5 Improvement of the Mass Fragmentographic Technique for Quantification of Tetrahydrocannabinol in Human Blood Plasma A G N E T A O H L S S O N and STIG A G U R E L L — D e p a r t m e n t of Pharmacognosy, Faculty of Pharmacy, B M C , Box 579, S-751 23 Uppsala, Sweden J A N - E R I K L I N D G R E N — A s t r a Lakemedel A B , S-151 85 Sodertalje, Sweden K U R T L E A N D E R — D e p a r t m e n t of Organic Chemistry, Arrhenius-laboratoriet, University of Stockholm, Fack, S-104 05 Stockholm, Sweden
In 1973 we i n v e s t i g a t e d a mass fragmentographic method to determine Δ - t e t r a h y d r o c a n n a b i n o l (THC) i n plasma from cannabinis smokers ( 1 , 2 ) . This method was used f o r pharmacokinetic studies of Δ -THC i n man a f t e r smoking, where the plasma l e v e l s were c o r r e l a t e d with p h y s i o l o g i c a l and p s y c h o l o g i c a l e f f e c t s ( 3 ) . To im prove the sensitivity and to give an a l t e r n a t i v e route if i n t e r f e r i n g endogenous lipids or other contaminants appear, we have now developed a d e r i v a t i z a t i o n proce dure for the determination of Δ -THC and Δ -THC by t h i s nanogram-sensitive technique. 1
6
1
6
METHODS The s y n t h e t i c procedures for deuterium l a b e l l e d THC used to have been published elsewhere ( 4 , 5 ) . d -Labelled Δ - and Δ -THC were synthesized s t a r t i n g from 3,5-dimethoxybenzoic a c i d . The content of u n l a b e l l e d THC i n the final product was determined from mass spectroscopy t o be 10%. To increase the number of deuterium atoms in the molecule and to obtain a lower content of u n l a b e l l e d THC we therefore, with some m o d i f i c a t i o n s , used the method of Pitt et al, ( 6 ) . For the synthesis of d - or d -labelled THC 5-(3,5-dimeth2
1
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0-8412-0488-8/79/47-098-073$05.00/0 ©
1979 A m e r i c a n C h e m i c a l Society
CANNABINOID ANALYSIS IN PHYSIOLOGICAL FLUIDS
74
o x y - p h e n y l ) - p e n t a - 2 , 4 - d i e n o a t e was used a s s t a r t i n g material. The d e u t e r i u m c o n t e n t was f o r t h e d3«anal o g u e (m/e 314, 317) : d 0.7%, d 100% and f o r t h e d - a n a l o g u e (m/e 314, 321) : d 2%, d 100%. The com pounds were found t o be more than 9 5% p u r e a c c o r d i n g t o GLC. The p o s i t i o n s o f t h e d e u t e r i u m i n A -THC and Ai-THC and Δ -THC a r e shown i n F i g . 1. Q
3
7
7
1
6
Δ'-THC
A 6 -THC
D Figure 1.
2"
4"
D
D D
D
D
Formulas of ùJ-THC and A -THC and deuterium containing internal standards 6
ANALYSIS OF THC IN BLOOD PLASMA 1
The f o l l o w i n g p r o c e d u r e was used f o r A -THC (1,2) and f o r A -THC ( 3 ) . I n a d d i t i o n a s i l y l a t i o n s t e p was introduced. 6
OHLSSON E T A L .
5.
Mass Fragmentographic Technique
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BLOOD SAMPLES Plasma samples were o b t a i n e d from s u b j e c t s a f t e r smoking c a n n a b i s samples c o n t a i n i n g 5-20 mg Δΐ-THC o r A -THC. I n a n o t h e r t e s t , s u b j e c t s were g i v e n o r a l doses o f up t o 50 mg A -THC. B l o o d samples (10 ml) from smokers were c o l l e c t e d as needed i n h e p a r i n i z e d tubes. Plasma (5 ml) o b t a i n e d by c e n t r i f u g a t i o n was s t o r e d i n s i l a n i z e d g l a s s tubes a t -20°C u n t i l a n a l y sis. The plasma samples (1-4 ml) o b t a i n e d from sub j e c t s g i v e n o r a l doses o f A -THC were f r o z e n a f t e r c e n t r i f u g a t i o n and s t o r e d a t -20°C f o r f o u r months be fore analysis. 6
1
1
EXTRACTION To a 1-4 ml plasma sample i s added 10 ng o f deu t e r a t e d i n t e r n a l s t a n d a r d (A -THC-d3) d i s s o l v e d i n 50 y l e t h a n o l . The e x t r a c t i o n p r o c e d u r e and t h e l i q u i d c h r o m a t o g r a p h i c p u r i f i c a t i o n on Sephadex LH-20 columns are described i n d e t a i l i n our previous s t u d i e s (1-3). The p u r i f i e d sample was d i s s o l v e d i n a b s o l u t e e t h a n o l (10 y l ) and k e p t c o l d (-20°C) and dark u n t i l a n a l y s i s . T h i s s o l u t i o n can be s u b j e c t e d t o mass fragmentography d i r e c t l y or after s i l y l a t i o n . 1
SILYLATION On t h e day o f a n a l y s i s t h e samples were d r i e d un der n i t r o g e n and d i s s o l v e d i n 25 y l d r y a c e t o n i t r i l e , mixed w i t h 10 y l s i l y l a t i n g agent B S A — N , 0 , b i s - t r i m e t h y l s i l y 1 ) - a c e t a m i d e — o r BSTFA—Ν,0-bis-(trimethy1s i l y l ) - t r i f l o u r o a c e t a m i d e — a n d k e p t a t 50-60° f o r 10 m i n u t e s . The s o l u t i o n s were e v a p o r a t e d t o d r y n e s s under a s t r e a m o f n i t r o g e n and r e d i s s o l v e d i n 10 y l d r y a c e t o n i t r i l e and 2 y l was s u b j e c t e d t o mass fragmentography. MASS FRAGMENTOGRAPHY Mass fragmentography was c a r r i e d o u t u s i n g an LKB 2091 GC-MS i n s t r u m e n t . The column was a 1.4x2mm i . d . s i l a n i z e d g l a s s column c o n t a i n i n g 3% OV-17 o r 3% SE-30 on Gas Chrom Q 100/120 mesh. Temperatures were i n t h e column 210°C, f l a s h h e a t e r 250°C and i o n s o u r c e 290°C. H e l i u m was c a r r i e r gas (25 ml/min) and t y p i c a l r e t e n t i o n times were: Δβ-THC 4.0 m i n . and A!-THC 4.5 min. Lower column t e m p e r a t u r e s (180°C) was used f o r t h e s i l y l a t e d cannabinoids. F o r mass fragmentography a m u l t i p l e i o n d e t e c t o r was added ( 7 ) . F o r Δΐ-THC-TMS
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CANNABINOID ANALYSIS IN PHYSIOLOGICAL FLUIDS
( T M S = t r i m e t h y l s i l y l ) t h e mass s p e c t r o m e t e r was s e t t o c o n t i n u o u s l y r e c o r d t h e i n t e n s i t y o f m/e 38 6 ( m o l e c u l a r ion) and f o r t h e i n t e r n a l s t a n d a r d Δ -THC-d3~TMS t h e i n t e n s i t y o f m/e 389 ( m o l e c u l a r i o n ) . The i o n i z i n g p o t e n t i a l was o p t i m i z e d a t 50 eV f o r b o t h t h e s i l y l a t e d and u n d e r i v a t i z e d THC. The r e l a t i v e s t a n d a r d d e v i a t i o n when 1 ng was i n j e - t e d was 2.7% (n=5) and w i t h 0.5 ng i n j e c t e d 5.7% (n=5). The s t a n d a r d c u r v e s were p r e p a r e d by a d d i n g known amount o f A -THC (0.5 - 20 ng/ml) t o b l a n k plasma sam p l e s and c a r r y i n g o u t t h e d e s c r i b e d p r o c e d u r e ( F i g . 2 ) . The c o r r e c t n e s s o f t h e s t a n d a r d c u r v e s was checked dur i n g t h e day. 1
1
PEAK HEIGHT RATIO
A'-THC-TMS (m/e 386) A-THC-d3-TM5 (m/e 389)
10
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20 η g Δ'-THC
Figure 2.
Standard curve for A^THC-TMS (0-20 ng/mL) in plasma
RESULTS AND DISCUSSION 1
6
The e x t r a c t i o n p r o c e d u r e f o r b o t h A -THC and Δ THC, as r e v e a l e d by e x p e r i m e n t w i t h t r i t i u m l a b e l l e d
5. OHLSSON E T A L .
Mass Fragment ο graphic Technique
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compounds i s q u i t e e f f i c i e n t a n d t h e r e c o v e r y a f t e r b o t h e x t r a c t i o n and l i q u i d c h r o m a t o g r a p h i c p u r i f i c a t i o n i s u s u a l l y o v e r 80%. The p e r t i n e n t f r a c t i o n 5 ml from the Sephadex LH-20 column c o n t a i n s o v e r 90% o f the peak, b u t a 7-8 ml f r a c t i o n i s c o l l e c t e d . The s e n s i t i v i t y i n the f i n a l mass f r a g m e n t o g r a p h i c assay may be l i m i t e d by the amount o f n o n - l a b e l l e d ( d ) compound p r e s e n t i n t h e d e u t e r a t e d i n t e r n a l s t a n d a r d . T h i s i n t e r f e r e s w i t h the n o n - l a b e l l e d THC i n the plasma. Thus, we have t r i e d t o m i n i m i z e t h i s i n t e r f e r e n c e by i n c r e a s i n g the number o f hydrogens s u b s t i t u t e d w i t h d e u t e r i u m , and by l i m i t i n g the amount o f i n t e r n a l s t a n d a r d i n t h e samples c o n t a i n i n g low amounts o f THC. Thus, the p r e s e n t l i m i t o f s e n s i t i v i t y i s p a r t l y due t o t h e amount o f THC-d i n the i n t e r n a l s t a n d a r d a n d n o t due t o the c h r o m a t o g r a p h i c o r mass s p e c t r o m e t r i c problems p e r se. A s e x p e c t e d , d 3 - c o n t a i n i n g THC showed, t o g e t h e r w i t h A -THC-d7, t h e l e a s t contamination w i t h d -analogues (0.7-2%). The p r e s e n t s e n s i t i v i t y f o r u n d e r i v a t i z e d THC i s 0.3 ng/ml plasma. This s e n s i t i v i t y i s s t i l l not s a t i s f a c t o r y t o f o l l o w Δΐ-THC l e v e l s i n plasma from s u b j e c t s , more than 12-24 h o u r s . I n o r d e r t o improve t h e s e n s i t i v i t y i t may a l s o be n e c e s s a r y t o r e d u c e t h e b l o o d sample volumes o r t o e l i m i n a t e compounds i n t e r f e r i n g w i t h mass fragmentography. A f t e r s i l y l a t i o n , t h e sen s i t i v i t y i n c r e a s e d down t o 0.1 ng/ml plasma. With a s e n s i t i v i t y o f 0.1 ng/ml plasma i t may perhaps be p o s s i b l e t o e s t a b l i s h a t r u e e l i m i n a t i o n phase f o r A -THC i n man. Lemberger a n d c o - w o r k e r s (8) have e s t i m a t e d e l i m i n a t i o n phase h a l f - l i v e s i n man o f 1-2 days. T y p i c a l mass fragmentograms from the d e t e r m i n a t i o n s o f A!-THC l e v e l s i n man a f t e r o r a l a d m i n i s t r a t i o n a r e shown i n F i g . 3. The plasma l e v e l o f 0.2 ng/ml was d e t e r m i n e d from a t o t a l volume o f 2.25 ml plasma. We have so f a r e n c o u n t e r e d l i t t l e i n t e r f e r e n c e i n the mass f r a g m e n t o g r a p h i c d e t e r m i n a t i o n o f THC p r o v i d e d r e d i s t i l l e d s o l v e n t s , p a r t i c u l a r l y e t h a n o l , and a l l g l a s s a p p a r a t u s a r e used. I f i n t e r f e r e n c e does o c c u r , the s i l y l a t i o n t e c h n i q u e p r o v i d e s an a l t e r n a t i v e . T h i s method can a l s o be used t o d e t e r m i n e b o t h u n d e r i v a t i z e d o r s i l y l a t e d c a n n a b i d i o l and c a n n a b i n o l from plasma. Q
Q
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CANNABINOID ANALYSIS IN PHYSIOLOGICAL FLUIDS
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Figure 3. Mass fragmentograms of ^-THC-TMS (m/e 386) with A -THC-d TMS as internal standard (m/e 389) from purified plasma extracts. Plasma levels of 0.0, 0.2, 6.6, and 19 ng/mL. 6
3
ACKNOWLEDGMENTS The s u p p o r t o f the Swedish M e d i c a l Research Counc i l i s appreciated. A.O. wishes t o e x p r e s s h e r g r a t i tude t o the A p o t e k a r s o c i e t e t e n f o r awarding a s c h o l a r ship .
REFERENCES (1)
Agurell, S., Gustafsson, B . , Holmstedt, B . , Leander, K . , Lindgren, J-E., Nilsson, I . , Sand berg, F . , and Asberg, M., J. Pharm. Pharmac. 25, 554 (1973).
5.
(2)
(3)
(4) (5)
(6) (7) (8)
OHLSSON E T A L .
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A g u r e l l , S . , "The Poisoned P a t i e n t : The Role of the Laboratory", Ciba Roundation Symposium 26, E l s e v i e r - E x c e r p t Medica-North-Holland, AmsterdamOxford-New York, 1974, p . 125. A g u r e l l , S . , Levander, S . , Binder, Μ., BaderB a r f a i , Α . , Gustafsson, B., Leander, K., Lindgren, J-E., Ohlsson, Α . , and Tobisson, B., in "Pharmacology of Cannabis", Eds. S. Szara and M. Braude, Raven Press, New York, 1974. Ohlsson, Α . , Lindgren, J-E., Lean der, K., A g u r e l l , S . , "Cannabinoid Assays i n Humans, NIDA Research Monograph S e r i e s , No. 7, 1976. Ohlsson, Α . , Lindgren, J-E., Leander, K., A g u r e l l , S . , in "Mass Spectroscopy i n Drug Meta b o l i s m " , Eds. A . Frigerio and E. Chisalberti, Plenum Press, New York, 1976. P i t t , C. G., Hobbs, D. T., Schran, H., Twine Jr., C. E . and W i l l i a m s , D. L., J. Label. Comp. 11, 551 (1975). E l k i n , K . , P i e r r o u , L., Ahlborg, U . G . , Holmstedt, B., Lindgren, J-E., J. Chromatogr. 81, 47 (1973). Lemberger, L., A x e l r o d , J., and Kopin, I. J., New York Acad. Sci. 191, 142 (1971).
RECEIVED
December 12, 1978.
