Life Sciences, Vol. 51, pp. 1823-1832 Printed in the USA
THE
ACCUMULATION IN RAT HAIR
Andrew
P.
Pergamon Press
AND DISAPPEARANCE OF COCAINE AND FOLLOWING PROLONGED ADMINISTRATION
F e r k o I, E d w a r d J. B a r b i e r i , and E i l e e n K. R u c h
G.J.
BENZOYLECGONINE OF COCAINE
DiGregorio,
D e p a r t m e n t of P h a r m a c o l o g y H a h n e m a n n U n i v e r s i t y , S c h o o l of M e d i c i n e Philadelphia, Pennsylvania (Received in final form September 30, 1992)
summary H a i r s a m p l e s w e r e o b t a i n e d at v a r i o u s t i m e p e r i o d s f r o m male Sprague-Dawley rats following the injection of c o c a i n e h y d r o c h l o r i d e in d o s e s of 5, i0, a n d 20 mg/kg, ip, for 28 days. Hair samples were also taken continually a f t e r t h e d o s i n g w a s s t o p p e d u n t i l t h e p r e s e n c e of c o c a i n e and benzoylecgonine w e r e no l o n g e r detected in hair. Cocaine and benzoylecgonine in h a i r and plasma were a n a l y z e d by gas c h r o m a t o g r a p h y / m a s s spectrometry. Both c o c a i n e and b e n z o y l e c g o n i n e w e r e f o u n d in h a i r s a m p l e s 4 d a y s a f t e r t h e i n i t i a t i o n of c o c a i n e a d m i n i s t r a t i o n . When c o c a i n e d o s i n g w a s s t o p p e d a f t e r 28 days, a p p r o x i m a t e l y 25 to 30 d a y s w e r e r e q u i r e d for c o c a i n e and b e n z o y l e c g o n i n e to d i s a p p e a r f r o m rat h a i r in the g r o u p of a n i m a l s t h a t r e c e i v e d t h e h i g h e s t d o s e of cocaine. The disappearance of cocaine and benzoylecgonine followed first-order kinetics. T h e m e a n r a t e c o n s t a n t a n d m e a n h a l f - l i f e for c o c a i n e d i s a p p e a r a n c e f r o m h a i r w e r e 0.212 ± 0.005 d a y -I a n d 3.31 ± 0.09 days, r e s p e c t i v e l y , and the mean rate constant and mean half-life for benzo~lecgonine d i s a p p e a r a n c e f r o m h a i r w e r e 0.098 ± 0.006 d a y -± and 6.90 ± 0.28 days, r e s p e c t i v e l y . The mean plasma concentrations of c o c a i n e on D a y 25 for t h e 5, i0, a n d 20 m g / k g d o s e s of c o c a i n e w e r e 508 ± 42, 852 ± 95, a n d 2027 ± 75 ng/mL, respectively, and the mean plasma benzoylecgonine levels for t h e 5, i0, a n d 20 m g / k g d o s e s of c o c a i n e w e r e 49.9 ± 7.0, 103.3 ± 9.3, and 191.0 ± 16.0 ng/mL, r e s p e c t i v e l y . There was a positive correlation b e t w e e n the d o s e s of cocaine hydrochloride a d m i n i s t e r e d and t h e p l a s m a l e v e l s of b o t h c o c a i n e a n d b e n z o y l e c g o n i n e . This study showed t h a t c o c a i n e and b e n z o y l e c g o n i n e c a n be m e a s u r e d in rat h a i r f o l l o w i n g the a d m i n i s t r a t i o n of c o c a i n e a n d t h a t it w a s p o s s i b l e to c o r r e l a t e t h e c o n c e n t r a t i o n s of c o c a i n e and benzoylecgonine found in h a i r w i t h the doses of cocaine that were administered.
Icorrespondence to: A n d r e w P. Ferko, Ph.D., Department of P h a r m a c o l o g y , H a h n e m a n n U n i v e r s i t y S c h o o l of M e d i c i n e , P h i l a d e l p h i a , Pennsylvania 19102. 0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd All rights reserved.
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C o c a i n e is a c e n t r a l n e r v o u s s y s t e m s t i m u l a n t t h a t is w i d e l y a b u s e d in o u r s o c i e t y and has become a major public health problem. E f f o r t s h a v e b e e n m a d e to i d e n t i f y i n d i v i d u a l s w h o are c o c a i n e users. S e l f - r e p o r t e d d r u g h i s t o r i e s by t h e s e i n d i v i d u a l s m a y be unreliable. A n a l y s i s of t h e i r b l o o d a n d u r i n e s h o w o n l y r e c e n t c o c a i n e use, s i n c e c o c a i n e and its m e t a b o l i t e s a r e e l i m i n a t e d w i t h i n h o u r s or days, d e p e n d i n g on t h e a m o u n t of c o c a i n e t h a t w a s t a k e n (i, 2). R e p o r t s h a v e a p p e a r e d in t h e l i t e r a t u r e t h a t t h e a n a l y s i s of h a i r m a y m o r e a c c u r a t e l y r e f l e c t t h e u s a g e of c o c a i n e by an i n d i v i d u a l , s i n c e b e n z o y l e c g o n i n e a n d c o c a i n e are d e p o s i t e d in h a i r a n d a p p e a r to r e m a i n in t h e h a i r (3-5). One r e p o r t (6) h a s i n d i c a t e d t h a t c o c a i n e c o n c e n t r a t i o n s in h a i r f r o m c o c a i n e a d d i c t s w e r e 0.6 to 6.4 n g / m g of h a i r a n d a f t e r 3 m o n t h s of a b s t i n e n c e f r o m t h e d r u g by t h e s e s u b j e c t s t h e level of c o c a i n e w a s 0.3 to 0.6 n g / m g of hair. C o c a i n e a n d b e n z o y l e c g o n i n e are a l s o r e p o r t e d to be f o u n d in s h e e p (6), rat (7), a n d m o u s e (8) hair. O n l y one study, in w h i c h a n i m a l s w e r e i n j e c t e d w i t h 2 m g / k g of cocaine, m o n i t o r e d t h e a c c u m u l a t i o n of c o c a i n e , b u t n o t b e n z o y l e c g o n i n e , in h a i r o v e r 40 d a y s (6). W e are u n a w a r e of a n y p u b l i s h e d i n v e s t i g a t i o n s in w h i c h t h e a c c u m u l a t i o n a n d d i s a p p e a r a n c e of b o t h c o c a i n e and b e n z o y l e c g o n i n e w e r e e x a m i n e d in h a i r f o l l o w i n g t h e a d m i n i s t r a t i o n of v a r i o u s d o s e s of cocaine. It w a s t h e i n t e n t of t h i s s t u d y to add f u r t h e r i n f o r m a t i o n on t h e p r e s e n c e of c o c a i n e and b e n z o y l e c g o n i n e in h a i r by t h e u s e of an a n i m a l model. R e c e n t i n v e s t i g a t i o n s in o u r l a b o r a t o r y h a v e u s e d t h e r a t to d e t e r m i n e t h e p r e s e n c e of c o c a i n e a n d b e n z o y l e c g o n i n e in p a r o t i d saliva, plasma, and u r i n e as w e l l as the d e p o s i t i o n of c o c a i n e a n d b e n z o y l e c g o n i n e in p r e g n a n t and n o n p r e g n a n t f e m a l e r a t s (9, i0). T h e p r e s e n t s t u d y w a s d e s i g n e d to a s c e r t a i n w h e t h e r the concentrations of c o c a i n e and benzoylecgonine in r a t h a i r are r e l a t e d to t h e d o s e s of c o c a i n e t h a t are a d m i n i s t e r e d . In a d d i t i o n , the d i s a p p e a r a n c e of c o c a i n e and b e n z o y l e c g o n i n e from hair was m e a s u r e d a f t e r t h e d i s c o n t i n u a t i o n of c o c a i n e t r e a t m e n t s .
Methods Animals. M a l e S p r a g u e - D a w l e y r a t s (150 - 180 g) w e r e o b t a i n e d f r o m Charles River Laboratories, Inc. ( W i l m i n g t o n , MA) a n d w e r e h o u s e d for 1 w e e k p r i o r to e x p e r i m e n t a t i o n at 22 ± 1 °C w i t h a l i g h t c y c l e f r o m 6:00 A M to 6:00 PM. T h e a n i m a l s h a d f r e e a c c e s s to P u r i n a R o d e n t L a b o r a t o r y C h o w 5001 (Ralston P u r i n a Co., St. Louis, MO) and water.
Cocaine Experiments.
Animals were injected intraperitoneally with c o c a i n e h y d r o c h l o r i d e (5, i0 or 20 mg/kg) for 20 d a y s o v e r a 2 8 - d a y period. T h e d r u g w a s a d m i n i s t e r e d on M o n d a y t h r o u g h F r i d a y ; no d o s i n g o c c u r r e d on w e e k e n d s ; t h e f i r s t i n j e c t i o n w a s g i v e n on D a y 1 (a T u e s d a y ) a n d t h e last on D a y 28 (a M o n d a y ) . Hair samples were r e m o v e d w i t h an e l e c t r i c s h a v e r f r o m a s m a l l a r e a on t h e b a c k s of t h e a n i m a l s t w i c e a week, b e g i n n i n g on D a y 4 a n d c o n t i n u i n g e v e r y T u e s d a y a n d F r i d a y u n t i l c o c a i n e a n d b e n z o y l e c g o n i n e w e r e no l o n g e r d e t e c t e d in r a t h a i r (Day 53); h a i r s a m p l e s w e r e n e v e r t a k e n f r o m a p r e v i o u s l y s h a v e d area. A p p r o x i m a t e l y 50 m g of h a i r w a s u s e d f r o m e a c h s a m p l e a n d a n a l y z e d for c o c a i n e a n d b e n z o y l e c g o n i n e by gas chromatography/mass s p e c t r o m e t r y (GC/MS). B l o o d s a m p l e s (300 ~L), t a k e n f r o m t h e o r b i t a l s i n u s of t h e r a t s on D a y s 25, 32, 39, a n d 46 w e r e a n a l y z e d by GC/MS. T h r e e r a t s w e r e h o u s e d p e r c a g e in t h i s
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experiment. In a n o t h e r experiment, all a n i m a l s w e r e h o u s e d individually in metabolic c a g e s so t h a t u r i n e samples, uncontaminated by feces, could be o b t a i n e d . These animals were injected with cocaine h y d r o c h l o r i d e , 20 mg/kg, ip, as w a s d o n e in t h e e x p e r i m e n t d e s c r i b e d above, e x c e p t t h a t o n l y i0 i n j e c t i o n s w e r e g i v e n a n d t h e last d o s e of c o c a i n e w a s g i v e n on Day 14. Hair samples were obtained during t h i s e x p e r i m e n t in t h e s a m e m a n n e r and t i m e p e r i o d s as p r e v i o u s l y d e s c r i b e d , up to D a y 15. S a m p l e Preparation and Analysis. In o r d e r to p r e v e n t t h e loss of c o c a i n e d u e to h y d r o l y s i s , all c o l l e c t i n g t u b e s for b l o o d and u r i n e s a m p l e s c o n t a i n e d 30 m g of s o d i u m f l u o r i d e (11). The collecting t u b e s for b l o o d a l s o c o n t a i n e d 30 u n i t s of s o d i u m h e p a r i n to p r e v e n t c o a g u l a t i o n of blood. T h e b l o o d s a m p l e w a s c e n t r i f u g e d at 2000 x g for 10 m i n and p l a s m a removed. For t h e a n a l y s i s of c o c a i n e and benzoylecgonine, 100 ~ L of p l a s m a a n d u r i n e w e r e u s e d (9, 10). Cocaine and benzoylecgonine were extracted from hair by a modification of t h e p r o c e d u r e s described by V a l e n t e et al (3). A f t e r w a s h i n g t h e h a i r s a m p l e w i t h d i s t i l l e d w a t e r to r e m o v e a n y possible external contamination on the h a i r shaft, the s a m p l e of h a i r ( a p p r o x i m a t e l y 50 m g d r y weight) w a s i n c u b a t e d o v e r n i g h t w i t h 1 m L of 0.I M h y d r o c h l o r i c a c i d at 45 °C. T h e a c i d e x t r a c t was n e u t r a l i z e d w i t h i00 ~ L of 1 N s o d i u m h y d r o x i d e s o l u t i o n a n d 0.9 m L of 0.05 M p h o s p h a t e b u f f e r solution. T h e i n t e r n a l s t a n d a r d for the GC/MS analysis, scopolamine (50 ~ L of a i0 ~ g / m L s o l u t i o n (or 500 ng)) w a s a d d e d to t h e m i x t u r e a n d t h e e n t i r e s o l u t i o n w a s e l u t e d on a c o n d i t i o n e d C-18 e x t r a c t i o n column. Cocaine and benzoylecgonine w e r e r e m o v e d f r o m t h e c o l u m n by 600 ~ L of a c i d i c m e t h a n o l . The e l u a n t w a s d r i e d at 70 °C u n d e r a n i t r o g e n a t m o s p h e r e . A f t e r the centrifuge tube cooled to r o o m t e m p e r a t u r e , derivatization was accomplished by the addition of 50 ~L of silylation grade acetonitrile and i00 ~L of N-methyl-N-trimethylsilyltrifluoroa c e t a m i d e (MSTFA, P i e r c e C h e m i c a l Co., R o c k f o r d , IL) and h e a t i n g the centrifuge t u b e for 15 m i n at 70 °C (Taylor a n d Jain, 1987). F o l l o w i n g t h e c o o l i n g of the sample, 1 ~ L w a s i n j e c t e d into a gas chromatograph/mass spectrometer system for identification and quantitation of c o c a i n e and benzoylecgonine. All reagents and s o l v e n t s u s e d in t h e e x t r a c t i o n p r o c e d u r e w e r e of a n a l y t i c a l grade. The a n a l y s i s for c o c a i n e and b e n z o y l e c g o n i n e in blood, urine, and h a i r w a s d o n e by GC/MS. T h e G C / M S s y s t e m w a s a V a r i a n 3400 gas chromatograph/Finnegan M A T ion t r a p d e t e c t o r (ITD). The capillary column was a J&W DB-5 bonded-phase, 30 m, 0.25 m m id., a n d 0.25 micron coating, coupled to t h e ITD t h r o u g h a direct capillary interface. The injection port was a capillary split/splitless i n j e c t o r (in t h e s p l i t l e s s mode) w i t h a s i l a n i z e d g l a s s insert. The c a r r i e r gas, helium, w a s at a f l o w r a t e of 1.0 m L / m i n t h r o u g h the column. The temperature p r o g r a m w a s 170 °C w i t h a 1 m i n hold, i n c r e a s i n g to 250 °C at 30 ° C / m i n w i t h a 6 m i n hold. The injector a n d i n t e r f a c e t e m p e r a t u r e s w e r e 250 °C each. T h e ITD S c a n M o d e w a s a full scan, 72 to 190 ~ and at one s c a n / s e c . Compounds were identified by l i b r a r y search (Finnegan-Toxicology) of t h e m a s s s p e c t r u m t o g e t h e r w i t h r e t e n t i o n time. S t a n d a r d s of c o c a i n e and b e n z o y l e c g o n i n e w e r e o b t a i n e d f r o m the R e s e a r c h T e c h n o l o g y B r a n c h of NIDA (Rockville, MD). Cocaine and the MSTFA derivative of benzoylecgonine exhibited retention times of 448 and 482 sec,
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respectively. The internal standard, the M S T F A derivative of scopolamine, h a d a r e t e n t i o n t i m e of 538 sec on t h e c h r o m a t o g r a m . T h e l o w e s t s e n s i t i v i t y of the m e t h o d for c o c a i n e and b e n z o y l e c g o n i n e is i00 n g / g w h e n 50 m g of a h a i r s a m p l e is u s e d and the c o r r e l a t i o n c o e f f i c i e n t is 0.22. Calculations and S t a t i s t i c s . The pharmacokinetic parameters for cocaine and benzoylecgonine were determined using standard pharmacokinetic mathematical calculations as u s e d by K h a n et al (12). First-order disappearance d a t a w e r e g r a p h e d by the least s q u a r e method. Statistically significant differences in g r o u p e d d a t a w e r e d e t e r m i n e d by an a n a l y s i s of v a r i a n c e (ANOVA). Multiple comparisons of g r o u p s were analyzed by A N O V A followed by the S c h e f f e ' s Test. Results
F i g u r e s 1 shows the a c c u m u l a t i o n and the d i s a p p e a r a n c e of c o c a i n e t h a t was f o u n d in rat h a i r d u r i n g the 5 3 - d a y e x p e r i m e n t a l period. In general, c o c a i n e a p p e a r e d to c o n t i n u e to a c c u m u l a t e in the h a i r u n t i l a p p r o x i m a t e l y D a y 18, r e m a i n e d at a s t e a d y - s t a t e level f r o m Days 18 to 29, e x c e p t for t h e h a i r c o c a i n e c o n c e n t r a t i o n s w h e n the
7,
7000
6O0O A
o
4ooo
o
rr 2ooo
lOOO
4
8
12
16
20
24
28
32
TIME ( days
FIG.
36
40
44
48
52
56
60
)
1
T h e a p p e a r a n c e and d i s a p p e a r a n c e of c o c a i n e in rat h a i r f o l l o w i n g t h e ip i n j e c t i o n of c o c a i n e h y d r o c h l o r i d e in d o s e s of 5 ( : : ), i0 ( 0 .... 0 ), and 20 ( 0 0 ) mg/kg.
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20 m g / k g d o s e of c o c a i n e w a s g i v e n , a n d t h e n t h e c o n c e n t r a t i o n of d r u g b e g a n to f a l l as t h e d o s i n g w a s d i s c o n t i n u e d . Using the data f r o m D a y 29 as an e x a m p l e , t h e m e a n ± SE c o n c e n t r a t i o n s of c o c a i n e w e r e 1752 ± iii, 2 5 6 8 ± 174, a n d 4 5 1 3 ± 185 n g / g of h a i r w h e n 5, i0 or 20 m g / k g of c o c a i n e h y d r o c h l o r i d e was administered, respectively. These data were all significantly different from each other (p < 0.05) a n d t h e r e w a s a s t a t i s t i c a l l y significant correlation between t h e d o s e s of c o c a i n e h y d r o c h l o r i d e administered and the hair cocaine concentrations t h a t w e r e a s s a y e d (r = 0.99, p < 0 . 0 1 ) .
The a p p e a r a n c e and d i s a p p e a r a n c e of b e n z o y l e c g o n i n e in rat hair samples from the same animals is shown in Figure 2; the pattern of the c o n c e n t r a t i o n s of this cocaine m e t a b o l i t e in hair was similar to that o b s e r v e d with cocaine itself, as d e s c r i b e d above. Again, using the data from Day 29, the mean ± SE levels of b e n z o y l e c g o n i n e achieved by the a d m i n i s t r a t i o n of 5, i0, and 20 mg/kg of cocaine h y d r o c h l o r i d e were 354 ± 25, 437 ± 17, and 687 ± 42 ng/g of hair, respectively. These data were all s i g n i f i c a n t l y d i f f e r e n t from each other (p < 0.05); the c o r r e l a t i o n coefficient (r = 0.99) between the doses of cocaine hydrochloride and the concentrations of b e n z o y l e c g o n i n e in the hair was highly significant (p .--4) 1o m~Jkg ~.,.....@
20
0o,.
coc.,~.
¢
5 mg/kg
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z
THE
ACCUMULATION IN RAT HAIR
Andrew
P.
Pergamon Press
AND DISAPPEARANCE OF COCAINE AND FOLLOWING PROLONGED ADMINISTRATION
F e r k o I, E d w a r d J. B a r b i e r i , and E i l e e n K. R u c h
G.J.
BENZOYLECGONINE OF COCAINE
DiGregorio,
D e p a r t m e n t of P h a r m a c o l o g y H a h n e m a n n U n i v e r s i t y , S c h o o l of M e d i c i n e Philadelphia, Pennsylvania (Received in final form September 30, 1992)
summary H a i r s a m p l e s w e r e o b t a i n e d at v a r i o u s t i m e p e r i o d s f r o m male Sprague-Dawley rats following the injection of c o c a i n e h y d r o c h l o r i d e in d o s e s of 5, i0, a n d 20 mg/kg, ip, for 28 days. Hair samples were also taken continually a f t e r t h e d o s i n g w a s s t o p p e d u n t i l t h e p r e s e n c e of c o c a i n e and benzoylecgonine w e r e no l o n g e r detected in hair. Cocaine and benzoylecgonine in h a i r and plasma were a n a l y z e d by gas c h r o m a t o g r a p h y / m a s s spectrometry. Both c o c a i n e and b e n z o y l e c g o n i n e w e r e f o u n d in h a i r s a m p l e s 4 d a y s a f t e r t h e i n i t i a t i o n of c o c a i n e a d m i n i s t r a t i o n . When c o c a i n e d o s i n g w a s s t o p p e d a f t e r 28 days, a p p r o x i m a t e l y 25 to 30 d a y s w e r e r e q u i r e d for c o c a i n e and b e n z o y l e c g o n i n e to d i s a p p e a r f r o m rat h a i r in the g r o u p of a n i m a l s t h a t r e c e i v e d t h e h i g h e s t d o s e of cocaine. The disappearance of cocaine and benzoylecgonine followed first-order kinetics. T h e m e a n r a t e c o n s t a n t a n d m e a n h a l f - l i f e for c o c a i n e d i s a p p e a r a n c e f r o m h a i r w e r e 0.212 ± 0.005 d a y -I a n d 3.31 ± 0.09 days, r e s p e c t i v e l y , and the mean rate constant and mean half-life for benzo~lecgonine d i s a p p e a r a n c e f r o m h a i r w e r e 0.098 ± 0.006 d a y -± and 6.90 ± 0.28 days, r e s p e c t i v e l y . The mean plasma concentrations of c o c a i n e on D a y 25 for t h e 5, i0, a n d 20 m g / k g d o s e s of c o c a i n e w e r e 508 ± 42, 852 ± 95, a n d 2027 ± 75 ng/mL, respectively, and the mean plasma benzoylecgonine levels for t h e 5, i0, a n d 20 m g / k g d o s e s of c o c a i n e w e r e 49.9 ± 7.0, 103.3 ± 9.3, and 191.0 ± 16.0 ng/mL, r e s p e c t i v e l y . There was a positive correlation b e t w e e n the d o s e s of cocaine hydrochloride a d m i n i s t e r e d and t h e p l a s m a l e v e l s of b o t h c o c a i n e a n d b e n z o y l e c g o n i n e . This study showed t h a t c o c a i n e and b e n z o y l e c g o n i n e c a n be m e a s u r e d in rat h a i r f o l l o w i n g the a d m i n i s t r a t i o n of c o c a i n e a n d t h a t it w a s p o s s i b l e to c o r r e l a t e t h e c o n c e n t r a t i o n s of c o c a i n e and benzoylecgonine found in h a i r w i t h the doses of cocaine that were administered.
Icorrespondence to: A n d r e w P. Ferko, Ph.D., Department of P h a r m a c o l o g y , H a h n e m a n n U n i v e r s i t y S c h o o l of M e d i c i n e , P h i l a d e l p h i a , Pennsylvania 19102. 0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd All rights reserved.
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C o c a i n e is a c e n t r a l n e r v o u s s y s t e m s t i m u l a n t t h a t is w i d e l y a b u s e d in o u r s o c i e t y and has become a major public health problem. E f f o r t s h a v e b e e n m a d e to i d e n t i f y i n d i v i d u a l s w h o are c o c a i n e users. S e l f - r e p o r t e d d r u g h i s t o r i e s by t h e s e i n d i v i d u a l s m a y be unreliable. A n a l y s i s of t h e i r b l o o d a n d u r i n e s h o w o n l y r e c e n t c o c a i n e use, s i n c e c o c a i n e and its m e t a b o l i t e s a r e e l i m i n a t e d w i t h i n h o u r s or days, d e p e n d i n g on t h e a m o u n t of c o c a i n e t h a t w a s t a k e n (i, 2). R e p o r t s h a v e a p p e a r e d in t h e l i t e r a t u r e t h a t t h e a n a l y s i s of h a i r m a y m o r e a c c u r a t e l y r e f l e c t t h e u s a g e of c o c a i n e by an i n d i v i d u a l , s i n c e b e n z o y l e c g o n i n e a n d c o c a i n e are d e p o s i t e d in h a i r a n d a p p e a r to r e m a i n in t h e h a i r (3-5). One r e p o r t (6) h a s i n d i c a t e d t h a t c o c a i n e c o n c e n t r a t i o n s in h a i r f r o m c o c a i n e a d d i c t s w e r e 0.6 to 6.4 n g / m g of h a i r a n d a f t e r 3 m o n t h s of a b s t i n e n c e f r o m t h e d r u g by t h e s e s u b j e c t s t h e level of c o c a i n e w a s 0.3 to 0.6 n g / m g of hair. C o c a i n e a n d b e n z o y l e c g o n i n e are a l s o r e p o r t e d to be f o u n d in s h e e p (6), rat (7), a n d m o u s e (8) hair. O n l y one study, in w h i c h a n i m a l s w e r e i n j e c t e d w i t h 2 m g / k g of cocaine, m o n i t o r e d t h e a c c u m u l a t i o n of c o c a i n e , b u t n o t b e n z o y l e c g o n i n e , in h a i r o v e r 40 d a y s (6). W e are u n a w a r e of a n y p u b l i s h e d i n v e s t i g a t i o n s in w h i c h t h e a c c u m u l a t i o n a n d d i s a p p e a r a n c e of b o t h c o c a i n e and b e n z o y l e c g o n i n e w e r e e x a m i n e d in h a i r f o l l o w i n g t h e a d m i n i s t r a t i o n of v a r i o u s d o s e s of cocaine. It w a s t h e i n t e n t of t h i s s t u d y to add f u r t h e r i n f o r m a t i o n on t h e p r e s e n c e of c o c a i n e and b e n z o y l e c g o n i n e in h a i r by t h e u s e of an a n i m a l model. R e c e n t i n v e s t i g a t i o n s in o u r l a b o r a t o r y h a v e u s e d t h e r a t to d e t e r m i n e t h e p r e s e n c e of c o c a i n e a n d b e n z o y l e c g o n i n e in p a r o t i d saliva, plasma, and u r i n e as w e l l as the d e p o s i t i o n of c o c a i n e a n d b e n z o y l e c g o n i n e in p r e g n a n t and n o n p r e g n a n t f e m a l e r a t s (9, i0). T h e p r e s e n t s t u d y w a s d e s i g n e d to a s c e r t a i n w h e t h e r the concentrations of c o c a i n e and benzoylecgonine in r a t h a i r are r e l a t e d to t h e d o s e s of c o c a i n e t h a t are a d m i n i s t e r e d . In a d d i t i o n , the d i s a p p e a r a n c e of c o c a i n e and b e n z o y l e c g o n i n e from hair was m e a s u r e d a f t e r t h e d i s c o n t i n u a t i o n of c o c a i n e t r e a t m e n t s .
Methods Animals. M a l e S p r a g u e - D a w l e y r a t s (150 - 180 g) w e r e o b t a i n e d f r o m Charles River Laboratories, Inc. ( W i l m i n g t o n , MA) a n d w e r e h o u s e d for 1 w e e k p r i o r to e x p e r i m e n t a t i o n at 22 ± 1 °C w i t h a l i g h t c y c l e f r o m 6:00 A M to 6:00 PM. T h e a n i m a l s h a d f r e e a c c e s s to P u r i n a R o d e n t L a b o r a t o r y C h o w 5001 (Ralston P u r i n a Co., St. Louis, MO) and water.
Cocaine Experiments.
Animals were injected intraperitoneally with c o c a i n e h y d r o c h l o r i d e (5, i0 or 20 mg/kg) for 20 d a y s o v e r a 2 8 - d a y period. T h e d r u g w a s a d m i n i s t e r e d on M o n d a y t h r o u g h F r i d a y ; no d o s i n g o c c u r r e d on w e e k e n d s ; t h e f i r s t i n j e c t i o n w a s g i v e n on D a y 1 (a T u e s d a y ) a n d t h e last on D a y 28 (a M o n d a y ) . Hair samples were r e m o v e d w i t h an e l e c t r i c s h a v e r f r o m a s m a l l a r e a on t h e b a c k s of t h e a n i m a l s t w i c e a week, b e g i n n i n g on D a y 4 a n d c o n t i n u i n g e v e r y T u e s d a y a n d F r i d a y u n t i l c o c a i n e a n d b e n z o y l e c g o n i n e w e r e no l o n g e r d e t e c t e d in r a t h a i r (Day 53); h a i r s a m p l e s w e r e n e v e r t a k e n f r o m a p r e v i o u s l y s h a v e d area. A p p r o x i m a t e l y 50 m g of h a i r w a s u s e d f r o m e a c h s a m p l e a n d a n a l y z e d for c o c a i n e a n d b e n z o y l e c g o n i n e by gas chromatography/mass s p e c t r o m e t r y (GC/MS). B l o o d s a m p l e s (300 ~L), t a k e n f r o m t h e o r b i t a l s i n u s of t h e r a t s on D a y s 25, 32, 39, a n d 46 w e r e a n a l y z e d by GC/MS. T h r e e r a t s w e r e h o u s e d p e r c a g e in t h i s
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experiment. In a n o t h e r experiment, all a n i m a l s w e r e h o u s e d individually in metabolic c a g e s so t h a t u r i n e samples, uncontaminated by feces, could be o b t a i n e d . These animals were injected with cocaine h y d r o c h l o r i d e , 20 mg/kg, ip, as w a s d o n e in t h e e x p e r i m e n t d e s c r i b e d above, e x c e p t t h a t o n l y i0 i n j e c t i o n s w e r e g i v e n a n d t h e last d o s e of c o c a i n e w a s g i v e n on Day 14. Hair samples were obtained during t h i s e x p e r i m e n t in t h e s a m e m a n n e r and t i m e p e r i o d s as p r e v i o u s l y d e s c r i b e d , up to D a y 15. S a m p l e Preparation and Analysis. In o r d e r to p r e v e n t t h e loss of c o c a i n e d u e to h y d r o l y s i s , all c o l l e c t i n g t u b e s for b l o o d and u r i n e s a m p l e s c o n t a i n e d 30 m g of s o d i u m f l u o r i d e (11). The collecting t u b e s for b l o o d a l s o c o n t a i n e d 30 u n i t s of s o d i u m h e p a r i n to p r e v e n t c o a g u l a t i o n of blood. T h e b l o o d s a m p l e w a s c e n t r i f u g e d at 2000 x g for 10 m i n and p l a s m a removed. For t h e a n a l y s i s of c o c a i n e and benzoylecgonine, 100 ~ L of p l a s m a a n d u r i n e w e r e u s e d (9, 10). Cocaine and benzoylecgonine were extracted from hair by a modification of t h e p r o c e d u r e s described by V a l e n t e et al (3). A f t e r w a s h i n g t h e h a i r s a m p l e w i t h d i s t i l l e d w a t e r to r e m o v e a n y possible external contamination on the h a i r shaft, the s a m p l e of h a i r ( a p p r o x i m a t e l y 50 m g d r y weight) w a s i n c u b a t e d o v e r n i g h t w i t h 1 m L of 0.I M h y d r o c h l o r i c a c i d at 45 °C. T h e a c i d e x t r a c t was n e u t r a l i z e d w i t h i00 ~ L of 1 N s o d i u m h y d r o x i d e s o l u t i o n a n d 0.9 m L of 0.05 M p h o s p h a t e b u f f e r solution. T h e i n t e r n a l s t a n d a r d for the GC/MS analysis, scopolamine (50 ~ L of a i0 ~ g / m L s o l u t i o n (or 500 ng)) w a s a d d e d to t h e m i x t u r e a n d t h e e n t i r e s o l u t i o n w a s e l u t e d on a c o n d i t i o n e d C-18 e x t r a c t i o n column. Cocaine and benzoylecgonine w e r e r e m o v e d f r o m t h e c o l u m n by 600 ~ L of a c i d i c m e t h a n o l . The e l u a n t w a s d r i e d at 70 °C u n d e r a n i t r o g e n a t m o s p h e r e . A f t e r the centrifuge tube cooled to r o o m t e m p e r a t u r e , derivatization was accomplished by the addition of 50 ~L of silylation grade acetonitrile and i00 ~L of N-methyl-N-trimethylsilyltrifluoroa c e t a m i d e (MSTFA, P i e r c e C h e m i c a l Co., R o c k f o r d , IL) and h e a t i n g the centrifuge t u b e for 15 m i n at 70 °C (Taylor a n d Jain, 1987). F o l l o w i n g t h e c o o l i n g of the sample, 1 ~ L w a s i n j e c t e d into a gas chromatograph/mass spectrometer system for identification and quantitation of c o c a i n e and benzoylecgonine. All reagents and s o l v e n t s u s e d in t h e e x t r a c t i o n p r o c e d u r e w e r e of a n a l y t i c a l grade. The a n a l y s i s for c o c a i n e and b e n z o y l e c g o n i n e in blood, urine, and h a i r w a s d o n e by GC/MS. T h e G C / M S s y s t e m w a s a V a r i a n 3400 gas chromatograph/Finnegan M A T ion t r a p d e t e c t o r (ITD). The capillary column was a J&W DB-5 bonded-phase, 30 m, 0.25 m m id., a n d 0.25 micron coating, coupled to t h e ITD t h r o u g h a direct capillary interface. The injection port was a capillary split/splitless i n j e c t o r (in t h e s p l i t l e s s mode) w i t h a s i l a n i z e d g l a s s insert. The c a r r i e r gas, helium, w a s at a f l o w r a t e of 1.0 m L / m i n t h r o u g h the column. The temperature p r o g r a m w a s 170 °C w i t h a 1 m i n hold, i n c r e a s i n g to 250 °C at 30 ° C / m i n w i t h a 6 m i n hold. The injector a n d i n t e r f a c e t e m p e r a t u r e s w e r e 250 °C each. T h e ITD S c a n M o d e w a s a full scan, 72 to 190 ~ and at one s c a n / s e c . Compounds were identified by l i b r a r y search (Finnegan-Toxicology) of t h e m a s s s p e c t r u m t o g e t h e r w i t h r e t e n t i o n time. S t a n d a r d s of c o c a i n e and b e n z o y l e c g o n i n e w e r e o b t a i n e d f r o m the R e s e a r c h T e c h n o l o g y B r a n c h of NIDA (Rockville, MD). Cocaine and the MSTFA derivative of benzoylecgonine exhibited retention times of 448 and 482 sec,
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respectively. The internal standard, the M S T F A derivative of scopolamine, h a d a r e t e n t i o n t i m e of 538 sec on t h e c h r o m a t o g r a m . T h e l o w e s t s e n s i t i v i t y of the m e t h o d for c o c a i n e and b e n z o y l e c g o n i n e is i00 n g / g w h e n 50 m g of a h a i r s a m p l e is u s e d and the c o r r e l a t i o n c o e f f i c i e n t is 0.22. Calculations and S t a t i s t i c s . The pharmacokinetic parameters for cocaine and benzoylecgonine were determined using standard pharmacokinetic mathematical calculations as u s e d by K h a n et al (12). First-order disappearance d a t a w e r e g r a p h e d by the least s q u a r e method. Statistically significant differences in g r o u p e d d a t a w e r e d e t e r m i n e d by an a n a l y s i s of v a r i a n c e (ANOVA). Multiple comparisons of g r o u p s were analyzed by A N O V A followed by the S c h e f f e ' s Test. Results
F i g u r e s 1 shows the a c c u m u l a t i o n and the d i s a p p e a r a n c e of c o c a i n e t h a t was f o u n d in rat h a i r d u r i n g the 5 3 - d a y e x p e r i m e n t a l period. In general, c o c a i n e a p p e a r e d to c o n t i n u e to a c c u m u l a t e in the h a i r u n t i l a p p r o x i m a t e l y D a y 18, r e m a i n e d at a s t e a d y - s t a t e level f r o m Days 18 to 29, e x c e p t for t h e h a i r c o c a i n e c o n c e n t r a t i o n s w h e n the
7,
7000
6O0O A
o
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o
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lOOO
4
8
12
16
20
24
28
32
TIME ( days
FIG.
36
40
44
48
52
56
60
)
1
T h e a p p e a r a n c e and d i s a p p e a r a n c e of c o c a i n e in rat h a i r f o l l o w i n g t h e ip i n j e c t i o n of c o c a i n e h y d r o c h l o r i d e in d o s e s of 5 ( : : ), i0 ( 0 .... 0 ), and 20 ( 0 0 ) mg/kg.
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20 m g / k g d o s e of c o c a i n e w a s g i v e n , a n d t h e n t h e c o n c e n t r a t i o n of d r u g b e g a n to f a l l as t h e d o s i n g w a s d i s c o n t i n u e d . Using the data f r o m D a y 29 as an e x a m p l e , t h e m e a n ± SE c o n c e n t r a t i o n s of c o c a i n e w e r e 1752 ± iii, 2 5 6 8 ± 174, a n d 4 5 1 3 ± 185 n g / g of h a i r w h e n 5, i0 or 20 m g / k g of c o c a i n e h y d r o c h l o r i d e was administered, respectively. These data were all significantly different from each other (p < 0.05) a n d t h e r e w a s a s t a t i s t i c a l l y significant correlation between t h e d o s e s of c o c a i n e h y d r o c h l o r i d e administered and the hair cocaine concentrations t h a t w e r e a s s a y e d (r = 0.99, p < 0 . 0 1 ) .
The a p p e a r a n c e and d i s a p p e a r a n c e of b e n z o y l e c g o n i n e in rat hair samples from the same animals is shown in Figure 2; the pattern of the c o n c e n t r a t i o n s of this cocaine m e t a b o l i t e in hair was similar to that o b s e r v e d with cocaine itself, as d e s c r i b e d above. Again, using the data from Day 29, the mean ± SE levels of b e n z o y l e c g o n i n e achieved by the a d m i n i s t r a t i o n of 5, i0, and 20 mg/kg of cocaine h y d r o c h l o r i d e were 354 ± 25, 437 ± 17, and 687 ± 42 ng/g of hair, respectively. These data were all s i g n i f i c a n t l y d i f f e r e n t from each other (p < 0.05); the c o r r e l a t i o n coefficient (r = 0.99) between the doses of cocaine hydrochloride and the concentrations of b e n z o y l e c g o n i n e in the hair was highly significant (p .--4) 1o m~Jkg ~.,.....@
20
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coc.,~.
¢
5 mg/kg
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