Analtical

Ana1yticaI Profiles
of
Drug Substances
Volume 13
EDITORIAL BOARD
Abdullah A. Al-Badr Klaus Florey

Steven A. Benezra Salvatore A. Fusari
Rafik Bishara Lee T. Grady
Gerald S. Brenner Boen T. Kho
Glenn A. Brewer, Jr. Joseph A. Mollica
Nicholas DeAngelis James W. Muiison
John E. Fairbrother Milton D. Yudis
Academic Press Rapid Manuscript Reproduction

Analytical Profiles
of
Drug Substances
Volume 13
Edited by
Klaus Florey
The Squilh Institute for Medical Research
New Rriiiiswick, N e w Jersey
Contrilmting Editors
Abdullah A. Al-Badr Glenn A. Brewer, Jr.
Norman W. Atwater Salvatore A. Fusari
Steven A. Benezra Lee T. Grady
Rafik Bishara James W. Munson
Gerald S. Brenner
Compiled tinder the auspices of tlw
Pliuriiiaceutical Analysis (ind Control Section
APhA Ac(idcmy of P l w t-titcrcentical Scimces
ACADEMIC PRESS, INC. 1984

(Harcourt Brace Jovanovich, Publishers)
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United Kingdom Edition published by

ACADEMIC PRESS, INC. LONDON) LTD.
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Library of Congress Cataloging in Publication Data

Analytical profiles of drug substances.
Compiled under the auspices of the Pharmaceutical
Analysis and Control Section, Academy of Pharmaceutical
Sciences.
Includes bibliographical references and indexes.
1. Drugs--Analysis. I. Florey, Klaus. 11. Brewer,
Glenn A. 111. Title: Academy o f Pharmaceutical Sciences.
Pharmaceutical Analysis and Control Section.
[DNLM: 1. Drugs--Analysis--Yearbooks. QV740 AAl A551
RS189.A58 615l.1 70- 187259
ISBN 0-12-260813-5 ( V . 13)
PRINTED IN THE UNITED STATES OF AMERICA
84 85 86 81 9 8 7 6 5 4 3 2 1
CONTENTS
Affiliations of Editors, Contributors, and Reviewers vii

Preface ix
Atenolo1 1
Vesna caplar, Zooniinira MokotiC-Milaun, Hrvoje Hofinan,
Josip Kuftinec, Franjo KajfeZ, Antun Nagl, and Nikola BlaZeviC
Camphor 27
Jaber S . Mossa and M u h o u d M . A . Hassan
Chloroy uine 95
Molaammad Tariq and Abdullalz A. Al-Badr
Cimetidine 127
P. M . G. Bazjin, Alex Post, arid John E . Zarenibo
Disopyramide Phosphate 183

Alan Wickinan and Patricia Finnegan
Indomethacin 211
Matthew O’Brien, laines McCauley, and Edward Cohen
Ketotifen 239
Zvonirnira MikotiC-Mihun, Josip Kuftinec, Hrcoje Hofman,
Mladen ZiniC, Franjo Kajfei, and Zlatko MeiC
Melphalan 265
L . Valentin Feyns
Moxalactam Disodium 305

Leslie J . Lorenz and Patricici N . Thomas
V
vi CONTENTS
Oxyphenbutazone 333
Abdullah A . Al-Badr and Hunieidn A . El-Obeid
Pentazocine 361
Terry D . Wilson
Yhen ytoin 417

Jose Philip, Ira J. Hokoinh, and Snhatare A. Fusnri
Pyridoxine Hydrochloride 447

Hassun Y. Aboul-Eneiii and Mohaninied A . Lout$/
Saccharin 487
Muhammad Uppnl Zubnir and Mahinoud M. A. Hnssan
Salicylamide 52 1
Salini A. Babhair, Abdullnh A . Al-Buds.
and Hassan Y. Aboul-Enein
Silver S ulfadiazine 553

Auke Bult and Cees h4. Plug
Sulindac 573
Fotios M . Plakogiannis and James A. McCnuley
Tetracycline Hydrochloride 597

Syed Laik Ali
Vitamin D3 (Cholecalciferol) 655

K . Thonias Koshy and Willianc F . Beyer
PROFILE SUPPLEMENTS
Tolbu tamide 719

Said E . lbrahiin and Abdullah A. Al-Badr
Reserpine 737
Farid j . Muhtadi
ERRATUM
Phenylpropanolamine Hydrochloride 767
Cumulative Index 769

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

A . A . Al-Badr, King Saud University, Riyadh, Saudi Arabia
S , L. Ali, Zentrallaboratoriuin Deutscher Apotheker e. V . , Eschborn, Federal
Republic of Germany
N . W. Atwater, E. R. Squibb & Sons, Princeton, New Jersey
S. A . Babhair, King Saud University, Riyadh, Saudi Arabia
P. M . G. Bauin, Smith Kline & French Research, Ltd., The Frythe, Welwy11,
Hertfordshire, United Kingdom
S . A . Benezm, Wellcoine Kesearch Laboratories, Research Triangle Park,
North Carolina
W. F . Beyer, The Upjohn Company, Kalamazoo, Michigan
R. Bishara, Lilly Research Laboratories, Indianapolis, Indiana
N . BlaZeuiC, Institute for the Control of Drugs, Zagreb, Yugoslavia
G. S. Brenner, Merck Sharp & Dolime Research Laboratories, West Point,
Pennsylvania
G . A. Brewer, J r . , The Squibl) Institute for Medical Research, New Bruns-
wick, New Jersey
A . Bult, State University of Leiden, Leiden, The NetherIands
V. Cuplnr, Podravka-Institute, Zagreb, Yugoslavia
E . Colzen, bferck Sharp & Dohme Rcscwdi Laboratories, West Point, Penn-
sylvania
N . DeAngelis, Wyeth Laboratories, Philadelphia, Pennsylvania
H . A . El-Obeid, King Saud University, Riyadh, Saudi Arabia
J. E. Fairbrother, Stiefel Lahratories Ltd., Sligo, Ireland
L. V. Feyns, The United States Pharmacopeia, Rockville, Maryland
P. M . Finnegan, G . D. Searle and Co., Skokie, Illinois
K . Florey, The Squibb Institute for Medical Kesearch, New Brunswick, New
Jersey
vii
...
Vlll AFFILIATIONS OF EDITORS, CONTRIBUTORS, AND REVIEWERS
S. A . Fusari', Warner-Lambert Company, Morris Plains, New Jersey

L. T. Grady, The United States Pharinacopeia, Rockville, Maryland
M . M . A. Hnssan, King S a d University, Riyadh, Saudi Arabia
H . Hofinan, Podravka-Institute, Zagreb, Yugoslavia
I . J . Holcomb, Warner-Lambert Research Institute, Morris Plains, New Jersey
S. E . Ibrahim, King Saud University, Riyadh, Saudi Arabia
F . Kajfei , Podravka- Inst it u tc , Zagreb , Yugoslavia
B . T. Kho, Ayerst Laboratories, Rouses Point, New York
K . Thomas Koshy, The Upjohn Company, Kalamazoo, Michigan
J . Kuftinec, Podravka-Institute, Zagreb, Yugoslavia
L. J . Lorens, Lilly Research Laboratories, Indianapolis, Indiana
M . A . Loutfy, King Saud University, Kiyadh, Saudi Arabia
J . A . McCauley, Merck Sharp & Dohme Research Laboratories, Rahway, New
Jersey
Z. MeiC, Institute Ruder Boskovii., Zagreb, Yugoslavia
Z. MikotiC-Mihun, Podravka-Institute, Zagreb, Yugoslavia
J . A . Mollica, CIBA-GEIGY Corporation, Summit, New Jersev
J . S. Mossa, King Saud University, Riyadh, Saudi Arabia
F . J . Muhtadi, King Saud University, Riyadh, Saudi Arabia
J . W. Munson, The Upjohn Company, Kalainazoo, Michigan
A . Nagl, University of Zagreb, Zagreb, Yugoslavia
M . O'Brien, Merck Sharp & Dohme Research Laboratories, West Point, Penn-
sylvania
J . Philip, Warner-Lambert Company, Morris Plains, New Jersey
F . M . Plakogiannis, Arnold and Marie Schwartz College of Pharmacy and
Health Sciences, Brooklyn, New York
C. M . Plug, State University of Leiden, Leiden, The Netherlands
A . Post, Smith Kline & French Laboratories, Philadelphia, Pennsylvania
M. Tariq, King Saud University, Riyadh, Saudi Arabia
P. N . Thomas, Lilly Research Laboratories, Indianapolis, Indiana
A. Wickman, Searle Laboratories, Skokie, Illinois
T.D. Wilson, Sterling-Winthrop Research Institute, Rensselaer, New York
M . D.Yudis, Shering-Plough Inc., Bloomfield, New Jersey
J . E . Zarembo', Smith Kline & French Laboratories, Philadelphia, Pennsyl-
vania
M . &nit, Podravka-Institute, Zagreb, Yugoslavia
M . U . Zubair, King Saud University, Riyadh, Saudi Arabia
'Present address: 22625 St. Joan, St. Clair Shores, Michigan 48080.
'Present address: Revlon Health Care Group, Tuckahoe, New York 10707
PREFACE
The compilation ofdnnlyticcil Profiles of Drug Szibstunces to supplement the

information contained in the official compendia is now a well-established
activity.
That we are able to publish one voluine per year is a trilxite to the diligence
of the editors to solicit articles and even more so to the enthusiastic re-
sponse of our authors, an international group associated with pharmaceutical
firms, academic institutions, and coinpendial authorities. I would like to ex-
press my sincere gratitude to them for making this venture possible.
Over the years, we have had queries concerning our publication policy. Our
goal is to cover all drug sulxtaiices of medical valiie, and therefire, we have
welcomed any papers of interest to an individual contributor. We also
have endeavored to solicit profiles of the most useful and used medicines, hut
inany in this category still need to be profiled.
In the preface to the eleventh voliime, I announced that we would try to
supplement previously published profiles with new data. Unfortunately, most
of the original contributors are no longer available to undertake this task, and it
has proven difficult to find other volunteers. We shall continue to pursue the
updating program, but it will not be as comprehensive as originally envisioned.
Again, I would like to request those who have found these profiles useful to
contribute papers of their own. We, the editors, stand ready to receive such
contributions.
ix
This Page Intentionally Left Blank
ATENOLOL
Vesna Caplar, ' Zvonimira Mikotid-Mihun,
Hrvoje Hofman, Josip Kuftinec, Franjo Kajfei
Podravka-I nstitute
Zagreb, Yugoslavia
Antun Nag1
IJnioerszt!l of Zagreb
Zagreb, Yzigoslacia
Nikola Blaievid
lnstitiite for the Control of Drugs
Zagreb, Ytigoslnoin
1. Foreword, History, Therapeutic Category 2

2. Description 2
2. I Name, Formula, Molecular Weight 2
2.2 Appearance, Color, Odor, Taste 2
3. Synthesis 2
4. Physical Properties 5
4.1 Spectra 5
4.2 Solid Properties 15
4.3 Solution Properties 19
5. Methods of Analysis 20
5.1 Elemental Analysis 20
5.2 Titrimetric Determination-Electrochemical 20
5.3 Chromatographic Methods 20
5.4 Determination of Impurities 22
6. Stability-Degradation 22
7. Drug Metabolism. Pharrnacokinetics, Bioavailability 22
8. Identification and Determination in Body Fluids and Tissues 23
9. Determination in Pharmaceuticals 23
References 24
'Correspondence,
ANALYTrCAL PROFILES OF DRUG SUBSTANCES Copyright 01984

VOLUME 13 1 by the i\rnencan Pharmaceutical A\\ociation
ISBS 0-1?-?60813-5
2 VESNA CAPLAR ET AL.
1. Foreword, History, Therapeutic Category

Atenolol is a &adrenolytic, cardioselective
drug, having no intrinsic sympathomimetic activity.
J3-Receptor blocking drugs were introduced in 1966,
t o treat cardiovascular disorders, These drugs are
especially efficient in cases of coronary failure
(angina pectoris), arterial hypertension and cardi-
ac arrhythmia. The s thesis o f atenolol was first
reported in 1970, (lyand the first pharmacological
and clinical studies were made in 1973. and 1974.
(2-4).
2. Description
2.1 Hame, Formula, Molecular Weight
b
Atevlolol is 4-/2-hydroxy-3-/(1-methylethyl)-
amino/-propoxy/-benzenacetamide and is also known
as 2-/p-/2-hydroxy-3- (isopropylamino)propoxy/phe-
nyl/acetamide or l-p-carbamoylmethylpheno~-3-iso-
propylamino-2-propanol,
CH2CO NH2
I
OCH2CHCH2
I NHCH(CH3)z
OH
‘1 4H22”203
Mole Wt;. 266.33
2.2. Appearance, Color, Odor, Taste
Atenolol is a white powder. It is odorless
and h a s a slightly bitter taste.
3 . Synthesis
Atenolol is synthetized starting from comer-
cially available 4-hydroxyphenylacetaide (&) and
epichlorohydrine (2) in 15-fold molar excess (1)
with piperidine as a catalyst (see Scheme 1.j. zhe
reaction mixture is stirred 4-6 hours at 95-100 C,
then allowed to cool to room temperature and left
t
0
G
+
I"
z
0
0
I
0
t n
(Y
i?
0
Y
I
0
I
2
I"
2
0
I"
z 0 0
0
I I
0-0
I"
g o 0 hl
I
0 (Y
n
X I m
0-0 I
+
i? 0
Y
I
0
0 4 N
-r
4
I" 0 2
0 0--0
z '
0 I
\I
0 -0
0
ATENOLOL 5
standing overnight. The precipitate is collected by

filtration and thoroughly trashed with methanol to
remove unreacted epichlorohydrine. The product, 1-
-(4-carbarnoylmethylphenoxy)2,3-epoxy-propane ( )
may be recrystallized from methanol, water or 2i:-
xane. The recrystallized compound 2 is reacted
with isopropylamine (24-fold molar exoess), in me-
thanol as solvent, at the boiling point, f o r 5-30
minutes. The solvent and excess of isopropylamine
are distilled o f f , and the crude atenolol recrys-
tallized from water. The use of isopropylamine in
the equimolar amount, or in a small excess, resul-
ted in formation of an impurity, namely the product
of a reaction of atenolol 2 and its precursor, 2,
which had the structure of di-/3-(4*-carbamoylme-
(3)
7.
thylpheno )-2-hydroxy-propyl/-isopropylamine
(3cheme 2.
The infrared spectrum o f atenolol, presented

in Fig. l., w a s recorded with a R3r pellet, The-l
bands a r e assigned as f o l l o w s : 3340 and 3160 cm
-CO-YH), 2940 (=CH), 1625 (-C=O, amide I), 1500
-N-'J=O, amide 11), 1400 (H N-CO-) 1385 (i-APr),
1390, 1235 (arylether) 117g (i-Prj, 1105, 1080,
1030, 910, 880, 810, 740, 700, 660. (5) The ir spe-
ctrum of the "dimeric" compound 5 exhibits the same
bands wit5 the same intensities in the region 4000-
-1100 cm- as does coypound2. However, t h e bands at
1085, 910 and 880 cm (weak t o medium) present in
the spectrum of a t e n o l o l are lacking in the spect-
rn of compound 2-
4.1.2. Ultraviolet
The ultraviolet spectrum of atenolol is pre-
sented in Fig, 2. It was recorded2with a methanolic
solution at the concentration lo- g/L. The
values of bands of atenolol and compound-5 lie
at the same wavelenths (i.e. 225, 275 and 283 nm),
but their intensities differ slightly.
.
4.1 3 . Proton Nagnetic R e s o n a E
The proton magnetic resonance spectrum of a-
tenolol in GD OD is presented. in Fig. 3. The spec-
3
10 0
80
CI
\
L 60
w
0
z
U
LO
-
I
v)
z
U
=
c 20
0' 1 I I I I r I I I a
LOO0 3000 2000 1800 1600 1200 cm-l 800

F i g . 1. I n f r a r e d s p e c t r u m of a t e n o l o l ( K B r p e l l e t ) .
I n s t r u m e n t : Pye Unicam SP3-200.
ATENOLOL
A
0.4
0.3
0.2
0.1
nm 200 300
Fig. 2. Ultraviolet spectrum of' atenolol. Instru-
ment: Pye Unicam SP8-loo UV-VIS
I
% L
I
8 7
I I
6 5
I I
4
1
3
I
2
Fig. 3. 80 MHz proton MR spectrum of atenolol.
’I
6 (pprn,d
Instrument:
Bruker WP 80 DS at 80 MHz (8 K points).
ATENOLOL 9
Table 1. Assignment of proton MR s i g n a l s in t h e

spectrum of a t e n o l o l
F r o t o m Chemical s h i f t Type Inten- Coupling const.
Ha
d cppm)
I
. d(A9)
s i tg
-
? H
Ja,b
J (Hz)
8 '(95 .
% 7-05 d(AB) 2 H J 8.755
b ,a
% 4. 99 S 2+1+1 H -
Hd 4.28-4.iO m 1 H -
He 4-12 S 2 H -
Hf 3.59 S 2 H -
H 3-03 dd JgA,gB 12.5
gA
JgA, d 5.00
HgB 2.81 dd J g ~ , g12.5
~
5.00
JgB, d
Hh
3 14-2.65 m .1 B -
i' 1.25 d 6 R Ji t 6.254
t r a l assignments are shown i n !Table 1. (5) Charac-

t e r i s t i c peaks of t h e proton PYX s p e c t r u a of compo-
und 2 (which may be p r e s e n t as an impurity i n cru-
de a t e n o l o l ) i s presented i n Table 2.
13C-hT!R s p e c t r a o f a t e n o l o l and compound 5

(presented i n Fig. 4.) were run in p u l s e FT mode on
a J e o l PX-loo spectrometer o p e r a t i n g a t 25-05 FlHz.
The samples were measured i n 5 mm t u b e s with TIMS as
an i n t e r n a l standard. An 8 R computer memory and i n -
t e r n a l deuterim lock ygre used., (5)
The broad-band C' Nl'JR s p e c t r a o f DMSO-d so-
l u t i o n s o f atenolol and i t s " d i n e r " 2 a r e ShOiA i n
0
Ea
I
rg
0
N
0
*
0
u)
0
OD
0
c
0
s!
0
3
0
0
a
0
s
0
-nk
10
0
(v
0
-l
0
(0
0
co
0
0
c
0
(v
c
0
*
c
0
P
0
W
c
Table 2. Assignment of p r o t o n MR s i g n a l s i n the

spectrum o f compound 2
(H* Nc 0c NCH(CH3)
\c h i
eotons
hemical s h i f t
6 (ppm) Type ;&
n en- Couplin
H 4 Y
a '? 36 d Ja.b
Hb 7,02
HC
4.98
Id
'
4.2-4. o m 2 13 -
He 4.13 s 4 rj -
9f 3.59 s 4 B -
9
&.A
2 875 d 2 R JgA, gB 4.0
H@ 2.98 d 2 H JgB, $4 5.0
Hh
2.8-3. o m 1 H -
H, 1.20 d 6 H J 6.504
Fig. 4. S i n c e t h e s o l v e n t peaks c o v e r one carbon

s i g n a l , t h e complementary CD OD s o l u t i o n s were a l s o
used i n making t h e complete assignment. I n t h i s way
an unambiguous assignment was achieved, a p p l y i n g
off-resonance and g a t e d decoupling (?JOY) s p e c t r a ,
as well as t a b u l a t e d data of chemical s h i f t s f o r
r e l a t e d c o n s t i t u t i o n a l p a r t s c o n t a i n e d i n t h e ate-
nolol molf3ule. ( 6 )
The C ITIIR s p e c t r a of t h e S D OD s o l u t i o n s r e -
v e a l two signals belonging t o noneauivalent methyl
carbons o f t h e i s o p r o p y l group. I n t h e Y313 spectrum
o n l y one complex q u a r t e t was produced correspon-
d i n g l y , s o t h a t o n l y one f i r s t o r d e r 3-€1 c o u p l i n g
c o n s t a n t was determined, t h e o t h e r one was o n l y es-
t i a a t e d , as being h i g h e r by a3out 4 Hz (see Table
3 . ) . The o t h e r m u l t i p l e t s were a l s o v e r y complex,
due t o s e v e r a l long-range c o u p l i n g s and o v e r l a p s ,
with both t h e s o l v e n t s used. l e v e r , a r a t h e r com-
p l e t e a n a l y s i s of t h e coupled9E'C MT4R spectrum w a s
made w i t h numerous f i r s t and h i g h e r C-H c o u p l i n g
ATENOLOL 13
T a b l e 3.13C Chemical s h i f t s a and I3C-'H coupling

constantsb f o r a t e n o l o l dissolved i n CD,OD
2
C-atom d (PPd lJCH
~~ ~
JcR JCH
C-lC 2.0;1.5 -
c-2
c-3
50.75 (t)
69.59
71.78
(d)
(t)
132.6
1+2 6
144.o
. 2.4;2.4
2.4
-
-
22.61 (a_) 129d - -
22.42 (9) 125.5 5.4 2.0
49.68 (d) 131e
c-1' - 2 9.7
C-2' '6' 115.36 (d 160.1 4.9 -
c-3' '5' 130.84 ( d ) 158.7 2.4 7.1;4.9
c-4' 128.83 ( s ) - 5.7 7.8
C-1' ' 176.80 ( s ) - 6.6;4.4 -
(3-2'' 42.41 ( t ) 128.4 - 3.9
+d i n pprn
-0.02
downfield from i n t e r n a l TKS; accuracy
ppm; off-resonance m u l t i p l e t s a r e given i n
parentheses
+
n ~ C Hi n HZ; accuracy -0.5 HZ
nJCHv a l u e s determined from t h e DMSO-d6 s o l u t i o n
estimated t o 21 Hz
4-
estimated t o -2 Hz; t h e long range coupling con-
s t a n t s can not be determined due t o very complex
multiplets
c o n s t a n t s which may be u s e f u l f o r a f u r t h e r s t r u c -
t u r a l study. The complete data a r e l i s t e d i n Table
3.
I n t h e DMSO-d s o l u t i o n only one CH - s i g n a l
was found i n t h e spgctrum o f a t e n o l o l , an8 t h e s o l -
vent caused a s l i g h t downfield e f f e c t upon he che-
mical s h i f t . On t h e o t h e r hand, a l l o t h e r I3C che-
mical s h i f t s were moved s u b s t a n t i a l l y u p f i e l d , t h e
changes rangin f r o m 0.60 f o r C-4' t o 4.27 ppm f o r
t h e C=O group ?Table 4.). The NOIT spectrum shows
r a t h e r smeared m u l t i p l e t s which were not suitable
for p r e c i s e determination of coupling constants,
except i n very few cFges.
The broad band C NPlR spectrum of t h e compo-
und 5 dissolved i n DFE0-d (Fig. 4 B ) shows t h e do-
ubling o f t h e s i g n a l s a r o h d t h e c h i r a l c e n t r e (Ta-
b l e Yo), which demonstrates t h e presence of d i a s t e -
reomers. I n comparison with t h e a t e n o l o l carbon
Table 4. 1 3 C Chemical s h i f t s a f o r a t e n o l o l and com-

pound 5 d i s s o l v e d i n DMSO-d,
C-atom 'me Atenolol Compound 2
b
c-3 t 70 32b
70 71 70.32
CH3 9 .
22 83
18. 66c
17 77
CH3 22.83 17 77c
16.76
CH 48.12 51.41
50.78
c-1 ' 157.15 157.25
C-2'' 6'
C-3' '5'
c-4'
114.08
123.87
128.23
.
114.08
129 91
128.16
C-1' ' 172.57 172.90
c-2' ' ~c.1.27 41.27
a +& i n ppm downfield from i n t e r n a l T X 3 ; accrJracy
-0.02 ppm; off-resonance r n u l t i p l e t s a r e given
b 9 c broadened s i g n a l i n d i c a t e s t o chemical s h i f t s
separated by l e s s t h a n 0.02 ppm
chemical s h i f t s , t h e methyl s i g n a l s a r e s i g n i f i c a n -
t l y moved u p f i e l d , and those belonging t o isopropyl
CH and 2-1 a r e moved downfield. A l l o t h e r s i g n a l s
a r e c l o s e t o those of a t e n o l o l .
4.1.5. Nass
The mass s p e c t r a of a t e n o l o l and compound 2
were obtained by d i r e c t i n s e r t i o n of t h e sample ia-
t o CEC 21-110 I3 mass spectrometer. C h a r a c t e r i s t i c s
of t h e s e mass s p e c t r a a r e summarized i n Tables 5.
and06, and Fig. 5, The i o n source temperature was
150 C and 200-250 C , r e s p e c t i v e l y , and t h e i o n i z i n g
e l e c t r o n beam ener,gy was 70 eV.
Atenolol gave a molecular i o n , with very l o w
i n t e n s i t y , a t m/e 266. Other s i g n i f i c a n t peaks are
due t o t h e following fra ments: isopropyl group (PI-
-43), carboxamide group $M-44) and carboxamidome-
thylene group (M-56). The base peak i n t h e a t e n o l o l
ATENOLOL 15
Table 5. Peak assignments in the mass spectrum of

atenolo1
,*
.J m/e fragment
1.2 266
24.5 222
3.9 223
12.9 116
43.1 107 01'
14.3 107- C HO
m
3.7 151
lo 0 72 - C H 2 N HC H(C H3)2
23.5 73 - HC H H C H2N H-
OH
spectrum is due to isopropylaminomethylene group

(m/e 7 2 ) . The base peak in the spectrum of compound
2 corresponds to radical-ion of protonated p-methy-
lenequinone (m/e 107).
4.2. Solid Properties
4.2.1. Melting Range
und
Atenolol melts begween 150:
5 melts between 154 and 156 .and 1 5 2 O C ; compo-
4.2.2. Crystal Properties

X-Ray diffraction data of atenolol are pre-
sented in Table 7.(5) The diffraction spectrum w a s
produced by onochromatic radiation from the CuK
line (1.542 !!
) which was obtained by excitation at
35 kV and 20 mA, Recording condhtions were as fol-
l g w s . Optics: detector slit 0.2 ; I"I.R6 soller slit,
3 ; beam slit, 0.0007"; N& filter, 3 take o f f an-
gle. Goniometer: scan at 2 , 2o/min. Detector: am-
plifier gain 16 coarse, 3.1 fine. Scintillation co-
0)
\
E
0
In
N
0
0
h(
0
5
0
5
0
In
17
18 VESNA CAPLAR ET AL
Table 6. Peak assignements in the mass spectrum of

compound 2
/??
m/e fragment
Table 7. X-Ray diffraction data of atenolol crystal

20 Ia db(interplanar distance)
9.62 43 .
9 1936
12.84
16.04
17.74
6
20
54
.
6.8943
5 5254
4.9996
18.22 61 4.8689
19.22 74 4.6178
20.60
22.24
23 80.
loo
91
81
.
4.3115
3 9971
3 7385
3 .3759
24.36 72 3.6538
26.40 92
29. lo 11 3.0686
31.84 42 2.8105
34.58 14 2 5938
35 94 17 2.49~7
40.46 15 2.2296
a based on the highest intensity which is selected

as unity
d = nk/2 sin@
ATENOLOL 19
unter tube and DC voltage at plateau. Fulse light

selection 9V, En out. Rate meter: T.C. 1.0, 5000
CPS. (5)
q . 3 . Solution Properties
4.3.1. Solubility
All solubilities were determined at room tem-
perature and are presented in Table-8. The solub4.
lities are reported in accordance with U.S.P.(XX
ed.) definitions.(5)
Table 8. Solubilities of atenolol
Solvent Solubility
methano 1 freelg soluble
acetic acid soluble
dirnethylsulfoxyde so 1ub1e
96$ ethanol sparingly soluble
water slightly soluble
isopropanol slightly soluble
acetone very slightly soluble
dioxane very slightly soluble
aceto n i t r i l e insoluble
ethylacetate insoluble
chloroform insoluble
The negative logarithm of the proton dissoci-

ation constant for atenolol was determined as pRa=
=8.6. (7)
4.3.3. Partition Coefficient
Atenolol has a l o w partition coefficient f o r
n-octanol-phosphate buffer (0.16 M). Its values we-
re 0.008 at pH 7.0 and 0.052 at pH 8.0.(7)
4.3.4. Dipole Moment
The dipole moment of atenolo& was determined
in propionic acid solutions, at 20 C, using a Dipol-
meter DM 01 (Wiss. -Techn. Werks-$Btten, D 812 Weil-
.
heim) The value found was 5.71-0.20 D. (5)
4.3.5. Optical Rotation

The atenolol molecule contains an asymmetric
carbon atom. The commercial product, however, is a
racemic mixture and its resolution has not been re-
ported so f a r .
. Methods
.lo
of Analysis
Elemental Ana1.y l’s
s
Atenolol C14K22Nz03 (266.33) calc.: C 63.13%
H 8.33%
N 10.52%
0 18.02$
loo 00%
-
0
3.2. Titrimetric Determination Electrochemical

Atenolol may be assayed in glacial acetic
acid/acetic anhydride 15:2 by titration with 0.1 PI
perchloric acid. The endpoint may be determined po-
tentiometrically using a glass/calomel electrode
pair. (5)
2.3. Chromatographic Methods
2.3.1. Thin Layer
Thin-layer chromatography was performed using
silica gel 60 F plates (Nerck); the solvent sys-
tem was dioxane228etonitrile-methanol-conc ammonia
(25f;) (60:36:5:4). One hundred mg of the sample was
.
dissolved in lo ml of methanol, the solution spot-
ted on the plate and subjected to ascending chroma-
tography. After a front migration f o r at least 16
cm the plate was air-dried and sprayed with Dragen-
dorff’s reagent. Atenolol and compound 2 appear as
orange-brown spots, on a yellow background, at Rf
0 . 3 and 0.6, respectively.(5)
High performance thin-layer chromatography
(HPTLC) on silica %el plates was used for the sepa-
ration A-adrenoceptor blocking drugs. The detecti-
on limit for atenolol was 25.0 ng, and the absorp-
tion wavelength selected f o r its determination was
205 nm.(8)
3.3.2. Gas-Liquid
For gas-liquid chromatographic determinations
a 1 m x 4 m (I.D.) g l a s s column packed with 3$ OV-
lo1 on 80-loo mesh Gas Shrom Q was used. (5) The in-
ATENOLOL 21
jector tempBrature was set at 30Ooc, oven tem era-

ture at 190 C, Bad detector (flame ionization7 tem-
perature at 300 C. The sample was pretreatred with
trifluoroacetic acid anhydride as f o l l o w s : to 20 mg
of atenolol in lo-ml volumetric flask was added 5
ml of dioxane and 0.5 r n l of trifluoroacgtic acid
anhydride; the mixture was heated to 40 C and kept
5 min. at this temperature. The flask was then co-
o l e d to room temperature and the contents made up
to lo r n l with dioxane. One ml of this solution was
mixed with 0.2 r n l of internal standard solution (2
mg/ml of methyl ester of margarinic acid in dioxa-
ne) and 1 ,AL~ of the mixture forced into the injec-
tion block. The relative weight response of the de-
tector was calculated according to equation (1):
A* x cs
RWR= A x CA (1)
s
in which symbols have the following meanings:
AA = atenolol peak area of standard
AS = internal standard peak area
C, = concentration of atenolol (mg/ml)
Cs = concentration of methyl margarinate ( m g / m l )
In actual analyses the content of atenolol is

calculated from the areas of atenolol and internal
standard according to eq. (2):
AA x CS x 1000
mg of atenolol = AS Rm
in which the meanings of symbols are following:
AA = atenolol peak area of sample
AS = internal standard peak a r e a
Cs concentration of methyl margarinate (mg/ml)
=
RWR= relative weight response
W = weight of the sample (ng)
5 . 3 . 3 . Hiah Pressure Liquid
The apparatus used for high pressure liquid

chromatography was by Fye-Unicam (Cambridge), LPU-
-4011 equipped with a PU-4020 UV detector. The co-
22 VESNA CAPLAR ET A L
lumn was prepacked with Partisil l o p m ODS (25 cm;

4.6 mm ID, 6 mm OD). The mobile phase was acetoni-
trile-dist. water-orthophosphoric acid (35:loo:o.l)
having a pH of 3.5. In some analyses benzimidazole
was used as an inOernal standard. The flow rate was
1.0 ml/min. and the detector monochromator was ad-
justed at 222 nm; the amplifier gain was set at
0.08 A.U.F.S. The retention time of atenolol was
5.48.(5) In another case, 8 LiChrosorb 3 (RD-2,
ethylsilaniaed) column and a mobile phase consis-
ting of 351.;' MeOH, and 65,: of an aqueous solution
0.0005 M in HCl and 0.05 M in NaC1 were used. The
analyte was detected at its peak absorption 220 nm,
using a variable-wavelength detector.(9)
5.4. Determination of Impurities
s
The "dimeric" com3ound , which may be pre-
sent i n atenolol can be detec ed by all chromato-
graphic methods and determined by Sas-liquid and
I3PL chromatography.
6. Stability - Degradation
Atenolol, as the powder and in the form of
tableljs was kept at 50-60;L relative hunidity and
45-50 C for seven days. No changes were observed i n
HPL chromatogram, neither did the color and appear-
ance of the material show change.
7. Drug Metabolism, Pharmacokinetics, Bioavailabi;
litg
Owing to l o w hydrophilicity only a small
of atenolol is metabolised (about lo$ of a dose
The d r u g is also poorly bound to plasma protein
7Y
(less 5% of the amount in blood). (lo) Plostly of the
drug is eliminated, in its unchanged form, by seve-
ral routes, but prevailing via the kidney.(ll) Af-
ter oral administration atenolol is excreted with
mine to the extent of about 4073; (12-14), after
intravenous administration to the total urinary ex-
cretion encompasses 75-1007 of the dose (about l o -
-14% appeared in t h e form of catabolites).(l2,15,
16) Urinary pH variations do not change the extent
ob excretion with the wine.(17) M d e n c e s was put
forward to show that atenolol is not eliminated ex-
clusively by glomerular filtration.(ll) Atenolol is
also excreted with the faeces. After intravenous
ATENOLOL -.
77
administration, about lo$ of the dose excreted un-

changed; after oral administration, 40-50;: of the
dose appears in faeces, of which 6-8;; accounts for
catabolites.(E)
Atenolol is characterized by a low hepatic
clearance; for the most part o f body clearance is
extrahepatic.(l8) The hepatic biotransformation of
atemlo1 gives rise t o the pharmaceutically inacti-
ve catabolites, such as compound hydroxylated at
the methylene group attached to the benzene ring
and compouEds conjugated with glucuronic acid. ( 7 j
Only a s m a l l fraction of a dose of atenolol
reaches the brain and there is ratio of 2:lo bet-
ween the brain and plasma concentration. Atenolol
does not accumulate in tissues, such as lungs, he-
art and others.(7)
The pharrnacokinetics of atenolol was studied
in young and o l d subjects: no age-related differen-
ces were found with respect to volume of distribu-
tion, and bioavailability.(ll) The absolute bio-
availability (E), defined as the fraction of an or-
al dose which reaches the systemic circulation in
unchanged form, was 0.55-0.56.(11) Other authors
report bioavailabilities of 50-63,;for oral dosage
forrns.(15,16,19-22) However, a given oral prepara-
tion possesses unifhrm bioavailability, and exhi-
bits but little variation in neak plasma concentra-
tion.(l8) Postprandial intake reduces the bioava-
ilability of atenolol.(l9) Several investigators
reported less than loo;: bioavailabilities of injec-
table preparations: with intravenously administered
dosage forms, bioavailabilities between 85-98;s were
obtained.(15,16,21,22)
8. Identification and Determination in Body Fluids
and Tissues
The concentrations of atenolol in various bi-
ological material was determined by gas chromato-
graphy.(23-25) Another method, EELC, w a s used f o r
determination in whole blood,(26) plasma, (27,281
and urine.(28)
9. Determination in Pharmaceuticals
In tablets atenolol is determined titrimetri-
cally as follows: A sufficient number of tablets
are powdered in a mortar, and a powder portions
corresponding to one-half of average tablet mass
24 VESNA CAPLAR ET AL
a r e t r m s f e r e d t o t h e t i t r a t i o n v e s s e l containing
14 ml of g l a c i a l a c e t i c acid. Two m l o f a c e t i c a c i d
anhydride and 2 m l o f a s a t u r a t e d s o l u t i o n of mer-
cury a c e t a t e i n a c e t i c a c i d a r e added per v e s s e l ,
and t h e r e s u l t i n g suspension i s t i t r a t e d with 0.1
M HC104 using a g l a s s electrode as endpoint indica-
t o r (VS. a satd. calomel reference electrode). The
a t e n o l o l content i n one t a b l e t i s calculated by
using equation ( 3 ) . One ml of 0.1 PI HC104 corres-
ponds t o 26.63 m g o f atenolol.
V x f x 26.63 x WA
mg of a t e n o l o l i n one t a b l e t =
W
where V = volume of 0.1 M IIC10,: consumed, (3)
f = normality f a c t o r ,
WA= .average mass of a t a b l e t ,
W = mass of t h e powder sample.
Acknowledgement. 'The authors a r e thankful t o dr. Z.
,lei&, "Ruder Ro%ovib " I n s t i t u t e , Zagreg Yugosla-
;a, f o r h i s h e l p i n i n t e r p r e t a t i o n o f 3C PR4R spe-
ctra.
l o . References
1. A.M. B a r r e t t , R. Hull, 3.J. LeCount, C.J. Squire,
J. C a r t e r ( I C I L t d . ) , Ger. Offen. 2,007.751
(1370); Z.A. 22, 1 2 0 3 1 8 ~(1970).
2. J.F. GiudicelB, J.3. 3 o i s s i e r , Y. Dunas, C. Ad-
venier, Compt. Rend. SOC. B i o l . ,
3. L. Hansson, H. Aberg, S. Jameson,
%,232 (1973)-
Karlberg
R. Malmcrona, Acta Pled. Scand. 549 (1973).
4. A. Amery, L. B i l l i e t , R. k g l . J. Ned,
2 o 284 (1974).
5. &8aplar, 2. Mikoti6-P?5.hunC H. Eofman, J. Kuft-
inec, PI. Bkreblin, F. Kajfez, A. ITa I, IT. Blaze-
v i 6 , Acta Pharm. Jugoslav., 2 ( 2 ) , 1983) i n
press,
7
6. EfidBreitmaier, 'iJ. Voelter, l 3 C "IR Spectroscopy,
2 %I.Verlag
., Chemie, !.Jeinheim, 1978.
7. E. F'Jarmo, Drugs Bcptl. Clin. Ses. 6 , 639 (1980).
8. %. Zhou, C.F. Poole, J. Triska, A.-Zlatkis, J.
Chromatogr., Chromatogr. Commun.
Kirschbaum, X.E. Poet, J. Pharm,
Sci., 2, 336 (1981).
lo.iJ. Kirch, K.G. G B r g , W r o
macokinetics, 5,
81 (19827.
.
J. Drug Metab. Phar-
11.P.S. Rubin, P. .1W. S c o t t , K. McLean, A. Pearson,
ATENOLOL 25
D. Ross, J.L. Reid, Br. J. c l i n . Pharmac. 3,

. 235 (1982)-
.. -
12 J. McAinsh, Postgrad. Med. J.
74 (1977).
z,(suppl. 3),
13. J. 6Ieffih, S.R. Harapat, D.C. Harrison, Res.

Commun. Chem. Pathol. Pharmacol. I + 31 (1976).
-
14. Ha. Ochs, D.J. Greeblatt, T.11. Smi h, "Farma-
c o l o g i a c l i n i c a e t e r a p i a " , 'a.Medico-Scienti-
f i c h e s.r.l., Torino, 1978.
15 H.C. Brown, 3.G. Carruthers, G.D. Johnston, J.
K e l l y , J. McAinsh, D.G. NcDevitt, R.G. Shan
G.
Shanks, Clin. Pharmacol. Ther. 20 524 (1976).
16. J.D. F i t z g e r a l d , R. R u f f i n , I<. GTimedsted, R.
Roberts, J. McAinsh, Europ. J. Clin. Pharmacol.
,
81 (1978).
?i(1974)
17 .Ma Kaye, B r i t . J. Clin. Pharmacol. 1,513
.
18 A.J. NcLean, 3. I*Iilhelm, B.G. Seinzow, %ugs
(suppl. 21, 131 (1983).
2
9
19 .J. Conway, J.D. F i t z g e r a l d , J. McAinsh, D.J.
R o w l a n d , W.T. Simpson, B r i t . J. Clin. Pharmacol.
2, 267 (1976).
20. S.H. \Jan, R.Z. Koda, R.F. Maronde, 3rit. J.
Clin. Pharmacol. 2 , 569 (1979).
. 3,
21 W.D. Mason, 21. 'diener, G. Kochak, I. Cohen R.
Vell, Clin. Pharmacol. Ther. 4-08 (1'3793.
.
22 rjJ. Kirch, H. Kdhler, 3. Mutsch e r , M. Schgfer,
Ebr. J. Clin. Pharmacol. 3, 65 (1981).
23 . Scales, P.B. Copsey, J. Iharm. Pharmacol.
B
a,4-30
24. J.O.
(1975).
Malluca, K.R. Monson, J. Pharmacol. Sci.
-
25 . S.H. Yan, R.P. Tlaronde, S.B. Matin, J. Pharma-
64, 1992 (1975).
col. Sci. 42, 1340 (1978),
26. Y.-G. Yee, P. Rubin T.F. Blaschlce, J. Chroma-
tog. 1 1, 357 (1979).
27 4
74.A. Le ebvre, J. G i r a u l t , J.B. F o u r t i l l a n , J.
Liquid Shromatog. 4, 483 (1981).
28. 0.Y. 'deddle, T . N . xmick, 7.D. Yason, J. Pharma-
c o l . Sci. 9,1033 (1978).
This
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CAMPHOR
Jaber S. Mossa and Mahinoud M. A. Hassail
King Satid Uiiiwrsity
Rirlcicllz, S n d i Amliia
I, Description 28
1, I Nomenclature 28
1.2 Formulae 28
1.3 Molecular Weight 31
1.4 Elemental Composition 31
1.5 Appearance, Colour. Odour. and Taste 31
31
2. Physical Properties
2.1 Melting Range 31
2.2 Solubility 31
2.3 Non-Volatile Matter 31
2.4 Optical Rotation 32
2.5 Crystal Struclure 32
2.6 Spectral Properties 34
3. Preparation of Camphor 48
4. Synthesis of Camphor 51
4.1 Total Synthesis 51
4.2 Partial Synthesis 55
4.3 Commercial Synthesis 59
5, Biosynthesis of Camphor 61
6. Metabolism of Camphor 64
7. Pharmacology 64
7.1 Action and Use 64
7.2 Toxicity 64
7.3 Dosage 66
7.4 Preparations 66
8, Methods of Analysis 66
8.1 Identification 66
8.2 Gravimetric Methods 6X
8.3 Titrimetric Methods 72
8.4 Refractometric Method 13
8.5 Nephlometric Method 14
8.7 Spectroscopic Methods 80
References 82
ANALYTICAL PROFILES OF DRUG SUBSTANCE5 Copyright G 1984

VOLUME 13 27 hy thc Arncrican Pharnmxutical Association
ISBN 0-12-?60813-5
28 JABER S . MOSSA A N D MAHMOUD M . A . HASSAN
1. DESCRIPTION
1.1. Nomenclature
1.11 Chemical Names
2-0X0-1,7 ,T-trirnethyl b i c y c l o ( 2 , 2 , 1 ) h e p t a n .
1,7,7-Trimethylbicgclo( 2 , 2 , 1 ) heptan-2-one.
2-Keto-l,7 ,T-trimethyl norcamphane.
Bicyclo ( 2 , 2 ,l) hept an -2- one ,1,7 ,T-trirnethyl.
(4,7,7-Trimethyl b i c y c l o 1 , 4 ) heptan -5-one.
2-Bornanone.
d-2-Camphanone. (1,2)
1 . 1 2 Generic Names
Camphor.
1.13 Trade Names
a ) Formosa camphor
b ) L a u r e l camphor
c ) Alcanfor
d) Camphre du Japon ( N a t u r a l )
e ) Camphre D r o i t ( N a t u r a l )
f ) Gum camphor.
1 . 2 Formulae
1 . 2 1 BnpiricaL
1.22 S t r u c t u r a l
Camphor i s a s a t u r a t e d ketone, C 1 0 H 1 6 0 , which on

r e d u c t i o n y i e l d s t h e corresponding hydrocarbon
camphane, C H Camphor must t h e r e f o r e be
10 18'
CAMPHOR 29
bicyclic. The c a r b o n y l group i s f l a n k e d by only

one r e a c t i v e CH2 group, s i n c e camphor forms a
monobenzylidene d e r i v a t i v e o n l y , i n r e a c t i o n w i t h
benzaldehyde. The presence of a -CO.CH2-group i s
confirmed by t h e formation of an i s o n i t r o s o -
d e r i v a t i v e with n i t r o u s a c i d . On d i s t i l l a t i o n w i t h
phosphorus pentoxide camphor y i e l d s p-cymene. The
formation of which s u g g e s t s t h a t t h e b a s i c s k e l e -
t o n of camphor i s a s i x membered carbon-ring, sub-
s t i t u t e d i n t h e 1- and 4- p o s i t i o n s , and s i n c e
camphor i s b i c y c l i c it i s probable t h a t t h i s 1:4
s u b s t i t u t i o n t a k e s t h e form of a b r i d g e ( 5 , 6 ) .
A tremendous amount o f work was done b e f o r e t h e

s t r u c t u r e of camphor w a s s u c c e s s f u l l y e l u c i d a t e d .
Bredt (1893) w a s t h e f i r s t t o a s s i g n t h e c o r r e c t
formula to camphor. The s t r u c t u r e w a s confirmed
by t o t a l s y n t h e s i s , which w a s achieved by s e v e r a l
a u t h o r s . The most important a s p e c t s of t h i s work
a r e s m a r i s e d ( 5 ) below:
Camphor Camphor j.c Camphoric

acid anhy d r i d e
Homoc amphoric Homocamphoric

Campholi d e
nitrile acid
Camphor
30 JABER S. MOSSA AND MAHMOUD M . A . HASSAN
1 . 2 3 CAS R e g i s t r y Number
a ) Natural camphor [76-22-2]

b ) S y n t h e t i c camphor [ 76-22-21 (2)
1 . 2 4 Wiswesser Line Notation
a ) N a t u r a l camphor
L55A CVTJA
AB DX
L5 5A CVTJA
b ) S y n t h e t i c camphor
AB DXLV (7)
1.25 S t e r e o c h e m i s t r y
The s t e r e o c h e m i s t r y of camphor i s w e l l summarised

by F i n a r and Pinder ( 5 , 8 , 9 ) .
Camphor has two d i s s i m i l a r c h i r a l c e n t r e s ( C - 1

and C-4) and t h e r e f o r e f o u r enantiomorphs might
be expected. But only one p a i r of enantiomers i s
known. This i s due t o t h e f a c t t h a t o n l y t h e cis-
form i s p o s s i b l e ; trans f u s i o n of t h e gem-dimethyl-
methylene b r i d g e t o cyclohexane r i n g i s impossible.
Thus o n l y t h e enantiomers of cis-isomer a r e known.
Camphor and i t s d e r i v a t i v e s e x i s t i n t h e boat con-

formation. S i n c e t h e gem-dimethyl b r i d g e must be
cis, t h e cyclohexane r i n g must have t h e b o a t form.
4 0
The m a s s spectrum of camphor shows t h e common

peaks of i s o p r e n e : m/e 27, 29, 41, 53, 67, 68.
There are a l s o t h e m o l e p l a r i o n (M+ 1 5 2 ) and t h e
base peak m / e 95 ( C T H l l ) . T h e base peak i s pro-
bably formed as f o l l o w s :
[CloH160]k+ C H f. + CH3+ C2H20.

7 11
CAMPHOR
1.3 Molecular Weight
a ) N a t u r a l camphor 32.24
b ) S y n t h e t i c camphor 1.52.24 (10)
1 . 4 Elemental Composition
c , 78.89%; 11, 10.60%;

0, 10.51%. (2)
1 . 5 Appearance , Colour , Odoiir and Taste.
C o l o u r l e s s , t r a n s p a r e n t c r y s t a l s , c r y s t a l l i n e masses,
b l o c k s of tough c o n s i s t a n c e o r p u l v e r u l a n t masses known
as 'Flowers o f Camphor'; t r a n s l u c e n t m a s s w i t h c r y s t a l -
l i n e f r a c t u r e , Rhombohezlral c r y s t a l s from a l c o h o l , c u b i c
c r y s t a l s by m e l t i n g and c h i l l i n g ; odour, f r a g r e n t ,
p e n e t r a t i n g and c h a r a c t e r i s t i c ; t a s t e , pungent, a r o m a t i c ,
s l i g h t l y b i t t e r followed by a s e n s a t i o n o f c o l d ( 2 , h ) .
2. PHYSICAL PROPERTIES
2 . 1 Melting Range
a ) N a t u r a l Camphor 1 7 9 . 8 O C Sub. 204OC
b ) S y n t h e t i c Camphor 178'C. (10,ll)
2.2 S o l u b i l i t y
S o l u b l e i n 700 o f water ( g i v i n g c o l l o i d a l s o l u t i o n ) ,
1 i n 0.25 of c h l o r o f o n r , 1 i n 1 o f a l c o h o l ( 9 5 % ) . 1 i n 1
of e t h e r , 1 i n 0.4 of benzene, 1 i n 0.4 of a c e t o n e ,
1 i n 1 . 5 o f t u r p e n t i n e o i l and 1 i n 4 of o l i v e o i l ,
v e r y s o l u b l e i n carbon d i s u l p h i d e , l i g h t petroleum
e t h e r , f i x e d and v o l a t i l e o i l s . Also s o l u b l e i n con-
c e n t r a t e d m i n e r a l a c i d s , i n phenol, i n l i q u i d ammonia,
and i n l i q u i d s u l p h e r dtioxide. It i s i n s o l u b l e i n
g l y c e r i n . It l i q u i f i e s when t r i t u r a . t e d w i t h c h l o r a l
hydrate,menthol, r e s o r c i n o l , s a l o l , & n a p h t h o l ; thymol,
phenol and u r e t h a n e ( 2 . , U - l 5 ) .
2 . 3 Non V o l a t i l e Matter
When d i s t i l l e d a t l O 5 O , l e a v e s not more t h a n 0.1% of

residue ( 4 ) .
2.4 O p t i c a l R o t a t i o n
Camphor o c c u r s i n n a t u r e i s o p t i c a l l y a c t i v e ( d a n d l
f o r m s ) , whereas t h e s y n t h e t i c camphor i s i n racemic
from. The following o p t i c a l r o t a t i o n s were r e p o r t e d .
f o r (+)-Camphor.
[a]," Solvent Ref.
[a]:? + 4 1 t o 4 3 O 95% Alcohol (C=1.0 i n a l c o h o l ) (4)
+ 42.200 90% Alcohol ( C = 5 i n 1 0 0 m l ) . (16)

+ 42.12O Benzene (17)
[a]:' + 43.8' Absolute a l c o h o l (C=7.5 in 100 m l ) (2)
The s p e c i f i c r o t a t i o n of camphor a s 5% s o l u t i o n i n
chloroform has been determined by u s i n g a P e r k i n Elmer
Polarmatic Model 2 4 1 MC and found t o be :
2 - 5 Crystal Structure
A l l e n and Rogers (18) have redetermined t h e c r y s t a l

s t r u c t u r e of ( + ) -3-bromocamphor t o elucidate i t s
a b s o l u t e s t e r e o c h e m i s t r y and which i s known t o have
t h e same s t e r e o c h e m i s t r y o f (+)-camphor. The determi-
n a t i o n w a s achieved by t h e u s e of new t h r e e dimensional
x-ray d a t a .
The c r y s t a l s o f ( + )-3-bromo camphor C l o H 1 5 O Br , are

monoclinic, space grooup PZ1, w i t h a = 7.36, b = 7.59,
c = 9.12 l ; = 94.1; Z = 2. This work has unambig-
uously d e f i n e d t h e a b s o l u t e s t e r e o c h e m i s t r y of (+)-3-
bromo camphor and hence t h e (+)-camphor. The l a t t e r
may be r e p r e s e n t e d as [l] w i t h t h e gem-dimethyl
b r i d g e below t h e plane. The s u b s t i t u t i o n of bromine
a t (C-3) i n (+)-camphor g i v e s r i s e t o a t h i r d asym-
& t r i c c e n t r e . The [lOO]-projection of t h e s t r u c t u r e i n
F i g . 1 shows t h a t bromine i s trans t o t h e gem-dimethyl
b r i d g e ( e n d o - c o n f i g u r a t i o n ) as it i s i n t h e (+)-3-
bromo ca1nphor-9-~-sulphonate anion [ 2 ] ( 1 9 ) and i n
bromoisofenchone ( 2 0 ) . This c o n t r a s t s w i t h t h e cis-
CAMPHOR 33
-4c
&
Fig. 1. A view of t h e s t r u c t u r e down t h e

a-axis .
p o s i t i o n (eX0-configuration) adopted by t h e bulky h a l o -

gen atom i n (-)-2 bromo-2-nitrobornane [ 31 ( 2 1 ) and
(+)-l0-bromo-2-chloro-2-nitrosobornane [4] ( 2 2 ) .
10
I
I
Bond l e n g t h s and v a l e n c e a n g l e s a r e a l s o given i n

Tables 1 and 2 .
Table 1
Bond l e n g t h s / A , e s t i m a t e d s t a n d a r d d e v i a t i o n s i n
parentheses r e f e r t o t h e l e a s t s ig n if ic a n t f i g u r e ( s ) :
2.6 S p e c t r a l Properties
2.61 U l t r a v i o l e t Spectrum
The UV spectrum of camphor i n chloroform ( F i g . 2 )

w a s scanned from 200 t o 400 nm u s i n g DMS 90 V a r i a n
spectrophotometer. It e x h i b i t e d a h a x a t 290
nm. Also it w a s scanned i n methanol from 200 t o
400 nm u s i n g Varian Carry 219, and e x h i b i t e d a
hax a t 289 nm. Other r e p o r t e d W s p e c t r a l d a t a
36 JABER S . MOSSA AND MAHMOUD M . A . HASSAN
Table 2: Valency angles/O: estimated s t a n d a r d d e v i a t i o n s i n

parentheses r e f e r t o t h e l e a s t s i g n i f i c a n t f i g u r e ( s ) .
102.8(1.4)
100.8(1.4)
112.3( 1.2)
102.6(1.4)
115.7( 2.1)
120.5(1.8)
105.6(1.0)
127.7(1.2)
126.6(1.2)
102.o ( 1.1)
112.6(1.1)
llg.l(l.3)
lll.h(l.6)
103.2(1.2)
98.3(1.4 )
102.3(1.6)
104,1(1.6)
94 9(1 . 3 )
.
I
112.3(1.4)
112.2(1,5)
112.6( 1 5 )
113.8(1.3)
110.3(1.5)
CAMPHOR 37
f o r camphor i n methanol (7) : h a x , a t 289 nm;

camphor i n e t h a n o l (1) : h a x , a t 289 nm (El%,
1 cm 2 ) ; a s 0.25 p e r c e n t w/v s o l u t i o n i n a l c o h o l
(95 p e r c e n t ) e x h i t ) i t s a hmax a t 289 nm ( E 1 . 0 6 )
[4) and i n chlorof'orm h a x , a t 292 nm 1 2 ) .
2.62 I n f r a r e d Spectrum
The I R spectrum of camphor as KBr d i s c and Nujol

m u l l w e r e r e c o r d e d 0 n . a P e r k i n Elmber 580 B
I n f r a r e d spectrophotomet e r t o which I n f r a r e d d a t a
s t a t i o n i s a t t a c h e d ( F i g . 3 ) . The s t r u c t u r a l
a s s i g n m e n t s have been c o r r e l a t e d w i t h t h e f o l l o w -
ing frequencies ("able 3 ) .
Table 3 : I R C h a r a c t e r i s t i c s of Camphor.
-I
Frequency cm Assignment
3450 b OH o r H20
1744 s C = 0 Stretch.
O t h e r c h a r a c t e r i s t i c bands are:
2959, 2873, 1 4 7 1 , lj51, 1418, 1391, 1 3 7 2 , 1 3 2 4 ,

1 2 7 8 , 1094, 1 0 4 6 , 1021, 751, and 521. The I n f r a -
r e d d a t a f o r ( + ) , (.-) and (*)-Camphor are also
r e p o r t e d ( 7 , 2 3 ) . T.ne r e p o r t e d d a t a are i n a g r e e -
ment w i t h o u r d a t a 'except f o r t h e hydoxyl band a t
3450 cm-l.
2 . 6 3 Nuclear Magnetic Resonance S p e c t r a
2 . 6 3 1 P r o t o n Spec=
The PMR spectrum o f camphor i n C D C l 3 w a s r e -

corded on a V a r i a n T - ~ O A , 60 MHz NMR s p e c t r o -
m e t e r u s i n g TMS ( T e t r a m e t h y l s i l a n e ) as an i n -
t e r n a l r e f e r e n c e ( F i g . 4 ) . The f o l l o w i n g
s t r u c t u r a l a s s i g n m e n t s have been made
(Table 4 ) .
6"'
'CH3* 7
I 1 1 I 1 I . 1 1
1 . * 1 . . . . 1 . . . . 1 . . . . 1 . . . . 1 . . . . 1 . . . . 1 ' . . . . 110 , 0
8B 70 60 5 0 fPM(b) 44 44 20
Fig. 4. PMR spectrum of camphor in CDCl
3'
40 J A B E R S . M O S S A A N D M A H M O U D M . A . HASSAN
Table 4. PMR C h a r a c t e r i s t i c s o f Camphor
Group Chemical s h i f t (ppm)

Methylene a t ( C-2, C-4 and C-5 ) . 2.5 m
2.07 m
and Methine a t C-3 p r o t o n s . 1.43 b t
C-9 M e 0.93 s
C-8 M e 0.88 s
C-10 M e 0.83 s
s = singlet ; t = t r i p l e t ; b t = b r o a d t r i p l e t ; rn = m u l t i p l e t .
Our assignment o f t h e methyl p r o t o n s s i n g l e t s i s
b a s e d on t h e a n i s o t r o p y of t h e c a r b o n y l f u n c t i o n
o f camphor. The 9-methyl p r o t o n s are f a l l i n g w i t h -
i n t h e d e s h i e l d i n g zone o f t h e c a r b o n y l group and
i s e x p e c t e d t o be t h e most lower f i e l d w h i l e C-10
m e t h y l p r o t o n s are away from t h e c a r b o n y l f u n c t i o n
and i s e x p e c t e d t o s u f f e r no d e s h i e l d i n g e f f e c t .
Our Model i s shown i n F i g . 5 . I n c o n t r a s t C-8
m e t h y l p r o t o n s w i l l have an i n t e r m e d i a t e chemical
s h i f t value (24).
Other r e p o r t e d d a t a (25-29) are i n agreement w i t h
o u r d a t a e x c e p t f o r t h e a s s i g n m e n t s o f C-8 and
C-10 m e t h y l p r o t o n s r e s o n a n c e s and which a r e l i s -
t e d below:
c-8 c-9 c-10
Our v a l u e s . . . .............. 0.88 0.93 0.83

R e f . 7 and 8 . . . . . . . . . . . . . . . 0.85 0.92 0.98
Ref. 9 (deuterium
substitution) ...... 0.85 0.98 0.92
R e f . 10 (using metal
Reagent s h i f t s ) .... 0.83 0.95 0.86
2.632 l3C-NMR Spectra
The l3C-NMR noise-decoupled and off-reso-

nance s p e c t r a ( F i g . 6 and 7 ) , w e r e recorded
o v e r 5000 Hz w i d t h i n C D C l on V a r i a n FT.80,
80 MHz NMR s p e c t r o m e t e r , d i n g a 1 0 mm sample
t u b e and TMS as a r e f e r e n c e s t a n d a r d a t 20’.
The carbon chemical s h i f t s a r e a s s i g n e d on
t h e b a s i s of t h e off-resonance s p l i t t i n g
p a t t e r n and a d d i t i v i t y p r i n c i p l e s and i s
CAMPHOR
F i g . 5. Camphor model.
'i'
H
L
Fig. 7 . 13C-NMR off-resonance spectrum of camphor in CDC13.
44 JABER S. MOSSA AND M A H M O U D M . A . HASSAN
shown i n t h e s t r u c t u r e below. Other repor-

t e d data are a l s o a v a ila b le (7,23,30).
27.10 (d)
s = singlet; t = t r i p l e t ; q = quartet
2.64 Mass Spectrum
The mass spectrum of camphor o b t a i n e d by

e l e c t r o n impact i o n i z a t i o n ( F i g . 8 ) which
w a s recorded on a Varian Mat 1 1 2 s mass spec-
trometer. The spectrum w a s scanned
from 40" up t o 250 8.m.u. E l e c t r o n energy
w a s 70eV. The mass s p e c t r a l d a t a a r e shown
i n Table 5. (31-33).
Table 5 : The Most Promiment Fragnents of Camphor

Table 6: P r i n c i p a l Mass S p e c t r a Peaks of Camphor
and Deuterated Analogs.
Isotopic r m/e (% shift) \

Compound
purity,% M - 15 M - 42 M - 43 M - 57 M - 57 M - 69 M - 71
137 110 109 108 95 83 81
P
0
.
cb 82(93)
The symbol q r e f e r s t o a q u a n t i t a t i v e t r a n s f e r ( i . e . , > 95%).

+0 -t
t
111I
c,
h
Ip
a
k
\
47
48 JABER S. MOSSA AND MAHMOUD M . A. HASSAN
Reed ( 3 4 , 3 5 ) and Von Sydow ( 3 6 ) have examined

r e c e n t l y t h e m a s s spectrum of Camphor and
offered a highly speculative explanation of
t h e fragmentation o f camphor on e l e c t r o n im-
p a c t . Weinberg ( 3 7 ) have s t u d i e d t h e m a s s
f r a g m e n t a t i o n of camphor and i t s d e u t e r a t e d
d e r i v a t i v e s and proposed fragmentation r e a c -
t i o n s ( T a b l e 6 and Scheme 1) based on labe-
l i n g and h i g h - r e s o l u t i o n r e s u l t s .
3. PREPARATION OF CAMPHOR
Camphor i s t h e main c o n s t i t u e n t of camphor o i l which i s

o b t a i n e d from t h e wood and t h e l e a f o f camphor t r e e ,
Cinnamomwn camphora ( L i n n e ) Nees e t Ebermaier, family
Lauraceae. The p l a n t grows w e l l i n Japan, China, Formosa,
I n d i a , Burma and Malaysia. Camphor a l s o o c c u r s i n c e r t a i n
s p e c i e s of A r t e r n i s i a , (Compositae) chrysanthemum ( c o m p o s i t a e ) ,
S a l v i a ( L a b i a t a e ) , Ocimum ( L a b i a t a e ) ; Lavander ( L a b i a t a e ) ,
Pinus ( P i n a c e a e ) . It i s a l s o p r e s e n t i n Rosemarinus
o f f i c i n a Z i s ( L a b i a t a e ) , AristoZochia indica ( A r i s t o l o c h i -
a c e a e ) , BLwnea balsamifera (Campbreaceae) , PruneZZa vulgaris
L a b i a t a e ) , Cinnamomwn gandu2ifen.m ( L a u r a c e a e ) e t c (38-55).
Commercially camphor i s produced from cinnamomwn Camphor%.

The p r o d u c t i o n of camphor w a s s t a r t e d much e a r l i e r , probably
a t t h e end of 13th c e n t u r y . I n t h e middle of t h e 15tL
c e n t u r y , numerous camphor i n d u s t r i e s were e s t a b l i s h e d i n
China, Japan and Formosa. Any how t h e major production of
camphor w a s o b t a i n e d from Japan and Formosa. The p r o d u c t i o n
of camphor from t h e n a t u r a l source i s w e l l summarised belbw:
The t r e e s over 40 y e a r s old a r e f e l l e d , t h e i r r o o t s a r e

dug out. The r o o t s and t h e t r u n k s o f t h e camphor t r e e s a r e
brought t o t h e f a c t o r y where t h e y a r e reduced t o small
c h i p s . The camphor o i l i s t h e n d i s t i l l e d w i t h steam i n
s p e c i a l , r a t h e r wooden s t i l l ( F i g . 9). The camphor o i l
which accumulates i n t h e condensing v e s s e l s i s u s u a l l y
emptied once a month. About 113 of t h e pure camphor, pre-
s e n t i n t h e o i l i s c r y s t a l i z e d out and it i s s e p a r a t e d from
t h e l i q u i d by s t r a i n i n g . The camphor remaining i n t h e o i l
i s recovered e i t h e r by f r a c t i o n a l d i s t i l l a t i o n o r by f r e e z -
i n g out or by t h e formation o f complexes w i t h t h e s t r o n g
acids.
The camphor t h u s o b t a i n e d i s f u r t h e r p u r i f i e d by mixing

w i t h soda lime, sand, c h a r c o a l and s u b j e c t e d t o s u b l i m a t i o n
.. ... .. ... I.. ... .. ..1.... ..-.
...I.. ... .. U , , .
;. . - . .
camphor wooddisti//ationpost.
A . Retod("Koshiki"). E . 3rd condenser.
8.Kettk . E Liebiy condenser.
C. 1st condenser. 6.A p e Foor,rerumofdistr'ilatlonwater.
D . nndcondenser. ( r o h ~ b r h o nw r r h r p i p c ) .
F i g u r e 9.
so JABER S. MOSSA AND MAHMOUD M. A . HASSAN
50Y, 46%
Crudekmphor campior oil

(Recovered Camphor) or Bppmduct oil
o r “0” Ca (T( p hor (Dccanphorlzed (Oil)
(Sublimcltion)
Refined Camphor(2nd class)

or “00“ Camphor
I (Sublimution and Pressing)
Refined Camphor(f St class)

O r “A“ Camphor
Figure 1 0 .
CAMPHOR 51
where camphor w i l l sublime i n p u r e s t a t e (56,57).

The p r o c e d u r e o u t l i n e i s p r e s e n t e a i n F i g u r e 1 0 .
4 . SYNTHESIS OF CAMPHOR
Many methods of p r e p a r i n g camFhor are d e s c r i b e d i n t e c h n i -
c a l j o u r n a l s and i n t h e p a t e n t l i t e r a t u r e s ( 5,8,58-62).
4 . 1 Total Synthesis
Route I : The b i c y c l i c k e t o n e s t r u c t u r e o f camphor

proposed by B r e d t i n 1893 w a s confirmed by t o t a l
synthesis (58,59)as follows:
Komppa ( 1 9 0 3 ) f i r s t s y n t h e s i s e d (+)-Camphoric a c i d
f o l l o w e d by s y n t h e s i s o f ( + ) camphor i n 1908.
Komppa (1889) f i r s t s y n t h e s i s e d 3 , 3 - d i m e t h y l g l u t a r i c
e s t e r s t a r t i n g w i t h m e s i t y l o x i d e and e t h y l m a l o n a t e .
The p r o d u c t o b t a i n e d was 6,6-dimethylcyclohexane-
2 , 4 - d i o n e - l - c a r b o x y l i c e s t e r [l]. ( T h i s i s produced
f i r s t by a Michael c o n d e n s a t i o n , f o l l o w e d by a
Dieckrnann r e a c t i o n ) . On h y d r o l y s i s f o l l o w e d by
o x i d a t i o n w i t h sodium hypobromite, 3 , 3 d i m e t h y l g l u -
t a r i c a c i d [ 2 ] w a s o b t a i n e d . E s t e r i f i c a t i o n of which
w i t h e t h a n o l and H C l a f f o r d e d d i e t h y l Oa-dimethylglu-
t a r a t e [ 3 ] . D i e t h y l o x a l a t e and d i e t h y l @@-dimethyl
g l u t a r a t e condense t o y i e l d t h e c y c l o p e n t a n e d i o n e [ 41 ,
which b e i n g a B-Ketoester, can be C-methylated t o
d i k e t o c a m p h o r i c e s t e r [ 5 ] . Sodium amalgam r e d u c t i o n
c o n v e r t s t h e d i k e t o n e [ 51 t o t h e g l y c o l [6]. T r e a t -
ment w i t h h y d r o i o d i c a c i d e f f e c t s a r e d u c t i o n and
d e h y d r a t i o n t o c y c l o p e n t e n e d i e s t e r [ T I . The bromo
d e r i v a t i v e [8] o f which y i e l d s d i e t h y l c a m p h o r a t e [ 9 ]
on d e b r o m i n a t i o n . A f i n a l h y d r o l y s i s a f f o r d s ( + ) -
camphoric a c i d [ l o ] a s a m i x t u r e of c i s - and f2an.S-
forms. The Cis-forrn i s s e p a r a t e d by c o n v e r s i o n t o
i t s a n h y d r i d e [ll], dl-camphoric a n h y d r i d e i s redu-
ced by sodium and a b s o l u t e e t h a n o l or hydrogen and
n i c k e l t o dl-campholide [12], which on t r e a t m e n t
w i t h H B r and AcOH g i v e s dl-bromcampholic a c i d
[13]. The camphohalide and KCN form dl-cyanocam-
p h o l i c a c i d [14]. 1141 when b o i l e d w i t h KOH it i s
t r a n s f o r m e d i n t o dl-homocamphoric a c i d [15]. D i s -
t i l l a t i o n o f ( 1 5 ) w i t h Sa(OH)p, g i v e s dl-camphor
[161.
52 JABER S. MOSSA A N D MAHMOUD M . A . HASSAN
Route I
CO,Et
C02Et
CHBr3 +
Y-
[21
co2a.y. ’
!O E t
2
+ E t ONa CO2Et
I02Et C02Et
0
CAMPHOR 53
[ 51 61
KCN
H+ b
Br
C a salt
heat
CAMPHOR 55
Route 11: By P e r k i n and Thorpe (60,61).

as f o l l o w s :
CE-COOC H
2 5
C O + CH COOC H
-H20
---+ CH-
IIC -CH NaCH( C02Et ) 2
3 2 5 3 3
t Mic haeJ
m
CZ3 reaction.
d i e t h y l 66-dimethylgutarate.
Route 111: More recent:Ly a n e a t a l t e r n a t i v e s y n t h e s i s

of camphoric a c i d has been d e s c r i b e d by Alder ( 6 2 ) .
1:1:2-Trimethylcyclopentadiene [l] and d i e t h y l -
a c e t y l e n e d i c a r b o x y l a t e [ 21 undergo a d i e n e a d d i t i o n
reaction t o give the d i e s t e r [ 3 ] . P a r t i a l reduction
y i e l d s [4], which i s o x i d i s e d t o t h e b i s - g l y o x a l a t e
[ 5 ] by n i t r i c a c i d . The l a t t e r on d e c a r b o n y l a t i o n
y i e l d s d i e t h y l campho:?ate [ 61 which on h y d r o l y s i s
a f f o r d s camphoric a c i d [TI. Camphoric a c i d i s con-
v e r t e d t o camphor as d e s c r i b e d i n Route I .
4.2 P a r t i a l Synthesis
Many r o u t e s f o r t h e p a r t i a l s y n t h e s i s of camphor a r e
d e s c r i b e d ( 5 , 8 , 63,64).
H a l l e r , 1896 s t a r t e d w i t h camphoric a c i d prepared by

t h e o x i d a t i o n of camphor as i l l u s t r a t e d i n Scheme 11.
This i s not unambiguous s y n t h e s i s , s i n c e t h e compholide

o b t a i n e d might have had t h e s t r u c t u r e of t h e B-campho-
l i d e [l] which i n t u r n w I L l 1 g i v e homo-camphoric a c i d
[ 2 ] and t h i s would have given camphor w i t h s t r u c t u r e s
[ 3 ] which w a s r e j e c t e d (Scheme I I a ) .
Sauers (1959) has now o x i d i s e d camphor d i r e c t l y t o t h e
a-campholide by means o f p e r a c e t i c a c i d . It i s a l s o o f
i n t e r e s t t o n o t e t h a t Otvos e t a1 (1960) have shown, u s i n g
l a b e l l e d - CH2C3C02H( 1 4 ~ 1 ,t h a t i n t h e p y r o l y s i s o f t h e
calcium s a l t o f homocamplioric a c i d t o camphor, it i s t h e
l a b e l l e d carboxyl group t h a t i s l o s t .
Route 111
+ ,y,.COOEt ,
C .C O O E t
\ COOEt
[21
&o.cooEt *
CO. C O O E t
HB03 bC COOEt
COOEt COOH
KOH
> COOH
OOEt
CAMPHOR
kCO
~-
Scheme I1
$-yo lo]+
HN03 COOH
Camphoric acid
Camphor
a-Campholide
0 t-
Na--Hg
G
Camphoric \ O
anhydride
eCN
( i )KCN ( i i )H+
&
N i t r i l e of horno-
camphoric a c i d
-
; ~ Hornocamphoric COOH
acid
Scheme I I a .
-- ., 0
0 121 [31
Ill
Scheme I11
MeC(=CH2)0AC
lsopropenyl acetate
____Ic
[31
[ll
[41
CAMPHOR 59
Money e t a1 (1969) (65)IlaYe now c a r r i e d o u t a two s t e p

s y n t h e s i s of (*)-camphor from dihydrocarvone.
(Scheme 111).
Dihydrocarvone [l] w a s t r e a t e d w i t h i s o p r o p e n y l a c e t a t e
i n t h e presence o f p - t o l u e n e s u l p h o n i c a c i d and conver-
t e d i n t o a m i x t u r e of e n o l a c e t a t e s [ 2 ] and [ 3 ] , sepa-
r a t e d by GLC Treatment o f [ 2 ] w i t h boron t r i f l u o r i d e
i n methylene c h l o r i d e a t room t e m p e r a t u r e f o r 1 0 minu-
t e s gave ( + ) Camphor [4]. This s y n t h e s i s i s p a r t i c u -
l a r l y i n t e r e s t i n g i n t h a t it is a chemical analogy f o r
t h e b i o s y n t h e s i s conversion o f a monocyclic i n t o a b i -
c y c l i c monoterpenoid.
4.3 Commercial S y n t h e s i s
Camphor i s o f considerab1Le importance t e c h n i c a l l y , be-

i n g used i n t h e manufacture of c e l l u l o i d and m e d i c i n a l
p r o d u c t s . It i s manufactured i n d u s t r i a l l y from a-pinene,
o b t a i n e d from t u r p e n t i n e , , by s e v e r a l p r o c e s s e s (66-107)
which d i f f e r mainly i n d e t a i l . S y n t h e t i c camphor i s
u s u a l l y o b t a i n e d as t h e racemic m o d i f i c a t i o n . The f o r -
m a t i o n of camphor i n v o l v e s t h e Wagner-Meerwein r e a r r a n g e -
ments, e . g . :
( i )a-Pinenc HC1 gas* B o r n y l c h l o r i d e AcoNar Camphene

10% ( -HC1)
-
Ac OH
H2S04
Isobornylacetate Nas
-Isoborneol
PhNo2
pp Camphor
( i i )a-Pinene HC1 gas- Bornylchloride Camphene

10°C (-HC1)
HCooH, Isobornylformate ’* O2
IsoborneolNi ,2000c* Camphor
( i i i )a-Pinene Isomerization>g a p h e n e AcOH

______) Isobornyl-
(rearrange-
acetate NaoH > Isoborneol

ment. )
Catalytic
dehydrogenation
Camphor. -
One such process i s d e s c r i b e d i n d e t a i l i n Scheme I V .
Scheme I V
Saturated at Meerwein Base-catalysed

rearrangement elimination
131
Il51
+
1 Sod. A c e t a t e - g l a c i a l a c e t i c acid.
CAMPHOR 61
The most s t r a i g h t f o r w a r d method i n v o l v e s t h e conver-

s i o n of p i n e n e [ I ] i n t o 13inene h y d r o c h l o r i d e [ 2 ] ,
b o r n y l c h l o r i d e [ 3 ] and hence i n t o b o r n e o l [6] (or i s o -
b o r n e o l ) from which cam3hor [7] i s o b t a i n e d by o x i d a -
t i o n . Also b o r n y l c h l o r i d e [ 3 ] i s c o n v e r t e d t o i s o b r o -
n y l a c e t a t e [ 5 ] upon h e a t i n g w i t h sodium a c e t a t e and
g l a c i a l a c e t i c a c i d . A l L e r n a t i v e l y , camphene [ 4 ] i s
o x i d a t i v e l y h y d r a t e d t o camphor [ T I .
By s t a r t i n g w i t h o p t i c a l l y a c t i v e a - p i n e n e , o p t i c a l l y
a c t i v e camphor can be s y n t h e s i s e d . Commercial camphor
is b e s t p u r i f i e d by s u - b l i m a t i o n .
CH
WOH
1 3 ICH3
I I
I I
I
I i
H
H
Borneo1 [OI Isoborneol
5. BIOSYNTHESIS OF CAMPHOR
The b i o s y n t h e s i s of m o n o t e r p m o i d s and camphor h a s been

d e s c r i b e d by s e v e r a l a u t h o r s (108-114). Ruzicka (115,116)
proposed a u n i f i e d b i o g e n e t i c scheme for t e r p e n e s . The
biosynthetic building blocks f o r t h e s e terpenes a r e iso-
p r e n e u n i t s . The b i o s y n t h e 1 ; i c a l l y a c t i v e i s o p r e n e u n i t s
a r e i s o p e n t e n y l pyrophosphate [ 1 1 and d i m e t h y l a l l y l
pyrophosphate [ 2 ] t h e compounds t h a t are d e r i v e d from
a c e t a t e v i a mevalonic a c i d (Scheme V ) , Geranyl pyrophosphate
[ 3 ] i s t h e C-10 p r e c u r s o r for t h e t e r p e n e s (117). Banthorpe
and Baxendale (118) confirmed t h e b i o s y n t h e t i c pathway o f
Camphor v i a a c e t a t e mevalonate by c o n d u c t i n g d e g r a d a t i o n
s t u d y o f camphor, biosynthesfized from 14c l a b e l l e d mevalo-
n i c a c i d . The b i o s y n t h e s i s of camphor i s summarised i n
Scheme V I .
The i s o p e n t e n y l pyrophosphate [ 1 3 d e r i v e d from mevalonic

a c i d i s o m e r i z e d i n t o dimethy:L a l l y l pyrophosphate [ 2 ] and
(R-mevdlonk aid1
CHzOPP
/ \ /
CH3 c
I
-OPP=OP,O,H3 Scheme V
CAMPHOR 63
Scheine
- - VI
POH
64 JABER S. MOSSA A N D M A H M O U D M . A . HASSAN
t h e subsequent condensation y i e l d g e r a n y l pyrophosphate

[ 3 ] . The g e r a n y l pyrophosphate a g a i n isomerized i n t o
n e r y l pyrophosphate [4]. The n e r y l pyrophosphate w i l l be
converted i n t o a common enzyme bound c y c l i c i n t e r m e d i a t e ,
a - t e r p i n e y l c a t i o n [ 5 ] which would undergo proton l o s s
from C-5 and 1-2 h y b r i d e s h i f t w i t h t h e expulsion o f
enzyme t o y i e l d borneyl c a t i o n [6]. T h i s on f u r t h e r
c y c l i z a t i o n g i v e s borneol [7] and t h e n f i n a l l y camphor [8].
6. METABOLISM OF CAMPHOR
( - ) and ( + ) Camphor a r e m e t a b o l i s e d by h y d r o x y l a t i o n of a
methylene group acd subsequent g l u c u r o n i d e c o n j u g a t i o n .
Hydroxylation produced t h e 3 s n d o d e r i v a t i v e [ 2 ] and t h e
5-endo-derivative [ 31, t h e l a t t e r predominating. These
r e s u l t s a r e c o n s i s t e n t w i t h t h e more s t r a i n e d n a t u r e of
t h e 5-versus t h e 3 - p o s i t i o n o f camphor. The two hydroxyl
d e r i v a t i v e s c a n j u g a t e w i t h g l u c u r o n i c a c i d t o g i v e t h e 3-
and 5-glucuronides r e s p e c t i v e l y and e x c r e t e d as such. Also
it i s hydroxylated i n t h e 2 - p o s i t i o n t o g i v e ( + ) b o r n e o l
[11 ( 1 1 9 , 1 2 0 ) -
Metabolism o f Camphor i s p r e s e n t e d i n Scheme V I I .
7 . PHARMACOLOGY
7.1 Action and Use
Taken i n t e r n a l l y camphor i s i r r i t a n t and c a r m i n a t i v e .
It has been used as a mild e x p e c t o r a n t and r e l i e v e s
g r i p p i n g . Applied e x t e r n a l l y , camphor a c t s as a
r u b e f a c i e n t and m i l d a n a l g e s i c and i s employed i n l i n i -
ments as a c o u n t e r - i r r i t a n t i n f i b r o s i t i s , n e u r a l g i a
and s i m i l a r c o n d i t i o n s . Camphor h a s been a d m i n i s t e r e d
a s a s o l u t i o n i n o i l by subcutaneous o r i n t r a m u s c u l a r
i n j e c t i o n as a c i r c u l a t o r y and r e s p i r a t o r y s t i m u l a n t ,
but t h e r e i s l i t t l e evidence o f i t s v a l u e f o r t h i s
purpose (121-125).
7.2 Toxicity
Poisoning has occured through t h e a c c i d e n t a l adminis-

t r a t i o n of camphor l i n i m e n t to young c h i l d r e n by m i s -
t a k e f o r c a s t o r o i l . The symptoms are nausea, vomit-
i n g , c o l i c d i s t u r b e d v i s i o n , d e l i r i u m , mental confusion
and e p i l e p t i c c o n v u l s i o n s . Recovery i s t h e r u l e . But
i n rare c a s e s d e a t h may occur f r o m r e s p i r a t o r y f a i l u r e
( 126-128) .
CAMPHOR
65
H OOC
Glucuronic Acid Glucuronic Acid
OH H
S c h e m e .EZ
7.3 Dosage
120 t o 300 mg ( 2 t o 5 g r a i n s ) ; 60 t o 200 mg (1-3 g r a i n s )

by i n j e c t i o n s ( 1 2 ) .
7.4 Preparations
The f o l l o w i n g are t h e p r e p a r a t i o n s of camphor o f f i c i a l

i n Pharmacopoeas:
a) Camphor Liniment ( 4 )
b) Camphorated opium T i n c t u r e (126)
c) W r p e n t i n e Liniment ( 4 )
d) Camphor Ointment ( 1 2 6 )
e) Camphor S p i r i t ( 1 2 6 )
f ) Camphor Water ( 1 2 6 )
g ) Camphor I n j e c t i o n ( 1 2 6 )
h ) Camphorated Parachlorophenol ( 1 5 2 ) .
8. METHODS OF ANALYSIS
8.1 I d e n t i f i c a t i o n
8.11 Pharmacopeial T e s t
The following t e s t s have been d e s c r i b e d i n B r i t i s h
Pharmacopoeia for t h e i d e n t i f i c a t i o n o f clamphor ( 4 ) :
a ) Burns r e a d i l y w i t h a b r i g h t smoky flame, and

v o l a t i l i s e s a t ordinary temperature.
b ) The l i g h t a b s o r p t i o n i n t h e range 230 t o 350 nm,

of a 2-cm l a y e r of a 0.25 p e r c e n t w/v s o l u t i o n
i n a l c o h o l ( 9 5 % ) e x h i b i t s a maximum o n l y a t
289 nm; e x t i n c t i o n a t 289 nm, about 1 . 0 6 .
8.12 Colour Tests
The following c o l o u r t e s t s have been d e s c r i b e d for

t h e i d e n t i f i c a t i o n of camphor:
a ) Bohrisch camphor r e a c t i o n : When 0.5g of natu-

ral camphor i s t r e a t e d w i t h 1 c c . of v a n j - l l i n
h y d r o c h l o r i d e s o l u t i o n (1 p a r t o f v a n i l l i n
d i s s o l v e d i n 1 0 0 p a r t s of 25% h y d r o c h l o r i c
a c i d ) and h e a t e d on a water-bath t o 75-100°,
a b l u e c o l o u r p a s s i n g t o green i s o b t a i n e d .
S y n t h e t i c camphor g i v e s no colour (129-131).
CAMPHOR 67
Bohrisch ( 1 3 2 ) i n reexamining t h e o f f i c i a l t e s t s
f o r t r u e camphor, s u b s t i t u t e s t h e following :
To 0 . 1 g. o f powliered camphor i n t e s t t u b e i s
added 2 c c . of 1% vanillin-hydrochloride solu-
t i o n and t h e m i x t u r e p u t i n t o a beaker w i t h
water which i s warmed g r a d u a l l y . A t 30° t h e
c o l o u r i s yellow, a t 60° b l u i s h g r e e n , a t 75-
80' indigo-blue. The l a t t e r c o l o u r p e r s i s t s
f o r s e v e r a l h o u r s , even a f t e r c o o l i n g . Synthe-
t i c camphor by t h e same t r e a t m e n t g i v e s only a
yellow colour.
b ) When 1 g o f n a t u r a l camphor i s t r e a t e d w i t h 1 0
drops of Rosentk.al's Reagent ( e q u a l volume of
v a n i l l i n - h y d r o c h l o r i d e and c o n c e n t r a t e d s u l -
p h u r i c a c i d ) a & u l l g r e e n c o l o u r changing in
7-8 hours t o indigo-blue i s o b t a i n e d ( 1 2 9 ) .
c ) With barium peroxide-sulphuric a c i d r e a g e n t ,
camphor g i v e s a yellow c o l o u r ( 1 3 3 ) .
d) When 2 m l of a 0.5% o f a l c o h o l i c s o l u t i o n s o f
camphor i s trealied w i t h 5 ml o f 5% phosphomo-
l y b d i c a c i d s o l u t i o n and 5 m l of s u l p h r i c a c i d ,
a greenish-yellow p r e c i p i t a t e i s o b t a i n e d
which r a p i d l y t u r n s g r e e n and d i s a p p e a r s on
shaking ( 1 3 4 ) .
e ) With f u r f u r a l and s u l p h u r i c a c i d camphor g i v e s

r e d d i s h brown l i q u i d which slowly t u r n s purp-
lish-violet f o r s y n t h e t i c and p u r p l e f o r
natural (134).
f ) Fluorescence t e s t : When camphor i s i r r a d i a t e d

by x-rays it e x h i b i t s a b l u i s h v i o l e t f l u o r e -
scence i n t h e v i s i b l e r e g i o n ( 1 3 5 ) .
8.13 D e t e c t i o n of S y n t h e t i c Camphor i n Natural Camphor:
a ) About 1 g of camphor i s thoroughly mixed w i t h

2 g of l i m e , t h e mixture heated u n t i l t h e
camphor i s completely v o l a t i l i z e d , t h e r e s i d u e
i s e x t r a c t e d wi.th water, and t h e s o l u t i o n f i l -
t e r e d . The f i l t r a t e , a c i d i f i e d w i t h n i t r i c
achd BhOuld give no p r e c i p i t a t e w i t h AgNO i f
s y n t h e t i c camphor i s a b s e n t from t h e o r i g i n a l
3
s u b s t a n c e (136, 137).
b) 5 g o f t h e camphor t o be t e s t e d i s d i s s o l v e d
i n 50 c c 90% a l c o h o l , an aqueous s o l u t i o n o f
5 g hydroxylamine h y d r o c h l o r i d e and 8 g .
sodium hydroxide are added, and t h e n enough
a l c o h o l t o produce a c l e a r s o l u t i o n . The s o l u -
t i o n i s h e a t e d for 90 min. on a water-bath,
water i s t h e n added, and i f t u r b i d i t y a p p e a r s ,
t h e presence o f i s o b o r n e o l o r camphene i s proved.
I f t h e t u r b i d s o l u t i o n i s now n e u t r a l i z e d w i t h
h y d r o c h l o r i c a c i d , t h e p r e c i p i t a t e which forms
should be s o l u b l e i n excess of h y d r o c h l o r i c
a c i d and a l s o i n sodium hydroxide (136).
8.2 Gravimetric Methods
I n o r d e r t o determine t h e camphor c o n t e n t of e s s e n t i a l
o i l s (which c o n t a i n no o t h e r carbonyl compounds) t h e
g r a v i m e t r i c d e t e r m i n a t i o n proposed by Aschan (138) may
be employed:
Procedure: I n t r o d u c e about 1 g . o f t h e o i l , a c c u r a t e l y
weighed, i n t o a t e s t t u b e and d i s s o l v e t h e o i l i n
2 g. o f g l a c i a l a c e t i c a c i d . Add 1 g. of semicarba-
z i d e h y d r o c h l o r i d e and 1 . 5 g . o f f r e s h l y f u s e d an-
hydrous potassium a c e t a t e . T r i t u r a t e thoroughly
with a g l a s s r o d , s t o p p e r t h e t u b e w i t h a plug of
absorbent c o t t o n and h e a t t h r e e hours i n a water
b a t h a t 70'. Cool t h e m i x t u r e , add 1 0 t o 15 c c . of
water, s t i r t h o r o u g h l y and t r a n s f e r t h e p r e c i p i t a t e
q u a n t i t a t i v e l y t o a t a r e d 4 t o 5 em. f i l t e r . Wash
w i t h water u n t i l a l l water s o l u b l e m a t t e r i s removed,
a i r d r y , wash w i t h petroleum e t h e r and dry i n a i r t o
c o n s t a n t weight. Determine t h e weight of semicar-
bazone from t h e i n c r e a s e i n t h e weight of t h e f i l -
t e r . C a l c u l a t e t h e c o n t e n t of camphor i n t h e o r i -
g i n a l o i l by means of t h e f o l l o w i n g formula:
P e r c e n t a g e of camphor = V.7P
S
where: p = weight o f semicarbazone i n grams;

s = weight o f o i l i n grams.
Other methods f o r d e t e r m i n a t i o n of camphor v i a s e m i -

c a r b a z o n e , i n pharmaceutical p r e p a r a t i o n s were a l s o
r e p o r t e d (139, 1 4 0 ) .
Estimation of camphor i n camphor s p i r i t ( 1 4 1 ) :

The method isbased on t h e a b i l i t y of camphor t o enter
CAMPHOR
i n t o molecular combination w i t h s a l o l , t o y i e l d
salolcamphor, C ~ O H ~ ~ O . C ~ ~ H ~ O O ~ .
I n t o a 1 0 0 cc .weighed Erlenmeyer f l a s k introduce about
log. ( a c c u r a t e l y weighed) camphor s p i r i t and 70 c c of
water. Add e x a c t l y 8 g. d e s i c c a t e d chemically pure
s a l o l , shake t h e f l a s k , f i n a l l y allowing t h e camphor
and s a l o l t o c o l l e c t on t h e bottom of t h e c o n t a i n e r .
Pass t h e c l e a r superr.atant l i q u i d through a weighed
Gooch c r u c i b l e ( t h e bottom of which c a r r i e s 2 s m a l l
f i l t e r s covered by a p r o c e l a i n n e t ) . Wash t h e cru-
c i b l e with a few drops of c o n c e n t r a t e d s a l o l s p i r i t .
P l a c e t h e Erlenmeyer f l a s k i n a water b a t h a t 50-60'
whereby t h e e n t i r e c o n t e n t o f t h e f l a s k m e l t s t o a
uniformly o i l y l i q u i d c o n s i s t i n g o f salol-camphor
and s a l o l . Remove t h e c o n t a i n e r from t h e b a t h , s e t
a s i d e 1 h r . i n a cold room i n o r d e r t h a t t h e s a l o l
excess may c r y s t a l l i z e . Should t h i s not t a k e p l a c e ,
shake t h e f l a s k , whereupon r a p i d c r y s t a l l i s a t i o n
u s u a l l y r e s u l t s . T r a n s f e r t h e e n t i r e c o n t e n t of t h e
f l a s k t o t h e c r u c i b l e s e p a r a t i n g t h e o i l y salolcamphor
from t h e c r y s t a l s s a l o l by s u c t i o n . Wash t h e l a t t e r
by s u c t i o n w i t h 5 c c . c o n c e n t r a t e d s a l o l s p i r i t .
Remove t h e combined p o r t i o n s o f s a l o l t o a s h e e t of
smooth paper. Wash t h e i n s i d e o f t h e f l a s k w i t h a
few cc o f e t h e r , r e c e i v i n g and e v a p o r a t i n g t h e
l a t t e r on a watch g l a s s . P r e s s t h e s a l o l r e s i d u e
on t h e watch g l a s s 'c,etween 2 s h e e t s o f w h i t e paper
u n t i l no f u r t h e r g r e a s e s p o t forms. Add t h i s s a l o l
t o t h e main p o r t i o n , t h e n d r y a t f i r s t i n t h e a i r
u n t i l t h e odor of csmphor i s no l o n g e r p e r c e p t i b l e ,
t h e n i n a d e s i c c a t o r t o c o n s t a n t weight. The per-
centage of camphor j.s t h e n determined according t o
t h e following formula:
% camphor = ( a 7 1 . 0 6 ) / b
a = i s t h e amount of camphor combined w i t h
salol.
b = t h e amount of sample t a k e n for a n a l y s i s
and t h e v a l u e 71.06 = t h e molecular weight of

s a l o l i n t o t h a t of zamphor m u l t i p l i e d by 1 0 0 .
Good r e s u l t s a r e r e p o r t e d .
Another method u t i l i z i n g hydroxylamine h y d r o c h l o r i d e

( 1 4 2 , 143) i s as f o l l o w s :
70 JABER S. MOSSA AND MAHMOUD M. A. HASSAN
1 0 g . of s p i r i t of camphor, 1 g . o f hydroxylamine
h y d r o c h l o r i d e and 2 g. o f sodium hydroxide i n 20 c c
d i s t i l l e d water are h e a t e d i n a f l a s k provided w i t h
a r e f l u x condenser on a w a t e r b a t h f o r t h r e e hours.
After c o o l i n g d i l u t e s u l f u r i c a c i d i s added, phe-
n o l p h t h a l e i n being used as i n d i c a t o r . The l i q u i d
i s t h e n e x t r a c t e d w i t h e t h e r t h r e e t i m e s . The
e t h e r e a l s o l u t i o n o f camphor oxime i s d r i e d w i t h
anhydrous sodium s u l p h a t e and poured i n t o a weighed
f l a s k . After d i s t i l l i n g t h e g r e a t e r p a r t o f e t h e r ,
t h e remaining e t h e r i s removed w i t h a i r a t room
t e m p e r a t u r e , u n t i l a c r y s t a l mass i s formed which
i s d r i e d i n a d e s s i c a t o r and weighed. The r e s u l t s
o b t a i n e d by t h i s method a r e s a t i s f a c t o r y .
Hampshire and Page ( 1 4 4 ) evolved a u s e f u l p r a c t i -

c a l method f o r t h e d e t e r m i n a t i o n o f camphor i n
g a l e n i c a l s u s i n g 2,h-dinitrophenylhydrazine as
a p r e c i p i t a n t . The procedure used i s as f o l l o w s :
D i s s o l v e about 0 . 2 g of camphor, a c c u r a t e l y weighed

by d i f f e r e n c e , i n 25 m l aldehyde-free e t h a n o l i n a
300 m l c o n i c a l f l a s k and slowly add w i t h c o n s t a n t
shaking, 75 m l of t h e r e a g e n t ( p r e p a r e d by d i s s o l v -
i n g 1 . 5 g of 2,4-dinitrophenylhydrazine i n a m i x t u r e
of 1 0 m l o f c o n c n e t r a t e d s u l p h u r i c a c i d and 1 0 m l
o f water, d i l u t i n g w i t h w a t e r t o 100 m l and f i l t e r -
i n g ; t h e r e a g e n t decomposes on s t a n d i n g and must be
made up j u s t b e f o r e u s e ) . Then h e a t t h e m i x t u r e on
a water-bath under a r e f l u x condenser f o r f o u r h o u r s ,
a l l o w it t o c o o l , d i l u t e w i t h 2 p e r c e n t v/v sulphu-
r i c a c i d t o 200 m l (weaker a c i d allows c r y s t a l l i s a -
t i o n of t h e excess o f base from t h e s o l u t i o n ) and
a l l o w t o s t a n d f o r twenty-four hours. C o l l e c t t h e
p r e c i p i t a t e on a weighed gooch c r u c i b l e w i t h paper
m a t , o r on a s i n t e r e d - g l a s s f i l t e r , wash it w i t h
s u c c e s s i v e q u a n t i t i e s of 10 ml of c o l d d i s t i l l e d
w a t e r u n t i l t h e washings are no l o n g e r a c i d , and
d r y at 80'; 1 g o f camphor 2,h-dinitrophenylhydra-
zone corresponds t o 0.458 g o f camphor.
A comparison o f v a r i o u s methods for d e t e r m i n a t i o n

o f camphor and o t h e r compounds u t i l i z i n g 2,k-di-
n i t r o p h e n y l h y d r a z i n e were a l s o r e p o r t e d (145-149).
A vacuum oven method and a comparison w i t h t h e

U . S . P , X . method as w e l l as a m o d i f i c a t i o n , u t i l i z i n g
a n t i - o x i d a n t s such a s p y r o g a l l o l , a-naphthol,
CAMPHOR 71
hydroquinone and ParaFhenylenediamine f o r t h e d e t e r -

m i n a t i o n of camphor i n camphor l i n i m e n t were des-
c r i b e d by Poe ( 1 5 0 , 1 5 1 ) .
The U.S.P. X I X ( 1 5 2 ) d e s c r i b e d a method f o r t h e

d e t e r m i n a t i o n of camphor i n camphorated p a r a c h l o r e -
phenol.
Procedure: T r a n s f e r about 300 mg of Camphorated Para-

chlorophenol, a c c u r a t e l y weighed, t o a 200 m l
p r e s s u r e b o t t l e c o n t a i n i n g 50 m l o f f r e s h l y prepared
dinitrophenyl-hydrazire TS. Close t h e p r e s s u r e
b o t t l e , immerse it i n a w a t e r b a t h , and m a i n t a i n
it a t about 75' f o r 4 hours. Cool t o room tempera-
t u r e , t h e n t r a n s f e r t h e c o n t e n t s t o a beaker w i t h
t h e a i d o f 1 0 0 m l of d - i l u t e s u l f u r i c a c i d (1 i n 12),
and a l l o w it t o s t a n d o v e r n i g h t . C o l l e c t t h e pre-
c i p i t a t e on a t a r e d f i l t e r c r u c i b l e , wash w i t h 1 0 0 ml
o f d i l u t e s u l f u r i c a c i d (1 i n 1 2 ) and t h e n w i t h 75
ml of c o l d w a t e r , i n d i v i d e d p o r t i o n s , t o remove t h e
a c i d . Dry a t 80' f o r 2 h o u r s , c o o l and weigh. The
weight of t h e p r e c i p i t a t e so o b t a i n e d , m u l t i p l i e d
by 0.4581, r e p r e s e n t s t h e weight of C H 0 i n t h e
sample t a k e n .
l o 16
Another o f f i c i a l method d e s c r i b e d by t h e pharmaco-

p e i a of Japan I X (153:i f o r t h e a s s a y o f camphor i s
as f o l l o w s :
T r a n s f e r about 0.2 g o f d-camphor, a c c u r a t e l y weigh-

e d ¶ t o a 300-ml g l a s s s t o p p e r e d f l a s k , and d i s s o l v e
i n 25 m l o f aldehyde-j7ree e t h a n o l . Add slowly 75 m l
o f 2,4-dinitrophenylhydrazine TS w h i l e shaking, and
h e a t f o r 4 hours on a water b a t h under a r e f l u x con-
d e n s e r . Cool, add 1 0 0 m l of d i l u t e d s u l f u r i c a c i d
(l+ 5 0 ) , and a l l o w t o s t a n d f o r 24 hours. C o l l e c t
t h e p r e c i p i t a t e so obliained on a g l a s s f i l t e r ( G 3 ) ,
wash t h e r e s i d u e on t h e f i l t e r w i t h c o l d water un-
t i l t h e washings become n e u t r a l , d r y f o r 2 hours a t
105' and weigh as t h e 2,h-dinitrophenylhydrazone o f
d-camphor ( C 1 6 ~ 2 0 ~ 4 0: 4 332.36) .
Amount (mg) of d-camphor (C10H160) = amount (mg) of
2,4-dinitrophenylh:~drazoneof d-camphor
( C16H20N404 ) X 0.,4580.
The o f f i c i a l method O F NF X I V (154) f o r t h e a s s a y of

camphor i n camphor s p i r i t i s g i v e n below:
JABEK S. MOSSA A N D MAHMOUD M . A. HASSAN
Assay: T r a n s f e r 2 . 0 ml of camphor s p i r i t t o a s u i t -
a b l e p r e s s u r e b o t t l e c o n t a i n i n g 50 m l of f r e s h l y
prepared d i n i t r o p h e n y l h y d r a z i n e TS. Close t h e
p r e s s u r e b o t t l e , immerse it i n a water b a t h , and
m a i n t a i n a t about 75' f o r 1 6 hours. Cool t o room
t e m p e r a t u r e , and t r a n s f e r t h e c o n t e n t s t o a beaker
w i t h t h e a i d o f 1 0 0 ml o f d i l u t e s u l f u r i c a c i d (1
i n 1 2 ) . Allow t o s t a n d not l e s s t h a n 1 2 hours
a t room t e m p e r a t u r e , t r a n s f e r t h e p r e c i p i t a t e t o
a t a r e d f i l t e r c r u c i b l e , and wash w i t h 100 m l of
d i l u t e s u l f u r i c a c i d (1 i n 1 2 ) followed by 75 ml
of c o l d water i n d i v i d e d p o r t i o n s . Continue t h e
s u c t i o n u n t i l t h e excess water i s removed,dry t h e
c r u c i b l e and p r e c i p i t a t e a t 80' f o r 2 h o u r s , c o o l ,
and weigh. The weight o f t h e p r e c i p i t a t e so o b t a i n -
ed, m u l t i p l i e d by 0.4581, r e p r e s e n t s t h e weight o f
C H 0 i n t h e sample t a k e n .
10 16
8.3 T i t r i m e t r i c Methods
8.31 Aqueous
Mital and Gaind (155) claim good r e c o v e r i e s f o r
camphor i n pharmaceutical p r e p a r a t i o n s w i t h a
volumetric method i n which hydroxylamine hydro-
c h l o r i d e i s used. For s p i r i t , l i n i m e n t and solu-
t i o n of camphor i n t u r p e n t i n e , t h e following pro-
cedure i s a p p l i c a b l e :
Reflux t h e sample ( c o n t a i n i n g 0 . 2 g of camphor)

f o r f o u r hours w i t h 20 m l of aldehyde-free 95 p e r
c e n t e t h a n o l , 1 0 ml of a 4 p e r c e n t s o l u t i o n of
hydroxylamine h y d r o c h l o r i d e i n aldehyde-free 90 per
c e n t e t h a n o l and 0.3 g o f sodium b i c a r b o n a t e . Cool,
r i n s e t h e condenser w i t h 20 m l o f l i g h t petroleum
( b . p . 50' t o 60°), c o l l e c t i n g t h e r i n s i n g s i n t h e
f l a s k and t i t r a t e t h e m i x t u r e f i r s t w i t h 0.2N hydro-
c h l o r i c a c i d t o dimethyl yellow and t h e n with 0.2N
potassium hydroxide t o p h e n o l p h t h a l e i n . Carry
o u t a c o n t r o l o m i t t i n g t h e camphor and addicg 5 m l
of s t e a m - d i s t i l l e d t u r p e n t i n e o i l b e f o r e r e f l u x i n g
f o r t h e s p i r i t and s o l u t i o n and 1 g of a r a c h i s o i l
before refluxing f o r t h e liniment. Calculate t h e
amount o f camphor p r e s e n t from t h e d i f f e r e n c e i n
t h e volume of 0.2R potassium hydroxide used i n
t h e two t i t r a t i o n s . 1 m l of 0.2N KoH = 0.0304 g
camphor .
CAMPHOR 13
Other r e p o r t e d procedures w i t h minor m o d i f i c a t i o n s

and u s e of d i f f e r e n t i n d i c a t o r s have been a l s o
r e p o r t e d (156-165). A method w a s a l s o d e s c r i b e d
f o r d e t e r m i n a t i o n of camphor i n d i l u t e s o l u t i o n s
by photometric t i t r a t i o n u s i n g bromophenol b l u e
as t h e i n d i c a t o r (166).
8.32 Vanadometry
An i n d i r e c t VasladOmetriC method of a s s a y f o r camphor

w a s developed. The method i n v o l v e s t h e formation o f
2,4-dinitrophenylhydrazone of camp$or. The n i t r o
groups i n t h e hydrazone a r e t h e n reduced t o m i n e s
by t r e a t m e n t w i t h vanadium s u l f a t e (VSO4) and t h e
excess o f t h e r e a g e n t i s back t i t r a t e d w i t h sodium
dichromate (Na2C1-207). The e n t i r e procedure i s
c a r r i e d o u t i n a modified s e p a r a t o r y f u n n e l i n t o
which s t a n d a r d v a n a d a t e , 6N H2SO4, and z i n c amalgam
a r e i n t r o d u c e d . A carbondioxide c u r r e n t i s t h e n
passed and t h e m i x t u r e i s shaken t o o b t a i n a b l u e
c o l o u r a f t e r which t h e z i n c amalgam i s drawn o f f .
Then s u l f u r i c a c i d i s added t o g e t h e r w i t h s e v e r a l
drops o f phenosafranine and excess vandate i s
t i t r a t e d with standazd sodium dichromate s o l u t i o n .
8.33 Polarographic
Schwabe and Berg ( 1 6 7 ) have s t u d i e d t h e polaro-

graphy of t e r p e n e d e r i v a t i v e s . The s o l v e n t s were
80% e t h a n o l , 80% dioxane, 80% acetone o r water
and t h e s u p p o r t i n g e l e c t r o l y t e s were 0 . 1 M L i C 1 ,
L i O H , EtkNI, o r Eti+rlOH. Camphor was found t o
d e p r e s s t h e polarogi-aphic maxima and t h i s could
be used f o r i t s d e t e r m i n a t i o n .
8.4 R e f r a c t o m e t r i c Method
Rapaport and Solyanik (168) have developed a r a p i d

method of r e f r a c t o m e t r i c d e t e r m i n a t i o n of 23 m i x t u r e s
c o n t a i n i n g a n e s t h e s i n , barbamyl , bromcanphor, bro-
misoval, camphor , a n t i p y - r i n e, amidopyrine , a c e t a l s a l i -
c y l i c a c i d , b a r b i t a l , codeine, s a l o l , t e r p i n h y d r a t e ,
hexamethylenetetramine, znd p h e n o b a r b i t a l . The met hod
d e s c r i b e d i s s u i t a b l e f o r a n a l y s i s o f pharmaceutical
m i x t u r e s c o n t a i n i n g cornpxnds which a r e i n s o l u b l e i n
water and s o l u b l e i n e t h a n o l . A m i x t u r e of 2 pharma-
c e u t i c a l s ( 0 . 1 g ) i s d i s s o l v e d i n 1 m l . e t h a n o l and
nD of t h i s s o l u t i o n i s determined. One component i s
14 JABER S . MOSSA A N D MAHMOUD M. A . HASSAN
chemically determined i n a n o t h e r 0 . 1 g of t h e sample.

The amount o f such component i s c a l c u l a t e d from formulas
A = [(n-no) - (CxF)] P/100F1P1
C = VT 1 0 0 , B = VTP/P1 where A i s wt. o f t h e 1st

component i n grams, V i s t h e volume o f 0.1N s o l u t i o n
used for t i t r a t i o n o f t h e 2nd component, T i s t h e g .
e q u i v a l e n t o f t h e 1 s t compound, B is t h e w t . of t h e
2nd component ( g . ) , n i s t h e r e f r a c t i v e index o f t h e
s o l u t i o n , no i s t h a t o f e t h a n o l , C i s t h e amount of
t h e 2nd component i n 1 0 0 m l . s o l u t i o n ( c h e m i c a l l y
d e t e r m i n e d ) , F and F1 a r e c o n s t a n t s determined e x p e r i -
m e n t a l l y , P i s t o t a l wt. of t h e m i x t u r e and P1 i s wt.
of sample (0.1 g . ) .
Other r e p o r t e d procedures f o r t h e d e t e r m i n a t i o n o f
camphor, by u s i n g r e f r a c t i v e index and o t h e r p h y s i c a l
c o n s t a n t s have a l s o been r e p o r t e d (169,170).
8.5 Nephlometric Method
Nephlometric methods f o r d e t e r m i n a t i o n o f camphor

vapors i n a i r (171) and camphor i n s p i r i t s were des-
c r i b e d ( 1 7 2 ) . Optimum r e s u l t s w e r e o b t a i n e d w i t h a
m i x t u r e c o n s i s t i n g o f e t h e r , 9676, e t h a n o l and w a t e r
i n t h e p r o p o r t i o n 0 . 1 : 0.9 : 3. The t u r b i d i t y of t h e
m i x t u r e i s i n c r e a s e d by t h e presence of camphor, i n
p r o p o r t i o n t o t h e c o n c e n t r a t i o n o f camphor i n s o l u t i o n .
Approximately, 0.2 mg of camphor can be determined i n
2 ml o f t h e m i x t u r e ( 0 . 0 1 mg /L can be determined i f
100 L of a i r and 1 0 m l of t h e s o l u t i o n a r e u s e d ) .
8.6 Chromatographic Methods
8.61 Thin Layer Chromatography
Miller and Kirchner (173) have developed a n analy-

t i c a l method f o r v o l a t i l e c o n s t i t u e n t s i n c l u d i n g
camphor. They used s i l i c i c a c i d c o a t e d - g l a s s
c h r o m a t o s t r i p s . E t h y l a c e t a t e hexane, chloroform,
benzene, i s o p r o p y l e t h e r and t h e i r m i x t u r e s were
used as d e v e l o p e r s and t h e s t r i p s were examined
w i t h v a r i o u s r e a g e n t s l i k e bromine, f l u o r e s c e n c e ,
0 - a n i s i d i n e , Bulphuric a c i d - n i t r i c a c i d , and
c o n c e n t r a t e d s u l p h u r i c a c i d . The R f v a l u e s were
calculated.
CAMPHOR 75
Table 7: R f v a l u e of Camphor on S i l i c i c a c i d u s i n g
different solvents.
Colour of s p o t Df
w i t h H2SO)++For- Value.
I\L
S.
Solvent System.
No. maldehyde
Reagent.
1. n-Hexane. Light green. 0.00

2. Carbon t e t r a c h l o r i d e 0.31
3. C yc l o hexane 0.33
4. Benzene 0.10
5. Chloroform 0.61
6. Ethyl acetate Spot a p p e a r s
a t t h e sol-
vent f r o n t .
7. Benzene : Carbon t e t r a c h l o r i d e
(50 : 50) 0.62
8. E t h y l a c e t a t e : n-Hexane (1.O:gO) 0.38
9. E t h y l a c e t a t e : n-Hexane (15:85) 0.39
10. E t h y l a c e t a t e : Carbon
t e t r a c h l o r i d e (1j:8j) 0.42
11. E t h y l a c e t a t e : Cyclohexane (15:85) ,, 3 3 0.42
12. E t h y l a c e t a t e : Benzene (1j:85) 9 9 3 3 0.37
Conditions
Glass p l a t e s 10.5 x 25 cm. (coated with s i l i c i c a c i d

(Mesh No.100).
Temperature 2 5 O C .
R e l a t i v e humidity 25 5%.+_
Normal Chromatographic Chamber, s a t u r a t e d f o r

15 minutes b e f o r e u s e .
76 JABER S. MOSSA AND MAHMOUD M. A . HASSAN
Egon S t a h l (174) h a s invented a s p e c i a l chromato-

g r a p h i c d e v i c e for t h e s e p a r a t i o n of v o l a t i l e
o i l s u s i n g uniform t h i c k n e s s of adsorbent on p l a t e .
El-Deeb, e t a1 (175) have r e p o r t e d t h e s e p a r a t i o n

and e v a l u a t i o n o f camphor by TLC t e c h n i q u e on s i l i -
c i c a c i d p l a t e . T h e behaviour of camphor on chro-
matoplates w a s studied using d iffe re n t solvents of
i n c r e a s i n g p o l a r i t y and t h e i r m i x t u r e s as deve-
l o p e r s . The s p o t s were l o c a t e d by s p r a y i n g t h e
p l a t e s f i r s t w i t h s u l p h u r i c a c i d and t h e n w i t h 40%
s o l u t i o n of Formaldehyde and h e a t i n g t h e p l a t e t o
100°C f o r 3 t o 5 minutes. The R f v a l u e s were c a l -
c u l a t e d . It i s given i n Table 7.
8.62 Column Chromatography

The column t e c h n i q u e has been used for t h e separa-
t i o n and d e t e r m i n a t i o n of t h e p u r i t y of camphor.
Montes (176) analysed t h e e s s e n t i a l o i l c o n s t i -

t u e n t s u s i n g t h e p r i n c i p l e o f chromatographic
a d s o r p t i o n w i t h t h e a i d of columns of s i l i c i c
a c i d and b e n t o n i t e . The compounds were t h e n
c r y s t a l i z e d and t h e i r m e l t i n g p o i n t s were d e t e r -
determined.
Gustave Vavon and Bernard Gastambide (177) and

Bernard Gastambide (178),s t u d i e d t h e e f f e c t s o f
s t e r i c h i n d r a n c e of camphor and fenchone on chro-
matographic alumina column.
Eero Sjostrom (179) h a s d e s c r i b e d a method of t h e

s e p a r a t i o n of camphor and o t h e r ketones u s i n g
a n i o n exchange r e s i n s column.
A s u c c e s s f u l attempt of 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 camphor from m i x t u r e s w a s achieved by
Karawya (180) and by Karawya and El-Deeb (181)
u s i n g alumina column.
El-Deeb, e t a1 (175) have r e p o r t e d t h e comparative

s t u d y of d i f f e r e n t i a l a d s o r p t i o n o f camphor on
both aluminium oxide and s i l i c i c a c i d . The r e t e n -
t i o n volumes were determined by u s i n g Petroleum-
e t h e r and e t h e r as s o l v e n t s. 100 mg of m a t e r i a l
w a s chromatographed on column o f 1 em diameter
and 1 0 g of e i t h e r Alumina or s i l i c i c a c i d s ( m e s h
No. 3 0 ) . The r e s u l t s are p r e s e n t e d i n Table 8.
CAMPHOR 17
Table 8: R e t e n t i o n of Volume of Camphor on

d i f f e r e n t columns.
R e t e n t i o n volume
S. Adsorbent. -
No. Petroleum E t h e r Ether
1. S i l i c i c a c i d More t h a n 25 m l . 20 m l .
2. Alumina 100 m l . 15 m l .
8.63 G a s Liquid Chromatography
A GLC method f o r t h e d e t e r m i n a t i o n of camphor

( s y n t h e t i c and n a t u r a l ) has been c a r r i e d out i n
o u r l a b o r a t o r y ( 1 8 2 ) , u s i n g a Varian GC-3700 g a s
chromatograph equipped w i t h Varian CDS 111
integrator.
Column c o n d i t i o n s : 3 % OV1 on chromosorb W-Hp (80-

1 0 0 mesh s i z e ) ; g l a s s column (2% x h m ) . The
column r u n i s o t h e r m a l l y a t 100' + 2OO0C f o r 2 min-
u t e s ( t h e t e m p e r a t u r e i s i n c r e a s e d at t h e r a t e of
10' p e r m i n u t e ) , C a r r i e r g a s : Nitrogen, flow r a t e
w a s a d j u s t e d t o 40 ml/minute. D e t e c t o r : FID,
hydrogen and a i r , flow r a t e s were a d j u s t e d t o
30 ml/minute and 300 ml/minute r e s p e c t i v e l y .
E t h a n o l w a s used as s o l v e n t . The i n j e c t i o n t e m -
p e r a t u r e was 150OC and t h e c h a r t speed w a s a d j u s -
t e d t o g i v e 1 cm/minute. The r e t e n t i o n t i m e =
1 . 4 5 minutes. Camphx c o n t e n t of t h e sample i s
c a l c u l a t e d from t h e 2hromatogram by u s i n g peak a r e a
r a t i o method. The GLC of camphor i s p r e s e n t e d i n
F i g . 11.
Camphor has been determined by Baines and P r o c t o r

( 1 8 3 ) i n a number of e s s e n t i a l o i l s and pharmaceu-
t i c a l p r e p a r a t i o n s u s i n g g a s chromatographic t e c h -
nique. The a p p a r a t u s employed has a t h e r m a l con-
d u c t i v i t y d e t e c t o r w i t h 4 i n . platinum w i r e 0 . 0 0 1
i n . d i a m e t e r of nominal r e s i s t a n c e 250 Ohms. The
w i r e s i n t h e channel:; being matched t o w i t h i n 0 . 1
Ohm. The b r i d g e c u r r e n t w a s 200 mA and t h e o u t p u t
r e c o r d e d on a 2 . 5 mV r e c o r d e r . 2% ethylbenzene
w a s added t o t h e s t a n d a r d s and samples as an i n t e r -
n a l s t a n d a r d . The o p e r a t i n g c o n d i t i o n s w e r e as
78 JABER S. MOSSA A N D MAHMOUD M . A . HASSAN
Fig. 11: GLC of Camphor.
( a ) : Natural Camphor.
( b ) : S y n t h e t i c Camphor.
.
follows: Column l e n g t h 7 f t Column diameter 4
t o 5 mm. Column temperature 13OoC. S t a t i o n a r y
phase 20% of squalane on 100 t o 1 2 0 mesh c e l i t e .
Sample s i z e 30 pl, C a r r i e r gas 4:1 hydrogen and
n i t r o g e n mixture a t 100 ml/min. The r e t e n t i o n
volumes were camphor1200 m l , ethylbenzene 270 m.
camphor content of t h e sample i s c a l c u l a t e d from
t h e chromatogram by using peak a r e a r a t i o method.
Yoshio Hanada and Masayoshi Kitajima (184) have

r e p o r t e d t h e gas chromatographic determination of
camphor using naphthalene as t h e i n t e r n a l
standard .
GLC procedure f o r t h e s e p a r a t i o n of camphor and
o t h e r t e r p e n e s on polypropyleneglycol-impregnated
CAMP H 0R 79
column w a s a l s o d e s c r i b e d by Egon S t a h l and

Trennheuser ( 1 8 5 ) . El-Deeb, - et - a 1 (1.75)
h a v e r e p o r t e d G L C t e c h n i q u e f o r determin-
i n g t h e c o n s t i t u e n t s of v o l a t i l e o i l u s i n g s i l i -
con o i l DC 550 as a s t a t i o n a r y phase.
8.64 High-Performance Liquid Chromatography
Q u a n t i t a t i v e ana1ysj.s of b o t h camphor and para-

chlorophenol i n chaniphorated parachlorophenol by
h i g h - p r e s s u r e l i q u i d . chromatography i s d e s c r i b e d
(186).
The HPLC chromatograph (Waters A s s o c i a t e Model
A . I . C . 202) was used w i t h s o l v e n t system o f
60% hexane and 40% chloroform. A UV d e t e c t o r a t
254 nm was used a t a n a t e n u a t i o n of x8 f o r cam-
phor and x 64 f o r pa.rachlorophenol and phenol.
The flow r a t e was 2 . 5 ml/minute a t an i n l e t p r e s -
s u r e of 0.131 Nrn-2. The c h a r t speed w a s 0 . 5 cm/
min. The column (Hi--Eff M i c r o p a r t ) w a s 0.64 cm
0.d X 1 5 em l o n g , packed w i t h 5 Y m s i l i c a g e l
(Applied S c i e n c e L a b o r a t o r i e s ) . The i n j e c t i o n
volume w a s 7-15 1 arid t h e e l u t i o n over was cam-
phor ( 2.76 min.
phenol (12.61 min) .
!
parachlorophenol ( 10.64 min) and
The r e s u l t s i n d i c a t e d t h a t camphor and parachloro-

phenol can be s i m u l t a n e o u s l y assayed by simple
d i l u t i o n s of t h e sample and i n j e c t i o n i n t o a h i g h
p r e s s u r e l i q u i d chromatograph. The l i n e a r i t y
c u r v e i n d i c a t e d t h a t t h e components have been ana-
l y s e d g i v e l i n e a r response. R e s u l t s a r e shown i n
Table 9.
Table 9: Assay R e s u l t s on USP Camphorated

Parac h:Lorophenol
Assay Camphor, 7; Parachlorophenol,%
1 64.3 34.7
2 65.6 34.9
3 65.0 35.4
4 64.6 35.6
SD 0.56 0.42
cv 0.86 1.19
Average devia- 0.43 0.35
tion.
80 JARER S MOSSA A N D MAHMOIJD M . A . HASSAN
8 . 7 S p e c t r o s c o p i c Methods
8.71 C o l o r i m e t r i c
Camphor can be determined c o l o r i m e t r i c a l l y w i t h
s u l f u r i c a c i d and f u r f u r a l (187). To l c c of cam-
phor s o l u t i o n , add 3 cc of e t h a n o l ( 9 5 % ) and 1 0
drops of 1% s o l u t i o n o f f u r f u r a l i n 95% e t h a n o l .
S t i r t h e m i x t u r e , add two drops of c o n c e n t r a t e d
s u l f u r i c a c i d and h e a t i n a water b a t h . Then c o o l
and add 5cc. of e t h a n o l and compare t h e r e d t o
v i o l e t c o l o u r produced w i t h a s t a n d a r d . The
amount p r e s e n t can be determined w i t h i n 10%.
The c o l o u r r e a c t i o n s o f camphor w i t h aldehydes

have been a p p l i e d w i t h p -dimethylaminobenzaldehyde
i n s u l f u r i c a c i d (188). The yellow c o l o u r formed
conforms t o B e e r ' s law and checks c l o s e l y with t h e
o f f i c i a l N F method ( 1 5 4 ) . The a s s a y is c a r r i e d
out a s follows :
Dissolve 0.125 g of pdimethylaminobenzaldehyde

i n 65 ml o f c o n c e n t r a t e d s u l p h u r i c a c i d and 35 m l
of water. Add 7 ml of t h i s r e a g e n t by b u r e t t e
t o an a l i q u o t o f a d i l u t i o n of t h e sample i n
chloroform t o c o n t a i n between 0 . 6 and 7 . 5 mg o f
camphor. S t o p p e r , shake and allow t o s t a n d two
hours f o r c o l o u r development and measure t h e
e x t i n c t i o n a t 460 mp.
P r e p a r e a s t a n d a r d curve i n a s i m i l a r manner from

a l i q u o t s o f a prepared s o l u t i o n of camphor i n
chloroform i n t h e r a n g e 0 . 6 t o 7.5 mg of camphor.
A method has been d e s c r i b e d f o r d e t e r m i n a t i o n o f

camphor i n d i l u t e s o l u t i o n s and i n p l a n t s (189).
The a s s a y i s based on t h e formation o f a yellow
c o l o u r w i t h phenylhydrazine (Camphor phenylhydra-
z o n e ) . The c o l o u r produced a f t e r a b s o r p t i o n on
Whatman No.120 paper and t h e i n t e n s i t y o f t h e
yellow c o l o u r produced was compared with t h a t
produced from a sample of known c o n c e n t r a t i o n .
A m i c r o a n a l y t i c a l procedure w a s r e p o r t e d f o r
d e t e r m i n a t i o n o f camphor ( 1 9 0 ) . The method i s
based on t h e formation o f camphor oxime. The
oxime i s s u b j e c t e d t o a c i d h y d r o l y s i s and t h e
r e s u l t i n g hydroxylamine r e a c t e d w i t h f o r m a l i n and
CAMPHOR 81
f e r r i c ammonium. The r e s u l t i n g f e r r i c formohydro-

xamate i s t r e a t e d w i t h ammonium p e r s u l p h a t e and
t h e c o l o u r produced i s t h e n determined.
8.72 Nuclear Magnetic Resonance
Hassan - et - a 1 (29) r e c e n t l y have d e s c r i b e d a proton

magnetic resonance a n a l y t i c a l procedure f o r t h e
d e t e r m i n a t i o n of camphor a s b u l k drug and t h e
simultaneous a s s a y o f camphor and p-chlorophenol.
The method i s based on t h e i n t e g r a t i o n of t h e
n i n e p r o t o n s of t h e t h r e e methyl groups of
camphor c e n t r e d a t 0.88 ppm and t h e t e n aromatic
p r o t o n s of benzophenone c e n t r e d a t 7.55 ppm
employed as an i n t e r n a l r e f e r e n c e s t a n d a r d . The
method i s s p e c i f i c and a c c u r a t e w i t h s t a n d a r d de-
v i a t i o n o f 2 0.86 and an average r e c o v e r y of
100.28%. I n c a s e of camphor parachlorophenol
a s s a y , i n t e g r a t i o n of t h e n i n e protons o f t h e
t h r e e methyls o f camphor c e n t r e d a t 0.83 ppm,
t h e f o u r aromatic p r o t o n s of parachlorophenol
c e n t r e d at 6.97 ppm, and t h e two o l e f i n i c pro-
t o n s s i n g l e t of maleic a c i d appearing a t 6.37 ppm
were c a r r i e d o u t . The method w a s s p e c i f i c and
a c c u r a t e with s t a n d a r 5 d e v i a t i o n s of t 1.13, 2 1.01
f o r camphor and t 0.62, t 0.89 f o r parachlorophenol,
i n s t a n d a r d mixtures and i n camphorated para-
chlorophenol r e s p e c t i v e l y . The PMR spectrum i n
a d d i t i o n , provides a very s p e c i f i c means f o r
i d e n t i f i c a t i o n of camphor and parachlorophenol.
ACKNOWLEDGEMENT
The a u t h o r s g r a t e f u l l y thank M r . K.U. Abdul Hameed,

L e c t u r e r , Department of Pharmacognosy, College of Pharmacy,
King Saud U n i v e r s i t y , Riyadh, Saudi Arabia f o r doing t h e
l i t e r a t u r e survey and f o r h i s ef:eort and hard work which have
been undertaken d u r i n g t h e prepa:-ation of t h e manuscript and
a l s o acknowledge Mr. A l t a f Hussain Naqvi, f o r t y p i n g t h e
manus c r ip t .
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CAMPHOR xi
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-
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-
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CAMPHOR x9
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--
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CAMPHOR
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CAMPHOR 93
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Agentina. 43, 62 ( 1 9 5 8 ) .
CHLOROQUINE
Mohammad Tariq and Abdullah A. Al-Badr
King Satad Utrirersity
Riycidh, Saudi Arabia
I . History 96
2. Description 96
2.1 Nomenclature 96
2.2 Formulae 97
2.5 Appearance, Color, Odor, and Taste 97
3.1 Melting Point 97
3.2 Boiling Point 98
3.3 Solubility 98
3.4 Acidity 98
3.5 Moisture Content and Hygroscopicity 98
3.6 Dissociation Constants 98
3.7 Stability 98
3.8 Storage 98
3.9 Sterilization 98
4. Synthesis Ill
5. Pharmacokinetics I I3
5.1 Absorption. Distribution, Metabolism, and Excretion I I3
5.2 Distribution and Metabolism in Neonate:; 114
6. Therapeutic Uses 115
7. Toxicity 1 I5
8.1 Identification 1 I5
8.2 Microbiological Assay 116
8.3 Spectrophotometric Methods 1 I6
8.4 Chromatographic Methods 1 I8
References 123
ANALYTICAL PROFILES OF DRUG SUBSTANCES Copyright 0 1984

VOLUME 13 95 by the American Pharmaceutical Association
ISBN 0-12-260813 5
96 M O H A M M A D TARIQ A N D ABDULLAH A . AL-BADR
1. History
Chloroquine i s one of a l a r g e series of 4-aminoquinolines

investigated i n connection with t h e extensive cooperative
program of a n t i m a l a r i a l r e s e a r c h i n t h e United S t a t e s
durinE World War 11. The o b j e c t i v e w a s t o d i s c o v e r more
e f f e c t i v e and l e s s t o x i c s u p p r e s s i v e a g e n t s t h a n quina-
c r i n e . Although t h e 4-aminoquinolines had p r e v i o u s l y been
d e s c r i b e d a s p o t e n t i a l a n t i m a l a r i a l s by Russian i n v e s t i -
g a t o r s , s e r i o u s a t t e n t i o n w a s n o t p a i d t o t h i s chemical
c l a s s u n t i l t h e French r e p o r t e d t h a t 3-methyl-7-chloro-4-
(4-diethylamino-1-methylbutylamino) q u i n o l i n e (SN-6911;
S o n t i c h i n , Sonotoquin) w a s w e l l t o l e r a t e d and had h i g h
a c t i v i t y i n human m a l a r i a s . Beginning i n 1943, t h o u s a n d s
of t h e s e compounds were s y n t h e s i z e d and t e s t e d f o r a c t i v i t y
i n a v i a n malaria and f o r t o x i c i t y i n m a m m a l s ; t e n of t h e
s e r i e s were t h e n examined i n human v o l u n t e e r s w i t h e x p e r i -
m e n t a l l y induced m a l a r i a s . O f t h e s e , c h l o r o q u i n e proved
most promising and w a s r e l e a s e d f o r f i e l d t r i a l . When
h o s t i l i t i e s c e a s e d , i t w a s d i s c o v e r e d t h a t t h e chemical
had been s y n t h e s i z e d and s t u d i e d under t h e name of Resochin
by t h e Germans a s e a r l y as 1934. (1)
2. Description
2.1 Nomenclature
2. I. 1 Chemical N a m e s
4
N -(7-chloro-4-quinolinyl~-N’, MI-diethyl-1 ,
4-pentanediamine.
7-Chloro-4-(4-diethylamino-l-methylbutyl-
amino) q u i n o l i n e .
The CAS R e g i s t r y No. i s [54-05-71. (2 & 3)
2.1.2 Generic Name
Ch l o r o q u i n e
RP 3377, SN 7 , 618. (2)
2.1.3 Trade N a m e s
-
Base: Aralen, Nivaquine B , Sanquine,
A r t r i c h i n , B i p i q u i n e , Reumachlor, Bemaphate,
Tanakan, Resoquine. (3)
CHLOROQUIN E 97
Diphosphate: Valaquine, Resochin, S i l b e s a n ,

T r e s o c h i n , Arechin, A v l o c l o r , Imagon. ( 2 , 3)
Sulphate: Nivaquine and Remasulph. ( 2 , 3 & 8)
2.2 Formulae
2.2.1 Empirical
1gH2 6'IN3
2.2.2 Structural
,
CH
1 3 / C2H5
NH-CH-CH2CH 2CH2-N
JQJ@
C2H5
c1 8 1
N
Chlor oqu i n e
2.2.3 ldiswesser L i n e N o t a t i o n
T66 R N J EM & 3
N2 & 2 1GSQP
QQ0 2 DL (4)
319.89
2.4 Elemental Composition
C 6 7 . 5 9 % , H 8 . 1 9 % , C 1 11.08% N 13.14%. (2)
2.5 Appearance, C o l o r , Odor and T a s t e
White o r s l i g h t l y y e l l o w c r y s t a l l i n e powder, o d o r l e s s
and b i t t e r i n t a s t e . ( 5 & 6 ) .
3.1 Melting Point
M e l t s between 87' and 97'. (6)

98 M O H A M M A D TARIQ A N D ABDULLAH A . AL-BADR
3.2 Boiling Point
212' t o 214' a t 0.2 mm Hg. (7)
3.3 Solubility
Very s l i g h t l y s o l u b l e i n water; s o l u b l e i n d i l u t e
a c i d s , chloroform and e t h e r . (7)
3.4 Acidity
Chloroquine phosphate h a s a pH of 3.5 t o 4.5 of 10%

s o l u t i o n . While t h e 10% s o l u t i o n of c h l o r o q u i n e
s u l p h a t e h a s a pH of 4 t o 5 . (7)
3.5 M o i s t u r e Content and H y g r o s c o p i c i t y
Not more t h a n 1.5% determined by d r y i n g a t 105'. It

a b s o r b s i n s i g n i f i c a n t amounts of m o i s t u r e a t tempera-
t u r e s up t o 37O a t r e l a t i v e h u m i d i t i e s up t o about
80%. (7)
3.6 Dissociation Constants
pKa Values a r e 8 . 4 , 10.8 (20'). (7)
3.7 Stability
S o l u t i o n s of pH 4 t o 6 a r e s t a b l e when h e a t e d b u t
sensitive to light. (7)
3.8 Storage
I t should be p r o t e c t e d from l i g h t . (7)
3.9 Sterilization
S o l u t i o n s f o r i n j e c t i o n a r e s t e r i l i z e d by h e a t i n g i n
a n a u t o c l a v e o r by f i l t r a t i o n . ( 7 ) .
3.10 S p e c t r a l P r o p e r t i e s
3.10.1 U l t r a v i o l e t Spectrum
The u l t r a v i o l e t spectrum of c h l o r o q u i n e i n
n e u t r a l methanol s o l u t i o n i n t h e r e g i o n of
200 t o 400 nm e x h i b i t s m a x i m a a t 218 nm,
253 nm and 328 nm and minima a t 243 and
CHLOROQUINE 99
275 nm. The spectrum i s shown i n Fig. I .

The spectrum wasobtained on Varian AG U V - V i s
Spectrophotoineter model DMS 90.
3.10.2 I n f r a r e d Spectrum
The i n f r a r e d spectrum of c h l o r o q u i n e i s pre-

s e n t e d i n F i g . 2. The spectrum w a s o b t a i n e d
from K B r d i s c on a Perkin-Elmer I n f r a r e d Spec-
trophotometeir model 580 B , i n t h e r a n g e of
4000 - 400 CIn-1. It shows t h e f o l l o w i n g :
-
Frequency (cm-l) Assignments
3260 NH group
1540
1580 quinoli n e
1610
I n a d d i t i o n t o o t h e r bending and s t r e t c h i n g
v i b r a t i o n bands € o r t h e a l i p h a t i c moiety of
t h e molecule.
C l a r k e (8) r e p o r t e d t h a t t h e p r i n c i p a l peaks
f o r c h l o r o q u i n e b a s e i n K B r d i s c a r e 1573,
1538, 1372 o r 1 4 4 8 .
3.10.3 Mass Spectrum
The chemical i o n i s a t i o n (CI) m a s s spectrum

F i g . 3 , w a s r e c o r d e d on a Finnigan 4000 Mass
Spectrometer w i t h i o n s o u r c e p r e s s u r e 0.3
T o r r , i o n s o u r c e t e m p e r a t u r e 15OoC, e m i s s i o n
c u r r e n t 300 VA, e l e c t r o n energy 100 e V u s i n g
methane a s a r e a g e n t g a s . The e l e c t r o n
impact ( E I ) m a s s spectrum F i g . 4 , w a s
r e c o r d e d on Varian MAT 311 S p e c t r o m e t e r , w i t h
a n i o n s o u r c e p r e s s u r e 10-6 T o r r , i o n s o u r c e
t e m p e r a t u r e 180, e m i s s i o n c u r r e n t 300 UA
and e l e c t r o n energy of 7 0 e V .
Table 1 and t h e f o l l o w i n g f i g u r e i l l u s t -
r a t e t h e prominent f r a g m e n t s i n t h e E I spec-
trum and t h e i r proposed a s s i g n m e n t s .
L
i
100.0- r
50.0 - -
Bc
211
207’
so
100.0-
320 -
50.0- -
284 I 348
Fig. 3. Mass spectrum of chloroquine, CI.

m
a
r,
L
I03
104 MOHAMMAD TARQ AND ABDULLAH A . AL-BADR
Table 1 E I Mass Spectrum Assignments of Chloroquine.
mfe R e l a t i v e Abundance I o n o r Fragment
319 13 M+'.
M-CHT t
304 3
290 M - C ~ H ~ +
247 M - N E ~ +
231 M-CH2-NEg+
219 5 M-( (XI2) 2-Nhq+

M-(CFT2) 3NEt2
--I+
205 5
126 5 C ~ H ~ N E ~ J
+
112 13 C ~ H , N E ~ +
99 13 c 2~ 3 ~ ~ t 2 )
+
86 100 CH 2~~;-;1+
58 40 H-N -Et
CHLOROQUINE I05
3.10.4 'H-NMR Spectrum

1
The H-NMR s p e c t r a of c h l o r o q u i n e b a s e and
p h o s p h a t e a r e shown i n F i g . 5 and 6 and
were o b t a i n e d o n V a r i a n T6-A NMR S p e c t r o m e t e r
w i t h CDC13 and D:,O r e s p e c t i v e l y as s o l v e n t s
and t e t r a m e t h y l s i l a n e a s a n i n t e r n a l r e f e r -
ence. The s i g n a h a r e a s s i g n e d i n T a b l e 2 .
Table 2 . 'H-NMR S p e c t r a l Assignments of C h l o r o q u i n e .
Chemical S h i f t (ppm)
and M u l t i p l i c i t y [
Proton
Base Phosphate
- 1.5 [m]
- -
CH3CH2; CH3-CH 0.83 -. 1.33 [m] 1.2
--
-CH CH2CH2 CH2- 1.66 [ t ] 1.82 [ b s ]
-CH2-N 2.33 -' 2.67 [m] 3.2 [ 4 ]

- \
CH2-
F 3
-CH -
I
3.66 [41 4.00 [ b s ]
NH 5.64 [ d ] Exchanged
Aromatic C H- 8.43 [ d ] 8.2 [ d ]

2 I_
Aromatic C H- 6.4 [d] 6.65 [ d ]

-3
Other Aromatic P r o t o n s 7.2 - 7.96 [m] 7.15 - 7.9 [m]
s = singlet d = doublet t = triplet q = quartet
b s = broad s i n g l e t b m = broad m u l t i p l e t .
MOHAMMAD TARIQ AND ABDULLAH A. AL-BADR
3.10.5 C-13 NMR Spectrum
The noise-decoupled and o f f - r e s o n a n c e C-13

NMR s p e c t r a of c h l o r o q u i n e a r e p r e s e n t e d i n
F i g u r e s 7 and 8 r e s p e c t i v e l y . The s p e c t r a
were o b t a i n e d on Varian FT-80A S p e c t r o m e t e r .
S p e c t r a l a s s i g n m e n t s are l i s t e d i n T a b l e 3.
9
2 1
CH3
I 7 6 5 /CH2-CH3
NH -CH- CH -CH -CH2 -N
2 \
m2 -(=H3
3 4
12
14
Table 3 . Carbon-13 NMR S p e c t r a l Assignment of Chloroquine.
Carbon No. Chemical S h i f t (ppm) Mu1t i p l i c i t y

r e l a r i v e t o 'INS
1, 4 11.7 quartet
2, 3 46.27 triplet
5 52.25 triplet
6 23.60 triplet
7 33.46 triplet
8 47.72 doublet
9 19.83 quartet
10 149.60 singlet
11 98.79 doublet
12 151.85 doublet
13 149.47 singlet
14 127.55 doublet
15 133.36 singlet
16 123.71 doublet
17 124.40 d oub 1e t
18 117.67 singlet
Fig. 7 . I3C-NMR spectrum of chloroquine in DMSO-D6, noise-decoupled.
. .
l ~ l l ~ ~ l l ~ l l l ~ l ~ l l l ~ l ~ l ~ l ~ l l ~ ' l ~ i ~
Fig. 8. I3C-NMR spectrum of chloroquine in DMSO-D o f f -resonance.

6'
CHLOROQUINE Ill
4. Synthesis
a) One of the earliest and most effective antimalarials

is "chloroquine", in whose synthesis 4,7-dichloro-
quinoline, prepared from '7-chloro-4-quinolone, is key
intermediate. (9, 10).
250' >
&
Cl NHCH=C(C02C2H5)2 Cl
oo+ COOEt
(1) NaOH
( 2 ) HC1
c1
HO@'& 250' ~ d
c1
,
00 'OC13 ~
fH3 Et
Chloroquine
112 MOHAMMAD TARIQ AND ABDULLAH A. AL-BADR
b) In another synthesis methyl acrylate is reacted with

m-chloroaniline,the resulting methyl m-chloroanilino-
propionate is tosylated and then hydrolyzed to the
acid. The acid is converted to acid chloride and
cyclized to give 4-keto-7-chloro-1,2,3,4-tetrahydro-
quinoline reaction of this compound with 4-diethyl-
amino-1-methylbutylamine under the dehydrogenating
influence of nitrobenzene gives chloroquine in about
252 overall yield (11 6 12).
COOCH3 COOH
I I
Tos H
-m C
n
H
5
-.
$3
nitrobenzene
Bt
N H ~ ~ ( c H3~;j3~
~)
Chloroquine
c) Chloroquine is obtained by coupling 4,7-dichloroquino-

line with 5-diethylamino-2-aminopentane. (13)
c1
YH3 /Et
NH2-CH-(CH 2) 3N,Et
t Chloroquine
CHLOROQUINE I13
5. Pharmacokinetics
5.1 A b s o r p t i o n , D i s t r i b u t i c i n , Metabolism and E x c r e t i o n
Chloroquine i s r e a d i l y absorbed from t h e g a s t r o -

i n t e s t i n a l t r a c t and about 50% i n t h e c i r c u l a t i o n i s
bound t o plasma p r o t e i n . I t d e l a y s g a s t r i c emptying.
I t a c c u m u l a t e s i n h i g h c o n c e n t r a t i o n s i n some tis-
s u e s , such as t h e k i d n e y s , liver l u n g s and s p l e e n
and i s s t r o n g l y bound i n m e l a n i n - c o n t a i n i n g c e l l s
such a s t h o s e i n t h e e y c s and t h e s k i n ; i t i s a l s o
bound t o double s t r a n d e d DNA, p r e s e n t i n r e d blood
c e l l s c o n t a i n i n g s c h i z o i i t s ( 1 ) . A t a d o s e of 310 mg,
plasma c o n c e n t r a t i o n s r e a c h a p l a t e a u a t 10 d a y s w i t h
a c o n c e n t r a t i o n of about: 120 ng/ml; a f t e r a 500 mg
d o s e , c o n c e n t r a t i o n s of about 700 ng!ml are a t t a i n e d
i n haemolysed blood i n 4. h o u r s and a f t e r s i n g l e o r a l
d o s e s of 400 t o 500 mg, peak plasma c o n c e n t r a t i o n s
of 35 t o 220 ng/ml a r e a t t a i n e d i n 2 t o 6 h o u r s ( 6 ) .
About 7 0 4 5 % of t h e t o t a l whole blood c o n t e n t of
c h l o r o q u i n e and i t s m e t a b o l i t e d e s e t h y l c h l o r o q u i n e
were r e c o v e r e d i n blood c e l l s i s o l a t e d from whole
b l o o d , i n d i c a t i n g t h a t t h e s e compounds have a c e l l /
plasma c o n c e n t r a t i o n r a t i o ( 1 4 ) . It h a s been
r e p o r t e d t h a t small amounts of c h l o r o q u i n e may b e
found i n t h e blood and u-rine of p a t i e n t s f o r as l o n g
a s 5 y e a r s a f t e r t h e lasir known a d m i n i s t r a t i o n , and
t h e e x i s t e n c e of l a r g e t i s s u e r e s e r v o i r s h a s been
p o s t u l a t e d (15 ti 1 6 ) . Other s t u d i e s are p u b l i s h e d
on u n i d e n t i f i e d metabo1it:es p r e s e n t i n plasma and
u r i n e of t h e t r e a t e d s u b j e c t s . The p o l a r and non-
p o l a r m e t a b o l i t e s of chlclroquine are shown below:
NHR
Fig. 9
c1
I R = CH(CH3)-(CH2)3-N-(C2H5)2
I1 R = CH(CH3)-(CH2)3-NH-C2H5
I11 R = CH(CH3)-(CH2)3-NH2
IV R = CH(CH~)-(C:H~)~-~JC~H~)~
V same a s I V , b u t r i n g N i s a l s o N@ --+ Oe VI R = H
I14 MOHAMMAD TARlQ AND ABDULLAH A. AL-BADR
F i g . 9 C h l o r o q u i n e ( I ) , d e s e t h y l - ( 1 1 ) and d i d e s -
e t h y l c h l o r o q u i n e (111). S i d e - c h a i n N-oxide ( I V ) , d i -
N-oxide (V) , and 7-chloro-4-aminoquinoline (VI) .
IV
and V , p o l a r m e t a b o l i t e s ; I1 and 111, r e l a t i v e l y non-
polar metabolites. (17).
Chloroquine and i t s m e t a b o l i t e d e s e t h y l c h l o r o q u i n e
found i n thrombocytes and g r a n u l o c y t e s i n v i v o and
--
i n v i t r o . C h l o r o q u i n e i s c h a r a c t e r i z e d by i t s
e x t e n s i v e t i s s u e d i s t r i b u t i o n which c a u s e s a slow
e l i m i n a t i o n from t h e body. Plasma h a l f - l i f e i s v a r i -
a b l e 21/,-7 d a y s . B r o h u l t e t a 1 (18) o b t a i n e d a v a l u e
f o r plasma h a l f - l i f e of c i i l o r o q u i n e i s 4 d a y s , w h i l s t
Frisk-Holmberg e t a1 (19) found h a l f - l i v e s of 3.1
h o u r s t o 13 d a y s depending upon t h e dose. M e t a b o l i c
r e a c t i o n s i n v o l v e o x i d a t i v e N - d e a l k y l a t i o n and o x i d a -
t i v e d e a m i n a t i o n and a l s o c o n j u g a t i o n , p o s s i b l y w i t h
g l u c u r o n i c a c i d , of t h e c a r b o x y l i c a c i d m e t a b o l i t e s
d e r i v e d from d e a l k y l a t i o n and d e a m i n a t i o n ; t h e meta-
b o l i t e s i n c l u d e mono- and b i s d e s e t h y l c h l o r o q u i n e , 4 -
(7-chloroquinol-4-ylamino) pentan-1-01, and 4-(7-
chloroquinol-4-ylamino) p e n t a n o i c a c i d and i t s con-
j u g a t e (7). Chloroquine i s e l i m i n a t e d v e r y s l o w l y
from t h e body and i t may p e r s i s t i n t i s s u e f o r a pro-
longed p e r i o d . About 55% i s e x c r e t e d i n t h e u r i n e
and a b o u t 10% i n t h e f a e c e s by 90 d a y s f o l l o w i n g
t h e r a p y w i t h 310 mg f o r 1 4 d a y s ; t h e u r i n a r y excre-
t i o n o f t h e unchanged d r u g i s dependent upon u r i n a r y
pH and l a r g e r amounts are e x c r e t e d i n a c i d u r i n e t h a n
i n a l k a l i n e u r i n e ; of t h e m a t e r i a l e x c r e t e d i n t h e
u r i n e , a b o u t 70% i s unchanged, 23% i s d e s e t h y l c h l o r o -
q u i n e , 1 t o 2% i s b i s d e s e t h y l c h l o r o q u i n e and an
u n i d e n t i f i e d m e t a b o l i t e , and 1 t o 2% i s e x c r e t e d a s
c a r b o x y l i c a c i d m e t a b o l i t e s i n c o n j u g a t e d form. (21 &
22).
5.2 D i s t r i b u t i o n and Metabolism i n Neonates
C h l o r o q u i n e , i t s N-dealkylated m e t a b o l i t e s , and
c h l o r o q u i n e N-oxides were d e t e c t e d i n t h e u r i n e of
p r e g n a n t women who were r e c e i v i n g c h l o r o q u i n e medi-
c a t i o n whereas c h l o r o q u i n e and i t s n o n p o l a r m e t a -
b o l i t e s , d e s e t h y l and d i d e s e t h y l c h l o r o q u i n e and 7-
chloro-4-aminoquinoline have been found i n t h e neo-
n a t e s ’ u r i n e , b l o o d and cord blood. T h a t c h l o r o q u i n e
and i t s r e l a t i v e l y n o n p o l a r m e t a b o l i t e s ( i n c l u d i n g
one w i t h o u t t h e a l k y l s i d e - c h a i n , 7-chloro-4-amino-
q u i n o l i n e ) c r o s s t h e p l a c e n t a i s d e m o n s t r a t e d by t h e
CHLOROQUINE 115
p r e s e n c e of t h e s e compounds i n t h e c o r d blood, neo-

n a t a l s y s t e m i c b l o o d , and n e o n a t a l u r i n e . The
s e l e c t i v e t r a n s f e r of t h e compounds a c r o s s t h e p l a -
c e n t a h a s a l s o been r e p o r t e d ( 1 7 ) .
Both i n v i t r o and i n -- I
v i v o m e t a b o l i c s t u d i e s have
shown t h a t some drug metabolism o c c u r s o r can be
induced i n p l a c e n t a l t i . s s u e s ( 2 0 ) . F u r t h e r degrada-
t i o n of p a r e n t c h l o r o q u i n e t h a t r e a c h e s t h e p l a c e n t a
would r e s u l t i n t h e appearance of less c h l o r o q u i n e
and more of t h e n o n p o l a r m e t a b o l i t e s i n t h e f e t a l o r
n e o n a t a l c i r c u l a t i o n . (117).
6. Therapeutic Uses
I t i s h i g h l y e f f e c t i v e i n t e r m i n a t i n g a c u t e v i v a x and
f a l c i p a r u m malaria. I t i s a l s o v a l u a b l e i n t h e t r e a t m e n t
of i n t r a i n t e s t i m l a m e b i a s i s and some cases of g i a r d i a s i s
(1). Chloroquine a l s o produce symptomatic improvement i n
B a r b e s i a i n f e c t i o n s . It can b e used a s an a n t i r h e u m a t i c
agent (23).
7. Toxicity
I n t h e r a p e u t i c d o s e s c h l o r o q u i n e may c a u s e headache,
v i s u a l d i s t u r b a n c e s , g a s t r o i n t e s t i n a l u p s e t and p r u r i t i s .
I n some c a s e s i t may c a u s e d i s c o l o r a t i o n of n a i l b e d s and
mucous membranes. Prolonged t r e a t m e n t may c a u s e l i c h e n o i d
s k i n e r u p t i o n , v i s u a l b l u r r i n g and b l e a c h i n g of h a i r .
Prolonged l a r g e d o s e s (250 t o 750 mg d a i l y ) may c a u s e l o s s
of c e n t r a l v i s u a l a c u i t y , g r a n u l a r p i g m e n t a t i o n of macula
and r e t i n a l a r t e r y c o n s t r i c t i o n . The v i s u a l l o s s a p p e a r s
t o be i r r e v e r s i b l e ( 2 4 ) . O t o t o x i c i t y h a s been r e p o r t e d
i n few c a s e s . Hart and Naunton, (25) have i m p l i c a t e d
c h l o r o q u i n e i n f e t a l a b n o r m a l i t y . Blood d y s c r a s i a s have
been r e p o r t e d r a r e l y . They i n c l u d e r e v e r s i b l e agranu-
l o c y t o s i s , thrombocytopenia, and n e u t r o p e n i a ( 6 ) .
8. Methods of A n a l y s i s
8.1 Identification
a ) D i s s o l v e a b o u t 25 mg i n 20 m l of water and add

8 m l of t r i n i t r o p h e n o l s o l u t i o n ; a p r e c i p i t a t e i s
produced which, a f t e r washing s u c c e s s i v e l y w i t h
w a t e r , a l c o h o l ( 9 5 % ) , and e t h e r , m e l t s a t about
207O. (8).
1I6 MOHAMMAD TARlQ AND ABDULLAH A. AL-BADR
b) Micro: P i c r i c a c i d - r o s e t t e s of p l a t e s ( s e n s i -
t i v i t y : 1 i n 1000); s t y p h n i c a c i d -
r o s e t t e s of
p l a t e s ( S e n s i t i v i t y 1 i n 1000). (8).
8.2 M i c r o b i o l o g i c a l Assay
A r a p i d semiautomated m i c r o d i l u t i o n method f o r t h e
m i c r o b i o l o g i c a l a s s a y of t h e c h l o r o q u i n e h a s been
developed by Desgardins ( 2 6 ) . A n t i m a l a r i a l a c t i v i t y
of c h l o r o q u i n e may be s t u d i e d a g a i n s t c u l t u r e d
Plasmodium f a l c i p a r u m , m i c r o p l a t e s a r e used t o pre-
p a r e s e r i a l d i l u t i o n of t h e drug. P a r a s i t e s o b t a i n e d
fromcontinuous s t o c k c u l t u r e s a r e s u b c u l t u r e d i n t h e
m i c r o - p l a t e s f o r 4 2 h. I n h i b i t i o n of u p t a k e of a
r a d i o l a b e l e d n u c l e i c a c i d p r e c u r s o r by p a r a s i t e s
serves a s t h e i n d i c a t o r o f a n t i m i c r o b i a l a c t i v i t y .
8.3 Spectrophotometric Methods
8.3.1 U l t r a v i o l e t Spectrophotometric A n a l y s i s
8.3.1.1 Ap, Method

J
Chloroquine h a s been assayed s p e c t r o -
p h o t o m e t r i c a l l y ( 2 7 ) u s i n g t h e Apj
method (28) t h a t depends on d i f f e r e n c e s
i n orthogonal function c o e f f i c i e n t s .
The l a t t e r method i s based on t h e f a c t
t h a t t h e peaks of c e r t a i n compounds
may s p l i t i n t o s u b s i d i a r y peaks without
any a p p r e c i a b l e change i n i n t e n s i t y by
changing t h e pH i n a s u i t a b l e i n t e r v a l .
The b e h w i o u r of t h e s e compounds
r e s t r i c t s t h e a p p l i c a t i o n of t h e AA
method ( 2 9 & 30) because AA may n o t
f u l f i l t h e requirements specified f o r
its successful application. I n these
c i r c u m s t a n c e s , t h e n p j method o f f e r s a
s o l u t i o n f o r t h e d e t e r m i n a t i o n of such
compounds i n t h e presence of i r r e l e v a n t
absorption. Thus, t h e c o n t r i b u t i o n of
a pH-insensitive i r r e l e v a n t absorption
may be c a n c e l l e d by means of
AP. .=[ajia Cx
J1
+ P..(Z)I
11
- [ajih Cx + pji ( Z > I
and C =Ap../Aa..
x 31 11
where p j i s t h e c o e f f i c i e n t o f t h e
polynomial, p . ( 4 ) a i i s p j ( I X , 1 cm),
t h e c o e f f i c i e i t f o r A (151, 1 cm) o f the
CHLOROQUINE I17
p u r e compound, x ; C, i s t h e c o n c e n t r a -
t i o n ; pji(Z) d e n o t e s t h e c o n t r i b u t i o n
from i r r e l e v a n t a b s o r p t i o n ; t h e sub-
s c r i p t s i , a , and b d e n o t e t h e wave-
l e n g t h r a n g e and t h e two d i f f e r e n t
s o l u t i o n s , r e s p e c t i v e l y . Thus, by
choosing P . and a l s o t h e s e t of wave-
l e n g t h s , i: s o t h a t p j i i s optimum i n
one s o l v e n t and n e g l i g i b l y small i n
t h e o t h e r s o l v e n t , Apji can be used t o
e v a l u a t e compound x.
The a s s a y p a r a m e t e r s u s i n g t h e Ap.
J
method f o r c h l o r o q u i n e :
Solvents : 0.1 N H2S04 & 0.1 N

NaOH .
Concentration : 1 . 6 mg/100 m l s o l -
vent .
P : P (the quartic
j polynomial).
Number of : 12-point o r t h o g o n a l
Points polynomials.
Wave 1en g t h : 324-346 nm each 2 nm.

range
*p4
: - 0.00170
: 8008
N;
1,
(Ap,. N')"* : 0.160
4
fc N4 = normalizing f a c t o r f o r P
4'
f 5 ~ should exceed 0.14 f o r t h e c o e f f i -
c i e n t of v a r i a t i o n t o be l e s s t h a n
17.
I18 MOHAMMAD TARIQ AND ABDULLAH A . AL-BADR
8.3.2 F l u o r i m e t r i c Method
A h i g h l y s e n s i t i v e , a c c u r a t e and improved
f l u o r i m e t r i c method h a s been developed f o r
a n a l y s i s of c h l o r o q u i n e i n b i o l o g i c a l samples
(31). The c h l o r o q u i n e i s e x t r a c t e d w i t h
h e p t a n e o r methylene d i c h l o r i d e from a n
a l k a l i medium b u f f e r e d w i t h b o r a t e , pH 9.5.
Chloroquine i s r e e x t r a c t e d i n t o 0.1 N H C 1
which i s t h e n mixed w i t h an e q u a l volume of
0.2 M a l c o h o l i c NaOH and f l u o r e s c e n c e i s
r e a d a t a c t i v a t i o n and emission wavelengths
of 335 and 400 nm r e s p e c t i v e l y .
8.4.1 Paper Chromatography
Several s o l v e n t systems have been d e s c r i b e d ,

which are used f o r t h e i d e n t i f i c a t i o n of
c h l o r o q u i n e ( 8 ) a s shown i n t h e f o l l o w i n g
table:
S o l v e n t Systems V i s u a l i z i n g Agents (Rf)
C i t r i c a c i d : water: n- Ultraviolet, iodoplatinate

b u t a n o l 4.8 g : 103 m l : s p r a y ( s t r o n g react i o n ) ,
870 m l . bromocresol g r e e n s p r a y
( s t r o n g r e a c t i o n ) (0.19).
A c e t a t e b u f f e r (pH 4 . 5 8 ) Ultraviolet, iodoplatinate

spray ( p o s i t i v e r e a c t i o n ) .
(0.70).
Phosphate b u f f e r (pH 7 . 4 ) Ultraviolet, iodoplatinate

spray. (0.14).
8.4.2 Thin-Layer Chromatography
Clarke ( 8 ) d e s c r i b e d t h e f o l l o w i n g system
f o r t h e i d e n t i f i c a t i o n of c h l o r o q u i n e .
S o l v e n t System: S t r o n g ammonia s o l u t i o n :
methanol ( 1 . 5 : 100) should be changed
a f t e r two r u n s .
Absorbent: Silica G e l G
CHLOROQU INE I I0
V i s u a l i z i n g Agent : A c i d i f i e d i o d o p l a t i n a t e
spray ( p o s i t i v c r e a c t i o n ) .
Rf : 0.31.
8.4.3 Gas Chromatography
a) C l a r k e (8) h a s d e s c r i b e d t h e f o l l o w i n g
g a s chrom5,tographic method € o r t h e
i d e n t i f i c a t i o n of c h l o r o q u i n e .
Column: 1Z SE-30 on 100-120 mesh Arkrom

ABS. 6 f t X 4 m i n t e r v a l diameter
b o r o s i l i c a t e g l a s s column.
Col.umn Temperature .: 2500 .
Carrier Gass Argon.
G a s Flow: 80 m l p e r min.
D e t e c t i o n and R e t e n t i o n Time: Argon

i o n i s a t i o n d e t e c t o r or flame i o n i s a t i o n
d e t e c t o r . R t 1.44 r e l a t i v e t o c o d e i n e .
b) C h u r c h i l l e t a 1 1 9 8 3 (23) h a s
developed two gas chromatographic
methods u s i n g Tandem f u s e d - s i l i c a
c a p i l l a r y f o r t h e d e t e r m i n a t i o n of
c h l o r o q u i n e and i t s m a j o r m e t a b o l i t e
d e s e t h y l c h Loroquine. The method I
employed a s i n g l e e x t r a c t i o n s t e p
and i n t e r n a l s t a n d a r d i z a t i o n to
permit r a p i d p r e c i s e a n a l y s i s f o r
c h l o r o q u i n e i n whole blood. Whereas
method I1 employes d e r i v a t i z a t i o n
with pent:afluoropropionic a n h y d r i d e
b e f o r e e x t r a c t i o n and e s t i m a t i o n of
c h l o r oqu i n e and d e se t hy l c h l o r o q u i n e
t h e d e t e c t i o n l i m i t f o r chloroquine
i n blood by b o t h methods are 5 ng/ml
and f o r d e s e t h y l c h l o r o q u i n e i s 1 5 ng/
ml. Following s p e c i f i c a t i o n column;
I20 MOHAMMAD TARlQ AND ABDULLAH A . AL-BADR
1.83 m X 2 mm I D g l a s s column packed

w i t h OV-101 on 100-120 macsh g a s
chrome Q and a helium f l o w r a t e
15 ml/min.
Temperature : 250°C.
E l e c t r o n impact i o n i s a t i o n performed
a t 70 eV.
8.4-3.1 L i q u i d Chromatography
The l i q u i d chromatographic system

c o n s i s t e d of an M45 pump a U6K
i n j e c t o r . The e x c i t a t i o n wavelength
w a s 335 nm and a 370 nm f i l t e r w a s
used. The column (0.15 m X 4.6 mm
I . D . ) w a s s l u r r y - packed w i t h
L i c h r o s o r b S i 6 0 , 5 pm and e l u t e d .
The flow r a t e of t h e e l u e n t w a s 1 m l /
min. A f t e r a d d i t i o n of water and
e x t r a c t i o n with d i e t h y l e t h e r , t h e
o r g a n i c phase w a s d r i e d (sodium
su1phate)and t h e s o l v e n t evaporated ,the
p r o d u c t could be used d i r e c t l y . The
l i m i t of d e t e c t i o n of t h i s method w a s
1 ng/ml of plasma f o r c h l o r o q u i n e and
0.5 ng/ml f o r d e s e t h y l c h l o r o q u i n e .
The p r e c i s i o n of t h e method w a s 3.5
of (n=lO) a t 50 ng/ml plasma ( u r i n e )
f o r c h l o r o q u i n e and 5% (n=10) a t t h e
25 ng/ml f o r d e s e t h y l c h l o r o q u i n e .
(32).
8.4.4 High-pressure L i q u i d Chromatography
8.4.4.1 Reversed-Phase HPLC A n a l y s i s
B e r g q v i s t and Frisk-Holmberg, (33)

r e p o r t e d a r a p i d and s e n s i t i v e method
f o r t h e a n a l y s i s of c h l o r o q u i n e and
i t s metabolite desethyl chloroquine
i n plasma, blood and u r i n e u s i n g
high-performance l i q u i d chromatogra-
phy. Chloroquine i s e x t r a c t e d from
the alkalinized biological f l u i d with
e t h y l e n e d i c h l o r i d e and r e - e x t r a c t e d
w i t h d i l u t e a c i d . It i s t h e n chromato-
CHLOROQUINE 121
graphed on a r e v e r s e d - p h a s e column.
U l t r a v i o l e t a b s o r p t i o n ( 2 5 4 o r 340
nm) o r f l u o r e s c e n c e d e t e c t o r s are
used. C h l o r o q u i n e and d e s e t h y l -
chloroquine concentrations i n t h e
r a n g e of 10 n m o l / l (UV-detection)
and of 0.5 n mol/ 1 ( f l u o r e s c e n c e
d e t e c t i o n ) c o u l d be a c c u r a t e l y
measured w i t h a r e l a t i v e s t a n d a r d
d e v i a t i o n of 12%.
8.4.4.2 S t r a i g h t - P h a s e HPLC A n a l y s i s
B e r g q v i s t and O l i n ( 3 4 ) h a v e d e s -
c r i b e d a s t r a i g h t - p h a s e HPLC method
f o r t h e a n a l y s i s of c h l o r o q u i n e a s
follows :
LiChrosorb S i 100 ( 7 um) w a s u s e d a s

support:. The m o b i l e and s t a t i o n a r y
p h a s e s were e q u i l i b r a t e d b e f o r e u s e .
A t u b e ( l e n g t h 1.5 m and volume 4 m l )
w a s i n s e r t e d i n f r o n t of t h e i n j e c t o r
and p l a c e d i n a t h e r m o s t a t i n o r d e r
t o e n s u r e good t e m p e r a t u r e c o n t r o l
of t h e m o b i l e p h a s e . 25 m l of a n
aqueous p e r c h l o r a t e s o l u t i o n was
p r e s e n t as a s e p a r a t e l a y e r i n t h e
s o l v e n t r e s e r v o i r i n o r d e r t o keep
t h e mobile phase s a t u r a t e d . The
column c o a t i n g w a s a c h i e v e d by t h e
pumping t e c h n i q u e . Columns w e r e
f i r s t t e s t e d i n t h e a d s o r p t i o n mode
w i t h n-hexane + 1 - b u t a n o l (199 + 1)
as t h e m o b i l e p h a s e . The v o i d volume
V,, w a s 2.86 m l . It w a s d e t e r m i n e d
w i t h t o l u e n e , which i s n o t r e t a i n e d .
The c o n c e n t r a t i o n of 1 - p e n t a n o l i n
t h e m o b i l e phase w a s c o n t r o l l e d by
g a s chromatography on a Chromosorb
102 cclumn w i t h 1 - b u t a n o l a s i n t e r n a l
standard.
8.4.4.3 I o n - P a i r HPLC A n a l y s i s
R e c e n t l y a method f o r d e t e r m i n a t i o n
of chl.oroquine and i t s m e t a b o l i t e s
h a s been d e s c r i b e d by Brown ( 3 5 ) .
I22 MOHAMMAD TARlQ A N D ABDULLAH A . AL-BADR
T h i s method i s s i m p l e s p e c i f i c and
s e n s i t i v e f o r s e p a r a t i o n and q u a n t i -
f i c a t i o n of c h l o r o q u i n e d e s e t h y l c h l o -
r o q u i n e and b i d e s e t h y l c h l o r o q u i n e
i n biological f l u i d s using ion p a i r
r e v e r s e phase HPLC procedure. I t can
d e t e c t amount as low as 5 ng and
a n a l y s i s t i m e i s 12 minutes. The
f o l l o w i n g c o n d i t i o n s have been u s e d .
Column 30 mm X 3.9 mm I . D .
packed w i t h 10 Um
U Bondspak CI8.
Mobile Phase : 0.02 m l I-heptane

s u l p h o n i c a c i d and
acet o n i t r i l e .
PH 3.4
Pumping r a t i o : 6 6 ~ 3 4of P I C B-7 to

acetonitrile.
Column P r e s - : 62-76 b a r .
sure
8.5 Photolysis Analysis
Aron and Fidanza 1982 h a s d e s c r i b e d a photochemical

method f o r d e t e r m i n a t i o n and s e p a r a t i o n of c h l o r o -
q u i n e a t nanogram level c o v e r i n g a r a n g e between 4
and l o 4 ng. They showed t h a t r e v e r s i b l e photo i s o -
m e r i z a t i o n of c h l o r o q u i n e t a k e s p l a c e and y i e l d s a
f l u o r e s c e n t photoproduct. The c h l o r o q u i n e s o l u t i o n
a r e s p o t e d on s i l i c a g e l p l a t e s and developed i n
a l c o h o l ammonia m i x t u r e . The d r i e d p l a t e s a r e
i r r a d i a t e d w i t h t h e image of mercury a r e focused on
c h l o r o q u i n e s p o t s . The f l u o r e s c e n c e i n t e n s i t y w a s
r e c o r d e d . For p h o t o l y s i s s t u d i e s c h l o r o q u i n e zone
w e r e s c r a p e d from t h e p l a t e s and mixed w e l l w i t h t h e
4 m l of water. The c l e a r s u p e r n a t a n t decanted and
s t u d i e d by a b s o r p t i o n photometry and s p e c t r o f l u o r o -
metry ( 3 6 ) .
Acknowledgements
The a u t h o r s are t h a n k f u l t o M r . Syed R a f a t u l l a h f o r h i s

s k i l l f u l a s s i s t a n c e and M r . Tanvir A. B u t t f o r t y p i n g
t h e manuscript.
CHLOROQUINE 123
References
1. Goodman and Gilman's The Pharmacological Basis of

Therapeutics, A.G. Gilman, L.S. Goodman and Alfred
Gilman, 6th Ed., Macmillan Publishing Co., Inc. New
York, page 1044 (1980).
2. Merck Index, An Encyclopedia of Chemicals and Drugs,

Motha Windholz et al, IX Ed., Merck and Co. Inc.,
II
Rahway, N.J., U.S.A. page 2143 (1976).
3. Remington's Pharmaceutical Sciences, A. 0 ~ 0 1 ,15th Ed.,

Mack Publishing Co., Easton, Pennsylvania, U.S.A.
4. CRC, Atlas o f Spectral Data and Physical Constants at

Organic Compounds, edited by J.G. Grasselli and W.M.
Ritchey, 2nd Ed. Published, CRC Press, Inc. c,
1975.
5. Unites States Pharmacopoeia XX, Twenteeth Edition,

United States Pharmacopoeia1 Convention, Inc., page 627
(1980).
6. Martindale, The Extra Pharmacopoeia, 27th Ed., The

Pharmaceutical Press, 1 Lambth High Street S E I , London,
page 343 (1977).
7. The Pharmaceutical Codex, 11th Ed., The Pharmaceutical

Press, London, page 176 (1979).
8. E.G.C. Clarke, Isolation and Identification o f Drugs,

The Pharmaceutical Press, London, page 252 (1971).
9. Heterocyclic Compounds, "hinolines", G. Jones, John

Wiley 6 Sons Inc., New York, page 391 (1977).
10. Organic Synthesis, Collective. 1701. 3 , C.C. Price and

R.M. Roberts, John Wiley & Sons Inc., Mew york, page
272 (1955).
11. Medicinal Chemistry, 2nd Ed., A. Burger, Wiley Inter-

science, New York, page 836 (1960).
12. W.S. Johnson and B.G. Bull, 2. &. Chem., 74, 4513,
(1952).
13. The Chemistry of Organic Medicinal Products, G.L.

Jenkins --
et al, 4th Ed., John Wiley 6 Sons Inc., New
York , page 360 (1957).
I24 MOHAMMAD TARIQ AND ABDULLAH A. AL-BADR
14. Y. B e r g q v i s t and B. Domeij-Nyberg, J. Chromatogr.,

-
272, 137 (1983).
15. pI.1.Rubin, H.M. B e r n s t e i n and N . J . Zvaifler, Arch.

Opthalmol. -
7 , 474 (1963).
16. N.J. Z v a i f l e r and M. Rubin, A r t h e r i t i s Rheum, 5, 330

(1962) .
17. E.E. E s s i e n and G.C. Afamefuna, -
Clin. -
Chem., 285, 1148
(1982).
18. J. B r o h u l t , L. Rombo, V. S i r l e a f and E. Bengtsson: &.

--
Trop. Med. P a r a s i t . , 73,401 (1979).
19. M. Frisk-Holmberg, Y. B e r g q v i s t , B. Domeij-Nyberg, L.

H e l l s t r o m and F. Jamson, C l i n . Pharmac. Ther. 25, 345-
-I
350 (1979).
20. B. Testa and P. J e n n e r , I n Drug Metabolism: Chemical and

Biochemical A s p e c t s , J.J. S w a r d r i k , Ed., Marcel Dekker
I n c . N . Y . , 397-404 (1976).
21. S . A . Adeluse and L.A. S a l a k o , Gen. pharmac. 2, 433

(1982).
22. E.W. McChesney, M . J . F a s c o and W.F. Banks, Jr., 2. Pharm.

-
Exp. Therap. 158, 323 (1967).
23. F.C. C h r u c h i l l , D.L. Mount and I . K . Schwartz, -

J.
Chromatogr., 274, 111 (1983).
24. H.N. B e r n s t e i n , N . J . S v a i f l e r , F. Robin and A.M. Mansour,

--
I n v e s t . Ophthalmol V i s u a l S c i . , - 2 , 384 ( 1 9 6 3 ) .
25. C.W. H a r t and R.F. Naunton, Arch. O t o l a r y n g o l , 80,

407 (1964).
26. R.E. D e s g a r d i n s , J. C a n f i e l d , J . D . Haynes and J . D .

Chulay, A n t i m i c r o b i a l Agents
~- - Chromotherapy,
and 16,
710 (1979).
27. A.M. Wahbi, H. Abdine and S.M. R l a i l , Die Pharmazie,

33, 96 (1978).
I
28. H . Abdine, A.M. Wahbi and M.A. Korany, J. Pharm. Pharmac.

-
24, 518 (1972).
CHLOROQUINE I25
29. G . Erdtman, Chem. I n d . , 74, 581 ( 1 9 5 5 ) .
30. R.A. F i s h e r and F. Yates. S t a t i s t i c a l Tables f o r

B i o l o g i c a l , A g r i c u l t u r a l and Medical Research, 6 t h Ed.,
-
9 8 , O l i v e r b Boyd, E d i n b u r g h , ( 1 9 7 4 ) .
31. S . A . Adeluse and L.A. Salako, Gen. Pharmac., 13,433

(1982).
32. G. Alvan, L. Ekman and B. L i n d s t r o m , -

J. Chromatogr.,
2 2 9 , 241 ( 1 9 8 2 ) .
-
33. Y. B e r g q v i s t and M. Frisk-Holmberg, -
J. Chromatogr.,
221, 119 ( 1 9 8 0 ) .
34. Y . B e r g q v i s t and A . O l i n , Acts Pharmaceutica S u ecica,

1 9 , 161 (1982).
I
35. N.D. Brown, B.T. Poon and J . D . Chulay, 2. Chromatogr.,

-
2 2 9 , 248 ( 1 9 8 2 ) .
36. J . J . Aaron and J . F i d a n z a , T a l a n t a , -

2 9 , 383 ( 1 9 8 2 ) .
CIMETIDINE
P. M . G. Bavin
Smith Kline cind French Rewnrch, Ltd
The Frytlze, Welwyn
Hertfordy h ire, United Kingdom
Alex Post and John E. Zarembo'

Smith Kline cind French Laboratories
Pliilacielphici, Peniisyltjnnin
Introduction i28
1. Description i28
1.1 Nomenclature 129
1.2 Formula, Molecular Weight, Structure 129
1.3 Appearance, Color, Odor 129
2.2 Physical Properties of the Solid 152
2.3 Solubility of Cimetidine 158
2.4 Physical Properties of Cimetidine Solutions 161
3. Synthesis 161
3.1 Synthesis of Cimetidine 161
3.2 Synthesis of Cimetidine Hydrochloride 164
4. Stability 164
4.1 Cimetidine 164
4.2 Cimetidine Hydrochloride 166
5. Analytical Chemistry 166
5.1 Identity Tests 166
5.2 Non-Aqueous Titration 167
5.3 Spectrophotometric Procedure 168
5.4 Thin Layer Chromatography 169
5.5 High Pressure Liquid Chromatographic Procedure 171
5.6 Determination of Cimetidine Sulfoxide Content 176
6. Metabolism and Pharmacokinetics 176
6.1 Determination of Cimetidine and Its Metabolites in Biological Fluids 177
References 181
'Present address. Revlon Health Cure Group, Tuckuhoe, Neu' York 17070.
ANALYTICAL PROFILES OF DRUG SUBSTANCES Copyright C 1984

VOLUME 13 127 by the American Pharmaceutical Asaocidtion
ISBN 0-12-260813-5
i 28 P. M . G . BAVIN Er AL.
INTRC)DUCTION
Cimetidine i s a histamine H2-receptor

antagonist which inhibits the secretion
of basal and gastric hydrochloric acid
secretion and also reduces the output
of pepsin. The drug has been widely
used i n the treatment o f ducdenal and
gastric ulceration. It has also been
used i n the t r e a h e n t of reflux
esophagitis and f o r the reduction of
gastric acid secretion and for the
managanent of Zollinger-Ellison syndrcxne.
The ccBnpound is given orally a s a tablet,
syrup, i.v., and as an injection fluid.
1. Description
1.1 Ncpnenclature
1.11 Ch&cal Names
(a) N”-cyano-N-methyl-N’- [ 2- [ (5-methyl-1~-
imidazol-4-yl)mthyll thiol ethyll guanidine
(c.a. name)
(b) N”-cyanc-N-mthyl-N’- [ 2- [ (5-methyl-
imidazol-4-yl)methylthiolethyll guanidine
1.12 Trade Name
Marketed by SnithKline Corporation under the
trade nanie, Tagatnet.
CIMETIDINE 129
1.2 Formula, Molecular Weight Structure

1.21 mirical F o d a , Pblecular Weight
ci oHi 6 N 6 S 252.352
1.22 Structure
dN-CSN
H3C) (CHZSCH~MZNHC
b m 3
v
1.23 Hydrochloride S a l t
C1 I ) H ~ ~ NHC1
~S 288.816
1.3 Ap- ance, Color, odor
The f r e e base and hydrochloridesalt are w h i t e
c r y s t a l l i n e s o l i d s with l i t t l e or no odor. A
slight sulfur-mercaptan odor may be present.
2.1 Spectral Properties
2.11 Infrared Spectra
Figure 1 i s the infrared spectrum of
cimetidine f r e e base. Figure 2 i s the
infrared spectrum of cimetidine hydrochloride.
The spectra were obtained as mineral o i l
dispersions from 400-625 an-’ on a Perkin-
Elmer Model 457A infrared spectrometer. The
s i g n i f i c a n t absorption bands in t h e spectra
are :
Cimetidine
wavelength (an-’) Assignment
3220-3150 NH s t r e t c h
2180 - E N stretch
1620 >C=N-, 9~10-
guanidine
1590 C-C, C-N, armtic
I 1 I I I I I t 1 1 I I 1 1 II
4000 2500 Boo 400 250
WVENUMBER (CW) WVENUMBER (CM')
Fig. 1. Infrared spectrum of cimetidine.

I
01
w
0
5 02
m
g 03
3 04
0-5
14J
0
)o 35m 3ooo 2500 2mo leoo 1600 1- 1200 loo0 800 io

WVENUMBER (CM') WVENUMBER (CW)
Fig. 2. Infrared spectrum of cimetidine hydrochloride.

132 P. M . G . BAVIN E T A L
C h t i d i n e €iydrochloride:
wavelength (un-l) Assignment
3418 NH stretch
3280 NH stretch
3130 NH stretch
2780-2600 NH2+
2175 >N-C!% stretch

1705-1595 X - N stretch
I infrared study
& of the i n t r a m o l e c u l a r
hydrogen bonding i n cimetidine and several
related H2-receptor antagonists has been
reported by R. C. Mitchell.' H i s study
shaws that c d t i d i n e can form intramolecular
bonds in solution and that it can adopt a ten-
membered ring conformation i n which the basic
imidazole nitrogen atan is believed to be
intrmlecular hydrogen bonded t o the NH group
furthest f m the imidazole ring.
2.12 Ultraviolet Spectrum
The ultraviolet absorption spectrum of
cimetidine i n 0.lN aqueous sulfuric acid
is sham in Figurz 3. A sumnary of the
ultraviolet absorbance characteristics
obtained i n several solvents is shown in
Table 1.
Table 1 S- of Cimetidine Ultraviolet Absorption Data
Solvent x max (m) E max-&260 log E
0.1N- aqueous H2m4 218 20,530 4.31

95% ethanol 220 22,600 4.35
0.1N- aqueous HC1 216 19,360 4.29
0.6-
0.5-
0.4 -
w 0.3-
18 0.2-
0.1-
0.0- I 5
1 1 1 1 1
220 230 240 250 270 280 290 300
NANOMETERS
Fig. 3. Ultraviolet absorption spectrum of cimetidine in
0.1 N aqueous sulfuric acid.
I34 P. M. G. BAVIN E T A L
The absorption maxirmnn is due primarily to

the T -f T* transition of t h e cyanoimino
( ~ N - C Z N ) grow with a p a r t i a l contribution
frcan the conjugated double bond system of
the imidazole ring. The pH absorption
dependency of c k t i d i n e frm pH 1.36 t o
10.35 has been described by Kajfez, e t al.
2.13 Nuclear Magnetic Resonance Spectra
2.131 Proton Magnetic Resonance of Spectnm
Cimetidine
The proton magnetic resonance spectrum
(Figure 4 ) of cimetidine was
recorded for a deuterated dimethyl
sulfoxide solution containing
approximately 100 mglrril of the ccmpclund
with tetramethylsilane as t h e internal
standard. The spectrum i l l u s t r a t e d w a s
obtained using a P e r k i n - E m R32
proton magnetic resonance (CW)
.
spectraneter The resonances are:
I
H
h
Protons Chtsnical Shift ZJUtlkr
-
At (HE) Multiplicity of Protons lnteqral
2 -50-2.58 multiplet 2 2
ca
2.6C singlet 3 3
cc 2.65 doublet 3 3
‘d 3.09-3.44 multiplet 2 2
ce 3.60 singlet 2 2
6.45-7.34* multiplet 2 2
Nf“9
ch 7.43 singlet 1 1
‘i 11.7 singlet
(broad)
*quartet and t r i p l e t overlapped
0 1 2 3 4 5 6 7 8 9 10
I I I 1 I I I w
I I I I I 1 1 I I
PPM 9 0 7 6 5 4 3 2 1
Fig. 4. Proton magnetic resonance spectrum of cimetidine in

deuterodimethylsulfoxide.
I36 P. M. G . BAVIN E T A L .
2.132 Proton Spectrum-cimetidine

Hydrcchloride 3'4
The proton magnetic resonance spectrum

of cimetidine hydrochloride was
obtained by preparing a solution of
approximately 100 mg/ml of the chemical
in deuterated dimethyl sulfoxide
containing tetramethylsilane as the
internal standard. The chemical s h i f t
values of the hydrochloride s a l t d i f f e r
f r m those of the free base and are:
&N-C-sN
H
g
Proton Chem. Shift
~
No. of
Position Structure (ppn) Multiplicity Protons
a ringG3 2.29 singlet 3
b s a 2 2.60 t r i p l e t over- 2-
lapped by mso
m l t i p l e t at
2.5 p p & a
doublet a t
2.7 PPn
c -ma33 2.70 doublet over- 3.
lapped bY
-SCH2 t r i p l e t
d -2CH2NH 3.32 multiplet . 2
e ring-CH2S 3.87 singlet 2
$7 N-CH=N 8.99 singlet 1

h -NH 9.0-10 .o broad singlet 1
The chemical p f t s at positions d and fwere verified by
E. S. Pepper using spin decoupling and spin-decouplhg
CIMETIDINE 137
with D20 exchange, respectively. These shifts were later

cmf- by mjfez, et a l .
2.133 Double Resonance Spin Decouplinq and
Deuterium Exchange Studies of
Cimetidine
Spin Decoupled at: 7.12 p p
Results: 2.69 ppn doublet collapses to
a singlet. Shms that a
coupling exists between
NH-cH3.
Spin Decoupled at: 7.1 ppn
Results: 2.09-3 -44 mltiplet (2H) is
reduced to a less ccanplex
pattern. This is assigned to
4X2NH< (=NCN) protons.
Deuterium (D20)exchange causes the
chmical shifts at 7.18 and 9.0-10.0 to
disappar. These resonances are
assigned to three exchangeable hy&ogens
attached to nitrogen on the imidazole
ring and in the side-chain.
2.134 Carbon-13 NMR Spectrum
The broad band decoupled carbon-13 NMR
spectrum of cimetidine hydrochloride
(Figure 3) was obtained by using a
solution of approximately 100 mg/ml in
deuterated dimethylsulfoxide. The
deuterium signal of dimethylsulfoxide
was used as the internal reference and
the spctrum was obtained on a Varian
Associates Model ET-80 fourier
transform NMR spectrcmeter. The
chemical s h i f t assignments are:
1 4 2 E
H-N .N
91
H
P. M . G . BAVIN E T A L .
Postition of carbon Chemical Shift, p p

1 9.68
2 26.01
28.17
4 30.05
5 40.81
6 118.18
7 124.91
8 130.00
9 133.34
10 159.96
2.14 Mass Spectrum
2.141 Electron-Ionization Impact specrtrum 3,4
The mass spectrum of cimetidine using

a Hitachi Perkin-Elmer W - 7 E medium
resolution mass spectrmeter and a
direct insertion probe of the sample is
shown in Figure 6. m e mass spectrum
was recorded on magnetic tape and
relative abundances were calculated
with a PDP-8/Idigital ccrmputer coupled
to the instrument. The results are
presmted in tabulated fonn in Table 2.
Table 2 Cimetidine Mass Spectral Fragment Ions By
-
Electron-Ionization Irnpact
5
m
- ion
252 M+
+
158
157
L
(continued)
CIMETIDINE 139
Table 2 Continued
m
- -
ion
128
127
126
125
116
115
NCN
111 cH&kCH3
95
NCN
II
82 c==fiHcH3
(continued)
140 P. M. G. BAVIN E T A L .
Table 2 Continued
m
- ion
-
69 CH2=&-C-NCH3
30 CH2-h2
The fragmentation of cimetidine base

appears t o follcm t h e three major
pathways. Sham in Schmes 1, 2, and
3. The mlecular ion is observed a t
m/e = 252.
2.142 Mass Spctrum-Field Desorption
The f i e l d desorption mass spectrum of
cimetidine base and a table of the
fragmntation peaks are presented i n
Figure 7 and tabulated in Table 3.
The spectrum was obtained using a
V a r i a n MAT M-5 D F mass spectrometer.
The &tter w a s loaded by t h e dipping
techniques fran an acetone solution. An
e m i t t e r m e n t of 20 m i l l i a n p r e s was
used t o obtain the spectrum. The
spectrum shcms a m l e c u l a r ion,,'M at
m / e = 252 and a MH+ a t m / e = 253. The
peak a t m / e = 258 is due t o acetone
used for instrument tuning purposes.
Several low intensity f r a q e n t ions
are found and are related t o the
electron-ionization fragmentation
mechanisn described above.
2.143 Mass Spectrum42hmical Ionization (CI)
The chemical ionization mass spectrum
of cimetidine and the major fragmenta-
t i o n ions are presented in Figure 8
and Table 4. The spectrum was
obtained using a Finnigan H e 1 3200
quadruple mass spectrmter f i t t e d
with a chemical ionization source. The
sample, applied t o the probe f r m an
acetone solution, was introduced v i a
t h e d i r e c t inlet system. Methane w a s
used as the reactant gas.
The spectrum shcrws an (M+1)+ peak f o r
t h e m l e c u l a r ion and characteristic
(200)ppm
'H-
3 1
I I
Fig. 5. Carbon-13 NMR spectrum of cimetidine hydrochloride.

125
kwlralion Wlage. 7OeV

Source Temperalure. 200°C
Direct Sample Inlet Temperalure. 300°C
Inslrwnenl: Hilachi-Perkln Elmer RMU 6E I
C
c)
130 170 210 250

m/e
Fig. 6. Electron-ionization mass spectrum of cimetidine.
Fig. 7. Field desorption mass spectrum of cimetidine.
I r 5.W
2: 1
I: 1
117.6
28.4
I
Fig. 8. Mass spectrum cimetidine- chemical ionization- methane reactant gas.

ClMETlDINE I45
Table 3 Field &sorption Mass Spectrm of

C h e t i d i n e . Peak Intensity D a t a .
04116/811SPEC# 185lKMIFD 20 MA
BASE SUM
11306 27545
PEAK ItBASE MASS

1 0.30'10 43.8
2 12.48% 58.0
3 4.49 '10 59.0
4 0.26% 77.9
5 0.09% 81.O
6 3.02% 95.1
7 0.44 '10 96.1
8 0.14% 97.0
9 2.98% 105.9
10 0.09% 106.3
11 0.12O/O 117.0
12 0.62% 125.9
13 0.64% 127.0
14 0.38% 141.9
15 0.92% 157.0
16 1.78% 158.0
17 1.27% 158.9
18 0.37% 188.0
19 0.35% 188.1
20 0.13% 197.0
21 1.71% 21 1.1
22 0.78% 226.9
23 1.4O '10 228.0
24 0.84% 249.0
25 1.63/o' 250.0
26 6.O9% 251.O
27 69.95 /'o 252.1
28 100.00% 253.1
29 16.45% 254.0
30 5.98% 255.0
31 0.51O/o 256.1
146 P. M . G . BAVIN E T A L .
peaks a t (M+29)+ and (M+41)+ when using

methane as the reactant gas a t 1 1-1 t o r r
pressure. The major fragwntation ions
are given in Table 4. An INCOS
ccknputer system coupled w i t h the mass
spectrometer w a s used t o obtain,
normalize, and plot the spectral data.
Table 4 Chemical Ionization Mass Spectral Fragrtlent
Ions of Cimetidine (CHs-Reaction Gas)
m ion
293.2 (m + 41)
281.2 (m + 29)
253.1
159.1
117.0
+H
[Em2a2-v*Nm3 I+
95.1
68.9 CH2=h-C-NCH3
2.144 Negative Ion Mass Spectrum

The negative ion mass spectrum of
cimetidine was obtained using a
Finnigan We1 3200 quadrupole mass
SpeCtKHneter quipped with an INCOS
data system. The mass spectrum is
presented in Figure 9 and the major
fragnents are tabulated in Table 5. A
caparison of the mlecular ion peak
intensity obtained in the negative ion
mde w i t h that obtained in the chemical
ionization mode shows the former t o be
11.4 times mre intense. As the
fragwntation i n the negative ion mode
is less, identification of snaller s i z e
samples of c k t i d i n e can be made.
2:
I: 9
53.9
I
'! E I50 -.s

;.& l -:no
Fig. 9. Negative ion-mass spectrum of cimetidine.

Illdl28 mle 126 mle 127
Scheme 1. Fragmentation of cimetidine.

[C.H.N-J 1: mle 158
We 68
NCN
t II
HSCHaCH = NHCNHCH,
m/e 157
/I CHa
s "'
NCN
*+ YN
I
NHCNHCH,
rn'e
II t
C = NHCH,
mle 82
+ r
CH~SCHaCHaNHCNHCHa
m/e 120
mle 158
'
-HSCHaCHaN I'
= C = NCH,
mla 116
+
*

CIMETIDINE 151
Table 5 Negative Ion Mass S p c t r a l Fracpent Ions of

Cimetidine
m ion
251.0 [M-HI-
N-CN
II
156.9
-
93.9 M3wm Nk9N

2.15 Photoelectron Spectrum of C i m e t i d i n e
The photoelectron spectrum of cimetidine has
ken reported2. The folbwing bands w e r e
found and their assignrents have been
described:
Band Assignmen t
8.41 Ionization potential of both
imidazole TT electrons and the
lone electron p a i r of imidazole
N (3) atcsn.
9.03 I o n i z a t i o n potential of
unshared s u l f u r e l e c t r o n pair.
9.4 IT^ electrons of imidazole r i n g
and TT electron pairs of three
nitrogen atcans in guanidinium
unit.
11.57, 12.08 n electrons -CW
1
13.03, 13.36,
14.20, 14.95, N o t assigned
15.88
The photoelectron spectrum w a s obtained4 with
a Vacuum Generator W 63 instrumnt a t l a v
r e s o l u t i o n (35 eV) using He e x c i t a t i o n a t 140'
and xenon internal c a l i b r a t i o n .
2.16 X-Ray D i f f r a c t i o n
S i n g l e c r y s t a l x-ray diffraction s t u d i e s of
I52 P. M . G . BAVlN E T A L .
cimetidine w e r e carried out by Hadicke,

Frickel, and F'ranke.6 " C i m e t i d i n e was found
to form mnoclinic crystals (space group
P21/C). The molecule is internally hydrcqen
bnded by the N.. .N-N b n d between the
imidazole and guanidine residue t o form a ten-
-red ring system."2 The X-ray m er
diffraction spectnrm of cimetidine is sham
in Figure 10. The spectnrm was obtained using
a General E l e c t r i c XRD-5 @er diffractmeter
using CuKci irradiation. Table 6 lists the
d-spacings (interplanar distances), the
diffraction angle, 28, and the relative peak
intensities. Crystals w e r e found t o be
prisnatic and monoclinic. Prodic-Kojic , et
a17 have reported the cell constants t o be
a = 6.82 ( l ) ,b = 18.813 (3), and c = 10.374
(2) A
,
' B = 106.42 (1)'.
2.2 Physical P r o p e r t i e s of the Solid
2.21 Identity T e s t s
2.211 Thin Layer Chrmtography/Color T e s t s
This test may be used with the chemical,
tablet, or liquid dosage form containing
cimetidine o r cimetidine hydrochloride.
A methanol extract of the sample is
prepared and chram-aphed on a
Silica G e l GF c h r m t o p l a t e with ethyl
acetate, mthanol, m n i m hydroxide
(100, 1 0 , 1 0 , v/v) m b i l e phase. The
Rf comparison t o a reference similarily
chrcanatqraphed, the r e s p n s e to
diazotized p-nitroaniline (imidazole
nucleus detection), U.V. (254 nm), and
I p vapors serves to identify cimetidine.
2.212 Infrared Identification
The infrared spectrum of a mineral o i l
dispersion of the sample, previously
dried a t 105OC for four hours,
corresponds t o the reference sample.
Refer t o Section 2.11.
Fig. 10. X-ray powder diffraction pattern of cimetidine.
IS4 P. M . G . BAVIN ET AL
Table 6 - - Diffraction Pattern o f Cimetidine

X-Ray
20 d, i* Relative Intensity
~~
7 -20 12.27 5.7

9.40 9.40 7.4
10.10 8.75 2.8
13.00 6.80 14.5
14.75 6 .OO 31.9
16.58 5.34 38.3
16.80 5.27 58.2
17.80 4.98 24.1
18.46 4.80 32.3
19.10 4.64 4.6
19.72 4.50 39.0
20.58 4.31 4.3
21.26 4.18 4.3
23.58 3.77 100 .o
26.06 3.42 24.1
26 -90 3.37 6.4
27.30 3.26 14.2
28.10 3.17 5.7.
28.50 3.13 19.1
29.10 3.07 4.3
29.96 2.98. 0.7
30.50 2.93 7.1
31.62 2.83 1.4
32.60 2.74 7.8
33.92 2.64 14.9
34.60 2.59 9.2
35 .OO 2.56 2.8
36.06 2.49 2.1
37.64 2.39 1.4
38.80 2.32 2.1
40.50 2.22 2.1
41.40 2.18 2.1
42.64 2.12 5.3
43.40 2.08 4.3
44.80 2.02 2.1
46.40 1.96 2.1
47 -30 1 .92 1.4
nX
* d = interplanar distance =
ClMETlDlNE IS5
2.213 Ultraviolet Absorption Identification

The ultraviolet absorption spectrum of
a solution of cimetidine in 0.1N
sulfuric acid o r 0.1N HC1 exhib% a
maximum between 217-319 and a minimum
between 210 m and 212 m, (Figure 4 ) .
Refer t o Section 2.12.
2.214 Color Identification Test I
'Ib 0.1 m l of solution of the sample
prepared by dissolving 1 mg of
cimetidine i n 1 m l of ethanol add 5 ml
of a solution of 1 g citric acid in
acetic anhydride to make 50 ml,
(freshly prepared). Heat the mixture
on a waterbath f o r ten to fifteen
minutes. A red violet color develops.
2.215 Color Identification T e s t I1
'Ib 0.1 g of sample add 5 ml of 1 N
hyckochloric acid, and heat gentiy.
Add 5 m l of sodium hydroxide solution
(3 g in 1 0 ml H20) t o the solution and
heat additionally. The reaction mixture
evolves an amnonia odor. When a piece
of mistened red l i t m u s paper is
exposed t o the evolved gas, it tums
blue.
2.22 Them1 Properties
2.221 Melting Range
Cimetidine m e l t s between 140 t o 143.5OC
using the USP procedure for class 1
substances.
2.222 Differential Scanning Calorimetry (DSC)
The DSC thesmogram for cimetidine is
shown h Figure 11. A single endothem
i s found a t 142OC. As it does not
recrystallize f r m its m e l t a f t e r
prolonged coolincj a t room temperature,
seeding, scratching, or annealing, it
is considered an mrphous glass.
The hydrochloride s a l t of cimetidine
shows a single endothem a t 193O C
(Figure 12) .
156 P. M. G.BAVIN E T A L .
Fig. 11. Differential scanning calorimetric

thermogram of cimetidine.
167 177 187 "C 197 207 217 207
Fig. 12. D i - f f e r e n t i a l scanning calorimetric

thermogram o f cimetidine hydrochloride.
158 P. M . G. BAVIN E T A L .
2.23 Densitv/Specific Surface Area'

Density measurments were carried out by the
use of an air canparison pycnoweter and t h e
specific surface area was determined on
milled and unmilled sample using a Stroklein
areameter. The data are sumnarized in Table 7.
Table 7
Property Units Unmilled Milled
Mean Particle Microns 19.0 7.8
Size 12.1
Specific
Surface Area m2/g 0.666 1.304
Absolute q/cc 1.2815 -

Density
Tappea g/cc 0.40 0.51
Density
2.24 Themcgravimetric Analysis (TGA)
The themqravimetric curves for cimetidine
and cimetidine hydrochloride are sham in
Figure 13 and 14, respectively. The curves
shaw that the compounds are stable up to a
tenperatwe of approximately 175-185OC and
then decompse. The therrmgrams were obtained
at a heating rate of lO"C/minute.
2.3 Solubility of Cktidine
2.31 Solubility in Various Solvents
The solubility of cimetidineg * was determined
in a variety of solvents using essentially the
mthod of Schefter and Higuchi.
Solvent Solubility (mg/ml)
-2OoC
- - _ 24OC
_ _ _25OC
- 37.0"C 37.5OC
Acetonitrile 2.7
Chlorofo m 1 .o
Cyclohexane <o .Ol
Ethanol (USP) 58.0 64.5
Isopropanol 13.2
Diethyl Ether 0.01
&than01 122 144
Polyethylene Glycol 400 51
Water 5 6.15 11.4
0.m- HC1 >250
ClMETlDlNE 161
A. Prdic-Kojec , e t a17 reported a high

dependence of equilibrium solubilities of
cinetidine which ranged f r m 0.75% a t pH 8.92
t o 2.72% a t pH = 2.26 when determined a t
35.0 f 0.1OC. Similar data was obtained by
Mitchell.
2.311 Aqueous Solubility of C i m e t i d i n e
HydrocNoride
I n a study of apparent equilibrium
solubility cimetidine hydrochloride
is approximately 1.0 g/ml a t 25OC. The
equilibrium solubility i s approximately
160 mg/ml.
2.4 Physical Properties of Cimetidine Solutions1
Aqueous solutions of cimetidine and cimetidine
hydrochloride are usually colorless t o a very pale
s t r a w color. A saturated solution of the free base
has a pH of approximately 9.0 and a 15%solution
of the hydrochloride salt, a pH of approximately
3.7.
2.41 Dissociation Constants
The apparent pKa of cimetidine is 7.11 t 0.04
at 25OC masured potentimetrically in 0.1N-
aqueous KCll * . In water, the pKa of the
imidazole ring nitrogen (-NH-) is 6-80?' At
pH 7.4, 20.7% of cimetidine is present as
cations. The nitrogen a t m on the side chain
t o which the cyan0 (-EN) group is attached is
essentially neutral and has a pKa = -0.4?3
It is essentially non-ionized i n a broad pH
range ( ~ 2 - 1 2 ) .
2.42 Partition Coefficient
The "Apparent Partition Coefficient" for
cimtidine in an cctanol/pH 7.4 phosphate
buffer system a t 25OC and in an octanol/water
system a t 37OC w e r e found t o be 2.0 and 2.5
respectively.
3. synthesis
3.1 Synthesis of C h t i d i n e
Synthesis of cimetidine has been described by
Durant, G. J., e t a1.13r14
I62 P. M G . BAVIN E T A L .
ClMETlDlNE 163
Route 2
CIMETIDINE
164 P. M . G . BAVIN E T A L .
3.2 sy"thesis of C i m e t i d i n e Hydrochloride

N"-cyano-N-Inethyl-N ' - [2- [ (5-~thyl-l.~-imidaZ01-4-yl)
mthyll thiol ethyl] guanidine, hydrochloride
To an ethanolic suspension of cimetidine,
concentrated hydrocholoric acid and ethyl acetate
are added, the product is collected, washed w i t h
ethyl acetate, and dried. The schematic is
illustrated k l w :
II
CHzSCH,CHzNH-C-NHCH3 -HC1
4. Stability
4.1 Cimetidine
4.11 D r y State
C i n e t i d i n e in the dry s t a t e , stored i n a
closed container a t temperature shwed
no d e c m p s i t i o n a f t e r five years when
examined by high pressure liquid c h r m t o -
graphy, thin layer chrmtography, infrared
spectrophotmetry, and mass spectranetry. It
is stable for a t least 48 hours a t 100OC.
4.12 Acid Hydrolysis
Acid hydrolysis of cimetidine13 follows the
folloWing scheme:
A
ClMETlDLNE I65
The formation of the amide is pH and t-a-

ture depmdmt. Solutions of cimetidine in
hydrochloric acid a t p H %5.4 showed no
d e c a p s i t i o n a t 50' over a period of t h i r t y
days. Hydrolysis t o the guanylurea (B)
occurred when the caarq?ound w a s heated a t 45'
for 36 hours w i t h an excess of 1N hydrochloric
acid a t p H 4.The guanidine was formed by
heating cimetidine for tm hours a t l0O'C
with concentrated hydrochloric acid.
4.13 Effect of Ultraviolet Light
N o damnpsition of cimetidine was noted 1s
a f t e r several mnths exposure t o U.V. light.
4.14 Effect of Hydrogen Peroxide and Oxyqen
When cimetidine i s treated w i t h 3% H202 a t
room t m p r a t u r e or exposed t o oxygen a t 50'C
for short time periods, formation of sulfoxide
is observed. Refer t o the following:
I66 P. M . G . BAVlN E T A L .
4.2 C i m e t i d i n e Hydrochloride
The s t a b i l i t y of cimetidine hydrcchloride w a s
reported by Rosenberg, e t al.
4.21 Aq~musSolutions
pqueous solutions (vials for injection) shmed
a shelf l i f e of a t least two years when
examined by high pressure liquid and e x e e d
by high pressure liquid and thin layer
chrmtography .
4.22 Infusion Fluids
Solutions i n comnon infusion fluids a t
concentrations of 120 o r 500 mg/lOO m l w e r e
found t o be stable for a t l e a s t one week a t
rocan temperature when examined by high
pressure liquid and thin layer chranatography?6
5. Analytical Chemistry
5.1 Identity T e s t s
5.11 Identification T e s t s for C h e t i d i n e
A.
- Dissolve about 20 mg of sample 2 m l of
distilled w a t e r with gentle warming. Add 1 m l
of 5% aqueous sodium n i t r i t e and allm t o
stand for approximately three minutes. Add
1 m l of sulfanilic acid test solution
(250 mg of sulfanilic acid dissolved in 25 ml
d l 0 8 v/v aqueous HC1) and allow t o stand
f o r a b u t five minutes. The formation of a
yellaw-orange t o reddish brown color indicates
the presence of cimetidine.17
B.
- Place about 20 mg of sample in a s m a l l
test tube. Add one drop of concentrated
hydrochloric acid, 5 m l of glacial acetic
acid, stir t o dissolve, and thm add 1 ml of
mrcuric acetate test solution (USP).
formation of a white precipitate indicates
the presence of cimetidine.
-
C. A solution of the sample of the hydro-
chloride s a l t responds positively t o the test
for chloride.
5.12 Identity T e s t s by I R , U V , TLC and HPLC
-
A. Cimetidine and cimetidine hydrochloride
have characteristic spectra (Figures 1 and 2 ) .
CIMETIDINE 167
-
B. The ultraviolet absorption spectrum of a
solution of cimetidine or cimetidhe
hydrochloride in 0.1g H2%4 exhibits a maximum
between 217 and 219 nm and a minimum a t 210-
212 nm (Figure 3 ) .
-
C. Characteristic TLC and HPLC retention
times are described for cimetidine in the
text and may be used a s confirmatory tests
for c h e t i d i n e (Section 5.4, 5.5).
-
D. Single crystals or trace amounts of
cimetidine respond t o color tests and w i l l
give a positive mass spectral peak for the
mlecular ion. These have been described
above.
5.2 Non Aqyeous Titration
5.21 Chemical
Dissolve about 240 mg of sample, accurately
weighed, in 75 ml of glacial acetic acid.
T i t r a t e with standardized 0.1 N perchloric
acid in acetic acid t o a potenFianetric end-
pint using glass-calm1 electrodes. Each
ml of 0.1N perchloric acid is equivalent t o
25.234 mgof cimetidhe.
5.22 Tablet
Determine the average tablet weight of twenty
tablets. Powder and weigh accurately a sample
equivalent t o about one gram of cimetidine
into a 200 ml volumetric flask. Add about
150 m l of methanol, and shake mechanically for
t h i r t y minutes. Dilute a v o l m with methanol,
mix and f i l t e r through a Whatxan #1 f i l t e r
paper, discarding the f i r s t 15 m l of the
f i l t r a t e . To 50 ml add 25 ml of glacial acetic
acid and t i t r a t e with 0.1N acetous perchloric
acid t o a potenticmetric endpoint using glass-
calmel electrodes.
Calculation:
mg Cimetidine tablet =
ml x N x 252.34 x 200 x Average Tablet Weiqht
Sample Weight (mg) x 50
168 P. M . G . BAVIN ET AL.
5.3 spectrophanetric Procedure

5.31 Chemical
Transfer about 65 mg of sample, accurately
weighed, t o a 200 m l v o l m t r i c flask.
Dissolve in and d i l u t e t o volume w i t h 0.1N-
s u l f u r i c acid. Transfer 5.0 ml of t h i s
solution t o a 200 ml volurnetric flask, and
d i l u t e t o volume w i t h the same solvent.
Record the ultraviolet absorption spectrum
of t h i s solution i n 1 cm cells on a suitable
spectrophotawter fran 320 t o 210 nm using
0.1N sulfuric acid as a blank. Measure the
abs&ance difference ( PA) between the
absorbance a t 218 nm (maximum) and that a t
260 nm.
Calculation:
% Cimetidine =
A??, x 0.01212* x Dilution Factor x 100
Weight of Sample (mg)
*0.01212 = mg cirrretidine/ml/unit AA
This absorptivity factor was experimentally
determined using accurately weighed samples of
cimetidine reference standard carried through
the sarne procedure.
5.32 Tablet
Weigh and finely p d e r twenty tablets. Weigh
accurately a portion of the p d e r , equivalent
to a b u t 100 mg of cimetidine and transfer
t o a 200 m l voluretric flask. Add 75 ml of
0.1N sulfuric acid, and shake mechanically
for-thbrty minutes. Dilute t o volm w i t h the
same solvent, mix, and f i l t e r through
Whatman #1 f i l t e r paper, discardjng the f i r s t
15 m l of f i l t r a t e . P i p e t 5.0 m l of f i l t r a t e
into a 200 volurnetric flask, add 0.1N
sulfuric acid t o v o l w , and mix. G o r d the
ultraviolet absorption spectrum of t h i s
solution frm 300 t o 215 nm in a 1 cm cell
v e r s u s 0.1N s u l f u r i c a c i d on a s u i t a b l e
spectrophotometer. S u b t r a c t the a b s o r b a n c e
a t 260 nm from the a b s o r b a n c e o f the maximum
a t a b o u t 218 nrn *
CIMETIDINE I69
Calculation:
mg c h t i d i n e / t a b l e t =
AA x 0.01212" x Average Tablet Weight (mg) x
Dilution Factor
Sample Weight (mg)
5.4 Thin Layer Chrmtqraphy
Adsorbent: Silica G e l GF, 250 microns
Eluting Solvent: ethyl acetate, methanol,
concentrated m n i u m hydroxide
( l O : l : l / v/v)
Ethyl acetate must be freshly
distilled.
Equilibration: 15 minutes in a closed paper lined
tank
Concentration: 50 mg/ml mthanol
Detection: iodine, W (254 m)
spotting: 100 ?Jg
The solvent i s allowed t o rise t o the 15 cm line,
the plate i s remved frcnn the chrmtographic
chamber and dried in a wrent of a i r u n t i l no
solvent odor is detected. The plate is examined
under W light a t 254 nm. To obtain maximum
sensitivity the plate is placed i n a chamber
containing iodine crystals for about t h i r t y t o forty
minutes o r u n t i l maximum contrast of the spots i s
obtained. The R f of c k t i d i n e is approximately
0.35.
Several additional thin layer chrmtography
systems have been reported by Kajfez, e t a l .
These systems separate the sulfoxide of cimetidine
f r m the free base. A l l work was carried out using
Merck-Kieselgel 60F254 plates. Visualization of
the spots W 254 and iodine absorption.
0
d:
0
0
'9
0
..
h
N
W
Y
!
A..
B
n
u
CIMETIDINE 171
5.5 20
High PressiJre Liquid Chrmatoqraphic Procedure
Adsorption and reverse phase systems have been used
t o evaluate cimetidine. The procedures are rapid,
have high s p e c i f i c i t y and are amenable t o the
analysis of cimetidine and c h t i d i n e formulations.
Table 9 contains the parameters of several c i t e d
investigations.
5.51 Analysis of Cimetidine
Mobile Solvent:
Mix 975 m l of a c e t o n i t r i l e , 20 ml of
d i s t i l l e d water, and 5 m l of concentrated
m n i u m hydroxide.
Assay Preparation:
Accurately weigh about 50 q of sample i n t o a
50 ml volumetric flask. Dissolve i n and
d i l u t e t o volume with mobile solvent.
Standard Preparation:
Accurately weigh about 50 nq of cimetidine
reference standard i n t o a 50 ml volumetric
flask. Add 25 m l of mobile solvent, shake f o r
one-half hour, d i l u t e to volume with mobile
solvent, and mix.
Procedure :
I n j e c t 20 p 1 of Assay and Standard Preparations
i n t o a l i q u i d chromatograph adjusted t o t h e
f o l l w i n g operating conditions:
Instrment: C h r m t r o n i x 3100
Column: Zorbax-Sil ( W o n t )
Column D i a m e t e r : 2.1 mn i . d . and 0.25 inch
0.d.
Column Length: 25 cm
Column Temperature: A m b i e n t
Column Pressure: 500 psig
Attenuation: 16
F l w Rate: 0.2 ml/minute
Detector: W (254 nm)
NOTE: Under the above operating conditions,
cimetidine has a retention time of about
172 P. M . G. BAVIN E T A L .
1 0 minutes.
Calculation :
C x AU x dilution factor x 100
%Cimetidine= As x Sample Weight (q)
Where: A u = area under the peak relating t o
the Assay Preparation
A s = area under the peak relating t o
the Standard Preparation
C = concentration of c h t i d i n e
reference standard in q/ml
5.52 Analysis of Tablets:
Wbile Solvent:
Mix 975 ml of acetonitrile, 20 m l of
d i s t i l l e d water, and 5 m l of concentrated
ammonium hydroxiae.
Assay Preparation :
Weigh and finely M e r twenty tablets.
Weigh accurately a portion of powder
(equivalent t o 80 mg of c k t i d i n e ) into a
50 ml volumetric flask. Add 25 m l of m b i l e
solvent, shake for one-half hour, d i l u t e t o
volume with m b i l e solvent, and m i x .
Transfer about 80 mg of c k t i d i n e reference
standard accurately weighed, into a 50 m l
v o l m t r i c flask. Add 25 ml of m b i l e solvent,
shake for one-half hour, and dilute to volume
with m b i l e solvent, and m i x .
Procedure:
Inject 20 1.11 of Assay and Standard Preparations
into a l i q i d c h r m t q r a p h adjusted t o the
following operating conditions:
Instrument: Qrrcanatranix Liquid Chranatcqaph
o r equivalent
Column: Zorbax-Sil (DuPont), o r equivalent
Column Diameter: 2.1 mn i.d. and 0.25 inch
0.d.
ColumnLength: 2 5 m
Column Temper ature: Ambient
CIMETIDINE I73
Colurrm Pressure: 1200 p s i

Attentuation: x 16
Flow Fate: 0.54 ml/minute
- Under the operating conditions,
NOTE:
cimetidine has a retention t i m e of
about 10 minutes.
Calculation:
q cimetidine/tablet =
C x Au x dilution factor x Av Tablet W t (q)
As x S q l e Weight (mg)
Where: Au = area under the peak relating t o
Assay Preparation
As = a-rea under the peak relating t o
the Standard Preparation
C = concentration of cimetidine
reference standard in mg/ml
5.53 Analysis of Capsules
Pbbile Phase (0.05M borax) :
Dissolve 39.1 g of Na2B,+07-10 H 2 0 in
d i s t i l l e d water and d i l u t e t o 2000 ml. Adjust
the pH t o 7.5 w i t h formic acid.
Internal Standard Solution:
Dissolve about 120 mg of benzoic acid
(ACS grade) in 100 m l of mobile phase.
Assay Preparation:
Transfer as ccanpletely as possible the
contents of not less than twenty capsules t o
a tared container, determine the average
content weight per capsule, and mix the
cmbined contents thoroughly. Transfer an
accurately weigh& portion of the pwder,
equivalent t o about 50 mg of c h t i d i n e , t o
a 25 ml volumetric flask. Add about 8 m l of
methanol, and shake mechanically for 10
minutes. Add 15.0 ml of Internal Standard
Solution, d i l u t e t o volurne w i t h methanol and
mix.
Table 9 HPIC Parameters For C i m e t i d i n e
COlUmn Mobile Phase F l w Rate W Detector 'LRT (min.) Reference
W/min. 1 Inm)
Lichrosorb Si 60 CH 3CN ,H 2 0 , CH 30H, 0.8 228 10.0 40
conc. NH40H (1000,

20,60,5)
Lichrosorb Si 60 0.1% ~ M L + O H / C H ~ ~ 40
0.8 228 40.0
Zorbax S i l 38
m3at a30H,H20, 3.0 228 3.4
coric. NH40H (1000,
500,20,2)
Zorbax S i l CH3CN,H20,conc. 0.5 228 10.0 37
NH4OH (975,20,5)
p Bondapak C18 CH3C", lorrfil potas- 2.0 220 4.0 39
sium phospnate
buffer (pH 3.0) (90,910)
p Bondapak C18 4 1
0.3% 2 m 4 , 1.0 254 16.5
CH3OH (450,550)
Partisil sex 0.9% ( M 4 ) 2m4, 1.0 228 3.5 40
a30H (200,800)
P a r t i s i l SAX 0.M Borax Buffer 42
0.75 254 2.8
(pH-8 .5 )
P a r t i s i l 1O-ODS CH3CN,H2O, NH40H 2.5 228 - 43
(1000,50,1)
CIMETIDINE 175
Transfer %50 mg of cimetidine reference
standard, accurately weighed, into a 25 m l
volumetric flask. Dissolve i n 8 ml of
methanol, add 15.0 ml of Internal Standard
Solution, dilute t o voluw with methanol,
and mix.
Procedure:
Using a loop injector, inject 20 $ of
Sample and Standard Preparation.
Instrumental Conditions:
The follming instrumental conditions are
typically employed when a Chranatronix Liquid
Chrmtograph is employed:
Instrument: Chrmatronix, p/lodel 3100
Column Packing: Strong Anion Exchange Resin
Column D i m t e r : 2.1 m (i.d.)
ColmLength: 1 mter
Column T a p e rature: Ambient
Colm Pressure: %500 p s i
Flaw Rate: d.40 ml/minute
Attentuation: x 64
Qlart Sped: 4 minutes/cm
Using the ahwe conditions, the peaks are
recorded in the order: cimetidine (approxi-
mately 4 minutes) and benzoic acid (approxi-
mately 8 minutes).
Calculation :
Calculate the ration 'R' for each chranatcqam
where :
h t i d i n e Peak Height
R=C
Benzoic Acid Peak Height
q cimetidine/capsule =
% x Wt of Std (9) x 1000 x Av N e t Contents (9)
% x W t of Sample (9)
176 P. M. G . BAVlN ET AL.
where: 5 = Ratio of Assay Preparation
% = Ratio of Standard Preparation
5.6 Determination of C i m e t i d i n e Sulfoxide Content

White21 used the previously cited procedure
(Section 5.5) to separate and estimate the m u n t of
the sulfoxide of cimetidine in the new drug
substance. With sample concentration of 1 m g / d
and an injection of 20 1.11,as l i t t l e as 1 ppn of
the 'sulfoxide' could be detected and estimated a t
the FT of about 13 minutes.
t
0 N-CTJ
+ 11
CH2SCH2CH2NHCNHCH3
m*N
'Sulfoxide '
white4 also detennined the presence of the
'sulfoxide' using a P a r t i s i l SCX column (25 cm x
4.6 m i.d.); Pbbile Phase: 80% CH30H and 20% H z 0
containing 1.8 g ( N H 4 ) 2 = 4 / 1 , a flaw r a t e of about
1.5 rnl/minute; Detection: W a t 228 m a d an
injection of 25 plof a 25 mg/ml solutian of sample.
6. Metabolism and Phamxokinetics
The m e t a b o l i s m and pharmacokinetics of cinetidine has
been studied and reviewed by several investigators.
The CcBnpOund is absorbed rapidly whether given orally
or i.v.. About 15-20% of cimetidine appears t o be
plasna protein bound and this is r e p r t e d t o be of no
pharmarological significance. In r a t s and dcgs,
c k t i d i n e is readily absorbed and has a plasna half-
l i f e of about one-half t o t m hours. In human studies,
cinetidine was Shawn to have a half-life of 123 f 1 2
minutes in the blood.23 Cimetidine has been found
f r m radioactive trace studies w i t h 2-1 4 C - c i m e t i d i n e
in healthy humans as w e l l as dogs and r a t s t o be
mainly excreted in the urine. It is widely distributed
throughout a l l the tissues and is rapidly eliminated
w i t h the exceptim of the liver, kidney and adrenal
cortex. TLC, HPLC , and radioautographic studies
indicated 56-85% of cimetidine (I) was unchanged, up
to 30% was excreted as the sulfoxide (11), 5-8% as the
&droxymz?thyl (111) ccmpund, approximately 2% as the
CIMETIDINE 171
guSnylurea (IV) and 7417% a s unidentified material. '-"

The r e p r t e d metabolism products are i l l u s t r a t e d in
Figure 15.
6.1 Determination of Cimetidine and its Metabolites i n
Biological Fluids
C h t i d i n e may be assayed i n blood, urine, plasna,
30,31,32,33,38
serum, b i l e , pancreatic fluid, gastric
fluid, plueral fluid, ceribrospinal fluid, a s c i t i c
fluid and body tissues by HPLC procedures? I The
major identified metabolite, cirnetidine sulfoxide'
which is eliminated f r m the M y mainly by renal
excretion has been determined in blood and
urine by Lee and O s b r n e using a high pressure
liquid chrcmtcqraphic procedure. Their procedure
allows the operator to cleanly separate creatinine,
which i s frequently present in c l i n i c a l samples of
patients suffering f r m renal failure and t o
determine both the c h t i d i n e a s w e l l as the
cimetidine sulfoxide.
The separation of cimetidine and its metabolites i s
usually carried out by extraction of the biological
medium with 1-octanol fran an aqueous alkaline
pH %9 solution followed by mixing, addition of an
internal standard and centrifugation. The
extraction with octanol i s repeated and the
combined extracts are re-extracted with dilute
hydrochloric acid. The aqueous acid solution is then
separated, ethanol is added and mixed. This is then
followed by saturating the mixture with a large
munt of potassium o r sodium c a r b n a t e to " s a l t
out" the ethanol layer which contains the cimetidine
and its metabolite, the sulfoxide. Several different
internal standards have been used: Metiamide,
1-nrtthyl-3- [ 2- [ [ ( 5 - ~ t h y l - ~ d a ~ o l e - 4 - y-rraethyl]
l)
thio] ethyl] -2-thiourea, (~-cyan~-~'-~~thyl-
N"- (3- (4-imidazolyl) -yopyl) guanidine and B-
hydroxy-theophylline. After extraction the samples
are either evaporated t o dryness and reconstituted
w i t h a hm m u n t of ethanol, injected directly o r
dissolved in the m b i l e phase for the HPLC analysis.
The c o l m s used for HPIC w e r 29,31,32,34 and
a reverse phase C18 B E?~ndapa@'? The separation
of cimetidine sulfoxidell and the guanidine deriva-
t i v e of cimetidine , N-methyl-N ' - [ 2- ( (4-methyl-5-
imidazolyl)methyl] thio) ethyl]guanidine , (VI) plus
the polar decmposition and metabolic products of
CIMETIDINE I79
cimetidine may be carried out using a P a r t i s i l SC@

(whatman Inc. ) strong ion exchanqe column using the
fcdlowing set of conditions : '
Column: P a r t i s i l 5cx
@ 1 0 p (25 cm x 4.6 rm i.d.)
Whabnan Inc.
While Phase: weigh 1.8 grams of ammnium sulfate
into a one liter volumetric flask.
Add 200 m l of d i s t i l l e d water t o
dissolve, then make up t o volume w i t h
methanol.
Flow Rate: %l ml/minute
Sensitivity: Variable
Sample Diluent: Mobile Phase
Retention Times:
Minutes
man01 (carboxamide) Derivative (IV) -10
SK&F 92422
Guanidine Derivative (VI) $15
SK&F92408
'Guanidine' Derivative of C h t i d i n e
VI
In the recent rwthcd of Z i M a k , e t a133 , a liquid
chrmatoqraphic procedure has been developed which
separates and permits quantitative analysis of
cimetidine ( I ) ,c k t i d i n e sulfoxide (11), its
h y d r o p t h y l 013 and guano1 urea derivatives (IV) .
Metimide, SK&F 92058, i s used as the internal
standard. Their procedure involves the precipitation
of protein with acetonitrile, addition of anhydrous
K2J3E04, extraction of the separated aqueous phase
w i t h mthylene chloride and KH2Po4 t o saturate and
salt out the solution. The methylene chloride is
evaporated t o dryness, the sample is reconstituted
with mobile phase (CE3CN:CH30H:H20:NH,+OH,
P. M. G . BAVIN ET AL..
1000:50:50:2). Detection is by W a t 228 nm and a

Zorbax S i l Column, 4.6 mn x 25 cm (DuPont Instru-
m t s , Wilmington, DE) equipped w i t h a Whatman HC
Pellosil p r e c o l m (ma- Inc., Clifton, N J ) is
used. An apparent pH of 10.5 has been used, and
according t o the authors column performance was
satisfactory for more than a five mnths period in
spite of the high alkalinity.
Identification of d e a m p s i t i o n products and
mtablic products is mst conveniently done by
their isolation using thin layer chramtoqraphy
and identification by use of f i e l d desorptim
mss spectrcanetry.
The authors muld like to achowledge and thank

minkers of SnithKline Corporation’s Analytical &
Physical C h b s t r y staff and other scientific
staff a t Philadelphia, Guayama , Welwyn, Tonbridge,
Ireland, and France for t h e i r assistance, advice,
recarmendations and their personal efforts. The
authors would further like t o thank SmithKline
Corporation for s u p p r t h g them t o carry out t h i s
mrk .
CIMETIDINE 181
7. References
1
Mitchell, R. C., J.C.S., Perkin 11, 915 (1980).
2
Kajfez, F., e t a l , Acta. Pharm. Yugoslav., 27, 199 (1977).
3 Warren, R. J . , smith Kline & French Labs., Phila., PA.
4
Pepper, E. S., Smith K l h e & French Labs., Ltd., Welwyn
Garden C i t y , England.
5
Darkin, D., Smith K l h e & French Labs., Ltd., Welwyn
Garden City, England.
6
Hadicki, E., e t a l , Chem. Ber. -
111, (9), 3222 (1978).
7
Prodic-Kojiz, B., et a l , Gaz. Chbn. I t a l . , -
109, 535
(1979).
8
Leonard, G., smith Kline & French Labs., Ltd., Welwyn
Garden C i t y , England.
9
Mitchell, R. C., Smith K l h e & French Lab., Ltd., Welwyn
Garden City, England
10
Baldinus, J., Smith Kline & French Labs., Phila., PA.
11 Schefter, E. and Higuchi, T., J. Pharm. S c i . , -
52, 781
(1963) .
12
Graham, M. J., Smith K l h e & French Ltd., Unpublished
results.
13 Durant, G. J., e t a l , J. W. Chem., 20, 901 (1977).
14 Smith K l h e & French Labs., German Patent 2,344,799
c.a. 80,1461681.
15
Z a r a t b , J. E., Smith K l i n e & French Labs., Phila., PA,
unpublished data.
16
Rosenberg, H. A., e t a l , Am. J. Hosp. Pharm., 37, 390
(1980).
17 Eqosh, P. and smith, M., Smith K l k e & French Labs.,

Phila., PA, unpublished data.
18 United States Pharmacopeia, XX, p. 727.
182 P. M. C. BAVIN ET AL
19 l3eqosh, P., Smith K l i n e and French Labs., Phila., PA,

m d l i s h e d data.
20
aid.
21 White, E. R., Smith K l i n e & French Labs., Phila., PA,
unpublished data.
22 Pedersen, P. V. arid Miller, R. J., Pharm. Sci., 69, 394
(1980).
23 Kinetics
G r i f f i t h s , R.; Lee, R. M., and Tavlor, D. C.,
of Cimetidine'in M k and &p=r&tal'Animals in Proc.
Sec. Int. Symp. of H i s t a m i n e H2-Receptor Antagonists
FCxerpta M i c a , Amsterdam4xford, p. 38 (1977).
24 Brimblecombe, R. W.; Duncan, W. A. M.; Durant, G. J.;
Bnnett, J. C.; Ganellin, G. R., and Parsons, M. E.,
J. Int. M e d . Res., 3, 86 (1975).
25
Taylor, D. C. and C r e s s w e l l , P. R., Biochem. Soc. T r a n s .
-
3, 884 (1975).
26 Jacobs, R. S. and Catania, H., Drug. I n t e l l . C l h .
Pharmac., 11, 723 (1977).
27
Taylor, D. C.; Cresswell, P. R., and Bartlett, D. C . ,
D r u g . %tab. D i s p . , 6, 21 (1978).
28
Burland, W. L.; Duncan, W. A. M.; HesselbO, T . ;
Mills, J. G., and Sharpe, P. C., Br. J. C l b . Pharmac,,
-
2, 481 (1975).
29 Soldin, S. J., et a l , Therap. D r u g Monitoring, I,371
(1979)
30 Larsen, N. E., et a l , J. Chromatog. ( B i d . Applic.),
163, 57 (1979).
31 m d o l p h , W. C.; O s b o m e , V. L.; Walkensteb, S. S.,
and Intoccia, J., Pharm. Sci., - 66, 1148 (1977).
32 Ice, R. M. and Osborne, P. M., J. QvQMtog., 146,
354 (1978).
33 Ziermnak, J. A.; Chiannmte, D. A., and Schentog, J. J.,
Clin. Chem. -
27, 272 (1981).
34
Chianmnte, D. A. and Schentaq, J. J., Therap. Drug
plbnitoring, I,
545 (1979).
DISOPYRAMIDE PHOSPHATE
Alan Wickman and Patricia Finnegan
G. D. Seurle and Co.
Skokie, Illinois
I . Description 184
1. I Name. Formula, Molecular weight i84
1.2 Color, Odor, Appearance 184
2. Synthesis 184
3. Physical Characteristics 186
3.1 Infrared Spectrum 186
3.2 Proton Magnetic Resonance Spectrum 188
3.3 Carbon Magnetic Resonance Spectrum 190
3.4 Ultraviolet Spectrum 193
3.5 Thermal Analysis 194
3.6 Mass Spectrum 194
3.7 Solubility 194
5 . Methods of Chemical and Dosage Form Analysis 199
5.1 Titrimetric Analysis 199
5.2 Spectrophotometric Analysis 199
5.3 Chromatographic Analyses 199
6. Methods of Analyses in Biological Fluids 204
6.2 Spectrofluorometric Analysis 204
6.3 Chromatographic Analyses 205
References 208

ISBN 0-12-260813-5
184 ALAN WICKMAN AND PATRICIA FINNEGAN
1. Description
1.1. Disopyrami.de Phosphate C21H29N30'H3P04
Mol, Wt. 437.5
0I t
a-[2-(Diisopyropylamino~ethyll-a-phenyl-
2-pyridineacetamide phosphate (1:1)
1.2. Color, Odor, Appearance
Disopyramide phosphate is an odorless
white, or slightly off-white free
flowing powder.
2. Synthesis
Nitrilopyramine is heated with concentrated
sulfuric acid for 4 hours on a steam bath, the
mixture is poured into ice after which it is
made alkaline with 10 normal sodium hydroxide.
The pH of the solution is then adjusted to 6
with acetic acid and the solution is washed
with toluene. The mixture is again made alka-
line with 10 normal sodium hydroxide and ex-
tracted with toluene, The toluene is evapora-
ted and the residue dissolved in ethanol and
treated with activated carbon. The ethanol is
then evaporated and the residue recrystallized
from hexane to give disopyramide. The phos-
phoric acid salt of disopyramide is prepared
by reacting disopyramide with a phosphoric
acid solution.' The synthesis pathway is
illustrated in Figure 1.
DISOPYRAMIDE PHOSPHATE I85
Nitrilopyramine
oFNH
1 CH,N CH{CH,} 2
2
D i sopy r am ide
Disopyramide Phosphate
Figure 1 - Synthesis of Disopyramide Phosphate

I86 ALAN WICKMAN AND PATRICIA FINNEGAN
3. Physical Characteristics
3.1. Infrared Spectrum
The infrared spectrum of disopyramide
phosphate in a Nujol and Fluorolube
split mull is shown in Figure 2.' Band
assignments are summarized below.
(cm-l) Assisnment
3480, 3290 amide N-H stretch
23 00, broad N+-H stretch
1678, 1640 amide C=O stretch
and NH2 deformation
1590, 1560 benzene and pyridine

1480, 1460 ring vibrations
13 95 CH -N+ methylene
dezormation
1263 C-N+ stretch
1065, 940 H2POa stretches
760 4 adjacent H wag

(a - substituted
pyr idine 1
7 4 0 , 695 5 adjacent H wag
(mono-substituted
benzene1
C
Y
c
N
n c
Y c
9
n
C
-
U c
c
c
c
D:
C
c
(I
0
c
0
e
u) 0
c
m
e
W
E C
E U
P
;
0
c
m
d
C c
c
c
N
c c
c
n
Y
c
c
c
c
e
3
3
0
m LD
D *
0 N
0 0
f /.*? NOISSIWSNML
188 ALAN WICKMAN AND PATRlClA FINNEGAN
3.2. Proton Magnetic Resonance Spectrum

The PMR spectrum of disopyramide phos-
phate in deuterated water is shown in
Figure 3.* Shifts are reported in pprn
downfield from the methyl signal of 3-
trimethylsilyl propanoic acid-d sodium
salt which was used as an interial ref-
erence. Band assignments are summarized
below.
0
I I
d 2 "CHC
Assiunment
a
2.93 singlet b
3.67 septet C
7.10 - 7.55 multiplet d

7.88 triplet of
doublets e
8.57 doublet of
doublets f
aJ
c,
(d
.c
a
m
0 0
c
pc
a,
-
a a
-4
5k
h
0 a
N
0
m
.d
a
a
D
w
0
5
si
3
9 u
u
a,
a
ZI]
3
"p a
u
0,
.5 d
(d
c
D
0
0
(I)
a,
p:
a u
c v i
c,
a,
I:
tr
D
ID
c
0
u
3
m 0
k
PI
I
3
3 m
a,
$4
3
D
.4
L4
3.3. Carbon Magnetic Resonance Spectrum

Noise and single frequency off-resonance
decoupled CMR spectra of disopyramide
phosphate in deuterated water solution
are shown in Figures 4 and 5.2 Shifts
are reported in ppm downfield from the
shift of tetramethyl silane as refer-
enced to a shift of 67.4 ppm for dioxane
which was used as an internal reference.
Band assignments are given below.
0
H H,PO,
2
18.7, 17.1 a
35.4 b
45.2 C
55.8 d
62.7 e
125.4, 124.5 f
130.0, 129.2 9
139.4 h
141.8 1
149.6 j
160.6 k
178.2 1
3
3
n
3
3
4
E
PI
3
n
-I
3
3
v
3
n
u
3
3
n
3
3
-4
E
P4
5
n
-4
3
3
u
3
n
u
DISOPYRAMIDE PHOSPHATE 193
1.0
0.9
0.8
0.7
0.6
w
U
2
m
0.5
a
$ 0.4
m
a:
0.3
0.2
0.1
210 230 250 270 290 310 330 350 370
WAVELENGTH (nm)
Fig. 6. Ultraviolet spectrum of disopyramide

phosphate.
3.5. Thermal A n a l y s i s
T h e DSC thermogram of disopyramide phos-

p h a t e o b t a i n e d u s i n g a h e a t i n g r a t e of
10°C/minute i s shown i n F i g u r e 7.2 The
endothermic change of 1 3 8 J / g a t a b o u t
213OC i s due t o m e l t i n g and, a s can be
s e e n by t h e TG i n F i g u r e 8 , 2 i s accompa-
n i e d by weight l o s s .
T h e TG was o b t a i n e d u s i n g a h e a t i n g r a t e
of 20°C/minute. The maximum r a t e of
w e i g h t l o s s i s a t 236OC. The t o t a l
weight l o s s i s 82.4% of t h e sample
weight.
3.6. Mass Spectrum

A chemical i o n i z a t i o n mass spectrum of
disopyramide phosphate c o n t a i n s a b a s e
peak a t M/z = 340 which i s due t o t h e
p r o t o n a t e d f r e e base of disopyramide
phosphate. No peaks g r e a t e r t h a n 1 0 % of
t h e base peak a r e o b s e r v e d below M/z =
340. No peak due t o t h e s a l t i s p r e -
sent. *
3.7. Solubility
The s o l u b i l i t y of disopyramide phosphate
i n v a r i o u s o r g a n i c s o l v e n t s i s summa-
r i z e d i n Table 1 belowO3
Table 1
S o l u b i l i t y i n Organic S o l v e n t s *
Solvent s, 01 u b j J i . Q (a/J.L
Ethanol 1.089
Isopropanol 0.103
Chloroform 1.413 x 1 0 -2
Cyclohexane 6.524 x lo-)
* S o l u b i l i t y d e t e r m i n e d a t 24 - 26°C
The solubility of disopyramide phosphate

free base in various aqueous media is
summarized in Table 2 below.3
Table 2
Solubility in Various Aqueous Media**
Aqueous Molar i ty pH*** Solubility

Medig .o 0
Acetate 0.1 3 -93 62.0
Phosphate 0.1 5.85 32.0
Phosphate 0.16 7.45 8.71
Phosphate 0.1 7.98 4.32
Glyce r ine 0.1 9.86 3.78
0.1 N NaOH 0.1 12.7 1.29
** Solubility at 27OC
*** Represents pH of Aqueous Media prior to
Addition of Disopyramide
4. Metabolism and Pharmacokinetics

After oral administration of 100 mg of the
drug to healthy men and w ~ m e n ~ ,peak
~ , plasma
levels of 2.4 ug/ml were obtained 2 hours af-
ter administration. The drug had an absorp-
tion rate of 1.35 hours-’. The drug was eli-
minated with a biological half life of 7
hours. Twenty four hours after administra-
tion, plasma levels were. low but urinary ex-
cretion was still appreciable. An average of
60% of the oral dose was recovered unchanged
in the urine within 48 hours, with 50% being
recovered within 24 hours of administration.
After oral administration of the drug to dogs
and rats, the biological half life, after peak
plasma levels were obtained, is 1.5 hours and
1.4 hours respectively. Urinary excretion
after 24 hours was much lower for dogs (20%)
and rats (35%) than for man. It is assumed
that the rapid disappearance of disopyramide
from the blood of both dog and rat is due to
both urinary excretion and formation of meta-
bolites which undergo biliary excretion.
Karim et a16 determined that the main pathway
of disopyramide metabolism involved N-dealky-
lation of the isopropyl group and arylhydroxy-
lation, with a marked species difference in
biotransformation between dog, rat, and man.
Oral administration of I b C labeled disopyra-
mide to dogs yielded 79% of radioactive com-
pound in the urine after 72 hours. Of this
17% was the unchanged compound, 12% the mono-
N-dealkylated species 2, 29% the pyrrolidone
species 4, and 18% a water soluble conjugate
that gave the pyrrolidone upon acid hydroly-
sis. Figure 9 is the proposed metabolic path-
way for disopyramide in dogs. (See Figure 9)
Intraperitineal administration of “C labeled
disopyramide to rats yielded 44% of the radio-
active compound in the urine after 72 hours.
Of this, 80% was the unchanged disopyramide 1.
The minor metabolic products recovered were
two phenolic compounds 5 and 6 arising from
arylhydroxylation. (See Figure 10.1
Oral administration of disopyramide 1 to man

yielded 56% of disopyramide and 4 % of the
mono-N-dealkylated species 2 in the urine
after 24 hours (refer to Figure 9).
5. Methods of Chemical and Dosage Form Analysis
5.1. Titrimetric Analysis
Accurately weigh a portion of the diso-
pyramide phosphate and add it to 50 mL
of glacial acetic acid in a 250 mL
Erlenmeyer flask. Titrate with 0.1 N
perchloric acid to a potentiometric end-
point. A blank determination is per-
formed and any necessary corrections
made. Each mL of 0.1 N perchloric acid
is equivalent to 21.87 mg of disopyra-
mide phosphate.’
5.2. Spectrophotometric Analysis
The analysis of disopyramide phosphate
may be performed by W spectrophotomet-
ric analysis employing 0.1 N sulfuric
acid in absolute methanol as solvent.
The ultraviolet absorption maximum is at
about 268 nm.O
5.3. Chromatographic Analyses
5.3.1. Thin Layer Chromatography
Several TLC systems are available for
the analysis of disopyramide phos-
phate.s Solvent, adsorbent, and de-
tection parameters are summarized in
Table 3.
0 0
1
c
0 0 1
-
I -1
IH
Of Q L
Figure 9 - Proposed Metabolic Pathway

of Disopyramide in Dogs
DlSOPYRAMIDE PHOSPHATE 20 1
HO
Figure 10 - Proposed Metabolic Pathway

of Disopyramide in Rats
202 ALAN WICKMAN A N D PATRICIA FINNEGAN
Table 3
~~ ~
Solvent System Adsorbent Detection

~~~ ~
Rf
Benzene:ethanol Silica Gel 1,2 0.39
2B:ammonium 0.25 mm
hydroxide 170:
28:2 (v/v/vi
To1uene:ethanol Silica Gel 1 .34
2B :ammonium 0.25 mm
hydroxide 170:
28:2 (v/v/v)
Methylene chlo- Silica Gel 2 0.65
ride:ethanol 0.25 mm
2B:ammonium
hydroxide 70:
28:2 (v/v/v)
Detection Systems
1. Spray with Dragendorff Reagent
2. Color test the chromatographic plate
by exposure to excess t-butyl hypo-
chlorite. Evaporate the excess rea-
gent (20 minutes in fume hood) and
then spray the plate with 0.5% (w/v)
potassium iodide and 0.5% (w/v) starch
in water.
5.3.2. High Performance Liquid Chromatography
Disopyramide phosphate may be chro-
matographed under the following HPLC
conditions.10
System 1
Column: u-Bondapak C18 (30 cm
x 4 . 0 mm i.d.1
Mobile Phase: 70% triethyl ammonium
phosphate buffer/30%
methanol (v/v)
Flow Rate: 2 mL/min
Detection: 254 nm
Temperature: Ambient
Retention Time: Approximately 6
minutes
System 2
Column: DuPont Zorbax C8 (15
cm x 4.6 mm i.d.1
Mobile Phase: 65% TEAP Buffer pH
3.7/35% MeOH
Flow Rate: 2 mL/min
Temperatur e : Ambient
Detection: 254 nm
Retention Time: Approximately 5
minutes
5.3.3. Gas Liquid Chromatography
Disopyramide phosphate can be chroma-
tographed as the free base. The free
base is obtained by dissolving diso-
pyramide phosphate chemical or capsule
in an aqueous base medium and extract-
ing into an organic solvent such as
chloroform, ethyl ether, methylene
chloride, etc.. Disopyramide is chro-
matographed under the following condi-
tionsl l.
System 1
Co1umn : 3% OV-1 on Gas Chrom Q,
80/100 mesh (1.8 m x 2
mm i.d., glass column)
Oven Temp.: 21 o o c
Carrier: Helium
Flow: 50 mL/min
Detection: Flame Ionization

System 2
Column: 3% Silar 1 O C on Gas
Chrom Q 1 0 0 / 1 2 0 (1.8 m
x 2 mm i.d., glass
column)
Oven Temp.: 25OOC
Carrier: Helium
Flow Rate: 50 mL/min
Detection: Flame Ionization
6. Methods of Analyses in Biological Fluids
6.1. Spectrophotometric Analysis
A portion of the plasma/serum is added
to 0.5 M phosphate buffer pH 7.5 and
extracted with methylene chloride. The
organic portion is washed with potassium
hydroxide and then extracted with 0.1 N
sulfuric acid. The ultraviolet absorp-
tion of the aqueous extract is measured
at about 2 6 0 nm to quantitate the diso-
pyramide12.
6.2 Spectrofluorometric Analysis
A portion of the sample fluid is added
to sodium hydroxide solution and ex-
tracted with methylene chloride. The
methylene chloride solution is then
extracted with 50% sulfuric acid.
To quantitate disopyramide, the fluores-
cence of the sulfuric acid extract is
determined at hemission = 410 nm,
3c excitation = 275 nm5.
DISOPYRAMIDE PHOSPHATE 20s
6.3. Chromatographic Analyses

6.3.1. Gas Liquid Chromatography
Many investigators have used gas chro-
matography to analyze disopyramide
l 3 - 2 0 , and to a lesser extent diso-
pyramide and its mono-N-dealkylated
metabolite * l - 2 3 , in biological
fluids.
The majority of the methods involve
basifying the sample fluid and ex-
tracting the free base into an organic
medium. This organic medium may then
be evaporated and the sample reconsti-
tuted in ethanol or another solvent,
or the sample can be injected directly
into the chromatograph. Some of the
methods described use a double extrac-
tion where the organic medium contain-
ing disopyramide is extracted with
acid solution, which is then made
basic and again extracted with organic
solvent. Many of the methods also
employ an internal standard for quan-
titation. Internal standards commonly
used are p-chlorodisopyramide, and
aminopentamide. Table 4 gives a sum-
mary of the gas chromatographic condi-
tions employed.
Table 4
Column Column Detector Carrier Plow Ref.

Temp.
~
1.58 BE30 on Gas 230.C FID 30 mL/mln N2 13

Chrom Q (1.8 m x
2 mm, g l a s s )
38 OV-17 on Gas 255.C Nitrogen 20 mL/min N2 14
c h r w Q 100/200 mesh
(1 m x 2 mm, g l a s s )
38 OV-101 on Gas 23 O*C PID 30 mL/min 2' 15
Chrom Q 80/100 mesh
(1.8 I x 6 m e g l a s s )
38 OV-1 on Supel- 240.C PI0 4 0 mL/min N2 16
c o p o r t 80/100 mesh
(1.5 D X 4
silanised glass)
28 SP2250, 2) OV-101 230.C Nitrogen 30 mL/min 2' 17
on Ehromosorb w-BP
100/120 mesh (1.2 D
x 2 mm, g l a s s )
38 ocw 98 on as 210-C PID 100 mL/min 2' 18
Chrom Q 100/120 mesh
(1.7 m x 2.6 mm, g l a s s )
38 OV-1 Gas Chrom Q 200.C Nitrogen 40 a h i n N2 19
100/100 mesh ( 2 m x
2 mm, g l a s e )
38 OV-17 on Gas 260.C PID 30 W m l n 2
' 20
Chrol Q 100/120 mesh
( 6 0 cm x 2 m, g l a s s )
38 OV-17 on Chrooo- 250.C PID 2 1 pLL/min 2

' 21
s o r b W 100/120 mesh
(1 x 2 mm, g l a s s )
38 OV-17 on Gas 245.C Nitrogen 25 a h i n 22
Chrom Q 100/120 mesh
(60 cm x 2 mm, g l a s s )
2.68 OV-17 on Chromo- 21 0-3 PI0 60 W m f n Be 23
sorb W-BP 80/100 mesh 230.C
(.6 D x 2 me g l a s s )
6.3.2. High Performance Liquid Chromatography

Several HPLC methods have been report-
ed that can be used to analyze disopy-
ramide phosphate a l o n e Z b - 2 8 or in
combination with its mono-N-dealkylat-
ed metabolitez9 - 3 2 . Sample prepara-
tion is similar to that for gas chro-
matography. For most methods the sam-
DISOPY RAM1 DE PHOSPHATE 207
Table 5
5 oicron ketonitri1e:potasrium 254 --- 24

ODs-18 phosphate (monobasic)
buffer
10 b ODS-18 1t Acetic acid:methanol: 254 1 25
(25 cm x triethylamine (34.5145:
3 nun i.d.1 .5)
pBondapak Ilethano1:heranczsulfonic 254 1 26
Cl8 (30 cm acid (pE 4-51 (60r401
x 4 QIP i.d.1
Lichrosorb Dich1orocthane:methanolr 26 5 1 21
S160 (15 cm percbloric acid (95.7:41
x 4 mm i.d.1 .3)
pBondapak - 0 5 I4 Potassium phosphate 258 2 28
C18 (30 cm (debasic1:acetonitrile
a 4 mm i.d.1 (65:35)
pBondapak Acetonitriler.01 H sodium 254 1.2 29

CN (30 cm acetate tpH 4 ) (50:50)
x 4 mm i.d.1
PBondapa k .06 H Sodium acetate, 4.78 254 1.9 30
CN 125 cm acetic acid CpR 3.5):
x 5 mm i.d.1 methanol (85:15)
ODs C18 Hethanolrwater with 254 1 31
(25 cm x .005 H heptanesulfonic
5 am i.d.l acid ( 5 3 ~ 4 7 1
Lichrosorb 0.5 H sodium phosphate% 254 1.8 32
RP-8 10 acetonitrile (73x27)
micron (25
cm x 4.6 nm
i.d.1
ple fluid is basified and extracted

into an organic medium. The medium is
then evaporated and the sample recon-
stituted in mobile phase solution or
other organic medium. Table 5 gives a
summary of different HPLC Systems.
Acknowledgments
The authors wish to thank Dr. J. Witt, Dr. R.
Bible, Dr. A. Karim, Dr. J. Hribar, C. Seul,
and E. Hajdu for their contributions and
suggestions and to A. Fenton for her
secretarial assistance in preparing this
manuscript.
References
1. U.S. Patent 3,225,054 C.A. 58, 12522 (1963)
2. Physical Methodology Group - G. D. Searle &
Co. - Internal Communication
3. Y. W. Chien - G . D. Searle & Co. - Internal
Communication
4. Norpace/Investigational Brochure - G. D.
Searle & Co.
5. Ranney, R. E., Dean, R. R., Karim, A,,
Radzialowski, F. M., Arch Int. Pharmacodyn
191 (1971) 162-188
6. Karim, A., RanneyI R. E., Kraychy, S., J
Pharm Sci 61 No. 6 (1972) pp. 888-893
7. USP xx
8. G. D. Searle & Co. - Internal Communication
9. C. Wohlt, J. Drury, C. Murphy - G. D. Searle
& Co. - Internal Communication
10. G. D. Searle b Co. - Internal Communication

11. G. D. Searle & Co. - Internal Communication
12. Martin, D., Burke, L., Norden, W., Chen, S.,
Clinical Chemistry 24 No. 6 (19781 991
13. Daniel, J. W., Sobramanian, G., J Inst. Med.
Res. 4 , Supp (1) (1976) 2
14. Duchateau, A.* Merkus, F., J Chromatog 109
(1975) 432-5
15. Johnson, A., McHaffie, D., J. Chromatog 152
(19781 501-506
16. Hayler, A . * Flanagan, R.? J Chromatog 153
(1978) 461-471
17. Vasiliades, J., Owens, C., Pirkle, D., Clin
Chem 25 (1979) 311-313
18. Foster, E. , Reid, P. , J Chromatog 178 (1979)

571-574
19. Gai, J., Brady, J., Kett, J., J Anal Toxic01
4 (1980) 15-19
20. Doedens, D., Forney, R., J Chromatog 161
(1978) 337-339
21. Aitio, M. L., J Chromatog 164 (1979) 515-520
22. Bredesen, J., Clin Chem 26 (1980) 638-640
23. Hutsell, T., Stachelski, S., J Chromatog 106
(1975) 151-158
24. Clark, C., Lankford, G., Am Chem SOC Abstr
176 Anal 57 (1978)
25. Broussard, L., Frings, S., Clin Chem 24
(1978) 1007
26. Draper, P., Shapcott, D., Leonieux, B., Clin
Biochem 12 11979) 52-55
27. Lagerstrom, P. O., Perpson, B. A., J
Chromatog 149 (1978) 331-40
28. Ilett, K., Hackett, L., Dusci, L.,
Jokrosetio, R., J Chromatog 154 (1978) 325-29
29. Nygard, G., Shelver, W., Wahba Khalil, S., J
Pharm Sci 68 (1979) 1318-20
30. Lima, J., Clin Chem 25 (1979) 405-8
31. Meffin, P., Harapet, S., Harrison, D., J
Chromatog 132 (1977) 503-11
32. Ahokas, J., Davies, C., Ravenscroft, P. J., J
Chromatog 183 (1980) 65-71
INDOMETHACIN
Matthew O’Brien
Merck Sharp 6 Dolime Research Laboratories
West Point, Pennsylcania
James McCauley
Merck Sliarp 6 D o h e Research Laboratories
Rahway, New Jersey
Edward Cohen
Merck Sharp 6 Dolinie Research Lahoratories
West Point, Pennsylcania
1. Introduction 212
1.1 Historical 212
I .2 Name, Formula, Molecular Weight 212
1.3 Definition 213
I .4 Appearance, Color, Odor 213
2. I Ultraviolet Absorbance 213
2.3 Nuclear Magnetic Resonance 214
2.4 Crystal Properties 220
2.5 Infrared Absorbance 222
2.6 Thermal Behavior 222
2.7 Solubility 226
2.8 Dissociation Constant 221
2.9 Partition Behavior 221
3. Synthesis 227
4. Stability 228
5.3 Fluorescence Analysis 229
5.4 Polarographic Analysis 230
5.5 Mass-Transport Techniques 230
5.6 Titrimetric Analysis 234
References 235
Copyright 0 1984
ANALYTICAL PROFILES OF DRUG SUBSTANCES
VOLUME 13
21 1 by the American Pharmaceutical Association
ISBN 0-12-260813-5
212 MATTHEW O'BRIEN ET AL
1. Introduction
1.1 Historical
Indomethacin is a non-steroidal, anti-inflamatory
agent with anti-pyretic and analgesic properties discovered
and developed by the Merck Sharp and Dohme Research
Laboratories(1).
Indomethacin has been used effectively in the management of
patients with moderate to severe rheumatoid arthritis, ankylosing
spondylitis, osteoarthritis, acute painful shoulder (bursitis
and/or tendinitis) and acute gouty arthritis(2-6). Recently,
indomethacin has been found effective in the treatment of
neonates with patent ductus arteriosus and in patients with
acute cystoid macular edema follcwing cataract surgery(7-9).
Worldwide, indomethacin has been formulated into many dosage
forms, including formulations designed for long duration of
activity. The discovery of this compound continues to provide
new insights into medical treatment of disabling diseases(l0).
1.2 Name, Formla, Molecular Weight
Indomethacin (I) is l-(p-chlsrobenzoyl)-5-methoxy-2-

mthylindole-3-acetic acid. Other names for the compound
include (l-p-chlorobenzoyl-5-methoxy-2-methylindol-3-yl)
acetic acid(l0). Proprietary names for indomethacin include
AMUNO INDACID, INDACIN, INDOCIN, INDOCIN-SR, INCKIN-IV,
INEOMEE, INDOPTIC, METINDOL, and MEZOLIN. Additional
chemical and proprietary names are listed in the monograph
for indomethacin in the Merck Index(l1).
CH,O CHzCOOH
Official monographs for indomethacin are given in U.S.P. XX

B.P. 1980 , European Ph.1980 ,and the Nord. Ph.1969 ,
(12-15)
INDOMETHACIN 213
1.3 Definition
Indomethacin is defined as the Form I crystalline, non-
solvated free acid moiety of the compound unless otherwise
noted. Indomethacin as described below can exist as several
crystalline forms but has been used in pharmaceutical prep-
arations principally as Form I and less frequently as the
crystalline sodium trihydrate.
1.4 Appearance, Color, Odor
Pale yellcw to yellow-tan, crystalline powder that is
odorless or almost odorless.
2.1 Ultraviolet Absorbance

Indomethacin was first characterized by Shen --
et al. as
having ultraviolet absorbance maxima at 319 and 230 nm with
an inflection at 260 nm in ethanol. The corresponding
values reported were 6290, 20800 and 16200,
respectively(1).
U.S.P.XX lists a W absorbance maximum at 318 nm

in methanolic 0.1N hydrochloric acid. Figure 1 shows
an ultraviolet absorption spectrum of indomethacin (Merck
Standard 6375-66-1) in methanolic 0.1N HC1 at a concentration
of 1.429mg/100 ml. Absorbance maximum is at 318 with E l % l c m
value of 182 ( =6510).
2.2 Mass Spectrum
The mass spectrum of indomethacin obtained on an LKB-
5000 at 70 eV ionization energy is found in Figure 2.
Important Features of the spectrum include:
(1) Molecular ion peak at 357 (m/e).

(2) Peak at m/e 312 ( M . W . 4 5 ) attributed to loss of C02H.
(3) The most intense peak of the spectrum occurs at m/e 139
which corresponds to p-chlorobenzoyl(16).
2 I4 MATTHEW O’BRlEN ET AL.
2.3 Nuclear Magnetic Resonance

2.3.1 The proton NMR spectrum of indomethacin in deuterated
dimethylsulfoxide (d6 DMSO) at a concentration of
20% (w/v) is reproduced in Figure 3. A tabulation of
the assignments and chemical shifts is found in Table
I( 17).
The carboxylic proton is not visible in the above
spectrum due to exchange broadening involving the
water in the solvent.
2.3.2 Carbon 13 Spectrum
The I3C magnetic resonance spectrum is given in
Figure 4 and was obtained using a Varian CFT-20
(FT mode) spectrometer with d DMSO as the solvent at
a concentration of 10% w/v. $he spectrum is
consistent with the structure and the assignments are
in Table I1 (17).
CH
INDOMETHACIN 215
Wavelength (nm 1
F i g u r e 1. U l t r a v i o l e t A b s o r p t i o n Spectrum of Indomethacin
i n M e t h a n o l i c H y d r o c h l o r i c Acid a t 0.00143% C o n c e n t r a t i o n ;
max @ 318 nm; E l % l c m = 182. Merck S t a n d a r d 6375-66-1
A
h
M
u
a,
8
C
0
.rl
u
rd
N
-4
C
0
*rl
3
a,
0
b
I
0
0
0
In
INDOMETH ACIN 217
Table I. Proton NMR Spectrum of Indomethacin

Merck Standard 6375-66-1
Solvent: d6 DMSO; Conc: 20% w/v; Inst.: JEOL C-60 Hz
Chemical. Shift Integral Rel. No.

(PIa (m) Proton Assigmnt
6.55-7.10
7.45-7.80
(m)
(m)
44 1/2
61 1/2
[2*97I
[4.11I c1 w-
H q r H6 and H7
H H
a - TMS used as internal reference.
Table 11. Carbon 1 3 Chemical Shifts for Indomethacin
Shift d 3 C
(PP) Assignment*
172.0
167.8
155.5
137.6
135.1
134.1
1.31.1
130.7
130.2
129.0
114.5
113.4
111.2
101.7
55.3
29.5
13.1
I I I I I 1 I I
5ppm Offset
f
4
1 AMPL. 2.5 x I 0
L
I I I I I I I I
8.0 7.0 6.0 5.0 4.0 3.0 2.0 i.0 0
Chemical Shift (ppm)

F i g u r e 3. P r o t o n N4R Spectrum of I n d o m e t h a c i n i n d -DMSO.
6
Merck Standard 6375-66-1
200 100 0
Chemical Shift 6 13C (ppm)
Figure 4 . 13C NMR Spectrum of Indomethacin. Merck Standard 6375-66-1.

2.4 Crystal Properties
Tndomethacin is known to exist in more than one

nonsolvated crystalline modification. Yamamto described
three polynorphs oc,\B and 7with melting point, TR and x-ray
powder diffraction data(l8). Monkhouse and Lach reported
two modifications characterized by melting point and IR(19).
Borka found four and reported hot-stage microscopic melting
point, IR and solubility data (solubility on only
three)(20). Others have also observed four crystalline
polymrphs(21). While there is disagreement as to the
existence of some of the polymrphs, all agree on two of the
crystalline modifications. There are consistent melting
point, IR and x-ray powder diffraction data for these two
modifications. Most authors refer to these polymorphs as
Form I and Form 11. Yamamoto, however, uses 7-type and
a-type, respectively. Form I is the highest melting (see
2.6 k l w ) and lowest- solubility (see 2.7 below) polymorph
and is, therefore, the thermodynamically stable crystalline
modification of indomthacin. However, from a practical
view, both Form I and I1 are equally biologically available
and active (22).
The x-ray powder diffraction data for Forms I and I1 of
indomethacin are found in Table 11. The d-spacings and
relative intensities for Form I are consistent with those
cited in the National Formulary X I V (1975) (13).
Kistenmacher and Marsh determined the crystal and
molecular structure of indomethacin by single crystal x-ray
diffraction methods(23). Although not explicitly stated by
Kistemcher and Marsh, the crystals grown from anhydrous
acetonitrile are most likely Form I. They reported that
the crystals are triclinic, space group P1 with cell
constants a = 9.295(2)A, b = 10.979(1)A, C = 9.742(1)A,
a=69.38(1)', g3 = 110.79(1)',
3
V = 9 .78(1 and 2 = 2.
The calculated density i3 1.37 gm/cm ; the observed
density is 1.38(1) gm/cm .
In addition to the polymorphism described above,
indomethacin is known to form solvates with benzene and t-
butyl alcohol(21). Borka has also observed solvates with a
number of different solvents(20).
INDOMETHACIN 22 I
Table I11
X-Ray Powder Diffraction of Indomethacin Forms I and I1

Sample Merck Standard
Cu & Radiation
Form I Form I1
Rel. Re1 .
2 ('1 Intensity 2 ('1 d(g) Intens ity
10.18 8.69 22 4.85 18.22 14

11.60 7.63 100 6.89 12.83 43
12.75 6.94 15 8.80 10.05 100
16.65 5.32 54 10.20 8.67 36
17 .OO 5.21 89 11.35 7.80 32
17.30 5.13 25 11.95 7.46 77
18.55 4.78 19 13.85 6.39 34
19.30 4.60 36 14.19 6.24 80
19.60 4.52 60 14.45 6.13 79
20.30 4.37 10 14.70 6.03 30
20.90 4.25 15 14.90 5.95 30
21.80 4.08 89 16.05 5.52 31
22.90 3.88 17 17.50 5.07 45
23.15 3.84 15 18.00 4.93 50
24.00 3.71 22 18.90 4.70 55
25.50 3.49 13 19.00 4.67 16
26.62 3.35 42 19.70 4.51 54
27.50 3.24 15 20.15 4.41 39
28.3 3.15 11 20.60 4.31 46
28.9 3.09 23 21.10 4.21 29
29.35 3.04 31 22.02 4.04 77
30.40 2.94 14 22.60 3.93 68
33.60 2.67 10 23.35 3.81 52
34.15 2.63 11 24.01 3.71 39
34.75 2.58 8 24 .50 3.63 57
37.50 2.40 13 24.90 3.58 23
25.28 3.52 38
26.25 3.40 36
27.20 3.28 14
28.35 3.15 39
3 1 .O 2.89 25
222 MATTHEW O'BRIEN ET A L
2.5 Infrared Absorbance

The infrared absorption spectrum for Form I of
indomethacin in KEr is given by Hayden --
et al. and
Sadtler(24,25). Monkhouse and Lach, and Borka, in addition
to IR spectrum for Form I, give IR spectra for the other
crystalline modifications of indomethacin that they
observed(l9,20).
The IR spectrum which is consistent with the literature
for Form I of indomethacin is found in Figure 5. Some band
assignments for the solid state infrared absorption spectrum
are given belw(26).
Wavelength (an-')
*3400-2500 Aromtic C-H stretch Carboxylic acid
0-H stretch
1715, 1695 C=O stretch
1600 Aromatic C=C stretch
1450 0-CH3 deformation
1230 ? (C-0) stretch plus 0-H deformation
925 Carboxylic 0-H out of plane deformation
900-600 Various C-H out of plane deformation for
substituted aromatic
750 c-c1 ?
2.6 Thermal Behavior
Shen -
et -
al. initially reported a melting point of 153-
154O for indomethacin(1). The N.F. XIV states that the
melting point of indomethacin is 162°C while N.F. XI11
gives 1550 and 1620 for the melting points of two
polymrphs of indomethacin. The British Pharmacopoeia
-
1980 specifies a ntelting point of 158u to 162uC(14).
Tabulated in Table IV are the melting points observed
for the various polymorphs previously reported in the
literature.
INDOMETH AClN 223
Table N
Melting Points of Indomethacin Polymorphs
Form Melting Point (OC)
Form I (type y ) 160-161.5( 18)

160(20,18)
158( 19)
Form I1 (typeo ( ) 154.5-155.5(28)
154(20,28)
152( 19)
Form I11 148( 21)
Form IV 134(21)
Type 4 158-160.5(18)
A LYTA curve for indomethacin (Form I - Merck Standard)

obtained on a du Pont 990 thermal analyzer is given in
Figure 5. Observed DTA peak temperature is 162OC.
0 0 0 0 0 0
a0 W U cu 0
0 % 3
u
1 I
0 0 0 c,
a0 W d N
224
3
LD
N
3
N
N
0
00
v
*0
Y
0
0
7
0
(D
0
N
22s
226 MATTHEW O'BRIEN ETAAL.
2.7 Solubility
~
The following s o l u b i l i t y d a t a have been reported:
Sol vent Temp ( OC ) Sol ubi li ty Reference
Water 25 0.40 mg/100 m l a 20

Water 25 0.52 mg/lOO mlb 20
Water 25 0.88 mg/lOO m l c 20
Water RT P r a c t i c a l l y Insoluble 13
Phosphate Buffer pH 5.6 25 3 mg/100 m l a 28
Phosphate Buffer pH 5.6 25 5 mg/100 m l b 28
Phosphate Buffer pH 6.2 25 11 mg/IOO m l a 28
Phosphate Buffer p U 6.2 25 16 mg/lOO rnlb 28
Phosphate Buffer pH 7.0 25 54 mg/100 m l a 28
Phosphate Buffer pH 7.0 25 80 mg/lOO nilb 28
Ethyl alcohol (95%) RT 1 i n 50 13
Chloroform RT 1 i n 30 13
Ether RT 1 i n 45 13
Metl-ranol. 25 32 mg/gm 29
Benzene 25 4 mg/W 29
n-butanol 25 19 W/gm 29
sec-butanol 25 27 mg/gm 29
aForm I, bForrn 11, 'Form 111

INDOMETHACIN 221
2.8 Dissociation Constant

A pKa of 4.5 for the carboxyl group of indomethacin
was calculated from aqueous solubility data(30). Potentiometric
titration data for indomethacin in 50% methanol-water yielded
pKa of 4.5 using a correction factor for the solvent(31).
2.9 Partition Behavior
Apparent distribution coefficients for indomethacin are
tabulated below(31). Additional information is given in the
references.
Solvent Pai.rs K*
Methylene Chloride/pH 7.1 Phosphate Buffer 16.3

Ether/pH 7.1 Phosphate Buffer 8.2
K* - conc. in organic phase

conc. in aqueous phase
3.Synthesis
3.1
Indomethacin(1) was originally prepared as shown
in the scheme below by conversion of 5-methoxy-
2-methylindole-3-acetic acid to its anhydride using
dicyclohexylcarbodiimide in THF(1). Treatmnt of the anhydride
in the presence of zinc chloride and butanol produced the
butyl ester, which was then acylated with p-chlorobenzoic
acid to yield indomethacin butyl ester. The ester was then
pyrolyzed and purified to produce indomthacin.
Indomethacin ethyl ester is synthesized by the reaction of
N-p-chlorobenzoyl-N-p-methoxyphenylhydrazine with l-hydroxy-
2-propanone and 2-bromoethylacetate after the method of
Yamamto et.al. .(32)
Indomethacin has also been synthesized from sodium p-methoxy-
phenylhydrazine sulfonate and from saffrole(33,34).
. ~.DCHC/THF
2. ZnC12/BuOH
3
4. Stability
In general, the integrity of indomethacin powder and formulate(

products exists for at least five years at room temperature(35,36).
Exposure to strong direct sunlight induces an increase in the color
of indomethacin(37);however,degradation is slight, but the
precaution of employing light resistant containers should be taken
to minimize discoloration of solid. Indomethacin undergoes alkalinc
hydrolysis to p-chlorobenzoate and 2-methyl-5-methoxy-indole-3-
acetate. These transformation products are also primary metabolic
products(section on metabolism and pharmacokinetics).
-
co2
6-0
I
c1
The half-life at room temperature is about 200 hours in pH 8.0

buffer and about 90 minutes in pH 10.0 solutions(36). In a patent
specification, Sumitom Chemical Co. Ltd. reports stability
conditions for seven different injectable formulations of
indomethacin(38). Several of these formulations were stable after
four months storage at 5OoC.
Formulations of indomethacin as the free-acid and as the sodim
salt are available in various parts of the world. They include:
Capsules 25mg and 50mg

Time Release Capsules 75mg
Injection l.Omg/ml
Ophthalmic Suspension lOn!q/ml
Oral Suspension lOmg/ml
Suppositories 5Omg and lOOmg
INDOMETHACIN 229
5. Methods of Analysis
5.1 Elemental Analysis
Analysis of Merck Sharp & Dohme reference lot 590,226-0A38
for C, H, N and 0 was reported as follows:
Theory Observed
C 63.78 63.87
H 4.51 4.44
N 3.92 4.09
0 9.91 10.01
Reference material is obtainable from both the U.S.

Pharmacopeia and the British Pharmacopeia (13-14).
5.2 Spectrophotometric Analysis
Analysis of capsules containing indomethacin is described
in the USP/NF and the British Pharmacopeia (13,141.
After an extraction into Ethylene chloride from a methanolic
pH 7.2 phosphate buffer, the ultraviolet alxorbance is measured
near 318 nm. This analysis measures intact indomethacin in the
presence of its hydrolytic degradation products. If
esterification has occurred, an ether wash prior to extraction
from phosphate buffer has been used to prevent interference
caused by esters(39). This procedure is a l s o applicable to
injections, suppositories, suspensions and tablets.
Allesandro and co-workers have also described analysis of
indomethacin by formation of a nitroso derivative which has an
ultraviolet absorption maximum at 317 nm(40).
5.3 Fhorescence Analysis
The indomethacin hydrolysis product 2-methyl-5-methoxy

indole acetic acid fluoresces at 385 nm after excitation at 300
nm in 0.1N NaOH,(41) and at 387 nm after excitation at 312 nm
in pH 11.6 buffer(42). The latter procedure claimed a three-
fold increase in detectability. Neither method distinguishes
indomethacin from salicylates. Clinical studies employing
subjects administered aspirin must use a separation prior to
fluorescence analysis. Without adequate separation the indole
metabolites as well as salicylate, produce a positive assay
bias.
230 MATTHEW O'BRIEN ET AL.
5.4 Pol.arographic Analysis
In aqueous methanol, indomethacin exhibits a half-wave

potential (El 2) at the dropping mercury electrode which is
dependent up& pH. In 0.1M methanolic lithium chloride,
indomthacin has two waves between -1.4~and -1.6~ (vs.
S.C.E.). The first step height is diffusion controlled and
corresponds to a two electron reduction of the amide carbonyl.
The second wave is believed to be a kinetic wave. The method
as described is specific for nonhydrolyzed indomethacin and is
suitable for analysis of capsules, suppositories and
suspensions with precision of +1.2%, -+0.7% and -
+1.2% for
the respective formulations(.)
3
4
5.5 Mass-transport Techniques

5.5.1 Liquid-liquid Extraction
Distribution ratios of indomethacin and its
metabolites, N-deschlorobenzoylindomethacin and 0-
desmethylindomethacin, have been described for heptane
and aqueous solutions of buffers between pH 5.0 and
7.0(44). In addition, a comparison was made on samples
extracted from sera of man, dog and rat in the above
pH range.

Harman and co-workers described a series of solvent
systems for ascending chromatography on Whatman 3MM
paper(45). The following table compares the R values
of indomethacin ( T) , N-deschlorobenzoyl j ndomet6acin (I1)
and 0-desmethylindomethacin (111).
System I
- I1
- I11
-
A 0.75 0.40 0.66
B 0.95 0.88 0-92
C 0.95 0.95 0.95
System A - isopropyl alcohol - 15N arnmonium hydroxide-
water (8:l:l)
B - methanol-water-n-butyl alcohol-benzene
(2:1:l: 1)
C - acetic acid - isopropyl alcohol (5:95)
INDOMETHAClN 23 I
Other workers achieved separation of I, 11, and

I11 using as a developing system Xylene-toluene-dioxane-
isopropanol-20% morpholine (1:1:3:3:2)(46).
Sondergaard and Steines measured indomethacin in
plasma and urine by direct quantitative TLC using
chloroform-methanol (30:6) on silica gel.(47). Reliable
quantitation was achieved at the 30 ng/ml level(+l2&).
-
In addition to the systems described above, Harman

and co-workers developed TLC systems on silica
gel (45).
-Rf
System I
- I1
- -
I11
D 0.50 0.55 0.65
E 0.57 0.37 0.15
D ethyl acetate - isapropyl alcohol-10%
m n i u m hydroxide (5:4:3)
E acetic acid - chloroform (5:95)
Allessandro and co-workers using silica gel G were
able to separate indomethacin from drugs of similar
pharmacological properties(40). Likewise Thompson and
Johnson reported Rf values for a variety of
analgesics, antipyretics and anti-inflmatory drugs
which were separable from indomethacin(48). Polyamide
gels were also used by Hsiu -et -
al. to resolve a series
of antipyretics including indomethacin(49).
Curran,et.al. , used TLC to resolve and identify

impurities in formulations(50). The systems employed
had detection limits on the order of 0.1 to 0.2 ug.
-
Rf
System I I1 PCB W V
A 0.80 0.75 0.90 0.24 0.35
R 0.40 0.38 0.50 0.33 0.28
PCB = parachlorobenzoic acid

IV = alpha monoglyceride of I from suppositories
V = alpha monoglyceride of PCB from suppositories
232 MATTHEW O’BRIEN ET Af
5.5.4 Gas-Liquid Chromatography

GC-MS characterization of indomethacin has been
reported using trimethylsilyl esters separated on
SE-52 liquid phase(51). The same paper also reports
precision and accuracy using an electron capture
detector, which for plasma was 92219%( 5ng/ml)
and 9651.5% ( 1000ng/ml). For aqueous humors , the
values were 9755.6% and 99+2.2%,respectively.
Indomethacin has been determined without any
preparatory work by Saito and Hara on 2% (w/w) CN-17
supported on Diasolid L and on 1.5% (w/w) SE-52
supported on high purity Chromsorb W(52).
Indomethacin has also been determined as its ethyl
ester on a lm x 2.5 mm column packed with 100-120 mesh
ChromosorbW (AW-DMCS) coated with 2% (w/w) OV-1(53).
Under optimum conditions utilizing an electron capture
detector, a recovery from spiked serum of 96 2 3% was
attained. Formation of the ethyl ester prevents assay
inclusion of O-desmethylindomethacin, a metabolite which
after methyl.ation would be identical with the methyl
ester of indomethacin.
Measurement with an electron capture detector has
been made using the pentafluoropropyl derivative in
the plasma of neonates with comparable precision and
linearity over the range 10 to 1000 ng(54).
5.5.5 Liquid-Sol id Chromtography

Separation of salicylic acid and indomethacin was
accomplished by packing 6 gms. of kieslguhr ground with
citrate buffer on top of 2 gms. of kieslguhr mixed with
pH 7.0 phosphate buffer. Material was eluted with
heptane(42).
5.5.6 Hiqh Performance Liquid Chromatography

Table V contains references for chromatographic
systems used to measure indomethacin in various
matrices.
Table V SEPARATION SYSTEMS FOR INDOMETHACIN BY HPLC
Sample Colurrm Mobile Phase Detection -

REF
Biological Hypersil ODs 76% methanol in 0.025M Fluorometric 55

pH4 Phosphate Buffer exc @ 295nm
emm @ 340nm
1.5ng/ml
Dosage Form Ultrasphere ODs Methanol-water-acetonitrile- Ultraviolet@254 56
acetic acid(55:35:10:1)
Biological Zorbax ODs Mobile phase optimizatiom Ultraviolet@235 57
Dosage Form Silica,lOum Gradient:
A:8% acetic acid in heptane Ultraviolet@254 58
B:8% acetic acid and 20%
ethanol in heptane
Dosage Form C18uBondapak 27%acetonitrile in 1% aqueous Ultraviolet@254 59
formic acid
Dosage Form C18uBondapak 60% methanol in 2.5% phos- Ultraviolet@240 60
phoric acid
Bulk Drug C18uBondapak Gradient:
A:250ml acetonitrile in 750 Ultraviolet@254 61
phosphate buffer ( PH 7, 0 . 0 2 ~ )
B:700ml acetonitrile in 300
234 MATTHEW O’BRIEN ET A L
5.6 Titrimetric Analysis

Assessment of indomethacin p d e r had been specified in
the U.S.P XIX and the British Pharmacopeia,l980 as a
back-tTtyazn with hydrochloric acid after alkaline
hydrolysis(l3,14). This method can attain a precision of 2
0.8%(62). A direct titration for tablets and capsules is
described using sodium hydroxide. If performed rapidly with
phenolphtha in indicator, a precision of 50.3% is
attainable. & The latter procedure serves to differentiate
ester formation as well as hydrolysis products from intact
material. The presence of parachlorobenzoic acid and 5-methoxy-
2-methylindole-3-acetic acid are cause for positive error.
6. Metabolism ant3 Pharmacokinetics
The only known routes of indomethacin elimination are renal.,

biliary and metabolic(46,63). Unchanged indomethacin (I), 0-
desmethyl-indomethacin (DMI), N-deschlorobenzoylindomethacin (DBI)
and 0-desmethyl-N-deschlorobenzoylindomethacin (DMBI) and their
respective acyl glucuronides account for all drug-related moieties
in plasma urine and feces. I, DMI and DBI are the major components
in plasm and urine: they appear predominantly as conjugates in
urine but totally unconjugated in plasma. DMBI is the major
chemical species in the feces.
In a l l animal species studied, all four chemical moieties
undergo enterohepatic circulation to varying degrees. The same can
be inferred from the time courses of each in man. Quantitatively,
it has been estimted that approximately 50% of an intravenous dose
undergoes enterohepatic circulation as I.
Because of enterohepatic circulation the amount of I available

to the systemic circulation can exceed the administered dose. On
the average, the bioavailability of orally and rectally adminstered
I is 100 and 80% relative to an intravenous reference dose.
Within the therapeutic range, there is no evidence that the
disposition of I is route- or dose-dependent. The mean plasma,
renal, biliary and metabolic clearances of I are 107, 28, 60, 68
ml/min., respectively. Because of the sporadic and variable nature
of gall bladder discharge and consequently the ensuing reabsorptior
attempts to determine plasma half-life directly tend to be futile.
Thus, values ranging from 90 minutes to 16 hours have been reportec
in the literature. An effective half-life of about 4.5 hours has
been estimated from the time course of I accumulation during
repeated drug administration and is the mean value which must have
prevailed for the observed accumulation to obtain.
INDOMETHACIN 235
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236 MATTHEW O'BRIEN ET Af
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INDOMETHACIN 137
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238 MATTHEW O'BRIEN ET A L
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E. Duggan,J. Pharmacokin. Biopharm.,A ,255(1976).
KETOTIFEN
Zvoniinira Mikotib-Mihun', Josip Kuftinec,
Hrvoje Hofman, Mladen Zinik, Franjo Kajfei
Podrackn-lnstitute
Zagreh, Iligorlatki
Zlatko Meie '

Institute Rtider BoikoLiC
Zagreb, k'rigoslaaici
I. Foreword, History, Therapeutic Category 240

2. Description 240
2.2 Formula 240
2.3 Molecular Weight 24 I
2.4 Appearance, Color, Odor 24 1
3. Synthesis 24 1
4.1 Spectra 243
4.2 Solid Properties 256
4.3 Solution Properties 251
5.3 Titration 260
6. Stability and Degradation 260
7. Drug Metabolism, Pharmacokinetics, Bioavailability 24 I
9. Determination in Pharmaceuticals 262
References 262
'Correspondence.

ISBN 0-12-260813-5
240 ZVONIMIRA MIKOTIC-MIHUN ET AL
1. Foreword, History, Therapeutic Category

Ketotifen is a nonspecific oral, mast cell
stabilizer introduced in 1972 (1j. The prominent
biochemical-pharmacological activities of ketotifen
are H -receptor antagonism, phosphodiesterase inhi-
bitioh, inhibition of the formation SRS-A, and in-
hibition of calcium f l u x in smooth muscle prepara-
tions: a l l these actions are suited to prevent a
development of asthmatic conditions. Promising re-
sults vere obtained in early clinical trials: keto-
tifen was equipotent with disodimn cromoglycate in
prevention of asthma induced by spontaneous excer-
cise or by antigens. Also, ketotifen is more speci-
fic than clemastine as a H -receptor antagonist.
Rowever, more recent, contholed trials failed to
substantiate the early therapeutic optimism. The
beneficial effect of ketotifen in the treatment
of asthma is only small; it was noted that this
effect is associated with pronounced sedation (2).
2. Description
2.1. Nomenclature
2.1.1. Chemical T?ame
4-(1-Methyl- iperidylidene)-4R-benzo- [4,5] -
-cyclohepta- [l, 2-bj)-thiophene-lo (9H)-one hydrogen-
f umarate
2.1.2. Generic Name
Ketotifen
2.1.3. Trade Name
Zaditen
2.2. Formula
2.2.1. Zmpirical
C19H19NOS.C4CF-ILF04
KETOTIFEN 24 1
2.2.2. Structural
0
HOOC
x 1cCOOH
CH, J
2 . 3 . Molecular Weight
425.504
2.4. Appearance. Color. Odor
The drug is marketed as the salt, either con-
taining 2.5 molecule of water, or anhydrous. In
either form it is a white, odorless crystalline
powder.
Ketotifen base: a yellow, odorless crystalline pow-
der.
3 . Synthesis
The reaction sequence leading to ketotifen
hydrogen fumarate is shown in sheme I.
2-Thenylchloride (11), prepared by chlorome-
thylation of thiophene (Ia) or 2-hydroxymethylthio-
phene (Ib), was subjected to Arbuzov’s rearrange-
ment to give 2-thenyl-diethylphosphonate (111).
This product was reacted with 2-carbolrybenzaldeh de
whereupon 2-/2- (thieny1)-vinyl/-benzoic acid (IVY
was obtained; IV was h drogenated t o 2-/2-(thienyl)-
ethyl/-benzoic acid (VT. Cyclisation under the in-
fluence of polyphosphoric acid, (PPAJ resulted in
phene-4-one (VI) .
9,lo-dihydro-4H-benzo[4,~]-cyclohepta~l, 2-bl-thio-
Alternatively (V) may be prepared by reducti-
on of 2-thenylidene-phthalide obtained from 2-thi-
CH
OCH3 A
Scheme 1.
KETOTIFEN ’743
engl acetic-acid (Ic) and sodium ethylate ( 7 ) .

Bromination of (VI) gives the dibromo deriva-
tive which, on refluxing in methanol, followed by
dehydrobromination with-KOH in refluking methanol,
gave lo-methoxy-413-benzo- [ 4,5]-cyclohepta- [l, 2-51 -
-thiophene-4-one (IX). This compound was condensed
with t h e magnesium derivative of 4-chloro-1-methyl-
piperidine in tetrahydrofurane, and the resultin
lo-methoxy-4-(l-methyl-4-piperidyl)-49-benzo[4,~
-cyclohepta- [1,2-b]-thbhene-%-one (X) was fin 11
7-
dehydrated and demethylated with HC1 at 95-100Bflw ,
to give the base of ketotifen ( q ) .
The IR absorption spectrum of ketotifen base,

and that of ketotifen hydrogen fumarate with 2.5
molecules of water, a r e shown in Fig. 1, These spe-
ctra were recorded with K3r-pelleted samples usin
a m e Unicam SP-200 Infrared Spectrophotrometer (57.
Some of the major frequencies and band assignements
are given in Tables 1, and ,2.
Table 1. Characteristic IR bands of ketotifen base
frequency (crn-l) assignment
- ~ ~ - -~ ~ ~ ~
3100 thiophene C-Ii stretching

3000-2840 aromatic and CH stretchings
1640 C=O stretching 3
1620 C=C (ring), stretching
1400 and 1280 C-C-H, interaction
1450 C-11, bending
1120 C-C(=O)-C, stretching and
bending
lo60 C-R, in-plane bending
935-700 thiophene
4.1.2, Ultraviolet
Fig. 2. shows the UV spectra (me-Unicam SP-
-8-100 Spectrophotoaeter) of ketotifen base (E)
and ketotifen hydro en fumarate with 2.5 molecule
of crystal water (27 both compounds being in me-
thanolic solution 75).
Fig. 1. I n f r a r e d absorption spectra o f k e t o t i f e n

base ( A ) and k e t o t i f e n hydrogen fumarate
w i t h 2.5 H20 (B). Instrummt: Pye-Unicam
SP-200.
KETOTIFEN 24s
200 300 nm 400
2. U l t r a v i o l e t s p e c t r a o f k e t o t i f e n base (L),
and k e t o t i f e n hydrogen fumarate with 2.5
-
c r y s t a l H20 (2). Instrument: Pye-Unicam SP-
8-100.
t
309(H' I
100-
>
c
5
I-
E
W
>
-
2 60-
_I
W
e 58
70
98 2%
165 108 237
1 II 11111 1
mle
50 100 150 2 00 2 50 300
Fig. 3. Mass spectrum of ketotifen hydrogen fumarate. The

electron energy was 70 eV and the temperature of the ion source was
25OoC. Instrument: mass spectrometer CEC-21-110 B.
KETOTIFEN 247
Table 2. C h a r a c t e r i s t i c I R bands of k e t o t i f e n hyd-

rogen Pumarate containing 2.5 molecules of c r y s t a l
water
frequency (crn-l) assignment
3550-3450 OH, broad band
3080-3030 aromatic CH, s t r e t c i n g
3095-3075 =CB s t r e t c h i n g
3100 th&hene C9, s t r e t c h i n g
2800-2200 ZOOH and YH
1710 COOH (fumaric a c i d )
1640 C=C ( r i n g ) , s t r e t c h i n g
1390 C-11, s t r e t c h i n g
1280 -C-0-H, interaction
4.1.3. Mass
The mass spectrum (CEC-21-110 B Mass spectro-
meter) of k e t o t i f e n hydrogen fumarate i s shown i n
Fig. 3 . The s t r o n g e s t peak corresponds t o t h e mole-
c u l a r i o n o f k e t o t i f e n hydrogen fumarate (m/e 309).
Fragmentation a p p m e n t l y s t a r t s by a s e p a r a t i o n of
t h e methyl group (m/e 15) from t h e p i p e r i d y l i d e n e
moiety, and t h e remainder of t h e o r i g i n a l molecule
(m/e 294) does not fragmentate f u r t h e r , a t l e a s t
not markedly. The h e t e r o c y c l i c p a r t s o f t h e nolecu-
l e , thiophene and p i p e r i d i n e , should g i v e t h e f r a g -
ments (m/e 58) and CH2-;-CH3 (m/e 5 7 ) , respec-
t i v e l y S (5).
CH2
4.1.4. Proton Magnetic Resonance
.The proton MR s p e c t r a ( J e o l PX-loo Spectrome-
t e r ) were recorded i n d i f f e r e n t s o l v e n t s : t h a t of
k e t o t i f e n base i n d e u t e r a t e d chloroform, and t h a t
o f k e t o t i f e n hydrogen fumarate i n d e u t e r a t e d dime-
t h y 1 sulphoxide ( i n e i t h e r case a g a i n s t TPMS as i n -
t e r n a l s t a n d a r d ) (5). These s p e c t r a a r e presented
i n Pigs. 4. and 5. C h a r a c t e r i s t i c f e a t u r e s of t h e
s p e c t r a a r e given s e p a r a t e l y i n Tables 3 . and 4.
The proton ME spectrum of k e t o t i f e n base was p a r t -
l y described by Waldvogel e t a l . (6).
Vhen k e t o t i f e n hydrogen f u n a r a t e was shaken
with I) 0 , t h e qarboyylic group protons of fumaric
a c i d &d t h e N H protons were exchanged f o r deute-
r o n s from D 0, a s shown by t h e absence of t h e broad
peak a t 9.12 ppm (see Table 4.) (5).
Table 3 . 'H-NMR spectrum of ketotifen base; charac-

teristics and assignments
I
CH3
h
chem. shift intens. multipli- coupling assign-

d' (ppm) city const. J @z) ment
7.50 1 H doublet J = 4.9 *a
a,c
7.36-7.10 4H nuliplet Hb
7-05 1 H doublet Jc,a= 4.9 HC
4.24 1R doublet Jd,e=13.18 d'
3-74 1 3 doublet Je,d=13.18 He
2.94-2.58 2H multiplet *f
2 49-2-39 2H multiplet H
63
2.28-1.99 7H multiplet Hh, N-CH
4.1.5. 13C-Huclear Magnetic Resonance

13C-TTb7R spectra were recorded with a Jeol FX-
-loo spectrometer at 25.05 NHz. The sample of keto-
tifen base was dissolved in both CDCl and DMSO-d6,
and hydrogen fumarate only in DWO-d 3 (5). The
samples were measured in 5 mm tubes h t h TMS as in-
ternal standard and uy3ng internal deuterium lock.
The broad band C-NNR spectra of >?GO-d so-
lutions of base and the hydrogen fumarate are ghown
in Figs. 6. and 7. In both cases all the signals
were clearly resolved, The assignments of saturated
carbon a t o m can be made on the basis of their well
1
a,
A 0
0
KETOTIFEN 15 I
T a b l e 4. 'H-NMR spectrum of ketotifen hydrogen fu-

marate
n
chem. shift intens, multipli- coupling ass 1gn-

(ppm) city const. J (Hz) ment
9.14 2 H broad singlet COOH
7.98 1 H doublet Ja,c= 4.9 Ha
7.43-7.26 1H multiplet HC
7.26-7.21 4H singlet Hb
6.60 2 H doublet Jd,,=13.17 i'
(fummic ac.)
4.37 1 H doublet J, ,d=13.i 7 d'
3.72 1H multiplet He
3 17-3 03 2H multiplet H
g
2.80-2.66 2H multiplet Hf
2.54-2.51 8H multiplet ;"Ih
defined absorption regions ( 7 ) , but due to very
closely spaced chemical shifts of numerous aromatic
and two quaternary olefinic carbons, the assignment
was possible only by means of gated decoupled (NOE)
spectra. Eecause of the solute-solvent interaction
in DMSO-d solutions these spectra were not quite
suitable $or an unambiguous interpretation. There-
in CDCl .
fore we have recorded the 130E spectrum of the base
3From the multiplets of the aliphatic part
of the completely coupled spectrum, it was possible
to assign the signals belonging to C - 2 ' , C-3' and
Table 4. I 3 C Chemical shiftsa and I3C-'H coupling

'1-
constants" for ketotifen base dissolved

in C D G l
C-atom
c-2
(ppm)
lJCH
185,l
JCH
6.3
'-
JCH 4JCH
c-3 169.9
- 3.9
- -
c-4 137.73 ( s ) 7.8
c-5 161.1 i1.0 7.5
C-6 159.0 < l o o 6,8
c-7 157.6 =2.0 4.0
C-8 158.0 *loo 5.0
(3-9 134.9 - 3.9
125.9 - 3.9
(2-10 189.05 ( s ) -- 8.0 -
c-11 147.95 ( s ) - 9.3
c-12 132.39 ($1 - overlaped with
C-7 multiplet
c-13 141.02 (s)' - - -
(2-14
c-2'
c-3 '
c-4'
GH,
a $in ppm downfield from internal TMS; precision
-0.05 ppm; off-resonance multiplets a r e given in
parentheses in the second column.
+
J in Hz; the precision is -0.5 Elz.
C
The assignment can be interchanged. The long-ran-
ge couplings give complicated multiplets.
methyl group of the N-methyl-piperidine part of the

molecule, as well as the methylene carbon ((3-9) of
the cyclic ketone part. The latter carbon atom dis-
plays a doublet of doublets indicating a pronounced
departu&e of the CH group from the planarity. Two
different first ord& C-H coupling constants were
deteymined (Table 5.1, which is in agreement with
the H-NMR spectrum (Fig. 4, Table 3 . ) which shows
the typical pattern for geminal protons.
The assignment of the aromatic-olefinic part
o f the spectrum was not possible using only known
substitution rules (8) due to simultaneous interac-
tion of few different moieties. However, the NOE
spectrum enabled a sound interpretation based on
the long-range carbon-hydrogen couplings. The corn-
a,
11)
rd
Q
c
.d
..
KETOTIFEN 25.5
Table 6. I3C Chemical s h i f t s a f o r k e t o t i f e n base

and hydrogen fumarate i n IlMSO-d, s o l u t i o n
C-atom base f umarat e Adb
c-2 0
G-3 +O .54
c-4 -0.84
c-5
C-6
c-7
+0.07
+O
+o. 23
.4 1
C-8
C-9
c-lo
+o. 23
+o 02
+o. 16
.
5-11 -0 62
’5-12 +o. 21
(2-13’ 140.89 ( s ) -0 46
C-14’ 138.94 ( s ) -1.96
c-2 ’ 56.21 ( t ) -2.07
c-3 ’ 30.92 (t) -2.10
c-4’ +0.9
C%/ -2.15
c-1’ ’ (C=C
c-2’ ’(c=o
a +d i n ppm downfield f r o m i n t e r n a l TWS; p r e c i s i o n
-0.05 ppm; o f f resonance m u f t i p l e t s a r e given i n
parentheses.
Changes i n chemical s h i f t s are going from base t o
fumarate; + denotes a downfield, and - an u p f i e l d
shift .
The assignments can be interchanged.
p l e t e assignment i s given i n Table 5. i n c l u d i n g a
nimber of long-range coupling c o n s t a n t s up t o t h e
f o u r t h order.
Figs. 6. and 7. show Che broad-band decoupled
s p e c t r a o f k e t o t i f e n base an9 i t s fumarate i n DT4SO-
-d , and Table 6. l i s t s t h e 3C chemical s h i f t s .
Thg assignment i s made on t h e b a s i s of a comparison
with t h e values from Table 5. The most remarkable
changes (an u p f i e l d s h i f t o f about 2.1 ppm) a r e ob-
served f o r t h e s a t u r a t e d carbons of t h e heterocyc-
l i c moiety, The t h i r d column i n Table 6. i n d i c a t e s
t h e changes i n chemical s h i f t s f o r a l l carbon atoms
common t o b o t h species.
It should be also mentioned t h a t t h e a d d i t i o n
256 ZVONlMlRA MIKOTIC-MIHUN ET AL
of D 0 to the C D C l solution cau es not only the

vanishing of the 08 peak in the fH-IWR spectrum,
but the diminishing of the signal at 49.67 ppm be-
longing to C-9 as well. This indicates the exchan-
ge of protons by deuterons causing the splitting of
t h e signal into a multiplet.
4.2. Solid Properties
4.2.1. Melting Characteristics
The melting range of ketotifen base is 156-158
OC,that ofoketotifen hydrogen fumarate with 2.5 H20
is 124-130 G, whereas the anhydrousohydrogen fuma-
rate decomposes between 184 and 200 C.
4.2.2. X-Ray Diffraction
X-Ray diffraction patterns were determined
with a Nod. XRD-6 Spectrogoniometer (General Elec-
tric, Schenectady) (5). Pertinent data are presen-
ted in Table 7.
Table 7. X-Ray diffraction data of ketotifen hydro-
gen fumarate
da (2) I/Iob
@<"> interplanar
- distance relative
intensitg
4.94 8-95 3
5.37 8.24 6
6.08 7-28 14
6.42 6.89 9
7-05 6.28 12
7.76 5.71 23
8.79 5.04 17
9.00 4.93 20
9.61 4.62 67
9-90 4.48 93
10 79 4.12 26
11.46 3.88 14
12.02 3.70 48
12.42 3.58 loo
12 99 3.43 16
14.32 3.12 12
15.65 2.86 lo
3-7 4.5 2- 57 9
17.76 2.53 11
based on the highest relative
KETOTIFEN 251
4.3. Solution Properties

%.3.1. Solubility
Solubilities of ketotifen base and ketotifen
hydrogen fumarate with 2.5,H 0 in various solvents,
at room temperature (18-20 C3, are summarized in
Tables 8. and 9. (5)
Table 8. Solubilities of ketotifen base
Solvent Solubility
water insoluble
methanol soluble
ethanol soluble
isopropanol sparingly soluble
acetone sparingly soluble
1,4-dioxane sparingly soluble
diethyl ether sparingly soluble
ethyl acetate sparingly soluble
chloroform freely soluble
dichloromethane freely soluble
casbon tetrachloride slightly soluble
toluene sparingly soluble
n-hexane insoluble
Table 9. Solubilities of ketotifen hydrogen fumara-

te hydrate (2.5 H,O)
Solvent Solubility
water slightly soluble
methanol freely soluble
ethanol freely soluble
isopropanol sparingly soluble
acetone sparingly soluble
1,4-dioxane sparingly so luble
diethyl ether sparingly soluble
ethyl acetate very siightly soluble
chloroform very slightly soluble
dichloromethane very slightly soluble
n-hexane insoluble
4.3.1. Dipole Moment

The dipole moment of ketotifen base was de-
termined with a Dipolmeter DY 01 (Yiss. Techn.
WerkstEitten, Weilheim), using 1,4-dioxane as the
solvpt ( 5 ) . The value obtained at 20.0~o.l°C was

3.55-0.12 D.
..I.Nethods of Analysis
Elemental Analysis
Elemental analysis of ketotifen hydrogen fu-
marate: C H NO S, calc.: C 64.90% (theor.)
23 23 5 H 5.45%
N 3029%
0 18.81%
s 7.54%
5.2. Chromatographic Methods
,5.2.1. Thin-Layer (TLC)
Thin layer chromatographic systems are given
in Table lo. Spots were visualized under UV irradi-
ation (5).
Table lo. Solvent systems f o r thin-layer chromato-
graphy of ketotifen base on silica gel
plates
Solvent system Rf
methanol 0.2
ethylacetate 0.3
methanol :ammonia (19:1) 0.9
ethy1acetate:ammonia:methanol (1:0.1:0.9) 0.8
ethy1acetate:ammonia:methanol (8:0.l:0.9) 0.5
ethano1:chloroform (1:5) 0.8
3 . 2 . 2 . Gas (GC)
Ketotifen hydrogen fumarate w a s chromatogra-
phed on a glass capillary column 15 m x 0.32 mm I.
D. with SE-30. The carrier gas was H , inlet pres-
sure 0.45 b a r . Th& tempegature progrgm used in ana-
lysis was 120-240 C at 6 /min. rate. The concentra-
tion of ketotifen hydrogen fumarate (so1vent:etha-
nol) in the sample that gave the record shown i n
Fig. 80 was 1 mg/nl and the retention time was 12.3
min (5).
5.2.3. High Performance Liquid (HPLC)
Table 11. gives the €IPIJC conditions used f o r
the analysis of ketotifen hydrogen fumarate ( 5 ) .
The column was Supelcosil LC-8 (14 cm x 4 mm I.D.),
detection UTT-230 nm, 0.16 A.U.F.S. (5).
KETOTIFEN 259
h
2 6 10 min.
Fig. 8. The gas chromatogram of ketotifen base.

Instrument: we-Unicam Mod. 204.
260 ZVONIMIRA MIKOTIC-MIHUN ET AL.
Table 11. HPLC conditions for ketotifen hydrogen

fumarate
Retention
Mobile phase rate B essure
'low
time
(ml/min) (bar) (min)
.
acetonitrile-dist water-
-acetic acid 70:10:0.5 15 60 22.6
(PH 3.5)
acetonitrile-dist. water-
-methanol 120:20:30 0.8 25-30 12.3
b H 7.5)
5.3. Titration
Fifty mg of ketotifen base was weighed (50.1
mg) into a titration vessel and dissolved in 20 ml
of glacial acetic acid. The resulting solution was
titrated potentiometrically using a glass S.C.E.
pair: 1.0 ml of 0.1 M HC104 is equivalent with
30.94 mg of ketotifen base (5). The content of ke-
totifen base in the sample was calculated b sub-
stituting numerical values into equation (1 : 3
content of ($1 = V x f x 30.94 loo
ketotifen base W
where V = volume of 0.1 M HClO consumed (ml)
f = normality factor of $.I PI H C ~ O ~
W = mass of the sample (mg).
The limits allowed are 98-102$.
Potentiometric titration of fumaric acid:
fifty rng of ketot$fen hydrogen fumarate with 2.5
H 0 was weighed (-0.1 mg) into a titration vessel
agd dissolved in lo ml of ethanol and lo m l of wa-
ter ( 4 ) . The resulting solution was titrated poten-
tiometrically with 0.1 P I RaOIT is equivalent with
5.8 mg of fumaric acid. The content of fumaric acid
in the sample is calculated by using equation (2):
$ fumaric acid = x loo
where V = volume of 0.1 M NaOH consumed (nl)
f = its normality factor
!.I = weight of the sample (mg).
The limits allowed are 24-25.2g.
6. Stability and Dearadation
Tablets and the pure substance of ketotifen
hydrogen fumarate with 2.5 H20 were tested in the
KETOTIFEN 26 I
following way: one seriesof samplesowas kept at

elevated temperatures, up to the 60 C and the rela-
tive moisture of 50% for seven days, and the other
was exposed to daylight, Individual samples were
withdrawn at 24-hr intervals, and subjected to HPLC
analysis for degradation products. No changes were
observed in the tablets; the pure substance assumed
a slight yellow coloration at the end of the test-
ing period.
7. D r u ~Metabolism, Pharmacokinetics, Bioavailabi-
litg
Ketotifen appears to be a potent drug acting
in the skin, nose and airways. There is little evi-
dence from acute experiments to suggest that this
drug has substantially different properties from a
typical H1 antagonist, e.g. clemastine (9). Admini-
stered to allergic patients, ketotifen decreased
the required aller en challenge and showed antihi-
7
staminic activity lo), It was ineffective in redu-
cing excercise-induced bronchoconstriction in 23
asthmatic children (ll), so a higher dose of keto-
tifen is necessary to obtain a beneficial effect
(12)
To study the bioavailability of ketotifen
from sustained-release oral formulations, deutera-
ted ketotifen (N-CD3) was used. The kinetic profi-
les of ketotifen in the plasma and in the urine
shows that any isotopic effect due to the presence
of deuterium is lacking (13).
80 Identification and Determination in Body Fluids
and Tissues
Selective and sensitive gas-chromatographic
methods have been developed for quantitative deter-
mination of ketotifen and its desmethyl metabolite
in biological fluids. The ketotifen is detected by
a nitrogen detector, or by mass fragmentometry,
which allows determination at concentration down
to 1 ng/ml and 0.05 ng/ml, respectively (14). The
N-glucuronide of ketotifen is measured as ketotifen,
after enzymatic cleavage. The nor-ketotifen is
quantitated, after derivatisation with heptafluoro-
butyric anhydride, by gas chromatography with elec-
tron capture detection. On chromatograms obtahned
from urines of subjects who had taken ketotifen,
peaks occured. The structures of metabolites sepa-
262 ZVONIMIRA MIKOTIC-MIHUN ET AL.
rated by chromatography were ascertained by high

and low-resolution mass spectrometry (14). The te-
chnique described was applied to establish the me-
9.
tabolic spectrum of ketotifen in monke s (rhesus,
baboon, gibon, chimpanzee) and men (14
9. Determination in Pharmaceuticals
The tablets were crushed in a mortar. A mass
of powder equivalent with the average weight of one
tablet was transferred into a loo m l volumetric
flask and 50 m l of methanol was added. The flask
was then shaken automatically for 20 min, the re-
sulting solution made up to the mark with methanol,
and mixed thoroughly. A 20 ml aliquot was centrifu-
gated at 4000 r.p.m. The absorbance of the clear
supernatant was measured at 298 nm against metha-
nol.. A reference solution of ketotifen hydrogen
fumar9te 2.5-hydrate was prepared by dissolving
15.2(-0.1) me; of the salt in methanol (lo-ml volu-
metric flask; concentration of ketotifen base, abo-
ut 1 mg/ml). One ml of the standard solution was
pipetted into a loo-ml volumetric flask, the solu-
tion made up to the mark with methanol, and the ab-
sorbance measured at 298 nin against methanol.(5).
The ketotifen content in one tablet was calculated
by using equation ( 3 ) :
mg ketotifen base- As x Cr x A.W.
-
in one tablet
where A, = absorbance of the sample solution
A, = absorbance of the standard solution
Cp = concentration of ketotifen base in the
standard solution
A.W.= average mass of one tablet (mg)
W = mass of powdered sample (mg)
The limits allowed are 90-110%.
Acknowledgments
The authors are thankful to dr N. Blazevie
for his help in many useful discussions and dr A.
Nag1 f o r X-ray diffraction spectral data.
References
1. Sandoz Ltd., G e r . Offen. 2,302,944 (1972).
2. M. Bser, Pharmac. Ther., 12, 245 (1982).
KETOTIFEN 263
3 . J.M. Bastian, A. FDnGther, E. Jucker, E. Rissi,

A.P, S t o l l , Helv. Chim. Acta ,
214 (1966).
4. Sandoe AGt Ger. Offen. 2,302,
5. 2. Mikotic-mihun, J. Kuftinec, 8. Bofman, M.
Zinib, F. KajfeZ, Z. Mei6, Acta Pharm. Jugos.
( 2 ) , (1983), i n press.
6. E.
lave 3
Wal vogel, G. Schwarb, J.M. Bastian, J.?,
Bourquin, 3elv. C h i m . d c t a 866 (1976).
7. 3. Brgktmaier, W. Voelter, 'k-?@IR Spectrosco-
py, 2 M., Verlag Zhemie, 'Jeinheim 1978.
8. E. P r e t s c h , T* Clerc, J. S e i b l , W. Simon, 'Pa-
b e l l e n XUT StrukturaufklElrung organischer ver-
bindungen m i t spektroskopischen Methoden, 2.
A u f . , Springer Verlag, Berlin-Heidelberg-New
York, 1981.
9 - R.J. Davies M.J. P h i l l i p s , Res. Clin. Porums
-4,
lo . K.
67 (19823.
Aas, Allergy 4 1 2 1 (1979).
11 . nes,
J.D. Kennedy, $.%:sham, P?I.J.D.
B r . Pled. J. 251, 1458 (1980).
Clay, R.S. Jo-
1 2. G.R. Kennedy, R e s z l i n . Forums 2, 17 (1982)-
1 3 C. Julien-Larose, R. Voges, D. Lavene, M.F.
Guillaume, J.R. Kiechel, E.-R.-Congr. Eur. Bio-
-
pharm. Pharmacocinet., '1 , 1, 326 (1981), edi-
t e d by J.M. Aiache, J. Tlirtz, Tech, Documenta-
3,
.
t i o n , Paris.C.A. 209527h (1981).
14 $1. Guerret, C. Julien-Larose, D, LavenegtC.-R.-
-Congr. Eur. Biopharm. Pharmacocinet. 1 -2
9
317 (19811, C.A. 96,2 8 1 8 7 ~(1982).
MELPHALAN
L. Valentin Feyns
I. Foreword. History, Therapeutic Category 266

1. Description 267
2.2 Formula. Molecular Weight 268
2.3 Appearance. Odor, Color 268
3. Synthesis 26X
4.1 Spectra 270
4.3 Melting Range 28 I
4.4 Solubility, Partition. Dissociat~onConstants 282
ti. Methods of Analysis 2x2
5.2 Spectrophotometric Methods 282
5.4 Titration 288
5.5 Identification and Purity Tests i n Official Compendia 288
6. Stability. Degradation. Mechanism of Action 289
7. Toxicity, Distribution. Metabolism. Excretion 292
7.1 Toxicity 292
7.2 Carcinogenicity 293
7.3 Distribution 293
7.4 Metabolism 293
7.5 Excretion 294
8. Determination in Body Fluids and Tissues 294
9. Identification and Determination in Pharmaceutical Preparations 29 5
9.1 Tablets 295
9.2 Melphalan Injection 295
References 291
ANALYTICAL PROFILES OF DRUG SUBSTANCES Copyright G 1984

VOLUME 13 265 hy the Aincrican Pharmaceutical Association
ISBN 0-1?-260813-5
266 L. VALENTIN FEYNS
1. Foreword, H i s t o r y , T h e r a p e u t i c Category.
Melphalan i s an a n t i n e o p l a s t i c d r u g , l i s t e d a l s o as a
Class I immunosuppressive agent ( e f f e c t i v e o n l y when given
p r i o r t o t h e immune s t i m u l u s ) [ l ] . It i s used f o r t h e
t r e a t m e n t of m u l t i p l e myeloma, o v a r i a n carcinoma, tumors of
t h e t e s t e s , c h r o n i c g r a n u l o c y t i c Leukemia, c h r o n i c lympho-
c y t i c leukemia, seminoma, Ewing's sarcoma, r e t i c u l u m c e l l
sarcoma, and thymoma [ 1 , 2 ] . I t s u s e as an a d j u v a n t t o
s u r g e r y i n t h e management of primary b r e a s t c a n c e r w a s one
of t h e f i r s t i l l u s t r a t i o n s of t h e t h e r a p e u t i c p o t e n t i a l of
combined m o d a l i t i e s of t r e a t m e n t 131.
Chemically, i t i s a bis(2-chloroethy1)amine ( a n i t r o g e n
m u s t a r d ) , and i t s b i o l o g i c a l a c t i v i t y i s r e l a t e d t o t h e
a b i l i t y t o f u n c t i o n a s an a l k y l a t i n g .gent i n p h y s i o l o g i c a l
conditions.
Attempts t o use s u l f u r mustard, t h e chemical weapon of

World War I, a g a i n s t e x p e r i m e n t a l tumors o r i n t h e c l i n i c
were abandoned because of t h e h i g h t o x i c i t y of t h e compound
[ 4 ] . E x t e n s i v e s t u d i e s of t h e b i o l o g i c a l and chemical
a c t i o n s of a l i p h a t i c n i t r o g e n m u s t a r d s , d i r e c t e d mainly t o
t h e i r p o t e n t i a l m i l i t a r y u s e , were conducted b e f o r e and
d u r i n g World War 11. Noting t h e marked c y t o t o x i c a c t i o n of
n i t r o g e n mustards on lymphoid t i s s u e s and r a p i d l y d i v i d i n g
c e l l s , Goodman and Gilman i n i t i a t e d s t u d i e s on e x p e r i m e n t a l
tumors and, i n 1942, t h e f i r s t c l i n i c a l t r i a l s on p a t i e n t s
i n t h e t e r m i n a l s t a g e s of v a r i o u s neoplasms [ 2 , 5 ] . The
p u b l i c a t i o n of t h e r e s u l t s had t o w a i t f o r t h e d e c l a s s i f i -
c a t i o n of t h e m i l i t a r y r e s e a r c h a t t h e end of t h e war. The
s e r i e s of papers published i n 1946 i n Science and i n t h e
. .
J o u r n a l of t h e American Medical A s s o c i a t i o n marked t h e emer-
gence of chemotherapy as an a l t e r n a t i v e t o s u r g e r y and
i r r a d i a t i o n i n cancer treatment [5 - 4 1 . This emergence was
followed by t h e s y n t h e s i s and e v a l u a t i o n of thousands of
n i t r o g e n mustards, i n a s e a r c h f o r a s e l e c t i v e , less t o x i c
tumor i n h i b i t o r .
The s y n t h e s i s of Melphalan, i n 1954, w a s t h e r e s u l t of

two premises: (1) a r o m a t i c n i t r o g e n mustards should be l e s s
r e a c t i v e , t h u s less t o x i c , t h a n the a l i p h a t i c a n a l o g s [9] ;
(2) a t t a c h i n g t h e n i t r o g e n mustard moiety t o a " c a r r i e r "
s e l e c t e d from normal c e l l u l a r c o n s t i t u e n t s might c o n f e r
g r e a t e r s e l e c t i v i t y t o t h e molecule. Bergel and Stock a t
t h e Chester B e a t t y Research I n s t , i t u t e i n London d e s c r i b e d
t h e s y n t h e s i s of t h e n i t r o g e n mustards of DL-, D-, and L-
p h e n y l a l a n i n e [ l o ] ; t h e l a t t e r i s Melphalan. Shortly a f t e r
t h a t , a Russian team r e p o r t e d t h e s y n t h e s i s and e v a l u a t i o n
MELPHALAN 267
of t h e h y d r o c h l o r i d e of t h e racemic d e r i v a t i v e , which they

c a l l e d S a r c o l y s i n [Larionov, 111.
The s u c c e s s of t h e s t u d i e s on e x p e r i m e n t a l tumors and

of t h e c l i n i c a l t r i a l s prompted t h e s y n t h e s i s of a wide
range of Melphalan-related compounds such a s t h e o r t h o
isomer [12--151, t h e meta isomer [16--20], B-alanine a n a l o g s
[ 191 , esters , N-acyl d e r i v a t i v e s , and p e p t i d e s . D e s p i t e
c o n t r o v e r s i a l r e p o r t s of g r e a t e r a c t i v i t y o r b e t t e r t h e r a -
p e u t i c a l index, none of t h e s e analogs r e p l a c e d Melphalan i n
c 1i n i c a l use.
The h i g h e r i n h i b i t o r y e f f e c t of t h e L-isomer on most of

t h e e x p e r i m e n t a l tumors r e p r e s e n t e d one of t h e f i r s t exam-
p l e s of s e l e c t i v i t y of a c t i o n through o p t i c a l isomerism i n
cancer chemotherapy [ 10, 21--231.
2. Description
2.1 Nomenclature
Chemical A b s t r a c t s Names
Current:
L-Phenylalanine, 4-[bis(2-~hloroethyl)amino]-
Pre-1972:
Alanine, L-3-[p-[Bis(2-chloroethyl)amino]-
phenyl 1 -
Other Names: Alkeranm, L-Sarcolysine, L-PAM, L-

P h e n y l a l a n i n e Mustard, Compound CB 3025, Compound NSC-8806.
D-isomer : Medphalan
Racemic: Merphalan, S a r c o l y s i n , S a r c o c h l o r i n
Chemica 1 A b s t r a c t s Reg i s t r y Numbers
Melphalan (L-form, f r e e base) : 148-82-3

L-hyd r och 1o r i d e : 3223-07-2
D-free base : 13045-94-8
D-hydrochlor i d e : 4213-32-5
DL-free base : 531-76-0
DL-hydrochloride : 1465-26-5
2.2 Formula, Molecular Weight
C13H18C12N202 Molecular weight 305.20

2.3 Appearance, Odor, Color
In t h e o r i g i n a l p u b l i c a t i o n [ l o ] and i n t h e
Merck Index [ 2 4 ] t h e monosolvated p r o d u c t o b t a i n e d by
c r y s t a l l i z a t i o n from methanol i s d e s c r i b e d as small
c o l o r l e s s needles.
The compendia1 a r t i c l e is d e s c r i b e d as a n
". . .off-white t o buff powder, h a v i n g a f a i n t odor. . ."
. ." ". .
[ 2 5 , 2 6 ] o r as
less. (271.
.a w h i t e o r almost w h i t e powder; odour-
3. Synthesis
Melphalan and t h e racemic a n a l o g have been p r e p a r e d by

two g e n e r a l r o u t e s (Scheme I). In Approach (A) t h e amino
a c i d f u n c t i o n i s p r o t e c t e d , and t h e n i t r o g e n mustard moiety
is p r e p a r e d by c o n v e n t i o n a l methods from a r o m a t i c n i t ro-
derivatives. Thus, t h e e t h y l e s t e r of N-phthaloyl-
p h e n y l a l a n i n e w a s n i t r a t e d and reduced cat a l y t i c a l l y t o
amine I. Compound I w a s r e a c t e d w i t h e t h y l e n e o x i d e t o form
t h e c o r r e s p o n d i n g bis(2-hydroxyethy1)amino d e r i v a t i v e 11,
which w a s t h e n t r e a t e d w i t h phosphorus o x y c h l o r i d e o r
thionyl chloride. The b l o c k i n g groups were removed by
acidic hydrolysis. Melphalan w a s p r e c i p i t a t e d by a d d i t i o n
of sodium acetate and was r e c r y s t a l l i z e d from methanol. No
r a c e m i z a t i o n w a s d e t e c t e d [ 10,28--301. The h y d r o c h l o r i d e
was o b t a i n e d i n p u r e form from t h e f i n a l h y d r o l y s i s m i x t u r e
by p a r t i a l n e u t r a l i z a t i o n t o pn 0.5 [ 3 1 ] . V a r i a n t s of t h i s
. a p p r o a c h , u s e d f o r t h e p r e p a r a t i o n of t h e racemic compound,
f o l l o w e d t h e same r o u t e v i a t h e a-acylamino-a-p-aminobenzyl
malonic ester I11 [10,28--30,32,33] o r t h e hydantoin I V
(121
In Approach (B), a n i l i n e is c o n v e r t e d i n t o t h e
c o r r e s p o n d i n g n i t r o g e n mustard, which i s f o r m y l a t e d t o t h e
benzaldehyde n i t r o g e n mustard V. The a l a n i n e moiety is
c o n s t r u c t e d v i a t h e Erlenmeyer r e a c t i o n w i t h h i p p u r i c a c i d ,
r e d u c t i o n , and h y d r o l y s i s [23,34--381.
High y i e l d s of o p t i c a l l y p u r e p r o d u c t s a r e c l a i m e d f o r
t h e r e s o l u t i o n of N-f ormyl-DL-melphalan t h r o u g h t h e b r u c i n e
s a l t , f o l l o w e d by H C 1 h y d r o l y s i s (391.
f
0
8
&
s
Q H
H
N
E
0
c
3.1
[I]
l-l
X"
U
$0
U
Q
0
=*' "
0
f 'I
m Q z,
S y n t h e s i s of Melphalan l a b e l l e d a t t h e n i t r o g e n mustard
f u n c t i o n [40--421 o r a t t h e b e n z y l i c methylenic group [43]
were r e p o r t e d . D e u t e r a t i o n a t t h e p o s i t i o n s o r t h o t o t h e N-
mustard s u b s t i t u e n t was r e p o r t e d when melphalan was r e f l u x e d
i n deuterium c h l o r i d e i n D 2 0 (20% w/v) [ 4 4 ] .
4.1 Spectra
4.1.1 Ultraviolet
The u l t r a v i o l e t spectrum of a 1-in-

100,000 s o l u t i o n of melphalan i n a l c o h o l e x h i b i t s an
absorbance maximum at about 260 nm ( a b s o r p t i v i t y about 72)
and a minimum a t about 226 nm [ 4 5 ] . The spectrum of one
compendia1 a r t i c l e i n methanol (0.0005 percent w/v s o l u t i o n )
has been d e f i n e d a s showing a maximum a t 260 nm and a less
well-defined maximum a t 301 nm [ 2 7 ] ; t h e wavelength of t h e
second maximum i s sometimes r e p o r t e d a s 310 w [ 2 5 , 4 6 ] . The
301 nm v a l u e appears t o be t h e c o r r e c t one ( F i g u r e 1 ) .
4.1.2 Infrared
S p e c t r a of Melphalan and Melphalan

Hydrochloride i n K B r d i s p e r s i o n s a r e shown i n F i g u r e s 2 and
3.
The f o l l o w i n g prominent bands a r e

present i n the spectra:
( a ) broad bands i n $he 2900--3100

cm-' r e g i o n , assigned t o CH and NH ( i n NH3) s t r e t c h i n g
v i b r a t i o n s ; ( b ) a c o t i n u o u s series of bands and s h o u l d e r s
i n t h e 2400-2900 cm-' r e g i o n , c h a r a c t e r i s t i c f o r aminoac i d s
and t h e i r h y d r o c h l o r i d e s ; ( c ) t h e asymmetrical and
symmetrical v i b r a t i o n s of t h e COO- group a t about 1590 and
1400 cm-l i n t h e spectrum of Melphalan ( I n t h e spectrum of
t h e h y d r o c h l o r i d e , i n which t h e i o n i z a t i o n of t h e c a r b o x y l i c
group is suppressed, t h e s e bands a r e re laced by a s t r o n g
c a r b o x y l i c a b s o r p t i o n a t about 1740 cm-I.); ( d ) a s t r o n g
band a t about 1620 cm-l t h a t can be assigned t o an a r o m a t i c
r i n g s t r e t c h i n g v i b r a t i o n w i t h some c o n t r i b u t i o n from t h e
deforma ion amino a c i d band 1; and ( e ) t h e m e d i m band n e a r
730 cm-' a r i s i n g from t h e C-C1 ( t r a n s ) s t r e t c h i n g .
The assignments a r e i n agreement with
t h e g e n e r a l I R l i t e r a t u r e and with d a t a r e p o r t e d f o r t h e
meta isomer of Melphalan [ 1 7 , 2 0 , 4 9 ] .
MELPH ALAN 271
0.8
O.?
0.6
0.5
W
0
z
a
a
a 0.4
0
v)
m
a
0.3
0.2
0. I
I l l I I 1 1 1 1 1 I l 1 1
I 240 270 300 330 3
WAVELENGTH
Pfgurc 1. Ultravfolct Spectrum of Melphalan fa Xethanol (*lo ug/ml).
0
0
0
rl
a,
0
a
2
L
0
0
‘0
QJ
h
1
M
212
0
0
Q
0
0
W
r\
a
a,
.d
0 M
0 a
a 5
W
0 a,
0 a
2
0
s-
0
s
0
0
P
N
0
0
N
a0
0
R
0
m
a,
0 Ll
0 1
.Q
0 bc
.rl
k
0
-0
8
273
4.1.3 Nuclear Magnetic Resonance
Proton magnetic resonance spectra of

melphalan in dimethylsulfoxide (DMSO)-d6 and D 0--DC1
solutions and of melphalan hydrochloride in DM'h-d6 are
shown in Figure 4. The spectra were run on a Varian FT-80A
spectrometer at 80 MHz. The assignments of the chemical
shifts are tabulated in Table I.
They are within reasonable agreement

with the literature data reported for phenylalanine [50] and
melphalan [51], with one exception. Hsieh [33] reported for
melphalan in D20--DC1 the following assignments: 9 C 1 ,
4.10 ppm; S N , 3.60 ppm. Based on a comparison of the
spectra in DMSO-d6 and D20--DC1, we reversed the assign-
ments; our assignment was confirmed by a two-dimensional
proton--carbon correlation experiment run by D r . James
Shoolery of Varian, Palo Alto, California.
Fully decoupled 13C Spectra of

Melphalan, recorded on a Varian FT-80A spectrometer are
shown in Figure 5 . The chemical shift assignments are
presented in Table 11.
Integration of the signals in the

proton NMR spectra and multiplicity in the coupled 13C NMR
spectra support the assignments in Tables I and 11.
In both types of spectra, the marked

influence of the ionization form of the molecule on the
chemical shifts is apparent.
4.1.4 Mass spectra
A mixture of melphalan and its hydro-

lytic decomposition products was silylated and separated by
gas chromatography; the peaks were identified by mass
spectrometric analysis [52]. GC-MS was also used for the
identification of the HPLC melphalan peak separated from
plasma or hydrolysis mixtures [40,53,54].
A mass spectrum of the methyl ester of

N-trifluoroacetylmelphalan was reported, without analysis of
the fragmentation pattern [41]. The mass spectrum of mel-
phalan-dz (direct insertion mode) showed a molecular ion at
m/e 306 (3% relative intensity) and a base peak at m/e 232
(M-CH(NH2)COOH)+. A linear relationship was demonstrated
between the composition of melphalan/melphalan-d mixtures
and the peak intensities at m/e 230 and m/e 232 f441.
G
3
.ri
a,
a
ah
4
c
c.
6
rl
a
c
0 m
-+
Table I. Chemical S h i f t Assignments i n t h e P r o t o n NMR S p e c t r a of Melphalan
. ..
5
Melphalana Melphalan Hydrochloridea Melphalanb

Proton # (DMSWd6 ) (DMS @d6) (conc. DC1-D20 1 :4, v/v)
1 7.12 ( d , J1.2 = 8.7 Hz) 7.12 ( d , 51.2 = 8.7 Hz) 7.69 ( d , 51.2 = 9.2 Hz)
2 6.67 ( d ) 6.70 ( d ) 7.61 ( d )

I\)
a.
4 3 3.70 (s) 3.71 (s) 4.16 ( t , 53.4 6.0 Hz)
4 3.70 ( s ) 3.71 (8) 3.67 ( t )
5 3.36 (J5.6 4.25 HZ)' 4.04 ( t , J5,6 = 6.2 HZ) d 4.45 ( t , J 5 , 6 6.5 Hz)
6 3.03 ( J 6 , 7 14 HZ)' 3.02 ( d ) d 3.42 (die
7 2.77 (J5,7 = 7.75 H Z ) ~ 3.02 ( d , J5,7 = 6.2 Hz) d 3.41 ( d , J5,7 = 7.0 Hz)
a
i n ppm, downfield from TMS;
For p h e n y l a l a n i n e i n 2NDC1 i n D 2 0 , J
5,6
'
i n ppm, downfi I d from DSS
ABC system, assignments a c c o r d i n g t o [Sob]; probably u n r e s o l v e d ABC system
= 5.5 Hz, J5,7 = 7.7 Hz, J
6 97
= -14.6 Hz [50a]
a
277
Table I1
Chemical S h i f t Assignments i n t h e 13C NMR S p e c t r a of

Melphalan
b
Carbon Melphalana Melphalan Hydrochlorideb Melphalan
atom # D20--DC1 DMSO-db DMSO-d6
172.5 170.2 170.0

139.9' 122.8 125.4
136.2' 145.5 145.1
134.2 130.5 130.4
125.3 112.0 112.1
61.4 52.2 52.2
55.8 53.4 55.5
39.5 41.1 41.2
37.1 34.6 35.9
~ ~~
a) i n ppm, downfield from DSS, assignments from [5Obl

b, i n ppm, downfield from TMS
c, i n t erchangeable assignment
Using chemical i o n i z a t i o n with

i s o b u t a n e and s e l e c t e d i o n monitoring of t h e M + H peak, t h e
d e t e c t i o n l i m i t of t h e methyl ester of N - t r i f l u o r o a c e t y l -
melphalan was less t h a n 500 pg 1411.
The e l e c t r o n impact i o n i z a t i o n spectrum

of melphalan is shown i n F i g u r e 6. It was o b t a i n e d by
direct-probe i n t r o d u c t i o n of t h e sample on a Hewlett-Packard
5995A mass s p e c t r o m e t e r (70 e V ) . The r e l a t i v e i n t e n s i t i e s
of t h e most prominent fragments are given i n Table 111; t h e
abundance cut-off w a s 1%.
4.2 Optical Rotation
The f o l l o w i n g data were r e p o r t e d f o r t h e

monosolvated c r y s t a l s o b t a i n e d by c r y s t a l l i z a t i o n from
methanolic s o l u t i o n [ l o ] :
r
n
a,
rl
z
(I)
I
W
W
0
-.
a r E U
E *
r
I
r
I
r
r
I
Table I11
Fragmentation pattern of melphalan
Ion Probable Structure m/e Relative Abundance, %
304 2.6
306 1.4
M+-COOH 259 2.3

261 1.8
M+-CH~C~ 255 1.2
M+-CH(NH~)COOH 230 100

232 64.8
234 10.6
+
I -CH2C1 181 15.9
3
183 6.1
+
I -CH2=CHC1 168 7.4
3
170 2.2
+
I -CH2C1 119 23.9
5
+
14-CH2CH2C1
118 70.7
MELPHALAN 28 I
22
Solvent Concent rat ion, [ a1
g/100 ml D
1 N HC1 1.33 +7.5" 0.5"

MeFhano 1 0.67 -31.5" f 0.5"
22
-
The D-isomer in 6 N HC1 had [aID - 4.5" f 0.25'.
The USP XX monograph requires the specific

rotation to be between -30" and -36", calculated on the
dried basis and determined at 7 mg/ml in methanol solution
[25]. In the British Pharmacopeia the levorotatory optical
activity is used as an identification test [27].
The following specific rotations of melphalan

were determined at 25" in methanolic solution at a
concentration of 7 mg/ml [47]:
-
A, nm -
[ a ] ,( " )
589 -32.1 i 0.6

578 -33.5 * 0.8
546 -38.3 f 0.7
436 -66.7 * 1.0
365 -102.4 f 1.6
4.3 Melting range
All the melting ranges indicated below are

uncorrected and are reported to occur with decomposition:
Me 1ph a1an.MeOK 182--183" [ 101 (from methanol)

D-Melphalan.MeOH 181.5--182" [lo] (from
me thano1)
DL-Melphalan 180--181" (from methanol)
[10,24]; 172--174" (from
methanol) 1291 ; 167--170"
(from aqueous alcohol [ 111
DL-Melphalan.HC1 182--185" (from 95% ethanol)
[111.
The official compendia require melting points of

about 177" [27] or 180" [26].
4.4 Solubility, Part it ion, Dissociation Constants
Approximate solubilities:
(a) Practically insoluble (1 part in more than

10,000 parts of solvent) in water [25,27,55], in chloroform
[25,$7], and in ether [25,27]; (b) slightly soluble (1 part
in 100 to 1000 parts of solvent) in alcohol [25]; soluble in
150 parts of methanol [27]; (c) soluble (1 part in 10 to 30
parts of solvent) in dilute mineral acids [25,27]; and (d)
soluble in propylene glycol [241.
The logarithm of the part it ion coefficient

between benzene and pH 7.4 phosphate buffer is -1.70 [56].
A partition coefficient of 0.1 was reported for the heptane-
-water distribution 1571.
The following
- pKa
- values were reported:
approximately 2.5 in water; 5.9 in acetone--water, 9:l (v/v)
[581.
5.1 Elemental Analysis
The empirical formula C13H18C12N202 implies the

following percentage compositions: C, 51.16; H, 5.94; N,
9.18; C1, 23.3; and 0, 14.48%.
The following percentages were reported for the

racemic compound: C, 51.1; H, 6.0; N, 8.9; and C1, 23.2%
[lo]. Melphalan has been isolated from methanol as
monosolvated crystals exhibiting N (8.3%) and C1 (21.1%)
[lo]; C13H18C12N202.CH40 implies N (8.3%) and c1 (21.0%).
The USP XX limits f o r the nitrogen content of
melphalan are 8.90--9.45% [25]. Several compendia1 assays
are based on the determination of the chlorine liberated by
alkaline hydrolysis (see 5.4).
5.2 SDectroDhotometric Methods
5 .2.1 Co lor imet r y
The use of 4-(p-nitrobenzyl)pyridine

(NBP) as an analytical reagent for the determination of
alkylating agents [59,60] is based on the series of
reactions presented in scheme IIA. Melphalan is treated at
80--100" with a large excess of the chromogenic reagent to
I
I
f-j
.-'
I8
L
0
ll
+
I
I
I
t$ 0
L
I1
z
X
a!
8 H
H
+
Q
X
0
11
z
t
2
8
prevent competing reactions from traces of water or other

nucleophilic reactants. Addition of base produces a purple
color, the absorbance of which is read immediately at 565 nm
[60]. The intensity of the color produced on alkalinization
is affected by the pH and/or the nature of the buffer used
in the first step. The improvements of the original
procedure [60] were aimed at a better reproducibility and
increased sensitivity (up level) [51,61]. The procedure has
been used to follow the kinetics of hydrolysis [62].
A similar method was described for the

quantitative determination of Melphalan on TLC plates
[63b]. The plate is sprayed with an acidic solution of 4-
pyridinecarboxaldehyde 2-benzothiazolylhydrazone and heated
over an acetophenone bath at 200". After cooling, the plate
is sprayed with triethylamine, and the red spots are
measured at 530 nm with a densitometer. The detection limit
is 0.2 ug. The reactions are shown in scheme 11 B.
Sarcolysin fused with 2-nitroindan-l,3-

dione and dissolved in acetic acid o r 95% ethanol gives an
orange--red color. Sarcolysin heated for 1--2 min with
acetic anhydride gives a yellow color [64].
5.2.2 U 1 trav iolet
Melphalan has been determined

spectrophotometrically at 262 nm in methanol solution
[65]. The assay is linear in the 3 to 15 ug/ml range, with
precision of i-2%. In contrast with the above mentioned
colorimetric methods, the procedure is not stability-
indicating; the major contaminant and degradation product,
the bis(2-hydroxyethyl)derivative, has a W spectrum very
similar to that of Melphalan [481. Ultraviolet spectro-
photometric determinations in aqueous solution were also
reported [66].
5.2.3 Fluorescence
In freshly prepared aqueous solutions

the fluorescence intensity of Melphalan was found to be
proportional to the concentration in the range of 0.05 to 5
pg/ml, but the method does not discriminate between the
parent compound and some of its hydrolytic products [48].
MELPHALAN 285
The solvent systems used for the

analysis of Melphalan are listed below. The composition of
the mixtures is given in volume to volume ratios. In all
cases Whatman paper No. 1 was used.
Developing solvent (v/v) 2

- Reference
-
n-Butanol--conc’d HC1--water 2:1:1 67
sec-Butanol--AcOH--water 5:1:4 >0.9 68
Phenol--wat er 4:l >0.9 68
-
n-But ano 1--(water-saturated) 0.43 43
-
n-But anol--AcOH--wat er 5:2:3 0.87 43
-
n-Butanol--AcOH--water
i-Propanol--ammonia--water
12:3:5
20:1:2
0.80
0.53
48
48
Visualization of the spots was done by examination under W-

light or spraying with ninhydrin. The color-forming
reaction with nitrobenzylpyridine (NBP) (see 5.2.1) has been
used by dipping the chromatograms in solutions of NBP
followed by heating at 90” and treating with triethylamine
[681.
5.3.2 Thin-layer Chromatography
The TLC systems reported in the

literature for the analysis of melphalan are reported in
Table IV.
The spots were located by examination

under W light or spraying with one of following solutions
(detection limits in pg): fluorescamine (0.5), ninhydrin
(l), NBP followed by heating and treatment with a base (5)
[51,63a] , and 0.5% iodine in chloroform. Visualization
using 4-pyridine-carboxaldehyde 2-benzothiazolyl hydrazone
has been described under 5.2.1.
Quantitative determinations were

reported by densitometry [63,72] or, in the case of I4C-
labelled compounds, by scraping or cutting the chromatograms
into strips and measuring the radioactivity in a liquid
scintillation spectrometer [44,70].
Table IV
Thin-layer Chromatography of Melphalan
Plate Solvent (v/v) Re f erence
Silica gel -n-Butanol--acetic acid--water

(7:2:1) or (4:l:l) or (13:3:5)
47,51,69
70,71
or (4:1:5, the upper phase)
Silica gel -
n-Butanol--pyridine--acet ic acid-- 51
water (8:1 :2:9, the upper phase)
Silica gel
Silica gel
-
sec-Butanol--3% armnonia (100:44)
Chloroform--methanol (9:1)
51
51
Silica gel n-Butanol--0.1 -
- N ammonium hydroxide 47
(1:l)
Silica gel
Silica gel
-
n-Butanol--Acetic acid (2:l)
Methanol
47
47
Silica gel Chloroform-methanol--acet ic acid 63a
(75 :20:5)
Silica gel Ethyl acetate--acetone--tetrahydro- 63a
furan--water (4:2:2:2, upper
phase)
Silica gel
Silica gel
-
n-But anol water saturated
-
i-Propanol--water (1:L)
63a
63a
Silica gel Benzene--methanol--acetone--acetic 63a
acid (7:2:6:5)
Kieselguhr -
n-Butanol--water 86:14
HPTLC Silica Ch loroform--me thano1--ace t ic
54
72
ge1 acid (35:17:3)
Cellulose -
n-Butanol--Ethanol--Propionic 73
acid--water (10 :5 :2 :5)
MELPHALAN 287
Melphalan has been converted to its

trimethylsilyl derivative with bis(trimethylsily1)acetamide
and has been analyzed by GC on a 1.8 m x 3 nun column packed
with 2.5% (w/w) SE-54 on acid-washed, silanized Chromosorb W
(80-100 mesh) at 210" (injector temperature, 250"; flame
ionization detector temperature, 215") using nitrogen as the
carrier at 30 ml/min. The order of elution from a partly
hydrolyzed mixture was: melphalan, mono-hydroxy-derivative
VI and di-hydroxy-derivative VII (Scheme 111). The same
elution order was obtained on a SE-30 column; it was
reversed on a more polar liquid phase (OV-17). Identi-
fication of the peaks was done by mass spectrometry [ 5 2 ] .
In another procedure, melphalan was

derivatized by successive acylation with trifluoroacetic
acid and eserification with diazomethane. The samples were
analyzed on a 3% OV-101 Supelcoport (80--100 mesh) 5 foot x
2 nun glass column with temperature programming from 210" to
280" at 8"/min with a helium flow of 20 ml/min. Analysis of
samples extracted from plasma was performed by mass
spectrometry using chemical ionization with isobutane gas
and me1~halan-d~ as internal standard. Selected-ion
monitoring at the M + H peak of derivatized melphalan gave
linear calibration curves over a range of 2 to 200 ng of
drug per ml of plasma. The detection limit was less than
500 pg 1411.
5.3.4 High-pressure Liquid Chromatography
The HPLC procedures reported in the

literature (see Table V) were developed mainly for
biological and stability studies , and they do separate
Melphalan from its hydrolytic degradation products.
Detect ion was performed by W absorbance measurements at
254--263 nm [40,53,74,75,76] or by fluorescence with
excitation at 256 or 260 nm and emission at 350 or 360 nm
[ 7 7 , 7 8 ] . For the W measurements a linear standard curve
was reported from 10 to 500 ng per injection [ 7 5 ] . With the
fluorescence monitoring the detection limit was about 500 pg
of drug injected 1781.
Table V
HPLC Methods
Column Mobile phase ( v / v ) R e f er enc e
lO-prn p-Bondapak c18 2-methoxyethanol--O.l% 75

AcOH g r a d i e n t from
33.7:66.3 t o 51.3:48.7
10- pm S p h e r i s o r b ODS Methanol--0.01 M NaH2P04
L
53
(pH 3) ( 1 : l )
10- um HCODS/SIL x-c18 Acetonitrile--0.0175 M AcOH 76
concave g r a d i e n t from 12:88
t o 80:20
10- pm p-Bondapak c18 Water-methanol ( 1 :1) , 40,74,79
c o n t a i n i n g 1%a c e t i c a c i d
10-pm p-Bondapak c18 2% a c e t i c a c i d - - a c e t o n i t r i l e 54
(78:22)
5-pm S p h e r i s o r b ODS Methanol--water (40 :60) 78
5-~RIS p h e r i s o r b ODS -
Methanol--0.01 M NaH2P04 77
(pH 3 ) ( 7 : 3 )
5.4 Titration
T i t r a t i o n with s i l v e r n i t r a t e i s used both t o

confirm t h e l a c k of c h l o r i d e ions i n t h e sample and, a f t e r
a l k a l i n e h y d r o l y s i s , as an a s s a y f o r t h e drug. The
compendia1 a s s a y s a r e performed p o t e n t i o m e t r i c a l l y u s i n g
s i l v e r and calomel e l e c t r o d e s , t h e l a t t e r modified t o
c o n t a i n s a t u r a t e d potassium s u l f a t e s o l u t i o n [25--271.
V i s u a l t i t r a t i o n was a l s o r e p o r t e d , t h e excess of s i l v e r
being t i t r a t e d with potassium t h i o c y a n a t e i n t h e presence of
f e r r i c ammonium s u l f a t e i n d i c a t o r [51]. Mercuric n i t r a t e
t i t r a t i o n s were a l s o r e p o r t e d [ 6 2 ] .
P o t e n t i o m e t r i c and thymol b l u e i n d i c a t o r t i t r a -
t ions of s a r c o l y s i n i n dimethylformamide s o l u t i o n s were
performed using a 0.1 - N sodium methoxide s o l u t i o n 180).
5.5 I d e n t i f i c a t i o n and P u r i t v Tests i n O f f i c i a l

Compendia
S e v e r a l of t h e f o l l o w i n g t e s t s a r e included i n
melphalan monographs: ( a ) t h e W spectrum of a methanolic
s o l u t i o n should have maxima at 260 and 301 nm [25,27] o r be
i n agreement with t h e spectrum of a r e f e r e n c e s t a n d a r d [26] ;
( b ) t h e IR spectrum i n K B r d i s p e r s i o n should e x h i b i t maxima
only a t t h e same wavelengths as a s i m i l a r p r e p a r a t i o n of a
MELPHALAN 289
r e f e r e n c e standard [26,79]; ( c ) a p o s i t i v e color r e a c t i o n

w i t h 4-(p-nitrobenzyl)pyridine [ 25--27,791; ( d ) a 0.5% (w/v)
s o l u t i o n i n methanol should be l e v o r o t a t o r y ; ( e ) a m e l t i n g
p o i n t of about 177" [ 2 7 ] or about 180" [ 2 6 ] with decomposi-
t i o n ; ( f ) t h e loss on d r y i n g i n vacuum a t 105" t o c o n s t a n t
weight should not be more t h a n 7.0%, and t h e r e s i d u e on
i g n i t i o n should n o t be more t h a n 0.3% [251 or 0.1% 1261; ( g )
a l i m i t t e s t f o r " i o n i s a b l e c h l o r i n e , " performed by d i r e c t
p o t e n t i o m e t r i c t i t r a t i o n of t h e sample 125,271; and (h) a
p o s i t i v e test f o r c h l o r i d e s a f t e r a l k a l i n e h y d r o l y s i s of t h e
sample [25--27,79].
6. S t a b i l i t y , Degradation, Mechanism of Action
I n d i c a t i v e of t h e l a c k of s e l e c t i v i t y of n i t r o g e n
m u s t a r d s , t h e i r d e g r a d a t i o n and b i o l o g i c a l a c t i o n proceed
through a common mechanism, an o u t l i n e of which i s p r e s e n t e d
i n Scheme 111.
According t o t h e n a t u r e of X, Scheme I11 i l l u s t r a t e s a

wide range of r e a c t i o n s , such as t h e h y d r o l y t i c d e g r a d a t i o n
of t h e compound o r b i o l o g i c a l l y s i g n i f i c a n t a l k y l a t i o n s of
r e a c t i v e groups i n p r o t e i n s ( c a r b o x y l , s u l f h y d r y l ) and i n
n u c l e i c a c i d s ( p h o s p h o r y l , h e t e r o a r o m a t i c amino, h e t e r o -
a r o m a t i c h y d r o x y l ) . The o v e r a l l k i n e t i c s of t h e a l k y l a t i o n
depend on t h e r e l a t i v e r a t e s of t h e r e a c t i o n s involved. For
t h e a l i p h a t i c n i t r o g e n m u s t a r d s , t h e f a s t f o r m a t i o n of t h e
c y c l i c immonium i o n V I I I and t h e o v e r a l l second-order
k i n e t i c s f o r t h e r e a c t i o n have been supported by s e v e r a l
a n a l y t i c a l t e c h n i q u e s i n c l u d i n g N M R [ 8 1 ] . There i s l e s s
agreement on t h e r e a c t i o n mechanism f o r t h e aromatic n i t r o -
gen mustards [ 4 ] . The reduced b a s i c i t y of t h e a r o m a t i c
n i t r o g e n mustards r e t a r d s t h e i o n i z a t i o n s t e p and makes i t
rate-determining. It w a s i n i t i a l l y p o s t u l a t e d t h a t t h i s
i o n i z a t i o n i n v o l v e s t h e format i o n of t h e c a r b o c a t i o n I X
[ 9 , 8 2 , 8 3 ] , but t h i s view has been c h a l l e n g e d by a u t h o r s who
c l a i m t h a t t h e c y c l i c immonium i o n is common t o a l l n i t r o g e n
mustards [ 4 0 , 6 2 , 8 1 ] .
The h y d r o l y s i s of melphalan (and of most aromatic

n i t r o g e n m u s t a r d s ) r e p r e s e n t s t h e most important d e g r a d a t i o n
mechanism and has been e x t e n s i v e l y s t u d i e d . The f i r s t
r e p o r t s showed a 22% h y d r o l y s i s under t h e s t a n d a r d
c o n d i t i o n s designed by Ross f o r t h e assessment of aromatic
n i t r o g e n mustard r e a c t i v i t y (30 min r e f l u x i n acetone--water
1:1, v / v ) [ 1 0 , 8 4 ] . Rate c o n s t a n t s f o r t h e d i s a p p e a r a n c e of
melphalan i n v a r i o u s media a r e l i s t e d i n Table V I .
Scheme I11
Helphalan VI VII
A. Hydrolysis of melphalan.
N
0
\o
CHZCHzC1 VIII
X-H
R-N' c R-N-CHzCH2X
I
] : :-
'CH~CH~CL
CHZCHZCl
;i!-R[
B . P o s s i b l e mechanism of a l k y l a t i o n .
Table V I
Rate Constants For t h e Disappearance of Melphalan i n Various Media
Solvent TemDerature. " C k. h-l Reference
Water 0 0.04 40
Water 23 0.13 40
Water 37 0.83 40,90
0.01 M C i t r a t e b u f f e r (pH 3) 25 0.132 53
0.01 3 Phosphate b u f f e r (pH 5) 25 0.140 53
0.01 Z P h o s p h a t e b u f f e r (pH 7 ) 25 0.146 53
0.01 Phosphate b u f f e r (pH 7 ) 41 0.994 53
0.01 tris(hydroxymethy1)- 25 0.176 53
N amiFomethane (pH 9)
2 pH 7 Buffer + 0.019 M NaCl 25 0.103 53
pH 7 Buffer + 0.15 M-NaC1 25 0.043 53
0.013 M HC1 (pH 2.4T 37 0.44 a7
0.013 KH3P04 (pH 2.6) 37 0.78 87
0.156 NaCl (pH 5.4) 37 0.28 87
Burroughs-We1 lcome i n j e c t a b l e 25 0.0057 53
kita
Human plasma 37 0.31--0.45 40
a Three component k i t . Melphalan (100 mg) i s d i s s o l v e d i n 1 m l of 92% e t h a n o l c o n t a i n i n g 2%

(w/v> HC1. This s o l u t i o n i s d i l u t e d with 9 m l of a s o l u t i o n c o n s i s t i n g of 1.2% (w/v) K2HP04
and 60% (w/v> propylene g l y c o l i n water. The f i n a l s o l u t i o n has a pH of about 7.
The d i s a p p e a r a n c e of melphalan from aqueous s o l u t i o n s

h a s been shown t o f o l l o w f i r s t - o r d e r k i n e t i c s [62,82,85] and
t h e c o n c e n t r a t i o n p r o f i l e d u r i n g h y d r o l y s i s is c o n s i s t e n t
w i t h a mechanism c o n s i s t i n g of two c o n s e c u t i v e pseudo f i r s t -
o r d e r r e a c t i o n s (Scheme I11 A) 1531.
Of t h e systems l i s t e d i n Table VI, melphalan i s most

s t a b l e i n t h e Burroughs-Wellcome i n j e c t a b l e k i t . The
i n s t r u c t i o n s recommend t h e use of t h e s o l u t i o n w i t h i n 15--30
minutes. According t o t h e m a n u f a c t u r e r , 8.5% h y d r o l y s i s
t a k e s p l a c e i n 24 h o u r s a f t e r mixing 1861. The s t a b i l i t y o f
melphalan i n c r e a s e s i n a c i d i c s o l u t i o n s and h i g h e r tempera-
t u r e s a c c e l e r a t e t h e h y d r o l y s i s . The t y p e and c o n c e n t r a t i o n
of a n i o n i c s p e c i e s p r e s e n t i n t h e s o l u t i o n a l t e r t h e
h y d r o l y s i s r a t e . The formation of t h e i n t e r m e d i a t e i o n i z e d
s p e c i e s i s r e t a r d e d a n d / o r r e v e r s e d by t h e presence of
c h l o r i d e ions [9,82,87] , r e s u l t i n g i n a s i g n i f i c a n t l y
increased s t a b i l i t y [53,87,88]. The s t a b i l i t y of melphalan
is i n c r e a s e d i n t h e p r e s e n c e of bovine serum albumin, human
plasma, b i l e a c i d s , and b i l e , probably by hydrophobic i n t e r -
a c t i o n s t h a t make t h e c h l o r o e t h y l moiety less a c c e s s i b l e t o
n u c l e o p h i l i c a t t a c k [40,62,74,87,89--911 .
S o l u t i o n s i n methanol were found t o r e t a i n f u l l i n t e g -
r i t y f o r more t h a n 1 2 hours a t room t e m p e r a t u r e and f o r a t
l e a s t 4 weeks at -20" [ 7 8 ] . Melphalan is s t a b l e i n s o l u -
t i o n s of d i m e t h y l s u l f o x i d e a t -20" f o r over a month even
w i t h f r e q u e n t thawing [ 511.
Melphalan i s l i g h t - s e n s i t i v e and t h e packaging and

storing instructions call for tight, light-resistant
containers.
7. T o x i c i t y , D i s t r i b u t i o n , Metabolism, E x c r e t i o n
7.1 Toxicity
D e t a i l e d d a t a on t h e t o x i c o l o g y and pharmacology
of melphalan (and o t h e r a n t i t u m o r a g e n t s ) were r e p o r t e d i n
1965 by Schmidt and c o l l a b o r a t o r s [ 2 1 ] .
The LDIO and LD50 v a l u e s f o r mice and r a t s show

c o n s i d e r a b l e v a r i a t i o n s among s t r a i n s ; i n t r a v e n o u s and
i n t r a p e r i t o n e a l a d m i n i s t r a t i o n s were twice as t o x i c as t h e
o r a l d o s i n g - The L D l o v a l u e s ranged from 4 t o 15 mg/kg f o r
mice and from 1 . 3 t o 3 mg/kg f o r r a t s 13,211. LD values
of 2 t o 16 mg/kg were r e p o r t e d f o r r a t s [ 2 1 , 7 7 , 9 2 j . It w a s
a l s o r e p o r t e d t h a t t h e LD50 v a l u e s i n mice v a r i e d with t h e
time of dosing [ 9 3 ] .
MELPHALAN 293
The c l i n i c a l t o x i c o l o g y is m o s t l y h e m a t o l o g i c a l
[21.
7.2 Carcinogenicity
Melphalan induced cancer at t h e s i t e of

i n j e c t i o n ( p e r i t o n e a l c a v i t y ) i n mice and r a t s given t h r e e
i n j e c t i o n s per week a t t h e maximally t o l e r a t e d dose [ 9 4 ] .
It was a l s o r e p o r t e d t o i n c r e a s e t h e r i s k f o r development of
leukemias [ 3 ] .
7.3 Distribution
Following o r a l a d m i n i s t r a t i o n t o human s u b j e c t s ,
peak blood l e v e l s for melphalan were seen a t 2 hour [ 4 4 ] ,
much slower than i n animals [ 3 , 9 1 , 9 5 ] . The drug i s w e l l
absorbed when given t o animals by t h e o r a l r o u t e [ 3 , 9 1 ] , but
c o n s i d e r a b l e v a r i a b i l i t y i n a b s o r p t i o n by humans was
reported [41,96,97].
A f t e r i n t r a v e n o u s a d m i n i s t r a t i o n , melphalan
d i s a p p e a r s from t h e plasma with h a l f - l i v e s of 67 min and 160
hour [ 4 4 ] . I n a p a t i e n t administered a 5-minute i n t r a v e n o u s
bolus of 140 mg of melphalan p e r m 2 , t h e a l p h a and b e t a
h a l f - l i v e s i n plasma were found t o be 6.2 and 53.5 minutes,
r e s p e c t i v e l y [ 5 4 ] . Binding of t h e drug t o plasma p r o t e i n s
i s e x t e n s i v e [ 4 4 ] , confirming t h e i n - v i t r o s t u d i e s
[ 4 0 , 4 8 , 7 4 , 8 9 , 9 8 ] ; t h i s might have a profound e f f e c t on t h e
in-vivo s t a b i l i t y of t h e drug [ 9 7 ] . The dihydroxy d e r i v a -
t i v e VII is d e t e c t a b l e i n plasma more than t h r e e weeks a f t e r
a d m i n i s t r a t i o n [ 7 6 ] . It was suggested t h a t t h e prolonged
decay phase [541 i s due t o h y d r o l y s i s products and
m e t a b o l i t e s r a t h e r than t h e p a r e n t compound [ 4 1 ] .
The a b s o r p t i o n from t h e p e r i t o n e a l c a v i t y one

hour a f t e r a d m i n i s t r a t i o n was 25% [ 5 7 ] .
The kidney, l i v e r , and s p l e e n had t h e h i g h e s t

s p e c i f i c a c t i v i t y a f t e r a d m i n i s t r a t i o n of l a b e l e d melphalan
t o r a t s and mice [ 6 8 , 9 8 ] , while i n dogs and monkeys t h e
h i g h e s t l e v e l s of drug were i n t h e b i l e [ 3 , 7 5 , 9 9 ] . No
s i g n i f i c a n t c o n c e n t r a t i o n s i n t h e tumor were n o t e d , and t h e
i n h i b i t i o n of t h e tumor growth could not be a t t r i b u t e d t o
i n t e r a c t i o n s with p r o t e i n s , DNA, o r RNA w i t h i n t h e tumor
c e l l [ 6 8 ] . C e l l u l a r t r a n s p o r t of melphalan by amino a c i d
c a r r i e r systems has been r e p o r t e d [ 100--1051.
7.4 Metabolism
Only t h e mono- and d i h y d r o x y - d e r i v a t i v e s V I and

VII were identified in plasma and urine [ 8 5 , 9 9 ] , and it is

difficult to distinguish between m tabolic changes and
chemical degradation. Up to ten “C-labelled products of
metabolism or hydrolysis of melphalan were detected without
identification in the serum, urine, and bile of experimental
animals. Metabolic pathways similar to that of phenyl-
alanine were suggested [ 3 1 .
7.5 Excretion
In man, urinary excretion accounts for most drug

elimination after intravenous administration ( 5 0 % within 24
hr, 7 5 % over 7 days), and only small amounts are recoverable
from the stool [ 4 4 , 8 6 ] . Following oral administration of
labelled melphalan, 30% of the label was recovered in urine
over 9 days and 20--50% in feces over 6 days [ 4 4 ] .
In animals also, urinary excretion appears to be

the major means of elimination of the drug from the body
[3,68,91,99].
8. Determination in Body Fluids and Tissues
The separation and measurement of melphalan in

biological samples are hampered by its instability and by
the amphoteric nature of the molecule. Some of the earlier
publications did not make corrections for reversible protein
binding and decomposition during the work-up of the sample.
They also lacked sensitivity and specificity.
The following techniques were reported: administration

of labelled melphalan, followed by radioactivity measure-
ments [ 4 4 , 9 1 , 9 8 ] ; formation of a colored complex by oxida-
tion with FeCl and H202 [ 1 0 6 ] ; colorimetric assay with 4-
(p-nitrobenzyljpyridine (detection limit--5 pg/ml of plasma)
[60,61,107,108]; fluorescence measurements (interference by
tissue constituents) [ 4 8 ] ; thin-layer chromatography and
densitometry (detection limit--20 ng/ml of plasma) [ 7 2 ] ;
thin-layer chromatography followed by radioactivity
measurements or derivatization and mass spectrometry [ 4 4 ] ;
high-pressure liquid chromatography (detection limit with
fluorescence detection--less than 5 ng/ml of plasma)
[3,40,54,74,76--78,91,99]; and derivatization--gas
chromatography--chemical ionization mass spectrometry
(detection limit--less than 2 ng/ml of plasma) [ 4 1 ] .
MELPH ALAN 295
9. I d e n t i f i c a t i o n and Determination i n Pharmaceutical

Preparations
9.1 Tablets
Identification
The I d e n t i f i c a t i o n T e s t s i n t h e o f f i c i a l com-
pendia a r e s i m i l a r t o t h o s e used f o r t h e drug s u b s t a n c e :
( a ) e x t r a c t i o n of t h e drug with methanol, t r e a t m e n t with 4-
( p - n i t r o b e n z y 1 ) p y r i d i n e a t 80" and a l k a l i n i z a t i o n w i t h
potassium hydroxide o r ammonia a f t e r c o o l i n g , producing a
v i o l e t t o r e d - - v i o l e t c o l o r [ 2 5 , 4 6 , 7 9 ] ; ( b ) t h e W spectrum
of a methanolic e x t r a c t e x h i b i t s a maximum a t 260 nm and a
l e s s w e l l d e f i n e d maximum a t 301 mn [ 2 5 , 4 6 ] ; and ( c ) t h e
r e t e n t i o n t i m e of t h e major peak i n t h e HPLC chromatogram of
t h e Assay P r e p a r a t i o n is t h e same a s t h a t of a Standard
P r e p a r a t i o n of Melphalan Reference Standard [ 7 9 ] .
4 ssay
The following procedures were r e p o r t e d f o r t h e

a s s a y of Melphalan T a b l e t s : ( a ) h y d r o l y s i s w i t h aqueous
potassium hydroxide under r e f l u x and p o t e n t i o m e t r i c
t i t r a t i o n of the l i b e r a t e d c h l o r i d e i o n s w i t h s i l v e r n i t r a t e
i n t h e presence of n i t r i c a c i d . (Corrections for "ionisable
c h l o r i n e " a r e made by t i t r a t i n g under t h e same c o n d i t i o n s a
sample t h a t i s not s u b j e c t e d t o h y d r o l y s i s [ 2 5 , 4 6 ] . 1; ( b )
HPLC Assay of an e t h y l a l c o h o l - a c e t i c a c i d e x t r a c t a g a i n s t a
Reference Standard m a t e r i a l (chromatographic c o n d i t i o n s :
reversed-phase mode; c18 column; methanol--water--acetic
a c i d , 50:49:1 ( v / v ) , mobile phase; d e t e c t i o n a t 254 nm
[ 7 9 ] ) ; and ( c ) e x t r a c t i o n of t a b l e t s w i t h methanol or
e t h a n o l and UV measurements a t 260 o r 262 nm. L i n e a r i t y i n
t h e 3--15 pg/ml and s t a n d a r d e r r o r s of l e s s t h a n 2% were
r e p o r t e d [65,109,110]. A s i m i l a r procedure i s used f o r t h e
d e t e r m i n a t i o n of t h e Content Uniformity of t h e t a b l e t s [ 7 9 ] . .
The c o n c e n t r a t i o n p r o f i l e d u r i n g t h e d i s s o l u t i o n
t e s t i n g of melphalan t a b l e t s i n water and simulated g a s t r i c
f l u i d was determined by WLC. A method of d a t a a n a l y s i s was
developed t o t a k e i n t o account t h e spontaneous drug degrada-
t i o n [111].
9.2 Melphalan I n j e c t i o n [46]
Melphalan I n j e c t i o n i s d e f i n e d a s a s t e r i l e
s o l u t i o n of melphalan h y d r o c h l o r i d e . It i s a v a i l a b l e
commercially as a k i t comprising melphalan i n a s e a l e d
296 L. VALENTlN FEYNS
c o n t a i n e r , a d i s s o l v i n g s o l v e n t , and a d i l u t i n g s o l v e n t ( s e e
f o o t n o t e i n Table VI).
The i d e n t i f i c a t i o n r e q u i r e m e n t s i n c l u d e examina-
t i o n of t h e W spectrum of a methanolic s o l u t i o n , p o s i t i v e
r e a c t i o n with 4-(p-nit robenzyl ) pyr i d i n e , p o s i t i v e r e a c t i o n
for chlorides a f t e r alkaline hydrolysis, levorotatory
o p t i c a l a c t i v i t y f o r a s o l u t i o n i n methanol, and a m e l t i n g
p o i n t of about 177" w i t h decomposition.
The a s s a y i s performed by p o t e n t i o m e t r i c
t i t r a t i o n of t h e c h l o r i d e i o n s l i b e r a t e d by a l k a l i n e
h y d r o l y s i s , a s d e s c r i b e d under 9.1 Assay ( a ) .
Other p u r i t y r e q u i r e m e n t s i n c l u d e a c l a r i t y and
a c i d i t y of s o l u t i o n t e s t , and l i m i t s f o r i o n i z a t i o n
c h l o r i n e , loss on d r y i n g , and s u l p h a t e d ash.
Acknowledgments
The a u t h o r wishes t o thank D r . Richard Lindauer of t h e

United S t a t e s Pharrnacopeia, R o c k v i l l e , Maryland f o r h i s
h e l p f u l comments on t h e manuscript and f o r p e r m i s s i o n t o use
d a t a o b t a i n e d i n t h e Drug Research and T e s t i n g Laboratory of
USP. The h e l p of D r . James Shoolery of Varian A s s o c i a t e s ,
P a l o A l t o , C a l i f o r n i a and of D r . J i m K e l l e y of t h e N a t i o n a l
Cancer I n s t i t u t e , Bethesda, Maryland i n t h e i n t e r p r e t a t i o n
of t h e NMR and mass s p e c t r a , and t h e t e c h n i c a l a s s i s t a n c e of
M r . Anthony J . Manna and M r . G. S c o t t Kobler a r e g r a t e f u l l y
acknowledged. C r e d i t f o r p r o c e s s i n g t h e manuscript belongs
t o Mrs. P a t r i c i a Perando and Mrs. Barbara Bowman.
MELPHALAN 297
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88. A. D. Chinaeva and A. Y. B e r l i n , Zh. Obshch. Khim. 33 -

-
( 9 ) 3082 (1963); C.A. 60, 1552f (1964).
89. H. Ehrsson and U. Lonroth, J. Pharm. S c i . 71 ( 7 ) 826

(1982).
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90. S. Y. Chang, T. L. Evans, D. S. A l b e r t s , and I. G.
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S i p e s , L i f e Sciences 23 1697 (1978).
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Rep. 61 1637 (1977).
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Y. Oketani, et a l . , Oyo Yakuri 13 ( 6 ) 899 (1977); C.A.
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88 487 (1978).
93. N. A. Lesnaya and T. G. Lobova, B u l l . Exp. B i o l . Med.

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76, 837 (1972); c i t . by 1601.
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(1964).
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96. D. S. A l b e r t s , e t a l . , Proc. Am. Assoc. Cancer Res. 19
334 (1978).
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97. D. S. A l b e r t s , e t a l . , C l i n . Pharm. Exp. Therap. 26,
737 (1979).
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98. -
P. Cohn, B r . J . Cancer 11, 258 (1957).
99. R. K. Brown, G. Duncan and D. L. H i l l , Cancer T r e a t .

Rep. 64,
643 (1980).
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(1979).
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Cancer R e s . 39 ( 2 , p t . 1) 353
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L e t t . 6 ( l ) , 7 (1979).
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Pharmacol. 27 (24) 2865 (1978).
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B i o l . Chem. 254 ( 4 ) , 1057 (1979).
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D. T. V i s t i c a , Biochim. Biophys. Acta 550 ( 2 ) 309
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For t h i s p r o f i l e , t h e l i t e r a t u r e h a s been searched

through Chemical A b s t r a c t s Vol. 96 (1982).
MOXALACTAM DISODIUM
Leslie J. Lorenz and Patricia N. Thomas
Lilly Research Laborntvries
Indititiapvlic, Indiana
1. Description 306
1.1 Name 306
1.2 Structure 306
1.3 Molecular Formula 306
I .5 Appearance 307
2.2 Nuclear Magnetic Resonance Spectrum 307
2.6 Differential Thermal Analysis 315
2.7 Thermogravimetric Analysis 315
2.9 Solubility Properties 315
3. Chemical Synthesis 316
4. Stability 318
4.1 Bulk Stability 318
4.2 Solution Stability 318
5. Bacteriology and Pharmacokinetics 318
5.I Bacteriology 319
5 . 2 Pharmacokinetics 320
6.1 ldentification Tests 322
6.2 Quantitative Tests 323
6.3 Related Substance Assays 326
7. Analysis of Biological Samples 327
7.1 Microbiological Assay 321
7.2 Chromatography 327
8. Analysis of Pharmaceutical Formulations 327
References 329
Copyright C 1984
VOLUME 13
305 hy the American Pharmaceutical Association
ISBN 0-12-260813-5
306 LESLIE J. LOREN2 AND PATRICIA N. THOMAS
1. Description
1.1. Name
Moxalactam disodium is a drug entity which was discovered
at Shionogi and Company, Limited, Osaka, Japan, and codevel-
oped with Eli Lilly and Company. The drug is marketed under
the trade names of MOXAM" and LAMOXAMTN. The chemical entity
is the disodium salt of (6R,7R)-7-[[carboxy(4-hydroxy-phenyl)-
acetyl]amino]-7-methoxy-3-[ [ l-methyl-1H-tetrazole-5-yl )thiol-
methyl ]-8-oxo-5-oxa-l-azabicycl o[ 4.2.O]oct-2-ene-2-carboxyl i c
acid.
1.2. Structure
H OCH3H
.NI-~++JH
E O H 1 TET
HO
N1 2/ S k N
0 C6*
3'
'=YN
CH3
As seen in the structure, moxalactam disodium has three

asymmetric centers. Two centers in the ring system, C6 and
C7, are stereospecifically defined during the biosynthesis of
the penicillin used to produce the compound. A third asym-
metric center exists adjacent to the amide carbon on the side
chain of the antibiotic. The configuration o f this center is
free to equilibrate and thus a pair of diasterioisomers is
possible for moxalactam disodium. The rate of interconversion
between the isomers increases with increasing acidity, with
the maximum rate occurring at a pH of about 2.5. For solu-
tions with pH lower than 2.5, the rate of interconversion is
decreased only slightly from the maximum rate.
1.3. Molecular Formula
The molecular formula for moxalactam disodium i s
c2 oH2 0N609SNa2.
MOXALACTAM DISODIUM 307
1.4. Molecular Weight

The molecular weight f o r moxalactam disodium i s 564.44.
1.5. Appearance
Moxalactam disodium i s a white t o s l i g h t l y cream-colored
amorphous powder.
The infrared spectrum of moxalactam disodium i n a potas-
sium bromide p e l l e t i s presented i n Figure 1. The assign-
ments for the spectral peaks a r e given i n Table 1.
Table 1
~~~ ~~
IR Absorption Band (cm-l) Assignment

3600-2500 g-OH, H20, OH
(broad multiple bands) stretching, s t r o n g
hydrogen bonding
1770 8-lactam, C=O stretching
1680 Amide I , C=O stretching
1610 C O T , asymmetrical
stretching
1515 Amide I1
1410 CO;, symmetrical
stretching
1380, 1350 Tetrazol e , N-CH3 , CH2
(C-H bending)
1250 (broad) @-OH C-0 stretching
820 p - d i s u b s t i tuted phenyl
(C-H bending )
2.2. Nuclear Magnetic Resonance Spectrum

Figure 2 shows the proton magnetic resonance spectrum
f o r moxalactam disodium. The spectrum was recorded on a
60 MHz instrument. An interpretation o f the spectrum i s pre-
sented i n Table 2. The spectrum i s complex, f o r the config-
uration a t s i t e 01 undergoes rapid equilibrium and t h e proton
a t cc i s exchanged w i t h deuterium. A t h i g h resolution such a s
a t 360 MHz every resonance i s s p l i t except f o r that of Ho.
1
1.282 \I \ I 1
" V
.ooo -
I 1 I I I 1 I I I I I I I
4000 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400
I/CM
Figure 1. The Infrared Spectrum of Moxlactam Disodium i n a
KBr Pellet
E
cv .-3
ir
0
c3
V
a,
Q
Ln
S
0
42
0
L
a
310 LESLIE J . LORENZ AND PATRICIA N . THOMAS
Table 2
Proton Magnetic Resonance Spectrum
H
0
H oCH3
r I I H H
- 0 Na+ I U I
Na+ -0
2=*
€!E Multiplicity
3.5 two singlets
4.0 two singlets
4.1 complex AB patterns
4.5 complex AB patterns
5.1 two singlets
6.83 doublet j-8Hz
not vi sibl e
fully deuterium
exchanged
Figure 3 shows the carbon-13 nuclear magnetic resonance
spectrum obtained for moxalactam disodium. The spectrum was
run in deuterium oxide with dioxane present as a spectral
marker. Moxalactam again has a complex spectrum, for the
configuration at the C(11) carbon undergoes inversion. This
tends to cause doublipg of peaks; therefore, assignments for
the aromatic resonances are difficult. Table 3 provides the
assignments for the carbon-13 spectrum.
!
L I I I I I I I I I I I I I 1 I I I I I 1 1
200 150 100 50 0
PPM (6)
-
F i sure 3. The Carbon-13 Nucl ear Magnetic Resonance
S ectrum for Moxalactam Disodium in D20
Paus Dioxane
312 LESLIE J . LORENZ AND PATRICIA N. THOMAS
1 TET
HO 4"\ /1" 7
k N
1
3'
'VN CHS
Table 3
p pm Assignment
32.4 N-CH3
34.8 C(3' 1
53.8 OCH 3
61.5 C(11)
65.9 C(4)
67.5 Dioxane marker
83.5 C(6)
94.9 C(7)
116.n
124.4 aromatic carbons
124.6 C(2") ,C(3"),
129.5 C(5") ,C(6"),
C(2), and C(3)
154.8 C (1-TET)
155.8 C(4")
163.2 C(8)
167.2 2c01
174.6 C(12)
176.7 C(10)
2.3. Mass Spectrum
Moxalactam disodium can y i e l d a mass spectrum when r u n
i n the f i e l d desorption mode. The major ions f o r moxalactam
disodium are a t m/z o f 116 and 130. These are a t t r i b u t e d t o
1+' 1 +'
N-N I
I
CH,
derived from t h e t e t r a z i n e moiety a r i s i n g from p y r o l t i c

processes.
The f i e l d desorption mass spectrum o f moxalactam d i a c i d

gives i o n s a t m/z o f 543, 521, 477, and 361. The majortion
i s a t m/z o f 521 which i s t h e quasi-molecular i o n [ M t H ] A .
small i o n i s observed a t m/z o f 543 which i s t h e c a t i o n i z e d
molecular i o n o f moxalactam [MtNa]'. The i o n a t m/z o f 477
i s t h e r e s u l t o f t h e decarboxylation o f t h e parent compound.
The i o n observed a t m/z o f 361 i s l i k e l y caused by t h e e l i m i -
n a t i o n o f C o n and
N-N
" A
N\N s t i
I
CH,
from t h e parent compound.
2.4. U l t r a v i o l e t Spectrum
F i g u r e 4 shows t h e u l t r a v i o l e t spectrum f o r moxalactam
disodium. The major chromophore c o n t r i b u t i n g t o t h e u l t r a -
v i o l e t absorbance spectrum i s t h e 3-oxacephem nucleus. This
e n t i t y leads t o peaks a t 271 nm (~=12,100) which i s assigned
t o a r-m* t r a n s i t i o n and a t 229 nm (~=18,000) which i s a
n-m* t r a n s i t i o n .
253-
.ooo -
I I I I I I I I
210.0 235.0 260.0 285.0 310.0 335.0 360.0 385.0 410.0
NM
Figure 4. The U l t r a v i o l e t Spectrum of Moxalactam Disodium
i n Water
2.5. Optical Rotation

In section 1.2. moxalactam disodium i s described as con-
sisting of a pair of diasterioisomers which can interconvert
in solution. The observed value for optical rotation is
therefore a function of the isomer distribution in solution at
the time of measurement. No experimental data areavailable
for the rotation of the pure isomers.
2.6. Differential Thermal Analysis
The thermogram for moxalactam disodium shows an exotherm
at about 170°C. This exotherm indicates the decomposition of
the substance.
2.7. Thermogravimetric Analysis
The thermogram for moxalactam disodium shows a weight
loss throughout the curve beginning at about 30°C. A major
loss in mass occurs near 170°C which corresponds to the exo-
therm in the DTA curve for moxalactam disodium. Moxalactam
disodium is thermally labile and undergoes increased decompo-
sition with increasing temperature.
2.8. Dissociation Constants
The following dissociation constants have been deter-
mined for moxalactam disodium:
Solvent pKa
Carboxyl 1 Carboxyl 2 Phenol
H 20 2.4 3.5 9.95
66% DMF 4.9 6.1 12.9
2.9. Solubility Properties
The solubility properties of rnoxalactam disodium are
described in Table 4.
316 LESLIE J . LOREN2 AND PATRICIA N . THOMAS
Table 4
Sol vent Sol ubi 1i t y (mg/ml )
Water >loo.
-
pH 1.2 (USP XIX) >loo.
-
pH 4.5 (USP XIX) goo.
pH 7.0 (USP XIX) >loo.
-
Methanol -
>50. b u t <loo.
Octanol <5.
Isopropanol 4.
Diethyl e t h e r <0.5
Ethyl a c e t a t e <0.5
Chloroform <0.5
Benzene <0.5
Cycl ohexane <0.5
2.10. Crystal Properties
S a l t s of moxalactam have been prepared w i t h cations such
a s sodium, potassium, and ammonium. These s a l t s tend t o
c r y s t a l l i z e , w i t h the preferred form of moxalactam having t h e
R configuration.
3. Chemical Synthesis
Otsuka -et -
a l . (1) have provided several synthetic routes
t o moxalactam and other s i m i l a r types of compounds. A flow
sheet indicating a s y n t h e t i c route t o moxalactam i s shown i n
Figure 5.
6-Aminopenicillanic acid ( I ) i s acylated, oxidized and

e s t e r i f i e d t o give the sulfoxide ester (11). Sulfoxide ester
(11) i s converted t o i t s C-6 epimer (111) by treatment w i t h
trirnethylchlorosilane and triethylamine. Rearrangement of
I11 using triphenyl phosphine y i e l d s the epioxazol ine (IV).
The epioxazol ine i s successively t r e a t e d w i t h chlorine,
sodium iodide, cuprous o x i d e and boron t r i f l u o r i d e t o produce
.
the exomethylene compound (V) Methoxylation u s i n g chlorine
w i t h a methoxide followed by reaction with l-methyl-1H-
tetrazol e-5-thiol and p a r t i a l de-blocking u s i n g phosphorous
pentachloride converts exomethylene ( V ) i n t o the nucleus (VI).
Acylation of the nucleus with an appropriately blocked side-
chain (VII) followed by f i n a l de-blocking using aluminum
chloride gives moxalactam acid ( V I I I ) .
MOXALACTAM DISODlUM 317
VII Vlll
Figure 5. The Chemical Synthesis of Moxalactam Disodium

318 LESLIE J. LORENZ AND PATRICIA N . THOMAS
4. Stability
4.1. Bulk Stability
When moxalactam undergoes degradation, two major pro-

ducts a r e observed. These substances a r e the decarboxylated
product o f moxalactam and the t h i o t e t r a z o l e ; the s t r u c t u r e s
a r e given be1 ow.
N-N
XN,i
C=O I
Na+ - 0' CH,
DecarboxylatedMoxalactam
\
C"3
Thiotetrazok
Decarboxylation o f moxalactam i s the dominant degrada-

tion pathway i n the dry s t a t e . Elimination o f the t h i o t e t r a -
zole sidechain i s the dominant degradation pathway i n solu-
tion.
4.2. Solution S t a b i l i t y
Moxalactam formulations i n s t e r i l e v i a l s remain s t a b l e
for u p t o 24 months when stored a t 25°C o r below (38).
MOXAMmreconstituted i n various commonly used intramuscular
and intravenous d i l u e n t s i s s t a b l e f o r 96 hours a t room
temperature ( 2 ) .
5. Bacteriology and Pharmacokinetics
The s t r u c t u r a l design of moxalactam includes four
elements which contribute t o i t s unique biological properties.
The oxygen atom, at a key position in the nucleus, gives

moxalactam greater antibacterial activity than its cephalo-
sporin analogue. The methyl tetrazolethio moiety tends to
maximize activity of the drug. The 7(a)-methoxyl substi tuent
confers .6-lactamase stabil i ty to the drug. The p-hydroxy-
phenylmalonyl group positively influences the 8-lactamase
stability and antibacterial spectrum of the drug as well as
influencing the pharmacokinetic parameters leading to a half-
life without high serum binding (3,14,38).
5.1 . Bacteri o 1 ogy
The bactericidal activity of moxalactam results from
inhibition of cell wall synthesis.
5.1.1. Susceptibi 1i ty Tests
Susceptibility testing for moxalactam disodium can be
accomplished through the use of diffusion discs and solution
M I C determinations which may include the use of systems such
as the AutobacB. For moxalactam, susceptibility is defined
as a minimum inhibitory concentration (MIC) of 16 mcg or less
per mL; resistance is defined as an MIC value of 64 mcg or
more per mL (55). Disc diffusion methods that require
measurement of zone diameters give the most precise estimate
of antibiotic susceptibility. A procedure has been recom-
mended for use with discs to test the susceptibility o f
various gram-positive and gram-negative organisms to moxalac-
tam (53). Highly susceptible organisms produce zones of
20 mm or greater while zones of 15-19 mm indicate organisms
susceptible to higher dosages of moxalactam. Resistant
organisms produce zones of 14 mm or less. Organisms should
be tested against moxalactam discs since moxalactam is active
against several strains found resistant to other 8-lactam
antibiotic discs in in vitro testing. The agar-dilution
method of Sutter et a 1 . 0 is recommended for susceptibility
testing of anaerobic organisms.
5.1.2. In Vitro Susceptibility
Moxalactam is active against most commonly occurring
gram-negative bacteria (4-34). Studies showed, for example,
that 90-100% of Enterobacteriaceae were susceptible at 4 mcg
or less per mL, including many strains resistant to the
cephalosporins, cephamycins, semi-synthetic penicillins, or
aminoglycosides (9 ,10,13 ,16 ,18,22,24,26). Moxal actam
inhibited most strains of gram-positive aerobic organisms at
concentrations of 8 mcg/mL or less (6,10,14,22,24-30).
320 LESLIE J . LORENZ AND PATRICIA N. THOMAS
Moxalactam is active against most commonly encountered anaero-

bic bacteria (6,10,14,23-25,29,30,35-37) and is more active
against certain strains than cefotaxime, cefoperazone, and
cefoxitin. For many isolates, the concentration of moxalac-
tam required for bactericidal activity is the same as or two-
fold greater than the MIC (23,27,28). For most bacterial
strains, increasing the inoculum size has little or no effect
on the MIC of moxalactam (6,9,10,13,16,21).
5.1.3. Resistance to 6-lactamase
Because of its unique chemical structure, moxalactam is
not only resistant to hydrolysis by 6-lactamase, but acts as
a potent inhibitor of several of these enzymes (29,39,40).
5.1.4. Protein Binding
By the dialysis method, the protein binding of moxalac-
tam was 43 percent and by the agar-diffusion method, 40 per-
cent (6). By an ultrafiltration method, the binding of moxa-
lactam in pooled fresh human plasma was 50.7 percent at
physiologic temperature and pH (38).
5.1.5. Synergism
Moxalactam in combination with several aminoglycosides,
9.gentamycin or tobramycin, has been shown to be syner-
gistic in vitro when tested against certain bacterial
strai n s 7 1 m 21,24 ,2 6 ,38 ,4 1,42 ) .
5.2. Pha rmacokineti cs
High concentrations are achieved in the serum and urine
after intravenous and intramuscular administration of moxa-
lactam. Significant concentrations also are obtained in
certain body tissues and fluids, including the cerebral spinal
fluid.
5.2.1. Serum Concentrations - Intravenous and
Intramuscular
After rapid intravenous infusion (2 minutes) of 500 mg
and 1 g doses of moxalactam in normal adults, mean peak serum
levels of 57 mcg/mL and 95 mcg/mL, respectively, were achieved
(38). Peak serum concentrations at the end of single 20-min-
Ute infusions were: 24.1 mcg/mL after 250 mg; 47.8 mcg/mL
after 500 mg; 101.2 mcg/mL after 1 g; and 204.3 mcg/mL after
2 g 138). After doses of 1 g or more, concentrations of
moxa actam persist in the serum for 12 hours (38), but
MOXALACTAM DlSODlUM 32 I
intravenous administration of 4 g doses, every 8 hours f o r 7

doses, produced no evidence of accumulation. Following i n t r a -
muscular administration of 250 mg, 500 mg, and 1 g doses of
moxalactam t o normal adults, the mean peak serum concentra-
tions were 10, 16, and 27 mcg/mL, respectively, occurring a t
60-120 minutes (38).
5.2.2. Serum Half-life
The h a l f - l i f e a f t e r an intravenous dose i s approximately
1.9 hours 114 minutes); a f t e r intramuscular injection, the
half-1 i f e s about 2 . 1 hours (126 minutes) (38,43-45). In
pat i en t s w t h reduced renal function, the serum h a l f - l i f e of
moxal actam i s s i g n i f i c a n t l y prolonged.
5.2.3. Urine Concentrations, Excretions and
Renal Clearance
Moxalactam i s excreted primarily by the kidneys; small
amounts are excreted i n the stool via the bilary t r a c t , b u t
non-renal clearance appears t o be low (46). Urinary concen-
t r a t i o n s of moxalactam were highest d u r i n g the f i r s t 2-4
hours a f t e r intravenous administration: 170 mcg/mL f o r
250 mg; 446 rncg/mL f o r 500 mg; 1820 mcg/mL f o r 1 g; and
4220 mcg/mL f o r 2 g doses. The same dosages produced 24 hour
recoveries ranging from 60-90 percent (38). Intramuscular
administration a1 so produced highest urinary concentrations
during the f i r s t 2-4 hours w i t h mean recoveries of 349 mcg/mL
for 250 mg, 469 mcg/mL f o r 500 mg, and 586 mcg/mL f o r 1 g
doses. From 55-65 percent of the d r u g was excreted i n the
f i r s t 24 hours a f t e r intramuscular injection (38). No
detectable breakdown products o r metabolites have been
detected in the urine (46,48). The mean renal clearance of
moxalactam was approximately 78 mL/minute, and i t was found
t h a t probenecid d i d n o t s i g n i f i c a n t l y a f f e c t the elimination
(38). In patients w i t h renal impairment, d r u g clearance was
s i g n i f i c a n t l y reduced (48).
5.2.4. Body Fluid and Tissue Concentrations
Moxalactam readily diffuses i n t o the cerebral spinal
f l u i d (CSF) of patients w i t h and w i t h o u t meningitis (49-52).
Distribution of the d r u g following therapeutic dosages has
been determined i n CSF (49-52), b i l e (38,54,55), aqueous
humor (53,54) , peritoneal f l u i d , pleural f l u i d , p r o s t a t i c
f l u i d , sputum, and several other tissues and f l u i d s . Body
f l u i d and t i s s u e analyses give primarily q u a l i t a t i v e data as
t o the presence o r absence of the d r u g a t a particular s i t e ,
and therapeutic efficacy cannot be predicted from these data.
5.2.5. Pediatric Pharmacology

Serum concentrations of moxal actam were determined i n
newborns, including prematures, and infants being treated f o r
bacterial infections ( 4 9 ) . After a 10 minute intravenous
administration, individual peak serum concentration ranges
were 28.6 mcg/mL (the lowest following the 25 mg/kg dose) t o
260 mcg/mL (the highest following t h e 100 mgfkg dose). The
mean h a l f - l i f e was 5.4 t o 7.6 hours i n neonates l e s s than one
week of age, 4.4 hours i n those one t o four weeks old, and
1.6 hours i n infants over one month of age. The calculated
plasma clearance r a t e s , expressed i n relation t o body surface
area were: from 16 t o 31 mL/min/1.73m3 i n neonates and
137 mL/min/1.73m3 i n infants over one month of age. Serum
values f o r moxalactam given intravenously and intramuscularly
were similar f o r infants l e s s than 7 days old. Intravenous
infusion of moxalactam f o r 30 minutes i n doses of 50 mg/kg
o r l e s s t o children and infants w i t h bacterial meningitis
produced mean serum concentrations and half-1 i f e values
similar t o those i n adults following 1 g doses (50).
6.1. Identification Tests
6.1.1. Infrared
The infrared spectrum of a sample i n a potassium bromide
p e l l e t may be used as an identity t e s t . In such cases, the
infrared spectrum of the sample should compare favorably w i t h
the moxalactam disodium reference spectrum over the range of
2.5 t o 16 microns.
6.1.2. Nuclear Magnetic Resonance
The NMR spectrum may be used a s a means of identification
f o r moxalactam disodium. In such cases, the NMR spectrum of
the moxalactam sample dissolved i n deuterium oxide should
have chemical s h i f t s and integrations over the range of 0 t o
10 ppm which compare favorably t o those of the moxalactam
disodium reference spectrum.
6.1.3. High Performance L i q u i d Chromatography

HPLC i s a means f o r the identification of moxalactam
materials. In such cases, the retention c h a r a c t e r i s t i c s of
b o t h the R and S isomers of moxalactam must compare favorably
t o the elution of the two isomers present i n a reference
sampl e of moxal actam.

6.1.4. T h i n Layer Chromatography
T h i n layer chromatography i s y e t another means f o r
determining i d e n t i t y of moxalactam materials. The mobility
of the moxalactam sample must be identical t o the mobility of
the moxalactam reference standard which i s r u n on the same
TLC plate. The developing solvent f o r TLC consists of 42
parts ethyl acetate: 14 parts glacial a c e t i c acid: 14 parts
a c e t o n i t r i l e : and 18 parts water. A s i l i c a gel type F plate
i s used and i s viewed under short wavelength UV l i g h t t o
detect the p o s i t i o n of the components on the developed plate.
6.2. Quantitative Tests

Amorphous disodium moxalactam and crystal1 ine diammonium
moxalactam s a l t s have been used as standard materials i n the
control of moxal actam products. Special hand1 i n g i s required
f o r a l l moxalactam standards used i n quantitative t e s t i n g of
moxalactam. Amorphous moxalactam disodium i s somewhat unsta-
ble and must be stored i n a dry s t a t e . The dry material i s
hydroscopic and therefore d i f f i c u l t t o weigh. The diammonium
s a l t of moxalactam is a s t a b l e c r y s t a l l i n e substance; however,
i t e x i s t s almost completely i n the R configuration. For most
assay procedures the required R t o S r a t i o i s about one;
therefore, the material must be treated t o e q u i l i b r a t e the
r a t i o of the isomers.
Moxalactam disodium reference standards are equilibrated
under controlled humidity conditions t o r a i s e the moisture
content t o a level where the material can be handled by
ordinary means. T h i s i s accomplished by spreading a t h i n
layer of material i n a wide mouth weighing b o t t l e which i s
placed i n a 42% r e l a t i v e humidity chamber f o r about 16 hours
before use. The 42% r e l a t i v e humidity chamber i s prepared
by storing a saturated aqueous solution of potassium carbon-
a t e i n a desiccator or closed chamber held a t room tempera-
ture. After a 16 hour equilibration period, the water content
of the standard i s determined by Karl Fischer t i t r a t i o n . The
a c t i v i t y of the standard i s t h e n corrected f o r t h e water con-
t e n t of the equilibrated reference material. Experience
indicates t h a t the water content of a properly equilibrated
reference material should be in the range o f 10 t o 12%.
When the c r y s t a l l i n e diammonium moxalactam i s used a s a
standard, the R form of the substance must be equilibrated t o
yield a product which has nearly equal concentrations of both
324 LESLIE J. LOREN2 AND PATRICIA N. THOMAS
t h e R and S isomers. T h i s i s achieved by e q u i l i b r a t i n a

weiqhed sample o f moxalactam diammonium s a l t i n a 0.04fi s o l u -
t i o i o f h y d r o c h l o r i c a c i d a t room temperature f o r 90 t o 120
minutes. The s o l u t i o n i s then b u f f e r e d t o a pH o f 6 w i t h a
potassium phosphate b u f f e r .
HPLC i s t h e technique o f choice f o r determining t h e

p u r i t y o f moxalactam disodium i n raw m a t e r i a l s , f o r m u l a t e d
products, and i n body f l u i d s . Moxalactam i s determined i n a
system c o n t a i n i n g 0.05 t o 0.1 M ammonium a c e t a t e w i t h about
6 percent methanol present. An ES I n d u s t r i e s Chromegabond"
C18 column o r o t h e r a l t e r n a t i v e column w i t h s i m i l a r r e t e n t i o n
c h a r a c t e r i s t i c s i s used t o determine t h e p u r i t y o f t h e moxa-
lactam sample. The substance may be monitored a t 254 nm o r ,
when a v a i l a b l e , a v a r i a b l e wavelength d e t e c t o r can be opera-
t e d a t 271 nm f o r assay. The sample i s d i s s o l v e d i n water o r
i n 0.1 M ammonium a c e t a t e s o l u t i o n . Under c o n d i t i o n s o f t h i s
method, t h e assay should be completed w i t h i n 4 hours o f sample
dissolution.
The R isomer o f moxalactam w i l l be t h e f i r s t t o e l u t e

w i t h a k ' o f about 4.5. The S isomer o f moxalactam w i l l e l u t e
a t a k ' o f about 7.0. For q u a n t i t a t i v e purposes, t h e
responses o f b o t h peaks must be combined t o p r o v i d e a v a l i d
potency f o r t h e moxalactam standard o r sample.
6.2.2. I o d o m e t r i c Assay
The i o d o m e t r i c assay f o r moxalactam i n v o l v e s t h e h y d r o l y -

s i s o f moxalactam i n aqueous base which r e s u l t s i n t h e c l e a v -
age o f t h e B-lactam r i n g . T h i s i s f o l l o w e d by o x i d a t i o n o f
t h e h y d r o l y s i s products w i t h i o d i n e , and t h e t i t r i m e t r i c
d e t e r m i n a t i o n o f t h e i o d i n e consumed. Since t h e unhydrolyzed
drug does n o t r e a c t w i t h i o d i n e , an unreacted sample i s used
as a b l a n k t o compensate f o r i o d i n e consuming i m p u r i t i e s .
T h i s assay i s s p e c i f i c f o r an i n t a c t B-lactam r i n g and w i l l
g i v e a r e s u l t f o r any e n t i t y having such a moiety present.
T h i s procedure t h e r e f o r e may n o t be a s t a b i l i t y i n d i c a t i n g
assay f o r t h e drug. T h i s i s a v e r y r e a l problem f o r moxalac-
tam f o r one o f t h e major degradation products i s t h e decar-
b o x y l a t e d product o f moxalactam which r e t a i n s t h e B-lactam
moiety and i s i t s e l f a compound w i t h c o n s i d e r a b l e a n t i b i o t i c
a c t i v i t y (61-63).
MOXALACTAM DlSODlUM 325
6.2.3. Hydroxyl ami ne Assay

Moxalactam i s also amenable t o the popular hydroxylamine
assay f o r p l a c t a m a n t i b i o t i c s . In t h i s procedure the 8-
1actam i s reacted w i t h hydroxyl ami ne t o cl eave the 8-1 actam
moiety and form a hydroxamic acid. The hydroxamic acid will
in t u r n r e a c t w i t h acidified f e r r i c ion t o form a colored
complex which i s measured a t 480 nm. A blank correction f o r
non-8-lactam impurities which r e a c t w i t h hydroxylamine i s
incorporated by adding hydroxylamine t o an a c i d i f i e d sample
where the acid i s used t o destroy a l l B-lactam species.
A g a i n , t h i s t e s t i s n o t s p e c i f i c f o r moxalactam and will
react w i t h a l l B-lactam moieties. This t e s t would therefore
include the decarboxylated product of moxalactam which may
be present i n the sample, and i s not a good s t a b i l i t y i n d i -
cating assay f o r moxalactam (59,60).
62.4. Microbiological Assay
The microbiological assays recommended for moxalactam
are performed w i t h gram-negative organisms. Specifically,
Escherichia coli i s used f o r potency determinations w i t h the
bulk material and the f i n a l dosage forms, Providencia
s t u a r t i i w i t h biological f l u i d s and t i s s u e s , and Pseudomonas
aeruginosa f o r the assay o f moxalactam i n s u s c e p t i b i l i t y
discs.
6.2.4.1. Turbidimetric Assay
A turbidimetric assay w i t h &. gYJ ATCC 10536 may be
used f o r the bulk d r u g substance and the dosage forms. On
the day prior t o assay, a n t i b i o t i c medium 3 (64) i s inoculated
w i t h the organism and the b r o t h culture i s incubated f o r
approximately 16 hours on a rotary shaker a t room temperature.
Immediately prior t o assay, a s u f f i c i e n t volume of medium 3
i s inoculated w i t h 0.05 mL of the broth c u l t u r e f o r each
100 mL of medium. Samples are diluted w i t h O.lM, pH 6 potas-
sium phosphate buffer and 0.1 mL of each sample i s added t o
assay tubes containing 10 mL of inoculated medium. Dose
response concentrations a r e i n the range of 0.02 t o 0.04
mcg/mL of assay medium. The t e s t i s incubated in a 37°C water
bath until t u b e s containing no a n t i b i o t i c have a l i g h t
transmittance of 20% a t 530 nm (usually 3 t o 4 hours). The
t e s t i s terminated by heating f o r 2 t o 3 minutes i n an 80°C
water bath, and the percent l i g h t transmittance (530 nm) i s
determined f o r each tube a f t e r they a r e cooled and shaken.
326 LESLIE J. LORENZ AND PATRICIA N . THOMAS
6.2.4.2. Agar Diffusion Method

For agar diffusion assays of bulk materials o r f i n a l
dosage forms, E. coli ATCC 10536 i s used. W i t h the agar
p l a t e system c c n s m n g of 10 mL of agar medium 2 (65) a s
base layer and 5 mL of medium 1 (66) a s seed layer, dose
response concentrations w i t h i n the range of 1.0 t o 20.0
mcg/mL a r e appropriate. The agar f o r the seed layer i s inocu-
l a t e d w i t h 0.5 mL of an overnight culture of E. c o l i per each
100 mL of medium. Tests a r e incubated a t 30°C f 5 6 t o 18
hours.
6.3. Related Substance Assays
6.3.1. Decarboxylated Moxalactam

The decarboxylated product of moxalactam (see Section 4 )
is determined by an HPLC technique. A reverse phase HPLC
system consisting of 80 parts of 0.1M ammonium a c e t a t e and
20 parts of methanol is used w i t h a Dupont ZorbaxTMC8 o r other
suitably similar column t o determine the decarboxylated pro-
duct. In this system, the decarboxylated moxalactam should
e l u t e w i t h a k ' of about 6.5. The decarboxylated moxalactam
i s quantitatively determined by comparing the peak response
f o r the sample w i t h a peak response calibration curve of the
authentic decarboxylated moxalactam reference standard
material.
6.3.2. l-Methyl-5-me'rcapto-l,2 ,3,4-tetrazol e
l-Methyl-5-mercapto-l,2,3,4-tetrazol e (see Section 4.1)
is a possible impurity and degradation product of moxalactam
w h i c h can be determined by polarographic techniques (67). I t
is oxidized a t a dropping mercury electrode i n a pH 5.8 buf-
f e r . The concentration of the impurity t e t r a z o l e i s propor-
tional t o the sum o f the currents f o r the adsorption prewave
and the main wave (half-wave potentials of about -0.20 V and
-0.05 V vs SCE, respectively).
6.3.3. Analysis of Other Related Materials
Gradient HPLC procedures can be used f o r the determina-
t i o n of other impurities i n moxalactam. This determination
is usually run on a Spherisorb"0DS column o r other s u i t a b l e
similar column. The solvent system consists of 0.05M ammonium
acetate i n water f o r the weak solvent and 0.05M ammonium ace-
t a t e i n 10 parts water and 90 parts methanol f o r the strong
solvent. A gradient is r u n from the weak solvent t o the
strong solvent in two varying l i n e a r ramps. Most of the

materials of interest e l u t e i n the weak end o f the solvent
gradient so a l i n e a r gradient i s r u n w i t h a change of 2% per
minute for 25 minutes. To remove any strongly retained com-
ponents, the second p o r t i o n of the gradient curve i s run from
50% t o 100% of the strong solvent a t a r a t e of change of 5%
per minute.
Figure 6 shows a typical chromatogram obtained from such
a procedure. In this chromatogram, the elution of the thio-
t e t r a z o l e , the R and S isomers of moxalactam, and the moxa-
lactam decarboxylated product are shown. In t h i s t e s t f o r
other related substances, only those substances which are n o t
d e a l t w i t h by s p e c i f i c tests a r e considered. Quantitative
information i s obtained by summing the response f o r a l l the
extraneous peaks and comparing them t o the sum of the response
f o r the R and S isomers o f a diluted moxalactam reference
standard. The assumption i s made t h a t a l l of these materials
have similar spectral response c h a r a c t e r i s t i c s a t 254 nm.
7. Analysis of Biological Samples
7.1. Microbiological Assay
T h e microbiological assay f o r moxalactam i n serum, urine,
and t i s s u e s i s w i t h Providencia s t u a r t i i ATCC 33700 a s the
assay organism. The agar plate system i s a 10 mL single layer
plate consisting of trypticase soy agar which has been modi-
f i e d by the addition of 1 g of dextrose and 10 g of sodium
acetate per l i t e r of medium. The agar medium i s inoculated
a t 0.1% ( v / v ) with a b r o t h culture which has been adjusted t o
25% 1i g h t transmittance (530 nm) . Standard curve concentra-
t i o n s a r e prepared i n the same diluent a s i s used f o r samples
and a r e w i t h i n the range of 0.1 t o 1.0 mcg/mL. Incubation i s
a t 30°C f o r 16 t o 18 hours.
7.2. Chromatography
HPLC techniques can be used t o determine the levels of
moxalactam i n various biological f l u i d s . Where appropriate,
the protein i s removed from the sample by addition of i c e cold
methanol and centrifugation. The resulting solutions are
evaluated under conditions described i n Section 6.2.1.
8. Analysis of Pharmaceutical Formulations

Pharmaceutical formulations of moxalactam are handled i n
a manner analogous t o the substances described i n Section 6 .
Tetrazole Thiol
Moxalactam R Isomer
i
1
Moxalactam S Isomer
- Decarboxylated Product
I 2
N u1 -4
u1 0 Ln
8
8
m
The o n l y d i f f e r e n c e i s t h a t t h e pharmaceutical f o r m u l a t i o n
c o n t a i n s mannitol which i s r e l a t i v e l y innocuous t o a l l o f t h e
t e s t procedures described.
Acknowledgements
The a u t h o r s wish t o express t h e i r s i n c e r e thanks t o t h e

f o l l o w i n g people who have p r o v i d e d i n f o r m a t i o n f o r s p e c i f i c
p o r t i o n s o f t h i s chapter: D. E . Dorman, J. W. Paschal, and
A. D. Kossoy f o r t h e NMR i n t e r p r e t a t i o n ; A. Hunt f o r t h e
u l t r a v i o l e t s p e c t r a l i n t e r p r e t a t i o n ; B. W. Jackson f o r t h e
s y n t h e t i c p r e p a r a t i o n ; A. D. Kossoy f o r t h e degradation work;
J . L. Occolowitz f o r t h e mass s p e c t r a l i n t e r p r e t a t i o n ;
L. G. Tensmeyer and C. D. Underbrink f o r t h e i n f r a r e d assign-
ments; and C. L . Winely f o r t h e m i c r o b i o l o g i c a l assay
portions.
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62. J. F. Alicino, @. Eng. Chern. Anal. Ed. , 18,619
(1946).
63.
64.
j . F.'Alicino, Anal,
CFR §436.102(b)(3).
m.,33, 648 (1961).
65. CFR §436.102(b)(2).
66. CFR §436.102(b) ( 1 ) .
67. E. C. Rickard and G. G. Cooke, J. Pharm. Sci., 61,
379 (1977).
OXYPHENBUTAZONE
Abdullah A. Al-Badr and Humeida A. El-Obeid
King Sotid Uriioersity
Ri!/ndli, Saudi Arabia
1. Description 334
1 . 1 Nomenclature 334
1.2 Formulae 334
I .4 Elemental Composition 335
2.2 Solubility 335
3. Synthesis 344
4. Absorption, Metabolism, and Excretion 348
5. Methods of Analysis 35 I
5.1 Titrimetric Methods 35 1
5.2 Electrochemical Methods 352
References 358
ANALYTICAL PROFILES OF DRUG SUBSTANCES Copyright iJ 19x4

VOLUME 13 333 by thc American Phannaccut~calAswciation
ISBN 0-12-2hOX13-5
334 ABDULLAH A . AL-BADR AND HUMEIDA A . EL-OBEID
1. Description
1.1 Nomenclature
1.1.1 Chemical Names
4-Butyl-l-(4-hydroxyphenyl)-2-phenyl-3,5-pyra-
zolidinedione .
4-Butyl-l-(p-hydroxyphenyl)-2-phenyl-3,5-pyra-
zolidinedione.
4-Butyl-2-(4-hydroxyphenyl)-l-phenylpyrazolidine-
3 ,5-dione.
4-Butyl-2-(p-hydroxyphenyl)-l-phenylpyra~olidine-
3,5-dione.
4-Butyl-l-(p-hydroxyphenyl)-3,5-dioxo-2-phenyl-
pyrazolidine .
4-Butyl-l-(4-hydroxyphenyl)-3,5 dioxo-2-phenyl-
pyrazolidine.
3,5-Dioxo-l-phenyl-2-(p-hydroxyphenyl)-4-butyl-
pyrazolidine.
l-(p-Hydroxyphenyl)-2-phenyl-4-~~tylpyrazolidine-
3,5-d ione.
l-Phenyl-2-(p-hydroxyphenyl)-3,5-dioxo-4-n-butyl-
pyrazolidine.
1.1.2 Generic Names
Oxyphenbutazone, G27202, Hydroxyphenylbutazone.
1.1.3 Trade Names
Crovaril, Flogitolo, Flogoril, Frabel, Neo-

Fannadol, Oxalid, Tandacote, Tandearil, Tanderil,
Visubutina.
1.2 Formulae
1.2.1 Empirical
C
19 20 2 3
1.2.2 Structural
2o ''
H N 0 (anhydrous), C H N 0 -H 0 (mono-
hydrate)
OXY PHENBUTAZONE 33s
1.2.3 CAS no.
[ 129-20-41 ( a n h y d r o u s ) , [ 7081-38-11 (monohydrate).
324.38 ( a n h y d r o u s ) , 342.39 (monohydrate).
C 70.35%, H 6.22%, N 8.64%, 0 14.80%.
1.5 Appearance, Color, Odor and Taste
White c r y s t a l l i n e powder, o d o r l e s s o r almost o d o r l e s s

with b i t t e r taste.
2. P h y s i c a l P r o p e r t i e s
2 . 1 Melting Point
Anhydrous c r y s t a l s from e t h e r and petroleum e t h e r ,

m.p. 124-125O (1). Monohydrate c r y s t a l s m . p . 96
(1,2>.
P r a c t i c a l l y i n s o l u b l e i n water, s o l u b l e i n 3 p a r t s of
a l c o h o l ( 9 5 % ) , i n 20 p a r t s of chloroform, i n 20 p a r t s
of s o l v e n t e t h e r and i n 6 p a r t s of a c e t o n e ; a l s o
s o l u b l e i n s o l u t i o n s of a l k a l i h y d r o x i d e s ( 3 ) .
2.3.1 I n f r a r e d S p e c t r o s c o p i c Test
The i n f r a r e d a b s o r p t i o n c h a r a c t e r i s t i c s of
oxyphenbutazonehave been used by t h e B.P. (3)
and U.S.P. ( 4 ) as a meansfor i d e n t i f y i n g t h e
d r u g . The i n f r a r e d a b s o r p t i o n spectrum e x h i b i t s
m a x i m a which a r e only a t t h e same wavelengths a s ,
and have s i m i l a r r e l a t i v e i n t e n s i t i e s t o , t h o s e
i n t h e spectrum of a s i m i l a r p r e p a r a t i o n of a
s t a n d a r d oxyphenbutazone. The i n f r a r e d charac-
terist'ics of oxyphenbutazone s h a l l be d i s c u s s e d
l a t e r i n t h e s p e c t r a l p r o p e r t i e s of t h e drug.
336 ABDULLAH A. AL-BADR AND HUMEIDA A . EL-OBELD
2.3.2 U l t r a v i o l e t S p e c t r o s c o p i c T e s t
The B.P. and U.S.P. a l s o recommend t h e u s e of

u l t r a v i o l e t c h a r a c t e r i s t i c s of oxyphenbutazone
as an i d e n t i f i c a t i o n t e s t . A s o l u t i o n of t h e
d-.,- I n sodium hydroxide e x h i b i t s maxima and
LUh
minima a t t h e same wavelengths a s t h a t of a

similar s o l u t i o n of a s t a n d a r d d r u g . The u l t r a -
v i o l e t a b s o r p t i o n c h a r a c t e r i s t i c s of oxyphenbuta-
zone s h a l l b e d i s c u s s e d l a t e r i n t h e s p e c t r a l
p r o p e r t i e s of t h e d r u g .
2.3.3 Color Tests
Table 1 l i s t s some of t h e c o l o r t e s t s r e p o r t e d
f o r t h e i d e n t i f i c a t i o n of oxyphenbutazone.
Table 1. Color T e s t s f o r Oxyphenbutazone.
Reagents Color Ref.
i) Hot s o l u t i o n of t h e (3)
drug i n g l a c i a l a c e t i c
and h y d r o c h l o r i c a c i d s .
Cool and f i l t e r .
F i l t r a t e p l u s sodium Yellow c o l o r
nitrite
A d d i t i o n of 2-naphthol B r i g h t orange p p t .
solution
P p t + 95% a l c o h o l Orange s o l u t i o n
i i ) Drug i n a m i x t u r e of Solution c l e a r with (3)
sodium hydroxide and a n e x t i n c t i o n of 4-
aminoacet i c a c i d . cm l a y e r a t 420 nm
i s n o t more t h a n 0.40.
i i i ) S o l u t i o n of d r u g i n Cherry r e d p p t . (4)
methanol p l u s M i l l o n ' s
Reagent, h e a t .
iv) Sulfuric acid - formal- P a l e yellow (sensi- (2)
dehyde t i v i t y : 1.0 pg).
v) Ammonium molybdate Pale orange ( s e n s i - (2)
t i v i t y : 1.0 ug).
vi) Vitali's Test Pale yellow o r red (2)
( s e n s i t i v i t y : 0.25 pg) .
OXYPHENBUTAZONE 337
2.3.4 C r y s t a l T e s t s
Crystal tests reported (2) include:
a ) Treatment w i t h p l a t i n i c i o d i d e s o l u t i o n g i v e s
r o s e t t e s of p r i s m s , forming o v e r n i g h t . The
s e n s i t i v i t y of t h e t e s t i s 1 i n 100.
b ) Treatment w i t h p o t a s s i u m i o d i d e s o l u t i o n
g i v e s s h e l l - l i k e c r y s t a l s , forming o v e r n i g h t .
The s e n s i t i v i t y of t h e t e s t i s 1 i n 100.
2.3.5 Chromatographic T e s t s
a) C l a r k e ( 2 ) d e s c r i b e d a p a p e r c h r o m a t o g r a p h i c
method f o r t h e i d e n t i f i c a t i o n of t h e d r u g
u s i n g t h r e e d i f f e r e n t s o l v e n t systems.
System I. C o n s i s t s of c i t r i c a c i d : water:
n - b u t a n o l ( 4 . 8 Em: 130 ml : 870 m l ) . Detec-
t i o n c a r r i e d out using u l t r a v i o l e t l i g h t
a b s o r p t i o n . p-Dimethylaminobenzaldehyde o r
p o t a s s i u m permanganate can b e used a s s p r a y
t o l o c a t e t h e d r u g . Rf 0.96.
System 11. A c e t a t e b u f f e r (pH 4.58). Detec-

t i o n i s e f f e c t e d by u l t r a v i o l e t l i g h t a b s o r p -
t i o n . Rf 0.04,
System 111. p h o s p h a t e b u f f e r (pH 7 . 4 ) . Detec-

t i o n i s e f f e c t e d by u l t r a v i o l e t l i g h t a b s o r p -
t i o n . Rf 0.58.
b) An i d e n t i f i c a t i o n t e s t f o r t h e d r u g by t h i n -
l a y e r chromatography w a s a l s o r e p o r t e d by
Clarke (2). The s o l v e n t system c o n s i s t s o f :
s t r o n g ammonia s o l u t i o n : m e t h a n o l (1.5 : 100).
D e t e c t i o n i s e f f e c t e d by p o t a s s i u m permanga-
n a t e s p r a y . Rf 0 . 7 7 .
338 ABDULLAH A . AL-BADR AND HUMEIDA A. EL-OBEID
The u l t r a v i o l e t spectrum of oxyphenbutazone i n

n e u t r a l methanol u s i n g Cary, 219 Spectrophoto-
meter i s shown i n F i g u r e 1. The spectrum of t h e
d r u g e x h i b i t s two maxima a t 243 and 265 nm and a
minimum a t 221 nm. The u l t r a v i o l e t a b s o r p t i o n
c h a r a c t e r i s t i c s of t h e drug a r e used by t h e B.P.
( 3 ) and U.S.P. ( 4 ) , as an i d e n t i f i c a t i o n t e s t .
A 0.001 % w/v s o l u t i o n of oxyphenbutazone i n 0.01
N sodium hydroxide s o l u t i o n e x h i b i t s a maximum
o n l y a t 254 nm w i t h an e x t i n c t i o n of about 1.5.
Oxyphenbutazone i n e t h a n o l w a s a l s o r e p o r t e d ( 2 )
t o have a maximum a t 242 nm ( E l % 1 cm 564) and an
i n f l e x i o n a t 275 nm.
The i n f r a r e d spectrum of oxyphenbutazone i n n u j o l

m u l l i s p r e s e n t e d i n F i g u r e 2. An i n t e r p r e t a t i o n
of t h e spectrum i s g i v e n below. The i n f r a r e d
a b s o r p t i o n spectrum of oxyphenbutazone i s used
a s an i d e n t i f i c a t i o n t e s t by t h e B.P. and U.S.P.
-1
Frequency (cm ) Assignment
1288 C-N s t r e t c h
1702
C=O s t r e t c h
1748
3220 0-H s t r e t c h
1180 C-0 s t r e t c h of
phenol
Reported (2) p r i n c i p a l i n f r a r e d a b s o r p t i o n peaks

of oxyphenbutazone i n potassium bromide d i s c s
are 1683, 1725, 1275 and 1518 cm-1.
1 1
2.4.3 H Nuclear Magnetic Resonance ( H NMR) Spectrum
1
The H NMR Spectrum of oxyphenbutazone i s pre-
s e n t e d i n F i g u r e 3 . The drug was d i s s o l v e d i n
acetone-d6 and i t s spectrum determined on a
Varian-TGOA NMR Spectrometer u s i n g TMS as t h e
OXYPHENBUTAZONE 339
T
C
c
Fig. 1. Ultraviolet spectrum of oxyphenbutazone

in neutral methanol.
rl
rl
;
rl
0
-n
I
z
a,
.
c
0
N
id
4J
I
Q
.
c
a,
A
a
h
X
0
w
0
5
k
4J
u
a,
i?
a
a,
Ll
id
k
w
c
H
(u
m
v i
h
340
'A
P
Fig. 3 . 'H NMR spectrum of oxyphenbutazone in acetone-d6

with TMS as internal standard.
342 ABDULLAH A . AL-BADR AND HUMElDA A. EL-OBEID
i n t e r n a l standard. The s t r u c t u r a l assignments

a r e shown below.
P r o t o n Assignments* Chemical S h i f t s (6) Mu 1t i p 1i c it y
a 7.33 broad s i n g l e t
b, c 7.00 AB q u a r t e t
d 3.57 triplet
e 2.04 mu 1t i p l e t
€ 1.41 multiplet
a 0.92 quartet
0-H 3.14 broad s i n g l e t
*See s t r u c t u r e i n F i g u r e 3 f o r p r o t o n a s s i g n e m t s .
2.4.4 I3C Nuclear Mangetic Resonance (I3C NMR) Spectrum
The I 3 C NMR spectrum of oxyphenbutazone i n

acetone-dg u s i n g TMS as an i n t e r n a l r e f e r e n c e w a s
o b t a i n e d u s i n g a J e o l FX 100 MHz Spectrometer a t
an ambient t e m p e r a t u r e u s i n g 1 0 mm sample and i s
p r e s e n t e d i n F i g u r e 4 . The carbon chemical
s h i f t v a l u e s are d e r i v e d from t h e o f f - r e s o n a n c e
spectrum and are l i s t e d below. The r e s u l t s a r e
consistent with those reported earlier ( 5 ) .
Carbon No.* Chemical S h i f t s Carbon No. Chemical S h i f t s
(PPm) (PPd
1' 137.2 6 28.7
3 170.9 6' 126.4
3' 129.3 7 28.0
4 46.4 7' 116.1
4' 124.3 8 20.0
5 171.5 8' 157.1
5' 127.2 9 13.9

*See s t r u c t u r e i n F i g u r e 4 f o r carbon assignment.
I
~ ~~
, I - I I I
Fig. 4. 13NMR spectrum of oxyphenbutazone in acetone -d

with TMS internal reference. 6
344 ABDULLAH A . AL-BADR AND HUMEIDA A. EL-OBEID
2.4.5 Mass Spectrum and Fragmentometry
The f r a g m e n t a t i o n p a t t e r n s of oxyphenbutazone
and o t h e r p y r a z o l i d i n e d i o n e s were d e s c r i b e d ( 6 )
b u t t h e v e r i f i c a t i o n by e v i d e n c e of m e t a s t a b l e
i o n s , a c c u r a t e mass d e t e r m i n a t i o n s o r u s e of
l a b e l e d d e r i v a t i v e s w a s p r e s e n t e d by Locock et
-
a1 ( 7 ) .
The mass s p e c t r a of oxyphenbutazone i s r e p r o -

duced i n F i g u r e 5. According t o Locock e t a1 L-
( 7 ) t h e drug undergoes McLafferty rearrangement

t o g i v e a r a d i c a l i o n a t m / e 268. A m e t a s t a b l e
i o n is p r e s e n t i n t h e spectrum t o i n d i c a t e t h a t
t h i s i o n i s a d i r e c t f r a g m e n t a t i o n from t h e
m o l e c u l a r i o n w i t h t h e l o s s of t h e e l e m e n t s of
b u t e n e (Scheme I ) .
The l o s s of a p r o p y l r a d i c a l from t h e b u t y l s i d e
c h a i n of t h e m o l e c u l a r i o n r e p r e s e n t s a minor
bathway t o g i v e an i o n a t m / e 281.
The most c h a r a c t e r i s t i c f r a g m e n t s i n t h e m a s s
spectrum of oxyphenbutazone w e r e a s e r i e s of
p e a k s a t m / e 198, 199 and 200. The peak a t m / e
198 i s d u e t o t h e f o r m a t i o n of t h e azobenzene
.
r a d i c a l i o n ( C ~ H ~ N ~ C G H , ) ~The r e m a i n i n g p e a k s
a t m / e 199 and 200 t o g e t h e r w i t h o t h e r p e a k s
r e s u l t i n g from t h e f r a g m e n t a t i o n o f oxyphenbuta-
zone are e x p l a i n e d by Scheme I.
Other p e a k s i n t h e low m a s s r a n g e , below m / e 100,

a r e common t o s u b s t i t u t e d a r o m a t i c s y s t e m s
(Fig. 5 ) .
3. Synthesis
Oxyphenbutazone w a s s y n t h e s i z e d (8) b y c o n d e n s a t i o n of t h e
p r o t e c t e d aminophenol ( I , Scheme 2 ) w i t h n i t r o b e n z e n e (11).
Reduction of t h e r e s u l t i n g azo-compound (111) gave t h e
hydrazobenzene ( I V ) which when condensed w i t h d i e t h y l b u t y l -
Fig. 5 . Mass spectrum of oxyphenbutazone (7).
..
346
al
\
F
0
1 I
347
malonate (V) gave t h e h e t e r o c y c l e ( V I ) . Removal of t h e

b e n z y l group was a c h i e v e d by h y d r o g e n o l y s i s .
6 0
I I
,co OEt
- @
N
I +
6 0
Scheme 2. Synthesis o f oxyphenbbutatone.
4 . Absorption, Metabolism and E x c r e t i o n
Oxyphenbutazone i s one of t h e main m e t a b o l i t e s of phenyl-

butazone and i s c o n s i d e r e d t o be more t o x i c . It is rapidly
absorbed from t h e g a s t r o i n t e s t i n a l t r a c t and slowly meta-
b o l i z e d and e x c r e t e d mainly i n u r i n e . The r a t e of i t s
metabolism v a r i e s i n d i f f e r e n t s p e c i e s a s r e f l e c t e d by t h e
great variance i n half-lives.
I n h i s s t u d y on the metabolism of b u t a z o l i d i n e , Herrmann

(9) measured t h e h a l f - l i f e of oxyphenbutazone i n r a t s and
r a b b i t s a f t e r i n t r a v e n o u s a d m i n i s t r a t i o n of t h e d r u g . They
w e r e 8 and 4-5 h r s r e s p e c t i v e l y . Weiner e t a1 (10) s t u d i e d
t h e d r u g d i s p o s i t i o n p a t t e r n s i n subhuman p r i m a t e s a s com-
pared t o humans and o t h e r s p e c i e s . I n t h e babooqoxyphen-
OXY PHENBUTAZONE 349
b u t a z o n e h a s a s h o r t h a l f - l i f e , w h i l e i n man it h a s a l o n g
half-life. The plasma b i n d i n g and a p p a r a n t volumes o f
d i s t r i b u t i o n of t h e d r u g are s i m i l a r i n t h e baboon and
man. T h e r e i s s u g g e s t i v e e v i d e n c e t h a t a b s o r p t i o n may n o t
b e a s complete i n t h e baboon a s i t i s i n man. Studies
i n dogs i n d i c a t e d t h a t t h e d r u g is n o t absorbed i n t h e
l y m p h a t i c s . Burns (11) compared t h e r a t e s o f metabolism of
oxyphenbutazone i n man, r h e s u s monkey and dogs. The h a l f -
l i v e s showed g r e a t v a r i a t i o n s . P r e v i o u s s t u d i e s i n d i c a t e d
t h a t metabolism of t h i s and o t h e r compounds may b e accom-
p a l i s h e d by d i f f e r e n t mechanisms s o t h a t s p e c i e s d i f f e r e n -
ces become b o t h q u a n t i t a t i v e and q u a l i t a t i v e . P e r e l -- e t a1
( 1 2 ) c o r r e l a t e d t h e p h y s i c o c h e m i c a l p r o p e r t i e s of phenyl-
butazone a n a l o g s w i t h t h e i r p h y s i o l o g i c a l d i s p o s i t i o n , i n
p a r t i c u l a r w i t h r e f e r e n c e t o s p e c i e s d i f f e r e n c e s . While
i n man t h e r e e x i s t s a d i r e c t r e l a t i o n between pKa and h a l f -
l i f e , no such c o r r e l a t i o n w a s o b s e r v e d i n dogs. The h a l f -
l i f e i n d o g s a p p e a r s t o depend on f a c t o G such as f a t l b u f f e r
p a r t i t i o n c o e f f i c i e n t , plasma p r o t e i n b i n d i n g , t i s s u e d i s -
t r i b u t i o n and d r u g m e t a b o l i z i n g enzyme a c t i v i t y . The r a t e
of metabolism of t h e a n a l o g s , based on plasma l e v e l
d e c l i n e , v a r i e d e x t e n s i v e l y i n b o t h s p e c i e s . Oxyphenbuta-
zone h a s a h a l f - l i f e of 0.5 h r i n t h e dog, whereas i n man
it i s 7 2 h r s . The r e s u l t s of b i n d i n g of oxyphenbutazone
t o human and dog plasma i n d i c a t e t h a t t h e d r u g i s h i g h l y
bound ( 9 8 % ) . T h i s h i g h e r b i n d i n g o f t h e d r u g t o human
plasma compared t o dog plasma s h o u l d be a s i g n i f i c a n t
f a c t o r i n a c c o u n t i n g f o r some of t h e s p e c i e s d i f f e r e n c e s .
In t h e i r s t u d y of t h e mechanism of h e p a t i c d r u g o x i d a t i o n
and i t s r e l a t i o n t o i n d i v i d u a l d i f f e r e n c e s i n r a t e s of
o x i d a t i o n i n man, Davies and T h o r g e i r s s o n (13) s u g g e s t e d
t h a t plasma h a l f - l i v e s of a n t i p y r i n e d e t e r m i n e d f o l l o w i n g
a s i n g l e d o s e w e r e a good measure of a n i n d i v i d u a l ' s
a b i l i t y t o m e t a b o l i z e p h e n y l b u t a z o n e and oxyphenbutazone,
i f t h e h a l f - l i v e s of t h e s e two d r u g s were d e t e r m i n e d u n d e r
c o n d i t i o n s which d i d n o t change michrosomal enzyme a c t i v i t y .
Human l i v e r microsomal p r o t e i n and cytochrome P-450 con-
c e n t r a t i o n s d i d n o t show l a r g e i n d i v i d u a l d i f f e r e n c e s and
were s i m i l a r t o v a l u e s f o r a d u l t m a l e r a t s . Hoffmann et
a1 (14) c a r r i e d o u t s t u d i e s on t h e o r g a n d i s t r i b u t i o n and
I
s i d e e f f e c t s of oxyphenbutazone and p h e n y l b u t a z o n e u s i n g
r a d i o a c t i v e i s o t o p e s . No i n f l u e n c e on t h e h e m o p o i e t i c
s y s t e m w a s d e m o n s t r a t e d by oxyphenbutazone o r by phenyl-
butazone. A m i l d e f f e c t on oxyphenbutazone metabolism w a s
seen. I n d i r e c t r e l a t i o n of t h e c o n c e n t r a t i o n of oxyphen-
b u t a z o n e and phenylbutazone a d e c r e a s e of oxyphenbutazone
u p t a k e b y t h e t h y r o i d was n o t e d , w i t h an i n c r e a s e i n i t s
p r o t e i n binding. I n t h e presence of f e v e r , organs w i t h a
u
-L.
B
d*c
d
L
Q t
d
-4
I
0 k
I
0 (i
cu
I
F
V u
r
0
N
Z
0
v)
-8
3
(0
d
OXYPHENBUTAZONE 35 1
r e t i c u l a r e n d o t h e l i a l c e l l system, e s p e c i a l l y t h e l i v e r ,
had a h i g h e r u p t a k e of oxyphenbutazone w h i l e muscle t i s s u e
showed a d e c r e a s e a t t h a t t i m e . S t i e r l i n and Saubermann
(15) s t u d i e d t h e t r a n s p o r t of l o c a l l y a d m i n i s t e r e d r a d i o -
a c t i v e oxyphenbutazone i n t o t h e eye t i s s u e s of t h e r a b b i t .
The r e s u l t s showed t h a t t h e c o r n e a l - s c l e r a l b a r r i e r can be
passed by t h e drug. The c o n c e n t r a t i o n of oxyphenbutazone
l o c a l l y a d m i n i s t e r e d i n t h e t i s s u e and f l u i d s of t h e eye
w a s h i g h e r t h a n w i t h a s i n g l e 10 mg/kg i.v. dose. The
d i r e c t a p p l i c a t i o n of oxyphenbutazone on t h e eye d o e s n o t
r e s u l t i n a n accumulation of t h e d r u g w a s demonstrated by
t h e f a c t t h a t no oxyphenbutazone could be d e t e c t e d i n t h e
e y e a f t e r 24 h r s .
The o x i d a t i o n of oxyphenbutazone by sheep v e s i c u l a r gland

microsomes and l i p o x y g e n a s e w a s s t u d i e d by P o r t o g h e s e et
a 1 ( 1 6 ) . Oxyphenbutazone w a s o x i d i z e d when i n c u b a t e d w i t h
I
a c e t o n e powder p r e p a r e d from sheep v e s i c u l a r g l a n d micro-

somes o r w i t h l i p o x y g e n a s e a t pH 4 o r 5. Oxidation a l s o
occured a t pH 8 o r 9 , i f a r a c h i d o n a t e o r l i n o l e a t e w a s
added t o e i t h e r of i n c u b a t i o n m i x t u r e s . The oxygenated
p r o d u c t w a s found t o b e i d e n t i c a l w i t h 4-hydroxyoxyphen-
butazone (Scheme 3 ) which w a s s y n t h e s i z e d and a n a l y s e d by
g a s - l i q u i d chromatography and mass s p e c t r o m e t r y . The oxy-
genated compound w a s n o t an i n h i b i t o r of p r o s t a g l a n d i n
biosynthesis. P e r e l e t a1 (12) found t h a t oxyphenbutazone
w a s s l o w l y m e t a b o l i z e d i n man and r a p i d l y m e t a b o l i z e d i n
dogs. When t h e d r u g i s a d m i n i s t e r e d i n t r a v e n o u s l y t o d o g s ,
10-15% of unchanged d r u g and 18-28% of i t s g l u m r o n i d e were
e x c r e t e d i n 24 h r s . When e i g h t t i m e s t h i s d o s e of t h e drug
w a s given o r a l l y t o human s u b j e c t s , t h e y e x c r e t e d 1-5% of
t h e d o s e as g l u c u r o n i d e s i n 24 h r s .
The s u l f a t e of oxyphenbutazone i s p r o b a b l y n o t a major

metabolite. It h y d r o l y s e s i n water a t room t e m p e r a t u r e
and s i n c e less t h a n 2% of oxyphenbutazone i s e x c r e t e d i n
24 h r s (17) t h i s would r e p r e s e n t t h e maximal c o n v e r s i o n of
oxyphenbutazone t o s u l f a t e .
5 . Methods of A n a l y s i s
a ) The BP 1980 ( 3 ) d e s c r i b e s a t i t r i m e t r i c method i n

which oxyphenbutazone solution i n acetone is
t i t r a t e d w i t h s t a n d a r d sodium hydroxide s o l u t i o n
u s i n g bromothymol b l u e a s i n d i c a t o r u n t i l a b l u e
c o l o r p e r s i s t s € o r a t l e a s t t h i r t y seconds. The
352 ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID
operation is repeated without t h e drug; t h e d i f f e -

r e n c e between t h e t i t r a t i o n s r e p r e s e n t s t h e amount
of a l k a l i r e q u i r e d by oxyphenbutazone.
b) The U S P 1980 ( 4 ) a l s o recommends a s t a n d a r d sodium

hydroxide t i t r i m e t r i c method b u t t h e d r u g s o l u t i o n
i s made i n methanol and t h e end p o i n t i s determined
p o t e n t i o m e t r i c a l l y u s i n g a calomel-glass e l e c t r o d e
system w i t h a s a t u r a t e d s a l t b r i d g e of potassium
c h l o r i d e i n methanol. A blank determination is
necessary.
c ) Walash and Rizk (18) determined oxyphenbutazone i n

c e r t a i n dosage forms by a nonaqueous t i t r a t i o n pro-
c e d u r e i n t e t r a m e t h y l u r e a u s i n g s t a n d a r d sodium
methoxide a s a t i t r a n t . Recovery from compound
p r e p a r a t i o n s of t h e a n a l g e s i c w a s q u a n t i t a t i v e and
t h e end p o i n t could be l o c a t e d e i t h e r p o t e n t i o m e t r i -
c a l l y u s i n g glass-colomel e l e c t r o d e system o r
v i s u a l l y u s i n g thymol b l u e as i n d i c a t o r .
5.2 E l e c t r o c h e m i c a l Methods
a ) Fogg and Ahmed (19) adopted a B.P. 1980 ( 3 ) t e s t

f o r t h e i d e n t i f i c a t i o n of oxyphenbutazone t o t h e
q u a n t i t a t i o n of t h e drug by d i f f e r e n t i a l p u l s e
polarography. The drug s o l u t i o n i n a c e t i c and hydro-
c h l o r i c a c i d s w a s t r e a t e d w i t h sodium n i t r i t e and
coupled w i t h 1-naphthol. The azo-dye formed w a s
determined by d i f f e r e n t i a l p u l s e polarography.
Polarograms were reproduced f o r t h e d e t e r m i n a t i o n
of t h e drug a t c o n c e n t r a t i o n s i n t h e r a n g e s 0 t o
0.9 UM and 0 t o 70 uM. The c a l i b r a t i o n g r a p h s w e r e
rectilinear. The p r o c e d u r e , however, does n o t d i s -
t i n g u i s h between oxyphenbutazone and phenylbutazone.
--
b) P e l i n a r d e t a 1 (20) s t u d i e d t h e e l e c t r o c h e m i c a l
b e h a v i o r of a n t i i n f l a m m a t o r y d e r i v a t i v e s of pyrazo-
l o n e and t h e a p p l i c a t i o n t o d e t e r m i n a t i o n i n medi-
caments. Oxyphenbutazone and o t h e r 3,5-pyrazoline-
d i o n e s w i t h mobile hydrogen atoms i n p o s i t i o n 4 were
d i r e c t l y reduced e l e c t r o c h e m i c a l l y i n n e u t r a l e t h a -
n o l . Monoketones, phenazone and aminophenazone,
were n o t reduced under s i m i l a r c o n d i t i o n s . The
a p p l i c a t i o n of t h e method t o t h e d e t e r m i n a t i o n of
3 , 5 - p y r a z o l i d i n e d i o n e s , w i t h l a b i l e hydrogen i n t h e
4 - p o s i t i q g a v e a p r e c i s i o n of about 3% when 2 X 10-6
mole of a s u b s t a n c e were used.
OXYPHEN BUTAZON E 353
5.3 S p e c t r o p h o t o m e t r i c Methods
5.3.1 U l t r a v i o l e t S p e c t r o p h o t o m e t r i c Methods
a ) Herrmann ( 9 ) r e p o r t e d a n u l t r a v i o l e t method
f o r t h e d e t e r m i n a t i o n of p h e n y l b u t a z o l i d i n e
together with i t s metabolic products, chiefly
oxyphenbutazone, i n serum. The serum i s shaken
w i t h 3N h y d r o c h l o r i c a c i d i n 3% isoamyl
alcohol i n heptane. After c e n t r i f u g a t i o n an
a l i q u o t of t h e o r g a n i c p h a s e i s shaken w i t h
2.5 N sodium h y d r o x i d e and t h e e x t i n c t i o n of
t h e a l k a l i n e e x t r a c t i s d e t e r m i n e d a t 265 nm.
A s i m i l a r method h a s a l s o been r e p o r t e d (21).
b ) Fuchs ( 2 2 ) d e s c r i b e d a method f o r t h e blood

l e v e l d e t e r m i n a t i o n of t a n d e r i l [ oxyphenbuta-
zone] u s i n g a s i n g l e d r o p of c a p i l l a r y b l o o d .
The sample of t h e serum i s mixed w i t h water
and 3N h y d r o c h l o r i c a c i d . The m i x t u r e i s
shaken with p u r e 1,2-dichloroethylene. After
c e n t r i f u g a t i o n , a n a l i q u o t of t h e o r g a n i c
p h a s e i s added t o a s e p a r a t i n g f u n n e l c o n t a i n -
i n g 1 m l of 2.5 N sodium h y d r o x i d e and shaken.
The aqueous e x t r a c t of T a n d e r i l i s s e p a r a t e d
by c e n t r i f u g a t i o n and d e t e r m i n e d by measuring
a t 240, 245, 250, 252.5, 255, 257.5, 260, 265
and 270 nm. The e r r o r w a s < 4%.
5.3.2 C o l o r i m e t r i c Methods
a ) S v a t e k and Hradkova (23) d e t e r m i n e d oxyphen-

b u t a z o n e and r e l a t e d k e t o d e r i v a t i v e s i n serum
using a c o l o r i m e t r i c method i n v o l v i n g t h e
r e a c t i o n of t h e d r u g s w i t h d i a z o t i z e d s u l f a -
n i l i c a c i d . According t o t h e procedure, t h e
d i l u t e d serum is shaken w i t h N h y d r o c h l o r i c
a c i d , h e p t a n e and isoamyl a l c o h o l . The h e p t a n e
p h a s e i s shaken w i t h 0.1 N sodium h y d r o x i d e .
The a l k a l i n e e x t r a c t i s shaken with a m i x t u r e of
1%s u l f a n i l i c a c i d , N h y d r o c h l o r i c a c i d , 1%
sodium n i t r i t e and methanol. The a b s o r b a n c e
of t h i s s o l u t i o n i s measured a t 525 nm a g a i n s t
sodium h y d r o x i d e b l a n k . The e r r o r was 3-5%.
Oxyphenbutazone combines w i t h d i a z o t i z e d s u l -
f a n i l i c a c i d i n a l k a l i n e medium w i t h o u t need
f o r preliminary hydrolysis. The m e t h y l e n e
group of t h e s i d e c h a i n i s a c t i v a t e d by t h e
y k e t o group w i t h t h e f o r m a t i o n o f a formazan
derivative. The formazan d e r i v a t i v e of s u l f a -
n i l i c a c i d i s v e r y s o l u b l e i n w a t e r and h a s a
maximum a t 5 2 5 nm. The c o l o r i s s t a b i l i z e d
by t h e presence of 25% methanol. The method
i s s e n s i t i v e t o 0.5 y/ml and t h e l i n e a r r a n g e
i s 0-15 y/ml.
b ) Wahbi -- e t a 1 (24) used sodium c o b a l t i n i t r i t e

as a r e a g e n t f o r d e t e r m i n i n g some p h e n o l i c
d r u g s . When oxyphenbutazone is r e a c t e d w i t h
t h e r e a g e n t i n aqueous a c e t i c a c i d a yellow
c o l o r measurable a t 3 2 0 nm i s produced. Sub-
s t i t u t i n g sodium hydroxide f o r a c e t i c a c i d
g i v e s a c o l o r measurable a t 3 8 0 nm. The
c o l o r e d product i s e x t r a c t a b l e w i t h c h l o r o -
form w i t h a maximum a b s o r p t i o n a t 3 2 5 nm.
The method i s a p p l i e d t o t h e d e t e r m i n a t i o n
i n t a b l e t form. The r e s u l t s o b t a i n e d are
reasonably reproducible with a coefficient
of v a r i a t i o n < 2%.
c ) Sanghavi e t a 1 (25) t r e a t e d oxyphenbutazone

w i t h a m i x t u r e of anhydrous a c e t i c a c i d and
h y d r o c h l o r i c a c i d . The product of t h e h e a t e d
mixture i s reacted with v a n i l l i n or 4-
dimethylaminobenzaldehyde i n a c e t i c a c i d . The
r e s u l t i n g c o l o r i s measured a b s o r p t i o m e t r i c a l -
l y o r a t 4 2 4 nm w i t h v a n i l l i n o r a t 406 nm
with 4-dimethylaminobenzaldehyde. Beer’s Law
i s obeyed f o r 8 t o 12 y/ml when measured
s p e c t r o p h o t o m e t r i c a l l y o r 5 0 t o 2 5 0 y/ml when
measured a b s o r p t i o m e t r i c a l l y .
5. 3.3 S p e c t r o f l u o r o m e t r i c Method
Miller e t a 1 (26) s t u d i e d t h e luminescence pro-

p e r t i e s of oxyphenbutazone and some o t h e r a n t i -
inflammatory and a n t i p y r e t i c d r u g s . Although
oxyphenbutazone showed no s i g n i f i c a n t f l u o r e s -
cence a t room t e m p e r a t u r e i t w a s s t r o n g l y f l u o -
r e s c e n t a t 77K. Submicrogram q u a n t i t i e s of t h e
drug could be r e a d i l y d e t e c t e d . The wavelength
of e x c i t a t i o n maximum i n e t h a n o l was 2 8 0 nm and
t h a t of t h e f l u o r e s c e n c e maximum w a s 4 3 0 nm.
OXYPH EN B UTAZONE 355

5.4.1 Thin-Layer Chromatography (TLC)
S e v e r a l TLC methods a r e a v a i l a b l e f o r t h e s e p a r a -
t i o n , i d e n t i f i c a t i o n and d e t e r m i n a t i o n of oxy-
phenbutazone and i t s m e t a b o l i t e s .
Schmidt (27) a n a l y s e d oxyphenbutazone t o g e t h e r

w i t h o t h e r p h a r m a c e u t i c k l s by s t a i n i n g on s i l i c a
g e l GF254 u s i n g W l i g h t v i s u a l i z a t i o n . An
A v i c e l l a y e r chromatography w a s developed by L e e
(28) 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
s e v e r a l k i n d s of d r u g s . A c i d i c m a t e r i a l s w e r e
s e p a r a t e d by a c i d i c s o l v e n t s and b a s i c materials
w e r e s e p a r a t e d s a t i s f a c t o r i l y by b a s i c s o l v e n t s
u s i n g mixed A v i c e l k i e s e l g e l p l a t e s . The pro-
c e d u r e w a s a p p l i e d t o t h e d i f f e r e n t pharmaceuti-
c a l f o r m u l a t i o n s . MoYe TLC methods are i n c l u d e d
i n T a b l e 2.
Table 2. TLC Methods f o r t h e D e t e r m i n a t i o n of Oxyphenbutazone.
Adsorbent S o l v e n t System Visualizing R value Ref.

f
agent
S i l i c a g e l MethanollCHC131
G-0.06 M Aq. NH3
KH2PO4 (100:300:1)
b u f f e r (1:2)
dry p l a t e
and a c t i v a t e
at llOo.
Silica gel HexaneIAcetone UV r a d i a t i o n 0.76 (30)

(1:l)
HF254 + 366
Silica gel CHC13/Ethylace- UV r a d i a t i o n 0.28 (30)
tate
HF254 + 366
Silica gel S t r o n g NH3 KMn04 0.77 (2)
solution/Metha-
no1 (1.5:lOO)
Silica gel CyclohexaneIAce- W radiation 0.58 (31)

sheets tone/Acetic acid a t 254 nm
(40: 50:l)
A paper chromatographic method w a s d e s c r i b e d by

C l a r k e ( 2 ) and i s o u t l i n e d above under Chromato-
graphic T e s t s .
A g a s chromatographic method w a s r e p o r t e d by
Hasegawa e t a 1 (32) f o r t h e q u a n t i t a t i v e d e t e r -
m i n a t i o n of p y r a z o l i d i n e d i o n e a n a l g e s i c s p r e s e n t
i n blood plasma u s i n g a n e l e c t r o n c a p t u r e t e c h -
nique. The method i s claimed t o b e s i m p l e and
a b l e t o d e t e c t low c o n c e n t r a t i o n s . The g a s
chromatographic r e t e n t i o n i d e x e s of several com-
pounds of t o x i c o l o g i c a l i n t e r e s t i n c l u d i n g
oxyphenbutazone were determined ( 3 3 , 3 4 ) i s o t h e r -
m a l l y a t 180° on SE30, O V 1 , O V 1 7 and QF4. The
d a t a f o r many of t h e s e s u b s t a n c e s w e r e compared
s t a t i s t i c a l l y among themselves when a p p r o p r i a t e
(SE30 and O V l ) and w i t h l i t e r a t u r e d a t a . I n
g e n e r a l , no s t a t i s t i c a l l y s i g n i f i c a n t d e v i a t i o n s
were found i n t h e series compared. The v a l u e s
of t h e r e t e n t i o n i d e x e s w e r e r e p r o d u c i b l e t o a
good d e g r e e which means t h a t t h e s e i n d e x e s a r e
of c o n s i d e r a b l e d i a g n o s t i c v a l u e s i n chemical
toxicological investigations.
Bruce -- e t a1 ( 3 5 ) determined oxyphenbutazone and

phenylbutazone i n plasma and u r i n e by g a s - l i q u i d
chromatography. While phenylbutazone w a s d e t e r -
mined d i r e c t l y a f t e r e x t r a c t i o n from blood
plasma o r u r i n e , oxyphenbutazone w a s r e a c t e d
with heptafluorobutyric anhydride p r i o r t o gas
chromatography. Tanimura -- e t a 1 (36) d e s c r i b e d
a g a s - l i q u i d chromatographic method f o r t h e
d e t e r m i n a t i o n of phenylbutazone and i t s metabo-
l i t e s , oxyphenbutazone and y-hydroxyphenylbuta-
zone i n human and r a b b i t f o l l o w i n g a d m i n i s t r a t i o n
of phenylbutazone. A modified Herrmann’s extrac-
t i o n method h a s been used and coupled w i t h t h e
g a s - l i q u i d chromatographic p r o c e d u r e w i t h o u t
d e r i v a t i v e f o r m a t i o n f o r phenylbutazone and
using trimethylsilylation f o r the metabolites.
The procedure w a s d e s c r i b e d as a c c u r a t e and
s u f f i c i e n t l y s e n s i t i v e f o r use i n routine c l i n i -
c a l a s s a y and t h e e s t i m a t i o n of t h e pharmacoki-
n e t i c p a r a m e t e r s of phenylbutazone and i t s
metabolites.
OXYPHENBUTAZONE 3.57
5 . 4 . 4 High Performance L i q u i d Chromatography (HPLC)
Pound and S e a r s (37) d e s c r i b e d a s e n s i t i v e ,

s p e c i f i c , high-speed l i q u i d c h r o m a t o g r a p h i c pro-
c e d u r e f o r t h e s i m u l t a n e o u s d e t e r m i n a t i o n of
phenylbutazone and i t s m e t a b o l i t e , oxyphenbuta-
zone i n blood plasma. According t o t h i s proce-
d u r e , a c i d i f i e d plasma i s p a r t i t i o n e d w i t h
c y c l o h e x a n e : e t h e r (1:l) c o n t a i n i n g t h e 2,4-
d i n i t r o p h e n y l h y d r a z o n e o f 3,4-dimethoxybenzalde-
hyde as an i n t e r n a l s t a n d a r d . The o r g a n i c
e x t r a c t i s reduced t o d r y n e s s , t h e r e s u l t i n g
r e s i d u e i s r e d i s s o l v e d i n c h l o r o f o r m and a l i -
q u o t s of t h i s s o l u t i o n a r e chromatographed on an
a d s o r p t i o n column, u s i n g a m o b i l e p h a s e of 0.002%
a c e t i c a c i d and 23.0% t e t r a h y d r o f u r a n i n n-hexane
a t 35OC. Use of a UV d e t e c t o r p e r m i t s q u a n t i t a -
t i v e a n a l y s i s of samples c o n t a i n i n g less t h a n
0.25 ug/ml of p h e n y l b u t a z o n e o r oxyphenbutazone.
H a r z e r a n d B a r c h e t (38) s e p a r a t e d v a r i o u s combi-
n a t i o n s of a n a l g e s i c s by HPLC, u s i n g b o t h
r e v e r s e - p h a s e and a d s o r p t i o n columns and UV
d e t e c t i o n a t 254 nm. For t h e r e v e r s e - p h a s e
t e c h n i q u e , v a r i o u s p r o p o r t i o n s of methanol-water
were used a s e l u a n t , whereas t h e a d s o r p t i o n
system r e q u i r e s a ) c h l o r o f o r m - m e t h a n o l - a c e t i c
a c i d and b) chloroform-C7H16 - a c e t i c a c i d . The
a d v a n t a g e of t h e r e v e r s e - p h a s e column w a s t h a t
i t r e q u i r e d changes o n l y i n column t e m p e r a t u r e
and s o l v e n t p r o p o r t i o n s i n o r d e r t o e f f e c t a l l
s e p a r a t i o n s . U s e of a v a r i a b l e wavelength
d e t e c t o r enhanced t h e s e n s i t i v i t y by p e r m i t t i n g
a d j u s t m e n t t o t h e a b s o r p t i o n maximum of e a c h
drug.
Oxyphenbutazone, and o t h e r n o n - n a r c o t i c a n a l g e -
s i c s are s c r e e n e d by r e v e r s e - p h a s e h i g h - p r e s s u r e
l i q u i d chromatography ( 3 9 ) . The column i s
L i C h r o s o r b RP8 and t h e m o b i l e p h a s e i s a a c e t o -
n i t r i l e - w a t e r mixture. Blood and o r g a n s a r e
homogenized i n 4% p e r c h l o r i c a c i d b e f o r e e x t r a c -
t i o n w i t h t h e s o l v e n t s . U r i n e i s e x t r a c t e d with-
out primary treatment.
D i e t e r l e e t a1 ( 4 0 ) used a p r e p a r a t i v e r e v e r s e -
p h a s e chromatography f o r s e p a r a t i o n of p o l a r and
n o n p o l a r m e t a b o l i t e s , i n c l u d i n g t h o s e of phenyl-
358 ABDULLAH A . AL-BADR A N D HUMEIDA A . EL-OBEID
b u t a z o n e , on column packed w i t h m i c r o n i z e d XAD-

2 r e s i n . By e q u i l i b r a t i o n w i t h s i n g l e - p h a s e
m i x t u r e s of water, a lower a l i p h a t i c a l c o h o l and
a hydrophobic s o l v e n t a r e v e r s e - p h a s e p a r t i t i o n
system i s formed i n s i t u . The c h r o m a t o g r a p h i c
technique is claimed t o b e c h a r a c t e r i z e d by a
l a r g e c a p a c i t y , a h i g h power of r e s o l u t i o n and
a h i g h d e g r e e of s e l e c t i v i t y and r e p r o d u c i b i l i t y
and v e r s a t i l e a p p l i c a t i o n i n t h e i s o l a t i o n of
p o l a r and n o n p o l a r d r u g m e t a b o l i t e s from complex
mixtures.
Acknowledgement
A sample of oxyphenbutazone w a s k i n d l y d o n a t e d by Ciba-

Geigy L i m i t e d , Basle, S w i t z e r l a n d . The a u t h o r s would l i k e
t o t h a n k M r . T a n v i r A. B u t t f o r t y p i n g t h i s m a n u s c r i p t .
Reference
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N.J., U.S.A., p . 903 (1976).
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The P h a r m a c e u t i c a l P r e s s , London, p . 462 (1969).
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O f f i c e , London, U.K., p. 321 (1980).
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P u b l i s h i n g Co., E a s t o n , P e n n s y l v a n i a , p. 575-6 (1980).
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J. H e t e r o c y c l . Chem., 1 5 ( 1 ) , 1 3 (1978).
-
6. B. U n t e r h a l t , -Arch.
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J. --
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( 1 957) .
9. B. Herrmann, Med. E x p t l . , 1,170 (1959).
OXYPHENBUTAZONE 250
10. M. Weiner, P.G. Dayton and A.G. Hendrickx i n " U s e Non-

human P r i m a t e s Drug Eval., Symp." 1967 (pub. 1968),
3-22. E d i t e d by Vagtborg, H a r o l d . Univ. of Texas P r e s s :
A u s t i n , Tex.
11. J.J. Burns, Ann. N.Y. Acad. S c i . , 151 ( A r t . 21, 959

(1968).
12. J . M . P e r e l , M.M. S n e l l , W. Chen and P.G. Dayton, Biochem.

Pharmacol, , 13(9) , 1305 (1964).
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179, 411 (1971).
14. G . Hoffmann, H. Heimpel and P. P f a n n e n s t i e l i n "Nucl.
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15. H. S t i e r l i n and G. Saubermann i n "Nucl. Med., Suppl.",

1968 (Pub. ,1970) , No. 8, 183.
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Biophys. x. Commun., 63(3), 748 (1975).
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L.E. Sicam and J.J. Burns,
(1960).
&. J. K . , 2, 1017
18. M . I . Walash and M. R i z k , --

I n d i a n J. Pharm., __I
39(4), 82
(1977).
19. A.G. Fogg and Y.Z. 94, 453

Ahmed, Anal. Chim. A c t a , -
(1977).
20. C. P e l i n a r d , E. Nivaud and P. Boucly, T a l a n t a , 25, 119

(1978).
21. F. F r e y t a g , A r z n e i m i t t e l - F o r s c h . ,-
20, 1273 (1970).
--
22. W. Fuchs, Muench. Med. Wochschr., 107,1267 (1965).
23. E. S v a t e k and A. Hradkova, Cesk. Farm., 15,76 (1966).
24. A.M. Wahbi, H. Abdine, M. Korany and M.H. Abdel Hay,

-
J. -Assoc. - - -.'-
O f f . Anal. Chem 61, 1113 (1978).
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I n d i a n J.
-'Pharm
* -
39, 87 (1977).
26. J . N . Miller, D.L. P h i l l i p p s , D.T. Burns and J . W . Bridges,

T a l a n t a , 25, 46 (1978).
27. F. Schmidt, -
Dtsch. - Z t g . , 114, 1593 (1974).
Apoth. -
28. S.C. Lee, -- 4, 117 (1973).

Hua Hsuch, -
29. F. Gouezo, J . P . Roman and B. C r i s t a u , Annls. Pharm. E.,

-
26, 301 (1968).
Acta P o l . -
30. T. Pomazanska-Kolodziejska, -- Pharm., 29, 389
(1972).
-
31. R.D. Thompson and G.L. Johnson, -
J. Chromatogr., 88, 361
(1974).
32. M. Hasegawa, T. Maeda, H. Takenaka, T. Noguchi and Y.

Hirayama, J a p a n Kokai, 76 25, 194 ( C l , GOlN31/08). 01
Mar. 1976. Appl. 74/97, 698. 26 Aug. 1974.
33. E . Marozzi, V. Gambaro, F. L o d i and A. P a r i a l i , Farmaco.

-
Ed. -P r a t-
. 31, 180 (1976).
34. Idem., -
Ibid., 32, 330 (1977).
35. R.B. Bruce, W.R. Maynard and L.K. Dunning, 2. Pharm. g.,
63, 446 (1974).
I
36. Y. Tanimura, Y. S a i t o h , F. Nakagawa and T. S u z u k i , Chem.

- B u l l . , 23, 651 (1975).
Pharm. -
37. N.J. Pound and R.W. S e a r s , J. Pharm. S c i . , 64,248 (1975).
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D1. Apoth-Ztg., 116,1226
(1976).
39. H. K a e f e r s t e i n and G. S t i c h t , Beitr. G e r i c h t l . -

Med.,
-
36, 457 (1978).
40. W. Dieterle, J . W . F a i g l e and H. Mory, J. Chromatogr.,

-
168, 27 (1979).
PENTAZOCINE
Terry D. Wilsoii
I. Foreword 362
2. Description 362
2.2 Formula 362
2.4 Structure 362
2.5 CA Registry Number 362
2.6 Appearance, c o k x , Odor 362
2.7 Recognized Dosage Forms 365
3. Synthesis and Resolution 365
3.1 Synthesis 365
3.2 Isomers and Resolution 365
4. I Polymorphism 365
4.3 Differential Scanning Calorimetry 366
4.6 Ionization Constant 366
4.7 Partition Coefficient 372
4.8 Solubility 372
4.9 Spectral 37 5
4.10 X-Ray Diffraction 382
4.1 I Dissolution 382
5. Stability 388
5. I Hydrolysis 3x8
5.2 Dosage Form Stability 389
6. I Titrimetric 389
6.2 Ultraviolet Spectrophotometry 389
6.3 Fluorescence Spectrophotometry 389
6.4 Radioimmunoassay 390
6.5 Chromatography 39 I
7. Biological Disposition and Pharmacokinetics 40 I
7.1 Disposition 40 I
7.2 Pharmacokinetics 40 I
8. Determination in Body Tissues and Fluids 406
References 41 1
ANALYTICAL PROFILES O F DRUG SUBSTANCE Copyright IC 1984

VOLUME 13 36 I by the American Pharmaceutical Association
ISBN 0- 12-260813-5
362 TERRY D. WILSON
1. Foreward
Pentazocine i s a p o t e n t a n a l g e s i c o f t h e benzo-
morphan s e r i e s w i t h a 50 m g o r a l dose e q u i v a l e n t t o
60 m g o f codeine. I t i s a l s o a weak n a r c o t i c a n t a g -
o n i s t w i t h about o n e - f i f t i e t h t h e a c t i v i t y o f
n a l o r p h i n e . Pentazocine i s i n d i c a t e d i n t h e t r e a t -
ment o f moderate t o s e v e r e p a i n . I t i s a l s o used
a s a p r e a n e s t h e t i c and a n e s t h e t i c supplement (1,2).
2. Description
2.1 Nomenclature
Pentaz c i n e
Talwin 8
Fortral ++
(2d P 6 0 1 , l l R ) - ( !> -1,2,3 ,4,5,6-hexahydro-
6,11-dimethyl-3-(3-methyl-2-bu ?nyl)-2,6-methano-
3-benzazocin-8-01
2.3 K o l e c u l a r Weight
2a5*43
2.4 Structure
2.5 CA R e g i s t r y Number
base 7 3-83 -11
h y d r o c h l o r i d e [68964-90-9]
Pentazocine i s w h i t e t o p a l e t a n , odor-
l e s s , c r y s t a l l i n e powder.
PENTAZOCINE 363
- @z$
- WoMe
Me Me
NaBH4
I HBr - Me
Me
HO
CN
/
-
1) HCI
HO
Me
Br C H2CH = C (C H3)2
DMF
HO
--Me
Me
NORPENTAZOCINE PENTA ZO C1NE
Scheme 1. Synthesis of pentazocine.

Ap
I
z
a
\
PENTAZOCINE 365
2.7 Recognized Dosage Forms

The forms o f p e n t a z o c i n e r e c o g n i z e d by t h e
U.S. P . i n c l u d e p e n t a z o c i n e b a s e , h y d r o c h l o r i d e - and
l a c t a t e s a l t s i n t h e f o l l o w i n g f o r m u l a t i o n s (1 ,3 , & I .
p e n t a z o c i n e h y d r o c h l o r i d e t a b l e t s 50 mgbase
p e n t a z o c i n e l a c t a t e i n j e c t i o n 30 m g base/mL
p e n t a z o c i n e h y d r o c h l o r i d e and a s p i r i n
tablets 12.5 mg base
325 mg a s p i r i n
3. S y n t h e s i s and R e s o l u t i o n
3.1 Synthesis
The s y n t h e s i s o f p e n t a z o c i n e was o r i g i n a l -
l y accomplished by Archer and coworkers (5,6,7)and
h a s been reviewed p r e v i o u s l y ( 8 , 9 , 1 0 , 1 1 ) . The
i n t e r m e d i a t e : 1,2,3,4,5,6-hexahydro-8-hydroxy-
6,11-dimethyl-2,6-methano-3-benzazocine ( n o r p e n t a z -
o c i n e ) w a s s y n t h e s i z e d e a r l i e r by Nay and Eddy (12)
as shown i n Scheme 1. Treatment o f n o r p e n t a z o c i n e
with l-bromo-3-methyl-2-butene i n DNF gave p e n t -
azocine. A v a r i e t y o f a l t e r n a t e r o u t e s t o pent-
a z o c i n e have been demonstrated. Included a r e meth-
ods employing: a 3-benzyl i n t e r m e d i a t e (13,141,
an i n i t i a l r e a c t i o n o f 2-lithio-2-butene with f o r -
maldehyde (151, a thermal rearrangement o f amino-
methylcyclopropyl ketone (16), a n abnormal Hof-
mann d e g r a d a t i o n o f N-(&-hydroxybenzyl)-3-benz-
azociniurn h a l i d e s ( 1 7 ) and a B i s c h l e r - N a p i e r a l -
ski reaction(l8).
3.2 Isomers and R e s o l u t i o n

Geometrical ( c i s - t r a n s ) i s o m e r s o f benz-
omorphans r e s u l t from t h e c y c l i z a t i o n s t e p i n
t h e i r s y n t h e s i s when t h e bond t o C-6 i s formed
(Scheme 2). When t h i s i s c o n s i d e r e d a t r a n s -
a d d i t i o n t o t h e double bond t h e preponderance o f
t h e c i s o r d form i s e a s i l y explained ( 1 0 ) .
R e s o l u t i o n o f o p t i c a l i s o m e r s f r o m b o t h c i s and
t r a n s f o r m s has been r e p o r t e d (7,19,20). The
n o r p e n t a z o c i n e p r e c u r s o r i n t h e c i s form c r y s t a l -
l i z e d f i r s t as t h e ( - ) base w i t h ( + ) t a r t a r i c a c i d
which would g i v e t h e t w e n t y - f o l d more a c t i v e ( - 1
p e n t a z o c i n e . The ( + ) base ( - ) t a r t a r a t e was r e c -
overed f r o m r e s o l u t i o n l i q u o r s . Q u i n i c a c i d s a l t s
were formed f o r r e s o l u t i o n o f t h e t r a n s isomers.
4. Ph s i c a l P r o p e r t i e s
h y m o m h i s r n
366 TERRY D. WILSON
Two d i f f e r e n t i n t e r c o n v e r t i b l e c r y s t a l
polymorphs o f p e n t a z o c i n e h y d r o c h l o r i d e a r e known
w i t h m e l t i n g p o i n t s o f 2180 and 254' ( 2 , 2 1 ) .
4.2 Melting P o i n t
The r e p o r t e d m e l t i n g p o i n t s o f p e n t a z o c i n e
base and h y d r o c h l o r i d e salts a l o n g w i t h t h o s e o f
r e s o l v e d o p t i c a l isomers a r e shown i n Table 1.
4.3 D i f f e r e n t i a l Scanning C a l o r i m e t r y
A D.S.C. curve f o r p e n t a z o c i n e i s shown
i n F i g u r e 1. It w a s c a r r i e d o u t on a P e r k i n Elmer
D.S.C. 1B a t a s c a n r a t e o f 2.5O,per minute, a
range s e t t i n g o f 4 and a n i t r o g e n \ e r g e r a t e o f
20 mL p e r minute ( 2 3 ) . ---

Table 2 i n c l u d e s r e s u l t s for o p t i c a l
r o t a t i o n o f r e s o l v e d o p t i c a l isomers o f Pentazocine
c a r r i e d o u t by T u l l a r et g ( 1 9 ) . A l s o i n c l u d e d
i s t h e v a l u e o b t a i n e d by Nambara - et -
a1 for ( - )
pentazocine (22).
4.5 Elemental_ A n a l y s i s
R e s u l t s o f e l e m e n t a l a n a l y s i s f o r pentazo-
c i n e and p e n t a z o c i n e h y d r o c h l o r i d e a r e shown i n
Table 3 .
4.6 Ionization Constant
The PKa v a l u e for p e n t a z o c i n e l i s t e d i n
g e n e r a l t e x t s as 8.76 ( 2 4 ) or 8.95 ( 2 ) i s t h e
t e r t i a r y amine p r o t o n a t i o n c o n s t a n t . Borg and
Nlikaelsson however have shown t h a t t w o i o n i z a t i o n s
f o r pentazocine s h o u l d be cDnsidered. F i r s t t h e
pentazocinium i o n l o s e s t h e p r o t o n f r o m t h e n i t -
rogen and t h e n t h e p h e n o l i c p r o t o n i s l o s t a t
h i g h e r pH. T h i s i s p i c t u r e d i n Scheme 3 where
two s e p a r a t e i n t e r m e d i a t e s a r e p o s s i b l e ; t h e
n e u t r a l p e n t a z o c i n e b a s e (111) and a z w i t t e r i o n i c
form (11).
The f o u r microscopic d i s s o c i a t i o n c o n s t a n t s
were determined u s i n g 0.1 i o n i c s t r e n g t h c a r b o n a t e /
b i c a r b o n a t e b u f f e r s with t h e v a l u e s : p k = 9.74,
pk2= 10.56, pk12 = 11.17 and pk21 = 10.3). From
t h e s e t h e macroscopic d i s s o c i a t i o n c o n s t a n t s were
determined from t h e r e l a t i o n s :
H+
+ Me
Me > H+
+ Me
H+
HO
HO
Iu
Scheme 3. Pentazocine ionization.
TABLE 1
MELTING POINTS OF PENTAZOCINE
Isomer
Form Optical Geometrical Melting Point OC Reference
base (3 cis 145.4-148.6 5
base (+I cis 179 -182 7
base (-1 cis 176 -179 7
base (-1 cis 164 -170 22
base (+> cis 180 .4-182.0 19

base (-1 cis 180.6-182.2 19
hydrochloride (+) trans 254.5-255.0 19
hydrochloride (-1 trans 246.0-254.0 19
PENTAZOCINE 369
1 1 1 1 1 1 1 1 1 1 I I
n n n
0 0 0
0 0 0
(D 0 m
-
d-
Y
-
0
Y
-
In
Y
Fig. 1. Differential scanning calori-

metry curve f o r pentazocine.
TABLE 2
OPTICAL R O T A T I O N OF PENTAZOCINE ISOMERS
Fo m Isomer [d]o; Solvent Reference

base cis (+) +I35* 5 CHC13 19
W
base c i s (-) -138.0 CHC13 19
d
base cis (-) -131 CHC13 22
hydrochloride trans(+) +115.4 2% HOAc 19
hydrochloride trans(-) -116 3 2% HOAc 19
TABLE 3
PENTAZOC INE ELEMENTAL ANALYSIS
$I Carbon $I Hydrogen %' N i t r o g e n % Chlorine"
Form Isomer C a l c ' d Found C a l c ' d Found C a l c ' d Found C a l c ' d Found Ref.
base (T) c i s 79.95 79.84 9.34 9.28 4.91 5.23 - - 5
HC1 (+)trans 70.89 70.62 8.77 8.46 4.35 4.25 11-01 11.25 1 9
* ( - ) t r a n s isomer
312 TERRY D.WILSON
aH+ [Iv]
K2=
[II] + LII]
Thus pKI was f o u n d t o e q u a l 9.68 and pK2 e q u a l s
11.23 (25).
4.7 Partition Coefficient
The p a r t i t i o n c o e f f i c i e n t f o r p e n t a z o c i n e
h a s been measured between v a r i o u s o r g a n i c and
aqueous phases. The r e s u l t s o f f o u r s t u d i e s a r e
shown i n Table 4. The f i r s t v a l u e was o b t a i n e d by
a s i n g l e e x t r a c t i o n u s i n g 50 mL o f each phase
while t h e second value i s from a 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 s t u d y with pentazocine added t o t h e
aqueous phase and e x t r a c t e d e i g h t t i m e s i n t o ben-
zene. The t h i r d value was o b t a i n e d by t h e s i n g l e
e x t r a c t i o n method with t h e a d d i t i o n a l d e f i n i t i o n o f
a d i s t r i b u t i o n r a t i o , DNOH, f o r p e n t a z o c i n e u s i n g
t h e p a r t i t i o n c o e f f i c i e n t kd(NOH) i n t h e r e l a t i o n :
1 - 1
+-
DNOH kd ( N O H )
Here kl, k2 and k2 are t h e microscopic ion-

i z a t i o n c o n s t a n t s d e f i n k d i n s e c t i o n 4.6. From
t h i s it was p o s s i b l e t o c a l c u l a t e and p l o t v a r i a t i o n
i n DNOH with pH f o r pentazocine a s shown i n F i g u r e
2. The f o u r t h p a r t i t i o n c o e f f i c i e n t w a s o b t a i n e d
by s t r u c t u r a l group c o n t r i b u t i o n t o t h e measured
p a r t i t i o n c o e f f i c i e n t o f a r e f e r e n c e compound
(morphine). T h i s w a s done f o l l o w i n g t h e Hansch
approach.
4.8 Solubility
The s o l u b i l i t y o f pentazocine base i s
TABLE 4
PARTITION COEFFICIENTS FOR PENTAZOCINE

-~
0r g a n ic. Aqueous Coefficient Reference
CHC13 0 . 1 M pH 7.35 25.4 26

phosphate b u f f e r
benzene 0.2 M pH 6.7 1.17 27

phosphate b u f f e r
benzene 0.1 i o n i c s t r e n g t h 630 25

pH 6.9-8.2 phos-
phate b u f f e r
octanol water 1.07 104 28
3 74 TERRY D. WILSON
I
2 2.0 -
n
1.0 -
s
Fig. 2. Relationship between log (benzene/water)

distribution ratio and p H for pentazocine (after
Borg and Mikaelsson) .
-- 40 cI 0
c
E
\
- 30
0
2 4 6 8 10 12
PH
Fig. 3. Relationship between aqueous solubility

and pH for pentazocine.
PENTAZOCINE 315
shown for v a r i o u s s o l v e n t s i n Table 5. I n a d d i t i o n

a pH/ e q u i l i b r i u m s o l u b i l i t y s t u d y h a s been c a r r i e d
o u t i n which s a t u r a t e d s o l u t i o n s o f p e n t a z o c i n e
were prepared. u s i n g H C 1 and NaOH t o a d j u s t pH.
A p l o t o f t h e pH/ s o l u b i l i t y r e l a t i o n s h i p i s shown
i n F i g u r e 3(30).
IC*’ m s s Spectrum
A d i r e c t i n l e t e l e c t r o n impact mass
spectrum o f p e n t a z o c i n e ( S t e r l i n g - W i n t h r o p )
o b t a i n e d on a HP 5980 i s shown i n F i g u r e 4(31).
The e l e c t r o n energy, emission c u r r e n t and s o u r c e
t e m p e r a t u r e were 150 e . v , 0.42 mA and 185.6O
r e s p e c t i v e l y . The m o l e c u l a r i o n i s found a t m/e
o f 285.1, w i t h t h e b a s e peak a t 217.1 ( 1 0 0 % ) .
G.C./N.S. r e s u l t s f o r p e n t a z o c i n e p r e v i o u s l y
r e p o r t e d i n c l u d e b o t h d e r i v a t i z a t i o n and nonder-
i v a t i z a t i o n procedures, Underivatized pentazocine
gave a molecular i o n a t m/e 285 and t h e nor-ion
a t m/e 217 ( 3 2 ) while t h e 217 base Peak and a 202
peak were observed by a m u l t i p l e i o n d e t e c t o r (33).
Chemical i o n i z a t i o n w i t h methane gave a molecular
i o n o f m/e 286 and fragments shown below (34).
m/e
230 (M+ - 55) 30% (M+ - CH-CH-( CH3)z)
217 (M+ - 68) 155 (M+ - CH2CH-C(CH3)2)
270 (M+ - 15) 10% (M* - CH3)
The most f r e q u e n t l y encountered d e r i v a t i v e of
p e n t a z o c i n e i n GC/NS w o r k h a s been t h e t r i f l u o r o -
a c e t y l with a molecular i o n a t m/e 381 (35,36,37).
A p e n t a f l u o r o b e n z y l bromide was r u n g i v i n g a mol-
e c u l a r i o n o f m/e 4-65 (38). A t r i m e t h y l s i l y l
d e r i v a t i v e gave a M+ o f 357 w i t h a base peak o f
45 m/e f o r p e n t a z o c i n e (39), while a permethylated
p e n t a z o c i n e g l u c u r o n i d e gave a m o l e c u l a r i o n a t
517, a n aglycone a t 217 and a b a s e peak a t m/e 201.
H y d r o l y s i s o f t h e g l u c u r o n i d e f o l l o w e d by s i l a t i o n
gave t h e 357 monoTMS d e r i v a t i v e ( 4 0 ) .
4.92 Nuclear Ma n e t i c Resonan=
The p r o t o__&--r
n and 3C nmr s p e c t r a
o f p e n t a z o c i n e ( S t e r l i n g - W i n t h r o p ) a r e shown i n
F i g u r e s 5 and 6 r e s p e c t i v e l y . Interpretations are
g i v e n i n Tables 6 and 7 . The pmr spectrum was
r u n o n a Varian HA-100 on a 10% s o l u t i o n i n
316 TERRY D.WILSON
TABLE 3
PENTAZOCINE SOLUBILITY
Solvent S o l u b i l i t y (%w/v)
Reference: 29 2
CHC13
95% e t h a n o l
ethyl acetate 2.94 -
benzene 1.25 -
ac e t one 3.3 -
ether 3 03-5 0 2.4
water 0.005 0.1
1 N HC1 3 -
1 N lactic acid 15 -
lnt
378
t
a,
c
4
u
0,
a
4J
c
a,
a
w
0
5
k
4J
u
a,
a
[O
/ / I a,
u
c
rd
c
0
[O
a,
k
u
-ti
4J
a,
c
5l
2
k
rd
a,
rl
u
3
c
u
m
d
a;
' 5
w -ti
k
- 0
5l-l
-4 c
h u
- 0
E k
25
h
:.c
379 E
380 TERRY D. WILSON
TABLE 6
PENTAZOCINE 1 H NMR ASSIGNMENTS
Chemical S h i f t (ppm) No. H Assignment
1.07 3H CH3-CH
1 m53 3H CH3-C -
1.92, 1.85 6 H (CH3)2C=
1.4-3.6 5H CH2x2 , CH
3.8-4.0 4H NCH2x2
5.45 1 H N-CH
6.86-7.25 3H aromat ic s
7.5 1 H OH, p a r t i a l l y
exch * d
11.36 2 H exch'd H
PENTAZOCINE 38 1
TABLE 7
PENTAZOCINE I 3 C NMR ASSIGNMENTS
CHZ-CHZCH-CH~
d 1 * HC1
CH3
HO
9, h P
Carbon PPdTMS Multiplicity

from partial off
resonance dec oupling
a 156.1 S
b 140.9 S
C 139.6 S
d 127.9 d
e 122.5 S
f 113.7 d, d
g 111.7 d
h 57.3 d
i 50.8 t
j 44.8 t
k 38.1 t
1 37.2 d
m 34.6 S
n 25.1 9"
0 23.7 9"
P 21 .a t
sr 17.7
12.3
9
9
"values may be interchanged

382 TERRY D. WILSON
CF3COOD. The l 3 C nmr w a s o b t a i n e d on a J e o l FX 60

f o r a 10% DMSO dg s o l u t i o n (41). P r e v i o u s p r o t o n
nmr data h a s been p u b l i s h e d f o r p e n t a z o c i n e ( 3 2 ) ,
N-benzylpentazocinium bromide (42) and p e n t a z o c i n e
c h l o r o a c e t i c e t h e r ( 2 2 ) . These a r e summarized i n
Table 8.
The i n f r a r e d suectrum o f Dentazocine
( S t e r l i n g - W i n t h r o p ) , 1/2 7% i n - KBr, taken- on a P e r k i n
E l m e r 467 i s shown i n F i g u r e 7 (43). The previous-
l y noted a l l y l i c (C = C ) s t r e t c h i n g a t 1670 cm-1
i s a l s o seen (32).
An u l t r a v i o l e t spectrum o f pentazo-
c i n e ( S t e r l i n g - W i n t h r o p ) was o b t a i n e d on a Cary
15 and i s shown i n F i g u r e 8 . The r e l a t i v e
f o r t h i s 0.0318 g/L s o l u t i o n i n 95% a l c o h o l i s
s e e n a t 283 nm. The c a l c u l a t e d a b s o r p t i v i t y t h e r e
i s 2052 ( 4 4 ) .
A normalized powder x-ray d i f f r a c t i o n p a t -
t e r n f o r p e n t a z o c i n e ( S t e r l i n g - W i n t h r o p ) i s shown
i n Figure 9 . I t w a s r e c o r d e d on a Rigaku X-ray
D i f f r a c t o m e t e r M i n i f l e x w i t h Cu K a , Ni f i l t e r e d
r a d i a t i o n a t 4000 c p s , TC/1, ss/s, 0 . 5 2 e/ min.
The s c a n w a s done from 400 t o 3.50 2 8 with 1/1
peak h e i g h t r a t i o s r e l a t i v e t o t h e 1 2 . 4 O 2 0 100%
peak. The c a l c u l a t e d d-spacings a s s o c i a t e d w i t h
t h i s p a t t e r n a r e l i s t e d i n Table 9 ( 4 5 ) .
4.11 D i s s o l u t i o n
The d i s s o l u t i o n method f o r p e n t a z o c i n e
h y d r o c h l o r i d e t a b l e t s u t i l i z e s a UV d e t e r m i n a t i o n
o f pentazocine i n f i l t e r e d t e s t samples a t 278 nm.
The a p p a r a t u s used i s USP number two a t 50 rprn with
water a s t h e d i s s o l u t i o n medium. The s o l u t i o n s are
measured i n h y d r o c h l o r i c a c i d d i l u t e d with d i s s o l -
u t i o n medium t o g i v e 0.01 N HC1 and q u a n t i t a t i v e l y
compared t o a s u i t a b l e s t a n d a r d ( 4 6 ) .
A d i s s o l u t i o n procedure f o r p e n t a z o c i n e hydro-
c h l o r i d e and a s p i r i n t a b l e t s i s s e t f o r t h i n Adden-
dum a , Second Supplement t o t h e USP XX. The pro-
cedure i s c a r r i e d o u t w i t h a p p a r a t u s number one a t
80 rpm u s i n g w a t e r as t h e d i s s o l u t i o n medium.
??
5
v
a
0
I
%
k \ o
P -
O &
..
coo
I I
a 3 1
*
u \ o
a
E h
7 0
c2 b
.rl
c k
w
.r(
oc
w
2 A
H \o
u II
0 %
N I -
E
E m . . .
a % a 3
nw m m u l
..
a,
0
5
k
(v
n
W
4-(
a,
I2
u
N
p: v
u
II
u
a;
a
-d
k
0
rl
c
0
0
k
a
h
,G
a,
c
-4
u
0
N
rd
4
c
a,
a
w
0
5
k
c,
u
a,
a
In
aa,
k
rd
k
w
c,
H
r-
m
-4
E
p
PENTAZOCINE 385
I.o
0.9
0.8
0.7
8 0.6
z
a
0.5
0
cn
m
a 0.4
0.3
283 nm
0.2
0.I
0.0
210 240 270 300 330
WAVELENGTH (nm)
Fig. 8. Ultraviolet absorption

spectrum of pentazocine.
I
u
a
k
w
w
k
a,
a
50a
001 x O1/1
PENTAZOCINE 387
TABLE 9
PENTAZOCINE d SPACINGS
d(i) =
x
- sin 8 = 1.54
0
A
2
2 eo d(1) 1/10
11 8.03 69 69
11.5 7.68 13.13
12.4 7.12 100
13.15 6.72 18.18
13 95 6.34 60.6
15 95 5-54 34 3 4
16.85 5.25 81.81
17.1 5.17 71.71
17.75 4.99 20.2
18 35 4.82 12.12
20.0 4.43 31 - 3 1
20.45 4.33 50.5
21 *55 4.11 33 33
22.85 3.88 17 1 7
25 75 3.45 19 1 9
25.85 3.44 15.15
28.5 3.12 12.12
29.9 2.98 6.06
34.6 2.58 9.09
36.05 2.48 6.06
388 TERRY D.WILSON
Twenty-five mL of t e s t s o l u t i o n i s f i r s t shaken with

a s t r o n g l y basic a n i o n exchange r e s i n . Five mL o f
t h e s u p e r n a t a n t i s t h e n e x t r a c t e d with a mixture o f
20 mL chloroform and 1 0 mL o f 1 i n 4000 bromocresol
purple i n g l a c i a l acetic acid. A spectrophotometric
d e t e r m i n a t i o n o f t h e i o n p a i r formed between pen-
t a z o c i n e and t h e i n d i c a t o r i n t h e chloroform l a y e r
i s t h e n made a t 408 nm and compared t o a s i m i l a r l y
e x t r a c t e d pentazocine standard (4).
4.12 Identification
Pentazocine i s i d e n t i f i e d a c c o r d i n g t o
USP XX by u s e o f i n f r a r e d o r u l t r a v i o l e t s p e c t r o -
photometric methods. I n t h e f i r s t a d i s p e r s i o n i n
KBr of t h e d r i e d d r u g e x h i b i t s maxima o n l y a t t h e
same wavelengths as a similar p r e p a r a t i o n o f USP
Pentazocine Refemnce Standard. I n t h e u l t r a v i o l e t
method a 1 i n 12,500 s o l u t i o n o f p e n t a z o c i n e i n
0 . 0 1 N H C 1 e x h i b i t s maxima and minima a t t h e same
wavelengths as r e f e r e n c e standard. C a l c u l a t e d ab-
s o r b a n c e s a t 278 nm o f d r i e d drug and s t a n d a r d do
n o t d i f f e r by more t h a n 3.0 % (3).
Pentazocine i s i d e n t i f i e d i n t h e combination
p r o d u c t : p e n t a z o c i n e h y d r o c h l o r i d e and a s p i r i n
t a b l e t s by a method g i v e n i n Addendum a , 2nd Sup-
plement o f USP XX. A s i l i c a g e l TLC system i s used
w i t h t h e s o l v e n t system: e t h y l a c e t a t e : methanol
: formic acid ( 9 0 : 5 : 5 ) . D e t e c t i o n i s by UV, i o d i n e
vapor and i o d o p l a t i n a t e s p r a y ( 4 ) .
5. Stability
5.1 H y d r o l y s i s
The h y d r o l y s i s r e a c t i o n of p e n t a z o c i n e i s
t h e o n l y c h e m i c a l l y d e g r a d a t i v e pathway which h a s
been observed. An e a r l y r e p o r t d e s c r i b e d t h e
r e a c t i o n and c h a r a c t e r i z e d t h e p r o d u c t : 1,2,3,4,
5,6-hexahydro-6,11-dimethyl-3-(3-hydroxy-~-~ethyl-
butyl)-2,6-methano-3-benzazocin-8-ol which w a s
o b t a i n e d when p e n t a z o c i n e w a s h e a t e d a t 90° f o r
two h o u r s i n 1 N HC1. A c i d i c h y d r o l y s i s o f pen-
t a z o c i n e g1ucuroni.de a l s o y i e l d e d t h i s p r o d u c t
while p e n t a z o c i n e i n b i o l o g i c a l f l u i d s was found
t o be s t a b l e ( 3 2 ) . T h i s h y d r o l y s i s r e a c t i o n was
i n f a c t used f o r t h e d e t e r m i n a t i o n o f p e n t a z o c i n e
i n human u r i n e (34). The r e a c t i o n was e l u c i d a t e d
by a k i n e t i c s s t u d y which showed t h a t a carbonium
i o n p s e u d o - f i r s t o r d e r mechani m w a s involved.
The kobs a t 80° a t a. 3.9 x lo-’ M H C 1 c o n c e n t r a t i o n
PENTAZOCINE 389
w a s 3.412 x hr w i t h an a c t i v a t i o n energy o f
35.9 k c a l mol-1 and a n a c t i v a t i o n e n t r o p y o f 27.3
c a l ( m o l deg1-l ( 4 7 ) .
5.2 Dosage Form S t a b i l i t y
The s t a b i l i t y o f repackaged p e n t a z o c i n e
h y d r o c h l o r i d e i n j e c t i o n w a s s t u d i e d t o determine
t h e e x p i r a t i o n d a t e . An e x t r a p o l a t i o n o f t h e Ar-
r h e n i u s p l o t o b t a i n e d u s i n g 40: 50'an 600
r a t e c o n s t a n t s gave a k o f 5.448 x 10-3 day-l a t
250. T h i s meant t h a t a 10% l o s s o f p o t e n c y occur-
r e d i n 1909 days a t 25' ( 4 8 ) .
6. Methods o f A n a l y s i s
6.1 T i t x m e t r i c
Pentazocine can b e determined by a t i t r i -
m e t r i c procedure i n which t h e compound i s d i s s o l v e d
i n g l a c i a l a c e t i c a c i d . C r y s t a l v i o l e t i s used
as i n d i c a t o r and t h e s o l u t i o n i s t i t r a t e d t o a g r e e n
end-point w i t h 0 . 1 N p e r c h l o r i c a c i d . Each mL of
0.1 N perchloric acid a t t h i s point i s equivalent
t o 28.54 m g o f p e n t a z o c i n e base ( 3 ) .
6.2 E r a v i o l e t Spectrophotometry
Pentazocine can be determined i n p e n t a z -
o c i n e h y d r o c h l o r i d e t a b l e t s by a UV s p e c t r a l
method. The sample i s e x t r a c t e d w i t h s u l f u r i c
a c i d , b a s i f i e d , e x t r a c t e d w i t h e t h e r and back-
e x t r a c t e d i n t o 1 i n 7 0 d i l u t e s u l f u r i c a c i d . The
absorbance o f t h e s o l u t i o n i n 0 . 5 N H2SO i s d e t -
ermined a t 278 nm and compared t o t h a t o f t h e
s t a n d a r d p r e p a r a t i o n . A c a t i o n exchange column
i s o l a t i o n method can b e u t i l i z e d t o determine
pentazocine i n pentazocine l a c t a t e i n j e c t i o n .
P e n t a z o c i n e i s e l u t e d from t h e column with methanol:
6 N HC1 ( 1 : l ) . Sample absorbance i s r e a d a t 278 nm
and compared t o a p e n t a z o c i n e s t a n d a r d a t approx-
i m a t e l y 1 2 0 a g / mL ( 3 ) .
6.3 Fluorescence Spectrophotometry
Highly s e n s i t i v e s p e c t r o f l u o r o m e t r i c mea-
surements have been used e x t e n s i v e l y i n p e n t a z o c i n e
a n a l y s i s e s p e c i a l l y i n measurements from b i o l o g i c a l
s o u r c e s . Themethod developed e a r l y by Berkowitz,
Way and coworkers ( 2 7 , 4 9 , 5 0 , 5 1 , 5 2 ) h a s been modi-
f i e d and extended by o t h e r i n v e s t i g a t o r s (53,511,
55,56). Pentazocine l e v e l s i n plasma, u r i n e , b r a i n ,
and i n t e s t i n e have been determined by t h i s method.
Briefly the t i s s u e o r f l u i d is b a s i f i e d with a
390 TERRY D. WILSON
carbonate/bicarbonate buffer at pH 8-9 and the

pentazocine is extracted into benzene. An aliquot
is washed with 0.2 M sodium phosphate buffer,
pH 6.7 and is then back-extracted into 0.2 N H C 1 .
The fluorescence excitation wavelength used most
commonly has been 278 nm with emission measured
at 310 nm.
An extraction, ion-pair partition chromatog-
raphy separation method was developed by Borg and
Nikaelsson for pentazocine using fluorescence det-
ection ( 2 5 ) and has been used by others (57.58).
The tissue, plasma, brain or serum was extracted
with benzene following basification with a car-
bonate/bicarbonate buffer at pH 10.5. An ethanol-
ized cellulose column was used with a stationary
phase of 0.1 PI HC1 and a mobile phase: cyclohex-
ane: 1-pentanol (614) followed by extraction into
0.1 M phosphoric acid for fluorescence measurements.
Other fluorescence methods for pentazocine have
been used on plasma ( 5 9 ) and for TLC quantitation
(47). A fluorescent 2-p-chlorosulfophenyl-3-phen-
ylindone ( D I S - C 1 ) derivative of pentazocine has
been measured by TLC (60) while another extraction
method using heptane with 0.5:’. isoamyl alcohol on
basified plasma has been developed. In the latter,
fluorescence was measured after back-extraction into
0.1 N HC1 (61).
6.4 Radioimmunoassay
Radioimmunoassay methods have been devel-
oped for pentazocine which were shown to be both
sensitive and specific. An early method utilized
tritium labeled pentazocine at 1.5 Ci/mrnol. Anti-
bodies were produced in rabbits to both an azoben-
zoic acid and a carboxymethyl derivative of pen-
tazocine coupled by carbodiimide condensation to
poly-L-lysine. No cross reactivity was found for
metabolites and benzomorphan analogs to the former
antiserum with only conjugated pentazocine cross-
reacting with the latter antiserum (62).
In a second method rabbit antibodies were
produced against a butanoic acid derivative of
pentazocine which was conjugated to bovine se
albumin via a carbodiimide condensation. An € 8 1
labeled methyltyrosine pentazocine giving 25,000-
PENTAZOCINE 39 1
30,000 cpm/ 1 0 0 d was used in the assay. An ap-

proximate detection limit of 1 ng/mL was measured
(63)
Antiserum specific for the biologically more
active 1-pentazocine has been produced in rabbits
using an&pentazocine-2’-carboxymethyl ether-bovine
serum albumin conjugate. A low 0.084% cross-reac-
tivity was found for d-pentazocine while 51.2% was
measured for dP -pentazocine (22).
6.5 Chromatography
6.51 Thin L?yer Chromatography
Extensive use has been made of TLC
separation for pentazocine on both qualitative and
quantitative levels. Table 10 indicates mobile
and stationary phases used and Rf values obtained.
6.52 Gas Chromatography
Gas chromatography has been the most
frequently used method for pentazocine analysis as
indicated by published accounts. It has found
especially wide spread application in the field of
biological disposition of pentazocine. Table 11
summarizes column packing, detection, derivati-
zation, percent recoveries and detection limits
where applicable.
6.53 High-Performance LiquidEhromatoPraphy
Several reports of high-performance
liquid chromatographic analysis of pentazocine have
appeared. The separations have been made using
both normal phase and reversed-phase systems.
Detection was by ultraviolet, fluorescence and elec-
trochemical means. W measurements were generally
made at 278-280 nm on underivatized pentazocine
although a greater sensitivity was found using a
2-p-chlorosulfophenyl-3-phenylindonederivative (91).
Recoveries found for particular processes described
include 78% by extraction from blood (91) and 98.6
and 98.9% on pentazocine tablet extractions by
normal and reversed-phase methods respectively ( 9 2 ) .
Fluorescence measurements were made on a dimer
of pentazocine produced by on-column oxidation with
0.04 M K3Fe(CN)6. This product was separated from
a dihydromorphine fluorescent dimer and a pentaz-
ocine-dihydromorphine product (93).
TABLE 10
THIN LAYER CHROMATOGRAPHY OF PENTAZOCINE
Stationary Mobile Visualization Rf Re ference
Phase Phase
Silica gel glass Et0Ac:cyclo- iodoplatinate .35-.42 64
microfiber sheets hexanerMe0H: and iodine/
MI OH KI
( 52 :40r 0.8 t
0.4)
Silica gel nBuOH:KOAc:H20 Folin-Ciocal- .60 50
W
\o
N ( 4 11:2) teau and 20%
benzene:MeOH:. Na26O3 35
28%NH3 (79:21:1)
Silica gel G EtOAcrtriethyl- Dragendorff .62 32
amine (911) and iodine
HOActEtOAcracet- vapor 9 33
one (3:5:5)
Kieselgel 60 EtOAcretherrEtz- fluorescence .4 47
F254 NH (10110:1) (280/380nm)
iodine vapor
Silica gel G benzenetMeOH:28% Folin-Ciocalteau 30 49
NH4OH (79:20:1) 20% Na2C03
nBuOH :HOAc I H20 .62
((hlr2)
Silica gel Me0H:HzO ( 9 5 : 5) 2-p-chlorosulfo- 27 60
-
ph e nyl -3- ph enyl
indone derivative
Na in MeOH:DMSO
(8g:100:8)
a
w
Silica gel G- EtOAc:MeOH:NH4- ninhydrin 34
'& 254 OH (85t10:5) iodoplatinate .68
EtOAc:MeOHi H20 fluo Ee scamine
(80:15:5)
- - .
hexanerEt0Ac:EtOH: 57
NHbOH (45:50:5:2)
Silica gel EtOAc :MeOH:formic iodine vapor - 4
acid ( 9 0 : 5 15) iodoplatinate
(Continued)
T A B L E 1 0 (Continued)
THIN LAYER CHRONATOGRAPHY OF P E N T A Z O C I N E
Stationary Kobile Reference
Phase Phase Visualization Rf
Silica gel G MeOH:NH3 (100: radiochromatog. 67 65

1.5) Folin-Ciocalteau
benzeneiMe0H: diazotized p-nitro- .53
28%NH3 ( 7 9 : 20I 1 )aniline, naphtha-
reso rc ino1
Silica gel benzene:MeOH:iso- liquid scintil- - 66
\o
W
P
F-254 propylamine ( 9 5 : lation
513)
Preparative dioxane :MeOH: radiochromatog. - 67
Silica gel G formic acid
(100:5: 5 )
benzene iMeOH :
isopropylamine
(95I 5 :3 1
Silica gel benzene SlKeOH I liquid scintil- 68
F-254 isopropylamine lation
( 9 5 : 5: 3)
Silica gel benzene:MeOH:NHb- radiochromatog. 52 26
OH (80:20:1)
EtOAc :st3N (90: - -
10)
Silica gel glass EtOAcrcyclohex- ninhydrin - 69
microfiber sheets aneiNH4OH:MeOH: 0 . 5 % H2SO4
H20 (70:1512: io do plat inate
8:0.5) ammoniacal
AgN03
w
\D
ul Silica gel G benzene:MeOH:iso- UV 65 70
and GF propylamine(95:5: iodine vapor
3) Dragendorff
d i o xane :H20 I HO Ac -45
(100: 2 I5)
dioxane:H20:NH3 87
(100:15:15)
dioxanerMe0H: formic 70
acid (100:5: 5)
EtOAc:Et3N (9O:lO) 57
~~ ~
(Continued)
TABLE 10 (Continued)
THIN LAYER CHROMATOGRAPHY OF PENTAZOCINE
Stationary Mobile Visualization Rf Referenc e
Phase Phase
Silica gel glass EtOAcrcyclohex- ninhydrin .60 71
micro fiber ane :MeOH :NH iodoplatinate
( 7 0 : 15: 10: 57 i o d i ne/KI
develop to 9 cm
then:
EtOAcrcyclohex-
ane:NH (50:40:0.1)
to 14.3 c m
Kieselgel 60/ benzenerMe0H:isopro-UV 9 73 39
Kieselgur FZ54 pylamine (95:5:3) iodoplatinate
dioxanerMe0H:formic Dragendorff 79
acid (100:5:5) Folin-Ciocal-
EtOActEt N (90110) teau 9 65
benzene I J e O H :NH3 72
( 7 9 :20: 1
TABLE 11
GAS CHROIViATOGRAPHY OF PENTAZOCINE

Col.
Column Detection Derivatization Temp.oC D e t e c t i o n L i m . %Recovery Ref.
1.5% OV-I mass frag- t r i f l u o roac e t - -

3% O V - 1 7 mentography yl
3% O V - 1 7 nitrogen - 220 0 . 5 -ug/m~ 81 72
1.5% OV-101 F.I.D. t r i m e t h y l s i l y l 100-320 - - 40
mass s p e c t - permethyl
ral
w
5 3% O V - 1 7 mass s p e c t - trifluoroacet- 180-260 1 0 ng/mL 89 36
ral Yl
ov-1 6 3 N i E.C. pentafluoropro - 220 - - 73
pionyl
3% ov-1 F.I.D. - 210 25 ng 96-100 74
2,S!%,SE-30 F.I.D. - 200 - 93-98 75
2,5%SE-30 F.I.D. - 220 5 M g/mL 97 76
5% QF-1 nitrogen - 206 2.4 ng 100.3 57
(Continued)
TABLE l l ( C o n t i n u e d )
GAS CHRONATOGRAPHY OF PENTAZOCINE

Col.
Column Detection Derivatization Temp.OC D e t e c t i o n L i m . $Recovery Ref.
5% O V - 1 7 63Ni E.C
F.I.D.
. pentafluoro-
benzyl
235
220
3 Pg - 38
3PDexs i 1 6 3 N i E.C. p e n t a f 11-10ro- 265 5 ng - 77

300 benzyl
10% OV-1 3 H E.C. h e p t a f l u o r o b u t y r y l 215 5 ng

w
\D
P F.I.D. trimethylsilyl 245 0.5~g/mL 66.4-95 78
2.5%SE-30 F.I.D. ace t y l 200 - - 32
4%Apiezon F. I .D. -
L
5% QF-1 nitrogen - 206 2.4 ng 100.3 65

3% O V - 1 7 F.I.D. trimethylsilyl 90-220 10 ng 79.9 80
3% ov-1 F.I.D. trimethylsilyl 225 - - 81
3% SE-30 mass frag- - 21 0 2 ng/mL 92-94 33
mentography
cu c7 =t u\ 0 u\
a3 a3 a3 a3 b cl
co
m
I I I I I I I
n 0
m aJ
u\
N
hl)
.c
0
u\
I
s
4
I I
0
4 4 (\I
0
u\
N
0 0 u\ 0 0 I 0 u\
u'\ 4 cl 0 0 0 4 cl
N Lu cu Lu Lu c? cu (v
cu
I I
2
a,
rl
h
P rl
0 .A
k rn
rl
h
c
I 1 I P I
a,
&
.d
k
P
V V
w
r=l
w n n d n
H H H H H
.ri
B4 z L k k 4 F
c?
v3 v3
399
TABLE l l ( C o n t i n u e d )
GAS CHROMATOGRAPHY OF PENTAZOCINE
Col.
Column Detection Derivatization Temp.oC D e t e c t i o n L i m . $Recovery Ref.
3% SE-30 F.I.D. 130-290
3% OV-7 150-290
3% OV-17
3% ov-1 F.I.D. trimethylsilyl 230 39
mass s p e c t -
ral
5% SE-30 F.I.D. 260
ov-101 nitrogen pentafluoropro- 220

pionyl
3% Dexsil 63Ni E.C. pentafluorobenz- 265 89
300 Yl
3% SE-30 F.I.D. - 130-290 90
PENTAZOCINE 40 I
Glassy carbon working e l e c t r o d e s were used i n

electrochemical d e t e c t i o n o f pentazocine while no
response w a s measured i n one s t u d y a t an o p e r a t i n g
p o t e n t i a l o f 0.8 V (94). Table 12 summarizes t h e
HPLC column and mobile phase c o n d i t i o n s u t i l i z e d i n
reported investigations.
7. B i o l o g i c a l D i s p o s i t i o n and Pharmacokinetics
7.1 Disposition
The a b s o r p t i o n . d i s t r i b u t i o n . metabolism
and e x c r e t i o n of pkntazocine have been widely s t u d -
i e d i n both man and animal s p e c i e s . Early work w a s
c a r r i e d out by t h r e e major groups: Pfttman and
coworkers, Beckett and coworkers and Berkowitz,
Way and coworkers, Reviews o f metabolism (51) and
pharmacokinetics (97) o f pentazocine have been
published. The e f f e c t o f r o u t e of a d m i n i s t r a t i o n
on t h e d i s p o s i t i o n of pentazocine h a s been explored
(27,49,75,85,86). The metabolic pathways a v a i l a b l e
t o pentazocine a r e o u t l i n e d i n Figure 10. Also
shown a r e u r i n a r y e x c r e t i o n data f o r t h e metab-
o l i t e s expressed as p e r c e n t o f administered dose.
A r h e s u s monkey study i s a l s o included (66) which
s p e c i e s shows t h e t r a n s - a l c o h o l m e t a b o l i t e excreted
as well. Urinary e x c r e t i o n c o n s i s t s of unchanged
pentazocine and m e t a b o l i t e s p l u s phenolic glucu-
r o n i d e s o f each. Fecal e x c r e t i o n c o n s i s t s of l e s s
t h a n 1%administered dose ( 8 6 ) .
D i f f e r e n c e s i n pentazocine d i s p o s i t i o n i n
smokers and nonsmokers h a s been observed with t h e
former metabolizing 40% more rapidly(98).The e f f e c t
o f c i r r h o s i s on pentazocine b i o a v a i l a b i l i t y h a s also
been s t u d i e d . A 46% decrease i n c l e a r a n c e and a
233-2’785 i n c r e a s e i n b i o a v a i l a b i l i t y o f pentazocine
for t h e s e p a t i e n t s was found (84,89). Other d i s -
p o s i t i o n s t u d i e s have been done on p o s t - o p e r a t i v e
p a t i e n t s (591, mother and i n f a n t a t time o f b i r t h
(99) and normal s u b j e c t s with c i r c a d i a n v a r i a t i o n
(36)
7.2 Pharmacokinetics
The uharmacokinetics of uentazocine have
been i n v e s t i g a t e d i n humans (11,76,86). Table 13
shows values f o r h a l f - l i f e , volume of d i s t r i b u t i o n ,
b i o a v a i l a b i l i t y and c l e a r a n c e c a l c u l a t e d by var-
i o u s a u t h o r s . The plasma h a l f - l i f e values obtained
range from 2 t o 5.7 hours. The p o s s i b i l i t y t h a t
TABLE 12
HPLC ANALYSIS OF PENTAZOCINE
Mobile Phase
Detection Column Mobile Phase pH Reference
UV-278 nm ABondapak C18 methanoltwater (66: 3.5 48
34) PIC B7
W-280 nm ABondapak c18 acetonitrile:0.7% 8.0 91
NH4Cl (80:20)
UV-254 nm /lh Bondapak C18 methanolrwater (28: 7.0 28
0
P 12) (0.0048 M K2HPO4,
N 0.0077M KH2PO4)
UV-254 nm & Bondapak c18 methano1:water (20: 6.1 94
SO), 0.05 M tetra-
butylammonium hydrox-
ide, H3PO4 pH adjust-
ment
W-280 nm Partisil 5& CHC13 :methanol:iso- - 92
Silica propylamine ( 960:40:
2)
UV-280 nm JU Bondapak c18 sodium octanesul- 2.4 92
fonate ( 0 . 0 0 5 M) :
methanol: H3PO4 ( 6 0 0 :
400:1
fluore sc enc e Partisil por- methanol: 2N NH4OH: 9.5 93
320/436 nm ous silica 1 N NH4C1 (3O:zO:
10)
electrochem. Syloid 74 methano1:ammonium 10.2 95
0.6 V, 50 nA/V silica nitrate buffer ( 9 :
1)
P
0
W
electrochem. M Bondapak C18 methano1:water (20: 6.1 94

0.8 v, 5-20 n ~/ 8 0 ) , 0 . 0 5 M tetra-
v butylammoni um hydrox -
ide, H3P04 pH adjust-
ment
elec trochem. LiChrosorb ODS methanol :0.01 M 4.6 96

1.0 v KH2PO4 (85:15)
404 TERRY D.WILSON
URINARY EXCRETION
( % DOSE 1
C" 3
\ 'I 3.0 6.9
UNC HA NG ED
)- 9.5 (5-20)
no
62) -N
CH3
dpCH3 GLUCURONIDE
UNIDENTIFIED
METABOLITES
13 20.0
PENTAZOCINE
CH20H
>-)cH~ UNCHANGED 2.3

11.6 - II
GLUCURONIDE 19.0
HO
-
CIS ALCOHO L
1
fpG
2 CH20H UNCHANGED
GLUCURONIDE
- CH3
HO
TRANS-ALCOHOL
UNCHANGED
38.9 - 39 24
GLUCURONIDE
HO
TRANS-ACID
REFERENCE 81 70 51 66*
)c Rhesus Monkey
Fig. 10. Pentazocine metabolic pathways and

urinary excretion products in man and monkey.
TABLE 13
HUMAN P E N T A Z O C I N E F'HARMACOKINETICS PARAMETERS

Half -1if e Volume o f Oral B i o a v a i l - Clearance Reference
0-4 Distribution (L) a b i l i t y ($> (L/rnin)
3 04 396 18.4 1.38 100
3.8 342 18 1.25 84
2.41 2 58 1.741 day 36

2.20 26 5 1.672 n i g h t
2.2 208 - 33
2 - - 59
406 TERRY D. WILSON
t h e i n t e s t i n e m e t a b o l i z e s p e n t a z o c i n e t o account
f o r t h e l o w b i o a v a i l a b i l i t y was shown i n a r a b b i t
jejunum s t u d y . Here s u l f a t i o n and g l u c u r o n i d a t i o n
o c c u r r e d by s a t u r a b l e p r o c e s s e s ( 1 0 1 ) . Data i n
r e f e r e n c e 76 was f i t t o a t h r e e comDartment open
model w i t h a d e t e r m i n a t i o n o f m e t a b o l i c , a b s o r p t i o n
and e l i m i n a t i o n r a t e c o n s t a n t s from u r i n a r y
excretion rates. Data c a l c u l a t e d t o f i t
t h e equation:
Cp=Ae e a t + B e- 8 t
f o r a two compartment open model f o r p e n t a z o c i n e
i s shown i n Table 1 4 . I t w a s shown t h a t c i r -
c u l a t i n g p e n t a z o c i n e is d i s t r i b u t e d w i t h
48% i n blood c e l l s , 33% bound t o plasma p r o t e i n
and 19% i n plasma w a t e r ( 6 7 ) .
S t u d i e s o f pentazocine b i o l o g i c a l d i s p o s i t i o n
i n a n i m a l s c o n s i s t of t i s s u e uptake ( 2 6 , 5 2 , 5 8 , 6 5 ,
6 8 , 7 9 ) , blood l e v e l ( j 0 , 6 1 , 7 4 ) and u r i n e l e v e l
d e t e r m i n a t i o n s ( 8 8 ) . While animal m e t a b o l i c
pathways i d e n t i f i e d ( 3 9 , 4 0 , 7 O ) a r e similar t o t h o s e
found i n man, a d d i t i o n a l p r o d u c t s have been demon-
s t r a t e d . I n t h e r a t 9-methoxypentazocine ( o r i t s
8-methoxy-9-hydroxy i s o m e r ) , 8 , 9 ( o r 9,8)-methoxy-
hydroxy m e t a b o l i t e o f t h e c i s - a l c o h o l p r o d u c t and
8 , 9 ( o r 9,8)-methoxyhydroxy m e t a b o l i t e o f t h e t r a n s -
a l c o h o l p r o d u c t were found. Pharmacokinetic s t u d i e s
have been done i n r h e s u s monkey ( 6 6 ) and i n t h e
d o g ( 3 5 , 5 4 , 6 3 ) . T y p i c a l plasma h a l f - l i v e s measured
i n dog plasma a r e 1.2-1.6 h o u r s . A day-night v a r -
i a t i o n w a s a l s o observed i n t h e c a l c u l a t e d p a r a -
meters ( 3 5 ) .
8, Determination i n Body T i s s u e s and F l u i d s
Pentazocine a n d i t s m e t a b o l i t e s have been d e t -
ermined i n human and animal t i s s u e s and f l u i d s by
a v a r i e t y o f a n a l y t i c a l methods. References f o r
t h e s e t e c h n i q u e s a r e shown i n Table l 5 A f o r
humans and Table l 5 B f o r animal s t u d i e s ,
Acknowledgement
The a u t h o r wishes t o e x p r e s s h i s t h a n k s t o
members of t h e P h y s i c a l Chemistry S e c t i o n , Anal-
y t i c a l c h e m i s t r y Department, Sterling-Wjnthrop
Research I n s t i t u t e e s p e c i a l l y A l l a n G. Hlavac;
a l s o t o E . L . P r a t t , L.J.Dombrowski, A.V.R.Crain,
R.Lorenz and J . Edelson f o r reviewing t h e manuscript.
TABLE 14
TWO COMPARTNENT OPEN NODEL PARAMETERS FOR PENTAZOCINE
553 102 8.82 0.0942 5.81 1.638 1.668 33
495 108 11.39 0.357 7 357 2.74 1.653 36*

324 98 10.58 0.349 6.65 2.98 1.32 36**
~~ ~~~
* day
** night
TABLE 15
REFERENCES F O R PENTAZOCINE DETERMINATION I N BODY T I S S U E S AND F L U I D S
A) HUMAN S T U D I F S
Method I GC GC/MS TLC HPLC f 1u o r e sc enc e RIA
Tissue/Flui d
62
Cerebro- - 33 - - -
spinal
Fluid
B) ANIMAL S T U D I E S
M et h o d I GC GC/NS TLC f 1u o r e sc e n c e RIA

Species Tissue/Fluid
baboon brain 79 -
plasma 79 -
cat bile - - 26 -
plasma - - - 61
P
0
\o
urine - - 26 -
C SF - 54
plasma - 56 54 23
monkey brain 66 - 68 -
plasma 66 - 68 -
urine - - 66 -
(Continued )
TABLE 1 5 (Continued )
REFERENCES FOR PENTAZOCINE DETERPINATION I N BODY TISSUES AND FLUIDS
B) ANIP!AL STUDIES
Method: GC GC/M TLC f 1uo r e sc e nc e RIA
Species Tissue/Fluid
mouse bile 65 -
brain -
gut 65 -
plasma - -
serum - -
Pony plasma - - - 61 -
rabbit
rat bile 40 50
brain 4; - -
50
52
- - 53
gut
plasma 4; - - 52
urine 80 39 39
wbc 103 - I
swine plasma - - - 61 -
PENTAZOCINE 41 1
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The p r e s e n t l i t e r a t u r e review i n -
cludes m a t e r i a l published through J u l y ,
1982.
PHENYTOIN
Jose Philip, Ira J. Holcomb, and
Salvatore A. Fusari'
Wurrier-Lumbert Compuny
Morris Plains, New Jersey
I. General Information 418

1.2 Formulas and Molecular Weight 41 8
1.3 Description 418
1.4 Forms in Official Compendia 418
2.1 Spectra 419
2.3 Solubility 425
2.5 Partition Coefficients 426
3. Synthesis 427
4. Stability and Degradation 429
6. Identification 43 1
7. Methods of Analysis 43 1
7.1 Differential Thermal Analysis 43 1
7.2 Thermo-Gravimetric Analysis 43 I
7.3 Elemental Analysis 43 I
7.4 Colorimetric 433
7.5 Spectrophotometric 433
7.6 Polarographic 435
7.7 Titrimetric Assay 435
7.8 Chromatographic 436
7.9 Immunoassay 439
References 440
'Present address: 22625 St. Joan, St. Clair Shores, Michigan 48080.

VOLUME 13 417 hy the American Pharmaceutical Association
ISBN 0-12-260813-5
418 JOSE PHILIP ET AL.
1. General I n f o r m a t i o n
1.1 Nomenclature
1.1 1 Chemical N a m e s
5,5-Diphenyl-2,4-imidazolidinedione
5,5-Diphenylhydantoin
1 .12 Generic Names
Phenytoin, diphenylhydantoin
1 .13 Trade Names - Dantoin, Diphentyn,

D i v u l s a n , Novod i p h e n y l ( a l l Canad. ) :
D i f h y d a n , F e n a n t o i n ( b o t h Swed.);
Di-Hydan, P y o r e d o l , S o l a n t y l ( a l l
F r . ) ; D i l a b i d , Ekko, K e s s o d a n t e n
( a l l USA); D i l a n t i n ( A u s t r a l . ,
C a n a d . , USA); D i t o i n , P h e n t o i n
( b o t h A u s t r a l . ) ; Phenhydan,
Z e n t r o p i l ( b o t h Ger.); T o i n
U n i c e l l e s (S. A f r . )
1.2 Formulas and M o l e c u l a r Weight
C15H12N202 Molecular W e i g h t = 2 5 2 . 2 6
NH
1.3 Description
W h i t e o r almost w h i t e c r y s t a l l i n e p o w d e r .
1.4 Forms i n O f f i c i a l Compendia
T h e USP c o n t a i n s P h e n y t o i n , P h e n y t o i n
Sodium and t h e f o l l o w i n g f o r m u l a t i o n s :
P h e n y t o i n Oral S u s p e n s i o n
Phenytoin Tablets
E x t e n d e d P h e n y t o i n Sodium C a p s u l e s
P r o m p t P h e n y t o i n Sodium C a p s u l e s .
USP r e f e r e n c e s t a n d a r d s f o r P h e n y t o i n
and P h e n y t o i n Sodium are a v a i l a b l e ,
PHENYTOIN 419
2. P h v s i c a l P r o D e r t ies
2.1 Spectra
2.11 Infrared: T h e i n f r a r e d spectrum of
phenytoin is presented i n Figure 1.
The band a s s i g n m e n t s 1 are summarized
i n T a b l e 1.
T a b l e 1: I n f r a r e d Band A s s i g n m e n t s
f o r Phenytoin.
Wavenumber Assignment
(cm-1)
3275, 3205 N-H

st r e t c h i n g
3064 a r o m a t i c C-H
stretch
1774, 1740, 1719 carbonyl

stretching
vibrations
o f hyd a n t o i n
ring
phenyl r i n g
breathing
vibrations
1403 C-N s t r e t c h i n g
v ibrat ion
7 4 7 , 690 C-H o u t of
plane
vibrations
o f mo nosub-
s t i t u t e d phenyl
I I 1 I 1
S8 0
PHENYTOIN 42 1
2.1 2 Nuclear Maqnet ic Resonance
The Proton NMR spectrum of phenytoin is shown

in Figure 21 and t h e spectral assignments are
i n Table 2.
Table 2: NMR spectral assignments for
Phenytoin
Resonance Mult ipl i c i t y I n t q r a t ion Assiqnment

-
(PM)
7.3 singlet 10 phenyl
protons
9.2 singlet 1
10.9 very broad 1 amide
singlet protons
2.13 Mass Spectrum
"he mass spectrwn is shown i n f i g u r e 31.

These are t h e p r i n c i p a l ions observed.
Table 3: -
m/e Fragmentat ion
252 molecular ion

223 M-CHO
209 M-HNCO
180 M-HNCDCHO
175 6H5
165 C13H9
147 HgHg-Co
104 C6H5a
77 C6H5
2.14 Ultraviolet Spectrun
The w spectrun o f phenytoin i n methanol

is given i n Figure 4'. 'Ihe a b s o r p t i v i t y ,
a, a t 258 nm is 2.93.
L
I
\
-- --==D
422
N
R
In
U
423
424 JOSE PHILIP ET AL
220 260 300 340

WAVELENGTH (nm)
Fig. 4. Ultraviolet absorbance spectrum

of phenytoin in methanol.
PHENYTOIN 425

2.2 1 C r y s t a l Morphology
M i c r o c r y s t a l l o g r a p h i c c h a r a c t e r i s t i c s of
P h e n y t o i n were d e t e r m i n e d and r e p o r t e d
b y G . L. Keenan*. R o d l i k e p l a t e s and
f i n e n e e d l e s of P h e n y t o i n were examined
w i t h a p o l a r i z i n g microscope. I n par-
a l l e l p o l a r i z e d l i g h t t h e e x t i n c t i o n is
p a r a l l e l and t h e s i g n o r e l o n g a t i o n
n e g a t i v e . In convergent p o l a r i z e d l i g h t
no i n t e r f e r e n c e figures are observed.
R e f r a c t i v e i n d i c e s : a = 1.600 2 002;
y = 1.635 -+ 0.002.
2.22 M e 1 t i n g Range
P h e n y t o i n melts a t 2 9 5 - 2 9 8 ' .
2.3 Solubility - Phenytoin is p r a c t i c a l l y i n s o l -
u b l e i n water, 1 g. d i s s o l v e s i n about 6 0 ml
a l c o h o l , a b o u t 30 m l a c e t o n e . The compound
is s o l u b l e i n a l k a l i h y d r o x i d e s .
Hong e t al3 s t u d i e d t h e s o l u b i l i t y o f
p h e n y t o i n i n b u f f e r s of d i f f e r e n t pH v a l u e s .
pH S o l u b i l i t y (mq/ml)
1.6 0.02
4.4 0.02
5 .O 0.01
5.9 0.02
6.9 0.02
8.0 0.01
9.0 0.10
10.0 0.96
1 1 .o 9.6
12.0 96.0
2.4 D i s s o c i a t i o n Constant
The i o n i z a t i o n c o n s t a n t , pKa, of p h e n y t o i n
was d e t e r m i n e d t o b e 8.31 by u l t r a v i o l e t
s p e c t r o p h o t o m e t r y and 8.33 by p o t e n t i o m e t r i c
t i t r a t i o n 4 . A v a l u e of 8.06 was r e p o r t e d
by S c h w a r t z e t a 1 . 5
2.5 Partition Coefficients
Riedel et a16a determined p a r t i t i o n
c o e f f i c i e n t s ( K ) f o r p h e n y t o i n b e t w e e n a few
s e l e c t e d o r g a n i c s o l v e n t s and aqueous
s o l u t i o n s a t t h r e e d i f f e r e n t p H v a l u e s . The
r e s u l t s a r e t a b u l a t e d i n T a b l e 4.
T a b l e 4. P a r t i t i o n c o e f f i c i e n t s ( K ) o f
p h e n y t o i n ( D P H ) , b e t w e e n aqueous l a y e r s
p H 4-10 and o r g a n i c s o l v e n t s ;
DCE = d i c h l o r o e t h a n e , K = C o r g a n i c
solventfaqueous layer.
K (DPH) i n :
pH CH7C17 CHCi-( DCE
4 26.0 10.1 13.5

5 26.0 9.9 13.5
6 15.7 3.2 10.1
7 4.6 4.3 9.0
a 3.0 2.0 9.0
9 1 .o 1 .o 3.2
10 0.37 0.56 0.70
K v a l u e s o b t a i n e d by P h i l ip6b between
c h l o r o f o r m and a q u e o u s b u f f e r s o f p H 4, 5
and 6 a r e g i v e n below:
Solvent K
pH = 4.0 p H = 5.0 pH = 6.0
Chloroform 28.9 24.1 18.9
PHENYTOIN 427
I n Table 5, t h e p a r t i t i o n r a t i o s o f p h e n y t o i n w i t h
s i x s o l v e n t s and a q u e o u s b u f f e r s a t t h r e e d i f f e r e n t
pH v a l u e s d e t e r m i n e d b y G o d h a r t e t a 1 6 c a r e g i v e n .
T a b l e 5. P a r t i t i o n r a t i o s K ( c o n c e n t r a t i o n i n
s o l v e n t / c o n c e n t r a t i o n i n aqueous p h a s e ) .
Solvent
p H 3.4 pH 7 . 4 pH 1 2 . 4
Hexane 0.008 0.004 0.0001

Toluene 4.1 1 2.59 0.001
Chloroform 28.8 25.4 0.007
Dichloroethane 28.7 26.0 0.02
Ether 40.8 30.3 0.009
Ethylacetate 197 178 -
S i m i l a r r e s u l t s were o b t a i n e d b y G o l d b e r g and
T o d o r o f f 6 d i n 0.1 M p h o s p h a t e b u f f e r of p H 7 . 0 ,
(Table 6).
T a b l e 6. P a r t i t i o n c o e f f i c i e n t s ( K ) i n p H 7.0.
Chloroform 25.5
Dichloroethane 24.1
Toluene 4.3
Hexane 0.02
3. Synthesis
P h e n y t o i n c a n b e s y n t h e s i z e d i n s e v e r a l ways:
U.S. P a t e n t 2 , 4 0 9 , 7 5 4 ( 1 9 4 6 ) t o H . R. Henze,
P a r k e - D a v i s and C o . i n v o l v e s r e a c t i n g
b e n z o p h e n o n e , p o t a s s i u m c y a n i d e and ammonium
c a r b o n a t e i n 6 0 % e t h a n o l . I t c a n - a l s o be
prepared b y r e a c t i n g b e n z o p h e n o n e , s o d i u m
c y a n i d e and ammonium b i c a r b o n a t e a c c o r d i n g t o
Scheme 1 .
SCHEME 1 - Synthesis of Phenytoin (Henze)
SCHEME 2 - Synthesis of Phenytoin (Biltz)
I1
SCHEME 3 - Hydrolysis of Phenytoin

PHENYTOIN 429
B i l t z 7 p r e p a r e d p h e n y t o i n by h e a t i n g urea
with a previously prepared a l k a l i n e a l c o h o l i c
s o l u t i o n of b e n z i l . S i k d a r a n d Ghosh8
s t u d i e d t h e m e c h a n i s m of t h e B i l t z t e c h n i q u e
a n d h a s shown t h e r e a c t i o n t o t a k e place a s
shown i n Scheme 2.
4. S t a b i l i t y a n d D e g r a d a t i o n
P h e n y t o i n is v e r y s t a b l e e v e n u n d e r c o n d i t i o n s
of e x t r e m e stress. A f t e r r e f l u x i n g p h e n y t o i n
i n 2.5 N h y d r o c h l o r i c a c i d f o r s e v e n h o u r s ,
e s s e n t i a l l y complete r e c o v e r y o f s t a r t i n g
m a t e r i a l was o b t a i n e d b y F u ~ a r i . ~D u s c h i n s k i l
reported t h a t p h e n y t o i n , h e a t e d 24 h o u r s a t
170-180" i n 2 0 % s o d i u m h y d r o x i d e (5N) g a v e a n
8 2 % y i e l d of d i p h e n y l g l y c i n e . Hong3 d e t e r m i n e d
a 75% c o n v e r s i o n t o d i p h e n y l y c i n e for a s o l u t i o n
o f p h e n y t o i n s o d i u m h e a t e d a t r e f l u x i n 2N s o d i u m
hydroxide for f i v e days. He f o u n d d e t e c t a b l e
amounts o f benzhydrol i n phenytoin s o l u t i o n s
h e a t e d i n 0.2 N s o d i u m h y d r o x i d e i n a m p u l e s i n
a 105" o v e n a f t e r 7 5 d a y s . S t a b i l i t y s t u d i e s
on Dilantinm Infusion concentrate, a s o l u t i o n
of p h e n y t o i n s o d i u m i n t e t r a g l y c o l t r o m e t h a n a -
m i n e - w a t e r , h a v e shown t h e p r e s e n c e o f d i p h e n y l -
hydantoic a c i d as a decomposition product
along with d iphenylglycine. Hydrolysis s t u d i e s
of p h e n y t o i n i n 0.1M p h o s p h a t e b u f f e r of pH 1 1
a n d 1 2 a t 7 0 , 8 0 a n d 9 0 " showed d e c o m p o s i t i o n
t o f o l l o w p s e u d o - f i r s t o r d e r k i n e t i c s . Extrapo-
l a t i o n of t h e A r r h e n i u s p l o t s a t 25°C i n d i c a t e d
t h a t t h e times f o r d e c o m p o s i t i o n o f 1 0 % of t h e
d r u g a t room temperature, t 0 . g would b e a b o u t
61 y e a r s a t pH 11 a n d 35 y e a r s a t p H 12.11.
A l k a l i n e d e g r a d a t i o n of p h e n y t o i n was i n f e r r e d
t o proceed v i a h y d r o l y s i s t o d iphenylhydantoic
a c i d , a r e v e r s i b l e r e a c t i o n , and i r r e v e r s i b l e
h y d r o l y s i s of t h e l a t t e r t o d i p h e n y l g l y c i n e
(Scheme 3 ) .
A l k a l i n e p e r m a n g a n a t e o x i d a t i o n of p h e n y t o i n
w i l l r e s u l t i n q u a n t i t a t i v e y i e l d o f benzophenone.
5. Metabolism and Pharmamkinetics
Phenytoin is p a r t i a l l y metabolized and p a r t i a l l y

excreted unchanged i n t h e urine. About 4% of
t h e dose is excreted i n t h e urine unchanged;
t h e excretion of unchanged drug is increased
when t h e urine is alkaline.12
The principal metabolic products of phenytoin

are phydroxy-phenyl d e r i v a t i v e (HPPH) and a
conjugate of HPPH with glucuronic acid ( 1 3-21).
The most l i k e l y pathways involved i n t h e
metabolic d i s p o s i t i o n of DPH are sumnarized
belaw:
--- >Diphenylhydantoic acid
(minor canponent)
DPH-->meta or para-HPPH----> (glucuronic acid

I conjyates)
I----- > I (Epox ide? )-->3,4 ,-Dihydrod iol-->catecho1
I
(Con] ugates)
1
3+Methyl-
catechol
Effective serum concentrations are i n t h e range

10 to 20 ug/ml; a t concentrations i n t h e range
20 t o 40 ug/ml, s i d e e f f e c t s may occur.
Plasma h a l f l i f e v a r i e s considerably and is i n t h e

approximate range of 7 to 40 hours; a t doses above
100 mg, t h e h a l f l i f e .is dose dependent. Fhenytoin
is widely and r a p i d l y d i s t r i b u t e d throughout t h e body;
it is secreted i n t h e milk, accunulates i n red blood
cells and amounts secreted i n s a l i v a show a l i n e a r
r e l a t i o n s h i p t o nowprote i n bound concentrations
i n serum.
PHENYTOIN 43 1
6. Identification
1. T h e i n f r a r e d a b s o r p t i o n spectrum o f a po-
t a s s ium bromide d ispers i o n of p h e n y t o i n i s
i d e n t i c a l t o t h a t of a r e f e r e n c e s t a n d a r d ,
s i m i l a r l y prepared ( F i g u r e 1) .
2. D i s s o l v e a b o u t 100 mg i n a m i x t u r e of
0 . 5 m l of 1N s o d i u m h y d r o x i d e a n d 2 m l of
a 1 0 % s o l u t i o n of p y r i d i n e , a d d a f u r t h e r
8 m l s o l u t i o n of p y r i d i n e , s h a k e , a d d 1 m l
of copper s u l f a t e w i t h p y r i d i n e s o l u t i o n
and allow t o s t a n d f o r 1 0 m i n u t e s ; a b l u e
p r e c i p i t a t e is producedl2. Pennington,
e t a1 h a v e d e s c r i b e d i d e n t i f i c a t i o n d a t a ,
u s i n g m i c r o c r y s t a l 1i n e , c h r o m a t o g r a p h i c ,
s p e c t r o p h o t o m e t r i c methods and t e c h n i q u e s
f o r color t e s t s . 2 2 .
7. M e t h o d s of A n a l y s i s
7.1 D i f f e r e n t i a l Thermal A n a l y s i s
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 was c a r r i e d
o u t on a Perkin-Elmer d i f f e r e n t i a l scanning
colorimeter, m o d e l DSC-2C, a t a h e a t i n g
r a t e o f 1 0 " / m i n , i n a n a t m o s p h e r e of n i t r o -
gen. I t g a v e a s i n g l e s h a r p m e l t i n g endo-
t h e r m i c p e a k a t 298.2"C, f o l l o w e d b y decom-
p o s i t i o n ( F i g u r e 5 ) .23
7.2 Th e r m o - G r av i m e t r i c A n a l y s is
TGA a n a l y s i s 2 3 was c a r r i e d o u t o n a
P e r k i n - E l mer T h e r m o g r a v i m e t r i c a n a l y z e r ,
model TGS-2 a t a h e a t i n g r a t e o f 1 0 " m i n .
I t showed n o w e i g h t l o s s u p t o 2OOoC,
i n d i c a t i n g n o n - v o l a t il i t y a n d a b s e n c e of
s o l v e n t of c r y s t a l l i z a t i o n .
7.3 Elemental Analvsis
Element % Calculated
C 71.42
H 4.80
N 11.10
NORMALIZED PHENYTOIN USP
YTr 4.75 m g FIGURE 5
SCAN RATL 10.00 drg/min
PEAK FROMc 288.01

TOt 381
ONSETi 295.98
CAL/CRAMi 31.8
FXLEI PMNZ M TEMPERATURE CC) DSC

PERKIMLMER [Thermal Analysis
DATES 83/02/06 TIMES 03138
PHENYTOIN 433
7.4 Colorimetric
A colorimetric p r o c e d u r e f o r p h e n y t o i n d e v e l -
oped by D i l l e t a124, p r o vi d e d t h e f i r s t
r e l i a b l e means f o r d e t e r m i n i n g b l o o d a n d
t i s s u e l e v e l s of t h i s d r u g . The p r o c e d u r e
involves quantitative n i t r a t i o n , reduction
o f t h e n i t r o group t o a primary amine,
d i a z o t i z a t i o n and c o u p 1 i n g w i t h t h e B r a t t o n -
Marshall reagent.
7.5 Spectrophotometric
7.51 Ultraviolet
U l t r a v i o l e t absorption i n methyl alcohol:
maximum a t 2 5 8 nm; a = 2.93.
T h e l a c k of a w e l l d e f i n e d UV a b s o r p t i o n
s p e c t r u m f o r p h e n y t o i n makes i t s d e t e r -
m i n a t i o n s b y d i r e c t UV s p e c t r o p h o t o m e t r y
d i f f i c u l t : nevertheless, t h e literature
d e s c r i b e s s e v e r a l m e t h o d s which d e p e n d
upon t h e s p e c t r u m o f t h e unchanged
d r u g 2 5 . Wallace e t a126 and
F e l l e n b e r g e t a127 reviewed t h e u s e of
UV s p e c t r o p h o t o m e t r i c m e t h o d s f o r t h e
a n a l y s i s o f phenytoin. A spectrophoto-
metric method f o r t h e q u a n t i t a t i v e
d e t e r m i n a t i o n of phenytoin h a s been
d e s c r i b e d b y Bgorge e t a12* b a s e d on
t h e d i f f e r e n c e i n absorbance of a n
a l c o h o l i c s o l u t i o n a t 233 nm b e f o r e a n d
a f t e r conversion of phenytoin t o t h e
s o d i u m s a l t . Methods t h a t r e q u i r e
c o n v e r s i o n o f t h e d r u t o benzophenone
h a v e b e e n r e p o r t e d 2 9 I 3 0 I 31 I 3 2.
A m e r e t a133 have d e s c r i b e d a s i n g l e
u l t r a v i o l e t s p e c t r o p h o t o m e t r i c method
f o r t h e d e t e r m i n a t i o n of p h e n y t o i n i n
pharmaceutical preparations. The mean
w a s 100.9% ( s i x d e t e r m i n a t i o n s ) and t h e
r e l a t i v e s t a n d a r d d e v i a t i o n was 1.2%.
T h e method was a p p l i e d s u c c e s s f u l l y t o
t h e d e t e r m i n a t i o n of p h e n y t o i n i n
c a p s u l e s and s u s p e n s i o n s .
434 JOSE PHILIP ET A t .
Cunningham and c o w o r k e r s 3 4 h a v e
d e s c r i b e d a column c h r o m a t o g r a p h i c method
f o r t h e d e t e r m i n a t i o n of p h e n y t o i n i n
c a p s u l e s . The method is b a s e d o n t h e
a c i d i c n a t u r e of p h e n y t o i n and p e r m i t s
t h e s e p a r a t i o n and d i r e c t UV q u a n t i t a t i o n
o f t h e d r u g . The sample i s d i s s o l v e d i n
d i m e t h y l s u l f o x i d e and h y d r o c h l o r i c a c i d ,
and t h e n d i l u t e d t o volume w i t h s o d i u m
c a r b o n a t e . An a l i q u o t o f t h i s is mixed
w i t h C e l i t e and a d d e d t o a c h r o m a t o g r a p h i c
column c o n s i s t i n g o f 0.5 M s o d i u m c a r b o n a t e
a b s o r b e d o n C e l i t e . A p r e w a s h of 30 p e r c e n t
c h l o r o f o r m - i s o o c t a n e removes i n t e r f e r i n g
s u b s t a n c e s and 75 m l c h l o r o f o r m c o m p l e t e l y
removes t h e d r u g from t h e column. The e l u a t e
i s e v a p o r a t e d and t h e r e s i d u e is d i s s o l v e d i n
e t h a n o l i c HC1 a n d d e t e r m i n e d s p e c t r o p h o t o m e t -
r ically.
F u s a r i and c o w o r k e r s 3 5 h a v e d e v e l o p e d a
similar method f o r t h e a s s a y o f p h e n y t o i n
a n d p h e n o b a r b i t a l i n d o s a g e f o r m s . They
h a v e u s e d S i l a n i z e d C e l i t e 545 a s t h e s u p p o r t
m a t e r i a l , 25% amyl a l c o h o l i n c h l o r o f o r m as
t h e s t a t i o n a r y p h a s e a n d 0.05M b o r a t e b u f f e r
o f pH 9.5 a s t h e m o b i l e p h a s e . P h e n o b a r b i t a l
was e l u t e d w i t h t h e m o b i l e p h a s e and p h e n y t o i n
e l u t e d with absolute ethanol. U l t r a v i o l e t
a b s o r p t i o n w a s u s e d t o q u a n t i t a t e t h e sub-
stances .
7.52 Fluorometric
A simple f l u o r o m e t r i c a s s a y f o r p h e n y t o i n i n
plasma was r e p o r t e d b y D i l l e t a136.
PHENYTOIN 435
The assay w a s done d i r e c t l y on 0.2 m l o f

plasma by t r e a t i n g with a l k a l i n e potassiun
permanganate to form benmphenone, e x t r a c t i n g
with heptane and shaking t h e heptane layer
with s u l f u r i c acid. me fluorescence of t h e
r e a c t i o n product was measured with e x c i t a t i o n
a t 360 nm and emission a t 440 nm. Detection
of 1 ug phenytoin per m l o f plasma was
reported.
Abrahamsson e t a137 described a f l u o r i -

metric microdetermination o f phenytoin i n
plasma. The phenytoin is e x t r a c t e d from
a c i d i f i e d plasma and oxidized to benzomenone
which is then condensed with N ' ~ e t h y 1 n i m t i t ~
amide. The r e s u l t i n g product is s t r o n g l y
f l u o r e s c e n t with e x c i t a t i o n a t 440 nm and
emission a t 500 nm.
7.53 phosphor imetric
The method38 is based on Wallace method f o r

phenytoin, conversion to benzophenone i n an
a l k a l i n e permanganate medium, e x c i t i n g a t
260 nm and emitting a t 446 nm. ?he limit of
d e t e c t i o n is 0.05 ug/ml.
7.6 Polarograph ic
Brooken e t a139 described a s e n s i t i v e d i f -

f e r e n t i a l pulse polarographic assay for
phenytoin i n blood. It involves e x t r a c t i o n
of phenytoin i n t o chloroform followed by
n i t r a t i o n . The a n a l y s i s of t h e n i t r o
d e r i v a t i v e is then acccmpl ished .
7.7 T i t r i m e t r i c Assay
weigh a c c u r a t e l y about 500 mg o f phenytoin

and transfer t o a 125-1111c o n i c a l f l a s k .
Dissolve i n 50 ml of dimethylformamide, add
3 drops of a s a t u r a t e d s o l u t i o n o f a z o v i o l e t
i n benzene, and titrate with 0.1N sodim
methoxide to a blue end-pint. Ferform a
blank determinat ion , and make any necessary
correction. Each m l of 0.1 N sodium methoxide
is equivalent to 25.23 mg of C15H12~202.40
Numerous assay procedures for phenytoin i n pharma-

c e u t i c a l preparations have been devised, based on t h e
s e p a r a t i o n of phenytoin fran phenobarbital by ion
exchange chranatography or s o l v e n t e x t r a c t i o n
procedure, followed by nowaqueous t i t r a t i o n . 4 1
7.8 Chrmatographic
7.81 T h i w l a y e r (TLC)
The TIC system f o r i d e n t i f i c a t i o n of

phenytoin i n capsules and t a b l e t s 4 2 is
ccnnpsed o f s i l i c a g e l G plate (Analtech)
and c h l o r o f o r m isopropyl alcohol-ammonia
(45-45-10). Rf = 0.6. Mas0nd43 h a s
given t h e Rf values for Phenytoin i n t h e
following systems on silica g e l GF plate:
Chlorofomether-methanol-NH40H( 75-25-5-1 ) 0.48

Ethylacetatel-propanol-NH40H (40-30-3) 0.83
MethanOl-NH4OH ( 100-1.5) 0.86
Ethanol-Acetic acid-H20 (60-20-1 0 ) 0.95
Benzene 0
Detection of phenytoin on t h e plate can be

achieved by W l i g h t , iodine exposure and
Ehrlich's s p r a y followd by Iodoplatinate
spray.
Wad e t a144 described a rapid q u a n t i t a t i v e

method for t h e determination of phenytoin i n
serum by TLC.
Perhaps t h e most exacting TLC s t u d i e s were
c a r r i e d o u t by Simon e t a145. Fhenytoin
w a s e x t r a c t e d f r m plasma, concentrated by
evaporation, and subjected t o TLC on s i l i c a
g e l plates. After t h e phenytoin spot was
scraped off t h e colorimetric procedure of
D i l l et a146 was applied d i r e c t l y t o t h e
scrapings. Phenytoin l e v e l s of 5 ug/ml or
more could be recognized by v i s u a l inspection
of t h e plates under u l t r a v i o l e t l i g h t , and
serum l e v e l s of 1 ug/ml were d e t e c t a b l e with
t h e c o l o r i m e t r i c procedure.
PHENYTOIN 431
7.82 Gas Chranatography ( G K )

Glazko47 reviewed sane e a r l y s t u d i e s on t h e
g a s chranatographic assay of phenytoin. These
included d e r i v a t i z a t ion with d iazanethane48,
t h e use o f t r i m e t h y l s i l y l derivative49r50,
on colunn methylation using tetramethyl anmnium
hydroxide51 and trimethylanil inium hydroxide52.
A large number o f GKC procedures have been reported
i n which phenytoin is chrcnratographed d i r e c t l y a t
high temperatures without d e r i v a t i v e formation.53~54955
I n general t h e direct GLC methods without

d e r i v a t i v e formation tend t o produce asymmetric
peaks which increase t h e d i f f i c u l t y of q u a n t i t a t i o n
by measurement o f peak heights. me on-colm
methylation techniques appear t o be convenient to
run and sharper peaks are obtained a t lower
temperatures.
Cramers e t a156 described high resolution
g a s chranatography f o r t h e q u a n t i t a t i v e
determination of underivatized anti-convulsant
drugs w i t h support coated open tubular c o l m s .
A s i l i c e o u s support material (-Sil) is
deactivated with benzyltriphenyl phosphcnium
c h l o r i d e and deposited on t h e inside w a l l
of a glass c a p i l l a r y colm. After additional
coating with OV-225, a nmber of anticonvul-
s a n t drugs were run.
Vandemark e t a157 described a procedure for
determining phenytoin i n 50 u l volurne of
serum by u s e of GLC and a detector with
heightened s e n s i t i v i t y and s e l e c t i v i t y for
nitrogenous compounds. mtal a n a l y s i s time
for a s i n g l e s q l e is 15 minutes.
7.83 High Performance Liquid Chranatography (HPLC)

Holccsnb e t a1 58 developed a s t a b i l i t y
indicating HPLC procedure f o r t h e simul-
taneous determination of phenytoin and pheno-
barbital i n formulations. ?hey used a Waters
Associates, 2 f t x 1/8 in. O.D. s t a i n l e s s
steel column packed with u Bondapak C18 on
Corasil 11. ?he mobile phase was a mixture
of m e t h a n o l - w a t e r - a c e t i c a c i d i n t h e r a t i o
25-75-0.1. W i t h t h e d e v e l o p m e n t o f new
c o l u m n s , t h i s p r o c e d u r e was l a t e r r e v i s e d b y
Fusari et al. 59 T h e c u r r e n t met h o d uses
a Waters Associates, 1 0 u , 3.9 mm x 30 c m u
Bondapak C18 c o l u m n ; UV d e t e c t i o n a t
254 nm, t h e mobile p h a s e i s a m i x t u r e o f
methanol-water-acetic acid i n t h e ratio
40-59.5-0.5. A t a f l o w r a t e of 1.5 m l p e r
m i n u t e , p h e n o b a r b i t a l e l u t e d a t 6.6 m i n u t e s
and p h e n y t o i n a t 13.8 m i n u ts .
A t w e l l e t a 1 6 0 d e s c r i b e d a n HPLC p r o c e d u r e
f o r t h e s i m u l t a n e o u s a n a l y s i s of p h e n y t o i n
a n d p h e n o b a r b i t a l i n plasma.
S l o n e k e t a16I d e v e l o p e d a rapid and s i m p l e

HPLC m i c r o a n a l y t i c a l m et h o d f o r t h e
d e t e r m i n a t i o n of c l i n i c a l l y e n c o u n t e r e d
plasma p h e n y t o i n l e v e l s . T h i s method is
a c c u r a t e down t o a b o u t 1 ug of p h e n y t o i n / m l
of plasma a n d requires a s l i t t l e a s 1 0 u l o f
sample.
C h a f e t z e t a l l 1 u s e d a n HPLC met h o d t o
s t u d y t h e h y d r o l y s i s of p h e n y t o i n s o d i u m i n
a q u e o u s s o l u t i o n s a t pHs 1 1 a n d 12. T h ey
used a n A 1 t e x U 1 t r a s p h e r e m r e v e r s e - p h a s e
bonded o c t a d e c y l s i l a n e column,
4.6 mm x 25 c m ; t h e mo bile p h a s e was a
m i x t u r e of 550 m l of m e t h a n o l w i t h 450 m l
of a n a q u e o u s s o l u t i o n a t p H 3.2 c o n t a i n i n g
30 m l o f 0.067 M p o t a s s i u m p h o s p h a t e a n d
1 g of h e x a n e s u l f o n i c a c i d s o d i u m s a l t ;
t h e UV detector was s e t a t 220 nm. Under
t h e s e conditions, phenytoin e l u t e d a t
10.3 m i n , d i p h e n y l g l y c i n e a t 4.0 m i n ,
d i p h e n y l d y d a n t o i c a c i d a t 5.6 m i n ,
b e n z h y d r o l a t 2 1 .6 m i n a n d b e n z o p h e n o n e
a t 37.0 m i n .
PHENYTOIN 439
7.9 Immunosassay
I m m u n o l o g i c m e t h o d s f o r q u a n t i t a t i n g t h e plasma
c o n c e n t r a t i o n s of many h o r m o n e s a n d a f e w d r u g s
have been d e s c r ibed62 4. Tigelaar
e t a165 h a v e developed a r a d i o i m m u n o s a s s a y
for phenytoin. Cook e t a 1 6 6 s t u d i e d t h e
s a l i v a a n d plasma l e v e l s o f p h e n y t o i n i n a
s e r i e s of e p i l e p t i c p a t i e n t s b y m e a n s o f r a d i o -
immunoassay t h a t r e q u i r e d o n l y 1 0 u l of s a l i v a
o r plasma. The r a d i o immunoassay i s v e r y
s e n s i t i v e , q u a n t i t a t i o n of s u b s t a n c e s p r e s e n t
i n c o n c e n t r a t i o n s a s l o w as a few p i c o g r a m s per
m i l l i l i t e r of a b i o l o g i c a l f l u i d c a n be
achieved. The d e v e l o p m e n t of s imple r a d io-
immunosassy is r e v i e w e d b y Cook e t d7.
P i p p e n g e r e t a 1 6 8 r e p o r t e d a homogenous
immunoassay s y s t e m w h i c h h a s t h e a d v a n t a g e s
o f r a d i o i m m u n o a s s a y w i t h o u t t h e u s e of r a d i o -
a c t i v e compounds. Quantitative correlation to
GLC w a s o b t a i n e d . P h e n y t o i n i n human s e r u m w a s
determined by spinimmunoassay t e c h n i q u e by
Montgomery e t a l 6 9 . A s p e c i f i c radioimmune-
a s s a y f o r p h e n y t o i n , t h a t w i l l measure
t h e r a p e u t i c l e v e l s d i r e c t l y i n 1 u l of p l a s m a
is d e s c r i b e d by C h r i s t e n s e n 7 0 .
C a s t r o e t a17’ made c o m p a r a t i v e d e t e r m i n a t i o n s
of p h e n y t o i n b y v a r i o u s m e t h o d s . Serum from
p a t i e n t s b e i n g t r e a t e d w i t h p h e n y t o i n were
analyzed f o r t h e drug by spectrophotometry,
g a s c h r o m a t o g r a p h y , r a d i o i m m u n o a s s a y , enzyme
immunoassay a n d l i q u i d c h r o m a t o g r a p h y . The
a s s a y v a l u e s o b t a i n e d were c o m p a r e d s t a t i s -
t icall y . En zyme immunoassay a n d 1i q u i d
c h r o m a t o g r a p h y appear t o b e a t t r a c t i v e
a l t e r n a t i v e s t o t h e more t r a d i t i o n a l m e t h o d s
of s p e c t r o p h o t o m e t r y a n d g a s c h r o m a t o g r a p h y .
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PYRIDOXINE HYDROCHLORIDE
Hassan Y. Aboul-Enein and
Mohammed A. Loutfy
K i n g Soud University
Riyndh, Saudi Arabia
1, Description 448
1.2 Formulae 448
I .3 Molecular Weight 449
2.2 Solubility 450
2.3 X-Ray Diffraction 450
2.4 Stability 450
2.5 Identification 45 I
2.6 Spectral Properties 45 I
3. Biosynthesis 462
4. Synthesis 463
5. Metabolism 466
6.1 Titrirnetric 468
6.2 Chromatography 469
6.3 Spectrophotometry 413
6.4 Microbiological 417
6.5 Enzymatic 41I
References 478

lSBN 0-12-260813-5
448 HASSAN Y. ABOUL-ENEIN AND MOHAMMED A. LOUTFY
1. D e s c r i p t i o n
1 . 1 Nomenclature
5-Hydroxy-6-rnethyl-3,4-pvr i d ined irnethanol hydro-

chloride ;
2-Methyl-3-hydroxy-4,5-bis (hydroxymethyl) p r y i -
dine hydrochloride;
5-Hvdroxy-6-methyl-3,4-pvridinedicarbinol hydro-
chloride;
3-Hydroxy-4,5-dirnethyl-ol-ci-picoline hydrochlo-
r i d e (1);
3-Hydroxy-4,5-d i (hydroxymethyl) -2-methylpyr i d i n e
h y d r o c h l o r i d e (2) ;
4,5-Bis (hydroxymethy1)-3-hydroxy-Z-methylpyri-
dinium c h l o r i d e (3) ;
3,4-Pyridinedimethanol, 5-hydroxy-6-methyl-,
hydrochloride ( 4 ) ;
3-Hydroxy-4,5-bis (hpdroxymethyl)-2-methylpyri-
dinium c h l o r i d e ( 5 ) .
1 . 1 . 2 Generic N a m e s
P y r i d o x i n e h y d r o c h l o r i d e ( 5 , 6) ; P y r i d o x i n i -
chloridum; P y r i d o x i n i hydrochloridum; Pyridoxi-
nium c h l o r i d e ; P y r i d o x o l h y d r o c h l o r i d e ; Vitamin
$6; kdermine h y d r o c h l o r i d e ; p i r i d o s s i n a c h l o r i d -
r a t o ( 2 ) ; Vitamin 8 6 h y d r o c h l o r i d e (1); Hexa-
bione hydrochloride.
1.1.3 Trade N a m e s
Beadox; Benadon; Hexa-Betalin ( 7 ) ; Hexavibox,

Hexabion; Pyr i v e l ; Pyr i d i p c a ; B e c i l a n , Hexermin;
Campoviton 6 (1); Comploment; Ancoloxin (with
meclozine hydrochloride) ( 3 ) .
1 . 2 Formulae
1 . 2 . 1 Empirical
C8H1 1N03, HC1.
1.2.2 S t r u c t u r a l
P y r i d o x i n e i s o n l y one of t h r e e s i m i l a r compounds
PYRIDOXINE HYDROCHLORIDE 449
t h a t may b e r e f e r r e d t o as v i t a m i n B 6 , t h e o t h e r
two a r e p y r i d o x a l and pyridoxamine ( 8 ) . Only
p y r i d o x i n e h y d r o c h l o r i d e , however, i s used i n
pharmaceutical preparations (9).
c1-
H3cQ
HO CH20H
1 . 2 . 3 Wiswesser L i n e N o t a t i o n
T6 N J B C Q D I
OEIQ and GH (10)
1 . 2 . 4 Chemical A b s t r a c t R e g i s t r y Number
58-56-0 (5 , 10)
1 . 3 M o l e c u l a r Weight
205.64.
1 . 4 E l e m e n t a l Composition
C, 46.72%; H , 5.88%;
C1, 17.24%; N , 6.81%, 0 , 23.34%.
1 . 5 Appearance, C o l o r , Odor and Taste
A w h i t e o r a l m o s t w h i t e , c r y s t a l l i n e powder, p l a t e l e t s
o r t h i c k b i r e f r i n g e n t r o d s (from a l c o h o l + acetone);
odorless, with a s l i g h t l y b i t t e r s a l i n e taste, acid.
1 . 6 Dissociation Constants
pKa 5.0, 9 . 0 (25O) (3).
2.1 M e l t i n g P o i n t
About 205O ( 3 ) ; 202-206O ( 5 ) ; 205-212' (I), w i t h decom-

position. I t sublimes. The f r e e b a s e m e l t s a t 160°

(1).
F r e e l y s o l u b l e i n w a t e r (1 gm : 5 m l ) , s l i g h t l y s o l u -
b l e i n a l c o h o l (1 gm : 100 ml) , s o l u b l e i n propylene
glycol; sparingly soluble i n acetone; insoluble i n
e t h e r and i n chloroform.
2 . 3 X-Ray D i f f r a c t i o n
X-ray d i f f r a c t i o n p a t t e r n s of v i t a m i n B 6 and o t h e r
v i t a m i n s by a p r e c i s i o n x-ray camera, have been d e t e r -
mined. Elementary cell-dimension s t u d i e s have been
r e p o r t e d (11).
2.4 S t a b i l i t y
P y r i d o x i n e , H C 1 i s more s t a b l e t h a n ppridoxamine and

p y r i d o x a l . Acidic aqueous s o l u t i o n s of v i t a m i n B6 a r e
s t a b l e d e f i n i t e l y a t room t e m p e r a t u r e and may be heated
a t 120° f o r 30 m i n u t e s w i t h o u t decomposition ( 1 3 ) .
P y r i d o x i n e i s d e s t r o y e d by W r a d i a t i o n i n n e u t r a l o r
a l k a l i n e solutions, but not i n a c i d i c solution (9).
P y r i d o x i n e i s u n s t a b l e toward v a r i o u s food, d r u g , and

cosmetic c o l o r s , because of t h e p o s s i b i l i t y of t h e
formation of complex a d d i t i o n p r o d u c t s between t h e
c o l o u r s and p y r i d o x i n e (14).
S e v e r a l s t a b i l i t y s t u d i e s of p y r i d o x i n e , when added t o
b r e a d , f l o u r ( 1 5 ) , cereals (16) and d u r i n g canning and
f r e e z i n g of sweet c o r n (17), have been p u b l i s h e d .
S h i r o i s h i and Hayakawa (18) have d e s c r i b e d t h e e f f e c t

of s u n l i g h t i r r a d i a t i o n on p y r i d o x i n e and r e l a t e d com-
pounds i n aqueous s o l u t i o n a t v a r i o u s pH's. Pyridoxine
and pyridoxamine a r e r e l a t i v e l y s t a b l e i n a c i d i c
medium, w h i l e p y r i d o x a l decomposes independently of pH.
The a u t h o r s suggested t h a t t h e a l d e h y d i c 0 p a r t i c i p a t e s
i n t h e p h o t o l y s i s of p y r i d o x a l . The p r e s e n c e of Cu*
had no e f f e c t on t h e s t a b i l i t y of t h e s e s u b s t a n c e s . P.
b o r a t e complex of p y r i d o x i n e i s s t a b l e t o s u n l i g h t
i r r a d i a t i o n , h e a t i n g , o r a u t o c l a v i n g . Acetic a c i d and
a r e d d i s h brown s u b s t a n c e were s e p a r a t e d as t h e decom-
p o s i t i o n p r o d u c t s from an i r r a d i a t e d s o l u t i o n of
p y r i d o x i n e (18).
PYRIDOXINE HYDROCHLORIDE 45 1
The e f f e c t of s h e l f - l i f e on t h e s t a b i l i t y of vitamin
B6, i n o r a l pharmaceutical p r e p a r a t i o n s , h a s been
r e p o r t e d (19).
The f o l l o w i n g t e s t s have been d e s c r i b e d f o r t h e i d e n t i -

f i c a t i o n of p y r i d o x i n e hydrochloride:
a. To 0.1 m l of a 5% w/v s o l u t i o n add 10 ml of a 5%

w / v s o l u t i o n of sodium acetate, 1 ml of w a t e r and 1
m l of a 0.5% w/v s o l u t i o n of 2,6-dichloroquinone-4-
chloroimine i n e t h a n o l (96%) and shake; a b l u e
c o l o r is produced which r a p i d l y f a d e s and t u r n s t o
brown. R e p e a t t h e o p e r a t i o n adding 1 m l of a 3X
w/v s o l u t i o n of b o r i c a c i d i n p l a c e of t h e 1 m l of
w a t e r ; no b l u e c o l o r i s produced (3-5, 1 2 ) .
b. To 2 ml of a s o l u t i o n (1 i n 200) add 0.5 m l of

phosphotungstic a c i d , a w h i t e p r e c i p i t a t e i s formed
(4)*
c . A 5% w/v s o l u t i o n a c i d i f i e d w i t h 2H n i t r i c a c i d
y i e l d s r e a c t i o n s c h a r a c t e r i s t i c of c h l o r i d e s .
d. pH of a 1% s o l u t i o n , 2.5-3.2 (12); pH. of a 5% w/v

s o l u t i o n s , 2.3-3.5 ( 7 ) .
2.5.1 Micro-Color Tests
a. Ammonium molybdate t e s t g i v e s f a i n t b l u e
c o l o r ( s e n s i t i v i t y : 0.25 yg) ( 7 ) .
b . Ammonium v a n a d a t e t e s t produces b l u e then

grey-green c o l o r ( s e n s i t i v i t y : 0.25 yg).
2.5.2 Micro-Crystal Tests
a. P l a t i n i c i o d i d e s o l u t i o n forms r e c t a n g u l a r
c r y s t a l s ( s e n s i t i v i t y : 1 i n 1000) ( 7 ) .
b. Potassium bismuth i o d i d e s o l u t i o n g i v e s
bunches of p l a t e s o r b l a d e s ( s e n s i t i v i t y : 1
i n 200) ( 7 ) .
The UV s p e c t r u m of p y r i d o x i n e , HC1 i n n e u t r a l
e t h a n o l i s shown i n F i g . 1. I t e x h i b i t s a maxi-
mum a t a b o u t 291 nm ( E l % , 1 cm = 523 nm).
I n a b a s i c s o l u t i o n , p y r i d o x i n e g i v e s two maxima
a t 245 nm ( E l % , 1 cm = 219), and a t 307 nm a s
shown i n F i g . 2 . These are i n agreement w i t h
p r e v i o u s l y p u b l i s h e d d a t a ( 7 , 20-21). I n phos-
p h a t e b u f f e r of pH = 7 , two maxima are observed
a t 324 and 254 nm ( E l X , 1 c m = 345 and 183,
r e s p e c t i v e l y ) (9).
M e t z l e r and S n e l l (22) have r e p o r t e d t h e UV

a b s o r p t i o n s p e c t r a and i o n i s a t i o n c o n s t a n t s of
v i t a m i n B 6 group. The a u t h o r s have c a l c u l a t e d
t h e e q u i l i b r i u m c o n s t a n t s between d i p o l a r i o n i c
and uncharged n e u t r a l forms of t h e 3-hydroxy-
pyridines investigated.
The I R spectrum of p y r i d o x i n e , H C 1 i n KBr-disc

i s shown i n F i g . 3. Major band a s s i g n m e n t s a r e
g i v e n i n T a b l e 1.
T a b l e 1. I R S p e c t r a l Assignments of P y r i d o x i n e , HC1.
Frequency, cm-l Assignment
3340 OH p h e n o l i c
3250 OH a l c o h o l i c
1630
1550 1 C=C and C=N
aromatic r i n g
Other f i n g e r - p r i n t bands c h a r a c t e r i s t i c of
p y r i d o x i n e , H C 1 are: 1275, 1220, 1100, and 1570
cm-1. These d a t a are i n agreement w i t h p r e -
v i o u s l y r e p o r t e d o n e s ( 7 , 20, 2 3 ) .
2.6.3 N u c l e a r Magnetic Resonance Spectrum
The PMR spectrum of p y r i d o x i n e , H C I i n d e u t e r a t e d

water w a s r e c o r d e d on a V a r i a n T-60AY 60-m~
NMR s p e c t r o m e t e r . The spectrum i s shown i n F i g .
4. The f o l l o w i n g s t r u c t u r a l a s s i g n m e n t s have
been e l i c i t e d from F i g . 4 .
PYRIDOXINE HYDROCHLORlDE 453
wave fength
Fig. 1. Ultraviolet spectrum of pyri-
doxine hydrochloride in ethanol solution.
Fig. 2. Ultraviolet spectrum of

pyridoxine in basic ethanol solution.
-
c,
a,
rl
rl
a,
P
k
m
x
Y
a,
a
.d
k
0
rl
c
V
0
k
a
h
c
a,
c
-d
x
0
a
-4
k
h
a
v-4
0
s
k
4
u
a,
a
z
n
a
a,
k
rd
k
Icl
G
H
m
bl
-4
l
h
454
P
LA
1
Fig. 4. 'H-NMR (6OMHz) spectrum of pyridoxine hydrochloride in D 0
2 -
Chemical S h i f t (6)
- P r o t o n Assignments
2.73 singlet CH3 a t C 2
5.06 singlet CH2 a t C4 and C

5'
8.13 singlet aromatic proton a t
'6'
When t h e spectrum i s d e t e r m i n e d i n DMSO-d6, t h e
hydroxymethyl groups a r e r e s o l v e d i n t o two s i n g -
l e t s a t 4.7 6 ( f o r CH20H a t C5) a t 4.8 6 ( f o r
CH20H a t C4).
These s p e c t r a are i n agreement w i t h p r e v i o u s l y

published d a t a (24).
The e l e c t r o n i c p r o p e r t i e s of v a r i o u s i o n i c forms
of v i t a m i n B6 group have been s t u d i e d and
r a t i o n a l i z e d u s i n g PMR ( 2 5 ) .
F u r t h e r m o r e , t h e PMR s p e c t r o s c o p y of v i t a m i n
B6 compounds h a s been s t u d i e d by Korytnyk and
Paul (26).
2.6.4 Mass Spectrum
The mass spectrum of p y r d o x i n e i s shown i n F i g .

5. The most prominent i o n s a r e shown in T a b l e
2.
T a b l e 2. Mass S p e c t r a l F r a g m e n t a t i o n of p y r i d o x i n e .
m le Fragment
169 M o l e c u l a r i o n M+.
151
122
H3
+ H
106
94
H3
HB Y
I!
a,
c
-4
x
0
a
-4
k
h
a
5
k
+,
u
a,
a
m
m
m
2
c
0
A L
0
m
a,
k
3
0
L A I4
r +
Ho\ 1'
H*o 0
1
H3C &: m / e 169
H3CO f i 2 0 H
m / e 151 J
l o s s of l o s s of
CO and co
HO'
possible H3c gcH 'OH
m / e 106 i
Azatropylium ion m / e 106
t
CHZOH Y
c,
H
H
m / e 123
-H'
*I H
H
N
m / e 122 H
m / e 94
( b a s e peak)
Scheme 1. Mass S p e c t r a l F r a g m e n t a t i o n of P y r i d o x i n e .
PYRIDOXINE HYDROCHLORlDE 459
The mechanism by which t h e major fragments a r e

formed, i s shown i n Scheme 1. The d e t a i l e d
mechanism i s r e p o r t e d by D e Jongh e t a l . (27)
w i t h t h e a i d of m e t a s t a b l e peaks, deutrium
l a b e l i n g , and a n a l y s i s w i t h s i m i l a r system.
2.6.5 13C-NMR
The 13C-nuclear map,netic resonance s p e c t r a of

p y r i d o x i n e h y d r o c h l o r i d e , o b t a i n e d i n D20 a t
ambient temperature u s i n g dioxane a s r e f e r e n c e
l i n e , i s shown i n F i g . 6 (off-resonance) and
F i g . 7 (lH-decoupled?.
The s p e c t r a have been recorded a t 20 MHz f o r

carbon-13 on a Varian FT80A spectrometer. The
chemical s h i f t s and s p e c t r a l assignments a r e
given i n Table 3 .
Table 3. 13C-NMR of P y r i d o x i n e , H C 1 i n D20.

H
c1-
HO CH20H
7
CH20H
Chemical S h i f t (ppm) 8 Carbon N o .
153.52 S 3
143.42 S 2
141.32 S 4
137.62 S 5
130.57 d 6
58.92 7 and 8.
57.88 t
15.23 Q 9
s = singlet d = doublet
t = triplet 4 = quartet
P
0
01
1 1 t 1 " 1 1 1 1 " 1 1 ' 1 I I ' I ' I I I , I l ~ l ~ l ' l ' l r' I 1
Fig. 6. 13C-NMR Off -Resonance Spectrum of Pyridoxine Hydrochloride in D20.

46 I
462 HASSAN Y. ABOUL-ENEIN AND MOHAMMED A . LOUTFY
These d a t a w e r e r e f e r e n c e d from dioxane which

a p p e a r s a t 67.39 ppm. The assignments were
based on p u b l i s h e d d a t a of s e v e r a l p y r i d i n e
d e r i v a t i v e s (28).
3 . Biosynthesis
Yoshiki (29) h a s s t u d i e d t h e b i o s y n t h e t i c pathways of

v i t a m i n B6, i n c l u d i n g t h e discGvery of t h e p r e c u r s o r gly-
c o l a l d e h y d e , g l y c o l a l d e h y d e dehydrogenase, and i s o l a t i o n
of microorganisms d e f i c i e n t i n r e g u l a t i o n mechanism of Bf,
biosynthesis.
The s t a t u s of g l y c o l a l d e h y d e i n t h e b i o s y n t h e s i s of v i t a -
min Bg h a s been a l s o d e s c r i b e d by Vella, e t a l . (30). Com-
p e t i t i o n e x p e r i m e n t s , employing 14C-labeled samples of
g l y c e r o l and glycoaldehyde, i n d i c a t e t h a t i n E. C o l i B
ware 2 independent pathways l e a d i n g t o p y r i d o x a l . I n
mutant WG2 t h e major pathway u t i l i z e s g l y c e r o l and r e l a t e d
t r i o s e s as t h e s o l e C s o u r c e i n t h e c o n s t r u c t i o n of t h e
Cg N s k e l e t o n of p y r i d o x o l . I n t h e minor pathway glyco-
aldehyde, and n o t g l y c e r o l , s u p p l i e s C-5 and C-5’ of p y r i -
d o x o l , w h i l e g l y c e r o l i s t h e s o u r c e of t h e o t h e r 6 carbon
atoms.
Pope and Smith (31) have r e p o r t e d t h e s y n t h e s i s of p y r i -

doxine by t u b e r c l e b a c i l l i when grown on s y n t h e t i c media.
P y r i d o x i n e h a s been s y n t h e s i z e d , t o g e t h e r w i t h o t h e r B-
complex v i t a m i n s , by H37 and Ravenel s t r a i n s of t h e t u b e r -
cle bacillus.
Tani, e t a l . (32) have d e s c r i b e d a paper d i s c a s s a y method

t o s c r e e n t h e b i o s y n t h e t i c i n t e r m e d i a t e s of v i t a m i n B6.
Dempsey ( 3 3 ) h a s p u b l i s h e d t h e b i o s y n t h e s i s and c o n t r o l of
v i t a m i n B6 i n E s c h e r i c h i a c o l i .
Yamada, e t a l . ( 3 4 ) have s t u d i e d t h e v i t a m i n I36 b i o s y n t h e -

sis of a pdx B m u t a n t , E s c h e r i c h i a c o l i R WG3, i n amino
a c i d - supplemented medium. An amino a c i d m i x t u r e and
g l y c o l a l e d h y d e , which s a t i s f y t h e v i t a m i n B 6 requirement
of E . C o l i B WC, 3, a pdx B m u t a n t , have been examined f o r
t h e i r e f f e c t on growth of t h e mutant i n l i q u i d c u l t u r e .
The amount of B6 s y n t h e s i z e d by t h e mutant i n t h e amino
a c i d - supplemented medium i s lower t h a n t h e amount syn-
t h e s i z e d i n t h e g l y c o l a l d e h y d e - supplemented medium.
4. S y n t h e s i s
S e v e r a l r o u t e s have been d e s c r i b e d f o r t h e s y n t h e s i s of
pyridoxine hydrochloride.
Route 1:
By t h e condensation of cyanoacetamide and e t h o x y a c e t y l a c e -

t o n e (35-38). The r e s u l t i n g 3-cyano-4-ethoxymethyl-6-
methyl-2-pyridone ( I ) w a s n i t r a t e d t o g i v e n i t o r p y r i d o n e
(11). The sequence of r e a c t i o n s of t h i s r o u t e may be
r e p r e s e n t e d g r a p h i c a l l y (Route 1 ) .
Some v a r i a t i o n s and improvements i n t h i s r o u t e have been

r e p o r t e d (37).
Route 2:
A l k y l - s u b s t i t u t e d o x a z o l e s have been found t o react w i t h

maleic a c i d o r i t s anhydride i n a d i e n e s y n t h e s i s t o y i e l d
s u b s t i t u t e d p y r i d i n e r e a d i l y converted t o p y r i d o x i n e ( 3 9 ) .
I n t h i s r o u t e , e t h y l d , 1 - a l a n i n a t e h y d r o c h l o r i d e i s treat-
ed w i t h f o r m i c - a c e t i c anhydride t o y i e l d e t h y l N-formyl
d , l - a l a n i n a t e (78X). T h i s compound i s r e f l u x e d i n c h l o r o -
form w i t h phosphorous p e n t o x i d e ( 4 0 ) , quenched w i t h
aqueous potassium hydroxide, and t h e o r g a n i c l a y e r d i s -
t i l l e d t o g i v e 4-methyl-5-ethoxyoxazole ( I ) ( 6 0 % ) . The
r e s u l t i n g oxazole ( I ) i s condensedreadily w i t h a number
of a p p r o p r i a t e d i e n o p h i l e s t o form 2-methyl-3-hydroxy-4,5-
d i s u b s t i t u t e d - p y r i d i n e s c o n t a i n i n g s u b s t i t u e n t s (111, a ,
b , c ) which could be converted t o p y r i d o x i n e as f o l l o w s :
1. A m i x t u r e of the oxazole ( I ) w i t h two moles of d i e t h y l

m a l e a t e ( I I a ) i s heated and t h e adduct cleaved with
e t h a n o l i c hydrogen c h l o r i d e t o form t h e d i e t h y l ester
of 2-methyl-3-hydroxy-pyridine-4,5-dicarboxylic acid
h y d r o c h l o r i d e ( I I I a ) ( 8 5 X ) . T h i s d i e s t e r i s reduced
w i t h l i t h i u m aluminum h y d r i d e t o p y r i d o x i n e (41-42)
i s o l a t e d a s i t s hydrochloride.
2. A m i x t u r e of the oxazole ( I ) w i t h one mole of fumaro-

n i t r i l e ( I I b ) i s r e f l u x e d i n methanol and t h e adduct
cleaved w i t h 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 t o g i v e
c r y s t a l l i n e 2-methyl-3-hydroxy-4,5-dicyanopyridine
( I I I b ) (75X). Hydrogenation of I I I b i n m e t h a n o l i c
hydrogen c h l o r i d e over 5% palladium on c h a r c o a l g i v e s
2-me t h y 1-3 -hydroxy-4,5 -d lam inomethyl pyr i d i n e tr ihydr o-
c h l o r i d e (111, Y=CH2NH2) ( 7 0 % ) , which on d i a z o t i s a t i o n
464 HASSAN Y.ABOUL-ENEIN A N D MOHAMMED A. LOUTFY
0 0 0
II II
CH3-C-CH2-C-CH 0C H + N.CCH2-C-NH2
It Ethano 1
2 2 5 P i p e r i d i n e as'
Catalyst
CH OC H
* 2 2 5
H3C 0: 0 I
I
H
H N O /~A C ~ O
>
CH20Et
I
02N.
H3C ' \-N HI
I1
CN
H 2 / P t and
\
PhCl
c1 Pd/Charcoal
HONO
F
CH20Et CH2Br
Ho'@cH20HE:i 28120/AgC1
____1_3
H3 H3C
HBr
Pyridoxine, HC1
R o u t e 1. S y n t h e s i s of P y r i d o x i n e H y d r o c h l o r i d e .
PYRIDOXINE HYDROCHLORIDE 46.5
CH -CHC00C2H5, HC1 + CH3 CO,

CH CH-COOC2H5
/x3
3 1
00 31
NH2 H - CO NH-CHO
+ YCH=CHY >--
\ I1
Y V
CH20H
I I11
CH20H
Pyridoxine
a. Y = - COOC2H5
b. Y = - CN
C. Y-Y= - CH -0-CH -
2 2
R o u t e 2. S y n t h e s i s of P y r i d o x i n e .
i n w a t e r a t 85' y i e l d s pyridoxine hydrochloride (797).
3. A s o l u t i o n of t h e oxazole ( I ) i n a 20-mole e x c e s s of
2,5-dihydrofuran ( I I c ) c o n t a i n i n g 1%of t r i c h l o r o a c e t i c
a c i d is h e a t e d t o g i v e 2-methyl-3-hydroxy-4,5-epoxy-
d i m e t h y l p y r i d i n e ( I I I c ) of 95% p u r i t y (58Z). The
c y c l i c e t h e r ( I I I c ) i s cleaved w i t h 48% hydrobromic
a c i d t o t h e dibromide hydrobromide (111, Y=CHZBr) and
hydrolysed t o p y r i d o x i n e (35-36).
S e v e r a l r e p o r t s and p a t e n t s a r e l i s t e d i n t h e l i t e r a t u r e
f o r t h e s y n t h e s i s of t h e drug (43-54).
5. Metabolism
Pyridoxine, p y r i d o x a l , and pyridoxamine, which occur i n

f o o d s t u f f s , a r e c o l l e c t i v e l y known a s v i t a m i n B6. I n t h e
body, a l l t h r e e are converted t o p y r i d o x a l phosphate which
i s t h e coenzyme f o r amino-acid d e c a r b o x y l a s e and f o r
transaminase. The s t r u c t u r e s of t h e t h r e e a c t i v e forms of
v i t a m i n B6 and t h e p y r i d o x a l phosphate, a r e shown below
(55).
R
Pyridoxine CH20H
Pyridoxal CHO
Pyr idoxamine CH2NH2
0
II
CHO
P y r i d o x a l phosphate
COOH
4-Pyridoxic a c i d
Although, owing t o t h e wide d i s t r i b u t i o n of v i t a m i n B6 i n

n a t u r e , c l i n i c a l d e f i c i e n c y symptoms are seldom o b s e r v e d ,
t h e r e i s l i t t l e doubt t h a t p y r i d o x i n e i s e s s e n t i a l i n
human n u t r i t i o n . P y r i d o x i n e i s absorbed from t h e g a s t r o -
i n t e s t i n a l t r a c t and i s c o n v e r t e d t o t h e a c t i v e form p y r i -
d o x a l phosphate. Absorption i s d e c r e a s e d i n g a s t r o i n t e s t i -
n a l d i s e a s e s and a l s o i n s u b j e c t s t a k i n g i s o n i a z i d ( 3 ) . It
i s e x c r e t e d i n t h e u r i n e a s 4-pyridoxic a c i d ( 2 ) . The
metabolism of v i t a m i n B6 i n human b e i n g s h a s been i n v e s t i -
g a t e d (56).
Moller (57) h a s r e c o g n i z e d p y r i d o x i n e , p y r i d o x a l , pyridoxa-

mine, and 4-pyridoxic a c i d a s t h e e x c r e t i o n p r o d u c t s of
v i t a m i n Bg. Complete b a l a n c e s t u d i e s have been made i n
p i g s and i n b a b i e s on a l l t h e known v i t a m i n B6 m e t a b o l i c
compounds. I n b a b i e s , t h e t o t a l o u t p u t exceeded t h e
i n t a k e . The assumption t h a t a l i m i t e d s v n t h e s i s of v i t a m i n
R6 o c c u r s seems j u s t i f i a b l e .
Lumeng e t a l . (58) have r e p o r t e d t h e plasma c o n t e n t of B6

v i t a m e r s and i t s r e l a t i o n s h i p t o h e p a t i c v i t a m i n B6 meta-
bolism. O r a l l y i n g e s t e d p y r i d o x i n e i s r a p i d l y m e t a b o l i s e d
i n l i v e r and i t s p r o d u c t s are r e l e a s e d i n t o t h e c i r c u l a -
t i o n i n t h e form of p y r i d o x a l phosphate, p y r i d o x a l , and
pyridoxic acid.
Johansson e t a l . (59) have s t u d i e d t h e metabolism of

l a b e l e d p y r i d o x i n e i n man. 15-20% of t h e l a b e l e d
adminstered pyridoxine i s excreted i n t h e u r i n e during t h e
f i r s t day. The r a t e of e l i m i n a t i o n of v i t a m i n B6 from t h e
body r e s e r v o i r i s 2.3% /day.
Wozenski e t a l . (60) have r e p o r t e d t h e metabolism of s m a l l

d o s e s of v i t a m i n B(j i n men. It h a s been found t h a t res-
ponses t o pyridoxamine are g e n e r a l l y slower t h a n f o r p y r i -
d o x i n e o r p y r i d o x a l , s u g g e s t i n q t h a t pyridoxamine i s
absorbed more s l o w l y o r m e t a b o l i z e d d i f f e r e n t l y , o r b o t h ,
t h a n p y r i d o x a l o r p y r i d o x i n e . A d o s e of a t least 1 mg of
B6 i s n e c e s s a r y t o o b t a i n measurable changes i n v i t a m i n B6
metabolism.
Cox, e t a l . (61) have d e s c r i b e d t h e metabolism of tritium-

l a b e l e d p y r i d o x i n e i n rats. U r i n e , blood, and f e c a l
samples from r a t s i n j e c t e d i n t r a v e n o u s l y w i t h l a b e l e d
p y r i d o x i n e are c o l l e c t e d f o r e x c r e t i o n and chromatographic
s t u d i e s . Approximately 70% of t h e l a b e l i s e x c r e t e d i n t h e
u r i n e i n 5 hours. F e c a l e x c r e t i o n i s less t h a n 0.03 X i n
468 HASSAN Y. ABOUL-ENElN AND MOHAMMED A . LOUTFY
96 h o u r s . Chromatographic s t u d i e s of u r i n e have showed

t h a t p y r i d o x i n e i s e x c r e t e d p r i m a r i l y unchanged w i t h a
small amount of o n l y one m e t a b o l i t e , probably 4-pyridoxic
acid.
Johansson, e t a l . (62) have r e p o r t e d t h e metabolism of

t r i t i u m - l a b e l e d p y r i d o x i n e i n r a t s and humans. 4-pyri-
d o x i c a c i d accounted f o r 25% of t h e t o t a l i s o t o p e i n t h e
u r i n e of r a t s and humans.
Ham (63) h a s d e s c r i b e d t h e t r a n s p o r t and i n t e r c o n v e r s i o n

of Bg v i t a m e r s i n t h e p e r f u s e d i n t e s t i n e and kidney of t h e
rat.
It h a s been r e p o r t e d t h a t v i t a m i n B6 metabolism becomes

a l t e r e d w i t h age ( 6 4 ) .
Some a b n o r m a l i t i e s of v i t a m i n R 6 metabolism i n human b e i n g s

have been d e s c r i b e d (65-66).
The m e t a b o l i c s i g n i f i c a n c e of v i t a m i n B 6 and i t s r o l e t o
t h e growth and f u n c t i o n a l development have been published
(67-78).
S e v e r a l o t h e r i n v i t r o and i n v i v o m e t a b o l i c s t u d i e s of
v i t a m i n B6 have been p u b l i s h e d (79-86).
6.1 T i t r i m e t r i c
6.1.1 Non-Aqueous
P y r i d o x i n e , H C 1 has been assayed by non-aqueous

t i t r a t i o n s , u s i n g 0.1 N p e r c h l o r i c a c i d i n t h e
p r e s e n c e of m e r c u r i c a c e t a t e and c r y s t a l v i o l e t
i n d i c a t o r ( 4 , 6 , 10, 8 7 ) .
6.1.2 Complexometric
The drug h a s been determined by complexometric

t i t r a t i o n of t h e Cuu l i b e r a t e d by p a s s i n g t h e
s o l u t i o n through a column of c a t i o n exchange
r e s i n i n t h e cupper form. A s o l u t i o n of 0.005
o r 0.0025 M of disodium EDTA i s t h e t i t r a n t and
u s i n g murexide a s an i n d i c a t o r (88).
6 . 1 . 3 Amperometric
Lemahieu, e t a l . (89) have r e p o r t e d an ampero-

m e t r i c method f o r t h e d e t e r m i n a t i o n of hydrochlo-
r i d e s of several o r g a n i c b a s e s , i n c l u d i n g
pyridoxine. T i t r a t i o n , w i t h AgN03 s o l u t i o n i n
d i m e t h y l s u l p h o x i d e , of s u c h h y d r o c h l o r i d e s i n
d i m e t h y l s u l p h o x i d e g i v e s a n end-point c o r r e s -
ponding t o t h e f o r m a t i o n of AgC12- and a l e s s
s h a r p end-point f o r t h e p r e c i p i t a t i o n of AgC1.
U s e of t h e f i r s t end-point and two p l a t i n u m
indicator electrodes with a potential difference
of 100 mV a l l o w s t i t r a t i o n down t o a 0 . 2 mM
c o n c e n t r a t i o n of p y r i d o x i n e , H C 1 .
6.1.4 Polarographic
Manousek and Zuman (90) have d e s c r i b e d a d i r e c t

p o l a r o g r a p h i c d e t e r m i n a t i o n of p y r i d o x a l i n t h e
p r e s e n c e of p y r i d o x a l 5-phosphate. The s i m u l t a n -
e o u s d e t e r m i n a t i o n of b o t h compounds i s p o s s i b l e
o n l y when t h e c o n c e n t r a t i o n s a r e comparable. The
same a u t h o r s a l s o have r e p o r t e d t h e p o l a r o g r a p h y
of p y r i d o x i n e and several a n a l o g u e s i n b u f f e r e d
s o l u t i o n s . A 2 - e l e c t r o n wave w a s observed which
corresponds t o a diffusion-controlled reduction
o f t h e a c t i v a t e d C-OH bond i n p o s i t i o n 4 .
Nakaya (91) h a s d e s c r i b e d p o l a r o g r a p h i c s t u d i e s
o f p y r i d o x i n e , FC1 and several p y r i d i n e d e r i v a -
t i v e s a t v a r i o u s pH's.
Another p o l a r o g r a p h i c method f o r t h e e v a l u a t i o n
of v i t a m i n B6 and i t s m e t a b o l i t e s h a s been
r e p o r t e d (92).
6.2 Chromatography
6 . 2 . 1 P a p e r Chromatography
S e v e r a l s o l v e n t s s y s t e m s (93-97) have been u s e d

f o r t h e i d e n t i f i c a t i o n , s e p a r a t i o n , and q u a n t i -
t a t i o n of p y r i d o x i n e i n p h a r m a c e u t i c a l s and b i o -
l o g i c a l f l u i d s . These a r e p r e s e n t e d i n T a b l e 4 .
Table 4. P a p e r Chromatowaphv of Pyridoxine.

Solvent System Localising Ref.
F-f
agent
1. Citric a c i d : UV l i g h t ( b l u e 0.18 (7)
water: n-buta- fluorescence) ,
no 1 iodo p l a t i n a t e
(4.8 gm : 130 spray (white),
m l : 870 m l ) . bromocresol g r e e n
spray (strong
r e a c t ion) .
2. Acetate b u f f e r UV l i g h t ( b l u e 0.90 (7)
(pH=4.58) fluorescence)
3 . Phosphate UV l i g h t ( b l u e 0.86 (7)
buffer(pH=7.4) fluorescence).
4 . Two dimensional
chromatography
consists of:
a. Collidine: 0.88 (98)
l u t i n e (1:l)
saturated with
water.
b. Butanol: 0.63 (98)
a c e t i c acid:
w a t e r (40:10:50)
5. Amy1 a l c o h o l - (99)
a c e t o n e : water
(2:l:Z).
Isoamyl a l c o h o l :
p y r i d i n e : water
(2:1:2).
6. Two dimensional
chromatography
consists of:
a. Propanol: UV l i g h t
10% formic a c i d
(80:20).
b. Butanol -
saturated with
1%ammonia.
The s e p a r a t i o n of w a t e r - s o l u b l e v i t a m i n s ,
i n c l u d i n g v i t a m i n B 6 , on ion-exchange paper h a s
been d e s c r i b e d by K l o t z and H u e t t e n r a u c h , (101)
u s i n g Amberlite SB-2, Amberlite WB-2, and
Amberlite WA-2.
For t h e s e p a r a t o r y d e t e r m i n a t i o n of p y r i d o x i n e
i n multi-vitamin preparations, o t h e r vitamins
are s e p a r a t e d by paper chromatography i n which
t h e upper l a y e r of butanol-ammonium hydroxide-
water (4:1:5) are used a s t h e d e v e l o p e r , and by
t h e one-dimensional a s c e n d i n g method (102).
C l a r k e (7) h a s d e s c r i b e d a s o l v e n t system con-

s i s t i n g of s t r o n g ammonia solution-methanol (1.5:
100) and potassium permanganate s p r a y a s a
detecting agent.
Lyle and T e h r a n i (103) have s e p a r a t e d s e v e r a l

vitamins, i n c l u d i n g v i t a m i n Bf,, by TLC, u s i n g
anhydrous a c e t i c a c i d - a c e t o n e - methanol -
benzene (1:1:4:14).
A r a p i d TLC method f o r t h e r o u t i n e i d e n t i f i c a t i o n
of v i t a m i n B 6 , and o t h e r d r u g s , i n b i o l o g i c a l
f l u i d s , h a s been d e s c r i b e d (104).
6.2.3 Electrophoresis
A r a p i d e l e c t r o p h o r e t i c method f o r t h e s i m u l t a n -
eous s e p a r a t i o n and e s t i m a t i o n o f v i t a m i n B6 and
d e r i v a t i v e s from v a r i o u s t i s s u e s h a s been des-
c r i b e d (105). The method i s based on h i g h
v o l t a g e e l e c t r o p h o r e s i s of s m a l l a l i q u o t s of
t i s s u e e x t r a c t s , a p p l i e d on c e l l u l o s e - c o a t e d
p l a t e s . The method i s s e n s i t i v e u p t o 1 pg o f
v i t a m i n B6.
6.2.4 Column Chromatography
S e v e r a l methods have been r e p o r t e d f o r t h e

s e p a r a t i o n of water s o l u b l e v i t a m i n s i n c l u d i n g
vitamin B6. Some of t h e s e systems a r e d e s c r i b e d
as f o l l o w s :
T a b l e 5. Gas-Chromatography of P y r i d o x i n e , H C 1 .
Column Cond it i o n s Comment Ref.
1. 3% SE-30 100-270° tempera- Derivatized using (108)

t u r e programming. NO-bis(trimethy1-
s i l y l ) acetamide.
2. 152 DON - Column t e m p e r a t u r e D e r i v a t i z e d as ( 1 09)

Corning h i g h - 175O, w i t h t h e r m a l above.
vacuum s i l i - conductivity
cone g r e a s e d e t e c t i o n and H e
on Gas-Chrom as carrier g a s .
Q (60-80
mesh).
3 . 3% SE-52 on Column t e m p e r a t u r e D e r i v a t i z e d as (110)

silanized 165O, N2 a s above.
Gas Chrom P c a r r i e r gas.
(60-80 mesh).
4 . 3% OV-101 on Column t e m p e r a t u r e D e r i v a t i z e d w i t h (111)

Gas Chrom Q 160'. diazomethane and
(100-200 butaneboronic a c i d
mesh). a s p y r i d o x i n e 0-
methyl e t h e r c y c l i c
butaneboronate.
r e a c t i o n h a s been u t i l i z e d f o r t h e q u a n t i t a t i o n
of t h e d r u g (122-126).
R e c e n t l y , Moussa ( 1 2 2 ) h a s d e s c r i b e d a m o d i f i e d
p r o c e d u r e t o improve t h e s t a b i l i t y of the c o l o r ,
t h e s p e e d , and t h e s e n s i t i v i t y of a s s a y . I n t h i s
p r o c e d u r e , t h e r e a c t i o n i s c a r r i e d o u t i n 2-
p r o p a n o l medium ( i n s t e a d of w a t e r ) , t r i e t h a n o l -
amine i s used as a b u f f e r , and i o d i n e i s used
as an oxidant. The d e t e c t i o n l i m i t of t h i s
method i s 10 pg of v i t a m i n B 6 .
a . Amberlite WA-2, c a r b o x y l i c a c i d r e s i n , u s i n g
a c e t a t e b u f f e r (pH=4.62) a s a n e l u e n t (106).
b. SD c a t i o n i t e , u s i n g c o n c e n t r a t e d aqueous
ammonia a s a n e l u e n t ( 1 0 7 ) .
Another system c o n s i s t i n g of Amberlite IRA-400,

u s i n g 80% a c e t i c a c i d a s a n e l u e n t , h a s been
a l s o described (107).
6.2.5 Gas-Chromatography
S e v e r a l gas-chromatographic procedures have been

r e p o r t e d f o r t h e d e t e r m i n a t i o n of v i t a m i n R6 i n
pharmaceutical f o r m u l a t i o n s and i n b i o l o g i c a l
fluids. Table 5 summarises some of t h e s e
methods.
A p p l i c a t i o n of p y r o l y s i s - g a s chromatography f o r
t h e d e t e r m i n a t i o n of pyridoxone, H C 1 and o t h e r
w a t e r - s o l u b l e v i t a m i n s , h a s been r e p o r t e d (103).
Id en t i f i c a t i o n and s e m i -quan t i t a t i v e e s t imat i o n

of v i t a m i n B 6 , u s i n g microphase e x t r a c t i o n and
gas-chromatography, h a s been d e s c r i b e d ( 1 0 4 ) .
6.2.6 High-Performance Liquid Chromatography
S e v e r a l HPLC procedures have been published f o r

t h e d e t e r m i n a t i o n of p y r i d o x i n e , H C 1 and o t h e r
water-soluble vitamins, i n b i o l o g i c a l f l u i d s
and i n f e e d s (112-1.16).
Table 6 summarises, some of t h e HPLC systems

used f o r t h e a n a l y s i s of v i t a m i n B6.
6.3 Spectrophotometry
6.3.1 C o l o r i m e t r i c
S e v e r a l c o l o r i m e t r i c a s s a y s have been developed

f o r p y r i d o x i n e . The U.S.P. method ( 4 ) f o r t h e
d e t e r m i n a t i o n of p y r i d o x i n e H C 1 , i s based on
c o u p l i n g of p y r i d o x i n e w i t h 2,6-dichloroquinone
chloroimide t o give a blue c o l o r , according t o
t h e method d e s c r i b e d by Kuhn and Low (121). The
absorbance of t h e b l u e c o l o r i s measured a t 650
nm and compared w i t h s t a n d a r d s o l u t i o n s . The
Table 6. HPLC systems of p y r i d o x i n e .
Column Mobile Phase Detection Comment Ref.
1. LiChrosorb Methanol-phos- uv The method i s (116)

RP-8 phate buffer applicable i n
(1 :1) c o n t a i n i n g a n a l y s i s of
0.002 M C TAB. v i t a m i n B6 i n
u r i n e but not
i n blood.
2. TSK-Gel Sodium p e r c h l o - mr A p p l i c a b l e i n (117)

LS-160 rate u r i n e and
- potassium blood analy-
dihydrogen sis. Detection
phosphate l i m i t 30 ng.
- Sodium chlo-
ride.
3. Octadecyl - Acidic potas- Fluores- Recovery (118)

silica s i u m phosphate cence 93.9 ? . 7 . 6 %
4. LiChrosorb Acetonitrile uv A p p l i c a b l e f o r ( 119)

NH2 - phosphate the analysis
buffer of v i t a m i n B6
i n i.v. solu-
t i o n s , and
beverages.
Standard d e v i -
t i o n s l-2X.
5. U l t r a s p h e r e 0.033 M p o t a s - Fluores- Recovery 82.4- (120)

IP sium phosphate cence 105.1X.
b u f f e r (pH=2.2) Applicable f o r
c o n t a i n i n g 2.5% the analysis
acetonitrile. of v i t a m i n B6
i n animal
t i s s u e s and
i n milk.
Among other colorimetric methods for the deter-

mination of vitamin B6, are the following:
a. Pyridoxine and other derivatives give a stable,

alcohol-soluble, red-violet color with zinc
chloride-stabilized diazotised sulphathiazole
at pH 6.5-7.0 (127).
b. Murai (102) has determined vitamin B6 by its

reaction with dimethyl-p-phenylenediamine,
HC1 and sodium hypochlorite in a buffered
solution (pH=8). The resulting color is
extracted with isobutyl alcohol and the
absorbancy of the solution is measured at
625 nm.
c. A colorimetric method similar to that des-

cribed in b, using N,N-diethyl-p-phenylene-
diamine, HC1 and potassium ferricyanide at
pH 7 (128).
Other colorimetric methods for the analysis of

vitamin B6 have been reported (9, 129).
6.3.2 Ultraviolet
Bosch (130) has published a spectrophotometric

determination of vitamin R6, and other vitamins,
in pharmaceutica1,preparations with N-bromo-
succinimide, in methanol at 370-380 nm. The
method allows determination of 100-1000 pg of
vitamin B6. Vitamin B 1 , BIZ, A , and menadione
bisulphite interfere with this method.
Vitamin B6 has been spectrophotometrically deter-

mined at 290 run, after chromatographic separation
from other related analogues(131).
A survey of normal and derivative spectra of

numerous vitamins, including vitamin B6, has been
reported. The second derivative spectra of these
vitamins are especially suitable for their
quantitative determinations (132). Quantitative
determination by derivative spectroscopy is
superior to direct spectrophotometric measurement
in many problem situations.
476 HASSAN Y. ABOUL-ENEIN A N D MOHAMMED A . LOUTFY
Barrosa (133) h a s r e p o r t e d t h e a n a l y s i s of a
m i x t u r e of p y r i d o x i n e and i s o n i a z i d i n t a b l e t
f o r m u l a t i o n . P y r i d o x i n e i s determined by
d i f f e r e n t i a l spectrophotometry a t 324 nm.
6.3.3 I n f r a r e d
P y r i d o x i n e , H C 1 has been determined by I R spec-

trophotometry, u s i n g t h e r e g i o n 6-11 u , and
water a s a s o l v e n t ( 1 3 4 ) .
6.3.4 Nuclear Magnetic Resonance
A PMR s p e c t r o m e t r i c method h a s been d e s c r i b e d

(135) f o r s i m u l t a n e o u s d e t e r m i n a t i o n of v i t a m i n
B1 and Bg. The r e l a t i o n s h i p of t h e peak a r e a s
of t h e s i g n a l s a t 6 2.55 ppm ( v i t a m i n B1) and
a t 6 2.65 ppm ( v i t a m i n B1 + B6) t o t h a t a t
6 6.6 ppm (maleic a c i d ) a r e d e r i v e d , whereby t h e
compounds of t h e m i x t u r e s can be r e a d i l y
quantitated.
Recently, a n o t h e r PMR method f o r t h e s i m u l t a n -

eous a n a l y s i s of v i t a m i n B 6 and B 1 , u t i l i s i n g
s i m p l e r e q u a t i o n f o r c a l c u l a t i o n , h a s been
developed (136). The mean p e r c e n t r e c o v e r y of
v i t a m i n B6 and B1 i n t h e i r dosape forms i s
97.42 f 2.99 and 97.57 t 1.52, r e s p e c t i v e l y .
T h i s method a v o i d s o v e r l a p i n t h e peak a r e a s
used i n t h e former method.
6.3.5 Absorption-Luminescence
The t e c h n i q u e of absorption-luminescence s p e c -
t r o s c o p y h a s been r e c e n t l y a p p l i e d f o r t h e analy-
s i s of v i t a m i n B 6 and r e l a t e d compounds (137-
138).
Razhulina and Morozov (137) have i n v e s t i a g e d

t h e e q u i l i b r i u m c o n s t a n t s between v a r i o u s ions and
t h e t a u t o m e r i c forms of v i t a m i n B6 t o g e t h e r w i t h
t h e e f f e c t of t e m p e r a t u r e , s u b s t i t u t i o n , and
medium on t h e a b s o r p t i o n p r o p e r t i e s of t h e s e
forms. The e x p e r i m e n t a l s p e c t r o s c o p i c d a t a a r e i n
good agreement w i t h quantum c a l c u l a t i o n .
6.3.6 Spectrofluorimetric
A l e K s e i c h i k , --
e t a 1 (139) have p u b l i s h e d q u a l i t a -
t i v e a n a l y s i s of v i t a m i n B6 and o t h e r d r u g
preparations. The s p e c t r a are d e t e r m i n e d i n
e t h a n o l , h y d r o c h l o r i c a c i d , and sodium h y d r o x i d e ,
u s i n g v i t a m i n B6 c o n c e n t r a t i o n s from 0.01-r).001
mg/ml.
F u j i t a , 5 a l . (140) have r e p o r t e d f l u o r o m e t r i c
d e t e r m i n a t i o n o f v i t a m i n B6. The a u t h o r s have
made e x t e n s i v e s t u d y i n t h i s r e s p e c t ( 1 4 1 ) .
F u j i n o (142) h a s improved a method f o r t h e d e t e r -

m i n a t i o n of v i t a m i n B6, b y u t i l i s i n g t h e f l u o r e s -
c e n c e of 4 - p y r i d o x i c a c i d which i s produced by
o x i d a t i o n of v i t a m i n €56 w i t h permanganate. T h i s
method i s a p p l i e d f o r t h e d e t e r m i n a t i o n of
v i t a m i n B6 i n b i o l o g i c a l f l u i d s (85, 143-144).
6.4 Microbiological
S e v e r a l m i c r o b i o l o g i c a l methods f o r t h e a n a l y s i s of
v i t a m i n B6 i n b i o l o g i c a l materials and o t h e r p r o d u c t s
have been r e p o r t e d (145-147).
The growth r e s p o n s e of some m i c r o o r g a n i s m s , e . g . ,

Streptococcus faecal& ( 1 48) and Saccharomyces
carzsbergensis (99, 1 4 9 ) , p r o v i d e a r e l i a b l e estimate
of v i t a m i n B6 c o n t e n t i n n a t u r a l p r o d u c t s . The v i t a m i n
B6 c o n t e n t i s measured n e p h e l o m e t r i c a l l y a f t e r t h e
p r o p e r t r e a t m e n t of t h e sample.
Gregory (118) h a s d e t e r m i n e d v i t a m i n B6, u s i n g

Saccharomyces uvarum. The method i s used f o r t h e
e s t i m a t i o n of v i t a m i n B6 i n f o r t i f i e d b r e a k f a s t cereals.
6.5 Enzymatic
Wada and S n e l l (150) have developed a method f o r t h e

a s s a y of p y r i d o x i n e and pyridoxamine p h o s p h a t e , based
on e n z y m a t i c o x i d a t i o n t o p y r i d o x a l which i s allowed
t o r e a c t with phenylhydrazine.
478 HASSAN Y. ABOUL-ENEIN A N D MOHAMMED A . LOUTFY
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SACCHARIN
Muhammad Uppal Zubair and
Mahmoud M. A. Hassan
King Snud Unimrsity
Riyadh, Saudi Arabia
1. Description 488
I,1 Nomenclature 488
1.2 Formula 488
I.5 Appearance. Color, Taste, Odor 489
2.2 Solubility 490
2.3 pK,, Dissociation Constant 490
2.4 Spectral Properties 49 1
3. Synthesis 50 1
4. Metabolism 504
5.1 Detection and Identification 506
5.3 Gravimetric 508
5.4 Spectrophotometric 508
References 5 I4

lSBN 0-12.260813-5
488 MUHAMMAD UPPAL ZUBAIR AND MAHMOUD M. A. HASSAN
1. Description
1.1 Nomenclature
a. 1,2-Benzisothiazole-3 (2H)-one 1, 1-
dioxide.
b. 1,2-Benzisothiazol-3-one 1, 1-dioxide.
c. 2,3-Dihydro-2-ketobenzisosulfonazole.
d. l,2-Dihydro-2-ketobenzisosulfonazole.
e. Benzosulphimide.
f. o-Benzoicsulphimide.
g. o-Sulfobenzimide.
1.1.2 Generic Names
Benzoic sulfimide, benzosulfimide, benzoic

sulfimide, o-sulfobenzimide , o -sulfobenzoic
acid imide , saccharin, saccharin insoluble.
1.1.3 Trade Names
Garantose, Glucid, Gluside, Hermesetas,

Sacarina, Saccarina, Saccharinol, Saccharinose,
Saccharol, Saxin, Sykose, Zaharina.
1.2 Formula
1.2.1 Empirical
C H N O S
75 3
1.2.2 Structural
SACCHARIN 489
1.2.3 CAS' No.
81-07-2
1.2.4 Wiswesser Line Notation
T56 BSWMVJ
183.18
1.4 Elementa1 Composition
C, 45.89%; H, 2.75%; N, 7.65%;
0, 26.20%; S, 17.50%.
1.5 Appearance, Colour, Taste, Odor
White odorless or faintly aromatic crystals or

crystalline powder (l).In dilute aqueous solution
it is 500 times as sweet as sugar, the sweet taste is
detectable even at 1:100,000 dilution. (2)
2.1.1 Crystallinity
The crystal structure of saccharin was

determined (3) by 3-dimensional integrated
intensity data collected on a computer-
controlled diffractometer operated by
an IBM 1620, in a closed-loop manner. The
crystals were found to be monocligic with
a 9.55*53, b 6.91923, c 11.80324 A, and
f3l03.g0, and the space group is 'p21/c.
The H atoms were also located and are included
in the refinement. The centrosymmetric dimer
(C6H4C0 NHS02)* mols. are formed by hydrogen
bonds between the N of imide and 0 of the
.
carbonyl atoms, (NH.. .OC), of the five
membered ring system of saccharin. The
six-sided ring formed by the hydrogen bonds
around the center of symmetry is completely
planar. The location of the H atom eliminates
the possibility of the lactim structure for
490 MUHAMMAD UPPAL ZUBAIR A N D MAHMOUD M . A . HASSAN
t h e molecule. The c o n t a c t mode of phenyl

r i n g s i s normal. A c h a r a c t e r i s t i c feature
of t h e molecular c o n f i g u r a t i o n i s t h e narrow
C-S-N a n g l e of 92.2' i n t h e five-membered
ring.
The narrow a n g l e diminishes s t r a i n from t h e
r i n g and t h u s makes it p o s s i b l e f o r t h e e n t i r e
molecule t o assume a p l a n a r shape. Other bond
a n g l e s and bond d i s t a n c e s were found t o be
normal.
Luigi e t a1 ( 4 ) have a l s o s t u d i e d t h e c r y s t a l
and molecular s t r u c t u r e s of s a c c h a r i n , and
observed some n d e l o c a l i z a t i o n i n t h e C-0 and
C-N bonds [ t r i c l i n i c (Pi), k 8 2 O 4 5 ' ( 2 0 ' ) ,
k82O 1 7 1 ( 1 0 1 ) , ~ = 6 5 O4 0 ' ( 1 2 ' ) , Z=4]. The
C-C, C-S and S-N bonds i n t h e i s o t h i a z o l e
r i n g are s i n g l e , and s t r o n g hydrogen bonding
(NH ... 0 ) g i v e r i s e t o centrosymmetrical dimers,
as was observed w h i l e determining t h e c r y s t a . 1
s t r u c t u r e of s s c c h a r i n .
2.1.2 Melting Range
The m e l t i n g ranges of s a c c h a r i n are

( a ) 228.8-229.7 ( 2 , 5 ) and ( b ) 226-230(1,6,7)
dec. 228.8-229.7 (8).
2.2 Solubility
Saccharin i s s o l u b l e a t 20°, i n 290 p a r t s of water,

i n 30 p a r t s o f a l c o h o l , i n 1 2 p a r t s o f a c e t o n e , i n
50 p a r t s of g l y c e r o l , i n about 25 p a r t s of b o i l i n g
water. It i s v e r y s l i g h t l y s o l u b l e i n e t h e r and
chloroform; s o l u b l e i n d i l u t e ammonia s o l u t i o n , a n d i n
s o l u t i o n s of a l k a l i b i c a r b o n a t e s w i t h e v o l u t i o n of
carbon dioxide ( 9 ) . I t s aqueous s o l u t i o n i s a c i d
t o l i t m u s ( 2 ) . On a l k a l i n e and a c i d h y d r o l y s i s
s a c c h a r i n g i v e s r i s e t o 0-sulphamoyl-benzoic a c i d
and ammonium 0-sulphobenzoic a c i d r e s p e c t i v e l y ( 2 ) .
2.3 pKa , D i s s o c i a t i o n Constant
The d i s s o c i a t i o n c o n s t a n t of s a c c h a r i n a t 25' has

been r e p o r t e d t o be pKa 1.6.
SACCHARIN 49 I
2.4.1 Infrared Spectrum
The IR spectrum of saccharin as KBr disc was

recorded on a Perkin Elmer 580-~Spectropho-
tometer and shown in Fig 1 (10). The struc-
tural assignments have been correlated with
band frequencies and are given in Table 1.
Table 1. IR Characteristics of Saccharin
-1
Frequency Cm Assignment
3410, 3100 -co-x-
2970, 2700 -C-H frequencies
0
1722 -
-8-NH-
1595 aromatic- C=C-
1340
1165, 1180, 1188 -so2 - N -
700-900 C-H out of plane
deformation showing
1,2-disubstituted
benzene ring.
760 Four adjacent hydrogens,
ortho-disubstituted
benzene.
Other characteristic bands are:

2010, 1980, 1870, 1462, 1300, 1260, 1200,
1140, 1122, 1055, 1020, 1010, 975, 902,
688, 632.
Other IR studies were also reported (11,121.
2.4.2 Ultraviolet Spectrum
The W spectrum of saccharin in ethanol was

scanned from 200 to 400 nm using Varian DMS 90
spectrophotometer. It exhibits a characteris-
tic UV spectrum Fig 2 with maxima at 283, 275,
225 and 208 nm (13).
Other UV spectral data of saccharin have also
been reported (14).
492
493
494 MUHAMMAD UPPAL ZUBAIR AND MAHMOUD M . A. HASSAN
-
Xmax -&
285 775
276 951
226 8570 in methanol.
1%
-
hax
-
Elcm
234.5 351 in 0.01 N NaOH (5,9)
268 89
284 Inflexion
-
Amax
267.5
-
69
1%
1cm -
244.5 minima saccharin sodium

in water (5)
2.4.3 Nuclear Magnetic Resonance Spectra
2.4.3.1 Proton Spectrum
The PMR spectrum of saccharin in

DMSO-d6 was recorded on a Varian
FT80 NMR spectrometer using tetra-
methylsilane as a reference standard,
Fig 3 (15). The following structural
assignments have been made.
Table 2. PMR Characteristics of Saccharin
Assignments Chemical Shifts

Aromatic protons 8.11 (m)
- NH 12.45 (s)
m = multiplet; s = singlet
Natural abundance l5N NMFt data has been

reported (16). N-H chemical shift is
215.4 ppm.
2.4.3.2 l 3 C NMR S p e c t r a
The 13C-NMR n o i s e decoupled and

off-resonance s p e c t r a a r e shown i n
F i g 4 and F i g 5 r e s p e c t i v e l y (17).
Both w e r e recorded over 5000 Hz range
i n DMSO-d6 on FT 8 0 , 80 MHz NMR
s p e c t r o m e t e r , u s i n g 1 0 m l sample t u b e ,
and t e t r a m e t h y l S 1 a n e as r e f e r e n c e
s t a n d a r d a t ambient temperature. The
carbon chemical s h i f t s a s s i g n e d on t h e
b a s i s of a d d i t i v i t y p r i n c i p l e and o f f -
resonance s p l i t t i n g p a t t e r n a r e given
i n Table 3.
Table 3. Carbon Chemical S h i f t s of Saccharin
Carbon no. Chemical S h i f t (ppm)

c-1 127.57 ( s )
c-2 139.75 ( s )
c-3 124.53(d)
c-4 134.72(d)
c-5 135.55( d)
C-6 121.18(d)
c-7 160.79 ( s )
d = doublet; s = s i n g l e t
2.4.4 Mass Spectrum
The m a s s spectrum of s a c c h a r i n o b t a i n e d by
electron impact i o n i z a t i o n and which w a s r e c o r -
ded on a Ribermag R-10-10 m a s s spectrometer
equipped w i t h d i r e c t i n l e t probe and a d a t a
a
a
d
a
?
0
u
a,
a
a,
(I)
-d
0
d
0
2
n
W
a
0
m
2
c
-d
c
-4
k
rd
.c
u
u
rd
m
w
0
5
k
+r
0
a,
a
m
a,
u
c
rd
c
0
m
a,
k
I
w
w
0
i2
SACCHARIN 499
system ( 1 8 ) . The spectrum ( F i g 6 ) shows a

molecular i o n peak a t m / e 183 and a base peak
m/e 184 (M+l). The most prominent fragment,
t h e i r r e l a t i v e i n t e n s i t i e s and p o s s i b l e s t r u c -
t u r e s a r e l i s t e d i n Table 4 .
Table 4. Mass Fragments of S a c c h a r i n
m/e Relative Intensity Fragment
184 100 M+1
183 30.7 M+
168 6.9
120 54
GY
CONH2
119 59
+ 9
[ ( g C O N H
1
104 25.6
I(Continued)
I
SACCHARIN SO I
Table 4 (continued)
m le Relative Intensity Fragment
103 11.8
+'
92 46
+.
76 51 H +'
Other mass s p e c t r a l d a t a a r e a l s o r e p o r t e d ( 1 9 ) .
3. Synthesis
Route 1
Saccharin w a s f i r s t s y n t h e s i s e d i n 1879 from o-sulph-
anoylbenzoic a c i d ( 2 0 ) . The s t a r t i n g m a t e r i a l i s o b t a i n e d
from t h e m i x t u r e o f o-and p - s u l f o n i c a c i d s , r e s u l t i n g from
s u l f o n a t i o n of t o l u e n e . The s u l f o n i c a c i d s are t h e n con-
v e r t e d t o t h e s u l f o n y l c h l o r i d e s by t h e a c t i o n o f phos-
phorous p e n t a c h l o r i d e . The p - t o l u e n e s u l f o n y l c h l o r i d e
i s l a r g e l y removed by f r e e z i n g , and t h e l i q u i d r e s i d u e
c o n t a i n i n g t h e compound i s t r e a t e d w i t h ammonia t o g i v e
t h e o-toluenesulfonamide which on o x i d a t i o n w i t h a l k a l i n e
potassium permanganate s o l u t i o n g i v e o-sulfonamidobenzoic
a c i d which undergoes spontaneous l o s s of water t o y i e l d
s a c c h a r i n which can be o b t a i n e d on a c i d i f i c a t i o n of t h e
r e a c t i o n mixture.
Bo2c1
Sac c h a r i n
Route I1
It may be prepared ( 2 1 ) by t r e a t i n g t o l u e n e w i t h
c h l o r o s u l f o n i c a c i d and s e p a r a t i n g t h e c- and p-toluene-
sulfowlchloride. The o -compound i s t h e n t r e a t e d with
ammonia and t h e r e s u l t i n g sulfonamide i s o x i d i z e d w i t h
potassium permanganate i n a l k a l i n e s o l u t i o n y i e l d s a
s a l t of S s u l f a m o y l b e n z o i c a c i d . The f r e e a c i d which i s
l i b e r a t e d on a c i d i f i c a t i o n l o s e s w a t e r spontaneously t o
give saccharin.
SACCHARIN 503
acH
aCH3 s02c1
NH3 ~
S02NH2
COOH
S02NH2
Saccharin
Route I11
Saccharin i n h i g h y i e l d may be o b t a i n e d by o x i d i s i n g
o-toluenesulfonamide with dichromate and s u l f u r i c a c i d .
Saccharin
Route IV,
Many o t h e r i n d u s t r i a l methods are now employed e.g.

one s t a r t s ( 2 2 ) w i t h a n t h r a n i l i c a c i d which i s d i a z o t i s e d
and t r e a t e d w i t h l i q u i d s u l f u r d i o x i d e i n presence of
copper as c a t a l y s t . The s u l p h i n i c a c i d d e r i v a t i v e t h e r e b y
obtained i s t r e a t e d with chlorine i n a lk a lin e solution
and sulfonyl c h l o r i d e so o b t a i n e d i s t r e a t e d w i t h m o n i a
and heated.
COOH
NH2 N2HS04
NaOH
4. Metabolism
Early s t u d i e s r e p o r t e d (23) t h a t 90-95% of an i . v . dose

of saccharin could be recovered from t h e u r i n e of a
c a t h e t e r i z e d dog. Staub ( 2 4 ) r e p o r t e d t h a t o r a l l y admin-
i s t e r e d saccharin w a s excreted unchanged i n t h e u r i n e .
Herter ( 2 5 ) demonstrated t h e presence of s a c c h a r i n i n
various organs of a r a b b i t a f t e r an o r a l dose. I n recent
s t u d i e s u t i l i z i n g r a d i o - l a b e l l e d saccharin ( 26-28 ) t h e
compound w a s shown t o be metabolised t o a s l i g h t e x t e n t
i n r a t s and o t h e r animals. Lethio and Wallace ( 2 9 ) have
used ( 3 - 1 4 C ) saccharine ( I ) i n r a t , dog, r a b b i t , guinea
p i g and hamster, i n d i c a t e d t h a t t h e r e w a s l i t t l e d i f f e r -
ence i n p a t t e r n of metabolic p r o f i l e s due t o animal spec-
i e s or dose l e v e l . They a l s o showed t h a t l a b e l l e d C 0 2
i n t h e expired a i r i n d i c a t e d t h a t s a c c h a r i n w a s decarbo-
x y l a t e d t o a s l i g h t degrec. (11), and GEAE chromatographic
s e p a r a t i o n and i s o l a t i o n of l a b e l l e d compounds i n d i c a t e d
t h a t more t h a n 99% of t h e u r i n a r y 1 4 C w a s unchanged
SACCHARIN 505
saccharin and up to 1% of 1 4 C was a metabolite identified

as o-sulphamoylebenzoic acid (111) (Fig 1, Scheme ).
Scheme
*
I11
99%
I
Saccharin
2
a I1
S02NH2
Fraction Number
Fig. 1. Elution diagram of the chromatography
on DEAE cellulose of urine from a rat which
received [ 3 1 4 C ] saccharin at 50 mg/kg. Each
fraction contained 10 m l . Peak I: carbonate;
Peak 11: sulfamoylbenzoic acid; Peak 111:
saccharin.
506 MUHAMMAD UPPAL ZUBAIR AND MAHMOUD M. A . HASSAN
I n man s a c c h a r i n i s r a p i d l y absorbed from t h e g a s t r o -

i n t e s t i n a l t r a c t and e x c r e t e d unchanged mostly i n t h e
u r i n e w i t h i n 24 h o u r s ( 5 ) .
5.1 D e t e c t i o n and I d e n t i f i c a t i o n
a. Saccharin i s h e a t e d w i t h r e s o r c i n o l and s u l p h u r i c
a c i d over a low flame; on d i l u t i o n and a d d i t i o n of
sodium hydroxide s o l u t i o n , a f l u o r e s c e n t green l i q u i d
i s o b t a i n e d (5,30,31).
b. Saccharin i s f u s e d w i t h sodium hydroxide t i l l t h e
e v o l u t i o n of ammonia c e a s e s . N e u t r a l i s e d s o l u t i o n
on a d d i t i o n o f f e r r i c c h l o r i d e g i v e s r i s e t o v i o l e t
c o l o u r (30 J1).
C. Thermal a n a l y s e s ( 3 2 ) and microchemical r e a c t i o n s
of s a c c h a r i n w i t h Mn, Co, N i and Cu have been
employed f o r t h e c h a r a c t e r i s a t i o n of s a c c h a r i n ( 3 3 ) .
d. Saccharin can a l s o b e i d e n t i f i e d by i t s r e a c t i o n
w i t h H202 and NaNO2 and subsequent b a s i f i c a t i o n
w i t h a l k a l i a f f o r d s dark brown-red c o l o u r ( 3 4 ) .
e. I d e n t i t y t e s t s f o r s a c c h a r i n p r e s e n t i n food have
a l s o been based on column and t h i n l a y e r chromato-
graphy ( 3 5 ), m a s s s p e c t r o m e t r y (36) , spectrophoto-
metry ( 3 7 ) and m i c r o a n a l y t i c a l methods ( 3 8 ) .
5.2.1 Aqueous
The USP XIX(7) and B.P 1980 ( 6 ) methods

c o n s i s t of d i s s o l v i n g an a c c u r a t e l y weighed
sample of s a c c h a r i n i n h o t water and t i t r a t i n g
w i t h 0 . 1 N sodium hydroxide, u s i n g phenolph-
thalein as indicator.
Another o f f i c i a l procedure ( 3 0 ) i n v o l v e s
d i g e s t i o n of an a c c u r a t e l y weighed sample
o f s a c c h a r i n i n h y d r o c h l o r i c a c i d . The
r e s u l t i n g ammonium s a l t i s r e a c t e d w i t h
sodium hydroxide and t h e l i b e r a t e d ammonia
i s d i s t i l l e d i n t o 0 . 1 N s u l p h u r i c a c i d . The
excess of t h e a c i d i s determined by t i t r a t i n g
w i t h . sodium hydroxide u s i n g methyl r e d solu-
t i o n as i n d i c a t o r .
SACCHARIN 507
5.2.2 Non-aqueous
A non-aqueous differentiating titration

procedure (39) involves passage of the
artificial sweeteners mixture through a
cationic exchange resin , Dowex 50-x8,
to separate and convert saccharin to its
acid form. The solution obtained is titrated
potentiometrically in methyl isobutyl ketone
with 0.1 N sodium methoxide. The method
has been applied in presence of cyclamate and
benzyl alcohol.
5.2.3 Ion Selective Electrode
Hazemoto et a1 (40) developed an ion-

selective electrode sensitive to saccharin,
by establishing an ion association between
Fe2+-bathophenanthroline chelate and saccharin
in nitrobenzene. The electrode developed
could measure saccharin ion in presence of
other sweetening agents e.g., sucrose, glu-
cose, sodium cyclamate and sorbitol in the
Concentration range of 10-1to 10-5 M.
5.2.4 Polarography
Polarographic methods of analysis have been

applied to samples of foods containing sacc-
harin (41-44). In a procedure (44) saccharin
is extracted into organic solvents in an
acidic medium. Further purification is
achieved by column chromatography. The
residue obtained is dissolved in 0.1 N NaOH
and an aliquot is polarographed in a support-
ing electrolyte of 0.1 N HC1, 0.1 N KC1 and
0.1% Bu4 N Br.
Nasierowska (45) has applied alternate-
current oscillopolarographic method to
pharmaceuticals containing saccharin and
sodium saccharin. Both forms a well develo-
ped oscillopolarographic curve in MH2SO4 in
air with mercury anode and dropping-mercury
cathode, and the results are comparable to
those obtained by UV spectrometry.
508 MUHAMMAD UPPAL ZUBAIR AND MAHMOUD M . A . HASSAN
5.3 Gravimetric
Saccharin has been determined gravimetrically by

the oxidation of its sulfur to sulfate ion (46).
An accurately weighed amount of the sample is
boiled with concentrated nitric acid and the
vapours evolved are reacted with sodium hydroxide
in a tube. The sulfate content is then determined
gravimetrically .
5.4 Spectrophotometric
5.4.1 Colorimetric
A method involving salt formation between
saccharin and methylene blue has been deve-
loped (47)and its concentration as low as
10 p M can be determined by this method.
Taneka et a1 (48) developed another proce-
dure in which saccharin is reacted with
phenothiazine and copper acetate in 50%
acpeous ethanol. The resulting complex is
extracted into xylene and its absorbance is
measured at 510 nm.
Belagy (49) analysed saccharin by forming
a complex with safranine and measuring its
absorbance at 510 nm.
5.4.2 Ultraviolet
Several procedures have been developed for
the extraction and isolation of saccharin
when present in beverages (50-53), and food
( 54-57) before its estimation spectrophoto-
metrically. The absorbance was measured in
chloroform at 278 nm ( S O ) , in ethanol at
271 nm (55) and in 1% aqueous sodium carbo-
nate at 235 and 244 nm (52 1.
5.4.3 Infrared
A procedure has been developed (58) for

the determination of saccharin by IR spectro-
photometric method. Saccharin is separated
from cyclamate by extracting it into a mixture
of benzene and ether from an acid medium.
SACCHARIN 509
The IR absorption at 1721 cm-l is then

measured. Saccharin up to 0.2 mg can be
determined by this method.
5.4.4 Spectrofluorometric
Nakamura (59) had developed a procedure for
the isolation and determination of saccharin
in foods by measuring fluorescence at 410 nm
(excitation at 277 nm) . However recovery of
saccharin added to 23 types of food ranged
from 82 to 98%.
5.4.5 PMR Spectrometry
A PMR procedure was described (60) involving

the integration of the aromatic protons sing-
let of benzisothiazoline ring occurring at
8.00 ppm and that singlet due to protons of
the internal standard maleic acid occurring
at 6.25 ppm. The method was employed for
saccharin sodium tablets resulted in an
average of 97.1% ? 3.15 and for saccharin-
lactose mixture with an average of 98.92 2
3.5 for saccharin.

Saccharin has been detected and estimated

after extraction from food samples on Whatman
No. 1 filter paper (61,62). The sample in
solution form, such as carbonated water can
be used for paper chromatography. The spotted
paper is developed in BuOH-ACOH-H20 (40:10:22)
for 18 hours; and sprayed with a solution of
phthalic acid and aniline for spot develop-
ment. Saccharin with an Rf 0.17 can be
estimated colorimetrically after elution
of the spot with 60% AcOH (62).
5.5.2 Thin Layer Chromatography
Thin layer chromatography has been used in

qualitative and quantitative analysis of
saccharin, when present in artificial sweet-
ening agents , beverages , food and pharmaceu-
ticals. Several systems have been used and
are listed in table 5.
510 MUHAMMAD UPPAL ZUBAIR AND MAHMOUD M . A. HASSAN
Table 5
Thin Layer Chromatography Systems f o r Saccharin Analysis

~~ ~ ~ ~~ ~
S t a t i o n a r y Phase Developing Phase Detection Reference

10%-Acetylated S h e l l s o l A- U.V. and
Cellulose- propanol- m u l t ip l e 63
Polyamide ( 3 :2 ) a c e t i c acid- colorimetric
formic a c i d methods
(45:6:7: 2 )
S i l i c a gel chloroform- U.V. 64
acetic acid (9:l)
Kieselgel G acetone-ammonia, silver n i t r a t e ,
ethanol-ammonia peroxide-ferric
and benzene c h l o r i d e and 65
2,6-dichloro-
quinone
chloroimide
spray reagent
Wakogel B-5 multiple 66
plates colour
react ion
Acetyl cellulose- xylene-propanol- 67
polymi.de powder a c e t i c acid-
(3:2) formic a c i d
(45: 6 : 7 : 2 )
Silica gel but anol-et hanol
( Adsorbosil-1 ) (95%)-ammonia
(28%)-water, 254 m 68
(40:4:1:9 ) irradiation
Avicel SF ethanol- spray with
ammonia-acetone, 0 .l% 69
(1:1:8) pina c r y s t a l
dime t h y l yellow i n
formamide- 95% e t h a n o l
ethanol-water , and i r r a d i a t e
(5:4:1) a t 365OAO.
dioxane-
pyrimidine-
water. ( 7 : 2 : 1 )
Polyamide benzene- methyl r e d 70
ethyl acetate- bromocresol
formic a c i d green
( 5 :10:2)
(Continued)
SACCHARIN 51 1
Table 5 (continued)
S t a t i o n a r y Phase .Developing Phase Detection Reference
A1 0 /Polyamide toluene- multiple
2 3
propionic acid- colour 71
formic a c i d r e a c t ions
(5:4:2)
Silica gel G but anol- U.V. 72
ethanol-
25% aqueous
ammonia
(40 :4 :5 )
5.5.3 G a s Chromatography
Saccharin has been d e t e c t e d and analysed by

gas chromatography, when p r e s e n t i n pharma-
c e u t i c a l s and food. It i s u s u a l l y , f i r s t
e x t r a c t e d from t h e sample w i t h an o r g a n i c
s o l v e n t and t h e n converted t o i t s e s t e r , such
as methyl or s i l a l y l t o make it more v o l a t i l e .
Table 6 shows v a r i o u s systems t h a t have
been used.
5.5.4 High-Performance L i q u i d Chromatography
High-Performance Liquid Chromatography h a s

been used e x t e n s i v e l y f o r a n a l y s i n g s a c c h a r i n ,
p r e s e n t i n pharmaceutical s , beverages and
food. The d i f f e r e n t HPLC systems used for
t h e a n a l y s e s are given i n Table 7.
l n w 0 cu
t - t - co a3
I
cu cu
2 2
0
0 0 0
co 0 ri
cu
ri (u
X
ffl
k
a,
c,
2
a
3
d;J.
. .
rlmN
I
0
0
rl
I I I I
0
co
U
P
k E
0 0
k
ffl
z
0
0
0
cu
e
k m
.c V Ld
t7a
V !2
a,
a
c
u cd
.rl ?3? F
4 W R ri d
m m
-rl
m &
0
W
0 %
ri m
SACCHARIN 513
Table 7. HPLC of Saccharin

__
Analyte or Parameters and Comments Reference
Matrix
Pharmaceuticals Micronized silica gel 83
treated with 3-amino-
propyltriethoxysilane;
0.04 M phosphate buffer;
254 nm.
Beverages p Bondapak/C18(S/S 30 cm x 84
4 m m ) ; 5% acetic acid, -1
at 2000 p.s.i, (2 ml min ) ;
254 nm.
Alcoholic Partisil-10 ODS ( S / S , 85
products 25 cm x 4 mm); 0.01 M '
KH2P04 at pH 2.2 (1.2
ml min-l); 254 nm.
Food Permaphase AA X or 86
Zipax SAX; 10 nm-KClO4
in 10 nm-Na2B407 buffer
of pH 8.0;
Pharmaceuticals ODS-Sil-X -1, 400- 87
and 500 p.s.i.; aqueous
non-alcoholic 45% methanol or
drinks phosphate buffer
solution (pH 3.0) and
methanol ( 3 :2 ) ;
U.V. detection.
Pharmaceuticals 1-1 Bondapak cl8; 88

acetic acid-methanol-
H2O (1:60:139);
280 nm
Plasma and Partisil PXS 89
urine ODS-2 (10 m)
(30 cm x 4 m m ) , 1.2 ml
min-l ; H20-methanol-
anhyd. acetic acid
(399:lOO:l);
fluorimetric detection
(excitation at 230 nm;
emission at 350 nm)
Beverages HC ODS Sil-X 90 919
(25~0.26cm);
1M CH3COOH
References
1. Mart indale (The Extra-pharmacopceja) 27th Edition,

The Pharmaceutical Press, London, (1977)p.612.
2. The Merck Index, 9th Edition, p . 8075, Merck & Co.,

Inc., Rahway, N.J., U.S.A. (1976)p. 8075.
3. Y. Okaya, Acta Crystallogr., Sect. B, 25 ( R . 11),

2257 (1969).
4. C. Luigi, M. Alessandro, and P. Giancarlo, Gazz. Chim.
Ital., 100(7), 629 (1970).
5. Isolation and Identification of Drugs, E d . , E.G. Clarke,

The Pharmaceutical Press, London, (1974)p. 538.
6. British Pharmacopoeia, V o l . I, Her Majesty's Stationery

Office, London, (1980), p. 394.
7. The United States PharmacopoeiaXIX, Mack Publishing

Company, Easton, PA, (1975),p. 56.
8. "Atlas of Spectral and Physical Constants of Organic Comp-

ounds", J. Grasselli and W. Ritchy ( E d s . ) , Vol. I , 2nd
Edn., CRC Press, Inc., Ohio, (1975), p. 498.
9. The Pharmaceutical Codex, 11th Edition, The Pharmaceutical

Press, London, (1979), p. 799.
10. M.M.A. Hassan and M. Uppal Zubair, unpublished work

(1980).
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-
43(25), 4693 (1978).
SACCHARIN 515
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516 MUHAMMAD UPPAL ZUBAlR AND MAHMOUD M. A. HASSAN
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48. A. Tanaka, N. Nose, T. Suzuki, S. Kobayashi and
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50 G. Bosile, Chim. Prov., 17(4), 554 (1966).
SACCHARlN 517
A. Vincerzo and D. Vincerzo, Chim. Unione Ital. Lab.

w.,
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52 - G. Krueger , Lebensmittelindestrie, 27(6), 264 (1980).
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24(1), 36 (1976).
58. D. Coppini and A. Albasini, Mitt. Geb. Lebensmittelun-

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37, 302 (1982).
61. W. Mis'kiewicz, Roczn-pah. Zakt. Hig. , &(1), 33
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62. D. Das and T. Mathew, J. Inst. Chem. , Calcutta,
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207(4), 269 (1965).
65. G. Dick&, J. Ass. Publ. Analyst, 3(4), 118 (1965).
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67. H. Woidich, H. Gnauer and E. Galinosky, Z. Lebensmittel-
unters. U. Forsch, 139(2), 142 (1969).
518 MUHAMMAD UPPAL ZUBAIR A N D MAHMOUD M. A . HASSAN
68. T. Korbelok and J. Bartlett; J. Chromat. 41(1),

124 (1969).
69. K. Nagasawa, H. Yoshidome and K. Anryo, J. Chromat.

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70* T. Ryuzo, J. Chromat. 6 6 ( 2 ) , 283 (1972).
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~ ( 3 1 153
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66(7),223 (1970).
75. R. Gerstl and K. Ranfft, Z. analyst. chem., 2 5 3 ( 2 ) ,

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78 - H. Youji, T. Suzuki, Y. Kikuchi, N. Norihide, W.

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79. P. Hartvig, 0. Gyllenhaal and M. Hammerlund, J.

Chromat . 1 5 1 ( 2 ) , 232 (1978).
80. T. Sumimoto, M. Imaida, M. Yoshida and A. Kuwano,

Osaka-furitsu koshu Eisei Kenkyusho Kenkyu Hokoku
Shokuhin risei Hen, 9,13 (1979).
81. K. Karlowski, Rocz Panstw. Zakl. Hig; z ( 6 ) 581 (1980).
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K. Szczawinska; J. Pharm. Sci., 7 0 ( 8 ) , 871 (1981).
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Acta mat., 12(3),195 (1976).
84. D. Smyly, €3. Woodward and E. Conrad; J. Ass. Off.

Analyt. Chem. , 59(1), 1 4 (1976).
SACCHARIN 519
85. T. Milton, M. Glenn, J , Assoc. Off. Analyt. Chem. ,

, 1321 (1977) -
86. U. Masahiko and M. Masanori; Shokuhin Eiseigaku Zasshi,
18(3),278 (1977).
87. L. Ciraolo, R. Calapaj and M. Clasadonte; Rass Chim,
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88. P. Palermo and P. Tsai; J. Pharm. Sci., 68(7), 878
(1979).
89. I. Bekersky and W. Colburn, Anal. Lett., Part B,
13(10), 805 (1980).
90. E. Gillyon, Chromat. Newsl. , 8(2), S O ( 1980).
91. G. Seroussi, R. Barbes, J. Dauchy, D. Daniel,

I. Xavier, M. Laparra; Spectra 2000, 9,29 (1981).
Acknowledgement
The authors would like to thank Mr. Mohammad

Saleem Mian of Pharm. Chem. Dept., College of
Pharmacy, King Saud University for his assis-
tance in this work.
SALICYLAMIDE
Salim A. Babhair, Abdullah A. Al-Badr,
Hassan Y. Aboul-Enein
King Sotid Unizjer,sity
1. Description 522
1.2 Formulae 522
1.5 Appearance, Color, and Odor 523
2.2 Solubility 523
2.3 pK, 524
3. Synthesis 53 1
4. Stability 534
6.1 Non-Aqueous Titration 535
6.2 Colorimetric Analysis 536
6.4 Spectrophotofluorimetric Analysis 537
6.5 Infrared Spectrophotometric Analysis 538
6.6 Chromatographic Analysis 538
6.7 Radiochemical Analysis 542
6.8 Flame Emission Spectrometry 542
6.9 Thermomicroscopic Method 547
6.10 The Ring-Oven Technique 547
References 548
Copyright 0 1984
VOLUME 13 521 by the Amencan Pharmaceutical Association
ISBN 0-12-260813-5
522 SALIM A . BABHAIR ET AL
SALICYLAMIDE
1. Description
1.1 Nomenclature
1.U Chemical Names
o-Hydroxybenzamide, 2-Hydroxybenzamide.
1 . 1 2 Generic Name
Salicylamide .
1.13 Trade Names
Afko-sal, Amid-sal, Doldram, Drosprin, L i q u i p r i n ,

Raspberin, S a l r i n , Uromide, Salamide, Samid,
Acket, Algiamida, Cidal, Oramid, P a n i t h a l ,
S a l i z e l l , Novecyl, Dolomide, S a l i p u r , Salymid,
Saliamin, u r t o s a l , Algamon, Benesal (1,2).
1.14 Chemical A b s t r a c t R e g i s t r y Number

[ 65-45-21.
1 . 2 Formulae
1 . 2 1 Emperical
C H NO
7 7 2
1.22 S t r u c t u r a l
4
137.13
SALICYLAMIDE 523
C , 61.27%; H, 5.11%; N, 10.28%; 0, 23.34%.

1 . 5 Appearance, Color, and Odor
White o r yellowish white c r y s t a l l i n e powder or c r y s t a l s
darkens on exposure t o a i r , o d o r l e s s .
2.1 Cryst a1 P r o p e r t i e s
2 . 1 1 C r y s t a l System
Monoclinic, Rods from s o l v e n t s o r sublimation.
C e l l Dimensions a = 12.93 A., b = 5.02 A.,

c = 24.80 0.2A.
Formula Weight per c e l l . 8 (8.05 c a l c u l a t e d

from X-ray d a t a ) .
Density. 1.320 ( f l o t a t i o n and pycnometer) ;

1.308 (X-ray).
Optical Properties.
R e f r a c t i v e i n d i c e s (5893 A , , 25OC) a = 1.595 ?r
0.002. b = 1.637 0.001, q = 1.727 2 0.005.
Optic Axil Angles (5893 A., 25'C) 2H = (+) 82.5'
2 V = ( + ) 76' ( c a l c u l a t e d from b and 2H), 2V =
75' ( c a l c u l a t e d from a , b and q) (3).
2.13 Melting Point and Fusion Data
Salicylamide sublimes b e f o r e melting (142OC;

h o t s t a g e ) . A s t h e m e l t c o o l s , broad and un-
s t a b l e form appears. This form i s m e t a s t a b l e
below t h e melting point. When seeded o r on
standing t h e s t a b l e form grows slowly through
t h e u n s t a b l e form. The r a t e of transformation
i s very slow a t room temperature (3).
One gram i s s o l u b l e i n about 500 m l o f water, about

1 5 ml o f a l c o h o l , about 35 m l o f e t h e r , about 100 ml
524 SALIM A. BABHAIR ET AL.
of chloroform and about 20 m l of propylene g l y c o l .
I n s o l u b l e i n benzene, carbon t e t r a c h l o r i d e and xylene.

Soluble i n s o l u t i o n of a l k a l i e s . A s a t u r a t e d s o l u t i o n
of salicylamide i n water has a pH 5.2-6(1,4,5).
2 . 3 pKa
8.1 ( 6 ) .
2.4 Identification
Salicylamide g i v e s a b l u e v i o l e t c o l o r w i t h f e r r i c chlo-
r i d e solution, a color reaction indicative t o s a l i c y l a t e
derivatives.
T r i n d e r ' s reagent g i v e purple c o l o r w i t h salicylamide

(5).
Matta and Nunes ( 7 ) i d e n t i f i e d salicylamide from chemi-
c a l l y r e l a t e d analogues by t h e determination of i t s
r e f r a c t i v e index at t h e melting p o i n t which w a s found
t o be 1.55.
Furthermore, salicylamide could be c h a r a c t e r i z e d

through t h e formation of diphenylmethyl d e r i v a t i v e
prepared by r e f l u x i n g with diphenylmethanol and
p-toluene sulphonic a c i d i n a c e t i c a c i d ( 8 )
A f u l l r e p o r t f o r t h e i d e n t i f i c a t i o n of salicylamide
through melting p o i n t , r e a c t i o n s of t h e a i d e group,
t h e phenolic group, t h e benzene r i n g , and t h e forma-
t i o n of c h e l a t e compounds has been published ( 9 ) .
Salicylamide can be d e t e c t e d microchemically by means

of t h e formation of t h e dibromo-derivative ( 9 ) . Fur-
thermore, t h e i d e n t i f i c a t i o n of salicylamide by chro-
matographic a n a l y s i s a r e discussed f u r t h e r i n t h e
monograph.
2.51 U l t r a v i o l e t spectrum
The u l t r a v i o l e t spectrum of salicylamide i n metha-

n o l and water shows maxima a t 235 nrn and 302 nm.
The u l t r a v i o l e t spectrum i n e t h a n o l i s shown i n
Figure [l] and i s recorded on Varian AG UV-VIS
spectrophotometer model DMS 90 with maxima a t
S ALICYLAMIDE 5 25
F i g . 1. The UV spectrum of s a l i c y l a -
mide i n e t h a n o l .
I
f’
I
’,
Q,
2
cd
P
k
0
(0
5
2
Fig. 2 . The UV spectrum of salicylami.de

i n 0 . 1 N NaOH s o l u t i o n .
526 SALIM A . BABHAIR ETAL.
235 nm (El%, 1 cm 543) and 302 nm (E;$, 1 cm 295).

I n a l k a l i n e medium, t h e spectrum e x h i b i t s two
maxima a t 242 nm ( E l % , 1 cm 536) and 328 nm ( E l % ,
1 cm 435) as shown i n f i g u r e [ 2 ] .
2.52 I n f r a r e d spectrum
The i n f r a r e d of salicylamide i n KBr d i s c i s pre-

sented i n f i g u r e [ 3 ] and i s recorded i n Perkin-
E l m e r spectrophotometer model 580 B. The frequen-
c i e s and t h e i r s t r u c t u r a l assignment a r e shown
below:
Frequency( cm-1) Assignments
3400 OH s t r e t c h
3200 JTH s t r e t c h
1680 CO (amide)
:760
;:I C=C aromatic
CH out-of -plane
750 bending aromatic .
Other c h a r a c t e r i s t i c f i n g e r p r i n t bands a r e 1500,
1450, 1430, 1360, 1310, 1250 cm-1.
2.53 Mass Spectrum
The m a s s spectrum of salicylamide i s shown i n

f i g u r e [ 4 ] . The spectrum w a s c a r r i e d out on
Varian MAT 311 spectrometer with an i o n i s i n g
energy of 70 e V . The sample w a s introduced by
d i r e c t probe a t 12OoC. A molecular ion peak
w a s observed a t m/e 137. Some c h a r a c t e r i s t i c
peak observed a r e l i s t e d below:
Mass (m/e) Species or Fragment
137 M"
+=
120
d-O
4 -1
'k
v)
2
100 0
. 500
Fig. 4. The mass spectrum of salicylamide deter-

mined by electron impact.
SALICY LAMIDE 529
92
65
39 I aromatic
fragmentations.
2.54 Nuclear Magnetic resonance spectrum

1
2.541 H-NMR Spectrum
1
The 60 MHz H-NMR spectrum o f s a l i c y l a m i d e
i n DMSO-Db i s shown i n f i g u r e [ 5 ] . The spec-
t r u m w a s recorded on Varian T ~ O - ANMR spec-
t r o m e t e r u s i n g TMS as t h e i n t e r n a l s t a n d a r d .
The f o l l o w i n g s t r u c t u r a l assignments have
been e l i c e t e d from f i g u r e [51.
Chemical s h i f t (ppm1 Assignments
Multiplet centered at Aromatic p r o t o n s

6.83 a t C3 and C 5
Multiplet centered at Aromatic p r o t o n

7.36 at C 4
Mult i p l e t c e n t e r e d a t Aromatic p r o t o n
7-90 at Cg
Broad s i n g l e t a t 8.33 NH2 amide( exchang-

a b l e w i t h D~o).
S i n g l e t a t 13.00 OH phenolic (ex-

changable w i t h
D20, Fig. [ 6 1 ) .
Long range coupling between p r o t o n s a t C 3

C5, C 4 and C 6 i s observed.
2.542 l3C-NMR Spectrum
The Cwbn-13 NMR spectrum o f Salicylamide have

been determined on Varian FT 80 Spectrometer. The
,.. ....
I '
.... .,,. ,... , . . . ..,.
I I . I I " " I '
0
/ r
A A
, . a0 ZO
1
l . . . . l . . . . l . . . . l . . . . I . . . . I
6.0 $U PPw(I)+R 3.0
1 .
.... I
1 1 . . . 1 . . . ,
20 1.0
I
Fig. 5. 'H-NMR spectrum of salicylamide in

DMSO-D6.
1
6 r
I I I . 1
1 . . . . 1 . . . . 1 . . . . 1 . . . . 1 . . . . 1 . . . . 1 . . . . 1 ,
,
8.0 TO 6.0 5.0 w(r)*.O 35 2.0 t.0 0
Fig. 6. IH-NMR spectrum of salicylamide in

DMSO-D6, showing D 0 exchange.
2
SALICYLAMIDE 53 1
sample w a s d i s s o l v e d i n DMSO-DG i n a tube

with a diameter of 1 0 mm. S p e c t r a l width:
4000 Hz, a c q u i s i t i o n time: 1.023 seconds,
p u l s e width 6 seconds, and number of d a t a
p o i n t s 8192. The noise-decoupled spectrum
i s shown i n f i g u r e [ 7 ] shows seven s i n g l e t s .
The complete o f f - r e s o n w e e spectrum i s shown
i n f i g u r e [ 8 ] and s p e c t r a l assignments a r e
l i s t e d below:
XY0*
6 5
Chemical s h i f t Assignment
( i n ppm r e l a t i v e t o TMS)
114.60 (s) c2
161.31 ( s ) c3
117.39 ( d ) c4
134.16 (a) c5
118.49 ( a ) c6
128.31 ( d ) c7
172.39 ( s ) c=o
s = singlet.
d = doublet.
The assignments a r e i n agreement w i t h r e c e n t l y

published d a t a (11).
3. Synthesis
Although s e v e r a l methods were published f o r t h e s y n t h e s i s

of salicylamide, y e t t h e main r o u t e i s through t h e amino-
l y s i s of methyl or e t h y l s a l i c y l a t e ( 1 2 , 13, 1 4 , 15,
16, 17) as shown i n t h e following scheme:
532
533
534 SALIM A . BABHAIR ET AL.
0 =C-OR 0c , -N H,
4. Stability
Salicylamide i s s t a b l e i n s o l i d s t a t e , however, it i s
recommended t o be s t o r e d a t room temperature i n a dry-
dark container. The hydrolysis of salicylamide under experi-
mental conditions designed t o simulate t h e g a s t r o i n t e s t i -
n a l environment proved t h a t t h e amide bond i s r e s i s t a n t
t o a c i d cleavage (18). The k i n e t i c s and mechanism of
a l k a l i n e hydrolysis was reported and found t o follow
pseudo f i r s t order k i n e t i c s ( 1 9 ) .
5. Metabolism and pharmacokinetics

Salicylamide i s r e a d i l y absorbed from t h e g a s t r o i n t e s t i n a l
t r a c t and d i s t r i b u t e d t o a l l body t i s s u e s but t h e drug
does not bind appreciably t o plasma p r o t e i n s . Several
s t u d i e s had been r e p o r t e d i n l i t e r a t u r e s d e a l i n g with t h e
absorption of salicylamide and i t s plasma concentration ( 2 0 ) ,
( 2 1 ) , (22), ( 2 3 ) . It i s r a p i d l y excreted i n t h e u r i n e
mainly as t h e glucuronide and sulphate conjugates. S a l i -
cylamide i s metabolized i n human e n t i r e l y as shown i n
Scheme 1.
The percentage o f t h e u r i n a r y conjugated m e t a b o l i t e s

v a r i e s according t o animal s p e c i e s ( 2 4 ) , ( 2 5 ) , age ( 2 6 )
and r o u t e of administration ( 2 7 ) and ( 2 8 ) .
SALICYLAMIDE 535
4% I
oc, 9'
Hr Sulphate
6'
Glucuronide
Gen tisamide
Schemei, Metabolic pathways OFsalicylmnide in humans.
Tne proportion of a dose of salicylamide excreted as sul-
phate decreased with i n c r e a s e d doses ( 2 9 ) . Peak serum
l e v e l s are reached between 30 minutes and 1 hour a f t e r
o r a l a d m i n i s t r a t i o n ( 5 ) . I n v e s t i g a t i o n has revealed t h a t
s a l i c y l a m i d e i n h i b i t s t h e a c y l a t i o n of sulphonamides lead-
ing t o i n c r e a s e i n t h e i r c o n c e n t r a t i o n ( 3 0 ) .
6.1 Non-aqueous T i t r a t i o n
Rhodes e t al (31) determined a mixture containing ace-
t y l s a l i c y l i c a c i d , acetaminophen and salicylamide,
p o t e n t i o m e t r i c a l l y by non-aqueous t i t r a t i o n . The tit-
r a n t w a s 0 . 1 N tetrabutylammonium hydroxide i n benzene/
methanol mixture and t h e t i t r a t i o n solvent w a s dimethyl-
formamide. The d i f f e r e n c e i n t h e pKa values f o r t h e s e
weak a c i d i c drugs ( a s p i r i n pKa 3.5, acetaminophen pKa
8.31) w a s s u f f i c i e n t t o permit u s e f u l d i f f e r e n t i a t i o n .
Walash and R i z k ( 3 2 ) r e p o r t e d a non-aqueous t i t r a t i o n

o f several a n a l g e s i c s i n dosage form including s a l i c y l a -
mide i n t e t r a m e t h y l u r e a using 0.1 N sodium methoxide as
t h e t i t r a n t , t h e end point w a s measured e i t h e r p o t e n t i o -
m e t r i c a l l y or with thymol b l u e as i n d i c a t o r . The r e -
sults were comparable with t h o s e obtained using dimethyl-
formamide as a s o l v e n t .
536 SALIM A. BABHAIR ET A t .
6.2 Colorimetric Analysis
Rutkowski ( 3 3 ) r e p o r t e d t h e simultaneous c o l o r i m e t r i c
assay of salicylamide, s a l i c y l i c a c i d and g e n e t i s i c
a c i d i n pharmaceutical p r e p a r a t i o n s . These compounds
g i v e b l u e c o l o r complexes with f e r r i c s a l t ( n i t r a t e i n
ethanol s o l u t i o n ) . The b l u e c o l o r complex of s a l i c y l -
amide i s e x t r a c t e d with e t h e r while t h e o t h e r s a r e n o t .
Another method r e p o r t e d by Souza (34) for t h e determi-

n a t i o n of s e v e r a l phenols i n c l u d i n g salicylamide and
aminopyrazoles i n pharmaceutical products, where a solu-
t i o n of 2% potassium f e r r i c y a n i d e g i v e s a colored indo-
phenol d e r i v a t i v e which i s measured a t 500 nm.
6.3 Spectrophotometric Analysis
Salicylamide had been estimated spectrophotometrically

as a s i n g l e component, i n complex pharmaceutical mix-
t u r e and a n a l g e s i c t a b l e t s by s e v e r a l procedures among
them a r e t h e following:
a ) The treatment of salicylamide with chloramine a t pH

8.00 i n t h e presence of thiobromine sodium s a l i c y -
l a t e , c a f f e i n e , s a l i c y l i c a c i d , acetaminophen, and
methanamine. The method could be used t o determine
as low as 0 . 1 mg o f salicylamide i n t h e mixture ( 3 5 ) .
b ) The mixture of salicylamide, papaverine H C 1 , pheno-

b a r b i t a l and g l y c e r y l g u a i a c o l a t e i n glycol-glycerol
aqueous s o l u t i o n w a s determined i n 0 . 1 M H2S04 at
250, 273 and 299 nm ( 3 6 ) -
c ) Soliman and Salaheldin ( 3 7 ) r e p o r t e d a simple r a p i d
method f o r r o u t i n e a n a l y s i s of salicylamide i n anal-
g e s i c t a b l e t containing acetaminophen, phenobarbital,
c a f f e i n e , codeine phosphate, ascorbic a c i d , chloro-
amine phosphate and prednisone. The method does not
r e q u i r e preliminary s e p a r a t i o n of salicylamide from
o t h e r c o n s t i t u e n t s p r i o r t o determination. The
absorbance w a s l i n e a r f o r concentration of s a l i -
cylami.de up t o 4 mg/lOO r r i i s o l u t i o n a t 308 nm.
d ) Salicylamide may be estimated as s a l i c y l i c a c i d i n

b i o l o g i c a l f l u i d s following t h e a d d i t i o n of T r i n d e r ' s
reagent ( 3 8 ) o r a f t e r e x t r a c t i o n i n t o 0.4% malonic
acid i n butyl ether (39).
SALICYLAMIDE 537
6.4 Spectrophotofluorimetric Analysis

The spectrophotoflourimetric determination of s a l i c y l -
amide i n b i o l o g i c a l f l u i d s (blood, serum and u r i n e ) w a s
r e p o r t e d by Vercsh e t a1 ( 4 0 ) . The procedure involves
t h e simultaneous determination of salicylamide and s a l i -
c y l i c a c i d a t pH 11. The method i s s e n s i t i v e and cap-
a b l e of estimating a c o n c e n t r a t i o n of salicylamide and/
o r i t s m e t a b o l i t e s as low as 0 . 1 pg/ml blood, serum o r
o t h e r b i o l o g i c a l fluids. The method may a l s o be used
i n monitoring t h e b i o a v a i l a b i l i t y o f salicylamide pro-
vided i n d i f f e r e n t dosage forms. The fluorescence of
salicylamide w a s measured d i r e c t l y a t maximum a c t i v a -
t i o n and e m i s s i o n wavelengths of 340 and 430 nm res-
p e c t i v e l y while f o r s a l i c y l i c a c i d a t 310 and 435 nm
r e s p e c t i v e l y . Lange e t a1 ( 4 1 ) employed t h e f l o u r e s -
cent p r o p e r t y of salicylamide i n b a s i c s o l u t i o n t o
determine t h e drug and o t h e r s a l i c y l a t e s following t h e
g e l f i l t r a t i o n separation.
Barr and Riegelman ( 4 2 ) d e t a i l e d a s p e c t r o f l o u r i m e t r i c

procedure f o r t h e determination of salicylamide and i t s
m e t a b o l i t e s i n b i o l o g i c a l f l u i d s following h y d r o l y s i s
as described by Levy and Matsuzawa ( 2 9 ) . The method
involves t h e h y d r o l y s i s of a l l m e t a b o l i t e s t o s a l i c y l a t e
and determining them at emission t o e x c i t a t i o n wave-
l e n g t h of 350 and 430 nm r e s p e c t i v e l y . T o t a l s a l i c y l a -
mide w a s determined by hydrolysing a l l m e t a b o l i t e s t o
s a l i c y l i c a c i d i n aqueous b a s i c s o l u t i o n a t a maximum
a c t i v a t i o n and e m i s s i o n wavelengths of 350 and
430 nm r e s p e c t i v e l y .
Recently, S t r e e t and Schenk ( 4 3 ) reported a s p e c t r o f l u o -

r i m e t r i c a n a l y s i s of salicylamide i n t h e presence of
a c e t y l s a l i c y l i c a c i d and s a l i c y l i c a c i d as i m p u r i t i e s
by d i r e c t and i n d i r e c t methods. S a l i c y l i c a c i d i s
determined i n t h e range of 10-7M concentration a f t e r
s e p a r a t i o n from salicylamide. The instrumental condi-
t i o n s f o r t h e s p e c t r o f l u o r i m e t r i c determination of
salicylamide, a c e t y l s a l i c y l i c a c i d and s a l i c y l i c a c i d
i s shown i n Table 1.
538 SALIM A. BABHAIR ET AL
Table 1
Ingredient Wavelength nm S l i t Width nm

determined
Excita- -is- Excita- -is-
tion sion tion sion
Salicylamide
i n ethanol. 300 42 5 10 10
i n c,hloroform 315 445 10 20
Acetylsalicylic acid 280 335 10 20

Salicylic acid 315 445 10 10
The presence of o t h e r medicinals such as phenacetin,

ascorbic a c i d , methapyreline hydrochloride among o t h e r
a n a l g e s i c s does not a f f e c t t h e determinations of
salicylamide, a c e t y l s a l i c y l i c a c i d and s a l i c y l i c a c i d .
6.5 I n f r a r e d Spectrophotometric Analysis

Salicylamide i n mixed pharmaceutical p r e p a r a t i o n s w a s
determined by i n f r a r e d spectrophotometry using t h e
amide absorption a t 3425 cm-l as t h e key band ( 4 4 ) .
The absorbance measured with a base l i n e at 3300 em-l
o r 3470 cm-l. The c a l i b r a t i o n curves were l i n e a r i n t h e
c o n c e n t r a t i o n range between 0.5 t o 2 mg/ml and t h e
r e s u l t s were i n good agreement with t h o s e determined
c o l o r i m e t r i c a l l y . The determination w a s not i n t e r f e r r e d
w i t h t h e coexistance of a l i m i t e d amount of 40 p o s s i b l e
drug c o n s t i t u e n t s e.g., phenacetin and p y r a b i t a l ( 4 4 ) .
Another I R spectrophotometric method for t h e determina-

t i o n of salicylamide among o t h e r medicinally used
amides w a s reported ( 4 5 ) . The amide carbonyl absorp-
t i o n band a t 1732 cm-I w a s used as t h e b a s i s of quan-
t i t a t i v e assay.
6.6 Chromatographic Analysis

6.61 Paper Chromatography
1) Clarke ( 5 ) described a method f o r t h e separa-
t i o n of s a l i c y l a t e s using t h e following t.wo
systems :
SALICYLAMIDE 539
a ) Strong ammonia: n- butanol: water 33:100:66

by descending technique. The l o c a l i z i n g
agent used w a s f e r r i c c h l o r i d e spray o r ul-
t r a v i o l e t l i g h t a t 254 mu..
b ) Phosphate b u f f e r (pH 7.4) and d e t e c t i n g agent
bromine-starch potassium i o d i d e .
2 ) Other method was described by Wagner ( 4 6 ) f o r

t h e a n a l y s i s and d e t e c t i o n of s a l i c y l i c a c i d
and d e r i v a t i v e s including salicylamide. Deve-
lopment w a s e f f e c t e d by using c o l o r r e a c t i o n s ,
w i t h f e r r i c ammonium s u l p h a t e , ammoniacal s i l -
ver n i t r a t e s o l u t i o n , potassium n i t r a t e , HC1
and 5% KOH through d i a z o t i z a t i o n and coupling
with, 2-naphthol and coupling with d i a z o t i z e d
sulphanilic acid.
3 ) It w a s r e p o r t e d ( 4 7 ) t h a t salicylamide and o t h e r
aromatic hydroxy carboxylic a c i d amides under-
go autooxidation, when chromatographed i n
a l k a l i n e media. This autooxidation products
hinder t h e development of t h e complex colora-
t i o n with f e r r i c ion. However, paper chromato-
graphic s e p a r a t i o n of salicylamide and analogs
i n a l k a l i n e b u f f e r media can be accomplished by
ionophoresis ( 4 8 ) .
6.62 Column Chromatography
Column chromatography has been a p p l i e d t o s e p a r a t e

and analyse a mixture of salicylamide and a c e t -
minophen i n t a b l e t s and capsules ( 4 9 ) . Strongly
a c i d i c and b a s i c c e l i t e columns a r e used t o sepa-
r a t e acetaminophen and salicylamide r e s p e c t i v e l y .
Q u a n t i t a t i o n i s performed using u l t r a v i o l e t
spectrophotometry .
Cataionic and anionic ion-exchange r e s i n s ( 5 0 ) were

used as s t a t i o n a r y phases f o r t h e s e p a r a t i o n of
s e v e r a l analgesic drugs including salicylamide
using aqueous ethanol as t h e mobile phase.
6.63 Thin Layer Chromatography
S e v e r a l r e p o r t s have been published on t h e i d e n t i -

f i c a t i o n and s e p a r a t i o n o f salicylamide which a r e
summarized i n Table 2.
Table 2: Thin Layer Chromatography of Salicylamide.
S t a t i o n a r y Phase Solvent System Detecting Agent References

'A
0
P
1) S i l i c a g e l Butyl a c e t a t e : Chloroform : 85% 5% s o l u t i o n o f f e r r i c c h l o r i d e i n (51)

formic a c i d (6:4:2) acetone followed 1% a l c o h o l i c
o-phenanthroline.
2 ) Polyamide a ) Isopropanol : Water : 90% f o r - -
resin mic acid. ( 1 . 5 : 6 : 0.1)
b ) Chloroform : benzene : 90% Formic
a c i d . ( 5 : 1 : 01).
c ) Chloroform : 90% Formic a c i d .
(20 : 0.1).
d) Cyclohexane : Chloroform :
a c e t i c a c i d . ( 4 : 5 : 1).
3 ) SGF p l a t e s Benzene : Acetic a c i d : Methanol U l t r a v i o l e t l i g h t 254 nm. (53)
Chloroform : Petroleum e t h e r
( 3 0 - 7 0 ' ) (10:5:5:10:70).
4) Silica Methanol 10% Copper s u l p h a t e , 2% ammonium (54)
g e l G. hydroxide, d a r k yellow c o l o r .
5) S i l i c a gel a ) Chloroform : E t h e r : Acetone F e r r i c c h l o r i d e and 2,6-dibromo- (55)
( 6 0 : 20 : 2 0 ) . quinone-4-chloroimide.
b ) Chloroform : Benzene : Acetone
( 6 0 : 20 : 2 0 ) .
6) S i l i c a A c e t i c a c i d : Carbon t e t r a c h l o r i d e :
g e l G. Chloroform : Water (100:60:90:50)
Kieselguhr G a ) Benzene : Chloroform (30:120) s a t u -

impregnated r a t e d w i t h formamide.
with
f ormamide .
'A b ) Carbon t e t r a c h l o r i d e s a t u r a t e d
-
c.
w i t h f ormamide .
6.64 Gas Liquid Chromatography

Various gas l i q u i d chromatographic methods have
been developed f o r t h e i d e n t i f i c a t i o n and determi-
n a t i o n of salicylamide i n pharmaceuticals and bio-
l o g i c a l f l u i d s . It i s of i n t e r e s t t o mention t h a t
salicylamide could be gas chromatographed without
d e r i v a t i z a t i o n . Some of t h e d a t a b t a i n e d from
t h e l i t e r a t u r e a r e summarized i n Table 3.
The a n a l y s i s of s e v e r a l drugs including s a l i c y l -

a i d e using semiatoumated gas l i q u i d chromato-
graphy has been r e p o r t e d ( 5 7 ) .
Nieminen (58) described a gas chromatographic

method f o r simultaneous s e p a r a t i o n and q u a n t i t a -
t i o n of some a n t i p y r e t i c s including salicylamide
containing phenobarbital, c a f f e i n e and codeine
phosphate. Codeine phosphate w a s f i r s t separated
from t h e samples as t h e base. Although t h e separa-
t i o n w a s e x c e l l e n t f o r a l l components , y e t a c e t y l -
s a l i c y l i c a c i d i n t e r f e r e s with t h e s e p a r a t i o n of
sa1icylami.de.
6.65 High-pressure Liquid Chromatography

High-pressure l i q u i d chromatography has been u t i -
l i z e d f o r t h e d e t e m i n a t i o n of salicylamide i n
blood, serum and o t h e r b i o l o g i c a l f l u i d s as w e l l
as pharmaceutical formulations. Some of r e c e n t l y
reported methods are summarized i n Table 4.
6.7 Radiochemical Analysis

The radiochemical a n a l y s i s of salicylamide i n plasma
using t h e r i n g - l a b e l l e d t r i t i a t e d compound was r e p o r t e d
by S t e l l a e t al ( 5 9 ) . The t r i t i a t e d salicylamide w a s
synthesized by r e a c t i n g c o l d salicylamide with t r i t u i m
oxide i n t h e presence o f h e p t a f l u o r o b u t y r i c a c i d . This
procedure i s e f f e c t i v e f o r t h e a n a l y s i s of salicylamide
and i t s m e t a b o l i t e s ( t h e s u l p h a t e and glucuronide) i n
t h e presence of s i m i l a r phenolic compounds.
6.8 Flame m i s s i o n Spectrometry

Dialonzo and S i g g i a r e p o r t e d a method f o r determining
several compounds including salicylamide based on t h e
use of Hofmann r e a c t i o n f o r t h e s y n t h e s i s of m i n e s
Table 3: Gas Chromatography o f Salicylamide
Column Carrier Gas Detector Remarks Reference
27% OV-225 Helium Flame Quantitation was carried out by (60)

+1% OV-17 ionization combined GC/MS.
Gas Chrom-Q Sensitivity ranges from 1 to 25 u g / d
(whole blood plasma and saliva) and
5-100 ug/50 ul urine.
1% Silicon Helium Thermal Applied for simultaneous determination of (61)

Gum SE-30 on conducting dextromethorphan, chlorpheniramine nore-
Chromosorb-G phedrine and salicylamide.
8% OV-101 - Flame ioni- Simultaneous analysis of salicylamide, (62)

zation phenylpropanolamine HC1, caffeine, chlor-
pheniramine maleate phenylephrine HC1 and
pyrilamine maleate, the sample should be
treated with b-(dimethylamino)-pyridine in
pyridine-acetic anhydride 1:l.
2% SP-2501 Helium Flame ioni- Detection limits 5 mg/l. (63)

DA zation.
(continued)
Table 3 (continued)
Column C a r r i e r Gas Detector Remarks. Reference
Mixture of Helium Flane ioniza- Detection l i m i t s 5 mg/l. (63)

g:1 2% t ion.
2 methyl/phenyl
P
s i l i c o n (SP
2110) and 1%
SP 2510 on
100-120 mesh
a c i d washed
dimet h y l d i -
ch l o r o s i l a n e
t r e a t e d dia-
t o m i t e support.
Table 4: HPLC for Salicylamide
Column Mobile Phase Detect i o n Remarks Reference
RPz Methanol : Water w L i m i t of d e t e c t a b i l i t y i s 0 . 2 Vg/ml. (64)

(60: 40) Caffeine and paracetamol do not i n t e r f e r e
i n t h e determination of salicylamide.
RP-5c18 Methano1:Buffer pH 3 w Method i s s u i t a b l e f o r c l i n i c a l determina- (65
(10: 50) n a t i o n and t o x i c o l o g i c a l use.
0 . 0 1 M potassium a c i d uv Simultaneous q u a n t i t a t i o n of acetaminophen, (66
phosphate i n w a t e r a s p i r i n , c a f f e i n e , codeine phosphate,
with 19% v/v methanol, phenacetin and salicylamide i s a f f e c t e d .
pH w a s a d j u s t e d
t o 2 . 3 with 80% aq.
phosphonic a c i d Solvent system with pH 2 . 3 a f f e c t s b e t t e r
s o l u t ion. separation.
Same as above except Reverse-HPLC technique w a s used. (66)
pH w a s a d j u s t e d t o 4.85
1% a c e t i c a c i d i n 25% uv LOW concentration of unconjugated s a l i c y l a - (67)
v/v methanol / w a t e r mide i n human serum and s a l i v a were
solution. assayed.
3 mM t e t r a b u t y l - w Reverse phase ion-pair chromatography (67)
ammonium hydroxide i n i s used.
a mixture of 8 p a r t s
methanol and 92 p a r t s Salicylamide and i t s conjugated m e t a b o l i t e s
v/v a c e t i c a c i d 7%. were assayed.
(continued)
Table 4 (continued)
Column Mobile Phase Detect ion Remarks Reference
p-Bondapak 1% Acetic a c i d i n W G e n t i s a i d e and i t s conjugates i s (67)

'18 15% v/v methanol/Water/ assayed.
solution,
f-
-Bondapak 0.15 M Sodium Flourometric Can d e t e c t salicylamide as l o w as (68)

oDS-cl8 d i b a s i c phosphate detector 5 m g / d i n plasma.
pH 8 : Methanol
( 7 0 : 30).
LFS P e l l i - One molar T r i s w Applied f o r r o u t i n e s e p a r a t i o n of (69)

cuar anion- (pH 9 ) a t 60' common components o f a n a l g e s i c
exchange tablets.
resin.
SALICYLAMIDE 541
w i t h one l e s s of carbon atom t h a n t h e s t a r t i n g amide.

B a r i u m n i t r a t e i s used as reagent. B a r i u m carbonate
formed i n t h e r e a c t i o n i s d i s s o l v e d i n n i t r i c a c i d and
i s determined by flame atomic emission spectroscopy;
t h e p r e c i s i o n w a s t 1 . 5 t o 6.5% i n t h e range 1 t o 4 mM
(4 t o 9 r e p l i c a t e r e s u l t s f o r each compound). The mean
r e c o v e r i e s were g e n e r a l l y between 2 95 and 105% (70).
6.9 Thermornicroscopic Method

Analysis o f t e r n a r y mixtures of c e r t a i n drugs includ-
ing salicylamide were s t u d i e d by p l o t t i n g t h e r e f r a c -
t i v e index isotherms of t h e fused m a s s and t h e
isotherms of t h e primary c r y s t a l s on t r i a n g u l a r con-
c e n t r a t i o n diagrams. The i n f l u e n c e of each f a c t o r
upon t h e q u a n t i t a t i v e determination could be found. I n
each case t h e r e w a s an a r e a u n s u i t a b l e f o r measurement
where t h e isotherms were p r a c t i c a l l y p a r a l l e l ; when
t h i s w a s not t h e c a s e , q u a n t i t a t i v e determination w a s
p o s s i b l e . Precaution should be taken t o prevent l o s s
of highly v o l a t i l e components ( 7 1 ) .
6.10 The ring-oven Technique.

Salicylamide w a s determined a t concentrations of 10-
400 ng w i t h a m a x i m u m e r r o r o f 7.656 by t h e ring-oven
technique. Drugs such as paracetamol and c a f f e i n e
d i d not i n t e r f e r e i n t h e determination ( 7 2 ) .
Acknowledgement
The a u t h o r s uould l i k e t o thank Mr. Altar H . Naqvi f o r

t y p i n g t h e manuscript.
548 SALIM A . BABHAIR ET AL.
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S ALICYLAMIDE 549
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SALlCY LAMlDE 55 1
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M. Hanif, S . Parveen, F. Jamshaid, S. Shahnawaz, Z . Sheikh,

Chem.
J. -
- SOC. Pak., 2, 67 (1980).
SILVER SULFADIAZINE
Auke Bult and Cees M. Plug
1. History 554
2. Description 554
2.1 Name, Formula, Molecular Weight 554
2.2 Appearance, Colour, Odour 554
3 . Synthesis 554
4.2 Nuclear Magnetic Resonance (NMR) Spectrum 557
4.5 Melting Range 560
4.6 Differential Scanning Calorimetry 560
4.7 Crystal Structure and X-Ray Powder Diffraction 562
4.8 Stability Constant 563
4.9 Conductivity 564
4.10 Solubility 565
5 . Methods of Analysis 566
5.2 Identification Tests 566
5.3 Purity Tests 567
5.4 Volumetric Methods 561
5.5 Spectroscopic Methods 568
5.6 Thin Layer Chromatography 568
5.7 High Performance Liquid chromatography 568
6 . Stability 569
7. Pharmacology 569
8. Identification and Determination in Body Fluids and Pharmaceuticals 569
8.1 Determination in Body Fluids 569
8.2 Identification and Determination in Pharmaceuticals 570
References 570

ISBN 0-12-2608 13-5
554 AUKE BULT AND CEES M . PLUG
1. HISTORY
Silver sulfadiazine was first described in 1943 by Wruble
(1) and was found to be mildly antiseptic. Fox (2)rediscovered
the compound in 1968 in his search for a suitable substance for
topical treatment of burns. His philosophy was to combine the
oligodynamic effect of the silver ion with the antibacterial
activity of sulfadiazine. The compound has been in clinical use
in the USA since 1973 and appears to be active against a wide
range of micro-organisms (3). The usual application form is a
1% w/w oil-water cream.
2. DESCRIPTION
2.1 Name, Formula, Molecular Weight
Generic name - Silver sulfadiazine
Nomenclature -
The following nomenclature is used in
Chemical Abstracts:
monosilver(1) salt of 4-amino-N-2-pyrimidinylbenzenesulfon-
amide 122199-08-21
Synonyms - Silver compound (salt) of N1-2-pyrimidinyl-
sulfanilamide
Structure
Molecular Weight: 357.14
2.2 Appearance, Colour, Odour

White or creamy-white micro-crystalline powder which
is odourless or almost odourless. It is stable in air but
slowly becomes yellow on exposure to light.
3. SYNTHESIS
The synthesis of silver sulfadiazine is based on the
AgN03 + NaSD -
following reaction scheme (1,4):
AgSD+ + NaN03
Mixing of equimolar amounts of silver nitrate and sodium sulfa-
diazine (NaSD), both dissolved in water, results in the almost
SILVER SULFADIAZINE 555
q u a n t i t a t i v e p r e c i p i t a t i o n of s i l v e r s u l f a d i a z i n e . An a l t e r n a -
t i v e procedure makes u s e of ammoniacal s o l u t i o n s of s i l v e r
n i t r a t e and s u l f a d i a z i n e ( 5 ) . R e c r y s t a l l i z a t i o n o f t h e compound
can t a k e p l a c e from 25% ammonia s o l u t i o n ( 4 ) .
D e r i v a t i v e s of s i l v e r s u l f a d i a z i n e can be prepared accord-
ing to:
AgSD + nY- AgSD.Yn
Examples of such d e r i v a t i v e s a r e : Y = m o r f o l i n e , n = 1 (6) and
Y = imidazole, n = 2 ( 7 ) .
4. PHYSICAL PROPERTIES
4.1 Infrared Spectrum
The i n f r a r e d spectrum g i v e n i n F i g u r e 1 w a s determined f o r
a KBr p e l l e t p r e p a r a t i o n of s i l v e r s u l f a d i a z i n e w i t h t h e u s e o f
a Beckman I R 1 0 s p e c t r o m e t e r . The assignments f o r i m p o r t a n t ab-
s o r p t i o n bands are p r e s e n t e d - as f a r as p o s s i b l e - i n Table I.
The assignments a r e based on v a r i o u s l i t e r a t u r e r e f e r e n c e s (4,
8,s). I n p r a c t i c e i t i s found t h a t d i f f e r e n t b a t c h e s of s i l v e r
s u l f a d i a z i n e do produce s l i g h t l y d i f f e r e n t I R s p e c t r a , w h i l e
t h e batches assay closely t o the t h e o r e t i c a l values (4,5,9).
The d i f f e r e n c e s o c c u r i n t h e r e g i o n s above 3000 c m - l , between
1630 and 1660 c m - l , n e a r 1600 cm-l and 1360 c m - l , and between
1200 and 1300 cm-I and 650 and 750 cm-I ( 9 ) . The SO2 s t r e t c h i n g
v i b r a t i o n s a r e known t o show a m u l t i p l e s t r u c t u r e w i t h sub-
bands on t h e s i d e s of t h e main peaks u s u a l l y ( 1 0 ) . The o r i g i n
of t h e s e sub-bands i s n o t clear. Wysor and S c o v i l l (5) d i s t i n -
g u i s h e d two forms of s i l v e r s u l f a d i a z i n e based u p o n d i f f e r e n c e s
i n t h e I R s p e c t r a . Both forms of t h e compound were a c t i v e
Table 1. Infrared Assignments for Silver Sulfadiazine

~~ ~
Wavenumber (cm-I) Assignment
3390
3340
1655 6 (NH2)
1595 phenyl s k e l e t a l v i b r a t ' i o n s
1500 phenyl s k e l e t a l v i b r a t i o n s
1560 pyrimidine s k e l e t a l vibration
1230 V a s y m (SO2)
1125 vsym (332)
1070 aromatic v i b r a t i o n
0
0
m
0
0
U
0
0
u)
0
0
03-
I
0 5
0
Z L0,
n
E
8 2
Pa2
>
0 ;
<
7
0
0
e
0
0
2
0
0
0
hl
0
0
m
hl
$4
a,
0 ?
.o 4
0 -4
m rn
w
0
0
0
Ln
m -cI
0
0
0
U
SILVER SULFADlAZINE 557
a g a i n s t b a c t e r i a , b u t only one form was a c t i v e a g a i n s t trypano-

somes. Evidence of polymorphism was n o t found w i t h t h e u s u a l
p h y s i c a l methods. The two t y p e s of s i l v e r s u l f a d i a z i n e r e s u l t e d
from s y n t h e s i s a c c o r d i n g t o a p p a r e n t l y i d e n t i c a l methods.
4.2 Nuclear Magnetic Resonance (NMR) Spectrum

The p r o t o n NMR spectrum was r e c o r d e d i n DMSO-db c o n t a i n i n g
t e t r a m e t h y l s i l a n e as i n t e r n a l r e f e r e n c e and w i t h t h e u s e of a
Bruker WM-300 s p e c t r o m e t e r a t frequency 300.13 MHz. The spec-
trum i s p r e s e n t e d i n F i g u r e 2 and t h e s p e c t r a l assignments are
summarized i n T a b l e I1 (11). The chemical s h i f t s roughly a g r e e
w i t h t h o s e r e p o r t e d f o r s u l f a d i a z i n e ( 1 2 ) . The change i n chemi-
c a l s h i f t s ( t o h i g h f i e l d ) f o r s i l v e r s u l f a d i a z i n e compared t o
s u l f a d i a z i n e i s 0 . 3 ppm (NH2) o r less ( 1 1 ) .
The n a t u r a l abundance carbon-13 NMR spectrum was r e c o r d e d
under t h e same e x p e r i m e n t a l c o n d i t i o n s e x c e p t t h a t t h e frequen-
cy was 75.46 MHz. Because of t h e low s o l u b i l i t y of s i l v e r s u l -
f a d i a z i n e i n DMSO-d6 an i n c o m p l e t e spectrum was o b t a i n e d . The
chemical s h i f t s observed i n t h e p r o t o n - n o i s e d e c o u p l e d s p e c t r u m
are g i v e n i n T a b l e I1 ( 1 1 ) . Again t h e chemical s h i f t s of s i l v e r
s u l f a d i a z i n e compared t o s u l f a d i a z i n e are s h i f t e d s l i g h t l y t o
h i g h f i e l d , e.g. 4.5ppm and less ( 1 1). Carbon-13 NMR d a t a of s u l f a -
d i a z i n e are r e p o r t e d by Chang e t a l . (13).
Table 11. lH and I 3 C NMR Assignments for Silver S u l f a -

di azi ne
Chemical s h i f t Multiplicity Number of Assignment

6 (ppd atoms
5.69 singlet 2 NH2

6.52 doublet 2 H- 4
6.81 triplet 1 H- 1
7.65 doublet 2 H- 3
8.42 doublet 2 H-2
111.2 1 c-4 '
111.8 2 c- 3
129.6 2 c- 2
151.4 1 c-4
159.0 2 c-3'
7 u
a)
a
v
Ll
a)
?
rl

The ultraviolet spectra for silver sulfadiazine were deter-
mined in very dilute aqueous ammonia solutions. One set of data
was obtained with 0.0010% silver sulfadiazine in 0.005% NH3
1 .o
solvent: 0.005% NH3
concentration : 0.001 %
pathlength: 1.0 cm
08
06
OL
02
220 2LO 260 280 300 320 3LO 360 380

wavelength ( n m )
Figure 3
W spectrum of silver sulfadiazine
solution and recorded with an Uvikon 810/820 spectrophotometer

(14); the second set of data was obtained with 0.0015% silver
sulfadiazine in 0.05% NH3 solution and recorded with an Unicam
SP 800 spectrophotometer (9).
A summary of the data is presented in Table 111. The data
in 0.005% NH3 solution are the average values of three samples
of silver sulfadiazine from different origin and/or batches
(14).
The ultraviolet spectrum for 0.001% silver sulfadiazine in
0.005% NH3 solution is presented in Figure 3 (14).
560 AUKE BULT AND CEES M . PLUG
Table I I I . U1 t r a v i 01 e t Spectral Val ues f o r Si 1ver

Sulfadiazine
0.005% NH3 24 1 616 22,000

254.5 614 21,930
0.05% N H 3 240 62 1 22,170
254 617 22 ,050
4.4 Mass Spectrum

The mass spectrum of s i l v e r s u l f a d i a z i n e ( F i g u r e 4 ) was
o b t a i n e d w i t h a K r a t o s MS 9/50 m a s s s p e c t r o m e t e r i n t h e elec-
t r o n impact mode w i t h an i o n i z i n g energy of 70 e V and equipped
w i t h a d i r e c t i n l e t p r o b e , probe t e m p e r a t u r e 25OoC (15).
The b a s e peak i s found a t m / e 185, w h i l e t h e m o l e c u l a r i o n
peak i s v e r y weak, v i a . m / e 250, r e l a t i v e i n t e n s i t y 0.3%.
The m a s s spectrum of s i l v e r s u l f a d i a z i n e corresponds i n peak
p o s i t i o n s and r e l a t i v e i n t e n s i t i e s w i t h t h e spectrum of s u l f a -
d i a z i n e (12). A peak c o r r e s p o n d i n g w i t h Ag’ (m/e 108) , i f
p r e s e n t , i s masked by a r e l a t i v e l y i n t e n s i v e peak of s u l f a -
diazine.
4.5 Me1 t i n g Range

S i l v e r s u l f a d i a z i n e m e l t s w i t h decomposition. A summary of
t h e d a t a o b t a i n e d from t h e l i t e r a t u r e i s l i s t e d below.
Me l t i n g Range/Point (“C) Reference
27 1 (8)
277 (5)
286-288 (6)
286-290 (9)
290 (16)
4.6 D i f f e r e n t i a l Scanning Calorimetry

The DSC thermogram f o r s i l v e r s u l f a d i a z i n e ( F i g u r e 5 ) w a s
determined w i t h a Mettler TA 3000 equipped w i t h a DSC 30
measuring c e l l and alumina pan w i t h p i e r c e d l i d sample h o l d e r .
The sample s i z e w a s a b o u t 3 mg and t h e h e a t i n g r a t e was5K/min
(17)
I n a i r an e x o t h e r m i c double-peak a p p e a r s a t about 29OoC.
In n i t r o g e n o r h e l i u m atmosphere an endothermic peak a p p e a r s
between about 283 and 3 O O 0 C , b u t a g a i n an exothermic peak
m
a,
3
r(
rd
P
0 0
0 -N a,
U N '
E
5
0
Q)
-g '5
0 N m
0
u3
m
0
52
0
U
m
0 0
,N U
m 7
0
0
m m
07-
N
0
Q)
N
0 0
u3 03
N
0
ID-
N
0
' U
N
562 AUKE BULT AND CEES M. PLUG
0
X
W
0
n
Z
W
I I 1 I I
2 60 2 70 2 80 2 90 3 00
Temperature ('C)
Figure 5
DSC thermogram of silver sulfadiazine
occurs at about 2 9 O o C . The exothermic effect is not found with

sulfadiazine or sodium sulfadiazine and is most probably con-
nected with a chemical reaction of silver or catalyzed by
silver. The endothermic process coincides with the melting
range of silver sulfadiazine.
4.7 Crystal Structure and X-ray Powder Diffraction

Two independent single-crystal structure determinations of
silver sulfadiazine have been reported (18,19) which mutually
agree in the description of the main features of the structure.
Each silver atom is coordinated by three separate sulfadiazine
moieties, resulting in a distorted tetrahedral coordination by
6&
10 - S - N
' 0
1
1
- Ag
/ 31,
-N \
Figure 6
C r y s t a l s t r u c t u r e of s i l v e r
s u l f a d i a z i n e ( 18)
t h r e e N-atoms and one 0-atom ( F i g u r e 6 ) . The c r y s t a l d a t a are

given i n Table I V .
Table IV. Crystal Data for Silver Sulfadiazine
Space group PZ1/c ( r e f . 19) PZ1/c ( r e f . 18)
a (8) 6.173(2) 6.172(5)

b (1) 9.600 (5) 9.605 (8)
c (8) 20.30 (2) 20.33(2)
B ("1 96.22(8) 96.60(8)
The X-ray powder d i f f r a c t i o n p a t t e r n was recorded by means of

a P h i l i p s d i f f r a c t o m e t e r , t y p e PW 1025125, u s i n g Cu-Ka r a d i a -
t i o n ( A = 1.5418 8 ) . The r e s u l t s are i n good agreement w i t h t h e
r e p o r t e d s i n g l e - c r y s t a l d a t a . A s t r o n g r e f l e c t i o n w a s observed
a t 20 = 10.2'. A l l o t h e r r e f l e c t i o n s have an i n t e n s i t y less t h a n
20% r e l a t i v e t o t h i s s t r o n g (011) r e f l e c t i o n . The d a t a f o r t h e
major l i n e s i n t h e d i f f r a c t i o n p a t t e r n of s i l v e r s u l f a d i a z i n e
are given i n T a b l e V .
4.8 Stability Constant

The thermodynamic s t a b i 1i t y cons t a n t of s i l v e r s u l f a d i a z i n e
i n w a t e r has been measured by Boelema e t a l . (20). They made
use of a microcomputer-controlled t i t r a t o r and measured simul-
t a n e o u s l y t h e pH v a l u e w i t h a Radiometer G 2 0 4 0 C g l a s s elec-
t r o d e and t h e s i l v e r i o n c o n c e n t r a t i o n w i t h an Orion r e s e a r c h
941600 i o n - s e l e c t i v e s i l v e r s u l f i d e e l e c t r o d e . The measured
s t a b i l i t y c o n s t a n t and s t a n d a r d d e v i a t i o n were l o g K = 3 . 6 2 2
0.05; n = 9 , temperature 25OC, i o n i c s t r e n g t h 0.1 (sodium
n i t r a t e ) . The c a l c u l a t e d c o n d i t i o n a l s t a b i l i t y c o n s t a n t logK'
a t pH: 7.4 w a s 3.57.
Table V. X-ray Powder Diffraction Pattern of Silver

Sul f a d i azi ne
*
28 Degrees d (1) IDo
8.80 10.05 20
10.21 8.664 100
16.20 5.505 6
18.17 4.882 3
18.49 4.798 10
19.91 4.459 7
20.05 ( s ) 4.43 2
20.6 ( s ) 4.32 4
20.66 4.299 6
23.67 3.759 5
24.28 3.666 12
25.62 3.477 2
26.69 3.340 2
27.91 3.197 8
28.11 3.174 5
28.85 3.094 4
30.91 2.893 2
31.51 2.839 4
33.20 2.698 9
33.60 2.667 2
34.63 2.590 2
36.06 2.491 2
36.33 2.473 3
37.17 2.419 5
37.44 2.402 5
38.53 2.337 6
*Relative intensity in percentage of the strongest signal.

Reflections with intensities less than 2% have been
excluded.
(s) Shoulder or poorly resolved from adjacent peak.
4.9 Conductivity
Bult and Klasen (4) have performed conductivity measure-
ments of silver sulfadiazine with a Radiometer conductivity
meter, type CDM2d, and conductivity cell, type CDC 104.
The molar conductivity AM in DMSO was 2.2 Q-1.cm2.mole-1
(measured at 1.10-3 M). Considering the standard range of an
1 : 1 electrolyte in DMSO, AM=23-42 (21), the conclusion was
drawn t h a t t h e compound i s almost u n d i s s o c i a t e d i n t h i s

solvent.
4.10 Solubility
The s o l u b i l i t y of s i l v e r s u l f a d i a z i n e has been determined
by e q u i l i b r a t i n g t h e compound i n doubly d i s t i l l e d w a t e r a t 25OC
and t h e measurements of t h e s a t u r a t e d s o l u t i o n f o r s u l f a d i a z i n e
by UV s p e c t r o m e t r y and f o r s i l v e r by atomic a b s o r p t i o n s p e c t r o -
scopy ( 7 ) . N e s b i t t and Sandmann ( 2 2 ) measured t h e e q u i l i b r a t e d
solution f o r s i l v e r using a silver-ion s e l e c t i v e electrode.
T h e i r measurements w e r e performed a t 25OC i n n i t r i c a c i d -
potassium n i t r a t e b u f f e r s (pH 2 - 3 ) and i o n i c s t r e n g t h 0.1.
The d a t a a r e summarized i n Table V I .
Table VI. Solubility o f Silver Sulfadiazine
S o l v e nt Solubility pH Reference
(mg/100 ml)
~ ~~
water 0.34 6.8

water 0.2 --
water, : 0.1 20 2.13
w a t e r , p : 0.1 2.3 3.85
water* 0.19 6
water
* 0.11 7
dime t h y 1 s u l f o x i de >35
10%w/vNH3 s o l u t i o n >2.10
25%w/vNH3 s o l u t i o n >5. lo3
* c a l cu l a t e d
A s can be seen from t h e d a t a , t h e s o l u b i l i t y of s i l v e r
s u l f a d i a z i n e i n c r e a s e s w i t h t h e d e c r e a s e of pH. A s o l u b i l i t y
of 0.34 mg/ 100 m l corresponds w i t h a 1. M solution.
The degree of d i s s o c i a t i o n , a, can be c a l c u l a t e d from t h e
e q u a t i o n K.c = ( 1-a)/a2, where K i s t h e s t a b i l i t y c o n s t a n t and
c i s t h e molar c o n c e n t r a t i o n . For s i l v e r s u l f a d i a z i n e t h e c a l -
c u l a t e d a i s about l a t pH 7 , which means t h a t t h e d i s s o l v e d
f r a c t i o n of s i l v e r s u l f a d i a z i n e i n water i s almost completely
d i s s o c i a t e d i n t o t h e i o n s s i l v e r and s u l f a d i a z i n e . This com-
p l e t e d i s s o c i a t i o n h a s been confirmed by experiment ( 2 2 ) .
The s o l u b i l i t i e s of s i l v e r s u l f a d i a z i n e i n v a r i o u s s o l v e n t s
a r e only known approximately. The compound i s v e r y s l i g h t l y
s o l u b l e i n a c e t o n e , p r a c t i c a l l y i n s o l u b l e i n e t h a n o l , chloro-
form and d i e t h y l e t h e r ( 1 4 ) , f r e e l y s o l u b l e i n 30% ammonia
s o l u t i o n , and decomposes i n moderately s t r o n g m i n e r a l a c i d s (9).
Broad information about the solubility of silver sulfadiazine

in a number of solvents may be derived from identification and
quantitative determination procedures. Some of the solubilities
collected in this way are presented in Table VI.
5. METHODS OF ANALYSIS
5.1 Elemental A n a l y s i s
The results from an elemental analysis of silver sulfadi-
azine are listed below (19).
EZementaZ AnaZysis of SiZver SuZfadiazine

Element % Theory % Found
C 33.63 33.3
H 2.54 1.9
N 15.69 15.7
Ag 30.20 30.22
The assay limits demanded in a draft of a monography on silver

sulfadiazine for the Dutch Pharmacopoeia Ed. IX (14) are: Ag
29.6 - 30.3% and sulfadiazine 6 9 . 1 - 71.2%. Indemans (14)
assayed three different samples of silver sulfadiazine and
found contents within the ranges Ag 29.8-30.1% and sulfadi-
azine 69.3-69.8%. Horlington (9) reported the results of five
batches of the compound: Ag 29.6 - 30.08% and sulfadiazine
69.3-70.0%.
5.2 I d e n t i f i c a t i o n Tests
The identification of silver sulfadiazine is based on the
detection of the silver ion and the sulfadiazine moiety.
5.2.1 SiZver
The compound is dissolved in 35% ammonia solution or in
diluted nitric acid. Upon addition of hydrochloric acid a
curdled, white precipitate is formed. The precipitate dissolves
on addition of 10% ammonia solution.
5.2.2 Sulfadiazine
The identification can be based on the following tests:
a) Detection of the primary aromatic amine group: the compound
is (partly) dissolved into dilute hydrochloric acid. Upon
addition of a 10% sodium nitrite solution, followed after
two minutes by the addition of an alkaline 5% 2-naphtol
solution, an orange or red colour is formed (25).
b) Detection of the 2-aminopyrimidine moiety: the compound is
suspended in a 5% resorcinol solution in ethanol 94% v/v.
Upon addition of 95% sulphuric acid a deep red colour is
formed (26).
c) D e t e c t i o n of s u l f a d i a z i n e : an ammoniacal s o l u t i o n of the
compound shows two a b s o r p t i o n maxima a t 255 nm and 240 nm.
The s p e c i f i c absorbance (Bizcm) a t b o t h m a x i m a i s between
610 and 6 6 0 . The r a t i o of A!%cm ( 2 5 5 nm)/A;%cm ( 2 4 0 nm)
i s 0.97 t o 1.00 ( 1 4 ) .
O t h e r u s e f u l i d e n t i f i c a t i o n methods are t h e I R spectrum and
t h i n l a y e r chromatography. These methods demand t h e a v a i l a b i l i -
t y of a r e f e r e n c e sample of s i l v e r s u l f a d i a z i n e .
5 . 3 Purity Tests
I n a d r a f t of a monography on s i l v e r s u l f a d i a z i n e f o r t h e
Dutch Pharmacopoeia Ed. I X ( 1 4 ) t h e f o l l o w i n g p u r i t y t e s t s are
included.
2.5 gram of t h e f i n e l y ground s i l v e r s u l f a d i a z i n e i s shaken
f o r 5 min. w i t h 50 m l of water. The s o l u t i o n i s f i l t e r e d o f f
and t h e r e s i d u e i s washed w i t h 50 m l of water. The combined
f i l t r a t e i s used f o r t h e t e s t s on pH, n i t r a t e and f r e e s i l v e r .
pH: 4.5 t o 6 . 5 . N i t r a t e : 50.1%; based on c o l o u r f o r m a t i o n w i t h
a 0.05% s o l u t i o n of t h e sodium s a l t of chromotropic a c i d i n
s u l p h u r i c a c i d . Free s i l v e r : 5 100ppm; t u r b i d i t y measurement i n
a hydrochloric acid solution.
O t h e r p u r i t y t e s t s are:
Loss on drying: n o t more t h a n 0.5% a t 100 t o 105OC.
TLC: t h e t h i n l a y e r chromatogram of t h e t e s t e d compound
may n o t d i f f e r from t h e chromatogram of t h e r e f e r e n c e compound;
s t a t i o n a r y phase, s i l i c a g e l GF254R, mobile phase: chloroform -
methanol - ammonia 25% ( 7 0 :40 : 10 v o l . p a r t s ) , s a t u r a t e d chamber,
d e t e c t i o n : 254 nm.
Light-absorbing i m p u r i t i e s : t h e absorbance of a 5% s o l u t i o n
of s i l v e r s u l f a d i a z i n e i n 35% ammonia, measured a t 400 nm, may
n o t exceed 0.20. T h i s demand seems t o b e s h a r p b e c a u s e h d e m a n s
( 1 4 ) r e p o r t e d f o r t h r e e b a t c h e s an absorbance between 0.199 and
0 . 2 7 3 , w h i l e t h e t h r e e b a t c h e s meet t h e o t h e r r e q u i r e m e n t s .
P a r t i c l e s i z e : t h e p a r t i c l e s i z e of t h e h i g h l y i n s o l u b l e
s i l v e r s u l f a d i a z i n e i s thought t o be i m p o r t a n t f o r i t s a v a i l -
a b i l i t y under wound c o n d i t i o n s i n t h e t o p i c a l t r e a t m e n t of
b u r n s . T h e r e f o r e , t h e micronized form i s an i m p o r t a n t q u a l i t y
c r i t e r i o n . A c u r r e n t demand i s t h a t 90% of t h e p a r t i c l e s s h o u l d
measure 10 pm o r l e s s i n g r e a t e s t dimension, 99% n o t o v e r 20pm
and 100% n o t over 50 pm ( 2 4 ) .
5.4 Volumetric Methods

5 . 4 . 1 SiZver Content
S i l v e r s u l f a d i a z i n e i s d i s s o l v e d i n 65% n i t r i c a c i d and t h e
s o l u t i o n i s d i l u t e d w i t h water t o a t e n f o l d volume. The s i l v e r
i s a s s a y e d by t h e Volhard p r o c e d u r e : t i t r a t i o n w i t h thiocya-
n a t e , i n d i c a t o r Fe3'.
5.4.2Sulfadiazine Content
Silver sulfadiazine is dissolved in dilute hydrochloric
acid and determined by titration with sodium nitrite solution
(assay of the primary aromatic amine function). The endpoint
detection is commonly biamperometric (27).
5.5 Spectroscopic Methods

Both silver and sulfadiazine of silver sulfadiazine have
been determined by spectroscopic methods. An appropriate quan-
tity of the compound is dissolved in concentrated ammonia and
diluted with water to the desired concentration.
5.5.1 S i l v e r Content
Determination with atomic absorption spectroscopy with the
use of an acetylene-air flame and hollow-cathode lamp, e.g.
3 UAX/Ag Cathodeon Ltd.; measurement at 328.1 nm (7,9).
5.5.2SuZfadiazine Content
a) Determination with UV absorption spectrophotometry at
254 nm (9,28).
b) Application of the Bratton-Marshall reaction (29):
sulfadiazine is diazotized with sodium nitrite and then
coupled with N-( I-naphthy1)-ethylenediamine dihydrochl0ri.de.
The pink colour is measured at 545 nm (9).
5.6 T h i n Layer Chromatography
The sulfadiazine moiety of silver sulfadiazine has been
analyzed by thin layer chromatography. The compound is dis-
solved in 35% ammonia solution and the solution is diluted, if
necessary, with methanol. The separation is performed on silica
gel F254 plates with one of the following mobile phases:
A. Ethanol-25% ammonia (95:5); Rf about 0.5 (28)
B. Chloroform-methanol-25% ammonia (70:40: l o ) ; Rf 0.3 (14,24)
C. Chloroform-methanol-25% ammonia (30: 10:2); Rf 0.12 (24)
D. Ethyl acetate; Rf 0.42 (24)
E. 2-Propanol-2-butanol-ethyl acetate-35% ammonia (1:l:Z:I);
Rf about 0.25 (9)
The spot can be located by one of the following methods:
1 ) UV detection at 254 nm
2) I2 vapour (28)
3) 5% potassium dichromate in sulphuric acid (40% w/w) (24)
4) 0.1% N ,N-dimethylaminobenzaldehydehyde in an ethanol-conc.
hydrochloric acid (99: 1 ) mixture (9)
The detection limit with spray reagent (4) is about 0.2 pg (9).
5.7High Performance Liquid Chromatography

The only reported separation of silver sulfadiazine is
based on reverse phase high performance liquid chromatography
( 9 ) . A C18-type bonded column, e.g. Partisil 10 ODs, was used

as stationary phase and 1% acetic acid-methanol (80:ZO) as
mobile phase. The detection was performed at 254 nm and o-cresol
was used as internal standard. This quantitation is based on
detection of the sulfadiazine moiety of silver sulfadiazine.
6. STABILITY
Silver sulfadiazine in the solid state turns into slightly
yellow within one day upon exposure to light and remains in
that state for at least two years ( 2 4 ) . N o subsidiary spots
were found in TLC separation, nor changes in silver and sulfa-
diazine contents. The compound remains unchanged for four years
at 2OoC in the dark ( 2 4 ) . Preservation in the dark at 20°C and
at a high relative humidity (90%) results in a light yellow
powder after two years ( 2 4 ) . Exposure to a temperature of 5OoC
in the dark results in a light yellow-brown product after two
years ( 2 4 ) . The extent of colour formation increases with rise
of the temperature, but no subsidiary TLC spots or changes in
silver and sulfadiazine contents were found.
7. PHARMACOLOGY
Silver sulfadiazine is more of less active against both
gram-positive and gram-negative bacteria, fungi, treponema and
viruses. The compound is in use as a 1% w/w oil-water cream in
topical burn treatment and has favourable clinical results
(first choice drug).
In the environment of the burn the compound slowly dis-
sociates into silver and sulfadiazine (30). The silver ion is
the antimicrobially active part of the compound and acts by
interaction with the micro-organism. Possibly sulfadiazine has
a supportive antibacterial effect, but the concentration seems
to be subinhibitory (30). The role of sulfadiazine may be to
create a sustained delivery of silver ions into the wound
environs.
Almost the total concentration ( > 9 9 % ) of silver remains
located in the wound environment (31). The sulfadiazine is ab-
sorbed to about 10% into the general circulation ( 3 2 ) . Depend-
ing on the burnt surface, blood levels up to about 3 mg/100 ml
and renal excretion to 2 g / 2 4 hours have been reported ( 3 2 ) .
Details about the metabolism of sulfadiazine have been reported
before (12).
8. IDENTIFICATION AND DETERMINATION IN BODY FLUIDS AND PHARMA-

CEUT I CALS
8.1 Determination in Body Fluids
570 A U K E BULT AND CEES M. PLUG
From the topically applied silver sulfadiazine only sulfa-

diazine is absorbed to some extent (about l o x ) . The analysis of
sulfadiazine in biological fluids can be based on the methods
described by Stober and De Witte ( 1 2 ) . Delaveau and Friedrich-
Noue (33) employed the Bratton-Marshall reaction for the
measurement of sulfadiazine levels in plasma, serum and urine
after topical application of silver sulfadiazine cream.
8.2 Identification and Determination in Pharmaceuticals

The common application form of silver sulfadiazine is a I %
w/w oil-water cream. Prior to the identification and determi-
nation of silver sulfadiazine the cream basis can be removedby
dissolution into a suitable organic solvent (or mixture of
solvents). The following extraction media are described:
a) a mixture of ethanol 94% and chloroform ( I : ] ) and
b) the successive extraction with n-butanol and diethyl ether
( 9 , 2 8 ) . The identification and/or determination of the isolated
silver sulfadiazine can be based on the methods of analysis
described in sections 5.2 - 5.7.
Direct treatment of the whole cream with 2 M hydrochloric
acid by refluxing for 30 minutes followed by filtration results
in isolation of sulfadiazine. The sulfonamide present in the
filtrate is determined by biamperometric titration ( 2 4 ) .
Treatment of the cream with concentrated ammonia followed
by acidification with 1% acetic acid is suitable as a sample
preparation method for the high performance liquid chromato-
graphic determination ( 9 ) . The clear supernatant is used for
the analysis.
ACKNOWLEDGEMENT
The authors are indebted to Dr J.M. Gillingham, Dr A. van

der Hoek, Dr M. Horlington and Prof. A.W.M. Indemans for con-
tributing relevant analytical information, to Mr C . Erkelens,
Drs H. Jousma, Mr B.E. Pareraand DrJ. vanThuij1 for performing
the physical measurements, to Mr A.G. Huigen for drawing the
figures and to Miss S. Oosterwijk for preparing the manuscript.
REFERENCES (the literature is cited through 1983)
1 . M. Wruble, J. Am. Pharm. Ass. 32, 80 (1943).

2. Ch.L. Fox, Arch. Surg. 96, 1 8 4 7 1 9 6 8 ) .
3. J.J.M. van Saene, A. B u z and C.F. Lerk, Pharm. Weekblad,
Sci. Ed. 5, 61 (1983).
4. A. Bult azd H.B. Klasen, J. Pharm. Sci. 67, 284 (1978).
5. M.S. Wysor and J.P. Scovill, Drugs ExptlyClin. Res. VIII,
155 (1982).
6 . B.J. Sandmann, R.U. Nesbitt and R . A . Sandmann, J. Pharm.
SILVER SULFADIAZINE 57 I
S c i . 63, 948 (1974).

7. A. B u C and H.B. K l a s e n , Arch. Pharm. (Weinheim) - 313, 1016
(1980).
8. K.K. Narang and J . K . Gupta, C u r r . S c i . 45, 744 (1976).
9. M. H o r l i n g t o n , Smith & Nephew R e s e a r c h E d . , Harlow,
p e r s o n a l communication.
10. L. J. Bellamy, Advances in Infrared Group Frequencies, Chap-
man and H a l l , London (1968), p . 219.
11. P.P. d2 W i t , H. van Doorne and A. B u l t , Pharm. Weekblad,
S c i . Ed., i n p r e s s .
12. H. S t o b e r and W. DeWitte, Analytical Profiles of D m g Sub-
stances, Vol. 1 1 , Ed. K. F l o r e y , Academic P r e s s , New York
( 1 9 8 2 ) , p . 523.
13. C. Chang, H.G. F l o s s and G.E. Peck, J . Med. Chem. - 18, 505
(1975).
14. A.W.M. Indemans, L a b o r a t o r y of t h e Dutch P h a r m a c i s t s , The
Hague, p e r s o n a l communication.
15. J . van T h u i j l , S u b f a c u l t y o f C h e m i s t r y , L e i d e n , p e r s o n a l
communication.
16. P. Gundu Rao, P. Ranganatham, K.K. S r i n i v a s a n and N.R.
S h a r a d a , C u r r e n t S c i . I n d i a 48, 99 (1979).
17. H. Jousma, S u b f a c u l t y of Pharmacy, L e i d e n , p e r s o n a l com-
munication.
18. D.A. Cook and M.F. T u r n e r , J. Chem. SOC., P e r k i n T r a n s 11,
1021 (1975).
19. N . C . B a e n z i g e r and A.W. S t r u s s , I n o r g . Chem. - 15, 1807
(1976).
20. G.J. Boelema, A . B u l t , H . J . M e t t i n g , B.L. Bajema and D . A .
Doornbos, Pharm. Weekblad, S c i . Ed. 4 , 38 (1982).
21. W. J . Geary, Coord. Chem. Rev. 7, 81 T1971).
22. R.U. N e s b i t t and B . J . Sandmanny J. Pharm. S c i . - 66, 519
(1977).
23. Ch.L. Fox, S. Modak, J . W . S t a n f o r d and P.L. Fox, Scand. J .
P l a s t . R e c o n s t r . Surg. 13, 189 (1979).
24. A. v a n den Hoek, ACF C h z i e f a r m a NV, Maarssen, p e r s o n a l
communi c a t i o n .
25. European Pharmacopoeia, 2nd E d i t i o n , P a r t I, C o u n c i l of
Europe, V . 3 . 1 . 1 . (1980).
26. European Pharmacopoeia, Ed. I , P a r t 111, C o u n c i l of Europe,
p. 347 (1975).
27. European Pharmacopoeia, 2nd E d i t i o n , P a r t I , C o u n c i l of
Europe, V.3.5.1. (1980).
28. Anonymous, Pharm. Weekblad, 109, 884 (1974).
29. A.C. B r a t t o n andF.K. M a r s h a l r J . B i o l . Chem. 128, 537 (1939).
30. A . B u l t , Pharmacy I n t e r n a t i o n a l 2, 400 (1982).
31. H.N. H a r r i s o n , Arch. Surg. (Chicago) 114, 281 (1979).
32. A.R. Grossman, Am. Fam. Phys. 1 , 69 (1970).
33. P. Delaveau and Ph. Friedrich-Goue, T h g r a p i e - 32, 563 (1977).
SULINDAC
Fotios M. Plakogiannis
Arnold and Marie Schwarts College
of Pharmucy and Health Sciences
Brooklyn, New York
James A. McCauley
Merck Sharp G Dokine Research Laboratories
Rnhtoay, New Jersey
1, Foreword, History, Therapeutic Category 574

2. Description 574
2.1 Name, Formula, Molecular Weight 574
2.2 Appearance, Color, Odor 574
3 . Synthesis 575
4.3 Nuclear Magnetic Resonance Spectra 577
4.4 Ultraviolet Absorption Spectrum 582
4.5 Solubility 582
4.7 Partition Behavior 584
4.8 Thermal Behavior 584
4.9 Polarographic Behavior 586
4.10 Polymorphism 586
5. Methods of Analysis 59 1
5.1 Identification 59 1
5.3 Chromatographic Analysis 59 1
5.4 Titrimetry 592
6. Drug Metabolic Products, Pharmacokinetics, Bioavailability 593
References 595
Copyright 0 1984
ISBN 0-12-260813-5
514 FOTIOS M . PLAKOGIANNIS AND JAMES A. McCAULEY
1. Foreword, History, Therapeutic Category.

Sulindac is a new nonsteroid anti-inflammatory drug
with analgesic and antipyretic activity1,2.
Sulindac and indomethacin had similar pharmacologic
profiles in laboratory animals but sulind c showed
less gastrointestinal bleeding and ulcers’, 3.
2. Description
2.1 Name, Formula, Molecular Weight
1H-Indene-3-acetic acid, 5-fluor0-2-methyl-lI4-
(methyl-sulfinyl) phenyllmethylene -, (2)-;CIS-5-
fluoro-2-methyl-1- (p-methyl-sulfiny1)benzyl-idenel
indene-3-acetic acid.
Empilrical formula C20H17F03S
Molecular weight 356.41
The CAS Registry number is CAS-38194-50-2

Sulindac is an odorless, yellow, crystalline powder.
SULINDAC S75
3. Synthesis
A
synthesis4r5 of sulindac is summarized as.
follows:
SULINDAC
4.1 Infrared Spectrum
The infrared spectrum of sulindac, run as a
nujol mull, is reproduced in Figure 1 and is con-
sistent with the structure. Band assignments
which may be made with confidence are tabulated
below:
TABLE I
Band Assiqnment Infrared Spectrum of Sciliiidac

v (crn-l) Assignment
2 500-2 650 -CO H dimer
1700 v c=o
1600
1580 Aromatic ring n d e s
1270 -CO H dimer
1155 v C-F
1 0 0 0-10 20 v s-0
In addition to these, sharp bands between 8 0 0

rl
0 rl
0 7
cu E
rl
0
0 -n
0 7
ro z
c
-4
0 u
0 a
aD a
c
-4
rl
0 7
0 cn
w w
0
-
0
0
d
5
k
4J
u
a,
-
0
a
v)
0
Ic c
0
-4
4J
0
0
0
e
0
cu
0
0
i
a
rT)
cu a,
k
a
0 k
0 Icl
0
M
c
H
0
0 4
v)
M
a,
0 k
0 b,
0 -4
0 0 0 0-J-
CD d cu F
SULINDAC 517
aqd 900 c m
'
l are expected from the ArH out-of-plane
modes in both rings. The nujol mulling agent, of
course, accounts for the very strong band from
2 8 2 5 to 2 9 7 5 cm , as well as for all or part of
the bands at 1 4 6 0 and 1 3 7 5 cm-l.f6)
4 . 2 Mass Spectrum
The electron impact ionization spectrum is

given in Figure 2. A LKB model 9 0 0 0 mass spectro-
meter was used with ionization potential of 70eV
and accelerating voltage of 3.5kv. The mass spec-
trum shows a molecular ion at 3 5 6 (m/e) with two
relatively intense peaks at 3 4 1 and 2 3 3 (m/e).
Loss of a methyl group from the parent molecule
will account for the 3 4 1 m/e fragment. Loss of
CH COOH, CH SO n hydrogen will account for the
other fragment. 7 7 7
4.3 Nuclear Magnetic Resonance Spectra

4.3.1 Proton Spectrum
The proton NMR spectrum is found in Figure 3
and assignments to the characteristic protons are
recorded below. The complex pattern located at
6.4 to 7 . 2 ppm is consistent with aromatic 1,2,4
substitution with fluorine splitting. The singlet
with weak satelites ( ' ~ 7 . 7 4 ppm) is consistent with
an aromatic AA'BB' pattern which is nearly col-
lapsed into a singlet. A solvent (DMSO-ds) reso-
nance appears at approximately 2.5 ppm ( 6 ) .
u
KJ
a
.
(v
578
f
519
580 FOTIOS M. PLAKOGIANNIS A N D JAMES A . MCCAULEY
TABLE I1
H
' NMR Sulindac Assignments
Chemical Shift
ppm ( 6 1 Pattern Proton Assignment
2.17 singlet
2.83 singlet
3.60 singlet
~6.4 complex
to
7.2
7.36 singlet
singlet
7.74 with weak
satelites
4.3.2 Carbon 13 Spectrum
The I3C magnetic resonance spectrum is given
in Figure 4 and was obtained using a Varian CFT-20
(FT mode) spectrometer with CDC13/CK30D (9:l) as
the solvent at a concentration of 0.5M. The spec-
trum is consistent with t e structure and the as-
signments are as follows:?8)
I I I I I I I I I I I I I I I I I I I 1 I I I I I I I I I 1 I I I r I I 1 . 1 7
c2;6 c33
CH,DH
(Solvent 1
CO,H
(Solvent 1
Chemical Shift Si3C ( ppm)

Figure 4. 1 3 C NMR of S u l i n d a c
582 FOTIOS M. PLAKOGlANNlS AND JAMES A. McCAULEY
TABLE I11
Carbon-13 Chemical Shifts for Sulindac
Shift, 613C
PPm) Assignment
141.9 C
138.2 C
132.4 C
106.2 C
163.4 C
110.7 C
123.7 C
147.1 C
129.7 C
128.0 c (Benzal)
140.1 C1 '
130.5 CZ', c6'
124.1 C3', C5'
144.2 c4 1
10.4 2 - c ~ ~
31.7 3-CH2
172.9 Carboxyl
43.2 SCH3
4.4 Ultraviolet Absorption Spectrum

The ultraviolet absorption spectrum of su-
indac in methanolic 0.1N hydrochloric acid is
found in Figure 5 and is characterized by maxima at
327, 284, 258 and 228 nm with El%lcm values of ap-
proximately 370, 420, 405 and 540 respectively.
4.5 Solubility
Sulindac is sparingly soluble in methanol and
95% ethyl alcohol, slightly soluble in ethyl ace-
tate, and practically insoluble in water. The
water solubility increases with increasing pH as
indicated in the following table. (9)
SULINDAC 5x3
1 I 1 I
0.9
0.8
0.7
0.6
0)
0
C
0
n
0.5
L
a
n
a 0.4
0.3
0.2
0.1
250 300 350 400
Wavelength ( n m )
Figure 5. U l t r a v i o l e t A b s o r p t i o n S p e c t r u m of
S u l i n d a c i n M e t h a n o l i c 0.1N H C 1
1 . 6 2 5 mg/100 m l
584 FOTIOS M. PLAKOGIANNIS AND JAMES A . McCAULEY
TABLE IV
Solubilitv of Sulindac
Solvent Temperature Solubility

("C) (mg/ml)
0.1N IlCl 25 0.003
Water 25 0.003
p H 7.0 (Phosphate) 25 2.7
pH 7 . 4 (Phosphate) 25 3.4
p H 3.9 (Citrate) 37 0.01
pH 4.8 (Citrate) 37 0.05
p H 5.9 (Citrate) 37 0.35
pH 6.4 (Citrate) 37 1.0
Acetone 25 18
Chloroform 25 25
Ethanol 25 15
Ethyl Acetate 25 4
Hexane 25 1
Isopropanol 25 7
Methanol 25 30
4.6 Dissociation Constant

The pKa of sulindac is 4.7 at 25°C.
4.7 Partition Behavior
The distribution coefficient for sulindac be-
tween phosphate buffer pH 7 . 0 and octanol at 25°C
is 0.28. Sulindac can be extracted quantitatively
from aqueous acidic media into the following or-
ganic solvents: methylene chloride, chloroform,
ethyl acetate and 3 volume % isoamyl alcohol in
heptane. Sulindac aan be back extracted into di-
lute alkali from these solvents.
4.8 Thermal Behavior
Sulindac melts with decomposition and there-
fore the observed temperature is dependent upon
the conditions of the observation. Under USP Class
la con it'ons, the temperature ranges from 182-
185°C. The DTA curve (Fig. 6) f o r s u l i n d a c at
20°C/minis characterized b y a single endotherm with
c rn
peak temperature at approximately a186 C.

4.9 Polarographic Behavior
In 0.1M tetraethylammonium perchlorate in ace-
tonitrile, half-wave potentials of -1.38 and -1.52
were observed for sulindac. The reductions involve
the C=C bonds and not the sulfur functionalities by
analogy to the work of Hawson et a1 (I1 and 1 2 ) *
4.10 Polymorphism
Sulindac is a polymorphic substance and can

exist in two nonsolvated enantiotropic crystal
forms designated Forms I (191°C) and I1 (186°C).
Form I1 is the more stable polymorph at room temp-
erature and temperatures below the transition
point which is estimated to be 157°C from heats of
fusion and melting points of the polymorphs. The
transition temperature has a l s o been estimated to
be 165°C by extrapolations of solubility measure-
ments of the two forms over the temperature range
of approximately 20 to 80°C. The average of the
two determinations yields a value fo 161 f 7°C for
the transition point.
Form I is the more stable form above the tran-
sition point up to its melting point of 191°C which
is about five degrees ab0v.e the metastable melting
point of Form 11. Although Form I is metastable
with respect to Form I1 at room temperature, the
rate of conversion of Form I to Form I1 is depend-
ent upon the conditions. With solid Form I alone,
the rate is so slow that it cannot be conveniently
measured. In contact with water or aqueous buf-
fered systems, the conversion requires weeks or
months. In contact with ethanol Form I converts to
Form I1 within 16 hours or less.
Form 11, the more stable form at room temper-
ature, is the preferred form. X-ray powder dif-
fraction (Figs. 7 and 8), infrared absorption (Figs.
1 and 9) and differential thermal analysis (Figs.
6 and 10) are capable of distinguishing the two
forms .
c
0
U
(d
a
c
b
rd
k
w
w
a,
a
3
0
PI
I
x
h
IZ
0
-4
4J
E
d
3-
0
-4
4J
0
fd
k
..
Ill
a
3
c 0
PI
h
2I
x
03
.
a,
k
7
b
-4
I%
Iv
0
Endotherm
0
Exot her m,
0
&
0
0
A
P
0
A
03
0
Iv
N
0
Iv
0
0
Wavelength Microns
2.5 3 4 5 6 7 8 9 10 12 14 18 22 3.550
100
80
60
40
20
0
4000 3500 3000 2500 2000 1700 1400 I100 800 500 200
Frequency (cm-'1
Figure 1 0 . I n f r a r e d A b s o r p t i o n Spectrum of S u l i n d a c Form I in Nujol Mull
SULINDAC 59 1
5 . 0 Methods of Analysis
5 . 1 Identification
Comparison of infrared and absorption spectra

with those of reference standard are me hods of
identifications specified by the USP-NF413) .
5.2 Elemental Analysis ( 1 0 )
The following elemental composition was ob-

served for sulindac when approximatley 2 mg of sam-
ple was analyzed with a Perkin-Elmer, Model 2 4 0 CHN
Analyzer.
Element Theory % Funds %
C 67.40 67.63
H 4.81 4.72
5 . 3 Chromatoqraphic Analysis
5.3.1 Thin Layer Chromatography

Several TLC systems have been used for sulin-
dac. Table V lists the solvents and the sulindac
RF’s.
TABLE V
TLC Solvents and Rf for Sulindac
Solvent Rf
1. Chloroform/Ethyl Acetate/Acetic Acid 1 6 : 5 : 1 -0.4
2 . Ethyl Acetate/Acetic Acid 9 7 : 3 and 9 4 : 6 -0.4
3. Chloroform/Acetic Acid 9 5 : 5 ~ 0 . 3
4 . Ethyl Acetate/Chloroform 1:1 QO. 1
5 . Carbon Tetrachloride/Acetic Acid 9 : l s0.2
6 . Chloroform/Dioxane/Acetic Acid 2 5 : l : l -0.7
7 . Benzene/Methanol/Ammonia 50:20:1 s0.2
Silica gel GF absorbents are used with all the

solvent systems and detection is with short wave UV
light ( ~ 2 5 4nm) or with exposure to iodine vapor.
Solvent system #1 has been used for quantita-

tive determination of impurities in sulindac.
Quantum Q-7G plates are used and the plate is
592 FOTIOS M. PLAKOGIANNIS A N D JAMES A. McCAULEY
s c a n n e d a t 280 nm w i t h a s u i t a b l e d e n s i t o m e t e r (13) .
Approximate R f ' s f o r s u l i n d a c r e l a t e d compounds
f o r s o l v e n t s y s t e m s #1 and # 2 a r e l i s t e d below.
Rf
-
Compound #1 #2
-
Sulfone analoq %0.6 %O. 7
S u l f i d e analog -0.7 -0.8
E t h y l ester s u l i n d a c %Lo. 5 %0.5
T r a n s isomer 'LO. 3 %O. 3
5 . 3 . 2 G a s ChromatouraDhv
S u l i n d a c h a s been c h r o m a t o g r a p h e d a s the m e t h y l
ester ( d i a z o m e t h a n e ) o n a 6 ' x 1 0 . 2 5 " 1% SE-30 on
S u p e l c o n 80/100 column u n d e r i s o t h e r m a l a t 2 6 2 ° C
w i t h n i t r o g e n as a c a r r i e r gas a t + l o 0 ml/min. De-
t e c t i o n w a s w i t h e l e c t r o n c a p t u r e and s a m p l e s i z e s
r a n g e s from 450 t o % l o 0 ng. The r e t e n t i o n t i m e of
s u l i n d a c w a s a p p r o x i m a t e l y 7 m i n u t e s . The s u l f i d e
and s u l f o n e a n a l o g s h a v e r e t e n t i o n t i m e s o f 1 . 5 and
5 m i < y i f 3 s r e s p e c t i v e l y u n d e r t h e same c o n d i -
tion
5.3.3 -
L i q u i d Chromatography
S e v e r a l HPLC s y s t e m s h a v e b e e n u s e d f o r s u l i n -
d a c i n c l u d i n g o n e employing c h l o r o f o r m / e t h y l ace-
t a t e / a c e t i c a c i d ( 8 0 0 : 2 0 0 : 2 ) as t h e mobile phase
w i t h a LI P o r a s e l , 30 c m , 1 0 p p a r t i c l e s i z e column;
UV d e t e c t i o n a t 280 nm and a f l o w r a t e of 2.0 m l /
min (500-3000 p s i ) . Under t h e s e c o n d i t i o n s s u l i n -
d a c c h r o m a t o g r a p h e d w i t h a r e t e n t i o n t i m e o f approx-
i m a t e l y 6 . 3 m i n u t e s . The s u l f i d e and s u l f o n e ana-
l o g s chromatographed w i t t i m e s of %1.8
2 . 5 minutes r e s p e c t i v e l y
O t h e r HPLC s y s t e m s u s e Zorbax ( s i l i c a g e l )
column 0 . 5 m x 8 mm o r 0.25 m x 2 . 1 mm w i t h m o b i l e
phases of chloroform/ethyl acetate/acetic a c i d
38 :5 :1( 8 ) o r m e t h y l e n e chloride/hexane/isobutanol/
a c e t i c a c i d / w a t e r 50:50:10:4:.046.
5.4 T i t r i m e t r y
S u l i n d a c c a n be q u a n t i t a t i v e l y a s s a y e d by po-
t e n t i o m e t r i c t i t r a t i o n . Methanol i s u s e d a s a
SULINDAC 593
s o l v e n t f o r s u l i n d a c and t h e s o l u t i o n i s t i t r a t e d
w i t h s t a n d a r d i z e d sodium h y d r o x i d e ( 0 . 1 N ) t o a
p o t e n t i o m e t r i c end p o i n t using a qlass-calomel
electrode system(l5).
6. Drug M e t a b o l i c P r o d u c t s , P h a r m a c o k i n e t i c s , B i o -
availability
The major u r i n a r y m e t a b o l i t e s are s u l i n d a c and i t s

g l u c u r o n i d e , s u l f o n e and i t s g l u c u r o n i d e and i n
t r a c e amounts t h e i n s o l u b l e s u l f i d e m e t a b o l i t e and
i t s g l u c u r o n i d e . The major b i o t r a n s f o r m a t i o n
i n v o l v e s i r r e v e r s i b l e o x i d a t i o n of t h e s u l f o x i d e
g r o u p o f s u l i n d a c t o s u l f o n e and a r e v e r s i b l e r e d u c -
tion t o the sulfide. Studies i n healthy subjects
revealed t h a t a f t e r oral administration of sulindac
a t l e a s t 88% i s a b s o r b e d . A f t e r a s i n g l e 2 0 0 mg
o r a l dose t h e peak plasma c o n c e n t r a t i o n s o f s u l i n d a c
and i t s s u l f o n e and s u l f i d e m e t a b o l i t e s were 4 , 2 ,
and 3 mg/ml r e s p e c t i v e l y , and w e r e a t t a i n e d a f t e r
1 hour f o r t h e p a r e n t d r u g and a f t e r 2 h o u r s f o r
each of the metabolites.
The s u l f o n e and i t s c o n j u g a t e s are t h e major p r o d -

u c t s e x c r e t e d i n u r i n e , and a c c o u n t f o r 27.6 + 2 . 8 %
o f t h e a d m i n i s t e r e d dose. S u l i n d a c and i t s g i u c u -
r o n i d e accounted f o r 2 0 . 2 + 0 , 2 % o f t h e dose e x c r e -
ted i n the urine. Mean reFal c l e a r a n c e o f s u l i n d a c
and i t s s u l f o n e m e t a b o l i t e w a s 4 5 . 1 - + 1 6 . 2 and
33.2 + 1 2 . 2 ml/min r e s p e c t i v e l y ( l 6 ) . The e f f e c t i v e
h a l f - i i f e f o r accumulation i s about 7 hours f o r
s u l i n d a c ( l 7 ) . The l o n g h a l f - l i f e i s d u e t o e x t e n -
s i v e e n t e r o h e p a t i c r e c i r c u l a t i o n (18).
There a r e no r e p o r t e d b i o a v a i l a b i l i t y p r o b l e m s w i t h
sulindac tablets. No s i g n i f i c a n t d i f f e r e n c e i n
plasma c o n c e n t r a t i o n s o r u r i n a r y e x c r e t i o n o f
s u l i n d a c w a s found f o l l o w i n g a d m i n i s t r a t i o n of
t a b l e t s or s o l u t i o n ( l 7 ) .
7. Identification and determination in body fluids

and tissues.
The following methods have been used for the assay

of sulindac in body fluids and tissues.
Methods References
Radioactivity (17)
Isotope dilution-radioimmunoassay (191
Ultraviolet (171
Thin-layer chromatography (16)
Gas-liquid chromatography (161
Mass spectrometry (16)
HPLC (20)
SULINDAC 595
References
1. T . Y . Shen, B . E . W i t z e l , H. J o n e s , B.O. L i n n ,
J. McPherson, R. G r e e n w a l d , M . F o r d i c e and A.
Jacobs: Fed. Proc. - 31, 5 7 7 ( 1 9 7 2 ) .
2. C.G. V a n A r m a n , E.A. R i s l e y , and G.W. Nuss: -

Fed.
Proc. 31, 5 7 7 ( 1 9 7 2 ) .
3 . R.N. B r o g d e n , R.C. H e e l , T.M. S p e i g h t and G . S .

Avery: D r_
_ u g_s -1 6 , 9 7 ( 1 9 7 8 ) .
4. T.Y. Shen and C.A. Winger i n " A d v a n c e s i n Drug

R e s e a r c h " , vol. 1 2 , N . J . Harper and A.B. S h m o n s ,
E d . , A c a d e m i c Press, New Y o r k , N . Y . 1 9 7 7 .
89-228.
5 . S h u m a n , R.F. et-al., -
JOC, -
42, 1914 (1977).
6. D o u g l a s , A.W., Merck Sharp & Dohme R e s e a r c h

L a b o r a t o r i e s , Rahway, N . J . , Personal Communica-
tion.
7. Smith, J.L., Merck Sharp & Dohme R e s e a r c h L a b o r -
atories, Rahway, N . J . , Personal C o m m u n i c a t i o n .
8 . D o u g l a s , A.W., C a n . J. C h e m . , 56, 2129(1978).
9 . B r e n n e r , G . , Merck Sharp & D o h m e R e s e a r c h L a b o r -

atories, Rahway, N . J . , Personal C o m m u n i c a t i o n .
10. S h u m a n , R.F. et.al., JOC, -
- 42, 1 9 1 4 ( 1 9 7 7 ) .
11. T a i t , W.E., Merck Sharp & D o h m e R e s e a r c h L a b o r a -

t o r i e s , Rahway, N . J . , Personal C o m m u n i c a t i o n .
1 2 . N e w t o n , P . , Merck Sharp & D o h m e R e s e a r c h L a b o r a -

tories, Rahway, N . J . , Personal C o m m u n i c a t i o n .
13. M a n c i n e l l i , R . J . , Merck Sharp & D o h m e R e s e a r c h
Laboratories, R a h w a y , N . J . , Personal Communica-
tion.
1 4 . T h e U n i t e d S t a t e s P h a r m a c o p e i a , 2 0 t h rev.,
A d d e n d u m a t o S u p p l e m e n t 2 , t h e U.S. P h a r m a c o -
p e i a l C o n v e n t i o n I n c . , 1 9 8 0 , page 2 9 5 .
596 FOTIOS M . PLAKOCIANNIS AND JAMES A. McCAULEY
15. The United States Pharmacopeia, 20th rev.,

Addendum a to Supplement 1, the U.S. Pharmaco-
peial Convention Inc., 1980, page 151.
16. H.B. Hucker; S.C. Stauffer; S.D. White et.al.,
Drug Metab. and Disp. 1:721(1972).
17. D.E. Dugqan, L.E. Hare, C.A. Ditzler, B.W. Lei,
and K.CI-Kwan: Clinical Pharmacoloqy and Thera-
peutics. 21, 326 (1977a).
18. R.W. Walker et.al., Anal. Biochem. -
95, 579
(1979).
19. L.H. Hare, C.A. Dirtzler, M. Hichens, A .
Rosejay, and E.E. Duggan: J. Pharm. Sci. -66,
414 (1977).
20. W.O.A. Thomas, T.M. Jeffenes, and R.T. Parfitt.
J. Pharmacy and Pharmacol. 31, 91P(1979).
TETRACYCLINE HYDROCHLORIDE
Syed Laik Ali
Zentrallaborntoriul,1 Deutscher Apotlzeker e . V
Eschborn, Feder(11 Republic of Germiny
1. History 598
2. Nomenclature 598
3 . Description 598
3.1 Name, Formula. Molecular Weight 598
3.2 Appearance, Colour, Odour 600
4. Synthesis 600
5. Physical Properties 60 I
5. I Solubility 60 1
5.2 Loss on Drying 602
5.4 Specific Optical Rotation 602
5.5 pH Value 602
5.8 Nuclear Magnetic Resonance Spectrum 606
5.9 I3C NucIear Magnetic Resonance Spectrum 606
5. I0 Mass Spectrometry 608
5.1 I Circular Dichroism Spectra 610
5.12 Differential Thermal Analysis 613
6. Colour Reactions 613
7. Degradation and Stability 613
8. Related Impurities 620
9. Toxicity 620
10. Dissolution 62 1
1 I . Microbiological Methods 622
11. I Agar-Diffusion Method 623
I 1.2 Turbidimetric Method 623
12.1 Titrimetry 623
12.2 Visible and UV Spectrophotometry 623
12.3 Fluorimetry 625
12.5 Polarographic 642
13. Drug Metabolism and Pharmacokinetics 642
References 645

VOLUME 13 591 by the American Phanndccuticai Associdtlon
ISBN O-L2-ZM)X13-5
598 SYED LAlK ALI
Te t racycl ine Hydrochloride
Tetsacycline and tetracycline hydrochloride

have been mostly used synonymously in this
monography. Literature cites quite often te-
tracycline but it is presumed to be valid al-
s o for tetracycline hydrochloride. Generally
solutions of tetracycline are made in dilute
acids. Physical properties, spectra etc are
given f o r tetracycline hydrochloride.
1. History
During the course of experiments for the elu-
cidation o f the structure of the two earlier
discovered compounds chlortetracycline (CTC)
and oxytetracycline (OTC) it was found that
hydrogenation of chlortetracycline resulted
in halogenolysis and the product tetracycline
(TC) retained the useful activity spectrum of
the first two members of the family. TC ap-
pears to represent the first clinically suc-
cessful antibiotic produced by shere chemical
manipulation of preexisting antibiotic. TC
was found to be present in fermentations of
both cultures streptomyces aureofaciens and
streptomyces rimosus as well as in strepto-
myces viridofaciens (1).
2. Nomenclature
The name tetracycline derives from the naph-
thacene nucleus that it possesses. The rings
are lettered A through D from right to left
and the numbers start at the bottom o f ring
A . The enolized form as illustrated on next
page has been selected arbitrarily and is
used most exclusively in the literature. It
is further defined as hydrochloride of 4F-di-
methylamino-1,4,4a,5,5a,6,ll,l2a-octahydro-3,6
o(,lO ,12,12a -pentahydroxy-6-methyl-l,11 -
R
dioxonapht acene-2-carboxamide.
3. Description
3.1. Name, Formula, Molecular Weight
Tetracycline hydrochloride
480.90
0
1
600 SYED LAIK ALI
3 . 2 . Appearance, Colour, Odour

A yellow crystalline powder, odourless, with
a bitter taste.
4. S nthesis
h c a l total synthesis of tetracycline
hydrochloride is economically not feasible.
The original process involved catalytic hy-
drogenolysis of CTC to TC and then a directed
biosynthesis using chlorine-free-culture-me-
diums ( 2 , 3 ) . In fact the removal of chlorine
from the medium o r decreasing chlorine in-
corporation with a wide variety of chlorina-
tion inhibitors is technically quite impor-
tant in order to facilitate processing with-
out the need of expensive chromatographic
steps as otherwise undesirable quantities of
CTC are coproduced along with TC ( 4 ) .
Fermentation and Isolation ( 5 ) : Various te-
e m ] fer-
mentation of different streptomyces species
isolated in soil screening programms and then
nutritionally and genetically manipulated to
optimize yields. The fermentations are done
on a very large scale of about 20,000 to
50,000 gallons. The organism and strain cho-
sen not only determine maximal yields but al-
s o the identity of the antibiotic formed.
Chloretracycline fermentations frequently
have up to 10% of tetracycline present. A
wide variety of media have been employed for
TC production. Fermentation starts with a
tube of lyophilized culture which must be
opened aseptically and transferred to agar
slants for incubation. The cell population is
then built up in stages. The pH of the fer-
mentation is generally closely controlled
between 6 and 7 by the periodic addition of
the base, usually solid CaC03 and the tem-
perature is maintained between 2 5 0 and 30
OC. Many energy and carbon sources such as
starch, dextrin, maltose, glucose and nitro-
gen sources including amino acids, casein,
meat extracts, nitrates and ammonium salts
have been employed. A variety of inorganic
ions must be present and calcium
TETRACYCLINE HYDROCHLORIDE 60 I
carbonate is specially noteworthy as it sta-

bilizes the tetracyclines formed and may help
increase yields by precipitating the antibio-
tics as they are formed and thus decreases
their autotoxic effect on the producing o r -
ganism. Several patents dealing with the fer-
mentative production of tetracyclines are re-
ported in literature. The yields of tetra-
cyclines are reported to lie about in the
range of 10 g/liter ( 6 ) . There are a variety
of procedures which have been employed for
isolating the various tetracyclines from
high-yielding fermentations. The processes in
use are governed by the physical and chemical
properties of the antibiotics and by economic
factors. Tetracycline is an amphoteric sub-
stance and has an isoelectric point at 4.8
and its water solubility near neutral pH is
about 1 mg/ml. The solubility in water is
about ten times greater at pH 1 - 3 than at
pH 5 - 6 . The isolation of TC can be affected
through the use of a liquid ion exchange pro-
cess, extraction from filtered beer at pH 8 . 5
with n-butanol or M I B K and precipitation with
a non-polar solvent as the free base ( 7 1 ,
fractional precipitation of the less soluble
TC-HC1 from CTC-HC1 (coproduced in the fer-
mentation) (8) and precipitation as the cal-
cium-complex ( 9 ) . The tendency of TC fermen-
tations to produce sufficient quantities of
CTC also renders refining difficult on a
commercial scale (10). TC-oxalate is less
soluble than CTC-oxalate s o that fractional
crystallization is quite effective (11). Pre-
cipitation at about pH 4 in the presence of
citrate (121, urea (13) and phosphate (14)
has also been reported. Various cultures
known to produce major fermentation-derived
TC are also mentioned in literature ( 5 ) .
5.1. Solubility: It is freely soluble in water
giving a clear solution' which becomes turbid
on standing, soluble in aqueous solutions of
alkali hydroxides and carbonates, slightly
soluble in alcohol and practically insoluble
in various organic solvents (15,16).
602 SYED LAIK ALI
Following solubility parameters are given in

Table 1.
Table 1
Solubility of TC-HC1 and TC in various solvents in
mg/ml at 28 OC I17).
TC-HC1 TC
Water 10.9 1.7
Methanol 2o+ 20+
Ethanol 7.9 20+
Benzene 0.3 1.0
Petroleum ether 0.0 0.005
Carbon tetrachloride 0.1 0.3
Ethyl acetate 0.8 17.3
Acetone 0.8 17.4
Ether 0.6 3.7
Ethylene chloride 0.8 11.3
Dioxane 7.7 14.6
Chloroform 2.9 13.8
Pyrid ine 20+ 20+
Benzyl alcohol 10.8 14.4
5.2. Loss on Drying (15)
Not more than 2.08, determined with 1.00 g by
drying for 3 hours at 60 0 over phosphorus
pentoxide at a pressure not exceeding 5 Torr.
5.3. Dissociation Constant
The macroscopic PKa values of tetracycline
hydrochloride in water at 25 OC are repor-
ted to be 3.30, 7.68 and 9.69 (18, 19). The
most acidic value is attributable to the un-
usual 8-tricarbonyl system in ring A , the
middle PKa is associated with the vinylo-
gous acidic 8-dicarbonyl system o f rings B
and C and highest PKa is then assigned to
the protonated dimethylamino function at
C4. A fourth PKa value of 10.7 has also
been reported and is also attributed to the
phenolic OH group at C 10 (20).
5 . 4 . Specific Optical Rotation (15)
- 239 0 to - 258 0 , determined in a 1.00%
w/v solution in 0.01N HC1 and calculated with
reference to the dried substance.
5.5. pH Value (15)
The pH of a 1.0% w/v solution is between 1.8
and 2.8.
TETRACYCLINE HYDROCHLORIDE 603
5.6. Ultraviolet Spectrum

The quaternary carbon at C 12a separates the
chromophores of the tetracyclines into two
relatively distinct regions. The 13-tricarbo-
nyl system of ring A contributes a strong ab-
sorption band at about 260 nm while the aro-
matic ring D conjugates with the 13-diketone
moiety in rings B and C to produce a visible
band at about 360 nm (21). tjte molecular ex-
tinction coefficients and %(,,of TC-HC1 in
different solvents are rep0 ed to be (22):
Methanol 0.1 N HC1 0.1N NaOH
Absorption 363 nm 356 nm 380 nm
maximum 270 nm 269 nm 268 nm
A1 % 331 301 366
1m 345 382 299
15940 14480 17580
€ 16590 18380 14360
The UV spectra are given in Fig. 1.
The infrared spectrum of TC-HC1 is given in
Fig. 2. The spectrum was obtained with a Per-
kin-Elmer Spectrophotometer 257 from a KBr
pellet. The several carbonyl groups of the TC
are either amide in character o r conjugated
to double bond system o r strongly hydrogen
bonded. Thus the amide band at about 1670
cm-1 and the large carbonyl envelope near
1620 cm-1 are usually identical for almost
all the tetracyclines whose spectra then dif-
fer mostly in the finger print region (21).
The structural assignments may be correlated
with the foll wing band frequencies:
Frequenc.(cm-?) Assignment
3350 streching vibrations of NHz
3100 stretching vibrations of OH
2600-2800 Amine halide salt bands
1670 stretching vibrations of
amide group
1620 stretching vibrations of car-
bony1 group
1450 characteristic vibrations of
the aromatic ring
604 SYED LAlK ALI
El
220 240 260Ainnm#300 320 340

- Methanol
-- OlN-HCI
--- - 0.1N- NaOH
Fig. 1
UV Spectrum Of Tetracycline Hydrochloride
In Different Solvents
606 SYED LAlK ALI
Nuclear Magnetic Resonance Spectrum

The nuclear magnetic resonance spectrum of
TC-HC1 as shown in Fig. 3 was obtained on a
Varian T-60 NMR Spectrometer in deuterated
methanol containing 1% tetramethyl silane as
the internal standard. The following spectral
assignments are made f o r Fig. 3.
Chemical Shift Ass i gnment
1.72 singlet CH3 at C 6
3.10 singlet N(CH3)2 at C 4
7.20 doublet H a t C 7
7.58 doublet H a t C 8
6 . 9 5 doublet H a t C 9
They are approximately in agreement with va-
lues reported in literature, but the spectrum
there was obtained with a HA-100 (23). The
nmr shifts appear to be general regardless of
solvent. Similar shifts are obtained in pyri-
dine, trifluoroacetic acid and dimethyl sul-
foxide. Particularly notable is the substan-
tial downfield shift in the resoncance due to
the C 4 proton when the tetracyclines epime-
rize at C 4 . This is especially convenient as
both isomers are usually available and can be
distinguished easily in this way. NMR was
used to monitor the epimerization o f TC since
the dimethylamino resonance of TC and its C 4
epimer differ by 0.1 ppm ( 2 4 ) . Formation of
anhydrotetracycline could also be readily
detected through NMR (23).
5 . 9. 13C Nuclear Magnetic Resonance Spectrum
carbon magnetic resonance measurements have
been performed using the natural 13C abun-
dance. Many more resonances are present in
these spectra compared with the proton nmr.
The spectrum of TC-HC1, dissolved in D 2 0 ,
i s presented in Table 2 . The signals are re-
ported to be slightly different in DMSO ( 2 5 ) .
500 400
I , . . .. . .
I , . I , I . . . . I . ...
I . . . . 1 . . . . 1 . . . . l . . . . l . . . . l . . . . , . . . . 1 . . . . 1 . . . . ,
I . . . . I . . . . I . . . . I . . . . l . . . . I
80 70 60 5 0 FPM(0 40 30 20 10 0
Fig. 3
N M R Spectrum Of Tetracycline Hydrochloride,
Varian T 6 0 Spectrometer
608 SYED LAIK ALI
Table 2
13C NMR Spectrum o f TC-HC1 in D 2 0 (25)
Carbon Assignment Chemical Shift
c 1 193.9
c 2 97.1
CONH 2 173.0
c 3 187.3
c 4 70.7
HN+(CH3)2 43.1 (quartet)
C 4a 35.0 (doublet)
c 5 27.3 (triplet)
C 5a 42.3 (doublet)
C 6 70.4
C - CH3 22.1
C 6a 146.7
c 7 118.6
C 8 138.4
c 9 116.8
c 10 161.9
C 10a 115.1
c 11 194.3
C lla 107.3
c 12 172.3
C 12a 74.3
5.10. Mass Spectrometry
Due to its non-volatility and thermal
instability the electron-impact mass spectra
o f tetracyclines is rich in fragment ions
and weak in molecular ion intensity (26). In
the highest mass region one sees m/e 426 for
dehydration followed by ions 409 and 391
resulting from loss of NH3 and water from
the carboxamide function as well as 381 for
l o s s of the whole group a s CONH3. Most of
the fragments seen involve rings A and B
because rings C and D become s o quickly
naphthalenoid. The mass spectrum of TC-HC1
is given in Fig. 4.
Instrument: Varian Mat 44
Sample temperature: (direct inlet)
50OC - 250OC in 5 minutes
Source temperature: 2 0 0 OC
Electron energy: 80 eV
The prominent ions o f this spectrum can be
correlated to the structure as following:
M-HC1 H20=426; M-HC1 H20 NH3=409;
M-HC1 2H20 NH3=391; M-HC1 H20 CONH3 = 381;
-
2.
3 .
6
E 50-
300
lAi1
400
,i, , , , , , ,
500
Fig. 4
Mass Spectrum Of Tetracycline Hydrochloride
610 SYED LAIK ALL
5.11. Circular Dichroism Spectra

TC-HC1 gives useful ORD-CD spectra. The in-
tensity of the A ring band and its relative
absence in 4-epitetracycline provides a sen-
sitive means of assay for epimeric mixtures.
Mixtures of tetracycline and 4-epitetra-
cycline were assayed utilizing the large
difference in their circular dichroism
spectra at 2 6 2 nm. The method is very rapid
and requires only the experimental measure-
ments of ellipticity at 2 6 2 nm and absor-
bance at 3 5 6 nm ( 2 7 ) . The disadvantages of
the assay are its inability to determine the
content of anhydrotetracycline if it is pre-
sent in large amounts. In Fig 5 - 6 molar
ellipticity is illustrated graphically ( 2 7 ) .
The ability of this technique to reflect
subtle conformational shifts in dilute
hydroxylic solvents under a wide variety of
conditions is a particular strong point.
Interactions with complexing ions gave dra-
matic changes in the spectral bands from
both chromophoric regions providing evidence
for chelation in both areas. Quantitative
studies of ion-binding led to the conclusion
that little if any binding takes place below
pH 3 , that the binding of B C D rings takes
place at pH 5 and the A ring binds at phy-
siological pH's ( 2 8 ) . The study of C a z L
and M g 2 L complexes of tetracycline in buf-
fered solution was undertaken to determine
their stoichiometry and the chelation sites.
Circular dichroism was used to follow com-
plex formation. Circular dichroism spectra
of calcium and magnesium complexes of tetra-
cycline in aqueous and methanol-water
(9O:lO) systems are reported. Fig. 7-9 show
these spectra as published by Newman and
Frank (29). Calcium formed a 2:l metal ion
to ligand complex at C 10 and C 12 sites,
while magnesium formed a 1:l ratio complex,
the chelating site being at C 11 ( 2 9 ) . Cir-
cular dichroism spectra of TC-calcium
complexes in different organic solvents and
the effect o f barbital sodium and L-trypto-
phan on it in alkaline and other buffer s o -
lutions are also known ( 3 0 ) .
250 300 350

Wavelength, nm.
Fig. 5 Molar ellioticity ( 8 ) of tetracycline
hydrochloride (-) & 4-erpitetracycline ammonium
salt (---+ as a function of wavelength in 0.03 N
HC 1
250 300 350 400 450

Wavelength, nm.
Fig. 6 Molar ellipticity ( 8 ) of anhydrotetracycline

hydrochloride (----) and 4-epianhydrotetracycline
(-1 as a function of wavelength in 0.03 N H C 1
612 SYED LAlK ALI
-T
-5
Fig. 7 Circular dichroism spectrum of

tetracycline in methanol-water ( 9 O : l O )
lot
Fig. 8 Circular dichroism spectrun of the calcium

complex of tetracycline in methanol-water ( 5 0 : 5 0 )
5.12. Differential Thermal Analysis

Stability studies of TC have been done with
differential thermal analysis (DTA) (31,
32). DTA was employed to study the effect of
certain additives, magnesium stearate, talc
and lactose on the stability of tetracy-
cline. The incorporation of magnesium stea-
rate has a profound influcence upon the cha-
racteristic thermogram features of TC (Fig
10). The analysis was performed with a ther-
mal analyzer with a heating rate of 50/min
(33). DTA was further used to identify some
vitamins a s the inactivating components in
formulations containing TC-HC1 and lactose
with uncoated and coated vitamins' granules.
The thermal analysis was done employing a
Mettler T.A. 2000 differential thermal ana-
lyser with a constant heating rate of
lOO/min (34).
6. Colour Reactions
To about 0 . 5 mg TC-HC1 when conc. sulfuric
acid is added a purplish-red colour is pro-
duced. On addition of 1 ml water the colour
changes to deep yellow (15). When a solution
of 2 mg TC-HC1 in 2 m l water is reacted with
5 drops of iodine solution, a brown volumi-
nous precipitate of periodide is obtained. 2
mg TC-HC1 dissolved in 1 ml dilute sodium
hydroxide are treated with 1 m l sulfanilic
acid and few drops of sodium nitrite solu-
tion. A brown-orange coloured solution is
obtained ( 3 5 ) .
7. Degradation and Stability
The dimethyl amino function at C 4 of TC-HC1
stands 1,3-diaxially eclipsed with respect
to C 12a OH group in acidic solutions. Epi-
merization takes place and the product
4-epitetracycline has no significant anti-
bacterial activity. The reaction takes place
most rapidly between pH 2 - 6 in aqueous solu-
tions and is a reversible first-order reac-
tion (36-42). In alkaline solutions using
precise conditions epitetracyclines are con-
verted almost comletely back to the bioac-
tive isomer in the presence o f chelating
metals (43). This is due to the complexing
metals tying together the C 4 -
614 SYED LAlK ALI
-tk---
i7L
I \
300 A(nm) 50
\ VI -41
.5 1
Fig. 9 Circular dichroism spectrum of the
magnesium complex of tetracycline in aqueous
solution
I At
100 200 300 400°C

Fig. 10 Thermograms of tetracycline 8, mixtures
with magnesium stearate; l.tetracycline, 2.
tetracycline + Mq. stearate (freshly prepared),
3. tetracycline + Mg.stearate (stored for 1
year)
dimethyl amino and C 12a - hydroxy functions

thus overcoming their steric repulsion (44).
Other groups which influence the epimeriza-
tion include phosphate (41), citrate (411,
urea (45) and some pharmaceutical ingre-
dients (46). Additionally the reaction is
catalysed by light. The equilibrium is
reached, depending on the reaction-medium,
at the level of about 40-50 % 4-epitetra-
cycline following the 1st order reaction ki-
netics. Further epimerization is strongly
dependent on pH, temperature and the buffer
system used (47). In a solution o f 0 . 2 mg
TC-HC1 in 10 ml water at pH 3.5 about 7%
epitetracycline is formed in 48 hours (48).
The C 6 - OH group is axial and antiperi-
planar trans to the H at C 5a s o that it is
sterically ideal for an elimination reaction
o f second order. The anhydrotetracycline is
produced due to dehydration at C 5a, 6. It
is progressively formed at pH's lower than
2 , specially upon heating. Anhydrotetra-
cycline possesses in vitro bioactivity but
has not found clinical applications (49-55).
The degradation of tetracycline itself is
superimposed by the concurring degradation
reactions of the primary tetracycline de-
gradation products. Epimerization of anhy-
drotetracycline (ATC) is faster and the de-
hydration of 4-epitetracycline (ETC) is
slower than the corresponding degradation
reactions of the intact tetracycline. Kine-
tics of dehydration of epitetracycline in
solution to form epianhydrotetracycline
(EATC) were studied using UV and visible
spectrophotometry. The reaction was found to
be first order with respect to epitetra-
cycline and hydronium-ion concentrations
(56). Degradation pathway of TC to ETC, ATC
and EATC is presented in Fig. 11 as reported
by Leeson (77). I n alkaline medium tetra-
cycline decomposes at the C 6 - OH site and
isoderivatives with phthalide structure are
obtained. The tetracyclines are stable in
neutral o r mildly acidic solutions. At pH
extremes, specially at elevated temperatures
bioactivity i s progressively lost (57).
Table 3 gives relevant stability data for
-N
I
z
r o a,
a
I
___,
11 11
the fermentation derived tetracycline.

Table 3 ~~
Stability Data for the Fermentation-derived

Tetracycline in Solution
Half-life Conditions Reference
2 min 0.2 N H2SO4/lOOO 58
1 min 1.0 N H2S04/1OOO 59
15.5 hr 1.0 N H2S04/250 40
6.8 min 0.1 N NaOH/1000 59
11 hr pH 10 buffer/220 58
101 min 0.1 N NaOH/600 40
12 hr pH 8.8 buffer/240 60
12 hr pH 8 . 8 5 phosphate buffer 61
The stability of TC-HC1 injection solutions
can be enhanced through the addition of sta-
bilising agents such as magnesium gluconate
(62). Generally bivalent and trivalent ca-
tions such as magnesium, calcium, mangnese,
copper, nickel, iron and aluminium form
chelates with TC which are mostly microbio-
logically inactive. This sort o f com-
plex-formation can be eliminated through the
addition of citric acid, polyphosphates and
glucoseamine hydrochloride (48). TC does not
decompose in glucose solutions in the pH
range 2-7.5 during a period of 6 hours. Pro-
pylene glycol, pyridin derivatives, pato-
thenic, lactic and tartaric acids may be
used as stabilising agents and the TC solu-
tions should be stored cool (47). Diffe-
rential thermal analysis was empolyed to
study the effect of certain additives such
as magnesium stearate. It was found that
magnesium stearate affects the stability o f
TC even in freshly prepared formulations
(33). Photodecomposition o f TC at oil-water
interfaces has been studied. When the oils
were irradiated in presence of TC, much lar-
ger amounts of peroxide were formed. This
increase in the peroxide content was presu-
mably due t o the formation of the oil-solu-
ble anhydrotetracycline derivatives (63).
The stability of TC and some TC-complexes in
suspensions has been tested.
618 SYED LAIK ALI
Maximum stability of TC and its complexes

with CaC12, urea and sodium hexameta-
phosphate has been reported in weakly acidic
medium (pH 4-5). Ascorbate anion influences
the stability negatively. In non-aqueous
vehicles the TC-urea-complex shows better
stability than TC itself. Sodium metabisul-
fite and sodium hydrogensulfite influence
the stability positively. Further it can be
increased by treating the TC-suspensions
with an inert gas (64). Studies of TC sta-
bility in aqueous solutions and the in-
fluence of pyridin derivatives, magnesium
salts and aliphatic and aromatic acids has
been extensively investigated by Kassem and
coworkers (62, 65). Commercial tetracycline
samples and formulations have been the sub-
ject of stability studies by various authors
(66, 67, 68). It was found that newly manu-
factured TC preparations contain only small
amounts of anhydrotetracycline and 4-epi-
tetracycline. Storage under adverse condi-
tions markedly increase the amount of de-
gradation products, but storage under normal
conditions results in only a slow increase
in anhydrotetracycline and 4-epitetra-
cycline. In syrups this can be correlated
with loss in tetracycline potency. Citric
acid was found to increase greately the ten-
dency of tetracycline degradation. TC-HC1 is
more stable than TC-phosphate (68). Epian-
hydrotetracycline could not be found over 1%
in any of the commercial formulations tested
( 6 6 ) . Problems of TC stability in eye-drops,
injections and ways and means to increase
this has been reported (69, 70, 71, 72).
Powder o r crystaline TC and TC-HC1 samples
darken in strong sunlight in a moist
atmosphere. In aqueous solutions TC and
TC-HC1 degrade by epimerisation which leads
to further decomposition products. It is ra-
pidly inactivated at a pH less than 2 and is
slowly destroyed at pH 7 and above. The rate
of degradation of TC to epianhydrotetra-
cycline is increased in the presence of cit-
ric acid (16). Influence of temperature on
the stability of solid TC-HC1
was investigated by Dihuidi and coworkers

( 7 3 ) . At 3 7 0 and 70 OC the stability was
studied over a period of 2 7 months and at 5 0
OC over a period of 16 months. At 37 OC
and 5 0 OC no decomposition is observed for
TC nor for its related substances. At
7 0 OC a distinct decrease in TC is obser-
ved as well as a small increase in anhydro-
tetracycline. It is inferred from these va-
lues that at 2 0 OC and under conditions of
low humidity TC-HC1 is a very stable product
having a tgo of at least 50 years but pro-
bably of about 3 5 0 years ( 7 3 ) . The stability
of solid samples of TC-HC1, TC and
TC-phosphate aged at room temperature for up
to 8 years has been followed. No appreciable
decomposition was observed except for
TC-phosphate which showed a significant
increase of 4-epitetracycline and anhydro-
tetracycline after a period of 4 years ( 7 4 ,
7 5 ) . Dony-Crotteux ( 7 6 ) reported that
aqueous TC solutions containing riboflavin
degrade in the presence of air and light. In
addition suppression of degradation by the
antioxidant ascorbic acid was observed. Some
of the potency loss encountered results from
epimerization catalysed by the buffer
system. Parenteral TC-formulations contai-
ning ascorbic acid may be mixed with ribo-
flavine for intravenous administration
without concern for antibiotic stability.
Although ascorbic acid itself appears to in-
duce another degradation pathway, the loss
is adequately covered by normal product
overage ( 7 6 , 7 7 ) . The dehydration of TC at
the C 5a-6 position as a function of acidity
was investigated at various temperatures
( 7 8 ) . TC epimerization kinetics has also
been investigated using NMR spectrometry
( 2 3 ) . The concentration of TC, ETC (epi-
tetracycline), ATC (anhydrotetracycline) and
EATC ( e p i a n h y d r o t e t r a c y c l i n e ) were studied
in pH 1.5 phosphate solution at four tempe-
ratures in a degradation-pattern investiga-
tion. The study showed that epimerization of
TC and ATC can take place at a low pH ( 7 9 ) .
Stability of TC and TC-HC1 in methanol at
26 OC has also been performed (130).
620 SYED LAlK ALI
Siewert and coworkers made a thorough study

of TC stability in large number of formula-
tions such as ointments, creams and hydro-
gels. All formulation showed a relatively
quick TC decomposition at temperatures above
50 OC. Preparations containing non-aqueous
ointment bases demonstrated a higher degree
of stability than aqueous ones like creams
and hydrogels. TC-HC1 decomposed quicker
than TC-base in aqueous systems (79a). In
another study of the same authors 14 sus-
pensions and 8 solutions were subjected to
an accelerated stability test. 1 2 out of 2 2
preparations were calculated to be stable
for a period of over three years under nor-
mal storage conditions. Two formulations de-
composed to an extent of more than 10% at 61
OC within one day. Suspensions did not
show a better stability than TC-containing
solutions (79b).
8. Related Impurities
Impurities of 4-epitetracycline, anhydro-
tetracycline and 4 - e p i a n h y d r o t e t r a c y c l i n e
have been limited in tetracycline hydro-
chloride by european pharmacopoeia (15),
british pharmacopoeia ( 8 0 ) to 4 % (ETC) and
0.5% each for ATC and EATC. USP XX (81)
allows the content of EATC up to 2 % . The
permissable content o f EATC in various TC
formulations such as capsules and injections
has been even increased to 3 . 0 % by USP X X .
’*
w a c y c l i n e s have a safety margin which
is quite satisfactory under most circum-
stances. Acute toxicity data f o r mice is
given for TC as following:
Route of Administration Acute Toxicity
LD50 (mg/kg)
oral 6300
IV 170
IP 2 80
Overaged TC preparations degrading t o EATC

show a definite toxicity. Fatal reactions
have been reported, although withdrawal of
the drug usually causes a slow reversal of

the damage. Expiration dates should then be
respected and TC should not be stored under
conditions of excess heat and humidity. The
LD50 of EATC is 4.8 times higher than that
of unchanged TC. The immunosupressive effect
is even reported to be 40-100 times higher
(83). A comparative toxicity of ATC has not
been reported. But the further degradation
of ATC to EATC takes place much faster than
the epimerisation of tetracycline.
10. Dissolution
The dissolution of TC was investigated using
in vitro dissolution system of Stricker.
Simulated gastric and intestinal juices were
used as dissolution media separately. A good
in vitro - in vivo correlation for TC was
obtained (84). The aminomethylation of TC, a
known process to make tetracyclines water-
soluble and useful for parenteral admini-
stration has no significant influence on the
bioavailability of the orally administered
antibiotic. The in vitro adsorption of
TC-HC1 was studied on various antacids,
namely magnesium trisilicate, magnesium
oxide, calcium carbonate, bismuth oxycar-
bonate, aluminium hydroxide and kaolin. The
reversibility of the adsorption process was
studied in different media and at pH values
similar to those of the gastrointestinal
tract. In general 0.0143 n NaHC03 was
found to possess higher eluting properties
than 0.01 N HC1 (85). The effect of various
additives, PEG 6000, PVP, CMC on the ad-
sorption of TC-HC1 on magnesium trisilicate
and milk was also studied. Conclusions were
made that the incorporation of some of these
additives may possibly reduce TC-antacid
interactions and consequently improve the
antibiotic availability when coadministered
with antacids ( 8 6 ) . The influence of addi-
tives on the in vitro release of drugs from
hard gelatin capsules has been studied. A
factorially designed experiment was used to
assess the total percentage of TC-HC1 re-
leased from hard gelatine capsules as the
function of a) diluent - the diluents being
622 SYED LAlK ALI
lactose, primojel and dry-flow starch - b)

the quantity of diluent c) the presence and
absence of magnesium stearate and d) the
presence and absence of sodium lauryl sul-
phate (87). In a further study in vitro re-
lease of TC-HC1 from hard-gelatine, soft-
gelatine capsules and TC-HC1 tablets in a
number of commercial formulations was in-
vestigated. Majority of the commercial pre-
parations conformed to the FDA regulations.
The paddle and the flow-through-cell systems
were used for the in vitro release of the
drug (88). BP 80 prescribes a dissolution
test for TC-tablets using a basket method
and 0.1 M HC1 as medium (88a). According to
FDA regulations (89) tablets and hard-gela-
tine capsules containing 250 and 500 mg
TC-HC1 should release in first 30 minutes
more than 60 and 50% of the drug repecti-
vely.
11. Microbiological Methods
'fn vitro antimicrobial spectrum of TC as mi-
nimum inhibitory concentration in pg/ml can
be given a s following (89a):
Ps.aeruginosa A.aerogenes Shigella Sonnei
10 1.66 2.5
S . typhosa Vibrio comma Kl.pneumoniae
1.56 0.58 1.56
S.aureus 5 S.aureus TC resist. Ps.vulgaris
0.21 60loo+
S.pyogenes Ps.Morganii E.coli
0.06 2 0.73
The antibacterial activity of TC expressed

in MIC-values (minimum inhibitory concen-
tration) for a large number of gram-positive
and gram-negative coccis, gram-negative
bacteria and other microorganisms have been
given in detail (89b). Microbiological assay
methods are used due to their sensitivity
but they are correspondingly nonspecific.
The method is based upon a comparison of the
inhibition of the growth of bacteria by
measured concentrations of the antibiotic to

be examined with that produced by known con-
centrations of a reference standard of known
activity. The assay for TC-HC1 may be car-
ried either by diffusion method or by turbi-
dimetric method.
11.1. Agar-Diffusion Method
The micro-organism used for TC is Bacillus
pumilus NCTC-8241 and Bacillus cereus ATCC
11778. Buffer solutions of pH 4.5 in both
cases and the incubation temperatures of 37
- 390 and 35 - 3 7 OC were used respec-
tively (90).
11.2. Turbidimetric Method
The growth of the test organism is measured
by means of a suitable optical apparatus.
The microorganism used for tetracycline are
Staphylococcus aureus NCTC 6751 and Staphy-
lococcus aureus ATCC 6538 P. The buffer s o -
lution with a pH of 4.5 and the incubation
temperature between 35 - 37 OC are used
(90, 91). Additives in TC formulations can
influence the results obtained by microbio-
logical methods (92). Comparison of the
microbiological results has been made with
those obtained through other methods (93,
94). Effects of certain tablet-formula-
tion-additives such as starch, bentonite,
veegum F , talc, liquid paraffin, stearic
acid etc. on the antimicrobial activity of
TC-HC1 has been investigated (95).
12. Methods of Analvsis
12.1 Titrimetry
Titrations of TC-HC1 in non-aqueous medium
are known. TC-HC1 is dissolved in glacial
acetic acid, 6% mercuric acetate solution
added and then titrated potentiometrically
with 0.1 N perchloric acid in dioxane (96,
97, 9 8 ) . Indirect volumetric determination
of TC-HC1 after formation of a tetra-
cycline-thiocyano-chrom (111)-complex
through oxidation with KMn04, KBr03 and
KIO3 has also been reported (99).
12 2 Visible and UV-Spectrophotometry
TC-HC1 can be determined after dissolving i t
in 0.25 N NaOH. The yellow colour of the s o -
lution has an absorption maximum at
624 SYED LAlK ALI
3 8 0 nm. Another method is the addition of

ferric chloride solution to a dilute HC1 s o -
lution of TC-HC1 which gives an orange-brown
colour with an absorption maximum at 4 9 0 nm
(100, 101). Another procedure, originally
developed f o r chlortetracycline, in which TC
is dehydrated by heating in acid and the re-
sulting solution is examined at 440 nm. This
is preferred to direct spectrophotometric
determination at 3 6 0 nm because there are
fewer interferences at longer wavelengths
(102, 103). The simplest method o f all is to
measure absorbance directly at 355 nm in di-
lute acid solutions (0.01N HC1) when rela-
tively "clean" pharmaceutical formulations
are involved. If dimethylformamide is used
as solvent, the method can also be applied
for TC determination in dragees (104). A
spectrophotometric method for determining
the amount of 4-epitetracycline is also
known ( 4 0 ) . The content of TC in presence of
ATC can be determined through the appli-
cation of absorbancy ratios, but the results
are higher compared to those obtained in
other studies (105). TC forms a rose colour
with diazobenzene sulfonic acid in acid s o -
lutions which can be measured spectrophoto-
metrically without interference from CTC and
OTC (106). Another dye reaction used for
analysis is the butanol soluble blue colour
developed by TC with p-aminodimethyl aniline
and sodium hypochlorite in neutral solutions
(107). A spectrophotometrically screening
method was used to screen powder, tablet and
capsule samples for the contents of ATC and
EATC in TC samples ( 6 8 , 108). TC and its de-
gradation products ATC and EATC have been
determined spectrophotometrically as the me-
tal complexes of titanium (1111, zirconium
(IV) and thorium ( I V ) salts at different
wavelengths (109). The method has some ad-
vantages in respect to sensitivity and
rapidity of measurements. Colorimetric de-
terminations of TC after chemical reactions
such as treatment with boric acid in sulfu-
ric acid (110) o r heating it with a mixture
o f sodium tungstate,
sodium carbonate and hydrogen peroxide (111)

or reacting it with zirconium oxychloride in
methanol and diazobenzoic sulphonic acid
(106) are known. Other colorimetric methods
for the determination of TC-HC1 involve the
use o f ferric thiocyanate reagent, nitro-
sation method and an iodobismuthate reagent.
TC-HC1 was determined in various formula-
tions such as powder, capsules and tablets
(1121, utilising these reagents. Spectro-
photometric determination of TC and its de-
gradation products ETC, ATC and EATC in
synthetic mixtures and some solid-dosage
forms on account of the differences in molar
absorptivities at the wavelengths 254, 260,
267, 357 and 390 nm has been reported (113).
A sensitive colorimetric method of analysis
for TC-HC1 and other antibiotics involves
the formation of a coloured complex between
TC and thorium (IV). The influence of pH and
time upon the stability of the complexes was
investigated. The method was applied to 29
different pharmaceutical dosage forms (114).
Complex formation of uranyl acetate with TC
has been utilized for its microdetermina-
tion. The mean stability constant of the 1:l
complex with absorption maxima at 404, 350
and 270 nm, as determined spectrophoto-
metrically, amounted to 1.2 x lo5, thus
permitting the use of this procedure for TC
+
microdetermination ( 1 1 5 ) .
12.3 Fluorimetr
quantum corrected fluorescence spectrum of
TC HC1 (11 mg/50 ml) in 0.05 N NaOH taken
with a Perkin-Elmer spectrofluorometer LS 5
is given in Fig. 12. Various fluorimetric
methods of determination of tetracycline
antibiotics are reported in literature (116,
117, 118, 119, 120). TC can be dehydrated to
ATC by heating in acid solutions and taking
the advantage of the greater lipophilicity
of ATC i t is extracted at about pH 4 - 5
with chloroform and then the fluorescence of
aluminium-chelate is measured (116, 121).
The content of TC has been determined
fluorimetrically in partially decomposed
aqueous solutions of different pH values.
Calcium and diethyl barbituric acid com-
plexes of TC were extracted into an
626 SYED LAIK ALI
E
3
Z I
EXCITATION
SPECTRUM
60
NANOMETERS
Fig. 12
Fluorescence Spectrum Of Tetracycline Hydro-
chloride in 0.05 N NaOH, Perkin-Elmer
Spectrofluorometer LS 5
organic solvent and the fluorescence inten-

sity was determined by using an excitation
wave length of 365 nm mercury line and an
emission wave length of 530-550 nm (glass
filter). Strongly fluorescent extractable
complexes of calcium and barbituric acid are
formed only with undecomposed biologically
active TC. Decomposition products of TC do
not form such complexes o r they are not
extractable with an organic solvent (122).
Spectrofluorimetric determination of TC in
serum and urine is performed after isolation
through adsorption on A1203 at an ex-
citation wavelength of 335 nm and emission
wavelength of 414 nm. The assay is based on
the strong fluorescence of a degradation
product o f TC formed on heating in alkaline
solution. 0.2 - 5 g TC/ml in serum and 5 -
P
5 0 ug/ml in urine can be determined (123).
Fluorimetric assay of TC in mixtures of an-
tibiotics is performed by formation of
strongly fluorescent aluminium-TC complex
without prior extraction o r separation of
individual antibiotics. Estimation was done
by measuring fluorescence at 465 nm excita-
tion and 555 nm emission wavelengths (124).
TC-HC1 is determined fluorimetrically after
addition of perchloric acid t o its solution
in acetic anhydride as well as after ex-
traction of ATC at pH 4 . 5 with chloroform.
The excitation wavelength of 365 nm and
emission wavelength 5 3 0 - 5 5 0 nm (glass fil-
ter) were used for measurements (125). TC
formed ternary calcium complexes with bar-
bital sodium and tryptophan in aqueous s o -
lutions. The fluorescence of TC and its com-
plexes was measured at the emission maximum
of 5 1 2 nm with the excitation wavelength of
390 nm (30). Fluorimetric determination o f
TC in biological materials is based on sol-
vent extraction with ethyl acetate of mixed
tetracycline-calcium-trich-
loroacetate-ion-pairs from aqueous solution.
The excitation and emission maxima are re-
ported to be 400 nm and 500 nm respectively.
Detection limits of 0.05 and 0.125 g/ml
were obtained (126, 127). I"
628 SYED LAIK ALI

Paper Chromatography
Paper partition chromatographic methods have
been widely applied to the analysis of te-
tracyclines (128, 129). Pharmaceutical
aqueous suspensions for oral use are acidi-
fied with HC1 and diluted with methanol.
Crystalline formulations are dissolved only
in methanol. A paper chromatographic method
for TC determination in pharmaceutical pre-
parations is based on the complexation of
the antibiotic with a mixture of urea and
disodium edetate on paper at pH 7.4. Urea
helped in the separation of degradation pro-
ducts and led to the formation of well de-
fined spots (130). Samples from fermenta-
tions must be acidified with oxalic acid to
liberate TC from the mycelium. TC in fil-
trates may be precipitated in saturated s o -
lution of sodium tetraphenyl borate, preci-
pitate dissolved in ethyl or butyl acetate
and applied for paper chromatography. Va-
rious solvent systems and hRF values for
paper chromatography are given in Table 4 .
Table 4
Solvent System hRF Reference
n-butanol-acetic acid- 68 TC 131
water (4:1:5)
n-butanol-conc. NH4OH- 24 TC 132
water (4:1:5)
chloroform-nitromethane-
pyridine (10:20:3) on
paper impregnated with 28 TC 133
Mcllvaine's citrate-phos-
phate buffer pH 3.5
chloroform-n-butanol (4:l) 49 TC 134
saturated with Mcllvaine's
buffer pH 4.5
n-butanol-acetic acid 65 TC
water (4:1:5) on paper 65 ETC 135
impregnated with EDTA and 87 ATC
dried 87 EATC
(continued)
TETRACYCLINE HYDROCHLORIDE
Table 4 (continued)
n-butanol-ammonia-water 39 TC 135
(4:1:5) on paper impreg 15 ETC
nated with EDTA and dried 62 ATC
40 EATC
n-butanol-chloroform (9:l)
saturated with O.15M 34 - 42 TC 136
buffer pH 3.0, Whatman
no. 4 paper impregnated
with a 3.7% solution of
chelaton I11 and wetted
with aqueous phase
ethyl acetate-acetone (4:l)
saturated with disodium
edetate and urea; What 37 TC
man no. 1 paper impreg-
nated with a mixture 45 TC-!-ICl 130
(1:3) of 10% urea in
Mcllvaine's buffer pH 98 ATC
7.4 and 5% disodium ede-
tate in the same buffer 2 EATC
Detection is made in UV light. The yellow
fluorescence of tetracyclines and their epimers in
UV light is greatly enhanced by exposing the paper
to ammonia vapours. They may also be detected by
Ehrlich's reagent or by starch-iodine following
N-chlorination (137).
Column Chromatography
A partition chromatographic procedure was develo-
ped for the assay of tetracycline mixtures. Washed
diatmaceous earth impregnated with buffered sta-
tionary phases containing EDTA have been used to
separate and quantify TC mixtures in presence of
degradation products. The compounds are eluted
from a column of Celite 545 coated with a pH 7
buffer containing EDTA, glycerine and PEG 400 and
then determined spectrophotometrically (138, 139,
140). ATC and ETC were determined in commercial
and aged TC formulations through column chroma-
tography using Celite 545 o r Kieselgur moistened
with EDTA in buffer solution as stationary phase
and chloroform, methanol and n-butanol etc as
eluents (68, 141, 142, 143).
630 SYED LAIK ALI
Sephadex G-25 buffered at alkaline pH's has

been found to possess excellent resolving
power in some cases (144, 145, 146). A co-
lumn liquid chromatographic method f o r de-
termining TC in various dosage forms was
automated. Each chromatogram consisted of a
continuous flow separation of components
followed by a spectral determination of the
column eluate at a rate o f 12 samples per
hour (147). Gel chromatography has also been
applied to the analysis of TC antibiotics
using a Bio-Gel P - 2 column with 0.1M acetic
acid as an eluent (148).
Thin Layer Chromatography
TLC h a s been applied in european pharma-
copoeia for the identification of TC-HC1 as
well as for detection of impurities and re-
lated products. TLC plates were prepared
with a slurry of kieselguhr and sodium ede-
tate solution adjusted to pH 7 o r 7.5 and
mobile phases of chloroform + ethyl acetate
+ acetone (4:4:2), previously saturated with
0.1M sodium edetate,and water + ethyl ace-
tate + acetone (1:3:23) were used for iden-
tification and detection of impurities re-
spectively (15). TLC has been widely used
for the analysis of TC and related products
(ETC, ATC, EATC) with various stationary
phases and solvent systems. Stationary
phases impregnated with EDTA solutions have
been chosen often to avoid the complexation
of TC with metal cations. Lloyd and Cornford
(149) described an improved TLC limit test
for the detection of ATC and EATC in TC
using cellulose layer as stationary phase,
buffered before development by spraying with
a buffer solution (0.1 M disodium EDTA - 0.1
NH4C1), and chloroform as mobile phase,
previously saturated with the same buffer
solution. The Rf-values for TC, ATC, EATC
were found to be 0.25, 0.93 and 0.4% re-
spectively (149). A method for quantitative
analysis of TC-HC1 on microcrystalline cel-
lulose thin layer plates is described (150).
Determination of ATC and EATC in microgram
range in degraded TC tablets through TLC is
also reported (151).
Table 5 gives a number of stationary phases,

solvent systems and where available
hRf-values for TC and related products for
TLC.
TLC has been used for qualitative and quan-
titative assay of degradation products of TC
in TC-HC1, commercial formulations of va-
rious dosage forms such as powder, tablets
and capsules etc. Determination has been
performed after scratching the zone from TLC
plates, elution of the drug and spectro-
photometric measurement at 430 nm (155). Di-
rect fluorimetry of the spots on TLC plates
applying a 360 nm excitation and 540 nm
emission wavelengths has been also perfor-
med. A linear relationship was found between
the concentration of TC and related com-
pounds in the range of 0.05-1 g and
P
fluorescence intensity (157, 59, 160, 161,
162, 163). Chromatograms may be detected by
bioautography with Bacillus subtilis using
microbiological techniques (137, 169, 170,
171). Spraying with methanolic solution of
ferric chloride (dark greyish spots), with
antimony trichloride solution, MgC12 in
ethanol or methanolic triethanolamine solu-
tions have been reported (137). Aqueous fast
blue solution, diazotized p-nitroaniline,
modified Sakaguchi reagent (boric acid in
conc. sulfuric acid) and diphenyl picryl-
hydrazyl spray reagents have also been used
for detection of TC and degradation products
on TLC plates. Coloured spots on white back-
ground or yellow spots on a bluish-white
background were obtained (156). The tetra-
cyclines have been most commonly detected by
their fluorescence under long wavelength UV
light at 365 nm either with or without ex-
posure of the chromatograms to ammonia va-
pours ( 8 0 , 162, 164, 165, 168). TC-HC1 was
allowed to epimerize in phosphate and ace-
tate solutions at room temperature and the
reaction products were studied at suitable
time intervals by TLC (156).
High Performance. Liquid Chromatography
HPLC can seDarate and resolve TC-HC1 from
its degradaiion products~ETC,ATC, EATC sen-
sitively and reproducibly. The technique
Table 5
Stationary phase Mobile Phase References
hRF-values
Kieselguhr, impregnated with dichloromethane - ethanol 95 % 152
citrate-phosphate buffer pH (9:l); TC:55-65; ETC:10;
5 . 5 containing 10% glycerine ATC:100; EATC:85;
Silica Gel G n-butanol-oxalic acid-water 153
(100:6:100); TC:38;
Silica Gel G n-butanol-tartaric acid- 153
water (100:6:100); TC:26;
Silica Gel impregnated with n-butanol saturated with 153
EDTA-water and activated water; TC:23
Kieselguhr G, impregnated Chloroform-acetone(1:l) 154
with glycerol-phosphate- saturated with the same
citrate buffer, pH 3.7 buffer
Kieselguhr impregnated with Acetone-water (1O:l) 155
5% aqueous EDTA solution TC:69; ETC:29
adjusted to pH 9 with NaOH ATC:84; EATC:55;
The same sorbent as above, acetone-ethyl acetate- 155
but adjusted to pH 7 . 5 water (20:10:3); TC:71
ETC:38; ATC:88; EATC:46;
Kieselguhr G impregnated methyl ethyl ketone saturated 156
with EDTA+PEG 400+glycerine with Mcllvaine's buffer pH 4.7
TC:53; ETC:20; ATC:93; EATC:47;
Same as above dichloromethane-ethyl formate- 156

ethanol (9:9:2) saturated with
buffer pH 4 . 7 ; TC:36; ETC:12
ATC:83; EATC:50;
Kieselguhr Impregnated with ethylene glycol-water-acetone- 157
mobile phase ethyl acetate (2:2:15:15)
TC:50; ETC:28; ATC:95; EATC:30;
?
'd
Cellulose TLC plates impreg- n-butanol saturated with water 158
nated with EDTA, pH adjusted TC:18; ETC:10; ATC:23; EATC:23;
to 9.0
Kieselguhr G impregnated MIBK-ether-water-methanol- 159
with 0.1M EDTA ethylene glycol (50:60:3:7.5:2.5)
TC:16; ETC:4; ATC:83; EATC:22;
Microcrystalline cellulose aqueous 0.25M MgCl2 160

plates, Merck TC:72; ETC:68; ATC:29; EATC:16;
Same a s above aqueous 0.20M CaC12 160

TC:67; ETC:63; ATC:25; EATC:25;
Table 5 continued
~ Same as above aqueous 0.25M BaC12 160
w
P TC:63; ETC:60; ATC:24; EATC:17;
Same as above aqueous 0.15M ZnCl2 160
TC:65; ETC:65; ATC:22; EATC:18;
Microcrystalline cellulose twice in ethyl acetate 161
impregnated with phosphate- saturated with water
citrate buffer and subseque- TC :35
ntly submerged in methanolic
ethylene glycol solution
Powdered cellulose m TLC plates chloroform saturated with 162
impregnated with 0.1M 0.1M EDTA, after develop-
EDTA rnent exposed t o ammonia
TC:6-23(band); ETC:3.5;
ATC:92; EATC:52;
Kieselguhr plates impreg- acetone-ethyl acetate-water 163
nated with EDTA, adjusted (20:10:3); TC:10; ETC:40;
to pH 7 . 5 ATC:95; EATC:50;
Kieselguhr G impregnated ethyl methyl ketone saturated 164, 165
with 0.1M EDTA with Mcllvaine buffer pH 4 . 7
or alcohol; later plates
exposed to ammonia; TC:56; ETC:30;
Microcrystalline cellulose 0.1M EDTA with 0.1% NH4C1; 166, 167
on TLC plates chloroform saturated with
EDTA-NH4Cl solution;
TC:72; ATC:36; EATC:52;
m
Kieselguhr G treated with ethyl acetate saturated with 168
a buffer containing 0.1M 0.1M EDTA at pH 7.0
EDTA at pH 7.0, glycerine
and PEG 400
636 SYED LAIK ALI
has been successfully applied for the quan-

titative assay of TC and related compounds
in all sorts of commercial solid and liquid
dosage forms, stability studies, in body
fluids, pharmacokinetics and drug metabolism
investigations. Quite often tests for iden-
tity, potency and assay determination are
performed with this method. Micro to sub-
microgram amounts could be determined with
ease. Different modes of HPLC, adsorption,
reversed phase, ion-exchange and paired-ion
chromatography have been applied. Butter-
field separated various tetracyclines using
an ion-exchange system (172). Later workers
included EDTA in the system to prevent bin-
ding with the metal columns (75). Advantages
of reversed phase HPLC over ion-exchange
techniques have been given (173). Serum le-
P
vels of as little as 0 . 3 g/ml TC have been
determined through HPLC 174). A preliminary
study of the effectiveness of ion-exchange,
adsorption, liquid-liquid partition and re-
versed phase ion-pair chromatography indi-
cated that only the last method showed pro-
P
mise. A 18 m partisil column gave plate
heights in the range of 0.15 - 0.3 mm. The
method was used to quantify the impurities
in TC at round the 1% level (175). T C s may
be separated with high efficiency (plate
heights around 0.05mm) using SASand ODs-Hy-
persils in the pH range 3-5. Within this pH
range the presence of EDTA at a concentra-
tion of about M is essential for s a -
tisfactory chromatography. EDTA is thought
to act as a zwitterion pairing agent with
the zwitterionic form of te TCs (176). Table
6 gives various columns, mobile phases etc.
reported in literature for the analysis of
TC-HC1 and TC.
Concentrations as low as 4 g/mg TC in urine
were quantitated accuratelK TC was extrac-
ted from urine as calcium comlex ( 1 8 3 ) . Cal-
cium complex of TC was extracted from urine
and plasma with ethyl acetate and then re-
extracted into HC1. Concentrations of less
than l p g / m l in urine and 1.5 pg/ml in
plasma were determined with a relative stan-
dard deviation of less than 5 % (184).
rETRACYCLINE HYDROCHLORIDE 637
The addition of phenylbutazone to urine o r

plasma enhances the extraction of these com-
pounds by ethyl acetate. The formation of
p h e n y l b u t a z o n e - t e t r a c y c l i n e ion pairs and
their role in extraction process is dis-
cussed (185). C 18 reversed phase column was
used with the mobile phase water-acetoni-
trile-perchloric acid in two steps with
varying amounts of acetonitrile. Six diffe-
rent TC preparations analysed contained ETC,
ATC and EATC from undetectable to 7.78%
(186). Limits of detection for ATC and EATC
in TC were found to be 12 and 7 ng respec-
tively through HPLC analysis (187). TCs can
be extracted from serum o r organ homogenates
by means of acetonitrile containing buffers.
The lowest concentrations of the intact mo-
lecule detectable are 0.2 pg/ml serum o r
zr
blood and 0.4 g/g organ. HPLC analysis
yielded the s me results as microbiological
method (191). Reversed phase ion-pair
chromatography with the use of tertiary
amines as counter ions has been applied for
the separation of TC analogs and their po-
tential impurities (192). Retention of TC
can be regulated by the addition of organic
ammonium compounds to the mobile phase in
reversed phase ion-pair chromatography
(194). The addition of inorganic anions
C1-, Br-, ClOT as their sodium salts
to the mobile phase and their influence on
the retention of TC has been studied (194).
TC has been extracted from plasma as an
ion-pair with tetrabutyl ammonium hydrogen
phosphate into chloroform-1-heptanol and af-
ter reextraction into an acidic aqueous
phase the separation was performed through
reversed phase HPLC. Analysis of urine was
done by direct injection (193).
Table 6
Column Mobile Phase Detection Application References
Pellicular 0.003M EDTA in water-
cation-ex- ethanol (60:40) TC in formu- 177
change resin 254 nm lat ions
6 ;ygaPa;k 0.02M phosphate buf-
fers, pH 2 - 5 in water- TC im pharm. 178
P acetonitrile gradient 280 nm preparations
TMS bonded 0.1M HClO4 in water- separation 179
WLCU Si gel acetonitrile gradient o f TC, ETC,
w
QI
m
85:15 to 70:30 280 nm ATC, EATC
Micropak CH water-methanol-0.02M
phosphate buffer (pH
7.6) and 1mM EDTA, gra- TC from peni-
dient 25-50% v / v methanol 267 nm mocycline 180
Vydac RP 18
and Lichrosorb 0,15M (NHq12C03-
RP 8 acetonitrile (96:4) pH
adjusted t o 8 . 4 with
ammonia; citrate-phos- separation of
phate buffer (pH 2.20)- TC from rela-
acetonitrile ( 6 5 : 3 5 ) 350 nm ted compounds 181
lo rm
Vydac RP 18 isopropanol-diethano-
lamine-phosphate ammo-
nium EDTA-water
254,280,365 TC from deg-
405 nm radation
products
182
TMS-bonded 0.1M HC104 o r O.lM 2 8 0 nm separation 175

partisil 18 H2SO4 or 0.05M of TC, ETC
HNO3 - acetonitrile
P" (85:15 t o 70:30)
ATC, EATC
Zipax HCP 0.001M EDTA, 0.01M 2 8 0 nm separation 75

polymer H3PO in water-metha- of TC, ETC,
lon 487313, v / v > ATC, EATC
ODs-Sil-X-1 0.005M EDTA, 0.05M 2 8 0 nm TC 173
(NH4)2C03 in water-
methanol ( 9 2 : 8 )
Pellionex CP 0.1M Na+, 0.002M EDTA in TC, ETC,
172
water-ethanol (70:30) ATC, EATC
ODs-Hypersil, water-acetonitrile-dime- 272 nm TC, ETC, 176
thyl-formamide-0.001M 2 8 0 nm ATC, EATC
Na2EDTA-citric acid
P and acetic acid, pH a d -
justed to 3.0 or 5.0
continued
Table 6 continued
Ion-X SA, 0.005M EDTA, 0.05M NaCl 375 nm TC in urine 183
conditioned in water-methanol, 95:s extracts as
with EDTA and 70:30 calcium corn-
plex
0.01M NaH2P04 in 355 nm TC in urine 184+185
water-acetonitrile 60:40 plasma,
pH 2.4 tissues as
calcium com-
plex
a.
P
O ODs-Sil-X-1 Water-acetonitrile-per- 254 nm TC, ETC, 186
chloric acid 76:22:1.8; ATC, EATC
lo r"" 61:37:1.8
Zipax SCX 0.3M EDTA buffer, pH ad- 429 nm ATC, EATC 187
justed to 7.0 in TC
lo P"
Lichrosorb 10% acetonitrile in 0.1M 357 nm TC in mix- 188
NHz, RP 8, phosphoric acid with tures
5 and 10 im
Lichrosorb
t 1 0 - 3 ~HIBS
acetonitrile-0.1M citric 3 5 0 nm Re tent i on 189
RP 8, 5 m acid 25:75 mechanism
r" of TCs
Bondapack a step gradient of 12-22% ETC, ATC, 190
{henyl C 18, acetonitrile in 0.2M EATC in TC
phosphate buffer at pH 2.2 powder and
capsules
Nucleosil 25% acetonitrile-0.01M 270, 357 nm TC in blood 191
C 8, 10 m NaHzP04 , p1-i adjusted and organs
I" to 2.4 o f animals
L ichrosorb phosphate buffer, tri- 280 nm separation 192

r
RP 8 , 5 m propylamine o r N,N-di-
methyloctylamine, pH 8.0,
with 20-35% acetonitrile
of TC and
analogs and
their impu-
rities
Lichrosorb 0.01M phosphoric acid in 357 nm ion-pair LC 193,194
RP 2, 8, 18, water-35% acetonotrile of TC, TC in
organic ammonium compounds plasma and
in mobile phase urine
methanol-water-1 M phos- 280 nm influence o f 195
phoric acid, gradient temperature
elution on solid TC
642 SYED LAIK ALI
Gas Chromatography
The GLC determination of TC, ETC, ATC and
EATC in aged TC samples has been reported.
Trimethylsilyl derivatives of TCs were formed
and gas chromatographed on six different co-
lumns, 3% OV-1, 3% JXR, 3 8 SE 52, 3% OV-17,
10% OV-25 and 10% OV-210. A 6 feet 3% JXR co-
lumn at 260 OC isotherm separated most of
the compounds. Different TC-HC1 powder
samples were analysed through GLC for their
degradation products and the results compared
with those obtained from microbiological and
UV assay methods (74).
12.5 Polarogra h
T Z T Z d & $ antibiotica have been determined
qualitatively and quantitatively through po-
larographic methods (196, 197). Polarographic
behaviour of tetracycline and some of its de-
gradation products has been investigated in
phosphate-acetate buffer solutions. The first
reduction wave is attributed to the carbonyl
group at C 1 and the second reduction step
corresponds to the conjugated system at C 11
- C 12 (198). Oscillopolarographic methods
have been also applied for the analysis of
tetracycline and its degradation products
(199, 200). Optimum conditions for the dc
polarographic reduction of TC were studied. A
boric acid-sodium borate buffer provided the
best conditions for the elctro-reduction o f
TC. Quantitative determination in various
dosage forms were carried on (201). TC can be
analysed through polarography and differen-
t i a l - c a t h o d e - r a y p o l a r o g r a p h y in a supporting
electrolyte o f 0.1M diethyl barbituric acid
in 40% methanol and 0.1M lithium chloride at
pH 4.7 (202). TC-HC1 gives a polarographic
step in a phosphate buffer solution, pH 6.2,
with a half-wave potential of -1.45 to -1.51
V (203). A method involving ac polarography
is also reported in literature (204). Elec-
trochemical methods for the determination of
TC have been reviewed by Unterman and Hughes
(205, 206).
13, Drug Metabolism and Pharmacokinetics
TC-HC1 is incompletely and irregulary absor-
bed after oral administration. The
presence of metal ions such as calcium, mag-

nesium, aluminium, zinc and iron reduce its
absorption due to formation of chelates and
coordination compounds. Alkalis also lower
the serum concentration. Rectal absorption
from suppositories is unsatisfactory. Ab-
sorption from stomach is minimal. Absorption
from the intestines is incomplete and subject
to considerable variation between individuals
and is affected substantially by the contents
of GI tract. Milk and food reduce TC absorp-
tion, phosphates may enhance it. Some prac-
tical improvement in absorption appears to
have resulted from the exclusion of "inert"
magnesium stearate or calcium carbonate from
TC capsules (207, 208, 209, 210). Therapeutic
blood concentrations are achieved more rea-
dily by increasing the frequency of admini-
stration than by increasing the dose. Thera-
peutic serum concentrations lie in the range
g/ml whilst the toxic concentration
Of "-"9
is abo t 12 g/ml o r over. Serum concentra-
tions of l-tyg/ml are maintained when oral
doses of 250 500 mg are administered daily.
An intramuscular dose of 100-250 mg brings
about serum concentration of 2 - 4 g/ml during
P
1-2 hours. A serum concentration of 5-30
g/ml is reached 30 minutes after an intra-
P
venous dose of 250-300 mg TC. Serum half-life
is about 9 hours when given orally in a sing-
le dose (16). A mean serum half-life is about
11 hours when administered orally in divided
doses (211). TC is secreted in milk and sa-
liva and crosses the placents. It is readily
taken up by newly formed bones and teeth in
which it forms a TC-calcium orthophosphate
complex. It remains bound to malignant cells
for a longer time than to non-malignant
cells. TC is excreted both in urine and bile.
The concentration in bile may reach 10 to 20
times that of blood (212, 213). About 208 is
excreted in urine in 24 hours, mainly as
unchanged drug, after oral administration and
50% after intravenous doses. The average con-
centration of TC in urine after oral and im +
i v administration is in the range of 100-300
rg/ml. Urinary excretion is increased when
634 SYED LAIK ALI
urine is alkaline (16). TC appears to be

excreted in the urine by glomerular
filtration as its half-life is strongly
dependent upon the extent of pro-
tein-binding. TC bounds to plasma proteins in
the range of 20-70% (16). It is also bound
reversibly to serum proteins. In serum 53% TC
is in the form of complexes with protein.
Among the bound TC, 54% is with albumin, 1 3
and 19% with low and high density lipopro-
teins respectively, 6% with very high density
lipoprotein and 8% is bound with other serum
proteins. TC appears to dissolve in the lipo-
philic portion of the lipoprotein molecule,
rather than t o be associated with specific
binding sites (214).
Acknowledgement
The author thanks Mr. Martin Siewert, Pharma-
cist, f o r providing some useful literature
and Mrs. Monica Hediger f o r typing the manus-
cript.
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VITAMIN D3
(CHOLECALCIFEROL)
K. Thoinas Koshy and William F. Beyer
The Upjohn Coinpaizy
Kalnmazoo, Michigan
I. Description 656
1.1 Name 656
1.2 Formula and Molecular Weight 656
1.3 Storage and Handling 656
I .4 Brief History 656
1.5 Biosynthesis 658
1.6 Potency Definition 658
2.1 Spectra 660
2.3 Solubility 672
3. Chemical Properties 672
3. I Chemical Synthesis 672
3.2 Previtamin D3-Vitamin D3 Equilibrium 677
3.3 Cis-Trans Equilibrium 677
3.4 Other Chemical Reactions 679
4. I Identification Tests 679
4.2 Quantitative Methods 680
5. Metabolites of Vitamin D3 70 1
5.1 Primary Metabolites 70 1
5.2 Secondary Metabolites 704
5.3 Metabolites of Very Low or Unknown Biological Activity 705
6. Vitamin D3 as Prohormone 705
References 707
ANALYTICAL PROFILES OF DRUG SUBSTANCE5 Copyright 0 1984

VOLUME 13 655 by the Americae Phannaceutlcal Association
ISBN 0-12-260813-5
656 K . THOMAS KOSHY AND WILLIAM F. BEYER
1. Description
1.1 Name: V i t a m i n D
Generic Name: C2olecal c i f e r o l
Chemical Name: 9,lO-Secochol esta-5,7 , l o ( 1 9 ) - t r i en-
38-01 ,(38,52,7E)-,
1.2 Formula and M o l e c u l a r Weight
The chemical s t r u c t u r e o f v i t a m i n 03 i s c l o s e l y
r e 1ated t o i t s p r e c u r s o r , 7-dehydrochol ester01 , from which
i t i s produced by a photochemical r e a c t i o n . Therefore,
vitamin D i s closely related s t r u c t u r a l l y t o the four-ring
nucleus o! s t e r o i d s d e r i ved from t h e c y c l opentanoperhydro-
phenanthrene r i n g system. No v i t a m i n D a c t i v i t y i s n o t i c e d
u n t i l t h e B r i n g o f 7-dehydrocholesterol i s opened between
C-9 and C-10. Thus, v i t a m i n D3 i s a 9,lO-seco s t e r o i d and
i t s carbon s k e l e t o n i s numbered a c c o r d i n g l y (Scheme I ) . The
i m p o r t a n t aspects o f i t s c h e m i s t r y c e n t e r about t h e 5,6,7-
c i s - t r i e n e s t r u c t u r e . The formula f o r v i t a m i n D3 i s C27H44O
and i t s formula weight i s 384.64.
1.3 Storage and Handling
C r y s t a l l i n e v i t a m i n D3 i s s t o r e d i n h e r m e t i c a l l y
sealed c o n t a i n e r s under n i t r o g e n i n a cool p l a c e and pro-
t e c t e d from l i g h t . Under such c o n d i t i o n s , d e y r a d a t i o n i s
n e g l i g i b l e f o r one year. V i t a m i n D 3 e x i s t s i n thermal
e q u i l i b r i u m w i t h p r e v i t a m i n D3 and t h e r a t e o f e q u i l i b r a t i o n
markedly i n c r e a s e s w i t h temperature and t i m e ( 1,Z).
1.4 Brief History
The symptoms o f v i t a m i n D d e f i c i e n c y disease has

been documented i n 1 6 t h c e n t u r y l i t e r a t u r e . A c l e a r p i c t u r e
o f t h e b a s i s f o r t h e disease and methods o f t r e a t m e n t were
u n c l e a r u n t i l t h e experiments by S i r Edward Mellanby
(3,4). I n t h e e a r l y 1920's, cod l i v e r o i l was known t o c u r e
r i c k e t s and xerophthalmia. The name v i t a m i n D was g i v e n t o
VITAMIN D3 (CHOLECALCIFEROL) 657
SCHEME I: Vitamin D3 structure with hydrocarbon

number assignments.
t h e a n t i r a c h i t i c substance i n cod l i v e r o i l ( 5 - 7 ) and d i f -

f e r e n t i a t e d from v i t a m i n A by t h e f a c t t h a t v i t a m i n A a c t i -
v i t y i n t h e o i l was l o s t by h e a t i n g w h i l e t h a t o f D was
not . Hess and Steenbock d i s c o v e re d , i n d e p e n d e n t l y ( 8 - l l ) ,
t h a t t h e a n t i r a c h i t i c a c t i v i t y o f t h i s v i t a m i n c o u l d be
induced by UV i r r a d i a t i o n o f r a c h i t i c animals o r t h e i r
food. Steenbock's d i s c o v e r y t h a t a n t i r a c h i t i c a c t i v i t y
c o u l d be induced by i r r a d i a t i o n o f foods, p a r t i c u l a r l y o f
the sterol fraction, ultimately led t o the i d e n t i f i c a t i o n o f
t h e s t r u c t u r e o f v i t a m i n D and t o t h e e r a d i c a t i o n o f r i c k e t s
as a major medical problem (9,10,12).
658 K . THOMAS KOSHY A N D WlLLIAM F. BEYER
It was l a t e r d i s c o v e r e d t h a t v i t a m i n D occurs i n two

a c t i v e forms, e r g o c a l c i f e r o l and c h o l e c a l c i f e r o l . Ergocal-
c i f e r o l i s t h e s y n t h e t i c form d e r i v e d by t h e i r r a d i a t i o n o f
e r g o s t e r o l and i s designated as v i t a m i n D2 (13,14). Chole-
c a l c i f e r o l , t h e n a t u r a l form, was i d e n t i f i e d (15,16) and
designated as v i t a m i n D3. Vitamins D and D3 a r e e q u a l l y
a c t i v e i n humans and o t h e r mammals, a u t D2 i s v i r t u a l l y
inactive i n poultry.
1.5 B i osynthesis
It has been known f o r a l o n g t i m e t h a t v i t a m i n D3

i s synthesized i n t h e s k i n when exposed t o s u n l i g h t from 7-
d e h y d r o c h o l e s t e r o l (7-DHC, P r o v i t a m i n D ). The sequence o f
steps l e a d i n g t o t h e cutaneous photosyn?hesis o f v i t a m i n D3
from 7-DHC was r e c e n t l y demonstrated (17). I n Caucasian
s k i n , a l l of t h e epidermal s t r a t a c o n t a i n 7-DHC. When
ir r a d i ated, 7-DHC i s photochemical l y converted by a f a s t
r e a c t i o n t o p r e v i t a m i n D throughout t h e epidermis and t h e
dermis. Once formed i n ?he s k i n , p r e v i t a m i n D3 i s isomer-
i z e d t o D by a slow nonphotochemical rearrangement a t a
r a t e d i c t a z e d by s k i n temperature. V i t a m i n D3 i s then bound
t o a s p e c i f i c b i n d i n g p r o t e i n (DBP) and t r a n s p o r t e d from t h e
s k i n i n t o t h e c i r c u l a t i n g blood. Thus t h e s k i n serves as
t h e source o f 7-DHC, a r e s e r v o i r f o r t h e s t o r a g e o f t h e
p r i m a r y photoproduct, p r e v i t a m i n D3, and t h e organ where t h e
slow thermal conversion o f p r e v i t a m i n D3 t o D3 occurs. The
t h i r d process p e r m i t s t h e s k i n t o c o n t i n u o u s l y s y n t h e s i z e D3
and r e l e a s e i t i n t o t h e c i r c u l a t i o n f o r up t o 3 days a f t e r a
s i n g l e exposure t o s u n l i g h t . Scheme I 1 (17) i l l u s t r a t e s
t h i s sequence o f events.
1.6 Potency D e f i n i t i o n
The o r i g i n a l r e f e r e n c e s t a n d a r d o f v i t a m i n D was a
s o l u t i o n of i r r a d i a t e d e r g o s t e r o l i n o l i v e o i l . The present
i n t e r n a t i o n a l standard i s a s o l u t i o n o f p u r e c r y s t a l l i n e
c h o l e c a l c i f e r o l i n o l i v e o i l c o n t a i n i n g 0.025 pg/mg o f
solution. One i n t e r n a t i o n a l u n i t ( I U ) i s e q u i v a l e n t t o
0.025 pg o f c r y s t a l l i n e D3 o r 1 pg o f D3 i s e q u i v a l e n t t o 40
IU. The USP r e f e r e n c e standards a r e c r y s t a l 1ine e r g o c a l -
c i f e r o l o r c h o l e c a l c i f e r o l packaged i n sealed ampules. The
USP standards and t h e i n t e r n a t i o n a l standards a r e e q u i v a l e n t
i n t h a t 1 pg o f each i s e q u i v a l e n t t o 40 IU.
7-Dehydrocholesterol
HjC
Previtamin D3
A,c H?
Vitamin D3
Blood
DBP
SCHEME 11: Diagram of the formation of previtamin D3 in the skin, its thermal conversion to
vitamin D 3 , and transport by binding proteins ( D B P ) in plasma into the general
circulation (Reference 17). From ref. 17, copyright 1980 by the AAAS.
2.1 Spectra
F i g u r e 1 i s t h e u l t r a v i o l e t spectrum o f a 10
mcg/ml s o l u t i o n o f v i t a m i n D3 i n methanol. The spectrum was
o b t a i ned u s i n g a Cary Model 219 r e c o r d i ng spectrophotometer
( V a r i a n Instrument Co., Palo A l t o , CA). Vitamin D3 and
r e l a t e d compounds have a c h a r a c t e r i s t i c UV a b s o r p t i o n
maximum a t 265 nm and a minimum a t 228 nm. The e x t i n c t i o n
c o e f f i c i e n t a t 265 nm i s about 17,500 and 15,000 a t 254
nm. An i n d e x o f p u r i t y o f v i t a m i n D3 i s a value o f 1.8 f o r
t h e r a t i o o f t h e absorbance a t 265 t o t h a t a t 228 nm. The
h i g h absorbance a t 254 nm enables one t o use t h e most common
and s e n s i t i v e s p e c t r o p h o t o m e t r i c d e t e c t o r used i n h i g h
performance 1 i q u i d chromatography (HPLC) f o r t h e a n a l y s i s o f
v i t a m i n D3 i n m u l t i v i t a m i n p r e p a r a t i o n s , f o r t i f i e d m i l k ,
o t h e r food products, animal feed a d d i t i v e s e t c .
The i n f r a r e d spectrum o f a m i n e r a l o i l m u l l o f
v i t a m i n D3 recorded on a Model FTS 15E i n s t r u m e n t ( D i g i l a b ,
Cambridge, Mass.) i s shown i n F i g u r e 2 (18). Table I shows
a s s i ynments f o r t h e s i g n i f ic a n t in f r a r e d a b s o r p t i on bands
(18)
2.13 Nuclear Magnetic Resonance Spectram
P r o t o n and C-13 NMK s p e c t r a o f v i t a m i n D3

r e s p e c t i v e l y a r e shown i n F i g u r e s 3 and 4 (19). Spectra
were o b t a i n e d w i t h a Model XL-200 NMR spectrophotometer
(Varian, Palo A l t o , CA). Tables I1 and I11 a r e t h e
corresponding chemical s h i f t s i n t h e p r o t o n and C-13 NMR
s p e c t r a which are based on t h e assignments made by Wing e t
.
a1 , (20) and W i l l i a m s e t a1 (21) , r e s p e c t i v e l y .
'9
0 0 0
100 I I I I I I I I I I I I I I I I I I I I I I I
n
s
W
w
0
z
a
-B
k 50
I-
to
z
a
Pe
I-
0 I
I I I 1 I I I I 1 I I I 1
I
I I I I I
3800 3000 2000 1800 1600 1400 1200 1000 800 600
FREQUENCY (CM-')
FIGURE 2: I n f r a r e d spectrum o f a m i n e r a l o i l m u l l o f v i t a m i n D3.

663
0
0
cv
0
t
0
(0
0
Qo
0
0
~
' 5
n m
o a
cv
'
0
t
'
0
(0
'
0
Qo
r
0
0
cv
0
cv
cv
VITAMIN Di (CHOLECALCIFEROL) 665
TABLE I
I n f r a r e d Band Assignments f o r V i t a m i n D? (Ref. 18)
Structural
Wave Numbers cm-’ Feature Assignment
3289 a1 cohol 0-H s t r e t c h
3077, 3055, 3031 a1 kenes C-H s t r e t c h
1648, 1634, 1604 Conjugated C=C s t r e t c h

a1 kenes
1053 secondary a1 cohol C-0 s t r e t c h

(equitorial)
895 a1 kene C-H Out O f

R 1 R 2 C=CH2 t y p e plane bending
968, 942, 888 a1 kene C-H Out O f

and/or 860 p l a n e bending
666 K.THOMAS KOSHY AND WILLIAM F. BEYER
TABLE I 1
Proton NMR S p e c t r a l Assignment f o r V i t a m i n D, (Ref. 20)
Chemical
Assignment Shift Shape
18 methyl 0.54 Singlet
27 methyl 0.85 Doublet
4 a H 2.55 Dou b l e t
Doublet
9 B H 2.80 Doublet
Doublet
3 a H 3.92 Mu1t i p l e t
19 E H 4.83 Doublet
19 Z H 5.05 Doublet
of Triplet
7 H 6.03 Doublet
6 H 6.24 lloubl e t
VITAMIN D? (CHOLECALCIFEROL) 667
TABLE I 1 1
13C Chemical S h i f t s o f V i t a m i n D2 a t 200 MHz i n 070

( R e f . 21) -
Carbon Chemi c a l Carbon Chemical

No. Shifts No. Shifts
1 31.95 14 56.33
2 35.15 15 23.58
3 69.17 16 27.66
4 45.90 17 56.56
5 145.05 18 12.00
6 122.41 19 112.41
7 117.47 20 36.14
8 142.26 21 18.84
9 29.02 22 36.14
10 135 '04 23 23.86
11 22.25 24 39.49
12 40.52 25 28.01
13 45.83 26 22.83
27 22.56
668 K. T H O M A S K O S H Y AND WILLIAM F. BEYER
2.14 Mass Spectrum
The e l e c t r o n impact mass spectrum o f v i t a m i n

D3 o b t a i n e d w i t h a CEC Model 21-llOB i n s t r u m e n t ( C o n s o l i -
dated Electrodynamics Corporat ion , Pasadena, CA) by d i r e c t
probe sample i n t r o d u c t i o n i s shown i n F i g u r e 5 (22). It
shows a s t r o n g m o l e c u l a r i o n a t m/z 384 and a f r a g m e n t a t i o n
p a t t e r n c h a r a c t e r i s t i c o f v i t a m i n s D2 and D3, t h e i r p r i m a r y
metabol it e s and o t h e r chemical l y re1 a t e d compounds. The
fragment i o n assignment i s shown i n Scheme I11 (22).
2.21 M e l t i n g Range
C r y s t a l l i n e v i t a m i n D3 o b t a i n e d as f i n e need-
l e s from d i l u t e acetone m e l t s i n t h e range o f 84-88".
2.22 C r y s t a l Conformation
Trinh-Toan e t a l . ( 2 3 ) have shown by x-ray

d i f f r a c t i o n a n a l y s i s t h a t v i t a m i n D3 c r y s t a l l i z e s i n an
equimolar r a t i o of t h e a c h a i r f o r m i n which t h e 19-CH2
group i s s i t u a t e d below t h e mean A r i n g p l a n e and t h e p
c h a i r form i n which t h e 19-CH2 group i s s i t u a t e d above t h e
mean A r i n g plane. The 3-OH s u b s t i t u e n t occupies an equi-
t o r i a l p o s i t i o n i n t h e a form and an a x i a l p o s i t i o n i n t h e p
form. F i g u r e 6 shows t h e two independent molecules o f
v i t a m i n D3 d i s p l a y i n g d i f f e r e n t s o l i d - s t a t e conformations o f
the A ring. These s o l i d - s t a t e conformations i n t h e A r i n g
correspond t o t h o s e i n f e r r e d from t h e ' H NMR i n v e s t i g a t i o n s
on t h e s o l u t i o n conformations o f v i t a m i n s D2 and D3 (24) and
several metabol i t e s i n c l u d i n g 1 ~ , 2 5 - ( 0 H ) ~ - v i t a r n i n D ~(25-
27). The o b s e r v a t i o n t h a t b o t h t h e mean t o r s i o n a l angles o f
53.8 and 50.1 i n r i n g A o f molecules a and @, r e s p e c t i v e l y ,
a r e small e r t h a n t h e correspondi ny experimental v a l u e o f
55.9" found i n cyclohexane i s a t t r i b u t e d t o t h e f l a t t e n i n y
e f f e c t caused by t h e two e x o c y c l i c double bonds (23).
Trinh-Toan e t a l . (23) have a l s o r e p o r t e d t h a t t h e r e i s
s t r o n g hydrogen bonding between t h e a and p c o n f o r m a t i o n a l
forms. The molecules are w e l l separated i n t h e u n i t c e l l
w i t h a l l i n t e r m o l e c u l a r nonhydrogen c o n t a c t s b e i n g g r e a t e r
t h a n 3.4 A except f o r t h e r e l a t i v e l y s h o r t O-H--0 d i s t a n c e s
o f 2.71 t o 2.73 A which i n d i c a t e reasonably s t r o n g OH--0
bonds. The a u t h o r s conclude t h a t t h e c r y s t a l packing o f
v i t a m i n D3 i n c l u d i n g t h e presence o f b o t h conformers i s
m a i n l y d i c t a t e d by s t e r i c c o n s t r a i n t s imposed by t h e i n f i n -
it e he1 ic a l hydrogen-bonded oxygen chain.
S
0 0
0 0 .C
In Low
m d l n
sce
OaJ
L L
LI
-
0
0
V E
aJ0
7
wce
L
I
0 0 0 0 0
5: m Im 3 N
IlISN31NI 3 h I l M 3 U
- - tl- - - - 1
I
I
1
I
I
I
I
I
1
I
i ,136 I
Scheme 111: Mass s p e c t r a l fragmentation p a t t e r n of vitamin

D (from r e f e r e n c e 22. Copyright 0 1972. Reprinted by per-
3
mission of John Wiley & Sons, Inc.)
U Q
t s
4-u
cr)
n .
m
C N
*r
E -
54-
4aJ
-7L
>
E
- 0
O L
4-
v)
E S
L O
0 .-
2.23 Thermal A n a l y s i s
D i f f e r e n t i a l scanning c a l o r i m e t r i c (DSC) and
t h e t h e r m o g r a v i m e t r i c (TGA) curves f o r v i t a m i n D3 a r e shown
i n F i g u r e s 7 and 8 ( 2 8 ) . The curves were generated on a
DuPont thermal a n a l y z e r (Model No. 1090, DuPont DeNemours
and Co., Wilmington, Del .). The sample was c o n t a i n e d i n
aluminum pans and t h e a n a l y s i s was conducted under N2 atmos-
phere. The h e a t i n g r a t e f o r b o t h was 5"Imin. There i s a
p r e c i s e m e l t i n g endotherm i n t h e DSC c u r v e which peaks a t
86.3'. The exotherm a t 206' may be due t o decomposition.
The TGA curve shows a l i t t l e w e i g h t g a i n i n t h e m e l t i n g
range o f 84-88' when t h e sample volume i s reduced and t h e
buoyancy e f f e c t o f N i s a l t e r e d , The sharp drop i n w e i g h t
a t 128' may be due f o b o i l i n g and p o s s i b l e s p l a t t e r i n g of
t h e sample.
F i g u r e 9 i s t h e x-ray powder d i f f r a c t i o n
p a t t e r n o f c r y s t a l 1i n e v i t a m i n D3 (USP r e f e r e n c e standard)
( 2 9 ) . The values f o r t h e 2 8 angles and t h e corresponding
"d" spacings a r e shown i n Table I V .
2.3 Solubility
The s o l u b i l i t y o f c r y s t a l l i n e v i t a m i n D (99 + %
pure, A l d r i c h Chemical Company, I r ~ c . , Milwaukee, d i s c o n s i n )
was determined i n our l a b o r a t o r i e s i n commonly used l a b o r -
atory solvents. It had o v e r 10% s o l u b i l i t y i n methanol,
ethanol, acetone, e t h y l a c e t a t e , hexane, chloroform, methy-
l ene c h l o r i de, d i met hyl f ormami de, c y c l ohexane, t e t rahydro-
f u r a n , m e t h y l e t h y l ketone, i s o p r o p a n o l and cyclohexanone.
The o n l y s o l v e n t w i t h low s o l u b i l i t y was a c e t o n i t r i l e i n
which t h e s o l u b i l i t y was about 0.18%.
The s p e c i f i c r o t a t i o n o f a f r e s h l y prepared 5 mg/ml

ethanol s o l u t i o n o f v i t a m i n D3 i s between +100.5' and +112"
(30)
3. Chemical P r o p e r t i e s
3.1 Chemical Synthesis
V i t a m i n D3 i s c h e m i c a l l y synthesized by t h e i r r a d i -
ation of 7-DHC and subsequent c o n t r o l l e d h e a t i n g o f t h e
1
2 -t
0
A
3
Y
E -1
%
0
-I
I& -2
l-
a
I -3
-4
-5
40 80 120 160 200 240 280 320 360 400 440 480
TEMPERATURE ( " C )
FIGURE 7: D i f f e r e n t i a l scanniny c a l o r i m e t r i c curve f o r v i t a m i n D3.

0
d
0
0
r
0
0
r -
0
0
>
L
0
Ic
0
(D
P
L
=I
v
0
v)
0
e
0
0
..
W
w
Y
3
W
U
614
8
N
.r
>
mi
W L
W 0
rc
K
c
L
a,
c,
c,
V
5
L
0
d
AlISN31NI 3 A I l V l 3 t l
675
TABLE I V
X-Ray Powder D i f f r a c t i o n P a t t e r n o f V i t a m i n D2 (Ref. 29)
Re1a t i ve
28 d-spaci ng ( A ) Intensity
4.89 18.06 4
5.05 17.48 3
6.60 13.38
8.60 10.27
8.85 9.98
9.20 9.60
10.15 8.71
13.00 6.80
13.59 6.51
14.25 6.21
14.95 5.92
15.15 5.84
15.75 5.62
16.15 5.48
16.65 5.32
18.05 4.91
18.65 4.75
19.35 4.58
21.79 4.08
22.85 3.89
23.35 3.81
23.65 3.76
25.10 3.54
25.60 3.48
27.05 3.29
27.75 3.21
29.80 3.00
31.30 2.86
31.90
33.65
38.45
VITAMIN D j (CHOLECALCIFEROL) 677
previtamin. A number o f by-products a r e formed, most o f

which a r e removed d u r i n g t h e clean-up steps. Scheme I V
shows t h e s t r u c t u r e s o f t h e known i m p u r i t i e s and d e g r a d a t i o n
p r o d u c t s (31-33) t h a t may be p r e s e n t i n t h e s y n t h e t i c v i t a -
min D3 concentrates. Some o f t h e by-products have
b i o l o g i c a l a c t i v i t y b u t o t h e r s such as l u m i s t e r o l , t h e
s u p r a s t e r o l s , and t h e pyro- and isopyrocal c i f e r o l s have no
anti rachitic activity. T a c h y s t e r o l and t r a n s - v i t a m i n D3
have o n l y s l i g h t a n t i r a c h i t i c a c t i v i t y . Thus, the
b i o l o g i c a l a c t i v i t y i s p r i m a r i l y due t o v i t a m i n D and
p r e v i t a m i n 0. The b i o l o g i c a l a c t i v i t y o f p r e v i t a m i n 3I? i s
a t t r i b u t e d t o i t s i n v i v o c o n v e r s i o n t o v i t a m i n D3. It may
be mentioned t h a t analogous compounds are formed d u r i n g t h e
s y n t h e s i s o f v i t a m i n U2 from e r g o s t e r o l . The two forms
d i f f e r o n l y i n t h e s i d e chain. V i t a m i n D2 has an e x t r a
methyl group on C-24 and a double bond between C-22 and 23.
3.2 P r e v i t a m i n D-,- - -.
V i t a m i n D-, E q u i l i b r i u m
The thermodynamics and k i n e t i c s o f t h e thermal

e q u i l i b r i u m between p r e v i t a m i n D3 and v i t a m i n D3 have been
s t u d i e d (34,35). The i s o m e r i z a t i o n o f p r e v i t a m i n D3 t o
v i t a m i n D3 i s an exothermic f i r s t o r d e r r e a c t i o n . The
v i t a m i n D 3 / p r e v i t a m i n D3 e q u i l i b r i u m r a t i o depends on t h e
t e m p e r a t u r e and can be c a l c u l a t e d from t h e a p p r o p r i a t e
e q u i l i b r i u m and k i n e t i c c o n s t a n t s r e p o r t e d by Hanewald e t
a l . (36). The r a t e c o n s t a n t s f o r t h e e q u i l i b r i u m have been
shown t o be independent o f t h e n a t u r e o f t h e s o l v e n t , o f
a c i d i c o r b a s i c c a t a l y s i s and o f f a c t o r s known t o a f f e c t
f r e e r a d i c a l process (37,38). The percentages o f v i t a m i n D3
i n e q u i l i b r i u m w i t h p r e v i t a r n i n D3 ranges from 98% a t -20" t o
78% a t 80'. Thus, when v i t a m i n D3 i s s t o r e d i n t h e c o l d ,
t h e e q u i l i b r i u m c o n s t a n t h i n d e r s t h e conversion t o p r e v i t a -
min D3.
3.3 Cis-Trans E q u i l i b r i u m
The 5,6,7-cis-triene configuration of vitamin D i s

i m p o r t a n t f o r i t s b i o l o g i c a l a c t i v i t y as t h e 5,6-trans ?arm
has v e r y low a c t i v i t y . Exposure t o i o d i n e i n non-polar
s o l v e n t s under d i f f u s e l i g h t (39,40) or t o mild a c i d i c
c o n d i t i o n s (41) a f f o r d s f o r m a t i o n o f t h e 5,6-trans isomer.
The r e v e r s e t r a n s f o r m a t i o n occurs p h o t o c h e m i c a l l y (42). The
5,6-trans isomer can undergo f u r t h e r i s o m e r i t a t i o n s upon
exposure t o heat (43) o r a c i d s o r t r e a t m e n t w i t h antimony
trichloride. The c i s and t h e t r a n s forms o f v i t a m i n D3
d i s p l a y c h a r a c t e r i s t i c u l t r a v i o l e t p r o p e r t i e s . The c i s form
as shown e a r l i e r has a UV maxima a t 265 nm. The t r a n s form
has a UV maxima a t 273 nm.
lsotachysterol D 3 Transvitamin D3 lsovitamin D3
no no
Provitamin D3 Prewitomin D 3 t Pholoisopyro D3 Pholopyro D3
Vitamin D3
no no HO
Tachysterol D3 Lumisterol D 3 Pyro D3 lsopyro D3 Suprasterol D3
Scheme I V . P h o t o c h e m i c a l t h e r m a l , and c h e m i c a l r e a c t i o n pathways

i n t h e s y n t h e s i s o f v i t a m i n D3 ( r e f e r e n c e s 31,33). From J . Pharm. S c i .
71, 137 (1982). Reproduced w i t h p e r m i s s i o n o f t h e c o p y r i g h t owner.
VlTAMlN Dz (CHOLECALCIFEROL) 679
3.4 Other Chemical Reactions
V i t a m i n D3 can undergo v e r y complex chemical reac-

t i o n s upon exposure t o heat, l i g h t and chemical reagents.
It i s beyond t h e scope o f t h i s a r t i c l e t o r e v i e w t h e s e
r e a c t i o n s . The reader i s r e f e r r e d t o r e c e n t reviews on t h e
s u b j e c t (99,100).
4. Methods o f A n a l y s i s
The a n a l y s i s o f v i t a m i n O3 i s d i f f i c u l t f o r t h e f o l l o w -
i n g reasons:
a. As shown i n Scheme I V a number o f by-products a r e

formed d u r i n g t h e chemical s y n t h e s i s o f v i t a m i n D3, some o f
which may be p r e s e n t i n t h e f i n i s h e d b u l k form.
b. V i t a m i n D3 c o n c e n t r a t e s are commerci a1 l y a v a i 1a b l e
i n a r e s i n form i n peanut o i l and as a d r y g e l a t i n bead-
let. The v i t a m i n D3 c o n c e n t r a t e s u s u a l l y c o n t a i n o t h e r o i l
s o l u b l e v i t a m i n s , e s p e c i a l l y v i t a m i n A. The v i t a m i n D3,
t h e r e f o r e , has t o be r e l e a s e d from i t s m a t r i x which i s
accomplished by s a p o n i f i c a t i o n and e x t r a c t i o n . It t h e n has
t o be analyzed by a method t h a t would d i s t i n g u i s h i t from
a1 1 i n t e r f e r i n g isomers and o t h e r i n g r e d i e n t s .
c. Vitamin D e x i s t s i n thermal e q u i l i b r i u m w i t h
p r e v i t a m i n D3 (See s e c t i o n 3.2). There i s always u n c e r t a i n -
t y o f t h e e x t e n t o f t h i s r e a c t i o n even when a v i t a m i n D3
s t a n d a r d i s t r e a t e d under t h e same c o n d i t i o n s as t h e sample.
d. The c o n c e n t r a t i o n s o f v i t a m i n D3 i s low i n food

p r o d u c t s such as f o r t i f i e d m i l k , m i l k formulae, b r e a k f a s t
c e r e a l s , animal feeds e t c . These samples, t h e r e f o r e , need
e x t e n s i ve c l ean-up p r i o r t o a n a l y s i s .
4.1 I d e n t i f i c a t i o n Tests
The USP XX (30) d e s c r i b e s t h e f o l l o w i n g i d e n t i f i c a -

t i o n t e s t s f o r v i t a m i n D3, which a r e reproduced verbatum:
a. The i n f r a r e d a b s o r p t i o n spectrum o f a potas-

sium bromide d i s p e r s i o n o f it, i n t h e range o f 2 p t o 12
pm, e x h i b i t s maxima o n l y a t t h e same wavelengths as t h a t o f
a s i m i l a r p r e p a r a t i o n o f USP C h o l e c a l c i f e r o i RS.
b. The u l t r a v i o l e t a b s o r p t i o n spectrum o f a 1 i n
100,000 s o l u t i o n i n a l c o h o l e x h i b i t s maxima and minima a t
t h e same wavelengths as t h a t o f a s i m i l a r s o l u t i o n o f USP
Cholecal c i f e r o l RS, c o n c o m i t a n t l y measured, and t h e respec-
t i v e a b s o r p t i v i t i e s a t t h e wavelength o f maximum absorbance
a t about 265 nm do n o t d i f f e r by more t h a n 3.0%.
c. To a s o l u t i o n o f about 0.5 my i n 5 m l of
c h l o r o f o r m add 0.3 m l o f a c e t i c anhydride and 0.1 m l of
s u l f u r i c acid, and shake v i g o r o u s l y ; a b r i g h t r e d c o l o r is
produced, and i t r a p i d l y changes through v i o l e t and b l u e to
green.
d. Prepare w i t h o u t h e a t i n g , and handle w i t h o u t

delay, a 1 i n 100 s o l u t i o n o f squalane i n c h l o r o f o r m con-
t a i n i n g 50 mg o f c h o l e c a l c i f e r o l p e r m l , and p r e p a r e a
Standard s o l u t i o n o f USP C h o l e c a l c i f e r o l RS i n t h e same
s o l v e n t and having t h e same c o n c e n t r a t i o n . Spot 10 p1 o f
t h e t e s t s o l u t i o n and 10 p l o f t h e Standard s o l u t i o n on a
l i n e p a r a l l e l t o and about 2.5 cm from t h e bottom edge o f a
t h i n - 1 ayer chromatographic p l a t e (See <621>) coated w i t h a
0.25 mn l a y e r o f chromatographic s i l i c a gel m i x t u r e . Place
t h e p l a t e i n a developing chamber c o n t a i n i n g and e q u i l i -
b r a t e d w i t h a m i x t u r e o f equal volumes o f cyclohexane and
d i e t h y l ether. Develop t h e chromatogram u n t i l t h e s o l v e n t
f r o n t has moved about 15 cm above t h e l i n e o f a p p l i c a t i o n .
Perform t h e development and subsequent o p e r a t i o n s i n t h e
dark. Remove t h e p l a t e , a l l o w t h e s o l v e n t t o evaporate, and
spray w i t h a 1 i n 50 s o l u t i o n o f a c e t y l c h l o r i d e i n antimony
t r i c h l o r i d e TS: t h e chromatogram o b t a i n e d w i t h t h e t e s t
s o l u t i o n shows a y e l l o w i s h orange area ( c h o l e c a l c i f e r o l )
h a v i n g t h e same Rf v a l u e as t h e area o f t h e Standard s o l u -
t i o n , and may show below t h e c h o l e c a l c i f e r o l area a v i o l e t
area a t t r i b u t e d t o 7-dehydrocholesterol .
4.2 Q u a n t i t a t i v e Methods
Since v i t a m i n D3 i s i n c o r p o r a t e d i n t o a number o f
food a d d i t i v e s f o r human and animal consumption, t h e r e i s no
one method t h a t may be b e s t s u i t a b l e f o r a l l such prod-
ucts. Therefore, several a n a l y t i c a l systems are d e s c r i b e d
here t o f i t t h e need f o r a s p e c i f i c s i t u a t i o n .
4.2.1 B i o l o g i c a l Methods
B i o l o g i c a l methods a r e t h e o l d e s t f o r t h e
a n a l y s i s o f v i t a m i n D3 and t h e one t h a t i s a p p l i c a b l e f o r
a l l t y p e s o f samples. The b i o l o g i c a l methods can be d i v i d e d
VITAMIN D3 (CHOLECALCIFEROL) 68 I
i n t o t h r e e groups: c u r a t i v e , p r o p h y l a c t i c , and t h o s e based

on c a l c i u m a b s o r p t i o n i n t o t h e b l o o d stream. A l l are based
on t h e a d m i n i s t r a t i o n o f measured doses o f a standard v i t a -
min D3 p r e p a r a t i o n t o a group o f t e s t animals and comparison
o f t h e b i o l o g i c a l responses w i t h a s i m i l a r group g i v e n t h e
substance under t e s t . A t h i r d group o f t e s t animals i s used
as c o n t r o l s .
The b i o l o g i c a l methods are p o p u l a r f o r t h r e e reasons: 1)

i n most b i o l o g i c a l samples t h e c o n c e n t r a t i o n of v i t a m i n D3
i s v e r y low and i s n o t s u i t a b l e f o r more s p e c i f i c methods
because o f i n t e r f e r i n g m a t e r i a l s even a f t e r e x t e n s i v e c l e a n -
up; 2) v i t a m i n D3 i s e f f e c t i v e b i o l o g i c a l l y i n t r a c e amounts
making i t more s e n s i t i v e than t h e b e s t chemical methods; and
3) t h e b i o l o g i c a l methods a r e s p e c i f i c f o r v i t a m i n D3 and
its biologically active metabolites. The chief
disadvantages o f t h e b i o l o g i c a l methods a r e i n t h e h i y h
i n d i v i d u a ? v a r i a t i o n s i n t h e responses o f t e s t animals, t h e
high cost, t h e t i m e f a c t o r and t h a t t h e y do n o t
d i f f e r e n t i a t e v i t a m i n D3 f r o m i t s b i o l o g i c a l l y a c t i v e
impu r it ie s o r met a bo 1it es .
The USP XX (30) and t h e O f f i c i a l Methods o f A n a l y s i s o f
t h e A s s o c i a t i o n o f O f f i c i a l A n a l y t i c a l Chemists (AOAC) (44)
d e s c r i b e i n d e t a i l t h e c u r a t i v e method more commonly known
as t h e r a t l i n e method. This method i s n o t a p p l i c a b l e t o
p r o d u c t s o f f e r e d f o r p o u l t r y feeding. For p o u l t r y feeds,
and f i s h l i v e r o i l s and t h e i r e x t r a c t s , t h e AOAC (44) a l s o
d e s c r i b e s i n d e t a i l t h e c h i c k bone ash method. I n addition
t o t h e above two methods, t h e r e i s a method based on t h e
comparative measurement o f serum c a l c i u m i n r a t s r e f e r r e d t o
as I n t e s t i n a l Calcium T r a n s p o r t Assay and another method
based on t h e comparative amounts o f c a l c i u m absorbed by
c o n t r o l and t e s t c h i c k e n r e f e r r e d t o as Calcium A b s o r p t i o n
Test. These methods are d e s c r i b e d by DeLuca and B l u n t (45).
4.2.2 Chemical Methods
The most w i d e l y used chemical method i s t h e

antimony t r i c h l o r i d e c o l o r i m e t r i c method. The method i s
a p p l i c a b l e t o v i t a m i n s D2 and D3 f o r t h e i r a n a l y s i s i n
pharmacopeial p r e p a r a t i o n s . The r e a c t i o n product o f v i t a m i n
D w i t h antimony t r i c h l o r i d e i s b e l i e v e d t o be i s o v i t a m i n D
( i 6 , See Scheme I V ) . Antimony t r i c h l o r i d e r e a c t s w i t 8
v i t a m i n A also. V i t a m i n A occurs a l o n g w i t h D3 i n many
b i o l o g i c a l samples and i s a l s o an i n g r e d i e n t i n many
commerci a1 products. Therefore, i t i s necessary t o remove
i t and o t h e r i n t e r f e r i n g substances p r i o r t o r e a c t i o n w i t h
t h e reagent. This i s g e n e r a l l y achieved by s a p o n i f i c a t i o n

w i t h a l c o h o l i c potassium hydroxide, e x t r a c t i o n o f t h e
u n s a p o n i f i a b l e f r a c t i o n w i t h petroleum ether, and clean-up
on chromatographic columns. The procedure f o r t h e
d e t e r m i n a t i o n o f v i t a m i n D i n pharmaceutical p r e p a r a t i o n s i s
d e t a i l e d i n t h e USP ( 3 0 ) .
This method i s a p p l i c a b l e w i t h s u i t a b l e m o d i f i c a t i o n s t o
b i o l o g i c a l samples l i k e f i s h l i v e r o i l s , f o r t i f i e d m i l k ,
etc., b u t t h e c o l o r i m e t r i c procedures are being replaced
when p o s s i b l e by more s p e c i f i c i n s t r u m e n t a l methods, u s u a l l y
HPLC.
Paper chromatographic s e p a r a t i o n o f some

s t e r o l s , provitamins and v i t a m i n s D and D has been r e p o r t -
ed by Peereboom e t a l . (47). ?he b e 4 chromatographic
s e p a r a t i o n was achieved by reverse phase chromatography on
paper impregnated w i t h reagents shown i n Table V, which a l s o
gives t h e R f values obtained. D e t a i l s r e g a r d i n g t h e prepar-
a t i o n and development o f t h e paper are given. A number o f
d e t e c t i o n systems are described which i n c l u d e d exposure t o
U V l i g h t and r e a c t i o n s w i t h phosphomolybdic acid, antimony
t r i c h l o r i d e , bismuth c h l o r i d e , phosphotungstic acid, s i l i c o -
t u n y s t i c acid, a m i x t u r e o f dimethyl-p-phenylenediamine and
m-to1 uenedi ami ne, M i 11ion's reagent and sodi um iodate.
Paper chromatographic systems are a1 so d e s c r i bed (48) by
manufacturers o f r a d i o l a b e l e d v i t a m i n D3 f o r m o n i t o r i n g
purity The systems r e p o r t e d a r e reverse phase chromato-
graphy w i t h 95% aqueous methanol on paper t r e a t e d w i t h 10%
minera o i l i n benzene and w i t h 1 O : l a c e t i c acid-water on
p a r a f f n coated paper.
4.2.4. Thin Layer Chromatography
Thin Layer Chromatography i s used by major

manufacturers o f r a d i o l a b e l e d v i t a m i n D3 (48) f o r m o n i t o r i n g
p u r i t y . The systems described are; 1) s i l i c a gel G w i t h 10%
acetone i n hexane; 2) s i l i c a gel G w i t h acetone-hexane 1:l
and acetone-chloroform 1:l; 3) s i l v e r n i trate-impregnated
s i l i c a gel G w i t h c h l o r o f o r m o r chloroform-acetone 9 : l ; and
4) s i l i c a gel s a t u r a t e d w i t h s i l i c o n e o i l u s i n g 4:l acetone-
water. It may be mentioned t h a t these operations art? car-
r i e d o u t under very c a r e f u l l y c o n t r o l l e d c o n d i t i o n s t o
prevent o r minimize decomposition. TLC i s an e x c e l l e n t
technique f o r s e p a r a t i n g v i t a m i n D from i n t e r f e r i n g mater-
i a l s , b u t i t has t o be used w i t h due care i n t h e q u a n t i t a -
TABLE V
3
- (Ref. 47)
S t a t iona ry Mobi 1e
Phase Phase Rf x 100
Q u i1on MeOH-H20-ethyl ene 76
g l y c o l monomethyl e t h e r
(65:20: 20)
Qui1on MeOH-H20 (95: 5 ) 91
Paraf f in A c e t i c Acid-H20 (84: 16) 36
Paraf f i n Ethylene g l y c o l monoethyl 46

ether-n-propanol-MeOH-
H20 (35:10:30:25)
Para f f i n N-propanol -MeOH-H20 68
(15:82:3)
Paraffin MeOH-H20 (85: 1 5 ) 68

684 K . THOMAS KOSHY AND WlLLlAM F. BEYER
t i v e a n a l y s i s o f v i t a m i n D , e s p e c i a l l y i n b i o l o g i c a l sam-
p l e s c o n t a i n i n g small q u a n j i t i e s o f t h e v i t a m i n . The R f
values f o r v i t a m i n D3 on S i l i c a Gel GF i n common o r g a n i c
s o l v e n t s as determined i n t h e authors'*j!boratory a r e shown
i n Table V I (49). These values are r e p o r t e d t o enable one
t o develop s u i t a b l e systems i n unknown m a t r i c e s .
Hashmi e t a l . (50) have r e p o r t e d R f values f o r b o t h

water and o i l s o l u b l e v i t a m i n s on s i l i c a gel and alumina
p l a t e s by c i r c u l a r t h i n l a y e r chromatography. These systems
a r e f a s t ( < 2 minutes development t i m e ) and convenient f o r
d e t e c t i n g and e s t i m a t i n g t h e amounts o f v i t a m i n D3 i n d i f -
f e r e n t matrices. The authors have a l s o l i s t e d d e t e c t i o n
systems f o r a l l t h e vitamins. The chromatographic systems
d e s c r i b e d i n t h i s r e p o r t a r e shown i n Tables V I I and V I I I .
4.2.5. Gas L i q u i d Chromatography (GLC)
Although GLC procedures have been used

e x t e n s i v e l y f o r t h e determi n a t i on o f numerous p h y s i o l o g i -
c a l l y i m p o r t a n t s t e r o i d s and hormones, o n l y a few i n v e s t i y -
a t o r s have attempted t o develop s i m i l a r techniques f o r
v i t a m i n D3. The major problem w i t h t h e GLC a n a l y s i s o f
v i t a m i n D3 i s due t o i t s open r i n g s t r u c t u r e i n c o r p o r a t i n g
t h r e e Conjugated double bonds i n a 5,6-cis configuration.
The 5,6-cis double bond causes l e s s c o n t r o l l a b l e thermal
c y c l i z a t i o n i n t o t h e pyro- and i s o p y r o c a l c i f e r o l s a t opera-
t i n g GLC temperatures, r e s u l t i n g i n two peaks. Another
reason f o r t h e l a c k of i n t e r e s t i n GLC a n a l y s i s i s t h a t
v i t a m i n D3 c o n c e n t r a t i o n i n b i o l o g i c a l systems i s low and
t h e samples g e n e r a l l y c o n t a i n s t r u c t u r a l l y s i m i l a r compounds
r e q u i r i ny e x t e n s i v e sample c l ean-up.
Murray e t a l . ( 4 6 ) overcame t h e d i f f i c u l t y o f t h e
thermal i s o m e r i z a t i o n o f v i t a m i n D3 i n t o two peaks by con-
v e r t i n g i t i n t o i s o v i t a m i n D3 by t r e a t m e n t w i t h antimony
t r i c h l o r i d e which gave a s i n g l e peak by GLC. They r e p o r t e d
s u c c e s s f u l a p p l i c a t i o n o f t h e method f o r t h e d e t e r m i n a t i o n
o f v i t a m i n D3 i n b i o l o g i c a l samples. Sheppard e t a l . ( 5 1 )
t r e a t e d v i t a m i n D3 w i t h a c e t y l c h l o r i d e t o c o n v e r t i t quan-
t i t a t i v e l y t o i s o t a c h y s t e r o l D3 (See Scheme I V ) which gave a
s i n g l e peak by GLC. N a i r and deLeon ( 5 2 ) prepared t h e 5,6-
t r a n s v i t a m i n D3 by t r e a t i n g v i t a m i n D3 w i t h i o d i n e f o l l o w e d
by exposure t o UV l i g h t . They then prepared t h e penta-
f 1u o r o p r o p i onyl o r t h e h e p t a f l u o r o b u t y r y l e s t e r s o f t h e
trans-D3 which chromatographed as t h e i s o t a c h y s t e r o l D3
e s t e r s as a s i n g l e peak. Using 63Ni e l e c t r o n c a p t u r e detec-
TABLE V I
R f Values o f Vitamin D on S i l i c a Gel GFZs4 TLC Plates'

f o r Pommon s o l v e n t s
Sol vent Rf
Hexane (1.9) 0
1,4-Di oxane (2.2) 0.67
Benzene (2.3) 0.10
Toluene (2.4) 0.07
E t h y l Ether (4.3) 0.55
Chl o r o f o n (4.8) 0.23
E t h y l Acetate (6.0) 0.70
Methylene C h l o r i d e (9.1) 0.23
Cyclohexanone (18.3) 0.90
Acetone (21) 0.78
Ethanol , Absol Ute (24.3) 0.73
Methanol ( 32.6) 0.67
Acet o n i t r i 1e ( 38.8) 0.78
Hexane-Acetone, 9 : l 0.27
Hexane-Acetone, 1: 1 0.75
Acetone-Chloroform, 1:l 0.70
'Anal t e c h Inc., Newark, DE.
2The numbers i n parentheses are t h e d i e l e c t r i c constants o f

t h e s o l v e n t s a t 2OOC.
686 K . THOMAS KOSHY AND WILLIAM E BEYER
TABLE V I I
C i r c u l a r Thin La.yer Chromatographic Systems f o r

Vitamin D3 on S i l i c a Gel D-0. Camag Without Binder
(Ref. 50)
Mobile D e t e c t i n g Reagents
Phase Rf x 100 and Colors
A B C
Benzene-Petrol eum 33 Grey Bluish Greyish

Ether ( 4 : l ) (0.7)’ Grey Green
(0.7)l (0.8)l
II 11 II
Cycl ohexane-Methyl 35
E t h y l Ketone (21:4)
II II
To1 uene-Methyl 40
E t h y l Ketone ( 9 9 : l )
11
Cycl ohexane-Cyclo- 48
pentanone (24: 1)
-
Cy c 1oh exa ne Et hy 1 35 II
Ether ( 4 : l )
II
Cyclohexane-Methyl Iso- 50 II
p r o p y l Ketone ( 9 : l )
11
To1 uene-Chl oroform 32
(9: 1)
II
Cycl ohexane-Acet ic 66
Acid (22:3)
II
Benzene-Cycl ohexane 34
(9: 1)
11
Toluene-Cyclohexane 23
(9:l)
II
Cycl ohexane-Methanol 32
(43: 7 )
(Continued)
TABLE V I I - Continued
Phase Rf x 100 and Colors
A B C
Cycl ohexane-Methyl 50 Grey Bluish Greyish

E t h y l Ketone (21:4) (0.7)l Grey Green
(0.7)' (0.8)'
I1 I1 I1
Cycl ohexane-Ethyl 52
Ether ( 4 : l )
A 70% P e r c h l o r i c A c i d
B Concentrated S u l f u r i c A c i d
C S a t u r a t e d S o l u t i o n o f antimony p e n t a c h l o r i d e i n carbon
tetrachloride.
The numbers i n parentheses r e p r e s e n t t h e minimum amounts

(11.9)o f v i t a m i n D3 t h a t can be d e t e c t e d and i d e n t i f i e d on
the plates.
68 8 K . THOMAS KOSHY A N D WILLIAM F. BEYER
TABLE VIII
C i r c u l a r T h i n Layer Chromatographic Systems f o r V i t a m i n
D3 on Aluminum Oxide G (E. Merck)
(Ref. 5 0 )
Phase R f x 100 and C o l o r s
A B C
Benzene-Pet r o l eum 53 Brown Grey L iyht

Ether ( 4 : l ) (0.6)’ (0.3)’ Brown
(0.4)’
II
Cycl ohexane-Met h y l 53 11 11
E t h y l Ketone (21:4)
11
To1 uene-Methyl 52 11
E t h y l Ketone ( 9 9 : l )
II
Cycl ohexane-Cyclo- 47 II II
pentanone (24: 1)
-
Cyc 1 o hexa ne E t hy 1 30 11 II
Ether (4:l)
Cy cl o hexane -Met hy 1 31 II 11 II
I s o p r o p y l Ketone (9: 1)
II
To1 uene-Chl o r o f o r m 45 II II
(9:l)
II
Cyclohexane-Acetic 63 II
Acid (22:3)
II II
Benzene-Cycl ohexane 59
(9:l)
II
To1 uene-Cycl ohexane 38
(9:l)
Cyclohexane-Methanol 47 II II II
(43: 7 )
(Continued )
VITAMIN Di (CHOLECALCIFEROL) 689
TABLE V I I I - Continued
Phase Rf x 100 and C o l o r s
A B C
Methanol - g l a c i a l 82 Brown Grey Light

A c e t i c Acid (49:l) (0.6)l (0.3)l Brown
(0.4)'
A S a t u r a t e d S o l u t i o n o f antimony p e n t a c h l o r i d e i n carbon
tetrachloride.
B 70% p e r c h l o r i c acid.
C Concentrated s u l f u r i c a c i d .
The numbers i n parentheses r e p r e s e n t t h e minimum amounts

(pg) o f v i t a m i n D3 t h a t can be d e t e c t e d and i d e n t i f i e d on
the plates.
690 K. THOMAS KOSHY AND WILLIAM F. BEYER
t o r , t h e y were a b l e t o d e t e c t nanogram q u a n t i t i e s o f v i t a m i n
D3-
I n r e c e n t y e a r s GLC methods have been superceded by HPLC
methods. There has n o t been much i n t e r e s t i n c a p i l l a r y GLC
a n a l y s i s o f v i t a m i n D3. C a p i l l a r y column technology,
however, has improved so much i n r e c e n t y e a r s t h a t t h i s
t e c h n i q u e may have a p p l i c a b i l i t y i n t h e d e t e r m i n a t i o n o f
t r a c e amounts o f v i t a m i n D3 i n b i o l o g i c a l m a t r i c e s
e s p e c i a l l y i n c o n j u n c t i o n w i t h an e l e c t r o n c a p t u r e d e t e c t o r .

(HPLC)
I n r e c e n t y e a r s HPLC procedures have

evolved as t h e methods o f c h o i c e f o r t h e a n a l y s i s o f v i t a m i n
D i n b u l k d r u g (32,33,53-61), pharmaceutical p r e p a r a t i o n s
(22-73), f o r t i f i e d m i l k (74-82), animal feed supplements
(83-89), margarine (79), i n f a n t formulae (79), cod l i v e r o i l
(90-93), and chicken egg (96). It may be p o i n t e d o u t t h a t
these reports represent t h e l a t e s t i n t h e state-of-the-art
i n t h e HPLC analyses o f v i t a m i n s D2 and 03.. In s p i t e of t h e
i n t e n s i v e e f f o r t by a number o f i n v e s t i g a t o r s and a few
c o l l a b o r a t i v e s t u d i e s , t h e r e i s no consensus on w i d e l y
acceptable HPLC methods f o r v i t a m i n D a n a l y s i s i n t h e b u l k
drug, pharmaceutical p r e p a r a t i o n s , and f o r t i f i e d m i l k .
4.2.6.1 HPLC A n a l y s i s o f Bulk V i t a m i n D3
B u l k v i t a m i n D may c o n t a i n some o f
s y n t h e t i c by-products shown i n Scheme 1".
Tartivita et al.
(33) have r e p o r t e d an excel 1e n t chromatographi c system which
showed r e s o l u t i o n of most o f t h e photochemical isomers and
r e a c t i o n by-products. The chromatograms o b t a i n e d on a 30-cm
x 4-mn i.d. commercial m i c r o p a r t i c u l a t e s i l i c a column u s i n g
a 70:30:1 m i x t u r e o f c h l o r o f o r m ( f r e e f r o m ethanol and
water), n-hexane, and t e t r a h y d r o f u r a n a t a f l o w r a t e of 1
ml/min i s shown i n F i g u r e 10. The d e t e c t i o n was by a 254 nm
UV d e t e c t o r . Using t h i s system, v i t a i n D3 was q u a n t i t a t e d
i n a r e s i n sample c o n t a i n i n g 20 x 10' IU/g w i t h a r e l a t i v e
standard d e v i a t i o n o f 1.37%. T h i s procedure i s e s s e n t i a l l y
t h e b a s i s f o r t h e USP XX (30) procedure f o r t h e a n a l y s i s of
b u l k v i t a m i n D3 which i s reproduced below, i n i t s e n t i r e t y .
Standard P r e p a r a t i o n - T r a n s f e r about 30 my o f USP

C h o l e c a l c i f e r o l RS, a c c u r a t e l y weighed, t o a l o w - a c t i n i c ,
25-ml v o l u m e t r i c f l a s k , add i s o o c t a n e t o volume, mix, and
s o n i c a t e f o r 5 minutes. S t o r e a t 0 f 5O, and use t h i s s t o c k
VITAMIN D j (CHOLECALCIFEROL) 69 1
5
2
L
w
v)
z
2
v)
w
a
J I 1 I 1 1 I I I I I I I 1
0 2 4 6 8 1 0 12 14 1 6 18 20 2 2 2 4 2 6
RETENTION TIME (minutes)
1 I I I I 1 1 I I I
0 2 4 6 10 12
8 14 16 18 20
RETENTION TIME (minutes)
FIGURE 10: High Performance Liquid Chromatograms: ( A ) A

synthetic mixture of photochemical isomers and reaction
products of vitamin D3. Key: ( 1 ) unknowns,; ( 2 ) trans-
vitamin D3; (3) previtamin D3; (4) lumisterol3; (5) iso-
tachysterol3; ( 6 ) p-dimethylaminobenzaldehyde (internal
standard); ( 7 ) tachysterol3; (8)vitamin D3; (9) 7-dehydro-
cholesterol. ( B ) A typical vitamin D3 resin. Key: (1) resin
impurities; ( 2 ) p-dimethylaminobenzaldehyde (internal
standard); (3) tachysterolj; ( 4 ) vitamin D3. (Both from
Reference 33; reproduced with permission of the copyright
owner. 1
s o l u t i o n w i t h i n 3 days. Pipet 5 ml o f t h i s stock s o l u t i o n

i n t o a l o w - a c t i n i c , 50-1111 v o l u m e t r i c flask, d i l u t e with
i s o o c t a n e t o volume, and mix t o o b t a i n a s o l u t i o n having a
known c o n c e n t r a t i o n o f about 120 pg per ml.
Assay P r e p a r a t i o n - T r a n s f e r about 30 my o f Cholecal-

c i f e r o l , a c c u r a t e l y weighed, t o a l o w - a c t i n i c , 25-1111 v o l u -
m e t r i c f l a s k , and proceed as d i r e c t e d f o r Standard Prepar-
a t i o n , b e g i n n i n g w i t h "add i s o o c t a n e t o volume," t o o b t a i n a
s o l u t i o n having a c o n c e n t r a t i o n o f about 120 11.4 per m l .
A l c o h o l -Free C h l o r o f o r m - Prepare 2 Chromatographic

columns by packing 2 chromatographic tubes w i t h an amount o f
dry, a c t i v a t e d , 80- t o 200-mesh alumina s u f f i c i e n t t o h a l f -
f i l l each t u b e (See Column A d s o r p t i o n Chromatography under
Chromatoyraphy <621>), and mount one column above t h e o t h -
er. E x t r a c t 500 m l o f c h l o r o f o r m w i t h t h r e e 75-ml p o r t i o n s
o f water, and d i s c a r d t h e aqueous e x t r a c t s . Pass t h e c h l o -
r o f o r m l a y e r through t h e Chromatographic columns, c o l l e c t
t h e e l u a t e i n a glass-stoppered f l a s k , i n s e r t t h e stopper,
and mix. Prepare t h i s s o l v e n t on t h e day o f use.
M o b i l e Phase -
Prepare a f i l t e r e d and de-gassed m i x t u r e
of a l c o h o l - f r e e c h l o r o f o r m ,
n-hexane, and t e t r a h y d r o f u r a n
(about 70:30:1 by volume). The r a t i o o f components and t h e
f l o w r a t e may be v a r i e d t o meet system s u i t a b i l i t y r e q u i r e -
ments.
Chromatographic System - Typically, a high-pressure

l i q u i d chromatograph, operated a t room temperature, i s
f i t t e d w i t h a 30-cm x 4-mm s t a i n l e s s s t e e l column packed
w i t h chromatographic column p a c k i n g L3*. An u l t r a v i o l e t
d e t e c t o r t h a t m o n i t o r s a b s o r p t i o n a t t h e 254-nm wavelength
i s used.
System S u i t a b i l i t y P r e p a r a t i o n - P i p e t 5 m l o f t h e stock
s o l u t i o n , prepared as d i r e c t e d under Standard p r e p a r a t i o n ,
i n t o a 25-11-11 v o l u m e t r i c f l a s k , d i l u t e w i t h i s o o c t a n e t o
volume, and mix. Heat t h i s s o l u t i o n a t r e f l u x f o r 2 hours,
cool , and i r r a d i a t e f o r 1 hour under lony-wavelength and
short-wavelength u l t r a v i o l e t l i g h t . This s o l u t i o n contains
.
c h o l e c a l c i f e r o l , p r e - c h o l e c a l c i f e r o l , and t a c h y s t e r o l
System S u i t a b i l i t y l e s t - Chromatograph f i v e i n j e c t i o n s
o f t h e h e a t - e q u i l ib r a t e d Standard P r e p a r a t i o n , and measure
*Porous s i l i c a m i c r o p a r t i c l e s , 5 t o 10 pm i n diameter.
t h e peak response as d i r e c t e d under Procedure. The r e l a t i v e

s t a n d a r d d e v i a t i o n f o r t h e peak response does n o t exceed
2.0%. The r e t e n t i o n t i m e s observed f o r t h e System S u i t -
a b i 1 ity P r e p a r a t i o n , chromatographed as d i r e c t e d f o r Proce-
dure, a r e between 10 and 11 minutes f o r p r e - c h o l e c a l c i f e r o l ,
between 14 and 16 minutes f o r t a c h y s t e r o l , and between 16
and 18 minutes f o r c h o l e c a l c i f e r o l . The r e s o l u t i o n between
t a c h y s t e r o l and c h o l e c a l c i f e r o l i s n o t l e s s t h a n 1.0.
Procedure - Equi 1ib r a t e t h e Standard P r e p a r a t i o n and t h e

Assay P r e p a r a t i o n i n t h e dark a t 80' f o r 2.5 hours, accur-
a t e l y timed. Cool, and i n t r o d u c e equal volumes ( 5 t o 10 k l )
o f t h e h e a t - e q u i l ib r a t e d Standard P r e p a r a t i o n and Assay
P r e p a r a t i o n i n t o t h e high-pressure l i q u i d chromatograph (See
chromatography <621>) by means o f a s u i t a b l e sampling
valve. Measure t h e peak responses o b t a i n e d f o r t h e Assay
P r e p a r a t i o n and t h e Standard P r e p a r a t i o n , and c a l c u l a t e t h e
q u a n t i t y , i n mg, o f C27H440 i n t h e p o r t i o n o f C h o l e c a l c i -
f e r o l t a k e n by t h e f o r m u l a 0.25C(A /As), i n which C i s t h e
c o n c e n t r a t i o n , i n p g per m l , o f UP! C h o l e c a l c i f e r o l RS i n
t h e Standard Preparation, and A, and As are t h e peak r e s -
ponses f o r c h o l e c a l c i f e r o l o b t a i n e d f o r t h e Assay Prepar-
a t i o n and t h e Standard P r e p a r a t i o n , r e s p e c t i v e l y .
4.2.6.2. HPLC A n a l y s i s o f v i t a m i n D? i n
Pharmaceutical P r e p a r a t i o n s
HPLC methodology p e r m i t s r a p i d and

g e n e r a l l y q u i t e simple a n a l y s i s o f v i t a m i n D3 i n pharmaceu-
t i c a l b u l k drugs, o c c u r i n g as o i l s o l u t i o n s , beadlets, o r
resins. However, f o r many m u l t i v i t a m i n products, e x c i p i e n t
components w i t h d i f f e r i n g degrees o f p u r i t y , o t h e r v i t a m i n s
and t h e i r d e g r a d a t i o n products, and whether v i t a m i n D i s
p r e s e n t as beadlets, o i l , o r r e s i n , can make HPLC methoqogy
a c h a l l e n g i n g procedure. Since t h e e x t i n c t i o n c o e f f i c i e n t
f o r D3 a t 254 nm i s very h i g h (-15,000) standard, s i n g l e
wavelength d e t e c t o r s a t 254 nm a r e g e n e r a l l y used.
Reverse-phase HPLC procedures f o r v i t a m i n Dg i n b u l k

d r u g were r e p o r t e d i n t h e e a r l y 70's by W i l l i a m e t a l . ( 5 4 )
u s i n g a DuPont Permaphasee ODs column (DuPont , W i l m i ngton,
Del.) and 78% methanol i n water as t h e m o b i l e phase. A
s i m i l a r column and m o b i l e phase (DuPont's Zorbax" ODS Column
and 95% methanol i n w a t e r ) were r e p o r t e d t o g i v e i n c r e a s e d
r e s o l u t i o n o f t h e two forms o f v i t a m i n D (62) i n m u l t i v i t a -
min f o r m u l a t i o n s . Complete s e p a r a t i o n o f v i t a m i n s D2 and D3
by reverse-phase chromatography i n model m u l t i v i t a m i n prepa-
r a t i o n s were r e p o r t e d by Osadca and A r a u j o (63). In this
694 K. THOMAS KOSHY A N D WILLIAM E BEYER
a n a l y s i s t h e y used a Vydac column ( S e p a r a t i o n s Group, Hes-

pera, CA.) and a m o b i l e phase o f 90% methanol i n water.
Tscherne and Capi t a n 0 (64) a l s o r e p o r t e d complete s e p a r a t i o n
o f D2 and D3 w i t h a 12 Bondapake C column (Waters, M i l f o r d ,
Mass.) and a m o b i l e phase o f 86!!!?% methanol i n water w i t h
added s i l v e r n i t r a t e . S e p a r a t i o n o f t h e two v i t a m i n s was
s a t i s f a c t o r y , however, t h e presence o f s i l v e r n i t r a t e i n
i t s e l f can g i v e r i s e t o a m u l t i t u d e o f problems.
A reverse-phase HPLC assay, as p a r t of t h e A s s o c i a t i o n

o f O f f i c i a l A n a l y t i c a l Chemists r e p o r t on a n a l y s i s o f f a t -
s o l u b l e v i t a m i n s , was d e s c r i b e d by DeVries e t . a l . (65).
A n a l y s i s were made w i t h a Merck LiChrosorb RP-18 column
( M a n u f a c t u r i n g Chemists, Inc., Cincinnati, OH) and a
a c e t o n i t r i 1 e : p r o p i o n i t r i 1e:water (79:15:6) m o b i l e phase.
A1 though adequate chromatography was r e a l i z e d , t h e a u t h o r s
were concerned t h a t problems arose concerning i n f l u e n c e o f
temperature, d i s s o l u t i o n of sample and p u r i f i c a t i o n of s o l -
vents i n t h e m o b i l e phase. F o r these reasons they recom-
mended normal -phase chromatography. Separation o f v i tami ns
D2 and D3 w i t h t h e i r systems was n o t discussed.
Normal-phase HPLC on s i l i c a columns a r e a l s o used

e x t e n s i v e l y i n D3 a n a l y s i s o f v i t a m i n products w i t h non-
p o l a r m o b i l e phase c o n t a i n i n g p o l a r m o d i f i e r s . Krol e t al.
(66) separated D3 from pre-D3 and from a m i x t u r e o f o t h e r
v i t a m i n s u s i n g an a d s o r p t i v e s i l i c a support i n t r o d u c e d i n
1972 (Vydaco, s u p p l i e d a t t h a t t i m e by A p p l i e d Science
l a b o r a t o r i e s , Inc. S t a t e Col 1ege , Penn. ) . The hand-packed
column was used i n c o n j u n c t i o n w i t h a m o b i l e phase o f pen-
t a n e : t e t r a h y d r o f u r a n (97.5:2.5). S t e r u l e (32) used aluminum
o x i d e as column support w i t h c h l o r o f o r m as t h e m o b i l e
phase. Separation o f O3 and i t s isomers and from v i t a m i n A
a c e t a t e was achieved.
DeVries e t a l . (67) r e p o r t e d t h e sumnary o f s t u d i e s by a

number o f c o l l a b o r a t i n g l a b o r a t i e s f o r t h e HPLC assay o f
v i t a m i n D i n m u l t i v i t a m i n p r e p a r a t i o n s . S a p o n i f i c a t i o n and
a reverse-phase Merck LiChrosorb RP-8 column were used f o r
sample cleanup. The a n a l y t i c a l column was a P a r t i s i l a , 5 pm
column (Whatman, C l i f t o n , N.J.) w i t h hexane-amyl a l c o h o l
(99.65:0.35%) as t h e m o b i l e phase. The cleanup procedure
a l t h o u g h a deparature from t h e usual a n a l y t i c a l methods, was
incorporated to ensure predictable, interference-free
v i t a m i n D assays (D2 and D3 c o - e l u t e ) .
Normal-phase columns, e i t h e r Water's p P o r a s i l o o r

DuPont's Zorbax-sil@, were used f o r t h e data o f Sheridan's
VITAMIN DI(CHOLECALCIFEROL) 695
(68) f i n a l r e p o r t o f t h e Pharmaceutical M a n u f a c t u r e r ' s

Associ a t i on-Qua1 it y C o n t r o l S e c t i o n (PMA-QC) .
covered c o l l a b o r a t i v e s t u d i e s on v i t a m i n D u s i n g HPLC.
The d a t a
A
m o b i l e phase o f ch1oroform:hexane:tetrahydrofuran (70:30:1)
was used w i t h d e t e c t i o n a t 254 nm. The HPLC method
separated v i t a m i n D from i n t e r f e r i n g compounds t h a t c o u l d be
present, namely 7-dehydrocholesterol , t a c h y s t e r o l , i s o -
t a c h y s t e r o l p r e - v i t a m i n D, t r a n s - v i t a m i n D, l u m i s t e r o l , and
e r g o s t e r o l , from o t h e r v i t a m i n s ( A a c e t a t e , A p a l m i t a t e ,
t o c o p h e r o l , t o c o p h e r y l acetate, and phytonadione), and from
antioxidants. Samples and standards were t r e a t e d i d e n t -
i c a l l y as t o t i m e and temperature so t h a t o n l y v i t a m i n D
need be measured, s i n c e f o r p r a c t i c a l purposes p r e v i t a m i n D
would be t h e same f o r sample and standard. Vitamins D3 i n
r e s i n and v i t a m i n D2 i n b e a d l e t s were t r e a t e d d i f f e r e n t l y
f o r sample p r e p a r a t i o n s . The r e s i n was d i s p e r s e d i n i s o -
octane, t r e a t e d w i t h u l t r a s o n i c a t i o n , and d i l u t e d W i t h
isooctane. Beadlets were added t o a m i x t u r e o f DMS0:water
(3:1), shaken v i g o r o u s l y , and heated a t 5OoC f o r 20 min-
utes. A f t e r c o o l i n g t o room temperature, hexane was added,
t h e m i x t u r e was t h e n shaken and c e n t r i f u g e d . The hexane
l a y e r was removed and t h e e x t r a c t i o n procedure w i t h hexane
was c a r r i e d o u t t h r e e more t i m e s . The combined hexane
e x t r a c t s were t h e n d r i e d w i t h anhydrous sodium s u l f a t e and
reduced t o dryness w i t h a r o t o - e v a p o r a t o r . The d r y r e s i d u e
was t h e n d i s s o l v e d i n i s o o c t a n e f o r i n j e c t i o n on t o t h e
column. The HPLC procedure was considered advantageous
because m i c r o - p a r t i c u l a t e columns were r e p r o d u c i b l e and
a v a i l a b l e commercially, ambient temperature requirements
reduced thermal breakdown o f 0, and time-consuming s a p o n i f i -
c a t i o n was n o t necessary. The method was considered
s u p e r i o r t o methods o f t h e USP (30) and AOAC (44).
Based on t h e r e p o r t o f Sheridan (68), Walker e t a1 (69) .

were a b l e t o assay a v a r i e t y o f pharmaceutical p r o d u c t s f o r
D (and r e p o r t e d l y D3, a l t h o u g h no d a t a were given). A S i -
5g s i l i c a column (Brownlee Labs., Santa Clara, C a l i f . ) was
used i n c o n j u n c t i o n w i t h a m o b i l e phase comprised o f a
m i x t u r e o f ch1oroform:water-saturated hexane: hexane: t e t r a -
h y d r 0 f u r a n : a c e t i c a c i d (60:15:25:1.5:0.4). As w i t h t h e HPLC
assay o f t h e PMA-QC r e p o r t (68), D2 and D3 co-eluted.
Comments were made concerning t h e a d d i t i o n o f p o l a r modi-
f i e r s o r changes i n t h e r a t i o of m o b i l e phase t o enhance
chromatography.
B u i l d i n g on p u b l i s h e d l i t e r a t u r e and based on e m p i r i c a l
t r i a l s , an e f f i c i e n t reverse-phase HPLC system and sample
p r e p a r a t i o n scheme has been developed f o r v i t a m i n D3 i n
m u l t i v i t a m i n products i n t h e author's laboratory f o r i n -

house use (70). Although t h e method does n o t appear i n open
l i t e r a t u r e , i t has been used e x t e n s i v e l y i n our l a b o r a t o r i e s
and w i l l be d e s c r i b e d i n some d e t a i l . It i s f e l t t h a t t h e
sample p r e p a r a t i o n and t r e a t m e n t scheme, t h e use o f two
i n t e r n a l standards, and t h e c a p a b i l i t y t o s w i t c h m o b i l e
phases t o r i d t h e column o f v e r y slow e l u t e r s a r e h e l p f u l i n
r o u t i n e q u a l i t y c o n t r o l assays and f o r s t a b i l i t y e v a l u a t i o n
o f v i t a m i n D3 i n a v a r i e t y of pharmaceutical products.
I n t h i s procedure, t h e HPLC a n a l y s i s i s c a r r i e d o u t on a
DuPont Zorbax ODs@ column u s i n g methanol , acetoni t r i l e and
water 10:2:1 a t t h e m o b i l e phase. T h i s system r e s o l v e s
v i t a m i n s D3 from D2 and from d e g r a d a t i o n products and o t h e r
f a t s o l u b l e vitamins. D i d e c y l and d i n o n y l p h t h a l a t e were
used as t h e i n t e r n a l standards. The l a t t e r was i n c l u d e d f o r
use i n t h e event t h a t extraneous peaks i n t e r f e r e d w i t h t h e
d i d e c y l p h t h a l a t e peak. F i g u r e 11 shows a chromatogram o f
an e x t r a c t o f a m u l t i v i t a m i n f o r m u l a t i o n w i t h t h e two added
i n t e r n a l standards.
M u l t i v i t a m i n l i q u i d suspensions were prepared f o r assay

u s i n g a hexane e x t r a c t i o n o f t h e sample from d i m e t h y l s u l -
foxide:lO% g l a c i a l a c e t i c a c i d ( 9 : l ) . A small volume o f
hexane c o n t a i n i n y t h e i n t e r n a l standards was added quan-
t i t a t i v e l y t o t h e e x t r a c t i n g s o l u t i o n . P r o v i s i o n s were made
t o a u t o m a t i c a l l y r i n s e t h e column between i n j e c t i o n s w i t h
methanol : t e t r a h y d r o f u r a n : d i m e t h y l s u l f o x i d e (25:25:1) to
remove any slow e l u t i n g m a t e r i a l s . Column r i n s i n g was
i n i t i a t e d and c o n t r o l l e d ( D i g i t a l Valve Sequence Programmer,
Valco, Houston, Texas), by an automatic sampler. Mobile
phase and column r i n s e s w i t c h i n g was made w i t h a v a l v e
(Model 5302 Rheodyne, C o t a t i , C a l i f . ) , f i t t e d w i t h an a i r
a c t i v a t o r (Model 5300, Rheodyne). A i r t o t h e v a l v e was
c o n t r o l l e d by a s o l e n o i d v a l v e (No. 062E1-3-10-20-35, Hum-
phrey Products, Kalamazoo, Mich.). With t h i s chromato-
graph c system d i n o n y l p h t h a l a t e e l u t e d i n about 15 minutes,
v i tam n D i n 25 minutes, and d i d e c y l p h t h a l a t e i n 30 min-
Utes. Co3umn r i n s e was complete i n approximately 10 min-
Utes, p e r m i t t i n g an a n a l y s i s t i m e o f about 40 minutes.
0 1 f o r m u l a t i o n s o f m u l t i v i t a m i n Droducts were d i l u t e d
w i t h hexane, passed t h r o u g h a SEP-PAK@ s i l i c a c a r t r i d g e
(Waters, M i l f o r d , Mass.) where t h e o i l was r e t a i n e d . The
c a r t r i d g e was t h e n f l u s h e d w i t h methanol t o e l u t e D3 i n t o a
c o n t a i n e r h a v i n g t h e a p p r o p r i a t e amount o f i n t e r n a l stan-
dard, p r e v i o u s l y evaporated t o dryness. The c a r t r i d g e ,use
was based on t h e r e p o r t s f r o m Water's ( 7 1 ) and from R.A.
RESPONSE, 0.0032 AUFS
-Injection
n
m
5
n
S
A.
a
Dinonyl Phthalate
0
-h
nJ
A.
2
3
S
4.
0
S
Pask-Hughs and D.H.Ca1 am (72).
HPLC procedures f o r v i t a m i n D i n most i n s t a n c e s have

s o l v e d problems o f i n t e r f e r e n c e f r o m t h e m a t r i x and
d e g r a d a t i o n products, presence of isomers and homo1ogues,
o t h e r f a t - s o l u b l e v i t a m i n s and t h e i r companion compounds.
S t a b l e and h i g h l y e f f i c i e n t columns, an e a s i l y o b t a i n a b l e
and s t a b l e d e t e c t o r operated a t 254 nm ( n e a r absorbance
maximum of 265 nm) , and r e 1ia b l e sample p r e p a r a t i v e
techniques and apparatuses have made HPLC a most appeal ing
technique. A choice of techniques w i t h o r w i t h o u t i n t e r n a l
standards i s available, depending on t h e amount o f
development t i m e t h a t can be committed and t h e presence o r
absence o f s u i t a b l e areas on t h e chromatogram f o r t h e
i n s e r t i o n of an i n t e r n a l standard peak. The a d d i t i o n o f an
i n t e r n a l standard a t t h e e a r l i e s t t i m e p o s s i b l e i n t h e
sample p r e p a r a t i o n scheme can add c o n s i d e r a b l y t o t h e
accuracy o f t h e HPLC method. It can a i d i n v a r i o u s ways,
namely; c o r r e c t f o r recovery, negate b i a s f r o m l o s s e s i n
i s o l a t i o n steps, improve p r e c i s i o n a s s o c i a t e d w i t h a l i q u o t
removal and d i l u t i o n , and i m p o r t a n t l y , compensate f o r
changes i n i n s t r u m e n t a l performance, p a r t i c u l a r l y , long
chromatographic runs. Valve s w i t c h i n g f o r columns and
m o b i l e phases can decrease chromatographic t i m e by d i v e r t i n g
unwanted peaks o r f l u s h i n g t h e column a f t e r d e s i r e d peaks
have been obtained. I n essence, HPLC has been u t i l i z e d as
one o f t h e most powerful a n a l y t i c a l t o o l s a v a i l a b l e f o r
v i t a m i n D a n a l y s i s i n pharmaceutical products. Even g r e a t e r
u t i l i t y o f HPLC can be expected as h i g h e r e f f i c i e n c y columns
and e x t r a c t i o n procedures, b e t t e r column hardware, and
in c r e a s i n y l y e f f ic i e n t in s t r u m e n t a t i on a r e developed and
made a v a i l a b l e .
4.2.6.3 A n a l y s i s V i t a m i n D? i n M i l k and M i l k
Powder
M i l k s o l d i n t h e USA and Canada i s

r e q u i r e d by law t o be f o r t i f i e d w i t h e i t h e r v i t a m i n D o r D
a t t h e l e v e l o f 400 I U / q u a r t which i s e q u i v a l e n t t o 16 ng/m!?
o r 10 ppb. It i s i m p o r t a n t f o r n u t r i t i o n a l reasons t h a t
compliance w i t h t h e r e g u l a t i o n be checked by a n a l y z i n g t h e
f o r t i f i e d milk. Moreover, i t i s q u i t e p o s s i b l e t h a t a
harmful excess c o u l d be added by mistake, and t h e r e f o r e i t
i s i m p e r a t i v e t h a t e r r o r s i n f o r t i f i c a t i o n be d e t e c t a b l e .
O f a l l t h e methods a v a i l a b l e HPLC appears t o be t h e most
r e l i a b l e one even though i t r e q u i r e s c a r e f u l h a n d l i n g o f t h e
sample. A number o f methods have been r e p o r t e d f o r t h e
a n a l y s i s o f v i t a m i n D3 i n whole m i l k (74-79), f o r t i f i e d
d r i e d m i l k s (76,77,80), skim and c h o c o l a t e m i l k (81), i n -

s t a n t n o n f a t d r i e d m i l k (82) and i n f a n t formula (79). There
i s s t i l l no w i d e l y accepted method b u t t h e general approach
( 7 6 ) i n v o l v e s s a p o n i f i c a t i o n and e x t r a c t i o n o f t h e unsaponi-
f i a b l e r e s i d u e f o l l o w e d by an HPLC cleanup on a n i t r i l e
bonded s i l i c a column t o separate v i t a m i n D3 and i t s isomers
from i n t e r f e r i n g substances. The f r a c t i o n corresponding t o
v i t a m i n D3 i s c o l l e c t e d and q u a n t i t a t e d on a m i c r o p a r t i c u -
l a t e s i l i c a column u s i n g 0.35-1% amyl a l c o h o l i n n-hexane as
t h e m o b i l e phase. The HPLC method i s s u i t a b l e f o r t h e
d e t e r m i n a t i o n o f e i t h e r v i t a m i n D or D3 i n m i l k . It i s
remarkable t h a t i t i s p o s s i b l e t o teetermine f a i r l y accurate-
l y t h e amounts o f v i t a m i n D p r e s e n t i n whole m i l k and i n
d i f f e r e n t forms o f r e c o n s t i t u t e d m i l k .
4.2.6.4 D e t e r m i n a t i o n o f V i t a m i n D2 i n
Animal Feeds
The b u l k use o f commercially pro-

duced v i t a m i n s D2 and D a r e f o r t h e supplementation o f
p o u l t r y , c a t t l e , swine an$ p e t feeds. The c o n c e n t r a t i o n o f
v i t a m i n D i n animal feed premixes ranges from 30,000 t o
150,000 I i / k g . It i s i n t h e range o f 500 t o 4000 IU/kg i n
t h e f i n a l feed. The animal f e e d composition v a r i e s
depending on t h e a v a i l a b i l i t y and p r i c e s o f t h e feed
ingredients. Therefore i t i s a very c h a l l e n g i n g problem t o
develop an acceptable method f o r t h e a n a l y s i s o f v i t a m i n D
i n animal feeds. However, t h e r e a r e a l r e a d y r e p o r t s (83-897
o f p a r t i a l l y successful e f f o r t s i n t h i s regard. A
c o l l a b o r a t i v e study (88) was conducted under t h e auspices o f
Associ a t ion o f O f f ic i a1 Ana 1y t ica 1 Chemi s t s , Wash in g t on,
D.C. The procedure based on t h e one developed by Knapstein
.
e t a1 (89) i n v o l v e s s a p o n i f i c a t i o n o f 25 g o f t h e powdered
sample, e x t r a c t i o n o f t h e u n s a p o n i f i a b l e s , cleanup on an
a1 umi na column u s i ng ether-hexane m i x t u r e s , a d d i t i o n a l
cleanup on a c18 bonded m i c r o p a r t i c u l a t e s i l i c a column u s i n g
CH$N-MeOH-H20 (50:50:5) as t h e m o b i l e phase and f i n a l
q u a n t i t a t i o n on a m i c r o p a r t i c u l a t e s i l i c a column (250 x 4.6
( i d ) mn) u s i n g 0.35% amyl a l c o h o l i n n-hexane as t h e m o b i l e
phase. R e s u l t s are very encouraging and i t can be expected
t h a t p r a c t i c a l methods w i l l be a v a i l a b l e i n t h e near f u t u r e .
4.2.6.5. D e t e r m i n a t i o n o f V i t a m i n D2 i n Cod
Liver O i l
P r i o r t o t h e a v a i l a b i l i t y o f synthe-
t i c v i t a m i n s D2 and U3, f i s h l i v e r o i l s were t h e p r i m a r y
sources of v i t a m i n D3. The commonly used o i l i s t h a t from
t h e cod l i v e r which c o n t a i n s about 100-150 I U o f v i t a m i n D

per gram. F i s h l i v e r o i l s a l s o c o n t a i n s l a r g e amounts o 9
v i t a m i n A and c h o l e s t e r o l . I n cod l i v e r o i l t h e w e i g h t
r a t i o may be 1:100:3000 f o r v i t a m i n D3, v i t a m i n A and
c h o l e s t e r o l . The main d i f f i c u l t y i n a n a l y z i n g cod l i v e r o i l
f o r v i t a m i n D3 i s i n s e p a r a t i n g i t from t h e l a r g e excess o f
cholesterol. Even though p r e c i p i t a t i o n of c h o l e s t e r o l w i t h
d i g i t o n i n or by f r e e z i n g a r e s t a n d a r d procedures, t h e s e
methods a r e p h y s i c a l l y cumbersome and a l s o r e s u l t i n l o s s o f
v i t a m i n D by o c c l u s i o n . Therefore, t h e a n a l y s i s o f v i t a m i n
D3 i n c o d f i v e r o i l i s a d i f f i c u l t problem. A l i (90) has
r e p o r t e d a method based on a column chromatographic clean-up
f o l l o w e d by a t h i n l a y e r chromatography and spectrophoto-
metry. HPLC procedures have been r e p o r t e d by A l i (91),
Egaas and Lambersten (92) and Stancher and Sonta (93). 3f
these t h e procedure by Stancher and Zonta (93) appears t o be
t h e simplest. The procedure i s r e p o r t e d l y a p p l i c a b l e f o r
t h e simultaneous d e t e r m i n a t i o n o f v i t a m i n s D3 and E, b u t
t h e recoveries o f t h e p r e l i m i n a r y e x t r a c t i o n step using
e i t h e r hexane o r e t h y l e t h e r i s i n t h e range 60-75% only.
The a u t h o r s do n o t r e p o r t t h e o v e r a l l recovery f o r t h e
procedure.
From t h e above d i s c u s s i o n i t i s c l e a r t h a t t h e r e i s
scope f o r more work i n d e v e l o p i n g a b e t t e r method f o r v i t a -
min D3 i n cod l i v e r o i l .
4.2.6.6. D e t e r m i n a t i o n o f Vitamin D2 i n
Chicken Eaa Yolk
The c h i c k e n egg y o l k , as deduced

from i t s a n t i r a c h i t i c a c t i v i t y , c o n t a i n s 1-2 pg o f c h o l e c a l -
c i f e r o l and t h i s can be r a i s e d f u r t h e r by i n c r e a s i n g t h e
i n t a k e o f v i t a m i n D3 o f t h e l a y i n g hen (94). The concen-
t r a t i o n i n y o l k i s 5-10 t i m e s h i g h e r than t h a t o f t o t a l
v i t a m i n D3 p l u s i t s m e t a b o l i t e s i n b l o o d plasma o f normal
chickens and 50-100 t i m e s h i g h e r than t h e c o n c e n t r a t i o n i n
any o t h e r t i s s u e (95). Consequently, t h e chicken egg y o l k
i s a p o t e n t source o f v i t a m i n U3.
Because several o f t h e m e t a b o l i t e s o f v i t a m i n D3 a r e
b i o l o g i c a l l y a c t i v e , t h e i n o l e c u l a r species o f v i t a m i n D3
which passes i n t o t h e y o l k cannot be determined j u s t from
measurement o f a n t i r a c h i t i c a c t i v i t y . Consequently, a
r e l i a b l e and s e n s i t i v e method f o r d e t e r m i n i n g t h e amount o f
t h e unchanged form o f v i t a m i n D3 would be e x t r e m e l y
benef ic i a1 t o those i n t e r e s t e d i n t h e metabol ism and o t h e r
f a c t o r s t h a t i n f l u e n c e t h e c h i c k e n t o d e p o s i t v i t a m i n D3 i n
VITAMIN Dj (CHOLECALCIFEROL) 70 I
t h e egg y o l k .
Packson e t a l . ( 9 6 ) have r e c e n t l y r e p o r t e d an HPLC

method f o r t h e a n a l y s i s o f v i t a m i n D3 i n egg y o l k . They use
v i t a m i n D2 as an i n t e r n a l s ta n d a rd which i s added t o 50 g of
t h e f r e e z e - d r i e d egg a t t h e b e g i n n i n g o f t h e a n a l y s i s t o
compensate f o r l o s s e s d u r i n g t h e s e v e r a l m a n i p u l a t i v e s t e p s
i n t h e procedure. The sample i s s a p o n i f i e d f o r 30 m i n u t e s
i n a N2 atmosphere. The u n s a p o n i f i a b l e s a r e e x t r a c t e d w i t h
1:l m i x t u r e o f p e tro l e u m e t h e r and d i e t h y l e t h e r . The ex-
t r a c t a f t e r washing f r e e o f base i s evaporated. The excess
s t e r o i d s a r e removed by p r e c i p i t a t i o n by c h i l l i n g from 9O:lO
methanol-water s o l u t i o n and removed by f i l t r a t i o n . The
e x t r a c t i s evaporated t o dryness, d i s s o l v e d i n a s m a l l
volume of l i g h t p e tro l e u m e t h e r and s u b j e c t e d t o TLC on
s i l i c a gel p l a t e s . The band r e p r e s e n t i n g v i t a m i n D i s
scraped, e l u t e d w i t h HPLC grade methanol , c o n c e n t r a t e d and
s u b j e c t e d t o HPLC on a 250 x 4 mm i.d. stainless steel
column packed w i t h a C reversed-phase p a c k i n g (Magnusil
C 5x) u s i n g e i t h e r 9$:5 o r 9O:lO methanol-water as t h e
m % i l e phase. Under t h e HPLC c o n d i t i o n s v i t a m i n D2 e l u t e d
b e f o r e v i t a m i n D3 i n about 14-17 minutes. The two peaks
were n o t c o m p l e t e l y re s o l v e d . Replicate analysis o f 5
samples showed v i t a m i n D3 c o n t e n t i n t h e range 1.3-1.9
pg/lOO g (mean 1.6 f 0.2 pg/lOO g) o f whole f r e e z e - d r i e d
sample. T h i s i s e q u i v a l e n t t o 0.32 pg/20 g o f whole egg.
T h i s v a l u e i s c o n s i d e r a b l y l o w e r t h a n t h e c u r r e n t l y accepted
v a l u e of 1-2 p g p e r egg y o l k w e i g h i n g 15-20 g (94).
5. Metabolites o f Vitamin D3
5.1 Pri m a ry Metabol it e s
F o r n e a r l y 30 y e a r s f o l l o w i n g t h e d i s c o v e r y o f
v i t a m i n D3, r e l a t i v e l y l i t t l e was known about i t s m e t a b o l i c
f a t e i n man. It was th o u g h t t h a t v i t a m i n D3 a c t e d d i r e c t l y
on t h e t a r g e t t i s s u e s o f i n t e s t i n e and bone. However, t h e
time lag reported by Carlsson (97) between the
admi n i s t r a t ion o f v i ta m i n D3 and i t s p h y s i o l o g i c a l response
led investigators to suspect that vitamin D3 was
m e t a b o l i c a l l y a l t e r e d b e f o r e i t became b i o l o g i c a l l y a c t i v e .
Major e v e l o p y g n ts i n t h e m e t a b o l i c s t u d y came a f t e r
s e l e c t i v e H' and C labeled vitamin D w i t h high specific
a c t i v i t i e s were s y n t h e s i z e d i n t h e mid-?960's. S i n c e then,
massive e f f o r t s by a number o f i n v e s t i g a t o r s have l e d t o t h e
d i s c o v e r y o f a l a r g e number o f m e t a b o l i t i e s , t h e hormonal
n a t u r e of t h e p r i m a r y m e t a b o l i t i e s i n t h e maintenance o f
calcium homeostasis, t h e n a t u r e o f t h e s p e c i f i c p r o t e i n s
t h a t c a r r y t h e m e t a b o l i t e s t o t a r g e t t i s s u e s , t h e enzymes
i n v o l v e d i n t h e t r a n s f o r m a t i o n s and t h e i n t r i c a t e r o l e
played by o t h e r endocrine organs i n t h e c o n t r o l o f t h e
metabolism o f v i t a m i n D
e x t e n s i v e l y (98-100).
. These f i n d i n g s have been reviewed
?he c u r r e n t l y known metabol i c pathway
o f v i t a m i n D3 i s shown i n Scheme V. Vitamin D from t h e
d i e t o r t h a t generated i n t h e s k i n i s absorbed i n t o t h e
blood. The c o n c e n t r a t i o n o f v i t a m i n 03 i n t h e blood i s very
low, but excess v i t a m i n i s s t o r e d i n t h e f a t depots. Deple-
t i o n o f v i t a m i n from f a t t y t i s s u e s depends on t h e t u r n o v e r
o f f a t which i s g e n e r a l l y a slow process.
Lund and DeLuca (101) administered C3H] v i t a m i n 03 t o

r a t s and found b i o l o g i c a l l y a c t i v e m e t a b o l i t e s i n t h e bone,
1iver and serum. The aqueous-sol u b l e metabol it e s f rom t h e
t i s s u e s and t h e feces d i d not have v i t a m i n D a c t i v i t y . A t
l e a s t t h r e e b i o l o g i c a l l y a c t i v e m e t a b o l i t e s were i s o l a t e d
from t h e c h l o r o f o r m - s o l u b l e p o r t i o n o f t h e e x t r a c t . One o f
these was found i n l a r g e amounts i n t h e l i v e r , blood and
bone. I n 1968, B l u n t et. a l . (102) proved c o n v i n c i n g l y t h a t
t h i s major m e t a b o l i t e i s 25-hydroxyvitamin D3. (25-OH-0 ).
Two o t h e r groups o f i n v e s t i g a t o r s (103,104) independenhy
found c l u e s t o t h e metabolic h y d r o x y l a t i o n o f v i t a m i n D
It was soon e s t a b l i s h e d t h a t 25-hydroxylation o f v i t a m i n
takes p l a c e p r i m a r i l y i n t h e l i v e r (105,106) and t h a t 25-OH-
a;
D i s t h e major form o f c i r c u l a t i n g v i t a m i n D3 i n human
p?asma (107).
I n i t i a l l y , 25-OH-D3 was considered t o be t h e main b i o -

l o g i c a l l y a c t i v e m e t a b o l i t e o f v i t a m i n 0. But soon i t was
discovered t h a t p h y s i o l o g i c a l c o n c e n t r a t i o n s o f Z5-OH-D3,
l i k e v i t a m i n D , are incapable o f s t i m u l a t i n g e i t h e r i n t e s -
t i nal c a l c i um %ransport or bone c a l c i urn y o b i 1 iz a t i on (108-
110). E a r l i e r work (111,112) w i t h [ l a - H I v i t a m i n D3 had
shown t h a t one o f t h e unknown m e t a b o l i t e s had l o s t i t s
t r i t i u m from t h e C - 1 p o s i t i o n . Fraser and Kodicek (113)
e s t a b l i s h e d t h a t t h i s a c t i v e m e t a b o l i t e i s 1-oxygenated 25-
OH-D3 and t h a t it was produced i n t h e kidney. A s h o r t t i m e
l a t e r Lawson e t a l . (114) i d e n t i f i e d t h i s m e t a b o l i t e t o be
l a , 25-dihydroxyvitamin D3 (1,25-(OH)2D3) which was confirm-
ed by o t h e r i n v e s t i g a t o r s (115,116).
The endogenous l e v e l o f 25-OH-03, t h e primary m e t a b o l i t e

o f v i t a m i n 03, has been determined by a number o f i n v e s t i -
gators and found t o be i n t h e 15-30 ng/ml i n normal Cauca-
s i a n plasma. The c o n c e n t r a t i o n o f 25-OH-03 increases w i t h
increased v i t a m i n D3 i n t a k e and t o extended exposure t o
\
lo-OH-03 1,25-~OH1203- 2 6 . 2 3 - l o c l o n e
(Ref 130,1311
no"
Tn U"k"O.".i
\\
25-(OH1-24-ora D3 25,26(OH1203 25-01-03-26,23- loctone
( R e f 133) ( R e 1 1251 ( R e f 128.1291
t
Unknowns 25-OH-D3 (Ref.1321
( R e f . 1021
c."nnn
&on
Unknowns
8n the b t l i
( R e f . 127)
AZ5 -Ia-OH-D3 A Z 4 - .1 - O H - 0 3
( R e f . 1271 ( R e f 127)
Scheme V . M e t a b o l i c pathway f o r v i t a m i n D3. From J . Pharm.

-
S c i . 71, 137 (1982). Reproduced w i t h p e r m i s s i o n o f t h e copy-
r i g h t owner.
sunlight. As p o i n t e d o u t e a r l i e r , i t i s t h e major c i r c u l a -
t i n g and r e a d i l y a v a i l a b l e form o f v i t a m i n D The endo-
genous l e v e l o f 1,25-(OH) D , on t h e o t h e r %nd, i s very
low, between 20 and 40 pgfm? o f plasma. The f o r m a t i o n o f
1,25-(OH)2D3 i s feedback r e g u l a t e d depending on t h e need f o r
c a l c i u m i n t h e blood. A t t h e present time, 1,25-(OH)2D3 i s
c o n s i d e r e d t o be t h e most p o t e n t o f a l l t h e known metabo-
1it e s o f v i t a m i n D3 f o r c o n t r o l 1ing c a l c i u m and phosphorus
a b s o r p t i o n f r o m t h e in t e s t i ne and mobi 1iz a t ion o f these
elements from t h e bone when necessary.
5.2 Secondary Metabol it e s
It became known d u r i n g t h e work on t h e i d e n t i f i c a -

t i o n o f 25-OH-D3 and 1,25-(0H)2D3 t h a t t h e r e were o t h e r
m e t a b o l i t e s some o f which were i d e n t i f i e d i n q u i c k succes-
sion. Of these t h e i m p o r t a n t ones from a b i o l o g i c a l stand-
p o i n t a r e 24R,25-dihydroxyvitamin D3 [24,25-(OH)3D ] and
1,24,25-t r i h y d r o x y v i tami n D3 [ 1,24,25-( OH) 3D3]. Omd&l and
DeLuca r e p o r t e d (117) t h a t when t h e s y n t h e s i s o f 1,25-(OH)
D3 was suppressed by a b l o c k o f t h e kidney hydroxylase whic i
produces 1,25-(OH)2D3 from 25-OH-D , a new m e t a b o l i t e
appeared. T h i s m e t a b o l i t e i s made e x c j u s i v e l y i n t h e kidney
(118,119) and was i d e n t i f i e d as 24,25-(OH)?D3 (120). The
c o n c e n t r a t i o n o f t h i s m e t a b o l i t e i n human i s low b u t much
h i g h e r t h a n t h a t o f 1,25-(OH) D3. T a y l o r e t a l . (121)
r e p o r t e d a value o f 1.68 f 0.8 8 ng/ml among several normal
volunteers. It was demonstrated (122,123) t h a t under normal
o r hypercalcemic c o n d i t i o n s , 24,25-(OH) D-3 i s t h e major
c i r c u l a t i n g m e t a b o l i t e o f 25-OH-D3. L i k e $b-OH-D3 and 1,25-
(OH)2D3, i t i s capable o f e l e v a t i n g serum c a l c i u m and sup-
p o r t i n g bone growth on a normal calcium, normal phosphorus
diet. However, u n l i k e 1,25-(OH) D i t has l i t t l e a b i l i t y
t o m o b i l i z e c a l c i u m f r o m t h e bon$.3’Boyle e t a l . (122,123)
had a l s o shown t h a t 24,25-(OH)2D3 has t o be metabolized t o a
more p o l a r m e t a b o l i t e i n t h e k l d n e y b e f o r e i t became b i o l o y -
i c a l l y active. This m e t a b o l i t e was i s o l a t e d by H o l i c k e t
a l . (124) i n pure form from c h i c k e n kidney homogenates and
i d e n t i f i e d as 1,24,25-(OH)3D3. The b i o l o g i c a l a c t i v i t y o f
1,24,25-(OH) D3 p a r a l l e l s t h e b i o l o g i c a l a c t i v i t y o f 24,25-
(OH)2D3 (1233. There i s no c o n c l u s i v e d a t a on t h e concen-
t r a t i o n o f 1,24,25-(OH)3D3, It i s known t o be lower than
t h a t o f 1,25-(OH) D3 and, t h e r e f o r e , i s i n t h e very low
picogram per m i l l i ? i t e r range i n t h e plasma.
5.3 M e t a b o l i t e s o f Very Low o r Unknown B i o l o g i c a l

Activity
A number o f m e t a b o l i t e s o f v i t a m i n D3 w i t h extreme-
l y low o r o f unknown b i o l o g i c a l a c t i v i t y have been d i s c o v e r -
ed. Scheme V shows t h e s t r u c t u r e s o f these m e t a b o l i t e s ,
t h e i r o r i g i n and a1 so a p p r o p r i a t e 1 it e r a t u r e c i t a t i o n s o f
t h e i r discovery. The m e t a b o l i t e s a r e 25,26-di h y d r o x y v i t a m i n
D3 d i s c o v e r e d by Suda e t a l . (125), la-OH-24,25,26,$i-
t e t ranor-D3-23-carboxyl ic a c i d c a l c i t r o i c a c i d ) ( 126), A -
l a - h y d r o x y v i t a m i n D (127), b2'-la-hydroxyvitamin D (127),
25-hydroxyvitamin d -26,23-lactone (128,129) , la-25-dihy-
d r o x y v i t a m i n D3-26,2\-lactone (130,131), 25,26,27-tris-nor-
v i t a m i n D -24-carboxyl ic a c i d ( 132), 25-hydroxy-24-oxocho1 e-
c a l c i f e r o ? (133), and p o s s i b l y 25-hydroxyvi tami n D3-25-
g l u c u r o n i d e (134,135). I n i t i a l experiments w i t h r a d i o l a b e l -
ed v i t a m i n D3 showed very l i t t l e r a d i o a c t i v i t y i n t h e aqu-
eous p o r t i o n s a f t e r t h e t i s s u e s were e x t r a c t e d w i t h a mix-
t u r e o f c h l o r o f o r m and methanol. However, t h e presence o f
w a t e r s o l u b l e m e t a b o l i t e s has always been suspected. The
presence o f v i t a m i n D s u l f a t e i n human m i l k was r e p o r t e d by
a few i n v e s t i g a t o r s ( h 6 - 1 3 8 ) . .
R e c e n t l y H o l l i s e t a1 (139)
used a more s p e c i f i c HPLC method f o r v i t a m i n D s u l f a t e i n
human m i l k whey and d i d n o t f i n d any ( d e t e c t i o n l i m i t 1
ng/ml). However, i t i s e s t a b l i s h e d t h a t human m i l k has
antirachitic activity. Lakdawala and Widdowson (137) have
r e p o r t e d t h a t even i n w i n t e r i n t h e U n i t e d Kingdom, b r e a s t
m i l k p r o t e c t s i n f a n t s from r i c k e t s . Therefore, t h e metabo-
1 it e t h a t p r o t e c t s in f a n t s f rom r i c k e t s whi l e on b r e a s t m i 1k
remains t o be c l e a r l y i d e n t i f i e d .
Leading i n v e s t i g a t o r s a r e c e r t a i n t h a t t h e r e a r e many
u n i d e n t i f i e d m e t a b o l i t e s o f v i t a m i n 03. For example, ac-
c o r d i n g t o Onisko e t a l . (127), t h e r e a r e s u b s t a n t i a l
amounts o f unknown m e t a b o l i t e s o f 1,25-(OH)2D3 i n b i l e .
These a r e water-sol u b l e, n e g a t i v e l y charyed compounds which
a r e rendered c h l o r o f o r m - s o l u b l e a f t e r m e t h y l a t i o n suggesting
t h a t t h e s e are unknown c a r b o x y l ic a c i d metabol it e s . Admi n-
i s t r a t i o n o f r a d i o a c t i v e v i t a m i n D3 d a i l y f o r 1-2 weeks
(140) have l e d t o t h e d i s c o v e r y o f a few unknown metabo-
lites. I t i s n o t known i f any o f these have i m p o r t a n t
b i 01o y i c a l a c t iv i t y
6. V i t a m i n D1 As A Prohormone
V i t a m i n 03 i s unique i n t h a t under normal circumstances
it i s formed i n t h e s k i n in s u f f i c i e n t q u a n t i t i e s when
humans and animals a r e exposed t o sunshine, and e x t r a sup-
706 K. THOMAS KOSHY AND WILLIAM E BEYER
plementation i s unnecessary. The p r e v i t a m i n D3 t h a t i s

formed i n t h e s k i n i s s l o w l y released i n t o t h e body as
v i t a m i n D3. Vitamin D3 i s a l s o unique i n t h a t i t has t o be
metabolized i n sequence t o 25-OH-D3 and t o 1,25-(OH) D3 o r
t o some as y e t unknown m e t a b o l i t e b e f o r e i t becomes pEysio-
l o g i c a l l y a c t i v e . Since 1,25-(OH)*D3 a c t s on t i s s u e s remote
from i t s p r o d u c t i o n s i t e , it meets t h e c r i t e r i a and d e f i n i -
t i o n o f a hormone. I n t r u e hormonal form, i t s biogenesis i s
r e g u l a t e d by hypocalcemia o r hypophosphatemia. Then v i t a m i n
D3 can be considered a prohormone and 25-OH-D3 as a
prehormone.
Acknowledgement
The authors acknowledge t h e excel l e n t s e c r e t a r i a l support o f

Mrs. Becky B. Hughson throughout t h e p r e p a r a t i o n o f t h i s
manuscript .
VITAMIN Dz (CHOLECALCIFEROL) 701
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65, 624 (1982).
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49, 35 (1979).
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S t e r o i d Hormone", Academic Press, New York, San Fran-
c i sco, London (1979) .
100. D.E.M.Lawson i n " V i t a m i n D", Academic Press, New York,
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101. J.Lund and H.F.DeLuca, J. L i p i d Res., I, 739 (1966).
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7, 3317 (1968).
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115, 269 (1969).
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106. M.Horsting and H.F.DeLuca, Biochem. Biophys. Res.

Commun., 36, 251 (1969).
107. E.B.Mawer, G.A.Lumb, and S.W.Stanbury, - -222, 482

Nature,
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Luca, Endocrinology, -90, 605 (1972).
109. M.F.Holick, M. Garabedian, and H.F.DeLuca, Science,

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176, 1146 (1972).
110. R.G.Wong, J.F.Myrtle, H.C.Tsai, and A.W.Norman, J.

B i o l . Chem., 247, 5728 (1972).
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Ser. B., 164, 1 (1966).
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171 (1969).
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714 K. THOMAS KOSHY A N D WILLIAM F. BEYER
114. D.E.M.Lawson D.R.Fraser, E.Kodicek, H.R.Morris, and

D.H.Williarns, i b i d . , 230, 228 (1971).
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147, 143 (1971).
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Acad. Sci. USA, 68, 803 (1971).
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174, 949 (1971).
118. I.T.Boyle, R.W.Gray, J.L.Omdah1, and H.F.DeLuca, in

I 1I, I n t e r n a t i o n a l Syrnposi urn Endocri nology , Wm. Hei ne-
mann Medical Books, L t d , London, England, pp. 468-476
(1972).
119. J.L.Omdah1, R.W.Gray, I.T.Boyle, J.Knutson, and H.F.De-

Luca, Nature New B i o l . , - 237, 63 (1972).
120. M.F .Hol ick, H.K.Schnoes , H.F .DeLuca , R.W .Gray,

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B i o l , Chem., 248, 4174 (1973).
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M.F.Holick, B i o c h e m i s t r y , 2, 4776 (1970).
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3977 (1979).
127. B.L.Onisko, R.P.Esvelt, H.K.Schnoes, and H.F.DeLuca,

ibid., 19,4124 (1980).
128. J.K.Wichmann, H.F.DeLuca, H.K.Schnoes, R.L.Horst,

R.M.Shepard, and N.A.Jorgensen, ibid., 18,4775 (1979).
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Res. Commun., p5, 520 (1980).
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H.F.DeLuca, Proc. N a t l . Acad. Sci., USA, 77, 6411
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A.W.Norman, Arch. Biochem. Biophys., -
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F o u r t h Workshop on V i t a m i n D," Walter deGruyter, Ber-
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T.Suda, S.Yamada, H.Takayama, H.Hori kawa, T.Masumura
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20. 222 (1981).
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m i no1 , 33 (1967).
Higaki, and T.Asano,
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Lancet, 1, 167 (1977).
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(1980).
139. t3.W .Hol 1is , B.A.Roos , D.H. Draper,

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Nutr.,- 111, 384 (1981).
and P.W. Lambert , -
J .
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__
188, 145 (1978).
PROFILE SUPPLEMENTS
TOLBUTAMIDE
Said E. Ibrahiin and Abdullah A. Al-Badr
King Saud University

1. I Crystal Shape 720
I .2 Crystal Structure 720
1.4 Carbon-I3 Nuclear Magnetic Resonance Spectrum 72 1
3. Pharmacokinetics 73 1
4. Identification 733
References 734
ANALYTICAL PROFILES OF D R U G S U B S T A N C E S Copytghr 0 19x4

VOLUME 13 719 by [he American Phannaceutical Asrociation
ISBN 0-I?-260813-5
720 SAID E. IBRAHIM AND ABDULLAH A . AL-BADR
TOLBUTAMIDE
Tolbutamide, N- [ (butylamino) c a r b o n y l ] -4-methylbenzenesulphona-

mide; N-(butylamino) carbonyl-p-toluene sulphonamide is a
s u l p h o n l y l u r e a t h a t i s o r a l l y a c t i v e a s a hypoglycemic a g e n t .
The drug s t i m u l a t e s t h e p o n c r e a t i c i s l e t b e t a c e l l s t o
release e x t r a i n s u l i n . I t a l s o i n h i b i t s p h o s p h o d i e s t e r a s e ,
which p r e s e r v e s c y c l i c AMP and t h u s f a v o r g l y c o g e n o l y s i s i n a
number of t i s s u e s .
0
II
CH3
-NH - C - NH- CH -CH2-CH2-CH
2 3
Tolbutamide
1.1 C r y s t a l Shape
Tolbutamide e x i s t s i n two polymorphs, one w a s o b t a i n e d

e i t h e r by c r y s t a l l i z a t i o n of t o l b u t a m i d e from benzene
s o l u t i o n a f t e r a d d i t i o n of hexane, o r by p r e c i p i t a t i o n
from s o l u t i o n i n aqueous ammonia by a d d i t i o n of a c e t i c
a c i d , and t h e o t h e r , m e t a s t a b l e form w a s o b t a i n e d from
e t h a n o l i c s o l u t i o n a f t e r a d d i t i o n of water. The two
forms w e r e c h a r a c t e r i z e d by i n f r a r e d s p e c t r o s c o p y , x-
r a y d i f f r a c t i o n and d . t . a . ( l )
1.2 C r y s t a l S t r u c t u r e
Nirmala and Gowda ( 2 ) have r e p o r t e d t h a t t o l b u t a m i d e

c r y s t a l l i z e s i n t h e orthorhombic s p a c e group P n 2 l a ,
w i t h a=20.16 (2), b=9.05 ( l ) , C=7.85 (1) Ao, 2=4,
D c = l .25, h=l. 24 Mgm-3 and (CuKa) = l .968 mm-1. The
s t r u c t u r e w a s s o l v e d by t h e P a t t e r s o n s e a r c h method and
r e f i n e d t o a n R f a c t o r of 0.083 f o r 615 v i s u a l l y
measured r e f l e c t i o n s . The s t r u c t u r e i s s t a b i l i z e d by
hydrogen bonding between t h e p o l a r group and Van d e r
waals i n t e r a c t i o n s between t h e non p o l a r groups. The
T'OLBUTAMIDE 12 I
hydrogen-bond network and t h e dynamics of t h e p r o t o n s

a r e of g r e a t importance i n e x p l a i n i n g t h e p r o p e r t i e s
of t h e c r y s t a l s and t h e i r phase t r a n s i o n s .
The f o l l o w i n g f i g u r e r e p r e s e n t s a p r o j e c t i o n of t o l -
butamide molecule showing t h e atom numbering and bond
lengths (Ao). The a u t h o r s excluded t h e hydrogen atoms
f o r c l a r i t y (2).
1.3 Mass Spectrum
The e l e c t r o n impact ( E I ) m a s s spectrum a t 70 eV r e c o r d -

ed on Varian Mat 311 m a s s s p e c t r o m e t e r and t h e methane
d e r i v e d chemical i o n i z a t i o n (CI) m a s s spectrum o b t a i n e d
w i t h F i n n i g a n 4000 m a s s s p e c t r o m e t e r are shown i n
F i g u r e s 1 and 2 r e s p e c t i v e l y . The ( E I ) spectrum F i g .
1 shows a b a s e peak a t m / e 91. Other major fragment
a t m / e ( r e l a t i v e abundance) 108(71) , 1 5 5 ( 4 8 ) , 65(27) ,
and 1 9 7 ( 1 5 ) . The ( C I ) spectrum F i g . 2 i s s u r p r i s i n g -
l y s i m p l e , t h e base peak m/e 271 c o r r e s p o n d s t o E.t' i o n .
Other major f r a g m e n t s a r e a t m / e 117, 172, 200, 61 and
m f e 74.
1 . 4 Carbon-13 Nuclear Magnetic Resonance Spectrum
The carbon-13 NMR c o m p l e t e l y decoupled and off-resonance

s p e c t r a are shown i n Fig. 3 and 4 r e s p e c t i v e l y .
Both were r e c o r d e d o v e r 5000 Hz r a n g e i n d e u t e r a t e d
d i m e t h y l s u l p h o x i d e on FT-SO A-80 MHz NMR s p e c t r o m e t e r
u s i n g t e t r a m e t h y l s i l a n e a s r e f e r e n c e s t a n d a r d . The
carbon chemical s h i f t v a l u e a r e a s s i g n e d on t h e b a s i s
of s i g n a l m u l t i p l i c i t y , chemical s h i t s and t h e com-
p a r i s o n w i t h t h e chemical s h i t s of model compounds.
Table 1 summarizes t h e carbon chemical s h i f t s of
.
t o 1b u t a m i d e
a
a,
c
.;i
E
a,
4
a,
a
-w
h
rl
a,
4
at s
a
-4
5
u
7
Q
rl
0
c,
w
0
122
IOO.0-
50.0 -
100.0- 271 -
4
w
N
50.0- -
'
325 344 353 351 849 378 337 395
l.'**'"''I
Fig. 2. Mass spectrum of tolbutamide (C1) deter-

mined by direct probe insertion.
7
1
124
Fig. 4. C-13 nuclear magnetic resonance spectrum ( o f f -
resonance) of Tolbutamide.
726 SAID E. IBRAHIM A N D ABDULLAH A . AL-BADR
Table 1. Carbon Chemical S h i f t s of Tolbutamide.
H3C "0; 8' 7'

0
-NH
0
!I 4 3
- 5C - IW-CH 2-CH 2-CH2-CH
2 1
3
Carbon Multiplicity Chemical S h i f t (ppm)
(9) 13.26
c2 (t) 19.30
(t> 20.90
c3
(t1 38.98
c4
(s) 151.45
c5
'6 (s) 148.36
(d) 127.09
c7
(d) 127.09
c7 '
(d) 129.23
'8
'8 ' (d) 129.23
(s) 137.62
c9
(9) 31.28
clo
2 . Methods of A n a l y s i s
2.1.1 Aqueous T i t r a t i o n
The methods of E l - F a t a t r y -- e t a1 (3) i n v o l v e s

measurements of t h e pKa v a l u e s of t o l b u t a m i d e
i n d i f f e r e n t s o l v e n t s and t i t r a t i o n of t h e d r u g
i n a s u i t a b l e medium w i t h s t a n d a r d a l k a l i t o t h e
TOLBUTAMIDE 121
pH v a l u e c o r r e s p o n d i n g t o t h e pKa of i t i n t h e
medium. Accurate and p r e c i s e r e s u l t s were
o b t a i n e d i n t h e c o n c e n t r a t i o n r a n g e 1 t o 10 mg
m l - 1 i n aqueous 60% a c e t o n e .
2.1.2 Non-Aqueous T i t r a t i o n
Tolbutamide i n t a b l e t s and p u r e forms w a s d i s -

s o l v e d i n t e t r a m e t h y l u r e a and t i t r a t e d w i t h 0.1 N
l i t h i u m methoxide i n benzene-methanol medium,
t h e end p o i n t w a s determined v i s u a l l y w i t h 0 . 2 %
a z o - v i o l e t i n t o l u e n e as i n d i c a t o r ( 4 ) .
2.2 SDectroDhotometric Methods
2.2.1 U l t r a v i o l e t
a. Abdel Hady -- et - a l ( 5 ) r e p o r t e d two s p e c t r o p h o t o -

m e t r i c methods f o r t h e q u a n t i t a t i o n of t o l -
butamide w i t h o u t i n t e r f e r e n c e from t h e t a b l e t
e x c e p i e n t s . I n t h e f i r s t (Glenn's) method,
t h e absorbance of t o l b u t a m i d e i n 952 e t h a n o l
w a s measured a t 250-270 nm a t 4 nm i n t e r v a l s
and the p2 c o e f f i c i e n t c a l c u l a t e d . The co-
e f f i c i e n t w a s l i n e a r l y r e l a t e d t o concentra-
t i o n w i t h i n a r a n g e of 0.1-0.4 mg/ml. The
second method i s based on t h e f o r m a t i o n of a
complex of a r a t i o 1:l w i t h b a s i c dye b r i l l i -
a n t c r e s y l b l u e o r s a f r a n i n e T , t h e complex
w a s e a s i l y e x t r a c t e d w i t h chloroform and t h e
absorbance of t h e chloroform e x t r a c t w a s
measured a g a i n s t e i t h e r a b l a n k o r r e f e r e n c e
experiment a t 615 o r 510 nm f o r b r i l l i a n t
c r e s y l b l u e o r s a f r a n i n e T methods r e s p e c t i v e -
l y . The r e s u l t s of t h e s e methods a r e more
a c c u r a t e t h a n t h o s e of t h e t r a d i t i o n a l u l t r a -
v i o l e t s p e c t r o p h o t o m e t r i c methods.
b. Tolbutamide w a s determined i n p r e s e n c e of
t h i a m i n e h y d r o c h l o r i d e and p y r i d o x i n e hydro-
c h l o r i d e , by measuring t h e d i f f e r e n c e i n
absorbance a t 274 and 276 nm i n 95% e t h a n o l
medium, t h e i n t e r f e r e n c e by t h i a m i n e hydro-
chloride o r pyridoxine hydrochloride i s
n e g l i g a b l e . Mean r e c o v e r i e s (10 determina-
t i o n s ) w e r e 100.0 t o 100.8 and t h e s t a n d a r d
d e v i a t i o n = 0.5% ( 6 ) .
728 SAID E. IBRAHIM A N D ABDULLAH A . AL-BADR
c. Tolbutamide w a s determined i n c i t r a t e d blood

contaminated w i t h i t s m e t a b o l i t e s l-butyl-3-
(4-carboxybenzenesulphonyl) u r e a and l - b u t y l -
3-(hydroxymethylbenzenesulphonyl) u r e a ( 7 ) .
The haemolyzed blood i n aqueous phosphate
b u f f e r of pH 5 was shaken w i t h heptane-chloro-
form ( 4 : l ) f o r 20 m i n u t e s , t h e o r g a n i c phase
was shaken w i t h 0.1 M sodium hydroxide s o l u -
t i o n f o r 20 minutes t h e n t h e aqueous a l k a l i
phase w a s mixed ( 2 0 : l ) w i t h 3M h y d r o c h l o r i c
a c i d s o l u t i o n and t h e e x t i n c t i o n w a s measured
a t 230 nm. Blank s o l u t i o n c o n t a i n i n g unconta-
minated drug w e r e used i n a l l i n s t a n c e .
2.2.2 Spectrofluorimetry
Among o t h e r a n t i d i a b e t i c d r u g s , t o l b u t a m i d e w a s
determined i n f o r m u l a t i o n s and i n b i o l o g i c a l
f l u i d s by a c i d h y d r o l y s i s , r e a c t i o n of t h e
r e s u l t i n g amine w i t h a c e t y l a c e t o n e and fonnal-
dehyde, and f l u o r i m e t r i c d e t e r m i n a t i o n of t h e
r e s u l t i n g s u b s t i t u t e d d i h y d r o p y r i d i n e a t 600 nm,
w i t h e x c i t a t i o n a t 480 nm (8).
2.2.3 P r o t o n Magnetic Resonance Spectrometry
Al-Badr and Ibrahim (9) r e p o r t e d a n a c c u r a t e ,

r a p i d and p r e c i s e method f o r t h e d e t e r m i n a t i o n
of t o l b u t a m i d e and some o t h e r hypoglycemic a g e n t s
i n p u r e and t a b l e t forms u s i n g p r o t o n magnetic
resonance spectroscopy. The method i n v o l v e s
comparing t h e i n t e g r a t i o n of t h e a r o m a t i c doub-
l e t s of t o l b u t a m i d e a t 7.33-7.8 ppm w i t h t h a t of
t h e s i n g l e t of hexamethylenetetramine ( i n t e r n a l
s t a n d a r d ) a t 4.61 ppm u s i n g d i m e t h y l s u l f o x i d e as
s o l v e n t . Recovery i s 1 0 0 % f 1 . 5 and 9 9 . 6 % ? 1 . 4
f o r p u r e t o l b u t a m i d e and i t s t a b l e t dosage form
respectively .
2.2.4 Mass Spectrometry
a. Tolbutamide and i t s m e t a b o l i t e s i n plasma and

u r i n e were determined u s i n g chemical i o n i z a -
t i o n mass spectrometry. The d r u g s and i t s
m e t a b o l i t e s were e x t r a c t e d w i t h e t h y l e t h e r
from samples of plasma and u r i n e t o which 2H-
l a b e l e d i n t e r n a l s t a n d a r d s have been added.
The extract w a s t h e n t r e a t e d w i t h e x c e s s
TOLBUTAMIDE 129
diazomethane. Quantilative measurements were

made on protonated molecular ion (which is
free from interference), with use of isobutane
as reagent gas, by single ion monitoring for
determination of single component or in scan
mode for multi-component mixtures. Levels
down to 0.5 ug ml in plasma can be determined
by this method (10).
b. A high resolution mass spectroscopic analysis

of methylated tolbutamide derivatives was
reported by Sabih (11). The methyl derivative
of tolbutamide obtained by treatment with
dimethyl sulphate was subjected to g.1.c. on
a stainless steel column ( 6 ft. X 0.125 in)
packed with 1% OV-1 on Gas-Chrom Q , tempera-
ture programmed from 150 to 1900 at 20 per
minute and operated with helium as carrier gas
(30 ml per minute). A mass spectrum of the
column effluent was obtained at 70 eV with a
double-focussing instrument linked to a digi-
tal data system.
c. Braselton et a1 (12) determined tolbutamide

and its metabolites in human serum by the
formation of thermally stable derivatives (N-
methyl-N--trifluoroacetyl) and analyzing this
derivatives by g.1.c.-m.s. method. The g.1.c.
was performed on a glass column (1.5 m X 2 nun)
operated at 170° with helium as carrier gas.
The g.1.c.-m.s. system was coupled with PDP-8
computer for data processing, a detection
ion limit of 1 p mol is claimed.
2.3.1 Gas Chromatographic
Gas chromatography has frequently been used for

the analysis to tolbutamide. The type of column,
carrier gas and other experimental conditions are
listed in Table 11.
2.3.2 Liquid Chromatography
Tolbutamide, among other sulphonylureas, was

determined in pharmaceutical products by high-
speed liquid chromatography. The column used
Table I1
G a s Liquid Chromatography of Tolbutamide.
Drug Tempera- D e r i v a t i v e Internal

Phase and Column Carrier g a s Detector Ref.
Source t u r e (OC) used Standard
Serum 1%SP-2100 on s u p e l c o p o r t n i t r o g e n 165 N-Methyl- FID* Chlorpropa- (12)
(100-120 mesh). Glass (30 m l m i r r l ) N--tri- midie.
Column (1.8 m X 2 mm). f luoro-
acetyl.
Plasma 3% OV-17 on Gas-Chrom 0 nitrogen 220 methyl EC** Hexadocosane (13)
(100-120 mesh). Glass
column 2 . 1 m X 0.2 m.
Serum 3% OV-17 on Chromosorb nitrogen 190 methyl FID 4-chloro-N- (14)
W-HP (100-120 mesh), ( 50 m l /min-l) propylbenzene
shaped g l a s s column sulphonamide .
( 6 f t . X 0.29 i n ) .
Plasma 10%W-98 on Chromosorb W- helium 150 methyl FID Chlorpropa- (15)
6 u r i n e HP(80-100 mesh) , s t a i n l e s s (30 ml/mi<l) mide.
s t e e l column ( 6 f t . X
0.125 i n ) .
Plasma 3.8% UC W-98 on d i a t o p o r t n i t r o g e n 220 methyl FID Chloropropa- (16)
S (80-100 mesh) g l a s s (25 ml/min-') mide.
column ( 4 f t . X 0.25 i n ) .
* Flame I o n i z a t i o n D e t e c t o r ** E l e c t r o n Capture
TOLBUTAMIDE 73 I
(100 c m X 2.1 mm) w a s packed w i t h 1% e t h y l e n e -

propene copolymer on Zipax w i t h a m o b i l e phase
of 0.01 M - disodium hydrogen c i t r a t e c o n t a i n i n g
15% of methanol (pH 4 . 4 ) . D e t e c t i o n a t 254 nm
w a s used and peak area w e r e i n t e g r a t e d . The
p r o c e d u r e w a s a p p l i e d t o compressed t a b l e t s . ( 1 7 ) .
2.3.3 High-Perf ormance L i q u i d Chromatography
S e v e r a l high-performance l i q u i d c h r o m a t o g r a p h i c
methods f o r t h e q u a n t i t a t i o n of t o l b u t a m i d e w i t h
o r w i t h o u t i t s m e t a b o l i t e s i n body f l u i d s h a v e
been r e p o r t e d . The c h r o m a t o g r a p h i c s y s t e m s
i n v e s t i g a t e d f o r t h e a n a l y s i s of t h i s d r u g are
p r e s e n t e d i n T a b l e 111.
3 . Pharmacokinet i c s
Pond -- e t a1 ( 2 3 ) r e p o r t e d t h a t t h e r a t e of metabolism of
t o l b u t a m i d e w a s d e c r e a s e d by c h r o n i c a d m i n s t r a t i o n of
c e r t a i n drugs, they claimed t h a t t h e tolbutamide h a l f - l i f e
w a s i n c r e a s e d by c h r o n i c a d m i n s t r a t i o n of s u l p h a p h e n a z o l e
( 9 . 5 h r s t o 28.6 h r s ) , p h e n y l b u t a z o n e ( 7 . 9 h r s t o 2 3 . 1 h r s ) ,
and oxyphenbutazone (8.1 h r s t o 3 0 . 2 h r s ) . The r a t e of
e l i m i n a t i o n of t o l b u t a m i d e w a s d e c r e a s e d w i t h i n one t o two
h o u r s a f t e r a s i n g l e d o s e of s u l p h a p h e n a z o l e and t h e h a l f -
l i f e w a s i n c r e a s e d from 9.2 h r s t o 25.7 h r s . I n contrast,
p h e n y l b u t a z o n e and oxyphenbutazone, a d m i n s t e r e d a s a s i n g l e
d o s e 8 0 0 mg have no immediate e f f e c t on t o l b u t a m i d e e l i m i -
nation. I t i s s u g g e s t e d t h a t p h e n y l b u t a z o n e and oxyphen-
b u t a z o n e a c t by i n d u c i n g a form of a cytochrome P-450 w i t h
low a c t i v i t y f o r t o l b u t a m i d e h y d r o x y l a t i o n , w h e r e a r e ,
s u l p h a p h e n a z o l e a c t s by d i r e c t i n h i b i t i o n of t h e microsomol
mixed f u n c t i o n o x i d a s e system.
Another group of i n v e s t i g a t o r s ( 2 4 ) showed t h a t t o l b u t a m i d e

w a s eliminated f a s t e r i n p a t i e n t s with h e p a t i t i s than i n
t h o s e without liver d i s e a s e .
During c h o l e s t a s i s , t h e m e t a b o l i s m of t o l b u t a m i d e i s
g r e a t l y a l t e r e d , t h u s , t o l b u t a m i d e (100 mg/kg) i n j e c t e d
i . v . i n t o rats with experimental c h o l e s t a s i s w a s eliminated
more r a p i d l y t h a n i n c o n t r o l a n i m a l s , t h i s i n c r e a s e w a s
owing t o t h e i n c r e a s e i n l i v e r weight and microsomal pro-
t e i n ( 2 5 ) . In p a t i e n t s w i t h r e c u r r e n t i n t r a h e p a t i c c h o l e s -
t a s i s no d i f f e r e n c e s i n t o l b u t a m i d e m e t a b o l i s m were n o t e d
a s compared t o t h e c o n t r o l group. In c h o l e s t a t i c h e p a t i t i s
t h e plasma h a l f - l i f e of t o l b u t a m i d e a p p e a r e d unchanged. The
Table I11
HPLC of Tolbutamide.
Drug
Column Mobile Phase Internal Detect o r Remarks Ref.
Source Standard ^___
_I
Plasma p Bondapack A c e t o n i t r i l : 0.05 M Chlorpropa- 254 nm C a l i b r a t i o n curve was (18)

H3PO4 b u f f e r pH 3.9 mide r e c t i l i n e a r f o r 2-80
‘18
(7:13) 2.5 m l min-1. 1Jg m1-1
Serum Silica 1%a c e t i c a c i d 3-isopentyl 254 nm The l i m i t of d e t e c t i o n i s (19)

and beads ( a d j u s t e d t o pH 5.5) 1-(toluene i s 6 mg 1-l and r e c o v e r y
plasma coated w i t h : a c e t o n i t r i l (18:7) p-sulpho- is 95%.
p bondapack 2.2 m l min-l nyl) urea.
c18.
Plasma ybondapack A c e t o n i t r i l : 0.05% - 200 nm The c a l i b r a t i o n graphs (20)

H3P04 ( 9 : l l ) . 1.5 m l cover t h e pages of 5-300
‘18
min-1 . 1Jg m1-1
Plasma ODS-Sil-X-1 22% aqueous a c e t o - Chlorpropa- 223 nm The c a r l i b r a t i o n graphs (21)

nitril. mide. r e c t i l i n e a r f o r 25-500
mg 1-1.
T a b l e t s L i Chrosorb 0.06% a c e t i c a c i d i n Prednisone 254 nm The r e s u l t s of undegraded (22)

S i 60 ethanol : tetrahydro- t a b l e t s agreed w i t h i n
f u r a n : hexane 1:2:22 0.3% w i t h r e s u l t s by
U.S.P.
-
TOLBUTAMIDE 133
most s t r i k i n g v a r i a t i o n s were observed i n t h e p a t i e n t s w i t h

e x t r a h e p a t i c b i l i a r y o b s t r u c t i o n , i n such c a s e s , t h e meta-
b o l i s m of t o l b u t a m i d e was a c c e l e r a t e d as evidence by a
s i g n i f i c a n t d e c r e a s e of plasma h a l f - l i f e (165 m i n u t e s
v e r s u s 384 of t h e c o n t r o l . However, t h e metabolism of
t o l b u t a m i d e i n v i t r o d i d n o t show any d i f f e r e n c e between
normal and c h o l e s t a t i c l i v e r . P r e l i m i n a r y r e s u l t s i n v i t r o
s u g g e s t e d t h a t t h e b i l e s a l t could d i s p l a c e t o l b u t a m i d e
from albumin b i n d i n g t h u s i n c r e a s i n g t h e amount of t h e f r e e
drug a v a i l a b l e f o r b i o t r a n s f o r m a t i o n by t h e l i v e r . (26)
Darby e t a1 (27) have r e p o r t e d t h a t t h e r e i s no s i g n i f i c a n t

L-
c o r r e l a t i o n s w e r e found between t h e h e p a t i c microsomal

k i n e t i c c o n s t a n t s f o r 14C-tolbutamide metabolism -- in vitro
and t h e plasma h a l f - l i v e s and plasma c l e a r a n c e of t h e drug
adminstered o r a l l y .
4 . Identification
The B.P. (1980) (28) d e s c r i b e s t h e f o l l o w i n g i d e n t i f i c a t i o n

test f o r tolbutamide:
A . D i s s o l v e 25 mg i n s u f f i c i e n t methanol t o produce 100 m l .

The l i g h t a b s o r p t i o n , i n t h e r a n g e 220 t o 350 nm,
e x h i b i t s a band a t about 228 nm and maxima a t 258 nm,
263 nm and 275 nm. The d i l u t e d s o l u t i o n of t o l b u t a m i d e
i n methanol (0.001% w/v) e x h i b i t s a maximum o n l y a t 228
nm; A ( l % , 1 cm) a t 228 nm, about 490.
734 SAID E. IBRAHIM A N D ABDULLAH A. AL-BADR
References
1. Simmons, D.L.; Ranz, R . J . ; Gyanchandani, N.D.; and

P i c o t t e , P.; --
Can. J. Pharm. S c i . ; 7,
1 2 1 (1972).
2. N i m a l a , K.A.; and Gowda, D.S. Sake; Acta C r y s t . ; B,

1597 (1981).
3 . El-Fatatry, H.M.; S h a r a f , M.K.; and h e r , M.M.; Pharmazie,

I34, 543 (1979).
4. Agarwal, S.P.;
(1972).
and Walash, M . I . ; x. J . Pharm. 34, 109
5 . Abdel-Hady, E.M.; B e l a l , S . F . ; El-Walily, A.M.; and

Abdine, H.; 2. Assoc.
I _ -
O f f . Anal. Chem.; 62, 533 (1979).
- -
6. Abdel-Hady, E.M.; B e l a l , S.F.; El-Walily, A.M.; and

Abdine, H . ; 2.--
Pharm. S c i . ; 68, 739 (1979).
7. S h i b a s a k i , J ; K o n i s h i , R . ; Ueki, T . ; and M o r i s h i t a , T.;

- --
Chem. Pharm. B u l l . , Tokyo; 2,1747 (1973).
8. Narnigohar, F . ; Makhani, M.; and Radparvar, S . ; Trav. S O ~ .

Pharm. M o n t p e l l i e r ; 40,
55 (1980).
9. Al-Badr, A.A.; and I b r a h i m ; S . E . ; Pharmazie; 37, 378

(1982).
10. Weinkam, R . J . ; Rowland, M.; and M e f f i n , P . J . ; Biomed.

Mass Spectrom; 4, 42 (1977).
11. S a b i h , K . ; Biomed. Mass Spectrom; -

1, 163 (1974).
12. B r a s e l t o n , W.E.J.; Ashline, H.C.; and Bransome, E . D . J . ;

~-
Analyt. L e t t . ; - 8, 301 (1975).
13. H a r t u i g , P . ; F a g e r l u n d , C . ; and G y l l e n h a a l , 0; J.
Chromatogr.; l& ,
Biomed. Appl.; 7, 17 (1980).
14. Simmons, D . L . ; Ranz, R . J . ; and P i c o t t e , P.; 2. Chromatogr.;

71, 421 (1972).
I
15. Aggarwal, V. ; and S u n s h i n e , I.; C l i n . Chem. ; 20, 200

(1974).
1 6 . P e r s c o n , L.F.; and Redman, D . R . ; J. Pharm. Pharmac.; 24,

713 (1972).
TOLBUTAMIDE
17. B e y e r , W.F.; A n a l y t . Chem. 44, 1312 (1972).

18. Raghow, G . ; and Meyer, M.C.; 2. Pharm. g . ; 70,1166
(1981).
19. H i l l , R.E.; and C r e s h i o l o , J.; J.Chromatogr.; 145;

Biomed Appl.; 2, 165 (1978).
20. N a t i o n , R.L.; Peng. G.W.; and Chiou, W.L.; J. Chromatogr.

-
146; Biomed. Appl.; 2, 1 2 1 (1978).
21. P e c o r a r i , P.; A l b a s i n i , A , ; M e l e g a r i , M.; Vampa, G . ;

and Manenti, F . ; Farmaco. Ed. E.; 36, 7 (1981).
22. R o b e r t s o n , D.L.; B u t t e r f i e l d , A . G . ; K o l a s i n s k i , H.;
L o v e r t i n g , E.G.; and M a t s u i , F.F.; J. Pharm. S c i . ; 68,
577 (1979).
23. Pond, S.M.; B i r k e t t , D . J . ; and Wade, D.N.; E.Pharmcol.

Ther. ; 22, 573 (1977).
~
24. Von O l d e r s h a u s e n ; H.F.; Schoene, B . ; Held, H . ; Menz, H.P.;

Fleischmann, R.A.; and Remmer, H.; Z. G a s t r o e n t e r o l ; 11,
403 ( 1 9 7 3 ) .
25. Gallenkamp, H . ; Wilhelm, T . ; Z i l l y , W . ; and R i c h t e r E . ;

I_ -
Verh. Dtsch. -
Ges. --Inn. Med.; 82, 297 (1976).
26. C a r u l l i , N . ; M a n e n t i , F . ; Ponzde Leon, M . ; F e r r a r i , A . ;

S a l v i o l i , G . ; and G a l l o , M . ; K. J . C l i n . I n v e s t . , 5,
455 (1975).
2 7 . Darby, F . J . , and Grundy, R.K. , Life Sci.; 13,97 (1973).
2 8 . I h r i t i s h Pharmacopiea”, London Her M a g e s t y ‘ s S t a t i o n a r y

O f f i c e , Page 458 (1980).
RESERPINE
Farid J. Muhtadi
King Saud Unioersity
1, Description 738
1 . 1 Nomenclature 738
1.2 Formulae 738
1.5 Appearance, Color, Odor. and Taste 740
2.1 Eutectic Temperature 740
2.2 Solubility 740
2.3 Loss on Drying 740
2 . 4 Spectral Properties 74 I
3. The Total Synthesis of Reserpine 749
4. Biosynthesis of Reserpine 754
5. Drug Metabolism and Pharmacokinetics 756
6.1 Identification Tests 756
6.2 Microcrystal Tests 758
6.3 Titrimetric Method 758
7. High Performance Liquid Chromatography (HPLC) 76 1
References 163
Copyright 0 1984
ISBN 0-12-260813-5
738 FARID J. MUHTADI
1. Description
1.1 Nomenclature
11, 17 a-Dimethoxy-18 B-[ (3,4,5 -

trimethoxybenzoyl) oxyl-3 B , 20 a-
yohimban-16 B -carboxylic acid methyl
ester.
Yohimban-16-carboxylic acid, 11, 17-
dimethoxy-18-[ ( 3,4,5-trimethoxybenzoyl)
oxyl-, methylester, (3B,16B,17ay18B,20a).
Methyl 18 a-hydroxy-11 ,l7 a-dimethoxy-3f3 ,
20ccyohimban-16 a-carboxylate 3,4,5-
trimethoxybenzoate (ester).
3,4,5-trimethoxybenzoyl methyl reserpate.
Methyl 18-0-(3,h ,5-trimethoxybenzoyl)
reserpate.
1.1.2 Generic Names
Reserpine
1.1.3 Trade Names
Austrapine; Bioserpine; Crystoserpine;

Eskaserp; Hiserpia; Orticalm; Quiescin;
Rau-sed; Reserpex; Reserpoid; Rivasin;
Roxinoid; Sandril; Sedaraupin; Serfin;
Serolfia; Serpanary; Serpasil, Serpasol;
Serpate; Serpen; Serpine; Serpiloid.
1.2 Formulae
1.2.1 hpirical
C33H40N209
1.2.2 Structural
The structure (facing page) was confirmed by

the total synthesis of Reserpine ( 1 - 3 ) .
M
X
I
--u
0 a!
c
.ri
a
k
a!
X
740 FARlD J. MUHTADI
1.2.3 CAS R e g i s t r y No.
[ 50-55-5 I
1.2.4 Wiswesser Line Notation
T F6 D5 ~ 6 6 6E M ON
&& TTTJ-HO1 SOW C
01 DO1 E01 &-TO1 UVOl (4)
608.70
1.5 Appearance, C o l o r , Odor and T a s t e
Long prisms from d i l u t e a c e t o n e .

White to p a l e fawn s m a l l c r y s t a l s o r c r y s t a l l i n e
powder, darkening slowly on exposure t o l i g h t .
Odorless and h a s a b i t t e r t a s t e .
2.1 E u t e c t i c Temperature
Ac e t a m i n o s a l o l
D i cyandiami de
Phenolphthalein
k:225'
2z ] h o t s t a g e method (5)
2.2 Solubility
Very s p a r i n g l y s o l u b l e i n water, s l i g h t l y s o l u b l e
i n e t h y l a l c o h o l and methyl a l c o h o l .
S o l u b l e a t 20' i n 6 p a r t s of chloroform, i n
90 p a r t s of a c e t o n e and i n 2000 p a r t s o f e t h a n o l .
2.3 Loss on Drying
When d r i e d f o r 3 hours a t 60' a t a p r e s s u r e n o t

exceeding 0.7 kPa (about 5 t o r r ) , loses n o t more
t h a n 0 . 5 p e r c e n t of i t s weight; use 0.5g. (6)
RESERPINE 74 1
2.4.1 Infrared Spectrum
The IR spectrum of reserpine as KBr-disc

was recorded on a Perkin Elmer 580~Infrared
Spectrophotometer to which Infrared Data
station is attached (Fig. 1).
The structural assignments have been correla-
ted with the following frequencies (Table 1).
Table 1. IR Characteristics of Reserpine

-1
Frequency c m Assignment
3430 N-H stretch of indole ring
2940 C-H stretch
2840 CH3, CH2-stretch
0
1732 0-C-CH3
1710
0
0-C-aromatic
1625 C-N stretch, C=C alkene
1
1588
1500 C=C aromatic
1455
139 CH2-bending
1275
1250
1225 1 c-0-c
Other characteristic absorption bands are:

1410, 1188, 1120, 1060, 1030, 1005, 975,
940, 872, 820, 800, 763, 740, 710, 615 cm-l.
The IR data of reserpine have been also
reported by several authors (7-11).
2.4.2 13C-NMR Spectrum
The 13C-NMR noise decoupled and off resonance

spectra are presented in Fig. 2 and Fig. 3
respectively. Both were recorded over
4000 Hz range in deuterated chloroform on a
Varian ~ ~ A-80
8 0MHz spectrometer, using
10 rm. sample tube and tetramethyl silane
as a reference standard at 2 2 ' .
142
743
RESERPINE 145
The carbon chemical shifts are assigned on the

bases of the additivity principals and o f f resonance
splitting pattern (Table 2).
Table 2. Carbon Chemical S h i f t s of Reserpine
Carbon no. Chemical s h i f t Carbon no. Chemical s h i f t

[ PPm 1 [PPml
C 1'72.84( s ) 60.86(~)
32 '30
c20
165~56 (s) '27 "29 56A (9)
C 156.09(s) '28 "33 55.67(9)
9
'23 "25 153.02(s) c2 53.87( a )
142.36(s) 51.80(d)
c24 '14
136.50(s1 51.80(q )
51 '31
130.61(s) C 51.39(t)
3
(Continued)
146 FARID J . MUHTADI
Table 2 (continued)
‘21
125.38( s ) C 49.01(t )
19
‘6 122.22( s ) ‘18 33.99( d )
C 118.49 ( a ) 32.36(d)
7 ‘13
‘8 108.89(d ) 29.85(t)
‘1’7
C 24.31 ( t)
5 107.76(s) ‘12
‘22 “26 106.89(d)
c4 16.84(t)
‘10 95*33(d)
‘15 ] 78.88 i d )
78.06 ( d )
‘16
s = s i n g l e t ; d = doublet ; t = t r i p l e t ;
q = quartet.
Other 13C-NMR d a t a f o r r e s e r p i n e have been a l s o

r e p o r t e d (12-14) .
2.4.3 Mass Spectrum
The mass spectrum of r e s e r p i n e i s p r e s e n t e d

i n Fig. 4. This w a s o b t a i n e d by e l e c t r o n
impact i o n i z a t i o n and run on a Varian MAT 311
by d i r e c t i n l e t probe a t 1 8 0 O c . The spectrum
w a s recorded w i t h t h e a i d of t h e Incos d a t a
system. The e l e c t r o n energy w a s 70 eV and
t h e a c c e l e r a t i n g v o l t a g e w a s 2.4 KV.
The spectrum (Fig. 4 ) shows a molecular i o n
peak M+ a t m / e 608 w i t h a r e l a t i v e i n t e n s i t y
100%.
The most prominent fragments, t h e i r r e l a t i v e
i n t e n s i t i e s and some proposed i o n fragments
are given i n t a b l e 3.
Table 3. Mass fragments of r e s e r p i n e
m/e relative intensity % Ions

608 100 Mi
609 36.0 [ M+H] -k
607 45.3 [M-HI+
(Continued)
hl
d
N
Y)
z.!
c, . .
4' w
m
, . ,
?
+
$
A E
\
f w
398 21.3
0 OCH3
L -
397 20.0
396 41.3
381 20.0
251 18.7
+
195 33.3
Other mass spectral data for reserpine have been also

reported (4,15,16).
RESERPINE 749
3. The Total Synthesis of Reserpine
The total synthesis of reserpine was achieved by

Woodward et al. (1-3) in 1956. The remarkable Wood-
ward's synthesis of reserpine is summarised as follows:-
p-Benzoquinone [ 11 is condensed with vinylacrylic
acid [2] to give the adduct [ 3 ] which is reduced by
sodium borohydride to the alcohol [4]. This is oxidised
by perbenzoic acid in benzene-dioxane to the oxide [51.
The corresponding lactone [6] obtained from [5] by the
action of acetic anhydride and sodium acetate in benzene.
[61 is transformed by aluminium isopropoxide in hot
isopropyl alcohol into the ether [TI. This by the action
of sodium methoxide in methanol gives the methoxy-ether
[8]. [8] is treated with N-bromosuccinimide in warm
aqueous solution in the presence of sulfuric acid to
give the bromohydrine [ 9 ] which is oxidized by chromium
trioxide in acetic acid to the corresponding ketone,
and this is transformed by short treatment with zinc in
cold glacial acetic acid to the hydroxy-acid [lo]. The
latter is treated with diazomethane in dioxane to give
the methyl ester [ll]. This is converted to the acetate
[12] by acetic anhydride in pyridine and then to the
diol [13] upon treatment with aqueous osmium tetroxide,
followed by potassium chlorate. The diol [13] is
esterified with diazomethane to give the methylester
[141. Condensation of [ 141 with 6-methoxytryptamine [ 15 ]
in benzene to give the condensate [16] which is reduced
with sodium borohydride in methanol to give the lactam
[l-i]. This is cyclized with phosphorous oxychloride
and reduced with sodium borohydride to dl-methyl-0-
acetyl-isoreserpate [18]. Upon saponification and
treatment with hydrochloric acid and warmed with
N-N-dicyclohexylcarbodiimide in pyridine [18] is
transformed into isoreserpic acid lactone [19]. This
is isomerized with ljivalic acid in xylene to give the
more stable reserpic acid lactone [20]. Methclysis is
followed to give methyl reserpate [21] which is condensed
with 3,4,5-trimethoxybenzoylchloride [ 221 in pyridine to
give dl-reserpine [23]. This is readily resolved via
the highly crystalline 1-reserpine d-camphor-10-sulfo-
nate to give 1-reserpine, identical in respects with
natural reserpine.
The total synthesis of reserpine is presented in
scheme I.
A simplified method for the synthesis of reserpine has
been patented (17).
Scheme1:Total Synthesis of Reserpine
HOO
I
H
'H -
NaBH4
HOOC [41
[31
C H COOOH
6 5
RESERPINE 75 1
Br
I
HO
-
[91
0
0 -5?J
i$$ OCH3
I [I01 LOCH3 OH
0ch3
1
0ch3
H3C0
-
OAc
1
OCH3
RESERPINE 753
H3C0 Pivalic
H' T
acid
[191
I
OCH3
H3C0 Metholysis
I
I
OCH3
H3C0
OH
+
Reserpine [ 23 1 a CCH3
1221
4. Biosynthesis of Reserpine
The b i o s y n t h e t i c pathways of r e s e r p i n e and o t h e r

a l k a l o i d s of Rauwalfia. have been e x t e n s i v e l y s t u d i e d
by s e v e r a l authors. Leete (18, 19) f e d DL-[2-l4C]-
tryptophan i n t o Rauwolfia s e r p e n t i n a (3 y e a r s old p l a n t s )
which l e d t o t h e formation of r a d i o a c t i v e ajmaline,
s e r p e n t i n e and r e s e r p i n e (18). L a t e r , he found t h a t
r a d i o a c t i v e s e r p e n t i n e w a s l a b e l e d s o l e l y a t C-5 i n d i c a t -
i n g t h a t tryptophan w a s a d i r e c t precursor o f t h e B-
c a r b o l i n e moiety of t h i s a l k a l o i d (19). Other radio-
a c t i v e precursors have been administered t o Rauwolfia
p l a n t s such as [l-lk] a c e t a t e (20), [2-14C] a c e t a t e (21),
[2-14~1a l a n i n e (20) and [2-14~1glycine (21, 22). AU
incorporated i n t o ajmaline and r e s e r p i n e .
Thomas (23) p r e d i c t e d t h a t t h e non-tryptamine moiety of
t h e i n d o l e a l k a l o i d s i s derived from a cyclopentanoid
monoterpene precursor. Wenkert (24) independently
reached to t h e same conclusion.
Battersby and Co-workers (25-28), Money e t a1 (29) and
Leete e t al (30) a l l reported t h e s p e c i f i c incorporation
of [ 2-l4C] mevalonic a c i d and [ 2-%] geraniol i n t o
r e p r e s e n t a t i v e examples of t h e corynanthe, aspidosperma
and iboga groups of a l k a l o i d s , and i n each case t h e
d i s t r i b u t i o n of r a d i o a c t i v i t y w a s i n f u l l agreement with
t h e monoterpene hypothesis developed by Thomas (23) and
Wenkert (24), and according t o t h e biogenetic isoprene
r u l e t h a t monoterpene are formed i n n a t u r e by s u i t a b l e
modification of geranyl pyrophosphate (31).
The Biosynthetic pathway of r e s e r p i n e i s presented i n
scheme 11.
a,
a,
G z
o=v
0
t
(J-O 0
..
H
H
ov
I
V
5. Drug Metabolism and Pharmacokinetics

Reserpine i s absorbed r a p i d l y 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 . Maximum blood c o n c e n t r a t i o n s are reached i n
approximately 2 hours w i t h r e p o r t e d v a l u e s of 0.14 to
0.18 mcg/dl i n whole blood and 0.13 t o 0.15 mcg/dl
i n plasma ( 3 2 ) .
Reserpine i s metabolised by t h e l i v e r b 2 - 3 4 ) w i t h more t h a n
90% e x c r e t e d as m e t a b o l i t e s ( 3 3 ) . The major u r i n a r y
m e t a b o l i t e s are methyl r e s e r p a t e and 3,4,5-trimethoxy-
benzoic a c i d . Other m e t a b o l i t e s are shown i n S c h e m e 3 ( 3 5 ) .
Reserpine has a h a l f - l i f e range of 50 t o 100 hours (34 ) .
A b i o l o g i c a l h a l f - l i f e i n whole blood of 386 hours and a
b i o l o g i c a l h a l f - l i f e i n plasma of 271 hours have been
r e p o r t e d ( 3 2 ) . D e t e c t a b l e l e v e l s of r e s e r p i n e may be
found a f t e r 11 days from a d m i n i s t r a t i o n of t h e drug.
Reserpine i s not removed e i t h e r by hernodialysis o r by
peritoneal dialysis(36).
6.1 Identification tests
The f o l l o w i n g i d e n t i f i c a t i o n t e s t s a r e mentioned
i n t h e B r i t i s h Pharmacopoeia ( 6 ) .
1. 20 Mg o f r e s e r p i n e t o be d i s s o l v e d i n 1 0 ml
o f chloroform and 1 m l of t h i s s o l u t i o n i s d i l u t e d
t o 100 ml w i t h e t h a n o l (96%). The l i g h t a b s o r p t i o n
i n t h e range 230 t o 350 nm, e x h i b i t s a maximum a t
268 nm; absorbance a t 268 nm, i s about 0.55.
Absorbance over t h e range 288 nm t o 295 nm i s
about 0.34.
2 . Upon t h e a d d i t i o n of 1 m l o f a 0.1% w/v
s o l u t i o n of sodium molybdate i n s u l f u r i c a c i d t o
1 mg r e s e r p i n e ; a yellow c o l o r i s produced immedia-
t e l y which changes t o b l u e w i t h i n two minutes.
3. Upon t h e a d d i t i o n of 0 .2 ml o f a f r e s h l y
p r e p a r e d 1% w/v s o l u t i o n of v a n i l l i n i n hydro-
c h l o r i c a c i d ; a r o s e pink c o l o r i s produced w i t h i n
two minutes.
4. Upon mixing 0.5 mg of r e s e r p i n e w i t h 5 mg of
4-dimethylaminobenzaldehyde, 0.2 m l of g l a c i a l
a c e t i c a c i d and 0.2 ml of s u l f u r i c a c i d ; a green
c o l o r i s produced. Upon t h e a d d i t i o n of 1 ml
g l a c i a l a c e t i c a c i d ; t h e c o l o r changes t o r e d .
Other i d e n t i f i c a t i o n t e s t s ( 7 ) a r e as follows:
1. Reserpine i n e t h a n o l e x h i b i t s i n t h e ultra-
v i o l e t region m a x i m a a t 267 nm (E 1%, 1 em 239)
and 294 nm ( E 1%, 1 em 1 5 0 ) .
2. When drops of s u l f u r i c acid-formaldehyde are
added t o r e s e r p i n e ; a grey-green c o l o r i s produced
which changes t o brown.
3. When a s o l u t i o n of ammonium vanadate i s added
t o r e s e r p i n e ; a green c o l o r i s formed.
4. V i t a l i ' s t e s t gives w i t h r e s e r p i n e purple f l a s h
c o l o r which changes t o orange t h e n t o brown c o l o r .
6.2 Microcrystal t e s t s
A s o l u t i o n of 0.1% w/v of r e s e r p i n e i n concentrated

hydrochloric a c i d w a s used f o r t h e following micro-
c r y s t a l t e s t s :-
1. A s o l u t i o n of 1% w/v m o n i u m thiocyanate w a s
added t o t h e above s o l u t i o n ; a s t e l l a t e l i k e cry-
s t a l s were formed ( F i g . 5 ) ( 7 , 3 7 ) .
2. A s o l u t i o n of 0.5% w/v potassium cyanide w a s
added t o t h e above r e s e r p i n e s o l u t i o n ; small
r o s e t t e c r y s t a l s were formed ( F i g . 7 ) ( 7 ) .
3 . Hager's reagent w a s added t o t h e above
r e s e r p i n e s o l u t i o n t o give i r r i g u l a r b l a d e
c r y s t a l s (Fig. 8 ) ( 3 7 ) .
4. To a s o l u t i o n of r e s e r p i n e i n acetone, a
s o l u t i o n of 0.5% w/v potassium cyanide w a s added
t o give r a d i a t i n g rod c r y s t a l s (Fig. 6 ) ( 3 7 ) .
6.3 T i t r i m e t r i c Method
A ncn-aqueous t i t r a t i o n method i s described f o r

t h e determination of some a l k a l o i d s i n c l u d i n g
r e s e r p i n e and t h e i r dosage forms using O.OO5M
c h l o r a n i l i c a c i d s o l u t i o n i n 1 , b d i o x a n e as t h e
t i t r a n t . The end p o i n t i s determined by measuring
t h e change i n absorbance of t h e sample a t 535 nm.
Q u a n t i t a t i v e r e c o v e r i e s with good r e p r o d u c i b i l i t y
a r e reported f o r r e s e r p i n e and o t h e r a l k a l o i d s ( 3 8 ) .
RESERPINE 159
Fig. 5. Microcrystals of reserpine with

ammonium thiocyanate.
Fig. 6. Microcrystals of reserpine in ace-

tone with potassium cyanide.
6
%
€9
Fiq. 7. Microcrystals of reserpine with

potassium cyanide.
Fig. 8. Microcrystals of reserpine with

Hager's reagent.
RESERPINE 76 I
7. High Performance Liquid Chromatography (HPLC)
Reserpine can be determined i n pharmaceutical prepara-

t i o n s by HPLC(39) as follows:-
Reserpine i s e x t r a c t e d from powdered t a b l e t s w i t h w a t e r
s a t u r a t e d with e t h y l a c e t a t e , a f t e r addition of a solution
of propiophenone i n e t h y l a c e t a t e s a t u r a t e d w i t h w a t e r (as
internal standard). A f t e r centrifugation, the ethyl
a c e t a t e l a y e r i s a n a l y s e d by HPLC on a 1 0 pm Lichrosorb
RP-8 column u s i n g methanol/O.O5M sodium phosphate mono-
b a s i c mixture (1:l) as t h e mobile phase. The f l c w r a t e
i s a d j u s t e d t o 2 ml/minute. D e t e c t i o n i s c a r r i e d o u t
under UV a t 254 nm. ( F i g . 9 ) .
Another HPLC system to a n a l y s e r e s e r p i n e and hydrochloro-
t h i a z i d e i n two-component t a b l e t f o r m u l a t i o n s (40): 0.1-0.2
mg of r e s e r p i n e and 25-5Omg of h y d r o c h l o r o t h i a z i d e i n t a b -
l e t form a r e shaken w i t h 0.05% p o l y t h i a z i d e s o l u t i o n i n
t e t r a h y d r o f u r a n (10 m l ) f o r 20 m i n u t e s , c e n t r i f u g e d f o r
f i v e minutes a t 2000 r.p.m. and 7 ml of t h e s u p e r n a t a n t
l i q u i d i s i n j e c t e d t o HPLC u s i n g Lichrosorb S i 60 column
and a mixture of 77% hexane, 18% i s o p r o p y l a l c o h o l , 5%
chloroform and 0.01% diethylamine as t h e mobile phase.
The flow r a t e i s a d j u s t e d t o 1 . 5 m l minute. D e t e c t i o n i s
performed under W a t 254 nm.
A t h i r d method i s d e s c r i b e d f o r t h e q u a n t i t a t i v e determi-
n a t i o n o f c h l o r t h a l i d o n e i n pharmaceutical dosage forms
c o n t a i n i n g r e s e r p i n e (41).
Powdered t a b l e t s of c h l o r t h a l i d o n e and r e s e r p i n e a r e
e x t r a c t e d w i t h a c e t o n i t r i l e / w a t e r (9:l), c e n t r i f u g e d and
t h e s u p e r n a t a n t l a y e r i s i n j e c t e d t o HPLC u s i n g a column
of Pellamidon a t 35Oc and t h e s o l v e n t i s o p r o p a n o l / a c e t i c
acid/water/hexane (60:3 :1:36) as t h e mobile phase. Detec-
t i o n i s c a r r i e d out under W a t 254 nm.
Reserpine and o t h e r a n t i h y p e r t e n s i v e drugs can be analysed
by r e v e r s e phase HPLC(42) as follows:-
0.5% drug sample i n t h e mobile phase i s i n j e c t e d i n t o HPLC
f i t t e d w i t h e i t h e r octadecyltrichlorosilane ( c l 8 ) column
or w i t h d i p h e n y l d i c h l o r o s i l a n e ( p h e n y l ) column. S e v e r a l
mobile phases a r e used. A c e t o n i t r i l e or a b s o l u t e methanol
mixed w i t h aqueous s o l u t i o n s of 0 . 1 o r 1 . 0 % ammonium ace-
t a t e , 0 . 5 o r 1% ammonium c h l o r i d e and 0.2 or 1% ammonium
carbonate,
A flow r a t e of 1 . 4 ml/min i s maintained. D e t e c t i o n i s
c a r r i e d u s i n g UV d e t e c t o r .
Reserpine in plasma can also be determined by HPLC(43)

as follows:-
Equine plasma (2 m l ) saturated with aqueous sodium borate
(3 ml) and benzene (2 ml) are mixed and centrifuged until
phases separated. The benzene layer is collected and
evaporated to dryness at 50°c under nitrogen. The reser-
pine so isolated is oxidised with N205-H3P04 reagent f o r
10 minutes. 2 m l is injected t o HPLC apparatus fitted
with Bondapak cl8 column using methanol/aqueous 0.01M
sodium heptanesalfonate (13:7) as the mobile phase with
f h w rate of 2.5 &/minute. Detection is carried out
under fluorescence.
Propiophenone
Reserpine
0.25 mg
TABLET
Solvent
I l l I 1 I 1
0 5 10 15 20 25 30
TIME (min)
Fig. 9 HPLC of Reserpine in Tablets ( 3 9 ) .

RESERPINE 763
References
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R.W. Kierstead, J . Am. Chem. SOC., 78, 2023 (1956).
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R.W. Kierstead, Tetrahedron 2, 1 (1958).
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RESERPINE 165
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ACKNOWLEDGEMENT
The author would like to thank Mr. Uday
C. Sharma of Pharmacognosy Dept., College
of Pharmacy, King Saud University for his
secretarial assistance in the reproduction
of the manuscripts.
ERRATUM
PHENYLPROPANOLAMINE HYDROCHLORIDE
Volume 12, p. 358.
2.1 Name, Formula, Molecular Mass
The structural formula should be replaced by the following:
e
y OH H
I -y-NH*=HCl
I
H CH3
CUMULATIVE INDEX
Bold numeral\ refer to volume numbera
Acetaminophen. 3, 1 Chlordiazepoxide hydrochloride, 1, 39: 4, 5 18

Acetohexamide, 1, 1; 2, 573 Chloroquine, 13, 95
Allopurinol. 7, I Chloroquine phosphate, 5, 61
Alpha-tocopheryl acetate, 3, 11 1 Chlorpheniramine maleate, 7, 43
Amantadine, 12, I Chlorprothixene. 2, 63
Amikacin sulfate, 12, 37 Chlortetracycline hydrochloride. 8, 101
Arninophylline. 11, 1 Cholecalciferol, see Vitamin D3
Aminosalicylic acid, 10, 1 Cinietidine, 13, 127
Amitriptyline hydrochloride. 3, 127 Clidinium bromide, 2, 145
Anioxicillin. 7, 19 Clindamycin hydrochloride. 10, 75
Anlphotericin B, 6 , I ; 7, 502 Clofibrate. 11, 197
Anipicillin. 2, 1; 4, 518 Clonazepam, 6, 61
Ascorbic acid. 11, 45 Clorazepate dipotassium. 4, 91
Aspirin. 8, I Clotrimazole. 11, 225
Atenolol. 13, I Cloxarillin sodium, 4, I 13
Azathioprine. LO, 29 Codeine phosphate. 10, 93
Bacitracin, 9, I Colchicine. 10, 139
Bendroflumethiazide. 5, 1; 6, 597 Cyanocobalamin. 10, 183
Benzocaine. 12, 73 Cyclizine. 6, 83: 7. 502
Benzyl benzoate. 10, 55 Cycloserine, 1 , 53
Betamethasone dipropionate. 6, 43 Cyclothiazide. 1, 66
Bretylium tosylate, 9, 7 I Cypropheptadine, 9, 155
Brvmocriptine methanesulfonate, 8, 47 Dapsone. 5, 87
Calcitriol, 8, 83 Dexamethaaone. 2, 163: 4, 5 19
Camphor. 13, 27 Diatrizoic acid. 4, 137: 5, 556
Captopril. 11, 79 Diazepam, 1, 79: 4, 518
Carbamazepine. 9, 87 DibenLepin hydrochloride. 9, I8 1
Cefaclor. 9, 107 Dibucaine and dibucaine hydrochloride.
Cefamandole nafate. 9, 125: 10, 729 12, 105
Crfazolin. 4, I Digitoxin. 3, 149
Cefbfaxime. 11, 139 Digoxin, 9, 207
Cefoxitin, sodium. 11, 169 Dihydroergotoxine methanesulfonate, 7 , 8 I
Cephalexin. 4, 21 Dioctyl sodium sulfosuccinate. 2, 199: 12. 713
Cephalothin sodium. 1, 319 Diperodon, 6, 99
Cephradine. 5, 21 Diphenhydramine hydrochloride. 3, I73
Chloral hydrate, 2, 85 Diphenoxylare hydrochloride, 7, 149
Chloramphenicol, 4, 47, 5 I8 Disopyrainide phosphate. 13, 183
Chlordiazepoxide. 1, 15 Disulfiram. 4, 168
769
770 CUMULATIVE INDEX
Dobutamine hydrochloride, 8 , 139 Ketotifen, 13, 239

Dopamine hydrochloride, 11, 257 Khellin, 9, 371
Doxorubicine, 9, 245 Leucovorin calcium, 8, 315
Droperidol, 7, 171 Levallorphan tartrate, 2, 339
Echothiophate iodide, 3, 233 Levarterenol bitartrate, 1, 49; 2, 573: 11, 555
Emetine hydrochloride, 10, 289 Levodopa, 5, 189
Epinephrine, 7, 193 Levothyroxine sodium, 5 , 225
Ergonovine maleate, 11, 273 Lorazepam, 9, 397
Ergotamine tartrate. 6, 113 Melphalan, 13, 265
Erythromycin, 8 , 159 Meperidine hydrochloride. I, I75
Erythromycin estolate, 1, 101; 2, 573 Meprobamate. I, 209; 4, 520: 11, 587
Estradiol valerate, 4, 192 6-Mercaptopurine. 7, 343
Estrone, 12, 135 Mestranol. 11, 375
Ethambutol hydrochloride, 7, 23 I Methadone hydrochloride, 3, 365; 4, 520;
Ethynodiol diacetate. 3, 253 9, 601
Etomidate, 12, 191 Methaqualone, 4, 245, 520
Fenoprofen calcium, 6, 161 Methimazole, 8 , 351
Flucytosine, 5, 115 Methotrexate, 5 , 283
Fludrocortisone acetate, 3, 28 1 Methoxsalen. 9, 427
Flufenamic acid, 11, 313 Methyclothiazide, 5 , 307
Fluorouracil, 2, 221 Methylphenidate hydrochloride, 10, 471
Fluoxymesterone. 7, 25 1 Methyprylon. 2, 363
Fluphenazine decanoate, 9, 275: 10, 730 Metoprolol tartrate. 12, 325
Fluphenazine enanthate, 2, 245; 4, 524 Metronidazole. 5, 327
Fluphenazine hydrochloride. 2, 263: 4, 5 19 Minocycline, 6, 323
Flurazepam hydrochloride, 3, 307 Moxalactam disodium, 13. 305
Gentamicin sulfate. 9, 295; 10, 731 Nabilone, 10, 499
Clibenclaniide, 10, 337 Nadolol, 9, 455; 10, 732
Gluthethimide, 5, 139 Nalidixic acid. 8, 371
Gramicidin, 8, 179 Natamycin. 10, 513
Griseofulvin, 8, 219; 9, 583 Neomycin, 8, 399
Halcinonide, 8, 251 Nitrazepam, 9, 487
Haloperidol. 9, 341 Nitrofurantoin. 5 , 345
Halothane, 1, 119; 2, 573 Nitroglycerin, 9, 519
Heparin sodium, 12, 215 Norethindrone. 4, 268
Heroin. 10, 357 Norgestrel, 4. 294
Hexestrol, 11, 347 Nortriptyline hydrochloride, I , 233; 2, 573
Hcxetidine, 7, 277 Noscapine. 11, 407
Hydralazine hydrochloride, 8, 283 Nystatin. 6, 341
Hydrochlorothiazide, 10, 405 Oxazepam. 3, 441
Hydrocortisone. 12, 277 Oxyphenbutarone. 13, 333
Hydroflumethiazide. 7, 297 Oxytocin. 10, 563
Hydroxyprogesterone caproate, 4, 209 Penicillamine. 10, 601
Hydroxyzine dihydrochloride, 7, 31 9 Penicillin-G benzothine. 11, 463
Indomethacin, 13, 2 I I Penicillin-V. 1, 249
lodipamide, 2, 333 Pentazocine, 13, 361
Isocarboxazid. 2, 295 Phenazopyridine hydrochloride. 3, 465
Isoniazide. 6, 183 Phenelzine sulfate. 2, 383
Isopropamide. 2, 315; 12, 721 Phenformin hydrochloride, 4, 319: 5, 429
lsosorbide dinitrate, 4, 225: 5 , 556 Phenobarbital. 7, 359
Kanamycin sulfate, 6, 259 Penoxymethyl penicillin potassium. 1, 249
Ketamine. 6, 297 Phenylbutazone. 11, 483
Ketoprofen. 10, 443 Phenylephrine hydrochloride. 3, 483
CLiMULATlVE INDEX 77 1
Phenylpropanolamine hydrochloride, 12, 357: Sulfamethoxazole, 2, 467; 4, 521

13, 771 Sulfasalazine. 5, 515
Phenytoin. 13, 417 Sulfisoxazole. 2, 487
Pilocarpine, 12, 385 Sulindac, 13, 573
Piperazine estrone sulfate, 5 , 375 Sulphamerazine, 6, 5 15
Primidone. 2, 409 Testolactone. 5 , 533
Probenecid, 10, 639 Testosterone enanthate. 4, 452
Procainamide hydrochloride, 4, 333 Tetracycline hydrochloride, 13, 597
Procarbazine hydrochloride, 5 , 403 Theophylline. 4, 466
Promethazine hydrochloride. 5 , 429 Thiostrepton. 7, 423
Proparacaine hydrochloride, 6, 423 Tolbutamide. 3, 5 13: 5 , 557: 13, 7 I9
Propiomazine hydrochloride, 2, 439 Triamcinolone. 1, 367: 2, 571; 4, 521. 524;
Propoxyphene hydrochloride, 1, 301: 4, 520: 11,593
6, 598 Triamcinolone aceronide. 1, 397, 416; 2, 571:
Propylthiouracil, 6, 457 4, 521: 7, 501: 11, 615
Pseudoephedrme hydrochloride. 8, 489 Triamcinolone diacetate. 1, 423; 11, 651
Pyrazinamide. 12, 433 Triamcinolone hexacetonide. 6, 579
Pyridoxine hydrochloride, 13, 447 Triclobisonium chloride, 2, 507
Pyrirnetharnine. 12, 463 Trifluoperazine hydrochloride. 9, 543
Quinidine sulfate. 12, 483 Triflupromazine hydrochloride, 2, 523; 4, 521:
Quinine hydrochloride, 12, 547 5, 557
Reserpine. 4, 384; 5, 557; 13, 737 Trimethaphan camsylate, 3, 54.5
Rifampin. 5, 467 Trimethobenzamide hydrochloride, 2, 55 I
Rutin, 12, 623 Trimethoprim, 7, 445
Saccharin, 13, 487 Trimipramine maleate. 12, 683
Salbutamol. 10, 665 Trioxsalen. 10, 705
Salicylamide, 13, 521 Triprolidine hydrochloride, 8, 509
Secobarbital sodium, 1, 343 Tropicamide, 3, 565
Silver sulfadiazine. 13, 553 Tubocurarine chloride. 7, 477
Sodium nitroprusside. 6, 487 Tybamate, 4, 494
Spironolactone, 4, 43 I Valproate sodium and valproic acid. 8, 529
Succinylcholine chloride, 10, 691 Vinblastine sulfate, 1, 443
Sulfadiazine. 11, 523 Vincristine sulfate, 1, 463
Sulfamethazine. 7, 401 Vitamin D,. 13, 655

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Ana1yticaI Profiles

Abdullah A. Al-Badr Klaus Florey

Academic Press Rapid Manuscript Reproduction

ACADEMIC PRESS, INC. 1984

ACADEMIC PRESS, INC.

United Kingdom Edition published by

Library of Congress Cataloging in Publication Data

PRINTED IN THE UNITED STATES OF AMERICA

Affiliations of Editors, Contributors, and Reviewers vii

Disopyramide Phosphate 183

Moxalactam Disodium 305

Yhen ytoin 417

Pyridoxine Hydrochloride 447

Silver S ulfadiazine 553

Tetracycline Hydrochloride 597

Vitamin D3 (Cholecalciferol) 655

Tolbu tamide 719

Phenylpropanolamine Hydrochloride 767

Cumulative Index 769

H . Y. Abod-Enein, King Saud University, Riyadh, Saudi Arabia

S. A . Fusari', Warner-Lambert Company, Morris Plains, New Jersey

The compilation ofdnnlyticcil Profiles of Drug Szibstunces to supplement the

1. Foreword, History, Therapeutic Category 2

ANALYTrCAL PROFILES OF DRUG SUBSTANCES Copyright 01984

1. Foreword, History, Therapeutic Category

standing overnight. The precipitate is collected by

The infrared spectrum o f atenolol, presented

LOO0 3000 2000 1800 1600 1200 cm-l 800

Table 1. Assignment of proton MR s i g n a l s in t h e

F r o t o m Chemical s h i f t Type Inten- Coupling const.

t r a l assignments are shown i n !Table 1. (5) Charac-

13C-hT!R s p e c t r a o f a t e n o l o l and compound 5

Table 2. Assignment of p r o t o n MR s i g n a l s i n the

Fig. 4. S i n c e t h e s o l v e n t peaks c o v e r one carbon

T a b l e 3.13C Chemical s h i f t s a and I3C-'H coupling

Table 4. 1 3 C Chemical s h i f t s a f o r a t e n o l o l and com-

Table 5. Peak assignments in the mass spectrum of

43.1 107 01'

spectrum is due to isopropylaminomethylene group

4.2.2. Crystal Properties

Table 6. Peak assignements in the mass spectrum of

Table 7. X-Ray diffraction data of atenolol crystal

a based on the highest intensity which is selected

unter tube and DC voltage at plateau. Fulse light

The negative logarithm of the proton dissoci-

4.3.5. Optical Rotation

3.2. Titrimetric Determination Electrochemical

jector tempBrature was set at 30Ooc, oven tem era-

In actual analyses the content of atenolol is

5 . 3 . 3 . Hiah Pressure Liquid

The apparatus used for high pressure liquid

lumn was prepacked with Partisil l o p m ODS (25 cm;

administration, about lo$ of the dose excreted un-

D. Ross, J.L. Reid, Br. J. c l i n . Pharmac. 3,

13. J. 6Ieffih, S.R. Harapat, D.C. Harrison, Res.

ANALYTICAL PROFILES OF DRUG SUBSTANCE5 Copyright G 1984

1.11 Chemical Names

1.13 Trade Names

Camphor i s a s a t u r a t e d ketone, C 1 0 H 1 6 0 , which on

bicyclic. The c a r b o n y l group i s f l a n k e d by only

A tremendous amount o f work was done b e f o r e t h e

Camphor Camphor j.c Camphoric

Homoc amphoric Homocamphoric