Chapter 4 Tropane Alkaloids

249

Chapter 4 TROPANE ALKALOIDS 4.1. Tropine a l k a l o i d s 4.1.1. Ion-exchange HPLC.. 4.1.2. Reversed-phase HPLC.. 4.1.3. I o n - p a i r HPLC.. 4.1.4. Straight-phase HPLC.. 4.1.4. Detection 4.2. Pseudotropine a l k a l o i d s 4.2.1. Ion-exchange HPLC.. 4.2.2. Reversed-phase HPLC.. 4.2.3. I o n - p a i r HPLC.. 4.2.4. Straight-phase HPLC 4.2.5. Detection.. References

........................................................ ...................................................... ............................................................ ...................................................... .................................................................. ........................................................ ...................................................... ............................................................ ........................................................ ................................................................ ........................................................................

4.1.

249 249 250 252 252 260 260 26 1 261 262 262

TROPIYE ALKALOIDS*

Most o f the work performed so f a r on t r o p i n e a l k a l o i d s concerns the a n a l y s i s o f such a l k a l o i d s i n pharmaceutical preparations (Table 4.5).

Special methods have been i n v e s t i g a t e d

i n order t o lower the d e t e c t i o n l i m i t o f such alkaloids13’16’28’33’35’37’41’43’45. on the a p p l i c a t i o n o f d e r i v a t i z a t i o n techniques i n LC given by F r e i and S a n t i ”

Reviews

and F r e i 39 ,

include some examples o f t r o p i n e a l k a l o i d s . The a n a l y s i s o f atropine and i t s degradation products has a l s o been i n v e ~ t i g a t e d ~ ~ * ~ ~ ’ ~ ~ * ~ ~ . 4.1.1.

ION-EXCHANGE HPLC

Ligand-zxchange chromatography o f a l k a l o i d s by means o f ion-exchange m a t e r i a l s loaded w i t h metal ions was described by Walton and M ~ r g i a ~ ’ ~ ’ ’The ~ ~ technique . i s discussed i n Chapter 7. Instead o f a HPLC system using a d i o l c ~ l u m n ~ Huen ~ ’ ~ and ~ , T h e ~ e n i nfound ~ ~ a micropartic u l a t e s u l f o n i c a c i d cation-exchange m a t e r i a l t o be b e t t e r f o r a s e l e c t i v e separation o f t r o pane a l k a l o i d s p r i o r t o post-column f l u o r i m e t r i c i o n - p a i r d e r i v a t i z a t i o n (Fig.4.1).

Atropine

and scopolamine could be separated from i t s major decomposition products: apoatropine, t r o p i c acid, t r o p i n e and scopoline. 4.1.2.

REVERSED-PHASE HPLC

Honigberg e t a1 .15 described the HPLC analysis o f antispasmodic mixtures, c o n t a i n i n g i . a . atropine and scopolamine. The r e t e n t i o n behaviour was studied by changing three o p e r a t i n g parameters, i . e . the s t a t i o n a r y phase, the methanol

-

water r a t i o , and the pH o f the mobile

phase. A p e l l i c u l a r octadecyl and a phenyl column were used. I t was found t h a t more b a s i c mob i l e phases

-

containing ammonium carbonate - l e d t o t a i l i n g and m u l t i p l e peaks f o r a t r o p i n e

and scopolamine. Generally b e t t e r r e s u l t s were obtained on a phenyl column. Lund and Hansen3’ studied the separation o f a t r o p i n e and some o f i t s decomposition products using m i c r o p a r t i c u l a t e medium p o l a r i t y reversed-phase m a t e r i a l s as s t a t i o n a r y phase, i . e . s t a t i o n a r y phases containing chemically bonded cyano o r alkylamino groups. Atropine and apoatro*Because atropine and 1-hyoscyamine behave s i m i l a r i n a l l HPLC systems, the term a t r o p i n e w i l l be used i n the t e x t and i n a l l tables and f i g u r e s t o describe both a l k a l o i d s . Refcrenea p. 262

250

p i n e c o u l d be s e p a r a t e d on a cyano column, whereas t r o p i c a c i d and a t r o p i c a c i d were s e p a r a t e d o n a n a l k y l a m i n o column. C o u p l i n g o f a cyano and an a l k y l a m i n o column i n s e r i e s enabled t h e sep a r a t i o n o f a l l t e s t compounds ( T a b l e 4.1). A mixed bed column (cyano : a l k y l a m i n o 2 : l ) was found t o a l l o w f a s t s e p a r a t i o n ; however, a t r o p i n e c o u l d n o t be determined q u a n t i t a t i v e l y because i t was e l u t e d c l o s e t o t h e s o l v e n t f r o n t . The problem c o u l d be s o l v e d by i n c r e a s i n g t h e l e n g t h o f t h e cyano column t o 15 cm. Column s w i t c h i n g was used t o improve t h e d e t e c t i o n l i m i t o f a p o a t r o p i n e and s t i l l a l l o w a q u a n t i t a t i v e d e t e r m i n a t i o n o f a t r o p i n e ( F i g . 4 . 2 ) . G f e l l e r e t a1 . 3 5 * 4 1 used a m i c r o p a r t i c u l a t e c h e m i c a l l y bonded d i o l s t a t i o n a r y phase i n comb i n a t i o n w i t h an e x c l u s i v e l y aqueous m o b i l e phase. I t a l l o w e d post-column i o n - p a i r d e r i v a t i z a t i o n o f t h e separated a l k a l o i d s . By changing t h e pH and t h e i o n i c s t r e n g t h o f t h e b u f f e r , t h e c a p a c i t y f a c t o r s o f t h e a l k a l o i d s c o u l d be v a r i e d . A t r o p i n e was i n c l u d e d i n a s e r i e s o f a l k a l o i d s w h i c h were s e p a r a t e d on a macroporous s t y r e n e - d i v i n y l b e n z e n e copolymer47 (Chapter 8. Table 8 . 4 ) . 4.1.3.

ION-PAIR HPLC

F o r most o f t h e HPLC analyses o f t r o p i n e a l k a l o i d s , i o n - p a i r chromatography has been a p p l i e d . 26 analyzed methylscopolamine u s i n g sodium d e c y l s u l f a t e as i o n - p a i r i n g r e a g e n t . W a l t e r s

Burdo"

used a s i m i l a r 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 scopolamine i n t a b l e t s . Octanesulf o n i c a c i d (0.01 M) s e r v e d as t h e p a i r i n g - i o n i n an a c e t a t e b u f f e r o f pH 3.5 c o n t a i n i n g 34% a c e t o n i t r i l e . The a n a l y s i s was performed on an o c t a d e c y l column. F o r q u a l i t a t i v e work a decrease o f t h e percentage o f a c e t o n i t r i l e t o 28 was p r e f e r e d ( T a b l e 4 . 2 ) . Brown e t a l . 2 7 ' 3 0 used h e p t a n e s u l f o n i c a c i d (0.01 M) as p a i r i n g - i o n and an aqueous m o b i l e phase o f pH 3.40 cont a i n i n g 35% a c e t o n i t r i l e i n t h e a n a l y s i s o f a n t i c h o l i n e r g i c drugsz7, and a t r o p i n e 3 ' on m i c r o p a r t i c u l a t e o c t a d e c y l columns. Hartmann3* c o n t i n u e d t h e work s t a r t e d by Burdo22. mentioned above, and used sodium d e c y l s u l f a t e and d i o c t y l s u l f o s u c c i n a t e as i o n - p a i r i n g r e a g e n t s i n t h e a n a l y s i s o f methylscopolamine i n neomycine c o n t a i n i n g v e t e r i n a r y p r e p a r a t i ons ( F i g .4.3). A c h a r i and Jacob5'

s t u d i e d s e v e r a l parameters t h a t have an i n f l u e n c e on t h e r e t e n t i o n o f

b a s i c drugs i n i o n - p a i r HPLC, i . a .

a t r o p i n e and scopolamine. Some g e n e r a l c o n c l u s i o n s were

drawn concerning t h e n a t u r e o f t h e p a i r i n g - i o n , t h e t y p e o f c h e m i c a l l y bonded s t a t i o n a r y phase and t h e c o m p o s i t i o n o f t h e m o b i l e phase (Chapter 2 ) . F o r t h e a n a l y s i s o f a t r o p i n e and i t s m a j o r a c i d i c decomposition products, K r e i l g a r d Z 5 used 0.01 M tetrabutylammonium s u l p h a t e as p a i r i n g - i o n i n a m o b i l e phase o f a c e t o n i t r i l e

-

0.05 M

a c e t a t e b u f f e r (pH 5 . 5 ) ( 1 : 4 ) ( T a b l e 4 . 3 ) . S a n t i e t a l . 1 3 t e s t e d v a r i o u s s t a t i o n a r y phases f o r s t r a i g h t - p h a s e i o n - p a i r p a r t i t i o n chromatography. P i c r i c a c i d served as precolumn p a i r i n g - i o n 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 , scopolamine and ergotamine because o f i t s s t r o n g UV-absorption, which enabled a 100-300 t i m e s i m provement o f t h e d e t e c t i o n l i m i t o f t h e p o o r l y UV-absorbing t r o p i n e a l k a l o i d s (see d e t e c t i o n ) . Good r e s u l t s were o b t a i n e d w i t h m i c r o p a r t i c u l a t e K i e s e l g u h r , b u t t h e r e p r o d u c i b i l i t y o f t h e column performance was d i f f i c u l t because o f v a r i a t i o n i n t h e q u a l i t y o f t h e K i e s e l g u h r . M i c r o p a r t i c u l a t e s i l i c a g e l w i t h a pore s i z e o f 100 o r 1000 the pairing-ion,

8 was

found t o be b e t t e r as s u p p o r t f o r

b u t low f l o w r a t e s had t o be used ( 0 . 2 m l / m i n ) ( F i g . 4 . 4 ) .

S i l i c a gel w i t h a

s m a l l e r pore s i z e gave p o o r r e s u l t s , due t o t h e mechanical i n s t a b i l i t y o f t h e system a t t h e r a t h e r h i g h l i n e a r v e l o c i t i e s used.

261 TABLE 4 . 1 RETENTION DATA AND DETECTION LIMITS FOR ATROPINE AND I T S DEGRADATION PRODUCTS3' Compound

k'

Atropine Apoatropine Tropic acid Atropic acid d e l ladonnine

0.57 2.0 2.4 3.0 3.5

minimum d e t e c t a b l e amount ( n g ) 5 40 2

Compound

k'

6-Be1 l a d o n n i n e 6-Isatropic acid Scopolamine Homatropi ne

5.9 3.0 0.36 0.44

Column N u c l e o s i l 5 CN ( 5 0 ~ 4 . 6 nnn I D ) and N u c l e o s i l 5 NH ( 5 0 ~ 4 . 6mn I D ) connected i n s e r i e s , m o b i l e phase 0.05 M sodium a c e t a t e b u f f e r (pH 5) - methino1 (3:1), d e t e c t i o n UV 254 nm. TABLE 4.2 SEPARATION

OF SOME TROPANE ALKALOIDS'~

Compound

k'

Compound

Tropic acid Scopol ami ne Homatropine Scopolamine N-oxi de Methylscopolamine

0.65 2.70 2.76 2.88 3.24

Methyl a t r o p i ne Atropine Hyoscyami ne Cocaine Benztropine

~

~~

~~

~

k' 3.63 4.00 4.00 11.6 not eluted

~~

Column UBondapak C18 ( 3 0 0 ~ 3 . 9 mm I D ) , m o b i l e phase 28% a c e t o n i t r i l e i n 0 . 0 1 M aqueous octanes u l f o n i c a c i d a d j u s t e d t o pH 3.5, f l o w r a t e 1 ml/min, d e t e c t i o n UV 230 nm. TABLE 4.3 SEPARATION OF SOME TROPANE ALKALOIDSz5 Compound

k'

Compound

k'

Atropine Belladonnine Tropic acid Apoatropine

0.2 0.3 1.5 2.5

Atropic acid 8-Isatropic acid 4-Methylbenzoic a c i d ( i n t e r n a l standard)

4.5 6.2 6.5

Column L i c h r o s o r b RP8, 5 p m ( 1 0 0 ~ 4 . 6 mm I D ) , m o b i l e phase 0.01 M tetrabutylammonium s u l p h a t e i n 0.05 M a c e t a t e b u f f e r - a c e t o n i t r i l e ( 4 : l ) a t pH 5.5, f l o w r a t e 1 ml/min, d e t e c t i o n UV 254 nm

.

Huen e t a1.28 found t h e optimum pH f o r i o n - p a i r s e p a r a t i o n s w i t h p i c r i c a c i d t o be 5-6 i n t h e i r i n v e s t i g a t i o n s , when t h e e f f e c t o f t h e v a r i a t i o n o f p i c r i c a c i d c o n c e n t r a t i o n and temp e r a t u r e was s t u d i e d . G f e l l e r e t a1 . 3 3 3 3 7 d e s c r i b e d a u t o m a t i z a t i o n o f precolumn d e r i v a t i z a t i o n f o r t h e systems mentioned above. Post-column d e r i v a t i z a t i o n w i t h i o n - p a i r i n g t e c h n i q u e has a l s o been used t o improve t h e de43

t e c t i b i l i t y o f t r o p i n e a l k a l o i d s ( s e e d e t e c t i o n ) . The t e c h n i q u e was used by Lawrence e t a l . after

s e p a r a t i o n o f t h e a l k a l o i d s on a m i c r o p a r t i c u l a t e s i l i c a g e l column w i t h a m o b i l e phase

o f c h l o r o f o r m and methanol, c o n t a i n i n g t h e weakly i o n - p a i r i n g b u t y r i c a c i d . C r ~ m m e ni n~v~e s t i g a t e d t h e r e t e n t i o n o f o r g a n i c compounds on s i l i c a g e l u s i n g aqueous mob i l e phases. A r e t e n t i o n model was presented, based o n t h e d i s t r i b u t i o n o f i o n - p a i r s . A p p l i c a t i o n s t o t h e s e p a r a t i o n o f some t r o p i n e a l k a l o i d s a r e shown i n Fig.4.5.

References p. 262

4.1.4.

STMIGHT-PHASE HPLC

The f i r s t HPLC separation of t r o p i n e a l k a l o i d s was performed on a s i l i c a gel column by means o f tetrahydrofuran

- 28% ammonia (100:l)’. Verpoorte and Baerheim Svendsen” separated some - methanol - diethylamine (9O:lO:l) on m i c r o p a r t i c u l a t e

tropine alkaloids with diethyl ether s i l i c a gel (Fig.4.6).

Rather l a r g e amounts o f a l k a l o i d s had t o be i n j e c t e d due t o the poor

chromophore. which caused severe t a i l i n g on p e l l i c u l a r s i l i c a gel columns. Chloroform cont a i n i n g mobile phases d i d n o t p e r m i t useful separations o f the a l k a l o i d s because o f l a r g e d i f ferences i n capacity f a c t o r s o f a t r o p i n e and scopolamine. Also, by means o f a mobile phase cons i s t i n g o f d i e t h y l ether and diethylamine. separations were achieved4’

-

( Chapter 7,Fig.7.14).

dichloromethane ( 3 : l ) . t o which 1%o f 29% ammonia was added. as mobile phase and a m i c r o p a r t i c u l a t e s i l i c a gel column. Atropine, homatropine and Achari and TheimerL4 used methanol

scopolamine showed d i f f e r e n t capacity f a c t o r s . Straight-phase separations by means o f p o l a r mobile phases were used by Jane

12

(Chapter 7.

Table 7.8). Aigner e t a1.18 separated some drugs, i n c l u d i n g tropane a l k a l o i d s . on s i l i c a gel columns impregnated w i t h s i l v e r iodide. By using gradient e l u t i o n , multicomponent mixtures o f drugs could be separated. 4.1.5.

DETECTION

A major problem i n the analysis o f t r o p i n e a l k a l o i d s by HPLC i s t h e i r poor chromophore. The decomposition products t r o p i n e and scopoline completely lack any chromophore. UV d e t e c t i o n a t 254 nm has. according t o Stutz and Sass

1 a d e t e c t i o n l i m i t o f about 1 pg, and R I d e t e c t i o n o f

about 50 ug. WaltersL6 found a wavelength o f 230 nm t o be t h e optimum compromise between great e r a b s o r p t i v i t y o f the a l k a l o i d s and increasing background absorbance o f the mobile phase (detection l i m i t 0.5 ug). Brown and S1eeman3’ reported a d e t e c t i o n l i m i t o f 200 ng f o r a t r o pine a t 254 nm. Because o f the low absorbance o f t r o p i n e a l k a l o i d s , Santi e t al.13’28

developed an i o n - p a i r

separation using the strong UV absorbing p i c r i c a c i d as p a i r i n g - i o n . Compared t o a reversed-phase separation followed by d e t e c t i o n a t 210 nm, a f i f t y - f o l d enhancement o f the d e t e c t i o n l i m i t was observed using the p i c r i c a c i d i o n - p a i r technique and d e t e c t i o n a t 254 nm. The det e c t i o n l i m i t f o r a t r o p i n e was 5 ng and f o r scopolamine 50 ng. The non UV absorbing scopoline could be detected a t a l e v e l as low as 2.5 ng. By measuring a t the UV maximum o f p i c r i c a c i d

-

345 nm - interference o f the s i g n a l s o f non-pairing compounds was suppressed. G f e l l e r e t a1 .33*37 developed the technique t o an automated pre-column d e r i v a t i z a t i o n method.

G f e l l e r e t a1 .35941 a l s o developed an automatic post-column d e r i v a t i z a t i o n method. A f l u o rescent pai r i n g - i o n

-

9,lO-dimethoxyanthracene-2-sul fonate (DAS)

-

was used t o improve the

detection l i m i t o f atropine. The best r e s u l t s were obtained w i t h an e x c l u s i v e l y aqueous mobile phase, t o which the reagent was added as an aqueous s o l u t i o n . Subsequently the i o n - p a i r was extracted w i t h chloroform o r dichloromethane. An increase i n the percentage o f methanol i n the mobile phase reduced the s e n s i t i v i t y . The method was found t o be 200 times more s e n s i t i v e than UV detection a t 208 nm; the minimum detectable amount was 200 pg. Peak broadening caused by

the post-column r e a c t o r was about 40%. To meet the requirement o f a s e l e c t i v e separation w i t h a mobile phase containing only small amounts o f organic solvents, Huen and T h e ~ e n i nprefer~~ red ion-exchange separation (Fig.4.2).

263

Lawrence e t a1 .43 described a simpler post-column ion-pair derivatization technique. whereby the alkaloids were separated by means of an organic mobile phase on s i l i c a gel. The column eluate and the aqueous OAS solution were mixed and the two imniscible phases separated. About 50% of the organic phase was led t o the fluorimetric detector. Various parameters influencing the bandwidth were investigated, i.a. the influence of methanol in the mobile phase. An increasing methanol content deteriorated the signal t o noise r a t i o , and the most useful range was 0-15% methanol. The detection l i m i t f o r atropine was found t o be 40 ng. Hashimoto e t al.36 described a capacitance-conductance detector f o r HPLC; i t had a detection l i m i t of 5 p g f o r scopolamine.

References p. 262

264

NH?+CN NHZ

-

1

'

20

min

"

'

I

10

'

'

-

~

I

0

-

0

5

10

15 m\

Fig. 4.1. HPLC analysis a t r i E i n e w i t h post-column f l u o r i m e t r i c i o n - p a i r d e r i v a t i z a t i o n t o improve the detection l i m i t Column Nucleosil 10 SA (300x4 mm I D ) , mobile phase methanol 0.2 M aqueous diammonium hydrogen phosphate (pH 3)(1:9), f l o w r a t e 2 ml/min, column temperature 85 C. d e t e c t i o n UV 254 nm (1). f l u o r i m e t r i c d e t e c t i o n a f t e r post-column d e r i v a t i z a t i o n w i t h Na-DAS ( e x c i t a t i o n 383 nm, emission 446 nm) (2). Peaks: 1, scopolamine; 2, atropine; 3, apoatropine. (Reproduced w i t h permission from r e f . 45) 32 Fig. 4.2. HPLC analysis atropine and i t s degradation products usinq column s w i t c h i n g Columns Nucleosil 5NH ( 5 0 ~ 4 . 6mn 10) and Nucleosil 5CN(100x4.6 mn I D ) , mobile phase methanol - 0.05 M sodium azetate b u f f e r (pH 5)(1:3), d e t e c t i o n UV 254 nm.(see a l s o Table 4 . 1 ) . Peaks: 1, t r o p i c acid; 2, a t r o p i c acid; 3, atropine; 4, apoatropine.

-

i

1

A

i2

1'6

min

38 Fig. 4.3. HPLC analysis methylscopolamine and some added i m p u r i t i e s Column UBondapak C18 ( 3 0 0 ~ 3 . 9mn I D ) , mobile phase 0.01 M d i o c t y l s u l f o s u c c i n a t e sodium s a l t water (55:45). pH adjusted t o 3.5 w i t h g l a c i a l and 0.01 M anonium n i t r a t e i n 95% ethanol a c e t i c acid, f l o w r a t e 1 ml/min, d e t e c t i o n UV 254 nm. Peaks: 1, t r o p i c acid; 2, a t r o p i c acid; 3, DOSS a r t i f a c t ; 4, scopolamine; 5, methylscopolamine; 6, aposcopolamine; 7. methylaposcopolamine. (Reproduced w i t h permission from r e f . 38, by courtesy o f Journal Association of o f f i c i a l a n a l y t i c a l chemists).

-

255

2

i i1

0.001A

5

I

min 10 8 6 L 2 Fig. 4.4. HPLC analysis t r o p i n e a l k a l o i d s 28

Column S i l i c a gel S i l o 0 5 p m (100x3 m I D ) impregnated w i t h 0.06 M p i c r i c a c i d (pH 6). mobile phase chloroform saturated w i t h the s t a t i o n a r y phase 0.06 M p i c r i c acid, f l o w r a t e 0 . 2 ml/min, detection UV 345 nm. Peaks: 1. dodecylbenzene ( t o ) ; 2, apoatropine; 3, ergotaminine; 4 , a t r o pine; 5, ergotamine; 6, scopolamine. 44 Fig. 4.5. HPLC analysis a n t i c h o l i n e r g i c s Column Lichrospher S i l o 0 10 pm (200x4 nm ID), mobile phase 0.1 M sodium phosphate b u f f e r pH 2 . 2 containing 1.9% n-amylalcohol, l i n e a r v e l o c i t y 1 . 6 nm/sec., d e t e c t i o n UV 254 nm. Peaks: 1. scopolamine; 2 , afropine; 3, benactyzine; 4, adiphenine; 5, methylatropine; 6, oxypyrronium; 7, oxyphenonium.

h

Rafaence# p. 262

F i g . 4 . 6 . HPLC analysis o f some t r o p i n e a l k a l o i d s 1 9 Column P a r t i s i l 5 pm ( 3 0 0 ~ 4 . 6mn I D ) , mobile phase d i ethyl ether methanol diethylamine (90:10:1), f l o w r a t e 2.29 ml/min, d e t e c t i o n UV 254 nm. Peaks: 1, scopolamine; 2. apoatropine; 3, atropine.

-

-

N

8:

TABLE 4.4 HPLC ANALYSIS OF VARIOUS COMPOUNDS INCLUDING TROPINE ALKALOIDS ~~

~

Aims

Stationary Phase

Column Dim. LxID mn Mobile Phase

A,S,22 other alkaloids

Analysis alkaloids

Merckosorb Si60. 5pm

300x2

CHC13-HeOH( 9: 1) .(8:2) ,( 7:3) Et20-MeOH(8:2),(7:3),(6:4) 5

A.opium alkaloids, quinine.cinchonine.strychnine, n i c o t i ne, coc

Separation on ionexchange resins (ligand-exchange LC)

Hydrolyzed Portgel PT loaded w i t h Cu Bio-Rad2$C20 .loaded w i t h Cu

470~6.3

0.06M NH OH i n 33% EtOH 0.2M NH 8H i n 33% EtOH 0.05M NA OH i n 33% EtOH 0.03M NHdOH i n 33% EtOH

A,benztropine. various alkaloids

Analysis drugs o f abuse(Tab1e 7.8)

P a r t i s i l 6 um

250~4.6

Detection w i t h conductance detector

S i l i c a gel 10

A1 k a l o i d *

Other Compounds

Various drugs o f forensic interest

S,Cinchona a1 k a l o i ds .bruc i ne.strychnine, emeti ne ,reserp i ne,yohimbine. caffeine

470~6.3

6.10, 17

MeDH-2M NH OH-1M NH N03(27:2:1) MeOH-O.2M AH4N03()3 !: 12 CHC13-MeOH-hexane(7:3: 10)

Vm

36

A.S,codeine, papaveri ne .qui nine,caffeine, ephedrine

Various drugs

Retention behaviour basic drugs i n ionp a i r HPLC

pBondapak C18 VBondapak Phenyl pBondapak CH VBondagel Chromegabond C8 Chromegabond C6Hl,

300x4

0.005M heptanesulfonic acid i n H O-MeOH-AcOH(50:49:1) (PH a.0,

A,codeine.morphine,dihydrocodeine,quinine, q u i n i d i ne,caffeine ,theophyl1ine ,ajmal ine

Various basic drugs

Separation o f basic drugs on s i l i c a gel w i t h non-aqueous i o n i c eluents

Spherisorb S5W S i l i c a

250~4.9

MeOH-hexane(85:15)containing 0.10% HC104

*

Ref.

For abbreviations see footnote Table 4.5

50

66

k?

H

i P N

TABLE 4.5 HPLC ANALYSIS TROPINE ALKALOIDS I N PHARMACEUTICAL PREPARATIONS

(0

A1 k a l o i d *

Other Compounds

A.H .S .apoA ,t r o p HMe,codeine,dihydrocodeinone,ephedrine, strychnine

S t a t i o n a r y Phase

Column Dim. LxID mn Mobile Phase

Ref.

Separation

Sil-X

1000~4.5

1

C o r a s i l C18

1220~2.3

Various analgesics. Determination i n a n t i h i s t a m i n i c s and cough-cold a n t i t u s s i ves mixtures

Chlordiazepoxide, propanthel i n e . i s o propamid,clidinium, phenobarbital ,prochlorperazine

Determination o f antispasmodic mixtures

THF-28% NH40H( 1OO:l) ACN-1%NH40Ac(3: 2) (pH 7.4)

7

Separation w i t h i o n p a i r LC S p h e r o s i l XOB.5-1@m 1 0 0 ~ 2 . 8 loaded w i t h 0.03M p i c r i c a c i d and b u f f e r DH 5 S i l i c a g e l 100.5pm 100~2.8 loaded w i t h 0.06M p i c r i c a c i d and b u f f e r PH 5

A,S .ergotami ne

~~

Aims

C o r a s i l C18 o r Corasi 1 Phenyl

1220~2.3

CHCl s a t . w i t h 0.05M p i c r i c a c i d 3 i n pH 5 b u f f e r CHCl s a t . w i t h 0.06M p i c r i c a c i d 3 i n pH 6 b u f f e r

13.16 MeOH-l% aq.(NH ) H P04(6:4) (pH 5.85),(1:17(~4 5.50). (2:3)(pH 5.50). MeOH-1% aq. (NH )H PO -1%aq. (NH ) HPO ( 3 : 1 : f ) ( i H 4.20). (2:f : (pa 7. go), (4:3: 3) (pH 7.60) MeOH-0.5% aq. (NH ) CO (3:2) 8.74) (pH 8.65),(1:1)(pa (2:3)(pH 8.80) 15

3)

A.S,ergotamine.er-

B u t a l b i t a l .pheno-

Separation on s i l -

Lichrosorb Silo0

gotami n i ne , c a f f e i ne

barbital

v e r impregnated s i l i c a gel

5,,m,imp. with 1.09% AgI

Separation

P a r t i s i l 5um

A. S ,apoA

n o t given

CHCl -DEA(99.99:0.01)

A C H h -hexane(l:l) B CHCl 3-MeOH-DEA(90: 1 O : O . 5) 300~4.6

l i n e a r 3 g r a d i e n t 16-92% B i n A (1.5-2.5 min.)

18

Et20-MeOH -OEA(90: 10: 1 )

19 N

3

N 01 W

A,H,S,quini ne,qui n i dine.dihydroquinidine ,xanthi nes .strychnine.ephedrine,codeine ,papaverine

Various drugs

A.apoA.trop ac.atrop ac .B ,8-i s a t r o p i c a c

.

Analysis i n pharmaceuticals

P a r t i s i l 10pm

250~4.6

CH C1 -MeOH(1:3)with NH~OH~

1%29%

24

Separation a t r o p i n e L i c h r o s o r b RP8.5pm and degradation' products (Table 4.3)

100~4.6

0.01M tetrabutylamnonium i n 0.05M aq. a c e t k e buffer-ACN (4:l)(pH 5.5)

25

A.H ,S AMe .SMe, trap ac,S N-ox,coc,benztropine

L i d o c a i ne

Determination i n tablets(Tab1e 4.2)

UBondapak C18

300~3.9

0.01M 1 - o c t a n e s u l f o n i c a c i d (pH 3.5)-ACN(66:34),(72:28)

A

Various drugs

Separation a n t i chol in e r g i c drugs

pBondapak C18

300~3.9

0.01M h e p t a n e s u l f o n i c a c i d -ACN(65:35)

A.S,B ,apoA. scop ,ergotami ne ,di hydroergotami ne . c a f f e i ne

Barbiturates, pizotifene

Separation w i t h i o n - p a i r HPLC ( F i g . 4.4)

L i c h r o s o r b Si100.5pm 150x3 loaded w i t h 0.06M p i c r i c acid(pH=6)

CHCl s a t . w i t h 0.06M p i c r i c acid3(pH 6 )

Sepa r at ion

pBondapak C18

300~3.9

0.01M h e p t a n e s u l f o n i c a c i d (pH 3.40)-ACN( 65 :35)

50x4.6

eOH-O.025M NaOAc b u f f e r (PH 5)(1:3)

A,trop

ac

A,H .S .a-B, 8-B .aPoA, t r o p ac,atrop ac. 8 - i s a t r o p i c ac

Determination a t r o - N u c l e o s i l 5CN and p i n e and i t s degra- N u c l e o s i l 5NH2 i n d a t i o n products series (Table 4.1 ,Fig.4.2) Various drugs

Post-column d e r i v a - L i c h r o s o r b DIOL.lOpm 250x4 t i z a t i o n (fluoroL i c h r o s o r b RP8,lOpm 1 0 0 ~ 4 . 6 rescent ion-pairs)

sk,S ,apoS .apoSMe.

Neomycin,benzphetami ne

Determination i n pBondapak C18 v e t e r i n a r y formulat i o n s (Fig.4.3)

A.H.S ,various a1 k a l o i d s

A.ergotami ne A,

S ,AMe

A S , apoA

S u l f a n i l a m i d e .phenytoine,phenobarbital Hydroxyatrazine Various drugs

Identification pharmaceuticals ( F i g . 7.14) Post-column d e r i v a t i z a t i on

P a r t i s i l PXS 5/25 L i c h r o s o r b Si60,5pm

I o n - p a i r chromato- Lichrospher S i 100, 5pm graphy(Fi g .4.5) Post-column d e r i Nucleosi 1 l O S A v a t i z a t i o n ( F i 9.4.1)

27 28.33, 37

30

32

A,emtine,ephedrine, d i hydroergotami ne, bromocrypti ne t r o p ac,atrop ac

26

300~3.9

0.1M phosphate b u f f e r ( p H 3) MeOH-0.02M phosphate b u f f e r (PH 3)(3:2) 0.01M Na-decylsulfate,O.OlM NH NO i n MeOH-H 0(3:2)

35,41

nate,O.OlM NH NO i n 95% EtOH-H20( 55:49) ($H 3.5)

38

0.~lM3Na-dioctyl~ulfosucci-

250~4.6

E t 0 s a t . w i t h 50-100% H20 +0?05-0.8% DEA

60x3

0.1M b u t y r i c a c i d i n CHC13 -MeOH (9: 1)

200x4

0.1M Na-phosphate b u f f e r ( p H 2. .*A4 +1.3% AmOH MeOH-0.2M aq. (NH4)2HP04(1:9) 45

300x4

42 43

? 3

HMe,codeine.morphine, noscapine,papaverine, thebai ne

p

SMe A,S,theophyl l i n e

I Q N N

Determination i n N u c l e o s i l 5C8 pharmaceutical preparations

120~4.6

ACN-O.01M phosphate b u f f e r (pH 5.0)(2:3)

Pyri1amine.phenirami ne

Analysis i n t a b l e t s P a r t i s i l 10 ODS

250~4.6

ACN-2.85mM ethylenediamine b u f f e r ( p H 7.44)(1:1)

Phenobarbital

A n a l y s i s i n pharma- Spherisorb 5pm c e u t i c a l preparations

250x4

MeOH-O.05M tetramethylamm n i u m phosphate b u f f e r ( p H 2.0) 68.69 (21:lO)

* A b b r e v i a t i o n s used i n Tables 4.4 and 4.5 A Me apoA a t r o p ac

B

COC

H HMe S SMe S N-OX apoS s cop trop t r o p ac

a t r o p i n e .hyoscyamine methyl a t r o p i ne apoatropi ne atropic acid belladonnine cocaine homatropine methylhomatropine scopolamine methyl scopol ami ne scopolamine N-oxide aposcopolamine scopoline tropine tropic acid

54 61

4.2.

PSEUDOTROPINE ALKALOIDS

Most of the i n v e s t i g a t i o n s on HPLC analysis o f cocaine and r e l a t e d compounds concern the abuse o f cocaine, A series o f methods has been described f o r the i d e n t i f i c a t i o n o f s t r e e t drugs

-

i n c l u d i n g cocaine (Table 4.11). HPLC systems used f o r the a n a l y s i s o f drugs o f abuse are Tables 7.6, 7.8 discussed i n the Chapter 7 4s9,ii I12,20,23.30~53,58959,60965 966 D67 (see also

A review on the analysis o f cocaine has been given64. Although HPLC should be a

and 7.11).

w e l l s u i t e d method f o r the analysis o f cocaine metabolites i n b i o l o g i c a l m a t e r i a l , o n l y two papers seem t o have been published on t h i s matter31y48. The separation o f the f o u r p o s s i b l e 49 diastereoisomers o f cocaine was achieved by Olieman e t a1.46 and Lewin e t a l . . 4.2.1.

ION-EXCHANGE HPLC

Ion-exchange chromatography has been used f o r the analysis o f s t r e e t drugs caine

-

i n a number o f i n v e s t i g a t i o n s 3 ~ g s 2 0 s * 1 Walton . e t a l .6’10’17

-

i n c l u d i n g co-

a p p l i e d ligand-exchange

chromatography f o r alkaloids, i . a . f o r cocaine. None o f the methods mentioned was designed e s p e c i a l l y f o r cocaine, b u t r a t h e r f o r s t r e e t drugs i n general. They are discussed i n more d e t a i l i n Chapter 7. 4.2.2.

REVERSED-PHASE HPLC

Reversed-phase HPLC was used by Jatlow e t a1.31 f o r the a n a l y s i s o f cocaine and i t s metab o l i t e s i n u r i n e (Fig.4.7).

An amount o f 0 . 1 pg/ml cocaine could be detected by using a micro-

p a r t i c u l a t e octadecyl column and an a c i d i c mobile phase c o n s i s t i n g of 0.25 M potassium dihydrogen phosphate (pH 2.7) containing 17% a c e t o n i t r i l e . For the analysis o f cocaine i n plasma, an octadecyl column has been employed i n combination w i t h the mobile phase methanol potassium phosphate b u f f e r (pH 6.6)(3:1),

-

0.05 M

using t e t r a c a i n e as i n t e r n a l standard48. Noggle and

Clark63 reported a method f o r the i d e n t i f i c a t i o n of

cis- and trans-cinnamoylcocaine

in illicit

cocaine. The a l k a l o i d s and some o f t h e i r degradation products were separated on an octadecyl type o f column, using methanol

-

aqueous phosphate b u f f e r (pH 3)(1:2) as mobile phase.

T r i n l e r and R e ~ l a n dused ~ ~ a chemically bonded d i p h e n y l s i l y l s t a t i o n a r y phase f o r the ident i f i c a t i o n o f cocaine and some l o c a l anaesthetics, comnonly used as adulterants i n cocaine samples. A mobile phase o f a c e t o n i t r i l e

-

water (85:15) containing 1%ammonium carbonate was

used. The same system was a l s o used f o r semi preparative work

-

t o allow further i d e n t i f i c a t i o n

o f the separated drugs by means o f I R spectroscopy. To study the h y d r o l y s i s o f cocaine i n u r i n e and plasma samples, F l e t c h e r and H a n ~ o c kused ~ ~ an octadecyl column and methanol

-

water con-

t a i n i n g phosphoric a c i d t o g i v e pH 3.8 as mobile phase. Above pH 7 a s i g n i f i c a n t h y d r o l y s i s o f cocaine t o benzoylecgonine took place.This may e x p l a i n the d i f f e r e n c e which has been reported i n the l i t e r a t u r e i n the estimations o f unchanged cocaine i n body f l u i d s . Jane e t a l .56 obtained u n s a t i s f a c t o r y r e s u l t s f o r analysis o f cocaine and r e l a t e d compounds when using s i l i c a gel o r octadecyl modified s i l i c a gel as s t a t i o n a r y phase. However, good separations and peak performance were obtained w i t h a chemically bonded d i m e t h y l s i l y l phase and methanol

-

aqueous 0.1 M amnonium n i t r a t e (2:3)(pH 4.3) as mobile phase (Table 4.6).

261 TABLE 4.6 HPLC RETENTION DATA OF COCAINE AND RELATED ( r e l a t i v e r e t e n t i o n times(RRT) were calculated w i t h respect t o c o c a i n e ( r e t e n t i o n time 2.7 min)) Compound

RRT

Compound

RRT

Procaine Chloroprocaine L i gnocai ne Pyrrocai ne Benzoylecgonine Dimethocai ne Octacai ne Propoxycaine Prilocaine Mepivacaine Orthocaine Cocaine Benzocaine Butani 1icai ne P i perocai ne cis-Cinnamoylcocaine Leucinocaine Proxymetacai ne Amylocaine

0.65 0.67 0.70 0.74 0.77 0.77 0.77 0.80 0.83 0.83 0.89 1.00 1.13 1.13 1.17 1.18 1.26 1.32 1.41

Butacaine trans-cinnamoylcocaine Amydri c a i ne Phenacaine Cinchocaine Cy c 1omethyca ine

1.47 1.49 1.69 1.72 3.00 3.00

Morphine Codeine 0-acetylmorphi ne Heroin Acetyl codeine

0.45 0.52 0.61 0.91 0.92

Ephedrine Caffeine Amphetamine Me thy1amp het ami ne Cycl i z i ne D i p i panone

0.60 0.69 0.69 2.58 2.58

0.58

Column Lichrosorb RP2, 5 p m ( 1 5 0 ~ 4 . 6m ID), mobile phase methanol - 0.1 M ammonium n i t r a t e (2:3) adjusted t o pH 4.3 w i t h 2 M h y d r o c h l o r i c acid, f l o w r a t e 1.5 ml/min, d e t e c t i o n UV 279 nm. TABLE 4.7 RETENTION VOLUME OF COCAINE AND RELATED COMPOUNDSZ3 ( r e t e n t i o n volume (Rr) r e l a t i v e t o cocaine, see a l s o Table 7.3) Compound

Rr

Compound

Rr

Anti p y r i ne Procaine Benzocai ne Lidocaine

0.27 0.34 0.45 0.62

Cocai ne Methaqualone Mecl oqual one Tetracaine

l.OO(26.4 m l ) 1.11 1.31 2.60

Column pBondapak C18 (300x4 m I D ) , mobile phase methanol - a c e t i c a c i d - water (40:1:59) (pH 3.5) containing 0.005 M n-heptanesulfonic acid, f l o w r a t e 2 ml/min, d e t e c t i o n UV 254 nm. 4.2.3.

ION-PAIR HPLC

L u r i e and co-workers 23’58’59*60’67

used i o n - p a i r chromatography f o r the a n a l y s i s o f s t r e e t

drugs. The r e s u l t s obtained f o r cocaine and some l o c a l anaesthetics on an octadecyl column using heptanesulfonic a c i d as p a i r i n g - i o n i n a mobile phase o f methanol (40:1:59)

-

water

-

acetic acid

are given i n Table 4.7 (see a l s o Table 7.6). Olieman e t a l . 4 6 separated t h e f o u r

cocaine diastereoisomers on a m i c r o p a r t i c u l a t e octadecyl column by means o f a mobile phase o f tetrahydrofuran

-

water (1:4) containing 0.005 M _I-heptanesulfonic a c i d (Fig.4.8).

Lichrosorb

RP18 was found t o be less s u i t a b l e as s t a t i o n a r y phase than Nucleosil C18 because o f t a i l i n g and peak broadening. 4.2.4.

STRAIGHT-PHASE HPLC

Lewin e t a l .49 separated the f o u r isomeric cocaines by straight-phase chromatography

Referenca p. 262

262

(Fig.4.9).

HPLC was prefered over GLC because o f the decomposition o f some o f the a l k a l o i d s

during GLC. Some o t h e r straight-phase systems f o r drugs o f abuse are d e a l t w i t h i n Chapter 74.12 4.2.5,

DETECTION

The UV spectrum o f cocaine shows maxima a t 229. 274 and 281 nm ( i n ethanol). The l a t t e r two maxima have o n l y low i n t e n s i t y and are, therefore, n o t s u i t a b l e f o r a s e n s i t i v e detection.

A detection a t 230-235 nm has u s u a l l y been prefer re^?^'^^'^^'^^, thus l i m i t i n g the choice o f the mobile phase. Olieman e t al.46 found the d e t e c t i o n l i m i t o f cocaine a t 235 nm t o be 2.8 ng i n t h e i r system. Jatlow e t a1.31 detected cocaine both a t 200 nm and a t 235 nm. The former wavelength gave a 2.5 times more s e n s i t i v e d e t e c t i o n o f cocaine. The debenzoylated metabolites (ecgonine, norecgonine and methylecgonine) had i n s u f f i c i e n t UV absorption f o r UV d e t e c t i o n even a t 200 nm. Comparison o f peak h e i g h t absorbance a t the wavelength o f d e t e c t i o n could be

-

used f o r f u r t h e r v e r i f i c a t i o n o f the i d e n t i t y . Jane e t a l ? p r e f e r r e d UV d e t e c t i o n a t 279 nm, because i t allowed d e t e c t i o n o f r e l a t i v e l y small amounts o f t h e strong UV absorbing cinnamoylcocaine i n cocaine samples. Baker e t a1.40 used the r a t i o o f absorbance a t 254 and 280 nm t o characterize drugs o f f o rensic i n t e r e s t

-

and a l s o cocaine. S i m i l a r l y , L u r i e e t a1.66 used the 220/254 absorbance r a -

t i o (Table 7.6). Noggle and Clark63 applied t h i s method t o the i d e n t i f i c a t i o n o f trans-cinnamoylcocaine i n i l l i c i t cocaine samples (254/280 r a t i o ) .

-

cis- and

REFERENCES 1 M.H. Stutz and S.'Sass, Anal. Chem., 45 (1973) 2134. 2 C.Y. Wu. S. Siggia, T. Robinson and R.D. Waskiewicz. Anal. Chim. A c t a , 63 (1973) 393. 3 J.D. Wittwer, J . Forensic S c i . , 18 (1973) 138. 4 M.L. Chan, C. Whetsell and J.D. McChesney, J . Chromatogr. S c i . , 12 (1974) 512. 5 R. Verpoorte and A. Baerheim Svendsen, J . Chromatogr., 100 (1974) 227. 6 H.F. Walton. J . Chromatogr.. 102 (1974) 57. 7 I.L. Honigberg, J.T. Stewart and A.P. Smith, J . Pharm. S c i . , 63 (1974) 766. 8 V. Quercia, B. Tucci Bucci and A.R. La Tegola, F i t o t e r a p i a , 46 (1975) 3. 9 P.J. Twitchett. J . Chromatogr., 104 (1975) 205. 10 E. Murgia and H.F. Walton. J . Chromatogr., 104 (1975) 417. 11 P.J. T w i t c h e t t and A.C. Moffat, J . Chromatogr., 111 (1975) 149. 12 I . Jane, J . Chromatogr., 111 (1975) 227. 13 W. Santi. J.M. Huen and R.W. Frei. J. Chromatogr., 115 (1975) 423. 14 W.A. T r i n l e r and D.J. Reuland, J . Forensic sci. Soc., 15 (1975) 153. 15 I.L. Honigberg, J.T. Stewart, A.P. Smith, R.D. P l u n k e t t and E.L. Justice, J . Pharm. sci., 64 (1975) 1389. 16 R.W. F r e i and W. Santi, 2. Anal. Chem., 277 (1975) 303. 17 E.O. Murgia, D i s s . A b s t r . I n t . B , 36 (1976) 3911. 18 R. Aigner, H. S p i t z y and R.W. F r e i . J . Chromatogr. S c i . , 14 (1976) 381. 19 R. Verpoorte and A. Baerheim Svendsen, J . Chromatogr., 120 (1976) 203. 20 P.J. Twitchett. A.E.P. Gorvin and A.C. Moffat, J . Chromatogr., 120 (1976) 359. 21 M. Deki and K. Mizuki. Kanzei Chuo Bunsekishoho, 16 (1976) 23. CA 87 (1977) 16622m. 22 T.G. Burdo, 4 t h Ann. Fed. Anal. Chem. and S p e c t r . SOC. Meeting, Detroit,paper no 176 (1977). 23 I . S . Lurie, J . ASSOC. Off. Anal. Chem., 60 (1977) 1035. 24 R.G. Achari and E.E. Theimer, J . Chromatogr. Sci., 15 (1977) 320. 25 8. Kreilgard, Arch. Pharm. Chem., 6 (1978) 109. 26 M.J. Walters, J . ASSOC. Off. Anal. Chem., 6 1 (1978) 1428. 27 N.D. Brown. L.L. H a l l . H.K. Sleeman, B.P. Doctor and G.E. Demaree. J . Chromatogr., 148 (1978) 453. 28 J.M. Huen, R.W. Frei. W. Santi and J.P. Thevenin. J . Cbromatogr., 149 (1978) 359. 29 K. Sugden. G.B. Cox and C.R. Loscombe, J . Chromatogr., 149 (1978) 377.

263

30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69

Brown and H.K. Sleeman, J . Chromatogr., 150 (1978) 225. P . I . Jatlow, C. Van Dyke, P. Barask and R. Byck, J . Chromatogr., 152 (1978) 115. U. Lund and S.H. Hansen, J . Chromatogr., 161 (1978) 371. J.C. G f e l l e r , J . Huen and J.P. Thevenin, J . Chromatogr.. 166 (1978) 133. W.A. T r i n l e r and D.J. Reuland, J . F o r e n s i c S c i . , 23 (1978) 37. J.C. G f e l l e r , G. Frey, J.M. Huen and J.P. Thevenin, HRC 8 cc., J . High R e s o l u t . Chromatoqr., Chromatogr. Commun., 1 (1978) 213. Y . Hashimoto, M. Moriyasu, E. Kato, M. Endo, M. Miyamoto and H. Uchida. Mikrochim. d c t a 2 (1978) 159. J.C. G f e l l e r , J.M. Huen and J.P. Thevenin, Chromatographia. 12 (1979) 368. P.A. Hartmann. J . ASSOC. off. A n a l . Chem...~ 62 (19791 1099. R.W. Frei, J . Chromatogr., 165 (1979) 75. J.K. Baker, B.E. Skelton and Ch.Y. Ma, J . Chromatoqr., 168 (1979) 417. J.C. G f e l l e r , G. Frey, J.M. Huen and J.P. Thevenin, J . Chromatogr., 172 (1979) . . 141. R. Gimet and A. F i l l o u x , J . Chromatogr., 177 (1979) 333. J.F. Lawrence, U.A.T. Brinkman and R.W. Frei. J . Chromatogr.. 185 (1979) 473. J. CrOIIBnen, J . Chromatogr., 186 (1979) 705. J.M. Huen and J.P. Thevenin, HRC & CC., J.High Resol. Chromatogr. ,Chromatogr.Commun., 2 ( 1 9 7 9 ) 1 5 4 . C. Olieman, L. Maat and H.C. Beyenan, Recl. T r a v . Chim. Pays-Bas, 98 (1979) 501. K. Aramaki, T. Hanai and H.F. Walton. Anal. Chem., 52 (1980) 1963. A.N. Masoud and D.M. Krupski, J . A n a l . T o x i c o l . , 4 (1980) 305. A.H. Lewin, S.R. Parker and F.I. C a r o l l , J . Chromatogr.. 193 (1980) 371. R.G. Achari and J.T. Jacob, J . L i g . Chromatogr., 3 (1980) 81. G.K. Poochikian and J.C. Cradock, J . Pharm. s c i . . 69 (1980) 637. A . I . Da Rocha, A . I . Reiz-Luz and F. Marx, d c t a dmazonica, 11 (1981) 661. J.D. Wittwer, F o r e n s i c s c i . I n t . . 18 (1981) 215. P. Majlat. P. Helboe and A.K. Kristensen, r n t . J . Pharm., 9 (1981) 245. S.M. Fletcher and V . S . Hancock. J . Chromatogr., 206 (1981) 193. I. Jane, A. Scott, R.W.L. Sharpe and P.C. White, J . Chromatogr., 214 (1981) 243. G. Hoogewijs. Y . Michotte, J. Lambrecht and D.L. Massart. J . Chromatogr.. 226 (1981) 42. I . S . L u r i e and S.M. Demchuk, J . L i q . Chromatogr., 4 (1981) 337. I . S . L u r i e and S.M. Demchuk, J. L i q . Chromatogr., 4 (1981) 357. I . S . Lurie, J . L i q . Chromatogr., 4 (1981) 399. D.R. Heidemann, J . Pharm. S c i . , 70 (1981) 820. V. Das Gupta. r n t . J . Pharm., 10 (1982) 249. F.T. Noggle and C.R. Clark. J. ASSOC. Off. A n a l . Chem., 65 (1982) 756. T.A. Gough and P.B. Baker. J . Chromatogr. s c i . , 20 (1982) 289. J.G. Umans, T.S.K. Chiu, R.A. Lipman, M.F. Schultz and S.U. Shin, J . Chromatogr., 233 (1982) 213. R.J. Flanagan, G.C.A. Storey, R.K. Bhamra and I. Jane, J . Chromatogr., 247 (1982) 15. I.S. Lurie, S.M. Sottolano and S. Blasof, J . F o r e n s i c S c i . , 27 (1982) 519. L.J. Pennington and W.F. Schmidt. J . Pharm. s c i . , 7 1 (1982) 951. W.F. Schmidt and L.J. Pennington. J . Pharm. S c i . , 71 (1982) 954.

N.D.

\

I

TABLE 4.8

N

a P

HPLC ANALYSIS COCAINE I N COMBINATION WITH OTHER COMPOUNDS Alkaloid

*

Aims

S t a t i o n a r y phase

Column Dim. Mobile phase LxID mn

Coc.opiun a1 kaloids,quinine, cinchonine,caffeine

Separation by means o f dynamic c o a t i n g HPLC

C o r a s i l I and II,dyn a m i c a l l y coated w i t h P o l y 6-300(2%)

1000x1

Coc.23 o t h e r a l k a l o i d s

Analysis a l k a l o i d s

Merckosorb Si60.5pm

300x2

C0c.a tropine,opi um a1ka-

loids,nicotine.strychnine,

Separation on ion-exchange resins(1igand-exchange LC)

Coc,amyl o c a i ne .benzocai ne, b u t a c a ine

Separation b a s i c drugs on s i l i c a g e l w i t h aqueous s o l v e n t s

Hydrolyzed Porpqel PT, loaded w i t h Cu 470~6.3 Bio-Rad+$CZO,loaded w i t h Cu 470~6.3 L i c h r o s o r b Si100,5,,m 150x5

Coc.benzoy1ecgonine.morphi-

S t a b i l i t y COC and h e r o i n i n pharmaceutical dosage form

q u i n i n e .cinchoni ne

ne.heroin.0Ac-morphine, benzoic a c i d

pBondapak C1B

300x4

Ref.

Heptane-EtOH(1:l) w i t h d i f f e r e n t percentage s a t u r a t i o n w i t h P o l y 6-300 CHCl -MeOH( 9: 1) ,(8: 2) ,( 7: 3) Et203MeOH(8: 2), (7: 3), (6:4)

0.o6M 0.2M 0.05M 0.03M

NH OH i n 33% EtOH NH40H i n 33% EtOH NH40H i n 33% EtOH NHfOH i n 33% EtOH

2

5 6.10. 17

MeOH-H 0(7:3) c o n t a i n i n g NH - f o r mate.Nfi NO o r Na-fonnate v a r i ~ u s ~ c o ~ c e n t r a t i oand n s pH 29

i4

ACN-0.015M

Na2HP04(pH 3.0)(1:3) 51

TABLE 4.9 HPLC ANALYSIS OF COCAINE AND RELATED ALKALOIDS Alkaloids* Coc.pseudococ.al allopseudococ

lococ.

Coc.pseudococ,allococ. allopseudococ

Aims

S t a t i o n a r y phase

Column Dim. Mobile phase

Separation o f d i a s t e r e o i s o mers ( F i g .4.8)

N u c l e o s i l C18,km

150~4.0

THF-H 0(1:4) c o n t a i n i n g 0.005M h e p t a g e s u l f o n i c a c i d and 2% AcOH

Separation o f d i a s t e r e o i s o mers ( F i g .4.9)

P a r t i s i l 10 PXS

250~4.6

Heptane-isoprOH-DEA(25:75:0.1)

Analysis i n Erytroxylum p l a n t material F o r a b b r e v i a t i o n s see f o o t n o t e Table 4.11

COC

S i l i c a g e l ODS C18.8um 250x4

Ref.

46 49

MeOH-0.05M phosphate b u f f e r ( p H 7 ) (32:68) 52

Coc.benzoylecgonine, e t h y l -p-ami nobenzoate N N

S t a b i l i t y COC i n phannaceutic a l p r e p a r a t i o n s a t v a r i o u s pH

Bondapak CN

300x4

MeOH-O.02M NH40Ac(3: 1)

62

TABLE 4.10 HPLC ANALYSIS OF COCAINE AN0 RELATED ALKALOIDS I N BIOLOGICAL MATERIAL

*

-

Aims

S t a t i o n a r y phase

Coc,morphine,codeine.caffeine, theophylline

Analysis u r i n e

BOP(no f u r t h e r d e t a i 1s)

COC .benzoyl ecgonine.norcoc,benzoylnorecgonine

Determination i n u r i n e ( F i g .4.7)

P a r t i s i l 10 ODS

Various l o c a l Coc,benzoylecgon i n e .A.S,morphi ne, anaesthetics and caffeine analgesics

Analysis

Coc ,benzoyl ecgonine

Alkaloids

Other comounds

Column Dim. M o b i l e phase LxID mn

Ref.

Heptane-prOH(9:l) 8 250~4.6

ACN-0.25M KH2P04(pH 2.7)(17:83)

_..

JA

ODS-HC SIL-X-1

250~2.6

MeOH-O.05M phosphate b u f f e r ( p H 6.6)(3:1)

H y d r o l y s i s COC i n b i o l o gical fluids

H y p e r s i l 5 ODS.5pm

100~4.6

MeOH-H20(55:45).pH

Various drugs Coc,papaverine, yohimbi ne .heroi n, s t r y c h n i n e , c a f f e i ne

Analysis papaverine i n blood

Micropak CN-10

300x4

Coc.opium alka1o i ds . c a f f e i ne, a u i n i ne

Determination h e r o i n i n blood

L i c h r o s o r b S i 6 0 . 5 ~ m 300x4

Various drugs

*For a b b r e v i a t i o n s see f o o t n o t e Table 4.11

COC

i n plasma

4a

3.8 w i t h H PO 455 Hexane-CH C1 -ACN-propylamine (50: 25: 25?0. 57

I)

ACN-MeOH-(MeOH-NH OH(2:1))-(AcOH -MeOH( 1: 1)) (75: 25?0.040:0.216)

__

b5

TABLE 4.11

EJ

m

HPLC ANALYSIS OF COCAINE I N CONNECTION WITH THE ANALYSIS OF DRUGS OF ABUSE Ref.

A1 kaloids*

Other compounds

Aims

S t a t i o n a r y phase

Column Dim. M o b i l e phase LxIO mn

Coc,various opium a1 k a l o i d s . q u i n i nine.quinidine

Procaine.ani l e r id i ne .methapyrilene

A n a l y s i s drugs o f abuse

Zipax SAX

1ooox2.1

A 0.01M b o r i c a c i d b u f f e r pH 9.5 w i t h 1M NaOH 6 0.01M KH PO b u f f e r pH 6.0 w i t h 1M NaOH g r a d i e n t A+6(85:15) t o 6 , l i n e a r 50r 10% p e r min 3

Coc.various opium alkaloids ,quinine.LSD,rnescal ine

Procaine,benzoc a i ne ,various o t h e r drugs

Identification street drugs

C o r a s i l II,37-50pm,

500~2.3

A1 0 .Woelm 618, 1823dpm

500~2.3

Cyclohexane-MeOH-cyclohexylamine (98.3: 1.5: 0.2) ,(94.5: 4.5: 1) A S k e l l y 8-952 EtOH-dioxane-cyclohexylamine(991.3:50:25:13) 6 idem (686:100:200:14) l i n e a r g r a d i e n t from A t o 6 Cyclohexane-cycl ohexyl ami ne (98.8: 0.2)

4 A 0.2M b o r i c a c i d buffer.pH 9.3 w i t h 40% NaOH B 0.2M b o r i c a c i d buffer-ACN-prOH (86:12:2),pH 9.8 w i t h 40% NaOH l i n e a r g r a d i e n t 0-100% B i n 6 min 9 MeOH-2M NH OH-1M NH NO (27:2:1) MeOH-0.2M 4NH4N03(3?2)3

Coc,various opium alkaloids,caffeine,s t r y c h n i ne, quinine,ephedrine

Procaine.1 ignocaine,barbiturat e ,paracetam01

Analysis i l l i c i t heroin p r e p a r a t i o n s ( F i g . 7.2)

Zipax SCX

12oox2.1

Coc,various drugs o f abuse(Tab1e 7.8)

Local anaesthetics.various o t h e r drugs

Separation drugs o f abuse

P a r t i s i l 6pm

250~4.6

Screening o f drugs o f abuse

Bondapak C18/Corasi1

610x2

ACN-H O(9:l) ,(65:35) ,( 1: 1 ) a1 1 contaqning 0.1%( N H ~ ) ~ c o ~

Eva1u a t i o n ion-exchange ana reversed-pnase columns f o r t h e a n a l y s i s o f drugs

P a r t i s i l SCX 10 m

250~4.6

UBondapak C18

300x4

0.5.0.1.0.05.0.01M (NH )H PO b u f f e r s o f pH 3.5 o r 7,wi?h 6.28,40 o r 60% MeOH 0.025M NaH PO o r Na HPO b u f f e r s of pH 3,5,3 o$ 9, wi?h 0?20,40,60 o r 60% MeOH 11.20

Identification

Zipax SCX

Coc,morphine.heroin.methadone

-

Coc .morphine ,n i cotine.ephedrine. c a f f e i n e , q u i n i ne, tubocurarine

Coc,various opium a1k a l o i d s

Various drugs

12

0.2M NaOH+5% prOH,l% KN03 and

2% ACN(pH 9 )

14

21

R1 a I 69

P N

N

0.005M h e p t a n e s u l f o n i c a c i d i n MeOH-AcOH-H20(40:1:59)(pH 3.5)

Coc.various opium Local anaesthet i c s .various and e r g o t a1 kaloids,caffeine, o t h e r drugs theophylline, q u i n i n e . s t r y c h n i ne COC Local anaesthetics

I o n - p a i r chromatography f o r the separation o f drugs o f abuse(Tab1e 4.7)

I d e n t i f i c a t i o n s t r e e t drugs Bondapak PhenyllPo- 1 2 0 0 ~ 3 . 2 rasil 8

ACN-H 0(85:15) by we?ght

Coc.various a l k a loids(Tab1e 2.2 and 2.3)

Various drugs

I d e n t i f i c a t i o n by means o f dual wavelength d e t e c t i o n

UBondapak C18

300~3.9

0.25M NaH2P04 i n MeOH-H20(2:3).

PPorasi 1

300~3.9

Coc,various opium a1 k a l o i d s , c a f f e i ne.quinine,qui n i d i n e , s t r y c h n i ne

Local anaesthetics,hypnotics, analgesics

Analysis h e r o i n seizures (Table 7.11)

pPorasi 1

300x4

MeOH-2M NH OH-1M NH NO (27:2:1) CH2C12-con?.NH40H( d o : $ ) Cyil ohexane- (CHCl 3-MeOH-NH40H (800:200:1))(3:1) conc. NH40H: 28, 14 o r 7%

COC, benzoylecgonine,cinnamoylcoc,opium a l k a l o i ds . c a f f e i ne

Local anaesthetics

A n a l y s i s i l l i c i t cocaine samples(Tab1e 4.6)

L i c h r o s o r b RP2.5pm

150~4.6

Coc,various o p i um Local anaesthea l k a l o i d s and t i cs ,amphetami other alkaloids nes ,barbi t u r a t e s

I o n - p a i r HPLC o f drugs o f forensic i n t e r e s t

pBondapak C18, 300~3.9 UBondapak Phenyl o r UBondapak CN

COC,transand c i s - c i nnamoyl coc, benzoylecgoni ne, benzoic a c i d

A n a l y s i s i l l i c i t cocaine samples

pBondapak C18

300~3.9

56 0.005M a l k y l s u l f o n a t e ( C ,C C ) i n MeOH-H O-A~OH(40:59:1),~’ 58.59, (30:69:1)?(20:79:1), pH 3.5 60 MeOH-phosphate b u f f e r ( p H 3.0)(1:2)

Analysis h e r o i n seizures (Table 7.6)

pBondapak C18 o r P a r t i s i l 10-ODs-3

300~3.9 250~4.6

ACN-H 0-H PO (12:87:1) c o n t a i n i n g 0 . 0 2 M 2 m e t ~ a n ~ s u l f o n iacid, c pH 2.2

Coc,tropacoc,A, o p i um a1 k a l o i ds , various alkaloids

COC

S

300x4

23 w i t h 0.1% (NH ) CO

334

DH 7 . 0

MeOH-O.1M NH NO (2:3),pH w i t h 2M HC1

40

53

4.3

63 Local anaesthet i c s ,various o t h e r drugs

*Abbreviations used i n Tables 4.8 A

VBondapak C18

cocaine atropine scopolamine

-

4.11

67

268

31 Fig. 4.7. HPLC analysis cocaine and metabolites i n u r i n e Column P a r t i s i l 10-005 ( 2 5 0 ~ 4 . 6m ID), mobile phase 0.25 M potassium dihydrogen phosphate (pH 2.7) c o n t a i n i n g 17% aceton i t r i l e . f l o w gate 2 ml/min, d e t e c t i o n UV 200 and 235 nm, column temperature 40 C. Peaks: 1, benzoylecgonine; 2, cocaine; 3, benzoylecgonine 2 - e t h y l e s t e r ( i n t e r n a l standard).

‘I,, 1 0 8 6

2,L

L

3

1

L

0.005 A

I 1

0

,

2

1

L

1

6

1

8

1

10 min

1

12

1

14

1

16

1

18

1

20

I J

r

16

12

min

8

L

I

I,

0

F i g . 4.8. HPLC separation o f isomeric cocaines 49 isopropanol - diethylamine Column P a r t i s i l lOPXS ( 2 5 0 ~ 4 . 6mn I D ) , mobile phase heptane (75:25:0.1), flow r a t e increasing e x p o n e n t i a l l y (see dashed l i n e ) , d e t e c t i o n UV 230 nm. Peaks: 1, N,N-dibenzylbenzamide ( i n t e r n a l standard); 2, cocaine; 3, allococaine; 4, pseudococaine; 55 Zllopseudococaine. 46 Fig. 4.9. Separation o f isomeric cocaines Column Nucleosil C18 5 pm ( 1 5 0 ~ 4 . 0mn ID), mobile phase tetrahydrofuran water (1:4) cona c i d and 2% a c e t i c acid, f l o w r a t e 1.0 ml/min, d e t e c t i o n t a i n i n g 0.005 M !-heptanesulfonic UV 235 nm. Peaks: 1, cocaine; 2, pseudococaine; 3, allococaine; 4, allopseudococaine. (Reproduced w i t h permission from r e f . 46, by courtesy o f Recueil des travaux chimiques des Pays-Bas)

-

-