Analytical Profiles Drug Substances and Excipien T S: Harry G. Brittain

Analytical Profiles
of
Drug Substances
and
Excipients
Volume 21
Edited by
Harry G. Brittain
Bristol-Myers Squibb
Pharmaceutical Research Institute
New Brunswick, New Jersey
Founding Editor
Klaus Florey
ACADEMIC PRESS, INC.

Harcourt Brace Jovanovich, Publishers
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EDITORIAL BOARD
Abdullah A. Al-Badr George A. Forcier

Gerald S. Brenner Lee T. Grady
Glenn A. Brewer David J. Mazzo
Harry G. Brittain Thomas W. Rosanske
Klaus Florey Timothy J. Wozniak
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AFFILIATIONS OF
EDITORS AND CONTRIBUTORS
Mohummud A . Abounussf, Pharmaceutical Chemistry Department, College of Phar-

macy, King Saud University, Riyadh 11451, Saudi Arabia
Abdul Furruh A . A . Ajfy, Department of Pharmacognosy, College of Pharmacy, King
Saud University, Riyadh 11451, Saudi Arabia
Iqbal Ahmad, Pharmaceutical Chemistry Department, Faculty of Pharmacy, Univer-
sity of Karachi, Karachi 75270, Pakistan
Tuuqir Ahmud, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Uni-
versity of Karachi, Karachi 75270, Pakistan
Abdulluh A . Al-Budr, Pharmaceutical Chemistry Department, College of Pharmacy,
King Saud University, Riyadh 11451, Saudi Arabia
Fuhud J. Al-Shammury, Clinical Laboratory Sciences Department, College of Ap-
plied Medical Sciences, King Saud University, Riyadh 11433, Saudi Arabia
Silvia Alessi-Severini, Faculty of Pharmacy and Pharmaceutical Sciences, University
of Alberta, Edmonton, Alberta T6G 2N8, Canada
Syed Laik Ali, Zentrallaboratorium Deutscher Apotheker, 6236 Eschborn, Germany
Adnun A . Budwun, The Jordanian Pharmaceutical Manufacturing Company, Naor,
Jordan
Gary Burberu, Bristol-Myers Squibb, Pharmaceutical Research Institute, New
Brunswick, New Jersey 08903
Gerald S. Brenner, Merck Sharp & Dohme Research Laboratories, West Point, Penn-
sylvania 19486
Glenn A . Brewer, Bristol-Myers Squibb, Pharmaceutical Research Institute, New
Hurry G . Brirruin, Bristol-Myers Squibb, Pharmaceutical Research Institute, New
Marvin A . Brooks, Merck Sharp & Dohme Research Laboratories, West Point, Penn-
sylvania 19486
Robert A . Curr, Faculty of Pharmacy and Pharmaceutical Sciences, University of
Alberta, Edmonton, Alberta T6G 2N8, Canada
Owen 1. Corrigan, School of Pharmacy, University of Dublin, Dublin 4, Ireland
Ronald T. Coutts, Faculty of Pharmacy and Pharmaceutical Sciences, University of
vii
...
Vlll AFFILIATIONS OF EDITORS AND CONTRIBUTORS
Joseph D. DeMarco, Merck Sharp & Dohme Research Laboratories, West Point,
Pennsylvania 19486
Joseph DeVincenfis, Bristol-Myers Squibb, Pharmaceutical Research Institute, New
Humeida A . El-Obeid, Pharmaceutical Chemistry Department, College of Pharmacy,
King Saud University, Riyadh 11451, Saudi Arabia
Dean K. Ellison, Merck Sharp & Dohme Research Laboratories, West Point, Penn-
sylvania 19486
Klaus Florey, Bristol-Myers Squibb Company, Lawrenceville, New Jersey 08543
George A . Forcier, Central Research Division, Pfizer, Inc., Groton, Connecticut
06340
Robert T. Foster, Faculty of Pharmacy and Pharmaceutical Sciences, University of
Lee T. Grady, The United States Pharmacopeia, Rockville, Maryland 20852
Dominic I? Zp,Merck Sharp & Dohme Research Laboratories, West Point, Pennsyl-
vania 19486
Fakhreddin Jamafi, Faculty of Pharmacy and Pharmaceutical Sciences, University
of Alberta, Edmonton, AlbertaT6G 2N8, Canada
Eric C. Jensen, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis,
Indiana 46285
Michael J. KauJinan, Merck Sharp & Dohme Research Laboratories, West Point,
Pennsylvania 19486
David J. M a u o , Department of Analytical & Physical Chemistry, RhBnC-Poulenc
Rorer Recherche-Development,92165 Antony Cedex, France
Michael J. McLeish, School of Pharmaceutical Chemistry, Victorian College of Phar-
macy, Monash University, Parkville, Victoria 3052, Australia
Mohammad SafeemMian, Pharmaceutical Chemistry Department, College of Phar-
macy, King Saud University, Riyadh 11451, Saudi Arabia
Neelofur Abduf Aziz Mian, Clinical Laboratory Sciences Department, College of
Applied Medical Sciences, and Department of Pharmacognosy, College of
Pharmacy, King Saud University, Riyadh 11433, Saudi Arabia
Ann W. Newman, Bristol-Myers Squibb, Pharmaceutical Research Institute, New
CaifrionaM . O’Driscoll, School of Pharmacy, University of Dublin, Dublin 4, Ire-
land
Mahmoud A1 Omari, The Jordanian Pharmaceutical Manufacturing Company, Naor,
Jordan
Franco M . Pasutto, Faculty of Pharmacy and Pharmaceutical Sciences, University
of Alberta, Edmonton, Alberta T6G 2N8, Canada
Thomas W Rosanske, Marion Merrell Dow, Inc., Kansas City, Kansas 64134
Charles M . Shearer, Wyeth-Ayerst Research, Rouses Point, New York 12979
Delores J. Sprankle, Lilly Research Laboratories, Eli Lilly and Company, Indianapo-
lis, Indiana 46285
AFFILIATIONS OF EDITORS AND CONTRIBUTORS ix
K . Usmanghani. Department of Pharmacognosy, Faculty of Pharmacy, University of

Karachi, Karachi 75270, Pakistan
G.Michael Wall, Alcon Laboratories, Inc., Fort Worth, Texas 76134
Titnothy J. Wozniak, Eli Lilly and Company, Lilly Corporate Center, Indianapolis,
Indiana 46285
Muhammad B . Zughul, Department of Chemistry, Faculty of Science, University of
Jordan, Amman, Jordan
PREFACE
The profiling of drug compounds as to their physical and analytical characteristics

has been the focus of the preceding twenty volumes in the Analytical Profiles series,
and the need for this information is as important today as it was when the series was
first initiated. The compilation of concise summaries of physical and chemical data,
analytical methods, routes of compound preparation, degradation pathways, and the
like, is a vital function to both academia and industry. Under the editorship of Klaus
Florey, the Analytical Profiles has met this need over its twenty year history.
With the publication of Volume 21, the editorship has been assumed by Harry
Brittain. The focus of the chapters will remain unchanged, but the scope of the
Analytical Projiles series has expanded to include profiles of excipient materials, and
this has led to a modification of the series title. The series will henceforth be known
as the Analytical Profiles of Drug Substances and Excipients. The first excipient
profile (anhydrous lactose) appeared in Volume 20, and a profile on titanium dioxide
is included in the present volume.
The success of the series will continue to be based on the contributions of the
chapter authors, and on the quality of their work. We seek profiles of new drug
compounds as they come to markets but we also wish to profile important older
compounds that have escaped attention thus far. A complete list of available
candidates can be obtained from the editor by any prospective author. We look
forward to hearing from new and established authors and to working with the
pharmaceutical community on the Analytical Profiles of Drug Substances and
Excipients.
Harry G . Brittain Klaus Florey

Editor Founding Editor
xi
ACETOHEXAMIDE
Abdullah A. Al-Badr and Humeida A. El-Obeid
Pharmaceutical Chemistry Department
College of Pharmacy
King Saud University
Riyadh, Saudi Arabia
ANALYTICAL PROFILES OF DRUG SUBSTANCES Copyright 0 1992 by Academic Press, Inc.

-
AND EXCIPIENTS VOLUME 21 1 All rights of reproduction reserved in any form
2 ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID
C O N T E N T S
1. DESCRIPTION
1 . 1 Nomenclature
1.1.1 Chemical Names
1.1.2 Genermic Names
1.1.3 Trade Names
1 . 2 Formulae
1.2.1 Empirical
1.2.2 Structural
1.2.3 GAS No.
1.3 Molecular Weight
1 . 4 Elemental Composition
1 . 5 Appearance
2. PHYSICOCHEMICAL PROPERTIES
2 . 1 Melting Range
2.2 S o l u b i l i t y
2 . 3 Polymorphism
2 . 4 Thermal Analysis
2 . 5 X-ray Powder D i f f r a c t i o n
2.6 Spectral Properties
2.6.1 U l t r a v i o l e t Spectrum
2.6.2 I n f r a r e d Spectrum
2.6.3 Proton Nuclear Magnetic Resonance (PMR)
Spectrum
2.6.4 lac-Nuclear Magnetic Resonance (‘SC-NMR)
Spectrum
2.6.5 Mass Spectra
3. SYNTHESIS
4. METHODS OF ANALYSIS
4 . 1 T i t r i m e t r i c Methods
4.1.1 Nonaqueous
4.1.2 Gravimetric
4.1.3 Campleximetric
4.2 Spectrometric Methods
4.2.1 Colorimetric
4.2.2 U1t r a v i o l e t
4.2.3 Infrared
4.2.4 Fluormetric
4.2.5 Proton Magnetic Resonance
ACETOHEXAMIDE 3
4.3 Chromatographic Methods

4.3.1 Thin-Layer Chromatography (TLC)
4.3.2 Gas-Liquid Chromatography (GLC)
4.3.3 High-Performance Liquid Chromatography
(HPLC)
5. PHARMACOKINETICS
5 . 1 Introduction
5.2 Mechanism o f Action
5.3 Onset and Duration o f Action
5.4 Absorption
5.5 Distribution
5.6 Metabol ism
5.7 Excretion
5.8 Half-Life
ACKNOWLEDGEMENT
REFERENCES
4 ABDULLAH A. AL-BADR AND HUMEIDA A . EL-OBEID
ACETOHEXAMIDE
1. DESCRIPTION
-
1 1 Nomenclature
4-Acetyl-N-[(cyclohexylamino)carbonyl]benzenesul-
fonamide
l-[(pAcetylphenyl)sulfonyl]-3-cyclohexylurea.
3-Cyclohexyl-l-(pacetylphenylsulfonyl)urea.
N-(pAcetyl benzyl sul fonyl l-N -cyclohexyl urea.
1.1.2 Generic Names
Acetohexamide, Acetohexamidum
-
1 1.3 Trade Names
Cycl am1de , Dime 1in , Dime1o r , Dyme 1o r , Gamadiabet,

Metaglucina, Ordimel, Tsiklamid.
1.2 Formulae
1.2.1. EmDlriCal
Ct sHzoNz04S
1.2.2 Structural
1.2.3 CAS No.
[968-81-01
324.42 (1)
1.4 Elemental ComDosltion
C 55.54%, H 6.21%, N 8.64%, 0 19.73%, S 9.89% (1).

ACETOHEXAMIDE 5
1.5 Armearance
A w h i t e , c r y s t a l l i n e powder; o d o r l e s s o r almost
odorless (2).
2. PHYSICOCHEMICAL PROPERTIES
2.1 M e l t i n g Range
C r y s t a l s from 90% aqueous e t h a n o l m e l t between

188-190" ( 3 ) . Crystals from d i l u t e ethanol m e l t between
175-177 (4).
2.2 Solubility
Soluble i n p y r i d i n e , s l i g h t l y soluble i n alcohol

and chloroform. I n s o l u b l e i n water and ether ( 1 ) .
2.3 P01YmOrDh'ism
The l i t e r a t u r e r e p o r t s i n d i c a t e t h a t acetohexamide
e x i s t s as more t h a n one polymorphic forms ( 5 - 1 5 ) .
G i rgis-Takla and Chroneos (5) prepared acetohexamide
polymorphs A and B by h e a t i n g t h e drug ( 1 gm) w i t h
g l a c i a l a c e t i c a c i d o r chloroform respectively, before
c r y s t a l l i z a t i o n a t 1 0 5 ' and room t e m p e r a t u r e
respectively. While acetohexamide polymorph A showed a
m e l t i n g range o f 180"-183', t h e acetohexamide polymorph
B melted a t 183'-185". D i f f e r e n t i a l scanning
calorimetry and I R spectroscopy showed t h a t c r y s t a l s o f
polymorph B were converted t o polymorph A by grinding.
A c c o r d i n g l y , t h e s e r e s u l t s i n d i c a t e t h a t any
i d e n t i f i c a t i o n t e s t u t i l i z i n g g r i n d i n g may f a i l to
i d e n t i f y t h e two polymorphs. I n t h e i r phystco-chemical
studies on t h e polymorphism o f acetohexamide, Kuroda e t
a7 (6) obtained t h r e e polymorphs o f acetohexamide by
r e c r y s t a l l i z a t i o n from d i f f e r e n t solvents. These are
f o r m I,f o r m I 1 and CHC13-11. A l t h o u g h t h e X-ray
d i f f r a c t i o n p a t t e r n s , I R s p e c t r a and d i f f e r e n t i a l
scanning calorimeter curves o f t h e CHC13-I1 polymorph
were i d e n t i c a l w i t h those o f polymorph 11, t h e CHC13-I1
t y p e c o n t a i n e d a C H C l j molecule which c o u l d n o t be
removed by normal d r y i n g condition. Polymorph CHC13-I1
seemed t o be unsuitable f o r medicinal use. Form I 1 i s
1.2 times more soluble than form I.
6 ABDULLAH A. AL-BADR AND HUMElDA A. EL-OBEID
Burger ( 7 ) c h a r a c t e r i z e d t h e t h r e e p o l y m o r p h i c
m o d i f i c a t i o n s o f acetohexamide by thermomicroscopy,
d i f f e r e n t i a l scanning calorimetry and I R spectroscopy.
The s o l u b i l i t y behavior o f the three modifications o f
the drug i n butanol and buffer solutions i s described
and d i s c u s s e d i n r e l a t i o n t o thermodynamics and
pharmacological parameters such as b i o a v a i l a b i l i t y from
t a b l e t s and USP X I X d i s s o c i a t i o n t e s t . M u e l l e r and
L a g a s ( 8 ) h a v e c o n f i r m e d t h e e x i s t e n c e and
characterized two polymorphic forms o f acetohexamide
using d i f f e r e n t i a l scanning calorimetry, thermogravi-
metric analysis, scanning e l e c t r o n microscopy as we1 1
as I R , NMR and X-ray analysis. The study has pointed t o
the u n s u i t a b i l i t y o f phosphate b u f f e r s o l u t i o n which i s
sometimes prescribed f o r use i n the d i s s o l u t i o n t e s t s
o f the drug since the s a l t o f the drug c r y s t a l l i z e s out
during the t e s t . I n another study (9) the same authors
reported t h a t form Idecomposed during melting and form
I1 melted a t 180" and then r e c r y s t a l l i z e d t o form I.A t
a heating r a t e o f lO'/minute melting points o f 193.6"
and 180.5" were found f o r forms Iand 11, respectively.
No morphological differences were observed between the
two forms. I n s o l u b i l i t y and d i s s o l u t i o n r a t e studies
i n sodium potassium b u f f e r , potassium acetohexamide
c r y s t a l l i z e d e x h i b i t i n g a lower s o l u b i l i t y than
acetohexamide. I n t h i s respect, form I 1 was transferred
t o potassium acetohexamide more quickly than form I.
Yokoyama e t a7 (10) calculated the thermodynamic values

o f forms I and I 1 o f acetohexamide from s o l u b i l i t y
measurements. The t r a n s i t l o n temperature and the heat
o f t r a n s i t i o n were 154" and 230 cal/mole, respectively.
I t i s found t h a t the polymorphic forms o f acetohexamide
d i d n o t a f f e c t i t s b i o a v a i l a b i l i t y when i n v i v o
absorption studies o f form I & I 1 were c a r r i e d out i n
b e a g l e dogs. The p r e p a r a t i o n o f f o u r c r y s t a l l i n e
modifications o f acetohexamide was reported (11). Their
thermograms, I R s p e c t r a , X-ray d i f f r a c t i o n and
s o l u b i l i t y are also reported. Two o f the forms reverted
t o the most stable form on storage i n solution.
S o l i d dispersion o f acetohexamide was studied by Graf

e t a7 (12-14) u s i n g d i f f e r e n t polymers and v a r i o u s
ratios. C o p r e c i p i t a t e s o f acetohexamide w i t h
polyethylene g l y c o l (PEG 6000) were prepared by t h e
s o l v e n t method w i t h ethanol ( c r y s t a l l i n e form I)or
with chloroform ( c r y s t a l 1ine form 111). Phase diagrams
ACETOHEXAMIDE I
o f form I-PEG and form 111-PEG coprecipitates were o f

the p e r i t e c t i c type and the molecular compounds were
formed i n the r a t i o o f 1 mole o f acetohexamide t o 4
moles o f PEG. The e u t e c t l c t e m p e r a t u r e , e u t e c t i c
composition and the end o f melting o f the two binary
system were, however, d i f f e r e n t ( 1 2 ) . Both the
s o l u b i l i t y and t h e s o l u t i o n r a t e were increased by PEG.
S i m i l a r r e s u l t s were o b t a i n e d by s u b s t i t u t i n g
p o l y ( v i n y l p y r r o 1 i d o n e ) (PVP) f o r PEG ( 1 3 ) . Also,
c o p r e c i p i t a t e s o f acetohexamide-PVP ( i n e t h a n o l )
containing drug concentrations o f 60% o r more showed
the same X-ray d i f f r a c t i o n pattern as t h a t o f form I.
Increasing the PVP concentration ( > 55%) d i d n o t show
any c r y s t a l behavior i n the X-ray analysis. I n another
r e p o r t Graf e t a7 ( 1 4 ) d e s c r i b e d t h e methods o f
p r e p a r a t i o n and t h e e f f e c t o f t h e s o l v e n t s on t h e
acetohexamide-PVP coprecipi tates. They were obtained
from ethanol o r chloroform by evaporating the solvent
a t room temperature, under vacuum or by spray drying.
Changing t h e s o l v e n t and/or i t s e v a p o r a t i o n r a t e
affected the polymorphic form, the c r y s t a l l i n i t y and
the s o l u t i o n r a t e o f acetohexamide i n coprecipitates
containing less than 70% PVP.
Kassem e t a7 (15) studied the enhancement o f the r a t e

o f release o f acetohexamide from i t s t a b l e t s by t h e
f o r m a t i o n o f s o l i d d i s p e r s i o n s w i t h each o f f o u r
water-sol uble pol ymers prepared in d i f f e r e n t r a t i0s.
The polymers were r a t e d i n t h e o r d e r o f decreasing
r e l e a s e r a t e s as f o l l o w s : PEG 6 0 0 0 , PVP,
hydroxypropylmethylcellulose, methylcellulose.
2.4 Thermal Analysis
The h e a t o f f u s i o n and m e l t i n g p o i n t o f
acetohexamide were done u s i n g DuPont TA 9900 on t h e
DSC- u n i t a t a temperature range i n d i c a t e d i n t h e
thermogram (Figure 1). Sample i s done i n duplicate and
the average o f t h e value i s reported as follows:
AHf = 63.7 kJ/mOle Purity = 99.82% Tm = 187.45 C
2.5 X-ray Powder D i f f r a c t i o n
The X-ray powder d i f f r a c t i o n p a t t e r n o f acetohexa-

mide was determined using P h i l i p s f u l l automated X-ray
d i f f r a c t i o n spectrogoniometer equipped w i t h PW1730/10
PURITY v l . l A
F i g u r e 1. Thermal cu rve o f acetohexamide.

ACETOHEXAMIDE 9
Figure 2 . X-Ray powder d i f f r a c t i o n p a t t e r n of acetohexamide.

generator. Radiation was provided by a copper t a r g e t

(Cu anode 2000W, X = 1.5480 A), high I n t e n s i t y X-ray
tube operated a t 40 kV and 35 mA. The monochromator was
a curved s i n g l e c r y s t a l one (PW1752/00). Divergance
s l i t and t h e r e c e i v i n g s l i t were 1 and 0.1
r e s p e c t i v e l y . The scanning speed o f t h e gonlometer
(PW1050/81) used was 0.02 2 8 p e r second. The
instrument i s combined w i t h P h i l i p s PM8210 p r i n t i n g
r e c o r d e r w i t h b o t h analogue r e c o r d e r and d i g i t a l
p r i n t e r . The goniometer was a l i g n e d using s i l i c o n
sample before use.
The X-ray pattern o f acetohexamlde I s presented i n

Figure 2. The interplanar distance d(A) and r e l a t i v e
i n t e n s i t i e s 1/10 are shown i n Table 1.
2.6 Spectral ProDerties
2.6.1 U l t r a v i o l e t Spectrum
The u l t r a v i o l e t a b s o r p t i o n s p e c t r u m o f
acetohexamide i n methanol was obtained on a Cary 219
spectrophotometer. The spectrum, shown i n Figure 3, i s
characterized by two maxima. The one w i t h a Xmax a t 247
nm i s t y p i c a l o f s u b s t i t u t e d acetophenones. The
absorption a t X m a x 283 nm represents a conjugated
aromatic r i n g system.
2.6.2 I n f r a r e d SDectrum
The i n f r a r e d absorption spectrum o f acetohexamide,

obtained from a potassium bromide d i s p e r s i o n , was
recorded on a Pye Unicam SP 1025 spectrometer and i s
shown i n Figure 4. The assignment o f the c h a r a c t e r i s t l c
bands are shown i n Table 2.
t I 1 1 i
220 XO nm 300 3 50 400 450

Figure 3 . U l t r a v i o l e t spectrum o f acetohexamide in methanol.
v,.
m.
c
N.
C V
cn
c -I-
I-- U
6,. L
m
Y
al. "
Q.
z!
W
E
N
Y
W
c
t-. 0
c,
W
V
tcl
'*-
0
f
L
c,
V
W
0
cn
*I
v), U
E
fu
I
U
ACETOHEXAMIDE 13
Table 1: X-ray d i f f r a c t i o n p a t t e r n o f acetohexamfde
d(A) 1/10 d(A) 1/10
15.74 31.25 2.55 1.82

9.47 30.04 2.49 1.75
7.85 6.89 2.40 6.19
7.21 2.25 2.36 4.44
5.30 100.00 2.31 5.26
4.99 8.28 2.29 2.20
4.93 10.95 2.27 1.81
4.55 4.71 2.24 2.54
4.30 5.30 2.18 2.04
4.19 15.19 2.15 2.38
4.08 23.07 2.13 1.20
3.92 5.44 2.09 2.56
3.78 2.82 2.04 4.51
3.60 24.35 1.99 1.69
3.50 4.52 1.95 2.91
3.28 23.29 1.94 4.16
3.26 9.83 1.89 1.50
3.15 5.72 1.81 1.48
3.07 9.36 1.77 1.29
3.01 1.26 1.72 1.77
2.91 7.99 1.66 1 .oo
2.88 2.79 1.64 1.18
2.74 4.08 1.61 1.16
2.65 1.51 1.57 1.32
2.61 2.15 1.47 0.85
2.58 1.80 1.35 0.80
d = Interplanner distance
1/10 = r e l a t i v e i n t e n s i t y (based on highest i n t e n s i t y of
100).
Table 2: I n f r a r e d c h a r a c t e r i s t i c bands and t h e i r

assignments.
Frequency (cm- Assignment
3340, 3270 Amide N-H s t r e t c h
2980, 2940 Aromatic C-H s t r e t c h

0
1710, 1680 Conjugated - E-

1602 , 1600 Aromatic C s t r e t c h
1455 C - CH3 bending

0
1345
Aromatic C-H out o f plane

780, 760
bending .
2.6.3 Proton Nuclear Magnetic Resonance ( W R l

Spectrum
Acetohexamide s o l u t i o n i n DMSO-de was used t o

obtain the PMR spectrum on a Varian XL 200 MHr FT NWR
spectrometer u s i n g TMS as i n t e r n a l reference. The
spectrum i s shown i n Figure 5. The number o f protons i s
established by both i n t e g r a t i o n o f the area under the
curve and t h e m u l t i p l i c i t i e s o f t h e peaks. Table 3
a s s i g n s t h e chemical s h i f t s t o t h e i r r e s p e c t i v e
protons. F u r t h e r evidence f o r p r o t o n assignment i s
obtained from the HETCOR pulse sequence (Figure 9).
Figure 5. PMR spectrum of acetohexamide i n DMSO-dG using TMS
as internal reference.
F i g u r e 6. 1 3 C NMR spectrum o f acetohexzmide i n DMSO-ds using
TMS as internal reference.
Figure 7. 1 3 C NMR spectrum o f acetohexarnide u s i n g DEPT
.
ex P e r iment
F i g u r e 8. 1 3 C I M R sDectrum of acetohexanide u s i n g APT
expe r irnent .
Figure 9. 13C N M R spectrum of acetohexamide using HETCOR
experiment.
Table 3: Assignment o f the NMR chemical s h i f t s t o the

d i f f e r e n t protons
Chemical s h i f t Multiplicity Proton No. o f

(6) assignment protons
1.09 - 1.71 mu1ti p l e t Cyclohexyl 11

ring3
2.66 singlet -
CH3-0 3
6.45 doublet -
CH-NH 1
8.06 - 8.19 mu1t ip l e t Aromat ic d 4
2.6.4 13C-Nuclear Magnetic Resonance (13C NHR)

SDect rum
The 1 3 C NMR spectra o f acetohexamide i n DMSO-ds

using TMS as i n t e r n a l reference are obtained using a
V a r i a n XL 200 MHz p u l s e FT s p e c t r o m e t e r and a r e
p r e s e n t e d i n F i g u r e s 6-9. The assignment o f t h e
chemical s h i f t s and the degree o f carbon protonation,
presented i n Table 4, are achieved u s i n g t h e DEPT
(Figure 7) and APT (Figure 8) experiments as well as
t h e HETCOR p u l s e sequence ( F i g u r e 9 ) and t h e
approximate a d d i t i v e e f f e c t s o f substituents.
2.6.5 Mass SDectra
The 70 eV e l e c t r o n impact mass spectrum o f

acetohexamide, presented i n Figure 10, was obtained on
Varian MAT 311 mass spectrometer u s i n g i o n source
pressure o f 10-0 Torr, i o n source temperature o f 180'C
and an emission current o f 300 M. The molecular i o n i s
detectable a t m/e 324 and the base peak a t m/e 56. A
proposed fragmentation p a t t e r n and t h e mass/charge
r a t i o s o f the major fragments are shown I n Scheme 1.
ACETOHEXAMIDE 21
Table 4: Assignment o f the carbon chemical s h i f t s .
Chemical s h i f t Carbon assignment Number o f Protons

(PHI attached
24.26 d 2
25.07 C 2
26.99 e 3
32.33 b 2
48.30 a 1
127.73 i 1
128.73 j 1
140.00 k 0
143.93 h 0
150.45 9 0
197.30 f 0
The chemical i o n i z a t i o n spectrum, shown i n Figure 11,

was obtained on Finnigan 4000 mass spectrometer using
methane gas as a reagent with ion electron energy o f
100 eV, ion source pressure o f 0 . 3 T o r r , i o n source
temperature o f 150’C and emission current o f 300 pA.
The spectrum i s dominated by a quasimolecular i o n (M +
1 ) . Two peaks appearing a t m/e 353 and m/e 365 are
a t t r i b u t a b l e t o the t r a n s f e r o f carbocations from the
c a r r i e r gas. The mass s p e c t r a l assignment o f t h e
N
N
F i g u r e 10. Electron impact mass spectrum o f acetohexamide.

Figure 11. Chemlcal ionization mass spectrum o f
acetohexarnide.
prominent ions under the chemical i o n i z a t i o n conditions

are presented i n Table 5.
Table 5: Mass spectral assignment o f acetohexamide

using chemical ionization.
M/e Species
365 [M t C3H5]+
353 [M t CzHs]+
325 [M t H (MH)1+
324 [MI+
3. SYNTHESIS
Marshall e t a7 ( 4 ) reported a method o f synthesis

o f acetohexamide which i n v o l v e s t h e r e a c t i o n o f t h e
diazonium s a l t from paminoacetophenone w i t h s u l f u r
dioxide t o a f f o r d the sulfonyl chloride which i s then
converted t o the sulfonamide by reaction w i t h a m n i a .
E l a b o r a t i o n v i a t h e carbamate w i t h cyclohexylamine
a f f o r d s acetohexamide. Another r e p o r t e d method ( 1 6 )
uses p-chloroacetophenone as t h e s t a r t i n g m a t e r i a l .
Both methods are o u t l i n e d i n Scheme 2.
ACETOHEXAMIDE 25
Scheme 1: Proposed mass fragmentation pattern o f

acetohexamide
n
0 0
W-Q-
I
O H
mle 3 2 4
+
0-H NH
I1
m/e 243
Scheme 1 Continued ...
mle183
m l e 324
I -CH,-CO
m /el41
mle 324 m l e 200
I
- YN0,S
0
C H3I; 0 -i
mle76 m l e 104 mle 119

ACETOHEXAMIDE 21
Scheme 1 Continued ...
1
[ O N H I +
m/e 324
i
0
II
2 68
+
28 ABDULLAH A . AL-BADR A N D HUMEIDA A. EL-OBEID
Scheme 3: Synthesis o f acetohexamide

Method 1 (4)
SO,-NH-C-NH
Method 2 (16)
0 0
CH,t@ S03Na p0c13*
c H 3 - ! G so, CI SO,NH,-
ACETOHEXAMIDE 29
4.1 T i t r i m e t r i c Methods
4.1.1 Nonaaueous
A non-aqueous t i t r a t i o n method f o r t h e d r u g and

other hypoglycemic and d i u r e t i c agents was reported by
Agarwal and Walash (17). The drug i n t a b l e t o r pure
form was d i s s o l v e d i n t e t r a m e t h y l urea and t i t r a t e d
w i t h 0.1 N l i t h i u m m e t h o x i d e i n benzene-methanol
medium. The end p o i n t was determined u s i n g 0.2% azo
v i o l e t i n toluene as i n d i c a t o r . Recovery ranged from
98.8% t o 101.6%.
A n o t h e r non-aqueous t i t r a t i o n p r o c e d u r e , f o r t h e
q u a n t i t a t i v e a n a l y s i s o f t h e d r u g and o t h e r
h y p o g l y c e m i c s u l f o n y l u r e a s u s i n g HC104 t i t r a t i o n
method, was a l s o reported (18).
4.1.2 Gravimetric
Amer and Walash (19, 20a) r e p o r t e d a method f o r

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 o f acetohexamide by
treatment w i t h 2,4-dinitrophenylhydrazine t o
p r e c i p i t a t e t h e h y d r a z o n e (19). A m i x t u r e o f
acetohexamide, tolbutamide and chlorpropamide was a l s o
determined g r a v i m e t r i c a l l y (20a).
4.1.3 Compleximetric
G u e r e l l o and Dobrecky (21) have d e s c r i b e d a

procedure f o r t h e compleximetric e v a l u a t i o n o f
medications w i t h hyoglycemic a c t i o n i n c l u d i n g
acetohexamide. The procedure permits the determination
o f t h e hypoglycemic sulphonylureas. A weighed amount o f
drug was h y d r o l y s e d by h e a t i n g f o r 30 minutes w i t h
d i l u t e aqueous sodium h y d r o x i d e and t h e s o l u t i o n
n e u t r a l i z e d w i t h 0.1 N HC1, t r e a t e d w i t h 0.1 M CuSO4,
then w i t h b u f f e r s o l u t i o n t o pH 6, and f i l t e r e d . The
excess C U + ~ i n t h e f i l t r a t e was d e t e r m i n e d by
complexometric t i t r a t i o n w i t h 0.02 M EDTA disodium s a l t
u s i n g 1-(2-pyridylazo)-2-naphthol as i n d i c a t o r . The
method i s applicable t o evaluate drugs i n t a b l e t .
30 ARDUI.1.AH A. AL-BADR AND HUMEIDA A. EL-OBEID
4.2 SDect romet r i c
4.2.1 Colorimetric
Reaction o f acetohexamide w i t h 2,4-dinitropheny’l-

hydrazine t o produce t h e colored hydrazone was used by
Amer and Walash (19) t o determine t h e drug c o l o r i m e t r i -
c a l l y . The c o l o r e d p r o d u c t was d i s s o l v e d i n KOH and
determined a t 480 nm. The accuracy o f the method was
claimed t o be 100%. A n i n h y d r i n c o l o r i m e t r i c method f o r
some o r a l hypoglycemic agents was a l s o reported (20b).
Meier e t a7 ( 2 2 ) analysed acetohexamide and o t h e r

h y p o g l y c e m i c a g e n t s by d i s s o l v i n g t h e d r u g i n
chloroform, adding calcium acetate (1% i n methanol),
propylamine (5% i n methanol), d i l u t i n g w i t h chloroform
and reading the absorbance a t 565 nm a f t e r 15 minutes.
Pharmaceutical preparations may be estimated s i m i l a r l y .
4.2.2 U l t r a v i o l e t (UV)
Solomonova and D v o r n i t s k a y a ( 2 3 ) d e t e r m i n e d
acetohexamide by measuring t h e absorbance a t 229 nm i n
ethanol or 0.1 M sodium hydroxide. Other UV t e s t s f o r
t h e drug are a l s o reported (24, 25).
4.2.3 Infrared (IR)
Acetohexamide and o t h e r s u l p h o n y l u r e a s were

analysed by IR (22). A t e s t have a l s o been described
(24). Lazaryan (26) determined t h e d r u g and o t h e r
h y p o g l y c e m i c a g e n t s by i n f r a - r e d a b s o r p t i o m e t r i c
determination. A sample i s t r e a t e d with chloroform and
t h e s o l u t i o n from t h e t a b l e t sample i s f i l t e r e d . A
p o r t i o n o f s o l u t i o n i s d i l u t e d w i t h chloroform and t h e
absorbance i s measured a t 1722 t o 1715 cm-1 i n 0.25 m
NaCl c e l l against chloroform.
4.2.4 F1uoromet r ic
G i r g i s - T a k l a and Chroneos ( 2 7 ) d e s c r i b e d a
s e n s i t i v e method f o r t h e f l u o r o m e t r i c determination o f
t h e d r u g i n plasma o r i n t a b l e t s by means of i t s
r e a c t i o n w i t h 1 - m e t h y l n l c o t l n a m i d e . The l i m i t o f
d e t e c t i o n was approximately 0.2 Mg o f t h e drug/mL and
t h e r e l a t i v e standard d e v i a t i o n was 31% f o r 2 Ng/ml i n
ACETOHEXAMIDE 31
plasma. The method i s s u i t a b l e f o r plasma samples

containing 0.5-2.5 Mg o f the drug/ml.
4.2.5 Proton Magnetic Resonance
Al-Badr and Ibrahim (28) described a simple, r a p i d

and accurate method f o r t h e assay o f the drug and other
hypoglycemic agents u s i n g p r o t o n magnetic resonance
spectrometry. The pure drug o r i n t a b l e t form, can be
determined using DMSO-ds as solvent and maleic a c i d as
i n t e r n a l standard.
The reported recovery i s 100 f 1.5% f o r pure drug and

98 t o 99.6 f 1.4% f o r t a b l e t s .
4.3 ChromatonraDhic Methods
4.3.1 Thin-Layer ChromatonraDhY (TLC]
Gergis-Takla and Josh1 (29) reported a TLC method

f o r the i d e n t i f i c a t i o n , assay and p u r i t y determination
o f t h e drug and other hypoglycemic agents i n powder and
i n t a b l e t f o r m u l a t i o n . The d r u g was d e t e c t e d by
d i s s o l v i n g powdered t a b l e t s o r powder i n
dichloromethane-acetone m i x t u r e (2: 1) and chromato-
graphing t h e s o l u t i o n on s i l i c a gel F 2 5 4 p l a t e s with
cyclohexane-chloroform-acetic a c i d and e t h a n o l
(10:7:2:1). For q u a n t i t a t i v e determination, t h e spots
were s e p a r a t e d , e l u t e d w i t h m e t h a n o l i c sodium
hydroxide, d i l u t e d w i t h m e t h a n o l i c HC1 and t h e
absorbance was measured.
Surborg and Roeder (30) have recommended c o n s t a n t -

b o i l i n g s o l v e n t m i x t u r e s f o r t h e development o f
chromatograms on s i l i c a gel f o r acetohexamide and o t h e r
a n t i d i a b e t i c drugs: propanol-cyclohexane (37:163),
propanol-benzene-cyclohexene (9:14:27), and cyclohexane
- isopropanol (177:23). The R f values o f t h e drugs a r e
t a b u l a t e d , s p o t s were l o c a t e d by v i e w i n g i n 254 nm
radiation.
4.3.2 Gas L i a u i d ChromatonraDhY (GLC)
Kleber e t a l . (31) determined acetohexamide and

hydroxyhexamide i n b i o l o g i c a l f l u i d s u s i n g GLC.
Tolbutamide was used as an i n t e r n a l standard and M-HC1
was added t o the sample o f plasma o r urine, the m i x t u r e
was shaken w i t h t o l u e n e and was c e n t r i f u g e d . The

separated organic phase was shaken w i t h 7.5% KzC03
s o l u t i o n and centrifuged again. The aqueous phase was
heated a t 6 0 " f o r 1 0 m i n u t e s w i t h methanol and
dimethylsulphate, cooled and M-acetate b u f f e r s o l u t i o n
was added t o a d j u s t t o pH 5.2. The m e t h y l a t e d
sulphonylureas were e x t r a c t e d w i t h hexane and t h e
e x t r a c t was evaporated t o dryness a t 50' i n a stream o f
nitrogen. The residue was dissolved i n CS2-CHC13 (l:l),
25 u l and 2 p l was submitted t o GLC on a glass column
(61 cm X 3 mn) containing 0.5% o f PEG 20 M on Gas-Chrm
Q (80 t o 100 mesh) and the temperature was programed
f o r 190 t o 240' a t 5 min-1, w i t h helium as c a r r i e r gas
(90 m l min-1) and flame i o n i s a t i o n d e t e c t i o n . Peak
heights were compared. A t concentrations o f 10 t o 40 ug
m l - 1 i n plasma. The mean recoveries (8 determination)
were : f o r acetohexamide 9.9 and 39.4 c(g m l - 1 ; f o r the
metabolite hydroxyhexamide 14.1 and 40 c(g m l - 1 .
Fricke (32) presented a GLC method f o r t h e analysis o f

t h e drug and o t h e r drugs i n pharmaceuticals, u s i n g
s i m p l e e x t r a c t i o n s and semiautomated g a s - l i q u i d
chromatography, using Ddxil 300 as the l i q u i d phase and
an automatic sample i n j e c t o r . Results by t h i s method
and t h e o f f i c i a l and o t h e r a p p l i c a b l e methods a r e
compared. Content uniformity analysis can be made by
u s i n g t h i s procedure. The e x t r a c t i o n and chromato-
graphic conditions were standardized t o make possible a
successful interlaboratory study.
4.3.3 Hinh-Performance L i a u i d ChrmatoqraDhy

( HPLCl
A simple HPLC assay o f t h e drug i n plasma was

developed by T a k a g i s h i e t a7 ( 3 3 ) . A sample was
extracted w i t h a mixture o f benzene and e t h y l acetate
a t pH 5 and t h e organic phase was evaporated. A 50%
s o l u t i o n i n CH3CN o f the residue was chromatographed
u s i n g a Lichrosorb RP-8 reversed-phase column and a
mobile phase composed o f 0.2% a c e t i c a c i d - methyl
c y a n i d e ( 1 : l ) . The m e t h o d c a n be u s e d f o r
b i o a v a i l a b i l i t y and c l i n i c a l pharmacokinetic studies o f
acetohexamide.
Beyer (34) used high speed l i q u i d chromatography f o r

analysis o f the drug and other a n t i d i a b e t i c agents. The
reocvery o f the drug from i n e r t t a b l e t ingredients by
ACETOHEX AM I DE 33
t h i s method was near 100%. A column (100 cm X 2 . 1 mm)

packed w i t h 1% o f ethylene-propene copolymer on Zipax
was used w i t h mobile phase o f 0.01 M disodium hydrogen
c i t r a t e containing 15% o f methanol (pH 4 . 4 ) . Detection
was c a r r i e d o u t a t 254 nm and pack a r e a s were
integrated.
Testosterone, chlorpropamide i n 95% ethanol were used

i n t e r n a l s t a n d a r d s . The p r o c e d u r e was a p p l i e d t o
compressed t a b l e t s , t h e powdered sample being e x t r a c t e d
w i t h t h e i n t e r n a l standard s o l u t i o n . Recoveries o f
added sulphonylurea were 98.9% t o 100.2%.
5. PHARMACOKINETICS
5.1 Introduction
Acetohexamide i s used as an o r a l a n t i d i a b e t i c
agent f o r t h e t r e a t m e n t o f k e t o a c i d o s i s - r e s i s t a n t
d i a b e t e s . I t i s an i n t e r m e d i a t e a c t i n g s u l f o n y l u r e a
d e r i v a t i v e . The c l i n i c a l e f f e c t s o f lowering elevated
blood glucose l e v e l s i s s i m i l a r f o r a l l o f t h e
sulfonylurea d e r i v a t i v e s . Acetohexamide, however, i s
t h e only one t o a l s o possess u r i c o s u r i c a c t i v i t y and
t h e r e f o r e i s a p r e f e r a b l e agent t o t r e a t d i a b e t i c
p a t i e n t s w i t h gout.
The d u r a t i o n o f a c t i o n o f acetohexamide (12-24 hours)

permits once o r t w i c e d a i l y dosage. The crossover study
o f Fox e t a7. (35) conducted i n 36 p a t i e n t s w i t h
m a t u r i t y onset diabetes m e l l i t u s i n d i c a t e d t h a t both
chlorpropamide and acetohexamide gave s i m i l a r responses
based on f a s t i n g blood sugar. Acetohexamide was used i n
a dose range o f 500-3,000 mg/day and i t i s i n d i c a t e d
t h a t primary f a i l u r e on acetohexamide i s more l i k e l y t o
respond t o chlorpropamide and v i c e versa. Appropriate
dosing r e q u i r e i n d i v i d u a l i z a t i o n o f therapy t i t r a t e d t o
t h e d e s i r e d t h e r a p e u t i c e f f e c t . The usual PO dosage
range i s 250-1500 mg/day i n s i n g l e o r d i v i d e d doses
(36,37), w i t h a maximum recommended dose o f 1500
mg/day. The 250 mg dose o f acetohexamide i s equivalent
t o 500 mg t o l b u t a m l d e , 100 mg tolazamide, o r 100 mg
chlorpropamide (36). The o r a l a n t i d i a b e t i c agents prove
more u s e f u l when d i e t a r y r e s t r i c t i o n and w e i g h t
reduction accompany t h e i r use.
34 ABDULLAH A. AL-EADK AND HUMEIDA A. EL-OBEID
Acetohexamide i s l a r g e l y metabolized t o an a c t i v e
metabolite which is excreted i n t h e u r i n e (see below).
Therefore, dosage adjustment o r t o t a l avoidance i s
necessary i n c e r t a i n cases. One such case i s t h e renal
f a i l u r e . Azotenic p a t i e n t s may experience prolonged
hypoglycemia. A t w i c e d a i l y dose i s recommended f o r
p a t i e n t s w i t h m i l d r e n a l f a i l u r e and p a t i e n t s w i t h
moderate t o severe renal f a i l u r e should not receive t h e
drug (38,39).
Dosage adjustment may a l s o be required i n p a t i e n t s with

1i v e r i n s u f f i c i e n c y since acetohexamide i s e x t e n s i v e l y
metabolized i n t h e l i v e r . Prolonged hypoglycemia may
r e s u l t i n p a t i e n t s w i t h severe l i v e r impairment (36).
Dosage r e d u c t i o n may be r e q u i r e d i n e l d e r l y o r
d e b i l i t a t e d p a t i e n t s , due t o renal o r l i v e r impairment
o r hyperresponsiveness (36).
I t i s recommended by Bennett e t a7. (39) t h a t no dosage

supplementatlon i s r e q u i r e d i n p a t i e n t s f o l l o w i n g
.
p e r i t o n e a l d i a1y s i s
L i k e other o r a l a n t i d i a b e t i c agents, acetohexamide may

be used i n combination w i t h i n s u l i n t o reduce i n s u l i n
r e q u i r e m e n t s i n i n s u l i n dependent m a t u r i t y o n s e t
d i a b e t i c s and t o r e d u c e t h e p o t e n t i a l f o r a
hypoglycemic reaction.
5.2 Mechanism o f Action
Acetohexamide i s a sulfonylurea d e r i v a t i v e , t h a t
produces i t s hypoglycemic e f f e c t by s t i m u l a t i n g t h e
i s l e t t i s s u e t o s y n t h e s i z e and r e l e a s e endogenous
i n s u l i n ( 4 0 ) . The h y p o g l y c e m i c e f f e c t s a r e a l s o
a t t r i b u t e d t o an increased s e n s i t i v i t y o f i n s u l i n
receptors as w e l l as improved peripheral u t i l i z a t i o n o f
i n s u l i n (37).
A r e p o r t by Lebowitz and Feinglos (41) i n d i c a t e s t h a t ,

d u r i n g chronic administration, p a r t o f t h e hypoglycemic
action o f the sulfonylureas i s e x t r a pancreatic.
Peripheral t i s s u e s may become more s e n s i t i v e t o a f i x e d
dose o f an a d m i n i s t e r e d hormone p o s s i b l y due t o an
increase i n the number o f i n s u l i n receptors.
A study on the mode o f a c t i o n o f t h e sulfonylureas (42)

has shown t h a t acetohexamide increased glucose uptake
ACETOHEXAMIDE 35
by r a t diaphragm, i n h i b i t e d the a c t i v i t y o f glucose-6-

phosphatase, triosephosphate isomerase and l i p o p r o t e i n
1ipase .
5.3 Onset and Duration o f Action
B r e i d a h l e t a 7 . ( 4 3 , 4 4 1 r e p o r t e d a peak
hypoglycemic e f f e c t t o occur between 8 t o 10 hour post
ingestion o f acetohexamide.
A duration o f action o f 12 t o 24 hours i s reported by

Breidahl et a7. (43,44) and Galloway et a7. (45) which
i s s i m i l a r t o t h a t o f tolazamide (up t o 24 hours), less
than t h a t o f chlorpropamide (60 hour) and greater than
t h a t o f tolbutamide (6 t o 12 hours) (37).
The serum c o n c e n t r a t i o n s i n d i a b e t i c p a t i e n t s
responding w e l l t o t h e drug had mean acetohexamide
l e v e l s o f 3.7 mg/dL w i t h a ragne f o 2.5 t o 4 . 9 mg/dL
f o l l o w i n g dosage regimens o f 0.5 t o 3 g/day (46). No
good c o r r e l a t i o n between b l o o d c o n c e n t r a t i o n s o f
acetohexamide and therapeutic e f f e c t i s established.
However, f a s t i n g blood glucose concentrations a r e
decreased i n a dose-dependent f a s h i o n i n t h e dosage
range between 250 mg t o 1,000 mg (47).
5.4 AbsorDtion
O r a l l y a d m i n i s t e r e d acetohexamide i s almost
completely absorbed (47). I t i s reported t o appear i n
the blood w i t h i n 30 minutes a f t e r PO administration and
peak l e v e l s occur a f t e r 3 t o 5 hours (43,44). Galloway
et a7. (45) reported t h a t , f o l l o w i n g single PO doses o f
1 g o f acetohexamide, mean peak blood l e v e l s o f t h e
drug t o be 47 mcg/ml and f o r hydroxyhexamide mean
l e v e l s o f 60.3 mcg/ml were achieved. These peak l e v e l s
occurred w i t h i n 1.5 t o 2 hours f o r the parent compound
versus 2 t o 6 hours f o r th e a c t i v e metabolite,
hydroxyhexamide.
5.5 Distribution
J u d i s ( 4 8 , 4 9 ) r e p o r t e d t h a t acetohexamide
extensively binds t o plasma proteins t o the extent o f
65 t o 90%.
5.6 Hetabol ism

A c e t o h e x a m i d e is mainly metabolized b y
hydroxylation reactions in the liver to inactive and
active metabolites. The primary metabolite (47 to 60%)
is hydroxyhexamide (47,501. It is an active metabolite
and is reported (45,50) to be excreted unchanged in the
urine, as well as metabolized to the inactive
dihydroxyhexamide (38).
Hydroxyhexamide, like acetohexamide, possesses both
hypoglycemic and uricosuric properties (51,52), but it
is 2.5 times as potent as its parent d r u g (36).
Impairment of hydroxyhexamide’s el imination has been
reported (51) to result in severe hypoglycemia.
Kojima et a7. (53) investigated the effect of various
drugs on the i n v i v o metabolic reduction of
acetohexamide. Most of the nonsteroidal anti-
inflammatory drugs inhibited the acetohexamide
reduction in liver, kidney and heart cytosol from
rabbits. Ketone-containing drugs including warfarin
also inhibited the reduction reaction in both the liver
and the kidney; in the heart, acetohexamide reduction
was inhibited only by warfarin.
Species differences in the i n v i t r o metabolic reduction
of acetohexamide were studied (54) in rabbit, guinea
pig, hamster, rat and mouse. The rabbit exhibited the
highest acetohexamide reductase activity in the cytosol
of the liver and kidney among the species tested. The
sensitivity to specific inhibitors of cytosolic
acetohexamide reductase in the liver and kidney of the
rabbit were different from those of the rat. Only rats
and guinea pigs showed significant activity of
acetohexamide reductase activity in the microsomes of
the liver and kidney.
Nagamine e t a7 (55) estimated the rates of available
fraction for 4-acetamidoacetophenone, 4-acetylbenzene-
sulfonamide, and acetohexamide and their respective
reduced compounds, 4-substituted a-hydroxyethylphenyl
derivatives, in rats. The study indicated that the
compounds are i n a reversible drug-metabolite
relationship. The pharmacokinetic profiles o f the
agents were studied after an intraportal administration
ACETOHEXAMIDE 37
i n comparison w i t h those a f t e r I . V . administration

using an interconversion model.
5.7 Excretion
Acetohexamide and i t s m e t a b o l i t e s a r e m a i n l y
e x c r e t e d b y t h e k i d n e y s . The u r i n a r y r e c o v e r y o f
radioactivity a f t e r the administration o f oral
14C-labeled acetohexamide averaged 71.6% i n 24 hours
(45). Approximatley one-half t o two-third o f t h e drug
was r e p o r t e d t o be excreted i n u r i n e as t h e a c t i v e
metabolite, hydroxyhexamide (45,501. Fecal excretion o f
r a d i o a c t i v i t y f o l l o w i n g o r a l administration o f t h e drug
i n one p a t i e n t was 15%. Even a f t e r 1 g I . V . dose
u r i n a r y recovery was o n l y 85% ( 4 5 1 , suggesting t h a t
b i l i a r y e x c r e t i o n represents a secondary r o u t e o f
e l i m i n a t i o n o f acetohexamide and/or i t s metabolites.
However, more data are needed t o confirm the occurrence
o f b l l i a r y excretion.
5.8 Half-Life
F o l l o w i n g o r a l a d m i n i s t r a t i o n o f 14C-labeled
acetohexamide t o human subjects, a mean blood h a l f - l i f e
o f t h e drug o f 1 . 6 hours was determined, using isotope
d i l u t i o n a n a l y s i s , w i t h a range o f 0 . 8 - 2 . 4 hours
(45,56).
F i e l d e t a l . ( 5 1 ) , however, reported a range o f 21-70

minutes averaging t o a value o f 55.8 minutes. The
combined h a l f - l l f e o f t h e parent compound and i t s
a c t i v e metabolite, hydroxyhexamide, i s reported t o be
5.3 hours (43-45). The h a l f - l i f e o f acetohexamide i s
reported be prolonged i n renal f a i l u r e ( 3 8 ) .
The a c t i v e m e t a b o l i t e , hydroxyhexamide i s r e p o r t e d
(45,561 t o have a mean h a l f - l i f e o f 5.3 hours with a
range o f 3.7-6.4 hours. The average value o f 5.3 hours
agrees w i t h t h e f i n d i n g o f F i e l d e t a l . ( 5 1 ) who
reported a range o f 3.2-7.6 hours.
The blood and u r i n e data reported by Galloway et a l .

(45) agree w i t h those reported by Scheldon et a l . (46)
and confirm the report by Smith e t a l . ( 5 6 ) , t h a t the
combined half-1 i f e o f acetohexamide and hydroxyhexamide
i s comparable w i t h t h a t o f tolbutamide.
ACKNOWLEGEMENT
The authors would l i k e t o thank M r . Tanvir A. B u t t

f o r t y p i n g t h i s manuscript.
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29, 640 (1977).
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28,
13. E. Graf, C. Beyer and 0. A b d a l l a h , ibid, a,131

(1982).
14. E. G r a f , C. Beyer and 0. A b d a l l a h , m, 28, 225

(1982).
ACETOHEXAMIDE 39
15. A.A. Kassem, A.M. F o u l i , S. Said and E. Shehata, B u l l .

Fac. Pharm. (Cairo Univ.), 19, 309 (1982).
16. Mfg. Chem., 34, 454, 467 (1963).
17. S u r a j P. Agarwal and Mohammed I.Wa ash; I n d i a n J.

Pharm., 3 4 ( 5 ) , 109-111 (1972).
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Farm., [Univ. Cent. Ecuador], El(16); 44 49 (1969).
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21. L i l o 0. Guerello and Jose Dobrecky; Rev. Asoc. Bioauim.

Argent. 33(178-1791, 185-8 (1968).
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Rev. Real. Acad. Cience. Exactas. Fis. Natur. Madrid,
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6 5 ( 3 ) , 653-674 (1971).
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(Kiev), 2, 80-82 (1977).
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385-386 (1967).
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Vlachopoulas and A. Lobenwein; S c i . Pharm., 38(3)
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(1980).
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(1235) 117-123 (1979).
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(1982).
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Anal. 1 ( 2 ) , 189-193 (1983).
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u,
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1162-1 167 (1972).
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Sakamoto; Yakusaku Zasshi, B ( 9 ) , 961-963 (1979).
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35. O.J. Fox e t a l , J. Med. Assoc. Alabama, 31, 1155

(1968).
36. Product Information: Acetohexamide, E l i L i l l y & Co.,

Indianapolis, 1N: 1983.
37. AMA Department o f Drugs: AMA Drug Evaluations, 4 t h ed.

American Medical Association, Chicago, I L , 1980.
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Med. Sci., 254, 608 (1967).
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Kidney Dis., 3, 155 (1983).
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Gillman’s The Pharmacological Basis o f TheraDeutics,
MacMillan & Co. New York. 1980.
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(1978).
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36 (1969).
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79 (1972).
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204 (1972).
45. J.A. Galloway, R.E. McMahon, H.W. Culp, F.J. Marshall

and E.C. Young , Diabetes, 16, 118 (1967).
ACETOHEXAMIDE 41
46. J. Scheldon, J. Anderson and L. Stoner, m, l4, 362

(1965).
47. R.E. Ferner and S. Chaplin, Clin. Pharmacokinet., 2,

379 (1987).
48. J. Judis, J. Pharm. Sci., 6 l , 89 (1972).
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50. R . E . McMahon, F . J . M a r s h a l l and H.W. Culp, J.

Pharmacol. EXD. Ther., 149, 272 (1965).
51. J.B. Field, M. Ohta, C. Boyle e t a l , N. Ensl. J. Med.,

277, 889 (1967).
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(1968).
53. Y. Kojima, Y. Imamura and M. Otagiri, Yakusaku Zasshi,

108(1), 66 (1988).
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Bull., 36(1), 4199 (1988).
55. S. Nagamine, T . Otawa, H. Nakae and S. Asada, M,

36(11), 4612 (1988).
56. D.L. Smith, T.J. Vecchio and A.A. Forist, Metabolism,

-
14, 229 (1965).
AMODIAQUINE HYDROCHLORIDE
Iqbal Ahmad and Tauqir Ahmad
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,

University of Karachi, Karachi-75270, Pakistan.
K. Usmanghani
Department of Pharmacognosy, Faculty of Pharmacy,

University of Karachi, Karachi-75270, Pakistan.
1. INTRODUCTION
2. DESCRIPTION
2.1 Name, Formula, Molecular Weight
2.2 Appearance, Color, Odor and Taste
2.3 Proprietary Names
3. SYNTHESIS
4. PHYSICAL PROPERTIES
4.1 Melting Point
4.2 Solubility
4.3 Completeness of Solution
4.4 Acidity
4.5 Water Content
4.6 Residue on Ignition
4.7 Chromatographic Purity
4.8 Ultraviolet Spectrum
4.9 Infrared Spectrum
4.10 Nuclear Magnetic Resonance Spectrum
4.11 Mass Spectrum
4.12 Complex Formation
5. QUALITATIVETESTS
5.1 Identification
5.2 Color Tests
5.3 Field Test
ANALYTICAL PROFILES OF DRUG SUBSTANCES Copyright G 1992 by Academic Press, Inc
AND EXClPlENTS - VOLUME 21 43 All rights of reproduction reserved in any form
44 IQBAL AHMAD, TAUQIR AHMAD. AND K . USMANGHANI
5.4 Impurity Test for 4-(7-Chloro-4-quinolylamino)phenol Hydrochloride

6.1 Titrimetric Analysis
6.2 Spectrophotometric Analysis
6.3 Fluorometric Analysis
6.4 Chromatographic Analysis
7. METABOLISM AND PHARMACOKINETICS
7.1 Metabolism
7.2 Pharmacokinetics
8. TOXICITY
ACKNOWLEDGEMENT
REFERENCES
1. INTRODUCTION
Amodiaquine is a congener of chloroquine and is employed for the

treatment of overt malarial attacks and for suppression. Although it is
more active than chloroquine both in vitro and in vivo against certain
strains of Plasmodium f’cipamm with decreased sensitivity to
chloroquine, amodiaquine is not recommended for routine use in the
treatment of such infections (1). It appears that phenolic hydroxyl is
essential to the activity of amodiaquine since the removal of this group
depresses, and its methylation completely destroys antibacterial activity
(2). Amodiaquine has been synthesized and patented under the name of
Camoquin by Parke, Davis and Company in 1949 (3). It is used medicinally
in the form of its dihydrochloride.
2. DESCRIPTION
2.1 Name, Formula, Molecular Weight
Amodiaquine hydrochloride is 4-(7-chloro-4-quinolylamino)-2-

(diethylaminomethyl) phenol dihydrochloride dihydrate (4).
AMODIAQUINE HYDROCHLORIDE 45
CaH2zCIN30,2HC1, 2H20 = 464.8
The CAS registry No. is 6398-98-7.
Official monographs for amodiaquine hydrochloride are given in

Argentinian (1966), British (1988), Brazilian (1977), Egyptian (1984),
French (1982), Indian (1985), International (1981) and United States
(1990) Pharmacopeias.
2.2 Appearance, Color, Odor and Taste
A yellow, odorless or almost odorless, crystalline powder with a bitter

taste (5).
2.3 Proprietary Names
CAMAQI, Camoquin, Flavoquine, Miaquin (6,7).
3. SYNTHESIS
Burckhaiter et al. (8) synthesized amodiaquine (111) in 1948 by

condensing 4,7-dichloroquinoline (I) with 4-amino-2-diethylamino-
methylphenol (11) in dilute hydrochloric acid (Figure 1). In a later method
(9), the alkylamino group was added as a last step.
,q+
46 IQBAL AHMAD. TAUQIR AHMAD, AND K. USMANGHANI
C1 H2N a c H 2 N ( q H d 2
OH
c1
II
I
~ ,@2N(c2H5)2
0". 2 HCl
dilute
10 hours
OH
111
Figure 1. Synthesis of Amodiaquine
The free base was recrystallized from absolute ethanol and converted
into the dihydrochloride by treating with hot concentrated hydrochloric
acid.
4.1 Melting Point

It melts at about 158OC (7).
4.2 Solubility
It is soluble in 22 parts of water and in 70 arts of ethanol (96%),
practically insoluble in chloroform and ether &).
4.3 Completeness of Solution

A solution of 200 mg in 10 ml of water is clear (10).
4.4 Acidity
The pH of a 2.0% w/v solution is 3.6 to 4.6 (4).
4.5 Water Content

Not less than 7.0% and not more than 9.0% (10)
4.6 Residue on Ignition

Not more than 0.2% (10)
4.7 Chromatographic Purity

Chromatographic purity of amodiaquine hydrochloride can be
examined on thin-layer plate coated with a 0.25 mm layer of silica gel G
using solvent system chloroform (saturated with ammonium hydroxide):
dehydrated alcohol (9: 1). Under short-wavelength ultraviolet light, the
chromatogram shows principal spot at about the same Rf value, and no
secondary spot, as obtained with the USP Amodiaquine Hydrochloride RS
(10).
4.8 Ultraviolet (UV) Spectrum

The ultraviolet spectra of amodiaquine and amodiaquine
hydrochloride have been reported by Sunshine (12) and Clarke (7)
respectively. The ultraviolet absorption characteristics are used for the
identification of these drugs (4,10,13). The absorption spectrum of
amodiaquine hydrochloride as a function of pH in the range 1-11.8 shows a
hypsochromic effect at 343 nm, a hyperchromic effect at 305 nm and the
isosbestic point at 323 rim (14). The effect of solvents and substitution on
the ultraviolet spectra of amodiaquine has been studied and the changes of
absorption bands E,K, and B discussed in detail (15).
The ultraviolet spectrum of amodiaquine hydrochloride in 0.1 M
hydrochloric acid was recorded on a Shimadzu 240 UV-Visible
spectrophotometer and is shown in Figure 2. The uv spectral data
reported for amodiaquine and amodiaquine hydrochloride are listed in
Table 1.
48 IQBAL AHMAD, TAUQIR AHMAD. AND K. USMANGHANI
1.50 !r
1
1
3 .0
WAVELENGTH Cnm’l
Figure 2. Ultravlofet Spectrum of Amodluquinc Hydrochloride
InO.l M HCI
AMODIAQUINE HYDKOCHLORIDE 49
Table 1
UV Spectral Data for Amodiaquine and Amodiaquine Hydrochloride

Compound Solvent Amax, nm A (l%,Icm) Molar Ref.
Absorptivity
Amodiaquine 0.1 M HCl 283 890 41370 12

237 530 24630
247 470 21850
Amodiaquine Water 224 6
hydrochloride 342 394- 18310-
410 19060
0.1 M HCI 343 366 17010 4
0.1 M HCl 223 11
237
343 366 17010
Aq. acid 237 600 27890 7
343
Aq. alkali 273 7
287
0.1 M HCl 223 836 38850
237 489 22710
342.5 369 17160
*Values determined by the authors.
4.9 Infrared (IR)Spectrum

The infrared spectrum of amodiaquine has been determined in KBr
disc (4). The principal peaks in the infrared spectrum of amodiaquine
hydrochloride (KBr disc) are reported at 1565, 815, 1535, 1255, 869, 847
cm" (7). Attenuated total reflectance infrared spectrum is used to detect
amodiaquine hydrochloride in the solid state as a layer of crystals on
adhesive tape. The method has been applied to the identification of the
drug in tablet formulations. Common excipients such as starch, and
lactose (absorption in the 1000 to 1200 cm-' region) do not interfere with
the method (16).
The infrared spectrum of amodiaquine hydrochloride as KBr disc was
obtained with a Shimadzu IR 460 Infrared spectrophotometer and is
shown in Figure 3. The assignments for characteristic bands are given in
Table 11.
, 5.a
.. 4.0 5 o 60 7.0 8.g 5.0 10 0 15.0 zoo
I I 1 I I I I 1 I :oo.o
0
0
L
m
80.0
0
0
0
I
I 60-0
0
0
0
ID
0
0
1
40 0
*
0
0
I
0
20.0
I
0.0
0
0
0
a
000
I
3000
I 2000 I
'1500 'I 00"
I
500
Wave number (em-')
Figure 3. Infrared Spectrum of Amodiaquine Hydrochloride (KBr disc).
Table I1
IR Spectral Assignments for Amodiaquine Hydrochloride

Frequency, cm-1 Assignment
3420 - NH stretching
3 170 - OH stretching
1615 C = C stretching (aromatic)
1585,1540,1505 C =C, C = N stretching
in disubstituted quinoline
1448 - CH2-N- deformation
1265,1207 C-OH stretching (aromatic)
1095 C-Cl stretching (aromatic)
852,840 isolated CH deformation
in disubstituted quinoline
4.10 Nuclear Magnetic Resonance (NMR) Spectrum
The 'H-NMR and 13C-NMR spectra of amodiaquine hydrochloride in

DMSO-d6 were determined at 300 MHz and 75.4 MHz respectively on a
Bruker AM-300 NMR spectrometer using tetramethylsilane as reference
standard. The 'H-NMR determinations included spin decoupling
experiments, 2D J-resolved and COSY-45 measurements (Figure 4-6).
The 13C-NMR spectra comprised DEFT and hetero-nuclear (C-H
correlation) measurements (Figure 7-9). The spectral assignments are
listed in Table 111.
h
d,
.I'
7 11-1'
L
I , . . :. ,. .
11 0 I0 ..,. ,...9 0 .. 8 0 , 1 0 P O
.. ..
: . . .. -* . .
. ...
4 PTL
-,rPPM
F l p r c I. Homonuclear Chemlul Shin Cormlaled tCoS)-Ul ~ I I - N S I R

Spectrum or Amodisqulnc 1i)dmchloridc
I .
I
w’
e
I
1
,
‘ I
. . ,. . .. - , ._.. * ‘Iv, h ?,
1 ..
, ,1, 1, , , , , , , ( 1 ” ’ , ’ , , , , , , , , r*~ , , , , , , , , ( , , , , , , , , , ,
-
a L --
“LPr2
I! 1 -3r . BIO 7.0 6,O 5 0 4.0 I 0 2 0 1 0
PAI
b u r r 6. Homonuclcar 211 J-Rcsolvcd N M R Spcclrum of Amodiaquinc It)drorhloridr..
CH: CHdCH2
14 1
l,,c.3tii
C.ll..ll
C.5
I ICI. cd‘
C.Y
C.2’
I I
-I--
11 1
C4
1 A L
Figure R. 75 M l l r "c-NMR Off Resonance Decoupled Spectrum ofAmodiaquine Hydrofhloride
t'
-==i I
t
7'1
-2
L.
'4
I
i
58 IQBAL AHMAD. TAUQIR AHMAD. AND K. USMANGHANI
Table I11
‘H- and 13C-NMR Chemical shifts and Coupling Constants for

Amodiaquine Hydrochloride
‘H-NMR coupling ”C-NMR
Chemical shift Proton constant Chemicalshift Carbon
(PP4 (J in Hz) (PPd
156.04 1
138.87 2
7.67 (1H,d) 3 25 l30.00 3
115.60 4
7.35 (lH,dd) 5 8.6’2.5 128.30 5
4.22 (lH,d) 6 8.6 116.85 6
4.22 (lH,s) 7 49.11 7
3.09 (4H,q) 9’10 6.7 46.19 9’10
1.27 (6H,t) 11’12 1.2 8.41 11’12
8.44 (lH,d) 2’ 7.0 142.91 2’

6.82 (1H’d) 3’ 7.0 100.41 3’
117.54 4’
8.% (lH,d) 5’ 9.1 126.19 5’
7.78 (lH,dd) 6‘ 9.1’2.1 127.04 6‘
138.15 T
8.17 (lH,d) 8’ 2.1 118.97 8’
154.85 9’
c“
127.92 10’
NH/OH
10.89
4.11 Mass Spectrum
The electron impact ionization spectrum of amodiaquine

hydrochloride obtained at 70 eV using a solid probe insertion is shown in
Figure 10. The spectrum was run on a Finnigan Mat 112s double focusing
mass spectrometer connected to a PDP 11/34 (DEC) computer system. It
shows a molecular ion peak M + at m/z 355. Since the molecule contains
one chlorine atom, M+ 2 peak appears at m/z 357. The proposed
fragmentation pattern and prominent ions are given in Table IV.
Figure 10. Electron Impact-Mass Spectrum of Amodiaquine Hydrochloride
60 IQBAL AHMAD. TAUQIR AHMAD. A N D K.USMANGHANI
Table 4
Proposed Fragmentation Pattern of Amodiaquine Hydrochloride
d z Relative intensity % Ion
355, 357 55.77, 16.83
NH
283 43.05
@CH2
OH
282 99.00
c'w
@CH
OH
I-
-
253 43.56
179 8.18
177 5.81
4.12 Complex Formation
Amodiaquine hydrochloride forms 1:l and 1:2 complexes with ferrous

sulphate. The infrared spectra indicate that amodiaquine hydrochloride is
bonded to iron via N and 0 and that water molecules are coordinated to
iron (17). It forms a 1:2 complex with silver nitrate in alcoholic solutions.
The average stability constant, log K, for the complex is 7.7 and A E is
about 10.8 kcal/mol. (18).
The formation of 1:l ion association complex between amodiaquine
and Fast Green FCF or Orange I1 dye has been reported (19).
5. QUALITATIVE TESTS
5.1 Identification (4)
5.1.1 Dissolve 0.1 g of amodiaquine hydrochloride in 10 ml of water and

add 2 ml of 2 M sodium hydroxide. Extract with two 20 ml quantities of
chloroform, wash the combined chloroform extracts with 5 ml of water, dry
with anhydrous sodium sulphate and evaporate to dryness. Dissolve the
residue in 2 ml of chloroform. The infrared absorption spectrum of the
resulting solution is concordant with the reference spectrum of
amodiaquine.
5.1.2 The light absorption in the range 240 to 360 nm of a 0.003% w/v
solution of amodiaquine hydrochloride in 0.1 M hydrochloric acid exhibits
a maximum only at 343 nm. The absorbance at 343 nm is about 1.1.
5.1.3 To 1 ml of a 2% w/v solution of amodiaquine hydrochloride add 0.5
ml of cobalt thiocyanate reagent. A green precipitate is produced.
5.1.4 Amodiaquine hydrochloride yields the reactions characteristic of
chlorides.
The identification tests of amodiaquine hydrochloride based on
comparison of infrared and ultraviolet absorption spectra, and reactions
of chloride are reported in USP (10).
5.2 Color Tests (7)
Amodiaquine hydrochloride gives a blue color with Folin-Ciocalteu

reagent. The Liebermann’s test yields a black color. An orange color is
62 IQBAL AHMAD. TAUQIR AHMAD, AND K. USMANGHANI
produced when amodiaquine hydrochloride is treated with Millon's

reagent.
5.3 Field Test (20)
Amodiaquine base is extracted from urine into amyl acetate

immediately after alkalinization. The addition of bromophenol blue in 5%
boric acid to the organic phase causes a green to blue coloring, depending
on the concentration of the drug. The sensitivity of the test is 0.8 mg%.
5.4 Impurity Test for 4-(7-Chloro-4-quinolylamino)phenol Hydrochloride
(4)-
Carry out thin-layer chromatography, using silica gel G as the coating
substance, spread in a layer about 0.5 mm thick, and a solvent system
chloroform: butan-2-one: diethylamine (50:40: 10). Apply separately to the
chromatoplate 5 pl of each of two solutions in methanol containing (1)
10.0% w/v of the substance being examined and (2) 10.0% w/v of
amodiaquine hydrochloride BPCRS and 0.020% w/v of 4-(7-chloro-
4-quinolylamino) phenol hydrochloride BPCRS. After development
remove the plate, heat it at 105' for 10 minutes, spray with a freshly
prepared mixture of equal volumes of a 10% w/v solution of iron (111)
chloride and a 1% w/v solution of potassium hexacyanoferrate (111) and
examine immediately. Any spot corresponding to 4-(7-chloro-4-
quinolylamino) phenol in the chromatogram obtained with solution (1) is
not more intense than the spot with lower Rfvalue in the chromatogram as
obtained with solution (2).
6.1 Titrimetric Analysis
6.1.1 Nonaqueous titration
The BP method (4) for the assay of amodiaquine hydrochloride as pure

drug and in dosage forms is based on nonaqueous titration. A 0.2 g
quantity of amodiaquine hydrochloride is dissolved in a suitable volume of
anhydrous glacial acetic acid, 7 ml of mercury (11) acetate solution is
added and the solution titrated with 0.1 M perchloric acid to a green end
point using 1-naphtholbenzoin solution as indicator. In dosage forms, a
quantity of the powdered material equivalent to about 0.2 g of

amodiaquine hydrochloride is dissolved in 30 ml of water and 5 ml of 2 M
sodium hydroxide is added. Amodiaquine base is extracted with three 30
ml quantities of chloroform, the combined chloroform extracts are washed
with 10 ml of water and evaporated to a volume of about 10 ml. To the
chloroform extracts, 40 ml of anhydrous glacial acetic acid is added and the
solution titrated with 0.1 M perchloric acid using 1-naphtholbenzoin
solution as indicator. Each ml of 0.1 M perchloric acid is equivalent to
0.02144 g of CaHzCIN30,2HCl.
Wu et d. (21) have described a simple, rapid and accurate method for
the nonaqueous titration of amodiaquine in dosage forms. A powdered
sample of 5 milliequivalent weight is dissolved in 7 ml of N hydrochloric
acid, made alkaline with 3 ml of 6 N sodium hydroxide, shaken with 30 ml
of chloroform for 10 minutes and with 1 g of tragacanth for another 2
minutes, filtered through adsorbent cotton, and titrated (20 ml) with 0.1 N
acetic perchloric acid to blue or green end point using crystal violet
solution as indicator. For pure chemicals, the digestion with acid and
alkali could be omitted. The results agree with those obtained by the
official method.
6.1.2 Titration with brominating agents
Amodiaquine can be determined in bulk and in dosage forms by a

titrimetric method based on reaction with 1,3-dibromo-5,5-
dimethylhydantoin or N-bromosuccinimide as the titrant. The mixture is
later treated with potassium iodide solution and the liberated iodine
titrated with sodium thiosulphate solution. The recovery is about 100%
(22).
A method for the determination of amodiaquine hydrochloride in
tablets by titration with N-bromosuccinimide has been developed (23).
The sample is dissolved in water, treated with an acetic acid solution of the
reagent and mixed with potassium iodide. The iodine released is titrated
with sodium thiosulphate solution. The relative standard deviation for the
titration is 2.12% and the recovery is 99.4 - 101.0%.
6.1.3 Titration with vanadium (V)
The determination of amodiaquine hydrochloride by oxidation with

ammonium metavanadate solution and back titration of the unconsumed
reagent with acidic iron (11) ammonium sulphate solution, using
64 IQBAL AHMAD, TAUQlR AHMAD. A V D K. USMANGHANI
N-phenylanthranilic acid as indicator has been reported (24). The

recovery of amodiaquine hydrochloride in the pure form and in
pharmaceutical preparations is 99.83% (standard deviation 0.49%) and
99.69% (standard deviation 0.78%) respectively. The method is of general
applicability and is quick and simple compared with the official methods.
6.2.1 Ultraviolet spectrophotometry
The USP assay (10) of amodiaquine hydrochloride in pure form and in

tablets involves ultraviolet spectrophotometric determination. A quantity
of the drug equivalent to about 300 mg is dissolved in dilute hydrochloric
acid (1:lOO) to obtain a concentration of about 15 pg/ml. The absorbance
of this solution, along with a solution of undried USP Amodiaquine
Hydrochloride RS in the same medium having a known concentration of
about 15 pg/ml, is determined at 342 nm using dilute hydrochloric acid
(1:lOO) as the blank. The quantity, in mg, of CmH22CIN30, 2HC1 in the
portion of amodiaquine hydrochloride taken is calculated by the formula
20C (Ad&), in which C is the concentration, in pg/ml, calculated on the
anhydrous basis, of USP Amodiaquine Hydrochloride RS in the standard
solution and AU and As are the absorbances of the solution of
amodiaquine hydrochloride and the standard solution respectively. The
same method is applied to the assay of amodiaquine hydrochloride in
tablets after extraction of the base into chloroform and then re-extraction
with dilute hydrochloric acid (1:lOO).
Amodiaquine and primaquine can be quantitatively separated by
selective precipitation with 4 N ammonium hydroxide, followed by
determination of the two compounds at 342 and 282 nm respectively. The
method is valid upto primaquine - amodiaquine ratio of 1:40. Recoveries
of 98.30 - 100.11% have been reported (25). The presence of higher
amounts of amodiaquine yields low results in respect of primaquine as on
precipitation with ammonium hydroxide, the primaquine is trapped into
the precipitate of amodiaquine (26).
Hassan et al. (27) have developed a method for the simultaneous
determination of amodiaquine - primaquine mixtures in dosage forms.
The drugs are extracted with 0.1 N hydrochloric acid and absorbance of
the mixture is measured at 342 and 282 nm. The concentration of each
compound is calculated by solving two simultaneous equations. Excellent
AMODlAQUlNE HYDROCHLORIDE 65
recoveries from authentic samples are obtained and the method is suitable
for routine analysis.
6.2.2 Colorimetry
Amodiaquine hydrochloride is determined colorimetricallyby complex

formation, in aqueous solution, with bromophenol blue, bromocresol
green, bromocresol purple, and methyl orange, respectively. The complex
with bromophenol blue has the highest molar absorptivity. Recoveries are
more than 98.6% for all complexes, and the absorbance is linear with
concentration in the range 1-11 pglml. The absorption maxima for the
complexes occur at 420 nm except for the bromocresol purple complex
which exhibits maximum at 415 nm. The various complexes are extracted
with chloroform and absorbance is measured at the respective maxima for
quantitative determination (28).
A simple, sensitive, and selective method for the determination of
amodiaquine hydrochloride in tablets has been developed. It is based on a
color reaction with chloramine-T in the pH range 7.4- 8.0. The chromogen
is extracted with chloroform and the absorbance is measured at 442 nm.
Beer’s law is obeyed in the concentration range 1-200 p g / l . The
coefficient of variation has been found to be 0.64% and the recovery
ranges between 100.3 and 102.5%. Chloroquine phosphate or primaquine
phosphate do not interfere with the method (29).
Amodiaquine reacts with cobalt and thiocyanate to yield stable ternary
complexes. These complexes are readily extractable in nitrobenzene to
give a greenish-blue color with maximum absorption at 625 nm that can be
used for quantitative determination. The mean recoveries for authentic
samples of amodiaquine hydrochloride are 100.81 & 1.77% (p = 0.05).
Alternatively, determination of the cobalt content of nitrobenzene extract
by atomic absorption spectroscopy provides an indirect method for the
determination of the drug with a mean recovery of 99.99 2 2.16%. Both
the methods have been successfully applied to the assay of the drug in
pharmaceutical preparations (30).
A colorimetric method for the determination of amodiaquine in tablets
or powders has been reported (31). The drug is dissolved in 0.1 N
hydrochloric acid, treated with acidic ammonium reineckate, the
precipitate dissolved in acetone, and the absorbance measured at 525 nm.
The results compare favourably with those obtained by the official
methods.
66 IQBAL AHMAD. TAUQlK AHMAD. AND K. USMANGHANI
Amodiaquine hydrochloride has been determined in tablets by

dissolving it in water and treating with an acetic acid solution of
N-bromosuccinimide. An orange-yellow color is produced, whose
absorbance is measured at 450 nm. Beer’s law is obeyed in the
concentration range 15-160 pglml. The relative standard deviation for the
method is 1.44%, and the recovery is 99.7-100.9% (23).
Amodiaquine hydrochloride tablets have been assayed by a method
based on the reaction of the drug with 2,3-dichloro-S, 6-dicyano-
p-benzoquinone and measurement of the absorbance at 460 nm. The
color attains its maximum intensity after five minutes and remains stable
for at least one hour. Beer’s law is valid in the concentration range 1-4
mg/100 ml, and the recovery is 99.9-102.6% (32). Another colorimetric
method for the determination of amodiaquine in tablets depends on its
reaction with chloranilic acid in aqueous solution and measurement of the
absorbance at 522 nm. The absorbance is linear over the concentration
range 0.04 -0.20 mdml, and the recovery is 99.9-101.3% (33).
A simple, rapid and sensitive method for the colorimetric
determination of amodiaquine in bulk and in pharmaceutical preparations
has been reported by Sastry et al. (34). It is based on the reaction of
amodiaquine with potassium dichromate at pH 1.1 in the presence of
sulphanilamide, and measurement of the absorbance of resulting solution
at 510 nm. The color is stable for twenty-four hours. Beer’s law is obeyed
in the concentration range 20-120pg/ml. The relative standard deviation of
the method is 0.94%, and the recovery is 99.0-101.0%. Chloroquine
present even in ten-fold excess does not interfere with the determination.
A highly sensitive method is based on the complexation of
amodiaquine with ammonium molybdate. The bound molybdenum is
converted into its thiocyanate, reduced, and the absorbance of the colored
solution measured at 465 nm. The Beer’s law limits, molar absorptivity
and Sandell’s sensitivity for the amodiaquine complex are 50-300pg/25 ml,
1.75 x lo4 M 1cm-l and 0.026 &cm2 / 0.001 absorbance unit, respectively.
Recovery ranges from 98-101%. The color obtained is stable for
twenty-four hours and common excipients do not interfere with the
method (35).
Amodiaquine forms a colored ion association complex with Fast Green
FCF or Orange I1 dye. The stoichiometric ratio of the drug-dye complex
has been shown to be 1:l. The method can be applied to the assay of
amodiaquine in bulk and in pharmaceutical preparations. Sulphur
containing drugs do not interfere with the determination (19).

6.3 Fluorometric Analysis
A fluorometric method for the determination of amodiaquine in

serum, plasma, or red cells has been reported (36). Amodiaquine is
extracted from alkalinized biological fluids, buffered, and heated to
produce a species with marked increase in fluorescence, which could be
measured. Standard curves prepared in serum and red cells are linear
between 50 and 3000 pgll. Reproducibility of the assay and recovery of
amodiaquine from serum and red cells is satisfactory. The specificity of
the assay and the nature of the induced fluorophor are not known.
6.4.1 Thin-layer chromatography (TLC)
Amodiaquine can be separated and identified on silica gel G plates

using a number of solvent systems. The spots are visualized under
short-wavelength ultraviolet light or by spraying with acidified
iodoplatinate solution. The following Rr values (Table V) have been
reported (37).
Table V
Solvent Systems for TLC of Amodiaquine
Adsorbent Solvent system Rr
Silica gel G F w dipped Methanol: ammonia 0.62

in 0.1 M KOH and dried (1rn1.5)
Cyclohexane :toluene : 0.08
diethylamine
(751510)
Cbloroform:methanol 0.40
( 91)
Acetone 0.37
The application of principal components analysis to the TLC behaviour

of a large number of basic drugs including amodiaquine has been studied
(38). A two-component model explains 77% of the total variance in four
68 IQBAL AHMAD, TAUQIR AHMAD. AND K. USMANCHANI
eluting mixtures. For the identification of unknowns, the method provides

a drastic reduction of the range of possibilities to a few drugs.
6.4.2 High-performance liquid chromatography (HPLC)
A variety of HPLC packing materials have been prepared and their

chromatographic properties evaluated for separating amodiaquine and
other basic drugs using a single mobile phase. The three most promising
packing materials are silica, a mercapto Pr modified silica and a Pr sulfonic
acid modification (39).
A simple and precise HPLC assay for quantitating amodiaquine in
tablets and biological fluids involves acid extraction of the drug from
tablets and chloroform extraction of its base from the biological fluids
after treatment with ammonia. A p-Bondapak Ph column is employed for
separation with a mobile phase comprising methanol : water: acetic acid
(25:25:1) (pH 2.3), using quinidine as the internal standard. The mean
recovery of the drug from tablets is 102.03%, while in the biological fluids,
it ranges from 85.2 to 104.6%. Interference from tablet excipients or
biological fluids is negligible (40).
A column liquid chromatographic method for the simultaneous
determination of chloroquine, amodiaquine and their monodesethyl
metabolites in human plasma, red blood cells, whole blood and urine has
been developed (41). The drugs and internal standards are extracted as
bases with dichloromethane and then re-extracted into an acidic aqueous
phase. Separation is achieved using a reversed-phase column and a mobile
phase of phosphate buffer (pH 3.0) : methyl cyanide (88:12). The
absorbance of the drugs is monitored at 340 nm with a sensitivity limit of
10 pmoVml. The mean overall recovery from each biological fluid is more
than 75%. This method can be applied to therapeutic, pharmacokinetic,
and epidemological studies.
7. METABOLISM AND PHARMACOKINETICS
7.1 Metabolism
Churchill et al. (42) have isolated four metabolites of amodiaquine in

humans using a reversed-phase HPLC method. The two major metabolites
have been identified as desethylamodiaquine and 2- hydroxy-
desethylamodiaquine. The importance of these metabolites in the

antimalarial effect of amodiquine in humans and on the in vitro sensitivity
of persons dosed with amodiaquine is discussed. 2-Hydroxy-
desethylamodiaquine has been isolated from urine and characterised by
HPLC and NMR spectroscopy. The presence of three additional
metabolites of this drug in humans has been suggested and
chromatographic confirmation for one of these obtained. The in vitro
activity of 2-hydroxydesethylamodiaquine is shown to be 1% that of
amodiaquine for two chloroquine sensitive Plasmodiumfdcipamm strains
(43). The metabolism of 2-amino-4- quinoline derivatives of chloroquine
and amodiaquine in humans has been compared by Pussard et al. (41).
7.2 Pharmacokinetics
Amodiaquine hydrochloride is readily absorbed from gastro-intestinal

tract after oral administration, and higher concentrations occur in
erythrocytes, kidney, liver, lungs and spleen than in the plasma. After
absorption it is slowly released into the blood and excreted in the urine for
at least seven days after a single dose. The rate of excretion is increased in
acid urine (5,7). Amodiaquine is altered rapidly in vivo to yield products
which appear to be excreted slowly, and thus have a prolonged suppressive
activity (44).
Following a single oral dose of 10 mgkg of amodiaquine to five human
subjects, serum concentrations of 0.30 to 0.68 pg/ml (mean 0.5) have been
reported after four hours; the ratio of erythrocyte to serum concentration
varies with time and between individuals, but erythrocyte concentrations
are generally higher than the serum concentrations after forty-eight hours
(36).
The metabolic transformation of amodiaquine to monodesethyl-
amodiaquine, and its pharmacokinetics in humans have been reported
(41).
8. TOXICITY
Amodiaquine hydrochloride is an antimalarial of low toxicity and is

three to four times as active as quinine as a suppressive drug against
Plasmodium vivav and Plasmodium fakiparum infections (44,45,46). Jn
therapeutic doses amodiaquine hydrochloride is generally well tolerated
but may occasionally give rise to side-effects, including nausea, vomiting,
70 IQBAL AHMAD. TAUQIR AHMAD. AND K . USMANCHANI
diarrhoea, insomnia, vertigo, and lethargy (5).

The prolonged use of amodiaquine hydrochloride in the dosages
necessary to treat lupus erythematosus and rheumatoid arthritis is not
recommended, for corneal opacities and retinopathy, peripheral
neuropathy, fatal blood dyscrasias, and fatal hepatitis have been reported
after these large dosages (47). Patients have experienced involuntary
movements, usually with speech difficulty, after large but not excessive
doses of amodiaquine (48). It may cause birth defects if taken during
pregnancy (49).
A method is described for evaluating the relative toxicity of
amodiaquine in rats on the basis of effect on growth, lethal effects,
production of pathological changes, and the concentration of drug in blood
or plasma. The test can be completed in fourteen days (50).
ACKNOWLEDGEMENT
The authors wish to thank the United States Pharmacopeial
Convention, Inc., for donating a sample of amodiaquine hydrochloride.
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12. Sunshine, I. (1981). "Handbook of Spectrophotometric Data of
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CLOFAZIMINE
Caitriona M . O’Driscoll and Owen I . Corrigan
University of Dublin
Department of Pharmaceutics
School of Pharmacy
Trinity College, Dublin, Ireland
ANALYTICAL PROFILES OF DRUG SUBSTANCES Copyright 0 1992 by Academic Press, Inc

AND EXClPlENTS - VOLUME 21 75 All rights of reproduction reserved in any form
76 CAITRIONA M. O'DRISCOLL AND OWEN 1. CORRIGAN
CLOFAZIMINE
Caitriona M. ODriscoll and Owen I. Corrigan
University of Dublin, Department of Pharmaceutics,

School of Pharmacy, Trinity College Dublin, Ireland.
1. Introduction
2. Description
2.1 Structural and Molecular Formulas and Molecular
Weight
2.2 Nomenclature
2.3 Official Compendia
2.4 Other Compendia
3. Synthesis
4, Physical Properties
4.1 Ultraiiolet Absorbance Spectrum
4.2 Infrared Absorbance Spectrum
4.3 Mass Spectrum
4.4 Proton Nuclear Magnetic Resonance Spectrum
4.5 Carbon-13 Nuclear Magnetic Resonance Spectrum
4.6 X-Ray Diffraction
4.7 Melting Point
4.8 Differential Scanning Calorimetry
4.9 Dissociation Constants
4.10 Solubilities
4.11 Par tition Coefficients
5. Methods of Analysis
5.1 Elemental Analysis
5.2 Identification
5.3 Ultraviolet and Visible Spectrophotometry
5.4 Spectrofluorometric Analysis
CLOFAZIMINE
5.5 Thin Layer Chromatography

5.6 High Pressure Liquid Chromatography.
6. Pharmacokinetics
6.1 Bioavailability Considerations
6.2 Distribution, Metabolism and Elimination
7. Pharmacology
7.1 Mechanisms of Action
7.2 Structure - Activity Relationships
7.3 Toxicity
7.4 Dose Schedules
Acknowledgements
References
1. INTRODUCTION
Clofazimine is active against Mycobacterium leprae and is used

clinically to treat leprosy (Hansen's disease). It was synthesised in
1957 by Barry et al., Laboratories of the Medical Research Council
of Ireland, Trinity College Dublin. The precise mechanism of the
antileprotic action of clofazimine has not been established. The
World Health Organisation classify clofazimine as an "essential
drug" and recommend its use, in combination, with other agents
to treat all cases of leprosy (WHO, 1982).
Clofazimine is also used to treat Mycobacteriurn avium
infections which frequently occur in patients with AIDS (acquired
immunodeficiency syndrome), (Masur et al., 1987; Woods and
Washington, 1987; Gangadharam et al., 1988; Lindholm - Levy
and Heifets, 1988; Young, 1988).
Clofazimine also displays anti inflammatory activity which is
clinically useful in controlling erythema nodosum leprosum
(ENL) reactions which occur in multibacillary forms of leprosy
(Gidoh and Tsutsumi, 1979; Yawalkar and Vischer, 1979; Browne
et al., 1981). A study using animal models of rheumatoid arthritis
has indicated that clofazimine may be potentially useful to treat
this disease (Currey and Fowler, 1972). Although the exact
mechanism of clofazimine mediated anti-inflammatory activity is
unknown, it may be related to the ability of the drug to increase
78 CAITRIONA M. O’DRISCOU AND OWEN 1. CORRlGAN
the synthesis of anti-inflammatory immunosuppressive

prostaglandin E2 (PGE2) by human polymorphonuclear leucocytes
(Anderson, 1985;Zeis et al., 1987;Yawalkar, 1988).
2. DESCRIPTION
Clofazimine is a dark red or orange - red fine powder, odourless

or almost odourless.
2.1 Structural and Molecular Formulas and Molecular Weight
Q CI
Molecular Formula: C27H22C12N4
Molecular Weight: 473.4

CLOFAZIMINE 19
2.2 Nomenclature
2.21 Generic Name
Clofazimine (BAN, USAN, rI")
2.22 Chemical Names
O-dihydrophenazin-2-
3-(4-chloroanilino)-10-(4-ehlorophenyl)-2,1
ylideneisopropylamine.
N,5-Bis(4-chlorophenyl)-3,5-dihydro-3-[(l-methylethyl)iminol-2-
phenazinamine,
or 3-(p-chloroanilino)-1O-(p-chlorophenyl)-2,10-dihydro-
2(isopropylimino) phenazine,
or 2-(4-chloroanilino)-3-isopropylimino-5-(4-chlorophenyl)-3,5-
dihydrophen azine,
or 2-p-chloroanilino-5-p-chlorophenyl-3,5-dihydro-3-
isopropy liminophenazine.
2.23 Trade name
Clofazimine is marketed by Ciba Geigy under the proprietary

name "Lamprene".
2.24 Other Names, Abbreviations and Drug Codes
Riminophenazine, 8663, G30320, NSC 141046, chemical abstracts

service registry number (CAS no.) 2030 - 63-9.
2.3 Official Compendia
A monograph on clofazimine is included in the British

Pharmacoepia and the Indian Pharmacoepia.
2.4 Other Compendia
Clofazimine is included in the Merck Index (19891, the

Pharmaceutical Codex (1979), and in Martindale (1989). Clarke
(1986) gives a useful summary of physical and chemical data.
80 CAITRIONA M. O'DRISCOU AND OWEN 1. CORRIGAN
NHz
+
NHR
i, ii ~
NHR
-
iii
(3)
NHR
('1 R = aryl
1
R = Ph, 4-CI-C6H4-
iv
I V
Reagents: i, FeCl3, H+; ii, NH3; iii, R*NH2, alkyamines;

iv, benzoquinone/carbonyl compound RkOR3; v, Pt @/H or Pt/C (lO%)/H2;
vi, air; vii, Pd/C (lO%)/Hz.
Figure 1. Principal synthetic routes to riminophenazines (Hooper,

1987)
3. SYNTHESIS
The original synthetic routes to riminophenazines (Barry et al.,

1956a; 1956b; 1957 and 1958 ) have been modified (O'Sullivan,
1984) to give reproducible high yields. The modifications have
been summarised by Hooper (1987) (Figure I) as follows; N-aryl
ortho -phenylenediamines (1) undergo regiospecific oxidative
dimerization to yield the parent iminophenazines (2) which react
further with alkylamines to give substituted iminophenazines (3).
Alternatively, oxidation with benzoquinone in the presence of a
CLOFAZIMINE 81
carbonyl compound gives an imidazolophenazine (4) which may

be reduced with cleavage of the imino substituent (5) followed by
subsequent aerial oxidation to the parent iminophenazine (2). A
more selective reduction results in an alternative cleavage of the
imidazoline ring (6) which after oxidation gives a substituted
iminophenazine (7). The type of catalyst used in the reduction of
these compounds is crucial and allows full control of the reactions.
4.1 Ultraviolet Absorbance Spectrum
The ultraviolet spectrum of clofazimine (0.001% w/v) is shown

in Figure 2. The spectrum was obtained using a Hewlet Packard
845 2A diode array UV visible spectrophotometer and 1 em quartz
cells. The spectrum, in the range 230 to 600nm, in 0.01m
methanolic hydrochloric acid, exhibits two maxima, at 284nm and
486nm. The absorbance at 284nm is about 1.30 and at 486nm is
about 0.64.
I I I 1
220 300 400 500 600
WAVELENGTH
Figure 2. Ultraviolet spectrum of clofazimine.

82 CAlTRlONA M. O’DRISCOLL AND OWEN I. CORRIGAN
4.2 Infrared Absorbance Spectrum
The infrared absorbance spectrum of clofazimine is shown in

Figure 3. The spectrum was recorded with a Nicolet 5ZDX FT-IR
spectrophotometer, from a compressed potassium bromide disc.
Structural assignments for some of the characteristic absorption
bands in the spectrum are listed in Table I.
Table I. Infrared assignments for clofazimine
W avenumber (cm-l) Assignment
1587,1560,1510,1460,1300 aromatic CH stretching

1389,1360,1130 CH(CH3)2 stretching
4.3 Mass Spectrum
The mass spectrum of clofazimine, shown in Figure 4, was

obtained using a Finnigan Quadrupole mass spectrometer, by
electron - impact at 70 electron volts. The molecular ion (M-H) at
m / z 473 was observed. Major peaks were detected at m / z (%) 474
(66.17),473 (36.22), 472 (1001,457 (93.04), 455 (70.87), 456 (24.13),431
(19.57), 414 (30.43), 380 (22.17), 345 (17.83), 331 (30.43).
4.4 Proton Nuclear Magnetic Resonance Spectrum (IH-NMR)
The IH-NMR spectrum of clofazimine, shown in Figure 5, was

obtained in deuterated chloroform containing tetramethylsilane
(TMS) as internal standard, using a Joel GX- 270 MHz instrument.
A 2D COSY spectrum was also obtained (Figure 6a and 6b). Figure
6b is an expansion showing coupling in the aromatic regions.
4.5 Carbon - 13 Nuclear Magnetic Resonance Spectrum
The carbon-13 NMR spectrum of clofazimine was obtained in

deuterated chloroform containing TMS as internal standard using
a Joel GX-270 MHz instrument at a frequency of 67 MHz. The
carbon-13 NMR spectrum, with DEPT, is shown in Figure 7.
CLOFAZIMINE 83
Figure 3. Infrared spectrum of clofazimine.
0 . . .
2000
: .
I BOO
. . : .
1so0
. . : .
1400
. . : .
1200
. . : .
1000
. . : . .
800
- : . . .
SO0
I
400
W ~ v m u n b r r Cpm-1)
84 CAlTRlONA M . O'DRISCOLL AND OWEN 1. CORRIGAN
Figure 4. Electron-impact mass spectrum of clofazimine.

CLOFAZIMlNE 85
Figure 5 . Proton nuclear magnetic resonance spectrum of

clofazimine.
86 CAITRIONA M. O'DRISCOLL AND OWEN I. CORRICAN
Figure 6a. 2-D proton nuclear magnetic resonance spectrum of

clofazimine.
CLOFAZIMINE 87
Figure 6b. 2-D proton nuclear magnetic resonance spectrum of

clofazimine.
88 CAITRIONA M. O'DRISCOLL AND OWEN I. CORRIGAN
Figure 7. l3c1 nuclear magnetic resonance spectrum of clofazimine,

with DEPT.
CLOFAZIMINE 89
4.6 X-Ray Diffraction
The powder X-ray diffraction pattern of clofazimine was

obtained on a Siemens D-500 X-ray diffractometer, using a Cu
X-ray tube, at 40 kV and 40 mA. The diffraction pattern is shown
in Figure 8, indicating the crystalline nature of clofazimine.
Rychlewska et al. (1985) reported two different crystalline forms
of clofazimine, a monoclinic form with a density of 1.3 g cm-3, and
a triclinic modification with a density of 1.29 g cm-3. The former
was prepared by recrystallization from acetone, and the latter by
recrystallization from 12 N-methylformamide/acetone. Cell
constants were also calculated. The values obtained for the
monoclinic form were a = 7.788 A, b = 22.960 A, c = 13.362 A, p =
98.580. The values for the triclinic form were a = 10.507 A, b =
12.852 A, c = 9.601 A, a = 95.960, p = 97.220, y = 69.730.
4.7 Melting Point
Melting points reported in the literature are in the temperature

range of 210 - 215OC, with degradation (Barry et al. 1956a; Clarke
1986; Merck Index 1989; Pharmaceutical Codex 1979).
4.8 Differential Scanning Calorimetry (DSC)
The DSC thermogram of clofazimine obtained using a Mettler

DSC 20, scan speed lOOC min-1, is shown in Figure 9. A single
sharp melting endotherm was obtained with onset temperature at
214OC. This value is in good agreement with the melting points
previously published (Section 4.7). The estimate of the heat of
fusion (AH) was 740 joules/gram. However, with some samples
there was evidence of degradation on melting.
4.9 Dissociation Constant
The values for the dissociation constant reported for

clofazimine are summarised in Table 11.
YO CAlTRlONA M . O'DRISCOLL AND OWEN 1. CORRIGAN
Figure 8. X-ray powder diffraction pattern of clofazimine.
TWO - THETA IOEGREESI

CLOFAZIMINE 91
Figure 9. Differential scanning calorimetry thermogram of

clofazimine.
200.0- I 2 0 . 0 0 0 nU
- I
-
-
2lO.O-
-
-
-
220.0-
-
--
-
230.0-
-
-
-
-
240.0-
--
-
-
Table 11. Dissociation constant of clofazimine (pKa)
PKa Method of determination Reference
8.35 Not stated Morrison a n d Marley

(1976a)
8.37 Potentiometric Canavan et al. (1986)
8.51 Spectropho tome tric Fahelelbom et al. (1989)
4.10 Solubilities
Clofazimine is practically insoluble in water, estimates in the

range of 1.03 - 0.49 pg ml-1, at 37OC, have been reported
(Fahelelbom, 1989; OReilly, 1991). It is soluble 1 in 700 of ethanol,
1 in 15 of chloroform, and 1 in 1000 of ether. It is also soluble in
dilute acetic acid and dimethylformamide (Clarke, 1986).
U I I I I
5 6 7 8
PH
Figure 10. pH solubility profile of clofazimine.
CLOFAZIMINE 93
The effect of pH (range 5.15 - 7.8) on the solubility of

clofazimine, shown in Figure 10 (OReilly, 19911, is consistent
with the basic nature of the compound. The solubility of the drug
is 5.68 and 0.278 mg ml-1 x 10 -3 at pH 5.15 and 7.8 respectively.
Values for intrinsic solubility in the range of 2.0 - 2.3 x 10-5mg
ml-1 (Fahelelbom, 1989; OReilly, 1991) have been reported.
The solubility of clofazimine was enhanced in aqueous micellar
systems, containing both naturally occuring surfactants e.g bile
salts, and synthetic surfactants, e.g the non ionic Cremophor EL
and Triton X100, and the anionic sodium dodecyl sulphate. The
incorporation of fatty acids to form mixed micelles brought about a
further enhancement in drug solubility in the case of naturally
occuring surfactants (approximately 300 fold with sodium cholate:
linoleic acid relative to buffer). In contrast, with synthetic
surfactants this enhancement decreased (Fahelelbom et al., 1991;
ODriscoll et al., 1991).
4.11 Partition Coefficients (Log P)
Partition coefficients for clofazimine have been determined

using different solvents and temperatures. The data is
summarised in Table 111.
Table 111. Partition coefficients of clofazimine
Solvents Temp (OC) LogP Reference
Octanol: water - +7.48* Morrison and

Marley
(1976a,b)
Isooctane: buffer pH 5.15 20 5.01 Canavan et al.
(1986)
N-octanol: buffer pH 5.15 20 4.30 Quigley
et al. (1990)
N-octanol: buffer pH 5.15 37 4.40 Ibid
* Estimated
94 CAITRIONA M.O'DRISCOLL AND OWEN 1. CORRIGAN
% Calculated % Found
Carbon 68.50 68.68
Hydrogen 4.68 4.52
Nitrogen 11.83 11.48
Chlorine 14.98 15.32
5.2 Identification
The B.P. (1988) outlines three methods of identification:

(A) By the infrared absorption spectrum, outlined in section 4.2.
(B) The light absorption, the UV spectrophotometry is described
in section 4.1.
(C) A colour test, dissolve 2mg clofazimine in 3ml of acetone
and add O.lml of hydrochloric acid, an intense violet colour is
produced. Add 0.5ml of 5M sodium hydroxide, the colour changes
to orange - red.
5.3 Ultraviolet and Visible Spectrophotometry
Quantitative ultraviolet analysis of clofazimine has been

performed, in a range of aqueous and nonaqueous media, at
280nm (Canavan et al., 1986; O'DriscolI et al., 1990a,b), and
colorimetrically at 482nm (Quigley et al., 1990).
A colormetric assay was developed by Barry et al. (1960) and
modified by Mansfield (1974) to analyse plasma and tissue levels of
clofazimine. The drug was extracted using benzene and
concentrated hydrochloric acid, and the absorption read at 540nm.
The limit of detection reported was 0.2pg/ml in plasma and
O.lmg/gram in tissue.
5.4 Spectrofluorometric Analysis
A fluorescent derivative of clofazimine was formed following

reduction with titanous chloride (Dill et al., 1970). The
fluorescence was measured at 366mp emission. The limits of
CLOFAZIMINE 95
detection reported for this method were in the range of 0.1 - 0.2
pg/ml in plasma (Banerjee et al., 1974; Levy, 1974).
A thin layer chromatographic (TLC) system suitable for

determination of clofazimine in plasma has been developed
(Lanyi and Dubois, 1982). The plasma samples were acidified
using acetate buffer pH 5 and extracted with toluene, evaporated to
dryness under nitrogen, reconstituted in toluene and applied to
the TLC plate. The adsorbent used was HPTLC silica gel 60. The
plates were developed in toluene - acetic acid - water (50 : 50 : 4),
allowed to stand for 30 min at room temperature, the Rf value of
clofazimine was 0.36. Detection and quantitation is carried out
using a densitometric method. The limit of detection reported for
this method was 5ng/g.
5.6 High Performance Liquid Chromatography
Gidoh et al. (1981) developed a high performance liquid

chromatographic (HPLC) method with ultraviolet detection to
separate and quantify clofazimine (287nm) from other antileprosy
drugs, dapsone and rifampicin, in serum on a pBondapak c18
column. This method involved a complicated extraction
procedure with the switching of 2 different mobile phase (i.e
acetonitrile - water, 20 : 80; and tetrahydrofluran - water containing
PIC B-5,50 : 50, the latter reagent contains 1 - pentanesulfonic acid
and glacial acetic acid) in order to allow complete resolution of
clofazimine from related components. The limit of detection for
this method was long ml-1. Recently a modification of this
technique was used to study clofazimine and its derivatives
(O'Sullivan et al., 1990).
Another HPLC method, was described by Peters et al. (1982), for
measuring clofazimine in plasma, with a limit sensitivity of 10 ng
ml-1. This method involved extraction of clofazimine into
organic solvents and quantifation on a reversed-phase Ultrasphere
- octyl column, using a mobile phase of 0.0425M phosphoric acid
in 81% methanol and UV detection at 285nm.
The gastrintestinal absorption of clofazimine, using a rat gut
perfusion technique, was determined by HPLC (O'Driscoll et al.,
1990a,b). The column used was Partisil lOPAC, the mobile phase
was ethanol : N-heptane (50 : 50) and detection was by UV at

283nm.The limit of sensitivity was 0.1pg ml-I.
6 . PHARMACOKINETICS
6.1 Bioavailability Considerations
Clofazimine absorption following oral administration is

incomplete and varies significantly from patient to patient.
Following administration as coarse crystals only about 20% is
absorbed, if however, the drug is given as a microcrystalline
suspension in an oil wax base an absorption rate of 70% can be
achieved (Yawalkar and Vischer, 1979).
The gastrointestinal absorption of clofazimine in the
anaestheised rat, using an in situ rat gut perfusion model (Komiya
et al., 19801, was enhanced by co-administration of simple and
mixed micellar systems (O'Reilly et al., 1988; O'Driscoll et al.,
1990a,b). The simple micellar systems included various bile salts,
and the synthetic emulgents, Cremophor EL (non ionic) and
sodium dodecyl sulphate (anionic). The mixed micelles were
formulated by the incorporation of various fatty acids. A mixed
micellar system containing sodium cholate: linoeleic acid
enhanced the rate of absorption of clofazimine by a factor of 840
compared to a buffered solution of the drug. The enhancements
were due to a combination of increased solubility and increased
membrane permeability. There is also evidence that clofazimine is
transported in part via the lymphatic system (Barry et al., 1960;
Atkinson et al., 1967).
Clofazimine has a reported pKa of 8.35 and consequently it is
highly ionised under physiological conditions. This high degree
of ionization, together with its high molecular weight, may be
significant factors in the poor oral bioavailability.
Schaad - Lanyi et al. (1987) studied the pharmacokinetics of
single oral doses of clofazimine over 11 days following
administration. They examined the effect of food on the
bioavailability. Following administration with food the area
under the plasma concentration versus time curve (AUC) and the
peak plasma concentration C, were 62 and 30% higher
respectively compared to results obtained in the fasted state. The
CLOFAZIMINE 97
median time (tmx) to reach Cmaxwas 8 hours with food and 12

hours without food.
6.2 Distribution, Metabolism and Elimination
Plasma levels of the drug are approximately 0.5mg 1-1 but

increase with the dose and at 300mg daily levels of 1.0 - 1.5 mg 1-1
have been achieved (Banerjee et al., 1974;Levy,1974).
Administration of 50mg of clofazimine daily for 8 days did not
achieve steady state (Schaad - Lanyi et al., 1987). The time to reach
steady state has been theoretically estimated to be in the range of 30
- 70 days (Schaad - Lanyi et al., 1987; Holdiness, 1989). There is no
data available on loading doses. Likewise, there is no information
currently available on the pharmacokinetics of clofazimine
following intravenous administration.
The appearance of clofazimine in the plasma following
absorption is short lived (Banerjee et al., 19741,it rapidly passed
out of the circulation and is deposited in various tissues and
organs, particularly the fatty tissue, the spleen, lymph nodes, and
the cells of the reticulo - endothelial system. Concentrations of 2.1-
5.3 mg g-1 have been reported in the subcutaneous fat (Mansfield,
1974),and 0.6-1.0mg g1 in the spleen (Desikan and Balakrishnan,
1976;Mansfield, 1974). It is taken up by the macrophages
throughout the body (Conalty et al., 1971;Yawalkar and Vischer,
1979). Electrophoretic studies of serum from orally treated mice
have shown almost complete binding of clofazimine to the
lipoproteins of the a and globulin fractions, these lipoprotein are
then phagocytosed by the macrophages (Conalty et al., 1971).
Clofazimine crystals have been found at autopsy in the small
intestine and in the macrophages of mesenteric lymph nodes
(Conalty et al., 1971;Aplin and McDougall, 1975;Desikan and
Balakrishnan, 1976;Jopling, 1976).Clofazimine does not appear to
cross the intact blood-brain barrier (Mansfield, 1974;Desikan and
Balakrishnan, 1976). It does, however, appear to cross the placenta
causing pigmentation of the foetus (Holdiness, 1989). There is no
data available on the volume of distribution of clofazimine.
Feng et al. (1981;1982)have used mass, ultraviolet and visible
spectrometry to identify three metabolites in the urine of leprosy
patients (Figure 11). Metabolite I is the unconjugated compound 3
(p-hydroxyanilino)-lO-(p-chlorophenyl)-2,lO-dihydro-2-
98 CAITRIONA M. O’DRISCOLL AND OWEN 1. CORRIGAN
isopropyliminophenazine, the other two metabolites are

conjugated, metabolite 11 is 3-(P-D-glucopyransiduronic acid)-lO-(p-
chloropheny1)-2, 10-dihydro-2- isoproyliminophenazine), and
metabolite 111 is 3 - (p-chlorani1ino)-10- (p-chlorophenyl) - 4, 10-
dihydro - 4 (PD-glucopyranosiduronic acid) -2-
isopropyliminophenazine. Metabolite I is reported to be formed
by a hydrolytic dehalogenation reaction, metabolite 11by hydrolytic
deamination followed by glucuronidation, and metabolite III by
hydration followed by glucuronidation.
Following administration of 300mg/day of clofazimine, 0.2% of
metabolite I, 0.25% of metabolite 11, and 0.2% of metabolite I11 were
recovered in the urine over 24 hours (Feng et al., 1981; 1982). No
information is available on the pharmacological activity of these
metabolites, or whether they are found in faeces or bile. The
authors have shown that metabolite I11 may be produced in the
laboratory through metabolism by liver enzymes. However, they
were unable to demonstrate the same hepatic conversion of
clofazamine to metabolites I and 11. In contrast, they suggest that
these metabolites are produced by bacterial degradation in the
intestine prior to absorption and urinary excretion.
Clofazimine accumulates in certain tissues throughout the body
(fatty tissue, skin, lymph nodes, macrophages etc.) and is
eliminated very slowly. The kinetics of the drug has been
described by both one and two compartment models. Data
obtained with relatively low dose, short term administration
indicated a one compartment model, with a a plasma tipof
approximately 7 days (Levy, 1974; Hastings et al., 1976; Holdiness,
1989). A second compartment is evident with long term, high dose
administration and appears to have a ttp of at least 70 days
(Banerjee et al., 1974; Levy, 1974).
Following oral administration of 50mg/day of clofazimine to
health volunteers Schaad - Lanyi et al. (1987) predicted that steady
state (SS) plasma concentrations would occur after approximately
30 days. They calculated an accumulation factor for the drug from
the ratio of AUCss: AUC. A value of 4.85 was obtained suggesting
a slow accumulation towards steady state. The authors suggest
that this may be avoided by administering higher loading doses,
followed by daily maintenance doses.
CLOFAZIMINE 99
CI
N.CH(CH,),
Metabolite I
1. Hydrolytic
N.CH(CH,), - - -deamination
-- -- ---
2. Glucuronation
Clofazimine
Metabolite II
Metabolite 111
Figure 11. Metabolic pathways of clofazimine in humans (Feng et

al., 1981; 19821)
Up to 50% of a dose of clofazimine is excreted unchanged in the

faeces, indicative of poor oral absorption (Banerjee et al., 1974).
However, high concentrations of the drug have been found in bile
and in the gall bladder. This suggests that part of the ingested drug
recovered from the faeces may represent excretion by means of the
bile rather than simply the failure of absorption from the
gastrointestinal tract (Mansfield, 1974). Urinary excretion in
leprosy patients is negligable accounting for an average of 0.1%
I00 CAITRIONA M. O'DRISCOLL AND OWEN I. CORRIGAN
(range 0.01 - 0.43%)of the dose in 24 hours (Levy, 1974). A small

amount of the drug is excreted in the sebum and sweat (Vischer,
1969).
7. PHARMACOLOGY
7.1 Mechanisms of Action
Although the precise mechanism of the antileprotic activity of

clofazimine has yet to be determined several explanations have
been proposed (Hooper 1987).
(a) The drug has been shown to bind to cytosine - guanine DNA
base pairs in vitro (Morrison and Marley, 1976a,b). The binding is
specific for guanine residues only. The DNA of M. Zeprue has a
high guanine - cytosine content, consequently this binding may
disrupt the template function of the DNA, causing inhibition of
protein synthesis.
(b) The redox properties of clofazimine can divert up to 20% of
cellular oxygen (Barry et al., 1957) and thus disrupt normal
mitochondria1 oxidation processes (Rhodes and Wilkie, 1973). In
addition, it has been suggested that cytotoxic oxygen species,
hydrogen peroxide and superoxide, are generated as a result of the
presence of the drug (Hooper and Purohit, 1983; Savage et al.,
1989). If such a reaction occurred within the macrophages it will
enhance the killing of the bacilli which are also found inside the
macrophages.
(c) In addition, it has been suggested that the antileprotic effect
of clofazimine may be due to its action on the macrophage
lysosomal apparatus (Sarracent and Finlay, 1984).
7.2 Structure Activity Relationships (SAR)
The earlier SAR studies, reviewed by Hooper and Purohit (1983),

concentrated on three main areas of molecular modification
(Figure 12). Firstly, the structure of clofazimine was varied by
introducing additional chlorine atoms at positions 4, 7,8 and 9.
This resulted in loss of activity, except for the 7- chloro derivative,
with was equipotent with clofazimine. The second series was
CLOFAZIMINE 101
based on triaryl derivative. A variety of derivatives with a chloro-

or methoxy substituent in various positions showed only modest
activity. The third series involved variations at R2 coupled with
changes at R1 and R3, and the introduction of various substituents
at positions 7 and 9. In general for optimum activity R2 had to be
alkyl or cycloalkyl, and R1/R3 aryl or substituted aryl. When
hydrophilic salt forming groups were introduced at R2 activity was
greatly reduced.
R’
9 1 1
Figure 12. Basic structure of iminophenazines
An X-ray crystallographic study (Rychlewska et al., 1985)

described the crystal and molecular structures of two crystal forms
of clofazimine and of its inactive isomer, isoclofazimine (B3857).
The geometric differences between clofazimine and isoclofazimine
were probed by CND0/2 molecular orbital calculations. The
geometry at the exocyclic amino nitrogen atom N(3) is
significantly different in isoclofazimine from that in both forms of
clofazimine and in other active analogues (Figure 13). The
authors suggest that the value for the intramolecular angle a at
N(3) (defined by C(3) - N(3) - C(21)in clofazimine) may play a
significant role in the activity. Molecules with values of a in the
range 125.5 & 10 were inactive, while those with expanded a angles
(i.e 131f 10) were active in vitro. The larger angle in the active
compounds is thought to favour intramolecular hydrogen
bonding between N(3)-H ... N(2). The capacity to form an
intramolecular hydrogen bond was interpreted as evidence of a
capacity for intermolecular hydrogen bonding in solution e.g
between guanine in DNA and clofazimine.
I02 CAlTRlONA M. O’DRISCOLL AND OWEN 1. CORRICAN
Figure 13. Crystal structure of clofazimine (Rychlewska et al., 1985)
A wide range of clofazimine analogues have been designed as

follows; (a) to be active against resistant organisms, (b) not to
accumulate in adipose or other tissues, (c) to be rapidly and
adequately absorbed from the gastrointestinal tract, and (d) not to
crystallize within cells (Barry et al., 1959; Franzblau and
OSullivan, 1988; OSullivan et al., 1988; Byrne et al., 1989). These
structural modifications generally involve substitution at the
imino nitrogen atom by an unbranched alkyl or branched alkyl
chain containing a primary, secondary, tertiary, or alicyclic amino
group. Frequently the pKa values of these amine containing side
chains are approximately 9.5 - 10.5 thus ensuring that these
molecules will be substantially ionized under physiological
conditions. To counter act this increased hydrophilicity the
aliphatic part of the substituents usually contain 6 - 8 hydrophobic
methylene groups (Hooper, 1987).
A study, (Canavan et al., 19861, on the influence of lipophilic
and stearic properties on the distribution of a range of clofazimine
analogues to the spleen of mice following oral administration,
CLOFAZIMINE I03
indicated that lipophilicity of the molecule is a significant factor

whereas the stearic properties of the N2 - substituents are not.
The structural features of phenazine derivatives which
contribute to stimulation of PGE2 production by
polymorphonuclear leucocytes (Zeis et al., 1987) and pro-oxidative
interactions with neutophils (Savage et al., 1989), have also been
investigated.
7.3 Toxicity
Clofazimine is a relatively non-toxic drug W.S. Leprosy panel,

1976). The acute LD50 was found to be >5 g/kg in mice rats and
guinea pigs. It was 3.3 g/kg in the rabbit. Daily oral doses of 30 and
50 mg/kg given for six months were generally well tolerated by
monkeys and rats. Reddish discolouration of the skin, faeces and
urine was observed. Temporary diarrhoea was occassionally
reported in rats (Stenger et al., 1970). Experimental studies in
animals did not show any evidence that clofazimine possesses a
primary embryotoxic or teratogenic action (Stenger et al., 1970).
The drug does not exhibit mutagenic activity (Morrison and
Marley, 1976a).
A long term study on 51 patients receiving clofazimine for
periods up to 8 years showed that, despite the deposition of the
drug in various tissues, it appears to be remarkably free from
serious side effects in clinical use (Hastings et al., 1976). Although
clofazimine crosses the placenta, no evidence of teratogenicity has
been found (Schulz, 1972). The most frequently reported side
effects of clofazimine therapy are red-brown hyperpigmentation of
the skin and conjunctiva, and abdominal pain (Hastings et al.,
1976; Jopling, 1976; Yawalkar and Vischer, 1979; Granstein and
Sober, 1981; Moore, 1983; Negrel et al., 1984; Venencie et al., 1986).
Cutaneous pigmentation normally fades within 6 to 12 months-
Generalised dryness of the skin (xeroderma) ichthyosis, puritis,
phototoxicity, acneiform eruptions, exfoliative dermatitis and non
specific skin rashes have been reported (Yalwalkar and Vischer,
1979; Pavithran, 1985). Discolouration of sweat, hair, sputum,
urine and faeces have also been observed (Yalwakar and Vischer,
1979). Apart from subepithelial pigmentation in the cornea no
other side effects on the eye were recorded. Clofazimine crystals
were found in the tears of 82% of patients studied (Negrel et al.,
1984).
I 04 CAITRIONA M. O'DRISCOLL AND OWEN I. CORRIGAN
Gastrointestinal side effects, nausea, diarrhoea, anorexia,

constipation and weight loss have also been reported (Hastings et
al., 1976; Moore, 1983). These symptoms have been associated with
the deposition of clofazimine crystals in the submucosa of the
small intestine and in the mesenteric lymph nodes (Jopling, 1976;
Harvey et al., 1977).
The occurence of drug interactions involving clofazimine have
also been investigated. Most of the studies show that clofazimine
does not exert any effect on dapsone excretion in leprosy patients
(Balakrishnan and Seshadri, 1981; Zuidema et al., 1986).
Clofazimine has been shown to significantly reduce the absorption
of simultaneously administered rifampicin, resulting in delayed
time to reach peak serum concentration and increased t; . No
significant changes were seen in C,,, or AUC (Mehta et al., 1986).
7.4 Dose Schedules
A dose of 300mg once montly plus 50mg daily or lOOmg on

alternative days has been recommended to treat multibacillary
forms of leprosy (Martindale, 1989).
The World Health Organisation (1982) has published guidelines
for the treatment of leprosy. Dosage schedules are generally not
based on serum/plasma concentrations, or pharmacokinetic data.
Clofazimine is usually used in combination with other
antileprotic agents e.g dapsone and rifampicin, to prevent the
emergence of resistance. It is usually given with food in doses
adjusted according to body weight and the activity of the disease.
The therapeutic activity of clofazimine depends on the
concentration of drug in the immediate environment of M.leprae
in the tissues and not on the serum level. Since the drug in not
evenly distributed through out the tissues it is impossible to
calculate the minimal inhibitory concentration (MIC) in animals
(Yawalkar and Vischer, 1979).
CLOFAZIMINE 10.5
ACKNOWLEDGEMENTS
The authors wish to thank Dr. J. F. OSullivan, formerly of the

Health Research Board, Trinity College, Dublin, Dr. Helen
Sheridan, Department of Pharmacognosy and Dr. Mary Meegan,
Department of Pharmaceutical Chemistry, Trinity College, Dublin
for their advice and assistance, Ciba Geigy, England, for the supply
of clofazimine, Ms. Mary Lally and Ms. Mary Reilly for technical
assistance.
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CLONIDINE HYDROCHLORIDE
Mohamrnad A. Abounassif,' Mohammad Saleem Mian,'
and Neelofur Abdul Aziz Mian'
(1) Pharmaceutical Chemistry Department

College of Pharmacy
(2) Clinical Laboratory Sciences Department

College of Applied Medical Sciences

AND EXCIPIENTS - VOLUME 21 109 All rights of reproduction reserved in any form.
110 M.A. ABOUNASSIF, M.S. MIAN, AND N.A.A. MIAN
Contents
1, Introduction
2. Description
2.1 Nomenclature
2 . 1 . 1 Chemical Names
2 . 1 . 2 Generic Names
2 . 1 . 3 Trade Names
2.2 Formulae
2 . 2 . 1 Empirical
2 . 2 . 2 Structural
2 . 2 . 3 CAS (Chemical Abstract Service Registry
Number)
2.4 Elemental Composition
2.5 Appearance, Color, Odour and Taste
3. Physical Properties
3.1 Melting Range
3.2 Solubility
3.3 PH
3.4 Loss on drying
3.5 Sulphated Ash
3.6 Clarity and Color of Solution
3.7 Stabi1 ity
3.8 PK
3.9 LD5 o
3 . 1 0 Action
3 . 1 1 Half Life Plasma
3 . 1 2 Volume of Distribution
3 . 1 3 Protein Binding
3 . 1 4 Storage
3 . 1 5 X-ray Powder Diffraction
3 . 1 6 Crystal Structure
3 . 1 7 Spectral Properties
3 . 1 7 . 1 Ultraviolet Spectrum
3 . 1 7 . 2 Infrared Spectrum
3 . 1 7 . 3 Nuclear Magnetic Resonance Spectra
3 . 1 7 . 4 Mass Spectrum
4. Synthesis
5. Phnrmacokirietics
5.1 Absorption and Distribution
5.2 Uses and Administration
5.3 Adverse Effects
5.4 Precautions
6.1 Identification
6.2 Colorimetric
6.3 Fluorimetric
6.5 Radio-Immunoassay
6.6.2 High-Performance Liquid Chromatography
(HPLC) .
7. Acknowledgements
8. References
Clonidine Hydrochloride
1. Introduction
Clonidine hydrochloride is an imidazoline

derivative hypotensive agent ( 1 ) which is thought to
act through the central nervous system to elicit a
hypotensive response ( 2 ) . Although the locus of action
in the central nervous system is unlcear, clonidine
has been shown to be a potent a-adrenergic agonist in
both central and peripheral systems ( 3 ) .
The commercially available transdermal system of

clonidine consists of an outer layer of pigmented
polyester; a drug reservoir of clonidine, mineral oil,
polyisobutylene, and colloidal silicon dioxide; a
microporous polypropylene membrane that controls the
rate of diffusion of the drug; and a final adhesive
layer that provides an initial release of drug and
contains those ingredients found in the reservoir. The
adhesive layer is covered by a protective strip which
is removed prior to application ( 1 ) .
2. Description
2.1 Nomenclature
[2-(2,6-Dichlorophenylimino)imidazolidine
hydrochloride (2,4);
2-(2,6-Dichloroanilino)-2-imidazoline
hydrochloride ( 4 ) ;
2,6-Dichloro-N-(imidazolidine-2-ylidene)aniline
hydrochloride (4);
2-(2,6-Dichlorophenylamino)-2-imidazoline
hydrochloride (5).
2.1.2 Generic Names
Clonidine hydrochloride.
CLONIDINE HYDROCHLORIDE I13
2.1.3 Trade Names
Catapres, Catapresan, Clonistada; Dixarit,

Drylon, Hyposyn, Ipotensium, Isoglaucon,
Tenso-Timelets.
2.2 Formulae
2.2.1 Emoirical
CgHgClzN3 (Clonidine).
CsHsCIzN3 .HC1 (Clonidine hydrochloride).
2.2.2 Structural
2.2.3 CAS (Chemical Abstract Service Registry

Number 1
4205.90.7 (clonidine) (4).

4 2 0 5 . 9 1 . 8 (Clonidine hydrochloride) ( 4 ) .
2.3 Molecular Weipht
230.10 (Clonidine) ( 6 , 7 ) .
266.6 (Clonidine hydrochloride) ( 4 ) .
Clonidjne (7): C 46.98%; H 3.94%;

ci
30.82%; N 18.26%.
Clondine hydrochloride: C 40.51%; H 3.75%
C1 3 9 . 9 8 % ; Nz 15.75%.
I14 M.A. ABOUNASSIF, M.S. MIAN. AND N.A.A. MIAN
2.5 ADpearance, Color, Odour and Taste
A white o r almost white crystalline powder (8)

which has a bitter taste ( 1 ) .
3.1. Melting Range
Clonidine 130'C ( 7 ) .
Clonidine hydrochloride 305'C ( 7 ) .
Clonidine hydrochloride 300'C with decomposition (9).
3.2 Solubility
Soluble in 1 3 parts of water, soluble in

absolute ethanol, slightly soluble in chloroform ( 8 ) .
1 g soluble in 6 m l H 2 O ( 6 0 ' C ) , about 13 m l H 2 0
( 2 0 ' C ) , about 5 . 8 m l C:H3OH, about 2 5 ml C z H 5 0 H and
about 5000 ml of CHCln (9). Practically insoluble in
ether (6).
5% solution in H2O has a pH of 4 . 0 to 5 . 0 ( 8 ) .
3.4 Loss on Drying
When dried to constant weight at 1 O O ' C to 105-C,

loses not more than 0 . 5 % of its weight use 1 g (8).
3.5 Sulphated Ash
Hot more than 1% (8).
3.6 Clarity and Color of Solution
A 5% w/v solution in carbon dioxide free water

is clear (8).
3.7 Stability
Stable in light air and room temperature (9).
3.8 pk
The drug has a Pk of 8.2 ( 6 , 9 ) .

CLONIDLNE HYDROCHLORIDE I I5
The acute toxicity for clonidine in related

species is as follows: ( 7 )
Species LD5o (mg/kg)

Oral I.V.
Mouse 328 18
Rat 270 29
Rabbit 80 45
Dog 30-100 6
Monkey 150-267
3.10 Action
Clonidine hydrochloride is an antihypertensive

agent, whose mechanism of action appears to be central
a-adrenergic stimulation. This result in the
inhibition of bulbar sympathetic cardioaccelarator and
sympathetic vasoconstrictor centers, therapy causing a
decrease in sympathetic outflow from the brain.
Initially drug stimulates pheripheral a-adrenergic
receptors producing transient vasoconstriction ( 5 ) .
3.11 Half-Life Plasma ( 6 )
Pl.asma half-life, 10 to 25 hours.
3.12 Volume of Distribution ( 6 )

2 to 4 litres/kg.
3.13 Protein Binding ( 6 )
About 20 to 40%.
3.14 Storage
The drug should be kept in a well-closed

containers ( 8 ) and protect from sun light ( 4 ) .
3.15 X-ray Powder Diffraction
The X-ray diffraction pattern of clonidine

hydrochloride was determined using philips full
automated generator. Radiation was provided by a
copper target (Cu annode 2000W, Y = 1.5480 A ) . High
intensity x-ray tube operated at 40 kv and 35 Mv was
used. The monochromator was a curved single crystal
one (Pw 1752/00). Divergence slit and the receiving
slit were 0 and 0 . 1 " , respectively. The scanning speed
of the goniometer (Pw 1050/81) used was 0 . 0 2 - 20 per
second. The instrument is combined with philips PM
8210 printing recorder with both analogue recorder and
digital printer. The goniometer was aligned using
silicon sample before use. The x-ray pattern of
clonidine hydrochloride is presented in Fig. ( 1 ) . The
values of scattering angle 2 0 interplanner distance
dA and relative intensity 1/10 are shown in the table
(1).
3.16 Crystal Structure
Gudmund et a1 ( 1 0 ) have determined the crystal

structure of clonidine hydrochloride by x-ray
diffraction methods using 3209 observed reflections
collected on a counter diffractometer. The crystals
are monoclinic, space group ( 2 / c with unit cell
dimensions a = 1 7 . 9 5 7 ( 2 ) A b = 1 1 . 9 5 0 ( 1 ) A , c =
13.664 ( 1 ) A and I3 = 128.64 ( 1 ) O ; (t = 18 k 1 . C ) ; V =
2290.2 A , M = 2 6 6 . 5 6 , Z 8 ; F(OOO) = 1088; Dcalc =
1 . 5 4 6 g cm-3; p = 7 . 6 7 cm-1,
Selected interatomic distances and bond angles

are listed in Table ( 2 ) . Perspective view of the
molecule showing bond lengths is presented in Fig.
( 2 a ) . The stereoscopic view of the crystal structure
of clonidine is shown in Fig, ( 2 b ) .
Cody et al. (11) also determined the crystal

structure of clonidine hydrochloride in order to
determine the conformation of protonated clonidine and
to el ucjdate the relationships between its structure
and that required for binding to the a-adrenergic
re1,epl UI",
T a b l e ( 1 ) Characteristic lines of x-ray diffraction of

clonidine hydrochloride
29 d(A) I/Io%
9.281 9.5283 20.533 46.963 1.9347 2.943

9.779 9.0446 58,521 47.821 1.9020 5.133
12.463 7.1020 22.587 50.711 1.8002 7.734
13.032 6.7932 72.142 51.129 1.7865 5.886
14.625 6.0569 8.213 52.072 1.7563 8,008
16.801 5.2768 16,974 52.667 1.7379 12.388
17.676 5.0176 4.859 53.648 1.7084 3.901
19.723 4.5012 16.563 54.372 1.6873 6.365
22.232 3.9986 33,812 54.903 1.6722 5.817
22.684 3.9199 11.362 56.132 1.6385 2.395
23.093 3.8514 10.814 56.476 1.6293 7.665
24.634 3.6138 46.406 60.422 1.5320 3.080
25.308 3.5191 41.204 61.337 1.5114 3.011
25.872 3.4436 12.662 65.051 1.4338 2.806
26.334 3.3842 100 65.578 1.4235 3.080
27.032 3.2985 48.665 66.518 1.4057 6,707
28.182 3.1664 6.639 67.342 1.3905 2.258
29.088 3.0698 31.211 70.397 1.3341 2.327
29.828 2.9953 21.697 72.236 1.3078 3.832
30.660 2.9159 6.981 72.918 1.2973 2.121
30.889 2.8948 9.582
33.515 2.6738 4.859 20 = scattering angle
33.813 2,6509 8.213
34.788 2.5788 4.996 dA = Interplanner distance.
35.335 2.5401 2.943
36.073 2,4898 5,612 I/Io = relative intensity
36.754 2.4452 6.433 distance ( b a s e d on
37.214 2.4161 5,270 h i g h e s t i n t e n s i t y as
38.105 2.3616 8.008 100.
38.104 2.3439 10.746
39.260 2.2947 2.943
39.861 2.2615 4.585
40.252 2.2394 6.228
40.851 2.2089 8.418
41.889 2.1566 4.175
42.175 2.1426 5.133
42.471 2.1284 9.924
43.419 2,0841 4.517
43.747 2.0692 4.106
44.235 2.0475 3.764
44.586 2,0322 5.544
45.623 1.9884 3.285
(20 value)
Fig. (1) The X-ray diffraction p a t t e r n o f Clonidine HC1.

CLONIDINE HYDROCHLORIDE 1 I9
Table (2). Distances ( A ) and angles ( " ) in the crystals of

clonidine hydrochloride
Bond Length Bond angles
C6-Cl-C2 117.3
Cl-C2 1.391 Cl-C2-C3 121.5
C2-C3 1.382 c2-c3-c4 119.8
c3-c4 1.377 C3-C4-C5 120.2
c4-c5 1.371 C4-C5-C6 119.8
C5-C6 1.385 C5-C6-C1 121.4
C6-C1 1.392 Cl-C2-C12 120.0
C1-N1 1,418 c3-c2-c12 118.5
C2-Cl2 1.733 Cl-CG-Cl3 118.9
C6-Cl3 1.724 C5-C6-C13 119.7
N1-C7 1 328 C2-C1-N1 121.4
N2-C7 1.322 C6-Cl-N 1 121.3
N3-C7 1.321 Cl-Nl-C'I 123.0
N2-C8 1.450 Nl-C'?-NB 123.1
N3-C9 1.447 Nl-C7-N2 125.2
C8-C9 1.533 C7-N2-C8 110.6
N2-C8-C9 103.5
C8-C9-N3 102.6
C9-N3-C7 111.5
N2-C7-N3 111.8
Torsional angles
(positive for a clockwise rotation)
C2-Cl-Nl-C7 - 76.5
Cl-Nl-C7-N2 0.0
Cl-Nl-C7-N3 178.1
C6-Cl-bil-C7 105.2
Hydrogen bonds
Cll-Nl(i - X, - t t y, g - Z) ( A ) 3.094
C11-HN1 ( A 1 2.25
C11-HN1-N1 ( " ) 161.2
Cll-NX(x,y,~)( A ) 3.193
C11-HN2 ( A ) 2.38
Cll-HN2-N2 ( " ) 163.4
I20 M.A. ABOUNASSIF. M.S. MIAN. AND N.A.A. MIAN
F i g (2b) S t e r e o s c o p e v i e w o f t h e crystal
s t r u c t u r e of C l o n i d i n e .
Fig (2a) Perspective view o f Clonidine

m o l e c u l e s h o w i n g b o n d lengths.
CLONIDINE HYDROCHLORIDE 121
Crystals of clonidine hydrochloride [2,6-dichlo-

rophenylamino)-2-imidazoline HCl] , CgHioN3C13 , were
grown by slow evaporation from aqueous solution. The
crystals are of exceptional quality. A crystal of
columnar shape, 0.2 x 0.2 x 0.6 mm, was screened
o p t j cally and by X-ray Weissenberg photography for
quality and assignment of space group. The refined
cell constants, obtained by a least-squares fit of the
I values of 73 high-angle reflections measured
( = 0.707 A ) automatically on a kappageometry
diffractometer, are listed in table 3 along with other
crystal data. Intensity data were measured in theQ-28
s c a n m o d e u s i n g Mo h'a r a d i a t i o n a n d a
dispersion-corrected scan sidth of ( 0 . 8 t 0.2tane ) "
to a. SinQ,/A maximum of 0.70 8 - l . Of the 3335 unique
reflections measured, 2112 are greater than or equal
to twice their estimated standard deviations.
Table 3. Crystal data for clonidine HC1.
Molecular formula CgHgN3Clz .CH1

Molecular weight 266.56
Crystal system Monoclinic
Space group C2/c
2 8
Cell dimensions a = 17.962(3) A
b = 11.976(2) W
c = 13.672(2) A
I3 = 128.62(1)'
Cell volume 2298.2 R
Density (calc. ) 1.541 g ~ m - ~
(obs. ) 1.543 g cm-3
Crystal size 0.2 x 0.2 x 0.6 mm
Final R index 0.05 (1223 data)
3.17.1 Ultraviolet SDectrun (UVY
The UV spectrum (12) of clonidine hydrochloride

in H2O (8 mg%) was scanned from 200-600 nm (Fig. 3)
using LKB 4054 UV/Vis spectrometer. Clonidine
0
0
(D
0
In
m
0
0
In
0
m
U
0
0
U
0
m
0
0
0
m
0
Ln
CJ
I
0
- I
0
I
0
I
0
I
0
I
0
0
0
cv
0 0 0 0 0 0
In 0 m 0 In 0
hl (v F c 0 0
hydrochloride exhibited the following U V data

(Table 4 ) .
Table 4: UV data of clonidine hydrochloride
\ax nm Absorbance Molar absorptivity A(1%, 1 cm)

(,E) cm-1 gm mol./L
213 2.488 8290,327 311
271 0.214 713.074 26.75
302 0.102 339.876 12.75
3.17.2 Infrared SDectrum
The I R spectrum ( 1 2 ) of clonidine hydrochloride

as KBr disc was recorded on a Perkin Elmer 1310
Infrared spectrometer. Fig. (4) shows the infrared
absorption spectrum of clonidine hydrochloride. The
structural assignments of clonidine hydrochloride have
been correlated with the following frequencies
(Table 5).
3.17.3 Nuclear Magnetic Resonance Spectra
4.17.3.1. 'H-NMR Spectrum

The 1 H - N M R spectrum ( 1 2 ) of clonidine
hydrochloride in DMSO-ds (Fig. 5 - 6 ) was recorded on a
Varian X L 200 M H z NMR spectrometer using TMS as an
internal reference, The following structural
assignments have been made (Table 6 ) .
0
0
to
0
0
.o
7
0
0
m
c
5 0
0
0
hl
8
d
m
0
0
1 JJ I A I 0
*
0
3 0 0
3 aD (0 U hl
I24
Fig. (5) PMR s p e c t r u m o f C l o n i d i n e HC1 i n DMS0.D6.
Fig. (6) PMR spectrum o f Clonidine HCl in DMSO.D6 (DiO Each.)
Table 5: IR characteristics of clonidine HC1.
Frequencies Approximate description

of vibrational modes
3320 NH stretch
3000-3080 Chlorophenyl CH stretch
1650, 1600, 1565 Iaidazolidine ring

stretch
1440, 1400 Phenyl ring stretch
1330, 1280 Chlorophenyl C-H planar

bend
1190, 1100 Chlorophenyl C-C1

stretch bends.
790, 780 -
Table 6: FMR characteristics of clonidine HC1

structure
Protons 6 (PPM) Multiplicity
g, d (two protons) 8.618 singlet
a,b,c (three protons) 7.439-7.661 multiplet
e,f ( four protons) 3.672 singlet

I28 M.A. ABOUNASSIF. M.S. MIAN, ANDN.A.A. MIAN
3.17.3.2 13C-NMEI S p e c t m
13C-NMR spectrum (12) of clonidine hydrochloride

Fig. (7-9) was recorded in DMSO-d6 by Varian XL-200
MHz NMR spectrometer. The multiplicity of the
resonances was obtained from DEPT (Distortionless
enhancement by polarization transfer) and APT
(attached proton test), The carbon chemical shifts are
presented in Table ( 7 ) .
Table 7: C-13 chemical shifts of clonidine HC1.
Carbon assignment Chemical shift 6 (ppm)
cs, c9 42.647
c1, c 3 129.121
CS, c 4 133,987
cz 130.772
c5 130.262
CI 157.919
3.17.4 Mass SDectrum
The mass spectrum ( 1 2 ) of clonidine hydrochlo-

ride obtained by electron impact ionization (Fig. 10)
was recorded on a Finnigan MAT 90 mass spectrometer.
The spectrum was scanned from 50 to 500 8.m.a. The
electron energy W A S 70 ev. Emission current 1 mA and
ion source pressure 10-6 t o r r . The spectrum shows a
Fig. (7) 13C.NMR spectrum of C l o n i d i n e HC1 in DMSO-D6.
Fig. (8) 13C,NMR spectrum of C l o n i d i n e H C 1 i n DMS0.D6 (APT)
Fig. (9) 13C.NMR spectrum of Clonidine HC1 in DMS0.D6 (DEPT).
100.0
50.
I
I ' I I
50 100 150
100.01
50.
Fig. (10) M a s s s p e c t r u m o f C l o n i d i n e HCJ,

molecular ion M + at m/z 229 with a relative intensity

100%. The most prominent fragments and their relative
intensities are listed in Table 8 .
4. Synthesis
Clonidine is synthesized (13) by the
condensation of 2,6-dichloroaniline and imidazoline.
2,6-Dichloro- Imidazoline Clonidine

ani1ine
5. Pharmacokinetics
5.1 AbsorDtion and Dietribution

Clonidine hydrochloride is readily absorbed by
o r a l route with an absorption time of 2 to 4 hours
(9). Drug is well absorbed from the gastro-intestinal
tract. I t may also be absorbed when applied topically
to the eye, clonidine is well absorbed percutaneously
following topical application of a transdermal system
t o t h e arm or chest. Plasma clonidine concentrations
of 2 ng/mL have been detected one hour after
administration of a single 0.39 mg oral dose of
radiolabeled drug. Peak plasma concentrations
following oral administration occur in approximately
3-5 hours (1).
Reduction in blood pressure is maximal at plasma
clonidine concentrations less than 2 ng/mL. Blood
pressure begins to decrease within 30-60 minutes after
an oral dose of clonidine hydrochloride, the maximum
decrease occurs in approximately 2-4 hours. The
hypotensive effect lasts up t o 8 hours. Following
administration of clonidine by slow intravenous
injection in patients with acute hypertensive crises,
a hypotensive effect occurred within minutes, peaked
in 30-60 minutes and lasted more than 4 hours (1).
I34 M.A. ABOUNASSIF, M.S. MIAN, AND N.A.A. MlAN
Table 8: The mass fragments of clonidine HC1
m/z Relative intensity % Fragment
230 65
229 100
221 10
207 12
200 20
196 22
194 52
193 17
186 11
174 45
172 54
165 20
147 18
124 18
109 20
73 17
Animal studies indicate that clonidine is widely

distributed into body tissues, tissue concentration of
the drug are higher than the plasma concentration.
After oral administration highest concentrations of
the drug are found in the kidneys, liver, spleen, and
GI tract. High concentrations of the drug also appear
in the lacrimal and parotid glands. Clonidine is
concentrated in the choroid of the eye and is also
distributed into the heart, lungs, testes, adrenal
glands, fat and muscle. The lowest conc. occurs in the
brain. Clonidine is distributed in CSF. It is not
known whether the drug crosses the placenta. Clonidine
is distributed into milk (1).
The plasma half life of clondine is 6-20 hours

in patients with normal renal function. The half life
in patients with impaired renal function has been
reported t o range from 8-41 hours. The elimination
half life of the drug may be dose dependent,
increasing with increasing dose ( 1).
The drug is metabolized in the liver. In humans,

4-metabolites have been detected but only one, the
inactive p-hydroxylated derivative, has been
identified ( 1 ) .
In humans 65 % of administered dose of clonidine

hydrochloride is excreted by the kidneys, 3 2 X as
unchanged drug and the remainder as inactive
metabolites. Approximatly 20 % of dose is excreted in
feces, probably via entrohepatic circulation.
Approximately 85 % of a single dose is excreted with
72 hours and excretion is complete after 5 days (1).
5.2 Uses and Administration
Clonidine is an antihypertensive agent which

appears to act centrally by stimulating az-adrenergic
receptors and producing a reduction in sympathetic
tone, resulting in a fall in diastolic and systolic
blood pressure and a reduction in heart rate. It also
acts peripherally, and this peripheral activity may be
responsible for the transient increase in blood
pressure seen during rapid intravenous administration
as well as contributing to the hypotensive effect
during chronic administration. Peripheral resistance
is reduced during continuous treatment. Cardiovascular
reflexes remain intact so postural hypotension occurs

infrequently. When given by mouth its effects appear
in about 30-60 minutes reaching a maximum after 2-4
hours as lasting up to 8 hours ( 4 ) .
Clonidine hydrochloride is used in the treatment

of grades of hypertension. The usual initial dose of
clonidine hydrochloride is 50 to 100 pg orally thrice
daily increased every second or third day according to
the response of the patient. The usual maintenance
dose is 0.3 to 1 . 2 mg daily but doses of up to 1.8 mg
o r more daily may be required. To reduce side effects
a similar dose of clonidine may be given in
conjunction with a thiazide diuretic but combination
w i t h a Ij-blocking agent should be avoided where
possible clonidine may also be given in a sustained-
release formulation which enables twice-daily dosage,
or by a transdermal delivery system which is applied
once a week and delivers 100-300 pg daily at a
constant rate ( 4 ) .
Drug may be given by slow intravenous injection

in hypertensive crises usually in doses of 1 5 0 to
300 l.lg (419
It is also used in lower doses for the

prophylaxis of migrane or recurrent vascular headaches
and in the treatment of menopausal flushing ( 4 ) .
Clonidine hydrochloride has been used topically

to reduce intraocular pressure in the treatment of
open angle (chronic simple) and secondry glaucoma and
hemorrhagic glaucoma associated with hypertension (1).
B e c a u s e of i t s GI e f f e c t s c l o n i d i n e
hydrochloride has been used with some success in a
limited number of patients for the management of
diarrhea of various etiologies (e.g. narcotic bowel
syndrom, idiopathic diarrhea associated with diabetes)
(1).
5.3 Adverse Effects
Serious toxic effects have been reported after

ingestion of doses of 0.4 to 4 mg by children and 4 to
11 mg by adults. However, recovery is usually rapid
(6).
CLONIDLNE HYDROCHLORIDE 137
Drowsiness, dry mouth, dizziness and headache

commonly occur during the initial stages of therapy
with clonidine. Fluid retention is often transient but
may be responsible for a reduction in the hypotensive
effect during continued treatment. Constipation is
also common and other adverse effects which have been
reported include depression, anxiety, fatigue, nausea,
anorexia, parotid pain, sleep disturbances, vivid
dreams, impotence, urinary retention or incontinence,
slight orthostatic hypotension, and dry itching or
burning sensations in the eye. Rashes and pruritus may
occur and are more common with the use of transdermal
delibery systems. Less frequently, bradycardia,
including sinus bradjcardia with atrioventricular
block, hallucinations, and transient abnormalities in
liver function tests have been reported large doses
have been associat.ed with initial increases in blood
pressure and persist during continued therapy ( 4 ) .
Symptoms of overdosage include transient

hypertension or profound hypotension, bradycardia,
sedatjon, miosis, respiratory depression, and coma.
Treatment consists of general supportive measures. An
(1-adrenoceptor blocking agent may be given if
necessary ( 4 ) ,
Clonidine withdrawal may result in an excess of

circulating catecholamines. Therefore, caution should
be exercised in concomitant use of drugs which effect
the metabolism o r tissue uptake of these amines
(monoamine oxidase inhibitors or tricyclic antidepres-
sants, respectively) ( 1 ) .
5.4 Precautions
Clonidine should be used with caution in

patients with cerebral, or coronary insufficiency,
Raynaud’s disease or thromboangitis obliterans, or
with a history of depression. The hypotensive effect
may be antagonised by tricyclic antidepressants, and
enhanced by thiazide diuretics. Clonidine cause
drowsiness and patients should not drive or operate
machinery where loss of attention could be dangerous.
The effect of other cent.ra1 nervous system depressants
mag be enhanced, withdrawal of clonidine therapy
should be gradual as sudden discontinuation may cause
rebound hypertension which may be severe. Agitation,
I38 M.A. ABOUNASSIF. M.S. MIAN, AND N.A.A. MIAN
sweating, tachycardia, headache, and nausea may also

occur. 0-blockers can exacerbate the rebound
hypertension and if clonidine is being given
concurrently with a D-blocking agent, clonidine should
not be discontinued until several days after the
withdrawal of the B-blocker. It has been suggested
that patients should be warned of the risk of missing
a dose or stopping the drug without consulting their
doctor and should carry a reverse supply of tablets
(4).
Although hypotension may o c c u r d u r i n g

anaesthesia in clonidine-treated patients clonidine
should not be given intravenously during the operation
to avoid the risk of rebound hypertension. Intravenous
injections o f clonidine should be given slowly to
avoid a possible transient pressor effect especially
in patients already receiving other antihypertensive
agents such as guanethidine or reserpine ( 4 ) .
Abrupt withdrawal of oral clonidine therapy may

result in a rapid increase of systolic and diastolic
blood pressure, with associated symptoms as
nervousness, agitation, restlessness, anxiety,
insomnia, headache, sweating, palpitation increased
heart rate, tremor, hiccups, stomach pains, nausea,
musc1.e pains, and increased salivation (1).
6.1 Identification
1) Dilute a volume containing 0.3 mg of clonidine

hydrochloride to 5 m l with 0.01 M hydrochloric acid.
The light absorption of the resulting solution in the
range of 245 to 350 nm exhibits maxima at 272 nm and
279 nm and inflection at 265 nm ( 8 ) .
2) To a volume containing 0.15 mg of clonidine

hydrochloride add 1 ml of a 10% w/v solution of
ammonium reineckate and allow to stand for 5 minutes.
A pink precipitate is produced (8).
3) The drug gives Libermann’s color test yellow to

orange at 100°C ( 6 ) .
4) Gives characteristics reaction of chlorides (8).

CLONlDlNE HYDROCHLORIDE I39
5) The infrared absorption spectrum is concordant

with the spectrum of clonidine hydrochloride (8).
6) Dissolve 0 . 2 g in 70 ml of ethanol ( 9 6 % ) and

titrate with 0 . 1 M ethanolic sodium hydroxide vs
determining the end point potentiometrically. Each ml
of 0 . 1 M ethanolic sodium hydroxide vs is equivalent
to 0.02666 g of C6HgClzN3.HCl ( 8 ) .
6.2 Colorimetric
Tawakkol et al, ( 1 4 ) developed a method for the

colorimetric determination of colonidine in which
clonidine reacts with sodium nitroprusside in presence
of sodium hydroxide, and on treatment with saturated
boric acid it gives a violet color which was measured
a t 570 nm.
1 . 0 mg of powdered tablets were shaked with 10

ml of H20 and centrifuged, decant the clear solution
into a 100 ml separating funnel. Repeat two times each
Kith 1 5 m l of distilled H2O collecting in the same
separating funnel, then add 1 ml of sodium carbonate
and extract three times with chloroform, extract on a
water bath and few drops of HC1 were added, evaporate
and extract with few ml of distilled H2O. Then
standard or test solution was treated with 0.8 ml of 1
N NaOH s o l u t i o n f o l l o w e d by 1 m l o f sod.
nitroprusside, mixed and leave for 10 min, then 2 ml
of 4% boric acid solution was added leave in an ice
bath for 10-15 minutes. Complete up to the mark and
measure the violet color of both the standard and the
test at 570 nm.
Sane (15) developed a method for the estimation of

clonidine hydrochloride in pharmaceutical preparations
by ion pair extraction and colorimetric method. An
acid-dye complexing method with bromophenol blue,
broaocresol purple and methyl-orange was used for the
ion-pa.ir extraction and colorimetric determination of
clonidine hydrochloride in pharmaceuticals containing
100 mg of clonidine hydrochloride was 98.9% and
relative standard deviation 0.89%.
I40 M.A. ABOUNASSIF, M.S. MIAN, AND N.A.A. MIAN
6.3 Pluorimetric
A very sensitive fluorimetric method ( 1 6 ) based

on the reaction of clonidine hydrochloride with
1-dimethylaminonaphthalene-5-sulphonyl chloride (dan-
syl chloride) to give a highly fluorescent derivative.
Dissolve 50 mg of clonidine hydrochloride in a

mixture of 10 ml of acetone and 40 ml of 0.5 M sod.
carbonate solution. Transfer to a 50 ml flask make up
to the mark with dansyl chloride and acetone was added
and then 4-methyl pentan-2-one was added. Fluorescence
intensity was measured after 10 minutes at 455 nm
using an excitation wavelength of 345 nm.
A simple and rapid spectrophotometric method

( 1 6 ) based on the reaction of clonidine hydrochloride
with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone to form
a colored product with maximum absorption at 455 nm.
Accurately weighed amount of clonidine
hydrochloride equivalent to 50 mg of the base was
dissolved in about 20 m l distilled HzO, made alkaline
with few drops of 10% w/v NaOH solution and extract
with five successive 10 ml portions of chloroform.
Pass the chloroform extracts sequentially over
anhydrous sod. sulphate and collect the combined
chloroform extracts in 50 ml flask make up to volume
with chloroform. Heat and dissolve in acetonitrile and
2,3-dichloro-5,6-dicyano-1,4-benzoquinone and measured
at 455 nm against. blank.
An other simple and sensitive spectrophotometric

method based 011 color reaction with bromocresol green
WAS studied by Zivanov-Stakic et a l . ( 1 7 ) for the
determination of colonidine hydrochloride in tablet
form.
Another method ( 1 8 ) reported the spectroscopic

determination of clonidine hydrochloride.
6.5 Radio-Immunoassag
Radio-immunoassay for clonidine in human plasma

a.nd urine using a s o l i d phase second-antibody
separation was studied by Farina et al. ( 1 9 ) . In which
plasma was incubated for 1 8 hour at 4" with 0 . 1 M

sodium phosphate buffer (pH 7.4). 1251-labeled
4-carboxyclonidine-tyrosine and antibodies raised in
rabbit against 4-carboxyclonidine N-hydrosuccinimide
ester conjugated with bovin serum albumin. After a
second incubation with immuno beads of goat
anti-rabbit immunoglobulin for 2 hours at room
temperature, the mixture was centrifuged and
radioactivity in the pallets was counted. The
detection l i m i t was 10 pg per m l of clonidine, the
within and between assay coefficient of variation were
2.8 to 9 and 10 to 13% respectively.
A newly developed and precise and sensitive

radio-immunoassay f o r clonidine was done by Arndts et
al. ( 2 0 ) . The antiserum is raised in rabbits injected
with the pcarboxy derivative of clonidine and is used
in the radioimmunoassay for clonidine in the residues
of ethyl ether extracts (pH 9.5) of 0.2 ml of plasma
[3H] clonidine being used as a tracer in a phosphate
buffer medium (pH 7 . 4 ) . After incubation for 18 hours
at 4'C unbound antigen is adsorbed on the
dextran-coated charcoal, and the bound 3H is
determined. A calibration graph is constructed for 0 . 1
to 10 ng per ml of clonidine in plasma.
Another method ( 2 1 ) of clonidine in rats as

determined by radio-immunoassay. In which an antigen
prepared by reacting 4-hydroxyclonidine with
4-carboxybenzene diazonium chloride and coupling the
product t o bovin serum albumin was used to raise an
antiserum in rabbits. [3Hl Clonidine or [ 1 4 C ! 1
clonidine was used as tracer and separation of the
free and bound forms of the antigen was carried out.
The sensitivity was 1 0 pg with f3H1 clonidine and 600
pg with [14C] clonidine.
I t has been possible to measure plasma and
tissue levels after the administration of rather high
doses of radiolabelled clonidine to humans and animals
(22-25).
1) Determination of submicrogram quantities of

clonidine in biological fluids by Chu (26). Clonidine
is extracted from plasma, extract is purified by

solvent extraction and column chromatography and
clonidine is converted to heptaf luorobutyryl
derivative for g.1.c at 175' on a partially
inactivated column cntaining 3% OV-17 on Chromosorb W
AW DMCS with electron capture detection. The 4-methyl
analogue of clonidine is used as internal standard.
The limit of determination was 2 5 pg m l - 1 , and the
coefficient of variation at the level of 60 pg ml-1
was approximately 8%.
2) The method (27) describes the measurement of

clonidine in human plasma and urine by combined gas
chromatography-mass spectrometry with ammonia chemical
ionization. Addition of [2H4] clonidine to plasma or
urine is followed by ethylacetate extraction of
clonidine from alkaline medium, back extraction into
acid extraction into ethyl ether from alkaline medium
and evaporation of the extract to dryness.
Trirnethylanilinium hydroxide is added to the residue,
and dimethyl derivatives of clonidine are formed by on
column methylation with an injection-port temperature
of 250' for g.c. -70-eV m , s , , the glass column (1.8 m
x 2 mm) packed with 3% of OV-17 on Gas-Chrom Q (100 to
120 mesh) is operated at 245". With He as carrier gas
(15 m l min-l); NH3 is admitted to an ion-source
pressure of 0.2 Torr, and ions are monitored at m/e
258 and 264. Graphs of peak height ratios (n/e 258 to
2 6 4 ) vs amounts of clonidine in urine (up to 4 0 ng
ml-l) and in plasma (up to 5 ng ml-1) are rectilinear.
The precision for assay of clonidine in plasma is 11%
at 0.25 ng ml-1 and 5% at 0 . 5 ng ml-l and the lower
limit of determination is 0.1 ng ml-1.
3) A simple and sensitive gas-liquid chromatogra-

phic method ( 2 8 ) has been developed for the
quantitative determination of clonidine and some
structurally related imidazolidines in rat brain
tissues. The aqueous brain homogenates are first
purified and t,hen extracted into benzene. Samples are
injected directly to GLC column ( 2 m x 2 mm I.D.)
pa.cked with 3% OV-17 on chromosorb 750 (80-100 mesh)
was used at an oven temperature of 200-270' and an
injector temperature of 280" the carrier gas was
helium; flow rate 30 ml/min,
4) A gas chromatography assay for clonidine in

human plasma has been developed by per Olof Edlund
(29). The buffered serum is extracted on silica
columns, alkylated with pentrafluorobenzyl bromide,
clertned up by extractions and analysed by glass-needle
injection and electron-capture detection. The packed
column (2 m x 3 m m I.D.) was silanized glass and was
packed with 3% of OV-17 on 80-100 mesh. Gas-Chrom Q.
5) Another method (30) for the determination of

clonidine in plasma by G . L . C . 2-(2,4-dichloroani-
line)-2-imidazoline is used as internal standard. The
column (WCOT: 30 m x 0 . 3 5 mm) was operated at 250'C
with H2 as carrier gas.
Other methods used for the GLC determination of

clonidine hydrochloride in biological materials
(31,321. Recently GC was applied to the measurement of
picogram levels of clonidine hydrochloride after
derivatization ( 3 3 ) also by ( 3 4 - 3 6 ) .
Advantages o f fused silica capillary gas

chromatography (FSCC) for conventional GC method (37).
6.6.2. High-Performance Liauid ChmmatograDhp

( HPLC )
1) A rapid, reversed-phase high-performance liquid

chromatography (HPLC) method (38) is described for the
determination of clonidine in tablets. Individual
tablets or composite samples were sonicated in water,
diluted with methanol and filtered. Clonidine
formulated at 0.1 or 0.2 mg/tablet was chromatographed
on trimethylsilyl-bonded, 5 to 6-ym spherical silica
with 65% methanol in pH 7.9 phosphate buffer as mobile
phase detection at 254 nm. Mean recovery from 6
synthetic tablet samples was 99.7% (at 0.1 mg/tablet
level) with relative standard deviation of 1.55%.
2) A sensitive, selective and reproducible assay

for clonidine hydrochloride in tablets and eye drops
were described (39). A Nucleosil 5 Cis colum (125 mm x
4 . 6 mm - I.D.) with methanol-water 80:20 containing
0.005% of TEA as the mobile phase at a flow rate of 1
ml per minute at 240 nm U V detection and attenuation,
0.02 a . u . f . s . in tablets and 0.16 a.u.f.s. in eye
drops and recorder chart speed, 0.5 crn min-l.
144 M.A. ABOUNASSIF, M.S. MIAN. A N D N.A.A. MIAN
7. Acknowledgements
The authors are highly thankful to Mr. Babkir

Awad Mustafa, College of Applied Medical Sciences f o r
his efforts in drawing the spectras and figures, The
authors also would like to thank Mr. Tanvir A. Butt,
College of Pharmacy, King Saud University, for his
valuable and professional help in typing the
manuscript.
8. Beferences
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Pharmacists Inc. 4630 Montgomery Avenue, Bethesda M.D.
20814, p. 910-915.
2. Vivian Cody, presented in part at the American

Crystallographic Association Meeting, Clemenson, South
Carolina, January 1976,
3. Goodman, L . S. and Gi lman, A . "The Pharmacological

Basis of the Therapeutics", 4th ed., 1970, p. 7 3 5 .
4. "Martindale", The Extra Pharmacopoeia, 29th Ed. Editor

James E.F. Reynolds, The Pharmaceutical Press, London,
p. 472 (1989).
5. "Physician's Desk Reference", 42nd E d . , Medical

Economics Company, USA, p. 718 (1988).
6. "Clarke's Isolation and Identification of Drugs" 2nd

ed., A.C. Moffat Edit., p. 481-482. 'The
Pharmaceutical Press' London (1986).
7. "The Merck Index", 11th Ed., Merck and C o . , Inc.,

Rahway, N.J., p. 374 (1989).
a. "The British Pharmacopoeia", Her Majestys Stationary

Office, London, p. 147 (1988).
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Publishing Company Easton, Pennsylvania, 18042, p. 785
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Chemica Scandinavica, m,
p. 843-846 (1976).
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Molecular Structure, 9 ( 1 ) , p . 33-43 ( 1 9 7 9 ) .
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unpublished data ( 1 9 9 2 ) .
13. "The Organic Chemistry of Drug Synthesis" Vol. 1, p.

2 4 1 , by Daniel Lednicer, Lester A. Mitscher, John
Wiley dr Sons, New York.
14. M.S. Tawakkol, A.I. Jado and H.Y. Aboul-Enein,

Arzniem.-Forsch 31, 1064-66 ( 1 9 8 1 ) .
15. Sane, R.T. , Thombare, C.H. Indian D r u m , 1 8 ( 9 ) , 335-7

(1981).
16. Fawzy, A,, El-Yazbi, Mona Badair, and Mohamed A.

Analyst, Vol. III ( 1 9 8 6 ) .
17. Zivanov-Stakic, D., Panic, L . J . and Agbaba, G . Farmaco

45, 381-383 ( 1 9 9 0 ) .
18. Kovar, K.A. and Abdel-Hamed, M., Arch. Pharm., 317,

246 ( 1 9 8 4 ) .
19. Farina, P.R., Homon, C . A . , Chow, C.T., Keirns, J . J . ,

Zavorskas, P.A., Esber, H.J. Ther. Drug. Mointo.,
7 ( 3 ) , 344-350 ( 1 9 8 5 ) .
20. Arndts, D., Staehle, H. and Struck, C.J.,

Arznei,-Forsch., 2 9 ( 1 ) , 532-538 ( 1 9 7 9 ) .
21. Jarrot, Revyn, and Spectro, Sydney. J. Pharmacol. EXP.

Ther. 2 0 7 ( 1 ) , 195-202 ( 1 9 7 9 ) .
22. D. Rehbinder and W. Deckers. Arzneim.Forsch. (Drug

Rea), l9, 169 ( 1 9 6 9 ) .
23. D. Rehbinder, in A. Zahchett and M. Envico (Editors)

Ipertension Arteriosa, Boehringer Ingelheim, p . 3
( 1973).
24. J . P . Fillastre, D , Dubois and P. Brunelle, in A.

Zanchett, and Enrico (Editors) IDertension Arteriosa
Boehringer Ingelheim, p . 8 1 ( 1 9 7 3 ) .
25. S. Darda, in P. Millies and M. Safar (Editors). Recent

Advances in Hypertension, Boehringer, Ingelheim, p .
375 ( 1 9 7 5 ) .
26. Chu, L.C. , Bayne, W.F. , Tao, F.T., Schmitt, L.G. and
Shaw, J.E. J. Pharm. Sci. 6 8 ( 1 ) , 72-74 ( 1 9 7 9 ) .
27. Murray, S., Waddeil, K . A . and Davies, D.S. Biomed.

Mass SDectrom, 8 ( 1 0 ) , 500-502 ( 1 9 8 1 ) .
28. P.B.M.W.M. Timmermans, A. Brands and P.A. Van Zwieten.

J. Chromatogr., 144, 215-222 ( 1 9 7 7 ) .
29. Per Olof Edlund. J. Chromatogr., 187, 161-169 ( 1 9 8 0 ) .
30. Hiltunen, R . , Maryola, M., Hirsjarvi, P. and Raisanen,

S. Acta Pharm. Fenn., 8 8 ( 4 ) , 161-167 1 9 7 9 ) .
31. A.K. Cho and S.H. Curry, Biochem. Pharmacol., l8, 5 1 1

(1969).
32, C.T. Dollery, D.S. Davies, G.H. Draffan, H.J. Dargie,

C.R. Dean, J.L. Reid, R . A . Clare and '3. Murray, Clin.
Pharmacol. Ther., l8, 11-17 ( 1 9 7 6 ) .
33. S. Murray and D.S. Davies. Biomed. Mass Spectrom, 11,

435 ( 1 9 8 4 ) .
34. P.O. Edlund and L . K . Paalzow, Acta Pharmacol.

Toxicol., 40, 145-152 ( 1 9 7 7 ) .
35. A . K . Cho and S.H. Curry. Biochem. Pharmacol. 18, p.

511 (1966).
36. A. Frydman, Y. Weiss, M. Safar and J.M. Alexandre, in

P. Millies and M. Safar (Editors), Recent Advances in
Hypertension, Boehringer, Ingelheim, p . 369 ( 1 9 7 5 ) .
37. J . Settlage and H. Jaeger. J. Chromatogr. Sci. 22,

192-197 ( 1 9 8 4 ) .
CLONlDINE HYDROCHLORIDE I47
38. Walters, Stephen, M., Stonys, Dalia, B. J. Chromatom.

&, 2 l ( l ) , 43-5 ( 1 9 8 3 ) .
39. I. Wilezynska-Wojtulewicz and N. Sadlej-Sosnowska. JI

Chromatogr., 367, 434-437 (1986).
CYCLANDELATE
Charles M. Shearer
Wyeth-Ayerst Research
Rouses Point, NY 12979
ANALYTICAL PROFILES OF DRUG SUBSTANCES Copyright Q 1992 by Academic Press, Inc.

I50 CHARLES M.SHEARER
1. Description
I . 1 Name Formul a Mol ecul ar Weight
1.2 Appearance, Color and Odor
2. Synthesis
3.1 Nuclear Magnetic Resonance Spectra
3.4 Mass Spectrum
3.5 Melting Point
3.6 Di fferential Scanning Calorimetry
3.7 Solubility
3.8 Crystal Properties
4. Stability and Degradation
5. Metabolism
6. Analysis
6.2 U1 traviolet Spectrophotometry
6.3 Titrimetry
6.4 Gas Chromatography
6.5 High-Performance Liquid Chromatography
7. Identity
8. References
CY CLAN DELATE 151
1. Description
1.1 Name. Formula. M o l e c u l a r Weiqht
The name used by Chemical A b s t r a c t s f o r
c y c l a n d e l a t e i s a-hydroxybenzeneacetic a c i d , 3,3,5-
trimethylcyclohexyl ester. It i s a l s o c a l l e d mandelic acid,
3 , 3 , 5 - t r i m e t h y l c y c l ohexyl e s t e r ; 3 , 3 , 5 - t r i m e t h y l c y c l ohexyl
mandel ate; 3,3,5-trimethyl c y c l ohexyl amygdal a t e ; and 3,3,5-
t r i m e t h y l c y c l ohexanol a-phenyl -a-hydroxyacetate. Trade names
in c l ude, Cycl ospasmol , Nat i1, Novodi 1 , P e r e b r a l , and
Spasmocyclon (1). The Chemical A b s t r a c t s number i s 456-59-7.
1.2Appearance, C o l o r and Odor

C y c l a n d e l a t e i s a w h i t e t o o f f - w h i t e amorphous
powder w i t h a s l i g h t m e n t h o l - l i k e odor.
M. W. 276.36
'17"24'3
2. Synthesis
T r i m e t h y l c y c l o h e x y l mandelate was f i r s t s y n t h e s i z e d by
r e a c t i n g a - m a n d e l ic - a c i d w i t h 3 , 3 , 5 - t r i m e t h y l c y c l ohexanol
( c o n s i s t i n g o f c i s and t r a n s isomers) (2,3,4). C y c l a n d e l a t e
i s now s y n t h e s i z e d u s i n g o n l y t h e l o w m e l t i n g ( c i s ) isomer o f
3,3,5-trimethylcyclohexanol (5,6). E s t e r s o f m a n d e l i c a c i d
w i t h t h e h i g h e r m e l t i n g 3,3,5-trimethylcyclohexanol a r e t w i c e
as t o x i c as t h o s e made w i t h t h e l o w m e l t i n g isomer ( 7 ) . The
m a j o r s i d e r e a c t ion p r o d u c t , tri met h y l c y c l ohexyl phenyl
g l y o x a l a t e , can be removed d u r i n g t h e s y n t h e s i s by t r e a t i n g
t h e c r u d e c y c l a n d e l a t e w i t h aqueous sodium b o r o h y d r i d e (8) o r
z i n c and h y d r o c h l o r i c a c i d ( 9 ) .
T h i s s y n t h e s i s , u s i n g o n l y t h e c i s isomer, r e s u l t s i n
f o u r isomers as d e s c r i b e d i n t h e n e x t s e c t i o n .
152 CHARLES M.SHEARER
Figure 1 - Proton NMR Spectrum of Cyclandelate

(Wyeth-Ayerst Reference Standard No.
1361) in deuterated chloroform
CYCLANDELATE 153
Figure 2 - Carbon -13 NMR Spectrum o f Cyclandelate

1361) in deuterated chloroform
I54 CHARLES M.SHEARER
3. Phvsical ProDerties
3.1 Nuclear Maanetic Resonance SDectra
The f o u r isomers which make up c y c l a n d e l a t e a r i s e
i n t h e synthesis from t h e r e a c t i o n o f a - m a n d e l i c a c i d w i t h
-
cis-3,3,5-trimethylcyclohexanol and a r e described i n Table I
(taken from Nakamichi (10)).
Table 1
Isomers o f Cvcl andel a t e
Absolute c o n f i g u r a t i o n a Absol Ute c o n f i g u r a t i o n

Isomer o f mandelic a c i d moiety o f c y c l ohexanol moiety
Position 1 Position 5
A R R
B S S
C R R
D S S
a) The cyclohexanol m o i e t i e s o f A,C and B,D are l e v o r o t a t o r y
and d e x t r o r o t a t o r y , r e s p e c t i v e l y (11). The absolute
c o n f i g u r a t i o n o f ( - ) - c i s - 3 , 3 , 5 - t r i m e t h y l c y c l ohexanol i s
assigned as R on t h e basis o f i t s chemical c o r r e l a t i o n w i t h
p u l egone (12).
The p r o t o n NMR sample (Wyeth-Ayerst Reference Standard No.
1361) was d i s s o l v e d i n deuterated c h l o r o f o r m c o n t a i n i n g
tetramethyl s i l a n e as an i n t e r n a l standard. The spectrum was
obtained (13) on a 400 MHz Bruker spectrometer and i s
presented as Figure 1. The s p e c t r a l assignments are l i s t e d
i n Table 11. The C-13 NMR sample was a l s o prepared i n
deuterated chloroform and i t s spectrum obtained (13) on a
100 MHz Varian spectrometer. The spectrum i s presented as
F i g u r e 2 and t h e s p e c t r a l assignments are l i s t e d i n
Table 111. The spectra are i n agreement w i t h those o f
Nakamachi (10).
3.2 I n f r a r e d Soectrum
The i n f r a r e d spectrum o f a K B r p e l l e t o f
c y c l andel a t e (Wyeth-Ayerst Reference Standard No. 1361) was
obtained (14) on a N i c o l e t 20 DX instrument and i s presented
as Figure 3. The s p e c t r a l band assignments are g i v e n i n
Table I V .
CYCLANDELATE
4000 3000 2000 1500 1000 500

Wavenumber (crn-1)
Figure 3 - Infrared Spectrum o f Cyclandelate

1361) KBr pellet
156 CHARLES M. SHEARER
Table I 1
P r o t o n NMR S p e c t r a l Assisnments o f Cvcl a n d e l a t e
Chemical S h i f t Number o f Assignment

(ppm f r o m TMS) Protons
7.4 5 Aromatic CH
5.10 d 1 -H-C-OH
4.95 m 1 -H-C-OC
3.47 exchangeabl e 1 -H-0
2.1 - 0.6 17 A l i p h a t i c CH, CH CH3
0.94 s gem C I - J ~ (AB p a i g j
0.88 s gem CH3 (CD p a i r )
0.84 d ( J = 6) HC-CI-J3 (AB p a i r )
0.91 d (J = 6) HC-Cti3 (CD p a i r )
Table I11
Carbon-13 NMR S p e c t r a l Assisnments f o r Cvcl andel a t e
Carbon PPm
1 73.3
2 43.7 (AB) 43.2 (CD)
3 32.2 (AB) 32.1 (CD)
4 47.3
5 27.0 (AB) 26.9 (CD)
6 39.7 (AB) 40.1 (CD)
7 32.9 (AB) 32.8 (CD)
8 25.4 (AB) 25.3 (CD)
9 22.0 (AB) 22.1 (CD)
1 173.1
2 72.8
1 138.6
2, 6 126.3
3, 5 128.4
4 128.1
Table I V
I n f r a r e d S p e c t r a l Assisnments f o r Cvcl andel a t e
Wavenumber ( C m - l ) V i b r a t i o n Mode
3460 OH s t r e t c h
3100 - 2800 CH s t r e t c h
1730 CEO s t r e t c h
1212, 1192 C-0-C s t r e t c h
730, 695 o u t - o f - p l a n e bending of
monosubstituted aromatic
CYCLANDELATE 157
3.3 U l t r a v i o l e t SDectrum
The u l t r a v i o l e t spectrum o f c y c l a n d e l a t e (Wyeth-
A y e r s t Reference Standard No. 1361 r e c r y s t a l l i z e d t o remove
0.1% 3 , 3 , 5 - t r i m e t h y l c y c l ohexyl phenyl g l y o x a l a t e ) i n USP
e t h a n o l i s presented as F i g u r e 4. The a b s o r p t i v i t i e s a r e as
f o l l ows :
X max(nm) a €
269 0.57 1575
258 0.73 2020
251 0.59 1630
3.4 Mass SDectrum

The mass spectrum o f c y c l a n d e l a t e was o b t a i n e d (15)
by e l e c t r o n impact i o n i z a t i o n u s i n g a Finnegan MAT 8230
spectrometer and i s g i v e n as F i g u r e 5. I d e n t i f i c a t i o n o f t h e
p e r t i n e n t masses i s presented i n Table V .
Table V
Mass Spectrum Fraqmentation P a t t e r n o f Cvcl andel a t e
m/e Species
276 Mt
125 '9"17'
107 C6H5CHOHt
83 CH2CHCH2C ( CH3) *t
79 '6"5'
69 CH2CHCH2CHCH3t
55 ( CH3) C C H 2 t
3.5 M e l t i n g Ranqe
Observed (16) m e l t i n g range (USP I a ) f o r
c y c l a n d e l a t e (Wyeth-Ayerst Reference Standard No. 1361) i s
55.0" - 56.5"C.
I58 CHARLES M. SHEARER
Figure 4 - Ultraviolet Spectrum o f Cyclandelate

(Wyeth -Ayerst Reference Standard No.
1361) i n USP alcohol
CYCLANDELATE
20
111
0
50 100 150 200 250
mie
Figure 5 - Mass Spectrum o f Cyclandelate

1361)
3.6 Di fferenti a1 Scanninq Calorimetry

The DSC thermogram (14) for cycl andel ate (Wyeth-
Ayerst Reference Standard No. 1361) is presented as Figure 6.
The thermogram was obtained at a heating rate of lO'C/minute
in a nitrogen atmosphere utilizing a Perkin-Elmer DSC-2. The
thermogram exhibits no endotherms or exotherms other than
that associated with the melt.
3.7 Solubility
The following s lubi ties at room temperature have
been observed (16).
USP Classificat ons :
Sol vent Solubil itv
Water i nsoubl e
Methanol very soluble
Acetonitrile freely soluble
Ethyl acetate freely soluble
Di met hyl formami de freely soluble
To1 uene freely soluble
Chloroform very soluble
3.8 Crystal ProDerties

The X-ray powder diffraction pattern of
cycl andel ate (Wyeth-Ayerst Reference Standard No. 1361)
obtained (14) with a Phillips diffractometer using copper Ka
radiation is presented as Figure 7. The calculated "d"
spacings are given in Table VI.
Table V I
X-Ray Diffraction Pattern
d mo -d
m;
19.04 100 4.72 69
11.72 4 4.56 11
9.55 5 4.42 14
7.80 40 3.99 32
7.34 34 3.90 15
6.77 15 3.85 13
6.11 21 3.77 17
5.59 13 3.71 15
5.27 9 3.57 8
4.97 21 2.65 8
CYCLANDELATE 161
I I I I
20 40 60 80 100 120
Temperature (C)
Figure 6 - Differential Scanning Calorimetric

Thermogram of Cycl andelate (Wyeth-Ayerst
Reference Standard No. 1361)
I62 CHARLES M. SHEARER
4 13 22 31 40
DEGREES 2 THETA
Figure 7 - X-Ray D i f f r a c t i o n Pattern o f Cyclandelate

(Wyeth-Ayerst Reference Standard No. 1361)
CYCLANDELATE 163
4. Stability and Desradation

Cycl andel ate can decompose by hydrolysis to mandel ic
acid and 3,3,5-trimethylcyclohexanol (17). It is oxidized to
3,3,5-trimethyl cyclohexyl phenylglyoxal ate (18).
A study of the formation of 3,3,5-trimethylcyclohexanol
in cyclandelate capsules concluded that less than 5% of the
cycl andel ate degraded in 66 months at ambient temperatures
(17) *
5. Metabol i sm
The metabolites of cyclandelate are mandelic acid,
phenylglyoxyl ic acid and 3,3,5-trimethylcyclohexanol. These
are detectable in the urine of rabbits and humans in less
than two hours after oral administration (19,20). The
ratio o f mandelic acid to phenylglyoxylic acid increases with
increased dosage (21). Another metabolic study in humans
showed that the maximum blood levels of mandelic acid were
reached in 0.5 to 1.5 hours after oral dosing (22).
A pharmacokinetic study using tritiated cyclandelate
shows that most organ specimens took up the radioactivity
rapidly; usually reaching a maximum within one hour. The
brain, diaphragm, stomach and vein specimem showed a maximum
level at 24 hours. The levels gradually declined in a non-
linear manner over 28 days (23).
6. Analysis
Element Theory Found (24)
C 73.88% 73.95%
H 8.75% 8.55%
6.2 Ultraviolet SDeCtrODhOtOmetrY
Di rect determination of cycl andel ate by UV
spectrophotometry is not practical since the oxidative
degradation product, 3,3,5-trimethyl cycl ohexyl
phenylglyoxalate has about 55 times the absorptivity (25).
Spectrophotometri c determinations of cycl andel ate after
hydrolysis to mandel ic acid and oxidation to benzaldehyde
have been reported (26,27).
164 CHARLES M.SHEARER
6.3Titrimetrv
Cycl andel ate can be determined by hydrolyzing the
ester in 0.5 N NaOH under reflux for 0.5 hours, then
backtitrating the excess base with 0.1 N HC1 (28,29).
6.4 Gas ChromatoqraDhv
Gas chromatography has been used to analyze
cycl andel ate and to separate it from its degradation products
and impurities as well as from other pharmaceuticals. Table
VI gives column conditions and other necessary data for the
various methods.
Table V I
Gas ChromatoqraDhv of Cvcl andel ate
Column Oven Reference
Temoera t ure
2 rn x 4 mm i.d.; 5% Q F - 1 on 160" (30)
Chromosorb W(HP) 100/200 mesh
6 ft x 1/8 in; 3% QF 1 t 0.5% 200 O
(31)
HiEFF 8BP on GasChrom Q
25 m x 0.3 mm i.d.; deactivated, 125" for (32)
coated w/S E - 3 0 13 min; 3'/min
to 180", hold
1 min.
30 m x 0.28 mm i.d.; FFAP 170" (10)
6 f t x 1/4 in i.d.; 15% Dexsil 220' (33)

300 on HP Chromosorb W 80/100 mesh
1 m x 3.2 mm; Tenax GC 60/80 mesh 140" for (34)

5 min., 20'/min
to 240", lO'/min
to 280"
6 ft x 4 mm i.d.; 2.5% SE30 on 200 O (35)
80/100 mesh Chromosorb G
CYCLANDELATE I65
6.5 Hiqh-Performance L i a u i d ChromatoqraDhv

An HPLC system c o n s i s t i n g o f a Microbondapak CN (30
x 0.39 cm.) column, 65% methanol, 35% sodium a c e t a t e b u f f e r ,
a d j u s t e d t o pH 3.7 as t h e e l u a n t : and 254 nm UV l i g h t f o r
d e t e c t i o n has been used (36).
6.6 T h i n Laver ChromatosraDhy

The f o l l owing TLC systems have been r e p o r t e d :
P1 a t e s S o l v e n t Svstem R f Value Reference
S i l i c a Gel 254 Benzene (37)
S i l i c a Gel 254 Hexane 55 0.09 (38)

C h l o r o f o r m 45
Silica G Chloroform 4 0.74 (39)

Acetone 1
Silica G Ethyl Acetate 0.71 (39)

7. Identity
C y c l a n d e l a t e can be i d e n t i f i e d amongst many o t h e r drugs,
p o i s o n s and b i o g e n i c compounds by gas chromatography ( 3 3 ) .
D e t a i l s f o r t h i s procedure a r e g i v e n i n S e c t i o n 6.4. Several
odor and c o l o r i d e n t i f i c a t i o n t e s t s a r e g i v e n by Doorenboos
and coworkers (28).
8. References
1. The Merck Index, 1 1 t h ed., Merck and Co., Rahway NJ,

(1989) page 421.
2. N. V . K o n i n k l i j k e Pharmaceutische Fabrieken voor.

Brocades-Stheeman & Pharmacia, Dutch P a t e n t 68,704.
3. K. J. H. van S l u i s , Chemical Products, 11, 374(1954).

4. N. V . K o n i n k l i j k e Pharmaceutische F a b r i e k e n voorheen
Brocades-Stheeman & Pharmacia, B r i t i s h P a t e n t
707,227.
5. A. B. H. Funcke, M. J . E. E r n s t i n g , R. F. Rekker, and

W . Th. Natua, A r s n e i m i t t e l - F o r s c h . , 3, 503(1953).
6. N. V . K o n i n k l ij k e Pharmaceutische F a b r i e k e n voorheen
Brocades-Stheeman & Pharmaci a, B r i t i s h P a t e n t
810,888.
7. N. V . K o n i n k l i j k e Pharmaceutische Fabrieken voorheen
Brocades-Stheeman & Pharmacia, Dutch P a t e n t 88,249.
8. D. F l i t t e r , U n i t e d S t a t e s Patent 3,663,597.
9. H. Takahashi, U n i t e d S t a t e s P a t e n t 3,673,239.
10. T. Amano, T. Kasahara and H. Nakamachi, Chem. Pharm.

B u l l . , 3, 1106(1981),
11. M. J . E. E r n e s t i n g and W. Th. Nauta, Rec. Trav. Chim.

Pay-Bas., 8 l , 751 (1962).
12. N. L. A l l i n g e r and C . K. Riew, J . Org. Chern.,
-
40, 1316 (1975).
13. B. Hofmann, Wyeth-Ayerst L a b o r a t o r i e s , Personal
Communication.
14. C. L o n g f e l l o w , Wyeth-Ayerst L a b o r a t o r i e s , Personal

Communication,
15. J . Cantone, Wyeth-Ayerst L a b o r a t o r i e s , Personal
Communication.
16. D. Berg, Wyeth-Ayerst L a b o r a t o r i e s , Personal

Commun ic a t ion,
17. 3. R i c h a r d and G. Andermann, Pharm. A c t a Helv.,

-
57, 116(1982).
18. M. J . E. E r n s t i n g , R. F . Rekker, J . H. Bos and
W . Th. Nauta, Pharm. Weekblad, a,605(1953).
19. M. J . E. E r n s t i n g , R. F . Rekker, A. 6. H. Funcke,
H. M. Tersteege, and W . Th. Nauta, A r s n e i m i t t e l -
Forsch, 6 , 245(1956).
20. M. J . E. E r n s t i n g , R. F. Rekker, H. M. Tersteege, and

W. Th. Nauta, A r s n e i m i t t e l - F o r s c h , l2, 632(1962).
CYCLANDELATE I67
21. M . J . E. E r n s t i n g , R. F . Rekker, H. M . Tersteege and

W . Th. Nauta, A r s n e i m i t t e l - F o r s c h , l2, 853(1962).
22. K. Kojima, Y, Uezono, T. Takahashi and Y. Nakanishi,

J . Chromatogr., 425, 203(1988).
23. A. O r r and J. R. W h i t t i e r , I n t . J . Nucl. Med. B i o l . ,
-4, 205( 1974).
24. C. Kraml, Wyeth-Ayerst L a b o r a t o r i e s , Personal
Communication.
25. R.F. Rekker, H. J . Doorenbos and W. Th. Nauta, Pharm.

Weekbl ad, lO2,946( 1967).
26. L. Chafetz, J . Pharm. S c i . , 53, 1192(1964).
27. 6. Andermann, M. D i e t z , and D. Mergel, J. Pharm.

B e l g . , 3 4 , 233( 1979).
28. H. J . Doorenbos, H. J . van d e r Pol, R. F . Rekker and

W. Th. Nauta, Pharm. Weekblad, 100, 633(1965).
29. J . Zhou, and C . Zhou, Yiyao Gongye, l7, 369(1986)

f r o m CA( 26) :232528t.
30. R.T. Sane, V.B. Malkar, and V . G . Nayak, I n d i a n Drugs,

-
22, 321(1985) f r o m CA103(16):129151z.
31. D. Rodgers, Wyeth-Ayerst L a b o r a t o r i e s , Personal

Communication.
32. G. Andermann and M. D i e t z , J Chromatogr., 223,

365 (1981) .
33. B. Kaempe, Arch. Pharm. Chem , S c i . Ed., 2,

145(1974).
34. M. D i e t z and G. Andermann, J . High. Resol.

Chromatogr. & Chromatogr. Comm., 2, 635(1979).
35. B. S . F i n k l e , E. J . Cherry and D. M. T a y l o r ,

J . Chromatogr. S c i . , 9, 393(1971).
36. R. T . Sane, S . V . Desai and R. S . Samant, Indian

Drugs, a, 42(1986).
37. M. He and X . L i , Yaowu Fenxi Zazhi, 4, 40(1984) from
CAlOO(18): 1 4 5 0 7 5 ~ .
38. B. Kennedy, Wyeth-Ayerst L a b o r a t o r i e s , Personal
Communication.
39. A. H. Stead, R. G i l l , T. Wright, J . P. Gibbs and
A. C . Moffat, Analyst, 107, 1106(1982).
FLECAINIDE
Silvia Alessi-Severini, Ronald T.Coutts,
Fakhreddin Jamali, and Franco M. Pasutto
Faculty of Pharmacy & Pharmaceutical Sciences
University of Alberta
Edmonton, Alberta, Canada, T6G 2N8
ANALYTICAL PROFILES OF DRUG SUBSTANCES Copyright D 1992 by Academic Press, Inc.

AND EXCIPIENTS - VOLUME 21 169 All rights of reproductionresewed in any form.
SILVIA ALESSI-SEVERINI ET AL.
TABLE OF CONTENTS
1. Description
1.1 Nomenclature, Formula and Molecular Weight
1.2 Appearance, Color, and Odor
1.3 History
2. Synthesis
2.1 Synthesis of Flecainide Acetate
2.2 Preparative Separation of Flecainide Enantiomers
3.2 NMR Spectra
3.3 Mass Spectrum
3.5 Optical Rotation and Absolute Configuration
3.6 Melting Point
3.7 Ionization Constant
3.8 Distribution Coefficient
3.9 Solubility
3.10 Stability
4.1 Elemental
4.2 Spectrophotofluorometry
4.3 Fluorescence Polarization lmmunoassay
4.4 Chromatographic Assays
4.4.1 Stereospecific
4.4.2 Non-stereospecific
5. Pharmacodynamics and Pharmacokinetics
6. References
FLECAlNlDE 171
1. DescriDtion
1.1 Nomenclature, Formula and Molecular Weiaht
(f)-Flecainide acetate, USAN, INN; described as R-818

in the early literature; ( f )-N-(2-piperidinylrnethyl)-2,5-bis(2,2,2-
trif1uoroethoxy)benzamide acetate. The terms flecainide and
flecainide acetate refer to the respective racemates unless
otherwise specified.
Registry No.: ( f 1-flecainide acetate 99495-88-2;( f 1-

flecainide free base 99495-87-1 +
; ( )-flecainide acetate 99495-
93-9;( + )-flecainide free base 99495-92-8; (-1-flecainide acetate
99495-94-0; (-)-flecainide free base 99495-90-6.
Free base c17H20F6N203 M.W. 414.36

Acetate c17H20F6N203 C2H402 M.W. 474.40
1.2 Amearance. Color. and Odor
Free base: white granular solid from

isopropanol/isopropyI ether; odorless. Acetate: white crystalline
solid.
1.3 History
Flecainide acetate is a class Ic antiarrhythmic agent

which was developed in the Riker Laboratories as part of a
broad-based project investigating the effect of fluorine
substitution on local anaesthetic or antiarrhythmic activity. The
details concerning the development of this drug have been
reported l .
I72 SILVIA ALESSI-SEVERINIET AL.
2. Svnthesig
2.1 Svnthesis of Flecainide Acetate (Fiaure 11
Trifluoroethylation of 2,5-dihydroxybenzoic acid affords

2,2,2-trifluoroethyl 2,5-bis(2,2,2-trifluoroethoxy)benzoate. This is
slowly added to a solution of 2-aminomethylpyridine in glyme,
under N2, at 25OC. After stirring for 20 hr the reaction mixture is
refluxed (3 hr), cooled, and the solvent evaporated in vacuo.
Recrystallization of the residue from benzene-hexane gave N42-
pyridylmethyl)-2,5-bis(2,2,2-trifluoroethoxy)benzamide as an off-
white solid (map103-105°C) in 91 % yield. This was dissolved in
acetic acid and reduced over Pt02 in a Parr hydrogenator.
Filtration of the catalyst and evaporation of the filtrate gave a
viscous syrup which solidified on trituration with isopropyl ether.
Crystallization from isopropanol-isopropyl ether gave flecainide
acetate as a white granular solid (m.p. 145-147OC) in 75%
yield2.
FIGURE 1. Synthesis of Flecainide Acetate
2.2 PreDarative SeDaration of Flecainide Enantiomers
+
( )-Flecainide has been obtained as a salt of ammonium
( + )-ar-bromocamphor-7-sulfonate while (-1-flecainide was isolated
FLECAINIDE I73
with ammonium (-)-a-bromocamphor-?r-sulfonate. In both cases

salt formation was accomplished in methanol. The respective
enantiopure flecainide free bases are readily recovered by
treatment of the salts with dilute alkali. Subsequent reaction with
acetic acid affords the enantiopure flecainide acetate$.
Flecainide has also been similarly resolved by preparation of
diastereomeric salts (in ethyl acetate) of enantiopure mandelic
acids followed by fractional crystallization4,
3. Phvsical ProDertieg
3.1 Infrared Soectrum
The infrared spectrum of flecainide free base (KBr disc)

was recorded on a Nicolet 7199 Fourier Transform spectrometer
and is presented in Figure 2. The principal absorption bands
include (cm-l):
3427 1291
3358 1221
2927 1169
1637 1154
1606 1083
1549 978
1500 863
1458 657
Some suggested assignments include: broad peak

centered at approximately 3400 cm-1 (N-H), 2927 (aliphatic C-
HI, 1637 (amide I band), 1606 (C=C), 1549 (amide II band),
1500 (C=C str). Aryl ether and CF3 absorptions appear in the
same general region; CF3 groups typically show absorptions
between 1120-1350 and 690-770. Ar-0-CH2 is usually evident
as absorptions between 1200-1275 and 1020-1075 cm-1.
3.2 NMR SDectra
Proton, carbon-1 3, and fluorine-1 9 spectra of flecainide

free base, in CDC13, were obtained on a Bruker AM-300 FT NMR
spectrometer. The respective spectra are illustrated in Figures 3,
FIGURE 2. IR Spectrum of Flecainide Base. KBr Pellet.
FLECAINIDE I75
4, and 5 and assignments presented in Tables 1, 2, and 3.
TABLE 1
300 MHz Proton NMR Spectrum of Flecainide Base in CDC13
Chemical Shift Number of Assignment

(ppm from TMS) Protons
7.76 broad 1 CONH

7.76 d (J= 3) 1 aromatic C6 H
7.09 d,d (J= 9, 3) 1 aromatic C4 jj
6.9 d (J= 9) 1 aromatic C3 li
4.44 q (JH,F = 8) 2 c2 o c y 2
4.36 q (J,,,= 8) 2 c5 ocy2
3.48 m 1 CONHCH2
3.30 m 1 CONHCH2
3.06 m 1 piperidine C H
2.75 m 1 piperidine NCH2
2.62 m 1 piperidine NC&
2.1-1.08 m 7 NH, piperidine (CH2)3
TABLE 2
75 MHz Carbon-13 NMR Spectrum of Flecainide Base in CDC13 a
14 16 3
OCH~CF~
16 17
Chemical Shift b Assignment C
24.34 3
26.49 2 or 4
30.57 2 or 4
PPM
FIGURE 3. Proton NMR Spectrum of Flecainide Base in CDCJ3.
FLECAINIDE 177
46.04 1 or6
46.68 1 or6
55.83 5
65.72
66.19
66.52
66.67 1 4 a n d 16
67.00 (C-C-F coupling)
67.14
67.47
67.95
77 CDC13
114.88 12
1 1 7.22 9
120.49 11
1 17.47
1 1 7.67
121.15
121 -36 15 and 17
124.84 (C-Fcoupling)
125.04
128.52
128.73
124.31 8
150.23 10
153.00 13
163.99 7
a In Figure 4 the CH3 and CH groups are shown as signals

possessing an anti-phase with respect to the CDC13 signal,
while quaternary carbons, CH2 and carbonyls are in phase.
b ppmfromTMS
C carbon numbering as shown in structure

FIGURE 4. Carbon-13 NMR Spectrum of Flecainide Base in CDC13.
E9.S 89.0 88.5 8B.m 87.5 E7.m 86.5 86.8 85.5
PPM
FIGURE 5. Fluorine 19 NMR Spectrum of Flecainide Base in CDC13.

I80 SILVIA ALESSI-SEVERINI ET AL.
TABLE 3
282 MHz Fluorine-19 NMR Spectrum of Flecainide Base in CDC13
Chemical Shift a Assignment
87.1 1
87.14 CF3
87.17
87.44
87.46 CF3
87.49
a external standard CgFg
3.3 Mass SDectrum
The positive ion electron impact mass spectrum was

recorded on a Kratos MS 50 double focusing magnetic sector
mass spectrometer. Operating conditions: mass range 31.01 84-
415.1471, sampling rate 25, signal level threshold 1, minimum
peak width 7, scan rate (sec/dec) 10.0, # of scans averaged 9.
High resolution MS: M + calculated, 4 14.1378; found,
41 4.1351, Mass spectral data and suggested structures for
fragment ions are shown in Figure 6.
Significant Ions Measured Mass %Relative

Abundance
c 1 7H19N203F6 413.1290 0.17
c1 1H7°3F6 301.0295 3.64
CgH1 1 97.0889 10.17

FLECAlNlDE
96.081 2 2.08
91.0546 2.51
84.0814 100.00
5 6.0520 6.26
CEO+
I
H
m h 301
mlz 97
0 N
1
H
N
I
H
m h 84 mlz 5 6
FIGURE 6. Mass Spectral Data
3.4 Ultraviolet Saectrum
The ultraviolet spectrum of flecainide base in ethanol

(0.0016 g m / l 0 0 ml) is shown in Figure 7. The absorptivities are:
hmax E (1%: 1 cml
205 521
230 (shoulder) 219
300 59
3.5 Ootical Rotation and Absolute Confiauration
Optical rotations (sodium D line, 1 dm cells, methanol as

solvent) were obtained with a Perkin Elmer Model 241
182 SILVIA ALESSI-SEVERINI ET AL.
+ +
+ +
350. + f + +
4 68
FIGURE 7. Ultraviolet Spectrum of Flecainide Base.

FLECAINIDE I83
polarimeter. Optical purity of flecainide free base enantiomers

was >99% as determined by 100 MHz NMR using the chiral
shift reagent tris[3-heptafluorobutyryl-d-camphoratoleuropium
1113.
( +
1-flecainide free base [a]26, +3 . 4 O
(-1-flecainide free base -3.3O
( +)-flecainide acetate [aI2'D +4.6O

(-1-flecainide acetate [aI27, -4.50
The absolute configurations of flecainide enantiomers

have been determined on the basis of CD spectra of the N-chloro
+
derivatives. Thus, ( )-flecainide has the S-configuration while
the antipode is R-(-)-flecainide, The optical rotations of
hydrochloride salts were also reported4.
+
( 1-flecainide HCI [a12036s + 20.0°
(- 1-f Ieca in ide HC I [a]
20365 -2O.OO
3.6 Meltina Points
( f 1-flecainide freebase 105-107°C

( + 1-flecainide freebase 104-105°C 3
(-1-flecainide free base 102-104OC 3
( f )-flecainide acetate 145-147OC

( + )-flecainide acetate 153-155OC 3
(-)-flecainide acetate 152.5-1 54OC 3
( + 1-flecainide HCI 222-225OC 4

(-1-flecainide HCI 223-225OC
3.7 Ionization Constanf
The pKa of flecainide acetate has been determined5 as

9.3.
3.8 Distribution Coefficient
The octanoVwater partition coefficient was determined6

to be 11.4at pH 7.4and logP calculated7 as 4.50.
3.9 Solubility
The solubility of flecainide acetate at 37°C is 48.4

mg/ml in water and 300 mg/ml in alcohol5.
3.10 Stabilitv
A solution of flecainide acetate in water has been
reported to be very stable at room temperatures. The stability in
biological fluids seems t o be significantly decreased over a period
of 3 months even under storage at -2O"Cg. The tablet
formulation must be stored in light resistant containers at 15-30'
c5.
4. f Anal is
4.1 Elemental
The calculated elemental analysis for flecainide

[Cl7H2oF$J2031:
C 49.28%
H 4.87%
N 6.76%
0 11.58%
F 27.51%
The calculated elemental analysis for flecainide acetate

[C17H20FgN203. C2H402I:
C 48.10%
H 5.10%
N 5.91%
0 16.86%
F 24.03%
FLECAINIDE 185
A method for the determination of (*I-flecainide in

plasma utilizes the natural fluorescence of the molecule.
Flecainide is extracted from plasma with heptane after addition
of 0.5 mol/L Na3P04 and triethylamine. The organic phase is re-
extracted with 0.25 mol/L NaH2P04 and the aqueous phase is
read in the spectrophotofluorometer (300 nm excitation
wavelength, 370 nm emission). The sensitivity is reported to be
25 ng/ml per 2 ml of plasmalo.
4.3 Fluorescence Polarization lmmunoassay
Direct determination of ( f )-flecainide in plasma is

possible through the utilization of a commercially available
fluorescence polarization immunoassay (Abbott). The reaction is
based on the competitive binding of free and fluorescein-labeled
flecainide to specific antibodies. Fluorescence polarization
measurements are dependent upon the concentration of the free
drug in the sample. The method can be performed on 5 0 pL of
plasma, with a sensitivity of 0.1 pg/ml, and is very convenient
for therapeutic drug monitoring’ l .
4.4 Chromatoarmhic Assavs
4.4.1 StereosDecific
Sample preparation and chromatographic

conditions are summarized in Table 4.
4.4.2 Non-stereomecific
Sample preparation and chromatographic

conditions are summarized in Table 5.
5. Pharmacodvnamics and Pharmacokinetics
The pharmacodynamics and pharmacokinetics of ( f )-

flecainide acetate have been studied extensively in animal models
and in humans. This drug exhibits potent antiarrhythmic effects
TABLE 4: STEREOSPECIFIC ANALYTICAL METHODS
EXTRACTIONlDERlVATlZATION COLUMNlMOBlLE PHASE DETECTION a R REF

APPROXIMATE RETN TIME SENSITIVITY
( + 1-1-phenylethyl isocyanate silica (250 x 4.6 mm) 1.19 2.37 4

hexane:EtOAc (55:45)
1. tetra-O-acetyl-P-D-glucopyranosyl isothio- C18 1. 1.16 12

cyanate MeOH:H20 2. 1.05
2. S-l-(l-naphthyl)ethyl isothiocyanate 3. 1.00
3. R-l-(2-naphthyl)ethyl isothiocyanate 4. 1.04
4. R-or-methylbenzylisothiocyanate
serum mixed with MeCN, supernatant C18 (100 x 3mm) fluorescence (300ex. baseline 13
evaporated; R( + 1-1 -phenylethyl isocyanate MeOH:H20:HOAc (60:40:1) 370em); 0.05 mg/L resolved
20 min
plasma with butyl chloride:2-propanol silica (250 x 4.6 mm) fluorescence (305ex, 1.08 14
(95:5); (-)-menthy1chloroformate hexane:EtOAc:Et3N(84:16:O.l) 340em); 2.5 ng/ml
22 min UV (298); 40 ng/ml
plasma and urine with diethyl ether; 1-1(4- C18 (300 x 3.9 mm) UV (280); 50 ng/ml 1.07 1.25 15
nitrophenyl)sulfonyll-L-prolyl chloride MeCN:H,O:Et,N (45:55:0.2)
3 0 min
plasma with 1% 2-propanol in n-hexane; SE 3 0 fused silica1 GC capillary negative ion chem- 1. 1.39 16
1. RWphenylbutyric anhydride column (25 m) ical ionization mass 2. 1.43
2. R( + 1-1 -MeO-1(CF31phenylacetylchoride spectrometer; 0.41 3.3.38
3. N-trifluoroacetyl-L-prolyl chloride ng/ml 4. 1.14
4. f-butyloxycarbonyl-L-alanine
plasma with 1% 2-propanol in n-hexane; Chirasil-L-Val fused silica GC negative ion chem- R = 1. l - 1 .6 16
pentafluoropropionic anhydride capillary column (25 m); XE- ical ionization mass on either col-
60-(R)-phenylethylamide glass spectrometer; < 0.4 umn
capillary column (29 m) ng/ml
urine with EtOAc; (-)-menthy1chloroformate silica (250 x 4.6 mm); hexane: fluorescence (290ex, baseline 17
2-butanol:MeCN(98.75:1:0.25) 340em); 25 ng/ml resolved
20 min
TABLE 5 : NON-STEREOSPECIFIC ANALYTICAL METHODS
EXTRACTlONlDERlVATlZATlON COLUMNlMOBlLE PHASE DETECTION REF

APPROXIMATE RETN TIME SENSITIVITY
plasma deproteinized, pH adjusted, super- C18 pBondapak (150 x 4 mm) fluorescence (300ex, 18
natant injected ammonium phosphate buffer:MeOH 370em); 50 ng/ml
(60:40); 6 rnin UV (280)
plasma or urine washed and extracted with Zorbax TMS (150 x 4.6 mm) UV (308); 22 ng/ml 19
hexane MeCN:l% HOAc in 0.01M pentane-
sulfonate (45:55); 5 min
plasma or serum extracted with methyl r- Spherisorb S5W silica (125 x 5 mm) fluorescence (200ex. 20
butyl ether MeOH:2,2,4-trimethylpentane (80:20) no emission filter);
containing d-10-camphorsulfonic acid; 20 pg/L
4 rnin
plasma extracted with hexane pBondapak phenyl (300 x 3.9 mm) fluorescence (300ex. 21
MeCN:0.06% H,P04 (40:60); 5.5 rnin 370em); 3 ng/ml
solid phase extraction of plasma with C8 pBondapak phenyl (300 x 3.9 mm) fluorescence (300ex. 22
adsorbent MeCN:0.06% H,P04 (40:60); 5.5 rnin 370eml; 3 ng/ml
UV (298); 50 ng/ml
diethyl ether extraction of plasma then back pBondapak C18 (300 mm) UV (214); <30 ng/ 23
extracted into dilute phosphoric acid MeCN:H20 (30:70) containing dibutyl- 0.5 ml
amine phosphate; 7 min
solid phase extraction of plasma with C8 Radial-Pak C18 (100 x 8 mrn) fluorescence (293ex, 24
adsorbent MeOH:25% ammonia (99.9:O.l); 7 340em); 10 ng/ml
min
serum or plasma extracted with methyl r- octyl microbore column (250 x 2 mm) UV (298); 80 pg/L 25
butyl ether 0.05% triethylamine in MeCN:O. 1M (250 pL plasma)
sodium acetate (45:55); 11 min
plasma extracted with 1-chlorobutane then phenyl reverse phase (300 x 3.9 mm) UV (297); 0.033 26
back extracted into dilute phosphoric acid MeCN:20 mM sodium acetate mg/L
(42:58); 10 min
serum or plasma extracted with methyl t- octyl microbore column (250 x 2 mm) fluorescence (300ex, 27
butyl ether 0.05% triethylamine in MeCN:O.lM 370em); 20 pg/L
sodium acetate (40:60); 8 rnin (100 pL plasma)
microscale protein precipitation of serum Nucleosil 5 C18 (150 x 4.2 mm) fluorescence (285ex. 28
with Zn sulfate/MeCN, supernatant injected 300 ml MeCN, 1 ml H,PO,, 0.5 ml 370em); 30 ng/ml
diethylamine in 1L H,O; 5 min
-
m
rD
plasma, urine or saliva extracted with diethyl 3% SP-2250 GC column (180 cm x 2 electron capture 29
ether, back extracted into 0.5M HCI, mm); 16 min detection; 12.5
basified, derivatized with pentafluorobenzoyl ng/ml
chloride, extracted into hexane
by blocking the cardiac sodium channels and by stabilizing the

myocyte membrane without affecting the repolarization30-34, It
is used in the suppression and control of ventricular and
supraventricwlar a r r h y t h m i a ~ 3 0 - ~and
~ it has shown some
effectiveness in the treatment of atrial arrhythmia~3~-39. The
drug is not recommended in non-symptomatic post-infarction
patients because of potentially life threatening t o ~ i c i t y . ~ 0 # ~ 1
Flecainide acetate is rapidly and almost completely absorbed
from the GI tract following oral administration. Absolute
bioavailability of the commercailly available tablets averages 85-
90%. First pass metabolism is negligible. Plasma concentrations
must be kept within the 200-1000 ng/ml range in order t o
maximize the therapeutic effect and minimize the risk of serious
side e f f e c t ~ 3 0 - 3 ~Plasma
. levels and dose show a linear
correlation42, however considerable inter- and intra-individual
variations have been observed and, most recently, evidence on
non-linear kinetics has been reported43. After i.v. administration
to humans, flecainide is rapidly and apparently widely distributed
(Vd ranges from 5-13.4 L/kg; average 5.5-8.7 L/kg). After an
oral dose the Vd has been determined t o be 10 L/kg44. After i.v.
administration t o rats, flecainide is distributed extensively t o
many tissues, including the heart, but only minimally into the
CNS, and dose-dependent tissue uptake has been shown in
rabbits after chronic a d m i n i ~ t r a t i o n ~ 5 , ~The
6 . in vitro protein
binding (mainly a1 -acid glycoprotein) is approximately 40-50%
and is independent of the drug plasma concentration30-34, The
pharmacokinetic profile after i.v. administration follows a two-
compartment open model with tr/,ar = 3-6 min and a tr/,B = 11-
14 hr (range 7-19 hr)44. Elimination half-life is prolonged in
arrhythmias ( 19 hr), in renal and hepatic impairment (26-49 hr),
and in congestive heart failure (up to 50 hrl44f47-52. Urinary pH
affects elimination half-life, prolonging it when alkaline (pH 7.2-
8.3) and reducing it when acidic (4.4-5,8)53-55.
Flecainide is extensively metabolized by the liver. The t w o
major metabolites are m-0-dealkylated flecainide and the m-0-
dealkylated lactam derivative. These are formed by preferential
0-dealkylation at the meta-position of the benzamide ring and by
subsequent oxidation of the piperidine ring of m-0-dealkylated
flecainide, respectively. Both metabolites undergo extensive
sulfate and glucuronide conjugation at the m-0-dealkylated
FLECAINIDE 191
position. About 80-90940 of a dose is recovered in the urine (30%

as unchanged drug, the rest as metabolites and their
conjugates). Only 5% is excreted in the faeces and 3% of the
total excretion is attributed to at least three unidentified
metabolites44.
Hemodialysis removes about 1% of a dose as unchanged
drug. Total apparent plasma clearance of flecainide, by healthy
subjects, following oral administration, has been reported to be
in the range of 4-20 ml/kg. Renal clearance of the drug is 25-
40% of total plasma clearance. Total apparent plasma clearance
is decreased in arrhythmias, congestive heart failure and renal
impairment30-34,47-50,52,
There are comparatively few reports concerned with the
stereoselective pharmacodynamics and pharmacokinetics of
flecainide enantiomers. ln vivo studies have revealed equipotency
and equiactivity for the two enantiomers in mouse and dog
models of arrhythmia3. ln vitro tests on canine Purkinje fibres
have also shown that the enantiorners have comparable
electrophysiologic a~tivities5685~. The t w o enantiomers have
also shown similar affinities to a receptor site associated with
cardiac sodium channels in isolated rat cardiac m y o c y t e ~ ~ ~ .
The pharmacokinetic patterns of the enantiomers following
oral administration in humans appear to be essentially parallel.
The plasma R/S ratio seems to range from 0.67-1.44 in different
patient populationsl3#56,59, Stereoselective elimination has been
suggested in healthy subjects, which have been classified as
poor metabolizers of the sparteine debrisoquine type; R-flecainide
is predominant in plasma after oral administration o f the
racemate. The oral AUCs of the enantiomers as well as the
elimination half-lives were slightly, but significantly, different.
This has been interpreted as being the result of stereoselective
hepatic metabolism1 7.
6. References
1. J.M. Hudak, E.H. Banitt, J.R. Schmid, Am. J. Cardiol., 53,

17B-20B (1984).
2. E.H. Banitt, W.R. Bronn, W.E. Coyne, J.R. Schmid, J. Med.
Chern., 20, 821-826 (1977).
192 SlLVlA ALESSI-SEVERINI ET AL.
3. E.H. Banitt, J.R. Schmid, R.A. Newmark, J. Med. Chem.,

-
29, 299-302 (1986).
4. G. Blaschke, U. Scheidemantel, B. Walther, Chem Ber 118
461 6-4619 (1985).
5. American Formulary Service. Drug Information '88,
American Society of Hospital Pharmacists, Bethesda, MD,
832-840.
6. L. Lie-A-Huen, J.H. Kingma, Eur. J. Clin. Pharmacol., 35 89-
91 (1988).
7. Comprehensive Medicinal Chemistry, Vol. 6, pg. 526, C.
Hansch (Chairman, Editorial Board), Pergamon Press,
Oxford, U.K., 1990.
8. Selvi-3M, Milan, Italy. Personal communication (1988).
9. Unpublished data from our laboratories.
10. S.F. Chang, A.M. Miller, J. Jernberg, R.E. Ober, G.J.
Conard, Arzneim.-Forsch./Drug Res., a
251-253 (1983).
11. R.E. Coxon, A.J. Hodgkinson, A.M. Sidki, J. Landon, G.
Gallacher, Ther. Drug Monitor., 9 478-483 (1987).
12. D. Desai, S. Meyer-Lehnert, J. Gal, 193rd American
Chemical Society National Meeting, Denver, CO, April 5-10,
1987, Abstract #I 99, Division of Analytical Chemistry.
13. L. Lie-A-Huen, R.M. Stuurman, F.N. Ijdenberg, J.H. Kingma,
D.K.F. Meijer, Ther. Drug Monitor., 708-711 (1989).
14. J. Turgeon, H.K. Kroemer, C. Prakash, I.A. Blair, D.M.
Roden, J.Pharm.Sci., 79 91-95 (1990).
15. S. Alessi-Severini, F. Jamali, F.M. Pasutto, R.T. Coutts, S.
Gulamhusein, J. Pharm. Sci., 79 257-260 (1990).
16. C. Fischer, F. Schonberger, C.O. Meese, M. Eichelbaum,
Biomed. Environ. Mass Spec., 19.256-266 (1990).
17. A.S. Gross, G. Mikus, C. Fischer, R . Hertrampf, U. Gundert-
Remy, M. Eichelbaum, Br. J. Clin. Pharmacol., 28 555-566
(1989).
18. J.W. DeJong, J.A.J. Hegge, E. Harmsen, P.Ph. DeTombe,
J. Chromatogr., 229 498-502 (1982).
19. S.F. Chang, T.M. Welscher, A.M. Miller, R.E. Ober, J.
Chromatogr., 272 341-350 (1983).
20. K.K. Bhamra, R.J. Flanagan, D.W. Holt, J. Chromatogr.,
307 439-444 (1984).
21. S.F. Chang, A.M. Miller, J.M. Fox, T.M. Welscher, J. Liq.
Chromatogr., 7 167-176 (1984).
FLECAINIDE 193
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FLECAINIDE 195
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Chern., aZ 886 ( 1991 1.
GLAFENINE
Adnan A . Badwan,' Muhammad B. Zughul,2
and Mahmoud A l Omari'
(I ) The Jordanian Pharmaceutical Manufacturing Co.

Naor, Jordan
(2) Department of Chemistry

Faculty of Science
University of Jordan
Amman, Jordan

AND EXCIPIENTS - VOLUME 21 197 All rights of reproducilon reserved In any form.
198 ADNAN A. BADWAN. MUHAMMAD B. ZUGHUL,AND MAHMOUD AL OMARI
CONTENTS
1 - DESCRIPTION
1.1. Nomenclature.
1.1.l. Chemical Names.
1.1.2. Generic Name.
1.1.3. Registry Number.
1.1.4. Wiswesser Line Notation.
1.2. Formulae.
1.2.1. Emperical Formula.
1.2.2. Molecular Weight.
1.2.3. Structural Formula.
1.3. Colour, Appearance and Odour.
1.4. Therapeutic Use.
2 - SYNTHESIS
2.1. Synthetic Route (I).
2.2. Synthetic Route (11).
2.3. Synthetic Route (111).
2.4. Synthetic Route (IV).
3 - PHYSIC0 - CHEMICAL PROPERTIES

3.1. Melting Range.
3.2. Differential Scanning Calorimetry.
3.3. Solubility.
3.4. DissociationConstant.
3.5. Partition Coefficients.
3.6. Spectral Properties.
3.6.1. UltravioletSpectra.
3.6.2. Fluorescence Spectrum.
3.6.3. Single Photon Counting Spectrofluorometry.
3.6.4. -
lnfra Red Spectrum.
3.6.5. Nuclear Magnetic Resonance Spectrum.
3.6.6. Mass Spectrum.
3.6.7. X - Ray Powder Diffraction.
GLAFENINE 199
4 - METHODS OF ANALYSIS
4.1. Starting Material and PharmaceuticalDosage Forms.
4.1.1. Elemental Composition.
4.1.2, Related Materials.
4.1 2 . 1 4,7 - dichloroquinoline.
4.1.2.2 Anthranilic acid esters.
4.1.2.3. N - (7 - chloro - 4 - quinolyl) anthranilic acid (glafenic acid).
4.1 -3. Titrations.
4.1.3.1 I Non - Aqueous Titration.
4.1.3.2. Alkalimetric Titration.
4.1.4. GravimetricAnalysis.
4.1.5. Spectrophotometric Methods.
4.1 5 . 1 . Ultraviolet Absorption.
4.1.5.2. SpectrofluorometricAnalysis.
4.1.6. Chromatographic Methods.
4.1.6.1. Thin Layer Chromatography.
4.1-6.2. High Performance Liquid Chromatography.
4.2. Body Tissues and Fluids.
5 - STABILITY
5.1. Stability of The Solid.
5.2. Stability in The Solution.
6 - PHARMACOKINETICS
6.1. Absorption.
6.2. Bioavailability.
6.3. Distribution.
6.4. Metabolism.
6.5. Excretion.
6.6. Half - Life.
200 ADNAN A. BADWAN. MUHAMMAD B. ZUGHUL, AND MAHMOUD AL OMARl
1. DESCRIPTION
1.l.Nomenclature
1.1.1, Chemical names
- - - -
1.1.1.1 a - glyceryl or 2', 3' dihydroxypropylN - (7 chloro 4 quinolyl)
anthranilate.
-
1.1.1.2 2',3' dihydroxypropyl2"-(7 - chloro - 4 aminoquinolyl) benzoate.
-
1.1.1.3 4 - (2" (2', 3' - dihydroxypropyl carboxyphenyl) amine) 7 --
chloroquinoline.
1.1.2. Generic name
Glafenine (Listed in The French Pharmacopoeia)
1.1.3. Registry number
-
CAS 3820 - 67 - 5
1.1.4. Wiswesser line notation
T66 BNJ EMR 8 V1 YQ1 Q and JG
1.2. Formulae
1.2.1. Emperical formula
1.2.2. Molecular weight
372.8
GLAFENINE 20 I
1.2.3. Structural Formula
1.3. Colour, Appearance and Odour.
Pale yellow, crystalline powder, odourless.
1.4. Therapeutic Use
Glafenine is an analgesic.
2. SYNTHESIS
Different routes of synthesis were proposed. A brief of these is described

and a schematic presentation is illustrated.
2.1. Synthetic Route 1.
Reacting of isoatoic anhydride with glycerol to produce glyceryl

anthranilate, followed by reacting with 4, 7 - dichloroquinoline to produce
glafenine (1).
202 ADNAN A. BADWAN, MUHAMMAD B. ZUGHUL. AND MAHMOUD AL OMARl
Scheme (I) GlafenineSynthesis, Route (I)
2.2. Synthetic Route II.
Condensation of 2 - chlorobenzoate, glyceryl ester with 7 - chloro - 4 -

aminoquinoline (1).
I--\
Scheme (/I) Glafenine Synthesis, Route (11)

GLAFENINE 203
2.3. Synthetic Route 111.
Condensation of 4, 7 - dichloroquinoline with methylanthranilate. The

resulting methyl ester is transesterified with glyceryl acetonide which is further
hydrolysed to glafenine (1).
HN -
glvceryl acetonide I
Scheme (111) Glafenine Synthesis, Route (111).

204 ADNAN A. EADWAN. MUHAMMAD B. ZUGHUL, AND MAHMOUD AL OMARI
2.4. Synthetic Route IV
Esterification of 2 - nitrobenzoicchloride with glyceryl acetonide, followed

by a reduction of the nitro group to the amino group. The resulted ester
is condensed with 4, 7 - dichloroquinoline, followed by hydrolysis of
acetonide (2).
HO OH
Scheme (IV) Glafenine Synthesis, Route (IV).

GLAFENINE 205
3. PHYSIC0 - CHEMICAL PROPERTIES

3.1. Melting Range
The French Parmacopoeiaspecifies that the melting range of glafenine is

between 170°C - 174 "C, (3).
3.2. Differential Scanning Calorimetry
Glafenine was recrystallized from ethanol, butanol, hexanol and

acetonitrile. Thermograms of these crystals were obtained using Mettler TA
3000 - DSC - 20 unit. The heating rate was 10"C. min-' and the sample size was
ranging from 3 - 10mg. The recrystallized glafenine showed a single sharp
peak without any decomposition at melting. Melting range of obtained crystals
from different solvents was between 170 - 174°C. Recrystallization from
hexanol yielded sharper thermogram peak, figure (1). The heat of fusion of
these crystals was in the vicinity of 43.8 KJ. mole-' (4).
Heat Flow Exothermal c
E
l-
180
Figure ( 1 ) The DSC Profile of Glafenine Recrystallized

from Hexanol
206 ADNAN A. BADWAN. MUHAMMAD B. ZUGHUL, AND MAHMOUD AL OMARI
3.3. Solubility
Equillibrium solubility of glafenine was determined by shaking an excess

of glafeninewith the solvent requiredin a water bath at 30 “C for 48 hours. Table
( I) presents glafenine solubility in commonly used solvents (4).
Table ( I )
Glafenine EquillibriumSolubility in Commonly Used Solvents
Solvent gm/lOOml at 30°C
Hexane <0.001
Water 0.001
Chloroform 0.260
Acetone 0.297
Ethanol 0.700
0.1N HCI 1.295
3.4. Dissociation Constant
The pKa was determined spectrophotometricallyat 20 “C in accordance

with an earlier reported method (5). Stock solution of glafenine in 10” N HCI
was prepared, and diluted with suitable buffer solutions rangingfrom pH 6 - 10
to obtain afinal glafenine concentrationof 1Oug. mL-’. The absorbance of these
solutions were measured at the maximumat 342.5nm. This method yielded 7.2
as pKa of glafenine at 20 “C (4).
GLAFENINE 207
3.5. PartitionCoefficients
The partition coefficients of glafenine between n - decanol and aqueous

buffers of different pH values were determined at room temperature. Different
pH values were obtained by using 0.1 N HCI for pH 1.O, acetate buffers for pH
3.0,4.0,4.5 and 5.0 and phosphate buffers for pH 6.0,7.0 and 8.0. Figure (2)
shows the plot of pH against the partition coefficients (5).
20
15
10
2 4 6 8
PH
Figure (2) The Plot of Partition Coefficient of Glafenine

Against the pH
208 ADNAN A. BADWAN, MUHAMMAD B. ZUGHUL. AND MAHMOUD AL OMARI
3.6. Spectral Properties
3.6.1. Ultraviolet spectra
The absorption spectra of glafenine dissolved in methanol and 0.1 N HCI

are shown in figure (3). It exhibits maximum at 356nm, 225nm and 255nm for
methanol and maxima at 342nm, 223nm and a shoulder at 252 nm for 0.1N
HCI. These were recorded using Beckman DU - 7 spectrophotometer.
Wavelength (nm)
Figure (3) The UV absorption Spectra of Glafenine
(70 ug.mL-’) in Methanol (-) and 0.7N HCl (...).
GLAFENINE 209
Table (11) repesents the ultraviolet absorption spectral bands of glafenine

in various commonly used solvents (4 , 7).
Table ( I1)
The UV Absorption Spectral Bands of Glafenine in Different Solvents
Solvent W8Velength (Extinction Coefficient)

nm (M-' cm-')
Chloroform 360 (22,900), 255 (18,100)

Benzene 360 (14,900), 255 ( 4,100)
Methanol 356 (21,OOO), 255 (1 8,800)
Ethanol 355 (19,600), 255 (1 7,000)
Acetone 355 (18,700), 255( 5,600)
Ether 351 (19,300), 255 (1 9,000)
O.1NHCI 342 (18,300), 252 (Shoulder)
3.6.2. Fluorescencespectra
The excitation and emission spectra of glafenine in ether is shown in

figure (4). The excitation and emission maxima of glafenine in different
solvents are presented in Table (Ill). Aqueous solutions having pH values lower
than 4 do not fluoress while solutions having higher pH values exhibit
fluorescence which intensifies with the pH increase. This intensity is
maximized in solutions having pH 9 - 10. (7).
210 ADNAN A. BADWAN. MUHAMMAD 8 . ZUGHUL. AND MAHMOUD AL OMARI
60
L.
.-
u)
C
Q,
-
4-
C
40
9
.-
-Q0
c
U
20
Wavelength (nrn)
Figure (4) The Fluorescence Excitation and Emission
Spectrum of Glafeninein Ether (10 ug. mL-';
Aexc= 250,327 and 7em = 400 nm).
0 2 4 6 8 1 0 l 2 1 4 1 6
Figure (5) The Fluorescence Decay Curve of Glafeninein
Ethanol at hexc=340nmand-hem=475nm.
GLAFENINE 21 I
Table ( 111 )
The Fluorescence Characteristics of Glafenine in Various Solvents
(5 ug. mL-’)
Solvent exc. (nm) em. (nm) Intensitya
Benzene 330 392 20

Ether 250b,327 400 17
Chloroform 250b,340 436 10
Ethanol 245b,336 439 6
2 - Propanol - 10% (v/v) buffer pH9 340 410 6
(0.1N glycine-sodium hydroxide).
Methanol 245b,336 425 2
2 - Propanol - 10% (v/v) buffer, pH4 26!jb,355 450 2
(0.1N citrate-hydrochloric acid).
Acetone 330 430 0.5
Methanol - 10% (v/v) ammonia 273b,365 455 0.5
(28% w/w).
2 - Propanol - 10% (v/v) buffer, pH1 (0.2N 300 407 0.5
potassium chloride - hydrochloric acid).
Sulfuric acid (10- 0.005N). No signal
a: Referred to a solution of quinine sulfate in a concentration of 1ug.mL-’ in 0.5N sulfuric acid, of which the
relative fluorescence intensity is 100, measured simultaneously.
b: Secondary excitation value with much less intensity.
3.6.3. Single photon counting spectrofluorometry.
A single photon counting spectrofluorometry was used to measure the

decay life time of glafenine in absolute ethanol (20ug. mL-’). A hydrogen lamp
was used as monochromatic radiation source while the excitation and emission
wavelengths were 340nm and 475nm, respectively figure (5). The fluorscence
decay curve was obtained using Edinibrugh Instruments single photon
counting model 199 - spectrofluorometer. This figure shows that the decay life
time of glafenine in ethanol is 0.88 n - sec.(8).
212 ADNAN A. BADWAN. MUHAMMAD 8. ZUGHUL. A N D MAHMOUD AL OMARI
3.6.4. Infrared spectrum
Glafenine infrared spectrum is shown in figure (6). This is obtained by

screening glafenine - KBr dispersion disc using Perkin Elmer 598
spectrophotometer. Table (IV) shows the band assignments of infrared
spectrum of glafenine (4).
Table ( IV )
The I.R. Spectral Assignments of Glafenine
Wavenumber, cm-' Vibration Mode
3500 - 3220 0-H (stretching),N-H (stretching)

3100 C-H (stretching), aromatic
2900 C-H (stretching),aliphatic
1680 C =0 (stretching)
1620,1580,1530 C=C (stretching), aromatic
1260,1050 C-0 (stretching), ester
1 100,980 C - 0 (stretching), alcohol
1450 C-H (bending), aliphatic
870,830,750,600 C-H (out of plane bending), aromatic
3.6.5. Nuclear magnetic resonance spectrum
NMR spectrum of glafenine is presented in figure (7). This spectrum was

recorded on Varian T60 NMR spectrometer. Table (V) lists the spectral
assignmentsof glafenine in DMSO (1,8).
Table ( V )
The NMR Spectral Assignments of Glafenine
Chemical Shlft Number of Proton Assignment

(Multiplicity)
10.1
8.7- 7.0
4.4
3.8
3.5
Wavenumber (Cm-')
Figure (6) The 1.R. Spectrum of Glafenine.
figure (7) The N.M.R. Spectrum of Glafenine in DMSO.
GLAFENlNE 215
3.6.6. Mass spectrum.
The mass spectrum of glafenine was obtained using mass spectrometer

MAT - 112. Some characteristic peaks are listed in Table (VI). Figure (8)
representsthe mass spectrum of glafenine, (4).
Table ( VI )
The Mass Spectrum of Glafenine
m/z Species I/ I,%
372 M+ 40.0
298 372 - (CHZ= CHOHCHZOH) 27.1
280 298 - (HZO) 62.9
253 280 - (CO) 71.4
21 7 253 - (CI) 34.3
121 (C&COOH)' 40.0
104 (C6H4CO)+ 100.0
76 (c6H4)+ 70.0
74 (CH2=CHOHCH20H) 15.7
I / lo = relative intensity (Eased on the highest intensityof 100.0).
The molecular ion peak appeared at m/z ratio of 372. Glafenine mass
spectrum showed a distinct peak at m/z 298. This peak corresponds to
Maclafferty rearrangement.The cleavage of oxygen - carbonyl bond is evident
at m/z 280. Further, the removal of C = 0 group and CI atom is shown at m/z
253 and 217, respectively. The pattern of glafenine mass fragmentation is
presented in scheme (V).
Figure (8) The Mass Spectrum of Glafenine
GLAFENINE 217
-
QtJ
-CHFCHOHC H,OH
mlz =74
C L U d m/z=298
m/z = 372
cl$
CL
m/z = 280 c,QI$ m/z = 253
QQ
m/z =29 8 m/z = 177 m/z =121
m/z =I21 m b =I 04
Scheme (V} The Mass Fragmentation Pattern of Glafenine.

218 ADNAN A. BADWAN. MUHAMMAD B. ZUGHUL, A N D MAHMOUD AL OMARl
-
3.6.7. X ray powder diffraction
The x - ray powder diffraction of glafenine powder was determined using

Phillips PW 1050 - 81 Goniometer with a PW 1729 Generator with Nickle
filtered copper radiation ( = 1.5418nm) as a source of radiation. The
scanning rate was 28. (2cm)-’. min-’. The interplanner distance and relative
intensity of the major peaks are listed in Table (VII). Figure (9) illustrates the x -
ray powder diffraction pattern of glafenine recrystallizedfrom 1 - hexanol (4).
Table ( VII )
The X - Ray Powder Diffractionof Glafenine
5.6 15.781 2.8 23.4 3.8015 32.4

7.6 11.632 17.3 24.1 3.6926 100.0
9.9 8.9341 5.6 25.3 3.5201 13.4
11.4 7.7617 70.9 26.0 3.4269 16.8
14.9 5.9454 16.8 27.6 3.2318 30.7
15.5 5.7166 30.7 29.8 2.9980 11.1
16.6 5.3402 51.4 30.9 2.8938 6.7
17.6 5.0390 4.5 34.1 2.6292 7.8
19.2 4.6226 5.6 35.8 2.5081 10.6
20.4 4.3532 79.9 37.9 2.3738 22.3
21.2 4.1907 14.0 40.6 2.2220 5.0
22.2 4.0042 50.3 41.7 2.1659 8.4
22.8 3.9001 22.3 44.1 2.0534 21.2
d = Interplanner Distance, l / l o = Relative Intensity (based on the highest intensity of 100).

44 38 32 26 20 14 8 2
Diffraction Angle (28)
Figure (9) The Powder X - Ray Diffraction Pattern of
Glafenine Recrystallized from Hexanol.
220 ADNAN A. BADWAN, MUHAMMAD B. ZUGHUL, AND MAHMOUD AL OMARI
4.1. Starting Material and PharmaceuticalDosage Forms
4.1 .l.Elemental Composition
Calculated Percentage
C 61.21
H 4.60
CI 9.51
N 7.51
0 17.17
4.1.2. Related Materials
4.1.2.1. 4,7 - dichloro - quinoline : 10 mg glafenine are dissolved in 0.5rnLof

hydrochloric acid solution (1 % v/v hydrochloric acid in ethanol). 0.5 rnLof
paranitrophenyl hydrazine solution (0.1 % millimole in ethanol) is added. The
mixture is cooled in air and then warmed in a water bath at 80°Cfor one hour in
darkness. The mixture is further cooled to 20 "Cf 1 "C. 0.25rnbftriethylamine
is added, shaken and followed by the addition of 4 ml of dimethyformamide and
shaken until a homogenous liquid is obtained. This solution is compared in
colour intensity with a standard solution of 0.5rnLof 4,7 - dichlocoquinoline(10
ug permL)in 1 % v/v hydrochloric acid in ethanol. The percent concentrationof
4,7 - dichloroquinolinein glafenine should not exceed 0.05% (3).
4.1.2.2. Anthranilic acid esters: 50mg of glafenine are dissolved in 5mL of

ethanol. 1mL of diluted sulphuric acid is added. The mixture is cooled for 2
minutes in an ice bath. 1mL of 1% millimole per volume of sodium nitrite
solution is added and the mixture is left for 10 minutes in the ice bath. 1 mL of
freshly prepared and filtered B - naphthol solution of 10% millimole per volume
in concentrated ammonia, is added. The obtained solution is compared in
colour intensity with a simultaneously prepared standard solution of a mixture
of 4.8mLof ethanol and 0.2 mL of methyl anthranilate solution (0.05% millimole
per volume in alcohol). The percent concentrationof anthranilic acid esters in
glafenine should not exceed 0.2% (3).
GLAFENINE 22 I
4.1.2.3. N - (7 - chloro - 4 - quinolyl) anthranilic acid (glafenic acid): Thin layer

chromatography can be used to detect glafenine and glafenic acid in the bulk
material. The system consists of a plate of silica gel HF 254 impregnated with
sodium acetate. The mobile phase is a mixture of chloroform - methanol -
glacial acetic acid and water mixed in volumetric proportions of 85:12:2:1,
respectively. The developed spots are identified by exposure to U.V. lamp
(254nm). Glafenine and glafenic acid solutions having a known concentration
are prepared in a mixture of chloroform - methanol and water mixed in
volumetric proportions of 3.0:3.0:0.5, respectively. These solutions include
0.5% w/v glafenine sample (solution a), 0.5%, 0.1 %, 0.0025% w/v glafenine
standard solutions (b, c and d), 0.0025% w/v glafenic acid standard (solution
e), a mixture of 0.5 w/v glafenine standard and 0.0025% w/v glafenic acid
standard (solution f). If a secondary spot appears in the chromatogram of
solution a with an Rf value slightly inferior to the principal spot of the same
chromatogram, it should not be more intense than the principal spot in the
chromatogram obtained from solution c. If a secondary spot appears
corresponding to glafenic acid, it should not be more intense than the spot
obtained from solution e. If secondary spots other than the previous two appear
in the chromatogram of solution a, it should not be more intense than the
principal spot of solution d. This test could be adopted only if glafenine and
glafenic acid were separated in two spots in the developed chromatogram of
solution f (3).
4.1.3. Titrations.
4.1.3.1. Non - aqueous titration: Glafenine (300 mg) is dissolved in 30 mL of

acetic acid. The end point is detected potentiometrically. When glafenine
hydrochloride is used, mercuric acetate is to be added to the titration medium.
Each mL of 0.1 M perchloric acid volumetric solution is equivalent to 37.28mgof
glafenine. This method is applied for the drugs determination in tablets and
suppositories (9).
4.1.3.2. Alkalimetric titration: Powdered tablets equivalent to 50 - 200 mg of

glafenineare placed in 150 mL conical flask. 25 mL of 0.1N HCI are added and
shaken for 2 - 3 minutes. The bromocresol green indicator is added to the
mixture (2 - 3 drops) and titration is carried out with 0.1 N NaOH. The indicator
colour changes from yellow to bluish green at the end point. This method could
be adopted for different dosage forms determination. Each mL of 0.1 N HCI
volumetric solution is equivalent to 37.28 mg of glafenine (10).
4.1.4. Gravimetricanalysis
Glafenine is determined in tablets and suppositories by adding 1 mL of 1N

HCI, 10 mL of diluted acetic acid and 6 mL of 0.25M KBi14with stirring, to 10 mL
portions of sample solution prepared from tablets or suppositories described,
containing 50mg glafenine. After 30 minutes, the precipitate is filtered off on a
sintered glass filter and washed with diluted acetic acid and water. The filter is
then dried at 105°C for 60 to 90 minutes, cooled and weighed. Each gram of the
precipitate is equivalent to 0.3728 gm of glafenine(l1).
4.1.5. Spectrophotometricmethods
4.1 5 1 . Ultraviolet absorption: Glafenine (50 mg) is dissolved in 0.1 N HCI and
the volume is made up to 250mL. 5 mL of this solution is further diluted to
100 mL with the same solvent. The solution exhibits a maximum absorption at
about 343 nm. The specific absorbanceat this maximum is about 490 (4).
4.1 5.2. Spectrofluorometric analysis: Glafenine is determined in tablets by

transfering a quantity of fine powder equivalent to 25 mg of glafenine into
500 mL conical flask. About 450 mL of ether are added and the mixture is
stirred with a magnetic stirrer for 2 hours. After filtration on a paper filter, the
filtrate is diluted to 500 mL with ether. Further 10 mL of this solution is dilutedto
100 mL with ether. An analogous standard solution having the same
concentration of the sample solution is prepared. It is advisable to extract the
tablets simultaneouslywith the preparationof the standard solution or during at
least the same period to avoid incomplete extraction. Pure ether is used as the
blank solution. Fluorometric measurement is performed at 327 nm excitation
and 400 nm emission (7).
GLAFENINE 223
4.1.6. Chromatographic Methods
4.1.6.1. Thin - layer chromatography (TLC): Glafenine (I), glafenic acid (11)
(major metabolite and major photoproduct in the solid state) and methyl N - (7 -
chloro - 4 - quinolyl) anthranilate (111) (minor photoproduct in the solid state) can
be identifiedby TLC method using silica gel 60 F254 (2Ox20cm) with thickness
of 0.2 mm as stationary phase. 1OuL of 0.20% and 0.01 % of glafenine and
glafenic acid in chloroform are spotted. Methyl N - (7 - chloro - 4 - quinolyl)
anthranilate is detected after storing a solution of 0.20% of glafenine in
methanolfor 24 hours under ambient conditions. The system is equillibratedfor
15 minutes before the development. The development distance is 10 cm and
the plate is air dried. The detection method is UV lamp (254 nm) or by naked
eye (yellow colour spots). Table (VIII) lists the Rf values of glafenine and its
photodegraded products in different mobile solvents (4).
Table ( Vlll )
The Thin layer Chromatography of Glafenine and its Photodegraded
Products in the Solid State (4).
~ ~~~~
Mobile Phase Rf Value Rf Value Rf Value

of (1) of (11) of (111)
Ethylacetate - Chloroform (70/30by Volume) 0.06 0.00 0.37

Chloroform- Methanol - Acetic Acid 0.30 0.22 0.80
(85/12/3by Volume)
Ethylacetate- Methanol - 33% Ammonia 0.45 0.15 0.75
(85/10/5by Volume)
Chloroform - Methanol (80/20by Volume) 0.57 0.24 0.77
224 ADNAN A. BADWAN. MUHAMMAD B. ZUGHUL, AND MAHMOUD AL OMARI
4.1.6.2. High performanceliquidchromatography:HPLC profile of glafenine is

shown in figure (10). This profile was obtained using Beckman HPLC system.
-
An R Sil C18column (150 mm x4.6 mm I.D.) with a particle size of 5 um was
used. The mobile phase consisted of a mixture of methanol, water and acetic
acid (64:27:9 by volume). The chromatographicsystem was operated at room
temperature with an eluent flow rate of 1.OmL.min-’. It has a sensitivity of 0.01
absorbance unit, attenuation of 64 and chart speed of 0.5 cm.min-’. The
wavelength of the detector was set at 344 nm. This method is stability indicating
and may be used for tablets, capsules and suppositories (8).
r, 4
I Rt (min)
Figure (1 0) The HPLC Profiles of Glafenine Dissolved in

Ethanol (10 ug. mL-’).
GLAFENINE 225
4.2. Body Tissues and Fluids
Spectrophotometric methods for the determination of glafenine and its

metabolitesin the body fluids were insensitive and unspecific. More recent and
specific methods using HPLC were reported:
- Determinationof glafenineand its metabolitesinvolveda separation and
extraction procedure in human plasma. Floctafenine was used as internal
standard for both the drug and its metabolites. Chromatographic conditions
were 5 um R - Sil Cle column, solvent mixture consisted of methanol - water -
acetic acid (67:23:10),respectively.pHwas adjusted to 4.3 by the addition of
ammonia. The flow rate was 0.5 mL.min-’ and the detector was set at 360 nm.
For 1 mL plasma, the detection limit was 0.5mg. L-’ for glafenine and
hydroxyglafenicacid, and 0.2mg. L” for glafenic acid. This method allowedthe
deduction of some primary pharmacokineticparameters (12).
- For determination of glafenine (I): Plasma (1mL) containing 50uL of
floctafenine (11) as internal standard solution, was made alkaline with glycine
buffer of pH 11 (1 mL) and extracted with CHCl3 (2x5 mL).After centrifugation,
the combined organic layer was evaporated to dryness at 37OC and the residue
was dissolved in the mobile phase (100 uL). For determinationof glafenic acid
(111) plasma (1mL) containing (11) solution (50 uL) was acidified with 0.1 N HCI
(200uL) and extracted as above. Sample solutions (20uL) were analysed by
HPLC on acolumn (1 5 cm x 4.6mm) of SpherisorbCe(5 um) with acetonitrile -
water - diethylamine (550:400:3) adjusted to pH 4.5with anhydrous acetic acid
as mobile phase (1 mL.min-’), detection was at 362,358and 364 nm for I,II and
111, respectively. Calibration graphs were rectilinear from 0.05(detection limit)
to 2.5mg.L-’, and 0.25(detection limit) to 2.0 mg. L” for I and Ill, respectively.
The coefficients of variation (n = 10)were 8.1to 13.7and 7.7to 10.8% for Iand
Ill, respectively (13).
5. STABILITY
5.1. Stabillty of The Solid
Glafenine powder is stable against heat and moisture.The powdered drug

was stable when stored at 40 "Cin the dark for 180 days (14). Glafenine in the
-
solid form readily undergoes photodegradation when exposedto UV visible or
solar radiation. The photodecomposed products are similar regardless of the
method of radiation used. The two photodecomposition products were
- -
identified as N - (7 chloro 4 - quinolyl) anthranilic acid and methyl N - (7 -
chloro - 4 - quinolyl) anthranilate. The first was separatedand identifiedas solid
while the second was identified in solution of isopropanol and was found to be
present in trace amounts. These were cross checked with similar prepared
photoproducts. It seems that intramolecular H - abstraction leads to the
formation of these photoproducts. Glafenine formulated into solid dosage
forms has to be guarded against sources initiating photochemical
degradation(8).
5.2. Stability in The Solution
Glafenineis insoluble in water. Heating the drug's suspension at 50 "Cfor

several hours produced no degradation. However, boiling the same solution
yielded hydroysedforms of glafenine. In neutral alcoholic solution, galfenine is
unstabletowards UV/visible radiationwhere it photodecomposes into two main
products, namely glyceryl anthranilate and 7 - chloroquinoline. Glafenine
photodegradation is suggested to occur via intermolecular H - abstraction in
the presence of proton donor solvents. The rate of photodecomposition in
neutralalcoholic solution was found to decrease with the polarity of the solvent,
while the increase in viscosity of the solvent was found to be impededprobably
due to the cage effect. In nonpolar solvents such as benzene,
photodecomposition is very low. In acidic alcoholic and aqueous solutions,
glafenine proved to be quite stable (8).
GLAFENINE 227
6. PHARMACOKINETICS
6.1. Absorption
Glafenine is well absorbed from the intestinalwall in gastrointestinaltract.

Following oral administration of glafenine, peak concentration of the main
metabolite, glafenic acid, is reached after about one hour, The decline in
plasma concentration is multiphasic and incompatible with one compartment
model. In view of the lack of free glafenine in the central compartment, a
substantialfirst - phase elimination by liver or gut wall can be assumed. Rectal
absorption of glafenine or glafenine hydrochloride is extremely slow and
incomplete due to the slight water solubility of glafenine at the prevailing pH in
the rectum lumen (15).
6.2. Bioavailability
Glafenine suspension was administered and compared with glafenine

suppositories and enemas. It is clear from Table (IX) that rectal administration
of micro - enemas or suppositories containing this drug is not bioequivalentwith
oral dosage form (15).
6.3. Distribution
It seems that the glafenic acid is deposited in the kidney. Such deposition
is manifested by yellow colouration which disappears with biochemical
disturbances(16).
6.4. Metabolism
Comparative studies suggest that analgesic activity of glafenine is due to

one of its metabolites. The glycerol liberated in vivo following administration of
glafenine does not appear to be responsible for the effect of the drug since
Table ( IX )
Absorption Characteristics, Relative Bioavailability and Urine Excretion
Pattern of Glafenic Acid (Mean _+ S.D.), After Oral and Rectal Administrationof
400mg Glafenine (439mg Glafenine HCI).
Plasma concentation
(ug.mL-') at t:
30 (min) 9.2f3.4 0.20*0.02
60 12.8k2.7 0.36k0.05
120 4.6k0.7 0.40&0.07
1 80 1.6k0.5 0.37+0.05
240 1.1k0.2 0.34k0.06
300 0.6k0.1 0.30i~0.02
Number 7 7 7
Cll, (ug.mL-') 12.6k3.1 0.44k0.09
tmaw (min) 50+27 125&28
AUCo.5 (ug.rnin". mL-') 1308k68 98+9
Frei 1 .oo 0.075
Urine concentation
(mg) at t:
60 (min) 31.126.7 3.8k0.4 0.8k0.07
120 44.2f8.2 5.1i-0.3 2.120.2
I80 41.0f6.7 4.8k0.4 1.7k0.1
240 12.1k3.1 4.7f0.5 1 -5k0.2
300 9.20k2.7 3.5f0.2 0.2k0.04
GLAFENINE 229
equimolar doses of glycerol did not induce any of glafenine characteristic

effects. It was reported (17) that glafenine occurs in human urine mainly as
-
correspondingfree acid N - (7 - chloro - 4 quinolyl) anthranilic acid; the process
of hydrotysis (enzymatic or not) being still unknown. Glafenine does not seem
to be metabolized into simpler molecules such as 4 - amino - chloroquinoline
and anthranilic acid. The structure of glafenine and its metabolites are shown in
metabolicpathway establishedin the rat scheme (VI). The excretion patterns in
rat and human urine are very similar indicating that the metabolic pathway
should be similar in the two species (17).
Scheme (vl) The Metabolism Pathways of Glafeninein Rat.

6.5. Excretion
In normal subjects 70% of the product is eliminated through the biliary

tracts. The remaining 30% are eliminated in the urine. The urine elimination is
early, the maximum is between 2 - 4 hours and it is also rapid as about 70% of
the quantity eliminated in the urine is achieved in the first 6 hours. It is
eliminated almost totally in the form of metabolites and the major metabolite is
free glafenic acid. Glafenineis rapidly eliminated from the body even in cases
of high doses of renal insufficiency,plasma half - life of glafenine though longer
than in normal subjects stays sufficiently short. After the absorption of a 400 mg
dose, the serum level after 24 hours is zero or extremely low (16).
-
6.6. Half Life
The distribution half - life was reported as 75 minutes (15) and the
elimination half life was 3.03 hours (18).
GLAFENINE 23 1
REFERENCES
1 . Gilbert Mouzin, Henri Cousse and Jean Marie Autin; Synthesis, 1;54- 55
(1980).
2 . Netherlands Appl. Patent 296,793 (CI. C07d), Roussel- Uclaf (1 965),C.
A., 64,3504e (1966).
3 . French Pharmacopeia. Glafenine Monograph.
4 . A.A. Badwan and M.M. AL - Omari; Unpublished Data. The Jordanian
PharmaceuticalManufacturingCompany, Jordan.
5 . Pamela Girgis Takla and Christos J. Dakas; Int. J. of Pharm., 43,225- 232
(1988).
6 . Nadia Ghazal; Unpublished Data, The Jordanian Pharmaceutical
ManufacturingCompany, Jordan.
7 . W. Baeyens and P. De Moerloose; J. of pharm. Sci., 66 (12),1771 - 1773
(1 977).
8 . M.M. Omari (1987);M.S. Thesis. Universityof Jordan, Jordan.
9 . Mostafa S.Tawakkol and Mohamed E. Mohamedi Analytical Letters14
(BlO),763 - 770 (1981).
10. Mostafa S.Tawakkol, Mohamed E. Mohamed and Mahmoud A. Ibrahim;
Pharmazie, 36 (H.2), 163 (1981).
11. S. A. Ismaiel, Abdel - Moety, E.M.; Zentralbl. Pharm., Pharmakother
57 - 59 (1988).
Laboratoriumsdiagn,127 (2),
12. Marie Christine Tournet, Catherine Girre and Pierre Etienne Fournier;J. of
Chromatography, 224,348- 352 (1 981).
13. Ennachachibi, A,, Nicolas P., Fauvelle F., Perret G., Petitjean 0;J.
Chromatogr. Biomed. Appl., 3 June, 71 (2),(J. Chromatog, (427),307 -
314(1988).
14. A.A.Badwan; Stability Data on Glafenine, Unpublished Data, The
Jordanian PharmaceuticalManufacturing Company, Jordan.
15. F. Moolenaar, J. Visser and T. Huizinga; Int. J. Pharm. 4,195 - 203 (1980).
16. Pharmacology File on Glafenine - JPM.
17. J. Pottier, M. Busigny and J.P. Raynaudi Eur. J. Drug Metab. 4 (2) 109 -
115 (1979).
18. M. C. Tournet, S. Giudicelli, C. Girre, J. Crouzetle and P. E. Fournier; C. -
R. - Congr. Biopharm. Pharmaco Kinet. lst, 2,288 - 301, (1981). Editedby
J. M. Aiache and J. M. Hirtz.
LISINOPRIL
Dominic P. Ip, Joseph D. DeMarco
and Marvin A . Brooks
Merck Sharp & Dohme Research Laboratories
West Point, PA 19486

AND EXCIPIENTS- VOLUME 21 233 All rights of reproduction W e N e d in any form.
234 DOMINIC P. IP, JOSEPH D. DEMARCO, AND MARVIN A. BROOKS
LlSlNOPRlL
Dominic P. Ip, Joseph D. DeMarco and Marvin A. Brooks
1. History and Therapeutic Properties
2. Description
2.1 Nomenclature
2.1.1 Chemical Name
2.1.2 Generic Name
2.1.3 Laboratory Codes
2.1.4 Trade Names
2.1.5 CAS Registry Number
2.2 Formula and Molecular Weight
2.3 Appearance, Color, Odor
3. Synthesis

4.2 'H - Nuclear Magnetic Resonance Spectrum
4.3 I3C - Nuclear Magnetic Resonance Spectrum
4.5 Mass Spectrum
4.6 Specific Rotation
4.7 Thermal Behavior
4.8 Solubility
4.1 1 Hygroscopicity
4.12 Partition Coefficient
LISINOPRIL 235
5 . Methods of Analysis

5.2 Chromatographic
5.2.1 Thin-Layer Chromatography
5.2.2 High Performance Liquid Chromatography
5.2.2.1 Bulk Drug Analysis
5.2.2.2 Lisinopril in Formulation
5.3 Titration
5.4 Other Methods
5.5 Identification Tests
6. Stability
6.1 Solid State Stability

6.2 Solution Stability
7. Determination in Body Fluids and Tissues
7.1 Radioimmunoassay
7.2 Competitive Inhibitor Binding Assay
7.3 Fluoroentymatic Assay
8. Drug Metabolic Products, Pharrnacokinetics and Bioavailability
9. References
1. History and TheraDeutic ProDerties
Lisinopril, a lysine analogue of enalaprilat, is a long-acting

angiotensin converting enzyme inhibitor which differs from captopril
by lacking the sulfhydryl group. Lisinopril, discovered and developed
by the Merck Sharp & Dohme Research Laboratories (l), is indicated
for the treatment of hypertension and congestive heart failure.
Several review articles give a detailed account of the history, design,
chemistry and pharmacology of the drug (2-6).
2. Description
2.1 Nomenclature
2.1.1 Chemical Name
(a) L-Proline, 1-[N2-(1-carboxy-3-phenylpropyl)-L-

h
lysyl -dihydrate, (S)-
(b) 1-[N -[(S)-1-carboxyl-3-phenylpropyl]-L-lysyl]-L-
proline dihydrate
2.1.2 Generic Name
Lisinopril
L-l54,826-000T, MK-0521
2.1.4 Trade Names
Prinivil, Zestril, Carace, Novatec
2.1.5 CAS Reqistry Number
93015-83-7
2.2 Formula and Molecular Weight
'21 H31 N3°5 ' 2H20

Molecular Weight 441.52
LISINOPRIL 231
H H H O
T I T I I 2H20
CH2CH2 ..- C- - N- - C- -C-N
A
COOH AI
(CH2)4
NH2 H?OOH
Lisinopril is a white to off-white crystalline, odorless powder.
3. Synthesis
Lisinopril has been prepared by the scheme outlined in Figure 1

(7,8). The dipeptide, N,-trifluoroacetyl-L-lysyl-L-proline c1) is
subjected to reductive alkylation with ethyl 2-oxo-4-phenylbutanoate
(J over Raney Nickel via a Schiff base 3
2 (J to yield a diastereomeric
mixture 4 (SSS and RSS). Hydrolysis of the N,-trifluoroacetyl moiety
and saponification of the ethyl ester followed by crystallization in
ethanol/water and final recrystallization in water yield lisinopril (SSS,
J of greater than 98% purity in about 65% yield (based on 3. In
5
addition to this synthetic route, others have also been described in
the literature (9-12).
4.1 Infrared Spectrum (13)
The infrared spectrum of lisinopril as shown in Figure 2 was

obtained in a potassium bromide pellet using a Perkin-Elmer
Model 281-B spectrophotometer. Assignments for the
characteristic bands in the spectrum are listed in Table 1.
4.2 ’ H-Nuclear Maanetic Resonance Spectrum (14)
The proton magnetic resonance spectrum of lisinopril is shown

in Figure 3. The spectrum was obtained using a Bruker
Instruments Model WM250 spectrometer and a 10% W N
solution of lisinopril in ,
lJsolution of deuterium chloride in
deuterium oxide. The reference compound (internal) was p-
-1 -
2
R=CF 3CONHCH 2(CH 2)3
N
m
W Ph
(1) OH-
*2H20 *
(2) Crystallization E~O,C
H
0 CO,H
-5, Lisinopril 4
R'=H 2NCH ,(CH 2)3
Figure 1
Synthesis of Lisinopril
Wsvelenglh (rnlcrons)
239
N
w
10
Frequency (CM.'I
Figure 2. Infrared Absorption Spectrum of Lisinopril

240 DOMINIC P. IP. JOSEPH D. DEMARCO, AND MARVIN A. BROOKS
dioxane. An expansion of the spectrum in the 0-6 ppm region

is shown in Figure 4. Chemical shifts and assignments for the
numbered structure shown below are tabulated in Table II.
COOH
1
Table I
Lisinopril Infrared Band Assianmentsa
Wavenumber (cm-') Assiqnment
3545 OH stretching vibrationb (dihydrate

H 0)
Near 3370 813290 (broad) 06 stretching vibration
3090 - 2860 C-H stretching region
-2800 - -2100 stretching region
1655
1609 Asymmetric -C02: stretch
1570 Asymmetric -C02 stretch
1541 'NH or N ' H, bending
1450, -1 443 (strong) CH2%ending
1388 Symmetric -C02- stretch
1340, 1299 Not assigned
741, 732,692 Phenyl out-of-plane bending (2
bands plus -(CH2)4-r~~k)
a These bands are subject to a reading error range of 25 cm-' above
2000 cm-' and +3 cm-' below.
These bands disappeared in a dehydration experiment monitored by
infrared spectrometry.
Figure 3. The Proton Magnetic Resonance Spectrum of Lisinopril
Figure 4. The Proton Magnetic Resonance Expanded Spectrum of Lisinopril
LISINOPRIL 243
Table II
Lisinopril, Proton Maqnetic Resonance Assiqnments
Chemical Shift, SH (ppml Assiqnmenta
7.35 Phenyl ring protons

5.06 HCI/HD0
4.44 C, protonb
4.35 ,, protonb
C
3.84 C:, proton
3.75 p-dioxane (reference)
3.62 C, protons
3.04 C6, protons
2.85 C
, protons
2.33 C
, protons; C, proton (one of
the two)'
2.03 C3, protons; C, protons; C
proton (other of the twoyc
1.73 C5, protons
1.60 C,, protons
a Assignments refer to number structure above.
b As in the case of aqueous solutions of captopril (15) and
enalapril (16), signals attributable to rotamers (rotation about
the lysyl-proline tertiary amide bond) are observed in the
spectrum of lisinopril. The triplets at 4.62 ppm and 4.13 ppm
represent, respectively, the C, and C2, protons in the minor
rotamer.
C Private communication with Dr. B. J. Woodhall, Pharmaceutical
Division, Imperial Chemical Industry.
4.3 I3C Nuclear Maqnetic Resonance Spectrum (12)
The carbon-13 magnetic resonance spectrum of lisinopril

shown in Figure 5 was obtained using a Varian Associates
Model XL-1OOA spectrometer and a 10% (WN) solution of
lisinopril in 1N deuterium chloride in deuterium oxide. The
reference compound (internal) was p-dioxane. An expansion
of the spectrum in the 12.5-75 ppm region is shown in Figure
6. Chemical shifts and assignments for the numbered
structure shown below are tabulated in Table Ill.
244
N
3
3
3
3
:
0
rl)
D
3
Q
5
Figure 5. The Carbon-13 Magnetic Resonance Spectrum of Lisinopril
N
P
VI
75 62.5 50 37.5 25 12.5

PPM
Figure 6. The Carbon-13 Magnetic Resonance Expanded Spectrum of Lisinopril

246 DOMINIC P. IP, JOSEPH D. DEMARCO. AND MARVIN A. BROOKS
NH
COOH
1
Table Ill
Lisinomil, Carbon-13 Maanetic Resonance Assianments
Chemical Shift, 6, (ppmla Assianmentb-

21.57 (22.04) " c43
25.39 (22.51)
27.20 :;:
29.49 (31.38)
30.02 (30.58)
31.21
:;:
C
,
31.92 (32.24) C
.,
39.89 '6'
48.70 (48.1 6) c5
59.73 (60.29) c21
60.1 1 C
.,
60.53 (59.64) c2
67.40 p-dioxane (reference)
127.58
cP
129.510~ crn
129.6Eid
140.53 (140.44)
167.32 (1 67.80)
:;
CIS
171.44 (1715 5 ) c, I.
175.71 (175.14) Cl
Values in parentheses are due to a minor conformational
isomer, and many of the assignments for this component
are tentative.
Assignments refer to numbered structure above.
Private communication with Dr. 8 . J. Woodhall,
Pharmaceutical Division, Imperial Chemical Industry.
These assignments could be reversed.
LISINOPRIL 241
The ultraviolet absorbance spectra of lisinopril shown in Figure

7 (17) were obtained using a Perkin-Elmer Lambda 5 UV-VIS
scanning spectrophotometer. The spectrum in 0.1N sodium
hydroxide solution is characterized by low intensity maxima at
-246 nm, -254 nm, -258 nm, -261 nm and -267 nm with
respective A l % 1 cm values of -4.0, -4.5, -5.1, -5.1 and -3.7.
The spectrum in 0.1N hydrochloric acid is characterized by
maxima at -246 nm, -253 nm, -258 rim, -264 nm and -267
nm with respective A l % 1 cm values at -3.2, -3.9, -4.5, -3.0
and -2.8.
The ultraviolet absorbance arises from the unconjugated

phenyl ring in lisinopril molecule.
4.5 Mass Spectrum (18)
The mass spectrum of lisinopril shown in Figure 8 was

obtained by direct probe-electron impact (70 eV) method using
an LKB Model 9000 mass spectrophometer. The spectrum
shows no molecular ion peak. A pseudo-molecular ion peak at
m/e 387.2160 is attributed to the diketopiperazine formed
during vaporization. Mass fragment assignments are given in
Table IV.
4.6 Specific Rotation (19)
Lisinopril contains three chiral centers and is

The specific rotation values [a]25 and [a]
405 nm 436 nrn
0.25M pH 6.4 zinc acetate) are respectively --120" and --96".
4.7 Thermal Behavior (20)
Differential thermal analysis under vacuum (DTA heating rate =

2OoC/rnin.) as shown in Figure 9 indicated three endotherms
with peak temperatures at -98"C, -1 22°C and -1 82°C. The
first two endotherms correspond to loss of water of hydration
and the one at 182°C to melting.
0.80
0.64
8 0.48
C
m
e
2!
0.32
0.16
0
220 240 260 280 300 320 340
Wavelength (nrn)
Figure 7. The Ultraviolet Absorption Spectrum of Lisinopril in

(a) 0.1NSodium Hydroxide; Concentration: 1.374 mg/ml and
(b) 0.1NHydrochioric Acid; Concentration: 1.374 mg/ml
Figure 8. The Direct Probe-Electron Impact (70 eV) Mass Spectrum of Lisinopril
Table IV
Lisinopril, Mass Spectrum Assianments
-
M/e Assiqnment
1.
387 C
,, H2,N304 (M' minus H,O)
+
369 m/e 387 minus HO

,
358 m/e 387 minus CH3N
342/343 m/e 387 minus CO,(H)
329 m/e 387 minus C3H,N
315/316 m/e 387 minus C,H9,0N
313 m/e 358 minus C0,H
296 m/e 387 minus benzyl radical
283 C,3H,1 N304 (m/e 387 minus styrene)
265 m/e 283 minus H20
0
252 m/e 296 minus CO,
245
CH&H?-N=CH(CH,),NH,
c=o
€B
LISINOPRIL 25 I
Table IV (Continued)
Lisinopril, Mass Spectrum Assiqnrnents
-
Mle Assiqnment
224 aN32
1' 1Hl '
CH&HzCHzC&NH, CHCH$&CHzNH2
d e 224 rnle 224
CYCH,CH,CH,NH,
.,,dly"
tj we224
207 C, HN
,O
,, (rn/e 224 minus NH,)
179
H Jaco-g '
e
H
84
QHH
Table IV (Continued)
Lisinopril, Mass SDectrum Assianments
-
M/e Assianment
70
W" n
4.7 Thermal Behavior (continued)
The thermogravimetric analysis (TGA) curve of lisinopril shown

in Figure 10 depicts three inflections corresponding to the loss
of free water and the first and second moles of water of
hydration. The excess unbound water was 0.6% over the
theory of 8.2% for the dihydrate indicating lisinopril is
somewhat hygroscopic.
4.8 Solubility (20)
The following approximate solubility data were obtained at

ambient temperature.
Table V
Solvent Solubility (mq/ml)
Water 97
Methanol 14a
Ethanol <0.1
Acetone <o. 1
Acetonitrile <0.1
Chloroform <o. 1
N,N-Dimethylformamide <0.1
a Upon dissolution of lisinopril in methanol, changes in X-
ray diffraction patterns indicative of loss of water of
hydration were observed. The solubility value obtained
becomes dependent upon the water content of the
solution.
I ~ I ~ I ~ I ~I I II I I~ I ~ l ~
04 - -
00 - I
-0.4 - -
Q
2
r
g -0.8 - -
0
0 L
5
e0
c.
-1.2 - -
0. -
E
-16 - -
20 - c
-
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 l 1 1 l l ~
-2 4
Figure 9. DifferentialThermal Analysis (DTA) Curve of Usinopril

Temperelure ('C)
Figure 10. Thermogravimetric Analysis Curve of Llsinopril

LISINOPRIL 255
4.9 Dissociation Constants (20)
Aqueous acidichasic potentiometric titration at 25°C yielded

four pKa values of 2.5, 4.0, 6.7 and 10.1 for lisinopril.
4.1 0 Crvstal ProDerties (20)
Lisinopril is crystalline as determined by X-ray powder

diffraction. A typical X-ray powder diffraction pattern obtained
using a Philips APD 3720 X-ray powder diffractometer is
shown in Figure 11. A monohydrate which is crystalline, also
exists. The monohydrate is readily distinguishable from the
dihydrate by its X-ray powder diffraction pattern (Figure 12).
4.1 1 Hvcrroscopicity (21)
Lisinopril is slightly hygroscopic. A sample stored for three

months at 98% relative humidity and room temperature
showed an increase of 1.1Yo in the total volatiles content by
TGA.
4.1 2 Partition Coefficient (17)
The partition coefficient of lisinopril in the phosphate buffer

(O.lM, pH 7)h-octanol system was determined to be 10.2
0.5 at room temperature.
5. Methods of Analvsis
5.1 Elemental Analysis (22)
The elemental analysis found for a reference lot of lisinopril L-

154,826-00T031 was
/Elemental) Analvsis
'21 H31 N3°5 ' 2H20
% Theow* % Found*
Carbon 57.13 56.89

Hydrogen 7.99 7.69
Nitrogen 9.52 9.49
*Anhydrous basis
256
Figure 11. Powder X-Ray Diffraction Pattern of Lisinopril

LU
Figure 12. Powder X-Ray Diffraction Pattern of Lisinopril Monohydrate

2% DOMINIC P. IP, JOSEPH D. DEMARCO. AND MARVIN A. BROOKS
5.2 Chromatoqraphic
Chromatographic procedures have been developed to separate

lisinopril from its principal decomposition product, the
diketopiperazine (DKP), a product of intramolecular
dehydration. Since there are three optical centers in the
molecule, isomers of lisinopril and its DKP degradate are
possible. The sequence of thermal decomposition for lisinopril
to form isomers of DKP is shown in Figure 13.
The conversion of SSS DKP to SSR DKP has been

demonstrated (23) by heating SSS DKP for 15 minutes at
160°C.
Chromatographic procedures also separate in some cases a

process impurity, 4-phenyl-2-aminobutyric acid (APBA) and the
RSS isomer of lisinopril (Figure 14).
5.2.1 Thin Laver Chromatoqraphy (TLCl(19)
Four TLC systems have been developed and are listed

below. Systems (I) and (11) separate lisinopril from its
RSS isomer, and APBA. Systems (Ill) and (IV) are of
somewhat limited value since they do not separate
lisinopril from the RSS isomer. Systems (I) and (11)
utilize E. Merck Silica Gel G-60 while systems (Ill) and
(IV) use Analtech Silica Gel G-60. Visualization of
spots is accomplished by reaction with ninhydrin.
Lisinopril (SSS) Lisinopril SSS DKP Lisinopril SSR DKP
Figure 13. Thermal Decomposition of Lisinopril to Its Diketopiperazine

Isomers
H H O
V H V I I
HOOC- -C--N--C--C-N
A A
CH2 (CH2)4
I
6
I H
NH2
RSS Isomer
8
CHzCHpCHNH2
Ao2H
2-amino-4-phenyl butyric acid

(AP BA)
Figure 14
LISlNOPRlL 26 I
Table VI
TLC Systems
Lisinopril
Solvent Systems Rf (Approximate)
(1) n- Butanol/tol uene/glacial 0.34

acetic acid/water/acetone
(1:l:l:l:l)
(11) n-Butanol/water/glacial 0.22

acetic acid (3:l:l)
(111) n-Butanol/water/glacial 0.43

acetic acid/ethyl acetate
(1:l:l:l)
(1V) Chloroforrn/rnethanol/conc. 0.14 to 0.39

ammonium hydroxide (4:4:1)
262 DOMINIC P. IP. JOSEPH D. DEMARCO. AND MARVIN A . BROOKS
5.2.2 Hiqh Performance Liquid Chromatoqraphv (HPLC)
5.2.2.1 Bulk Druq Analysis (24)
Methods of analysis for lisinopril in bulk drug

are summarized in Table VII. Method (I)
which utilizes a linear gradient is capable of
separating lisinopril from two potential process
impurities, the RSS isomer of lisinopril and
the 2-amino-4-phenylbutyric acid (APBA), and
the diketopiperazine SSS and SSR isomer
degradation products (see Figures 13 and
14). Under isocratic conditions, method (I)
also separates lisinopril from its RSS isomer
and APBA. Both gradient and isocratic
procedures use a Zorbax (DuPont) RP-8
column at a pH of 5.0 and a column
temperature of 50°C. The isocratic procedure
has been published in the USP (25).
Lisinopril exhibits typical chromatographic
behavior (peak broadening) attributed to
rotational isomers (26) of proline-containing
dipeptides. Higher column temperatures of
950°C appears to be necessary to minimize
this effect for acceptable chromatography.
Method (11) which utilizes a PRP-1 column

(Hamilton Co.) has also been used for the
evaluation of lisinopril bulk drug. This method
is, however, more cumbersome to use than
method (I) which was found to offer better
resolution for compounds of interest.
Detection for all methods is by UV at 210 to
215 nm.
5.2.2.2 Lisinopril in Formulation (27)
Methods for the analysis of lisinopril in

dosage forms are summarized in Table VIII.
Methods (I), (11) and (111) have been developed
to separate lisinopril from its principle
degradation product, the SSS
diketopiperazine (DKP) and a process
impurity APBA (see Section 5.2.1). Method
LlSlNOPRlL 263
(IV) was developed to include the separation

of hydrochlorothiazide as well. These
methods (I-IV) all utilize a Lichrosorb RP-8
column, 10 pm at 40°C or 50°C. Method (IV)
uses a somewhat longer column (300 x 4.6
mm) and it was found that as a result of the
increased length, column temperature could
be reduced to 40°C while maintaining good
peak efficiency (see Section 5.2.2.1). This
method is also capable of separating lisinopril
diketopiperazine SSS from the SSR isomer as
well.
The retention times of both lisinopril and SSS

DKP have been found to be a function of the
phosphate concentration in methanol (see
Figures 15 and 16). Similar data have been
obtained in acetonitrile.
The retention of lisinopril and SSS DKP also

decreased as the % organic modifier
increased. Thus by varying both the molarity
of phosphates and % organic modifier in the
mobile phase, the resolution of these
compounds can be optimized. Methods (V)
and (Vl) were developed to quantitate
lisinopril for content uniformity and dissolution.
Detection in all of these methods as in the

methods designed for the bulk drug is by UV
at 210 nrn to 215 nm.
A stability-indicating HPLC method for

lisinopril tablets has been published in the
USP (28). This compendia1 method employs
the same mobile phase composition as
method (111) but contains an ion-pair reagent.
264 DOMINIC P.IP.JOSEPH D. DEMARCO. AND MARVIN A. BROOKS
Table VII
HPCC Systems for Lisinqxil Bulk DruQ
-
Merhcd Column Chromatographic Conditions Separation
(1) Zorbax (DuPont) RP-8 A:Acetonitrile a.b,c,d,e

250 x 4.6 mm, 5 p n B:0.02M NaH2P04 pH 5.0
T = 50%
-
Gradient:
0% A to 30% A linear over
35 minutes
-
Isocratic: 96% Solvent B
4% Solvent A
a,b,e
PRP-1 (Hamilton Co.) A:O.O2M NaH2P04 at pH 6.8

250 x 4.6 mm. B:O.O15M NaH2P04at pH 3.0
10 km, T = 50°C C:Acetonitrile
-
Isocratic: 96% Solvent A
4% Solvent C
Gradient (11
Solvent NSolvent C ( 9 7 . 5 2 5 ) for 10
minutes, then linear gradient to
Solvent NSolvent C (70:30) in 30 minutes
Gradient [ZJ
Solvent &Solvent C (95:s)
for 10 minules then linear gradient to
Solvent BlSolvent C (70:30)
in 30 minutes.
Legend
a = Lisinopril
b = RSS Isomer
C = SSS DKP
d = SSR DKP
= APBA
LISINOPRIL 265
Table Vlll
HPLC Systems for Lisinopril in Solid Dosage Formulations
ChromatographicConditions
Purpose Column Mobile Phase (Conditions) Separation
Stability Lichrosorb RP-8 Acetonim’le/0.004Mphosphate, a,c,e

Single entity (Hewlen Packard) pH 2.0
200 x 4.6 mm, 10 pm 45:55
T = 50%
Stability Lichrosorb RP8 MethanoVO.WMphosphate, pH 2.0 a.c.e

Single entity (Hewlen Packard) 45:55
200 x 4.6 mm. 10 pn
T = 50°C
Stability Lichrosorb RP-8 Acetonitrile/O.O3M phosphate, a.c,e

Single entity (Hewlea Packard) pH 2.0
200 x 4.6 mm. 10 pm 2o:ao
T = 50°C
Stability Lichrosorb RP-8 AcetonitriIe/O.O05Mphosphate, a.c,d,e.f

HCTZ Combination (E.S. Industries) pH 2.0
300 x 4.6 mm. 10pm 35:65
T = 40%
Content Uniformity Hypersil ODs MethanoVO.02M phosphate, pH 2.0 a

Dissolution (Shandon) 12:88
Single entity 50 x 4.6 mm, 5 pn
T = 60°C
Content Uniformity Lichrosorb RP-8 Acetonitrile10.04M phosphate, a

Dissolution Hewlen Packard pH 2.0
HCTZ Combination 200 x 4.6 mm, 10 pm 15:85
T = 50°C
Legend
a = Lisinopril
b = RSS Isomer
-
C = SSS DKP
d SSR DKP
E = APf3A
f = Hydrochlorothiazide(HCTZ)
Phosphate Molarity
Figure 15. Effect of Phosphate Concentration on Retention of Lisinopril
Figure 16. Effect of Phosphate Concentration on Retention of SSS DKP
5.3 Titration (17)
Lisinopril can be determined by potentiometric titration with

aqueous sodium hydroxide and non-aqueous perchloric acid.
The sodium hydroxide titration is carried out by titrating the
lisinopril potentiometrically with carbonate free 0.1 N NaOH to
one endpoint using a combination electrode. Lisinopril can
also be determined by titration potentiometrically with 0.1 N
perchloric acid in acetic acid to one endpoint. The electrode
system consists of a glass electrode (such as a Metrohm
Model EA 107 vs. a silver/silver chloride reference electrode
such as a Metrohm Model EA 432 filled with 0.1N lithium
perchlorate in glacial acetic acid.
5.4 Other Methods
Lisinopril has also been determined by radioimmunoassay

(RIA), fluoroenzymatic assay (FEA) and competitive inhibitor
binding assay (CIBA). These procedures are described in
Section 7.
Identification of lisinopril can be carried out by infrared

absorption (see Section 4.1), TLC (see Section 5.2.1) and by
HPLC (see Section 5.2.2).
Supportive evidence for identification can be obtained by

differential thermal analysis (DTA) (see Section 4.7).
6. Stability
6.1 Solid State - Thermal (29)
Lisinopril is stable as a solid at ambient temperatures (20-

25°C). Degradation can be induced when the solid is stressed
at the severe thermal stress conditions of 105°C. HPLC
studies have demonstrated that intramolecular dehydration to
DKP is the primary degradate. The yield to the
diketopiperazine as % of the total chemical loss of lisinopril is
higher in nitrogen flushed closed container (80%) than in non-
flushed closed container (50-60%) or in open container (6%).
Lisinopril, when stressed under these severe conditions, yields
LISINOPRIL 269
additional degradates, the identity of which has not been

determined.
Studies have demonstrated that the lisinopril diketopiperazine

isomer initially formed is the SSS isomer which can degrade
further to the SSR isomer (see Section 5.2).
6.2 Solid State - Photochemical (30)

Very slight surface discolorations have been observed when
lisinopril is exposed to intense UV radiation for 24 hours.
6.3 Solution Stability (29)
The solution stability of lisinopril at a concentration of 0.2

mg/ml was studied at pHs ranging from 2.7 to 10.0 and at a
constant ionic strength (p = 0.3). Figure 17 depicts a plot of
the zero order rate constants from 60" and 80°C data vs. pHs.
Lisinopril decomposition proceeds rapidly in acidic media with
the major decomposition product being the diketopiperazine.
The rate of formation of the diketopiperazine was found to be
linear with time. In neutral and basic >pH 7.0, the
decomposition rate is minimal.
Figure 17. Plot of Rate Constants at 60" and 80°C as a Function of pH
LISlNOPRlL 27 I
7. Identification and Determination in Body Fluids and Tissues
The quantitation of lisinopril in biological fluids has been reported by

standard radioimmunoassay (RIA), competitive inhibitor binding assay
(CIBA) and fluoroenzymatic assay (FEA).
Radioimmunoassay procedures for the drug in plasma and

urine have been developed utilizing a polyclonal antiserum
produced to a conjugate in which lisinopril is linked to albumin
via a dinitrophenylene bridge (2,31) or succinoylated keyhole
limpit haemocyanin (32). In the former procedure, the
radiolabel was introduced via radio-iodination of a p-
hydroxybenzamidine derived from lisinopril, whereas in the
latter procedure, the radiotracer was prepared by acylation of
the epsilon amino group of the lysyl side chain of lisinopril with
N-~uccinimidyl-(2,3-~H)-proprionate. The limit of sensitivity for
the RIA procedures is approximately 0.2-0.4 ng/ml (0.5-1nM).
7.2 Competitive Inhibitor Bindinq Assay
Binding assays were first described as a technique for the

measurement of angiotensin converting enzyme inhibitor (ACE
inhibitor) activity (33). This procedure requires incubation
(37°C for 2 hours) of serum samples with 1251-labeledACE
inhibitor (p-hydroxybenzamidinederivative of lisinopril) followed
by a charcoal precipitation and gamma counting of the
precipitate. The ACE value is then determined from a
standard curve of inhibitor binding vs. ACE activity determined
by an enzymatic kinetic assay (34). The principle of this assay
was then extended to a competitive inhibitor binding assay in
which the 1251-labeled inhibitor (see above) is displaced from
isolated ACE by lisinopril in the biological sample (35-36). The
free label is separated by adsorption onto charcoal and related
to lisinopril drug concentration. The sensitivity of the assay is
reported to be 2-4 ng/ml (35) and correlates well with specific
radioimmuno-assays and ACE enzymatic activity (37).
7.3 Fluoroenzymatic Assay
The determination of ACE enzymatic activity via the

measurement of the rate of cleavage of the substrates
hippuryl-L-histidyl-L-leucineor hippuryl-L-glycyl-L-glycine to
212 DOMINIC P. IP. JOSEPH D. DEMARCO. AND MARVIN A. BROOKS
hippuric acid with quantitation by fluorometric, radioassay,

liquid chromatographic, radioimmunoassay and enzyme linked
immunoassay methods has been reviewed (33). Recent assay
procedures have incorporated ACE inhibitors into these
enzymatic assays with radioassay (38) or fluorometric (39)
measurement. The percent of ACE activity inhibited is
correlated with drug (inhibitor) concentration. These
procedures (38-39) require extraction of the drug from plasma
or urine with methanol to separate the drug (inhibitor) from
endogeneous ACE. The methanolic supernatant is
evaporated, and then reconstituted in a solution of exogenous
ACE containing the substrate. The fluorometric assay (39)
utilizes the substrate N-benzoyloxycarbonyl-L-phenylalanyCL-
histidyl-L-leucine which has been previously utilized to
measure enalaprilat in biological fluids (40). The enzymatic
reaction is quenched with ice, o-phthaldialdehyde added, and
spectrofluorometric measurement of the derivative is performed
at excitation/emission wavelengths of 365490 nm. A logit-log
relationship between drug (inhibitor) concentration and percent
enzyme inhibitor is used to calculate drug (inhibitor)
concentration. The assay has demonstrated a sensitivity limit
of 0.7 ng/ml with an RSD of 3-10% over the standard
concentration range of 0.4-35 ng/ml for enalaprilat. The assay
clearly has the advantage over RIA and ClBA as described of
not requiring radiolabel technique to perform the assay.
However, utilizing the lengthy sample preparation, assay
capacity is limited.
8. Druq Metabolic Products, Pharmacokinetics and Bioavailability
Utilizing the RIA procedure for serum and urine and in vitro isotope
dilution procedure for feces, the absorption and elimination profile of
lisinopril was determined in 12 healthy male volunteers following oral
administration of a 10 mg capsule (2). The observed peak serum
concentration was 95 +. 55 nM with a time to peak of 7 +. 1 hours and
an AUC (0-72 hours) of 1694 +. 808 nmol liter-’ hr. The serum
concentration vs. time profile was polyphasic and the terminal half-life
was approximately 30 hours. The renal clearance was 106 2 13
ml/min wit4 urinary and fecal recovery of 29% 2 15% and 69%
23%, respectively, indicating the drug was excreted unchanged.
The half-life for the terminal phase (approximately 40 hours) was not
predictive of steady state parameters when 10 daily doses of lisinopril
were administered orally to healthy subjects. The mean effective
LISINOPRIL 213
half-life of accumulation was 12.6 hours. The mean accumulation

ratio was 1.38 with steady state attained after the 2nd dose (41).
The drug is not metabolized but is eliminated via the kidneys.
Lisinopril probably undergoes glomerula filtration, tubular secretion
and tubular reabsorption (42).
The close correlation between serum concentration of drug and the

degree of inhibition of ACE has been demonstrated. Furthermore,
there was a close inverse relationship between plasma levels and the
ratio of angiotensin II:i, the latter parameter being a measure of the
conversion of angiotensin I to II (43).
Age and cardiac failure are reported to be associated with reduced

renal clearance of lisinoprii (44). The plasma concentration and drug
half-life in patients with chronic renal failure (creatinine clearance 5
30 ml/min) are generally higher than those seen in patients with
normally functioning kidneys (45). Food intake had no effect on the
pharmacokinetics of lisinopril (42,46).
Acknowledcrements
The authors wish to thank Mrs. Laurie Rittle for typing the manuscript
and Mrs. Florence Berg for conducting the literature search.
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Taub, E.R. Peterson, T.J. Ikeler, J. tenBroeke, L.G. Payne, D.L.
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Therapeutic Approaches", A.E. Doyle and A.G. Bearn, Editors,
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274 DOMINIC P. IP, JOSEPH D. DEMARCO. AND MARVIN A. BROOKS
5. C.S. Sweet and E.H. Ulm, Cardiovasc. Drua Rev. 6, 181 (1988).
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Springer, B. Arison and A.A. Patchett, 2816
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H. Joshua and A.A. Patchett, J. Pharm. Sci. 74, 352 (1985).
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Rahway, NJ.
15. D.L. Rabenstein and A.A. Isab, Anal. Chern. 54, 526 (1982).
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16,207 (1987).
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18. G.A. Schonberg, Merck Sharp & Dohrne Research Laboratories,

Rahway, NJ.
19. R.B. Waters, Merck Sharp & Dohrne Research Laboratories,

Rahway, NJ.
20. J.A. McCauley, Merck Sharp & Dohme Research Laboratories,

Rahway, NJ.
LISINOPRIL 215
21. R.J. Magliette, Merck Sharp & Dohme Research Laboratories,

Rahway, NJ.
22. J. Perkins, Merck Sharp & Dohrne Research Laboratories,

Rahway, NJ.
23. C. Bell, Merck Sharp & Dohme Research Laboratories, West

Point, PA.
24. T. Novak, Merck Sharp & Dohme Research Laboratories,

Rahway, NJ.
25. The United States Pharrnacopeia XXII, 2355 (1990).
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Laboratories, West Point, PA.
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30. J. DeMarco, Merck Sharp & Dohrne Research Laboratories, West

Point, PA.
31. M. Hichens, E.L. Hand and W.S. Mulcahy, Liqand Quarterv 4, 43

(1981).
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Hichens, Clin. Chern. 30, 696 (1984).
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Pharmacoloqy 36, 1357 (1987).
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37. B. Jackson, R. Cubela and C.I. Johnston, J. Cardiovasc.

Pharmacol. 9,699 (1987).
38. B.N. Swanson, K.L. Stauber, W.C. Alpaugh and S.H. Weinstein,
Anal. Biochem. 148,401 (1985).
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and Biomed. Awl. 6,241 (1988).
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and I.L. Junggren, Biopharm. Druq Dispos. 10,397 (1989).
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929 (1987).
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LOVASTATIN
Gerald S. Brenner, Dean K. Ellison,
and Michael J . Kaufman
Merck Sharp & Dohme Research Laboratories
West Point, PA 19486
ANALYTICAL PROFILES OF DRUG SUBSTANCES Copyright @ 1992 by Academic Press, Inc.

278 GERALD S. BRENNER, DEAN K . ELLISON, AND MICHAEL J. KAUFMAN
LOVASTAT IN
Gerald S. Brenner
Dean K. Ellison
Michael J. Kaufman
1. History and Therapeutic Properties

2. Description
2.1 Nomenclature
2.1.l Chemical Name
2.1.2 Generic Name (USAN)
2.1.4 Trade Names
2.1.5 Trivial Names
2.1.6 Chemical Abstract Services (CAS)
2.2 Structure, Formula and Molecular Weight
2.3 Appearance
3. Synthesis
4.2 Proton Nuclear Magnetic Resonance Spectrum
4.3 Carbon-13 Nuclear Magnetic Resonance Spectrum
4.5 Mass Spectrum
4.6 Optical Rotation
4.8 Solubility
4.11 Partition Behavior
LOVASTATIN 219

5.2 Chromatography
5.2.1 Thin Layer Chromatography
5.3 Flow Injection Analysis

7. Pharmacokinetics and Metabolism

7.2 Metabolism
7.3 Excretion
8. Determination in Biological Fluids
9. References
280 GERALD S . BRENNER, DEAN K. ELLISON, AND MICHAEL I. KAUFMAN
1. Historv and TheraDeutic ProDerties
It was discovered by the Merck Sharp & Dohme Research

Laboratories that a strain of Aspergillus terreus obtained from a soil
sample produced the cholesterol lowering fungal metabolite lovastatin
(initially named mevinolin). Details of the isolation, structural
characterization and biochemical properties of lovastatin have been
summarized by Alberts et al. (1). Lovastatin is identical to monacolin
K isolated independently from Monascus ruber by Endo
(2).
Lovastatin is a prodrug. After oral administration, the inactive parent
lactone is hydrolyzed to the corresponding hydroxyacid form. The
hydroxyacid is the principle metabolite and a potent inhibitor of 3-
Lactone Hydroxyacid
hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase. This

enzyme catalyzes the conversion of hydroxymethylglutarate to
mevalonate, which is an early and rate limiting step in the
biosynthesis of cholesterol. The effectiveness of lovastatin in
lowering cholesterol has been confirmed clinically and it is approved
for the treatment of primary hypercholesterolemia.
Several review articles give a detailed account of the discovery,

preclinical evaluation, mechanism of action, biological profile, and
clinical evaluation of the drug (3-7).
2. DescriDtion
2.1 Nomenclature
2.1.1 Chemical Name

S-[
[l 1a(R*),3a,7P,8P(2S*,4S*),8a~]]-2-Methylbutanoic
acid 1,2,3,7,8,8a-hexahydro-3,7-dirnethyl-8-[2-
LOVASTATIN 28 I
(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethyl]-1-
naphthalenyl ester; (1S,3R,7S,8SI8aR)-1 ,2,3,7,8,8a-
hexahydro-3,7-dimethyl-8-[2-[(2R,4R)-tetrahydro-4-
hydroxy-6-0~0-2H-pyran-2-ylJethyl]-1-naphthatenyl (S)-
2-methylbutyrate; 1,2,6,7,8,8a-hexahydro-P,6-
dihydroxy-2,6-dimethyl-8-(2-methyl-l-oxobutoxy)-1-
naphthaleneheptanoic acid Glactone; PP,Ga-dimethyl-
8a-(2-methyl-l -oxobutoxy)-mevinicacid lactone.
2.1.2 Generic Name (USAN1
Lovastatin
2.1.3 Laboratow Codes
L-l54,803-000G
MK-0803
2.1.4 Trade Names
Mevacor; Mevinacor; Mevlor
2.1.5 Trivial Names
Mevinolin
Monacolin K
3-Methyl Compactin
2.1.6 Chemical Abstracts Services GAS1
Registry Number: 75330-75-5
2.2 Structure, Formula, and Molecular Weiaht
Structure:
282 GERALD S . BREWER, DEAN K. ELLISON, AND MICHAEL I. KAUFMAN
Molecular Formula: C OH,,

Molecular Weiaht: 40&5
Lovastatin is a white, crystalline powder.
3. Synthesis
There have been numerous approaches to the total synthesis of

lovastatin (8-10); however, lovastatin is produced commercially via a
multi-stage fermentation process which originates from cultures of a
strain of Aspergilks ferreus. The complete details of the isolation
and identification of lovastatin from the fermentation media have
been described (1). Synthetic approaches have been reviewed (1 1).
4. Phvsical Properties
The infrared spectrum of lovastatin is shown in Figure 1 (12).

The spectrum was obtained as a potassium bromide pellet
using a Nicolet Model 7199 FT-IR spectrophotometer.
Assignments for the characteristic absorption bands are shown
below.
Wavenumber (cm-’ 1 Assignment

3542 Alcohol 0-H stretch
3016 Olefinic C-H stretch
296 7 Methyl C-H asymmetric stretch
2929 Methylene C-H asymmetric stretch
2866 Methyl and methylene C-H asymmetric stretch
1725 Lactone and ester carbonyl stretch
1711 (hydrogen bonded for 1711 and
1700 1700 cm-’ )
1460 Methyl asymmetric bend
1384 Methyl symmetric bend
1359 Methylene symmetric bend
1260 Lactone C-0-C asymmetric bend
1222 Ester C-0-C asymmetric bend
1072 Lactone C-0-C symmetric stretch
1056 Ester C-0-C symmetric stretch
969 Alcohol C-OH stretch
87 1 Trisubstituted olefinic C-H wag
LOVASTATIN 283
.49
a 37
'31
.25
.la
.12
.O6
0 1 I 1 I 1
4000 3600 3200 2800 2400 2000 1600 1200 800

1 I d
Wavenumbers
Figure 1. Infrared Absorption Spectrum of Lovastatin

2R4 GERALD S. BRENNER. DEAN K. ELLISON. AND MICHAEL J. KAUFMAN
4.2 Proton Nuclear Mametic Resonance Spectrum
The proton magnetic spectrum is shown in Figure 2 (13). This

spectrum was obtained on a Bruker Instruments Model AM-300
NMR spectrometer using a 4% w h solution of lovastatin in
deuterated chloroform. Chemical shifts (6) are expressed as
ppm downfield from tetramethylsilane (internal standard). The
tabulated signal assignments refer to the numbered structure
of lovastatin shown below.
6 (mml '
Multiplicity /J Assianment
0.88 t/J = 7.6 HZ

0.89 d/J = 7.3 HZ
1.08 d/J = 7.4 HZ
1.11 d/J = 7.0 HZ
1.20-2.05 Overlapping
Multiplets
2.20-2.50 Overlapping
Multiplets
2.55-2.77 Overlapping Multiplets
4.37 m
4.64 m
5.38 m
5.53 Broad t
5.78 d Of d/J =
6.1, 9.6 Hz
6.00 dN = 9.6 HZ
7.27 S
1 Multiplicity: s, singlet; d, doublet; t, triplet; m, multiplet

PPM
Figure 2. Proton Nuclear Magnetic Resonance Spectrum of Lovastatin
286 GERALD S. BRENNER, DEAN K . ELLISON, AND MICHAEL J. KAUFMAN
4.3 Carbon-13 Nuclear Maanetic Resonance Spectrum
The carbon-13 nuclear magnetic resonance spectrum of

lovastatin shown in Figure 3 was obtained using a Bruker
Instruments Model AM-300 NMR spectrometer and an
approximately 4% w/v solution of the compound in
deuterochloroform. Signal assignments are tabulated below
and refer to the numbered structure shown in Section 4.2
Chemical Shift (61, Dpm Assianment
11.69
13.83
16.21
22.79
24.23
26.78
27.39
30.63
32.62
32.90
36.06
36.55
37.24
38.55
41.46
62.52
67.86
76.37
77.00
128.26
129.58
1315 3
133.03
170.50
176.88
In recent publications, the 1H and 13C NMR spectra of

lovastatin were fully assigned by the use of selective
homonuclear and heteronuclear decoupling and two
dimensional techniques (14,15).
Figure 3. Carbon-13 Nuclear Magnetic Resonance Spectrum of Lovastatin
288 GERALD S. RRENNER. DEAN K . ELLISON. AND MICHAEL J . KAUFMAN
The ultraviolet (UV) absorption spectrum of lovastatin is

characterized by absorption maxima at 231,238, and 247 nm
with A l % values of 538, 629, and 424, respectively. The
absorption maxima at 238 nm is typical for a trisubstituted
heteroannular diene chromophore (16). A UV spectrum of
lovastatin (c = 0.015 mg/mL in acetonitrile) is shown in Figure
4.
4.5 Mass Spectrum
The mass spectrum of lovastatin is shown in Figure 5. This

spectrum was obtained by the direct probe electron impact (90
eV) method using a Finnigan MAT 212 mass spectrometer
(17). The spectrum exhibits a weak molecular ion signal at
m/z = 404 (C H 0 , exact mass calculated = 404.2563;
observed = 461.2%6lf. Other pertinent fragment ions are at
m/z = 302, 284, and 159; these ions can be rationalized by the
fragmentation pattern shown in Figure 6.
4.6 ODtical Rotation
Lovastatin has eight chiral centers and is optically active. The

specific rotation a
[2
,]5 is +330" for a 5.0 mg/mL solution in
acetonitrile.
The differential scanning calorimetry (DSC) curve for lovastatin

at a heating rate of 2"/min under a nitrogen atmosphere is
shown in Figure 7. The thermogram is characterized by a
single melting endotherm with an extrapolated onset
temperature for melting of 175°C which is independent of
heating rate from 2-2O0C/min. In contrast, the DSC
thermogram for lovastatin obtained at a heating rate of 2"/min
in air (Figure 8) exhibits an exotherm at 154°C which is
attributed to oxidative reactions occurring in the non-inerted
atmosphere.
The thermal properties of lovastatin, in particular those derived

from DSC experiments, have been used to assess the
oxidative stability of the compound (18,19).
Wavelength (nm)
Figure 4. Ultraviolet Absorption Spectrum of Lovastatin

I/.
159
loo]
15; 2 84
60 302
198
I
''{ 143 172
105 I !85
200 404
20 224 hi 1
0
100 150 200 250 300 350 400
Figure 5 . Direct Probe Electron Impact Mass Spectrum of Lovastatin

LOVASTATIN 29 I
m/z 302
mlz 159 m/z 284
Figure 6. Proposed Fragmentation Pattern to Explain the Mass

Spectrum of tovastastin
292
Figure 7. DSC Therrnogram for Lovastatin under Nitrogen

a
L
.-C
.-
c
t
m
c
3
>
0
J
L
c
0
E
2
m
0
a
r
l-
0
m
n
cd
??
3
.-0)
LL
293
294 GERALD S . BRENNER. DEAN K. ELLISON, AND MICHAEL J. KAUFMAN
4.8 Solubility
Lovastatin is insoluble in water, and is sparingly soluble in the

lower alcohols (methanol, ethanol, and i-propanol). Solubility
data obtained at room temperature are tabulated below (20).
Solvent Solubility ImalmL)
Acetone 47
Acetonitrile 28
n-Butanol 7
i-Butanol 14
Chloroform 350
N,N-dimethylformamide 90
Ethanol 16
Methanol 28
n-Octanol 2
n-Propanol 11
i-Propanol 20
Water 0.4
4.9 Crvstal Properties
Lovastatin is a white, crystalline, non-hygroscopic solid. Single

crystal X-ray diffraction experiments on a sample crystallized
from ethanol indicate that the space group is P2,2 2, with a =
5.974A, b = 17.337A, and c = 22.148A. The calculated density
is 1.17 g/cm3. (1)
The X-ray powder diffraction pattern for lovastatin is shown in

Figure 9. This spectrum was obtained on a Phillips APD 3720
X-ray diffractometer using CuKa irradiation. No crystal forms
(polymorphs) other than that represented by the X-ray pattern
in Figure 9 have been observed.
4.1 0 Dissociation Constants
Consistent with the structure, lovastatin exhibits no acidhase

dissociation constants. Potentiometric titration of a sample in
50% aqueous methanol revealed no observable buffering
action in the pH range of 2-1 1.
0
*d
4
rc)
M
c!
0
M
9
m
N
0
d
N
9
In
c
5
8
c
9
In
I I I I I I 0
0 0
I
0 0
I
0
I
0 0
I
0 0 0 d
- m
o dc oo ~ or o ~ * r~? c o
r ? zo o
296 GERALD S. BRENNER, DEAN K. ELLISON, AND MICHAEL J. KAUFMAN
4.11 Partition Behavior
In the n-octanol/water test system, lovastatin partitions

quantitatively into the organic phase. At room temperature, the
partition coefficient is approximately K = 1.2 x 104. The
partition coefficient for the hydroxyaci82erivative (opened
lactone form of lovastatin) between n-octanol and a pH 7.4
phosphate buffer is KO,, = 14.1 (21).
Analysis of Merck Sharp & Dohme reference lot L-154,803-

000G102 for carbon and hydrogen gives values compared to
calculated values as given below:
Calculated Found
Carbon 71.25 71.26

Hydrogen 8.97 9.18
5.2 Chromatoqraphy
5.2.1 Thin-Layer Chromatoaraphv
Table I lists the thin layer chromatographic systems

which have been used for the analysis of lovastatin.
Table 1
Thin-Layer ChromatographicSystems for Lovastatin (221
Solvent System Plate Type -

Rf System
Toluenehnethanol Analtech@ Silica 0.77 1

70/30 Gel GF
Toluenelacetone Analtech@ Silica 0.48 2

70BO Gel GF
Cyclohexaneh-butanoI/ethyl acetate Analtech@ Silica 0.43 3

4:l:l Gel GF
Cyclohexane/chloroformlisopropanol E. Merck Silica 0.60 4

5:2:1 Gel 60
F254 High Performance
LOVASTATIN 291
Visualization is either by viewing the developed plate

under ultraviolet light or by spraying the developed
plate with a dilute methanolic sulfuric acid solution
and application of heat. System 4 with sulfuric acid
spray detection is the most useful system because
non-UV absorbing impurities are detectable.
5.3.2 Hiqh Performance Liquid Chromatoaraphv (HPLC)
A variety of gradient and isocratic reverse phase

HPLC systems have been used to chromatograph
lovastatin (see Table 2).
Table 2
Hiqh Performance Liquid Chromatoqraphic Systems
Application System No. Column Mobile Phase nm Detection Ref

Drug substance 1 Whatrnan A = Acetonitrile 238 (23)
purity Partisil C-8 B = 0.1% (v/v%)
H3P04 aqueous
A:B 70:30
Measurement of 2 Whatman Gradient 238 and (24)

low level impurities Partisil C-8 A = Acetonitrile 200 nrn
in drug substances B = 0.1% (vW/O)
H3P04 aqueous
Measurement in 3 Sepralyte C-18 lsocratic and 238 nm (25)

plasma and bile Gradient
M
A = 0.05 ( NH4)3P04 and
An
0.01 H3P04 Buffer
B = acetonltrile
A:B 5050 (isocratic)
Measurement of 4 DuPont A = acetonitrile 260 nrn (26)

low levels in Zorbax C-8 B = methanol
fermentation broth' C = water
A:B:C 62229
Measurement in 5 Waters A = acetonitrile 238 nm (27)

tablets B = water (0.04M
KH2P04 pH s 4)
60:40 A:B
298 GERALD S. BRENNER. DEAN K. ELLISON, AND MICHAEL J . KAUFMAN
Table 2 (Cont'd)
High Performance Liquid Chromatographic Systems
Application System No. Column Mobile Phase nm Detection Ref

Measurement 6 Hypersil 5 230 nm (28)
in tablets micron ODS A = 0.025M NaH2P04
pH = 4
B = CH CN
C = MebH
33:55:12 A:B:C
Derivatization of lovastatin described.
5.3 Flow lniection Analysis
A flow injection analysis system has been described by Mazzo

--
et al. to simultaneously monitor lovastatin and antioxidants in
tablets (29).
Three methods are routinely used to identify lovastatin: 1. the

infrared spectrum; 2. the ultraviolet spectrum; and 3. the
chromatographic retention time.
Crystalline lovastatin stored at room temperature yields with

time trace amounts of oxidation products. The oxidative
pathway for degradation has been supported with data
generated by chromatography, degradate isolation, and
identification, differential scanning calorimetry and heat
conduction calorimetry. No products of nonoxidative
degradation have been detected. HPLC and TLC studies
have demonstrated that samples stored in air generate a
complex mixture of largely unidentified trace polar products
(30). These products are essentially absent and drug loss
prevented in samples stored under nitrogen. For samples
stored in air, all isolated and identified degradates result from
oxidation and include the 4'-oxolactone which is the major
LOVASTATIN 299
degradate retaining the diene of the parent. The ultraviolet

absorption spectra of air degradates indicate
Oxolactone
that more than half of the mass has lost diene, suggesting that
oxidation takes place primarily at this site (e.g., epoxidation
and subsequent reactions of the resultant epoxides). Heat
conduction calorimetry (19) and differential scanning
calorimetry (18) also demonstrate the enhanced reactivity of
the compound in an air vs. a nitrogen atmosphere.
The hydrolysis of the lactone ring of lovastatin occurs readily in

aqueous solution especially under acidic or alkaline conditions
(31). The acid catalyzed hydrolysis is reversible leading to a
mixture of lactone and hydroxyacid, the equilibrium ratio of the
two species being pH dependent. The rate to equilibrium is
also pH dependent, being more rapid at acidic pH than near
neutrality. In alkaline solution, the lactone ring is irreversibly
converted to the hydroxyacid. Solutions of the hydroxyacid
demonstrate good stability.
Kaufman (32) has studied and determined the rate and

equilibrium constants for the acid catalyzed hydrolysis of
mevalonolactone, lovastatin and other structurally related HMG
CoA reductase inhibitors in pH 2.0 buffer at 37°C. Under
these conditions lactone concentrations decrease with time but
do not approach zero indicating that the hydrolysis is
reversible. The equilibrium nature of the reaction was further
confirmed by repeating the experiment with hydroxyacid as
starting material in the same system and demonstrating that an
equilibrium composition is achieved that is identical to that
achieved starting with lactone. Kinetic points in all studies
were carried out to 15 hours and data obtained indicate that
300 GERALD S. BRENNER, DEAN K . ELLISON. A N D MICHAEL J. KAUFMAN
there are no side reactions (e.g., oxidation) competing with

hydrolysis/lactonization during this time frame.
The solution phase oxidation of a number of HMG CoA

reductase inhibitors, including lovastatin, was studied in
aqueous surfactant solutions at 40°C (33). Reaction rate
constants were determined by monitoring oxygen consumption
using an oxygen electrode. In the absence of a free radical
initiator, there was no oxygen uptake indicating that the
spontaneous rate of oxidation at 40°C was too slow to be
detected. With an initiator present, all analogs consumed
oxygen with the exception of the one in which the diene is
saturated, demonstrating the diene functionality to be most
labile to oxidation.
Oxidation of lovastatin in aerated ethylene dichloride solution

at 35", containing a free radical initiator, has been monitored
kinetically using HPLC (34). The degradates formed in this
complex solution system, different from those in the solid state,
are primarily oligomers, with peroxide groups within the
backbone chain and hydroperoxide end groups. Also, some
monomeric epoxides are formed.
7. Pharmacokinetics and Metabolism
Lovastatin is an inactive prodrug which undergoes in vivo lactone

hydrolysis to give the hydroxyacid derivative which is an inhibitor of
HMG-CoA reductase. The pharmacokinetic and metabolic profile of
lovastatin has been described in detail (33,3537). In the sections
below, the absorption, distribution, metabolism, and excretion of
lovastatin are briefly reviewed. For this discussion it is helpful to
distinguish between active inhibitors (defined as the sum
concentration of the hydroxyacid derivative of lovastatin plus other
active hydroxyacid metabolites) and total inhibitors (the total
concentration of active inhibitors plus lactones and conjugates).
Active and total inhibitors can be separately quantitated by assaying
samples before and after ex vivo hydrolysis of plasma samples.
In studies in laboratory animals, the absorption of lovastatin

following oral administration is approximately 30% complete as
estimated relative to an intravenous dose of the hydroxyacid.
An intravenous formulation of lovastatin for human studies is
LOVASTATIN 30 I
not feasible due to its low aqueous solubility. In all species

studied, lovastatin is converted to the hydroxyacid form in viva
This conversion is apparently reversible since lovastatin is
found in the biological fluids of rats and dogs following
administration of the hydroxyacid.
In animals, lovastatin is more efficiently extracted by the liver

where it is converted to the active enzyme inhibitor.
Accordingly, the systemic bioavailability of active inhibitors is
less than 5% of an oral dose of lovastatin. The high hepatic
extraction and low systemic availability are desirable features
since the liver is the primary site of cholesterol biosynthesis.
Peak plasma concentrations of both active and total inhibitors

occur between 2-4 hours post dose, and the area under the
curve (AUC) increases proportionally with dose. The
hydroxyacid is rapidly cleared; plasma clearance and half-life
range from 300-1248 mUmin and 1.1-1.7 hrs, respectively.
When lovastatin is administered with food, a 50% increase in
AUC for inhibitory activity is attained relative to administration
in the fasted state.
The plasma protein binding of lovastatin and the hydroxyacid

form has been determined by equilibrium dialysis. Both forms
are greater than 95% protein bound.
7.2 Metabolism
Lovastatin is extensively metabolized to give both active and

inactive compounds. The major active metabolites present in
human plasma are the hydroxyacid of lovastatin and its 3-
hydroxy-, 3-hydroxymethyl, and 3-exomethylenederivatives.
The 3-hydroxylated metabolite is approximately 70% as active
as the non-hydroxylated metabolite. In human bile, the 3-
hydroxylated metabolite undergoes an allylic rearrangement to
give the 6-hydroxy isomer which is inactive (38):
"3C
302 GERALD S. BRENNER. DEAN K . ELLISON. A N D MICHAEL J . KAUFMAN
All of the hydroxyacid metabolites also exist in their

corresponding inactive lactone forms. After base hydrolysis to
convert lactones to active inhibitors, about 80% of the total
enzyme inhibitory activity in human plasma is accounted for by
these four lactonelhydroxyacidpairs.
7.3 Excretion
The excretion of lovastatin has been assessed following an

oral dose of 14C-labeled compound in man. Total recovery of
drug equivalents in urine and feces averaged 10% and 83%,
respectively. A substantial amount of radioactivity is also
recovered in the feces following intravenous dosing of 14C-
labeled hydroxyacid, indicating that biliary excretion is an
important elimination for orally administered lovastatin.
8. Determination in Bioloaical Fluids
An enzyme inhibition assay capable of measuring total HMG-CoA

reductase inhibitors in biological fluids has been described in the
literature (1). The basis of this assay is the in vitro inhibition of the
HMG-CoA reductase catalyzed conversion of 14C-HMG-CoA to 14C-
mevalonic acid. The concentration of inhibitors can be measured
before and after base hydrolysis of plasma samples. The
measurement before hydrolysis gives the concentration of inherently
active species (active inhibitors). Base hydrolysis irreversibly
converts inactive but potentially active species (lactones and
conjugates) to their corresponding active forms; the inhibition assay
of hydrolyzed samples thus provides the concentration of total
inhibitors. The enzyme inhibition assay is sensitive (detection limit of
ca. 5 ng/mL), but is not specific for lovastatin.
The determination of lovastatin and its hydroxyacid metabolite in

plasma and bile can be accomplished by high performance liquid
chromatography (25). Plasma samples are prepared for analysis by
solid phase extraction and are analyzed using isocratic elution on a
C18 column. Bile samples do not require any sample clean-up prior
to HPLC analysis, but do require the use of a gradient elution method
to separate the compounds of interest. The HPLC assay has a limit
of detection of 25 ng/mL.
An analytical method for the determination of lovastatin in serum

based on gas chromatography/massspectrometry has recently been
reported (39).
LOVASTATIN 303
Acknowledclements
The authors wish to thank Mrs. Laurie Rittle for typing the manuscript
and Ms. Agnes Hendrick for performing the literature search.
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LOVASTATIN 305
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NAPHAZOLINE HYDROCHLORIDE
G . Michael Wall
Alcon Laboratories, Inc.
6201 South Freeway
Fort Worth, Texas 76134

308 G.MICHAEL WALL
NAPHAZOLINE HYDROCHLORIDE
1. DESCRIPTION
1.1 Name, Formula and Molecular Weight
1.3 History
1.4 Pharmacology
2. SYNTHESIS
3.1 Spectroscopy
3.1.1 Infrared Spectrum
3.1.2 Ultraviolet Spectra
3.1.4 Mass Spectra
3.2 Thermal Properties
3.2.1 Melting Range
3.2.2 Differential Thermal Analysis
3.2.3 Thermogravimetric Analysis
3.3 X-Ray Crystallography and Powder Diffractometry
3.4 Partition Coefficients
3.5 Ionization Constant, pKa
3.6 Solubility
3.7 Solution Color, Clarity and pH
4. TYPICAL METHODS OF ANALYSIS
4.1 Identity
4.1.1 Infrared Spectrophotometry
4.1.2 Ultraviolet Spectrophotometry
4.1.3 Chloride Identity Test
4.1.4 Reaction with Bromine
4.2 Colorimetry
4.4 Titrimetry
4.5 Chromatography
4.5.2 High-pressure Liquid Chromatography
4.5.3 Gas Chromatography
5. STABILITY-DEGRADATION
5.1 Potential Routes of Degradation
5.1.1 Characterization of 1-Naphthylacetylethylenediamine
5.1.1.1 Thin-Layer Chromatography
5.1.1.2 Liquid Chromatography
5.1.1.3 Synthesis of 1-Naphthylacetylethylene-
diamine
NAPHAZOLINE HYDROCHLORIDE 309
5.1.1.4 Physical/Chemical Properties of 1-

Naphthylacetylethylenediamine
5.1.2 Synthesis and Analysis of l-Naphthylacetic Acid
5.2 Solid-state Stability
6. DISPOSITION AND TOXICITY
7. ACKNOWLEDGEMENTS
8. REFERENCES
1. DESCRIPTION
1.1 Name, Formula and Molecular Weight
Naphazoline hydrochloride is an u-adrenergic sympathomimeticagent used

in topical nasal or ophthalmic pharmaceutical formulations. Naphazoline has
been established as the International Nonproprietary Name (INN) by the
World Health Organization for the chemical compound, (2-(l-naphthyl-
methyl)-2-imidazoline1$2,which is typically used as either the hydrochloride
or nitrate salt. The hydrochloride salt has been given the USAN, naphazo-
line hydrochloride1. Other chemical names include: (a) 1H-imidazole, 4,s-
dihydro-2-(l-naphthalenylmethyl)-, monohydrochloridel, (b) 2-( l-naph-
thylmethyl)-2-imidazoline monohydrochloridel, and (c) 4,5-dihydro-2-(1-
naphthalenylmethy1)-1H-imidazole,monohydrochloride3. The CAS registry
number for naphazoline hydrochloride is 550-99-21; the CAS number for
the free base is 835-31-4l.
Empirical Formulal: C14H14N2 * HC1
Molecular Weightl: 246.74

Structure:
310 G . MICHAEL WALL

Naphazoline hydrochloride is a white to almost white, odorless, crystalline
powdefl with a bitter taste5y6.
1.3 History
An investigation of the vasoconstrictor activity of substituted imidazolines
by Fritz Uhlmann at Ciba in Basle, Switzerland during the early 1940’s re-
sulted in the introduction of the sympathomimetic drug, naphazoline; its
analogs, xylometazoline and oxymetazoline, used as decongestants; and
also the a-adrenoceptor antagonist, tolazoline798. Patents include: U.S
Patent 2,161,938 (1939) and Danish Patent 62,889 (1944)6. Naphazoline
has been marketed under a variety of trade names around the world29399,
1.4 Pharmacology
Naphazoline is a potent a-adrenergic sympathomimetic agent. It is a vaso-

constrictor with a rapid and prolonged action in reducing swelling and con-
gestion when applied to mucous membranes, hence, its use for the symp-
tomatic relief of rhinitis and sinusitis. Rebound congestion and rhinorrhea
are common after prolonged use. Nasal drops or spray are used as a 0.05%
aqueous solution of the hydrochloride or nitrate, with a usual recommended
dosage of 2 drops in each nostril every 3 hours. Aqueous solutions have
also been used as ophthalmic conjunctival decongestants4.
2. SYNTHESIS
Naphazoline hydrochloride has been prepared through a series of synthetic
chemical steps beginning with (1-naphthy1)-acetonitrile,I (Figure l)3910.
The starting material, I, is treated with ethanol and hydrochloric acid to ob-
tain the naphthyl-(1)-acetiminoethylether hydrochloride, II1o. A solution is
made of 2.7 parts II and 12 parts absolute alcohol3. One part of ethylenedi-
amine is then added and the mixture is heated to gentle boiling with stirring
under nitrogen until the evolution of ammonia ceases. The alcohol is then
distilled and the residue is dissolved in 40 parts of benzene and 1.8 parts of
caustic potash. The benzene is removed and the residue is recrystallized
several times from toluene. Reaction with hydrochloric acid gives naphazo-
line hydrochloride, III3. The preparation of radiolabelled naphazoline with
14Cin the 2-position of the imidazoline ring has also been reported11 using
a-chloromethylnaphazoline, potassium cyanide-14C, and ethylenediamine.
I I11
Figure 1. Synthesis of naphzolinc hpirochbr&ie.

312 G. MICHAEL WALL
3.1 Spectroscopy
The infrared spectrum of naphazoline hydrochloride was obtained. A mix-
ture of the drug substance and potassium bromide was pressed into a pellet
and analyzed using a Perkin-Elmer Model 1750 FTIR. The spectrum is
shown in Figure 2. The major absorption bands for the infrared frequencies
and the corresponding assignments are listed in Table I.
3.1.2 Ultraviolet Spectra
The ultraviolet absorption spectra of naphazoline hydrochloride in absolute
ethanol, pH 3 buffer (0.05M phosphate), pH 7 buffer (0.05M phosphate)
and pH buffer (0.05M borate) were obtained using a Perkin-Elmer 559A
W/VIS spectrophotometer and 1cm cells. A representative W spectrum in
ethanol is shown in Figure 3. Samples of naphazoline hydrochloride in
these solvents were scanned from 200 to 400 nm and the absorption coeffi-
cients at wavelengths of maximum absorption were calculated (Table II).
The lH-NMR spectrum (100 MHz) of naphazoline hydrochloride has been

reported and the chemical shifts have been assigned for the methylene
groups12. The IH-NMR spectrum (300 MHz) of naphazoline hydrochloride
(143 mgmL DMSO-d6 at 1OOOC) was obained using a Vanan VXR 300
spectrometer (Figure 4). In order to assign all of the aromatic proton
signals, a series of 2-D experiments were carried out: these spectra were not
shown but the assignments are listed in Table III.
The l3C-NMR spectrum (22.5 MHz) of naphazoline hydrochloride has

been reported and the chemical shifts have been assigned for the methylene
and imidazoline carbons12. The %NMR spectrum (75 MHz) of
naphazoline hydrochloride (143 mg/mL DMSO-& at 1OOOC) was obtained
using a Varian VXR 300 spectrometer (Figure 5 ) and the assignments are
listed in Table III. The Attached Proton Test (APT)(Figure 5 ) and extensive
2-D studies were performed in order to assign aromatic carbons.
The resonances for the methylene protons were shifted downfield for the
HC1 salt compared to the base: h 6 (ppm) (+ indicates downfield shift
compared to base); C&C&, +0.44 and aryl-C& +0.53)12. The reso-
Figure 2. In@zrd spctnm (KBr)Of Mphotolinc hyhxhbnkk.
3 14 G.MICHAEL WALL
I .E
0.c
I I I I 1
200 250 300 350 600
Wavelength (nm)
Figure 3. UVspctrum of naphamline hydnxhloride (0.019mghL in

ethanol).
Table I. Infrared spectral assignments for naphazoline hydrochloride.
Wavelength (cm-I) Assignment
3150-2500 C-H and N-H stretch

1618 Amine salt N-H
1302, 1198 20 N-H
801, 765 Imidazoline C-H
602, 561, 525, 481 Out of plane ring bend
Table II. Ultraviolet absorption of naphazoline hydrochloride.

E (l%,1 cm)
Solvent 223nm 270nm 280nm 287nm 291nm
Ethanol 3622 239 286 196 198
pH 3 Buffer 3214 246 287 198 193
pH 7 Buffer 3246 238 279 193 188
pH 9 Buffer 3294 236 274 191 187
I - - - . . - - - - - - - 1 - - - . . - - - I - . - . ..-.,.... ....,.... . . . . I . . . , ...,,..., rrr
....,.,.. 0
1)
F i R m 4. H-NMR Siuctn#n (300MHz) of Mphatolne h y h x h l o f i (143 m g h L in DMso-d6 at I W C ) .

I
Figun 5. JJC-NMRSpanun (75 MHz) of napAazoline hydrrnrhloride (143 m g h d inDMSOd6 a! IcK)DC).

Table III. lH- (300 M H Z ) and 1% (75 MHz) N M R Data for Naphazoline
HCl(l43 mg/mL in DMSO-& at 1OOOC).
1 128.71
2 7.64 (lH,d) 128.09
3 7.49 (lH,m) 125.61
4 7.91 (lH,d) 128.47'
5 7.96 (lH,d) 128.66*
6 7.54 (lH,m) 126.06
7 7.58 (lH,m) 126.83
8 8.15 (1H,d) 123.34
9 - 131.37
10 133.57
11 4.45 (2H,s) 29.41
12 169.78
14 & 15 3.82 (4H,s) 44.40
* Interchangeable assignments
nances for carbons attached to the imidazoline ring were shifted for the HC1
salt compared to the base: A 6 (ppm) (- and + indicate upfield and
downfield shifts respectively compared to base); zIHgH2, -5.09; aryl-
CH2, -3.63; and N-IZ-N, +3.9611.
3.1.4 Mass Spectra
Mass spectra were obtained for naphazoline hydrochloride using a Finnegan
MAT TSQ46 GC/MS/MS unit. A small amount of naphazoline hydrochlo-
ride was volatilized by heating at a linear rate of 5 mA/sec from 0 mA to
about 500 mA and ionized by either chemical ionization (CI, 0.3 Torr pres-
sure of isobutane) or by electron impact (EI) at 70 eV. The CI and EI mass
spectra were presented in Figures 6 and 7 and the interpretation presented in
Table IV.The fragmentation pattern was consistent with the chemical
structure of naphazoline hydrochloride (Figure 8).
3.2 Thermal Properties
3.2.1 Melting Range
The melting point of naphazoline hydrochloride has been reported as
257OC13 with a range of 255-60 (decomposition)5*6;the melting point for
the base has been reported as 115-120OC13.
3.2.2 Differential Thermal Analysis
A 2-mg sample of naphazoline hydrochloride drug substance was heated
from 40OC to 3000C at a linear rate of 200C/min using a Perkin-Elmer DSC-
4. One single, sharp endotherm was observed with an onset of 259OC and a
maximum of 261OC, corresponding to the melting range, after which
decomposition occurred (Figure 9).
3.2.3 Thermogravimetric Analysis
A 7-mg sample of naphazoline hydrochloride was heated using a Perkin-
Elmer System 4 ThermogravimetricAnalyzer from 4OOC to 298OC at a
linear rate of 200C/min. The drug substance exhibited a gradual weight loss
near the melting range (Figure 10).
3.3 X-Ray Crystallography and Powder Diffractometry
Naphazoline hydrochloride exists as a crystalline powde8. Podder et a1.15
described the crystal structure: Mr = 246.73, monoclinic, P21/c, a = 11.895
-
(3), b = 9.228 (2), c = 12.820 (3) A, J3 = 117.18 (2)0, V = 1252 813, 2 = 4,
D m 1.30, Dn= 1.29 Mg m-3, h (Cuka) = 1.5418 A,p = 2.48 mm-1,
F(OO0) = 524, T = 277 (1) K. Final R = 0.040 for 1291 observed reflec-
I
xP
ii
d
E
p
320
Figure Z EJ Mass spectrum of napfuwlituh@mhlon&.
322 C. MICHAEL WALL
Tablc I V . t I and CI Mass splrital Assignnrcnls for naphamline hydrochloridc

~~~
El Rel. Assignment Rel. CI
21 I 4
209 100
I95 7
181 6
I53 9
141 9
1 I5 12
11s I 153 I
--,-I ----
NAPHAZOLINE HCL
VT. 228 g
SCAN RATE. 20.00 w a i n
Figurc 9. DSC of naphazolinc hyhchloride.

IT, 7.290. ag RATE. 20.00 d.g/rln
m a
I#c .Q¶J
T Q -84
W
P
N
TEMPERATURE (C) TC
Figwv 10. Z A of naphawline hydrochlode.

x
U
.r(
u)
c
W
Y
N
Ln U
C
Y
5 10 15 28 2s 30 35 45
20
F i g u n 11. X-Ray pmvdPr difiaction pancrn of naphamline hyirochloride.

Table V. X-Ray powder diffraction data for naphazoline hydrochloride
- -
obtained using CuKa radiation and indexed on the basis of a monoclinic
cell: P2ljc, a = 11.895 (3), b 9.228 (2), c 12.820 (3) A, fl- 117.18
(2)O.
V h k 1 d Intensity
1 0 0 10.7 47
0 1 1 7.21 29 1 2 1
3.760 18
1 1 0 1 - 2 1 3 1
-1 1 1 )
6.97 13
1 2 2 J
'I
-1 0 2 6.38 41
0 0 2 5.71 3 -3 1 2 3.598 100
-3
2 0 0 ) 0 2 2 J
1 1 1
-2 0 -2 I
5.28 8
0 1 3
} 3.529 23
-2 1 1 5.01 25 3 1 0 3.300 4
0 1 2 4.87 29 -1 0 4 j
3.135 18
0 2 0 1 1 2 2 1
4.60 27
2 1 0 ) 2 2 1 3.091 6
0 2 1
1 0 2 1
I 4.29 30
2 1 2
-2 1 4
3.042
3.016
8
14
- 3 0 2 i
. 3.881 6
1 1 2 1
tions. The bond lengths of the N-C-N group of the imidazoline ring were
short and indicative of double bond character. One nitrogen atom was pro-
tonated and both nitrogen atoms participated in hydrogen bonding. Each
chlorine atom was involved in two intermolecular hydrogen bonds of the
form Nl-H-Cl-H-N2, that linked the molecules into continuous parallel
chainsls.
To obtain an x-ray powder diffraction pattern, a sample of the drug sub-
-
stance was irradiated using a Philips powder diffractometer equipped with a
diffracted beam graphite monochronometer. CuKa (1 1.5405 A) radiation
was used for obtaining the powder pattern (Figure 11).All of the diffraction
lines could be assigned hkl indicies on the basis of the unit cell parameters
proving that the material was single-phase (Table V).
3.4 Partition Coefficients
Partition coefficients were determined for naphazoline hydrochloride be-
tween pH 3 buffer (0.05 M phosphate), pH 7.0 buffer (0.05 M phosphate)
and pH 9 buffer (0.05 M borate) versus l-octanol. All solutions were pre-
pared using octanol-saturated buffers and buffer-saturated octanol. Tubes
containing 100 mg of naphazoline hydrochloride, 10 ml of buffer and 10 ml
of octanol were agitated for 2 hours at 23OC and allowed to partition
overnight. Analysis (HPLC) of the aqueous phases of each mixture revealed
the following partition coefficients: pH 3.0 = 0; pH 7.0 = 0; pH 9.0 = 7.4.
The pKa of naphazoline HC1 has been reported as 10.9 at 200C4, 10.35 k
0.02 at 25OC16, 10.13 ? 0.02 at 35*C16, and 9.92 f 0.03 at 450C16.
3.6 Solubility
The solubility of naphazoline hydrochloride in various solvents at room
temperature is presented in Table VI.
Table VI. Solubility of Naphazoline Hydrochloride

Solvent Solubility Reference
Water 1i n 6 13
Ethanol 1 in 15 13
Chloroform Very slightly soluble 4
Diethyl Ether Practically insoluble 4
3.7 Solution Color, Clarity and pH

An aqueous solution (1 in 100) of naphazoline hydrochloride in carbon
dioxide-free water is clear, colorless and exhibits a pH value between 5.0
and 6.617.
4. TYPICAL METHODS OF ANALYSIS
4.1 Identity
4.1.1 Infrared Spectrophotometry
The identity of naphazoline hydrochloride may be determined by compari-
son of its infrared spectrum (KBr) (see Figure 2) to an authentic reference
standardl7.
4.1.2 Ultraviolet Spectrophotometry
The identity of naphazoline hydrochloride may be confirmed by comparison
of its ultraviolet spectrum (1in 50,000) to that of an authentic standard and
the observation of a maximum at 280 nm17.
4.1.3 Chloride Identity Test
An aqueous solution of naphazoline hydrochloride (1 in 100) is treated with
6N ammonium hydroxide to precipitate naphazoline base. The filtrate then
yields a white, curdy precipitate upon the addition of 0.1N silver nitrate.
The precipitate is insoluble in nitric acid but is soluble in a slight excess of
6N ammonium hydroxide5~~~.
4.1.4 Reaction with Bromine
A 10-mL aliquot of an aqueous solution of naphazoline hydrochloride (1 in
100) when mixed with 5 mL of bromine-saturated water yields a yellow
precipitate. Upon boiling, a deep purple color is produced5.
4.2 Colorimetry
Naphazoline has been analyzed by colorimetry using reagents such as
sodium nitroprussidel8, ceric sulfatelg, chloranillg, bromocresol green20,
bromophenol blue20, bromothymol blue20, methyl orange20, cobaltous
acetate in chloroform-methanol21,iodine in chloroform22-25,and 2,6-
dichlorophenol-indophenolin CHC1326.

Elemental analysis of a sample of naphazoline hydrochloride was per-
formed: Anal. (C14H1&HCl) C (calcd 68.14; found, 68.52), H (calcd,
6.14; found, 6.28), N (calcd, 11.36; found, 11.48), C1 (calcd, 14.37;
found, 14.54).
4.4 Titrimetry
Naphazoline hydrochloride drug substance may be titrimetrically assayed as
described in the USP monographl7. The hydrochloride salt is dissolved in
glacial acetic acid with mercuric acetate and titrated with dilute perchloric
acid using crystal violet as the indicator. Also, nonaqueous titration of n a p
hazoline hydrochloride in acetic anhydride/glacial acetic acid with dilute per-
chloric acid and potentiometric detection has been described as an official
method in the Japanese Pharmacopoeias. In addition, nonaqueous titration
with dioctylsulfosuccinate sodium salt using 3'3"5'5"-tetrabromophenolph-
thalein as the indicator has also been reported25.
4.5 Chromatography
The USP describes a TLC method for ordinary impurities in naphazoline
hydrochloride drug substancel7. A sample of the drug substance dissolved
in methanol (10 mg/mL) is spotted on a silica gel TLC plate, eluted with a
mobile phase of methanol-glacial acetic acid-water (8:1:1, v/v/v), and
visualized with iodoplatinate spray.
4.5.2 High-pressure Liquid Chromatography
Naphazoline has been analyzed in ophthalmic preparations by HPLC using
a 10 pm octadecylsilane column (3.9 X 300 mm), a mobile phase of 0.08 M
HClO4 (PH 2.2)-methanol(7/3, v/v), a flow rate of 2 mL/min and UV de-
tection at 265 nm28; in ear and eye drops using a 10 pm octadecylsilane
column (4 X 250 mm), a mobile phase of methanol-water (40/60, vh), a
flow rate of 2 mL/min and UV detection at 279 nm29; in ophthalmic formu-
lations or raw material using a 5 pm cyano column (4.6 X 150 mm), a
mobile phase of dilute phosphate solution (PH 3)/-acetonitrile (60:40, v/v),
a flow rate of 2.0 mL/min and W detection at 225 nm30 or a 5pm octylsi-
lane column (4.6 X 250 mm), a mobile phase of 0.05 M phosphate solution
(pH 5,6)-acetonitrile (4:1, v/v) containing 0.07 M triethylamine, a flow rate
of 1.5 mL,/min and W detection at 270 nm3O; in tablets and capsules using
a 10 pm phenyl column (4 X 300 mm), a mobile phase of water-methanol-
glacial acetic acid (55:44:1, v/v/v) containing 0.005 M heptane sulfouic acid
sodium salt, at a flow rate of 2.0 d / m h and W detection at 254 nrn3l.

Naphazoline has been analyzed by gas chromatography using various sta-
tionary phases including OV-l3%, OV-3 3%, OV-7 3%,OV-17 256, and
QF-1 5%32*
5. STABILITY-DEGRADATION
5.1 Potential Routes of Degradation
Naphazoline has been shown to be relatively stable in acidic or neutral solu-
tions but readily prone to hydrolysis in basic solution. The first step in the
hydrolytic reaction33 results in the formation of l-naphthylacetylethylenedi-
amine which upon vigorous treatment34, undergoes further cleavage to form
1-naphthylacetic acid and ethylenediamine(Figure 12). The kinetics of this
reaction have been describedl6. The major degradation products of napha-
zoline, 1-naphthylacetylethylenediamineand 1-naphthylaceticacid, have
been prepared33 and investigated30*33*34.
5.1.1 Characterization of 1-NaphthylacetylethylenediamineHydrochloride
5.1.1.1 Thin-Layer Chromatographyof 1-Naphthylacetylethylenediamine
An adaptation of the USP TLC procedure17 for the determination of ordi-

nary impurities in naphazoline hydrochloride allowed for the detection of 1-
naphthylacetylethylenediaminein the presence of naphazoline. Using silica
gel 60 high-performance TLC plates (20 X 20 cm) and a mobile phase of
methanol-glacial acetic acid-purified water (8:1:1, v/v/v), spots were visible
-
after spraying with ninhydrin: naphazoline Rf= 0.54; l-naphthylacetyl-
ethylenediamine Rf 0,6330. A similar method has been described in the
European Pharmacopoeia for 1-naphthylacetylethylenediaminein napha-
zoline nitrate35.
5.1.1.2 Liquid Chromatography of 1-Naphthylacetylethylenediamine
1-Naphthylacetylethylenediaminehas been quantitated in the presence of
naphazoline and 1-naphthylacetic acid using column chromatography fol-
lowed by W assay33934. A modem HPLC procedure has been developed
for the analysis of 1-naphthylacetylethylenediaminein the presence of nap-
hazoline by HPLC using a 5 pm cyano column (4.6 X 150 mm), a mobile
phase of 0.025 M Na2HPO4 buffer (pH 7,4)-acetonitrile (35:65, v/v), a
flow rate of 2.0 mL,/min and W detection at 270 nm30. Retention times
were: naphazoline, 6.3 min; 1-naphthylacetylethylenediamine,3.1 min
(Figure 13).
d? N NH2
H
OH-
/ / HC'
Naphazolinc HCI
1-Naphthylacetyluhylenadiamine
1-Naphthylacdc Acid M y lmdiarnine
Figure 12. Lkgradaion producrs of nuphazolinc hydrochloridc undcr alkaline conditions.

c L i
Figure 13. HPLC of I-nriphrhylacerylerh~l~~diurnine HCI (5.2 pg), L and

naphamtine HCl (1.2 ps), 2 IS pm qano column, 4.6 X 150 nim, 5.025 M
phosphate bufler (pH 7.4)-acetonirrile (35:6S. vh). 2.0 mllinin, UV 2701.
5.1.1.3. Synthesis of 1-NaphthylacetylethylenediamineHydrochloride

The synthesis of 1-naphthylacetylethylenediaminewas described by
Schwartz et ~1.33using a modification of previous work by Miescher et
uP6. Five grams of naphazoline hydrochloridewere refluxed with 100 ml of
0.5N NaOH for 30 minutes. The mixture was then cooled, made alkaline,
and extracted with CHC13. The CHCl3 extract was evaporated, leaving a
yellowish oil which solidified upon chilling to give the base as an off-white
solid recrystallized from CHC13-petroleum ether (1:l) mp 93-95OC. The
base in chloroform was treated with HC1 gas to obtain the HCl salt as an
off-white solid mp 142-8%
5.1.1.4. Physical/Chemical Properties of 1-Naphthylacetylethylenediamine
Hydrochloride
A sample of 1-naphthylacetylethylenediaminehydrochloridewas prepared
and evaluated30. The substance appeared as off-white powder with a melt-
ing point of 153.8-154.2OC. The material was not hygroscopic. The IR
spectrum (Table W, Figure 14), U V spectrum (Figure 15), lH-NMR
spectrum (Table VIII, Figure 16), 13C-NMR and APT spectra (Table WI,
Figure 17), and mass spectrum (Table IX,Figure 18) were consistent with
the proposed chemical structure. The DSC (Figure 19) of l-naphthyl-
acetylethylenediaminehydrochloride was consistent with the metling range.
Table W. Infrared spectral assignments for l-naphthylacetylethylenedi-

amine HC1.
Wavelength (cm-1) Assignment
3392 10 amide N-H stretch

3220-2400 C-H and N-H stretch
1641 amide C-0
1599,1482,1438 aryl C-C stretch
1520 20 amide N-H
802,795,779 substituted aromatic
5.1.2. Synthesis and Analysis of 1-Naphthylacetic acid

The synthesis of another naphazoline degradation product, 1-naphthylacetic
acid, was described b Schwartz et aP3. Five grams of naphazoline HC1
were refluxed with 5(rml of 1N NaOH for 2 hours. The mixture was cooled
7-
ma I U 1 2 1 CI.
Figun 14. Inrfrand spanun (KBr) of I-~phrhylaccrykthyktudicdiamirv

HCI.
1.0
e
0
8
4
0.0
Figum 15. U V Spctrum o f l - M ~ h y l ~ ~ l ~ h HCI

y ~(0.02
M m i ~
m g M in ethanol).
W
01
W
Figure 16. IH-NMR (200 MHz) of l - ~ p h r h y l o c t r y l c t h y l e ~HCI

i ~ i(20
~ mg in DMSOdd.
W
41
Table WI. NMR assignments for 1-naphthylacetylethylenediamneHC1

(20 mg in DMSOQ6).
1
2
3 7.44-8.60
4 7.44-8.60 124.34J25.47,
5 7.44-8.60 P 125.59,125.96,
6 7.44-8.60 127.08,127.88,
7 7.44-8.60 128.31
8
9
10
11
12
13
14
15
16
and acidified with HCI, producing a flocculent white precipitate. The pre-
cipitate was filtered, washed with cold H20 and recrystallized from hot H20
mp 133-134OC; W spectrum, A max 283 pm, E (l%, 1 cm in CHC1-j) =
360. 1-Naphthylaceticacid has been quantitated in the presence of
naphazoline and 1-naphthylacetylethylenediamineusing column
chromatography followed by UV a~say3373~.
Table IX.EI Mass spectrum of 1-naphthylacetylethylenediamineHC1.

EI Relative
(We) Abundance (%) Assignment
228 4 [MI+
199 14 [M-NHCH$
185 73 [M-NHCH2CH2]+
141 100 [M-C3H7N20]+
128 14 [M-C4H8N2O]+
5.2 Solid-state Stability

Naphazoline hydrochloride drug substance has been shown to be stable for
at least 6 months under the conditions of room temperature, 4OC, 350C,
40OC at 75% relative humidity, 50OC and exposure to light (1000 foot-can-
dles) at room temperature. The drug substance was found not to be hygro-
scopic.
An investigation was performed to determine if naphazoline hydrochloride
would exhibit polymorphism. Samples of the drug substance were treated
with (a) heat at lOOOC for 4 hours or (b) vigorously ground with a mortar
and pestle. Analytical data from IR, DSC, and x-ray powder diffractometry
showed no changes compared to a control sample, suggesting no evidence
of polymorphism for naphazoline hydrochloride30.
The stability of naphazoline hydrochloride in aqueous buffers (PH 4.5,7.0,
9.0) and oxygen-saturated water was evaluated under the conditions room
temperature, 350C, 55OC, and light exp0sure3~.The drug was relatively
stable under all conditions at acidic and neutral pH and in oxygen-saturated
water for at least 26 weeks. The alkaline solutions, however, turned a range
of dark colors and showed severe degradation after only 4 weeks.
1m.a 7 I
n.0-
1s
T
F i p n 18. El Mars s p c m m of 1-naphrhylacctyltthyknedim~neHa.
u
f
6. DISPOSITION AND TOXICITY

Naphazoline hydrochloride is commercially available in concentrations of
0.01 to 0.1% as a nasal or ocular decongestant. Local effects are obtained
after topical administration to the eye and nasal passages. Metabolic studies
could not be found in the literature, despite the report of the synthesis of
14C-labelleddrug substance intended for that purpose1'. Dittgen et a1.37
studied the elimination of naphazoline from the isolated pig eye after topical
application. Reports of systemic effects of poisoning with naphazoline have
been scarce38. The LDSOS.C. in rats has been reported as 385 mgAcg6.
ACKNOWLEDGEMENTS
The author expresses his sincere thanks to the following persons who have
provided data and/or information for this chapter: R.E. Hall for solid-state
data; G. Havner for confirming the bromine ID test; D.D. Taylor for
partition coefficients, UV, MS, and NMR data; R. Conroe and S. Spruill
for the synthesis and B. Scott and P. Ritter for analytical data on 1-
naphthylacetylethylenediamine,all at Alcon Laboratories; Professor John
Baker, Department of Medicinal Chemistry, University of Mississippi,
Oxford, Mississippi for NMR data and interpretation; and Professor Hugo
Steinfink, Department of Chemical Engineering, University of Texas,
Austin, Texas for x-ray powder diffraction and crystallographic
information.
REFERENCES
1. Heller, W.M.; Fleeger, C.A. USAN and the USP Dictionary of
Drug Names, United States Pharmacopeial Convention, Inc.:
Rockville, Maryland; 1990, p. 405.
2. Pharmacological and Chemical Synonyms, Ninth Edition, Marler,
E.E.J., Ed., Elsevier: New York; 1990, p.380.
3. Sittig, M. Pharmaceutical Manufacturing Encyclopedia, Second
Edition, Noyes Publications, Park Ridge, N.J., 1988, p. 1058.
4. Martindale: the Extra Pharmacopeia, Twenty-ninthEdition,
Reynolds, J.E.F., Ed., The Pharmaceutical Press: London; 1989,
1470.
5. The Pharmacopeia of Japan, Eleventh Edition (English Version),
The Society of Japanese Pharmacopeia: Tokyo, Japan; 1986, p.761.
6. The Merck Index, Eleventh Edition, Budavari, S.; O’Neil, M.J.;

Smith, A.; Heckelman, P.E., Eds.; Merck & Co., Inc.: Rahway ,
N.J., 1989, p.1008.
7. Scholz, C.R. Ind. Eng. Chem., 1945,37, 120.
8. Hartmann, M.; Isler, H. Arch. Exptl. Path. Pharmakol., 1939,
192, 141,
9. Japta List: Japanese Drug Directory, Third Edition, Japan
Pharmacetucial Traders’ Association: Tokyo, Japan; 1987, p. 395.
10. Kleeman, V.A.; Engel, J. Pharmazeutische Wirkstoffe, Georg
Thieme Verlag Stuttgart: New York; 1982, p. 620.
11. Luu Duc, C.; Pera, M.H.; Fillion, H.; Delord, C.A. Bull. SOC.
Chim. Fr., 1976, (3-4 Part 2), 555.
12. Kountourellis, I.E. Pharmazie, 1988, 43, 26.
13. Clarke, E.G.C. Isolation and Identification of Drugs, The
Pharmaceutical Press: London; 1969, p. 435.
14. The United States Pharmacopeia, Twenty Second Revision, United
States Pharmacopeial Convention: Rockville, Maryland; 1990,
Naphazoline Ophthalmic Solution Monograph, p. 917.
15. Podder, A.; Mukhopadhyay, B.P.; Dattagupta, J.K.; Saha, N.N.
Acta Cryst., 1983, C39, 495.
16. Stern, M.J.; King, L.D.; Marcus, A.D. J . Am. Pharrn. Assoc.,
1959, 48, 641.
17. Reference 14, Naphazoline Hydrochloride Monograph, p. 916.
18. Ismaiel, A.; Twakkol, M. Pharmazie, 1974, 29, 54.
19. Belal, S.; Elsayed, A.H.; Abdel-Hamid, M.E.; Abdine, H. J .
Pharm. Sci., 1981, 70, 127.
20. Sane, R.T.; Sane, S. Indian Drugs, 1979,16, 239.
21. Bult, A.; Klasen, H.B. Pharm. Weekbld., 1974, 109, 513.
22. Kovar, V.K.A.; Abdel-Hamid, M. Arch. Pharm., 1984,317,
246.
23. Lajosne, S. Acta Pharm. Hung., 1980,50, 130.
24. Lajosne, S . Acta Phurm. Hung.,1980,50, 270.

25. Lajosne, S . Acta Pharm. Hung., 1982, 52, 61.
26. Salam, M.A.; Issa, A.S.; Mahrous, M.S. Anal. Lett., 1986, 19,
2207.
27. Solimar, S.A.; Abdine, H.; Mocos, M. Can. J . Pharrn. Sci., 1976,
11, 36.
28. Bauer, J.; Krogh, S . J . Pharm. Sci., 1983, 72, 1347.

29. Al-Kaysi, H.N.; Salem, M.S.; Al-Khalili, N . Dlrasat, 1985, 12,
101.
30. Alcon Laboratories, Inc., Unpublished data on fire.
31. Koziol, T.R.; Jacob, J.T.; Achari, N. 1. t'harm. Sci., 1979, 68,
1135.
32. Quaglio, M.P.; Cavicchi, G.S.; Cavicchioni, G. Boll. Chim.
Farm., 1973, 112, 760.
33. Schwartz, M.; Kuramoto, R.; Malspeis, L. J . Am. ktiarm. Assoc.,
1956,15, 814.
34. Stern, M.J. Drug Standards, 1958,26, 158.
35. European Pharmacopoeia, Second Edition, Maisontleuve S .A.:
Sainte-Ruffine, France; 1982, p. 147.
36. Miescher, K.; Marxer, A.; Urech, E. Helv. C h b . Acta, 1951,34,
1.
37. Dittgen, M.; Oe.stereich, S.; Eckhardt, D. F'harmazie, 1991,46,
716.
38. Montfrans, G.A.; van Steenwijk, R.P; Vyth, A.; Borst, C. Acta
Med. Scand., 1981, 209, 429.
NAYKOXEN
Fahad I . Al-Shamnlary .' Neelofur Abdul Aziz Mian.'

and Mcrhamrnad Saleem Mian'
( I ) Clinical Laboratory Sciences Department

(2) Pharmaceutical Chemistry Department

College of Pharmacy
ANALYTICAL PROFILES OF DRUG SUBSTANCES Copyright c 1992 by Academic Press, Inc

AND EXCIPIENTS - VOLUME 21 345 All rights of reproduction reserved in any form
346 F. I. AL-SHAMMAKY. N. A. A. MIAN. A N D M . S. MIAN
CONTENTS
1 Introduction
2 Description
2.1 Nomenclature
2.1 .l Chemical Names
2.1.2 Generic Names
2.1 .3 Trade Names
2.2 Formulae
2.2.1 Empirical
2.2.2 Structural
2.2.3 CAS (Chemical Abstract Service Registry Number)
2.2.4 Optical Rotation
2.5 Appearance, Colour, Odour and Taste
3 Physical Properties
3.1 Melting Range

3.2 Solubility
3.4 Loss on drying
3.5 Half-life
3.6 Volume of Distribution
3.7 LD50
3.8 Action
3.9 Sulphated Ash
3.10 Stability
3.11 X-Ray Powder Differaction
3.1 2 Spectral Properties
3.12.1 Ultraviolet Spectra (UV)
3.12.3.1 PMR Spectrum
3.12.3.2 13C-NMR Spectrum
3.12.4 Mass Spectrum
4 Synthesis
NAPROXEN 347
5 Pharmacokinetics

5.2 Adverse Effects and Precautions
5.3 uses
6 Methods of Analysis
6.1 Identification Methods

6.2 Spectrophotometric
6.3 Nuclear Magnetic Resonance Method
6.4 Titrimetric
6.5 Polarometric
6.6 Fluorometric
6.7.1 Thin Layer Chromatography (TLC)
6.7.2 High Performance Liquid Chromatography(HPLC)
7 Acknowledgements
8 References
348 F. J. AL-SHAMMARY. N. A. A. MIAN. AND M. S. MIAN
NAPROXEN
1 INTRODUCTION
Naproxen, a propionic acid derivative, is a nonsteroidal anti-

inflammatory agent (NSAIA). The drug is structurally and
pharmacologically related to fenoprofen and ibuprofen (1).
Naproxen is a nonsteroidal compound that has anti-inflammatory,

analgesic and antipyretic activities. The mode of action is unknown,
except that inhibition of prostaglandian synthesis may have an
action role. While various manifestations of anti-inflammatory and
analgesic actions are evident in patients with rheumatoid arthritis
under treatment with naproxen or its congeners, there is no
evidence that the progressive source of the underlying disease is
altered (2) I
2 DESCRIPTION
2.1
1) (S)-6-Methoxy- CY -methyl-2-naph?halene acetic acid (3)
(2) d-2-(6-methoxy-2-napht hy1)propionic acid(3)
(3) 2-naphthalenacetic acid (2,4)
(4) 6-methoxy- CY -methyl-,(+)(2,4)
(5) (+)-2-(6-Methoxy-2-naphthyl)propionic acid (5,6,7)
(6) (+)-6-Methoxy- CY -methyl-2-naphthaleneacetic acid

(2,8)
2.1.2 Generic Names
Naproxen
NAPROXEN 349
2.1.3 Trade Names
Axer; Bonyl; calasen; Diocadal, Dysrnenalgit N; Equiproxen; Floginex;

Laraflex; Laser; Naixan, Napren, E; Naprux; Naxen; Prexan; Prirneral;
Proxen; Reuxen; Veradol; Xenar.
2.2 I
2.2.1
..
Dirical (3)
c14 H14 0 3
2.2.2 Structural
2.2.3 GAS (Chemical Abstracf

Service Reaistrv Number)
(22204-53-11
2.2.4 Qptical Rotat i o n
[ a ] +~65.5O (C = 1 in chloroforrn)(9)
In 4% w/v solution in chloroform
+63.0° - 68.5' (6)
2.3 Jvlolecular Weiaht (3)
230.26
2.4 Elemental C o r n m s i t i o n (3)
C = 73.03% H = 6.13% 0 = 20.84%

350 F. J. AL-SHAMMARY. N. A . A. MIAN. AND M. S. MIAN
2 . 5 m e a r a n c e . Colour Odour and Taste
White to off white crystalline powder with bitter taste (2)

odourless or almost odourless (6).
3 PHYSICAL PROPERTIES
3.1
152 - 1540 (3) 155O (2) 155.30 (9)

about 156O (6,7)
..
3 . 2 Solubility
Practically insoluble in water. Soluble in 25 parts of

ethanoI(96%), in 20 parts of methanol, in 15 parts of chloroform
and in 40 parts of ether (6).
Practically insoluble in water at pH 2; freely soluble in water at

pH 8 or above, sparingly soluble in alcohol (2).
. .
3 . 3 Dissociation ComtaqJ
Pka = 4.2 (25O) (7)
3.4 Loss 0n drvina (6.8)
Dry it at 105O for 3 hours; it loses not more than 0.5% of its
weight.
3.5 Half-life
Plasma half fife, 10-20 hours (mean 14) (7)

. .
3 . 6 Volume of Distribution (7)
.
About 0.1 litre/kg
3.7 LDgo(3)
In mice (mg/kg): 435 intraveinous;

1235 orally
In rats (mg/kg): 575 ip;

534 orally
NAPROXEN 35 I
3.8 A C t i Q n (2,6)
Anti-inflammatory: analgesic and antipyretic activities.
3.9 Sulphated A s h (1 0)
Not more than 0.1%. Use 1.5 g and ignite at a temperature of about
6000.
3.1 0 Stabilitv
Commercially available preparations of naproxen should be stored at

room temperature, exposure of suspension to temperature exceeding
4OoC should be avoided (1). It should be kept in well-closed
containers, protected from light (10).
3.1 1 X-Rav Powder Differaction
X-ray powder differaction data for Naproxen is determined by De Camp,

W.H. (11). X-ray powder differaction pattern was obtained by the use
of Cu K a radiation on a Philips goniometer. The X-ray tube was
typically operated at 40 KV and 20 mA. Detection was effected with a
Nal (TI) scintillation counter coupled to a pulse-height analyser.
3.1 2 Sr>ectral PrsDertieS
3.12.1 Ultraviolet SDectra CUV)
UV spectra of Naproxen (12) in Ethanol (6 mg%) was scanned from

200-400 nm (Fig. 1) using LKB 4054 UV/vis spectrophotometer.
Naproxen exhibited the following UV data (Table 1).
3.12.2 Infrared SDec t r u m
The IR spectrum of Naproxen as KBr disc (12) was recorded on a

Perkin Elmer 1210 infrared spectrometer and is presented in Fig. (2).
The structural assignments of Naproxen have been correlated with the
following frequencies (Table 2).
3.12.3 Nuclear Maanetic Resonance Spectra
The PMR spectra of Naproxen (12) in BMSO-d6 (Fig. 3, 4) was

recorded on a varian XL 200 MHZ NMR spectrometer using TNS as an
internal reference. The following structural assignments have been
made (Table 3).
352 F.J. AL-SHAMMARY. N. A. A. MIAN. AND M. S. M I A N
Table 1: UV Data of Naproxen in Ethanol
Absorbance Molar Absorptivity A:

nrn (E )ci' am moVL
21 2 2.81 3 10795.355 468.833
21 5 2.812 10791.51 7 468.666
263 1.345 51 61.66 224.1 66
27 1 1.325 5084.907 220.833
31 7 0.467 1792.189 77.833
33 1 0.518 1987.91 86.333
Table 2: 1.R. Characteristics of Naproxen
Frequency Assignment
cm-1
31 80 (Carboxylic) -OH.
3000 Aromatic C=C stretch.
2940, 2930 Alipathic C-H stretch.
0
1730 (Carboxylic) II
6-
1600 Aromatic stretch.

0
0
U
0
a3
m
0
' W
0
0
a
m
0
(v
m
0
' 0
m
0
.%
0
. w
hl
0
- Y
hl
- 0
(v
hl
--
%_
-- -.
-cz
0
I I i I
0 0 0 0
0 0 0 0 0
0
-
0
m o Ln 0 Ln
N cv c 0 0
TRANSMITTANCE
0
0
u3
0
0
03
-
0
0
0
0
0
2
0
-
0
-4
-
0
0
lo
-
0
0
03
0
0
0
cy
.c.r
0 0
0 aJ
m Q
N
v,
0
0
w
0 Y
#
CI
0
0 N
L n
Y
cr,
0
.-Cj,
0
0
LL
.t
354
00
4
0
I
Fig. ( 3 ) PMR SPECTRUM OF NAPROXEN IN C D C l

3
.-
Fig. ( 4) PMR SPECTRUM OF NAPROXEN ( D20 Exchange 1

NAPROXEN 3.51
C
C H,
h I
I
CH-COOH
9 b a
i
C H,O
f e
Table 3: 'H-NMR Characteristics of Naproxen
Proton Assignment I Chemical Shift 6 (ppm)

I
6 H aromatics 7.087 - 7.136 (t)
(at d, e f, g, h, i) 7.656, 7699 (d)
3 H Cj) 3.869, 3.878 ( d )
5 H (at a, b, c ) 1.552 - 1.590 (d)
d = douolet, t = triplet.
3.12.3.2 1 3C-NMR Spectrum
I3C-NMR spectrum of Naproxen (12) in DMSO-d6 (Fig. 5, 6) was

recorded on varian XL-200 NMR-spectrometer. The multiplicity of
the resonance was obtained from APT (Attached Proton Test) program.
The BC-NMR spectrum displayed all the fourteen carbon resonances.
The narrow resonance range of some of the carbons makes the
spectrum rather complex. The carbon chemical shifts assignments
are presented in (Table 4).
3.1 2.4 Mass SDectrurn
The mass spectrum of Naproxen (12) obtained by electron impact

ionication (Fig. 7) was recorded on a Finnigen MAT 90
spectrometer.
The spectrum wc?s scanned from 50 to 500 a m a . Electron energy was
70 ev. Emission current 1 mA and ion source pressure torr.
The most prominant fragnents and their relative intensities are
presented in (Table 5).
358 F. J. AL-SHAMMARY, N. A. A. MIAN,AND M.S.MIAN
3
7 5
14
10 12
Table 4: Carbon-13 Chemical Shifts of Naproxen

~~~ ~~~ ~~
Carbon Assianment :hemica1 Shift (wml
18.1 15
45.306
55.267
128.867
133.818
134.825
157.696
181.052
105.559
119.027
129.292
126.137, 126.174
Linterchangable A
127.21 7
I A
180 160 140 1 3 100 80 60 40 2oPPM I

0
13
Fig. ( 5 ) C -NMR SPECTRUM OF NAPROXEN IN C D C l
3
mlr ’
-A- - -
;I I Ii I I I I t ) ’ ])IIII’III I I I I I I l q I I I I l I I I I IIII I I 1 I I Ill 1 1 ) I II I I l i l l l
I I I1
1-40 120 100 80 60 40 20 P P M

13
Fig. ( 6 1 C -NMR OF NAPROXEN I N C O C l (APT)
3
23
100.0
Fig. ( 7 ) MASS SPECTRUM OF NAPROXEN

362 F. 1. AL-SUMMARY. N. A. A. MIAN. AND M.S.MIAN
Table 5: Mass Spectrum of Naproxen
rnlz Relative intensitv % ions
230.1 100
21 5 2
185 58
169.9 10.2
153.2 4
141.1 7
NAPROXEN 363
4 SYNTHESIS
SCHEME
Naproxen is prepared (13) by the acylation of 6 substituted

naphthalenes by AcCl forming the 2-acetyl derivative, which is
further converted to 2-naphthyl acetic acid. Esterification and
alkylation of 2-naphthyl acetic acid in the prescence of H2S04,
MeOH, NaH, Me1 and with NaOH gave after hydrolysis the naphthyl
propionic acid. Resolution of 2-(6-methoxy-2-naphthyl)
propionic (Naproxen) was readily achieved by crystallization of the
cinchonidine salt.
SCHEME
6-substituted 2-acetyl d e r i v a t i v e
Naphthalene
(1) Morpholine, S
( i ) H2SO4, CH30H
CH, ( i i ) NaH, C H s I
I
( i i i ) NaOH
&bOH- D C O O H
C H30 CH,O
Naproxene. (6 s u b s t i t u t c d ) 2 - N a p h t h y l -
a c e t i c acid.
364 F. 1. AL-SHAMMARY. N. A. A. MIAN, AND M.S.MIAN
5 PHARMACOKINETICS
. .
5 . 1 Absorption and Distribut i a
When administered as the acid or the sodium salt, naproxen is

completely absorbed from the gastrointestinal tract; the sodium salt
is absorbed more rapidly than the acid (1). Peak plasma
levels (about 55 pg/ml) are reached in 2 to 4 hours after a 500 mg
dose, and steady state levels are attained after 4-5 doses at 12 hours
intervals. More than 99% is bound to serum albumin. The mean
plasma half life is about 13 hours (2). Approximately 95% of a
dose is excreted in the urine, principally as conjugates of naproxen
and it's inactive metabolite 6-desmethyl naproxen (2).
The apparent volume of distribution of naproxen averaged abut 8.3 L

in healthy adults and about 11.9 L in patients with severe renal
failure (serum creatinine 5.4-1 2.5 mg/dl)(2). In healthy adults,
plasma half-life of naproxen reportly ranges from 10-20 hours.
After entering the stomach naproxen sodium readily dissolves in

gastric juice and about 30% of dose of naproxen is metabolised in
the liver to 6-desmethyl naproxen, which is inactive. Most of the
drug is excreted in urine as unchanged naproxen (10%) and 6-
desmethyl-naproxen (5%) and their glucuronide or other
conjugates (82%). Some data, however suggest that renal excretion
of unchanged naproxen may be negligible or absent. In patients of
with severe renal failure, total body clearance of naproxen may
increase apparently because of decreased binding of the drug serum
proteins. A small amount (less than 5%) of the drug excreted in
feces (2), precipitates is out as fine particles of naproxen. These
particles provide a greater surface area for dissolution than the
larger particles that result from naproxen tablet disintegration
(14). When using conventional tablet formulation of naproxen
sodium and naproxen, the former consequently produces earlier and
higher plasma conc. of naproxen (15). The same holds true during
administration of suppositories of naproxen sodium and naproxen
(16). Mean time to peak plasma conc. (tmax) is about 1 hour for
naproxen sodium and 2 hours for naproxen when administered to
fasting subjects (14).
With respect to concomitant antacid administration past studies have

shown that sodium bicarbonate enhances the rate of naproxen
absorption, magnesium carbonate caused a slight reduction, and a
mixture of magnesium oxide and aluminium hydroxide gave a clear
reduction in the rate of naproxen absorption (17).
NAPROXEN 365
5 . 2 Adve rse Effects a nd Precautions
Adverse reactions to naproxen mainly involve the GI tract,

constipation, heartburn, abdominal pain, and nausea occur in about
3-9% of patients recieving the drug, less frequently, dyspepsia,
diarrhea, stomatitis, vomiting, anorexia, flatulence occur (1).
Adverse effects of naproxen is similar to that of ibuprofen. The

most frequent adverse effects occuring are gastrointestinal
disturbances. Peptric ulceration and gastro-intestinal bleeding
have been reported, other side effects include headache, dizziness,
nervousness, skin rash, pruritus, tinnitus, oedema, depression,
drowsiness, insomnia, and blurred vision and other occular
reactions. Hypersensitivity reactions, abnormalities of liver
function tests, impairment of renal function including interstitial
nephritis or the nephrotic-syndrome, agranulocytosis, and
thrombocytopenia have occasionally been observed (5).
Naprosyn (Naproxen) should not be used conmitantly with the

related rug anaprox (naproxen sodium) since they both circulate as
the naproxen anion (4). All aspirin-sensitive asthmatic patients
developed reactions such as rhinorrhoea, tightness of chest,
wheezing, dyspnoea, after taking naproxen in doses of 40-80 mg
(18).
Gastrointestinal reactions tended to be more frequent and severe

when naproxen dosage was increased from 750 mg/day to 1500
mglday in patients with rheumatoid arthritis. In studies involving
almost 500 children with juvenile arthritis the incidence of rash
and prolonged bleeding time was increased, gastrointestinal and CNS
reactions were reported at approximately the same rate, and other
adverse effects were observed less frequently in children than in
adults. However naproxen was not associated with a higher
frequency of adverse effects in elderly (> 65 years) patients with
rhematoid arthritis or osteoarthritis compared with younger
patients (19, 20).
Naproxen should be given with care to patients with asthma or

bronchospasm, bleeding disorders, cardiovascular disease, peptic
ulceration or a history of such ulceration, renal failure, and in
those who are recieving coumarin anticoagulants. Patients who are
sensitive to aspirin should generally not be given naproxen (5).
Naproxen may interfere with some tests for 17-ketogenic steroids

(5).
F. 1. AL-SHAMMARY, N. A. A. MIAN, AND M. S. MIAN
Naproxen has analgesic, anti-inflammatory, and antipyretic

properties; it is an inhibition of prostaglandin synthetase. The drug
is used in rheumatic disorders such as ankylosing spondylititis,
osteo arthritis, and rhematoid arthritis, in mild to moderate pain
such as dysmenorrhoea, migrane and some musclokeletal
disorders, and in acute gout (43).
Naproxen is used to relieve mild to moderately severe pain. The

drugs are also used for anti-inflammatory and analgesic effects in
the symptomatic treatment of mild to moderately severe, acute and
chronic muscleskeletal and soft tissue inflammation (1).
The usual dose of naproxen or naproxen sodium is the equivalent of

500 mg to 1 g of naproxen daily in 2 divided doses. A dose of 10 mg
per kg body-weight daily of naproxens in 2 divided doses has been
used in children over 5 years of age with juvenile rheumatoid
arthritis ( 5 ) . In painful conditions such as dysmenorrhoea the
usual initial dose is the equivalent of 500 mg of naproxen followed
by 250 mg every 6 or 8 hours. In accute gout an initial dose
equivalent to 750 mg of naproxen followed by 250 rng every 8
hours has been suggested (5). Rectal administration of naproxen is
sometimes employed naproxen has also been used orally as the
piperazine salt (5).
Naproxen is comparable to aspirin in controlling disease symptoms,

but with lesser frequency and severity of nervous system and
milder gastrointestinal adverse effects (2).
Naproxen has been used effectively to relieve pain, fever, redness,

swelling and tenderness in patients with accute gouty arthritis (1).
One study indicates that single oral dose of naproxen (2.5 or

7.5 mg/kg) was at least as effective as a single oral dose of
aspirin (15 mg/kg) in the reduction of fever in children. The
result of one study suggested that the combination of naproxen
sodium and ampicillin was more effective than ampiciline alone in
elleviating fever, dyspnea, and coughing associated with accute
respiratory infections in children (1). A favourable antipyretic
effect with naproxen 5 mg/kg twice daily in children with fever
caused by infection (21).
Naproxen 7.5 mg/kg twice daily in children (22) and 250-375 mg

twice daily adults (23) has been shown to be extremely effective
NAPROXEN 367
for controlling fever associated with malagnancy. Naproxen has

been proposed as a test to distinguish neoplastic fever from
infectious fever in cancer patients with fever of unknown origin
(23).
6 METHODS OF ANALYSIS
6 . 1 JDENTlFlCATION METHO DS
The infrared absorption spectrum of a potassium bromide

dispersion of its exhibits maxima only at the same wavelengths
as that of a similar preparation of USP Naproxen R.S. (8).
It gives liebermann's test black-green; Marquistes-brown.

Sulphuric acid-orange (7).
The light absorption in the range of 230 to 350 nm of a 0.004%

w/v solution in methanol, exhibits four maxima, at 262, 271,
316 and 331 nm. The absorbance at 262 nm is about 0.91, at
271 nm, about 0.92 at 316 nm, about 0.26 and at 331 nm,
about 0.30 (6).
It melts at 156OC (6).
Dissolve about 500 mg of Naproxen, accurately weighed in a

mixture of 75 ml of methanol and 25 ml of water that has been
previously neutralized to the phenolphthalein end point with
0.1 N NaOH. Dissolve by gentle warming, add phenolphthalein
and titrate with 0.1N NaOH VS. Each ml of 0.1N NaOH is
equivalent to 23.03 mg of C14H1403 (8).
6 . 2 SPECTROPHOTOMETRIC
Spectrophotometric determination of naproxen in tablets form

was done by Tosunoglu, S. (24). A portion of the crushed tablets
containing about 250 mgs of naproxen was shaken with 50 mi of
96% ethanol for 30 minutes and the mixture was diluted to
100 mi with 96% ethanol and filtered. A 1 ml portion of diluted
solution was mixed with 4 mM-rosaniline in 20% ethanol
(3 ml) and extracted with 5 mi of CHC13 and the absorbance was
measured at 545 nm against blank. The calibration range was 1
to 15 p g per ml. Recovery was 99.9%.
Quantitation of naproxen with other drugs in pharmaceutical

dosage forms by first and second derivative uv spectrometry by
368 F. J. AL-SHAMMARY, N. A. A. MIAN. AND M. S. MIAN
Mahrous, M.S. et al (25). Derivative uv spectroscopy was used

to determine the cited drugs in capsules and tablets. First
derivative spectroscopy was used to determine indomethacin in
0.1 N-HzS04 solution, and second derivative spectroscopy was
used to determine naproxen and iboprofen in O.1N-NaOH
solution. The method is rapid and accurate.
3 Powdered tablets (26) equivalent to 50 mg of naproxen were

dissolved in 10 ml of methanol and boiled under reflux with
20 ml of 5M-HCI for 45 mins. The solution was cooled and the
excess of HCI was removed under vacuum. The residue was
dissolved in 10 ml of methanol, adjusted to pH 7.0 with NaOH
solution and diluted to 100 ml with H20. To a portion of this
solution, were added 1 ml of 0.05% P-NN-dimethyl
phenylenediammonium chloride and 1 ml of aq. 0.2% K2Cr207
and H 2 0 to 25 ml. The absorbance was measured after 10
minutes (but with in 1 h) at 600 nm vs, a reagent blank.
Beer's law was obeyed from 5-40 pg ml-1 ( E = 2760).
6.3
Tosunoglu, S . ; et al (27) determined naproxen by NMR

spectrometry. Powdered tablets equivalent to 85 mg of naproxen
were mixed with acetanilide (40-45 mg; internal standard) and
extracted with CHC13 (25 ml). After ultrasonic agitation for 30
minutes, the mixture was filtered and a 10 ml portion of the filtrate
was evaporated to dryness in vacuo. The residue was dissolved in
0.8 ml of CHC13 and the spectrum was recorded on a Bruker NMR
spectrometer operated at 250 and 300 MHz with the probe of 370.
Chemical shifts were measured relative to tetramethylsilane at
1.58 and 2.09 ppm for naproxen and the internal standard
respectively.
6 . 4 TlTRlMETRlC DETERMINATION
The determination of naproxen involved oscillometric titration

(28) of its solution in aq. 20% acetone (10 ml, 10 mM in naproxen
and containing 2 ml of aq. 0.1 M-NH3) with O.1M-KOH, a Radelk is
type OK-302 apparaturs being used for determining the end point.
Results obtained for naproxen indicated the good precision of the
oscillometric technique.
6.5
Polarometric determination (29) of naproxen by dissolving in

various heterocyclic, aromatic and aliphatic basic solvents and the
NAPROXEN 369
optical rotation was determined at Na and Hg iines. The total specific

rotation was calculated, the results obtained showed variations of up
to 22O at the Na line and of 30° at the Hfg line with heterocyclic
solvents with aromatic amines the values obtained were lower than
those of published in U.S.P. and B.P; Negative values of optical
rotation were determined when naproxen was dissolved in aliphatic
amines.
6 . 6 FLUOAIMETR IC DETERMINAT l W
Naproxen contents in sugar coated tablets was determined by

fluorescence (30) spectrophotometry. Naproxen tablets with their
sugar-coating removed, were powdered and a sample equivalent to
about 25 mg of naproxen was dissolved in 5 ml of 1% NaOH and
diluted with Y20. A 0.5 ml portion of the supernatant solution was
mixed with 1 ml of 1M-HCI and dilute to 100 ml with H20 and the
fluorescence of the solution was measured at 356 nrn (excitation at
274 nm) vs. 0.01 M-HCI. Recovery was 39.5% with a coeff. of
variation of 0.8%
6 . 7 CHROMATOGRAPHIC METHODS
6.7.1 Thin laver c h r o m c r t o w h v (TLQ
Naproxen and its metabolites were measured (31)in urine. 1-

2 ml ot sample was acidified with 0.1 rnl of O.1M-HCI and
extracted with 5 rnl of CHCI3. The extract was evaporated, and the
residue was dissolved in 100 jd of ethanol. 10 pI portions were
spotted on to silica gel 60 F254 plates and TLC was carried out with
CHCl3:methanol (17:3) as mobile phase. Spots corresponding to
naproxen and its 6-demethylmetabolite (Rf 0.54 and 0.41,
respectively) were visualized under 254 nrn radiation, scraped off
and extracted with aq. 95% ethanol (4 x 5 ml). The extracts were
diluted to 25 ml, and the absorbance was measured at 232 nm. The
calibration graph covered the range 0.2-0.3pg m1-l in the final
solution.
6.7.2 Hiah Performa nce Liauid ChromatoaraDhy

w
* A summary of the sum of the HPLC methods for the analysis of
Naproxen are given in the Table (6).
ACKNOWLEDGEMENTS
The authors are highly thankful to Mr. Babkir Awad Mustafa, College of
TABLE ( 6 ) Summary of HPLC conditions of Naproxen.
-
Flow rate rnl/rnin. Ietectior Sample -
3ef.
t t f
240 nm 'lasma or blood 32
Dctadecy IsiIane acetic acid (1 125:1375:8)
(10 cm x 4.6 mm) of 25 um.amm.phosphate 254 nrn 'lasma 33

Brownlee RP 18 (5um) buffer (pH 3.0) in 75%
methanol
.**
(25 cm x 4 rnm) of acetonitrile-acetate buffc 240 nm 'lasrna or serur 34
Hypersil ODS (5 urn) (pH 4.8 or 4.2)
(22 cm x 4.6 mm) of 5mM-aq. sod. phosphate- 1 ml/min. uv >apsules or 35

underivetized 5-urn H3P04 buffer of pH 2.6 ablets
Brownlee silica with (19:l) containing 0.9%
7-um silica pre-column acetonitrile
(1.5 cm x 4.6)
Octadecylsilane column methanol.sod. acetate 1.5 ml/rnin. 235 nm Plasma 36

(25 cm x 4.5 mm i.d) buffer
NAPROXEN 37 1
Applied Medical Sciences for his efforts in drawing the spectrums and
figures. The authors also would like to thank Liberty S . Matibag,
College of Applied Medical Sciences for her valuable and professional
help in typing the manuscript.
REFERENCES
1 "Drug Information 91" p. 1124. American Society of Hospital

Pharmacists.
2 "Pharmaceutical Sciences" p. 1059. Mack publishing

Company Easton, Pennsylvania, U.S.A. (1980).
3 "The Merck Index" 11th ed. p. 1014 (1989).
4 Physician's Desk Reference 42nd ed. p. 2101 (988).
5 "Martindale" "The extra pharmacopeia" 29th ed. p. 28. The

Pharmaceutical Press, London (1 989).
6 "British Pharmacopoeia" Her Majesty's Stationary

Office London p. 384 (1988).
7 "Clark's Isolation and Identification of Drugs" 2nd ed.

The Pharmaceutical Press London (1986).
8 "T h e U n it ed States Ph a r ma cop o e ia" United States

Pharmacopoeia1 Convention, Inc., 12601. Twinbrook Parkway,
Rockville, M.D. 20882 p. 917 (1990).
9 "The Merck Index" 10th ed. p. 920 (1983).
10 '* Br iti s h Pha rma co poe ia" Her Majesty's Stationary

Office London p. 300 (1980).
11 De Camp, W.H. J. Assoc. Off. Anal. Chem. 67(5) 9 2 7 -

933 (1984).
12 Mohammad Saleem Mian, Neelofur Abdul Aziz Mian unpublished

data (1992).
13 Jan T. Harrison; Brian Lewis; Peter Nelson; Wendell Rooks;

Adolph Rostkowski; Albert Tomolonis and John H. Fried. J.
Med. Chem. 13 203-205 (1970).
14 A, Moyer S. Cephalgia 6 (Suppl. 4) 77-88 (1986).

372 F.J. AL-SHAMMARY. N. A. A. MIAN, AND M.S. MIAN
15 Sevelius H., Runkel R., Segre E., Bloomfield, S.S. British

Journal of Clinical Pharmacology 10 259-263 (1980).
16 Gamst, O.N., Vesje, A.K., Arabakke, J., International Journal

of Clinical Pharmacology, Therepy and Toxicoloty
22 99-103 (1984).
17 Weber, S.S., Bankhurst, A.D., Mroszczak, E., Ding, T.L.

Therapeutic Drug Monitoring 3 75-83 (1981).
18 A. Szczeklik, et. al. Br. Med. J. 2 231 (1977).
19 Geczy, M., Peltier, L., Wolbach, R. J. Rheumatology 14

348-354 (1987).
20 Husby, G. American J. Medicine 81(Suppl. S.B.) 6-10

(1986).
21 Szmyd, L., Perry, H.D. American Journal of Bpthamology

99 598 (1985).
22 Azeemuddin, S.K.; Vega, R.A.; Kim, T.H.; Ragab, A.H.; The Effect of
Naproxen on Fever in Children with Malignancies Cancers 59
1966-1968 (1987).
23 Peter A. Todd and Stephen P. Clissold Drugs 40(1} p. 91-

137 (1990).
24 Tosunoglu, S. Acta. Pharrn. Turc. 31(3) p. 119-122

(1 9 8 9 ) .
25 Mahrous, M.S.; Abdel-Khalek, M.; Abdel-Hamid, M.E.; J.

Assoc. Off. Anal. Chem. 68(3) 535-539 (1985).
26 Sastry, C.S.P.; Prasad, Tipirneni, A.S.R.; Suryanavayana, M.V.;

Aruna, M. India Drugs 26(11) 643-644 (1989).
27 Tosunaglu, S.; Buyuktimkin, N. Acta. Pharm. Turc. 31(4)

149-152 (1989).
28 Kolodziejska, T. Acta. Pol. Pharm. 40(3) 357-360

(1 983)(Pol.).
29 Ceccarin, G.; Maione, A.M. J. Pharm. Sci. 78(12) 1053-

1054 (1989).
NAPROXEN 313
3 0 Tang, H.; Wang, K. Yaown Fenxi Zazhi lO(6) 358-359

(1990) (Chinese)
31 Abdel.-Moety, E.M.; Al-Obaid, A.M.; Jado, A.I.; Lotfi, E.A. Eur.
J. Drug Metab. Pharmokinet. 13(4) 267-271 (1988).
32 Levine, B.; Caplan, Y.H. Clin. Chem. 31(2) 346-347

( 1 985).
33 Kazernifard, A.G.; Moore, D.E. J. Chromatogr. 533 125-
132 (1990).
3 4 Streete, P.J. J. Chromatogr. 495 179-193 (1989).
35 Larnpert, B.M.; Stewart, J.T.; J. Chromatogr. 504(2)
381 -389 (1990).
36 Shirnek, J.L.; Rao, N.G.S.; and Wahba Khalil, S.K. J. Phar. Sci.
71(4) 436-439 (1982).
PERGOLIDE MESYLATE
Delores J . Sprankle and Eric C. Jensen
Lilly Research Laboratories
Eli Lilly and Company
Indianapolis, IN 46285

AND EXClPlENTS -VOLUME 21 375 All rights of reproduction reserved in any form,
316 DELORES J. SPRANKLE AND ERIC C. JENSEN
TABLE OF CONTENTS
1. Description
1.1 Name, Formula, and Molecular Weight
1.2 Appekance and Color
1.3 History
2. Synthesis
3.1 Confirmation of Structure
3.1 1 SpectroscopicData
3.12 Potential Isomerism
3.4 Mass Spectrum
3.5 Fluorescence Identification
3.6 Fluorescence Spectrum
3.8 Melting Range
3.9 Differential Thermal Analysis
3.10 Thermogravimemc Analysis
3.13 Solubility
3.16 Color Identification Test
4. Methods
4.1 Identity
4.4 Chromatography
4.41 Thin Layer
4.42 High Performance Liquid
5 . Stability - Degradation
5.1 Degradation Profile
5.2 Stability in Dosage Form
6. Drug Metabolism and Pharmacokinetics
7. References
8 . Acknowledgments
PERGOLIDEMESYLATE 377
1. DESCRIPTION

Pergolide mesylate is marketed by Eli Lilly and Company under the trade
name Permax@. It is also commonly referred to as LY 127809.
Chemically, it is known as 8~-[(methylthio)methyl]-6-propylergoline,
monomethane sulfonate. The CAS Registry Number is 66 104-23-2.
Empirical Formula: C19H26N2SCQ03S
Molecular Weight: 410.6

Structure:
1.2 Appearance and Color

Pergolide mesylate occurs as white to off-white crystals or a crystalline
powder .
1.3 History
Permax (pergolide mesylate) is a dopamine receptor agonist at both DI
and D2 receptor sites.' Permax is indicated as adjunctive treatment to
levodopalcarbidopa in the management of signs and symptoms of Parkinson's
disease. Administration of Permax should be initiated with a 0.05 mg/day
dosage for the first 2 days. The dosage should then be gradually increased by
0.1 or 0.15 muday every third day over the next 12 days of therapy. The
dosage may then be increased by 0.25mg/day every third day until an
optimum therapeutic dosage is achieved. Permax is usually administered in
divided doses three times per day. During dosage titration, the dosage of
concurrent I-dopalcarbidopa may be cautiously decreased. 2
DEWRES J. SPRANKLEAND ERIC C. JENSEN
2. SYNTHESIS
Pergolide mesylate can be synthesized from dihydroelymoclavine.3 In the

first step of this method, dihydroelymoclavine is demethylated by von Braun
cleavage. The resulting cyanamide intermediate I is then cleaved to
intermediate I1 via sodium hydroxide. The Wallach reaction utilizing
propionaldehyde and formic acid then propylates N-6 to give intermediate n1.
A mesylate ester functional group is added to intermediate 111using
methanesulfonyl chloride in pyridine to give intermediate IV. Displacement of
the mesylate ester with methanethiol and sodium methoxide results in sodium
thiomethoxide to produce crude pergolide base, intermediate V, which is
isolated, and using methanesulfonic acid, is converted into its final form as
the crystalline mesylate salt from methyl alcohol. The isolated pergolide
mesylate is finally recrystallized from methanol.
Dlhydrodymoclavlna
cn2cn2cn3
CH,SH, NaOCH.
DMF
-
(IV)
Pergollde Mesllste
Figure I . Chemical synthesis for pergolide mesylate

PERGOLIDE MESYLATE 379
3.1 Confirmation of Structure

3.11 Spectroscopic Data
The chemical structure of pergolide mesylate was determined from the
data of synthetic method, elemental analysis, ultraviolet (UV) spectra, infrared
(IR) absorption spectra, hydrogen (1H) and carbon (13C) nuclear magnetic
resonance (NMR) spectra, and mass spectra. The following is a summary
discussion of the spectroscopic data and potential isomerism of pergolide
mesylate to support the confirmation of structure of this compound?
Pergolide mesylate (I), is an ergoline alkaloid that is structurally related
to lysergic acid and its derivatives.5-6 The structures of several lysergic acid
derivatives have been determined by X-ray crystallography 6, and the proton
NMR spectra of lysergic acid and iso-lysergic acid diakylamides have been
reported.5 The proof of structure for pergolide mesylate is provided by
spectroscopic data and supported by correlation with lysergic acid, its
derivatives, and other ergoline alkaloids.
SCH 3
I
CH - SO
3 3
The proton-decoupled 13C NMR spectrum of pergolide mesylate indicates

the presence of twenty carbons, which are further identified as four non-
protonated carbons, six methine carbons, and ten methyl and methylene
carbons via a 1% Distortionless Enhancement by Polarization Transfer
(DEFT) spectrum.7 The eight aromatic and olefinic carbon resonances in the
134 ppm to 106 ppm region have chemical shifts consistent with an indole
structure (2).8 The 1H NMR spectrum contains four aromatic proton
resonances, from 6.88 to 7.22 ppm, whose chemical shifts are also indicative
of an indole-type structure. The assignment of these carbon and proton
resonances were confirmed with IH correlation spectroscopy (COSY) and
W-1H heteronuclear correlation spectroscopy (HETCORR). The UV
spectrum also indicates the presence of an indole chromophore.
The NMR data shows the molecule contains a methyl with carbon and
proton shifts at 10.78 and 0.93 ppm, respectively. The COSY experiment
identifies the coupling of these methyl protons to a methylene site, with 13C
and 1H chemical shifts at 15.53 and 1.68 ppm, which in turn is coupled to
another methylene site. The 13C and 1H shifts of this last site (53.83 and
3.36 ppm) indicate this methylene is attached to a heteroatom. Correlation
experiments c o n f m the coupling to an NH+ site, and the IR absorption band
at 2556 cm-1 is indicative of MI+,allowing us to propose substructure 3.
H
3
The COSY experiment shows the protonated amine of 3 residing in a ring

system as shown in 4. The 13C, 'H, COSY, and HETCORR NMR data are
consistent with the structure proposed in 3. The l3C data indicate a
methylene carbon, located at 55.53 ppm, attached to a heteronuclear site. The
corresponding protons, located at 2.95 and 3.59 ppm, were assigned from the
HETCORR experiment. The COSY experiment shows that these two protons
are coupled to a methine site, whose proton is coupled to protons located at
1.44 ppm, 1.55 ppm, and 2.8 ppm. These protons were assigned to the
corresponding carbons using the HETCORR data, and these l3C resonances
were identified as methylenes from the DEPT spectrum. The COSY and
HETCORR experiments also show a methylene carbon whose protons are
coupled to a methine carbon bonded to a heteroatom, based upon IH and 13C
chemical shifts, 2.55 and 36.44 ppm, respectively.
PERGOLIDE MESYLATE 38 I
The MS spectrum of pergolide mesylate contains a fragment with m/z

267, which is consistent with the combination of substructures 2 and 4 to
yield 5.
CH
MS spectrometry also indicates the presence of an -S-CH3 group from the

m/z 285 fragment. The IH and l3C spectra also shows resonances, located at
2.08 and 15.19 ppm, which are consistent with the -S-CH3 group.
Attachment of the -S-CH3 group to a methylene follows from the 13C data of
the methylene. Presence of the mesylate moiety was confirmed by the carbon
resonance located at 39.64 ppm and its correlation to the protons at 2.36
ppm.8 This is supported by IR absorption bands for an S-0-C stretch at 1038
cm-1 and 0-S-0 stretches at 1157 cm -1 and 1331 cm-1.
The structure for pergolide mesylate (1) is supported by other
spectroscopic evidence. For example, aromaticity is observable in the IR
spectrum and in fragments ( d z 144,267,285) of the mass spectrum. The
mass spectrum fragmentation pattern also supports the connectivity of the ring
systems of the molecule.
3.12 Potential Isomerism

Protonation of the tertiary nitrogen to produce the quaternary amine salt
gives rise to the possibility of both an a and p isomer at the nitrogen position.
The 1H NMR spectrum indicates the presence of two NH+ protons,
confirmed by the results of a deuterium exchange experiment, which are
consistent with 6-a (6) and -p (7)isomers. These assignments are further
confirmed by the coupling of the 6-a-NH+and 6-p-NH+ protons to lH (the
protons attach to the carbon at the 1' position). In anhydrous solvents, such
as dimethylsulfoxide-ds, it is possible to observe both isomers.
In aqueous solvents, the rate of exchange is such that these isomers
rapidly interconvert, resulting in only one NH+ resonance.
SCH 3 SCH 3
I I
r2
- -
f"2 CH - SO
3 3 3 3
CH-So
N
6 7

The infrared spectrum for pergolide mesylate as a potassium bromide
pellet is illustrated in Figure 2. The spectrum was recorded on a Nicolet
Model 60SXB Fourier Transform infrared spectrophotometer. The infrared
spectrum of pergolide mesylate is positively identified with maxima at the
following approximate wave numbers: 3183 cm-l,2556 cm-1, 1456 cm-1,
1157 cm-l, 1038 cm-l, and 775 cm-l. The major absorption bands for the
infrared frequencies and the corresponding assignments are listed in Table I.
Table I. Infrared Band Assignments for Pergolide Mesylate
Wavenumber Assignment
(cm-1)
3183 N-H pyrole with hydrogen bonding: N-Hstretch
3040 Aromatic: C-Hstretch
2556 NH+: N-H stretch

1619, 1606 Aromatic: C-Cstretch
1456, 1443 Aliphatic: C-Hdeformation
1331, 1157 RS03-: 03-0 stretch
1038
RS03-: S-0-C
stretch
794.775
N-Hpyrole with hydrogen bonding: N-H
deformation
m
00 1 08 09 06 02 0
33NWLlIWSNtJklL %
The 300 MHz 1H spectrum of pergolide mesylate (10 mglmL) in

dimethylsulfoxide-dg (2.5ppm) is shown in Figure 3. The spectrum was
obtained on a Varian Unity spectrometer using the following instrumental
parameters: 5 mm 1H/13C dual probe; spectral width, 4416 Hz; 90" pulse;
64K time-domain data points; acquisition time, 7.421 seconds; 100 scans and
probe temperature, 35OC. The spectrum was provided with 0.1 Hz Lorentzian
line broadening.
The proton-decoupled 13C spectrum of pergolide mesylate (50mg/mL) in
dimethylsulfoxide-& (39.5 ppm) is shown in Figure 4. These data were
obtained using a 5 mm lH/13C dual probe; spectral width, 11 658.5Hz; 90"
pulse width; 64K time-domain data points; acquisition time, 5.49 seconds;
relaxation delay, 2.4 seconds; WALTZ- 16 proton decoupling; 4000 scans and
probe temperature, 35°C. The spectrum was processed with 1.0 Hz
Lorentzian line broadening followed by the addition of 64K zero-fill data
points.
Structural assignments for both proton and carbon NMR spectra are listed
in Table 11. Assignments are based on 'H, l3C, lH-lH COSY, DEPT, and
lH-13C HETCORR experiments.
8'
CHZ-S-CH,
I
13
1' 3'
N-
1 2
1 .Dm
Figure 3. IH NMR spectrum of pergolide msylate in dimethylsulfoxide-dg (35 "C)

W
i
m
4
w(
IY IU I 1 "I 11, ," s " n " Y * 1 e# I. F
.
Figure 4 . l3C NMR spectrum of pergolide mesylate in dimethylsdfoxide-dg (35 "C)

388 DELORES 1. SPRANKLE AND ERIC C . JENSEN
Table 11. NMR Chemical Shift Assignments of Pergolide Mesylate
d: doublet m: multiplet q: quartet t: triplet

s: singlet bd: broad doublet bs: broad singlet
3.4 Mass Spectrum

The electron impact mass spectrum of pergolide mesylate is shown in
Figure 5. The spectrum was obtained using a VG Model 7070E magnetic
sector instrument operating at 70 eV ionizing potential. A molecular ion for
the free base at m/z 314 was observed. Accurate mass measurements on these
fragments support these assignments. Several of these fragment assignments
are illustrated in Table 111.
IUk
9s.
98.
65.
88.
E.
78.
65.
68.
55.
5%.
15.
49.
35. 285
154
3% 4ie
Figure 5. Electron impact mass spectrum ofpergolide mesylate

390 DELORES J . SPRANKLE AND ERIC C. JENSEN
Table 111. Assignment of Ion Fragments of the EI-MS Spectrum

- Elemental
- - Parent ion
observec
m/z composition mass mmua D B E ~ relative Structure
- - - - intensities
SCH3
I
$-
CHZ
I
314 314.181 1.1 8.0 100
H’ N
&
--
S+
I
299 299.36C -2.1 8.5 4

0
N
H’
- --
SCH3
I
285 285.144 -1.7 8.5 34

Q$ “CH:
H*
--
Table 111. Assignment of Ion Fragments of the EI-MS Spectrum (continued)
Elemental 3bserved
- Parent ion
mmu Structure
dN-
composition mass relative
intensities
267.183 3 .O 8.5 11
N
H*
25 1 C17H19N2 251.151 3.3 9.5 6
- -
223 C15H15N2 223.123 0.7 9.5 5
@
H'
N
- -
Table 111. Assignment of Ion Fragments of the ELMS Spectrum (continued)
Elemental observed
-- Parent ion
Zompositior mass mmua D B E ~ relative structure
-- intensities
c 14H13N2 209.107
--
0.4 9.5 4
[@] H'
194.095 2.2 9.5 7

[&] CH2
-
+
c 12H 1oN 168.079 2.1 8.5 10

@
, H'
~~ --
167.071 2.1 9.0 12
-
observec
- - Parent ion
Elemental mass mmua D B E ~ rclative Structure
-
miz :ompositior
-- intensities
154 154.056 -3.5 9 .O
H-
- --
33
154 154.056 -0.1 4.0
- --
144 144.069 0.2 7.0 11

iJH2
- --
144 144.078 2.8 6.5
- --
394 DELORES J. SPRANKLE AND ERIC C . JENSEN
structure
a: milli mass unit (difference between observed b double bond equivalents

mass and elemental composition mass expressed
in thousandths of mass units)
3.5 Fluorescence Identification

When pergolide mesylate is viewed under long wavelength UV light (366
nm), the product is observed to fluoresce. Pergolide mesylate has a
characteristic, pale bluish-white fluorescence.
3.6 Fluorescence Spectrum

The fluorescence spectrum of pergolide mesylate in methanol is shown in
Figure 6. The fluorescence excitation and emission analyses of the compound
were obtained using a Perkin-Elmer MPF-66 fluorometer.
M
Wavelength (nm)
Figure 6 . Fluorescence spectrum of pergolide mesylate


The UV absorption spectrum of pergolide mesylate is consistent with the
absorption pattern for compounds with an indole chromophore. The
electronic transition of the indole chromophore of the pergolide mesylate
molecule is observed as follows: the absorption bands at 280 and 274 nm are
A to A* electronic transitions separated by l L b and 'La vibrational transitions.
Additional vibrational transitions for the indole chromophore in pergolide
mesylate are observed by the 0-0 'Lb transition at 291 nm and the 1B
transition at 224 nm.
The maximum absorptions (hm a ) and the molar absorption coefficients
(E m a ) of pergolide mesylate agree with those observed in 3-methylindole? a
model compound with an indole chromophore, and support the structure of
pergolide mesylate. A maximum absorption at or about 280 nm, a shoulder at
or about 290 nm, and a minimum at or about 244 nrn are useful for
identification of an indole structure. The UV absorption data of pergolide
mesylate and 3-methylindole are shown in Table IV.
Table IV. Maximum Absorption and Molar Absorption Coefficients
Maximum Molar absorption

Compounds absorption coefficient
(nm) W-lcm-1)
29 1 474 1
280 5667
274 5290
224 26930
pergolide mesylate
290 4700
282 5640
223 35500
3-methylindole
Spectral acquisition was performed on a Perkin Elmer Model Lambda 6

spectrophotometer using 1-cm quartz cells. The ultraviolet spectra of
pergolide mesylate in water, methanol, and dehydrated ethanol are shown in
Figure 7. UV absorption spectra of pergolide mesylate in water, methanol,
and dehydrated ethanol exhibit different absorbances but similar spectral
patterns.
The ultraviolet spectra of pergolide mesylate in buffers of various pH
values are shown in Figure 8. Nearly consistent spectra are observed from
pH 2-6. No absorption pattern is observed for pH values above 6. This is
due to the insolubility of pergolide mesylate in alkaline media.
1%
Solvents hmax (nm) Elcm E max
Water 219 155 6385
Methanol 280 170 6980
Dehydrated ethanol 28 1 170 6993
3.8 Melting Range

Pergolide mesylate melts between 258-260°C (decomposition).
3.9 Differential Thermal Analysis

The DTA thermogram for pergolide mesylate shows a large, sharp
endotherm at 263OC, indicating a melt (decomposition).
3.10 Thermogravimetric Analysis

The TGA thermogram for pergolide mesylate shows a weight loss starting
at 255°C followed by a continuous weight loss, indicating decomposition.
1.500
ABS
I.ooo
0.500
0.000
240 265 290 315 340
WAVELENGTH (nml
Figure 7. Ultraviolet absorption spectra of pergolide mesylate, in water, methanol, and ethanol
2.950
ABS
1.900
0.850
.200
240 265 290 315 340
WAVELENGTH Inn1
Figure 8. Ultraviolet absorption spectra of pergolide mesylate, obtained at pH 2-8

3.1 1 Optical Rotation

Pergolide mesylate contains three asymmetric carbon centers, all of which
are chiral in the drug. The absolute configurations at the three asymmetric
centers of the molecule, C5, C8, and C10, have been confirmed by
anomalous x-ray dispersion techniques.3>10The centers at positions C5 and
C10 are fixed in the starting material (dihydroelymoclavine)and are not
changed during the course of the synthesis. The third chiral site occurs at
position C8, where the methylthiomethyl side chain may occur in either an
alpha or beta position. The stereochemistry at this position is known based
on the known stereochemistry of dihydroelymoclavine, which contains a beta
hydroxymethyl group in the C8 position. The synthetic chemistry involved in
synthesis of the bulk drug is not expected to alter this configuration. The
results of the structural determination by x-ray confirm these results.
CH3S03-
Because the stereochemistry is fixed at all three chiral centers of the

molecule, one would expect to observe optical rotation by the molecule. The
optical rotation of pergolide mesylate was measured with the sodium d line
(589 nm) as the light source at a concentration of 10 mg/rnl in
dimethylformamide @MF) in a 100 mm cell using a Perkin-Elmer Polarimeter
Model 241MC. The specific rotation measured at 2OoC has been observed to
be between - 18.0 and -23.0'.

The X-ray powder diffraction pattern of pergolide mesylate, crystal Form
I, is shown in Figure 9. The pattern was obtained using a Nicolet powder
diffractometer using copper Ka irradiation (1.5418 A) with a graphite
monochrometer.
Pergolide mesylate has been observed to demonstrate two different
crystalline forms. Only one of these is observed routinely in the manufacture
of the bulk drug substance and has been designated Form I. A mixture of
2 0 0 . 7
4
180-
160-
z 140-
CI
-d
(0
C
a, 120-
c,
C
H
u 100-
Ill
(0
\
(0
4J
C
BO-
3
0
U
60-
201 1 1 . 1
5.0 10.0 15..0 20..0 25.0 30.0 35.0

Two-Theta
Figure 9. Powder X-ray diffraction pattern of pergolide mesylate (crystal form I )

Form I and Form I1 has been observed experimentally in laboratory lots

where the recrystallized solutions were cooled very rapidly. On larger scale
equipment where the cooling takes place more slowly, only Form I is
observed.
Form I and a mixture of Form I and I1 have been studied by x-ray
diffraction and by IR spectroscopy. Figure 10 shows the IR spectrum for
Form I. Figure 11 shows the IR spectrum for a mixture of Forms I and 11.
An additional peak at angle 5.788 degrees is noted in a mixture of Forms I
and I1 material, while this peak is absent in the pattern for the Form I material.
Bands appear to be sharper in Form I material, and subtle differences are
observed in the groups of peaks centered at about 775 cm-1,607 cm-l, and
544 cm-*. In the IR spectrum of Form I, these groups appear as a triplet, a
doublet, and a triplet, respectively. In the IR spectrum from the mixture of
Form I and 11, the groups of bands appear as a doublet, a singlet, and a
doublet, respectively.
3.13 Solubility
The solubility properties of pergolide mesylate are listed in Table VI.
The measurement was performed by adding an excess amount of sample to a
solvent, shaking for thirty seconds at five-minute intervals for a total of thirty
minutes, filtering the saturated solution, and determining the concentration of the
drug in the filtered solution with a UVNIS spectrophotometer. (For dehydrated
ethanol, ether, dimethylformamide, acetonitrile, dichloromethane, acetone, and
chloroform, the filtered solutions were evaporated to dryness and reconstituted
in methanol).
Table VI. Solubility of Pergolide Mesylate

i
i
I 1
4000 3600 3iOO 2800 2400 2bOO 1600 li00 800 4 00
WAVENUMBER
Figure 10. Infrared spectrum of pergolide mesylate (crystalform I)

I I I I
I0 3600 3200
I
2800 2400 2600 1600 1500 800 400
WAVENUMBER
Figure I I . Infrared spectrum of pergolide mesylute (crymlforms I & II)

The partition coefficient was determined as follows. Saturated aqueous

solutions of the product in various pH buffer solutions were prepared and an
equal amount of chloroform was added and the solutions were shaken at 25°C
for thirty minutes. The aqueous and organic layers were separated and filtered.
A portion of the organic layer was evaporated to dryness and reconstituted using
a mixture of equal parts of methanol and a solution of methionine (0.01 mg/ml)
in 0.01 N hydrochloric acid. A portion of the aqueous layer was prepared in a
mixture of equal parts of methanol and a solution of methionine (0.01 m g / d ) in
0.01 N hydrochloric acid. The test solutions were assayed by subjecting them
to the isocratic conditions described under high performance liquid
chromatography (see Section 4.42). The chlorofodwater partition coefficients
were calculated as the ratio of the concentration of pergoIide mesylate in the
organic phase to the concentration of pergolide mesylate in the aqueous phase.
The results are shown in Table VII. A partition coefficient was observed in
the acidic buffers. No partition coefficient was observed in alkaline buffers due
to the insolubility of the pergolide mesylate in alkaline media.
Concentration in Concentration in
System organic layer aqueous layer Partition coefficient
(rndml) (mdml)
pH 2.19 Buffer 1.42 0.23 6.14
pH 4.02 Buffer 1.93 0.02 119.6
pH 6.10 Buffer 1.322 0.00 -
pH 7.95 Buffer 0.02 0.00 -
The pKa of pergolide mesylate in a 66% dimethylformamide solution was

measured by potentiometric titration with 2 N potassium hydroxide. The pKa
of the secondary amine is 7.8.
3.16 Color Identification Test

Pergolide mesylate contains an indole ring system. Indole derivatives
with free 2- or 3- positions in the pyrrole ring condense with
p-dimethylaminobenzaldehydein a sulfuric acid solution to produce a deep
purplish blue color (Neubauer Rhode Reaction). Since the more reactive 3-
position in pergolide mesylate is blocked, the condensation reaction of the less
reactive 2-position results in a slow color development. The addition of femc
chloride catalyzes this color reaction.
A 0.1 mg/ml solution of pergolide mesylate is prepared in 2.5 N sulfuric
acid. The addition of 1 ml of a p-dimethylaminobenzaldehydeTS and 3 drops
of ferric chloride TS to 2 ml of the pergolide mesylate solution results in the
immediate formation of a dark purplish blue colored solution (the control
solution remains a pale yellow solution).
4. METHODS
4.1 Identity
The identity of pergolide mesylate is determined using the specificity of
infrared spectroscopy, which differentiates it from any synthetic
intermediates, process related substances or degradation products. Pergolide
mesylate is triturated with potassium bromide and pressed into a transparent
pellet for spectroscopic analysis. The identity is confirmed by comparison to
a reference standard spectrum obtained under similar conditions.

The following elemental composition was obtained using a Controlled
Equipment Corporation Elemental Analyzer Model 240XA.
Table VIII. Elemental Analysis
Element % Calculated 96 Found

C 58.51 58.26
H 7.37 7.46
N 6.82 6.89
S 15.62 15.49

The identity of pergolide mesylate can be determined by ultraviolet
spectroscopic assay at 280 nm. Using a 1 cm cell, the procedure is done at a
concentration of 0.026 mg/mL pergolide mesylate in a mixture of equal parts
of methanol and a solution of methionine (0.01 mg/ml) in 0.01 N
hydrochloric acid.
4.4 Chromatography
4.41 Thin Layer
A TLC method can be used for the identity and purity of raw material for
pergolide mesylate. Precoated silica gel 60 F254 TLC plates are used as the
stationary phase and a tertiary solvent system consisting of
chlorofomdrnethanolfethylacetate (90:10:10) is used as the developing
solvent. Visualization is performed by viewing the plate under long
wavelength UV light (366 nm) and also by exposing the plate to iodine vapors
prior to viewing under short wavelength UV light (254 nm). This developing
solvent system will resolve known process related substances and degradation
products from pergolide mesylate.
4.42 High Performance Liquid
Conditions for quantifying pergolide mesylate have been optimized using
isocratic reversed-phase HPLC. The mobile phase consists of a 1/1 mixture
of methanol and acetonimle added to an equal part of a 2 mg/ml
octanesulfonic acid, sodium salt buffer (0.1% glacial acetic acid v/v). A
DuPont Zorbax RX column (25 cm x 4.6 mm; 5 micron particle size) is used
in conjunction with a flow rate of 1.5 mL/min. Detection is obtained with an
UV detector set at 280 nm. The sample is analyzed at a concentration of
approximately 0.065 mg/mL,. The method is stability-indicating as indicated
by its ability to separate pergolide mesylate and known degradation products.
Figure 12 shows the separation of pergolide mesylate and its primary known
degradation product, the sulfoxide.
Gradient reversed-phase HPLC methodology is used to quantify pergolide
mesylate and its potential related substances (synthetic impurities and
degradation products). A Supelco LC-18-DB column (25 cm x 4.6 mm; 5
micron particle size) is used in conjunction with a flow rate of 1 mL/min.
Detection is obtained with a UV detector set at 280 nm. The mobile phase
components are a 0.5% morpholine buffer (v/v) in water (pH 7.0 with
phosphoric acid) (A) and HPLC-grade methanol/acetonitrile/tetrahydrofuran
(1:1:1) (B). A linear gradient is initiated at 30% (B) and increased 2%/minute
for 35 minutes to a final concentration of 100% (B); then returned to 30% (B)
and re-equilibrated for 20 minutes. The sample is analyzed at a concentration
of approximately 3 rngJmL.
Figure 12. HPLC chromatogramof stabiliry-indicating assayfor pergolide rnesylate and degradationproducts .
Peak identification: ( 1 ) pergolide mesylate sulfoxide, (2)pergolide mesylate
5. STABILITY - DEGRADATION
5.1 Degradation Profile

Pergolide mesylate is stable as the bulk drug substance stored at 25°Cfor
up to 2 years and is also stable under stress conditions (100°Cfor 4
weeksklosed container, 3 x 106 lux-hr. light exposure, 65'C for 3 months,
40°C/75% Wclosed container for 3 months, 25"C/75% Wclosed container
for 3 months, and 4@C/75% RWopen container for 6 months).I4 A trace
level of (8~)-8-[(methylsulfinyl)methyl]-6-propyl-D-ergoline(the sulfoxide,
I) is observed to remain relatively constant under both long term and stress
storage conditions.
Pergolide mesylate was also subjected to degradation experiments
involving water, acid, base, heat, and light to generate degradation profiles.
In all of these studies, the compound was evaluated by specific HPLC or GC,
and TLC procedures. The studies revealed the following resu1ts:ll
1. Pergolide mesylate is stable in base (a slurry in 0.1 N sodium
hydroxide at 40'C for 7 days).
2. Pergolide mesylate is stable in acid (a slurry in 0.1 N hydrochloric
acid at 40'C for 7 days).
3. Pergolide mesylate is considered unstable in water when exposed
to severe light and heat. When exposed to 3 x 106 lux-hr.,
pergolide mesylate undergoes degradation to yield two degradation
products, the sulfoxide (I) and (8~)-8-[(methylsulfonyl)methyl]-
6-propylergoline (the sulfone, 11). The sulfone probably results
from further oxidation of the sulfoxide. When exposed to 40°C
for 7 days in a slurry of water, a slight increase in the sulfoxide is
observed.
The following structures represent potential degradation products of
pergolide mesylate:
Sulfoxide (I) Sulfone (11)

The conclusions of these studies demonstrate that, while some

decomposition was observed under extreme conditions, pergolide mesylate
bulk drug substance is very stable.
5.2 Stability in Dosage Form

Pergolide mesylate is marketed as 0.05,0.25, and 1 mg Permax tablets.
The active ingredient in the formulated tablet for all three dosage strengths is
stable for up to two years when stored in a cold-formed aluminum blister
package. 12
6. DRUG METABOLISM AND PHARMACOKINETICS

Pergolide mesylate is a potent dopamine receptor agonist. Pergolide is 10
to 1,OOO times more potent than bromocriptine on a milligram per milligram
basis in various in vitro and in vivo test systems. Pergolide mesylate inhibits
the secretion of prolactin in humans; it causes a transient rise in serum
concentrations of growth hormone and a decrease in serum concentrations of
iuteinizing hormone. In Parkinson’s disease, pergolide mesylate is believed
to exert its therapeutic effect by directly stimulating postsynaptic dopamine
receptors in the nigrostriatal system.
Information on oral systemic bioavailability of pergolide mesylate is
unavailable because of the lack of a sufficiently sensitive assay to detect the
drug after the administration of single doses. However, following oral
administration of 14Cradiolabeled pergolide mesylate, approximately 55% of
the administered radioactivity can be recovered from the urine and 5 % from
expired C&, suggesting that a significant fraction is absorbed. Nothing can
be concluded about the extent of presystemic clearance, if any.
Data on post absorption dismbution of pergolide are unavailable.
At least 10 metabolites have been detected, including
N-despropylpergolide, pergolide sulfoxide, and pergolide sulfone. Pergolide
sulfoxide and pergolide sulfone are dopamine agonists in animals. The other
detected metabolites have not been identified and it is not known whether any
other metabolites are active pharmacologically.
The major route of excretion is via the kidneys.
Pergolide is approximately 90% bound to plasma proteins. This extent of
protein binding may be important to consider when pergolide mesylate is co-
administered with other drugs known to affect protein binding.
Permax is indicated as adjunctive treatment to levodopa/carbidopa in the
management of the signs and symptoms of Parkinson’s disease.
Evidence to support the efficacy of pergolide mesylate as an

antiparkinsonian adjunct was obtained in a multicenter study enrolling 376
patients with mild to moderate Parkinson’s disease who were intolerant to
I-dopalcarbidopa as manifested by moderate to severe dyskinesia and/or on-
off phenomena. On average, the sample of patients evaluated had been on
I-dopdcarbidopa for 3.9 years (range, 2 days to 16.8 years). The
administration of pergolide mesylate permitted a 5 to 30%reduction in daily
dose of I-dopa. On average, these patients treated with pergolide mesylate
maintained an equivalent or better clinical status than they exhibited at
baseline.
412 DELORES 1. SPRANKLE AND ERIC C. JENSEN
7. REFERENCES
1. Package Insert, PermaxB, Eli Lilly and Company.

2. Langtry, H.D. and Clissold, S.P. (1990). Drugs 39,493.
3. Kornfeld, E.C., Bach, N.J. (1979). U.S. patent 4,166,182.
4. McCune, K.A., Maple, S.R., Cooke, G.G., and Underbrink, C.D. (Eli
Lilly and Company, Lilly Research Laboratories) “Confirmation of
Structure of Pergolide Mesylate” internal report, 29 July 1991.
5. Bailey, K., Grey, A.A. (1972). Can J. Chem. 2,3876.
6. Baker, R.W., Chothia, C., Pauling, P., Weber, H.P. (1973). Mol.
Pharm. 9, 23.
7. Derome, A.D. (1987). in Modern Techniquesfor Chemistry Research;
Chapter 6. Pergamaon Press, New York.
8. Pretsch, E., Seibl, J., Simon, W., Clerc, T. (1989). in Tables of Spectral
Data for Structure Determination of Organic Compounds; p. C160.
Springer-Verlag, New York.
9. The Stadtler Handbook of Ultraviolet Spectra (1970). Stadtler Research
Laboratories, Philadelphia.
10. Ma, L., Camerman, N., Swartzendruber, J. Jones, N., and Camerman,
A. (1987). Can. J. Chem. 65, 256.
11. Jensen, E.C. et al. (Eli Lilly and Company, Lilly Research Laboratories)
“Physiochemistry of Pergolide Mesylate”, internal report, 1990.
12. Jensen, E.C. er af. (Eli Lilly and Company, Lilly Research Laboratories)
“Permax Stability Reports”, Japanese New Drug Application.
13. Jensen, E.C. et al. (Eli Lilly and Company, Lilly Research Laboratories)
U.S. New Drug Application for Pergolide Mesylate, 1985.
14. Jensen, E.C. et al. (Eli Lilly and Company, Lilly Research
Laboratories) “Pergolide Mesylate Stability Reports”, Japanese New
Drug Application.
8. ACKNOWLEDGMENTS
The authors wish to express their sincere thanks to the following
individuals who have provided information for portions of this chapter: K.A.
McCune, S.R. Maple, G.G. Cooke, C.D. Underbrink, and G. Stephenson
for the spectroscopic data ; A.G. Wich and T. Wozniak for chapter review;
and B.T. Farrell for method development.
PREDNISOLONE
Syed Laik Ali
Zentrallaboratorium Deutscher Apotheker
6236 Eschborn
Germany
ANALYTICAL PROFILES OF DRUG SUBSTANCES Copyright D 1992 by Academic Press, Inc

AND EXClPlENTS -VOLUME 21 415 All rights of reproduction reserved In any form
416 SYED LAIK ALI
Prednisolone
Syed Laik A l i
1. H i story
2. Nomenc 1ature
3. Description
3.1 Name, Formula, Molecular weight
3.2 Appearance I Colour Odour I Taste
4. Svnthesis
5. Phvsical properties
5.1 Solubi 1 ity
5.2 Loss on drying
5.3 Melting point
5.4 Specifical optical rotation
5.5 Residue on ignition
5.6 Selenium
5.7 Light absorption
5.8 Related impurities
5.9 Colour reactions
5.10 Ultraviolet spectrum
5.11 Infrared spectrum
5.12 Nuclear magnetic resonance spectrum
5.13 Mass spectrum
5.14 Crystal structure
5.15 Po 1 ymorp h i sm
5.16 Circu 1 ar dichroi sm
6. Stability and deqradation

PREDNISOLONE 417
7. Methods of analysis
7.1 Colorimetric and spectrophotometr ic determination
7.2 Polarography
7.3 Radiochemistry and radioimmunoassay
7.4 NMR determination
7.5 Chromatographic methods
7.5.1 Thin layer chromatography
7.5.2 High performance liquid chromatography
7.5.3 Gas chromatography-mass spectrometry
7.5.4 Supercritical fluid chromatography
a. I n vitro dissolution
9. Pharmacokinetics and druq metabolism
10. Acknowledqements
11. References
418 SYED LAlK ALI
Prednisolone
1. History
Hershberg and co-workers (1) observed at first that
the dihydroderivative of hydrocortison, prednisolon,
possesses a 4 to 5 times stronger antirheumatic and
antia 1 1 erg i c activity , showing simu 1 taneous1 y 1esser
undesired side effects.
2. Nomenclature
1 ,2-Dehydrohydrocortisone;
Pregna-1 ,4-diene-3,20-dione,11 , 17 21-tri- hydroxy-l1R-1
7a,21-Trihydro~y-l,4-pregnadien-3~2O-dion. The
formula is illustrated at the next page (Fig. 1).
3. Descriution
3.1 Name, Formula, Molecular weiqht

Prednisolone; C21H2805 360,45 (anhydrous)
3.2 Appearance, Colour, Odour, Taste

A white or almost white, crystalline hygroscopic,
odourless powder with a bitter taste.
4. Svnthesis
Prednisolone can be obtained with a chemical
dehydration of hydrocortisone with selendioxide in
tertiary butanol (2) or microbiological ly through
the dehydration action of corynebacterium simplex in
Al,Z-position ( 3 , 4). A methanolic solution of the
substrate was mixed with a 24 hours old bacteria
PREDMSOLONE 419
PREDNISOLON
ch20h
i
C=O
&-QH
0-
Fig. 1
S t r u c t u r a l Formula
420 SYED LAIK ALI
culture (0.1 % yeast extract, buffer, pH 7.0) and

was shaken for 3 to 34 hours at 28 "C. The contents
were then extracted with chloroform and prednisolon
could be crystalised from aceton in very good yield.
Wettstein and Co-Workers have further found that an

enzymatic introduction of a 1,Z-double bond in
hydrocortisone could best be obtained with mushrooms
of Genus Didy mella type ( 5 , 6).
The 1,Z-double bond could also be introduced in the

molecule of hydrocortisone chemically through
2,4-dibromination of 3-ketone and then the
subsequent dehydrobromination ( 7 ) . The yield is only
10 - 15 %- The squibb company used this classical
method for the production of 9 -Fluorprednisolon (8).
5. Physical properties
5.1 Solubility (9, 10)

Prednisolone is very slightly soluble in water,
soluble in 27 parts of absolute ethanol, in 30 parts
of ethanol, in 50 parts o f acetone and in 180 parts
o f chloroform. It is soluble in dioxane and methanol.
5.2 Loss on dryinq (10. 111

Anhydrous prednisolone loses not more than 1.0 % of
its weight when dried at 100 - 105 "C. Hydrous
prednisolone loses not more than 7.0 % of its weight.
5.3 Meltina point (10)

About 230 "C with decomposition.
PREDNISOLONE 42 1
5.4 Specific optical rotation (10)

In a 1 % m/v Solution in 1,4-dioxanet +96 to +120 '.
5.5 Residue on ignition (11)
Negligible, from 100 mg.
5.6 Selenium (11)

0.003 %, a 200 mg test specimen being used.
5.7 Liqht Absorption (10)

The A ( 1 %, 1 cm) in 96 % ethanol at the maximum of
240 nm i s between 400 to 430.
5.8 Related impurities (10)

Between 1 to 2 % using silica gel TLC plates
containing a fluorescent indicator and a mixture of
dichlormethane + ether + methanol + water,
77:15:8:1.2 as mobile phase and detection under UV
254 nm.
5.9 Colour reactions

The simplest reagent, used for more than 40 years in
steroid analysis is concentrated sulfuric acid. They
exhibit intense spectra in the range 220 - 600 nm.
Prednisolone shows 2 hours after dissolution in
concentrated sulphuric acid an absorption maximum at
470 nm with a specific extinction o f 89.
Prednisolone gives after dissolving in conc.
sulphuric acid ( 1 mg/ml) instantaneously a red
colour which after dilution with water changes to
violet-brown ( I ) . Prednisolone (1 mg in 5 ml
nitromethane or nitrobenzene) reacts with aluminium
422 SYED LAIK 4 L I
chloride ( 4 g anhydrous aluminium chloride in 10 ml

nitromethane or nitrobenzene, 2 ml reagent) to give
a weak orange colouration (12).
5.10 Ultraviolet spectrum

Prednisolone shows absorption maximum in methanol at
242 nm. The E 1 %, 1 cm in this solvent is reported
to be 416 and the molecular extinction coefficient
15000 (13). The UV spectrum is shown in Fig. 2.
5.11 Infrared spectrum

The infrared spectrum i s given in Fig. 3. The
spectrum was obtained with a Perkin-Elmer 1420 Ratio
Recording Infrared Spectrophotometer from a KBr
pel let.
5.12 Nuclear maanetic resonance spectrum

The nuclear magnetic resonance spectrum o f
prednisolone was taken with a Varian 60 MHZ
spectrometer i n deuterated dimethyl sulfoxide. The
spectrum is reproduced in Fig. 4.
5.13 Mass spectrum

The mass spectrum was recorded with a Varian Mat 311
mass spectrometer using direct inlet in EI-mode at
80 ev and source temperature of 300°C. The spectrum
is illustrated in Fig. 5.
PREDNISOLONE 423
F i g . 2 (13)
UV S p e c t r u m o f P r e d n i s o l o n e i n Methanol
Fig. 3
IR Spectrum o f Prednisolone, KBr P e l l e t
Perkin-Elmer 1420 Spectrophotometer
k
a,
c,
a,
E
0
k
c,
0
a,
n
v)
N
I
z
0
\o
C
0
*rl
k
0
w
W
.
C
0
4
0
u)
-4
C
0
W
k
L
+
0
E
2
k
c,
0
.z
a,
-tn
v)
mtx
-rl
LLZ
425
426 SYED LAlK ALI
Fig. 5
Mass Spectrum o f P r e d n i s o l o n e
PREDNISOLONE 427
5.14 Crystal structure

Inclusion complexation o f prednisolone with three
Cyclodextrins, a p and -phomologues, in aqueous
solution and in solid phase was examined using UV
absorption, CD, C 13 NMR, C 13-CP/MAS-NMRI X-ray
d iffractometry and thermal analysis. The
spectroscopic data suggested the different inclusion
mode of prednisolone within the three cyclodextrin
cavities. X-ray diffraction patterns of the
complexes differed significantly from those of the
physical mixtures (14).
5.15 Polvmorphism
Prednisolone shows the phenomenon of polymorphism
(15, 16). The results o f investigation on
po 1ymorph i sm and Pseudopo1 ymorph i sm (formation of
solvates) of about 100 steroids including
prednisolone is described. Prednisolone forms
solvates with water and chloroform. These
polymorphic forms have melting points between 218 -
234 "C and 210 - 225 "C. The hydrate always
represents the most stable form. The analytical
methods of thermomicroscopy, differential scanning
calorimetry (DSC) and infrared spectrophotometry
were applied for investigation (17, 18, 19, 20, 21).
The therapeutic activity varies for the different

polymorphs o f the same chemical substance. This
behaviour is attributed to various factors but
especially to the differences in the crystalline
structure (22, 23, 24).
428 SYED LAIK ALI
Veiga and co-workers have isolated three forms I ,

11, 111, of prednisolone which were identified by IR
spectroscopy and characterized by scanning electron
microscopy, X-ray powder diffraction and hot stage
microscopy (25). The study by optical microscopy as
well as scanning electron microscopy revealed that
the crystals have a very different appearance. Form
I has small, tridimensional crystals with irregular
shape and granular surface. Crystals of form I 1 have
a lamellar structure, well defined edges with
smaller and very differently shaped crystals grown
on their surfaces. Crystals of form I11 are reported
to be similar in size to those of form 11; they have
regular shape, but are formed by layers, so their
edges are not well defined and are rather opaque
(25). Diffractometer patterns are shown in fig. 6,
interplanar spacings are presented in table 1 and
unit cell parameters in table 2. The study was
carried out by a Phillips PW 1010 powder
diffactometer using the incident radiation Cu K (Y
(X=1.5418A0) filterd with Nickel. The unit-cell
parameters and the interplanar spacings were
determined using a counter diffractometer and
powdered samples containing si 1 icon powder as
internal standard.
Unit cell parameters have been assigned to forms I

and 111, automatic indexation tests have failed to
yield a solution for from I 1 (25). Table 3 shows the
results of hot stage study. During the heating
process forms I and I11 remained unchanged until the
melting point was reached. Form I 1 showed in the
PREDNISOIBNE 429
FORM I
x
L)
a
c
Y
3
c
H
FORM I t
FORM 111
Fig. 6 (25)
X-ray D i f f r a c t i o n P a t t e r n s o f P r e d n i s o l o n e
430 SYED LAIK ALI
- 16.9SO3 -
10.9743 10.9743 10.9743
85840 6.8044 8.3S40
6.3657 6.1672 6:4116
5.7120 5.9806 5.7304
5 m 3 56577 516043
5.4335 5.5546 5.4335
50924 50349 5.0349
5.0349 49239 4.5484
4.5484 4&670 43392
4.1874 4.5600 4.1726
41)188 45709 4.0188
3.9139 4.3080 3.9054
35729 4m70 3.7825
3.5173 3.9224 3.7048
5.4635 38471 3.587 I
3.3985 3.7124 3.524 I
52407 36897 3.4615
31)868 3.4635 3.3965
3M)55 3.4113 32465
2 m 5 33483 3.1951
2-7945 331 I 6 3.1079
2.7526 3246s 2.9956
2.7121 3m73 2.9144
2.5686 2 3 7 I2 250m
2.4693 2 m 2.5810
23599 2.8290 2.4959
233018 2.7363 25x29
22739 2.6685 23103
22176 26766 zm22
2-1392 261% I S772
2.1 157 25129
2M52 2.4662
2fi5I 2.4275
2.0128 2.-*2
I .9%0 22s22
I .979s 2 . ~ 6 51
I8827 2.0107
I .%33
1.9240
1S226
1.7730
PREDNISOLONE 43 I
TABLE I1 (25)
Unit cell parameters.
Momclinic &lonoclinic
Crystal S>1ICm
Form II
Form I
1 ID48 (A) 16.904 (A)
13.3% 13.167
6.3 I5 7.909
9(r 90-
9 1-76 s3m
90 90
TABLE 111 (25)
ram II Form I Form Ill
Partial melting and

recrystallization ienip. ("1 115-130
Melting point (") 2 10224 205-218 2 10-220

432 SYED LAlK ALI
range of 115 - 130 "C a partial, intracrystalline

melting with further recrystallization which then
remains unchanged up to their melting point. Veiga
and coworkers deduced from these facts that I and
I 1 1 are anhydrous polymorphic forms of prednisolone
while the form I 1 is a 1.5 hydrate and probably a
twin (26).
5.16 Circular dichroism

Circular dichroism (CD) is much more useful than any
other spectroscopic technique in control 1 ing the
sterospecificity of the reactions in the total
synthesis of steroids. Prednisolone can be
determined simultaneously along hydrocortisone.
Hydrocortisone can be measured selectively at 326 nm
where prednisolone does not show dichroism, while at
314 nm prednisolone can be measured on the basis of
its selective dichroism (27, 28).
6. Stabilitv and deqradation

One of the identified decomposition products formed
during the anaerobic decomposition of prednisolone
at pH 8 is 17-deoxyprednisolone. This decomposition
product differs from prednisolone only in the side
chain. The hydroxyl group at C 17 disappears thus
giving 17-deoxyprednisolone (29). Gutman and Meister
(30) found that with the dihydroxyacetone side chain
of prednisolone two reactions predominate which
yield the 17-ketosteroid and the hydroxy acid ( 3 0 ) .
The isolation through TLC and HPLC methods and
structural e 1 uci dat ion through po 1 arograph i c and
PREDNISOLONE 433
masspectrometric techniques is described. A

decomposition mechanism of prednisolone leading to
17-deoxyprednisolone is postulated.
Studies on the stability of corticosteroids and

degradation patterns in aqueous solution are
described ( 3 1 ) . Formation and degradation kinetics
of 2 1 - d e h y d r o c o r t i c o s t e r o i d s , key intermediates in
the oxidative decomposition of 21-hydroxycorticoids
(32) , kinetics and mechanism of the acid-catalyzed
degradation of corticosteroids are reported ( 3 3 ) .
Another decomposition product formed during the
anerobic decomposition of prednisolone is
17-deoxy-21-dehydroprednisolone. This can only be
detected when the decomposition of prednisolone
takes place at about pH 6 or lower pH-values. This
product has been isotaled through chromatographic
techniques and structure elucidated through mass
spectrometry ( 3 4 ) . This product differs only from
prednisolone in the side chain where the hydroxyl
group at C 17 has disappeared and at C 21 the
hydroxyl group has been changed to an aldehyde group
(341. Another decomposition product, the
17-Ketosteroid where at C 17 the hydroxyl group has
been converted to a keto group, is described. The
isolation and the structural elucidation of
17-Ketosteroid is reported as well as decomposition
mechanisms of prednisolone leading to the
17-ketosteroid are discussed ( 3 5 ) . The major
decomposition product of prednisolone phosphate
formed under anerobic decomposition conditions in
434 SYED LAIK ALI
aqueous solution at pH 8.3 i s identified as 17

a-h ydroxy- 1 7a- hydroxymethy 1 - 17- keto-D- homosteroid
phosphate. Isolation, structural elucidation and a
mechanism leading to this compound are postulated
(36).
In another publication the stability and

interactions between prednisolone and urea in
ointments and the influence of one component on the
other are discussed (37). Factors influencing
stabi 1 ity, the rate of disappearance of prednisolone
from aqueous solution have been investigated (38).
The rate exhibited a marked dependancy on buffer
concentration. Prednisolone is susceptible to
degradative reactions which involve the
17-dihydroxyacetone side-chain. Transformation and
elimination of the side-chain have been shown to
occur both in the presence and absence of oxygen.
Autoxidation, however, appears to be the mode o f
destruction which is most likely to be responsible
for stability problems in drug products. The
involvement of trace metals in catalyzing the
autoxidation i s an obvious possiblity. Trace-metal
impurities which were present in the buffer reagents
were catalytically involved in the degradation. Rate
constants were determined over a wide range o f pH in
borate and phosphate buffers and in the presence and
absence o f ethylenediamine tetraacetic acid. The
rate of the apparent metal-catalysed reaction was pH
dependant above pH 7 and below pH 5 and exhibited a
first-order dependency on the hydroxide-ion in the
PREDNISOLONE 435
intermediate range. In the presence of EDTA, the

rate of reaction was strongly dependant on
OH-concentration above pH 8 but exhibited only
slight dependency at lower pH values. Addition of
EDTA provided a method to isolate and quantitate the
rate o f the apparent metal-catalyzed reaction (38).
The stability of prednisolone in an aqueous-organic

solvent system (40 % lI2-propandiol + 38 %
tetraglycol + 30 % water) under accelerated
conditions has been studied. It was possible to
demonstrate six different decomposition products.
The four major products were identified by TLC. The
accelerated stability tests were evaluated using a
stability-indicating assay procedure. Although
decomposition of prednisolone in solution is
complex, stability prediction via Arrhenius plotting
is possible. The degradation of prednisolone in this
system is a first order kinetic reaction. The
temperature dependence of degradation process in
this organic-aqueous system is illustrated in fig. 7
and the Arrhenius plot of this diagramm is shown in
fig. 8 (39).
7. Methods of analvsis
7.1 Colorimetric and spectrophotometric determination

Blue tetrazolium (BT) and Phenylhydrazine H2SO4 (PH)
reagents react with the intact side chain at C17 o f
prednisolone while isonicotinic acid hydrazide (INH)
and UV-absorption depend upon conjugation in Ring A
at the other end of the prednisolone molecule. Since
I 7.
100-
90
e
m
w
eo
f\sr Y -4
70.
Fig. 7 (39)
Temperature-Dependence of Degradation o f P r e d n i s o l o n e
a,
K
0
4
0
v)
-4
5
73
a,
k
n
k
0
Lc
a,
E
E
0
k
m
0
2
+J
0
431
438 SYED LAIK ALI
the reactions in these four methods occur with

different portions of the molecule, they can be used
to detect and distinguish between decomposition
products (40). The PH method is described by Silber
and Porter (41) and the I N H method is given by
Umberger (42). The Porter-Silber reaction for the
colorimetric determination of corticosteroids is
applicable only to steroids with a side chain at
position 17 or their derivatives that are readily
hydrolysed in the strongly acid medium (43). Prior
to the determination by BT, PH, I N H and UV method a
column chromatographic clean-up and removal of
decomposition products and interfering substances
has been performed (44). The method of tetrazolium
assay of prednisolone consists of reaction of the
substance (34 - 36 ug/ml) with a 0.5 % solution of
triphenyl-tetrazolium chlorid (TTC) solution in
aldehyde-free ethanol (96 %) and measuring the
extinction at 485 nm (45). A colorimetric method of
determination of prednisolone in powder and tablet
forms using ammonium molybdate has been described.
Prednisolone gives a blue colour with an absorption
maximum at 655 nm. The range of sensitivity for
prednisolone is given between 5 - 30 ug ( 4 6 ) . The
prednisolone contents in low concentrated ointments
and creams were measured with blue tetrazolium
reaction after several steps of extraction and
clean-up ( 4 4 ) . A large number of prednisolone dosage
forms have been examined using tetrazolium blue
method ( 4 8 ) . A review of methods of practical
importance for the determination of steroids in
PREDNISOLONE 439
pharmaceutical formulations i s given (49). The most

generally used method for the assay o f formulations
containing unsaturated 3-Ketosteroids is their
condensation with isonicotinoyl hydrazide ( I N H ) and
measurement of the formed hydrazones in strongly
acid medium at 410 nm (E 17000). For the
determination of prednisolone in dosage forms on the
basis of their side chain at C-17 tetrazolium
methods based on the reducing properties of the side
chain are stability indicating. Both the triphenyl
tetrazol ium chloride and tetrazolium blue methods
are fairly sensitive having molar absorptivities of
16200 and 24000 respectively (49, 50). Condensation
of the glyoxal, obtained by cupric acetate oxidation
of prednisolone, with aqueous phenylhydrazine
reagent affords a near UV chromophore at 366 nm with
a molar extinction coefficient of 17000 ( 5 1 ) . The
TTC and BT methods have a relative standard
deviation o f less than 1 % in the assay o f bulk
corticosteroids and not more than 2 % for dosage
forms ( 5 2 ) . The TTC method has been used for the
determindtion of prednisolone in tablets (53, 54) ,
ointments (55, 56) and for the kinetic investigation
of its decomposition in alkaline media (57). The
sodium borohydride method has been appl ied by Gorog
for the analysis of prednisolone in ointments (58).
An alcoholic solution of ointment containing 10 - 15

mg prednisolone is treated with 1 N Sodium hydroxide
followed by the addition of 100 mg sodium
440 SYED LAIK ALI
borohydride. The mixture is refluxed for 1 h, cooled

and treated with 1 N HC1. The absorption of the
resulting solution is measured at 243 nm against a
corresponding blind solution.
7.2 Polaroaraphv
The electoanalyt ical behaviour of predn i solone along
with other corticosteroids has been studied in
supporting electrolytes. Dependence of the peak
potentials on the structure of the steroids at
concentrations of 10-4 M in 0.03 M tetramethyl
ammonium hydroxide (TMAH) in methanol, in
Britton-Robinson buffer pH 10 (50 % V/V in methanol)
and in 0.02 M TMAH in dimethyl formamide (87 % V/V)
has been studied. In prednisolone the reduction of
C-3 and C-20 keto groups takes place. Both reduction
steps can be used for analytical purposes. The
differential pulse peak height i s linear with the
concentration down to 10-6 M. The wave pattern of
the differential pulse polarography in methanol
shows for prednisolon reduction at -1.60 V and an
additional peak at -1.76 V versus SCE. The reduction
in DMF is similar to that in methanol. The peak
potential in a methanol-buffer mixture at pH 10 for
prednisolone is given as -1.50 V . Prednisolone has
also been analysed by constant potential coulometry
(59). The differential pulse polarographic
determination of prednisolone in single component
tablets is described. After extraction o f
prednisolone with methanol from tablets it was
PREDNISOLONE 44 1
analysed in a supporting electrolyte of 0.03 M TMAH

in methanol with a dropping mercury electrode, a
Ag/AgCl reference electrode and a platinum wire
auxiliary electrode. The concentration of
prednisolone to be determined was in the range of
10-3 - 10-5 M (60). Prednisolone gives waves in d.
C. and normal pulse polarography and peaks in
differential pulse polarography which correspond to
a one-electron uptake. Mechanism of polarographic
electroreduction of prednisolone is described. The
effect of pH using different buffers (acetate,
phosphate, borate and ammonia buffers) on half-wave
potential and limiting current for prednisolone is
given. The half-wave potential of the prednisolone
wave remains pH-independent up to pH 10.3 but is
shifted towards more negative potentials at higher
pH-values. Dependence of the peak-heights in pulse
polarography on pH for the first wave for
prednisolone closely resembles the pH dependence of
these waves obtained by d-c-polarography. In linear
sweep vol tammetric curves the dependence of the
values of peak potentials of prednisolone on the
logarithm of the scan rate was linear over a wide pH
range (61).
A differential pulse polarographic method for the

determination of prednisolone in tablets is
described. The method is more sensitive than dc
polarography and the measurement of diffusion
current is greatly simp1 if ied. Sorenson phosphate
buffer pH 5.6 was used as the supporting
442 SYED LAlK ALI
electrolyte. The peak potential was found to be

-1.19 V versus SCE for prednisolone. The position of
the peak was independent of concentration and peak
heights were linear over the 5 - 20 ug/ml range
(62). Predni so 1 one and predn i sone can be determined
in tablets using ethanol as an organic solvent,
acetate buffer pH 5.6 as a supporting electrolyte
and polarographing between -1 and -1.5 V ( 6 3 ) .
7.3 Radiochemistrv and radioimmunoassav

Strict limits on allowable residual quantities of
ethylene oxide and its major reaction products have
been imposed due to their possible mutagenic and
cancerogenic properties. Co-60 irradiation is a
major goal o f a sterilization alternative programme.
The cobalt 60 radiolytic degradation products have
been identified for many corticoids including
prednisolone. Two major types o f degradation
processes have been identified: loss of the
corticoid side chain on the D ring to produce the
C-17 ketone and conversion of C 11 alcohol, if
present, to C 11 ketone. Minor degradation products
derived drom the other changes affecting the side
chain are also identified. These compounds are
frequently associated in corticoids as process
impurities or degradation compounds. No new
radiolytic compounds unique to Co-60 irradiation
have been found. Through cobalt 60 radiolytic
degradation pathway of prednisolone prednisone and
llB-hydroxy-l,4-androstadiene 3,17-dione are formed.
PREDNISOLONE 443
Conditions for isolation of cobalt 60 radiolytic

degradation products as well as paths and schemes of
degradation are described. For prednisolone
methylchloride was used as enrichment solvent, a
HPLC Brownlee RP 18 column C 18 bonded phase and a
mobile phase o f methanol-water, 55:45 were used. The
rate of radiolytic degradation in prednisolone is
given as 0.7 %/Mrad (64).
Radioimmunoassay has been used for the estimation of

prednisolone after prednisone intake. Taking
advantage of the similarity of structure of
prednisone to cortisone and of prednisolone to
cortisol , urinary prednisolone was estimated by
radioimmunoassay for cortisol and found to be
linearly correlated with the dose o f prednisone
administered. Free prednisolone in urine was
estimated by the "clinical Assay" R1A kit for
cortisol, which gave a very high cross-reactivity
with prednisolone. In estimating free prednisolone
by RlA, cortisol cross reactivity was found to be
91,6 % in the range of 2-100 ug prednisolone. When
RIA kits with differing specificity of the antibody
to cortisol were used, the cross-reactivity also
differed as expected. The less specific the
antibody, the higher were the results obtained with
the same blood sample (65).
The predn i solone rad ioimmunoassay developed by

Colburn and Buller (66) used an antiserum raised
against prednisolone-21-hemisuccinate conjugated to
444 SYED LAIK ALI
bovine serum albumin (BSA) and showed roughly 10 %

cross-reactivity with endogenous cortisol. The
plasma samples were treated at 70 "C for 30 min
prior to radioimmunoassay so as to eliminate
interference by endogenous corticosteroid binding
globulin with the binding of prednisolone to the
antiserum ( 6 7 ) .
The smallest amount of prednisolone which can be

assayed by radioimmunoassay with confidence was 0.5
ng giving a usable range for the assay of 5 - 400
ng/ml in the plasma. The within-batch precision of
the assay was between 3 - 5 %.The cross-reactivity
of the antiserum with various metabolites of
prednisolone and some endogenous steroids was
between 6.4 and less than 1 %. The crossreaction
with cortisol and corticosterone was 6.4 % and 3.8 %
respectively (68). A method for measuring
prednisolone using an antiserum raised against
dexamethasone-21,-hemisuccinat-bovine serum albumin
conjugate in sheep is reported, which reacts poorly
with endogenous steroids. The results of
radioimmunoassay were compared with those obtained
by using competitive protein binding method (69).
This method is based on the high affinity of
predn i solone for p 1 asma corticosteroi d binding
globulin (69, 70). The data show that binding of
prednisolone to dexamethasone antiserum is
sufficient for this to be used as the basis for a
prednisolone radioimmunoassay as we1 1 as perfectly
adequate for the measurement o f prednisolone plasma
PREDNISOLONE 445
levels in routine clinical investigations.

Nevertheless, the technique is less sensitive than
that which uses an antiserum raised against a
predn i solone-21-hemi succinat-bov i ne serum a1 bumine
conjugate. The dexamethasone antiserum used did not
cross-react significantly with any of the several
cortisol metabolites tested (68). Comparison of the
results obtained by the competitive protein binding
method and the radioimmunoassay method showed good
agreement, although over the whole range of
concentrations the latter technique always gave
slightly lower values. This discrepancy was
attributed either to the presence of cortisol in the
pooled plasma used for preparing the standard curve
or to some metabolite of prednisolone which
cross-reacts in protein binding method. The method
permits measurement o f prednisolone in the presence
of prednisone, since the latter does not cross-react
with the antiserum (68).
7.4 NMR determination

A highly selective NMR method for the determ nation
of prednisolone in tablets i s described. After
extraction of prednisolone with 95 % ethano , the
solvent is evaporated and the residue is dissolved
in diemthyl sulphoxide containing fumaric acid as
internal standard. The NMR spectrum of the resulting
solution is recorded with a 60 MHZ instrument. The
prednisolone content is calculated from the integral
of the signal of the C 1 proton at 7.9 ppm w i t h the
aid of the integral of the signal of internal
standard at 6.9 ppm (71).
446 SYED LAIK ALI
7.5 Chromatographic methods
7.5.1 Thin layer Chromatography (TLC)

All important parameters of TLC separation are given
in table 4. Prednisolone has been separated from its
oxidation product through TLC on fluorescent silica
gel plates. In some cases the quantity of the
oxidation product could amount upto 2 %. By applying
100 ug of corticosteroid, the oxidation products can
be detected at 0.4 % level and their presence is
easily demonstrable at the 1 % level (72, 73). Knopp
(74) applied three different mobile phases and HPTLC
plates for the detection of decomposition products
of prednisolone through UV 254 nm and INH reagent. 5
- 50 ul o f 0.1 % ethanolic solution of prednisolone
were applied on TLC plates (74). Prednisolone could
also be determined through densitometry at 250 nm
( 7 4 ) . A simple TLC screening procedure was developed
for the detection of prednisolone as adulterant in
Chinese herbal preparations. Depending on its
complexity the sample may be directly extracted into
aqueous ethanol, or stepwise fractionated into
acidic, basic and neutral components. Extracts were
analysed on silica gel TLC plates with fluorescent
indicator with the aid of four solvent systems and
detected under short and long wavelength UV light
and iodine vapour (75). Prednisolone and
chloramphenicol in oily 1 iquids could be separated
through TLC (76). Quantitative HPTLC coupled with
densitometry was developed for the determination of
PREDNISOLONE 441
prednisolone in human plasma, saliva and urine. For

HPTLC analysis the residues after extraction were
reconstituted in 10 ul acetone and 5 ul were applied
on TLC plates and developed with two solvent
systems. The plates were then sprayed with a mixture
o f sulfuric acid-ethanol and heated at 60°C for 45
min. The fluorescent intensity of the steroid bands
were determined by densitometry at a wavelength of
598 nm, with excitation at 254 nm. The method allows
simultaneous measurement of endogenous cort is01 in
plasma following administration of prednisolone. The
calibration curve was linear over a wide range of
concentration in all biological fluids (0.025 - 4
ug/ml). The limit of detection was 10 ng/ml in
plasma and saliva and 25 ng/ml in urine. The method
was reproducible with an inter- and intra-assay
coefficient of variation of < 10 %. No interference
from endogenous steroids was found (77). TLC
behaviour of prednisolone in creams is described. As
the content of corticosteroids like prednisolone in
creams is generally low, the detection of these
active components can be a problem. Detection was
not sufficiently sensitive for creams containing
about 0.1 % or less of a particular corticosteroid
(78). In B.P. 88 and european pharmacopeia a TLC
method for identification of prednisolone and for
the examination of related substances is given (79)
K. Macek has tabulated the chromatographic data with
number of mobile phases for the identification and
separation of prednisolone from innumerable
corticosteroids and related substances (80).
448 SYED LAIK ALI
Levarato has developed a series of quantitative

methods for the determination of prednisolone along
other corticoids after extraction from preparations
and separation by TLC on silica gel plates and
determination by spectrophotometric (absorption at
240 nm) or colorimetric methods (INH-hydrazone
formation, tetrazol ium reaction) (81). A simple,
fast and quantitative TLC-method for the
determination of prednisolone in tablets is
described. The method is stability-indicating with
respect to accuracy, specificity, sensitivity and
precision. The coefficient of variation was between
1.26 and 1.96 % and the sensitivity was about 25 ng.
The chromatographic separation was performed on a
silicagel plate using two step development of the
plate ( 8 2 ) . A simple TLC method of separation of
prednisolone from other corticosteroids is reported
(83). Prednisolone and the dephosphorylated
D-homosteroid can be separated on silanised silica
gel TLC plates (36).
Fluorimetry is a useful method for the direct

determination of steroids on chromatographi c p 1 ate.
The method is based on the measurement of the
fluorescence produced on irradiation of the
chromatogram with UV light. The quenching of the
fluorescence of various activated layers by
unsaturated ketosteroids 1 ike prednisolone can also
be used for quantitative measurements. The basis of
the quantitative evaluation is the difference
between the fluorescence of the background and that
PREDNISOLONE 449
of the dark spots. A direct densitometric evaluation

o f prednisolone has been performed (84) * The method
for detecting prednisolone on TLC plates i s assigned
according to the functional group involved in the
reaction. The name and composition of the reagent,
the colour o f fluorescence observable on spraying
and the sensitivity is given. Detection of
predn i solone with sulphur ic ac i d , orthophosphoric
acid and antimony (111) chloride reagents is
described ( 8 5 ) . The limit of detection of
prednisolone on silica gel fluorescent TLC plates
with a mobile phase acetone-cyclohexane-ethyl
acetate, 1:l:l in UV light 254 nm is given as 0.3 ug
(85). Compernolle and coworkers (86) reported on the
purity checking of commercial prednisolone samples
by TLC, where several impurities such as
hydrocortisone, prednisone etc. could be detected.
Excellent separations could be achieved by TLC with
solvent mixtures such as dichlormethane-dioxane-
water (2: 1 : l ) or dichlormethane-diethylether-
methanol-water (77:15:8:1). The Rf-values for
prednisolone 0.42 and for prednisone 0.62 were found
with this system ( 8 7 ) .
Table 4
Stationary Phase Mobile Phase Detect ion References
hRf -values
Silica gel 60 with methylene chloride- UV 254 nm

fluorescence indicator dioxane-water 100:50:50
prednisolone: 34
oxidation product: 77
P
VI
3
same as above same as above UV 254 nm (73)
120:30: 50
prednisolone: 13
oxidation product: 55
HPTLC plates, Merck Methylene chloride-ether- UV 254 nm (74)

pre washed with methanol- methanol-water INH reagent
chloroform (80+20); 77:15:8:1.2
15 cm prednisolone: 29
decomposition products: 0-71
m
a,
V
c
a,
L
a,
n
d
h
*
h
h
rc v W
a,
p:
a, a, L
> > S
0 0 0
n n 9.
S la 5 5
0 >
*r wl m
c, 5 l-0 a,
V c
a, a, a, .r
c,
a,
n
5
m
5
m
U
0
*r
h
Lo
N I
h 0
..
I N
..
0
d wl
m c,
a, CI V
7 V S
n 7 S U M
.-
5 U 0
+ 0
c h L M L M
.. .. ..
la N Q N
..
L D Q
r Ln
c, c c
a 0 a 0
..
a, a, h a ,
m c c N e
.e
I *r *r
5
c
m
a, E 2o .m
? 2o m -
0
Q
a,
a
7
la
0
rc
0
m
‘7
o
Q
m
*r
o
Q
m
2s: .r
>
2 ?N? GLa E ,
7 L e E L C
.r I - 0 0 a,u
n rc , v a , v c , a ,
0 CL T a , L a , 5 L
r T v m n o Q U 3 Q
a,
m
la
T a,
Q >
0
h n
L la
la
S wl m
0 la la
.r
c, a,
IU
c,
m
5
wl
45 I
Table 4
Stationary Phase Mobile Phase Detection References
hRf-values
same as above ethyl acetate-toluene-formic same as above (75)

acid-dimethyl formamide-water
75:75:2:4:4; prednisolone: 12
same as above ethyl acetate- to1 uene-formi c same as above (75)
acid-dimethyl formamide
P
N
VI
75:75:2:4; prednisolone: 15
Silica gel UV 254 Ether; prednisolone: 1 1 UW 254 nm (76)

Table 4
hRf -values
HPTLC plates without Chloroform-ethano 1 -water H2SOq-ethano1, 6.5:3.5

fluorescent indicator BDH 90:10:2; prednisolone: 25 spray reagent; densi-
10 x 20 cm tometric determina-
tion at 598 nm with
excitation at 254 nm
e
W
?n
same as above Ch loroform-ethano 1 -water same as above (77)

45:5:7.5; prednisolone: 29
Silica gel 60 F HPTLC n-butan01-water-g1 ac i a 1 UV 254 nm (78)

plates 10 x 10 cm Merck, acetic acid, 20:5:2
distance o f 7 cm under prednisolone: 72
saturated conditions
Table 4
Stationary Phase Mobile Phase Detect ion References
hRf-values
Silica gel 60 F 254 TLC methyl ene ch 1 or i de-ether- UV 254 nm, spraying (79)
plates methanol-water; 77:15:8:1.2 with ethanolic sul-
furic acid (20 % ) ,
heat at 120°C for
same as above ether-toluene-butano1 10 min and UV 365 nm (79)
e
P
VI
saturated with water, 80:15:5
Silica gel 60, a) to1uene-ether-acetone 250 nm, densitometry; (82)

20 x 20 cm Merck 18:19:3 development first with
without fluorescent b) methylene chloride-ethyl mobile phase a, drying
indicator acetate-ether-formic acid the plate with air and again
10:10:10:0.3 development with mobile
prednisolone: 25 phase b
Table 4
hRf -values
Silica gel 60 F 254 methy 1 ene ch 1 or ide-acetone UV 254 nm, spraying (83)
plates Merck 75:25; prednisolone: 20 with 20 % H2S04 in ethanol
and drying at 120°C 10 min
reddish brown spot
%
VI
Silanised silica plates methanol-water - 0.4 M sodium UV 254 nm; blue
60 F 254 Merck, 0.25 nm phosphate solution 50:50:1 colouration with
prednisolone: 54 tetrazolium blue
456 W E D LAlK ALI
7.5.2 Hiah performance liquid chromatoaraphy

A sensitive, specific, HPLC procedure for the
determination of prednisolone in plasma is
described. The organic solvent extract from plasma
is chromatographed on a silica gel column using a
mobile phase of 0.2 % glacial acetic acid, 6 %
ethanol and 30 % methylene chloride in n-Hexane at
254 nm. Quantitation of plasma samples containing 25
ng/ml prednisolone is reported. Metabolites and
endogenous hydrocortisone do not interfere with
prednisolone (88). For the simultaneous analysis of
prednisone and prednisolone in plasma the internal
standard was dexamethasone and the mobile phase
consisted of glacial acetic acid-ethanol-
methylene ch 1 or ide-n-hexane (0.2 :3.5 :30 :66.3) (89,
90). A brief discussion of the merits and
limitations of HPLC relative to other
chromatographic methods and special problems in the
application to steroids are discussed (91). A
sensitive, specific and reproducible HPLC assay for
the simultaneous determination of prednisone,
prednisolone and cortisol in biological fluids was
developed with dexamethasone as the internal
standard. Samples were extracted with methylene
chloride, washed with sodium hydroxide and then
water and chromatographed on a microparticulate
silica gel column with a mobile phase
methanol-methylene chloride, 3:97 at a flow rate of
2 ml/min and detected at 254 nm. Sensitivity was
greater than 15 ng for all four steroids. A constant
ratio of peak height of a steroid at the wavelengths
PREDNISOLONE 451
280 and 254 nm served as an added measure of

specificity o f the assay. The 280:254 nm ratio for
prednisolone was 0.09 ( 9 2 ) . While monitoring at dual
wavelengths improves assay specificity, the 254 nm
wavelength yields nearly optimum absorbance (92).
Determination of prednisolone in plasma by HPLC
using a water saturated mobile phase of equal
volumes o f ethanol and methylene chloride and 1 %
glacial acetic acid was used on a stainless steel 30
cm x 3.9 cm porasil column (10um porous silica) with
a flow rate of 2 ml/min and detection at 254 nm. A
1inear relationship exists over the concentration
range 25 - 150 ng/ml. The effects of sample storage
on reproducibility of results were examined. The
samples were stored at -20°C for upto four weeks.
The mean recovery was between 101.6 to 103.9 % for
prednisolone concentrations between 20 and 100 ng/ml
respectively (93). A normal phase micro-bore column
packed with 10 um microsphere silica (50 cm x 1 mm
i .D.), mobile phase o f water-saturated butyl
chloride-buty 1 ch lor ide-THF-met hano 1-g laci a1 acetic
acid (450:450:105:53:44) , and the detection at 254
nm were used for the separation of prednisolone from
other corticosteroids (94). HPLC retention values of
prednisolone along various other corticoids on a
Bondapak C18/corasil column using methanol or
acetonitrile of different compositions in water at a
flow-rate of 1.5 ml/min and detection at 254 nm were
evaluated. The values for prednisolone lie between
1.06 t o 3.35 i n relation to acetone with a value
1.00 (95). An improved HPLC separation of
458 SYED LAIK ALI
decomposition products of prednisolone by adding

sodium sulphite to the mobile phase is reported. To
a methanol-water, 1:1, solution 1 % of 0.4 M sodium
phosphate solution pH 7.0 was added. Sodium sulphite
was optionally added t o give a concentration of 0.1
% w/w. The flow-rate was 1.0 ml/min and the
detection was performed at 240 nm. A C18 M Bondapak
co 1 umn was used. With this system
21-dehydro-prednisolone, a decomposition product of
prednisolone is separated and detected (96).
Prednisolone was separated along other
corticosteroids in topical pharmaceuticals on a
reversed phase microparticulate HPLC column Zorbax
c8, Dupont (25 cm x 4.6 mm) with a mobile phase of
THF-methanol-water, 25:12.5:62.5 with a flow-rate of
1 ml/min and at a detection wavelength of 254 nm.
Prednisolone had a relative retention value of 0.94
with respect to hydrocortisone (value 1 .OO). Various
commercial topical formulations of these
corticosteroids were prepared by both simple
dilution and by extraction for analysis by the
proposed HPLC procedure, by the blue tetrazolium
procedure and by the isoniazid procedure and/or by
phenylhydrazine method (97). Prednisolone and
chlorhexidine have been separated on a Nucelosil C18
10 um column, 30 cm x 4 mm along with a Vydac 201 RP
guard column 5 cm x 4 mm with a mobile phase
methanol-water, 120:80 containing the PIC reagent
87, Waters with a flow-rate of about 1.35 ml/min at
50 "C and detected at 240 nm. Prednisolone and
chlorhexidine dichloride were thus well separated in
hydrophilic emulsions and lipophilic ointments (98).
PREDNISOLONE 459
A normal phase HPLC method for the determination of

prednisolone in tablets and bulk drugs was studied.
The HPLC system consisted of the mobile phase
methanol-water washed ethylene dichloride-acetic
acid ( 6 + 94 + O . l ) , 25 cm x 4.6 mm column packed
with 5-6 um porous spherical particles (Du Pont),
flow-rate 1.5 ml/min, detection at 254 nm and
injection volume 10 - 15 ul. The bulk drugs and
tablets containing prednisolone were extracted with
a mixture of methanol and methylene chlorid (4:96)
and an internal standard of 1 mg/ml solution of
f luoxymesteron was used. The coefficient of
variation of the analysis results ranged from 1.34 %
for bulk drugs to 2.14 % for tablets ( 9 9 ) .
Extraction-monitoring and rapid flow fractionation
for determination of serum corticosteroids is
described. The HPLC system applied for the analysis
of serum corticosteroids including prednisolone
consisted of a mobile phase 0.1 % water, 4 %
methanol, 30 % methylene chloride in n-hexane,
Lichrosorb Si 60.5 um, 25 cm x 4 mm column
pretreated with 5 % H2SO4, flow-rate 2 ml/min and
240 nm UV detection (100). A HPLC analysis of
prednisolone and endogenous cortisol is described in
plasma samples of kidney transplantation patients
using dexamethasone as an internal standard. A glass
column (15 cm x 3.1 rnm filled with Separon Six, 5
urn, Laboratorni Pristroe, Prag, CSSR), a mobile
phase methylene chloride-methanol, 9 7 : 3 , flow-rate 1
ml/min and detection at 254 nm were the HPLC
parameters. The calibration curve for prednisolone
460 SYED LAIK ALI
(Y = 0.0046x) is linear up to 500 ng/ml and the

detection limit is given between 2-5 ng/ml. The use
of glass column permitted a higher sensitivity and
less consumption of mobile phase (101).
Application of HPLC procedure to the determination

of binding of prednisolone to high-affinity binding
sites in human serum is reported. Prednisolone binds
to globulin and albumin in human serum. The binding
affinity of the steroid for globulin is high whereas
the capacity is low. In contrast, albumin has a low
affinity for the drug but the binding capacity is
high. The method describes a HPLC gel permeation
procedure which a1 lows prednisolone bound to albumin
to completly dissociate during chromatography while
the binding of the drug to high affinity proteins is
unaffected. The column (Bio-Sil TSK-250, 3 0 0 ~ 7 . 5mm,
10 um, Bio-rad Labs) with a molecular mass range of
1000-300,000 was preceded by a guard column. The
mobile phase consisted of 0.1 M Sodium sulfate and
0.02 M Sodium phosphate monobasic adjusted to pH 6.8
with 0.1 M NaOH. The flow-rate was 5.4 ml/h and
detection was performed at 280 nm. The data about
the effect of prednisolone concentration on the
binding in serum and comparison o f binding of
prednisolone by HPLC and equilibrium dialysis is
given (102).
HPLC determination of prednisolone incorparated in

gel ointment i s reported. The gel ointment is
composed of carboxy vinyl polymer (1.3 % W/W) and a
PREDNISOLONE 46 1
large amount of an aqueous organic solvent. A

methanol extraction system offered simultaneous
advantages of the removal of the polymer and the
recovery of active ingredients from the gel phase.
The recovery of the drug was 100 %. The following
chromatographic conditions were used: mobile phase
methanol-water, 6:4, reversed phase, Bondapak C 18,
10 um column, 30 cm x 3.9 mm, flow-rate 1 ml/min,
detection at 254 nm. The prednisolone content in gel
ointment was well maintained for 3 months or longer
when stored at 5 "C (103).
Retention data of 12 corticosteroids including

prednisolone is given on dynamically modified silica
by cetyltrimethylammonium bromide added to the
eluent with various organic modifiers. Separation
factors between hydrocortisone and 11 other
corticosteroids including prednisolone measured on 8
different silica columns and six different
ODs-sil ica columns are presented. The variations in
selectivity were found to be substantially smaller
than those of chromatographic systems based on
chemically bonded ODs-silicas from the same sources
(104).
A HPLC method for the simultaneous determ nation of

predn i so one a ong other corticosteroids in swine
plasma i s described. Extraction of the steroid
mixture from swine plasma with dexamethasone as
internal standard was accompl ished by sol id-phase
(SPE) extraction or by the more traditional
462 SYED LAIK ALI
liquid-liquid extraction (LLE) techniques. A

Lichrosorb Si 60, 5 um silica column, 25 cm x 4.6
mmI mobile phase methylene chloride-water
saturated methy 1ene chlor ide-tetrahydrofuran-
met hano 1 -g 1 ac i a 1 acetic acid (664.5 :300:10:25 :0.5) ,
flow-rate 0.8 ml/min and detection at 254 nm were
used. Calibration curves were found to be linear
between 10 and 100 ng/ml by the LLE technique.
Within-day and inter-day variability for the
measurement of the plasma samples spiked with
prednisolone is given (20 ng/ml, 10 % and 100 ng/ml,
8.4 % ) . The average recovery of prednisolone at 20
ng/ml is between 70 and 90 % (105). Cox et a1 (106)
used a weak cation-exchange column and a mobile
phase of 0.05 M ammonium formate in 2.5 % aqueous
ethanol, flow rate 0.5 ml/min, detection 240 nm, to
separate prednisolone from prednisone. The retention
times were about 18 and 23 min for prednisone and
prednisolone respectively (106). Prednisolone and
prednisone could not be resolved with a ODS 10 cm
column, methanol-water (1:l) mobile phase, flow rate
1 ml/min, detection 240 nm (107). Retention values
of prednisolone along with a number o f other
steroids relative to acetone on a u Bondapak C 18
column using methanol-water and acetonitrile-water
mobile phases o f different compositions at a flow
rate of 1.5 ml/min are reported (108). The
extraction efficiency of ethyl acetate, diethyl
ether and dichlormethane in extracting prednisolone
from pooled plasma is reported (109). Dichlormethane
appears to be the best solvent for extraction of
corticosteroids from plasma (109). Trefez et a1
PREDNISOLONE 463
(110) showed that prednisolone could be separated

from other corticosteroids in plasma samples on a 25
cm Zorbax Sil (Dupont) column. Two techniques, HPLC
and HPTLC were developed for the determination o f
prednisolone in human plasma, salvia and urine. Both
methods shared a single and simple step o f an
organic extraction procedure and separation using a
normal phase column or HPTLC plates. A normal phase
HPLC column (0.45 x 20 cm) packed with Zobrax SIL 5
um, mobile phase dichlormethane-methanol-acetic
acid, 95:1:3:75, flow-rate 2.5 ml/min and detection
at 254 nm were the chromatographic parameters. The
method allows simultaneous measurement o f endogenous
cortisol in plasma following administration of
prednisolone and methyl prednisolone. The
calibration curves of steroids in a1 1 biological
fluids were linear over a range of concentration o f
0.025 - 4 ug/ml. The limit o f detection for
prednisolone was 10 ng/ml in plasma and salvia and
25 ng/ml in urine. The method was reproducible with
an inter- and intra-assay coefficient o f variation
of < 10 % over a wide range of concentration in all
biological fluids. No interference from endogenous
steroids was found (111).
7.5.3 Gas chromatoaraphy - mass spectrometrv

Pentafluorobenzyl hydroxylamine has been used as a
derivatization reagent in the analysis of
corti costeroids inc1 uding predn i solone by gas
chromatography-negative ion chemical ionization mass
spectrometry (NCI). The resulting
pentaf luorobenzyloxime (PFBO) trimethylsi lyl (TMS)
464 SYED LAlK ALI
derivatives were generally formed in moderate yield

but, despite this, the use of these derivatives
resulted in a 10-fold improvement in the capability
of identification of corticosteroids by GC/NCI mass
spectrometry in comparison with the methoxime/TMS
derivatives. The NCI mass spectra of PFBO/TMS
dervatives were simple with most o f the ion current
being carried by the (M-CfjF6CH2)- or (M-PFB)- - ion
and by a reagent-specific peak at m/z 196.
The PFBO/TMS derivatives are suitable for the

analysis o f pi cogram quantities o f corti costeroi ds
in biological media by GC/NCI mass spectrometry
(112). The negative ion chemical ionization mass
spectra of the methoxime-TMS dervatives of the
corticosteroids including predni solone have been
obtained using capi 1 lary co 1 umn gas
chroamtography-mass spectrometry. Fig. 9 shows a NCI
spectrum of the methoxim-TMS derivative of
prednisolone. The spectra showed abundant diagnostic
ions at m/Z greater than 300 allowing for clear
discrimination between prednisolone and other
steroid dervatives (113). A capi 1 lary GC-MS method
using negative ion chemical ionization mass
spectrometry has been developed to confirm the
presence of the parent steroids in horse urine
following the administration of proprietary
preparations o f prednisolone and betamethasone. For
this purpose standard steroids (40 ug) were treated
with methoxylamine hydrochloride in dry pyridine (8
% w/v; 100 ul) and heated at 80°C for 30 min. The
solvent was removed under N2 and
Fig. 9 (113)
NCI Spectrum o f Methoxime - TMS D e r i v a t i v e o f P r e d n i s o l o n e
441
512
300 400
466 SYED LAIK ALI
trimethylsilylimidazole (50 ul) was added and sily-

lation was carried out at 80°C for 2 hours. Excess
dervatization reagents were removed by filtration
through a 2 cm Sephadex LH-20 column using chloro-
form-n-hexane (1 :1) as eluent. The steroid MO-TMS
derivatives were eluted in the first 2 ml of eluent.
The solvent was removed under nitrogen and the
residue was dissolved in n-hexane (100 ul) for
analysis by GC and GC/MS. The base peak in the
spectrum o f prednisolone-Mo-TMS occured at m/Z 457.
The abundant diagnostic ions in higher mass regions
of the spectra render these derivatives amenable to
analysis by SIM. Capillary GC/MS-NCI analysis of a
mixture of 1 ng derivatives of prednisolone Mo-TMS
and dexamethasone Mo-TMS and monitoring ions 441,
457, 473 and 489 demonstrates the applicability of
this technique. The sensitivity that can be achieved
by this technique is 250 pg of each steroid deriva-
tive (113). The use of capillary GC-MS/NCI for the
confirmatory analysis of corticosteroids in horse
urine is more sensitive than the liquid chromato-
graphy-mass spectrometry method (114). The combina-
tion of HPLC and mass spectrometry for the analysis
of prednisolone has been reported (115). Mass
spectra for prednisolone has been obtained in the
thermospray discharge mode. Thermospray is a reli-
able HPLC/MS interface. The spectra obtained are
reproducible but fragmentation is not predictable.
The sensitivity o f the technique i s compound-depen-
dent and variable. Because of the dependence of ion
production on solvent composition, it is not easy to
use the interface with gradient elution (115).
PREDNISOLONE 461
7.5.4 Supercritical fluid chromatoqraphy (SFC)

The coupling of supercritical fluid chromatography
(SFC) with mass spectrometry (MS) seems to be easier
than liquid chromatography (LC) and MS. This follows
from consideration of the facility of the mass
spectrometer vacuum system pumping excess carbon
dioxide from SFC eluent rather than aqueous
reversed-phase eluents from HPLC. The other
important factor in SFC/MS is whether capillary or
packed columns are used. A synthetic mixture of five
corticosteroids including prednisolone was analysed
by packed-column E 1 SFC/MS (Fig. 10). The
separation was accomplished on a 2 mm x 250 mm, 3 um
S3CN spherisorb column maintained at 70°, a
flow-rate of 0.8 ml/min 92:8 C02-methanol and an
inlet head pressure of 3000 Psi. The corticosteroids
are difficult to analyse even by GC/MS because they
are relatively polar and thermally labile. Although
they can be characterised by capillary GC/MS either
as parent drugs or TMS derivatives, their long
retention time and thermal instability suggest a
need for alternative means of confirmation.
Prednisolone along with other corticosteroids could

be separated within 6 min by EI SFC/MS. The packed
column SFC/MS was applied for the analysis o f a TLC
scrape of a urine sample collectd two hours after
the intramuscular administration of 100 mg of
prednisolone to a horse. The extracted ion current
profile for the abundant fragment ion at m/z 122 and
the molecular ion at m/z 360 easily identify the
prednisolone in this sample. Packed-column SFC/MS
468 SYED LAIK ALI
1- UELEWOESTROL ACEPITE
2. CORT180NE
8. PREONISONE
4- H YDROCORT180N E
6 . PREOHl8OLONE
6- BETAUETHASONE
- 1
Fig. 10 (116)
2
Packed Column SFC/MS

3 4 5 6 MIN
Separation o f Prednisolone
100 - Fig. 10 (116)

80 EI Mass Spectrum of
60 - Prednisolone
40.
20.
0
80 100 720 140 160 180 200 220
80.
60 - P R E O H t 8 0 LO H E
40.
20
~.- .
1
0
'
8 . .- 1 . . . . . a 1
PREDNISOLONE 469
with a two-stage momentum separator is feasible for

obtaining E I mass spectra of compounds amenable to
chromatographic separation by this route. The
sensitivity afforded by this approach is
nevertheless not suitable for trace analysis (116,
117).
Prednisolone has been analysed by capi 1 lary

supercritical fluid chromatography in equine urine
extract and was identified by matching retention
time of pure standard. Supercritical fluid carbon
dioxide was used as the mobile phase in conjunction
with a methylpolysiloxane stationary phase capillary
column and a flame ionization detector. SFC can thus
be successfully applied for the estimation of
prednisolone without derivatization (118).
8. In vitro dissolution
USP X X I I requires that not less than 70 % of the

labelled amount of prednisolone is dissolved in 30
minutes in dissolution medium water (900 ml) with
paddle stirring element test apparatus (apparatus 2)
at 50 rpm (119). I n vitro dissolution profiles of
sustained release formulations of prednisolone are
given. For each tablet formulation 20 tablets were
placed in a 100 ml beaker of 5.5 cm diameter; 35 ml
of distilled water were added and the contents were
stirred for one hour, at 37°C. Sustained-release
formulation gives a more uniform blood level of
prednisolone and avoids high peaks of plasma
prednisolone (120).
470 SYED LAIK ALI
Two nonporous and three porous-amorphous silicas

were used as dispersion media to convert corticoid
solutions into free flowing powders. Prednisolone
was dissolved in N,N-dimethylacetamide- polyethylene
glycol 400, 7:3, and their 10 % (w/v) solutions were
mixed with silicas (1:3 V / W ) . Dissolution rate from
such powdered solution was more rapid than those of
their micronised powders in various aqueous media.
Dissolution in simulated gastrointestinal media of
solution of prednisolone dispersed on various
silicas is reported (121).
Prednisolone tablets, enteric coated with

neutral ised hydroxypropyl methylcel luolsoe phthalate
(HPMCP) were compared with Delta cortril tablets
(Pfizer) by compendia1 in vitro testing. For this
study tablets were tested using the disintegration
and gastroresistance tests o f both the USP and
european pharmacopeia. The dissolution of
prednisolone from coated tablets followed the USP
X X I procedure which involved monitoring drug release
in a pH 6.8 phosphate buffer after two hours in 0.1
M HC1. Percent of drug (prednisolone) released in
different pH media for neutral ised HPMCP coated
tablets is illustrated in fig. 11. The dissolution
performance closely reflected the disintegration
characteristics and was independent o f coating
weights between 5 and 25 mg for the neutralised
HPCMP tab ets (122).
A solid d spersion technique with PEG, PVP, urea,

sorbitol , mannitol and cremophor has been used for
Percent of Drug Released i n Different pH Media for
Neutralised HPMCP Coated Tablets
412 SYED LAIK ALI
improving prednisolone dissolution. The optimum

di ssolution-rate composition was for dispersions
containing 10 % w/w prednisolone (123). A marked
increase in the dissolution rate of prednisolone in
solid dispersion was observed compared with that of
drug alone or with that of a physical mixture with a
carrier (123). Prednisolone, which is poorly soluble
in water, was chosen to prepare solid dispersion
systems with water-soluble carriers. I t was further
determined whether the quantities of these carriers
and their chemical structure influenced the
dissolution rate of prednisolone from such systems.
The results of the studies showed that the
dissolution rate of prednisolone from all solid
dispersions increased markedly from those o f the
physical mixture and the drug alone. Nevertheless,
there was no observed relationship between the
higher dissolution rate and chemical structure of
the carriers, Nor was it possible to predict
quantitatively to what extent any carrier would
improve the dissolution rate o f the drug in solid
dispersion. For example sorbitol and mannitol, which
are chemically similar, produced different effects
on dissolution rate. Results indicated that sorbitol
was one of the better carriers and the maximum drug
dissolved (100 %) was achieved after 3 h. Yet at the
same time only 63.17 % prednisolone was dissolved
from the mannitol solid dispersion. When PEG, PVP
and urea were used as carriers, they gave similar
results. The amount of prednisolone dissolved from
the solid dispersions with above carriers was twice
as great as that from the drug alone. Similar
PREDNISOLONE 413
investigations with physical mixtures showed that

smaller amounts of drug were dissolved than solid
dispersions. Comparison of these results with those
of the dissolution rate of prednisolone in carrier
solutions showed that a solubilizing effect had
taken place and this had evoked a better dissolution
rate. The mechanism of the enhanced dissolution
properties of prednisolone in solid dispersion with
different carriers could not be explained. The
results showed that improved wettability of drug
molecules and their solubilization by the carriers
were not basic processes. Molecular dispersion of
drug through the matrix o f the carriers was of
greater importance. A1 though changes in
crystalographical structure of the drug during
preparation of solid dispersion were evident, x-ray
diffraction studies nevertheless indicated an
amorphous form of prednisolone in solid dispersion
with PVP. In contrast the presence of identical
prednisolone diffraction peaks in the spectrum of
pure drug and solid dispersion systems with PEG,
urea, mannitol, and sorbitol showed that these solid
dispersions contained prednisolone in crystalline
form (123).
The dissolution behaviour of ground mixtures of

prednisolone with chitin and chitosan were prepared
by co-grinding in a ball mill. The x-ray diffraction
patterns and results of differential scanning
calorimetry suggested that the size of prednisolone
crystals decreased in the ground mixtures. The
dissolution rate o f prednisolone from the ground
414 SYED LAIK ALI
mixtures was significantly greater than that from

the physical mixtures or from intact prednisolone
powder. These results indicate that chitin and
chitosan can improve the dissolution properties of
prednisolone. The ground mixture with chitosan gave
slightly greater dissolution than that with chitin
and this difference reflected the reducing effect of
chitosan on the relative enthalpy change o f
prednisolone (124). The dissolution profiles of a
model formulaion of prednisolone tablets containing
different disintegrants have been investigated.
Marked increase was observed in disintegration and
dissolution rate with increased concentration of
microcrystalline cellulose, methylcellulose, maize
starch, whereby a decrease in dissolution rate was
recorded with increasing concentration of sodium
carboymethylcellulose and pregelatinized starch
(125).
9. Pharmacokinetics and drua metabolism
Prednisolone is efficiently absorbed through the

gastrointestinal tract, with approximately 75 - 98 %
o f the dose given being abosorbed. Inactivation of
prednisolone is achieved mainly in the liver through
reduction of the double bonds in ring A and the Keto
groups to form tetrahydroprednisolone which
conjugates with glucoronic acid and sulphate groups
to form water-soluble compounds that are excreted in
the urine (126, 127). Prednisolone plasma
concentrations are commonly determined by either
radioimmunoassay or competitive protein binding
PREDNISOLONE 415
techniques. Prednisolone is absorbed completely and

rapidly after oral administration reaching peak
plasma conentrations after 1 to 3 hours. The
bioavailabiltiy of prednisolone afte oral prednisone
administration is approximately 80 % of that after
prednisolone. A wide intersubject variation in
prednisolone concentration is evident, which may
suggest impaired drug absorption in some
individuals. Prednisolone shows dose-dependent
pharmacokinetics, where an increase in dose leads to
an increase in volume o f distribution and plasma
clearance. This can be explained in terms of the
non-linear binding of the drug to plasma proteins.
The degree of binding will determine the
distribution and clearance of free drug.
Prednisolone pharmacokinetics is also dependent on
age, the half-life being shorter in children. Liver
disease prolongs the prednisolone half-life and also
increases the percentage of unbound drug. In these
cases prednisolone rather than prednisone is the
drug of choice in active liver disease owing to the
poor conversion of prednisone to prednisolone.
However, the reduced plasma concentration of
prednisolone in such patients is compensated for by
delayed clearance. Thus, there is little advantage
of one preparation over the other (126).
Hepatic conversion o f prednisone to prednisolone i s

extensive and the two compounds are generally
considered to be therapeutically equivalent when
used systemically. It is however suggested that
orally dosed prednisone resulted in lower
416 SYED LAIK ALI
circulating prednisolone concentrations compared

with equivalent oral doses of prednisolone. The
results provided evidence that oral prednisone
products may not be bioequivalent to oral
prednisolone products and suggest that substitution
of one drug from for another can result in marked
changes in circulating concentrations o f active
steroid (128). The half-life of prednisolone was
found to be about 4.2 h , the apparent volume of
distribution in the B-phase was about 0.6 l/kg and
the systemic clearance about O.ll/l.h.kg (111). In
Fig. 12 concentration-time profile of prednisolone
in biological fluids is illustrated (111).
Prednisolone is cleared from the body primarily by
hepatic metabolism and greater than 90 % of
radioacti vt i y admin i stered ora 1 1 y or intravenous1 y
as 4-C14-prednisolone is recovered in urine (127,
128). Only approximately 7 - 15 % of an oral dose of
prednisolone i s excreted as unchanged prednisolone
in the urine, the rest being recovered as a variety
of metabol ites (70).
The plasma half-life of prednisolone following the

oral administration of prednisone to normal subjects
ranges form 2.5 to 3.5 h (126, 130, 131). Similar
half-1 ife values for prednisolone were observed
after oral prednisolone is administered (126, 132,
133, 134). Nugent et a1 (135) found after an
intravenous dose of 1 mg/kg body weight of
predn i so 1 one ( sodium succ i nate sa 1 t ) an average
half-life o f 3.5 h. After an intravenous dose of 0.3
mg/kg prednisolone as phosphate the mean plasma
PREDNISOLONE 411
URINE
2 4 6 8 lo 12
TIME (hr)
Fig. 12 ( 1 1 1 )
Concentration-Time Profile o f Prednisolone in
Biological Fluids Following Intravenous
Administration o f 64 mg o f Prednisolone
478 SYED LAIK ALI
half-life was 4.2 h, plasma clearance 97.3

ml/min/1.73 m2, volume of the central compartiment
28.2 1/1.73 m2 and that of peripheral compartiment
36.9 1/1.73 m2 (136). In another study eight
subjects received an intravenous bolus dose of 12 mg
prednisolone phosphate and four received 48 mg dose.
Similar pharmacokinetic parameters were found.
Neither half-life nor clearance was statistically
different between the two dose levels (137). In a
later study the mean metabolic clearance rate was
measured as 1.16 ml/min/kg, mean half-life 3 . 2 h and
the values for the apparent volume o f distribution
were 0.14 l/kg for V and 0.15 l/kg for V2 (138).
The plasma clearance, half-life and volume of

distribution of prednisolone is reported to be
independent in the range o f the doses 10, 20 and 30
mg prednisolone adminstered orally (139). Pickup et
a1 (140) studied pharmacokinetics of prednisolone at
different levels in ten subjects, four normal
subjects and six patients with osteoarthritis after
intravenous administration o f prednisolone. Average
prednisolone half-lives were found to be between 2.6
to 3.8 h , mean volume distribution between 0.22 to
0.64 l/kg, and plasma clearance between 1.02 to 2.0
ml/min/kg following the tracer 0.15 mg/kg and 0.3
mg/kg doses. This data showed sginificant increases
in volume of distribution and plasma clearance of
prednisolone with inceasing dose. An increase in
half-life was also observed. Pickup et al. (140)
thus postulate that the observed dose-dependent
kinetics is primarily due to the non-linearity in
PREDNISOLONE 479
plasma protein binding of prednisolone. The

percentage of unbound prednisolone increases with
increasing dose and results in a larger apparent
volume of distribution and plasma clearance. The net
effect of these changes causes the observed
prolonged prednisolone half-life following larger
doses (140).
In another study the area under the plasma

concentration-time curve for prednisolone for the 20
mg dose was 77.89 % of that calculated for the 10 mg
dose. This change in area represented an increase in
prednisolone clearance from 1,7 ml/min.kg to 2.2
ml/min.kg when the dose was increased (141). Rose et
a1 . (142) found dose-dependent pharmacokinetics of
prednisolone where the plasma half-life increased
from 3 to 5 h as the oral dose of prednisone was
increased from 5 t o 50 mg. T.anner et a1 (143)
reported the pharmacokinetics of prednisolone at
different dose levels in 43 subjects. Each subject
received only a single dose, 5 - 200 rng of oral
prednisolone. Kinetic parameters of oral
prednisolone are presented in table 5 and fig. 13
illustrates concentration-time profile of
prednisolone. The mean half-life of prednisolone
remained fairly constant between 3.4 to 3.8 h.
Bioavailability of prednisolone was 98.5 2 4 %.
Furthermore as the prednisolone dose increased, the
area under the curve increased but not
proportionally to the dose, such that a fivefold
increase in dose from 20 to 100 mg resulted in only
a two-to threefold increase in area under the curve.
Table 5 (143)
Kinetic parameters for oral prednisolone, 6 to 200 rng
I07 467 2-9 45 10.7

198 2 9 1,079 f 108 3,7 2 0 , 2 49 ,c 5 9.3 f 0.8
250 2 21 1,243 2 85 3.8 t 0.2 66 f 6 12.1 2 0.9
320 2 24 1,739 f 150 3.5 2 0.2 58 2 5 11.5 t 0.9
a
0
299 f 27 1,664 2 52 3.7 2 3.3 I60 t I 1 30.0 2 2.6
625,549 3 , ~ m s 3.4,3.4 19,23
W
99,109
761 -c 177 4,361 f 1141 3.7 2 0. I 1.72 5 40 22.9 t 5.2
676,661 4,694,4186 3,6,3.6 134,146 26,28
I409 8,692 3,8 I26 23.0
* 20 mo of thle preparation waa equlvalent to 18 mo

prednleolone and 100 mg woe equlvslent to 90 mg prednleolone;
numbere In Parentheeee lndlcate the number of eublects
atudled a t that pertloular doaage and from whloh the data
are derlved.
Fig. 13 (143)
Concentration-Time Relationship o f Prednisolone
and Prednisone Following the Oral Administration of
100 mg Prednisolone PREDN180LON 0- 0
PREDNISON O--o
48 1
482 SYED LAIK ALI
The apparent volume of distribution divided by the

extent of avai labi 1 ity (Vd/F) and clearance (CL/F)
for prednisolone was found to increase with dose.
For example Vd/F at 10 mg was 49 1 increasing to 132
1 if 100 mg dose was given and CL/F increased from
155 ml/rnin to 382 rnllrnin. The authors (143)
postulated that since they found no change in the
plasma protein binding of prednisolone over the dose
range studied, the increase in volume may be due to
increasing binding of prednisolone at extravascular
sites. There was a constant amount of prednisolone
bound to cortisol-binding-globulin (CBG; 145 2 16
ng/ml ) .
It appears that prednisolone may exhibit

dose-dependent pharmacokinetics, so that with
increasing dose values volume of distribution,
plasma clearance and half-life may increase. It is
believed to be related to changes in the plasma
protein binding of prednisolone. Prednisolone
appears to bind to plasma proteins in a non linear
manner over the range of doses used (131).
It is believed that it is the free non-protein-bound

fraction of the circulating steroid wich is
biologically active and which is metabolised.
Prednisolone has been shown to bind to albumin and
to specific a- and P-globuline in plasma. Human
corticosteroi d-bind ing g lobu1 in (CBG , transcortin)
binds prednisolon with a high affinity but low
capacity due to relatively low concentrations of
PREDNISOLONE 483
about 10-7 in plasma. Thus there is a saturation in

protein binding as the steroid concentration
increases above physiological levels. However,
albumin, although it has a lower affinity for
prednisolone, has a larger capacity for binding due
to its higher plasma concentrations o f about 10-4 M.
It is therefore suggested that changes in
predn isolone plasma protein binding are responsible
for the varience in volume of distribution,
half-life and metabolic clearance (144). At low
prednisolone concentrations binding to CBG is
important, which becomes then saturated so that a
higher concentration of albumin plays the major role
(145). In another study it is suggested that
variations in the level of circulating cortisol
could cause variation in the protein binding o f
prednisolone (146). Uribe et a1 (147) determined the
effect of a liquid diet on the serum protein binding
o f prednisolone in normal healthy subjects. It was
observed that the percentage of prednisolone bound
to plasma proteins measured at the time of peak
levels was 80 %, whether administered with the meal
or with water.
Prednisone and prednisolone tablets are on the list

o f drugs with high risk potential for therapeutic
inequivalence because of differences in
bioavailability. Levy et a1 reported a case of a
patient with arthritis who was successfully treated
with a proprietary brand o f prednisolone 5 mg
tablets. Subsequently when prednisone 5 mg tablets
484 SYED LAIK ALI
as generic brand was given even at fourfold dose,

but the response failed. It appears that prednisone
tablets with low dissolution rates may be clinically
ineffective (148).
Gambertoglio et a1 (131) have given in a detailed

review the pharmacokinetics of prednisolone in
healthy volunteers, patients with different diseases
as well as effect of other drugs such as
barbiturates , phenytoi n , r ifampi n and oral
contraceptives.
Much attention has been focussed on comparisons

between standard and susstained-release preparations
(70, 149) and between standard and enteric-coated
tablets (150). Enteric coated prednisolone has been
introduced to reduce gastrointestinal distress
(151). The ability of enteric coated and
sustained-release preparations to produce prolonged
plasma concentrations were considered to be of no
greater value than conventional tablets (152). The
distribution and elimination of prednisolone have
been described in terms of a 2-compartiment open
model, with rapid distribution within the first
half-hour followed by a slower terminal elimination
phase (126).
The absolute bioavailability of prednisolone from a

rectal capsule was tested in 12 healthy volunteers.
the bioavailability from this dosage form was
PREDNlSOLONE 485
calculated with about 48 % compared to a short

infusion. The maximum plasma levels occured between
1.2 to 3.4 h (153).
10. Acknowledqement
The author is indebted to Miss Michaela Schiavulli

who has taken great pains in typing this manuscript.
Mrs. Petra Grotsch kindly assisted in drawing the
figures.
486 SYED LAIK ALI
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SOTALOL
Robert T. Foster and Robert A. Carr
Faculty of Pharmacy & Pharmaceutical Sciences
University of Alberta
Edmonton, Alberta, Canada, T6G 2N8

AND EXClPlENTS - VOLUME 21 501 All rights of reproduction reserved in any form.
502 ROBERT T. FOSTER AND ROBERT A. CARR
1. Description
1.1 Nomenclature
1.1.2 Nonproprietary Names
1.1.3 Proprietary Names
1.2 Formula
1.2.1 Empirical
1.2.2 structural
2. Synthesis
3.1 Infrared Spectra
3.2 NMR Spectra
3.2.1 Proton NMR
3.2.2 13C N M R
3.3 Mass Spectra
3.4 Ultraviolet Spectra
3.6 Melting Point
3.7 Ionization Constants
3.9 Solubility
4.1 Elemental
4.2 Chromatographic
4.2.1 Thin-Layer
4.2.2 Gas
4.2.3 High-PerformanceLiquid
5. Pharmcokinetics
5.1 Absorption
5.2 Distribution
5.3 Metabolism
5.4 Excretion
6. References
SOTALOL 503
1. DESCRIPTION
1.1 Nomenclature
N-[4-[ 1-Hydroxy-2-[ (1-methylethyl)amino]ethyl]phenyl]-

methanesulfonamide; 4 ' -[1-hydroxy-2-(isopropylamino)ethyl]-
methanesulfonanilide (1,2); MJ-1999 (2). Chemical abstracts registry
no. : 3930-20-9, sotalol; 959-24-0. sotalol hydrochloride (2).
1.1.2 Nonproprietary Name
Sotalol (1)
Beta-Cardone, Betacardone, Betades, Sotacor, Sotalex, Sotapor

(192).
1.2 Formula
1.2.1 Empirical
C ~ ~ H ~ O Nsotalol
~ O ~base;
S , C12H21ClN203S, sotalol
hydrochloride
1.2.2 structural
Figure 1 depicts the structure of sotalol.
C Hg SO 2 N H <ob* !!C HC HZN H C H ( C Hg ) 2
FIGURE 1. Structure of sotalol, where the asterisk denotes the

chiral center.
272.36, sotalol base; 308.82, sotalol hydrochloride
An odorless, white, crystalline solid (1).
2. SyNntIESIs
The synthesis of several sulfonamidophenethanolamines,

including sotalol, has been described previously (3). The synthesis of
the sulfonamidophenethanolamine compounds relied upon firstly
obtaining a 2-aminoacylsulfonanilideprecursor. The alcohol formed
from the ketone precursor is via either palladium-catalyzed low-pressure
hydrogenation or sodium borohydride chemical reduction (3).
Two schemes outlining the synthesis of the 2-
aminoacylsulfonanilideprecursor have been reported (3). The first
scheme (Figure 2) introduces the amino moiety last, whilst holding the
suIfonamido constant. The second scheme (Figure 3) introduces the
sulfonamido last, thus introducing the amine moiety first and holding it
constant.
3. PEYSICAL
PROPERTIES
3.1 Infrared Spectra
The infrared spectrum of sotalol hydrochloride is depicted in

Figure 4. The spectrum was obtained on a KBr disk using a Nicolet 20
SX Fourier Transform infrared spectrometer. Diagnostic peaks were
observed at 3570 cm-l (secondary alcohol, free); 3410 cm-1 (secondary
alcohol, H-bonded); 2700-2800 cm-l and 2950-3200 cm-1
(hydrochloride); 1325 cm-1 (S =O asymmetric stretch); 1154 cm-1
(S=O symmetric stretch). The peaks are presented in Table I.
SOTALOL 505
0
It
0 0
II I1
pczr2
NHS02R I Br2
t R ,S02CI
0
II
NHS02R
NH2
FIGURE 2. Synthetic Pathway for Ketone Precursor to Sotalol (from

ref. 3). R1 =CH3; R2=H; R3Rq=CH(CH3)2
0
II
Fci:NR
0
3 4
H2N
NHSO R
2 1
- HNR3R4
FIGURE 3. Synthetic Pathway for Ketone Precursor to Sotalol (from

ref, 3). R1 =CH3; R2=H; R3Rq=CH(CH3)2
FIGURE 4. Infrared Spectrum of Racemic Sotalol Hydrochloride.
Instrument: Nicolet 20 SX Fourier Transform infrared spectrometer
Table I. I.R. Spectrum of (+)-Sotalol HC1. KBr pellet. Instrument:

Nicolet 20 SX Fourier Transform I.R.
Wavenumber cm- Relative Intensit I
s broad
2700-3 100 s broad
bl
1585 m
1508 S
1460 m
,1393 m
1325 S
1225 m
1201 W
1071 m
1016 m
985 m
962 W
904 m
864 W
837 m
,793 W
773 m
689 S
655 W
1 635 Iw
8, s=strong; m=medium; w=weak.
SOTALOL 509
3.2 NMR Spectra
3.2.1 Proton NMR
The 300 MHz proton NMR spectrum of (+)-sotalol in CD30D is

described in Table 11. The spectrum was obtained on a Bruker AM-300
spectrometer. Instrumental settings were: time domain (data points),
FT NMR 16K; aquisition time, 1.819 sec.; spectral width, 4504.51;
receiver gain, 32; line broadening, 0.200. The spectrum is shown in
Figure 5.
The spectra for S( +)- and R(-)-sotalol are depicted in Figures 6
and 7, respectively. There is no difference between the spectra for
either pure enantiomer of sotalol and that of the racemate (all run in
CD30D). It is worth noting that the coupling of protons
(e.g., -CHC&NH, -CH(C&)2, and -Cg(OH)CH2-) is altered
probably as a function of the chiral center of sotalol.
The D 2 0 exchange NMR spectrum of sotalol is shown in Figure
8. The exchangeable protons (OH and NH) are absent, and are replaced
by a single HOD peak at 4.886.
3.2.2 13C NMR
The 300 MHz 13C NMR spectrum of sotalol in CD30D is

described in Table 111. The spectrum was obtained on a Bruker AM-300
spectrometer. Instrumental settings were: time domain (data points),
FT NMR 16K; aquisition time, 0.4424 sec.; spectral width, 18518.52;
receiver gain, 400; line broadening, 2.00. The spectrum is shown in
Figure 9.
3.3 Mass Spectra
Mass spectra were obtained on a AEI MS9 (Manchester, U.K.)

instrument equipped with a fast atom bombardment source (Figures 10
and 11). The medium was either glycerol or Cleland and the sample was
introduced by means of direct insertion. Instrument settings were: 92-
963; total scans in run, 4; sampling rate, 256; signal level threshold,
30; minimum peak width, 5; scan rate (sec/dec), 10.0.
Table II. 300 MHz Proton NMR of (f)-Sotalol in CD30D.

i
J-
FIGURE 5 .
i
r
1 6 5
1,L 9
Prn
Proton NMR Spectrum of Racemic Sotalol.
3 2 1
I
-
Instrument: Bruker AM-300 FT NMR spectrometer

I
JL
1
I I
9 8 7 6 5 3 2 1
PPfl
FIGURE 6. Proton N M R Spectrum of S( +)-Sotalol.
Instrument: Bruker AM-300 FT Nh4R spectrometer
-4 -I I
I
9 8 7 6 5 9 3 2 1
PPW
FIGURE 7. Proton NMR Spectrum of R(-)-Sotalol.
Instrument: Bruker AM-300 FT N M R spectrometer
I
I !I
I 4
7 6 5 3 2 I
PPV
FIGURE 8. Proton N M R Spectrum of Racemic Sotalol. D20 Exchange.
Instrument: Bruker AM-300 FT N M R spectrometer
SOTALOL 515
Table III. 300 MHz l3C NMR of (+)-Sotalol in CD30D.
b
Chemical Shift - 7
139.22 1 c-4
140.81 1 c-1
I wa It m 1mm am 6m rm 21
PP 11
FIGURE 9. l3C N M R Spectrum of Racemic Sotalol.

Instrument: Bruker AM-300 FT NMR spectrometer
SOTALOL 517
Regardless of the medium, MH+ peaks were found at m/z 273. Both
spectra also exhibited peaks at m/z of 545, corresponding to M2H+; at
m/z of 581, corresponding to M2H+ + HC1; and at m/z of 255,
corresponding to MH+-H20. The spectrum with glycerol as the
medium showed a base peak at m/z of 93, which suggests CH3NSOZ.
Table IV summarizes the fast atom bombardment spectral data and
suggests the structures for the fragments.
Additionally, a positive ion electron impact mass spectrum was
obtained on a Kratos MS 50 double focusing magnetic sector mass
spectrometer. The sample was introduced by means of direct insertion.
Instrument settings were: mass range, 5 1.0235-279.1606, total scans in
run, 1; sampling rate, 25; signal level threshold, 1; minimum peak
width, 7; scan rate (seddec), 10.0, number of scans averaged, 11. The
calculated M+ is at m/z 272.1195; a M+ was found at m/z 272.1196.
Furthermore, diagnostic peak (100% relative abundance) was found at
m/z 72.0817 which suggests a C ~ H ~ O fragment.
N
Table IV. FAB Mass Spectral Data of Sotalol HCl.
Ton Measured Mass 9% Relative Abundance
C12H21N203S 272.93 100.00 (Cleland)

272.93 97.05 (glycerol)
C12H19N202S 255.01 62.77 (Cleland)
255.02 56.46 (glycerol)
CH3NS02 93.04 100.00 (glycerol)
Previously, spectral data for sotalol were reported using negative

ion chemical ionization mass spectrometry (4). The base peak (M-79)-
corresponded to the loss of (-S02CH3). Other characteristic ions were
found at m/z 163 (C2FsCOO)-; m/z 147 (C2F5CO)-; m/z 144
(C2F4COO)-; and at (M-147)- and (M-166)-.
Figure 12 depicts the ultraviolet spectrum of sotalol free base in

chloroform. The spectrum was obtained using a Phillips PU8700 series
UV/VIS scanning spectrophotometer (Cambridge, U.K.). Qualitative
results depict maximal wavelengths at 242.2 and 275.2 nm.
FIGURE 10. Positive Ion FAB Mass Spectrum of Racemic Sotalol.
Instrument: AEI, MS9. Medium: glycerol
80
- 255
60 - 119
- 33 195
-
w’
20
I,, .#.ul 155
./< I
177
l
213 238
1.. il. I, .
FIGURE 11. Positive Ion FAB Mass Spectrum of Racemic Sotalol.

Instrument: AEI, MS9. Medium: Cleland
+ +
+ +
FIGURE 12. Ultraviolet Spectrum of Racemic Sotalol Base in

Chloroform.
Instrument: PU8700 series scanning UV/VIS
spectrophotometer
SOTALOL 521
Optical rotations of the two pure enantiomers of sotalol HC1 were

obtained using a Perkin Elmer Model 241 polarimeter. The rotations
were measured in a 10 cm cell (water as solvent) at the sodium D-line
(589 nm). The optical rotations (specific rotations) of sotalol HCl were:
(+)-Sotalol HC1 [ c Y ] ~ ~ D +35.80"

(-)-SOtalol HC1 -34.75 "
The specific rotations of sotalol HC1 in methanol were reported

(5) as:
(+)-sotal~iHCI +39.9"
(-)-SOtalol HC1 [ c Y ] ~ ~ D -36.3"
3.6 Melting Points
Utilizing a Uni-Melt capillary melting point apparatus (Arthur H.

Thomas Company, Philadelphia, PA), the melting points of racemic
sotalol HCl, S-and R-sotalol HCl were 218 to 219, 210 to 211 and 204
to 205 "C, respectively. The melting point of racemic sotalol HCl has
previously been reported as being within the range of 206.5 to 207 (1).
3.7 Ionization Constants
The pka values for sotalol are 9.8 and 8.3 for the amine and the
sulfonamide, respectively (6).
The watedn-octanol partition coefficient (log P value) has been

reported to be 0.24 (7). Using octan-1-ol/phosphate buffer @H 7.4) at
37" C, sotalol was reported to have a partition coefficient of 0.09 (8).
3.9 Solubility
Sotalol HC1 is freely soluble in water and only slightly soluble in

chloroform (1).
4.1 Elemental
The elemental analysis of sotalol(1) is:
C 52.92%
H 7.40%
N 10.28%
0 17.62%
S 11.77%
4.2.1 Thin-layer
A number of methods have been reported for the analysis of

sotalol(9-12). These methods are summarized in Table V.
Table V. Rf Values of Sotalol under Various Thin-Layer
-
c Conditions
Solvent-System Rf Value Ref.
methano1:ammonium 01 962 3,lO
hydroxide (100:1.5) where both
cyclohexane: constants
toluene:diethylamine represent
(75: 15:10) principle
chloroform:methanol component
(9:1) scores.
acetone
ethyl
acetate:methanol: 30
% ammonia
(85: 105)
continued.. .
SOTALOL 523
Table V continued.. .
Silica Gel 60 ethyl 22 11

F254 acetate:methanol:
ammonia (85: 105)
methanol:ammonia 56
(100:1.5)
methanol:butanol 75 (bad spot
(60:40 and 0.1 M shape)
NaBr)
methanol:water:HCl 71
(5050: 1)
Polygram Sil ethyl 0.7 12
N-HR UV acetate:methanol:
254 concentrated
4.2.2 Gas
The use of gas chromatography has been reported by others (4,

13-15). Generally, these methods have only been utilized for the
analysis of sotalol in urine. Table VI summarizes most gas
chromatographic methods reported to date.
4.2.3 High-Performance Liquid
a. Nonstereospecific. Numerous HPLC methods have been

reported for the analysis of sotalol (16-23). Generally, most
nonstereospecific HPLC assays utilize reverse-phase chromatography
with isocratic flow. Table VII summarizes the more recently reported
methods.
b. Stereospecific. The enantiomers of sotalol have been reported

utilizing HPLC methods (24-28). These methods employed either chiral
stationary phases (24,25), or pre-column derivatization with a
homochiral reagent and subsequent separation utilizing either reverse-
phase (26) or normal-phase (27) chromatography. For the most part,
however, chiral columns have primarily been used for preparative-scale
Table VI. Conditions of Reported Gas ChromatographicAnalyses.
Extraction I Derivatization I C o l d
Retention Time
diethyl ether; pentafluoro-propionic capillary, fused silica electroncapture 4
diethyl ether: anhydride:pyridine (1=25 m, 0.32 mm i.d.) detector (ECD)
dichlorometh- (2: 1) and SE-30 methylsilicone
ane (1:l) bonded phaseI8.64 min
two acetic capillary, cross-linked flame ionization 13

extractions; anhydride:pyridine methyl-silicone (1 = 12 m, detector and a
dichlorometh- (3:2) 0.2 mm i.d.), 0.33 pm nitrogen-sensitive
ane:isopro- film thickness/ detector
panol :ethyl ret. index, 2675 (see ref.
acetate (1:1:3) 12)
continued.. .
Table VI continued.. .
~
diethyl ether N-methyl-N- capillary, J & W Finnigan-MAT ion 14

followed by t- trimethylsilyl- Durabond 1 (1=30 m, trap detector
butyl trifluoracet-amide, 0.25 mm i.d.), 25 pm
alcohol: diethyl followed by N- film thickness/ 3.3 min.
ether (15 ) methylbistri-
diethyl ether, fluoracetamide
followed by trifluoroacetic
chloroform- anhydride:ethyl
pentanol (3:l) acetate (2: 1)
Bond-Elut C2, dried 1-butaneboronic
C18 and CN acid in ethyl acetate
Ln
N
Ln
solid-phase
diethyl ether none capillary, fused silica, Ion trap detector 15
SE54 (1=25 m, 0.32 mm gn>
i.d.), 0.3 mm film
thickness/ret. time not
reported
Table VII. Conditions of Reported Non-Stereospecific High-Performance Liquid Chromatographic Analyses.
Extraction Mobile Phase Composition; Retention Time Detector; Ref.

Flow Rate Sensitivity
no extraction, methano1:hexane (85:15) with rel. ret. time, 0.68 UV, 215 nm; 16
qualitative 0.02 % perchloric acid (1.85 (rel. to prazepam, qualitative use
mM); 2mVmin where rel. ret. time
of 1.0=9.20 min)
benzyl alcohol: methanol:water: acetonitrile approx. 5 min. U V , 227nm; 17
chloroform (55:45:20) with 1% acetic acid 10 ng/ml
(60:40) and 0.005 M dodecyl sodium
sulphate; 1 mVmin
1-butanol: 0.01 M phosphate buffer @H 10 min W, 226 nm; 18
chloroform 3.2):acetonitrile (20:80) with 3 0.03 pmoV1
(20: 60) mM n-octylsodium sulphate;
1.5 ml/min
Baker- 10 SPE water:methanol:acetonitrile: 0.1 8.3 min fluorescence, 19
Octyl, elution M dibasic ammonium phosphate 240/310 nm
with ethyl (45:48:6: 1); 1.5 mVmin (ex., em.);
acetate: 10 ng/ml
acetonitrile (1:2) (plasma), 0.5
p g / d (urine)
continued.. .
Table VII continued.. .
25 cm Altex, Baker-10 SPE 0.01 M potassium phosphate 4.5 min diodearray, 20

ODs-5-pm Octyl, elution dibasic buffer @H 2.4) 235nm; 20
with containing 0.002 M ng/ml
acetonitrile: nonylamine; 2 mYmin
ethyl acetate
(2: 1) -
1
25 cm n-pentanol: acetoniae: water:acetic acid approx. 5 min fluoreGnce, 21
Hypersil, chloroform (1:3) (20:79: l), adjusted to pH 2.5 235 nm/no
ODs-5-pm by NaOH; 0.005 mol/l emission
heptanesulfonic acid and 0.0005 filter; 50
moYl sodium dodecylsulfate ng/ml
added; 1 mYmin (plasma), 2
pg/ml (urine)
25 cm ethyl acetate methanol:2-propanol: 1.16 M 4.4 min fluorescence, 22
LiChrosorb, perchloric acid (75:25:0.5); 235/310 nm;
CN 10-pm 2.5 mYmin 2 ng/ml
22 cm 1-pentanol: water:acetonitrile (60:40) with 9.3 min fluorescence, 23
Brownlee chloroform (1:3) 1%
! heptanesulfonic acid:glacial 235
Labs, ODS acetic acid (75); 1 ml/min (excitation)/
5-pm no emission
filter; 25
ng/ml
Table VIII. Conditions of Stereospecific High-Performance Liquid Chromatographic Analyses for Sotalol.
Column Extraction Mobile Phase Retention Time Detector; Ref.

Composition; Flow (d); Sensitivity
Rate
25 cm amylose tris No extraction; hexane:2- (+)- and (-)- UV, h not 24
(3,5-dimethyl- qualitative propanol:diethyl- sotalol at approx. specified;
phenylcarbamate) analysis mine (80:20:0.1); 17 and 23 min, sensitivity not
0.5 mYmin respectively reported
10 cm alphal-acid (+)- and (-)- U V , 230 nm; 25
glycoprotein quantitation acid in 0.02 M sotalol at approx. sensitivity not
(Enantiopac, LKB) phosphate buffer @H 15 and 10 min, reported
respectively.
10 cm, Partisil ODS acetonitrile (-)- and (+)- fluorescence, 232 26
5-pm (60:40); 1 mYmin sotalol at approx. nm ex/no
28 and 30 min, emission filter;
respectively. sensitivity not
~ reported
25 cm, Partisil5-pm (+)- and (-)- fluorescence, 220 27
methanol (65:33: 2); sotalol at 7.5 and nm ex/no
2 mYmin 8.7 min, emission filter;
respectively. I 20 ng/ml
SOTALOL 529
enantiomer separations of sotalol. Table VIII summarizes the stereospecific

HPLC assays for sotalol.
5 . PaARMAcoKINEllcs
5.1 Absorption
Although the lipid solubility of sotalol is relatively low compared

with other P-blocking adrenoceptor drugs (28), oral bioavailability is
deemed to be 100%. Sotalol is absorbed somewhat slower than most
other @-blockers,with peak concentrations occuring within 2-3 hours
(29). Although food may impair the absorption of sotalol (28),
administration of either calcium carbonate or aluminum hydroxide
antacids has little effect on absorption (30). After administration of a
single 160 mg oral dose of sotalol, both enantiomers reached maximal
plasma concentrations in approximately 3 hours (31) and, hence, did not
exhibit stereoselective absorption.
5.2 Distribution
Sotalol is only negligibly bound (28) to plasma proteins (albumin

and alphal-acid glycoprotein). The volume of distribution of sotalol is
1.3 L/kg. As expected, the more lipophilic @-blockingdrugs, including
metoprolol and propranolol, have greater reported volumes of
distribution of 5.5 and 2.8-5.5 Wkg, respectively (28). Interestingly,
the volume of distribution appears to be somewhat reduced in elderly
hypertensive subjects (32). For example, values of 3.55k0.51 and
2.22k0.28 Wkg were reported for healthy young and elderly
hypertensive subjects, respectively. As sotalol has a very low lipid
solubility compared with other P-blocking drugs, there is slow entry of
drug into brain; the brain:plasma ratio was determined as 0.52 in
anesthetized cats (29). At present, there is no evidence for
stereoselective distribution of sotalol after administration of the racemate
(31).
5.3 Metabolism
Sotalol does not undergo first-pass metabolism after oral

administration (29). Following intravenous administration of 3H-sotalol
to dogs, over 90% of the drug was excreted renally; less than 1% of the
drug was excreted in bile (33). In a stereospecific study of sotalol(31),
nonrenal clearance constituted a mean of approximately 23% of the oral
clearance. As sotalol does not a D m u to be metabolized in man. it was
suggested that either biliary excretion and/or direct secretion of drug

across gut wall may occur in humans (31).
5.4 Excretion
Sotalol is excreted by glomerular filtration with approximately

75% of the drug being excreted within 72 hours (29). The reported
elimination half-life ranges from 7-18 hours (29). As expected, reduced
renal function (i.e., reduced creatinine clearance) results in reduced renal
clearance values of sotalol. For example, renal clearance has been
reported (34) to be reduced from a mean of 4.99 Wh (creatinine
clearance > 80 ml/min) to a mean of 0.27 L/h (creatinine clearance C
10 ml/min). In fact, after chronic administration of sotalol, the serum
half-life was reported to be 69 hours in an anuric patient (35). Although
there is no difference in the enantiomeric clearance of sotalol (31), it has
been suggested that the clearance of (+)-sotalol after administration of
such may be reduced (36) as compared to its clearance when
administered with an equal proportion of (-)-sotalol (i.e., when
administered as racemate).
The disposition of sotalol appear to be comparable between obese
individuals and control subjects (37). In elderly hypertensive subjects,
however, renal clearance was reduced from a value of 4.10f0.60
ml/min/kg which was observed in healthy young subjects, to 1.93f0.32
ml/min/kg (32). Presumably, the reduction in sotalol renal clearance in
the elderly is a reflection of the changed physiology in the elderly (e.g.,
reduced glomeruIar filtration).
Finally, sotalol is excreted in breast milk, whereby mi1k:serum
concentration ratios ranged from 2.43-5.64 (38). Consequently, breast-
fed infants may be exposed to relatively large sotalol concentrations.
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SOTALOL 533
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35. Berglund G, Descamps R, Thomis JA: Pharmacokinetics of sotalol
after chronic administration to patients with renal insufficiency.
Eur. J. Clin. Pharmacol. 1980;18:321-326.
36. Carr RA, Foster RT: Enantiospecific study of sotalol in rats.
Pharm. Res. 1991;8:S265.
37. Poirier JM, Le Jeunne C, Cheymol G, Cohen A, Barre J, Hugues
FC: Comparison of propranolol and sotalol pharmacokinetics in
obese subjects. J. Pharm. Pharmacol. 1990;42:344-348.
38. Hackett LP, Wojnar-Horton RE, Dusci LJ,Ilett KF, Roberts MJ:
Excretion of sotalol in breast milk. Br. J. Clin.
Pharmacol. 1990;29:277.
THIOPENTAL SODIUM
Michael J . McLeish
School of Pharmaceutical Chemistry
Victorian College of Pharmacy (Monash University)
Parkville, Victoria, Australia

536 MICHAEL J. MCLEISH
1. Description
1.1 Nomenclature
1.1.2 Nonproprietary Names
1.2 Formulae
1.2.1 Empirical
1.2.2 CAS Registry Numbers
1.2.3 Structural
2.1 Melting Range

2.2 Solubility Data
2.4 pHRange
2.5.3 Nuclear Magnetic Resonance Spectrum
2.5.4 Mass spectrum
3. Synthesis
4. Stability
THIOPENTAL SODIUM 531
5.1 Extraction
5.2 Identification
5.2.1 USP Analysis
5.2.2 BP Analysis
5.3 Colorimetric,Spectrophotometric and Fluorimetric Analysis
5.4 Chromatography
5.4.1 Paper, Thin-layer and Column Chromatography
6. Metabolism
7. Uses, Administration and Contraindications
8. Pharmacokinetics
9. Acknowledgements
10. References
1. DESCRIPTION
1.1 Nomenclature
(a) (rt)-5-Ethyldihydro-5-(1-methylbutyl)-2-thioxo4,6(lH,5H)-
pyrimidinedione monosodium salt [1,2]
(b) (f)-S-Ethyl-5-(1-methylbutyI)-2-thiobarbituricacid sodium salt

[1,2,3,41
1.1.2 Nonproprietaw Names
Thiopental sodium, Thiopentone sodium, thionembutal, thiomembumal

sodium, penthiobarbital sodium [lJ, soluble thiopentone [3J.
Pentothal Sodium, Nesdonal Sodium, Intraval Sodium, Trapanal,

Thiothal Sodium, Farmotal, Hypnostan, Sandothal[ 1,3].
1.2 Formulae
1.2.1 Empirical
1lH17N2Na02S (Thiopental sodium)

cl 1H18N202S (Thiopental)
1.2.2 CAS Registry Numbers
71-73-8 (Thiopental sodium) [1,2,3]

76-75-5 (Thiopental) [3]
1.2.3 structural
H H
I I
NaS N yJ ,CH,
CHCH,CH,CH, CHCH,CH,CH,
0 I 0 I
CH, CH,
Thiopental Sodium Thiopental
264.31 (Thiopental Sodium)

242.33 (Thiopental)
Thiopental Sodium:
C. 49.98% H. 6.48% N. 10.60%

0. 12.11% Na. 8.70% S. 12.13%
Thiopental:
C. 54.52% H. 7.49% N. 11.56%

0. 13.21% S. 13.23%
S40 MICHAEL J. MCLEISH
Thiopental sodium is a yellowish-white, crystalline powder or pale

greenish hygroscopic powder with an alliaceous, garlic-like odor
[1,3,4]. Thiopental sodium for injection is a sterile mixture of
thiopental sodium and anhydrous sodium carbonate as a buffer [1,2,4]
2. PHYSICAL PROPERTLES
2.1 Melting Range
The free acid (thiopental) melts at 158-160 OC [5,6].
2.2 Solubility Data
At 20 "C, thiopental sodium is soluble in 1.5 parts water [3,4,5]. It is

partially soluble in alcohol [1,3,4,5] and is practically insoluble in
ether [I ,3,4,5], benzene [l] and petroleum ether [ 1,3].
The partition coefficent of unionized thiopental between isoamyl

alcohol and water at 37 "C, is 991 [7].
pK, 7.6 at 20 OC IS].
2.4 pH Range
An 8% solution for injection has a pH of 10.2 to 11.2 [2,5].

THIOPENTAL SODIUM 54 1
2.5.1 Ultraviolet Spectrum
The ultraviolet absorption spectra of thiopental sodium, in both 0.02M

HCl and 0.02M NaOH, were obtained on a Shimadzu UV-160A
recording UV-Vis spectrophotometer. The spectra (shown in Figure
One) exhibited maxima at 295 and 305nm, respectively. The
pH-dependence of the W absorption spectrum has also been
determined 181.
The infrared spectrum of thiopental sodium and/or thiopental has

undergone considerable investigation [S-1 11. The infrared spectrum of
thiopental sodium and thiopental, both as KBr disks, were obtained on
a Hitachi 270-30 infrared spectrophotometer. The spectra, presented
as Figure Two and Three, respectively, display absorption
characteristics in good agreement with those previously reported.
Frequency assignments for some of the characteristic bands are given
in Table 1.
Frequency cm-I Infrared Assignment

3270 N-H stretch
2940 C-H stretch
1720 C=O stretch
1655 C=O stretch
1525 N-C=S
1420 NCS stretch (asymm)
1350 C-N stretch
1300 C-N
1220 NCS stretch (symm)
T a b l e 1. IR characteristics of Thiopental
E
I .
W
a
f
-
3
I
0
\
E
z
v
m
a
In
m
m
n,
W
a
m
W
6,
4
..
a a
m W
m
v)
m
+
542
0
0
0
0
W
0
m
0
0
0
.-I
0
cy
rl
h
7
:g
0 1
v
z f t)
l
2 E
3
C
0,
Q
i
0
0
0
N
0
0
n
N
0
0
F1
0
-\
0
m
ul
0
0
*
0
0
543
0
0
-4
0
0
W
0
m
0
0
0
d
0
Y
h
Y
8 'E
2s
04
% E
::z
d 1
C
2 3
0
0
N
0
m
(Y
0
0
m
0
0
m
m
0
0
*
0
2.5.3 Nuclear Magnetic Resonance Spectrum
The 'H nmr spectrum of thiopental in dimethyl SdphOXide-d6 has been

recorded at 60 MHz [121. The spectrum showed considerable overlap
which made assignment difficult. A later study, carried out in CDCL3,
showed that lanthanide shift reagents could be used to simplify the
spectrum [13].
The 'H nmr spectrum of thiopental sodium in DMSO-d6 has been

recorded on a Bruker AMX300 nmr spectrometer and is presented in
Figure Four. Spectral assignments for both thiopental sodium and
thiopental are given in Table 2. Initial assignments were based on
integrals and expected splitting patterns, and were later c o n f i i e d
using two dimensional proton correlation spectroscopy (COSY).
Figure Five shows the proton decoupled 13Cnmr spectrum of

thiopental sodium in DMSO-d6. Spectral assignments for thiopental
sodium and thiopental are provided in Table 3. The assignments are
based on those of Fratiello et al. [14].
2.5.4 Mass Spectrum
The fast atom bombardment (FAB) mass spectrum of thiopental

sodium was recorded using a JEOL JMS-DX 300 mass spectrometer
and is presented in Figure Six. The spectrum shows an (M+H)+peak
at m/z 265 (relative intensity lo%), a peak at m/z 287 (45%)
corresponding to (M+Na)+,and a further peak at m/z 309 (28%)
corresponding to (M+2Na-H)+. The base peak is at m/z 115.
The electron impact (EI) mass spectrum of thiopental was also

obtained, at an ionization voltage of 70eV, on the same instrument, and
is presented in Figure Seven. The spectrum corresponds to that
Figure Four. '8 nmr spectiurn of thiopental sodium in D M S O - 4
'1
ppm 12 10 8 6 4 2
Table 2 . 'H NMF4 Assignments
a c d s
CHCH,CH2CH,
0 I b
Thiopental Sodium Thiopental Multiplicity 'H

6 @pm) 6 (PPW (No. H) Assignment
0.583 0.696 triplet (3H) H,

0.785 0.807 triplet (3H) He
0.825 0.906 doublet (3H) Hb
0.990 1.090 multiplet (2H) H,

1.310 1.340 muliplet (2H) H,
1.730 1.900 approx quartet Ha, H,
on multiplet (3H)
10.42 12.56 broad singlet 4
Figure Five. Proton &coupled *C nmr spectrum of thiopental sodium
I " " I ' l ' ' ' ' l ~ ' i ' I

ppm 150 100 50
Table 3. 13C NMR Assignments
H
I
0 11 12 13
H’
CHCH,CH,CH,
O 10
I
CH,
Carbon Thiopental Sodium Thiopental

Assignment 6 (ppm) 6 @pm)
185.6 178.8
177.0 171.0
176.5 170.6
58.3 59.9
41.3 41.8
33.6 33.4
27.4 27.5
20.5 20.1
14.3 14.1
14.1 13.8
9.8 9.4
*
May be interchanged
Figure Six. FAB mass spectrum of thiopental sodium
loo r 115
+
(M-Na)
1
LA
0
LA 287
(M+2Ya-H)+
309
100 150 200 250 300 350
M/Z
Figure Seven. Electron impact (EI) mass spectrum of thiopental
100
50
172
157
0
50 100 150 200 250 300
M/Z
Figure Eight. Fragmentation pathway for thiopental
H
I
CH3 m/z 242
%
/ ~-c5xlo
-
H H H
I
OH OH OH
+ +' +
m/z 173 m/z 172 m/z 157
reported by Maurer [151. The most prominent ions were at m/z 242
(%), 173 (21%), 172 (42%), 157 (26%) and 29 (100%). A possible
fragmentation pathway is shown in Figure Eight.
3. SYNTHESIS
Two methods of preparing thiopental are outlined in Figure Nine.
In method A, ethyl (1-methylbutyl) malonate (I) was condensed with

thiourea (II) in the presence of sodium ethoxide in absolute alcohol.
After heating at reflux for several hours the solvent was removed and
the residue dissolved in cold water. Thiopental (III) was precipitated
by the addition of dilute hydrochloric acid. Further purification could
be accomplished by dissolving III in dilute sodium hydroxide and
precipitating with carbon dioxide [6].
In method B, the appropriately substituted nitrile (IV)was condensed

with thiourea (II), again in the presence of sodium ethoxide in absolute
alcohol. Heating at reflux for several hours resulted in the 4-imino
compound (V) which was hydrolysed to thiopental[16].
Thiopental sodium can be prepared by treating an alcoholic solution of

thiopental with one equivalent of sodium hydroxide and removal of the
solvent [6].
These two methods formed, respectively, the basis of the American

and British patents for the production of thiopental[17]. Upon
comparison of the two methods under laboratory conditions it was
concluded that method A was preferable [17].
Figure N i n e . Synthesis of thiopental
- 'YJ
Method A.
H
I
c H 3 c H 2 2 H 3 i) NaOCH2CH3
N ,CH,
CH,CH, CHCH,CHzCH3 ii) H+
0 CH, H' CHCH,CH,CH,
I 11 0 I
CH,
III
-
Method B.
H H
'YJ
I I
NaOCH,CH3 CH,CH, H20 ,CH,

CH3CH20
N
CN CH, IV H' CHCH,CH,CH, H' CHCH,CH,CH,
+ o I 0 I
.NH
.. .
CH, CH,
V 111
4. STABLLITY
According to the Codex [5], aqueous solutions of thiopental sodium

decompose upon standing and solutions should not be used if they have
become cloudy or contain precipitates or crystals. A shelf life of 5
days has been reported for thiopental in glycine/NaOH buffer at pH 9.0
[18] while a thiopental sodium for injection solution (2.5%in water) is
stable for at least 10 days at 25 O C [19].
In normal saline (0.9%), when stored in plastic infusion bags,

thiopental sodium loses up to 23%of its activity in one day [20]. This
loss was attributed to sorption onto the polyvinyl chloride (PVC) of the
infusion bags [20]. Furthermore, absorption onto the plastic matrix of
an intravenous delivery system and concomitant loss of activity, has
also been observed [21]. Conversely, a solution of thiopental sodium
stored in disposable plastic syringes showed only negligible loss of
potency after 5 days at 25 OC and 45 days at 5 "C [22]. A recent study
has examined the interaction between thiopental sodium and infusion
containers and found no decrease in potency when stored for 24 hours
at 21 OC in the dark [23]. The initial study [20] was carried out at pH
6.0 while the more recent study was carried out at pH 9.1 [23]. The
higher sorption rate at low pH was attributed to a greater fraction of
thiopental being nonionized [23]. This is consistent with no loss of
thiopental activity being seen in the study using plastic syringes 1221
which was also carried out at high (10.1) pH.
The effect of y-irradiation on thiopental has also been investigated

124). No evidence of decomposition was observed with a 2.5 Mrad
radiation dose. It was concluded that thiopental, in powder form, may
be sterilised by y-irradiation [24].
556 MCLEISH
MICHAEL .I.
5.1 Extraction
Thiopentone sodium, although soluble in water, in acid solution is

converted to the free acid (thiopental) which is water insoluble. The
assay of thiopental sodium in both biological fluids and proprietary
preparations takes advantage of these acid-base characteristics.
Extraction is accomplished by acidification followed by shaking with
organic solvents. Solvents commonly employed include chloroform
[25-271, methylene chloride [28-311 and diethyl or petroleum ether
[32-361. Less frequently used are benzene [37], toluene [38,39], ethyl
acetate [40,41], n-hexane [15,28] and n-butyl chloride [42]. Additional
selectivity and sensitivity was obtained by back-extraction into sodium
hydroxide [25,28,35,36,42,43].
More recently an extraction procedure using a solid phase column

(Bond-Hut C18)has been reported [MI.
5.2 Identification
5.2.1 USP Analysis 121
Dissolve about 500 mg thiopental sodium in 10 mL water in a

separator, add 10 mL of 3 N hydrochloric acid, and extract the
liberated thiopental with two 25 mL portions of chloroform. Evaporate
the combined chloroform extracts to dryness. Add 10 mL of ether,
evaporate again and dry at 105 OC for 2 hours: the infrared absorption
spectrum of a potassium bromide dispersion of the residue so obtained
exhibits maxima only at the same wavelengths as that of a similar
preparation of USP Thiopental reference standard.
5.2.2 B.P. Analysis 141
A. Acidify 10 mL of a 10%w/v solution of thiopental sodium in

carbon dioxide free water with 2 M hydrochloric acid. The solution
effervesces. Shake the solution with 20 mL of ether, separate the ether
layer, wash with 10 mL of water and dry over anhydrous sodium
sulphate. Filter, evaporate the filtrate to dryness and dry the residue at
100 to 105 OC. The infrared absorption spectrum of the residue is
concordant with the spectrum of thiopental EPCRS.
B. Determine the melting point of the residue obtained in test A

and of a mixture of equal parts of the residue and thiopental EPCRS.
The difference between the melting points, which are about 160 OC, is
not greater than 2 O C .
5.3 Colorimetric, Spectrophotometricand Fluorimetric Analysis
Early estimations of thiopental and other thiobarbiturates depended on

color reactions with either cobalt [27] or copper [34]. These
estimations were neither accurate nor particularly sensitive and were
superseded by ultraviolet spectrophotometricmethods [33,35,45].
Thiopental has W absorption maxima at 290 nm and 305 nm in acidic

and alkaline media, respectively. In contrast, the oxobarbiturates have
absorption maxima at 220 nm and 255 nm, respectively 1461. These
differences provided a means of distinguishing thiopental from its
major metabolite, pentobarbital, and as a consequence most
determinations were carried out at around 280 nm [33,35,45]. In one
case greater selectivity was provided by a change in extraction solvent,
which permitted the determination of the carboxylic acid metabolite of
thiopental[35]. Additional sensitivity could be provided by the use of
back-extraction methods (vide supra).
The UV methods reported minimumdetectable limits of approximately

0.5 pg/mL. The development of a spectrofluorimetricmethod (hex=
305 nm,=,A 505 nm) lowered this limit to 0.1 pg/mL [36].
5.4 Chromatography
5.4.1 Paper, Column and Thin-Layer Chromatography
Raventh 1461 lists a number of methods using paper chromatography

for the identification of thiopental and other barbiturates. These
methods all show relatively poor sensitivity with a minimum of 50 pg
required for the positive identification of most barbiturates.
Alumina column chromatography using 2% methanol in chloroform as

eluant was employed in the determination of thiopental in tissues, urine
and blood 1341. Thiopentone (>2 mg) showed as a dark band under
ultraviolet light.
Thin-layer chromatography using silica get has been used to isolate

and identify thiopental[36,47]. Elution from silica gel was achieved
with benzene-glacial acetic acid (1:9). The Rf under these conditions
was 0.47, the recovery better than 95% and the sensitivity as low as 0.5
pg [36]. Other solvents have included chloroform-acetone(9:l) and
dioxan, benzene and aqueous ammonia (20:75:5)[47]. Using a
potassium permanganate spray, thiopental could be identified as a
yellow spot on a purple background [47].
Table 4, although by no means exhaustive, provides a summary of the

numerous methods that have been developed for the gas
T a b l e 4. GC Methods for the determination of thiopental
~~
COLUMN / SUPPORT DETECTOR DEIUVAT'IZATION SENSITIVITY REF.
3% Neopental Adipate FID none 25
3% Poly A-103 on Gas Chrom Q Alkali - FID none 37
3% SE-30 on HP Chromsorb WP FID TMPAH methylation 38
3% OV-17 on Gas Chrom Q ECD Th4AH methylation 40
5% OV-1 on HP Chromosorb W m none 28
3% OV-17 on Gas Chrom Q NP-FID Iodomethane methylation 43
2% SP2110 - 1% 2510 DA F!ID none 29

on Supelcoport
5% OV-101 on HP Chromosorb G FID none 15

chromatographicanalysis of thiopental [15,25,28,29,37,38,40,43].

Generally these methods have achieved much greater selectivity and
sensitivity than colorimetric methods, with low nanogram levels being
measured using alkali-flame or nitrogen-phosphorus detectors [37,43].
However, the lowest detection limit (100 pg) was obtained using an
electron capture detector [40].
In most cases the extraction of thiopental was achieved using

procedures described in section 5.1. When required, methylation was
the favored method of derivatizationwith reagents including
trimethylphenyl ammonium hydroxide F/IpAH, 381,
trimethylaniliniumhydroxide [TMAH, 401 and iodomethane [43].
Gas chromatography has also been combined with mass spectrometry

to develop a computerised general screening procedure for
barbiturates, including thiopental [151.
In recent times HPLC appears to have become the method of choice

for the assay of thiopental. As detailed in Table 5 all the methods have
employed reversed phase columns and ultraviolet detection. The
variation in mobile phases and detector wavelength has been primarily
to enable determination in different body matrices or to permit the
simultaneous determination of thiopental and either its metabolites or
another drug. For example, particular attention has been paid to the
simultaneous measurement of thiopental and its active metabolite,
pentobarbital [26,31,42,44,48 1.
When developing these assays much attention was focussed on sample

preparation. For many of the methods sensitivity and selectivity were
of prime importance and consequently extraction methods were
Table 5. Conditions employed for the HPLC determination of thiopental
COLUMN MOBILE PHASE DETECTOR INTERNALSTANDARD SENSITIVlTY REF.
c18 -5p MeCN / H,O w,254nm Secobarbital 1oong 48

Nucleosil (32 : 68)
c
8 -lop -04 ( pH 7.7) / MeoH / THF W, 254nm none 125ng 44
Radial-Pak (13 : 7 : 4)
cg -lop MeOH / H20 W, 29Onm Phenolphthalein n.s. 30

(60 : 40)
c18 - 7 p NaP04 (0.05M, pH 4.6)MeCN W, 195nm Hexobarbital n.s. 39

LiChroCart (1 : 1)
c8-5 p
1 MeOH / KPO, (O.OlM, pH 4.4) W, 284nm 5-Ethyl-5-p-tolylbarbituric 30Ong 31
pBondapak (1 : 1) acid
c18 5p MeOH / H20 W, 280nm Phenolphthalein 30Ong 49

Spheri-5 (60 :40)
--------------
continued
c6 - 5 p NaOAc (O.OlM,pH 3.6) / MeCN U V , 28Onm Flunitrazepam 50Ong 50
Spherisorb (70 :30)
c
1 8 -5 p NaP04 (0.16M, pH 6.6) /THF W, 24Onm Barbital l0Ong 42
(86 : 14)
c18 - 1op-n KP04(pH 7.8) / MeCN / THF W, 254nm Pentobarbital l0Ong 26

@ondapak (78 : 22 : 4)
ci8 - 5cUn Do4 (O.OMM, pH 6.5) / MeOH W, 28Onm Phenolphthalein n.s. 51

Spheri-5 (52 : 48)
low MeOH / H,O W, 2541x11 Methohexitone n.s. 32

Spherisorb (1 : 1)
c18 KCL (0.2M, pH 2.0) / MeOH W, 254nm none 90% 52

Wondapak (1 : 1)
CIS- l o p NaCit (0.1%,pH 6.5) / MeOH W, 254nm Quinoline 50Ong 53

Partisil10/25 (55 : 45)
KPO4 (0.2M,pH 4.0) / MeCN W, 205nm Bupivicaine 5M 41

sil-x-1 (9: 1) 0
favored. However, others have concentrated on the rapidity and ease

of sample preparation. In these cases the preparation was limited to
the precipitation of plasma proteins with either acetonitrile [49-511or
ethanol [52],with the supernatant being injected directly on the
column. In the most extreme example untreated plasma was also
injected directly onto the column [53].Not unexpectedly, this method
suffered in that column efficiency was rapidly lost.
The sensitivity of most methods was 300 ng/mL, or better, which is

ample for monitoring plasma levels during thiopental infusion. During
continuous treatment plasma levels of even unbound thiopental are
generally greater than 500 n g h L [541.
5.5 Radioirnmunoassay
Flynn and Spector [55]developed a radioimmunoassay for a number of

barbiturates, including thiopental. The sensitivity for the latter was
100 ng, a figure tenfold higher than for its oxo-analogue, pentobarbital.
This indicated that the urea portion of the ring was critical in
determining antibody specificity [%I.
6. METABOLISM
The metabolism of thiopental and other thiobarbiturates has been

reviewed extensively [46,56-581.In mammals the biotransformation
of thiobarbiturates appears to take place by up to four different
pathways [46,56-581:
i) Side-chain oxidation
ii) Desulphuration
iii) Hydrolysis of the thiobarbiturate ring
564 MICHAEL J . MCLEISH
iv) N-dealkylation
In man, hydrolysis of the thiobarbiturate ring of thioperital does not

occur [58], and biotransformation by the liver microsomal oxidase
system appears to be the main route of elimination from the body [59].
However, in spite of the many studies of thiobarbiturate metabolism,
the fate of the majority of the administered dose is yet to be identified
[58]. What is clear is that less than 0.5% of the dose is excreted as
unchanged thiopental [35,60].
The major known pathways [58] of thiopental metabolism in humans

are shown in Figure Ten. Of these, conversion to pentobarbital seems
to be of minor significance, with only a small proportion of the
administered dose being excreted as the desulphurated metabolite
[25,60]. Oxidation of the side-chain appears to be the major pathway
for metabolism in humans, 10-25% of the administered dose being
excreted in urine as the carboxylic acid metabolite [35,60]. Carroll et
al. 1601 also demonstrated the formation of the hydroxy metabolite,
albeit in small amounts.
7. USES, ADMINISTRATION and CONTRAINDICATIONS
Thiopental sodium is a barbiturate which is administered intravenously

for the induction of general anaesthesia or for the production of
complete anaesthesia of short duration [3]. Other uses include the
supplementationof regional anaesthesia or low potency agents such as
nitrous oxide, the control of convulsive states and as a hypnotic [3,61].
In psychiatry it has found some use as an aid in diagnosis, and as a
treatment of some disorders 1611.
Thiopental sodium is administered intravenously as a 2.5% or 5%

Figure Ten. Metabolism of thiopental in humans
H
I
ST-@---.
N CH2CH3
H’ CHCH2CH2CH3
0 I
H
I
/ I \ H
I
CH3
H
I
oyJ N H2CH3 syJ N H2CH3 sTJ N CH2CH3
H/
H/ CHCH2CH2CH3 H/ CHCH2CH2COOH CHCH2CH(OH)CH3
0 I 0 I 0 I
CH3
Pentobarbital Thiopental Carboxylic Acid Thiopental Alcohol

566 MICHAEL I. MCLEISH
solution, the dose for induction being 100-250mg administered over

10-20 seconds [3,61]. For longer operations it may be given as an
intravenous drip, or additional injections of 50-100 mg may be given
as required [3].
The physical incompatabilities of thiopental sodium which are

sometimes observed [62] have been attributed to:
a. acidic solutions that precipitate the free acid (thiopental)
b. calcium or magnesium solutions that form insoluble carbonates
c. amine salts that liberate the free base in alkaline solutions
There are few absolute contraindicationsto the use of thiopental

sodium, but porphyria is generally considered to be completely
restrictive [61]. Extra care with both dosage and rate of administration
is requited in cases of severe haemorrhage, burns dehydration, severe
liver disease, status asthmaticus, severe anaemia, raised intracranial
pressure, and some metabolic diseases such as thyrotoxicosis and
diabetes 1611.
8. PHARMACOKINETICS
Thiopental sodium is rapidly and efficiently absorbed following either

oral or rectal administration [63,64]. However, clinically thiopental is
administered as an intravenous injection whereupon it is extremely
rapidly taken up by the brain. Equilibrium of brain and plasma
thiopental is achieved within about one minute 165,661 and is followed
by a speedy decrease in the brain concentration, approximately half the
maximal concentration remaining after five minutes [65,66]. The rapid
decline in brain and plasma concentration has been attributed to the
redistribution of the drug to other body tissues [58, and references
therein] and is responsible for its extremely short duration of action.
The plasma concentration curve for thiopental shows phases

corresponding to its distribution to lean tissue, to adipose tissue and to
its elimination from the body [67]. Although early studies showed the
elimination half-life to be of the order of 2-5 hours [35,68,69], these
studies were carried out using inadequate sampling times (less than
three half-lives). Recent studies have determined the elimination
half-life to be approximately 12 hours [67,70,71], although the half-life
has been shown to be significantly longer in babies [72,73] and the
elderly [74-761. Conversely, in patients aged between 5 months and 13
years the elimination half-life is considerably shorter than in adults
[77], an observation attributed to children having a relatively higher
hepatic mass [59].
To produce surgical anaesthesia, it has been seen that a plasma

thiopental concentration of 39-42 pg/mL is necessary [78]. The
average dose required for induction is essentially independent of age in
patients between 20 and 60 years [59]. A reduction in dose may be
required for patients over the age of 60; with severely deteriorated
hepatic function, with moderately affected kidney function [59], or
those heavily prernedicated with narcotics and other central
depressants [61].
In some cases thiopental is used as a primary hypnotic and is

administered as an infusion, over several days. If the plasma
concentration does not exceed 15-20 p g / d the pharmacokinetics are
essentially the same as those for bolus administration [54,79].
9. ACKNOWLEDGEMENTS
The author would like to thank Abbott Australia Pty.Ltd. for providing
568 MICHAEL .I.MCLEISH
the sample of thiopental sodium (Lot No: 58355WB) used for spectral
analyses. Thanks are also due to Denis Morgan and Malea Kneen for
critical reading of the manuscript.
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67,32-41.
TICLOPIDINE HYDROCHLORIDE
Fahad J . Al-Shammary and Neelofur Abdul Aziz Mian
Clinical Laboratory Sciences Department

514 F.J. AL-SHAMMARY AND N.A.A. MIAN
CONTENTS
1 Introduction
2 Description
2.1 Nomenclature
2.1 .1 Chemical Names
2.1 .2 Generic Names
2.1.3 Properietary Names
2.2 Formulae
2.2.1 Empirical
2.2.2 Structural
2.2.3 CAS (Chemical Abstract Service Registry
Nu mber)
2.5 Appearance, Colour and Odour
3.1 Melting Range
3.2 Solubility
3.3 Action
3.4 Indications
3.6 LD50
3.7 Compression Properties
3.8 X-Ray Powder Differaction
3.9.1 Ultraviolet Spectrum (UV)
3.9.3.1 1H.NMR.Spectrum
3.9.3.2 13C .NMR .Spectrum
3.9.4 Mass Spectrum
4 Synthesis
TICLOPIDINE HYDROCHLORIDE 575
5 Pharmacokinetics
5.2 Metabolism
5.3 Elimination and Excretion
5.5 uses
6 Methods and Analysis
6.2 Spectrophotometric Determination
6.3.1 Gas Liquid Chromatography (GLC)
(HPLC)
7 Acknowledgements
8 References
TICLOPIDINE HYDROCHLORIDE
1 INTRODUCTION
Ticlopidine ( l ) is an inhibitor of platelet action that has been

used in the treatment of a variety of disease states in which
platelet play a prominent role. Studies in animal and in man
have demonstrated that ticlopidine is a potent inhibitor of
platelet aggregation induced by adenosine diphosphate (ADP)
and variably inhibits aggregation due to collagen, adrenaline
(epinephrine), archidonic acid, thrombin and plate activating
factor. Inhibition of platelet aggregation is both dose and
time-related, with it's onsset of activity being 24 to 48 hours.
It's maximal activity occuring after 3-5 days and its activity
still being present 72 hours after a final dose.
Ticlopidin (2) is potent and specific platelet aggregation

inibitor and antithrombotic agent, exhibiting a sustained effect
and wide spectrum of activity.
Ticlopidine (3) is superior to aspirin and dipyridamole as

anti-thrombotic agent towards different kinds of experimental
thrombosis .
2 DESCRIPTION
2.1 m e nclature
(a) 5- [ (2-c h Ior op h e ny I)met hy I]-4 ,5,6,7- tetra h y d ro -

ethieno [3,2-C] pyridine (2.4);
(b) 5- (0-chloro b e n t y I)- 4 ,5,6,7-t et r a h y dro t h ie no
[3,2-C} pyridine (1, 2, 4)
(c) 5-(2-chlorobenzyl)-4,5,6,7-tetrahydrothieno
[3,2-C] pyridinehydrochloride (5)
2.1.2 Generic Names
Ticlopidine hydrochloride, Ticlopidina.

Anagregal, Aplaquette, Caudaline, Opteron, Panaldine, Ticlid,

Ticlodix, Ticlodone, Tiklyd, Ticlosan.
2.2.1 E m D i r i c a L (4,5)
C14H14CI N S (Ticlopidine)
C14HlqCI N S.HCI (Ticlopidine Hydrochloride)
2.2.2 Structural
Tic l o pidine Hydrochloride (1)
2.2.3 CAS (Chemical Abs-

Service Reaistrv Number1 (2, 5)
-
15 5 1 4 2 85 -3) (Ticlopidine)
[ 5 3 8 8 5 - 3 5 - 1] (Ticlopidine Hydrochloride)
2.3 Molecular Weiaht
263.78 (Ticlopidine) (4)

300.2 (Ticlopidine Hydrochloride) (5)
Ticlopidine:
C 63.75% H 5.35%
CI 13.44% N 5.31 %
S 12.1 5 %
578 F.J.AL-SHAMMARY AND N.A.A. MIAN
Ticlopidine Hydrochloride:
C 56.01 % H 5.03%
CI 23.65% N 14.006%
S 10.68%
2.5 &Jl!m3nce.Color.-~
Ticlopidine hydrochloride is a white, odorless crystalline
powder.
3 PHYSICAL PROPERTIES
3.1 Meltina Range (4)

MP = 18900 for Ticlopidine Hydrochloride
3.2 A c t i o n (2)
Potent and specific platelet aggregation inhibitor and

antithrombotic agent, exhibiting a sustained effect and wide
spectrum of activity.
3.3 Jndicatlons (2)

Prevention and correction of platelet disorders in surgical
patients undergoing extracorporal circulation and in long term
hemodialysis patients.
3.4
.. ..
p a r t i w n Coefficient
The PKa of Ticlopidine is 7.64 (6)
3.5 LR50 ( 4 )
55 mg/kg/24 hrs (IV in mice).
>300 mg/kg/24 hrs (orally in mice).
3.6 S o l u u
..
Almost soluble in water, soluble in 95% alcohol also soluble
in methanol, chloroform and insoluble in ether.
Comparative evaluations of aqueous film coated tablet

formulations by high humidity aging was studied by Chowhan,
Z. T; et a1.(7) Dissolution rate studies of 3 film coated
formulations of ticlopidine. HCI compared by storage under
95% relative humidity at 23 and 37% showed that tablets

coated with a formulation containing Eudragit E 30D dissolved
more slowly before storage and the dissolution became very
slow after storage. Tablets coated with 10% hydroxypropyl
Methyl cellulose or Ethyl. Cellulose colloidal dispersion also
dissolved slower after storage except that the dissolution rate
of tablets coaled with 10% hydroxy propyle-Me-Cellulose
increased af'ter 12-15 week storage at 25%. In general the
decrease in dissolution rate is related to the nature of the film
coating temp. of storage, amount of moisture gain, and tablet
core formulation. Thus to maintain good dissolution
throughout the shelf life of film coated tablets they should be
protected from high relative humidity.
3.7 Comp ression Proberties
Z.T. Chowhan and Y.P. Chow (8) studied the role of the
granulation moisture content on compression properties of
granules made with selected binders. The results suggested
that at lower pressures, higher moisture containing granules
were slightly more compressible than lower moisture-
containing granules. However at higher pressures, the
reverse was true because of the water lubrication effect. At
lower moisture levels, the crushing strength of the tablets was
dependent on the binder, at higher moisture levels, binder
differences became less significant.
3.8 X-rav Powder Differaction
The X-ray differaction pattern of ticlopidine hydrochloride

was determined using Philips full automated x-ray differ-
action spectrogoniometer equipped with PW 1730/10
generator. Radiation was provided by a copper target (Cu
annode 2000W, y = 1.5480 Ao). High intensity x-ray tube
operated at 40 Kv and 35 Mv was used. The monochromator
was a curved single crystal one (Pw 1752/00). Divergence
slit and the receiving slit were 0 and 0.10 respectively. The
scanning speed of the goniometer (Pw 1050/81) used was
0.02-2 0 per second.
The instrument is combined with Philips PM 8210 printing
recorder with both analogue recorder and digital printer. The
goniorneter was aligned using silicon sample before use. The
x-ray pattern of Ticlopidine hydrochloride is presented in
Fig,(l). The values of scattering angle 2 8 interplanner
distance dAO and relative intensity 1/10 are shown in the table
(1).
( z e - VALUE)
Fig. ( 1 1 X-Ray powder Diffraction of Ticlopidine Hydrochloride
TICLOPIDINE HYDROCHLORIDE 58 I
Table (1): Characteristic Lines of the X-ray Powder

Diffraction of Ticlopidine Hydrochloride.
20 dA I/lo% 20 dA I/lo%
9.236 9.5746 7.193 36.859 2.4385 2.843

11.817 7.4889 10.973 37.725 2.3845 4.884
12.622 7.0127 0.057 38.192 2.3564 2.877
14.422 6.1416 100 39.461 2.2835 3.278
16.233 5.4602 9.836 40.261 2.2400 3.445
16.466 5.3834 5.386 40.710 2.2163 2.375
17.658 5.0227 11.408 42.240 2.1395 2.107
18.825 4.7139 19.003 42.960 2.1053 2.944
19.009 4.6686 14.319 43.496 2.0805 2.475
19.847 4.4734 12.345 44.423 2.0393 3.479
20.073 4.4234 4.884 44.824 2.022 2.107
22.578 3.938 10.806 46.72 1.9442 3.21 1
23.043 3.8596 14.084 48.418 1.8799 3.613
23.876 3.7268 25.46 48.719 1.8690 3.41 2
24.929 3.5717 12.947 50.099 1.8207 3.178
25.673 3.4698 24.422 50.288 1.8143 3.579
26.425 3.3728 16.627 52.095 1.7556 1.706
27.128 3.287 8.999 52.61 1.7396 2.643
27.91 3.1966 3.029 53.453 1.7141 2.810
28.566 3.1247 20.809 53.773 1.7047 1.806
29.213 3.057 3.345 55.35 1.6598 1.873
29.751 3.0028 2.709 56.273 1.6347 2.576
30.037 2.975 3.412 56.99 1.6159 2.074
30.800 2.9029 3.479 58.879 1.5685 1.304
31.75 2.8183 9.769 61.613 1.5052 11.572
32.974 2.7164 3.512 64.749 1.4397 1.706
33.981 2.6381 9.501 70.693 1.3325 1.438
34.416 2.6058 2.408 77.876 1.2266 1.271
25.578 2.5233 5.319 78.818 1.2143 1.237
26.010 2.494 2.843 83.013 1.1 633 1.572
36.436 2.4658 4.583
- anale.
2 0 = scatterina - dA = intemlanner distance.
I/lo% = relative intensity based on highest as’ 100.
582 F.J.AL-SHAMMARY AND N.A.A. MIAN
3.9
3.9.1 Ultraviolet Spectrum tuvl

The UV spectrum (9) of ticlopidine hydrochloride in H 2 0
(7 mg %) was scanned from 200 to 400 nm (Fig. 2) using
LKB 4054 UV/Vis spectrophotometer. Ticlopidine
hydrochloride exhibited the following UV data (Table 2).
Table (2) UV Data of Ticlopidine
n.m max
x Absorbance
(€1
Molar Absorptivity A'
1
cm-1 gm mol/L
21 4 2.127 9121.79 303.8
268 0.092 394.5 13.14
295 0.014 60.04 2
3.9.2 Infrared Seect r u q
The 1R spectrum (9) of Ticlopidine hydrochloride as KBr disc

was recorded on a Perkin Elmer 1210 Infrared Spectrometer.
Fig. (3) shows the infrared spectrum of Ticlopidine
hydrochloride. The structural assignments of Ticlopidine
hydrochloride have been correlated with the following frequencies
(Table 3).
3.9.3 Nuclear Maanetic Resonnance SDectra
The PMR spectra (9) of Ticlopidine HCI in D M s 0 - d ~

(Fig. 4-6) was recorded on a varian XL 200 MHZ NMR
spectrometer using TMS as an internal reference. The
following structural assignments have been made (Table 4).
0
0
-#
aJ
0 E
OD
m B
-c
0
0
u)
L
0
m U
r
0 I
-s aJ
m
.-
C
0
2n
(Y
m 0
.-0
A
0 I-
0
m rc
0
0
@
E
3
N L
l5
aJ
0
lo c1
N
v,
7
0
Y
3
N
c
0
(Y
(Y
-
c\
.-
0
LL
0
0
cy
I I
4 000
I
3000
I
2000
I
1500 1 boo
jb
6r 0
WAVEN UM6 ER S
Fig. ( 3 1 Infra Red spectrum of Ticlopidine Hydrochloride
LD
V
I
0
r/)
x
0
c
.C
aJ
.-
U
L
d
0
rU
0
L
U
%
11
aJ
.-.-Ua
c
.--
0
U
6.908 7 I-
%-
0
5
L
t
U
a,
P
v)
a
z
z I
I
7
CI
4
--c
.-m
u,
1 (Expansion
Fig. ( 5 1 H-NMR Spectrum of Ticlopidine Hydrochloride of peak
N
m
s-
w
I
4.JI
1
1
12 10 a L
I I I
2
I I 1 1
0 PPM 6’
I I 1 I 1
1
Fig. ( 6 1 H-NMR spectrum of Ticlopidine Hydrochloride in DMSO- d 6
( DzO Exchange )
Table (3) IR Characteristics of Ticlopidine Hydrochloride
Frequency cm-1 Assignment
3400 NH stretch and plannar

bend
3020, 3040 Chlorophenyl CH Stretch
2260 C-S-C stretch
1590, 1560 Chlorophenyl ring stretch
1430, 1425 Pyridine methylene wag
1280 Methylene twist
1220, 1200 Chlorophenyl C-CI stretch

and bends
1160 Pyridine ring stretch
1080, 1060, 1020, 1000 Pyridine-methylene rock
750, 560, 720 Chlorophenyl spatial bend
3.9.3.2
l3C-NMR spectrum (9) of ticlopidine in DMSO-de

(Fig. 7-9) was recorded on varian XL-200 NMR-spectro-
meter. The multiplicity of the resonances was obtained
from APT (Attached Proton Test) and DEPT (Distortionless
Enchancement by Polarization Transfer) programs. The 13C-
NMR spectrum displayed all the fourteen carbon resonances.
The narrow resonance range of some of the carbons makes the
spectrum rather complex. The carbon chemical shifts
assignments are presented in table (5).
I
L'J
I
180 160 140 120 100 80 60 40 20 0 P PM
13
Fig. ( 7 ) C- NMR spectrum of Ticlopidine Hydrochloride
065
13
Fig. ( 8 1 C - NMR Spectrum of Titlopidine Hydrochloride in D M SO -d 6 (APT)
CH 3
c MX
13
Fig, ( 9 ) C-NMR SPECTRUM OF TICLOPIOINE H d IN DMSO-d 6 (DEPTI
TABLE (4 ): PMR Characteristics of Ticlopidine HCI
Structure
g C
____------__________--__----__----____
Protons I (PPm) I Multiplicity
a,b 8.086 - 8.131 m

g.h,i,j 7.457 - 7.577 m
f 6.908 - 6.934 d
d 4.609 S
C 4.277 S
e 3.424 S
The mass spectrum (9) of Ticlopidine HCI obtained by electron

impact ionization (Fig. 10) was recorded on a Finnigen MAT
90 spectrometer.
The spectrum was scanned from 50 to 500 a.rn.a. Electron

energy was 70 ev. Emission current 1 mA and ion source
pressure 10-6 torr. The most prominent fragments and their
relative intensities are presented in Table (6 ).
4 SYNTHESIS
4.1 Scheme I
Ticlopidine Hydrochloride is prepared (10) by the treatment

of 4, 5, 6,7-tetrahydrothieno [3,2-C] pyridine with
0
c
0
0
0
m
0
Fig. (10 1 Mass spectrum o f Ticlopidine Hydrochloride

594 F.J.AL-SHAMMARY AND N.A.A. MlAN
Table (5) Carbon-13 Chamical Shifts of TiclqpLQUle

..
Chemical Shift
21.529
48.91 1
49.698
54.336
134.61 9
131.401
127.726
127.763
127.581
124.898
133.863
129.793
131.333
125.257
2-chlorobenzyl chloride in the presence of Pot. Flouride. The

reaction mixture is stirred at 50% for three hours in THF.
(Scheme I).
4.2 Scheme II
Yamanochi et al (11) developed a method for the preparation

of 4, 5, 6, 74etrahydrothieno [3,2,-C] pyridine by treating
2-(24hienyl)ethylarnine and HCHO at 9OOC for three hours.
The reaction mixture was extracted with CgHg which was
recrystallised with CgHe/hexane mixture to give 1, 3, 5-
tris(thieny1 ethyl) triazine. A solution of 1, 3, 5-

tris(thieny1 ethy1)triazine in (CH3)zCHOH was added
dropwise to (CH3)zCHOH containing HCI at 50oC and reaction
mixture was stirred at 50oC for 5 hours to give 81% of
1,3,5-tris(thienyI ethy1)triazine. (Scheme 11).
4.3 Scheme Ill
Ticlopidine HCI has also been synthesized (12) by the reaction

of 4, 5, 6, 7-tetrahydrotheno[3,2-C] pyridine with
O-CIC6H6COCI in CHCl3 - aq. NaOH at room temperature for
overnight. Which was further treated with AIH(CH&HMe2)2
in toluene at 90-95OC for 2 hours.
Ticlopidine has also been synthesised by other methods (13-

16).
TABLE (6): The Mass fragments of Ticlopidine HCI
m/z Relative Ions

Intensity
110.4 100
125.2 25%
Scheme I
OS)+
CI
&cH2c'
Ticlopidine
Scheme I1
r;Sf'ziW
for 90°C for 3h.,~
4 , 5, 6, 7, tetrahydrothjeno [3,2-C] pyridine

Scheme 111
1
5- (O-~hlorobenzoy1)-4,5,6,7, tetrahydrothieno
[ 3,2-C] pyridine
/ CH3
in toluene
A1..H(CH2CH 12
' CH3
Ticlopidine
598 F.J. AL-SHAMMARY A N D N.A.A. MIAN
About 80-90% of an oral dose of the drug absorbed

after oral administration in rat or man (17, 18). After a
single dose in rats or man, peak plasma concentrations occured
at 1-3 hours (17, 18, 19, 20).
In human volunteers and patients given single doses of

500 mg, peak plasma ticlopidine concentrations were 0.61 and
0.82 mg/L respectively. Accumulation was not noted in
multiple-dose studies (18). In volunteers given a single oral
dose of 1000mg, peak plasma concentrations were 2.13 mg/L
those given repeated doses of 250 mg twice daily for 21 days.
had peak concentrations of 0.90 mg/L (18).
In rats given single or repeated doses, highest ticlopidine

concentrations were measured in the liver, kidneys, duodenum
and fat tissues. In pregnant rats, conc. in fetal blood were 40-
90% of those in maternal blood, and fetal, placental and
amoniotic conc. were appreciable (20). Plasma protein
binding has not been studied in vivo, but in rats 60% of
circulating radiolabelled ticlopidine was distributed to plasma
and 40% blood cells (1).
Ticlopidine HCI (21) is readily absorbed from the gastro-

intestinal tract after oral dosing.
The oral bioavailability of ticlopidine was increased by 20%

when taken after a meal. In contrast, absorption of ticlopidine
administered after antacid treatment was approximately 20%
lower than under fasting conditons. Administration of drug
with food is recommended to maximize gastrointestinal
tolerance (22).
5.2 Metabolism
The metabolic disposition of ticlopidine is complex with at

least four metabolites isolated in man and thirteen in rats
(23). The rate of metabolism in man is rapid as even shortly
after dosing, when ticlopidine concentrations are at their peak,
only 22% of the total radioactivity in plasma represents
unchanged ticlopidine (23), and by 15 hours past dose,
unchanged ticlopidine represents 6% or less of the total dose
in man (17).
The main quantitative metabolic route in man is

N.dealkylation, followed by oxidation with opening of the
thiophene ring (17) but another metabolic pathway is
responsible for the 2-keto derivative of ticlopidine called
PCR-3787. This metabolite, which has been found in small
concentrations in rat bile, has been found to be 5 to 10 times
more potent than ticlopidine itself as an antiplatelet agent,
although its potential contribution to ticlopidine's effect is
as yet uncertain (24).
Anne Tuang et al (21) has been studied the metabolism of

Ticlopidine on rats, the compound was quickly absorbed as
evaluated by the time of the peak plasma concentration. The
metabolism of drug involved N-oxidation, cleavage of the N-C
bond oxidation of aliphatic carbon followed by glycine
conjugation. Urinary excretion took place essentially in the
first 24 hours and biliary excretion was most pronounced in
the hour following dosing. Small amounts of drug were
excreted unchanged. The major urinary metabolites were 2-
chlorohippuric acid (16% of the dose) and
tetrahydrothienopyridine (8%) while Tic1opidine.M
predominated in the bile (2% in 0-5 h). The peak plasma
concentration of Ticlopidine occured at 0-5 hour. The plasma
concentrationAime curve displayed a biphasive profile and the
terminal half life of Ticlopidine was tentatively estimated to
be 6-10 h in the rat.
Metabolic Path of Ticlopidine
Unchanged Ticlopidine (25) and three metabolites Ticlopidine

N-oxide, (T-NO), Tetrahydrothieno pyridine (THTP), and
2,chloro-hyppuric acid (CI-HPA) were isolated from rat
urine by differencial solvent extraction and characterized by
their behaviour on TLC and GLC. Their identities were
confirmed by comparison with authentic standards. A fourth
metabolite (T-M) gave rise upon acid hydrolysis to a
compound, which co-chromatographed with authentic (T),
both on TLC and GLC. The original structure of this
metabolites is not yet elucidated. (T) and (T-M) were also
found in bile wxtracts, whereas (T-NO), (THTP), and (CL-
HPA) were not detected in the bile, under the conditions used.
Urine and bile samples were assayed for the supposed

intermediates of (CI-HPA), i.e. 2-chlorobenzyl alcohol (CI-
BzOH), 2-chloro-benzaldehyde (CI-BzAld), and 2-
chlorobenzoic acid (CI-BzA), however, under the conditions
used, only trace amounts of (CI-BzA) were detected by GLC.
F.J. AL-SHAMMARY AND N.A.A. MIAN
Glucuronides or sulphate conjugated metabolites may account

for only insignificant amounts, as the TLC patterns of enzyme
hydrolyzed and untreated samples were quite similar.
Following acid hydrolysis, however, the TLC patterns showed
the presence of several though minor compounds. Attempts to
characterize these (except T-M) or to make derivatives
suitable for GLC have so for failed.
The possibility of hydroxylated metabolites was investigated

using various TLC spray-techniques and spot-test reactions
upon silicagel eluted materials, but no net reactions resulted.
A scheme for the known metabolic pathway of Ticlopidine is

shown in Fig. (11).
5.3 Elimination and Excretion
In man, approximately 60% a radiolabelled dose is

recoverable in urine, and 25% infaeces following oral
administration (17). Ticlopidine concentrations measured as
detectable nitrogen by gas chromatography (thus, probably not
specific for the parent compound) dropped rapidly from
0.70 mg/L at 2 hours post-dose to 0.15 mg/L at 6 hours
post-dose following a single 500 mg oral dose (18). Plasma
concentration of unchanged ticlopidine fell rapidly after oral
administration of a single 750 mg dose in volunteers (18).
After repeated doses of 250 mg twice daily for 21 days, peak

concentrations of 0.90 L 0.18 mg/L fell to trough
concentrations of 0.20 L 0.07 mg/L. Elimination half-lives
of 24 2 7.5 hours and 33.2 f 3.8 hours have been reported
(18).
Ticlopidine plasma or blood concentrations do not correlate
with ex vivo activity as an antiaggregant of platelets (17, 18).
Anne Tuong et al (21) have studied that also high

concentrations of unchanged ticlopidine were found in
various organs (liver, kidney, and adipose tissues mainly)
although only minute amounts of drug were excreted in urine
(0.1% of the dose) and in bile (0.02% of the dose).
Approximately 10-15% of patients receiving ticlopidine have

experienced side effects, the most common of which have been
TICLOPIDINE HYDROCHLORIDE 60 I
Ticlopidine (T)
(T-NO) (C1 - B ZOH)

-----, (THTP)
I 1
+ acid
T
C1 -BzAld.
Cl
C1-BzA
1 + Glycine
H O O C H2C H N O C
ti
C1-HPA
Fig. (11) Metabolic pathway of Ticlopidine.

602 F.J. AL-SHAMMARY AND N.A.A. MlAN
gastrointestinal complains and skin rash. About 10%

experience gastrointestinal discomfort, dyspepsia, abdominal
pain, nausea and diarrhoea (1).
Gastro-intestinal disturbances and skin rashes are the most

commonly reported side-effects associated with Ticlopidine
therapy. Blood dsycrasias, particularly serious in elderly
patients have been reports of vertigo and occasional reports of
cholestatic jaundice (5)- Gastrointestinal distress may
necessitate discontinuation of the medication but may be
markedly reduced if the drug is given after meals (26).
Bleeding during ticlopidine therapy is an unusual side effect,

but is dangerous in patients who must undergo surgery or
another invasive procedure (1). In patients undergoing AV
access insertion, there has been no increase in bleeding (27).
But in patients undergoing open heart surgery the risk of
bleeding may be increased with ticlopidine (28).
Agranulocytosis. neutropenia, thrombocytopenia, and

erythroleu-kaemia have been reported during therapy with
ticlopidine. Elevation of liver function tests are unusual
with ticlopidine therapy, but occasionally cholestatic
jaundice or hepatitis have been reported. Drug may increase
total serum cholesterol, as well as LDL- and VLDL-cholesterol
and other lipoproteins, without effecting HDL-cholesterol (1).
Ticlopidine should not be administered to patients with

haemorrhagic diathesis, gastrointestinal ulcers or severe
liver dystfunction. It should not be given to patients receiving
aspirin, anticoagulants or corticosteroids (5).
5.5 Uses
Ticlopidine is an inhibitor of platelet aggregation. It has been

given in the treatment of atherosclerotic disease and
intermittent claudication in doses of 250 mg once of twice
daily by mouth, with meals. Regular haernotological
monitoring has been recommended (5).
Ticlopidine (29) is equally effective in both men and woman

and also improves symptoms of claudication in patients with
peripheral arterial disease and appears to reduce anginal
pain. Patients with subarchnoid haemorrhage and sickle cell
disease have shown some improvement with ticlopidine
ad minist ration.
Giuffetti, G; et al (30) studied the treatment with ticlopidine

improved the neurologic outcome and the hemorheologic
pattern in the postacute phase of ischemic stroke.
The drug (31) is to be effective in influencing the rheological

measures of red cell filteribility and membrane
microviscosity filteribility was increased and microviscosity
was decreased.
Davi G., et al (32) concluded that in schemic hear disease

patients the association of ticlopidine and low dose aspirin
seems superior toe each drug alone in inhibiting platelet
activity and according to Uchiyama S ; et al (33) combination
of aspirin plus ticlopidine is a potent antiplatelet strategy, in
ischemic attach or cerebral infarction. Balsano F; et al (34)
concluded that long term treatment with ticlopidine improves
walking ability and ankle systolic blood pressure in patients
with claudication.
6 METHO DS OF ANA LYSlS
6.1 Elemental Analvsis
The elemental analysis of ticlopidine is as reported (4).
Element Composition
C 63.75%
H 5.35%
CI 13.44%
N 5.31%
S 12.15%
For Ticlopidine Hydrochloride:
Element Composition
C 56.01 %
H 5.03%
CI 23.65%
N 4.01 yo
S 10.68%
6.2 SDectroDhotornetric Determination
A spectrophotometric study of ticlopidine was carried out by

Sanchez Perez (35). Ticlopidine reacts slowly with iodine in
CHC13 forming a mol. complex with two change transfer bands
(hmax = 295 nm and Amax = 360 nm) which was also

observed after the extraction, into CHC13 of the ticlopidine-
iodine complex formed in aqueous solution. Two spectrophoto-
metric methods for the determination of drug based on the
formation of the ticlopidine-iodine complex were studied. The
first involves the extraction of ticlopidine base from aqueous
samples into CHC13 and addition of a solution of ticlopidine.
The second involves the formation of the mol. complex in
aqueous solution (pH = 7.0) over 90 minutes and its later
extraction into CHC13. In both procedures Beer's Law was
followed with the ticlopidine concentration rage of 1.6 x l o m 6
- 1.6 x 10-5 M for the two max.
6.3
6.3.1 Gas Liauid Chromatoaraphv (GLCL
1. Ticlopidine and its metabolites in biological fluids are

being analysed by GLC.
Ticlopidine (T), and Ticlopidine N-oxyde (T-NO), were

simultaneously solvent extracted and separated by column
chromatography (T-NO) was converted to (T) by reduction
with SO2 (36) before analysis due to degradation of
(T-NO) within the injection port of gaschromatograph.
Ticlopidine-M (T-M) was processed as (T) after acid
hydrolysis of the aqueous phase. GLC analysis was
performed on Hewlett Packard model 5710
gaschromatograph equipped with a nitrogen-phosphorous
detector and a HP 3352 data system using a 6 ft x 2 mm ID
glass column, packed with 3% OV 17 on Chromosorb WHP
100/120. Injection port temp. 2500, detector temp.
3 0 0 0 , oven temp. 2000. He flow 25 ml/min. The
retention times for Ticlopidine was 6.1 min and for
internal standard was 3.2 min (25).
2. Another method used to analyse the drug and its metabolites

ie. 2-chloro-hippuric acid (CI-HPA) after extraction and
methylation of the dry residue with diazomethane, GLC
analysis was performed on a Hewlett-Packard model 5830
gaschromatograph equipped with a flame-ionisation
detector and an automatic sampler, using a 4 ft. x 2 mrn ID
glass column, packed with 1% OV 25 on Chromosorb WHP
100/120, injection port temp. 2400, detector 2500, oven
temp. 210°,for 5 minutes then raised to 230° at 10°/rnin.
The methylated derivatives of CI-HPA and the internal

standard had the reaction times of 4.0 min. and 8.4 min.,
respectively (25).
6.3.2 Jhin Laver Chromatoaraphv fTLC)
Folowing extraction and coupling in aqueous medium with

sodium naphtoquinone-sulfonate (37) the tetrahydrothie-
nopyridine (THTP) derivative extracted into methylene
chloride. The residue after solvent evaporation was
quantitatively applied on a silica gel plate (Merck 60F
254, 0.25). and the plate was developed in chloroform-
methanol (9O:lO v/v). The orange coloured derivative
migrated as a single spot (Rf 0.69) well resolved from co-
extracted, absorption measured at 480 nm (25).
2. Giuseppe Musumarra et al (38) analysed Ticlopidine HCI

by T.L.C. The drug dissolved in methanol (5 ml) or
extracted from an alkaline aqueous solution with ethyl
acetate and prepared as a solution containing about
2 rng/ml of drug. The freshly made drug solution were
applied approximately 1 cm apart to 20 x 10 cm silica gel
60 F254 HPTLC plates (Merck).
6.3.3 ah Performance L i a U
. .
ChrornatoaraDhvl [HPLC)
An HPLC (39) method was developed for determination of the

drug and its metabolites in human and rat bile. A stainless-
steel column (15 cm x 4.6 mrn I.D.) packed with LiChrosorb
RP-8 (Pore size 5pm) or Nucleasil C i 8 (pore size 5pm) was
used. The columns were packed by means of a balanced density
slurry method specially developed for the ammonia elution
system. Gradient elution was performed with water (0.005 M
ammonia) to which methanol was added, according to the
desired programme. The final elution was usually effected
with 100% methanol. Flow rate was lml/min. A wavelength
of 235 nm was found suitable for the detection of drug and its
metabolites.
7 ACKNOWLEDGEMENTS
The authors are highly thankful to Liberty S. Matibag and

Mr. Babikir Awad Mustafa, College of Applied Medical
Sciences, King Saud University for their Secretarial and
technical assistance respectively in preparing the manuscript.
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Ghijsen and R.A. De Zeeuw. J. of Chrom. 267 329-345
(1 9 8 3 ) .
VINBLASTINE SULFATE
(SUPPLEMENT)
Farid J . Muhtadi and Abdul Fattah A. A . Afify
Department of Pharmacognosy
College of Pharmacy
ANALYTICAL PROFILES OF DRUG SUBSTANCES Copyright 0 1992 by Academic Press. Inc.

AND EXClPlENTS -VOLUME 21 611 All rights of reproduction reserved in any form.
612 FARID J. MUHTADI AND ABDUL FATTAH A. A. AFIFY
V INB LASTINE SU LFAT E
Contents
Foreword
1. Description
1.1 Nomenclature
1.2 Empirical Formulae
1.4 Structure
1.6 CAS Registry Number
2.1 Melting Range
2.2 Solubility
2.3 Specific Optical Rotation
2.4 pH Range
2.5 Loss on Drying
2.7.1 Ultraviolet Spectrum
2.7.3 'H-NMR Spectrum
2.7.4 Carbon-I3 Spectrum
2.7.5 Mass Spectrum
3. Isolation of Vinblastine
4. Total Synthesis of Vinblastine
4.1 Total Synthesis of ( 2 ) - Vindoline

4.2 Total Synthesis of ( 2 ) - Catharanthine
4.3 Total Synthesis of Vinblastine
5. Biosynthesis of Vinblastine
6. Pharmacokinetics
6.1 Drug Absorption

6.2 Drug Distribution
6.3 Metabolism
VINBLASTINE SULFATE 613
6.4 Drug Excretion

6.5 Half-Life
7. Preparation and Preservation
8. Uses of Vinblastine Sulfate
8.1 Precautions
8.2 Contra-indications

9.2 Titrimetric Determinations
9.3 Voltametric Determination
9.4 Spectrophotometric Determinations
9.4.1 UV Spectrophotometry
9.4.2 Colorimetric Determinations
9.5.1 Paper Chromatography

9.5.2 Thin Layer Chromatography
9.5.3 Gas Liquid Chromatography
9.6 Radioimmunoassay Methods
Acknowledgement
References
614 FARID I. MUHTADI AND ABDUL FATTAH A. A. AFlFY
Foreword
Vinblastine or vincaleukoblastine is an indole alkaloid
obtained from Madagascan periwinkle, Catharanthus roseus G.
Don., (FamiZy Apocynaceae) which has been formerly designated
Vinca rosea L . Vinblastine is one of the antineoplastic
agents and is mainly used for the treatment of Hodgkin's
disease and other lymphomas as well as choriocarcinoma (1).
It is used as vinblastine sulfate which is formulated as IV
injections .
1. Description
1.1 Nomenclature
Vinblastine; vincaleukoblastine; VBL; 29060 - LE.

(The Base).
Vinblastine sulfate; vincaleukoblastine sulfate;
vincaleukoblastine sulfate ( 1 : 1) (salt) ; Exal;
Velban; Velbe (The Salt).
1 . 2 Empirical Formulae
C46H58N409 (Vinblastine) .
C46H58N409 .H2S04 (Vinblastine sulfate) .
1 . 3 Molecular Weight
810.98 (Vinblastine) .
909.06 (Vinhlastine sulfate).
1 . 4 Structure
The following is the absolute configuration of

vinblastine ( 2 ) .
The structure of vinblastine was deduced by a combi-
nation of chemical degradation and spectral data which
indicated that the molecule is a dimeric indole-
indoline (bisindole) and thus composed of two parts,
vindoline which i? connected through a carbon to
carbon bond to 16 B-carbomethoxyvelbanamine.
The X-ray c r y s t a l - s t r u c t u r e d e t e r m i n a t i o n o f v i n c r i s -
t i n e methiodide d i h y d r a t e (3) defined t h e a b s o l u t e stereo-
chemistry of v i n c r i s t i n e ; v i n b l a s t i n e should t h e r e f o r e h a s
t h e above a b s o l u t e s t r u c t u r e i n view of t h e known r e l a t i o n -
s h i p between t h e s e two a l k a l o i d s .
C, 68.13%; H, 7.21%; N, 6.91%; 0, 17.75% ( V i n b l a s t i n e ) .

C, 60.78%; H, 6.65%; N , 6 . 1 6 % ; S, 3.53%; 0, 22.88%
(Vinblastine s u l f a t e ) ,
1.6 CAS R e g i s t r y Number
[ 865 -21 -4 ] Vinb 1ast i n e .

[143-67-91 Vinblastine sulfate.
The base o c c u r s as s o l v a t e d n e e d l e s from methanol ( 4 ) .

Small c o l o r l e s s n e e d l e s from e t h a n o l (5) o r a white
c r y s t a l l i n e powder o r white t o s l i g h t l y yellow amor-
phous powder; o d o r l e s s ; very hygroscopic (1, 6) (The
sulfate salt).
2.1 Melting Range
V i n b l a s t i n e m e l t s a t 211-216' (4).
V i n b l a s t i n e s u l f a t e m e l t s a t 284-285' (4,7).
616 FARID 1. MUHTADI AND ABDUL FATTAH A. A. A F I R
2.2 Solubility
Vinblastine p r a c t i c a l l y insoluble i n water, s o l u b l e

i n alcohols, acetone, e t h y l a c e t a t e and chloroform ( 4 ) .
One p a r t of v i n b l a s t i n e s u l f a t e i s soluble i n 10 p a r t s
of water; i n 50 p a r t s of chloroform; very s l i g h t l y
soluble i n ethanol (96%); p r a c t i c a l l y insoluble i n
e t h e r (6).
2.3 S p e c i f i c Optical Rotation
[a]DZ6 + 42" ( i n CHC13) f o r v i n b l a s t i n e (4,7).

The following data have been reported f o r v i n b l a s t i n e
s u lf a t e :
[a]D26 - 28' ( c = 1.01 i n methanol) (4,7).
[a]D - 28" t o - 35' i n a 2% w/v s o l u t i o n i n methanol
(6)
[u]D between -
28' and -
35', c a l c u l a t e d on t h e d r i e d
b a s i s , determined i n a solution of methanol containing
200 mg i n each 10 m l (8).
2.4 PH Range (The s u l f a t e s a l t )
Between 3.5 and 5.0 i n a s o l u t i o n prepared by d i s s o l v -

ing 3 mg i n 2 m l of water (8).
3 . 5 t o 5 . 0 i n a s o l u t i o n of 0.15% w/v (6).
2.5 Loss on Drying
When v i n b l a s t i n e s u l f a t e i s d r i e d a t 60' a t a pressure

not exceeding 0.7 kPa f o r 16 hours, l o s e s not more
than 17.0% of i t s weight (6).
The USP (8) r e q u i r e s t h e determination t o be perform-
ed by thermogravimetric a n a l y s i s ,
pKa 5.4, 7.4 ( 1 , 7 ) .

2.7.1 Ultraviolet Spectrum (UV)

The UV absorbance spectrum of vinblastine sulfate in
methanol was scanned from 200 to 400 nm using a Pye-
Unicum SP 8-100 Spectrophotometer. The spectrum is
shown in Figure 1. Vinblastine sulfate exhibited the
following absorptivity values (Table 1).
Table 1 : UV Absorptivity Values
X max. nm log E A ( l % ,lcm)
212 4.75 627.50

262 4.28 209.25
284 4.22 185.0
292 4.18 167.50
Other reported UV data for vinblastine
Solvent X max. nm (Ref.)

Ethanol 214 (log E 4.74)
259 (log E 4.22)
(7)
288 (log 4*151 shoulder
296 (log E 4.12)
Aqueous
acid 268 (A = 176) (9)
2.7.2 Infrared Spectrum (IR)
The IR absorption spectrum of vinblastine sulfate as

a KBr-pellet (1%)was recorded on a Pye-Unicum SP 3-
300 Infrared Spectrophotometer. The spectrum is pre-
sented in Figure 2 .
Assignment of the functional groups have been correla-
ted with the following frequencies (Table 2 ) .
Table 2 : IR Characteristics of Vinblastine
-1
Frequency cm Functional Group
3420 (very broad) Free OH
3035 N-H stretch of indole ring
2950 C-H stretch
1725 Ester C=O (acetoxy)
618 FARID J. MUHTADI AND ABDUL FAITAH A. A. AFIFY
FIGURE 1 : UV SPECTRUM OF VINBLASTINE.

60
40.
20-
0*4000 3500 3000 2500 2000 1800 1600 ti00 1200 1000 800 600 400 20'0
FIGURE 2 : IR SPECTRUM O F VINBLASTINE.

620 FARID J. MUHTADI AND ABDUL FATTAH A. A. AFlFY
Frequency ern-' F u n c t i o n a l Group
1610 Lactam C=O

1580, 1500, 1455 Aromatic C=C
1225 c-0-C
The f o l l o w i n g p r i n c i p a l peaks a t wave numbers

1227, 1136, 1111, 1724, 1176, 1613 cm-1 were
reported f o r vinblastine sulfate as KBr d i s c (9).
Other I . R . d a t a have been a l s o r e p o r t e d (10-12).
2.7.3 'H-NMR Spectrum
The proton magnetic resonance spectrum of v i n b l a s t i n e

s u l f a t e i s shown i n Figure 3. I t was o b t a i n e d on a
Varian XL 200 NMR spectrophotometer f o r a s o l u t i o n i n
D20. The proton chemical s h i f t s a r e p r e s e n t e d i n
Table 3.
1
Table 3 : H-NMR Assignment t o V i n b l a s t i n e
Chemical S h i f t 6 (ppm) Assignment
7.18-7.48 (m) 4 , H aromatic p r o t o n s o f

c a t h a r a n t h i n e ( a t C9'
9
ll', 1 2 3 .
6.68(s) H , aromatic proton of vind-
o l i n e ( a t Cg).
6.42 (s) H, a r o m a t i c proton of v i n d -
o l i n e ( a t C12).
3.878(s) 3H, e s t e r p r o t o n s of
c a t h a r a n t h i n e ( a t Cl6/).
3.867 ( s ) 3H, methoxy p r o t o n s of
vindoline ( a t C )
11
.
3.692 (s) 3H, ester: p r o t o n s o f
v i n d o l i n e ( a t C16).
2.755 (s) 3 H , N-methyl p r o t o n s of
vindoline.
2.123 (s) 3H, ester p r o t o n s o f v i n d -
oline (at C ) .
17
s = s i n g l e t , m=multiplet
1
Other H-NMR s p e c t r a f o r v i n b l a s t i n e have been
r e p o r t e d (13-16).
FIGURE 3 : 'H - NMR SPECTRUM OF VINBLASTINE.
622 FARID J. MUHTADI AND ABDUL FA'ITAH A. A. AFlFY
2.7.4 13C-NMR
The carbon-13 NMR spectra of vinblastine and some

derivatives have been exhaustively studied and com-
plete assignments for all the 46 carbon atoms in the
structure have been made (17-19).
These are presented in Table 4. This table includes
the reported 13C-chemical shifts of vinblastine, its
sulfate salt and two of its derivatives i.e. the desa-
cetylvinblastine and vinblastine N-oxide.
Figure 4 represents the reported proton decoupled
l3C-NMR spectrum of vinblastine which was measured
on a Jeol PFT-100 Spectrometer (19).
a r - T r i t i a t e d v i n b l a s t i n e (C9,12,91,101,11',121-3H6 ) v i n b l a s -
t i n e has been prepared and a n a l y z e d b y means of t r i t i u m NMR
spectroscopy, this technique p r o v i d e s a r a p i d , nondestruc-
t i v e and d i r e c t method for the a n a l y s i s of t r i t i u m on a v e r y
small s c a l e and c a n be a p p l i c a b l e t o the a n a l y s i s of vinblas-
t i n e r e c o v e r e d from animal t i s s u e s i n b i o l o g i c a l e x p e r i m e n t s
(20).
The NMR data f o r vinblastine are considered to be consis-
tents with the conformation shown in structure below f o r the
piperidine ring in the velbanamine residue (18).
' HO COOCH,
FIGURE 4 : 13C - NMR SPECTRUM OF VINBLASTINE.
624 FARID I. MUHTADI AND ABDUL FATTAH A. A. AFlFY
Table 4 : Carbon-13 Chemical Shifts o f Vinblastine and

Derivatives 6 (ppm) .
Carbon Vinblastine VLB Desacetyl VBL
W B I H2S04 VLB N-Oxide
Vindoline 1(18) 2(19) (19)

Moiety
c2 83.1 83.3a 80.7 82.8 83.0

c3 50.0 50.2 50.4 50.4 50.4
c5 50.0 50.2 50.4 49.8 50.4
‘6 44.3 44.6 44.4 44.7 44.5
c7 52.8 53.2 53.9 53.2 53.2
122.6 122.6 124.1 122.8 123.6
c9 123.1 123.5 122.5 123.9 123.1
clo 120.4 121.1 120.7 120.9 120.5
c1 1 157.8 158.0 159.4 158.0 157.7
2 93.8 94.2 95.5 93.9 93.8
‘13 152.5 152.5 153.7 152.5 153.0
‘14 124.3 124-4 124,l 124.2 124.6
‘15 129.7 129.9 131.0 130.0 130.0
‘16 79.3 79.7 80.7 80.7 79.7
‘1 7 76.1 76.4b 75.6 74.1 76.4
c18 8.1 8.3 7.9 8.6 8.1
c19 30.5 30.8 31.7 32.9 30.7
c20 42.3 42.7 43.2 42.4 42.7
c21 65.2 65.5‘ 66.6 66.4 65.5
COOCH3
- 170.6 170.8 172.9 173.1 170.9
-
COOCH3 51.8 52.la 52.9 52.8 52.2
-
ArOCH3 55.3 55.8a 56.8 55.8 55.8
NCH3
- 38.0 38.3‘ 38.6 38.6 38.0
OCOCH3
- 171.4 171.6 173.4 - 171.6
-
OCOCH3 20.7 21.1 20.9 - 21.1
contd ... ,.
Table 4 contd... .
Carbon Vinblastine VLB Desacetyl VBL

VLB N-Oxide
WB) H2S04
Velbanamine
Moiety
ci 130.9 131.4 131.5 131.3 123.6

47.5 48.0 61.1 48.1 64.0
c; 55.5 55.8 54.5 55.8 67.9
cs' 28.7 28.2 26.9 28.7 21.4
c; 115.9 117.0 114.5 117.0 113.9
cs' 129.0 129.5 128.7 129.4 129.7
cs' 118.1 118.4 121.1 118.4 119.1
clo' 122.2 122.1 121.1 122.2 123.9

cli 118.8 118.7 119.0 118.7 119.2
cli 110.2 110.4 112.3 110.4 110.1

134.7 135.0 135.9 134.9 134.4
cl<
'
1
4
' 29.2 30.1 35.8 30.2 30.5
cis' 40.0 41.4 45.8 41.4 39.1
clB 55.3 55.8 56.1 55.8 56.1
1' '7 34.1 34.4 34.8 34.3 35.5
cli 6.7 6.9 6.9 6.9 7.1

34.1 34.4 36.1 34.3 35.5
c19'
c2d 68.6 69.4 68.9 69.5 71.9
63.1 64.2 61.1 64.3 77.8
c21'
COOCH3
- 174.6 174.9 175.2 175.1 175.1
52.0 52.3 52.8 52.3 52.7
Specific decoupling frequency (sdf) a = 3.76; h = 5.46 6;

c = 2.7 6.
626 FARlD J . MUHTADI AND ABDUL FATTAH A. A. AFIFY
2.7.5 Mass Spectroscopy
Conventional mass spectra (21) as well as high resolu-

tion mass spectra of vinblastine and vinblastine hydra-
zide have been reported (22).
High resolution mass spectrometry has established the
correct elemental composition of vinblastine, provi-
ded completely independent additional information
regarding the point of attachment of the two parts
(vindoline - velbanamine) and showed that this alka-
loid is thermally labile. Some characteristic ion
peaks, their corresponding element composition and
element lost have been reported (22).
3. Isolation of Vinblastine
Initial methods for the isolation of vinblastine

from the periwinkle plants ( v i n c a r o s e a ) had been descri-
bed (5,7,23-25) and well documented in several texts inclu-
ding the previous profile of vinblastine sulfate (12).
Isolation of vinblastine and vincristine from C a t h a r a n t h u s
r o s e u s continues to receive attention, and several proce-
dures have been reported (mainly in the patent literature)
for the isolation and separation of these alkaloids (24-29).
Extracts of C a t h a r a n t h u s r o s e u s have been found to contain
N-demethylvinblastine and this can be used to prepare vin-
cristine by formylating the alkaloid mixture before separa-
tion and purification (30).
In summary, vinblastine is extracted from C a t h a r a n t h u s
r o s e u s plants with aqueous acid or with aqueous alcoholic-
acid, isolating the alkaloids from the extracts by the
usual precipitation and solvent techniques, followed by
purifying by chromatography (usually on alumina oxide
columns), vinblastine is then obtained (31).
Vinblastine sulfate
Conversion to the (1:l) sulfate is effected by dissolving
the alkaloid in an equimolar quantity of dilute sulfuric
acid and either evaporating to dryness o r precipitating
with a suitable organic solvent (31).
4. T o t a l S y n t h e s i s of V i n b l a s t i n e
S i n c e v i n b l a s t i n e i s a d i m e r i c a l k a l o i d , c o n s i s t s of
v i n d o l i n e moiety and carbomethoxyvelbanamine p a r t , schemes
f o r t h e t o t a l s y n t h e s i s of b o t h a r e r e q u i r e d followed by
j o i n i n g t h e two monomeric u n i t s t o produce t h e d i m e r i c
alkaloid.
The t o t a l s y n t h e s e s o f v i n d o l i n e and d i h y d r o c a t h a r a n t h i n e
( a d e r i v a t i v e o f c a r b o m ethoxyvelbanamine) have been r e -
p o r t e d (32-34).
4.1 Total Synthesis of (?) -Vindoline (32)
6-Benzyloxyindole [ 11 underwent Mannich condensation

with dimethylamine [ 2 ] and formaldehyde [3] i n aqueous
a c e t i c a c i d t o g i v e t h e condensate [ 4 ] . T h i s a f t e r
q u a t e r n i z a t i o n w i t h dimethyl s u l f a t e , was t r e a t e d
with aqueous sodium c y a n i d e t o g i v e t h e n i t r i l e [ 5 ] .
Methylation of [51 w i t h methyl iodide-sodium h y d r i d e
i n dimethylformamide, followed by hydrogenation o v e r
Pd/C i n methanolethyl a c e t a t e a t 50 p s i , gave t h e
phenol [ 6 ] . T h i s was t r e a t e d with t o s y l c h l o r i d e -
sodium h y d r i d e i n t e t r a h y d r o f u r a n followed by hydro-
g e n a t i o n o v e r platinum i n aqueous e t h a n o l - e t h y l a c e t a t e
c o n t a i n i n g h y d r o c h l o r i c a c i d t o produce t h e t r y p t a -
mine [ 7 ] . The h y d r o c h l o r i d e o f [7] was condensed
with 1-chloro-3-ketobutene-1 i n e t h a n o l - t r i e t h y l a m i n e
provided t h e l i q u i d Z-enamino ketone [ 8 ] ( i n 83%
yield). [ 8 ] was converted t o i t s E-acetamide [ 9 ] by
t r e a t m e n t with a c e t y l chloride-sodium h y d r i d e i n
t e t r a h y d r o f u r a n ( i n 89% y i e l d ) . [9] was s u b j e c t e d
t o c y c l i z a t i o n by h e a t i n g a t 90' i n boron t r i f l u o r i d e
e t h e r a t e f o r 16 minutes t o a f f o r d t h e amine [ l o ] i n
89% y i e l d . The l a t t e r was t r e a t e d w i t h 20% potassium
hydroxide i n methanol-water a t r e f l u x , t o g i v e t h e
phenol which was h e a t e d with dimethyl s u l f a t e i n ace-
t o n e o v e r suspended potassium c a r b o n a t e t o a f f o r d t h e
methyl e t h e r [ l l ] i n q u a n t i t a t i v e y i e l d . Removal of
t h e a c e t y l group i n [ l l ] was accomplished with t r i e t h y -
loxonium f l u o r o b o r a t 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 o v e r suspended sodium b i c a r b o n a t e t o pro-
v i d e t h e amine [ 1 2 ] i n 82% y i e l d . Condensation of
[ 1 2 ] with a c r o l e i n i n methanol c o n t a i n i n g sodium meth-
o x i d e followed by d e h y d r a t i o n with methanesulfonyl
c h l o r i d e i n p y r i d i n e gave t h e u n s a t u r a t e d ketone [13]
i n 60% y i e l d . E t h y l a t i o n of [13] w i t h e t h y l i o d i d e
i n t e r t - b u t y l alcohol-dimethylformamide c o n t a i n i n g
potassium t e r t - b u t o x i d e y i e l d e d t h e e t h y l u n s a t u r a t e d
ketone [14] i n 53% y i e l d . Condensation o f t h e sodium
628 FARlD .I.
MUHTADI AND ABDUL FATTAH A. A. AFIFY
Scheme I : Total Synthesis of (*)-Vindoline.

'15' I
630 FARID J. MUHTADI AND ABDUL FAITAH A. A. AFlFY
h y d r i d e g e n e r a t e d e n o l a t e o f ketone [14] w i t h dime-

t h y l c a r b o n a t e gave t h e k e t o e s t e r [15]. Hydroxylation
o f t h i s with 38% hydrogen p e r o x i d e i n t e r t - b u t y l
alcohol-dimethoxyethane c o n t a i n i n g potassium t e r t -
butoxide a f f o r d e d t h e 8-hydroxy ketone [16] i n 76%
y i e l d . [16] was t r e a t e d with aluminum c h l o r i d e (-2S0,
t e t r a h y d r o f u r a n ) followed by r e d u c t i o n with sodium
b i s (2-methoxyethoxy) aluminum h y d r i d e (-20°) t o g i v e
s i n g l e epimer a l c o h o l . A c e t y l a t i o n o f t h i s a l c o h o l
w i t h a c e t i c anhydride-sodium acetate a f f o r d e d (?) -
v i n d o l i n e [ 171.
T h i s s y n t h e s i s i s presented in scheme I .
4.2 T o t a l S y n t h e s i s of (+)-Dihydrocatharanthine (33,34)
E t h y l 2-carbethoxy-4, 4-diethoxy b u t a n o a t e [ l ]
(prepared from dimethylmalonate ( 35 ) underwent
condensation with 0.5 molar excess of methyl-a-
e t h y l a c r y l a t e [ 21 (prepared from methyl -2-carboxybuta-
n o a t e ( 36,37) i n t h e p r e s e n c e of f r e s h l y p r e p a r e d
sodium e t h o x i d e as t h e c a t a l y s t t o g i v e t h e condensate,
methyl-2-ethyl-4, 4-dicarbethoxy-6, 6-diethoxy hexan-
o a t e [3] i n 86% y i e l d . T h i s was r e f l u x e d w i t h 1.5
e q u i v a l e n t s of d r y sodium cyanide i n dry dimethyl
s u l f o x i d e t o a f f o r d [4] i n 70% y i e l d . Substance [ 4 ]
was d i r e c t l y condensed with t r y p t a m i n e [S] by r e f l u x -
i n g i n aqueous acetic a c i d under Nz f o r 6 h o u r s t o
produce t h e lactam e s t e r [63. Product [6] was redu-
ced by r e f l u x i n g a s o l u t i o n o f i t i n t e t r a h y d r o f u r a n
(THF) with LAH t o g i v e t h e amine a l c o h o l [ 7 ] . Mesyla-
t i o n of [ 7 ] with anhydrous methane s u l f o n y l c h l o r i d e
and trimethylamine in anhydrous e t h e r , followed by
r e f l u x i n g t h e mesylate i n anhydrous a c e t o n i t r i l e f o r
several hours t o y i e l d t h e q u a t e r n a r y s a l t [ 8 ] . T h i s
s a l t was h e a t e d a t 200° KCN i n d i g o l , conversion t o
16-cyanodihydro cleavamine [9] was e f f e c t e d . Methan-
o l y s i s of [ 9 ] under mild c o n d i t i o n s by u s i n g anhyd-
r o u s methanol and bubbling d r y HC1 gas a t 25' a f f o r d e d
(+)-16-methoxycarbonyldihydrocleavamine [ l o ] . S u b s t -
ance [ l o ] was s u b j e c t e d t o o x i d a t i o n with m e r c u r i c
a c e t a t e t o g i v e (+)-dihydrocatharanthine [ l l ] .
T h i s t o t a l s y n t h e s i s is presented i n scheme 11.
Dihydrocatharanthine [ l l ] can be converted i n t o c a t h -

a r a n t h i n e [ 1 2 1 by an e s t a b l i s h e d method (38).
Other s y n t h e s e s o f v i n d o l i n e (39,40) and o f c a t h a r a n -
t h i n e (41,42) have been r e p o r t e d .
VINBLASTINE SULFATE 63 I
S c h e m e I1 T o t a l S y n t h e s i s of ( + ) - D i h y d r o c a t h a r a n t h i n e
/\i
( E t O ) 2CH
C02Et
+ H2C=C-C02CH3
I
Et -
C02Et
(EtO) 2 C H q ^ ( c 0 2 c H 3
C02Et C02Et
I
[I1 [21
NaCN DMSO
[31
PI [41
- LAH
THF
1. C H 3 S 0 2 C 1
2 . CH3CN
q \ T-
CH2OH
(-1
OS02CH3 [71
H Digoi
KCN
[81
MeOH/HCl
25'
632 FARID I. MUHTADI AND ABDUL FATTAH A. A. AFIFY
4.3 T o t a l S y n t h e s i s of V i n b l a s t i n e (43-47)
C a t h a r a n t h i n e [ l ] underwent o x i d a t i o n with m-chloro-

perbenzoic a c i d t o g i v e t h e N-oxide.
C a t h a r a n t h i n e N-oxide [ 2 ] was t r e a t e d with v i n d o l i n e
i n methylene chloride-trifluoroacetic anhydride a t -
5 0 ° , c o u p l i n g o c c u r r e d , t o g i v e t h e immonium i o n [3]
which wa r,edu,ced with sodium borohydride t o p r o v i d e
t h e A15' q 2 0 1 2 0 - d e o x y v i n b l a s t i n e ( a n h y d r o v i n b l a s t i n e )
[ 4 ] . This upon t r e a t m e n t with t h a l l i u m t r i a c e t a t e
followed by borohydride r e d u c t i o n a f f o r d e d v i n b l a s -
tine [ S ] .
T h i s s y n t h e s i s i s p r e s e n t e d i n scheme III.
A h i g h l y e f f i c i e n t and commercially important

s y n t h e s i s o f v i n b l a s t i n e from c a t h a r a n t h i n e and v i n -
d o l i n e h a s r e c e n t l y been d e s c r i b e d ( 4 8 ) .
Other s y n t h e t i c methods have a l s o been r e p o r t e d ( 49,SO).
Scheme 111 : Total Synthesis of Vinblastine

-0
Vindol ine
coup 1ing
/
Vindoline [41
i) T l ( 0 A c )
3 I
ii) NaBH4
3. H3C
H : OCOCH3
CH3 ~ O ~ C H ~
H3COOC
/
/
/ [51
Vindoline
634 FARID I. MUHTADI AND ABDUL FATTAH A. A. AFIFY
5. B i o s y n t h e s i s of V i n b l a s t i n e
I t h a s long been proposed t h a t t h e i n d o l i c moiety of t h e

i n d o l e a l k a l o i d s i s d e r i v e d from t h e aminoacid "tryptophan"
( 51-53). T h i s h a s been j u s t i f i e d when r a d i o a c t i v e t r y p t o -
phan o r t r y p t a m i n e (decarboxytryptophan) were i n c o r p o r a t e d
i n t o s e v e r a l i n d o l e a l k a l o i d s ( 5 4 - 5 7 ) . I t h a s a l s o been
p r e d i c t e d t h a t t h e non-tryptophan p o r t i o n s of t h e s e a l k a l -
o i d s a r e formed from two mevalonate u n i t s t o a f f o r d a c y c l o -
pentane monoterpenoid p r e c u r s o r (58,59). T h i s was proved
upon f e e d i n g d l - [2-I4C] -mevalonic a c i d l a c t o n e , and sodium
(?)-[2-14C] mevalonate i n t o V i n c a r o s e a p l a n t s and r e s u l -
t e d i n t h e i s o l a t i o n of r a d i o a c t i v e v i n d o l i n e , c a t h a r a n -
t h i n e and a j m a l i c i n e ( 60-64).
I t was f u r t h e r p r e d i c t e d t h a t t h e monoterpenoid p r e c u r s o r
c o u l d w e l l b e "the g l u c o s i d e loganin" ( 6 5 ) . I t i s now
known t h a t l o g a n i n a r i s e s i n t h e p l a n t s from two mevalonate
u n i t s . One of which i s transformed by a s e r i e s of s t e p s
i n t o i s o p e n t e n y l d i p h o s p h a t e (66) and t h e o t h e r i n t o dime-
t h y a l l y l p y r o p h o s p h a t e ( 6 7 ) . Combination of t h e s e two
u n i t s l e a d s t o g e r a n i o l (68-73), t h e n t o l o g a n i n (74-76)
and f i n a l l y i n t o s e c o l o g a n i n (77, 7 8 ) .
Evidence s u g g e s t s t h a t t r y p t a m i n e ( o r L-tryptophan) [ 11
r e a c t s w i t h s e c o l o g a n i n [2] t o form s t r i c t o s i d i n e ( i s o -
v i n c o s i d e ) [3] ( 66,79-84).
I t h a s been observed t h a t l a b e l e d s t r i c t o s i d i n e [ 3 ] ; g e i s -
s o s c h i z i n e [4] ( 8 0 , 8 5 , 8 6 ) ; stemmadenine [7] ( 8 4 , 8 7 ) and
t a b e r s o n i n e [7b,9] (86-88) were a l l i n c o r p o r a t e d i n t o b o t h
c a t h a r a n t h i n e [8] and v i n d o l i n e [ 101 i n C a t h a r a n t h u s r o s e u s
p l a n t s , i n d i c a t i n g t h a t t h e s e a r e t h e main p r e c u r s o r s i n
t h e b i o s y n t h e t i c pathway t o t h e Aspidosperma-Iboga alkaloids.
Other i n t e r m e d i a t e s such as g e i s s o s c h i z i n e o x i n d o l e [ 5 ] ,
preakuammicine [6] have been d e t e c t e d 28-40 h o u r s a f t e r
germination of C. r o s e u s s e e d s (85,87,89) provided s t r o n g
evidence f o r t h e f o r m a t i o n of c a t h a r a n t h i n e [8] and v i n d o -
l i n e [ l o ] as p r e s e n t e d i n schemes I and 11.
Feeding r a d i o a c t i v e [8] as [3H-C02CH3] and [ l o ] as [14C-
OCOCH31 i n t o a p i c a l c u t t i n g of 3-4 month-old C. r o s e u s
p l a n t s a f f o r d e d low b u t d e f i n i t e i n c o r p o r a t i o n s of b o t h
a l k a l o i d s i n t o v i n b l a s t i n e [12] d e m o n s t r a t i n g t h a t t h e s e
monomeric a l k a l o i d s a r e t h e p r e c u r s o r s of [ 1 2 1 ( 9 0 ) . Feed-
i n g b o t h [acetyl-14C] v i n d o l i n e and [OC3H3] c a t h a r a n t h i n e
t o 6 week-old d i f f e r e n t i a t e d c. r o s e u s p l a n t s f o r 6 d a y s ,
l a b e l l e d a n h y d r o v i n b l a s t i n e [ 111 was i s o l a t e d ( 91) . T h i s
was i n c o r p o r a t e d i n t o v i n b l a s t i n e by c e l l - f r e e p r e p a r a t i o n s
Of C a t h a r a n t h u s r o s e u s ( 9 2 , 9 3 J .
Later it was found t h a t a n h y d r o v i n b l a s t i n e [ l l ] can b e con-
v e r t e d i n t o v i n b l a s t i n e [ 1 2 ] by c e l l - f r e e homogenates of
VINBLASTINE SULFATE 63.5
Scheme I : B i o s y n t h e s i s of C a t h a r a n t h i n e
+-
I
[51
-
C02CH3 CH20H
636 FARID 1. MUHTADI AND ABDUL FATTAH A. A. AFIFY
Scheme 11: B i o s y n t h e s i s of C a t h a r a n t h i n e and Vindoline
0-B
0N
H I
1
[91 C02CH3
(-)-form COZCHS
(+)-form
[7b1
VINBLASTINE SULFATE
Scheme 111: Biosynthesis of Vinblastine
Catharanthine [S] + Vindoline [ l o ]
Cell-free extracts
from Catharanthus
roseus plants
Vindoline
Cell-free extracts -
or Cell-free homoyenates
leaves C. roseus of C. roseus cell suspen-
1 sion cultures
638 FARlD J. MUHTADI AND ABDUL FATTAH A. A. AFlFY
C . roseuscell suspension cultures ( 3 4 ) , thus administra-

tion of [21’a-3H] anhydro-VLB to a cell-free homogenate of
suspension culture cells C. roseus afforded radioactive
[3H]-vinblastine ( 94 ) .
The biosynthesis of vinblastine is presented i n scheme III.
6. Pharmacokinetics
6.1 Drug Absorption
Vinblastine is poorly absorbed after oral administra-
tion (9,951.
It is readily absorbed after intravenous administra-
tion (IV) or intraperitoneal injection (IP).
6.2 Drug Distribution

Vinblastine is rapidly distributed with high tissue
binding, readily binds to platelets, red blood cells
and white blood cells; subject to enterohepatic cir-
culation; volume of distribution, 86 to 111 liters
( 1,961.
After IV radioactively labeled, vinblastine is detec-
ted mostly in the liver in less than an hour (97).
Protein binding: It is highly protein bound ranging
from 98 to 99.7% ( 9 8 ) . Binds in plasma to a- and 8 -
globulins (1,96). The drug does not penetrate the
CNS o r other fatty tissues ( 99).
Drug concentration levels: continuous infusions of
vinblastine (2 mg/square meter) will produce cyto-
toxic concentrations of approximately 2 ng/mL ( 100).
After an IV dose of 15 mg, a plasma concentration of
about 16 ng/mL is obtained in 24 hours; an additional
dose of 15 mg at this time produces a plasma concentra-
tion of about 55 ng/mL 4 hours later ( 1,96).
6.3 Metabolism
Vinblastine is metabolized in the liver.
Metabolic reactions in rats is deacetylation to give
desacetylvinblastine which is the major metabolite of
vinblastine ( 1,9).
A significant amount of vinblastine is metabolized in
the liver to the active metabolite desacetylvinblas-
tine ( 95).
6.4 Drug Excretion

In 72 hours, 25 to 40% of an intravenous dose is
excreted in the feces and 19 to 23% is excreted in
the urine, most of the urinary excreted material is
unchanged, w h i l s t t h a t i n t h e f a e c e s i s i n t h e form
of m e t a b o l i t e s (1,961.
About 14% of a r a d i o a c t i v e l y l a b e l e d dose i s e x c r e t e d
i n t h e u r i n e i n 72 hours and 10% i s e l i m i n a t e d i n t h e
f a e c e s i n t h e same p e r i o d ( 9 ) .
Following I V v i n b l a s t i n e d o s i n g depending upon t h e
r a d i o a c t i v e l a b e l t e c h n i q u e used o n l y 13.6 t o 23%
of t h e t o t a l dose was e x c r e t e d i n t h e u r i n e and t h a t
e x c r e t e d i n t h e f e c e s ranged from 9.9 t o 41% w i t h 72
hours ( 101) .
6.5 Half-Life
Plasma h a l f - l i f e ( t o t a l a c t i v i t y ) , about 20 hours
(9) *
In whole blood, a - p h a s e , about 4 minutes and 8-phase,
about 190 minutes, f o r drug p l u s m e t a b o l i t e s ( 1 ) .
V i n b l a s t i n e f i t s a 3-compartment pharmacokinetic
model w i t h a l p h a , b e t a and gamma ( t e r m i n a l p h a s e ) ,
h a l f - l i v e s of 0.062, 0.164 and 25 hours r e s p e c t i v e l y
were o b t a i n e d ( 9 9 ) .
7. P r e p a r a t i o n and P r e s e r v a t i o n
V i n b l a s t i n e s u l f a t e should be s t o r e d i n a i r t i g h t c o n t a i -
n e r s , a t a t e m p e r a t u r e between 2" and 10" p r o t e c t e d from
light.
V i n b l a s t i n e s u l f a t e i s a d m i n i s t e r e d by t h e IV i n j e c t i o n
o f a s o l u t i o n o f 1 mg p e r mL i n water f o r i n j e c t i o n o r i n
sodium c h l o r i d e i n j e c t i o n ( p r e s e r v e d w i t h phenol o r benzyl-
a l c o h o l ) . Usually t h e ampoule c o n t a i n s 10 mg s t e r i l e v i n -
blastine sulfate.
The drug d i s s o l v e s i n s t a n t l y t o g i v e a c l e a r s o l u t i o n
having a pH i n t h e range of 3.5-5.0. V i a l s of v i n b l a s t i n e
s u l f a t e should be s t o r e d i n a r e f r i g e r a t o r between 2 O and
8°C t o a s s u r e extended s t a b i l i t y . After r e c o n s t i t u t i o n
w i t h 10 mL b a c t e r i o s t a t i c Sodium C h l o r i d e I n j e c t i o n USP
( p r e s e r v e d with b e n z y l a l c o h o l ) , s o l u t i o n may kept i n a
r e f r i g e r a t o r a t 2" t o 8OC f o r 30 days without s i g n i f i c a n t
l o s s of potency ( 1 0 2 ) .
If t h e i n j e c t i o n c o n t a i n s no b a c t e r i o c i d e , i t should be
used a s soon a s p o s s i b l e a f t e r p r e p a r a t i o n , and i n any
c a s e w i t h i n 4 days. In t h e p r e s e n c e of a s u i t a b l e b a c t e -
r i o c i d e such a s 0.5% phenol, i t may be used f o r up t o a
one month when s t o r e d a t 2 O t o 10°C ( 1 ) .
640 FARID J . MUHTADI AND ABDUL FATTAH A. A. AFIFY
8. Uses of Vinblastine Sulfate
Vinblastine is an antineoplastic drug which apparentlyacts

by binding to the microtubular proteins of the spindle and
arresting mitosis at the metaphase.
It also interferes with aminoacid metabolism and nucleic
acid synthesis. It has some immunosuppressant activity,
as it suppresses the immune response and in high doses it
is neurotoxic. Like other cytotoxic drugs it is teratog-
enic (95).
Vinblastine sulfate is mainly used in association with
other antineoplastic agents, in the treatment of Hodgkin's
disease and other lymphomas including mycosis fungoides.
It is also of use in the treatment of some inoperable
malignant neoplasms including those of the breast, female
genital tract, testis, lung, gastrointestinal tract and
in neuroblastoma, choriocarcinoma, Kaposi's sarcoma and
histiocytosis X ( 9 5 ) .
In the treatment of Hodgkin's disease, it is often given
with cyclophosphamide or mustine, procarbazine and pre-
dnisone or with doxorubicin, bleomycin and dacarbazine.
In carcinoma of testis, vinblastine is given with bleomy-
cin and cisplatin (95).
I n clinical dosage it depresses bone-marrow activity,
affecting mainly the white cells, with relative sparing of
the erythroid elements. The bone-marrow depression is
reversible on stopping the drug (1).
8.1 Precautions
Vinblastine sulfate should be used with care in cach-

ectic patients. Its use in pregnancy is not advised
as it is teratogenic.
Care should be applied when it is injected intraven-
ously as perivenous infiltration may cause cellulitis,
phlebitis and venous thrombosis ( 1).
8.2 Contra-indications
Vinblastine sulfate should not be given if the white-

cell count is below 4000 per cubic millimeter, if
bacterial infection is present, or if the bone-marrow
is infiltrated with neoplastic cells ( 1).
VlNBLASTlNE SULFATE 64 I
9. Methods o f A n a l y s i s
9.1 I d e n t i f i c a t i o n Tests
The following identification tests are mentioned in

the B P (6).
To lmg v i n b l a s t i n e s u l f a t e add 0.2 m l of 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 h y d r o c h l o r i c a c i d . A pink
c o l o r i s produced i n about 1 minute ( d i s t i n c t i o n from v i n -
cristine sulfate).
Mix 0.5 mg v i n b l a s t i n e s u l f a t e w i t h 5 mg of 4-dimethylamino-
benzaldehyde and 0.2 ml o f 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 reddish-brown c o l o r i s produced. Add
1 m l of g l a c i a l acetic a c i d ; t h e c o l o r changes t o p i n k .
The following identification tests are mentioned in
the USP (8).
The i n f r a r e d a b s o r p t i o n spectrum o f a pot.assium d i s p e r s i o n
of v i n b l a s t i n e s u l f a t e , p r e v i o u s l y d r i e d i n vacuum a t 60'
f o r 16 h o u r s , e x h i b i t s maxima only a t t h e same wavelengths
a s t h a t of a s i m i l a r p r e p a r a t i o n of USP v i n b l a s t i n e s u l f a t e
RS .
Other i d e n t i f i c a t i o n t e s t : Marquis T e s t ( s u l f u r i c a c i d -
formaldehyde) g i v e s wine-red c o l o r w i t h v i n b l a s t i n e (103).
9.2 T i t r i m e t r i c Determinations
Non-Aqueous T i t r a t i o n s
Q u a n t i t a t i v e d e t e r m i n a t i o n of t h e a l k a l o i d s i n c l u d i n g
v i n b l a s t i n e i n d r u g s and e x t r a c t s a r e r e p o r t e d as f o l l o w s
( 104).
A e r i a l p a r t s of Vinca r o s e a were ground, t r e a t e d w i t h 25%
NH40H f o r 30 minutes, and e x t r a c t e d with MeOH. The e x t r a c t
was evaporated and t h e r e s i d u e was d i s s o l v e d i n 2% H2SO4 on
a w a t e r b a t h . The H2S04 e x t r a c t was a l k a l i n i z e d with NH40H,
r e e x t r a c t e d w i t h CHC13, t h e e x t r a c t was d r i e d , and evapora-
t e d . The r e s i d u e was d i s s o l v e d i n HOAc and t i t r a t e d w i t h
0 . 1 ~ ~ 1 0 4 .
A e r i a l p a r t s of v i n c a minor were t r e a t e d w i t h 25% NH40H f o r
30 minutes, e x t r a c t e d with CHC13, t h e c o n c e n t r a t e d e x t r a c t
was r e e x t r a c t e d with 2% t a r t a r i c a c i d a d j u s t e d t o pH 9 . 0
with 25% NH40H, and a l k a l o i d s were e x t r a c t e d w i t h CHC13,
d i s s o l v e d i n HOAc and t i t r a t e d w i t h H C l O 4 by u s i n g c r y s t a l
violet indicator.
- A mixture of v i n b l a s t i n e s u l f a t e and t e s t o l a c t o n e i s d e t e r m i -
ned by p r e c i p i t a t i o n a t pH 3.7 w i t h a measured volume o f Na
t e t r a p h e n y l b o r a t e s o l u t i o n , and t i t r a t i o n of unconsumed r e a -
gent with hexadecylpyridinium c h l o r i d e u s i n g bromophenol
b l u e a s an i n d i c a t o r ( 1 0 5 ) .
9.3 Voltametric Determination
Ultrasensitive voltammetric measurements based on

coupling hydrogen catalytic systems with controlled inter-
facial accumulation of the catalyst has been reported (106).
By combining adsorptive stripping voltammetry with a cata-
lytic hydrogen process, trace Pt can be measured with a
detection limit of -1X10-12M. The supporting electrolyte
(1.08 M H2SO4) contains 0.04% (wt./vol.) formaldehyde and
0.001% (wt./vol.) hydrazine. The adsorption of the Pt-
formazone complex (the catalyst) results in a well-defined
catalytic H peak at - 0.92 V, with a peak half-width of
60 mV. The peak height increases rapidly with increasing
preconcentration time, indicating a large enhancement of
the complex on the surface of the hanging Hg drop electrode
(SDS) and gelatin cause serious interference by competing
with the catalyst on adsorption sites. By using 0.25 M
ammoniacal buffer (pH 9 . 3 ) , the vinca alkaloids vinblastine
and vincristine can be determined at subnanomol levels (106)
9.4 SDectroDhotometric Determinations
9.4.1 UV Spectrophotometry
The official methods for determination of vinblastine

sulfate are UV spectrophotometric techniques(6,8).
The BP ( 6 ) recommends the following procedure:
Dissolve 10 mg of vinblastine sulfate in sufficient
methanol to produce 500 ml and measure the absorbance
of the resulting solution at the maximum at 267 nm.
Calculate the content of C46H58N409, H2SO4 taking 185
as the value of A (1%, 1 cm) at the maximum at 267 nm.
The USP (8) recommends the following procedure:
Dissolve about 5 mg of vinblastine sulfate, accurately
weighed in methanol and dilute quantitatively and step-
wise with methanol to obtain a solution containing
about 20 pg per ml on the dried basis.
Dissolve an accurately weighed quantity of USP Vinblas-
tine Sulfate RS in methanol and dilute quantitatively
and stepwise with methanol to obtain a standard solu-
tion having a known concentration of about 20 pg per
ml on the dried basis. Concomitantly determine the
absorbances of both solutions in 1-cm cells at the
wavelength of maximum absorbance at about 267 nrn, with
a suitable spectrophotometer, using methanol as the
blank.
Calculate the quantity in mg of C46H58N40g. H2S04 in
the portion of vinblastine sulfate taken by the formula
0.25 C (Au/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 pg

p e r m l of USP V i n b l a s t i n e S u l f a t e RS i n t h e S t a n d a r d
s o l u t i o n and Au and As a r e t h e absorbances of t h e s o l u -
t i o n o f v i n b l a s t i n e s u l f a t e and t h e Standard s o l u t i o n
respectively.
The USP r e q u i r e s t h a t t h e weighings t o be performed r a p i -
d l y and w i t h a minimum of exposure of t h e s u b s t a n c e t o a i r .
9.4.2 C o l o r i m e t r i c Determinations
A c o l o r i m e t r i c method was f i r s t deviced f o r t h e a s s a y of

p u r e v i n b l a s t i n e s u l f a t e ( 107). The method depends on
t h e f o r m a t i o n of a deep r o s e c o l o r upon h e a t i n g v i n b l a s -
t i n e s u l f a t e a t 80' w i t h a r e a g e n t c o n s i s t i n g of p y r i -
d i n e (35 ml) , c o n c e n t r a t e d s u l f u r i c a c i d (1 m l ) and
a c e t i c anhydride ( 3 5 ml) c o n t a i n i n g 0.05% a c e t y l c h l o r i d e .
The c o l o r so produced i s measured a t 574 nm.
The s e n s i t i v i t y i s 5 t o 7 0 pg of v i n b l a s t i n e s u l f a t e p e r
ml .
To check p u r i t y o f t h e sample, t h e absorbances of t h e
c o l o r produced a r e measured i n 1 cm c e l l s a t 574 and
538 nm a g a i n s t w a t e r a s a r e f e r e n c e . The r a t i o of A574
nm/A538 nm should b e i n t h e range of 1.20 - 1.25 ( 1 0 7 ) .
Another c o l o r i m e t r i c method h a s been r e p o r t e d 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 v i n b l a s t i n e and l e u r o s i n e
i n Vinca r o s e a ( 1 0 8 ) .
The s e q u e n t i a l s t a g e s used were - ( i ) s e l e c t i v e e x t r a c -
t i o n o f t h e a l k a l o i d s i n t o benzene o r t o l u e n e ( w e t t i n g
t h e raw m a t e r i a l with aq. 5% Na a c e t a t e improved t h e
e x t r a c t i o n ) , ( i i ) b a c k - e x t r a c t i o n of t h e a l k a l o i d s i n t o
2% c i t r i c o r t a r t a r i c a c i d , ( i i i ) adjustment of pH t o
6 . 0 w i t h aq. 5% NH3 and r e - e x t r a c t i o n of t h e a l k a l o i d s
i n t o t o l u e n e , ( i v ) s e p a r a t i o n o f t h e a l k a l o i d s on LH-20
with methanol - CHC13 ( 7 : 3 ) , s e p a r a t i o n of t h e d i m e r i c
a l k a l o i d f r a c t i o n on s i l i c a g e l , with CHC13 - benzene -
a c e t o n e - e t h y l a c e t a t e - methanol (20:20:15:5:3) ( v i )
e l u t i o n of t h e a l k a l o i d s w i t h 1%H C 1 , and ( v i i ) a d d i t i o n
of 0.2 m l of 1%t r o p a e o l i n 000-1 (C.I. Acid Orange 20)
and t h e n CHC13. The c o l o r so produced i s measured a t
490 nm.
644 FARlD J . MUHTADl AND ABDUL FATTAH A. A. AFlFY
9.5.1 Paper Chromatography
Clarke ( 1 0 3 ) described the following system for the

identification of vinblastine.
System : Reversed phase, Whatman no. 1 or no. 3 paper
chromatography, impregnated by dipping in 10% solu-
tion of tributyrin in acetone and drying in air.
Sample : A 5 1-11 of 1 to 5% solution in ethanol o r
chloroform.
Solvent : Acetate buffer (pH 4.58).
Location : Under UV light gives purple fluorescence.
Location spray : Iodoplatinate.
Rf value : 0.10.
The following TLC systems were recommended for the

identification and separation of vinblastine.
i
Chromatograni Solvent System Rf value Ref.
1
~~
1. Silica gel G, 250 methanol-strong

1-1m thick, dipped ammonia (100:l.S)
in or sprayed with Cyclohexane-toluene-
0.1 M KOH in diethylamine (75:15:10) 0.10
methanol and dried chloroform-methanol
(90:10) Oe60
0.60 (9
2. Silica gel Gf 254 toluene-chloroform-
diethylamine (80:40:6) - (6)
3 . Silica gel G ethylacetate-absolute
alcohol (3 :1) 0.21 (1091
4. Silica gel G ethylacetate-absolute
alcohol ( 1 :1) 0.33 (110)
5. Alumina ethylacetate-absolute
alcohol ( 3 : 1) 0.66 (110)
6. Silica Gel G n-butanol-acetic acid-
water (4:1 :1) 0.19 (109)
7. Silica Gel G methano 1 0.46 (109)
8. Silica gel G chloroform-methanol
(95 :5) 0.24 (111)
9. Alumina chloroform 0.17 (110)
10. Alumina chloroform-ethyl-
acetate (1:l) 0.25 (110)
Detection : The spots can be detected by:

1- Under short UV light ( 2 5 4 nm)
2- Spraying with:
a) Dragendorff's reagent ( 103)
b) Acidified iodoplatinate solution (103)
s) 1% Ceric ammonium sulfate in 85% phosphoric
acid (110)
-A double development technique was recommended f o r the

separation of vinblastine from the other dimeric cath-
aranthus alkaloids on alumina layers(ll2).
The loaded chromatoplates were first developed in the
solvent ethylacetate, then after drying, a second deve-
lopment in ethylacetate-absolute alcohol (3: 1) was carried
out. Vinblastine in this technique gave Rf value of 0.73
( 112).
- Two dimensional TLC technique was reported for the separa-
tion of more complex mixture of catharanthus alkaloids (115) .
.- The BP (6) adopted a TLC technique to test for the pres-
ence of related alkaloids in vinblastine sulfate sample
(checking the purity of the sample):
TLC chromatoplates are coated with silica gel GF 254. 5r.tl
of the following three solutions in methanol are separately
applied to one chromatoplate.
1- 1.0% w/v of the substance being examined.
2 - 0.02% w/v of standard vincristine sulfate BPCRS.
3- 1.0% w/v of standard vinblastine sulfate BPCRS.
The chromatoplate is then developed in the solvent toluene-
chloroform-diethylamine (80:40:6). After development, the
plate is allowed to dry in air and examined under UV light
(254 nm). Any secondary spot in the chromatogram obtained
with solution (1) is not more intense than the spot obta-
ined with solution (2).
- A TLC - Densitometric determination of vinblastine was
described. Two dimensional TLC is performed followed by
densitometric scanning of the spots at 289 nm. The C O -
efficient variation of the method was 7.5% ( 114).
9.5.3 Gas Liquid Chromatography (GLC)
The following GLC system has been reported for the identi-
fication and separation of vinca alkaloids including vin-
blastine ( 115).
Column condition: Glass column (lm x 3.2mm), precoated
with hexamethyldisilazane and packed with 3% OV-101 on
Gas chrom Q (80-100 mesh), with temperature programmed
from 200' to 300' at 5' min.-l
Carrier gas: Nitrogen at a flow rate of 30 ml/min.-l
Detection: F.1.D
Condition: Vinca alkaloids including vinblastine were
derivatized before application by heating for 5 minutes
at room temperature with t r i f l u o r o b i s - ( T r i m e t h y l s i l y l )
acetamide-pyridine (1 : 1).
9.5.4 High Performance Liquid Chromatography (HPLC)
Several HPLC methods have been employed to determine

vinblastine and its metabolites in biological fluids
and tissues. Some of these methods are as follows:
System 1: The following system has been recommended
for quantitative determination of vinblastine
and other vinca alkaloids in plasma and urine
( 116).
Conditions:The drugs are extracted from biological mate-
rials by an ion pair extraction with sodium
octylsulfate as counter ion at pH 3.0. The
extracts are injected onto a reversed-phase
system with a cyano column as stationary phase.
Mobile MeCN -phosphate buffer pH 3.0 (65:35).
phase :
System 2: The following system is a reversed-phase with
electrochemical detection. It is employed for
quantitative determination of vinblastine and
its metabolites in plasma and urine. Quantifi-
cation of substances in human plasma and urine
is possible down t o 1 ng/ml ( 117).
Column : Hypersil ODs.
Mobile Methanol - 10m M phosphate buffer pH 7.0.
phase :
System 3: This system is employed for the analysis of

Catharanthus alkaloids including vinblastine
(118).
Column: A stainless steel (25cm x 4mm), packed with
Li-Chrosorb RP-8; operated at ambient temp-
erature.
Mobile 0.01M ammonium carbonate-acetonitrile (53:47).
phase :
Flow rate: 1.5ml min.-l
Retention 1 2 . 3 7 minutes forvinblastine.
time :
Detection: UV at 298 nm.
System 4 : This system has been employed f o r the separa-
tion, detection and correlation of plate
height and molecular weight of vinblastine
and other Vinca alkaloids (119).
Column: 25cm x 4.6mm, packed with R SiL C1g HL-D
octadecyl-silica gel.
Mobile Gradient elution with aqueous 50 to 85%
phase : methanol containing 0.1% ethanolamine.
Flow rate: 2 ml min.-l
Detection: UV at 290 nrn.
System 5: The following reversed-phase system has been
used for the analysis of Catharanthus alkaloids
including vinblastine by thermospray liquid
chromatography-mass spectrometry (120).
Column: p Bondapak C18 (30cm x 3.9mm), reversed-phase
column.
Mobile Isocratic solvent, 0.1M ammonium acetate
phase : (pH 7.2) - MeCN (51:49).
Flow rate: 1 ml min.-l
Detection: Electrochemical and UV (The limit o f detection
being 4 ng/injection €or each alkaloid).
System 6 : The following reversed-phase system has been
reported f o r the determination of vinblastine
and other alkaloids of Catharanthus roseus
leaves ( 121).
Column: p Bondapak Cis.
Mobile 0.1M diammonium hydrogen orthophosphate - MeCN
phase : (25:75), pH 7.0.
Detection: UV at 254 and 280 nm.
648 FARID J . MUHTADI AND ABDUL FATTAH A. A. AFIFY
System 7: This reversed-phase system i s d e s c r i b e d f o r

t h e d e t e r m i n a t i o n o f v i n b l a s t i n e and o t h e r
Catharanthus a l k a l o i d s . 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 , t h e l i m i t of d e t e c t i o n and
t h e r e c o v e r y were 1.63-3.52%, 1 0 ug/ml and
96.6-102% r e s p e c t i v e l y ( 1 2 2 ) .
Column: A c a r t r i d g e column packed w i t h Spheri-5RP.
Mobile 2 g r a d i e n t systems c o n t a i n i n g MeOH, MeCN,
phase : 0.025M ammonium a c e t a t e and t r i e t h y l a m i n e
i n different ratios.
Internal 5-Methoxytryptamine.
standard :
D e t e c t i o n : UV a t 280 and 254 nm.
System 8 : This i s a l s o a r e v e r s e d - p h a s e system which
has been a p p l i e d f o r s e p a r a t i o n and q u a n t i t a -
t i o n o f a l k a l o i d s from c e l l suspension c u l t u -
res of Catharanthus r o s e u s i n c l u d i n g v i n b l a s -
t i n e (123).
Column: 1.1 Bondapak C18.
Mobile A mixture of methanol and (NH4)2HP04 i n w a t e r .
phase :
D e t e c t i o n : UV a t 298 nm.
Other HPLC systems f o r v i n b l a s t i n e have a l s o been r e -
p o r t e d (124-127).
9.6 Radioimmunoassav Methods
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 v i n b l a s t i n e i n t i s s u e c u l -
t u r e s of Catharanthus r o s e u s by radioimmunoassay was
r e p o r t e d ( 128).
Antibody was o b t a i n e d by t h e immunization of r a b b i t s a g a i -
n s t a c o n j u g a t e of v i n b l a s t i n e w i t h bovine serum albumin.
The a n t i b o d y had a h i g h a f f i n i t y (Ka = 1.2 x 109 L/mol)
and s p e c i f i c i t y f o r v i n b l a s t i n e . The u s a b l e range o f
s t a n d a r d curve f o r a s s a y was 0.5-10 ng/ml. Crude a l k a l o i d
e x t r a c t s of t i s s u e c u l t u r e s c o u l d be assayed and many sam-
p l e s could be p r o c e s s e d i n one time. The v i n b l a s t i n e con-
t e n t s o f m u l t i p l e shoot c u l t u r e s were lower t h a n t h a t of
i n t a c t p l a n t s b u t much h i g h e r t h a n t h a t of c a l l u s c u l t u r e s .
Another Radioimmunoassay method was r e p o r t e d f o r v i n b l a s -
t i n e and v i n c r i s t i n e a s f o l l o w s ( 1 2 9 ) : Radioimmunoassay
developed f o r d e t e r m i n i n g t h e neoplasm i n h i b i t o r s v i n b l a s -
t i n e ( I ) and v i n c r i s t i n e (11) i n blood i n v o l v e s t h e u s e of
antiserum r a i s e d i n a r a b b i t immunized with ( I ) bovine
serum albumin c o n j u g a t e . D e t e c t i o n l i m i t s (ng ml-1) o r

2 . 1 f o r ( I ) and 3.8 f o r (11) w i t h u s e o f t r i t i a t e d ( I )
under n o n - e q u i l i b r i u m a s s a y c o n d i t i o n s . The a n t i s e r u m
showed no c r o s s - r e a c t i v i t y w i t h 25 o t h e r a l k a l o i d s and
c y t o t o x i c drugs used t h e r a p e u t i c a l l y i n combination w i t h
( I ) and (11).
A t h i r d method o f Radioimmunoassay for Vinca a l k a l o i d s
v i n b l a s t i n e and v i n c r i s t i n e was r e p o r t e d ( 130) a s f o l l o w s :
A n t i s e r a were r a i s e d i n r a b b i t s by immunisation a g a i n s t
compounds p r e p a r e d by c o u p l i n g c a r b o x y l i c a c i d d e r i v a t i -
v e s of v i n b l a s t i n e and v i n c r i s t i n e t o human serum albumin.
For a s s a y , a n t i s e r u m was i n c u b a t e d with t h e sample, e.g.
p l a n t e x t r a c t and t h e a p p r o p r i a t e t r i t i a t e d a l k a l o i d f o r
1 h a t 37', and t h e m i x t u r e was allowed t o r e a c t w i t h a
goat a n t i - r a b b i t serum o r w i t h polyoxyethylene g l y c o l
o v e r n i g h t a t 4', and t h e n c e n t r i f u g e d ; t h e p r e c i p i t a t e
was d i s s o l v e d i n NaOH s o l u t i o n f o r s c i n t i l l a t i o n c o u n t i n g .
E i t h e r compound could be determined i n amounts down t o <1
pmol. The s p e c i f i c i t y o f t h e a n t i s e r a i s d i s c u s s e d ; t h a t
r a i s e d a g a i n s t v i n c r i s t i n e bound t h i s a l k a l o i d 200 times
more e f f e c t i v e l y t h a n it bound v i n b l a s t i n e . S e v e r a l o t h e r
compounds, i n c l u d i n g a n t i n e o p l a s t i c d r u g s , showed no c r o s s -
r e a c t i v i t y . The a s s a y s were a p p l i e d t o t h e blood o f rabb-
i t s i n j e c t e d w i t h t h e d r u g s , and a l s o t o e x t r a c t s o f Vinca
r o s e a a f t e r p r e l i m i n a r y f r a c t i o n a t i o n by HPLC.
A s e n s i t i v e radioimmunoassay method f o r v i n b l a s t i n e and
v i n c r i s t i n e was a l s o r e p o r t e d a s f o l l o w s ( 131 ) :
Antiserum which was used was r a i s e d i n r a b b i t s a g a i n s t a
4 - d e a c e t y l v i n b l a s t i n e carboxazide-bovine serum albumin
c o n j u g a t e ; [3H] - v i n c r i s t i n e o r r3H] - v i n b l a s t i n e was used
as r a d i o - l i g a n d . Rates of b i n d i n g of v i n c r i s t i n e and
v i n b l a s t i n e t o t h e a n t i s e r u m were s i m i l a r . A f t e r incuba-
t i o n f r e e and bound l i g a n d were s e p a r a t e d w i t h u s e o f a
dextran - charcoal suspension; a f t e r c e n t r i f u g a t i o n t h e
a c t i v i t y of t h e s u p e r n a t a n t s o l u t i o n was measured by l i q u i d
s c i n t i l l a t i o n c o u n t i n g . S e n s i t i v i t y was improved by a
s e q u e n t i a l s a t u r a t i o n procedure ( i n c u b a t i o n w i t h u n l a b e l -
l e d d r u g , followed by i n c u b a t i o n with r a d i o l i g a n d ) . O f
t h e drugs t e s t e d , only bleomycin ( > 0 . 1 u n i t ) i n t e r f e r e d .
650 FARlD J. MUHTADI AND ABDUL FATTAH A. A. AFlFY
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ACKNOWLEDGEMENT
The a u t h o r would l i k e t o thank Mr. Uday C . Sharma,

Dept. o f Pharmacognosy, College of Pharmacy, Riyadh,
Saudi Arabia f o r h i s v a l u a b l e and s i n c e r e e f f o r t s i n
t y p i n g t h i s manuscript.
TITANIUM DIOXIDE
Harry C. Brittain, Gary Barbera,
Joseph DeVincentis, and Ann W. Newman
Bristol-Myers Squibb Pharmaceutical Research Institute
Bristol-Myers Squibb Company
New Brunswick, NJ 08903

AND EXClPlENTS - VOLUME 21 659 All rights of reproduction reserved in any form.
660 HARRY G . BRlTTAlN ET AL.
CONTENTS
1. Description
1.2 Appearance
1.3 General Chemical properties
1.4 Uses and Applications
2. Method of Preparation
3.1 Particle morphology
3.2 Crystallographic Properties
3.3 Thermal methods of analysis
3.4 Particle size distribution
3.5 Surface area
3.6 Density
3.7 Powder Flow characteristics
3.8 Hygroscopicity
3.9 Solubility
3.10 Spectroscopy
4.1 Compendia1 Tests
4.2 Identification
4.4 Spectrophotometric Methods of Analysis
4.5 Thin Layer Chromatographic Methods of Analysis
4.6 Gas-Liquid Chromatographic Methods of Analysis
4.7 High Performance Liquid Chromatographic
Methods of Analysis
5. Stability
5.1 Stability
5.2 Incompatibilities with functional groups
6. References
TITANIUM DIOXIDE 66 1
1. Description
1.1 Name. Formula. and Molecular Weight
Titanium dioxide is the most stable oxide of titanium, and can be

obtained from either natural or synthetic sources. The material exists
naturally in three crystal modifications, known as rutile, anatase, and
brookite [I].
The Chemical Abstracts identification number is CAS- 13463-67-7. In

the United States, it is identified in the Color Index as 77891, and is
denoted as C.I. Pigment White No, 6. It is also identified as EEC No.
E 171 [2].
The chemical formula is TiO,, which corresponds to a formula weight

of 79.90 Daltons. The elemental composition is Ti 59.95% and 0
40.05% .
1.2 Appearance
Naturally occurring titanium dioxide may appear red, or reddish brown

to black. The color is normally due to the presence of iron, chromium,
or vanadium contamination, which may amount to 10% of the total
titanium content [3].
Synthetically purified titanium dioxide exists as a white, odorless,

tasteless powder.
L3 General Chemical Properties
A very detailed description of the chemistry associated with titanium

oxygen systems is available [4]. The material is very thermally stable,
and extremely resistant toward chemical degradation. It can be partially
reduced when heated in the presence of hydrogen or carbon monoxide,
the products being either lower oxides or mixtures of titanium carbide
and lower oxides. Reduction by active metals (Na, K, Ca, or Mg) can
662 HARRY G . BRIITAIN ET AL.
only be partially effected, and chlorination of the oxide phase is

possible only in the presence of a reducing agent.
The reactivity of titanium dioxide toward acids is very dependent on the

temperature of the reaction mixture. It may be slowly dissolved by
boiling concentrated sulfuric acid, with the dissolution rate being
promoted by the addition of ammonium sulfate. Titanium dioxide may
be readily dissolved by hydrofluoric acid. The material is completely
insoluble in aqueous alkalies, but is readily dissolved in molten sodium
(or potassium) hydroxide, carbonate, or borate. An equimolar molten
mixture of sodium carbonate and sodium borate is partially effective as
a dissolution medium.
The most important use of titanium dioxide is as a white pigment,

owing to its very high reflectance at visible and ultraviolet wavelengths
[ 5 ] . The refractive index of this material is so extremely high that fine
particles scatter light with almost total efficiency. At the same time,
the films are almost totally opaque. The ability of industry to produce
titanium dioxide in appropriate particle size ranges has made it the most
important white pigment in existence.
In ointments or lotions, titanium dioxide is a very efficient reflector of

sunlight [6]. Its ability to act as a sunblock has led to its widespread
use as a protective agent toward sunburn.
2. Method of Preparation
Although titanium dioxide can be obtained naturally as one of three

crystal polymorphs (anatase, rutile, or brookite), pharmaceutically
acceptable material is produced synthetically.
The bulk of pure titanium dioxide is obtained from purification of the

abundant ore, illmenite (FeTiO,), using the su&m process [7]. In this
TITANIUM DIOXIDE 663
procedure, the raw ore is first digested with concentrated sulfuric acid.
The product of this step is termed the "sulfate cake", which is then
leached with water to produce a mixture of FeSO,, Fe;?(SOJ,, and
TiOSO,. At this point, scrap iron is used to reduce all Fe(IT1) to
Fe(II), whereupon the FeSO, is removed by filtration. The TiOSO,
solution is boiled to hydrolyze the solute into a suspension of hydrated
TiO,. This material is filtered, and finally calcined at 800-900°C to
produce the final product. When calcined at 8OO0C,the hydrous oxide
normally converts to the anatase phase.
The other important process for production of titanium dioxide is

termed the chloride process [7]. The raw material used in this process
is natural rutile, which is first heated at 950°C in the presence of
carbon (in the form of coke) and chlorine. This produces crude TiCI,,
and this product is heated at 1OOO"C in the presence of oxygen to
produce the final titanium dioxide product. Under these conditions, the
final product is the rutile phase.
The chloride process accounts for over half of the TiOz production in
the United States, and is the most economical when high-grade ores are
available. The sulfate process cannot use rutile as its starting material,
but is able to make use of low-quality ores (or slags remaining after
iron processing is complete) as input materials.
The physical properties of the titanium oxide pigments can be further

improved by slurrying the products in water, and then selectively
precipitating a surface coating of either S i 9 , A1203, or TiO, itself on
the fine particles.
A concise summary of the physical properties of titanium dioxide is

available in the Handbook of Excipients [S]. Also summarized in this
publication are commercial availability, methods of manufacture, and
various pharmacopeial specifications.
664 HARRY G. B R I T A I N ET AL
For the present work, three representative samples of U.S.P. grade

titanium dioxide were characterized. Two of these were obtained from
Warner Jenkinson, and were described as being either oil ("Atlas
White" material, lot 402239) or water ("Kowett" material, lot 401398).
The third lot was obtained from Spectrum Chemical Co., and was also
certified to be U.S.P. grade (lot HB098). In addition to these U.S.P.
materials, a non-U.S.P. grade of titanium dioxide (obtained from
Johnson Matthey, lot MTI50W) was also studied as part of the
crystallographic characterization.
Photomicrographs of the titanium dioxide samples are shown in Figures

1-4. All materials were found to exist as aggregate species which were
built up from the consolidation of exceedingly fine subparticles. When
viewed at 2O,OOOx, the subparticles of the U.S.P. materials are
uniformly round in nature, and appear to be approximately 100 nm in
diameter. No difference in the subparticle size was noted for materials
prepared for different applications, or obtained from alternate vendors.
This would indicate that the subparticle size was determined by the
method of manufacture. The aggregates formed from these subparticles
were very compact in nature, indicating efficient close-packing of the
subparticles. The particle size of the aggregate species did vary among
vendors, and this difference will be discussed in a different section.
The subparticles of the non-U.S.P. grade material were found to be

much coarser. The size distribution of these was also quite variable,
but averaged approximately 1 pm in diameter. These subparticles were
also spherical in their appearance, and the aggregate species formed
from these appeared to be looser than those of the U.S.P. materials.
Titanium dioxide is known to crystallize in three polymorphic forms,

anatase, brookite, and rutile. While the rutile and anatase polymorphs
are commonly encountered, the brookite phase is quite rare. The
Figure 1. Scanning electron photomicrographs of U.S.P. grade

titanium dioxide ("Atlas White" material, Warner
Jenkinson), obtained at 500x (upper photo) and 3 0 0 0 ~
(lower photo).
666 HARRY G . BRIlTAIN ET AL.
Figure 2. Scanning electron photomicrographs of U.S.P. grade

titanium dioxide ("Kowett" material, Warner Jenkinson),
obtained at 500x (upper photo) and 3OOOx (lower photo).
Figure 3. Scanning electron photomicrographs of U . S .P. grade

titanium dioxide (Spectrum Chemical Company),
obtained at 505x (upper photo) and 3OOOx (lower photo).
668 HARRY G.BRI'ITAIN ET AL.
Figure 4. Scanning electron photomicrographs of non-U .S.P. grade

titanium dioxide (Johnson Matthey), obtained at SoOx
(upper photo) and 3000~(lower photo).
relative stabilities of rutile and anatase are almost equivalent, but it

appears that rutile is the most stable polymorph of the two. The crystal
structures of these three polymorphs are well known, and the important
properties summarized in Table I 191.
Although anatase and rutile are both tetragonal, they are not
isomorphous. Anatase is usually obtained in near-regular octahedra,
which has given it the alternate name of octahedrite. Rutile is found as
slender prismatic crystals, which often grow as twins.
The rutile structure has been discussed in great detail, since it is

commonly taken as one of the prototype crystal structures [9]. The unit
cell is depicted in Figure 5. The structure consists of chains of TiO,
octahedra, in which each octahedron shares a pair of opposite edges and
vertices with neighboring octahedra. Another way to envision the
overall structure is to consider it as a slightly distorted hexagonally
close packed array of oxygen atoms with half the octahedral interstices
being occupied by titanium atoms.
Powder x-ray diffraction can be used to easily differentiate between the

polymorphs of titanium dioxide. For the anatase phase, the most
intense diagnostic scattering peaks correspond to d-spacings of 3.52 and
1.89 A, with relative intensities of 100:4. For rutile, the most intense
scattering peaks correspond to d-spacings of 1.69, 3.26, and 2.49 A,
and exhibit relative intensities of 100:97:70. Although brookite is
never encountered in synthetically produced samples of titanium
dioxide, its three most intense scattering peaks would correspond to d-
spacings of 3.47, 2.90, and 1.88 A (relative intensities of 100:85:75).
The U.S.P. grade of titanium dioxide most commonly marketed is the

anatase phase. The x-ray powder pattern for this material is shown in
Figure 6, while the scattering angles, d-spacings, and relative intensities
are found in Table 11. For comparison purposes, a powder pattern
obtained for the rutile phase (the non-U.S.P. material described earlier)
is shown in Figure 7, and its corresponding crystallographic information
is presented in Table 111.
670 HARRY C . B R I T A I N ET AL.
Table I
Crystallographic Data for the Three Polymorphs of Titanium Dioxide
Property Anatase Brookite Rutile
Crystal system orthorhombic tetragonal tetragonal
Space group 14, Pcab P4/mnm
Number Ti02 in
unit cell 4 8 2
Cell dimensions (nm)

a 0.3785 0.5456 0.4594
b 0.9182
C 0.9514 0.5143 0.2962
Cell volume
(mL x i d 4 ) 136.3 257.6 62.5
anisotropic refractive indices

"1 2.554 2.583 2.616
n2 2.586
n3 2.493 2.741 2.903
density (glmL) 3.893 4.1 19 4.245

TITANIUM DIOXIDE 67 I
Figure 5 . Structure of the unit cell of titanium dioxide, rutile

phase.
@-
/ I
0
I
I
I
I
I
I
- l r\ I
I I
Oo @ Ti
612 HARRY G.BRITTAIN ET AL.
Figure 6. Powder x-ray diffraction pattern of titanium dioxide,

anatase phase. The intensity scale is presented in
arbitrary units.
0.0 10 .a 20.0 30.0 40.0 50.0

4
60.0
1 70.0
Degrees 2-8
Table II
Crystallographic Data for the 10 Most Intense Scattering Peaks of

Titanium Dioxide, Anatase Phase
Angle D-Spacing Relative Intensity

(degrees 2-0) (Angstroms) wnax)
25.2625 3.5226 100.00

36.9150 2.4330 7.12
37.7600 2.3805 27.59
38.5500 2.3335 8.41
48.0100 1.8935 42.40
53.8500 I .7011 28.61
55.0200 I .6677 28.32
62.6150 1.4824 23.58
62.82OO 1.4817 11.98
68.6800 I .3655 11.14
674 HARRY G . BRI'ITAIN ET AL.
Figure 7. Powder x-ray diffraction pattern of titanium dioxide,

rutile phase. The intensity scale is presented in arbitrary
units.
0.0 10.0 20.0 30 .O 40.O 50 .O 60.O 70.0
Degrees 2-8
Table 111
Crystallographic Data for the 1 1 Most Intense Scattering Peaks of

Titanium Dioxide, Rutile Phase
Angle D-Spacing Relative Intensity

(degrees 2-8) (Angstroms) (1 4naJ
27.3025 3.2638 97.07

36.0200 2.4914 69.69
39.0275 2.3061 4.64
41.1400 2.1924 37.65
43.8625 2.0624 10.34
54. I700 1.6918 100.00
56.3075 1.6325 23.69
62.7175 1.4802 23.69
63.8100 1.4575 13.60
68.7850 1.3637 37.29
69.7850 1.3466 30.43
616 HARRY G . BRIITAIN ET AL
The rutile polymorph of titanium dioxide is refractory, and melts

around 1850°C. Melting points for the anatase and brookite
polymorphs have not been established, since these materials convert to
the rutile phase at elevated temperatures.
3.4 Particle size distribution
The particle size distributions of the three U.S.P. titanium dioxide

samples were obtained using optical microscopy and image analysis.
This method provides information on the size of the aggregate species
only, and does not contain any data pertinent to the subparticles from
which the aggregates are composed. Full particle size distributions for
these three Ti@ lots are provided in Table IV. The particle diameters
were obtained from analysis of the particle cross-sectional areas
(provided by the image analysis system). All materials were found to
be composed of very small particles, the basic units of which were
smaller than 10 pm. The average particle size (shown in Table V) of
the two Warner Jenkinson lots were equivalent, while the material
obtained from Spectrum Chemical Co. was slightly coarser in nature.
32 surface a r a
The surface area of the three titanium dioxide lots was determined using
a five-point B.E.T. analysis procedure, and the results of this study are
found in Table V. The three materials exhibit fairly high surface areas
(approximately 10 m2/g), and were found to be mutually equivalent.
The true density (measured by helium pycnometry) of titanium dioxide

differs with the polymorphic state of the material. Rutile is the most
dense (4.25 g/mL), followed by brookite (4.12 g/mL) and anatase (3.89
g/mL).
Table IV
Particle Size Distributions for Various Titanium Dioxides, Obtained

Using Optical Microscopy (aggregate species having diameters larger
than 10 pm have been excluded)
Band Size
(Pm) 401398 402239 HB098
0.0 - 0.5 12.7 9.6 8.0

0.5 - 1.0 45.4 52.2 15.4
l . 0 - 1.5 26.5 26.7 14.1
1.5 - 2.0 8.2 5.4 14.6
2.0- 2.5 4.0 3.6 14.9
2.5 - 3.0 l .3 1.1 7.5
3.0- 3.5 1.1 0.0 8.8
3.5 - 4.0 0.0 0.5 5.3
4.0- 4.5 0.5 0.0 4.8
4.5 - 5.0 0.0 0.4 1.6
5.0 - 5.5 0.0 0.2 0.5
5.5 - 6.0 0.3 0.0 1.9
6.0 - 6.5 0.0 0.0 1.1
6.5 - 7.0 0.0 0.2 0.8
7.0 - 7.5 0.0 0.0 0.8
7.5 - 8.0 0.0 0.0 0.0
678 HARRY 0. BRITTAIN ET AL.
Table V
Micromeritic Properties Obtained for Various Titanium Dioxides
property 401398 402239 HB098
Average Particle
Size (pm) 1.05 1.02 2.19
Surface Area
(m2/s) 10.5 8.2 9.0
Bulk Density
(g/mL) 0.4 0.4 0.5
Tap Density
0.7 0.6 0.8
Compressibility
39 37 35
The bulk densities of the commercially supplied U.S.P. grade materials

were found to average around 0.4 g/mL (Table V). The tap densities
of these lots were found to increase to approximately 0.7 g/mL,
corresponding to a compressibility factor of approximately 37%. No
significant difference among the three lots studies was evident when
comparing the micromeritic properties.
3.7 HVgroscoDicin!
Titanium dioxide is not hygroscopic, and does not form true hydrate
phases. It is possible to prepare hydrated titanium oxide materials
through the addition of alkali-metal hydroxides to a solution of a Ti(I1)
or Ti(II1) salt. The resulting titanium hydroxide precipitate is extremely
unstable, is a powerful reducing agent, and rapidly converts to a
hydrated oxide material [4].
If the precipitation is performed at room temperature, one obtains a

compound known as orthotitanic acid, and which has the approximate
formula of Ti0;2H20’Ti(0H),. If the suspension is boiled, or if the
precipitation is effected from a hot solution, a compound known as
metatitanic acid is obtained. This less hydrated oxidic compound has
the approximate formula of TiOz-H20TiO(OH)2,Metatitanic oxide is
commonly obtained in the colloidal state, and is the preferred
intermediate in the manufacture of titanium dioxide pigments.
U Solubility
Titanium dioxide is completely insoluble in water, dilute acids, or

common organic solvents. It can be dissolved in concentrated sulfuric
or hydrofluoric acids at elevated temperatures, with the accompanying
production of salt species.
322 Spectroscopy
Titanium dioxide transmits through the visible and near infrared regions
680 HARRY G. BRlTTAlN ET AL.
of the spectrum, having no absorption bands in these regions. It

becomes completely opa ue at wavelengths below 400 nm, and at
9
energies below 2000 cm- . The reflectivity of TiO, is approximately
90% of that of a MgO standard [4].
4.1 Compendia1 Tests
The U.S.P. compendia1 requirements [ 101 for titanium dioxide are that
it cannot contain less than 99.0% and not more than 100.5% TiO,,
when calculated on a dried basis. In addition, the material may not
contain more than 0.001 % of lead, not more than 2 ppm of antimony,
and not more than 1 ppm of mercury.
The compound is tested as to its identification, loss on drying, loss on

ignition, water-soluble substances, acid-soluble substances, and arsenic
content. A full method is provided for the potency assay. The details
of these tests are as follows:
Identification: The compound is suspended in hot concentrated

sulfuric acid, and diluted with water. Undissolved solid is
filtered off, and a few drops of hydrogen peroxide test solution
are added to the clear filtrate. The positive identification
consists of an orange-red color which develops immediately.
Loss on drying: The general test method <731> is followed.

After being dried at 105°C for 3 hours, the material cannot lose
more than 0.5% of its weight.
Loss on ignition: Following general test method <733 > , the

material is ignited at 800 k 25°C to constant weight. The
material cannot lose more than 0.5% of its weight.
Water-soluble substances: The sample is suspended in water,

mixed, and allowed to stand overnight. Ammonium chloride
test solution is used to clarify the suspension, which is then

filtered. A 100 mL portion of the filtrate is collected, dried,
and ignited to constant weight. The residue cannot amount to
more than 0.25% of the original sample weight.
Acid-soluble substances: The solid is suspended in 0.5 N

HCI, and heated on a steam bath. The suspension is filtered,
with the filtered solids being washed with 0.5 N HCI. These
washings are combined with the original filtrate, dried, and
ignited to constant weight. The residue cannot weigh more than
0.5% of the original sample weight.
Arsenic: The arsenic content is determined according to

general test <211> . The solid is suspended in water, to which
is added appropriate amounts of hydrazine sulfate, potassium
bromide, sodium chloride, and sulfuric acid. Any evolved
arsine is collected, and determined. The limit is 1 ppm.
Assay: The initial sample is dissolved in a mixture of hot

sulfuric acid and ammonium sulfate. After the dissolution is
complete, the mixture is allowed to cool, and diluted with water.
The suspension is then filtered, and neutralized with ammonium
hydroxide. This filtrate is reduced in a Jones reductor (making
use of a zinc amalgam), and then titrated with 0.1 N potassium
permanganate volumetric reagent. IJnder these conditions, each
mL of 0.1 N potassium permanganate reagent is equivalent to
7.988 mg of TiO,.
All identification tests for titanium dioxide first require solubilization of

the oxide, typically by concentrated acid solutions. After production of
aqueous solutions of titanium ions, a number of colorimetric reactions
may be used for identification purposes.
Hydrogen peroxide causes a yellow color to develop in acidic solutions

containing dissolved titanium [111. In solutions containing sulfuric
682 HARRY G . BRITTAIN ET AL.
acid, the color is due to a complex peroxidic anion formed from free
peroxodisulfatotitanic acid. This reaction is the one used for the
cornpendial identification test since it is not interfered with by common
metallic ions,
Several other color reactions may be used for the identification of

titanium dioxide, after solubilization of the material. Pyrocatechol
yields a yellowish-red color with weakly acidified solutions of titanium
salts [12]. When chromotropic acid is reacted with solubilized titanium
between pH 2.5 and 5.0,a wine red colored solution is obtained 1131.
An intense brown color is given by the addition of acidic solutions of
3,5,7,2’,4’-pentahydroxyflavone(morin) to titanium salts, although the
composition of the product is unknown [ 141,
Disodium- 1,2-dihydroxybenzene-3,5-disulfonate(tiron) yields a strong

yellow color when reacted with titanium salts in the pH range of 4.3-
9.6 [15]. Titanium salts also react with 5-sulfosalicylic acid at pH 3-5
to yield yellow solutions [16]. Other reagents which have found limited
use in identification testing are thymol [17], gallic acid [18], and
salicylic acid [ 191.
4J Gravimetric Methods of Analysis
As one of the transition elements, titanium can be readily precipitated

with ammonium, sodium, or potassium hydroxide. The hydrated oxide
is ignited to constant weight at any temperature exceeding 350°C. The
method is not useful for complicated samples containing other cations
which could be precipitated by hydroxide, but is very applicable to the
analysis of U.S.P. grade titanium dioxide.
Titanium salts can also be determined using cupferron (the ammonium

salt of nitrosophenylhydroxylamine) [20] or tannin [21]. In addition,
2’-hydroxy-4’-methylpropiophenoneoxime has been shown to form an
insoluble 1:l complex with Ti salts [22].
-
4.4 Titrimetric Methods of Analysis
Most titrimetric methods for titanium depend on the reduction of Ti(1V)

to Ti(III), followed by subsequent titration with a standard oxidizing
solution [23]. The methods vary with the choice of reductant, the
titrant, and with the method of detecting the endpoint.
The standard dissolution procedure for titanium oxides is the ammonium

sulfate-sulfuric acid mixture developed by Rahm [24]. The most
commonly used method in industry is based on the use of metallic
aluminum as the reductant, and ferric ammonium sulfate as the titrant.
The use of ferric ion as the reagent is preferred, since relatively few
species will interfere with its reaction with reduced titanium solutions.
Other reductor systems can be used, which will yield equally

satisfactory results [25]. These can be the Jones, lead, cadmium, iron,
nickel, or bismuth reactors, with the Jones reactor being chosen for use
in the compendia1 assay method. Liquid mercury amalgams can also be
used as reductors, being prepared with zinc, cadmium, bismuth, lead,
or tin. While the liquid amalgams are easier to handle, and are more
rapid than are column reactors, none of these is as simple as the
aluminum foil reductor.
Besides ferric ammonium sulfate, permanganate solutions can be used to

titrate the reduced titanium. Although many cations interfere with the
permanganate titration, this reagent is still useful in the assay of highly
purified titanium dioxide materials. Potassium dichromate can be used
to titrate Ti(II1) solutions (with the aid of 0.2% indigo as the indicator),
and Ce(1V) reagents can also be used as titrants.
Ethylenediaminetetraacetic acid can be used as a reagent for the titration

of Ti(1V). However, the reaction proceeds slowly, and the Ti(1V)
species tend to hydrolyze during the titration, so that a back-titration
method is necessary to make the complexometric method work properly
[261.
684 HARRY G.BRI7TAIN ET AL.
U Polarographic Methods of AM lysis
It has been demonstrated that the half-wave potential for the reduction
of Ti(1V) to Ti(I1I) is -0.81 V (against the standard calomel electrode)
in O.1M HCI [27]. The further reduction of Ti(II1) to Ti(l1) can be
observed in alkaline media, but this reaction has no useful analytical
significance. In these methods, oxalate, tartrate, or citrate buffer
systems are used as supporting electrolytes to prevent the hydrolytic
precipitation of hydrated titanium oxides. In the presence of tartrate
buffer, well defined waves are obtained only at pH values less than 2,
or between 6 and 7. The Ti(1V)-Ti(II1) couple is reversible only in
tartrate buffer at pH values less than 1.
When 0.4M citrate ion is used as the medium, the reduction is well
defined at all pH values, but the reduction potential was found to vary
with the solution pH [27]:
PH E,,, V.S. S.C.E.
0.0 -0.28
3.0 -0.80
7.0 -0.95
11.5 -1.49
Ethylenediaminetetraacetic acid has been found to be satisfactory as a

supporting electrolyte [28]. It was demonstrated that the half-wave
potential for a 0.4M solution of Ti(1V) in 0.25N EDTA varied from -
0.22 V (vs. S.C.E.) at pH 3.0 to -0.82 V at pH 8.7. The half-wave
potential was found to be independent of the solution acidity below pH
2.
AC voltammetry using the fundamental and second harmonic wave of

Ti at a semistationary mercury drop electrode has been used for the
direct determination of Ti [29]. This method has the distinct advantage
of being able to tolerate large quantities of metal ion impurities. In
another method, Ti salts were chelated with dihydroxyazo dyes,
adsorbed onto a hanging mercury drop electrode, and then determined
by cyclic voltammetry [30]
-
4.6 Atomic Spectroscopic Methods of Analysis
Historically, emission spectrography has been very important in the

quantitative assay of titanium-containing materials. It should be pointed
out that where Ti is a major constituent of the material (as would be the
case for titanium dioxide), the spectrophotometric and titrimetric
methods of analysis are more appropriate. Nevertheless, a variety of
quantitative methods have been developed for the determination of Ti in
oxidic phases.
After spark-source excitation, the Ti lines at 3239.04 or 3349.41 A are

A
determined against an internal standard. The 3239.04 Ti line is
A
normally quantitated against the 3232.61 line of Li, while the
A
3349.41 Ti line is quantitated against the 3126.11 A line of Cu.
After dissolution of the oxide, Ti may be directly determined by atomic

absorption using either the nitrous oxide-acetylene [3I] or oxygen-
nitrogen-acetylene [32] flame systems. The method is straight-forward,
and no interference has been noted from Cr, Co, Mn, Mo, Nb, W, Ta,
or Cu [33]. An improvement in the determination of Ti using the
nitrous oxide-acetylene flame system was noted when the analysis was
performed in a buffered HF-boric acid mixture [34].
X-ray fluorescence has been found to be useful in the quantitation of

titanium oxides, with internal standards also being used [35]. The
methods all make use of the Ti K a emission at 2.750 A, and differ in
their choice of internal standards.
Atomic absorption spectroscopy has been used to determine the trace

quantities of other metal contaminants in titanium dioxide pigments
[36]. Auger electron spectroscopy has been used to directly determine
the levels of Ti in oxide layers [37].
686 HARRY G.BRITTAIN ET AL.
4.7 Spectrophotometric Methods of Analysis
Theoretically, any colorimetric method useful for identification purposes

can be developed into a quantitative spectrophotometric assay. The
number of reagents proposed for use in photometric assay methods is
extensive, and interested readers should consult appropriate review
articles. Although many of these reagents have been developed for use
in areas relating to nonferrous metallurgy [38], they may be applied to
the spectrophotometricassay of Ti in TiO,. Assays involving
spectrophotometric reagents probably represent the most extensive range
of methods developed for determination of Ti in any sample matrix.
The peroxide method has proven to be the most useful for this purpose,
owing to the high acidity of the medium in which the reaction is
conducted. Interferences are observed only in the presence of V, Mo,
or F, but these species are not normally present in U.S.P. grade
titanium dioxide. In the spectrophotometric assay method, the
absorption maximum at 410 nm is used to determine the titanium
concentration after the oxide is dissolved [39]. The spectrophotometric
endpoint of the peroxide method has been combined with flow injection
analysis techniques to yield an automated procedure “1.
Reagents containing oxygen-donor atoms (phenolic or alcoholic hydroxy

groups) are most suitable as spectrophotometric reagents, but nitrogen-
donor functional groups can also be used. A detailed review of
photometric reagents for Ti has been written by Sommer [41].
The yellow colors developed by titanium salts with tiron [15] and
sulfosalicylic acid [16,42] have also been used to develop quantitative
spectrophotometricassay methods. Other useful reagents include
tichromin and dibromotichromin [43], chromotropic acid [MI,
chlorophosphonazo I [45], and diantipyrylmethane [46]. The
diantipyrylmethane reagent has also been used to measure Ti salts after
these have been stripped off a silica gel column [47].
4J3 Chromatographic Methods of Analvsis
Chromatographic methods for titanium dioxide have not been

investigated vigorously, owing to the wealth of alternate methods
available. Spectrophotometric or atomic spectroscopic measurements
can be conducted more rapidly, are more sensitive, and are not affected
by the oxidation state of the metal. A chromatographic method would
be appropriate only if the Ti species was to be separated from a
complicated sample matrix, but this is not anticipated in the analysis of
U.S.P. titanium dioxide.
In addition, Ti salts are prone to hydrolysis, and ultimately form

insoluble hydrated oxides. For a chromatographic procedure to be
developed, the eluent would have to contain strongly coordinating
agents capable of preventing hydrolysis. Although such agents have
been proposed for the ion chromatographic assay of other transition
elements [48], they do not appear to have been applied to the analysis
of titanium salts.
5. Stability
Stability
Titanium dioxide reacts only with hot, concentrated mineral acid

solutions. It is completely stable with respect to light, oxidation,
changes in pH of suspensions, and microbiological attack. It has been
found to be stable at all temperature values up to its melting point
(1850°C). If heated strongly under vacuum, there is a slight loss of
oxygen corresponding to a change in composition to TiO,.,. This
product is dark blue, but reverts to the original white color when heated
in air.
Incompatibilities with functional srouDS
Titanium dioxide is chemically unreactive, and its only chemistry takes

place after the oxide has been dissolved in strong acids. It is a known
688 HARRY G . BRlTTAlN ET AL
catalyst, however, and is capable of inducing solid state chemical

reactions under certain conditions. However, these reactions require the
application of temperatures exceeding lOO"C, which should not be
encountered in the normal range of pharmaceutical formulations.
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Chemistry", 4Ih ed., John Wiley & Sons, New York, 1980, pp.
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2. L.C. Pakenham and E.G. Murphy, "CTFA International Color

Handbook, Cosmetic, Toiletry, and Fragrance Assoc.,
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3. C.S. Hurlbut, "Dana's Manual of Mineralogy", IS* ed., John

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6. A.R. Gennaro, "Remington's Pharmaceutical Sciences", 17*

ed., Mack Publishing Co., Easton, PA, 1985, p. 790.
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330.
9. A.F. Wells, "Structural Inorganic Chemistry", 5thed.,

Ciarendon Press, Oxford, 1984, pp. 247-252, 560-565.
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Pharmaccopeial Convention, Inc., Rockville, MD, 1990, p.
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11. J.L. SChoM, Z. Anal. Chem., 9, 330 (1870).
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(1913).
18. P.N. Das-Gupta, J. indiun chem. Soc., 6, 855 (1929).
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Nostrand Pub., New York, 1947.
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22. L.C. Jhaveri, G.S. Patel, and V.M.Thakor, J. Indian Chem.

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23. I.M.Kolthoff, R. Belcher, V.A. Stenger, and G. Matsuyama,

"Volumetric Analysis", vol. 111, Interscience, New York,
1957.
690 HARRY G. BRITTAIN ET AL.
24. J.A. Rahm,Anal. Chem., 24, 1823 (1952).
25. J.M. Thompson, And. Chom., 24, 1632 (1952).
26. H. Flaschka, A.J. Barnard, and W.C.Broad, Chemist Analyst,

47, 79 (1958).
27. I.M. Koltoff and J.J. Lingane, "Polarography," vol. 11, 2"d
ed., Interscience, New York, 1952.
28. R.L. Pecsok and E.F. Maverick, J. Am. Chem. SOL'., 76,358
(1954).
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60,2402 (1988).
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Electrochem., 208,383 (1986).
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(1966).
33. J.B. Headridge and D.P. Hubbard, And. Chim. Actu, 37, 151
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34. 1. Janousek, Chem. Listy, 82, 549 (1988).
35. G.J. Lewis and E.D. Goldberg, Anul. Chem., 28, 1285 (1956).
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38. H.J. Seim and R.C. Calkins, Anul. Chem., 51(5), 170R (1979).
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John Wiley & Sons, New York, 1985.
CUMULATIVE INDEX
Bold numerals refer to volume numbers.
Acehutolol, 19. 1 Benzyl benzoate, 10.55

Acetaminophen, 3, I; 14.551 Betamethasone dipropionate, 6,43
Acetohexamide, 1.1; 2,573;21,I Bretylium tosylate, 9.71
Allopurinol, 7,1 Bromazepam, 16.1
Alpha-tocopheryl acetate, 3.111 Bromocriptine methanesulfonate, 8,47
Amantadine, 12.1 Bupivacaine, 19.59
Amikacin sulfate, l2.37 Busulphan, 16.53
Amiloride hydrochloride, IS, I Caffeine, 15,71
Aminoglutethimide, 15,35 Calcitriol, 8, 83
Aminophylline, 11, 1 Camphor, 13,27
Aminosalicylic acid, 10, 1 Captopril, ll, 79
Amiodarone, 20.1 Carbamazepine, 9,87
Amitriptyline hydrochloride, 3, 127 Cefaclor, 9,107
Amoharbital, 19,27 Cefamandole nafate, 9.125; 10,729
Amodiaquine hydrochloride, 21,43 Cefazolin, 4,1
Amoxicillin, 7,19 Cefotaxime, 11. 139
Amphotericin B,6,I; 7,502 Cefoxitin, sodium, ll, 169
Ampicillin, 2,I; 4.518 Ceftazidime, 19.95
Apomorphine hydrochloride, 20,121 Cefuroxime sodium, 20,209
Ascorbic acid, ll,45 Celiprolol hydrochloride, 20,237
Aspirin, 8,1 Cephalexin, 4.21
Astemizole, 20,173 Cephalothin sodium, 1,319
Atenolol, 13, 1 Cephradine, 5,21
Atropine, 14,32 Chloral hydrate, 2, 85
Azathioprine, 10,29 Chlorambucil, 16,85
Azintamide, 18,1 Chloramphenicol, 4.47,518;15,701
Aztreonam, 17,l Chlordiazepoxide, 1, 15
Bacitracin. 9,1 Chlordiazepoxide hydrochloride, 1,39;4,518
Baclofen, 14,527 Chloroquine, 13,95
Bendroflumethiazide, 5, I; 6,597 Chloroquine phosphate, 5, 61
Benperidol, 14,245 Chlorothiazide, 18.33
Benzocaine, 12.73 Chloropheniramine maleate, 7.43
693
Chlorprothixene, 2, 63 Enalapril maleate, 16, 207
Chlortetracycline hydrochloride, 8, 101 Ephedrine hydrochloride, 15, 233
Chlorthalidone, 14.1 Epinephrine, 7, 193
Chlorzoxazone, 16,119 Ergonovine maleate, 11,273
Cholecalciferol, see Vitamin D, Ergotamine tartrate, 6 , 113
Cimetidine, U,127; 17, 797 Erythromycin, 8, 159
Cisplatin. 14.77; L5,7% Erythromycin estolate, 1,101; 2,573
Clidinium bromide, 2,145 Estradiol, 15,283
Clindamycin hydrochloride, 10.75 Estradiol valerate, 4, 192
Clioquinol, 18.57 Estrone, 12, 135
Clofazamine, 18,91 Ethambutol hydrochloride, 7,231
Clofazimine, 21,75 Ethynodiol diacetate, 3,253
Clofibrate, 11,197 Etomidate, 12, 191
Clonazepam, 6,61 Etoposide, 18, 121
Clonidine hydrochloride. 21, 109 Fenoprofen calcium, 6, 161
Clorazepate dipotassium, 4,91 Flecainide, 21, 169
Clotrirnazole, ll, 225 Flucytosine, 5, 115
Cloxacillin sodium, 4, 113 Fludrocortisone acetate, 3,281
Cocaine hydrochloride, 15,151 Flufenamic acid, ll.313
Codeine phosphate, 10,93 Fluorouracil, 2,221: 18.599
Colchicine, 10, 139 Fluoxetine, 19, 193
Cyanocobalamin, 10,183 Fluoxymesterone, 7,251
Cyclandelate, 21,149 Fluphenazine decanote, 9,275; 10,730
Cyclizine, 6, 83; 7, 502 Fluphenazine enanthate, 2,245; 4,524
Cyclobenzaprine hydrochloride, 17,41 Fluphenazine hydrochloride, 2,263; 4, 519
Cycloserine, 1.53; 18,567 Flurazepam hydrochloride, 3,307
Cyclosporine, 16,145 Folic acid, 19,221
Cyclothiazide, 1,66 Furosemide, 18, 153
Cypropheptadine, 9, 155 Gentamicin sulfate, 9,295; 10,731
Dapsone, 5.87 Glafenine, 21, 197
Dexamethasone, 2,163; 4,519 Glibenclamide, 10,337
Diatrizoic acid, 4, 137: 5,556 Gluthethimide, 5, 139
Diazepam, 1, 79: 4, 518 Gramicidin, 8, 179
Dibenzepin hydrochloride, 9,181 Griseofulvin, 8,219; 9,583
Dibucaine and dibucaine hydrochloride, l2.105 Guanabenz acetate, 15,319
Diclofenac sodium, 19, 123 Halcinonide, 8, 251
Diethylstilbestrol, 19, 145 Haloperidol, 9 , 341
Diflunisal, 14,491 Halothane, 1,119; 2,573: 14,597
Digitoxin, 3.149 Heparin sodium, 12,215
Digoxin, 9,207 Heroin, 10,357
Dihydroergotoxine methanesulfonate, 7 , 81 Hexestrol, ll,347
Diwtyl sodium sulfosuccinate, 2.199; 12,713 Hexetidine, 7.277
Diperodon, 6 , 9 9 Homatropine hydrobromide, 16,245
Diphenhydramine hydrochloride, 3,173 Hydralazine hydrochloride, 8,283
Diphenoxylate hydrochloride, 7,149 Hydrochlorothiazide, 10,405
Disopyramide phosphate, W , 183 Hydrocortisone, 12,277
Disulfiram, 4,168 Hydroflumethiazide. 7,297
Dobutamine hydrochloride, 8,139 Hydroxyprogesterone caproate, 4,209
Dopamine hydrochloride, 11,257 Hydroxyzine dihydrochloride, 7,319
Doxorubicine, 9,245 Impenem, 17.73
Droperidol, 7, 171 Imipramine hydrochloride, 14,37
Echothiophate iodide, 3, 233 Indomethacin, 13,211
Emetine hydrochloride, 10,289 Iodamide, 15,337
694
Iodipamide, 2,333 Minocycline, 6,323
lodoxamic acid, 20,303 Minoxidil, 17, 185
Ioparnidol. 17,115 Mitomycin C, 16,361
Iopanoic acid, 14, 181 Mitoxantrone hydrochloride, 17,221
Iproniazid phosphate, 20, 337 Morphine, 17,259
Isocarboxazid, 2, 295 Moxalactarn disodium, W , 305
Isoniazide, 6,183 Nabilone, 10,499
Isopropamide, 2,315; 12,721 Nadolof, 9,455; 10,732
Isoproterenol. 14,391 Nalidixic acid, 8,371
Isosorbide dinitrate, 4, 225; 5, 556 Naloxone hydrochloride, 14,453
Ivermectin, 17,155 Nalorphine hydrobromide, 18, 195
Kanamycin sulfate, 6, 259 Naphazoline hydrochloride, 21,307
Ketamine, 6,297 Naproxen. 21,345
Ketoprofen, 10,443 Natamycin, 10,513
Ketotifen, 13, 239 Neomycin, 8 ,3 9 9
Khellin, 9, 371 Neostigmine, 16,403
Lactose, anhydrous, 20,369 Nicotinamide, 20,475
Leucovorin calcium, 8,315 Nifedipine, 18,221
Levallorphan tartrate, 2,339 Nitrazepan], 9 ,4 8 7
Levartereno1bitartrate, 1 ,4 9 ;2,573; 11,555 Nitrofurantoin, 5, 345
Levodopa, 5 , 189 Nitroglycerin, 9, 519
kvothyroxine sodium, 5,225 Nizatidine, 19,397
Lidocaine base and hydrochloride, 14,207; 15, Norethindrone, 4,268
76 I Norfloxacin, 20,557
Lisinopril, 21, 233 Norgestrel, 4, 294
Lithium carbonate, 15, 367 Nortriptyline hydrochloride, 1,233; 2,573
Lobeline hydrochloride, 19,261 Noscapine, 11,407
Lomustine, 19.315 Nystatin, 6,341
Loperamide hydrochloride, 19,341 Oxamniquine, 20,601
Lorazepam, 9, 397 Oxazepam, 3,441
Lovastatin, 21, 277 Oxyphenbutazone, 13,333
Maprotiline hydrochloride, 15,393 Oxytocin, 10,563
Mebendazole, 16,291 Papaverine hydrochloride, 17,367
Mefloquine hydrochloride, 14, 157 Penicillarnine, 10,601
Melphalan, l3,265 Penicillin-G, benzothine, 11,463
Meperidine hydrochloride, 1, 175 Penicillin-G, potassium, 15,427
Meprobamate, 1,209; 4,520; 11,587 Penicillin-V, 1,249; 17,677
6-Mercaptopurine, 7,343 Pentazocine, W, 361
Mestranol. ll, 375 Pergolide mesylate, 21, 375
Methadone hydrochloride. 3,365; 4,520; 9,601 Phenazopyridine hydrochloride, 3 ,4 6 5
Methaqualone, 4,245,520 Phenelzine sulfate, 2,383
Methimazole, 8,351 Phenformin hydrochloride, 4,319; 5,429
Methotrexate. 5, 283 Phenobarbital, 7,359
Methoxamine hydrochloride, 20, 399 Phenolphthalein, 20, 627
Methoxsalen, 9,427 Phenoxymethyl penicillin potassium, 1,249
Methyclothiazide, 5, 307 Phenylbutazone, l l .4 8 3
Methylphenidate hydrochloride, 10,473 Phenylephrine hydrochloride, 3 ,4 8 3
Methyprylon, 2, 363 Phenylpropanolarnine hydrochloride, 12,357; 13,
Metipranolol, 19, 367 77 1
Metoclopramide hydrochloride, 16.327 Phenytoin, 13,417
Metoprolol tartrate, 12,325 Physostigmine salicylate, 18,289
Metronidazole. 5.327 Phytonadione, 17,449
Mexiletine hydrochloride, 20,433 Pilocarpine, 12,385
695
Piperazine estrone sulfate, 5, 375 Terazosin, 20.693
Pirenzepine dihydrochloride, 16,445 Terbutaline sulfate, 19,601
Piroxicam, 15,509 Terfenadine, 19,627
Polythiazide, 20,665 Terpin hydrate, 14,273
Pralidoxine chloride, 17,533 Testolactone. 5,533
Prazosin hydrochloride, 18,361 Testosterone enanthate, 4,452
Prednisolone, 21.415 Tetracaine hydrochloride, 18,379
Primidone, 2,409;17,749 Tetracycline hydrochloride, W, 597
Probenecid, 10,639 Theophylline, 4,466
h-ocainamide hydrochloride, 4,333 Thiabendazole, 16,611
Procarbazine hydrochloride, 5,403 Thiamine hydrochloride. 18,413
Promethazine hydrochloride, 5,429 Thiopental sodium, 21,535
Proparacaine hydrochloride, 6,423 Thioridazine and Thiondazine hydrochloride, 18,
Propiomazine hydrochloride, 2,439 459
Propoxyphene hydrochloride, 1,301;4,520;6, Thiostrepton, 7,423
598 Thiothixene, 18.527
Propylthiouracil, 6,457 Ticlopidine hydrochloride, 21,573
Pseudoephedrine hydrochloride, 8,489 Timolol maleate, 16,641
Pyrazinamide, 12,433 Titanium dioxide, 21.659
Pyridoxine hydrochloride, 13,447 Tolbutamide, 3,513;s.557;13,719
Pyrimetharnine, 12,463 Trazodone hydrochloride, 16,693
Quinidine sulfate, 12,483 Triamcinolone, 1,367;2,571;4,521,524;ll,593
Quinine hydrochloride, 12,547 Triamcinolone acetonide, 1,397.416;2,571;4,
Ranitidine, 15,533 521; 7,501 ; U, 615
Reserpine, 4,384;5,557;W,737 Triamcinolone diacetate, 1,423;11,651
Riboflavin, 19,429 Triamcinolone hexacetonide, 6,579
Rifampin, 5,467 Triclobisonium chloride, 2.507
Rutin, 12,623 Trifluoperazine hydrochloride, 9,543
Saccharin, 13,487 Triflupromazine hydrochloride, 2,523;4,521;5,
Salbutamol, 10,665 557
Salicylamide, 13,521 Trimethaphan camsylate, 3,545
Scopolamine hydrobromide, 19,477 Trimethobenzamide hydrochloride, 2,551
Secobarbital sodium, 1,343 Trimethoprim, 7,445
Silver sulfadiazine, 13,553 Trimipramine maleate. 12, 683
Sodium nitroprusside, 6,487;15,781 Trioxsalen, 10,705
Sotalol, 21,501 Tripelennamine hydrochloride, 14,107
Spironolactone. 4,431;18,641 Triprolidine hydrochloride, 8,509
Streptomycin, 16,507 Tropicamide, 3, 565
Strychnine, 15,563 Tubocurarine chloride, 7,477
Succinycholine chloride, 10,691 nbamate, 4,494
Sulfadiazine, ll.523 Valproate sodium and valproic acid, 8,529
Sulfadoxine, 17,571 Verapamil, 17,643
Sulfamethazine, 7,401 Vidarabine, 15,647
Sulfamethoxazole, 2,467;4,521 Viblastine sulfate, 1,443;21,611
Sulfasalazine, 5,515 Vincristine sulfate, 1,463
Sulfisoxazole, 2,487 Vitamin D,, 13,655
Sulfoxone sodium,19,553 Warfarin, 14,243
Sulindac, 13,573 Xylometazoline hydrochloride, 14,135
Sulphamerazine, 6,515 Yohimbine, 16,731
Sulpiride. 17,607 Zidovudine, 24,729
Teniposide, 19,575 Zomepirac sodium,15.673
696

Analytical Profiles Drug Substances and Excipien T S: Harry G. Brittain

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Analytical Profiles

ACADEMIC PRESS, INC.

Abdullah A. Al-Badr George A. Forcier

This book is printed on acid-free paper. @

Copyright 0 1992 by ACADEMIC PRESS, INC.

Academic Press, Inc.

United Kingdom Edition published by

International Standard Serial Number: 0099-5428

International Standard Book Number: 0-12-260821-6

PRINTED IN THE UNITED STATES OF AMERICA

Mohummud A . Abounussf, Pharmaceutical Chemistry Department, College of Phar-

K . Usmanghani. Department of Pharmacognosy, Faculty of Pharmacy, University of

The profiling of drug compounds as to their physical and analytical characteristics

Harry G . Brittain Klaus Florey

Abdullah A. Al-Badr and Humeida A. El-Obeid

Pharmaceutical Chemistry Department

King Saud University

Riyadh, Saudi Arabia

ANALYTICAL PROFILES OF DRUG SUBSTANCES Copyright 0 1992 by Academic Press, Inc.

4.3 Chromatographic Methods

1.1.1 Chemical Names

1.1.2 Generic Names

Cycl am1de , Dime 1in , Dime1o r , Dyme 1o r , Gamadiabet,

1.2.3 CAS No.

1.3 Molecular Weight

1.4 Elemental ComDosltion

C 55.54%, H 6.21%, N 8.64%, 0 19.73%, S 9.89% (1).

C r y s t a l s from 90% aqueous e t h a n o l m e l t between

Soluble i n p y r i d i n e , s l i g h t l y soluble i n alcohol

Yokoyama e t a7 (10) calculated the thermodynamic values

S o l i d dispersion o f acetohexamide was studied by Graf

o f form I-PEG and form 111-PEG coprecipitates were o f

Kassem e t a7 (15) studied the enhancement o f the r a t e

AHf = 63.7 kJ/mOle Purity = 99.82% Tm = 187.45 C

2.5 X-ray Powder D i f f r a c t i o n

The X-ray powder d i f f r a c t i o n p a t t e r n o f acetohexa-

F i g u r e 1. Thermal cu rve o f acetohexamide.

Figure 2 . X-Ray powder d i f f r a c t i o n p a t t e r n of acetohexamide.

generator. Radiation was provided by a copper t a r g e t

The X-ray pattern o f acetohexamlde I s presented i n

2.6 Spectral ProDerties

The i n f r a r e d absorption spectrum o f acetohexamide,

220 XO nm 300 3 50 400 450

Table 1: X-ray d i f f r a c t i o n p a t t e r n o f acetohexamfde

d(A) 1/10 d(A) 1/10

15.74 31.25 2.55 1.82

Table 2: I n f r a r e d c h a r a c t e r i s t i c bands and t h e i r

Frequency (cm- Assignment

3340, 3270 Amide N-H s t r e t c h

2980, 2940 Aromatic C-H s t r e t c h

1710, 1680 Conjugated - E-

1455 C - CH3 bending

Aromatic C-H out o f plane

2.6.3 Proton Nuclear Magnetic Resonance ( W R l

Acetohexamide s o l u t i o n i n DMSO-de was used t o

Table 3: Assignment o f the NMR chemical s h i f t s t o the

Chemical s h i f t Multiplicity Proton No. o f

1.09 - 1.71 mu1ti p l e t Cyclohexyl 11

8.06 - 8.19 mu1t ip l e t Aromat ic d 4

2.6.4 13C-Nuclear Magnetic Resonance (13C NHR)

The 1 3 C NMR spectra o f acetohexamide i n DMSO-ds

2.6.5 Mass SDectra

The 70 eV e l e c t r o n impact mass spectrum o f