Cannabinoid Analysis in Physiological Fluids - American Chemical

2 G L C and H P L C Analyses of Cannabinoids in Biological Fluids and Applications E . R.

GARRETT , 1a

A . J.

GOUYETTE , 1b

and C . A .

HUNT

1c

Downloaded by UNIV OF MASSACHUSETTS AMHERST on March 1, 2016 | http://pubs.acs.org Publication Date: April 10, 1979 | doi: 10.1021/bk-1979-0098.ch002

College of Pharmacy, University of Florida, Gainesville, FL 32610

Pharmacological and metabolic studies on cannabi noids ( F i g . 1) have suffered from a lack of knowledge of t h e i r physico-chemical p r o p e r t i e s and the insensi tivity of assays o f Δ - t e t r a h y d r o c a n n a b i n o l 1, and its metabolites i n biological fluids. Unambiguous, sensi tive, s p e c i f i c and accurate q u a n t i f i c a t i o n is required for forensic t o x i c o l o g y and pharmacokinetic studies which can be c o r r e l a t e d w i t h the time course of the psychoactive e f f e c t s ( 2 ) . The determination (3) of p h y s i c a l and chemical p r o p e r t i e s such as solubility, stability, pKa, glass - b i n d i n g and p r o t e i n - b i n d i n g of Δ - t e t r a h y d r o c a n n a b i n o l 1 and c o r r e l a t e d congeners were only p o s s i b l e a f t e r the development of new high pressure liquid chromatography (HPLC) techniques and the m o d i f i c a t i o n o f gas liquid chromatography (GLC) t e c h n o l o g i e s . Clinical investigations of Δ -tetrahydrocannabinol (4,5) and 1 1 - h y d r o x y - Δ - t e t r a h y d r o c a n n a b i n o l (6) have relied upon a n a l y s i s by r a d i o a c t i v e l a b e l i n g . However, the study of distribution, metabolism and e x c r e t i o n of the drug and its metabolites under chronic or "street" conditions demands nonradioactive a n a l y t i c a l proced ures. When plasma suspensions of C-Δ -tetrahydrocannabinol were administered intravenously to three dogs at doses o f 0.1 - 2.0 mg/kg and plasma l e v e l s of 1 were followed f o r up to 7000 minutes, no s i g n i f i c a n t differences were seen in 1 plasma l e v e l s as determined by liquid scintillation and e l e c t r o n capture d e t e c t i o n (GLC) a f t e r HPLC collection. 9

9

9

9

14

9

0-8412-0488-8/79/47-098-013$06.25/0 © 1979 American Chemical Society

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

ANALYSIS

IN

PHYSIOLOGICAL


CANNABINOID

Figure 1.

Structural formuhs of some cannabinoids


FLUIDS

2.

GARRETT

E T

AL.

15

GLC and HPLC Analyses

PHYSICOCHEMICAL PROPERTIES OF Δ 9-TETRAHYDROCANNABINOL


Solubility Δ^-tetrahydrocannabinol i s a l i q u i d and i s h i g h l y i n s o l u b l e i n w a t e r ( 8 - 1 0 ) . T h i s c a n be a c r i t i c a l f a c t o r i n i t s b i o a v a i l a b i l i t y , p h a r m a c o k i n e t i c s and p h a r m a c o l o g i c a l a c t i o n . Large d i f f e r e n c e s i n the b i o a v a i l a b i l i t y o f t e t r a h y d r o c a n n a b i n o l from v a r i o u s s o l u t i o n s and a d m i n i s t r a t i v e r o u t e s have been r e p o r t e d ( 9 , 1 0 ) . E v i d e n c e has been p r e s e n t e d (8) t h a t t e t r a h y d r o c a n n a b i n o l ' s s o l u b i l i t y may be exceeded i n p l a s m a , r e s u l t i n g i n i t s p o s s i b l e p r e c i p i t a t i o n and f o r t u i t o u s l o c a l i z e d a c c u m u l a t i o n i n body o r g a n s . The s o l u b i l i t i e s o f A - t e t r a h y d r o c a n n a b i n o l were d e t e r m i n e d (7) by two methods: 1) B e e r ' s l a w p l o t s were o b t a i n e d by p l o t t i n g t h e absorbance a t one w a v e l e n g t h (225 nm) v e r s u s t h e amount o f t e t r a h y d r o c a n n a b i n o l added. The s o l u b i l i t y o f t e t r a h y d r o c a n n a b i n o l was de t e r m i n e d as t h a t c o n c e n t r a t i o n a t w h i c h d e v i a t i o n from B e e r ' s l a w was o b s e r v e d ; 2) A m o d i f i c a t i o n o f t h e p r o cedure o f Saad and H i g u c h i (11) f o r d e t e r m i n i n g s o l u b i l i t y by u s i n g a p a r t i c l e - s i z e c o u n t e r was a l s o u s e d ; 1_ formed m i c e l l e s when i t s s o l u b i l i t y was exceeded and the appearance o f t h e s e m i c e l l e s was m o n i t o r e d by t h e counter. The s o l u b i l i t y o f 1 i n c r e a s e d as a l i n e a r f u n c t i o n of ethanol concentration (at constant i o n i c strength) and d e c r e a s e d w i t h t h e square r o o t o f t h e i o n i c strength (at constant ethanol c o n c e n t r a t i o n ) . " S a l t i n g o u t " c o e f f i c i e n t s were d e t e r m i n e d . The s o l u b i l i t y o f 1 i n 0.15 M N a C l was 0.77 m g / l i t e r a t 23° and e x t r a p o l a t i o n e s t i m a t e d a s o l u b i l i t y o f 2.8 m g / l i t e r i n p u r e water. The p a r t i c l e - s i z e c o u n t i n g p r o c e d u r e o v e r e s t i m a t e d the t r u e s o l u b i l i t y s i n c e t h e r e i s a l i m i t below w h i c h p a r t i c l e s i z e c a n n o t be c o u n t e d . The mean s o l u b i l i t y e s t i m a t e was 1.05 m g / l i t e r by t h i s method i n 0.9% N a C l (0.154 Μ ) , i n good agreement w i t h t h e s p e c t r o p h o t o m e t r i c method. 9

Spectrophotometric

Determination o f the pK' (7) a

The p K ' o f t e t r a h y d r o c a n n a b i n o l was c a l c u l a t e d by e m p l o y i n g t h e m o d i f i e d form o f t h e H e n d e r s o n - H a s s e l b a c h equation (12): a

log { ( e

b

- ε)

/

(ε - c ) } a

λ

= H - pK' P

a


16

CANNABINOID

ANALYSIS

IN

PHYSIOLOGICAL

FLUIDS

where e ( 5 , 8 5 0 ) and e ( 1 2 , 2 2 6 ) a r e t h e m o l a r a b s o r b t i v i t i e s o f t h e u n i o n i z e d and c o m p l e t e l y i o n i z e d d r u g measured a t t h e g i v e n w a v e l e n g t h λ (240 nm), r e s p e c t i v e l y , and ε i s t h e a p p a r e n t m o l a r a b s o r p t i v i t y o f a s o l u t i o n measured a t a p a r t i c u l a r pH. The a b s o r p t i v i t i e s o f t h e i o n i z e d and u n i o n i z e d s p e c i e s were e s t i m a t ed u s i n g 0.5 Ν HC1 and 0.5 Ν NaOH. The p K ' was 10.56 ± 0.16 Τ σ ) i n a c c o r d a n c e w i t h E q u a t i o n 1 ( F i g . 2 ) , a

b


a

Journal of Pharmaceutical Sciences Figure 2. Spectre-photometric determination of the pK' of A -THC in accord ance with log {(e — e)/(e — € )} = pH — pK!' , where c and e are the mohr absorptivities of the unionized and completely ionized drug at 240 nm, respec tively, and e is the apparent mohr absorptivity at the stated pH value (7). a

b

a

a

0

9

b


2.

GARRETT

ET

AL.

GLC

and

17

HPLC Analyses

T h i s pK' i s h i g h e r t h a n would be a n t i c i p a t e d f o r o r t h o - and meta- s u b s t i t u t e d p h e n o l s s i n c e the ο metho x y p h e n o l has a pK' o f 9.98, m-methoxyphenol a pK' of 9.65 and r e s o r c i n o l a pK' o f 9.81 ( 1 3 ) . A m o l e c u l a r model o f t h e d r u g shows t h a t t h e f r e e r o t a t i o n o f t h e p h e n o l i c hydrogen i s h i n d e r e d by the A - h y d r o g e n and can e x p l a i n A ^ - t e t r a h y d r o c a n n a b i n o l s h i g h e r p K * . Of course, i f solvated dimers, t r i m e r s , e t c . , e x i s t with h y d r o p h o b i c b o n d i n g , the o b s e r v e d pK' c o u l d be a hy brid pK f o r a s o l u t i o n o f such p o l y m e r s . a

a

a

a

9

1

a

a


f

a

Glassbinding The r a t e and e x t e n t o f t e t r a h y d r o c a n n a b i n o l ( i n aqueous s o l u t i o n ) b i n d i n g t o g l a s s depend on the s u r f a c e a r e a o f the g l a s s , t h e p r e t r e a t m e n t o f t h e g l a s s , and the c o n c e n t r a t i o n o f t h e d r u g . About 20 and 40%, r e s p e c t i v e l y , o f t e t r a h y d r o c a n n a b i n o l b i n d i n 50 ml v o l u m e t r i c f l a s k s a t c o n c e n t r a t i o n s o f 0.1 and 0.05 yg/ml. A t h i g h d r u g c o n c e n t r a t i o n s (0.5 - 1.0 yg/ml) and a f t e r f u l l e q u i l i b r a t i o n , l e s s t h a n 7% o f the amount i n an aqueous s o l u t i o n was bound t o g l a s s from v a r i o u s f l a s k s and t e s t t u b e s . Preconditioning glass with strong a l k a l i or a c i d d i d n o t d e c r e a s e t h e e x t e n t o f b i n d i n g . New, unused g l a s s w a r e bound the d r u g t o a s i m i l a r e x t e n t . Treat ment o f the f l a s k s w i t h a w a t e r s o l u b l e s i l i c o n e con c e n t r a t e i n c r e a s e d the e x t e n t o f b i n d i n g . However, vigorous shaking immediately p r i o r to sampling r e duced the degree o f b i n d i n g , b u t no more t h a n 50% i n any c a s e . T r i m e t h y s i l y l treatment of glassware s i g n i f i c a n t l y r e d u c e d b i n d i n g . However, a t room t e m p e r a t u r e the bound f r a c t i o n o f a 0.1 yg/ml s o l u t i o n i n c r e a s e d t o t h e p r e t r e a t m e n t l e v e l a f t e r 300 m i n u t e s . P o l y c a r b o n a t e , p o l y p r o p y l e n e , T e f l o n , and s t a i n l e s s s t e e l c o n t a i n e r s showed more e x t e n s i v e b i n d i n g t h a n g l a s s ( F i g . 3 ) . S i g n i f i c a n t r e t e n t i o n on g l a s s p i p e t s was o b s e r v e d . Of g r e a t i m p o r t a n c e i n the l i g h t o f normal h a n d l i n g o f samples o f b i o l o g i c a l f l u i d s i s the f a c t t h a t 70 - 97% o f the d r u g i n t h e v i a l s was l o s t t o the r u b b e r s t o p p e r a f t e r one hour o f s h a k i n g o f the stoppered v i a l s . T e t r a h y d r o c a n n a b i n o l i n whole b l o o d o r plasma a l s o b i n d s t o g l a s s . However, s i n c e t e t r a h y d r o c a n n a b i n o l p r o t e i n binding i s very l a r g e , glass binding i s s i g n i f i c a n t l y r e d u c e d . The b i n d i n g o f d r u g from plasma t o the s i l y l - t r e a t e d g l a s s w a r e was n e g l i g i b l e . R


18

CANNABINOID

ANALYSIS

IN

PHYSIOLOGICAL

FLUIDS

•ih


100

o-o—o-o

O-

•

B

•o—o

c

:

20

40

60 Time, min.

80

D

100

300

Journal of Pharmaceutical Sciences

Figure 3. Ghss binding of Δ -ΤΗΟ showing the percent of drug added that re mained in solution at various times for: (A) TMS-treated 50-mL volumetric flasks from an aqueous drug concentration of 0.1 pg/mL; (B) an aqueous drug concen tration of 0.1 (xg/mL in untreated 50-mL volumetricflasks;(C) an aqueous drug concentration of 0.1 ^g/mL in water-soluble silicone concentrate-treated 50-mL volumetric flasks; (D) an aqueous drug concentration of 0.05 \x%/mL in untreated 50-mL volumetric flasks; and (E) an aqueous drug concentration of 0.1 pg/mL in a 20-mL stainless steel ultracentrifuge tube. Each point is the mean of four sepa rate determinations (7). 9


2.

GARRETT

ET

GLC

AL.

and

19

HPLC Analyses

Protein-Binding When t e t r a h y d r o c a n n a b i n o l i n i s o t o n i c phosphate b u f f e r was d i a l y z e d a g a i n s t i s o t o n i c phosphate b u f f e r , 50 - 100% of the d r u g was bound t o the t u b i n g used as the membrane. A l l o f the d r u g was bound below 0.05 yg/ml. U l t r a f i l t r a t i o n was e q u a l l y u n s u c c e s s f u l , s i n c e o n l y 0 - 5% o f the d r u g i n i s o t o n i c phosphate b u f f e r was r e c o v e r e d i n the u l t r a f i l t r a t e . The o n l y o t h e r known s t u d y on the b i n d i n g o f Δ t e t r a h y d r o c a n n a b i n o l t o plasma p r o t e i n s was p e r f o r m e d w i t h e l e c t r o p h o r e t i c t e c h n i q u e s on t r i t i u m - l a b e l e d m a t e r i a l and human plasma ( 1 4 ) . The compound was 90 95% a s s o c i a t e d w i t h l i p o p r o t e i n s . Because o f t h i s e x t e n s i v e plasma p r o t e i n b i n d i n g , a method o f v a r i a b l e plasma c o n c e n t r a t i o n s was d e v i s e d (7) w h i c h t o o k advantage o f the c o m p e t i t i o n between r e d b l o o d c e l l s and plasma p r o t e i n f o r the f r e e d r u g i n plasma w a t e r . The r e d b l o o d c e l l d i s t r i b u t i o n c o e f f i c i e n t , D = ( A j ^ ^ O / i A ^ ) , was 12.5 f o r the dog r e d b l o o d c e l l s where (AJ^Q) i s the c o n c e n t r a t i o n o f drug i n the r e d b l o o d c e l l s and (A ) i s t h e c o n c e n t r a t i o n o f un bound d r u g i n p l a s m a . The f u n c t i o n o f t e t r a h y d r o c a n n a b i n o l bound t o plasma p r o t e i n was i n d e p e n d e n t o f drug c o n c e n t r a t i o n . The f r a c t i o n 0.9 72 o f t e t r a h y d r o c a n n a b i n o l i n plasma was bound a t n o r m a l p r o t e i n c o n c e n t r a t i o n s . The d e v e l o p e d p r o c e d u r e can be used r o u t i n e l y t o d e t e r m i n e the p r o t e i n b i n d i n g o f i n d i v i d ual subjects. I t p e r m i t s more a c c u r a t e e s t i m a t e s a t h i g h degrees o f p r o t e i n b i n d i n g w h i c h the n o r m a l e r r o r s i n c l a s s i c a l methods w o u l d n o t a l l o w .


9

u

SEPARATION AND

ANALYSES

P u r i f i c a t i o n o f C a n n a b i n o i d s by H i g h L i q u i d Chromatography (HPLC) (15,16) 1 4

Pressure

9

The C-A -tetrahydrocannabinol used as s u p p l i e d by NIDA was checked f o r p u r i t y on r e v e r s e and n o r m a l phase HPLC and was c o n t a m i n a t e d by two compounds w h i c h were not f u r t h e r analyzed. The q u a l i t y o f the w a t e r used i n the e l u t i n g s o l v e n t a c e t o n i t r i l e - w a t e r i n r e v e r s e phase (column: Bondapack C-18 ; e l u e n t : 45% a c e t o n i t r i l e i n w a t e r a t 2.5 ml/min) was an i m p o r t a n t f a c t o r i n main t a i n i n g t h e r e p r o d u c i b i l i t y o f the p e r c e n t r a d i o a c t i v i t y recovered f o r a given c o l l e c t i o n range. On n o r m a l phase HPLC (column: y - P o r a s i l , e l u e n t : 5% t e t r a h y d r o f u r a n i n n-hexane a t 0.5 ml/min) Δ and R

R

8



20

CANNABINOID

ANALYSIS

IN

PHYSIOLOGICAL

FLUIDS

A ^ - t e t r a h y d r o c a n n a b i n o l were q u a n t i t a t i v e l y s e p a r a t e d w i t h r e s p e c t i v e r e t e n t i o n volumes o f 6.37 and 6.62 ml (the dead volume between t h e UV d e t e c t o r and t h e c o l l e c t i o n p o i n t was 0.46 ml as d e t e r m i n e d by t h e i o d o f o r m test). C a n n a b i d i o l i n t h e same system had a r e t e n t i o n volume o f 6.06 m l . The e t h a n o l i c s t o c k s o l u t i o n o f t r a n s - c a n n a b i d i o l 10, o r i g i n a l l y a n a l y z e d by gas l i q u i d chromatography TGLC) (column: 3% OV-17 on 100-120 mesh Gas Chrom Q, 1.8 m, 240) showed c o n t a m i n a t i o n w i t h t r a c e s o f h e x a h y d r o c a n n a b i n o l 1JL, A ^ - t e t r a h y d r o c a n n a b i n o l 1 (12.2%), c a n n a b i n o l 12 (0.65%) and t h r e e u n i d e n t i f i e d compounds ( t o t a l 0.66%T. T h i s s o l u t i o n was p u r i f i e d on two y - P o r a s i l column i n s e r i e s , u s i n g 5% t e t r a h y d o f u r a n i n n-hexane (0.5 m l / m i n ) , where t h e r e t e n t i o n volume o f c a n n a b i n o l 10. A - t e t r a h y d r o c a n n a b i n o l ,2/ ~ t e t r a h y d r o c a n n a b i n o l 1, were 13.3, 14.0 and 14.6 ml r e s p e c t i v e l y ( F i g . 4 ) . GLC a n a l y s i s o f t h e c a n n a b i n o l c o l l e c t i o n s showed g r e a t e r t h a n 99.5% p u r i t y w i t h l e s s t h a n 0.3% c o n t a m i n a t i o n by t e t r a h y d r o c a n n a b i n o l and n e g l i g i b l e other peaks. A n o t h e r s o l v e n t system was a l s o used t o r e s o l v e t e t r a h y d r o c a n n a b i n o l , c a n n a b i n o l and c a n n a b i d i o l by t h e n o r m a l phase HPLC (column: y - P o r a s i l ; e l u e n t ; 20% c h l o r o f o r m i n heptane a t 2.5 m l / m i n ) . However, t h e chosen c o l l e c t i o n range w o u l d a l s o c o l l e c t c a n n a b i n o l and c a n n a b i d i o l i f 98% o f t h e t e t r a h y d r o c a n n a b i n o l were t o be c o l l e c t e d . The r e t e n t i o n volumes o f c a n n a b i d i o l and c a n n a b i n o l r e l a t i v e t o 1 s i g n i f i c a n t l y d i v e r g e d w i t h i n c r e a s i n g percentages of increased percent of c h l o r o f o r m i n heptane t o g i v e b e t t e r s e p a r a t i o n . The 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 had l a r g e r e t e n t i o n volumes (> 15 ml) on t h e n o r m a l phase column when 20% c h l o r o f o r m i n heptane was t h e s o l v e n t and c o u l d be c o m p l e t e l y s e p a r a t e d from t e t r a h y d r o c a n n a b i n o l on t h i s system. They were r e s o l v e d from each o t h e r w i t h a more p o l a r s o l v e n t , 80% c h l o r o f o r m i n h e p t a n e . A ^ - T e t r a h y d r o c a n n a b i n o l and l l - h y d r o x y - A - t e t r a h y d r o c a n n a b i n o l were q u a n t i t a t i v e l y s e p a r a b l e on t h e r e v e r s e phase HPLC system a t 47% (or l e s s ) a c e t o n i t r i l e i n water. The c o l l e c t i o n e f f i c i e n c i e s i n t h e chosen r a n g e s were 98% o f the r e c o v e r a b l e r a d i o a c t i v i t i e s o f H - l l - h y d r o x y - A - t e t r a h y d r o c a n n a b i n o l and 14c-A t e t r a h y d r o c a n n a b i n o l ( F i g . 5 ) . Δ - and Δ^-Tetrahydroc a n n a b i n o l were n o t r e a d i l y r e s o l v a b l e i n any o f t h e s e s y s t e m s . However, t h e p o o l e d f r a c t i o n o f 1 and _2 c o u l d be r e s o l v e d ( F i g . 4) on t h e n o r m a l phase HPLC (5% t e t r a h y d r o f u r a n i n n-hexane). R

8

a n d

δ 9

R

9

3

9

9

8


ET

AL.



GARRETT

Figure 4. Normal phase HPLC separation of cannabinoids (two μ-Porasïl® columns in series) using 5% THF in n-hexane at aflowrate of 0.5 mL/min



22

CANNABINOID

ANALYSIS IN

PHYSIOLOGICAL FLUIDS

ω 2 Z> -J Ο >

Ζ ο Ul or

47

49

51

%ACETONITRILE- WATER Journal of Pharmaceutical Sciences

Figure 5. Retention volumes for the peak amounts of (Φ), A -THC and (0)> Hhydroxy-& -THC for reverse-phase HPLC vs. solvent composition. Each point represents the mean peak retention volume for two determinations. The vertical bars represent the ranges of retention volumes that contained approximately 98% of the area under the plot of recovered radioactivity vs. retention volume (15). 9

9


2.

GARRETT

ET

GLC

AL.

and HPLC Analyses

23

C a n n a b i n o l , Δ - and Δ - t e t r a h y d r o c a n n a b i n o l had the same r e t e n t i o n volumes o f 0.05 ml on a y - P o r a s i l column w i t h 30% t e t r a h y d r o f u r a n i n n-hexane and were s e p a r a t e d from the a c i d d e g r a d a t i o n p r o d u c t s w i t h r e s p e c t i v e r e t e n t i o n volumes o f 9.5 and l l . t H m l . The c o l l e c t i o n f r a c t i o n c o n t a i n i n g the t e t r a h y d r o c a n n a b i n o l c o u l d be p u r i f i e d l a t e r u s i n g the 5% t e t r a h y d r o f u r a n hexane s o l v e n t system. 8

9


R

E f f e c t o f HPLC S e p a r a t i o n on GLC A n a l y s i s o f Δ T e t r a h y d r o c a n n a b i n o l i n Plasma (15)

9

-

An e q u a l amount o f Δ - t e t r a h y d r o c a n n a b i n o l and l l - h y d r o x y - Δ ^ t e t r a h y d r o c a n n a b i n o l i n 2 ml dog plasma (the pH was a d j u s t e d between 9.5 and 11.0 by a d d i t i o n o f 0.1 Ν Na2CU3 p r e p a r e d from water p u r i f i e d u s i n g a Bondapak C 1 8 column) was e x t r a c t e d i n a s i l y l a t e d tube by heptane w i t h 1.5% i s o a m y l a l c o h o l . In t h i s e x t r a c t , the compounds were s e p a r a t e d from a m a j o r i t y o f e x t r a c t e d components by r e v e r s e phase HPLC. The r e d u c t i o n i n p o t e n t i a l c o n t a m i n a n t s from plasma ob s e r v a b l e on GLC was demonstrated by flame i o n i z a t i o n GLC a n a l y s i s (17,18) both b e f o r e and a f t e r HPLC t r e a t ment ( F i g . 6 ) . The normal phase HPLC (20% c h l o r o f o r m i n heptane) c o u l d s e p a r a t e Δ - t e t r a h y d r o c a n n a b i n o l from monohydroxy l a t e d m e t a b o l i t e s and from l l - h y d r o x y - Δ - t e t r a h y d r o cannabinol. However, a minor o v e r l a p c o u l d be a v o i d e d by c o l l e c t i n g the t e t r a h y d r o c a n n a b i n o l 1 i n a s l i g h t l y narrower volume range. The p r i o r heptane e x t r a c t i o n o f a l k a l i n i z e d plasma had s e p a r a t e d these n o n - p o l a r c o n s t i t u e n t s from any a c i d i c m e t a b o l i t e . This separa t i o n o f plasma e x t r a c t s and normal phase HPLC c o l l e c t i o n o f volumes i n the a p p r o p r i a t e range r e s u l t e d i n a s u b s t a n t i a l r e d u c t i o n i n GLC background from plasma components f o r d e r i v a t i z e d t e t r a h y d r o c a n n a b i n o l ana l y z e d w i t h e l e c t r o n c a p t u r e (63^1) d e t e c t i o n . Plasma samples o b t a i n e d from dogs, a d m i n i s t e r e d Δ - t e t r a h y d r o c a n n a b i n o l s o l u t i o n s i n t r a v e n e o u s l y , were a n a l y z e d by the e l e c t r o n c a p t u r e GLC i n a c c o r d a n c e w i t h the m o d i f i e d p r o c e d u r e s d e s c r i b e d h e r e i n t h a t i n c l u d e d e x t r a c t i o n , normal phase HPLC s e p a r a t i o n and d e r i v a t i z a t i o n except t h a t no i n t e r n a l s t a n d a r d was added. These p r o c e d u r e s would have i n c l u d e d any c a n n a b i n o l o r c a n n a b i d i o l i n the HPLC c o l l e c t i o n volume range used. However, no peaks were seen a t the r e t e n t i o n times o f c a n n a b i n o l or c a n n a b i n o l p e n t a f l u o r o b e n z o a t e and no s i g n i f i c a n t amounts o f c a n n a b i n o l o r c a n n a b i d i o l c o u l d be d e t e c t e d as m e t a b o l i t e s o f 1 i n the dog. Thus 9

R

9

9

9



24

CANNABINOID

ANALYSIS

I N PHYSIOLOGICAL

FLUIDS

RETENTION TIME : MINUTES

Journal of Pharmaceutical Sciences Figure 6. GLC (FID) of an extract of 2 mL of plasma containing A - T H C , Peak I (200 ng/mL), and ll-hydroxy-A -THC, Peak II (200 ng/mL), before (A) and after (B) reversed-phase HPLC separation of both cannabinoids over a range of predetermined collection volumes. 9

9

(A) A 1 mL aliquot of 18 μL of extract was injected into the gas chromât ο graph prior to HPLC. (B) A 10 mL aliquot of 18 μL of the extract was injected into the liquid chromât ο graph; the collected fraction was reconstituted in 10 μL of chloroform, and 1 μL was injected into the gas chromât ο graph. Peak III is the solvent; Peak IV is an unknown from plasma. For HPLC, the column was Corasil C ; the eluent was 51 % acetonitrile in water at 1.5 mL/min. For GLC, the column was 1.5 m (5 ft) X 2 mm, 1.9% OV-225 at 245°, with a nitrogen flow of 24 mL/min. An attenuation of 8 X 10' was used for both chromatograms. The initial baselines and injection times for both chromatograms are superimposed for comparison (15). 18

12



2.

GARRETT

E T AL.


25

e i t h e r compound, when p u r i f i e d s h o u l d s e r v e as an ap propriate i n t e r n a l standard i n pharmacokinetic studies. S i n c e c a n n a b i n o l has been r e p o r t e d as a m i n o r metabo l i t e (19,20), c a n n a b i d i o l p e n t a f l u o r o b e n z o a t e was chosen as t h e i n t e r n a l s t a n d a r d . I t must be r e a l i z e d t h a t c a n n a b i n o l , now, i s known t o be a c o n t a m i n a n t o f degraded A ^ - t e t r a h y d r o c a n n a b i n o l (16,18). The GLC methodology p r e s e n t e d h e r e i n d i f f e r e d from p r i o r s t u d i e s (17) i n t h a t t h e s h o r t 30 cm column o f 3% OV-225 was s u p p l a n t e d by a l o n g e r OV-17 packed column t o be c o n s i s t e n t w i t h t h e d a t a i n t h e l i t e r a t u r e accumulated f o r t h e r e s o l u t i o n o f t h e c a n n b i n o l s (23-25) . However, t h e e f f i c i e n c i e s o f t h e s e p a r a t i o n o f t h e s e two t y p e s o f columns (OV-17 and OV-225) were compared f o r a m i x t u r e and t h e HPLC p u r i f i e d p r o d u c t s o f t h e a c i d d e g r a d a t i o n (15) o f 1 by flame i o n i z a t i o n d e t e c t i o n . The r e t e n t i o n t i m e s r o r b o t h columns a r e g i v e n i n T a b l e I and c l e a r l y show t h e s u p e r i o r i t y o f OV-225 i n peak s e p a r a t i o n o f t h e s e p a r t i c u l a r com pounds . TABLE

I

Comparison of Retention Times of a Mixture Purified Cannabinoids and Products of Acid of Different Columns* (From R e f e r e n c e

OV- -225

Compounds ΔΗ-Tetrahydrocannabinol 2

of HPLCDegradation

16)

OV-17

Ret. time Ret. time Retention r e l a t i v e Retention r e l a t i v e t i m e (min) t o 4 t i m e (min) t o 4 4.93

0.38

4.65

0.58

A -Tetrahydrocannabinol 1

5.53

0.41

5.19

0.64

C a n n a b i n o l 12

8.45

0.64

6.43

0.80

9

9,10-Dihydro9-hydroxyisocannabidiol 6

10.62

0.81

7.35

0.91

9-Hydroxyhexahydrocannabinol 4

13.14

1.00

8.07

1.00

*3% on Gas Chrom Q (100-120 mesh); column, l e n g t h 1.8m, t e m p e r a t u r e 235°; d e t e c t o r and i n j e c t o r t e m p e r a t u r e , 260O; h e l i u m f l o w , 35 ml/min; hydrogen f l o w , 30 ml/min; a i r f l o w , 300 ml/min. Journal of Pharmaceutical Sciences


26

CANNABINOID

ANALYSIS

I N PHYSIOLOGICAL

FLUIDS

APPLICATIONS S t a b i l i t y o f T e t r a h y d r o c a n n a b i n o l i n Aqueous S o l u t i o n s (16,18)


9

P r e v i o u s s t u d i e s (18) on t h e s t a b i l i t y o f Δ t e t r a h y d r o c a n n a b i n o l i n a c i d i c media below pH 4 moni t o r e d by GLC demonstrated an a p p a r e n t b i p h a s i c s e m i l o g a r i t h m i c p l o t o f undegraded 1 a g a i n s t t i m e ; e i t h e r t h e r e was formed an intermedTate w h i c h has t h e same r e t e n t i o n time as 1 t h a t a l s o gave r i s e t o t h e o b s e r v e d products o r t h e r e was a r e l a t i v e l y r a p i d e q u i l i b r a t i o n o f A 9 - t e t r a h y d r o c a n n a b i n o l 1 w i t h a n o t h e r compound and s l o w e r f u r t h e r i r r e v e r s i b l e d e g r a d a t i o n o f one o r a l l o f t h e s e compounds. (The s t u d i e s were performed w i t h C - l a b e l e d and n o n - l a b e l e d 1 ) . S i n c e t h e p o s s i b l e r e a s o n s why t h i s problem was n o t s o l v e d were t h a t t h e s p e c i f i c a c t i v i t y o r t h e c o n c e n t r a t i o n were t o o l o w , a d d i t i o n a l s t u d i e s (16) a t c o n c e n t r a t i o n s about 10 mg/1 o f HPLC p u r i f i e d 1 were c a r r i e d o u t i n 20% e t h a n o l i c s o l u t i o n s (0.1N HC1) . The GLC (OV-17) a n a l y s e s o f t h e degraded p r o d u c t s a r e summarized i n T a b l e 2. The r e t e n t i o n t i m e s o f t h e d i f f e r e n t compounds were t h e 1 4

TABLE a

GLC Characterization of Acid Purified t\ -Tetrahydrocannabinol in 0.1 Jl HC1 (from d

Retention 4.00

Percent o f Total Area 0.66

4.90

1.49

5 .44 6.66

81.53 9.62

7.43

2.57

8.43

1.55

9.18

2.16

c

2 Degradation^ of HPLCin 20% Ethanol reference 16)

Compound C a n n a b i n o l 10_ Δ^-Tetrahydrocannabinol ,2 A - T e t r a h y d r o c a n n a b i n o l 1_ 9

C a n n a b i n o l 12^ 9,10-Dihydroxy-9-hydroxyisocannabidiol £ 9-Hydroxyhexahydrocannabinol 6

£

,12-Dihydro-6-hydroxycannabidiol 8

h e l i u m , 35 ml/min; a i r , 300 ml/min; h y d r o g e n , 30 m l / min. b 100 m l 1.0 Ν HC1, 200 m l EtOH, 700 ml H 0 a t 60° f o r 30 m i n . c The t o t a l i s n o t 100% s i n c e t h e r e a r e some minor u n i d e n t i f i e d peaks. Journal of Pharmaceutical Sciences 2



2.

GARRETT

E T

AL.


27

same as f o r p u r i f i e d a u t h e n t i c m a t e r i a l s . The i d e n t i f i c a t i o n o f c a n n a b i d i o l 1£ a t r e t e n t i o n t i m e o f 4.00 m i n u t e s and 6 , 1 2 - d i h y d r o - 6 - h y d r o x y - c a n n a b i d i o l IB, a t 9.18 minutes was done by GC-mass spectrometry. Equili b r i a c o u l d t h e n be p o s t u l a t e d among Δ^-tetrahydrocann a b i n o l 1, c a n n a b i d i o l 10, 6,12-dihydro-6-hydroxycann a b i d i o l 8_ and p o s s i b l y ~ T s o c a n n a b i d i o l 9^ (Scheme I) . T h i s was c h a l l e n g e d by s u b j e c t i n g HPLC p u r i f i e d cannab i d i o l 10_ ( w i t h a A 9 - t e t r a h y d r o c a n n a b i n o l c o n t e n t l e s s t h a n 0.3%) t o d e g r a d a t i o n under t h e same c o n d i t i o n s and a s s a y i n g t h e r e s u l t a n t p r o d u c t s by GLC ( T a b l e 3 ) . The appearance o f 1, £ and $_ was c o n f i r m a t o r y . Furthermore, a k i n e t i c study o f c a n n a b i d i o l degra d a t i o n u s i n g 4-androsten-3,17-dione as an i n t e r n a l s t a n d a r d showed a b i p h a s i c d e c l i n e i n s e m i l o g a r i t h m i c p l o t t i n g a g a i n s t time ( 1 6 ) . Concomitant w i t h t h e i n i t i a l d e c l i n e i s an appearance o f A ^ - t e t r a h y d r o c a n n a b i n o l 1, u n d o u b t l y due t o a c i d - c a t a l y z e d c y c l i z a t i o n . The a l m o s t s i m u l t a n e o u s appearance o f £ can be due t o t h e r e v e r s i b l e a c i d - c a t a l y z e d hydration o f the e x o c y c l i c d o u b l e bond o f c a n n a b i d i o l 1£, o r t h e a c i d i c c l e a v a g e o f t h e e t h e r l i n k a g e o f 1^ i n t h e e q u i l i b r i u m . The r e t a r d e d appearance o f 6_ i n d i c a t e s t h a t a c i d - c a t a l y z e d h y d r a t i o n o f t h e e n d o c y c l i c bond may p r e f e r e n t i a l l y be e f f e c t e d o n l y when t h e i n t a c t e t h e r l i n k a g e o f 1^ e x i s t s . The f i n a l e q u i l i b r i a must f a v o r 4, 6_ and A - t e t r a h y d r o c a n n a b i n o l 2_ where d e h y d r a t i o n oT 4_ f a v o r s t h e Δ configuration. The f o r m a t i o n o f c a n n a b i n o l i n n i t r o g e n purged a c i d i c s o l u t i o n s was d i f f i c u l t t o e x p l a i n i n t h e absence of o x i d i z i n g a g e n t s ( 1 8 ) . The p r o b a b l e r o u t e i s by a d i s p r o p o r t i o n a t i o n o f .1 t o h e x a h y d r o c a n n a b i n o l 11^ and c a n n a b i n o l 12^. GLC a n a l y s e s (16) o f t h e c o n t e n t s o f a c i d aqueous s o l u t i o n s o f 1 ( r e a c t e d under d i f f e r e n t c o n d i t i o n s ) gave s i g n i f i c a n t a r e a s under t h e peaks a s s i g n a b l e t o h e x a h y d r o c a n n a b i n o l 11 and c a n n a b i n o l . Both peaks were p r e s e n t t o g e t h e r ; Eoth were a b s e n t when d e g r a d a t i o n s were e f f e c t e d i n HPLC p u r i f i e d w a t e r and when t h e g l a s s w a r e was p r e v i o u s l y s i l y l a t e d ( R e g i s i l ) . The use o f TLC p u r i f i e d 1_ showed b o t h p r o d u c t s 11 and 12. A l s o , when t h e w a t e r used was p r e - e x t r a c t e c T w i t h chloroform, f o r both untreated o r aqueous-silicone ( S i l i c l a d ) coated glassware, both of these products were p r e s e n t i n h i g h e v i d e n c e . Thus i t c a n be c o n c l u d e d t h a t t r a c e s o f c h l o r o f o r m i n t h e r e a c t i o n m i x t u r e and t r a c e s o f s i l i c i c a c i d from TLC p l a t e s c a t a l y z e t h e f o r m a t i o n o f f r e e r a d i c a l s and l e a d t o t h e d i s p r o p o r t i o n a t i o n o f 1. t o 11 and 12 (Scheme I I ) . The r a d i c a l Γ3 i s s t a b i l i z e d by t h e ben zene r i n g and t h e a l l y l i c a c t i v a t i o n o f t h e C-10 benz l i c hydrogen. 8

8

R

R



*

^rz ζ ι *

«

paths

reaction

reaction

hypothetical

actual paths

Scheme I

|


H

a

g

ο

§

δ

Η

Β

S

*
>

> >

η

to oo


9

— —

0.25 0.30

0.80 0.83

0.88 1.00

6.95 7.27

7.68

8.73

unknown (7 ?)

6,12-Dihydro6-hydroxyc a n n a b i d i o l 8_ 4-Androsten-3,17-dione

c

5.16

1.54

_ —

—

—. —

0.57

99.30

—

— - -

—

— —

— •-

- - •-

2. 71

—

97. 11

Journal of Pharmaceutical Sciences

ml/min; a i r , 300 ml/min. b m aqueous H C l (pH 0.3 f o r 20 h r s ; pH 1.1 f o r 3 h r s ) o r i n 0.05 M phosphate b u f f e r f o r 200 m i n . c The t o t a l i s n o t 100% s i n c e t h e r e a r e some m i n o r u n i d e n t i f i e d p e a k s .

2.02

8.13

9-Hydroxyhexahydrocannabinol £

0.70

6.10

1 .19

1.48

9,10-Dihydro9-hydroxyisocannabidiol £

3 .88

8.87

0.63

-•—

94 .71

5.48

61.71

C a n n a b i n o l 12

10.61

5.65

72.33

0.50

0.45

from

Percent of T o t a l A r e a pH 0.3 pH 1.10 pH 5.72 pH 7.03 pH 9.12

4.34

3.97

Products

(From r e f e r e n c e 16)

A -Tetrahydrocannabinol 1

9

A -Tetrahydroc a n n a b i n o l 2_

3

of Degradation pH Values^

TABLE

Ret. t i m e Retention r e l a t i v e to t i m e (min) the s t e r o i d Compounds 0.36 3.19 C a n n a b i d i o l 10 0.37 H e x a h y d r o c a n n a b i n o l 11 3.23

GLC Characterization Cannabidiol at Different

a


In Cannabinoid Analysis in Physiological Fluids; Vinson, Joe A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979. Scheme II


S

g

r

>

Ω

δ r ο ο

3

C/3

C/î

r

Β > >

Ο

3

w

>

>

Ο

03

Ο

2.

GARRETT

E T A L .


31


Equivalency o f Radiochemical Analyses o f 1 4 Ο Δ 9 T e t r a h y d r o c a n n a b i n o l and GLC E l e c t r o n C a p t u r e D e t e c t i o n o f D e r i v a t i z e d M a t e r i a l a f t e r Normal Phase HPLC i n Dog Plasma ( 1 4 ) The heptane e x t r a c t i o n e f f i c i e n c y from plasma was h i g h l y r e p r o d u c i b l e o v e r a wide range o f plasma concent r a t i o n s d - 1 0 0 ng/ml) : 9 0 . 6 ± 0 . 7 % (SEM) . The r e c o v e r y o f 1^ from t h e heptane e x t r a c t o f dog plasma by normal phase HPLC was r e p r o d u c i b l e o v e r t h e range o f plasma c o n c e n t r a t i o n s s t u d i e d . E q u i v a l e n t o v e r a l l r e c o v e r i e s were o b t a i n e d by b o t h r a d i o c h e m i c a l a n a l y s i s ( 8 3 . 7 ± 1 . 8 % SE) and e l e c t r o n - c a p t u r e GLC a n a l y s i s ( 8 4 . 0 ± 4 . 9 % SE) o f t h e d e r i v a t i z e d t e t r a h y drocannabinol . B o t h methods p e r m i t t e d e s t i m a t i o n o f a 9 2 . 5 % r e c o v e r y o f t h e amount i n t h e heptane e x t r a c t i n j e c t e d on HPLC and c o l l e c t e d i n t h e chosen r a n g e . A s i m i l a r study o f t h e r e p r o d u c i b i l i t y o f c o l l e c t i o n o f C - A - t e t r a h y d r o c a n n a b i n o l i n plasma assayed by l i q u i d s c i n t i l l a t i o n a f t e r e x t r a c t i o n and r e v e r s e phase HPLC was a l s o c o n d u c t e d . The amounts r e c o v e r e d were p r o p o r t i o n a l t o t h e amounts i n j e c t e d , and t h e HPLC r e c o v e r y e f f i c i e n c y o f t h e d r u g i n t h e heptane e x t r a c t 1 4

was

9

95.7%.

The plasma o f a dog i n t r a v e n o u s l y a d m i n i s t e r e d s o l u t i o n s o f l ^ c - A ^ - t e t r a h y d r o c a n n a b i n o l was m o n i t o r e d w i t h time a f t e r heptane e x t r a c t i o n by b o t h r a d i o c h e m i c a l a n a l y s i s and e l e c t r o n - c a p t u r e GLC o f t h e de r i v a t i v e o f the appropriately collected eluate f r a c t i o n from normal phase HPLC. T y p i c a l p l o t s o f t h e t i m e c o u r s e o f t h e r e s u l t s from b o t h methods a r e g i v e n i n Figure 7 . The p r o c e d u r e f o r GLC a n a l y s i s gave a l o w e r l i m i t f o r q u a n t i t a t i o n o f 1 i n plasma o f a p p r o x i m a t e l y 1 ng/ml from t w i c e t h e s t a n d a r d d e v i a t i o n ( 0 . 3 2 ng) o b t a i n e d f o r t h e amount o f 1^ r e c o v e r e d from 2 . 2 5 ng i n 2 ml o f plasma. S i m i l a r l y , t h e procedure f o r r a d i o c h e m i c a l a n a l y s i s gave a l o w e r l i m i t o f a p p r o x i m a t e l y 0 . 2 ng/ml from t w i c e t h e s t a n d a r d d e v i a t i o n ( 0 . 0 8 4 ng) . A s t a t i s t i c a l a n a l y s i s o f t h e a p p a r e n t d i f f e r e n c e s be tween t h e t e t r a h y d r o c a n n a b i n o l a s s a y s a t a g i v e n time from b o t h a n a l y t i c a l methods showed no s i g n i f i c a n c e . This demonstrated t h a t a l l o f the recovered r a d i o a c t i v i t y from t h e HPLC s e p a r a t i o n p r o c e d u r e c o u l d be a s s i g n e d t o 1, assayed s p e c i f i c a l l y by e l e c t r o n - c a p t u r e GLC, and thus no s i g n i f i c a n t amounts o f r a d i o l a b e l l e d m e t a b o l i t e s were i n t h e c o l l e c t e d HPLC f r a c t i o n s .


32

CANNABINOID

ANALYSIS

IN

PHYSIOLOGICAL

10

12

FLUIDS

I0J

8·

σ

Ο

Ε


(Λ

σ Ο Ι Ο

•

ω (Λ Ο

0

2

4

6

8

ο Λ

14

Ο Ο

ο

- ιο*

s.

ο,

-Ω Ο C C

ο ο ο

ο ·

οο ©

ο •

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0

1 0 0

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Minutes Journal of Pharmaceutical Sciences Figure 7. Semilogarithmic plots of fraction of the A -THC, 2.0 mg/kg dose/mL of plasma, vs. time for Dog A from (O), the liquid scintilhtion analysis of the total C collected as A -THC on normal-phase HPLC and from (Φ), the electroncapture GLC of the derivatized HPLC collected fraction. The values were cor rected for the fractions of extracts and total collection range used (15). 9

14

9


2.

GARRETT

ET

AL.

GLC

Pharmacokinetic

and HPLC Analyses

S t u d i e s i n Dogs 1 4


33

9

When plasma s u s p e n s i o n s o f C-A -tetrahydrocannabi n o l 1 were a d m i n i s t e r e d i n t r a v e n o u s l y t o t h r e e dogs a t doses o f 0.1 - 2.0 mg/kg and plasma l e v e l s o f ]L were f o l l o w e d f o r up t o 7000 m i n u t e s , no s i g n i f i c a n t d i f f e r ences were seen i n 1^ plasma l e v e l s as d e t e r m i n e d by l i q u i d s c i n t i l l a t i o n and e l e c t r o n c a p t u r e GLC a f t e r HPLC c o l l e c t i o n . The t i m e c o u r s e s f o r t h e f r a c t i o n o f t h e dose p e r ml o f plasma f o r each s t u d y was f i t by a sum o f f i v e e x p o n e n t i a l s . The f i t s were n o t s t a t i s t i c a l l y d i f f e r e n t e i t h e r among dogs o r between doses and t h e r e f o r e , no dose-dependency was c o n c l u d e d f o r t h e dose range s t u d i e d . The mean a p p a r e n t volume o f d i s t r i b u t i o n o f 1_, r e f e r e n c e d t o t o t a l d r u g i n p l a s m a , V = 1.31 ± 0.07 l i t e r s , was s l i g h t l y g r e a t e r t h a n plasma volume. The mean, o v e r a l l m e t a b o l i c c l e a r a n c e , C I = 124.0 ± 3 m l / min, was a p p r o x i m a t e l y 50% o f t h e e s t i m a t e d h e p a t i c plasma f l o w , i n d i c a t i n g t h a t b o t h unbound (3%) and plasma p r o t e i n bound (-97%) d r u g were c l e a r e d by t h e liver. The mean, f i r s t - o r d e r m e t a b o l i c r a t e c o n s t a n t , k = 0.1 ± 0.005 m i n " ( t ^ = 6.93 ± 0.3 m i n ) , demons t r a t e d t h e r a p i d m e t a b o l i s m o f 1^. R e t u r n o f 1 from t i s s u e s became t h e dominant r a t e d e t e r m i n i n g p r o c e s s a f t e r 300 m i n . The mean c a l c u l a t e d h a l f - l i f e o f !L i n p l a s m a , t ^ = 8.2 ± 0 . 2 3 d a y s , was a r e s u l t o f a slow r e t u r n o f 1 from t i s s u e s and r e s u l t e d from a s u b s t a n t i a l a c c u m u l a t i o n o f 1_ i n deep compartments. An a v e r age o f 24% o f t h e dose remained u n m e t a b o l i z e d i n t i s sues a f t e r 5 d a y s . An i n s i g n i f i c a n t amount o f 1_ (0.01%) was e l i m i n a t e d unchanged. The major f r a c t i o n o f t h e dose (40 - 45%) was e l i m i n a t e d as m e t a b o l i t e s i n f e c e s w i t h i n 5 d a y s , w i t h 14 - 16.5% e l i m i n a t e d as m e t a b o l i t e s i n u r i n e f o r t h e same i n t e r v a l . When t h e b i l e d u c t o f one o f t h e dogs was c a n n u l a t e d and no b i l e was a l l o w e d t o r e c i r c u l a t e , 55% o f t h e dose was e l i m i n a t e d as m e t a b o l i t e s i n b i l e w i t h i n 5 d a y s , i n d i c a t i n g an e n t e r o h e p a t i c r e c i r c u l a t i o n o f 10 - 15% o f t h e dose as m e t a b o l i t e s . I n t r a v e n o u s l y a d m i n i s t e r e d b i l i a r y m e t a b o l i t e s were r a p i d l y e l i m i n a t e d i n b o t h b i l e and u r i n e , s u p p o r t i n g the p r o p o s i t i o n t h a t t h e r e t u r n o f !L from t i s s u e s was the r a t e determining step process a f t e r i n i t i a l d i s tribution. c

1

M


34

CANNABINOID

ANALYSIS

IN

PHYSIOLOGICAL

FLUIDS


CONCLUSIONS I t i s c l e a r t h a t a l l r e l e v a n t physicochemical prop e r t i e s o f a d r u g s h o u l d be o b t a i n e d p r i o r t o i n i t i a t i n g d e t a i l e d pharmacokinetic i n v e s t i g a t i o n s (3). The e x t r e m e l y low s o l u b i l i t y o f 1^ (2.8 mg/1 i n w a t e r and 0.77 mg/1 i n 0.15 M N a C l a t 23°) must c e r t a i n l y a f f e c t i t s b i o a v a i l a b i l i t y on o r a l d o s i n g o f amounts w h e r e i n t h e s o l u b i l i t y i s exceeded. Tetrahy drocannabinol i n excess of i t s s o l u b i l i t y i n s t a n t a n e o u s l y forms a s t a b l e e m u l s i o n o r m i c e l l a r d i s p e r s i o n . Advantage can be t a k e n o f the h i g h p r o t e i n b i n d i n g o f _1 to a d m i n i s t e r p l a s m a - s o l u b i l i z e d s o l u t i o n s o f _1 i n t r a venously. The r a p i d d i f f u s i o n o f Δ^-tetrahydrocannabinol i n t o the p l a s t i c o f c o n t a i n e r s and i n t o the r u b b e r s t o p p e r s n o r m a l l y used as c l o s u r e s f o r plasma v i a l s (70 - 96%) and the s i g n i f i c a n t b i n d i n g t o g l a s s a t low t e t r a h y d r o c a n n a b i n o l c o n c e n t r a t i o n s (20 and 40% a t 0.1 and 0.05 yg/ml, r e s p e c t i v e l y , i n 50 ml v o l u m e t r i c f l a s k s ) d e f i n i t e l y demand c a r e f u l t e c h n i q u e s i n the h a n d l i n g , s t o r a g e , and a s s a y o f t h i s compound from aqueous and biological fluids. I n f a c t , the r e s u l t s o f any p e r t i n e n t s t u d y where t h e s e c o n d i t i o n s were n o t h e l d i n a c c o u n t s h o u l d be q u a n t i t a t i v e l y s u s p e c t . The h i g h degree o f p a r t i t i o n i n t o l i p i d phases and o f a b s o r p t i o n t o a l l and any s u r f a c e by t e t r a h y d r o c a n n a b i n o l ( l i p o p r o t e i n b i n d i n g may be i n c l u d e d i n these c a t e g o r i e s ) i m p l i e s t h a t o r a l a d m i n i s t r a t i o n of the d r u g i n l i p i d v e h i c l e s t h a t a r e r e l a t i v e l y immis c i b l e w i t h aqueous f l u i d s would d r a s t i c a l l y reduce the b i o a v a i l a b i l i t y o f t h e d r u g . A common p r a c t i c e o f ad m i n i s t e r i n g t e t r a h y d r o c a n n a b i n o l admixed w i t h l i p i d c o n t a i n i n g f e e d i n a n i m a l e x p e r i m e n t s would c a s t s u s p i c i o n on t h e d o s e - r e s p o n s e r e l a t i o n s h i p s p r o p o s e d from the r e s u l t s o f such s t u d i e s . The h i g h s e q u e s t e r i n g and b i n d i n g o f A - t e t r a h y d r o c a n n a b i n o l a r e c o n s i s t e n t w i t h the p r o p o s a l o f a p h a r m a c o k i n e t i c a l l y deep compartment, e s p e c i a l l y w i t h the known r a p i d i t y o f i t s m e t a b o l i s m . The p r o l o n g e d b u t lowered b l o o d l e v e l s o f a d m i n i s t e r e d d r u g (17) must be r a t i o n a l i z e d by i t s slow r a t e - d e t e r m i n i n g r e l e a s e from such compartments. The l a c k o f s i g n i f i c a n t r e n a l e x c r e t i o n o f unchanged d r u g ( 1 7 ) i s r e a d i l y u n d e r s t a n d able since i t s high l i p o p h i l i c i t y should r e s u l t i n complete t u b u l a r r e a b s o r p t i o n . The p a r t i t i o n i n g o f t e t r a h y d r o c a n n a b i n o l from p l a s ma w a t e r i n t o r e d b l o o d c e l l s i s enormously h i g h s i n c e D = 12.5. T h i s may a l s o be a s c r i b e d t o t h e h i g h s u r 9



2.

GARRETT

ET

AL.

GLC

and

35

HPLC Analyses

f a c e a f f i n i t y o f the d r u g . However, t h e c o m p e t i t i o n o f a l a r g e degree o f u n s a t u r a b l e b i n d i n g (97%) t o plasma p r o t e i n s m i n i m i z e s t h e amounts i n t h e r e d b l o o d c e l l s o f whole b l o o d , a l t h o u g h p o s s i b l y l a r g e adherence o f t h e d r u g t o t h e w a l l s o f b l o o d v e s s e l s and t o t h e s u r f a c e s o f t h e t i s s u e s must be a n t i c i p a t e d . The f a c t t h a t the plasma b i n d i n g i s l a r g e l y a s s i g n e d t o t h e l i p o p r o t e i n f r a c t i o n (14) may r e s u l t i n l a r g e i n d i v i d u a l and s p e c i e s v a r i a t i o n s i n l i p o p r o t e i n and f a t cont e n t . Thus, the p r o p o s e d method (7) o f v a r i a b l e plasma c o n c e n t r a t i o n may be u s e f u l i n t h e r o u t i n e d e t e r m i n a t i o n of p r o t e i n b i n d i n g of i n d i v i d u a l s u b j e c t s . The l a r g e i n s t a b i l i t y o f A - t e t r a h y d r o c a n n a b i n o l i n a c i d s o l u t i o n (16,18), i m p l i e s t h a t the d r u g may be s i g n i f i c a n t l y degraded i n the normal stomach. Again, t h i s i n t i m a t e s t h a t o r a l a d m i n i s t r a t i o n may n o t be an optimum r o u t e on w h i c h t o e s t a b l i s h d o s e - r e s p o n s e c o r r e l a t i o n s . Furthermore, the c h o i c e of a cannabinoid as an i n t e r n a l s t a n d a r d i s v e r y c r i t i c a l s i n c e i t must n o t g i v e any i n t e r f e r e n c e w i t h t h e o t h e r r e l a t e d compounds, u n l e s s a HPLC p u r i f i c a t i o n s t e p i s i n c l u d e d i n the p r o c e d u r e . C a n n a b i n o l w h i c h , a t t i m e s , has been t a k e n as a p o s s i b l e m e t a b o l i t e (19-21) o f A ^ - t e t r a h y d r o c a n n a b i n o l , may o n l y be an a r t i f a c t o f the a n a l y t i c a l procedure s i n c e d i s p r o p o r t i o n a t i o n of 1 occurs readily. - T e t r a h y d r o c a n n a b i n o l can be e x t r a c t e d from plasma and o t h e r b i o l o g i c a l f l u i d s . I t can be separ a t e d on HPLC from t h e s i m u l t a n e o u s l y e x t r a c t e d b i o l o g i c a l l y endogenous m a t e r i a l s and m e t a b o l i t e s t h a t would i n t e r f e r e w i t h a chosen h i g h l y s e n s i t i v e a n a l y t i c a l method, such as e l e c t r o n - c a p t u r e gas l i q u i d chromatography. I t i s not necessary t o c o l l e c t a l l the materi a l t o be a n a l y z e d ; a s s u r a n c e t h a t a r e p r o d u c i b l e o r known f r a c t i o n o f the t o t a l m a t e r i a l i n j e c t e d on HPLC i s recovered i s a l l t h a t i s necessary s i n c e i t i s d i r e c t l y p r o p o r t i o n a l to the t o t a l drug c o n c e n t r a t i o n . I f u n l a b e l l e d tetrahydrocannabinol i n a s o l u t i o n of plasma were a n a l y z e d , the c a l c u l a t e d r e c o v e r y o f known amounts o f l a b e l l e d t e t r a h y d r o c a n n a b i n o l added e i t h e r t o plasma p r i o r t o e x t r a c t i o n o r t o heptane e x t r a c t subsequent t o e x t r a c t i o n would p e r m i t c a l c u l a t i o n o f the e x t r a c t i o n and/or HPLC c o l l e c t i o n e f f i c i e n c i e s f o r t h a t p a r t i c u l a r b i o l o g i c a l sample. These known e f f i c i e n c i e s would p e r m i t t h e c a l c u l a t i o n o f the o r i g i n a l plasma c o n c e n t r a t i o n s . I f a l a b e l l e d C-A -tetrahyd r o c a n n a b i n o l were used i n p h a r m a c o k i n e t i c s t u d i e s , e x t r a c t e d and s e p a r a t e d on the HPLC, a t r i t i u m - l a b e l l e d H - A - t e t r a h y d r o c a n n a b i n o l c o u l d be used as t h e approp r i a t e i n t e r n a l standard to monitor recover e f f i c i e n cies . 9

1 4

3

9

9


36

CANNABINOID ANALYSIS IN PHYSIOLOGICAL FLUIDS

F i n a l l y , t h e c o m b i n e d u s e o f HPLC t e c h n i q u e s a n d GLC m e t h o d o l o g i e s h a v e g i v e n t h e same r e s u l t a s t h e r a d i o a c t i v i t y monitoring during the pharmacokinetic s t u d i e s , b a s e d upon t h e knowledge o f t h e p h y s i o c o c h e m i cal properties of Δ9-tetrahydrocannabinol.


ACKNOWLEDGMENTS S u p p o r t e d i n p a r t b y g r a n t s I R 0 3 MH 1 9 2 6 8 , D A 00743-01 and DA-0073 from t h e N a t i o n a l I n s t i t u t e o f M e n t a l H e a l t h , B e t h e s d a , M d . 20014 a n d i n p a r t b y a NATO g r a n t f o r o n e o f u s ( A J G ) . REFERENCES

(1)

(2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13)

(14)

(a) (b)

To whom i n q u i r i e s should be d i r e c t e d ; P o s t d o c t o r a l f e l l o w from C.E.R.C.O.A. -C.N.R.S., 2 ā 8, Avenue H e n r i , Dunant, 94320, T h i a i s (France); (c) School o f Pharmacy, U n i v e r s i t y o f Califor nia, San F r a n c i s c o , CA. 94143. Braude, M. C., Acta Pharm. Suecica 8, 674 (1971). G a r r e t t , E . R . , Int. J. Clin. Pharmacol. Ther. Toxicol. 4, 6 (1970). Lemberger, L., Silberstein, S. D., A x e l r o d , D., and I. J. Kopin, Science 170, 1320 (1970). Lemberger, L., Tamarkin, N . R., A x e l r o d , J., and I. J. Kopin, Science 173, 72, (1971). Lemberger, L., Crabtree, R. E., and Η. M. Rowe, Science 177, 62 (1972). G a r r e t t , E . R., and Hunt, C . Α . , J. Pharm. Sci. 63, 1056 (1974). Freudenthal, R. I., M a r t i n , J., and W a l l , M . E., Brit. J. Pharmacol. 44, 244 (1972). Borgen, L. Α . , and Davis, W. M., J. Pharm. Sci. 62, 479 (1973). Peres-Reyes, M., L i p t o n , Μ. Α., Timmons, M. C., W a l l , M. E., B r i n e , D. R., and Davis, Κ. H., Clin. Pharmacol. Ther. 14, 48 (1973). Saad, Η. Υ . , and H i g u c h i , W. I., J. Pharm. Sci. 54, 1205 (1965). M a r t i n , A . N., Swarbrick, J., and Cammarata, Α . , " P h y s i c a l Pharmacy", Lea and F e b i g e r , P h i l a d e l p h i a , Pa., 1969, p. 237. Kortum, G., V o g e l , W., and Andrussow, K., " D i s s o c i a t i o n Constants o f Organic Acids in Aqueous S o l u t i o n " , I n t e r n a t i o n a l Union of Pure and A p p l i e d Chemistry, Butterworth, London, England, 1961. Wahlquist, M., N i l s s o n , I. M., Sandberg, F., and A g u r e l l , S . , Biochem. Pharmacol. 19, 2579 (1970).


2.

GARRETT

(15) (16) (17)


(18) (19) (20) (21) (22) (23) (24)

E T A L .


37

G a r r e t t , E . R., and Hunt, C. Α . , J. Pharm. Sci. 66, 20 (1977). G a r r e t t , E . R . , Gouyette, A . J., and Roseboom, H., J. Pharm. Sci., submitted (1977). G a r r e t t , E . R . , and Hunt, C. Α . , J. Pharm. Sci. 62, 1211 (1973). G a r r e t t , E . R . , and Tsau, T . , J. Pharm. Sci. 63, 1563 (1974). McCallum, Ν. Κ., J. Chromatogr. 11, 509 (1973). McCallum, Ν. Κ . , Yagen, Β . , Levy, S . , and Mechoulam, R . , Experientia 31, 520 (1975). Widman, M., Nordquist, M., Agurell, S . , L i n d g r e n , J. E . and Sandberg, F., Biochem. Pharmacol. 23, 163 (1974). Fetterman, R. S . , and Turner, C . E., J. Pharm. Sci. 61, 1476 (1972). Turner, C . E., and Hadley, K . W., J. Pharm. Sci. 62, 251 and 1083 (1973). Turner, C . E., Hadley, K . W., Henry, J., and Mole, M. L., J. Pharm. Sci. 63, 1872 (1974).

RECEIVED

December 12, 1978.


Cannabinoid Analysis in Physiological Fluids - American Chemical

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