Feb 3, 2011 - synthesis of atropine from tropine and tropic acid, thus proving ..... propoxide behaved as a monobasic acid when undergoing salt formation ...
ATROPINE Abdullah A. Al-Badr and Farid J. Muhtadi King Saud University Riyadh, Saudi Arabia 326 326 326 328 328 328 328 328 329 329 329 329 329 330 340 340 340 340 352 352 354 355 357 358 359 359 360 360 365 366 373 378 380
1. Description 1.1 Nomenclature 1.2 Formulae 1.3 Molecular Weight 1.4 Elemental Composition 1.5 Appearance, Color, Odor, and Taste I .6 Dissociation Constant 1.7 pH range 2. Physical Properties 2. I Melting Point 2.2 SublimationRange 2.3 Solubility 2.4 X-Ray Crystallography 2.5 Spectral Properties 3. Isolation 4. Synthesis 4.1 Partial Synthesis 4.2 Total Synthesis 5 . Biosynthesis 5.1 Biosynthesis of Tropine 5.2 Biosynthesis of Tropic Acid 6. Metabolism 7. Pharmacokinetics 8. Therapeutic Uses of Atropine 9. Methods of Analysis 9.1 Identification Tests 9.2 Microcrystal Tests 9.3 Titrimetric Methods 9.4 Polarographic Methods 9.5 SpectrophotometricMethods 9.6 Chromatographic Methods 9.7 Radio-immunoassay References
ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 14
325
Copyright 0 1985 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 oct3-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
7 6
The structure was confirmed by the total synthesis of atropine which was achieved by several authors ( 1-4)
.
.
327
ATROPINE
1.2.3
CAS Registry No.
51-55-8 1.2.4
I
Wiswesser Line Notation
~ 5 A6 ANTJ A -GOVYR & 1Q- DL 1.2.5
(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-substitue 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 great 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
0-C-CH
NCH~OH \
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 C-=O and N---H-O were i n d i a c t i o n s of t h e t y p e N--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 broadening 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 magnet 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, 1.5
70.56%; H , 8.01%; N ,
4.84%; 0 , 16.59%
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 ) .
329
ATROPINE
2.
Physical P r o p e r t i e s
2.1 Melting P o i n t 114 114 2.2
- 116' - 118'
(15)
(16)
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 U l t r a v i o l e t Spectrum
2.5.1
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
20 5
246 251.5 257 263.5 271
A ( 1 % , 1 cm)
-
-
147.6 175.1 209.8 143.3 24.6
5.1 6.05 7-25 4.95 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 stati 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
3070 2930
2810
1725 1595, 1580
Assignment OH (hydrogen bonded) CH ( s t r e t c h )
N-CH
3
B 0-C - ( e s t e r ) C=C aromatic
FIG, 1, THE UV SPECTRUM OF ATROPINE I N ETHANOL
44.
i &O#
WAVE##H#FR
#o
asM
2060
fm
YO0
1400
FIG. 2 , THE I R SPECTRUM OF ATROPINE AS KBR-DISC
1000
800
680
&
333
ATROPINE
-1 Frequency cm
Assignment
1155, 1030 770,725,690
C-0-C
(ether)
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 ) .
8
0- C9
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
I LO
TO
I I
..
I 40
1
I
. I
.,
. . uI .
a# Rn(,)
. .I .
F I G . 3 I A ) . TPE PYR SPECTnWi OF ATROPI3E Ill C D C L ~
.
I
B.#
....i
1..
, I , .
i
1.0
,
,
335
ATROPINE
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
Chemical S h i f t (ppm)
Group
5 aromatic protons 1 3 , l b ,15,16,17 H-3 CH2 CH2
CDC13
TFA
7.23(s)
7.36( s 1
- OH
- 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 m=mult iplet 2.5.3.2
, bs=broad
singlet ,
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 assigned 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 ) . 8
11 15
4
10
17
16
336
ABDULLAH A. AL-BADR AND FARID J . MUHTADI
1 1 1
d
FIG. 4 .
THE I3C-NER NOISE DECOUPLED SPECTRUE OF ATROPINE
I
337
ATROPINE
4.
Table Carbon no. C
Chemical S h i f t [PPd
Carbon no.
171.92( s )
9
‘17
‘13’
127.48 ( d )
‘15 3
67.63 ( d )
cll
63.54(t)
s = s i n g l e t , d=doublet
c4
c23
59.54(d)
40.08(q)
‘8
128.11 ( d )
Chemical S h i f t [ P P ~
54.94 ( d )
‘10
128.66( d )
‘14’ ‘16
c5
c1
136.17 ( s 1
5 2
C
Carbon Chemical S h i f t s of Atropine
36.04(t
7
25.31(t)
‘6
24.93(t
C
t=triplet
q=quartet
1
.
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 .
339
ATROPINE
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* 125- H
100.00
6.35 96
-
r-
10.67 c
4
95 94
8.60
96-H
22.66
954
83
18.87
82
25 * 97
67
14.78
44
5-15
42
21.83
41
8.36
i
-
-
:-CH2=N=CH2 r
+
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 ).
1
i I
340
3.
ABDULLAH A . AL-BADR AND FARID I. MUHTADI
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 racemization 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 aqueous 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 , dehyd 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 mixt 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
341
ATROPINE
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 reported. 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 modif 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 dimethylaminocyclohept-2-ene [ 51. The l a t t e r i s converted t o cyclohepta-lY3-diene [61 by exh 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-dibromocyclohept2-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 quino l i n e t o give cycloheptatriene [S]. Substance [8] i s t r e a t e d with hydrogen bromi 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-5dimethylamino-cycloheptane [ 111. This i s warmed i n e t h e r when intramolecular alkylat i o n occurs t o give 2-bromotropane methobromide [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-bromotropane [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 pseudot 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
15ooc
*
HBr
____)
[SI
Br
[I11
343
ATROPINE
OH N-CK3
Scheme 11: Robinson's total synthesis of CHO
-t
CH-OH CH3NH2
[21 CHO
[11
tropine
cond.
*
(
Ci-OH
[ 31
+
N-CH3
/
CH3
\
/"=O
CH3
[41
344
ABDULLAH A. AL-BADR AND FARID J . MUHTADI
This ketone i s reduced with zinc and hydriodic 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 methylamine [ 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 acet 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 temp 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 hydri 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 methylamine [21 t o give biscarbinolamine [31. 131 i s condensed w i t h calcium acetonedicarboxyl 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-methylpyrr 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-methylpyrrolidinediacetate [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 reduced 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:
345
ATROPINE
(
Scheme 11: Robinson's s y n t h e s i s ( y i e l d improvement) CH-OH
tNH2CH3
)-CH3
cond.
~
CH2COOCa
+ \ ,C=O
[21
CH-OH
[11
[31
\4
CH2COOCa
-
[41 COOCa
COOCa
Scheme 111: Willstatter's second s y n t h e s i s
I
CH=
CH-
CH2-COOC
H 2 5
CH=
CNI
CH,-COOC H 2 5 CH3-
CH=
C-
CH2-COOC
H
2 5
[11 H I
CH2- C-
1 F1
H3C$
CH2-
H
CH2-COOC
1
H
2 5
y=O CH
COOC H
2-
Na/p-c ymene 4 -
i"
-C-
CH2-?-
2 5
CH2-COOC H h-CH3 25 I I
CH2-COOC
H
CH
C-CH-COOH
31
2
- C1
I CH2
______t
H
2 5
3%
ABDULLAH A . AL-BADR A N D FARID J . MUHTADl
CH
1 2
-CCF-CClr! \- I 2
CH - - ~ H - - C H ~ 2
CH2 - CH - CH2
"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-cyanoethylbenzene [ 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 dlt 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
[41
[ 31 CH2
II
C
- COOH
_____)
CH20H
@
6~-COOH I
Scheme 11: McKenzie and Wood's s y n t h e s i s
KCN
[ 31 CH2CI I
CH20H
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
Zn Br
I
I
CH-COO% Zn
CH2OH I
CH-COOEt HCHO
b
______r
CH2OH
I
349
ATROPINE
Scheme V:
Blicke's synthesis (34).
Phenylacetic a c i d [l] i s b o i l e d w i t h isopropylmagnesium 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 Grignard 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 toget 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 S y n t h e s i s of Labeled Tropic a c i d
4.2.3.1
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 formaldehyde 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 ). S y n t h e s i s of Labeled Tropine
4.2.3.2
- 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 reduct 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 Robi 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 succin-dialdehyde and t h e n t o 1-or 5-14Ct 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 , 7l'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 )
.
351
ATROPINE
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
5.
ABDULLAH A . AL-BADR AND FARID J . MUHTADI
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 precur 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-Nm e t h y l o r n i t h i n e [ 2 ] (47-49) as [ methyl-l4C ] 6 -Nmethyl-[ 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 hyoscyamine 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 enzymes 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 4methylaminobutanal [ 5 ] . This i s c y c l i z e d t o give Nmethyl- A l-pyrrolinium s a l t [ 6
-
I.
353
ATROPINE
Leete's Scheme: Biosynthesis of Atropine
COOH
COOH
-7
E N C H 3
x-
LrnCH3
+--
' 7 NHCH3
i
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 acet 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 prec 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 *CH2 [I31 +h-mH2 I * COOH
JrJ
/
+ I
HOH2C-*C-H I
.COOH [12]
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
6.
355
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. Hydrol 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 metabol 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, approximately 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
( aldehyde )
Noratropine Apoatropine
Tropine Tropic a c i d
i n v o
R a t liver
4 -
in vitro
Man
ATROP 1 NE > -
Mouse
Tropine , Modified+Tropic atropines acid (10%) ( 1%)
Mouse
p-hydroxyatropine (2%)4 m,p-Dihydroxyatropine
\
p-Glucuronosidoatropine (5%)
my-Hydroxy-p-glucuronosidoatropine (27%)
m,p-DiglucuronosidoatropineC-p-hydroxy-m(20%) glue urano s idoatropine
ATROPINE
7.
357
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 followi 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 decrease 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 volun 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 cyclop 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
8.
ABDULLAH A. AL-BADR AND FARID J . MUHTADI
Therapeutic Uses of Atropine 1.
(69)
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 anasthetics.
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
359
ATROPINE
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 potassium 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 charac 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:Gerrard 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.
- The
-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 sulf 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
Microcrystal tests
9.2
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 microcrys 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 hexagons 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
9.3 9.3.1
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 . Kitrimetric Methods
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 misoh 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 ) .
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361
~~
FIG. 7, MICROCRYSTALS OF PICRIC ACID,
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5 b
FIG, 8, MICROCRYSTALS
OF ATROPINE WAGNER'S REAGENT,
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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.
363
ATROPINE
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 nonaqueous 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 eyedrops c o n t a i n i n g a t r o p i n e or belladonna. The endp 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 nonaqueous media by means o f t h e h y d r o c h l o r i c a c i d
ABDULLAH A . AL-BADR AND FARID J . MUHTADI
363
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 reagent 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 solut 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 determinat 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 cont 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
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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 determinat 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 sleevet 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 phosp 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 determinat 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.
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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 hydrox 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 3%. probable e r r o r i s
*
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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-lnaphthylethylenediamine d i h y d r o c h l o r i d e solut 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 pdimethylaminobenzaldehyde. 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 chloroform, 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-dimethylaminobenzaldehyde 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
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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-catechola 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 compared 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-aminobenzaldehyde 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
369
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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 compar 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 , tungstophosp 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 microdetermination 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 photom 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 irri 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 NH2SO4. 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 chromatography and determined by a modified V i t a l i procedure. 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
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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 respect 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 reprod 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).
371
ATROPINE
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 pres 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 appropr 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.
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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 To and a l k a l o i d s i n g a l e n i c a l compositions. 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 atrop 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 proport 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 .
313
ATROPINE
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. Locat 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 highspeed, h i g h p r e s s u r e l i q u i d chromatography of
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ATROPINE
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 , recover 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 chromatography. 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 prod 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 apoatropine.
e.
V a n Buuren et (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 chromatographytechnique (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
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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 desc 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 morp 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 boros 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:
W AW.
5% SE-30 on 60-80 mesh Chromosorb 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.
311
ATROPINE
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 homa 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 chloroform 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 pharmac 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).
379
ATROPINE
-
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 radioimmunoa 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.
ABDULLAH A. AL-BADR AND FARlD J . MUHTADl
3 80
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7
