abdullah a. al-badr and farid j. muhtadielbdisliker.at.ua/jedy/albadr1985.pdf · abdullah a....

$: Abdullah A. Al-Badr and Farid J. Muhtadielbdisliker.at.ua/Jedy/albadr1985.pdf · ABDULLAH A. AL-BADR AND FARlD J. MUHTADI H 0 11 NCH~OH 0-C-CH N-CH3 \ C6H5 A detailed review is available$
ATROPINE

1.

2.

3. 4.

5 .

6. 7. 8. 9.

Abdullah A. Al-Badr and Farid J. Muhtadi King Saud University Riyadh, Saudi Arabia

Description 1.1 Nomenclature 1.2 Formulae 1.3 Molecular Weight 1.4 Elemental Composition 1.5 I .6 Dissociation Constant 1.7 pH range Physical Properties 2. I Melting Point 2.2 Sublimation Range 2.3 Solubility 2.4 X-Ray Crystallography 2.5 Spectral Properties Isolation Synthesis 4.1 Partial Synthesis 4.2 Total Synthesis Bios ynthesis 5.1 Biosynthesis of Tropine 5.2 Biosynthesis of Tropic Acid Metabolism Pharmacokinetics Therapeutic Uses of Atropine Methods of Analysis 9.1 Identification Tests 9.2 Microcrystal Tests 9.3 Titrimetric Methods 9.4 Polarographic Methods 9.5 Spectrophotometric Methods 9.6 Chromatographic Methods 9.7 Radio-immunoassay References

Appearance, Color, Odor, and Taste

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

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.

8-methyl-8-azabicyclo [ 3.2.11 oct-3-yl ester, *-( +)-

la H,-:a H-tropan-3a -ol( 2)-tropate.

b) Benzene-acetic acid a-( hydroxymethy1)-,

c)

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 ) .

ATROPINE 327

1.2.3 CAS Registry No.

51-55-8 I 1.2.4 Wiswesser Line Notation

~ 5 6 A ANTJ A -GOVYR & 1Q- DL ( 5 )

1.2.5 Stereochemistry

Examination of t h e NMR spectra of some tropane deuterohalides has shown t h a t t h e N-substituent i n tropanes i s predominantly equator ia l (6 1. X-ray analysis of t ropine hydrobromide has shown the presence of chair conformation ( 7 ) . Study of t h e dipole-moment and Kerr-constant measurements of a number of tropane der iva t i - ves has shown t h a t t h e piperidine r i n g i s i n t h e chair form with t h e N-methyl equator ia l (8). NMR spectra of some tropane der ivat ives have confirmed t h a t t h e piperidine r ing i s i n t h e chair conformation with t h e N-methyl group predominantly equator ia l ( 9 ) . In t ropine, however, t h e predominant conformation i s the piperidine r ing i n a deformed chair form to- gether with a minor amount i n t h e boat form

Another study of t h e dipole-moments and

(10).

HO

Tropine

In atropine, t h e a-3-substituent i s of grea- t e r bulk than t h e hydroxyl, and the boat form may w i l l be favored because of t h e increased in te rac t ions involving t h e dimethylene bridge i n the chair confirmation (11).

ABDULLAH A. AL-BADR A N D FARlD J. MUHTADI

H 0 11 N C H ~ O H

0-C-CH N-CH3

\ C6H5

A de t a i l ed review i s ava i l ab le for t h e boat or cha i r conformation i n t rop ines ( 1 2 ) .

Other PMR study suggested a preference f o r t h e boat conformation i n several tropane de r iva t ives . This study showed s t rong cross-ring intramolecular i n t e r - act ions of t h e type N--- C-=O and N---H-O were indicated by t h e broadening of t he proton s igna l due t o the coupling between 1(5)-H and 2(4)-H protons i n the boat conformer compared with t h e cha i r . This broadening a r i s e s as a consequence of ec l ip s ing of these protons i n t h e boat conformer (13). t i c resonance study has a l s o suggested a non-chair conformations i n tropane de r iva t ives (14) .

Carbon-13 magne-

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

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

1 . 6 Dissociation Constant

PKa 5.93

1.7 BH range

pH of 0.0015 molar so lu t ion i s 10.0 (15),approximate pH of saturated aqueous so lu t ion i s 9.5 (16) .

ATROPINE

2. Physical Propert ies

329

2.1

2.2

2.3

2.4

Melting Point

114 - 116' (15) 114 - 118' (16)

Sublimation range

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

Solub i l i t y

One gram dissolves i n 460 m l water, i n 90 m l water at 80°, i n 2 ml alcohol, 1.2 ml alcohol a t 60°, i n 27 ml glycerol , 25 m l ether . Soluble i n benzene and d i l u t e acids.

X-ray crystallography

The X-ray crystallography of t ropine hydrobromide ( 7 ), t ropine ethobromide (17) pseudotropine (18) hyoscine hydrobromide (19 ) and t r o p i c ac id i n hyoscine N-oxide (20 ) have been reported.


2.5 Spectral Propert ies

2 .5 .1 Ul t rav io le t Spectrum

The W spectrum of at ropine i n ethanol (Fig.1) was scanned from 200 t o 400 nm using DMS 90 'Varian Spectrophotometer. It exhibited t h e following W data (Table 1).

Table 1. UV c h a r a c t e r i s t i c s of a t ropine

A m a . a t nm E A ( 1 % , 1 cm) - - 20 5

246 147.6 5 .1 251.5 175.1 6.05

263.5 143.3 4.95 271 24.6 0.85

257 209.8 7-25

Other reported W s p e c t r a l da ta f o r a t ropine i n 0 . 1 N s u l f u r i c acid ( 2 1 ) :

h max at . 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 Infrared Spectrum

The I R spectrum of a t ropine as KBr-disc was recorded on a Perkin Elmer 580 B Infrared Spectrophotometer t o which Infrared Data stat- ion i s attached (Fig. 2 ) .

The s t r u c t u r a l assignments have been correla- t e d with t h e following frequencies (Table 2 ) .

Table 2. I R Character is t ics of Atropine

-1 Frequency cm Assignment

3070 OH (hydrogen bonded) 2930 CH ( s t r e t c h )

3 2810 N-CH

B 1725 0-C - (ester)

1595, 1580 C=C aromatic

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

44.

i

&O# WAVE##H#FR #o asM 2060 fm Y O 0 1400 1000 800 680 &

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

ATROPINE 333

Assignment -1 Frequency cm

1155, 1030 C-0-C (e ther )

770,725,690 5 H (mono subst i tuted aromatics)

The I R exhibited t h e following other charac te r i s t ic bands : -

1450,1420,1370,1355,1335,1270,12~5,~230,~220,~205 , 1190,1165,1132,1108,1065,975,920,845,805,515 cm-1.

Other I R data f o r atropine (5,21) have been a l s o reported.

2.5.3 Nuclear Magnetic Resonance Spectra

2.5.3.1 Proton Spectra

The PMR spectra of both atropine i n C D C 1 3 and i n TFA (Trif luoroacet ic 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 - na l reference. These are shown i n Fig. 3 (a) and 3{b) respectively. The following s t r u c t u r a l assignments have been made (Table 3) .

8

0- C- 9

Other PMR data f o r atropine are a l s o reported (5,9, 13,22).

3 34 ABDULLAH A. AL-BADR AND FARID J. MUHTADI

I 1 I . I I . . I I . I . , . . I . . . . I . . . . i , I , . i , ,

L O TO 4 0 a# Rn(,) u B.# 1.. 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 atropine

Group

5 aromatic protons 13, lb ,15,16,17

3-H CH2 - OH CH2 - OH, 10 CH

1 3 5 H -

8-N-Me

2,4,6,7 H

Chemical Sh i f t (ppm)

CDC13 TFA

7.23(s) 7.36( s 1

s=singlet , d=doublet , t=triplet , bs=broad s ing le t , m = m u l t i p l e t

2.5.3.2 13C-NMR

The I3C-NMR noise decoupled and off resonance spectra a r e presented i n Fig. 4 and Fig. 5 respectively. Both were recorded over 4000 Hz range i n deuterated chloroform on a Varian FT 80 A-80 MHz spectrometer, using 10 mm sample tube and tetramethyl s i l - ane as a reference standard a t 21'. The carbon chemical s h i f t s a r e assi- gned on t h e bases of the a d d i t i v i t y p r inc ipa l s and off resonance s p l i t t - ing pa t t e rn (Table 4 ) .

8

4

11

1 5 10

1 7 16


d 111

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

I

ATROPINE

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

337

Carbon no. Chemical Shi f t Carbon no. Chemical S h i f t [PPd [ P P ~

171.92( s ) c1 c5 59.54(d)

136.17 ( s 1 ‘10 54.94 ( d)

9 C

5 2

40.08(q)

36.04(t

25.31(t)

24.93(t 1

‘8 ‘13’ ‘17 128.66( d )

c23 c4 128.11 ( d ) ‘14’ ‘16

‘15 C

7

3 67.63 ( d ) ‘6

127.48 ( d) C

cll 63.54(t)

s=singlet , d=doublet t = t r i p l e t q=quartet . Other l3C-NMR data f o r atropine ( 14,23 ) hydrochloride ( 1 4 ) and atropine methoiodide ( 1 4 ) have a l s o been reported.

atropine

2.5.4 Mass Spectrum

The mass spectrum of atropine i s presented i n Fig. 6. This was obtained by electron i m - pact ionizat ion on a Varian MAT 1020 by d i r e c t i n l e t probe a t 270’~. The electron energy was 70 eV. The spectrum scanned t o mass 300 amu. The spectrum (Fig. 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 is 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 re given i n t a b l e 5.

.T CJ ...

ATROPINE 339

Table 5 . Mass Fragments of Atropine

96

95 94

83

82

Relative i n t e n s i t y % Ions

9-50 M+

7.79 - 9.34 See below*

100.00

6.35

10.67

125- H -

r-

c 4

8.60 96-H 22.66 9 5 4

18.87

25 * 97

i

67 14.78 -

44

42

5-15

21.83

- i I

r + : - CH2=N=CH2 1 41 8.36 42-H

Other reported mass spectra of atropine ( 2 4 ) : Base peak 124 , m/e: 42, 55, 67, 82, 94, 104, 124 , 1 4 0 , 272, 289. Tropine fragmentations a re a l so reported ( 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 several solanaceous p l an t s these include species of Atropa, Datura, Hyoscyamus, Duboisia, Mandragora and Scopolia ( 26). It i s claimed t h a t a t ropine does not occur as such i n the p l a n t s , but 2-hyoscyamine present i n p l a n t s , (27) and during ex t r ac t ion process, 2-hyoscyamine undergoes race- mization t o give atropine. i s t he preferred source for t h e manufacture of a t ropine because of i t s high a lka lo id content , with stramonium next i n order ( 28 ) .

Hyoscyamus muticus from Egypt

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

The powdered drug i s throughly moistened with an aqueous so lu t ion of sodium carbonate and ex t r ac t ed with e the r or benzene. The a lka lo ida l bases a r e ex t r ac t ed from t h e solvent with water a c i d i f i e d with a c e t i c acid. The acid solut ion i s then shaken with e the r as long as t h e lat ter takes up color ing matters. The a lka lo ids a r e precepi ta ted with sodium carbonate, f i l t e r e d o f f , washed and dried. The dried p r e c i p i t a t e i s dissolved i n e t h e r or acetone, dehy- drated with 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 concentrated, cooled, when crude hyoscyamine and atropine c r y s t a l l i z e from t h e so lu t ion . 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 off and dissolved in a lcohol , sodium hydroxide solut ion i s added and t h e mix- t u r e i s allowed t o stand u n t i l recemization of hyoscyamine t o a t ropine i s completed (as indicated by t h e absence of o p t i c a l a c t i v i t y ) .

The crude atropine 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 synthesis of a t ropine from t ropine and t r o p i c a c i d , thus proving atropine t o be t h e t rop ine e s t e r of t r o p i c acid. Tropine and t r o p i c a c i d a r e heated i n the presence of hydrogen chlor ide t o give atropine.

b . 2 Total Synthesis

Since atropine i s the t rop ine e s t e r of t r o p i c

ATROPINE 341

acid, schemes f o r t h e t o t a l synthesis of t ropine and the t o t a l synthesis of t rop ic acid were reported.

4.2.1 Total Synthesis of Tropine

Four schemes fo r t h e t o t a l synthesis of t ropine a re 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 yield. Scheme I: W i l l s t a t t e r ' s t o t a l synthesis of t ropine ( 2 ) .

Suberone (cycloheptanone) [ 1 1 i s reduced t o suberol which i s t r e a t e d with 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- haustive methylation. [ 6 ] i s brominated a t lY4-posi t ions 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 effected by quinol ine 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 reacted with dimethylamine t o give dimethyl aminocyclohepta-2 ,b-diene [lo 1. The l a t t e r i s t r e a t e d with sodium i n ethanol followed by bromination t o give 1,2-dibromo-5- dimethylamino-cycloheptane [ 111. This i s warmed i n e ther when intramolecular alkyla- t i o n occurs t o give 2-bromotropane methobromide [12]. Hydrogen bromide i s eliminated from [12] by t h e act ion of a l k a l i t o y i e l d t ropidine methobromide [13]. This i s t rans- formed t o t ropidine methochloride El41 by t h e act ion of potassium iodide followed by t h e ac t ion of s i l v e r chloride. Substance [ 1 4 1 i s pyrolized t o give t ropidine [IF]. Hydrogen bromide i s added t o an ace t ic acid solution o f t ropidine [15] t o y i e l d 3-bromotropane [I61 which i s hydrolysed with 10% s d f u r i c acid at 200-210' t o give pseudo- t ropine [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

( i i ) H I

exhaust. + methyln.

quinol i ne

15ooc * 0 [SI

HBr ____)

Br [I11

Qr [4 1 Br

B r

0

ATROPINE 343

OH N-CK3

Scheme 11: Robinson's total synthesis of tropine

C H 3 CHO CH-OH

cond. N-CH3 + \

/"=O C H 3

[ 4 1 / * ( Ci-OH

-t CH3NH2

[21

CHO [ 1 1 [ 31


This ketone i s reduced with zinc and hydrio- d i c ac id t o t rop ine [ l g ] .

Scheme 11: Robinson's synthesis ( 3 )

Succindialdehyde [ 11 i s condensed with methylamine [2 ] t o give t h e condensate biscarbinolamine [ 31. This i n t u r n condensed with acetone [ h ] t o give tropinone [ 5 ] (This mixture i s allowed t o s tand i n water a t ordinary temperature f o r ha l f an hour) . Tropinone [ 5 ] i s reduced with zinc and hydr- i od ic ac id t o t ropine [61.

The y i e l d can be improved by subs t i t u t ion of t h e more r eac t ive acetone dicarboxylate or i t s e s t e r f o r acetone. Succindialdehyde [ 11 i s condensed with methylamine [21 t o give biscarbinolamine [31. 131 i s condensed with calcium acetonedicarboxy- la te [ 4 ] t o a f fo rd t h e condensate [5] . This i s warmed with hydrochloric ac id t o give t r o - pinone [ 6 ] . Tropinone [ 6 ] i s reduced with zinc and hydriodic a c i d t o t rop ine [TI.

Scheme 111:

Succinyldiacetic e s t e r [l] i s condensed with methylamine [ 21 t o give diethyl-N-methylpyr- ro l ed iace t a t e [ 3 ] . This i s reduced (H2+Pt) t o a f fo rd diethyl-N-methylpyrrolidinediace- t a t e [ 4 ] . t h e presence of N a and p-cymene t o give ethyl- tropinone-2-carboxylate [ 51. Hydrolysis of [ 5 ] with 10% s u l f u r i c a c i d gives e thy l t rop 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 tropinone [ T I which i s reduced with zinc and hydriodic a c i d t o t rop ine [8]. Scheme IV:

Tropinone can a l s o be synthesized ( 2 9 ) using methylamine hydrochloride,acetondicarboxylic acid and generating succindialdehyde i n s i t u by t h e ac t ion of acid on 2,5-dimethoxy t e t r a - hydrofuran as follows :

W i l l s t a t t e r ' s second synthesis( 4 )

The c& form of [ 4 ] i s cycl ized i n

--

CHO

ATROPINE 345

Scheme 11: Robinson's synthesis ( y i e l d improvement)

CH-OH CH2COOCa c: CH-OH CH2COOCa

( )-CH3 + \ ,C=O t NH2CH3 cond. ~

[ 21

[41

[31 \ [11

COOCa 4

- COOCa

Scheme 111: Willstatter's second synthesis

I

CH= CH- CH2-COOC H 2 5

[ 1 1

H I

CH2- C- CH2-COOC H 1 1 2 5

2- 2 5

1 H3C$ y=O

H

CH2- F- CH COOC H

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

CH= C- CH2-COOC H 2 5

-C- CH2-COOC H Na/p-c ymene i" h-CH3 2 5

4 - I CH2-?- CH2-COOC H

I 2 5 H

- C-CH-COOH

3 1 I ______t

CH2 CH - C- 2 1

3% ABDULLAH A . AL-BADR A N D FARID J . MUHTADl

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

CH - - ~ H - - C H ~ 2

CH2 - CH - CH2

"71 181

4.2.2 To ta l Synthes is of Tropic ac id

Severa l schemes f o r t h e t o t a l syn thes i s of t r o p i c a c i d are known (Scheme I t o V ) .

Scheme I: Landenburg's syn thes i s ( 3 0 ) . Acetophenone [l] i s converted i n t o a ,a-dichloro- ethylbenzene [2 ] by t h e a c t i o n of phosphorous pentachlor ide . 121 i s r e a c t e d wi th potassium cyanide and e thano l t o f u r n i s h ct-ethoxy-a-cyano- ethylbenzene [ 3 ] . This i s hydrolysed wi th barium hydroxide s o l u t i o n t o g ive a t r o l a c t i c e t h y l e t h e r [4]. The la t te r i s heated wi th hydrogen ch lo r ide to y i e l d a t r o p i c a c i d [ 5 ] which i s converted t o t r o p i c a c i d [61. Scheme I1 : McKenzie and Wood's syn thes i s (31). Acetophenone [l] i s converted by t h e a c t i o n o f potassium cyanide t o acetophenone cyanohydrine [ 23. This upon hydro lys is i s converted 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 heated under pressure t o y i e l d a t r o p i c a c i d [4]. Atropic ac id [4] i s t r e a t e d with hydrogen ch lo r ide i n e t h e r e a l s o l u t i o n t o form 6-chlorohydratropic a c i d [ 5 ] . This upon b o i l i n g wi th aqueous sodium carbonate 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 wi th e thyl - formate t o g ive e t h y l a-formyl a c e t a t e [ 2 ] . This on reduct ion wi th aluminium amalgam y i e l d s dl- t r o p i c ester [3] which upon hydro lys i s g ives t r o p i c a c i d 141.

Scheme IV: Chambon's syn the i s (33). Ethyl a-bromophenylacetate [l] is t r e a t e d wi th Zn t o g ive ethyl+-zincbromophenylaceate [ 2 ] which i s t r e a t e d wi th formic a c i d t o g ive d l - t r o p i c es ter [ 3 ] which upon dydro lys i s y i e l d s t r o p i c a c i d [4].

Scheme I: Landenburg's synthes is

CH3 I

KCN

[ 31

CH2 II C - COOH

_____)

Scheme 11: McKenzie

KCN

[41

CH20H I

6~-COOH

@ and Wood's synthes is

[ 31

CH2CI I

CH20H I

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

Scheme I11 : Miiller I s synthesis

C HO

CH2OH CH2OH I I

CH-COOEt

hydrolysis

Scheme IV: Chambon's synthesis

I Zn Br Br

CH-COO%

Zn b

I CH-COOEt

HCHO ______r

CH2OH CH2OH I I

ATROPINE 349

Scheme V: Blicke's synthesis (34) . Phenylacetic acid [l] i s boiled with isopropyl- magnesium chloride i n e therea l solution 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 ropic acid [ 3 ] .

Scheme V: BLicke's synthesis

Tropine f i n a l l y can be combined with t rop ic acid t o give atropine. This can be done by heating the two toge- t h e r i n the presence of hydrogen chloride (Fischer-Speier e s t e r i f i c at ion) .

atropine


4.2.3 Synthes is of Labeled Atropine

4.2.3.1 Synthes is of Labeled Tropic a c i d

Benzylmagnesium c h l o r i d e [l] i s trea- t e d wi th I4CO2 followed wi th magnesium ch lo r ide t o g ive t h e condensate [2 ] . This upon t h e a d d i t i o n of formaldehyde g ives l a b e l e d t r o p i c a c i d [3]. Synthes is of l abe led t r o p i c a c i d i s presented i n scheme VI ( 35 ).

4.2.3.2 Synthes is of Labeled Tropine

- Synthes is of t rop ine-6 , 7 T has been achieved 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 dihydrofuran and fol lowing Robinson's r o u t e t o tropinone-6, 7 T , by subsequent reduc- t i o n wi th hydrogen over Raney n i c k e l

- Synthes is of methyl-14C l abe led t r o - (36).

pine i s c a r r i e d out from Na 1 4 C N (37 ) v i a 1nethylamine-~4C and based on Rob- i n s o n ' s rou te ; inethyl-l4C t ropinone i s obtained i n 70% o v e r a l l y i e l d and tropine-14C i n 68% y ie ld .

- Synthes is of b i 4 C t r o p i n e can be s t a r t e d wi th arabinose-5-l4C [ 11 conversion i n t o furan [ 2 ] and appl ica- t i o n o f t h e Clauson-Kaas r o u t e t o suc- cin-dialdehyde and then t o 1-or 5-14C- t rop inone 31 ( 38) . U i n g arabinose-3, 4-14C gives 6 , 7- l'C-tropinone ( 3 9 Labeled a t rop ine can be then obtained by e s t e r i f i c a t i o n of l abe led t r o p i c a c i d o r l abe led t r o p i n e t o give e i t h e r l abe led a t rop ine or double l abe led 7 . k a t rop ine ( a r i s e d from l abe led 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 ) .

Scheme V I I . 4.2.3.3

ATROPINE 351

Scheme V I : Synthesis of Labeled Tropic acid

H [21

Scheme VII : Labeled t ropine

' CH20H

Double labeled atropine


5 . Biosynthesis of Atropine

Most s tud ie s on t h e biosynthesis of a t ropine and of i t s isomer hyoscyamine have been performed on various species of Datura, but a l l t h e ava i l ab le evidence suggests t h a t s i m i l a r pathways occur i n other tropane alkaloid- 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 lkal- oids of t h e group are e s t e r s of hydroxylamines and various ac ids ( t r o p i c , t i g l i c , e t c . ) t he re a r e , for each a lka lo id , two d i s t i n c t biosynthet ic routes ( 26 ).

5 . 1 Biosynthesis of t rop ine

Ornithine and the r e l a t e d aminoacids (glutamic ac id , p r o l i n e ) have been proved t o be t h e precursors of t h e pyrrol idine r i n g of t ropine ( 40-45 ). It w a s found t h a t feeding [2-l4C] o rn i th ine t o Datura stramoniwn r e su l t ed i n radioact ive hyoscyamine l abe l - l ed only a t C - 1 bridgehead carbon of t rop ine (46).

N-CH3 COOK

And t h a t [ 5 - 1 4 C ] prol ine r e s u l t e d i n radioact ive hyoscyamine l a b e l l e d only t h e C-5 pos i t i on of trop- i ne ( 4 4 ) . It w a s a l s o reported t h a t [2-l4C, 6 - 1 5 N I o rn i th ine incorporated i n t o t rop ine moiety of hyoscyamine and the 6-aminogroup of o rn i th ine is an e f f i c i e n t precu- r s o r of t h e t rop ine nitrogen (44,46). The incorporation of glutamic a c i d and p ro l ine i s considered t o occur v i a orni thine ( 46 ) . Ornithine [l] i s incorporated i n t o t rop ine v i a 6-N- m e t hylorni thine [ 2 ] (47-49) as [ methyl-l4C ] - 6 -N- methyl-[ 2-&] o rn i th ine w a s incorporated i n t o hyoscyamine l a b e l l i n g C - 1 and the N-methyl group. 121 i s decarboxylated t o y i e l d N-methylputrescine [ 41 ( 50 ,51) .

Putrescine [ 3 ] has a l s o been shown t o be a precursor of t he t ropine alkaloids (43 ,52-5&). It w a s suggested ( 4 6 ) t h a t putrescine [ 31 i s converted by c e r t a i n enz- ymes i n Datura p l an t s t o N-methyl putrescine [4 ] . Oxidation o f t h e primary alcohol of [ 4 ] a f fo rds 4- methylaminobutanal [5] . This i s cyclized t o give N- methyl- A l-pyrrolinium sal t [ 6 I.

ATROPINE 353

Leete's Scheme: Biosynthesis of Atropine

COOH

-7

E N C H 3 +-- x-

COOH

LrnCH3 '7 NHCH3

i

F y O H f -atropine

1141

354 ABDULLAH A . AL-BADR A N D FARID J . MUHT.4DI

Carbons 2, 3 and 4 of t rop ine are derived from ace- t a te ( 55,56 ) and it is assumed t h a t t h e ace t a t e i s incorporated v i a acetoacet ic ac id or some su i t ab le ac t iva t ed der ivat ive such as coenzyme A e s t e r ( 46 ). [ 6 ] is t he re fo re condensed with acetoacetate t o give hygrine- a -carboxylic a c i d [ 71. Decarboxylation of [7] a f fo rds hygrine [8] which i s an establ ished precursor of t ropine ( 56,57 ). [8] i s dehydrogenated t o give dehydrohygrine [g]. The l a t t e r i s cyclized t o y i e l d tropinone [lo]. Stereospecif ic reduction of [lo] a f fo rds t ropine [ll].

5.2 Biosynthesis of t r o p i c ac id

Tropic a c i d 1121 i s formed by t h e intramolecular rearrangement of phenylalanine I131 (58 ) . Compounds which a r e metabolically r e l a t e d t o phenylalanine such as phenylpyruvic ac id are a l s o incorporated 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 with t r o p i c ac id [12] t o give atropine [14].

ATROPINE 355

6. Metabolism of Atropine

Atropine i s rapidly absorbed from the g a s t r o i n t e s t i n a l t r a c t and r ead i ly absorbed from t h e mucous membranes and the skin (21,151 ).Absorption from the 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 atropine c i r - cu l a t e s i n the f r e e form i n t h e blood and the other ha l f i s bound by t h e plasma proteins ( 21). t he c i r cu la t ion when applied l o c a l l y t o mucosal surfaces of t h e body (61) . a t ropine i s considerable. pine i s absorbed and excreted following subconjunctival i n j e c t i o n i n t h e r abb i t ( 62). v a r i e s considerably from one species t o another. Hydro- l y s i s t o t ropine and t r o p i c a c i d i s not thought t o be a major metabolic route since only t r a c e s of t r o p i c a c i d a re recovered i n the urine ( 21). Atropine disappears rapidly 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 (21) . lung and pancrea.s a r e the most important organs t h a t t ake up t h e labeled atropine (62) . Most i s excreted i n t h e ur ine within the f i r s t 12 hours, i n p a r t unchanged (21 ) . Following intra-mascular administration of a s ing le 2 mg doses of I4C-labelled atropine i n man, Gosselin etal . (63) found t h a t 85 t o 88% of t h e r ad ioac t iv i ty w a s excreted i n t h e ur ine within 24 hours, only a t r a c e could be extracted from t h e faeces; about 50% of t h e dose appeared i n t h e ur ine unchanged, over 30% w a s excreted as unknown metabo- l i t e s and l e s s than 2% appeared as f r e e t rop ic acid. A f t e r intravenous in j ec t ion of a t ropine i n the mouse, approximately 25% of the dose i s excreted i n the urine as atropine, more than 50% as conjugates with glucuronic ac id and t h e remaining 20-25% as intermediate oxidation products (probably p-hydroxyatropine and 3,44ihydroxyatropine ) and tropine-modified atropines (62). The metabolism of a t ropine is presented i n scheme I

Atropine a l s o en te r s

The t ransconjunct ival absorption of About 95% of radioact ive a t ro -

The metabolism of a t ropine

The l i v e r , kidney,

[ after (62) 1.

ABDULLAH A . AL-BADR AND FARID J . MUHTADI

SCHEME 1; THE METABOLISM OF ATROPINE

C02 + Noratropine (2%)

Rabbit, Guinea p i g

Tropine ( aldehyde ) i n v o Tropic acid

R a t l i v e r Man Tropine , Noratropine 4- ATROP 1 NE -> Modified+Tropic Apoatropine i n v i t r o Mouse atropines ac id

(10%) ( 1 %)

Mouse

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

p-Glucuronosidoatropine (5%)

xyatropine

my-Hydroxy-p-glucuronosidoatropine (27%)

\

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

a t ropine

ATROPINE 357

7. Pharmacokinetics

The pharmacokinetics of atropine were reported by several authors. Peak serum l eve l s occur approximately 30 minutes following intramuscular ( I . M . ) administration of 1 mg dose of a t ropine (64 ). Serum l e v e l s following intravenous (I.V. ) administration of a t ropine drop within t h e f i r s t 10 minutes and then decrease more gradually. Levels one hour following e i t h e r I.V. or I.M. administrations a r e very similar (64). Following I.M. administration of 2 mg atropine, t h e onset and duration of e f f e c t on hear t r a t e a r e reported (65) t o be m a x i m u m a t 15-50 minutes and up t o 5 hours, respect i - v i t y . Following endotracheal administration of 1 mg atropine s u l f a t e , serum l e v e l s of a t ropine were l e s s than 5pg/ml at 30 seconds and U p g / d a t 10 minutes (66 ). Atropine's ha l f - l i f e i s reported t o occur a t two r a t e s , with 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 ropine i s 4.125 hours following a s ing le 1 mg intravenous dose of atropine i n humans (67). The mean t o t a l plasma clearance of s i x normal human volu- n t ee r s following a s ing le 1 mg intravenous dose of a t ro - pine i s reported t o be 533.35 ml/minute (67). Maximum cycloplegia usually occurs within several hours of administration of t o p i c a l a t ropine, though e f f ec t ive cyclo- p l eg ia 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 ac t ion may las t for 5 days (68).


8. Therapeutic Uses of Atropine (69)

1. Pre-anaesthetic medication :

- t o decrease sec re t ions of s a l iva ry , naso-pharyngeal and bronchial glands.

- t o prevent r e f l e x brancho-spasm.

- t o reduce r e f l e x bradycardia of i nha la t iona l anasth- e t i c s .

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 purgatives.

3. Vaso-Vagal syncope due t o r e f l e x lowering of blood pressure and severe Bradycardia.

4. Nocturnal enuresis and urgency of mic tu r i t i on t o decrease ur inary bladder r e f l e x i r r i t a b i l i t y .

5. I n Parkinsonian disease t o reduce r e g i d i t y ( c e n t r a l ac t ion ) .

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 :

I r i t i s

Kerat it i s

Corneal u l ce ra t ions or i n j u r i e s

ATROPINE 359

9. Methods of Analysis

9.1 Iden t i f i ca t ion 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 the B r i t i s h Pharmacopoeia of 1963 ( 7 0 )

-1 mg of a t ropine i s added t o 4 drops of fuming n i t r i c ac id and t h e mixture i s evaporated t o dryness on a water bath; a yellow residue is obtained. 2 ml of acetone and 4 drops of a 3% w/v solut ion of potassium hydroxide i n methyl alcohol a r e added t o t h e cooled residue; a deep v i o l e t color i s produced.

-50 mg of a t ropine i s dissolved i n 5 m l of water acid- i f i e d with hydrochloric acid, gold chlor ide solut ion 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 ap id ly 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 bo i l ing water a c i d i f i e d with hydrochloric ac id , 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 pulverulent when dry, and has a melting point 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:- - The Gerrard react ion (71 ) .

To about 6 mg of a t ropine, 1 m l of 2% solut ion o f mercuric chlor ide i n 50% aqueous methanol i s added; a deep red color i s produced.

-To a t r a c e of atropine i n an evaporating dish, drops of t h e p-dimethylaminobenzaldehyde reagent ( 2 g of p-dimethylaminobenzaldehyde i s dissolved i n 6 gm sul- f i c a c i d ) are added as wel l as 0.4 m l of water. The r e s u l t i n g mixture is heated on a bo i l ing water bath; an intense red color i s produced which changing t o Permanent cherry red on cooling.

(can be performed on young c a t s , dogs and r a b b i t s ) . An aqueous, alcohol free solut ion of a t ropine or i ts s u l f a t e i s dropped i n t o t h e conjunctival sac of t h e eye and held so t h a t non i s l o s t by overflow of t e a r s . It has been reported (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 ropine 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 he pupi l of t he eye i n 1 hour.

-Physiological t es t : Induction of mydriasis

360 ABDULLAH A. AL-BADR A N D FARID J . MUHTADI

9.2 Microcrys ta l tests

100 mg of a t rop ine d i s so lved i n 5 m l water a c i d i f i e d with d i l u t e s u l f u r i c ac id . The fol lowing microcrystals were performed.

(21 ) . The c r y s t a l s are shown i n Fig. 7.

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 Fig. 8.

- Dragendorff ' s reagent wi th a t r o p i n e g ives i r r i g u l a r r ec t ang le s as shown i n F ig . 9.

- Mercuric c h l o r i d e wi th a t rop ine g ives long prisms as shown i n Fig. 10.

- P i c r i c a c i d wi th a t rop ine g ives bunches of p l a t e s

- Wagner's reagent wi th a t rop ine g ives i r r i g u l a r hexa-

9 .3 Kitrimetric Methods

9.3.1 Aqueous T i t r a t i o n s Bobtelsky and Barz i ly (72 ) have repor ted a miso- he te rometr ic t i t r a t i o n of l a r g e , o rganic , ni t rogen- conta in ing compounds inc luding a t rop ine . Micro amount of a t r o p i n e i s t i t r a t e d he t e romet r i ca l ly with t u n g s t o s i l i c i c a c i d , tungstophosphoric ac id or molybdophosphoric at pH 1 or 7 .

Other t i t r i m e t r i c methods for t h e a s say of a t rop ine have been publ ished:

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 ac id i n decomposed a t r o p i n e products (73) .

The a p p l i c a t i o n of sodium dodecyl s u l f a t e t i tr i- metr ic s o l u t i o n i n t h e a n a l y s i s of a t rop ine i n j e c t i o n s (74 ) . The in f luence of s a l t s , polyhydric compounds and absorbents on t h e de te rmina t ion of organic bases by an ionic s u r f a c t a n t i n two-phase systems. The method was appl ied t o a t r o p i n e among o t h e r organic bases ( 7 5 ) .

Atropine i n ae roso l has 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 ) through a s tandard 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 P I C R I C A C I D ,

5 b

FIG, 8, MICROCRYSTALS OF ATROPINE WAGNER'S REAGENT,


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

-/- -- FIG, 10. MICROCRYSTALS OF ATROPINE

WITH MERCURIC CHLORIDE.

ATROPINE 363

e ) The inf luence of a t rop ine among o the r organic bases on t h e p a r t i t i o n of i nd ica to r a c i d s i n a w a t er-chloroform system (77 ) .

f ) Atropine w a s de tec ted and q u a n t i t a t i v e l y de te 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 using l e a d n i t r a t e w a s descr ibed f o r drug products including a t rop ine s u l f a t e (79 ) .

9.3.2 Non-Aqueous Titrat ion

The USP XX 1980 ( 8 0 ) descr ibed a non-aqueous t i t r a - t i o n f o r t h e assay of a t rop ine as follows:

Dissolve about 400 mg of a t rop ine , accu ra t e ly 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 wi th 0 .1 N pe rch lo r i c a c i d VS t o a green end-point. Perform a blank determinat ion and make any necessary co r rec t ion .

Each m l of 0 .1 N pe rch lo r i c a c i d i s equivalent t o 28.94 mg of a t rop ine ( C H NO ).

The B r i t i s h Pharmacopoeia 1980 (81) descr ibes a non- aqueous t i t r a t i o n f o r t h e assay of a t rop ine as fol lows :

1 7 23 3

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 with 0.1 M pe rch lo r i c ac id VS and determine t h e end-point potent iomet r i c a l l y . Dzyuba and Shra iber ( 82 ) have q u a n t i t a t i v e l y de te r - mined a t rop ine by t i t r a t i o n i n non-aqueous so lven t s . The t o t a l a lka lo ids of t h e a t rop ine group ( a t r o p i n e p lus hyoscyamine p lus hyoscine) are determined by tit rat ion aga ins t HC104 i n anhydrous a c e t i c ac id . The method i s appl ied 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 , suppos i to r i e s and eye- drops conta in ing a t rop ine or belladonna. The end- poin t i s determined potent i ome t r i ca l ly ( quinhydrone e l ec t rode wi th S.C.E. as comparison e l ec t rode ) o r wi th c r y s t a l v i o l e t as ind ica to r .

Symoni and Tokar (83 ) have repor ted new reagent for t i t r a t i o n s of a t rop ine and o the r a lka lo ids i n non- aqueous media by means of t h e hydrochlor ic ac id

363 ABDULLAH A . AL-BADR AND FARID J . MUHTADI

colrplex of aluminium isopropoxylate . The s tandard s o l u t i o n conta in ing t h e H C l complex o f aluminium chloroisopropoxylate i s prepared by d i s so lv ing aluminium chloro isopropoxyla te i n chloroform and pass ing t h e c a l c u l a t e d amount of H C 1 gas i n t o i t , o r by adding t h e s to iche iometr ic amount o f chloroform s o l u t i o n of aluminium chloroisopropoxylate t o a s tandard ized s o l u t i o n of H C 1 (3% t o 4 % ) i n dry chloroform. The s o l u t i o n must be kept very dry .

The above au thors (84) have a l s o r epor t ed a new reagent f o r t i t r a t i o n s i n non-aqueous media. The de ter - mination of 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 hydrochlor ic a c i d complex of ch loro aluminium isoproxide . Resu l t s w e r e d i scussed which have been shown t h a t t h e H C 1 complex of ch loro aluminium isopropoxide behaved as a monobasic a c i d when undergoing s a l t formation wi th var ious a l k a l o i d s . The au thor have given a method f o r t h e de te rmina t ion of a t rop ine (and o t h e r a l k a l o i d s ) wi th 0 .1 N ch loro aluminium isopropoxide i n chloroform. The dev ia t ion was C 1% i n t h e range 38 t o 245 mg of a l k a l o i d .

Simon et +- (85) have descr ibed a method f o r t h e determinat ion of t r a c e amounts of a t rop ine by t i t r a - t l o n i n anhydrous so lven t s . For s o l i d a t rop ine 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 wi th 0.005 N - H C l O 4 u n t i l t h e c o l o r changes from yel low t o pink. For aqueous solu- t ion of a t r o p i n e su lpha te , make a l k a l i n e wi th aqueous sodium bicarbonate , e x t r a c t w i th chloroform and t i t r a t e t h e e x t r a c t a s descr ibed above.

9.3.3 Gravimet r i c T i t rat ion

Poethke and Traber t (86) have u t i l i z e d potassium iodobismuthate f o r t h e de te rmina t ion of small quant i - t i e s of 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 of 8-hydroxyquinoline (87) is descr ibed . 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 determining it g rav ime t r i ca l ly .

The above au thors (88) have a l s o determined a t rop ine i n ampules, eye ointment , p i l l s and e x t r a c t s of bel ladonna, and i n t a b l e t s and stomach powders con- t a i n i n g bel ladonna. Good results were obtained when

ATROPINE 365

assaying comparatively small amounts of t h e drug.

Van Pinxteren et a1 (89) have repor ted t h e determina- t i o n of a t rop ine by means of te t raphenylboron (Kal ignos t ) . boron method (90,91) f o r t h e determinat ion o f a t rop ine 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 , recover ies var ing f r o m 8 l . g t o 99.6% were obtained according t o t h e volume of s o l u t i o n analysed. Reasonable r e s u l t s were obtained by reducing t h e volume of so lu t ion t o 25 m l and wi th 1 0 t o 25 mg o f a t rop ine . g rav imet r ic method t o 50 t o 100 ml samples of Maceratum Radic is Belladonnae, accura te results were obtained over t h e range o f about 0.020 t o 0.035% of a t ropine .

By us ing Flaschkas sodium te t raphenyl -

By applying t h e

9.3.4 Potent iometr ic Ti t ra t ion

Pernarowski and Blackburn (92 ) have c a r r i e d out a poten t iomet r ic t i t r a t i o n of a t rop ine . The t i t r a t i o n i s c a r r i e d out i n chlorobenzene wi th g l a s s and sleeve- type calomel e l ec t rodes ; 0.05 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 b lue i s a s u i t a b l e ind ica to r 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 po in t . The r e s u l t s of s i x t i t r a t i o n s of a t rop ine showed an average recovery of 99.7% and s tandard devia t ion of 0.55%.

9 .4 Polarographic Methods

Souckova and Zyka (93,94) have repor ted two polarographic t i t r a t i o n methods for t i t r a t i o n o f organic bases inc luding a t rop ine . The f irst method i s t h e t i t ra- t i o n wi th 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 wi th tungstophosphoric and molybdo phos- phoric ac ids . The l a t t e r method i s repor ted t o be unsa t i s f ac to ry f o r a t rop ine . The f irst method al lows accura te determinat ion of 1 0 t o 20 mg of a base.

Novotny (95) have published a polarographic determina- t i o n of a t rop ine i n mixtures . The drug i s ex t r ac t ed from a l k a l i n e so lu t ion wi th chloroform, evaporated and a t rop ine i s n i t r a t e d with HNO3-H2SOq mixture ( > 1O:l) on water ba th for 30 minutes. The mixture i s made a l k a l i n e and, af ter removing oxygen by means of n i t rogen , polarography o f t h e so lu t ion i s c a r r i e d ou t . The polarogram i s compared with one prepared from a s i m i l a r sample t o which a known amount of a t rop ine i s added.

366 ABDULLAH A . AL-BADR A N D FARID J . MUHTADI

An Osi l lopolarographic s tudy of a t rop ine and o t h e r a l k a l o i d s i s repor ted by Habersberger and Zyka ( 9 6 ) . Os i l lopolarographic curve of a t r o p i n e w a s s tud ied wi th a dropping mercury e l ec t rode . A carbon electrode was used a r e fe rence e l ec t rode .

Some aspec t s of t h e polarographic determinat ion of a t r o p i n e i s repor ted 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 of a t r o p i n e a t t h e droping mercury e l ec t rode i n 0 .1 N L i C l i s a simple reduct ion process .

9.5 Spectrophotometric Methods

9.5 .1 Colorimetry

Atropine has 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 ropa a l k a l o i d s , by t h e use of new reagents . An absorpt iomet r ic method i s descr ibed ( 9 8 ) f o r t h e de te rmina t ion of a t ro - p ine 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 nves t iga t ed wi th a view t o improving t h e s t a b i l i t y o f t h e co lored 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 obta ined wi th tetraethylammonium hydro- x ide as t h e base and dimethylformamide as t h e so lvent . 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 dryness , n i t r a t e d with 0 . 2 t o 0 .3 m l o f fuming HNO3, aga in evaporated, d i sso lved i n dimethylformamide, t r e a t e d wi th 0.3 ml of 25 percent aa. t e t r a e - thylammonim hydroxide and d i l u t e d t o 1 0 ml with dimethylformamide. The o p t i c a l dens i ty i s determined at 540 mu i n . 1-cm c e l l s aga ins t dimethylformamide and t h e a l k a l o i d a l content i s a sce r t a ined from a c a l i b r a t i o n graph which i s l i n e a r .

Simonyi and Tokar (99 ) have repor ted a new co lo r ime t r i c method f o r t h e determinat ion of s m a l l amounts of t r o p i c a c i d and i t s es ters . Atropine w a s n i t r a t e d f o r 1 5 minutes at 50' wi th 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 wi th hot 18 t o 20% N a O H , a c o l o r develops i n 30 minutes . This i s es t imated by us ing 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 sens i - t i v i t y i s 50 and 60 ug of a t rop ine per ml. The probable e r r o r i s * 3%.

ATROPINE 367

Nir-Grosfeld and Weissenberg (100) have repor ted two co lo r ime t r i c methods f o r t h e determinat ion of a t rop ine i n pharmaceutical p repara t ions . Recovery experiments i n d i c a t e an accuracy of ? 1%. The results agree with t h e s e obtained by t h e method of USPXV.

In 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 ba th . N i t r i c ac id (fuming) w a s added, and heated till fuming ceased, d r i ed a t l O 5 O f o r 1 5 min and allowed t o cool . The r e s idue obtained w a s d i sso lved i n acetone and d i l u t e d t o 25 m l . An a l iquo t ( 5 m l ) w a s mixed wi th isoproprylamine and 0.1% methanolic KOH and t h e e x t i n c t i o n at 540 mu w a s measured after one minute.

I n method 11. The compound is n i t r a t e d as i n method I and d isso lved i n 50% e thanol (10 m l ) . Heated on a water bath wi th 1% HC1 and z inc dus t f o r 1 0 minutes, cooled and f i l t e r e d . The z inc r e s idue w a s washed with H2O and t h e washings were added t o t h e f i l t r a t e . NaN02 i s added, mixed and allowed t o s tand for 10 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 and f o r 1 0 minutes. N - l - naphthylethylenediamine dihydrochlor ide solu- t i o n was added, d i l u t e d with water t o 25 ml and a f te r 30 min, t h e ex t fnc t ion a t 550 mu w a s measured.

1% of

Pohm (101) repor ted a micro-determination of a t rop ine co lo r ime t r i ca l ly , by means of p- dimethylaminobenzaldehyde. Atropine i s mixed wi th e t h e r and aq. N H 3 and s i t a s ide f o r two hours and f i l t e r e d . The f i l t e r e d ex t r ac t i s extra.cted wi th 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 ex t r ac t ed wi th chloroform, evaporated t o dryness . Three drops of aq. bromine are added and evaporated o f f . The r e s idue i s d isso lved i n methanol and again evaporated with 3 drops of aq. bromine. A f t e r drying f o r 2 hours over P2O5, t h e r e s idue i s t r e a t e d wi th 7 drops of Wasicky reagent ( a s o l u t i o n o f 1. gm of p-dimethylaminobenzaldehyde i n 9 of 88% H2SO4) and s i t a s i d e f o r 2 minutes. It i s then heated f o r 3 minutes i n


a b o i l i n g water ba thand cooled i n i c e f o r 1 5 seconds. Acet ic anhydride i s added with s t i r r i n g and a f t e r 30 minutes , t h e ex t inc t ion i s measured at 500 mu.

Atropine has been determined @02) color imet r i - c a l l y by means of Reineck's sa l t . Ammonium re inecka te w a s used f o r t h e de te rmina t ion of a t r o p i n e i n 1% H2SO4. s o l u t i o n (0 .5%) w a s added t o t h e t e s t so lu t ion , t h e mixture was placed 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 wi th cooled water and d isso lved i n acetone. The e x t i n c t i o n i s then measured aga ins t a reagent b lank .

Ammonium re inecka te

The e x t r a c t ion-spectrophotometric determination method f o r t h e assay of a t r o p i n e wi th t h e use of vanadium ca techo la t e has been repor ted by Shesterova e t a l . (103). The method involves formation of a water- insoluble V'v-catechol- a t r o p i n e (1:2:1) complex ( I ) i n an aq. medium ad jus t ed t o pH 3 t o 4 with hydrogen p h t h a l a t e b u f f e r s o l u t i o n conta in ing a 200-fold molar excess ( r e l a t i v e t o I ) o f VO; and an 8000-f01d molar excess of ca t echo l and e x t r a c t i o n of t h i s complex i n t o chloroform. The complex e x h i b i t s max. absorp t ion at 620 nm.

Semenicheva Qo4) r epor t ed a method f o r t h e de te rmina t ion o f a t r o p i n e su lpha t e i n eye drops. Atropine su lpha te i n n e u t r a l s o l u t i o n is t r e a t e d wi th 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 wi th 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 wi th sodium su lphide s o l u t i o n and t h e c o l o r o f t h e sodium picramate formed i s compared wi th s tandard prepared by reducing p ic r ic ac id s o l u t i o n i n t h e same way.

9 .5 .2 Photometric Analysis

Akopyan (105) has r epor t ed a photometric method for t h e de te rmina t ion of a t r o p i n e and o t h e r t ropan a l k a l o i d s i n pharmaceutical mixtures . The determinat ion i s based on t h e r e a c t i o n of t h e a l k a l o i d ( a t r o p i n e ) wi th p-aminobenzalde- hyde on concentrated su lphur ic a c i d . The

ATROPINE 369

i n t e n s i t y o f t h e co lo r produced being measured i n a photometric absorpt iometer wi th a green f i l t e r . Fahmy e t a l . (106,107) have publ ished a compa- ra t ive s tudy of t h e d i f f e r e n t photometric methods of determinat ion of a t ropine:-

--

I. The t u n g s t o s i l i c i c a c i d , tungstophosphoric a c i d , copper su lpha te , 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 of a t rop ine i n tox ico log ica l samples. Vitali 's method i s p re fe r r ed .

11. The use of bromothymol b lue , bromocresol purp le , Metani l yellow (C.I. ac id yellow 36) and methyl orange, and var ious organic so lven t s , i n t h e alkaloid-dye method of determinat ion has been s tudied . The combination of Metani l yellow and chloroform i s most convenient.

The use of ammonium re ineckate i n t h e photo- met r ic determinat ion of a t rop ine , has been descr ibed (108). The procedure i s as follows:

To t h e s o l u t i o n conta in ing from 2 t o 1 0 mg of a t r o p i n e add 0.5 N H$O4 ( 2 drops) and sa tu- r a t e d ammonium re ineckat e so lu t ion , with st irr- ing . Col lec t t h e p r e c i p i t a t e on a s in t e red - g l a s s f i l t e r (Gb), wash it wi th co ld water, and d i s so lve it i n dioxan a c i d i f i e d with 0.5 N- H2SO4. s o l u t i o n at 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 rop ine i n t a b l e t s .

Levine and Roe 609) have descr ibed a method for t h e determinat ion of a t rop ine and t r o p i c ac id . Atropine and t r o p i c ac id were separa ted from each o the r and from p rese rva t ives such as benzyl a lcohol 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 , wi th Celite 545 as support ing phase.

Measure t h e e x t i n c t i o n of t h e dioxan

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 wi th N NaHC03 (1 m l ) absorbed on Cel i te (4 g + 1 g ) , cons 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 Cel i te ( 3 g + 1 g ) . On washing t h e columns wi th chloroform (100 m l + 25 m l through 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 preserva- t i v e s pass both columns. Tropic a c i d i s e l u t e d from column A wi th e i t h e r a f t e r ac id i - f i c a t i o n of t h e column wi th 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 wi th chloroform after n e u t r a l i z a t i o n of t h e column wi th aqueous ammonia. Atropine and t r o p i c a c i d are converted i n t o t h e i r sa l t s by add i t ion of HC1 and aqueous ammonia respec- t i v e l y , and evaporated t o dryness . For the modified co lo r ime t r i c procedure, t rea t t h e d ry r e s idue on a steam ba th f o r 30 minutes wi th fuming n i t r i c ac id (1 m l ) i n a covered f l a s k . Add water ( 1 0 m l ) , aqueous ammonia, ( 2 m l ) , sodium d i t h i o n i t e (about 50 mg) and 5% NaN02 s o l u t i o n ( 5 m l ) and hea t f o r a f u r t h e r f i v e minutes , 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 cu r ren t of air. Add 25 mg of s o l i d N-1-naphthylethylenediamine dihydrochlor ide , make up t o volume, set a s i d e f o r 0 .5 t o 4 hours , and compare t h e e x t i n c t i o n at 550 mu wi th va lues obtained from s tandards 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 at least 4 mg recover ies were from 100 t o 103%.

Febvre (11.0) r epor t ed t h a t Vitali-Morin reac- t i o n f o r a t r o p i n e i s modified t o g ive a repro- duc ib le co lo 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 dryness under vacuum i n a cen t r i fuge tube , t hen t r e a t e d wi th a f e w drops of a mixture 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% abso lu te e tha- n o l i c KOH ( t h e presence 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 solu- t i o n i s n e u t r a l i z e d , when t h e co lo r appears at once. Af te 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 acetone and making up t o 10 ml with acetone; t h e e x t i n c t i o n i s measured (Filter 63 of 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 only f o r concent ra t ion from 5 t o 20 vg per m l , but f o r h igher concentrat i on , a c a l i b r a t i o n curve can be used. Above 100 ug per 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%. products o f a t rop ine . No co lo r i s given by t h e hydro lys is

9.5.3 U l t r a v i o l e t Spectrophotometric Methods

Systematic t ox ico log ica l ana lys i s by spectrophotometric methods have been publ ished (111). The sample of t i s s u e i s homogenized with 25 m l of 0 .1 N HC1; t h e homogenate i s ex t r ac t ed on a wa te rba th wi th 75 m l 95% e thanol and 2 m l , 10% Na2W04. m l of Mcl l r a ins ' s b u f f e r at pH 7 and ex t r ac t ed wi th chloroform (50 m l ) . The separa ted chloroform l a y e r i s then ex t r ac t ed 100 m l of 0 .1 N HC1. The c h a r a c t e r i s t i c U.V. absorp t ion curves f o r 30 a l k a l o i d s i n d i l . HC1 a r e presented; a t rop ine can be determined quant i ta - t i v e l y by t h i s method.

The res idue i s being d isso lved i n 50

Cross e t a l . Q12) have determined some alka- l o i d s inc luding a t rop ine spec t rophotometr ica l ly and descr ibed i t s a p p l i c a t i o n t o pharmaceutical p repara t ions . To determine a t rop ine , 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 of a t rop ine (1 mg) i n phosphate bu f fe r s o l u t i o n (pH 7 ) (20 m l ) , e x t r a c t with chloroform, shake t h e e x t r a c t with phosphate bu f fe 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 wi th t h e same buf fe 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 descr ibed t h e fol lowing procedure, for t h e u l t r a v i o l e t detenninat i on of apoatropine and be l ladonine i n a t ropine :

"Prepare a so lu t ion of t h e mixture i n 0 . 1 N - H2S04 conta in ing 15% of e thanol , 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". Calcu la te t h e content of each a l k a l o i d by so lu t ion o f t h e t h r e e approp- r i a t e simultaneous equat ions. The e x t i n c t i o n c o e f f i c i e n t of each compound at each wavelength i s given.


Atropine w a s determined spec t rophotometr ica l ly i n eye drops by Zabrak and Farkas (114). The absorp t ion s p e c t r a of a t rop ine show a m a x i m a at 186 mu. D i l u t e 1 m l of t h e sample t o 100 ml and 5 ml of 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 with w a t e r and measure t h e e x t i n c t i o n at 186 m u aga ins t water. l a w i s obeyed over t h e range 0 t o 8 pg pe r ml. The r e s u l t s ob ta ined by t h i s method are wi th in 1% of those obta ined by e x t r a c t i o n methods.

Beer's

Uhlmann (115) repor ted a spectrophotometr ic assay method f o r a t rop ine 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 assay t h e drug i n aq. s o l u t i o n of i t s s a l t , t h e e x t i n c t i o n of t h e d i l u t e d sample i s determined at t h e wavelength f o r maximum absorp t ion (257 t o 286 nm) and compared with t h a t of p rog res s ive ly d i l u t e d samples o f s tock so lu t ion . The method i s c h i e f l y designed f o r use on aq. p repa ra t ions ( ampoules).

9.5.4 In f r a - r ed Spectrophotometr ic Method

The a p p l i c a t i o n of i n f r a - r ed spectrometry t o q u a n t i t a t i v e a n a l y s i s of a t r o p i n e i n t h e s o l i d phase has been r epor t ed by Browning e t a l . (116). t echnique has been success fu l ly appl ied as an a i d i n t h e q u a n t i t a t i v e de te rmina t ion of a t ro - p ine by I R spectrophotometry.

The pressed potassium bromide b e l l e t

9.5.5 Fluorometr ic Analysis

Laugel (119 have publ i shed a method f o r t h e de te rmina t ion 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 luorescence of compounds of t h e type a c i d dye-azo base. The concent ra t ion of a t r o p i n e i n pharmaceutical p repa ra t ion i s determined ( t o wi th in 4%) by measuring t h e f luorescence of t h e complex formed quan t i t a - t i v e l y , i n chloroform s o l u t i o n , by a t rop ine wi th a dihydroxyl luran a c i d dye, e . g . cos in . The concent ra t ion which i s d i r e c t l y propor- t i o n a l t o t h e f luorescence (measured at 550 m u ) , i s obtained from a s tandard c a l i b r a t i o n curve for a t rop ine . Beer's l a w being obeyed f o r 10 t o 60 pg of a t rop ine .

ATROPINE 313

Shuntaro Ogawa e t a l . (118) have determined t h e fluorimetry of atropine with eosin yellowish ( C . I . Acid Red 87) . The method which i s simple and rapid i s based on t h e formation of fluores- cent complex between atropine and eosine. To a solut ion of atropine i n chloroform ( 9 m l ) i s added 0.1% eosine solut ion (1 m l ) , t h e mixture is 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 at 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 ropine per m l ; t h e coe f f i c i en t of va r i a t ion i s 2.6%.

9.5.6 Phosphorimetric Analysis

Winefordner and Tin (119) have determined atropine i s urine. A rapid method w a s described f o r t h e ex t r ac t ion of a t ropine from body f lu ids ; t h e concentration of t h e drug i s determined by phosphoresence measurement and comparison with standard solut ion.

9.6 Chromatographic Methods

9.6.1 Paper Chromatography

Clarke (21) described two systems:

Whatman No. 1, sheet 1 4 X 6 i n , buffered by dipping i n a 5% solut ion of sodium hydrogen c i t r a t e , b l o t t i n g and drying at 25' f o r one hour. It can be s tored indefi- n i t e l y . A sample of 3 1.11 1% solut ion i n 2 N ace t i c acid o r i n ethanol i s used. Solvent system: 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 several weeks i f water i s added from t i m e t o t i m e t o keep t h e spec i f i c gravi ty ak 0.843 t o 0.844). The chromatogram i s developed, ascending i n a tank 8 X 11 X 15% in . 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 loca t ion reagent i s iodoplat inate spray,Rf = 0.37.

4.8 gm of c i t r i c acid i n

2 ) Whatman No. 1 o r No. 3, sheet 17 X 19 cm, impregnated by dipping i n a 10% solut ion

3 74 ABDULLAH A. AL-BADR AND FARID J . MUHTADI

of t r i b u t y r i n i n acetone and drying i n a i r . i n e thanol or chloroform, us ing a c e t a t e b u f f e r (pH 4.58) as so lven t . The beaker conta in ing t h e so lvent i s e q u i l i b r a t e d 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, ascending, where t h e paper is fo lded i n t o a cy l inde r and c l ipped . The cy l inde r i s i n s e r t e d i n t h e beaker containing t h e so lvent which i s not 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 wi th s i l i c o n e grease may se rve as a cover . T i m e run 1 5 t o 20 minutes. The 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 and Rf = 0.94.

A sample o f 5 1.11 of 1 t o 5 % so lu t ion

Other paper chromatography systems have been publ ished (120-136).

9.6.2 Thin-Layer Chromatography

Clarke (21) descr ibed t h e fol lowing system f o r t h e sepa ra t ion of a t rop ine :

Glass p l a t e s 20 X 20 em, coated wi th a s l u r r y cons i s t ing 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 ive a l a y e r 0 .25 mm t h i c k and d r i e d at llOo f o r 1 hour. A sample of 1 .0 1-11 of 1% s o l u t i o n i n 2 N a c e t i c a c i d , t aken by a micro drop, i s used. The so lvent system c o n s i s t s of s t rong ammonia s o l u t i o n : methanol (1.5 : 1 0 0 ) . It should be changed a f te r two runs . Solvent i s allowed t o s tand i n t h e tank f o r 1 hour. The ascending chromatogram i s developed i n a tank 21 X 2 1 X 10 em, t h e end of t h e tank being covered wi th f i l t e r paper t o assist evapora t ion . Time of run 30 minutes . The l o c a t i o n reagent 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 value i s 0.18.

Other TLC systems have been publ ished (133435, 137-140) for t h e sepa ra t ion of a t rop ine .

9 .6 .3 Eigh Pressure Liquid Chromatography

S t u t z and Sass (141) have descr ibed a high- speed, h igh p res su re l i q u i d chromatography of

ATROPINE 375

9.

a t rop ine and o t h e r t ropane a lka lo ids . The compound w a s separa ted on a s t a i n l e s s - s t e e l column (1 meter X 4.6 mm) packed with s i l - X absorbent wi th 28% aq. NH3-tetrahydrofuran (1:lOO) as so lvent and wi th a column i n l e t p ressure of 500 l b per sq. i n . A d i f f e r e n t r e f r a c t i v e index de tec to r and a W de tec to r opera t ing at 254 nm were used t o monitor t h e e lua t e . When appl ied q u a n t i t a t i v e l y , recove- r ies of a t rop ine su lpha te added t o var ious a lka lo id samples were between 88 and 94.5% at t h e = 25 pg l e v e l .

F e l l e t a l . (142) have repor ted an ana lys i s of a t rop ine su lpha te and i t s degradat ion products by reversed-phase high-pressure l i q u i d chromatography. Atropine was determined on a column of Hypersi l ODS ( 5 pm) w i l l 50 mM. Sodium a c e t a t e i n 1 0 mM -tetrabutylammonium sulphate (pH 5 . 5 ) - a c e t o n i t r i l e ( 3 : l ) a s mobile phase and de tec t ion a t 254 nm. The i n t e r n a l s tandard was p- toluic ac id . Atropine w a s w e l l separa ted from it degradat ion pro- duc t s , t r o p i c ac id a t r o p i c ac id and apoatro- p ine .

V a n Buuren et e. (143) have published a reversed-phase l i q u i d chromatography of bas i c drugs including a t rop ine - with a f luorogenic ion-pair ex t r ac t ion de tec to r .

Lawrance e t al. (144) have separated a t rop ine from o the r bas i c organic compounds by cont i - nuous post-column ion-pair e x t r a c t i o n detect ion i n normal-phase chromatography. The column ( 6 cm X 3 mm) of LiChrosorb S i 60 ( 5 pm) wi th a mobile phase (1 m l min-l) of 10% methanol s o l u t i o n i n chloroform conta in ing 0 .1 M - bu ty r i c ac id . Detect ion o f t h e f luorescence of t h e organic phase w a s measured at 452 nm.

6.4 Ion-Exchange Chromatography

Morphine su lpha te w a s separa ted from a t rop ine su lpha te 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 absorp t ion at 258 mp E 1% 1 cm = 40. The two drugs cannot be separa ted


on a weakly b a s i c r e s i n , which conver t s both t o t h e f r e e a l k a l o i d s , but t h e a l k a l o i d s can be separa ted on a s t rong ly bas i c r e s i n which r e t a i n s only t h e (pheno l i c ) morphine. The procedure o f t h e sepa ra t ion have been des- c r ibed as fol lows:

D i l u t e t h e sample ( con ta in ing 400 mg of morphine su lpha te and 10 mg o f a t r o p i n e su lpha te , t o 50 m l wi th 75 per cent methanol. To de t e r - mine t h e concent ra t ion o f morphine su lpha te , d i l u t e a 10 m l a l i q u o t t o 1000 m l wi th water and measure t h e e x t i n c t i o n a t 285 mp. To determine t h e concent ra t ion of a t r o p i n e s u l - pha te , pass a 25 m l a l i q u o t through a two-bed column c o n t a i n i n g h b e r l i t e IR-4B (10 m l ) above Amberlite IRA-410 (10 m l ) , e l u t e w i th 7 5 percent methanol (4 X 10 ml) and t i t r a t e t h e e l u t e wi th 0.02 N H C 1 wi th bromothymol b lue as i n d i c a t o r .

9 .6 .5 Gas Chromatography

Clarke ( 2 1 ) desc r ibes t h e fol lowing t h r e e systems f o r t h e sepa ra t ion of a t rop ine : -

Column: 1% SE-30 on 100-120 mesh Anakrom ABS. 6 f t X 4 mm i n t e r n a l diameter boro- s i l i c a t e g l a s s column. Column temperature: 180°. Carrier gas : Argon. Gas flow: 6 5 m l pe r minute at 180. Detector : 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 . Retent ion 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 temperature: 200'. Carrier gas : Argon. Gas flow: 80 ml per minute. Other condi t ions are as i n system a. Retent ion t i m e : 3.80 min. r e l a t i v e t o diphenhydramine.

Column: W AW. 5 f t X 1/8 inch i n t e r n a l diameter s t a i n l e s s s t ee l column. Column temperature: 230'. Carrier gas: n i t rogen . Gas flow: 30.7 m l per minute. Detector: flame ioniza- t i o n d e t e c t o r , hydrogen 22 m l per minutes. Retent ion t i m e : 0 .59 min r e l a t i v e t o codeine.

5% SE-30 on 60-80 mesh Chromosorb

ATROPINE 311

Santoro e t al,. Q46) have repor ted a s e l e c t i v e determinat ion of belladonna a lka lo ids by gas l i q u i d chromatography. Atropine w a s de te r - mined i n pharmaceutical p repa ra t ions in t h e presence of c e r t a i n m i n e s . t h e res idue i s d isso lved 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) containing 3% OV-17 on Gas-Chrom Q (80 t o 100 mesh) operated at 210' with H e l i u m as c a r r i e r gas (50 m l per min) and flame ioniza- t i o n de tec t ion and measure t h e peak h igh t s .

A f t e r e x t r a c t i o n ,

Nishimoto e t a l . &47) have descr ibed a s impli- f i e d q u a n t i t a t i v e ana lys i s of a t rop ine and o the r a lka lo ids i n scopol ia e x t r a c t . Analysis i s c a r r i e d out by g l c on columns (1 mm X 3 mm) packed wi th 0.75% of Dexsi l 300 GC on Gas Chrom Q, with n i t rogen (40 m l min-1) as c a r r i e r gas; wi th t h e column at ( u s u a l l y ) 180", a t ro - p ine (as i t s t r i m e t h y l s i l y l d e r i v a t i v e i s separa ted from hyoscine, apoatropine and hom- a t rop ine ( t h e i n t e r n a l s tandard. column at 90' and t h e c a r r i e r gas flow a t 30 m l min-l, a t rop ine i s e lu t ed 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 tandard. The method is appl ied 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 of scopol ic r o o t s . The peak h igh t r a t i o vs a t rop ine content i s r e c t i l i n e a r f o r 25 t o 75 ng of a t rop ine .

Other GC methods

Atropine t a b l e t s were ex t r ac t ed wi th chloroform i n an a l k a l i n e media and analyzed us ing a GC method wi th diphenhydramine as t h e i n t e r - n a l s tandard (148).

With t h e

9.6.6 Colwnn Chromatography

Kamienski and Puchalka (149) have repor ted t h e separa t ion of a t rop ine and hyoscyamine by a pot-entiometric chromatographic method. The a l coho l i c e x t r a c t s from t h e leaves Datura stramonium and t h e r o o t s of Atropa belladonna were d i l u t e d u n t i l t h e i r a lka lo id concent ra t ion approximately reached 0 .001 M. The separa t ion of a t rop ine and hyoscyamine i n t h e s e so lu t ions

378 ARDULLAH A. AL-BADR AND FARlD J . MUHTADI

w a s s tud ied . Four m l o f each s o l u t i o n were placed on alumina columns and e l u t e d wi th e i t h e r aqueous e thanol (60 o r 80%) or a mixture of benzene; water and e thanol (14.5%, 8.5% and 77% r e s p e c t i v e l y ) , t h e antimony microelectrode being 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 aga ins 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 epa ra t ion was achieved wi th t h e benzene - e thano l e l u t i n g s o l u t i o n , and a 20 cm column of Merck's alumina.

9.6.7 Paper Electrophoreses

Atropine and hyoscine were separa ted quant i - t a t i v e l y by paper e lec t rophoreses (150). They were separa ted wi th 0 . 1 N aq. N H 3 a s t h e e l e c t r o l y t e , and de tec t ed as brown spo t s by exposure t o iod ine vapour. After e l u t i o n o f t h e spo t s , t h e so lvent w a s evaporated, t h e r e s idue w a s n i t r a t e d with fuming H N O 3 , t hen d i s so lved i n dimethylformamide and t e t r a e t h y l - ammonium hydroxide w a s added according 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 concent ra t ion 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 equat ion.

9.7. Radio-immunoassay

~y us ing 3~ a t rop ine as t r a c e r , an ant iserum w a s r a i s e d by immunisation of r a b b i t s wi th an immunogen prepared by coupl ing t o human serum albumen. The de tec t ion of down t o 9 nM a t r o p i n e i n 0 .1 m l of serum or plasma i s poss ib l e . The recovery of a t r o p i n e added a t var ious concent ra t ion t o pooled normal human plasma w a s near 100%. Atropine r e a c t s wi th t h e a n t i - bodies; 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 ropine- hydro lys i s products ( 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 usefu lness of t h i s method i n pharma- cok ine t i c s s t u d i e s have been demonstrated i n assays of a t rop ine i n ser ia l serum samples from two p a t i e n t s who rec ieved 1 . 3 mg o f a t r o p i n e i n connect ion wi th anaes thes i a (151).

- A p rec i se , s e n s i t i v e and r a p i d radioimmunoassay f o r t h e ana lys i s of a t r o p i n e from unpur i f i ed e thano l i c e x t r a c t s of a t r o p i n e bel ladonna i s descr ibed (152).

ATROPINE 379

- Fasth e t a l . (153) descr ibed t h e f irst radioimmunoassay for a t rop ine using rabbit antiserum.

- Wurzburger e t a l . (154) repor ted and s e n s i t i v e and s p e c i f i c radioimmunoassay for a t rop ine and showed c learance curve f o r drog plasma.

- Radioimmunoassay (RIA) w a s appl ied t o measure a t ro - p ine i n himan plasma using ant iserum, t h e plasma c learance of a t rop ine i n four adul t vo lunteers w a s measured.

The measurement accomplished by a competi t ive R I A using r a b b i t an t i -a t ropine antibody. T r i t i a t e d a t rop ine is used as t h e r ad ia l igand (155).

Acknowledgement

The au thors would l i k e t o thank Mr. Uday C. Sharma and Tanvir A. Bu t t , both of College of Pharmacy, King Saud Univers i ty f o r t h e i r va luable s e c r e t a r i a l a s s i s t ance i n typ ing of t h i s manuscript.

3 80 ABDULLAH A. AL-BADR AND FARlD J . MUHTADl

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