t i v e i n j e c t i o n s w i t h a p r e c i s i o n loop i n j e c t o r gave a c o e f f i c i e n t of v a r i a t i o n of 0.41%. I n j e c t i n g h i g h e r c o n c e n t r a t i o n s of s t e r o i d , u s i n g a more s e n s i t i v e d e t e c t o r , p e r m i t s t h e q u a n t i t a t i o n o r estimaa t i o n of trace amounts of u l t r a v i o l e t - a b s o r b i n g i m p u r i t i e s t h a t do n o t e l u t e e i t h e r w i t h fluoxymesterone o r a t t h e v o i d volume.
4.3
FLUOXYMESTERONE
269
Figure 6: Reverse Phase High Pressure Liquid Chromatographic Separation of Fluoxymesterone, Norethindrone (Internal Standard) and Related Compounds. See text for details.
3A Alcohol
Solvent (2 ~ 1 )
$ 1 1 -owldo-1 7-methyl testosterone

(0.16 mg)
d
9
LJ
270
JOEL KIRSCHBAUM
4 . 3 3 Thin Layer Chromatography Fluoxymesterone chromatographed59 on silica gel, G.F., as support, with a mobile phase of butyl acetateacetone (8.2) gave a Rf of 0 . 4 8 . Visualization was with short wavelength ultraviolet light. The purity of one lot is 9 9 . 7 % , with one impurity, Rf 0 . 5 7 , accounting for 0 . 3 % . With benzene-ethyl acetate, (CAUTION), 1:1, fluoxymesterone remained at the origin, but a trace impurity migrated. The steroid is quantitated after elution from the plate with ethanol, by ultraviolet spectrometry.
Kieselguhr G plates, impregnated with 10% propylene glycol solution in acetone, were used to chromatograph fluoxymesterone with toluene as mobile phase.60 Visualization utilized a 20% solution of p-toluenesulfonic acid in 94% ethanol as spray, followed by heating at 120" for 10 minutes, and then examination under visible and long wavelength ultraviolet light. Fluoxymesterone was chromatographed on silica gel, F-254, plates and visualized either by ultraviolet light or spraying with 40% methanolic sulfuric acid. Fluoxymesterone gave one spot in four mobile phases29: benzene-acetone, 4 l (CAUTION) , Rf 0.22: chloroform-methanol (93:7) , Rf 0.41; : ethanol-chloroform-benzene (CAUTION) ( : : ) 2 2 1 , Rf 0.89, and methanol-ethanol-chloroform-acetone ( 3 : 3 : 3 : 1 ) , Rf 0.70. Model studies for gas-liquid chromatography56 involved thin layer chromatography to determine polarity. With benzene-ethyl acetate, 1:l (CAUTION), the Rf of fluoxymesterone is 0.12. Testosterone and epitestosterone have an Rf of 0.27 and androsterone has an Rf of 0 . 3 8 . In ligrointoluene-methanol-water (5:5:7:3) fluoxymesterone, testosterone and epitestosterone are not separated, Rf 0.42. Androsterone has an Rf of 0 . 3 9 . In benzene-ether (l:l), fluoxymesterone remains at the origin, testosterone has an Rf of 0.16, epitestosterone 0.15, and androsterone 0 . 2 3 . Reverse-phase thin layer chromatography, using silica gel plates impregnated with silicone oil, was utilized to study the lipophilic character of 101 steroids,61 including fluoxymesterone. Relative mobilities (R,) in 15 to 65% acetone were determined and extrapolated to 0% acetone. For fluoxymesterone Rm is -0.5%. %I values, using 45% acetone as the mobile phase, generally correlate with steroidal partition coefficients in ether-water, hemolytic activity and membrane and protein binding. The effect on A R , of substituting or derivatizing groups could also be estimated.
FLUOXYMESTERONE
271
4.34 Paper Chromatography Various hormonal s t e r o i d s were s p o t t e d on Whatman62 No. 1 paper. A f t e r being dipped i n t o 50% m e t h a n o l i c propylene g l y c o l , chromatography w a s w i t h e i t h e r h e p t a n e s a t u r a t e d w i t h propylene g l y c o l (46 hours) o r heptane-methanolwater, 10:8:2 (3 h o u r s ) . V i s u a l i z a t i o n w a s w i t h e i t h e r 20% antimony t r i c h l o r i d e i n chloroform, 20% phosphoric a c i d , o r s u l f u r i c a c i d - a c e t i c a c i d ( 1 : 9 ) , under normal and u l t r a v i o l e t l i g h t . Relative t o t e s t o s t e r o n e , t h e m o b i l i t y of f l u o x y mesterone is 0.0 i n b o t h systems. Whatman No. 1 paper h a s a l s o been used i n two o t h e r systems63 t o d e t e r m i n e t h e homogeneity of fluoxymesterone. Twenty p e r c e n t formamide i n methanol a s s t a t i o n a r y phase, and methyl i s o b u t y l ketone-formamide ( 2 0 : l ) as mobile phase; 25% p r o p y l e n e g l y c o l i n chloroform as s t a t i o n a r y phase, and t o l u e n e s a t u r a t e d w i t h propylene g l y c o l , as mobile phase, were once r o u t i n e l y used f o r d e t e r m i n i n g p u r i t y . 4.35 Column Chromatography A diatomaceous e a r t h column w a s used t o separ a t e fluoxymesterone from e x c i p i e n t s . 6 4 Aqueous methanol w a s t h e s o l v e n t f o r t h e f o r m u l a t i o n and chloroform w a s used t o e l u t e t h e s t e r o i d . Q u a n t i t a t i o n involved u l t r a v i o l e t s p e c t r o metry. Fluoxymesterone i n T i s s u e s and Body F l u i d s B i o l o g i c a l a s s a y s f o r fluoxymesterone i n lower a n i mals i n c l u d e e f f e c t s on seminal v e s i c l e w e i h t , 6 5 sperm mobili t y 6 6 and r a t e of mounting dnd e j a c ~ l a t i o n . ~ 7I n man, t h e s e e f f e c t s can be evidenced by i n c r e a s e s i n f e r t i l i t y . 4.4
A radioimmunoassay h a s been developed68 i n which t h e d e r i v a t i v e of fluoxymesterone i s conj u g a t e d t o bovine serum albumin. Hapten i s prepared i n rabb i t s w i t h t h e a i d of F r e u n d ' s Adjuvant. The r e s u l t i n g a n t i body b i n d s 75% of 6,7-3H-fluoxymesterone a t a 1:5000 d i l u t i o n . Twenty-five picograms of fluoxymesterone c a n be d e t e c t e d i n 0 . 1 m l of u n e x t r a c t e d serum. No endogeneous human s t e r o i d s c r o s s react a p p r e c i a b l y w i t h t h e primary a n t i s e r u m . Specif i c i t y is d e r i v e d mainly from t h e 116-hydroxyl and 17-methyl group, s i n c e t h e r e i s c o n s i d e r a b l e c r o s s r e a c t i o n w i t h l-dehydrofluoxymesterone, 1 7 - m e t h y l t e s t o s t e r o n e and 118-hydroxy-17m e t h y l t e s t o s t e r o n e . I n j e c t i n g 2 , 5 o r 1 0 m of fluoxymesterg one i n t o dogs gave peak c o n c e n t r a t i o n s , a f t e r 1-2 h o u r s , of 120, 195 and 505 ng/mL, r e s p e c t i v e l y .
3-[0-(carboxymethoxime)]
272
JOEL KIRSCHBAUM
Using gas chromatographys6 (of. Section 4 3 ) less .1, than 5% of injected fluoxymesterone (plus closely related compounds) was found in human urine. Stability: Effects of Temperature, Time and Light Fluoxymesterone, formulated into tablets, was stored at 22" or 40' for 28 months. After extraction with chloroform, drying, and rediesolution in ethanol, the steroid was assayed with isonicotinic acid hydrazide (nydrazid) and blue tetrazolium reactions (cf.Section 4.2) or by paper chromatography followed by nydrazid. Results within 6% of label were found, 42 indicating stability.
5.
Storage of solid fluoxymesterone, at 50' for 3 weeks, showed neither a loss in content nor the appearance of new peaks, on comparison with refrigerated fluoxymesterone. Analyses utilized high pressure liquid chro~natography~~. (f c Section 4.32)
Exposure of fluoxymesterone to approximately 900 footcandles of light for 3 weeks caused a 2% loss in content, as determined by reverse-phase high pressure liquid chromatogra~h.5~ new peak eluted prior to fluoxymesterone and was A equivalent to 0.3%, assuming similar absorbtivity as fluoxymesterone, and correcting for diffusion. Photolytic degradation of the A-ring is expected since hydrocortisone and prednisolone undergo rearrangements69-71 when alcoholic solutions are exposed to ultraviolet radiation or ordinary fluorescent laboratory lighting. Acknowledgements The author gratefully acknowledges the assistance of the many contributors cited as "personal communication." In many instances, the information was especially obtained for this summary. Special thanks go to R.M. Johnson and W. Beyer of the Upjohn Company, Kalamazoo, Michigan, for their help; to R. Poet, G. Brewer and S. Perlman of E.R. Squibb, New Brunswick, New Jersey, for their critical reading of this MS., and to my family, for their patience and understanding shown during the compiling of this analytical profile.
6.
FLUOXYMESTERONE
273
7.
References
1. P h y s i c i a n s ' Desk Reference, 3 1 t h E d i t i o n , Medical Econnomics Co. I n c . , O r a d e l l , New J e r s e y , 1 9 7 7 , p . 1 6 2 7 . Edward H. Fr i e d e n . i n "Chemical Endocrinology" , Academic Press, New York, 1 9 7 6 , p. 6 1 . 2. J . F r i e d and E . Sabo, J . Amer. Chem. Soc., 7 5 , 2273 (1953). 3. USAN and t h e USP D i c t i o n a r y of Drug N a m e s , 1 9 7 7 , p. 1 3 2 . 4. Merck Index, 9 t h E d i t i o n , Merck and Co., I n c . , Rahway, N e w J e r s e y , p. 541, no. 4 0 7 0 . 5. D. Lednicer and L.A. M i t s c h e r i n "Organic Chemistry of Drug S y n t h e s i s , ' ' John Wiley & Sons, New York, 1 9 7 7 , p. 1 7 6 . 6. U.S. P a t e n t 3 , 1 6 0 , 6 4 4 , December 8, 1 9 6 4 t o Upjohn Co., CA 62, 7837f ( 1 9 6 5 ) . 7 . D.H. P e t e r s o n , H.C. Murray, S . H . E p s t e i n , L.M. Reinecke, A.. Weintraub, P.D. Meister, and H.M. Leigh, J . Arner. Chem. Soc., 7 4 , 5 9 3 3 ( 1 9 5 2 ) . 8. U.S. P a t e n t 3 , 3 5 1 , 6 4 2 , November 7 , 1 9 6 7 , t o Upjohn Co., CA, 68, 1 1 4 8 5 4 r ( 1 9 6 8 ) . 9. J . F r i e d and E.F. Sabo, J . Amer. Chem. Soc., 7 6 , 1 4 5 5 (1954). 10. French P a t e n t 1 , 3 7 4 , 5 9 1 , October 9 , 1 9 6 4 , t o C I B A , L t d . , CA 62, 9 2 1 2 a ( 1 9 6 5 ) . 11. Swiss P a t e n t , 3 3 1 , 9 8 3 , September 3 0 , 1 9 5 8 , t o C I B A L t d . , CA 53, 8 2 1 3 b ( 1 9 5 9 ) . 1 2 . U.S. P a t e n t 2 , 8 3 7 , 5 1 7 , J u n e 3, 1 9 5 8 , t o Upjohn Co., CA 53, 3 3 0 1 e ( 1 9 5 9 ) . 13. U.S. P a t e n t 2 , 7 9 3 , 2 1 8 , May 2 1 , 1 9 5 7 , t o Upjohn Co., CA 52, 1 2 9 8 d ( 1 9 5 9 ) . 1 4 . M.E. Herr, J . A . Hogg and R.H. Levin, J . Amer. Chern. SOC., 78, 500 (1956). 15. A. Cooper, C.T. Lu and D.A. Norton, J . Chern. Soc., B , 1228 (1968). 1 6 . P. Meulman, Upjohn Company, p e r s o n a l communication. 1 7 . R . S . Mesley and C.A. Johnson, J . Pharm. Pharmacol., 17, 329 (1965). 18. R.S. Mesley, Spectrochimica Acta, 22, 8 8 9 ( 1 9 6 6 ) . 1 9 . O.R. Sammuel, W.L. Brannon and S.L. Hayden, J . of t h e AUAC 4 7 , 9 1 8 ( 1 9 6 4 ) . 20. W. Beyer, Upjohn Company, p e r s o n a l communication. 21. M. P u a r , E.R. Squibb p e r s o n a l communication. 22. P. Funke, E.R. Squibb, p e r s o n a l communication. 23. A. Cohen, E.R. Squibb, p e r s o n a l communication. 24. Q. Ochs, E . R. Squibb, p e r s o n a l communication. 25. J . P a r s o n s , W.T. Beher and G . Baker, Henry Ford Hosp. Med. B u l l . , 9 , 54 ( 1 9 6 1 ) .
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26. 27. 28. 29. 30.
31. 32. 33. 34.

35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47.
48. 49. 50. 51. 52. 53. 54. 55.
B. Kabadi, E.R. Squibb, p e r s o n a l communication. C. Djerassi, R. R i n i k e r and B. R i n i k e r , J . Amer. Chem. Soc., 7 8 , 6377 (1956). United S t a t e s Pharmacopeia, 2 8 , 936 (1970) L. Grady, United States Pharmacopeia N a t i o n a l Formulary p e r s o n a l communication. B. Steinmetz, V . Beach, B. Dubnick, A. Meli and G . Fujimoto, Steroids, 2 , 395 (1963). United S t a t e s Pharrnacopeia. 29, 651 (1975). H. Jacobson, E.R. Squibb, p e r s o n a l communication. S. Perlman, E.R. Squibb, p e r s o n a l communication. M. Kuhnert-Brandstxtter, E . Junger and A. K o f l e r , Microchern. J . , 9, 105 (1965). G. Vinjamuri, E.R. Squibb, p e r s o n a l communication. A. Dhruv, E.R. Squibb, p e r s o n a l communication. United S t a t e s Pharmacopeia, 29, 6 (1975). National Formulary, 24, 942 (1975). J . A l i c i n o , E.R. Squibb, p e r s o n a l communication. N. T r a n d i f i r , E.R. Squibb, p e r s o n a l communication. United S t a t e s Pharmacopeia, 29, 620 (1975). G. Brewer, E.R. Squibb, p e r s o n a l communication. United S t a t e s Pharmacopeia, 29, 619 (1975). A. Niedermayer, E.R. Squibb, p e r s o n a l communication, United S t a t e s Pharmacopeia, 24, 206 (1975). J . Kirschbaum, J . Pharm. S c i . , 6 7 , 275 (1978). J. B a r t o s and M. Pesez, in "Colorimetric and F l u o r i m e t r i c A n a l y s i s of S t e r o i d s , " Academic P r e s s , New York, 1976, and M. Pesez and J . B a r t o s in "Colorimetric and F l u o r i metric A n a l y s i s of Organic Compounds and Drugs," Marcel Dekker, New York, 1974. A.A. F o r i s t and J.L. Johnson i n "Pharmaceutical Analysis," T. Higuchi and E . Brochmann-Hanssen, E d i t o r s , I n t e r s c i e n c e P u b l i s h e r s ( J . Wiley), New York, 1961, p . 71. M. Kimura, T . Nishina, and T . Sakamoto, Chem. Pharm. Bull. (Tokyo), 25, 454 (1962). E. Cingolani, G. Cavina, and V. Amormino, Farmaco (Pavia) Ed. Prat., 2 5 , 301 (1960). E.P. Schulz, M. Diaz, G . Lopez, L . Guerrero, H . B a r r e r a , A. Pereda, and A. A q u i l e r a , Anal. Chem., 36, 1624 (1964). J. Cross, H. E i s e n and R . Kedersha, Anal. Chern., 24, 1049 (1952). M. Pesez and J . B a r t o s i n "Colorimetric and F l u o r i m e t r i c A n a l y s i s of Organic Compounds and Drugs," Marcel Dekker, New York, 1974, p. 481. S. B e r s t e i n and R. Lenhard, J . Org. Chem., 28, 1146 (1953). S. GtirUg and gy. Szdsz i n A n a l y s i s of S t e r o i d Hormone Drugs," Akadgmiai Kiadd, Budapest, 1978.
FLUOXY MESTERONE
275
56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71.
M. Ruchelman, CZin. Chim. Acta., 26, 253 (1969). H. Abdou and T . A s t , E.R. Squibb, p e r s o n a l communication. V . Gibbs and J . Kirschbaum, E . R . Squibb, p e r s o n a l communica t i o n . P. T a y l o r , E.R. Squibb, p e r s o n a l communication. D. Sonanini and L . Anker, Pharm. Acta. HeZv., 4 2 , 54 (1965). G.L. B i a g i , A . Barbaro, 0. G a n d o l f i , M. Guerra, and G. C a n t e l l i - F o r t i , J . Med. Chem., 18, 873 (1975). G. Cavina, E . Cingolana and A . G i r a l d e z , Farmaco (Pauia) Ed. Pract., 27, 149 (1962).
H. Roberts, Squibb, personal communication. R. Graham and C. Kenner, J . Pharm. Sci., 62, 1845 (1973). W. Johnson and L. T i e f e r , Endrocinozogy, 95, 912 (1974). J. Brown, J . FertiZ. and SteriZ., 26, 305 (1975). R. Dorfman, Acta. Endrocin., 42, 265 (1962). W. Coburn, Steroids, 2 5 , 43 (1975). D.H.R. Barton and W.C. T a y l o r , J . Chem. Soc., 2958 2500. B i d , J . Amer. Chem. Soc., 80, 244 (1958).
W. Hamlin, T . C h u l s k i , P.A.
Johnson and J . G . Wagner, J . Arner. Pharm. Assoc. ( S c i . E d . ) , 49, 253 (1963).
L i t e r a t u r e reviewed t o J a n u a r y , 1978.
HEXETIDINE
Gerhurd Satzinger, Wo&ung Herrmann. and Friedrich Zimmermunn
277
27M
GERHARD SATZINGER et al.
TABLE OF CONTENTS 1. 2. 3. 4. Introduction Description Preparation and Stability Physical Properties 4.1 Refractive Index and Density 4.2 Partition Coefficients 4.3 Dissociation Constant 4.4 IR Spectrum 4.5 NMR Spectrum 4.6 Mass Spectrum Analytical Methods 5.1 Elemental Analysis 5.2 Chromatographic Analysis 5.2.1 TLC Analysis 5.2.2 GLC Analysis 5.3 Titration in Nonaqueous Medium Experimental Acknowledgement References
5.
6.
7.
8.
HEXETIDINE
279
Introduction Hexetidine (1,3-bis(2-ethylhexy1)-5-amino-Smethyl-hexahydropyrimidine) can be prepared in pure state with difficulty only. For both the preparations HexoralR 1 and HexoralR-Spray') a purified hexetidine is used which is prepared from commercial hexetidine according to a process (1) patented to Goedecke Company, Berlin. Structurally, hexetidine occupies a special position among the chemotherapeutics; even among the heterocycles with biocidal activity there is no chemically comparable substance.
1.
Hexetidine is a highly substituted derivative of hexahydropyrimidine. As is well known, the parent compound "pyrimidine" possesses an extraordinary biological importance as structural element of the purines, thiamine, uric acid, uracil and others. Therefore, it is of special interest that the "synthobiotic" activity of hexetidine could be traced back to its interaction with metabolic processes of vital importance for the growth of pathogenic organisms ( 2 ) . The broad spectrum of antibacterial and fungicidal activity of hexetidine is a result of this property. Another striking characteristic of hexetidine is its unusual tissue affinity. Topically applied, hexetidine is not displaced too early from the active site by either physiological or pathological secretions. Purified hexetidine differs from commercial hexetidine ( 3 ) mainly by its thin-layer chromatographic and gas chromatographic purity. As yet, only a few analytical data of commercial hexetidine In the have been reported in the literature (4,5) following, we report mainly on physico-chemical properties of purified hexetidine and on spectroscopic data pertaining to its structure.
Description Hexetidine (I) is 1,3-bis (2-ethylhexyl)-5amino- 5 -methyl-hexahydropyrimidine.

2.
Purified hexetidine is a clear, colorless, oily liquid of weak, amine-like odor. 'Manufacturer: Goedecke AG, Berlin
280
(1)
ICH2
I
Hexetidine
Empirical formula : C2 IH45H3 Molecular w e i g h t : 3 3 9 . 6
Hexetidine can be distilled in high vacuum (b.p. 160 C/0.4 Torr (4); 140 C/0.3 Torr ( 6 1 1 , but purification cannot be achieved in this way, Hexetidine is readily soluble in organic solvents such as acetone, ethanol, benzene, chloroform, n-hexane and methanol, but is practically insoluble in water (~lO-~g/ml)
Preparation and Stability Hexetidine is synthesized technically according to a three-step process (see Formula Scheme 1) starting from nitroethane, formaldehyde and 2-ethyE. hexylamine (6).
3.
HEXETIDINE
281
Step 1
Step 2
+CH20
Step 3
(1)
Formula Scheme 1
During the course of our studie- n hex-tidine, some new findings as to the chemical behavior of I and related compounds were obtained. Thus, we have found, for example, that contrary to the literature ( 4 ) the hexahydropyrimidinering system in hexetidine is not particularly stable; it can be cleaved thermally and hydrolytically giving rise mainly to the open-chain compound I1 and to the condensated heterocycle 111. This process is some kind of disproportionation which can be reversible under certain conditions.
C H CH- CH2 -N- CH, -
'-1
C2H5
I NH2
P3
- CH2-N- CH2 -CH- C 4Hg

H
11
C2H5
N 1,N3-Bis-(Q-ethylhexy1)-2-methyl-propane= triamine- (1,2,3) ("triamine")
282
I11
2,6-Bis-(f3-ethylhexyl)-hexahydro-7a-methyllH-imidazo[1,5-c]imidazole ( "hexedine")
I1 and I11 are formed also under the conditions of the technical synthesis and subsequent distillation according to the process of the Commercial Solvents Corporation so that commercial hexetidine always is a mixture of about 7 5 % of hexetidine and of about 10% of I1 and I11 each. Moreover, additional amines can be detected in smaller amounts by thin-layer chromatography.
With the aim of using a drug as pure as possible for Hexoral, we have developed a process that allows to remove the by-products contained in commercial hexetidine. This process essentially consists of selectively separating I from the mixture of the amines with the aid of an aromatic disulfonic acid at low temperatures. On the subsequent recovery, I is obtained with a purity of about 99%. The amines I1 (7) and I11 (8) (both colorless oils of slight amine odor) have not been characterized in detail in the literature up to now. Their most important data are listed in Table 1. Table 1 Physical Properties of Triamine (11)
Triamine (11) Empirical formula Mol. wt.
B.p.
na O
C20H4SN3
&
Hexedine (111)
Hexedine ( I I I
c2 zH4 SN 3
327.6 L30-132C/0.05 Torr 1.4580 0.884

9.1
351.6 125OC/O.O2 Torr 1.4660 0.853

6.0
dB ,"
PKa
HEXETIDINE
283
Table 1 (continued)
I
Ti tra tion
Triamine (11) corresp. to I eq w t 1/ Bmol.w t
Hexedine
( III
. .. ,
corresp. t o I e q w t = 1/ mo 1 ,w t
. .
IR (neat)
3360 cm- ' N , )str, ) (H 3300 c m - l (NH,, NH str.) 2810 e m e 1 (N-CH, str.) 1570 cm- ' N , (H 1115 cm- '(C-N 2790 cm(N-CH, str.)
d. )
str. )
1115 c m- 1 (C-N str.)
Purified Hexetidine n2 D d !
O
C o m m e r c i a l Hexetfdine
1.4640 0.8697
1,4668 1.4653 0.8889 0.868
1.4686 0.8832
4.2
Pnrt ition Coefficients (purified

hexetidine) K C C
H 0 ,
CHCl
=
3
low3
1 x lo'
buffer 7.4
284
K =
Ha 0
'n- oc t a n 0 1
c c
buffer 7.4
n-octanol
1 x lo-*
The partition coefficients were determined according to a method described in detail in the literature ( 9 ) (c = 1.5 x 10 mole/l).
4.3
Dissociation Constant
PKa = 8 . 3 (purified hexetidine) Due to the sparing solubility of the bases in water (see also 11, 111) the dissociation constants were determined by titration with 0.1 M H C 1 in 50% aqueous ethanol (c = 8 mMole/l).
Method: pK
= p H
- l o g rundiss* c a t i o n ] [ base
= p H
; if [ B]-[ B H ' ] ,
K then p
[lo].
4.4
IR Spectrum
The IR spectrum of purified hexetidine (Fig. 1) exhibits the characteristic bands of the nitrogen groups (see Table 3 ) .
0 1 4ooo
3xK)
?no0
2500
Moo
leoo,-l
I600
Moo
1200
mo
600
400
250
Figure 1:
IR Spectrum of Purified Hexetidine (neat on CsJ).
286
Table 3 Band Positions and Vibrational Modes of the IR Spectrum of Purified Hexetidine
V i b r a t i o n a l mode
3360
3300
2920 2860 2800
asym. sym.
SP.
N-H s t r e t c h i n g of NH,
C-H s t r e t c h i n g of N-
C-H s t r e t c h i n g of CH,
and CH,
1570 1460 1380
N-H d e f o r m a t i o n of NH,
asym.
Sym.
C-H d e f o r m a t i o n s of CH, and CH,
C-N s t r e t c h i n g
N-H d e f o r m a t i o n s of NH2
The LR spectrum of commercial hexetidine shows an additional band at 1600 cm which is to be assigned predominantly to the dehydrohexetidine according to our investigations.
4.5
NMR Spectrum
The NMR spectrum (Fig. 2) confirms the structural formula. The assignment of the signals (chemical shift 6 in ppm) to the individual molecular groups (for numbering, see structural formula) is given in Table 4 . A complete analysis including 270-MHz proton spectrum and 13C-spectrumgives a number of information concerning structural details. After completion, these studies will be the subject of a separate publication.
E
U U
c1
4
*
-4
W
a a,
84
288
GERHARD SATZINGER et a].
Table 4 Aasignments in the NMR Spectrum
Mass Spectrum The mass spectrum of purified hexetidine (Fig. 3) shows along with the molecular ion at m/e 339 at M-1 peak at m/e 338 and characteristic ions at m/e 226, 197 and 142 (base peak) which can be accounted for by the fragmentations as represented in Formula Scheme 2. 5. Analytical Methods 5.1 Elemental Analysis (Purified Hexetidine) C H N Calc. : 74.28 13.35 12.37 Found : 74.06 13.29 12.44 5.2
4.6
Chromatographic Analysis 5.2.1 TLC-Analysis The following experimental conditions are especially suitable f o r the purity control of I:
Layer : S i l i c a g e l F 2 5 4 (Merck p r e c o a t e d p l a t e ) 10 x 20 c m ( P r e t r e a t m e n t w i t h ammonia vapor) Benzene/methanol ( 8 : 2 ) ( s a t u r a t e d atmosphere) c a . 15 c m ca. 1 h
Solvent system: Running d i s t a n c e : Running t i m e :
i d
Q,
Ef
b
Q,
I..
a
I
.. a
rl
rl
Fr
HEXETIDINE
291
Detection: Sample solution: Sample size:
n-hexane 1 pl (corresp. 500 p g )
1 0 mg of hexetidine/ml of 0
Iodine vapor (ca. 30 min)
According to the thin layer chromatogram (cf. Fig. 4 ) commercial hexetidine contains at least 3 additional accompanying compounds (amines), each in amounts of more than 1%. These were isolated and their structure, unless known, was elucidated. As may be seen from the TLC, the spots at hRf 65 and at hRf 15 correspond to the described amine I11 (hexedine) and to the base I1 (triamine), respectively. The spot at hRf 35 could be assigned to an oxidation product of hexetidine, named by us dehydrohexetidine (IV) the properties and structure of which will be reported on in a separate publication.
Fig. 4 :
Thin layer chromatogram of purified hexetidine and commercial hexetidine as compared to 1%-spots of 11, I11 and dehydrohexetidine (IV) each.
292
The concentrations of spots present in the TLC in addition to hexetidine (I) can be estimated semiquantitatively as follows: hRf hRf hRf hRf hRf
65: (ca. 12%), 111 45: (ca. 70%), I 35: (ca. 5%), IV 15: (ca. lo%), I1 75-70 and 10: (each l%), unknown.
5.2.2
GLC Analysis
The gas chromatographic separation of amines I1 and I11 and of additional unknown accompanying compounds of hexetidine and the reproducible quantitative determination of the components without thermal isomerisation and without formation of artifacts can be achieved on a few stationary phases and on glass columns only. The following operative GLC conditions proved advantageous (see Fig. 5): Apparatus : Column : Packing material: Carrier gas: Hydrogen: Synth. air: Oven temperature: FID detector and temperature programming 1.0 m,glass; 2 mm internal diameter. 1% OV 7 on 80-100 mesh Chromosorb G HP 4 0 ml N2/min. 30 ml/min. 350 ml/min. Program: 0-15 min: 15OoC. isothermal, 15-27 min: 4zC/min 27-38 min: 198 C. 0 24OOC. 240 C. 20 cm/h. 0 . 5 p1 of a solution in n-hexane ( a 5 ug)
Injection port: Detector: Chart speed: Sample injected:
1 I
a a (D r w
'C II r.
rt 0
Y O(DQ 0
r rx
(D(D9
X rta 10 r-P r t a Y
r-(D o 3 3
r-r-ra3 rn
(D
A A
P-
294
Rt- times
Commercial hexetidine
~
Values of one batch (see Fig.5) Hexetidine (I): 755 sec Triamine (11): 961 sec Hexedine (111): 1173 sec De hydr o== hexetidine(1V): 1382 sec Unknown 2076 5.3
(72.2%)
Var i a t ion
(8.5%) (14.4%) (3.7%) (1.2%)
70085% 5- 10% 5-15% 1- 5% max. 2%
Titration in nonaqueous medium Contrary to the literature ( 5 1 , the content of hexetidine can be determined without difficulties by nonaqueous titration in glacial acetic acid with perchloric acid either potentiographically or with quinaldine red or crystal violet as indicator. Equivalent weight = 1/2 mol. wt., i.e. 1 ml of 0.1 N HClO4 is equivalent to 16.98 mg of hexetidine. Experimental Spectra and chromatograms were recorded using the following instruments:
6.
W: IR: NMR : MS :
GLC :
Titration: 7.
Beckman Acta V Perkin-Elmer 457 Varian HA 100 Varian MAT CH4 Varian Aerograph 1840 Metrohm Potentiograph E 436
Acknowledgement The authors are grateful to Prof. Dr. H. Achenbach, Chemical Institute of the University of Freiburg/Brsg., for recording and interpreting the mass spectra.
HEXETIDINE
295
References
1.
2.
3.
4.
5. 6.
7. 8. 9.
10.
DBP 20 11 078 (Goedecke AG, Berlin), date of application: March 9, 1970. Corresponding patents in 18 countries. Church, B.D., Read, R.R., Sanders, R.G., Abstr. Papers Division Medicinal Chemistry, p . 2 N, Meeting of American Chemical Society, April 1957. Manufacturer: Commercial Solvents Corporation, New York (USA). The Merck Index (USA), 8th ed.p. 530 (1968). Breinlich, J., Pharm. Ztg. 46, 1774 (1971). Senkus, M., J. Amer. Chem. S O C . 68, 1611 (1946). Mulinos, M.G., Erger, B.D., Ostashever, A . S . , Antimicrob.-Ag. Chemother. p . 857 (1961). McMillan, F.H., Fr. 4501 (Nov. 14, 1966). Ullmann's Encyklopzdie der techn. Chemie, Vol. 2/1, p . 79, Verlag Chemie, Weinheim/ Bergstr. (1961). Abrahamczik, E., Houben-Weyl, 4th ed. Vol. 3/2, p. 166, Verlag G. Thieme, Stuttgart (1955).
HYDROFLUMETHIAZIDE
Chester E . Orzech, Norris G . Nash, and Raymond D . Daley
297
Copyright @ 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-2608070
298
CHESTER E. ORZECH et al.
CONTENTS
1.
DESCRIPTION 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor
PHYSICAL PROPERTIES 2.1 Infrared Spectra 2.2 Nuclear Magnetic Resonance Spectrum 2.3 U l t r a v i o l e t Spectre 2.4 Mass Spectrum 2.5 D i f f e r e n t i a l Thermal Analysis (mA) 2.6 Crystal Properties 2.7 S o l u b i l i t y 2.8 Ionization Constant 2.9 Fluorescence Spectra 2.10 Melting Points
2.
3.
SYNTHESIS STABILITY-DEGRADATION M T B LS E A O IM M T O S OF ANALYSIS EH D 6.1 I d e n t i f i c a t i o n Tests 6.2 Elemental Analysis 6.3 Color iw tr ic Method 6 4 T i t r a t i o n Methods . 6.5 Paper Chrometography 6.6 Column Chromatography 6.7 Ultraviolet Methods 6.8 Polarography 6.9 Thin Layer chromatography REFERENCES
5.
6.
7.
HY DROFLIJMETHIAZIDE
299
DESCRIPTION
1.1 Name, Formula, Molecular Weight Hydrof lume t h i a t i d e is 3,4-dihydro-6-( t r i f luorome thy1)2H-1,2,4-benzothiad iaz ine- 7-sulfonamide 1,l-dioxide , also known as 6-trifluoromethyl-3,4-dihydro-7-sulfamoyl-2H-1,2,4benzothiadiazine 1,l-dioxide and as 3,4-dihydro-7-sulfamyl-6tr i f luorome thyl-l,2,4-benzothiadiaz ine 1,l-dioxide. Chemical Abstracts indexes i t a s 2H-1,2,4-benzothiadiazine-7-sulfonamide, 3,4-dihydro-6- ( t r i f luorome thy1)-, 1,l-dioxide. The CAS Regis t r y Number is [ 135-09-11.
Mol. Wt.:
331.29
1.2 Appearance, Color, Odor Hydrof lume t h i a z i d e is white t o cream colored, odorl e s s , powder o r c r y s t a l s .
2.
PHYSICAL PROPERTIES
2.1 I n f r a r e d Spectra The i n f r a r e d spectrum of hydrof lumethiazide N ReferF ence Standard Lot No. 69178 is s h o k i n Figure 1. The spect r u m was obtained i n a potassium bromide dispersion, using a B e c h n Model IR-12 spectrophotometer. A similar spectrum is observed when a mineral o i l or o t h e r d i s p e r s i n g medium is used, except f o r absorption bands of the medium. The spectrum is similar t o those a l r e a d y published (1,2). In addition to the absorption bands shown here, hydroflumethiazide exhibits weak but s h a r p bands a t 382, 338, and 315 cm-1, and very weak bands a t 267, 242, 230, and 218 cm-1, when the spectrum is obtained as a f a i r l y thick mineral o i l dispersion. Some of the absorption bands may be assigned as follows (3):
Figure 1.
Infrared Spectrum of Hydroflumethiaeide, Potasrium Braaide Pellet.
HYDROFLUMETHIAZIDE
301
Absorption Bands, cm 3380, 3260, 3170 1300 t o 1350 1140 t o 1200
Assignment
N-H s t r e t c h
-SO2- s t r e t c h ; -CF3 de format ion

-SOz-
s t r e t c h ; -CF3 d e f o r m t ion
2.2 Nuclear Magnetic Resonance Spectrum The NER spectrum shown i n Figure 2 was obtained by dissolving A y e r s t- house reference standard hydroflume t h i a z i d e d59-1 i n ace tone-d6 containing te tram t h y l s i l a n e as i n t e r n a l reference. The s p e c t r u m was produced using a Varian EM-360 N M spectrometer. The peaks a t 7.42 ppm and 8.37 ppm a r e due t o the aromatic protons. The methylene protons are a t 5.00 ppm and the -NH protons a r e the broad peaks a t 6.70, 6.90, and 7.45 ppm. The s e r i e s of peaks a t 2.10 and 3.07 ppm are due t o the solvent. 2.3 U l t r a v i o l e t Spectra Figure 3 is the u l t r a v i o l e t absorption spectrum of NF Reference Standard hydrof lume t h i a z i d e , Lot No. 69178, in methanol. The spectrum was obtained on a Cary Model 14 spectrophotometer a t a concentration of 19.3 mg per l i t e r . Disc o n t i n u i t i e s i n the spectrum a r e due t o changes i n the absorbm ance range. The 277 t o 267 nm and the 222 t o 210 n regions of the scan a r e on the 1.0 t o 2.0 absorbance range; the r e m i n d e r of the scan is on the 0.0 t o 1.0 range. Pilsbury and Jackson (4) compare u l t r a v i o l e t s p e c t r a
of hydroflumethiazide with those of other thiazides. They r e p o r t E ( l a , 1 cm) for hydroflumethiazide of 450 a t 276 nm a t
p 10, 580 a t 273 nrn a t p 2. H H
Kracmar and Las tovkova (5,6) compare u l t r a v i o l e t s p e c t r a of hydroflumethiazide i n methanol, ethanol, 0.1E hydrochloric a c i d , and 0.lE potassium hydroxide t o the s p e c t r a of other thiazides. The a b s o r p t i v i t y (log 6 ) of hydroflumethiazide is reported a s 4.182 a t 275 nm i n 0.1E potassium hydroxide, 4.290 a t 273.5 n i n 0,lE hydrochloric a c i d , 4.229 m a t 272.5 nm i n ethanol, and 4.286 a t 272.5 i n methanol.
Other u l t r a v i o l e t s p e c t r a l d a t a a r e those of DePaulis and Dipietromaria (7), who r e p o r t E ( R , 1 cm) of 560 a t 274 nm i n 70 percent ethanol; Fazzari ( 8 ) , r e p o r t i n g a n absorpt i v i t y of 45.4 l i t e r l g cm a t 273 nm i n 0.2E sodium hydroxide; and Sunshine (9), with s p e c t r a i n 0.lg hydrochloric a c i d and in 0.1E sodium hydroxide solution. The B r i t i s h Pharmacopeia
9
Figure 2.
Proton Mopetic Resonance Spectrum of Hydroflumethiazide, in Deuterated Acetane,
Figure 3.
Ultraviolet Spectrum of Hydroflumethiazide, 19.3 - / l i t e r ,
in Methanol.
304
1973 u l t r a v i o l e t i d e n t i t y test (10) corresponds t o an absorpt i v i t y of about 46 l i t e r / g cm a t 274 nm i n 0.OlK sodium hy-
droxide
Figure 4 is the low resolution mnss spectrum of hydroflumethiazide NF Reference Standard Lot No. 69178, obtained with an LKB 9000s mass spectromtter, ionization voltage 70 eV, source temperature 25OoC. Some of the peaks m y be assigned as follows (11): m/e 331 m/e 331 m/e 303 m/e 303 m/e 239 Molecular ion EH
2.4 Mnss Spectrum
-CHNH
m/e 303 (*277.3)

b
-so
-SO2
m/e 255 m/e 239 m/e 222 (*206) m/e 174 m/e 158
b
b
-NM
m/e 222
m/e 222
-so
-SO2
m/e 69
2.5 Differential Thermal Analysis (DTA) The DTA curve i n Figure 5 was obtained w i t h a DuPont Model 900 instrument. The curve shows only a malting endothem a t 274OC with subsequent decomposition,
2.6 Crystal Properties
The powder d i f f r a c t i o n pattern is given i n Table I. This pattern was obtained w i t h 8 Norelco diffractometer, using nickel-filtered copper radiation. The pattern is similar t o one published by Corrigan and Timoney (12); they a l s o report the existence of a I t 1 c r y s t a l l i n e ethanol solvate. Kuhnert-Brandsti'tter e t a1 give information for the microscopic character iea tion of hydrof lume thiazide (13), as does Groenewegen ( 4 . 1)
0
I -
a a
W
a a a
I
tn tn
I:
() I
O -
o m a o r c c o m o n c u AlISN31NI 3 A I l W l 3 t l
I -
r--
0 4-
Figure 5.
Differential Thermal Analysis Curve of Hydroflumethiazide.
HYDROFLUMETHIAZIDE TABLE I
307
X-RAY P W E DIFFRACTION O DR PATTERN OF HYDROFLUMETHIUIDE
5.44 5.08 4.90 4.66 4.58 4.34 4.27 4.05 3.93 3.69 3.60 3.48 3.37 3.32 3.25 3.23 3.14 3.06 3.01 2.88 2.77 2.72
30
1
4 41 100 17 22
5
2 16 3 5 5 3 6 5 14 1 1 6 2 2 3
2.59 2.54 2.50 2.44 2.41 2.35 2.31 2.25 2.22 2.13 2.08 2.05 2.02 2.01 1.96 1.94 1.92 1.90 1.87 1.83 1.79 1.77
1 1 3 10
2 4 2 1
5 2 1 2 1 1 1 1 2
2 4 2 1
2.7 S o l u b i l i t y Some room temperature s o l u b i l i t i e s a r e a s followst Solvent Approximate S o l u b i l i t y , mg / m l 58 21.
Me thano 1 Ethanol (957.)

Ethyl Acetate Ethyl Ether (anhydrous) Chloroform Ben2 ene Water Ace toni tr i l e Ace tone
. .
11.
>loo.
0.2 0.1 CO. 1 0.3 ( 1 5 ) 43
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 n and an emiosioa maximum a t 393 nm. Fluorm ercence 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
Figure 6. Synthesis of Hydrof lume thiazide
/.
clso/
2. NQOH
1 (HCHOJn, .
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.
M T O S OF ANALYSIS EH D
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). The elemental composition of hydroflume thiazide is a8 follass :
6.2 Elemental Analysis
HYDROFLUMETHIAZIDE
Element Carbon Hydrogen F luor ine N i tr ogen Oxygen Sulfur 7 Theory . 29.00 2 -43 17.21 12.68 19.32 19.36
311
6.3 Colorimetric Method Bermlo (46) determined hydroflumethiazide by hydrolyzing i t to ~,4-disulfamyl-5-trifluoromeh y l a n i l i n e , diazot t 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. 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 , H developing f o r 20 minutes a t 9OoC with p 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
6.5 Paper Chromatography
312
CHESTER E. ORZECH et
01.
with thiazides
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.
6 6 Column Chromatography Fazzari ( 8 9 48) used a &sic celitt@ column to purify
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 ID .. Me thanol-cyclohexane-chloroform-ace t i c acid (40r30t30:l vol/vol) Elution Volum: 72 t o 117 m l Column: Eluant: Quantitation: Sample:
Fluorescence 333 nm excitation, 380 n emission m About 100 u g of hydroflumethiazide
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 c x 4 m ID m .. 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 23 8nd 6l 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.
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
6.8 Polarography
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. AK O LD MNS C N WE G E T 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
TABLE XI
THIN LAYER CHROMATOGRAPHY SYSTEMS FOR HYDROFLUMETHIAZIDE
Absorbent Alumina G S i l i c a Gel G 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
Solvent Sys tern 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:
u
0.36
Reference
40 40 40 41 41
0.41 04 .6 05 .2
Oa
53
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
3:2)
04 .3
42 43 43 54 44 45
55
G25-22
Benzene-B thyl Acetate (2:8) Ethyl AcetateM thanol- Anmonia e (85:10:5) Ethyl AcetateBenzene (8:2) Ethyl Acetate Ethyl Acetate E t h y l AcetateMethanol-0. !l Annnonia (96:2:2)
NR*
NR*
07 .8 0.45 0.45
05 .2
*Not Recorded
HYDROFLUMETHIAZIDE
315
REFERENCES
1.
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).
2.
3. 4. 5. 6.
7.
8. 9.
10.
11.
12. 13. 14. 15. 16. 17. 18. 19. 20.
21. 22.
316
CHESTER E. ORZECH et 01.
23 24. 25. 26. 27. 28
F.
29. 30. 31, 32. 33 34 35 36 37. 38
. . ..
39. 40 41. 42. 43
44. 45. 46. 47.
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, 255-64 (1961); 4 . C.A. 56, 10286e. H. C. Chiang, J. Pharm. Sci. 5 0 , 885-6 (1961).
fi,
HYDROFLUMETHIAZIDE
317
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.
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
1 . 2.
Introduction Description 21 Name, Formula, Molecular Weight . 22 Appearance, Color, Odor . 3. Physical Properties 3 1 Infrared Spectrum . 3 2 Nuclear Magnetic Resonance Spectrum . 33 Ultraviolet Spectra . 3 4 Acidity Constant . 35 Melting Range . 3 6 Thermal Analyses . 3 . 7 Crystal Properties and X-Ray Powder Diffraction 3 8 Solubility . 39 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 . 84 Chromatographic Methods . 84 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) . .
9 References .
HYDROXYZINE DIHYDROCHLORIDE 1 . Introduction
321
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 21 . 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
22 .
2HC1
Mol. Wt. = 447.83
Appearance, Color, Odor Hydroxyzine dihydrochloride is a white, odorless crystalline powder.
3.
Physical Properties 31 . 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
OF4
..
rn
ri
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) 3100-3700 Assignment
0-H stretching
C-H stretching tertiary amine salt6 aromatic C=C in plane skeletal vibrations C-0-C stretching aromatic CH out-of-plane bending
2800-3100
2410 1596, 1495, 1455 1090 700, 760, 805 32 .
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) protons all methylene protons (16) methine proton (1) all aromatic protons (9)
6064 .-.
7.2-8.3
3-0-4-4
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,
. . . I . . U
..
. I . . . 2 . . I . . . . % . . . 1 . . . . 1 . . . . 1 . . . . 1 . . . . 1 . . . . 1 . . l . . . . I . . . . l . .. . 1 . . . . 1 . . . .
I d . . . , . .I . . . . , . .
1 U
1
1
1
a
6
?a
11181
Figure 2-NMR Spectrum of Hydroxyzine Dihydrochloride, NF .. Reference Standard LOT NO. 6818. Solvent: Deuterated Dimethylsulfoxide, Internal Standard: tetramethylsilane. Instrument: Varian A 60.
325
3.3
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 p 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 H 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 p 1.83. H 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 W found t h a t t h a t p a r t of t h e spectrum e 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 1 s a t about 200 C t with d e c o m p o ~ i t i o n . ~ t m 1 ing range i s reported I El elsewhere t o be 192-3OC.
3,18,
3.6
Thermal Analyses The D C thermogram of hydroxyzine dihydrochloride S obtained a t a scan r a t e of 10C/min. exhibited a wide range endotherm from 190C 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 150C t o 200C 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
327
F'i
I: e
4-UV Spectra of Hydroxyzine DihydI-ochlor i.de Instrument: Cary 14
328
JOSEFTSAU AND NICHOLAS DEANGELIS
37 .
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 14.28 10.79 7.49 71 .3 6.59 60 .9 5.78 52 .6 5.13 4.53 4.37 41 .3 39 .6 37 .6 35 .8 32 .8 32 .0 30 .4 29 .4

=
I/I0 19 14 6
6
62 . 82 . 1. 18 12.4 13.4 1. 45 1. 53 1. 68 17.2 1. 95 2. 02 21.4 22.3 2. 35 2. 47 2. 69 27.6 2. 91 30.0

d (interplanar distance)
50 100 18 56 30 39 64 89
4 6
45 43
4 6
31 26
54
1/10 = relative intensity
L sin
38 .
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.
330
39 .
Mass Spectrum The electronic impact ( . . EI) 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 200C 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 (... ., C1) Table I11 High Resolution Mass Spectrum of Hydroxyzine Assignment
% Relative Intensity
375 ( . . c1) 374.1732 (E.I.) (Calc. 374.17611
MH+
90
M+
299
M+- CH, OCH, CH, OH 18

Cl-(4-c-g+ d-LCH,+
201
173
100 10
4 .
Synthesis 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
ud:w
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,
Lo
ii
W
k CI
U QI
Lo
Figure 7
A Synthetic Route or Hydroxyzine Dihydrochloride
p-chlorobenzhydryl chloride
piperazine
p-chlorobenzhydryl p iperaz ine
"A 'n
I
wcH-NW
N-CHZ CHZ OCH2 CH, OH CHCl, ref Zuxing acid-base extract ion
C 1CH2CH, OCH, CH, OH
hydroxyz ine base HCl/THF hydroxyzine dihydrochloride
333
pH
Percent of initial concentration
3 5
10 1
51
93
100
7 2
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 N=6.26%,
The theory elemental analysis is: C=56.3%, H=6.53%,

CL23.7570, 0=7.14"h.
334
8 Methods of Analysis .
81 . 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.
82 .
Formation of Color Derivatives With concentrated % SO, hydroxyzine develops a yellow color with a maximum absorbance at 460 ,2,3 .22 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
83 .
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
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 300C 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 carLsoprod0 . 1 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 300C 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 2 % (NH, ),C03 in water .5
(5:3)
flow rate: 1 5 ml/min .
336
JOSEFTSAU AND NICHOLAS DEANGELIS

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) acetone ch1oroform:methanol (90:10) 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)
cyc1ohexane:benzene:diethylcyc1ohexane:diethylamine: 0 benzene ( 75 :2 :15)
Ad sorbent Silica Gel G

II
Impregnated with
Rf
06 .8
Ref 32
I1
04 .
0.1N NaOH
I1
11
02 .2 0.33 05 .9
It II 11
Silica Gel
11 II
0.1M KOH
11
11
II
II
0.1M NaHSOG
II
00 .8 0.59 0.39
0.56
34
I1 11
II
0.25
I1
Silica GelgYPsm Silica Gel G W254 Silica Gel G W254

II II
0.1N Na, CO, 01 .N

I1
II
0.45
35 33
11
Na,CO,
04 .0
0.48 0.53
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 Impregnated with 0.1N
9
04 .9
Ref -
Na, CO,
33
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)
0.64
36
04 .1 08 .
0.68
16
It
It
0.65
0.30
0
11
II
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
Color Room temp 100C white bluish ye 1low flesh
Fluorescence 100C Room temp
Ref
32
gold violet
B
C
yellow brown violet brown
D
E
37
II
F
G
green-yellow
II
32
A.
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
B .
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 .

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. E.
%Mn04 s o l u t i o n (1%) Dragendorff r e a g e n t Iodop l a t i n a t e r e a g e n t I o d i n e chamber
F.
G.
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 InterestYffAOAC, J .... . Washington, DC , 1972. .. .. W E Thompson. R J Warren, IB 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); - 75 C A -9 .. 41145 t H G Morren, Synthesis-Belgium, 523, 899 (1954); . . . CA - 18072 d .. - 53, 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. -'- 79 (5), 70(August 4, 1956). Week J Pawlaczyk, P T P Nauk and Wyd. Lek., Pr. Kom. . . . . Farm - 117(1966); CA 5, .. 67, 5679 w. -0s J Pawlaczyk, Ann. Pharm. I , 127 (1969); CA 72, . .. 103676 r . . S. F Pong and C L Huang, J Pharm. Sci., 63, . . . 1527 (1974). .. G Cannizaro, Boll. Chim. Farm. 104, 39 (1965); CA . 63, 2264 g . J A Close, J G. Gobert and L A. M Rodriguez, . . . . . Proc. Eur. Soc. Study Drug Toxicity, 9, 1 4 ( 9 8 . 4 16) 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, - 261 . . 18, (1962); CA 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); CA .. 79, 49044 n . I M Roushdi, SA Soliman, and M H Sadek, . . .. . . UARJ .... Pharm. Sci., 12, 353 (1971). K Nikolic and 2. Cupic, Acta Pharm. Jugoslav., . . 21, 159 (1971); - - 103816 U C A 76, ..
2 . 3 .
z,
4 .
5 .
6 .
7 .
8 . 9 .
10
11. 12. 13. 14. 15. 16. 17. 18. 19
--
2. 0
21.
--
22. 23.
--
2. 4
25.
--
341
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 l , 79 (1958). 8 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); --64, 6405 h. C.A. 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,
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
344
STEVEN A. BENEZRA AND PENELOPE R. B. FOSS
Table of Contents
1.
Description
1.1 Name, Formula, Molecular Weight 1 . 2 Appearance, Color, Odor

2. 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 Synthesis Stability
3.
4.
5.
Methods of Analysis 51 . 5.2 5.3 5.4 5.5 5.6 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
6.
Pharmacokinetics and Metabolism 6.1 Excretion Rate 6.2 Tissue Distribution 6.3 Metabolism
6-MERCAPTOPURINE
345
1. Description
1.1 Name, Formula, Molecular Weight Mercaptopurine is purine-6-thiol monohydrate
SH
C5H4N4S * H 2 0
170.19
1.2 Appearance, Color, Odor Mercaptopurine is a yellow, practically odorless, crystalline p w e ' odr. 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
*rl
k 7
cd
\D
k a,
w
0
al
rn
a
b)
348
Proton S-H C-H (aromatic) C-H (aromatic) N-H
Chemical Shift (ppm) 2.75-3.75 broad singlet 8.15 singlet 8.35 singlet 11.0-12.0 broad singlet
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 0.1N HC1 Methanol
)i
max (nm) 230 312 222 327 216 329
(3) 14000(5) 19600 924012; 3 21300( ) 84( 903 19300)
2.4 Mass Spectrum The low r solution mass spectrum of mercaptopurine is shown in Figure 4% I was obtained with a Varian-MAT model t 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%)
y> > ;
m/e 119 ( 16%)
H2N
H2NXLJ7+
N
CzNSH'
m/e71 ( 1 1 % )
m /e 98 ( 10%)
(CN?
m/e 26 (20%)
6-MERCAPTOPURINE
349
I .4
I .2
I .o
0.8
0.6
0.4
0.2
0
I.4
W 0
I .2
I .o
a m
0 2
0.8 0.6
0.4
rn
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 1
nm
Figure 3- Ultraviolet Spectra of 6-Mercaptopurine Top-O.1N HC1, Middle-O.1N NaOH, Bottom-Methanol
100
> I cn
I-
80
60
w
A
z -
> - 40 I a
w
20
0
SO
I00
m /e
Mass Spectrum of 6-Mercaptopurine
Figure 4-
6-MERCAPTOPURINE
351
2.5 Melting Point Mercaptopurine decomposes above 308C1
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 tiometrically fs 7.77 ani 11.17 respectively
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.
I& *
?=J
d-
I z
6-MERCAPTOPURINE
353
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) Element X calculated
C -
35.28
H -
3.55
N -
32.92
S -
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.
5.3 Spectrophotometric Analysis The official USP analysis of mercaptopurine in tab. 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 1 x 100 cm colunm packed with .8 and animal tissues Beckman M71 strong cation exchange resin was eluted with 0.4M
354
ammonium formate (pH 4.6) at 8 ml/hr. The column was kept at 50C. 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 ( : ) 82. 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
6-MERCAPTOPURINE
355
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)
8. U.S. Patent 2,697,709
So,
9. U.S. Patent 2,721,866

1 . U.S. Patent 2,724,711 0 1 . U.S. Patent 2,756,828 1
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., 16. A.H. Chalmers, Biochem. Med.,
80, 9
(1977) (1975)
12, 234
17. D.G. Bailey, T.W. Wilson, G.E. Johnson, J. Chrom., 305 (1975) 18. M.J. Harber, J . L . Maddocks, J. Chrom.,
11 1,
1 1 231 0,
(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. 22. 23.
G.B. Elion, S Bieber, G H Hitchings, Ann. N . Y . Acad. . .. Sci., 60, 297 (1954)
M.G. Donelli, T. Columbo, A. Porgione, S. Garattini, Pharmacol., 2, 311 (1972)
R.H. Adamson, Ann. N.Y. Acad. Sci.,
179, 432
(1971)
PHENOBARBITAL
Marcus K . C . Chao. Kenneth 5. Albert, und Salvatore A . Fusari
3 59
360
MARCUS K. C. CHAO et al.
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
7.1 7.2 7.3 7.4
- Microchemical Tests
Differential Thermal Elemental Colorimetric Spectrophotometric
PHENOBARBITAL
361
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. 9.
Automated Analytical System References
362
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.
2.
Physical Properties
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)
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.
3080-3220 1710, 1770 1500, 1590
1250-1450
830 720, 770
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 ~ ~ - K B ~ ) and (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)
Assignment C=O stretching. Ring "breathing" vibration. Mono-substituted phenyl group.
1690, 1740 630 620, 1000, 1040, 1590
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
m<r>
2.2 7.8 7.3 2.7 11.6 -1.6
0.8
Chem ica1 Shift
Relative Intensity
3 2 5
Mu1tip1icity triplet quartet singlet singlet
Assignment
9.2
- 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. Instrument: Bruker WH-90
.,k
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
217
Re1ative Intensity 16.2 4.0 100.0 6.9 13.5

10.8 4.0
M +
Structure Assignment
M-(CH ) M- (COY M- (HNCO) M- (HNCONH) M- (CONHCO) M- (CONHCONH) M- (C H5) - (CONHCONH) C6H5
232
204 161 146

117 77 189 174
23.2
8.4
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.
372
MARCUS K. C. CHAO et a].
Figuro 7. Ultraviolot Abaorbonco Spoctrum of Phonobarbital (Lot 597821) in aolutiona with difforont pH. Inatrumont: Porkin- Elmor 124
0.6
0.5
y1
0.4
2 a
03 .
0.2
0.1
220
240
260
280
300
320
340
Wavolongth (nm)
PHENOBARBITAL
373
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) .
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
3 74
MARCUS K. C. CHAO et 01.
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: Organic Solvents 1-Chlorobutane Benzene Dichloromethane Diethyl Ether Where
=
to be 7.3 and hi.8 by Krahl2I and Butlerz2 respec-
K 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-workers measured the K values for 23 three organic solvents and the results are presented below: K Values in Various Solvents at Different pH
PH
4
6 7 8 9 10
5
CH2C12
19.0 11.5 4.9 3.0 1.10
0.35
CHC13
4.0
DCE 10.1 8.1

5.7
3.6 3.6 2.7 0.54 0.14 0.08
0.01
DCE
=
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
pH 7 . 4 0.005 0.552 2.35 2.85
pH 12.4
0.0004 0.0006
0.0014 0.003
0.004
Hexane 0.006 Toluene 0.538 Chloroform 4.16 1,2-Dichloroethane 5.26 Ether 16.4 Ethyl Acetate 57.3 Acetone
17.4
762
52.0
3.
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 1 following steps: ( ) 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
376
Scheme I
HI 0
Figure 8. Synthetic Procedure for phenobarbital
PHENOBARBITAL
377
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
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-workers reported that about 39 11 to 25% of the dose of phenobarbital was found in the patient's urine in the form of unchanged drug.
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
(I) is the major metabolite of phenobarbital. On
Butler38 found that the p -hydroxyphenobarbi tal
PHENOBARBITAL
379
Scheme
Scheme
O Y
GH.5
'\
J
=O
$-1/
O H
-0
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 . Also blades and dark splinter-rods in clusters5iarge red 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
7.1 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
382
is obtained for Phenobarbital Lot 597821. Element

% Found
7, Theory
62.06 12.06
C H N
62.54 5.30 11.97
5.21
7.3
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 Glbc!: odah) determined phenobarbital by precipitating it with mercury and y complex spectrophotometrically measuring th 5f:5 modified with dithizone. A few laboratories5 ? g y 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 .
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 6 for phenobarbital as presented by Kucharsky!! , 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.
384
MARCUS K. C.CHAO et al.
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 ( ml) and using a water-saturated solution of 1 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
386
MARCUS K. C.CHAO et
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 N.M. Column Length Base 2 m 3.175 mm Stationary Phase Support Detector
F.I.
Ref. 92
Chromosorb W 4% SE-30 8 0 / 100 6% QF-1 on acid washed DMCS 2% QF-1

3% QV-17
TMAH
(Flash Heater)
200 cm
6 ft,
3mm 0.25 in. 0.25 in. N.M. 2 m
Chromosorb W
60/80
F.I. E.C.D.
F,I.
93 94 95 96 97
TAAH (Flash Heater)

TMAH
Gas Chrom Q 100/120 Chromosorb W

60/80
(On Column) (Pre Column)
5 ft.
N.M. 200 cm
5% OV-17
3% XE-60
F.I.
TMAH
Gas Chrom Q l o o / 120 Supelcoport methyl-phenyl (50 : 50) 100/120
N.M.
F.I.
TMPAH
3% SP2250
TABLE 5 (Continued) Derivatization N.M. Column Length Base

6 ft.
2mm
Stationary Phase
3% OV-17 or SP2250
3% ov-101
Supp ort
Detector
Ref.
98
Deactivated E.C.D. Chromosorb 750

100/120
TBAH (on Column) TMAH N.M.
1.8 m
6 ft. 4 ft.
2mm
Chrom G HP 100/120 Chromosorb W HP/80/100 High Performance Chromosorb G Celite 545

100/120
F.I.M.
99
100
101
1/8 in.
4mm
10% UV-W98
1 OV-17 %
F.I.
F.I.
N.M.
213 cm
4 mm
3% OV-17
102
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
TABLE 6 DETERMINATION OF PHENOBARBITAL BY MODERN LIQUID CHROMATOGRAPHY* Mobile Phase NaCl Retention Volume (m1)
11
Column
Flow Rate (ml/hr)

26
Remark
8OoC
Ref. 103
Anion Exchange, Pellicular Type LSF (0.04 in. i.d. x 120 in. 1 )
SAX (Du Pont) ( 2 . 1 mm i.d. x
(a) 9.36(c)
2.25(')
1m 1 )
0.01 M Na3B03 0.01 M NaN03
28.8 28.8
() b (b)
(b)
104
0.01 M Na3B03 0 . 0 5 M NaN03

0.01 M Citric Acid
21.6 24
4.59(c)
1.17(C)
ETH Permaphase (Du Pont) ( 2 . 1 mm i.d. x 1 m 1)
d.d. Water
(b)
104
*UV detector ( 2 5 4 nm) is used for the measurement unless otherwise mentioned in the remark.
TABLE 6 (Continued) Column Micro Pak 10 p (Varian) (2 mm i.d. x 50 cm 1) VBondapak c18 4 (Waters) ( mm i.d. x 30 cm 1 ) Silica Gel (h) Bondapak C18 on Corasil I1 (1/8 in. i.d. x 2 ft. 1) Corasil c18 (Waters) (h) Corasil Phenyl (Waters) (h) Mobile Phase CH2C12-CH30HNHbOH ( 9 2 : 7 : 1 CHgOH-Buffer pH 3 (60:40) d.d. Water CH OH-HzOHOic (25:75: 0.1) Absolute Methanol 1% (NH4)H2PO4 (40: 60) Flow Rate (ml/hr) 45 120 Retention Volume (ml) 3
(d)
Remark
Ref. 105 106 107 108 109
4.4
3 4.4 2.15
W detection
(e)
at 230 nm
(f )
50
120
3OoC
(g)
84
(el
Absolute Methanol1% (NH4)H2P04 (40:60)
84
1.6
(e)
109
TABLE 6 (Continued)
Column
1~.
Mobile Phase Chloroformdioxane 2-propanolacetic acid (310:9.7 :l.0: 0.1)
Flow Rate (ml/ 1 hr
Retention Volume (ml)

4.5
Remark (i)
Ref.
Porasil (Waters) ( 4 mm i.d. x 30 cm 1)
90
77
SAX (Permaphase AAX) (2.1 m i.d. x 50 cm 1)
4 mM (NH412HP04 pH 6.2
24
1.7
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.
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. Pippenger18 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.
394
MARCUS K. C. CHAO et a].
References
1. "The Merck Index", 9th Edition, Merck & Co., Inc., Rahway, N.J., 1 9 7 6 , p. 9 3 9 . 2 . "The United States Pharmacopeia XIX", Mack Printing C o . , Easton, Pa., 1 9 7 5 . 3 . Gardner-Thorpe, C. and Schneider, H. : "Clinical Pharmacology of Anti-Epileptic Drugs", Schneider, H . ; Janz, D.; Gardner-Thorpe, C.; Meinardi, H. and Sherwin, A. (Eds.), Springer-Verlag, New York, 1 9 7 5 , Chap. F. 4 . Schoeb, E.; Warner-Lambert/Parke-Davis, Ann Arbor, MI, Personal Communication. 5 . "Infrared and Ultraviolet Spectra of Pharmaceutical Compounds", Revised Edition, Association of Official Analytical Chemists, Washington, D.C., 1 9 7 2 , Spectrum No. 441, 4 4 2 , 4 4 3 . 6 . Clark, E.G., "Isolation and Identification of Drugs", The Pharmaceutical Press, London, 1 9 6 9 , p. 7 5 4 . 7 . Mesley, R.J. and Clements, R.L., J. Pharm. Pharmac. 2 0 , 3 4 1 (1968). 8 . Willis, J T . ; Cook, R . B . and Jankow, R., Anal. Chem. 44, 1 2 2 8 (1972). 9 . Scott,R., Warner-Lambert/Parke-Davis, Ann Arbor, MI, Personal Communication, 10. Elvidge, W.F., Quart. J. Pharm. and Pharmcol,
11. m m i d t , G. : "Methods of Forensic Science", Landquist, F. (Ed.), Vol. 1, Interscience Publishers, New York, 1 9 6 2 , p. 3 7 3 . 1 2 . Mesley, R.J.; Clements, R.L.; Flaherty, B. and Goodhead, K., J. Pharm. Pharmac. 3, 3 2 9 ( 1 9 6 8 ) . 1 3 . Winchell, A.N., "The Optical Properties of Organic Compound", 2nd Edition, Academic Press Inc., New York, 1 9 5 4 , p. 2 3 4 . 1 4 . "Official Methods of Analysis", 12th Edition, Horwitz, W. (Ed.), Association of Official Analytical Chemists, Washington, D.C., 1 9 7 5 . 1 5 . Williams, P.P., Anal. Chem. 3 1 , 1 4 0 ( 1 9 5 9 ) . 1 6 . Huang, T.Y., Acta. Pharm. Intern. 2, 4 3 ( 1 9 5 1 ) . 17. Tabern, D.L. and Shelberg, E.F., JT Am, Chem. SOC. 5 5 , 3 2 8 ( 1 9 3 3 ) . 18. Krausq G.M. and Cross, J.M., J. Am. Pharm. Assoc. Sci. Ed., XL, 1 3 7 (1951). 1 9 . Aldrich, B . and Watson, T.R., The Australasian J. Pharm. -, 1271 ( 1 9 5 5 ) . 30
1 3 , 219 ( 1 9 4 0 ) .
PHENOBARBITAL
395
2 0 . Urdang, A. and Leuallen, E.E., J . Am. Pharm. Assoc. 4 5 , 5 2 5 ( 1 9 5 6 ) . 2 1 . Krahl. K E . , J. Phys. Chem. 4 4 , 4 4 9 ( 1 9 4 0 ) . 2 2 . Butler, T.C.;Ruth, J.M. andTucker, G.F., J. Am. Chem. SOC. 7 7 , 1 4 8 6 ( 1 9 5 5 ) . 2 3 . Biggs, A.I., J. Ch=. SOC., 2 4 8 5 ( 1 9 5 6 ) . 2 4 . Bush, M.T. and Sanders-Bush, E.: "Antiepileptic Drugs", Woodbury, D.M.; Penry, J . K . and Schmidt, R.P. (Eds.), Raven Press, New York, 1 9 7 2 , p. 293-302. 2 5 . Riedel, E.; Krzger, U.; Bayer, H. and Horak, H.:
26. 27. 28. 29.
D.M.;Penry, J.K. and Schmidt, R.P., (Eds.), Raven Press, New York, 1 9 7 2 , Chap. 3 0 . 4 0 . Harvey, D.J.; Glazener, L.; Stratton, C . ; Nowlin, J . ; Hill, R.M. and Horning, M.G., Res. Comm. Chem. Pathol. Pharmacol. 3 , 557 ( 1 9 7 2 ) . 41. Freudenthal, R.I. and Carroll, P . 1 . : "Drug Metabolism Reviews", Vol. 2 , D1 Carlo, F.J. (Ed.), Marcel Dekker Inc., New York, 1 9 7 4 ,
p. 2 6 5 .
(1935). 3 0 . Rotondaro, F.A., J . Assoc. Offic. Agr. Chem. 38, 809 ( 1 9 5 5 ) . 3 1 . Dunker, M.F., J. Am. Pharm, Assoc. Sci. Ed. 3 8 , 409 ( 1 9 4 9 ) . 3 2 . Kapadia, A.J., Goyan, J.E. and Austin, A , , J. Am. Pharm. ASSOC.,Sci. Edition, 407 ( 1 9 5 9 ) . 3 3 . Goyan, J.E.; Shaikh, Z . I . and Austin, A.; ibid, 627 ( 1 9 6 0 ) . 3 4 . Tishler, F.; Sinsheimer, J.E. and Goyan, J.E., J. Pharm. Sci. 5 1 , 2 1 4 ( 1 9 6 2 ) . 3 5 . Maulding, H.V.;Tolesuk,J. and Rosenbaum, D., J. Pharm. Sci. 6 4 , 2 7 2 ( 1 9 7 5 ) . 3 6 . Schmitz, R . E . a s Hill, J.S., J. Am. Pharm. Assoc. Pract. Ed. 11, 5 0 0 ( 1 9 5 0 ) . 3 7 . Raun-Jansen, A.; Lzding, M. and Secher, O., Acta Pharmacol. Suec. 2 7 . 1 9 3 ( 1 9 6 9 ) . 3 8 . Butler, T.C., J. PharmEol. Exp. Ther. 116, 326 ( 1 9 5 6 ) . 3 9 . Maynert, E.W.: "Antiepileptic Drugs", Woodbury,
"Chemical Pharmacology of Antiepileptic Drugs", Schneider, H.; Janz, D.; Gardner-Thorpe, C.; Meinardi, H. and Sherwin, A.L. (Eds.), SpringerVerlag, New York, 1 9 7 5 , p. 3 1 4 . Meijer, J.W.A., ibid, p. 2 8 9 . Inman, M.T. and Butler, W.P.; U.S. Pat. 2 , 3 5 8 , 0 7 2 , 1 9 4 4 (Kay-Fries Chem. Co.), Hoerlein, H.; U.S. Pat. 1 , 0 2 5 , 8 7 2 ( 1 9 1 2 ) . Chamberlain, J.S., J . Am. Chem. SOC. -, 3 5 2 57
396
MARCUS K. C. CHAO
et
al.
42. Heinze, E. and Kampffmeyer, H,G.,Klin. Wochenschr 9 , 1146 (1971). 43. Garrettson, L.K. and Dayten, P.G., Clin. Pharmacol. Ther. 11, 674 (1970). 44. Rose, S.W.; Smith7I.D. and Penry, J.K.: "Blood Level Determinations of Antiepileptic Drugs. Clinical Value and Methods", Washington, D.C., U.S. Dept. of Health, Education and Welfare, 1971. 45. Buchthal, F. and Lennox Buchthal, M.A. : "Antiepileptic Drugs", Woodbury, D.M.; Penry, J.K. and Schmidt, R.P. (Eds.), Raven Press, New York, 1972, p. 193. 46. Kutt, M., Clin. Pharmacol. Ther. 16, 243 (1974). 47. Vaj da, F. ; Williams, F. ; DavidsonFS , ; Falconer, M. and Breckenridge, A., Clin. Pharmacol. Ther. 15, 597 (1974). 48. m n , Y.J.; Awazn, S.; Hanano, M. and Nagami, H., Chem. Pharm. Bull. 21, 2749 (1973). 49. McArthur, J.N.; DawEns, P.D. and Smith, M.J., J. Pharm. Pharmacol. 23, 32 (1971). 50. Kramer. R.L. and Richzs. A.. Br. J. Pharmacol. 45, 184P (1972). 51. vfficial Methods of Analysis", 12th Edition, Horwitz. W. (Ed.). Association of Official Analytical Chemists, Washington, D.C. , 1975, p. 673, 677. 52. Davis. J.E.. J. Criminal Law. Criminal Police Sci. 52, 459 (1961). 53. C h n E L , oe,.. Am. J. Pharm., 118,40 (1946). 54. Pfeil, E. and Goldbach, H.T., Z. Physiol. Chem. 302, 263 (1955). 55. m m e n , E.Z., Clin. Chem. 16, 797 (1970). 56. Chromy, V. and Babjuk, J S ., Clin. Chem. Acta. 37, 547 (1972). 57. E h r , F., Clin. Chem. 21, 1609 (1975). 58. Curry, A.S., "Toxicology, Mechanisms and Analytical Methods", Vol. 2, Stewart, C.P. and Stolman, A. (Ed.), Academic Press, New York 1961, Chap. 5, p. 153. 59. Jain, N.C. and Cravey, R.H., J. of Chrom. Sci. 12, 228 (1974). 60. Xtlow, P., Am. J. Clin. Pathol. 59, 167 (1973). 61. Winefordner, J.D. and Tin, M., An=. Clin. Acta. 31, 239 (1964). 62. menfriend, S.; Duggan, D.; Vasta, B. and Brodie, B., J. Pharmacol. Exptl. Therap. 120, 26 (1957).
PHENOBARBITAL
397
Group Analysis by Micro and Semimicro Methods", Interscience Publishers, New York, 1 9 6 4 , Chap. 8 , p. 2 7 9 . 6 5 . Kucharsky, J. and Safarik, L . , "Titrations in Non-Aqueous Solvents", Elsevier Publishing Co., New York, 1 9 6 5 , p. 2 2 6 . 6 6 . Agarwal, S.P. and Blake, M.I., Anal. Chem. -, 41
1104 ( 1 9 6 9 ) . 6 7 . The Pharmacopeia of the United States of
6 3 . Brooks, M.A.; DeSilva, J.A.F. and Hackman, M.R., Anal, Chim. Acta. 6 4 , 1 6 5 ( 1 9 7 3 ) . 6 4 . Cheronis, N.D. and-a, T.S., "Organic Functional
America, 15th Revision, Mack Co., Easton, Pa., 1 9 5 5 , p. 5 3 1 . 6 8 . Walls, H.J., J. Forensic Med. 5 , 27 ( 1 9 5 8 ) . 6 9 . Clark, E.G.C., "Isolation and Tdentification of Drugs", The Pharmaceutical Press, London, 1 9 6 9 . 7 0 . Jain, N.C. and Cravey, R.H., J. Chromatog. Sci.
1 2 , 228 ( 1 9 7 4 ) . 7 1 . ZZeeuw, R.A., "Progress in Chemical Toxicology", V o l . 4 , Stolman, A. (Ed.), Academic Press, New York, 1 9 6 9 , p. 5 9 . , 7 2 . Kirchner , J .A. "Thin-Layer Chromatography", Interscience Publishers, New York, 1 9 6 7 , p . 5 0 4 . 7 3 . Stahl , E. , "Thin-Layer Chromatography" Springer-Verlag, New York, 1 9 6 9 , Q. 5 3 1 . 7 4 . Gardner-Thorpe, C.; Parsonage, M.J. and Toothill, C., Clin. Chim. Acta. 3 5 , 39 ( 1 9 7 1 ) . 7 5 * Itiaba, K.; Crawhall, T C . and Sin, C.M., Clin. BAochem. 3 , 287 ( 1 9 7 0 ) . 7 6 . Dunges, WT and Peter, H.W., "Methods of Analysis
8 2 , 143 ( 1 9 7 3 ) . 7 9 . Ksari, S . , Parke, Davis & Co., Personal Communication, 1 9 6 8 . 8 0 . Kullberg, M.P.; Miller, W.L.; Megowan, F.J. and Doctor, B.P., Biochem. Med. 7, 3 2 3 ( 1 9 7 3 ) . 8 1 . Hetland, L.B.; Knowlton, D.A, and Couri, D., Clin. Chim. Acta. 36, 4 7 3 ( 1 9 7 2 ) . 8 2 . Brochmann-Hanssen, E., "Gas Chromatography", Grune and Stratton, Inc., New York, 1 9 6 8 , p . 182,
of Anti-Epileptic Drugs", Meijer, J.W.; Meinardi, H.; Gardner-Thorpe, C. and van der Kleijn, E. (Ed.), American Elsevier Co., Inc., New York, 1 9 7 2 , p. 1 2 7 . 7 7 . Atwell, S.H.; Green, V.A. and Hanfy, W.G., J. Pharm. Sci. 6 4 , 8 0 6 ( 1 9 7 5 ) . 7 8 . Kammerl, E. and Schlemmer, W., J. Chromatog.
398
MARCUS K. C. CHAO et ol.
83. Gudzinowicz, B.J., "Gas Chromato ra hic Analysis of Drugs and Pesticides', iarcel Dekker, New York, 1967, Chap. 4. 84. "Methods of Analysis of Anti-Epileptic Drugs", Meijer, J.W.; Meinardi, H.; Gardner-Thorpe, C. and van der Kleijn, E. (Ed.), American Elsevier Co., Inc., New York, 1973. 85. Berry, D.J., J. Chromatog. 86, 89 (1973). 86. Ehrsson, H., Anal. Chem. 42 62 , 9 (1974). 87. Toseland, P.A.; Albani, Myand Ganchel, F.D., ClAn. Chem. 21, 98 (1973). 88. Brugmann, G.and Nalte, R., "Clinical Pharmacology of Anti-Epileptic Drugs", Schneider, H.; Janz, D.; Gardner-Thorpe, C.; Meinardi, N. and Sherwin, A.L. (Ed.), Springer-Verlag, New York, 1975, p. 317. 89. Gyllenhaal, 0.;Brglell, H. and Sandgren, B., J. Chromatogr. - 471 (1976). 122, 90. Walle, T., J. Chromatogr. 114, 345 (1975). 91. Horning, M.G. and Nowlin, J., Clin. Chem. 2 , 805 (1973); - 1282 (1975). 21, 92. Hatch, R.C., Am. J. Vet. Res. 33, 203 (1972). 93. Miyamato, M.; Seino, M. and Ikaa, Y., "Chemical Pharmacology of Anti-Epileptic Drugs", Schneider, H.; Janz, D.; Gardner-Thorpe, C.; Meinardi, H. and Sherwin, A.L. (Ed.), SpringerVerlag, New York, 1975, p. 323. 94. Pecci, J. and Giovanniello, T.J., J. Chromatogr. 109, 1963 (1975). 95. =is, H.L.; Falk, K.J. and Bailey, D., J. Chromatogr. 107, 61 (1975). 96. Gupta, R.N. 3 Keane, P.M., Clin. Chem. 21, 1346 (1975). 22, 97. Abraham, C.V. and Jaslin, H.D., Clin. Chem. 769 (1976). 98. Hall, R.C. and Risk, C.A., J. Chromatogr. Sci. 13, 519 (1975). 99. Goper, W.D.; Dubetz, D.K.; Eadie, M.J. and Tyrer, J. Chromatogr. - 206 (1975). 110, 100. Osiewicz, R.; Aggarwal, V.; Young, R.M. and Sunshine, I., J. Chromatogr. 88, 157 (1976). 101. Baumel, I, ; Gallagher, B.B,a n d Mattson, P. R.H., Arch. Neurol. 27, 34 (1972). 102. Cooper, R.G.; Greaves, M.S. and Owen, G., Clin. Chem. 18, 1343 (1972). 42, 103. Anders, M.W.and Latorre, J.P., Anal. Chem. 1430 (1970). 104. ROSS, R.W., J. Pharm. Sci. 61, 1979 (1972). 105. Evans, J.E., Anal. Chem. 45F2428 (1973).
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. - 31 (1976). 73, 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. - 869 (1974). 20, 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. - 1301 (1974). 75, 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.
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
SULFAMETHAZINE
403
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.
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
c,
rl rl
a,
a,
a,
0 E 7 k c,
a,
a a,
k
m
+ I
rl
a,
-I4
t P
F
SULFAMETHAZINE
405
Nuclear Maqnetic Resonance Spectrum: The 60 M z NMR spectrum ( F i g u r e 2 ) H 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 T S a s M 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.
2.2
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 ) .
12-
Sulfamethazine: Sulfamerazine:
R = CH3
R = H
1-n
G d
I,
im o
Figure 2.
NMR spectrum of sulf amethaz i n e .
SULF AMETHAZINE
407
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
2.3
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 ) .
m/e
H2N
as02/mf1
92 (333
m/e
2.4
156
m/e 1 2 3
H ?
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'
1m c
= 670) w i t h a s h o u l d e r
o n e peak a t 2 4 1 nm i s o b s e r v e d a t 255 nm.
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 n w i t h El%- o f 765 a n d 776 m 1m c 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 . 1m c
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.
2.5
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
108 MF12043
90
80
70
60
50
40
30
20
10
0
Q
CU
3-
CD
03
8 c U 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
=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 sulfamethazine ( ) in w a t e r , (, ) O . 01N H C 1 ,
...
(---)
0.01N NaOH.
410
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. m however, show a phosphorescence peak a t 410 n when e x c i t e d a t 310 n a t 77OK. The l i f e t i m e i s 0.8 m 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
2.9
D i f f e r e n t i a l Thermal A n a l y s i s
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 0
d
(g)
Relative Intensity
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
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
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
hY-3-
....--
2.y
F
m. u
_ I -
Figure 5 .
X-ray d i f f r a c t i o n pattern o f sulfamethaeine.
SULFAMETHAZINE
413
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).
2.11
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
(332
A 0
CH3 -2 H20 CH3
NH2
Sulfanilylguanidine
Acetylacetone
Sulfamethazine
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)
- O SO3H N
(1)
H2N <3 $ CH3

(11)
Druq Metabolism Sulfamethazine is rapidly absorbed from the gastro-intestinal tract so that regular oral administration of the drug will produce blood concentrations of 5 to 10 mcg %. Sulfamethazine is highly acetylated, about 30% in the blood and 60% in the urine being in the form of the acetyl derivative. A correlation appears to exist with the acetylator phenotype as determined with procainamide/N-acetylprocainamide and isonicotinic acid hydrazide. Sulfamethazine is firmly bound to plasma proteins (18,19), The metabolism of sulfamethazine has been studied in various species: Species References Human 18,19,20,2 1,22 Cattle 23,24,25,26 Hen 27 Rabbit 28,29 Swine 30 Horse 31 Goat 32 Monkey 33 Rat 34,35
5.
SULFAMETHAZINE
415
6.
Method of A n a l y s i s : 6.1 Elemental a n a l y s i s 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 s u l f a m e t h a z i n e a r e l i s t e d i n T a b l e I11 ( 3 6 ) . T a b l e I11 Elemental A n a l y s i s of Su 1fame.thazine

E l emen t
% Theory
51.78 5.07 20.13 11.52
% Found
51.53 5.00 19.90 11.42
C
H
N
S
Volumetric methods Various t i t r i m e t r i c methods can be used f o r t h e r o u t i n e a s s a y o f sulfonamides o f pharmaceut i c a l importance. T i t r a t i o n w i t h sodium n i t r i t e s o l u t i o n , t o d e t e r m i n e t h e a r o m a t i c amine f u n c t i o n , i s t h e most w i d e l y u s e d a s s a y procedure ( 3 7 , 3 8 ) .
6.2
Agarwal e t a 1 (39) d e s c r i b e a s p e c t r o p h o t o metric t i t r a t i o n procedure f o r t h e a n a l y s i s o f a number of sulfonamide d r u g s , The drug d i s s o l v e d i n a m i x t u r e o f c o n c e n t r a t e d h y d r o c h l o r i c a c i d and g l a c i a l a c e t i c a c i d (2:8) i s t i t r a t e d w i t h a 0.15 bromate-bromide mixture. The end-point i s determined s p e c t r o p h o t o m e t r i c a l l y a t 345 nm. Thermometric t i t r a t i o n methods have been used f o r t h e d e t e r m i n a t i o n of sulfonamides i n aqueous s o l u t i o n s . The r e a c t i o n s employed i n c l u d e d i a z o t i z a t i o n w i t h 0.1M sodium n i t r i t e ( 4 0 ) , o x i d a t i o n w i t h 0.5M sodium h y p o c h l o r i t e (41), t h e formation of s i l v e r d e r i v a t i v e s w i t h 0.3M s i l v e r n i t r a t e ( 4 2 ) , and c a t a l y t i c thermometric t i t r i m e t r y i n which t h e a c i d i c f u n c t i o n of s u l f a m e t h a z i n e i s determined by t i t r a t i n g a s o l u t i o n of t h e drug i n dimethylformamide w i t h tetra-n-butylammonium hydroxide. The h e a t evolved during t h e a l k a l i - c a t a l y z e d a n i o n i c polymerization of a c r y l o n i t r i l e ( i n d i c a t o r ) i s used t o e s t a b l i s h t h e end-point ( 4 3 ) .
416
Colorimetric Analysis One of the most widely used methods for the determination of sulfamethazine is the Bratton-Marshall test (44) which is based on diazotization and coupling with N-(l-naphthyl ethylenediamine dihydrochloride).
6.3
Davis et a1 describe a method for the determination of sulfonamides by the formation of an indophenol dye and measuring the absorbance at 7 2 5 nm, (45).
6 4 Column Chromatosraphv . Banes and Riggleman describe a chromatographic method for the assay of trisulfapyrimidine 4) tablets and oral suspensions ( 6 . Total sulfonamides are measured colorimetrically by the Bratton-Marshall procedure, Sulfamethazine is separated from its homologs by column partition chromatography and is determined by UV spectrophotometry Rader (47) describes a method for the operation and determination of individual sulfonamides in mixtures. These procedures are based on the ion-pair formation of the sulfonamides with tetrabutylammonium ion, followed by separation on partition chromatographic columns. The separated sulfonamides are then determined by UV spectrophotometry.
Hiqh-Pressure Liquid Chromatoqraphy Sharma et a1 (48) describe a method for the separation and quantitative determination of sulfamethazine, sulfamerazine, sulfathiazole, and their N4-acetylated metabolites on an amino-bonded reversed-phase column. The method is suitable for the analysis of these compounds in pure solutions as well as cattle urine.
6.5
Poet and Pu (49)and Kram (50) also describe methods for the separation of sulfa drugs using HPLC. Karger & (51) have developed a method combining HPLC and high selectivity ion-pair
SULFAMETHAZINE
417
p a r t i t i o n t o s e p a r a t e m i x t u r e s o f s u l f a drugs. Gas Chromatography S u l f a p y r i m i d i n e s have been a n a l y z e d by g a s chromatography a f t e r an a c i d h y d r o l y s i s t o o b t a i n s u l f a n i l i c acid and t h e i r r e s p e c t i v e h e t e r o c y c l i c amines u s i n g a column packed w i t h 5% SE-30 and 5% carbowax 20M on chromosorb W w i t h a column t e m p e r a t u r e o f 150 ( 5 2 ) . Sulfamethazine p r e s e n t i n f e e d s h a s a l s o been a n a l y z e d by GLC ( 5 3, 54) 6.6
Paper Chromatography S u l famethaz i n e and o t h e r s u l fonamides have been a n a l y z e d by paper chromatography i n s i x s o l v e n t systems and t h e spots l o c a t e d w i t h a v a r i e t y o f v i s u a l i z i n g r e a g e n t s (55).
6.7
The s o l v e n t systems u s e d w e r e : A: Methyl i s o b u t y l ketone-formic a c i d water (10 p a r t s k e t o n e s a t u r a t e d w i t h 1 p a r t 4% formic a c i d ) B: Chloroform-methanol-formic a c i d - w a t e r (10 p a r t s chloroform s a t u r a t e d w i t h a m i x t u r e of 1 p a r t methanol and 1 p a r t 4% formic a c i d ) C: Benzene-methyl e t h y l ketone-formic acid-water (a m i x t u r e o f 9 p a r t s benzene and 1 p a r t k e t o n e s a t u r a t e d w i t h 1 p a r t 2% formic a c i d ) D: Benzene-formic a c i d - w a t e r (10 p a r t s benzene s a t u r a t e d w i t h 1 p a r t 2% formic a c i d ) E: Methyl e t h y l ketone-acetone-formic acid-water (40:2:1:6) The chromatography was performed on S u l f a m e t h a z i n e gave Whatman N o . 1 f i l t e r paper. R f v a l u e s o f 0.91, 0.46, 0.88, 0.83, 0.54 and 0 . 2 2 w i t h s o l v e n t systems A t o F r e s p e c t i v e l y . The spots w e r e l o c a l i z e d w i t h t h e u s e o f either:
1) W l i g h t 2 ) Potassium permanganate s p r a y r e a g e n t
418
(1% aqueous s o l u t i o n ) Bromophenol b l u e s p r a y r e a g e n t (0.05% i n e t h a n o l ) Ehrlich reagent spray reagent (1% p-dimethylaminobenzaldehyde i n 1g HC1) p-Dimethylaminocinnamaldehyde s p r a y r e a g e n t (0.1% i n 1N_ HC1)
Thin-Layer Chromatography S u l f a m e t h a z i n e has an Rf o f 0.65 when s p o t t e d on s i l i c a g e l p l a t e s and developed w i t h a m i x t u r e of e t h y l a c e t a t e and methanol (9O:lO). The s p o t can be l o c a t e d u s i n g t h e f o l l o w i n g r e a g e n t s : (1) copper a c e t a t e i n methanol ( s a t u r a t e d ) : y e l l o w i s h g r e e n c o l o r : ( 2 ) 5% copper s u l f a t e i n water:orange c o l o r ; ( 3 ) 2% c e r i c s u l f a t e i n w a t e r u s i n g 5 m l conc. s u l f u r i c a c i d : y e l l o w i s h p u r p l e ( 5 6 ) .
6.8
A TLC system based on polyamide o r s i l i c a g e l G l a y e r s and a n aqueous mobile phase was a l s o u s e d f o r t h e s e p a r a t i o n of a s e r i e s of sulfonamides, ( 5 7 ) . T h e mobile phase was v e r o n a l a c e t a t e b u f f e r a t pH 7.4, a l o n e o r mixed w i t h d i f f e r e n t amounts o f a c e t o n e . L o c a l i z a t i o n w a s o b t a i n e d by s p r a y i n g w i t h p-dimethylaminobenzaldehyde (0.1% i n e t h a n o l ) / c o n c e n t r a t e d h y d r o c h l o r i c a c i d (99: 1 ) . Sulfamethaz i n e gave an RM v a l u e o f -0.18 on t h e s i l i c a g e l G p l a t e s (0% a c e t o n e ) and RM: 0.26 and 0.13 on polyamide p l a t e s (PA a c e t o n e and 10% a c e t o n e r e s p e c t i v e l y ) . (RM = l o g [ r - 1-7).
The chromatographic b e h a v i o r o f s u l f a m e t h a z i n e and o t h e r sulfonamides h a s a l s o been i n v e s t i g a t e d on s t r o n g and weak c a t i o n and a n i o n exchange TLC p l a t e s w i t h p o l y s t y r e n e , p a r a f f i n and c e l l u l o s e m a t r i c e s u s i n g aqueous and aqueous-organic eluents (58).
A method h a s a l s o been d e s c r i b e d f o r t h e a n a l y s i s of mixed sulfonamides by TLC and UV spectrophotometry ( 5 9 ) . The samples a r e s p o t t e d on f l u o r e s c e n t s i l i c a g e l H p l a t e s and developed i n
SULFAMETHAZINE
419
chloroform-methanol (88 :1 2 ) , The developed s p o t s a r e d e l i n e a t e d under W l i g h t , s c r a p e d from t h e p l a t e and e x t r a c t e d w i t h 1g NaOH. The W absorptions of t h e c e n t r i f u g e d extracts a r e r e a d w i t h a r e c o r d i n g spectrophotometer.
C i e r i ( 6 0 ) d e s c r i b e s a method f o r t h e d e t e c t i o n o f s u l f a m e t h a z i n e and o t h e r sulfonamides i n animal f e e d s . The sulfonamides a r e e x t r a c t e d from animal f e e d samples w i t h a l c o h o l o r a c e t o n e and c l e a n e d up by a c e l i t e column chromatographic technique. The e l u a t e i s s p o t t e d on a n e u t r a l A b s o r b o s i l - 1 TLC p l a t e , which i s developed i n chloroform-methanol ( 9 5 : 5 ) and t h e n sprayed w i t h an a l c o h o l i c s o l u t i o n of p-dimethylaminobenzaldehyde
M i c r o b i o l o g i c a l Assay S u l f a drugs and a n t i b i o t i c s have been d e t e c t e d i n milk u s i n g B a c i l l u s meqaterium ATCC 9855 a s t h e t e s t organism and Mueller-Hinton I n c u b a t i o n was a t 37O agar a s t h e t e s t s u b s t r a t e . f o r 4 t o 5 hours.
6.9
6 . 1 0 Assay i n Body F l u i d s and T i s s u e s S e v e r a l methods have been u s e d f o r t h e d e t e r m i n a t i o n of s u l f a m e t h a z i n e i n serum, u r i n e and t i s s u e s . The r e f e r e n c e s mentioned i n s e c t i o n 5 (Drug Metabolism) d e s c r i b e t h e most commonly used procedures.
420
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-, 128
58. 59. 60. 61.
B r a t t o n , A.C. & M a r s h a l l , F.K.; J. B i d . Chem., 537 ( 1 9 3 9 ) . D a v i s , D.R. ; Fogg, A.G. & Thorburn Burns, D. ; And&,, 99, 1 2 ( 1 9 7 4 ) . Banes, D. & Riggleman, O.H.; J. of t h e A.O.A.C., 54, 1195 ( 1 9 7 1 ) . Rader, B.R. ; J. Pharm.Sci., 62,1148 ( 1 9 7 3 ) . Sharma, J . P . : P e r k i n s , E . G , & B e v i l l , R.F. : Pharm. S c i . , 65, 1 6 0 6 ( 1 9 7 6 ) . Poet, R.B. & Pu, H.H.; J.Pharm.Sci., 62, 809 ( 1 9 7 3 ) . K r a m , T.C.; J.Pharm. S c i . , 61, 2 5 4 ( 1 9 7 2 ) . Karger, B.L. : s u , S.C. : Marchese, S. & Persson, B. : J. Chromatog. S c i . , 12,678 (1974). Turczan, J . W . : J. Pharm. S c i . , 57, 142 ( 1 9 6 8 ) . F r a v o l o n i , A , & B e g l i o m i n i , A . ; J. of t h e A.O,A.C., 2 767 (1969). Daun, R. J. : J. of t h e A.O.A. C., 54, 1 2 7 7 ( 1 9 7 1 ) , Reio, L. : J. Chromatoq., 88, 1 1 9 ( 1 9 7 4 ) . Walash,M. I. & Agarwal, S. P. ; J. Pharm.Sci., 61,277 ( 1 9 7 2 ) . B i a g i , G.L. : Barbaro, A.M. : Guerra,M. C. : C a n t e l l i - F o r t i , G. & G a n d o l f i , 0. : J. Chromatoq. , 1 6 349 ( 1 9 7 5 ) . 0, L e p r i , L . ; D e s i d e r i , , P.G. & T a n t u r l y , G . : Chromatoq., 93, 201 ( 1 9 7 4 ) . C i e r i , U.R.: J. Chromatog., 49, 493, ( 1 9 7 0 ) . C i e r i , U.R.: J. of t h e A.O.A.C.; 59, 56 (1976) Read, R. B. : Bradshaw, J . G . : Swartzentruber,A.A. & B r a z i z , A . R . : A p p l i e d M i c r o b i o l o g y , -, 21 806 ( 1 9 7 1 ) .
J.
J.
L i t e r a t u r e s u r v e y e d up t o December 1976.
THIOSTREPTON
Klaus Florey
423
424
KLAUS FLOREY
TABLE OF CONTENTS
1.
Introduction 1.1 History 1.2 Formula and Molecular Weight 1.3 Appearance, Color, Odor 1.4 Salts and Complexes Physical Properties 2.1 Infrared Spectrum 2.2 NMR Spectra 2.3 Mass Spectrum 2.4 Ultraviolet Spectrum 2.5 Optical Rotation 2.6 Melting Range 2.7 Differential Thermal Analysis 2.8 Phase Rule Analysis 2.9 Solubility 2.10 Crystal Properties Synthesis 3.1 Fermentation-Isolation 3.2 Structure Stability-Degradation Drug Metabolism, Pharmacokinetics Methods of Analysis 6.1 Elemental Analysis 6.2 Microbiological Analysis 6.3 Nonaqueous Titration 6.4 Color Reactions 6.5 Colorimetric Analysis 6.6 Chromatographic Analysis 6.61 Paper 6.62 Thin-layer 6.7 Counter Current Distribution Determination in Pharmaceutical Preparations References
2.
3.
4. 5.
6.
7.
8.
THIOSTREPTON
425
Introduction 1.1 History Thiostrepton, a polypeptide antibiotic of yet not fully known structure, was isolated and characterized by scientists of the Squibb Institute for Medical Research'in 1954. It was first obtained from a fermentation broth, that had been inoculated with Streptom ces azureus, isolated from a soil sample co ected in New Mexico. Thiostrepton is active against gram-positive bacteria and has found use in veterinary med.icine.6 The antibiotics thiactin and bryamcin were found to be identical with thiostrepton.4
1.
Formula and Molecular Weight No exact formula or molecular weight can be given. The formula probably ranges between C6gHglN18022Sg and C72H83N19017Sg and the molecular weight is approximately 16005 (see section 3 ) . 1.3 Appearance, Color, Odor White to light yellow, odorless crystals.
1.2
Salts, Complexes and Esters Thiostrepton is a weak base which forms salts. The hydr~chloride,~" phosphate' and sulfate' are described as are complexes with calcium7'' and sodium chloride.' The hemisuccinate ester has also been disclosed.g Physical Properties 2.1 Infrared Spectrum The infrared spectrum of thiostrepton is presented in figure 1 The following assignments . can be made:*' cm-1
1650) 1550
3400 (broad)
1.4
2 .
Assignment OH,NH stretch Amide, C=O, NH C=N stretch
2920) 2970
rl
al
.J l
r)
al k
i n
THIOSTREPTON
427
NMR Spectra The 100 MHz NMR spectrum of thiostrepton was studied3' in DMs0-d~containing TMS as internal reference utilizing a Varian XL-100-15 NMR spectrometer (Figures 2 and 3 ) . For the purposes of assignment, the NMR spectrum may be divided into three parts: a) 0-3 ppm consisting of methyl and me thylene protons , b) 3-6 ppm consisting of methylene and methine protons attached to the heteroatoms, and c) 6-10 ppm consisting of aromatic, heterocyclics and exchangeable protons. The addition of D20 exchanged several resonances at 6 7.70, 8.20, 9.20, 9.45 and 9.95 (Figure 3). 1 has not been possible to make 7 specific assignments. The I3C NMR spectrum of thiostrepton (Figure 4) was obtained at 600 in CDC13-CD30D (8:2 v/v) utilizing Varian XL-100-15 spectrometer, equipped with Fourier Accessories obtained from Nicolet Corporation. The system was locked to 2H frequency of 15.4 MHz and operated at a frequency of 25.2 MHz. After the expansion, it was possible to account for 71 carbons out of a total of 72. The chemical shifts (Table 1) are comparable to ones known in the literature.33
2.2
I\ II
Figure 2.
Proton NMR Spectrum of Thiostrepton in DMSO-d6. Instrument: Varian XL-100
Figure 3 .
Proton NMR Spectrum of Thiostrepton in DMSO-D20. Instrument: Varian XL-100
II
I
" " " I
" '
' " ' 1 ' '
b
* %
,
I
I I 1
1
I
I
I I
I
I
I
I
I
I I I
I
I
I
c,
Figure 4 .
1 3 C NMR Spectrum of Thiostrepton in CDC13-CD30D (8:2) Instrument: Varian XL-100
THIOSTREPTON
431
Table 1 13C Chemical Shift and Tentative Assignments C-CH3 C (S)-CH2 C-CH N (0) CH
(N (0) -C C=CH2 C=CH 11.0, 15.1, 15.3, 15.9, 17.7 22.4, 18.5, 18.8, 18.9 24.6, 24.8, 29.1 34.7, 38.5 49.1, 51-71 51.8, 53-01 55.8, 57.4, 59.1 64.2, 66.0, 67.2, 67.7, 71.9, 79.0, 66.5 57.4, 77.2 102.3, 103.3, 104.3 118.0, 122.1, 123.0, 125.0, 125.1, 127.3, 128.4 127.0, 129.9 132.3, 133.1, 134.4, 143.4 146.4, 150.0, 153.1, 154.5, 157.1, 159.5 160.8, 161.5, 161.9, 162.1 163.0, 165.4, 166.0, 166.3, 168.2, 168.5 169.7, 170.1(2), 171.8, 173.1, 174.0
c=c C=C-N
co -
N=C N=C S
--
- CDC13 = 6
2.3
The chemical shifts are relative to 76.9 ppm from TMS. Mass Spectrum Attempts to obtain a mass spectrum of thiostrepton or silanized derivatives were unsuccessful.26 Ultraviolet Spectrum The ultraviolet spectrum presents no maxima but shows characteristic shoulders at 225(Ei 5201, 250 (Ei 380) and 280 (E: 225) nm.2 2.4
432
KLAUS FLOREY
Optical Rotation The s p e c i f i c r o t a t i o n v a r i e s w i t h t h e s o l v e n t as f o l l o w s :

= 1.0
2.5
g l a c i a l acetic a c i d dioxane py r i d i ne
2.6
-98.5' -61. Oo -20. oo
M e l t i n g Range
T h i o s t r e p t o n m e l t s w i t h decomposition over t h e r a n g e 2 4 6 t o 2560 C.'" D i f f e r e n t i a l Thermal A n a l y s i s DTA w a s performed o n a sample of t h i o s t r e p t o n (house s t a n d a r d ) under s t a t i c a i r a t a h e a t i n g r a t e o f 15O C / m i n , u s i n g c a p i l l a r i e s (Figure 5 ) .
A broad endotherm w a s observed w i t h a peak t e m p e r a t u r e of 1 2 0 C . , p r o b a b l y r e p r e s e n t i n g t h e o d e h y d r a t i o n . A r e l a t i v e l y s h a r p exotherm a t 232 C . p r o b a b l y r e p r e s e 2 t s an o x i d a t i v e m e l t i n g and d e g r a d a t i o n p r o c e s s .
2.7
2.8
Phase Rule A n a l y s i s
The p u r i t y o f t h i o s t r e p t o n c a n be determined by p h a s e r u l e a n a l y s i s , u s i n g c h l o r o form-carbon t e t r a c h l o r i d e 3 : l a s g o l v e n t system and e q u i l i b r a t i n g f o r 168 h o u r s a t 25 C . The p u r i t y o f one sample w a s found t o be 98.6%. 3 5 2.9 Solubility The s o l u b i l i t o f t h i o s t r e p t o n was determined as f o l l o w s , 2 y e x p r e s s e d as mg/ml:
Water 0.088
E t h a n o l 0.990 Isoamyl Alcohol 5.250 Benzene 1.352 Isooctane 0.0 E t h y l Acetate 0.685 Acetone 0.492 D i e t h y l E t h e r 0.032
Methanol 0.285 I s o p r o p a n o l 0.595 Cyclohexane 0.030 Petroleum E t h e r 0.018 Carbon T e t r a c h l o r i d e 0.055 Isoamyl Acetate 0.285 Methyl E t h y l Ketone 0.720 E t h y l e n e C h l o r i d e 0.182
t,'c (CHROYEL: ALUMEU
*.lrrunuruNmu-
Figure 5.
DTA of Thiostrepton (house standard) Heating Rate: Instrument: DuPont 900
15O C/min.
434
KLAUS FLOREY
1 ,4 Dioxane >20 Carbon Disulfide 0.032 Formamide >20 Propylene Glycol 17.360 0.1 N NaOH 4.986
Chloroform >20 Pyridine >20 Ethylene Glycol 5.840 Dimethylsulfoxide >20 0.1 - HC1 10.055 N
In contrast to the last two values, Vandeputte and Dutcher2 claim that thiostrepton is not dissolved in dilute aqueous acid or base, but is dissolved in methanolic acid or base. The latter solvent, however, rapidly inactivates the antibiotic. Crystal Properties The powder x-ray diffraction pattern of thiostrepton is presented in Table 2 and Figure 62' No polymorphs of thiostrepton are known.
2.10
Table 2 Powder X-Ray Diffraction Pattern of Thiostrepton Batch #7H10-1

20 4.7 5.7 6.7 7.5 8.1 9.5 10.2 10.7 12.5 13.8 14.4 15.1 15.3 16.0 3.
'd'Val-A
18.7 15.5 13.1 11.8 10.8 9.3 8.7 8.3 7.1 6.4 6.15 5.87 5.77 5.53
Relative Intensity
.2836 1.0000 .3134 .3880 .5373 .2388 .lo45 .1343 .2985 .3134 .3283 .3880 .3731
120 16.4 17.5 18.4 19.7 20.7 21.5 22.8 23.9 25.0 26.1 26.6 127.2
'd'Val-A
5.40 5.05 4.81 4.50 4.27 4.13 3.89 3.72 3.50 3.42 3.35 3.28 3.08
Relative Intensity
.6268 .5074 .6418 .3433 .6866 .4179 .3283 .5074 .2537 .1940 .2388 .2388 .1045
Synthesis 3.1 Fermentation, Isolation Thiostrepton is produced by fermentation' using the micro-organism Stre tom ces azureus. The yield can be improved y t e addition of heavy metal cations to the fermentation
128*o
'
l-
i-
8
2
G
0
'
..
L
X
8
ot
i n
0
PI
Id
-4
8
2 P
436
KLAUS FLOREY
Actinomvces species also produce thiostrepton.12 Thiostrepton is found mainly in the mycelium while at least two other antibiotics are found in the supernatant broth. One of the companion antibiotics has been identified as ~ i o m y c i n . ~ ~ is It chemically very similar to thiostrepton; however, one isoleucine unit is replaced by valine, and it also has one less alanine unit. Thiostrepton is extracted from the mycelial cake by suitable organic solvents such as chloroform, dioxane, Further dimethyl formamide or benzyl alcohol. purification is carried out by carbon treatment and repeated crystallization from chloroform or dioxane-methanol.2
The preparation of sulfur labeled thiostrepton has also been described. Structure The structure of thiostrepton is not yet fully known. Pioneering studies in the elucidation of structure have been carried out by and Kenner 22-23 with their Bodanszky 5 - 2 collaborators. A significant contribution to clarify the structure was made by Anderson, Hodgkin and Viswamitra who examined thiostrepton by single crystal x-ray diffraction.
3.2
The principal fragments obtained on hydrolysis of thiostrepton are (-)-alanine, (-1-threonine, (-1-isoleucine, (+)-cysteine, pyruvic acid, also CH-OH
4-(a-Hydroxyethyl)-8-hydroxyquinaldic
acid.
THIOSTREPTON
437
CH2I
CH -fNYC.O2H
1 NH2
Thiostreptoic acid.
CH3
CH3-THOH
c I-
OH
Thiostreptine.
CH3-
CH2-
C 11
0
2-Propionylthiazole-4-carboxylic acid.
NH2-
CH-TC2 I (0 $H
YH2 C02H
2-(l-Amino-2-carboxyethyl)thiazole4-carboxylic acid.
438
KLAUS FLOREY
All these fragments fit in the structure as determined by single crystal x-ray diffraction (Figure 7 ) . 5 However, Figure 7 does not yet represent the total structure. The side chain (top of Figure 7 ) probably contains two additional dehydroalanine and one a,a-diaminopropionic acid residue? ,33 The structure as presented in Fiqure 7 also does not fully disclose the conformation of the molecule. As a whole, the molecule appears roughly globular with the two large rings folded over one another and the long side chain protruding from their junction. The conformation is maintained by a number of intramolecular hydrogen bonds.'
Thiostreptoic Acid
Pyruvic acid
Threonine
)+
"
-Cy s t e i n e
A
Thios treptine
Quinaldic Precursor
0 Nitrogen 0 Oxygen 0Sulphur
Figure 7.
Structure of Thiostrepton, as Determined by Single Crystal X-ray Diffraction.'
THIOSTREPTON
439
Stability-Degradation Thiostrepton is stable as a crystalline powder. In N,N -dimethyl acetamide it retains its potency for at least 16 days at 37* C. and for at least three months under refrigeration. The antibiotic is also stable at 30 C. for at least one week in 50% aqueous dioxane at a pH of 7 , although it rapidly loses potency in the same solvent systems at a pH of 2 or ll.41 For hydrolytic degradation products, see section 3.2.
4.
The acid sulfate and hydrochloride salts of thiostrepton in alcoholic solution soon lose their antibiotic activity. Thiostrepton base is precipitated when water is added to these alcoholic solutions. 4l Drug Metabolism, Pharmacokinetics No drug metabolites are known. Apparently, the antibiotic is not absorbed from the intestinal tract since systemic activity cannot be demonstrated following oral administration of the drug. Parenterally, however, thiostrepton is quite active. Following intramuscular administration, thiostrepton may lie in the local tissues a s a depot. No significant blood or urine levels could be demonstrated.27
5.
6.
Methods of Analysis 6 1 Elemental Analysis . Element C H N

S
T r T *
5.4 14.6 7.4
Ref. 2
Found Ref 7
5.3
9.22
Ref. 17
9.2-10.2%
Due to the capacity of the molecule to retain solvents tenaciously and to absorb moisture from the air after careful drying, the elemental analysis is difficult to perform.17 Microbiological Analysis A microbiological agar diffusion assay, using Micrococcus pyogenes var. aureus (ATCC6538P) as the test organism, has been described in detail?9 This assay can be used to determine thiostrepton in 6.2
440
KLAUS FLOREY
ointments and milk. An automated turbidimetric assay system, using streptococcus faecalis (ATCC10541) has also been used.3o The activity of thiostrepton is measured in units. One microgram of thiostrepton is equal . to 1 1 units.29 Nonaqueous titration Nonaqueous titration with perchloric acid in glacial acetic acid gives an equivalent weight of 420.*
6.3
Color Reactions Thiostrepton gives the following color reaction: 36

6.4
Folin-Ciocalteau: Ehrlich reagent: (p-dimethylaminobenzaldehyde in dilute acid) Alkaline hypoiodite: Ninhydrin: Biuret :
6.5
Intense blue color Strong violet color (see also 6.5) Small amount of iodoform Negative (positive after hydrolysis) Positive2
Colorimetric Analysis Thiostrepton in dimethylformamide can be reacted with p-dimethylaminobenzaldehyde in the presence of alcoholic hydrochloric acid. The resulting color formation can be used for quantitation, by reading the absorption at 535 nm.36 This method can be used for the determination of thiostrepton in mycelial cake36 and ointments.37 Chromatographic Analysis 6.61 Paper The following systems have been used in the ascending mode:l
6.6
THIOSTREPTON
441
Dev. time (Hrs.) Acetone:water (70:30) Acetone:propanol:water

( 4 0 : 40: 20)
5
Rf 0.50
0.80
7
6
0.2N Acetic acid
0.08
Paper chromatography,followed by bioautograph has also been used with the following conditions: 3 x Ethanol washed Whatman # 5 4 paper impregnated with propylene glycol Benzene saturated with Mobile phase : propylene glycol Development time: Sixteen hours, descending Drying conditions: 20 minutes at 90C. Detection: a. U.V. light b. Bioautography with Staph. aureus 209P. Exposuretrips to culture: 15 min. Incubation for 18 hours at 37OC. Thin-Layer The following methods separated thiostrepton from siomycin:
6.62
Stationary phase:
a.38 Analtech Silica Gel G.F. Developing Solvent: Chloroformmethanol (90:10) Rf Thiostrepton 0.36 Rf Siomycin 0.43 Visualization: Short-wave U.V. b.39 Neutral grade IV alumina Chloroform-methanol ( 9 7 : 3 ) Counter Current Distribution When thiostrepton was subjected to 16transfer counter current distribution in a system of
6.7
toluene-chloroform-methanol-water
(1:2:2:1),
442
KLAUS FLOREY
thiostrepton was found in tubes 0-5. Redistribution in a toluene-chloroform-methanol-water (5:5:8:2) system and a 350 tube transfer concentrates thiostrepton in a band with K=0.3. 4 0 Determination in Pharmaceutical Preparations Thiostrepton is determined in pharmaceutical preparations (ointments) by microbiol~gical~~ or c o ~ o r i m e t r i cassay. ~~ References 1. 2. 3. 4. 5. 6. 7. 8. 9.
10.
J. F. Pagano, M. J. Weinstein, H. A. Stout and
7.
R. Donovick, Antibiotics Ann., 1955/56, 554.
J. Vandeputte and J. D. Dutcher, Antibiotics Ann., 1955/56, 560.

. B. A. Steinberg, W. P. Jambor, L. 0 Suydam and A. Suriano, Antibiotics Ann., 1955/56, 562.
M. Bodanszky, J. D. Dutcher and N. J. Williams, J. Antibiotics, - 76 (1963). 16, B. Anderson, D. C. Hodgkin and M. A. Viswamitra, Nature, 225, 233 (1970).
Federal Register, 1971, 36(78), 7583. R. Donovick, J. F. Pagano and J. Vandeputte, U. S . Patent 2,982,689 (1961). B. Heinemann, I. R. Hooper and M. J. Cron, Brit. Patent 790,521 (1958).
M. Bodanszky and J. Fried, U. S . Patent 3,181,995 !1965). T. B. Platt and W. R. Frazier, Antimicrobial Agents, Chemotherapy, 1961, 205. T. B. Platt, U. S. Patent 2,982,698 (1961).
11. 12.
V' I. Frolova, S . M. Rudaya, G. S. Katrukha . and A. D. Kuzovkov, Antibiotiki, - 707 (1972), 17, C.A. - 338391-1(1973). 78,
THIOSTREPTON
443
13. 14. 15.
M. L. Sopori and P. Lengyel, Biochem. Biophys. Res. Comun., 46, 2 3 8 ( 1 9 7 2 ) .
J. H. Highland, G . A. Howard, J. Gordon, Eur. J. Biochem., 53, 3 1 3 (1975).
M. Bodanszky, J. T. Sheehan, J. Fried, N. J. Williams and C. A . Birkhimer, J. Chem. SOC., 82, 4 7 4 7 ( 1 9 6 0 ) .
Am.
16.
M. Bodanszky, J. Alicino, C. A. Birkhimer and N. J. Williams, J. Am. Chem. SOC., 84,

2003 ( 1 9 6 2 ) .
17.
M. Bodanszky, J. Fried, J. T. Sheehan, N. J. Williams, J. Alicino, A. I. Cohen, B. T. Keeler and C. A. Birkhimer, J. Am. Chem. SOC., 2, 2 4 7 8 ( 1 9 6 4 ) .
I. Muramatsu and M. Bodanszky, J. Antibiot.,
L
18.
19. 20. 21.
22,
287
(1969).
M. Bodanszky, J. A. Scozzie and I. Muramatsu, J. Am. Chem. SOC., 9 1 , 4 9 3 4 ( 1 9 6 9 ) .
M. Bodanszky, J. A. Scozzie and I. Muramatsu, J. Antibiotics, - 9 ( 1 9 7 0 ) . 23, C. N. C. Drey, G . W. Kenner, H. D. Law, R. C. Sheppard, M. Bodanszky, J. Fried, N. J. Williams and J. T. Sheehan, J. Am. Chem.
SOC.,
-, 83
3906 ( 1 9 6 1 ) .
22. 23. 24.
D. F. W. Cross, G . W. Kenner, R. C. Sheppard and C. E. Stehr, J. Chem. SOC., 1 9 6 3 , 2 1 4 3 .
M. A. Barton, G. W. Kenner and R. C. Sheppard, J. Chem. SOC. C., 1 9 6 6 , 2 1 1 5 . H. Nishimura, S. Okamoto, M. Mayama, H. Ohtsuka, K. Nakajima, K. Tawakara, M. Shimohira and N. Shimaoka, J. Antibiotics
c _
14A,
255 ( 1 9 6 1 ) .
25. 26.
J. R. Marsh and P . J. Weiss, J. Assoc. Offic. Anal. Chern., 50, 4 6 7 ( 1 9 6 7 ) .
P. Funke, The Squibb Institute, Personal Communication.
444
KLAUS FLOREY
27. 28. 29. 30. 31.

32.
B. A. Steinberg and J. F. Pagano, The Squibb Institute, unpublished data.

B. Toeplitz, The Squibb Institute, Personal Communication.
J. D. Levin, H. Stander and J. F. Pagano, Antibiotics and Chemotherapy, 10, 422 (1960).
T. B. Platt, J. Gentile and M. J. George, Ann, N. Y. Acad. Sci., 130, (2) 664 (1965).
H. Jacobson and Q. Ochs, The Squibb Institute, Personal Communication.
M. S. Puar, The Squibb Institute, Personal Communication.

K. Tori, K. Tokura, K. Okabe, M. Ebata, H. Otsuka and G. Lucacs, Tetrahedron Lett., 1976, 185.
33.
34. 35.
36.
H. Jacobson, The Squibb Institute, Personal Communication.
F. Russo-Alesi, The Squibb Institute, Personal Communication. J. Dutcher and M. Young, The Squibb Institute, Personal Communication.
37. 38. 39. 40. 41.
N. Coy and C. Fairchild, The Squibb Institute, Personal Communication.

H. R. Roberts and M. R. Siino, The Squibb Institute, Personal Communication.
E. J. Meyers, The Squibb Institute, Personal Communication.

J. T. Sheehan, The Squibb Institute, Personal Communication. J. Vandeputte, The Squibb Institute, Personal communication.
TRIMETHOPRIM
Gerald J . Manius
445
Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0- 12-260807-0
446
GERALD J. MANIUS
INDEX
1.
Description
1.1
1.2 2.
Name, Formula, Molecular Weight Appearance, Color, Odor
Physical Properties 2.1 2.2 2.3 2.4 I n f r a r e d Spectrum Raman Spectrum Nuclear Magnetic Resonance Spectrum U l t r a v i o l e t Spectrum Fluorescence Spectrum Mass Spectrum Optical Rotation M e l t i n g Range D i f f e r e n t i a l Scanning C a l o r i m e t r y Thermogravimetric A n a l y s i s Solubility Crystal Properties D i s s o c i a t i o n Constant
2.5
2.6 2.7 2.8 2.9 2.10 2.11 2.12 2.13
3.
Syn thes i s S t a b i l i t y , Degradation and D i s s o l u t i o n Drug M e t a b o l i c Products Toxicity Methods o f A n a l y s i s
4. 5.
6.
7.
7.1
7.2
Elemental A n a l y s i s Thin-Layer Chromatographic A n a l y s i s 7.3 High Performance L i q u i d Chromatographic A n a l y s i s 7.4 Gas-Li qu id Chroma tograph ic A n a l y s i s 7.5 Spectrophotofluorometric A n a l y s i s 7.6 D i r e c t Spectrophotometric A n a l y s i s 7.7 P o l a r o g r a p h i c A n a l y s i s 7.8 T i t r i m e t r i c A n a l y s i s 7.9 M i c r o b i o l o g i c a l A n a l y s i s 7.10 Phase S o l u b i l i t y A n a l y s i s Acknowledgements References
8.
9.
TRIMETHOPRIM
447
1.
Description
1.1
Name, Formula, Molecular Weight

T r imethopr im i s 2,4-d iami no-5- (3,4,5-t r imethoxybenzy 1 ) - p y r i m i d i ne
Trimethoprim
C14H18N403
1.2 Appearance, C o l o r , Odor
Molecular Weight: 290.32
White t o p a l e y e l l o w , o d o r l e s s , c r y s t a l l i n e
powder.
2 .
Physical Properties 2.1 I n f r a r e d Spectrum ( I R ) The i n f r a r e d spectrum o f t r i m e t h o p r i m i s presented The spectrum was o b t a i n e d on a i n Figure 1 (1). Perkin-Elmer Model 621 G r a t i n g I n f r a r e d Spectrophotometer as a s o l u t i o n i n c h l o r o f o r m i n a concent r a t i o n o f 14.8 mg/ml. The p a t h l e n g t h was 0.1 mm NaCI. The spectrum was r u n under survey c o n d i t i o n s ,
TRIMETHOPRIM
449
Table I
I R S p e c t r a l Assignments f o r Trimethoprim
Character i s t ic o f Asymmetric NH s t r e t c h i n g Symme t r ic NH22s t r e t ch ing Aromatic CH s t r e t c h i n g A l i p h a t i c CH s t r e t c h i n g NH d e f o r m a t i o n overlapped w i t h aromatic r i n g Aromatic r i n g CH d e f o r m a t i o n Aromatic methoxy
Frequency (cm
-1
3516 3414
3010 2966, 2940, 2840
1616, 1593
1506 1452 1236, 1129
2.2
Raman SDectrum The Raman spectrum o f t r i m e t h o p r i m i s shown i n I t was o b t a i n e d as a neat s g l i d on a F i g u r e 2. Spex 1401 Double Spectrometer a t 4880 A u s i n g an A r + l a s e r . A f 1 uorescence background was observed. Band assignments a r e l i s t e d i n Table I1 ( 2 ) . Table I 1 Raman S p e c t r a l Assignments f o r T r i m e t h o p r i m
Band
lntensi t y Strong Very Strong Med iurn Very s t r o n g
Assignment NH s t r e t c h i n g Aromatic C=C stretching 1.2.3.5 - benzene r I n g brea t h i ng
3500 c m ~ ~
1610 cm
1000 cm-l
785 cm-
L 3
b rea t h ing
E .-
oi
m E m
-8 m
IMETHOPRIM
451
2.3
Nuclear Magnetic Resonance Spectrum (NMR) The N M R spectrum shown in Figure 3 was obtained by dissolving 50 mg o f trimethoprim in 0.5 ml of DMSO-d containing tetramethylsilane as an Internal 6 reference. The N M R spectrum was determined on a Varian XL-100 Spectrometer at probe temperature ca. 3 1 C . Proton assignments are shown in Table 1 1 1
(3)
Table I l l
NMR Spectral Assignments for Trimethoprim Chemical Shift Coupling Constant 6 (ppm) Mu1 tip1 ici ty J (in Hz)
Protons
3.54 3.63 3.94

6.16 6.58 7.56
5.80
Sing let Sing let S i ng let Sing 1 et Sing 1 et Singlet Singlet
--------
TRIMETHOPFUM
453
2.4
U l t r a v i o l e t Spectrum (UV) The u l t r a v i o l e t spectrum o f trimethoprim i n the m region o f 400 t o 230 n i s shown i n Figure 4 ( 4 ) . The spectrum 5 x h i b i t s one maximum a t 287 2 2 n m ( E = 7.4 x 10 , a b s o r p t i v i t y = 25.5) and a minimum a t approximately 257 nm. The s o l u t i o n i s prepared by d i s s o l v i n g approximately 100 mg o f trimethoprim i n 25 m l o f USP ethanol and d i l u t i n g t o 100 m l volume w i t h 0.4% NaOH. Subdilutions a r e made i n 0.4% NaOH t o a f i n a l concentration o f 0.02 mg/ml. The reference c e l l contains 0.4% NaOH. The spectrum was obtained on a Cary 14 Spectrophotometer.
2.5
F1uorescence Spectrum
Trimethoprim e x h i b i t s s l i g h t fluorescence i n r e l a t i v e l y concentrated s o l u t i o n s o f chloroform (10 mgjrnl) and USP ethanol (5 rng/ml). Neither a c i d i f i c a t i o n nor b a s i f i c a t i o n o f the alcohol s o l u t i o n enhanced the fluorescence ( 5 ) . A i r oxidation over several days standing may i n t e n s i f y the fluorescence.
2.6
Mass Spectrum The low r e s o l u t i o n mass spectrum shown i n F i g u r e 5 was obtained using a Varian MAT CH 5 Mass Spectrometer, i n t e r f a c e d w i t h a Varian data system. The i o n i z i n g energy was 70 eV, source temperature, 150C and t h e probe temperature was 30 + 100OC. The f o l lowing fragmentations were observed (6). Molecular Ion (M ) a t m/e 290
A h i g h r e s o l u t i o n scan was run on the CEC-110 Mass Spectrometer a t 70 eV, i o n i z i n g energy and a t a source temperature of 210C. Table I V l i s t s the fragmentation ions (6)
454
GERALD J. MANIUS
250
300
150
400
NANOMETERS
FIGURE 4
UV Spectrum o f Trimethoprim
E .-
a 0 c
L)
E .W
\
0)
I -
LL-
al
456
GERALD J. MANIUS
Table I V
High Resolution Mass Spectrum o f Trimethoprim

Observed Mass Theoretical Mass Error!:
C 12
H ll
12
N -
0 -
Ion -
243.091 6 243.0882 258.092 1 258.0879 259.1221 275.1198 289.1327 290.1437 259.1 I96 275.1145 289. I301 290.1379
3.4
4.2
2.5
5.3
2.6
13 13 13 14
4 3
2 275-CH30H
3 275-NH3
2 H+-OCH 3 +
15
I5 17
18
5.8
14
4 4 4 4
3 M -CH3 3 M+-H 3 M+
*Mass Difference = Observed Mass 2.7 Optical Rotation
- Theoretical Mass
masses
in Milli-
Trimethoprim exhibits no optical activity. 2.8 Melting Range According to USP XVII, Class I Method, trimethoprim melts at approximately 201C (7). 2.9 Differential Scanning Calorimetry (DSC) DSC spectra showed a single endothermic transition at 199.6 2 0.2OC to 201.0 f O.2OCJ extrapolated onset to peak (8). Figure 6 represents the DSC spectrum obtained. 2.10 Thermogravimetric Analysis (TGA) Trimethoprim exhibited a single weight loss as follows (9):
145C
% Loss
0
-225OC
2.4
285C 36.0
325C
500"C*
69.0
80.0
nLimit of the instrument

The TGA spectrum i s shown in Figure
7.
220
210
cq
Q,
I I
E OH R XTEUENDOTHERM
FIGURE 7 Thermogravimetric Analysis of T r ime thopr im
TRIMETHOPRIM
459
2.11 S o l u b i l i t y
Solubi 1 it y data f o r trimethoprim a t 25C Table V Solvent Water Solubi 1 it y (g/IOO m l )
0.04
(7,lO).
95% Ethanol
3A Alcohol
M thano 1 e lsopropanol Ch 1o r o f orm E t h y l Ether Carbon T e t r a c h l o r i d e Petroleum Ether Benzene Ace tone Benzyl Alcohol
D i me thy 1ace tami de
0.81
0.35
1.21 0.12
1.82 0.003 0.002 0.02

0.002
0.35 7.29
13.86 2.57
Propylene Glycol
2.12 C r y s t a l Properties
The X-ray powder d i f f r a c t i o n p a t t e r n o f trimethoprim i s presented i n Table V I (11). Instrument Conditions Instrument Genera t o r Tube Target Optics GE Model XRD-6 D i f f r a c t o m e t e r 50 KV, 12.5 mA 0 Copper (CuKa = 1.5418 A) 0.05" Detector S l i t M.R. S o l l e r S l i t 3" Beam S l i t Ni Filter 4" Take O f f Angle Scan a t l " / i n c h 1.75 KV and 0.95 KV Pulse Height S e l e c t i o n 5 Volts Window Out
Detector
460
GERALD J. MANIUS
A t cps F u l l S c a l e 1K Time Constant 2.5 As i s
Sample Treatment
Table V I T r imet hop r im 20
("1
7.44
11.89
15.22 *16.22
5.82 5.47
5.05
0.44 0.21 0.74 0.48 1 .oo 0.36 0.39 0.75 0.34 0.69
0.94
17-55 18.65
*22.28 22.91 23.11 n23.81 25-37 25.85
4.76 3.99 3.88 3.85 3.74 3.51 3.45
*Lines broadened
1 / 1 1 = Relative I n t e n s i t y
2.13 D i s s o c i a t i o n Constant Due t o I t s low s o l u b i l i t y i n water, trimethoprim s o l u t i o n s were prepared i n ethanol-water mixtures a t v a r i o u s concentrations and then t i t r a t e d w i t h aqueous sodium hydroxide. The apparent pKa was p l o t t e d versus concentrations o f ethanol r e s u l t i n g i n a s t r a i g h t l i n e whose i n t e r c e p t i s the value o f pK i n water. The This value was pKa i n water was found t o be 6.6 confirmed by o t h e r s (12) who postulated a r e l a t i o n s h i p between d i s s o c i a t i o n constants and p h y s i o l o g i c a l a c t i v i t y , i n t h i s case the a b i l i t y t o i n h i b i t the enzyme d i h y d r o f o l a t e reductase.
(7).
3.
Synthesis Trimethoprim may be prepared by the r e a c t i o n scheme shown i n F i g u r e 8. A m i x t u r e o f methanol, sodium, 3,4,5-trimethoxybenzaldehyde and B-methoxypropionitrile Is a l l o w ed t o r e f l u x fpr several hours. The cooled product, 3,4, 5-trimethoxy-2 -methoxymethylcinnamonitrile, I s r e f l u x e d o v e r n i g h t w i t h methanol i n the presence o f sodium. The
TRIMETHOPRIM SYNTHESIS
3,4,5-TRlMElHOXYBEN~
3,4,5-TRIMOHOXY- 2'- METHOWMETHYL Cl"AM0NlTRlLE
H3COt=-'
CN
GUANIMNE
TRIMETHOPRIM
3.4,5-TRlMETHODCI - ' CWINO-DIHYDIWUNNAMALEHYDE 2DIMETHYL ACETAL
2.4- OIAMINO-5-(3,4,5 TRIMETHOXYBENZYLb

PYRIMIDINE
FIGURE 8
Synthesis o f Trimethoprim
462
GERALD J. MANIUS
product i s e x t r a c t e d w i t h toluene and r e c r y s t a l l i z e d t o form t h e d i m e t h y l a c e t a l . Trimethoprim i s then produced by r e f l u x i n g t h e d i m e t h y l a c e t a l w i t h guanidine i n methanol s o l u t i o n . The d r i e d c r y s t a l s a r e s l u r r i e d w i t h methanol, f i l t e r e d and d r i e d again (13).
4.
S t a b i l i t y , Degradation and D i s s o l u t i o n Trimethoprim, 100 mg t a b l e t s , packaged i n b o t h amber glass and opaque h i g h d e n s i t y p o l y e t h y l e n e b o t t l e s w i t h metal caps were s t o r e d a t room temperature f o r 58 months. No changes i n p h y s i c a l appearance were reported, no degradat i o n products were d e t e c t e d by t h i n - l a y e r chromatography and t h e p u r i t y was approximately a t claim, spectrophotoD i s s o l u t i o n s t u d i e s performed i n simum e t r i c a l l y (14). l a t e d g a s t r i c f l u i d on t h e s t o r e d t a b l e t s show t h a t a t l e a s t 60% i s d i s s o l v e d w i t h i n 30 minutes and 100% i s d i s solved w i t h i n 60 minutes. The i n v i t r o d i s s o l u t i o n procedure i s spectrophotometric ( 1 5 ) . D i s i n t e g r a t i o n times determined i n i t i a l l y and a f t e r 58 months a t room temperature show no s i g n i f i c a n t change (1%
5.
Drug M e t a b o l i c Products The m e t a b o l i c pathways o f t r i m e t h o p r i m a r e i l l u s t r a t e d i n F i g u r e 9. F i v e major m e t a b o l i t e s have been i s o l a t e d : M e t a b o l i t e 1 = 2,4-diamino-5-(4-hydroxy-3,5-dimethoxybenzy1)-pyrimidine; Metabol i t e I I = 2 , 4 - d i a m i n o - 5 - ( ~ hyd roxy-3,4,5- t r imethoxybenzy 1 ) - p y r im id ine ; Metabo1 it e s I I I (a and b ) , a = 2,4-diamino-5-(3,4,5-trimethoxybenzyl) - p y r i m i d i n e - l -oxide, and b = 2,4-d iami no-5- (3,4,5- t r i methoxybenzyl)-pyrimidine-3-oxide, and Metabol it e I V = 2,4-d iami no-5- (3-hydroxy-4,5-d imethoxybenzy l ) -pyr imid i ne. Studies by Schwartz (16) p o s t u l a t e d f o u r m e t a b o l i t e s , t o which a f i f t h , t h e N-3 o x i d e was l a t e r added (17). M e t a b o l i t e I V i s e x c r e t e d t w i c e as much as M e t a b o l i t e 1 . The N - l and N-3-oxide forms o f M e t a b o l i t e I l l a r e produced e q u a l l y (18). M e t a b o l i t e I I i s a minor metabol i t e b a r e l y d e t e c t a b l e i n t h e u r i n e of some i n d i v i d u a l s (19). M e t a b o l i t e s I I and I I I a r e unconjugated i n b o t h plasma and u r i n e , whereas M e t a b o l i t e s I and I V a r e conjugated and e x c r e t e d i n t h e u r i n e as glucuronides (161..
As a l l u d e d t o i n t h e s e c t i o n on d i s s o c i a t i o n constants (12) t r i r n e t h o p r i m ' s a c t i v i t y appears r e l a t e d t o i t s pKa. I t was p o s t u l a t e d t h a t protonated p y r i m i d i n e s i n t e r a c t w i t h t h e d i h y d r o f o l a t e coenzymes w i t h t h e b i n d i n g energy being pH dependent.
MElBBWTE II @ -3-OXIDE 2.4-01AMW) -5(p.- l m m X Y - 3 , 4 , 5 - T W ~ BENZW WR1MIDINE
METABOLITE H 2.4 DIAMINO 5(-3-HYDROXY-4.5-DIMETHo#yBENZYL) PYRIMIDINE
FIGURE
Metabol i c Pathways o f Trimethoprim
464
GERALD J. MANIUS
Trimethoprim is a sulfonamide potentiator which together act synergistically to deprive bacteria of folate coenzymes (20). They work in different parts of the bacterial protein synthesis chain to block purine and thymine production, and hence RNA and DNA formation (21).
6.
Toxicity Trimethoprim's mode of action, as indicated, is to have an inhibitive effect on the folate metabolism o f microorganisms. For most individuals having no deficiency in folic acid, trimethoprim therapy i s apparently quite safe, considering of course, length of treatment, amount of dose and potentiation by other drugs. Those who might be under folate stress such as the elderly, pregnant women, the chronically i l l and the malnourished, must be treated more carefully (22).
7.
Methods of Analysis
7.1
Elemental Analysis
A typical elemental analysis of a trimethoprim sample is presented in Table V I I (23).

Table V I I Elemental Analysis of Trimethoprim
E 1 ement
C H
% Theory
% Found
57.92
6.25
19.30
58.06 6.31 19.48
7.2
Thin-Layer Chromatographic Analysis Trimethoprim, and its immediate precursor 3,4,5trimethoxy-2 -cyano-dihydrocinnamaldehyde dimethylacetal have been separated by thin-layer chromatography using the following set of conditions (7): Adsorbent Solvent System Samp 1 e Silica Gel GF Benzene:diethyl ether:methanol: conc. NH40H (5050:10:I ) Dissolve 100 mg in 10 ml of methanol. Apply 0.05 ml (equivalent to 500 ug of trimethoprim)
TRIMETHOPRIM
465
Development
Detect i o n
Ascending f o r a t l e a s t 15 cm. Chromatography commences d i r e c t l y a f t e r the s o l v e n t has been poured i n t o the tank. (a) Shortwave u l t r a v i o l e t 1 i g h t (b) M o d i f i e d Dragendorff Reagent S o l u t i o n A: D i s s o l v e 0.8 gm o f Bi(N0 ) i n 10 m l o f g l a c i a l a c e t i c a c i d i n $ d i l u t e t o 40 m l w i t h water. S o l u t i o n B: D i s s o l v e 4 g o f K1 i n m 10 m l o f water. Spray Reagent: Mix 5 m l each o f t h e 2 s o l u t i o n s and d i l u t e t o 50 m l w i t h 10% H2S04. ( c ) Spray the p l a t e w i t h 10% H SO and then w i t h a 1 : l m i x t u r e of210k w/v f e r r i c c h l o r i d e and 5% w/v p o t a 9 sium f e r r i c y a n i d e . Table VI I I
Th in-Layer Chroma tog raphy o f T r imethopr im Approxi mate Compound
Rf 0.25
(a)
!! E .
0.1 10
S e n s i t i v i t y o f Detection
(b)
lie
0.05 20
T r imethopr im D imethy 1 a c h t a l
0.66
0.05
0.1
A t h i n - l a y e r chromatographic method has been r e p o r t e d (24) which separates t r i m e t h o p r i m and f o u r metabol i t e s as fluorescence spots on n o n - f l u o r e s c i n g s i l i c a gel plates. M e t a b o l i t e I I by t h i s method i s insepara b l e from M e t a b o l i t e 1 1 1 b, t h e N-3-oxide m e t a b o l i t e , whose presence has been q u a n t i t a t e d by an updated met hod emp 1 oy ing deve 1 opmen t i n ethanol /benzene ( 3 :7) p r i o r t o the chloroform/n-propano1/28% NH40H system below (19). E x t r a c t i o n s a r e made from b i o l o g i c a l f l u i d s and t i s s u e and 2 a l i q u o t s a r e drawn; one for t h e d e t e r m i n a t i o n o f t h e unconjugated m e t a b o l i t e s and t r i m e t h o p r i m and t h e o t h e r f o r the d e t e r m i n a t i o n of t h e conjugated m e t a b o l i t e s . The l a t t e r i s t r e a t e d w i t h Glusulase and i s developed u s i n g a s o l v e n t system o f chloroform, n-propanol and 28% NH40H (1OO:lO:l) Trimethoprim and M e t a b o l i t e s I I and 1 1 1 a r e
466
GERALD J, MANIUS
developed on separate p l a t e s from s u b d i l u t i o n s o f t h e a l i q u o t taken. The same combination o f developing s o l v e n t s i s used b u t t h e volume r a t i o s d i f f e r : 80:20:1 f o r trimethoprim: 25:20:1 f o r M I I I ( a ) and 100:10:1 f o l l o w e d by 80:20:1 f o r M I I . Each p a t e as i t a i r d r i e s shows a l i g h t - b l u e f l u o r e s c e n t spot v i s i b l e a t 360 nm.
7.3
High Performance L i q u i d Chromatographic Analys S
A h i g h performance l i q u i d chromatographic method has been developed (25) f o r t h e q u a n t i t a t i v e determinat i o n o f trimethoprim. The column was s t e e l , 0.5 m x 3 mm I .D., and packed w i t h Merckosorb S 1 60, whose p a r t i c l e s i z e was 5 pm. The mobile phase was 10% methanol, 89.9% c h l o r o f o r m ( c o n t a i n i n g 0.6-1% ethanol) and 0.1% concentrated ammonia, a t a f l o w r a t e o f 0.6 ml/rnin,, and a pressure o f 1150 p s i . A UV d e t e c t o r was used a t a wavelength o f 290 nm. As an i n t e r n a l standard, 2-amino-pyrimidine was found s u i t a b l e . A 2 P I methanolic s o l u t i o n c o n t a i n i n g 1 t o 2 pg o f each i s i n j e c t e d . F i g u r e 10 shows t h e s e p a r a t i o n o f t h e i n t e r n a l standard ( e l u t i n g f i r s t ) and t r i m e t h o p r i m a t a r e t e n t i o n time o f about 10 minutes.
7.4
Gas-Liquid Chromatographic A n a l y s i s
A g a s - l i q u i d chromatographic method has been r e p o r t e d f o r q u a n t i t a t i o n o f t r i m e t h o p r i m i n t i s s u e s (26). A 5% O V - 1 0 1 on Gas Chrom Q, pyrex column was used a t 250C i n c o n j u n c t i o n w i t h a n i t r o g e n thermionic d e t e c t o r . Acetone as a d i s s o l u t i o n s o l v e n t was found t o reduce trimethoprirn adsorption onto the N,N-d i-n-butyl-4-n-hexyloxy- 1 glass. naphthamidine was used as an i n t e r n a l standard. A g a s - l i q u i d chromatographic separation o f t r i m e t h o p r i m from i t s immediate precursors, the cinnamon i t r i l e and the d i m e t h y l a c e t a l can be achieved on a 4 f t x 2 mm I.D. g l a s s column packed w i t h 5% QF-l on 60/80 mesh Supelcoport. The column was programmed from 150C t o 25OoC a t 8"C/min. The c a r r i e r gas was n i t r o g e n a t a flow r a t e o f 30 ml/min (27). Figure 1 1 shows the separation.
TRIMETH0PRIM
467
t ( min.)
c----------l
5
0
F I G U R E 10
HPLC Analysis of Trimethoprim
U
i
3,4,5 -TRIMETHOXY -2-METHOXY-
METHYL CINNAMONITRILE
w F I CHLOROFORM SOLVENT
=)
m a
z z
ao ao
I
ln
0)
too too
I
I
90 90
70 70
60 60 50 50 40 40 RECORDER RESPONSE
30 30
20 20
1 1 0 0
0 0
08 08
ro o
TRIMETHOPRIM
469
7.5 Spectrophotofluorometric
Analysis
A semiautomated spectrophotofluorometric method o f a n a l y s i s of t r i m e t h o p r i m i n b i o l o g i c a l f l u i d s and t i s s u e has been r e p o r t e d (28). The method r e q u i r e s o x i d a t i o n w i t h a l k a l i n e potassium permanganate t o t h e f l u o r e s c e n t trimethoxybenzoic a c i d . The fluorescence i s measured a t an a c t i v a t i o n wavelength o f 275 nm, and a fluorescence wavel e n g t h o f 345 nm. A Technicon Sample I I i s used w i t h a Perkin-Elmer model 204 Fluorescence Spectrophotometer w i t h 150-W Xenon l i g h t source.
7.6
D i r e c t Spectrophotometric A n a l y s i s A d i r e c t spectrophotometric a n a l y s i s o f t r i m e t h o p r i m can be done by i o n - p a i r f o r m a t i o n w i t h bromc r e s o l green and measurement f o r the p r i m a r y amine a t 415 nm. Trimethoprim has been assayed i n t r i methoprim t a b l e t s and i n combination w i t h sulfamethoxazole i n t a b l e t s (29).
7.7
P o l arog raph i c Anal ys is
Trimethoprim has been analyzed on t h e PAR Model 174 P o l a r o g r a p h i c Analyzer u s i n g t h e f o l l o w i n g condit i o n s . Sample c o n c e n t r a t i o n was 0.090 vg/IO m l 0.1N H SO The temperature was 2'. 5C The v o l t a g e range i a ~ ~ l O .t8 -1.5 vs. Ag/AgCI r e f e r e n c e w i t h o t h e E = -1.02V. The c u r r e n t range was 5 pa f u l l scale! t h e scan r a t e 5 mv/sec over a c h a r t range o f 0-30 cm. The modulation amplitude was 50 mv. The Hg column h e i g h t was 60 cm, and t h e drop t i m e was 1/2 second. The o p e r a t i o n mode was d i f f e r e n t i a l p u l s e polarography u s i n g a low pass f i l t e r of 0.3. F i g u r e 12 shows t h e r e s u l t s o f t h e a n a l y s i s (30). A DPP method has a l s o been developed f o r t h e a n a l y s i s o f t h e two N-oxide m e t a b o l i t e s ( 1 8 ) .
7.8
T i t r i m e t r i c Analysis A t i t r i m e t r i c a n a l y s i s o f t r i m e t h o p r i m can be performed p o t e n t i o m e t r i c a l l y by t i t r a t i n g 0.8 0.9 grams o f t r i m e t h o p r i m i n 100 ml o f 9O:lO a c e t i c a n h y d r i d e : g l a c i a l a c e t i c a c i d w i t h 0.1N p e r c h l o r i c One m l o f 0.1N a c i d i n g l a c i a l a c e t i c a c i d (7). p e r c h l o r i c a c i d i s e q u i v a l e n t t o 29.03 mg o f t r i methoprim.
470
GERALD J. MANIUS
FIGURE 12 Polarographic Analysis o f T r i me thopr i m
TRIMETHOPRIM
471
m l HC104 x N HC104 x 0.2903 x 100
= % Trimethoprim
Sample W t .
(9)
F i g u r e 1 3 shows a t y p i c a l t i t r a t i o n . c u r v e (31) A M e t t l e r DK 10 was used w i t h a combination g l a s s calomel e l e c t r o d e a t a range o f 500 mv.
7.9
Microbiological Analysis
A m i c r o b i o l o g i c a l a n a l y t i c a l method f o r t r i m e t h o p r i m has been r e p o r t e d (32). An agar medium was prepared c o n t a i n i n g Evans peptone 0.5%, Lab-Lemco beef e x t r a c t 0.3%, New Zealand agar 1.51% i n d i s t i l l ed water, pH 7.5. The t e s t organism was B a c i l l u s pumilus. The t e s t b i o l o g i c a l f l u i d s were spiked w i t h 0.5 t o 0.005 pg/ml o f t r i m e t h o p r i m t o produce The p l a t e s were incubated a t 28Oc t h e standards. and were measured f o r s i z e o f i n h i b i t i o n as soon as growth was observed. A graph was c o n s t r u c t e d on semi-log paper o f t h e c o n c e n t r a t i o n s o f t h e standards versus zone s i z e s and t h e sample l e v e l s were then p i c k e d o f f from there.
7.10 Phase S o l u b i l i t y A n a l y s i s Phase s o l u b i l i t y a n a l y s i s may be c a r r i e d o u t u s i n g a 1 : l m i x t u r e by volume o f water and methanol as t h e s o l v e n t . A t y p i c a l example f o r t r i m e t h o p r i m i s shown i n F i g u r e 14 which a l s o l i s t s t h e c o n d i t i o n s under which t h e a n a l y s i s was performed ( 3 3 ) .
8.
Acknowledgements The author wishes t o thank t h e members o f t h e Research Records Department and S c i e n t i f i c L i t e r a t u r e Department o f Hoffmann-La Roche f o r t h e i r e f f o r t s . Sincere thanks a l s o t o D r . S. Moros and M r . R. Tscherne f o r t h e i r h e l p f u l advice. F i n a l l y , warm a p p r e c i a t i o n t o D r . P. Manchand f o r h i s c o n t r i b u t i o n s o f some o f t h e compounds necessary f o r t h i s monograph.
472
GERALD J. MANIUS
I
4
I
Titront Volume (ml)

9
I
FIGURE 13
Ti trimetric Curve for Trimethoprim
"
"
PHASE SOLUBILITY ANALYSIS

SAMPLE: TRlMETHOPRlM SOLVENT: Methanol:Water (I:I) SLOPE: 0.33% EQUILIBRATION: 20 Hu at 25.C an EXTRAPOLATED SOLUBILITY: 9.49 mg/g of Methanol:Water
20
40
60
80
loo
SYSTEM COMPOSITION mg of SAMPLE p g SOLVENT a
474
GERALD J. MANIUS
References
1.
2.
3.
4. 5.
6.
7.
8. 9.
10.
11.
12.
13. 14.
15. 16.
17.
18. 19.
20.
21.
22.
23.
Waysek, E., Hoffmann-La Roche Inc., Personal Communication. Chang , F., Hof fmann-La Roche Inc. , Personal Communication. Johnson, J. H., Hoffmann-La Roche fnc., Personal Commun i c a t ion. Winslow, W. C., Hoffmann-La Roche Inc., Personal Communication. Winslow, W. C., Hoffmann-La Roche Inc., Personal Communication. Benz, W., Hoffmann-La Roche Inc., Personal Communic a t ion. P i c c i o , E., Lau, E. and Senkowski, B. Z., HoffmannLa Roche Inc., Unpublished Data. Ramsland, A., Hoffmann-La Roche Inc., Personal Communication. Ramsland, A., Hoffmann-La Roche Inc., Personal Commun ica t ion. Heveran, J. E., Hoffmann-La Roche Inc., Unpublished Data. Stewart, D., Hoffmann-La Roche Inc., Personal Communication. Roth, B. and S t r e l i t z , J. Z., J. Org. Chem., 34 No. 4, 821-836 (1969). H o f f e r , M., U.S. Patent #3341541 (1967). Johnson, J. B., Hoffmann-La Roche Inc., Unpublished Data. U n i t e d States Pharmacopeia X I X , p . 651 (1974). Schwartz, D. E., V e t t e r , W., and E n g l e r t , G., Arzneim.-Forsch. 20, 1867 (1970). S i g e l , C. W., B r e z , D. A., J. Pharm. Sci., 62, 694-695 (1973). Brooks, M. A,, De S i l v a , J. A. F. and D'Arconte, L., J. Pharm. S c i I , 1395-1397 (1973). S i a e l . C. W.. Grace, M. E. and Nichol, C. J . o f I n f e c t i o u s Diseases, S580-S583 (1973). A.; Bushby, S.R.M., J. o f I n f e c t i o u s Diseases, S442-462 (.1 973). . _._ Then, R. and Angehrn, P., J. o f I n f e c t i o u s Diseases 1 8 S498-501 ( 1 973). 2, B r u m f i t t . W.. H a m i l t o n - M i l l e r , J. M. T., and Kosmidis; J.; J. o f I n f e c t i o u s Diseases;
62,
128,
128
S778-789 ( 1 973).
Scheidl, F., Hoffmann-La Roche Inc., Commun ic a t i on.
128,
Personal
TRIMETHOPRIM
475
24. 25. 26. 27 * 28. 29. 30 *
31.
32
33.
S i g e l , C. W. and Grace, M. E., J. Chromatogr., &, 111-116 ( 973). Oes t e rhe 1 , G., Schmidt, K. and Vecchi, M., Hoffmann-La Roche Inc., Unpublished Data. Flanagan, P., Chemistry and I n d u s t r y , 587-589, (1 975) * Manius, G., Hoffmann-La Roche Inc., Unpublished Data. Kaplan, S. A . , Weinfeld, R. E. and Lee, T. L., J. Pharm. S c i . , 62, 1865-1870 (1973). Weber, O., M o r i t c L. and G e l l e r , M., Hoffmann-La Roche Inc., Unpublished Data. Ross, A., Hoffmann-La Roche Inc., Personal Communic a t ion. S o k o l o f f , H., Hoffmann-La Roche Inc., Personal Communication. Bushby, S. R. M. and H i t c h i n g s , G. H., B r . J. Pharmac. Chemother., 33, 72-90 (1968). S o k o l o f f , H. and Umagat, H., Hoffmann-La Roche Inc., Personal Communication.
TUBOCURARINE CHLORIDE
Constantin Papastephanou
477
Copyright @ 1978 by Academic R e s s . Inc. All rights of reproduction in any form reserved ISBN 0-12-260807-0
470
CONSTANTIN PAPASTEPHANOU
TUBOCURARINE CHLORIDE Contents Description 1.1 Name, formula, molecular weight 1.2 Appearance, color, odor 2. 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 constant 3. Isolation and Purification 4 . Stability and Degradation 5. Drug Metabolism 6. Methods of 'Analysis 6.1 Identity tests 6.2 Elemental analysis 6.3 Ultraviolet absorption 6.4 Colorimetric analysis 6.5 Fluorescence analysis 6.6 Thin-layer chromatography 6.7 High-pressure liquid chromatography 6.8 Gas chromatography 6.9 Electrophoresis 6.10 Bioassay 6.11 Radioimmunoassay 6.12 Assays in body fluids and tissues 7. References
1.
479
TUBOCURARINE CHLORIDE Description 1.1 Name, formula, molecular weiqht Tubocurarium, 7' ,12'-dihydroxy-6,61dimethoxy-2,2' ,2l -trimethyl-, chloride, hydrochloride, pentahydrate. (+) -Tubocurarine chloride hydrochloride pentahydrate.
1.
c 1-
Appearance, odor, color White or yellowish white to grayish white, crystalline odorless powder.
1.2
480
2.
Physical P r o p e r t i e s
I n f r a r e d Spectrum The i n f r a r e d spectrum o f U . S . P . r e f e r e n c e s t a n d a r d t u b o c u r a r i n e c h l o r i d e is shown i n F i g u r e 1. The spectrum o b t a i n e d a s a KBr p e l l e t u s i n g a P e r k i n E l m e r 621 s p e c t r o m e t e r , i n d i c a t e s t h e following frequencies (1).
2.1
Frequency cm-1 34 1 ) 0 3330

2930
0-H,
C-H
Assignment
N-H
stretch
stretch
2500 ( b r o a d )
H-C1 Aromatic C = C s t r e t c h = C 0 ether and hydroxyl
1505
1220
-d
rl
ai
k 0
E
a,
.rl
m'
rcI
a, k m k
C H
482
Nuclear Magnetic Resonance Spectrum The 100 M z proton magnetic resonance H spectrum of d-tubocurarine (Figure 2) studied in DMSO-dG containing TMS as internal reference, and utilizing a Varian XL-100-15 NMR spectrometer, indicates the presence of the following resonances (2): chemical Shifts
(
2.2
Assignments
s
ppm)
-9
9.63 8.01
1H, D20 Exchanged OH,
g , lH,OH,D20 Exchanged
7.i a
6.18 7.00
6.60
d,J=8.0 Hz, 1H d,J=8.0 Hz, 1 d,J=8.5 Hz, d,J=8.5 Hz, 2H
H2C5+
7.08
other aromatic singlets
m,J=7.5
4.86 5*20)
Hz, l H }
3.96 3.36
3.12 2.94 2.75
s =
=,
s,
1H, 6H, OCH3
two
c-c
0
groups
5, 10H, 5 H20
2, 3H, 8 3 -
a 3
broad, g, d = doublet
singlet,
Figure 2.
NMR spectrum o f t u b o c u r a r i n e c h l o r i d e .
484
Some o f t h e s a l i e n t f e a t u r e s o f t h e NMR spectrum a r e changes, on D20 exchange, i n t h e resonances due t o water ( 5 moles) and N-methyl p r o t o n s : , t h e former moved downfield w h i l e t h e l a t t e r moved s l i g h t l y u p f i e l d . The resonance a t 6 2 . 7 5 also became s i g n i f i c a n t l y s h a r p e r . Mass Spectrum The mass spectrum o f t u b o c u r a r i n e c h l o r i d e ( F i g u r e 3 ) y i e l d s a molecular i o n of m / e 594, which i s c o n s i s t e n t f o r t h e f r e e base due t o e l i m i n a t i o n , b y e l e c t r o n - i m p a c t and/or t h e r m o l y s i s o f t h e elements of methyl c h l o r i d e and H C 1 . The molecular i o n a t m / e 50 s u g g e s t s t h a t CH3C1 i s eliminated thermally. I n a d d i t i o n a peak a t m / e 608 could be due t o e l i m i n a t i o n of two molecules of HC1 e i t h e r t h e r m a l l y and/or by e l e c t r o n - i m p a c t . Fragmentation a t t h e bonds @ - t o t h e n i t r o g e n w i t h t h e formation o f a tropylium ion s t r u c t u r e leads t o t h e i n t e n s e i o n a t m / e 298, d e p i c t e d below and a r i s i n g from t h e molecular i o n a t m / e 594.
2.3
HO
OCHq
m/e
298
The o t h e r h a l f of t h e molecule would t h u s be r e p r e s e n t e d by m/e 296. The r e a c t i o n o f t u b o c u r a r i n e c h l o r i d e w i t h t r i m e t h y l s i l y l imidazole r e a g e n t y i e l d s molecular i o n s a t m / e 738 and m / e 752 analogous t o t h e u n d e r i v a t i z e d compound. The m/e 370 fragment ion corresponds t o m / e 298 p l u s s u b s t i t u t i o n o f one s i l y l group confirming t h e p r e s e n c e of o n l y one hydroxyl group i n t h i s p o r t i o n of t h e molecule.
485
Likewise m / e 368 corresponds t o one s i l y l group s u b s t i t u t e d on t h e m/e 296 fragment. A l a r g e m / e 58 i o n i n b o t h t h e s p e c t r a of t h e compound and i t s s i l y l d e r i v a t i v e a r i s e s from t h e amino p o r t i o n of t h e molecule and p r o b a b l y h a s the elements C3H8N. A weak peak a t m / e 622 i n t h e spectrum of t h e u n d e r i v a t i z e d compound a s w e l l a s h i g h e r mass i o n s a t m/e 796, 810, 812 and 826 i n t h e spectrum of t h e s i l y l d e r i v a t i v e s u g g e s t s t h a t a t r a c e i m p u r i t y is p r e s e n t o r t h e r m a l r e a c t i o n s o c c u r i n t h e mass s p e c t r o m e t e r . The o r i g i n o f t h e s e i o n s h a s n o t been e s t a b l i s h e d (2). U l t r a v i o l e t Spectrum F i g u r e 4 shows t h e W spectrum o f a s o l u t i o n of t u b o c u r a r i n e c h l o r i d e i n w a t e r . One peak a t 280 n w i t h a n Eyern of 107 is seen. m The Same peak and El% a r e o b t a i n e d fOr a s o l u t i o n of t u b o c u r a r i n e c h l o r i % i n 0.01g HC1. The spectrum o f t h e drug i n 0 . 0 1 5 NaOH however, m changes t o one w i t h a peak a t 291 n and an E l % 1Cm of 156. The s p e c t r a i n t h e s e t h r e e s o l v e n t s remained unchanged a f t e r 2-1/2 h o u r s (3). 2.4
(D
m
N
.rl
ai a
k 0
al
.rl
k I U k
0 E
4J
k
.t 0
(D
a,
>
f
N
k 7
a,
.rl
bl
al 4J
-d
al
.d
r:
al
m
k
488
2.5
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 m i n g 1 0 m l o f water ( 4 ) . 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 m e x c i t a t i o n wavelength i s 375 n and t h e maximum emission wavelength is 425 nm ( 5 ) . 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 ) . 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 ) . 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 . 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 ) . 2.10 2.9 2.8 2.7 2.6
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
k a, c,
C
.d
.rl
0
CI 0
a,
rl
o N
0
k 0
E
. d
0
0 0
k rd k
0
CI W
o m
4
+ I
W
7u
0
0 u 0h
c
I
o r
u m N =
a, -9 U a,
w
2
4 >
O u 0
8 c
0 W
k k
7
0
E 7 k c,
m
u
0 I 0 n
%
a, U
0
a,
I 0 .
W 0
y1 0
u 0
"
0
N 0
y1 N
2 U
0
A l I S N 3 1 N I 33N33S380flld
rl
k 0 7
u) 0)
b.4
m
k
a,
.A
1 trr
b.4
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)
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
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
C
k m k
Q)
2
U
W
crc
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).
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.
3.
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.
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 . 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%i w a t e r ) . A f t e r mixing and warming on a steam n 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% / v ) , a w 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
6.
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 Element
C H N
% Found
58.08 6.65 3.62 8.99 6.3
% Calculated
57.58 6.79 3.63
c1
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 % 1m c 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 % lcm 2 8 0 n a s 118. m 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. 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
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. 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 -
6.5
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
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 p 6.5. H 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
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 1 Bioassay .0 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
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
499
References
1 .
2. 3 . 4 . 5.
6.
7.
8. 9.
10. 11. 12. 13.

14. 15. 16. 17. 1. 8
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).
500
23. 24. 25. 26. 27. 28. 29. 30. 31.

320 33.
34. 35. 36. 37. 38. 39. 4. 0
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, 9 . 237(1966). 5 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., 1 1 149 (1975). 1, 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,6 . 7 (1971) . 031 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,
ADDENDAANDERRATA
501
Copyright 0 1978 by Academic Press, h e . All rights of reproduction in any form reserved.
ISBN 0-12-2m7-0
502
ADDENDA AND ERRATA
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
CUMULATIVE INDEX
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
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

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Analytical Profiles of Drug Substances

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

W ACADEMIC PRESS, INC.

Academic Press Rapid Manuscript Reproduction

ACADEMIC PRESS, INC.

1250 Sixth Avenue, San Diego, California 92101

24/28 Oval Road, London N W l

United Kingdom Edition published by ACADEMlC PRESS, INC. (LONDON) LTD.

Library o Congress Cataloging in Publication Data f

Analytical profiles of drug substances.

AFFILLATIONS OF EDITORS, CONTRIBUTORS, AND REVIEWERS

N. W .Atwater, E. R. Squibb & Sons, Princeton, New Jersey

K . S. Albert, The Upjohn Co., Kalamazoo, Michigan

AFFILIATIONS OF EDITORS, CONTRIBUTORS. AND REVIEWERS

Analytical Profiles of Drug Substances, 7

Steven A. Benezra and T. Reney Bennett

STEVEN A. BENEZRA AND T. RENEY BENNETT

1 1 Name, Formula, Molecular Weight .

C5H4N40 1.2 Appearance, Color, Odor

Allopurinol is a white to off-white powder with a slight odor

Table I Infrared Spectral Assignments for Allopurinol Frequency (cm-l)

1700 1590 1245

Infrared Spectrum of Allopurinol

Nuclear Magnetic Resonance (NMR) Spectrum

Chemical Shift (ppm)

257 250 252

7200 7600 7600

STEVEN A. BENEZRA AND T.RENEY BENNETT

m/e 120 (4.2%)

Melting Point Allopurinol melts above 35OOC7

The solubility of ajlopurinol in various solvents at 25OC is given in Table 111

<0,01 0.60 0.30

The pka 9f allopurinol determined spectrophotometrically is 10.2

STEVEN A. BENEZRA AND T. RENEY BENNETT

Crystal and Molecular Structure

STEVEN A. BENEZRA AND T. MNEY BENNETT

Methods of Analysis 5.1 Elemental Analysis Element C H

2.96 41.17 11.76

Table V Paper Chromatography Systems for Allopurinol Mobile Phase

Paper Whatman 3MM Whatman 3MM Whatman 3MM

1-butano1:water 84:16 1-p ropanol:water 7 :3 1-butano1:water 84:16(NH3 atmosphere)

STEVEN A . BENEZRA AND T. RENEY BENNETT

from xanthine and uric acid16. with 1N hydrochloric acid.

Allopurinol was eluted

5.44 High Performance Liquid Chromatography

Spherisorb CH CN:H20: 10 &OH I 1060: 4 80: Aminex A-27 Whatman PA-3 8

Potassium Phosphate O.O15M, pH 5.7, 4OoC

Metabolism and Pharmacokinetics

STEVEN A. BENEZRA AND T. RENEY BENNETT

USP XIX, p. 18 (1975)

D.H. Mitchell, Burroughs Wellcome, personal cmunication

R Crouch, Burroughs Wellcome, personal communication .

D. Brent, Burroughs Wellcome, personal communication

P. Prusiner and M. Sundaralingam, Acta Cryst., 2148 (1972)

1 . U.S. Patent 3,694,484 0 11. U . S . Patent 3,864,341

14. P.D. Gressel and J.F. Gallelli, J. Pharm. Sci., (1968)

V. M i l l e r , V . Pac6kov6, and E. Smolkovb, J . Chrom., 216 (1976) W. Snedden and E.B.

Analytical Profiles of Drug Substances, 7

Pranah K . Bhnttacharyya and Winifred M . Cort

PRANAB K. BHATTACHARYYA AND WINIFRED M. CORT

Amoxicillin Stability Solubility

Methods of Analysis Pharmacological Evidence