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Analytical Profiles

of
Drug Substances
Volume 12

Edited by
Klaus Florey
The Squibb Institute for Medical Research
New Brunswick, New Jersey

Contributing Editors
Abdullah A. Al-Badr Glenn A. Brewer, Jr.
Norman W. Atwater Hans-Georg Leemann
Steven A. Benezra Joseph A. Mollica
Compiled under the auspices of the
Pharmaceutical Analysis and Control Section
APhA Academy of Pharmaceutical Sciences

ACADEMIC PRESS 1983


A Subsidiary of Harcourt Brace Jovanovich, Publishers
New York London
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EDITORIAL BOARD

Abdullah A. Al-Badr Klaus Florey


Norman W. Atwater Salvatore A. Fusari
Steven A. Benezra Lee T. Grady
Rafik Bishara Boen T. Kho
Gerald S. Brenner Hans-Georg Leemann
Glenn A. Brewer, Jr. Joseph A. Mollica
Nicholas DeAngelis James W. Munson
John E. Fairbrother Milton D. Yudis

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83 84 85 86 9 8 7 6 5 4 3 2 1
AFFILIATIONS OF EDITORS,
CONTRIBUTORS, AND REVIEWERS

H. Y. Aboul-Enein, King Saud University, Riyadh, Saudi Arabia


S. Ahuju, Ciba-Geigy Corporation, Summit, New Jersey
A. A. Al-Budr, King Saud University, Riyadh, Saudi Arabia
S. L. Ali, Zentrallaboratorium Deutscher Apotheker e.V., Eschborn Germany
N. Atwuter, E. R. Squibb & Sons, Princeton, New Jersey
G. Atzl, Sandoz Ltd., Basel, Switzerland
S. A. Benezru, Wellcome Research Laboratories, Research Triangle Park,
North Carolina
R. Bishuru, Lilly Research Laboratories, Indianapolis, Indiana
D. Both, The Squibb Institute for Medical Research, New Brunswick, New Jersey
G. Brenner, Merck Sharp & Dohme Research Laboratories, West Point,
Pennsylvania
G. A. Brewer, The Squibb Institute for Medical Research, New Brunswick, New
Jersey
R. D. Brown, Bristol Laboratories, Syracuse, New York
2. L. Chung, Abbott Laboratories, North Chicago, Illinois
J. Cohen, Ciba-Geigy Corporation, Summit, New Jersey
N. DeAngelis, Wyeth Laboratories, Philadelphia, Pennsylvania
R. Dowse, Rhodes University, South Africa
J. Fuirbrother, Stiefel Laboratories Ltd., Sligo, Ireland
E. Felder, Bracco Industria Chimica S.p.a., Milan, Italy
K. Florey, The Squibb Institute for Medical Research, New Brunswick, New Jersey
S. A. Fusuri, Warner-Lambert Research Institute, Morris Plains, New Jersey
L. T. Grady, The United States Pharmacopeia, Rockville, Maryland
J. M. Huigh, Rhodes University, South Africa
S.A. Hunnu, Bristol Laboratories, Syracuse, New York
M. M.A. Hassun, King Saud University, Riyadh, Saudi Arabia
I. Kunfer, Rhodes University, South Africa

vii
viii AFFILIATIONS OF EDITORS, CONTRIBUTORS, AND REVIEWERS

T. I . Khalifu, King Saud University, Riyadh, Saudi Arabia


B. T. Kho, Ayerst Laboratories, Rouses Point, New York
J . Kirschbuum, The Squibb Institute for Medical Research, New Brunswick,
New Jersey
H . G . Leemann, Sandoz Ltd., Basel, Switzerland
M. A. Loutfy, King Saud University, Riyadh, Saudi Arabia
J . R. Luch, Ciba-Geigy, Suffern, New York
J . B. Martin, Abbott Laboratories, North Chicago, Illinois
J . P. McGrot-y, Bristol Laboratories, Syracuse, New York
J . Mollicu, Ciba-Geigy Corporation, Summit, New Jersey
P. M. Monteleone, Bristol Laboratories, Syracuse, New York
N . Muhammed, Bristol Laboratories, Syracuse, New York
F. J . Miihtudi, King Saud University, Riyadh, Saudi Arabia
J . W. Munson, The Upjohn Company, Kalamazoo, Michigan
F. Nuchtmann, Sandoz Ltd., Bade, Switzerland
G. R. Padmanabhan, Ciba-Geigy Corporation, Suffern, New York
D. Pitre, Bracco Industria Chimca S.p.a., Milan, Italy
A. Post, Smith Kline & French Laboratories, Philadelphia, Pennsylvania
W. D. Roth, Sandoz Ltd., Basel, Switzerland
R. S. Suntoro, * Smith Kline & French Laboratories, Philadelphia, Pennsylvania
M. D. Yudis, Schering-Plough, Inc., Bloomfield, New Jersey

*Deceased
PREFACE

The compilationofAnalytica1Profiles of Drug Substancesto supplementthe infor-


mation contained in the official compendia is now a well-established activity.
That we are able to publish one volume per year is a tribute to the diligence of the
editors to solicit monographs and even more so to the enthusiastic response of our
authors, an internationalgroup associated with pharmaceutical firms, academic insti-
tutions, and compendia1authorities. I would like to express my sincere gratitude to
them for making this venture possible.
Over the years, we have had queries concerning our publication policy. Our goal is
to cover all drug substances of medical value, and therefore, we have welcomed any
monographs of interest to an individual contributor. We also have endeavored to
solicit profiles of the most useful and used medicines, but many in this category still
need to be profiled.
In the preface to the eleventh volume. I announced that we would try to supplement
previously published profiles with new data. Unfartunately, most of the original
contributors are no longer available to undertake this task, and it has proven to be
difficult to find other volunteers. We shall continue to pursue the updating program,
but it will not be as comprehensive as originally envisioned.
Again, I would like to request of all those who have found these profiles useful to
contribute monographs of their own. We, the editors, stand ready to receive such
contributions.

ix
AMANTADINE
Joel Kirschbaum

1. Introduction 2
1.1 History, Therapeutic Use, and Mechanism of Action 2
1.2 Nomenclature, Molecular Weight, and Structure 2
1.3 Appearance, Color, Odor, and Precautions 2
1.4 Synthesis 4
1.5 Reactions, Stability, and Metabolism 5
2. Physical Properties of Crystalline Amantadine 6
2.1 Single Crystal X-Ray Diffraction 6
2.2 X-Ray Powder Diffraction 7
2.3 Mass Spectrometry 9
2.4 Infrared Spectrometry 9
2.5 Electron Tunnelling and Photoelectron Spectrometry 11
2.6 Thermal Analysis 13
2.7 Microscopy 13
2.8 Surface Area 13
2.9 Hydration 13
2.10 Polymorphism 13
3. Spectrometryof Amantadine in Solution 14
3.1 Nuclear Magnetic Resonance Spectrometry (NMR) 14
3.2 Ultraviolet Spectrometry 16
1. Bulk Solution Properties 16
4.1 Solubilitiesin Aqueous and Nonaqueous Solvents 16
4.2 Ionization 18
4.3 Dipole Moments 19
4.4 Hydrodynamic Properties 19
5. Methods of Analysis 20
5.1 Compositional Analysis 20
5.2 Identity and Colorimetric Methods 20
5.3 Titration 21
5.4 Spectrometry 21
5.5 Gas-Liquid Chromatography 22
5.6 Thin-Layer Chromatography 22
5.7 High-Performance Liquid Chromatography 22
5.8 Electrochemistry 22
5.9 Fluorescence Spectrometry 29
5.10 Tissue Culture 30
5.11 Comparison of Methods 30
References 31

ANALYTICAL PROFILES OF DRUG SUBSTANCES 1 Copyright by the American Pharmaceutical Arwriation.


VOLUME 12 ISBN 0-12-260812-7
JOEL KIRSCHBAUM

1. Introduction

1.1 History, Therapeutic Use and Mechanism of Action

Amantadine is an orally active antiviral agent


(1,2). It was discovered by workers at DuPont via an
empiric screening program (3). Other than
vaccination, it is the only prophylactic drug
presently useful against many viral infections,
especially influenza A and C. Once administered, its
effect is immediate to reduce signs of infection
among 50% to 70% of individuals exposed to the virus.
A use panel recommended ( 4 ) it for individuals with a
high risk of serious morbidity or mortality due to
cardiovascular, immunodeficiency, metabolic, neuro-
muscular or pulmonary diseases, the elderly, and the
unvaccinated and the important (5). It is 91%
effective in preventing influenza. The antiviral
activity of amantadine hydrochloride appears at an
early phase of the infection (6). The mode of action
appears to be the inhibition of the uncoating of the
virus ( 7 ) once it has penetrated the host cell. Such
a failure prevents replication. Gene 7 , coding for
the virus matrix protein, carries the property of
amantadine resistance ( 8 ) , and can be transferred by
recombination between influenza viruses. It was
conjectured (9) that other highly symmetrical
hydrocarbons, perhaps in the shapes of the Platonic
solids like cubane and dodecahedrane, when
derivatized like amantadine, might have similar
properties to pass through the membrane of a cell and
destroy virus particles inside it (10).
Amantadine is also useful in treating
Parkinson's disease (2). This use was found by a
chance observation of a significant improvement in
such a patient taking 200 mg of amantadine daily for
flu prophylaxis. It also appears clinically
effective in the treatment of drug-induced
extrapyramidal symptoms (11).
Amantadine relieves Parkinson's disease
(including drug-induced Parkinsonism by
neuroleptics) , apparently by a mechanism involving
dopamine (12); indeed, amantadine enhances L-dopa
activation (2).
As expected, various investigators found
amantadine to have other uses; not only against other
AMANTADINE 3

viruses (12), but also in treating cancer (13),


aiding priapus (14), and inhibiting rust (15).
Rimantadine, an amino group analogue
[1-(1-aminoethyladamantane)] is also active against
virus (2,16). Rimantadine is 4-8 times more
effective than amantadine hydrochloride to protect
against influenza A virus infection, but it is more
toxic (17).

1.2 Nomenclature, Molecular Weight and Structure

Amantadine hydrochloride is the United States


adopted name (18). The preferred chemical name is
tricyclo r3.3.1. 13'7]decan-l-amine, hydrochloride.
Other names include 1-adamantanamine hydrochloride,
1-aminotricylo [ 3 . 3 . 1 . I. "1 decane hydrochloride,
1-adamantylamine hydrochloride, adamantylamine
hydrochloride, and 1-aminoadamantane hydrochloride,
and, less correctly, midantane and dimantane
hydrochloride (19). Its molecular weight is 187.71
daltons. Amantadine hydrochloride was given the
chemical abstracts service systematic number
665-66-7 ; the free base, amantadine , was numbered
CAS-768-94-5. It is currently marketed under the
name Symmetrel (Endo Laboratories). Other names
include EXP-105-1, Mantadix, Matadan, Mydantan and
Virafral. In Wiswesser notation it is L66 B6 A B- C
1B ITJ BZ &GH.
Amantadine hydrochloride can be represented a
variety of ways, as shown below:
Amantadine hydrochloride possesses a unique,
rigid, relatively unstrained ring system that is
composed of three fused cyclohexane rings in the
chair conformation (20). Amantadine is considered to
be the smallest repeating unit of the diamond lattice
(21). The symmetrical cage structure causes the
infrared, nuclear magnetic resonance and mass spectra
to be comparatively simple, 2s will be illustrated
later. As expected from this lack of asymmetry,
there is no observable optical rotation (22) using
the D lines of sodium, at a concentration of 1% in
water.

1.3 Appearance, Color, Odor and Precautions

Amantadine hydrochloride is a white, odorless,


free-fl owing crystalline powder. No precautions are
given for this relatively non-toxic compound.
4 JOEL KIRSCHBAUM

ia

HCH

5
4

NH2
7 HCI 3

5 10

1.4 Synthesis

Adamantane is found naturally at low


concentrations (approximately 0.02%) in various
petroleum fractions (23). However, it may be
synthesized by isomerization of ten carbon cyclic
hydrocarbons, the probable basis of the naturally
formed adamantane. A convenient starting material,
dicyclopentadiene (I) was hydrogenated quantitatively
to endo-trimethylnorbornane (11,
endo-tetrahydrodicyclopentadiene). After refluxing
overnight with such Lewis acids as aluminum
trichloride or tribromide, adamantane (111) was
found. The possible mechanism (24) is shown below.
Bromination to 1-bromoadamantane, an ionic
process, can he followed by a sequence of reactions
with either ammonia, methylcyanide, urea or thiourea
as sources of the amino group, to give amantadine
(25-30). More complicated reactions of the l-bromo-
compound involve dehalogenation, reaction with
methylcyanide and saponification (31,32). Other
syntheses utilize the 1-carboxylic acid (33) and the
1-nitrate (34).
AMANTADINE 5

Direct amination (35) of adamantane or


introduction of a source of an amino group during the
rearrangement of I1 (also known as
tricyclo[5.2. 1.02,6]decane) gives a yield of 75%
amantadine (36)
bridgehead carbon via >
The reaction precedes (37)
+ ~j
NC12 )NC12 *
-Cl+
+H+
Various other combinations of isomerization and
pH*.
the

conversion to amantadine have been described (38,39).


Amantadine can also be synthesized by the
photochemical reaction of chloramine with adamantane
(40).

I II

&- &+-&+

F(
+
"RH & m

1.5 Reactions, Stability and Metabolism

Possible reactions are substitution at the amino


group of amantadine, replacement of the amino group,
rearrangement of the cage structure or replacement of
the cage hydrogens, and have been discussed elsewhere
(20). A vast number of derivatives of the amino
group have been prepared (41). The amino group can
undergo all of the typical reactions of primary
amines, such as Schiff base formation (42),
alkylation (43,44), halogenation (45) and amination
(46). Deamination with sodium nitrite and acetic
6 JOEL KIRSCHBAUM

acid or nitrous acid gives 1-hydroxyadamantane in 97%


yield (20). The in situ reaction with
trichloroacetyl isocyanate in NMR tubes was used to
analyze for the amino function ( 4 7 ) . As expected,
various compounds like acid chlorides were reacted
with amantadine ( 4 8 , 4 9 ) to create potential drugs
with new properties. The relative stability of the
1-adamantyl cation ( 5 0 ) permits conversion of the
amino group to nitro, and then to a large series of
derivatives ( 5 1 ) .
The cage structure can be rearranged ( 5 2 ) in a
reversal of the synthesis. The cage hydrogens can be
replaced by fluorine, as induced by light ( 5 3 ) or by
perfluoridation ( 5 4 ; 19F-NMR, infrared and mass
spectra discussed), as well as by tritium ( 5 5 ) .
The amantadine structure has been characterized
as being extremely stable, as predicted from the
equatorial position of the amino group and the facile
rearrangement of ten carbon hydrocarbons to
adamantane ( 2 0 ).
After oral administration, amantadine was found
in the heart, kidney, liver and lungs ( 5 6 ) .
Concentration of the drug in the lungs may be part of
its prophylactic action. After an oral dose of 2.5
mgfkg, maximum concentration of 0 . 3 ug/mL was reached
in 1-4 hours ( 5 7 ) , with a plasma half-life of 9-15
hours ( 5 8 ) . The rate of excretion depends of the pH
of the urine; i . e . , at pH 5 . 0 , 5-7% per hour of body
content was excreted, but at pH 8 the excretion rate
was 4% per hour ( 5 9 ) . As expected, with patients
having negligible renal function, excretion was
impaired, with plasma concentrations reaching 4 . 4
UgfmL, and accompanied by toxic manifestations of the
drug (60).
In hepatic micro soma1 preparations,
N-hydroxy-1-aminoadamantane and 1-nitrosoadamantane
were identified as metabolites (61). Approximately
0.1% of the administered amantadine was found in
urine in the form of 1-amino-3-hydroxyadamantane
(62).

2. Physical Properties of Crystalline Amantadine

2.1 Single Crystal X-Ray Diffraction

Although the x-ray structure of amantadine


hydrochloride was not determined, the structure of
the parent compound adamantane was elucidated (63).
AMANTADINE I

The three-dimensional representation below is


reproduced with the permission of the
C r y s t a l l o g r a p h i c Data C e n t r e , Cambridge ( 6 4 ) .

E l e c t r o n d i f f r a c t i o n d a t a (65) f o r adamantane a g r e e d
w i t h t h e x-ray c r y s t a l l o g r a p h y . The band l e n g t h of
C-H and C-C (1.54 t 0.01A) a p p e a r normal, and t h e
C-C-C a n g l e s a r e t e t r a h e d r a l (109.5 +- 1.5O).

2.2 X-Ray Powder D i f f r a c t i o n

To observe x-ray d i f f r a c t i o n p a t t e r n s , a P h i l i p s
powder d i f f r a c t i o n u n i t e m i t t i n g CuKa r a d i a t i o n a t
1.54A was used w i t h a s c i n t i l l a t i o n c o u n t e r d e t e c t o r
(66). The r e l a t i v e l a c k of peaks s e e n i n F i g u r e 1
w a s e x p e c t e d from t h e h i g h l y symmetrical s t r u c t u r e of
amantadine h y d r o c h l o r i d e . Below a r e t h e s o r t e d d a t a
based on h i g h e s t i n t e n s i t y of 1.00 u s i n g CuKa
radiation.

20(Degrees) ' d' (Angstroms) R e l a t i v e Area

18.2 4.88 1.000


15.9 5.58 0.739
27.4 3.26 0.499
14.4 6.17 0.405
23.9 3.72 0.344
Figure 1. Powder X-Ray Diffraction Pattern of Amantadine Hydrochloride. See Text for Details
AMANTADINE 9

18.6 4.78 0.298


9.0 9.81 0.235

2.3 Mass Spectrometry

The mass spectrum (67) of amantadine


hydrochloride (Figure 2) shows that the amino
substituent was present as a major ionic species
(68). The molecular peak was at m/e 151, with an
intensity as great as 60% (69). Below is the
suggested fragmentation pathway (62).

Secondary ion mas? spectrometry $70) using


silver showed (M + H) and (Ag + M) adducts.
Protonated amfntadine gave rise to the fragment ion
( M + H - NH3)
Mass
.
spectrometry combined with gas
chromatography has been used to determine amantadine
in biological tissues and fluids, cf. section 5.5.

2.4 Infrared Spectrometry

Figure 3 shows the infrared spectra of a


commercial preparation of amantadine hydrochloride
using mineral oil and potassium bromide (71). The
instrument used was a Perkin-Elmer Model 983 Fourier
transform infrared spectrometer. The minor
differences in band intensities of the two spectra
could be due to either pressure effects in the
preparation of the potassium bromide pellet or
10 JOEL KIRSCHBAUM

5765 Q D Q M R N T R N R M I NE . H C L ( FlLClR I Cli 1 205C

90-

80-
>
t-
v)
70-
Z
60-
Z
H

50--
W
>
+ 48.-
a
30-
DL
20-

10-

0- T F V
3
D
d r l d r - i d

M F1 SS1'C H FIR C;E


INTENSITY SUM =56750 BFISE PERK % = 1 3 . 4 4

Figure 2: Mass Spectrum of Amantadine Hydrochloride,

Instrument AEI-MS 902.


AMANTADINE 11

polymorphism. Below are the interpretations (71, 72) aof


the absorbances of these relatively featureless specra.

Ab sorption ( cm- I ) Assignment

3000
+
NH3 stretching (broad)
2923 CH stretching (antisymmet ric)
2855 CH2 stretching (symmetric)
2700-2250 2+ stretching
NH3+
2000 NH3+ overtones
1600 NH3+ deformation
1500 NH3 deformation
1452 CH deformation
2
1365 NH deformation
1307 CH3 wag
1300 and below 2
Fingerprint region
a
zp absorbances at 3000-2800, 1460, 1377 and 123
cm were du to mineral oil. The absorbance at
3500-3400 cm-F was due to water from the KBr in the
pellet.
A diagnostic test for the presence of the
adamantane skeleton is the l.ow-intensity absorption
in the region 1017-1038 cm-l.
The far-in rared spectrum was determined from
- to 100 cm
6501 -5 .
The torsional vibration at 230
cm , which was identical in both the solid and in
cyclohexane solution, was assigned (73) to the amino
group. A barrier height of 2.00 kcal/molel was
calculated. The +band at approximately 490 cm- was
assigned to a NH torsional vibration.
3
2.5 Electron Tunnelling and Photoelectron
Spectrometry .
Inelastic electron tunnelling spectroscopy is a
non-optical vibrational spectroscopy used to study
the adsorption of adsorbates on barrier oxide films
grown on metals. The interpretation of the spectrum
(74) assigns peaks to C-C, -CH2, -CH and C-C-C to be
caused by scissoring, bending, twisting, wagging and
rocking. Vibrations associated with the amine
substituent are almost completely absent, probably
due t o interaction with the adsorbing oxide surface.
Photoelectron spectroscopy is used to determine
ionization potentials, which can test the theoretical
procedures used to predict orbital energies. The
12 JOEL KIRSCHBAUM

F i g u r e 3 . I n f r a r e d s p e c t r a of Amantadine h y d r o c h l o r i d e .
Upper p o r t i o n ; m i n e r a l o i l m u l l : Lower p o r t i o n , potassium
bromide p e l l e t , See t e x t f o r d e t a i l s .

li-
I
AMANTADINE 13

ionization potentials, 11, for a series of adamantane


derivatives are similar (75), 9.22 to 9.25 eV,
indicating that substituents have little effect.

2.6 Thermal Analysis

Thermal gravimetric analysis (76) of a


commercial preparation of amantadine hydrochloride,
using a heating rate of 20°/min., showed no loss in
weight until 190°, indicating a lack of volatile
solvents. Sublimation occurred at about 190" since
the inside of the apparatus was covered with powder,
These results are in good agreement with a melting
range (19) of 180-192'.
Differential thermal analysis and differential
scanning calorimetry (76) gave a series of endotherms
which may be due to sublimation. The parent
compound, adamantane, also sublimes. This unusual
(21) property for a hydrocarbon is considered due to
a face-centered cubic lattice with only forces
between the four molec 15s in a unit cell being
Y
effective (space group TdF43m, a = 9.426 2 0.008A).

2.7 Microscopy

A commercial preparation of amantadine


hydrochloride was found to contain irregularly shaped
crystals ranging from approximately 18 x 25 pm to
35 x 50 pm (76). There was no visual evidence for
polymorphism.

2.8 Surface Area

As measured by nitrogen gas adsorption (76), the


surface ar a of one lot of amantadine hydrochloride
9
was 0.73 m Ig.

2.9 Hydration

The crystals are not solvated with water, based


on the thermal gravimetric and differential thermal
analyses previously described, and the elemental
analysis (cf. section 5.1).

2.10 Polymorphism

There is weak evidence for polymorphism based on


infrared spectrometry (section 3.3) but none by
microscopy.
14 JOEL KIRSCHBAUM

3. Spectrometry of Amantadine in Solution

3.1 Nuclear Magnetic Resonance Spectrometry (NMR)


1
3.11 H-NMR

Figure 4 is the 100 MHz proton NMR spectrum


(77) of amantadine hydrochloride in
deuterochloroform, as obtained on a Varian
XL-100-15 spectrometer equipped to perform
Fourier transform spectrometry. Proton chemical
shifts were referenced to internal
tetramethylsilane (TMS) at 0 ppm. The high
degree of symmetry resulted in the simple
spectrum which was interpreted as follows:

Chemica1 Relative
Shift (ppm) Area Assignment

1.35 2H -NH
1.55 6H 8-Ci
1.62 6H 6-CH
2.05 3H y-CH

Chemical shifts of 1-substituted adamantane (78)


show large variations due to both the substituent
( 7 9 ) and the solvent.

3.12 13C-NMR

The exceptional structure of amantadine has


prompted many carbpj~l-13 NMR structural studies.
Figure 5 shows the C-NMR spectrum of a commercial
preparation of amantadine hydrochloride in
deuterochloroform run at 15 MHz on a Jeol FX 60Q NMR
system ( 7 7 ) . The natural abundance carbon shifts
were referenced to the center line of the CDCl
multiplet at 77.0 ppm fion tetramethylsilane, an3
interpreted as follows. The results are in excellent
agreement with values reported previously (80).

Chemical Shift (ppm) Assignment

29.7 6-C
36.2 B -C
46.2 Y-C
47.2 a-C
In addition, the calculated shift values agree
Figure 4 : Proton Magnetic Resonance Spectrum of Amantadine Hydrochloride

in Deuterochloroform, as Recorded at 100 MHz.


16 JOEL KIRSCHBAUM

with the experimental results, showing that no large


steric interactions ,ayd strain exist between the
carbon atoms (81). C Chemical shifts induced by
protonation of the amine showed the effects of charge
to be transmitted along the carbon skeleton (82) and
involve the next-nearest neighbor (6-ef fect) (83).
Substituent effects three bonds away (y-effect) have
also been studied (84).
Comparisons have been made of shifts in 1- and
2-substituted adamantanes (85) , and relaxation times
(86). Relaxation times are summarized in section
4.4, Hydrodynamic properties, lanthanide and other
shift reagents were used to study the structure (87,
88), donor strength (89), and 8- and y-effects (90).

3.13 15N-NMR

The natural abundance 15N nuclear magnetic


resonance shift was measured (91) for amantadine
P3drochlorideY and found to be 317.1 ppm relative to
N-nitric acid. In methanol, the shift of the free
base was 317.5, and in benzene the shift was 316.3.
The three y carbons of 1-aminoadamantane (see the
NMR section for designations of the various carbons
in the skeleton) appear to have no influence on the
shifts of the free amine or of the hydrochloride.

3.2 Ultraviolet Spectrometry

Below is the ultraviolet spectrum of a


commercial preparation of amantadine hydrochloride at
a concentration of 100 mg/mL method, obtained with
the aid of a Perkin-Elmer Model 320 Spectrophotometer
(92). At 226 nm, the molar absorptivity, E , was
0.128; at 222 nm, E was 0.351, and at 205 nv, E was
0.835. Using traditional nomenclature, the Eli
values are 0.0068, 0.0187 and 0.044, respective'fy.
4. Bulk Solution Properties

4.1 Solubilities in Aqueous and Nonaqueous Solvents.


Solubilities of a commercial preparation of
amantadine hydrochloride were determined (93) at room
temperature in various solvents with about one minute
of mixing.
Figure 5 : 13C-Magnetic Resonance Spectrum of Amantadine Hydrochloride in

Deuterochloroform, as Recorded at 15 MHz.


18 JOEL KIRSCHBAUM

So lvent Solubility (mg/mL)

Acetonitrile 4
Chloroform 75
Ethano1 200
Hexanes <1
Isopropanol 35
Methanol 250
Methylene chloride 10
Water-Methanol (1 :1) >250
Water >250
Hydrochloric acid solution 0.1M 350
Aqueous buffer, pH 2 75
Aqueous buffer, pH 4 50
Aqueous buffer, pH 7 -2
Aqueous buffer, pH 10 <1
Sodium hydroxide solution 0.1M <1

The high solubility in acidic aqueous solvents


precluded measuring an intrinsic dissolution rate,
since tablets compressed under 6000 p.s.i virtually
exploded into aqueous solution (94).

4.2 Ionization

Solutions of recrystallized amantadine


hydrochloride were titrated potentiometrically with
0.1M carbonate-free potassium hydroxide at various
temperatures and concentrations (95). Extrapolation
AMANTADINE 19

t o z e r o i o n i c s t r e n g t h gave pK v a l u e s of 10.71 f
0.01 a t 20', 10.58 f 0.07 a t 250aand 10.14 f 0.02 a t
37". A l i n e a r p l o t gave -d(pK ) / d T = 0.034. These
pKa r e s u l t s are similar t o s u c ! a l k y l a n a l o g u e s a s
2-amino-2-methylpropane (pKa = 10.68 a t 25') and
3-amino-3-ethylpentane (pKa = 10.59) , and s u p p o r t t h e
c o n c l u s i o n (96) t h a t t h e r e i s l i t t l e s t r a i n i n t h i s
a l i c y c l i c molecule.

4.3 Dipole Moments

Dipole moments were measured at various


c o n c e n t r a t i o n s i n benzene (97) f o r a series of
1 - s u b s t i t u t e d adamantanes. Amantadine h a s a d i p o l e
moment, of 1.38 Debyes. T h i s h i g h d i p o l e moment may
r e s u l t (98) from an enhanced p o s i t i v e i n d u c t i v e
e f f e c t of t h e adamantyl s k e l e t o n . I n solvents
forming hydrogen bonds w i t h t h e amino group,
anomalous v a l u e s a r e found ( 9 9 ) .

4.4 Hydrodynamic P r o p e r t i e s

R e l a x a t i o n times (TI) were ermined f o r


amantadine i n v a r i o u s s o l v e n t s usingdp'C-NMR (100).
V i s c o s i t i e s of t h e s o l u t i o n a r e a l s o t a b u l a t e d below.

1 3 ~ - ~(set)
1

Solvent Viscosity -
B 1 6
CD OD
cI 0.76 3.9 6.5 279
CDdlg 0.67 5.1 9.4 3.7
CC14 0.89 9.1 16.4 8.2

T v a l u e s a p p e a r t o be determined by t h e tumbling of
1
a monomer; any s o l u t e - s o l u t e o r s o l v e n t - s o l u t e p a i r s
t h a t are formed a r e t o o s h o r t - l i v e d t o b e measured by
t h i s technique.
I n deuteromethanol t h e moment o f i n e r t i a i s I
II'
300, I , 419 and p , which i s 1 1 / 1 , 1.4.
T t e d i f f u s i o n c o n s t a n t s a r e ' Liven below, where
R1 d e s c r i b e d t h e r o t a t i o n about t h e p r e f e r r e d C
a x i g , anClR2 i s t h e d i f f u s i o n c o n s t a n t , i n u n i t s 02
10 sec . The d a t a i n d i c a t e l i t t l e motion about
t h e C-NH2 bond.

Solvent
R1
-- y
CD OD 5.1 1.7
CD?13 7.2 2.2
20 JOEL KIRSCHBAUM

CC14 8.4 5.7


-0
The partial molar volume, V , in water at 25"
was determined (101) to be 138.9 2 0.3 cm3 mo1-l:
The discrepancy with the calculated value of 141.7
was assumed to be caused by interaction of the amino
group with water via hydrogen bonds.

5. Methods of Analysis

5.1 Compositional Analysis

The results of elemental analyses (102) of two


preparations of amantadine hydrochloride agreed with
theoretical results of carbon, 63.40%; chlorine,
18.89%; hydrogen, 9.13%, and nitrogen 7.46%.
Water content was measured by vapor phase
chromotography. A portion of a commercial
preparation of amantadine hydrochloride was dissolved
in methanol and, after retention on a precolumn,
water content of 0.07% was determined by comparison
with an external standard (103).
Emission spectrochemical analysis of a
commercial preparation (66) gave metallic impurity
contents of copper, 7 pg/g and aluminum, 45 pg/g.

5.2 Identity and Colorimetric Methods

A compendia1 method (104) involves reacting


amantadine hydrochloride in pyridine with acetic
anhydride to produce 1-acetamidoadamantane, which
should have a melting range between 147" and 150"
using the U . S . P procedure for class I compounds,
Alternatively, the characteristic infrared system can
be easily obtained for the solid.
Amantadine hydrochloride undergoes the
characteristic reactions of a primary amine. To 2 mL
of amantadine hydrochloride dissolved in
rnethanolic-acetonitrile (less than 0.3% water) add 1
mL each of 30% trichloroacetic acid and 0.2%
dimethylaminobenzaldehyde solution. The Schiff-base
condensation product forms within 10 min standing at
room temperature to give a bright yellow color (105).
Amantadine hydrochloride undergoes reaction with
l-fluoro-2,4-dinitrobenzene in acetonitrile solution
at an apparent pH of 9, at either 65" or on standing
overnight at room temperature, to give a yellow color
(106).
It also reacts with ninhydrin (107) to form the
AMANTADINE 21

iminoindandione, with acetic anhydride (104, 108) to


form 1-acetamidoadamantane (described in the above
identity test) and with isothiocyanate (log), as well
as forming the derivatives discussed in Section 1.5,
reactions, stability and metabolism; 5.4,
spectrometry, and 5.9, fluorescence.
Reactions of amantadine hydrochloride with
reagents on a microscope slide gave characteristic
crystals. A 1M solution of 1-adamantanamine
hydrochloride in 1M HC1 reacted with either palladium
chloride , gold chloride, iridium chloride, ruthenium
chloride, platinum chloride or potassium osmate to
give various shapes, colors and polarization colors
(109). Complexes were formed when one drop of
amantadine hydrochloride was reacted with one drop of
solutions of either 5-nitrobarbituric acid, HAuCl ,
HAuCl plus NaBr , chloroplatinic acid, chloroplatinfc
4
acid plus NaBr, mercuric chloride and platinum iodide
plus chloroplatinic acid with sodium iodide. Under
the microscope different colors and shapes were
visible (110). The original papers contain sketches
of the crystals.

5.3 Titration

The compendial method (104) uses perchloric acid


and either crystal violet indicator solution or
titrat ion to an endpoint determined
potentiometrically. The accuracy was found to be
within approximately 0.1% in various solvents (111).
Using the Kjeldahl procedure , amantadine was
distilled from amantadine hydrochloride, collected in
0.1 hydrochloric acid and determined by difference
after titration with base (112).

5.4 Spectrometry

Nuclear magnetic resonance (113) was used to


quantitate amantadine hydrochloride. Capsules were
dissolved in water, 30% sodium hydroxide was added
and, after extraction into benzene, acid was added to
a portion, then deuterium oxide and, finally, several
drops of hydrochloric acid. The characteristic
proton NMR spectrum (cf. section 3.1) of 0.4 mL of
purified compound was determined using succinimide as
NMR reference standard. The NMR and compendial
titration method, described in the previous section,
gave similar results. The relative standard
22 JOEL KIRSCHBAUM

deviation of the NMR procedure is 0.8%, it is,


however, an order of magnitude higher than
titrimetry.
Amantadine hydrochloride in biological fluids
was reacted with p-nitrobenzaldehyde (114) to give
1-(p-nitrobenzylidenamino)adamantane, which has
maximal absorption at aqproximately 292 nm (molar
absorptivity is 1.89 x 10 ) . The limit of detection
was approximately 1 pg per mL of blood and plasma.

5.5 Gas-Liquid Chromatography

The method preferred by the pharmaceutical


manufacturer for quantitat ing amantadine
hydrochloride is gas-liquid chromatography (115).
The various GLC systems are summarized in Table 1.
Figure 6 exemplifies gas chromatographic assays for
amantadine in plasma and urine, and were redrawn from
reference (120) with the permission of the Elsevier
Scientific Publishing Company.

5.6 Thin-Layer Chromatography

Table I1 summarizes TLC systems developed for


amantadine.

5.7 High-Performance Liquid Chromatography

The lack of an HPLC method in the literature


prompted development of an assay by the author. Both
reverse and normal phase systems gave multiple
irreproducible peaks for commercial amantadine
hydrochloride known to be of high purity. This
phenomenon was indicative of non-covalent
interactions. Derivatization similar to the U.S.P.
identity test (Section 5.2) but using phthalic
anhydride, gave a compound absorbing at 254 nm. An
octadecylsilane column was used with a mobile phase
of methanol-water-85% phosphoric acid (60:40:0.1)
flowing at 1 mL/min to obtain the chromatogram shown
on next page. Amantadine has negligible absorbance
at 254 nm. Reproducibility and linearity of the
amantadine peak were excellent. Details will be
published subsequently (126).

5.8 Electrochemistry

Oxidation (127) of amantadine in trifluoroacetic


acid gave several anodic waves. The voltammetric
AMANTADINE 23

1 2

- 1

0 4 8 0 4 8 12

Figure 6 .
0 4
1 2

8
Rerention

Gas-Liquid Chromatograms:
J
0
time
4
(min)
I
8

Upper P o r t i o n , A ,
c o n t r o l plasma; B , plasma e x t r a c t c o n t a i n i n g 457 ng
amantadine h y d r o c h l o r i d e (1) p e r mL: Lower P o r t i o n , C ,
C o n t r o l u r i n e ; D , Urine e x t r a c t c o n t a i n i n g 62.4 ug/mL drug
(1). Chlorphentermine i s t h e i n t e r n a l s t a n d a r d ( 2 ) .
Table I. Gas Liquid Chromatography of Amantadine

Bulk Amantadine
Column Carrier T(Co1umn) Detector Reference

5% by weight SE-30 on Chromaton N-AW-DMCS 125" Flame Ionization 116


(particle size 0.16-0.20 mm) N2

10% by weight 0s-124 on Chromaton-N-AW-HMDS 190" Flame Ionization 116


(particle size 0.16-0.20 mm) N2

5% by weight polyethylene glycol (M.W. 1SOO) N2


160" Flame Ionization 116
and 1% KOH on Chromaton N (particle size
0.20-0.25 IIIUI)

3% OV-17 on Chromosorb W He 180" Flame Ionization 117


20% Carbowax on Chromosorb W He 98" Scintillation - 57
Gas Flame

3% OV-1 on Chromosorb W-HP (100-120 mesh) N2,H2,Air 150-250" N-P Detector 118

3% OV-17 on Chromosorb W-HP (100-120 mesh) N 2 ,H2 ,Air 150-250" N-P Detector 118

Amantadine in Blood and Plasma


1.5% SE-30 on Anakrom AS (90-100 mesh) 220" Electron Capture 114
(claim linear response of 0.03-0.15 pg) N2
Table I GLC, continued

co l
m Carrier T(Co1umn) Detector Reference

2% Dexsil 300 on Gas Chrom P or 0 180-260" Mass Spectrometry 62


N2
(80-100 mesh)

Amantadine in Various Tissues and Body Fluids

20% Carbowax 20 M on base-treated Chromosorb W He, H


2'
95" Flame Ionization 123, 124
Air

3% OV-17 on Gas Chrom Q (100-120 mesh) Air, €? 0 150" Flame Ionization 61


2
N2
N
vI 7.5% PEG 20,000 on Gas Chrom Q (100-120 mesh) N2, He 170" Flame Ionization 61

5% Apiezon L o r Gas Chrom 0 (100-120 mesh) H2, Air 160" Flame Ionization 61
2% OV-101 on Chromosorb W (100-120 mesh) Air, H
2 130" Flame Ionization 61
N2
After conversion to isothiocyanate, 3% OV- He 170"or190" Mass Spectrometer 108
225 on Gas Chrom Q

After conversion to isothiocyanate He 180" Mass Spectrometer 108


3% OV-17 on Chromosorb W-HP

After conversion to isothiocyanate He 160" Ma ss Spectrometer 108


OV-101 or Gas Chrom Q
Table I. GLC, continued

COZUmn Carrier T(Colunm1 Detector Reference

After conversion t o N-trichloroacetyl N2


220" Electron Capture 119
derivative, 3% OV-17 on Chromosorb Q (100-
120 mesh). Claim linear response 25ng-ll~g/ml,
determined by MS.

Amantdine in Plasma and Urine

5% Apiezon on Gas-Chrom Q (100-120 mesh) 150" Flame Ionization 120


H2
N After conversion t o trichloroacetyl Ar-CH 200" Electron Capture 121
ch
derivative 5% SE-30 on Chromosorb W HP (90: 16)
(80-100 mesh)

Amantdine i n Urine
10% Apiezon L and 2% KOH on 80/100 145" Flame Ionization 122
N2
Chromosorb W AW

20% Carbowax 20 M on Gas Chrom P or Q 180-260" Mass Spectrometry 62


N2
20% Emulphor ON-870 on Gas Chrom P or Q 180-260" Mass Spectrometry 62
N2
1%SE 30 on Gas Chrom P or Q 180-260" Mass Spectrometry 62
N2
1%OV-17 on Gas Chrom P or Q 180-260" Mass Spectrometry 62
NH2
AMANTADINE 21

Pig 7. HPLC of Amantadine Hydrochloride.

data is E 1 / V = 2.30 and E 122/V=2.67.


Coulgkztry with bromge (128) was used to
quantitate amantadine hydrochloride in aqueous
solution at pH 8 using concentrations as low as 0.5 u
mole per liter.
The interaction of amantadine with lecithin
bilayers used as a membrane model was studied using
polarography (129). Amantadine affects permeability.

5.9 Fluorescence Spectrometry

The lack of natural fluorescence required


fluorescence of amantadine to be induced. Reaction
with fluorescamine (130) at a concentration of 1 pM,
followed by extraction at pH 5 into ethyl acetate,
gave a product with a maximum at 395ex/475em nm. The
limit of sensitivity is 1 ug/mL.
Reaction of 9 - i s o t h i o c y a n a t o a c r i d i n e ( 1 3 1) with
amantadine hydrochloride in ethanol, with heating,
yielded a product that was quantitated from 0.5-2
;g/mL using- 295ex/490em nm. Thin layer fluorimetry
was used.
Table 11. Thin-Layer Chromatography of Amantadine

Support Mobile Phase Detection Re ference

Silica gel Methanol-ammonia (100:1.5) Plate sprayed sequentially 125


with ninhydrin solution, Iron
(111) chloride-perchloric acid,
Dragendorff reagent (Bismuth
subnitrate in acetic acid then
aq. KI), and finally iodoplati-
Silica gel Cyclohexane-toluene-diethylamine nate solution. Second plate 125
(75: 15: 10) had Marquis reagent (formalde-
hyde in H SO ) poured over it.
2 4
Silica gel Chloroform-methanol ( 9 : 1) 125
0

Silica gel Acetone 125

Silica gel Butanol-acetic acid-water (4:1:5) Ninhydrin 57

Silica gel F254 Methanol-25% NH3 (100:1.5) Dragendorff 118

Silica gel F254 Dichloromethane-acetone (4:l) Chlorinate in C12; spray 118


with o-tolidine in ethanol

Silica gel G CHCl -methanol-benzene-25% NH3 Dragendorff 112


(50:20:40:2)
Silica gel G Lower phase HAc-CC1 -CHCl -H 0 61
3 2 3’
( 100 :60: 9 6 : 50) 5%; sulfanilic acid (0.5%)-1-
naphthylamine (0.1%)
Silica gel G n-Butanol-toluene-HAc-H 0 61
2
ninhydrin in n-butanol (98%)
Silica gel G n-Hep tane and HAc (2%) ; triphenyl- 61
tetrazolium reagent; or after
30 JOEL KIRSCHBAUM

5.10 Tissue Culture

Tissue culture and human trial methods for


measuring inhibition of viruses were reviewed
previously (1,2,132). Viral growth inhibition by
amantadine hydrochloride was determined by plaque
formation in Petrie plates with a gradient thickness
of agar plus compound (133). As for most
microbiological assays, analytical errors were
relatively large, typically averaging 22% (134).

5.11 Comparison of Methods

Infrared spectrometry is recommended for an


identity test. Gas-liquid chromatography was the
most frequently used procedure €or quantitating bulk
amantadine hydrochloride and amantadine in tissues
and body fluids. Although titrimetric methods are
more accurate, possible impurities may also be
titrated, limiting the usefulness of the simple
technique. No high-performance liquid
chromatographic assay was found in a search of the
literature, through written requests to several
investigators who published HPLC methods for
adamantanes or by contacting representatives of the
manufacturing pharmaceutical company. Accordingly,
an HPLC method was developed in this laboratory.

6. Acknowledgements

The author gratefully acknowledges the


assistance of the many contributors cited as
"personal communications". With one exception, this
information was especially obtained for this review
by investigators at the Squibb Institute for Medical
Research. The Crystallographic Data Centre,
Cambridge, gave permission t o reproduce the x-ray
derived structure. The Elsevier Scientific
Publishing Co., Amsterdam, gave permission to redraw
the gas-liquid chromatography figure. The figures
utilized the illustrative skills of Jose Alcantara
and the photographic talents of Charlotte Raymond.
The preparation of camera-ready copy was by
Lynn Mendenko. Eugene Ivashkiv provided translations
from Russian. The author appreciates the critical
reading and thoughtful comments of Dr. J. Adamovics,
Dr. G. Brewer and Mr. S . Perlman. Credit for
obtaining many of the references belongs to
Muriel George, Phyllis Gottstine, Letitia Fenwick and
AMANTADINE 31

Michael M a s i e l l o . The Chemical A b s t r a c t s c i t a t i o n s


were appended t o a r t i c l e s t h a t were d i f f i c u l t t o
o b t a i n o r where o n l y t h e a b s t r a c t was read.
L i t e r a t u r e was surveyed t o J a n u a r y , 1983.

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AMANTADINE 33

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Communication.
77. M. Porubcan, Personal Communication
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4593 (1971).
79. R.C. Fort, Jr., and P. von R. Schleyer, J . Org.
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80. H. Duddeck and P. Wolff, Org. Map. Reson, 8,
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81. T. Pehk, E. Lippmaa, V.V. Sevostjanova, M.M.
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84. J.B. Lambert and A. R. Vagenas, Org. Map.
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87. D.J. Chadwick and D.H. Williams, J . Chem. SOC.,
AMANTADINE 35

Perkin Trans., (1974) 1202.


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89. D.M. Rackam, Spectrosc. L e t t . , 12, 603 (1979).
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92. D. Sieh, Personal Communication.
93. L. Kerr and J. Kirschbaum, Personal Commun-
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94. E. Rudnic, Personal Communication.
95. D.D. Perrin and I. Hawkins, Experentia, 28, 880
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96. B.A. Korolev, A.P. Khardin, S.S. Radchenko, I . A .
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1632 (1978); CA, 89, 196776n (1978).
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by Consultants Bureau, J . Gen. Chem., 41,. 1640
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99. A.E. Lutskii and V.V. Prezhado, Zh. Fiz. Khim.,
4 5 , 1273 (1971); CA, 75, 102504h (1971).
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J . Chem., 5 , 1224 (1979).
101. F. Shahidi, P.G. Farrell and J.T. Edward, J .
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102. G. Conroy, Personal Communication.
103. M. Jemal and R. Mark, Personal Communication.
104. United S t a t e s Phamacopeia, 20, 26 (1980).
105. P. Stear, Personal Communication.
106. J. Kirschbaum, Personal Communication.
107. P.A. Crooks, Chem. Ind. (London), 1980, 467.
108. N. Narasimhachari, E. Helgeson and U. Prakash,
Chromatographia, 1 2 , 523 (1979).
109. H.F. Schaeffer, Microchem. J . , 9, 492 (1965).
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425 (1974).
36 JOEL KIRSCHBAUM
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Knqaku, 1 9 , 9 5 ( 1 9 7 0 ) ; CA, 73, 12723h ( 1 9 7 0 ) .
115. G. White, Endo L a b o r a t o r i e s , P e r s o n a l
Conyn:$ation.
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(1979). .
AMIKACIN SULFATE
Peter M . Monteleone, Naseem Muhammad,
Robert D . Brown, John P . McGrory,
and Samir A. Hanna

1, Historical and Description 38


1.1 Chemical Origin 38
1.2 Chemical Structure 39
1.3 Name, Formula, and Molecular Weight 39
1.4 Appearance 40
1.5 USP Standard 40
2. Physical Properties 40
2.1 Melting Range 40
2.2 Specific Rotation 40
2.3 Solubility 40
2.4 Electrometric Titration Curve and Apparent pK. Value 41
2.5 Spectra 41
2.6 X-Ray Powder Diffraction 49
2.7 Thermal Analysis (DSCand TGA) 49
3. Synthesis 56
4. Drug Metabolism and Pharmacokinetics 56
5. Stability-Degradation 58
6. Methods of Analysis for Bulk and Injection 59
6.1 Identification 59
6.2 Moisture Content 59
6.3 Specific Rotation 59
6.4 Microbiological Assay 59
6.5 Other Compendial Tests 60
6.6 Assay for Sulfate 60
7. Chromatographic Methods 60
7.1 Thin-Layer Chromatography 60
7.2 High-Pressures Liquid Chromatography (HPLC) 60
7.3 Gas-Liquid Chromatography (GLC) 60
8. Determination in Body Fluids 62
8.1 Microbiological Assay 62
8.2 Fluorescenceimmunoassay(FIA) 63
8.3 Radioimmunoassay (RIA) 63
8.4 Radioenzymatic Assay (REA) 63
8.5 Spectrophotometric-EnzymaticAssay 65
8.6 High-pressure Liquid Chromatography (HPLC) 66
8.7 Gas-Liquid Chromatography (GLC) 66
References 67

ANALYTICAL PROFILES OF DRUG SUBSTANCES 37 Copyright by [he American Pharmaceulical Association


VOLUME 12 ISBN 0-12-260812-7
38 PETER M . MONTELEONE E T A .

1.0 Historical and Description:


Amikacin is a semisynthetic aminoglycoside
produced by the strategic chemical alteration of
kanamycin A. The discovery of kanamycin was re-
ported in 1957 by Dr. Hamao Umezawa’. It is made
by fermentation and has been used to treat many
serious infections caused by gram-negative
bacteria. A s the use of Kanamycin A and other
aminoglycosides became widespread, resistant
strains of bacteria began to emerge. The insight
gained into the enzymatic mechanisms by which
resistant strains inactivated aminoglycosides and
the understanding of structural features related
to microbiological activity formed the basis for
synthesizing better aminoglycosides2. In 1972,
Dr. Hiroshi Kawaguchi at the Bristol-Banyu
Research Institute in Tokoyo described the dis-
covery of BB-K8 (amikacin) and demonstrated its
improved effectiveness3.
In 1976 the F . D . A . approved the marketing of
amikacin. Since then it has found worldwide use
in the treatment of serious gram-negative infec-
tions. The usual marketed forms are 50 and 250
mg/ml solutions designated Amikacin Sulfate
~njection~.
Several articles review the general micro-
biological5, pharmacological, and biochemical
properties6 of amikacin, as well as therapeutic
experience7, with Amikacin Sulfate Injection.
1.1 Chemical Origin:
-
Amikacin is prepared by acylation of
kanamycin A at the C-1 amino group of the
2-deoxystreptamine moiety with the I,(-1-y-
amino-a-hydroxybutyrl group. Acylation of
each of the four amino groups in kanamycin A
with the L(-)-y-amino-a-hydroxybutyrl sub-
stituent gives 4 different derivatives. A s
mentioned, amikacin, originally named BB-K8,
is the C-1 amino derivative. The other three
amino derivatives are BB-K6 (C-6’1, BB-K11
(C-3”) and BB-K29 (C-3) ’.
AMIKACIN SULFATE 39

1.2 Chemical Structure:


-
The structure of am kacin is shown in
Figure 1. The chemistry and proof of struc-
ture have been reported by H. Kawaguchi3.

L (-1
H-C0-C H-C
1
OH
OH

HO

FIGURE 1 STRUCTURE O F AMIKACIN

1.3 Name, Formula and Molecular Weight:


The USAN and USP Dictionary of Drug
Names lists the following names for amikacing:
o-Streptamine, 0-3-amino-3-deoxy-a-
o-glucopyranosyl (1-t6) -0- [ 6-amino-
6-deoxy-a-~-glucopyranosyl (1+4)1-
N1-(4-amino-2-hydroxy-l-oxobutyl)-
2-deoxy, ( S ) - ;
O-3-Amino-3-deoxy-a-D-glucopyranosyl
(1-+4)-0-[6-amino-6-deoxy-a-~-gluco-
pyranosyl (1+6)l-N3-(4-amino-~-2-
hydroxybutyryl)-2-deoxy-~-strept-
amine.
Cz2H43N5013 M.W. = 585.61
Amikacin sulfate is a 1:2 sulfate salt of
amikacin. The following are its formula and
formula weight.
C22H43N5013*2H2S04 F.W. = 781.75
40 PETER M. MONTELEONE E T A .

Amikacin h a s b e e n r e p o r t e d t o e x i s t a s a
f r e e b a s e (CAS Reg. N o . 37517-28-51, a 1 : 2
s u l f a t e (CAS Reg. N o . 39831-55-5), a 1:1
s u l f a t e (CAS Reg. N o . 56086-43-2) and a 1:1
c a r b o n a t e (CAS Reg. N o . 75282-58-5).

1.4 Appearance :

Amikacin i s a w h i t e , c r y s t a l l i n e p o w d e r 3 .
The u s u a l m a r k e t e d form i s a s t e r i l e , color-
less t o s t r a w yellow s o l u t i o n f o r i n j e c t i o n
p r e p a r e d by d i s s o l v i n g a m i k a c i n w i t h d i l u t e
s u l f u r i c a c i d a t a 1 : 2 r a t i o o f a m i k a c i n base
t o H2S04. The s o l u t i o n may c o n t a i n p r e s e r -
v a t i v e s , such a s b i s u l f i t e s and b u f f e r s , s u c h
a s c i t r a t e . The amount o f a m i k a c i n i s e i t h e r
50 o r 250 mg p e r m 1 4 r l o ,

1.5 USP S t a n d a r d :

The c u r r e n t USP s t a n d a r d f o r a m i k a c i n ,
L o t G-1, i s t h e f r e e base a n d h a s a n a s s i g n e d
p o t e n c y of 9 1 4 mcg/mg on a n " a s i s " b a s i s .

A n a l y s i s o f t h a t material g a v e a m o i s t u r e
c o n t e n t of 7 . 3 % 1 2 .

2.0 -
Physical Properties:

2.1 M e l t i n a Ranae:

H . Kawaguchi r e p o r t e d a m e l t i n g r a n g e
203-2040C3. commercial m a t e r i a l r a n g e s f r o m
201-204°C'
2.2 -
Specific Rotation:

The s p e c i f i c r o t a t i o n of a m i k a c i n h a s
been r e p o r t e d a s = +99O, (C= 1 . 0 , H 2 0 ) '.
The CFR r e q u i r e m e n t f o r t h e d r i e d s u b s t a n c e i s
+ 9 7 O t o +1O5Ol4

2.3 Solubilitv:

The e q u i l i b r i u m s o l u b i l i t y i n w a t e r a t
25OC h a s been r e p o r t e d a s 1 8 5 m g / m l 1 3 . Dosage
AMIKACIN SULFATE 41

solutions,prepared as the sulfate salt, con-


tain up to 250 mg/ml as amikacin.
2.4 Electrometric Titration Curve and
-
Apparent pKa Value:
Figure 2 is the electrometric titration
curve for amikacin. It was determined by
conditions which are similar to those reported
for k a n a m y ~ i n ’ ~ .Amikacin (0.1 m mole) was
dissolved in water, and 0.5 m mole of potas-
sium hydroxide was added. The solution was
titrated with 0.5N hydrochloric acid using
SCE/glass electrozes. Instrumentation in-
cluded a Radiometer recording titration
system with PM-64 p H meter TTTGO titrator,
REC-61 servograph and an ABU-13 autoburet16.
If the four amine groups in amikacin are
considered to be equivalent, then an apparent
pKa value of 8.1 may be estimated from the
half neutralization point’’.
2.5 Spectra:
2.5.1 Infrared
- Spectrum
Figure 3 is the infrared spectrum
for amikacin in a potassium bromide
pellet. Band assignments are listed in
Table 1”.
Ma’or bands reported by H.
Kawaguchi’ occurryd at 3350 , 2930 , 1640,
1580 and 1350 cm- .
2.5.2 Proton Nuclear Magnetic Resonance
-
SDectrum
The 90 MHz proton NMR spectrum for
amikacin in D20 is shown in Figure 4.
Table 2 lists the shift assignments”.
H. Kawaguchi has reported the 100 MHz
spectrum for amikacin3.
42 PETER M. MONTELEONE ETAL.

13

12

11

10

8
P"

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

FIGURE 2 ELECTROMETRIC TITRATION CURVE OF AMlKAClN


FIGURE 3 INFRARED SPECTRUM O F AMlKAClN
F I G U R E 4 P R O T O N NMR S P E C T R U M O F A M l K A C l N
d
.rl
0
Id
GI
m
u
&
0
c
ra
4-1 a,c
zrd
h CI)
0 n c,
ICC X c
z a,
[I)
-I-' n 0 !2
c
uI
N I
3: o=u
u I
a
2
rd 0
A c*l
d
0 o r l
co Lo
a3 L O -
O N d o
0
a,
Lo
m n
b rc,
n o @a,
PI m o 0 4 rlrl
rn N f A A
m a - 2 3
a cv r l o 0 0
LO nn
0
rd rl
F-l
ui
c
H
m LO
..
c
u
N
I
f
I

CDN
..
rlf
rl CD Lnm
a,
I+ ri N
A
rd
E-1
45
46 PETER M. MONTELEONE E T A .

2.5.3 Carbon-13 NMR Spectrum


The Carbon-13 NMR spectrum for
amikacin is shown in Figure 5. It was
obtained on a Bruker WM-360 WB instru-
ment at the following conditions: 90.6
MHz; spectral width, 15152 Hz; repetition
time, 0.6 sec.; solvent, D20; number of
scans, 2496.
The chemical shifts and spectral
assignments are listed in Table 3”.
These are in accord with the values
originally reported by S . Toda, who
studied the C-13 NMR spectra of amikacin,
its three positional isomers and four
related compounds2’.
2.5.4 Ultraviolet Spectrum
-
Amikacin in water at a concentra-
tion of 1.0 mg/ml shows only end absorp-
tion beginning at about 240 nmI6.
2.5.5 -
Mass Spectrum
The positive ion fast atom bom-
bardment (FAB) mass spectrum of amikacin
is presented in Figure 6. It was obtain-
ed on a VG-ZAB-2F mass spectrometer using
a solution prepared in thioglycero122.
The spectrum shows a strong protonated
molecular ion [M+H]+ which agreed with
the molecular ion with hydrogen loss
observed by negative ion FAB. Spectra
were intense and easy to observe with
most characteristic ions greater than
mass 210. Electron impact (EI) and
positive chemical ionization (PCI) mass
spectrometry failed to yield spectra
which included the molecular ion23.
Amikacin is quite labile in part
because the L-y-amino-a-hydroxybutyric
acid (L-HABA) side chain can fragment or
rearrange very readily. Structures are
given for some of the most prominent
FIGURE 5 CARBON - 13 NMR SPECTRUM OF AMlKAClN
48 PETER M. MONTELEONE ETAL.

Table 3: Carbon-13 chemical s h i f t s for amikacin.

CHEMICAL SHIFT
CARBON (pm) *
c-1 50.4
c-2 35.0
c-3 49.4
c-4 87.5
c-5 75.4
C-6 81.1
c-1’ 99.1
c-2’ 72.6
c-3’ 73.8
c-4’ 71.8
c-5’ 73.7
C-6’ 42.3
c-1- 100.2
c-2- 72.4
c-3- 54.9
c -4& 70.0
c-5- 72.8
C-6- 61.1
c= 0 177.2
Ca 70.7
C B 36.9
C Y 38.1

*Chemical shifts of free base i n ppm downfield from TMS (in D20)
AMIKACIN SULFATE 49

fragments present in the spectrumz3.


Most include some reaction of the L-HABA
moiety. The structures, given in Table 4
and Figure 7, are consistent with the
ions formed and result from very simple
cleavages. Other structures are also
consistent with some of these ions,
particularly because the two sugars are
structural isomers23.
2.6 X-Ray Powder Diffraction:
The x-ray powder diffraction pattern for
amikacin was obtained using a Rigaku Powder
Diffractometer. Copper Ka radiation ( A =
1.54051) was used with a nickel filter
(Figure 8). The results are listed in
Table 5'l.
2.7 Thermal Analysis (DSC & TGA)
-:
A thermogravimetric analysis curve was
obtained using a Perkin-Elmer TGS-2 thermo-
gravimetric analyzerz1. The analysis was
performed at a heating rate of 10°C/minute
from 4OoC to 300OC.
The TGA curve (Figure 9) shows a weight
loss of 8% from 5OoC to 100°C, due to loss of
water which is probably loosely bound and
evolved slowly. A second weight loss begins
at 22OOC and continues beyond the end of the
scan at 300OC. This is due to decomposition
and is apparently related to the last two
transitions observed by DSC.
A differential scanning calorimetry curve
was obtained (Figure 10) on a Perkin-Elmer
DSC-2 differential scanning calorimeter.
Using nitrogen as the purge gas, the scan was
performed at a rate of 10°C/minute from 4OoC
to 300OC. A broad endotherm was obtained from
119OC to 147OC, which was shown to be revers-
ible and may be due to a crystal phase transi-
tion or melting. Subsequent irreversible
endotherms were obtained at 187OC. 222OC and
260OC. These are attributed to decomposi-
tion2
a
I-
0
w
P
rn
rn
CI)
a
I
m
a
u.
(0
W
a
50
Table 4: F A B mass spectral ions and structural components for amikacin.

Ions
- Structure Formation
586 [M+H1 + Protonated molecular ion.

556 [M+H minus ( c H ~ - N H I~+) Cleavage from the L-HABA side


chain or from Ring A.

542 [M+H minus ( C H ~ - C H , - N H ~ ) I + Cleavage from the L-HABA side


chain.

512 [M+H minus ( C H O H - C H ~ - C H ~ - NH~)]+ Loss of all but the carbonyl of the
L-HABA side chain.

498 [M+H minus CHO-~OH-CH~-NH~) I+ Loss of C 4 ’ - C 5 - - C 6 ’ linked in Ring A.

440 [M+H minus L-HABA) minus (CH~=CH-OH)


I+ L o s s of the L-HABA side chain and
,.
elimination of c S ” - C ~ in Ring c.

429 [M+H minus 2 ( ~ ~ minus


0 ) ( N H ~ - C H ~ - C H O H - C H O H - C H ~ IO+H ) Loss of 2H20 and C S ’ - C / - C ~ ”o
-f
CK
Ring A or C with the ring oxygen and
H rearrangement.

382 [M+H minus (Ring A minus H) minus (CH2=CHNH2)]* Loss of Ring A with H rearrangement
and part of the L-HABA side chain.

324 [M+H minus (Ring A minus H) minus L-HABA + HI+ Simple cleavaqes with H rearrangement
leaving 2 rings.

217 [M+H minus (Ring A+H) minus (Ring C+H) minus Cleavage of both sugars and part of
(CHz=CH-NHz) 1’ the side chain.

215 [M+H minus (Ring A+H) minus (Ring C+H) minus A s above, yet H rearrangment is
(CH3-CHz-NHz) I+ reversed at one point.
52 PETER M. MONTELEONE E T A .

OH
$4
I
HO
bH
0

m l z 382 m l z 440

8 RING B

AMIKACIN

m l z 324 mfz 215

FIGURE 7 AMlKAClN FRAGMENTS


F I G U R E 8 X - R A Y D I F F R A C T I O N P A T T E R N OF AMlKAClN
54 PETER M. MONTELEONE ETAL.

Table 5: X-ray powder diffraction of amikacin.

d* (i) 1/ 109<$9 d (i) 1/10


-- d (i) 1/10
--
6.701 52 4.149 20 3.114 5

6.188 36 4.022 9 3.023 8

6.078 16 3.994 8 2.996 8

5.719 14 3.883 25 2.915 5

5.618 19 3.742 38 2.870 9

5.548 5 3.696 13 2.803 5

5.291 11 3.630 30 2.754 5

5.023 63 3.531 17 2.728 9

4.935 20 3.504 19 2.657 6

4.766 100 3.310 16 2.567 6

4.667 13 3.259 2 2.330 8

4.485 48 3.229 6 2.312 8

4.287 34 3.184 11

ftd = nX interplanar distance


2 sin 8
i'cf:I/Io- relative intensity (based on highest
intensity of 100)
F I G U R E 0 TGA C U R V E S F O R A M l K A C l N
56 PETER M. MONTELEONE ETAL.

3.0 Synthesis:
Amikacin (BB-K8) is a semisynthetic derivative
of kanamycin acylated with L(-)-y-amino-a-hydroxy-
butyric acid (L-HABA) at the C-1 amino group of the
2-deoxystreptamine moiety.
Amikacin (BB-K8) has been prepared by Hiroshi
Kawaguchi et a1.3 starting from kanamycin, I. The
carbobenzoxylation of kanamycin at the 6’-amino
group was achieved by the activated ester method
using N- (benzylcarbonyloxy)succinimide (CBZ-NOS),
11. The reaction product was purified by ion-
exchange chromatography on Amberlite CG-50 to give
6’-N-benzyloxycarbonyl kanamycin, 111, as shown in
Figure 11. The acylating agent was prepared by
protecting the y-amino group of L-HABA, IV, with
carbobenzoxy chloride, followed by reaction with
N-hydroxysuccinimide to yield active ester of
N-CBZ-protected-L-HABA, VI.
The acylating of 6’-N-Benzyloxycarbonyl kana-
mycin, 111, at C-1 amino group of the 2-deoxy-
streptamine moiety was carried out by an equimolar
reaction with Compound VI at room temperature. The
reaction mixture was subjected to hydrogenolysis
to remove protecting groups at both the 6’-amino
group of the 6’-amino-6’-deoxy-~-glucose moiety of
kanamycin and the y-amino group in the acyl side
chain. The crude amikacin (BB-K8) obtained was
purified by column chromatography on Amberlite
CG-50 with aqueous ammonia. The active fractions
were combined, evaporated in vacuo and the residue
was crystallized from methanol-isopropanol to yield
needle crystals of amikacin base, VII. Synthesis
of amikacin is also claimed in three U . S .
patents2 5, 6. The first of these is the one
claiming the product, while the latter two claim
alternative processes for its preparation.
4.0 Drug Metabolism and Pharmacokinetics:
Amikacin is usually given by intramuscular
injection or intravenous infusion. Protein
binding is minimal. Almost all of the dose is
excreted unmetabolized in the urine6f2’.
AMIKACIN SULFATE 57

L I-)
wM+ticy- $H-COOH
on

CbX-CJ

u-3
HHCO-CH-CH2-CHz
bn hz

VII Amikacin ( B B - K 8 )

Figure 11. Synthesis of Amikacin


58 PETER M . MONTELEONE ET AL.

The study published by H. Lode et a1.28 illus-


trates the pharmacokinetic properties of amikacin.
They reported a mean half-life of 114 2 1 6 . 7
minutes after a 1-hour continuous intravenous in-
fusion of amikacin at a dose of 7.5 mg/kg. Peak
serum levels during infusion reached 37.5 ? 4.9
mcg/ml. Eight hours later they dropped to 1.3 ?
0.5 mcg/ml. In the 24-hour period after dosing,
93.5% of the dose was recovered in the urine
(12 subjects). Using a single intramuscular
injection of 5 mg/kg, peak serum levels reached
21.4 f 5.4 mcg/ml, then declined to 2.4 f 0.9 mcg/
ml by 8 hours ( 3 0 patients).
Based on studies in beagle dogs and humans
after intravenous and intramuscular dosing,
B. C. Cabana and J. G. Taggart” concluded that
the pharmacokinetic profiles of amikacin and
kanamycin are similar.
Evaluating data from a crossover study in
humans, J. T. Clarke et a1.30 also reached the
conclusion that amikacin and kanamycin have similar
1

pharmacokinetics.
D. Zaske and K. CrossleyZ7, as well as K. A.
Kerridge6 have published articles which include
reviews for the pharmacokinetics of amikacin.
5.0 Stability - Degradation:
The stability of amikacin and amikacin sulfate
is reported in a series of papers by Kaplan
et al. 2, 3, These authors provide data on
the stability of amikacin base powder and solution,
aqueous amikacin sulfate solutions, and solutions
of amikacin sulfate admixed with various intra-
venous solutions with and without other therapeutic
agents present. Data given in these papers demon-
strate the chemical and thermal stability of
amikacin. Different lots of the free base powder
subjected to 56OC for four months showed an average
potency loss of 7.2 percent. At 25OC for 24 months
an average loss of 3.9 percent was observed. A
solution of the free base in distilled water at
approximately 185 mg per ml was autoclaved in a
closed vial for 30 minutes at 1 5 lb. pressure and
AMIKACIN SULFATE 59

1 2 1 O C ; no l o s s i n p o t e n c y was o b s e r v e d , a c o l o r
c h a n g e t o l i g h t y e l l o w was o b s e r v e d .

The s t a b i l i t y o f a q u e o u s s o l u t i o n s o f a m i k a c i n
s u l f a t e w a s summarized a s f o l l o w s : The a m i k a c i n
a c t i v i t y w a s maintained a t g r e a t e r than 90 percent
o f t h e o r i g i n a l l y p r e s e n t amount a f t e r e l e v a t e d
t e m p e r a t u r e s t o r a g e a t 56OC a n d 45OC f o r 4 m o n t h s ,
37OC f o r 1 2 months, a n d 25OC f o r up t o 36 months.
The p H o f t h e s o l u t i o n s w e r e 4 . 5 t o 6.8 f o r v a r i o u s
c o n c e n t r a t i o n s o f amikacin s u l f a t e w i t h and w i t h o u t
t h e presence of paraben p r e s e r v a t i v e s .

6.0 Methods o f A n a l y s i s f o r Bulk and I n j e c t i o n :

R e q u i r e m e n t s and methods a r e d e s c r i b e d i n t h e
1982 CFR, T i t l e 2 1 , p a r t s 444.6 and 444.206.

6.1 Identification:

I d e n t i f i c a t i o n i s p e r f o r m e d by c o n t i n u o u s
f l o w TLC ( 2 1 CFR 4 3 6 . 3 1 8 ) .

6.2 - oisture Content:


M

M o i s t u r e i s d e t e r m i n e d by K a r l F i s c h e r
t i t r a t i o n ( 2 1 CFR 4 3 6 . 2 0 1 ) . I t must n o t b e
more t h a n 8 . 5 % .
6.3 -
Specific Rotation:

The CFR r e q u i r e m e n t f o r s p e c i f i c r o t a t i o n
f o r a m i k a c i n b u l k i s n o t less t h a n + 9 7 O a n d
n o t more t h a n +105O ( a n h y d r o u s ) . The d e t e r -
m i n a t i o n i s made by t h e p r o c e d u r e d e s c r i b e d i n
436.20 u s i n g a n a q u e o u s s o l u t i o n c o n t a i n i n g
a m i k a c i n a t 2 0 mg/ml and a 1 d e c i m e t e r p o l a r i -
meter t u b e .
6.4 Microbiolosical Assav:

P o t e n c y i s d e t e r m i n e d by t h e m i c r o b i o -
l o g i c a l a s s a y f o r b o t h t h e b u l k and i n j e c t i o n .
I t is a t u r b i d i m e t r i c assay using Staphylo-
c o c c u s a u r e u s ATCC 6538P ( 2 1 CFR 4 3 6 . 1 0 6 ) .
Ti minimum p o t e n c y o f 9 0 0 mcg/mg, ( a n h y d r o u s )
i s r e q u i r e d f o r t h e b u l k and 9 0 % t o 1 2 0 % o f
t h e claimed value f o r t h e i n j e c t i o n .
60 PETER M. MONTELEONE ETAL.

6.5 Other Compendia1 Tests:


-
Amikacin bulk must additionally meet the
CFR requirements for safety, pH, residue on
ignition and crystallinity.
Amikacin injection must also pass the
test for safety, pyrogens, and sterility,
and have a pH of not less than 3 . 5 and not
more than 5.5.
6.6 Assay for Sulfate:
Although assay for sulfate in amikacin
injection is not a compendia1 requirement,
it may be determined by the gravimetric
method using barium sulfate p r e ~ i p i t a t i o n ~ ~ .
The procedure is very similar to that de-
scribed for gentamicin sulfate36.
7.0 Chromatographic Methods:
7.1 Thin-Layer Chromatography (TLC)
-
Table 6 summarizes TLC methods for
amikacin and related compounds.
7.2 High Pressure Liquid Chromatography
-
(HPLC):
Since amikacin has poor UV absorbance, it
is derivatized as part of the HPLC procedure.
S. M. Maitra et a1.42 separated amikacin
from its isomers by first forming o-phthal-
dehyde derivatives. They reported the
separation of amikacin from BB-K29, BB-K6,
and BB-K11 by this technique. Separation was
carried out on a C-18 column using a mobile
phase of methanol/water ( 7 0 : 3 0 ) containing
tripotassium EDTA. Retention times (minutes)
for the derivatives were amikacin, 6.6;
BB-K29, 7.1; BB-K6, 8 . 3 ; and BB-K11, 1 1 . 7 .

7.3 Gas-Liquid Chromatography (GLC):


The GLC procedure by J. W. Mayhew and
S. L. Gorbach for determination of amikacin
AMIKACIN SULFATE 61

Table 6: TLC conditions f o r amikacin and related compounds.

Rf Value
Plate Developer Detection F o r Amikacin Reference
Silica Gel A - 0.16 (a) 2
Silica Gel F2511 A - 0.16 ( b ) 37
Silica Gel F 2 5 4 B Ninhydrin 0.23 (c) 38
Silica Gel C Ninhydrin - (d) 39
Silica Gel D Bioautography .06 (el 40

Developers

A. Chloroform/methanol/28% ammonium hydroxide/water (1:4:2:1).


B. Tetrahydrofuran/methanol/ammonium hydroxide/water (1:l:l:l)
C. Methanol/amrnonium hydroxide/chloroform (60:30:25).
D. Chloroform/methanol/25% ammonium hydroxide (1:7:4).

NOTES

(a) Related compounds and Rf values reported as follows: BB-K6,


0.29; BB-K29, 0.211; BB-K11, 0.15; BB-K19, 0.17, and BB-K31,
0.16.
(b) System used to separate amikacin and w-amino-a-hydroxyalkanoic
acid derivatives homologous to amikacin.
(c) Related compounds chromatographed: BB-K11, 0 . 2 0 ; BB-K29, 0.36,
kanamycin A , 0.68, and y-amino-a-hydroxybutyric acid, 0.85.
(d) Continuous f l o w f o r 5% hours. Spots from Standard and Test
Solutions must correspond f o r Identification. Arnikacin
moves about 1.5 cm from the origin4* .
(e) In this system kanamycin A had an R f = 0.30.
62 PETER M. MONTELEONE ETAL.

in serum is described in section 8.7 of this


profile.
8.0 Determination in Body Fluids:
-
Aminoglycosides, such as amikacin, have the
potential to produce ototoxicity and nephrotox-
icity, if certain serum levels are exceeded for a
prolonged period of time4. However, minimum serum
concentrations are needed for these antibiotics to
be effective. By knowing the serum levels during
the course of treatment, the physician can provide
the safest and most effective therapy for the
patient. To meet this need, many rapid, sensitive
and precise assays have been developed. Some of
those for amikacin are described in this section.
8.1 Microbiological
- Assay:
P. Marengo and his c o - ~ o r k e r shave
~ ~ re-
ported a rapid, highly specific agar diffusion
method for the measurement of amikacin in body
fluids. The assay method uses a strain of
Pr0videncj-a stuartii resistant to multiple
antibiotics with the exception of amikacin.
D. Stevens -
et
- a1.44 described the stan-
dardization of a four-hour assay for amikacin,
gentamicin and tobramycin using Enterobacter
cloacae as the test organism. The method
-
performs well in the presence of other anti-
biotics including cefamandole and cefoxitin;
however, tetracycline and chloramphenicol will
interfere.
D. Buda et al.45 patented a microbiologi-
cal radioassay method to measure the serum and
plasma levels of amikacin and other amino-
qlycosides. The method measures the amount of
'CO2 produced during the incubation of an
aliquot of test sample in a "C urea test
medium containing Proteus rettgeri ATCC 31168
as the urease producing microorganism.
K. Price -
et
- a1.5 determined amikacin in
blood and urine samples by a 16-18 hour agar
diffusion plate method using B. subtilis
ATCC 6633 as the test organism.
AMIKACIN SULFATE 63

S. C. Edberg and A. M i ~ k i nused


~ ~ growth
curve analysis based on continuous measurement
of the turbidity of bacterial suspensions of
S. aureus MHMC386 grown at 37OC. A special
spectrophotometer with a two compartment cell
monitored the turbidity.
8.2 Fluorescenceimmunoassay (FIA):
The procedure reported by S. G . Thompson
d ~an~example of a homogene-
and J. F. B ~ r is
ous substrate-labeled FIA for amikacin. It
uses amikacin labeled by the fluorogenic
enzyme substrate 6-galactosylumbelliferone.
The labeled amikacin is non-fluorescent;
however, when it is hydrolyzed by B-galacto-
sidase, a fluorescent product results. As in
other immunoassays, labeled and unlabeled
amikacin compete for the antibody binding
sites. When labeled amikacin is bound to the
antibody, the hydrolysis of its fluorescent
label is inhibited. Enzymatic hydrolysis of
the unbound labeled drug releases a fluores-
cent product which can be related to the
original concentration of amikacin in the
sample.
The procedure requires two microliters
of serum. Good precision and recovery data
were shown over the range of 5 to 30 mcg of
amikacin per ml, and a comparison was made
with data from a radioimmumoassay. Specifi-
city in the presence of other aminoglycosides
and other antibiotics was discussed.
An example of a fluorescence polarization
immumoassay for certain aminoglycosides, in-
cludin amikacin, was reported by M. E. Jolley
et al. The fluorescent labeled aminoglyco-
side was prepared by reaction with 5-[4,6-
dichlorotriazin-2-yl)-amino] fluorescein.
Binding of the labeled compound to the anti-
bodies resulted in changes in its fluorescence
polarization thereby forming the basis for
quantitation. Good recovery data for amikacin
was obtained over the range of 3 to 50 mcg/ml
of serum. Specificity in the presence of
other drugs and antibiotics was discussed.
64 PETER M. MONTELEONE E T A .

8.3 Radioimmunoassay (RIA)


J. E. Lewis -
et
- a1.49 described an RIA for
amikacin requiring as little as 5 microliters
of serum, urine or cerebrospinal fluid. Assay
time was about 2 hours. A sensitivity of 5 ng
of amikacin was reported. Antisera were pro-
duced by immunizing rabbits with amikacin
conjugated to porcine thyroglobulin. Selec-
tivity in the presence of other aminoglyco-
sides was discussed, and a comparison was
made with results obtained by microbiological
and radioenzymatic assays.
C. Ashby et a1.” reported an RIA using

gentamicin. ’‘
three antiserum systems making possible the
specific assa of amikacin, tobramycin and
I-labeled aminoglycosides were
used. Serum samples ranging from 2 to 50 mcg
of amikacin per ml were used to demonstrate
accuracy.
8.4 - -:
Radioenzymatic Assay (REA)
J. 14. Broughhall and D. S. Reevess1 de-
scribed the 14C-acetyltransferase technique
for assay of aminoglycosides. The enzyme
transfers a 14C-labeled acetyl group from the
coenzyme substrate to the aminoglycoside.
Separation of the 14C-labeled aminoglycoside
is carried out by absor tion onto 9hospho-
cellulose paper. The “C-acetyl coenzyme
is removed by washing, and the 14C-labeled
aminoglycoside remaining on the paper is
counted. Assays can be completed within an
hour.
P. Botha -
et
- a1.52 reported that 2 years
experience in assaying serum for aminoglyco-
sides, including amikacin, had shown the
acetyltransferase method to be rapid and
accurate. The method uses 6’-N-transacetylase
prepared from E. coli CH15 and 14C-acetyl
coenzyme A to Tabel the amikacin prior to
isolating and counting. Standard curves,
shown for 3-13 mcg of aminoglycoside per ml,
were linear.
AMIKACIN SULFATE 65

P. Santanam and F. H. Kaysers3 described


the preparation of an aminoglycoside 6’-N-
acetyltransferase from E. coli A21218. The
improved enzyme extract acetylated amikacin
within 5 minutes at 35OC in human serum
buffered at pH 7.5 with trismaleate buffer.
Aminoglycoside calibration curves for concen-
trations of 2 to 20 mcg/ml of serum were shown
in their report. Phosphocellulose papers used
in the isolation step were pre-treated to
eliminate non-specific binding of 14C-acetyl
coenzyme A.
C. Manuel -
et
- a1.54 evaluated a REA to
show that accurate and precise assays could
be performed on capillary as well as venous
blood. Twenty microliters of plasma was re-
quired.
R. P. McNight and J. E. W i l l i ~de-
~~
scribed a procedure using kanamycin 6’-acetyl-
transferase (EC2.3.1.55) to transfer the
l4c-acety1 group from 14c acetyl coenzyme A
to the 6’ nitrogen of the aminoglycoside. The
reaction takes 10 minutes at 37’C to complete.
As little as 2 ng of amikacin could be
measured. Detailed data was presented to
demonstrate precision, accuracy and specifi-
city.
J. Bhattacharya et a1.56 described a way
to remove 67Ga interference in the radio-
enzymatic assay of amikacin and other amino-
glycosides.
8.5 Spectrophotometric-Enzymatic Assay:
-
E. Scarbrough et a1.57 reported a spec-
trophotometric-enzymatic assay for amikacin.
The method uses a purified kanamycin acetyl-
transferase. When the acetylation takes place,
a coenzyme A is produced. This is reacted
with 5,5’-dithiobis-(2,2’)-nitrobenzoic acid
(DNTB) to produce a disulfide and thionitro-
benzoic acid which absorbs at 412 nm. The
assay was reported to be linear from 0 to 60
mcg of amikacin with the lowest amikacin
standard shown to be about 2 - 3 mcg/ml of
66 PETER M. MONTELEONE ETAL.

serum. Reaction time is approximately


7 minutes.

8.6 High Pressure Liquid Chromatography


-(HPLC)
- :

S. K. Maitra et a1.58 detailed the HPLC


-1
procedure for amikacin in serum by the
o-phthaldehyde derivatization technique.
Amikacin was separated from serum on a small
silica column where it was reacted with
o-phthaldehyde to produce a fluorescent
derivative which was quantitated by HPLC.
Sensitivity for amikacin was about 1 mcg/ml
of serum.
J. P. Anhalt and S. D. Brown5' described
the procedure whereby the fluorescent o-
phthaldehyde derivative was formed using post
HPLC column continuous flow derivatization.
A CM-Sephadex (C-25) was used to extract the
aminoglycosides from serum.
8.7 Gas-Liquid Chromatography (GLC):
J. S. Mayhew and S . L. Gorbach6' publish-
ed a detailed description of a GLC method for
amikacin in serum. Interferences were removed
by acid precipitation. The amikacin was de-
rivatized in two steps using N-trimethylsilyl-
imidazole followed by N-heptafluorobutyryl-
imidazole. The derivative was extracted into
hexane before chromatographing. A Nickel-63
detector was used. Sensitivity for amikacin
was about 0.6 mcg/ml of serum. Time required
per assay was approximately 50 minutes.
9.0 Acknowledsements:
The authors thank Marie Foulks for typing the
manuscript, and Vilmars Sprancmanis and Dr. Ken A.
Kerridge for help in obtaining a literature search.
We gratefully acknowledge the work of Dave F.
Whitehead, Tim R. Marr, Dr. George R. Dubay, and
Dr. M. S. Balakrishnan in providing data on the
spectral and thermal properties of amikacin.
AMIKACIN SULFATE 67

10.0 References :

H. Umezawa, M. Ueda, K. Maeda,


K. Yagishita, S. Kondo, Y. Okami,
R. Utahara, Y. Osata, K. Nitta, and
T. Takeuchi, J. Antibiot. (Tokoyo),
-
10, 181-189 (1957).
H. Kawaguchi, J. Infect. Dis., 134,
S242-S248 (1976).
H. Kawaguchi, T. Naito, S. Nakagawa and
K. Fujisawa, -
J. Antibiot., -
25, 695-708
(1972).
Amiki@, Physicians Desk Reference,
721 (1982).

K. E. Price, D. R. Chisholm, M. Misiek,


F. Leitner and Y. M. Tsai, 2. Antibiot.,
25, 709-731 (1972).
-
K. A. Kerridge, Pharmcol. Biochem. Prop.
1, i25-53 (19.17).
Drug Subst., -
R. D. Meyer, Ann. Intern. Med., -
95,
328-332 (1981).
D. Zaske and K. Crossley, Minn. Med.,
61, 123-236 (1978).
-

USAN and USP Dictionary Drug Names, 31 c

(1981).
The United States Pharmacopeia XX, 28
(1980).
21 CFR 444.6 and 21 CFR 444.206 (1982).
W. C. Cantrell, Bristol Labs, Syracuse,
N.Y., data on file.
M. A. Kaplan, W. P. Coppola, B. C.
Nunning and A. P. Granatek, Curr.
Ther. Res. Clin. Exp., 2, 3 . 5 4 9 7 6 ) .
68 PETER M . MONTELEONE ET AL.

2 1 C F R 444.6 (1982).
1
P. J. Cloes, M. Dubost and H.
Vanderhaeghe, Analytical Profiles of
-Drug Substances, 5, 2 7 0 ( 1 9 7 7 ) .
D. A. Hull, Bristol Labs, Syracuse,
N.Y., data on file.
B. E. Rosenkrantz, J. R . Greco, J. G.
Hoogerheide and E. M. Oden, Analytical
Profiles of Drug Substances, -9, 3 1 0
Tr980) .
D. F. Whitehead, Bristol-Myers Pharma-
ceutical R. and D. Div., Syracuse,
N.Y., data on file.
M. S. Balakrishnan, Bristol-Myers
Pharmaceutical R. and D. Div.,
Syracuse, N.Y., data on file.
S. Toda, S. Nakagawa, T. Naito and
H. Kawaguchi, Tetrahedron Lett.,
No.
-- 41, 3 9 1 3 - 1 G ( 1 9 7 8 ) .
T. R. Marr, Bristol-Myers Pharmaceuti-
cal R. and D. Div., Syracuse, N.Y.,
data on file.
B. Green and V. Parr, V. G. Analytical
Instruments, Altrincham, England.
G. R. Dubay, Bristol-Myers Pharmaceuti-
cal R. and D. Div., Syracuse, N.Y.,
data on file.
H. Kawaguchi, T. Naito, and S.
Nakapany, U.S. Patent 3,781,268.
R. H. Schreiber and J . G. Keil, U.S.
Patent No. 3,974,137.
M. J. CrOn, J. G. Keil, J. S. Lin,
M. Ruggeri and D. Walker, U . S . Patent
No. 4,347,354.
AMIKACIN SULFATE 69

D. Zaske and K. Crossley, Minn. Med.,


61, 123-236 (1978).
H. Lode, K. Grunert, P. Koeppe and
H. Langmaak, Journal of InEectious
-
Diseases, -
134, S316-22 (19/6).
B. C. Cabana and J. G. Taggart,
Antimicrob. Agents Chemother., -
3,
478-83 (1973).
J. T. Clarke, R. D. Libke, C. Regamey
and W. M. Kirby, Clinical Pharmacology
and Therapeutics,- -
M. A. Kaplan, W. P. Coppola, B. C.
Nunning and A. P. Granatek, Current
Therapeutic Research, -
20, 352(1976).
B. C. Nunning and A. P. Granatek,
Current Therapeutic Research, -
20,
359 (1916).
B. C. Nunning, A. P. Granatek and
R. A. Ricci, Current Therapeutic
Research, -
- 20, 369 (1976).
B. C. Nunning and A. P. Granatek,
Current Therapeutic Research, -
20,
41/ ( 1976).
D. A. Hull, Bristol Labs, A.R. & D.
Report, October 3, 1974.
British Pharmacopeia, 208 (1980).
T. Naito, S. Nakagawa, Y. Narita,
S. Toda, Y. Abe, M. Oka, H. Yamashita,
T. Yamasaki, K. Fujisawa and
H. Kawaguchi, J. Antibiot., -27, 851-58
(1974).
W. H. Ellithorpe, Bristol Labs, A.R. &
D. Report, June 17, 1977.
The United States Pharmacopeia, -
3rd
Sypplement, 34, (1982).
70 PETER M . MONTELEONE ETAL.

J. Kadar Pauncz and 1. Harsanyi, J.


Chromatography, -
- 1 9 5 , 251-256 (1980).

D. A. Hull, Bristol Labs, personal


communication.
S. K. Maitra, T. T. Yoshikawa, C. M.
Stevn, L. B. Guze and M. C. Schotz,
& .

J. of Liquid Chromatography, -
- 2,
823-36 (1979).

P. B. Marengo, J. Wilkins and G. D.


Overturf, Antimicrob. Agents Chemo-
ther.,
-- 6 , 498-500 ( 1 9 7 4 ) .
-
D. L. Stevens, B. M. Page and C.
Adeniyi - Jones, Am. J. Chem. Pathol.,
-
70, 808-15 (1978):

D. Buda, R. L. Broman and J. R.


Walters, U.S. Patent No. 4 , 0 7 3 , 6 9 4 ,
Feb. 14, i 9 7 8 .
S. C. Edberg and A. Miskin, 2. Pharm.
Sci.,
- -6 9 , 1442-43 (1980).

S. G . Thompson and J. F. Burd, Anti- _-


microb. Agents Chemother., -18, 2 6 4 - 6 8
71980).

M. E. Jolley, S. D. Stroupe, C-H.J.


Wang, H. N. Panas, C. L. Keegan,
R. L. Schmidt. and K. S. Schwenzer,
Clin.
-- Chem. (Winston-Salem, N.C.)
1190-97 (1981).
,-c,
J. E. Lewis, J. C. Nelson and H. A.
Elder, Antimicrob. Agents Chemother.,
7, 4 2 - 4 5 ( 1 9 7 5 ) .
-
C. D. Ashbv, J. E. Lewis and J. C.
Nelson, Clin. Chem. (Winston-Salem,
2z, 1734-1737 71978) .
-- ) , -
N.C.

J. M. Broughall and D. S. Reeves,


Chemother. Proc. Int. Congr. Chemo-
ther.,
-- -
2, 159-164 ( 1 9 7 6 ) .
AMIKACIN SULFATE 71

(52) P. Botha, P. A. Berman, G. Elisha,


S. Afr. Med. J., 56,
J. J. Callanan, -
211-13 (1979).

(53) P. Santanam, F. H. Kayser, J. Anti-


microb. Chemother., -
5 , 477-79 ( 1 9 7 9 ) .

(54) C. Manuel, A. Rancononi and B. Pangon,


J. Antimicrob. Chemother., -
- 6, 560-561
(1980)

(55) R. P. McKnight and J. E. Willis, -


Clin.
-
Chem. (Winston-Salem, N.C.), -
27, 1256-
1261 (1981).

(56) I. Bhattacharya, R. Seligsohn and


S. A. Lerner, Antimicrob. Agents
Chemother., -
- 14; 448-53 ( 1 9 7 8 ) .

(57) E. Scarbrough, J . W. Williams and


D. B. Northrop, Antimicrob. Agents
Chemother., -
1 6 , 221-24 ( 1 9 7 9 ) .

(58) S . K. Maitra, T. T. Yoshikawa, C. M.


Steyn, L. B. Guze and M. C. Schotz
Antimicrob. Agents Chemother., -14,
880-85 (1978).

(59)
Chem. (Winston-Salem, N. C. 1
1940-7 (1978).
zF
J . P. Anhalt and S. D. Brown, Clin.

(60) J . W. Mayhew and S. L. Gorbach, J.


Chromatography, 1 5 1 , 133-146 (1978).
BENZOCAINE
Syed Laik Ali

1. History 14
2. Description 74
2.1 Name, Formula, Molecular Weight 74
2.2 Appearance, Colour, Odour 14
3. Synthesis 14
4. Physical Properties 15
4.1 Melting Range 15
4.2 Solubility 15
4.3 Dissociation Constant 15
4 . 4 Loss on Drying 76
4.5 Ultraviolet Spectrum 16
4.6 Infrared Spectrum 16
4.1 Nuclear Magnetic Resonance Spectrum 79
4.8 Mass Spectrum 19
4.9 Differential Thermal Analysis (DTA) 79
5 . Identification and Colour Reactions 82
6. Degradation and Stability 83
7. Dissolution, Liberation and Diffusion 87
8. Methods of Analysis 92
9 . Drug Metabolism and Pharmacokinetics 97
References 100

ANALYTICAL PROFILES OF DRUG SUBSTANCES 73 Copyright by the American PhamLlceuticd Aasocvatiun.


VOLUME 12 ISBN 0-12-2Ml812-7
74 SYED LAIK ALI

1. H i s t o r y . E t h y l p - a m i n o b e n z o a t e was f i r s t s y t h e -
s i s e d by a german chemist R i t s e r t i n 1 8 9 0 and d u e
t o i t s low t o x i c i t y and good l o c a l a n e s t h e t i c pro-
p e r t i e s was g i v e n t h e t r a d e name o f A n e s t h e s i n (1).
I n 1 9 0 1 t h e s u b s t a n c e was t e s t e d p h a r m a c o l o g i c a l l y
by K o b e r t - R o s t o d k ( 2 ) and was f o u n d t o p o s s e s s
remarkable a n e s t h e t i c p r o p e r t i e s . I n e a r l y 1902
d i f f e r e n t f o r m u l a t i o n s of e t h y l p-aminobenzoa t e ,
powder, p a s t e , l o z e n g e s , d r a g e e s and o i n t m e n t , were
b r o u g h t i n t h e m a r k e t u n d e r t h e t r a d e - n a m e o f Ane-
s t h e s i n ( 3 ) . L a t e r on i n 1 9 0 4 F. B u c k a ' s "Kopf-Phar-
macy" i n F r a n k f u r t am Main was f u r t h e r i n v o l v e d i n
marketing of A n e s t h e s i n p r o d u c t s .
2. Description
2.1. Name, Formula, M o l e c u l a r Weight.
E t h y l p-aminobenzoae; B e n z o c a i n e , b e n z o i c a c i d ,
4-amino e t h y l e s t e r ; E t h o f o r m ;
C9H1N02 165.19

NH2
2.2. A p p e a r a n c e , C o l o u r , Odour. White o r c o l o u r -
l e s s c r y s t a l s o r c r y s t a l l i n e powder w i t h a b i t t e r
t a s t e , f o l l o w e d by l o c a l a n e s t h e s i a o f t h e t o n g u e ,
almost odourless.
3 . S y n t h e s i s B e n z o c a i n e c o u l d be s y n t h e s i s e d i n
number o f ways. p - N i t r o b e n z o i c a c i d i s e s t e r i f i e d
and t h e r e s u l t i n g p r o d u c t i s r e d u c e d w i t h m e t a l l i c
t i n and h y d r o c h l o r i c a c i d ( 4 ) o r h y d r o g e n i n p r e -
s e n c e o f p l a t i n u m o x i d e ( 5 ) t o e t h y l p-aminoben-
z o a t e . O t h e r methods a r e t h e e s t e r i f i c a t i o n o f
p-aminobenzoic a c i d ( 6 ) o r t h e r e d u c t i o n o f e t h y l
p - n i t r o b e n z o a t e w i t h ammonium s u l p h i d e (7) .
S t a r t i n g w i t h n i t r o t o l u e n e benzocaine can a l s o be
s y n t h e s i s e d a s shown i n F i g . 1 ( 8 ) .
BENZOCAINE 75

Fig. 1

V a r i o u s s a l t s o f b e n z o c a i n e w i t h b e n z e n e - , naph-
t h a l i n a n d phenolsulphonic a c i d s have been synthe-
s i s e d and f o u n d t o b e o f t h e r a p e u t i c v a l u e ( 9 ) .

4. Physical Properties
4.1. M e l t i n g Range. B e n z o c a i n e melts b e t w e e n
88-92OCI b u t t h e r a n g e b e t w e e n b e g i n n i n g and end
of m e l t i n g d o e s n o t exceed 2O ( 1 0 ) .
4.2. S o l u b i l i t y (11) The s o l u b i l i t y o f b e n z o c a i n e
i n v a r i o u s s o l v e n t s a t 2OoC i s g i v e n i n Table 1
as 1 p a r t per s p e c i f i e d p a r t s - s o l v e n t .
Table 1
S o l u b i l i t y o f b e n z o c a i n e a t 20’C
Solvent Solubility
Water 2500
A 1c o h o 1 5
Chloroform 2
Ether 4
Almond o i l o r o l i v e o i l 30-50
Mineral a c i d s s o l u b l e under s a l t
f o r ma t i o n
4.3. D i s s o c i a t i o n C o n s t a n t . A c i d d i s s o c i a t i o n c o n -
s t a n t o f b e n z o c a i n e h a s b e e n g i v e n a s pK, 2.5
( 1 2 ) . E s t i m a t i o n o f m i c r o q u i l i b r i u m c o n s t a n t of
e t h y l p - a m i n o b e n z o a t e was d o n e s p e c t r o h o t o -
m e t r i c a l l y and a K v a l u e o f 4.17 x 10-0 was
o b t a i n e d . S p e c t r o p h o t o m e t r i c method a p p l i e d f o r
e s t i m a t i n g m i c r o e q u i l i b i r i u m c o n s t a n t s is simp-
l e r , f a s t e r and more a c c u r a t e t h a n t h e c o n v e n -
76 SYED LAIK ALI

t i o n a l method e m p l o y i n g t h e d i s s o c i a t i o n c o n s t a n t
o f an a l k y l a t e d d e r i v a t i v e ( 1 3 ) .
4.4. Loss on D r y i n q . When d r i e d t o c o n s t a n t w e i g h t
a t a p r e s s u r e n o t e x c e e d i n g 20 cm Hg l o o s e s n o t
more t h a n 0.5 p e r c e n t o f i t s w e i g h t , (11).
4.5. U l t r a v i o l e t S p e c t r u m ( 1 4 ) . B e n z o c a i n e i n
s o l u t i o n absorbs u l t r a v i o l e t r a d i a t i o n t o produce
s p e c t r u m w i t h d i f f e r e n t maxima i n d i f f e r e n t s o l u -
t i o n s . I n methanol o r e t h a n o l , 0 . 1 N H C l and 0 . 1 N
NaOH i t h a s a b s o r p t i o n maxima a t 220, 292, 270,
226 and 284 nm r e s p e c t i v e l y . The c o r r e s o n d i n g
m o l e c u l a r e x t i n c t i o n c o e f f i c i e n t s and APb v a l u e s
a r e reported as following ( 1 4 ) . 1C H

Methanol 0.1N H C l 0.1N NaOH


Absorption
maxima 292 nm 270 nm 284 nm
220 nm 226 nm
A 1b 1246 79
f Clm 538 770 1002

€ 20580
8890
1310
12720
16550

The UV s p e c t r a a r e g i v e n i n F i g . 2.
4.6. I n f r a r e d Spectrum. The i n f r a r e d s p e c t r u m of
b e n z o c a i n e is g i v e n i n F i g . 3. The s p e c t r u m was
o b t a i n e d w i t h P e r k i n - E l m e r 257 s p e c t r o p h o t o m e t e r
from a KBr p e l l e t . The s t r u c t u r a l a s s i g n m e n t s may
b e c o r r e l a t e d w i t h t h e f o l l o w i n g band f r e q u e n c i e s
e n c v ( cm - 1 1
Assignment
3200-3500 c h a r a c t e ristic s t r e t c h i n g
v i b r a t i o n s o f primary
amino g r o u p
1680 characteristic stretching
v i b r a t i o n s o f C=O g r o u p i n
ester
1 6 0 0 and 1 5 1 0 c h a r a c t e ristic skeletal
s t r e t c h i n g v i b r a t i o n s of
the aromatic ring
1250-1310; 1100-1150 c h a r a c t e r i s t i c C-0 s t r e t -
ching v i b r a t i o n s
BENZOCAINE 77

A i n nm-

Fig. 2 UV spectrum of benzocaine in


(methanol)
------ ( 0 . 1 N NaOH)
- - - -(0.1N H C 1 )

Fig. 3 IR Spectrum of benzocaine,


KBr pellet, Perkin-Elmer 257
Spectrophotometer
78 SYED LAIK ALI

Fig. 4 N M R Spectrum o f benzocaine,


Varian T 60 Spectrometer

Fig. 5 Mass Spectrum of benzocaine


BENZOCAINE 79

4.7. Nuclear M a g n e t i c R e s o n a n c e Spectrum.


The n u c l e a r m a g n e t i c r e s o n a n c e s p e c t r u m of b e n z o -
c a i n e a s shown i n F i g . 4 was o b t a i n e d on a V a r i a n
T-60 NMR s p e c t r o m e t e r i n d e u t e r a t e d c h l o r o f o r m
c o n t a i n i n g 1%t e t r a m e t h y l s i l a n e a s t h e i n t e r n a l
s t a n d a r d . The f o l l o w i n g s p e c t r a l a s s i g n m e n t s a r e
made f o r F i g . 4.

Chemical S h i f t ( ) Assignment
1 . 3 2 ppm m e t h y l p r o t o n s of e t h y l g r o u p
4.28 ppm p r o t o n s o f amino g r o u p
4.31 ppm m t h y l e n e p r o t o n s of e t h y l g r o u p
6.62 ppm two a r o m a t i c p r o t o n s a d j a c e n t
t o ester group
7.88 ppm two a r o m a t i c p r o t o n s a d j a c e n t
t o amino g r o u p
4.8. Mass S p e c t r u m .
Mass S p e c t r u m is g i v e n i n F i g . 5
I n s t r u m e n t : V a r i a n MAT 4 4
Sample t e m p e r a t u r e ( d i r e c t i n l e t ) : 8OoC
Source temperature: 2 0 0 0 ~
E l e c t r o n e n e r g y : 70 e V
The p r o m i n e n t i o n s of t h i s s p e c t r u m c a n be
c o r r e l a t e d t o t h e structure a s following:

m/e 1 6 5 = M ; 1 3 6 = M
120 = M -
- C2H5
C2H5O; 92 = M - COOC2H5

4.9. D i f f e r e n t i a l Thermal A n a l y s i s ( 1 5 , 1 6 , 1 7 )
The two-component s y s t e m b e n z o c a i n e - p i c r i c a c i d h a s
b e e n s t u d i e d i n o r d e r t o e l u c i d a t e some d i s c r e p a n -
c i e s c o n c e r n i n g t h e m e l t i n g p o i n t s of benzocaine
p i c r a t e . Two m o d i f i c a t i o n s , a m e t a s t a b l e o n e w i t h
m.p. 129O and a s t a b l e o n e w i t h m.p. 162O a r e
known. I t was shown by B o r k a ( 1 6 ) t h a t t h e e q u i -
molar p i c r a t e y i e l d s f o u r p o l y m o r p h i c f o r m s , and &
d i m o r p h i c i n h o m o g e n e o u s l y me1 t i n g p i c r a t e w i t h t h e
c o m p o s i t i o n o f 2 mole b e n z o c a i n e : 1 mole p i c r i c
a c i d . The s o l i d - s o l i d t r a n s f o r m a t i o n of t h e l o w
melting modification i n t o t h e high-melting stable
form i s r e p o r t e d . The s t a b l e m o d i f i c a t i o n c a n be
o b t a i n e d by e x c e s s i v e d r y i n g of t h e i s o l a t e d sub-
s t a n c e a t 105O. The t h e r m a l e n e r g y i n t r o d u c e d i s
SYED LAIK ALI

.-V
€ ................... .....................
2E .
Y
... ...
8 ...
...
w
AT
U

13 1 I
i
5 8b 120 160
Temperature "C

Fig. 6 Differential thermal analysis of


benzocaine picrate (Form I)
1st heating
------ reheating, after cooling to
room temperature

.-U
E
L
B
Y

2 ........................................
.... ::
w *.
AT ..
..
.-V ....
E
b, 5
50
'13
C
w

Fig. 7 Differential thermal analysis of


benzocaine picrate (Form 11) heated
to melting point
-- 1st heating (to 135OC)
------ reheating, after cooling to
room temperature
BENZOCAINE 81

c
0
u I
C 1 . 1
, . I I

W 100 130 160


Temperature "c
Fig.8 Differential thermal analysis of
benzocaine picrate ( F o r m 11) heated
above melting point.
1st heating (to 160°C)

------ reheating after cooling to


room temperature

Temperature "C
Fig. g Differential thermal analysis of
benzocaine picrate (Form 11) showing
crystal transformation to form I
82 SYED LAIK ALI

consumed by t h e c r y s t a l s which i n t u r n u n d e r g o
s o l i d - s o l i d t r a n s f o r m a t i o n from t h e l o w - m e l t i n g
metastable m o d i f i c a t i o n t o t h e high-me1 t i n g s t a b l e
form. The i n f r a r e d s p e c t r a of t h e two m o d i f i c a t i o n s
e x h i b i t some d i f f e r e n c e s , m a i n l y i n t h e 3500 cm'l
and t h e 1 5 0 0 - 1 7 0 0 cm'l r e g i o n .
D i f f e r e n t i a l t h e r m a l a n a l y s i s u s i n g a Mettler TA
2000 a p p a r a t u s was a p p l i e d f o r t h e i n v e s t i g a t i o n o f
b e n z o c a i n e p i c r a t e polymorphs. A p p r o x i m a t e l y 2 mg
o f t h e polymorphs were u s e d f o r e a c h a n a l y s i s . I n
F i g . 6 - 9 d i f f e r e n t i a l thermograms o f two polymor-
p h i c forms, o b t a i n e d under d i f f e r e n t c o n d i t i o n s a r e
g i v e n . The e n t h a l p h y c h a n g e 4 H f o r s t a b l e f o r m
( f o r m I ) was c a l c u l a t e d t o be 47.10 Cal g'l and
f o r t h e m e t a s t a b l e f o r m ( f o r m 11) 30.77 C a l 9-1.
5. I d e n t i f i c a t i o n and C o l o u r R e a c t i o n s .
B e n z o c a i n e g- i v e s c h a r a c t e r i s t i c r e a c t i o n s o f a n
a r o m a t i c amine. A f t e r d i s s o l v i n g i t i n water and
a d d i t i o n o f few d r o p s 3 N h y d r o c h l o r i c a c i d and
1 0 b sodium n i t r i t e f o l l o w e d b y 2 m l o f a s o l u -
t i o n o f 1 0 0 mg 2 - n a p h t o l i n 5 m l 1 N sodium hy-
d r o x i d e a n o r a n g e - r e d p r e c i p i t a t e i s formed ( 1 0 ) .
B e n z o c a i n e s o l u t i o n i n d i l u t e HC1 ( 2 0 mg i n 3 d r o p s
o f 2 M HC1) s p e a r a t e s on a d d i t i o n o f 1 m l water
and 5 d r o p s o f i o d i n e d a r k brown o i l d r o p s o f
per i o d i d e ( 1 8 ) . An i n t e n s e r e d - v i o l e t c o l o u r a t i o n
i s o b t a i n e d when 1 m l p h e n o l (1%) and 2 d r o p s o f
p o t a s s i u m b r o m a t e s o l u t i o n (0.1N) a r e added t o
b e n z o c a i n e s o l u t i o n i n d i l u t e HC1 ( 5 mg i n 2 m l
2 M HC1) ( 1 8 ) . B e n z o c a i n e g i v e s w i t h p i c r i c a c i d
s o l u t i o n a y e l l o w c r i s t a l l i n e p r e c i p i t a t e which
a f t e r washing w i t h water and d r y i n g a t 105OC
melts b e t w e e n 124-13OoC ( 1 9 ) . S a l t s o f b e n z o c a i n e
with v a r i o u s s u l f o n i c acids with d i f f e r e n t melting
p o i n t s a r e known. S a l t s w i t h p - t o l o u e n e s u l f o n i c
a c i d (m.P. : 185-187OC) , w i t h w - t o l u e n e s u l f o n i c
acid ( d e c o m p o s e s a t 235OC), w i t h b e n z e n e d i s u l -
f o n i c a c i d (decomposes a t 235OC), w i t h 2 and
4-phenol s u l f o n i c a c i d s r e s p e c t i v e l y (m.p. :
201-203O and 1 9 6 - 198OC), w i t h a n i s o l e s u l -
f o n i c a c i d (m.p. : 188OC) w i t h p h e n o l d i s u l f o n i c
a c i d (m.p. : 220 - 221OC) , w i t h 2 - o x y n a p h t h a l i n
s u l f o n i c a c i d and g u a j a c o l s u l f o n i c a c i d (m.p.
175OC) a r e known ( 2 0 ) .
BENZOCAINE 83

6. D e g r a d a t i o n and S t a b i l i t y . E s t e r h y d r o l i s e s of
b e n z o c a i n e i n a u u e o u s media i s well known ( 2 1 1 .
A t t e m p t s h a v e b e e n made t o decrease i t s d e g r a d a t i o n
t h r o u g h t h e use of c o m p l e x i n g a g e n t s and s u r f a c -
t a n t s . T h e a p p l i c a t i o n of m o l e c u l a r complex forma-
t i o n a s a means of d r u g s o l u b i l i z a t i o n and s t a b i l i -
s a t i o n h a s been s t u d i e d . C a f f e i n e i n i n c r e a s i n g
c o n c e n t r a t i o n s h a s b e e n used as complexing a g e n t t o
i n h i b i t the benzocaine d e g r a d a t i o n i n barium
h y d r o x i d e s o l u t i o n ( F i g . 1 0 ) (22,231. The i n f l u e n c e
of b e t a - c y c l o d e x t r i n on t h e base c a t a l y z e d d e g r a -
d a t i o n of b e n z o c a i n e h a s b e e n i n v e s t i g a t e d . K i n e t i c
d a t a p r e s e n t e d s u b s t a n t i a t e s t h e p o s t u l a t e d 1:l
s t o i c h i o m e t r i e r a t i o f o r t h e benzocaine-cyclodex-
t r i n i n t e r a c t i o n . T h i s shows t h a t t h e d e g r e e of
r e t a r d a t i o n of b e n z o c a i n e h y d r o l y s i s i s c o n s i d e -
r a b l e g r e a t e r than t h a t observed f o r t h e c a f f e i n e -
t y p e complexes i n d i c a t i n g a n i n t e r a c t i o n i n v o l v i n g
t h e i n c l u s i o n f o r m a t i o n and o t h e r a t t r a c t i v e f o r c e s
(24) ( F i g . 11 and 1 2 ) . C a t a l y s i s and i n h i b i t i o n of
t h e a l k a l i n e h y d r o l y s i s of b e n z o c a i n e and a n a l o g s
by c a t i o n i c s u r f a c t a n t s h a s a l s o b e e n r e p o r t e d (25).
S t a b i l i t y c h a r a c t e r i s t i c s of b e n z o c a i n e i n c l u d i n g
t h e e f f e c t s o f pH and p h o s p h a t e i o n s have b e e n
i n v e s t i g a t e d (26). B e n z o c a i n e d e g r a d a t i o n i s b o t h
a c i d and base c a t a l y z e d b u t is much s l o w e r i n t h e
p r e s e n c e of p h o s p h a t e i o n . S t a b i l i t y s t u d i e s were
p e r f o r m e d a t 25, 4 0 , 60 and 7OoC and a t pH v a l u e s
2, 7 and 11 (26). A t a l l t h r e e p H v a l u e s t h e d e g r a -
d a t i o n r a t e was s l o w e r i n 0.11 M p h o s p h a t e b u f f e r
t h a n i n water. T h u s b e n z o c a i n e was a p p r o x i m a t e l y
1 . 3 ( a t pH 7) 7.3 ( a t pH 2) and 8.0 ( a t pH 11)
times more s t a b l e i n p h o s p h a t e b u f f e r t h a n i n
n o n b u f f e r e d s o l u t i o n s of e q u i v a l e n t pH. E x t r a p o l a -
t i o n of t h e e l e v a t e d t e m p e r a t u r e d a t a t o room
temperature resul t e d i n a p r e d i c t e d f i r s t - o r d e r
r a t e c o n s t a n t of 0.0057 h r ' l , w h i c h compares
f a v o u r a b l y w i t h t h e r a t e c o n s t a n t o f 0.0051 h r - 1
o b s e r v e d a t room temperature. p-Aminobenzoic a c i d
was found t o be t h e o n l y d e c o m p o s i t i o n product.
E s s e n t i a l l y a l l t h e benzocaine l o s t due to degrada-
t i o n was a c c o u n t e d f o r b y t h e a p p e a r a n c e of p-amino-
b e n z o i c a c i d (26). B e n z o c a i n e i n a t h r o a t l o s z e n g e
f o r m u l a t i o n was f o u n d t o be u n s t a b l e w i t h v a r i o u s
e x c i p i e n t s . F r a c t i o n a l f a c t o r i a l experiments iden-
t i f i e d t h e e x c i p i e n t s c i t r i c a c i d , c o r n s y r u p and
SYED LAIK ALI
84

Fig. 10 I n f l u e n c e of i n c r e a s j n y c a f f e i n e -
c o n c e n t r a t i o n , 0 . 2 5 % (-0-1 ,
0.5% (-o-), 1.0% (-o-), 1.5%
(-m-), 2 . 0 % (-A-) and 2.5%
(-A-) o n t h e h y d r o l y s i s of
b e n z o c a i n e i n 0 . 0 4 n bar&um
h y d r o x i d e s o l u t i o n a t 30 C
L
c
C

2 4 6 8 10 2 4 6 0 10
Time Ch3 d l i m e Chi+
Fig. 1 1 Influence of beta Fig. 12 Influence of temperatures
cyclodextrin on benzocaine on benzocaine hydrolysks in 0.04N
hydro&ysis in 0.01N Ba(0H) Ba(OH)2, Key: A, with betacyclo-
at 30 C dex tr in :B, without bet acyc lodext P in
86 SYED LAIK ALI

n a t u r a l c h e r r y f l a v o u r a s t h e c a u s e s o f t h e incom-
p a t i b i l i t y . The p r i m a r y aromatic amine g r o u p i n g
i n s t e a d o f t h e e s t e r l i n k a g e o f b e n z o c a i n e was
involved i n t h e s t a b i l i t y problem ( 2 7 ) . I n a n o t h e r
i n v e s t i g a t i o n of 23 d i f f e r e n t b e n z o c a i n e f o r m u l a -
t i o n s some were 'found t o c o n t a i n n o t t h e l a b e l l e d
amount o f b e n z o c a i n e . p-Aminobenzoic a c i d c o u l d n o t
b e d e t e c t e d i n any o f t h e s u b s t a n d a r d b e n z o c a i n e
f o r m u l a t i o n s ( 2 8 ) . I n o n e of t h e s e f o r m u l a t i o n s a
compound h a v i n g t h e s t r u c t u r a l f o r m u l a

-CH=N COOC~HS
0

was i d e n t i f i e d t h r o u g h mass s p e c t r a a f t e r g a s
chromatographic s e p a r a t i o n (29) .
The i n f l u e n c e of a p r o t e c t i v e c o a t i n g o f b e n z o c a i n e
a s s u n s c r e e n i n g a g e n t upon t h e p h o t o s t a b i l i t y o f
dyes h a s been s t u d i e d . Colour s t a b i l i t y of t a b l e t s
c o a t e d w i t h c e r t i f i e d d y e s were t e s t e d . The p r o t e c -
t i v e e f f e c t o f t h e UV a b s o r b e r b e n z o c a i n e a p p e a r d
most e f f e c t i v e f o r F D and C y e l l o w no. 6 when
a p p l i e d a s t h e m o d i f i e d s u g a r c o a t or t h e f i l m c o a t
(30)
The e f f e c t o f p l a s t i c i z e r s on t h e i n t e r a c t i o n o f
PVC w i t h b e n z o c a i n e h a s b e e n i n v e s t i g a t e d (31).
R e s u l t s show t h a t d r u g p e r m e a b i l i t y i n c r e a s e s w i t h
p l a t i c i z e r c o n c e n t r a t i o n and t e m p e r a t u r e , w h i l s t
s o r p t i o n i n c r e a s e s with p l a s t i c i z e r c o n c e n t r a t i o n
and d e c r e a s e s w i t h t e m p e r a t u r e . The e f f e c t o f ATBC
( a c e t y l t r i - n - b u t y l c i t r a t e ) i s more p r o n o u n c e d
t h a n t h a t o f DEHP ( d i - 2 - e t h y l h e x y l p h t h a l a t e ) f o r
b o t h s o r p t i o n and p e r m e a t i o n p r o c e s s e s . The i n t e r -
a c t i o n o f PolyHEMA, a polymer w i d e l y u s e d i n t h e
m a n u f a c t u r e o f c o n t a c t l e n s e s , w i t h b e n z o c a i n e was
s t u d i e d a t 3OoC. b e n z o c a i n e was s o r p e d i n a r e v e r -
s i b l e manner and g a v e l i n e a r u p t a k e i s o t h e r m s (32).
The t r a n s p o r t of b e n z o c a i n e t h r o u g h a n y l o n 6
membrane was i n v e s t i g a t e d a s a f u n c t i o n of tempera-
t u r e ( 1 0 - 7OoC), c o n c e n t r a t i o n ( 0 . 6 , 1.1 and
6.0 x 10'3M), membrane a r e a and pH i n a s i m p l e
a l l - g l a s s p e r m e a t i o n c e l l . F i g . 1 3 shows t h e i n f l u -
e n c e of pH on t h e a p p a r e n t p e r m e a b i l i t y c o e f f i c i e n t
BENZOCAINE 87

c
t
n

I
ul
N
E
c!
I
Q
X
Y

PH -
Fig. 1 3 Influence of p H on t h e apparent
permeabflity coefficients for
6 x 1 0 M benzocaine trassfer across
a nylon 6 membrane at 50 C
f o r 6 x 10’3M b e n z o c a i n e t r a n s f e r a c r o s s a n y l o n
6 membrane ( 3 3 ) . The i n f l u e n c e o f p h a s e t r a n s i t i o n s
on t h e s o r p t i o n of b e n z o c a i n e b y p o l y a m i d e membra-
n e s h a s a l s o b e e n i n v e s t i g a t e d . The s o r p t i o n of
b e n z o c a i n e by non-or i e n t e d p o l y a m i d e membranes h a s
b e e n s t u d i e d a s ‘a f u n c t i o n o f temperature o v e r t h e
r a n g e 1 0 - 8OoC ( 3 4 ) .
7. D i s s o l u t i o n , L i b e r a t i o n and D i f f u s i o n
T h e i n c r e a s e d s o l u b i l i t y o f b e n z o c a i n e i n t h e pre-
s e n c e of v a r i o u s c o m p l e x i n g a g e n t s was a t t r i b u t e d
t o t h e f o r m a t i o n o f s o l u b l e c o m p l e x e s ( 3 5 ) . The
p o s s i b l e i n t e r a c t i o n s i n a mu1 t i c o m p o n e n t s y s t e m
c o n t a i n i n g b e n z o c a i n e , s o d i u m s a l i c y l a t e and PEG
300 were i n v e s t i g a t e d by d i s s o l u t i o n methods. D i s s o -
l u t i o n s t u d i e s i n d i c a t e t h e e x i s t e n c e of s y n e r g i s t i c
e f f e c t b e t w e e n sodium s a l i c y l a t e and PEG 300 on t h e
d i s s o l u t i o n r a t e of b e n z o c a i n e powder. The e f f e c t
is c o r r e l a t e d t o t h e s u r f a c e t e n s i o n measurement o f
sodium s a l i c y l a t e and PEG 300 s o l u t i o n s . D i s s o l u -
t i o n medium was s t i r r e d a t 7 5 rpm u s i n g a PTFE
20 40 60 20 40 60
Time L m i n J d Time Lminl-

Fig. 14 Effect of sodium salicylate on Fig. 15 Effect of PEG 300 o n


the dissolu&ion of benzocaine in the dissolution of bgnzo-
water at 37 C, 00.0% ; 0 1 . 0 % ; caine in water at 37 C,
A 2.0%; 0 5.0%;~10.0~/~. Key : see Fig. 14
BENZOCAINE 89

r e c t a n g u l a r b l a d e ( 4 . 5 cm by 1 . 7 cm) c o n n e c t e d by a
g l a s s rod ( d i a m e t e r : 6 mm) t o a c o n s t a n t s p e e d
motor. The r a t e o f d i s s o l u t i o n o f b e n z o c a i n e powder
is g r e a t l y i n c r e a s e d i n t h e p r e s e n c e o f e i t h e r
s o d i u m s a l i c y l a t e o r PEG 300. F i g . 1 4 and 1 5 i l l u -
s t r a t e t h e d i s s o l u t i o n p a t t e r n of b e n z o c a i n e ( 3 6 ) .
Spectral c h a n g e s observed i n b e n z o c a i n e - sodium
s a l i c y l a t e system have a l s o been r e p o r t e d ( 3 7 ) .
The i n v i t r o s t u d y o f d r u g r e l e a s e t h r o u g h s i l i -
c o n e r u b b e r membranes of b e n z o c a i n e s u s p e n d e d i n
carbomer h y d r o g e l s c o n t a i n i n g d i f f e r e n t concen-
t r a t i o n s o f low m o l e c u l a r w e i g h t p o l y o l s ( e t h y -
l e n e g l y c o l , p r o p y l e n e g l y c o l , g l y c e r o l and s o r -
b i t o l ) was s t u d i e d t o e s t a b l i s h g e n e r a l p r i n c i p -
l e s and p r o c e d u r e s f o r c o n t r o l o f t h e e f f e c t s o n
p e r c u t a n e o u s a b s o r p t i o n c a u s e d by c h a n g e s i n d r u g
s o l u b i l i t y and or d i f f u s i v i t y i n t h e v e h i c l e . Benzo-
c a i n e was s e l e c t e d a s a model o f n e u t r a l d r u g w i t h
low water s o l u b i l i t y . The e f f e c t o f t h e a d d i t i v e s
on t h e r e l e a s e is e x p r e s s e d i n terms o f t h e r e l a -
t i v e r e l e a s e d amount, i.e. t h e r a t i o of t h e amount
of drug r e l e a s e d from each a d d i t i v e - c o n t a i n i n g g e l
t o t h e amount released f r o m e a c h a d d i t i v e - f r e e g e l .
The e x p e r i m e n t a l v a l u e s a r e c o r r e l a t e d w i t h v a l u e s
c a l c u l a t e d by a s i m p l e e q u a t i o n i n v o l v i n g known
measurable parameters such a s t h e drug concentra-
t i o n i n t h e g e l , t h e d r u g s o l u b i l i t y i n t h e pure
l i q u i d p h a s e and t h e v i s c o s i t y o f t h i s p h a s e ( 3 8 ) .
The release of b e n z o c a i n e f r o m s u p p o s i t o r i e s h a s
b e e n i n v e s t i g a t e d u s i n g a c o n t i n u o u s Flow Bead-Bed
D i s s o l u t i o n A p p a r a t u s . B e n z o c a i n e was s e l e c t e d a s a
model compound w i t h b o t h a low m e l t i n g (33.5 - 35.5%)
and a h i g h m e l t i n g ( 3 7 - 39%) g l y c e r i d e - t y p e
s u p p o s i t o r y b a s e . I n v i t r o release of b e n z o c a i n e
d e c r e a s e d as t h e m e l t i n g p o i n t o f t h e s u p p o s i t o r y
i n c r e a s e d . The a p p a r a t u s c o n s i s t e d o f a g l a s s b e a d -
bed c o n t a i n i n g t h e s u p p o s i t o r y . A c o n t i n o u s f l o w o f
l i q u i d was p a s s e d t h r o u g h t h e bead-bed a t a c o n -
s t a n t r a t e . Direct c o n t a c t o f t h e s u p p o s i t o r y was
m a i n t a i n e d w i t h t h e d i s s o l u t i o n medium, c o n f i n i n g
t h e s u p p o s i t o r y w i t h i n t h e b e a d s . Drug release was
a f f e c t e d by t h e t e m p e r a t u r e o f t h e d i s s o l u t i o n
media, i n c r e a s i n g , d e c r e a s i n g a n d i n c r e a s i n g a g a i n
a t c e r t a i n t e m p e r a t u r e s . Dependence o f b e n z o c a i n e
release f r o m s u p p o s i t o r y b a s e a s a f u n c t i o n o f
90 SYED LAIK ALI

t e m p e r a t u r e was d e m o n s t r a t e d ( 3 9 ) ( F i g . 1 6 ) .
The r e l e a s e of b e n z o c a i n e from o i n t m e n t b a s e s and
t h e i n f l u e n c e of e m u l g a t i n g a g e n t s s u c h a s Span,
Tween and o t h e r l i p o p h i l i c a g e n t s , s u c h a s propy-
l e n e g l y c o l m o n o l a u r a t e and s o r b i t a n m o n o l a u r a t e
h a s been s t u d i e d ( 4 0 , 4 1 , 4 2 , 4 3 , 4 4 ) . D i s s o l u t i o n
s t u d i e s c o n d u c t e d i n m i c e l l a r s o l u t i o n c l e a r l y show
t h a t d i s s o l u t i o n r a t e is n o t p r o p o r t i o n a l t o t h e
a p p a r e n t s o l u b i l i t y of t h e d r u g . T h i s was f i r s t
d e m o n s t r a t e d by H i g u c h i ( 4 5 ) who found t h a t t h e
r a t i o of d i s s o l u t i o n of b e n z o c a i n e i n p o l y s o r b a t e
80 s o l u t i o n t o t h a t w i t h o u t t h e s u r f a c t a n t was
s u b s t a n t i a l l y lower t h a n t h e r a t i o p r e d i c t e d by t h e
Noyes-Whitney t h e o r y . Mechanism of s u r f a c t a n t
e f f e c t s on t h e drug a b s o r p t i o n h a s been r e p r o t e d i n
a review a r t i c l e ( 4 6 ) .
The d i f f u s i o n , p e n e t r a t i o n and s u r f a c e e f f e c t s o f
benzocaine i n c o r p o r a t e d i n v a r i o u s polyethylene
g l y c o l o i n t m e n t b a s e s t h r o u g h human s t r a t u m corneum
h a s been s t u d i e d . R e s u l t s showed a d e c r e a s e i n d r u g
d i f f u s i o n i n t h e p r e s e n c e of r e l a t i v e l y h i g h amounts
of t h e l o w e r m o l e c u l a r weight p o r t i o n s of p o l y e t h y -

Fig. 16 Dependence of benzocaine release from


suppositories as a funchion of
tegperatur8, Key : A 45,; 40°;
039 ; 37 ; and A 33 ;
BENZOCAINE 91

l e n e g l y c o l . Thermal a n a l y s i s i n d i c a t e d t h a t benzo-
c a i n e d i s s o l v e s i n p o l y e t h y l e n e g l y c o l . T h i s would
imply an a l t e r a t i o n of t h e r e l e a s e - p a t t e r n i f t h e
p o l y e t h y l e n e g l y c o l c o m p o s i t i o n is a1 tered ( 4 7 ) .
The i n f l u e n c e of f o r m u l a t i o n , m a n u f a c t u r i n g v a r i -
a b l e s and t e m p e r a t u r e on i n v i t r o r e l e a s e of benzo-
c a i n e d i s s o l v e d i n g e l - t y p e o i l y v e h i c l e s h a s been
repor ted (48) .
The t r a n s p o r t of benzocaine along w i t h o t h e r
p-aminobenzoate e s t e r s t h r o u g h a t u b u l a r d i m e t h y l
p o l y s i l o x a n e membrane i n t o a f l o w i n g l i q u i d was
examined. T h e o b s e r v e d t r a n s p o r t b e h a v i o u r r a n g e d
from c o m p l e t e c o n v e c t i v e d i f f u s i o n c o n t r o l f o r t h e
h e x y l ester t o c o m p l e t e membrane c o n t r o l f o r benzo-
c a i n e . The i m p l i c a t i o n s of convectiv e d i f f u s i o n a l
c o n s i d e r a t i o n s t o i n t e s t i n a l a b s o r p t i o n and d i s s o -
l u t i o n s t u d i e s have been d i s c u s s e d ( 4 9 ) .
The i n v i t r o r e l e a s e of b e n z o c a i n e from o l e a g i n o u s ,
a b s o r p t i o n , e m u l s i o n and w a t e r s o l u b l e o i n t m e n t
v e h i c l e s t h r o u g h a c e l l u l o s e membrane t o a n a q u e o u s
s i n k was s t u d i e d a t 37.5OC and was d e m o n s t r a t e d
t h a t v e h i c l e c o m p o s i t i o n markedly a f f e c t e d t h e r a t e
of r e l e a s e of t h e a c t i v e i n g r e d i e n t ( S O ) . The
e f f e c t of s u p p o s i t o r y v e h i c l e , drug c o n c e n t r a t i o n
and n o n i o n i c s u r f a c t a n t s on i n v i t r o d i a l y s i s
t h r o u g h a c e l l u l o s e membrane were s t u d i e d . I n v i t r o
d i a l y s i s correlated w e l l with i n vivo absorption
and d r u g r e l e a s e was g r e a t e r from w a t e r - s o l u b l e
v e h i c l e s t h a n from o l e a g n i o u s v e h i c l e s . I n c l u s i o n
of a n o n i o n i c h y d r o p h i l i c o r l i p o p h i l i c s u r f a c t a n t
( s o r b i t a n m o n o l a u r a t e , p o l y s o r b a t e 80) i n c o c a b u t -
ter resulted i n a s t a t i s t i c a l l y s i g n i f i c a n t i n c r e a s e
f o r i n v i t r o drug r e l e a s e , while a l i p o p h i l i c sur-
f a c t a n t showed l i t t l e e f f e c t i n v i v o and a hydro-
p h i l i c s u r f a c t a n t depressed r e l e a s e i n v i v o . Both
t y p e s of s u r f a c t a n t s had s m a l l e f f e c t s on r e l e a s e
from p o l y e t h y l e n e g y l c o l . Some f o r m u l a t i o n s f a c t o r
o t h e r t h a n t h e c o n c e n t r a t i o n of b e n z o c a i n e a f f e c t e d
t h e r e l a t i v e amounts of b e n z o c a i n e r e l e a s e d i n
v i t r o from t h e c o m m e r c i a l l y a v a i l a b l e p r o d u c t s
examined. The d i a l y s i s method is u s e f u l f o r e v a l u -
a t i n g t h e e f f e c t s of f o r m u l a t i o n on d r u g r e l e a s e
from s u p p o s i t o r i e s (51).
92 SYED LAIK ALI

8. Method o f A n a l y s i s
Titrimetry. T i t r a t i o n of benzocaine d i s s o l v e d
i n h y d r o c h l o r i c a c i d w i t h sodium n i t r i t e s o l u t i o n
using t h e s t a r c h - i o d i d e paper a s an e x t e r n a l i n d i -
c a t o r is t h e a s s a y method used i n U n i t e d S t a t e s
Pharmacopea (10)'. E n d p o i n t c a n a l s o be d e t e r m i n e d
p o t e n t i o me t r i c a l l y a p p l y i ng a c a l o me1 - p l a t i n u m
e l e c t r o d e system. I n assay methods o f BP 8 0 (52)
and e u r o p e a n pharmacopoea (53) b e n z o c a i n e is d i s s o l -
ved i n d i l u t e H C l , a b o u t 3 g p o t a s s i u m bromide
a d d e d , s o l u t i o n c o o l e d w i t h ice and t h e n t i t r a t e d
a m p e r o m e t r i c a l l y w i t h 0.1 M sodium n i t r i t e s o l u t i o n
u s i n g a d o u b l e p l a t i n u m wire e l e c t r o d e . B r o m i n a t i o n
o f b e n z o c a i n e i n a c e t i c a c i d s o l u t i o n u s i n g a mix-
t u r e of p o t a s s i u m bromide and 1 N potassium b r o m a t e
i n d i l u t e HC1 i s p e r f o r m e d a s a n i n d i r e c t a s s a y
method o f b e n z o c a i n e . On a d d i t i o n o f p o t a s s i u m
i o d i d e t h e e x c e s s of l i b e r a t e d bromine r e a c t s tr,
g i v e f r e e i o d i n e w h i c h is b a c k t i t r a t e d w i t h a s t a n -
d a r d t h i o s u l p h a t e s o l u t i o n . B e n z o c a i n e c a n be t h u s
e v a l u a t e d i n d i r e c t l y t h r o u g h t h e amount of s t a n d a r d
p o t a s s i u m bromate consumed (54). B e n z o c a i n e c a n
d i r e c t l y be t i t r a t e d w i t h p o t a s s i u m b r o m a t e a f t e r
d i s s o l v i n g i t i n 25 % H C l , a d d i n g potassium b r o m i d e
and u s i n g methyl red a s a n i n d i c a t o r (55) o r ampero-
m e t r i c a l l y u s i n g a d o u b l e p l a t i n u m e l e c t r o d e (56).
A non-aqueous t i t r a t i o n of b e n z o c a i n e w i t h
p e r c h l o r i c a c i d u s i n g c r y s t a l v i o l e t a s an i n d i -
c a t o r is a l s o known (57).
C o l o r i m e t r i c A n a l y s i s . A c o l o r i m e t r i c method f o r
t h e d e t e r m i n a t i o n of s u b s t a n c e s c o n t a i n i n g a
p r i m a r y aromatic m i n e s u c h a s b e n z o c a i n e i s based
upon d i a z o t i s a t i o n , c o u p l i n g w i t h N ( l - n a p h t y l )
e t h y l e n e d i a m i n e h y d r o c h l o r i d e and measuring a t 545
nm (58,59,60,61). O t h e r c o u p l i n g a g e n t s r e p o r t e d i n
l i t e r a t u r e a r e o( - n a p h t y l a m i n e thymol i n a l k a l i n e
s o l u t i o n (63) and 2 - n a p h t o l (61). C o l o r i m e t r i c
d e t e r m i n a t i o n of b e n z o c a i n e is a l s o p e r f o r m e d a f t e r
r e a c t i o n w i t h p-dimethylaminobenzaldehyde (65) A
c o l o r i m e t r i c d e t e r m i n a t i o n of benzocaine between
.
505-520 nm a f t e r r e a c t i n g w i t h h y d r o x y l a m i n e and
i r o n (111) s a l t s i s a l s o known (66). R e a c t i o n w i t h
sodium 1,2-naphthochinon-4-sulfonate and c o l o r i -
metric d e t e r m i n a t i o n b e t w e e n 460-480 nm is a l s o
r e p o r t e d (67,68). C o l o r i m e t r i c d e t e r m i n a t i o n s o f
BENZOCAINE 93

b e n z o c a i n e a f t e r r e a c t i o n w i t h : v a n i l l i n e and measu-
r i n g b e t w e e n 380-410 nm ( 6 9 1 , w i t h 9 - c h l o r o a c r i d i n e
and m e a s u r i n g a t 435 ( 7 0 1 , w i t h n i t r o u s a c i d and
m e a s u r i n g b e t w e e n 390-430 nm a r e known ( 7 1 , 7 2 ) . Use
o f Bindon 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 o f
benzocaine has a l s o been r e p o r t e d ( 7 3 ) .
Fluorimetry. Local a n e s t h e t i c s c o n t a i n i n g a
p r i m a r y a r o m a t i c amino g r o u p react w i t h 9 - c h l o r o c r i -
d i n e t o y i e l d a m i n o a c r i d i n e h y d r o c h l o r i d e s . The f o r -
mation of t h e s e d e r i v a t i v e s r e s u l t i n quenching of
fluorescence of the 9-chloracridine reagent solu-
t i o n . M o n i t o r i n g t h e f l u o r e s c e n c e a t a c t i v a t i o n and
e m i s s i o n w a v e l e n g t h s o f 385 and 4 2 0 nm r e s p e c t i v e l y
e n a b l e s to a n a l y s e drug i n t h e - 10-6M
r a n g e . An e t h a n o l i c s o l u t i o n o f b e n z o c a i n e i s
a d j u s t e d t o pH4.0 and acridine-tetrahydrofurane
s o l u t i o n e q u i v a l e n t t o a n a p p r o x i m a t e one t o f o u r -
f o l d molar e x c e s s is a d d e d . The m i x t u r e is h e a t e d
a t 600 f o r 1 0 min, c o o l e d and t h e d e c r e a s e i n t h e
f l u o r e s c e n c e i n t e n s i t y of t h e r e a c t i o n m i x t u r e i s
compared t o t h e r e a g e n t b l a n k . The method d i f f e r s
from o t h e r w i d e l y used t e c h n i q u e s i n t h a t i t i n v o l -
v e s quenching of f l u o r e s c e n c e rather than increased
fluorescence i n t e n s i t y as t h e b a s i s f o r t h e f l u o r i -
.
metric m e a s u r e m e n t s ( 7 4 ) Dambrowski and P r a t t ( 7 5 )
i n t r o d u c e d t h e u s e o f 2 , 6 - d i a m i n o p y r i d i n e a s a new
f l u o r e s c e n c e r e a g e n t and showed t h a t t h e s e n s i t i -
v i t y was i n t h e r a n g e of - 10'7M f o r benzo-
caine (75).
Phosphorimetry. Room-temperature phosphoresence
has been r e p o r t e d t o be u s e f u l f o r a n a l y t i c a l d e t e r -
m i n a t i o n o f v a r i o u s d r u g s . B e n z o c a i n e d i d n o t show
r o o m - t e m p e r a t u r e p h o s p h o r e s c e n c e when a d s o r b e d on
s o d i u m a c e t a t e . However i t was f o u n d t h a t b e n z o c a i n e
c o u l d be r e a d i l y h y d r o l y z e d by r e f l u x i n g i n d i l u t e
a c i d . The h y d r o l y s e d s o l u t i o n was s u b s e q u e n t l y
n e u t r a l i s e d w i t h aqueous a l k a l i s . Small a m o u n t s o f
t h i s s o l u t i o n when a p p l i e d to sodium a c e t a t e and
e v a p o r a t e d t o d r y n e s s showed t h e c h a r a c t e r i s t i c
p - a m i n o b e n z o i c a c i d p h o s p h o r e s c e n c e on e x c i t a t i o n
w i t h a UV lamp. Q u a n t i t a t i v e d a t a h a s n o t b e e n
gathered (76).
94 SYED LAIK ALI

Chroma t o g r a ph i c Method s
Sample P r e p a r a t i o n . Aqueous s o l u t i o n s c a n b e
a p p l i e d d i r e c t l y ( 7 7 ) o r t h e s o l u t i o n is f i r s t o f
a l l made a l k a l i n e w i t h NaOH and t h e l o c a l a n e s t h e t i c
t h e n e x t r a c t e d w i t h ether ( 7 8 ) . O i l s o l u t i o n s ,
s u p p o s i t o r i e s or o i n t m e n t a r e d i s s o l v e d f i r s t o f
a l l i n l i g h t p e t r o l e u m and t h e n e x t r a c t e d w i t h 1 N
HC1. The HC1 e x t r a c t is made a l k a l i n e and t h e n reex-
t r a c t e d w i t h c h l o r o f o r m . With some o i n t m e n t s i t i s
more a d v a n t a g e o u s t o d i s s o l v e t h e o i n t m e n t i n
b e n z e n e ( 7 2 ) . T a b l e t s c a n be e x t r a c t e d d i r e c t l y
w i t h m e t h a n o l a f t e r c r u s h i n g ( 7 7 ) . Drug f o r m u l a -
t i o n s a r e d i s p e r s e d i n 1%HC1, f i l t e r e d , f i l t r a t e
made a l k a l i n e w i t h ammonia, s o l u t i o n e x t r a c t e d w i t h
c h l o r o f o r m and t h e n r e e x t r a c t e d w i t h HC1 ( 7 9 ) .
P a p e r Chromatography. B e n z o c a i n e c a n be s e p a r a t e d
o n p a p e r , i m p r e g n a t e d w i t h formamide, u s i n g b e n z e n e
as m o b i l e p h a s e ( 7 7 ) . The s o l v e n t s y s t e m n - b u t a n o l -
a c e t i c a c i d - w a t e r , 4:1:5 ( 8 0 ) is a l s o w i d e l y u s e d .
Better results, p a r t i c u l a r l y f o r t h e s e p a r a t i o n of
decomposition products can be achieved i n s o l v e n t
s y s t e m c o n t a i n i n g n - b u t a n o l - HC1-water, 60:10:75
( 7 9 ) . I n p a p e r c h r o m a t o g r a p h y w i t h fromamide a s
s t a t i o n a r y p h a s e a l l s u b s t a n c e s w i t h a f r e e amino
group f l u o r e s c e i n UV-light a f t e r t h e chromatogram
h a s b e e n d r i e d a t 105OC.
T h i n l a y e r Chromatography. S i l i c a g e l G t h i n l a y e r
p l a t e s p r e p a r e d i n 0 . 1 N NaOH (811, s i l i c a g e l G F
254 p l a t e s ( 2 8 ) and a l u m i n a p l a t e s ( 8 2 ) h a v e b e e n
used f o r b e n z o c a i n e s e p a r a t i o n . The b e h a v i o u r o f
b e n z o c a i n e and i t s f u r t h e r homologues h a s a l s o b e e n
i n v e s t i g a t e d through p a r t i t i o n chromatography using
c e l l u s l o s e powder i m p r e g n a t e d w i t h o l e y l a l c o l h o l
and a q u e o u s b u f f e r s o l u t i o n s a s m o b i l e p h a s e s ( 8 3 ) .
Under t h e s e c o n d i t i o n s a n i n t e r e s t i n g r e l a t i o n s h i p
b e t w e e n t h e pH v a l u e o f t h e m o b i l e p h a s e and t h e
m o b i l i t y was d e r i v e d . Cyclohexane-benzene-diethyl-
amine, 75:15:10 ( 8 1 ) , b e n z e n e - e t h a n o l , 95:5 ( 8 0 )
and t o l u e n e - e t h a n o l , 95:s ( 2 8 ) h a v e b e e n u s e d a s
m o b i l e p h a s e s f o r t h e s e p a r a t i o n of b e n z o c a i n e .
Roeder and c o w o r k e r s ( 8 4 ) h a v e used mixtures o f
d i f f e r e n t homogeneous a z e o t r o p i c s o l v e n t s f o r t h e
t h i n - l a y e r chromatographic s e p a r a t i o n of benzocaine
from o t h e r l o c a l a n e s t h e t i c s . Chloroform-methanol,
BENZOCAINE 95

80 : 1 0 and e t h a n o l a s m o b i l e p h a s e s and s i l i c a g e l
t h i n l a y e r p l a t e s t r e a t e d w i t h 0.1 N NaOH h a v e b e e n
used f o r t h e s e p a r a t i o n o f b e n z o c a i n e f r o m o t h e r
l o c a l a n e s t h e t i c s ( 4 5 ) . Fuwa ( 8 8 ) r e p o r t e d t h e u s e
o f t h e s i l i c a g e l TLC p l a t e s and b e n z e n e - a c e t o n e -
ammonia, 80:20:T a s m o b i l e p h a s e f o r TLC s e p a r a -
t i o n . A TLC s y s t e m t h a t s e p a r a t e s b e n z o c a i n e f r o m
its h y d r o l y s i s product p-aminobenzoic acid h a s been
r e p o r t e d ( 8 7 ) . S i l i c a g e l TLC p l a t e s and a s o l v e n t
s y s t e m o f b e n z e n e - d i o x a n e - a c e t i c a c i d , 90:75:8
were a p p l i e d ( 8 7 ) .
D e t e c t i o n c a n b e made by q u e n c h i n g i n s h o r t wave-
l e n g t h UV l i g h t , a f t e r s p r a y i n g w i t h E h r l i c h ' s
r e a g e n t which g i v e s y e l l o w s p o t s ( 7 7 ) , d i a z o t i -
s a t i o n f o l l o w e d by c o u p l i n g w i t h & - n a p h t o l ( 8 0 )
and s p r a y i n g w i t h i o d i n e - p o t a s s i u m i o d i d e s o l u -
tion (82).
Q u a n t i t a t i v e d e n s i t o m e t r i c d e t e r m i n a t i o n of benzo-
c a i n e a t 285 nm a f t e r s e p a r a t i o n on t h i n l a y e r
p a l t e s h a s been r e p o r t e d ( 2 8 ) . Benzocaine h a s a l s o
b e e n d e t e r m i n e d a f t e r TLC s e p a r a t i o n by r e a c t i n g i t
w i t h d a n s y l c h l o r i d e r e a g e n t and t h e s u b s e q u e n t
d e t e r m i n a t i o n of f l u o r e s c e n c e i n t e n s i t y of a d d u c t s
t h r o u g h T L C - F l u o r i m e t r y ( 8 8 ) . Microgram q u a n t i t i e s
o f b e n z o c a i n e were d e t e r m i n e d t h r o u g h t h i s method.
R f - v a l u e s o f f l u o r e s c i n g a d d u c t s on d i f f e r e n t TLC
p l a t e s a r e r e p o r t e d ( 8 8 ) . TLC s e p a r a t i o n and s p e c -
t r o p h o t o m e t r i c d e t e r m i n a t i o n o f p r o c a i n e and b e n z o -
c a i n e i n v a r i o u s pharmaceutical p r e p a r a t i o n s h a s
a l s o been performed ( 8 9 , 9 0 ) .
High P e r f o r m a n c e L i q u i d C h r o m a t o g r a p h y .
B e n z o c a i n e and a n t i p y r i n e i n e a r d r o p s h a v e b e e n
s e p a r a t e d and d e t e r m i n e d t h r o u g h HPLC on /((-Ban-
d a p a c k C 1 8 column (Waters) u s i n g a m o b i l e p h a s e o f
0 . 0 2 M KH2P04 i n water c o n t a i n i n g 408 m e t h a n o l .
D e t e c t i o n was d o n e a t 254 nm ( 9 1 ) . HPLC h a s a l s o
been a p p l i e d f o r t h e s i m u l t a n e o u s a s s a y of benzo-
c a i n e and i t s h y d r o l y s i s p r o d u c t p - a m i n o b e n z o i c
a c i d . A C 1 8 m i c r o p a r t i c u l a t e column w i t h a m o b i l e
phase of methanol - a c e t i c a c i d - water, 33:4:63
were u s e d a t a m b i e n t t e m p e r a t u r e w i t h a f l o w r a t e
o f 2 ml/min and d e t e c t i o n w a v e l e n g t h 254 nm. The
r e t e n t i o n times f o r p - a m i n o b e n z o i c a c i d and b e n z o -
96 SYED LAIK ALI

c a i n e were r e p o r t e d t o be 2 min 28 sec and 7 min 1 0


sec r e s p e c t i v e l y . The s e n s i t i v i t y i s g i v e n a s
6-'
7 g/ml f o r b e n z o c a i n e and 0.3 g/ml f o r p-amino-
b"
b n z o i c a c i d f o r an i n j e c t e d v l u m e o f 1 0 1. The
s e n s i t i v e t y c a n be i n c r e a s e d f o u r f o l d i f 6"
detec-
t i o n wavelength o f 294 nm i s used ( 2 6 ) . A l i (28)
h a s g i v e n two modes of l i q u i d chromatography, ad-
s o r p t i o n and r e s e r v e d - p h a s e systems f o r t h e HPLC
a n a l y s i s of b e n z o c a i n e . I n a d s o r p t i o n mode a S i
60,25 cm column w i t h a mobiel p h a s e n-hexane-
c h l o r o f o r m , 30:70 a t a wavelength of 254 nm were
used. A RP-18, l o p column (Knauer) and a m o b i l e
phase of methanol-water-0.1N HC1, 54:36:10 were
used i n r e v e r s e d - p h a s e s y s t e m a t a d e t e c t i o n wave-
l e n g t h of 285 nm. F l o w - r a t e i n b o t h s y s t e m s was
k e p t a t 1 . 5 ml/min. Numerous b e n z o c a i n e f o r m u l a -
t i o n s i n d i f f e r e n t d o s a g e forms were assayed
through t h e s e two HPLC s y s t e m s ( 2 8 ) .
Gas-Liquid -Chr oma toq raphy
Ebel ( 9 2 ) h a s g i v e n a l i s t of d i f f e r e n t s t a t i o n a r y
p h a s e s and g a s - c h r o m a t o g r a p h i c c o n d i t i o n s f o r t h e -
s e p a r a t i o n o f s e v e r a l l o c a l a n e s t h e t i c compounds.
A l i (28) h a s d e t e r m i n e d b e n z o c a i n e t h r o u g h g a s
chromatography on a 3% SE 30 on V a r a p o r t column
w i t h a FID d e t e c t o r . 15OOC oven t e m p e r a t u r e i s o -
therm, 240 and 27OOC f o r i n j e c t i o n p o r t and
d e t e c t o r b l o c k t e m p e r a t u r e s r e s p e c t i v e l y were
used. G a s - l i q u i d chromatography of l o c a l a n e s t h e -
t i c s and r e l a t e d compounds on F l e x o l Chromosorb W
and S E 30 on g l a s s bead columns is a l s o r e p o r t e d
( 9 3 ) . F l e x o l on Chromosorb W column was p r e f e r r e d
f o r t h e l o w e r m e l t i n g compounds s u c h a s t h e amino-
c a l c o h o l s , w h i l e a low l o a d SE 30 on g l a s s - b e a d
column was p e r f e r r e d f o r h i g h e r m e l t i n g l o c a l
a n e s t h e t i c b a s e s and s a l t s . I d e n t i f i c a t i o n and
d e t e r m i n a t i o n of b e n z o c a i n e i n cosmetics h a s a l s o
been r e p o r t e d ( 9 3 a ) . A column packed w i t h 1 . 2 5 b
OV 1 7 + 1 . 2 5 b OV 25 on v a r a p o r t 30, 100-120 mesh
was used. The oven t e m p e r a t u r e programme p r o c e e d e d
from 16OoC t o 23OoC, a t r a t e of 8O/min; t h e
i n j e c t i o n and d e t e c t o r t e m p e r a t u r e s were a d j u s t e d
t o 25OoC and 3OO0C r e s p e c t i v l y ( 9 3 a ) .
BENZOCAINE 97

9. Drug Me t ab o l i s m and P h a r m a c o k i n e t i c s
An i n s i t u c o r n e a l p e r f u s i o n s y s t e m which e n a b l e s
q u a n t i t a t i o n of c o r n e a l d r u g u p t a k e h a s been u s e d
t o e v a l u a t e t h e u p t a k e o f b e n z o c a i n e i n r a b b i t s . By
determining t h e d e c l i n e i n drug c o n c e n t r a t i o n i n
t h e p e r f u s i o n a p p a r a t u s a s a f u n c t i o n o f time i t
was p o s s i b l e t o measure d r u g c l e a r a n c e from t h e
s y s t e m , t h e r e b y o b t a i n i n g a measure o f c o r n e a l d r u g
u p t a k e . Using t h i s method t h e c l e a r a n c e o f benzo-
c a i n e from t h e s y s t e m by r a b b i t c o r n e a s was exami-
ned. T h e i n s i t u p e r f u s i o n s y s t e m p r o v i d e s r e p r o -
d u c i b l e r e s u l t s and o b v i a t e s many o f t h e p r o b l e m s
associated w i t h isolated i n v i t r o corneal studies.
T h i s method c a n be u t i l i z e d t o s t u d y t h e e f f e c t of
p h y s i c o c h e m i c a l d r u g p r o p e r t i e s on c o r n e a l u p t a k e
a s well a s i n o c u l a r p h a r m a c o k i n e t i c modeling and
dosage form e v a l u a t i o n ( 9 4 ) .
E f f e c t s o f a n e s t h e t i c s l i k e b e n z o c a i n e etc. on
s u l f a t e t r a n s p o r t i n t h e red c e l l have been i n v e s t i -
gated. Experiments suggested t h a t a n e s t h e t i c s a c t
t h r o u g h a g e n e r a l mechanism and t h a t t h e s u l f a t e
t r a n s p o r t i n t h e red b l o o d c e l l i s a good model f o r
s t u d y i n g t h e mechanism of a c t i o n of a n e s t h e t i c s
(95)
Transport of benzocaine a c r o s s chromatophore has
b e e n s t u d i e d . T r a n s p o r t of u n p r o t o n a t e d l o c a l
a n e s t h e t i c , although electrically n e u t r a l , requires
t h e p r e s e n c e o f a membrane p o t e n t i a l ( 9 6 ) . Absorp-
t i o n of 3 H - l a b e l l e d b e n z o c a i n e from o i n t m e n t s
f o l l o w i n g a d m i n i s t r a t i o n i n r a t s h a s been s t u d i e d .
T o t a l r a d i o a c t i v i t y i n t h e blood of r a t s f o r f i v e
h o u r s f o l l o w i n g r e c t a l a d m i n i s t r a t i o n of 3H-ben-
z o c a i n e i n o l e a g e n o u s , a b s o r p t i o n e m u l s i o n (water
i n o i l and o i l - i n - w a t e r ) a n d water s o l u b l e o i n t m e n t
v e h i c l e s was measured. The release was g r e a t e s t
from t h e w a t e r - s o l u b l e v e h i c l e and f o l l o w e d t h e
same r e l a t i v e o r d e r a s i n a n i n v i t r o e x p e r i m e n t
( F i g . 1 7 ) ( 9 7 ) . No i n t a c t b e n z o c a i n e was found i n
t h e blood using radiochromatography. I n v i t r o hydro-
l y s i s o f b e n z o c a i n e by r a t b l o o d d i d n o t o c c u r . T h e
r a t e o f a p p e a r a n c e and amount of d r u g i n t h e blood
a f t e r r e c t a l a p p l i c a t i o n s h o u l d be a u s e f u l measure
of t h e r a t e and amount of d r u g t r a n s f e r from a
dosage form t o t h e s e n s o r y nerve e n d i n g s i n t h e
r e c t a l mucosa ( 9 7 ) . The a b s o r p t i o n of d e t e c t a b l e
SYED LAIK ALI
98

1 a 3 4 6
HOURS

Fig. 17 Blood radioactivity3after the


application of 20% H-benzocaine in
different ointment vehicles. Key :
1, white petroleum; 2, absorption
vehicle; 3, water-in-oil emulsion;
4, oil-in-water emulsion; 5, poly-
ethylene glycol. The standard error
of the mean is shown on one side of
each point.
BENZOCAINE 99

amounts of b e n z o c a i n e a f t e r a p p l i c a t i o n t o abroded
abdominal s k i n i n dogs and t h e a b i l i t y o f v a r i o u s
b e n z o c a i n e c o n t a i n i n g p r e p a r a t i o n s t o obtuned t h e
i t c h and b u r n i n g e l e c t r i c a l s t i m u l a t i o n i n humans
h a v e been r e p o r t e d ( 9 8 , 9 9 ) . A b s o r p t i o n of some
l o c a l a n e s t h e t i c s h a s been r e p o r t e d t o b e d e p e n d e n t
on t h e mucous s u r f a c e ( 9 8 ) . B e n z o c a i n e is c l e a r l y
degraded i n v i v o , however t h e d e g r a d a t i o n occurs
p r o b a b l y i n t h e l i v e r ( 1 0 0 ) . Rectal a d m i n i s t r a t i o n
of t h e same c o n c e n t r a t i o n of 3H-benzocaine i n t h e
same v e h i c l e t o male and f e m a l e r a t s r e s u l t s i n a
lower blood r a d i o a c t i v i t y v e r s u s time c u r v e s f o r
t h e male r a t s (51). No m e t a b o l i c s t u d i e s o f benzo-
c a i n e have been r e p o r t e d . M e t a b o l i c s t u d i e s of amino
a c i d d e r i v a t i v e s of b e n z o c a i n e r e v e a l e d t h e p o s s i b i -
l i t y of r a p i d h y d r o l y s i s o f t h e d e r i v a t i v e s t o
b e n z o c a i n e by t i s s u e s and plasma h y d r o l y z i n g
enzymes. They a r e c o n s i d e r e d a s p r o d r u g s (101).
C l i n i c a l p h a r m a c o k i n e t i c s of s e v e r a l l o c a l a n e s t h e -
t i c s h a s been examined by T u c k e r ( 1 0 2 ) . Blood con-
c e n t r a t i o n s of l o c a l a n e s t h e t i c s a f t e r p e r i n e u r a l
i n j e c t i o n s a r e n o t c l o s e l y r e l a t e d t o age, weight
or pregnancy b u t may be i n f l u e n c e d by d i s e a s e s
a s s o c i a t e d w i t h haemodynamic c h a n g e s and by o t h e r
d r u g s g i v e n a t o r around t h e time o f r e g i o n a l
b l o c k a d e ( 1 0 2 ) . The a b s o r p t i o n and u r i n a r y excre-
t i o n of b e n z o c a i n e were i n v e s t i g a t e d i n r a t s by t h e
method of mass fragmentography. The i n t a c t benzo-
c a i n e was found s l i g h t l y i n t h e serum a f t e r o r a l
d o s e . p-Aminobenzoic a c i d , h a y d r o l y z e d p r o d u c t of
b e n z o c a i n e r a p i d l y r e a c h e d t o t h e maximum c o n c e n -
t r a t i o n a t 1 0 min, t h e n soon d i s a p p e a r e d from t h e
serum ( 1 0 3 ) .

A c know1edg emen t
The a u t h o r t h a n k s D r . Hausser and D r . H.W. D i b b e r n ,
Hoechst, F r a n k f u r t , F e d e r a l R e p u b l i c o f Germany f o r
p r o v i d i n g some u s e f u l i n f o r m a t i o n .
100 SYED LAIK ALI

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DIBUCAINE AND DIBUCAINE
HYDROCHLORIDE
Gandharva R . Padmanabhan

1. Description 106
1.1 Name, Formula, Molecular Weight 106
1.2 Appearance, Color, Odor 106
1.3 Therapeutic Category 106
2. Physical and Chemical Properties 106
2.1 Infrared Absorption Spectra 106
2.2 Nuclear Magnetic Resonance Spectra (NMR) 110
2.3 Ultraviolet Absorption Spectra 110
2.4 Mass Spectra 114
2.5 Fluorescence Spectrum 117
2.6 Melting Range 118
2.7 Differential Scanning Calorimetry (DSC) 118
2.8 ThermogravimetricAnalysis (TGA) 118
2.9 Solubility 118
2.10 X-Ray Diffraction 120
2.1 1 Polymorphism 120
2.12 Partition Coefficient 120
2.13 Dissociation Constant 120
3. Synthesis 122
4. Stability-Degradation 122
5. Drug Metabolism and Pharmacokinetics 122
6. Toxicity 125
7. Methods of Analysis 125
7.1 Identification 125
7.2 Elemental Analysis 125
7.3 Nonaqueous Titration 126
7.4 Phase Solubility Analysis 126
7.5 Thin-layer Chromatography 126
7.6 High-PerformanceThin-layer Chromatography 129
7.7 High-pressure Liquid Chromatography 129
7.8 Gas Chromatography 130
7.9 Gas Chromatography-MassSpectrometry 130
7.10 Paper Chromatography 132
7.11 Polarography 132
7.12 Spectrophotometry 132
8. Miscellaneous 133
8.1 Dibucaine Number 133
References 133

ANALYTICAL PROFILES OF DRUG SUBSTANCES 105 Copyrightby the American Pharmaceutical Assuciatmn.
ISBN 0-12-260812-7
VOLUME 12
106 GANDHARVA R. PADMANABHAN

1. Description
1.1 Name, Formula, Molecular Weight
Dibucaine is 2-butoxy-N-[2-(diethylamino)-
ethyl]-4-quinolinecarboxamide

&'- 0CH2CH2CH2CH3

CONHCH~CH~N(C~HC,)
2

c2 OH29N302 Molecular Weight: 343.47


Other Names: Nupercaine, Percain, Cinchocain,
Cincain and Sovcain.
Dibucaine hydrochloride is 2-butoxy-N-[2-(diethyl-
amino)ethyl]-4-quinolinecarboxamide monohydro-
chloride

J
/
,J OCH2CH2CH2CH3
\ #
HC1

C20H2gN302'HCl Molecular Weight: 379.93


1.2 Appearance, Color, Odor
Dibucaine occurs as a white to off-white
powder, having a slight characteristic odor. It
darkens on exposure to light. Dibucaine hydro-
chloride occurs as colorless or white to off-
white crystals or as a white to off-white crystal-
line powder. It is odorless, is somewhat hygro-
scopic and darkens on exposure to light.

1.3 Therapeutic Category


Local Anesthetic

2. Physical and Chemical Properties


2.1 Infrared Absorption Spectra
The infrared spectra of mineral oil suspen-
sions of dibucaine and dibucaine hydrochloride are
shown in Figures 1 and 2. The spectral assignments
are listed in Tables 1 and 2.
DIBUCAINE AND DIBUCAINE HYDROCHLORIDE 107

TABLE I
I R Spectrum of Dibucaine

-1
Wavenumber, c m Assignment

3300 -NH
0
II
1665 -NH-C -
1605

1 550 Amide I1
1458)
Nujol
1378
1250 Aromatic e t h e r
7 80 0 - D i s u b s t i t u t e d phenyl

TABLE 2
I R Spectrum of Dibucaine HC1

-1
Wavenumber, c m Assignment

3205 -NH
2620
2500 Salt
H O
1670
I II
-N-C-

1605

1555 Amide I1
1458) Nuj 01
1378
1254 Aromatic e t h e r
765 0 - D i s u b s t i t u t e d phenyl
F i g u r e 1. I n f r a r e d Absorption Spectrum Dibucaine
Figure 2. I n f r a r e d Absorption Spectrum of Dibucaine Hydrochloride
110 GANDHARVA R. PADMANABHAN

2.2 Nuclear Magnetic Resonanace S p e c t r a (NMR)


The p r o t o n NMR s p e c t r a of d i b u c a i n e and
d i b u c a i n e h y d r o c h l o r i d e are shown i n F i g u r e s 3
and 4 . The s p e c t r a were determined on a Perkin-
E l m e r R-24B 60 MHz spectrometer a t ambient
temperature. The samples were d i s s o l v e d i n
d e u t e r a t e d chloroform c o n t a i n i n g t e t r a m e t h y l -
s i l a n e a s a n i n t e r n a l s t a n d a r d . The s p e c t r a l
assignment f o r d i b u c a i n e h y d r o c h l o r i d e i s shown
i n Table 3 .

TABLE 3

Chemical
No. of
Shift Multiplicity Assignment
Protons
6 (ppm)

8.7-9.1 Broad 1
0
-C-NH-CH 2 -
-
7.2-8.3 Multiplet 5 Aromatic E's
4.3-4.7 Triplet 2 -0-CE2-CH2-
9
3.6-4.3 Broad 2 -C-NH-CE2-CH2

, CE2
2.8-3.6 Multiplet 6 -CE2-N,
CE2-
/ cH2-c!!3
0.8-2.3 M u l t i p l et 13 N and
'CHz-CB3

13
The C- NMR spectra of d i b u c a i n e and d i b u c a i n e
h y d r o c h l o r i d e doterairled i n d e u t e r a t e d chloro-
form as s o l v e n t a r e shown i n F i g u r e s 5 and 6 .

2.3 U l t r a v i o l e t Absorption S p e c t r a
The UV s p e c t r a of d i b u c a i n e and d i b u c a i n e
hydrochloride (Figure 7 ) i n 1E H C 1 e x h i b i t
maxima and minima as shown i n Table 4 .
DIBUCAINE AND DIBUCAINE HYDROCHLORIDE 111

I
80
I
70
I
60
1
50
I
40
1
30
I
20
,
10
1
0

PPM

Figure 4. NMR Spectrum of Dibucaine Hydrochloride

f-

I 1 I T I I I I
90 80 70 60 50 40 30 20 10

PPM

Figure 3. NMR Spectrum of Dibucaine


112 GANDHARVA R. PADMANABHAN

I I I I I
175 150 125 100 75 50 25 0

PPM

l.3
Figure 5. C-NMR Spectrum of Dibucaine

I
I
150
1 I
100

PPM
I
50
nw

0
I

Figure 6. 13C-NMR Spectrum of Dibucaine Hydrochloride


DIBUCAINE AND DIBUCAINE HYDROCHLORIDE 113

10

09

08

07

06

Q)
0
C
0
05
2
0
a

04

03

0 2.

01

0 0- I 1 I I
250 300 350 400

Wavelength, Nanometers

Figure 7 . U l t r a v i o l e t Absorption Spectrum of Dibucaine


Hydrochloride
114 GANDHARVA R. PADMANABHAN

Dibucaine Dibucaine H C 1
'max, nm A ( 1% ,lcm) E A ( 1% ,lcm) E

247 7 30 25,070 650 24,700


320 260 8,930 232 8,810

2.4 Mass S p e c t r a
The low r e s o l u t i o n mass s p e c t r a of d i b u c a i n e
and d i b u c a i n e h y d r o c h l o r i d e o b t a i n e d
u s i n g a s o l i d probe i n s e r t i o n are shown i n
F i g u r e s 8 and 9. The s p e c t r a were run on a
Kratos MS 25 spectrometer i n t e r f a c e d w i t h a d a t a
handling system. Tables 5-6 i l l u s t r a t e t h e
fragments and t h e i r mass/charge r a t i o s .
TABLE 5
Fragmentation P a t t e r n of Dibucaine
DIBUCAINE AND DIBUCAINE HYDROCHLORIDE 115

100

90

80

70

60

50

40

30

20

10

0 1,
I 11 dl
L,
350

mfe

Figure 8
Low Resolution Mass Spectrum of Dibucaine
116 GANDHARVA R. PADMANABHAN

90 -

80 -

70-

60 -

50-

40 -

30-

20 -

10-

50

m/e

Figure 9
Low Resolution Mass Spectrum of Dibucaine Hydrochloride
DIBUCAINE AND DIBUCAINE HYDROCHLORIDE 117

TABLE 6
Fragmentation Pattern of Dibucaine Hydrochloride

m/e I Fragment

343

271

215

+
172

144

2.5 Fluorescence Spectrum


The following UV absorbance (Aa) and fluo-
rescence maxima (Af) of dibucaine in various
solvents have been reported (1-2)
Medium pH or Ho Species A ,nm
a
A f ,nm
Present ~~

H2S04 Ho=- 7 .9 325 508


2+
H2S04 H0 =-0.7 BH2 320 445
1+
Water pH=6.2 BH 328 409
1+
Ethanol - BH 328 400
1+
Chloroform - BH 329 392
Water pH=ll.5 B 328 403
Ethanol - B 327 390
Chloroform - B 328 388

Triply protonated cation

Doubly protonated cation

Mono protonated cation


Base
118 GANDHARVA R. PADMANABHAN

2.6 Melting Range


Dibucaine m e l t s between 64°C and 65°C when
t e s t e d according t o t h e USP XX, Class I a procedure.
Dibucaine h y d r o c h l o r i d e m e l t s between 95°C and
97.5"C when t e s t e d according t o t h e same procedure.

2.7 D i f f e r e n t i a l Scanning Calorimetry (DSC)


The DSC thermogram of d i b u c a i n e showed one
endotherm between 61" and 70°C w i t h a m e l t i n g
p o i n t of 64°C and a p u r i t y v a l u e of 98.9 mole
p e r c e n t when t h e thermogram w a s followed i n a
DuPont Model 900 instrument a t a scan r a t e of
20°C/minute (Figure 1 0 ) . S i m i l a r l y , t h e DSC
thermogram of d i b u c a i n e h y d r o c h l o r i d e showed one
endotherm between 94" and 104°C w i t h a m e l t i n g
p o i n t of 97°C. The p u r i t y of t h e h y d r o c h l o r i d e
was determined t o be 99.4 mole % (Figure 11).

2.8 Thermogravimetric Analysis (TGA)


The TGA of d i b u c a i n e e x h i b i t e d a weight
l o s s of 0.17% between 40°C and 130°C.
The TGA of t h e h y d r o c h l o r i d e s a l t e x h i b i t e d
a weight l o s s of 0.81% between room temperature
and 75"C, a loss of 0.14% between 75°C and 150°C
and a r a p i d l o s s above 150°C.

2.9 Solubility
Approximate s o l u b i l i t i e s i n d i f f e r e n t
s o l v e n i s were determined a f t e r e q u i l i b r a t i n g 10
mg (more, i f n e c e s s a r y , t o o b t a i n a s a t u r a t e d
s o l u t i o n ) of t h e drugs a t room temperature w i t h 1
mL of s o l v e n t .

S o l u b i l i t y , mg/mL
Solvent
Dibucaine Dibucaine H C 1
Water <0.1 >loo0
0.1z HC1 >33l >loo0
0.1E NaOH <0.1 KO.1
Methanol >loo0 >loo0
Ethanol, Absolute >loo0 >loo0
95% Ethanol >loo0 >loo0
Chloroform >loo0 >loo0
Ethyl Acetate 100- 1000 10-33
Ether 100-1000 0.1-1
Petroleum Ether 10-33 <0.1
Acetone 100-1000 100-1000
n-Hexane 10-33 <0.1
.1 g d i s s o l v e s i n 10-30 1
DIBUCAINE AND DIBUCAINE HYDROCHLORIDE 119

F i g u r e 10.DSC Scan of Dibucaine

F i g u r e 11. DSC Scan of Dibucaine H y d r o c h l o r i d e


120 GANDHARVA R. PADMANABHAN

2.10 X-Ray Diffraction


The x-ray powder diffraction pattern obtained
for dibucaine base is shown in Figure 12. The
data were collected on a GE Model XRD-spectrogonio-
meter using CuK, (1.542A') with a Ni filter as a
radiation source. The diffraction pattern obtained
for dibucaine hydrochloride is shown in Figure
13.

2.11 Polymorphism
No polymorphism has been reported for
dibucaine base and dibucaine hydrochloride.

2.12 Partition Coefficient


The following partition coefficient data
were obtained when 50 mL of 0.1 and 1.0 mg/mL
of dibucaine hydrochloride in 0.1N hydrochloric
acid solutions were partitioned individually
with 50 mL of ether and chloroform and when 50 mL
of 0.1 and 1.0 mg/mL of dibucaine in chloroform
and ether solutions were partitioned individually
at room temperature with 50 mL of pH 7 phosphate
buffer and 0.1N sodium hydroxide.

Aqueous Phase Organic Phase Partition Coefficient?

0.1N HC1 Chloroform 0.8-1.1


0.1N HC1 Ether -to
pH 7 Buffer Chloroform >15
pH 7 Buffer Ether >14
0.1N NaOH Chloroform >20
0.1N NaOH Ether >14

2.13 Dissociation Constant


The following pKa values have been reported (1):
pKBH+ = 8.31 (potentiometric titration)

pKBH;+ = 1.6 (absorption spectrophotometry)


psH;+ = - 5 . 3 (absorption spectrophotometry)
3+ 2+ +
BH3 , BH2 and BH are respectively triply pro-
tonated, doubly protonated and mono protonated
species.
DIBUCAINE AND DIBUCAINE HYDROCHLORIDE 121

I I I I
5 15 25 35

Degrees Two Theta

F i g u r e 1 2 . X-Ray Powder D i f f r a c t i o n P a t t e r n of Dibucaine


122 GANDHARVA R. PADMANABHAN

3* Synthesis
Dibucaine and dibucaine hydrochloride are prepared
by the following sequence of reactions (Figure 14)
starting with isatin (3). Isatin (I) is reacted with
malonic acid in the presence of an acid to form carbo-
styrilic acid (11). The acid is then treated with
phosphorous oxychloride to yield 2-chlorocinchoninic
acid chloride (111) in solution. The solution is then
reacted with diethylaminoethylamine to form 2-chloro-
N(2-diethylaminoethyl) cinchoninamide (IV) in solution.
The cinchoninamide solution is then treated with
sodium n-butylate to form dibucaine base(V). The base
is purified and then converted to the hydrochloride
salt (VI) by reacting with hydrogen chloride.

4. Stability-Degradation
Dibucaine hydrochloride (I) (Figure 1 5 ) , when
boiled for 4 hours in 2N hydrochloric acid, resulted
in complete hydrolysis to 2-hydroxyquinoline-4-carboxy-
lic acid diethylaminoethylamide (11) and 2-hydroxy-
quinoline-4-carboxylic acid (III)(4). Hydrolysis of
dibucaine hydrochloride, with pH = 5.45 and at a
temperature of 134°C for 40 hours resulted in the
formation of Compound I1 only Autoclaving of a solution
of dibucaine hydrochloride in a mixture of 10% sodium
hydroxide and ethanol at 120°C for 2 hours resulted in
the formation of 2-butoxyquinoline-4-carboxylic acid
(IV). Under the influence of an oxidizing agent such
as m-chloroperbenzoic acid, dibucaine can be oxidized
to its N-oxide analog (V). The N-oxide can further
react with reagents such as ferrous sulfate to yield
the desethyl analog (VI) of dibucaine and dibucaine
(5).

5. Drug Metabolism and Pharmacokinetics


An apparent half-life of approximately 11 hours
with a peak serum concentration at 2 hours after
administration was obtained following the administration
of single 5 mg dibucaine hydrochloride oral dose (15)
to human volunteers. Serum levels of dibucaine in
monkeys and dogs after intravenous administration
indicated an apparent elimination half-life of approxi-
mately one hour. Serum peak concentrations of dibucaine
were found after 1-6 hours in monkeys, dogs and humans
after rectal administration of an ointment formulation.
Rectal administration of the ointment formulation to
human volunteers at 0.2-0.6 mg/kg level, t.i.d. for 3
days resulted in peak serum level after the third or
fourth dose and declined to base-line within 48 hours
DIBUCAINE AND DIBUCAINE HYDROCHLORIDE 123

I I
5 1; 2; 35

Degrees Two Theta

Figure 1 3 . X-Ray Powder D i f f r a c t i o n P a t t e r n of


Dibucaine Hydrochloride
COOH

IV 111
NaOCgHg
124 GANDHARVA R. PADMANABHAN

\ II
0
0
t

IV

VI

Figure 15
Chemistry of Dibucaine and Dibucaine Hydrochloride
DIBUCAINE AND DIBUCAINE HYDROCHLORIDE 125

of t h e l a s t dose. D i s p o s i t i o n of d i b u c a i n e f o l l o w i n g
m u l t i p l e r e c t a l a d m i n i s t r a t i o n of 0.1% s u p p o s i t o r i e s
i s comparable t o ointment a d m i n i s t e r e d s i m i l a r l y
(6-7).

6. Toxicity
The a c u t e i n t r a p e r i t o n e a l LD50 v a l u e s of d i b u c a i n e
h y d r o c h l o r i d e i n male mice and female mice observed
over a p e r i o d of 15 days were found t o b e r e s p e c t i v e l y
7 1 mg/kg and 74 mg/kg. The a c u t e o r a l LD50 v a l u e s of
d i b u c a i n e h y d r o c h l o r i d e i n m a l e rats and female r a t s
observed o v e r a p e r i o d of 1 5 days w e r e found t o be
r e s p e c t i v e l y 371 mgfkg and 395 mg/kg (8).

7. Methods of A n a l y s i s
7.1 I d e n t i f i c a t i o n
Two i d e n t i f i c a t i o n tests are g i v e n i n t h e
USP XX f o r d i b u c a i n e , one a n i n f r a r e d a b s o r p t i o n
t e s t and t h e o t h e r a n u l t r a v i o l e t a b s o r p t i o n
t e s t . For d i b u c a i n e h y d r o c h l o r i d e , f o u r i d e n t i -
f i c a t i o n tests are g i v e n i n USP XX. The t e s t s
i n c l u d e d are i n f r a r e d a b s o r p t i o n , u l t r a v i o l e t
a b s o r p t i o n , m e l t i n g p o i n t of i s o l a t e d f r e e b a s e
and a t e s t f o r c h l o r i d e .

Methods t o i d e n t i f y and d i f f e r e n t i a t e
d i b u c a i n e from n i n e o t h e r l o c a l a n e s t h e t i c s have
been r e p o r t e d i n t h e l i t e r a t u r e ( 9 ) . The methods
are based o r t h e m e l t i n g , i n f r a r e d and photomicro-
g r a p h i c p r o p e r t i e s of t h e d e r i v a t i v e s o b t a i n e d
with styphnic acid, p i c r i c acid, c h l o r o p l a t i n i c
a c i d , p i c r o l o n i c a c i d , ammonium r e i n e c k a t e and
methyl i o d i d e .

7.2 Elemental A n a l y s i s
The f o l l o w i n g e l e m e n t a l compositions were
o b t a i n e d f o r d i b u c a i n e and d i b u c a i n e h y d r o c h l o r i d e
when 2 mg samples were employed f o r a n a l y s i s
w i t h a Perkin-Elmer Model 240 CHN Analyzer.
DIBUCAINE
Element Theory, % Found, %
Carbon 69.94 69.67
Hydrogen 8.51 8.60
Nitrogen 12.23 12.07
126 GANDHARVA R. PADMANABHAN

DIBUCAINE HYDROCHLORIDE
Element Theory, % Found, %
Carbon 63.22 62.99
Hydrogen 7.96 8.19
Nitrogen 11.06 10.88

7.3 Nonaqueous Titration


Dibucaine may be titrated in glacial acetic
acid with perchloric acid in glacial acetic acid
as titrant. The titration can be carried out
potentiometrically or with crystal violet as
indicator. Dibucaine hydrochloride may be titrated
similarly in glacial acetic acid containing
mercuric acetate with perchloric acid
in glacial acetic acid as titrant. Two equivalents
of acid are consumed in the titration of dibucaine
and dibucaine hydrochloride. The titration is not
specific for the drug in presence of some of
their degradation compounds.

7.4 Phase Solubility Analysis


Phase solubility analysis of dibucaine
hydrochloride has been carried out using the
following system (10):
Dibucaine Hydrochloride
Solvent Temperature Approx. Solubility
mdg
Ethyl acetate 25 15.4

7.5 Thin-layer Chromatography


A number of thin-layer chromatographic
systems have been developed for the identification
of the drug and for the determination of the
compounds related to the drug.
System I - The following system may be employed
particularly to control the impurities
likely to be present from the synthesis
of the drug.
Adsorbent : Silica Gel G plate, 20 cm x 20 cm
coated to a thickness of 250 microns
Mobile Phase: A mixture containing 30 mL of
acetone, 50 mL of toluene, 5 m L of
methanol and 1 m L of concentrated
ammonium hydroxide.
DIBUCAINE AND DIBUCAINE HYDROCHLORIDE 127

System I Continued
Detection
Systems: 1. Spray with 0.5% potassium
dichromate in 20% sulfuric acid
followed by heating at 140°C
for 10 minutes and viewing
under shortwave UV
2. Irradiate with high intensity
UV for 10 minutes followed by
visualization under long wave UV

System I1 - The following system may be employed


particularly when 3-chloro dibucaine
content in the drug has to be deter-
mined.
Adsorbent: Silica G plate, 20 cm x 20 cm,
coated to a thickness of 250
microns
Mobile Phase: A mixture of 35 mL of glacial
acetic acid, 55 mL of ethyl acetate,
5 mL of concentrated hydrochloric
acid and 5 mL of water
Detection
Systems : 1. Shortwave UV
2. Irradiation with high intensity
UV for 10 minutes followed by
visualization under longwave UV

System I11 - The following system may be employed


for the estimation of transformation
products in formulations
Adsorbent: Silica Gel G plate coated to a
thickness of 250 microns
Mobile Phase : A mixture of 80 mL of chloroform,
20 mL of methanol, 1 mL of
ammonium hydroxide and 1 mL of
water
Detection
System : Irradiate with high intensity
W for 10 minutes followed by
visualization under long-wave
w
128 GANDHARVA R. PADMANABHAN

Other Systems: The following systems have also


been employed for the analysis
of dibucaine or dibucaine hydro-
chloride.
System IV - Chloroform/Acetone/Diethylamine
(5:4:1); Silica Gel GF; Dragendorff
Spray, Iodoplatinic Acid Spray and
UV Detection Systems (11)
System V - Chloroform/Diethylamine ( 9 : l ) ;
Silica Gel GF; Detection Systems
same as in System IV
System VI - Methanol/Ammonium Xydroxide (100:
1.5); Silica Gel GF; Detection
Systems same as in System IV
System VII - n-Butanol/Acetic Acid/Water (5:3:2);
Silica Gel GF; Detection Systems
same as in System IV
. System VIII - Chloroform/Methanol (9:l); Silica
Gel GF; Detection systems same as
in System IV
System IX - Dioxane/Water ( 9 : l ) ; Silica Gel GF;
Shortwave W,Longwave UV, 0.5%
Iodine in Chloroform Spray,
Acidified Potassium Iodoplatinate
Spray and 40% Sulfuric Acid Spray
Followed by Heating and Longwave
UV Detection Systems (11)
System X - Dioxane/Water/Chloroform (8:l: 1);
Silica Gel GF; Detection Systems
same as in System IX
System XI - Dioxane/Water/Toluene (8:l:1);
Silica Gel GF; Detection Systems
same as in System IX
System XI1 - Acetone/Benzene/Methanol/Concentra-
ted Ammonium Hydroxide (30:50:5:1);
Silica Gel G; Longwave UV After 10
minute Irradiation with High
Intensity W
System XI11 - Chloroform/Methanol/Water (80:20:2);
Silica Gel G; Dichromate in 20%
Sulfuric Acid spray followed by
heating and visualization under
short-wave UV
DIBUCAINE AND DIBUCAINE HYDROCHLORIDE 129

7.6 High Performance Thin-layer Chromatography


The following system has been reported for
the quantitation of dibucaine in injectable
solutions and in plasma and serum samples (12).
Developing Solvent: Ether/Benzene/Cyclo-
hexane/Diethylamine
(20:12.5:10:3.5)
Adsorbent: HPTLC Silica Gel 6 0
F254 (Merck)
Chamber Saturation: 15 minutes
Development Distance: 4 cm
Time of Development: 5 minutes
Sample Volume 200 nL
Detection Mode: Reflectance (240 nm)

7.7 High Pressure Liquid Chromatography


The following systems have been reported for
the quantitation of impurities in dibucaine and
dibucaine hydrochloride samples (13).
System I
Column : 25 cm x 4.6 mm i.d. Zorbax C-8
stainless steel column with 6.5 cm
x 2.1 mm i.d. Whatman CO-Pel1 ODS
guard column
Detection: W-313 nm
Temperature: Ambient
Flow Rate: 1 mL/minute
Mobile Phase: Linear gradient from 100% A
to 95% B in 20 minutes. A = 1:l
methanol-water; B=0.2% ammonium
hydroxide in 2:8 methanol-aceto-
nitrile

System 11 (11)
Column : 25 cm x 4.6 mm Lichrosorb RP 8
column
Detection: UV 254 nm
Temperature: 25 C
Flow Rate: 2 mIJminute
Mobile Phase: A. Methanol-Water-Diethylamine
(90:10:0.02)
B. Methanol-Water-Diethylamine
(80:20:0.02)
C. Methanol-Water-Diethylamine
(75: 25:0.02)
130 GANDHARVA R. PADMANABHAN

System I1 Continued
Approximate
Retention A = 4.5 minutes
Time of B = 7.4 minutes
Dibucaine C = 10.9 minutes

System I11 (14)


Column: 50 cm x 2.1 mm (i.d.) stainless
steel column packed with Permaphase
ODS
Detection: W-254 nm
Fluorescence: Excitation-325 nm and
Emission-390 nm
Temperature: 40O C
Flow Rate: 0.85 mL/minute
Mobile Phase: 50% Isopropanol, 45% Methanol and
5% 0.001N NaOH

7.8 Gas Chromatography


The following system has been employed for
the analysis of dibucaine in the drug substance
and in a suppository formulation.
Column: 6 ft x 4 mm (i.d.) column with
3% OV-17 on Gas Chrom Q (100 x 120
mesh)
Temperature: -
Column 25OoC; Injector - 27OOC;
Detector - 300°C
Detector: Flame Ionization Detector
Carrier: Helium 60 cc/minute
Sample: Inject 2.0 pL of a 10 mg sample
in 1 mL of tetrahydrofuran

7.9 Gas Chromatography-Mass Spectrometry (GC-MS)


Sensitive methods €or the analysis of
dibucaine in serum samples have been reported
using GC-MS with selected ion-monitoring for
separation and detection. The following experi-
mental conditions were used for the analysis of
the drug in biological fluids.
Method I (15)
Column : 2.5 ft x 2 mm i . d . silanized glass
column packed with 1.5% OV-17 on
80/100 mesh Chemosorb W-HP
Detection: GC-MS selected ion monitoring
at m/e = 228 and at m/e = 237
DIBUCAINE AND DIBUCAINE HYDROCHLORIDE 131

Method I Continued
Temperature: I n j e c t o r - 260°C; Column - 250°C:
GC-MS I n t e r f a c e -250°C
Carrier: Helium
MS E I Source: 37 ev
Internal
Standard: Nonadeuterated d i b u c a i n e

Method I1 (15)
Column : 2.5 f t x 2 mm i . d . s i l a n i z e d g l a s s
column w i t h 1 . 5 % OV-1 on Chromasorb
W-HP 80/100 mesh
Detection: GC-MS (CI) s e l e c t e d i o n m o n i t o r i n g
a t m / e = 344 and m / e = 353
Temperature: Column - 215°C; I n j e c t o r - 260°C;
GC-MS I n t e r f a c e - 250°C
Carrier: Methane
MS C I Source: 55-85 ev
C I Reagent
Gas: Methane
Internal
Standard : Nonadeuterated d i b u c a i n e

Method I11 (16)


Column: 0.5 m x 3 mm i . d . g l a s s column
packed w i t h 3%Poly 1-110 on Gas
Chrom Q 80-100 mesh
Detection: E . I . S e l e c t e d ion-monitoring a t
m/ e=86
Temperature: Column - 260°C; I n j e c t o r - 350°C;
S e p a r a t o r - 3OO0C, I o n Source -
310 C
Carrier: Helium, 30 mL/min
In t e r n a 1
Standard : Chloropromazine H C 1 (m/e=86)

Method I V (17)
Column : 2 m x 2 mm g l a s s column packed
w i t h 3% OV-17 on 80-100 mesh Gas-
Chrorn Q
Detection : GC-MS s e l e c t e d i o n monitoring a t
m / e = 326 and a t m / e = 335 and 336
132 GANDHARVA R. PADMANABHAN

Method IV Continued
Temperature: Column: Programmed at G°C/minute
from 260-300°C; Injector - 300°C;
Ion Source - 300°C
Carrier: Not indicated; 30 mL/minute
MS-EI Source: 75 ev; ionizing current - 300 PA
Internal
Standard : Deuterium-labeled dibucaine (Dg
and Dlo)
7.10 Paper Chromatography
Stationary
Phase: Whatman #1 paper impregnated with
a 1:l solution of acetone and
.
formamide Formamide was adjusted
to pH 5.6 with benzoic acid before
mixing. Remove the excess of the
impregnated solution by blotting
between dry filter papers
Mobile Phase: 2% pyridine in 1:l benzene-
chloroform
Detection: Dragendorff spray reagent
Sample
Solution: Spot 10 pL of 1% dibucaine
hydrochloride in 1:l methanol-
chloroform (18)
R of
f
Dibucaine HC1: -0.75

7.11 Polarography
Polarography has been employed for the
analysis of dibucaine in soLutions and for the
identification of dibucaine (19-20). Polarography
was carried out using a borate-biphosphate buffer
with pH of 5 to 7.5 and measuring the reduction
current at -0.6 V vs calomel electrode. The method
was linear between 5 and 150 mg/100 mL.

7.12 Spectrophotometry
Dibucaine and dibucaine hydrochloride in
formulations can be analyzed by spectrophotometry
(21) by taking advantage of the maxima at 247 nm
and 320 nm in acidic solutions. The technique
when preceded by acid-ether and base-ether
extraction steps is selective for all products
discussed under Section 4 , Stability-Degradation,
except for compound VI.
DIBUCAINE AND DIBUCAINE HYDROCHLORIDE 133

8. Miscellaneous
8.1 Dibucaine Number
When succinylcholine, which is a neuromuscular
agent, was introduced for anesthetic procedures,
it was observed that certain individuals failed to
recover from the paralytic effects and this poor
recovery was attributed to the low activity of
the enzyme cholinesterase in plasma. The identi-
fication of the atypical enzyme activity has been
carried out by the selective inhibition of the
plasma esterase by dibucaine with benzoylcholine
as substrate. A quantitative measure of this
selective inhibition, expressed as a percent of
inhibition, is called the dibucaine number (22-25).

9. References
1. Martucci, J . D. and Schulman, S. G., Anal. Chim.
Acta, 77, 317 (1975)
2. Hrdy, 0. and Slouf, A., Cs. Pharm., 1,7 1 ( 1 9 5 2 ) ;
and Die Pharmaczie, 8, 1 5 9 ( 1 9 5 3 )
3. CIBA-GEIGY, Personal Communication
4 . Morch, J . , Dansk. Tidsskr. Farm., 27, 1 7 5 (1953)
5. Senn, H. and Kathriner, A., CIBA-GEIGY, Personal
Communication
6 . Alkalay, D., Carlsen, S., Khemani, L., Wagner
Jr., W. E . , and Le Sher, A., CIBA-GEIGY, Personal
Communication
7. Bartlett, M. F. and Egger, H., CIBA-GEIGY, Personal
Communication
8. Thomann, P. and Pericin, C., CIBA-GEIGY, Personal
Communication
9 . Rich, N. W. and Chatten, L. G., J . Pharm. Sci.,
-
5 4 , 9 9 5 (1965)
10. Grady, L. T., Pharmacopeial Formum, United States
Pharmacopeial Convention, Inc., p. 1 4 3 6 , Sept.-
Oct. 1 9 8 1
11 Grady, L. T., USP-NF Reference Standards Committee,
United States Pharmacopeia, Letter 9 9 , p. 432-
438, dated February 2 5 , 1 9 8 1
1 2 . Giibitz, G. and Wintersteiger, R., Sci. Pharm.,
4 6 , 275 (1978) (German)
-
13. Liu, R., CIBA-GEIGY, Personal Communication
1 4 . Takeoka, T., Kojima, T. and Kobayashi, H., Nippon
Hoigaku Zasshi, s, 20 (1979) (Japan)
15. Alkalay, D., Carlsen, S. and Wagner, W. E., Anal.
Letters, 14(B20), 1 7 4 5 (1981)
1 6 . Kageura, M., Totoki, K. and Nagata, T., Nippon
Hoigaku Zasshi, 2, 188 ( 1 9 7 8 ) (Jap. J . Legal
Med.) (English)
134 GANDHARVA R. PADMANABHAN

17. Shinka, T., Kuhara, T. and Matsumoto, T., Quant


Mass Spectrom. Life Sci., 2, 315 (1978)
18. Korzun, B. P., CIBA-GEIGY, Personal Communication
19. Dusinsky, G., Pharmazie, 9, 27 (1954)
20. Pech, J., Collection Czechosl. Chem. Communications,
6,
- 132 (1934)
21. United States Pharmacoepia, Twentieth Revision,
Mack Printing Company, Easton, PA, 1980, pages
226-228
22. Kalow, W. and Genest, K., Canad. J . Biochem.
Physiol., 35, 339 (1957)
23. Harris, H. and Whittaker, M., Nature, 191,496
(1961)
24. Brody, I. A., Resnick, J. S. and Engel, W. K.,
Arch. Neurol., 13,126 (1965)
25. Irwin, R. L. and Hein, M. M., Biochem. Pharmacol.,
-
15, 145 (1966)

10. Acknowledgement
The author expresses appreciation to Ingrid
Becue, Richard Brown, James B. Smith and Jane Johnson
f o r help in preparation of this manuscript.
ESTRONE
Douglas Both

I . Introduction 136
1.1 History 136
1.2 Structure, Nomenclature, and Molecular Weight 137
1.3 Biosynthesis and Metabolism 137
1.4 Synthesis and Commercial Production 142
2. Physical Properties 144
2.1 Crystal Structure 144
2.2 Powder X-Ray Diffraction 145
2.3 Melting Point 145
2.4 Thermal Analysis 145
2.5 Magnetic Susceptibility 149
3. Spectrometry 149
3.1 Proton Nuclear Magnetic Resonance 149
3.2 Carbon-I3 NMR Spectra 149
3.3 Mass Spectrometry 152
3.4 Infrared Spectrometry 154
3.5 Ultraviolet and Visible Spectrophotometry 156
3.6 Optical Rotatory Dispersion and Specific Rotation 156
3.7 Fluorescence and Phosphorescence 157
4. Solution Properties 158
4.1 Solubility 158
4.2 Partition Coefficients 158
4.3 Molecular Volume 158
4.4 Heat of Formation and Combustion 160
4.5 Acid Ionization Constant 160
4.6 Stability 160
5. Chromatographic and Other Separation-based Analysis 161
5.1 Column Chromatography 161
5.2 Thin Layer Chromatography 163
5.3 High-Performance Liquid Chromatography 165
5.4 Gas-Liquid Chromatography 167
5.5 Gas Chromatography-Mass Spectrometry 169
6. Radioassay 170
7. Colorimetric Analysis 172
8. Titrimetric Analysis 174
References 174

ANALYTICAL PROFILES OF DRUG SUBSTANCES 135 Copyrightby the American Pharmaceutical Association.
VOLUME 12 ISBN 0-12-260812-7
136 DOUGLAS BOTH

1.0 INTRODUCTION

1.1 HISTORY

In 1 8 9 6 l Y 2 , Dr. Emil Knauer, using ovarian transplants


into immature female animals, first demonstrated the
existence of sex hormones. In 1909, Henry H. Dale
developed a posterior pituitary gland extract which
stimulated uterine contraction that found great use during
complicated labor. This was the beginning of the steroid
era.

In 19273, two Berlin gynecologists, Dr. Selmar


Aschheim and Dr. Bernard Zondek, while developing a method
for the early diagnosis of pregnancy, established that the
urine of pregnant women contained a high concentration of
estrogen. Before this discovery, the isolation of
estrogens had been unsuccessfully pursued from placental
extracts.

Dr. E.A. Doisy and Dr. E. Allen, were working towards


the isolation, purification and crystallization of estrone
from pregnancy urine. Aldolf Butenandt, under the
direction of nobel prize winner Adolf Windaus of Gijttingen
University, was also independently working towards this
goal. After nearly two years, in August of 1929, Doisy4
and Allen reported the first account of the purification
and crystallization. A few months later in Octob%r 192g5,
Butenandt also reported its isolation. Butenandt claimed
that Doisy and Allen preempted him by virtue of an annual
cleaning of the Gijttingen Laboratory. The laboratory was
shut down for several weeks, just as he had obtained a
potent extract and was 10-14 days from purification.

At7 first, only the physiological effects and some


chemical properties were known. The first physico-chemical
characterization of the crystal and the correct empirical
formula were reported by Thayer in 1930. The name estrone
was not adopted until 1935. The first partial
of estrone was performed by Inhoffen in 1 9 4 1 and the first
total synthesis in 1948 by Anner.

The isolation of estrone was of great importance, for


it was the first estrogenic steroid hormone isolated. The
great interest generated by this compound was responsible
for the isolation of later steroid hormones. It was these
later steroids that revolutionized the treatment of many
different illnesses,
ESTRONE 137

1.2 STRUCTURE, NOMENCLATURE AND MOLECULAR WEIGHT

The basic skeletal structure of estrone is the


cyclopentanoperhydrophenanthrene nucleus. This nucleus is
composed of the four rings designated A,B,C and D. Each
carbon atom of the ring is numbered in rotation. The
methyl group is found in the beta position in naturally
occuring estrone. See figures 1 and 2.

Estrone is a white crystalline powder or a colorless


flat plate-like crystal. The powder is virtually odorless.
The name estrone is the offically adopted name, other
common synonymous names include: Oestrone,
3-hydroxyestra-1,3,5(10) trien-17-one, folliculin,
ketohydroxyestrin, menformon, theelin and follicular
hormone.

The empirical formula is C H O2 with a molecular


weight of 270.37. Estrone is gl!en2$he chemical abstracts
systematic number [53-16-71,

1.3 BIOSYNTHESIS AND METABOLISM

Estrone is resent in plants and animals. It has been


found in plantslg-14 such as the date, apple, pomegranate,
oat, apricot and in the oils of corn and olive. In
the major source is the ovary and to a lesser
extent, the adrenal cortex, feto-placental unit and Leydig
cells of the testis.

In Vitro s t u d i e ~ l ~
-- have
’ ~ ~shown that estrone arises
from acetate. After several Nicotinamide-Adenine
Dinucleotide Phosphate (NADPH) dependent condensation
reactions, acetate forms squalene, which subsequently
undergoes elimination and cyclization to yield cholesterol.
Cleavage of the side chain of cholesterol and several
rearrangements yield pregnenolone. Oxidation and
isomerization produce progesterone which leads to
4-androstene-3, 17-dione and finally to estrone. An
overview of the pathway can be seen in figure 3 .

Questionsz0 have been raised as to whether cholesterol


is a required intermediate in the synthesis. It seems that
several experiments have shown that the precursers of
cholesterol are incorporated into certain cells much more
readily. B. R. Bhavnani2 discusses other nonclassical
approaches to the biosynthesis of steroids which bypass
cholesterol as an intermediate.
138 DOUGLAS BOTH

ESTRONE
F i g u r e 1. The S t r u c t u r e of E s t r o n e

F i g u r e 2. Conformation Diagram o f E s t r o n e .
(Redrawn from R e f e r e n c e 82.)
ESTRONE 139

Figure 3 . Summary of the biosynthesis of estrone and other steroids.


(Adapted from references 18, 19.)
140 DOUGLAS BOTH

During the synthesis and metabolism, addition or


removal of hydrogen is catalyzed by enzymes found in the
ovary. Addition of oxygen is catalyzed by hydroxylases,
usually taking place in the liver and kidney, which are
important sites for the inactivation of steroids.
Hydroxylation reactions usually occur at the 2, 6 , 8 , 11,
16 or 18 carbon positions, and require molecular oxygen
while utilizing NADPH.

Omura22 worked out the electron transport system for


the 11-8 hydroxylation which occurs in the adrenal
mitochondria. Hydroxylation involves the transport of
hydrogen from NADPH to flavoprotein. The reduced
flavoprotein transfers electrons to a non-haem protein.
The reduced non-haem protein will transfer electrons to
cytochrome P-450. It is believed that this type of pigment
system exists for other biosynthesizing tissue such as the
feto-placental unit.

Estrone exists in bound and unbound forms. It is this


binding with protein that plays a role in the metabolism of
estrone. E ~ t r o n e binds
~ ~ ’ ~strongly
~ to red blood cells
which may increase its solubility, and thus, function to
regulate its distribution and availability to target
organs. ConjugationZ5 of estrone in the liver works to
inactivate the steroid while secretion of a sulfate
conjugate by the adrenal cortex serves as a reservoir for
later activation in target organs. Hydroxylation26 and
methylation appear to be major modes of estrone metabolism.
Both metabolites, as well as estrone, in the form of
glucuronic acid or sulphate conjugates, appear in
significant quantities in urine. Estrone can be readily
converted into a number of products as shown in figure 4.

During the metabolismz7 of estrone, a rapid


equilibrium is established between estrone, estriol and
178-estradiol. This equilibrium has been studied in vitro
in the liver and kidney by Rayan28 and Velle29. The
equilibrium ratios of estrone, estriol and 17B-estradiol in
urine are approximately 45:45:10. The rate constant for
the conversion of estrone to 176-estradiol in the uterus,
however, is 10-20 times larger than the rate constant for
the reverse reaction. MuseyZ6 discusses the pathways for
the metabolism of estrone sulfate and other conjugates in
greater depth; also discussed are conjugate transport
dynamics and hydrolysis.
ESTR 0NE 141

'2. HVOROXVESTRONE '2. YETHOXVESTRONE

-
Flgure 4 Summery of the metabollrm of emone.
(Adapted from reference 18, 10.)
142 DOUGLAS BOTH

Plasma 30’35 levels of estrone for men range form


0.1-0.42 ug/L. For women, levels range from 0.2-0.7 pg/L,
depending on the phase of the menstrual cycle. Urinary
excretion levels for men are 3-8.2 pg/24 hr, and for women
are 0.3-2.4 pg/24 hr.

1.4 SYNTHESIS AND COMMERCIAL PRODUCTION


The first partial synthesis36 of estrone was
accomplished by Inhoffen in 1941. The first total
synthesis was reported by Anner37 and Miescher in 1948.
Since that time, a multitude of starting products,
intermediates and routes have been reported. Estrone is an
important synthetic product, for it is a key intermediate
in the synthesis of many complex 19-nor steroids.

As early as 192838, Parke-Davis and Co. produced an


ovarian extract of estrone. Following the early crude
products were purer forms extracted from urine, as reported
by Schering-Kahlbaum and later Roussel and N.V. Organon.
After the discovery of the levels of estrone in the urine
of stallions and pregnant mares, Zondek, and later American
firms, produced estrone extracted from urine. While the
extraction process initially proved economically feasible,
improved partial or total synthesis became more cost
effective.

The in production of synthetic estrone was


Roussel who converted dehydroisoandrosterone to estrone.
Dehydroisoandrosterone can be produced from cholesterol
which, in turn, can be extracted from plant and animal
oils, grease, wool and waxes. Other methods of production
included: pyrolysis, microbiological reduction or
transformation. For a review of fermentation in industrial
applications see Reference 41, there are several papers
that review the general synthesis of estrone See References
42-46. Table (1) gives a listing of selected syntheses.

Table 1 - Selected Syntheses

REFERENCE METHOD

47 Regiospecific fuctionalization of 2,3,


BIS(trimethylsily1)estratrien -17-one using
cobalt catalyst
48 Regiospecific Diels-Alder using, 6-
methoxy-l-vinyl-3,4-dihydronapthalene
ESTRONE 143

49 Biomimetric polyene cyclization

50 From ethyl-l-carbethoxy-2-oxo-l-
cyclohexaneacetate

51 From methyl-l-keto-2-methyl-7-
methoxy-1,2,3,4,4A,9,10A-octahy-
dro-2-phenanthrenecarboxylate

52 From 1,4-androstandiene-l9-01-3,20-
dione-19-benzoate

53 From a mixture of androsta-1,4-


diene-3,17-dione-l7-(cyclic ethylene acetal)
and 20-(hydroxymethyl) pregna-lY4-diene-
3-one.

54 By irradiation of 3,3,17,17-bis
(ethylenedioxy)-19-hydroxy-5-androstene

55 From 4-pregnene-17aYl9,21-trio~-3,
20-dione-19,21-diacetate

56 From androsta-1,4-diene-3,17-dione-17-
ethylene acetal

57 From 3,17-dioxestra-4,9-diene by isomerizing


in the presence of an acylhalide

58 By fermentation using brevibacterium (ATCC


19653)

59 By asymmetric conversion of
2-methyl-2-carbethoxyethyl cyclopentane-
1 ,3-dione

60 By systematic degradation from


106-hydroxy-19-norperlplogenin

61 From 19-nor-4-androstene-3,17-dione with


p-cymene catalyzed by lead on carbon

62 An expeditious synthesis

63 From 5-androstene-3f3,19-diol-l7-one by
microbiological action of mycobacterium
phlei strain w
144 DOUGLAS BOTH

64 By oxidation of 19-nor-1(10),5-androstadiene
36-01-17-one
65 By steroselective intramolecular
cycloadditon of olefinic-o-quinodmethane

66 From andro-1,4-diene-3,17-dione by pyrolysis

67 From 17B-estradiol by enzymatic oxidation


and reduction using Pseudomonas testosteroni

68 Microbiological transformation with the use


of cholest-4-ene-3-one as a steroid inducer

69 By microbiological conversion of
19-hydroxy-androst-4-ene-3,17-dione

70 From m-CH OC H CH CH:CH2


3 6 4 2
71 By cationic olefinic cyclization

72 From 2B-hydroxy-3,17-dioxoandrost-
4-ene-19-al

73 From 19-hydroxycholesterol acetate by


mycobacterium conversion

74 By microbiologica~dehydrogenation of
3-keto-4-steroids

75 From l-hydroxy-4-androstene-3,17-dione by
fermentation with Penicillium Sp (ATCC
12,556)
76 From andro-1,4-diene-3,17-dione by
pyrolyzing with hot kerosine.

2.0 PHYSICAL PROPERTIES

2.1 CRYSTAL STRUCTURE

Estrone exists in three polymorphic crystalline forms.


The polymorphic form obtained depends on the mode of
crystallization. Two forms are orthorhombic; Form I is
stable and Form I1 is a metastable state. The third form,
Form 111, is monoclinic and metastable.
ESTRONE 145

Forms I and I11 are usually obtained by sublimation,


while Form I1 can be obtained by evaporation from a
solution of acetone or methanol. The crystalline cohesion
of the three forms are different. Forms I and 11177 have
layers of parallel molecules linked by hydrogen bonds and
Form I1 has a herring bone arrangement with weaker hydrogen
bonds and stronger dispersion bonds. Figure 5 7 8 is a
diagram of the two crystalline forms. Bernard B u ~ e t t a ' ~ - ~ ~
has studied the crystalline form of estrone quite
extensively. Table 2 is a summary of the parameters of the
three crystalline forms as measured by Busettair7.

2.2 POWDER X-RAY DIFFRACTION

Figure 6 is the x-ray powder diffractionB2 spectrum of


the U.S.P reference standard estrone. The spectrum was
obtained using a Philips powder diffraction unit utilizing
the Ka emission of copper at 1.54A. The sample was scanned
"as is" from 8 to 48 degrees ( 2 0 ) . The d spacing in
angstroms is given above each major peak in the diffraction
spectrum. The d spacings of the major peaks appear to be
consistant with published valuesM3.

2.3 MELTING POINT

AtH4 1 8 0 " C , unstable crystals in the shapes of rods


and prisms begin to sublime. At 22OoC, stable rectangular
shaped crystals sublime. At about 2 3 0 " C , some of Form I11
is converted into form I. The remaining portion of the
original substance melts at 254°C (Form 111) or at 256°C
(Form 11) while Form I melts at 259°C.

2.4 THERMAL ANALYSIS

Differential thermal a n a l y ~ i s ~of~ 'estrone ~~ shows a


small broad endotherm at about 230°C which seems to
indicate a crystalline transition described in section 2 . 3 .
A sharp strong endotherm at 258°C probably corresponding to
the melting process is also seen. The derivatogram shows
weight loss of 1 9 . 4 , 2 3 . 8 , 3 4 . 4 and 5 1 . 2 % at 3 5 0 , 3 6 5 , 395
and 4 2 5 " C , respectively.

The differential thermal analysis of the U.S.P.


reference standard estroneE7, scanned at 2"C/min, shows a
sharp endotherm at 264°C and a broad minor endotherm
between 2 3 1 and 237°C.

The U. S.P. reference standard estrone", dried at


105°C for 3 hours, lost 0.13% of its weight.
146 DOUGLAS BOTH

",
-
ORTHORHOMBIC FORM

Figure 5 - The two crystalline forms of estrone.


(Redrawn from reference 78.)
TABLE 2 - CRYSTAL DATA

FORM I FORM I1 FORM I11

Evaporation from
Mode of Crystallinzation Sublimation Acetone or Methanol Sublimation
State Stable Metastable Metastable
Crystal form Or thorhombic Orthorhombic Monoclinic

Space Group PZ1' zl, 2, PZ1' Z1' z1 p2 1


Molecules per Two independent
unit cell (Z) 4 4 (4) molecules
" 3 1481 1440 1461
Cell volume (A)
Cell dimensions:
0

+ 0.005 A
a - 12.188 10.043 9.271
0

+ 0.005 A
b - 16.301 18.424 22.285
0

c -
+ 0.005 A 7.463 7.787 7.610
+ 0.2"
a - 90 90 90
B 5 0.2" 90 90 111.45

y 0.2" 90 90 90
Fig. 6. Powder X-Ray Diffraction Spectrum of Estrone.
ESTRONE 149

2.5 MAGNETIC SUSCEPTIBILITY

The mean molar magnetic susceptibility88 of estrone


re r stallized from methanol was found to be -176.47 x 106
s y
cm /mole, when measured according to the Faraday method.
Values calculated according to the Pascal empirical
systematic, the revised Pascal systematic and the empirical
systematic f r bonds were rep0 ted to be -170.74 x 10 ,
8 6 3
-180.95 x 10 and -180.10 x 10 cm /mole respectively.

3.0 SPECTROMETRY

3.1 PROTON NUCLEAR MAGNETIC RESONANCE

The proton nuclear magnetic resonance spectrum of the


U.S.P. reference standard estrone is shown in figure 7 .
The spectrum was obtained on a Varian XL-100 at 100.1 MHZ
in deuterochloroform (CDC1 ) using tetramethylsilane (TMS)
as a internal reference. 3

Because of the similar magnitude of the coupling


constants and chemical shifts of the protons of the
aliphatic rings, normal splitting patterns are distorted.
Virtual coupling of these protons make spectral assignment
difficult. The spectrum shows only one singlet at 0.9 ppm
from TMS belonging to the axial methyl protons.

3.2 CARBON-13 NMR SPECTRA

The proton decoupled Carbon-13 nuclear magnetic


resonance spectrum89 of the U. S.P. Reference Standard
estrone is shown in figure 8. The spectrum was obtained on
a Varian XL-100 at 15.4 MHZ. The resonance assignments of
the carbons of estrone are shown in Table 3. The resonance
chemical shifts of the carbons from TMS are shown to range
from 13.3 to 219.4 ppm.

The Carbon-13 spectra of estrone and other Eteroid


hormones were studied in several recent papersg0 93. The
tritium nuclear magnetic resonance spectra of estrone and
estrone sulphate have also been studied, yielding
information concerning the distribution of tritium between
labeled sites on the steroidg4,
L
u
0,

Fig. 7. Proton NMR Spectrum of Estrone: Instrument: Varian XL-100


L
9

Fig. 8. Carbon-13 NMR Spectrum of Estrone. Instrument: Varian XL-100


152 DOUGLAS BOTH

TABLE 3
CARBON-13 RESONANCE ASSIGNMENTS
CARBON PPM DOWNFIELD FROM TMS

1 125.5
2 112.5
3 154.7
4 114.8
5 136.8
6 37.9
7 26.0
8 28.9
9 43.3
10 129.7
11 25.4
12 31.1
13 47.2
14 49.7
15 20.9
16 35.2
17 219.4
18 13.3
3.3 MASS SPECTROMETRY
Figure 9 gives the low-resolylion mass spectrum of the
U.S.P. reference standard estrone .
The high-resolution
spectrum is given in Table 4. The molecular ion was found
at m/z 270.1585 while the anticipated molecular ion is m/z
270.1620. The fragmentation seen in Table 4 appears to be
consistant with the structure and mass spectrum of estrone.

TABLE 4
HIGH RESOLUTION MASS SPECTRUM

Mass Found Mass Calc. Formula Compositional Loss

270.1585 270.1626 C18H2202


242.1298 242.1307 16H1802 C2H4
237.1306 237.1279 C17H170 CH50(CH3+H20)
226.1388 226.1357 16H180 C2H40
213.1232 213.1279
15H17' C3H50
UJ
ul
a
3
(3
154 DOUGLAS BOTH

211.1115 211.1123 C3H70 (H20+C3H5)


C15H150
199.1072 199.1123
1qH1'5 C4H70
185.0943 185.0966
13'1 3' C5H90
172.0913 172.0888 C6H100
C12H120
159.0776 159.0810 C7H110
c1lH1lo
146.0702 146.0732 'gH12'
CIOHIOo
144.0542 144.0524 'gH14'
1OH8O
133.0654 133.0653 'gH13'
C9H90
131.051 1 131.0497 'gHISO
C9H70
120.0603 120.0575
'sH8' 10H14'
107.0537 107.0497 'llH15'
C7H70
3.4 INFRARED SPECTROMETRY
The infrared spectrum of the U.S.P. reference standard
estrone is shown in figure 10. The spectrumg7 was obtained
as a solid sample disc composed of 1 mg of estronel300 mg
KBr. Table 5 gives a possible interpretation of the given
spectrum. The interpretation appears to be consistent with
a previous published paperg8.

TABLE 5
IR SPECTRAL INTERPRETATION
FREQUENCY (an-') Assignment

3325 0-H Stretch

3050-3000 C-H Aromatic Stretch


2990-2800 C-H Aliphatic Stretching

1725 C=O Stretch

1620-1580 A dpublet straddling 1600


cm-l and a single peak at 1500
cm indicative of C-C aromatic
stretching and endocyclic bonding
Fig. 10. Infrared Spectrum of Estrone.
156 DOUGLAS BOTH

1475-1350 CH3-C Bending

1285-1250 -
A dpublet straddling 1275
cm - OH bonding and C-0
stretching indicative of aromatic
OH

1200-850 C-C Stretching region


quite complex, showing large
numbers of H-present.

3.5 ULTRAVIOLET AND VISIBLE SPECTROPHOTOMETRY

The ultravioletg9 spectrum of estrone 3n p-dioxane


shows maxima a about 282 nm ( E = 2.37 x 10 ) and 296 nm
5
(E = 2.13 x 10 ). An ethanolic solution of the U.S.P.
reference standard8’ estrone at a concentration of 1 in
25,000 w/v exhibits a maxmium at about 280 nm with a
absorptivity of 2.72. In concentrated sulfuric acid, l o o
strong absorption occurs at about 300 and 450 nm, and in
0.1g sodium h droxide at about 239 and 293 nm. The far
ultravioletluY spectrum of estrone in n-hexane exhibits a
strong absorption at about 200 nm, a shoulder at about 225
nm and a broad band at 275 nm.

The visible spectrum’O 2 of estrone treated with


Engelbrecht-Mori-Anderson cholesterol reagent (E.M.A.) (1.0
g FeCl 6 H 0, 40 mL 85% H PO in 500 ml AcOH, 500 ml H2S04),
scannea a$ 2OoC, showed Lxfma at about 392 and 484 nm.

3.6 OPTICAL ROTATORY DISPERSION AND SPECIFIC


ROTATION

Several optical rotator dispersion studieslo3’ll4


have been reported. EstroneroS exhibits a maximum
absorption at 310 nm with molecular rotations of 157,000 in
a solution of methanol. Estrone shows a negative cotton
effect at 275 nm and a weak positive cotton effect at 225
nm with a molecular rotation of 33,750. The specific
rotation of a 1%solution of the U.S.P. reference standard
estrone determined in a solution of dioxane was +163O.

Estrone has been determined by differential-


spectropolarimetry.l o 6 This method is based on the
difference in the optical activity of estrone and sodium
borohydride reduced estrone (Estradiol). Estrone can be
determined in the range of 30-1200 ug/ml.
ESTRONE 157

3.7 FLUORESCENCE AND PHOSPHORESCENCE

The f l u o r e s c e n ~ eof~ estrone


~ ~ ~ ~ ~in
~ ethanol, when
excited at 280 nm, shows a sharp fluorescence emission peak
at 307 nm and a second, broad peak at approximately 410 nm
with a decay time of 3.8 ns. A broad, emission band at
410 nm is the fluorescence of the carbonyl group, while the
phenolic chromophore emission is at the shorter wavelength.

Most of the excitation energy that is absorbed is due


to the phenolic chromophore. A very efficient energy
transfer to the carbonyl group allows for its weak
fluorescence. In solid film, estrone exhibits no carbonyl
fluorescence. This is believed to be a result of hydrogen
bonding between the phenol hydroxyl and the carbonyl group
in the crystalline film.

The phosphorescence1O9 emission spectrum of estrone


consists of a single broad peak at 410 nm. The quantum
yield of the phosphorescence is 0.025 with a decay time of
2.5 S. The phosphorescence is due to only the phenolic
chromophore.

The fluorescence of estrone may be used to detect its


presence at higher concentrations. However, the intensity
of the fluorescence is not great enough for use in most
cases where estrone is in trace amounts.

Estrogens can produce fluorophores when placed in


solutions of strong acids such as sulfuric and
phosphoric1l0'lZ1. The reaction of estrogens with
1-dimethylaminonaphthalene-5-sulfonyl chloride (Dansyl
Chloride) usually results in a substitution at the carbon
3-position of most estrogens to yield a fluorescent
d e r i v a t i ~ e ' ~ ~ ' ~ This
~ ~ . is best carried out in
acetone-water mixtures at pn 11-12. Under these
conditions, most estrogens show maximum fluorescence
intensity in about 30 minutes at room temperature, with a
limit of detection of about 0.5 pg/ml. A semi-automated
fluorometric method for detecting the total estrogen
content of plasma during late pregnancy has been
reported124.
158 DOUGLAS BOTH

4.0 SOLUTION PROPERTIES

4.1 SOLUBILITY

Table 6 gives the ~ o l u b i l i t y ~


of~ estrone
~ ’ ~ ~ ~in
various solutions and solvents. E p ~ h t e i n lhas
~ ~ derived
equations that demonstrate the correlation between aqueous
solubility and Van der Waals molecular volume. The
solubilization128’129 of estrone in aqueous solutions of
different association colloids has been studied. The
solubilization of estrone in sodium dodecyl sulphate at
40°C and in Tween 20 at 2OoC were reported to be 0.014 and
0.0068 moles of estronel mole of micellar substrate
respectively.

TABLE 6
SOLUBILITY OF ESTRONE (BY G.L.C.)

Solvent Temp OC Solubility mg/mL


tetrahydrofuran 30 48.336
p-dioxane 30 19.200
acetone 30 11.535
chloroform 30 15.680
methylene chloride 30 6.384
absolute ethanol 30 3.516
95% ethanol 40 6.227
methano1 30 4.041
ethyl ether 30 2.127
toluene 30 1.011
cyclohexane 30 0.023
hexane 30 0.004
water 25 0.0008

4.2 PARTITION COEFFICIENTS

Table 7 gives selectedlUo partition coefficients for


estrone in various solvent systems. The partition
coefficient (K) is defined here as the concentration of
solute in the upper phase/concentration of solute in the
lower phase.

4.3 MOLECULAR VOLUME

The average apparent molecular volume130 of estrone in


a solution of methanol at a concentration of 0.0207
lpgles/1000 g of solvent was determined to be 372.5
A /molecule with a precision of 2.5%. It has been shown in
ESTRONE 159

steroids that the average molecular volume per atom of


carbon is constant and that the molecular volume decreases
as the number of substituted hydroxyl groups increases.

TABLE 7
PARTITON COEFFICIENTS
Partition
Solvent System Coefficient(K)

etherll.5M sulfuric acid 100


etherjwater 90
ether/pH 10 carbonate buffer 28
ether/l.OM sodium hydroxide 0.5-0.6
n-hexanejwater 6.8
petroleum etherjwater 3.34
petroleum ether/50% water-
50% methanol 0.06
petroleum ether/30% water-
70% methanol 0.56
40% ethyl acetate-60% n-hexanej
50% ethanol-50% water 2.2
10% ethyl acetate-90% cyclohexanej
40% ethanol-60% water 1.8
30% ethyl acetate-70% cyclohexanej
50% ethanol-50% water 2.1
40% ethyl acetate-60% cyclohexanej
50% ethanol-50% water 2.6
50% ethyl acetate-50% cyclohexanel
50% ethanol-50% water 4.2
10% methanol-90% waterjcarbon
tetrachloride 0.01
20% methanol-80% waterjcarbon
tetrachloride 0.04
30% methanol-70% waterjcarbon
tetrachloride 0.07
40% methanol-60% waterlcarbon
tetrachloride 0.15
50% methanol-50% waterjcarbon
tetrachloride 0.33
70% methanol-30% waterjcarbon
tetrachloride 1.3
90% methanol-10% waterjcarbon
tetrachloride 2.8
70% ethanol-30% waterjcarbon
tetrachloride 0.67
70% ethanol-30% water/5% chloroform-
95% carbon tetrachloride 0.40
70% ethanol-30% water/lO% chloroform-
160 DOUGLAS BOTH

90% carbon tetrachloride 0.31


70% ethanol-30% water/20% chloroform-
80% carbon tetrachloride 0.17

4.4 HEAT OF FORMATION AND COMBUSTION


The heat of combustion131 for estrone, using a
microbomb calorimeter, was determined to be AHo= 2355.3 2
3.1 Kcal/mole. Using this information the heaf of
formation was calculated to be -88.0 Kcal/mole.

4.5 THE ACID IONIZATION CONSTANT

The reported acid ionization constant (pK ) of estrone


shows great variation ranging from 9.36 to 11.8.
Previous132’1 3 3 methods of measurement included: back
titration, conductimetric and most recently U.V.
spectrophotometric methods. Recent workg9’1 3 4 places the
pKa between 10.34 and 10.914. The most recent
spectrophotometric determination135 reports the K to be
10.77 2 0.02 with seven determinations. Egorova1%a
discussed the correlation between structure and the
dissociation constant.

4.6 STABILITY
Estrone in most cases is a relatively stable compound.
A 0.1% wlw solution of estrone in chloroform was shown to
be stable for approximate1 3 years by TLC, using ten
different solvent s sternslg7. Estrone dissolved in sesame
oil and in showed little change in
physiological activity after six months of storage at room
temperature.

Four ml of blood140 were mixed with 1 ml ACD


stabilizer (2.13 g sodium citrate, 0.74 g citric acid, 2.0
g glucose1100 ml water), 1 ml of this mixture was then
incubated for 10 hours at 37’C with 1 pg of estrone and
estradiol and 10 mg glucose. The degradation of estrone
was twice that of estradiol with fresh blood and half that
of estradiolwith 42 day old blood. In the absence of
glucose degradation of estrone was half that of estradiol
with fresh as well as stored erythrocytes.
Estrone141 is not reduced by enzyme extracts of hog
ovaries, beef suprarenal glands or bull testes.
ESTRONE 161

Estrone dissolved in absolute alcohol decomposes when


exposed to ultraviolet radiation. When estrone in
d i o ~ a n e lis
~ ~irradiated with ultra-violet light at 313 nm
it forms 13a-estrone (lumiestrone), which is reversible
when unfiltered ultraviolet light is used. Creepage143 of
estrogens on glassware occurs only in uncovered vessels in
the presence of salts and absence of proteins. Larger
amounts of creepage occurs in freshly cleaned vessels and
when small volumes of concentrated solutions are placed in
larger vessels. No sorption onto glass from buffered
aqueous solution or decomposition in tightly closed
containers in the absence of proteins have been found.
Silanization of glassware can drastically reduce creepage.
The ~ t a b i l i t y ’ ~ ~of
’ ’estrone
~~ on TLC plates has been
studied. Estrone decomposes as a result of contaminants
present in air and not a result of the oxadative effect of
oxygen to any great extent.

5.0 CHROMATOGRAPHIC AND OTHER SEPARATION BASED


ANALYSIS

5.1 COLUMN CHROMATOGRAPHY

Estrone has been separated from other estrogens in a


variety of matrices using column chromatography. Column
chromatography has also been used to concentrate or
separate prior to analysis by HPLC, G.C., RIA or TLC.
Thus, column chromatography serves to remove interfering
compounds and to concentrate prior to detection by a more
sensitive method. Table 8 gives selected examples of
column chromatographic procedures.

TABLE 8
SELECTED COLUMN CHROMATOGRAPHIC PROCEDURES

Reference Description

145 Separation prior to G.C., TLC,


using anion exchange resin (AGl-X2 cl),
elution with a methanollwater solution.

146 Separation of estrogens prior to G.C.


on AGlX2 column, methanol water
elution.

147 Purification after hydrolysis on


Merckogel 6000, followed by separation
on Sephadex LH 20, elution with
162 DOUGLAS BOTH

heptane-chloroform-ethanol-water
mixture.

148 Separation of conjugated urinary


estrogens on Sephadex G-15, elution
with 0.01M ammonium formate.

149 Separation of estrogen conjugates on


DEAE-Sephadex using gradient elution
(0-0.4g) sodium chloride.
150 Separation of androgens, estrogens, and
progestrogens on Lipidex elution with
hexane-chloroform mixture.
151 Separation of Sephadex LH-20 using
methylene chloride elution.

152 Separation of 14 testicular steroids


using celite column prior to HPLC.

153 Analysis of steroid mixtures using


silicic acid column eluted with
gradient of ethyl ether.

154 Extraction of estrogen conjugates from


pregnancy urine using amberlite XAD-2
resin and elution with 30% ethyl
alcohol.

155 Separation of free estrogens on


Sephadex LH-20 eluting with
cyclohexane-benzene-methanol mixture,

156 Purification of urine for


quantification of complete estrogen
profile, using Sephadex G-25,
DEAE-Sephadex A-25, Sephadex LH-20 and
DEAE Sephadex A-25 followed by G.C. or
selective ion monitoring.
157 Separation of C CI9 and C steroids
using Sephadex 2A120 and eluied with
n-hexane-ethyl acetate-methanol
mixture.
ESTRONE 163

158 Separation of U.V. absorbing


constituents in urine on Diaion CDR-10
using a linear gradient of ammonium
acetate (0-6M).

159 A two step anion exchange separation


for the purification of estrogens using
DEAE-Sephadex A-25 prior to capillary
gas chromatography.

160 Separation of radioactive steroids and


steroid conjugates from urine using
Amberlite XAD-2 and DEAE- Sephadex A-25
with NaCl gradient elution.

161 Separation of conjugated estrogens in


urine using Sephadex G-25,
DEAE-Sephadex and Sephadex G-15.

162 Separation of estrone, estradiol, and


estriol on florisil 60 mesh column,
elution with methyl chloride- ethanol
solution.

163 Separation of steroid hormones from


plasma using a Merck extrelut column.
Elution with ethyl ether.

5.2 THIN LAYER CHROMATOGRAPHY

Thin layer chromatography (TLC) can be used to


separate complex mixtures or provide inital sample
purification for further more sensitive separation and
detection. Reviews of TLC steroid methods are found in
r e f e r e n ~ e s l ~ ~ ’Sander166
~~~. describes the theory and
applications of reverse phase TLC. Hais167 has studied the
relationship between chemical structure and TLC sequence in
single and multicomponent systems for the separation of a
group of 6-estrane and 10-androstane derivatives. Table 9
summarizes selected TLC separation methods.

TABLE 9
TLC SEPARATION METHODS

Reference Description

168 Separation and purification of urinary


estrogens on silica gel H-ascorbic
164 DOUGLAS BOTH

acid, developed in benzene containing


5 % ethanol, detection by gas
chromatography.

169 Silica gel containing ethanolic


ammonium bisulfate, development in
benzene-ethanol (85:15) using a
spectrodensitometer for detection.

170 Detection using an automatic


conductivity detector.

171 Separation of steroid mixtures on


silica gel G or aluminum oxide G with
phosphor 6-115 added to coating.

172 Separation of steroids on starch bound


silica gel using phosphomolybdic acid
visualization.

173 Separation of free estrogens and


estrogen acetates on silica g e l
containing dichlorofluoroscein, using a
15% ethanol in benzene and 10%
isopropyl ether in benzene system.

174 Separation of a variety of steroid


hormones on silica gel containing 5%
gypsum with development in a variety of
solvent systems.

175 Separation of free steroids and steroid


heptafluorobutyrates on silica gel G
and GF using benzene: ethyl acetate
(60:40), (9O:lO) and chloroform:
acetone (95:s).

176 Separation of steroids on Gelman sheets


in a chloroform: acetone (30:l) system
using silicotungstic acid
visualization.

177 Detection of estrogens on silica gel by


coupling the estrogens to the diazonium
compound fast dark blue R salt.
Development twice in diethyl
ether-cyclohexane (80:ZO).
ESTRONE 165

178 Simultaneoug separation of common


mammalian A -3-oxosteroids and
estrogens of adrenal, testicular,
ovarian and placental origin.

179 Separation of estrogens on silica gel G


using one and two dimensional develop-
ment, in five different solvent
systems.

180 Separation of antithyroid drugs and


estrogens from animal tissue extracts
by HPTLC with a detection limit of
10-200 ng.

181 Determination of separated estrogens on


silica gel by measurement of their
dansyl derivative fluorscence.

182 Identification of nine major components


of conjugated estrogens on Kieselguhr G
plates containing sodium hydroxide and
impregnated with formamide.

183 Separation of estrogens, androgens,


gestogens and corticoids on dimethyl-,
octyl- and octadecylsilyl silica gel
using a methanol-water solvent system.

5.3 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Estrone and many steroids exhibit rather strong U.V.


absorption. However, U.V. detection of these compounds in
physiological media is often limited because they are
present in low concentration, and the media often contain
other compounds which interfere with simple U.V. detection.
Thus for many other methods the sample must be pure or
highly concentrated. It is for these reasons the HPLC
analysis of steroid hormones is a preferred method. HPLC
analysis allows for sensitive detection on relatively
"dirty" samples. A simple preparative HPLC column
connected to a fraction collector can clean and concentrate
even the "dirtiest" of samples.

Derivatization can greatly enhance the sensitivity for


detection by resulting in improved U . V . absorption or
fluorescence, or altered retention time or oxidation
potential to allow for electrochemical detection. The
166 DOUGLAS BOTH

great number of combinations that can be achieved by


varying the parameters of mobile phase, column and detector
allow for the separation and detection gf complex mixtures
of steroid hormones. Several papersla4 186 review the
analysis of estrogens by HPLC. Table 10 gives selected
HPLC methods for the analysis of estrone and steroid
mixtures containing estrone.

TABLE 10
SELECTED HPLC METHODS

Reference Description

187 Analysis of estrogens in tablet and


injectable forms by measurement of
their dansyl derivatives.

188 Separation of esterified estrogens in


bulk mixtures and combination drug
preparations by reverse phase HPLC.

189 Analysis of estrogens in pregnancy


urine .
190 Separation of estrogen conjugates using
strong anion exchanger column.

191 Separation of 14 testicular steroids


including estrone by reverse phase
HPLC.
192 Determination of trace estrogenic
hormones using voltammetric detection.

193 Separation of various estrogen mixtures


by reverse and normal phase.

194 Separation of estrogen mixtures using a


mobile phase containing silver nitrate.

195 Separation of catechol estrogens and


detection with electrochemical
detector.

196 High speed L.C. separation of equine


estrogens including estrone.
ESTRONE 167

197 Purification of 19 hormonal steroids


prior to immunoassay.

198 Retention behavior for 43 steroids on


bonded reverse phase systems.

199 Reverse phase determination of


estrogens.

200 Separation of estrogens in primate


urine.

201 Determination of unconjugated estrogens


in amniotic fluid using an amperometric
detector.

5.4 GAS-LIQUID CHROMATOGRAPHY

Numerous analytical methods for the gas


chromatographic analysis of estrone and estrogen mixtures
containing estrone have been reported. Most methods
utilize a glass column typically 1-3 meters in length or a
glass capillary column 20-30 meters long. Estrone is
considered to be thermally stable, therefore instability on
the column appears to be of little concern. It is usually
the stability of other compounds of interest that quite
often limit the column operating temperature. Most
analyses are run at column temperatures of 140-260°C.

Estrone has been chromatographed in derivatized and


free forms. The derivatized forms offer improved thermal
stability, lower detection limits and decreased chance of
irreversible absorptivity losses. Therefore they are quite
suited for use with selective detectors such as electron
capture. Estrone has been frequently detected as
heptafluorobutyrate, chloroacetate, trimethylsilyl ethers,
trifluoroacetates and methoxamine-trimethylsilyl
derivatives.

Each derivative is useful in specific applications,


but not all derivatives are of equal value in a given
separation and quantitation. These same comments apply to
the many types of column supports and stationary phases.
In general, the high performance, acid washed, silanized
supports with particle sizes of 80-120 mesh having 2500 or
more theoretical plates appear to work well. Many
stationary phases which appear useful generally are those
168 DOUGLAS BOTH

of medium olarity and higher thermal stability. Several


papers202'506 review solid supports for steroid analysis.

Several papers185'207-214 review the separation and


identification of steroids. Papers which review retention
times of various steroid derivatives and derivative
applications include references 215-226. Table 11 gives
selected methods of analysis of various estrone containing
samples.
TABLE 11
SELECTED GAS-LIQUID CHROMATOGRAPHY METHODS
Reference Description

227 Estrogen separation from human urine as


-
0-methyloxime-heptafluorobutyrate
derivatives.

228 Separation of submicrogram amounts of


steroid hormones using non-selective
stationary phase.

229 Separation of estrogen stereoisomers as


heptafluorobutyric derivatives using a
completely automated splitless glass
capillary system.

230 Separation of estrogens using a solid


injection system.

23 1 Plasma estrogen measurement as


heptafluorobutyrate derivatives.

232 Urinary estrogen determination on


non-pregnancy urine. Extraction and
enzymatic hydrolysis followed by
silylation of concentrate.

233 Quantitation of urinary estrogens


throughout pregnancy. Enzymatic
hydrolysis and ion-exchange followed by
derivatization as heptafluorobutyric
anhydride derivatives.

234 Analysis of estrone in dermatological


products using internal-external
standard ratioing. Cream or lotion
ESTR0NE 169

analysis by hydroxide extraction and


filtration prior to G.C.

235 Analysis of conjugated estrogens using


dual derivatization and dual injection.
Enzyme hydrolysis and derivatization as
trimethylsilyl and methoxamine-
trimethylsilyl steroids.

236 Resolution of equine estrogens using a


short glass capillary column coated
with Silar 1OC. Detection of
oxime-trimethylsilyl ether derivatives
using a flame ionization detector.

5.5 GAS CHROMATOGRAPHY-MASS SPECTROMETRY

-
Combined gas chromatography mass spectrometry
(GC-MS) using the technique of selective ion monitoring or
fragmentography has been applied for the detection of
estrogens in the lower picogram range.

A typical method involves extraction and


derivatization of the estrogens followed by separation by
gas chromatography and detection using the mass
spectrometer as a detector by monitoring selected peaks of
the mass spectrum.

It was shown237 that the methyl and trimethylsilyl


ether estrogen derivatives of fully trimethylsilylated
estrogen derivatives are more suitable than methyl ether,
acetate or trifluoroacetate estrogen derivatives for use in
combined GC-MS. Several selected methods are listed in
Table 12.

TABLE 12
SELECTED COMBINED G.C.- M.S. METHODS

Reference Description

238 Determination of estrogens in the lower


picogram range using isotopically
labeled internal standards.

239 Comparison of a radio-gas


chromatography method for estrogens in
tissue to G.C. -
M.S. method.
170 DOUGLAS BOTH

240 Determination of steroid hormones in


plasma and urine.

241 Determination of unconjugated estrone,


178-estradiol and estriol in blood.

242 Identification of estrogens isolated


from pregnant mares' urine.

243 Determination of interfering estrogenic


compounds in tablet
preparations of conjugated and
esterified estrogens.

244 Determination of estrone and


178-estradiol in seminal plasma of man,
bull and boar.

6.0 RADIOASSAY

Radioimmunoassay (RIA) methods for the determination


of estrone in biological samples are quite sensitive.
Detection limits for estrone are in the picogram range,
with the use of high affinityfspecific antisera. There are
a great number of methods for the assay of estrone and
other steroid hormones in the literature, Table 13 lists
selected methods. Presently radioimmunoassay is the most
widely used technique for the quantitation of picogram
quantities of steroid hormones. However, because of the
inconveniences associated with the use and disposal of
radioactive compounds and the high per assay costs,
enzymeimmunoassay and fluorescence immunoassay methods have
been increasing in popularity. For recent reviews on
fluorescence-, enzyme- and radio-, immunoassay methods see
references (245-253).

TABLE 13
SELECTED RADIOASSAY METHODS

Referenre Comment

254 Non-chromatographic RIA for total


estrone in plasma.

255 Determination of estrone and


178-estradiol in pregnant and
non-pregnant plasma.
ESTRONE 171

256 Determination of estrone and


178-estradiol in urine.

257 Determination of total estrogens in


urine.

258 Non-chromatographic R I A for estrone and


17B-estradiol in plasma.

259 Solid phase R I A for estrone and


178-estradiol in plasma.

260 Total estrone in non-pregnant


peripheral plasma.

261 Determination of estrone in male


saliva.

262 Determination of estrone and equilin in


plasma after administration of a
conjugated equine estrogen preparation.

263 Determination of estrone and estradiol


in plasma.

264 Determination of estrone 178-estradio1,


estriol, testosterone,
5a-dihydrotestosterone and
androstenedione in plasma after
extraction and separation.

265 Sequential R I A for unconjugated and


conjugated estrone, 178-estradiol and
estriol in male plasma.

266 Determination of total urinary estriol.

267 Simultaneous R I A for progestins,


androgens and estrogens in rat testis.

268 Determination of oestrone in the plasma


of rhesus monkey.

269 Determination of estrone, equilin and


dehydroepiandrosterone in peripheral
plasma of pregnant pony mares.
172 DOUOLAS BOTH

270 Enzyme immunoassay for total estrogens


in pregnancy plasma.

271 Determination of estrone and estradiol


in bovine peripheral plasma.

272 RIA of estrone and 17B-estradiol


comparison of method with fluorimetry.

273 RIA of estrone in plasma comparison of


different methods.

274 Simultaneous determination of six


steroids in plasma.

275 Separation and extraction prior to RIA


for estrone, 17B-estradiol and
17a-estradiol in plasma.

276 Determination nf estrone, 17a-estradiol


or 176-estradiol in human and ruminant
plasma.

277 Simultaneous measurement of five


steroids in avian plasma.

278 Immunoenzymological assay of steroids


in plasma.

279 Non-chromatographic determination of


unconjugated estrone, 17B-estradiol and
estriol in plasma.

280 Determination of steroids in plasma of


the monkey.

281 Determination of conjugated estrogens


in plasma of cows.

7.0 COLORIMETRIC ANALYSIS

Colorimetric methods of analysis were once very


popular, but with the advent of more sensitive separative
and instrumential methods, colorimetric analysis today is
of less importance.

Table 14 gives s e l e ~ t e d ~colorimetric


~ ~ ’ ~ ~ ~ reactions.
It should be noted that these colorimetric reactions
ESTRONE 173

generally are not selective to estrone, and reaction with


other steroids is quite possible.

TABLE 14
SELECTED COLORIMETRIC REACTIONS

Reaction with 2,4-dinitrophehylhydrazine and nitromethane


at 100°C for 15 min, Xmax = 565 nm, 13 pg gives 0.3A.

Reaction with 4-nitrophenylhydrazine and


benzyltrimethylammonium hydroxide to give a pink color.
Xmax - 530 nm, 245 ug gives 0.3A.

Reaction of 2,4-dinitrophenylhydrazine and sodium


hydroxide. Amax = 420 nm, 64 pg gives 0.3A.

Reaction with 1:l mixture of 0.5% 1-nitroso-2-napthol in


ethanol and 0.05% sodium nitrate in 3.5 g nitric acid.
Xmax = 450 nm, 11Opg gives 0.3A.

Reaction of diazobenzene-p-sulfonyl chloride in alkaline


solution. Xmax = 510 nm.

Reaction with a 1:l mixture of 0.6% aqueous potassium


ferricyanide and 0.9% aqueous ferric chloride hexahydrate.
Xmax = 720 nm, 11 ug gives 0.3A.

Zimmerman reaction - 1% solution of 3,5-dinitrobenzoic acid


in 40% aqueous solution of benzyltrimethylammonium
hydroxide. Xmax = 530 nm, 175 pg gives 0.3A.

Reaction with salicyloylhydrazide to yield blue


fluorescence, excitation at 340 nm, emission at 420 nm.
Reaction with 1,3,5-trinitrobenzene Xmax = 475 nm, 110 Vg
gives 0.3A.

Reaction with 3,5-dinitrobenzoic acid in aqueous


benzyltrimethylammonium hydroxide solution, Xmax = 530 nm.

Reaction at 100°C in a 10% solution of potassium


guaiacolsulphonate in concentrated sulphuric acid. Xmax =
500 nm.

Reaction when heated in the presence of a 2% soluton of


hydroquinone in 66% sulphuric acid. Xmax = 500-550 nm.
174 DOUGLAS BOTH

8.0 TITRIMETRIC ANALYSIS

Estrone was determined285 in pharuceutical


preperations by titration after extraction with ethanol and
alkalinizaiton with sodium hydroxide to pH 11. It was
first complexed with lead citrate and then determined by
back titration with EDTA to a eriochrome black end point.

Estrone was also determined286 in the parts per


million range by titration with bromine in a methanol-water
solution containing hydrochloric acid and sodium bromide
using biamperometric end point detection.

9.0 ACKNOWLEDGEMENTS

I wish to thank Dr. Mira Szyper for her in-depth


critical reading and comments. The comments and reading of
Dr. Glenn Brewer, Mr. Ray Poet, Dr. Joel Kirschbaum,
Mr. Solomon Perlman, Dr. Jack Isidor, Dr. Mohammed Jemal
and Mr. Richard Koski are greatly appreciated.

Special thanks to Quentin Ochs for the x-ray


diffraction work, Dr. Michael Porubcan for the IR, Gloria
Jennings for the NMR and Dr. Phillip Funk for the mass
spectroscopy work. Special thanks to Dr. Sy-Rong Sun,
Director of the USP Drug Research and Testing Laboratory,
for suppling information concerning the USP Standard
Estrone.

Special thanks to Arminda Rubial for her translation


of several important papers. Thanks to Phyllis Gottstine
and Muriel George for getting those hard to get references.

10.0 References

1. A.Q. Maisel, "Hormone Quest," Random House, N.Y.


(1965)

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(1980)
ETOMIDATE
Zui L. Chang and Joseph €3. Martin

1. Description 192
I , 1 Nomenclature 192
1.2 Formulas and Molecular Weight 192
1.3 Appearance, Color, Odor 192
2. Physical Properties 192
2.1 Infrared Spectrum 192
2.2 Proton Magnetic Resonance Spectrum (PMR) 194
2.3 Mass Spectrum 196
2.4 Raman Spectrum 198
2.5 Ultraviolet Spectrum (UV) 20 1
2.6 Solubility 20 1
2.7 X-Ray Powder Diffraction 20 1
2.8 Melting Range 203
2.9 Differential Thermal Analysis 203
2.10 Specific Optical Rotation 205
3. Synthesis 205
4. Stability-Degradation 205
5. Method of Analysis 208
5.1 Identification 208
5.2 Elemental Analysis 209
5.3 Chromatographic Analysis 209
5.4 Titrimetry 210
6. Analysis of Pharmaceutical Formulations 21 1
6.1 Spectrophotometric Method 21 1
6.2 High Performance Liquid Chromatography (HPLC) 21 1
7. Drug Metabolism and Pharmacokinetics 211
8. Determination of Etomidate in Biological Fluids 212
References 213

ANALYTICAL PROFILESOF DRUG SUBSTANCES 191 Copyright hy the American Pharmaccurlcal Associatian.
VOLUME 12 ISBN 0-12-260812-7
192 ZUI L. CHANG AND JOSEPH B. MARTIN

1. Description

1.1 Nomenclature

1.11 Chemical Name

(K )- (+)e t h y l 1- (l-phenyle t h y l )-lH-imidazole-5-


car box y l a t e

1.12 Generic Name

E t omida t e .
1.2 Formulas and Molecular Weight

CHCH3
I

C14H16N202 M.W. 244.29

1.3 Appearance, C o l o r , Odor

Etomidate is a f i n e w h i t e powder w i t h no d i s c e r n i b l e
odor.

2. Physical Properties

2.1 I n f r a r e d Spectrum

The i n f r a r e d spectrum of Etomidate i s p r e s e n t e d i n


Figure 1. The spectrum was measured i n t h e s o l i d
s t a t e as a potassium brani.de d i s p e r s i o n . The fol-
lowing bands (cm'l) have been a s s i g n e d f o r F i g u r e
1 (1).
a. 3128 and 3100 cm'l Two bands due t o t h e C-H
s t r e t c h i n g v i b r a t i o n s of
t h e imidazole r i n g .
0
B
8
Y
0
0
0 -
C I -
-'s*
U
U
51s
0 s
* E
0
8
m
0
5:
m
0
0
0
9
0
e
0
t
0
9
0
094q*
0 - -
194 ZUI L. CHANG AND JOSEPH B. MARTIN

b. 3028 and 3062 cm-1 Two weak bands due t o t h e


C-H s t r e t c h i n g v i b r a t i o n s
of t h e benzene r i n g .

C. Between 3000 and Complex of weak t o medium


2850 cm-1 bands due t o t h e C-H
s t r e t c h i n g v i b r a t i o n s of
t h e methylene and methyl
g rou p s .
d. 1700 cm-1 Strong band due t o t h e C=O
s t r e t c h i n g v i b r a t i o n of
t h e a,$ u n s a t u r a t e d e s t e r
group.

e. 1597 and 1580 cm-1 Two week bands due t o t h e


skeletal s t r e t c h i n g v i b r a -
t i o n s of t h e benzene r i n g .

f. 1518 cm-1 Probably due t o t h e skel-


eta1 stretching vibration
of t h e imidazole r i n g .

g* 1205 cm-1 Strong band due t o t h e C-0


s t r e t c h i n g v i b r a t i o n s of
t h e a,$ u n s a t u r a t e d ester.

h. 765 and 710 cm'l Two bands due t o t h e C-H


o u t of p l a n e bending v i -
b r a t i o n s of t h e monosub-
s t i t u t e d benzene r i n g .

2.2 P r o t o n Magnetic Resonance Spectrum (PMR)

The proton magnetic resonance spectrum of e t o m i d a t e


as shown i n F i g u r e 2 w a s o b t a i n e d on a Varian As-
s o c i a t e s HA-100 PMR Spectrometer as a 10% w/v s o l u -
t i o n i n a s o l v e n t of d e u t e r a t e d chloroform. The
s p e c t r a l peak assignments ( 2 ) a r e presented i n Table
I.
Figure 2. Proton Magnetic Resonance Spectrum of Etomidate
196 ZUI L. CHANG AND JOSEPH B. MARTIN

Table I

PMR S p e c t r a l Assignments f o r Etomidate

Proton
Assignment Chemical S h i f t (ppm) Multiplicity

7.74 Doublet
7.78

7.1-7.4 Mu1t i p l e t

N-CH-Ar 6.37 Quartet

OCH2 4.23 Quartet

1.83 Doublet

1.27 Triplet

2.3 Mass Spectrum

The mass spectrum of etomidate as shown i n F i g u r e 3


was o b t a i n e d u s i n g a n Associated E l e c t r i c a l I n d u s t r i e s
Model MS-902 Mass Spectrometer w i t h t h e i o n i z a t i o n
e l e c t r o n beam energy of 70 eV. High r e s o l u t i o n data
were compiled and t a b u l a t e d w i t h t h e a i d of a n on-
l i n e PDP-11 Computer.

The mass spectrum assignments of t h e prominent i o n s


and subsequent fragments a r e shown i n Table I1 and
F i g u r e 4 (3).
8
cy
In
1
0
ul
0
AllSN3lNI 3hllVl311
198 ZUI L. CHANG AND JOSEPH B. MARTIN

T a b l e I1

High R e s o l u t i o n Mass Spectrum of Etomidate

Measured Mass (m/e) C a l c u l a t e d Mass

77.0392 7 7.0391

78.0467 78.0470

79.0545 7 9.0548

95.0242 95.0246

103.0543 103.0547

104.0621 104.0626

105.0699 105.0704

199.0884 199.0872

244.1218 244.1212

2.4 Raman Spectrum

The Raman s p e c t r u m of e t o m i d a t e as shown i n F i g u r e 5


was o b t a i n e d i n t h e s o l i d s t a t e on a Cary Model 83
S p e c t r o m e t e r . The f o l l o w i n g bands (cm-l) have been
a s s i g n e d f o r F i g u r e 5 (1).

a. 3135 and 3103 cm-1 Two bands due t o t h e C-H


s t r e t c h i n g v i b r a t i o n s of
t h e imidazole ring.

b. 3018 and 3070 cm-1 Two bands due t o t h e C-H


s t r e t c h i n g v i b r a t i o n s of
t h e benzene r i n g .

c. 3000 a n d 2850 cm-1 Complex of bands due t o


the stretching vibrations
of t h e methylene and
methyl groups.

d. 1710 cm-1 S t r o n g band due t o t h e C=O


s t r e t c h i n g v i b r a t i o n s of
t h e a,B u n s a t u r a t e d e s t e r
group.
Figure 4. Fragmentation Pathways of Etomidate
0 0 0 0
0
0 a 9 0 N 0
0
N
0
0
0
0
0
9
0
0
m
0
0
P
0
0
2
0 -
0
-cr
0
0
9
0
!i!
0
0
0
N
0
0
t
N
0
0
a
N
0
0
N
W
0
0
9
W
0
# 1 1 I 1 0
0
0 0 0 0 0 P
2 m 9 0 N
AlISN31NI
ETOMIDATE 201

e. 1610 and 1590 cm'l Two bands due t o t h e skel-


etal stretching vibrations
of t h e benzene r i n g .

f. 1528 cm'l Probably due t o t h e s k e l -


etal s t r e t c h i n g v i b r a t i o n s
of t h e imidazole r i n g .

g. 1009 and 992 cm'l Strong d o u b l e t c h a r a c t e r -


i s t i c of a m o n o s u b s t i t u t e d
benzene r i n g .

2.5 U l t r a v i o l e t Spectrum (UV)

When t h e UV spectrum of 0.001% s o l u t i o n of e t o m i d a t e


i n i s o p r o p a n o l s o l u t i o n was scanned from 400 t o 200
nm, one maximum a t 240 nm (€= 12,200) w a s observed
( F i g u r e 6).

The spectrum was o b t a i n e d w i t h a Beckman Acta V


Spectrophotometer.

2.6 Solubility

The f o l l o w i n g s o l u b i l i t y d a t a have been determined


f o r e t o m i d a t e a t room t e m p e r a t u r e (4):

Solvent mg Etomidate/100 m l S o l v e n t

Water 0.0045
0.01 N H C 1 0.30
0.1N-HC~ 2.09
Hexane 1.29
Chloroform Greater t h a n 100
Methanol Greater than 100
Ethanol Greater t h a n 100
I s o p r opanol 90-100
Propylene Glycol Greater t h a n 100
Diethyl Ether 23.8
Ace tone Greater t h a n 100
E t h y l Acetate 90-100
Benzene 90-100

2.7 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 of e t o m i d a t e


w a s determined by v i s u a l o b s e r v a t i o n of a f i l m
0.6

0.5

0A

b
'
z
b
Y)
m
0.3

0.2

0.1

200 250 300 350


WAVELENGTH (nm)

Figure 6. Ultraviolet Spectrum of Etomidate


ETOMIDATE 203

o b t a i n e d w i t h a 143.2 mi Debye-Scherrer Powder Cam-


e r a (Table I V ) . An Enraf-Nonius D i f r a c t i s 601 Gen-
e r a t o r ; 38 KV and 18 MA w i t h n i c k e l f i l t e r e d copper
r a d i a t i o n ; X = 1.5418, w a s employed ( 5 ) .

Table I11

X-Ray Powder D i f f r a c t i o n P a t t e r n of Etomidate


d-Spacings and I n t e n s i t i e s
0

d(A>* do* a**


a**
9.3 15 2.87 5
7.0 50 2.80 20
6.1 40 2.63 10
5.65 100 2.54 3
5.25 40 2.42 5
4.85 25 2.39 3
4.63 80 2.32 2
4.30 25 2.27 2
4.08 30 2.21 2
3.99 40 2.16 2
3.90 35 2.13 1
3.80 10 2.07 2
3.72 10 1.99 5
3.64 40 1.92 4
3.50 5 1.89 5
3.37 4 1.82 2
3.28 10 1.80B 2
3.20 5 1.74 2
3.03 20 1.71 2

*d = I n t e r p l a n a r d i s t a n c e .

**1/11 = R e l a t i v e i n t e n s i t y (based on t h e h i g h e s t
i n t e n s i t y of 1 0 0 ) .

2.8 M e l t i n g Range

Etomidate melts i n t h e range of 67.0 and 69.0"C.

2.9 D i f f e r e n t i a l Thermal A n a l v s i s

A s h a r p endothermic peak a t 66.5"C i s i n d i c a t i v e of


t h e m e l t i n g p o i n t of e t o m i d a t e ( F i g u r e 7 ) .
I I I I I
0 20 40 60 80 100 120 140 160 180 200
1 "C (CORRECTED FOR CHROME1 ALUMEL THERMOCOUPLES)

Figure 7. Differential Thermal Analysis C u r v e of Etomidate


ETOMIDATE 205

2.10 Specific Optical Rotation

The o p t i c a l r o t a t i o n s of 1%etomidate i n 12
s o l v e n t s measured w i t h a sodium lamp a t 589 nm
and with a mercury lamp a t 570, 546, 436 and 365
nm are summarized i n Table I V ( 4 ) .

Table I V

O p t i c a l Rotations* of Etomidate i n Various


S o l v e n t s a t Various Wavelengths

589 nm 578 nm 546 nm 436 m 365 nm


N a H g H g H g Hg
0.1 N HC1 +33.61 +3 5.31 + 40.56 + 73.17 +123.88
c kl0’;rofo nn +51.81 +54.55 + 62.89 +117.16 +208* 25
i so p r opanol +64.52 +67.77 + 78.21 +146* 9 8 +2 64.64
ethanol +69.49 +72.96 + 84.07 +15 7.63 +283.29
me t hanol +69.44 +72.94 + 84.23 +15 8.2 2 +284.75
hexane +7 3.04 +7 6.69 + 88.43 +163.37 +286.50
MIK +78.85 +82.80 + 95.64 +179.43 +320.69
ethylacetate +80* 42 +84.56 + 97.76 +183.32 +328.31
dime thyl- +80.83 +84.98 + 98.18 +185.86 +337.62
ace t amide
acetone +82.37 +86.52 + 99.74 +186.87 +334.74
d i e th y l e t h e r +84.89 +89.09 +102.49 +189.38 +332.57
benzene +90.01 +94.50 +108.94 +201.69 +3 54.29

*Mean value of 2 measurements.

3. Synthesis

Etoniidate may be s y n t h e s i z e d by t h e r e a c t i o n scheme shown


i n Figure 8 ( 4 ) .

4. Stability-Degradation

Etomidate has been r e p o r t e d t o hydrolyze i n s o l u t i o n t o


t h e f r e e a c i d ( 4 ) a s shown i n F i g u r e 9.
206 ZUI L. CHANG AND JOSEPH B. MARTIN

N , N-Dimethyformamide (C2H5)3N

0 ; H - N . - CH?- COOC2H5

CH3 R-(+)

Xylene
II HCOOH

t
0-- 7H
I
-

I
N CH2 COOC2H5

CH, R-(+)

Na O C 2H5 H C O O C 2H.j

H ‘c40
~ ~ H - N - C HI - C O O C ~ H S

1
CH3 o// C \ H R-(+l

Figure 8. Synthetic Pathway of Etomidate


ETOMIDATE 207

FIGURE 8 (Continued)

-N-I CH-
I
COOC~HS

iK.*H
CH3 CH2 0 C
I
H-C-CH3 R-(t)
I

$ -CH3
0
H- R-(t)

Etomidote
208 ZUI L. CHANG AND JOSEPH B. MARTIN

Figure 9 - Hydrolysis of Etomidate

CH 3CH 2OC q-JHt HOC y-3 I


I
CHCH, CHCH
I

R-(+) e t h y l 1-( l-phenylethyl- R- (+) 1-(1-phenylethy1)-


1H-imidazole-5-carboxylate 1H-imidazole-5-carboxylic
(E tomida t e ) acid
(Etomidate F r e e Acid)

Under mre d r a s t i c c o n d i t i o n s of r e f l u x i n a c i d systems,


t h e h y d r o l y s i s i s a c c e l e r a t e d . Almost t o t a l d e g r a d a t i o n
t o o t h e r products o c c u r s i n s t r o n g base r e f l u x . I n neu-
t r a l r e f l u x , a s w e l l as under exposure t o h e a t and l i g h t ,
very l i t t l e d e g r a d a t i o n (<O. 5%) occurs.

5. Method of A n a l y s i s

5.1 Identification

Etomidate may b e i d e n t i f i e d by i n f r a r e d spectropho-


tonretry and u l t r a v i o l e t spectroscopy. The charac-
t e r i s t i c bands i n t h e i n f r a r e d spectrum ( F i g u r e 1)
of etomidate are given i n S e c t i o n 2.1. The u l t r a -
v i o l e t spectrum of etomidate i s shown i n F i g u r e 6.
ETOMIDATE 209

5.2 Elemental A n a l y s i s

A t y p i c a l elemental a n a l y s i s of a sample of etomi-


d a t e i s p r e s e n t e d i n Table V I I .

Table V I I

Elemental A n a l y s i s of Etomidate

Element Theory Found

Carbon 68.83% 68.70%


Hydrogen 6.60 6.63
Nitrogen 11.47 11.35
Oxygen 13.10 13.23

5.3 Chromatographic A n a l y s i s

5.31 Thin-Layer Chromatography

A chloroform:methanol:ammonium hydroxide s y s -
t e m (100:100:2) h a s been used t o induce t h e
m i g r a t i o n of t h e compound and shortwave ul-
t r a v i o l e t l i g h t was used t o d e t e c t t h e etomi-
d a t e and d e g r a d a t i o n product. The Rf f o r
e t o m i d a t e was 0.74. The major d e g r a d a t i o n
product, e t o m i d a t e a c i d , i f p r e s e n t has a n Rf
of 0.45.

5.32 High Performance L i q u i d Chromatography

High performance l i q u i d chromatography (HPLC)


h a s been used t o q u a n t i t a t e t h e amount of ma-
j o r d e g r a d a t i o n p r o d u c t , etomidate a c i d i n
etomidate. The following a r e t y p i c a l chroma-
tographic conditions f o r analysis.

Column : Micro-Bondapak@/C18 ( o c t y l d e c y l
c h e m i c a l l y bonded t o s i l i c a )

Mobile Phase : A c e t o n i t r i l e :methanol :wa ter


(11:5: 8 4 ) c o n t a i n i n g 0.02 - M
c i t r a t e buffer.

Flow Rate: 2.0 m l / m i n

Detector: 254 nm
210 ZUI L. CHANG AND JOSEPH B. MARTIN

I n t e r n a l Standard: 3,4-Dimethoxybenzoic acid

5.33 Gas-Liquid Chromatography (GLC)

The p u r i t y of e t o m i d a t e can be determined by


d i r e c t i n j e c t i o n of e t o m i d a t e d i l u t e d i n a
s u i t a b l e s o l v e n t i n t o a chromatograph. Sev-
eral columns have been used t o determine t h e
p u r i t y of e t o m i d a t e (4).

The f o l l o w i n g are t y p i c a l chroma t o g r a p h i c


c o n d i t i o n s f o r t h e GLC d e t e r m i n a t i o n of
etomidate (4):

Column A: 3% OV-17 on Supelcoport 80/100


mesh, 1 m x 2 mm ( i - d . ) x 6 mm
(0.d.). U-shaped pyrex g l a s s
column.
Carrier Gas N2: 25 ml/min

Column B: 3% OV-17 on Supelcoport 80/100


mesh, 1 m x 3 mm ( i - d . ) g l a s s
column.
Carrier Gas N2: 70 ml/min

Column C: 3% SE-30 on Supelcoport 80/100


mesh, 2 m x 2 mm (i.d.) g l a s s
column.
Carrier Gas N 2 : 25 ml/min

Detect i o n : Flame i o n i z a t i o n

Temperature: I n j e c t o r : 290°C
D e t e c t o r : 290°C
Column: I s o t h e r m a l 190°C o r
programmed a t 20°C/min
I n i t i a l Temp.: 50°C
F i n a l Temp: 300°C

5.4 Titrimetrv

Etomidate e x h i b i t s b a s i c p r o p e r t i e s . I t can be po-


t e n t i o m e t r i c a l l y t i t r a t e d with s t a n d a r d i z e d 0. l N
p e r c h l o r i c a c i d u s i n g a modified glass-calomel eiec-
t r o d e system, i n which 0.1 N l i t h i u m p e r c h l o r a t e i n
a c e t i c a c i d h a s been s u b s t i T u t e d f o r potassium
c h l o r i d e , employing g l a c i a l a c e t i c a c i d as t h e sam-
p l e solvent.
ETOMIDATE 211

6. A n a l y s i s of P h a r m a c e u t i c a l Formulations

6.1 S p e c t r o p h o t o m e t r i c Method

Etomidate [ (R)- (+) e t h y l 1-(1-phenylethy1)-1H-imida-


zole-5-carboxylate] i s known t o hydrolyze i n t h e
p r e s e n c e of water, y i e l d i n g R-(+) 1-(1-phenylethy1)-
1H-imidazole-5-carboxylic a c i d . In s o l u t i o n t h e hy-
d r o l y s i s r e a c t i o n i s c a t a l y z e d by hydroxyl as w e l l
as by hydrogen ions. A n a l y t i c a l methodology i s
based on a n e x t r a c t i o n w i t h d i e t h y l e t h e r , f o l -
lowed by UV-measurement of t h e o r g a n i c l a y e r con-
t a i n i n g e t o m i d a t e and of t h e aqueous l a y e r contain-
i n g t h e h y d r o l y s i s product.

During t h e e x t r a c t i o n , t h e a c i d i c f u n c t i o n a l group
of t h e h y d r o l y s i s product, R-(+) 1-(1-phenylethy1)-
1H-imidazole-5-carboxylic a c i d , i s i o n i z e d by t h e
a d d i t i o n of t h e NaHCOg-solution and t h e r e f o r e re-
mains i n t h e aqueous l a y e r . On t h e o t h e r hand,
e t o m i d a t e i s n o t i o n i z e d and passes i n t o t h e o r -
g a n i c l a y e r , where i t i s measured spectrophoto-
.
m e t r i c a l 1y

6.2 High Performance L i q u i d Chromatography (HPLC)

Etomidate i n j e c t a b l e s o l u t i o n can be analyzed by a n


HPLC procedure using 2 -amino-5-chl o r 0benzo phenone as
t h e i n t e r n a l s t a n d a r d . The method uses a C 1 8 micro-
Bondapak column and 0.005 M sodium phosphate mono-
b a s i c : a c e t o n i t r i l e (60:40)-as t h e e l u e n t ( 5 ) .

Etomidate f r e e a c i d , t h e primary d e g r a d a t i o n product


of etomidate c a n be s p e c i f i c a l l y q u a n t i t a t e d i n
etomidate f o r m u l a t i o n u s i n g a n HPLC a n a l y s i s proce-
dure. The HPLC procedure u s e s a c18 microbondapak
column u s i n g acetonitri1e:methanol:water (11:5:84),
0.02 -
M i n c i t r a t e buffer, a s the eluent (6).

7. Drug Metabolism and Pharmacokinetics

The major m e t a b o l i c pathway of etomidate, (R)-(+)ethyl


l-(l-phenylethyl)-1H-imidazole-5-carboxylate, i s hydrolysis
of t h e ester t o produce p r i m a r i l y R-(+) 1-(1-phenylethy1)-
1H-imidazole-5-carboxylic a c i d . Oxidative N-dealkylation
occurs t o a s l i g h t e x t e n t , producing mandelic and benzoic
a c i d s , which a r e e x c r e t e d i n t h e urine. A l l t h e metabolic
p r o d u c t s a r e pharmacologically i n e r t (7).
212 ZUI L. CHANG AND JOSEPH B. MARTIN

Etomidate has a s h o r t d u r a t i o n of a c t i o n , presumably be-


cause of r a p i d h y d r o l y s i s i n t h e plasma (8).

L e w i ( 9 ) h a s r e p o r t e d t h a t t h e metabolism of e t o m i d a t e
i n t h e l i v e r i s a c a p a c i t y - l i m i t e d Michaelia Menten pro-
cess.

It has been suggested t h a t etomidate i s hydrolyzed a t


least i n p a r t i n plasma. The e a r l i e s t work on t h e a s s a y
of t h e drug advocated t h e a d d i t i o n of f l u o r i d e i o n t o
plasma s a m p l e s c o n t a i n i n g e t o m i d a t e t o i n h i b i t t h e p l a s -
ma esterases and t h u s , h y d r o l y s i s of t h e drug. Quick re-
covery with absence of hangover e f f e c t s and t h e absence
of cumulation w i t h r e p e a t e d doses of etomidate have been
explained by r a p i d h y d r o l y s i s i n t h e serum and l i v e r (10).

Meuldermans, e t . a l . (11) have s t u d i e d t h e i n - v i t r o meta-


bolism of e t o m i d a t e i n r a t l i v e r f r a c t i o n s . The main
metabolic pathway f o r t h e i n - v i t r o metabolism was t h e
h y d r o l y s i s of t h e e t h y l ester. Decarboxylation and oxi-
d a t i v e N-dealkylation were a l s o observed. The plasma
p r o t e i n binding and d i s t r i b u t i o n of etomidate i n dog, r a t
and human blood were a l s o reported by Meuldermans, et.
a l . (12).

The s i t e of h y d r o l y s i s of etomidate and e f f e c t s of b i s -


(p-nitrophenyl) phosphate (BNPP), a s p e c i f i c i n h i b i t o r
of t h e h e p a t i c rnicrosomal hydrolase, on t h e d u r a t i o n of
a c t i o n and e l i m i n a t i o n of e t o m i d a t e were r e p o r t e d by
Ghoneim and Van Hamme ( 1 3 ) .

The phannacokinetics of etomidate have been r e p o r t e d by


s e v e r a l i n v e s t i g a t o r s ( 9 , 13-18).

8. Determination of Etomidate i n B i o l o g i c a l F l u i d s

Etomidate i n blood plasma h a s been determined by mass


fragmentography (combined GLC-mass spectrometry w i t h
s i n g l e i o n monitoring). A 1.5 m x 2 mm ( i . d . ) g l a s s
column was packed w i t h 5% 08-225 on 100-120 mesh Supel-
coport. The column and i n j e c t i o n p o r t t e m p e r a t u r e were
230' and 250', r e s p e c t i v e l y . The method has a s e n s i t i -
v i t y of 1 n g / d and a c o e f f i c i e n t of v a r i a t i o n of 2.4%
(19).
A h i g h performance l i q u i d chromatography method f o r t h e
q u a n t i t a t i v e d e t e r m i n a t i o n of etornidate i n serum h a s
been r e p o r t e d by Uges and Bouma (20).
ETOMIDATE 213

Assay of e t o m i d a t e i n plasma by c a p i l l a r y g a s chromato-


graphy w i t h n i t r o g e n - s e l e c t i v e d e t e c t i o n h a s been re-
p o r t e d by DeBoer, A.G., e t . a l . (21).

References

1. W W
tion.
.
a shbur n, Abbot t Lab o r a t o r i e s , P e r s o n a l Commun i c a-

2. M. C i r o v i c and R. Egan, Abbott L a b o r a t o r i e s , P e r s o n a l


Communicat ion.

3. S. M u e l l e r , Abbott L a b o r a t o r i e s , P e r s o n a l Communica-
tion.

4. J a n s s e n Pharmaceu t i c a , Pe r s o n a l Communication.

5. J. Quick, Abbott L a b o r a t o r i e s , P e r s o n a l Communication,

6. J. Bawr, 2 . Chang and D. P a c e n t i , Abbott Laborator-


ies.

7. J. J. P. Heykants, e t . a l . : D i s t r i b u t i o n , metabolism
and e x c r e t i o n of e t o m i d a t e , a s h o r t - a c t i n g h y p n o t i c
drug, i n t h e rat. Comparative s t u d y of (R)-(+) and
(S)-(-)-etomidate. Arch I n t Pharmacodyn Ther, 216,
113-129, 1975.

a. M. Morgan, J. Lumley and J O G . Whitwam: Etomidate, a


new water s o l u b l e n o n - b a r b i t u r a t e i n t r a v e n o u s induc-
t i o n agent. Lannet 1, 955-956, 1975,

9. P. J. L e w i , e t . a l . : I n t r a v e n o u s p h a r m a c o k i n e t i c pro-
f i l e i n r a t s of e t o m i d a t e , a s h o r t - a c t i n g h y p n o t i c
drug. Arch I n t Pharmacodyn, 220, 72-86, 1976.

10. B. Kay: A dose-response r e l a t i o n s h i p f o r e t o m i d a t e


with some o b s e r v a t i o n on cumulation. Br. J. Anaesth
48, 213-216, 1976.

11. W.E.G. Meuldermans, e t . a l . : I n - v i t r o metabolism of


e t o m i d a t e by r a t l i v e r f r a c t i o n s . Arch I n t Pharma-
codyn Ther 221, 140-149, 1976.

12. W.E.G. Meuldermans, e t . al.: The plasma p r o t e i n


binding and d i s t r i b u t i o n of e t o m i d a t e i n dog, r a t
and human blood. Arch I n t Pharmacodyn Ther, 221,
150-162, 1976.
214 ZUI L. CHANG AND JOSEPH B. MARTIN

13. M.M. Ghoneim and M.J. Van Hamme: H y d r o l y s i s of


Etomidate. A n e s t h e s i o l o g y , 50, 227-229, 1979.

14. R.S. Reneman: E t o m i d a t e , a new, s a f e , s h o r t - a c t i n g


i n t r a v e n o u s h y p n o t i c - e x p e r i m e n t a l pharmacology a n d
interactions. Boerhaave S e r P o s t g r a d Med Educ, 1 2 ,
145-151, 197 6.

15. R. S. Reneman, e t. a l . : The e x p e r i m e n t a l pharmacol-


ogy of e t o m i d a t e , a new p o t e n t , s h o r t - a c t i n g i n t r a -
venous h y p n o t i c . A n a e s t h e s i o l Resusc, 106, 1-5,
1977.

16. J. J. Ambre, e t . a l . : P h a r m a c o k i n e t i c s of e t o m i d a t e ,
a new i n t r a v e n o u s a n e s t h e t i c . Fed P r o c 3 6 , 997,
1977.

17. M.M. Ghoneim, e t . a l . : P h a r m a c o k i n e t i c s of i n t r a -


venous a n a e s t h e t i c s : I m p l i c a t i o n s f o r c l i n i c a l use.
C l i n Pharmacokinet, 2, 344-372, 1977.

18. M.J. Van Hamme, e t . a l . : P h a r m a c o k i n e t i c s of e t o m i -


d a t e , a new i n t r a v e n o u s a n e s t h e t i c . Anesthesiology,
49, 274-277, 1978.

19. M.J. Van Hamme, e t . a l . : Mass f r a g m e n t o g r a p h i c de-


t e r m i n a t i o n of plasma e t o m i d a t e c o n c e n t r a t i o n s . J.
Pharm S c i , 66, 1344-1366, 1977.

20. D.R.A. Uges a n d P. Bourn: D e t e r m i n a t i o n of e t o m i -


d a t e i n seruni. Pharm Weekbl. Sci. Ed. 1, 459-460,
197 9.

21. A.G. DeBoer, e t . a l . : Assay o f e t o m i d a t e i n plasma


by c a p i l l a r y gas chromatography w i t h n t i r o g e n - s e l e c -
t i v e detection. J. Chromatogr. 1 6 2 ( 4 ) , 591-595,
1979.

Acknowlednements

The a u t h o r s wish t o thank Miss Diane Penza € o r t y p i n g


t h e manuscript.
HEPARIN SODIUM
Friedrich Nachtmann, Gunter Atxl,
and Wolf Dieter Roth

1. Description 216
1.1 Nomenclature 216
1.2 Occurrence 216
1.3 Structure 216
1.4 Molecular Weight 217
1.5 Elemental Composition 218
1.6 Appearance, Colour, Odour 218
2. Physical Properties 218
2.1 Infrared Spectrum 218
2.2 Ultraviolet Absorption 220
2.3 Circular Dichroism 22 1
2.4 'H-NMR 222
2.5 'T-NMR 224
2.6 Solubility 226
2.7 Viscosity 226
2.8 Optical Rotation 229
2.9 Molecular Weight 229
2.10 Titratable Groups 230
2.11 Dissociation Constant 232
2.12 Elemmlal Analysis 232
2.13 Crystal Properties 232
3. Production 233
4. Stability 234
4.1 Heparin Sodium 234
4.2 Aqueous Sotutions of Heparin ,Sodium (5000nJ/ml) 236
4.3 Combination of Heparin Sodium with Other Substances 237
5. Metabolism and Pharmacokinetics 238
6. Methods of Analysis 240
6.1 IdentificationTests 240
6.2 Colorimetric Analysis 240
6.3 Fluorimetric Analysis 242
6.4 Infrared Spectroscopy 242
6.5 Titrimetric Methods 242
6.6 Turbidimetric and Nephelometric Analysis 243
6.7 Polarographic Analysis 243
6.8 Electrophoretic Analysis 243
6.9 Chromatographic Methods 250
6.10 Amidolytic Methods 256
6.1 1 Esterolytic Methods 258
6.12 Anticoagulant and Allied Tests 259
References 263
ANALYTICAL PROFILES OF DRUG SUBSTANCES 215 Copyright by the American Pharmaceutical Association
VOLUME I2 ISBN 0-12-260812-7
216 FRIEDRICH NACHTMANN ETAL.

1. Description
H e p a r i n Sodium is the ccmrnercial sodium salt of the in-
stable heparinic acid, a n acidic rmcopolysaccharide. The name
is derived from the f i r s t isolation fran dog l i v e r by W a n
i n 1916 (1).
Besides the ccmrnercial sodium salt of heparin, calcium,
barium, lithium, potassium, amrmnium, zinc and lead salts are
also ham, of which, hawever, only heparin calcium is of can-
n-ercial importance. With organic nitrogen bases, heparin forms
s l i g h t l y soluble salts, which are of technological importance
as intennediate products i n the isolation of heparin.
1.1. Nomenclature
Generic name:
Heparin Sodium [9041-08-11
Brand names:
Lip-Hepinette, Liquemin, Pularin, 'Ihrcmbocid,
Thrantoliquine, Vetren
1.2. Occurrence
Heparin occurs in the body tissues of mmnals and could
be detected in the lung, l i v e r , spleen, heart, mcosa, skin,
plasm and lynph.
H e p a r i n is synthesized while bound to protein i n the gra-
nulae of the mast cells ( 2 ) . According to Jeanloz, heparin is
bound to O-L-serin-protein by mean of galactose and xylose ( 3 ) .
I n vim, the sugar is separated fran protein by means of pro-
tease. The release n-echanism of heparin is not ham.
On an industrial level, heparin is mainly isolated fran
t h e lung and nuccsa of the ox and pig, whereby that cbtained
fran porcine mccsa is of greatest importance.
1.3. Structure
For a long time, it was not possible to elucidate t h e
structure of heparin on account of the different origins and
due t o various acccmpanying substances such as heparan sul-
phate, dermatan sulphate and hyaluronic acid being present.
Heparin is a biopolymr belonging to the class of mco-
polysacdmrides. More than 70 % of the structure of "conventio-
nal" heparins can be accounted f o r by repeating disaccharide
units consisting of 1,4-linked L-idmnic acid and D-glucos-
amine. !The iduronic acid residues are O-sulphated a t position
2, and the glucosamine residues are N-sulphated and O-sulpha-
ted a t position 6 ( 4 ) .
HEPARIN SODIUM 217

a - L -I d A a-D-ClcAm

OSOj

The above regular blocks can be intemqted or extended


by residues of R-D-glucumnic acid and 6-0-sulphated N-acetyl-
a-D-glucosamine:
p-D-GlcA a - D - G l c kin

Heparin A, isolated fran porcine intestinal mcosa, is


characterised by a content of such residues that is several
times that of B-type heparin, which is isolated fran beef lung
(5). The location of these minor residues w i t h i n the p l y -
saccharide chain is not knm, ht it is clear that they co-
occur i n a t l e a s t some of the mlecules bonded glycosidically
t o residues of the m j o r , sulphated hexosamine and iduronic
acid, constibents.
Purified heparinase degrades heparin to a mixture of di-,
tetra-, hexa- and oligosaccharides ( 6 ) . The relative anrlunts
of di- and oligosaccharides abtained vary w i t h the source of
heparin used. The tetra- and higher oligosaccharides represent
areas which are strructurally different fran those degradable

iduronic acid
D-glucuronic acid
-
t o disaccharides.
Rosenberg and Lam suggest that a tetrasaccharide unit, L-
N-acetylated D-glucosamine-6-sulphate 4
N-sulphate D-glucosamine-6-sulphate
my represent the structural basis of heparin's anticoagulant
activity (7) .
As possible tertiary structure of hewin, Tabisch des-
cribes a helix, similar to that of peptides, with a pitch of
21 (8).

1.4. Molecular Weiqht


Heparin is a mixture of plyanions in a relatively wide
range of mlecular e i g h t s .
By mans of electqhoresis, 21 fractions =re detected in the
range 3,000 - 37,500 Daltons. (See chapter 2.9.).
218 FRIEDRICH NACHTMANN ETAL.

1.5. Elemental Compos i t i o n


Different results are obtained fran the e l m n t a l analy-
sis of heparin sodium depending on the origin and m u f a c t u -
ring procedure. A survey is given i n chapter 2.12.

1.6. Appearance, colour, odour


Heparin sodium is a vhite to creamy-white, mderately
hygroscopic powder. It is practically odourless .
2. Physical Properties

2.1. -
Infrared Spectrum
The infrared spectrum of heparin sodium isolated fran
porcine rmcosa w s recorded between 4000 and 600 cm-l using a
E3eckn-m Acculab apparatus. ?he spectrum of a KBr disc of the
product is given in figure 2.1. The d i s c was prepared with 1.5
mg of heparin sodium and 300 mg of potassium branide.
The spectm is i n good accordance with the l i t e r a t u r e
(9-11). Analogous spectra are obtained i n an aqueous solution
(D20). The following frequencies are given for the characte-
ristic bands (12,13)

' AS 1605 ? 5 cm-l % = 11.6)


cm
u s coo- 1420 ? 5 cm-l % = 3.8)
cm
p AmideI 1623 ? 3 cm-l

P Amide I1 1480 2 2 cm-I

AS s=o 1245 an-', 1222 cm-l


-1
u s=o
u Asc4-s
v s c-0-s
--
-1060

980 an-'

820 cm
QTI

-1

A-type heparins (isolated fran porcine mucosa) exhibit an -1


appreciable extra absorption-band in the region of 1130 cm ,
otherwise, the spectra of A- and B-type (or lung) heparins
shm little differences.
NOISSI!4SNVUl lN33t13d
. i0
o)
0
. (0
o
0
- 0
P
0
s
0
f
0
0
0 7
g z
[r
W
m
o x
0 3
$ 5
5
a
z
0
0
8
0
0
0
P
220 FRIEDRICH NACHTMANN E T A .

2.2. Ultraviolet Absorption


Bell and Krantz already examined the W absorption of
heparin sodium i n 1950 (14). They Cbtained different spectra
f o r low-, medium- and high-potency heparins. ?he spectra ex-
hibited absorption mirtla a t 245 nm and 292 nm and minima
between 240 and 260 nm. According to the authors, t h i s ab-
sorption behaviour is due to impurities. Further investiga-
tions later on by Awe and Stildemann (15) confirmed this hypo-
thesis: a minimurn a t 235 nm and a maximum a t 255 nm were found
f o r r a w heparins; these bands disappear a f t e r purification of
the product.
1.00

0.75

a
0
K
m
2
sf
2 0.50

0.25

Figure 2.2 ___* Wavelength [nm]

W-spectrum of heparin sodium (A type)


concentration: 0.8 mg/rnl H20; S p e c t m t e r : Zeiss DM 4
HEPARIN SODIUM 221

Ferrari and D e Ambrosi &ned 43 samples of cmm?rcial


.
heparin sodium (16) Different W spectra =re found depending
on the origin and activity. A correlation between the W spec-
trum and the a c t i v i t y could not been proven.
The W spectrum of heparin sodium, isolated f r m porcine
muma, is given i n figure 2.2. In conformity with the fin-
dings of Awe and Sti.Memann, the product exhibits no W rnaximurn
or mininaun. Between 300 - 230 nm, the W absorbance is very
poor, it is only frm this wavelength onwards that the absor-
bance increases strongly.
2.3. Circular D i c h r o i s m
Studies on the circular dichroism of heparin are descri-
bed by various authors i n the literature (17 - 22). The spec-
tra cbtained by V i l l i e r s e t al. (22) are given i n figure 2.3.
+20

+15

+ 10

+5
I

2
X
I
I1
L E I 0

-5

-10

- 15

Figure 2.3
CD spectrum of heparinic acid -( 1 , sodium heparinate
(-. - .-) , magnesium heparinate ( ...
.) and calcium heparhate
(----) in salt-free aqueous solution (TN = 26 mequiv/l) ; pub-
lished by V i l l i e r s e t al. (22)
-
Instrument : Jasco J-40 B d i c h r m t e r ; Temperature: 22 23OC
222 FRIEDRICH NACHTMANN E T A .

The characteristic range of wavelengths is bebeen 180


260 nm. The CD data of plymers are usually stated as nmo-
-
mlar e l l i p t i c i t y [el = e x m.r.w/l x c, where:
m.r.w. = mean residue mlecular w i g h t
1 =path length (am)
c = concentration (g/lOO rnl) .
On account of the differing data for the mean residue
mlecular weight in the literature, [G] = 10 x 0 / (1 x TN) was
used in figure 2.3 whereby Th is the normality of the solution,
determined fran titration curves.
The CD spectra of heparinic acid, and the sodium, calci-
um, and magnesium salts show two bands of opposite signs. The
first band (at 210 nm) is negative and its magnitude depends
on the counter-ion. "he second band is locaw a t lower nm
values. ?he corresponding maximum (at 195 nm) depends on the
counter-ion. The rtlaximum of the second CD band (below 185 nm)
was not reached for heparinic acid.
Unsubstituted polysaccharides do not shew any CD bands in
-
the 185 250 nm region of the spectrum. Therefore the bands
i n figure 2.3 result form the interaction of circularly pola-
rised electranagnetic waves w i t h chranophores of the substi-
tuent groups bund to the polysaccharide backbne. Heparin CD
bands are described as "amide - Cotton effects" (17) and
"amide-like Cotton effects" (18). The CD bands wre found to
be slightly dependent on temperature.
-
In the range of 20 6OoC, the changes are linear and in-
dependent of the munter-ion ( 2 2 ) .
2.4. 'H-NMR
The spectrum of heparin sodium, isolated fran porcine
mucosa, was recorded a t 360 MHz using a Bruker WH-360 spectro-
mter (figure 2 . 4 ) .
40 mg of the sample wre dissolved i n 0.25 m l of DS. Ihe
masuring temperature was 3 5 V , the pD value 6.7. Ihe spectrum
confinns the fact t h a t heparin sodium isolated fran mmsa or
ox lung is mainly canposed of repeating disaccharide sequences
in bhich a-L-idopyranuronic acid-2-sulphate and 2-deoxy-2-
s u l p ~ ~ - ~ g l u c q ? y r a n o s e - 6 - s u l p are
h a ~linked a t positi-
ons 1 and 4 (23, 24). Generally, this sequence accounts for
> 85 % of ox lung heparins and > 70 % of porcine maxal hep-
rins ( 2 3 ) .
of heparin sodium (A-type) in D S ; Instrument: B r u k e r WH-360
224 FRIEDRICH NACHTMANN ETAL.

The clearly detectable signal a t 2 . 1 p p i s due to acet-


&do-deoxy-hexose residues by means of which the mmsal A-
type heparin is characterised ( 2 5 , 2 6 ) . Unlike B-type heparin
(€ran ox lung), approx. 30 per cent of A-type heparin is can-
posed of residues of 2-acetamido-2-deoxy~-Pglucose and &&
glucurOnic acid ( 2 4 ) . For further interpretation of the spec-
t n n n , the reader should r e f e r to the literature (23-26).
2.5. 13C-NMR
The spectrum (figure 2 . 5 ) was recorded a t 90.5 MHz using
a B r u k e r WH-360 spectraneter.
190 mg of heparin sodium were dissolwd i n 2 ml of D20.
A t a pD value of 4.9, the temperature was 7OoC when recording
the spectrum. D i m , 8'= 67.8 ppn, was used f o r standardi-
sation. The signal a t 23 p p is due to a m t h y l carbohydrate
which is fran the 2-acetamido-2-deoxy-a-D-glumse i n the A-
type heparin. As regards the identification of the signals or
the interpretation of the spectrum, the reader should r e f e r to
the l i t e r a t u r e (24, 25, 27, 2 8 ) .

+) we are grateful to ~ r M.. wli, sand02 LU., f o r recor-


ding the spectrum.
Figure 2.5 +)
l3C-bM3 spectrum of heparin sodium (A-type) in D20
A : spectmm without decoupling
B : spectnrm w i t h decoupling
Instrument: Bruker -360
226 FRIEDRICH NACHTMANN ETAL.

2.6. Solubility
According to the Merck Index, 5 % of heparin sodium is
mluble in water (29). According to Martindale, The ]Extra
Pharmacopoeia, 1 part of heparir5 sodium dissolves i n 2.5
parts of water (30). Saturated solutions of heparin sodium,
i m l a t e d fran porcine numa, wre prepared in 4 d i f f e r e n t
solvents a t 2OoC. The concentrations of t h e solutions were
determined quantitatively by a coagulation mthod. The re-
s u l t s are given i n table 2.1.

Table 2.1
Solubility of heparin sodium (2OOC)

Acetone

2.7. Viscositv
For polymlecular substances such as heparin sodium,
there is a correlation between m l e c u l a r w i g h t and viscosi-
ty. The interdependence was examined f o r bovine heparin (31).
A graphical representation of the r e s u l t s is given in figure
2.6.
The two large peaks present i n the d i f f e r e n t i a l curve
suggest the p o s s i b i l i t y of two discrete species i n the unfrac-
tionated product. The detectable discontinuities in the inte-
gral d i s t r i b u t i o n curve indicate heterogeneity within a rela-
t i v e l y circwnscr- d i s t r i b u t i o n of molecular weights. The
discontinuities are of the order of a tetra-saccharide.
The viscanetric t i t r a t i o n of heparin with sodium hydro-
xide and calcium hydroxide was examined by V i l l i e r s e t al.
(22). The t i t r a t i o n curves are given in f i g u r e 2.7.
HEPARIN SODIUM 227

100 100

90 90

80 80

70 70

60 ' 60

50 50 5
v
X

40 ,40 U
?
30 30 Q
20 20

10 10

0 0
70 90 110 130 150 170 190 210
[TI x103

Figure 2.6
.Integral dispersion curve for heparin sodium iractionated
with alcohol. Abscissa: i n t r i n s i c viscosity, ordinate: m l a -
t i v e e i g h t recovered during fractionation; published by
Lasker (31).
+
For N a , the specific viscosity ( j l s p ) rises slightly as E
increases fran 0 to 0.6, i.e., when the strongly acidic groups
are neutralised. A t B -0.6 the carboxylic groups start to be
ionised. The new electric charges produce a further extension
of the already partially extended m a c m l e c u l e s . Beyond 5 = 1,
the specific viscosity decreases because of the salt effect of
the excess of alkaline reagent. me ca2+ viscmetric curve is
canpletely different. The specific viscosity decreases Mi-
ately as a increases fran 0. This trend indicates a strong
interaction that 61lows m c r m l e c u l e s to contract.
228 FRIEDRICH NACHTMANN ETAL.

F
w

Figure 2.7
V i s c a n e t r i c t i t r a t i o n curve of heparinic acid (TN 5 . 2 mequiv/
1) neutralised with sodium hydroxide (-.-.-#--- ) and calcium
hydroxide (---- ) a t 25OC i n salt-free w a t e r solution; pub-
lished by V i l l i e r s e t al. ( 2 2 ) .
Investigations rtFLde by Chung and Ellerton proved that the
viscosity of heparin salts is dependent on the size and &arge
of the ca ion 1 9 ) . ?hereby the following order was found:
5; 1+
Na+<K+<Mg<Sr <E3a2+<Ca2+. The viscosity of heparin is also
.
lowxed by periodate oxidation (19) The f l e x i b i l i t y of hepa-
r i n is increased by cleaving the vicinal hydmxyl groups.
Furthermore, a t constant ionic strength, the viscosity of
heparin in solution is dependent on the pH value. The viscosi-
t y increases between pH 2 and 5. This rise is due to the in-
crease in hydrodynamic wlum of the mlecule (19). The type
of solvent has a large influence on the viscosity of the
heparin solution. The viscosity increases linearly w i t h in-
crease in dielectric constant ( 3 2 ) .
Moreuver it was proved that the viscosity of heparin is
dependent on the shear stress and the ionic strength of the
solutions (19). The viscosity decreases w i t h increase in shear
stress and increase in ionic strength. Desulphatation also
leads to a decrease i n viscosity ( 3 2 ) .
HEPARIN SODIUM 229

2.8. Optical R o t a t i o n
O p t i c a l rotation values for heparin-sodium frm the li-
terature are given in Table 2.2.

Origin of sample Reference


+47.6O Bovine lung 9
+45.6O Bovine intestine 9
+46.3O P o r c i n e intestine 9
+61.6O Whale lung 9
+59.40 Whale intestine 9
+47.0° +) Not defined 10
+46.5O 2 7' V a r i o u s origin 15
+34.4O to +54.1° Various origin 33
+40.9O t o +49.5' Various origin 34
+48.7O Bovine 35
+65.4O Pihale 35
+48.4O 2 3.2' P o r c i n e intestine 36

2.9. Molecular Weight


On account of the varying origin and different procedures
for the m u f a c t u r e of heparin sodium, various values are
given in the literature for the w i g h t average mlecular
weight (table 2.3) .
The mlecular wights are distributed over a relatively
w i d e range. By means of electrofocusing on plyacrylamide gel
slabs, heparin-sodium was s p l i t up into 2 1 fractions ( 4 3 ) . The
mlecular weights of these fractions ranges between 3,000 and
37,500.
230 FXIEDRICH NACHTMANN E T A .

Table 2.3
Weight average mlecular weight of heparin sodium

Weight A v e r a g e Origin &thod of eference


lvblecular Weight Determination

16600 N o t defined Sedimentation 37


Diffusion
14200 Not defined Sedimentation 10
Diffusion
14400 W i n e intestine Sedimentation 9
13600 Porcine intestine Sedimentation 9
11600 Wale lung Sedimentation 9
10800 Wale intestine Sedimentation 9
11900 ? 200 Porcine intestine Sedimentation 38
Diffusion
15000 4 500 Porcine intestine L i q u i d chrana- 39
Ww?hY
22000 4 2600 +) Porcine intestine Liquid chrana- 40
togr@Y
17900 ? 1300 +) Wine lung Liquid chrana- 40
tOSraPhy
10600 4 400 Porcine intestine Liquid chrana- 41
tOSraPhY
Laser l i g h t
scattering
10500 ? 300 Wine lung Liquid chrana- 41
tographY
Laser l i g h t
scattering
11000 Porcine intestine Liquid chrana- 42
tWW?hY
10500 2 600 Wine lung Liquid chrana-
tosraphY

+) Dextrans =re used f o r calibration. The differences i n


mlecular shape of dextrans and heparin sodium can appear
as differences i n mlecular weight.
2.10. Titratable Groups
Heparinic acid originating f r a n porcine intestinal rmcosa
was t i t s a t e d with ptassium hydroxide (38), sodium hydroxide
and calcium hydroxide (22). A typical t i t r a t i o n curve w i t h
potassium hydroxide is given i n figure 2.8.
HEPARIN SODIUM 231

.-
-0

2 20
.-0
c
.-L
a
Q
a,
I
-
a,

8. Et OSO3
8 *O 0 0 0 0 0 O D 0

e
c
F
u)
Q

s2

0
Z 60
NotThrou h '.
I R A - ~ O O ?OH) '.- .-. - . -. -_
-
I I 1 1 1 1
0 2 4 6 8 1 0 1 2
PH

F i g u r e 2.8
Titration curves of heparinic acid before and after passage
through IRA-400 i n the hydroxyl fonn. Titration curve f o r the
amount of ethyl s u l p h r i c acid (EtOSO3H) equivalent to sul-
phate groups estimated to be present in one mle of heparinic
acid; pblished by Helbert and Marini ( 3 8 ) .
Frcm the curves one can identify 40 ionisable sulphate
g r q s , 20 ionisable &xyl groups (S-shaped part of the
t i t r a t i o n curve) and 1 ionisable amino group for the product
examined (38). Furthermore, 6 to 7 equivalents of sodium sul-
phate per mle of heparin s o d i u m were detected, which do not
represent an integral ccmponent of the mlecule and which
could be removed w i t h an anion exchanger (e.g.: IRA-400, - '
H
0
form, see figure 2.8.) .
By means of conductanetric tests, Casu
and Gennaro cbtained values fran 1.94 - 2.40 f o r the ratio of
sulphate to car33oxyl groups in heparin (44).
232 FRIEDRICH NACHTMANN E T A .

2.11. Dissociation Constant


The dissociation constants (%) of various heparin salts
w e r e determined by Herwats e t al. (45). A dissociation con-
stant of 10 k 5 mM was found f o r heparin sodium. Thereby one
presumed that sodium is only bound t o the carboxyl grou~+. I n
canparison, the bond between s o d i u m and citrate (Q = 300 nM)
is a least ten times maker. Py means of competition experi-
ments, the dissociation constant could be determined for
various other cations. The following order was found:

Of all the cations examined, calcium w a s the one rrost


strongly bound.
2.12. Elemental Analysis
Various data is given i n the literature, depending on the
origin, p l r i t y and mnufacturing procedure. A sumnary is given
i n table 2.4.

Table 2.4
Elemental analysis of heparin sodium

% C % H % N %S
2.2 13.3 15.1 Bovine 35
2.5 9.0 13.1 Whale 35
2.1 14.2 12.5 Bovine lung 9
2.6 11.3 11.5 Bovine intestine 9
2.1 12.4 11.7 Porcine intestin1 9
2.3 7.3 9.2 Whale lung 9
2.3 8.2 9.0 Whale intestine 9
23.4 3.3 2.2 11.6 12.4 Porcine intestinc 38
22.2 3.5 2.1 11.3 11.6 Porcine intestin( 36
18.2 - 3.6 - 2.3 - 9.3 - 11.6 - Various origin 34
21.7 4.4 3.6 10.4 16.8

2.13. C r y s t a l Properties
X-ray fibre diffraction d a t a wre phlished by Nieduszyn-
ski and Atkins (46,47). Orientated f i l m of heparin sodium
(origin: porcine intestinal rmcosa) c r y s t a l l i s e in a triclinic
unit cell with parameters a = 1.30 4 0.01 nm, b = 1.02 4 0.01
run, c (fibre axis) = 1.59 4 0.02 nm, a = 104 4 lo, R = 96 4 lo,
HEPARIN SODIUM 233

r= 116 i 1' and with volume = 1.79 m3.The density of a


crystalline sample was determined by flotation to be 1.72 g/
cm3. Heparin sodium mlecules require nearly their own volume
of w a t e r i n order t o crystallise. The crystallographic perio-
dicity ranges fran 1.65 to 1.73 m, which is consistent w i t h
a tetrasaccharide repeating unit.

3. Production
Heparin sodium is cbtained by extraction fran animal or-
gans. According to Scott and Charles, the lung, muscle and
liver are particularly suitable. Lately, it has also to an in-
creasing extent been abtained fran nucosa, whereby higher ac-
t i v i t i e s are attained (48). The mst recent development is to
use the k i n e obtained when pickling intestines as the r a w
mterial f o r the mufacture of heparin.
The prduction of heparin sodium takes place principally
i n two steps: isolation of a mre o r less p r e crude heparin
and subsequent p r i f i c a t i o n of the crude heparin t o obtain the
parenterally administrable p r d u c t .
The various procedures for the preparation of crude
heparin are based on the principle that heparin is withdrawn
fran the autolysed or digested organs by means of alkali and
a f t e r separation of the protein-lib acccmpanying substances,
precipita- with alcohols o r acetone as a barium or a l k a l i
salt.
Scott and Charles autolyse the frozen, d u t e d tissue
and extract the heparin with d i l u t e alkali (48). After pres-
sing out the protein mss coagulated on heating, the heparin
i s precipitated w i t h sulphuric acid a t pH 2.5. After removal
of f a t by means of alcohol and digestion with trypsin, the
crude heparin is then precipitated with t w i c e the munt of
alcohol.
Taylor imprwed t h i s mthod by using digestive enzyrnes
and carrying out several extractions w i t h alkali ( 4 9 ) . Taka-
m s a describes a deproteinisation of the mmsa extract w i t h
picric acid and subsequent precipitation with hydrochloric
acid a t pH 2.5 followed by precipitation of the heparin sodium
salt with n-ethanol (50). Thanpson precipitates the heparin out
of the filtered autolysate by means of cetylpyridinium chlo-
ride (51). Wins rmves the f a t fran the tempered, fermented
tissue azeotrqically by mans of dichloroethylene (52). Gede-
on Richter enriches the heparin i n a canplex w i t h protein by
means of heat coagulation and extracts i n a countercurrent of
saline solution (53). Riker uses proteolysis and the addition
of salt to release the heparin fran the tissue. It i s then ad-
234 FMEDRICH NACHTMANN ETAL.

so- on a strongly basic anion exchanger, eluted with sali-


ne solution and precipitated w i t h an organic solvent (54). As
starting m t e r i a l schering uses the brine produced when pre-
serving and dehydrating animal intestines w i t h sodium chlo-
ride (55). Heparin is c b t a i n d fran this brine, without pro-
teolytic digestion, by means of precipitation with methanol
and purification on a basic ion exchanger.
The crude heparin mnufactured fran other raw materials
than brine, as described above, must undergo deproteinisation,
decoloration and depyrogenisation processes. Further p r i f i -
cation is cbtained by dissolving and reprecipitating with al-
cohols or acetone or by precipitation of heparin as a slightly
soluble salt (barium, o q a n i c nitrogen bases) and reconverting
t o the sodium salt with soda or sodium chloride. Decoloration
of the product can be carried out by means of f i l t r a t i o n
through activated d~arcoalo r by oxidation with potassium per-
manganate, hydrogen peroxide o r -chlorite.

4. Stability
4.1. Heparin Sodium
Lyophilised, dry heparin sodium can be stored f o r several
years a t roan temperawe and 3OoC, without loss of activity,
i f kept in tightly closed containers (56).
Figure 4.1 shaws the biological activity (AFTT activity)
of heparin sodium (origin: mcosa) detennined over a period of
4 years as a percentage of the declaration.
The product is stable a t room temperature (RT, 15 - 25OC)
and 3OOC. The other quality characteristics (pH value after
dissolution, colour index, colour stability) and the content
of active ingredient remained unchanged throughout the storage
period.
HEPARIN SODIUM 235

I
110

- - - - - - -0- _ _ - - - - - -- _ _ _ _ -_- -_- -- - -


0
6
._ 100
c
---L---.
0
---___
0
E
a
-
_ - - - - - - 8- - - - - -
0
a,
n --- - - - _ _ _
c
0 - - - - _ _--
_
8 90

0 12 24 36 48
Months

Figure 4.1
S t a b i l i t y of heparin sodium (A type) a t roan temperature (RT)
and 3OoC
236 FRIEDRICH NACHTMANN ETAL.

4.2. Aqueous Solutions of Heparin Sodium (5000 I U / m l )


Figure 4.2 skm a graphical representation of the re-
sults.

L
0
$ 90 -
RT

110

100

c
0
s 90

80
0 12 24 36 48
Months
Fiqure 4.2
S t a b i l i t y of sterile aqueous solutions of heparin sodium
(5000 I U / m l ) a t roan temperature (RT) and 3OOC.
HEPARIN SODIUM 237

lhroughout the period of 36 mnths, no changes regarding


the efficacy, the pH value and the appearance of the solution
could be found to occur i n the ampule solution of heparin so-
dium (origin rmcosa) ready f o r administration (56).
Experimnts carried out by Goodall e t al. showed that
hqxtrin sodium undergoes no loss in quality i n the pH range
f r a n 4 to 9 (57). This has been confinned by other experiments
(56). According to other tests, injections of heparin sodium
are s t a b l e f o r mre than 7 years a t rcun temprature and for
6 - 8 years a t 37OC (30). ?he activities determined immdia-
t e l y after s t e r i l i s a t i o n tests (12loC, 5 or 10 minutes) shaved
no s i g n i f i c a n t changes in canparison w i t h t h e untreated solu-
tions so that any negative influence on the heparin sodium
solutions due to heating my be neglected (56).

4.3. Canbination of Heparin Sodium w i t h Other Substances


S t a b i l i t y tests carried out with heparin sodium in can-
bination with dihydrcergotamine rresylate both in the lyophili-
sed and dissolved form, produced equally good results (56). A t
roan temperature and 3OoC, heparin sodium does not undergo
s t a t i s t i c a l l y detectable changes in a c t i v i t y over a period of
4 years.
Many authors (57 -
64) conducted experiments on the e f f i -
cacy of heparin sodium infusions a f t e r various standing times.
Altbugh in the course of extensive studies, some authors
-
(57 62) did not ascertain any loss i n a c t i v i t y of heparin
sodium (20 -1000 I U / m l ) i n 5 per cent dextrose solution, 0.9
per cent NaCl solution or in g l a s s b o t t l e s within a period of
1 2 t o 24 hours, and Bowie e t al. (58) even speaks of stability
periods of up to 1 2 months, i n canparison with the initial
values, Jacobs (63) and Okuno (64) find a c t i v i t y losses of up
t o 50 per cent within 24 hours f o r heparin sodium in NaCl,
dextrose, Ringer's and Hartmann's solution.
238 FRIEDRICH NACHTMANN ETAL.

5. Metabolism and Pharmaoakinetics


The heparin activities found in the plasm of noml per-
sons, a f t e r hydrolytic destruction of the native heparin-pro-
tein ca~lplex,amount to 10 - 24 u n i t s / m l of plasm (65).
According to Heilbrunn, t h i s endogenous heparin serves to
mintain the protqlasmic flow equilibrium between the sol
and gel state, a fundamental function i n the cellular haneo-
stasis mechanism (66). Heparin, the mst effective anticoagu-
lant h m a t present, intervenes a t various pints in the
cascade of the oncping coagulation mechanism. Ihe aim of hepa-
rin therapy is, by activation of antithranbin 111, to fom an
inhibitor camlex hich circulates for a lonu tinre i n the

The heparin preparations on the market can be classified


into fractions of different affinities to antithranbin 111,
depending on their origin (mu-, lung) ; accOrding to Thanas
e t al., heparin procured fran the rmcosa possesses a higher i n
vitro activity than that abtained fran the lung (68). The same
authors found the contrary situation in vim (69). According
t o HLiLjk et al., the activities ik+&ned could be cansidered
equal to the biological efficacy (70). According to Rmen-
berg's model, the activity of heparin correlates with the pro-
portion of the linkage on the cofactor antithranbin I11 (71).
Y i n e t al. s h e d that this cofactor is lllaximally saturated
w i t h a p p o x . 0.3 U n i t s / m l although individual doses of mre
than 1 Unit/ml plasma are usually necessary in order to re-
main above the &sired time unit inhibition (72).
After i.v. application, heparin distributes i t s e l f wry
rapidly in the plasm mqxwtment and reacts as a carrier of
negative charge with nwnemus proteins - only 15 20 per cent-
of a i c h form a canplex with antithranbin I11 (73). Further-
mre, within approx. 40 minutes of injecting, 80 90 per cent-
of the injected heparin is m v e d frcm the system and metabo-
lised by the reticuloendothelial tissue of the liver and
spleen. The man half-life dxtm? in practically a l l human
cases m m t s to apprax. 1.5 hous (74 - 76) whereas the la-
test investigations carried out by Ciplle e t al. yielded a
man value of only 0.9 hours (77). Ihe ~ l u m eof distribution
m u n t s to apprax. 5.5 per cent of the body wight and there-
fore corresponds t o the plasm volume (74 - 77).
Many recent plblications prove that small subcutaneously
applied doses of heparin considerably decrease the danger of
-
deep-win thranbsis and p l m n a r y embolism (78 80). This
prophylactic effect of heparin is enhanced by the sinultaneous
administration of dihydergotamine (81 - 83). It is assum3d
that t h i s effect is based on an interaction between dihydro-
HEPARIN SODIUM 239

ergotamine and heparin, which leads to a higher and longer


lasting heparin plasma concentration (83). A study carried
out by Beennann and Lahnborg shows that the heparin plasma
level is the same f o r the canbination as f o r the pure heparin
i n normal persons, but that i n persons who are w e m i g h t ,
the levels are clearly elevated and the half-life increases
fran 1.4 to 2.1 hours (84).
There is no lack of investigations and clinical documen-
tation proving that heparin fran topical preparations pene-
trates to the blood circulation via the skin (85). One should
h a e v e r not expect any permeation rates for heparin which ex-
ceed an approx. threshold value of 1 per cent of the mterial
applied externally. I n a p b l i c a t i o n by Schaefer and Zech it
is state3 that, under suitable conditions (e.g. lanolin alco-
hol ointmnt), a penetration of up to 2 per cent of heparin
into the deeper layers of the skin takes place within 10 mi-
nutes (86). A l s o Kidron e t al. here able to detect absorption
of heparin from the gas-intestinal tract in connection with
a tenside (Cetanacrogol 1000) i n the case of rats (87). I n
s p i t e of t h i s success, one cannot imagine heparin therapy
without the parenteral form of administration.
Investigation of the metabolism of heparin have hitherto
only been carried out using radioactively marked substances or
by determination of the biological activities. chemical detec-
tion has not been carried out up till ncw since the different
mlar relationships of E-glucosamine, glucuronic acid, L-idu-
ronic acid and sulphide groups do not permit the use of the-
cal analysis or radioimnunological mthcds (88). U n t i l now,
exact statements about the mtabolism have only been possible
in a few points:

- R-dogenous heparin:
Rtiogenous h q a r i n released fran the roast cells is
immdiately taken up by macrophages and catabolysed. As a re-
sult, it loses its coagulation-inhibiting effect (89).
- &ogenous heparin:
Bparinase I and heparinitase I1 could be detected i n
the liver (90).
A heparin sulphamidase (91) and three different depoly-
merising enzymes (92) where also characterised. The degrada-
tion of heparin either takes place a t the heparin-antithrmbin
I11 canplex or a t the free substance i n the plasm.
Under the influence of disease ( l i v e r cyrrhosis), the
decrease in coagulation-inhibiting effect of i.v. administered
heparin is significantly slaver, since in such cases, the
240 FRIEDRICH NACHTMANN ETAL.

c l a r i f y i n g function of the liver cells f o r heparin is dis-


tu&ed (93). I n the case of p l m n a r y embolism and severe
p h l e b o t h r h s i s , the decrease in a c t i v i t y is about 30 per
cent quicker than i n the healthy body (94), whereas in the
case of chronic renal insufficiency, an approximtely 20 per
cent slower decrease in a c t i v i t y was found (95).

6. Methods of Analysis

6.1. I d e n t i f i c a t i o n %sts
Various tests can be used for the i d e n t i f i c a t i o n of
heparin:

6.1.1. Infrared Spectrwn


See chapters 2.1. and 6.4.

6.1.2. Chranatqraphic Methods


Paper chranatqraphy, thin-layer c h r m t q r a p h y and gel
chranatography (chapter 6.9.) are suitable.

6.1.3. Electrcphoresis
See chapter 6.8.
6.1.4. B i . o c h d c a l and B i o l o g i c a l Tests
The biochemical and biological rrethods given in chapters
6.10., 6.11. and 6.12. are selective f o r heparin and therefore
m y be used as i d e n t i t y tests.
6.2. C o l o r h t r i c Analysis
There are a large n h r of dyes which form a mtachro-
mtic colour a f t e r addition of heparin. Tests carried out by
Jaques e t al. shaved that BislMrCk hrown, azure A, b r i l l i a n t
cresol blue, cresol violet, brmcresol blue, neutral red,
basic fuchsin, pyronine and a c r i f l a v i n e a l l possess t h i s pro-
perty (96). Methylene blue (971, n i l e blue (98) or acridine
orange (99) are equally suitable. For the colorimetric deter-
mination of heparin there are three dyes which are mst often
used: toluidine blue (loo), alcian blue (101 - 103) and azure
A (103 - 106). ?he detection l i m i t f o r the determination of
heparin w i t h the dyes mentioned lies around 1 pg (103). I n the
presence of heparin i n d i l u t e , aqueous solution, t o l u i d i n e
blue e x h i b i t s 3 absorption bands (107). The a-band (620 nm)
corresponds t o t h e mnaneric dye, the &band (540 nm) t o the
dimeric dye and t h e p-band (500 nm) is produced by the binding
HEPARIN SODIUM 241

of the p l y a n i o n . Analogous relationships are found for the


s t r u c t u r a l l y very similar azure A; the canplex w i t h toluidine
blue is hawever mre i n t e n s e l y coloured than the ccanplex w i t h
a z u r e A. F'ractions of heparin possess differing properties:
mnosaccharide and disaccharide fragments only react slowly
w i t h o u t appearance of metachranasis whereas the f u l l meta-
chrcmatic activity i s produced by hexasaccharide units of he-
parin. The t o l u i d i n e blue mthod was described for the deter-
mination of heparin i n plasm (108 - 110), urine (111), mast
cells and granulocytes (112) and tissue (103, 113). The dyes
mentioned can also be used for the colouration of heparin
after separation by TLI= or electrophoresis.
Investigations carried o u t by Awe and StiElemann shcw
t h a t the values found by spectrophotanetry g r e a t l y depend on
t h e plrity of the active ingredient (114).
Anderson e t al. investigated the best measuring range,
l i n e a r i t y and standard d e v i a t i o n for the mst important dyes
(115). A ,summy is given in table 6.1.

Table 6.1.
Characteristic data for the determination of heparin - with
t o l u i d i n e blue ( A ) , azure A (B) and alcian blue (C) .
Method Heparin Useful
range of
heparin I Regression Standard Correla-
equation
Y =
deviatioi tion coef-
of ficient

I
scatter
about re
pg gression
A Porcine rmcous 0.042 0.995
Bovine lung 0.045 0.995
B Porcinermcous 0.016 0.966
W i n e lung 0.065~-0.011 0.024 0.971
C Porcine rmcous 0.030 0.993
Bovine lung 0.008 0.999

A canparison of Wo spectrophotanetric methods ( t o l u i d i n e


blue and m t h y l e n e blue) with the biological a c t i v i t y of hepa-
rin preparations was described by Marvola e t al. (116).
The spectrophotawtric methods cannot be used t o deter-
mine the biological activity of various preparations. ?he
correlation c o e f f i c i e n t s found m u n t to 0.386 - 0.702 only.
The colour reactions of sugars can also be used for the guan-
titative detennination of heparin. The mst significant is the
r e a c t i o n of m n i c a c i d s with carbazole. The mthod described
242 FRIEDRICH NACHTMANN E T A .

by Dishe (117, 118) was improved by B i t t e r and Muir (119) and


by Gauthier and Kenyon (120). The absorption mimum is
530 nm, linear c a l i b r a t i o n curves were found between 4 and
40 pg/ml of uronic acid. The reaction time a t 75OC m u n t s to
15 minutes. B i a n c h h i and Osima found no connection between
anticoagulant a c t i v i t y and content of uronic acid i n heparin
(121).

6.3. Fluorimetric Analysis


Phosphatd and sulphated p l y a n i o n s are bound by be&er-
i n e sulphate. The fluorescence of the r e s u l t i n g canplex can
be used f o r a quantitative determination of heparin (122).
Another dye which forms a fluorescent canplex with heparin is
acridine orange. The reaction conditions wre studied by Ca-
t i n i e t al. (123, 124). The emission wavelength of the com-
plex a t pH 2 to 10 is 640 nm. Linear c a l i b r a t i o n curves were
found f o r 7.8 - 303 pg of heparin/ml.
Fluorescent reactions of sugars can also be used for the
determination of heparin. The product of deamination of hepa-
rin with HNO2 reacts with 3,5-diami&nzoic acid and under
mild conditions (pH 3.0, 37OC), gives a stable derivative
(125). Less than 0.1 pg of 2-amin0-2-de~exose can be deter-
mined using standard cells.

6.4. Infrared Spectroscopy


The characteristic, a-tric S=O vibration (see chapter
2.1) can be used f o r quantitative sta-nts. For the measure-
mt of aqueous solutions of heparin sodium, cell w i n d m of
IRTRAN-2 or KSR-5 are reccrnmnded (12). Another msuring
tedmique consists of taking an internal standard such as po-
tassium thioqanate. The r e l a t i o n between the S=O vibrations
of heparin sodium (1240 m-l) and the CaN vibration of the in-
ternal standard (2065 m-l) is determined (126).
US' g D20 as the s o l w n t , spectra in the range 1500 -
1800 an3 can be recorded. &is permits t h e quantitative eva-
luation of the c a h x y l a t e band of heparin sodium (13).
6.5. T i t r b t r i c Methods

6.5.1. Potenticmetric t i t r a t i o n
The procedure was described by klbert and Marini ( 3 8 ) .
Heparin sodium is passed through a column of Dowex-50 in the
hydrogen form. An a l i q u o t of this is titrated for acid groups.
Another aliquot is first passed through a column of IRA-400 i n
the hydraxyl form to ratwlve inorganic anions, then titrated.
The difference is due to the p i o n acid groups.
HEPARIN SODIUM 243

6.5.2. C o n d u c t h t r i c t i t r a t i o n
For determination of the carboxyl content, the dialyzed
sample of heparin sodium is t i t r a t e d with 0.1M Hc1 with a con-
ductimeter and e s t h t e d by the m u n t of standard acid needed
up to t h e f i r s t i n f l e c t i o n p i n t ( 4 4 ) . For sulphate plus car-
boxyl groups, t h e dialyzed sodium salt is converted to the
f r e e acid w i t h A m b e r l i t e IR-120 ( H
'
) and titrated with 0.1M
NaOH. !the f i r s t pint of i n f l e c t i o n is equivalent t o the sul-
&ate content, t h e second t o the sulphate and carboxyl con-
tent.

6.5.3. Colorimtric t i t r a t i o n
A colorimetric t i t r a t i o n is described by Jaques (107).
Heparin sodium is added t o a toluidine blue solution u n t i l the
colour changes f r a n blue to plrple. The colour abtained is
m t c h d with that abtained with reference heparin. The proce-
dure is a f a s t , semiquantitative test and applicable f o r both
crude and F r i f i e d preparations.

6.5.4. W i d i n e t r i c titration
Some plysaccharides react with quaternary alkylammnium
halides resulting i n a precipitate. The reaction of heparin
and other plysaccharides w i t h cetyltrimethylammnium bromide
was studied by recording the t u r b i d i t y by means of an automa-
tic t i t r a t i o n device (127).

6.5.5. Colloid t i t r a t i o n
The plyrrrer p l y d i a l lyldimethylamrronium chloride (Cat-
Floc) binds heparin stochianetrically. After addition of an
excess of Cat-Floc, back t i t r a t i o n is carried out w i t h p-
tassium plyvinylsulphate. Wluidine blue is used as the indi-
cator (128).
6.6. Turbidimetric and N e p h e l m t r i c Analysis
Heparin sodium forms s l i g h t l y soluble canplexes with
various reagents and these can be used f o r a quantitative de-
termination. M a r b e t and Winterstein describe the canplex with
2-(dimethylaminanethyl)dibenzofuran (129). The method was used
t o determine heparin in urine. me t u r b i d i t y of t h e heparin-
protamine canplex i n an excess of protamine was suggested by
Fekete f o r the determination of heparin (130). Another pssi-
b i l i t y is t h e use of the canplex of heparin with cetylpyridi-
nium chloride (131, 132). High t u r b i d i t y values =re found i n
the presence of a 0.4 M solution of sodium chloride. !he
method can be autanated (132).
244 FRIEDRICH NACHTMANN ETAL.

6.7. Polarograpkic Analysis


An oscillopolarographic detennination of heparin and
other plyanions is described by Bohacek and Singh (133). The
optimal d i m f o r heparin was found to be 1M citric acid.

6.8. Electrophoretic Analysis


This analytical technique is mainly used f o r qualitive
detexnination of heparin. It is also possible to use it f o r
quantitative analyses.

6.8.1. Paper electrcphoresis


A determination of heparin in the presence of serum prc-
teins by mans of paper e l e c t q h o r e s i s is described by Ca-
selli (134). Basic dyes (basic fuchsin, gentian violet, mthy-
lene blue, toluidine blue) were used f o r staining. The mthod
m y he used i n the concentration range fran 10 - 100 p g of
plysaccharide.
Conti e t al. report on a separation of heparin on What-
man No. 1 electrqhoresis paper i n the presence of veronal
buffer pH 8.6 (135). Selective staining of heparin is possible
with auramine 0, malachite green or b r i l l i a n t green. These
dyes do not stain chondroitin sulphuric acid, hyaluronic acid
or nucleic acids.
A canbination of paper electrophoresis and gel electm-
phoresis has been used to identify heparin and other a c i d i c
mcqolysaccharides i n mammalian tissues (136).

6.8.2. Agarose gel e l e c t q h o r e s i s


The separation is based on the differences of the net
charge and a b i l i t y of acid mccplysaccharides to form cun-
plexes With different cations and 0c - amines. A g a r o s e
electrcphoresis i s mst useful for the characterisation of
sulphated mccplysaccharides. It accepts heavily contamina-
ted mixtures without change of pattern of fractionation (107).
A micrc-electqhoretic mthod on agarose f o r heparin and
relatsd canpounds was developed by Jaques e t 61. (107, 137,
138). Most of the samples of camercial heparin examined were
separated i n t o 2 fractions. 2 p l samples containing 0.1 t o 0.5
pg of heparin are applied t o the agarose gel on a microsccpe
s l i d e i n parallel with 0.8 pg of reference heparin. The elec-
t q h o r e s i s is conducted i n a Lucite chamber with three can-
partrnents and a rretal tray as l i d and cold finger (6.3 V/cm
for 10 t o 25 min a t 2OC). The nuccplysaccharide i n the gel is
fixed by immrsing the s l i d e i n 0.1 per cent cetyltrimethyl-
HEPARIN SODIUM 245

m n i u m branide or 0.1 per cent cetylpyridinium chloride so-


lution. The slide is stained with toluidine blue. The back-
ground i s cleared by washing w i t h 1 per cent acetic acid, and
t h e s l i d e is dried i n t h e air. ?he procedure separates heparin
f r a n rnmy other mterials present in tissue extracts. Optical
density of the spts is canpared w i t h that of s l i d e s prepared
with a range of concentrations of standard heparin, either
with a suitable d e n s i t c m t e r o r visually.
The e f f e c t s of d i f f e r e n t buffers on the r e l a t i v e electro-
phoretic migration of sulphated mccplysaccharides i n agarose
are summrised in T a b l e 6.2.
An e s s e n t i a l l y linear r e l a t i o n is found between log area
of t h e s p t and log of quantity of heparin applied up to 0.4
units. About 0.1 unit of heparin applied is q t i m a l f o r ma-
surmnt .
A r e l a t i v e standard deviation of 6.2 per cent f o r densi-
tanetric masurements could be achieved (139). The microelec-
trcphoresis procedure is mre sensitive than the colour tests
(107). It is 30 times as sensitive as the carbazole and 50
times as sensitive as the aqueous mtachranic test f o r h e p -
rin.
Kupchella and Curran separated heparin and a c i d i c glycos-
amino-glycans via cross-aver electrophoresis with t h e dyes
acridine orange and toluidine blue (140). Glycosaminoglycans
were applied to agarose gel films as 0.01 m l of 5 mg/ml
aqueous solutions and electrophoresis was carried o u t a t 200 V
(approximately 8.5 m A ) . Five minutes a f t e r starting, a dye-
soaked wick was applied t o the anode s i d e of the g e l , and
0.9 m l of dye was applied t o the wick. Electrophoresis was
continued u n t i l t h e dye-front passed t h e o r i g i n (about 15 min) .
The individual glycosaminoglycans are d i f f e r e n t i a l l y pigmen-
ted.
A discontinuous electrophoretic mthd f o r fractionation
of heparin into two or three d i f f e r e n t cayonents and of these
fran other a c i d i c mcopolysaccharides =re described by Bian-
c h i n i et al. (141). The mthod consists of electrophoresis
f i r s t i n agarose in barium acetate and then i n diaminopropane
h f f e r i n the sarne direction.
Table 6.2
Effects of Wfer on relative electropbretic migration of sulphated rmcapolysaccharides i n aga-
rose (values relative to migration of reference ox lung heparin), according to Jaques et al. (107)
Heparha
ox Forcine Heparitins Chonkoitins
Buffer M pH lung RUC. A B C D A B C KS
~~ ~~

Ba&ital 0.12 8.6 0.99 0.90 (0.81) 0.83 0.86/ 1.04 0.88 0.88 0.88 0.67
0.97 0.93
'&is 0.12 7.5 0.98 (0.76) 0.90/ 0.91/ 0.93 0.93 0.93 0.69
0.95 0.98
KCl-Hcl 0.02 2.0 1.00 0.87- 0.66 0.71- 0.61- 0.70
1.03 0.90 0.92 0.79 0.82
(2)
HAC-LiOH 0.35 3.0 1.00 0.93 0.72 0.76 0.88 1.04 0.84 0.82 0.84
1.00
Ethylene- 0.1 8.0 1.00 1.00 0.92 0.92 0.89 1.04 1.16 1.05 1.16
diamine
Prcpyl- 0.025 8.5- 1.00 0.98 1.11 1.12 0.92 1.08 1.35 1.25 1.35 1.20
diamine 10.0
Percentage rerrraining a f t e r fixation w i t h CPC (M NaC1)
100 100 39 70 0 0 0 0 0 ?

%ference: Ieo Heparin No. 4439 and Upjohn Heparin Lot No. ZX320. Muc. = intestinal mcosa; KS =
keratomlphate. / indicates alternative values depending on samples or runs; two values unconnec-
ted indicates two separate bands. Forcine intestinal nucosa heparin usually gives bm bands in
barbital and acid M f e r s in varying proportions. cpc = cetylpyridinium chloride
HEPARIN SODIUM 247

6.8.3. Cellulose acetate electruphoresis


The separation principle is the same as agarose electro-
phoresis. A disadvantadge is the small load capacity of the
cellulose acetate d r a n e s . The n-ethod is most useful f o r
identification of mll quantities of acid mcopolysaccharides
i n a relatively plre state (107). Cellulose acetate mmbranes
were used by Herd to identify mcopolysaccharides (142). Seven
ccnm?rcial heparin preparations were examined on cellulose
acetate d r a n e s by means of electrophoresis (143). The elec-
trolyte systems eXamined were: calcium acetate 0.1M and 0.3M,
zinc acetate 0. l M , El 0.1M and tris-ba&)iturate M f e r pH
8.8, I = 0.05. Heparin fractions were stained w i t h 0.5 per
cent alcian blue in 0.01M El. The use of the tris-barbiturate
buffer system allowed a l l samples except one to be resolved
i n t o two wll-defined zones a t 300 V f o r 50 min. The separa-
t i o n my he carried out using less than 1 pg of heparin.
A c o l o r k t r i c n-ethd for the quantitative determination
of heparin and other glycosaminoglycans based on the solubi-
l i s a t i o n of toluidine blue-polyanionk glycosamiraOglycans com-
plexes fran cellulose acetate membranes was described by Hsu
e t al. (144). The glycosaminoglycans were separated employing
three t y p s of electrolyte: 0.1N El, 0 . B htylamine and
0.05M calcium acetate containing 40 per cent of diethylene
qlycol .
A l l h a m glycosaminoglycans could be separated i n a
single mnodimensional electrophoresis on cellulose acetate by
Cappelletti e t al. (145). A very narrow s t a r t i n g band was ab-
tained by formation of a discontinuity in the electric f i e l d
and a l l ccmpounds wre then selectively resolved by combining
their migration properties i n a barium acetate buffer and
t h e i r differential sensitivity to precipitation by ethanol.
By wrking a t l o w current and temperature for longer
times, e l e c t r q h e r a y a n s are abtaincJ shawing well-defined
.
bands for heparin preparations (146) The electxphoreses were
perfomd i n barium acetate (0.1M) , adjusted to pH 5.8 w i t h
acetic acid, on cellulose acetate s t r i p s a t a voltage corres-
ponding to 0.4 mA/cm a t 4OC for 16 h. After staining w i t h
alcian blue, the densitanetric traces were recorded w i t h a
spectrqhotaneter a t 380 nm. Heparins w i t h similar profiles on
gel f i l t r a t i o n gave different electrophoretic patterns. It is
concluded that high-mlecular-weight species canplex Ba2+-ions
stronger than do low-mlecular-weight species.
6.8.4. Polyacrylamide gel electrophoresis
Polyacrylamide gel electrophoresis is a mthod which se-
parates acid nuccpolysaccharides as a function of their mle-
248 FEUEDRICH NACHTMANN ETAL.

cular sizes. The electrophoresis is performed in gel 0.2 an


thick on 5.0 x 7.5 or 10 can s l i d e s (107). After separation
(2 V / a n f o r 50 min with electrodes in b a r b i t a l l x f f e r ) , the
g e l is stained with 0.1 per cent toluidine blue in 1 per cent
acetic acid f o r 30 m i n and the stain reimved with 1 per cent
acetic acid. A p l o t of mlecular w i g h t x l o 3 on a log scale
versus relative distance f r a n the o r i g i n gives a s t r a i g h t
line. The mthod is very useful f o r the determination of t h e
mlecular sizes and degree of p l y d i s p e r s i t y of pure sulphated
muqlysaccharides.

6.8.5. Electrofocushg
A considerably greater heterogeneity of heparin is de-
tectable by mans of electrofocusing. Substances which show
2 bands in conventional electrophoretic systems can be resol-
ved into 21 bands by electrofocusing with ampblyte mixtures
(147). Polyacrylamide gel slabs (5.0 x 15 an), 0.2 an thick
containing 2 per cent of ampholyte (pH 3.0 to 5.0) f r a n LKB
(Sweden) were used. 100 t o 200 pg of heparin was applied to
the gel i n 2 p l volunrts a t 2 an f r a n t h e neyative end and
subjected to a p t e n t i a l of 15 V/cm f o r 18 hours. The 91 slab
was stained with toluidine blue. The 21 bands are d i s t r i b u t e d
between pH 4.2 and 3.2.
For 15 cannercia1 heparin samples tested, 21 f r a c t i o n s
were found by electrofocusing (148). These fractions shm a
mlecular-weight range from 3,000 t o 37,500 with a constant
i n t e r v a l between mlecular w i g h t s . me technique is s p e c i f i c
and unequivocally distinguishes heparin f r a n other acid mco-
polysaccharides. Results by f i g h e t t i and G i a n a z z a d m n s t r a t e
that t h e charge heterogeneity of heparin absented i n electro-
focusing is e l i c i t e d by strong interaction of t h i s p l y -
saccharide with Ampholine, the mixture of amphoteric sub-
stances used to generate and s t a b i l i z e the pH-gradient (149).
6.8.6. and lung o r i g i n
Ccinparison of heparin of mcosal
The differences wre summrised recently by W f i e and
Cuwie (150).
When electrofocused fractions are eluted f r a n the gel and
subsequently exposed to plyacrylamide electrcphoresis, a
range of mlecular w i g h t s is abtained. ?his is true of hepa-
rin of mcosal or lung origin. An important difference exists,
hawever, between lung and mcosal heparins. This is shown i n
figure 6.1. Unfractionated lung heparin y i e l d s a s i n g l e ccm-
ponent i n agarose, a d i f f u s e band i n plyacrylamide and 22
c c n p n e n t s upon electrofocusing. Unfractionated mcosal hepa-
rin yields two canponents in agarose, a d i f f u s e band in p l y -
HEPARIN SODIUM 249

acry1ami.de and 22 canponents upon electrofocusing. Lung hepa-


rin fractions derived fran the electrofucusing prccedure
yield a single band i n agarose, plyacrylamide and electro-
focusing. Mucosal heparin fractions derived fran the electro-
focusing procedure yield two bands i n agarose, two bands i n
polyacrylamide and a variable number of bands upon electro-
focusing.

-
ELECTROPHORETIC MIGRATION PATTERNS IN:
a b C

-
Heparin Agarose Polyacrylamide LKB Ampholyte

1(beef lung) I 11111111111111111111ll


(22)

2 (pork intestine) II 11111111111111111111l


(22)

Single Fraction
from l c I I I

Single Fraction
from 2c II II Ill

Separation on charge molecular size pH and chain len-


the basis of: density gth producing an
insoluble complex
with ampholyte.

Cathode and point of application considerably to left of each pattern;


anode to right.

Figure 6.1
Sumnary diagram of electrophoretic patterns abtained for dif-
ferent heparinS prior t o and following e l e c t r o f m i n g , accor-
ding to McDuffie and Cawie (150).
Mhen mlecular wights of the lung heparin fractions are
determined i n plyacrylamide gels, a family of different chain
lengths is &served. Molecular w i g h t determinations of mu-
ccsal heparin fractions, containing 2 canponents, yield 2
f d l i e s of differing &ain lengths w i t h a high degree of
parallelism.
250 FRIEDRICH NACHTMANN E T A .

6.9. Chranatographic Methods


6.9.1. Paper chromatography
Paper chrmtography is mostly used for the identifica-
t i o n of heparin.
Molho and Molho-Lacrout described a one-dimensional paper
partition chranatography of canmercial heparin with propanol/
water (1:1.5) by using toluidine blue as a detecting agent
(151). A very intense spot, Rf = 0.57 and a weaker spot = 0
w e r e found. Ihe former had strong anticoagulant activity,
whereas the latter had about 1/25 of the total activity.
Awe and Stiidemann reported the separation of heparin and
heparin-like canpounds by paper chranatography with aqueous
-
propano1 (30 45 per cent) as the solvent (152). The method
permits one to detect the degree of purity of heparin and dif-
ferentiation between heparin and synthetic sulphonated poly-
saccharides.
Another paper ChraMtographic system was developed f o r
t h e resolution of mixtures of sulphated mcoplysaccharides
by Spolter and Mam (153). The effect on t h e +
values of he-
parin and chondroitin sulphate of varying the ammnium forma-
te buffer/isoprqanol ratio i n the m b i l e phase was studied.
One of these solvent mixtures, amrmnium f o m t e buffer/isopro-
pan01 (65: 35) resolved ccnurercial bovine heparin into two
principal canponents.
Cenik and Kronberger used pper chrcmatography f o r the
quantitative and qualitative determination of heparin i n blood
(154). The chranatographic separation is performed i n propa-
nol/phosphate h f f e r of pH 6.4 (75:25). The Rf value f o r hepa-
rin was 0.90 - 0.93. For quantitative determination the CQ-
lour reaction w i t h toluidine blue was used.
Bayer described the separation of heparin and acidic
polysaccharides w i t h propano1 85 per cent/acetic acid/water
(30:10:40) as the m b i l e phase (155). The chranatcgram was
developed by the ascending technique f o r 6 -
16 hours and
sprayed with 0.05 per cent aqueous toluidine blue. Heparin
appears as a v i o l e t spot on a blue background, Rf = 0.45.
Castor and D o r s t e w i t z investigated the following solvents
f o r the paper chranatographic identification of heparin and
other acid mxopolysaccharides (156): 0.4M AlC13, 0.5M FeCl3,
0.1, 0.5, 1.OM MgC12 and 0.04M aqueous an-mnium acetate solu-
t i o n in methanol (45:55). Most of the k n m manutalian acid
muccplysaccharides could be identified. Protein contamina-
t i o n of significant degree did not interfere w i t h the method.
HEPARIN SODIUM 251

An improvement of the staining of plysaccharide sul-


phates was developed by Benaim (157). The mobile phase con-
sists of 5 m l of a 20 per cent solution of Cetavlon (hexa-
decyltrimethylamrrpnium branide) i n alcohol and 95 m l of a
0.2 per cent solution of toluidine blue or azure A. The paper
is dipped f o r 15 - 30 seconds, washed and dried.
6.9.2. Thin-layer chranatcgraphy
Like paper c h r a n a t q q h y , the i d e n t i f i c a t i o n of heparin
is the main use of thin-layer chranatography.
Giiven prepared thin-layers f r a n a mixture of 10 g of hy-
drated MgO, 1.5 g of CaC12 and 60 g of H20 and dried f o r 24
hours a t roan temperature (158). Heparin samples (about 50 I U )
were applied and developed f o r 3 hours i n mthanol/pyridine/
water (30:20:20). After spraying with 2 per cent sodium n i t r i -
t e and then with 0.5 per cent p n i t r o a n i l i n e in c o n c e n t r a t d
acetic acid, heparin appeared as a red s p t with an Rf value
of 0.7. The same author also used thin-layers prepared with
.
c e l l u l o s e (159) The developing systems armronia (25 per cent) /
pyridine/water (60:35:5) and butanol/acetic acid/water (3:l:l)
i n canbination w i t h t h e following colour reagents =re stu-
died: diazotized p-nitroaniline, ninhydrin, azure A, p d i -
methyl-aminchnzaldehyde, copper sulphate/mnia, eriochrcme
black T and water blue. The colour produced by h q a r i n on cel-
lulose thin-layer plates with azocarmine G, acridine orange
and brmcresol green w a s also investigated by Giiven and Er-
tan (160).
Thin-layer chranatography was applied by Bischel e t al.
f o r separating sulphated acid polysaccharide molecules (161).
Both cellulose and Sephadex 6 5 0 w e r e used as mdia f o r chro-
matography. Separation on cellulose p l a t e s is achieved with a
citrate bdfer p H 3.1.
mixture of n - p r o p a n o l / i ~ r ~ l / O . 0 3 7 M
For Sephadex plates, a mixture of n-propanol/isopropaml/etha-
no1/0.037 M citrate buffer pH 3.1 (1:4:2:13) is used. Deve-
loping time is approximately 18 - 36 h f o r Sephadex and 13 h
f o r cellulose plates. A f t e r staining with toluidine blue, as
little as 0.1 p g of plysaccharides can be detected.

6.9.3. Ion-exchange chranatoqraphy


Manley plblished a method f o r rapid separation of mco-
plysaccharides by salt-gradient, ion-exchange paper chranato-
graphy (162). The samples here separated on A m b e r l i t e SB-2
ion-exchange paper using ascending chranatography with a so-
dium chloride solution of s t e a d i l y increasing mlarity and &
tection of the nuccplysaccharides with alcian blue. h g e r t z
and &ichard fractionated
P
aluronic acid, chondroitin A and
heparin by e l u t i o n with NaC -El mixtures f r a n ECTEOLA-cellu-
252 FRIEDRICH NACHTMANN E T A .

lose columns (163). Similar methods using DEAEicellulose (164,


165) and D0we.x 1 X 2 (166) have been pblished.
A canbined chranatcgraphic fractionation for a canplete
separation of acid mcopolysaccharides using cetylpyridinium
chloride cellulose, DE-52 cellulose and Dohex 1 X 2 successi-
vely as column supports was described by Bohn and Kalbhen
(167).
Ion-exchange chranatography on DEAEiSephadex o r prota-
mine-Sephamse separated cmmrcial heparin along a continuum
of anticoagulant activities (168). 95 per cent of the applied
heparin was recovered from the DEAeSephadex column between
0.75 and 1 . 1 M NaCl. ?he specific anticoagulant activity of the
fractions rose steadily from less than 50 IU/mg to high levels
consistently greater than 250 IU/mg in fractions eluting close
t o 1.OM NaCl.
W r i m e n t s using DEAEiSephacel (DEAEicellulose i n bead
form) columns w i t h sodium chloride gradient eluent wen
carried out by Sache e t al. (169). ?he mst active anticoagu-
lant fraction was always associated with a heparin canponent
eluted a t high NaCl mlarity (approximately 0.8M).

6.9.4. G e l chranatography
G e l chranatography has been used f o r mny pars f o r frac-
tionating heparin. During the last few years an improvemnt
was made by introducing high pressure liquid chrcmtography
(WE)
Many authors describe separations using Sephadex-gels.
Skalka used Sephaaex G l O O and 200 (170). It was found that in
low ionicstrength eluents the mlecules of heparin are alte-
red to such an extent that they cannot penetrate into the
accessible pores of the gels. In solution of a higher ionic
strength, they are retarded even on 6 1 0 0 .
Porcine heparin was fractionated into 10 fractions of
varying mlecular w i g h t by gel f i l t r a t i o n on Sephadex G l O O
by Laurent et al. (171). Anticoagulant activity of the frac-
tions increased w i t h mlecular weight up to 18,000 and then
was f a i r l y constant.
Many ccnurercial heparin sodium injections (5 000 I U / m l )
wre fractional on a 2.5 x 45 cm column of Sephadex 6 5 0 , me-
dium grade (172). ?he heparin was eluted with d i s t i l l e d w a t e r
a t a flow rate of 36 ml/h. Each one m l fraction was mnitored
mtachranatically for heparin content by mixing 50 p l of the
fraction with 3 m l of an aqueous solution of 0.0025 per cent
toluidine blue and n-easuring the transmittance a t 500 m. X-k-
parin preparations of nucosal and lung origin gave similar re-
HEPARIN SODIUM 253

s u l t s in v i t r o except that the anti-Xa activation a c t i v i t y of


the lung preparation did not appear to increase w i t h decrea-
sing mlecular s i z e as was found with the mcosal heparins.
Rosenberg e t al. fractionated porcine heparin by cetylpy-
ridinium chloride precipitation and gel f i l t r a t i o n on Sephadex
G l O O (173). Additionnally, heparin fractions sere mixed with
antithranbin and fractionated by gel f i l t r a t i o n .
Recently, lrosito e t al. investigated a column chranato-
graphic technique f o r the fractionation of heparin using vari-
ous diameters of bead fonn cmss-linked dextran gels and an
autcmated apparatus (174). It was &served t h a t Sephadex 6 5 0
resulted i n the separation of three sell-formed peaks and pro-
vided superior resolution canpared to other gels. The s i z e of
the columns was 1 x 60 cm, the eluent 0.086M Tris-El, pH 8.0
containing lM sodium chloride a t 25OC.
Another gel type -
plyacrylamide agarose gel was used-
.
by Johnson and Mu1loy (175) Commrcially available heparins
were fractionated to give fractions whose mlecular w i g h t s
sere estimated by viscosimetry. A similar system was used by
O g m e t al. (176). !they mde experiments W i t h Ultrogel (AcA
44) agarose-acrylamide gel which was found to be favourable
f o r the separation of the higher mlecular w i g h t region of
heparin.
Gel f i l t r a t i o n experinlents w i t h Ultrogel (AcA 54) were
described by Sache et al. (169). Eorcine heparin sodium was
dissolved in 0.3M NaCl solution pH 6.8, applied to the column
(2.6 x 100 cm) and eluted w i t h the same solution. The anti-
thranbin specific a c t i v i t y was m w h a t syrmnetrically d i s t r i -
buted along the whole profile of the elution curve of heparin.
Gel f i l t r a t i o n w i t h ~ ~ particles -
1 1 (5 10 pn) becomes
increasingly important f o r the determination of m l e c u l a r
e i g h t distribution of heparin sodium. Rodriguez used 5 - 10
pn p a r t i c l e size controlled porous glass of different pore
diameters (40 A, 100 and 250 d) and simultaneous detection
with a d i f f e r e n t i a l r e f r a c t m t e r and ultraviolet detector a t
254 nm ( 4 2 ) . The column length was 10 - 60 cm, with 2.3 mrn in-
ternal diameter. The mobile phase was 0.1M sodium acetate
(0.01 per cent in sodium a i d e t o i n h i b i t bacterial qrowth) i n
b i d i s t i l l e d water. The columns were calibrated with different
molecular weight fractions of heparin sodium and baseline reso-
lution obtained between 6.5 k Daltons differences i n molecular
weight. Analysis time was 25 minutes per sample. Most effecti-
ve w a s the 250 8, pore diameter s i z e mterial.
A m d i f i c a t i o n of this technique was described by the
same author ( 4 1 ) . Instead of columns self-packed w i t h control-
254 FRIEDRICH NACHTMANN ETAL.

led porous glass, comnercially available columns consistinq of


30 an x 3.9 mn internal diameter stainless steel tubing packed
with micrqarticles (10 p) of a fully porous silica-ether
[si(W),CH3] could be used. Molecular w i g h t averages could be
determined accurately to about k 500 Daltons. A difference
between hovine lung and porcine intestinal mcosal heparin was
noticed i n this study: bovine lung heparin sodium gave a
f a i r l y syrrmtrical peak about the maxima whexeas mst mcosal
heparin gave a peak that was skewed taward low molecular
weight.
The calibration mthod proved to be wry critical. Dif-
ferences i n mlecular shape of ccrnpounds can appear as diffe-
rences i n mlecular weight in -1 chranatqraphy. Different
results are cbtained i f the columns are calibra- w i t h dex-
trans (40) instead of heparin sodium.
An HPK-system with W-detection a t 205 m, a differen-
tial refractaneter or a mving w i r e flame ionization detector
was described by Sugisaka and Petxacek (40). Columns of 2.3 mn
internal diameter, 1 m long, packed with controlled porous
glass and columns of 4.0 nun internal d i e t e r , 30 cm long,
packed with porous silica (particle size 10 prn) were studied.
The rmbile phase was sodium sulphate, 0.5M, pwnped a t a flow
rate of 1.0 rnl/min. The columns were calibrated by injecting
dextran solutions. Prabably due t o the calibration method, the
results for the fractionation of canmrcial heparins are dif-
ferent fran those of other investigations (41, 42). A similar
system f o r mlecular weight estimations of heparin fractions
was applied by Sache e t al. (169). 7Xm. stainless steel columns
(300 x 4.7 rmn internal diameter) packed with porous silica,
10 pn particle size, =re connected. The m b i l e phase was
0.02M sodium sulphate a t a flow rate of 1 rnl/min.

6.9.5. Affinity CsUraMtcgraphy


Affinity dwamtography is used for separation of high-
activity and low-activity heparin species. HLiijk e t al. coupled
p l r i f i d bovine antithranbin via amino groups to cyanogen bro-
mide-activated Sepharose (70). sarrp?les of heparin sodium,
dissolwd i n 0.2M NaCl - 0.lM Tris-HC1, pH 7.4 were applied to
a column containing 3 rnl of antithranbin-Sephamse gel. A f t e r
equilibration and mshing w i t h the same h f f e r at 4OC, the
sample was eluted with a linear salt gradient (sodium chloride
0.2M to 3 M i n 0.1M Tris-El, pH 7.4). The effluent MS analy-
sed by the carbazole reaction. A separation into a high-affi-
n i t y and a low-affinity heparin was achieved.
Bleyl and Raka determined the affinity between heparin
and a n t i t h r h i n by carrpetitive affinity chranatography (177).
HEPARIN SODIUM 255

They conclude that the chemical modification caused by cova-


l e n t linkage of antithrombin to a matrix alters t h e binding
characteristics between antithrombin and heparin.
Schrrrer patented an a f f i n i t y chranatography method for the
separation of high-activity heparin (178). The mthod employs
an a f f i n i t y column of protamine coupled to a water-insoluble
solid support such as agarose, and a series of sodium chloride-
imidazole e l u t i o n M f e r s (pH 6.5 - 7.5) varying in sodium
chloride m l a r i t y fran about 1.3 to about 2.0.
The fractionation of heparin by chranatography on imnnbi-
lized thranbin was plblished by Nordenman and Bjork (179). Bo-
vine a-thranbin was coupled to cyanogen branide-activated Se-
pharose in the presence of an excess of acetylated heparin.
The material was packed into a column, 1.6 x 3.4 cm. Elution
and detection were similar to techniques described by H W k e t
al. (70). The authors suggest that the separation of heparin
on matrix-linked thranbin into fractions with d i f f e r e n t anti-
coagulant activities is due m i n l y to ion-exchange chmmato-
graphy, the inmobilized thranbin a c t i n g as a weak anion ex-
changer-
A n w technique involves the binding of antithranbin I11
t o a concanavalin A-sepharose column, r e s u l t i n g i n a higher
capacity f o r high-activity heparin (180). Antithranbin I3 is
bound w i t h its &hydrate side chains to concanavalin A.
Chemical rrodification of the glycoprotein is not required and
active antithrcmbin m y be recovered. 40 per cemt of heparin
samples =re bound to t h e column with binding capacities of
15 mg/100 mg antithrcmbin. The peak of bound heparin e l u t e s a t
a sodium chloride concentration of 0.6M. The anticcagulant po-
tency was 270 unitdmg in the APTT assay.

6.9.6. HyJrophobic interaction chrunatoqraphy


Ogam et al. separated porcine nucosal heparin by hydro-
phobic i n t e r a c t i o n cfiramatography on phenyl-sepharose CG4B
i n t o two groups, one w i t h high a f f i n i t y and one w i t h low a f f i -
n i t y f o r t h e cpls (176). The former group i a s further separa-
t e d i n t o three fractions d i f f e r i n g i n hydmphcbicity. While
the N-acetyl content of the f r a c t i o n with the lowest hydro-
phabicity were 0.12 ml/ml of hexosamine, those of the frac-
tions with higher h y d q h o b i c i t y =re 0.15 - 0.17 ml/ml. ?he
anticoagulant activities of the fractions w i t h higher hydro-
phobicity ranged fran 210 to 254 units/mg, whereas that of the
f r a c t i o n with lower hydrophobicity was 100 units/mg. The
samples were loaded on a 2 x 43 cm column and eluted stepwise
w i t h mixtures of ammnium sulphate/hydrochloric acid a t roan
tgnperaixre.
256 FRIEDRICH NACHTMANN ETAL.

6.9.7. Countercurrent d’lranatography


This mthcd was described by Wst e t al. for continuous
flow fractionation of glycosaminoglycans by partition i n two-
phase systems (181). In the 1-butanol/aqueous N a C l two-phase
system containing hexadecylpyridinium chloride, fractionation
i s dependent primarily upon chemical canposition, o r charge
density, of the glycosaminoglycans and is relatively Mepen-
dent of molecular weight. Chondroitin 4-sulphate was fractio-
nated according to degree of sulphation and could be comple-
t e l y resolved frcm heparin. A heparin sample was shcrwn to con-
tain three discrete ccmponents differinq with respect to
sulphaminohexose and sulphate substitution.
6.9.8. Gas chranatography
Gas chranatcqraphy is used for the determination of dif-
ferent parts of the heparin mlecule. A differentiation bet-
ween mucous, lung and whale heparin is possible by gas chroma-
tographic determination of acetic acid obtained a f t e r hydro-
l y s i s of N-acetyl groups in heparin (182). The content of ace-
tic acid i n mcous, lung and whale heparin wre 1.52, 0.33 and
5.69 per cent.
The quantitative determination of hexuronic acids i n he-
parin using gas liquid chranatography was described (183). N-
Desulphation of heparin was achieved using 0.06N methanolic
HC1. The resulting N-desulphated heparin rrethyl ester was sa-
ponified to free acid w i t h 0.1N NaOH. It was then N-acetylated
with acetic anhydride. Trimethylsilylation of hexuronic acids
was carried out with hexamethyldisilazane and trimethylchlom-
silane. For gas chranatographic separation and quantification
a packed column f i l l e d w i t h Apiezon M on Gas-chran CLZ a t
190°C coupled t o a flame ionisation detector was used.
Another possibility was investigated by Inoue (184). He-
parin was d e a m i ~ t e dw i t h n-butyl n i t r i t e and rrethanolised.
The mnaneric silylated derivatives of uronic acids w e r e quan-
t i f i e d by gas chromatography. The separation was performed
with a packed column (200 an x 0.4 an internal diameter)
packed with 3.5 per cent SE30 on Gas-Chrm Q a t a l i n e a r tem-
perature programme fran 140 t o 2OOOC (l°C/min). A species-
specific difference i n the iduronic acid to glucwonic acid
r a t i o of heparin was noted.
Gas chranatography can also be applied for the quantifi-
cation on sulphate i n glycosaminglycans (185).
6.10. Amidolytic Methods
In standard procedures f o r the synthetic substrate rre-
thods, heparin is analysed as a heparin/antithranbin I11 ( A F
HEPARIN SODIUM 257

111) canplex. The pesence of AT-I11 in the test plasm is


crucial, either nonnal plasma or plrified AFIII can be used
to assure prcjpr canplexation. These assays can be adapted
for both end-point and kinetic mthods. Recently, test k i t s
containing the substrate, factor Xa, AT-111, Mfer, and nor-
ml human plasm in lyophilised fonn have becane available.
The procedure is as follows (186):
Heparin + AT-I11 d heparin/AFIII
Heparin/AFIII + factor Xa -~heparin/AFIII/Xa+residual Xa
Residual Xa + R-pNA _c,R-COOH + pNA
pNA = p-nitroaniline
Because the plrified Xa preparations are difficult to cb-

Residual thranbin + R-pm


-
tain, heparin assays involving thranbin were developed (186):
Heparin + AS111 4heparin/A?LIII
Heparin/AFIII + thranbin heparin/AFIII/thrallbhrcmbin t
residual thranbin
R-COOH + pNA
?he first chr-nic tripeptide, Bz-Phe-Val-Arg-pNA (S-
2160) was intrcduced by mndsen et al. as a specific substra-
te for thranbin (187). Many other similar peptides have been
described until nw. All of these substrates are coupled with
p-nitroaniline (pm) as a chrumphore at the terminal carboxyl
grow of the peptide and wntain arginine or lysine. The ab-
-
sorbance of F€W is measured at 400 410 nm. @lA bound to the
synthetic peptide does not absorb significantly at 400 - 410
nm, whereas the free form absorbs strongly here.
Nowadays fluorcgenic substrates are available for the de-
termination of serine protease enzymes such as thranbin or
factor Xa. An example is Pphenylalanine-proline-arqinine-5-
amido-isophthalic acid-dimethylester-diacetate (H-D-Phe-Pro-
Arg-AIE) the structure of which is given belw (188):

H-BPhe-PreArg .+N%<=’=3

thrallbin
5-43
0
?he excitation wavelength is 335 nm, the enission wave-
length 430 nm. The method is used for the determination of
heparin in humn plasm.
258 FRIEDRICH NACHTMANN ETAL.

A surmrary of the mst important synthetic substrates for


the determination of heparin is given i n table 6.3.
Table 6.3
Synthetic substrates for amidolytic determination of he-
parin
Substrate Detec- Literature
tion
S 2160 Bz-Phe-Val-Arg-pNA Thranbin Absorbance 187,189
S-2238 H-D-Phe-PipArg-pNA l%ranbin Absorbance 189 ,190,
191
ChrCfIlOZym Tos-Gly-Pro-Arg-pNA l % r d i n Absorbance 192,193,
TH 194
H-D-Phe-Pro-Arg-AIE Thranbin Fluores- 188
cence
t-Boc-Val-Pro-Arg- Thrdin Fluores- 195
MCA cence
s-2222 Bz-Ile-Glu-Gly-Arg- Factor Xi Absorbance 189,194,
pNA 197,198,
199
t-Boc-Ile-Glu-Gly- Factor Xi Fluores- 195
Arg-McA Cence
t-Boc-Ser-Gly-Arg- Factor Xi Fluores- 196
MCA CenW
AIE = amidoisophthalic acid-dimthylester-diacetate
MCA = methylcoumaryl-7-amide
t-m = tert,-butyloxycarbonyl

Synthetic substrate mthods will play an increasingly im-


portant part in the determination of heparin in clinical che-
mistry laboratories. k z p analysers can be used for these
analyses. Chranogenic and fluorogenic methods give canparable
results (186). The advantage of the fluorimetric mthods is
their higher sensitivity.

6.11. Esterolytic Methods


This assay of heparin is based on the heparin-accelerated
rate of a-thrcanbin inactivation by antithrcmbin I11 (200, 201).
After incubation, a-N-benzoyl-L-arginine-ethylester (BAEE) is
added, which canpletely inhibits further a - t h r d i n inactiva-
tion. The residual a-thranbin hydrolyses -+to produce etha-
nol, which is measured i n the presence of NAD and alcohol de-
hydrogenase by mnitoring NADH-formation a t 340 nm. ?he mthcd
was used for the determination of heparin in lmmn plasma.
There was found to be good correlation with an amidolytic
method.
HEPARIN SODIUM 259

6.12. Anticoagulant and Allied Tests


Reviews on these techniques W E written by Jaques (107)
and Utes (75). There are two applications of anticoagulant
tests: mnitoring drug dosage in heparin theram and standar-
dization of the drug f o r anticoagulant therapy. In a l l coagu-
lation tests involving heparin, extra attention rmst be qiven
to the special significance of pH and surfaces.
Values for clotting tines w i t h mst coagulation systems
are essentially the same between pH 5.5 and 7.6, but the anti-
coagulant effect of heparin is mrkedly pH dependent and is
minimal a t pH 6.4. A decrease i n the coagulation system of
only 0.1 pH u n i t can significantly decrease the anticoagulant
action of heparin. Lyophilisation gmerally results in a pH
shift, since b i c e n a t e , citrate, and ammnium salts decan-
pose. (31 reconstituting lyophilised plasm, a l l the material
must be redissolved, and the pH measured and adjusted. A l l
solutions must be ad-~ustedt o pH 7.0 t o 7.2. A t pH 7.0 t o 7.2,
heparin solutions can be sterilised by autoclaving.
Blood coagulation is activated by surface contact. The
surfaces w i t h which blood canes into contact thanselves af-
fect profoundly the inhibitory effectiveness of heparin on
blood coagulation. Therefore, spcial procedures f o r preser-
vation of test tubes used for the detemination of whole blood
clottinq t h e , Howell whole blood assay etc. are necessary
(107).

6.12.1. Hematological procedures


The hematological procedures are chiefly coagulation
tests carried out in the clinical laboratory for the investi-
gation of coagulation disorders. 'Ihe tests used are sunmrised
i n table 6.4.
Withhi ;Ihe range of measurable clotting times there is a
linear relation between log clotting time and heparin concen-
tration (107). This mans that phannacOaynamic parameters
(e.g., half-life) calculated from coagulation values W i l l not
agree w i t h those calculated fran concentrations of the drug i n
the blood. Clotting t i m s can be recorded by Semi-autanated
instruments. One possibility is the nephelometric masurement
of f i b r i n f o m t i o n which WIS described for the assay of
plasma heparin concentration by means of a thranbin time
methcd by Noren e t al. (220).
260 FRIEDRICH NACHTMANN ETAL.

Table 6.4
D i r e c t hematolcgical heparin tests on blood or plasma

Test Matrix Reference

Whole blood clotting time Fresh blood 202,203,107


S i l i c o n e c l o t t i n g tirre Fresh blood taken 203
i n silicone
P a r t i a l thranbplastin time Plasm 202,203
(PTT)
Activated PTT (APTT) Plasma 203,204,107
Thranbin t i m e Plasma 203,205,206
T i t r a t i o n with thranbin Plasm 207
Protamine titre Blood , plasma 203,208,209
210
Tbluidine blue titre Plasma 208,211
Factor Xa inhibitor Plasma 107,203,212,
213,214
‘Ihranbin inhibitor Plasma 219
Polyhrene n e u t r a l i s a t i o n Plasma 215 I 216 I 217
(titration)
P a r t i a l p l y b r e n e neutrali- Plasm 218
sation

Many s t u d i e s dealing w i t h a canparison of the different


tests were plblishea. ’kien and L i e canpared five mthcds
(221). The lowest detectable heparin concentration i n nonnal
plasma was: Factor Xa i n h i b i t i o n 0.012, calcium thranbin time
0.02, p l y b r e n e t i t r a t i o n 0.07 and APTT 0.08 I U / m l . The accu-
racy was lower in pathological than in n o m l plasm. A t a
heparin concentration of 0.5 I U / m l plasma, the coefficients
of v a r i a t i o n of eleven pathological plasmas were: Polybrene
-
t i t r a t i o n 3 per cent, factor Xa i n h i b i t i o n 15 20 per cent,
calcium thranbin time 35 per cent. W i t h the APTT method the
found heparin concentrations ranged frm 0.05 t o above 0.55
I U / m l plasma.
4 methods were canpared by Vinazzer (222). The lowest
s e n s i t i v i t y wis found for the thranbin time, s l i g h t l y better
r e s u l t s were abtained by the APTWr&hod. Addition of puri-
f i e d anthithranbin I11 t o the r e a c t i o n mixtures improved the
s e n s i t i v i t y of b t h mthods. l+henfactor Xa was used, the re-
s u l t s were canparable t o the APTCtest. The s e n s i t i v i t y was,
hawever, about 10 times higher h e n the remaining factor Xa
was d i r e c t l y measured w i t h a ckranogenic substrate.
Recently Phillips found, that plasm heparin levels mea-
sured using d i f f e r e n t mthods &wed a wide v a r i a t i o n (223).
HEPARIN SODIUM 261

I n particular, an anti-factor Xa clotting technique qave very


much higher results than thranbin t h e and partial thranbin
t k techniques. This was less marked when a synthetic ckrano-
genic substrate assay was used.
Whole blood coagulation t i m e , automated whole blood coa-
gulation time and APTT w x e cunpared by Sckriever et al. ( 2 2 4 ) .
Log APTT m s linearly correlated with blood heparin level and
with the 2 whole blood coagulation tests.

6.12.2. Phamacological procedures


Tests canparing the activity of two or more heparin pre-
parations i n a test system are designated "pharmacological
procedures". A review was given by Jaques (107), the most im-
portant coagulation tests are surrPMrised in table 6.5.
Tests of heparin preparations fran different sources with
different test systems give different anticoagulant a c t i v i t i e s
(107). Walton e t al. reported that when preparations of hepa-
rin from the lungs and mcosa of ox, pig, and sheep were
tested f o r potencies by the BP and USP assay prcedures, a
discrepancy of I+ to 4 0 per cent wis absenred &tween estima-
tes obtained by the two procedures on canparison of samples
of heparin derived fran lung and intestinal rmcosa (240) .
Shen and Barlow canpared human and sheep plasma ( 2 2 8 ) .
The specific activity of certain heparin fractions assayed i n
human plasma differ& fran that assayed i n sheep plasma. It
is concluded that the USP heparin assay does not necessarily
reflect the true activity in human blood clotting system.
The BP assay of heparin was canpared with som other
methods ( 2 4 1 ) . ?he APTT/BP potency ratios varied with the
preparations, lxlt was not dependent on the tissue source of
heparin. For rmcosal heparins, the anti-Xa/BP potency r a t i o s
were close t o 1, ht for heparin of lung origin the anti-Xa
potency was,1/4 of the BP potency. Heparin fractions prepa-
red by column gel chranatography showed a wide divergence bet-
wen the BP potency and those abtained with other lnethods.
Tests on camercial mcous heparin shaved that there is
no correlation between the potency found by the USP method and
an amidolytic method using Chranozym-TH as substrate ( 2 4 2 ) .
"he explanation of these results my be found i n the f a c t that
heparin preparations inhibit a number of steps i n the coagu-
lation sequence (107). Camxcial heparins are a mixture of
anticoagulant and nonanticoagulant heparins, with differing
relative proportions of the different &ain length heparins.
It appears most unlikely that the same specific heparin chain-
length provides the mst effective inhibitor for each step i n
the coagulation sequence. I n order t o investigate the problems
262 FRIEDRICH NACHTMANN ETAL.

of canparing different preparations of heparin, a collabora-


tive study was performed (243). Porcine nucosal heparin tested
versus i t s e l f by different methods proved a good accuracy of
the methods. However, when different heparin preparations were
canpared, estimates for the same method by different labora-
tories o r for different substrate batches varied by as much
as 40 t o 50 per cent.

Wle 6.5
Pharmacological coagulation tests

Coagulation tests Substrate Reference


i n vitro

Modified Howell Fresh whole blood 208,107


Fischer Whole chicken plasm and 208
thranboplastin
Jorpes e t al. Ox blood 208
USP &calcified citrated sheep 208,225,226,
plasm 227
Modified USP Recalcified citrated h m n 228
plasm
&calcified citrated p o r c h 229
plasm
Recalcified bovine plasm 230,231,232,
and thranbokinase 233,234
BP Sulfated whole blood and 208,235
thranbokinase
Antithranbin Oxalated ox plasm and 208,236
thranbin
Antilkranbin C i t r a t e d ox blood and 208
thranbin
APTT Different plasms 237
Protamine neutrali- 238,239
sation

Tests in vivo
Jorpes e t al. Sheep with i n 4 l l i n g 208
catheters
Kuo e t al. Blood samples fran 240,107
conscious, trained dogs

The mnplexity of heparin leads to the conclusion of


Jaques that anticoagulant activity cannot be equated w i t h
heparin, nor heparin defined by anticoagulant activity (107).
HEPARIN SODIUM 263

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HEPARIN SODIUM 265

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HEPARIN SODIUM 267

74 Estes J.W. and Poulin P.F.,


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75 Estes J . W . ,
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107 Jaques L.B. in G l i c k D. (ed.) ,
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HEPARIN SODIUM 271

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148 Eclc Duffie N.M., D i e t r i c h C.P. and Nader H.B.,
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152 Awe W. and Stijdemann D.,
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153 Splter L. and Marx W.,
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157 BeMh M.,
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159 G w e n K.C., Arabacicglu F. and Ertan G.,
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160 W e n K.C. and Ertan G.,
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162 Manley G.,
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272 FRIEDRICH NACHTMANN ETAL.

163 Ringertz N.R. and Reichard P.,


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177 B l e y l H. And Roka L.,
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178 Schmer G.,
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181 Hurst R.E., sheng I.Y.P. and It0 Y.,
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HEPARIN SODIUM 273

182 Himshi T. and 'Ibshio K.,


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183 Radhakrishnamrthy B., Dalferes Jr.E.R. and Berenson G.S.,
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184 b o u e S.,
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186 Fareed J., Wssmre H.L. and Bermes E.W.,
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188 Mitchell G.A., Gargiulo R.J., €fuseby R.M., Lawson D.E.,
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189 Barq N.U. and Mattler L.E.,
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190 Bhargava A.S., & h i c k J. and Giinzel P.,
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191 Larsen M.L., Abildgaard U., W i e n A.N. and G j e s d a l K.,
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192 Bart1 K., Dorsch E. and Z i e g e n h o m J.,
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193 Raka L. and B l e y l H.,
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197 Teien A.N., L i e M. and Abildgaard U.,
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198 Teien A.N., Abildgaard U., G j e s d a l K. and Hohn A.H.,
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199 Wien A.N. and L i e M.,
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274 FRIEDRICH NACHTMANN ETAL.

200 Kapke G.F., CwenW.G., W i t t e D.L. and Feld R.D.,


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208 Jaques L.B. and B e l l H.J. in G l i c k D. (ed.),
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HEPARIN SODIUM 275

217 bffmnn J.J.M.L. and Meulendijk P.N.,


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218 Talstad I.,
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219 Stavridis I.C. and Vorias N.I.,
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225 'Lhe United S t a t e s Phannacopeia, 20th Revision, United
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227 .%map 0.1. and Kuizenga M.H.,
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229 Benesova O., Spackova M., Zabrodska A. and Legerova A.,
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233 Peter H. and Messerschmidt W.,
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234 Messerschmidt W.,
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276 FRIEDRICH NACHTMANN E T A .

235 B r i t i s h P h a u n a c c p e i a 1980
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239 Lowary L.R., Smith F.A., rCryne E. and Dunham N.M.,
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240 Walton P.L., Ricketts C.R. and Bangham D.R.,
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242 Nachtrtlann F.,
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HYDROCORTISONE
Klaus Florey

1. Introduction 278
1.1 Foreword 278
1.2 Histoory 278
2. Description 278
2.1 Name, Formula, Molecular Weight 278
2.2 Appearance, Color, Odor 279
3. Isolation and Synthesis 279
3.1 Isolation 279
3.2 Chemical Synthesis 279
3.3 Biosynthesis 280
4. Physical Properties 28 I
4.1 Spectra 28 1
4.2 Solid Properties 289
4.3 Solution Properties 293
5. Methods of Analysis 294
5.1 Historical Synopsis 294
5.2 Elemental Analysis 296
5.3 Ultraviolet 296
5.4 Colorimetric 297
5.5 Fluorescence 299
5.6 Polarographic 300
5.7 Isotope Dilution 300
5.8 Chromatographic Methods 300
5.9 Electrophoretic 306
5.10 Bioassay-Enzymatic 308
5. I 1 Saturation Analysis 308
6. Stability-Degradation 309
7. Metabolic Products-Pharmacokinetics 310
8. Determination in Biological Fluids and Tissues 313
9. Determination in Pharmaceutical Formulation 3 14
References 315

ANALYTICAL PROFILES OF DRUG SUBSTANCES 277 Copyright by the American PharmaccuticalAssociation.


ISBN 0-12-260812-7
VOLUME 12
278 KLAUS FLOREY

1. Introduction
1.1Foreword
The compilation of an analytical profile
for hydrocortisone, also known as cortisol, turned
out to be a more ambitious undertaking than I had
expected. When scanning through Chemical Abstracts,
the literature is simply staggering. Fortunately,
there are quite a few very good and easily acces-
sible books and reviews available which will lead
the reader to secondary sources. First and fore-
most, there is the classic book on steroids by
Fieser and Fieser.' Helpful are also the more
modern books,on steroid analysis by Heftman* and
Gordg and Szaz.3 As in the case of aspirin, I have
endeavored to cover the newer literature as compre-
hensively as possible and have included only those
older references which are of historical interest.
Again, my apologies to all those scientists whose
publications in this field are not included here.
1.2History
Althouqh hydrocortisone is the major
hormone secreted by-the human adrenal cortex , it
was by no means the first adrenocortical hormone to
be isolated by the early investigators.
Reichstein14 the first to publish its isolation
from adrenal glands, gave it the letter M (see also
Section 3 ) . When Kendall and Hench made their
momentous discovery of the relief of the symptom of
rheumatoid arthritis5 in 1948, cortisone was the
first hormone to be made available by Merck and
Company. Only in the early fifties was hydrocorti-
sone also introduced into therapy, but was soon
eclipsed in potency and effectiveness by analogs
such as the A 1 - and 9a-halo compounds. Yet, it
still is much prescribed, since it is relatively
inexpensive. It is still the ultimate standard by
which the efficacy of other corticosteroids is
measured.
2. Description
2.1 Name, Formula, Molecular Weight
Hydrocortisone is pregn-4-3,20 dione,
11,17,21-trihydroxy-, ( 1 1 B l - i also 4-pregnene-118,
1701, 21-triol-3,20-dioneI 17-hydroxycorticosterone;
Reichstein's compound M; Kendall's compound F;
HYDROCORTISONE 219

cortisol Chem. Abstracts Registry Number [50-23-71.

21 CH20H
I
c=o

‘21H3005 M.W. 362.46

2.2 Appearance, Color, Odor, Taste


Bitter tasting, white to practically
white, odorless, crystalline powder.
3. Isolation and Synthesis
3.1 Isolation
In the late thirties, the isolation of
adrenocortical hormones was actively persued by
Pfiffner, Vars and Wintersteiner at Columbia, Mason,
Myers and Kendall at the Mayo Foundation, Cortland
and Kuizenga at the Upjohn Co. and Reichstein at
the University of Basle, Switzerland. It was
Reichstein4 who first described the isolation of
hydrocortisone and elucidated its structure. He
called it Substance M; but for several years, it
was better known as Kendall’s F. For details
relating to structure elucidation, I refer to
Fieser and Fieserl and also to Reichstein and
Shoppee.
3.2 Chemical Synthesis
As already mentioned (Section 1,2) ,
cortisone was the first corticosteroid introduced
into therapy, since the 11-keto group is more
readily accessible to synthesis. Hydrocortisone
was first synthesized in the Merck laboratories by
Wendler, Graber, Jones and Tishler7 from 20-cyano-
A17-pregnene-21-ol-3,11 dione, which in turn had
been prepared by Sarett8 in connection with his
cortisone synthesis. Introduction of oxygen at the
11 position has been crucial for the commercial
280 KLAUS FLOREY

production of corticosteroids from abundant natural


sources. The microbiological hydroxylation of
steroids at position 11, therefore, has been a
major breakthrough, pioneered in the Upjohn, Squibb
and Pfizer laboratories (for details, see
references 9 and 10) .The Pharmaceutical Manufac-
turinq Encyclopedia lists the microbiological
118 hvdroxvlation of Reichstein's compound S (11-
desox;- 17-hydroxy-corticosterone) to hydrocortisone
as a mode of manufacturing.

CH2OH CH2OH
I I
c=o C=O

However, I am not aware that this route is really


used on an industrial scale. The main commercial
routes to hydrocortisone are either based on
naturally occuring steroids, having an oxygen
function at carbon 12, or on llu-hydroxylation of
a suitable intermediate. In the first category are
the Merck process using bile acids and the process
starting with hecogenin, a constituent of sisal.
In the second category is the Upjohn process
starting with progesterone, derived from diosgenin,
a constituent of dioscorea or stigmasterol derived
from soybeans. Syntex played a leading role in the
development of suitable intermediates for the
diosgenin and hecogenin derived processes.
3.3Biosynthesis
The mammalian biosynthesis of hydrocortisone
and the other corticosteroids occurs in the adrenal
gland. The common precursor is cholesterol. The
immediate precursors of hydrocortisone are primarily
ll-desoxy-cortisol (Reichstein's compound S),which
is 118 hydroxylated enzymatically, and cortico-
sterone, which is hydroxylated at carbon 17.12 It
is curious to note that no mammalian lla-hydroxy-
lating enzymes are known which are the prevalent
HYDROCORTISONE 281

enzymes in microorganisms (see 3.2). The elucida-


tion of biosynthetic pathways was pioneered by
Hechter and Pincus and their collaborators at the
Worcester Foundation in the late forties.13 For
details on the biosynthesis of adrenal steroids,
I refer to an excellent review by Samuels and
Uchikawa14 and the book on steroid biochemistry by
Heftmann.15
4. Physical Properties
4.1 Spectra
4.11 Infared
The first spectrum of hydrocortisone,
prepared in a nujol mull was published by Collings-
worth, et a1 in 1953.16 A sDectrum of hvdrocorti-
.
sone dispersed in a potassium bromide disk was
recorded by Hayden17 in 1954. The classical atlas
of infrared spectra of steroids by Dobriner,
Katzenellenbogen and Jones18 contains only the
spectrum of hydrocortisone acetate. Mesleyl’
described infrared spectra of hydrocortisone
acetone, had no absorption near 760 cm-B.
polymorphs. Form A, evaporated from eth no1 or
Form B,
evaporated from chloroform, gave a double peak at
761 and 752 due to chloroform.
The spectrum of the USP reference
standard of hydrocortisone in a mineral oil
dispersion,218presented in Figure 1, essentially
agrees with the spectra in reference 16.
4.12 Ultraviolet Spectrum
Hydrocortisone exhibits the ultra-
violet absorption spectrum typical of a,B-unsatu-
rated ketones. Such a spectrum, with a maximum at
241 nm, was recorded already in 1936 for Reichstein4
by Mehler of Zurich, Switzerland on a Hilger quartz
spectrograph. The Merck Index gives a X maximum
242 nm Ei 445.20 The chapter on steroids by
A.A. Forist and J.L. Johnson in Pharmaceutical
Analysis2I gives a X maximum 242 nm, E maximum
16,200. See a l s o the review article on ultra-
violet absorption of steroids by L. Dorfman.22 A
A maximum 242 nm, E maximum 15,800 has also been
reported.23 It also has a weak maximum at 290 nm
(log E 2.O1Iz4 and inflections at:
WAVELENGTH (MICRONS)

0.2

0.4

0.6
0.8
1.o

Figure 1. Infrared spectrum of hydrocortisone (USP reference standard) in a


mineral oil dispersion (Instrument: Perkin-Elmer 621).
HYDROCORTISONE 283

x (nm)
- log E
315 1.82
324 1.75
332 1.73
338 1.65
346 1.56
357 1.23
4.13Fluorescence
Hydrocortisone does not have any
native fluorescence, but it can be induced by
reaction with concentrated acids (see Section 5.5).
4.14Phosphorescence
The singlet + triplet transitions by
phosphogescence excitation spectroscopy at both 77

1
and 4.2 K were studied for several steroids.25
The following data pertain to hydrocortisone:
Phosphorescence emission: 3880 origin; 4350 8
maximum; So+T absorption: 3826 ; S-tS (rl,II) dire t
absorption: 3,600, 3,440, 3,310 ; A E: 1600 cm E .
4.15 Nuclear Magnetic Resonance26
The 100 MHz proton NMR spectrum of
hydrocortisone in DMs0-d~is shown in Figure 2.
The spectrum contains characteristic resonances at
6 0.76 s (l8-CH3), 61.37 s (19-CH3), 65.55 s (C4-H),
64.26 M ((211-H) and a coupled A B quartet at 64.50,
4.05 (21-CH2). Three peaks that exchange with D20
were detected at 65.14, 4.64 and 4.26 (11, 17 and
21-OH). All proton chemical shifts are referenced
to internal TMS at 0.0 PPM.
The carbon-13 NMR spectrum of hydro-
cortisone (Figure 3) is summarized in Table 1.
Assignments of all 21 carbons are listed and are
consistent with those of Blunt and Stothers.27 The
reference peak in the carbon spectrum was the DMSO
multiplet which was assigned as 39.5 PPM from TMS.
The spectra shown in Figures 2 and 3
were obtained on a Varian XL-100-15 NMR spectro-
meter equipped with a Nicolet TT-100 data system.
4.16Mass28
The low-resolution mass spectrum of
hydrocortisone (see Figure 4) shows the expected M+
at m/z 362. Corticosteroids generally show frag-
Figure 2. Proton NMR spectrum of hydrocortisone. Instrument: Varian XL-100-15.
N
oo
01

Fig. 3. Carbon-13 NMR spectrum of hydrocortisone. Instrument: Varian XL-100-15


6394 SQ9318 B R T C H *NX001 200 DEG

01-MRY-80 MB0764
1001

90-

80.-
>
I-

II)
70.-
Z
I
z
U

W
>
U

l-
a
J
w
CY

MRSS/CHRRGE
I N T E N S I T Y SUM = 4 0 8 2 5 BRSE PERK Z = 3 . 4 3

Figure 4. Low Resolution Mass Spectrum of Hydrocortisone. Instrument: MS-9


HYDROCORTISONE 287

TABLE 1. CARBON-13 NMR DATA FOR HYDROCORTISONE


CARBON # CHEMICAL SHIFT ( 6 )
1 34.0
2 33.5
3 198.0
4 121.5
5 172.3
6 32.8
7 31.4*
8 31.2"
9 55.6
10 38.9
11 66.5
12 39.1
13 46.3
14 51.6
15 23.3
16 33.0
17 88.5
18 16.9
19 20.4
20 211.5
21 65.8
6 in PPM from TMS (ext): Reference peak DMSO at
39.5 PPM.
* These assignments may be interchanged.

mentation patterns resulting from the l o s s of


D-ring substituents combined with the loss of small
neutral molecules such as water. The principle
hicrh mass ions are thus formed from the loss of
these groups, as depicted below. *
a

m/z 313
M+ m/z 362

m/z 302 m/z 344 +H

m/z 329
\r m/z 267
* Scheme is intended to show losses rather than to
explicitly show fragmentation pathways.
** Rearrangement
288 KLAUS FLOREY

Ions of mass less than m/z 267 (CigH230) occur


throucrh the loss of carbons (and attached hydrogens)
progressing from the D-ring. .

L m/z 267 -

In contrast to 1,4-dien-3-ones, the 4-en-3-ones


yield less intense diagnostic A-ring ions of m/z123
and 124. The analogous 1,4-dien-3-one would have
had a much more intense A-ring m/z 121, 122 ions.
The mass spectrum and fragmentation
pattern presented here is in essential agreement
with data reported earlier.29
A comparison of field desorption,
chemical ionization and electron impact mass spectra
of some steroids30 revealed that, both in the field
desorption and chemical ionization mode, the
molecular ions ( (M)+ and (M+H)+, respectively) are
the base peaks for hydrocortisone, while m/e 302 is
the base peak in the electron impact mode.
The field desorption kinetics in the
mass spectrum of hydrocortisone indicate that the
(M-C2H302)+ ions are formed by the gas phase
decomposition of the hydrocortisone molecular ion
at times >10-9-8 s.31
HYDROCORTISONE 289

4.2 Solid Properties


4.21 Melting Range
The following melting ranges have
been reported:
OC . Reference
217-220 with some decomposition 20
from ethanol and isopropanol
212-213 (commercial samples) 20
About 214 with decomposition 32,33
207- 214 34
215-218 24
201 (from ethanol, micro rn.p.1 35
4.22Differential Thermal Analysis
Hydrocortisone (USP reference
standard) when heated at 15O/min. (Instrument:
Dupont 900) gave a single endotherm at 221.50.36
4.23Thermogravimetric Analysis
Hydrocortisone (USP reference
standard) when heated at 15O/min. (Instrument:
Dupont 900) had a 0% weight l o s s to a temperature
of 222O. 36
4.24Calorimetry
The heat of combustion of hydro-
cortisone and other steroids was determined in a
microcalorimeter.37
4.25Crystal Properties
Microscopic pictures of hydro-
cortisone crystals were already presented by
Reichstein4 in 1936. As most steroids, hydro-
cortisone crystallizes in polymorphic and solvated
modifications. These can be summarized as follows:
Solvent of
Modification Crystallization I R Characteristics

I Ethano1,acetone OH C=O and C=C Other :


3430 1715,1708,1645 902 (strong band) 19,38
3310 (sh) 1631,1611 887 (sh)

I1 Desolvation from 3435


chloroform 3350(sh) 1712,1643, 38
1630,1611

I11 Methanol Solvate Met hano1 3515 1722,1643 38


3405 1628,1607
3225

IV Chloroform Solvate Chlorofo m 3435 1712,1643 761 ,752 19,38,39


3350(sh) 1630,1609

The optical crystallographic properties of hydrocortisone crystal-


lized from ethanol were determined as follows:35
Crystal System and Habit; monoclinic plates;
Optic sign: +; Optic axial angle: 56O (58O calc.)
Dispersion: R>V; Refractive Indices: Nx: 1.540; Ny: 1.562; Nz: 1.638
Solvents, other than ethanol, gave hexagonal (dichloromethane) and
orthorhombic (methanol) crystals.42
Hydrocortisone was subjected to single crystal x-ray analysis and
HYDROCORTISONE 291

the established structure compared to that of 9a-


fluorohydrocortisone and other related steroids.41
Hydrocortisone methanol solvate (Cambridge Crystal
Library Code: (ORTPIS), when subjected to single
crystal x-ray analysis, was found to crystallize in
the orthorhombic space group P212121 with a=14.372
(4); b=18.400(5); c=7.706(2); 8,2=4. Ring A is a
distorted "sofa", rings B and C are in the "chair"
configuration, and ring D is close to a C(13)
envelope, with a pseudorotational parameter A of
26.1O. The ring junctions B/C and C/D are both
trans. The molecule as a whole is slighgly convex
towards the 8-side, with an angle of 10.9 between
the C(lO)-C(19) and C(13)-C(18) vectors.42 These
findings have been compared with energy calculations
according to the Westheimer The electronic
structures of hydrocortisone and other related
steroids was examined by extended Hueckel molecular
orbital calculations. Total and orbital energies,
Mulliken population, analytical data and dipole
moments were reported.44 The crystal structure of
the pyridine solvate (Cambridge Crystal Library
Code: CORTPY) has also been determined.45
The x-ray powder diffraction data
for hydrocortisone are presented in Table 2 and
Figure 5.46 An early x-ray powder attern was
presented by Collingsworth, et al. 1g Low micron
particles of hydrocortisone were obtained by ultra-
sonic irradiation of supersaturated solutions.47
TABLE 2

Powder X-Ray Diffraction


Pattern of Hydrocortisone (USP Reference Standard)
20 d (8) I 1/10
5.99 14.72 4.5 0.52
9.40 9.40 0.5 0.006
10.60 8.34 0.5 0.006
11.90 7.34 2.0 0.023
12.89 6.86 1.0 0.012
14.70 6.02 86.0 1.000
15.56 5.69 1.0 0.012
16.37 5.41 8.0 0.093
17.20 5.15 4.0 0.047
17.69 5.01 22.0 0.256
19.07 4.65 4.3 0.050
Figure 5. Powder X-Ray Diffraction Pattern of Hydrocortisone (USP Reference
Standard). Instrument: Norelco
HYDROCORTISONE 293

20 d (8) I
- 1/10
19. 80 4.48 4.5 0.052
20.49 4.33 2.0 0.023
20.98 4.23 1.0 0.012
23.52 3.78 2.5 0.029
24.50 3.63 0.5 0. 006
27.59 3.23 1.5 0.017

4.3 Solution Properties


4.31 Solubilitv
According to Clarke,48 hydrocortisone
has the following solubilities:
%

Water 0.029 (1 in 3 , 5 0 0 )
Ethano1 2.5 (1 in 4 0 )
Acetone 1.25 (1 in 8 0 )
Propylene glycol 1.0 (1 in 100)
Slightly soluble in chloroform, almost
insoluble in ether.
The solubility/tem erature profile
in ethanol ranges from 6.3% at 65' to 1 . 5 % at 25°.49
The solubility in water is increased by the addition
of surfactants such as Tween 20 ( 0 . 0 2 7 mol. of
steroid/mol. of Tween 20) .50 The solubility in
tris-buffered KC1 was found to be 161.0 1-1 molesfi
and,in egg lecithin liquid crystals, 1 5 . 5 n moles/
P mol. lipid.51 The solubilization of hydro-
cortisone and other steroids by long-chain polyoxy-
ethylene surfactantsS2 as well as in polyethylene-
glycol fatty acid esters53 has been studied.
Calculations on the dissolution of
hydrocortisone have been presented in connection
with comparing the measurement of the interfacial
energy between solid particles and a dissolving
solvent to the film theory of dissolution in which
diffusion is the principal mechanism.54 A non-sink
dissolution rate equation has also been applied to
hydrocortisone.55
4.32 Partition Coefficients, Diffusion
An extensive list of partition co-
294 KLAUS FLOREY

efficients (Table 3) were presented by Engel and


Carter. 5
For partition coefficients in
surfactants and solubilizers, see references 52
and 53 (Section 4.31). An estimation of hydro-
cortisone partition coefficients in ether:water
based upon molecular constitution has been
attempted.57 The absorption kinetics of hydro-
cortisone at the water-neutral oil interface has
been studiedI5*using the Gibbs equation59 and
verifying the Szyskowski law.60 The effect of
surfactants on the diffusion of hydrocortisone
across a cellulose acetate membrane has been
investigated.
Optical Rotation
4.33
The following values have been
reported:
Reference
( a )Do temp.
+167 (abs. ethanol) 22 20
+16 3O (methanol) 22 20
+150-15 6O (dioxane) 20 ,25 32,33 ,48
+162 24
Optical rotatory dispersion studies
were conducted by Foltz, Lippman and D J e r a ~ s i . ~ ~
The following values were obtained:
(a)700 + 113O; (a)589 + 162O; (a)300 +
12020; "max" (a)385 + 4560; "min" (a)367,5 +
312O; Ilmaxll(a1357.5 + 460°; 'lmin" (a!352,5 +
428O; "max" (a)315 + 3506O; c = 0.10 in dioxane;
temp. 23-25. Drude equation: (MI = 172/A2-
0.0551; A, 235 nm; % deviation (M) Obsd -
(M) calcd: -+ 1.0%; 620-410 nm; -+ 3.7%, 700-400
nm.
5. Methods of Analysis
5.1Historical Synopsis
As with all hormones which are present in
biological systems only in minute quantities, an
assay system, usually a biological one, is needed
as a guide for their detection and isolation. In
the early work leading to isolation of hydro-
cortisone and other corticosteroids from adrenal
HYDROCORTISONE 295

TABLE 3
Partition Coefficients for Hydrocortisone
(from reference 56)

1. Benzene/water 0.36
2. Benzene/50% methanol in water 0.14
3. Benzene/50% methanol in water 0.31
4. Petroleum ether/35% ethanol in water <o. 02
5. 25% Sec. butanol in n-hexane/water 0.24
6. 35% Sec. butanol in n-hexane/water 0.84
7. 25% Ethyl acetate in n-hexane/water 0.04
8. 50% Ethyl acetate in n-hexane/water 1.00
9. 30% Ethyl acetate in cyclohexane/30% ethanol
in water 0.31
10. 30% Ethyl acetate in cyclohexane/50% ethanol
in water 0.08
11. 30% Ethyl acetate in cyclohexane/70% ethanol
in water 0.04
12. 50% Ethyl acetate in cyclohexane/30% ethanol
in water 0.96
13. 50% Ethyl acetate in cyclohexane/50% ethanol
in water 0.39
14. 50% Ethyl acetate in cyclohexane/70% ethanol
in water 0.16
15. 70% Ethyl acetate in cyclohexane/30% ethanol
in water 2.7
16. 70% Ethyl acetate in cyclohexane/50% ethanol
in water 1.2
17. 70% Ethyl acetate in cyclohexane/70% ethanol
in water 0.02
18. Ethyl acetate/water 12.2
19. Water/carbon tetrachloride 0.16
20. 30% Methanol in water/70% chloroform in carbon
tetrachloride 0.70
21. 50% Methanol in water/50% chloroform in carbon
tetrachloride 2.6
22. 70% Methanol in water/30% chloroform in carbon
tetrachloride 5.9
23. 30% Ethanol in water/carbon tetrachloride 0.06
24. 0.05% NaCl in water/50% n-hexane in chloroform 2.57
25. 20% Ethanol in water/50% n-hexane in chloroform 2.57
26. 30% Ethanol in water/chloroform 0.06
27. 30% Ethanol in water/50% n-hexane in chloroform 1.63
28. 50% Ethanol in water/50% n-hexane in chloroform 0.61
29. 0.1N Acetate buffer/methylene chloride 0.12
30. 20% Methanol in water/50% n-hexane in chloroform 4.0
31. 80% Methanol in water/l.2-dichlvroethane 1.26
2% KLAUS FLOREY

tissue, the response of adrenalectomized animals


was used. Later on, corticosteroids were classi-
fied according to their relative activity in
promoting deposition of glycogen in the liver and
in controlling electrolyte balance in body fluids.
Hydrocortisone was found to have the highest gluco-
corticoid activitv and to be rather inactive
in controlling the electrolyte balance (cf. ref. 1,
p. 604).
When cortisone and hydrocortisone found
therapeutic applications, the Porter-Silber method62
permitted insight in the concentration of steroids
with the dihydroxyacetone side chain in biological
fluids. The blue tetrazolium method of blader and
provided a convenient and stability indica-
ting control procedure of these steroids in pharma-
ceutical preparations. Z a f f a r ~ n iapplied
~~ the
well-known color reaction of steroids with sulfuric
acid to hydrocortisone.
Paper chromatography, as an analytical
tool for steroids, was introduced in the fifties by
Zaffaroni and Bush, TLC and GLC followed in the
sixties, and HPLC in the seventies. For background
and discussion of methods, three excellent recent
books on steroid analysis are available. 2 ~ 3 ~ 6An
5
older book on pharmaceutical analysis also includes
a chapter on steroid analysis.21
5.2 Elemental Analysis
The elemental composition is as follows:20
C:69.59%; H:8.34%; 0:22.07%

5.3 Ultraviolet
The stronq abscmption band in the ultra-
violet ( A max 242 nm; Ei 445, see section 4.12) has
been used for quantitation of hydrocortisone itself
(cf. 21), but due to the interference of excipients,
it rarely can be used in the quantitative determi-
nation of dosage forms without difficulties. The
sodium borohydride method of GorZjg66 overcomes this
obstacle by using the reduced solution as a blank
in the reference cell. Kir~chbaurn~~ found that
addition of propylene glycol assures complete
reduction.
HYDROCORTISONE 297

5.4 Colorimetric
5.41 Sulfuric Acid
It was already known in the thirties
that steroids give color reactions with concentrated
sulfuric acid (cf. 3, p. 1 8 2 ) . A s already mentioned,
Z a f f a r ~ n iwas
~ ~ the first to apply it to hydro-
cortisone. He found absorption maxima at 2 8 0 , 3 9 5
and 4 7 5 mp. My personal prejudice concerning this
method coincides with that of Gorog and S z z z , who
....
state (cf. 3, p. 1 8 2 ) : I' spectra are not
reproducible as well as in the common solvents: the
positions and intensities of the maxima depend on
the time elapsed between dissolution of the steroid
and the recording of the spectrum, the temperature,
the quality of the sulfuric acid, the purity of the
sample, etc." The method today seems to be only of
historical interest, and I refer the reader to the
classical paper by Bernstein and Lenhard6* and the
chapter by Smith and Bernstein.69
They present the following data for
hydrocortisone:
1%
max (nm) Elcm
237 420
2 82 462
391 325
475 1 53

Sulfuric acid has also been used


diluted with other solvents, but rarely water. A
mixture of glacial acetic acid and concentrated
sulfuric acid ( 4 : 6 ) was found to give an absorption
maximum at 4 7 0 nm with hydrocortisone but not
cortisone.7 0
When hydrocortisone was heated in
sulfuric acid and treated with phosphorus
pentoxide, it gave an absorption maximum at 5 4 8 nm
(optical density 0 . 6 2 5 ) . 7 1
5.42 Other Acids
Hydrocortisone in orthophosphoric
acid (cf. 3, p. 1 8 6 ) gives spectra very similar to
sulfuric acid. The U.V. absorption maximum of
hydrocortisone in 7 2 % perchloric acid has been
given as 2 8 3 nm.72 Concentrated hydrochloric acid
has great specificity for the 116-hydroxyl group.
298 KLAUS FLOREY

Hydrocortisone gives a sharp, intense maximum at


465 nm in contrast to lla-h droxy, 11-keto, or
11-unsubstituted steroids.7 3

5.43 Porter-Silber
The principle of the Porter-Silber
method62 is the reaction of the dihydroxyacetone
side chain with phenylhydrazine in methanolic
sulfuric acid to produce a stable yellow color with
a maximum at 410 nm. This reaction is specific for
17-hydroxy steroids, and the sensitivity of the
method permitted its use to determine glucocorti-
costeroids in biological fluids. It was first
applied in 1952 to determine h y d r o c ~ r t i s o n ein
~~
urine and in 1957 to plasma75. With the advent of
chromatographic and RIA techniques, the method
may no longer have the importance it once had, but
it still is described in a modern (1975) book on
the determination of hormones.76
5.44Tetrazolium
The tetrazolium methods still belonq-
to the most important analytical techniques to
determine corticosteroids as such and in pharma-
ceutical preparations. For an excellent discussion
of mechanism and history, see reference 3 , p. 331.
It was first used for hydrocortisone as a spray
reagent for paper chromatography by Burton,
Zaffaroni and Keutman.77 Mader and Buck are
credited with extending the use of tetrazolium to
pharmaceutical steroid analysis but did not
describe the application to hydrocortisone in
their original paper.63 Hydrocortisone was first
quantitated by Henley,78 using 2-iodophenyl-3-
nitrophenyl-phenyl tetrazolium chloride. The USP33
favors the use of blue tetrazolium (B.T.; diani-
sole-bis-diphenyl tetrazolium chloride), the B.P.32
the use of triphenyl tetrazolium chloride (TZ) for
the determination of hydrocortisone and its dosage
forms. Absorbance maxima for the two variants are
at 525 nm and at 485 nm, respectively. The tetra-
zolium method is stability indicating, albeit an
indirect one measuring the reducing capacity of
the steroid side chain to form formazans. The
blue tetrazolium method has also been automated
for the assay of hydrocortisone in tablet^.^^^,^^
HYDROCORTISONE 299

5.45 Miscellaneous
For the determination of the free
21-hydroxy group of hydrocortisone in the presence
of its acetate,oxidation of the side chain with
cupric acetate to the glyoxal and condensation
with 0-phenylenediamine, (A max 331 nm, E 10,200) or
its 4,5-dimethyl derivative ( A max 351 nm, E 12,700)
have been used.80 Condensation with phenylhydrazine
gives an absorption maximum at 364 nm ( E = 19,000)
which has been used for the determination of hydro-
cortisone in tablets.81 For the determination of
residual hydrocortisone in prednisolone, the rate of
thiosemicarbazone formation was determined at
3 0 2 nm.82 A l s o used were reactions with Dische 83
reagent (diphenylamine in acetic and sulfuric acid),
bismuth oxidation,84 condensation with isonicotinic
acid hydrazide (370 nmIa5 also automated for
tabletsa6 and reaction with ammonium molybdate
(A max 655 nm) . 8 7
5.5 Fluorescence
Although hydrocortisone has no native
fluorescence, it can be induced by treatment with
concentrated sulfuric acid. This was already
observed by Wintersteiner and Pfiffners8 in 1936.
The various conditions of acid concentration and
temperature leading to slight variations of the
excitation wavelength (470 nm) and absorption
maximum (530 nm) as well as intensity of fluores-
cence are described by Goldzieher.89 The great
sensitivity of the reaction made it an ideal tool
to study the concentration of hydrocortisone in
biological fluids and tissues in a quantitative
fashion. It was first explored by Sweatgo in 1954.
He was able to determine as little as 5 nanograms
of hydrocortisone in 1 ml of ethanolic sulfuric
acid. The early work on the fluorimetric analysis
in several laboratories has been reviewed by
Silber.gl Phosphoric acid has also been used to
induce fluorescence,92r93as have been acetic acid-
antimony t r i ~ h l o r i d e ,aluminum
~~ salt and isonico-
tinylhydrazine (Ex.: 380 nm; Em: 495 nrnlg4 and
lithium hydroxide pellets (Ex.: 396/Em: 510) .95
A fluorescing derivative, amenable to TLC,96 has
been obtained by reactions with 4(6-methylbenzo-
thiazol-2yl) phenyl isocyanate.
300 KLAUS FLOREY

5.6Polarographic
Rased on the reduction of the 3-carbonyl
-
function, the polarographic reduction of hydro-
cortisone has been studied in well-buffered 50%
ethanol solutions.97 The halfwave potential ( E % )
was found to be dependent on pH, ranging from -1.26
for pH 2.8 to -1.74 for pH 10.8. A halfwave
potential of -1.50 was found in 90% ethanol at
pH 8.5.98 In DMF buffered at pH 9.15, a halfwave
potential of -1.64V was found.99 In acetonitrile-
tetrabutylammonium perchlorate, a halfwave potential
of -1.58 was found. The approximate detection limit
was determined as 3~10'~moles/liter . Polar-
ography of hydrocortisone has also been performed
with prior derivatization with Girard's reagent T
exhibiting a halfwave potential of 1.12lo1 and
betainyl hydrazine (E$ -1.42).98 Polarography has
been used for the determination of hydrocortisone
in ointments,99r102human bloodlol (see sections 8
and 9), and as a microbial conversion product after
TLC separations.lo3
5.7Isotope Dilution
Preparation of tritiated hydrocortisone
and its detection in fermentation brothlo4 and
adrenal sliceslo5 has been described.
5.8Chromatographic Methods
5.81 Paper
With the advent of TLC and HPLC, the
importance of paper chromatographic methods to
determine hydrocortisone declined steeply. It now
is only of historical interest. Yet, in the fifties
it was successfully used to enlarge the knowledge of
the distribution of hydrocortisone in body fluids
and tissues and to assay its purity, as well as
stability in dosage forms.
Both the Bushlo6 and Z a f f a r ~ n i ~ ~
type of chromatographic systems have been used for
hydrocortisone,and a typical sam ling is presented
in Table 4 according to Neher. o y Additional
systems can be found in references 108, 109, and
110. The use of fully acetylated paper,lll paper
impregnated with stearato chromic chloride for
reverse phase as well as centri-
fugal acceleration113has been applied to hydro-
cortisone.
HYDROCORTISONE 301

TABLE 4
Paper Chromatographic Systemslo7
Zaffaroni Type Systems:
Propylene/toluene 0.01
Formamide/chloroform 0.24
Formamide/ethyl acetate-butyl acetate-
water (15:85:1) 0.40
Formamide/butyl acetate:water (100:5) 0.42
Formamide/n-butanol-butyl acetate-
water (15:85:5) 0.60
Bush Type Systems:
Hexane-tert. butanol-methanol-water 0.19
Benzene-methanol-water (100:50:50) 0.25
Cyclohexane-dioxane-methanol-water
(4:4:2:1) 0.25
Toluene-ethyl acetate-methanol-
water (9O:lO: 50: 50) 0.37

For detection and quantitation,


ultraviolet light,ll1,ll4 blue tetrazolium,77
diphenylstyryl phenyltetrazolium chloride,l16 and
alkaline fluorescence117 have been used. Quantita-
tion of hydrocortisone by direct photometry of
paper chromatograms, using an adapter in a Beckman
DU spectrophotometer has been reported.ll* For
preparative paper chromatography to separate hydro-
cortisone from other adrenal constituents, see
reference 119.
5.82 Thin-Layer
Thin-layer chromatography started to
be applied to hydrocortisone in 1960. The two
earliest publications specifically mentionin hydro-
cortisone seem to be those b Carr and ReddyqPo and
by Cerny, Joska and Labler. 1 x 1 TLC is still an
important method for hydrocortisone. Until 1981,
the USP assay32 for hydrocortisone was based on
quantitative TLC,and the BP33 uses TLC for the
related steroid test. For general references, also
concerning quantitation, see references 3 , 107 and
110. Some typical systems have been summarized in
Table 5. While ultraviolet, blue tetrazolium and
sulfuric acid are employed as detection agents in
TABLE 5

SUPPORT SOLVENT SYSTEM Rf DETECTION REF.


-
Cellulose acetate Water-tert.butano1-pet.ether (2:1:3) - Fluorometric 120
Silica Gel Benzene-ethyl acetate (1:l) 0.22-0.36 Sulfuric acid 121
Celite Formamide/benzene-chloroform 0.13 Sulfuric acid, 125
(1:l) blue tetrazolium,
isonicotinic acid,
hydrazide
Silica Gel G Chloroform-methanol-water (188:12 :1) Sulfuric acid 126
Ethyl acetate-chloroform-water Ultraviolet
(90 :10 :1)
Silica Gel G Hexane-ethyl acetate (1:1) 0.06 Phosphomo1ybdic 127
(starch bound) Ethyl acetate 0.38 acid (10%in
Benzene-isopropanol (4:1) 0.55 ethanol)
Alumina
(acetic acid treated) Benzene-dioxane ( 2 : l ) 0.25 Ultraviolet 128
Benzene-dimethylformamide (9:1) 0.53
Chloroform-ethanol (96:4) 0.35
Diisopropylether 0.25
Silica Gel G Dichloroethane-methyl acetate- Diphenyl (styryl- 129
water (2:l:l) phenyl)-tetrazolium
Silica Gel G Chloroform 0.43 Toluene sulfonic acid 130
(impregnated with Methylene chloride-toluene (1:1! 0.10
formamide
TABLE 5 (Cont'd)

SUPPORT SOLVENT SYSTEM Rf DETECTION REF.


-
-
polyamide Resin Chloroform-ethanol (97:3) 0.50 Blue tetrazolium 131
Methylene chloride 0.23

Silica Gel G Cyclohexane-ethyl acetate- 0.23 Potassium periodate 132


ethanol (4.5 :4.5 :1) formaldehydog enic "
I'

Chloroform-ethanol (9:1) 0.18


Ethyl acetate-n-hexane-acetic
acid-ethanol (72:13.5:10:4.5) 0.44
Benzene-ethanol (4:1) 0.37
Silica Gel Butyl acetate-methanol (99:l) 0.48 Ultraviolet 107
W
W
0 Chloroform-methanol (9:1) 0.42 Ultraviolet
304 KLAUS FLOREY

most cases, Lisboa122 has described quite a number


of other "in situ" color reactions. Attention has
also been paid to the elution and uantitation of
steroids after chromatography. I g2 I Nanogram
amounts have been determined, and best recovery
was achieved by elution with methan01.l~~
A system for the separation of
hydrocortisone from its 17-keto oxidation product
has been described.133 Densitometry has also been
used for quantitation.134,135r136
5.83 Column
In the early years, column and
partition chromatography was used extensively for
the separation of hydrocortisone from adrenal gland
mixtures138 and the determination in blood.lol For
general reviews, see references 2, 3 and 119.
Diatomaceous earth (celite, superce1)101r138r139
silica ge1,140-143sephadex LH-20,144 G-50145 and
diatomaceous earth treated with a ~ e t o n i t r i l e l ~ ~
have been used as adsorbents with various solvent
mixtures.
5.84 High Performance Liquid
Chromatography (HPLC)
HPLC has swept the field of
pharmaceutical analysis. Shall wonder then that
HPLC is also rapidly replacing older chromato-
graphic methods for the control of the purity of
hydrocortisone and for its determination in form-
ulation and biological fluids (see section 8 and 9).
General reviews can be found in reviews by
Vestergaard,2 Di,! Smith,147 O'Hare and Nice,147
and Gorog and Szasz.3 The first reported use of
HPLC for h drocortisone seems to be by Siggia and
Dishman. 1 4 g Some typical data are summarized in
Table 6. A proposed selected method for the
analysis of hydrocortisone in serum has been
described.149
5.85 Gas Chromatography
A description of early attempts on
gas chromatography of hydrocortisone-can be-found ,
in the book b Wotiz and Clark.163 VandenHeuvel
and Horning16< already found in 1960 that gas
chromatography of underivatized hydrocortisone and
other steroids with the dihydroxy acetone side
TABLE 6
HPLC SYSTEMS

MOBILE PHASE COLUMN DETECTION -


REF.

Water and methanol, water (3:7) Trifluoroethylene beads coated Ultraviolet 148
with Amberlite LA-1
Ethyl acetate ( 5 % ), acetonitrile Zipax coated with oxydipropionitrile Ultraviolet 150
(0.2%) in hexane
1%Methanol in water Cyano ethyl silicone Ultraviolet 151

Chloroform, dioxane (20:l) Silica gel Ultraviolet 152

Ethylene chloride, ethanol, water Zorbax-Sil Ultraviolet 153

Aqueous ethanol with tetrapentyl Silica coated with octadecyl silane Ultraviolet 154
ammonium chloride
1.5% Methanol and 0.2% water Silica gel Ultraviolet 155
in chloroform
Methanol, water, acetic acid c18 Bondapak Ultraviolet 156
(53:42: 5)
Methylene chloride, methanol, Silica gel Ultraviolet 157
tetrahydrofuran, acetic acid
Acetonitrile, water, glacial ODS Ultraviolet 158
acetic acid (38:60:2)
Methanol-O.OlM ammonium acetate c18 Bondapak (gradient elution) Ultraviolet 159
pH 6.9 (10:90)
60% aqueous methanol c18 Lichrosorb Ultraviolet 160
TABLE 6 (Cont'd)

MOBILE PHASE COLUMN DETECTION REF.


-
*Ethylene dichloride, butanol, water Fluorescence 161
(91: 8 . 5 : 0.5)
Butyl chloride, tetrahydrofuran, s lica gel Ultraviolet 33
methanol, glacial acetic acid
(190:14:7:6)
Acetonitrile, methanol, water Silica coated with octadecylsilane Ultraviolet 33
(2:2:6)
Acetonitrile (20%) in 0.01M Lichrosorb RP-8 Ultraviolet 162
phosphate buffer pH 6 containing
0.01M tetrabutyl ammonium bromide
w
0
ch
dansyl hydrazine derivative

chain eliminated the side chain during vaporization and that the retention time
was that of the corresponding 17-keto compound. The main use of gas chromato-
graphy has been found in the determination of hydrocortisone in biological
fluids where it is competing with radioimmunoassays. Recently, it has been
coupled with mass spectrometry.165 GC/MS and RIA methods have been
Some typical data are presented in Table 7.
5.9 Electrophoretic
Hydrocortisone, being neutral, should not be moved by electrophoresis.
However, there is a report that hydrocortisone migrated in pH 7.0 phosphate buff-
er and pH 10 bicarbonate buffer when subjected to high voltage electroph~resis?~~
TABLE 7
GAS CHROMATOGRAPHIC SYSTEM
COLUMN
DERIVATIZATION COLUMN PACKINGS TEMP. DETECTOR REF.
-
- Silicone SE-30 on 222 Flame ionization 164
Chromosorb W
Trimethylsilyl ether 3% dimethylpolysiloxane - Flame ionization 167
on Gas Chrom. P
Methoxime-trimethylsilyl ether 1% SE-30 on Gas Chrom. P 250 Flame ionization 168
Methoxime-trimethylsilyl ether 3% OV-1 Diatomite CQ 240 Flame ionization 169
Periodate oxidation to SE-30 250 Flame ionization 170
w
eticholenic acid
0
rl llfksilylether and 17,21 1% OV-1 on Chromosorb G 240 Flame ionization 171
cyclic dimethyl
s iliconide
Trimethylsilyl ether-enol- 1% OV-1 on Gas Chrom. P 250 Flame ionization 172
trimethylsilyl ether
Chromium trioxi.de oxidation to 3% OV-17 on Chromosorb W 230 Electron capture 173
androstenetrione and forma-
tion of heptofluorobutyrate
3.20 Dimethoxime-11,17,21 tri- 3% SE-30 - Mass fragmentography 165
methylsilyl ether at m/e 636; m/e 638
(14C-hydrocortisone)
308 KLAUS FLOREY

5.10Bioassay-Enzymatic
The original workers, isolating hydro-
cortisone and other adrenal hormones from adrenal
tissue, used adrenalectomized animals and later on
such tests as the liver glycogen, e ~ s i n o p h i l , ~ ~ ~
thymus involution' and electrolyte-excret-
ing177 (cf. 1, 600ff). The enzyme 20-B-hydroxy
steroid dehydrogenase coupled with DPNH has also
been used to determine hydrocortisone in blood
plasma.178
5.11Saturation Analysis
The great importance of the accurate
determination of hydrocortisone body fluid levels
in normal and diseased states has led to the
development of the following techniques which can
be classified as saturation analysis.' Each of the
techniques has its proponents and pitfalls,179 but
at present, radioimmunoassay seems to be the most
useful.
5.111
Competitive Protein Binding
A review of this technique can be
found in the chapter by W. R. Slaunwhite, Jr. in
reference 2. Murphy first employed this technique
to study hydrocortisone levels in plasma and other
body fluids.lso,lB1 The competition of hydro-
cortisone in the plasma sample with added hydro-
cortisone-4-C14 for corticosteroid-binding globulin
(a1 fraction) is the basis of the method. The
method was extended to urine and the purification
prior to assay refined.l*' TLC has been used for
prepurification.183 Tritiated instead of carbon-14
labelled hydrocortisone has also been used.178
Horse transcortin was used to measure hydrocort-
isone in umbilical cord serum and amniotic f 1 ~ i d . l ~ ~
A commercial kit has been described.186 Ultra-
centrifugation was used to determine unbound
hydrocortisone.187
5.112 Double Isotope Derivative Assay
The double isotope derivative
assay is a modification of competitive protein
binding analysis where a trace amount of tritiated
hydrocortisone is added to determine recovery
values durin extraction. In a comparison of the
two methods,q 8 8 it was found that generally there
is good agreement; however, a large discrepancy
HYDROCORTISONE 309

was found in the values obtained in newborns and


patients with adrenogenital syndrome, where
competitive protein binding gives much higher
values, probably due to the presence of related
steroids competing with hydrocortisone.
5.113Radioimmunoassa
Ruder, Guy and ;ipsettlsg are
credited with the first description of a radio-
immunoassay for hydrocortisone in 1972, but others
were also hard at work. That this technique
offered advantages as to higher sensitivity and
greater specificity over protein-binding methods is
attested by the mushrooming literature which here
is reviewed very selectively. Brief reviews are
those by G. E. Abraham in references 2 and 192; a
detailed review is that of P. Vecsei in reference
193. Antigens used for immunization were the 21-
hemisuccinate, the 3-oxime-21-acetate, the 3-oxime,
the 3,20-dioxime and the 6a- or 66-hemisuccinoxy
derivatives to obtain antibody titers in sheep and
rabbits. These antibodies had varying percenta es
of cross reactivity to related steroids. 3H, 7zSe
and 125I-ligands are used, but in one comparison
study,lq4 tritiated hydrocortisone was found to
have the best sensitivity. Most laboratories use
dextran coated charcoal to separate protein-bound
and unbound radioactivity. The usual solvents for
extraction of plasma hydrocortisone are alcohol
and dichloromethane. Comparison of the radioimmuno-
assay with chemical ionization/mass spectrometry166
and with HPLCIq5 have been reported. An inter-
laboratory evaluation of four RIA kits has been
made.lg6 An automated assay method has been
evaluated.
5.114Other
A chemiluminescent immunoassaylg8
has been described. Enzyme-labelled immunoassays,
using alkaline phosphataselg9 and E. coli B-galac-
tosidase200 have also been developed.
6. Stability - Degradation
Hydrocortisone is very stable as a solid. In
aqueous and alcoholic solution, the dihydroxy-
acetone side chain, as in all such corticosteroids,
is prone to oxidative rearrangement and degradation
at very acid and particularly also alkaline pH's.
310 KLAUS FLOREY

For instance, it was reported in 1967201 that


hydro~ortisone-4-~~C in aqueous or methanolic
solution decomposed to products of lesser and high-
er polarity. The main decomposition product was
identified as 118-h droxyandrost-4-ene-3,17 dione.
In another study,20Y it was found that aqueous
solutions of hydrocortisone were spontaneously
oxidized to 21-dehydrocortisol and other products.
In pH 9.1 carbonate buffer, the conversion rate was
1.6-2.8%/hr. Addition of EDTA or high concentra-
tions of plasma or plasma protein fractions slowed
the degradation.
Recently, Hansen and Bundgaard162 have studied
the degradation of hydrocortisone in aqueous
solution in detail. They identified degradation
products by HPLC (see Section 5.84) and treated the
data kinetically. They confirmed results of
previous investigators, partially obtained with
other corticosteroids with a dihydroxyacetone side
chain. They identified the glycolic acid V, not
previously reported. Generally, they found
degradation patterns to be more complex in basic
solutions. Their results are summarized in
Figure 6.
The photolytic degradation of hydrocortisone in
alcoholic solutions when exposed to fluorescent
lighting was found to be first order. The half-
life of such a solution at room temperature was
found to be 160 days. The degradation involved the
A-ring of the molecule, as measured by a decrease
in U.V. absorption, but not the side chain as
determined by blue tetrazolium. No degradation
products were described.203
7. Metabolic Products - Pharmacokinetics
The metabolism of hydrocortisone is complicated
since it can be different both qualitatively and
quantitatively in health or disease. Much pioneer-
ing work has been done with and without the use of
14C-labeled hydrocortisone in isolated liver204 and
kidney205 in intact rats,206 and in man.207,208
However, the definitive quantitation study in
normal man is that of Fukushima, Bradlow, Hellman,
Zumoff and Gallagher,209 which is summarized in
Figure 7. It was found that 9 0 % of the radio-
activity in the neutral steroid extract would be
HYDROCORTISONE 311

HC=O c=o
I I
C-OH c=o

YT- IX
(minor)
IV
(major)

21 C H 2 0 H
IV
I (minor)
20 c=o

c=o
I
c=o

b'"
I11

I11
COOH
I (intermediate)
I
I
0 CHOH

+ y1 + others
C CHOH
VIII VI . ..OH
(major) (major) (minor) + others

(major) (major) (minor)

Fig. 6. Degradation pathways of hydrocortisone in aqueous


solution. The numbering is that of reference 162.
All oxidative pathways in neutral and alkaline
solution are metal (copper) catalyzed and can be
blocked by EDTA.
312 KLAUS FLOREY

CH20H CH20H
I I
c-0 c-0

68-Hydroxyhydrocortisone Cortisone

CH20H
l
Hydrocortisone

HO”

8
Hd
R-OH Tetrahydrocortisol; Allotetrahydrocortisol
R-0 Tetrahydrocortisonei Rllotetrahydrocortisona

CH20H
I OH HO&.
CHOH

’ __+

o*
HO‘ H HO’
li
20 o+R - Dihydrocortisol R-OH 20 o+B -- cortol 20 o+E -- allocortol
J R=O 20 a+B cortolone 20 a+B allocortolone

1
HO‘ @
116-Hydroxyetiocholanolone
+ HA .(y.p
;I
118-hydroxy androsterone
Gluconurides

Figure 7. Metabolic Pathways of Hydrocortisone.


HYDROCORTISONE 313

accounted for by the compounds depicted in Fig. 7,


and 70% were recovered in urine as the glucuronides.
By far, the largest fractions were the tetrahydro
derivatives both of hydrocortisone and cortisone
with the side chain intact, followed by the
cortolones and cortols. The formation of 6B-
hydroxycortisone and 17-keto compounds was found
to be minor.
Recently, the pharmacokinetics of orall
The
administered hydrocortisone was described. l X 0
mean elimination half-life of hydrocortisone from
plasma was found to be about 9 0 minutes.
Detailed reviews on the subject are available
available. I 210 I 2 2 0
8. Determination in Biological Fluids and Tissues
Since hydrocortisone is a hormone, determina-
tion in bioiogical fluids and tissues-has been of
particular importance, starting with the animal
assays leading to its isolation (cf. ref. 1,
600ff). A perusal of the general reviews,147
I I I mentioned in the preceding pages
may be useful. The following is a compilation of
references, most of them grouped according to
fluid or tissue:
Blood (Plasma, Serum) :
Porter-Silber: 7 5 , 7 6
Fluorometric: 91, 1 4 2 , 2 1 1 , 212
Paper Chromatography: 1 0 6 , 117
Polarographic: 1 0 1
HPLC: 1 6 1 , 1 4 9 , 1 5 7 , 1 5 6 , 1 5 3
Gas Chromatography: 173
Gas Chromatography/Mass Spectrometry:
165, 198
Competetive Protein Binding: 1 7 9 , 180,
181, 183, 186, 187, 219
Radioimmunoassay: 1 6 0 , 1 6 6 , 1 8 9 , 197
Chemiluminescent 1 A : 1 9 8
Enzyme 1 A : 1 9 9 , 2 0 0
Enzymatic: 1 7 8
Urine :
Porter-Silber: 74
Fluorometric: 213
314 KLAUS FLOREY

Paper Chromatography: 109, 207, 209, 214


TLC: 215, 216
HPLC: 155
Compet. Protein Binding: 181, 182, 184
Radioimmunoassay: 195
Reverse Isotope Dilution: 209
Adrenals :
Biological assays: cf. 1, p. 600ff
Paper Chromatography: 118, 217
Amniotic Fluid:
Compet. Protein Binding: 185
Liquid Chromatography: 40
Milk :
TLC: 137
Cerebral Spinal Fluid:
Compet. Protein Binding: 181
9. Determination in Pharmaceutical Formulation
The following tabulation highlights the methods
referenced previously which have been used in the
assay of pharmaceutical formulations:
Tablets:
Spectrophotometric (isonicotinic acid
hydrazide) : 78
Automated colorimetric: 79, 86, 176
Spectrophotometric (ammonium molybdate); 87
Tetrazolium: 33, 115
Paper Chromatography: 115
TLC: 33
Column: 146
HPLC: 33
Topicals:
Blue Tetrazolium: 33
Spectrophotometric (cupric acid ox.): 81
Polarography: 99, 102
TLC: 124, 136
Column Partition: 138
HPLC: 33, 147, 150
HYDROCORTISONE 315

Parenteral:
Blue Tetrazolium: 33
HPLC: 1 5 8

References
1. L.F. Fieser and M. Fieser, Steroids, Reinhold
Publishing Corporation ( 1 9 5 9 ) .
2. Modern Methods of Steroid Analysis,
E. Heftman, ed., Academic Press ( 1 9 7 3 ) .
3. I.S. G6rog and G . Sza'sz, Analysis of Steroid
Hormone Drugs, Elsevier ( 1 9 7 8 ) .
4. T. Reichstein, Helv. Chim. Acta, - 20, 9 5 3
(1937).
5. P.S. Hench, E.C. Kendall, C.H. Slocomb and
H.F. Polley, Proc. Staff Meetings, Mayo Clinic,
-
24, 1 8 1 (1949).
6. T. Reichstein and C.W. Shoppee, Vitamins and
Hormones, 1, 3 4 5 ( 1 9 4 3 ) .
7. N. L. Wendier, R.P. Graeber, R.E. Jones and
M. Tishler, J. Am. Chem. SOC., - 72, 5 7 9 3 ( 1 9 5 0 ) .
8. L.H. Sarett, J. Am. Chem. SOC., - 70, 1 4 5 4 (1948).
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10. S.H. Eppstein, P.D.-Meister, H.C. Murray and
D.H. Peterson, Vitamins and Hormones XIV, 3 5 9 ,
Academic Press ( 1 9 5 6 ) .
11. M. Sittig, Pharmaceutical Manufacturing
Encyclopedia, Noyes Data Corp. ( 1 9 7 9 ) .
12. D. Riad-Fahmy, G. Read and I.A. Hughes,
Hormones in Blood, Vol. 3, p. 1 8 0 , C.H. Gray
and V.H.T. James, editors, Academic Press
(1979).
13. 0. Hechter and G . Pincus, Physiol. Rev., -
34,
459 (1954).
14. L.T. Samuels and T. Uchikawa in Adrenal Cortex,
p. 6 1 , Little, Brown and Co. , Boston ( 1 9 6 7 ) .
15. E. Ileftmann, Steroid Biochemistry, Academic
Press, New York ( 1 9 7 0 ) .
16. D.R. Collingsworth, I.N. Karnemaat,
F.R. Hanson, M.P. Brunner. 1 C . M . Mann and
W.J. Haines, J. Biol. Chem., - 203, 807 ( 1 9 5 3 ) .
17. A.M. Hayden, Anal. Chem., 27, 1 4 8 6 ( 1 9 5 5 ) .
18. K. Dobriner, E.R. Katzenellenbogen and
R.N. Jones, Infrared Absorption Spectra of
Steroids, Interscience, New York ( 1 9 5 3 ) .
19. R.J. Mesley, Spectrochimica Acta, - 22, 8 8 9
(1966).
316 KLAUS FLOREY

20. The Merck Index, Ninth Edition, Merck and Co.,


Rahway, N.J. (1976).
21. A.A. Forist and J.L. Johnson, Pharmaceutical
Analysis, p . 69, T. Higuchi and E. Brochmann-
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METOPROLOL TARTRATE
James R. Luch

1. Description 326
1.1 Introduction 326
1.2 Formula, Name, Formula Weight 326
1.3 Appearance, Color, Odor 326
2. Physical Properties 326
2.1 Ultraviolet Absorption Spectrum 326
2.2 Infrared Absorption Spectrum 328
2.3 Proton Nuclear Magnetic Resonance Spectrum 330
2.4 Carbon-13 Nuclear Magnetic Resonance Spectrum 330
2.5 Mass Spectrum 334
2.6 Fluorescence Spectrum 334
2.7 Optical Rotation 337
2.8 Melting Range 337
2.9 Differential Scanning Calorimetry 337
2.10 Thermogravimetric Analysis 337
2.11 X-Ray Diffraction 337
2.12 Dissociation Constant 339
2.13 Solubility 339
2.14 Water Absorption Isotherm 339
2.15 Distribution Ratio 34 1
3. Synthesis 34 1
4. Stability 342
4.1 Solid State Stability 342
4.2 Solution Stability 342
5. Pharmacokineticsand Metabolism 342
6. Analytical Methods 343
6.1 Elemental Analysis 343
6.2 Nonaqueous Titration 343
6.3 Thin-Layer Chromatography 343
6.4 Gas Chromatography 346
6.5 Gas Chromatography-MassSpectrometry 349
6.6 High Pressure Liquid Chromatography 350
6.7 Ultraviolet Spectrophotometry 353
References 354

ANALYTICAL PROFILESOF DRUG SUBSTANCES 325 Copyright by ihe American Pharmaceutical AsSwafion.
VOLUME 12 ISBN 0- 12-260812-7
326 JAMES R.LUCH

1. Description
1.1 Introduction
Metoprolol tartrate is a synthetic, selective
beta 1-adrenoreceptor blocking agent ? - 3
1.2 Formula, Name, Formula Weight
OH
I
OCH2CHCH2NHCH(CH& COOH
I
H-C-OH
I
HO-C-H
I
COOH
2

Metoprolol tartrate
Formula Weight: 684.82 ( C I ~ H * ~ N O ~CqH6O6
)~
Chemical Abstracts Number: 56392-17-7
Metoprolol tartrate is a 2:1 salt consisting
of a racemic mixture of optical isomers of the
base and naturally occurring dex&o-tartaric acid.
The compound has been described by the following
chemical names.
i. 2-Propanol, 1-[ 4-(2-methoxyethyl)phenoxy]-3-
[ (1-methylethyl)amino]- , (+)- , [ R- (R* ,Rn) ] -
2,3-dihydroxybutanedioate (2:l)(salt)
ii. (+)-l-(Isopropylamino)-3-[p-(2-methoxyethyl)-
phenoxyl-2-propanol L-(+)-tartrate (2:l)(salt)
iii. 1-(1sopropylamino)-3- [p-(2-methoxyethyl)-
phenoxyl-2-propanol (2:l) dexho-tartrate salt
Trade Names: Betaloc, Lopresor, Lopressor, Seloken
1.3 Appearance, Color, Odor
Metoprolol tartrate is a white, virtually
odorless crystalline powder.
2. Physical Properties
2.1 Ultraviolet Absorption Spectrum
The ultraviolet absorption wavelength maxima
(Amax) and molar absorptivities ( E ) of metoprolol
tartrate in several solvents are listed in Table I.
A typical ultraviolet absorption spectrum of meto-
prolol tartrate in 0.1g HC1 obtained on a Hewlett-
Packard Model 8450A spectrophotometer is given in
Figure 1.
METOPROLOL TARTRATE 327

Figure 1: Ultraviolet Absorption Spectrum


of Metoprolol Tartrate in 0.1 N HCI

Wavelength (nm)
328 JAMES R. LUCH

TABLE I

Solvent

0.1g HC1 221 19.5


274 2.83
281 shoulder 2.31
4
Water 223 23.4
2 74 3.60
280 shoulder 2.94
4
- NaOH
0.01N 223 24.0
274 3.66
280 shoulder 3.00
Methanol 223 21.5
276 3.11
282 2.62
Chloroform 277 3.36
2 83 2.86

2.2 Infrared Absorption Spectrum


The infrared absorption spectrum of metoprolol
tartrate obtained as a Nujol mull on a Perkin-Elmer
Model 281B grating infrared spectrophotometer is
presented in Figure 2 . Spectral assignments €or
major absorption bands provided in Table I1 are
consistent with the structure of metoprolol
tartrate.
TABLE I1
~~~ ~~ ~~

-1
Wavenumber (cm ) Assignment ( s )

3600-2300 -fH2, -OH, Aliphatic and


Aromatic CH
1580 Carboxylic Acid Salt
1580, 1515 Aromatic Ring
1250, 1015 Aromatic Ether
1180 Isopropyl Group
1100 Aliphatic Ether, Secondary
A1coho 1
820 1,4-Disu~tituted Benzene
Figure 2: Infrared Absorption Spectrum of Metoprolol Tartrate
330 JAMES R.LUCH

2.3 Proton Nuclear Magnetic Resonance Spectrum


The 80 MHz proton nuclear magnetic resonance
(NMR) spectra of metoprolol tartrate5 obtained in
CDC13 at ambient temperature (lower trace) and 60°C
(upper trace) are given in Figure 3. The spectra
have been obtained on a Varian CFT-20 NMR instrument
using tetramethylsilane as an internal standard. The
chemical shifts, multiplicities and spectral assign-
ments are provided in Table 111.
TABLE I11

oioo
OCH2CHCH2NHCH(CH&
mo o COOH
@
0 1 63
H-C-OH
* @
HO-C-H
I 0
0 0 @ I @
COOH
2
Proton Chemical Shift Number of Multiplicity
Positi o n 6 (ppm) Protons
1 1.33 12 Doublet
2 2.80 4 Triplet
3 3.13-3.31 6 Broad
4 3.32 6 Singlet
5 3.53 4 Triplet
6 3.95 4 Broad
7 4.40 4 Singlet & Broad
8 6.77 4 p-Substituted
Rromatic
9 7.07 4 p-Substituted
Aromatic
10 7.25 8 Broad,
Exchangeables
Figure 3: 80 MHz 'H Nuclear Magnetic Resonance Spectra of Metoprolol Tartrate
at Ambient Temperature (lower trace) and 60°C (upper trace)
332 JAMES R.LUCH

2.4 Carbon-13 Nuclear Magnetic Resonance Spectrum


The 13C nuclear magnetic resonance (NMR)
spectra of metoprolol tartra e5 obtained in CDC13
at ambient temperature with FH-decoupling (lower
trace) and without '8-decoupling (upper trace)
are presented in Figure 4 . The spectra have been
recorded at 2 5 . 2 MHz on a Varian CFT-20 NMR
instrument. The chemical shifts, multiplicities
and spectral assignments are given in Table IV..
TABLE IV

OH
0 I0 @ O 63
OCH2CHCH2NHCH(CH3)2 COOH
10
H-C-OH
I@
HO-C-H
@@ @ I
COOH
@CH2CH20CH3
63
Carbon Multi licity Without
Position 'H-decoupling

1 18.67, 19.01 Quartet for each


2 35.09 Triplet
3 48.12 Triplet
4 50.37 Doublet
5 53.36 Quartet
6 65.50 Doublet
7 70.08 Triplet
8 73.60 Triplet
9 73.86 Doublet
10 114.37 Doublet
11 129.57 Doublet
12 131.35 Singlet
13 156.85 Singlet
14 178.35 Singlet
gure 4: I3C Nuclear Magnetic Resonance Spectra of Metoprolol Tartrate
ith 'H-decoupling (lower trace) and Without 'H-decoupling (upper trace)

Without 'H-decoupling

With 'H-decoupling

1 1 1 I I I I 1 I I 1 1 1 I I I 1 1 I 1
180 160 140 120 100 80 60 40 20 0
PPM
334 JAMES R.LUCH

2.5 Mass Spectrum


The low r e s o l u t i o n mass spectrum of m e t o p r o l o l
t a r t r a t e o b t a i n e d on a Kratos MS 25 s p e c t r o m e t e r
a t 7 0 ev u s i n g a s o l i d i n s e r t i o n probe a t a probe
temperature of 100°C is shown i n F i g u r e 5 . The
mass spectrum of metoprolol t a r t r a t e i s t h e
spectrum of i t s f r e e base r e s u l t i n g from thermal
d i s s o c i a t i o n when t h e compound i s v a p o r i z e d .
Prominent .fragments and t h e i r mass t o c h a r g e
r a t i o (m/e) a r e l i s t e d i n Table V .

TABLE V

m/ e

268, 267 [M + H]+, M + ( f r e e base)

252 [M - CH3]+

152
[ HO-o-CH2CH20CH3 ] +
OH
I
116 [CH2CHCH,NHCH(CH,) '
1
2

107

102 [HOCHCH*NHCH(CH3)2]+

77 C6H5+
72
+
CH2=NHCH(CH3)2

45 CH3-6=CH2

30 CH2=hH2

2.6 Fluorescence Spectrum


A s o l u t i o n of metoprolol t a r t r a t e i n w a t e r
o r methanol e x h i b i t s f l u o r e s c e n c e when e x c i t e d
w i t h u l t r a v i o l e t l i g h t . The c o r r e c t e d e m i s s i o n
s p e c t r a 6 of m e t o p r o l o l t a r t r a t e i n water ( s o l i d
t r a c e ) and i n methanol (dashed t r a c e ) , g i v e n i n
F i g u r e 6 , show a n emission maximum a t 298 nm and
a quantum y i e l d of approximately 0.3. These
s p e c t r a have been o b t a i n e d u s i n g a n e x c i t a t i o n
wavelength of 275 nm which corresponds t o t h e
maximum of t h e a b s o r p t i o n spectrum.
METOPROLOL TARTRATE 335

Figure 5: Low Resolution Mass Spectrum of Metoprolol Tartrate

100

80

=.
.-
c
6o
v)
c
al
c
-
C

.-9
c
-mal

r-
40

20

MassKharge Ratio
1 250
Figure 6: Fluorescence Spectra of Metoprolol Tartrate

2 . 3 ~-. .-.
'O0I ---------- Water
Methanol 3.2~1O-~M I
80-

60-

40-

20-

-
I I I I 1 I I
270 290 310 330 350 370

Wavelength (nrn)
METOPROLOL TARTRATE 337

2.7 Optical Rotation


Metoprolol tartrate contains three asymmetric
carbon atoms; one at the 2-propanol position of
the base and the other two in the tartaric acid
portion of the molecule. Optically active, natural
tartaric acid (dexZto-tartaric acid) is used to
prepare metoprolol tartrate and thus solutions of
the compound exhibit optical rotation even though
the base portion of the molecule is racemic. The
specific rotation determined at 20°C on a 2%
aqueous solution of metogrolol tartrate using
the sodium D line, [,]go c, is within the range
of + 6 . 5 " and +10.5". A value for [a]200c of
+8.5" has been reported €or a typica!? sample of
metoprolo1 tartrate. 5

2.8 Me1ting Range


Metoprolol tartrate has been observed to
melt over a 1-2 degree range between approximately
120-123°C when tested according to the USP XX
Class Ia procedure. For example, a sample of
metoprolol tartrate has been determined to melt
between 121.1 and 122.3"C by the USP Class Ia
procedure. 7
2.9 Differential Scanning Calorimetry
The differential scanning calorimetry curve
of metoprolol tartrate obtained7 on a DuPont Model
900 instrument at a scan rate of 10°C/minute
exhibits a sharp melting endotherm with an onset
temperature of 115°C and a peak temperature of
122.3OC as shown in Figure 7. A heat of fusion
value of 18,700 cal/mole has been obtained for a
metoprolol tartrate sample having a purity of 98.9
mole percent as determined by differential
scanning calorimetry.
2.10 Thermogravimetric Analysis
Thermogravimetric analysis of metoprolol
tartrate typically exhibits a weight l o s s of less
than 0 . 5 %between room temperature and 135°C. For
example, thermogravimetric data obtained7 for a
metoprolol tartrate sample on a Perkin-Elmer TGS-1
thermobalance at a scan rate of 10°C/minute showed
a weight l o s s of less than 0.2%.
2 . 1 1 X-ray Diffraction
The X-ray powder diffraction pattern obtained7
for metoprolol tartrate on a Diano Model 8235
diffractometer using the Cu K, line (1.542A) as
the radiation source with a Ni filter is shown in
Figure 8 .
JAMES R.LUCH

I I 1 I 1 I I
20 40 60 a0 100 120 140

1200

600

0 I ' I ' I ' I ' I ' I ' I ' I


7 11 15 19 23 27 31 35
Degrees Two Theta
Figure 8: X-ray Powder Diffraction Pattern of Metoprolol Tartrate
METOPROLOL TARTRATE 339

2.12 D i s s o c i a t i o n Constant
Dissociation constant data obtained f o r t h e
secondary amine of metoprolol t a r t r a t e by p o t e n t i o -
m e t r i c t i t r a t i o n a r e given i n Table V I . The pKa
v a l u e s r e p o r t e d i n The Merck Index8 f o r t a r t a r i c
a c i d a r e 2.93 and 4.23 a t 25OC.
TABLE V I

PG Conditions Reference

8.9 ? 0.2 - i n water a t 25OC


8 x 10-4M 4
9.68 ? 0.02 ionic strength, 0.1 9
9.5 ? 0.2 10

2.13 Solubility
The approximate s o l u b i l i t y of m e t o p r o l o l
t a r t r a t e i n s e v e r a l s o l v e n t s a t 25°C is g i v e n i n
Table V I I . The s o l u b i l i t y h a s been determined
a f t e r shaking a s a t u r a t e d suspension of t h e d r u g
f o r 2 hours.

Solvent S o l u b i l i t y (mg/ml)

Water > 1000


Methanol > 500
Chloroform 496
Acetone 1.1
Acetonitrile 0.89
Hexane 0.001
I

2 . 1 4 Water Adsorption Isotherm


Metoprolol t a r t r a t e i s a hygroscopic compound
a t high h u m i d i t i e s . The w a t e r a d s o r p t i o n i s o t h e r m 4
a t 25OC, given i n F i g u r e 9 , i n d i c a t e s t h e m a t e r i a l
r a p i d l y absorbs water a t r e l a t i v e humidities
g r e a t e r t h a n 70% and c o n v e r s e l y d e s o r b s w a t e r as
t h e r e l a t i v e humidity i s decreased. No h y d r a t e o r
change i n c r v s t a l form h a s been observed.
Figure 9: Water Adsorption Isotherm of Metoprolol Tartrate at 25°C
METOPROLOL TARTRATE 341

2.15 Distribution Ratio


Distribution ratio data, expressed as the
organic phase concentration divided by the aqueous
phase concentration, are summarized in Table VIII.
TABLE V I I I

Organic
Phase
Aqueous
Phase
Distribution
Ratio
I
Reference

1-Octanol 0.067M Phosphate 0.587 20.007


Buffer, pH 7 . 4
1-Oc tanol 0.0675 Phosphate 0.665 20.008
Buffer, pH 7.4
with 0.9% NaCl
Hexane 0.0675 Phosphate 0.0040 50.0005
Buffer, pH 7 . 4
Hexane 0.0675 Phosphate 0.0047 +0.0001
Buffer, pH 7.4
with 0.9% NaCl
Chloroform 0 . 1 5 NaOH 542 +16
Chloroform 0.15 HC1 0.0040 +0.0003

3. Synthesis

/O\
OCHZCH-CH~
?H

HzNCH(CHs)z
342 JAMES R. LUCH

4. Stability
4.1 Solid State Stability
Metoprolol tartrate stored at room tempera-
ture and at 3 5 O C for five years is physically and
chemically stable. After storage at 5 O o C for up to
thirty months, no degradation has been observed--
the only change has been that the material became
slightly off-white; at lower temperatures and at
shorter time intervals at 5 O o C , it has been
completely unchanged in color. 9 Under high
humidity metoprolol tartrate is hygroscopic and
rapidly adsorbs water at relative humidities
greater than 7 0 % ; however, upon drying and
reanalysis, the material is found to have retained
its chemical and physical integrity.

4.2 Solution Stability


No chemical change has been observed for
solutions of metoprolol tartrate buffered at pH
values of 4 , 7 and 9 which have been stored at
6 0 ° C for 10 days. l4 lletoprolol tartrate solutions
prepared in 0 . 1 E H C 1 , pH 7 phosphate buffer and
O . l N NaOH have been refluxed for 20 hours with no
evizence of chemical change. Ampuls containing an
aqueous solution of metoprolo1 tartrate, 1 mg/mI,
and 0.9% sodium chloride have not shown any
evidence of chemical change after storage for 7 7
months at room temperature and at 5 0 ° C . 9

5. Pharmacokinetics and Metabolism


An extensive review of the pharmacological pro-
perties and therapeutic efficacy of metoprolol tartrate
in hypertension and angina pectoris has been given by
Brogden &. l 5 Studies in rats and dogs indicate that
metoprolol is almost completely absorbed from the
gastrointestinal tract. l 6 Distribution is typical of
that of a moderately lipophilic, basic drug. Studies in
man show that metoprolol is readily and rapidly absorbed
after oral administration and is rapidly distributed to
body tissues. 3 Plasma levels vary considerably
between individuals, due partly to significant hepatic
first-pass elimination, which results in 50% of the
administered oral dose reaching the systemic circula-
tion. 18-20 Metoprolol is only slightly (12-14%) bound
to human serum protein, namely albumin, which is
reflected in its large volume of distribution (5.6
L/kg). 17,21,22
METOPROLOL TARTRATE 343

The elimination half-life of metoprolol is about


3 to 7 hours in most patients and is independent of
dose and duration of therapy. 1 8 * 2 0 * 2 3 The drug undergoes
extensive biotransformation and is excreted principally
via the kidneys; only about 3% of a dose is excreted
as unchanged drug after oral administration and about
10% after an intravenous dose. l 7 Three main metabolites
of metoprolol have been isolated in man as well as in
the rat and the dog. These three metabolites account
for 85% of the total urinary excretion in man.24
Arfwidsson d25 have reported an additional 4
urinary metabolites in the rat.

6. Analytical Methods
6.1 Elemental Analysis
The elemental composition determined for a
typical' sample of metoprolo1 tartrate on a
Perkin-Elmer Model 240 CHN Analyzer is given
below.

Element Theory (%) Observed ( X )

Carbon 59.63 59.90


Hydrogen
Nitrogen

6.2 Nonaqueous Titration


Metoprolol tartrate can be titrated in
glacial acetic acid with 0.11 perchloric acid in
glacial acetic acid or dioxane. The end point is
determined potentiometrically using a glass
electrode and a calomel electrode containing
glacial acetic acid saturated with lithium
chloride. Each ml of 0.lE perchloric acid is
equivalent to 3 4 . 2 4 mg of metoprolol tartrate.
A potentiometric titration with 1M_ sodium
hydroxide in a two phase system of water and 0 . e
pentachlorophenol in methylene chloride has been
used to determine metoprolol tartrate.26

6.3 Thin-layer Chromatography


Thin-layer chromatographic systems developed
for the identification and the determination of
metoprolol tartrate and related compounds are
summarized below.
344 JAMES R. LUCH

System 113 The following system is given as a


test for chromatographic purity in
the metoprolol tartrate monograph in
USP xx.27

Adsorbent: Silica Gel glass plate, 250 pm


layer
Solvent Chloroform (under NH3 atmosphere)
System :
Chamber : Saturate a filter paper lined
chamber containing the solvent
system and several beakers, each
containing 45 ml of concentrated
ammonium hydroxide, for 1.5 hours.

Detection Chlorine Gas-Starch: Dry the


System : developed plate until the odor of
ammonia is no longer perceptible and
place in a chamber of chlorine gas
(add 5 ml of 5N HC1 to a beaker
containing 0.5-g of potassium
permanganate) for 1 minute. Allow
the plate to stand in air for
several minutes and spray with
detecting reagent (mix 3 ml ethanol
with 10 ml of potassium iodide
solution, 1 g in 100 ml water, and
10 ml starch solution, 3 g soluble
starch triturated in 10 ml cold
water and then added to 90 ml
boiling water with constant
stirring).
Alternate Chromate-H?SOQ-UV 366 nm: 2 8 Dry the
Detection developed plate with warm air for
Systems: 10 minutes and spray with detecting
reagent (add 20 ml concentrated
sulfuric acid to 90 ml of water
containing 0.5 g of potassium
dichromate). Dry at 12OoC for 10
minutes and visualize the cooled
plate under long wave ultraviolet
light at 366 nm.
Anisaldehyde: Dry the developed
plate with warm air for 2-3 minutes
and spray with freshly prepared
anisaldehyde reagent (0.5 ml anisal-
dehyde in 10 ml glacial acetic
METOPROLOL TARTRATE 345

acid, 85 ml methanol and 5 ml concen-


trated sulfuric acid). Dry at 100°C
for 20 minutes and visualize under
daylight or under long wave ultra-
violet light, 366 nm.
Quantitative Determination: 2 a (The
following procedure has been used as
a dosage form stability method.)
Metoprolol tartrate is extracted from
the dosage form with chloroform-
methanol (1:l) and a portion of the
solution equivalent to 2 mg of
metoprolol tartrate is applied as a
band to a silica gel plate containing
a fluorescent indicator concomitantly
with a metoprolol tartrate standard
solution band. After development the
plate is dried and the silica gel
zones containing metoprolol from the
sample and standard are removed from
the plate after visualizing under
ultraviolet light (254 nm).
Metoprolol from the sample is eluted
from the silica gel with 0.15
ethanolic ammonia and quantitated by
ultraviolet absorption spectroscopy
vb a metoprolol standard solution
similarly prepared.
A number of additional systems have been
used for the analysis of metoprolol
tartrate.
System 1112,l 3 Chloroform-Methanol ( 9 5 : 5 ) , saturated
chamber with solvent and ammonia
atmosphere, Silica Gel P254 (Merck) ,
anisaldehyde or chl~rine-tolidine~~
detection
System 1 1 1 ~ 2 Methanol-Ethyl Acetate ( 4 0 : 2 0 ) ,
sandwich chamber, Silica Gel F254
(Merck), anisaldehyde detection
System 1v4 1-Butanol-Glacial Acetic Acid-Water
(71:7:22), Silica Gel with fluores-
cent indicator, iodine vapors or
ultraviolet light (254 nm) detection
346 JAMES R. LUCH

System v4 Methanol-Ethyl Acetate-Diethylamine


(60:35:5), Silica Gel with fluores-
cent indicator, iodine vapors or
ultraviolet light (254 nm) detection
System V130 Chlorofom-Methanol-Ammonium Hydrox-
ide (80:15:2), Silica Gel 60 F254
(Merck), chlorine gas-starch detec-
tion
System VI19 Benzene-Methanol (1:1), sandwich
chamber, Silica Gel F254 (Merck),
anisaldehyde detection
System VIII Benzene-Methanol-Glacial Acetic Acid
(30:30:5), sandwich chamber, Silica
Gel F254 (Merck), anisaldehyde
detection
System IX9 Benzene-Methanol (45 :5), sandwich
chamber, Silica Gel F25q (Merck),
anisaldehyde detection
System ~9 Benzene-Methanol-Glacial Acetic
Acid (40:20:5), sandwich chamber,
Silica Gel F254 (Merck), anisal-
dehyde detection
System X131 0.1% Ammonium Acetate in Methanol-
Water (56:44), reverse phase KC18F
plates (Whatman), ultraviolet light
(254 nm) or iodine vapor followed by
ultraviolet light (254 nm) detection

6.4 Gas Chromatography


Gas chromatographic systems reported for the
determination of metoprolol tartrate are summarized
below.
System 132 The following system has been used
for the determination of metoprolol
in human plasma.
Column : 6 ft x 4 mm i.d. glass column
packed with 3% OV-101 on Chromosorb
WHP (80-100 mesh)
Temperatures: For Hewlett Packard Model 76208
chromatograph; injector (25OoC),
column (195OC), detector (31OOC)
For Varian Model 3700 chromatograph;
injector (23OoC), column (206OC),
detector (31OOC)
METOPROLOL TARTRATE 347

Carrier: For Hewlett-Packard; helium at


approximately 60 ml/minute
For Varian; nitrogen at 28 ml/minute
Detection: Electron capture detection of the
pentafluoropropionyl derivative of
metoprolol
33
System I1 The following system has been
employed for the determination of
metoprolol in plasma and urine.
Column: 195 cm x 2 mm i.d. glass column
with 3% JXR on Gas Chrom Q (100-120
mesh)
Temperatures: Injector (not reported), column
(16OoC), detector (300°C)
Carrier: Argon-Methane (95:5) at 50 ml/minute
Detection: Electron capture detection of the
trifluoroacetyl derivative of
metoprolol
34
System I11 The following system has been used
to determine metoprolol in plasma.
Column : Glass column packed with 1% OV-17
on Gas Chrom Q (80-100 mesh),
column dimensions not reported
Temperatures: Injector (25OoC), column (185"C),
detector (300°C)
Carrier: Argon-Methane (95:5) at 60 ml/minute
Detection: Electron capture detection of the
pentafluoropropionyl derivative of
metoprolol
35
System IV The following system has been
reported for the determination of
metoprolol in blood or plasma.
Column: 150 cm x 2 mm i.d. glass column
packed with 3% JXR on Chromosorb G
(80-100 mesh)
Temperatures: Injector (220°C), column (2OO"C),
detector (350°C)
Carrier: Nitrogen at 30 ml/minute
348 JAMES R. LUCH

Detection: Electron capture detection of the


trifluoroacetyl derivative of
metoprolol
36
System V The following system has been used
for the determination of metoprolol
in plasma and cerebrospinal fluid.
Column: 150 cm x 3 mm i.d. glass column
packed with 4% OV-101 on Chromosorb
W (100-120 mesh)
Temperatures: Injector (210°C) , column (195OC) ,
detector (255°C)
Carrier: Nitrogen at 25 mllminute
Detection: Electron capture detection of the
trifluoroacetyl derivative of
metoprolol
30
System VI The following system has been used
for the determination of metoprolol
in pharmaceutical dosage forms.
Column: 210 cm x 5 mm i.d. glass column
packed with 3% J X R on Gas Chrom Q
(100-120 mesh)
Temperatures: Injector (25OoC), column (210OC) ,
detector (25OOC)
Carrier: Nitrogen at 45 ml/minute
Detection: Flame ionization detection of
trimethylsilyl derivative of meto-
prolol prepared using bis(trimethy1-
sily1)-trifluoroacetamide
In addition to the systems listed above,
metoprolol has been used as an internal
standard for the gas chromatographic deter-
mination of atenolol in plasma and urine
using electron capture detection of the
pentafluoropropionyl derivatives. 3 7 Alprenolol
and other 8-adrenorceptor blocking agents,
including metoprolol, have been derivatized
to form the 2,4-dichlorobenzeneboronate
adducts for gas chromatographic d ermination
using electron capture detection.'' Two
metabolites of metoprolol have been determined
in plasma and urine by gas chromatography
using electron capture detection. 39
METOPROLOL TARTRATE 349

6.5 Gas Chromatography-Mass Spectrometry


Sensitive methods reported for determining
metoprolol and its major metabolites in plasma and
urine using gas chromatography-mass spectrometry
(GC-MS) are given below.
40
System I The following GC-MS system used
selected ion monitoring €or the
quantitation of metoprolol and two
of its metabolites in plasma and
urine.
Column: 100 cm x 2 mm i.d. glass column
packed with 3% OV-17 on Gas Chrom Q
(100-120 mesh)
Detection: MS selected ion monitoring at m/e =
266, 270 and 2 7 1
Temperatures: Injector (250°C) , column ( 2 0 0 O C ) ,
separator and ion source (250OC)
Carrier: Helium at 15 ml/minute
MS Source: Electron impact at 60 eV
Sample: Trifluoroacetyl derivatives pre-
pared by using N-methyl-bis-(tri-
fluoroacetamide)
24
System I1 The fallowing GC-MS system has been
used for the determination of
urinary metabolites of metoprolol
tartrate.
Column: 6 ft x 2 mm i.d. glass column
packed with a mixture of 5% OV-1
and 5% OV-17 on Chromosorb W (80-
100 mesh)
Detection: MS using electron impact at 70 eV
Temperatures: Injector (24OoC), column and ion
source (not reported), separator
(210°C)
Carrier: Helium at 54 ml/minute
Sample: Trimethylsilyl derivatives prepared
by using bis-(trimethylsily1)-
acetamide
350 JAMES R.LUCH

6.6 High Pressure Liquid Chromatography


High pressure liquid chromatography (HPLC)
systems that have been used to determine metoprolol
tartrate and related compounds in various samples
are summarized below.
System I The following HPLC system is given
as an assay procedure in the mono-
graph for metoprolol tartrate tablets
in USP m.27
Column: pBondapak c18 column (30 cm x
3.9 mu i.d., Waters Associates), USP
column packing L1
Mobile Phase: A mixture of 550 ml methanol and
470 ml water containing 961 mg 1-
pentanesulfonic acid sodium salt
(monohydrate), 82 mg anhydrous
sodium acetate and 0.57 ml glacial
acetic acid
Flow Rate: 1 ml/minute
Detection: Ultraviolet absorption (254 nm)
Internal Oxprenolol hydrochloride
Standard :
The following two HPLC systems have been used
to determine metoprolol in animal feed samples.

System 1 1 ~ 1
Column: pBondapak c18 (30 cm x 3.9 mm
i.d. , Waters Associates)
Mobile Phase: A mixture of 520 ml methanol
and 480 ml water containing 1.10 g
1-heptanesulfonic acid sodium salt
(monohydrate) and 5.0 ml glacial
acetic acid
Flow Rate: 1.2 ml/minute
Detection: Ultraviolet absorption (274 nm)
Principle: Metoprolol tartrate is extracted
from feed samples with 0.1N HC1 con-
taining oxprenolol hydrochioride
(internal standard). Interfering
feed extractables are removed by
alkalizing the aqueous solution and
partitioning metoprolol into hexane-
methylene chloride ( 8 5 : 1 5 ) prior to
HPLC analysis.
METOPROLOL TARTRATE 351
42
System I11
Column: 15 cm x 4.5 m i.d. stainless steel
column packed with Partisil 10
(Whatman)
Mobile Phase: Methylene Chloride-Methanol-e
Diethylamine in Methanol (89:lO:l)
Flow Rate: 1 d/minute
Detection: Ultraviolet absorption (277 nm)
Principle: Metoprolol tartrate is extracted
from feed samples with 0.01E HC1,
the aqueous extract is made alkaline
and metoprolol is partitioned into
methylene chloride prior to HPLC
analysis.
43
System IV Metoprolol and one of its metabo-
lites (a-hydroxymetoprolol) have
been determined in plasma by the
following HPLC system.
Column: 25 cm x 4.6 nun i.d. stainless steel
column packed with 5 pm silica B/5
(Perkin-Elmer)
Mobile Phase: Hexane-Isopropanol-Methano1-
Concentrated Ammonium Hydroxide
(850:100:50: 1)
Flow Rate: 3 rdminute
Detection: Fluorescence, excitation wavelength
at 224 nm and no emission filter
Internal (+)-Ethyl-2- (4-(3-isopropylamino-
Standard : 2-hydroxypropoxy)phenyl)ethyl
carbamate
44
System V The following system based on ion-
pairing with a chiral counter ion,
(+)-10-camphorsulfonate, has been
used for the separation of meto-
prolol enantiomers.
Column: 10 cm x 3.2 mm i.d. or 15 cm x
3.2 mm i.d. stainless steel column
packed with 10 pm LiChrosorb-DIOL
(Merck)
352 JAMES R. LUCH

Mobile Phase: A mixture of methylene chloride-


l-p?ntanol (199:l) containing 2 . 2 x
10- -M (+)-10-camphorsulfonate
Detection: Ultraviolet absorption ( 2 5 4 nm)

System v19 The following system has been used


to investigate the purity of meto-
prolol tartrate.
Column: 15 cm x 4 mm i.d. stainless steel
column packed with LiChrosorb RP 8,
5 um or 10 pm particles (Merck)
Mobile Phase : A mixture of 100 ml of water
containing 1.8 ml perchloric acid
and 1 1 . 2 g sodium perchlorate
(monohydrate) with 200 ml aceto-
nitrile diluted to 1000 ml with
water,
Flow Rate: About 0.8-0.9 ml/minute
Detection: Ultraviolet absorption ( 2 7 0 nm)

System V I I ~ The
~ following HPLC system has been
reported for the determination of
several beta-blocking agents,
including metoprolol, in plasma and
urine.
Column: pBondapak c18 column (30 cm x
3.9 mm i.d., Waters Associates)
Mobile Phase: Methanol-Water-Acetic Acid (50: 4 9 :1)
containing 1-heptanesulfonic acid,
prepared by adding one bottle of PIC
B-7 reagent (Waters Associates) per
liter of mobile phase
Flow Rate: 1.3 mllminute
Detection: Fluorescence, excitation wavelength
at 2 2 2 nm and no emission filter
METOPROLOL TARTRATE 353

6.7 Ultraviolet Spectrophotometry


An ultraviolet spectrophotometric procedure
is used to determine content uniformity of
metoprolol tartrate tablets in the USP monograph
for this item.27 In this procedure metoprolol is
extracted from the dosage form with 0.1E H C 1 and
after alkalizing the aqueous extract metoprolol is
partitioned into chloroform for quantitation by
ultraviolet spectrophotometry. Assay and content
uniformity data for metoprolol tartrate tablets
also have been obtained using 0.1E H C 1 for
extraction and subsequent quantitation by ultra-
violet spectrophotometry.

The determination of amines and quaternary


ammonium ions by ion pair extraction with picrate
has been described and investigated by Gustavii
and S ~ h i 1 1 .
A ~stability
~ assay for metoprolol
tartrate active substance and dosage forms has been
developed using this approach.47 The active sub-
stance is dissolved in water and diluted with pH
6.5 phosphate buffer. Metoprolol is then extracted
into methylene chloride as a picrate ion pair which
is measured spectrophotometrically at approximately
347 nm. A similar procedure is used for the tablet
assay except 95% ethanol-water (30:ZO) is used to
extract the drug from the tablets.

Acknowledgment
The author expresses appreciation to Rebecca Yang, Donald
Kender, Jane Johnson and Richard Brown for their help in
preparing this manuscript and to Jijrgen Vessman for
reviewing the manuscript.
354 JAMES R. LUCH

References

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35th Edition, 906-908 ( 1 9 8 1 ) .
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cation.
5 . R. K. Rodebaugh, CIBA-GEIGY, Personal Communication.
6 . D. Bernstein, CIBA-GEIGY, Ltd., Personal Communi-
cation.
7 . H. Stober, J. Quitasol and R. Morris, CIBA-GEIGY,
Personal Communication.
8 . M. Windholz, Ed., "The Merck Index'', 9th Edition,
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cation.
10. K. Jake1 and P. Moser, CIBA-GEIGY, Ltd., Personal
Communication.
11. United States Patent N o . 3 , 6 7 4 , 8 4 0 ( 1 9 7 2 ) ; Swedish
Patent No. 7153169 ( 1 9 6 9 ) , AB Hassle.
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Switzerland), CIBA-GEIGY, Ltd., Personal Communi-
cation.
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S . Avery, otLugA, 14,321-348 ( 1 9 7 7 ) .
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Wallborg, A& p h m a c o l . et tutoxic~t., 36, suppl
V , 104-115 (1975).
17. C. G. Reggrdh, K. 0. Borg, R. Johansson, G.
Johnsson and L. Palmer, J. Phmacokinet. B&-
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SSilvell, A c b phanmacol. et t a x i c o l . , 36, suppl
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0
19. C. Bengtsson, G. Johnsson and C. G. Regardh,
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21. K. A. Johansson, C. Appelgren, K. 0. Borg and R.
Elofsson, A& p h m . duct., 11,333-346 ( 1 9 7 4 ) .
METOPROLOL TARTRATE 355

22. C. Appelgren, K. 0. Borg, R. Elofsson and K. A.


Johansson, A& phatun. huec., 11,325-332 (1974).
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Skgnberg, Xenobiofica, 5, 691-711 (1976).
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A& phahm,. hk&c., 13,391-406 (1976).
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29. C . G. Greig and D. H. Leaback, NUdhte, 188,310
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-
33. M. Ervik, A o t a p h m a c u l . QA: Zoaxicol., 36, s u p p l
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Dorrington, Cfin. Ctp. phU4mUcOl. phyAiOl., 3,
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356 JAMES R. LUCH

45. M. A . Lefebvre, J . G i r a u l t and J. B. F o u r t i l l a n ,


J. Liq. Ckrturnatugn., 4, 483-500 (1981).
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L i t e r a t u r e s e a r c h e d through March, 1982.


PHENYLPROPANOLAMINE
HYDROCHLORIDE
Isadore Kanfer, John M . Haigh, and Roslind Dowse

1. Introduction 358
2. Description 358
2.1 Name, Formula, Molecular Mass 358
2.2 Trade Names 358
2.3 Appearance, Odour and Colour 359
3. Synthesis 359
4. Physical Properties 359
4.1 Solubility 359
4.2 Melting range 359
4.3 Specific Rotation 359
4.4 Crystal Structure 360
4.5 Dissociation Constant 360
4.6 Infrared Spectrum 360
4.7 Differential Scanning Calorimetry 362
4.8 Proton Magnetic Resonance Spectrum 362
4.9 Ultraviolet Spectrum 364
4.10 Mass Spectrum 365
5. Methods of Analysis 365
5.1 Elemental Analysis 365
5.2 Ultraviolet Spectrophotometric Analysis 365
5.3 Colorimetric Analysis 367
5.4 Spectrofluorimetric Analysis 367
5.5 Titrimetric Analysis 367
5.6 Chromatographic Analysis 368
6. Stability 37 1
7. Absorption, Disposition, and Pharmacokinetics 377
8. Identification and Determination in Biological Fluids 377
References 380

ANALYTiCAL PROFILES O F DRUG SUBSTANCES 357 Copyright by the Anierican PharnYlceutical Association.
VOLUME 12 ISBN 0-12-260812-7
358 ISADORE KANFER E T A .

1. Introduction

Phenylpropanolamine hydrochloride belongs to the sympath-


omimetic mine class of drugs and is structurally related to
ephedrine hydrochloride. Its synthesis was first reported in
1910 (1 1 and the first American patent was registered in 1939.
The effects of phenylpropanolamine hydrochloride are largely
the result of alpha-adrenergic agonist activity resulting
from both direct stimulation of adrenergic receptors and
release of neuronal norepinephrine. The principal adverse
effect of phenylpropanolamine hydrochloride is dose-related
hypertension and ventricular arrhythmia has been described
(2). Phenylpropanolamine hydrochloride is widely used as a
decongestant and it has been used as an anorectic agent for
over 40 years ( 3 ) . A report in 1939 ( 4 ) described its effect
as an hypertensive agent when administered parenterally.

2. Description

2.1 Name, Formula, Elolecular Mass

Phenylpropanolamine hydrochloride, sometimes


referred to as dZ-norephedrine can also be named in a number
of ways :
(a) a-(1-aminoethy1)benzenemethanol hydrochloride
(b) a-(1-aminoethy1)benzyl alcohol hydrochloride
+
(c) (-)-2-amino-phenylpropan-l-ol hydrochloride
(d) 2-amino-1-phenyl-1-propanol hydrochloride
(e) a-hydroxy-B-aminopropylbenzene hydrochloride
(f) I-phenyl-2-amino-1-propanol hydrochloride

H CH3
MM 187.67
C9H13No

2.2 Trade Names

Propadrine, Control, Obestat and Dietac. Numerous


products containing phenylpropanolamine hydrochloride in
combination with other active ingredients are commercially
PHENYLPROPANOLAMINEHYDROCHLORIDE 359

a v a i l a b l e as a p p e t i t e s u p p r e s a n t s and c o l d and i n f l u e n z a
remedies.