A General Approach to the Analysis of Cannabinoids from

6 A General Approach to the Analysis of Cannabinoids from Physiological Sources J. R O S E N F E L D

Downloaded by FUDAN UNIV on February 20, 2017 | http://pubs.acs.org Publication Date: April 10, 1979 | doi: 10.1021/bk-1979-0098.ch006

Department of Pathology, McMaster Medical Centre, Hamilton, Ontario, Canada

It has been e s t a b l i s h e d that a f t e r smoking a pharmacological dose of Δ-THC the plasma concentra t i o n s o f that drug do not rise above s e v e r a l hundred ng/ml of plasma and that during the period of psycho logical e f f e c t the concentration is l e s s than 50 ng/ml. Since Δ -THC and many of its metabolites are lipophil ic, the general problem is summarized as the determina tion of nanogram q u a n t i t i e s of a lipophilic drug i n the lipid f r a c t i o n of plasma. Determination of concentration in the ng/ml range r e q u i r e s the h i g h l y s e n s i t i v e detectors o f gas chromatography, such as e l e c t r o n capture (EC), alkali flame (AF), and mass spectrometry (MS). The situation, how ever, is complicated by the l i p o p h i l e s present in the plasma and/or serum. This problem was h i g h l i g h t e d when EC was used to achieve the required sensitivity. Fenimore (1) and G a r r e t t (2) reported on EC methods for Δ -THC in human serum and dog plasma, r e s p e c t i v e l y . Both used a c y l a t i o n w i t h h i g h l y f l u o r i n a t e d reagents to prepare d e r i v a t i v e s f o r EC d e t e c t i o n . However, there was a dramatic change in the gas chromatographic t r a c e s and apparent interferences from lipophiles. The cause f o r the differences has not been determined but these r e s u l t s prompted Mechoulam to warn that i n the a n a l y s i s of Δ -THC and its metabolites it may not be p o s s i b l e to simply exchange plasma of d i f f e r e n t species but that each may, in f a c t , be d i f f e r e n t ( 3 ) . When the problem is expanded to include the determination of metabolites the situation becomes more complex ( F i g . 1 ) . 9

9

9

9

0-8412-0488-8/79/47-098-081$05.00/0 © 1979 American Chemical Society

Vinson; Cannabinoid Analysis in Physiological Fluids ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

82

CANNABINOID ANALYSIS IN PHYSIOLOGICAL FLUIDS

^O-^^CsHu Ο . .Λ 1 ' 33 Ο R2"H; C-CHg MONOHYDROXYLATED METABOLITES

"C5HH


DIHYDROXYLATED METABOLITES

B

m U

R

r

M

u

r

C M

C

C

C5H11

w

M

ΗγΟ

Ο

^

C5H11

6ketoA -THC

11 OXO Δ'-THC

9

C0 H 2

CO2H R -H R OH 1

r

R2-OH R -H 2

CABOXYUC ACID METABOLITES Figure I .

Metabolism of

Δ -ΤΗϋ 9

When we f i r s t approached the s t u d y on t h e d e t e r m i n a t i o n o f the c a n n a b i n o i d s by mass s p e c t r o m e t r y , we were somewhat i n t i m i d a t e d by the l a r g e number o f pos sible metabolites. We were a l s o c o n c e r n e d about t h e r e q u i r e m e n t o f d e u t e r a t e d a n a l o g s o f t h e compounds f o r mass s p e c t r o m e t r i c d e t e r m i n a t i o n . The d i f f i c u l t i e s i n v o l v e d i n the s y n t h e s i s o f n o n l a b e l e d com pounds, however, argued f o r an a p p r o a c h t h a t d i d n o t r e q u i r e t o t a l s y n t h e s i s o f the d e u t e r a t e d analogs. We t h e r e f o r e a p p l i e d Occham's r a z o r t o our de l i b e r a t i o n s and d e c i d e d t h a t , i n f a c t , w i t h o n l y one


6.

ROSENFELD

General Approach

83

o r two e x c e p t i o n s , we were d e a l i n g w i t h p h e n o l s . The p r o b l e m t h e n " r e d u c e d " t o the a n a l y s i s o f l i p i d s o l u ble phenols i n t h e l i p i d m a t r i x o f plasma.

+

S^^xK 1 1 1

+

^O-^vAcsHu

CsHfl Δ —THC

Ν,Ν,Ν, TRIMETHYL ANALINIUM HYDROXIDE

EXTERNAL STANDARD

4


PRESENT IN SOLUTION PRIOR TO INJECTION

PRESENT IN INJECTOR PORT Figure 2.

Derivatization of THC 9

Our o r i g i n a l method f o r A -THC e x p l o r e d t h i s p r o b l e m t o some e x t e n t . R a t h e r than a t t e m p t t h e s y n t h e s i s o f d e u t e r o l a b e l e d A -THC we d e c i d e d t o a n a l y z e A -THC a s i t s own m e t h y l e t h e r ( F i g . 2 ) . Our i n t e r n a l s t a n d a r d w o u l d be 1 - 0 - p e r d e u t e r i o m e t h y 1 - A - T H C . I t was p r o p o s e d t o c o n v e r t A -THC t o i t s 1-0-methyl e t h e r f o r t h e a n a l y s i s . T h i s was e f f e c t e d by t h e c o - i n j e c t i o n o f t r i m e t h y l a n i l i n i u m h y d r o x i d e and A -THC. A t the e l e v a t e d t e m p e r a t u r e s o f the i n j e c t o r p o r t t h e phenol i s converted t o i t s methyl d e r i v a t i v e . This c o n v e r s i o n i s b o t h r e p r o d u c i b l e and q u a n t i t a t i v e . I t i s t h e r e f o r e s u i t a b l e f o r use i n any a n a l y t i c a l t e c h nique . The p r o b l e m o f l i p o p h i l e s r e m a i n e d and h e r e a g a i n we c o u l d make use o f the a c i d f u n c t i o n a l i t y o f t h e phenols. W i t h l e s s l i p i d s o l u b l e p h e n o l s such a s t h e s t e r o i d s , s i m p l e back e x t r a c t i o n from o r g a n i c s o l v e n t i n t o s t r o n g base w o u l d have been s u f f i c i e n t . However, the h i g h l i p i d s o l u b i l i t y o f A -THC n e c e s s i t a t e d t h a t e x t r a c t i o n be c a r r i e d o u t w i t h B r o d i e ' s s o l v e n t (hexane and i s o a m y l a l c o h o l ) and t h a t back e x t r a c t i o n be done w i t h C l a i s e n ' s a l k a l i , w h i c h i s a m i x t u r e o f KOH, m e t h a n o l , and w a t e r . A f t e r a c i d i f i c a t i o n o f the C l a i s e n s a l k a l i , A -THC c o u l d be r e c o v e r e d by e x t r a c t ion. The e x t e r n a l s t a n d a r d and t h e t r i m e t h y l a n i l i n i u m h y d r o x i d e were added and t h e e x t r a c t e d p h e n o l ( i . e . A9-THC) was c o n v e r t e d t o t h e 1-0-methyl d e r i v a t i v e i n t h e i n j e c t o r p o r t and the d e t e r m i n a t i o n c a r r i e d o u t 9

9

9

9

9

9

f

9


84

CANNABINOID

ANALYSIS

IN PHYSIOLOGICAL

FLUIDS

by mass s p e c t r o m e t r y . U s i n g t h i s method we were a b l e t o o b t a i n plasma c o n c e n t r a t i o n p r o f i l e s from humans who h a d smoked c i g a r e t t e s dosed a t 88 μ g THC/kg. T h i r t y minutes a f t e r smoking; t h e c o n c e n t r a t i o n ranged from 5 t o 35 ng/ml w i t h an average o f 17 ng/ml ( 4 ) . These v a l u e s were s i m i l a r t o t h e c o n c e n t r a t i o n range r e p o r t e d by A g u r e l l (5) . We a t t e m p t e d t o approach t h e d e t e r m i n a t i o n o f l l - h y d r o x y - A - T H C i n t h e same way. P r e l i m i n a r y e x p e r i ments showed t h a t l l - h y d r o x y - A - T H C was n o t v e r y s o l u b l e i n B r o d i e ' s s o l v e n t a n d t h e m e t a b o l i t e was u n s t a ble t o methylation with t r i m e t h y l a n i l i n i u m hydroxide. I n t h e l a t t e r e x p e r i m e n t , we d e t e r m i n e d t h a t two p r o d u c t s were formed, b u t they were formed i n i r r e p r o ducible ratios. The major p r o d u c t was 1 , 1 1 , - d i m e t h y l ll-hydroxy-A9-THC. We d i d n o t i d e n t i f y t h e m i n o r product. I t was c l e a r t h a t a m i l d e r form o f d e r i v a t i z a t i o n was r e q u i r e d i f we were t o be s u c c e s s f u l i n c a r r y i n g on w i t h o u r o r i g i n a l p h i l o s o p h y o f a n a l y z i n g t h e c a n n a b i n o i d s as t h e i r 1 - 0 - a l k y l d e r i v a t i v e s . We found t h a t t h e t e c h n i q u e o f phase t r a n s f e r c a t a l y s i s s u i t e d our requirements. 9


9

AH AQUEOUS PHASE

(R) NOH — 4

A" + H

+

(R) N + 0 H μ 4

(R) NA~ 4

(R) NA~ 4

ORGANIC PHASE [(R) N+ A"]? 4

R X 2

A-R

2

R = HYDROCARBON X = I,Br. Figure S.

Phase transfer alkyhtion process

This technique i n v o l v e s the e x t r a c t i o n o f the a n i o n o f an o r g a n i c a c i d a s i t s i o n p a i r w i t h a l i p i d s o l u b l e q u a t e r n a r y ammonium c a t i o n ( i n o u r c a s e , t h i s


6.

ROSENFELD

General Approach

85

was t e t r a h e x y l ammonium h y d r o x i d e ) ; i f the o r g a n i c phase c o n t a i n s an a l k y l a t i n g a g e n t such as a bromo o r i o d o h y d r o c a r b o n t h e a n i o n becomes a l k y l a t e d as i n F i g . 3. The e x a c t mechanism i n v o l v e d i n t h i s a l k y l a t i o n i s s t i l l u n c e r t a i n . Whatever t h e a c t u a l mechanism b o t h m e t h y l and e t h y l i o d i d e were e q u a l l y r e a c t i v e , g i v i n g the 1 - 0 - a l k y l d e r i v a t i v e i n q u a n t i t a t i v e y i e l d . T h i s was i m p o r t a n t because the 1-0-methyl e t h e r o f l l - h y d r o x y - A - T H C has been shown t o be a m e t a b o l i t e o f l l - h y d r o x y - A - T H C ( 6 ) . Thus by d e r i v a t i z i n g t o t h e e t h y l e t h e r we were c e r t a i n t h a t we w o u l d be s p e c i f i c a l l y m e a s u r i n g l l - h y d r o x y - A - T H C and n o t an endogen o u s l y formed m e t a b o l i t e ( F i g . 4 ) . 9

9


9

Figure 4.

Analysis and metabolism of 11-hydroxy-A -THC 9

There was one f u r t h e r p r o b l e m , namely the 1-0e t h y l - l l - h y d r o x y - A - T H C was s u s c e p t i b l e t o p y r o l y s i s a t t h e e l e v a t e d t e m p e r a t u r e s o f the i o n s o u r c e . This r e s u l t e d i n i r r e p r o d u c i b l e mass s p e c t r a . Silylation o f t h e a l l y l i c a l c o h o l f u n c t i o n a l i t y overcame t h i s d i f f i c u l t y and the r e s u l t i n g e l e c t r o n i m p a c t fragment a t i o n p a t t e r n was q u i t e s i m p l e showing o n l y one m a j o r peak, base peak a t m/e = 327. The t r i m e t h y l s i l y l i o n appeared a t m/e = 7 3 (7). T h i s was i n t e r e s t i n g because t h e r e a r e s e v e r a l m o n o h y d r o x y l i c m e t a b o l i t e s o f A -THC. I f we were m o n i t o r i n g , s a y , the m o l e c u l a r i o n , we would d e t e c t a l l o f t h e s e m e t a b o l i t e s . S i n c e 327 r e s u l t s from a l o s s o f C - l l , t h i s i o n i s s p e c i f i c f o r the d e r i v a t i v e of ll-hydroxy-A -THC. T h i s i s p r o b a b l y a minor p o i n t , because the v a r i o u s m o n o h y d r o x y l a t e d m e t a b o l i t e s have been shown t o be s e p a r a b l e by gas chromatography. N e v e r t h e l e s s , i t does r e s u l t i n a h i g h l y s p e c i f i c det e r m i n a t i o n f o r t h e most d e b a t e d m e t a b o l i t e . Due t o the s p e c i f i c i t y o f the mass s p e c t r o m e t e r the p r o b l e m o f l i p o p h i l e s was n o t l i m i t i n g i n the 9

9

9


86

CANNABINOID ANALYSIS IN

PHYSIOLOGICAL FLUIDS

d e t e r m i n a t i o n o f t h e hydroxy m e t a b o l i t e . S i m p l e ex t r a c t i o n w i t h m e t h y l e n e c h l o r i d e gave an e x t r a c t s u f f i c i e n t l y clean to permit, a f t e r appropriate d e r i v a t i z a t i o n , determination of concentrations of s e v e r a l nanograms per m i l l i l i t e r . We c a r r i e d o u t a s t u d y i n t h e dog t o d e t e r m i n e t h e f o r m a t i o n o f l l - h y d r o x y - A - T H C from A -THC. I n t h e f i r s t s t a g e we i n j e c t e d l l - h y d r o x y - A - T H C i n o r d e r t o d e t e r m i n e t h e b e t a phase h a l f - l i f e o f t h e metabo l i t e ; the h a l f - l i f e was a p p r o x i m a t e l y 1.5 h o u r s . How e v e r , when Δ -THC was a d m i n i s t e r e d t o t h e same dog e i t h e r o r a l l y o r i n t r a v e n o u s l y t h e m e t a b o l i t e was n o t detectable. There a r e s e v e r a l p o s s i b l e e x p l a n a t i o n s f o r t h i s r e s u l t . The most o b v i o u s a r e t h a t t h e dog does n o t h y d r o x y l a t e A -THC a t t h e C - l l p o s i t i o n o r our method was i n s u f f i c i e n t l y s e n s i t i v e t o d e t e r m i n e t h e v e r y low c o n c e n t r a t i o n s i n t h e dog. I t was a l s o p o s s i b l e t h a t t h i s p a r t i c u l a r dog was a t y p i c a l . I n t h e case o f o r a l dosage i t was a l s o p o s s i b l e t h a t A -THC was n o t r e a d i l y absorbed. In o r d e r t o answer some o f t h e s e q u e s t i o n s we em b a r k e d on a second s t u d y . The purpose o f t h i s s t u d y was t o show t h a t A -THC was p r e s e n t i n t h e c i r c u l a t i o n of an a n i m a l and as a consequence, l l - h y d r o x y - A - T H C appeared i n t h e p l a s m a . F o r t h i s s t u d y we chose t h e r a b b i t as o u r a n i m a l model. S e v e r a l w o r k e r s have shown t h a t t h e r a b b i t o x i d i z e s A -THC a t t h e C - l l p o s i t i o n and a l s o t h a t r a b b i t l i v e r microsomes o x i d i z e A -THC a t t h e C - l l p o s i t i o n and a l s o t h a t r a b b i t l i v e r microsomes o x i d i z e A -THC t o l l - h y d r o x y - A - T H C . We were t h u s a s s u r e d t h a t we would n o t be d e a l i n g w i t h a s p e c i e s where t h e r e was doubt about o x i d a t i o n a t C - l l (3) . B e f o r e e n t e r i n g i n t o t h e s t u d y we f e l t t h a t i t would be u s e f u l t o have a method t h a t would d e t e r m i n e b o t h p a r e n t d r u g and m e t a b o l i t e s i m u l t a n e o u s l y . There were two r e a s o n s f o r t h i s ; f i r s t , t h e l o g i s t i c s o f t h e work would be g r e a t l y improved s i n c e o n l y one d e t e r m i n a t i o n would have t o be c a r r i e d o u t p e r sample o f plasma; second, o u r o r i g i n a l h y p o t h e s i s was t h a t , by e x p l o i t a t i o n o f t h e c h e m i s t r y o f t h e p h e n o l groups we c o u l d d e t e r m i n e A -THC and most o f i t s m e t a b o l i t e s . Our f i r s t two t e c h n i q u e s d e t e r m i n e d t h e s e compounds b u t by d i f f e r e n t e x t r a c t i o n and d e r i v a t i z a t i o n s . Therefore, these techniques lacked general a p p l i c a b i l i t y t o t h e c a n n a b i n o i d s . Our p h i l o s o p h y h e l d t r u e b u t our chemistry d i d n o t . A s i m p l e s t u d y on t h e s i m u l t a n e o u s e x t r a c t i o n o f r a d i o - l a b e l e d A -THC and l l - h y d r o x y - A - T H C once a g a i n 9

9

9


9

9

9

9

9

9

9

9

9

9

9

9


6.

ROSENFELD

General Approach

87

showed t h a t a n y t h i n g a l i v e o r even something once a s s o c i a t e d w i t h a l i v i n g e n t i t y can and u s u a l l y does throw a c u r v e a t t h e a n a l y t i c a l c h e m i s t . When A -THC was e x t r a c t e d from w a t e r , methylene c h l o r i d e and d i e t h y l e t h e r e x t r a c t e d t h e d r u g q u a n t i t a t i v e l y whereas t o l u e n e e x t r a c t e d o n l y 80% o f t h e A -THC. However, when e x t r a c t i o n s were c a r r i e d o u t from human plasma o r r a b b i t plasma b o t h m e t h y l e n e c h l o r i d e and e t h y l e t h e r e x t r a c t e d l e s s t h a n 45% o f t h e compounds. T o l u e n e , however, s t i l l e x t r a c t e d a p p r o x i m a t e l y 80% i n b o t h c a s e s . I n a d d i t i o n t h e y i e l d o f e x t r a c t i o n from r a b b i t plasma was s l i g h t l y h i g h e r t h a n t h a t found i n human p l a s m a . There a r e t h r e e t h i n g s t h a t a r e s t r i k i n g about t h e s e d a t a . F i r s t , d e s p i t e t h e acknowledged l i p i d s o l u b i l i t y o f A -THC, t h e d r u g , as documented by G a r r e t t and Hunt, i s t o some e x t e n t , s o l u b l e i n w a t e r (7). Consequently, i t i s not completely e x t r a c t a b l e in a l l organic solvents. In fact, only the polar s o l v e n t s such as d i e t h y l e t h e r and m e t h y l e n e c h l o r i d e a r e c a p a b l e o f q u a n t i t a t i v e l y e x t r a c t i n g A -THC from w a t e r . Second, i t i s s u r p r i s i n g t h a t t h o s e s o l v e n t s t h a t e x t r a c t A -THC e f f i c i e n t l y from w a t e r a r e n o t e f f i c i e n t a t e x t r a c t i n g i t from p l a s m a . I t i s a l s o i n t e r e s t i n g t h a t t h e r e i s no change i n t h e e x t r a c t i o n e f f i c i e n c y using toluene. Finally i t i s interesting that the e x t r a c t i o n e f f i c i e n c y f o r a l l three solvents i s s l i g h t l y h i g h e r from r a b b i t plasma t h a n from human plasma. I t i s n o t p o s s i b l e , c u r r e n t l y , t o o f f e r an e x p l a n a t i o n f o r t h e s e o b s e r v a t i o n s , b u t perhaps i t i s p e r missible to speculate. The low e f f i c i e n c y w i t h methyl e n e c h l o r i d e m i g h t be e x p l a i n e d by t h e f a c t t h a t p r o t e i n s p r e c i p i t a t e when plasma i s e x t r a c t e d w i t h t h i s s o l v e n t . S i n c e A -THC i s bound t o p r o t e i n , i t may c o - p r e c i p i t a t e and n o t be e x t r a c t e d from p r e c i p i t a t e . However, c o - p r e c i p i t a t i o n w i t h p r o t e i n s c a n n o t be invoked t o e x p l a i n the r e s u l t s w i t h d i e t h y l ether s i n c e t h i s s o l v e n t does n o t cause p r o t e i n p r e c i p i t a t i o n . Furthermore, i t i s a l s o necessary t o e x p l a i n the o b s e r v a t i o n on e x t r a c t i o n w i t h t o l u e n e and t h e d i f f e r e n c e s between r a b b i t and human p l a s m a . The c o p r e c i p i t a t i o n argument i s s i m p l y n o t a p p l i c a b l e t o t h e s e phenomena. We h y p o t h e s i z e t h a t a s u b t l e d r u g p r o t e i n i n t e r a c t i o n o c c u r s when p o l a r s o l v e n t s a r e used t o e x t r a c t h i g h l y l i p i d s o l u b l e d r u g s from p l a s m a . These s o l vents a r e capable o f d e l i p i d i z i n g l i p o p r o t e i n s . I t i s p o s s i b l e t h a t , when d e l i p i d i z a t i o n o c c u r s , t h e h y d r o p h o b i c r e g i o n o f t h a t p r o t e i n i s exposed. The h y d r o p h o b i c r e g i o n c o u l d t h e n b i n d A -THC and t h e b i n d i n g 9


9

9

9

9

9

9


88



f o r c e s m i g h t be s u f f i c i e n t l y g r e a t t h a t t h e y i n t e r f e r e w i t h the e x t r a c t i o n i n t o organic s o l v e n t . Since t o l u e n e i s n o t as e f f i c i e n t a s o l v e n t f o r l i p i d ex t r a c t i o n , i t i s p o s s i b l e t h a t t h i s s o l v e n t does n o t d e l i p i d i z e the l i p o p r o t e i n s t o the same e x t e n t as the more p o l a r s o l v e n t s . C o n s e q u e n t l y the e x t r a c t i o n e f f i c i e n c y w i t h t o l u e n e i s r e l a t i v e l y unchanged. We d i d n o t have s u f f i c i e n t r a d i o - l a b e l e d ll-hydroxy-Δ THC t o c a r r y o u t as e x h a u s t i v e a s t u d y w i t h t h a t com pound. Worst case a n a l y s i s showed t h a t the e x t r a c t i o n e f f i c i e n c y o f t h i s compound from human plasma was quite high. The phenomena t h e r e f o r e may be l i m i t e d t o A -THC and p o s s i b l y t o o t h e r d r u g s w i t h r e l a t i v e l y high l i p i d s o l u b i l i t y . The d e l i p i d i z a t i o n h y p o t h e s i s i s a t t r a c t i v e be cause i t can a l s o e x p l a i n the d i f f e r e n c e between t h e e x t r a c t i o n e f f i c i e n c y from r a b b i t and human plasma. A major d i f f e r e n c e between human and r a b b i t plasma i s t h a t t h e l a t t e r has l e s s and p o s s i b l y d i f f e r e n t l i p o proteins. I f the h y p o t h e s i s t h a t l i p o p r o t e i n s a f f e c t the e x t r a c t i o n e f f i c i e n c y f o r A -THC i s c o r r e c t , t h i s c o u l d e x p l a i n the somewhat h i g h e r e x t r a c t i o n e f f i c i e n c i e s from p l a s m a . These r e s u l t s s u p p o r t Mechoulam's argument t h a t , i n the a n a l y s i s o f A -THC i n p l a s m a , i t may be n e c e s s a r y t o c o n s i d e r t h e b i o l o g i c a l s o u r c e o f the plasma ( 3 ) . These o b s e r v a t i o n s d e f i n e one o f the problems i n e x t e r n a l c a l i b r a t i o n methods f o r the a n a l y s i s o f Δ THC. However, when t h i s d i f f i c u l t y i s r e c o g n i z e d and a c c o u n t e d f o r , e x t e r n a l c a l i b r a t i o n becomes a p l a u s i ble technique. H a v i n g d e t e r m i n e d the most a p p r o p r i a t e e x t r a c t i o n from plasma we i n v e s t i g a t e d the s i m u l t a n e o u s d e r i v a t i z a t i o n o f A -THC and 11-hydroxy-Δ -THC. We have claimed t h a t e t h y l a t i o n of ll-hydroxy-A -THC proceeded by phase t r a n s f e r c a t a l y s i s ( 7 ) . However, i t i s known t h a t q u a t e r n a r y ammonium h y d r o x i d e s a r e c a p a b l e o f catalyzing alkylations with a l k y l iodides i n aproptic s o l v e n t s ( 8 ) . F u r t h e r m o r e , we had n o t d e m o n s t r a t e d t h a t Δ -THC c o u l d be d e r i v a t i z e d under the same c o n d i t i o n s as l l - h y d r o x y - A - T H C . We found t h a t t h e minimum r e q u i r e m e n t f o r the r e a c t i o n t o p r o c e e d i s the p r e sence o f w a t e r , w h i c h p r o b a b l y i n c r e a s e s the degree o f i o n i z a t i o n o f the q u a t e r n a r y ammonium h y d r o x i d e . However, i n o r d e r f o r the r e a c t i o n t o go t o c o m p l e t i o n , a t l e a s t 0.1N NaOH i s n e c e s s a r y . T h i s s u p p o r t s t h e c o n t e n t i o n t h a t t h i s d e r i v a t i z a t i o n i s , t o some ex t e n t , a phase t r a n s f e r c a t a l y z e d a l k y l a t i o n . E t h y l a t i o n o f A -THC p r o v e d t o be f o r t u i t o u s . The base peak i n the mass s p e c t r u m o f t h i s compound i s a l s o m/e = 327. T h i s was i m p o r t a n t t o us because we 9


9

9

9

9

9

9

9

9

9

9


6.

ROSENFELD

89

General Approach

had a d u a l i o n d e t e c t i o n i n s t r u m e n t and i t was f o r t u n a t e t h a t b o t h p a r e n t drug and m e t a b o l i t e , when d e r i v a t i z e d , g e n e r a t e d t h e same peak. However, the f r a g m e n t a t i o n o f l-O-ethyl-Δ -THC was more e x t e n s i v e t h a n t h a t o f the m e t a b o l i t e s . C o n s e q u e n t l y , the s e n s i t i v i t y t o A -THC d e t e r m i n a t i o n on our i n s t r u m e n t was de creased. A t t h i s s t a g e we a l s o i n v e s t i g a t e d the f o r m a t i o n of d e r i v a t i v e s t h a t would p e r m i t a n a l y s i s by gas c h r o m a t o g r a p h i c t e c h n i q u e s o t h e r than gas c h r o m a t o g r a phy/mass s p e c t r o m e t r y . We f i r s t i n v e s t i g a t e d the r e a c t i o n w i t h p e n t a f l u o r o b e n z y l bromide (PFBB). We found t h a t PFBB i s a s r e a c t i v e as the s i m p l e a l k y l i o d i d e s ( F i g . 5) c o n s e q u e n t l y the p e n t a f l u o r o b e n z y l d e r i v a t i v e s o f THC a r e r e a d i l y p r e p a r e d . 9


9

1 NORMALITY NaOH

0.01 0.1 1 5 10 Figure 5.

>100 >100 >100 >100 >100

Conditions for the alkyhtion of A -THC with pentafluorobenzyl bro mide 9

We chose t h i s d e r i v a t i v e because we wanted t o ap p l y i t t o e l e c t r o n c a p t u r e d e t e r m i n a t i o n and p o s s i b l y h i g h p r e s s u r e l i q u i d chromatography d e t e r m i n a t i o n s (HPLC). A r e v i e w o f the l i t e r a t u r e s u g g e s t e d t h a t , w h i l e PFB d e r i v a t i v e s a r e d e t e c t a b l e a t the p i c o g r a m l e v e l by EC d e t e c t i o n , PFBB i s d i f f i c u l t t o remove and can cause i n t e r f e r e n c e s w i t h the d e t e r m i n a t i o n s . We therefore also investigated d e r i v a t i z a t i o n with t r i f l u o r o e t h y l i o d i d e a r e a g e n t w i t h a low b o i l i n g p o i n t (5 8°C). T h i s r e a g e n t was l e s s r e a c t i v e t h a n the a l k y l or b e n z y l h a l i d e s as seen i n ( F i g . 6 ) . T h i s i s p r o b a b l y due t o the e l e c t r o n w i t h d r a w i n g p r o p e r t i e s o f the t h r e e f l o u r i n e atoms, r e s u l t i n g i n a s t r o n g e r c a r b o n i o d i n e bond. N e v e r t h e l e s s , under p r o p e r c o n d i t i o n s , i t i s p o s s i b l e t o o b t a i n a h i g h degree o f d e r i v a t i z a tion. I t a p p e a r s , t h e r e f o r e , t h a t the phase t r a n s f e r c a t a l y z e d a l k y l a t i o n can be a p p l i c a b l e t o t h e d e v e l o p ment o f EC and p o s s i b l y HPLC as w e l l as mass s p e c t r o m e t r i c methods. F o r the p r e s e n t , however, we l i m i t e d


90



our s t u d i e s t o t h e GC/MS t e c h n i q u e . We had shown t h a t i t was p o s s i b l e t o e x t r a c t b o t h Δ -THC and l l - h y d r o x y - A 9 - T H C from plasma w i t h t o l u e n e . The n e x t q u e s t i o n was the p r o b l e m o f l i p o p h i l e s . Aqain we found t h a t i n o r d e r t o remove i n t e r f e r e n c e s t o Δ THC d e t e r m i n a t i o n s i t was n e c e s s a r y t o f r a c t i o n a t e the t o l u e n e e x t r a c t w i t h C l a i s e n ' s a l k a l i i n o r d e r t o ob t a i n the p h e n o l f r a c t i o n o f p l a s m a . We found t h a t the e x t r a c t i o n s o f Δ -ΤΗ0 f r o m t o l u e n e w i t h C l a i s e n ' s a l k a l i i s not q u a n t i t a t i v e , b u t i t i s r e p r o d u c i b l e . The l o s s o f Δ -ΤΗ0 i n t h i s r e a c t i o n however, i s compensated f o r by r e m o v a l o f i n t e r f e r e n c e s w h i c h were the l i m i t ing f a c t o r s i n the s i n g l e e x t r a c t i o n s t e p . 9

9

9


9

C5H11

C5H11

1

2

NORMALITY NaOH 0.01

0.06

0.1

0.65

1N

Figure 6.

RATIO 2/1

2.9

5N

10

10N

18

Conditions for the derivatization of A -THC with trifluoroethijl iodide 9

The f i n a l a n a l y t i c a l method f o r the s i m u l t a n e o u s d e t e r m i n a t i o n o f Δ -ΤΗ0 and i t s m e t a b o l i t e s c o n s i s t s o f the f o l l o w i n g sequence: the c a n n a b i n o i d s are ex t r a c t e d from plasma w i t h t o l u e n e ; t h e y a r e t h e n back e x t r a c t e d from t o l u e n e i n t o C l a i s e n ' s a l k a l i ; the Claisen's a l k a l i i s d i l u t e d with water, t e t r a h e x y l ammonium h y d r o x i d e i s added and the a l k a l i n e s o l u t i o n i s e x t r a c t e d w i t h methylene c h l o r i d e c o n t a i n i n g e t h y l i o d i d e . The o v e r a l l r e c o v e r i e s were 45% f o r A -THC and 8 3% f o r l l - h y d r o x y - A - T H C . E x t e r n a l s t a n d a r d s ( l - 0 - e t h y l - A - T H C and l-0-ethyl-ll-hydroxy-A -THC) were added t o the m e t h y l e n e c h l o r i d e phase f o l l o w e d by a s m a l l amount o f F l o r o s i l , w h i c h a b s o r b e d the t e t r a hexylammonium h y d r o x i d e and tetrahexylammonium i o d i d e . The m e t h y l e n e c h l o r i d e was d e c a n t e d and e v a p o r a t e d . The r e s i d u e was t a k e n up i n a m i x t u r e o f Ν,Ο-bis-(trim e t h y l s i l y l ) - a c e t a m i d e and t r i m e t h y l c h l o r o s i l a n e ( 9 / 1 ) . The f i n a l s o l u t i o n was used f o r GC/MS a n a l y s i s . T h i s t e c h n i q u e was used t o i n v e s t i g a t e the a p p e a r ance o f l l - h y d r o x y - A - T H C i n the plasma o f r a b b i t s t h a t 9

9

9

9

9

9


6. ROSENFELD

91

General Approach

had been dosed w i t h Δ -THC. The r a b b i t s r e c e i v e d 100 y g / k g , w h i c h was s l i g h t l y h i g h e r t h a n t h a t used i n the human s t u d y . ll-hydroxy-Δ -THC was p r e s e n t i n t h e c i r c u l a t i o n w i t h i n 5 m i n u t e s a t a c o n c e n t r a t i o n o f 10 ng/ml and r a p i d l y d e c l i n e d s o t h a t a t 45 m i n u t e s t h e r e were no d e t e c t a b l e c o n c e n t r a t i o n s . These d a t a do n o t deny o r c o n f i r m t h e h y p o t h e s i s t h a t ll-hydroxy-Δ -THC i s an a c t i v e m e t a b o l i t e . They do, however s u g g e s t a t y p e o f e x p e r i m e n t t h a t i s p o s s i b l e when methods o f determination are available. I f we t a k e t h e argument t h a t we a d m i n i s t e r e d a p h a r m a c o l o g i c a l l y a c t i v e dose t o t h e r a b b i t t h e n i t i s c l e a r t h a t i f ll-hydroxy-Δ -THC i s t o be c o n s i d e r e d an a c t i v e m e t a b o l i t e i t must be shown t o be a c t i v e a t c o n c e n t r a t i o n s l e s s t h a n 10 ng/ml. 9


9

REFERENCES

(1) (2) (3) (4) (5)

(6) (7) (8)

Fenimore, D. C., Freeman, R. R . , and Loy, P . R . , Anal. Chem. 45, 2331 (1973). G a r r e t t , E . R . , and Hunt, C. Α . , J. Pharm. Sci. 62, 1211 (1973). Mechoulam, R . , McCallum, Ν. Κ . , and B u r s t e i n , S . , Chem. Revs. 76, 75 (1976). Rosenfeld, J., Bowins, B., Roberts J., P e r k i n s , J., and Macpherson, A . S . , Anal. Chem. 46, 2232 (1974) A g u r e l l , S . , Gustafsson, B., Holmstedt, B., Lean der, K., L i n d g r e n , J., N i l s s o n , I., Sandberg, F., and Asberg, M., J. Pharm. Pharmac. 25, 554, (1973). Estevez, V . S., E n g l e r t , L . F., and Ho, B . T., Res. Commun. Chem. Path. Pharm. 6, 821 (1973). Rosenfeld, J. M. and Taguchi, V . Y., Anal. Chem. 48, 726 (1976). F i e s e r , L . F. and F i e s e r , M., "Reagents f o r Or ganic S y n t h e s i s " , John Wiley and Sons, New York, 1967, p . 1252.

RECEIVED December 12,

1978.


A General Approach to the Analysis of Cannabinoids from

Recommend Documents