ATROPINE

Abdullah A. Al-Badr and Farid J. Muhtadi

King Saud University
Riyadh, Saudi Arabia

1. Description 326
1.1 Nomenclature 326
1.2 Formulae 326
1.3 Molecular Weight 328
1.4 Elemental Composition 328
1.5 Appearance, Color, Odor, and Taste 328
I .6 Dissociation Constant 328
1.7 pH range 328
2. Physical Properties 329
2. I Melting Point 329
2.2 SublimationRange 329
2.3 Solubility 329
2.4 X-Ray Crystallography 329
2.5 Spectral Properties 330
3. Isolation 340
4. Synthesis 340
4.1 Partial Synthesis 340
4.2 Total Synthesis 340
5 . Biosynthesis 352
5.1 Biosynthesis of Tropine 352
5.2 Biosynthesis of Tropic Acid 354
6. Metabolism 355
7. Pharmacokinetics 357
8. Therapeutic Uses of Atropine 358
9. Methods of Analysis 359
9.1 Identification Tests 359
9.2 Microcrystal Tests 360
9.3 Titrimetric Methods 360
9.4 Polarographic Methods 365
9.5 SpectrophotometricMethods 366
9.6 Chromatographic Methods 373
9.7 Radio-immunoassay 378
References 380

ANALYTICAL PROFILES OF DRUG SUBSTANCES Copyright 0 1985

VOLUME 14 325 by the American Pharmaceutical Association
ISBN 0-12-260814-3
326 ABDULLAH A . AL-BADR AND FARID J . MUHTADI

1. Description

1.1 Nomenclature

1.1.1 Chemical Names

a) endo ( f)- a -( Hydroxymethyl) benzene-acetic

acid 8-methyl-8-azabicyclo [ 3.2.11 oct-
3-y1 ester.
b) Benzene-acetic acid a-( hydroxymethy1)-,
8-methyl-8-azabicyclo [ 3.2.11 oct-3-yl
ester, *-( +)-
c) la H,-:a H-tropan-3a -ol( 2)-tropate.

1.1.2 Generic Names

Atropine, dl-hyoscyamine, (2)-hyoscyamine,

tropic acid ester with tropine, tropine ( 2 )
tropate, dl-tropyl tropate , ( 2 ) tropyl tropate .
1.2 Formulae

1.2.1 Ebpirical

C H NO
17 23 3
1.2.2 Structural

The structure was confirmed by the total syn-

thesis of atropine which was achieved by
several authors ( 1-4) .
ATROPINE 327

1.2.3 CAS Registry No.

51-55-8 I
1.2.4 Wiswesser Line Notation

~ 5 A6 ANTJ A
-GOVYR & 1Q- DL (5)
1.2.5 Stereochemistry

Examination of t h e NMR s p e c t r a of some tropane

deuterohalides has shown t h a t t h e N-substitu-
e n t i n tropanes i s predominantly e q u a t o r i a l ( 6 1.
X-ray a n a l y s i s of t r o p i n e hydrobromide has
shown t h e presence of c h a i r conformation ( 7 ) .
Study of t h e dipole-moment and Kerr-constant
measurements of a number of tropane d e r i v a t i -
ves has shown t h a t t h e p i p e r i d i n e r i n g i s i n
t h e c h a i r form with t h e N-methyl e q u a t o r i a l
(8). Another study of t h e dipole-moments and
NMR s p e c t r a of some tropane d e r i v a t i v e s have
confirmed t h a t t h e p i p e r i d i n e r i n g i s i n t h e
c h a i r conformation with t h e N-methyl group
predominantly e q u a t o r i a l ( 9 ) . I n t r o p i n e ,
however, t h e predominant conformation i s t h e
p i p e r i d i n e r i n g i n a deformed c h a i r form t o -
gether with a minor amount i n t h e boat form
(10).

HO
Tropine

I n a t r o p i n e , t h e a-3-substituent i s of grea-
t e r bulk than t h e hydroxyl, and t h e boat form
may w i l l be favored because of t h e increased
i n t e r a c t i o n s involving t h e dimethylene bridge
i n t h e c h a i r confirmation (11).
 ABDULLAH A. AL-BADR A N D FARlD J. MUHTADI

H
N-CH3 0
11 NCH~OH
0-C-CH
\
C6H5

A d e t a i l e d review i s a v a i l a b l e f o r t h e boat or c h a i r
conformation i n t r o p i n e s ( 1 2 ) .

Other PMR study suggested a preference f o r t h e boat

conformation i n s e v e r a l tropane d e r i v a t i v e s . This
study showed s t r o n g c r o s s - r i n g i n t r a m o l e c u l a r i n t e r -
a c t i o n s of t h e t y p e N--- C-=O and N---H-O were i n d i -
c a t e d by t h e broadening of t h e proton s i g n a l due t o
t h e coupling between 1(5)-H and 2(4)-H protons i n t h e
boat conformer compared with t h e c h a i r . This broad-
ening a r i s e s as a consequence of e c l i p s i n g of t h e s e
protons i n t h e boat conformer (13). Carbon-13 magne-
t i c resonance study has a l s o suggested a non-chair
conformations i n tropane d e r i v a t i v e s ( 1 4 ) .

1.3 Molecular Weipht

289.38

1.4 Elemental Composition

c, 70.56%; H , 8.01%; N , 4.84%; 0 , 16.59%

1.5 Appearance, Color, Odor and Taste

C o l o r l e s s needle-like c r y s t a l s or white c r y s t a l l i n e
powder, o d o r l e s s and has a sharp b i t t e r t a s t e .

1.6 D i s s o c i a t i o n Constant

PKa 5.93

1.7 BH range

pH of 0.0015 molar s o l u t i o n i s 10.0 (15),approximate

pH of s a t u r a t e d aqueous s o l u t i o n i s 9.5 ( 1 6 ) .
ATROPINE 329

2. Physical P r o p e r t i e s

2.1 Melting P o i n t

114 - 116' (15)

114 - 118' (16)
2.2 Sublimation range

Atropine sublimes i n high vacuum a t 93-110'.

2.3 Solubility

One gram d i s s o l v e s i n 460 m l water, i n 90 m l water

a t 80°, i n 2 ml a l c o h o l , 1.2 ml alcohol a t 60°, i n
27 ml g l y c e r o l , 25 m l e t h e r . Soluble i n benzene
and d i l u t e a c i d s .

2.4 X-ray c r y s t a l l o g r a p h y

The X-ray c r y s t a l l o g r a p h y of t r o p i n e hydrobromide ( 7 ),

t r o p i n e ethobromide ( 1 7 ) pseudotropine (18) hyoscine
hydrobromide ( 1 9 ) and t r o p i c a c i d i n hyoscine N-oxide
(20 ) have been r e p o r t e d .
330 ABDULLAH A. AL-BADR AND FARID J . MUHTADI

2.5 Spectral Properties

2.5.1 U l t r a v i o l e t Spectrum

The W spectrum of a t r o p i n e i n e t h a n o l ( F i g . 1 )
was scanned from 200 t o 400 nm using DMS 90
'Varian Spectrophotometer. It e x h i b i t e d t h e
following W d a t a (Table 1).

Table 1. UV c h a r a c t e r i s t i c s o f a t r o p i n e

A m a . a t nm E A ( 1 % , 1 cm)
20 5 - -
246 147.6 5.1
251.5 175.1 6.05
257 209.8 7-25
263.5 143.3 4.95
271 24.6 0.85
Other r e p o r t e d W s p e c t r a l d a t a f o r a t r o p i n e
i n 0 . 1 N sulfuric acid ( 21 ) :

h max a t . 252 mu ( E 1%, 1 cm 5 ) , 258 mu

( E I%, 1 cm 6 ) and 264 mu ( E 1%, 1 cm 5 ) .

2.5.2 I n f r a r e d Spectrum

The I R spectrum of a t r o p i n e as KBr-disc was

recorded on a Perkin Elmer 580 B I n f r a r e d
Spectrophotometer t o which I n f r a r e d Data stat-
i o n i s a t t a c h e d (Fig. 2 ) .

The s t r u c t u r a l assignments have been c o r r e l a -

t e d w i t h t h e following frequencies (Table 2 ) .

Table 2. I R C h a r a c t e r i s t i c s of Atropine

-1
Frequency cm Assignment
3070 OH (hydrogen bonded)
2930 CH ( s t r e t c h )
2810 N-CH
3

1725
B
0-C - ( e s t e r )

1595, 1580 C=C aromatic

FIG, 1, THE UV SPECTRUM OF ATROPINE I N ETHANOL
44.

&O# WAVE##H#FR #o asM 2060 fm YO0 1400 1000 800 680 &

FIG. 2 , THE I R SPECTRUM OF ATROPINE AS KBR-DISC

ATROPINE 333

-1
Frequency cm Assignment
1155, 1030 C-0-C (ether)

770,725,690 5 H (mono s u b s t i t u t e d aromatics)

The I R e x h i b i t e d t h e following o t h e r c h a r a c t e r i s t i c
bands :-

1 4 5 0 , 1 4 2 0 , 1 3 7 0 , 1 3 5 5 , 1 3 3 5 , 1 2 7 0 , 1 2 ~ 5 , ~ 2 3 0 , ~ 2 2 0 , ~ 2,0 5
1190,1165,1132,1108,1065,975,920,845,805,515 cm-1.

Other I R d a t a f o r a t r o p i n e (5,21) have been a l s o

reported.

2.5.3 Nuclear Magnetic Resonance Spectra

2.5.3.1 Proton Spectra

The PMR s p e c t r a of both a t r o p i n e i n

C D C 1 3 and i n TFA ( T r i f l u o r o a c e t i c
a c i d ) were recorded on a Varian T-
~ O A ,60 MHz NMR Spectrometer using
TMS (Tetramethylsilane) as an i n t e r -
n a l reference. These are shown i n
Fig. 3 ( a ) and 3{b) r e s p e c t i v e l y . The
following s t r u c t u r a l assignments have
been made (Table 3 ) .

0- C-
9

Other PMR d a t a f o r a t r o p i n e are a l s o r e p o r t e d (5,9,

13,22).
334 ABDULLAH A . AL-BADR AND FARID J. MUHTADI

LO
I
TO
I
I
.. I
40
I
1
. I .,
a# Rn(,)
. . uI . . .I . .
I
B.#
....i
1..
, I , . i
1.0
, ,

F I G . 3 I A ) . TPE PYR SPECTnWi OF ATROPI3E Ill C D C L ~

ATROPINE 335

Table 3. PMR c h a r a c t e r i s t i c s of a t r o p i n e

Group Chemical S h i f t (ppm)

CDC13 TFA

5 aromatic protons 7.23(s) 7.36( s 1

1 3 , l b ,15,16,17
H-3
CH2 - OH
CH2 - OH, -
1 0 CH
135 H
8-N-Me
2,4,6,7 H

s = s i n g l e t , d=doublet , t = t r i p l e t , bs=broad singlet ,

m=mult iplet

2.5.3.2 13C-NMR

The I3C-NMR n o i s e decoupled and o f f

resonance s p e c t r a a r e presented i n
Fig. 4 and Fig. 5 r e s p e c t i v e l y . Both
were recorded over 4000 Hz range i n
d e u t e r a t e d chloroform on a Varian
FT 80 A-80 MHz spectrometer, u s i n g
1 0 mm sample tube and t e t r a m e t h y l s i l -
ane as a r e f e r e n c e standard a t 2 1
'
.
The carbon chemical s h i f t s a r e assi-
gned on t h e bases of t h e a d d i t i v i t y
p r i n c i p a l s and o f f resonance s p l i t t -
i n g p a t t e r n (Table 4 ) .

11

15
4 10

17 16
336 ABDULLAH A. AL-BADR AND FARID J . MUHTADI

d 111

FIG. 4 . THE I3C-NER NOISE DECOUPLED SPECTRUE OF ATROPINE

I
ATROPINE 337

Table 4. Carbon Chemical S h i f t s of Atropine

Carbon no. Chemical S h i f t Carbon no. Chemical S h i f t

[PPd [ P P ~
C 171.92( s ) c1 c5 59.54(d)
9
5 2
136.17 ( s 1 ‘10 54.94 ( d )
‘13’ ‘17 128.66( d ) ‘8 40.08(q)

128.11 ( d ) 36.04(t
‘14’ ‘16 c23 c4
127.48 ( d ) C 25.31(t)
‘15 7
C
3 67.63 ( d ) ‘6 24.93(t 1
cll 63.54(t)

s = s i n g l e t , d=doublet t=triplet q=quartet .

Other l3C-NMR d a t a f o r a t r o p i n e ( 14,23 ) a t r o p i n e
hydrochloride ( 1 4 ) and a t r o p i n e methoiodide ( 1 4 ) have
a l s o been reported.

2.5.4 Mass Spectrum

The mass spectrum of a t r o p i n e i s presented

i n Fig. 6. This was obtained by e l e c t r o n i m -
pact i o n i z a t i o n on a Varian MAT 1020 by d i r e c t
i n l e t probe a t 270’~. The e l e c t r o n energy was
70 eV. The spectrum scanned t o mass 300 amu.
The spectrum ( F i g . 6 ) shows a molecular ion
peak M+ a t m / e 289 with r e l a t i v e i n t e n s i t y
9.50%. The base peak i s 124 with r e l a t i v e
i n t e n s i t y 100%.
The most prominent fragments t h e i r r e l a t i v e
i n t e n s i t i e s and some proposed ion fragments
a r e given i n t a b l e 5.
...
CJ
T
.
ATROPINE 339

Table 5 . Mass Fragments of Atropine

Relative i n t e n s i t y % Ions
9-50 M+
7.79 -
9.34 See below*
100.00 125- H
6.35 -
r-

96 10.67

c 4

95 8.60 96-H
94 22.66 954

83 18.87

82 25 * 97
i

67 14.78 -
44 -
1
5-15 i
I

42 21.83 :-CH2=N=CH2
r +

41 8.36 42-H

Other r e p o r t e d mass s p e c t r a of a t r o p i n e ( 2 4 ) :
Base peak 1 2 4 , m/e: 4 2 , 55, 67, 82, 94, 1 0 4 ,
1 2 4 , 1 4 0 , 272, 289.
Tropine fragmentations a r e a l s o r e p o r t e d ( 25 ).
340 ABDULLAH A . AL-BADR AND FARID I. MUHTADI

3. I s o l a t i o n of Atropine

Atropine occurs i n s e v e r a l solanaceous p l a n t s t h e s e

include s p e c i e s of Atropa, Datura, Hyoscyamus, Duboisia,
Mandragora and Scopolia ( 26).
It i s claimed t h a t a t r o p i n e does not occur as such i n t h e
p l a n t s , but 2-hyoscyamine p r e s e n t i n p l a n t s , (27) and
during e x t r a c t i o n p r o c e s s , 2-hyoscyamine undergoes race-
mization t o give a t r o p i n e . Hyoscyamus muticus from Egypt
i s t h e p r e f e r r e d source for t h e manufacture of a t r o p i n e
because of i t s high a l k a l o i d c o n t e n t , w i t h stramonium
next i n order ( 28 ) .

One of t h e b e s t methods f o r t h e i s o l a t i o n of a t r o p i n e
i s as follows ( 2 8 ) .

The powdered drug i s throughly moistened w i t h an aqu-

eous s o l u t i o n of sodium carbonate and e x t r a c t e d w i t h e t h e r
or benzene. The a l k a l o i d a l bases a r e e x t r a c t e d from t h e
solvent with water a c i d i f i e d w i t h a c e t i c a c i d . The a c i d
s o l u t i o n i s t h e n shaken w i t h e t h e r as long as t h e l a t t e r
t a k e s up c o l o r i n g m a t t e r s . The a l k a l o i d s a r e p r e c e p i t a t e d
with sodium c a r b o n a t e , f i l t e r e d o f f , washed and d r i e d . The
d r i e d p r e c i p i t a t e i s d i s s o l v e d i n e t h e r or a c e t o n e , dehy-
d r a t e d w i t h anhydrous sodium s u l f a t e and f i l t e r e d . The
f i l t e r a t e i s c o n c e n t r a t e d , c o o l e d , when crude hyoscyamine
and a t r o p i n e c r y s t a l l i z e from t h e s o l u t i o n . The crude
c r y s t a l l i n e mass r e s u l t e d i s f i l t e r e d o f f and d i s s o l v e d i n
a l c o h o l , sodium hydroxide s o l u t i o n i s added and t h e mix-
t u r e i s allowed t o s t a n d u n t i l recemization of hyoscyamine
t o a t r o p i n e i s completed (as i n d i c a t e d by t h e absence of
optical activity).

The crude a t r o p i n e i s p u r i f i e d by c r y s t a l l i s a t i o n
from acetone.

4. Synthesis of Atropine

4.1 P a r t i a l Synthesis

Landenburg i n 1879 (1) accomplished t h e f i r s t

s y n t h e s i s o f a t r o p i n e from t r o p i n e and t r o p i c a c i d ,
t h u s proving a t r o p i n e t o be t h e t r o p i n e e s t e r of
t r o p i c a c i d . Tropine and t r o p i c a c i d a r e heated i n
t h e presence of hydrogen c h l o r i d e t o g i v e a t r o p i n e .
b.2 T o t a l Synthesis

Since a t r o p i n e i s t h e t r o p i n e e s t e r of t r o p i c
ATROPINE 341

a c i d , schemes f o r t h e t o t a l s y n t h e s i s of t r o p i n e
and t h e t o t a l s y n t h e s i s of t r o p i c a c i d were repor-
ted.

4.2.1 Total Synthesis of Tropine

Four schemes f o r t h e t o t a l s y n t h e s i s o f
t r o p i n e a r e known. Scheme I1 w a s a l s o modi-
f i e d t o give a much b e t t e r y i e l d .
Scheme I: W i l l s t a t t e r ' s t o t a l s y n t h e s i s of
tropine ( 2 ) .
Suberone (cycloheptanone) [ 1 1 i s reduced t o
suberol which i s t r e a t e d w i t h hydrogen iodide
t o give suberyl iodide [ 2 ] . This i s t r e a t e d
with potassium hydroxide i n ethanol t o give
cycloheptene [ 31. Cycloheptene i s brominated
t o give 1,2-dibromocycloheptane [ 41 which i s
t r e a t e d with dimethylamine t o y i e l d dimethy-
laminocyclohept-2-ene [ 51. The l a t t e r i s
converted t o cyclohepta-lY3-diene [61 by ex-
h a u s t i v e methylation. [ 6 ] i s brominated a t
l Y 4 - p o s i t i o n s t o give 1,4-dibromocyclohept-
2-ene [ T I . Elimination of two moles of t h e
hydrogen bromide of [ T I i s e f f e c t e d by quin-
o l i n e t o give cycloheptatriene [S].
Substance [8] i s t r e a t e d with hydrogen brom-
i d e t o give bromocyclohepta-3,5-diene [ g ]
which i s r e a c t e d with dimethylamine t o give
dimethyl aminocyclohepta-2 ,b-diene [lo1. The
l a t t e r i s t r e a t e d with sodium i n e t h a n o l f o l -
lowed by bromination t o give 1,2-dibromo-5-
dimethylamino-cycloheptane [ 111. This i s
warmed i n e t h e r when intramolecular alkyla-
t i o n occurs t o give 2-bromotropane methobro-
mide [12]. Hydrogen bromide i s eliminated
from [12] by t h e a c t i o n o f a l k a l i t o y i e l d
t r o p i d i n e methobromide [13]. This i s t r a n s -
formed t o t r o p i d i n e methochloride El41 by t h e
a c t i o n of potassium iodide followed by t h e
a c t ion of s i l v e r c h l o r i d e . Substance [ 1 4 1
i s pyrolized t o give t r o p i d i n e [IF].
Hydrogen bromide i s added t o an a c e t i c a c i d
s o l u t i o n o f t r o p i d i n e [15] t o y i e l d 3-bromo-
tropane [I61 which i s hydrolysed with 10%
s d f u r i c a c i d a t 200-210' t o give pseudo-
t r o p i n e [IT]. $-tropine [17J i s oxidized
with chromium t r i o x i d e t o give tropinone [18].
342 ABDULLAH A . AL-BADR AND FARlD J . MUHTADI

Scheme I: Willstatter's t o t a l synthesis of tropine

(ii)HI

+
exhaust.
methyln.
Qr [41 Br

0 0
Br

q u i n o l ine HBr
15ooc * [SI
____)

Br
[I11
ATROPINE 343

OHN-CK3

Scheme 11: Robinson's total synthesis of tropine

CHO CH-OH
CH3
+

(
-t CH3NH2
cond. N-CH3 \
/"=O

/
*
[21
CHO Ci-OH CH3
[11 [ 31 [41
344 ABDULLAH A. AL-BADR AND FARID J . MUHTADI

This ketone i s reduced with zinc and hydrio-

dic acid t o tropine [lg].
Scheme 11: Robinson's s y n t h e s i s ( 3 )
Succindialdehyde [ 11 i s condensed with methy-
lamine [ 2 ] t o give t h e condensate b i s c a r b i n -
olamine [ 31. This i n t u r n condensed w i t h ace-
t o n e [ h ] t o give tropinone [ 5 ] (This mixture
i s allowed t o s t a n d i n water a t o r d i n a r y tem-
p e r a t u r e f o r h a l f an h o u r ) .
Tropinone [ 5 ] i s reduced with zinc and hydr-
i o d i c a c i d t o t r o p i n e [61.
The y i e l d can be improved by s u b s t i t u t i o n
of t h e more r e a c t i v e acetone d i c a r b o x y l a t e or
i t s e s t e r f o r acetone.
Succindialdehyde [ 11 i s condensed w i t h methyla-
mine [21 t o give biscarbinolamine [31. 131
i s condensed w i t h calcium acetonedicarboxy-
l a t e [ 4 ] t o a f f o r d t h e condensate [ 5 ] . This
i s warmed w i t h hydrochloric a c i d t o give t r o -
pinone [ 6 ] . Tropinone [ 6 ] i s reduced with
zinc and hydriodic a c i d t o t r o p i n e [ T I .
Scheme 111: W i l l s t a t t e r ' s second s y n t h e s i s ( 4 )
S u c c i n y l d i a c e t i c e s t e r [l] i s condensed with
methylamine [ 21 t o give diethyl-N-methylpyr-
r o l e d i a c e t a t e [ 3 ] . This i s reduced (H2+Pt)
t o a f f o r d diethyl-N-methylpyrrolidinediace-
tate [4]. The c& form of [ 4 ] i s c y c l i z e d i n
t h e presence of N a and p-cymene t o give e t h y l -
tropinone-2-carboxylate [ 51. Hydrolysis of
[ 5 ] w i t h 10% s u l f u r i c a c i d g i v e s e t h y l t r o p i -
none-2-carboxylic a c i d [6]. The l a t t e r i s
heated t o y i e l d t r o p i n o n e [ T I which i s redu-
ced w i t h z i n c and hydriodic a c i d t o t r o p i n e [8].
Scheme IV:
Tropinone can a l s o be s y n t h e s i z e d ( 2 9 ) using
methylamine hydrochloride,acetondicarboxylic
a c i d and g e n e r a t i n g succindialdehyde - in -
situ
by t h e a c t i o n of a c i d on 2,5-dimethoxy t e t r a -
hydrofuran as follows :

CHO
c:
ATROPINE 345

(
Scheme 11: Robinson's s y n t h e s i s ( y i e l d improvement)
CH-OH CH2COOCa

tNH2CH3 cond. ~
)-CH3 + \
,C=O

[21
CH-OH CH2COOCa

\4
[11 [41
[31

-
COOCa

COOCa

Scheme 111: Willstatter's second s y n t h e s i s

CH= C- CH,-COOC H
I
N- CH3- 2 5

I
CH= CH- CH2-COOC H CH= C- CH2-COOC H
2 5 2 5
[11

i"
H
I
CH2- C- CH2-COOC H -C- CH2-COOC H

CH2-
1 F-
H3C$
1 1
y=O
CH COOC H
2 5

4 -
Na/p-c ymene

CH2-?-
h-CH3
I
CH2-COOC
25

H
2- 2 5 I 2 5
H H

- C-CH-COOH

31 I ______t
CH - C- CH2
2 1
3% ABDULLAH A . AL-BADR A N D FARID J . MUHTADl

CH -CCF-CClr!
1 2 \- I 2

CH - - ~ H - - C H ~ CH2 - CH - CH2
2

"71 181
4.2.2 T o t a l S y n t h e s i s o f Tropic a c i d

S e v e r a l schemes f o r t h e t o t a l s y n t h e s i s of
t r o p i c a c i d are known (Scheme I t o V ) .
Scheme I : Landenburg's s y n t h e s i s ( 3 0 ) .
Acetophenone [l] i s c o n v e r t e d i n t o a , a - d i c h l o r o -
ethylbenzene [ 2 ] by t h e a c t i o n o f phosphorous
pentachloride. 121 i s r e a c t e d w i t h potassium
cyanide and e t h a n o l t o f u r n i s h ct-ethoxy-a-cyano-
ethylbenzene [ 3 ] . T h i s i s hydrolysed w i t h barium
hydroxide s o l u t i o n t o g i v e a t r o l a c t i c e t h y l e t h e r
[4]. The l a t t e r i s h e a t e d w i t h hydrogen c h l o r i d e
to y i e l d a t r o p i c a c i d [ 5 ] which i s c o n v e r t e d t o
t r o p i c a c i d [61.
Scheme I1 : McKenzie and Wood's s y n t h e s i s (31).
Acetophenone [l] i s c o n v e r t e d by t h e a c t i o n o f
potassium cyanide t o acetophenone cyanohydrine
[ 23. T h i s upon h y d r o l y s i s i s c o n v e r t e d i n t o
a t r o l a c t i c a c i d [ 3 ] . The l a t t e r i s h e a t e d under
p r e s s u r e t o y i e l d a t r o p i c a c i d [4]. Atropic a c i d
[4] i s t r e a t e d w i t h hydrogen c h l o r i d e i n e t h e r e a l
s o l u t i o n t o form 6 - c h l o r o h y d r a t r o p i c a c i d [ 5 ] .
T h i s upon b o i l i n g w i t h aqueous sodium c a r b o n a t e
i s changed t o t r o p i c a c i d [ 6 ] .
Scheme 111: Miller's s y n t h e s i s (32).
Ethylphenyl a c e t a t e [l] i s condensed w i t h e t h y l -
formate t o g i v e e t h y l a-formyl a c e t a t e [ 2 ] . T h i s
on r e d u c t i o n w i t h aluminium amalgam y i e l d s dl-
t r o p i c ester [3] which upon h y d r o l y s i s g i v e s
t r o p i c a c i d 141.
Scheme IV: Chambon's s y n t h e i s (33).
E t h y l a-bromophenylacetate [l] is t r e a t e d w i t h
Zn t o g i v e ethyl+-zincbromophenylaceate [2]
which i s t r e a t e d w i t h formic a c i d t o g i v e d l -
t r o p i c e s t e r [ 3 ] which upon d y d r o l y s i s y i e l d s
t r o p i c a c i d [4].
 Scheme I: Landenburg's s y n t h e s i s
CH3
I

KCN

[ 31 [41

CH2 CH20H

@
II
C - COOH 6~-COOH
I

_____)

Scheme 11: McKenzie and Wood's s y n t h e s i s

KCN

[ 31

CH2CI CH20H
I I
348 ABDULLAH A . AL-BADR A N D FARlD J . MUHTADI

Scheme I11: Miiller I s synthesis

CHO

CH2OH CH2OH
I I
CH-COOEt

hydrolysis

Scheme IV: Chambon's synthesis

Br
I Zn Br
CH-COO% I
CH-COOEt

Zn HCHO
b ______r

CH2OH CH2OH
I I
ATROPINE 349

Scheme V: Blicke's synthesis (34).

Phenylacetic a c i d [l] i s b o i l e d w i t h isopropyl-
magnesium c h l o r i d e i n e t h e r e a l s o l u t i o n t o give
[ 2 ] and then t r e a t e d t h e product [ 2 ] , a Grig-
nard reagent with formaldehyde t o give t r o p i c
acid [ 3 ] .

Scheme V: BLicke's s y n t h e s i s

Tropine f i n a l l y can be combined with t r o p i c a c i d t o

give a t r o p i n e . This can be done by h e a t i n g t h e two toge-
t h e r i n t h e presence of hydrogen c h l o r i d e (Fischer-Speier
.
e s t e r if i c at i o n )

atropine
350 ABDULLAH A. AL-BADR AND FARID J . MUHTADI

4.2.3 S y n t h e s i s o f Labeled Atropine

4.2.3.1 S y n t h e s i s of Labeled Tropic a c i d

Benzylmagnesium c h l o r i d e [l] i s t r e a -
t e d w i t h I4CO2 followed w i t h magnesium
c h l o r i d e t o g i v e t h e condensate [ 2 ] .
This upon t h e a d d i t i o n of formaldeh-
yde g i v e s l a b e l e d t r o p i c a c i d [3].
Synthesis of labeled t r o p i c a c i d i s
p r e s e n t e d i n scheme VI ( 35 ).

4.2.3.2 S y n t h e s i s of Labeled Tropine

- S y n t h e s i s of t r o p i n e - 6 , 7 T h a s been
a c h i e v e d by c a t a l y t i c tritium a d d i t i o n
t o 2 , 5-dimethoxy-2, 5 d i h y d r o f u r a n
and f o l l o w i n g Robinson's r o u t e t o
tropinone-6, 7 T , by subsequent reduc-
t i o n w i t h hydrogen over Raney n i c k e l
(36).
- S y n t h e s i s o f methyl-14C l a b e l e d t r o -
p i n e i s c a r r i e d o u t from Na 1 4 C N ( 3 7 )
v i a 1nethylamine-~4C and b a s e d on Rob-
i n s o n ' s r o u t e ; inethyl-l4C t r o p i n o n e
i s o b t a i n e d i n 70% o v e r a l l y i e l d and
tropine-14C i n 68% y i e l d .
- S y n t h e s i s of b i 4 C t r o p i n e can be
s t a r t e d w i t h arabinose-5-l4C [ 11
conversion i n t o f u r a n [ 2 ] and a p p l i c a -
t i o n o f t h e Clauson-Kaas r o u t e t o suc-
cin-dialdehyde and t h e n t o 1-or 5-14C-
t r o p i n o n e 31 ( 3 8 ) .
U i n g arabinose-3, 4-14C g i v e s 6 , 7-
l'C-tropinone ( 39 Scheme V I I .
4.2.3.3 Labeled a t r o p i n e can be t h e n o b t a i n e d
by e s t e r i f i c a t i o n of l a b e l e d t r o p i c
a c i d or labeled t r o p i n e t o give e i t h e r
l a b e l e d a t r o p i n e or double l a b e l e d 7 . k
a t r o p i n e ( a r i s e d from l a b e l e d t r o p i c
a c i d and l a b e l e d t r o p i n e ) .
ATROPINE 351

Scheme V I : Synthesis of Labeled Tropic a c i d

H [21

Scheme VII : Labeled t r o p i n e

' CH20H

Double l a b e l e d a t r o p i n e
352 ABDULLAH A . AL-BADR AND FARID J . MUHTADI

5. Biosynthesis of Atropine

Most s t u d i e s on t h e b i o s y n t h e s i s of a t r o p i n e and of
i t s isomer hyoscyamine have been performed on v a r i o u s
s p e c i e s of Datura, b u t a l l t h e a v a i l a b l e evidence suggests
t h a t s i m i l a r pathways occur i n o t h e r tropane a l k a l o i d -
producing p l a n t s ( 26 ). Because t h e c h a r a c t e r i s t i c a l k a l -
o i d s of t h e group are e s t e r s of hydroxylamines and v a r i o u s
a c i d s ( t r o p i c , t i g l i c , e t c . ) t h e r e a r e , for each a l k a l o i d ,
two d i s t i n c t b i o s y n t h e t i c r o u t e s ( 26 ).

5.1 Biosynthesis of t r o p i n e

Ornithine and t h e r e l a t e d aminoacids (glutamic

a c i d , p r o l i n e ) have been proved t o be t h e p r e c u r s o r s
of t h e p y r r o l i d i n e r i n g of t r o p i n e ( 40-45 ).
It w a s found t h a t feeding [2-l4C] o r n i t h i n e t o Datura
stramoniwn r e s u l t e d i n r a d i o a c t i v e hyoscyamine l a b e l -
l e d only a t C - 1 bridgehead carbon of t r o p i n e (46).
COOK
N-CH3

And t h a t [ 5-14C] p r o l i n e r e s u l t e d i n r a d i o a c t i v e
hyoscyamine l a b e l l e d only t h e C-5 p o s i t i o n of t r o p -
ine ( 4 4 ) .
It w a s a l s o r e p o r t e d t h a t [2-l4C, 6 - 1 5 N I o r n i t h i n e
incorporated i n t o t r o p i n e moiety of hyoscyamine and
t h e 6-aminogroup of o r n i t h i n e i s an e f f i c i e n t precu-
r s o r of t h e t r o p i n e n i t r o g e n (44,46).
The i n c o r p o r a t i o n of glutamic a c i d and p r o l i n e i s
considered t o occur v i a o r n i t h i n e ( 46 ) .
Ornithine [l] i s i n c o r p o r a t e d i n t o t r o p i n e v i a 6-N-
m e t h y l o r n i t h i n e [ 2 ] (47-49) as [ methyl-l4C ] 6 -N- -
methyl-[ 2-&] o r n i t h i n e w a s i n c o r p o r a t e d i n t o hyos-
cyamine l a b e l l i n g C - 1 and t h e N-methyl group. 121
i s decarboxylated t o y i e l d N-methylputrescine [ 41
( 50,51).
P u t r e s c i n e [ 3 ] has a l s o been shown t o be a p r e c u r s o r
of t h e t r o p i n e a l k a l o i d s (43 ,52-5&). It w a s suggested
( 4 6 ) t h a t p u t r e s c i n e [ 31 i s converted by c e r t a i n enz-
ymes i n Datura p l a n t s t o N-methyl p u t r e s c i n e [ 4 ] .
Oxidation o f t h e primary a l c o h o l of [ 4 ] a f f o r d s 4-
methylaminobutanal [ 5 ] . This i s c y c l i z e d t o give N-
methyl- A l-pyrrolinium s a l t [ 6 I.
ATROPINE 353

Leete's Scheme: Biosynthesis of Atropine

COOH COOH

-7

E N C H 3
x-
+--
LrnCH3 '7
NHCH3

FyOH f -atropine
1141
354 ABDULLAH A . AL-BADR A N D FARID J . MUHT.4DI

Carbons 2 , 3 and 4 of t r o p i n e are d e r i v e d from ace-

t a t e ( 55,56 ) and it i s assumed t h a t t h e a c e t a t e i s
incorporated v i a a c e t o a c e t i c a c i d or some s u i t a b l e
a c t i v a t e d d e r i v a t i v e such as coenzyme A e s t e r ( 46 ).
[ 6 ] is t h e r e f o r e condensed w i t h a c e t o a c e t a t e t o give
hygrine- a -carboxylic a c i d [ 71. Decarboxylation of
[7] a f f o r d s hygrine [8] which i s an e s t a b l i s h e d pre-
c u r s o r of t r o p i n e ( 56,57 ). [8] i s dehydrogenated
t o give dehydrohygrine [g]. The l a t t e r i s c y c l i z e d
t o y i e l d tropinone [lo]. S t e r e o s p e c i f i c r e d u c t i o n
of [lo] a f f o r d s t r o p i n e [ll].

5.2 Biosynthesis of t r o p i c a c i d

Tropic a c i d 1121 i s formed by t h e i n t r a m o l e c u l a r

rearrangement of phenylalanine I131 (58) .Compounds
which a r e m e t a b o l i c a l l y r e l a t e d t o phenylalanine such
as phenylpyruvic a c i d are a l s o i n c o r p o r a t e d i n t o t r o -
p i c a c i d ( 59,60).

shikimic
acid (J JrJ + I
/

*CH2 HOH2C-*C-H
I
[I31 +h-mH2
I .COOH [12]
* COOH

Tropine [ll] i s f i n a l l y e s t e r i f i e d w i t h t r o p i c a c i d
[ 1 2 ] t o give a t r o p i n e [14].
ATROPINE 355

6. Metabolism of Atropine

Atropine i s r a p i d l y absorbed from t h e g a s t r o i n t e s t i n a l

t r a c t and r e a d i l y absorbed from t h e mucous membranes and
t h e s k i n (21,151 ).Absorption from t h e i n t e s t i n a l t r a c t i s
complete i n 2 hours. About one-half of t h e a t r o p i n e c i r -
c u l a t e s i n t h e f r e e form i n t h e blood and t h e o t h e r h a l f
i s bound by t h e plasma p r o t e i n s ( 21). Atropine a l s o e n t e r s
t h e c i r c u l a t i o n when a p p l i e d l o c a l l y t o mucosal s u r f a c e s
of t h e body ( 6 1 ) . The t r a n s c o n j u n c t i v a l absorption of
a t r o p i n e i s considerable. About 95% of r a d i o a c t i v e a t r o -
p i n e i s absorbed and e x c r e t e d following subconjunctival
i n j e c t i o n i n t h e r a b b i t ( 62). The metabolism of a t r o p i n e
v a r i e s considerably from one s p e c i e s t o another. Hydro-
l y s i s t o t r o p i n e and t r o p i c a c i d i s not thought t o be a
major metabolic r o u t e s i n c e only t r a c e s of t r o p i c a c i d
a r e recovered i n t h e u r i n e ( 21).
Atropine disappears r a p i d l y from t h e blood and i s d i s t r i -
buted throughout t h e e n t i r e body ( 2 1 ) . The l i v e r , kidney,
lung and pancrea.s a r e t h e most important organs t h a t t a k e
up t h e l a b e l e d a t r o p i n e ( 6 2 ) . Most i s excreted i n t h e
u r i n e w i t h i n t h e f i r s t 1 2 hours, i n p a r t unchanged ( 2 1 ) .
Following intra-mascular a d m i n i s t r a t i o n of a s i n g l e 2 mg
doses of I4C-labelled a t r o p i n e i n man, Gosselin e t a l . ( 6 3 )
found t h a t 85 t o 88% of t h e r a d i o a c t i v i t y w a s e x c r e t e d i n
t h e u r i n e w i t h i n 24 hours, only a t r a c e could be e x t r a c t e d
from t h e f a e c e s ; about 50% of t h e dose appeared i n t h e
u r i n e unchanged, over 30% w a s e x c r e t e d as unknown metabo-
l i t e s and l e s s than 2% appeared as f r e e t r o p i c a c i d .
A f t e r intravenous i n j e c t i o n of a t r o p i n e i n t h e mouse, app-
roximately 25% of t h e dose i s e x c r e t e d i n t h e u r i n e as
a t r o p i n e , more t h a n 50% as conjugates with glucuronic a c i d
and t h e remaining 20-25% as i n t e r m e d i a t e o x i d a t i o n products
(probably p-hydroxyatropine and 3 , 4 4 i h y d r o x y a t r o p i n e ) and
tropine-modified a t r o p i n e s ( 6 2 ) . The metabolism of a t r o -
pine i s presented i n scheme I [ after ( 6 2 ) 1.
 ABDULLAH A . AL-BADR A N D FARID J . MUHTADI

SCHEME 1; THE METABOLISM OF ATROPINE

C02 + Noratropine (2%)

Rabbit, Guinea p i g

Tropine
( aldehyde ) i n v o Tropic a c i d

R a t liver Man Tropine ,

Noratropine 4
- ATROP 1 NE >
- Modified+Tropic
Apoatropine in vitro Mouse atropines acid
(10%) ( 1%)

Mouse

p-hydroxyatropine (2%)4 m,p-Dihydro-

xyatropine

\
p-Glucuronosidoatropine
(5%)

my-Hydroxy-p-glucuronosidoatropine
(27%)

m,p-DiglucuronosidoatropineC-p-hydroxy-m-
(20%) glue urano s ido-
atropine
ATROPINE 357

7. Pharmacokinetics

The pharmacokinetics of a t r o p i n e were r e p o r t e d by

s e v e r a l authors.
Peak serum l e v e l s occur approximately 30 minutes follow-
i n g intramuscular ( I . M . ) a d m i n i s t r a t i o n of 1 mg dose of
a t r o p i n e (64).
Serum l e v e l s following intravenous (I.V. ) a d m i n i s t r a t i o n
of a t r o p i n e drop w i t h i n t h e f i r s t 1 0 minutes and t h e n decr-
ease more gradually. Levels one hour following e i t h e r I.V.
or I.M. a d m i n i s t r a t i o n s a r e very similar (64).
Following I.M. a d m i n i s t r a t i o n of 2 mg a t r o p i n e , t h e onset
and d u r a t i o n of e f f e c t on h e a r t r a t e a r e r e p o r t e d (65) t o
be m a x i m u m a t 15-50 minutes and up t o 5 hours, r e s p e c t i -
vity.
Following endotracheal a d m i n i s t r a t i o n of 1 mg a t r o p i n e
s u l f a t e , serum l e v e l s of a t r o p i n e were l e s s t h a n 5pg/ml
a t 30 seconds and U p g / d a t 10 minutes (66 ).
Atropine's h a l f - l i f e i s r e p o r t e d t o occur a t two r a t e s ,
w i t h an i n i t i a l fast r a t e of about 2 hours and a slow r a t e
ranges 12.5-38 hours (65).
The average h a l f - l i f e of a t r o p i n e i s 4.125 hours following
a s i n g l e 1 mg intravenous dose of a t r o p i n e i n humans (67).
The mean t o t a l plasma clearance of s i x normal human volu-
n t e e r s following a s i n g l e 1 mg intravenous dose of a t r o -
p i n e i s r e p o r t e d t o be 533.35 ml/minute (67).
Maximum c y c l o p l e g i a u s u a l l y occurs w i t h i n s e v e r a l hours of
a d m i n i s t r a t i o n of t o p i c a l a t r o p i n e , though e f f e c t i v e cyclo-
p l e g i a may occur i n 30 t o 40 minutes (68).
The mydriatic e f f e c t may p e r s i s t f o r up t o 10 days while
t h e cycloplegic a c t i o n may l a s t f o r 5 days (68).
358 ABDULLAH A. AL-BADR AND FARID J . MUHTADI

8. Therapeutic Uses of Atropine (69)

1. Pre-anaesthetic medication :
- t o decrease s e c r e t i o n s of s a l i v a r y , naso-pharyngeal
and b r o n c h i a l glands.
- t o prevent r e f l e x brancho-spasm.
- to reduce r e f l e x bradycardia of i n h a l a t i o n a l anasth-
etics.

2. Antispamodic i n :
Bronchial asthma.
Renal, b i l i a r y and i n t e s t i n a l c o l i c .
Peptic ulcer.
With p u r g a t i v e s .

3. Vaso-Vagal syncope due t o r e f l e x lowering o f blood

p r e s s u r e and severe Bradycardia.

4. Nocturnal e n u r e s i s and urgency of m i c t u r i t i o n t o

decrease u r i n a r y bladder r e f l e x i r r i t a b i l i t y .

5. I n Parkinsonian d i s e a s e t o reduce r e g i d i t y ( c e n t r a l
action).

6. Antidote for parasympathomimetic poisoning e.g.

organo-phosphorous i n s e c t i c i d e poisoning.

7. Mydxiatic and cycloplegic i n :

Iritis
Kerat it i s
Corneal u l c e r a t i o n s o r i n j u r i e s
ATROPINE 359

9. Methods of Analysis

9.1 I d e n t i f i c a t i o n Tests

The following i d e n t i f i c a t i o n t e s t s a r e mentioned i n

t h e B r i t i s h Pharmacopoeia of 1963 ( 7 0 )

-1 mg of a t r o p i n e i s added t o 4 drops of fuming n i t r i c

a c i d and t h e mixture i s evaporated t o dryness on a
water b a t h ; a yellow r e s i d u e i s obtained. 2 ml of
acetone and 4 drops of a 3% w/v s o l u t i o n of potas-
sium hydroxide i n methyl a l c o h o l a r e added t o t h e
cooled r e s i d u e ; a deep v i o l e t c o l o r i s produced.
- 5 0 mg of a t r o p i n e i s d i s s o l v e d i n 5 m l of water a c i d -
i f i e d w i t h hydrochloric a c i d , gold c h l o r i d e s o l u t i o n
i s added; a lemon-yellow o i l y p r e c i p i t a t e i s formed
which r a p i d l y c r y s t a l l i z e s . This p r e c i p i t a t e a f t e r
r e c r y s t a l l i z a t i o n from b o i l i n g water a c i d i f i e d w i t h
hydrochloric a c i d , has a minutely c r y s t a l l i n e chara-
c t e r , i s d u l l and p u l v e r u l e n t when dry, and has a
melting p o i n t about 136O.
Other i d e n t i f i c a t i o n t e s t s a r e as follows:-
- TheGerrard r e a c t i o n ( 7 1 ) .
To about 6 mg of a t r o p i n e , 1 m l of 2% s o l u t i o n o f
mercuric c h l o r i d e i n 50% aqueous methanol i s added;
a deep r e d c o l o r i s produced.
-To a t r a c e of a t r o p i n e i n an evaporating d i s h , drops
of t h e p-dimethylaminobenzaldehyde reagent ( 2 g of
p-dimethylaminobenzaldehyde i s d i s s o l v e d i n 6 gm sul-
f i c a c i d ) are added as w e l l as 0.4 m l of water. The
r e s u l t i n g mixture i s heated on a b o i l i n g water bath;
an i n t e n s e r e d c o l o r i s produced which changing t o
Permanent cherry r e d on cooling.
- P h y s i o l o g i c a l t e s t : Induction of mydriasis
(can be performed on young c a t s , dogs and r a b b i t s ) .
An aqueous, alcohol free s o l u t i o n of a t r o p i n e or i t s
s u l f a t e i s dropped i n t o t h e c o n j u n c t i v a l sac of t h e
eye and h e l d so t h a t non i s l o s t by overflow of t e a r s .
It has been r e p o r t e d (71) t h a t 1 p a r t i n 40,000 or
t h a t 0.000 ,000,427 g of a t r o p i n e s u l f a t e w i l l cause
a d i s t i n c t d i l a t i o n of t h e p u p i l of t h e eye i n 1 hour.
360 ABDULLAH A. AL-BADR A N D FARID J . MUHTADI

9.2 Microcrystal tests

100 mg of a t r o p i n e d i s s o l v e d i n 5 m l water a c i d i f i e d
w i t h d i l u t e s u l f u r i c a c i d . The f o l l o w i n g microcry-
s t a l s were performed.
- P i c r i c a c i d w i t h a t r o p i n e g i v e s bunches of p l a t e s
( 2 1 ) . The c r y s t a l s are shown i n F i g . 7.
- Wagner's r e a g e n t w i t h a t r o p i n e g i v e s i r r i g u l a r hexa-
gons i n c l u s t e r s (21). The shape of c r y s t a l s i s
shown i n F i g . 8.
- Dragendorff's reagent with a tr o p in e gives i r r i g u l a r
r e c t a n g l e s as shown i n F i g . 9.
- Mercuric c h l o r i d e w i t h a t r o p i n e g i v e s l o n g prisms
as shown i n F i g . 1 0 .
9.3 Kitrimetric Methods
9.3.1 Aqueous T i t r a t i o n s
Bobtelsky and B a r z i l y ( 7 2 ) have r e p o r t e d a miso-
h e t e r o m e t r i c t i t r a t i o n of l a r g e , o r g a n i c , n i t r o g e n -
c o n t a i n i n g compounds i n c l u d i n g a t r o p i n e . Micro
amount o f a t r o p i n e i s t i t r a t e d h e t e r o m e t r i c a l l y w i t h
t u n g s t o s i l i c i c a c i d , t u n g s t o p h o s p h o r i c a c i d or
molybdophosphoric a t pH 1 or 7 .

Other t i t r i m e t r i c methods for t h e a s s a y o f a t r o p i n e

have been p u b l i s h e d :

Determination o f a t r o p i n e , t r o p i n e and t r o p i c
a c i d i n decomposed a t r o p i n e p r o d u c t s ( 7 3 ) .

The a p p l i c a t i o n of sodium dodecyl s u l f a t e t i t r i -

metric s o l u t i o n i n t h e a n a ly s is of atropine
i n j e c t i o n s (74) .
The i n f l u e n c e of s a l t s , p o l y h y d r i c compounds and
a b s o r b e n t s on t h e d e t e r m i n a t i o n of o r g a n i c b a s e s
by a n i o n i c s u r f a c t a n t i n two-phase systems.
The method was a p p l i e d t o a t r o p i n e among o t h e r
organic bases ( 7 5 ) .

Atropine i n a e r o s o l h a s been determined t i t r i -

m e t r i c a l l y by slowly e J e c t i n g t h e sample ( 2 g )
t h r o u g h a s t a n d a r d s o l u t i o n of a c i d and t i t r a t -
ing t h e excess a c i d ( 7 6 ) .
ATROPINE 361

~~

FIG. 7, MICROCRYSTALS OF ATROPINE WITH

PICRIC ACID,

5
b

FIG, 8, MICROCRYSTALS OF ATROPINE

WAGNER'S REAGENT,
362 ABDULLAH A . AL-BADR AND FARID J . MUHTADI

F I G , 9, MICROCRYSTALS OF ATROPINE
DRAGENDORFF'S REAGENT,

-/- --
FIG, 10. MICROCRYSTALS OF ATROPINE
WITH MERCURIC CHLORIDE.
ATROPINE 363

e) The i n f l u e n c e o f a t r o p i n e among o t h e r o r g a n i c
b a s e s on t h e p a r t i t i o n o f i n d i c a t o r a c i d s i n a
w a t er-chloroform system (77 ) .
f) Atropine w a s d e t e c t e d and q u a n t i t a t i v e l y d e t e r -
mined i n decomposing t i s s u e s (78 ) .

A d i r e c t t i t r a t i o n method u s i n g l e a d n i t r a t e w a s
d e s c r i b e d f o r drug p r o d u c t s i n c l u d i n g a t r o p i n e
s u l f a t e (79 ) .
9.3.2 Non-Aqueous Titrat i o n

The USP XX 1980 ( 8 0 ) d e s c r i b e d a non-aqueous t i t r a -

t i o n f o r t h e a s s a y of a t r o p i n e as follows:

D i s s o l v e about 400 mg o f a t r o p i n e , a c c u r a t e l y weighed,

i n 50 m l of g l a c i a l a c e t i c a c i d , add 1 drop of
c r y s t a l v i o l e t TS, and t i t r a t e w i t h 0 . 1 N p e r c h l o r i c
a c i d VS t o a g r e e n end-point. Perform a blank
d e t e r m i n a t i o n and make any n e c e s s a r y c o r r e c t i o n .

Each m l of 0 . 1 N p e r c h l o r i c a c i d i s e q u i v a l e n t t o
28.94 mg of a t r o p i n e ( C H NO ).
1 7 23 3
The B r i t i s h Pharmacopoeia 1980 (81) d e s c r i b e s a non-
aqueous t i t r a t i o n f o r t h e a s s a y o f a t r o p i n e as
follows :

Dissolve 0 . 3 g i n 20 m l of anhydrous g l a c i a l a c e t i c
a c i d , and t i t r a t e w i t h 0 . 1 M p e r c h l o r i c a c i d VS and
determine t h e end-point p o t e n t iomet r i c a l l y .
Dzyuba and S h r a i b e r ( 82 ) have q u a n t i t a t i v e l y d e t e r -
mined a t r o p i n e by t i t r a t i o n i n non-aqueous s o l v e n t s .
The t o t a l a l k a l o i d s of t h e a t r o p i n e group ( a t r o p i n e
p l u s hyoscyamine p l u s h y o s c i n e ) are determined by
tit rat i o n a g a i n s t HC104 i n anhydrous a c e t i c a c i d .
The method i s a p p l i e d t o leaves, e x t r a c t and t i n c t u r e
of belladonna, and t o t a b l e t s , s u p p o s i t o r i e s and eye-
drops c o n t a i n i n g a t r o p i n e or belladonna. The end-
p o i n t i s determined p o t e n t i o m e t r i c a l l y ( quinhydrone
e l e c t r o d e w i t h S.C.E. as comparison e l e c t r o d e ) o r
w i t h c r y s t a l v i o l e t as i n d i c a t o r .

Symoni and Tokar ( 8 3 ) have r e p o r t e d new r e a g e n t for

t i t r a t i o n s o f a t r o p i n e and o t h e r a l k a l o i d s i n non-
aqueous media by means o f t h e h y d r o c h l o r i c a c i d
363 ABDULLAH A . AL-BADR AND FARID J . MUHTADI

colrplex of aluminium i s o p r o p o x y l a t e . The s t a n d a r d

s o l u t i o n c o n t a i n i n g t h e H C l complex o f aluminium
c h l o r o i s o p r o p o x y l a t e i s p r e p a r e d by d i s s o l v i n g
aluminium c h l o r o i s o p r o p o x y l a t e i n chloroform and
p a s s i n g t h e c a l c u l a t e d amount of H C 1 g a s i n t o i t , o r
by adding t h e s t o i c h e i o m e t r i c amount o f chloroform
s o l u t i o n of aluminium c h l o r o i s o p r o p o x y l a t e t o a
s t a n d a r d i z e d s o l u t i o n o f H C 1 (3% t o 4 % ) i n dry
chloroform. The s o l u t i o n must be k e p t v e r y d r y .

The above a u t h o r s ( 8 4 ) have a l s o r e p o r t e d a new rea-

gent f o r t i t r a t i o n s i n non-aqueous media. The d e t e r -
mination o f a t r o p i n e and o t h e r a l k a l o i d s by means of
t h e h y d r o c h l o r i c a c i d complex of c h l o r o aluminium
i s o p r o x i d e . R e s u l t s w e r e d i s c u s s e d which have been
shown t h a t t h e H C 1 complex of c h l o r o aluminium i s o -
propoxide behaved as a monobasic a c i d when undergoing
s a l t formation w i t h v a r i o u s a l k a l o i d s . The a u t h o r
have given a method f o r t h e d e t e r m i n a t i o n o f a t r o p i n e
( a n d o t h e r a l k a l o i d s ) w i t h 0 . 1 N c h l o r o aluminium
i s o p r o p o x i d e i n chloroform. The d e v i a t i o n was C 1%
i n t h e range 38 t o 245 mg o f a l k a l o i d .

Simon et +
- ( 8 5 ) have d e s c r i b e d a method f o r t h e
d e t e r m i n a t i o n of t r a c e amounts of a t r o p i n e by t i t r a -
t l o n i n anhydrous s o l v e n t s . For s o l i d a t r o p i n e
s u l f a t e , d i s s o l v e t h e sample i n anhydrous a c e t i c acid,
add 0.1% p-dimethyl aminoazobenzene s o l u t i o n i n
benzene, and t i t r a t e w i t h 0.005 N - H C l O 4 u n t i l t h e
c o l o r changes from y e l l o w t o pink. For aqueous solu-
t i o n o f a t r o p i n e s u l p h a t e , make a l k a l i n e w i t h aqueous
sodium b i c a r b o n a t e , e x t r a c t w i t h chloroform and
t i t r a t e t h e e x t r a c t a s d e s c r i b e d above.

9.3.3 Gravimet r i c T i t rat i o n

Poethke and T r a b e r t ( 8 6 ) have u t i l i z e d potassium

iodobismuthate f o r t h e d e t e r m i n a t i o n of small q u a n t i -
t i e s o f a t r o p i n e and o t h e r a l k a l o i d s . The method i s
based on t h e p r i n c i p l e s developed f o r t h e determina-
t i o n o f 8-hydroxyquinoline (87) i s d e s c r i b e d . The
drug i s determined by p r e c i p i t a t i n g i t s iodobismuthate
and, e i t h e r d e t e r m i n i n g it g r a v i m e t r i c a l l y .

The above a u t h o r s ( 8 8 ) have a l s o determined a t r o p i n e

i n ampules, eye o i n t m e n t , p i l l s and e x t r a c t s o f
b e l l a d o n n a , and i n t a b l e t s and stomach powders con-
t a i n i n g b e l l a d o n n a . Good results were o b t a i n e d when
ATROPINE 365

a s s a y i n g comparatively s m a l l amounts o f t h e drug.

Van P i n x t e r e n et a1 (89) have r e p o r t e d t h e determina-

t i o n o f a t r o p i n e by means o f t e t r a p h e n y l b o r o n
( K a l i g n o s t ) . By u s i n g Flaschkas sodium t e t r a p h e n y l -
boron method ( 9 0 , 9 1 ) f o r t h e d e t e r m i n a t i o n o f a t r o p i n e
i n a l k a l o i d a l s a l t s and g a l e n i c a l s , r e c o v e r i e s v a r i n g
f r o m 8 l . g t o 99.6% were o b t a i n e d according t o t h e
volume o f s o l u t i o n analysed. Reasonable r e s u l t s were
o b t a i n e d by reducing t h e volume o f s o l u t i o n t o 25 m l
and w i t h 1 0 t o 25 mg o f a t r o p i n e . By applying t h e
g r a v i m e t r i c method t o 50 t o 100 ml samples o f
Maceratum R a d i c i s Belladonnae, a c c u r a t e results were
o b t a i n e d over t h e range o f about 0.020 t o 0.035% of
atropine.

9.3.4 Potentiometric Titrat ion

Pernarowski and Blackburn ( 9 2 ) have c a r r i e d out a

p o t e n t i o m e t r i c t i t r a t i o n of a t r o p i n e . The t i t r a t i o n
i s c a r r i e d o u t i n chlorobenzene w i t h g l a s s and sleeve-
t y p e calomel e l e c t r o d e s ; 0 . 0 5 N H C l O 4 i n g l a c i a l
a c e t i c a c i d i s t h e most s u i t a b l e t i t r a n t . Bromophendl
blue i s a suitable indicator f o r t i t r a t i o n s i n
chlorobenzene t o a v i s u a l end p o i n t . The r e s u l t s o f
s i x t i t r a t i o n s o f a t r o p i n e showed a n average recovery
of 99.7% and s t a n d a r d d e v i a t i o n of 0.55%.

9 . 4 P o l a r o g r a p h i c Methods
Souckova and Zyka (93,94) have r e p o r t e d two p o l a r o g r a -
p h i c t i t r a t i o n methods f o r t i t r a t i o n o f o r g a n i c b a s e s
i n c l u d i n g a t r o p i n e . The f i r s t method i s t h e t i t r a -
t i o n w i t h t u n g s t o s i l i c i c a c i d , and t h e second i s
t i t r a t i o n w i t h tungstophosphoric and molybdo phos-
p h o r i c a c i d s . The l a t t e r method i s r e p o r t e d t o b e
u n s a t i s f a c t o r y f o r a t r o p i n e . The f i r s t method a l l o w s
a c c u r a t e d e t e r m i n a t i o n of 1 0 t o 20 mg of a base.

Novotny ( 9 5 ) have published a p o l a r o g r a p h i c determina-

t i o n o f a t r o p i n e i n m i x t u r e s . The drug i s e x t r a c t e d
from a l k a l i n e s o l u t i o n w i t h chloroform, evaporated
and a t r o p i n e i s n i t r a t e d w i t h HNO3-H2SOq mixture
( > 1O:l) on water b a t h for 30 minutes. The m i x t u r e
i s made a l k a l i n e and, a f t e r removing oxygen by means
o f n i t r o g e n , polarography o f t h e s o l u t i o n i s c a r r i e d
o u t . The polarogram i s compared w i t h one prepared
from a s i m i l a r sample t o which a known amount of
a t r o p i n e i s added.
366 ABDULLAH A . AL-BADR A N D FARID J . MUHTADI

An O s i l l o p o l a r o g r a p h i c s t u d y o f a t r o p i n e and o t h e r
a l k a l o i d s i s r e p o r t e d by Habersberger and Zyka ( 9 6 ) .
O s i l l o p o l a r o g r a p h i c curve o f a t r o p i n e w a s s t u d i e d
w i t h a dropping mercury e l e c t r o d e . A carbon e l e c t r o d e
was used a r e f e r e n c e e l e c t r o d e .

Some a s p e c t s of t h e p o l a r o g r a p h i c d e t e r m i n a t i o n o f
a t r o p i n e i s r e p o r t e d by Benraad and U f f e l i e ( 9 7 ) .
Experimental evidence i s produced which i n d i c a t e s t h e
r e a c t i o n o f a t r o p i n e a t t h e droping mercury e l e c t r o d e
i n 0 . 1 N L i C l i s a simple r e d u c t i o n p r o c e s s .

9.5 Spectrophotometric Methods

9.5 .1 Colorimetry
Atropine h a s been determined c o l o r i m e t r i c a l l y ,
among o t h e r a t r o p a a l k a l o i d s , by t h e u s e of
new r e a g e n t s . An a b s o r p t i o m e t r i c method i s
d e s c r i b e d ( 9 8 ) f o r t h e d e t e r m i n a t i o n of a t r o -
p i n e and r e l a t e d a l k a l o i d s . The well-known
Vitali-Morin r e a c t i o n w a s i n v e s t i g a t e d w i t h a
view t o improving t h e s t a b i l i t y o f t h e c o l o r e d
formed. It w a s found t h a t t h e b e s t r e s u l t s
were o b t a i n e d w i t h tetraethylammonium hydro-
x i d e as t h e b a s e and dimethylformamide as t h e
s o l v e n t . The s o l u t i o n (0.05-0.15 mg of
a l k a l o i d ) i s evaporated t o d r y n e s s , n i t r a t e d
w i t h 0 . 2 t o 0 . 3 m l o f fuming HNO3, a g a i n
e v a p o r a t e d , d i s s o l v e d i n dimethylformamide,
t r e a t e d w i t h 0.3 ml o f 25 p e r c e n t aa. t e t r a e -
thylammonim hydroxide and d i l u t e d t o 1 0 ml
w i t h dimethylformamide. The o p t i c a l d e n s i t y
i s determined a t 540 mu i n . 1-cm c e l l s a g a i n s t
dimethylformamide and t h e a l k a l o i d a l c o n t e n t
i s a s c e r t a i n e d from a c a l i b r a t i o n graph which
is linear.

Simonyi and Tokar ( 9 9 ) have r e p o r t e d a new

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 m a l l amounts o f t r o p i c a c i d and i t s e s t e r s .
A t r o p i n e w a s n i t r a t e d f o r 1 5 minutes at 50'
w i t h a s o l u t i o n of 20% mO3 i n conc. H2S04.
On making t h e product a l k a l i n e w i t h hot 18 t o
20% N a O H , a c o l o r develops i n 30 m i n u t e s . This
i s e s t i m a t e d by u s i n g an S42, S47 o r S50
f i l t e r i n t h e P u l f r i c h photometer. The s e n s i -
t i v i t y i s 50 and 60 ug o f a t r o p i n e p e r ml. The
probable e r r o r i s * 3%.
ATROPINE 367

Nir-Grosfeld and Weissenberg (100)have

r e p o r t e d two c o l o r i m e t r i c methods f o r t h e
d e t e r m i n a t i o n o f a t r o p i n e i n pharmaceutical
p r e p a r a t i o n s . Recovery experiments i n d i c a t e
an accuracy of ? 1%.The results a g r e e w i t h
t h e s e o b t a i n e d by t h e method o f USPXV.
I n method I , a chloroform e x t r a c t , prepared by
t h e USP method, i s evaporated t o dryness on a
water b a t h . N i t r i c a c i d (fuming) w a s added,
and h e a t e d t i l l fuming c e a s e d , d r i e d a t l O 5 O
f o r 1 5 min and allowed t o c o o l . The r e s i d u e
o b t a i n e d w a s d i s s o l v e d i n a c e t o n e and d i l u t e d
t o 25 m l . An a l i q u o t ( 5 m l ) w a s mixed w i t h
isoproprylamine and 0.1% methanolic KOH and
t h e e x t i n c t i o n a t 540 mu w a s measured a f t e r
one minute.
I n method 11. The compound i s n i t r a t e d as i n
method I and d i s s o l v e d i n 50% e t h a n o l ( 1 0 m l ) .
Heated on a water bath w i t h 1% HC1 and z i n c
d u s t f o r 1 0 minutes, cooled and f i l t e r e d . The
z i n c r e s i d u e w a s washed w i t h H2O and t h e
washings were added t o t h e f i l t r a t e . 1% o f
NaN02 i s added, mixed and allowed t o s t a n d
for 1 0 minutes. To t h i s 92.5% s o l u t i o n of
ammonium sulphamate was added, shaken and
allowed t o s t a n d f o r 1 0 minutes. N-l-
naphthylethylenediamine d i h y d r o c h l o r i d e solu-
t i o n was added, d i l u t e d w i t h water t o 25 ml
and a f t e r 30 min, t h e e x t f n c t i o n a t 550 mu w a s
measured.

Pohm (101) r e p o r t e d a micro-determination o f

a t r o p i n e c o l o r i m e t r i c a l l y , by means of p-
dimethylaminobenzaldehyde. Atropine i s mixed
w i t h e t h e r and aq. N H 3 and s i t a s i d e f o r two
hours and f i l t e r e d . The f i l t e r e d e x t r a c t i s
extra.cted w i t h 0.05 N H C 1 . The HC1 e x t r a c t i s
made a l k a l i n e (NaOH) and e x t r a c t e d w i t h chloro-
form, evaporated t o d r y n e s s . Three drops o f
aq. bromine are added and evaporated o f f . The
r e s i d u e i s d i s s o l v e d i n methanol and a g a i n
evaporated w i t h 3 drops o f aq. bromine. A f t e r
drying f o r 2 hours o v e r P2O5, t h e r e s i d u e i s
t r e a t e d w i t h 7 drops of Wasicky reagent ( a
s o l u t i o n o f 1. gm of p-dimethylaminobenzalde-
hyde i n 9 o f 88% H2SO4) and s i t a s i d e f o r
2 minutes. It i s t h e n h e a t e d f o r 3 minutes i n
368 ABDULLAH A . AL-BADR AND FARID J . MUHTADI

a b o i l i n g w a t e r b a t h a n d cooled i n i c e f o r 1 5
seconds. A c e t i c anhydride i s added w i t h
s t i r r i n g and a f t e r 30 m i n u t e s , t h e e x t i n c t i o n
i s measured at 500 mu.

Atropine h a s been determined @02) c o l o r i m e t r i -

c a l l y by means of Reineck's s a l t . Ammonium
r e i n e c k a t e w a s used f o r t h e d e t e r m i n a t i o n of
a t r o p i n e i n 1% H2SO4. Ammonium r e i n e c k a t e
s o l u t i o n ( 0 . 5 % ) w a s added t o t h e t e s t s o l u t i o n ,
t h e m i x t u r e was p l a c e d i n a r e f r i g e r a t o r f o r
30 minutes and t h e p r e c i p i t a t e i s c o l l e c t e d on
a g l a s s f i l t e r , washed w i t h c o o l e d water and
d i s s o l v e d i n a c e t o n e . The e x t i n c t i o n i s t h e n
measured a g a i n s t a r e a g e n t b l a n k .

The e x t r a c t ion-spectrophotometric determination

method f o r t h e a s s a y o f a t r o p i n e w i t h t h e u s e
o f vanadium c a t e c h o l a t e h a s been r e p o r t e d by
S h e s t e r o v a e t a l . (103). The method i n v o l v e s
formation o f a w a t e r - i n s o l u b l e V'v-catechol-
a t r o p i n e (1:2:1) complex ( I ) i n an aq. medium
a d j u s t e d t o pH 3 t o 4 w i t h hydrogen p h t h a l a t e
b u f f e r s o l u t i o n c o n t a i n i n g a 200-fold molar
e x c e s s ( r e l a t i v e t o I ) o f VO; and an 8000-f01d
molar e x c e s s o f c a t e c h o l and e x t r a c t i o n o f
t h i s complex i n t o chloroform. The complex
e x h i b i t s max. a b s o r p t i o n a t 620 nm.

Semenicheva Qo4) r e p o r t e d a method f o r t h e

d e t e r m i n a t i o n o f a t r o p i n e s u l p h a t e i n eye
drops. Atropine sulphate i n n e u t r a l s o lu tio n
i s t r e a t e d w i t h sodium p i c r a t e and t h e atropine
p i c r a t e formed i s e x t r a c t e d w i t h chloroform;
a f t e r removal o f chloroform, t h e p i c r a t e i s
t r e a t e d w i t h sodium s u l p h i d e s o l u t i o n and t h e
c o l o r o f t h e sodium p i c r a m a t e formed i s com-
pared w i t h s t a n d a r d p r e p a r e d by reducing p i c r i c
a c i d s o l u t i o n i n t h e same way.

9.5.2 Photometric A n a l y s i s

Akopyan (105) h a s r e p o r t e d a photometric method

for t h e d e t e r m i n a t i o n of a t r o p i n e and o t h e r
t r o p a n a l k a l o i d s i n pharmaceutical m i x t u r e s .
The d e t e r m i n a t i o n i s based on t h e r e a c t i o n o f
t h e a l k a l o i d ( a t r o p i n e ) w i t h p-aminobenzalde-
hyde on c o n c e n t r a t e d s u l p h u r i c a c i d . The
ATROPINE 369

i n t e n s i t y o f t h e c o l o r produced being measured

i n a photometric a b s o r p t i o m e t e r w i t h a g r e e n
filter .
--
Fahmy e t a l . (106,107)have p u b l i s h e d a compa-
r a t i v e s t u d y o f t h e d i f f e r e n t photometric
methods o f d e t e r m i n a t i o n o f a t r o p i n e : -

I. The t u n g s t o s i l i c i c a c i d , tungstophos-
p h o r i c a c i d , copper s u l p h a t e , sodium
p i c r a t e and p-dimethylaminobenzaldehyde
methods are s u i t a b l e f o r t h e microdeter-
mination o f a t r o p i n e i n t o x i c o l o g i c a l
samples. V i t a l i ' s method i s p r e f e r r e d .

11. The u s e of bromothymol b l u e , bromocresol

p u r p l e , M e t a n i l yellow (C.I. a c i d yellow
36) and methyl orange, and v a r i o u s
o r g a n i c s o l v e n t s , i n t h e alkaloid-dye
method of d e t e r m i n a t i o n h a s been s t u d i e d .
The combination of M e t a n i l yellow and
chloroform i s most convenient.

The u s e o f ammonium r e i n e c k a t e i n t h e photo-

m e t r i c d e t e r m i n a t i o n o f a t r o p i n e , h a s been
d e s c r i b e d (108). The procedure i s as follows:
To t h e s o l u t i o n c o n t a i n i n g from 2 t o 1 0 mg of
a t r o p i n e add 0.5 N H$O4 ( 2 d r o p s ) and s a t u -
r a t e d ammonium r e i n e c k a t e s o l u t i o n , w i t h s t irr-
i n g . C o l l e c t t h e p r e c i p i t a t e on a s i n t e r e d -
g l a s s f i l t e r (Gb), wash it w i t h c o l d water,
and d i s s o l v e it i n dioxan a c i d i f i e d w i t h 0.5 N-
H2SO4. Measure t h e e x t i n c t i o n o f t h e dioxan
s o l u t i o n a t 530 mu, and refer t h e r e s u l t s t o a
c a l i b r a t i o n curve. The method w a s used for
determining a t r o p i n e i n t a b l e t s .

Levine and Roe 609) have d e s c r i b e d a method

for t h e d e t e r m i n a t i o n o f a t r o p i n e and t r o p i c
acid.
Atropine and t r o p i c a c i d were s e p a r a t e d from
each o t h e r and from p r e s e r v a t i v e s such as
benzyl a l c o h o l or phenol by p a r t i t i o n chromato-
graphy and determined by a modified V i t a l i pro-
cedure. The chromatographic procedure employs
two columns connected i n s e r i e s , w i t h C e l i t e
545 as s u p p o r t i n g phase. I n column A t h e
370 ABDULLAH A . AL-BADR A N D FARID 3 . MUHTADI

sample ( 2 m l ) made a l k a l i n e w i t h N NaHC03

(1m l ) absorbed on C e l i t e (4 g + 1 g ) , c o n s t i -
t u t e s t h e s t a t i o n a r y phase and i n column 8 t h e
s t a t i o n a r y phase i s 0.2 N H2SO4 ( 2 ml) absorbed
on C e l i t e ( 3 g + 1 g ) . On washing t h e columns
w i t h chloroform ( 1 0 0 m l + 25 m l t h r o u g h column
B o n l y ) , t r o p i c a c i d remains on column A .
Atropine i s absorbed column B , and p r e s e r v a -
t i v e s p a s s b o t h columns. Tropic a c i d i s
e l u t e d from column A w i t h e i t h e r a f t e r a c i d i -
f i c a t i o n o f t h e column w i t h a c e t i c a c i d i n
e t h e r , and a t r o p i n e i s e l u t e d from column B
w i t h chloroform a f t e r n e u t r a l i z a t i o n o f t h e
column w i t h aqueous ammonia. A t r o p i n e and
t r o p i c a c i d are converted i n t o t h e i r s a l t s by
a d d i t i o n o f HC1 and aqueous ammonia respec-
t i v e l y , and evaporated t o d r y n e s s . For the
modified c o l o r i m e t r i c p r o c e d u r e , t r e a t t h e d r y
r e s i d u e on a steam b a t h f o r 30 minutes w i t h
fuming n i t r i c a c i d (1m l ) i n a covered f l a s k .
Add w a t e r ( 1 0 m l ) , aqueous ammonia, ( 2 m l ) ,
sodium d i t h i o n i t e ( a b o u t 50 mg) and 5% NaN02
s o l u t i o n ( 5 m l ) and h e a t f o r a f u r t h e r f i v e
m i n u t e s , add 5% sulphamic a c i d s o l u t i o n ( 1 0 m l )
and remove n i t r o u s fumes i n a c u r r e n t o f air.
Add 25 mg o f s o l i d N-1-naphthylethylenediamine
d i h y d r o c h l o r i d e , make up t o volume, s e t a s i d e
f o r 0 . 5 t o 4 h o u r s , and compare t h e e x t i n c t i o n
a t 550 mu w i t h v a l u e s o b t a i n e d from s t a n d a r d s
t r e a t e d s i m i l a r l y . Beer's l a w i s obeyed up t o
a t le a s t 4 mg r e c o v e r i e s were from 1 0 0 t o 103%.

Febvre (11.0) r e p o r t e d t h a t Vitali-Morin r e a c -

t i o n f o r a t r o p i n e i s modified t o g i v e a repro-
d u c i b l e c o l o r t h a t can be used q u a n t i t a t i v e l y .
A knotjnvolume o f t h e sample i s evaporated t o
d r y n e s s under vacuum i n a c e n t r i f u g e t u b e ,
t h e n t r e a t e d w i t h a f e w drops o f a m i x t u r e o f
7 rd. of H2SO4 (66" B e ' ) and 2 m l o f fuming H N O ~
and s t i r r e d t o make t h e s o l u t i o n homogenous.
Acetone ( 2 m l ) i s added qnd 10% a b s o l u t e e t h a -
n o l i c KOH ( t h e p r e s e n c e of water o r methanol
v i t i a t e s r e a c t i o n ) drop by drop u n t i l t h e s o l u -
t i o n i s n e u t r a l i z e d , when t h e c o l o r a p p e a r s a t
once. A f t e r c e n t r i f u g a t i o n t o remove s o l i d
p r e c i p i t a t e d by t h e a c e t o n e and making up t o
1 0 ml w i t h a c e t o n e ; t h e e x t i n c t i o n i s measured
(Filter 63 o f t h e Jobin - Yvon Spectrophotometer).
ATROPINE 371

The e x t i n c t i o n i s s t a b l e f o r 1 0 minutes at
20'. The Beer-Lambert's l a w i s followed
o n l y f o r c o n c e n t r a t i o n from 5 t o 20 vg p e r m l ,
but f o r h i g h e r c o n c e n t r a t i o n , a c a l i b r a t i o n
curve can be used. Above 100 ug p e r m l t h e
s e n s i t i v i t y f a l l s o f f . The mean e r r o r i s
about 1%. No c o l o r i s g i v e n by t h e h y d r o l y s i s
products of atropine .
9.5.3 U l t r a v i o l e t Spectrophotometric Methods

Systematic t o x i c o l o g i c a l a n a l y s i s by s p e c t r o -
photometric methods have been p u b l i s h e d (111).
The sample o f t i s s u e i s homogenized w i t h 25 m l
o f 0 . 1 N HC1; t h e homogenate i s e x t r a c t e d on a
w a t e r b a t h w i t h 75 m l 95% e t h a n o l and 2 m l , 10%
Na2W04. The r e s i d u e i s being d i s s o l v e d i n 50
m l o f M c l l r a i n s ' s b u f f e r a t pH 7 and e x t r a c t e d
w i t h chloroform ( 5 0 m l ) . The s e p a r a t e d
chloroform l a y e r i s t h e n e x t r a c t e d 1 0 0 m l o f
0 . 1 N HC1. The c h a r a c t e r i s t i c U.V. a b s o r p t i o n
curves f o r 30 a l k a l o i d s i n d i l . HC1 a r e pre-
s e n t e d ; a t r o p i n e can be determined q u a n t i t a -
t i v e l y by t h i s method.

Cross e t a l . Q12) have determined some a l k a -

l o i d s including atropine spectrophotometrically
and d e s c r i b e d i t s a p p l i c a t i o n t o pharmaceutical
p r e p a r a t i o n s . To determine a t r o p i n e , add 1%
sodium p i c r a t e s o l u t i o n ( 3 m l ) t o a s o l u t i o n o f
a t r o p i n e (1mg) i n phosphate b u f f e r s o l u t i o n
(pH 7 ) ( 2 0 m l ) , e x t r a c t w i t h chloroform, shake
t h e e x t r a c t w i t h phosphate b u f f e r s o l u t i o n ,
(pH 11.2 t o 1 1 . 5 ) ( 4 0 m l ) d i l u t e t h e aq. phase
w i t h t h e same b u f f e r s o l u t i o n t o 1 0 0 m l , and
measure t h e e x t i n c t i o n at 355 m u .

Waaler and Bjerkelund (ll3) have d e s c r i b e d t h e

f o l l o w i n g p r o c e d u r e , for t h e u l t r a v i o l e t
d e t e n n i n a t i o n o f a p o a t r o p i n e and b e l l a d o n i n e
i n atropine:
"Prepare a s o l u t i o n of t h e m i x t u r e i n 0 . 1 N -
H2S04 c o n t a i n i n g 15% o f e t h a n o l , and measure
t h e e x t i n c t i o n a t 261.5, 257.5 and e i t h e r
248.5 or 254.0 my". Calculate t h e content of
each a l k a l o i d by s o l u t i o n o f t h e t h r e e approp-
r i a t e simultaneous e q u a t i o n s . The e x t i n c t i o n
c o e f f i c i e n t o f each compound at each wavelength
i s given.
372 ABDULLAH A. AL-BADR AND FARID J . MUHTADI

A t r o p i n e w a s determined s p e c t r o p h o t o m e t r i c a l l y
i n eye drops by Zabrak and Farkas (114). The
a b s o r p t i o n s p e c t r a o f a t r o p i n e show a m a x i m a
a t 186 mu. D i l u t e 1 m l o f t h e sample t o 100
ml and 5 ml o f t h i s s o l u t i o n i s f u r t h e r
d i l u t e d t o 100 ml w i t h w a t e r and measure t h e
e x t i n c t i o n a t 186 m u a g a i n s t water. Beer's
l a w i s obeyed o v e r t h e r a n g e 0 t o 8 pg p e r ml.
The r e s u l t s o b t a i n e d by t h i s method are w i t h i n
1% o f t h o s e o b t a i n e d by e x t r a c t i o n methods.

Uhlmann (115) r e p o r t e d a s p e c t r o p h o t o m e t r i c
a s s a y method f o r a t r o p i n e and some n a r c o t i c s
and a l k a l o i d s i n g a l e n i c a l compositions. To
a s s a y t h e drug i n aq. s o l u t i o n o f i t s s a l t ,
t h e e x t i n c t i o n o f t h e d i l u t e d sample i s
determined at t h e wavelength f o r maximum
a b s o r p t i o n (257 t o 286 nm) and compared w i t h
t h a t o f p r o g r e s s i v e l y d i l u t e d samples o f s t o c k
s o l u t i o n . The method i s c h i e f l y designed f o r
use on aq. p r e p a r a t i o n s ( ampoules).

9.5.4 I n f r a - r e d S p e c t r o p h o t o m e t r i c Method

The a p p l i c a t i o n o f i n f r a - r e d s p e c t r o m e t r y t o
q u a n t i t a t i v e analysis of a tro p in e i n t h e s o l i d
phase h a s been r e p o r t e d by Browning e t a l .
(116). The p r e s s e d potassium bromide b e l l e t
t e c h n i q u e h a s been s u c c e s s f u l l y a p p l i e d as a n
a i d i n t h e quantitative determination of atro-
p i n e by I R spectrophotometry.

9.5.5 F l u o r o m e t r i c Analysis

Laugel (119have p u b l i s h e d a method f o r t h e

d e t e r m i n a t i o n o f a t r o p i n e and , o t h e r a l k a l o i d ,
based on t h e f l u o r e s c e n c e o f compounds o f t h e
t y p e a c i d dye-azo b a s e . The c o n c e n t r a t i o n of
a t r o p i n e i n pharmaceutical p r e p a r a t i o n i s
determined ( t o w i t h i n 4%) by measuring t h e
f l u o r e s c e n c e o f t h e complex formed q u a n t i t a -
t i v e l y , i n chloroform s o l u t i o n , by a t r o p i n e
w i t h a d i h y d r o x y l l u r a n a c i d dye, e . g . c o s i n .
The c o n c e n t r a t i o n which i s d i r e c t l y propor-
t i o n a l t o t h e f l u o r e s c e n c e (measured at 550
m u ) , i s o b t a i n e d from a s t a n d a r d c a l i b r a t i o n
curve for a t r o p i n e . Beer's l a w b e i n g obeyed
f o r 1 0 t o 60 pg o f a t r o p i n e .
ATROPINE 313

Shuntaro Ogawa e t a l . (118) have determined t h e

f l u o r i m e t r y of a t r o p i n e with e o s i n yellowish
( C . I . Acid Red 87). The method which i s simple
and r a p i d i s based on t h e formation of f l u o r e s -
cent complex between a t r o p i n e and eosine. To a
s o l u t i o n of a t r o p i n e i n chloroform ( 9 m l ) i s
added 0.1% eosine s o l u t i o n (1m l ) , t h e mixture
i s shaken thoroughly and t h e fluorescence
i n t e n s i t y at 556 mu ( e x c i t a t i o n a t 365 mu) i s
measured a f t e r 1 0 minutes. Beer's law i s
obeyed with 1 t o 5 pg of a t r o p i n e p e r m l ; t h e
c o e f f i c i e n t of v a r i a t i o n i s 2.6%.

9.5.6 Phosphorimetric Analysis

Winefordner and Tin (119) have determined a t r o -

p i n e i s u r i n e . A r a p i d method w a s described
f o r t h e e x t r a c t i o n of a t r o p i n e from body
f l u i d s ; t h e concentration of t h e drug i s
determined by phosphoresence measurement and
comparison w i t h standard s o l u t i o n .

9.6 Chromatographic Methods

9.6.1 Paper Chromatography

Clarke ( 2 1 ) described two systems:

Whatman No. 1, sheet 1 4 X 6 i n , b u f f e r e d

by dipping i n a 5% s o l u t i o n o f sodium
hydrogen c i t r a t e , b l o t t i n g and drying a t
25' f o r one hour. It can be s t o r e d i n d e f i -
n i t e l y . A sample of 3 1.111% s o l u t i o n i n
2 N a c e t i c a c i d o r i n ethanol i s used.
Solvent system: 4.8 gm of c i t r i c a c i d i n
a mixture of 130 m l of water and 870 m l of
n-butanol ( t h i s solvent may be used f o r
s e v e r a l weeks i f water i s added from t i m e
t o t i m e t o keep t h e s p e c i f i c g r a v i t y ak
0.843 t o 0.844). The chromatogram i s
developed, ascending i n a t a n k 8 X 11 X 15%
i n . 4 Sheets being run at a time. Loca-
t i o n i s done under u l t r a v i o l e t l i g h t and
t h e l o c a t i o n reagent i s i o d o p l a t i n a t e
spray,Rf = 0.37.

2) Whatman No. 1 o r No. 3, sheet 17 X 19 cm,

impregnated by dipping i n a 10% s o l u t i o n
3 74 ABDULLAH A . AL-BADR AND FARID J . MUHTADI

o f t r i b u t y r i n i n a c e t o n e and d r y i n g i n
a i r . A sample o f 5 1.11o f 1 t o 5 % s o l u t i o n
i n e t h a n o l or chloroform, u s i n g a c e t a t e
b u f f e r (pH 4.58) as s o l v e n t . The beaker
containing t h e solvent i s equilibrated i n
a t h e r m o s t a t i c a l l y c o n t r o l l e d oven a t 9 5 O
f o r 1 5 minutes. The chromatogram i s
developed, a s c e n d i n g , where t h e paper i s
f o l d e d i n t o a c y l i n d e r and c l i p p e d . The
c y l i n d e r i s i n s e r t e d i n t h e beaker c o n t a i n -
i n g t h e s o l v e n t which i s n o t removed from
t h e oven. A p l a t e - g l a s s d i s k t h i c k l y
smeared w i t h s i l i c o n e g r e a s e may s e r v e as
a c o v e r . T i m e run 1 5 t o 20 minutes. The
location reagent i s iodoplatinate spray
and Rf = 0.94.

Other paper chromatography systems have been

p u b l i s h e d (120-136).

9.6.2 Thin-Layer Chromatography

Clarke (21) d e s c r i b e d t h e f o l l o w i n g system f o r

t h e separation of atropine:

Glass p l a t e s 20 X 20 em, c o a t e d w i t h a s l u r r y
c o n s i s t i n g o f 30 g of s i l i c a g e l G i n 60 m l o f
water t o g i v e a l a y e r 0 . 2 5 mm t h i c k and d r i e d
at llOo f o r 1 hour. A sample o f 1 . 0 1-11of 1%
s o l u t i o n i n 2 N a c e t i c a c i d , t a k e n by a micro
d r o p , i s used. The s o l v e n t system c o n s i s t s
of s t r o n g ammonia s o l u t i o n : methanol (1.5 :
1 0 0 ) . It should be changed a f t e r two r u n s .
Solvent i s allowed t o s t a n d i n t h e t a n k f o r 1
hour. The ascending chromatogram i s developed
i n a t a n k 2 1 X 2 1 X 10 em, t h e end o f t h e t a n k
being covered w i t h f i l t e r paper t o a s s i s t
e v a p o r a t i o n . Time o f run 30 m i n u t e s . The
l o c a t i o n r e a g e n t i s an a c i d i f i e d i o d o p l a t i n a t e
spray: and t h e Rf v a l u e i s 0.18.

Other TLC systems have been p u b l i s h e d (133435,

137-140) for t h e s e p a r a t i o n o f a t r o p i n e .

9.6.3 Eigh P r e s s u r e Liquid Chromatography

S t u t z and S a s s (141) have d e s c r i b e d a high-

speed, h i g h p r e s s u r e l i q u i d chromatography of
ATROPINE 375

a t r o p i n e and o t h e r t r o p a n e a l k a l o i d s . The
compound w a s s e p a r a t e d on a s t a i n l e s s - s t e e l
column (1meter X 4.6 mm) packed w i t h s i l - X
absorbent w i t h 28% aq. NH3-tetrahydrofuran
(1:lOO) as s o l v e n t and w i t h a column i n l e t
p r e s s u r e o f 500 l b p e r s q . i n . A d i f f e r e n t
r e f r a c t i v e index d e t e c t o r and a W d e t e c t o r
o p e r a t i n g at 254 nm were used t o monitor t h e
e l u a t e . When a p p l i e d q u a n t i t a t i v e l y , recove-
r i e s o f a t r o p i n e s u l p h a t e added t o v a r i o u s
a l k a l o i d samples were between 88 and 94.5% a t
t h e = 25 pg l e v e l .

F e l l e t a l . (142) have r e p o r t e d an a n a l y s i s o f
a t r o p i n e s u l p h a t e and i t s d e g r a d a t i o n p r o d u c t s
by reversed-phase high-pressure l i q u i d chro-
matography. Atropine was determined on a
column o f H y p e r s i l ODS ( 5 pm) w i l l 50 mM.
Sodium a c e t a t e i n 1 0 mM -tetrabutylammonium
s u l p h a t e (pH 5 . 5 ) - a c e t o n i t r i l e ( 3 : l ) a s
mobile phase and d e t e c t i o n a t 254 nm. The
i n t e r n a l s t a n d a r d was p - t o l u i c a c i d . Atropine
w a s w e l l s e p a r a t e d from it d e g r a d a t i o n pro-
d u c t s , t r o p i c a c i d a t r o p i c a c i d and apoatro-
pine.

V a n Buuren et e. (143) have published a

reversed-phase l i q u i d chromatography of b a s i c
drugs i n c l u d i n g a t r o p i n e - w i t h a f l u o r o g e n i c
ion-pair extraction detector.

Lawrance e t al. (144) have s e p a r a t e d a t r o p i n e

from o t h e r b a s i c o r g a n i c compounds by c o n t i -
nuous post-column i o n - p a i r e x t r a c t i o n d e t e c t i o n
i n normal-phase chromatography. The column
( 6 cm X 3 mm) of LiChrosorb S i 60 ( 5 pm) w i t h
a mobile phase (1m l min-l) o f 10% methanol
s o l u t i o n i n chloroform c o n t a i n i n g 0 . 1 M -
butyric acid. Detection of t h e fluorescence
o f t h e o r g a n i c phase w a s measured at 452 nm.

9. 6.4 Ion-Exchange Chromatography

Morphine s u l p h a t e w a s s e p a r a t e d from a t r o p i n e
s u l p h a t e by t h e ion-exchange chromatographytech-
nique (145). Determination w a s done by measuring
t h e u l t r a v i o l e t a b s o r p t i o n a t 258 mp E 1%
1 cm = 40. The two drugs cannot be s e p a r a t e d
376 ABDULLAH A. AL-BADR AND FARID J . MUHTADI

on a weakly b a s i c r e s i n , which c o n v e r t s b o t h
t o t h e f r e e a l k a l o i d s , b u t t h e a l k a l o i d s can
be s e p a r a t e d on a s t r o n g l y b a s i c r e s i n which
r e t a i n s o n l y t h e ( p h e n o l i c ) morphine. The
procedure o f t h e s e p a r a t i o n have been des-
c r i b e d as f o l l o w s :

D i l u t e t h e sample ( c o n t a i n i n g 400 mg of mor-

p h i n e s u l p h a t e and 10 mg o f a t r o p i n e s u l p h a t e ,
t o 50 m l w i t h 75 p e r c e n t methanol. To d e t e r -
mine t h e c o n c e n t r a t i o n o f morphine s u l p h a t e ,
d i l u t e a 10 m l a l i q u o t t o 1000 m l w i t h w a t e r
and measure t h e e x t i n c t i o n a t 285 mp. To
determine t h e c o n c e n t r a t i o n o f a t r o p i n e s u l -
p h a t e , p a s s a 25 m l a l i q u o t t h r o u g h a two-bed
column c o n t a i n i n g h b e r l i t e IR-4B ( 1 0 m l ) above
Amberlite IRA-410 ( 1 0 m l ) , e l u t e w i t h 7 5 p e r -
c e n t methanol (4 X 1 0 ml) and t i t r a t e t h e
e l u t e w i t h 0.02 N H C 1 w i t h bromothymol b l u e as
indicator.

9.6.5 Gas Chromatography

Clarke ( 2 1 ) d e s c r i b e s t h e f o l l o w i n g t h r e e
systems f o r t h e s e p a r a t i o n o f a t r o p i n e : -

Column: 1% SE-30 on 100-120 mesh Anakrom

ABS. 6 f t X 4 mm i n t e r n a l d i a m e t e r boro-
s i l i c a t e g l a s s column. Column t e m p e r a t u r e :
180°. Carrier g a s : Argon. Gas flow: 6 5
m l p e r minute a t 180. D e t e c t o r : Argon
i o n i s a t i o n d e t e c t o r o r flame i o n i s a t i o n
d e t e c t o r . Retention t i m e : 3.22 rnin.
r e l a t i v e t o diphemhydramine.

Column: 3% Q?-1 on 100-120 mesh Anakran

ABS, Column t e m p e r a t u r e : 200'. Carrier
g a s : Argon. Gas flow: 80 ml p e r minute.
Other c o n d i t i o n s are as i n system a.
R e t e n t i o n t i m e : 3.80 min. r e l a t i v e t o
diphenhydramine.

Column: 5% SE-30 on 60-80 mesh Chromosorb

W AW. 5 f t X 1/8 i n c h i n t e r n a l d i a m e t e r
s t a i n l e s s s t e e l column. Column temperature:
230'. Carrier g a s : n i t r o g e n . Gas flow:
30.7 m l p e r minute. Detector: flame i o n i z a -
t i o n d e t e c t o r , hydrogen 22 m l p e r minutes.
R e t e n t i o n t i m e : 0 . 5 9 min r e l a t i v e t o codeine.
ATROPINE 311

Santoro e t al,. Q46) have r e p o r t e d a s e l e c t i v e

d e t e r m i n a t i o n o f belladonna a l k a l o i d s by g a s
l i q u i d chromatography. Atropine w a s d e t e r -
mined i n pharmaceutical p r e p a r a t i o n s i n t h e
presence o f c e r t a i n m i n e s . A f t e r e x t r a c t i o n ,
t h e r e s i d u e i s d i s s o l v e d i n dichloromethane
and i n j e c t e d g l c on a g l a s s column (4 f t X 4
mm) c o n t a i n i n g 3% OV-17 on Gas-Chrom Q ( 8 0 t o
100 mesh) o p e r a t e d at 210' w i t h H e l i u m as
c a r r i e r g a s (50 m l p e r min) and flame i o n i z a -
t i o n d e t e c t i o n and measure t h e peak h i g h t s .

Nishimoto e t a l . &47) have d e s c r i b e d a s i m p l i -

f i e d q u a n t i t a t i v e a n a l y s i s o f a t r o p i n e and
o t h e r a l k a l o i d s i n s c o p o l i a e x t r a c t . Analysis
i s c a r r i e d o u t by g l c on columns (1mm X 3 mm)
packed w i t h 0.75% o f D e x s i l 300 GC on Gas
Chrom Q, w i t h n i t r o g e n (40 m l min-1) as c a r r i e r
gas; w i t h t h e column at ( u s u a l l y ) 180", a t r o -
pine (as i t s t r i m e t h y l s i l y l derivative i s
s e p a r a t e d from hyoscine, a p o a t r o p i n e and hom-
a t r o p i n e ( t h e i n t e r n a l s t a n d a r d . With t h e
column at 90' and t h e c a r r i e r g a s flow a t 30
m l min-l, a t r o p i n e i s e l u t e d i n about 7
minutes. Thermon 1000 w a s a l s o used as a
s t a t i o n a r y phase and diphenhydramine i s used
a s i n t e r n a l s t a n d a r d . The method i s a p p l i e d
t o g a s t r o i n t e s t i n a l drugs as w e l l as e x t r a c t s
o f s c o p o l i c r o o t s . The peak h i g h t r a t i o v s
a t r o p i n e c o n t e n t i s r e c t i l i n e a r f o r 25 t o 75
ng o f a t r o p i n e .
Other GC methods
Atropine t a b l e t s were e x t r a c t e d w i t h chloro-
form i n an a l k a l i n e media and analyzed u s i n g
a GC method w i t h diphenhydramine as t h e i n t e r -
n a l s t a n d a r d (148).

9.6.6 Colwnn Chromatography

Kamienski and Puchalka (149)have r e p o r t e d t h e

s e p a r a t i o n of a t r o p i n e and hyoscyamine by a
pot-entiometric chromatographic method. The
a l c o h o l i c e x t r a c t s from t h e l e a v e s Datura
stramonium and t h e r o o t s o f Atropa belladonna
were d i l u t e d u n t i l t h e i r a l k a l o i d c o n c e n t r a t i o n
approximately reached 0 . 0 0 1 M. The s e p a r a t i o n
o f a t r o p i n e and hyoscyamine i n t h e s e s o l u t i o n s
378 ARDULLAH A . AL-BADR AND FARlD J . MUHTADI

w a s s t u d i e d . Four m l o f each s o l u t i o n were

p l a c e d on alumina columns and e l u t e d w i t h
e i t h e r aqueous e t h a n o l ( 6 0 o r 80%) or a mixture
o f benzene; w a t e r and e t h a n o l (14.5%, 8.5% and
77% r e s p e c t i v e l y ) , t h e antimony m i c r o e l e c t r o d e
b e i n g used t o measure p o t e n t i a l change i n t h e
e l u t e d s o l u t i o n a g a i n s t t h e volume of t h e
e l u a t e . The most e f f i c i e n t s e p a r a t i o n was
achieved w i t h t h e benzene - e t h a n o l e l u t i n g
s o l u t i o n , and a 20 cm column o f Merck's
alumina.

9.6.7 Paper E l e c t r o p h o r e s e s
Atropine and hyoscine were s e p a r a t e d q u a n t i -
t a t i v e l y by paper e l e c t r o p h o r e s e s (150). They
were s e p a r a t e d w i t h 0 . 1 N aq. N H 3 a s t h e
e l e c t r o l y t e , and d e t e c t e d as brown s p o t s by
exposure t o i o d i n e vapour. After e l u t i o n o f
t h e spots, t h e solvent w a s evaporated, t h e
r e s i d u e w a s n i t r a t e d w i t h fuming H N O 3 , t h e n
d i s s o l v e d i n dimethylformamide and t e t r a e t h y l -
ammonium hydroxide w a s added a c c o r d i n g t o t h e
method o f Freenan ( 9 8 ) . The e x t i n c t i o n ( y )
o f each s o l u t i o n at 545 mp w a s measured and t h e
c o n c e n t r a t i o n o f each a l k a l o i d i s c a l c u l a t e d
from a given e q u a t i o n .

9.7. Radio-immunoassay

~y u s i n g 3~ a t r o p i n e as t r a c e r , an a n t i s e r u m w a s
r a i s e d by immunisation o f r a b b i t s w i t h an immunogen
prepared by c o u p l i n g t o human serum albumen. The
d e t e c t i o n of down t o 9 nM a t r o p i n e i n 0 . 1 m l o f serum
or plasma i s p o s s i b l e . The r e c o v e r y of a t r o p i n e
added a t v a r i o u s c o n c e n t r a t i o n t o pooled normal human
plasma w a s n e a r 100%. Atropine r e a c t s w i t h t h e a n t i -
b o d i e s ; o t h e r s t r u c t u r a l l y r e l a t e d drugs and a t r o p i n e -
h y d r o l y s i s p r o d u c t s ( t r o p i n e and t r o p i c a c i d ) do not
i n t e r f e r e . The u s e f u l n e s s o f t h i s method i n pharma-
c o k i n e t i c s s t u d i e s have been demonstrated i n a s s a y s o f
a t r o p i n e i n s e r i a l serum samples from two p a t i e n t s
who r e c i e v e d 1 . 3 mg o f a t r o p i n e i n c o n n e c t i o n w i t h
a n a e s t h e s i a (151).
- A p r e c i s e , s e n s i t i v e and r a p i d radioimmunoassay f o r
t h e a n a l y s i s of a t r o p i n e from u n p u r i f i e d e t h a n o l i c
e x t r a c t s of a t r o p i n e b e l l a d o n n a i s d e s c r i b e d (152).
ATROPINE 379

- F a s t h e t a l . (153) d e s c r i b e d t h e f i r s t radioimmuno-
a s s a y for a t r o p i n e u s i n g rabbit antiserum.

- Wurzburger e t a l . (154) r e p o r t e d and s e n s i t i v e and

s p e c i f i c radioimmunoassay for a t r o p i n e and showed
c l e a r a n c e curve f o r drog plasma.

- Radioimmunoassay (RIA) w a s a p p l i e d t o measure a t r o -

p i n e i n himan plasma u s i n g a n t i s e r u m , t h e plasma
c l e a r a n c e of a t r o p i n e i n f o u r a d u l t v o l u n t e e r s w a s
measured.

The measurement accomplished by a c o m p e t i t i v e R I A

u s i n g r a b b i t a n t i - a t r o p i n e antibody. T r i t i a t e d
a t r o p i n e i s used as t h e r a d i a l i g a n d (155).

Acknowledgement

The a u t h o r s would l i k e t o thank Mr. Uday C. Sharma and

Tanvir A. B u t t , b o t h of College of Pharmacy, King Saud
University for t h e i r valuable s e c r e t a r i a l assistance i n
t y p i n g of t h i s manuscript.
3 80 ABDULLAH A. AL-BADR AND FARlD J . MUHTADl

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82. N.P. Dzyuba and M.S. S h r a i b e r , Aptechn. Delo, 5 ( 6 ) ,

17 (1957).
83. I , Simonyi and G. Tokar , Magyar K e . Foly. , &( 4 ) , 151
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84. I . Simonyi and G. Tokar (Verein. Arznel - Und Nahrmit-
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85. I . S . Simon, W.P. Dzyuba and Yu. V. Shostenko, Aptech.
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86. W. Poethke and H. T r a b e r t , Pharm. Z e n t r a l h , 94(6), 219

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87. Ibid, Pharm. Z e n t r a l h . , 91,284 (1952).

88. W. Poethke and H. Tabert , Pharm. Zentralh., 94(11) 432

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89. J.A.C. Van P i n x t e r e n , M.E. Verloop and D. Westerink,
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90. A.M. Amin and A. Holasek, Z. Anal. Chem. , 136 99 (1952).

91. Ibid; -
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93. M. Souckova and J. Zyka, Ceskosl. Farmac., 4(4),181

(1955).
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95. B. Novotny ( S t a t e I n s t . f o r Control of Drugs, Prague,

Czechoslovakia). Ceskosl. Farmac. , 9 ) 448 (1955). k(
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97. T . J . Benraad and O.F. U f f e l i e (Pharm. Lab., R i j k s u n i v . ,
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386 ABDULLAH A. AL-BADR AND FARlD J . MUHTADI

98 P.M. Freeman, Analyst, 80, 520 (1955).

99 I . Simony? and C. Tokar, Fagyar K e n . Foly, g(lO), 348

(1956).
100. I , Nir-Grosfeld and E. Weissenberg, Drug Standards,
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101. P. Pohm, Mikrochim. Acta, (L),120 (1958).
102. S. Bartkowicz, Dessert. Pharm. , E ( 2 ) , 113 (1960).
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104. A.A. Semenicheva; Aptechnoe Delo, 2, 18 (1953).

105. O.A. Akopyan, Aptechnoe Delo, 7 ( 2 ) , 19 (1958).

106. I.R. Fahmy, Z.F. Ahmad and S.A. Eid, J. Chem. U.A.R.,
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Ibid, 3 ( 2 ) , 241
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107. I.R. Fahmy, Z.F. Ahmad and S.A. Eid.
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109. J . Levine and J.E. Roe, J. A s s . Off. Agric. Chem., 42(4),

693 (1959).

110. M.?. Febvre, Ann. Pharm. France, %(11),635 (1957).

111. E. Berman and H.N. Wright, Arch. Ind. Hyc. Occ. Med.,
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112. A.H. J. Cross, D. McClaren and S.G.E. Stevens, J. Pharm.
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113. T. Waaler and E. Bjerkelund, Pharm. Acta Helv., 2 ( 3 ) ,

150 (1964).
114. E. Zabrak and S. Farkas, Acta Pharm. Hung, &(5), 207
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115. Uhlmann, Hans-Jocken, Pharm. Ztg, Ber., *(50), 2029
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ATROPINE 387

116. R.S. Browning, S.E. Wiberley and F.C. Nachod, Anal.

Chem., a(1) 7, (1955).
117. P. Laugel, Compt. Rend., 255(4), 692 (1962).
118. S. Ogawa, M. Morita, K. Nishiura and K. Fujiwara, J.
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119. L.D. Winefordner and M. Tin, Analytica Chim. Acta, %(1),

64 (1965).
120. H. Kaiser and H. Tori, Arch. Pharm., Berlin, 28J(4),
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121. A. Resplandy, Compt. Rend., *(26), 2527 (1954).
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124. A. Liukkonen, Farm. Aikakauslehtic, 69, 286 (1960).
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127. V. Koen, Farmatisiya, Sofia, g ( 3 ) , 36 (1962).

128. J. Jankulov, Compt. Rend. Acad. Bulg. Sci., l
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129. J. Zarnack and S. Pfeifer, Pharmazie, 19(2), 111 (1964).

130. H. Czlonkowska and I. Gradecka Pob, Farmacja Pol.,

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131. E. Soczewinski and B. Szabelska, Dissnes Pharm., Warsz,

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132. W. Deckers and A. Muller, J. Chromat. , x(3)
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388 ABDULLAH A . AL-BADR AND FARlD J. MUHTADI

13. 2 . Buchi and A. Zimmermann, Pharm. Acta Helv., &(7),

395 (1965).

134. ,J. Buchi and A. Zimmermann, Pharm. Acta Helv., & ( 6 ) ,

361 (1965).

135. 2 . Buchi and A. Zimmermann, Pharm. Acta Helv., 4 0 ( 5 ) ,

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137. C.A. T e i j g e l e r , Pharm. Weekbl., =(14), 507 (1962).

138. A. Negh, 2. Eudvari, G. S z a s z , A. B r a n t n e r and K.

Gracza. Ibid. , 3 3 ( 2 ) , 67 (1963).
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14'3. K. Karatodorov; R. Kolarova, IZV. Durzh. I n s t . -

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1L1. ?,!.H. S t u t z , and S. S a s s , Anal. Chem., 4 5 ( 1 2 ) , 2131

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1113. Van Buuren, J.F. Lawrance, U.A.T. Brinkman, I.L.

Ho9gberg and R.W. F r e i ; Anal. Chem. 52, 700 ( 1 9 8 0 ) .

144. J.F. Lawrance, U.A.T. Brinkman and R.W. F r e i ; J.

Chrornat. 185,
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1145. S.N. Blaug, Drug S t a n d a r d s , 23(4), 143 (1955).

11;6. R.S. S a n t o r o ; P.P. Progner, E.A. Ambush, and D.E.

Guttman, J. Pharm. S c i . , e ( 8 ) , 1346 (1973).

1h7. N. Yishimoto; R. Kato, ar.d S. Hayashi, Jakugaku Z a s s h i ,

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149. B. Kamienski and K. Puchalka, B u l l Acad Polon S c i . , 1

305-309 ( 1953).
ATROPINE 389

150. K. Yamaguchi and M. I t o , J. Pharm. SOC. Japan, & ( 2 ) ,

179 (1961)
151. R. Virtanem, J , Kanto, and E. L i s a l o ; Acta Pharmacol.
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152. T. L e h t o l a , A. Huhtikangas and R. Virtanem, P l a n t a Med.
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153. A. F a s t h , J. S o l l e n b e r g , B. Sorbo; "Production and

c h a r a c t e r i s a t i o n of a n t i b o d i e s t o Atropine", Acta Pharm.
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154. R . J . Wurzhurger, R.L. Miller Boxenbaum "Radioimmuno-

a s s a y of a t r o p i n e i n plasma", J. Pharm. Exper. Therp.
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155 * P.W. Hayden, S.M. Larson and S. Lakshminarayanan, J.
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