Tetrahydrocannabinol in Plasma by Dansylation and Double Labeling

12 Detection and Quantitation of Δ-Tetrahydrocannabinol in Plasma by Dansylation and Double Labeling

Downloaded by GRIFFITH UNIV on September 8, 2017 | http://pubs.acs.org Publication Date: April 10, 1979 | doi: 10.1021/bk-1979-0098.ch012

9

J. M. SCHERRMANN and R. BOURDON U. E. R. de Biologie Humaine et Experimentale, Laboratoire de Biochimie, Hopital Fernand Widal, Paris V, France H. HOELLINGER, NGUYEN-HOANG-NAM, and E. FOURNIER I.N.S.E.R.M. Unite de Recherche de Toxicologie Experimentale, Hopital Fernand Widal, 200, Rue du Faubourg Saint Denis, 75475 Paris Cedex 10, France

9

Δ-THC i s the major psychoactive constituent of Cannabis. Its detection and quantitation pose a d i f f i c u l t analytical problem because of i t s low concen tration in biological fluids. Much work has been done on the identification and quantitation of Δ -THC, i t s metabolites and cannabinoids by standard methods such as radio-immunoassay (1,2), gas chromatography, either alone (3-6) or coupled with mass spectrometry (7,8) and fluorometry (9-15). A l l these methods endeavor to satisfy two major criteria: specificity and s e n s i t i vity. Although radio-immunoassays are rapid and con venient to analyze large numbers of samples, they lack absolute s e n s i t i v i t y and specificity, since canna binoids cross-react within a given assay. Gas chroma tography is no more satisfactory. However, when com bined with mass spectrometry it i s far more specific and sensitive, although extremely costly. F i n a l l y , fluorometric techniques have proved suitable for many applications such as the one based on gallium chelate formation which can only be used for urine (9), and more especially for applications based on cannabinoid dansylation (16-18). However, these last techniques only allow qualitative determination of Δ -THC. 9

9

0-8412-0488-8/79/47-098-207$05.00/0 © 1979 American Chemical Society

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

CANNABINOID

208


Figure 1.

ANALYSIS

IN PHYSIOLOGICAL

FLUIDS

Chemical derivatization of A -THC 9

The method we have d e v e l o p e d f o r t h e d e t e c t i o n and q u a n t i t a t i o n o f A -THC i n plasma a l s o u t i l i z e s d a n s y l t e c h n o l o g y , b u t o u r methodology i s based on t h e use o f C DANS-Cl and, f o r 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 , on t h e u s e o f H 2 A -THC a s an i n t e r n a l s t a n d a r d (1922) . 9

1 4

3

9

PRINCIPLES OF THE METHOD The main s t e p s f o r d e t e c t i o n a r e 1) E x t r a c t i o n o f A -THC from plasma; 2) E s t e r i f i c a t i o n by 1 C DANSCl; 3) P u r i f i c a t i o n by TLC; 4) E l u t i o n o f t h e C DANS-Δ -THC s p o t and measurement o f C a c t i v i t y . In order t o perform q u a n t i t a t i o n , t h e four steps l i s t e d above a r e p r e c e e d e d by t h e a d d i t i o n o f H 2 A - T H C to the plasma. T h i s i s achieved i n o r d e r t o a l l o w a c c u r a t e d e t e r m i n a t i o n o f t h e q u a n t i t y o f A -THC i n i t i a l l y p r e s e n t , by c o r r e c t i n g t h e non n e g l i g i b l e l o s s e s o b s e r ved d u r i n g e x t r a c t i o n , e s t e r i f i c a t i o n a n d p u r i f i c a t i o n as shown s c h e m a t i c a l l y i n F i g u r e 2. 9

4

1 4

9

1 4

3

9

9

purification

Δ DANS

1

4

C

THC

DANS

1

C

4

L(T.C)

Δ

9

THC H DANS C 3

1

Figure 2.

4

Δ

9

THC H DANS C 3

1

4

Principles of the method of detecting and quantitating & -THC


9

12.

The

SCHERRMANN

E T

AL.

209

Dansylation and Double Labeling

i n i t i a l amount M o f Δ -THC i s g i v e n by t h e f o r m u l a : M = M

where M t c C

1

Τ

= = = = =

1

Χ - Χ £ c Τ amount o f H ? A - T H C added s p e c i f i c a c t i v i t y (SA) o f H A - T H C s p e c i f i c a c t i v i t y (SA) o f C DANS-C1 1 C a c t i v i t y measured a t t h e end o f a n a l y s i s (dpm) H a c t i v i t y a t t h e end o f a n a l y s i s (dpm) . 3

9

3

9

2

1 4

4

3


1

The known p a r a m e t e r s a r e : Μ , c and t . Measurement o f M i s o b t a i n e d by t h e d e t e r m i n a t i o n o f Τ and C i n t h e f r a c t i o n o f t h e p r o d u c t i s o l a t e d a t t h e end o f t h e t e s t . PROCEDURE A.

Extraction

1. Detection Four ml plasma a r e e x t r a c t e d t w i c e w i t h 4 ml o f the f o l l o w i n g s o l v e n t : methyl acetate/petroleum e t h e r / e t h a n o l (66:33:1.5 v / v ) . A f t e r c e n t r i f u g a t i o n , t h e organic e x t r a c t s a r e evaporated t o dryness, d i s s o l v e d i n t o 4 ml hexane and a g a i n e x t r a c t e d t w i c e w i t h 2 ml C l a i s e n ' s a l k a l i reagent. The a l k a l i n e s o l u t i o n s a r e a c i d i f i e d w i t h 1 ml Ν HC1 (pH = 1.5) a n d then e x t r a c t e d t w i c e w i t h 4 ml hexane. T h i s l a s t s o l u t i o n i s e v a p o r a t e d t o d r y n e s s and t h e r e s i d u e d i s s o l v e d i n t h e n e c e s s a r y amount o f a c e t o n e a n d t h e n t r a n s f e r r e d i n t o a hemolysis tube. 2. Q u a n t i t a t i o n The above s t e p s a r e p r e c e d e d by t h e a d d i t i o n o f 50 y l H2A -THC t o 4 ml plasma a n d by one h o u r s i n c u b a t i o n a t 37°C. 3

B.

9

1

E s t e r i f i c a t i o n , p u r i f i c a t i o n and c o u n t i n g 1 4

In a h e m o l y s i s t u b e , 60 nmoles C DANS-C1 s o l u t i o n i n 60 y l a c e t o n e , and 15 y l 0.5 M Na2CC>3 b u f f e r a r e added e i t h e r t o 50 y l o f t h e a c e t o n e s o l u t i o n ( e x t r a c t i o n ) o r t o a s o l u t i o n o f 3.18 nmoles A -THC ( y i e l d s t u d y ) . Simultaneously w i t h each t e s t , a blank i s made up under t h e same c o n d i t i o n s , b u t w i t h o u t Δ THC. The t u b e s a r e s t o p p e r e d , wrapped i n aluminum f o i l and i n c u b a t e d f o r one hour a t 40°C. A f t e r c o o l i n g , 18 nmoles o f u n l a b e l e d DANS A -THC ( c a r r i e r ) , 100 y l Ν NaOH a n d 500 y l d i s t i l l e d w a t e r a r e added t o the p r e p a r a t i o n . The s o l u t i o n i s t h e n s t i r r e d and 9

9

9


210

CANNABINOID

ANALYSIS

IN

PHYSIOLOGICAL

FLUIDS

e x t r a c t e d t w i c e w i t h 1 ml e t h y l a c e t a t e . The o r g a n i c phases a r e j o i n e d t o g e t h e r then e v a p o r a t e d t o d r y n e s s and d i s s o l v e d i n 200 y l e t h y l a c e t a t e . Then 20 y l o f t h i s s o l u t i o n a r e s p o t t e d on a Merck 60 F 254 s i l a n i z e d s i l i c a g e l p l a t e and d e v e l o p e d w i t h t h e s o l v e n t s y s t e m cyclohexane - e t h y l a c e t a t e (95:5). The DANS-Δ -THC s p o t i s d e t e c t e d w i t h UV, s c r a p e d and e l u t e d i n t o a c o u n t i n g v i a l c o n t a i n i n g 10 ml s c i n t i l l a t i o n l i q u i d . A c t i v i t i e s a r e e x p r e s s e d i n dpm a f t e r c o r r e c t i o n f o r q u e n c h i n g o f t h e a c t i v i t y o f e a c h sample from t h e b a c k ground due t o the b l a n k . 9


RESULTS A.

E x t r a c t i o n (See Table 14

I)

9

A f t e r studying CA -THC e x t r a c t i o n using f i v e s o l v e n t systems, e a c h w i t h a d i f f e r e n t c o m p o s i t i o n and n a t u r e , we c h o s e t h e f o l l o w i n g system: m e t h y l a c e t a t e / p e t r o l e u m e t h e r / e t h a n o l (66:33:1.5 v/v) w h i c h g i v e s 93% y i e l d a f t e r two e x t r a c t i o n s . As t h e o r g a n i c ex t r a c t i s r i c h i n l i p i d s , we t h e n p r o c e e d e d t o a p u r i f i c a t i o n based on s e l e c t i v e e x t r a c t i o n o f p h e n o l s by m o d i f i e d C l a i s e n ' s a l k a l i r e a g e n t , w h i c h g i v e s 70% y i e l d ( 8 ) . N o t e : t h e same t r e a t m e n t u s i n g aqueous sodium o r p o t a s s i u m h y d r o x i d e s o l u t i o n s does not p r o duce i d e n t i c a l r e s u l t s . B.

Esterification

D a n s y l a t i o n o f amines and p h e n o l s i s a c l a s s i c a l r e a c t i o n . However when a p p l i e d t o A -THC, i t c a l l s f o r s p e c i a l c o n d i t i o n s , f i r s t on a c c o u n t o f t h e use * C DANS-CI and second b e c a u s e o f t h e i n f l u e n c e o f v a r i o u s physico-chemical parameters. 9

4

1. Temperature and r e a c t i o n t i m e . The maximum y i e l d o f 14c DANS-Δ 9-THC (22.5 ± 0.6%) i s o b t a i n e d i n 120 m i n u t e s , b u t as e a r l y as t h e f i f teenth minute the r a t e of e s t e r i f i c a t i o n i s c o n s i d e r a b l y s l o w e d . A c o m p a r a t i v e s t u d y c o n d u c t e d f o r 24 h o u r s a t room t e m p e r a t u r e d i d n o t show how t h e y i e l d c o u l d be s i g n i f i c a n t l y i n c r e a s e d . The r e a c t i o n t i m e does n o t t h e r e f o r e have much e f f e c t on t h e d a n s y l a t i o n y i e l d . C o n s e q u e n t l y , i t i s p r e f e r a b l e t o o p e r a t e a t maximum t e m p e r a t u r e f o r a b r i e f p e r i o d , i . e . f o r one hour a t 40-45°C.



66 33

Methyl acetate Petroleum ether (40-60°C) Ethanol 1.5

67 33

Methyl acetate Petroleum ether 1/1

1/1

2/1

1/1

98.5 1.5

Heptane Ethanol

Ethanol

1/1

98.5 1.5

Volume of soIvents/ ρlasma

Study

Hexane Isoamyl a l c o h o l

Solvents

Extraction

9

1 from

85 90 87

1 3 1

93

83 2

2

80

3

%

Yield of Concentration

80

Plasma

4

Number of Extractions

of k -THC

TABLE


h-

1

to

οκ

S"

Γ—α

or

r—«ι

ο

Ο*

g4

Η

Ζ

>

M

Ο 33

to

212

CANNABINOID

ANALYSIS

IN

PHYSIOLOGICAL

FLUIDS

2.

pH, n a t u r e , volume and m o l a r i t y o f t h e b u f f e r ( F i g . 3, T a b l e s I I and I I I ) . DANS-Δ -THC s y n t h e s i s a t p r e p a r a t i v e s c a l e does n o t r e q u i r e a b u f f e r e d medium. On t h e o t h e r hand, t h e e s t e r i f i c a t i o n y i e l d a t t h e nanomole l e v e l n o t o n l y v a r i e s w i t h t h e pH b u t a c c o r d i n g t o t h e n a t u r e o f t h e buffer. The d i f f e r e n t pH r a n g e s t e s t e d showed t h a t maximum e s t e r i f i c a t i o n i s o b t a i n e d f o r 10.6 pH 12.3 w i t h an 0.5

Tetrahydrocannabinol in Plasma by Dansylation and Double Labeling

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